T cell interactions with tumor cells in the TME has the potential to result in T cell activation and spontaneous anti-tumor immunity (38). IL-35 can influence the transcription and expression of T cell immune response-related differentially expressed genes in tumor tissues (31), but the immunosuppressive activity of this cytokine is primarily attributable to its inhibition of CD4⁺ and CD8⁺T cells and of anti-tumor immune responses ( Figure 1 ) (39). IL-35 derived from Tregs within tumors can induce CD4⁺and CD8⁺ T cell exhaustion, reducing their effector functionality and impairing the generation of CTLs (31, 40). However, IL-35 does not directly suppress these CTLs, instead downregulating the costimulatory molecule CD28 on the surface of immature CD8⁺ T cells and thereby interfering with their ability to differentiate into anti-tumor CTLs. IL-35 can also disrupt Th1 cell activation to suppress differentiated anti-tumor CTL activity (41). IL-35 does not alter the survival of the A549 ADC cell line or the H520 SCC cell line cultured with IL-35, suggesting that the immunosuppressive functions of IL-35 necessitate the presence of the TME in vivo, as this cytokine does not seem to influence tumor cells in vitro (39). Together, these results suggest that IL-35 can suppress the activation of tumor-infiltrating T cells and anti-tumor CTLs, specifically promoting tumor development within the TME (31, 39, 41).

Tregs are key immunomodulatory cells within the TME, wherein the control tumor growth, migration, and local immunity (42, 43). Tregs have been shown to secrete both IL-10 and IL-35 (16, 44), and are present within tumor-draining lymph nodes wherein they can be stratified into Treg subsets secreting either IL-10 or IL-35. Cooperation between these two cytokines can drive conventional T cell failure and BLIMP1 upregulation-mediated transcriptional signal depletion in tumor-infiltrating CD8⁺T cell (45, 46). IL-35 can influence the function of T cells by modulating their expression of inhibitory cell surface receptors (IRs) and their ability to secrete effective cytokines (31). Inhibitory receptors including CTLA4 (cytotoxic T lymphocyte-associated protein 4), PD-1 (programmed cell death ligand 1 or CD279), Tim-3 (T-cell immunoglobulin and mucin-domain containing 3), and LAG-3 (lymphocyte activation 3) have been identified as hallmarks of dysfunctional T cells (47). IL-35 can specifically increase the expression of these receptors on CD4 and CD8⁺ tumor-infiltrating lymphocytes (TILs) within the TME, thereby depleting local T cells (45, 46). Tregs can also convert between the expression of IL-10 and IL-35, enabling them to adapt to the dynamic TME (40). Conventional T cells can differentiate into Tregs in response to IL-35-mediated STAT1/STAT3 signaling, yielding so-called iTr35 cells (48), which can mediate the relationship between IL-35 and tumor cells in TME, accelerating tumor cell growth and metastasis (22, 28). In addition, iTR35-derived IL-35 can drive the differentiation of other Tregs towards an iTr35 phenotype, creating a positive feedback loop that can control TME activity over extended periods of time (48, 49). Notch signaling blockade can also alleviate the inhibitory functions of purified Tregs from gastric carcinoma patients, which can increase the secretion of IL-35, confirming the close feedback relationship between IL-35 and Tregs (50).

Intriguingly, IL-35 improve anti-tumor activity via the WNT/β-catenin pathway, suggesting that IL-35 plays distinct roles in different signaling pathways (51). The activation of the β-catenin pathway has recently been shown to be associated with most human tumors exhibiting spontaneous T cell infiltration (52). β-catenin maintains Treg function and contributes to regulated IL-10 production within the TME, and its expression is inhibited by IL-35 (53–55). Overexpressing IL-35 inhibits colon cancer cell migration, invasion, proliferation, and colony formation by suppressing this β-catenin pathway (51). The WNT/β-catenin pathway may thus represent a novel mechanism whereby IL-35 can influence tumor progression, highlighting this as an additional avenue for the study of IL-35 use in the context of tumor immunotherapy.

The role of B cells within the TME remains a matter of controversy, with some evidence suggesting that these cells can promote or inhibit tumor progression via a range of immunomodulatory mechanisms (8, 56). B cells are known to regulate immunity by producing specific antibodies, forming antibody-antigen complexes, secreting cytokines, and serving as antigen-presenting cells to promote cytotoxic T cell responses (57, 58). Tumor-infiltrating B cells in the TME have been shown to promote tumor progression through interactions with macrophages, MDSCs, and other immune cells by secreting cytokines such as IL-10, TGF-β, and IL-35 (56, 59). B cells also exhibit distinct phenotypes under different TME conditions, with Bregs being a recently identified B cell subtype exhibiting immunosuppressive functions (60, 61). Much like Tregs, Breg recruitment can result in the modulation of local cellular responses through the production of IL-35 and other regulatory cytokines (62). In mouse models of breast cancer, Bregs induce T cell differentiation into Tregs and thereby accelerate tumor growth and lung metastasis (63).

Several early studies of Breg-like cells that produce IL-10 (B10 cells) were conducted, underscoring the regulatory roles of these cells in a range of different tissues and immunological contexts (64, 65). However, recent evidence suggests that IL-35 is a key Breg cytokine that can drive conventional B cells and B10 cells towards an IL-35-producing B cell (i35-Breg) phenotype (57, 66). In the TME associated with gastric and pancreatic tumors, these i35-Bregs are significantly more abundant and can promote tumor progression ( Figure 2 ) (37, 67). In pancreatic tumors, inhibition of the Bruton’s tyrosine kinase (BTK) signaling pathway reduces IL-35 and IL-10 expression and decreases i35-Breg cell function, suppressing tumor cell growth (67). Wang et al. determined that numbers of i35-Bregs were significantly increased in those with advanced gastric cancer, and found that the amount of these cells was associated with quantities of Tregs, MDSCs, B10 cells, and CD14⁺ monocytes in the patients (37). As a specific B cell subtype, i35-Bregs can regulate other stromal cells within the TME including effector T cells, tumor-infiltrating MDSCs, NK cells, and macrophages (35, 37). As the specific molecular markers of Bregs are unclear, however, future in-depth analyses of IL-35 and Bregs are required to understand the immunological effects of i35-Bregs (37, 62).

Dendritic cells (DCs) are key professional antigen-presenting cells (APCs) that promote antitumor immune responses by regulating T cell activation and proliferation, modulating immunological homeostasis within the TME. The migration of activated DCs into lymphatics can efficiently present antigens to T cells (68). DCs play a critical role in the initial activation of antitumor immunity by serving as an essential interface between antigen-independent innate immunity and antigen-specific adaptive immunity (69). Restoring the antigen-presenting activity of DCs thus represents an important approach to achieving efficacious tumor immunotherapy outcomes (69, 70). Seyerl et al. were the first to show that human rhinovirus-activated DCs (R-DCs) produced IL-35, driving CD4⁺ T cell development into ITR35 cells (33). DCs have also been shown to markedly upregulate IL-35 in response to lipopolysaccharide (LPS) stimulation, and there is evidence that IL-35 can suppress such LPS-triggered DC maturation and can influence the ability of DCs to produce factors including IL-10 and TGF-β (71, 72). IL-35⁺ DCs and interacting T cells enrich P35 and Ebi3 expression, and mice vaccinated using IL-35⁺ DCs exhibit enhanced tumor growth and reductions in T cells within the TME, consistent with the ability of this cytokine to constrain intratumoral immune responses (73, 74). Cytokine-induced killer (CIK) cells, a type of MHC-unrestricted CD3⁺CD56⁺ cytotoxic T lymphocytes, have also been found to exhibit anti-tumor activity as an immunotherapy target in certain malignancies (75, 76). Tregs and IL-35 levels increase in a time-dependent fashion in the context of CIK cell production, whereas DCs can inhibit such increases and enhance CIK cell cytotoxicity (77). As dendritic cell (DC)-CIK-mediated immunotherapy is an effective method for adoptive cell therapy, the potential role of IL-35 in combined CIK/DC-CIK therapy and chemotherapy warrants additional consideration (78, 79). IL-35 can also convert immunogenic CD8α-DCs into tolerogenic DCs. Such tolerance-induced DCs (TolDCs) also express IL-12p35 and Ebi3, and can upregulate Ebi3 and IL-12p35 when stimulated with IFN-γ, LPS, or CD40L (71). Such TolDCs may thus represent an important focus for future studies of interactions between IL-35 and DCs. Arginase-1 (Arg1) is a metabolic enzyme specifically expressed by DCs that functions as an immune checkpoint molecule associated with tumor immune escape, and it is also a downstream effector of IL-35 (39, 77). Downregulation of IL-35 within the TME can inhibit Arg1 expression and immunomodulatory activity, inducing immune escape and tumorigenesis (77).

Macrophages are among to most abundant immune cells within the TME, and they are broadly classified into the inflammatory, classically-activated M1 subtype and the pro-tumorigenic, alternatively-activated M2 subtype, which are referred to as tumor-associated macrophages (TAMs). TAMs, together with DCs, are members of APC populations in the TME and play important roles in tumor growth and progression, depending on their relative M1 or M2 polarization, respectively (80, 81). Relative to metastatic tumors, M2 markers are less abundant within primary tumors, wherein macrophages exhibit distinct activity. EBI3 and IL-12a, two subunits of IL-35, have been shown to be highly expressed in metastatic tumor-associated TAMs together. M2 cells in both humans and animal models express the IL-35 EBI3 subunit, which promotes macrophage differentiation, survival, and recruitment into the TME and maintains M2-like TAM cell functionality (20, 39, 82). Recruited by IL-35 from PDAC cells, macrophage express CXCL1 and CXCL8 to promote angiogenesis (83). Deletion of EBI3(EBI3^L/L-Tom) induced a decrease of M1-TAM population, while Sawant et al. observed a comparable advancement of it in IL-10-deficient mice (IL-10^L/L) (46). The biological interaction between IL-35 and M2 is also affected by some factors. IL-35 of M2 macrophages was suppressed by NRP1 expression in Stomach Adenocarcinoma (STAD), and the expressed cytokine was served as a major signal in the immune suppression mechanism of STAD (84). In the context of NSCLC, Heim et al. found that increased Arg1 mRNA expression was positively correlated with levels of IL-35⁺ M2 cells and Tregs, whereas TNF-α expression fell as IL-35 expression increased. This suggests that IL-35⁺ M2 cells produce Arg1 mRNA to induce or attract the production of iTr35 cells, thereby driving IL-35 production and associated responses (39). The number of CD68⁺ macrophages in tumors has also been shown to decline and to be positively correlated with IL-35 levels in normal tissue, suggesting that there may be a distinct subset of macrophages expressing IL-35 within the TME (39). TAMs within primary tumors mainly secrete TNF-α to induce the process of epithelial-mesenchymal transition (EMT) which is an indispensable process that enhances the migratory and invasive abilities of tumor cells (20, 85). At metastatic sites, in contrast, TAMs produce IL-35 to reverse the EMT by activating JAK2-STAT6, GATA3, and other signaling pathways, thereby facilitating metastatic tissue colonization (20). Such TAM-mediated activity is believed to be a primary driver of NSCLC metastatic colonization of distal tissue sites (20).

Angiogenesis, this process of tumor vascular growth is an inevitable program of tumor progression in the TME. It can promote the proliferation of epithelial cells by releasing several immunosuppressive cytokines and angiogenic growth factors. While secreted by endothelial cells and monocytes to less extent, the key role of IL-35 in angiogenesis should not be ignored. Wang et al. established a J558 mouse model and determined that IL-35 can induce the expression of CD31 and vascular endothelial growth factor (VEGF), inducing angiogenesis and endothelial cell activation (86).IL-35 also facilitates PDAC endothelial adhesion and transendothelial migration in vitro via the endothelial adhesion molecule, ICAM1 (29). With establishing a model system similar to the TME, Liao et al. confirmed that IL-35 from tumor cells, Tregs, and MDSCs promote the secretion of VEGF to attracts endothelial cells. In addition, monocytes are central players in angiogenesis and the source of pro-angiogenic cytokines. In early 2018, Huang et al. discovered that IL-35 increased the recruitment of monocytes to promote PDAC progression and monocyte-induced angiogenesis through IL-35-CXCL5 axis (C-C motif chemokine ligand 5) by RNA-seq and immunohistochemical analyses in xenograft mouse models (83). In PDAC samples from patients, they measured markers that can reflect the expression level of IL-35, such mRNA and protein. Not unexpectedly, the markers correlated with microvessel density and infiltration of monocyte lineage cells (83). Subsequently, Wang et al. identified that numbers of i35-Bregs were remarkably increased with the accumulation of CD14⁺ monocytes (37). Moreover, IL-35 suppressed CD14⁺ monocytes induced naive CD4⁺ T cell activation and the production of TNF-α and granzyme B, inhibiting cytotoxicity of them, whereas this effect has not been reported in TME (87). Further studies are needed to elucidate that function of IL-35 in the complex role of endothelial cells and monocytes during the angiogenesis in the TME.

Prior studies have explored the interactions between IL-35 and other intratumoral immune cell populations not discussed above, such as neutrophils, MDSCs, and NK cells (88–90), although more research on these interactions is warranted. Anti-tumorigenic N1 neutrophils and pro-tumorigenic N2 neutrophils are two different subsets of neutrophils identified in previous studies (91). IL-35 has been shown to promote N2 neutrophil polarization by increasing G-CSF and IL-6 production, thereby enhancing the ability of these N2 cells to promote angiogenesis and to suppress immune responses, thereby enabling invasion of N2 cells into tumor tissue (30). MDSCs within the TME can secrete a range of immunosuppressive factors including IL-35, and in PCA model mice, high IL-35 levels were associated with increases in MDSC and Treg levels and reductions in CD4⁺ and CD8⁺ T cell frequencies within the spleen, blood, and TME, and with alterations in tumor growth, metastasis, and worse survival outcomes (19). Moreover, tumor-cell derived IL-35 was reported to promote tumor growth and angiogenesis through the enhancement of myeloid cell accumulation in the TME (92, 93). NK cells are important mediators of anti-tumor immune responses, and cytokines including IL-35, IL-12, IL-23, and IL-27 can modulate DC, macrophage, and NK cell functionality to directly and indirectly control NK cell immune responses (36, 94). While the mechanistic basis for these interactions remains to be clarified, there is clear evidence that IL-35 is likely to regulate a range of immune cell functions within the TME.