T cell division using CFSE as a marker of allo-activation in acceptance and rejection of living related renal allografts

Debbie Watson, John Knight, Stephen Alexander.

Centre for Kidney Research, Children's Hospital at Westmead, Sydney.

T lymphocytes are involved in the initiation of allograft rejection. In order to study the frequency of allo-reactive CD4+ lymphocytes in two renal transplant patients, peripheral blood mononuclear cells (PBMC) were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE), then placed in a mixed lymphocyte culture with irradiated donor PBMCs. The in vitro effect of immunosuppression was studied using cyclosporine (500ng/ml) and anti-CD25 blocking antibodies (20ug/ml). After six days the cells were stained with PE labelled anti-CD4 antibodies (Becton-Dickinson) and then analysed using 2 colour flow cytometry. The cells were gated on CD4+ lymphocytes and analysed for proliferation using the intensity of CFSE staining. The number of cells in each generation of cell division was counted. Precursor frequencies were derived from these results. The first patient (on dialysis after rejecting a living related donor kidney) had 9.2% allo-reactive CD4+ precursor lymphocytes. The second patient (a recipient of a successful LRD kidney for > 5 years, on cyclosporine, azathioprine, prednisone) had 2.4% allo-reactive CD4+ precursor lymphocytes. Neither cyclosporine nor anti-CD25 antibodies in the culture altered the frequency of the precursor numbers. In a control unrelated MLR, using the above method, 2.8% of the control's CD4+ lymphocytes were allo-reactive. However this frequency was suppressed by cyclosporine to 1.4% and by anti-CD25 antibodies to 1.7%. These case studies suggest a high level of allo-reactive CD4+ T cells in patients with rejecting grafts and a lower level in patients with current functioning grafts similar to third party levels. Analysis of cell division allows quantification of the allo-reactive T cell population.

Presented at the Transplantation Society of Australia and New Zealand, Canberra, May 2001.

Correspondence
Dr Steve Alexander
StephenA@chw.edu.au