Racial differences in B cell receptor signaling pathway activation

Abstract

Background: Single-cell network profiling (SCNP) is a multi-parametric flow cytometry-based approach that simultaneously measures basal and modulated intracellular signaling activity in multiple cell subpopulations. Previously, SCNP analysis of a broad panel of immune signaling pathways in cell subsets within PBMCs from 60 healthy donors identified a race-associated difference in B cell anti-IgD-induced PI3K pathway activity.

Methods: The present study extended this analysis to a broader range of signaling pathway components downstream of the B cell receptor (BCR) in European Americans and African Americans using a subset of donors from the previously analyzed cohort of 60 healthy donors. Seven BCR signaling nodes (a node is defined as a paired modulator and intracellular readout) were measured at multiple time points by SCNP in PBMCs from 10 healthy donors [5 African Americans (36-51 yrs), 5 European Americans (36-56 yrs), all males].

Results: Analysis of BCR signaling activity in European American and African American PBMC samples revealed that, compared to the European American donors, B cells from African Americans had lower anti-IgD induced phosphorylation of multiple BCR pathway components, including the membrane proximal proteins Syk and SFK as well as proteins in the PI3K pathway (S6 and Akt), the MAPK pathways (Erk and p38), and the NF-κB pathway (NF-κB). In addition to differences in the magnitude of anti-IgD-induced pathway activation, racial differences in BCR signaling kinetic profiles were observed. Further, the frequency of IgD+ B cells differed by race and strongly correlated with BCR pathway activation. Thus, the race-related difference in BCR pathway activation appears to be attributable at least in part to a race-associated difference in IgD+ B cell frequencies.

Conclusions: SCNP analysis enabled the identification of statistically significant race-associated differences in BCR pathway activation within PBMC samples from healthy donors. Understanding race-associated contrasts in immune cell signaling responses may be one critical component for elucidation of differences in immune-mediated disease prevalence and treatment responses.

Figure 1

Gating strategy used to delineate the viable B cell subpopulation within PBMCs. Boolean logic was used to identify cells that were amine aqua negative, lymphocytes by light scatter, positive for CD20, and negative for c-poly(ADP-ribose) polymerase (c-PARP).

Figure 2

BCR signaling activation in AA and EA donors.A, Fold vs. time is shown for the 7 phospho-protein readouts for 9 of the 10 healthy donors (one AA donor was excluded due to an insufficient number of B cells (<200 events) collected for analysis). B, Race-associated differences were assessed by the Wilcoxon rank sum test (see Materials and Methods). Four phospho-protein readouts had a statistically significant race-associated difference (p < 0.05).

Figure 3

IgD+ B cell percentages in AA and EA donors. CD20- lymphocytes were used as an internal negative control to define the IgD+ region for each donor. The IgD+ region was applied to the viable B cell subpopulation to determine the IgD+ B cell percentage for each donor. Representative flow plots from one healthy donor are shown. IgD+ B cell percentages were assessed under the unmodulated condition. EAs had a higher percentage of IgD+ B cells than AAs and the race-associated difference was significant (p = 0.016, Wilcoxon test).

Figure 4

Correlations between IgD+ B cell percentages and BCR signaling pathway activation. The heatmap shows the magnitude of the Pearson correlation coefficients (r) between the IgD+ B cell percentage and the Fold for each signaling node at each anti-IgD modulation time point.

Figure 5

IgD+ B cell percentage vs. percentage of anti-IgD responsive (p-S6 “high”) B cells. Anti-IgD-induced p-S6 histograms are shown for 2 representative donors. p-S6 “high” and p-S6 “low” regions were established by estimating the location of the trough in the bimodal profile for one donor and the same regions were applied to all donors. The percentage of anti-IgD induced p-S6 “high” B cells had a statistically significant race-associated difference (p = 0.016, Wilcoxon test). The ratio of the MFI in the p-S6 “high” region to the MFI in the p-S6 “low” region did not differ between the races (p = 0.905, Wilcoxon test).