Stefani Spranger

Up-regulation of PD-L1, IDO, and T(regs) in the melanoma tumor microenvironment is driven by CD8(+) T cells.

CD8+ T cells induce immunosuppressive mechanisms in the tumor microenvironment. The Great Escape One of the long-standing questions in cancer biology is how tumors evade the patrolling immune response. Many potential explanations have been proposed, including that the tumor itself suppresses the immune response. Indeed, some of the most promising new immunotherapies work by blocking inhibitory molecules, which enhances immune activity against the tumor. Now, Spranger et al. show that immunosuppressive mechanisms in the tumor may involve negative feedback loops dependent on an infiltrating immune response. The authors noticed that even tumors that are infiltrated with CD8+ T cells are not rejected and that this correlated with expression of three types of immunosuppressives: indoleamine-2,3-dioxygenase (IDO), PD-L1/B7-H1, and regulatory T cells. Mechanistic studies in mice suggested that CD8+ T cells were required to be in the tumor microenvironment for the up-regulation of these immunosuppressives. Extending these studies to the clinic, patients with inflammatory tumor infiltrates will thus be more likely to benefit from immune checkpoint therapies. Tumor escape from immune-mediated destruction has been associated with immunosuppressive mechanisms that inhibit T cell activation. Although evidence for an active immune response, including infiltration with CD8+ T cells, can be found in a subset of patients, those tumors are nonetheless not immunologically rejected. In the current report, we show that it is the subset of T cell–inflamed tumors that showed high expression of three defined immunosuppressive mechanisms: indoleamine-2,3-dioxygenase (IDO), PD-L1/B7-H1, and FoxP3+ regulatory T cells (Tregs), suggesting that these inhibitory pathways might serve as negative feedback mechanisms that followed, rather than preceded, CD8+ T cell infiltration. Mechanistic studies in mice revealed that up-regulated expression of IDO and PD-L1, as well as recruitment of Tregs, in the tumor microenvironment depended on the presence of CD8+ T cells. The former was driven by interferon-γ and the latter by a production of CCR4-binding chemokines along with a component of induced proliferation. Our results argue that these major immunosuppressive pathways are intrinsically driven by the immune system rather than being orchestrated by cancer cells, and imply that cancer immunotherapy approaches targeting negative regulatory immune checkpoints might be preferentially beneficial for patients with a preexisting T cell–inflamed tumor microenvironment.

Melanoma-intrinsic β-catenin signalling prevents anti-tumour immunity

Melanoma treatment is being revolutionized by the development of effective immunotherapeutic approaches. These strategies include blockade of immune-inhibitory receptors on activated T cells; for example, using monoclonal antibodies against CTLA-4, PD-1, and PD-L1 (refs 3, 4, 5). However, only a subset of patients responds to these treatments, and data suggest that therapeutic benefit is preferentially achieved in patients with a pre-existing T-cell response against their tumour, as evidenced by a baseline CD8+ T-cell infiltration within the tumour microenvironment. Understanding the molecular mechanisms that underlie the presence or absence of a spontaneous anti-tumour T-cell response in subsets of cases, therefore, should enable the development of therapeutic solutions for patients lacking a T-cell infiltrate. Here we identify a melanoma-cell-intrinsic oncogenic pathway that contributes to a lack of T-cell infiltration in melanoma. Molecular analysis of human metastatic melanoma samples revealed a correlation between activation of the WNT/β-catenin signalling pathway and absence of a T-cell gene expression signature. Using autochthonous mouse melanoma models we identified the mechanism by which tumour-intrinsic active β-catenin signalling results in T-cell exclusion and resistance to anti-PD-L1/anti-CTLA-4 monoclonal antibody therapy. Specific oncogenic signals, therefore, can mediate cancer immune evasion and resistance to immunotherapies, pointing to new candidate targets for immune potentiation.

Loss of PTEN promotes resistance to T cell–mediated immunotherapy

UNLABELLED T cell-mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. We show that loss of PTEN in tumor cells in preclinical models of melanoma inhibits T cell-mediated tumor killing and decreases T-cell trafficking into tumors. In patients, PTEN loss correlates with decreased T-cell infiltration at tumor sites, reduced likelihood of successful T-cell expansion from resected tumors, and inferior outcomes with PD-1 inhibitor therapy. PTEN loss in tumor cells increased the expression of immunosuppressive cytokines, resulting in decreased T-cell infiltration in tumors, and inhibited autophagy, which decreased T cell-mediated cell death. Treatment with a selective PI3Kβ inhibitor improved the efficacy of both anti-PD-1 and anti-CTLA-4 antibodies in murine models. Together, these findings demonstrate that PTEN loss promotes immune resistance and support the rationale to explore combinations of immunotherapies and PI3K-AKT pathway inhibitors. SIGNIFICANCE This study adds to the growing evidence that oncogenic pathways in tumors can promote resistance to the antitumor immune response. As PTEN loss and PI3K-AKT pathway activation occur in multiple tumor types, the results support the rationale to further evaluate combinatorial strategies targeting the PI3K-AKT pathway to increase the efficacy of immunotherapy.

Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade involves restored IL-2 production and proliferation of CD8(+) T cells directly within the tumor microenvironment.

BackgroundBlockade of immune inhibitory pathways is emerging as an important therapeutic modality for the treatment of cancer. Single agent treatments have partial anti-tumor activity in preclinical models and in human cancer patients. Inasmuch as the tumor microenvironment shows evidence of multiple immune inhibitory mechanisms present concurrently, it has been reasoned that combination therapies may be required for optimal therapeutic effect.MethodsTo test this notion, we utilized permutations of anti-CTLA-4 mAb, anti-PD-L1 mAb, and/or the IDO inhibitor INCB23843 in the murine B16.SIY melanoma model.ResultsAll three combinations showed markedly improved tumor control over single treatments, with many mice achieving complete tumor rejection. This effect was seen in the absence of vaccination or adoptive T cell therapy. The mechanism of synergy was investigated to examine the priming versus effector phase of the anti-tumor immune response. Only a minimal increase in priming of anti-tumor T cells was observed at early time points in the tumor-draining lymph nodes (TdLN). In contrast, as early as three days after therapy initiation, a marked increase in the capacity of tumor-infiltrating CD8+ T cells to produce IL-2 and to proliferate was found in all groups treated with the effective combinations. Treatment of mice with FTY720 to block new T cell trafficking from secondary lymphoid structures still enabled restoration of IL-2 production and proliferation by intratumoral T cells, and also retained most of the tumor growth control.ConclusionsOur data suggest that the therapeutic effect of these immunotherapies was mainly mediated through direct reactivation of T cells in situ. These three combinations are attractive to pursue clinically, and the ability of intratumoral CD8+ T cells to produce IL-2 and to proliferate could be an important biomarker to integrate into clinical studies.

Cancer immunotherapy strategies based on overcoming barriers within the tumor microenvironment

For tumor antigen-specific T cells to effectively control the growth of cancer cells in vivo, they must gain access to, and function within, the tumor microenvironment. While tumor antigen-based vaccines and T cell adoptive transfer strategies can result in clinical benefit in a subset of patients, most of the patients do not respond clinically. Even for tumor-infiltrating lymphocyte (TIL)-based adoptive transfer for patients with metastatic melanoma, which can provide tumor shrinkage in around 50% of treated individuals, many patients are not eligible, in part because there are not sufficient TIL present in the resected tumor. Thus, the denominator is in fact larger, and it has been suggested that absence of TIL may be a marker for poor efficacy of immunotherapies in general. While qualitative and/or quantitative features of the T cells are important considerations for efficacy, a major component of primary resistance likely can be attributed to the tumor microenvironment. Data are accumulating suggesting that two major categories of immune resistance within the tumor microenvironment may exist: failure of T cell trafficking due to low levels of inflammation and lack of chemokines for migration, and dominant suppression through immune inhibitory mechanisms. New therapeutic interventions are being guided by these observations, and preliminary clinical success is validating this working model.

Pharmacologic inhibition of MALT1 protease by phenothiazines as a therapeutic approach for the treatment of aggressive ABC-DLBCL.

Proteolytic activity of the mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) paracaspase is required for survival of the activated B cell subtype of diffuse large B cell lymphoma (ABC-DLBCL). We have identified distinct derivatives of medicinal active phenothiazines, namely mepazine, thioridazine, and promazine, as small molecule inhibitors of the MALT1 protease. These phenothiazines selectively inhibit cleavage activity of recombinant and cellular MALT1 by a noncompetitive mechanism. Consequently, the compounds inhibit anti-apoptotic NF-κB signaling and elicit toxic effects selectively on MALT1-dependent ABC-DLBCL cells in vitro and in vivo. Our data provide a conceptual proof for a clinical application of distinct phenothiazines in the treatment of ABC-DLBCL.

MHC-restricted fratricide of human lymphocytes expressing survivin-specific transgenic T cell receptors

The apoptosis inhibitor protein survivin is overexpressed in many tumors, making it a candidate target molecule for various forms of immunotherapy. To explore survivin as a target antigen for adoptive T cell therapy using lymphocytes expressing survivin-specific transgenic T cell receptors (Tg-TCRs), we isolated HLA-A2-allorestricted survivin-specific T cells with high functional avidity. Lymphocytes expressing Tg-TCRs were derived from these T cells and specifically recognized HLA-A2+ survivin+ tumor cells. Surprisingly, HLA-A2+ but not HLA-A2- lymphocytes expressing Tg-TCRs underwent extensive apoptosis over time. This demise was caused by HLA-A2-restricted fratricide that occurred due to survivin expression in lymphocytes, which created ligands for Tg-TCR recognition. Therefore, survivin-specific TCR gene therapy would be limited to application in HLA-A2-mismatched stem cell transplantation. We also noted that lymphocytes that expressed survivin-specific Tg-TCRs killed T cell clones of various specificities derived from HLA-A2+ but not HLA-A2- donors. These results raise a general question regarding the development of cancer vaccines that target proteins that are also expressed in activated lymphocytes, since induction of high-avidity T cells that expand in lymph nodes following vaccination or later accumulate at tumor sites might limit themselves by self-MHC-restricted fratricide while at the same time inadvertently eliminating neighboring T cells of other specificities.

Dendritic cells pulsed with RNA encoding allogeneic MHC and antigen induce T cells with superior antitumor activity and higher TCR functional avidity.

Adoptive transfer of T cells expressing transgenic T-cell receptors (TCRs) with antitumor function is a hopeful new therapy for patients with advanced tumors; however, there is a critical bottleneck in identifying high-affinity TCR specificities needed to treat different malignancies. We have developed a strategy using autologous dendritic cells cotransfected with RNA encoding an allogeneic major histocompatibility complex molecule and a tumor-associated antigen to obtain allo-restricted peptide-specific T cells having superior capacity to recognize tumor cells and higher functional avidity. This approach provides maximum flexibility because any major histocompatibility complex molecule and any tumor-associated antigen can be combined in the dendritic cells used for priming of autologous T cells. TCRs of allo-restricted T cells, when expressed as transgenes in activated peripheral blood lymphocytes, transferred superior function compared with self-restricted TCR. This approach allows high-avidity T cells and TCR specific for tumor-associated self-peptides to be easily obtained for direct adoptive T-cell therapy or for isolation of therapeutic transgenic TCR sequences.

Density of immunogenic antigens does not explain the presence or absence of the T-cell–inflamed tumor microenvironment in melanoma

Significance The T-cell–inflamed tumor microenvironment correlates with efficacy of immunotherapy. It is critical to understand whether non–T-cell–inflamed tumors lack antigens for T-cell recognition. In melanoma, no difference between inflamed and noninflamed tumors for multiple antigen classes was observed. Synthesized peptides corresponding to predicted HLA-A2 binding epitopes showed no differences between inflamed and noninflamed tumors. Extrapolation of a T-cell signature across The Cancer Genome Atlas showed no correlation between gene expression and mutational burden in any cancer type. These results indicate that lack of spontaneous immune infiltration in solid tumors is unlikely to be due to lack of antigens. Rather, transcriptional profiling suggests lack of Batf3-lineage dendritic cells. Our data suggest that strategies to restore T-cell entry into noninflamed tumors should be developed. Melanoma metastases can be categorized by gene expression for the presence of a T-cell–inflamed tumor microenvironment, which correlates with clinical efficacy of immunotherapies. T cells frequently recognize mutational antigens corresponding to nonsynonymous somatic mutations (NSSMs), and in some cases shared differentiation or cancer–testis antigens. Therapies are being pursued to trigger immune infiltration into non–T-cell–inflamed tumors in the hope of rendering them immunotherapy responsive. However, whether those tumors express antigens capable of T-cell recognition has not been explored. To address this question, 266 melanomas from The Cancer Genome Atlas (TCGA) were categorized by the presence or absence of a T-cell–inflamed gene signature. These two subsets were interrogated for cancer–testis, differentiation, and somatic mutational antigens. No statistically significant differences were observed, including density of NSSMs. Focusing on hypothetical HLA-A2+ binding scores, 707 peptides were synthesized, corresponding to all identified candidate neoepitopes. No differences were observed in measured HLA-A2 binding between inflamed and noninflamed cohorts. Twenty peptides were randomly selected from each cohort to evaluate priming and recognition by human CD8+ T cells in vitro with 25% of peptides confirmed to be immunogenic in both. A similar gene expression profile applied to all solid tumors of TCGA revealed no association between T-cell signature and NSSMs. Our results indicate that lack of spontaneous immune infiltration in solid tumors is unlikely due to lack of antigens. Strategies that improve T-cell infiltration into tumors may therefore be able to facilitate clinical response to immunotherapy once antigens become recognized.

Molecular Pathways: Targeting IDO1 and Other Tryptophan Dioxygenases for Cancer Immunotherapy

Indoleamine 2, 3-dioxygenase 1 (IDO1), IDO2, and tryptophan 2, 3-dioxygenase (TDO) comprise a family of enzymes that catalyze the first- and rate-limiting step associated with the catabolic conversion of tryptophan (Trp) into kynurenine (Kyn). Through subsequent enzymatic and spontaneous reactions, Kyn is further converted into the energetic substrates, NAD+ and ATP, to fuel cellular metabolic functions. Coincidently, the depletion of Trp and accumulation of Kyn has been demonstrated to induce effector T-cell apoptosis/dysfunction and immunosuppressive regulatory T-cell induction, respectively. Similar to other immune checkpoints, IDO1 and TDO are suggested to be important targets for immunotherapeutic intervention. This is represented by the recent growth of efforts to inhibit the Trp-to-Kyn pathway as a means to control immunosuppression. Inhibitors currently in clinical trials, INCB024360, GDC-0919, indoximod, and an IDO1 peptide-based vaccine, are being evaluated for their efficacy against a wide range of cancers including melanoma, glioblastoma, non–small cell lung, pancreatic, and/or breast cancer, as well as metastatic disease. Despite the rapid development of potent clinical grade inhibitors, strategic questions remain. Here, we review the state of the literature with respect to current therapeutic inhibitors of tryptophan catabolism, evaluation of those efforts preclinically and clinically, compensatory changes that occur with therapeutic targeting, as well as newly recognized signaling features that raise critical questions to the field. Given the rapidly evolving interest in determining how IDO1/TDO, and to an unknown extent, IDO2, can be targeted for increasing cancer immunotherapeutic efficacy, we present a brief but comprehensive analysis that addresses critical questions, while highlighting the mechanics that remain to be explored. Clin Cancer Res; 21(24); 5427–33. ©2015 AACR.