Novel approaches to the characterization of antigen-specific CD4+ T cells in HIV-1 infection

Immune reconstitution is typical in chronic HIV-1 infected individuals who aretreated with combination anti-retroviral therapy (cART). The characteristics ofthis were monitored in 20 chronically infected subjects recruited through thePrIRIS study. Changes in CD4+ T cell function were characterized in the context ofincreases in CD4+ T-cell counts and decreases in plasma viral load over the firstyear of therapy using the CD25/OX40 assay and a range of phenotypic assays.The CD25/OX40 assay was used to identify antigen-specific CD4+ T-cells in vitro.In healthy individuals the majority of CMV-specific responses were from theeffector memory (Tem) subset, with TT-specific responses arising from the centralmemory (Tcm) subset. In chronic HIV+ subjects post-therapy, the ratio of theresponding memory subsets was inverted, with a greater gag response comingfrom the Tcm subset at week 48 compared to week 4. This result was concordantwith reduced antigen load. As activation decreased, the percentage of respondingTem cells decreased, but long-term Tcm responses were maintained.A qualitative multiplex single-cell RT-PCR assay was developed to be useddownstream of the CD25/OX40 assay. This assay has high specificity and a verylow cut-off of detection (down to 10 femtograms). Lineage specific transcriptionfactor profiling, allowed delineation of Th1 (Tbet), Th2 (Gata3), Th17 (Rorc), Tregs(Foxp3) and Tfh (BCL6) cells within an antigen specific population. Resultsdemonstrated >80% of activated Ag-specific single cells expressed only 1transcription factor. CMV-specific CD4+ T-cells displayed a dominant Th1-likeresponse, while TT-specific cells displayed a Th2-like response, additionally asmall percentage of polyfunctional CMV and TT-specific cells expressed ≥2transcription factors.PKC-θ a novel protein kinase plays an important role in T cell development,proliferation, differentiation and chromatin remodelling. In Ag-specific CD4+ Tcells,PKC-θ was bound in intronic regions of genes, where it was highly associatedwith permissive histone marks and enhancer-like chromatin state. NF-κB (p65)was identified as having common binding sites to PKC-θ. p65 motifs were foundmostly within the promoter, 5UTR and exons of inducible genes. This resulthighlights the possible involvement of NF-κB and PKC-θ as potential bindingpartners.In conclusion, antigen-specific CD4+ T-cells are very important for controllingsecondary infections and maintenance of immunological control. Thecharacterization of virus-specific CD4+ T-cells at the cellular and molecular levelare likely to reveal mechanisms for the manipulation of immunological memorydevelopment and its maintenance, allowing improved design of immunotherapiesand vaccines