Differentiation and Regulation of T_H Cells: A Balancing Act for Cancer Immunotherapy

Abstract

Current success of immunotherapy in cancer has drawn attention to the subsets of T_H cells in the tumor which are critical for activation of anti-tumor response either directly by themselves or by stimulating cytotoxic T cell activity. However, presence of immunosuppressive pro-tumorigenic T_H subsets in the tumor milieu further contributes to the complexity of regulation of T_H cell-mediated immune response. In this review, we present an overview of the multifaceted positive and negative effects of T_H cells, with an emphasis on regulation of different T_H cell subtypes by various immune cells, and how a delicate balance of contradictory signals can influence overall success of cancer immunotherapy. We focus on the regulatory network that encompasses dendritic cell-induced activation of CD4⁺ T_H1 cells and subsequent priming of CD8⁺ cytotoxic T cells, along with intersecting anti-inflammatory and pro-tumorigenic T_H2 cell activity. We further discuss how other tumor infiltrating immune cells such as immunostimulatory T_H9 and T_fh cells, immunosuppressive T_reg cells, and the duality of T_H17 function contribute to tip the balance of anti- vs pro-tumorigenic T_H responses in the tumor. We highlight the developing knowledge of CD4⁺ T_H1 immune response against neoantigens/oncodrivers, impact of current immunotherapy strategies on CD4⁺ T_H1 immunity, and how opposing action of T_H cell subtypes can be explored further to amplify immunotherapy success in patients. Understanding the nuances of CD4⁺ T_H cells regulation and the molecular framework undergirding the balancing act between anti- vs pro-tumorigenic T_H subtypes is critical for rational designing of immunotherapies that can bypass therapeutic escape to maximize the potential of immunotherapy.

CD4⁺ T Cells Classification

As immunotherapy emerges as an effective therapeutic strategy in cancer, T helper (T_H) cells have received widespread interest owing to their integral role in anti-tumor immune responses as has been demonstrated by diverse pre-clinical and clinical models (1, 2). While CD8⁺ cytotoxic T lymphocyte (CTL) function has been explored extensively in recent years in the context of immunotherapy (3), research shows the crucial role of CD4⁺ T_H cells and its interaction with dendritic cells (DC) to transmit necessary molecular help that stimulates CTL function (4). T_H1 and T_H2 subclasses of helper T cells engage in molecular crosstalk with multiple immune signaling pathways and have been investigated for their immunotherapeutic relevance in cancer. Considering the multidimensional role of CD4⁺ T_H cells, it is of utmost importance to understand the biology of these cells and how they contribute to tumor immune responses. Non-naïve CD4⁺ T cells are categorized as either effector or memory T cells. CD4⁺ memory T (T_M) cells constitute a subpopulation of CD4⁺ T cells crucial in the immune system response against infections and non-infectious antigen exposure. Detailed mechanisms of differentiation and function of each T_M cell subtype is not discussed in this review, since it has been extensively reviewed elsewhere (5, 6). T_M cells are broadly subclassified into T_RM (tissue- resident memory cells) cells which are thought to reside specifically in the area of previously infected tissue, while T_CM (T central memory) and T_EM (T effector memory) cells are found circulating in the blood (both subtypes), lymphoid organs (T_CM cells) and peripheral organs (T_EM cells) (7), and overall, has been shown to be critical for eliciting anti-tumor immune response (8).

CD4⁺ T Cells in Cancer

While distinct surface marker and functional profiles set T_M cell subtypes apart, T_RM cells have been crucial in anti-tumor immunity since a T_RM cell signature in the tumor has been associated with favorable prognosis in terms of disease-free survival and overall survival in breast cancer, ovarian cancer, cervical cancer, melanoma, lung cancer, head and neck squamous cell carcinoma, gastric cancer, bladder cancer and pancreatic cancer (5). Along with the expression of CD103 (integrin-αE) and CD69 surface markers, expression of immune checkpoint regulator genes such as PD-1, CTLA-4, TIM-3 and LAG-1 on T_RM cells obtained from solid tumors, clonal expansion of PD-1⁺TIM-3⁺ T_RM cells with high expression of proliferation and cytotoxicity markers, and enrichment of this specific cell type in lung cancer patients responding to PD-1 antibody therapy (9) suggest T_RM cells are a promising target for checkpoint inhibitor antibodies to offer therapeutic benefit in a myriad of solid tumor types.

In >300 patients with early stage triple negative breast cancer (TNBC), Savas et al. identified a gene signature of T_RM cells (high expression of the integrin αEβ7 αE chain (CD103) and significantly lower expression of SELL, KLRG1, KLF2, S1PR1 and S1PR5 genes) by single cell sequencing that shows significant positive association with reduced risk of recurrence and overall survival (10). In TNBC patients receiving combination therapy of chemotherapy with immunotherapy, specifically pembrolizumab, and/or targeted therapy, T_RM cell gene signature was associated with higher pathological complete response rate (pCR) in the I-SPY 2 neoadjuvant trials with 989 patients (11) and in the KEYNOTE-086 trial, in 200 patients with advanced-stage TNBC receiving pembrolizumab monotherapy (12–14). Compared to their CD8⁺ counterparts, CD4⁺ T_M cells appear to be persistent and regulated separately, independent of previous antigen exposure or homeostatic mechanisms (15). In the context of cancer therapy, long-lasting response to tumor antigen is critical, hence the importance of developing immunotherapies that stimulate these responses via CD4⁺ T_M cells.

T_H Cells: Functional Classification

Activated CD4⁺ T cells differentiate into several functional classes based on the cytokine milieu, antigen presentation, and expression of costimulatory molecules. Combinations of environmental stimuli and autocrine cytokine production lead to the induction of several signaling pathways to regulate the expression of lineage-specific transcription factors. CD4⁺ T_H cells are polarized to one of several effector types defined by cytokine profiles and immune functions: T_H1, T_H2, T_H17, T_H9, T_reg, and T_fh (16–20). Here we discuss the differentiation and secretion profile of each T_H cell subtypes (Figure 1), before delving deep into the molecular mechanism of signaling crosstalk between DC, CD4⁺ and CD8⁺ T cells and its therapeutic implication.

Figure 1

T_H1 Immune Response

T helper type 1 (T_H1) and type 2 (T_H2) are the two predominant classes of CD4⁺ T_H cells and were the first to be characterized by the production of interferon gamma (IFN-γ) and interleukin-4 (IL-4) cytokines, respectively (18). Specifically, generation of a T_H1 effector subset is dependent on IL-12 and IFN-γ cytokines. IL-12 recruits natural killer (NK) cells to produce IFN-γ and together leads to activation of the signal transducer and activator of transcription-1 (STAT1) and STAT4 signaling pathways to induce the expression of the major transcription factor T-box expressed in T cells (T-bet), which drives T_H1 differentiation by suppressing T_H2/T_H17 differentiation (17–20). Positive feedback regulation by IFN-γ secreted from these CD4⁺ T_H1 cells support further T_H1 differentiation (18, 19). The major cytokines and chemokines secreted from T_H1 immune cells are the primary effector molecules downstream of immune cell signaling and will be discussed in detail later in this review.

T_H2 Immune Response

Polarization to the T_H2 effector lineage is dependent on production of IL-4, stimulating STAT6 signaling to upregulate the GATA3 transcription factor (18, 20). Similar to T_H1 differentiation, a positive feedback loop is supported by autocrine IL-4 secreted from T_H2 cells, while combined IL-6 production by antigen presenting cells and GATA3 expression suppresses T_H1 differentiation (17–20). The balance between IFN-γ and IL-4 feedback loops is critical to the balancing act between T_H1 and T_H2 CD4⁺ T cell immune responses.

T_H2 differentiation has been shown to be dependent on IL-4 signaling via STAT6 signaling and transcriptional upregulation (21, 22). Once believed to solely derive from T_H2 cells, IL-4 has since been known to be secreted by B cells, natural killer T cells (NKT), naïve CD4⁺ T cells and mast cells that can induce T_H2 differentiation (23). Regulation of T_H2 differentiation and cytokine profile has been comprehensively discussed previously (22, 24, 25). Binding of IL-4 to IL-4 receptors on immune cells leads to STAT6 phosphorylation, nuclear translocation, and expression of GATA3 transcription factor, resulting in T_H2 cytokine secretion and eventual tumor growth and metastasis (26, 27). In studies ranging from lymphoma, melanoma, colorectal, breast, and lung cancer, STAT6 is overexpressed within the tumor microenvironment (TME) as an immunosuppressive signal to promote the function of M2 macrophages to assist in tumor growth and inflammation (28). To prevent dominance over each other, IL-12 expression from T_H1 cells inhibits the differentiation of T_H2 cells, while IL-4 inhibits T_H1 differentiation (29).

Following differentiation, T_H2 cells secrete IL-4, IL-5, IL-10, IL-13, and IL-17, not all of which are beneficial in cancer and have been shown to contribute to the tumor promoting role of this subtype. While IL-4, IL-5 and IL-13 have been documented to contribute to cancer growth and metastasis (21, 30, 31), a dual pro- and anti-tumorigenic role of IL-10 has been reported in recent literature, as reviewed elsewhere (32, 33). IL-10 elicits an anti-inflammatory immune response, downregulates T_H1 cytokine function and MHC class II antigen presentation (29). Simultaneously, binding of IL-10 with its cognate receptor activates STAT3 signaling and transcription of anti-apoptosis and cell cycle progression genes that further strengthen the protumorigenic effect (34).

T_H9 Immune Response

Expanding our view of CD4⁺ T_H1 and T_H2 cells, there are some less explored T_H cell subsets which have unique potential in adaptive anti-tumor immunity. T_H9 CD4⁺ T cells were once believed to be included within the T_H2 subset, before being recognized as an individual population (35). Differentiation from naïve T cells to T_H9 cells is facilitated by TGF-β and IL-4, mostly secreted from T_H2 cells and these T_H9 cells can stimulate uptake and presentation of antigens by DC for CD8⁺ T cell activation by secretion of IL-9 and signaling via CCL20-CCR6 axis (36, 37). While the functional profile of T_H9 cells appears like that of T_H1 cells, T_H9 cells were found to be less exhausted in the TME of lung carcinoma patients (38). This could offer a possible improvement to immunotherapy treatments if T_H9 cell proliferation can be increased, driven by the secretion of CCL20-CCR6 and IL-3. In tumor models, activation of this CCL20-CCR6 axis by T_H9 significantly drives DC generation and the proliferation of CD8⁺ T cells to attack cancer cells in the TME (35). IL-3 is also involved in the prevention of DC apoptosis, allowing prolonged CTL activation within draining lymph nodes (36). IL-21 secretion by T_H9 has also been shown to increase during anti-tumor response, which stimulates IFN-γ production by CD4⁺ T cells and is also involved in NK cell activation (36). An adoptive cell therapy study comparing the effects of T_H1, T_H17, and T_H9 cell transfer determined that T_H9 can induce a powerful enough immune response to fully regress B16 melanoma tumors in C57BL/6 mice. T_H9 cells outcompeted both T_H1 and T_H17 responses, both of which were able to temporarily regress the tumor, but eventually succumbed to tumor growth relapse (38).

T_H9 effector differentiation results from STAT6 signaling to express the IRF-4 transcription factor through TGF-β and IL-4 cytokine production (17, 20). Significance of Notch1 developmental signaling to induce T_H9 differentiation has been investigated in recent years. Notch1a activates the transcription factor SMAD3, which binds to the IL-9 cytokine promotor, and increases T_H9 proliferation (35). The primary concern regarding T_H9 function in the TME is the inconsistency within various tumor types. While the increase of the CCL20-CCR6 interaction is beneficial in antigen uptake by DC, the expression of CCL20 can also promote tumor cell migration as seen in a study involving lung carcinoma. IL-9 secretion can suppress immune cell response as well. However, a study found that the neutralization of secreted IL-9 limits the effect to only migration of the immune cells without affecting other immune functions (39).

As previously stated, T_H2 and T_H9 cells share many transcription factors involved in the differentiation of these subsets. While T_H2 and T_H9 both share the STAT6 signaling pathway, there are some other transcriptional differences that set them apart. For instance, PU.1, part of the EST family of transcription factors, is more highly expressed in T_H9 cells compared with T_H2 cells and is linked to targeted IL-9 secretion from T_H9 cells, while constraining T_H2 differentiation. On the same note, the IL-4R-STAT6-GATA3 axis in T_H9 cells is functionally different than in T_H2 cells. In T_H9 cells, its role is to act on FoxP3 expression induced by TGF-β, while the same axis drives IL-4 expression from T_H2 subsets (36). Therefore, despite shared transcription factors between the subsets of T_H cells, each one has a distinct role to play in one subset that is separate from the other, leading to polarization and functional differences.

T_H17 Immune Response

Following T_H1 and T_H2 effector classifications was the recognition of T_H17 and regulatory T cells (T_reg), which differentiate through similar cytokine production profiles. T_H17 lineage is characterized by the production of IL-17A-F, IL-21, IL-22, IL-10, IL23, and CCL20. Polarization proceeds through three stages with TGF-β and IL-6 driving T_H17 differentiation via STAT3 activation and expression of major transcription factor RORγt (18–20). Autocrine amplification by IL-21 production and secretion of IL-23 from antigen presenting cells (APC) stabilizes the T_H17 lineage (19, 20). IRF-4 is also important for T_H17 subtype induction, amplification and stabilization by IL-21 and subsequent IL-17 production (16). While TGF-β is important to T_H17 differentiation, high concentrations of TGF-β can result in the activation of STAT5 signaling and upregulation of the FOXP3 transcription factor to drive T_reg differentiation (18, 20). T_H17 immune cells display plasticity during immune response and induce immune regulatory functions (40), contributing to impaired immune functions by targeting granzyme B production, a dominant marker for cytolytic CD4⁺ activity (1, 41).

In the context of tumor immune response, T_H17 cells can not only use these similarities to T_H1 as an effector memory cell, but its stem-like properties can allow them to elicit immune response for a longer duration than T_H1 cells, positing the question of further research into their future use in cellular immunotherapy (42, 43). During tumor development, T_H17 promoting chemokines and cytokines are expressed within the TME, such as CCL4, CCL17, CCL22, IL-1β, IL-6, IL-23, and TGF-β (44). While T_H17 exhibits anti-tumor immune responses, the increase of these promotors is driven by tumor-associated macrophages (TAM) within the tumor to assist with tumor growth. Evidence of this can be found in melanoma, breast, ovarian, hepatocellular, pancreatic, and renal cancers and can be attributed to the role of cytokine IL-17 in angiogenesis by increasing VEGF and IL-6 production and myeloid-derived suppressor cells (MDSC) production resulting in immunosuppression within the tumor (45).

T Follicular Helper Cells

T follicular helper cells (T_fh) are considered the fifth major lineage of CD4⁺ T_H cells and are involved in the generation of high-affinity antibody responses by supporting B cell proliferation and helping to facilitate immunoglobulin class switching (19). The production of IL-6 and IL-21 induces the expression of the Bcl-6 transcription factor through STAT3 signaling and leads to the polarization to a T_fh effector class (19, 20). In systemically untreated breast cancer patients, CD4⁺ T cells were found to be the principal component of the tumor infiltrating lymphocytes (TIL) and along with T_H1, T_H2 and T_H17 subtypes, were also enriched for T_fh populations (46). Purified CD4⁺ T cells from a cohort of non-small-cell lung carcinoma (NSCLC) patients showed a T_fh signature associated with heightened CTL proliferation and adoptive transfer of T_fh cells in a murine tumor model augmented CTL function and inhibited tumor growth in vivo (47). Expression of ICOS and PD-1 as markers of activated T_fh cells in breast cancer has been reported while RNA analysis showed enhanced expression of IL-21, IFN-γ, and CXCL13 on sorted T_fh TIL, and only ICOS⁺PD-1⁺T_fh TIL from HER2⁺ and triple negative breast cancer were capable of inducing in vitro IgG secretion by B cells (46). Details of T_fh cells differentiation, signaling and functional profile has been reviewed comprehensively elsewhere (48, 49).

Regulatory T Cells

CD4⁺ regulatory T (T_reg) cells exhibit critical roles in maintaining self-tolerance and preventing various autoimmune diseases. In contrast, T_reg cells also play a detrimental role in promoting cancer progression via regulating immune surveillance and suppressing anti-tumor immune response (50). Elevated levels of T_reg cells is associated with disease progression and poor survival in patients with various types of cancer (51, 52) as it is postulated that the reduced efficacy of various targeted therapies and immunotherapies is hindered by the activation of T_reg cells. T_reg cells has the ability to limit the function of antigen presenting cells by CTLA-4 dependent downregulation of CD80 and CD86 expressions, thereby evading tumor antigen presentation and tumor-specific T cell activation (53). In addition, the interlink between the expression of PD-1 on T_reg cells was observed as a negative regulator, highlighting that PD-1 blockade therapy may not only recover dysfunctional CD8⁺ T cells, but also enhances the suppressive function of T cells in cancer (54, 55). T_reg cells may also regulate the anti-tumor effects of T cells via the secretion of important immune suppressive cytokines such as IL-10, IL-35 and TGF-β (56). TGF-β secretion from T_reg cells can regulate CTL function and reduce anti-tumor immunity, since an almost complete suppression of CD8-mediated cytolytic activity was found to be essentially dependent on TGF-β signaling, and CD8⁺ cells with a dominant negative TGF-β receptor were resistant to this suppression (57). Previous studies have shown that T_reg cells can prevent CD4⁺ and CD8⁺ T cells proliferation and function by decreasing the availability of IL-2 (58). STAT5 signaling activation in T_reg cells utilizing IL-2/IL-2 receptor signaling is necessary to acquire immunosuppressive function and control CD8⁺ T cell expansion (59). A recent study investigated the antigen specificity for T_reg cells in metastatic melanoma, gastrointestinal cancer and ovarian cancer and found that intratumoral T_reg cells reacted specifically to tumor antigen, resulting in activation and clonal expansion of T_reg cells (60).

Role of DC in CD4⁺ T_H Cell Differentiation and Function

DC can be characterized as conventional (cDC) and plasmacytoid DC (pDC) where classical DC include all DC except pDC even though they are derived from the same origin (2, 23, 61). Our lab has previously shown calcium mobilization induces mature, activated DC phenotype acquisition (i.e. CD83⁺and costimulatory molecule expression) and antigen sensitization in T cells through an apparently calmodulin-dependent mechanism in normal and transformed myeloid-derived cells (62). Our research group has also reported that in human PBMC-derived myeloid DC, presence of a calcium ionophore during DC maturation step antagonized IL-12 secretion in a calcineurin phosphatase-independent manner and showed preferential ability for T_H2 polarization (63). The inherent plasticity of DC further segregates these functional classes based on expression of surface receptors, secreted stimuli, and migratory capabilities. Plasmacytoid DC express surface markers including CD123, CD202, CD303 and CD304, which are absent from the surface of cDC, and function to monitor viral infections with capacity to secrete IFN-α and IFN-β (64, 65). Specifically, cDC are known for antigen presentation and classified as cDC1 and cDC2 based on functional activity, activation of adaptive T-cell response, and expression of MHC-I and MHC-II, respectively (2, 23, 61, 66). cDC1 express transcription factors IRF8, Btaf3, and Id2 in both human and mouse and exhibit CD141, CLEC9a, CADM1, BTLA, CD26, and XCR1 surface expression; while cDC2 polarization is driven by IRF4 and ZEB2 transcription factors and primarily express CD1c, CD11b, CD11c, CD2, FCεR1, SIRPA, and CLECL10A (64, 67, 68). As in humans, mice also demonstrate phenotypic distinction between cDC1 and cDC2 by CD8α+ (lymphoid) and CD103+ (migratory) expression on cDC1, and CD4+ (lymphoid) and CD11b+ (migratory) expression on cDC2 (65, 68, 69).

Role of DC in T_H1 and T_H2 Differentiation

Functionally, cDC1 are involved in antigen cross-presentation to stimulate CD8⁺ T cell cytotoxicity, and additionally play a role in CD4⁺ T_H1 differentiation and recruitment of NK cells through IL-12 production (23, 61). Expression of Notch ligand delta on DC upon LPS exposure has also been shown to stimulate T_H1 polarization, whereas exposure of DC to cAMP up regulators such as prostaglandin-E2 can direct CD4⁺ T cells towards a T_H2 phenotype in an IL-4 independent manner via expression of notch ligand Jagged. Similarly, CD70 expressed on mouse DEC-205+ DC can act as a T_H1 phenotype inducer, independent of IL-12 (70). Conventional DC activating CD4⁺ T cells is a controversial topic where recent studies have implicated cDC1 as being capable of activating CD4⁺ T cell responses in cancer (2, 61, 64, 71, 72); however, previously it has been understood that cDC1 secrete lower levels of IL-12 in comparison to cDC2, and cDC2 are recognized as the predominant activators of CD4⁺ T cell anti-tumor immunity (23, 64, 67, 72). This is in line with observations demonstrating that cDC2 are better equipped for CD4⁺ T cell differentiation due to preferential expression of MHC-II (64, 72, 73). Additionally, past research has demonstrated the preferential activation of CD8⁺ cytotoxic T cells by cDC1 through studies with Batf3-deficient mice unable to reject highly immunogenic tumor cell lines (74), while cDC2 have the capacity to stimulate CD4⁺ T cell differentiation and polarization into T_H1, T_H2, and T_H17 effector populations via production of an array of cytokines such as IL-23, IL-1, TNF-α, IL-6, and IL-10, cytokines (64, 67).

Role of DC in T_H9 Differentiation

Dectin-1 is a β-glucan receptor present on DC, macrophages and neutrophils and dectin-1-activated DC have been shown to secrete IL-6, IL-12p40 and TNF-α, leading to T_H1 and T_H17 polarization (75). However, Zhao et al. have described dectin-1-activated DC promote a potent T_H9 polarization in vitro by overexpression of TNFSF15 and OX40L via Syk, Raf1 and NF-κB signaling pathways. While they observed a significant increase in IL-9 and T_H9-associated transcription factor IRF4, no changes were observed for other T_H subtype-associated cytokines and transcription factors in vivo. Anti-tumor effects of Dectin-1-activated DC in melanoma and myeloma preclinical model relied heavily on this T_H9/IL-9 response while microarray analysis identified more than 40 cytokines, chemokines and costimulatory molecules such as TNF-a, TNFSF15, OX40L, TNFSF8 and low IL-12 (76, 77).

Role of DC in T_H17 Differentiation

Ability to produce cytokines IL-6, TGF-β, IL-1b and IL-23 support a critical role of DC in polarizing T_H17 phenotype, as observed ex vivo in human DC isolated from inflammatory fluids, equivalent to monocyte-derived DC in mice. Likewise, monocyte-derived DC cultured in vitro with lymphoid tissue-resident bacteria secrete T_H17-polarizing cytokines (78). In an experimental allergic encephalitis mouse model, CD11b⁺ myeloid DC in the central nervous system produce IL-23, TGF-β and IL-6, thereby inducing T_H17 cells. Similarly, stimulation of human monocyte-derived DC with intact E. coli and ATP stimulates IL-23 secretion that further activates IL-17 producing T_H17 cells (70). Significance of DC differentiation and antigen exposure on T_H cell polarization has been further highlighted in a study by Khayrullina et al. as DC differentiated in presence of prostaglandin E2 promotes an IL-17-producing T_H17 phenotype by inducing a modified IL-12/IL-23 balance and inhibition of T_H1/T_H2 polarization, both in vitro and in vivo.

Role of DC in T_fh Differentiation

Co-operation between DC and B cells induces and ensures differentiation into T_fh phenotype and lymph node-resident cDC2s in van Gogh mouse model has been shown to be sufficient for such T_fh priming. The unique localization of cDC2 in the interfollicular zone at the T cell-B cell border makes them ideally positioned to be the dominant T_fh-priming DC subset in both human and mouse, which is also consistent with preferential antigen presentation on MHC-II by cDC2s and stronger antigenic stimulation favoring T_fh cell differentiation. Mice deficient in cDC2 (Cd11cCre Irf4^−/−and Cd47^−/−), but not cDC1 (Batf3^−/−), demonstrate impaired T_fh responses to sheep RBCs and loss of DC in the T cell-B cell border leads to loss of T_fh polarization as well. However, cDC2 is not the sole determinant of the T_fh phenotype as a specific subset of cDC2 dependent on transcription factor krueppel-like factor 4 (KLF4) and expressing CD301b can induce only T_H2 but not T_fh polarization, highlighting the diversity of T cell fate determinants, that also includes recruitment of various cytokines such as IL-6, IL-12 and IL-21 not secreted by cDC2s (61, 79).

The role of DC to selectively develop adaptive regulatory T cells has been highlighted as an inducer of peripheral tolerance. Simultaneously, negative regulation by IL-10 resulting in downregulation of MHC-II expression, IL-12 secretion and maturation of DC leads to an indirect preference for immune tolerance, and can induce regulatory DC that promote an IL-10-producing T_reg phenotype (80).

Studies have noted the contribution of antigenic density in determining CD4⁺ T cell fate where higher antigen doses are associated with T_H1 differentiation in contrast to lower antigen doses leading to T_H2 differentiation (23, 61). Overall, antigenic stimulation combined with simultaneous interactions between costimulatory molecules and cytokine stimuli produced by DC induce downstream signaling pathways that lead to T cell effector differentiation as discussed above. As tumor cells more readily express MHC-I molecules, DC play a pivotal role in the activation and priming of CD4⁺ T cells to initiate the CD4⁺ anti-tumor response.

Molecular ‘HELP’ by CD4⁺ T_H Cells are Necessary for Cytotoxic Function of CD8⁺ T Cells

Cytotoxic and memory CD8⁺ T cell response as a principal component of immunity requires priming and expansion, both of which demand active help by CD4⁺ T cells. Even though the supporting role of CD4⁺ T_H cells to promote effector and memory function of CD8⁺ T cells have been well-established by late 1990s, recent research have generated crucial supporting evidence of the necessity of CD4⁺ T_H cells for anti-tumor CD8⁺ T cell function (81, 82). Neoantigen-specific vaccination has often showed largely CD4⁺ T cell response, and not CD8⁺ T cell response, in multiple pre-clinical models and clinical trials. In MMTV-PyMT spontaneous mammary carcinoma model, a unique T_H1 CD4⁺ subset was identified in non-tumor peripheral tissues that rendered protective benefit when transferred into treatment-naïve tumor hosts challenged with 4T1 tumors (83). In an aggressive B16F10 murine melanoma model, IL-21 secretion stimulated by CD4⁺ T_H cells drives CD8⁺ T cell differentiation towards CX3CR1⁺ cytotoxic effector phenotype and anti-tumor activity (84). T_H1 polarized CD4⁺ T cells offer long-term protection against tumor re-challenge and is required for response to immune checkpoint blockade therapy in a T3 murine sarcoma model (85). Based on their study with melanoma patients who showed prevalence of CD4⁺ neoantigen-reactive T cells, Ott et al. suggested two mechanisms underlying this unexpected dominance of CD4⁺ over CD8⁺ T cells, namely: 1) more efficient priming of CD4⁺ T cells compared to CD8⁺ T cells due to restriction of cross-presentation and 2) relatively higher promiscuity of MHC class II epitopes owing to relaxed binding requirements, unlike MHC class I epitopes (86).

Role of DC to Relay CD4⁺ ‘HELP’ to CD8⁺ T Cells

Priming of CD8⁺ T cells for effector function requires antigen cross-presentation with help from CD4⁺ T cells. The primary mechanism is via ‘licensing’ of DC that allows cross-presentation, essential for two-step priming of CD8⁺ T cells (2). To understand the spatiotemporal distribution and activation of CD4⁺ vs CD8⁺ T cells, in vivo imaging has demonstrated that after immunization, in the first priming step, CD4⁺ and CD8⁺ T cells encounter antigen in an independent and non-synchronous manner, presented by different subsets of cDC. Interaction between CD40 costimulatory protein on cDC and its cognate ligand CD40L (CD154) on CD4⁺ T cells is the key step in the licensing process that enhances antigen presentation on DC and allows direct interaction with CD8⁺ T cells.

The second step of priming these licensed type 1 cDC (cDC1) acts as a common platform where both CD4⁺ and CD8⁺ T cells encounter the same cDC1. XC-chemokine ligand 1 produced by CD8⁺ T cells recruits resident XC-chemokine receptor XCR1⁺ cDC in a prime position for receiving cross antigen presentation and thus, molecular help from CD4⁺ T cells is delivered to CD8⁺ T cells (2). Ahrends et al. demonstrated by RNAseq in ‘helped’ vs ‘non-helped’ CD8⁺ T cells that there is a differential expression of a multitude of genes associated with lymphocyte activation, differentiation, cell motility, and migration. Significantly enhanced mRNA and protein expression of cytotoxic effector molecules such as TNF-α, IFN-γ, FASL and granzyme B, as well as IL-2 and its receptor CD25, are regulators of CTL survival and memory. They also reported high levels of co-inhibitory immune receptors, e.g. PD-1, lymphocyte activate gene 3 (LAG3) and B and T lymphocyte attenuator (BTLA) on ‘helpless’ CTLs, rendering them unable to kill tumor cells even though they are able to exit the lymph node and enter circulation (87). These helpless T cells subsequently undergo activation-induced cell death due to TRAIL expression upon re-stimulation (88). In therapeutic pre-clinical models, vaccination with short MHC class I binding peptides hinders CTL priming and induce tolerance, whereas combination with CD40 agonist antibody infusion or DC stimulated in vitro with antigen-specific CD4⁺ T cell resulted in CTL-based anti-tumor immune response (2).

Secretion of CCL3 and CCL4 from the licensed DC guide the naïve CD8⁺ T cells to the site of antigen specific DC-CD4⁺ T cell interaction, that allows rapid interaction with the antigen presenting cDC1 even with a low frequency of both immune cell subtypes (89). CD4⁺ T cells also stimulate clonal expansion of antigen-specific CD8⁺ T cells and IFN-γ secretion, whereas ‘helpless’ memory CTLs primed without help from CD4⁺ T cells show deficiency in secondary expansion (90, 91). CD4⁺ T cells are a major source of IL-2, a key molecular help that is critical for imprinting the secondary responsiveness on CD8⁺ T cells. IL-15 is secreted from licensed DC and is considered to be necessary for imprinting secondary responsiveness even in absence of CD4⁺ T cells (92).

CD4⁺ Help in CD8⁺ T Cell Differentiation and Memory Function

Recent research has highlighted that the impact of CD4⁺ T cell help reflects on enhanced recruitment, proliferation, and effector function of CD8⁺ T cells intratumorally. In a murine tumor model, IL-2 secreted from tumor-resident CD4⁺ T cells increased CD8⁺ T cell proliferation and granzyme B expression (93). Poor survival and clonal expansion of CD8⁺ T cells in absence of CD4⁺ help has been reported, and the help was necessary for survival of memory T cells during recall expansion (81). During clonal expansion and differentiation of T cells into short-lived effector or persistent memory phenotypes, CD4⁺ T cells help in intrinsic function of CD8⁺ T cells by altering gene expression profile. The transcriptomic analysis by Ahrends et al. also showed CD4⁺ T cell help induces transcription factors and epigenetic modulators, such as T-BET, eomesodermin homologue, and inhibitor of DNA binding 3, in a preventive model that received vaccines encoding MHC class I vs MHC class II-restricted epitope-expressing HPV E7 protein. Elevated expression of CXCR4 and CX3CR1 chemokine receptors and matrix metalloprotease proteins on ‘helped’ CTLs augment their extravasation and infiltration into the tumor (87). Another study using a similar mouse model showed CD4⁺ ‘help’ has been shown to impact transcriptional landscape to support formation and maintenance of CD8⁺ effector and central memory phenotypes, and recall response in these memory T cells were help-independent (94). Defective recall response mounted by CD8⁺ memory T cells from a CD4^-/- mouse host indicated necessity of CD4⁺ help for CD8⁺ T cell functionality previously (91), and a recent study using an Influenza A virus infection model showed CD4⁺ T cell help promotes metabolic programming of CD8⁺ T cells to benefit recall response as well (95).

Molecular Nature of the ‘HELP’ Signal

Cytokine Signals

The key cytokine signals that deliver CD4⁺ T cell help to CD8⁺ T cells are IFN-γ and IL-12 secreted from conventional and CD40-stimultaed DC, respectively. It appears contribution of these two cytokines may work in a partially redundant manner, as they both promoted survival and differentiation of effector and memory CTLs by increased expression of transcriptional regulators in a mouse model (96). CD4⁺ T cells are a major source of IL-2, a key molecular signal that is critical for imprinting the secondary responsiveness on CD8⁺ T cells. IL-2 induces NAB2 protein expression by CD8⁺ T cells, inhibits TRAIL expression and promotes expansion (97). Simultaneously, IL-12p70 from licensed DC also upregulates IL-2Rα/CD25 expression on CD8⁺ T cells and therefore, enhances their responsivity to IL-2 (98). Our group reported a novel function of IL-12 to enhance recognition of tumor by T cells along with 10- to100-fold increases in peptide sensitivity and functional avidity (99). As reviewed by Kalia and Sarkar, IL-2 promotes differentiation into effector phenotype and contributes to the development and maintenance of short-lived effector responses by interaction with CD25 receptor (100). IL-15 is secreted from licensed DC and is considered to be necessary for imprinting secondary responsiveness even in absence of CD4⁺ T cells (92).

Co-Stimulatory Signals

Along with cytokines, costimulatory signaling between ligands and receptors expressed on DC, CD4⁺ and CD8⁺ T cells relay and implement CD4⁺ T cell help for T cell priming and effector function. Upregulated CD40L on CD4⁺ T cells interacts with its cognate receptor CD40 on DC and is the first step in relaying molecular help (4, 101, 102). Similarity of the cytokine profiles between CD4⁺ T cells and CD8⁺ T cells expressing CD40L has been reported and can potentially augment licensing of DC to enhance antigen cross-priming (103). CD70/CD27 costimulatory signaling has been reported to be the key mechanism to deliver CD4⁺ T cell help from DC to CD8⁺ T cells, and contribute to their clonal expansion and differentiation into effector and memory CTL in cancer and viral infections (104, 105). In a murine lung tumor model, CD27 agonism combined with anti-PD1 antibody treatment eradicated tumors and recapitulated CD4⁺ T cell help when vaccinated without helper epitopes (106), even though CD70/CD27 interaction alone may not stimulate sufficient CTL response and a combined, non-redundant role of CD27 and CD28 may contribute to the help. CD40-CD40L interaction stimulates CD80 and CD86 costimulatory molecule expression on DC and its subsequent binding with the CD28 costimulatory receptor on CD8⁺ T cells can deliver the CD4⁺ T cell help required for CTL activity, as observed in recent pre-clinical and clinical studies including anti-PD-1 and other immune checkpoint inhibitors (107, 108).

Opposing Action of Anti- and Pro-Tumorigenic CD4⁺ T_H Cells in Cancer

Research in past decades have revealed the critical and opposing role of T_H1 and T_H2 cells in determining the fate of intratumoral immune response, including therapies targeting oncodrivers and neoantigens. As shown in Figure 2, the regulatory network is multi-faceted and is governed by a range of cytokines and chemokines secreted from different T_H subtypes and hence, need to be coordinated in a balancing act for optimum efficiency of immunotherapy. We discuss the most well-known cytokines and chemokines secreted primarily from T_H1 and T_H2 cells that confer their anti- and pro-tumorigenic effects, respectively, to understand the mechanism of their opposing actions. The cytokines and chemokines secreted from the other T_H subsets have been summarized in Table 1.

Figure 2

Anti-Tumorigenic T_H Cytokines

Interferon-γ (IFN-γ)

CD4⁺ T_H1 effector cells are characterized by the production of dominant cytokines IFN-γ, TNF-α and IL-2. IFN-γ is a pleotropic cytokine and an important player in anti-tumor immunity with the ability to directly mediate tumor rejection as well as recruit and activate both innate and adaptive immune cells in the TME (109–112). The direct tumoricidal effects of IFN-γ result in cell death signaling and inhibition of angiogenesis. Increased expression of cell cycle regulators p21 and p27 induced by IFN-γ leads to cell cycle arrest, cell dormancy, and apoptosis in tumor cells via signaling pathways that induce the expression of tumor suppressor gene IRF-1, leading to caspase activation and apoptosis (109, 112, 113). Activation of the anti-proliferative STAT1 pathway by IFN-γ can lead to sensitization of tumor cells to FAS (CD95) and TRAIL resulting in apoptosis, and hindered tumor cell growth by inhibiting angiogenesis to induce a state of cellular dormancy (17, 109, 111–113). Our lab has recently elucidated a novel mechanism of IFN-γ action via ubiquitin proteasomal degradation pathway, mediated by zinc RING finger E3 ubiquitin ligase cullin-5, to facilitate proteasomal degradation of HER2 membrane receptor and improve response in HER2+ breast cancer in vitro and in vivo (114).

In the TME, IFN-γ enhances the immunogenicity of tumor cells by upregulating MHC class I and II expression to make them more susceptible to immune recognition (109, 111, 112) and influences the stromal cells in the TME including macrophages, myeloid-derived suppressor cells (MDSC), and DC (109). IFN-γ production leads to enhanced proinflammatory functions and tumoricidal activity of type I macrophages (M1) by increasing nitric oxide production and upregulates expression of MHC and costimulatory molecules on DC. Anti-tumor immune response by IFN-γ can also be elicited by recruitment of additional effector cells, namely NK cells and M1 macrophages to the TME, facilitating T-cell homing through CXCL9 and CXCL10 chemokines, and via enhanced CD8⁺ cytotoxicity in the TME (109, 111, 112).

Interleukin 2 (IL-2)

IL-2 plays a crucial role in driving T and NK cell proliferation and activation and in regulating their effector functions, such as cytolytic activity and cytokine production. IL-2 binds to IL-2 receptor (IL-2R), composed of three subunits: IL- 2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132) (115, 116). The heterotrimeric complex of IL-2αβγ is essential to regulate T cell expansion, are expressed on regulatory T cells, and binds IL-2 with the highest affinity, while T cells and NK cells express only the receptor dimer IL-2βγ (115–118). IL-2 is produced primarily by activated CD4⁺ T cells after antigen exposure, binds to its cognate receptors and drives differentiation to CD4⁺ T_reg immunosuppressive population that leads to immune tolerance. Research in the last decade has identified the role of IL-2 in promoting both T_H1 and T_H2 differentiation, and inhibiting T_H17 and T_fh development (Liao et a, 2013). IL-2 induced activation of AKT and mTORC1 signaling pathways have been shown to steer the differentiation preference towards T_H1 cells and away from T_fh subtypes (119). Binding of IL-2 to these receptor complexes induces signal transduction through three proliferative pathways: JAK/STAT, PI3K/AKT, and MAPK (115, 117, 118). Additionally, the positive feedback from CD4⁺ T_H1 produced IL-2 plays a crucial role in driving T cell effector differentiation and in the recruitment of activated cytotoxic NK and CD8⁺ T cells to the TME (116). While IL-2 was the first FDA approved immunotherapy for metastatic melanoma and metastatic renal cancer, the dual functionality of IL-2 has been reported to regulate immunosuppressive environment intratumorally (115–117). Low concentrations of IL-2 have been shown to promote T_reg function in the TME, whereby anti-tumor therapies that employ high dose IL-2 attempt to overcome this immunosuppressive role (115, 117).

Tumor Necrosis Factor-α (TNF-α)

Tumor necrosis factor-α (TNF-α) is one of the primary proinflammatory cytokines produced by CD4⁺ T_H1 cells and binds to two receptors, TNFR1 and TNFR2, that promote cell death and destruction of tumor vasculature. TNFR1 is expressed on various tumor and endothelial cells and is associated with pro-apoptotic signaling via MAPK and NFκB activation (111, 113, 120). Similar to the double-edged sword of IFN-γ, TNF-α has shown a dual tumor suppressing and tumor promoting role dependent on concentration and localization of the soluble cytokine. As TNFR1 is ubiquitously expressed on tumors as well as healthy endothelial cells and blood vessels, chronic exposure to TNF-α can cause non-specific tissue damage and has been linked to hemorrhagic necrosis (111). Additionally, TNFR2 is expressed primarily on immune cells including T_reg and MDSC (111, 120), where acute production of TNF-α is associated with T_reg expansion and increased infiltration of T_reg and MDSC populations in the TME, leading to tumor progression and decreased efficacy of immunotherapies (111, 121). Administration of even a low dose of TNF-α has shown increased expression of immunosuppressive molecules PDL-1 and TIM-3 on TIL and activate cell death pathways in tumor-infiltrating CD8⁺ CTL (121). Studies have shown that administration of TNF-α as an immunotherapy has resulted in high levels of toxicity, but localized delivery in isolated limb perfusion showed anti-tumor abilities in soft tissue sarcomas, melanoma, and hepatocellular carcinoma (111, 120).

Anti-Tumorigenic T_H Chemokines

In addition to T_H1 cytokines, the production of related chemokines has direct implications in shaping the immune landscape and TME of various cancer types. CD4⁺ T cell IFN-γ-inducible chemokines CXCL9, CXCL10, and CXCL11 recruit effector T cells to the TME, direct tumor infiltration, and are key players in T cell homing (122–125). CXCL9-11 bind to their cognate chemokine receptor CXCR3, which is expressed on cytotoxic CD8⁺ T cells, NK cells, and CD4⁺ T_H1 cells (123, 124). Upregulation of CXCR3 on activated CD4⁺ T cells is associated with optimal production of IFN-γ and a T_H1 effector phenotype (122). Additionally, CD40/CD40L signaling increases expression of CXCL10 and has been implicated in licensing DC and supporting the interactions of DC and naïve T cells in lymphoid organs (122, 126). CCL3 and CCL4 chemokines are released after interaction of DC with antigen specific CD4⁺ T cells and act as a chemoattractant for CCR5⁺ naïve CD8⁺ T cells for activation (89). Interaction of CCL19 and CCL21 with CCR7 receptor recruits T_regs, CD4⁺ T_H, T_CM, and monocyte-derived dendritic cells (mDC) to the TME. Upregulation of CXCR3 and CXCR5 chemokine receptors has been correlated with T_H1 differentiation while T_H2 cells express CCR4 receptors, induced by IL-4, to bind CCL17 and CCL22 chemokines (125). Simultaneously, CXCL9 and CXCL10 have been shown to increase levels of tumor infiltrating CD8⁺ effector T cells and NK cells, minimize metastasis, and are correlated with improved responses to checkpoint blockade and adoptive cell transfer therapies (124, 125).

Anti- vs Pro-Tumor T_H Immune Response in Cancer: Molecular Mechanism of Opposing Actions

T_H1 and T_H2 cells are often discussed in tandem in relation to cancer and tumor immune response, as T_H1/T_H2 balance, regulated by the factors summarized in Table 1, is paramount in tumor-specific immune response versus pro-tumor immune regulation. Typically, a shift in favor of T_H1 response results in dissipated T_H2 response and vice versa, resulting in either anti- or pro-tumorigenic consequences, respectively, and this shift is typically accomplished by antagonistic interaction of the cytokines produced by the T_H cells themselves (127). Depending on the TME and other external signals, the initial shift to either T_H1 or T_H2 can then become a positive feedback loop that continues to favor the specific T_H immune response (40).

T_H1 and T_H2 cells and their related cytokines have been studied in multiple cancer types and proved to play a pivotal role in prognosis, tumor fate, and patient disease-free survival. In one such study of patients with hepatocellular carcinoma, an increase in detectable IL-6 in whole blood after treatment with trans-arterial chemoembolization corresponded with a poorer prognosis and decreased overall survival rate. In the same study, a higher IFN-γ/IL-10 ratio increased overall survival rates, as did a higher IL-1/IL-10 ratio (128). In a similar study analyzing serum levels of cytokines in patients with invasive uterine cervical cancer, T_H2, T_H17, and T_reg cells were increased in peripheral blood mononuclear cells (PBMC) with a concurrent increase in their related cytokines IL-4, IL-10, IL-17, IL-23, and TGF-β (129). In a study using The Cancer Genome Atlas (TCGA) looking at glioblastoma multiforme, a low T_H2 balance correlated with better overall survival (130). These studies exemplify the importance of maintaining a T_H1-high/T_H2-low balance and the ability to use relative T_H cell prevalence and their related cytokine levels as a predictor of patient survival. However, incidence of cancer is not necessarily indicative of a pro-tumor high ratio of T_H2 over T_H1, as shown in a study looking at patients with ovarian cancer before receiving treatment. When T_H1 and T_H2 cytokine levels in the serum and cancer tissues were compared, T_H1 cytokines IL-2, TNF-α, IFN-γ, and IL-13 were significantly increased in patients compared to healthy controls. Additionally, IFN-γ/IL-4 and TNF-α/IL-4 ratios were significantly higher in cancer patients (131).

It has been previously shown that elevated T_H2 cytokines (IL-4, IL-10) and decreased T_H1 cytokines (IFN-γ, IL-2 and IL-12) correlate with poorer prognoses in breast cancer patients than those with elevated T_H1 and suppressed T_H2 cytokines. A recent study showed that alteration in T_H1/T_H2 cytokines can correlate with different subclasses of breast cancer as well. Shift in the T_H1/T_H2 balance towards a higher ratio of T_H2/T_H1 cytokines resulting in increased IL-4, IL-5, and IL-10 has been reported in TNBC. On the other hand, ER+ and luminal-like breast cancers were found to have lower T_H2 cytokines and a general shift towards T_H1 immune response. In the context of disease prognosis, such T_H1/T_H2 distribution is reflected in a better overall survival rate and prognosis in ER+ and luminal-like breast cancers (BC), and a worse prognosis in TNBC (132). T_H1/T_H2 balance in normal and cancer-associated immune response has been reviewed in general extensively elsewhere (29, 133). With respect to the cancer milieu, treatments that ensure a shift towards the anti-tumor T_H1 response are essential, while maintaining a low T_H2 response is critical to ensure a tumor-specific immune response is maintained.

In the context of pro-tumorigenic immune response in the TME, T_H2 response has been viewed as controversial, due to their possible role in tumorigenesis, along with another CD4⁺ T_H cell subset, T_H17 cells. T_H2 cells are responsible for the increase in population of tumor infiltrating M2 macrophages and eosinophils in the TME, via their expression of IL-5 and IL-13, which regulate TGF-β secretion and immunosuppressive responses (28). T_H2-induced tumorigenesis is further driven by their expression of IL-7, which can act as a pro-angiogenic factor, resulting in leaky vasculature and allowing the tumor microenvironment to expand and migratory tumor cells to enter the surrounding tissue (134). A study involving luminal breast cancer found that the presence of chemokine receptor CCR5 activates T_H2 differentiation, and the T_H2 cells in turn, help increase MDSC production within the TME, a characteristic feature shared by T_H17 cells as well but implemented via IL-17 secretion. A large enough population of MDSCmigrates to the edge of the tumor in order to prevent TIL from entering the tumor region and can severely diminish the immune response, allowing the tumor to thrive (135). A comprehensive understanding of how these T_H cells induce pro-tumorigenic immune response requires further research, to identify efficient strategies to repress the immunosuppressive populations and expand therapeutic benefit of T_H cell-based immunotherapy in cancer.

Regulation of B Cells by CD4⁺ T Cells: a Bidirectional System

Discovery of tumor infiltrating B cells (TIB) and tertiary lymphoid structures have reinforced interest in studying the significance of TIB subtypes and success of immunotherapy in cancer. Such studies have identified a dual role of TIB subtypes in stimulating or dampening of anti-tumor immune response, orchestrated by secreted antibodies, cytokines and chemokines, B cell receptor signaling, and interaction with T cells. CD4⁺ T cells act in a bidirectional regulatory network with B cells to induce differentiation of B cells which in turn, stimulates CD4⁺ T_H1 and T_H2 differentiation, suggesting the clinical significance of these immune cells for anti-tumor response. Similar to the conventional APC, B cells express MHC class II molecules on their surface and are, hence, capable of antigen presentation to CD4⁺ T cells for activation, and cognate interaction between T and B cells induce differentiation of anti-tumor T_fh cells (49, 136). Activated B cells secrete chemokines and costimulatory factors such as CCL2, CXCR4, CCL5, CXCL5, and CXCL10 to induce CD4⁺ and CD8⁺ T cell activation. Using B cell deficient and IFN-γ knockout mice along with other transgenic models of immune cell function and CD4⁺ and CD8⁺ depletion studies, Park et al. showed that anti-HER2/neu antibodies are necessary and sufficient for protection from tumor challenge, a temporary necessity for CD4⁺ T cells for 36-48h after immunization to provide help for B cells, and no requirement for CD8⁺ T cells at all. While tumor growth in immunized B cell-deficient mice was comparable to controls and showed no detectable antibodies in their serum, treatment of mice with anti-HER2 serum prevented tumor growth in vivo as effectively as adenoviral vaccination, supporting the necessity and sufficiency of antibodies for anti-tumor protection (137). In a later study, Berzofsky’s group demonstrated that even in a large and well-established subcutaneous TUBO tumor model (tumor size >2cm), vaccination with a recombinant adenovirus expressing a truncated ErbB2 antigen cured primary tumors and distant lung metastases in mice by antibody-mediated blockade of HER2/neu activity, in an Fc receptor-independent mechanism. Adoptive transfer of serum from vaccinated BALB/c mice to TUBO tumor-bearing mice resulted in significantly delayed tumor growth and showed considerable presence of anti-HER2/neu antibodies which were not observed upon deletion of CD4⁺ T cells (138), reinforcing the therapeutic benefits of anti-oncodriver antibodies and significance of CD4⁺ T cells.

On the other hand, inhibition of CTL activity in tumors by B cells can be associated with a subset of B regulatory cells (B_reg), that contribute to immunosuppression in the TME. B_reg inhibit proliferation of CD4⁺ T_H1 cells by secretion of suppressive cytokines, such as IL-10 and TGF-β, and promote conversion of CD4⁺CD25⁻ T cells to CD4⁺CD25⁺FoxP3⁺ T_reg with high expression of CTLA-4 and FoxP3, and the anti-tumor effects of B cell deficiency has been shown to be mediated by enhanced T cell and NK cell infiltration, vigorous T_H1 and CTL activity and reduced T_reg proliferation (139). These studies underline the relevance of B cell mediated antibody production and CD4⁺ T cell activation in anti-tumor immune response to encourage further research in understanding the complete therapeutic potential of B and T cell interaction.

Oncodriver-Specific and Neoantigen-Driven CD4⁺ T Helper Immune Response

Oncodrivers in Cancer

Dependence of cancer cells on oncodrivers present them as a promising candidate for targeted therapy development since these proteins are critical for survival and malignancy of tumor cells and are often overexpressed in tumors compared to healthy cells. While oncodrivers are sufficient and/or necessary for normal physiological cellular functions, their overexpression and hyperactivity often become the key regulator of tumor proliferation and escape from cellular death. Perhaps the most prominent oncodriver investigated in the context of breast cancer is human epidermal growth factor receptor 2/receptor tyrosine-protein kinase erbB-2 (HER2/Erbb2), and other members of the ERBB family of receptors, namely EGFR/HER1, HER3 and HER4, have been established as potent oncodrivers in breast cancer, along with lung, ovarian, gastric and bladder carcinoma (140–142). HER2 status has been correlated with poor recurrence-free survival and disease-specific survival in ER⁺/HER2⁺ BC (143–145), resectable gastric cancer (146, 147), and pancreatic cancer (148). Enhanced HER2-HER3 interaction and HER3 activity in breast cancer cells provide an escape route for HER2⁺ cancer cells to switch dependence, continue PI3K/AKT activity and induce trastuzumab resistance (149). In ER⁺ BC, HER3 emerges as a potent inducer of tamoxifen resistance (150), and as a prognostic marker, HER3 expression has been associated with poor survival in TNBC and HER2⁺ BC (151–153). In a cohort of 510 TNBC patients, immunohistochemistry and RNA sequencing revealed that the combined HER3-EGFR score is a more comprehensive prognostic marker than individual HER3 and EGFR scores and high HER3-EGFR score predicts worse breast cancer-specific and distant metastasis-free survival, suppressed apoptosis-inducer ATM activity, activation of EGFR, PARP1, and caspases, and inhibition of p53 and NFκB (154). Significance and current status of oncodriver-targeted immunotherapy have been reviewed previously (155).

Oncodriver-Specific T_H1 Immune Response in Breast Cancer: HER2-DC1 Vaccine

Interaction between oncodrivers and immune response has been documented in HER2⁺ BC, where trastuzumab induces antibody-dependent cell-mediated cytotoxicity (ADCC) by facilitating cross-link between tumor antigen with its antigen-binding fragment and recruitment of effector cells by interaction with the Fc region (fragment crystallizable region), resulting in cytokine release and cytotoxic cell death (156). Susceptibility to ADCC correlates with infiltration of CD16 and CD56-expressing lymphocytes in the tumor, suggesting recruitment of NK cells (157). Trastuzumab has also been shown to stimulate HER2 uptake by DC for enhanced antigen presentation and activation of antigen-specific T cells (158). Higher levels of chemokines, infiltration of T cells and monocytes, and PD-1 expression has been documented on trastuzumab sensitive breast tumors, compared to non-responding tumors (159). Our lab has reported a gradual and progressive loss of HER2-specific CD4⁺ T_H1 immune response in peripheral blood in HER2⁺ BC patients (160). Restoration of this T_H1 immune response with neoadjuvant HER2 peptide-pulsed type I DC (HER2-DC1) vaccination resulted in pathologic complete response in 30% of HER2⁺ DCIS patients in a randomized trial (161). Co-operation between CD4⁺ T_H1 cytokines IFN-γ/TNF-α and trastuzumab has been shown to be necessary for restoration of class I MHC molecule expression on HER2^high cells, critical for recognition and lysis of the cells by HER2-specific CD8+ T cells in these patients (162). In HER2⁺ IBC completely treated with trastuzumab and chemotherapy, anti-HER2 T_H1 immune responsivity independently correlates with disease recurrence and is mediated by anti-HER2 CD4⁺T-bet⁺IFN-γ⁺ (T_H1) phenotypes but not CD4⁺GATA-3⁺IFN-γ⁺ (T_H2) or CD4⁺CD25⁺FoxP3⁺ (T_reg) response (163). At the cellular level, T_H1 cytokine treatment up-regulated apoptosis and senescence in HER2⁺ BC cells (164), suggesting molecular communication between immune and oncodriver signaling. In a pre-clinical model of HER2⁺ BC, sequential anti-PD1 antibody treatment with murine HER2-DC1 vaccination significantly improves mouse survival and supports an essential role of CD4⁺ T_H1 immune response for the observed effect (165). Therapeutic success of HER2-DC1 vaccination in HER2⁺ BC supports the notion that targeting other oncodrivers employing the DC vaccine platform can have far-reaching beneficial effects in breast and other cancers dependent on oncodrivers, such as TNBC, which otherwise lacks effective therapeutic strategy. HER3 deserves attention in this context as our lab has reported progressive loss of HER3-specific T_H1 immune response in TNBC patients, and patients with residual or recurrent disease showed significant suppression of immune response compared to patients without recurrence or complete response after neoadjuvant chemotherapy (166). Future research will be essential for a comprehensive understanding of the interaction between oncodrivers and immune cell signaling in tumors, for developing efficient targeted immunotherapies with improved therapeutic success.

Neoantigens and Neoantigen-Driven Immune Response

While T cell activity towards tumor-derived neoantigen has been reported in mouse models as early as in the 1980s, they have gained renewed interest in recent years as significance of neoantigens to enhance ‘foreignness’ of tumors has been shown to be critical for success of immunotherapy, including immune checkpoint blockade therapeutics. Neoantigens are distinct from the tumor-associated antigens (TAA) which are proteins present in normal tissues and overexpressed in tumors, and therefore, peptides of TAA can be recognized by T cells following interaction with human leukocyte antigen (HLA). The most prominent TAA are HER2, MAGE, MUC1, NY-ESO-1, MART-1and mammaglobin-A, among others. Neoantigens, on the other hand, are unique non-autologous proteins expressed in tumor, due to somatic DNA alterations such as non-synonymous point mutations, insertion/deletion, gene fusion and frameshift mutations (167). Compared to TAA, neoantigens present a more appealing target for targeted immunotherapy development due to their higher immunogenicity that is enhanced because of increasing difference between the mutated and normal peptide sequence, strong individual tumor specificity, higher affinity towards MHC, and reduced risk of autoimmunity as they are recognized as foreign antigens and not affected by central immunological tolerance (168). Targeting TAA of low abundance and weak immunogenicity versus neoantigens that are abundant and highly immunogenic may not alter the intratumoral balance of anti- and pro-tumorigenic CD4⁺ T_H1 immune response, and the overall success of immunotherapy, to the same extent. As shown in Figure 3, low antigenic load presented by oncodrivers/self-antigen/TAA may require a more comprehensive shift in the balance, including both activation of anti-tumorigenic T_H1/T_H9/T_fh response and suppression of pro-tumorigenic T_H2/T_H17/T_reg/MDSC function, for effective immunotherapy; while the high abundance and immunogenicity of neoantigens may be enough to drive up one side of the balance, by either hyperactivating anti-tumorigenic response or severe suppression of immunoinhibitory populations in favor of anti-tumor immune response to result in superior therapeutic efficacy.

Figure 3

A series of studies have demonstrated correlation between tumor mutational burden and/or predicted neoantigen ‘load’ (abundance of neoantigens) and patient survival. Reports of a positive association between higher predicted neoantigen load and increased intratumoral lymphocyte infiltration (CD3⁺ and CD8⁺ T cells) and improved overall survival in colorectal, endometrial and ovarian cancer (169–171) led to studies addressing the relationship between neoantigen abundance and success of immune checkpoint blockade therapy in cancer. Indeed, in melanoma patients treated with anti-CTLA4 antibody, NSCLC patients receiving anti-PD1 antibodies and urothelial carcinoma patients receiving anti-PD-L1 therapy, the extent of DNA damage (that corresponds to tumor mutational burden and neoantigen load) correlates with therapeutic response (172–174). Even though a large number of studies have focused on teasing out the role of neoantigen-targeted CD8⁺ T cell activity in cancer, significant and preferential CD4⁺ T cell activation by neoantigens have been recognized in multiple pre-clinical and clinical studies (175). Current pre-clinical and clinical trials employ multiple platforms of neoantigen-targeted vaccines such as synthetic long peptide vaccine, DNA and RNA vaccine, and DC vaccine, along with adoptive T cell therapy. As reviewed previously, pre-clinical studies have demonstrated significant therapeutic benefit of neoantigen-targeted vaccines (167, 168). In three murine models of melanoma (B16F10), breast (4T1) and colon (CT26) cancer, the majority of the mutated neo-epitopes were recognized by CD4⁺ T cells, and vaccination with such mutations elicit robust tumor rejection (176). A recent study published with 4T1 and B16F10 murine models tested therapeutic efficacy of a novel cryo-thermal therapy with respect to conventional radiofrequency ablation and showed strong neoantigen-specific CD4⁺ T-cell response induced by cryo-thermal therapy, resulting in anti-tumor immune response and long-lasting protection against tumor re-challenge (177). Combination of local radiotherapy with an RNA-LPX vaccine that encodes CD4⁺ T cell-recognized neoantigens resulted in a poly-antigenic, potent CD8⁺ T cell response and memory that rejected CT26 tumor re-challenge, had higher number of polyfunctional IFN-γ⁺ CD4⁺ T_H1 cells specific for the immunodominant CD4 neoantigen ME1, elevated numbers of activated gp70-specific CD8⁺ T cells, and lower PD-1/LAG-3 expression. Follow-up immunotherapy with anti-CTLA4 antibody resulted in complete remission of gp70-negative CT26 tumors in all mice in this study (178). In an inducible lung adenocarcinoma mouse model, vaccination using the G12D KRAS mutations as neoantigens and a novel synthetic long peptide-containing cationic lipoplex-based delivery platform stimulated both CD4⁺ and CD8⁺ T cell response and suppressed tumor growth, while combination with checkpoint inhibitor furthered such suppression (179). Similarly, both CD4⁺ and CD8⁺ T cell response has been reported in recent clinical studies following neoantigen-specific vaccination, across multiple cancer types. Whole-exome sequencing demonstrated that TIL in metastatic cholangiocarcinoma contained CD4⁺ T_H1 cells specifically responsive against a mutation in ERBB2 interacting protein (ERBB2IP) and adoptive transfer of TIL containing mutation specific polyfunctional T_H1 cells resulted in a decrease in target lesions with prolonged survival (180). CD4⁺ T cells capable of recognizing the recurrent KRASG12V and the ERBB2 internal tandem duplication oncodriver mutations were identified in PBMC samples collected from a small cohort of NSCLC patients (181). Frequent recognition of neoantigens by CD4⁺ T_H1 cells have been reported in melanoma as well (182). In a phase I/Ib study reported last year, personalized neoantigen vaccination in glioblastoma patients increased tumor infiltrating cells, accompanied by a circulating polyfunctional neoantigen-specific CD4⁺ and CD8⁺ T cell responses enriched in memory phenotype, in patients who did not received dexamethasone (183). In treatment-naïve epithelial ovarian cancer patients, whole-exome and transcriptome sequencing analysis to identify neoantigen candidates and vaccination thereafter showed spontaneous CD4⁺ and CD8⁺ T-cell responses against neoepitopes from autologous lymphocytes in 50% of the patients, along with enhanced antigen processing and presentation machinery present in those specific tumors (184).

Therefore, along with further optimization of neoantigen prediction algorithm and targeting, a comprehensive understanding of the neoantigen recognition by CD4⁺ T cells and how that stimulates intratumoral effector and helper function of these T cells will be of utmost importance for the development of personalized immunotherapy targeting individual tumor neoantigens and demands extensive research.

Immune Checkpoint Modulators and T_H Cell Regulation

The tumor microenvironment weighs heavily on T cell differentiation. An intratumoral meshwork of regulatory immune cells and immunosuppressive cytokines/chemokines act as one of the central modulators of T cell differentiation and function. TGF-β produced by tumors can convert CD4⁺ T cells into T_reg cells in situ (185). Recruitment of MDSC in the TME aid in this suppression of T_H immune cells, where TNF-α, IL-1, IL-6, colony stimulating factor 1 (CSF-1), IL-8, IL-10, and type I interferons can also play a role in the regulation of T_H immune response to tumor cells. VEGF, IL-10, and TGF-β have been shown to inhibit DC maturation, leading to poor antigen presentation and co-stimulation of T cells, which favors T_H2 differentiation and shifts the balance from T_H1 to T_H2 phenotype (185). Manipulation of the TME, immune checkpoint regulation and cytokine levels may ensure long term tumor free survival in patients. Immune checkpoint blockades with anti-PD-1, anti-PDL1, and anti-CTLA4 antibodies to combat the inhibitory effects of TME on the immune system have been studied and showed promising therapeutic efficacy (186). Immune checkpoints are the gatekeepers of immune response and has garnered significant attention in the field of cancer immunosurveillance and immunotherapy in recent years. These inhibitory receptors/co-stimulatory molecules target T cell receptor (TCR) signaling activation, induce T cell exhaustion and anergy, and suppress proinflammatory cytokines (e.g. IFN-γ, TNF-α) secretion, ultimately resulting in immunosuppression in the TME and has been targeted with antibody-mediated checkpoint blockade therapy in recent years, as discussed comprehensively in recent reviews (187–189). Therefore, expression of these checkpoint regulators on T_H1 as well as cytotoxic T cells, while negatively impacting their proliferation and function, can be critical in determining success of checkpoint blockade therapy in high versus low density immune checkpoint-bearing tumors. In classic Hodgkin’s Lymphoma, where MHC-I expression is lost but MHC-II expression is intact, CD4⁺ T cell infiltration in tumor was correlated with better prognosis in patients and showed improved efficacy of a PD-1 blocking antibody in MHC-II-expressing lymphoma. In the same study, Nagasaki et al. showed that CD4⁺ T cell cytotoxicity played a critical role in delivering anti-tumor effects of anti-PD1 antibody which was observed in MHC-I⁻MHC-II⁺ tumors, but not on MHC-I⁻MHC-II⁻ tumors, in murine models of lymphoma and solid tumors (190). Kagamu et al. investigated NSCLC patients receiving nivolumab immunotherapy and found that treatment responders had higher circulating level of effector, CD62L^low CD4⁺ T cells prior to PD-1 blockade that correlated with effector CD8⁺ T cell abundance, and these cells expressed surface markers indicative of T_H1 phenotype (191). In a study with healthy subjects and glioblastoma multiforme (GBM) patients, PD1⁺ CD4⁺ T cells were found to be unable to proliferate but secrete IFN-γ and display exhaustion markers in RNA sequencing analyses. In GBM samples, enrichment of both PD1⁺ CD4⁺ and PD1⁺ TIM3⁺ CD4⁺ T cells suggest combined blockade of multiple checkpoints can be a requirement to tackle aggressive cancers like GBM (192). Varying levels of PD-1 expression on follicular lymphoma cells reflect on the T_fh phenotype of intratumoral CD4⁺PD-1^high T cells with no TIM3 expression that supports B cell growth, while CD4⁺PD-1^low T cells elicit an exhausted phenotype, express TIM3 with reduced cytokine secretion and cellular signaling, and significantly correlate with a reduced overall survival in follicular lymphoma patients (193). Another checkpoint modulator, CTLA-4, is constitutively expressed on CD4⁺CD25⁺ T_reg cells leading to trans-endocytosis of B7 ligands and interference with the CD28 co-stimulatory signaling, and has been deemed necessary for secretion of anti-inflammatory cytokines by T_reg cells (194, 195). Future research will be crucial to elaborate immune checkpoint regulation of CD4⁺ T_H cell differentiation and function and identify new nodes in the network for therapeutic targeting in cancer.

T_H Immune Cells in Cancer Cell Dissemination, Dormancy and Metastasis

It is widely accepted that cancer cells disseminate from non-invasive or primary tumor sites into the circulation and reach various distant organs to form overt metastasis (196). T_H1 cytokines such as IFN-γ, TNF-α and IL-2 produced by T_H1 cells contribute to inhibit tumor growth and activation of tumor-specific immune mechanisms (155, 197). On the other hand, T_H2 cytokines IL-10, IL-4 and TGF-β from T_H2 cells can promote dissemination of cancer cells dissemination and metastasis in various cancers (198). The imbalance between the ratio of T_H1/T_H2 cells and their associated cytokines correlates with decreased progression-free survival and overall survival in patients with breast, melanoma, ovarian, esophageal and colon cancers (199). Previous studies in breast cancer patients have shown that presence of cancer cells in the systemic circulation are associated with alteration of CD4⁺ T_H cells (200, 201). After dissemination, cancer cells can remain dormant for a prolonged period until they emerge for metastatic colonization in secondary organs (202, 203). T_H1 cells can reduce proliferation and mediate dormancy in these disseminated cancer cells (DCC) via IFN-γ dependent STAT1 signaling pathway activation and anti-tumor immunity (113). A mouse model of melanoma showed presence of DCC in various organs, such as the lungs, skin and reproductive tract, and regulation of their non-proliferative status by T_H1 immune cells (204). Another study using a mouse model for sarcoma also supports a role of CD4⁺ T cells to induce dormancy in cancer cells and tumor relapse (205). These reports suggest the regulatory role of T_H immune cells in controlling tumor dormancy and metastasis. Since maintaining T_H1/T_H2 immune cell balance is critical in anti-tumor immunity, therapeutics that enhance T_H1 response and prevent T_H2 activation and associated cytokines may simultaneously help to eradicate disseminated cancer cells, preventing recurrence and metastasis.

Tumor infiltrating T_H9 and T_H17 cells are observed to promote epithelial to mesenchymal transition (EMT) and migration potential of lung cancer cells and metastasis outgrowth. IL-9 and IL-17 cytokines from T_H9 and T_H17 cells can stimulate cytokine signaling and alter various genes linked to EMT and drive metastasis (39). In addition, high accumulation of T_H9 and T_H17 cells in lung cancer patients with poor survival further support their multifaceted role in cancer progression and metastasis (39). Another study has demonstrated high serum level of IL-9 and IL-17 cytokines with increased frequency of T_H9 and T_H17 cells in hepatic carcinoma patients with malignant ascites (206). This finding suggests that T_H9 and T_H17 cells may play a significant role in metastatic spread through IL-9 cytokine signaling.

Clinical Experience with CD4⁺ T_H Cells: Current Status

In the past decade, it has become clear that CD4⁺ T cells play a multifaceted role and are crucial for generating effective anti-tumor immunity. Therapeutic approaches designed to target CD4⁺ T cell responses can be broadly divided into passive immunotherapy (antibody-based therapies, adoptive cell therapy, and chimeric antigen receptor T cell therapy) and active immunotherapy (peptide vaccines, DC-based immunotherapies, immune checkpoint blockade). Here, we review current status of these immunotherapeutic approaches to stimulate tumor specific CD4⁺ T cell responses, focusing on peptide vaccines, adoptive T cell transfer and chimeric antigen receptor T cell therapy (Figure 4). Ongoing clinical trials utilizing these immunotherapy strategies have been summarized in Table 2.

Figure 4

Vaccines

Cancer vaccines were developed to stimulate specific anti-tumor T cell responses by (1) developing antigen-loaded DC ex vivo prior to vaccination (DC vaccines) and/or (2) directly administering immunogenic peptides (epitope-based vaccines). Antigens used in vaccination may span from use of peptide fragments or full proteins, DNA and mRNA, or even bulk cancer cell lysates to stimulate CD4⁺ T_H1 responses in vivo (207, 208). The first efforts to active CD4⁺ T_H1 anti-tumor immunity were actually implemented through the generation of peptide-based vaccines, whereby immunogenic class II peptide fragments from tumor associated antigens such as MUC1 (209), NY-ESO1 (210), MAGE-A3 (211), and HER2 (212) have been injected to induce antigen-specific CD4⁺ T_H1 response (1). Additionally, research into a universal cancer vaccine examined the efficacy of stimulating CD4⁺ T_H1 responses through vaccination with promiscuous epitopes from surviving and telomerase proteins (213, 214). Recently, research focus has shifted to generate cancer vaccines through stimulation of CD4⁺ T_H1 responses to mutated antigens, or neoantigens, selectively expressed by malignant tissue (168), and with the known synergistic effects of tumor specific CD4⁺ and CD8⁺ T cells, peptide epitopes capable of binding both MHC-I and MHC-II show potential to optimize vaccination efficiency (1, 207, 215). In 2017 Ott et al. demonstrated the efficacy of personalized neoantigen vaccines for the treatment of melanoma patients where specific mutations were identified in patients and synthetic long peptides were used in vaccination to stimulate both CD4⁺ and CD8⁺ responses. While CD4⁺ T_H1 cells demonstrated the highest rates of tumor specific response and targeted 60% of the unique neoantigens used across patients, resulting in no recurrence in four out of six patients 25 months after vaccination, two recurrent disease subsequently treated with anti-PD1 therapy led to complete tumor regression and expansion of the repertoire of neoantigen-specific T cells (1, 86). Similarly, Tondini et al. developed a poly-neoantigen vaccine composed of a fusion gene incorporating three neoepitopes derived from mouse colorectal tumor in combination PD-1 IBC to target both CD4⁺ and CD8⁺ responses in murine colorectal cancer models (216).

The pivotal role of DC to generate T cell mediated tumor immunity via activation, priming, and induced rapid expansion of antigen-specific T cells implicates therapeutic potential of DC-targeted immunotherapy development (215). Loading of patient autologous DC with previously identified immunogenic epitopes from tumor antigens and reinfusion of antigen-loaded DC can lead to the induction of specific anti-tumor CD4⁺ T_H1 responses (1). The first FDA approved DC vaccine (Sipuleucel-T) was approved in 2010 for metastatic prostate cancer (217). Following this, several trials have evaluated the therapeutic benefits of DC vaccines for the treatment of various cancer types (Table 2). Findings from multiple trials testing therapeutic efficacy of a DC vaccine primed with WT-1, a TAA overexpressed in glioblastoma, or autologous tumor lysate to treat patients with glioblastoma, has been reviewed by Eagles et al. (218). In 2016, De La Cruz et al. demonstrated the efficacy of a HER2-DC vaccine in HER2⁺ breast cancer patients, where treatment induced a significant increase in anti-HER2 CD4⁺ T_H1 response and improved rates of pathological complete response (160, 163, 219). A clinical trial (NCT00910650) adoptively transferring MART-1 T cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide-pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma showed evidence of tumor regression (220).

There are several cancer vaccines showing great promise for the treatment of different cancer types but there are still challenges in using this approach to treat advanced disease, and vaccination alone may be insufficient to control tumor progression. The efficacy of these vaccines is highly dependent on the identification of proper stimulatory antigens and functional status of the individual immune response in patients. Future research can optimize this immunotherapeutic strategy for eliciting tumor-specific CD4⁺ T cell responses by considering antigen dosing and immunogenicity, timing of the therapy, the role of adjuvants, immunosuppressive TME, and combinational strategies (221).

Adoptive Cell Therapy

Adoptive cell therapy (ACT) involves the generation of tumor specific T cells ex vivo that can be reinfused into patients. Clinical success of ACT depends greatly on the expansion of tumor specific T cells ex vivo, homing to the tumor site, and persistence following infusion. Although most cell therapies focus on CD8⁺ CTLs due to their tumor killing capabilities, considering the molecular ‘help’ by CD4⁺ T_H cells is required for CD8⁺ cytotoxicity and recruitment, adoptive transfer of CD4⁺ T cells may play an important role in overall tumor immune response (84). Interestingly, transfer of CD8⁺ T cells alone has shown to have low tumor free survival rates whereas transfer of both CD4⁺ T_H1 cells and CD8⁺ CTLs has shown a synergistic anti-tumor response resulting in complete regression in 80% of mice (222). Previous expansion methods focus primarily on the use of the cytokine IL-2 to expand T cells ex vivo. CD4⁺ T cells can be transformed into regulatory T cells in the presence of IL-2 and TGF-β secreted in the TME, allowing immune evasion. A study by K.L. Knutson et al. showed that IL-2 alone resulted in the loss of proliferation of antigen specific CD4⁺ T cells; however, addition of IL-12 was able to overcome the loss of proliferation (223). This suggests there is a pressing need to explore other cytokines that can successfully expand tumor specific CD4⁺ T cells without generating regulatory T cells. Elimination of immune suppressive cells such as MDSC has been shown to greatly enhance the efficacy of ACT (224), suggesting that ACT must also overcome the immunosuppressive effects of the TME and other innate immune cells to amplify the therapeutic efficacy.

Success of ACT also relies heavily on the generation of T cells that can persist long after infusion. K.A. Read et al. showed that IL-7 and IL-15 maintain important memory phenotypes in CD4⁺ T_H cells which aid in long term survival (225). IL-7 is also believed to have a role in T cell trafficking to secondary lymphoid organs. Peter Cohen and his group described tumor specific CD4⁺ and CD8⁺ T cells expansion from unfractionated PBMCs using an activator of innate immunity. Addition of toll-like receptor agonists, LPS and R848 (resiquimod), followed by addition of synthetic long peptides (>20aa) derived from widely expressed oncoproteins (MUC1, HER2/neu and CMVpp65) enhanced the processing and presentation of exogenous TAA. Addition of IL-7 enhanced the antigen-driven outgrowth of CD4⁺ and CD8⁺ T cells (226). Therefore, IL-7 and IL-15 can be potential alternatives to using IL-2 in the generation of tumor specific T cells.

Adoptive cell therapy alone is not enough in providing long term effects that can prevent relapse in patients. Combination therapies using immune checkpoint blockade, migratory molecules such as CXCR2, and stimulatory cytokines such as IFN-γ and ACT have been studied widely in melanoma models with great promise (186). Toxicity, however, can be a potential hinderance to combination therapies. Thus, finding a safe adoptive cell therapy that can harness the full effects of both CD4⁺ and CD8⁺ T cells is paramount in future immunotherapies.

Chimeric Antigen Receptor T Cell Therapy

Chimeric antigen receptor T cell (CAR-T) therapy entails genetic engineering of a patient’s own T cells to express membrane spanning fusion receptors with defined specificities for tumor associated antigens. In humans, CD4⁺ T cells as part of the CAR has been shown to induce target cell apoptosis in an MHC and Fas-independent manner, via cytolytic degranulation by perforin and granzyme (227, 228). However, reportedly low granzyme and perforin expression on CD4⁺ T cells, compared to CD8⁺ T cells, may contribute to their limited cytotoxicity (229). Equal tumor cell killing capacity of CD4⁺ and CD8⁺ CAR-T cells, albeit longer conjunction and delayed kinetics in CD4⁺ cells (230), and apoptosis and anergy in CD8⁺ T cells without the molecular help from CD4⁺ T cells in the vicinity suggest CD4⁺ CAR-T can potentiate the effects of the therapy in cancer (231). Between GBM-associated antigen-targeting CD4⁺ and CD8⁺ CAR-T cells, CD4⁺ CAR T cells showed effector persistency after tumor challenge and similarly in orthotopic GBM model, CD4⁺ CAR-T outcompeted CD8⁺ CAR-T in terms of durable anti-tumor response (232). In GBM in vitro and in vivo models, Brown et al. (233) tested anti-tumor effects of IL13Rα2-specific CAR T cells engineered from purified CD4⁺ or CD8⁺ T_CM pools and showed superior tumor killing by CD4⁺ CAR-T cells, along with higher cytokine production and persistent effector function upon tumor challenge, when compared with CD8⁺ CAR-T cells. Intracranial injection of CD4⁺ CAR-T in an NSG model of GBM showed durable anti-tumor efficacy and prolonged survival, while mice receiving CD8⁺ CAR-T cells recurred following an initial response (233). In a preclinical NSG mouse model, administration of CD19-CAR-T using CD4-targeted lentiviral vector (CD4-LV) displayed T_H1/T_H2 phenotype of the CAR-T, with a superior and faster tumor killing ability than CD8-LV CAR-T cells alone or in combination with CD4-LV. Such prolonged response by CD4⁺ CAR-T cells in preclinical and clinical models can be attributed to higher exhaustion in CD8⁺ cells (234). Overall, navigating the immune suppressive effects of the TME and reducing clinical toxicities, while maintaining a durable anti-tumor response, will be of paramount importance to successful CAR-T cell therapy for solid tumors.

Future Prospects of T_H Cells in Cancer Immunotherapy

Recent research has underscored the significance of CD4⁺ T_H cells as a component of anti-tumor immune response. In this review we discuss why CD4⁺ T_H cells are considered an integral component of current immunotherapy research and how the shifting balance between T_H1 and T_H2 cells, along with other T_H cell subtypes, modulate the intratumoral immune response. Notably, current research has pointed out how these other CD4⁺ T_H subtypes, such as immunostimulatory T_H9, T_fh and T_H17 while immunosuppressive T_H2, T_H17, T_reg cells and inhibitory function of MDSC, can sway the anti- vs pro-tumorigenic balance of T_H immune response and suggest the clinical relevance of targeting these CD4⁺ T_H subtypes. Therapeutic intervention to regulate not only T_H1 and T_H2 functional response, but other stimulatory/suppressive immune populations may be critical for more efficacious therapy design.

Review of recent literature points out the potential advantages of CD4⁺ T_H cell-based immunotherapy in comparison with strategies focused on CD8⁺ T cells. These cells are necessary and sufficient to activate CD8⁺ T cells for amplified anti-tumor response, along with their own contribution to cytotoxicity of tumor cells. The requirement for specific peptide recognition and HLA class matching can limit the therapeutic success of CD8⁺ T cells targeting neoantigens which are primarily derived by point mutations, since those mutations can significantly alter the interaction kinetics with CD8⁺ T cells and diminish CTL activity. The more promiscuous nature of CD4⁺ T cells permits interaction with a broader variety of neoantigens and mutated oncodrivers. Simultaneously, a reciprocal regulation of CD4⁺ T_H cells and B cells have been shown to be essential for both T_H1 and T_H2 and effector B cell differentiation and function, which hints at the therapeutic potential of stimulating CD4⁺ T_H immune response and indirectly boosting antibody secretion by B cells for enhanced anti-tumor response. However, studies in various pre-clinical models and clinical trials that have pointed out potential obstacles such as a hostile TME, presence of inhibitory T cell populations and immune checkpoint receptors suggest that stimulating a single subpopulation of CD4⁺ T_H function alone may not be adequate for robust anti-tumor response. Combinations of therapies that can drive multiple subtypes of CD4 T_H may better overcome this inadequacy and improve therapeutic efficacy in cancer. Targeted inhibition of oncodrivers by blocking/neutralizing antibodies and small molecule inhibitors, cell cycle kinases CDK4/6 inhibitors and standard-of-care therapeutics in combination with immunotherapy that drive CD4⁺ T_H cells are currently being tested in various stages of clinical trials and warrant future research to delve into the mechanism that these therapies have on anti-vs pro-tumorigenic CD4⁺ T_H responses for refined synergy. Developing DC-based vaccine platforms to stimulate oncodriver-specific T_H immune response can facilitate immunotherapy and future synergistic combination for effective cancer treatment. Understanding the role of immunosuppressive T_H cells, such as T_H2, T_H17 and T_reg, in the TME is equally critical to identify nodes in this regulatory network for therapeutic intervention. A successful cancer immunotherapy will require careful balancing of the CD4⁺ T_H compartment in order to orchestrate essential efforts that mediate tumor regression. A comprehensive overview of the CD4⁺ T_H cells, as discussed in this review, will help to elucidate the framework of CD4⁺ T_H function and highlight the clinical relevance of harnessing CD4⁺ T_H cells in cancer immunotherapy to encourage future translational research.

Funding

This work was supported by Department of Defense (Award# W81XWH-16-1-0385) awarded to BC, GK and MD. This work was also supported by Pennies in action to BC and GK. MD is also supported by the Helen B. Slonaker Endowed Professor for Cancer Research.

References

• 1

  TayRERichardsonEKTohHC. Revisiting the Role of CD4(+) T Cells in Cancer Immunotherapy-New Insights Into Old Paradigms. Cancer Gene Ther (2020) 1–2:5–17. doi: 10.1038/s41417-020-0183-x

  • CrossRef
  • Google Scholar

• 2

  BorstJAhrendsTBabalaNMeliefCJMKastenmullerW. Cd4(+) T Cell Help in Cancer Immunology and Immunotherapy. Nat Rev Immunol (2018) 18(10):635–47. doi: 10.1038/s41577-018-0044-0

  • CrossRef
  • Google Scholar

• 3

  RaskovHOrhanAChristensenJPGögenurI. Cytotoxic Cd8(+) T Cells in Cancer and Cancer Immunotherapy. Br J Cancer (2020) 124:359–67. doi: 10.1038/s41416-020-01048-4

  • CrossRef
  • Google Scholar

• 4

  BedouiSHeathWRMuellerSN. Cd4(+) T-cell Help Amplifies Innate Signals for Primary CD8(+) T-Cell Immunity. Immunol Rev (2016) 272(1):52–64. doi: 10.1111/imr.12426

  • CrossRef
  • Google Scholar

• 5

  ByrneASavasPSantSLiRVirassamyBLuenSJet al. Tissue-Resident Memory T Cells in Breast Cancer Control and Immunotherapy Responses. Nat Rev Clin Oncol (2020) 17(6):341–8. doi: 10.1038/s41571-020-0333-y

  • CrossRef
  • Google Scholar

• 6

  FarberDLYudaninNARestifoNP. Human Memory T Cells: Generation, Compartmentalization and Homeostasis. Nat Rev Immunol (2014) 14(1):24–35. doi: 10.1038/nri3567

  • CrossRef
  • Google Scholar

• 7

  MenaresEGalvez-CancinoFCaceres-MorgadoPGhoraniELopezEDiazXet al. Tissue-Resident Memory CD8(+) T Cells Amplify Anti-Tumor Immunity by Triggering Antigen Spreading Through Dendritic Cells. Nat Commun (2019) 10(1):4401. doi: 10.1038/s41467-019-12319-x

  • CrossRef
  • Google Scholar

• 8

  GrayJIWesterhofLMMacLeodMKL. The Roles of Resident, Central and Effector Memory CD4 T-Cells in Protective Immunity Following Infection or Vaccination. Immunology (2018) 154(4):574–81. doi: 10.1111/imm.12929

  • CrossRef
  • Google Scholar

• 9

  ClarkeJPanwarBMadrigalASinghDGujarRWoodOet al. Single-Cell Transcriptomic Analysis of Tissue-Resident Memory T Cells in Human Lung Cancer. J Exp Med (2019) 216(9):2128–49. doi: 10.1084/jem.20190249

  • CrossRef
  • Google Scholar

• 10

  SavasPVirassamyBYeCSalimAMintoffCPCaramiaFet al. Single-Cell Profiling of Breast Cancer T Cells Reveals a Tissue-Resident Memory Subset Associated With Improved Prognosis. Nat Med (2018) 24(7):986–93. doi: 10.1038/s41591-018-0078-7

  • CrossRef
  • Google Scholar

• 11

  YauCWolfDMCampbellMSavasPLinSBrown-SwigartLet al. Abstract P3-10-06: Expression-Based Immune Signatures as Predictors of Neoadjuvant Targeted-/Chemo-Therapy Response: Experience From the I-SPY 2 TRIAL of ˜1000 Patients Across 10 Therapies. Cancer Res (2019) 79(4 Supplement):P3–10-06. doi: 10.1158/1538-7445.SABCS18-P3-10-06

  • CrossRef
  • Google Scholar

• 12

  AdamsSSchmidPRugoHSWinerEPLoiratDAwadaAet al. Pembrolizumab Monotherapy for Previously Treated Metastatic Triple-Negative Breast Cancer: Cohort A of the Phase II Keynote-086 Study. Ann Oncol (2019) 30(3):397–404. doi: 10.1093/annonc/mdy517

  • CrossRef
  • Google Scholar

• 13

  AdamsSLoiSToppmeyerDCesconDWDe LaurentiisMNandaRet al. Pembrolizumab Monotherapy for Previously Untreated, PD-L1-positive, Metastatic Triple-Negative Breast Cancer: Cohort B of the Phase II Keynote-086 Study. Ann Oncol (2019) 30(3):405–11. doi: 10.1093/annonc/mdy518

  • CrossRef
  • Google Scholar

• 14

  LoiSSchmidPCortésJCesconDWWinerEPToppmeyerDet al. Abstract LB-225: RNA Molecular Signatures as Predictive Biomarkers of Response to Monotherapy Pembrolizumab in Patients With Metastatic Triple-Negative Breast Cancer: KEYNOTE-086. Cancer Res (2019) 79(13 Supplement):LB–225. doi: 10.1158/1538-7445.SABCS18-LB-225

  • CrossRef
  • Google Scholar

• 15

  SederRAAhmedR. Similarities and Differences in CD4+ and CD8+ Effector and Memory T Cell Generation. Nat Immunol (2003) 4(9):835–42. doi: 10.1038/ni969

  • CrossRef
  • Google Scholar

• 16

  ZhuJPaulWE. Cd4 T Cells: Fates, Functions, and Faults. Blood (2008) 112(5):1557–69. doi: 10.1182/blood-2008-05-078154

  • CrossRef
  • Google Scholar

• 17

  DobrzanskiMJ. Expanding Roles for CD4 T Cells and Their Subpopulations in Tumor Immunity and Therapy. Front Oncol (2013) 3:63. doi: 10.3389/fonc.2013.00063

  • CrossRef
  • Google Scholar

• 18

  SaraviaJChapmanNMChiH. Helper T Cell Differentiation. Cell Mol Immunol (2019) 16(7):634–43. doi: 10.1038/s41423-019-0220-6

  • CrossRef
  • Google Scholar

• 19

  ZhuJ. T Helper Cell Differentiation, Heterogeneity, and Plasticity. Cold Spring Harb Perspect Biol (2018) 10(10). doi: 10.1101/cshperspect.a030338

  • CrossRef
  • Google Scholar

• 20

  LuckheeramRVZhouRVermaADXiaB. CD4(+)T Cells: Differentiation and Functions. Clin Dev Immunol (2012) 2012:925135. doi: 10.1155/2012/925135

  • CrossRef
  • Google Scholar

• 21

  NappoGHandleFSanterFRMcNeillRVSeedRICollinsATet al. The Immunosuppressive Cytokine Interleukin-4 Increases the Clonogenic Potential of Prostate Stem-Like Cells by Activation of STAT6 Signalling. Oncogenesis (2017) 6(5):e342. doi: 10.1038/oncsis.2017.23

  • CrossRef
  • Google Scholar

• 22

  ZhuJ. T Helper 2 (Th2) Cell Differentiation, Type 2 Innate Lymphoid Cell (ILC2) Development and Regulation of Interleukin-4 (IL-4) and IL-13 Production. Cytokine (2015) 75(1):14–24. doi: 10.1016/j.cyto.2015.05.010

  • CrossRef
  • Google Scholar

• 23

  KumarSJeongYAshrafMUBaeYS. Dendritic Cell-Mediated Th2 Immunity and Immune Disorders. Int J Mol Sci (2019) 20(9):2159. doi: 10.3390/ijms20092159

  • CrossRef
  • Google Scholar

• 24

  WynnTA. Type 2 Cytokines: Mechanisms and Therapeutic Strategies. Nat Rev Immunol (2015) 15(5):271–82. doi: 10.1038/nri3831

  • CrossRef
  • Google Scholar

• 25

  WalkerJAMcKenzieANJ. TH2 Cell Development and Function. Nat Rev Immunol (2018) 18(2):121–33. doi: 10.1038/nri.2017.118

  • CrossRef
  • Google Scholar

• 26

  MaierEDuschlAHorejs-HoeckJ. STAT6-Dependent and -Independent Mechanisms in Th2 Polarization. Eur J Immunol (2012) 42(11):2827–33. doi: 10.1002/eji.201242433

  • CrossRef
  • Google Scholar

• 27

  MantovaniAAllavenaPSicaABalkwillF. Cancer-Related Inflammation. Nature (2008) 454(7203):436–44. doi: 10.1038/nature07205

  • CrossRef
  • Google Scholar

• 28

  FuCJiangLHaoSLiuZDingSZhangWet al. Activation of the IL-4/STAT6 Signaling Pathway Promotes Lung Cancer Progression by Increasing M2 Myeloid Cells. Front Immunol (2019) 10:2638. doi: 10.3389/fimmu.2019.02638

  • CrossRef
  • Google Scholar

• 29

  KiddP. Th1/Th2 Balance: The Hypothesis, its Limitations, and Implications for Health and Disease. Altern Med Rev (2003) 8(3):223–46.

  • Google Scholar

• 30

  LiZJiangJWangZZhangJXiaoMWangCet al. Endogenous Interleukin-4 Promotes Tumor Development by Increasing Tumor Cell Resistance to Apoptosis. Cancer Res (2008) 68(21):8687–94. doi: 10.1158/0008-5472.CAN-08-0449

  • CrossRef
  • Google Scholar

• 31

  BankaitisKVFingletonB. Targeting IL4/IL4R for the Treatment of Epithelial Cancer Metastasis. Clin Exp Metastasis (2015) 32(8):847–56. doi: 10.1007/s10585-015-9747-9

  • CrossRef
  • Google Scholar

• 32

  ManninoMHZhuZXiaoHBaiQWakefieldMRFangY. The Paradoxical Role of IL-10 in Immunity and Cancer. Cancer Lett (2015) 367(2):103–7. doi: 10.1016/j.canlet.2015.07.009

  • CrossRef
  • Google Scholar

• 33

  OftM. Il-10: Master Switch From Tumor-Promoting Inflammation to Antitumor Immunity. Cancer Immunol Res (2014) 2(3):194–9. doi: 10.1158/2326-6066.CIR-13-0214

  • CrossRef
  • Google Scholar

• 34

  Acuner-OzbabacanESEnginBHGuven-MaiorovEKuzuGMuratciogluSBaspinarAet al. The Structural Network of Interleukin-10 and its Implications in Inflammation and Cancer. BMC Genomics (2014) 15(4):S2. doi: 10.1186/1471-2164-15-S4-S2

  • CrossRef
  • Google Scholar

• 35

  SchmittEKleinMBoppT. Th9 Cells, New Players in Adaptive Immunity. Trends Immunol (2014) 35(2):61–8. doi: 10.1016/j.it.2013.10.004

  • CrossRef
  • Google Scholar

• 36

  ChenTGuoJCaiZLiBSunLShenYet al. Th9 Cell Differentiation and Its Dual Effects in Tumor Development. Front Immunol (2020) 11:1026. doi: 10.3389/fimmu.2020.01026

  • CrossRef
  • Google Scholar

• 37

  VegranFApetohLGhiringhelliF. Th9 Cells: A Novel CD4 T-Cell Subset in the Immune War Against Cancer. Cancer Res (2015) 75(3):475–9. doi: 10.1158/0008-5472.CAN-14-2748

  • CrossRef
  • Google Scholar

• 38

  LuYWangQXueGBiEMaXWangAet al. Th9 Cells Represent a Unique Subset of CD4(+) T Cells Endowed With the Ability to Eradicate Advanced Tumors. Cancer Cell (2018) 33(6):1048–1060.e7. doi: 10.1016/j.ccell.2018.05.004

  • CrossRef
  • Google Scholar

• 39

  SalazarYZhengXBrunnDRaiferHPicardFZhangYet al. Microenvironmental Th9 and Th17 Lymphocytes Induce Metastatic Spreading in Lung Cancer. J Clin Invest (2020) 130(7):3560–75. doi: 10.1172/JCI124037

  • CrossRef
  • Google Scholar

• 40

  TerhuneJBerkECzernieckiBJ. Dendritic Cell-Induced Th1 and Th17 Cell Differentiation for Cancer Therapy. Vaccines (Basel) (2013) 1(4):527–49. doi: 10.3390/vaccines1040527

  • CrossRef
  • Google Scholar

• 41

  TakeuchiASaitoT. Cd4 CTL, a Cytotoxic Subset of CD4(+) T Cells, Their Differentiation and Function. Front Immunol (2017) 8:194. doi: 10.3389/fimmu.2017.00194

  • CrossRef
  • Google Scholar

• 42

  MuranskiPBormanZAKerkarSPKlebanoffCAJiYSanchez-PerezLet al. Th17 Cells are Long Lived and Retain a Stem Cell-Like Molecular Signature. Immunity (2011) 35(6):972–85. doi: 10.1016/j.immuni.2011.09.019

  • CrossRef
  • Google Scholar

• 43

  WeiSZhaoEKryczekIZouW. Th17 Cells Have Stem Cell-Like Features and Promote Long-Term Immunity. Oncoimmunology (2012) 1(4):516–9. doi: 10.4161/onci.19440

  • CrossRef
  • Google Scholar

• 44

  BaileySRNelsonMHHimesRALiZMehrotraSPaulosCM. Th17 Cells in Cancer: The Ultimate Identity Crisis. Front Immunol (2014) 5:276–6. doi: 10.3389/fimmu.2014.00276

  • CrossRef
  • Google Scholar

• 45

  GueryLHuguesS. Th17 Cell Plasticity and Functions in Cancer Immunity. BioMed Res Int (2015) 2015:314620. doi: 10.1155/2015/314620

  • CrossRef
  • Google Scholar

• 46

  Gu-TrantienCLoiSGaraudSEqueterCLibinMde WindAet al. CD4(+) Follicular Helper T Cell Infiltration Predicts Breast Cancer Survival. J Clin Invest (2013) 123(7):2873–92. doi: 10.1172/JCI67428

  • CrossRef
  • Google Scholar

• 47

  SinghDGanesanAPPanwarBEschweilerSHanleyCJMadrigalAet al. CD4+ Follicular Helper-Like T Cells are Key Players in Anti-Tumor Immunity. bioRxiv (2020), 2020.01.08.898346. doi: 10.1101/2020.01.08.898346

  • CrossRef
  • Google Scholar

• 48

  QinLWaseemTCSahooABieerkehazhiSZhouHGalkinaEVet al. Insights Into the Molecular Mechanisms of T Follicular Helper-Mediated Immunity and Pathology. Front Immunol (1884) 2018:9. doi: 10.3389/fimmu.2018.01884

  • CrossRef
  • Google Scholar

• 49

  CrottyS. T Follicular Helper Cell Biology: A Decade of Discovery and Diseases. Immunity (2019) 50(5):1132–48. doi: 10.1016/j.immuni.2019.04.011

  • CrossRef
  • Google Scholar

• 50

  TogashiYShitaraKNishikawaH. Regulatory T Cells in Cancer Immunosuppression - Implications for Anticancer Therapy. Nat Rev Clin Oncol (2019) 16(6):356–71. doi: 10.1038/s41571-019-0175-7

  • CrossRef
  • Google Scholar

• 51

  NunezNGTosello BoariJRamosRNRicherWCagnardNAnderfuhrenCDet al. Tumor Invasion in Draining Lymph Nodes is Associated With Treg Accumulation in Breast Cancer Patients. Nat Commun (2020) 11(1):3272. doi: 10.1038/s41467-020-17046-2

  • CrossRef
  • Google Scholar

• 52

  TanakaASakaguchiS. Regulatory T Cells in Cancer Immunotherapy. Cell Res (2017) 27(1):109–18. doi: 10.1038/cr.2016.151

  • CrossRef
  • Google Scholar

• 53

  QureshiOSZhengYNakamuraKAttridgeKManzottiCSchmidtEMet al. Trans-Endocytosis of CD80 and CD86: A Molecular Basis for the Cell-Extrinsic Function of CTLA-4. Science (2011) 332(6029):600–3. doi: 10.1126/science.1202947

  • CrossRef
  • Google Scholar

• 54

  KumagaiSTogashiYKamadaTSugiyamaENishinakamuraHTakeuchiYet al. The PD-1 Expression Balance Between Effector and Regulatory T Cells Predicts the Clinical Efficacy of PD-1 Blockade Therapies. Nat Immunol (2020) 21(11):1346–58. doi: 10.1038/s41590-020-0769-3

  • CrossRef
  • Google Scholar

• 55

  KamadaTTogashiYTayCHaDSasakiANakamuraYet al. Pd-1(+) Regulatory T Cells Amplified by PD-1 Blockade Promote Hyperprogression of Cancer. Proc Natl Acad Sci USA (2019) 116(20):9999–10008. doi: 10.1073/pnas.1822001116

  • CrossRef
  • Google Scholar

• 56

  ChaudhryASamsteinRMTreutingPLiangYPilsMCHeinrichJMet al. Interleukin-10 Signaling in Regulatory T Cells is Required for Suppression of Th17 Cell-Mediated Inflammation. Immunity (2011) 34(4):566–78. doi: 10.1016/j.immuni.2011.03.018

  • CrossRef
  • Google Scholar

• 57

  ChenMLPittetMJGorelikLFlavellRAWeisslederRvon BoehmerHet al. Regulatory T Cells Suppress Tumor-Specific CD8 T Cell Cytotoxicity Through TGF-beta Signals In Vivo. Proc Natl Acad Sci USA (2005) 102(2):419–24. doi: 10.1073/pnas.0408197102

  • CrossRef
  • Google Scholar

• 58

  PandiyanPZhengLIshiharaSReedJLenardoMJ. Cd4+Cd25+Foxp3+ Regulatory T Cells Induce Cytokine Deprivation-Mediated Apoptosis of Effector CD4+ T Cells. Nat Immunol (2007) 8(12):1353–62. doi: 10.1038/ni1536

  • CrossRef
  • Google Scholar

• 59

  ChinenTKannanAKLevineAGFanXKleinUZhengYet al. An Essential Role for the IL-2 Receptor in Treg Cell Function. Nat Immunol (2016) 17(11):1322–33. doi: 10.1038/ni.3540

  • CrossRef
  • Google Scholar

• 60

  AhmadzadehMPasettoAJiaLDenigerDCStevanovicSRobbinsPFet al. Tumor-Infiltrating Human CD4(+) Regulatory T Cells Display a Distinct TCR Repertoire and Exhibit Tumor and Neoantigen Reactivity. Sci Immunol (2019) 4(31). doi: 10.1126/sciimmunol.aao4310

  • CrossRef
  • Google Scholar

• 61

  EisenbarthSC. Dendritic Cell Subsets in T Cell Programming: Location Dictates Function. Nat Rev Immunol (2019) 19(2):89–103. doi: 10.1038/s41577-018-0088-1

  • CrossRef
  • Google Scholar

• 62

  KoskiGKSchwartzGNWengDECzernieckiBJCarterCGressREet al. Calcium Mobilization in Human Myeloid Cells Results in Acquisition of Individual Dendritic Cell-Like Characteristics Through Discrete Signaling Pathways. J Immunol (1999) 163(1):82–92.

  • Google Scholar

• 63

  FariesMBBedrosianIXuSKoskiGRorosJGMoiseMAet al. Calcium Signaling Inhibits interleukin-12 Production and Activates CD83(+) Dendritic Cells That Induce Th2 Cell Development. Blood (2001) 98(8):2489–97. doi: 10.1182/blood.V98.8.2489

  • CrossRef
  • Google Scholar

• 64

  CollinMBigleyV. Human Dendritic Cell Subsets: An Update. Immunology (2018) 154(1):3–20. doi: 10.1111/imm.12888

  • CrossRef
  • Google Scholar

• 65

  AmonLLehmannCHKBaranskaASchoenJHegerLDudziakD. Transcriptional Control of Dendritic Cell Development and Functions. Int Rev Cell Mol Biol (2019) 349:55–151. doi: 10.1016/bs.ircmb.2019.10.001

  • CrossRef
  • Google Scholar

• 66

  MeradMSathePHelftJMillerJMorthaA. The Dendritic Cell Lineage: Ontogeny and Function of Dendritic Cells and Their Subsets in the Steady State and the Inflamed Setting. Annu Rev Immunol (2013) 31:563–604. doi: 10.1146/annurev-immunol-020711-074950

  • CrossRef
  • Google Scholar

• 67

  GardnerARuffellB. Dendritic Cells and Cancer Immunity. Trends Immunol (2016) 37(12):855–65. doi: 10.1016/j.it.2016.09.006

  • CrossRef
  • Google Scholar

• 68

  SchlitzerASivakamasundariVChenJSumatohHRSchreuderJLumJet al. Identification of cDC1- and Cdc2-Committed DC Progenitors Reveals Early Lineage Priming At the Common DC Progenitor Stage in the Bone Marrow. Nat Immunol (2015) 16(7):718–28. doi: 10.1038/ni.3200

  • CrossRef
  • Google Scholar

• 69

  QuCChenKChengS-Y. Conventional Type 1 Dendritic Cells in Protective Antitumor Immunity and its Potential in Hepatocellular Carcinoma. Hepatoma Res (2020) 6:38. doi: 10.20517/2394-5079.2020.12

  • CrossRef
  • Google Scholar

• 70

  KadowakiN. Dendritic Cells: A Conductor of T Cell Differentiation. Allergol Int (2007) 56(3):193–9. doi: 10.2332/allergolint.R-07-146

  • CrossRef
  • Google Scholar

• 71

  FerrisSTDuraiVWuRTheisenDJWardJPBernMDet al. cDC1 Prime and are Licensed by CD4(+) T Cells to Induce Anti-Tumour Immunity. Nature (2020) 584(7822):624–9. doi: 10.1038/s41586-020-2611-3

  • CrossRef
  • Google Scholar

• 72

  NoubadeRMajri-MorrisonSTarbellKV. Beyond Cdc1: Emerging Roles of DC Crosstalk in Cancer Immunity. Front Immunol (2019) 10:1014. doi: 10.3389/fimmu.2019.01014

  • CrossRef
  • Google Scholar

• 73

  PengQQiuXZhangZZhangSZhangYLiangYet al. Pd-L1 on Dendritic Cells Attenuates T Cell Activation and Regulates Response to Immune Checkpoint Blockade. Nat Commun (2020) 11(1):4835. doi: 10.1038/s41467-020-18570-x

  • CrossRef
  • Google Scholar

• 74

  HildnerKEdelsonBTPurthaWEDiamondMMatsushitaHKohyamaMet al. Batf3 Deficiency Reveals a Critical Role for CD8alpha+ Dendritic Cells in Cytotoxic T Cell Immunity. Science (2008) 322(5904):1097–100. doi: 10.1126/science.1164206

  • CrossRef
  • Google Scholar

• 75

  Acosta-RodriguezEVRivinoLGeginatJJarrossayDGattornoMLanzavecchiaAet al. Surface Phenotype and Antigenic Specificity of Human Interleukin 17-Producing T Helper Memory Cells. Nat Immunol (2007) 8(6):639–46. doi: 10.1038/ni1467

  • CrossRef
  • Google Scholar

• 76

  ZhaoYChuXChenJWangYGaoSJiangYet al. Dectin-1-activated Dendritic Cells Trigger Potent Antitumour Immunity Through the Induction of Th9 Cells. Nat Commun (2016) 7:12368. doi: 10.1038/ncomms12368

  • CrossRef
  • Google Scholar

• 77

  ChenJTZhaoYHChuXLuYWangSQYiQ. Dectin-1-activated Dendritic Cells: A Potent Th9 Cell Inducer for Tumor Immunotherapy. OncoImmunology (2016) 5(11). doi: 10.1080/2162402X.2016.1238558

  • CrossRef
  • Google Scholar

• 78

  AgaliotiTVillablancaEJHuberSGaglianiN. TH17cell Plasticity: The Role of Dendritic Cells and Molecular Mechanisms. J Autoimmun (2018) 87:50–60. doi: 10.1016/j.jaut.2017.12.003

  • CrossRef
  • Google Scholar

• 79

  KrishnaswamyJKAlsénSYrlidUEisenbarthSCWilliamsA. Determination of T Follicular Helper Cell Fate by Dendritic Cells. Front Immunol (2169) 2018:9. doi: 10.3389/fimmu.2018.02169

  • CrossRef
  • Google Scholar

• 80

  de JongECSmitsHHKapsenbergML. Dendritic Cell-Mediated T Cell Polarization. Springer Semin Immunopathol (2005) 26(3):289–307. doi: 10.1007/s00281-004-0167-1

  • CrossRef
  • Google Scholar

• 81

  NovyPQuigleyMHuangXYangY. Cd4 T Cells are Required for CD8 T Cell Survival During Both Primary and Memory Recall Responses. J Immunol (2007) 179(12):8243–51. doi: 10.4049/jimmunol.179.12.8243

  • CrossRef
  • Google Scholar

• 82

  LaidlawBJCraftJEKaechSM. The Multifaceted Role of CD4(+) T Cells in CD8(+) T Cell Memory. Nat Rev Immunol (2016) 16(2):102–11. doi: 10.1038/nri.2015.10

  • CrossRef
  • Google Scholar

• 83

  SpitzerMHCarmiYReticker-FlynnNEKwekSSMadhireddyDMartinsMMet al. Systemic Immunity is Required for Effective Cancer Immunotherapy. Cell (2017) 168(3):487–502 e15. doi: 10.1016/j.cell.2016.12.022

  • CrossRef
  • Google Scholar

• 84

  ZanderRSchauderDXinGNguyenCWuXZajacAet al. Cd4(+) T Cell Help is Required for the Formation of a Cytolytic Cd8(+) T Cell Subset That Protects Against Chronic Infection and Cancer. Immunity (2019) 51(6):1028–1042 e4. doi: 10.1016/j.immuni.2019.10.009

  • CrossRef
  • Google Scholar

• 85

  AlspachELussierDMMiceliAPKizhvatovIDuPageMLuomaAMet al. Mhc-II Neoantigens Shape Tumour Immunity and Response to Immunotherapy. Nature (2019) 574(7780):696–701. doi: 10.1038/s41586-019-1671-8

  • CrossRef
  • Google Scholar

• 86

  OttPAHuZKeskinDBShuklaSASunJBozymDJet al. An Immunogenic Personal Neoantigen Vaccine for Patients With Melanoma. Nature (2017) 547(7662):217–21. doi: 10.1038/nature22991

  • CrossRef
  • Google Scholar

• 87

  AhrendsTSpanjaardAPilzeckerBBabalaNBovensAXiaoYet al. Cd4(+) T Cell Help Confers a Cytotoxic T Cell Effector Program Including Coinhibitory Receptor Downregulation and Increased Tissue Invasiveness. Immunity (2017) 47(5):848–861 e5. doi: 10.1016/j.immuni.2017.10.009

  • CrossRef
  • Google Scholar

• 88

  SacksJABevanMJ. TRAIL Deficiency Does Not Rescue Impaired CD8+ T Cell Memory Generated in the Absence of CD4+ T Cell Help. J Immunol (2008) 180(7):4570–6. doi: 10.4049/jimmunol.180.7.4570

  • CrossRef
  • Google Scholar

• 89

  CastellinoFHuangAYAltan-BonnetGStollSScheineckerCGermainRN. Chemokines Enhance Immunity by Guiding Naive CD8+ T Cells to Sites of CD4+ T Cell-Dendritic Cell Interaction. Nature (2006) 440(7086):890–5. doi: 10.1038/nature04651

  • CrossRef
  • Google Scholar

• 90

  JanssenEMLemmensEEWolfeTChristenUvon HerrathMGSchoenbergerSP. Cd4+ T Cells are Required for Secondary Expansion and Memory in CD8+ T Lymphocytes. Nature (2003) 421(6925):852–6. doi: 10.1038/nature01441

  • CrossRef
  • Google Scholar

• 91

  ShedlockDJShenH. Requirement for CD4 T Cell Help in Generating Functional CD8 T Cell Memory. Science (2003) 300(5617):337–9. doi: 10.1126/science.1082305

  • CrossRef
  • Google Scholar

• 92

  OhSPereraLPTerabeMNiLWaldmannTABerzofskyJA. Il-15 as a Mediator of CD4+ Help for CD8+ T Cell Longevity and Avoidance of TRAIL-mediated Apoptosis. Proc Natl Acad Sci USA (2008) 105(13):5201–6. doi: 10.1073/pnas.0801003105

  • CrossRef
  • Google Scholar

• 93

  BosRShermanLA. Cd4+ T-cell Help in the Tumor Milieu is Required for Recruitment and Cytolytic Function of CD8+ T Lymphocytes. Cancer Res (2010) 70(21):8368–77. doi: 10.1158/0008-5472.CAN-10-1322

  • CrossRef
  • Google Scholar

• 94

  AhrendsTBusselaarJSeversonTMBabalaNde VriesEBovensAet al. Cd4(+) T Cell Help Creates Memory CD8(+) T Cells With Innate and Help-Independent Recall Capacities. Nat Commun (2019) 10(1):5531. doi: 10.1038/s41467-019-13438-1

  • CrossRef
  • Google Scholar

• 95

  CullenJGMcQuiltenHAQuinnKMOlshanskyMRussBEMoreyAet al. Cd4(+) T Help Promotes Influenza Virus-Specific CD8(+) T Cell Memory by Limiting Metabolic Dysfunction. Proc Natl Acad Sci USA (2019) 116(10):4481–8. doi: 10.1073/pnas.1808849116

  • CrossRef
  • Google Scholar

• 96

  AgarwalPRaghavanANandiwadaSLCurtsingerJMBohjanenPRMuellerDLet al. Gene Regulation and Chromatin Remodeling by IL-12 and Type I IFN in Programming for CD8 T Cell Effector Function and Memory. J Immunol (2009) 183(3):1695–704. doi: 10.4049/jimmunol.0900592

  • CrossRef
  • Google Scholar

• 97

  WolkersMCGerlachCArensRJanssenEMFitzgeraldPSchumacherTNet al. Nab2 Regulates Secondary CD8+ T-Cell Responses Through Control of TRAIL Expression. Blood (2012) 119(3):798–804. doi: 10.1182/blood-2011-08-373910

  • CrossRef
  • Google Scholar

• 98

  WieselMJollerNEhlertA-KCrouseJSpörriRBachmannMFet al. Th Cells Act Via Two Synergistic Pathways To Promote Antiviral Cd8+ T Cell Responses. J Immunol (2010) 185(9):5188–97. doi: 10.4049/jimmunol.1001990

  • CrossRef
  • Google Scholar

• 99

  XuSKoskiGFariesMBedrosianIMickRCheeverMet al. Rapid High Efficiency Sensitization of CD8+ T Cells to Tumor Antigens by Dendritic Cells Leads to Enhanced Functional Avidity and Direct Tumor Recognition Through an IL-12-Dependent Mechanism. J Immunol (Baltimore Md. 1950) (2003) 171:2251–61. doi: 10.4049/jimmunol.171.5.2251

  • CrossRef
  • Google Scholar

• 100

  KaliaVSarkarS. Regulation of Effector and Memory Cd8 T Cell Differentiation by IL-2-A Balancing Act. Front Immunol (2018) 9:2987. doi: 10.3389/fimmu.2018.02987

  • CrossRef
  • Google Scholar

• 101

  AraAAhmedKAXiangJ. Multiple Effects of CD40-CD40L Axis in Immunity Against Infection and Cancer. Immunotargets Ther (2018) 7:55–61. doi: 10.2147/ITT.S163614

  • CrossRef
  • Google Scholar

• 102

  SchoenbergerSPToesREvan der VoortEIOffringaRMeliefCJ. T-Cell Help for Cytotoxic T Lymphocytes is Mediated by CD40-CD40L Interactions. Nature (1998) 393(6684):480–3. doi: 10.1038/31002

  • CrossRef
  • Google Scholar

• 103

  FrentschMStarkRMatzmohrNMeierSDurlanikSSchulzARet al. CD40L Expression Permits CD8+ T Cells to Execute Immunologic Helper Functions. Blood (2013) 122(3):405–12. doi: 10.1182/blood-2013-02-483586

  • CrossRef
  • Google Scholar

• 104

  van de VenKBorstJ. Targeting the T-cell Co-Stimulatory CD27/CD70 Pathway in Cancer Immunotherapy: Rationale and Potential. Immunotherapy (2015) 7(6):655–67. doi: 10.2217/imt.15.32

  • CrossRef
  • Google Scholar

• 105

  WeltenSPRedekerAFrankenKLBenedictCAYagitaHWensveenFMet al. Cd27-CD70 Costimulation Controls T Cell Immunity During Acute and Persistent Cytomegalovirus Infection. J Virol (2013) 87(12):6851–65. doi: 10.1128/JVI.03305-12

  • CrossRef
  • Google Scholar

• 106

  AhrendsTBabalaNXiaoYYagitaHvan EenennaamHBorstJ. Cd27 Agonism Plus PD-1 Blockade Recapitulates Cd4+ T-cell Help in Therapeutic Anticancer Vaccination. Cancer Res (2016) 76(10):2921–31. doi: 10.1158/0008-5472.CAN-15-3130

  • CrossRef
  • Google Scholar

• 107

  HuiECheungJZhuJSuXTaylorMJWallweberHAet al. T Cell Costimulatory Receptor CD28 is a Primary Target for PD-1-mediated Inhibition. Science (2017) 355(6332):1428–33. doi: 10.1126/science.aaf1292

  • CrossRef
  • Google Scholar

• 108

  KimKHKimHKKimHDKimCGLeeHHanJWet al. PD-1 Blockade-Unresponsive Human Tumor-Infiltrating CD8(+) T Cells are Marked by Loss of CD28 Expression and Rescued by IL-15. Cell Mol Immunol (2020) 18(2):385–97. doi: 10.1038/s41423-020-0427-6

  • CrossRef
  • Google Scholar

• 109

  JorgovanovicDSongMWangLZhangY. Roles of IFN-gamma in Tumor Progression and Regression: A Review. Biomark Res (2020) 8:49. doi: 10.1186/s40364-020-00228-x

  • CrossRef
  • Google Scholar

• 110

  BenciJLXuBQiuYWuTJDadaHTwyman-Saint VictorCet al. Tumor Interferon Signaling Regulates a Multigenic Resistance Program to Immune Checkpoint Blockade. Cell (2016) 167(6):1540–1554 e12. doi: 10.1016/j.cell.2016.11.022

  • CrossRef
  • Google Scholar

• 111

  ShenJXiaoZZhaoQLiMWuXZhangLet al. Anti-Cancer Therapy With TNFalpha and Ifngamma: A Comprehensive Review. Cell Prolif (2018) 51(4):e12441. doi: 10.1111/cpr.12441

  • CrossRef
  • Google Scholar

• 112

  MojicMTakedaKHayakawaY. The Dark Side of IFN-γ: Its Role in Promoting Cancer Immunoevasion. Int J Mol Sci (2018) 19(1):89. doi: 10.3390/ijms19010089

  • CrossRef
  • Google Scholar

• 113

  AqbiHFWallaceMSappalSPayneKKManjiliMH. Ifn-γ Orchestrates Tumor Elimination, Tumor Dormancy, Tumor Escape, and Progression. J Leukocyte Biol (2018) 103(6):1219–23. doi: 10.1002/JLB.5MIR0917-351R

  • CrossRef
  • Google Scholar

• 114

  JiaYKodumudiKNRamamoorthiGBasuASnyderCWienerDet al. Th1 Cytokine Interferon Gamma Improves Response in HER2 Breast Cancer by Modulating the Ubiquitin Proteasomal Pathway. Mol Ther (2021) 29(4):1541–56. doi: 10.1016/j.ymthe.2020.12.037

  • CrossRef
  • Google Scholar

• 115

  MortaraLBalzaEBrunoAPoggiAOrecchiaPCarnemollaB. Anti-Cancer Therapies Employing IL-2 Cytokine Tumor Targeting: Contribution of Innate, Adaptive and Immunosuppressive Cells in the Anti-tumor Efficacy. Front Immunol (2018) 9:2905. doi: 10.3389/fimmu.2018.02905

  • CrossRef
  • Google Scholar

• 116

  SimGCRadvanyiL. The IL-2 Cytokine Family in Cancer Immunotherapy. Cytokine Growth Factor Rev (2014) 25(4):377–90. doi: 10.1016/j.cytogfr.2014.07.018

  • CrossRef
  • Google Scholar

• 117

  ChoudhryHHelmiNAbdulaalWHZeyadiMZamzamiMAWuWet al. Prospects of IL-2 in Cancer Immunotherapy. BioMed Res Int 2018 (2018) p:9056173. doi: 10.1155/2018/9056173

  • CrossRef
  • Google Scholar

• 118

  JiangTZhouCRenS. Role of IL-2 in Cancer Immunotherapy. Oncoimmunology (2016) 5(6):e1163462. doi: 10.1080/2162402X.2016.1163462

  • CrossRef
  • Google Scholar

• 119

  RayJPStaronMMShyerJAHoP-CMarshallHDGraySMet al. The Interleukin-2-mTORc1 Kinase Axis Defines the Signaling, Differentiation, and Metabolism of T Helper 1 and Follicular B Helper T Cells. Immunity (2015) 43(4):690–702. doi: 10.1016/j.immuni.2015.08.017

  • CrossRef
  • Google Scholar

• 120

  JosephsSFIchimTEPrinceSMKesariSMarincolaFMEscobedoARet al. Unleashing Endogenous TNF-alpha as a Cancer Immunotherapeutic. J Transl Med (2018) 16(1):242. doi: 10.1186/s12967-018-1611-7

  • CrossRef
  • Google Scholar

• 121

  MontfortAColaciosCLevadeTAndrieu-AbadieNMeyerNSeguiB. The TNF Paradox in Cancer Progression and Immunotherapy. Front Immunol (2019) 10:1818. doi: 10.3389/fimmu.2019.02515

  • CrossRef
  • Google Scholar

• 122

  GroomJRRichmondJMurookaTTSorensenEWSungJHBankertKet al. CXCR3 Chemokine Receptor-Ligand Interactions in the Lymph Node Optimize CD4+ T Helper 1 Cell Differentiation. Immunity (2012) 37(6):1091–103. doi: 10.1016/j.immuni.2012.08.016

  • CrossRef
  • Google Scholar

• 123

  MauldinISWagesNAStowmanAMWangESmolkinMEOlsonWCet al. Intratumoral Interferon-Gamma Increases Chemokine Production But Fails to Increase T Cell Infiltration of Human Melanoma Metastases. Cancer Immunol Immunother (2016) 65(10):1189–99. doi: 10.1007/s00262-016-1881-y

  • CrossRef
  • Google Scholar

• 124

  NagarshethNWichaMSZouW. Chemokines in the Cancer Microenvironment and Their Relevance in Cancer Immunotherapy. Nat Rev Immunol (2017) 17(9):559–72. doi: 10.1038/nri.2017.49

  • CrossRef
  • Google Scholar

• 125

  VilgelmAERichmondA. Chemokines Modulate Immune Surveillance in Tumorigenesis, Metastasis, and Response to Immunotherapy. Front Immunol (2019) 10:333. doi: 10.3389/fimmu.2019.00333

  • CrossRef
  • Google Scholar

• 126

  LebreMCBurwellTVieiraPLLoraJCoyleAJKapsenbergMLet al. Differential Expression of Inflammatory Chemokines by Th1- and Th2-cell Promoting Dendritic Cells: A Role for Different Mature Dendritic Cell Populations in Attracting Appropriate Effector Cells to Peripheral Sites of Inflammation. Immunol Cell Biol (2005) 83(5):525–35. doi: 10.1111/j.1440-1711.2005.01365.x

  • CrossRef
  • Google Scholar

• 127

  BretscherP. On Analyzing How the Th1/Th2 Phenotype of an Immune Response Is Determined: Classical Observations Must Not Be Ignored. Front Immunol (2019) 10:1234. doi: 10.3389/fimmu.2019.01234

  • CrossRef
  • Google Scholar

• 128

  LeeHLJangJWLeeSWYooSHKwonJHNamSWet al. Inflammatory Cytokines and Change of Th1/Th2 Balance as Prognostic Indicators for Hepatocellular Carcinoma in Patients Treated With Transarterial Chemoembolization. Sci Rep (2019) 9(1):3260. doi: 10.1038/s41598-019-40078-8

  • CrossRef
  • Google Scholar

• 129

  LinWNiuZZhangHKongYWangZYangXet al. Imbalance of Th1/Th2 and Th17/Treg During the Development of Uterine Cervical Cancer. Int J Clin Exp Pathol (2019) 12(9):3604–12.

  • Google Scholar

• 130

  TakashimaYKawaguchiAKanayamaTHayanoAYamanakaR. Correlation Between Lower Balance of Th2 Helper T-cells and Expression of PD-L1/PD-1 Axis Genes Enables Prognostic Prediction in Patients With Glioblastoma. Oncotarget (2018) 9(27):19065–78. doi: 10.18632/oncotarget.24897

  • CrossRef
  • Google Scholar

• 131

  HaoCJLiJLiuPLiXLHuYQSunJCet al. Effects of the Balance Between Type 1 and Type 2 T Helper Cells on Ovarian Cancer. Genet Mol Res (2016) 15(2). doi: 10.4238/gmr.15027936

  • CrossRef
  • Google Scholar

• 132

  HongCCYaoSMcCannSEDolnickRYWallacePKGongZet al. Pretreatment Levels of Circulating Th1 and Th2 Cytokines, and Their Ratios, are Associated With ER-negative and Triple Negative Breast Cancers. Breast Cancer Res Treat (2013) 139(2):477–88. doi: 10.1007/s10549-013-2549-3

  • CrossRef
  • Google Scholar

• 133

  RuterbuschMPrunerKBShehataLPepperM. In Vivo Cd4+ T Cell Differentiation and Function: Revisiting the Th1/Th2 Paradigm. Annu Rev Immunol (2020) 38(1):705–25. doi: 10.1146/annurev-immunol-103019-085803

  • CrossRef
  • Google Scholar

• 134

  ChenKKollsJK. Interluekin-17A (Il17a). Gene (2017) 614:8–14. doi: 10.1016/j.gene.2017.01.016

  • CrossRef
  • Google Scholar

• 135

  ZhangQQinJZhongLGongLZhangBZhangYet al. Ccl5-Mediated Th2 Immune Polarization Promotes Metastasis in Luminal Breast Cancer. Cancer Res (2015) 75(20):4312–21. doi: 10.1158/0008-5472.CAN-14-3590

  • CrossRef
  • Google Scholar

• 136

  CrottyS. T Follicular Helper Cell Differentiation, Function, and Roles in Disease. Immunity (2014) 41(4):529–42. doi: 10.1016/j.immuni.2014.10.004

  • CrossRef
  • Google Scholar

• 137

  ParkJMTerabeMSakaiYMunasingheJForniGMorrisJCet al. Early Role of CD4&lt;sup<+&lt;/sup< Th1 Cells and Antibodies in HER-2 Adenovirus Vaccine Protection Against Autochthonous Mammary Carcinomas. J Immunol (2005) 174(7):4228. doi: 10.4049/jimmunol.174.7.4228

  • CrossRef
  • Google Scholar

• 138

  ParkJMTerabeMSteelJCForniGSakaiYMorrisJCet al. Therapy of Advanced Established Murine Breast Cancer With a Recombinant Adenoviral ErbB-2/neu Vaccine. Cancer Res (2008) 68(6):1979–87. doi: 10.1158/0008-5472.CAN-07-5688

  • CrossRef
  • Google Scholar

• 139

  SchwartzMZhangYRosenblattJD. B Cell Regulation of the Anti-Tumor Response and Role in Carcinogenesis. J Immunother Cancer (2016) 4:40–0. doi: 10.1186/s40425-016-0145-x

  • CrossRef
  • Google Scholar

• 140

  MishraRPatelHAlanaziSYuanLGarrettJT. HER3 Signaling and Targeted Therapy in Cancer. Oncol Rev (2018) 12(1):355. doi: 10.4081/oncol.2018.355

  • CrossRef
  • Google Scholar

• 141

  ScharpenseelHHanssenALogesSMohmeMBernreutherCPeineSet al. EGFR and HER3 Expression in Circulating Tumor Cells and Tumor Tissue From non-Small Cell Lung Cancer Patients. Sci Rep (2019) 9(1):7406. doi: 10.1038/s41598-019-43678-6

  • CrossRef
  • Google Scholar

• 142

  MaennlingAETurMKNiebertMKlockenbringTZeppernickFGattenlohnerSet al. Molecular Targeting Therapy Against EGFR Family in Breast Cancer: Progress and Future Potentials. Cancers (Basel) (2019) 11(12):1826. doi: 10.3390/cancers11121826

  • CrossRef
  • Google Scholar

• 143

  CheangMCChiaSKVoducDGaoDLeungSSniderJet al. Ki67 Index, HER2 Status, and Prognosis of Patients With Luminal B Breast Cancer. J Natl Cancer Inst (2009) 101(10):736–50. doi: 10.1093/jnci/djp082

  • CrossRef
  • Google Scholar

• 144

  ToveySMBrownSDoughtyJCMallonEACookeTGEdwardsJ. Poor Survival Outcomes in HER2-positive Breast Cancer Patients With Low-Grade, Node-Negative Tumours. Br J Cancer (2009) 100(5):680–3. doi: 10.1038/sj.bjc.6604940

  • CrossRef
  • Google Scholar

• 145

  PernasSTolaneySM. HER2-Positive Breast Cancer: New Therapeutic Frontiers and Overcoming Resistance. Ther Adv Med Oncol (2019) 11:1758835919833519. doi: 10.1177/1758835919833519

  • CrossRef
  • Google Scholar

• 146

  GravalosCJimenoA. HER2 in Gastric Cancer: A New Prognostic Factor and a Novel Therapeutic Target. Ann Oncol (2008) 19(9):1523–9. doi: 10.1093/annonc/mdn169

  • CrossRef
  • Google Scholar

• 147

  OtsuHOkiEIkawa-YoshidaAKawanoHAndoKIdaSet al. Correlation of HER2 Expression With Clinicopathological Characteristics and Prognosis in Resectable Gastric Cancer. Anticancer Res (2015) 35(4):2441–6.

  • Google Scholar

• 148

  KomotoMNakataBAmanoRYamadaNYashiroMOhiraMet al. HER2 Overexpression Correlates With Survival After Curative Resection of Pancreatic Cancer. Cancer Sci (2009) 100(7):1243–7. doi: 10.1111/j.1349-7006.2009.01176.x

  • CrossRef
  • Google Scholar

• 149

  WatanabeSYonesakaKTanizakiJNonagaseYTakegawaNHarataniKet al. Targeting of the HER2/HER3 Signaling Axis Overcomes Ligand-Mediated Resistance to Trastuzumab in HER2-positive Breast Cancer. Cancer Med (2019) 8(3):1258–68. doi: 10.1002/cam4.1995

  • CrossRef
  • Google Scholar

• 150

  MaJLyuHHuangJLiuB. Targeting of erbB3 Receptor to Overcome Resistance in Cancer Treatment. Mol Cancer (2014) 13:105. doi: 10.1186/1476-4598-13-105

  • CrossRef
  • Google Scholar

• 151

  BaeSYLa ChoiYKimSKimMKimJJungSPet al. HER3 Status by Immunohistochemistry is Correlated With Poor Prognosis in Hormone Receptor-Negative Breast Cancer Patients. Breast Cancer Res Treat (2013) 139(3):741–50. doi: 10.1007/s10549-013-2570-6

  • CrossRef
  • Google Scholar

• 152

  GiltnaneJMMoederCBCampRLRimmDL. Quantitative Multiplexed Analysis of ErbB Family Coexpression for Primary Breast Cancer Prognosis in a Large Retrospective Cohort. Cancer (2009) 115(11):2400–9. doi: 10.1002/cncr.24277

  • CrossRef
  • Google Scholar

• 153

  RichardsL. HER3 Overexpression in Breast Cancer Conveys a Poor Prognosis. Nat Rev Clin Oncol (2010) 7(8):423–3. doi: 10.1038/nrclinonc.2010.106

  • CrossRef
  • Google Scholar

• 154

  OgdenABhattaraiSSahooBMonganNPAlsaleemMGreenARet al. Combined HER3-EGFR Score in Triple-Negative Breast Cancer Provides Prognostic and Predictive Significance Superior to Individual Biomarkers. Sci Rep (2020) 10(1):3009. doi: 10.1038/s41598-020-59514-1

  • CrossRef
  • Google Scholar

• 155

  BasuARamamoorthiGJiaYFaughnJWienerDAwshahSet al. Immunotherapy in Breast Cancer: Current Status and Future Directions. Adv Cancer Res (2019) 143:295–349. doi: 10.1016/bs.acr.2019.03.006

  • CrossRef
  • Google Scholar

• 156

  ZahaviDAlDeghaitherDO’ConnellAWeinerLM. Enhancing Antibody-Dependent Cell-Mediated Cytotoxicity: A Strategy for Improving Antibody-Based Immunotherapy. Antibody Ther (2018) 1(1):7–12. doi: 10.1093/abt/tby002

  • CrossRef
  • Google Scholar

• 157

  VarchettaSGibelliNOlivieroBNardiniEGennariRGattiGet al. Elements Related to Heterogeneity of Antibody-Dependent Cell Cytotoxicity in Patients Under Trastuzumab Therapy for Primary Operable Breast Cancer Overexpressing Her2. Cancer Res (2007) 67(24):11991–9. doi: 10.1158/0008-5472.CAN-07-2068

  • CrossRef
  • Google Scholar

• 158

  GallVAPhilipsAVQiaoNClise-DwyerKPerakisAAZhangMet al. Trastuzumab Increases Her2 Uptake and Cross-Presentation by Dendritic Cells. Cancer Res (2017) 77(19):5374–83. doi: 10.1158/0008-5472.CAN-16-2774

  • CrossRef
  • Google Scholar

• 159

  TriulziTForteLRegondiVDi ModicaMGhirelliCCarcangiuMLet al. HER2 Signaling Regulates the Tumor Immune Microenvironment and Trastuzumab Efficacy. Oncoimmunology (2019) 8(1):e1512942. doi: 10.1080/2162402X.2018.1512942

  • CrossRef
  • Google Scholar

• 160

  DattaJRosemblitCBerkEShowalterLNamjoshiPMickRet al. Progressive Loss of Anti-HER2 CD4(+) T-Helper Type 1 Response in Breast Tumorigenesis and the Potential for Immune Restoration. Oncoimmunology (2015) 4(10):e1022301. doi: 10.1080/2162402X.2015.1022301

  • CrossRef
  • Google Scholar

• 161

  LowenfeldLMickRDattaJXuSFitzpatrickEFisherCSet al. Dendritic Cell Vaccination Enhances Immune Responses and Induces Regression of HER2(pos) Dcis Independent of Route: Results of Randomized Selection Design Trial. Clin Cancer Res (2017) 23(12):2961–71. doi: 10.1158/1078-0432.CCR-16-1924

  • CrossRef
  • Google Scholar

• 162

  DattaJXuSRosemblitCSmithJBCintoloJAPowellDJJr.et al. Cd4(+) T-Helper Type 1 Cytokines and Trastuzumab Facilitate CD8(+) T-Cell Targeting of HER2/neu-Expressing Cancers. Cancer Immunol Res (2015) 3(5):455–63. doi: 10.1158/2326-6066.CIR-14-0208

  • CrossRef
  • Google Scholar

• 163

  DattaJFracolMMcMillanMTBerkEXuSGoodmanNet al. Association of Depressed Anti-Her2 T-Helper Type 1 Response With Recurrence in Patients With Completely Treated HER2-Positive Breast Cancer: Role for Immune Monitoring. JAMA Oncol (2016) 2(2):242–6. doi: 10.1001/jamaoncol.2015.5482

  • CrossRef
  • Google Scholar

• 164

  RosemblitCDattaJLowenfeldLXuSBasuAKodumudiKet al. Oncodriver Inhibition and CD4(+) Th1 Cytokines Cooperate Through Stat1 Activation to Induce Tumor Senescence and Apoptosis in HER2+ and Triple Negative Breast Cancer: Implications for Combining Immune and Targeted Therapies. Oncotarget (2018) 9(33):23058–77. doi: 10.18632/oncotarget.25208

  • CrossRef
  • Google Scholar

• 165

  KodumudiKNRamamoorthiGSnyderCBasuAJiaYAwshahSet al. Sequential Anti-PD1 Therapy Following Dendritic Cell Vaccination Improves Survival in a HER2 Mammary Carcinoma Model and Identifies a Critical Role for CD4 T Cells in Mediating the Response. Front Immunol (2019) 10:1939. doi: 10.3389/fimmu.2019.01939

  • CrossRef
  • Google Scholar

• 166

  FracolMDattaJLowenfeldLXuSZhangPJFisherCSet al. Loss of Anti-HER-3 Cd4+ T-Helper Type 1 Immunity Occurs in Breast Tumorigenesis and is Negatively Associated With Outcomes. Ann Surg Oncol (2017) 24(2):407–17. doi: 10.1245/s10434-016-5584-6

  • CrossRef
  • Google Scholar

• 167

  LiLGoedegebuureSPGillandersWE. Preclinical and Clinical Development of Neoantigen Vaccines. Ann Oncol (2017) 2812):xii11–7. doi: 10.1093/annonc/mdx681

  • CrossRef
  • Google Scholar

• 168

  PengMMoYWangYWuPZhangYXiongFet al. Neoantigen Vaccine: An Emerging Tumor Immunotherapy. Mol Cancer (2019) 18(1):128. doi: 10.1186/s12943-019-1055-6

  • CrossRef
  • Google Scholar

• 169

  HowittBEShuklaSAShollLMRitterhouseLLWatkinsJCRodigSet al. Association of Polymerase e-Mutated and Microsatellite-Instable Endometrial Cancers With Neoantigen Load, Number of Tumor-Infiltrating Lymphocytes, and Expression of PD-1 and PD-L1. JAMA Oncol (2015) 1(9):1319–23. doi: 10.1001/jamaoncol.2015.2151

  • CrossRef
  • Google Scholar

• 170

  StricklandKCHowittBEShuklaSARodigSRitterhouseLLLiuJFet al. Association and Prognostic Significance of BRCA1/2-mutation Status With Neoantigen Load, Number of Tumor-Infiltrating Lymphocytes and Expression of PD-1/PD-L1 in High Grade Serous Ovarian Cancer. Oncotarget (2016) 7(12):13587–98. doi: 10.18632/oncotarget.7277

  • CrossRef
  • Google Scholar

• 171

  MatsushitaHSatoYKarasakiTNakagawaTKumeHOgawaSet al. Neoantigen Load, Antigen Presentation Machinery, and Immune Signatures Determine Prognosis in Clear Cell Renal Cell Carcinoma. Cancer Immunol Res (2016) 4(5):463–71. doi: 10.1158/2326-6066.CIR-15-0225

  • CrossRef
  • Google Scholar

• 172

  SnyderAMakarovVMerghoubTYuanJZaretskyJMDesrichardAet al. Genetic Basis for Clinical Response to CTLA-4 Blockade in Melanoma. New Engl J Med (2014) 371(23):2189–99. doi: 10.1056/NEJMoa1406498

  • CrossRef
  • Google Scholar

• 173

  RizviNAHellmannMDSnyderAKvistborgPMakarovVHavelJJet al. Cancer Immunology. Mutational Landscape Determines Sensitivity to PD-1 Blockade in non-Small Cell Lung Cancer. Science (2015) 348(6230):124–8. doi: 10.1126/science.aaa1348

  • CrossRef
  • Google Scholar

• 174

  RosenbergJEHoffman-CensitsJPowlesTvan der HeijdenMSBalarAVNecchiAet al. Atezolizumab in Patients With Locally Advanced and Metastatic Urothelial Carcinoma Who Have Progressed Following Treatment With Platinum-Based Chemotherapy: A Single-Arm, Multicentre, Phase 2 Trial. Lancet (2016) 387(10031):1909–20. doi: 10.1016/S0140-6736(16)00561-4

  • CrossRef
  • Google Scholar

• 175

  SchumacherTNHacohenN. Neoantigens Encoded in the Cancer Genome. Curr Opin Immunol (2016) 41:98–103. doi: 10.1016/j.coi.2016.07.005

  • CrossRef
  • Google Scholar

• 176

  KreiterSVormehrMvan de RoemerNDikenMLowerMDiekmannJet al. Mutant MHC Class II Epitopes Drive Therapeutic Immune Responses to Cancer. Nature (2015) 520(7549):692–6. doi: 10.1038/nature14426

  • CrossRef
  • Google Scholar

• 177

  PengPHuHLiuPXuLX. Neoantigen-Specific CD4+ T-Cell Response is Critical for the Therapeutic Efficacy of Cryo-Thermal Therapy. J ImmunoTher Cancer (2020) 8(2):e000421. doi: 10.1136/jitc-2019-000421

  • CrossRef
  • Google Scholar

• 178

  SalomonNVascottoFSelmiAVormehrMQuinkhardtJBukurTet al. A Liposomal RNA Vaccine Inducing Neoantigen-Specific CD4(+) T Cells Augments the Antitumor Activity of Local Radiotherapy in Mice. Oncoimmunology (2020) 9(1):1771925. doi: 10.1080/2162402X.2020.1771925

  • CrossRef
  • Google Scholar

• 179

  ArbelaezCAEstradaJGessnerMAGlausCMoralesABMohnDet al. A Nanoparticle Vaccine That Targets Neoantigen Peptides to Lymphoid Tissues Elicits Robust Antitumor T Cell Responses. NPJ Vaccines (2020) 5(1):106. doi: 10.1038/s41541-020-00253-9

  • CrossRef
  • Google Scholar

• 180

  TranETurcotteSGrosARobbinsPFLuYCDudleyMEet al. Cancer Immunotherapy Based on Mutation-Specific CD4+ T Cells in a Patient With Epithelial Cancer. Science (2014) 344(6184):641–5. doi: 10.1126/science.1251102

  • CrossRef
  • Google Scholar

• 181

  VeatchJRJesernigBLKarglJFitzgibbonMLeeSMBaikCet al. Endogenous Cd4(+) T Cells Recognize Neoantigens in Lung Cancer Patients, Including Recurrent Oncogenic KRAS and ERBB2 (Her2) Driver Mutations. Cancer Immunol Res (2019) 7(6):910–22. doi: 10.1158/2326-6066.CIR-18-0402

  • CrossRef
  • Google Scholar

• 182

  LinnemannCvan BuurenMMBiesLVerdegaalEMSchotteRCalisJJet al. High-Throughput Epitope Discovery Reveals Frequent Recognition of Neo-Antigens by CD4+ T Cells in Human Melanoma. Nat Med (2015) 21(1):81–5. doi: 10.1038/nm.3773

  • CrossRef
  • Google Scholar

• 183

  KeskinDBAnandappaAJSunJTiroshIMathewsonNDLiSet al. Neoantigen Vaccine Generates Intratumoral T Cell Responses in Phase Ib Glioblastoma Trial. Nature (2019) 565(7738):234–9. doi: 10.1038/s41586-018-0792-9

  • CrossRef
  • Google Scholar

• 184

  LiuSMatsuzakiJWeiLTsujiTBattagliaSHuQet al. Efficient Identification of Neoantigen-Specific T-cell Responses in Advanced Human Ovarian Cancer. J Immunother Cancer (2019) 7(1):156. doi: 10.1186/s40425-019-0629-6

  • CrossRef
  • Google Scholar

• 185

  VinayDSRyanEPPawelecGTalibWHStaggJElkordEet al. Immune Evasion in Cancer: Mechanistic Basis and Therapeutic Strategies. Semin Cancer Biol (2015) 35 Suppl:S185–98. doi: 10.1016/j.semcancer.2015.03.004

  • CrossRef
  • Google Scholar

• 186

  FoleyKCNishimuraMIMooreTV. Combination Immunotherapies Implementing Adoptive T-cell Transfer for Advanced-Stage Melanoma. Melanoma Res (2018) 28(3):171–84. doi: 10.1097/CMR.0000000000000436

  • CrossRef
  • Google Scholar

• 187

  SharmaPAllisonJP. Dissecting the Mechanisms of Immune Checkpoint Therapy. Nat Rev Immunol (2020) 20(2):75–6. doi: 10.1038/s41577-020-0275-8

  • CrossRef
  • Google Scholar

• 188

  HeXXuC. Immune Checkpoint Signaling and Cancer Immunotherapy. Cell Res (2020) 30(8):660–9. doi: 10.1038/s41422-020-0343-4

  • CrossRef
  • Google Scholar

• 189

  KalbasiARibasA. Tumour-Intrinsic Resistance to Immune Checkpoint Blockade. Nat Rev Immunol (2020) 20(1):25–39. doi: 10.1038/s41577-019-0218-4

  • CrossRef
  • Google Scholar

• 190

  NagasakiJTogashiYSugawaraTItamiMYamauchiNYudaJet al. The Critical Role of CD4+ T Cells in PD-1 Blockade Against MHC-II-expressing Tumors Such as Classic Hodgkin Lymphoma. Blood Adv (2020) 4(17):4069–82. doi: 10.1182/bloodadvances.2020002098

  • CrossRef
  • Google Scholar

• 191

  KagamuHKitanoSYamaguchiOYoshimuraKHorimotoKKitazawaMet al. Cd4(+) T-cell Immunity in the Peripheral Blood Correlates With Response to Anti-PD-1 Therapy. Cancer Immunol Res (2020) 8(3):334–44. doi: 10.1158/2326-6066.CIR-19-0574

  • CrossRef
  • Google Scholar

• 192

  GoodsBAHernandezALLowtherDELuccaLELernerBAGunelMet al. Functional Differences Between PD-1+ and PD-1- CD4+ Effector T Cells in Healthy Donors and Patients With Glioblastoma Multiforme. PloS One (2017) 12(9):e0181538. doi: 10.1371/journal.pone.0181538

  • CrossRef
  • Google Scholar

• 193

  YangZZGroteDMZiesmerSCXiuBNovakAJAnsellSM. PD-1 Expression Defines Two Distinct T-cell Sub-Populations in Follicular Lymphoma That Differentially Impact Patient Survival. Blood Cancer J (2015) 5:e281. doi: 10.1038/bcj.2015.1

  • CrossRef
  • Google Scholar

• 194

  TaiXVan LaethemFPobezinskyLGuinterTSharrowSOAdamsAet al. Basis of CTLA-4 Function in Regulatory and Conventional CD4(+) T Cells. Blood (2012) 119(22):5155–63. doi: 10.1182/blood-2011-11-388918

  • CrossRef
  • Google Scholar

• 195

  TakahashiTTagamiTYamazakiSUedeTShimizuJSakaguchiNet al. Immunologic Self-Tolerance Maintained by CD25(+)CD4(+) Regulatory T Cells Constitutively Expressing Cytotoxic T Lymphocyte-Associated Antigen 4. J Exp Med (2000) 192(2):303–10. doi: 10.1084/jem.192.2.303

  • CrossRef
  • Google Scholar

• 196

  GoddardETBozicIRiddellSRGhajarCM. Dormant Tumour Cells, Their Niches and the Influence of Immunity. Nat Cell Biol (2018) 20(11):1240–9. doi: 10.1038/s41556-018-0214-0

  • CrossRef
  • Google Scholar

• 197

  ShowalterLCzernieckiBJKoskiGK. Th1 Cytokines in Conjunction With Pharmacological Akt Inhibition Potentiate Apoptosis of Breast Cancer Cells In Vitro and Suppress Tumor Growth In Vivo. Oncotarget (2020) 11(30):2873–88. doi: 10.18632/oncotarget.27556

  • CrossRef
  • Google Scholar

• 198

  BerraondoPSanmamedMFOchoaMCEtxeberriaIAznarMAPerez-GraciaJLet al. Cytokines in Clinical Cancer Immunotherapy. Br J Cancer (2019) 120(1):6–15. doi: 10.1038/s41416-018-0328-y

  • CrossRef
  • Google Scholar

• 199

  SpeiserDEHoPCVerdeilG. Regulatory Circuits of T Cell Function in Cancer. Nat Rev Immunol (2016) 16(10):599–611. doi: 10.1038/nri.2016.80

  • CrossRef
  • Google Scholar

• 200

  MegoMGaoHCohenENAnfossiSGiordanoASandaTet al. Circulating Tumor Cells (Ctc) Are Associated With Defects in Adaptive Immunity in Patients With Inflammatory Breast Cancer. J Cancer (2016) 7(9):1095–104. doi: 10.7150/jca.13098

  • CrossRef
  • Google Scholar

• 201

  MohmeMRiethdorfSPantelK. Circulating and Disseminated Tumour Cells - Mechanisms of Immune Surveillance and Escape. Nat Rev Clin Oncol (2017) 14(3):155–67. doi: 10.1038/nrclinonc.2016.144

  • CrossRef
  • Google Scholar

• 202

  MagbanuaMJMRugoHSHauraniehLRoyRScottJHLeeJCet al. Genomic and Expression Profiling Reveal Molecular Heterogeneity of Disseminated Tumor Cells in Bone Marrow of Early Breast Cancer. NPJ Breast Cancer (2018) 4:31. doi: 10.1038/s41523-018-0083-5

  • CrossRef
  • Google Scholar

• 203

  SosaMSBragadoPAguirre-GhisoJA. Mechanisms of Disseminated Cancer Cell Dormancy: An Awakening Field. Nat Rev Cancer (2014) 14(9):611–22. doi: 10.1038/nrc3793

  • CrossRef
  • Google Scholar

• 204

  EylesJPuauxALWangXTohBPrakashCHongMet al. Tumor Cells Disseminate Early, But Immunosurveillance Limits Metastatic Outgrowth, in a Mouse Model of Melanoma. J Clin Invest (2010) 120(6):2030–9. doi: 10.1172/JCI42002

  • CrossRef
  • Google Scholar

• 205

  KoebelCMVermiWSwannJBZerafaNRodigSJOldLJet al. Adaptive Immunity Maintains Occult Cancer in an Equilibrium State. Nature (2007) 450(7171):903–7. doi: 10.1038/nature06309

  • CrossRef
  • Google Scholar

• 206

  YangX-WJiangH-XHuangX-LMaS-JQinS-Y. Role of Th9 Cells and Th17 Cells in the Pathogenesis of Malignant Ascites. Asian Pacific J Trop Biomed (2015) 5(10):806–11. doi: 10.1016/j.apjtb.2015.07.013

  • CrossRef
  • Google Scholar

• 207

  MelssenMSlingluffCLJr.Vaccines Targeting Helper T Cells for Cancer Immunotherapy. Curr Opin Immunol (2017) 47:85–92. doi: 10.1016/j.coi.2017.07.004

  • CrossRef
  • Google Scholar

• 208

  SantosPMButterfieldLH. Dendritic Cell-Based Cancer Vaccines. J Immunol (2018) 200(2):443–9. doi: 10.4049/jimmunol.1701024

  • CrossRef
  • Google Scholar

• 209

  HiltboldEMCiborowskiPFinnOJ. Naturally Processed Class II Epitope From the Tumor Antigen MUC1 Primes Human CD4+ T Cells. Cancer Res (1998) 58(22):5066–70.

  • Google Scholar

• 210

  GnjaticSAtanackovicDJagerEMatsuoMSelvakumarAAltorkiNKet al. Survey of Naturally Occurring CD4+ T Cell Responses Against NY-ESO-1 in Cancer Patients: Correlation With Antibody Responses. Proc Natl Acad Sci USA (2003) 100(15):8862–7. doi: 10.1073/pnas.1133324100

  • CrossRef
  • Google Scholar

• 211

  FrancoisVOttavianiSRenkvistNStockisJSchulerGThielemansKet al. The CD4(+) T-Cell Response of Melanoma Patients to a MAGE-A3 Peptide Vaccine Involves Potential Regulatory T Cells. Cancer Res (2009) 69(10):4335–45. doi: 10.1158/0008-5472.CAN-08-3726

  • CrossRef
  • Google Scholar

• 212

  CliftonGTGallVPeoplesGEMittendorfEA. Clinical Development of the E75 Vaccine in Breast Cancer. Breast Care (Basel) (2016) 11(2):116–21. doi: 10.1159/000446097

  • CrossRef
  • Google Scholar

• 213

  AdoteviODossetMGalaineJBeziaudLGodetYBorgC. Targeting Antitumor CD4 Helper T Cells With Universal Tumor-Reactive Helper Peptides Derived From Telomerase for Cancer Vaccine. Hum Vaccin Immunother (2013) 9(5):1073–7. doi: 10.4161/hv.23587

  • CrossRef
  • Google Scholar

• 214

  BerinsteinNLKarkadaMOzaAMOdunsiKVillellaJANemunaitisJJet al. Survivin-Targeted Immunotherapy Drives Robust Polyfunctional T Cell Generation and Differentiation in Advanced Ovarian Cancer Patients. Oncoimmunology (2015) 4(8):e1026529. doi: 10.1080/2162402X.2015.1026529

  • CrossRef
  • Google Scholar

• 215

  Bou Nasser EddineFRamiaETosiGForlaniGAccollaRS. Tumor Immunology Meets...Immunology: Modified Cancer Cells as Professional APC for Priming Naive Tumor-Specific CD4+ T Cells. Oncoimmunology (2017) 6(11):e1356149. doi: 10.1080/2162402X.2017.1356149

  • CrossRef
  • Google Scholar

• 216

  TondiniEArakelianTOosterhuisKCampsMvan DuikerenSHanWet al. A Poly-Neoantigen DNA Vaccine Synergizes With PD-1 Blockade to Induce T Cell-Mediated Tumor Control. Oncoimmunology (2019) 8(11):1652539. doi: 10.1080/2162402X.2019.1652539

  • CrossRef
  • Google Scholar

• 217

  CheeverMAHiganoCS. Provenge (Sipuleucel-T) in Prostate Cancer: The First FDA-approved Therapeutic Cancer Vaccine. Clin Cancer Res (2011) 17(11):3520–6. doi: 10.1158/1078-0432.CCR-10-3126

  • CrossRef
  • Google Scholar

• 218

  EaglesMENassiriFBadhiwalaJHSuppiahSAlmenawerSAZadehGet al. Dendritic Cell Vaccines for High-Grade Gliomas. Ther Clin Risk Manag (2018) 14:1299–313. doi: 10.2147/TCRM.S135865

  • CrossRef
  • Google Scholar

• 219

  CostaRLBSolimanHCzernieckiBJ. The Clinical Development of Vaccines for HER2(+) Breast Cancer: Current Landscape and Future Perspectives. Cancer Treat Rev (2017) 61:107–15. doi: 10.1016/j.ctrv.2017.10.005

  • CrossRef
  • Google Scholar

• 220

  ChodonTComin-AnduixBChmielowskiBKoyaRCWuZAuerbachMet al. Adoptive Transfer of MART-1 T-Cell Receptor Transgenic Lymphocytes and Dendritic Cell Vaccination in Patients With Metastatic Melanoma. Clin Cancer Res (2014) 20(9):2457–65. doi: 10.1158/1078-0432.CCR-13-3017

  • CrossRef
  • Google Scholar

• 221

  KumaiTLeeSChoHISultanHKobayashiHHarabuchiYet al. Optimization of Peptide Vaccines to Induce Robust Antitumor Cd4 T-cell Responses. Cancer Immunol Res (2017) 5(1):72–83. doi: 10.1158/2326-6066.CIR-16-0194

  • CrossRef
  • Google Scholar

• 222

  LiKDonaldsonBYoungVWardVJacksonCBairdMet al. Adoptive Cell Therapy With CD4(+) T Helper 1 Cells and CD8(+) Cytotoxic T Cells Enhances Complete Rejection of an Established Tumour, Leading to Generation of Endogenous Memory Responses to non-Targeted Tumour Epitopes. Clin Transl Immunol (2017) 6(10):e160. doi: 10.1038/cti.2017.37

  • CrossRef
  • Google Scholar

• 223

  KnutsonKLDisisML. Il-12 Enhances the Generation of Tumour Antigen-Specific Th1 Cd4 T Cells During Ex Vivo Expansion. Clin Exp Immunol (2004) 135(2):322–9. doi: 10.1111/j.1365-2249.2004.02360.x

  • CrossRef
  • Google Scholar

• 224

  AlizadehDTradMHankeNTLarmonierCBJanikashviliNBonnotteBet al. Doxorubicin Eliminates Myeloid-Derived Suppressor Cells and Enhances the Efficacy of Adoptive T-cell Transfer in Breast Cancer. Cancer Res (2014) 74(1):104–18. doi: 10.1158/0008-5472.CAN-13-1545

  • CrossRef
  • Google Scholar

• 225

  ReadKAPowellMDMcDonaldPWOestreichKJ. Il-2, IL-7, and IL-15: Multistage Regulators of CD4(+) T Helper Cell Differentiation. Exp Hematol (2016) 44(9):799–808. doi: 10.1016/j.exphem.2016.06.003

  • CrossRef
  • Google Scholar

• 226

  PathangeyLBMcCurryDBGendlerSJDominguezALGormanJEPathangeyGet al. Surrogate In Vitro Activation of Innate Immunity Synergizes With Interleukin-7 to Unleash Rapid Antigen-Driven Outgrowth of CD4+ and CD8+ Human Peripheral Blood T-cells Naturally Recognizing MUC1, HER2/Neu and Other Tumor-Associated Antigens. Oncotarget (2017) 8(7):10785–808. doi: 10.18632/oncotarget.13911

  • CrossRef
  • Google Scholar

• 227

  DavenportAJJenkinsMRCrossRSYongCSPrinceHMRitchieDSet al. Car-T Cells Inflict Sequential Killing of Multiple Tumor Target Cells. Cancer Immunol Res (2015) 3(5):483–94. doi: 10.1158/2326-6066.CIR-15-0048

  • CrossRef
  • Google Scholar

• 228

  YasukawaMOhminamiHAraiJKasaharaYIshidaYFujitaS. Granule Exocytosis, and Not the Fas/Fas Ligand System, is the Main Pathway of Cytotoxicity Mediated by Alloantigen-Specific CD4(+) as Well as CD8(+) Cytotoxic T Lymphocytes in Humans. Blood (2000) 95(7):2352–5. doi: 10.1182/blood.V95.7.2352.007k40_2352_2355

  • CrossRef
  • Google Scholar

• 229

  HombachAKohlerHRapplGAbkenH. Human Cd4+ T Cells Lyse Target Cells Via Granzyme/Perforin Upon Circumvention of MHC Class II Restriction by an Antibody-Like Immunoreceptor. J Immunol (2006) 177(8):5668–75. doi: 10.4049/jimmunol.177.8.5668

  • CrossRef
  • Google Scholar

• 230

  LiadiISinghHRomainGRey-VillamizarNMerouaneAAdolacionJRet al. Individual Motile Cd4(+) T Cells Can Participate in Efficient Multikilling Through Conjugation to Multiple Tumor Cells. Cancer Immunol Res (2015) 3(5):473–82. doi: 10.1158/2326-6066.CIR-14-0195

  • CrossRef
  • Google Scholar

• 231

  BenmebarekMRKarchesCHCadilhaBLLeschSEndresSKoboldS. Killing Mechanisms of Chimeric Antigen Receptor (Car) T Cells. Int J Mol Sci (2019) 20(6):1283. doi: 10.3390/ijms20061283

  • CrossRef
  • Google Scholar

• 232

  WangDAguilarBStarrRAlizadehDBritoASarkissianAet al. Glioblastoma-Targeted CD4+ Car T Cells Mediate Superior Antitumor Activity. JCI Insight (2018) 3(10):e99048. doi: 10.1172/jci.insight.99048

  • CrossRef
  • Google Scholar

• 233

  BrownCEAguilarBStarrRYangXChangW-CWengLet al. Optimization of IL13Rα2-Targeted Chimeric Antigen Receptor T Cells for Improved Anti-Tumor Efficacy Against Glioblastoma. Mol Ther J Am Soc Gene Ther (2018) 26(1):31–44. doi: 10.1016/j.ymthe.2017.10.002

  • CrossRef
  • Google Scholar

• 234

  AgarwalSHanauerJDSFrankAMRiechertVThalheimerFBBuchholzCJ. In Vivo Generation of CAR T Cells Selectively in Human Cd4(+) Lymphocytes. Mol Ther (2020) 28(8):1783–94. doi: 10.1016/j.ymthe.2020.05.005

  • CrossRef
  • Google Scholar