Study On Immune Characteristics Of AIDS Non-progressive Rhesus Macaques

Most people develop a progressive decline in CD4+cell counts after infection with the human immunodeficiency virus(HIV)and progress to AIDS without highly active antiretroviral therapy(HAART).But a very small proportion of people,after acquiring HIV,are able to spontaneously control viral replication,maintain plasma viral load at an undetectable levels(viral load<50 copies per mL),have stable CD4+cell counts,do not progress disease,and do not develop to AIDS,and such patients are referred to as elite controllers.But the mechanisms by which such elite controllers spontaneously control virus replication are not very well defined,nor can predictions be made for virus elite controllers.Therefore,we used rhesus macaques as experimental animals to explore the immune characteristics of rhesus macaques spontaneous controllers,drawing on the reasons why human HIV elite controllers achieve spontaneous control of viral replication and providing new ideas for a functional cure of AIDS.Part 1:Reasons for achieving spontaneous virus control in SHIVSF162P3 infected rhesus macaques.The reasons why HIV elite controllers achieve spontaneous control of viral replication are highly complex and have not been fully explained.And now the vast majority of studies on elite controllers use human samples,but studies on HIV elite controllers in humans have certain limitations,such as the inability to monitor the full infection cycle,the lack of pre-infection data on immune levels,and the inability to make predictions for elite controllers.In this study,we used 11 SHIVSF162P3 infected rhesus macaques to explore the reasons why the infected macaques achieved spontaneous control of the virus through the detection of immune indicators before and after infection,transcriptome sequencing analysis.Found that PD-1,as an important immunosuppressive molecule,may be one of the reasons for the spontaneous control of the virus in rhesus macaques.Rhesus macaques in the nonprogression group had lower levels of PD-1 expression in CD8+TCM cells before infection,and low levels of PD-1 implied the activity of CD8+T cell function and enhanced the ability of CD8+T cells to kill target cells.And the PD-1 in CD4+TCM cells in non progression group decreased significantly less than that in progression group after infection.This phenomenon illustrates that following infection,immune activation of nonprogressive rhesus CD4+T cells may be more restricted,sparing more CD4+T cells from becoming infected by the virus,thereby suppressing disease progression.HLA-DR-CD38-CD4+T cells,that is,unactivated CD4+T cells,were also significantly higher in rhesus macaques from the non-progression group before infection than in those from the progression group,and were more significantly elevated later in infection.And at later stages of infection,the proportion of CD4+T and CD8+T cells that were HLA-DR-CD38+in the non-progression group decreased significantly.This change,and the elevated proportion of HLA-DR-CD38-T cells,allowed non-progression group rhesus macaques to avoid organismal damage from extensive immune activation.Transcriptome sequencing found that pro-inflammatory genes were mainly upregulated on both PBMCs and CD4+T cells in the progression group rhesus macaques,and a large number of inflammatory and immune molecules generated by the activation of the immune system tended to cause body damage,so that a high level of immunity during chronic infection tended to suggest a poor prognosis of viral infection.Whereas nonprogression group rhesus PBMCs and CD4+T cells upregulated mainly proinflammatory genes,genes involved in specific T cell responses,and genes inhibiting apoptosis.It can be seen that a higher level of specific T-cell response mainly existed in the non-progression group rhesus macaques,which inhibited the extensive immune activation and maintained the stable level of immunity in vivo.Taken together,this study suggests a certain correlation between PD-1 levels in CD8+TCM cells before infection and disease progression at the level of unactivated CD4+T cells,and low levels of immune activation may help achieve spontaneous control of the virus.Part 2:Effects of storage conditions on the activity of CD8+T cells in rhesus macaques.CD8+T cells play a very important role in the immune control of HIV infection,and it has been suggested that spontaneous control of virus in elite controllers(EC)may be mediated by HIV specific CD8+T cells.Therefore,the measurement of the intensity of the CD8+T cell response is of great importance in the study of HIV elite controllers,but because of the limited experimental conditions,usually the cells and tissues are often kept frozen(-80℃,liquid nitrogen,etc.),and the cryopreservation will affect the partial physicochemical state of the cells,but to what extent the cell activity is affected and the experimental basis is lacking.In this study,we use Intracellular cytokine staining(ICS)to detect the intensity of CD8+T cell response and explore the effects of different storage conditions on CD8+T cell activity.We collects Rhesus macaque’s peripheral blood,and separate peripheral blood mononuclear cells(PBMC),a part of fresh cells are directly used for detection,and the remaining cells are frozen at-80℃ and liquid nitrogen,and used for detection one week and one year later.In the detection,a positive stimulus is used to stimulate,flow cytometry is used to analyze the proportion of living cells,the secretion of MIP-1β,TNFα,IL-2,IFN-γ,and the expression of cell degranulation marker CD107a.We found that as the freezing time increases,the cell survival rate decreases,and liquid nitrogen freezing has a higher survival rate than freezing at-80℃.In the case of cytokine secretion and the expression of cell degranulation marker CD 107a,the secretion of MIP1β and TNF-α of cells cryopreserved for one year was significantly higher than that of normal cells,and the cell status changed.The status of cells frozen for a week are more similar to fresh cells.Therefore,in order to maintain low cell mortality and normal CD8+T cell response,fresh cells or short-term frozen cells should be used for experiments to measure the level of CD8+T cell response.