Vaccination with irradiated B16 melanoma cells expressing either GM-CSF (Gvax) or Flt3-ligand (Fvax) combined with antibody blockade of the negative T-cell costimulatory receptor cytotoxic T-lymphocyte antigen-4 (CTLA-4) promotes rejection of preimplanted tumors. Despite CTLA-4 blockade, T-cell proliferation and cytokine production can be inhibited by the interaction of programmed death-1 (PD-1) with its ligands PD-L1 and PD-L2 or by the interaction of PD-L1 with B7-1. Here, we show that the combination of CTLA-4 and PD-1 blockade is more than twice as effective as either alone in promoting the rejection of B16 melanomas in conjunction with Fvax. Adding αPD-L1 to this regimen results in rejection of 65% of preimplanted tumors vs. 10% with CTLA-4 blockade alone. Combination PD-1 and CTLA-4 blockade increases effector T-cell (Teff) infiltration, resulting in highly advantageous Teff-to-regulatory T-cell ratios with the tumor. The fraction of tumor-infiltrating Teffs expressing CTLA-4 and PD-1 increases, reflecting the proliferation and accumulation of cells that would otherwise be anergized. Combination blockade also synergistically increases Teff-to-myeloid-derived suppressor cell ratios within B16 melanomas. IFN-γ production increases in both the tumor and vaccine draining lymph nodes, as does the frequency of IFN-γ/TNF-α double-producing CD8 + T cells within the tumor. These results suggest that combination blockade of the PD-1/ PD-L1-and CTLA-4-negative costimulatory pathways allows tumorspecific T cells that would otherwise be inactivated to continue to expand and carry out effector functions, thereby shifting the tumor microenvironment from suppressive to inflammatory.he interaction between the T-cell receptor complex (TCR) and antigenic peptides bound in surface MHC molecules provides the specificity that defines adaptive T-cell immunity. In addition to TCR activation, costimulation via ligation of the coreceptor CD28 on T cells by B7 molecules on antigenpresenting cells (APCs) is required for optimal T-cell activation (1). Once mobilized, however, T cells begin to express other members of the CD28/B7 receptor family that attenuate the immune response through inhibition of proliferation and cytokine production (2). Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is rapidly up-regulated following T-cell activation and binds to B7 molecules with a higher affinity than does CD28. The receptor programmed death-1 (PD-1) is also expressed on T cells following activation, where, on binding to its ligands PD-L1 and PD-L2, it promotes T-cell anergy, apoptosis, and exhaustion. Recently, an additional coinhibitory ligand/receptor interaction has been described that involves binding of PD-L1 on T cells to B7-1 on APCs or vice versa (3). Although the existence of so many layers of T-cell inhibition may seem surprising, the severe and sometimes fatal autoimmunity that results when even one of these pathways is disrupted attests to their necessity.Malignant transformation was classically defined by the ability to avoid the norm...

Anti–4-1BB treatment of tumor-bearing or intracellular pathogen infected mice generates a population of Eomes+KLRG1+ tumor infiltrating T cells that have enhanced cytotoxic activity.

BackgroundThe co-inhibitory receptor Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) attenuates immune responses and prevent autoimmunity, however, tumors exploit this pathway to evade the host T-cell response. The T-cell co-stimulatory receptor 4-1BB is transiently upregulated on T-cells following activation and increases their proliferation and inflammatory cytokine production when engaged. Antibodies which block CTLA-4 or which activate 4-1BB can promote the rejection of some murine tumors, but fail to cure poorly immunogenic tumors like B16 melanoma as single agents.Methodology/Principal FindingsWe find that combining αCTLA-4 and α4-1BB antibodies in the context of a Flt3-ligand, but not a GM-CSF, based B16 melanoma vaccine promoted synergistic levels of tumor rejection. 4-1BB activation elicited strong infiltration of CD8+ T-cells into the tumor and drove the proliferation of these cells, while CTLA-4 blockade did the same for CD4+ effector T-cells. Anti-4-1BB also depressed regulatory T-cell infiltration of tumors. 4-1BB activation strongly stimulated inflammatory cytokine production in the vaccine and tumor draining lymph nodes and in the tumor itself. The addition of CTLA-4 blockade further increased IFN-γ production from CD4+ effector T-cells in the vaccine draining node and the tumor. Anti 4-1BB treatment, with or without CTLA-4 blockade, induced approximately 75% of CD8+ and 45% of CD4+ effector T-cells in the tumor to express the killer cell lectin-like receptor G1 (KLRG1). Tumors treated with combination antibody therapy showed 1.7-fold greater infiltration by these KLRG1+CD4+ effector T-cells than did those treated with α4-1BB alone.Conclusions/SignificanceThis study shows that combining T-cell co-inhibitory blockade with αCTLA-4 and active co-stimulation with α4-1BB promotes rejection of B16 melanoma in the context of a suitable vaccine. In addition, we identify KLRG1 as a useful marker for monitoring the anti-tumor immune response elicited by this therapy. These findings should aid in the design of future trials for the immunotherapy of melanoma.

CTLA-4, an Ig superfamily molecule with homology to CD28, is one of the most potent negative regulators of T-cell responses. In vivo blockade of CTLA-4 exacerbates autoimmunity, enhances tumor-specific T-cell responses, and may inhibit the induction of T-cell anergy. Clinical trials of CTLA-4–blocking antibodies to augment T-cell responses to malignant melanoma are at an advanced stage; however, little is known about the effects of CTLA-4 blockade on memory CD8+T-cell responses and the formation and maintenance of long-term CD8+T-cell memory. In our studies, we show that during in vivo memory CD8+T-cell responses toListeria monocytogenesinfection, CTLA-4 blockade enhances bacterial clearance and increases memory CD8+T-cell expansion. This is followed by an accumulation of memory cells that are capable of producing the effector cytokines IFN-γ and TNF-α. We also demonstrate that in a vaccination setting, blocking CTLA-4 during CD8+T-cell priming leads to increased expansion and maintenance of antigen-specific memory CD8+T cells without adversely affecting the overall T-cell repertoire. This leads to an increase in memory cell effector function and improved protective immunity against further bacterial challenges. These results indicate that transient blockade of CTLA-4 enhances memory CD8+T-cell responses and support the possible use of CTLA-4–blocking antibodies during vaccination to augment memory formation and maintenance.

Following treatment with 4-1BB agonist antibody, a novel population of KLRG1+ T-cells infiltrate murine melanoma. Compared to their KLRG1- counterparts, these T-cells express high levels of cytotoxicity associated genes in both the CD4 and CD8 lineages, and exhibit enhanced tumor-specific killing in vitro. The phenotype of these KLRG1+ cells is dependent on high expression of the T-box transcription factor Eomesodermin (Eomes). Interestingly, only activation of 4-1BB, not other TNFR family members generates this phenotype. The root of this difference appears to be that 4-1BB is expressed by antigen presenting cells (APC) which respond to its activation by producing cytokines which promote the development of these Eomes+KLRG1+ T-cells. By analyzing changes in APC cytokine production in vivo, and by using a series of gene knockout mice we have identified the factors necessary to generate this novel T-cell lineage. These T-cells represent a novel polarity we have termed ThEO (CD4) and TcEO (CD8) which resolve multiple questions associated with 4-1BB activation including how 4-1BB enhances tumor-specific cytotoxicity and how 4-1BB can promote tumor immunity while repressing autoimmunity. Understanding the nature of this novel lineage of highly tumoricidal T-cells in both tumor and pathogen-specific immunity may provide critical information for converting sub-optimal anti-tumor responses to therapeutically successful ones.

Following treatment with 4-1BB agonist antibody, a novel population of KLRG1+ T-cells infiltrate the tumors of mice. Compared to their KLRG1- counterparts, these T-cells express high levels of cytotoxicity associated genes in both the CD4 and CD8 lineages and also demonstrate enhanced tumor-specific killing in vitro. The phenotype of these KLRG1+ cells is dependent on high expression of the T-box transcription factor Eomesodermin (Eomes). The unique ability of α4-1BB to generate this phenotype stems from the expression of 4-1BB by antigen presenting cells (APC) which respond to its activation by producing cytokines which then drive the development of these Eomes+KLRG1+ T-cells. By analyzing changes in APC cytokine production in vivo, as well as by using a series of gene knockout mice we have begun to identify the factors necessary to generate this novel T-cell lineage. Among these factors, IL-27, IL-15, and IL-10 appear to be paramount. These T-cells represent a novel polarity we have termed ThEO (CD4) and TcEO (CD8) which resolves multiple questions associated with 4-1BB activation including how 4-1BB enhances tumor-specific cytotoxicity, and how 4-1BB can promote tumor immunity while repressing autoimmunity. Understanding the nature of this novel lineage of highly tumoricidal T-cells in both tumor and pathogen-specific immunity may provide critical information for converting sub-optimal anti-tumor responses to therapeutically successful ones.

Introduction: Immunotherapy with CTLA-4 blockade has resulted in dramatic responses in patients with advanced melanoma and prostate cancer. However, only 10-30% of patients have a response, therefore there is a need to increase the efficacy of CTLA-4 blockade. In this work we tested the hypothesis that combinatorial approaches incorporating tumor destruction with conventional chemotherapy and CTLA-4-blockade would synergize to significantly increase anti-tumor activity in vivo. Experimental Procedure: Mice with a transplantable prostate tumor (TRAMPC2) were treated with 5 doses of gemcitabine alone, or followed by immunotherapy with CTLA-4 blockade. Control mice were treated with isotype antibody. Tumor growth was measured by calipers and mice were sacrificed when tumors reached 15mm. Progression was followed over time and CD8 T cell responses to the tumor specific antigen SPAS were examined by tetramer staining and functional assays ex vivo. Data: Mice treated with the combination gemcitabine and α-CTLA-4 had improved survival over mice treated with gemcitabine alone. The majority of mice treated with control Hamster Ig or CTLA-4 blockade died (median survival 59 days for both groups). The median survival of mice treated with gemcitabine was 72 days with 50% having long-term survival. The addition of α-CTLA-4 to gemcitabine treatment increased median survival to > 125 days with 65% of mice with long term survival (p<0.05 for survival of gemcitabine versus gemcitabine + α-CTLA-4). The additive effect of α-CTLA-4 was lost when CD8 T cells were depleted. Analysis of draining lymph nodes revealed a reduced number of T regulatory T cells and myeloid suppressor cells with gemcitabine treatment. Mice receiving combination treatment had increased accumulation of tetramer positive CD8 cells for the SPAS peptide in the spleen and tumor draining lymph node. In addition, the combination treated mice had increased IFNγ production by ELISPOT in response to SPAS peptide re-stimulation ex vivo (p<0.05 for gemcitabine versus gemcitabine and α-CTLA-4). Conclusion: Combination chemo- and immunotherapy with gemcitabine and α-CTLA-4 improves survival. This is associated with an accumulation of CD8 cells that are tumor specific. Depletion of CD8 cells reduces the efficacy of this treatment. This study supports further investigation into the additive effects of chemotherapy with CTLA-4 blockade. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1786. doi:10.1158/1538-7445.AM2011-1786

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