Limiting dilution analysis of canine alloantigen-reactive T lymphocytes.
A sensitive limiting dilution assay for the determination of canine cytotoxic T lymphocyte precursors (CTL-P) has been developed. Murine second MLC supernatant as a source of Interleukin 2 (IL 2) was used to expand stimulated CTL-P to numbers that were easily detectable with the classic 51Cr lysis assay. The frequencies of CTL-P that reacted to allogeneic stimulator cells in canine peripheral blood ranged from 1:1000 to 1:2000 lymphocytes. During in vitro stimulation in a mixed leukocyte culture a rapid increase in frequency was noted, and at day 7 a frequency as high as 1:5.4 was found. The presence of irradiated stimulator cells during limiting dilution culture restricted proliferation of the CTL-P; only those which recognized the stimulator cells proliferated. The determination of cytotoxicity in large numbers of individual microcultures with CTL-P seeded at clonal conditions toward 2 allogeneic target cells permitted quantitation of the frequency of CTL-P with specificity for different alloantigens. These techniques greatly facilitate monitoring of cellular immune reactions after renal allografting because both determination of the influence of cytostatic drug treatment on CTL-P frequency, and analysis of the specificity and function of allograft infiltrating cells are now possible.
D000818:Animals; D004285:Dogs / Q000276:immunology*; D000939:Epitopes; D007519:Isoantigens; D007958:Leukocyte Count; D013601:T-Lymphocytes / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D000939:Epitopes; D007519:Isoantigens