Reporters of TCR signaling identify arthritogenic T cells in murine and human autoimmune arthritis

Abstract

How pathogenic cluster of differentiation 4 (CD4) T cells in rheumatoid arthritis (RA) develop remains poorly understood. We used Nur77-a marker of T cell antigen receptor (TCR) signaling-to identify antigen-activated CD4 T cells in the SKG mouse model of autoimmune arthritis and in patients with RA. Using a fluorescent reporter of Nur77 expression in SKG mice, we found that higher levels of Nur77-eGFP in SKG CD4 T cells marked their autoreactivity, arthritogenic potential, and ability to more readily differentiate into interleukin-17 (IL-17)-producing cells. The T cells with increased autoreactivity, nonetheless had diminished ex vivo inducible TCR signaling, perhaps reflective of adaptive inhibitory mechanisms induced by chronic autoantigen exposure in vivo. The enhanced autoreactivity was associated with up-regulation of IL-6 cytokine signaling machinery, which might be attributable, in part, to a reduced amount of expression of suppressor of cytokine signaling 3 (SOCS3)-a key negative regulator of IL-6 signaling. As a result, the more autoreactive GFP^hi CD4 T cells from SKGNur mice were hyperresponsive to IL-6 receptor signaling. Consistent with findings from SKGNur mice, SOCS3 expression was similarly down-regulated in RA synovium. This suggests that despite impaired TCR signaling, autoreactive T cells exposed to chronic antigen stimulation exhibit heightened sensitivity to IL-6, which contributes to the arthritogenicity in SKG mice, and perhaps in patients with RA.

Keywords: Nur77; T cells; antigen receptor signaling; autoimmunity; rheumatoid arthritis.

Fig. 1.

Nur77 reporter of TCR signaling marks the selection of an autoreactive repertoire during thymic development with further pruning in the periphery. (A) Assessment of Nur77-eGFP fluorescence in SKGNur CD4⁺CD25⁻ T cells freshly isolated from popliteal lymph nodes (LNs) ± phosphate-buffered saline (PBS) or zymosan (Zym) and arthritic joints (+Zym). Data are representative of 10 mice in 4 independent experiments. (B) Comparison of expression of Nur77-eGFP and CD5 in preselection (pre; CD69^loTCR-β^lo) and postselection (post; CD69^hiTCR-β^hi) double-positive (DP), CD4SP (CD4⁺CD3^hiTCRβ^hi) thymocytes, and peripheral CD4⁺CD25⁻ naive (CD62L^hiCD44^lo) and memory (CD44^hiCD62L^lo) and CD4⁺CD25⁺ Treg lymphocytes. Results are representative of 2 independent experiments with 6 mice in each group. (C) Depiction of AMLR workflow. (D and E) Sorted CD4⁺CD25⁻ naive (CD62L^hiCD44^lo) GFP^lo and GFP^hi T cells were loaded with CTV dye and cocultured with nonirradiated APCs from BALB/c mice for 5 d. (D) Cells were subsequently stained for T-cell markers and assessed for dye dilution by flow cytometry.*P < 0.05; **P < 0.01. ns, not significant. (E) Box plots represent percent (%) division of GFP^lo and GFP^hi CD4 T cells as assessed using the Flow Jo v9 proliferation platform (center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range). Representative histograms of CTV dilution (D) and % cell division (E) from 4 experimental replicates (each containing 2 to 3 pooled mice) are shown. (F) Histograms represent Ki67 proliferation marker staining in GFP^lo and GFP^hi CD4⁺CD25⁻ naive (CD62L^hiCD44^lo) or memory (CD44^hiCD62L^lo) T cells. In C and E, a 2-tailed Student’s t test was used.

Fig. 2.

Nur77-eGFP marks arthritogenicity of CD4 T cells in SKGNur mice. (A) The 10% highest and lowest Nur77-eGFP–purified CD4 T cells sorted on naive markers (CD62L^hiCD44^lo) and depleted of Treg marker CD25 were adoptively transferred into SCID recipients. To prevent complications of early-onset CD4 T cell inflammatory bowel disease after adoptive transfer of naive SKG T cells, WT Tregs were added back in a ratio of 1:3. (B) Joints were assessed by histological staining 14 d following adoptive transfer and scored in a blinded fashion. Black arrows indicate normal synovial lining, and white arrows indicate synovial proliferation and joint destruction. (Scale bar, 100 μm.) (C) Dot plot of the histological score from SCID recipients that received GFP^lo (black) or GFP^hi (green) CD4 naive T cells from WTNur (square) or SKGNur (circle) mice. Each dot represents 1 biologically independent sample pooled from 3 experiments (mean ± SEM). ns, not significant. (D) Dots represent percentage of IL-17–producing GFP⁺ CD4 T cells from SCID recipient splenocytes stimulated in vitro with PMA and ionomycin for 5 h (mean ± SEM).

Fig. 3.

Autoreactive Nur77-eGFP^hi SKG T cells signal poorly. (A) Integrated TCR signaling inputs reflect Nur77-eGFP levels. (B) TCR-induced calcium increases in GFP^lo and GFP^hi negatively selected CD4⁺CD25⁻ T cells were barcoded from WTNur and SKGNur mice. Data are representative of 4 to 5 mice in each group from 3 independent experiments. Indo-1 AM, fluorescent Ca²⁺ indicator. Histograms represent phospho-ERK (p-ERK) (C) and p-S6 (D) levels analyzed in GFP^lo and GFP^hi CD4⁺CD25⁻ T cells from WTNur and SKGNur mice by flow cytometry after stimulation with α-CD3ε. Data are representative of at least 8 mice in each group from 4 independent experiments. (E) Sorted CD4⁺CD25⁻ naive GFP^lo and GFP^hi T cells were loaded with CTV dye and stimulated with plate-bound α-CD3ε and 2 μg/mL αCD28 for 3 d. Cells were subsequently assessed for dye dilution by flow cytometry. Data are representative of 2 pooled mice in each group from 2 independent experiments.

Fig. 4.

Inhibitory receptors are up-regulated in GFP^hi CD4 T cells. (A) Expression levels of inhibitory receptors assessed by flow cytometry in memory CD4⁺CD25⁻ cells of GFP^lo and GFP^hi populations from WTNur or SKGNur mice. Results shown are representative of 2 independent experiments. % of max, percentage of maximum. (B) Quantification of percentage of positive inhibitory receptor levels by 2-tailed Student’s t test. Box plots describe the median and interquartile range (IQR); whiskers extend to the largest value, but no further than 1.5 * IQR; and data points beyond whiskers are outlying points. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.

Fig. 5.

Enhanced IL-6 response in GFP^hi CD4 T cells from SKGNur mice. (A) Levels of phospho-STAT3 (p-STAT) (pY705) in GFP^lo and GFP^hi total CD4⁺CD25⁻ T cell populations from WTNur and SKGNur mice ± IL-6 treatment for 15 min. % of max, percentage of maximum. (B) Quantification of p-STAT3 mean fluorescence intensity (MFI) upon IL-6 stimulation from A. Data are pooled from 2 independent experiments (n = 6 mice per group). **P < 0.01, ***P < 0.001. (C) Quantification of total STAT3 MFI in WTNur (n = 5) and SKGNur (n = 10) total CD4⁺CD25⁻ T cells. ns, not significant. (D) IL-6 signaling pathway. (E) Surface expression of CD130 and CD126 in GFP^lo and GFP^hi CD4⁺CD25⁻ T cells. PE, phycoerythrin. (F) Quantification of CD130 and CD126 MFI (n = 4 per group from 2 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (G) Real-time RT-PCR measurements of Jak1 and Jak2 messenger RNA levels in sorted GFP^lo and GFP^hi CD4 T cells from WTNur and SKGNur mice (n = 5 to 7 mice per group from 2 independent sorts). **P < 0.01, ***P < 0.001, ****P < 0.0001. In B, F, and G, all box plots represent quantification of MFI or RNA relative expression (center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range). (H) Immunoblot analysis of SOCS3 and β-actin in unstimulated, negatively selected CD4 T cells from SKG and WT mice. Data are representative of 3 experiments. (I) Quantification of SOCS3 protein levels normalized to β-actin pooled from 3 immunoblot experiments as shown in H, with 11 to 12 mice in each group. **P < 0.01.

Fig. 6.

Reporter of TCR signaling can be used to identify antigen-activated CD4 T cells in human RA synovium. (A) Depiction of Nur77 induction by TCR signal transduction, but not cytokine signaling. (B) Histogram shows the overlay of endogenous Nur77 protein levels in the infiltrating CD4⁺CD25⁻ T cells from joints of SKGNur mice treated with phosphate-buffered saline (PBS) or zymosan. Data are representative of >10 mice in at least 5 independent experiments. (C) Comparison of messenger RNA transcript levels of Nr4A1 (Nur77) in sorted CD4⁺CD25⁻ T cells from lymph nodes (LNs) or joints of SKGNur mice treated with either PBS or zymosan (Zym). Each dot represents a biologically independent sample pooled from 2 independent experiments with 3 mice per group (mean ± SEM), using repeated measures ANOVA. ***P ≤ 0.001. ns, not significant. (D, Top) Flow cytometry plots of RA and osteoarthritis (OA) synovial tissue T cells demonstrate T cell populations from LiveCD45⁺ RA and OA synovium. PerCP-Cy5.5, Peridin–Chlorophyll–protein Cyanine5.5. (D, Bottom) Histograms represent Nur77 expression in CD4 (gate I) and CD8 (gate II) T cells from synovial RA (red) and OA (green) tissue, RA PBMCs (blue), and isotype control (filled-in gray histogram). PE, phycoerythrin; % of max, percentage of maximum. Dot plots demonstrate percentage of Nur77⁺ CD4 T cells (gate I) from paired RA PBMCs and joints (n = 6) (E) and percentage of Nur77⁺ CD4 T cells from 8 RA and 8 OA synovial tissues (F). *P ≤ 0.05 and ***P ≤ 0.001 by 2-tailed Student’s t test. Each dot represents a biologically independent sample (mean ± SEM).

Fig. 7.

Comparison of gene expression levels in synovium from patients with RA. The gene expression values were log2-transformed and z-scaled. The gene expression levels are summarized in the form of mean ± SEM. (A) Synovial SOCS3 expression levels. (B) Table with summary statistics for genes in the IL-6 signaling pathway. FC, fold change. (C) Expression levels for IL6ST, IL6R, JAK1, JAK2, and STAT3 genes. P values were computed by performing the nonparametric 2-sample Mann–Whitney–Wilcoxon test. ****P < 0.0001. ns, not significant.