Identification Of The PBMC Lineage In HIV-1 Infected Long-Term Non-Progressive Using Single-Cell RNA Sequencing

ObjectiveLong term non-progressors(LTNPs)are a special group of HIV infection.It could maintain a CD4~+T cell counts at normal level and had undetectable plasma HIV RNA load over 10 years.Although previous studies have identified that the genetic background of the body,the immune response of the immune cells to HIV and the different HIV-1 strains can affect the progress of the disease,the current level of innate immune activation in LTNPs population has not been adressed.In this study,we perform single-cell transcriptome sequencing on long-term non-progressive population,typical progression population and healthy population.Through single-cell transcriptome sequencing,construct a cell map of peripheral blood mononuclear cells of typical progressors,long-term non-progressors and healthy people,group at the genetic level,and analyze whether there is a cell subgroup related to immune inflammation regulation.Analyze the gene expression differences of PBMC cell subsets and the involved signaling pathways in the three groups,and explore the potential role of PBMC cell subsets in the long-term progression of HIV-1infection.MethodsPBMCs were collected from 4 HIV-1 long-term non-progressors,4 HIV-1typical progressors and 4 healthy controls.single-cell transcriptome sequencing of PBMCs was performed by 10X Genomics chromium system.Strict multi-dimensional quality control,cell clustering and Identification the PBMC cell subsets.Screen up-regulated genes of each subgroup of PBMC to obtain specific expressed genes.The gene expression differences in the PBMC cell subsets of the three groups were analyzed,and the differential genes of CD4~+T cells and CD8~+T cells were subjected by GSEA enrichment analysis.In addition,the pseudotime analysis of cell trajectories was performed on CD4~+T cells and CD8~+T cells to analyze the differences in the differentiation status and related genes of CD4~+T cells and CD8~+T cells in the three groups.Results1.Identification of PBMC cell subpopulations:Cell cluster analysis divided25 clusters.According to the expression of PBMC marker genes,a total of 8 types of cells were defined.The average proportion of cells in each subpopulation of LTNPs was:CD4~+T cells(25.88%),CD8~+T cells(36.95%),B cells(9.12%),CD4~-CD8~-T cells(1.71%),dendritic cells(0.39%),macrophages/monocytes(15.31%),natural killer cells(2.09%)and natural killer T cells(8.54%).2.Differences in the proportion of PBMC cell subsets and gene expression among the three groups:Only differences in the proportion of CD4~+T cells in the three groups of people were statistically significant.The proportion of CD4~+T cells in TPs was lower than LTNPs and HCs(F=13.656,P=0.002).The differential gene analysis of the three groups showed that the number of differential genes of TPs and LTNPs in PBMC cell subsets was greater than the number of differential genes of HCs and TPs,HCS and LTNPs.The expression trends of PF4,GNG11 and TUBB1 in LTNPs in different cell subgroups are consistent,and the expression level is lower than the TPs.3.CD4~+T cell gene-set enrichment analysis:Compared with HCs,TPs chemokine signaling pathway and NK cell-mediated cytotoxicity levels are up-regulated;compared with TPs,LTNPs chemokine signaling pathway and NK cell-mediated cytotoxicity levels Down-regulation,including chemokines CCL4,CCL5,chemokine receptors CXCR1,CXCR2,CCR5 and cytotoxicity related genes GZMB,KLRD1,SOS1.4.CD8~+T cell gene-set enrichment analysis:Compared with HCs,TPs NK cell-mediated cytotoxicity levels were up-regulated;compared with TPs,LTNPs NK cell-mediated cytotoxicity levels were down-regulated.Compared with HC and LTNP,natural killer cell-mediated cytotoxicity in TPs is also up-regulated.5.Pseudotime analysis of CD4~+T cells:The transcription factor GATA3 is high levels expression in all states.ZBTB7B(th POK)and TBX21(T-bet)are in the middle abundance.RORC(RORγt)has low expression in all states.Compared with TPs,the proportion of LTNPs in state 2 is higher(F=4.670,P=0.041).In state 2,LTNPs express high levels of SOCS3 and low levels express GNLY and CCL5.6.Pseudotime analysis of CD8~+T cells:Transcription factor TBX21(T-bet),effector marker genes GZMB,KLRG1 and CD160,LAG3 are mainly expressed in state 2 and state 3.Compared with TPs,the proportion of LTNP in state 2 is higher(F=5.410,P=0.029).In state 2,TPs expressed CXCR3,GNG11,PF4and TUBB1 at low levels.Conclusion1.The proportion of CD4~+T cells of the Long term non-progressors in peripheral blood was higher than that in the TPs.The differential gene analysis of the three groups showed that the number of differential genes of TPs and LTNPs in PBMC cell subsets was greater than the number of differential genes of HCs and TPs,HCs and LTNPs.2.In the population of HIV-1 that has not progressed for a long time,it was found that the expression of chemokine signaling pathway in CD4~+T cells and cytotoxic signaling pathway mediated by NK cells and cytotoxic signaling mediated by CD8~+T cells may be regulated by these two signaling pathways.The immune response of T cells to HIV-1 virus plays a role in the long-term non-progression.It is found that CD4~+T and CD8~+T cells each have a characteristic cell differentiation cluster of LTNPs.