Single Cell Network Profiling (SCNP) Reveals Race-Associated Differences in B Cell Receptor Signaling Pathway Activation
Authors: Longo Diane , Louie Brent , Mathi Kavita , Pos Zoltan , Wang Ena , Hawtin Rachael E. , Marincola Francesco , Cesano Alessandra 
Published: October 2019

Abstract
Abstract 1125
Race-related differences have been documented in the incidence of autoimmune diseases such as systemic lupus erythematosus and multiple sclerosis, in the clinical response to immunotherapies [such as IFNα (in HCV infections) and belimumab (in systemic lupus erythematosus)] and to hematopoietic stem cell transplantation. However, the basis for such race-associated differences remains poorly understood. Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry based approach that simultaneously measures intracellular signaling activity in multiple cell subpopulations. Previously, SCNP analysis of peripheral blood mononuclear cells (PBMCs) from 60 healthy donors identified a race-associated difference in αIgD induced levels of pS6 and pAkt in B cells. 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.
35 BCR signaling nodes (a node is defined as a paired modulator and intracellular readout) were measured by SCNP in PBMCs from 10 healthy donors [5 African Americans (36–51 yrs), 5 European Americans (36–56 yrs), all males]. Cryopreserved PBMCs were thawed, modulated at 37°C in 96-well plates, fixed and permeabilized. Permeabilized cells were stained with fluorochrome-conjugated antibodies that recognize extracellular surface markers and intracellular signaling molecules. The levels of 7 phospho-proteins [pLck (Y505), pSyk (Y352), pAkt (S473), pS6 (S235/S236), p-p38 (T180/Y182), pErk (T202/Y204), and pNFκB (S529)] were measured in CD20+ B cells at 0, 5, 15, 30, and 60 mins post αIgD exposure. CD20 and IgD surface markers were used to determine the frequency of IgD+ B cells.
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 αIgD induced phosphorylation of multiple BCR pathway components, including the membrane proximal proteins Syk and Lck as well as proteins in the PI3K pathway (S6 and Akt), the MAPK pathways (Erk and p38), and the NFκB pathway (NFκB) (see example for αIgD induced pS6 levels in Fig. 1A). Overall, 4 (pSyk, pS6, pAkt, and pErk) of the 7 BCR pathway components tested (averaged over all timepoints for each donor) showed statistically significant differences in αIgD induced activation levels between racial groups (p=0.016, Wilcoxon test). Analysis of the frequency of IgD+ B cells showed that PBMCs from African Americans had a lower frequency of IgD+ B cells than PBMCs from European Americans [(p=0.016, Wilcoxon test), Fig. 1B], and that the frequency of IgD+ B cells had a strong positive correlation with BCR pathway activation (i.e. Pearson r>0.6 for most BCR signaling nodes). While race-associated differences in the frequency of IgD+ B cells were detected, the levels of IgD expression (as measured by the median fluorescence intensity) in the IgD+ B cell subpopulation did not differ between the races (p=0.286). Thus, the race-related difference in BCR pathway activation is attributable, at least in part, to a race-associated difference in IgD+ B cell frequencies.Fig. 1.A) αIgD induced pS6 signaling (based on the log2fold increase in MFI in αIgD treated cells relative to the untreated control (0 min)) over time are shown for the African American (AA) and European American (EA) donors. The difference in pS6 signaling (averaged over time points) between racial groups is statistically significant. B) The percentage of CD20+ B cells that were IgD+ is shown for the AA and EA donors. The difference in IgD+ frequency between racial groups is statistically significant. In both (A) and (B), one of the ten donors was excluded due to an insufficient number (<200) of B cells collected for analysis.Fig. 1. A) αIgD induced pS6 signaling (based on the log2fold increase in MFI in αIgD treated cells relative to the untreated control (0 min)) over time are shown for the African American (AA) and European American (EA) donors. The difference in pS6 signaling (averaged over time points) between racial groups is statistically significant. B) The percentage of CD20+ B cells that were IgD+ is shown for the AA and EA donors. The difference in IgD+ frequency between racial groups is statistically significant. In both (A) and (B), one of the ten donors was excluded due to an insufficient number (<200) of B cells collected for analysis.
In conclusion, SCNP analysis allowed for the identification of statistically significant race-associated differences in BCR pathway activation within PBMC samples from healthy donors. Further characterization of racial functional differences in additional immune signaling pathways using this assay in samples from both healthy and diseased individuals may be critical for elucidating the basis for race-related differences in immune-mediated disease prevalence and treatment responses.

Disclosures:
Longo: Nodality: Employment, Equity Ownership. Louie:Nodality: Employment, Equity Ownership. Mathi:Nodality: Employment. Hawtin:Nodality: Employment, Equity Ownership. Cesano:Nodality: Employment, Equity Ownership.