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. 2011 Mar 14;208(3):577–592. doi: 10.1084/jem.20100619

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© 2011 Wang et al.

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Figure 7. — VISTA-Ig–mediated suppression could overcome a moderate level of co-stimulation provided by CD28 but was completely reversed by a high level of co-stimulation, as well as partially rescued by exogenous IL-2. (A and B) CFSE-labeled CD4⁺ T cells were activated by 2.5 µg/ml plate-bound α-CD3 together with either VISTA-Ig or control-Ig at 1:1 and 1:2 ratios. (A) 40 ng/ml soluble mIL-2, mIL-7, mIL-15, or mIL-23 was also added as indicated to the cell culture. (B) 1 µg/ml α-CD28 was immobilized together with 2.5 µg/ml α-CD3 and Ig proteins at the indicated ratios. Cell proliferation was analyzed at 72 h by examining CFSE division profiles. (C and D) To examine the suppressive activity of VISTA in the presence of lower levels of co-stimulation, the indicated amounts of α-CD28 were coated together with 2.5 µg/ml α-CD3 and 10 µg/ml VISTA-Ig fusion proteins or control-Ig fusion protein to stimulate CFSE-labeled CD4⁺ T cells. Cell proliferation was analyzed at 72 h. Percentages of CFSE^low cells were quantified and shown as means ± SEM in D. Duplicated wells were analyzed in all conditions. Representative CFSE profiles from three independent experiments are shown.