Each symbol represents the mean of the counts per minute of triplicate wells for each individual patient

Each symbol represents the mean of the counts per minute of triplicate wells for each individual patient. (D) = 3 impartial experiments. (B and D) Two-tailed paired Students test. Open in a separate windows Physique 2 Immobilized CD96 mAbs enhance CD4+ and CD8+ T cell proliferation.(ACC) CFSE-labeled PBMCs were stimulated for 4 days with soluble OKT3 and plate-bound anti-CD96 mS2a antibodies or an isotype control (mS2a-IC), in the presence of (C) soluble blocking anti-CD155 mAb or an IC (m1-IC). Cell division among T cell subsets was analyzed by flow cytometry. (A) Representative examples of CFSE dilution. (B and C) Data show the mean SEM of the frequency of dividing cells, with each symbol representing the mean of triplicate wells for an individual HD. (D) CFSE-labeled CD3+ T cells purified from HDs were stimulated for 5 days with soluble anti-CD3/anti-CD28 tetrameric complexes and plate-bound anti-CD96 mS2a antibody (clone 19-134) or an IC. Each data point represents the mean of the frequency of dividing cells from triplicate wells for an individual HD. Data are combined from (B) = 6, = 4, and = 2 impartial experiments for clones 19-134, 19-14. and 4-31, respectively, and from (C) = 2 and (D) = 3 impartial experiments. * 0.05, ** 0.01, *** 0.001. BOP sodium salt (B and D) Two-tailed paired Students test; (C) 1-way ANOVA. Table 1 EC50 and IC50 values for D265A m2a anti-CD96 mAbs Open in a separate window To investigate if FcR engagement can substitute for the requirement for mAb immobilization, we isotype-switched FcR-disabled anti-CD96 mouse IgG2a (D265A) mAbs to FcR-competent human IgG1 and IgG2 isotypes. While human IgG1 exhibits binding to all FcRs, human IgG2 binds to FcRIIA and FcRIIIA, albeit with a lower affinity than IgG1 (24). For each antibody clone, we confirmed that the 2 2 isotypes displayed equivalent binding capacities to CD96, as exhibited by their comparable EC50 values (Table 2). Remarkably, soluble human IgG1, but not human IgG2 variants, augmented CD4+ and CD8+ T cell division in the BOP sodium salt PBMC proliferation assay, suggesting that this stronger and broader FcR binding activity BOP sodium salt of IgG1 was required for the observed costimulatory effects (Physique 3, A and B). To confirm that this costimulatory effects of the anti-CD96 IgG1 mAbs were dependent on coengagement of FcRs, we produced FcR-silent human IgG1 versions (N297S; refs. 25, 26) of anti-CD96 clones 19-134 and 19-14, which had the most potent effect on T cell proliferation. Table 2 shows that antibody binding to CD96 was not affected by the N297S mutation. Increased proliferation of CD4+ and CD8+ T cells elicited by soluble anti-CD96 IgG1 clones was completely abolished by the N297S mutation, demonstrating that coengagement of FcRs is essential for their costimulatory effects (Physique 3, C and D). To gain a better understanding of which FcR was required in mediating the activity of the anti-CD96 IgG1 mAbs, we generated a mutant (IgG1 V12) that possesses significantly reduced affinity to FcRI, FcRIIAH131, and FcRIIIA but stronger binding to FcRIIB (27). We evaluated 2 anti-CD96 clones (19-134 and 19-14) in the IgG1 V12 format, but neither mAb was active (Physique 3, C and D), corroborating the hypothesis that the higher affinity of IgG1 for FcRI, BOP sodium salt FcRIIA, and FcRIIIA was essential for antibody-mediated CD96 cross-linking and DDR1 subsequent T cell costimulation. To address the source of FcRs in the PBMC proliferation assay, we analyzed the expression of FcRI, FcRIIA/B, and FcRIIIA on various leukocytes from PBMCs. FcRI, FcRIIA/B, and FcRIIIA were expressed on monocytes, B cells, and NK cells (Supplemental Physique 3) in the expected pattern (28). In contrast, neither resting nor OKT3-activated T cells expressed these FcRs (Supplemental Physique 3), indicating that anti-CD96 mAb cross-linking was mediated through a = 4 impartial BOP sodium salt experiments and (D) from = 4 and = 3 impartial experiments for clones 19-134 and 19-14, respectively. * 0.05, ** 0.01. One-way ANOVA. Open in a separate window Physique 4 Cross-linking by FcRI enables the T cell stimulatory property of soluble anti-CD96 huG1 mAb.CFSE-labeled purified CD3+ T cells from HDs were stimulated for 4 days, and cell division was analyzed by flow cytometry. (A) T cells were.