Stimulation of thymocytes in vitro by spleen cells differing for the entire H-2 complex leads to a significant proliferative response without a significant cell-mediated lympholysis (CML) response. Addition of soluble cell-free supernates (SF), (taken from a 7-day mixed leukocyte culture) enables these cultures to develop CML response. For optimal CML response, the SF has to be added within 48 h of onset of cultures. Although with spleen cells as responding cells, SF could quantitatively replace I-region different stimulating cells for generation of CML responses, with thymocytes as responding cells, stimulation with I-region cells appeared obligatory for the generation of CML responses. The implications of these findings are discussed.

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