title: Limiting dilution analysis of the response of murine L3T4+ spleen cells to alloantigen.
abstract: 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.
mesh_terms: D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology*; D002469:Cell Separation; D002999:Clone Cells; D005434:Flow Cytometry; D006183:H-2 Antigens / Q000276:immunology*; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D007376:Interleukin-2 / Q000096:biosynthesis; D007377:Interleukin-3 / Q000494:pharmacology; D015847:Interleukin-4; D007378:Interleukins / Q000494:pharmacology; D008213:Lymphocyte Activation; D051379:Mice; D011971:Receptors, Immunologic / Q000502:physiology; D015375:Receptors, Interleukin-2; D013154:Spleen / Q000166:cytology / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*
chemical_list: D000945:Antigens, Differentiation, T-Lymphocyte; D006183:H-2 Antigens; D000949:Histocompatibility Antigens Class II; D007376:Interleukin-2; D007377:Interleukin-3; D007378:Interleukins; D011971:Receptors, Immunologic; D015375:Receptors, Interleukin-2; D015847:Interleukin-4