“In vivo MLR” analysis of lymphocytes from NIMA^d-exposed versus NIPA^d control offspring. Splenocytes were harvested from NIMA^d-exposed and NIPA^d control mice and labeled with CFSE. CFSE-labeled splenocytes (50 × 10⁶) were injected intravenously into a BDF₁ recipient, which is the maternal type in the F₁ backcross breeding system. After 3 days, splenocytes and ILN cells were harvested from the BDF₁ recipients. (A) Flow cytometric analysis of lymphoproliferation in BDF₁ hosts. H2K^d-negative CFSE-labeled splenic lymphocytes found in BDF₁ ILNs were gated as shown. Total lymphocyte proliferation from MMc⁺ mouse NIMA^d no. 9 was less than MMc^neg NIMA^d no. 10 and NIPA control (no. 2). (B) Inverse correlation between the level of MMc and percentage of proliferated responder lymphocytes recovered from BDF₁ lymph nodes (n = 22). (C) Gating strategy for H2K^d-negative (donor) CD4⁺ T cells. (D) Surface TGF-β and (E) intracellular Foxp3 staining of donor CD4⁺ T cells. NIMA^d no. 9–derived CD4⁺ T cells expressed a higher amount of surface TGF-β staining on nonproliferated CD4⁺ T cells than NIMA^d no. 10 and NIPA control (no. 2). The horizontal line indicates the level of staining with isotype control antibody. (F) Correlation between the level of MMc and percentage of donor CD4⁺ T cells expressing surface TGF-β (n = 15). The diagonal lines represent the best fit regression lines in panels B and F.