| Our human beings live in a world surrounded with various kinds of pathogenic microbes that may be harmful like virus, bacteria, gungus, protozoon, parasites and so on. Our immune system helps us to maintain homeostasis and uninterrupted by those kinds of prthogens. These harmful pathogenic microbes have proteins called antigens coating their surfaces. As soon as these antigens enter the body, the immune system recognizes that they are not from that person’s body and that they are "foreign," and attacks them. In transplantation, however, when a person receives an organ from someone else during transplant surgery, that person’s immune system may recognize that it is foreign, and the immune system may further attack the transplanted organ and cause rejection.Improved diagnosis of donor-specific antibodies has led to the current understanding that antibody-mediated rejection is the leading cause of organ allograft failure in the clinic. Antibody-mediated rejection manifests as microcirculation lesions and specific transcript changes that signify antibody-mediated endothelial injury, interferon-γ effects and the recruitment of natural killer cells. Because the main cause of late organ transplant failure is now correlated with antibody-mediated rejection, and T cell mediated rejection, which is common early but progressively disappears over time post-transplant, is not associated with graft failure. The working paradigm is that current immunosuppression is relatively ineffective in preventing antibody-mediated rejection, especially once donor-specific antibodies are detected. Donor-specific antibodies are produced by T-dependent alloreactive B cells that, upon encountering with alloantigen, differentiate into antibody-producing short-lived plasmablasts that are responsible for the acute production of antibodies, as well as long-lived plasma cells, which are responsible for serological memory. In addition, some activated alloreactive B cells differentiate into quiescent memory B cells that, upon antigen re-encounter, differentiate rapidly into plasmablasts capable of producing high affinity antibodies. Thus, donor specific B cells are very important in the process of allograft rejection.There is also emerging evidence that B cells may play an immunomodulatory role and facilitate the development of transplantation tolerance. In these studies, IL-10 produced by B cells have been shown to play a critical role, but the phenotype and the antigen-specificity of the IL-10 producing B cells and the micro-anatomical location of these IL-10-producing B regs that allow them to modulate T cell responses require further clarification. Additionally observations that operationally tolerant organ transplant recipients have enriched subsets of B cells compared to stable recipients on immunosuppression have lead some investigators to hypothesize a role for B cells, and potentially regulatory B cells, in clinical transplant tolerance. Given the dual roles of B cells as drivers and suppressors of the immune responses, there is a need to trace the fate of endogenous alloreactive B cells under different transplant scenarios.Part I. The preparation of MHC-II tetramer and the detction of endogenous donor specific alloreactive B cells.In this part, we employed FCS to confirm that some B cells in C57BL/6 mice can identify SAV-PE or SAV-APC, however, they can hardly identify these tow antibodies at the same time. Unlike SAV, The MHC-II tetramers double positive cells appearred gathered in draining lymph nodes, which means they can identify IEd-PE and IEd-APC simultaneouly. By using adoptive transfer, we collected the normal C57BL/6 mice spleen lymphocytes, and separate all that can identify IEd cells by magnetic beads. After that we injected the rest of the lymphocytes into the B cell deficiency mice. We found that after three weeks anti-IEd antibodies were largely decreased in B cell defectedμMT mice. The results suggested removing IEd cells can lead to reduced anti-IEd antibody, and these IEd-PE and IEd-APC double positive B cells are really the antibody-secreting B cells. At the same time, we also tested the sensitivity and specificity of double positive MHC-II tetramers positive method, finding that IEd-PE and IEd-APC double positive B cells barely express SAV, and no on natural cells that can combine to SAV or IEb cells.Part II. Using double positive MHC-II tetramers as a medthod to detect the differentiation, development, and evolution of donor specific B cells after subcutaneous sensitization or heart transplantionIn this section, we firstly examined the applicability of subcutaneous immunization by using BALB/c spleen cells to sensitize C57BL/6 mice, and collected serum to test the donor specific antibodies and anti-IEd antibodies at different time points. We found that the DSA and IEd peaked at day 14-21 after immunization. In the draining lymph nodes of C57BL/6 mice that immunized with BALB/c lymphocytes there is only 8% with Fas+Gl7+ germinal center phenotype at day 7, 35% at day 10, and 49% at day 14. These results suggested that while some antibodies with low specificity appeared in 7 da ys, most of the antibodies with high specificity were generated after a large number of B cells in draining lymph nodes showed Fas+Gl7+ germinal center phenotype. The numbers of IgDlo double positive MHC-II tetramers cell in draining lymph nodes are far more than those in the non-draining lymph nodes, which are about 10 times.Further study showed that compared with subcutaneous immunization, heart transplantation was more systemic, there are some double positive MHC-II tetramers B cells not only in draining lymph nodes, but also in spleens. But neither in the draining lymph nodes nor in the spleens have more double positive MHC-II tetramers B cells than subcutaneous immunization.Finally, we tested the percentages of Tfh cells in the spleens in recipients. In heart transplanted mice spleens, there were around 25% of T cells were CXCR-5+PD-1+ phenotype, of which about 9% are CXCR-5hiPD-1hi Tfh phenotype. This result further determined the time window of donor specific B cells got germinal center phenotype.Part III. Using double positive MHC-II tetramers as a medthod to detect the differentiation, development, and evolution of donor specific B cellsin recall responseIn this part, we found that in recall response, both donor specific B cells and donor specific germinal center phenotype B cells didn’t rise as much as primary immunization. The number of ASCs in recall response, by contrast, were much more than those in primary immunization. Memory B cells are the main part of the immune response in recall, and it can function with the help of non-sensitized T cells instead of sensitized T cells. The donor specific IgG rapidly peaked within 7 da ys after re-immunization, twice as much as the primary immunizaiton, and remain high in the next few weeks, droping slowly.By employing CTLA4-Ig, we further studied the activation of Tfh cells and the formation of germinal center. Application of CTLA4-Ig form day 0 can completely inhibit the generation of donor specific IgG, and also inhibit the generation of donor specific IgG during the recall response. In contrast, application of CTLA4-Ig form day 7 can mostly inhibit the generation of donor specific IgG, and also mostly inhibit the generation of donor specific IgG during the recall response. However, application of CTLA4-Ig form day 14 didn’t affect the generation of donor specific IgG. Further experiments revealed that the level of donor specific antibodies were consist with donor specific memory B cells.In summary, we use double positive MHC-II tetramers as a medthod to detect the generation time window of donor specific B cells, the changes of phenotype and scale in donor specific B cells, and generation of Tfh cells. In recall response, we employed CTLA4-Ig to inhibit germinal center formation and studied the changes of donor specific memory B cells. Our results revealed the formation time window of germinal center, and determined the role of donor specific B cells in trasnplantation rejection, putting forward a new understanding. |
PDF Full Text Request