Willem W. Overwijk

Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells.

Many tumor-associated antigens are derived from nonmutated “self” proteins. T cells infiltrating tumor deposits recognize self-antigens presented by tumor cells and can be expanded in vivo with vaccination. These T cells exist in a functionally tolerant state, as they rarely result in tumor eradication. We found that tumor growth and lethality were unchanged in mice even after adoptive transfer of large numbers of T cells specific for an MHC class I–restricted epitope of the self/tumor antigen gp100. We sought to develop new strategies that would reverse the functionally tolerant state of self/tumor antigen-reactive T cells and enable the destruction of large (with products of perpendicular diameters of >50 mm2), subcutaneous, unmanipulated, poorly immunogenic B16 tumors that were established for up to 14 d before the start of treatment. We have defined three elements that are all strictly necessary to induce tumor regression in this model: (a) adoptive transfer of tumor-specific T cells; (b) T cell stimulation through antigen-specific vaccination with an altered peptide ligand, rather than the native self-peptide; and (c) coadministration of a T cell growth and activation factor. Cells, vaccination, or cyto-kine given alone or any two in combination were insufficient to induce tumor destruction. Autoimmune vitiligo was observed in mice cured of their disease. These findings illustrate that adoptive transfer of T cells and IL-2 can augment the function of a cancer vaccine. Furthermore, these data represent the first demonstration of complete cures of large, established, poorly immunogenic, unmanipulated solid tumors using T cells specific for a true self/tumor antigen and form the basis for a new approach to the treatment of patients with cancer.

CD8+ T Cell Immunity Against a Tumor/Self-Antigen Is Augmented by CD4+ T Helper Cells and Hindered by Naturally Occurring T Regulatory Cells

CD4+ T cells control the effector function, memory, and maintenance of CD8+ T cells. Paradoxically, we found that absence of CD4+ T cells enhanced adoptive immunotherapy of cancer when using CD8+ T cells directed against a persisting tumor/self-Ag. However, adoptive transfer of CD4+CD25− Th cells (Th cells) with tumor/self-reactive CD8+ T cells and vaccination into CD4+ T cell-deficient hosts induced autoimmunity and regression of established melanoma. Transfer of CD4+ T cells that contained a mixture of Th and CD4+CD25+ T regulatory cells (Treg cells) or Treg cells alone prevented effective adoptive immunotherapy. Maintenance of CD8+ T cell numbers and function was dependent on Th cells that were capable of IL-2 production because therapy failed when Th cells were derived from IL-2−/− mice. These findings reveal that Th cells can help break tolerance to a persisting self-Ag and treat established tumors through an IL-2-dependent mechanism, but requires simultaneous absence of naturally occurring Treg cells to be effective.

T helper 17 cells promote cytotoxic T cell activation in tumor immunity.

Although T helper 17 (Th17) cells have been found in tumor tissues, their function in cancer immunity is unclear. We found that interleukin-17A (IL-17A)-deficient mice were more susceptible to developing lung melanoma. Conversely, adoptive T cell therapy with tumor-specific Th17 cells prevented tumor development. Importantly, the Th17 cells retained their cytokine signature and exhibited stronger therapeutic efficacy than Th1 cells. Unexpectedly, therapy using Th17 cells elicited a remarkable activation of tumor-specific CD8(+) T cells, which were necessary for the antitumor effect. Th17 cells promoted dendritic cell recruitment into the tumor tissues and in draining lymph nodes increased CD8 alpha(+) dendritic cells containing tumor material. Moreover, Th17 cells promoted CCL20 chemokine production by tumor tissues, and tumor-bearing CCR6-deficient mice did not respond to Th17 cell therapy. Thus, Th17 cells elicited a protective inflammation that promotes the activation of tumor-specific CD8(+) T cells. These findings have important implications in antitumor immunotherapies.

BRAF Inhibition Is Associated with Enhanced Melanoma Antigen Expression and a More Favorable Tumor Microenvironment in Patients with Metastatic Melanoma

Purpose: To evaluate the effects of BRAF inhibition on the tumor microenvironment in patients with metastatic melanoma. Experimental Design: Thirty-five biopsies were collected from 16 patients with metastatic melanoma pretreatment (day 0) and at 10 to 14 days after initiation of treatment with either BRAF inhibitor alone (vemurafenib) or BRAF + MEK inhibition (dabrafenib + trametinib) and were also taken at time of progression. Biopsies were analyzed for melanoma antigens, T-cell markers, and immunomodulatory cytokines. Results: Treatment with either BRAF inhibitor alone or BRAF + MEK inhibitor was associated with an increased expression of melanoma antigens and an increase in CD8+ T-cell infiltrate. This was also associated with a decrease in immunosuppressive cytokines [interleukin (IL)-6 and IL-8] and an increase in markers of T-cell cytotoxicity. Interestingly, expression of exhaustion markers TIM-3 and PD1 and the immunosuppressive ligand PDL1 was increased on treatment. A decrease in melanoma antigen expression and CD8 T-cell infiltrate was noted at time of progression on BRAF inhibitor alone and was reversed with combined BRAF and MEK inhibition. Conclusions: Together, these data suggest that treatment with BRAF inhibition enhances melanoma antigen expression and facilitates T-cell cytotoxicity and a more favorable tumor microenvironment, providing support for potential synergy of BRAF-targeted therapy and immunotherapy. Interestingly, markers of T-cell exhaustion and the immunosuppressive ligand PDL1 are also increased with BRAF inhibition, further implying that immune checkpoint blockade may be critical in augmenting responses to BRAF-targeted therapy in patients with melanoma. Clin Cancer Res; 19(5); 1225–31. ©2013 AACR.

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.

Persistent antigen at vaccination sites induces tumor-specific CD8+ T cell sequestration, dysfunction and deletion

To understand why cancer vaccine–induced T cells often do not eradicate tumors, we studied immune responses in mice vaccinated with gp100 melanoma peptide in incomplete Freunds adjuvant (peptide/IFA), which is commonly used in clinical cancer vaccine trials. Peptide/IFA vaccination primed tumor-specific CD8+ T cells, which accumulated not in tumors but rather at the persisting, antigen-rich vaccination site. Once there, primed T cells became dysfunctional and underwent antigen-driven, interferon-γ (IFN-γ)- and Fas ligand (FasL)-mediated apoptosis, resulting in hyporesponsiveness to subsequent vaccination. Provision of CD40-specific antibody, Toll-like receptor 7 (TLR7) agonist and interleukin-2 (IL-2) reduced T cell apoptosis but did not prevent vaccination-site sequestration. A nonpersisting vaccine formulation shifted T cell localization toward tumors, inducing superior antitumor activity while reducing systemic T cell dysfunction and promoting memory formation. These data show that persisting vaccine depots can induce specific T cell sequestration, dysfunction and deletion at vaccination sites; short-lived formulations may overcome these limitations and result in greater therapeutic efficacy of peptide-based cancer vaccines.

Plasmacytoid dendritic cells induce NK cell–dependent, tumor antigen–specific T cell cross-priming and tumor regression in mice

A prerequisite for strong adaptive antiviral immunity is the robust initial activation of the innate immune system, which is frequently mediated by TLR-activated plasmacytoid DCs (pDCs). Natural antitumor immunity is often comparatively weak, potentially due to the lack of TLR-mediated activation signals within the tumor microenvironment. To assess whether pDCs are capable of directly facilitating effective antitumor immune responses, mice bearing established subcutaneous B16 melanoma tumors were administered TLR9-activated pDCs directly into the tumor. We found that TLR9-activated pDCs induced robust, spontaneous CTL cross-priming against multiple B16 tumor antigens, leading to the regression of both treated tumors and untreated tumors at distant contralateral sites. This T cell cross-priming was mediated by conventional DCs (cDCs) and was completely dependent upon the early recruitment and activation of NK cells at the tumor site. NK cell recruitment was mediated by CCR5 via chemokines secreted by pDCs, and optimal IFN-gamma production by NK cells was mediated by OX40L expressed by pDCs. Our data thus demonstrated that activated pDCs are capable of initiating effective and systemic antitumor immunity through the orchestration of an immune cascade involving the sequential activation of NK cells, cDCs, and CD8(+) T cells.

BRAF Inhibition Increases Tumor Infiltration by T cells and Enhances the Antitumor Activity of Adoptive Immunotherapy in Mice

Purpose: Treatment of melanoma patients with selective BRAF inhibitors results in objective clinical responses in the majority of patients with BRAF-mutant tumors. However, resistance to these inhibitors develops within a few months. In this study, we test the hypothesis that BRAF inhibition in combination with adoptive T-cell transfer (ACT) will be more effective at inducing long-term clinical regressions of BRAF-mutant tumors. Experimental Design: BRAF-mutated human melanoma tumor cell lines transduced to express gp100 and H-2Db to allow recognition by gp100-specific pmel-1 T cells were used as xenograft models to assess melanocyte differentiation antigen–independent enhancement of immune responses by BRAF inhibitor PLX4720. Luciferase-expressing pmel-1 T cells were generated to monitor T-cell migration in vivo. The expression of VEGF was determined by ELISA, protein array, and immunohistochemistry. Importantly, VEGF expression after BRAF inhibition was tested in a set of patient samples. Results: We found that administration of PLX4720 significantly increased tumor infiltration of adoptively transferred T cells in vivo and enhanced the antitumor activity of ACT. This increased T-cell infiltration was primarily mediated by the ability of PLX4720 to inhibit melanoma tumor cell production of VEGF by reducing the binding of c-myc to the VEGF promoter. Furthermore, analysis of human melanoma patient tumor biopsies before and during BRAF inhibitor treatment showed downregulation of VEGF consistent with the preclinical murine model. Conclusion: These findings provide a strong rationale to evaluate the potential clinical application of combining BRAF inhibition with T-cell–based immunotherapy for the treatment of patients with melanoma. Clin Cancer Res; 19(2); 393–403. ©2012 AACR.

PD-1 BLOCKADE ENHANCES T CELL MIGRATION TO TUMORS BY ELEVATING IFN-γ INDUCIBLE CHEMOKINES

Adoptive cell transfer (ACT) is considered a promising modality for cancer treatment, but despite ongoing improvements, many patients do not experience clinical benefits. The tumor microenvironment is an important limiting factor in immunotherapy that has not been addressed fully in ACT treatments. In this study, we report that upregualtion of the immunosuppressive receptor programmed cell death-1 (PD-1) expressed on transferred T cells at the tumor site, in a murine model of ACT, compared with its expression on transferred T cells present in the peripheral blood and spleen. As PD-1 can attenuate T-cell-mediated antitumor responses, we tested whether its blockade with an anti-PD-1 antibody could enhance the antitumor activity of ACT in this model. Cotreatment with both agents increased the number of transferred T cells at the tumor site and also enhanced tumor regressions, compared with treatments with either agent alone. While anti-PD-1 did not reduce the number of immunosuppressive regulatory T cells and myeloid-derived suppressor cells present in tumor-bearing mice, we found that it increased expression of IFN-γ and CXCL10 at the tumor site. Bone marrow-transplant experiments using IFN-γR-/- mice implicated IFN-γ as a crucial nexus for controlling PD-1-mediated tumor infiltration by T cells. Taken together, our results imply that blocking the PD-1 pathway can increase IFN-γ at the tumor site, thereby increasing chemokine-dependent trafficking of immune cells into malignant disease sites.

Analysis of Immune Signatures in Longitudinal Tumor Samples Yields Insight into Biomarkers of Response and Mechanisms of Resistance to Immune Checkpoint Blockade

UNLABELLED Immune checkpoint blockade represents a major breakthrough in cancer therapy; however, responses are not universal. Genomic and immune features in pretreatment tumor biopsies have been reported to correlate with response in patients with melanoma and other cancers, but robust biomarkers have not been identified. We studied a cohort of patients with metastatic melanoma initially treated with cytotoxic T-lymphocyte-associated antigen-4 (CTLA4) blockade (n = 53) followed by programmed death-1 (PD-1) blockade at progression (n = 46), and analyzed immune signatures in longitudinal tissue samples collected at multiple time points during therapy. In this study, we demonstrate that adaptive immune signatures in tumor biopsy samples obtained early during the course of treatment are highly predictive of response to immune checkpoint blockade and also demonstrate differential effects on the tumor microenvironment induced by CTLA4 and PD-1 blockade. Importantly, potential mechanisms of therapeutic resistance to immune checkpoint blockade were also identified. SIGNIFICANCE These studies demonstrate that adaptive immune signatures in early on-treatment tumor biopsies are predictive of response to checkpoint blockade and yield insight into mechanisms of therapeutic resistance. These concepts have far-reaching implications in this age of precision medicine and should be explored in immune checkpoint blockade treatment across cancer types. Cancer Discov; 6(8); 827-37. ©2016 AACR.See related commentary by Teng et al., p. 818This article is highlighted in the In This Issue feature, p. 803.