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.

In view of the favorable outcome of CEA combined with curettage or Er:YAG laser ablation in the treatment of giant CMN, CEA is a safe and effective novel treatment adjunct that accelerates healing, with fewer side effects.

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.

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Antibodies which block the co-inhibitory receptor CTLA-4 or which activate the co-stimulatory receptor 4-1BB can promote the rejection of some murine tumors, but fail to cure poorly immunogenic tumors like B16 melanoma. We find that combining these two antibodies in the context of a Flt3-ligand based B16 melanoma vaccine promoted high CD8+ and CD4+ effector to FoxP3+ Treg ratios in tumors, highly elevated TH1 type cytokine production, increased T-cell proliferation and synergistic levels of tumor rejection. A hallmark of 4-1BB agonist antibody treatment is the upregulation of the lectin KLRG1 on tumor infiltrating CD8+ and CD4+ T-cells. We find that these KLRG1+ T-cells in the tumor express high levels of multiple cytotoxic molecules, kill tumor cells better than their KLRG- counterparts and may be responsible for the increased anti-tumor cytotoxicity attributed to α4-1BB treatment. We found that formation of these cells is driven by high-level expression of the master-regulatory transcription factor Eomesodermin (Eomes) in both the CD8+ and CD4+ T-cell compartments. These Eomes+KLRG1+ cytotoxic CD4+ and CD8+ T-cells represent a novel phenotype distinct from any described in the literature (KLRG1expression is not IL-12/T-bet dependent, they are not exhausted, and the Eomes expression exceeds that of normal TH1). The origins and applications of these novel T-cells have broad implications for tumor immunotherapy as well as basic T-cell immunology.

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.