Antibody capture beads

Flow cytometry staining of markers on CD4⁺ T cells was performed on Cyto-Chex-stabilized whole blood [31 (link)]. Preanalysis was conducted to confirm that Cyto-Chex-stabilized blood generated data of all tested markers equivalent to that obtained with fresh blood samples (data not shown). Stainings of blood were adopted from protocols used for peripheral blood mononuclear cells [32 (link)]. Red blood cells were lyzed and remaining cells were stained with fluorochrome-conjugated antibodies (Supplemental Digital Content Table S2). Cells were permeabilized and fixed with the FOXP3 staining kit (eBioscience, San Diego, California, USA). Within 6 h, cells were run on a LSR Fortessa (BD Biosciences), where minimally 600 000 events were collected per run. Antibody capture beads (BD Biosciences) were used for compensation and FlowJo 8.8.7 (Tree Star, Ashland, Oregon, USA) for analyses. All manual gatings were based on fluorescence-minus-one gating strategies as described [33 (link),34 (link)]. A typical gating strategy to distinguish CD4⁺ T cells is visualized in Fig. 1a.

Peripheral blood mononuclear cells were analyzed on a 4-laser LSR Fortessa (BD Bioscience). Approximately 1,500,000 events were recorded per specimen. In addition, antibody capture beads (BD Bioscience) were used for compensation and prepared individually by separate staining of all the antibodies used in the experiment. FlowJo X 10.0.7r2 (Treestar) was used for gating analysis, and statistical analysis was performed with GraphPad Prism 6.0. Analysis and graphical representation of cell surface inhibitory markers were conducted using data analysis program SPICE (version 5.35001) (28 (link)).

Data was entered in excel and exported to GraphPad prism 8 for analysis. CD4⁺ T cells were analyzed on an 8-laser FACS Canto II (BD Bioscience, Franklin Lakes, New Jersey, USA). Approximately 50,000 events were recorded per sample. In addition, antibody capture beads (BD Bioscience, Franklin Lakes, New Jersey, USA) were used for compensation and prepared individually by separate staining of all the antibodies used in the experiment. FlowJo X 10.6 (Treestar, Oregon, USA) was used for gating analysis. For mRNA quantification, relative quantification using the obtained CT value was done using the delta CT method. Statistical differences between the different groups were determined using the unpaired t test in Graph pad prism v8. Sequence analysis was done using Mutation Surveyor software to identify SNPs in the respective genes. Frequencies and percentages of the SNPs were determined. SNPs in the coding region were analysed using the gnomAD to determine the amino-acid change.

PBMCs were analyzed on a modified 4 laser LSR Fortessa (BD Biosciences). In total, approximately 1,000,000 events were collected per specimen. Antibody capture beads (BD Biosciences) were used to prepare individual compensation controls after separate stainings with all antibodies used in the experiments. FlowJo 8.8.7 (Treestar) was used for flow cytometric gating analyses. Most manual gatings were based on fluorescence minus one (FMO) gating strategies like previously described [35] , [36] (link). A response was considered positive if the frequency of IFNγ producing cells were >0.05% of total CD8+ T cells after background reduction and twice the negative background.

Stained PBMCs were acquired on an LSR Fortessa cytometer using FACSDiVa software (BD). Approximately 1,000,000 events of PBMCs were recorded per specimen. Antibody capture beads (BD) were used for single-stain compensation controls. Flow cytometry data were analyzed with FlowJo software v10.6.1, and fluorescence minus one (FMO) was used to set manual gates. We analyzed CD8+ T-cells by excluding dump and CD4+ T-cells. We excluded patients with <20% viability in lymphocytes and total CD8+ T-cells (Source data Figure 1A). As previously described, we measured by supervised and classical analyses the IRs expression in CD8+ T-cell subsets, including Naive, central memory, transitional memory, effector memory, and effector CD8+ T-cells (Breton et al., 2013 (link); Blanch-Lombarte et al., 2019 (link)). We performed two technical replicates for SEB-activated and HIV-1-specific CD8+ T-cell cytokine production. We considered the cytokine response positive after background subtraction (mean of two technical replicates) used as the cut-off value. For each independent sample, we recorded a median of 1,000 events and 50 events positive for cytokines for total and CD8+ T-cell subsets, respectively.