Pathogenic, glycolytic PD-1+ B cells accumulate in the hypoxic RA joint
Achilleas Floudas, Nuno Neto, Viviana Marzaioli, Kieran Murray, Barry Moran, Michael G. Monaghan, Candice Low, Ronan H. Mullan, Navin Rao, Vinod Krishna, Sunil Nagpal, Douglas J. Veale, Ursula Fearon
Achilleas Floudas, Nuno Neto, Viviana Marzaioli, Kieran Murray, Barry Moran, Michael G. Monaghan, Candice Low, Ronan H. Mullan, Navin Rao, Vinod Krishna, Sunil Nagpal, Douglas J. Veale, Ursula Fearon
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Research Article Cell biology Metabolism

Pathogenic, glycolytic PD-1+ B cells accumulate in the hypoxic RA joint

Abstract

While autoantibodies are used in the diagnosis of rheumatoid arthritis (RA), the function of B cells in the inflamed joint remains elusive. Extensive flow cytometric characterization and SPICE algorithm analyses of single-cell synovial tissue from patients with RA revealed the accumulation of switched and double-negative memory programmed death-1 receptor–expressing (PD-1–expressing) B cells at the site of inflammation. Accumulation of memory B cells was mediated by CXCR3, evident by the observed increase in CXCR3-expressing synovial B cells compared with the periphery, differential regulation by key synovial cytokines, and restricted B cell invasion demonstrated in response to CXCR3 blockade. Notably, under 3% O2 hypoxic conditions that mimic the joint microenvironment, RA B cells maintained marked expression of MMP-9, TNF, and IL-6, with PD-1+ B cells demonstrating higher expression of CXCR3, CD80, CD86, IL-1β, and GM-CSF than their PD-1– counterparts. Finally, following functional analysis and flow cell sorting of RA PD-1+ versus PD-1– B cells, we demonstrate, using RNA-Seq and emerging fluorescence lifetime imaging microscopy of cellular NAD, a significant shift in metabolism of RA PD-1+ B cells toward glycolysis, associated with an increased transcriptional signature of key cytokines and chemokines that are strongly implicated in RA pathogenesis. Our data support the targeting of pathogenic PD-1+ B cells in RA as a focused, novel therapeutic option.

Authors

Achilleas Floudas, Nuno Neto, Viviana Marzaioli, Kieran Murray, Barry Moran, Michael G. Monaghan, Candice Low, Ronan H. Mullan, Navin Rao, Vinod Krishna, Sunil Nagpal, Douglas J. Veale, Ursula Fearon

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Figure 6

PD-1+ RA patient B cells are dependent on glycolysis.

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PD-1+ RA patient B cells are dependent on glycolysis. (A) Representative...
(A) Representative flow cytometric analysis histograms and cumulative MFI of RA patient peripheral blood–derived PD-1– and PD-1+ B cell expression of phosphorylated AKT, mTOR, and S6 following stimulation (aCD40+aBCR+CpG) under normoxic (21% O2) and hypoxic (3% O2) conditions (n = 6/group, 3 independent experiments). Data are represented as a box-and-whisker plot, with bounds from 25th to 75th percentile, median line, and whiskers ranging from 5th to 95th percentile. (B) Representative flow cytometric analysis plots and MFI of RA patient–derived PD-1– and PD-1+ B cell expression of GLUT1 and STAT3 phosphorylation (pSTAT3) following stimulation under the indicated conditions (n = 4/group, 2 independent experiments). Data are represented as a box-and-whisker plot, with bounds from 25th to 75th percentile, median line, and whiskers ranging from 5th to 95th percentile. (C) Representative flow cytometric analysis histograms of glucose analog 2-NBDG uptake by RA patient–derived PD-1– and PD-1+ B cells under the indicated conditions, (n = 5/group, 2 independent experiments). Data are represented as a box-and-whisker plot, with bounds from 25th to 75th percentile, median line, and whiskers ranging from 5th to 95th percentile. Ordinary 2-way ANOVA with Holm-Šidák multiple-comparisons test was performed. *P < 0.05, **P < 0.01. (D) Representative flow cytometric analysis plots of PD-1 expression by RA patient–derived B cells following stimulation in the presence of glucose analog 2DG (n = 4, 2 independent experiments). (E) Frequency of PD-1 B cells following incubation with STAT3 small molecule inhibitor Stattic (n = 3). Data are presented as mean ± SEM. Ordinary 2-way ANOVA with Holm-Šidák multiple-comparisons test was performed. **P < 0.01. (F) Effect of PD-1/PD-L1 engagement on PD-1+ RA patient–derived B cell expression of CD80 and CD86 and phosphorylation of AKT, mTOR, and S6 under normoxic or hypoxic conditions (n = 9). Data are represented as a box-and-whisker plot, with bounds from 25th to 75th percentile, median line, and whiskers ranging from 5th to 95th percentile. Statistical analysis was performed by using paired standard Student’s t test.