Article Figures & Data
Figures
- FIGURE 1.
Flow cytometric detection of FcMR after culture with or without human serum.
PBMCs processed from whole blood were stained for FcMR before and after culture using multiple Abs. (A–D) Histogram overlays show FcMR fluorescence intensity after staining with mAb clones HM14 or HM7, or polyclonal rabbit anti-FcMR Abs (RpAbs). Bar graphs show mean fluorescence intensity (MFI) for each stain corrected to ΔMFI by subtracting fluorescence observed with isotype-matched controls for HM14 and HM7, or fluorescence-minus-one for RpAbs. Bar heights indicate average values measured from four donors in 10 experiments for HM14, three donors in 3 experiments for HM7, and three donors in 3 experiments for RpAbs; error bars denote SD. Independent experiments are represented with different symbol shapes. Welch and Brown–Forsythe one-way ANOVA with Dunnett T3 post hoc analyses were used to obtain adjusted p values. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001. n.s., not significant.
- FIGURE 2.
Surface FcMR is reduced by processing blood cells ex vivo.
Staining procedures of either minimal necessary manipulation (stain then lyse) or slightly increased manipulations (lyse once and then stain or lyse twice and then stain, respectively) were performed in parallel directly after peripheral blood collection to assess how cell processing affected FcMR abundance. (A) Bar graphs show FcMR MFI (HM14 mAb) corrected to ΔMFI by subtracting fluorescence observed with isotype-matched controls. Bar heights indicate average values measured from three donors in three experiments; error bars denote SD. Independent experiments/donors are represented with different symbol shapes. Welch and Brown–Forsythe one-way ANOVA with Dunnett T3 post hoc analyses were used to obtain adjusted p values. No significant differences were observed. (B) Representative histogram overlays showing FcMR fluorescence intensity for each cell type after staining with HM14 mAb versus isotype controls, which were repeated for each processing procedure.
- FIGURE 3.
Surface FcMR increases to similar levels after 24-h culture regardless of serum or IgM content.
PBMCs were processed from whole blood and cultured in medium supplemented with varying amounts of human serum prior to flow cytometric measurement of FcMR. (A) No correlation of IgM abundance in culture media with cell surface FcMR. Cell surface FcMR was measured after 24-h culture in 10% human serum media containing varying amounts of IgM, quantified by ELISA. PBMCs from the same donor were tested in five independent experiments. Human serum was derived from one of five sources with varying IgM amounts; two sources were repeated across experiments. Dotted lines show best fit for each cell type. Pearson two-tailed correlation analyses were performed to generate the p and r values depicted. (B) Cell surface FcMR as a function of serum content after 24-h culture. FcMR MFI (HM14 mAb) was corrected to ΔMFI by subtracting fluorescence observed with isotype-matched controls. Bar heights indicate average values measured from four donors in six experiments; error bars denote SD. Independent experiments are represented with different symbol shapes. Welch and Brown–Forsythe one-way ANOVA with Dunnett T3 post hoc analyses were used to obtain adjusted p values. No significant differences were observed. (C) Time course of surface FcMR on B, NK, and T cells cultured with varying serum amounts in media. Solid lines run through average values measured at each time point for three donors in three independent experiments (in one experiment, the 0.5-h time point was not collected). Dotted lines show ΔMFI surface FcMR for each cell type when whole blood was stained directly to minimize cell manipulation (see Fig. 2, stain then lyse). Two-tailed Welch t tests were performed to compare differences between the 0 and 40% serum cultures at each time point. *p < 0.05, **p < 0.01; Unlabeled differences are not significant (NS).
- FIGURE 4.
High cell density during culture suppresses surface FcMR expression.
PBMCs were processed from whole blood and cultured at varying cell densities prior to flow cytometric measurement of FcMR. (A) Effect of cell density during culture on FcMR expression. Scatterplots for different PBMC cell types show four donors in four experiments with FcMR MFI (HM14 mAb) corrected to ΔMFI by subtracting fluorescence observed with isotype-matched controls. Regression line, r2, and p values from linear regression analyses are depicted. Histogram overlays show representative FcMR fluorescence intensity after staining with HM14 mAb or isotype control. (B) Time course of surface FcMR on B, NK, and T cells cultured at varying cell densities. Solid lines run through average values measured at each time point for three donors in three independent experiments (in one experiment, the 0.5-h time point was not collected). Dotted lines show ΔMFI surface FcMR for each cell type when whole blood was stained directly to minimize cell manipulations (see (Fig. 2, stain then lyse). Two-tailed Welch t tests were performed to compare differences between the 0.2 and 2 M cells/well cultures at each time point. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001. Unlabeled differences are not significant (NS).
- FIGURE 5.
Surface FcMR changes after tonsil cell culture.
Whole tonsils from obstructive sleep apnea patients were processed to prepare and stain tonsil mononuclear cells (TMCs) to assess FcMR surface levels. (A) TMCs stained at 0 h or after culture for 24 h with varying amounts of human serum. FcMR MFI (HM14 mAb) corrected to ΔMFI by subtracting fluorescence observed with isotype-matched controls. Bar heights indicate average values measured from four donors in two experiments; error bars denote SD. Independent donors are represented with different symbol shapes. Welch and Brown–Forsythe one-way ANOVA with Dunnett T3 post hoc analyses were used to obtain adjusted p values. *p < 0.05, **p < 0.01. Unlabeled differences are not significant (NS). (B) The relationship of TMC surface FcMR after culture to cell culture density. Scatterplots for different TMC cell types show four donors in two experiments. Regression line, r2, and p values from linear regression analyses are shown.
- FIGURE 6.
The density-dependent regulation of surface FcMR does not depend on a specific cell type.
PBMCs processed from whole blood were depleted of various cell types, cultured at increasing cell densities, and stained for FcMR. (A–E) The effect of cell density on CD4 T cell surface FcMR is not attributable to B cells, CD8 cells, NK cells, CD14+ monocytes, or platelets. FcMR MFI (HM14 mAb) was corrected to ΔMFI by subtracting fluorescence observed with isotype-matched controls. Bar heights indicate average values measured from (A) four donors in four experiments, (B) three donors in three experiments, or (C–E) two donors in two experiments; error bars denote SD. Independent experiments/donors are represented with different symbol shapes. Welch and Brown–Forsythe one-way ANOVA with Dunnett T3 post hoc analyses were used to obtain adjusted p values for comparisons of similar cell densities between mock versus depleted groups. No significant differences were observed.
- FIGURE 7.
Surface FcMR is regulated at high densities in culture by cell–cell proximity but not a soluble factor.
PBMCs were cultured in varying formats to test for soluble factors versus cell-to-cell proximity effects. (A) PBMCs cultured at varying cell densities in flat- or U-bottom wells prior to FcMR staining. Solid lines run through average values at each cell density for flat-bottom (blue) or U-bottom (red) wells for five donors in five experiments (0.2 million to 2 million cells/well) or two donors in two experiments (4 million cells/well). Two-tailed Welch t tests were performed to compare differences between the flat- and U-bottom cultures at each cell density; *p < 0.05, **p < 0.01. Unlabeled differences are not significant (NS). (B) Cells were cultured in the tops or bottoms of transwell plates, allowing media but not cell communication, prior to staining for FcMR. Shaded cells in the table indicate cell populations whose FcMR expression is reported in the bars above. FcMR MFI (HM14 mAb) was corrected to ΔMFI by subtracting fluorescence observed with isotype-matched controls. Bar heights indicate average values measured from three donors in three experiments (for CD4 and CD8 T cells) or two donors in two experiments (for B and NK cells); error bars denote SD. Independent experiments/donors are represented by different symbol shapes. (C) Cells were cultured at 0.2 million cells/well in media with 50% cell-free SUPs from prior culture of 0.2 million or 2 million cells/well for 24 h. Positive effect control was PBMCs cultured at 2 million cells/well with no SUP; negative control was PBMCs cultured at 0.2 million cells/well with no SUP. Bar heights indicate average values measured from three donors in three experiments. For (B) and (C), independent experiments/donors are represented with different symbol shapes. Two-tailed Welch t test was used to compare means to first assess success of controls and separately assess significance between test groups. n.s., *p < 0.05, **p < 0.01. n.s., not significant.
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