Abstract
A growing body of evidence suggests that a major subset of patients with advanced solid tumors shows evidence for a T cell-inflamed tumor microenvironment. This phenotype has positive prognostic value for several types of early stage cancer, suggesting that the attempt by the host to generate an anti-tumor immune response reflects a biologic process associated with improved patient outcomes. In metastatic disease, the presence of this phenotype appears to be associated with clinical response to several immunotherapies, including cancer vaccines, checkpoint blockade, and adoptive T cell transfer. With the high rate of clinical response to several of these therapies, along with early data indicating that combination immunotherapies may be even more potent, it seems likely that effective immune-based therapies will become a reality for patients with a range of different cancers that physiologically support the T cell-inflamed tumor microenvironment in a subset of individuals. Therefore, one of the next significant hurdles will be to develop new therapeutic interventions that will enable these immunotherapies to be effective in patients with the non-T cell-inflamed phenotype. Rational development of such interventions will benefit from a detailed molecular understanding of the mechanisms that explain the presence or absence of the T cell-inflamed tumor microenvironment, which in turn will benefit from focused interrogation of patient samples. This iterative “reverse-translational” research strategy has already identified new candidate therapeutic targets and approaches. It is envisioned that the end result of these investigations will be an expanded array of interventions that will broaden the fraction of patients benefitting from immunotherapies in the clinic.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4555998/
The next hurdle in cancer immunotherapy: Overcoming the non-T cell-inflamed tumor microenvironment
Re: The next hurdle in cancer immunotherapy: Overcoming the non-T cell-inflamed tumor microenvironment
1. Characteristics of the T cell-inflamed tumor microenvironment. The molecular identification of tumor antigens that can be recognized by host T cells in cancer patients (1, 2) prompted the development of vaccination strategies to increase the frequency of tumor antigen-specific T cells as a therapeutic approach. In addition, it provided tools that could be utilized to quantify tumor antigen-specific T cells in the blood and also at tumor sites (3). During the course of this work, two major observations were made. First, a number of patients showed spontaneously elevated frequencies of tumor antigen-specific T cells at baseline, prior to immunization. Second, only a minor subset of patients showed clinical activity, despite induction of increased specific T cell frequencies after vaccination (4, 5). Together, these observations generated a new conceptual advance—the hypothesis that resistance mechanisms at the level of the target tissue (the tumor microenvironment) might remain dominant despite the presence of abundant anti-tumor T cells circulating in the host.
To pursue this question in patients, we began with studying baseline biopsies of melanoma metastases for interrogation by transcriptional profiling. This analysis revealed two major subsets of tumors that were largely characterized by the presence or absence of a gene expression profile indicative of a pre-existing T cell-inflamed tumor microenvironment. The T cell-inflamed subset of tumors showed presence of T cell transcripts, chemokines that likely mediate effector T cell recruitment, macrophage activation markers, and a type I IFN transcriptional profile (6). Immunohistochemistry confirmed the presence of CD8+ T cells, macrophages, and some B cells and plasma cells in these lesions (6). In several small series of HLA-A2+ patients, CD8+ T cells specific for melanoma differentiation antigens have been identified from tumor sites using peptide-HLA-A2 tetramer analysis. Therefore, at least a subset of T cells specific for tumor antigens is present among these infiltrates. In fact, this is arguably the starting point for adoptive T cell approaches utilizing tumor-infiltrating lymphocytes (TIL), which has consistently generated approximately a 50% response rate in patients with metastatic melanoma (7). However, functional analysis has indicated various degrees of dysfunction of these tumor antigen-specific T cells when analyzed directly ex vivo (8–10). These results suggest that the reason for tumor progression despite the presence of specific adaptive immunity in this subset of patients is likely secondary to immune suppressive mechanisms acting at the level of the tumor microenvironment. Interestingly, in some cases the presence of memory virus-specific CD8+ T cells also has been observed in these T cell-inflamed tumors. However, their function seems to be intact (8, 11), and these probably represent non-specifically recruited activated T cells migrating along chemokine gradients but not participating in tumor recognition. Thus, a component of antigen-specificity to the T cell dysfunction in the tumor microenvironment appears to be operational.
More detailed analysis of the T cell-inflamed subset of tumors revealed the presence of transcripts encoding indoleamine-2,3-dioxygenase (IDO), PD-L1, and FoxP3 (12, 13). Quantitative analysis of individual tumors revealed that the expression level of these three transcripts was positively correlated. IHC confirmed that PD-L1 and IDO protein expression, and also nuclear FoxP3+CD4+ cells, were found within T cell-inflamed tumors in the same region as CD8+ T cells. Mouse mechanistic studies confirmed that CD8+ T cells were required for the upregulation of all of these three factors within the tumor microenvironment. For PD-L1 and IDO induction, the requisite factor produced by the CD8+ T cells was IFN-γ. For FoxP3+ Tregs, production of the chemokine CCL22 was identified, which mediated Treg recruitment into tumor sites (13). Together, these data suggest that the involvement of these three immune-inhibitory mechanisms in T cell-inflamed tumors is not pre-existing and driven by the tumor cells, but rather is driven by the activated CD8+ T cells themselves.
The original hypothesis in the context of our melanoma vaccine studies was that the responding patients might have low expression of immune inhibitory mechanisms in the tumor microenvironment and resistant patients the highest expression. On the contrary, the opposite was the case. A baseline T cell-inflamed tumor microenvironment (that includes presence of PD-L1, IDO, and Tregs) was positively associated with clinical benefit from these vaccines (14–16). Thus, the ability of a tumor to produce chemokines and recruit activated T cells into the tumor microenvironment appears to be instrumental for clinical benefit. Similar results have been observed in patients treated with high-dose IL-2 (17) and also with the anti-CTLA-4 mAb ipilimumab (18). Most strikingly, clinical response with anti-PD-1 mAb, which is blocking PD-L1/PD-1 interactions directly within the tumor microenvironment, was found to occur almost exclusively in patients with pre-existing T cell infiltrates in the region of PD-L1 upregulation (19–21). Following anti-PD-1 administration, these CD8+ T cells seemed to proliferate and expand to penetrate throughout the tumor, which was correlated with tumor regression (21). These observations are consistent with our own mouse model data which demonstrated that tumor regression upon checkpoint blockade was almost completely mediated by re-activation of CD8+ T cells directly within the tumor site to be able to proliferate and produce IL-2 (22).
To pursue this question in patients, we began with studying baseline biopsies of melanoma metastases for interrogation by transcriptional profiling. This analysis revealed two major subsets of tumors that were largely characterized by the presence or absence of a gene expression profile indicative of a pre-existing T cell-inflamed tumor microenvironment. The T cell-inflamed subset of tumors showed presence of T cell transcripts, chemokines that likely mediate effector T cell recruitment, macrophage activation markers, and a type I IFN transcriptional profile (6). Immunohistochemistry confirmed the presence of CD8+ T cells, macrophages, and some B cells and plasma cells in these lesions (6). In several small series of HLA-A2+ patients, CD8+ T cells specific for melanoma differentiation antigens have been identified from tumor sites using peptide-HLA-A2 tetramer analysis. Therefore, at least a subset of T cells specific for tumor antigens is present among these infiltrates. In fact, this is arguably the starting point for adoptive T cell approaches utilizing tumor-infiltrating lymphocytes (TIL), which has consistently generated approximately a 50% response rate in patients with metastatic melanoma (7). However, functional analysis has indicated various degrees of dysfunction of these tumor antigen-specific T cells when analyzed directly ex vivo (8–10). These results suggest that the reason for tumor progression despite the presence of specific adaptive immunity in this subset of patients is likely secondary to immune suppressive mechanisms acting at the level of the tumor microenvironment. Interestingly, in some cases the presence of memory virus-specific CD8+ T cells also has been observed in these T cell-inflamed tumors. However, their function seems to be intact (8, 11), and these probably represent non-specifically recruited activated T cells migrating along chemokine gradients but not participating in tumor recognition. Thus, a component of antigen-specificity to the T cell dysfunction in the tumor microenvironment appears to be operational.
More detailed analysis of the T cell-inflamed subset of tumors revealed the presence of transcripts encoding indoleamine-2,3-dioxygenase (IDO), PD-L1, and FoxP3 (12, 13). Quantitative analysis of individual tumors revealed that the expression level of these three transcripts was positively correlated. IHC confirmed that PD-L1 and IDO protein expression, and also nuclear FoxP3+CD4+ cells, were found within T cell-inflamed tumors in the same region as CD8+ T cells. Mouse mechanistic studies confirmed that CD8+ T cells were required for the upregulation of all of these three factors within the tumor microenvironment. For PD-L1 and IDO induction, the requisite factor produced by the CD8+ T cells was IFN-γ. For FoxP3+ Tregs, production of the chemokine CCL22 was identified, which mediated Treg recruitment into tumor sites (13). Together, these data suggest that the involvement of these three immune-inhibitory mechanisms in T cell-inflamed tumors is not pre-existing and driven by the tumor cells, but rather is driven by the activated CD8+ T cells themselves.
The original hypothesis in the context of our melanoma vaccine studies was that the responding patients might have low expression of immune inhibitory mechanisms in the tumor microenvironment and resistant patients the highest expression. On the contrary, the opposite was the case. A baseline T cell-inflamed tumor microenvironment (that includes presence of PD-L1, IDO, and Tregs) was positively associated with clinical benefit from these vaccines (14–16). Thus, the ability of a tumor to produce chemokines and recruit activated T cells into the tumor microenvironment appears to be instrumental for clinical benefit. Similar results have been observed in patients treated with high-dose IL-2 (17) and also with the anti-CTLA-4 mAb ipilimumab (18). Most strikingly, clinical response with anti-PD-1 mAb, which is blocking PD-L1/PD-1 interactions directly within the tumor microenvironment, was found to occur almost exclusively in patients with pre-existing T cell infiltrates in the region of PD-L1 upregulation (19–21). Following anti-PD-1 administration, these CD8+ T cells seemed to proliferate and expand to penetrate throughout the tumor, which was correlated with tumor regression (21). These observations are consistent with our own mouse model data which demonstrated that tumor regression upon checkpoint blockade was almost completely mediated by re-activation of CD8+ T cells directly within the tumor site to be able to proliferate and produce IL-2 (22).
Debbie
Re: The next hurdle in cancer immunotherapy: Overcoming the non-T cell-inflamed tumor microenvironment
Combination immunotherapies are being developed and evaluated as an attempt to improve clinical benefit further (22–24). This is logical, as multiple immune inhibitory mechanisms appear to be present concurrently within the T cell-inflamed tumors, so blockade of two or more pathways could be synergistic. Preclinically, we have shown that concurrent doublets of anti-CTLA-4 +/− anti-PD-L1 +/− an IDO inhibitor each was synergistic in the B16 melanoma model in vivo (22). Interestingly, each of these combination therapies involved re-acquisition of IL-2 production and proliferation by CD8+ T cells directly within the tumor microenvironment. In fact, therapeutic effects were preserved even in the presence of FTY720 blockade, which prevents exit of new T cells from lymph nodes. Therefore, as clinical development of each of these doublets is proceeding in advanced cancer patients (25, 26), our working model suggests that they may indeed have increased clinical activity but that this clinical benefit may be restricted to the context of the T cell-inflamed tumor microenvironment phenotype. Therefore, new therapeutic approaches will likely be needed to support immunotherapy efficacy in patients with the non-T cell-inflamed tumor microenvironment. Rational development of such approaches will benefit from more detailed knowledge regarding the underlying mechanisms that explain the presence or absence of a spontaneous T cell infiltrate.
Debbie
Re: The next hurdle in cancer immunotherapy: Overcoming the non-T cell-inflamed tumor microenvironment
2. Mechanisms of innate immune sensing that translate into spontaneous T cell priming and T cell infiltration into tumors. As a first approach toward understanding the underlying mechanisms that control the T cell-inflamed tumor phenotype, we interrogated our melanoma gene expression profile data for evidence of innate immune activation pathways. In general, in order for adaptive T cell responses to be induced against an antigen, dendritic cells (DCs) or other involved antigen-presenting cells (APCs) need to be activated themselves by additional molecular entities. In the setting of infectious agents, this is often through stimulation of Toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs), such as endotoxin that is recognized by TLR4 (27). However, it had not been clear what factors might provide innate immune signaling in the context of sterile tumors in which infectious agents are not involved. Our melanoma data indicated that tumors that contained a T cell infiltrate also showed evidence for a transcriptional signature known to be induced by type I IFNs (6, 28). We therefore carried out mouse mechanistic experiments to determine whether type I IFN signaling on host cells was necessary for spontaneous priming of CD8+ T cells against tumor-associated antigens. This indeed was the case. Type I IFNR−/− mice, or mice deficient in the downstream transcription factor Stat1, showed markedly reduced T cell responses in multiple transplantable tumor models (28). The requirement for type I IFN signaling was mapped to the level of APCs, and indeed specific deletion of the type I IFNR in DCs was sufficient to reproduce this defect. Mixed bone marrow chimera experiments demonstrated that the specific subset of DCs involved in this effect was the Batf3-driven lineage that expresses CD8+ or CD103 (28–30). In addition, IFN-γ production was found to be induced in DCs upon implantation of a tumor in vivo. These data suggest that early innate immune recognition of cancer cells in vivo involved activation of a pathway that resulted in IFN-γ production, which in turn was necessary for complete DC activation and CD8+ T cell priming against tumor antigens to give rise to the T cell-infiltrated tumor microenvironment phenotype (31).
These observations led to interrogation of the next level of the problem, which is identifying the receptor system and putative ligands that induce IFN-γ production by host DCs in the context of a growing tumor in vivo. By using a series of knockout mice specifically lacking TLRs, the adaptors MyD88 or Trif, the intracellular RNA sensing pathway involving MAVS, or the extracellular ATP sensing receptor P2X7R, we were able to rule out most of the receptor systems that have been implicated in various infectious disease models. By process of elimination, this left us with the STING pathway as an important candidate. STING is an adapter that is activated by cyclic dinucelotides generated by cGAS, which in turn is directly activated by cytosolic DNA (32–34). This pathway has been implicated in the sensing of DNA viruses, but also in selected autoimmune models (35, 36). Moreover, activating mutations of STING have recently been identified in human patients with a vasculitis/pulmonary inflammation syndrome that is characterized by increased type I IFN production (37). Indeed, through the use of STING−/− mice we demonstrated that spontaneous T cell priming against tumor antigens was markedly reduced in vivo, and rejection of immunogenic tumors was ablated (38). We found evidence for tumor-derived DNA within the cytosol of a major population of tumor-infiltrating DCs, which was associated with STING pathway activation and IFN-γ production. Therefore, the host STING pathway appears to be a major innate immune sensing pathway that detects the presence of a tumor to drive DC activation and subsequent T cell priming against tumor-associated antigens in vivo. Recently, several additional tumor model systems have confirmed a role for the STING pathway in anti-tumor immunity in vivo (39–41). The realization of the importance of this particular innate immune pathway in the cancer context is generating new therapeutic strategies that might be utilized to activate or mimic this pathway for promoting immune-mediated tumor control, particularly in the non-inflamed tumor subset.
These observations led to interrogation of the next level of the problem, which is identifying the receptor system and putative ligands that induce IFN-γ production by host DCs in the context of a growing tumor in vivo. By using a series of knockout mice specifically lacking TLRs, the adaptors MyD88 or Trif, the intracellular RNA sensing pathway involving MAVS, or the extracellular ATP sensing receptor P2X7R, we were able to rule out most of the receptor systems that have been implicated in various infectious disease models. By process of elimination, this left us with the STING pathway as an important candidate. STING is an adapter that is activated by cyclic dinucelotides generated by cGAS, which in turn is directly activated by cytosolic DNA (32–34). This pathway has been implicated in the sensing of DNA viruses, but also in selected autoimmune models (35, 36). Moreover, activating mutations of STING have recently been identified in human patients with a vasculitis/pulmonary inflammation syndrome that is characterized by increased type I IFN production (37). Indeed, through the use of STING−/− mice we demonstrated that spontaneous T cell priming against tumor antigens was markedly reduced in vivo, and rejection of immunogenic tumors was ablated (38). We found evidence for tumor-derived DNA within the cytosol of a major population of tumor-infiltrating DCs, which was associated with STING pathway activation and IFN-γ production. Therefore, the host STING pathway appears to be a major innate immune sensing pathway that detects the presence of a tumor to drive DC activation and subsequent T cell priming against tumor-associated antigens in vivo. Recently, several additional tumor model systems have confirmed a role for the STING pathway in anti-tumor immunity in vivo (39–41). The realization of the importance of this particular innate immune pathway in the cancer context is generating new therapeutic strategies that might be utilized to activate or mimic this pathway for promoting immune-mediated tumor control, particularly in the non-inflamed tumor subset.
Debbie