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

While autoantibodies are used in the diagnosis of rheumatoid arthritis (RA), the function of 8 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% O-2 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, C080, CD86,IL-1 beta, 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.