The Role Of T Cell Adapter Protein ADAP In Murine Heart And Intestinal Transplantation

BackgroundsRejection remains to be the major problem to be overcome in organ transplantation. The immunological process of allograft rejection is characterized by several closely related events, including presentation and recognition of alloantigen, activation and expansion of host T cells, infiltration of lymphocytes into the graft, and ultimately, damaging of the graft by cytotoxic effector T cells. During this process T cells play a pivotal role and they use integrins in essentially all of their functions. Among all the integrins expressed on T cells αLβ2 (leukocyte function-associated antigen-1, LFA-1) is the most abundant and important one. LFA-1 take part in facilitating adhesion of T cell to endothelium, entering of T cell into the lymph node and contacting with APC, supplying the co-stimulatory signal for T cell activation, migration of lymphocytes into the graft.Since unstimulated endothelium constitutively expresses ICAM-1 and ICAM-2 at low levels, circulating T cells are continually exposed to LFA-1 ligands on the vasculature, and their ligand-binding activity needs to be tightly regulated. In addition to kinases and phosphatase, a recent major advance in our understanding of T cell signalling has been made with the identification of hematopoietic-specific adapters, proteins that lack intrinsic enzymatic activity or receptor function but contain domains that promote formation of multiprotein signaling complexes. A large body of evidence now indicates that at least four hematopoietic-specific adapter substrates of TCR-stimulated PTKs, including Src homology2 (SH2)-domain containing leukocyte phosphoprotein of 76 kDa (SLP-76), linker for activated T cells (LAT), Gads, and adhesion-and-degranulation-promoting adapter proteins (ADAP), play nonredundant roles in cellular activation.ADAP is expressed in T cells and myeloid cells that are phosphorylated upon TCR stimulation. Two isoforms have been cloned of 120 and 130 kDa, which differ in expression levels between thymocytes and peripheral T cells.The function of ADAP has most clearly been demonstrated in ADAP-deficient mice and by over-expression of ADAP in transgenic cells. The main function of ADAP is to positively couple the TCR engagement signal to LFA-1 activation, T cell activation, proliferation and β2 integrin-mediated cell adhesion. In addition, ADAP regulates the β1 integrin mediated cell adhesion and cytoskeletal remodeling.A large body of evidence has shown the important role of LFA-1 in the alloimmune responses. Blocking the activation of LFA-1 and its binding to the ligand leaded to the prolongation of allograft even the tolerance induction. Based on the role of ADAP in the LFA-1 activation and the T cell function, it is theoretically possible to expect that ADAP excerts key effect on the alloimmune responses in organ transplantation. Untill now there is no report on the relationship between ADAP and organ transplantation. The present study aim to explore the following questions applying the ADAP-deficient (ADAP-/-) mice :1. In order to mimic the in vivo situation in organ transplantation, responder lymphocytes were ioslated from ADAP-/- mice and allogenic dendritic cells (DC) were used as stmuli. The role of ADAP in T cell proliferation ability was studied in MLR in vitro.2. The role of ADAP in the effector phase of T cell function was studied in the in vitro and in vivo cytotoxic T lymphocyte( CTL) system.3. The role of ADAP in vascularised and non- vascularised organ transplantation was studied in murine heart and skin transplantation model.4. The role of ADAP in the small bowel transplantation and the synergistic effect of co-stimulatory blockade and ADAP deficiency were studied.Materials and methods1. T cell proliferation ability was studied by CFSE dilution method and 3H incorporation method in mixed lymphocyte reaction (MLR) system in vitro.2. Target cells were labeled by Cr-51 and CFSE. The lysis efficiency of target cells by effector lymphocyte was detected in CTL system in vitro and in vivo.3. Murine heart allograft was heterotopically transplanted in abdomen and the skin allograft was heterotopically transplanted on back.4. Two groups were made in heart and skin transplantation experiments: BALB/C mice (H-2d) served as donor, recepients were C57BL/6 mice (Wild type, WT) (H-2b)orADAP-/-mice.5. The heart allograft was analyzed by H.E staining, multi-immunofluorescence staining and immunohistochemical staining.6. Murine small bowel allograft was transplanted heterotopically in abdomen.7. Four groups in small bowel transplantation were divided according to various recepient and treatment: BALB/C mice (H-2d) served as donor, group 1: WT served as recipient, group 2: WT recepient received treatment of MR-1 (anti-CD40L monoclone antibody) , group 3: ADAP-/- mice served as recepient, group 4: ADAP-/- recepient received treatment of MR-1.500μg of MR-1 was administered by i.v. injection on the day of surgery, the same dosage was i.v. injected on day 2,4,7 additionally.8. The histological rejection score of intestinal allograft was valued on day 14 after transplantation by H.E staining.9. The infiltrating lymphocytes in heart and intestinal allograft were isolated by collagenase A-based protocol.10. The composition and activation status of infiltrating lymphocytes were determined by flow cytometry after incubation with fluorescence-conjugated rat anti mouse antibody.Results1. Compared with WT, ADAP-/- CD4+ and CD8+T cells had a prominant defect in proliferation and IL-2 production after stimulation of allogenic DC or anti-CD3/anti-CD28.2. There was no signifcant difference in CTL between ADAP-/- and WT T cells.3. The heart but not the skin allograft survival in ADAP-/- recepient was prolonged significantly compared with WT recepient.4. The infiltration of T cells, including CD4+ and CD8+T cell was reduced in heart graft in ADAP-/- recepient compared with WT. There was no difference in infiltration of B cell and granulocytes between two groups.5. The actively proliferating cells inside of the heart graft were decreased in ADAP-/- recepient compared with WT.6. In the heart transplantation, both the intragraft infiltration of CD4+ and CD8+T and the activation of CD8+ T cell was reduced in ADAP-/- recepient by flow cytometry method.7. The rejection severity of intestinal allogrfat was decreased a little in ADAP-/-recepient but the villi morphology was still destroied.8. Monotherapy of MR-1 has no protective effect on intestinal allograft.9. Combined therapy of MR-1 and ADAP deficiendcy inhibited the rejection of small bowel allograft and preserved the integrity of villi.10. Combined therapy of MR-1 and ADAP deficiendcy decreased the host CD4+ and CD8+ T in graft MLN and the percentage of activated host CD8+ T cells.11. ADAP deficiency and combined therapy decreased the ratio of activated/resting cells and the percentage of memory T cells in the graft Peyer's patch.12. ADAP deficiency and combined therapy decreased infiltration of host CD4+ and CD8+ T cells and increased the ration of donor/recepient cells.Conclusions1. The heart and intestinal allograft survival in ADAP-/- recepient was prolonged while a delayed rejection appeared ultimately.2. The mechanism of protection is mainly related with inhibition of T cell activation, proliferation, IL-2 production and infiltration into the graft by ADAP deficiency.3. There was a synergistic effect of MR-1 therapy and ADAP deficiency in inhibiting the rejection of small bowel allograft.4. The main function of ADAP in this setting appears to affect the induction phase rather than the effector phase of the alloimmune response of organ transplantation.