Differential expansion of T peripheral helper cells in early rheumatoid arthritis and osteoarthritis synovium

Objectives Programmed cell death protein 1 (PD-1)-expressing T cells are implicated in the pathogenesis of autoimmune inflammatory diseases such as rheumatoid arthritis. A subset of CXCR5− T cells, termed T peripheral helper (Tph) cells, which drive B cell differentiation, have been identified in ectopic lymphoid structures in established rheumatoid arthritis synovial tissue. Here, we aimed to characterise these in treatment-naïve, early rheumatoid arthritis to determine whether these cells accumulate prior to fully established disease. Methods Fresh dissociated tissue and peripheral blood mononuclear cell (PBMC) suspensions were stained with Zombie UV, followed by anti-CD45RO, PD-1, CD3, ICOS, CD8, CD4, CD20, CXCR5, TIGIT and CD38 antibodies prior to analysis. For histology, rheumatoid arthritis synovial sections were prepared for Opal multispectral immunofluorescence with anti-CD45RO, CD20, PD-1 and CXCR5 antibodies. Images were acquired on the Perkin Elmer Vectra V.3.0 imaging system and analysed using InForm Advanced Image Analysis software. Results Flow cytometry revealed T cell infiltration in the rheumatoid arthritis synovium with differential expression of PD-1, CD45RO, ICOS, TIGIT and CD38. We observed a higher frequency of PD1hiCXCR5− Tph in rheumatoid arthritis synovial tissue and PBMCs versus controls, and no significant difference in T follicular helper cell frequency. Microscopy identified a 10-fold increase of Tph cells in early rheumatoid arthritis synovial follicular and diffuse regions, and identified Tph adjacent to germinal centre B cells. Conclusions These data demonstrate that PD-1hi Tph cells are present in early rheumatoid arthritis, but not osteoarthritis synovium, and therefore may provide a target for treatment of patients with early rheumatoid arthritis.


INTRODUCTION
Programmed cell death protein 1 (PD-1) is an inhibitory immune checkpoint molecule with a prominent role in modulating T cell activity. 1 Upon T cell activation, PD-1 is upregulated where it then induces inhibitory signals that limit the magnitude of the T cell response. This is elicited through interaction with the ligands PD-L1 and PD-L2. 2 Normally, the expression of PD-1 is transient, and expression is downregulated during the resolution phase of the insult following clearing of antigen. 2 However, in states of chronic antigen exposure, such as in cancer and autoimmunity, high PD-1 expression is sustained, 3 and is typically associated with states of T cell dysfunction or exhaustion. 4 PD-1-expressing T cells have been implicated in the autoimmune pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis (RA) [5][6][7][8] and Sjogren's syndrome. 9 Particularly, CD4 + PD-1 + CXCR5 + ICOS + T follicular helper (Tfh) cells secrete interleukin-21 and provide CD40:CD40L co-stimulatory signals to germinal centre (GC) B cells. 10

WHAT THIS STUDY ADDS
⇒ We demonstrate differentially enriched Tph cells in the early, treatment-naïve RA synovium and periphery compared with healthy and osteoarthritis controls. ⇒ Within the synovium, Tph cells far outnumbered T follicular helper cells, were located in close proximity to B cells in follicular and diffuse infiltrates, and their numbers correlated with B cell numbers.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY ⇒ Our study, demonstrating the presence of pathogenic Tph cells in early RA synovium, lays the potential of using Tph-based therapeutic approaches for disease interception prior to fully established RA.

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recombination and somatic hypermutation of B cell receptor genes and the affinity maturation of the autoantibody responses to drive autoimmunity. 11 In chronic autoimmune diseases, these cognate T-B cell interactions are thought to occur within ectopic lymphoid structures of inflamed tissues. 12 In RA, this occurs within the synovial tissue (ST), where CD4 + ICOS + PD-1 + CXCR5 + Tfh cells were thought to provide important signals to autoreactive B cells in RA GCs within ectopic lymphoid structures. 13 14 However, in 2017, Rao et al 7 described a T cell subset devoid of CXCR5 expression, termed 'T peripheral helper (Tph) cells' present in the established RA ST. 7 These cells exhibit an overlapping transcriptional profile to Tfh cells and can stimulate B cell differentiation to plasma cells, which secrete immunoglobulin. 7 There has been described a 'window of opportunity' where superior clinical responses and increased potential for remission are observed when patients with RA are treated early. 15 Critically, however, it remains unknown whether Tph cells accumulate in treatment-naïve early RA and contribute to disease establishment, and whether they may present a target for early therapeutic intervention. Here, we identify Tph cells and examine their localisation and interaction within the early RA ST and demonstrate differential enrichment of PD-1 hi Tph cells in ST from patients with treatment-naïve early RA when compared with patients with osteoarthritis (OA).

Human subjects
Arthroscopic synovial biopsies and venous blood were collected from four treatment-naïve patients with rheumatoid factor and anti-citrullinated protein antibody (ACPA)-positive early RA (<12 months of symptom onset and fulfilling the 2010 American College of Rheumatology/EULAR classification criteria). Blood was collected from three additional patients with early RA. Four non-inflammatory OA ST, five healthy control blood samples and three tonsil samples were collected as controls.

Cell proximity analysis
Analysis of the cell segmentation files exported from InForm was performed using R V.4.0.0 16 and the 'phenoptr' package. 17 Cell co-localisation was examined using nearest neighbour distances. Briefly, the phenoptr 'compute_all_nearest_distance' command computes the distance to the nearest neighbour cell in each segment of the merged segmentation file for each of the included phenotypes (B cell, GC B cell, Tfh, Tph). The nearest neighbour distances from each Tph cell (n=57 994) or Tfh cell (n=2741), to either GC B cells or B cells were described further in Stata V.16 (StataCorp, Texas, USA), using kernel density plots of the distance distribution for each phenotype. Differences in distance distributions between cells were compared by the Kruskal-Wallis test.

Statistical analysis
GraphPad Prism V.8.0 was used for statistical analysis. Mean differences were compared using t-tests or one-way analysis of variance followed by multiple comparison tests (for comparisons of three of more groups), and correlations were analysed using non-parametric, two-tailed Spearman correlations. P values of <0.05 were considered to be statistically significant.

RESULTS
Tph cells are present within the ST and periphery of treatment-naïve early RA We analysed CD4 + T cell populations present in seropositive, treatment-naïve early RA PBMC samples (n=7, table 1) using flow cytometry. CD4 + T cells were gated (online supplemental figure 1), and visualisation using We observed a higher frequency of PD-1 hi CXCR5 − Tph cells in the peripheral blood of patients with early RA compared with healthy controls (HC) (figure 1B,C), consistent with previous observations in patients with established RA. 7 PD-1 hi CXCR5 + Tfh cells were comparable between RA and HC, as were PD-1 int and PD-1 − CD4 + T cells, regardless of CXCR5 expression (figure 1C). We next assessed for correlation of Tph and Tfh cell numbers with clinical parameters; however, due to low sample size, we did not observe any correlation between Tph numbers and Disease Activity Score (DAS28) (p=0.8587), C reactive protein (CRP) (p=0.7571), cyclic citrullinated peptide antibody (CCP) (p=0.9282) or erythrocyte sedimentation rate (ESR) (p=0.9857). Likewise, Tfh numbers did not correlate with DAS28 (p=0.

. We additionally observed higher numbers of Tph cells than GC B cells in follicular and diffuse infiltrates, higher Tph cells than B cells in diffuse infiltrates, and no significant difference between Tph and B cells in follicular infiltrates (p=0.3125). Spearman rank correlations (figure 2D) identified significant positive correlation between numbers of B cells and GC B cells, Tfh, and Tph cells, between GC B cells and Tfh and Tph cells, and negative correlation between Tfh and Tph cells in follicular infiltrates. In diffuse infiltrates, we observed significant positive correlation between Tph and GC B cells, and negative correlation between Tph and Tfh cells.
Tph and Tfh cells co-localise with B cells in treatment-naïve early RA ST Following InForm cell phenotype analysis, we measured the distance between identified Tph and Tfh cells and B cells in diffuse and follicular regions of early RA ST (n=4) (figure 3). Nearest neighbour analysis revealed that GC B cells ( figure 3A) and B cells (figure 3B) were present within 20 µm of Tph and Tfh cells in both diffuse and follicular regions. We observed a higher probability that GC B cells were located in proximity with Tph cells in diffuse regions (figure 3A, left); while in follicular regions, there was a higher probability that GC B cells were in proximity with Tfh cells ( figure 3A, right).

DISCUSSION
Here, we demonstrate Tph cell, rather than Tfh cell, accumulation in the ST of patients with treatmentnaïve, early RA. These Tph cells can be divided into three subsets based on surface PD-1 expression. PD-1 hi

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cleavage of cell surface receptors, 19 this is unlikely to be causing our observation of low Tfh cell numbers. As demonstrated in our control tonsil digestions (figure 1B), CXCR5 + Tfh cells were abundant and within intact tissue sections, Tfh cell numbers were similarly observed to be low in both follicular and diffuse regions ( figure 2C). Thus, the low levels of Tfh cells observed within the RA ST in disaggregated samples likely reflect the cellular composition of the ST, rather than enzymatic cleavage of the surface receptor CXCR5 ( figure 1B). Finally, the location of Tph cells was not restricted to diffuse or follicular regions of the tissue, indicating their ability to provide B cell help both within lymphoid aggregates and in the diffuse inflamed tissue. Meanwhile, we observed limited numbers of Tfh cells even in follicular regions, likely reflecting their specialised niche within lymphoid organs. 20 21 Thus, together, these data suggest that Tph rather than Tfh cells provide B cell help within the early RA ST, potentially driving disease progression. Tfh numbers within the lymphoid organs of patients with early RA have been demonstrated to correlate with B cell numbers within the same site. 21 In our study, we assessed Tph and Tfh cell numbers in the early RA ST and identified positive correlation between Tph and Tfh cells with B cells and GC B cells in follicular regions, and a negative correlation between Tph and Tfh cells

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in diffuse regions ( figure 2D). This, together with our observation of abundant Tph cells (figure 2C), suggests that adaptive immune cell interactions involving Tph cells may be increased locally within the early RA ST. In the GC of lymphoid organs, help provided by Tfh cells is usually limited as they are outnumbered by B cells, therefore promoting affinity maturation of B cells with the highest affinity for antigen. 22 Our finding of abundant Tph cells further suggests that in the inflamed early RA ST, B cell help is readily available, potentially allowing affinity maturation of autoreactive B cell clones with low affinity, driving production of autoantibodies locally in the inflamed tissue.
The current standard treatment regimen for patients with RA is inadequate in preventing progression from early to established disease. Our previous study has shown the existence of CD4 + PD-1 + T cells in synovial biopsies of patients with early and established RA. 8 The present study expands on this, further demonstrating the dominant presence of CD4 + PD-1 + CXCR5 − Tph cells over Tfh cells in early RA ST. Furthermore, our study elaborates the cognate interaction between Tph cells and GC B cells in the synovial tissue, suggesting the key role of this interaction to produce autoantibodies in driving disease progression and tissue damage. Thus, our findings of PD-1 hi Tph cell accumulation within the early RA ST suggest that PD-1 hi Tph cells may be targeted in early disease to prevent further progression.
Immune checkpoint blockade, particularly in the form of therapeutics that interfere with PD-1:PD-L1 signalling, has revolutionised cancer treatment. [23][24][25] However, by inhibiting the natural mechanisms limiting the magnitude and duration of the T cell response, these inhibitors are associated with a range of immune-related adverse events (irAEs), 26 including inflammatory arthritis resembling RA. 2 27 Our findings of Tph expansion in early, treatment-naïve RA suggest a role of these cells in driving the initial phase of disease. Thus, inflammatory arthritis as an irAE in patients with cancer treated with PD-1-targeting therapeutics may be a manifestation of enhanced Tph cell function.
However, despite the development of irAE following PD-1 pathway blockade, 28 PD-1 may yet serve as a therapeutic target in the early stages of autoimmunity through pathway agonism. 6 Our demonstration here of PD-1 hi Tph cells in early RA indicates that compared with existing therapeutics, targeting T cells agonistically through PD-1 may inhibit both T cell-mediated and autoreactive B cellmediated tissue damage during early RA disease progression. Tph cells from patients with established RA have previously been demonstrated to promote plasma cell differentiation and induce IgG production. 7 Thus, depletion and/or agonism of PD-1 hi Tph cells in situ may limit local autoantibody production. Therefore, therapeutic targeting of the PD-1 pathway locally in the RA ST has the potential to affect both B and T cells in the treatment of early RA and thus, may provide deeper responses and higher remission rates than the current generation of therapeutics. Indeed, investigation into the utility of bispecific antibodies to treat RA is already underway, and early studies have demonstrated successful targeting to the ST. 29

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In summary, our results indicate that PD-1 is a potential target for treatment of early RA and may prevent the progression of the disease to fully established RA.