Thread by @drmarglass

Good morning science Twitter 😁🧬🔬 Our deep profiling of human IL-10-producing B cells by single-cell, highly multiplexed cytometry analysis is live on @biorxivpreprint! #bcellsrule
1/n

As all you immunology researchers know well, Tregs are the 'popular' lymphocytes when it come to regulatory immune cells. Yet, there is provocative evidence that regulatory B cells (#Bregs) also mediate anti-inflammatory function, primarily through production of #IL10.
2/n

Many knowledge gaps remain with respect to the optimal signals to induce Bregs, their phenotypic and functional diversity across healthy humans, and the potential of canonical B cell subsets to give rise to IL-10-producing cells.
3/n

Where to start with so many Breg questions?! We first performed a massive screen of B cell activating conditions. #biorender
4/n

Our screen showed TLR9 activation with CD40 engagement and exogenous cytokines induced optimal production of IL-10 by B cells. P.S. I'm very proud of these biaxials!
5/n

Then, to capture the phenotypic heterogeneity and polyfunctionality of human IL-10-producing B cells, we developed a custom mass cytometry (#CyTOF) panel and analysis pipeline to deeply profile immunophenotype and regulatory protein expression.
6/n

A significant portion of IL-10+ B cells co-expressed proinflammatory cytokines.
7/n

Our also data showed IL-10-producing cells were not restricted to a single, unique phenotype that was consistent across stimulatory conditions.
8/n

To understand the temporal dynamics of IL-10 and proinflammatory cytokine co-expression, as well as how the immunophenotype of stimulated and IL-10+ B cells compare over time, we performed a timecourse of B cell activation.
9/n

Our timecourse data revealed that proinflammatory cytokine production precedes IL-10 expression in the course of B cell activation.
10/n

IL-10+ and total stimulated B cell phenotypes were highly overlapping across timepoints. When we compared IL-10+ and IL-10- populations, a handful of surface markers showed more significant upregulation by IL-10+ cells (but they are upregulated by both populations!).
11/n

But wait... what are these IL-10-producing cells?! Are certain functional B cell subsets more likely to express IL-10 upon activation? To clarify this, we isolated and labeled canonical B cell subsets, and traced them in our IL-10 production assay.
12/n

We found notable variation in the proportion of IL-10+ B cells by subset and individual (but the rank order was the same for each individual!), as well as in IL-10+ B cell TNFa and IL-6 expression levels by subset.
13/n

We also laid out how the IL-10-inducing activation process changes the resting immunophenotype of each B cell subset.
14/n

We determined the IL-10+:TNFa+ ratio for each subset, an analysis first employed by @AravindCheruku1 ! Transitional B cells had the highest ratio by a long stretch, meaning they were enriched for IL-10 production upon activation!
15/n

In summary, our healthy human B cell studies found that IL-10-producing cells constitute an induced, transient, yet multifunctional state, arising from a diversity of B cell subsets.
16/n

To identify unique characteristics of IL-10+ B cells in a context of immune tolerance, we applied our analytical framework to data for liver transplant recipients that maintained their graft function in the absence of immunosuppression, termed 'operationally tolerant'.
17/n

Our analysis revealed a significant enrichment of IL-10-producing B cells in the cohort of operationally tolerant transplant recipients, which was not the case for proinflammatory cytokine-producing B cells.
18/n

Collectively, the (cytometry) evidence points to these IL-10-producing B cells or Bregs representing a immunoregulatory cellular state, rather than a fixed/stable cell subset with a defined immunophenotype. #stopytryingtomakebregsasubset
19/n

We hope this work will serve as resource for future regulatory B cell studies. There is still much work to be done in the realm of IL-10-producing B cells in healthy humans and in disease states, and we look forward to seeing what more will be discovered from here!
20/n

Also, HUGE THANK YOU to our research team at #StanfordMed @TILStanford @Bendall_Lab @david_r_glass @jp_oliveria @BereniceMbiri @smkrams @martinezoliviam and many others who helped with this study 🙏
n/n

Our preprint: biorxiv.org/content/10.110…