CD11b^hiIa^lo DCs suppress mature DC-induced proliferation of peptide-specific CD4 T cells. (A,C,D) The influence of CD11b^hiIa^lo DCs on the antigen-presentation capacity of mDCs in vitro was assayed. CD11b^hiIa^lo DCs or LPS-treated CD11b^hiIa^lo DCs were added into CD4/mDC coculture system in the presence of OVA_323-339 peptide (CD11b^hiIa^lo DCs/mDCs = 1:1), compared with imDCs. After 5 days, the relative number of live CD4⁺ T cells in each well was counted by FACS (A), the levels of IFN-γ and IL-2 in the supernatant (C) and in the CD4 T cells (D) were assayed by ELISA and FACS, respectively. *P < .001. (B) The influence of CD11b^hiIa^lo DCs on T-cell division following antigen presentation was measured by CFSE-labeled peptide-specific CD4 T cells, as compared with imDCs. (E) The influence of CD11b^hiIa^lo DCs on the mDC-primed proliferation of activated peptide-specific CD4 T cells was assayed. Peptide-specific CD4 T cells were activated by mDCs for 24 hours in the presence of OVA_323-339 peptide, and then CD11b^hiIa^lo DCs, LPS-treated CD11b^hiIa^lo DCs, or imDCs were added. After 5 days, the relative cell number of viable peptide-specific CD4 T cells was measured by FACS. (F) The influence of CD11b^hiIa^lo DCs on the proliferation of peptide-specific CD4 T cells stimulated by mDCs in vivo was assayed. OVA_323-339 peptide–loaded CD11b^hiIa^lo DCs, imDCs, or mDCs were cotransferred with peptide-specific CD4 T cells into recipient mice. The ratio of the combination of APCs was 1:1. After 5 days, lymphocytes in spleen and mesenteric lymph nodes were double-stained with anti–CD4-FITC and KJ1-26-PE for FACS analysis. The percentage of peptide-specific CD4 T cells (KJ1-26⁺) among total CD4 T cells was calculated. One of at least 3 independent experiments with similar results was shown.