Pre and post transplant immune monitoring using the flow cytometric cytokine secretion assay to detect alloreactive T cells

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Abstracts

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PRE AND POST TRANSPLANT IMMUNE MONITORING USING THE FLOW CYTOMETRIC CYTOKINE SECRETION ASSAY TO DETECT ALLOREACTIVE T CELLS Yael D. Korin, Clara Lee, Leonard Liang, Albin Gritsch, Elaine F. Reed, Immunogenetic Center, Department of Pathology, University of California, Los Angeles, Los Angeles, CA, USA The direct and indirect allorecognition pathways play a role in graft rejection. To determine the contribution of the direct and indirect recognition pathways in renal allograft rejection, we have used a novel INF-g flow cytokine secretion assay for detecting alloreactivity via the direct recognition or indirect recognition pathways. Thirty-three renal allograft recipients were serially monitored, pre-transplant and at routine visits following transplantation. Five patients experienced acute cellular rejection (ACR), 2 had concurrent ACR and acute humoral rejection (AHR) and one showed only AHR. Serial studies revealed that 6 of the 7 patients with ACR showed T cell reactivity via the direct pathway prior to or at the time of rejection. Five of these seven patients also displayed T cell reactivity to donor antigens via the indirect pathway. However, indirect recognition was most often detected after the onset of ACR. All 3 patients with AHR displayed T cell alloreactivity via the indirect and direct pathways at the time of rejection. The average serum creatinine after the second month post transplantation in patients showing donor specific T cell reactivity was significantly higher than recipients without evidence of alloreactivity. This study reveals an association between T cell alloreactivity, ACR, AHR and early graft dysfunction. Our findings indicate that monitoring for T cell alloreactivity identifies patients at risk of graft rejection and early graft dysfunction.

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HUMAN DENDRITIC CELLS TREATED WITH INHIBITORS OF NF䊐B ABROGATE RESPONSES TO ALLOANTIGEN A. Hernandez,1 W.A. Ross,1 B.B. Blomberg,1,2 M. Burger,2 J.M. Mathew,1 A. Tzakis,1 J. Miller,1,2,3 V. Esquenazi1,2,3, 1Department of Surgery, University of Miami School of Medicine, Miami, FL, USA; 2Department of Microbiology Immunology, University of Miami School of Medicine, Miami, FL, USA; 3 VA Medical Center, University of Miami School of Medicine, Miami, FL, USA Dendritic cells (DC) prime T cells (Tc) but can also tolerize Tc and abrogate the immune response. Studies have shown that blocking NF-␬B during DC differentiation promotes allograft survival in mice. The aim is to assess the effect of blocking NF-␬B in human DC on Tc allostimulation and generation of regulatory cell populations. CD34⫹ BMC were incubated with PMA to produce DC (BMC-DC) in the presence (treated) or absence (untreated) of BAY11-7082 or ASA, both inhibitors of NF-␬B. Treated or untreated CD14⫹ PBMC were incubated with cytokines to promote differentiation/maturation into DC (PBMC-DC). Mismatched allogeneic purified Tc were cocultured with either (1) irradiated (IRR) BMC-DC (treated or untreated) (2) IRR PBMC-DC (treated or untreated) and Tc proliferation was measured. Tc were then isolated from these 1° cultures and used in a 2nd proliferation assay where they were combined with freshly isolated Tc from the same donor and cultured with allo (or third party) PBMC. Treated DC were far less potent than untreated DC in their ability to stimulate Tc proliferation. Treated DC were notably deficient in CD40. Cells in the treated cocultures produced more IL-10 and less IFN-䊐. Cultures with fresh Tc ⫹ Tc isolated from cocultures with treated DC proliferated less in response to allo and third party stimulation versus cultures with fresh Tc ⫹ Tc isolated from cocultures with control DC. These experiments show that human DC treated with inhibitors of NF-␬B may facilitate induction of tolerance in organ transplantation. DC lacking costimulatory molecules (ie, CD40) may cause responding Tc to become suppressors or render them anergic.