title: Combination CTLA-4 blockade and 4-1BB activation enhances tumor rejection by increasing T-cell infiltration, proliferation, and cytokine production.
abstract: BACKGROUND
  The 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.
  
  
  RESULTS
  We 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
  This 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.
authors.affiliation: Howard Hughes Medical Institute, Department of Immunology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Curran
authors.forename: Myoungjoo
authors.initials: M
authors.lastname: Kim
authors.forename: Welby
authors.initials: W
authors.lastname: Montalvo
authors.forename: Aymen
authors.initials: A
authors.lastname: Al-Shamkhani
authors.forename: James P
authors.initials: JP
authors.lastname: Allison
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D015551:Autoimmunity; D060908:CTLA-4 Antigen / Q000737:chemistry*; D049109:Cell Proliferation; D016207:Cytokines / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism; D037181:Lectins, C-Type; D008198:Lymph Nodes / Q000473:pathology; D008297:Male; D008546:Melanoma, Experimental; D051379:Mice; D008810:Mice, Inbred C57BL; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000276:immunology* / Q000628:therapy*; D010047:Ovalbumin / Q000737:chemistry; D061026:Programmed Cell Death 1 Receptor; D011971:Receptors, Immunologic / Q000378:metabolism*; D013601:T-Lymphocytes / Q000166:cytology*; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9 / Q000378:metabolism*
chemical_list: D000943:Antigens, Differentiation; D000970:Antineoplastic Agents; D060908:CTLA-4 Antigen; D016207:Cytokines; C451496:Klrg1 protein, mouse; D037181:Lectins, C-Type; C491383:Pdcd1 protein, mouse; D061026:Programmed Cell Death 1 Receptor; D011971:Receptors, Immunologic; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9; D007371:Interferon-gamma; D010047:Ovalbumin