Homeostatic signals, including IL-7 and self-MHC recognition, induce the development of peripheral helper T cells, which are enriched in the joints of rheumatoid arthritis

Abstract

Objective: Dysregulated T cell homeostasis has long been implicated in the pathogenesis of rheumatoid arthritis (RA), in the joint of which peripheral helper T (Tph) cells accumulate and form ectopic lymphoid organs. We examined whether homeostatic signals are involved in the development of Tph cells.

Methods: Human peripheral blood mononuclear cells were cultured with IL-7, the critical cytokine for T cell homeostasis. Development of Tph-like cells was assessed by flow cytometry, gene expression, and functional analysis. Chemotaxis of the Tph-like cells to RA synovial fluid (RASF) and the effect of RASF on the development of Tph-like cells was examined.

Results: PD-1^highCXCR5^- Tph-like cells developed from human peripheral blood CD4 T cells after proliferation in response to IL-7. Signals from self-MHC recognition and CD28 co-stimulation were also involved. The IL-7-induced Tph-like (IL-7-Tph) cells produced CXCL13 and IL-21 and helped B cells produce IgG. Comprehensive gene expression analysis further supported the similarity with Tph cells in RA joint. IL-7-Tph cells exhibited chemotaxis toward synovial fluid from RA patients (RASF), and RASF promoted the development of IL-7-Tph cells, which were also induced from CD4 T cells residing in non-inflamed joints.

Conclusions: Our results demonstrate an antigen-nonspecific developmental pathway of Tph cells triggered by homeostatic signals and promoted by the local environment of RA, which accounts for the accumulation of Tph cells in inflamed joints.

Keywords: Interleukin-7; Peripheral helper T cells; Rheumatoid arthritis.

Fig. 1

IL-7 induced the development of PD-1^highCXCR5- Tph-like cells from human PB CD4 T cells. (A) PBMCs from healthy human subjects were cultured with or without 50 ng/ml of IL-7 for 14 days. Representative dot plots showing the expression of PD-1 and CXCR5 on CD4 T cells are shown. The right panel (CD4 in RA joint) indicates the staining pattern of CD4 T cells isolated from RA joint. (B) The frequency (left) and cell number in a culture well (right) of PD-1^highCXCR5^- Tph-like cells induced with varying concentration of IL-7. (C) The frequency and number of PD-1^highCXCR5^- Tph-like cells induced from PBMC of HC and RA are shown (n = 6). (D) CTV-labelled PBMCs were cultured either with IL-7 (upper panels) or in plate wells precoated with anti-CD3 and anti-CD28 mAbs (lower panels). Expression of PD-1 and CTV staining of CD4 T cells cultured for the indicated days is shown. Induction of PD-1^highCXCR5^- Tph-like cells from PD-1-depleted (E) or CXCR5-depleted (F) PBMCs by IL-7. Left panels (d0) indicate the expression of PD-1 and CXCR5 on CD4 T cells before and after negative selection. ∗p < 0.05.

Fig. 2

Phenotypical and functional similarities between the IL-7-induced Tph-like (IL-7-Tph) cells and Tph cells in RA joint (RA-Tph cells). (A) Expression of surface molecules (ICOS, CD69, HLA-DR, CXCR3, and CCR6) on IL-7-Tph (n = 3) and RA-Tph cells (n = 3) are compared in histograms. Gray histograms indicate the staining with isotype controls. Graphs indicate the mean percentage of positive cells in PD-1^high CD4 T cells (n = 3). (B) Representative dot plots of cytokine production by the IL-7-Tph and RA-Tph cells with (lower panels) or without (upper panels) stimulation with PMA and ionomycin are shown. Graphs indicate the mean percentage of cytokine positive cells in PD-1^high CD4 T cells (n = 3). (C) Induction of plasmablasts (CD27⁺ CD38⁺) and IgG production from memory B cells cocultured with IL-7-Tph cells(n = 6). Representative dot plots of CD38 and CD27 expression on B cells are shown in left panels. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Fig. 3

Gene expression analysis of the IL-7-Tph cells. (A) Heatmap showing hierarchical clustering by DEGs among sorted naïve CD4 T (n = 3), IL-7-Tph (n = 3), and RA-Tph cells (n = 3). (B) Venn diagrams showing the number of the upregulated genes overlapped in IL-7-Tph and RA-Tph cells compared to naive CD4 T cells. (C) Volcano plots showing DEGs between IL-7-Tph and naive CD4 (upper), RA-Tph and naive CD4 (middle), and IL-7-Tph and RA-Tph (lower) cells. (D) GSEA analysis showing the pathway of genes upregulated in (C). (E) Expression levels of MAF, PRDM1, and PRDM1/BCL6 ratio in IL-7-Tph and RA-Tph cells by qRT-PCR analysis (n = 5). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 4

Signalling requirements for the development of IL-7-Tph cells. The frequency (left) and cell number in a culture well (right) of PD-1^high CTV^low cells after culturing PBMC with IL-7 in the presence or absence of anti-DR mAb (A) or CTLA-4 Ig (B) is shown (n = 9). (C) Effect of adding anti-IL-2 mAb and TGF-β was examined (n = 4). (D) Representative dot plots showing the induction of IL-7-Tph cells from either naïve (upper panels) or memory (lower panels) CD4 T cells as depicted by the expression of CD45RA and PD-1 (d0 and d14 left). Expression of CXCL13 and IL-21 in PD-1^high CTV^low cells after stimulation was shown in the right panels. (E) The effect of adding anti-DR mAb or CTLA-4 Ig on the induction of IL-7-Tph cells from naive (left) or memory (right) CD4 T cells (n = 5). ∗p < 0.05, ∗∗p < 0.01.

Fig. 5

Multiple mechanisms account for the accumulation Tph cells in the joint of RA. (A) Experimental system for the chemotaxis assay. (B) The frequency of PD-1^high CTV^low IL-7-Tph cells migrated toward wells with or without RA SF (n = 5). (C) Representative dot plots showing the development of IL-7-Tph cells with or without RASF. (D) The frequency of PD-1^high CTV^low IL-7-Tph cells induced with or without RA SF (n = 6). (E) Representative dot plots showing the expression of CXCL13 and IL-21 in the IL-7-Tph cells induced with RASF after stimulation. (F) PCA analysis on the gene expression of IL-7-Tph cells with or without RA SF, RA-Tph, and naive CD4 T cells (n = 3). (G) Representative dot plots showing the presence and absence of PD-1^highCXCR5- Tph cells in RA and OA joints, respectively. (H) Representative dot plots showing the development of IL-7-Tph cells from CD4 T cells in OA joints. Right panels show the expression of CXCL13 and IL-21 in PD-1^high CTV^low cells after stimulation.