The ability of CD8⁺ T cells to respond to cytokines in a bystander T cell receptor (TCR)-independent way has been studied for a long time. The work of Slifka et al. examined the effects of more than 1800 cytokine combinations on virus-specific CD8⁺ T-cell activation, demonstrating that certain cytokine combinations could synergize to induce antigen-independent IFNγ production, CD69 up-regulation and in some cases exhibit differential regulatory functions (1).

Interestingly, a recent review by Maier et al. has summarized data demonstrating that recognition of cancer cells is mainly restricted to a small subset of antigen (Ag)-specific tumor infiltrating leukocytes (TILs). In fact, many TILs are “cancer ignorant” and have been defined as “bystander T cells”, that recognize non-cancer peptides, including viral antigens (2). Although the most recent evidence has pointed to bystander T cells as being the majority of infiltrating T cells in tumors, there is still a lack of consensus regarding the activation status of these cells and if their presence in tumors plays a role in anti-cancer immunity.

Within the bystander T cell subset, virtual memory T cells are a population of CD8⁺ T cells that, despite their antigen inexperience, exhibit many hallmarks compatible with conventional Ag-specific memory T cells. By consensus, the lineage markers for T_VM cells are: CD8⁺ CD44^hi CD122^hi CD49d^lo. The most recent marker is integrin alpha 4 (CD49d) as it is highly expressed in T cells after a strong signal through the TCR, via the cognate Ag-TCR-MHC-I interaction. As a result, CD49d is highly expressed by T_MEM/T_EFF but not by T_VM cells, thus becoming an essential marker that discriminates between these 2 populations of CD8⁺ memory T cells (3–5). Also, a transcription factor, eomesodermin (Eomes), is highly expressed by T_VM cells and is associated with their functional capacity (6, 7).

During their thymic maturation these “memory-like” cells can be differentiated into two populations: T_VM and T_IM (innate memory) by the expression of different markers, including CD5^hi in T_VM and interleukin (IL)-4R^hi and CD49d^hi in T_IM cells. However, when they reach secondary lymphoid organs (SLO), T_IM cells downregulate CD49d, and as such, CD5 and IL-4R expression is not sufficient to discriminate between both cell types (8). As a result, they became indistinguishable from each other and compose a heterogeneous group of memory-like cells designated as T_VM cells (3, 8, 9).

In spite of never having contacted their specific Ags, T_VM cells are capable of developing a powerful cytotoxic response in a TCR-independent manner, mainly through mechanisms that may involve interaction through the receptor NKG2D (10, 11). Also, their lytic capacity is mediated by production of large amounts of the interferon gamma (IFNg), especially after IL-12 and IL-18 stimulation (3, 12–14) and by granzymes release (15, 16). Even though these cells have been described as being important in controlling infectious diseases through innate bystander mechanisms, the role of T_VM cells in cancer has not been widely studied.

Interleukin-12 and IL-18 are inflammatory cytokines mainly produced by activated macrophages and dendritic cells at the initiation of an immune response (17). It has been reported that IL-12 and IL-18 are capable of activating virtual memory CD8⁺ T cells leading to rapid production of IFNγ, resulting in crucial pathogen control during certain viral and bacterial infectious processes (10, 18–21). Moreover, we have previously described the role of T_IM cells during a murine model of parasitic infection with Trypanosoma cruzi ( 13).

Infiltration of CD8⁺ T cells into tumors is associated with a better prognosis for response in cancer patients (22, 23). Moreover, it has been long assumed that CD8⁺ T cells present in tumors are conventional memory CD8⁺ T specific for tumor antigens. In this context, the role of T_VM cells present in tumors and their Ag-independent antitumor mechanisms has not been thoroughly investigated.

The few reports that have addressed the role of T_VM cells (or cells compatible with T_VM phenotype, not identified by the current consensus lineage markers) mainly focus on the antitumor mechanism mediated by the receptor NKG2D (10, 11). However, the presence and activity of T_VM vs T_MEM cells in tumor growth control has not been thoroughly studied, especially with regard to the role of effector mechanisms. This is quite important, especially with respect to tumors that do not express the ligands for NKG2D. In this context, IFNγ is a cytokine highly produced by T_VM cells, able to promote a potent antitumor activity especially through anti-angiogenic mechanisms (24). Importantly, we have recently demonstrated that after IL-12 systemic expression, a significant reduction is observed in the number of blood vessels present in B16 and EL-4 tumors (25). This data is quite relevant to the biology of these tumors as they do not express NKG2D ligands (26) and their growth seems to rely upon neovascularization (24).

Based on this evidence, we decided to focus on the role of IFNγ in murine cancer models utilizing cell lines that lack NKG2D ligands, in order to avoid interference with this potential cytotoxic mechanism (26). Previously, our group demonstrated that systemic expression of IL-12+IL-18 was capable of inducing a strong antitumor effect against 2 different tumor cells lines in vivo (B16, melanoma and 3LL, lung carcinoma). However this occurred at the expense of toxic side effects resulting in only 50% survival over a period of 50 days post-treatment (27). Because toxicity was associated with IL-12 but not to IL-18 (27, 28), we used lower doses of IL-12 cDNA to minimize the side effects. Utilizing this lower dose, we demonstrated that survival reached 100% and the antitumor effect was still present (25). Interestingly, IL-12 systemic expression does not affect the total number of CD8⁺ T cells but induces an increase in the percentage of IFNγ⁺CD8⁺ infiltrating B16 and EL-4 tumors (25). As we have reported, a rapid and strong antitumor effect early after IL-12+IL-18 expression (27), we speculated that T_VM (CD8⁺ CD44^hi CD122^hi CD49d^lo) along with tumor Ag-specific CD8⁺ effector T cells (T_EFF) and T_MEM (CD8⁺ CD44^hi CD122^hi CD49d^hi) could be playing a role in tumor growth control in an Ag-independent manner.

In the present work we have evaluated the balance between these populations in SLO and tumors, both in steady-state conditions and after systemic stimulation with IL-12+IL-18. We observed that the number and the phenotypic characteristics of these T cells experienced changes from a normal state to inflammatory T helper 1 conditions following cytokine expression. Moreover, systemic expression of IL-12+IL-18 leads to a prevalence of T_VM cells expressing IFNγ in tumors and defines these cells as an essential factor for tumor growth control in the early stages of disease.

In the field of cancer immunology, the role of CD8⁺ T cells has been extensively addressed over the years. Solid tumors are usually infiltrated by different types of leukocytes and during the last 20-30 years, accumulating evidence has suggested that there is a positive correlation between tumor CD8⁺ T cells infiltration and a better prognosis for cancer patients. Moreover, this correlation is taken into account, both in the prognosis and in the choice of treatment for most cancers. For example, in colorectal cancer, Kirilovsky et al. have developed a prognostic score (‘Immunoscore’) in the classification system that takes into account the distribution of CD8⁺ cytotoxic T cells both in the tumor core and in the invasive margins that include tumor, lymph nodes and metastases (TNM) (22, 23).

Despite the clinical importance of the characterization of leukocytes in human and mouse tumors, there is a lack of information on the subtype of CD8⁺ T cells that infiltrate the tumors. For many years, it was assumed they are CD8⁺ T cells specific for Ags expressed by cancer cells. However, several reports suggest that CD8⁺ T cells with innate cell markers, not only infiltrate human and murine tumors, but also contribute to their eradication through mechanisms that are Ag-independent (11, 42, 43). Moreover, a recent review profoundly addresses the composition of tumor infiltrating CD8⁺ T cells and presents evidence demonstrating that a large fraction of human and murine tumor-infiltrating T cells are cancer unrelated and are designated as “bystanders T cells” (2). For example, Mognol et al. showed that, transferred activated “cancer-ignorant CD8⁺ cells” infiltrated murine tumors at levels comparable to those of cancer-specific CD8⁺ cells (44).

Comparative studies that simultaneously address T_MEM and T_VM cell differences and similarities in the cancer environment are rarely reported. Other than the work by Quinn et al. in humans (45), there is a complete absence of such reports in humans and the subject has only been approached by a few laboratories utilizing mouse models (4, 34, 46). However, it is important to note the work of Hussain et al. that presents a comprehensive comparative review of various aspects of the biology of both cell types (34). The appreciation of the role of T_VM cells as a first wave of immune protection, especially in infectious diseases, has become more obvious over the years. Moreover, the fact that T_VM cells can exhibit cytotoxicity more rapidly than naïve T cells, with similar kinetics and efficiencies of T_MEM cells (4, 45, 46), makes the investigation of T_VM cells very relevant in the cancer field.

Even though T_VM cells carry a functional TCR, these cells exhibit particularly high responsiveness to cytokines including IL-12, IL-18 and IL-15 as compared to specific Ag stimulation. Moreover, both Ag-inexperienced (T_VM) and Ag-experienced (T_MEM and T_EFF) cells proliferate in response to IL-15 and upon stimulation by IL-12 and IL-18 can elicit TCR independent proliferation, cytokine production and bystander cytotoxicity (4, 9, 34, 47, 48).

Based on these reports and the significant lack of information on the competitive role of T_VM vs T_MEM/T_EFF cells in cancer, we aimed to investigate the prevalence of T_VM vs T_MEM/T_EFF cells in SLO from mice bearing early established tumors, both in steady-state conditions and after systemic expression of IL-12, IL-18 and IL-15.

In our models, mice bear either B16 or KPC tumors for no longer than 8 to 10 days before cytokine induction. Moreover, the rapid antitumor response observed as early as 4 days (and up to 7 days) post-IL-12+IL-18 treatment leads us to speculate that the composition of the CD44^hiCD8⁺ T cell subset is a mix of T_VM cells (CD49d^lo) and Ag-specific CD8⁺ T effector cells and possibly early T_MEM cells (CD49d^hi). Thus, this provides us with a model where these memory/activated cells co-exist.

As previously reported, the constitutive high expression of IL-12R, IL-18R and CD122 situates T_VM cells in an advantagous position to rapidly respond to these cytokines (10, 49). Unexpectedly, our data demonstrate that systemic induction of IL-12+IL-18 (and IL-15) led to an enrichment of T_MEM/T_EFF cells with high expression of CD122 in SLO while T_VM cells remained at similar numbers and phenotype as compared to control mice. Even though we predicted the opposite results would be found, our data are similar to what is reported by Akue et al. The authors state that the frequency of pre-existing T_VM cells is stable in both steady-state conditions and after a greatly expanded Ag-driven memory CD8⁺ T cell immune response (47).

Another explanation for the prevalence of T_MEM cells after systemic IL-12+IL-18 expression could arise from the fact that T_VM cells in WT mice are a polyclonal population of memory-like CD8⁺ T cells. As such, after contacting their cognate Ag, these cells could convert to T_EFF and T_MEM cells as reported during influenza and listeria infections (4, 15). In our tumor models, the prevalence of T_MEM cells after Th1 systemic conditions could be the result of conversion of tumor-specific T naïve (T_N) cells plus tumor-specific T_VM cells to a T_EFF/T_MEM phenotype. To test this hypothesis, we performed similar experiments utilizing non-RAG OT-I mice. In this model system ( Supplementary Figure 4 ), we increased the number of T_VM cells (non-specific for tumor Ags) while a remnant number of polyclonal T_MEM/T_EFF are still present. Interestingly under these conditions, after IL-12+IL-18 expression we observed a significantly larger number of CD8⁺CD44^hi T cells in OT-I mice than what was observed in WT mice. This population was mainly composed of T_VM rather than T_MEM/T_EFF cells, especially in the spleen. An interesting finding was that after IL-12+IL-18 treatment, the levels of Eomes do not change compared to the levels in control mice both in T_VM and in T_MEM/T_EFF cells. More surprisingly, in OT-I mice, Eomes levels are significantly lower in T_VM and T_MEM/T_EFF from spleen but not in LNs as compared to WT mice. This is an unexpected finding since it has been reported that Eomes is able to bind to the il2rb promoter leading to increases in CD122 expression, thus driving increased T_VM cell sensitivity to IL-15 (6). This result led us to assume that up-regulation of CD122 in T_MEM/T_EFF cells after the cytokine treatment correlated with higher Eomes expression in this population. However, other stimuli could be responsible for CD122 up-regulation in T_MEM cells as it is reported that TCR signaling augments IL-2Rβ expression via both transcriptional and post-transcriptional regulation (50).

Interestingly, despite the higher proportion of T_MEM/T_EFF than T_VM cells in spleen and tumor-draining lymph nodes after IL-12+IL-18 expression, this is not what is observed on CD8⁺CD44^hi T cells infiltrating the tumors. These cells show a predominant presence of T_VM cells after cytokine expression. We could not determine if this effect is due to a preferential migration of T_VM cells to tumors under Th1 conditions or if the permanence/survival within tumors is favored by T_VM cells over T_MEM/T_EFF, similar to what has been reported by Miller et al. with T_N cells. Those authors demonstrated that when they co-transferred polyclonal CD8 T_N cells and polyclonal CD8 T_VM cells into TRAMP bearing mice, they found that donor T_VM cells constituted a substantial fraction of the tumor-infiltrating CD8⁺ T cells 4 months later (40).

The rapid antitumor immune response observed after systemic expression of IL-12+IL-18 points out to innate effector mechanisms rather than a TCR-mediated effect. The Ag-independent mechanisms achieved by bystander CD8⁺ T cells might rely on NKG2D-ligands recognition (10, 11), granzyme/perforin release (15, 16) and high IFNγ production (3, 12–14). The fact that B16 cells do not express ligands for NKG2D (51) may indicate that this pathway is not involved in the control of B16 tumor growth. Instead, rapid production of IFNγ, driven by IL-12+IL-18 could be responsible for the antitumor results observed in the experimental settings described here. Interestingly, we found that either IL-12 alone or IL-12+IL-18 systemic expression generated almost a complete disappearance of NK cells at 7-10 days post-treatment (25, 28) indicating that expression of IFNγ can largely arise from T cells. In this context we speculated that IFNγ produced by CD8⁺ T cells could be responsible for this early antitumor effect, especially since previous reports have demonstrated that IFNγ participates in different stages of tumor growth control (24, 52, 53). In our laboratory, we have previously documented that high expression of IFNγ is systemically induced after IL-12 or IL-12+IL-18 expression (27). Furthermore, there is a significantly higher number of IFNγ⁺CD8⁺ T cells infiltrating B16 and EL-4 tumors after cytokine treatment compared to tumors from control mice (25). Data presented in this work demonstrates that in the absence of IFNγ, the strong antitumor outcome observed after IL-12+IL-18 systemic expression is completely lost and this effect correlates with a significantly lower presence of T_VM cells in the tumors of IFNγKO mice compared to WT mice.

As mentioned earlier, IFNγ is able to induce different antitumor mechanisms. For example, interferon-inducible protein 10 (IP-10)-induced by IFNγ can exert a potent anti-angiogenic effect (53). Also, it is reported that IFNγ produced within tumors suppresses VEGFR3 expression by acting directly on tumor vessel endothelial cells and on the tumor-infiltrating lymphocytes to indirectly alter endothelial cells’ VEGFR3 expression (24). Consistent with this effect, previous work from our laboratory has demonstrated a significant reduction in the number of blood vessels present in B16 and EL-4 tumors after systemic expression of IL-12 (25). Additionally, Dangaj et al., using murine and human experimental systems, demonstrated that the initial recruitment of T cells to tumors is driven by tumor-derived CCL5 along with IFNγ-inducible CXCR3 ligands secreted by myeloid cells present in the tumors (54). Interestingly, not only do we report here that IFNγ KO mice show a reduced CD8⁺ T cell infiltration but we have also demonstrated that IFNγ is produced mainly by infiltrating CD8⁺ T cells rather than by NK cells in B16 tumors after IL-12 systemic expression (25). Furthermore, our preliminary data indicates higher CCR5 expression on CD8⁺ T cells infiltrating B16 tumors from 12 + 18-treated mice, as compared to control mice (unpublished data).

To further demonstrate the antitumor role of T_VM cells, we developed a model utilizing the KPC tumor cell line that shows poor T cell infiltration. In this murine pancreatic tumor model, most T cells are excluded (41). Moreover, contrary to most murine tumor models, KPC tumors recapitulate the structure of human cancers, with the presence of tumor islets surrounded by stromal cells. By using OT-I mice, our study demonstrated that CD8⁺ T cells (99-100% OVA-specific CD8⁺ T cells) not only highly infiltrate KPC tumors but also preferentially localized in the tumor islet after IL-12+IL-18 expression, contrary to the control OT-I mice where most CD8⁺ cells are found in the stromal areas. Consistent with our finding, Kantari-Mimoun et al. have reported that activated CAR T cells triggered the up-regulation of ICAM-1 on tumor cells in an IFNγ-dependent pathway that enabled T cell entry into tumor islets (52).

Based on this evidence from our and other laboratories, we hypothesize that systemic IL-12+IL-18 is able to trigger, in turn, expression of IL-15 and IFNγ. This pro-inflammatory environment could establish the perfect scenario for tumor eradication by different Ag-independent mechanisms mediated preferentially by IFNγ-producing T_VM cells. We speculate that in these conditions, CCR5⁺T_VM cells can preferentially migrate or selectively persist in tumors and locally produce IFN ҄γ. The IFNγ could then stimulate an anti-angiogenic effect along with the induction of adhesion molecules that permit the entry of T cells into the tumor islet. Even though we have presented solid evidence that tumor growth control is largely associated to the prevalence of T_VM cells in the tumor environment, the limitation of this work is to exclusively point to T_VM as the cells responsible for this effect. Currently experiments in our laboratory are focused on developing a more appropriate experimental strategy to substantiate this hypothesis.

The control of tumor growth through Ag-independent pathways is a topic of growing interest, especially considering that several tumors lose the expression of MHC type I as an immune evasion mechanism (55, 56). This loss of expression makes the tumor less susceptible to Ag-specific lysis, but more susceptible to innate control mechanisms such as the one exerted by T_VM cells. Furthermore, as proposed by White et al. and Drobek et al., the T_VM pool does not stochastically originate from the naïve T cell repertoire; instead, it is derived from a subset of naïve T cells that present a high affinity for self-ligands (visualized in mice as T cells with high levels of CD5) (40, 57). In this context, it is interesting to hypothesize that the continuous low level of homeostatic TCR signaling could result as an important signal for their anti-tumor responses. Accordingly, OT-I T cells are reported to be relatively highly self-reactive (40, 57). Even though systemic IL-12+IL-18 is normally triggered in infectious diseases, their use as an antitumor treatment has been difficult to accomplish, largely due to toxicity. However, type I IFNs, IL-12, IL-15 and IL-18 are produced by activated antigen presenting cells and other cell types that are often present in the tumor microenvironment, thus suggesting that an innate immune response may be important in the host anti-tumor response (58, 59). Moreover, BATF3⁺ dendritic cells are predominant producers of IL-15. Curiously, tumor-residing BATF3⁺ dendritic cells have been shown to be required for T cell trafficking (60) that can ultimately participate in the recruitment of T_VM cells to tumors due to high avidity of T_VM cells for IL-15. Based on this evidence, it could be possible that alternative therapies, able to induce expression of these cytokine in the tumor microenvironment, may become quite promising for the direction of future therapeutic strategies.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by Comité Institucional de Cuidado y Uso de Animales de Laboratorio of the Facultad de Ciencias Quı́micas of Universidad Nacional de Córdoba (CICUAL-FCQ).

CS-F: Methodology, validation, formal analysis, investigation. MV: Methodology, validation, formal analysis. NB: Methodology, investigation. NL: Methodology, validation. QF: Methodology, validation. HY: Conceptualization, original draft, review and editing. LV: Methodology, validation, formal analysis. ED: Conceptualization, investigation, funding acquisition. MR-G: Conceptualization, methodology, investigation, supervision, project administration, funding acquisition, writing. All authors contributed to the article and approved the submitted version.

This work was supported by Secretaría de Ciencia y Tecnología from Universidad Nacional de Córdoba (SECyT); Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT); Fondo para la Investigación Científica y Tecnológica (FONCyT); Fundación para el Progreso de la Medicina and P-UE 22920160100116CO - CONICET. This work was supported in part by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute (NCI), Cancer Innovation Laboratory (CIL) USA, under grant No. 1ZIABC009283-36. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government.

The authors thank Diego Luti, Victoria Blanco, Cecilia Noriega, Dr. Soledad Miro, Sergio Oms and Dr. Ivanna Novotny, for animal care. Dr. Pilar Crespo and Dr. Paula Abadie for FACS technical support. Dr. Laura Gatica, Lic. Gabriela Furlan and Dr. Noelia Maldonado for cell culture support and Paula Icely for overall experimental technical assistance. We acknowledge the NIH Tetramer Core Facility for provision of the PE and APC-labeled SIINFEKL/Kb tetramers.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.