Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis

Abstract

CD4⁺ T cells are central mediators of autoimmune pathology; however, defining their key effector functions in specific autoimmune diseases remains challenging. Pathogenic CD4⁺ T cells within affected tissues may be identified by expression of markers of recent activation. Here we use mass cytometry to analyse activated T cells in joint tissue from patients with rheumatoid arthritis, a chronic immune-mediated arthritis that affects up to 1% of the population. This approach revealed a markedly expanded population of PD-1^hiCXCR5^-CD4⁺ T cells in synovium of patients with rheumatoid arthritis. However, these cells are not exhausted, despite high PD-1 expression. Rather, using multidimensional cytometry, transcriptomics, and functional assays, we define a population of PD-1^hiCXCR5^- 'peripheral helper' T (T_PH) cells that express factors enabling B-cell help, including IL-21, CXCL13, ICOS, and MAF. Like PD-1^hiCXCR5⁺ T follicular helper cells, T_PH cells induce plasma cell differentiation in vitro through IL-21 secretion and SLAMF5 interaction (refs 3, 4). However, global transcriptomics highlight differences between T_PH cells and T follicular helper cells, including altered expression of BCL6 and BLIMP1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1, and CCR5, in T_PH cells. T_PH cells appear to be uniquely poised to promote B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues.

Extended Data Figure 1. Detection of PD-1^hi CD4⁺ T cells in RA synovial tissue and fluid by mass and flow cytometry

a) viSNE plots of mass cytometry of RA synovial tissue CD4⁺ T cells as in Fig. 1a from 2 additional donors. b) Gating strategy to identify synovial tissue PD-1^hi CD4⁺ T cell populations by mass cytometry. c) viSNE plots of flow cytometry of paired RA synovial fluid and blood memory CD4⁺ T cells. d) Gating strategy to identify synovial fluid PD-1^hi memory CD4⁺ T cells by flow cytometry. e) Examples of gating used to sort memory CD4⁺ T cell populations from patient samples. f) Detection of CXCR5 mRNA by RT-PCR in sorted memory CD4⁺ T cell populations from synovial tissue (n=3 donors, 2 of which provided sufficient PD-1^hi CXCR5⁺ cells for analysis), synovial fluid (n=3 donors, 1 of which provided sufficient PD-1^hi CXCR5⁺ cells for analysis), and blood (n=2 donors). Purple boxes indicate PD-1^- and PD-1^hi CXCR5⁺ cells sorted from human tonsil as controls. Lines in (f) indicate mean for synovial or blood samples.

Extended Data Figure 2. PD-1^hi CXCR5^- CD4⁺ T cells are expanded in circulation of patients with active, seropositive RA and decrease with response to therapy

a) Mean expression of MHC II and ICOS in memory CD4⁺ T cell populations from synovial tissue (n=10), synovial fluid (n=9), and blood (n=42) from seropositive RA patients. Mean ± SD shown. b) Flow cytometric detection of PD-1 and CXCR5 expression on blood memory CD4⁺ T cells. c-e) Frequency of PD-1^hi cells that co-express MHC II or ICOS (c), PD-1^hi CXCR5⁺ cells (d) or cells with intermediate PD-1 expression (e) within memory CD4⁺ T cells from blood of patients with seropositive RA (n=42), seronegative RA (n=16), spondyloarthropathy (SpA, n=11), and non-inflammatory controls (n=35). f) Correlation between age or disease duration and blood PD-1^hi CXCR5^- cell frequency in seropositive RA patients (n=38). g) PD-1^hi CXCR5^- cell frequencies in seropositive RA patients segregated based on sex or medication usage (n=38). h) Correlation between serum anti-CCP antibody titer and blood PD-1^hi CXCR5^- cell frequency in all RA patients (n=53, black line, p=0.0049) or in only anti-CCP antibody⁺ patients (n=29, green line, p=0.48). i) Correlation between fold change in CDAI and fold change in PD-1^hi CXCR5^- cell frequency in patients 3 months after addition of a new RA medication (n=23; methotrexate=11, anti-TNF=4, abatacept=4, tocilizumab=2, tofacitinib=2). j) Frequency of PD-1^hi T cell subpopulations in blood before and after RA treatment escalation in 18 patients with reduced disease activity after therapy. Median ± interquartile range in c,d,e; mean ± SD in a,g shown. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by Mann-Whitney (c,g), Kruskal-Wallis (d,e), Wilcoxon test (j). In f,h,i p-values calculated by Spearman correlation.

Extended Data Figure 3. Blood PD-1^hi CXCR5^- CD4⁺ T cells express factors associated with B cell help

a) RT-PCR for intracellular regulators in memory CD4⁺ T cell populations from RA synovial fluid (n=5 or 6 donors). b) RT-PCR for cytokines (n=10 donors, 6 RA patients (black), 4 controls (grey)) and intracellular regulators (n=4 or 5 donors) in memory CD4⁺ T cell populations from blood. c) Cytokine and transcription factor mRNA expression in blood PD-1^hi memory CD4⁺ T cell populations divided according to CXCR5 expression, relative to PD-1^- cells (n=6 donors). d) Flow cytometric quantification of Bcl6 and Blimp-1 in blood PD-1^hi memory CD4⁺ T cell subpopulations sorted according to chemokine receptor expression, then stimulated in vitro for 2 days with anti-CD3/CD28 beads. Representative data from 1 of 3 experiments using cells from different donors. Median ± interquartile range in a,b; mean ± SD in c,d shown. * p<0.05, ** p<0.01, *** p<0.001 by Friedman's test, compared to PD-1^- MHCII^- group (a,b).

Extended Data Figure 4. Identification and characterization of circulating PD-1^hi CXCR5^- and PD-1^hi CXCR5⁺ in mass cytometry and RNA-seq analyses

a) Gating of blood PD-1^hi memory CD4⁺ T cells in mass cytometry analyses. b) Flow cytometric detection of FoxP3 and PD-1 in blood memory CD4⁺ T cells from RA patients (black, n=5) and controls (grey, n=3). c) Flow cytometric detection of inhibitory receptors on blood CXCR5^- memory CD4⁺ T cells. Data from 1 of 3 RA patients with similar results. d) Sorting strategy for PD-1^hi CXCR5^- and PD-1^hi CXCR5⁺ cell populations for RNA-seq. e) Hierarchical clustering T cell subpopulations sorted as in (d), with clustering based on expression of Tfh-associated genes measured by RNA-seq. f) Chemokine receptor expression on blood memory CD4⁺ T cells from RA patients (black) or controls (grey) by flow cytometry. Mean ± SD shown. * p<0.05, **p<0.001, *** p<0.0001 by Kruskal-Wallis test compared to PD-1^- cells (b) or Wilcoxon test (f).

Extended Data Figure 5. Limited interconversion of PD-1^hi CCR2⁺ and PD-1^hi CXCR5⁺ T cells in vitro

a) Flow cytometry of CXCR5 and CCR2 on gated PD-1^hi memory CD4⁺ T cells from blood. b) Expression of CXCR5 and CCR2 on indicated sorted PD-1^hi T cell populations 7 days after in vitro stimulation with anti-CD3/CD28 beads. c,d) Percentage of cells from each sorted PD-1^hi population that expressed CXCR5 or CCR2 on day 2 (c) or day 7 (d) after in vitro stimulation. Naive CD4⁺ T cells are shown as control. Mean ± SD shown (n=3 donors from 3 separate experiments).

Extended Data Figure 6. SLAMF5 is required for B cell-helper function of PD-1^hi CXCR5^- CD4⁺ T cells

a) Flow cytometric quantification of SLAM, SLAMF5, and SLAMF6 expression on memory CD4⁺ T cells (n=10 donors; 5 RA patients, 5 controls). b) Quantification of frequency of memory B cells with plasma cell markers after co-culture with PD-1^hi CXCR5⁺ CD4⁺ T cells with addition of blocking antibodies against SLAMF5 and/or SLAMF6. c) IgG quantification by ELISA in co-cultures of memory B cells with PD-1^hi CXCR5^- or PD-1^hi CXCR5⁺ T cells with addition of blocking antibodies against SLAMF5 and/or SLAMF6. For b,c) 1 of 3 experiments with similar results (n=3 replicates shown). Mean ± SD shown. * p<0.05, ** p<0.01, *** p<0.001 by Kruskal-Wallis compared to PD-1^- CXCR5^- (a) or isotype control (b,c). d) Immunofluorescence microscopy of CD20 (green), CXCR5 (red), and PD-1 (blue), in seropositive RA synovial tissue. Arrows point to PD-1^hi CXCR5^- cells adjacent to B cells. Scale bar = 50 microns.

Figure 1. Expanded PD-1^hi CXCR5^- CD4⁺ T cells in joints and blood of seropositive RA patients

a) viSNE plots of mass cytometry of RA synovial tissue total CD4⁺ T cells. Color indicates cell expression level of labeled marker. Circle demonstrates PD-1^hi cells. Arrow indicates CXCR5⁺ cells. b) PD-1^hi T cell frequency in RA synovial tissue (n=6). c) PD-1^hi CD4⁺ T cell frequencies in synovial fluid from seropositive RA (n=9) and seronegative inflammatory arthritides (n=19). d) PD-1^hi cell frequencies in seropositive RA synovial fluid (n=9) and tissue (n=10). e) Percentage of PD-1^hi CXCR5^- cells within memory CD4⁺ T cells in seropositive RA (n=42), seronegative RA (n=16), spondyloarthropathy (SpA, n=11), and control (n=35) patient blood. f) PD-1^hi frequency in blood of seropositive RA patients with low (n=14) or moderate-high (n=28) disease activity. g) PD-1^hi CXCR5^- CD4⁺ T cell and plasmablast frequencies in blood before and after RA treatment escalation (n=18). Mean ± SD in b,c,d, median ± interquartile range in e,f shown. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by Mann-Whitney (c,d), Kruskal-Wallis (e,f), Wilcoxon test (g).

Figure 2. Synovial PD-1^hi CXCR5^- CD4⁺ T cells express factors associated with B cell help

a) RT-PCR for cytokines in memory CD4⁺ T cell populations from RA synovial fluid (n=7 donors). Median ± interquartile range. b) Cytokine production by synovial fluid memory CD4⁺ T cells (n=3 experiments using different donors). c) Transcription factor expression in synovial tissue memory CD4⁺ T cells by flow cytometry. d) Quantification of transcription factor expression in T cells from synovial fluid (blue, n=3 donors) or synovial tissue (green, n=3 donors). For b,d, mean ± SD shown. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by Friedman's test compared to PD-1^- MHC II^- cells (a) or one-way ANOVA comparing PD-1^-CXCR5^-, PD-1^hi CXCR5^-, and PD-1^- CXCR5⁺ (d).

Figure 3. High dimensional analyses of PD-1^hi CXCR5^- and PD-1^hi CXCR5⁺ cells identify shared and distinct features

a) viSNE plots of blood memory CD4⁺ T cells from an RA patient. Circle indicates PD-1^hi cells. b) Difference in expression of significantly altered proteins between PD-1^hi populations and PD-1^- CXCR5^- cells (n=14 RA patients). c) Expression of indicated proteins by mass cytometry (n=7 RA patients (black) and 7 controls (grey)). d) PCA of RNA-seq transcriptomes (n=4 RA patients). e,f) Heatmap of expression of Tfh-associated genes (e) or chemokine receptors (f). g) CCR2 expression on PD-1^hi CD4⁺ T cells by flow cytometry (blood n=20, fluid n=5, tissue n=10). Mean ± SD shown. ** p<0.001, *** p<0.0001 by Wilcoxon (c), Kruskal-Wallis test (g).

Figure 4. PD-1^hi CXCR5^- CD4⁺ T cells promote plasma cell differentiation via IL-21 and SLAMF5 interactions

a) Plasma cell frequency in T cell-B cell co-cultures using memory CD4⁺ T cells from indicated sources. Pooled data from 2 experiments (synovial tissue, n=3 replicates per experiment), 3 experiments (synovial fluid), or 6 experiments (blood) using different donors. b) Co-cultures as in (a) using blood T cell subpopulations. c) IgG in supernatants of co-cultures. d,e) Co-cultures with IL-21R-Ig fusion protein (d) or anti-SLAMF5/SLAMF6 antibody (e). For b-e) 1 of 3 experiments with different donors (n=3 replicates). f,g) Immunofluorescence microscopy of RA synovium showing PD-1^hi CXCR5^- cells (white arrow) and PD-1^hi CXCR5⁺ cell (grey arrow). Scale bar = 50μM. h,i) Quantification of PD-1^hi cells in RA synovium (n=5-8 HPF from 4 samples). Mean ± SD shown. * p<0.05, ** p<0.01, *** p<0.001 by Mann-Whitney (a[synovial tissue],d), Kruskal-Wallis compared to PD-1^- CXCR5^- (a[blood, synovial fluid],c,e), or Wilcoxon test (h,i).