Fig 1: Trogocytosed receptors are functional and CD32+ membrane patches on resting CD4 T cells preferentially bind virions and enhance HIV-1 fusion(A) SDF-1α (CXCL12)/CXCR4 migration assay. CXCR4 KO CD4 T cells were co-cultured with HeLa cells transiently co-expressing CD32B-GFP or H2B-GFP (control) together with CXCR4. Prior to co-culture, HeLa donor cells were pre-treated with or without anti-CD32 mAbs. One day after co-culture, CD4 T cells were placed into the top chamber of a Transwell and SDF-1α was added to the bottom chamber. Migrating cells collected were counted by flow cytometry. CXCR4 WT and KO CD4 T cells without co-culture were used as positive and negative control (mean ± SEM; n = 3). Asterisks indicate statistical significance by one-way ANOVA. p values were corrected for multiple comparison (Tukey).(B) RANTES (CCL-5)/CCR5 migration assay. HeLa cells transiently co-expressing CD32B-GFP and CCR5 were co-cultured with CD4 T cells and the latter analyzed for migration toward CCL-5 (assay setup as in A) (mean ± SEM; n = 3). Asterisks indicate statistical significance by one-way ANOVA. p values were corrected for multiple comparison (Dunnett).(C) CD11b binding assay. Following co-culture of M2 with autologous CD4 T cells for 48 h, T cells were sorted and cultured in plates coated with or without the ICAM-1 ligand. Attached cells were quantified by luminometry (mean cell binding ± SEM normalized to wells with input cells without washing; n = 3). Asterisks indicate statistical significance by two-way ANOVA. p values were corrected for multiple comparison (Tukey).(D) HIV-1 binding, fusion, or infection of (CellTrace+) CD4 T cells following co-culture with autologous M2. The illustration was created with BioRender.com.(E) HIV-1 binding assay. M2-co-cultured CD4 T cells were sorted and challenged with HIV-1 Vpr-GFP particles. Shown is GFP and CD32 positivity of target CD4 T cells (mean ± SEM; n = 4). Asterisks indicate statistical significance by two-way ANOVA (see Figures S14A–S14C for confocal microscopy images). p values were corrected for multiple comparison (Tukey).(F) CD4 T cells were co-cultured with biotin-xx-conjugated cholera toxin subunit-B (CT-B)-labeled M2, sorted, challenged with HIV-1 Vpr-GFP (see also Figures S14D–S14F), and stained for CD32 and fluorochrome-conjugated streptavidin. Shown are representative confocal microscope micrographs. White arrow heads: co-localization of CD32, HIV-1 Vpr-GFP, and CT-B. Scale bars, 5 μm.(G) HIV-1 fusion assay. CD4 T cells were co-cultured with autologous M2, isolated and used in an HIV-1 fusion assay using two multiplicities of infection (MOIs). Shown is the percentage of cells that allowed virion fusion (mean ± SEM; n = 5). Asterisks indicate significance by two-tailed paired t test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; n.s., not significant.(H) HIV-1 infection assay. CD4 T cells were co-cultured with autologous M2, isolated, and infected with HIV-1 at different MOIs. Shown is the percentage of infected cells (mean ± SEM; n = 7–11). Asterisks indicate significance by two-tailed paired t test.(I) M2 were pre-treated with alemtuzumab or an isotype control antibody and then co-cultured with autologous CD4 T cells. Sorted CD4 T cells were incubated with X4 HIV-1 (left panel) or R5 HIV-1 (right panel), carrying Vpr-BlaM, and virion fusion was quantified. Pearson correlations between CD32 positivity and HIV-1 fusion are shown. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; n.s., not significant.