Preliminary Study On T Cell Receptor Diversity In HIV-1 Infected Chinese With Different Disease Progression

BACKGROUND Though great achievements in HIV pathogenesis and antiretroviral therapy have been made after more than twenty-five years efforts by scientists throughout the world,the mechanism by which immune control of HIV infection can be achieved and the exact factors which effect on the disease progression are still poorly understood,These are the main obstacles in designing effective HIV-1 vaccines and the strategies of immunotherapy.Almost all people Who become infected with HIV-1 will be progressing to AIDS and death due to AIDS-associated opportunistic infection resulting from severe impairments of general immune function over time,unless they receive effective antiretroviral treatment.It is evidenced that CTLs play an important role in the control of HIV infection,but the precise mechanism by which they accomplish this protection is not clear.In recent years, most of studies relevant to HIV-1 specific T cell responses involve in T cell functions and frequencies,limited data is available on the changes of T cell receptor which has a critical role in immune system and the alteration of TCR diversity may closely correlate with immunodeficiency in HIV-1 infection.OBJECTIVES To characterize the immune status of HIV-1 infected individuals through analysis of TCRBV complementary determining region 3(CDR3) spectratype of CD4+ T lymphocytes and CDS+ T lymphocytes by performing PCR amplifications in CDR3 of 22 T cell receptor(TCR) gene families(not determined for nonfunctional Vβ10 and Vβ19) and to explore the potential relationship between the alterations of TCR spectratypes and the control of viral replication in HIV-1 infection.To investigate the difference of the alterations of TCR spectratypes between T helper lymphocytes(Th) and cytotoxic T lymphocytes(CTL) by analyzing TCR clonotypic diversity of CD4+ T lymphocytes and CD8+ T lymphocytes in HIV-1 infection.METHODS Separation of peripheral blood mononuclear cells(PMBCs) from EDTA anticoagulated whole blood was carried out by Ficoll-Hypaque density gradient centrifugation.Separation of CD8+ T cells and CD4+ T cells from PMBCs was carried out using dynal immunomagnetic beads coated with anti-CD8 antibody and anti-CD4 antibody,respectively.Total RNAs from the purified CD4+/CD8+ T lymphocytes were isolated and used to perform PCR amplifications in complementary determining region 3(CDR3) of 22 T cell receptor(TCR) gene families.PCR products were firstly analyzed by 1.5%agarose gel electrophoresis.After the electrophoresis by 6%denaturing polyacrylamide sequencing gel,the products with 6-FAM were analyzed by automatic sequencer and GeneScan software.The polymorphism and the distribution of CDR3 length of CD4+/CD8+ T lymphocytes were determined and the spectratyping and monoclonal/Oligoclonal proliferation of every singal Vβfamily were analyzed.CDR3 diversity and its association with viral load in individuals with HIV-1 infection were analyzed using SigmaPlot and SPSS software.RESULTS(1)For healthy controls,the complementary determining region 3(CDR3) of 22 T cell receptor(TCR) gene families displayed the profiles of gaussian distribution and the expressing frequency of each BV family was similar.(2)An average diversity for all CDR3 profiles in CD8+ T cells from HIV-infected individuals was significantly different as compared to healthy controls(P＜0.05) with the HIV-infected individuals losing diversity in the CDR3 profiles.There was positive correlation between alterations in TCR CDR3 diversity and viral load(R=0.640, P=0.008).The perturbation of TCR repertoires in typical progressors(TP group) is greater than that of long-term nonprogressors(LTNP group).The perturbation of TCR repertoires in individuals with viral load below 2000 copies/ml is less than that in individuals with viral load above 10000 copies/ml.The changes in CDR3 length diversity of Vβfamilies in TP group,particular in Vβ2,Vβ3,Vβ4,Vβ5,Vβ6,Vβ9, Vβ11,Vβ12,Vβ13,Vβ15,Vβ17,Vβ21,Vβ22,Vβ23,were statistically different from healthy controls;Vβ3,Vβ5,Vβ6,Vβ11,Vβ14 and Vβ23 in LTNP group were statistically different from healthy controls;Vβ2 and Vβ21 were statistically different between TP group and LTNP group.(3)An average diversity for all CDR3 profiles in CD4+ T cells from HIV-infected individuals was significantly different as compared to healthy donors(P＜0.05) with the HIV-infected individuals losing diversity in the CDR3 profiles.There was positive correlation between changes in TCR CDR3 diversity and viral load(R=0.676,P=0.003).The perturbation of TCR repertoires in TP group is greater than that in LTNP group.But there is no significant difference between healthy controls and LTNP group.The perturbation of TCR repertoires in individuals with viral load below 2000 copies/ml is less than that of individuals with viral load above 10000 copies/ml.The changes in CDR3 length diversity of Vβfamilies in TP group,particular in Vβ8,Vβ12,Vβ22,Vβ23 were statistically different from the healthy controls;Vβ12,Vβ22 and Vβ23 in LTNP group,were statistically different from the healthy controls;Vβ1,Vβ8,Vβ12 and Vβ13 were statistically different between TP group and LTNP group.(4)There was a positive correlation between the magnitude and breadth of specific CTL response and the TCR perturbation of CD8+ T lymphocytes,a negative correlation between the magnitude and breadth of specific CTL response and the TCR perturbation of CD4+ T lymphocytes.(5)The damage for the TCR clonotypic diversity of CD8+ T lymphocytes is much greater than that of CD4+ T lymphocytes.CONCLUSIONS(1)HIV-1 infection might induce the loss of TCR Vβrepertoire diversity and disrupt the CDR3 distributions within CD8+ T lymphocytes and CD4+ T lymphocytes;(2)The perturbation of TCR CDR3 clonotypic diversity was positively correlated with viral load,the better control of HIV-1 replication and disease progression,the less perturbation of TCR CDR3 clonotypic diversity;(3)The profiles of TCR CDR3 clonotypic diversity of CD4+ T lymphocytes is critical for the specific CTL response,a negative correlation between the magnitude and breadth of specific CTL response and the TCR perturbation of with CD4+ T lymphocytes was found in HIV-1 infection;(4)The perturbation of TCR CDR3 clonotypic diversity of CD8+ T lymphocytes is much greater than that of CD4+ T lymphocytes in HIV-1 infection.