Figure 2. Expanded cynomolgus Treg significantly down-regulate CD52 expression, are not bound by alemtuzumab, and show little change in CD46 expression.

(A) T cells from peripheral blood or LN were stained for CD3, CD4, CD25 and CD52 (n=3). CD52 MFI was analyzed for bulk T cells, fresh (unexpanded) Treg (gated on CD4⁺CD25^hiCD127⁻) and fresh (unexpanded) Teff (gated on CD4⁺CD25⁻). In the same experiment, Treg and Teff cells were analyzed for CD52 after round 1 and 2 of expansion (all conditions n=3). Expanded cells showed a decrease in CD52 expression after the first round of expansion, which was similar for Treg and Teff, with a further decrease after the second round (n=3 experiments for all conditions). *p<0.05. (B) Alemtuzumab does not bind to expanded Treg. Cells were incubated with alemtuzumab at concentrations from 0.001–100 µg/ml then stained with FITC-anti-human IgGγ, as described in the Materials and Methods to determine binding of alemtuzumab. Binding was expressed as relative MFI. Alemtuzumab exhibited concentration-dependent binding to freshly-isolated PBMC, as well as to freshly-isolated Treg and Teff (n=3 experiments). By contrast, alemtuzumab showed no binding to expanded Treg (n=3) or expanded Teff (n=3) at any concentration tested. *p<0.05; significance calculated between fresh T cells (n=3) and expanded Treg (n=3). (C), Representative histograms showing absence of binding to expanded cells. (D) Expanded Treg and Teff showed little change in surface expression of the complement inhibitory protein CD46 compared with fresh cells. Fresh and expanded Treg/Teff from the same monkey were stained for CD3, CD4, Foxp3, and CD46. Treg were gated on CD3⁺CD4⁺Foxp3⁺ while Teff were gated on CD3⁺CD4⁺Foxp3⁻. Compared to fresh cells, both Treg and Teff increased their expression of CD46 only modestly after two rounds of expansion. Data are representative of 3 separate experiments.