IN VITRO ANALYSIS OF INTRACELLULAR CYTOKINE PRODUCTION DURING ALLOGENEIC RESPONSE IN CLINICAL RENAL TRANSPLANTATION.

Abstract

A290 Aims: Previous analysis of cytokine production in allogeneic responses has shown predominance of type 1 cytokines with little type 2 cytokines. Here we report that in T cells from renal transplant patients stimulated in one-way mixed lymphocyte response (MLR), similar numbers of cells produce IFN-γ and IL-10, type 1 and type 2 cytokines respectively, with a small excess of IFN-γ producing cells. Methods: MLRs were set up to stimulate peripheral blood mononuclear leukocytes (PBML) from renal transplant patients using as stimulator cells T-cell depleted PBML. The MLRs were set up (i) in relation to the time of transplantation and (ii) according to the type of stimulator cells. The patients’ PBML were collected pre-transplant, 2 weeks, 4 weeks, 3 months and 6 months post transplant. MLRs were with recipient’s cells alone; DR matched and mismatched surrogate donor cells and cells from live donors if possible. Cells were permeabilized, stained and analyzed by flow cytometry, determining CD25+CD4+cells and CD25+CD8+cells. The proportion of the activated T cells (CD25+) that were producing IFN-γ or IL-10 was then calculated. Acute transplant rejection was biopsy proven reported independently by a skilled histopathologist. Results: A total of 174 MLRs were carried out with PBML from 16 patients. Nine patients showed evidence of T cell activation (CD25+) and the rest did not. Activation was not related to the type of the stimulator used, the presence or absence of rejection, infection or toxicity due to immunosuppressive medications and was consistent on repeat analysis. Where there was evidence of activation, the CD25+ T cells produced both type 1 (IFN-γ) and type 2 (IL-10) cytokine, albeit with an excess of IFN-γ producing cells. This appeared to be independent of the time of harvesting patient PBML. Also, cytokine production did not vary with the stimulator cells used. 3 out of 7 patients who did not show activation had biopsy proven rejection. The ratio of cells producing IFN-γ to those producing IL-10 was calculated, in the 9 patients with activation. If the ratio of IFN-γ to IL-10 producing cells was > 2 there was a trend towards the occurrence of rejection. 3 out of 4 patients with ratio > 2 had rejection; compared with 1 out of 5 with ratio < 2. The data were from a small number of patients and the p value was not found to be significant. Conclusions: T cell activation in renal transplant PBML stimulated with HLA mismatched donor cells show considerable variation in the degree of response between patients, not explained by the obvious clinical parameters. Though a higher production of intracellular IFN-γ suggests a trend towards the occurrence of rejection, it is not conclusive that the balance between Th1 and Th2 responses influence the outcome of transplantation and measurement of IFN-γ production is not likely to be clinically useful in the diagnosis of rejection.