Immune Monitoring And Biomarker Study Of Acute Rejection And Graft-versus-host Disease After Liver Transplantation

Chapter One Peripheral blood NK cells immune monitoring and biomarkers of acute rejection in liver transplantation recipientsPurposeNatural killer（NK）cells play a role in acute rejection after liver transplantation（LTx）.The characteristics and dynamics of peripheral blood NK cells in LTx recipients are still scarce.Analysis of the characteristics of peripheral blood NK cells in patients with rejection can provide potential predictive biomarkers for acute rejection after LTx,and is beneficial to deepen the understanding of the pathogenesis of acute rejection after LTx.MethodsWe constructed a longitudinal study cohort of patients undergoing LTx at the Liver Transplantation Center of Jilin University from March 2019 to July 2021,including 32patients with acute rejection and 30 patients with immune homeostasis.Peripheral blood of patients before and after transplantation was dynamically collected at multiple time points,peripheral blood mononuclear cells were extracted,and total NK cells and NK cell subsets(CD56^brightCD16^-NK cell subsets,CD56^dimCD16⁺NK cell subsets)in peripheral blood of LTx recipients were analyzed by multi-parameter flow cytometry during preoperative and one-year postoperative follow-up.And the expression of surface activated receptor（NKp30,NKp44,NKp46 and DNMA-1）and inhibitory receptor（NKG2A）also was analyzed.ResultsCompared with healthy controls,patients with AR post-LTx contained a higher proportion of CD56^brightCD16^-subsets in preoperative peripheral blood NK cells（19.53±15.42 vs 4.17±3.70,P<0.0001）and a lower percentage of CD56^dimCD16⁺subsets（76.76±16.74 vs 94.85±3.97,P<0.0001）.Compared with immune homeostasis patients,the rejection group also had a higher percentage of CD56^brightCD16^-subsets before LTx（19.53±15.42 vs 8.73±8.27,P<0.01）and a lower percentage of CD56^dimCD16⁺subsets（76.76±16.74 vs 87.79±11.83,P<0.05）.Two types of NK cell subsets expressed higher proportion of NKp30 in the rejection group.CD56^dimCD16⁺NK expressed a higher proportion of NKp46.Multivariate logistic regression analysis showed that the positive rate of NKp30 in preoperative peripheral blood CD56^dimCD16⁺NK cell subsets was an independent risk factor for predicting rejection（odds ratio:1.07,P=0.001）.During the one-year follow-up after transplantation,the composition of NK cell subpopulations in peripheral blood of AR group was consistent with that before LTx,that is,it consisted of a high proportion of CD56^brightCD16^-NK cell subsets and a low proportion of CD56^dimCD16⁺NK cell subsets.Both subsets maintained high positive rates of NKp30.CD56^dimCD16⁺NK cells also maintained a high positive rate of NKp46.After the occurrence of AR,the composition of NK subsets in peripheral blood remained stable,while the expressions of NKp30 and NKp46 were significantly up-regulated(NKp30:CD56^brightCD16^-57.86±4.24 vs 63.54±3.84,P<0.05,CD56^dimCD16⁺51.42±4.19 vs 58.17±4.08,P<0.05;NKp46:CD56^brightCD16^-57.86±4.24 vs 63.54±3.84,P<0.05,CD56^dimCD16⁺51.42±4.19 vs 58.17±4.08,P<0.05).Compared with patients in the immune homeostasis group,there were no significant differences in the expression levels of NKp44,DNAM-1 and NKG2A in peripheral blood of patients in the rejection group before and after LTx.ConclusionA high proportion of CD56^brightCD16^-NK cell subsets and a high proportion of positive rates of NKp30 and NKp46 were the characteristics of peripheral blood NK cells in patients with AR.The high positive rate of NKp30 on CD56^dimCD16⁺NK cells in the preoperative peripheral blood of recipients was an independent risk factor for AR.Chapter Two Dynamic analysis of GVHT cell clonal expansion and risk factors in peripheral blood of patients with graft-versus-host disease after liver transplantationPurposeGraft-versus-host disease（GVHD）is a rare immune complication after LTx.The disease is difficult to predict,diagnose and treat,and the mortality rate is extremely high.Donor-derived graft-versus-host（GVH）T cells are the main cells responsible for the pathogenesis of GVHD.However,the clones and amplification dynamics of GVHT cells in patients with GVHD have not been studied.In addition,known risk factors for GVHD in LTx have low specificity,and new risk factors need to be developed.In view of this,we integrated multi-parameter flow cytometry and immune repertoire sequencing technology to track the T cell phenotype,chimerism ratio of donor T cells,GVHT cell clones and amplification dynamics in postoperative peripheral blood of patients with GVHD,and analyzed the cytokine levels and TCR repertoire characteristics in peripheral blood of patients with GVHD before LTx.The aim is to provide a new idea for the prediction and treatment of GVHD following LTx.MethodsWe retrospectively selected preoperative and postoperative peripheral blood samples,donor peripheral blood and donor liver samples from 4 GVHD patients in the Liver Transplant Center of Jilin University from 2018 to 2021.A corresponding sample of 12 non-GVHD patients with similar age and MELD score was selected as controls.We applied multicolor flow cytometry to monitor the chimerism proportions of donor T,B and NK cells according to the HLA-mismatch of donors and recipients,and analyzed the differentiation dynamics of donor and recipient CD4 and CD8 T cells and the proportion changes of dendritic cells（DC）in peripheral blood of patients.The donor and recipient preoperative peripheral blood mononuclear cells were used as reaction cells and stimulator cells to establish mixed lymphocyte reaction,and the GVHTCR repertoire was established by T cell immune repertoire sequencing technology.The repertoire was used as biological fingerprint to track the clones and amplification dynamics of GVHT cells in postoperative peripheral blood of patients.The source of GVHT cells in preoperative donor liver samples was identified.Multifactor assay was used to quantify the serum cytokine content of patients before and after LTx.The width of peripheral blood TCR repertoire was measured by T cell immune repertoire sequencing technology.Donor-derived hematopoietic stem/progenitor cell differentiation in bone marrow samples from a patient with GVHD was analyzed by flow cytometry and full-length single-cell transcriptomic sequencing.ResultsPrior to the onset of GVHD,donor T cells had reached macrochimerism levels in the recipient’s peripheral blood,lasting for 3 weeks in one patient.The occurrence of large chimerism of donor T cells was accompanied by an increase in the proportion of CD4 and CD8 effector T cells in peripheral blood of patients.The chimerism ratio of donor T cells in peripheral blood of patients increased with clonal accumulation and clonal convergence of GVHT cells,as well as GVHD-related skew in the use frequency of TRV and TRJ genes in the overall TCR repertoire.The origin of the dominant GVHT cells in the donor liver before transplantation was traced,and it was found that these clones may also be derived from the residual circulating T cells in the allograft liver.After the onset of GVHD,the chimerism ratio of donor T cells increased further,and the macrochimerism of donor B cells and NK cells was detected.The macrochimerism of donor immune cells disappeared after the disease was cured.Before the emergence of large T cell chimerism,the proportion of recipient DC in the peripheral blood of the recipient continued to increase,and then decreased after the onset of the disease.Active Treg（aTreg）was present in peripheral blood of GVHD cured patients,while aTreg was absent in peripheral blood of deceased patients.The width of the TCR repertoire and the level of serum inflammatory cytokines in the preoperative peripheral blood samples of recipients were detected.The results showed that compared with the control group,the preoperative peripheral blood TCR repertoire of GVHD patients was narrowed,and this trend was more obvious in the patients who died.GVHD patients with preoperative serum TNF-αand IL-8 levels increased significantly.In addition,donor-derived hematopoietic stem/progenitor cells can migrate to the recipient bone marrow and have the potential to proliferate and differentiate.ConclusionDonor-derived GVHT cells,as the core of the pathogenesis of liver transplantation GVHD,accumulate in peripheral blood of patients before the onset of the disease.Preoperative TCR repertoire diversity of recipients decreases in peripheral blood and serum concentrations of TNF-αand IL-8 may be risk factors for the GVHD after liver transplantation.