Fig 1: CD32 and other receptors are transferred from macrophages to co-cultured CD4 T cells(A) Screening for surface receptors transferred from autologous M2 macrophages (M2) to CD4 T cells following co-culture for 2 days. Receptors most highly transferred were categorized into “top hits 1–10” and “top hits 11–20,” respectively. x axis, mean of the mean fluorescence intensity (MFI) ratio from a pool of three donors; y axis, MFI of receptor expression on M2 from a pool of three donors.(B) Peripheral blood CD4 T cells were stained for CD3, HLA-DR, and CD32 and analyzed by AMNIS Imagestream. Shown are bright-field and fluorescent images of cells gated for CD32 positivity. Upper panel, CD32+ HLA-DR+ CD4 T cells; lower panel, CD32‒ HLA-DR+ CD4 T cells (see Figure S1D for gating strategy).(C) HLA-DR expression on autologous wild-type (WT) or HLA-DR KO CD4 T cells (KO) and M2 after 2 days of co-culture (mean ± SEM; n = 2–8). Asterisks indicate statistical significance by one-way ANOVA. p values were corrected for multiple comparison (Tukey).(D) DC-SIGN expression on WT CD4 T cells after 2 days co-culture with M2, either non-targeting control (NTC) or DC-SIGN KO (KO) (mean ± SEM; n = 3). Isolated CD4 T cells served as control (-). Asterisks indicate statistical significance by one-way ANOVA. p values were corrected for multiple comparison (Tukey).(E) CD32 expression on PBMCs and CD4 T cells after 3 days of culture in presence of absence of PHA/IL-2 (median with 95% CI; n = 6). Asterisks indicate statistical significance by one-way ANOVA relative to unstimulated (Not stim.) CD4 T cells. p values were corrected for multiple comparison (Tukey).(F) CD32 expression on CD4 T cells, PBMCs depleted of CD14+ cells, PBMCs or co-cultures of autologous CD4 T cell/CD14+ cells (mean ± SEM; n = 6). Asterisks indicate statistical significance by one-way ANOVA. p values were corrected for multiple comparison (Tukey).(G) CD14+ monocytes were differentiated into the indicated myeloid lineages (see Figure S3B) and co-cultured with autologous CD4 T cells for 2 days with or without (Transwell) direct cell-cell contact. Bottom: CD4 T cells migrated to the Transwell bottom and thus had direct contact with differentiated myeloid cells. Mean ± SEM of CD32+ T CD4 cells are shown (n = 3). Asterisks indicate statistical significance by two-way ANOVA test. p values were corrected for multiple comparison (Tukey).(H) CD32 expression on CD14+ monocytes and cells derived by lineage-specific differentiation after 1 week of cultivation. One representative donor is shown (n = 3).(I) Pearson correlation plot for CD32 surface expression on monocyte-derived cells (MFI) and autologous CD4 T cells (percentage of CD32+ cells) after 2 days of co-culture.(J) CD32 expression on CD4 T cells residing in peripheral blood (n = 23), tonsil (n = 6), or lamina propria of jejunum or ileum (n = 6) was assessed by flow cytometry. Median with 95% CI are shown. Asterisks indicate statistical significance by one-way ANOVA. p values were corrected for multiple comparison (Dunnett). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.