Supplementary Figure 2: TNF-dependent regulation of PD-1 expression.

(a) Relative mRNA expression of CD4⁺ T cells from HIV^loPD-1^lo (n = 10) or HIV^hiPD-1^hi subjects (n = 10) for TNFRI and TNFRII by microarray analysis. (b) Expression of TNFRI and TNFRII on CD4⁺ T cells from HIV^loPD-1^lo (n = 5) or HIV^hiPD-1^hi subjects (n = 6). (c) Schematic representation of the human PDCD1 locus. The PDCD1 promoter predicted by analysis using Genomatix is shown in violet. (d) Luciferase reporter constructs driven by the Genomatix-predicted human PDCD1 promoter (-5.0 kb, red), the region directly upstream of the transcriptional start site (-0.5 kb, green), and an intronic enhancer in intron 4 (intron 4, blue)²¹ were transfected into HEK293T cells and luciferase activity was assessed after 24 hours in unstimulated cells and cells stimulated with TNF. Control represents the empty pGL4.24 construct. Mean ± s.d. of triplicate cultures are shown. Data are representative of three independent experiments. (e,f) Expression of PD-1 on memory CD4⁺ T cells from healthy donors pre-stimulated for 3 days with TNF (TNF) or medium alone (US), restimulated with (e) TNF or (f) anti-CD3, IL-2, and TNF (each n = 6). Left, exemplified flow cytometry data, right, cumulative data. (g) Differentiation of CD4⁺ T cells from HIV^loPD-1^lo (n = 5) or HIV^hiPD-1^hi subjects (n = 6) in naïve and memory CD4⁺ T cells as well as CD7⁺ or CD7^- memory CD4⁺ T cells. (e,f) *P < 0.05 (Student’s t-test). (a,b,e,f,g) Mean ± s.e.m. n.s. not significant.