Anti-CTLA-4 mAb is efficacious in enhancing tumor immunity in humans. CTLA-4 is expressed by conventional T cells upon activation and by naturally occurring FOXP3+CD4+ Treg cells constitutively, raising a question of how anti-CTLA-4 mAb can differentially control these functionally opposing T cell populations in tumor immunity. Here we show that FOXP3high potently suppressive effector Treg cells were abundant in melanoma tissues, expressing CTLA-4 at higher levels than tumor-infiltrating CD8+ T cells. Upon in vitro tumor-antigen stimulation of peripheral blood mononuclear cells from healthy individuals or melanoma patients, Fc-region-modified anti-CTLA-4 mAb with high antibody-dependent cell-mediated cytotoxicity (ADCC) and cellular phagocytosis (ADCP) activity selectively depleted CTLA-4+FOXP3+ Treg cells and consequently expanded tumor-antigen-specific CD8+T cells. Importantly, the expansion occurred only when antigen stimulation was delayed several days from the antibody treatment to spare CTLA-4+ activated effector CD8+T cells from mAb-mediated killing. Similarly, in tumor-bearing mice, high-ADCC/ADCP anti-CTLA-4 mAb treatment with delayed tumor-antigen vaccination significantly prolonged their survival and markedly elevated cytokine production by tumor-infiltrating CD8+ T cells, whereas antibody treatment concurrent with vaccination did not. Anti-CTLA-4 mAb modified to exhibit a lesser or no Fc-binding activity failed to show such timing-dependent in vitro and in vivo immune enhancement. Thus, high ADCC anti-CTLA-4 mAb is able to selectively deplete effector Treg cells and evoke tumor immunity depending on the CTLA-4-expressing status of effector CD8+ T cells. These findings are instrumental in designing cancer immunotherapy with mAbs targeting the molecules commonly expressed by FOXP3+ Treg cells and tumor-reactive effector T cells.

The generation of antigen-specific antibodies and the development of immunological memory require collaboration between B and T cells. T cell-secreted IL-4 is important for B cell survival, isotype switch to IgG1 and IgE, affinity maturation, and the development of germinal centers (GC). Fyn, a member of the Src family tyrosine kinase, is widely expressed in many cell types, including lymphocytes. This kinase is known to interact with both the B cell and T cell receptor (BCR and TCR, respectively). While Fyn deletion does not impair the development of immature T cells and B cells, TCR signaling is altered in mature T cells. The current study demonstrates that Fyn deficient (KO) B cells have impaired IL-4 signaling. Fyn KO mice displayed low basal levels of IgG1, IgE and IgG2c, and delayed antigen-specific IgG1 and IgG2b production, with a dramatic decrease in antigen-specific IgG2c following immunization with a T-dependent antigen. Defects in antibody production correlated with significantly reduced numbers of GC B cells, follicular T helper cells (TFH), and splenic plasma cells (PC). Taken together, our data demonstrate that Fyn kinase is required for optimal humoral responses.

A disintegrin and metalloproteinase 10 (ADAM10) is a zinc-dependent proteinase related to matrix metalloproteinases. ADAM10 has emerged as a key regulator of cellular processes by cleaving and shedding extracellular domains of multiple transmembrane receptors and ligands. We have developed B cell-specific ADAM10-deficient mice (ADAM10(B-/-)). In this study, we show that ADAM10 levels are significantly enhanced on germinal center B cells. Moreover, ADAM10(B-/-) mice had severely diminished primary and secondary responses after T-dependent immunization. ADAM10(B-/-) displayed impaired germinal center formation, had fewer follicular Th cells, decreased follicular dendritic cell networks, and altered chemokine expression in draining lymph nodes (LNs). Interestingly, when spleen and LN structures from immunized mice were analyzed for B and T cell localization, tissues structure was aberrant in ADAM10(B-/-) mice. Importantly, when ADAM10-deficient B cells were stimulated in vitro, they produced comparable Ab as wild type B cells. This result demonstrates that the defects in humoral responses in vivo result from inadequate B cell activation, likely because of the decrease in follicular Th cells and the changes in structure. Thus, ADAM10 is essential for the maintenance of lymphoid structure after Ag challenge.