Dynamics Of Peripheral B Cell Recovery And Analysis Of HLA Reactivity Following Tolerance Induction In Combined Kidney/bone Marrow Transplant Recipients

Background: The rejection of transplanted organs is an important cause of graft loss,and the side effects caused by long-term use of immunosuppressive drugs can seriously affect the graft survival rate as well as life quality of transplant recipients.Therefore,it is ideal for organ transplantation to make the allograft recipients reach the immune tolerance to donor organs.B lymphocytes are important subsets of lymphocytes in organ transplantation.Previous studies identified B cell gene expression signatures and predominance of specific B cell subsets as a marker of operational tolerance following kidney transplantation.These findings suggested a role for B cells in the establishment or maintenance of tolerance.In the field of organ transplantation,the related research and clinical trials of inducing immune tolerance have achieved good clinical efficacy.Among them,the Massachusetts General Hospital of Harvard University has performed combination of kidney and bone marrow transplantation using improvement program(ITN 036),some organ transplant recipients obtained good clinical results.The purpose of this paper is to study the reconstruction of peripheral B lymphocyte as well as serum reactivity of antibody to HLA at different time points in 4 cases of immune tolerance induced transplant recipients using kidney/bone marrow transplantation,and provide theoretical and experimental basis for the further study of the role of B lymphocytes in immune tolerance of organ transplantation.Methods: 5 cases were performed combined kidney/bone marrow transplants using improvement program.One receipient with thrombotic micro renal transplant vascular disease lost her kidney graft at 6 month post transplant,so this subject is not described further in the present report.For the four remaining subjects,immunosuppression was slowly tapered over several months and completely discontinued at 8 months.Graft function remained stable for 4~5 years for three subjects.One subject experienced acute TVI cell-mediated rejection approximately 2 months after immunosuppression was discontinued and following an episode of pyelonephritis treated with antibiotics.Immunosuppression was resumed but graft function never fully recovered.A total of 4 subjects were enrolled in this study.The distribution of peripheral B lymphocyte subsets was detected using flow cytometry in kidney/bone marrow transplant recipients at different time points post transplant.The distribution of immunoglobulin heavy chain variable region was analyzed using next generation sequencing technology.Luminex was used to detect the serum reactivity to HLA at different time points after transplantation.Results: B cell counts recovered around one year post-transplant except for one subject who experienced delayed reconstitution.This subject lost his graft to rejection at 10 months post-transplant while the other subjects achieved tolerance.B cell recovery was accompanied by a high frequency of CD20+CD24highCD38hightransitional B cells and a diversified clonal repertoire.Of note,all 4 subjects showed prevalence of CD20+CD27+ memory B cells around 6 months post-transplant when B cell counts were still low and the clonal B cell repertoire very limited.Through the calculation of Shannon diversity index of immunoglobulin heavy chain variable region at each time point post transplant,we found that Shannon diversity index of immunoglobulin heavy chain variable region for all transplant recipients at D182 post-transplant was significantly lower than that before transplant,and this index gradually recovered at approximately one year post-transplant.The calculation of somatic mutation of B lymphocyte IGHV gene sequence for kidney/bone marrow transplant recipients at different time points showed that the somatic mutation rate was elevated at day 182 for all patients except patient 7.Of note,subject 7 was the only subject with detectable transitional B cells at day 182,so the somatic mutation rate of B lymphocyte IGHV gene sequence for patient 7 at this time point showed decreased trend.Through the detection of serum reactivity to HLA in patients at different time points,we found that the serum of patient 6 at D182 post-transplant showd positive reactivity to multiple HLA class I antigen including the antigen expressed on donor cells.This phenomenon revealed that patient 6 generated donor specific antibodies.The serum of patient 7 at D14 and D20 post-transplant showd weak reactivity to multiple HLA class Iantigen,revealed that patient 7 generated non-donor specific antibodies.The serum of patient 9 at D121 post-transplant showd strong reactivity to multiple HLA class I antigen and HLA class II antigen including the HLA antigen expressed on donor cells as well as recipient cells.This phenomenon revealed that patient 9 generated donor specific antibodies and autoantibodiesConclusion: Our observations reveal the presence of mutated memory B cells early post-transplant that likely escaped the preparative regimen at a time consistent with the establishment of tolerance.Further studies are warranted to characterize the functional properties of these persisting memory cells and evaluate their potential contribution to tolerance induction.