Co-Inhibitors of Second Signal of Lymphocyte Response in Human Renal Transplants: PD-L2, GITR, and ILT-2/3/5 Positive Cells from Aspiration Biopsies Associate with Acute Rejection-Freedom

Following organ transplantation, the outcome of the encounter between an APC and a T lymphocyte is strongly dependent on the presence of costimulatory and co-inhibitory molecules, the former associated with allograft rejection and the latter with allograft acceptance. We evaluated the expression of PD-L2, GITR, ILT-2/3/5, and ILT-4 on graft-infiltrating cells procured by Fnab from human KTx under different immunosuppressive regimens. Methods: Fnab biopsies were performed on days 7 or 14 - 30 in stable KTx and on the day of acute rejection diagnosis. Cytopreparations were studied by the enzymatic avidin biotin complex staining. Results: Acute rejection group showed a significant down-regulated expression of PD-L2, GITR, and ILT-2/3/5 as compared to stable group, while for ILT-4 we did not find significant difference. Anti-IL2αR and rapamicyn treatment trend to down-regulate ILT-4 expression, although meaningless. A significantly positive correlation was observed between PD-L2 and GITR expression in Fnab. The PPV for acute rejection diagnosis for both PD-L2 and GITR was clearly above 0.8. Conclusions: Our findings point to an early entrance of cells expressing PD-L2, GITR and ILT-2/3/5 inside human KTx who are going to remain rejection-free. Both PD-L2 and GITR shared a high ability to rule-in and rule-out acute rejection.

CD16+ Cells and Costimulatory Molecules of Lymphocyte Activation Present inside Human Kidney Grafts and in Blood Circulation

Background: We studied the expression of important costimulatory molecules of lymphocyte activation and the presence of CD16+ cells on aspiration biopsies of kidney transplants, measured three soluble factors and when indicated tested their robustness in diagnosing acute rejection. Methods: Fine-needle aspiration biopsies were performed either on days seven or 14 - 30 post-transplantation among stable kidney transplants and on the day of acute rejection diagnosis, while a sample of peripheral blood was collected simultaneously. The cyto preparations were studied by the enzymatic avidin biotin complex staining. The immunocytochemistry was directed to CD16, CD28, CD152, ICOS, CD40, CD154, CD26 and CD27. We performed the analysis in the peripheral blood by ELISA for soluble(s) CD16, CD26, and CD154. Results: The group of acute rejection cases showed a significant up-regulated expression of CD16, CD26, ICOS and CD40 as compared to the group of stable cases. Both sCD16 and sCD154 were significantly higher in the blood samples of the group with acute rejection. Thymoglobulin down-regulated CD154 and sCD16. CD16, CD26 and ICOS exhibited very high sensitivity and specificity for acute rejection diagnosis. Conclusions: The presence of CD16+ cells inside the graft emerged as a distinct player in acute rejection, confirming other previous reports whereas we first document that in human kidney transplants, ICOS and CD26 are significantly up-regulated and both reached positive predictive values for acute rejection ≥ 80%. The other costimulatory molecules, with the exception of CD40, though widely known, did not show robust association with immune events.