A critical epitope in CD147 facilitates memory CD4+ T-cell hyper-activation in rheumatoid arthritis

Abstract

The abnormal activation of CD4⁺CD45RO⁺ memory T (Tm) cells plays an important role in the pathogenesis of rheumatoid arthritis (RA). Previous studies have shown that CD147 participates in T-cell activation. However, it remains unclear whether CD147 is involved in abnormal Tm-cell activation in RA patients. In this study, we demonstrated that CD147 was predominantly upregulated in Tm cells derived from RA patients. The anti-CD147 mAb 5A12 specifically inhibited Tm-cell activation and proliferation and further restrained osteoclastogenesis. Using a structural-functional approach, we depicted the interface between 5A12 and CD147. This allowed us to identify two critical residues, Lys63 and Asp65, as potential targets for RA treatment, as the double mutation K63A/D65A inhibited Tm-cell activation, mimicking the neutralization by 5A12. This study provides not only a theoretical basis for a "CD147-Tm/Osteoclast-RA chain" for the potential prevention and treatment of RA or other T-cell-mediated autoimmune diseases but also a new target for related drug design and development.

Keywords: CD147; CD4+ Memory T cell; Immunotherapy; Monoclonal Antibody; Rheumatoid arthritis.

Fig. 1

RA patients exhibit a higher number of Tm cells with high expression of CD147. a Representative FACS data show the expression of CD69 on CD4⁺ T cells in PBMCs from healthy donors (HD) and RA patients, and three samples of SF from the latter were detected. b Summary data of CD69 expression on CD4⁺ T cells in PBMCs from HD (n = 10) and RA patients (n = 15). Each symbol represents a value from a single donor. The horizontal lines denote the mean ± SD expression. c Summary data of CD69 expression on CD4⁺ T cells in PBMCs and SF from RA patients from three independent experiments are shown. d Detection of CD4⁺CD45RO⁺ T cells in PBMCs from HD (n = 7) and RA patients (n = 7). e Detection of CD4⁺CD45RO⁺ T cells in PBMCs and SF from RA patients (n = 3). f CD147 expression on different peripheral blood cells from HD and RA patients was determined by FACS. g Expression of CD147 on Tm and Tn cells in both RA patients and HD. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 2

Tm-cell activation and proliferation is significantly inhibited by CD147 mAb 5A12. a, b CD147 engagement by 5A12 resulted in the downregulation of TCR-induced CD69 and CD25 expression on Tm cells from RA patients (n = 8). The expression of CD69 at 6 h and CD25 at 24 h following activation of purified CD4⁺CD45RO⁺ T cells by anti-CD3/CD28 mAbs with or without 5A12 treatments was determined. c In vitro, 5A12 inhibited Tm-cell proliferation induced via TCR engagement by anti-CD3/CD28 mAbs for 72 h, as determined by CFSE staining and FACS. d Representative data from one of three experiments are shown. Histograms were gated on the Tm population. e-h Similar experiments were performed to observe the effects of 5A12 on Tn cells. i Effects of 5A12 on phosphorylation of T-cell activation by Luminex analyses. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

Effect of mAb 5A12 on activated Tm-mediated osteoclastogenesis. a, b Enhanced osteoclastogenesis by activated Tm cells in a co-culture system but not by activated Tn cells. The corresponding statistical results for TRAP⁺ cells are shown. c, d Effects of 5A12 on Tm-mediated osteoclastogenesis. 5A12 could significantly inhibit Tm-mediated osteoclastogenesis in the presence or absence of rhM-CSF and RANKL. The corresponding statistical results of TRAP⁺ cells are shown. *P < 0.05; **P < 0.01; ns, not significant

Fig. 4

Downregulation of CD147 inhibits Tm-cell activation and proliferation. a FACS analyses showed the suppression of CD147 expression on Tm cells via lentiviral interference. The black lines represent the negative control memory T cells (NC), and the red lines represent the CD147 knockdown of the T cells (KD). b Knockdown of CD147 significantly inhibited Tm-cell proliferation in vitro induced via TCR engagement by anti-CD3/CD28 mAbs for 120 h, as determined by CFSE staining and FACS. c, d Knockdown of CD147 resulted in a prominent decrease of CD69 in vitro induced via TCR engagement by anti-CD3 mAb for 6 h and of CD25 via TCR engagement by anti-CD3/CD28 mAbs for 24 h on Tm cells. The corresponding statistical results for the inhibition rate and representative data are shown. **P < 0.01

Fig. 5

Crystal structure of the CD147_ecto-5A12_Fab complex and function test of CD147-K63A/D65A mutation type. a Overall structure of the CD147-5A12 complex. The ribbon represents the CD147 ectodomain (magenta) and 5A12 Fab (heavy chain, green; light chain, cyan). b The CD147 footprint on 5A12. The 5A12 Fab structure (gray) is rendered to show the surface features that contribute to its idiotope by its CDRs. Both the heavy and light chains are involved in the interactions with CD147. In a head-on view with respect to the idiotope rotated 90°, a groove is formed among CDR-H1, -H2 and -H3. c The interactions between CD147 and the 5A12 heavy chain. Stick representations of the βC’ of CD147-D1 (carbon, magenta; nitrogen, blue; oxygen, red) and the heavy chain residues of 5A12 (carbon, green; nitrogen, blue; oxygen, red) interacting with it. Hydrogen bonds are indicated as dashed black lines. d The interactions between CD147 and the 5A12 light chain. Stick representations of the βE of CD147-D1 (carbon, magenta; nitrogen, blue; oxygen, red) and the light chain residues of 5A12 (carbon, cyan; nitrogen, blue; oxygen, red) interacting with it. e Representative sensorgram for 5A12 and CD147 interaction. f Identification of CD147 expression in transfected cells. CD147 expression was significantly downregulated in the KD group compared with the negative control (NC); rescue of CD147 mutant (MT) and revertant type (RT) was performed by RNAi-resistant gene re-expression. g CD147-K63A/D65A mutation resulted in a prominent decrease of CD69 expression on memory T cells upon single anti-CD3 stimulation for 6 h. h, i CD147-K63A/D65A mutation resulted in a prominent decrease of both CD69 and CD25 expression on memory T cells upon double anti-CD3/CD28 stimulation for 24 h. The corresponding statistical results of the inhibition rate and representative data are shown. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 6

CD147-Tm/Osteoclast-RA chain. CD147 is predominantly upregulated on Tm cells from PBMCs of active RA patients. Anti-CD147 mAb 5A12 specifically inhibits Tm-cell activation and proliferation by decreasing ZAP70, LAT, and Erk phosphorylation and further restricts Tm-mediated osteoclastogenesis. Knockdown of CD147 (KD) or CD147 mutation (MT, K63A/D65A) on Tm cells exhibits similar inhibitory effects to 5A12, compared with a CD147 revertant (RT). Importantly, the K63D65 residues are the most important epitope in the CD147_ecto-5A12_Fab complex and hence are a potential target for RA treatments