Background/Purpose: T peripheral helper (Tph) cells stimulate excessive B cell responses in the joints of patients with autoantibody-positive arthritis, including seropositive RA in adults and ANA-positive oligoarticular (oligo) JIA in children. Despite this potentially shared disease pathogenesis, a substantial proportion of children with oligo JIA enter long-term remission while this is rare in seropositive arthritis. To better understand these divergent outcomes, we studied the ability of regulatory T (Treg) cells to restrain Tph-B cell interactions in oligo and seropositive polyarticular (poly) JIA.

Methods: Synovial fluid (SF) from children with oligo and RF+ poly JIA (ILAR criteria) and peripheral blood (PB) from healthy controls were studied. Mononuclear cells were stained for surface markers, fixed/permeabilized for FOXP3 staining, and evaluated by flow cytometry. Bisulfite conversion pyrosequencing was done for methylation analysis of cytosine guanine dinucleotide (CpG) sites in the conserved noncoding sequence 2 (CNS2) region of FOXP3 (EpigenDx). SF Tph cells were co-cultured in the presence of SEB for 5 days with memory B cells (Bmem) obtained from controls with/without a given Treg population at a ratio of 1:10:1. The percentage of plasmablasts measured by flow cytometry and total IgG level measured by ELISA served as co-culture readouts.

Results: Table 1 summarizes the study participants. FOXP3, the lineage defining transcription factor for Treg cells, was measured in Treg and T effector (Teff) cells oligo JIA SF. We identified a population of SF Tregs that co-expressed Treg (CD4+CD25+CD127loFOXP3+) and B cell help (PD1int) markers, which we termed T peripheral regulatory (Tpr) cells (Fig 1A-B). By contrast, T cells in the Treg gate that were PD1hi expressed low levels of FOXP3. Similar to PB Tregs, Tpr cells maintained a Treg-specific methylation pattern while PD1hi T cells did not (Fig 1C-D). Confirming their suppressive capacity, the addition of Tpr cells to Tph-Bmem co-cultures reduced plasmablast differentiation and IgG production (Fig 1E-G). Next, we compared RF+ poly vs. oligo JIA and found that SF Tph cells were significantly more frequent in seropositive patients (Fig 2A-B). In the SF Treg compartment, PD1hi T cells predominated in RF+ poly JIA patients while PD1int Tpr cells were more plentiful in oligo JIA patients (Fig 2C-D). While not statistically significant, there was a trend towards reduced plasmablast differentiation in Tph-B cell co-cultures with PD1intTpr cells compared to PD1hi T cells from RF+ poly JIA patients (Fig 2E-F).

Conclusion: Tpr cells expressing intermediate levels of PD1 were enriched in the joints of oligo vs. RF+ poly JIA patients and displayed features of stable Treg cells, including robust FOXP3 expression, Treg-specific methylation patterns, and intact suppressive capacity. By contrast, the Treg compartment of seropositive JIA patients was characterized by PD1hi cells that had low FOXP3 expression, lack the Treg epigenic signature, and may be less suppressive. These findings suggest that Treg fitness and ability to suppress Tph-B cell interactions may play a role in disease course.

Figure 1. Tpr cells in synovial fluid from oligo JIA patients display features of stable Treg cells. A) Representative flow staining of T cells from the SF of oligo JIA patients. Histogram overlay showing the mean florescence intensity (MFI) of FOXP3 expression in the T cell subsets as indicated. B) Bar chart showing the % of FOXP3+ cells of the given Treg subsets in oligo JIA SF (n=8). CXCR5-expressing Treg subsets were excluded due to the insufficient cell number for analysis. C) Heatmap showing the % of methylation at 11 CpG sites in CNS2 of the FOXP3 locus in the sorted T cell populations. D) Bar charts showing the % of methylation in CpG sites of FOXP3 in the given T cell populations. For C-D) the following were studied: HC-PB Treg (CD4+CD127loCD25+) (n=3), oligo JIA-SF Tpr (CD4+CD127loCD25+PD1intCXCR5–) (n=5), oligo JIA-SF PD1hi T cells (CD4+CD127loCD25+PD1hiCXCR5–) (n=5), HC-PB Teff cells (CD4+CD25–) (n=3), oligo JIA-SF PD1neg T cells (CD4+CD25–PD1negCXCR5–) (n=5), oligo JIA-SF Tph cells (CD4+CD25–PD1hiCXCR5–) (n=5). An asterisk after the sample ID indicates male subjects. E) Representative flow staining showing % of plasmablast (CD38hiCD27+) of alive CD19+ B cells after co-culture of the given T cell populations with Bmem cells from the PB of a third party. Scatter plots showing F) the % of plasmablast and G) the level of total human IgG after co-culture SF Tph cells and an indicated Treg cell population from seroneg JIA-SF (n=8).

Mean ± SEM is shown. Statistical analysis: One-way ANOVA with Holm-Šidák ‘s correction for multiple comparisons. P-value: ns, >0.05; *, ≤0.05; **, ≤0.01; ***, ≤ 0.001; ****, ≤ 0.0001.

HC, healthy control; oligo JIA, oligoarticular juvenile idiopathic arthritis; poly, polyarticular; RF, rheumatoid factor; PB, peripheral blood; SF, synovial fluid; CpG, cytosine guanine dinucleotide; CNS2, conserved noncoding sequence 2; Bmem, memory B cells; Tph, T peripheral helper; Tpr, T peripheral regulatory cells; Teff, effector T; Treg, regulatory T.

Figure 2. Synovial fluid Treg compartment of RF+ poly JIA patients display features of destabilized Treg cells. A) Representative flow staining showing Tph cells (CD4+CD25–PD1hiCXCR5–) from the oligo JIA-SF and RF+ poly JIA-SF. B) Bar chart showing the % of Tph cells from oligo JIA-SF (n=12) and RF+ poly JIA-SF (n=4) compared to HC-PB (n=10). C) Representative flow staining showing Tpr (PD1intCXCR5–) and PD1hi T cells (PD1intCXCR5–) from the oligo JIA-SF and RF+ poly JIA-SF. D) Bar charts showing % of PD1hi and Tpr cells in SF from oligo JIA-SF (n=12) and RF+ poly JIA-SF (n=4) compared to HC-PB (n=10). E) Representative flow staining showing % of plasmablast (CD38hiCD27+) of alive CD19+ B cells after co-culture of the given T cell populations from a RF+ poly JIA patient with Bmem cells from the PB of a third party. F) Scatter plot showing the % of plasmablast after co-culture SF Tph cells and an indicated Treg cell population from RF+ poly JIA-SF (n=3) with Bmem from PB of a third party.

Mean ± SEM is shown. Statistical analysis: One-way ANOVA with Holm-Šidák ‘s correction for multiple comparisons. P-value: ns, >0.05; *, ≤0.05; **, ≤0.01; ***, ≤ 0.001; ****, ≤ 0.0001.

HC, healthy control; oligo JIA, oligoarticular juvenile idiopathic arthritis; poly, polyarticular; RF, rheumatoid factor; PB, peripheral blood; SF, synovial fluid; Bmem, memory B cells; Tph, T peripheral helper cells; Tpr, T peripheral regulatory cells; Teff, effector T cells; Treg, regulatory T cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/destabilized-treg-cells-predominant-in-severe-forms-of-juvenile-idiopathic-arthritis/