The Impacts Of Lymphocyte Migration On Allogeneic Immune Response

BackgroundsTransplant tolerance, defined as a state of donor-specific non-responsiveness without requirement for anti-rejection agents, has been the ultimate goal of transplantation research. Allograft rejection encompass of several closely linked events, including presentation and recognition of alloantigen, activation and expanding of host T cells, infiltration of lymphocytes into the graft. Since these processes occur in separate but mutually related sites, the trafficking of immune cells is an essential step for cellular interaction during the alloreactive immune response.In our knowledge, cell trafficking is controlled by quite complicated system, including adhesion molecules, cytokines and chemokines. Recently, chemokines and chemokine receptors have been shown to be critical components for modulating leukocyte trafficking.Chemokine and chemokine receptors have been found bearing various functions in the process of transplant rejection. The interaction of CCL19, CCL21 and their receptor CCR7 is the key point for the migration of peripheral lymphocytes into secondary lymphoid tissues. CCR7 deficient mice were generated in 1999 by F(o|¨)rsteret al. These mice show marked disturbances in the distribution of lymphocytes due to impairment of lymphocytes migration.It has been considered that secondary lymphoid organs are required for the initiation of allograft rejection. The most promising evidence comes from Lakkis et al's elegant study, which showed that mice lacking secondary lymphoid tissue were ignorant to vascularized organ transplants. However, recent work by Zhou et al in LTα^-/- or LTβR^-/- mice, which also lack secondary lymphoid organs, prompted opposite opinions. They pointed out that secondary lymphoid organs are important, but not critically required in the process of allograft rejection. To our knowledge, no investigation had been done from the complementary aspect, namely the lymphocyte's aspect, to answer the question. CCR7^-/- mouse is a well defined and therefore ideally suited model to clarify the definitive role of secondary lymphoid organ and lymphocyte trafficking in the process of allograft rejection.To investigate the impact of CCR7 deficiency on allograft survival, we designed 4 transplant combinations with CCR7 deficiency in donor and/or recipient, i.e. wild type (wt) to CCR7 knock out (KO,CCR7^-/-), KO to wt, and KO to KO. In the wt to KO combination, we tried to elucidate whether allograft rejection can be initiated in the host whose lymphocytes are unable to migrate into lymph nodes. In the KO to wt combination, the graft DCs can not traffic to host lymph nodes and may consequently influence the direct presentation of alloantigen, thereby alter the immune response against allograft. As in the KO to KO model, not only host lymphocytes can not migrate into LNs, but graft DCs are either excluded from LNs. We tried to test whether tolerance can be achieved by the double effects. Then, tolerance induction will be tested in KO mice to reveal the role of secondary lymph tissue in transplant tolerance.Intestinal transplantation is the life-saving alternative for patients with irreversible intestinal failure who have life-threatening total parenteral nutrition(TPN) complications. In spite of that, the clinical results are still far from acceptable compared to other solid organ transplants, plagued with refractory rejection. New strategies are urgently needed to overcome these immunologic and biologic challenges, for substantial improvements of the outcomes in SBTx.The CD40-CD40L pathway provides crucial costimulatory signals for activation of lymphocytes. However, in mouse SBTx it was found that CD40-CD40L blockade, either by monoclonal antibody (MR-1) or CD40-knockout, had no beneficial effect on intestinal allograft survival.FTY720 is a novel immunomodulator, which acts in a unique way compared to other immunosuppressants, by causing peripheral lymphocytopenia and lymphoid sequestration, but without inducing generalized immunosuppression. The feature of this agent enables us to use it in cell trafficking protocols.We tested the effect of combined therapy of MR-1 and FTY720 in mouse SBTx. As reported, anti-CD40 therapy and FTY720 acted in very distinguishable way in preventing rejections. Therefore, we tried to find out whether synergistic effects existed in a combined protocol. It was hypothesized that sequestration of lymphocytes in secondary lymphoid organs by FTY720 might enhance the efficiency of MR-1 to block the interaction of APCs and lymphocytes, thereby amplifying the effects of MR-1.Materials and MethodsPart I1. Animal and groups: CCR7^-/- mice were used as donors or recipients as designed.2. Heterotopic heart transplantation: Pulmonary artery and aorta were anastomosed to the recipient's inferior vena cava and abdominal aorta, respectively.3. Skin transplantation: Recipients were anesthetized, shaved, and grafted withtail skin on the back or flank. Bandages were removed after 7 days.4. Histological assessment of the heart graft: Hematoxylin-eosin staining or immunohistological staining with anti-CD4, anti-CD8 monoclonal antibodies were performed. High-power fields of 200× magnification were digitally scanned for counting immunostained cells.5. Analysis of draining lymph nodes: Axillary and brachial lymph nodes were harvested, and the isolated cells were stained with the antibodies. Multiple color analysis was performed on a FACSCalibur. Absolute and relative numbers of immunostained cells per lymph node were compared between ipsilateral and contralateral axillary and brachial lymph nodes.Part II1. Animal and groups: C3H (H-2k) mice were used as donor and C57B1/6(H-2b) mice as recipients. All recipients were divided into 4 groups, namely 1, non-treated group; 2, MR1 mono-treated group; 3, FTY720 mono-treated group; 4, FTY720 plus MR1 treated group.2. Heterotopic Small Bowel Transplantation: The graft portal vein and superior mesenteric artery were anastomosed to recipient's inferior vena cava and abdominal aorta in an end-to-side fashion.3. Histological Assessment of the intestinal graft: Recipient mice were sacrificed at the 6^th and 14^th day after transplantation and stained with hemotoxylineosin. All microscopic slides were observed and assessed blindly according to the histological criteria.4. Isolation of graft GALT: The intestines were split longitudinally and digested with Collagenase A and EDTA. The cells were suspended by Percol for purification.5. Flow cytometry: Four-color flow cytometric analysis was conducted on isolated cells from different regional LNs of the recipients and grafts.6. Immunofluorescent Staining: The frozen sections were incubated with monoantibody cocktail and subsequently incubated with secondary antibody. All slides were examined by using a motorized microscope and the image assembly by the KS300 MosaiX software.ResultsPart I1. Prolonged survival of skin and cardiac allografts were found in CCR7^-/- recipients.2. Heart allografts with a full MHC disparity were examined by immunohistology at day 4 and 6 after transplantation. At day 4, infiltration of CD4⁺ and CD8⁺ T cells was considerably reduced in CCR7^-/- recipients compared with WT controls.3. Lack of T cell expansion were found in draining lymph nodes of CCR7^-/- compared with WT recipients after fully mismatched skin transplantation.4. Subtherapeutic doses of CsA abrogated the CCR7^-/- effect of attenuating the rejection. In contrast, in the presence of CsA, skin graft survival was extended in both WT and CCR7^-/- mice5. Under the costimulation blockade, the grafts survived for 42-45 days but tolerance was not achieved, which indicated the resistance to CD40-CD40L blockade protocol in CCR7^-/- mice.PART II1. FTY720 plus MR1 markedly attenuate intestinal allografts rejection, whereas either of the monotherapy is not efficient.2. Combined therapy of FTY720 plus MR1 reduced the proportion of lymphocytes in the peripheral blood.3. FTY720 plus MR1 treatment results in reduced graft infiltration by host lymphocytes, particularly for CD8⁺ cells in the graft lamina propria.4. Host lymphocyte activation was inhibited by FTY720 plus MR1 treatment, as the CD44 level was profoundly reduced when compared to the other groups.5. Reduced infiltration of CD11b⁺ Gr1⁺ cells in the intestinal allografts was found in FTY720 treated groups.Conclusion1. The deficiency of CCR7 prolonged the allograft survival moderately, but did not abrogate the rejection. Tolerance induction by CD40-CD40L blockade and DST was resisted in CCR7^-/- mice.2. FTY720 plus MR1 markedly attenuate intestinal allografts rejection by inhibiting the host infiltration into the graft, particularly the CD8⁺ T cells in the lamina propria.