Thy-1+/L3T4-/Ly-2- spleen cells were purified from normal C57BL/6 (B6) and C,B-17 mice. Cells within this subset expressed the T cell receptor (TcR) for antigen: the majority of cells in this subset were CD3+; a fraction of the cells was stained with the monoclonal antibody (mAb) F23.1; and the TcR molecule was immunoprecipitable with mAb F23.1 from cells within this subset. In limiting dilution analyses, about 1/30 cells within this subset were growth inducible in vitro by stimulation with phorbol myristate acetate (PMA) plus ionomycin; conditioned media containing interleukin (IL) 1, IL2, IL3 or IL4 activity neither triggered nor promoted in vitro growth of these cells. The in vitro generated T cells displayed the Thy-1+/L3T4+/Ly-2- surface phenotype and were self-reactive, i.e., proliferated preferentially in response to syngeneic stimulator cells, and secreted IL2 and IL3 only in response to syngeneic but not allogeneic stimulator cells. The proliferative response of these cells to syngeneic stimulator cells was blocked by anti-self Ia mAb. This autoreactive helper T cell subset was not inducible in purified Thy-1+ spleen cell subsets from athymic nude mice or scid mice. Autoreactive helper T cells did not express detectable levels of the IL2 receptor (IL2R), and their proliferative response was not blocked by anti-IL2R mAb. From PMA plus ionomycin-stimulated double-negative Thy-1+ spleen cells, 14 T cell clones were established in long-term culture which displayed the CD3+CD4+CD8- surface phenotype and were self-reactive.

The cytotoxic response of cell sorter-purified small Lyt-2+ splenic cytotoxic lymphocyte precursors from 10 individual C57BL/6 mice to mutant class I H-2Kbm1 or H-2Dbm14 allodeterminants was analyzed under limiting dilution conditions. The cytotoxic activity of anti-bm1-specific or anti-bm14-specific cytotoxic T lymphocyte (CTL) populations (selected for a high probability of clonality) was tested against F23 hybridoma cells; F23+ CTL clones lysed F23 hybridoma targets but F23- CTL clones did not. In the C57BL/6 anti-bm1 mixed lymphocyte reaction, 36% (range 29-48%) of the generated CTL clones were F23+; in the B6-anti-bm14 mixed lymphocyte reaction, 45% (range 34-49%) of the generated CTL clones were F23+. Hence, a large fraction of the anti-bm1- or anti-bm14-reactive CTL clones from C57BL/6 mice use V beta.8 genes to construct these allospecific T cell receptor phenotypes, but no extensive variation in the use of V beta.8 genes in the construction of allospecific T cell receptor phenotypes of restricted heterogeneity is found in individual mice of the same strain.

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The cytotoxic response of splenic Lyt-2+ T cells to class I H-2 alloantigen-bearing stimulator cells was analyzed under limiting dilution conditions. One of 50 to one of 200 nylon wool-nonadherent (FACS-purified), small Lyt-2+ spleen cells of B6 origin gave rise in vitro to a cytotoxic T lymphocyte clone that specifically lysed targets bearing bm1 alloantigen. This population of alloantigen-specific cytotoxic lymphocyte precursors (CLP) was activated by different types of bm1 stimulator cells with different efficiency: 2 X 10(5) nonfractionated spleen cells, 5000 normal peritoneal cells, 400 to 10(4) L3T4+ helper T blasts, or 2000 to 10(4) Lyt-2+ T blasts induced clonal growth of this CLP pool. Irradiated or mitomycin-treated small (L3T4+ or Lyt-2+) bm1-derived T cells were inefficient stimulator cells for this response. Supplementation of culture medium with (recombinant) interleukin 2 was necessary and sufficient to support clonal development of alloantigen-triggered CLP in the presence of allogeneic T blasts. Under these limiting dilution conditions, we observed comparable cloning efficiencies for (wild-type) Kb-allospecific splenic Lyt-2+ CLP from bm1 mice generated in response to either irradiated B6 spleen cells or inactivated B6-derived T cell lines (EL4 and RBL-5 lymphoma cells). The data indicate that normal T lymphoblasts as well as tumor T cell lines stimulate clonal development in vitro of class I H-2-allospecific cytotoxic T lymphocytes in the presence of interleukin 2.

Cell sorter-purified small splenic L3T4+ cells from B6 mice were clonally expanded under limiting dilution (LD) conditions by coculture for 4-6 days with irradiated allogeneic stimulator cells in culture medium supplemented with various growth factor preparations. Proliferating L3T4+ cell clones were detected by [3H]thymidine uptake; interleukin 2 (IL-2) production of restimulated L3T4+ cell clones was measured in a sensitive colorimetric assay. IL-3 (but not IL-1 or IL-4) supported clonal expansion in vitro of many L3T4+ cell clones produced IL-2. The data were consistent with the hypothesis that only a single titrated precursor cell was limiting in the system. In the response to class II (bm12) H-2 alloantigen, 1 in 40-200 L3T4+ cells was induced to clonal growth; in the response to class I (bm1) H-2 alloantigen, a tenfold lower frequency (1 in 600-800) of inducible L3T4+ B6 cells was measured. A fraction of the generated L3T4+ cell clones showed IL-2-independent growth: anti-IL-2 receptor monoclonal antibodies (MoAb) (7D4 and PC61.5) blocked the proliferation of about 80% of the IL-2-producing L3T4+ cell clones, while about 20% of these clones seemed resistant to inhibition of proliferation by these MoAb. We have thus defined an LD system with high cloning efficiency for L3T4+ cells that does not depend on exogenous IL-2 supplements.