The secondary cytotoxic T cell response of BALB/c to B10.D2 or DBA/2 minor histocompatibility antigens in vitro requires the participation of an adherent cell. Nylon wool-passed spleen cells were only able to respond to nonadherent intact stimulator cells, or to membrane fragments derived from those cells, if a syngeneic adherent cell were present in the cultures. When the H-2 restriction properties of cytotoxic cells generated in response to various types of stimulation were analyzed, it was found that responses to B10.D2 or DBA/2 intact cells were always H-2 restricted. Responses to syngeneic adherent cells presenting B10.D2 or DBA.2 freeze-thaw antigen were either entirely or predominantly lacking in H-2 restriction as defined by efficient competition by B10 (H-2b) cold target cells. These unrestricted killers appeared to recognize minor histocompatibility as an independent determinant rather than as an H-2d/minors moiety cross-reaction with H-2b, because they were not absorbed by BALB.B (H-2b) macrophage monolayers, but were absorbed by B10 monolayers. Similarly, B10 but not BALB.B cold targets were able to compete for the anti-B10.D2 killers. These experiments eliminate the possibility that the lack of restriction was due to an H-2b restricted receptor cross-reactive with H-2b. Possible models to explain these findings are discussed.

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