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Newly Proposed B Cell Atlas Identifies 12 Unique Subsets

NEW YORK – Stanford University researchers have developed a new B cell atlas that segregates B cells from four lymphoid tissues into 12 unique subsets.

In a new paper published on Tuesday in the Cell Press journal Immunity, the researchers noted that B cells are capable of a wide range of effector functions including antibody secretion, antigen presentation, cytokine production, and generation of immunological memory. However, they added, a consistent strategy for classifying human B cells is needed.

They developed a highly multiplexed screen to quantify the co-expression of 351 molecules on the surfaces of millions of human B cells, identified differentially expressed molecules, and aligned their variance with isotype usage, VDJ sequence, metabolic profile, biosynthesis activity, and signaling response. Based on these data, the investigators then developed a classification structure to segregate B cells from peripheral blood, bone marrow, lymph node, and tonsil into 12 populations that included a CD45RB+ CD27- early memory population, a class-switched CD39+ tonsil-resident population, and a CD19hiCD11c+ memory population that potently responds to immune activation.

"This atlas of human B cell identity will enable further studies to interrogate functional B cell subsets in the context of homeostasis, vaccination, infection, autoimmunity, and cancer," the authors wrote.

For their multiplexed screen, the researchers designed 12 mass cytometry antibody panels using Fluidigm's CyTof2 mass cytometry platform, each consisting of nine conserved molecules and 30 variable molecules unique to each panel. The conserved molecules were selected to facilitate gating into four canonical B cell subsets: transitional, naive, non-switched memory, and switched memory. They identified 98 surface molecules expressed on human B cells.

Though the expression patterns largely recapitulated known biology, the investigators also observed some new mechanisms. For example, they found that CD73 was enriched in naive and switched memory cells, but low to absent in transitional and non-switched memory cells — this molecule has been used to subset murine memory B cells, although its expression was mostly associated with non-switched memory cells in contrast to its expression in switched memory cells in this human dataset.

After additional analyses allowed the investigators to classify the B cells they had identified, they further researched the cells' functional properties, asking whether the surface profiles denoted differences in other underlying functional cell processes. They stained healthy, human peripheral blood mononuclear cells (PBMCs) from additional donors with mass cytometry panels to interrogate B cell metabolic profiles, biosynthesis activity, and immune signaling. To assess single-cell metabolic profiles, the researchers quantified the expression of eight enzymes associated with four metabolic pathways: glycolysis or fermentation, ATP sensing, oxidative phosphorylation (ox-phos), and fatty acid oxidation.

They found that all subsets expressed all enzymes, but that expression levels varied by phenotype. For example, naive cells had the lowest expression of all subsets, whereas RB+ CD27- memory cells had an intermediate metabolic profile between naive and memory subsets. Plasma cells had the highest median expression for all enzymes and were significantly higher than other B cell subsets for molecules associated with both ox-phos and glycolysis. Outside of plasma cells, RB+ CD27+ CD73- memory and CD19hiCD11c+ memory cells had the highest median expression for all enzymes.

They next profiled bone marrow, tonsil, lymph node, and additional peripheral blood samples from a new cohort of healthy donors by mass cytometry, and found that the tonsil and lymph node samples were heavily enriched for B cells, as compared to the peripheral blood and bone marrow samples. To interrogate global differences in B cell expression between tissues, the researchers evaluated differences in expression between each pairwise combination of donor-pooled tissues for all molecules, and identified 21 molecules that were differentially expressed between at least one pair of tissues.

They observed that HLA-DR, which facilitates antigen presentation to T cells, was expressed at high amounts in tonsil and lymph node, and that CD32, an inhibitory Fc receptor, had low expression in tonsil and lymph node, as might be expected in a microenvironment promoting B cell activation. Conversely, several inhibitory molecules were enriched in the lymph node, including CD23, CD72, CD73, and CD305.

Within the tonsil, the researchers identified a B cell population uniquely characterized by high expression of CD39. Although this could indicate regulatory function, it's more likely that this expression is instead part of a larger expression program that balances immune sensitivity and activation. Because these cells were exclusive to the tonsil, the investigators didn't label them as conventionally circulating memory cells. Given that they shared phenotypic features with CD95+ memory, RB- memory, and CD19hiCD11c+ memory, they might represent a precursor to these populations.

"Here, our deep phenotypic profiling with multi-omic integration of numerous single-cell functional readouts in healthy individuals reveals the identity of new, more granular populations, comprehensively mapping B cell identity across blood and lymphoid tissues in the human," the authors concluded. "The quantitative assessment of the contribution of phenotype versus isotype usage across several cellular processes highlights the need for analysis beyond repertoire sequencing and isotype identity for understanding human B cell immune function."