The Kirby Institute
Level 6 Seminar Room
Wallace Wurth Building
Sydney NSW 2052
Kirby Institute is pleased to present:
Chan Phetsouphanh - "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 are treated with cART. The characteristics of this were monitored in 20 chronically infected subjects recruited through the PrIRIS study. Changes in CD4+ T cell function were characterized in the context of increases in CD4+ T-cell counts and decreases in plasma viral load over the first year of therapy using the CD25/OX40 assay and a range of other 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 the effector memory (Tem) subset, with TT-specific responses arising from the central memory (Tcm) subset. In chronic HIV+ subjects post-therapy, the ratio of the responding memory subsets was inverted, with a greater gag response coming from the Tcm subset at week 48 compared to week 4. As activation decreased, the percentages of responding Tem cells decreased, but long-term Tcm responses were maintained.
A qualitative multiplex single-cell RT-PCR assay was developed to be used downstream of the CD25/OX40 assay. Lineage specific transcription factor profiling, allowed delineation of Th1 (Tbet), Th2 (Gata3), Th17 (Rorc), Tregs (Foxp3) and Tfh (BCL6) cells within an antigen specific population. Results demonstrated >80% of activated Ag-specific single cells expressed only 1 transcription factor. CMV-specific CD4+ T-cells displayed a dominant Th1-like response, and TT-specific cells displayed a Th2-like response. HIV-specific CD4+ T cells displayed a Th1-like response at early time points.
PKC-θ a novel protein kinase plays an important role in T cell development, proliferation, differentiation and chromatin remodelling. In Ag-specific CD4+ T-cells, PKC-θ was bound in intronic regions of genes, where it was highly associated with permissive histone marks and enhancer-like chromatin state. NF-kB (p65) was identified as having common binding sites to PKC-θ. p65 motifs were found mostly within the promoter, 5’UTR and exons of inducible genes. This result highlights the possible involvement of NF-kB and PKC-θ as potential binding partners.
In conclusion, the characterization of virus-specific CD4+ T-cells at the cellular and molecular level are likely to reveal mechanisms for the manipulation of immunological memory development and its maintenance, allowing improved design of immunotherapies and vaccines.
Chan is a PhD candidate in the IVPP program under the supervision of Prof. Tony Kelleher. His project involves the characterization of antigen specific CD4+ T-cells in HIV-1 infection.