Fig. 1

Direct contact with CD4 + T cells drives EGFR dependent G0-G1 transition in macrophages a Differentiated macrophages were co-cultured with activated autologous CD4 + T-cells, or stimulated by LPS/Interferon gamma (to polarize cells into M1 macrophages), or IL-4/IL-13 (into M2 macrophages). CD4 + T-cells were washed off 2 days later and cell cycle, viral permissivity was analysed. a Macrophages were infected with VSV-G pseudotyped HIV-1 expressing GFP (left panel). Cells were fixed 2 days post-infection and percentage of GFP positive cells was quantified by using automated microscopic platform. Graphs represent average of n = 15 (M0, M0_CD4); n = 6 (M1-like, M2-like) biological replicates. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. ns, non-significant; ^∗p < 0.1; ^∗∗p < 0.01. Bars indicate mean with SD. Middle panel: Macrophages were fixed and stained for MCM2 (a marker of cell cycle entry) and percentage of positive cells was determined by using automated microscopic platform. Graphs represent average of n = 16 (M0, M0_CD4); n = 4 (M1-like, M2-like) biological replicates. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. ns, non-significant; ^∗p < 0.1. Bars indicate mean with SD. Right panel Macrophages were lysed and cell lysates from a representative donor were used for immunoblotting using antibodies against CDK1, MCM2, SAMHD1 and pSAMHD1 T592. b A diagram of cell cycle depicting an expression of several cell cycle markers. None of the markers are present in G0; MCM2 is expressed in G1, S, G2,M; Ki67 is expressed in late G1, S, G2,M; geminin is expressed in S, G2,M. Macrophages following cell-cell contact (M0_CD4) were fixed and stained for cell cycle markers. A representative acquisition field from microscopic platform is shown in middle panel. Scale bars: 40 μm. Right panel shows Percentage of positive cells. At least 10⁴ cells were acquired and used for analysis. n = 3 biological replicates; Ordinary two-way ANOVA with Sidak’s multiple comparisons test: ^∗∗∗∗p < 0.0001. Bars indicate mean with SD. c. Macrophages were plated at the bottom of transwell, CD4 + T-cells were added directly to macrophages to allow direct cell-cell contact or added in the top of transwell to prevent direct contact. CD4 + T-cell were washed off 2 days later, infected (HIV-1 GFP, left panel) or stained for MCM2 (middle panel). The percentage of GFP or MCM2 positive cells was quantified by using an automated microscopic platform. n = 3 biological replicates. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. ns, non-significant; ^∗∗∗∗p < 0.0001. Bars indicate mean with SD. Right panel: Macrophages were lysed and cell lysates from a representative donor were used for immunoblotting with antibodies to CDK1, MCM2, SAMHD1 and pSAMHD1 T592. d left panel: Volcano plot showing differentially expressed phosphoproteins in macrophages following CD4 + T cell contact from 0 vs. 120 min versus the absence of T cell contact. Coloured proteins were significantly up or down regulated with a significance level of 0.1; right upper panel. Macrophages were untreated (M0) or co-cultured with CD4 + T-cells for 2 days in the presence or absence (mock) of inhibitors. T-cells were washed off and macrophages infected with VSV-G pseudotyped HIV-1 expressing GFP. Percentage of GFP positive cells was quantified 2 days later. Macrophages were lysed and cell lysates from a representative donor were used for immunoblotting. CDK1, MCM2 and SAMHD1 phosphorylation are markers of cell-cycle re-entry. n = 3 biological replicates. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. ^∗∗p < 0.01; ^∗∗∗∗p < 0.0001. Bars indicate mean with SD. right bottom panel Macrophages were untreated (M0) or co-cultured with CD4 + T-cells for 2 days in the presence of control isotype IgG antibody or increasing concentration of anti-EGFR antibody. T-cells were washed off and macrophages lysed. Cell lysates from a representative donor were used for immunoblotting