Marilyn CD4 T cells capture MHC-II/H-Y in vitro, and induce death of memory but not naive T cells. (A) Purification of MHC-II–bearing T cells. Lymph node CD45.2 Marilyn cells were incubated with H-Y peptide–loaded (100 nM), CFSE-labeled, LPS-matured DCs. Twenty hours later, the T cells were FACS sorted according to CD11c and CFSE expression with exclusion of the doublets. (i) Postsort analysis of the CFSE^neg/CD11c^neg T-cell fraction. (ii,iii) Expression of A^b by the sorted T cells. (B) T cells that have captured MHC-II/H-Y complexes present them to naive and memory Marilyn T cells. (i,ii) CD45.2 fixed (as described in “In vitro experiments”) naive LN Marilyn cells (0.5 × 10⁶), (iii,iv) 0.5 × 10⁶ purified CD45.2 fixed T cells that had captured MHC-II/H-Y complexes, or (v,vi) 5000 purified H-Y–bearing fresh DCs were used to stimulate 2 × 10⁴ CFSE-labeled CD45.1 naive Marilyn LN cells or negatively purified memory Marilyn T cells. CD69 expression was measured the following day. Representative of 3 experiments. (C) T cells that have captured MHC-II/H-Y complexes suppress memory but not naive Marilyn T-cell proliferation. Purified CD45.2 fixed naive LN Marilyn cells (0.5 × 10⁶), or 0.5 × 10⁶ purified CD45.2 fixed T cells that had captured MHC-II/H-Y complexes (H-Y T cells), or not (unpulsed T cells), and/or various numbers of H-Y–bearing fresh DCs were used to stimulate 2 × 10⁴ CD45.1 naive or memory Marilyn T cells that had been negatively purified by FACS and then CFSE labeled. Proliferation was measured at day 3 by CFSE dilution. Percentage of undivided cells is indicated in each panel, and numbers of recovered cells/well are indicated at the bottom of each panel. Representative of 4 experiments. (D) Fresh DCs, able to stimulate naive cells, cannot relieve inhibition of memory T cells. B6 mice were injected with CD45.2 CFSE-labeled Marilyn cells and were immunized the next day with H-Y peptide–loaded LPS-matured DCs in one hind footpad. Naive or memory CD45.1 CFSE-labeled Marilyn cells were transferred 5 days later. Four hours later, some of the mice (bottom panels) received a second immunization with H-Y peptide–loaded DCs into both the original and the contralateral footpad. The CFSE profile is shown on the second cohort (TCR⁺ CD45.1⁺ cells) in the original DLN (left panels) or in the contralateral DLN (right panels) 6 days after they were injected. Representative of 2 experiments with 2 mice per group in each.