Characterisation of Intestinal Intraepithelial Lymphocytes by Flow Cytometry in Post-transplant Patients

Background: Intestinal transplant is indicated in individuals with irreversible intestinal failure and life-threatening complication related to parenteral nutrition. Post intestinal transplant risk includes sepsis, renal failure, graft-versus-host disease and allograft rejection. Currently, diagnosis the above complications are based on histological and clinical examination. Abnormal laboratory values and histological analysis are not specific to post-transplant complications. Intraepithelial lymphocytes (IELs) represent an abundant and heterogeneous population of T cells that reside within the intestinal epithelium. Increase in IELs is present in small bowel disorders. Flow cytometric analysis can be used to find the phenotypic expression of IELs, which can be diagnostic or prognostic in future.  Aim: The aim of this study is to compare the IEL subsets in pre and post-transplant patients.

Methods: The study included 14 intestinal biopsies (12 from terminal ileum and 1 from duodenum) from eight patients who underwent multivisceral transplantation between 2013 and 2018 at Addenbrooke’s Hospital, Cambridge. 10 biopsy samples from non-transplant patients (4 from duodenum and 6 from terminal ileum) are used as a control. All post-transplant patients were on standard rejection therapy. Intraepithelial lymphocytes were isolated from biopsies using dithiothreitol and ethylenediaminetetraacetic acid by a series of gentle and vigorous shaking. A cytospin slide and total nucleated cell count were performed to confirm the presence of intraepithelial lymphocytes. Flow cytometric estimations are performed in six colour BD FACS Canto II analyser using FACS DivaTM Software. Staining of IELs involves incubation of the sample with antibodies. Intracellular staining was performed after fixing and permiabilisation of cells. Statistics: GraphPrism 7.04 is used to do the statistical analysis.

Results: Percentage of lymphocytes were significantly different in duodenal and ileal biopsies. Increased cytoplasmic CD3 expressions were observed in post-transplant patients. A significant increase in CD38 expression was noted in post-transplant patients. CD3+ positive population from week-2 post-transplant to 220 weeks were found to have the similar pattern to another large study. Histological alterations were comparable in most of the cases including one acute rejection. Significant negative correlations were observed in double positive and double negative (sCD3+ and CytCD3+) populations. No significance was observed in CD4/CD8 ratio and CD56+ NK cells in pre and post-transplant patients.