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We have determined the frequencies and specificities of MHC-reactive and MHC-restricted cytotoxic T lymphocyte precursors (CTL-p) in mitogen (ConA)-activated splenocytes of normal unprimed mice. The limiting dilution (LD) system supported the growth of one out of three Lyt2 + T cell blasts. The generated CTL-populations lysed blast cell targets specifically as determined by split well analyses. MHC-gene product expression was necessary for lysis to occur, since MHC-negative F9 teratocarcinoma cells were not lysed. The frequency determinations and split well analyses revealed: 1) equally high numbers (∼ 1/100) of CTL-p that generated specific allo-MHC or self-MHC reactive CTL populations, 2) high frequencies of CTL-p which recognized hapten (TNP) or minor H (MH)-antigens in the context of self MHC or allo-MHC determinants. The results are discussed with respect to antigen, restriction and receptor specificities of mitogen-activated unprimed T cell blasts.

Polyclonally activated lymphoblasts, transferred to syngeneic recipient mice, elicited a host T-cell-mediated "response" in vivo. These T cells, which "responded" in vivo to syngeneic lymphoblasts (i.e. in vivo primed "responder" T-cell population), acquired the capacity to "stimulate" a "response" of syngeneic T cells in vitro in a syngeneic one-way mixed lymphocyte culture, S-MLC (i.e. in vitro primed "'responder" T-cell population). We now describe the presence of memory and specificity in these two types of "self-reactive""responder" T-cell populations. This is investigated in in vitro "restimulation" experiments with mitomycin-blocked syngeneic and allogeneic lymphoid cells of various origin. "Self-reactive" T cells could be restimulated repeatedly (over many weeks) with mitomycin-blocked syngeneic lymphoid-cell populations, but not with mitomycin-blocked allogeneic normal spleen cells. "Self-reactive" T cells "responded" to syngeneic large activated lymphoblasts, as well as to syngeneic small resting lymphocytes. We found no "responder" T-cell reactivity specific for the mitogen that induced syngeneic "stimulator" cell populations. Both populations of "self-reactive" T cells displayed reactivity to mitogen-induced allogeneic lymphoblasts.

Splenic T cells, engaged in an in-vivo "response" to transferred syngeneic lymphoblasts, displayed an extensive proliferative activity if cultured in vitro. These cells (blocked by mitomycin C) were "stimulator" cells in a one-way syngeneic mixed lymphocyte culture (S-MLC). "Responder" cells were derived form spleens of syngeneic (sex and age matched) mice. Fractionation studies showed that "stimulator" and "responder" cells in the investigated S-MLC were nylon-wool non-adherent and sensitive to treatment with anti-Thy-1 antiserum and complement, i.e. were cells of the T lineage. Coculture experiments demonstrated that direct cell contact is required to trigger a proliferative "response" in the S-MLC. Fetal calf serum-supplemented tissue-culture medium supported "responder" cell proliferation, normal mouse serum-supplemented medium did not. The in-vivo generation of a T-cell population, which could "stimulate" a syngeneic T-cell "response" in the S-MLC in vitro, was investigated.

The intravenous injection of polyclonally activated lymphoblasts elicited a proliferative T cell reaction in the spleens of syngeneic recipient mice. In the non-fractionated cell populations obtained from these spleens 6 days after lymphoblast transfer, the LPS-induced proliferation and differentiation of B cells in vitro was suppressed. This suppressive effect was mediated by T cells, as i) treatment with anti-Thy-1 antiserum plus complement restored responsiveness of B cells to LPS in the spleen cell population that had responded in vivo to a syngeneic lymphoblast graft, and ii) the responsiveness of B cells to LPS was not impaired in non-fractionated spleen cell populations of nu/nu mice injected with syngeneic lymphoblasts. The relationship of this nonspecific T suppressor cell activity to the previously described nonspecific T helper cell activity for B cell activation is discussed.

Polyclonally activated splenic lymphocytes (generated in mitogen-stimulated cultures) were transferred to syngeneic or autologous recipient mice. Injection of cells into the footpad of syngeneic recipients induced a regional response in the ipsilateral popliteal lymph node; intravenous cell transfer elicited a systemic splenomegaly reaction. These reactions displayed a linear log number of transferred cells/response relationship in syngeneic and autologous systems. The kinetic and magnitude of the regional response to syngeneic lymphoblasts and to allogeneic spleen cells were comparable. No difference was apparent in the phenomenology of the in-vivo responses to syngeneic lymphoblasts induced by various T- or B-cell mitogens. The in-vivo response was: 1. stimulated by large-size lymphoblasts; and 2. mediated by host T cells. Data excluded a direct involvement of mitogen or heterologous serum constituents in tissue culture medium in the described reaction. Experimental evidence argues against the involvement of virus components in the observed phenomenon.