Functional characteristics of 197+ CD4− CD8− sheep T lymphocytes: Expansion and differentiation of peripheral T cells

Abstract

This report describes the results of functional studies on the CD3⁺ 197⁺ CD4^- CD8^- TCR /⁺ peripheral T lymphocyte of sheep. Cell types were identified by immunofluorescent and immunoenzymic staining and separated by fluorescence activated flow cytometry. Newly synthesized DNA was labelled in vivo by incorporation of 5-bronio-2-deoxyuridine (BrdU), and total DNA by in vitro incubation with propidium iodide. Cells were challenged in vivo with alloantigens, in vitro with alloantigens or a range of mitogens, and activation/differentiation was assessed by determination of cell number, phenotype, and thymidine incorporation. In normal lambs the 197⁺ cells showed no in vivo DNA synthesis except at a low level in the ileocaecal lymph node. A similarly low level of synthesis in the prescapular node was induced by local allogeneic challenge. A higher proportion of 197⁺ cells than of other T cells isolated from blood and lymph had G2/M phase DNA content, while very few had S phase DNA content. The response of 197⁺ cells in terms of change in relative numbers following in vivo allogeneic challenge was quite different to that of CD4⁺ or CD8⁺ cells. Instead of rising to a peak at 5-8 days after challenge and then declining, the percentage of 197⁺ cells rose steadily with no evidence of decline by 14 days. Purified 197⁺ cells were activated in vitro by phytohaemagglutinin (PHA) and concanavalin-A (Con-A) but not by B cell mitogens; such activation was dependent on the inclusion of feeder cells or interleukin-2 (IL-2). PHA-induced proliferation was accompanied by altered expression of surface molecules by a proportion of the cells. Changes observed included loss of 197 antigen and gain of CD4, CD8 or MHC II antigens. In some cases coexpression of two or more of these surface antigens occurred. Thus the 197⁺ T-cell is as distinctive in the pattern of its functional behaviour as it is in its previously reported phenotype, distribution and ontogeny.