The Research Of Immune Atlas Of Patients With Chronic Graft-versus-Host Disease By Single-Cell Mass Cytometry

Background:Chronic graft-versus-host disease(cGVHD)remains the major complication leading to reduced patient-reported quality of life,significant non-relapse mortality(NRM)and increased incidence of secondary malignancies after allogeneic hematopoietic stem cell transplantation(Allo-HSCT).The incidence of cGVHD has been estimated to be between 30%-70%of patients after allo-HSCT.To date,the immune cells involved in cGVHD pathobiology remains unclear.The conventional flow cytometer,which is widely used nowadays,is a fluorescence emission-based detection system.The emission spectra of different fluorophores overlap,so the fluorescent signal markers that can be detected simultaneously are generally less than fifteen,and the signal markers easily interfere with each other,which have an impact on the accuracy of experimental results.Mass cytometry,or cytometry by time-of-flight(CyTOF),is a single cell-based platform that utilizes antibodies conjugated to rare heavy metal ions for analysis of cellular proteins by a time-of-flight mass spectrometer.It can simultaneously detect as many as 100 parameters in a single cell by single-cell mass spectrometry,we were able to study the whole immune cell subsets involved in the pathophysiological process of cGVHD and their corresponding characteristics at a single-cell level,and to reveal the pathogenesis of cGVHDObjective:In order to clarify the immunological profile of patients with cGVHD,this research explored the role of immune cell subsets imbalance in the development of cGVHD by single-cell mass spectrometry,and explored new strategies targeting specific immune mechanisms for the treatment of cGVHD.Methods:(1)Determined the 42 immunolabels required for single-cell mass spectrometry experiments and construct a preliminary computer program.Quality samples were collected in the early stage,and the quality of antibodies was inspected by comparison with traditional multi-color flow cytometry and single-cell mass spectrometry.After the quality detection,the chronic graft-versus-host disease CyTOF detection human immunoassay kit was officially constructed(including 42 CyrTOF antibodies);(2)According to NIH diagnosis and classification criteria,patients receiving allo-HSCT were divided into no cGVHD patients group,moderate cGVHD patients group,severe cGVHD patients group;and according to patients’rejection sites,patients were classified into two groups characterized with skin rejection symptoms or pulmonary rejection symptoms,respectively;(3)Collected peripheral blood samples from patients after allo-HSCT,and recorded their detailed disease information.At the same time,peripheral blood samples were also collected from 5 healthy individuals as a control.All participants were examined to be free of infection or autoimmune diseases.10 ml of fasting venous blood(EDTA anticoagulation)were drew and mononuclear cells were isolated by density gradient centrifugation within 24 hours.For each sample to be tested,3×106 mononuclear cells were selected for CyTOF antibody staining and detection.The remaining cells were frozen at 80 ℃ for 24 hours and then transferred to liquid nitrogen for storage;(4)Statistical analysis of all test data for subsequent experiments.Bioinformatics methods:(1)Raw data preprocessing and quality control:standardize the data,remove the noise effects such as batches,and obtain effective data of single、live immune cells for further analysis;(2)Cluster analysis:select the signal markers specified in the scheme and analyze by xshift clustering algorithm.When the number of single file cells was too large,random downsampling method was used to reduce the number of cells without adding artificial operation bias.Classification of immune cell subsetss of samples by high performance clustering algorithms.(3)Statistical analysis and visual display:visualize the results of t-SNE(t-distributed stochastic neighbor embedding)clustering,such as viSNE(visual stochastic network embedding),heatmap,etc.At the same time,the percentage of each subsets in each sample was analyzed.According to the clinical information of patients,the samples were classified and the differential expression of characteristic immune cell subsets in different groups was confirmed by bilateral t-test.Results:(1)We used mass cytometry with extensive antibody panels to perform in-depth immune profiling of peripheral blood samples from 31 patients following allo-HSCT,in which 12 patients were without cGVHD,7 patients experienced moderate cGVHD and 12 patients experienced severe cGVHD.The involved organs in patients with cGVHD were only skin(n=10),only lung(n=6),skin and lung(n=3).(2)This analysis showed a strong overlap between cGVHD of moderate and severe grades,but seperation from patients without cGVHD.(3)We found different cell subset distribution between patients with lung cGVHD and patients without cGVHD.(4)We found different cell subset distribution between patients with skin cGVHD and patients without cGVHD.(5)The percentage of myeloid-derived suppressor cells in peripheral blood mononuclear cells of patients with moderate cGVHD was higher than patients without cGVHD,the percentage of mononuclear phagocytes in peripheral blood mononuclear cells of patients with severe cGVHD was higher than patients without cGVHD.(6)The percentage of T cell subtype(C5)in peripheral blood mononuclear cells was higher in patients with lung cGVHD than patients without cGVHD or patients with skin cGVHD patients.Furthermore,the percentage of another T cell subtype(C21)in peripheral blood mononuclear cells also was higher in patients with lung cGVHD than patients with skin cGVHD.(7)The flow detection results of mouse cGVHD model showed that the severity of cGVHD was positively correlated with the proportion of CD11b+ myeloid cells in peripheral blood,which is consistent with the results obtained from CyTOF that the rate of granulocytes was higher in peripheral blood of patients with cGVHD.Conclusions:(1)Myeloid-derived suppressor cells and mononuclear phagocytes may be involved in the pathologic mechanisms of cGVHD.(2)Two T cell subtypes may be related to the pathologic mechanisms of lung cGVHD;(3)The results of mouse cGVHD model showed that CD11b+ myeloid cells may be related to the pathologic mechanisms of cGVHD.