Background:
HIV proviruses persist in a non-productive state in CD4+ cells. There is no individual cell surface biomarker that uniquely identifies infected cells. However, some cells contain proviruses that are transcriptionally active and translationally competent, leading to expression of viral proteins and T cell epitopes. We used soluble affinity-enhanced T cell receptors (TCR) to investigate the level of expression of viral peptides presented by HLA class I molecules.
Methods:
We developed T cell receptors with picomolar affinity (pM) for an HIV Gag epitope presented by HLA-A*02:01 and incorporated either a biotinylation tag or Fc fragment to enable fluorescence imaging. CD4+ T cell lines or primary cells with varying HLA expression levels were pulsed with peptide or infected with HIV and analysed for expression of viral peptide-HLA complexes by total internal reflection microscopy (TIRF; single molecule epitope counting).
Results:
HIV Gag peptide-HLA complexes could be identified on peptide pulsed cells using a labelled TCR and TIRF microscopy. The signal/noise ratio was improved using the TCR-Fc format. T2 cells pulsed with a titration of peptide were then used to quantify pHLA copies/cell and determine the dynamic range of the assay (~10-200 pHLA/cell). pHLA could also be detected on the surface of HIV-infected HLA-A*02-transduced C8166 cells, albeit at copies/cell near the lower limit of detection of the assay. Similar results were obtained with primary CD4+ T cells infected in vitro with HIV. In parallel we showed that the same TCR, when used in a bispecific format (Gag x CD3) could eliminate infected C8166 cells at picomolar drug concentrations in a T cell redirection assay.
Conclusions:
An affinity-enhanced TCR targeting a Gag peptide successfully detected pHLA complexes on the cell surface down to ~10 copies/cell and could redirect killing of infected cells when used in a bispecific format despite low pHLA expression, demonstrating its high sensitivity to antigen. Further work is ongoing to develop flow cytometry applications using labelled high affinity TCRs with the potential to isolate HIV-infected cells ex vivo for further analysis.