Study On The Relationship Between TCR Vα24+Vβ11+NKT Cells And The Progression Of HIV-1 Infection In Chinese Population

PrefaceAIDS(acquired immunodeficiency syndrome,AIDS)is a immune defect disease caused by HIV(human immunodeficiency virus,HIV)which still can not be completely cured and make breakthrough in the vaccine research right now.The basic reason lies in the lack of essential understanding of the HIV biology and the mechanism of human organism immune reactions and the interactions between them,especially the key regulator in the immune system is still barely known.In immune system,the NKT cell(nature killer T cell)express extremely restricted TCR repertoire,in human consisting of a Vα24 chain preferentially paired with a Vβ11 chain,which could activate T cells,B cells and NK cell,which is the bridge between innate and adaptive immune system,which is one of the key regular cells.Thus,though NKT cells are present at very small numbers in peripheral blood(between 0.01 and 1%of peripheral blood mononuclear cells),which play important roles in anti-virus,bacteria,fungus, parasite,tumor,and autoimmune diseases.Some reports suggest that due to the relative high level of CCR5 to the regular T cells,the perpetual activated state of NKT cells are more effective targets for R5-tropic HIV-1 infection in vivo.Co-administration ofα-GalCer with suboptimal doses of DNA vaccines greatly enhanced antigen-specific T cell and B cell responses.In all,NKT cells may play a role in the development of HIV infection disease progression.Due to the different heredity background between Chinese and the Westerner,the number of all kinds of cell and molecules nay be highly variable.In HIV infected Chinese,there has no study on the frequency of NKT cells according to disease progression.In the present paper,we studied the frequency and CCR5 level of TCR Vα24+Vβ11+NKT cells and their relationship to the disease progression of HIV-1 infected patients in China. Material and Methods1.Study objectsA total of 53 treatment naive HIV-infected patients from Henan provinces of China were enrolled(26 males,26 females;median age 40 years,range 30-68 years). HIV-infected subjects were classified into 3 stages.The first stage was long-term slow progressors(SPs)and included subjects with persistent CD4+ T-cell counts more than 500 cell/μl and no clinical sign of disease for at least 10 years.The second stage was chronical HIV-infected subjects and included patients who had CD4+ T-cell counts between 200-500 cell/μl,and no AIDS-defining condition(HIV group).The third stage was AIDS and consisted of patients with an AIDS-defining condition according to the World Health Organization classification,including CD4+ T-cells less than 200 cells/μl, presence or previous opportunistic infections or HIV-related neoplasms.There were 12 SPs,28 HIV and 13 AIDS in the HIV infected patients.Healthy control(NC)was randomly enrolled according to HIV negative,never exposed to HIV,and the age and gender are comparable with the HIV-infected,and there were 10 males and 9 females. Blood was drawn by venipuncture from each subject in EDTA tubes(Becton Dickinson) for FACS analysis and the detection of viral load.2.The absolute counts of T and NK cellThe 20μl TriTEST reagents CD4/CD8/CD3 or CD3/CD16CD56/CD45 were added to the CD4 absolute vials and 50μl whole blood was added then.After 15-min incubation at dark at room temperature,the erythrocytes were lysed by using FACS lysing solution.After 15-min incubation at room temperature again,the vials were taken to perform FACS analysis by using Multiset software.The absolute count and the percentage of CD4+,CD8+ and CD3+ T cells and NK cells were calculated automatically.3.Determination of the NKT cell and its CCR5 levelPBMCs were obtained from HIV-1 infected individuals and healthy controls by Ficoll-Hypaque density gradient centrifugation.Kits from Coulter and BD Pharmingen are used to defied Vα24+Vβ11+NKT cells.Briefly,100μL of prepared cells(1x106) were first stained with anti-Vα24-FITC,anti-Vβ11-PE(Coulter),anti-CD3-PerCP,anti-CCR5-APC,anti-CD4-PE-Cy7(BD Pharmingen),for 30 min,room temperature. Then the cells were washed with PBS and fixed in 1%polyformaldehyde.Cells were analyzed on a FACSAria(Becton Dickinson)with FACSDiva software.T lymphocytes were identified by gating on CD3+T cells and side scatter,then Vα24+Vβ11+NKT cells were gated and the percentage of CD4 and CCR5 expression were determined.4.Statistical analysisSAS9.0 software was used to assay the data.The frequencies of NKT subsets and its CCR5 levels were calculated by the Nemenyi test.The correlations were assessed by Spearman test.P values＜0.05 was considered significant.Results1.Frequency of NKT cell and its subsetsWe found that the NKT cell count of HIV infected patients is significantly lower than that of healthy controls,and the mean values continuously increase in AIDS, HIV,SP,and NC group,but only AIDS group is significantly lower than NC group (P＜0.05),other groups have no obviously differences.The CD4+NKT cell count of HIV infected patients is notably lower than NC,but only AIDS group is significantly lower than SP,HIV and NC group,there is no obviously difference in the later three groups.The trend of CD4-NKT cell is similar with total NKT cell,which only AIDS group is significantly lower than NC group.Among groups,there is no obviously difference in the NKT cell percentage of total CD3+T cell.2.The level of CCR5 on NKT cell and its subsetsOf notes,the percentage of CD4-NKT subset in CCR5+NKT cells is significantly higher than CD4+NKT subset in HIV infected patients.The percentages of the CD4+CCR5+NKT cell in HIV and AIDS groups were.significantly deceased, compared to the NC group(HIV group:1.23±2.34%;AIDS group:0.61±1.23%;NC group:1.64±2.22%).9.57±12.97%of NKT cells express CCR5 in HIV infected,which is not significantly different from NC(5.64±5.54%).4.42±7.16%of CD4+NKT cells in HIV infected patients express CCR5,which is also not notably different from NC (3.59±4.41%).3.The relationship between NKT subsets and the CD4+T cells, CD8+T cells,the ratio of CD4/CD8,and NK cells. We examined the relationship between NKT subsets and some important parameters.The frequency of NKT cells in HIV infected patients is closely related to CD4+T cells(r=0.33,P＜0.05).The frequency of CD4+NKT cells in HIV infected patients is closely related to CD4+T cells(r=0.51,P＜0.001).There is no notably relationship between CD4-NKT cells and CD4+T cells in HIV infected and NC.The CD4+NKT cells is obviously related with CD4-NKT cells in HIV infected patients (r=0.59,P＜0.001).There were no relationships between NKT cells and CD8+T cells, NK cells.Conclusion1.The frequencies of NKT subsets decreased significantly at the end stage of HIV disease progression,which has obviously relationship with HIV disease progression.2.The high level of NKT cell in SP patients may be the reason of their slow disease progression.