Figure 10.

VISTA blockade using a specific mAb enhanced CD4⁺ T cell response in vitro and in vivo. (A) An mAb clone 13F3 neutralized VISTA-mediated suppression in vitro. A20-RFP and A20-VISTA cells were used to stimulate CFSE-labeled DO11.10 CD4⁺ T cells in the presence of cognate OVA peptide. 20 µg/ml VISTA-specific mAb 13F3 or control-Ig was added as indicated. CFSE dilution was analyzed after 72 h, and percentages of CFSE^low cells are shown as mean ± SEM. Duplicated wells were analyzed for all conditions. (B and C) Total CD11b^hi myeloid cells (B) or CD11b^hiCD11c⁻ monocytes (C) and CD11b^hiCD11c⁺ myeloid DCs (C) sorted from naive splenocytes were irradiated and used to stimulate CFSE-labeled OT-II transgenic CD4⁺ T cells in the presence of OVA peptide. Cell proliferation was measured by incorporation of tritiated thymidine during the last 8 h of a 72-h culture period and shown as mean ± SEM. Triplicate wells were analyzed in all conditions. (D) Mean clinical scores and disease incidence of mice (n = 8 per group) that received suboptimal dosage of activated encephalitogenic CD4⁺ T cells (1.5 million). Recipient mice were treated with 400 µg 13F3 or control-Ig every 3 d, and disease course was monitored every day. Disease scores are shown as means ± SEM and are the representative results from three independent experiments. The statistical differences (p-values) were assessed with an unpaired Mann-Whitney test.