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 3

Involvement of CXCR3 in the migration of peripheral blood memory B cells to the synovial tissue.

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Involvement of CXCR3 in the migration of peripheral blood memory B cells...
(A) SPICE algorithm flow cytometric analysis of peripheral blood and synovial tissue RA patient B cell expression of the chemokine receptors CXCR3, CXCR5, CCR6, and CCR7. (B) Representative gating followed for the flow cytometric analysis and identification of CXCR3-expressing peripheral blood, SF, and synovial tissue B cells (at least 6 independent experiments were performed). FMO, fluorescence minus one. (C) Frequency of RA patient CXCR3-expressing B cells in the periphery (n = 12), SF (n = 7), and synovial tissue (n = 6). Data are presented as mean ± SEM. Statistical analysis was performed by using 1-way ANOVA with Tukey’s multiple-comparisons test. **P < 0.01, ***P < 0.001. (D) Representative flow cytometric analysis plots and frequency of RA patient switched memory and DN memory B cells within the CXCR3+ peripheral blood B cell compartment (n = 4). Data are presented as mean ± SEM. Ordinary 2-way ANOVA with Holm-Šidák multiple-comparisons test was performed. (E) Effect of CXCR3 expression change following incubation of isolated RA patient–derived peripheral blood B cells with the indicated cytokines (n = 6/group). (F) Representative flow cytometric analysis and CD19+/counting bead ratio for of invading B cells toward cRPMI (control), RA synovial biopsy-conditioned media (SP), or RA synovial biopsy-conditioned media following treatment of the B cells with the CXCR3 small molecule antagonist AMG487 (n = 7/group, 3 independent experiments); 1-way ANOVA with Tukey’s multiple-comparisons test; *P < 0.05. 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. (G) Linear regression analysis between the frequency of RA patient peripheral blood CXCR3+ B cells and DAS28; n = 11; each symbol represents an individual sample. Spearman r correlation analysis was performed.