Anti hla e

Cell proliferation was assessed using the carboxyfluoresceindiacetatesuccinimidyl ester (CFSE) dilution assay. Briefly, CD4⁺ T cells were stained with CFSE (Invitrogen, 1 µg/mL, 15 min at 37 °C), washed and cultured in RPMI medium supplemented with fetal bovine serum at 37 °C and 5% CO₂. Subsequently, T cells were seeded in 96 flat-bottom well plates (Costar Corning, 100,000/well) and stimulated with anti-CD3/anti-CD28 mAb coated beads (T cell activation/expansion kit, Miltenyi Biotec), in the presence or absence of hAEC (at hAEC:effector cell ratios ranging from 1:1 to 1:8). In the control group, hAEC were treated for 30 min with anti-HLA-G (#87G, Exbio, 10 µg/mL), anti-HLA-E (#3D12, Biolegend, 20 µg/mL) or anti-β2 microglobulin (kindly supplied by Dr. Fabio Malavasi, University of Turin, Italy).
In another set of experiments, 100,000 stimulated T cells were cultured in the presence of ssEV or lsEV derived from 40 mL of hAEC supernatant derived from 75 cm² at confluence (5 millions of cells) and resuspended in 100 µL of culture medium, or with hAEC (at hAEC:effector cell ratio 1:1) previously cultured in the presence of inhibitors of EV release, as described above.
Effector cells were harvested after 6 days, and stained with PE-conjugated anti-CD4 mAb (Beckman Coulter). CFSE/CD4 positive cells were analyzed using the Gallios cytometer and Kaluza software.

Peptide binding assay was performed in the HLA-E*0101 transfected K562 cell line (K562E*0101), kindly provided by Dr. Jakob Michaelsson (Karolinska Institutet, Center for Infection Medicine, Department of Medicine, Huddinge), and mycoplasma tested before use. These K562E*0101 cells have a constitutive HLA-E expression. Briefly, cells were re-suspended in a medium at 10⁶ cells/ml, and indicated peptides were added at a concentration titration of 0-100 µM. After an overnight incubation at 37°C, cells were washed with PBS to remove free peptides. Next, HLA surface expression was monitored after staining with anti-HLA-E (BioLegend) and viability dye. Analysis was done using flow cytometry as described above. Results are reported as flow cytometry histograms or mean fluorescence intensity (MFI) compared to Fluorescence Minus One (FMO) control.

Human tonsil and rhesus macaque lymph node cells were sorted using a MoFlo Astrios EQ. Cells were sorted into T_FH (CD3+CD8-CXCR5+), conventional CD8 (CD3+CD8+CXCR5-), CD8 T_FR (CD3+CD8+CXCR5^hiCD44^hi), and GC B cell (CD19+CD38+) populations. After sorting, all tonsil cell subsets were spinoculated with X4- or R5-HIV GFP reporter viruses and cultured in R10. All cell populations were spinoculated to mirror whole tonsil cell cultures and allow for all cell populations to be exposed to virus. For measurements of T_FH cytokine production, T_FH were seeded at 1 x 10⁵ cells in a 24 well plate and CD8 T_FR were added at ratios of 1:1, 1:10, and 1:50. After 2 days, T_FH were stimulated to measure IL-2 and IL-21 by ICS. For blocking experiments, T_FH and CD8 T_FR or conventional CD8 were cultured at a 1:1 ratio in the presence of 500 ng/μL anti-Tim-3 (Biolegend 345003) antibodies or 500 ng/μL anti-HLA-E (Biolegend 342602) antibodies for 2 days. In transwell assays, 4 x 10⁴ T_FH were cultured in the bottom compartment and 4 x 10⁴ CD8 T_FR or conventional CD8 T cells were added to the upper compartment of 96-well permeable support plates (Corning CLS3386) and cultured for 2 days.