Study On Virtual Memory CD8~+ T Cells In Shrinking HIV-1 Viral Reservoir: Effector Mechanisms And Influential Factors

BackgroundHuman immunodeficiency virus（HIV）infection is a worldwide epidemic.There were approximately 37.7 million people living with HIV infection,and 680 thousand people dying from HIV-related illness.Around the world,HIV could be divided into two major strains,HIV-1,and HIV-2.HIV-1 is the most widespread HIV strain.Anti-retroviral therapy（ART）could efficiently suppress HIV-1 replication and decrease the incidence of acquired immune deficiency syndrome（AIDS）,but could not eliminate HIV-1 reservoir,which has been integrated into host genome.Thus,lifelong ART is needed.Eradicating HIV-1 reservoir is necessary to achieve HIV-1 cure,but is too difficult for individuals with chronic HIV-1infection.Functional cure of HIV-1 is defined as suppressing of HIV-1 replication,maintaining HIV-1 viral load undetectable,and avoiding the disease progression（characterized with decrease of CD4 T cell count and immune dysfunction）following ART interruption.To achieve functional cure and ART-free remission,it is necessary to shrink HIV-1 reservoir.CD8⁺T cells are crucial in controlling HIV-1 viremia and limiting HIV-1 reservoir.However,owing to the heterogeneous nature of CD8⁺T cells,the cellular and molecular mechanisms underlying CD8⁺T cell-mediated HIV-1 suppression have not been fully clarified.Virtual memory CD8⁺T cell(T_VM)is a unique subpopulation of CD8⁺T cell with expression of multiple natural killing cell markers,such as killer Ig-like receptors（KIRs）and NKG2A.In mice,its phenotype is CD44^hi CD49d^lo CD8⁺T cells.In human,its phenotype is CD45RA⁺pan-KIR and/or NKG2A⁺CD8⁺T cells.It has been reported that T_VMcells could be expanded by interleukin-15（IL-15）and interferon alpha（IFNα）in both human and mice.Furthermore,IL-4 upregulation during helminth infection also causes T_VMcell expansion in mice.However,whether IL-4-mediated T_VMcell expansion exists in human is still unclear.Our previous study found that T_VMcells could sense HIV-1-infected cells independently on T cell receptor（TCR）signalling but dependently on KIR signalling.However,the exact molecular mechanisms underlying T_VM-mediated viral suppression,and influential factors regulating T_VMcell expansion in human have not yet been fully clarified.ObjectivesTo explore the cellular and molecular mechanisms underlying CD8⁺T cell-mediated viral suppression in HIV-1-infected individuals undergoing antiretroviral therapy（ART）,to explore the molecular mechanisms underlying T_VMcell-mediated viral suppression,and to clarify whether T_VMcells could be expanded by IL-4 during helminth infection.MethodsWe enrolled 60 HIV-1-infected individuals on ART for a median of 6.6 years,sorted peripheral blood mononuclear cells（PBMCs）,detected HIV-1 deoxyribonucleic acid（HIV-1DNA）and HIV-1 cell-associated unspliced RNA（HIV-1 CA us RNA）by polymerase chain reaction（PCR）,examined CD8⁺T cell subset fractions and effector molecule expression by flowcytometry,including cytotoxic molecules,cytokines,and beta-chemokines.Correlations of CD8⁺T cell subset fractions and effector molecule expression with HIV-1 DNA and HIV-1CA us RNA levels were assessed by nonparametric correlation analysis.The functional mechanisms underlying the antiviral effects of CD8⁺T and T_VM cells were confirmed using viral killing assays.According to clonorchis sinensis-specific Ig G examination,the enrolled 60 HIV-1-infected individuals were further divided into clonorchis sinensis co-infection group without anthelmintic treatment（Ig G+）and clonorchis sinensis uninfection group（Ig G–）.We assessed HIV-1 DNA and CA us RNA by PCR;detected serum IL-4 levels by Ella;examined T_VMcell count and percentage,Th1/2/17 fractions in CD4⁺T cells,and phosphorylated signal transducer and activator of transcription 6（p-STAT6）expression in T_VMcells following IL-4stimulation by flowcytometry.The comparisons of HIV-1 DNA and CA us RNA levels,T_VMcell count and percentage were performed between Ig G–and Ig G+groups.Determinants of T_VMcell count and percentage were analyzed by nonparametric correlation analysis and univariate/stepwise linear regressions.Results（1）CD8⁺T_CM ratio positively correlates with HIV-1 DNA and CA us RNA levels,whereas CD8⁺T_EMRA ratio negatively correlates with HIV-1 DNA and CA us RNA levels in ART individuals.（2）Fractions of C-C motif ligand 5（CCL5）and granzyme B（GZMB）-producing CD8⁺T cells negatively correlate with HIV-1 DNA and CA us RNA levels.In viral killing assays,CD8⁺T cells exert HIV-1 suppressive effects,which could by reversed by CCL5 blockade and GZMB inhibitor.（3）Fraction of CCL4⁺CCL5^–CD8⁺T cells positively correlates with HIV-1 DNA and CA us RNA levels,whereas fraction of CCL4^–CCL5⁺CD8⁺T cells negatively correlates with HIV-1 DNA and CA us RNA levels.（4）CCL4⁺CCL5^–CD8⁺cells are mainly distributed in CD8⁺T_CM cells,whereas CCL4^–CCL5⁺CD8⁺cells are mainly distributed in CD8⁺T_EMRAcells.CCL4⁺CCL5^–CD8⁺T_CM ratio positively correlates with HIV-1 DNA and CA us RNA levels,whereas CCL4^–CCL5⁺CD8⁺T_EMRA ratio negatively correlates with HIV-1 DNA and CA us RNA levels.（5）T_VMcells are enriched in CCL4^–CCL5⁺CD8⁺T_EMRA cells.Percentage of T_VM⁺ T_EMRAin CD8⁺T cells negatively correlates with HIV-1 DNA and CA us RNA,whereas that of non-T_VM T_EMRA doesn’t significantly correlate with HIV-1 DNA and CA us RNA.（6）T_VMcells are characterized with robust expression of functional molecules at transcriptional and protein levels,such as CCL5 and GZMB.T_VMcell count and percentage are negatively correlated with HIV-1 DNA and CA us RNA levels.In viral killing assays,T_VMcells inhibit HIV-1 more effectively than the non-T_VMCD8⁺cells.（7）Fractions of CCL5 and GZMB-producing T_VMcells negatively correlate with HIV-1DNA and CA us RNA levels.In viral killing assays,the HIV-1 suppressive effects of T_VMcells can be reversed by CCL5 blockade and GZMB inhibitor.（8）Compared to clonorchis sinensis Ig G–gourp,HIV-1 CA us RNA level,serum IL-15and CCL5 concentrations are increased in Ig G+group,but the HIV-1 DNA level does not differ between the two group.（9）Between Ig G–and Ig G+groups,comparable levels of T_VMcell count,percentage and functional molecule expression are found.No significant correlation between serum IL-4 or Th2 fraction with T_VMcell count and percentage is observed.Expression of p-STAT6 in T_VMcells following IL-4 stimulation significantly negatively correlates with T_VMcell count and percentage,whereas Th1 fraction significantly positively correlates with T_VMcell count and percentage.ConclusionsOur data indicate that,in ART-treated and virologically suppressed HIV-1-infected individuals,CD8⁺T and T_VMcells limit the size of the HIV-1 reservoir though granzyme B and CCL5 secretion.Expanding these cells might represent a promising strategy for shrinking HIV-1 reservoir and achieving functional cure of HIV-1 infection.In clonorchis sinensis Ig G+and HIV-1 co-infected individuals,T_VMcell count and percentage are not associated with Th2fraction and serum IL-4 concentration.The mechanisms regulating T_VM cell expansion in human might be different from those in mice,and need further exploration.Furthermore,Th1fraction positively correlates with T_VMcell count and percentage,indicating they might be regulated by the same upstream signaling.