In this report, we describe four findings that provide new insights into effector T cell populations within the tumor's microenvironment. First, a subpopulation of T cells defined as CD8+Foxp3+ T cells, are induced at the tumor site of regressing tumors but not in immune tolerant conditions. Second, despite having an effector phenotype with some features of other regulatory cells, CD8+Foxp3+ T cells can be associated with a desired anti-tumor immune response. Third, Foxp3 expression in TIL subsets is dependent on ongoing signals from the microenvironment. Fourth, only antigen-specific T cells that have not been actively tolerized can acquire effector function and Foxp3 expression.
For therapeutic vaccine strategies to achieve optimal benefit, it is essential to understand the elements necessary for adequate priming and expansion of tumor-specific T cells. It is also critical to understand the effects of vaccination strategies on T cell populations both at peripheral sites and within the tumor's microenvironment, and the complex interplay that occurs in the microenvironment where effectors must infiltrate and kill their targets. Our studies show for the first time that CD8+
T cells are localized to the TIL of regressing tumors and mark the presence of potentially functional tumor-specific T cells. In addition, induction of these T cells in TIL may serve as a marker for an effective T cell response. In both preclinical and clinical studies, vaccines have been able to induce robust T cell responses in the peripheral blood or lymph nodes. However, there has been a dissociation between systemic and intratumoral responses.16
Few studies have correlated vaccine-induced tumor regressions with tumor-specific T cell populations within the tumor's microenvironment. Yet, T cells must maintain their active state within the tumor's environment to serve as a true biomarker of tumor regression.
Some studies suggest that CD4+
Tregs in tumors contribute to immune tolerance and are a correlative marker of poorer patient outcomes in breast, ovarian and pancreatic cancers.17–20
Other studies suggest that it is Teff/Treg ratios that correlate with effective anti-tumor responses.21,22
In Hodgkin's lymphoma, Tregs in the tumor microenvironment are actually associated with improved clinical outcomes.23
In our model, the ratio of Tregs to effectors does not necessarily correlate with an active immune response as suggested by similar ratios of CD4Tregs/CD4effs in TILs isolated from both the FVB/N and neu-N
mice. In fact, by absolute numbers, the trend shows that CD4+
Tregs were more abundant in the actively regressing tumors (Supporting Information Fig. S3
). Foxp3 expression in the tumor microenvironment may serve as an activation marker in T cells. Another hypothesis is that they serve to dampen an anti-tumor response as it resolves. Interestingly, the transfer of Tregs into Apcmin/+
results in regression of intestinal tumors.24
This is primarily due to the anti-inflammatory role of the Treg in this model. In our model, the best correlative marker of an effective anti-tumor response is the presence of CD8+
The effector phenotype of a population with regulatory features is somewhat surprising. Activation markers such as CD25, GITR and CTLA4 are expressed on the majority of TILs as would be expected in ongoing immune responses. The use of Foxp3 as a Treg lineage marker has made it possible to distinguish Tregs from effectors cells. Studies currently show that Foxp3+ cells of any origin are suppressive. Even human CD8+ T cells that express Foxp3 transiently in vitro as a result of activation have suppressive capacity. CD8+Foxp3+ TILs also have potent in vitro suppressive capacity. Therefore, our report does not contradict prior studies on reportedly regulatory CD8+ T cell populations. Our data on vaccine-induced endogenously generated and adoptively transferred transgenic tumor-specific T cells which shows a correlation with an anti-tumor response stresses the importance that induction of Foxp3 expression has to be understood within the context of the host's immune response.
The interactions among the diverse components of the tumor microenvironment are becoming increasingly complex. Any one of the elements of the environment, from the diverse populations to the evolving cytokine milieu, could be critical in the induction or maintenance of the Foxp3 phenotype. Although most studies focus on the CD4 lineage, TGF-β induced Foxp3 induction and maintenance is not a novel concept. Priming and expansion of tumor-specific T cells under activating conditions appears to increase the pool of T cells that will subsequently enter the tumor microenvironment. Intact signaling through the T cell receptor and TGF-β/smad-3 pathway are likely necessary for Foxp3 induction. Because Foxp3 expression suggests active signaling through the TCR, CD8+Foxp3+ T cells signify a population of cells in which TCR signaling is not impaired. It is possible that these cells could be recruited to function as anti-tumor effectors. A pure population of clonotypic CD8+ effector T cells with tumor lysis capability can convert a proportion of active T cells into Foxp3+ T cells. We therefore propose a model in which effector CD8+ T cells initially traffic into the tumor's microenvironment and continue to expand to meet the demands of the antigen load within a T cell activating cytokine mileu. As the tumor begins to regress and the antigen load diminishes, changes in the cytokine mileu mediated by cytokines such as TGF-beta induce upregulation of Foxp3 expression within a subpopulation of the effector CD8+ T cells. Given that there is some suggestion that these cells acquire some suppressive capacity, a hypothesis generated from these findings is that these T cells then control the effector arm of the response, acting as a feedback mechanism to quickly turn off the activation status of the larger effector T cell population.
In conclusion, our findings identify a novel CD8+ T cell population within the tumor microenvironment that demonstrates tumor antigen specificity and effector function. Their Foxp3 expression depends on the cytokine mileu. We also show for the first time that the CD8+Foxp3+ TILs can serve as a potential marker for an effective anti-tumor immune response. The presence of Foxp3+ T cells as a marker of an active immune response may appear contradictory. However, these cells are present only in a host environment that is supportive of a functioning effector T cell population and appear to be antigen-specific. Thus, these findings provide new insights into the nature of Foxp3+ cells in the microenvironment and suggest caution in interpreting any analysis which involves the prognostic or predictive implications of the presence of Foxp3+ subsets.