A–C) Purified CD28-deficient CD4+ T cells were retrovirally transduced with either empty vector (RV), CD28-F51I, CD28-F51V or the CTLA4-CD28 chimera and cocultured with CHO cells expressing either IAd alone (panel A), or coexpressing human CD80 (panel B) or human CD86 (panel C) and stimulated with OVA peptide at the indicated doses. D and E) Purified CD4+ cells as described above were stimulated with plate bound anti-CD3 (.01 μg/ml) alone or in combination with graded doses of plate bound human CD80Ig or CD86Ig and proliferation determined. For all panels, the data shown are the mean ± standard deviation of triplicate wells and are representative of 3–5 independent experiments. Statistical comparisons were made comparing the mutant constructs to the wild type construct, and the wild type to empty vector. **= p<.001 by 2 tailed, multiple comparison, unpaired T testing. Differences were significant at all doses.