title: The immunologic function of 1B2+ double negative (CD4-CD8-) T cells in the 2C transgenic mouse.
abstract: BACKGROUND
  2C mice bearing the cytotoxic TCR for class I L(d) on a C57BL/6 (B6) background have a preponderance of 1B2+CD8+ T cells directed against L(d). These naive CD8+ T cells are not directly cytotoxic without prior in vivo or in vitro activation. However, after in vitro sensitization, they become highly cytotoxic and will acutely and specifically reject a tolerant L(d+) BALB/c heart graft. Anti-lymphocyte serum (ALS) treatment eliminates CD4+ and CD8+ cells and a large double negative (CD4-CD8-) 1B2+ non-cytotoxic transgenic cell population remains. The immunological function of this unique peripheral population of T cells is investigated in the 2C transgenic mouse.
  
  
  METHODS
  To determine the activation characteristics of the 2C CD4-CD8- T cells, 2C peripheral T cells were analyzed for 1B2+, CD8+, and CD4+ marker by FACS before and 48-h after 0.5 cc ALS i.p. Similarly, in vitro, the response of these 2C CD4-CD8- T cells remaining after deletion of mature CD4+ and CD8+ T cells with ALS plus complement were evaluated by mixed lymphocyte culture and cytotoxic T lymphocyte after 7 days culture with BALB/c, IL-2, or BALB/c + IL-2. Parallel experiments were performed with control non-transgenic B6 mice. Following in vitro culture with BALB/c + IL-2, 2C CD4-CD8- T cells were injected into B6 mice with a tolerant BALB/c heart (tolerization via anti-CD4 mAb and intrathymic BALB/c) to determine their immunogenicity.
  
  
  RESULTS
  While peripheral T cells in control B6 mice have <5% CD4-CD8- cells, transgenic 2C mice have a significantly increased percentage at 29 to 35% (P < 0.01). After the deletion of CD4+ and CD8+ T cells with either in vivo or in vitro ALS, 2C CD4-CD8- T cells increased to 96 to 99%. After 7-day culture, the 2C CD4-CD8- T cells decreased again to 33 to 38%. Simultaneously, 2C CD8+ T cells decreased from 56 to 62% to 0.1 to 3% after ALS treatment, but again increased to 61 to 70% after in vitro culture. Untreated 2C cells responded to IL-2 or BALB/c antigen equally well. However, after ALS treatment, CD4-CD8- T cells responded to IL-2 and IL-2 plus antigen, but not BALB/c antigen alone. Finally, CD4-CD8- T cells cultured for 7 days with BALB/c + IL-2 rejected the tolerant BALB/c heart in 5.3 +/- 0.3 days.
  
  
  CONCLUSIONS
  In the periphery of transgenic 2C mice is a unique CD4-CD8- population of T cells bearing the transgenic specific marker 1B2. These non-cytotoxic cells can be optimally stimulated to develop marked specific L(d) cytotoxicity in parallel with the expression of the CD8+ epitope.
authors.affiliation: Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Margenthaler
authors.forename: M Wayne
authors.initials: MW
authors.lastname: Flye
mesh_terms: D019264:Adoptive Transfer; D000818:Animals; D000961:Antilymphocyte Serum / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism*; D015704:CD4 Antigens / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects; D016827:CD8 Antigens / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects; D002478:Cells, Cultured; D002999:Clone Cells / Q000276:immunology; D005434:Flow Cytometry; D015321:Gene Rearrangement; D006084:Graft Rejection; D006183:H-2 Antigens / Q000276:immunology*; D016027:Heart Transplantation; D064532:Histocompatibility Antigen H-2D; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D023001:Transplantation Tolerance
chemical_list: D000961:Antilymphocyte Serum; D015415:Biomarkers; D015704:CD4 Antigens; D016827:CD8 Antigens; D006183:H-2 Antigens; D064532:Histocompatibility Antigen H-2D; D016693:Receptors, Antigen, T-Cell, alpha-beta