Supplementary Figure 1: Analysis of inhibitory signaling in T cells from human HIV-infected subjects.

(a) Workflow for screening of HIV-infected subjects. Boxes in grey indicate the subject groups chosen for further studies. Samples from subjects with at least 10⁶/ml CD4⁺ T cells were used for further studies (*). Subjects were excluded when RNA amount and quality did not reach necessary quality standards for genomic analysis (**). (b) Flow cytometric analysis of PD-1 expression on CD4⁺ T cells from HIV^loPD-1^lo or HIV^hiPD-1^hi subjects. Mean PD-1 expression of CD4⁺ T cells from HIV^loPD-1^lo (n = 26) or HIV^hiPD-1^hi subjects (n = 37). (c) Relative PDCD1 mRNA expression of CD4⁺ T cells from HIV^loPD-1^lo (n = 5) or HIV^hiPD-1^hi subjects (n = 7) by qPCR. (d) Representative flow cytometry dot plots from one HIV^loPD-1^lo and one HIV^hiPD-1^hi subject using current state-of-the-art methodology. (e-g) Flow cytometric analysis of PD-1 expression on CD8⁺ T cells from HIV^loPD-1^lo or HIV^hiPD-1^hi subjects. (e) Representative flow cytometry dot plots from one HIV^loPD-1^lo and one HIV^hiPD-1^hi patient. (f) Proportion of PD-1-expressing CD8⁺ T cells from HIV^loPD-1^lo (n = 10) or HIV^hiPD-1^hi subjects (n = 8). (g) Mean PD-1 expression of CD8⁺ T cells from HIV^loPD-1^lo (n = 10) or HIV^hiPD-1^hi subjects (n = 8). (h-j) Correlation between CD4⁺ T cell count of each subject and HIV-RNA (h), CD4⁺ T cell count and CD4⁺ T cell PD-1 expression (i), and HIV RNA and CD4⁺ T cell PD-1 expression (j). White circles: HIV^loPD-1^lo subjects; grey circles: HIV^hiPD-1^hi subjects. (k) Representative flow cytometry dot plots from one HIV^loPD-1^lo or one HIV^hiPD-1^hi subject for CTLA-4 expression on CD4⁺ T cells. (l) Proportion of CTLA-4-expressing CD4⁺ T cells from HIV^loPD-1^lo (n = 10) or HIV^hiPD-1^hi subjects (n = 8). (m) Mean CTLA-4 expression of CD4⁺ T cells from HIV^loPD-1^lo (n = 10) or HIV^hiPD-1^hi subjects (n = 8). (n) Representative flow cytometry dot plots from one HIV^loPD-1^lo or one HIV^hiPD-1^hi subject for CTLA-4 expression on CD8⁺ T cells. (o) Proportion of CTLA-4-expressing CD8⁺ T cells from HIV^loPD-1^lo (n = 10) or HIV^hiPD-1^hi subjects (n = 8). (p) Mean CTLA-4 expression of CD8⁺ T cells from HIV^loPD-1^lo (n = 10) or HIV^hiPD-1^hi subjects (n = 8). (q-s) Generation of RNA fingerprints. (q) Prior to assessment of transcriptional changes the functional impact of all components on purified CD4⁺ T cells was analyzed. Freshly isolated primary human CD4⁺ T cells were labeled with CFSE and left unstimulated or were stimulated as indicated. After 4 days, CFSE dilution was analyzed by flow cytometry. The overall percentage of dividing cells is displayed in the corresponding gate. For each condition at least four individual experiments were performed. Shown here are representative results. (r) CD4⁺ T cells were stimulated as above. After four days the concentration of IFN-γ was determined using flow cytometric bead assays. For each condition at least four individual experiments were performed. Mean ± s.d. (s) Visualization of fold changes and amount of genes significantly altered in CD4⁺ T cell transcription profiles after indicated stimulations of four different healthy blood donors defining the RNA fingerprint of the particular analyzed component. (t) Schematic overview of analysis of gene expression data for the contribution of RNA fingerprints to the differences between CD4⁺ T cells from HIV^loPD-1^lo (n = 10) or HIV^hiPD-1^hi subjects (n = 10). (u) Enrichment of inhibitory pathways in HIV-infected individuals. Gene set enrichment analysis (GSEA) using genes regulated by PD-1, CTLA-4, PGE₂, TGF-β, and IL-10 as the gene set in CD4⁺ T cells from HIV^hiPD-1^hi and HIV^loPD-1^lo subjects. ES: enrichment score, FDR: false-discovery rate. (b,c,g,m,p) Mean ± s.e.m. (b,c,f,g,l,m,o,p) *P < 0.05 (Student’s t-test). (f,l,o) Bounds of boxes denote interquartile range; lines within boxes denote mean; whiskers indicate interdecile range. Dots represent outliers.