Figure 4. CD4⁺CD25⁻Nrp1⁺ T cells augment CD4⁺Foxp3⁺ Treg accumulation in transplant recipients.

(A) Anti-CD4 and anti-Foxp3 intracellular staining were performed on spleen cells harvested from untreated mice on 7d or from Rapamycin and/or CD4⁺CD25⁻Nrp1⁺ T cells on 21d, 42d and 70d. (B) The percentages of CD4⁺Foxp3⁺ T cells were pooled from 4–6 mice from each group. (C) CD4⁺CD25⁺ T cells were purified from each group and used for suppression assays. 2×10⁴ CD4⁺CD25⁻ T cells (C57BL/6) were stimulated by the same amount of irradiated BALB/c splenocytes together with various doses of CD4⁺CD25⁺T cells purified from the indicated group. Cell proliferation was determined by ³H thymidine incorporation. Results are presented as mean ± SD values of triplicate wells, and are representative of 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001. Rapa  =  Rapamycin, Nrp1 = neuropilin-1, ³H-TdR = metabolic incorporation of tritiated thymidine, cpm = cells per million, Treg = T regulatory cells