Identification And Functional Study Of Myeloid-derived Suppressor Cells In Acute Graft-versus-host Disease

Background and objective: Hematopoietic stem cell transplantation(HSC T)is an established therapy to cure hematological disorders.Graft-versus-host disease(GVHD) is the most serious complication and one of the main causes of nonrelapse mortality(NRM) after transplantation.The clinical and pathophysiologic features of acute GVHD(a GVHD) are heterogeneous.It is necessary to further explore the stratification of a GVHD.With the development of omics technologies,new biomarkers and immune cells have been discovered to identify specific complications after HSCT.Myeloid-derived suppressor cells(MDSC) are a heterogeneous population of myeloid cells with immunosuppressive activity.MDSC is defined as a group of abnormally differentiated immature cells,including granulocytic MDSC (G-MDSC),monocytic MDSC(M-MDSC)and early-stage MDSC(e-MDSC).MDSC is characterized by its immunosuppressive effects.MDSC inhibits the allogeneic reaction of T cells and natural killer (NK) cells in many ways,which are the principal effector cells to inducea GVHD.Preclinical studies have confirmed that MDSC has certain anti-GVHD effect and can maintain graft versus leukemia (GVL) by selectively inducing NKG2D+CD8 memory T cells.In the tumor microenvironment,activation and amplification of MDSC promote the tumor immune escape,which is an adverse reaction of immune system.However,MDSC may have both proinflammatory and immunosuppressive effects in the inflammatory process.Immune monitoring of the body is enhanced by excessive inflammation and MDSC is produced to weaken the adaptive immune response.In this case,MDSC plays a role in the regulation of the immune response.Different from mice,MDSC in human still lacks the specific markers.The activation mechanism,phenotypic characterization and immunosuppressive activity of MDSC in a GVHD process have not been explored.Based on the pathological mechanism of a GVHD and the biological characteristics of MDSC,this study focused on the outcomes of HSCT recipients,clinical features and prognostic impacts of different a GVHD,MDSC recovery and its correlation with a GVHD.This study aims to clarify the role of MDSC in the a GVHD process and immune tolerance following HSCT.At the same time,the single-cell sequencing is used to define the MDSC subpopulations related to a GVHD and to explore the distinctive potential markers and signaling pathways of MDSC.Methods:Part I: The comorbidities,clinical features of a GVHD and clinical outcomes of patients who underwent HSCT in our center were analyzed.Adult recipients for the first HSCT were included.Patients with incomplete clinical data were excluded.The data of demography,primary disease,complication of vital organs,transplantation characteristics,donors,GVHD prophylaxis and treatment,GVHD development and transplant outcomes were obtained.Part II: Patients who were going to receive haploidentical HSCT(haplo-HSCT)were prospectively enrolled.MDSC recovery after transplantation was monitored regularly by flow cytometry.Once the patients developed grade II-IV a GVHD later,peripheral blood(P B)samples were obtained from the patients before starting immunosuppressive therapy.And CD45+ cells were separated from PB by magnetic-activated cell sorting for single-cell RNA sequencing(sc RNA-seq).The steps of sc RNA-seq analysis included aligning raw sequencing data to a reference,quality control and screening of data,data correction and integration,data standardization and normalization,dimensionality reduction analysis,dimensionality reduction and cluster analysis,and differential expression analysis.Part III: PB samples from patients who underwent haplo-HSCT were prospectively obtained for regular evaluation of MDSC reconstitution after transplantation.Flow cytometry was used to monitor MDSC recovery at different time points after transplantation.And in cases with a GVHD,MDSC frequency before and after a GVHD treatment(+3 days,+7 days,+14 days and+28 days)were also monitored.MDSC suppression assay was carried out to verify the inhibition of MDSC on T cell proliferation through the co-culture of purified CD8+ T cell and MDSCs isolated from the PB of patients.Results:Part I: A total of 329 patients who received HSC T were included and analyzed in this study.In the analysis of HSCT outcomes,allogeneic HSCT(allo-HSCT)was a stronger prognostic factor for overall survival(OS)and NRM(P < 0.001)than autologous HSCT(auto-HSCT).Among the 199 recipients of haplo-HSCT,the 1-year cumulative incidences of grade II-IV and III-IV a GVHD were 40.3%(95% C I: 2.3%-33.8%)and 8.0%(95% C I: 5.0%-12.8%),respectively.And there were 63 patients without a GVHD,91 with one episode of a GVHD and 45 with a GVHD recurrence.Recurrence of a GVHD was an independent risk factor for NRM(HR 3.2,P < 0.001).GVHD-related death was the leading cause of NRM in patients with recurrent a GVHD.R ecurrent a GVHD was also associated with inferior novel composite end points of GVHD relapse-free survival(GR FS)(HR 1.9,95% C I: 1.1-3.3,P = 0.020)and chronic GVHD relapse-free survival(CR FS)(HR 2.1,95% C I: 1.2-3.5,P = 0.007)Part II: ScRNA-seq confirmed that a subpopulation of neutrophils(Neutrophil1)in the PB of a GVHD patients,met the phenotypic criteria for G-MDSC with expressing CD11 b,C D15 and CD66 b,while not expressing CD14 and low in HLA-DR.AGVHD-associated G-MDSC also highly expressed ARG1,MPO,S 100A8,S100A9,MMP 8 and MMP9 without expressing C XCR2.Sc RNA-seq confirmed that a subpopulation of monocytes(Monocyte1)present in the PB of a GVHD patients,met the phenotypic criteria for M-MDSC with expressing CD11 b and CD14,while not expressing CD15 and low in HLA-DR.AGVHD-associated M-MDSC expressed high levels of CCR2,VNN2,IL-1β,C LEC4 D and C LEC 4E.Compared with the immune cell profile at the time of the first episode of a GVHD,the immune cell profile at the time of the recurrent a GVHD was dominated by T and NK cells with reduced neutrophil and monocyte subsets,with the most pronounced decrease being in G-MDSC(G-MDSC at recurrence / first onset of a GVHD = 0.045).Gene ontology(GO)analysis of differentially expressed genes(DEG)in G-MDSC revealed enrichment of biological processes in: neutrophil degranulation,positive regulation of reactive oxygen species metabolic process,negative regulation of the immune system,regulation of cell adhesion,differentiation of myeloid cells,regulation of leukocyte activation,tumor necrosis factor(TNF)-alpha/TNF-kappa B signaling pathway and regulation of TNF production.GO analysis of M-MDSC revealed DEG were abundant in biological processes including cell proliferation,neutrophil chemotaxis,activation of myeloid cells,positive regulation of leukocyte differentiation,interferon signaling pathway,signaling by interleukins,positive regulation of cytokine production and positive regulation of reactive oxygen species metabolic process.Part III: The ratio of MDSC to P B C D45+ cells after haplo-HSCT returned to baseline(pre-transplant)levels 7-14 days after HSCT.Compared with patients without a GVHD,patients with a GVHD had lower MDS C subpopulation ratios before transplantation,especially G-MDSC(42.88 ± 7.87 % vs.33.49 ±10.19 %,P = 0.034);G-MDSC were even lower 7-21 days after transplantation(+7 days,32.09 ± 29.80 % vs.10.64 ± 14.85 %,P = 0.026;+14 days,56.89 ±12.27 % vs.41.75 ± 19.40 %,P = 0.038);after 21 days G-MDS C was relatively higher(+28 days,39.64 ± 24.93 % vs.64.06 ± 15.09 %,P = 0.003;+60 days,15.04 ± 1.61 % vs 44.83 ± 15.70 %,P = 0.021).Before and 7-14 days after HSCT,the ratio of G-MDSC was lower in patients with grade II-IV a GVHD or a GVHD involving multiple organs(N ≥ 2)compared to controls(P < 0.05);the ratios of all MDSC subgroups were lower in patients with recurrent a GVHD,especially M-MDSC at 7 days after transplantation(P = 0.024)and G-MDSC at14 days after transplantation(P = 0.010).Compared to patients without a GVHD and steroid sensitive-a GVHD(SS-a GVHD),patients with steroid refractory-a GVHD(S R-a GVHD)had a relatively lower ratio of G-MDSC before and 14 days after transplantation(pre-transplant: 42.88 ± 7.87 % vs.24.36 ±14.84 % vs.37.26 ± 10.33 %,P = 0.009;+14 days: 56.89 ± 12.27 % vs.46.44 ±18.13 % vs.28.44 ± 17.77 %,P = 0.011).G-MDSC showed an increasing trend during a GVHD treatment,especially significant in the SS-a GVHD group,while the G-MDSC in the S R-a GVHD patients showed no significant increase.On day+3 after a GVHD therapy,the ratio of G-MDSC to baseline was significantly higher in the SS-a GVHD group than in SR-a GVHD group(MDSC at +3 days /baseline: 1.55 ± 0.31 vs.1.03 ± 0.08,P = 0.036).In contrast to G-MDSC,M-MDSC showed a decreasing trend after a GVHD treatment,with a greater decrease in the SS-a GVHD group than in the SR-a GVHD group at 7-28 days after treatment.In the MDSC suppression assay,MDSC significantly inhibited T cell proliferation(proliferation index: without MDSC,7.21 ± 4.19 vs.with MDSC,5.45 ± 4.05;P = 0.001).Conclusion:1.AGVHD increased the risk of NRM after allo-HSCT.AGVHD recurrence was a significant predictor for NRM and survival after transplantation.GVHD-related mortality was the leading cause of death in patients who developed recurrent a GVHD.Grade II-IV / III-IV a GVHD and recurrent a GVHD were significantly associated with higher NRM and inferior CR FS and GR FS.2.In the setting of a GVHD,there were G-MDSC and M-MDSC with specific phenotypes and gene expressions that were different from normal neutrophils and monocytes.The highly ex pressed genes in MDSC were responsible for MDS C-associated immunomodulatory functions(production of ROS and cytokines,regulation of cell proliferation and apoptosis),myeloid differentiation(regulation of leukocyte differentiation and hematopoiesis),cell migration and motility(neutrophil chemotaxis,Rho GTPase and cell adhesion).3.Compared with the previous a GVHD,the immune cells of recurrent a GVHD were significantly decreased in G-MDSC,slightly increased in M-MDSC,and dominated by effector T and NK cells.4.AGVHD-associated MDSC plays an immunomodulatory role mainly via mechanisms including production of AR G1,ROS,anti-inflammatory cytokines,degranulation of neutrophils and negative regulation of cell proliferation.5.MDSC recovery after transplantation was closely related to the development,recurrence and treatment response of a GVHD.C ompared with the controls,patients with lower G-MDSC levels at baseline and 7-21 days after HSCT were more likely to develop a GVHD grade II-IV,a GVHD with multi-organ involvement,a GVHD recurrence and SR-a GVHD.The kinetics of MDSC subpopulations during a GVHD treatment were associated with the intensity,duration and efficacy of immunosuppressive therapy.