Using HIV-1 Antigen Peptide-specific And HLA-class â… -restricted Tetramers To Characterize CTLs In HIV-1-infected Individuals

Objective Our study attempts to quantitate the frequency and characterize theproperty of multiple viral epitopes-specific cytotoxic T-lymphocytes (CTL) inperipheral blood of individuals with HIV-1 infection for evaluation of their clinicalimplications in antiviral effects and their association with disease progression. MethodWe synthesized the HLA-A*0201 tetramer loaded with HIV-1 Pol 294 - 302YTAFTIPSI peptide by using the molecular cloning and protein purification methods.Other two HLA-A*0201 tetramers complexed to Gag 77-85 SLYNTVATL, Pol 476-484 ILKEPVHGV were purchased from Prolmmune. All three HLA class I and HIV-1antigen epitope-specific Tetramers were used to characterize circulating specific CD8+Tcells in 14 HLA-A*02-positive HIV-1 infected subjects who were screened from 46individuals with HIV-1 infection using flow cytometry analyses.ELISPOT andintracellular cytokine staining (ICCS) assays were simultaneously employed toevaluate IFN-y-producing CTLs of PBMC from HIV-1 carriers through being pulsedwith SL9 peptide in vitro incubation. Result Circulating CD8+T cells specificallystained with aforementioned tetramers are able to be positively identified in 11 of 14HIV-1-infected individuals for HIV-1 Gag SL9 epitope tetramer, in 10 of 14 cases forHIV-1 Pol IV9 epitope tetramer, and in 5 of 7 for HIV-1 Pol YI9 epitope Tetrameramong all the HLA-A*02-positive subjects. The relative percentages of the three HIV-1Gag SL9-, Pol IV9- and Pol YI9-peptide specific Tetramers were 0.06%-3.27%,0.05%-0.57% and 0.09%-0.66%, respectively. There was a correlation found betweenthe frequency of HIV-1 Gag SL9-specific Tetramer and result of the ELISPOT assaywhich was performed after stimulation of PBMC with the Gag SL9 epitope for thestudied cases (r=0.8455, P<0.01). High frequencies of circulating HIV-specific CD8+ Tcells were observed to occur in some patients with a broad spectrum of plasma viralload. Furthermore, ELISPOT and ICCS analyses showed that the function in more than95% HIV-1-specific CTLs was probably impaired. Conclusion Our study suggests thatHIV-1 antigen epitope-specific Tetramer staining is a sensitive and productive tool toquantitatively analyze epitope-specific CTLs in individuals with HIV-1 infection. In addition, both ELISPOT and ICCS assays are also alterative approaches to evaluate the property of HIV-1 epitope-specific CTLs in peripheral blood of HIV-1-infected individuals. Combinational use of specific Tetramer staining, ELISPOT and ICCS assays can be more accurately evaluate the quantity and function of epitope-specific CTLs against HIV-1, which may help us to get more insight into the understanding of antiviral cellular immune responses and the underlying mechanism of pathogenesis of chronic HIV-1 infection in humans.