Several publications, however, have failed to support T reg cell depletion like a mechanism of action and have, to the contrary, proven that CCTLA-4 expands T reg cells in the secondary lymphoid organs (Quezada et al., 2006; Schmidt et al., 2009) and blood (Kavanagh et al., 2008) of both mice and humans, further assisting the notion that CTLA-4 restricts T cell proliferation. in trans inside a context-dependent manner. Our results reveal further mechanistic insight into the activity of anti-CTLA-4Cbased malignancy immunotherapy, and illustrate the importance of specific features of the local tumor environment on the final end result of antibody-based immunomodulatory therapies. The fully human being antiCcytotoxic T lymphocyteCassociated antigen 4 (CTLA-4) monoclonal antibody Ipilimumab represents the first of a PKC-IN-1 new class of malignancy therapies that function by enhancing immunological antitumor activity. Two pivotal phase III clinical tests demonstrated significant raises in survival in individuals with melanoma treated with Ipilimumab (Hodi et al., 2010; Robert et al., 2011), which led to its recent authorization from the FDA. Despite rigorous investigation, however, the mechanism of action remains unclear. Although the initial premise was that antiCCTLA-4 antibodies (CCTLA-4) function by obstructing inhibitory signals into effector T cells (T eff cell; Krummel and Allison, 1996; Sutmuller et al., 2001), the demonstration that CD4+Foxp3+ regulatory T cells (T reg cell) express high levels of CTLA-4 led to the suggestion that CCTLA-4 directly effects the T reg cell compartment, either by mediating depletion, or by influencing their suppressive activity (Go through et al., 2000, 2006; Takahashi et al., 2000; Wing et al., 2008). In this regard, we recently shown that CCTLA-4 needs to bind both T eff and T reg cells to elicit full tumor safety (Peggs et al., 2009). Several publications, however, possess failed to support T reg cell depletion like a mechanism of action and have, to the contrary, shown that CCTLA-4 expands T reg cells in the secondary lymphoid organs (Quezada et al., 2006; Schmidt et al., 2009) and blood (Kavanagh et al., 2008) of both mice and humans, further supporting the notion that CTLA-4 restricts T cell proliferation. The mechanisms by which CCTLA-4 directly affects the activity of the T reg cell compartment therefore remain obscure. A common feature associated with CCTLA-4Cmediated tumor rejection is an increase in the percentage of T eff to T reg cells within the tumor (T eff/T reg cell percentage; Shrikant et al., 1999; Quezada et al., 2006; Kavanagh et al., 2008; Liakou et al., 2008; Chen et al., 2009; Curran and Allison, 2009; Waitz et al., 2012). This increase is thought to arise from your preferential growth of T eff over T reg cells, although it remains unclear why this effect is restricted to the tumor microenvironment and why an antibody that simultaneously targets two cellular populations with opposing activities favors effector T cell function and promotes tumor rejection. Here, we further define the mechanism underlying the antitumor activity of CCTLA-4 by focusing on the factors controlling the selective increase in the T eff/T reg cell percentage within the tumor. By tracking tumor-specific CD4+ T cells, we display that CCTLA-4 increases the complete quantity of T eff and T reg cells in the lymph nodes and of T eff cells in the tumor, while selectively reducing the complete quantity of T reg cells in the tumor. The reduction in T reg cells was consistent with a mechanism including FcRIV-dependent depletion associated with the presence of FcR-expressing macrophages within the tumor, and elevated surface CTLA-4 manifestation by tumor-infiltrating T reg cells. Therefore, CCTLA-4 blocks inhibitory signals, resulting in the growth and build up of T eff and T reg cells in the lymph node and of T eff cells in the tumor, but in parallel depletes tumor-infiltrating T reg cells, leading to an increase in the T eff/T reg cell percentage within the tumor. Collectively, these data clarify the paradoxical effects of CCTLA-4 on T eff and T reg cell build up in the lymph nodes and tumor. More importantly, they spotlight the significant part played from the tumor microenvironment in determining the outcome of antibody-based immunotherapies, and how the impact on cellular compartments can differ in the periphery and in the tumor. Lastly, they suggest that methods leveraging the capacity PKC-IN-1 of the tumor microenvironment to deplete antibody-associated T PKC-IN-1 reg cells could be used to enhance the antitumor activity of immunotherapies. RESULTS GVAX+CCTLA-4 combination therapy protects against B16-BL6 melanoma through a CD4-dependent mechanism To establish the DFNA23 involvement of the CD4+ T cell compartment in tumor safety, C57BL/6 wild-type and I-A?/? mice (lacking a CD4+ T cell compartment) were challenged with the transplantable B16-BL6 melanoma collection. 3 d after implantation, mice were treated or not with an irradiated B16-BL6 tumor cellCbased vaccine that secretes GM-CSF (GVAX) in.
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