Figure 7.

Selective expansion of regulatory T cells in vivo with MH5A treatment. (A) Peripheral lymph nodes were isolated from untreated wt B6 mice and analyzed by flow cytometry. Surface staining was performed for CD4, CD25, and control Ab or PD-1H, followed by intracellular staining for FoxP3. Top panel is gated on CD4+CD25+ T cells. Bottom panel is gated on CD4+CD25(−) T cells. (B) Naïve CD4+CD25 negative T cells were isolated by MACS bead negative selection of CD4+ T cells followed by depletion of CD25+ cells with biotin labeled CD25 followed by anti-biotin MACS bead magnetic depletion. Cells were then cultured with anti-CD3, anti-CD28, recombinant IL-2 and titrated concentrations of TGF-β in the presence of soluble MH5A or control Ab for 5 days and analyzed for percentages of CD4+FoxP3+. (C) Treg cells were induced as in (B) but without control Ig or MH5A treatment. These cells were then added to 96-well plates with 5 ng/ml IL-2 and pre-coated with anti-CD3. MH5A or hamIg was added to wells at a final concentration of 10 ug/ml. Treg cell proliferation was analyzed 72 hours later. Experiment was repeated three times. (D) Treg cells were induced as in (B) and used in a mixed lymphocyte response (MLR) assay. Treg cells were pre-incubated with control IgG or MH5A, then washed and added at various ratios to an MLR of B6 responder T cells and irradiated BDF1 splenocytes as targets. Responder proliferation was analyzed 5 days later. (E–H) Total T cells (CD25 replete) (E,G) or CD25-depleted T cells (F,H) and TCD-BM from B6 mice were adoptively transferred to lethally irradiated BDF1 mice with MH5A or control Ab. Splenocytes were isolated at the indicated time points and absolute numbers of CD4+FoxP3+ T cells (E,F) and the ratio of absolute numbers of donor CD8+ to donor CD4+FoxP3+ T cells (G,H) were calculated. 3–5 mice per group repeated a minimum of three times.