Ly49D⁺ NK cells regulate alloreactive CD4 T-cell priming and polarization in response to allogeneic H-2^d DCs. (A) B6 CD8^−/− mice, treated or not with anti-NK1.1 PK136 mAb or anti-Ly49A/D 12A8 mAb, were injected subcutaneously with allogeneic BALB/c DCs. Six days after immunization, CD4⁺ T cells were purified from draining lymph nodes and restimulated (2 × 10⁵ cells/well) with irradiated allogeneic BALB/c splenocytes for 72 hours to measure proliferation and cytokine production. Data are mean plus or minus SEM of 3 mice per group. Data are from one representative experiment of 3 performed. (B) To evaluate the frequency of IL-4-producing cells, purified CD4⁺ T cells were cultured for 8 hours with T cell–depleted BALB/c splenocytes in the presence of anti-CD28 mAb. Intracytoplasmic staining for IL-4 was then performed. Data are percentage of CD69^pos CD4^pos T cells producing IL-4 (mean ± SEM of 3 mice per group). (C,D) B6 CD8^−/− mice, treated or not with anti-NK1.1 PK136 mAb or anti-Ly49D 4E5 mAb, were injected subcutaneously with DCs derived from either WT BALB/c mouse (C) or β₂-microglobulin^−/− BALB/c mouse (D). Six days after immunization, CD4⁺ T cells were purified from draining lymph nodes and restimulated (2 × 10⁵ cells/well) with irradiated allogeneic BALB/c splenocytes for 72 hours to measure proliferation and cytokine production. Data are mean plus or minus SEM of 3 mice per group. Background proliferation was less than 1500 cpm. Data are representative of at least 2 experiments performed.