CD4 T cell HIV infection and CTL response

HLA-B57+ primary CD4 T cells were either treated with LRA, stimulated with anti-CD3/CD28 or kept in culture with no stimulation for 48 hours. The cells were thoroughly washed and magnetic anti-CD3/CD28 beads were removed. For each condition, 2 million of CD4 T cells were infected with 20 ug HIV Gag p24 equivalent of NL4-3-Δenv-GFP virus pseudotyped with Vesicular Stomatitis Virus glycoprotein (VSV-g) in the presence of 5ug/mL polybrene (Sigma) by spinfection for 1.5 h at 2000x g as in [37 (link)]. At 24, 48 and 72 h post-infection, CD4 T cells were plated with epitope-specific CD8 T cells at a ratio of 1:4 (CD4:CD8) and an aliquot of CD4 T cells was also harvested to measure HIV-1 infection through GFP and HIV-1 p24 intracellular expression, as well as cellular activation and HLA class I surface expression by flow cytometry. After 30 min of co-culture, CD107a-Pe-Cy7 (BD Biosciences) and CD28/CD49d antibodies (BD Biosciences) were added to each well to measure CTL degranulation. After 6 h cells were harvested for surface staining.
The avidity of the CTL clones was assessed by performing a concentration-course titration. Target HLA-B57 CD4 T cells were incubated with increasing concentration of the cognate peptides (0.000002–2 ug/mL) before co-culture with the corresponding CD8 T cells as described above.