Limiting dilution analysis of canine alloantigen-reactive T lymphocytes

• Published in: Transplantation Vol. 35; no. 4; p. 363
• Main Authors: Buurman, W A, Daemen, A J, Groenewegen, G, Does, R J, van der Linden, C J, Vegt, P A
• Format: Journal Article
• Language: English
• Published: United States 01.04.1983
• Subjects: Animals; Dogs - immunology; Epitopes; Isoantigens; Leukocyte Count; T-Lymphocytes - immunology; T-Lymphocytes, Cytotoxic - immunology
• ISSN: 0041-1337; 1534-6080
• DOI: 10.1097/00007890-198304000-00020

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.