Expression Level And Clinical Significance Of PD-1 And Tim-3 In Human Immunodeficiency Virus-1 Infected Individuals

BACKGROUND A variety of studies have demonstrated that human leukocyte antigen(HLA)-mediated cytotoxic T-lymphocyte (CTL) responses play a pivotal role in the control of HIV replicatin as well as the disease progression. Though the close association of HIV-specific cellular immune response with viral replication and disease progression has been proved in HIV-1 natural infection, yet, most cases are incapable of long-term control of the virus replication and the majority of untreated-infectors develop AIDS in a period of 8-10 years without antiretroviral treatment. Under chronic antigen stimulation, HIV-specific CD4+T cells and CD8+T cells become dysfunctional (termed"exhaustion") and the overall impairments in proliferative capacity and cytokine production hasten disease progression. Likewise, the emergence of T cell deterioration invariably restrains attempts to boost the immune response using vaccines or other interventions. At present the exact mechanism by which cellular immune function occur exhaustion remains to be unclear, recently studies have shown that the inhibitory receptors programmed death 1(PD-1) and T-cell immunoglobulin domain and mucin domain 3(Tim-3) have been associated with exhaustion of CD8+ T cell response in various chronic viral infections, the activation of these coinhibitory moleculars may be one of the reasons which stir up cellular immune deterioration.OBJECTIVES To characterize the profiles of PD-1 and Tim-3 expression levels on T lymphocytes in human immunodeficiency virus-1 infected individuals with different clinical stages by flow cytometry and to explore the relationship between the PD-1 and Tim-3 expression levels and CD4+T cell counts as well as plasma HIV-1 viral loads. To investigate the potential role of PD-1 and Tim-3 on immune regulation during HIV infection through assessment for HIV-1-specific CTL response with multicolor intracellular cytokine staining(ICS) assay by using overlapping peptides(OLPs) covering the whole consensus clade B proteome, and the blocking effect by administration of specific antibodies to interdict PD-1/PD-L1 and Galectin-9/Tim-3 signaling pathways.METHODS Separation of peripheral blood mononuclear cells(PBMCs) from EDTA anticoagulated whole blood was carried out by Ficoll-Hypaque density gradient centifugation. Fresh PBMC from HIV-1-infected individuals and healthy donors were stained with fluorophore-conjugated monoclonal antibodies to CD3, CD4, CD8, PD-1 and Tim-3 to detect PD-1 and Tim-3 phenotype expression. Isotype control mAb was used to remove non-specific binding. Cellular immune function was measured by using ICS for 42 chronic infectors. Blocking effects were evaluated in 10 random-selected HIV-1 infectors. A total of>150,000 events were collected and analyzed with FlowJo software. SigmaPlot 10.0 and GraphPad Prism 5 were used to draw plots and to perform statistical analysis. The statistical differences between two groups were analyzed using nonparametric Mann-Whitney U test or student's t-test. Correlation analysis was calculated using Pearson correlation or Spearman rank correlation test. Comparing the levels of cytokines stimulated with HIV-1 genome between baseline and blockage were used with paired t-test or Wilcoxon matched pairs test. All tests were two-tailed and P value<0.05 was considered as significance.RESULTS (1) The mean frequencies of PD-1 and Tim-3 expression on T cells of healthy controls are as following:16.240% for PD-1 expressing CD4+T cells(PD-1/CD4),18.120% for Tim-3 expressing CD4+T cells(Tim-3/CD4),18.450% for PD-lexpressing CD8+T cells(PD-1/CD8),23.260% for Tim-3 expressing CD8+T cells(Tim-3/CD8), which are significantly lower than those of HIV-1 infected individuals (PD-1/CD4:20.240%; Tim-3/CD4:22.050%; PD-1/CD8:30.395%; Tim-3/CD8:25.370%). (2) The frequencies of PD-1+CD4+T cells correlated negatively with absolute CD4 counts(r=-0.27, P=0.0222), the frequencies of PD-1+CD8+T cells correlated positively with viral loads(r=0.247, P=0.0368) and negatively with absolute CD4 counts(r=-0.401, P<0.001) in 72 HIV-1 antiretroviral-naive infectors. Meanwhile, In 60 chronic HIV-1 infectors, the frequencies of Tim-3+CD8+T cells correlated positively with HIV-1 viral load (r=0.271, P=0.0364) and inversely with absolute CD4+T cell counts (r=-0.338, P=0.00851). Similarly, the frequencies of Tim-3+CD4+T cells were significantly correlated absolute CD4+T cell counts (r=-0.256, P=0.0482). (3) The HAART-untreated patients were further subdivided into four groups on the basis of CD4 count. The split points were 200,350 and 500 cells/μl. Along with the increase of CD4 groups, the expression levels of PD-1 and Tim-3 on CD4+T cells continually decreased(PD-1:29.90%,19.37%,18.76%,16.76%; Tim-3:31.40%,25.16%,22.14%,21.21%), the same trend of PD-1 and Tim-3 expression on CD8+T cells with CD4 groups was also found (PD-1:37.60%,32.40%,30.96%,20.92%; Tim-3: 33.55%,25.37%,22.90%,21.93%). (4) Based on CD4 absolute counts,HIV-1 viral loads and infection time, we owe these infectors to four disease progressive groups. We observed that elevated frequencies of PD-1 and Tim-3 expressed on CD4+ and CD8+T cells in primary infected-subjects and AIDS patients, but not in long-term nonprogressors. Chronic progressors possess high levels of PD-1 and Tim-3 on their T cells compared to healthy donors, yet some of them have no significant differences. (5) Because PD-1 and Tim-3 have been identified as markers of exhausted T cells in HIV-1 infection, we determined whether PD-1 and Tim-3 expression defines the same of a distinct population. Expression was analyzed by flow cytometry after gating on CD4+ or CD8+T cells. HIV-1 infectors displayed frequent PD-1+Tim-3- population (17.25%),PD-1-Tim-3+(20.25%) populations but retained 6.75% of PD-1+Tim-3+ population on CD4+T cells. The similar proportions were also found on CD8+T cells. (6) PBMC were stimulated with overlapping peptides(OLPs) covering the whole consensus clade B proteome. HIV-1 specific T cells(CD107a+ or IFN-y+) possess higher mean fluorescence intensity of PD-1 and Tim-3, especially of PD-1. (7) We detected CD107a and IFN-y production by CD4+ and CD8+ T cells stimulated with SEB and assessed the proportion of the cells that were able to produce any one of the two cytokines using flow cytometry, By and large, the results of experimentation showed the frequency of CD4+PD-1+T cells and CD4+Tim-3+T cells correlated negatively with cytokine production. (8) Interference on PD-1/PD-L1 and Galectin-9/Tim-3 pathways using administration of purified anti-PD-L1 and anti-Tim-3 partially restored the ability of PD-1 and Tim-3 cells to produce cytokines in vitro.CONCLUSIONS (1) Compared to healthy controls, the PD-1 and Tim-3 expression levels on CD4+/CD8+T lymphocytes were significantiy increased(P<0.05) in HIV-1 infected individuals; (2) The frequencies of PD-1+CD4+and PD-1+CD8+T cells correlated positively with HIV-1 viral load and negatively with absolute CD4+T cell counts, whereas, this correlation concerning to Tim-3 expression levels was only found on chronic infected individuals. Collectively, these data suggest that the expression of PD-1 and Tim-3 associated with disease progression and they could be be new independent markers for assessing disease progression; (3) PD-1 and Tim-3 expression on CD4+/CD8+T cells correlate with clinical status of progression in HIV-1 infected-individuals. Based on infection time,CD4 absolute counts and viral levels, we owe these infected individuals to four disease progressive levels:primary infectors,long-term nonprogressors,chronic HIV progressors and AIDS patients. We observed two dramatic increases of PD-1 and Tim-3 expression in early/acute infectors and AIDS patients, but the long-term nonprogressors are nearly the same, relative to uninfected individuals; (4) HIV-1 specific T cells, also interpreted as CD107a+ or IFN-y+T cells here, enhancely express PD-1 and Tim-3; (5) Expression of PD-1 and Tim-3 on CD4+T cells influences the abiliy of T cells to produce cytokines, and PD-1/PD-L1 and Galectin-9/Tim-3 manipulations partially restored virus-specific PD-1+/Tim-3+T cells to produce cytokines.