Defective CD8+CD28+ regulatory T cell suppressor function in rheumatoid arthritis is restored by tumour necrosis factor inhibitor therapy

Abstract

Balanced immunoregulatory networks are essential for maintenance of systemic tolerance. Disturbances in the homeostatic equilibrium between inflammatory mediators, immune regulators and immune effector cells are implicated directly in the pathogenesis of autoimmune diseases, including rheumatoid arthritis (RA). In this study we characterize the peripheral blood CD8(+) CD28(-) regulatory T cells (Treg) contribution to the immunoregulatory network in health and in RA. In health, CD8(+) CD28(-) Treg are suppressive but, unlike CD4(+) Treg , they function predominantly through the action of soluble mediators such as interleukin (IL)-10 and transforming growth factor (TGF)-β. Neutralization of TGF-β consistently reduced CD8(+) CD28(-) Treg suppressor function in vitro. RA, CD8(+) CD28(-) Treg are increased numerically, but have reduced expression of inducible co-stimulator (ICOS) and programmed death 1 (PD-1) compared to healthy or disease controls. They produce more IL-10 but autologous T cells express less IL-10R. This expression was found to be restored following in-vitro addition of a tumour necrosis factor inhibitor (TNFi). Deficiencies in both the CD8(+) CD28(-) Treg population and reduced sensitivity of the T responder cells impact upon their regulatory function in RA. TNFi therapy partially restores CD8(+) CD28(-) Treg ability in vivo and in vitro, despite the defects in expression of functionally relevant molecules by RA CD8(+) CD28(-) Treg compared to healthy controls. This study places CD8(+) CD28(-) Treg cells in the scheme of immune regulation alongside CD4(+) Treg cells, and highlights the importance of understanding impaired responsiveness to regulation that is common to these suppressor subsets and their restored function in response to TNFi therapy.

Keywords: CD8+ T cells; anti-TNF therapy; immunotherapy; regulatory T cells; rheumatoid arthritis.

Fig. 1

CD8⁺CD28⁻ regulatory T cells (T_reg) in healthy control (HC) and diseased groups. Peripheral blood (PB) samples were stained with antibodies. (a) CD3.peridinin chlorophyll cyanin 5·5 (PerCPCy5·5) and CD8. phycoerythrin (PE)-positive cells were live gated (R1) and analysed for CD28.fluorescein isothiocyanate (FITC)-negative cells (R2). (b) CD8⁺CD28⁻ T_regs from HC (n = 24), osteoarthritis (OA) patients (n = 17), rheumatoid arthritis (RA) patients treated with methotrexate(MTX); RA(MTX) (n = 60) or tumour necrosis factor (TNF)-α inhibitors RA(TNFi) (n = 57) and subsets co-expressing CD56 (c) or CD94 (d) was determined. Next, peripheral blood mononuclear cells (PBMC) were stimulated with anti-CD3antibodies (1:1000) for 48 h and 4-1BB (e), programmed death 1 (PD-1) (f) or inducible co-stimulator (ICOS) (g) on CD8⁺CD28⁻ T_regs was examined. In paired PB and synovial fluid (SF) samples, RA (MTX) (n = 3) CD8⁺CD28⁻ T_regs (h, A), subsets co-expressing CD56 (h, B), CD94 (h, C), 4-1BB (h, D), PD-1 (h, E) or ICOS (h, F) and RA (iTNF) (n = 3) CD8⁺CD28⁻ T_regs (iA), subsets co-expressing CD56 (iB) or CD94 (iC) were examined. Significant differences were determined using Student's unpaired and paired t-test. *P < 0·04; **P < 0·005.

Fig. 2

CD8⁺CD28⁻ regulatory T cells (T_reg) from rheumatoid arthritis (RA) patients treated with methotrexate (RA(MTX) are functionally defective. CD8⁺CD28– T_regs were isolated by immunomagnetic bead separation from the peripheral blood (PB) of (a) healthy controls (HC) (n = 11), (b) RA(MTX) (n = 6); and (c) RA patients on tumour necrosis factor (TNF)-α inhibitor therapy; RA(TNFi) (n = 12). Cells were co-cultured with autologous responder peripheral blood mononuclear cells (PBMC) in the presence of anti-CD3 antibody. CD8⁺CD28⁻ T_regs from (d) HC (n = 3), (e) RA(MTX) (n = 3) or (f) RA(TNFi) (n = 3) were co-cultured with autologous CD14⁺ monocytes in the upper chamber of a transwell with autologous responder PBMC in the lower chamber, or in direct contact at 1:1 and stimulated with anti-CD3 antibody. (g) To determine the effect of a TNF inhibitor in vitro, infliximab (IFX) was added. RA(MTX) CD8⁺CD28⁻ T_regs were co-cultured with autologous responder peripheral blood mononuclear cells (PBMC) (ratio 1:1) and stimulated with anti-CD3 antibody in the absence or presence of infliximab (IFX) (a representative figure from five experiments is shown). (h) To determine if transforming growth factor (TGF)-β is critical for suppressor function, CD8⁺CD28⁻ T_regs were co-cultured with autologous responder PBMC (ratio 1:1) stimulated with anti-CD3 antibody in the absence or presence of an anti-TGF-β antibody. Cell proliferation was determined by the uptake of [³H]-thymidine for the final 18 h of culture (mean cpm ± standard error of the mean). Statistical significance in functional assays and transwell experiments was determined by the Student's paired t-test (P < 0·05) and one-way analysis of variance (ANOVA) (*P < 0·04), respectively.

Fig. 3

Cytokine profiles of stimulated CD8⁺CD28⁻ regulatory T cells (T_reg), peripheral blood mononuclear cells (PBMC) and co-cultures. CD8⁺CD28⁻ T_regs were isolated by immunomagnetic bead separation from the PB of healthy controls (HC) (n = 9), RA patients treated with methotrexate, RA(MTX) (n = 9) or tumour necrosis factor (TNF)-α inhibitors, RA(TNFi) (n = 3) and cultured alone or in 1:1 co-culture with autologous PBMC and stimulated with anti-CD3 antibody. Supernatants from cultures were collected at 24 h. (a) Interferon (IFN)-γ and (b) interleukin (IL)-10 production was detected using FlowCytomix beads. IL-10 receptor (IL-10R) expression by CD3⁺ T cells (c) of PBMC was determined over 48 h culture with anti-CD3. (d) To determine the effect of in vitro addition, of blocking TNF in RA(MTX) cultures, infliximab (IFX) was added in vitro and IL-10R expression on RA(MTX) T cells was determined 24 h after stimulation.

Fig. 4

Responder T cells from rheumatoid arthritis (RA) patients treated with methotrexate(MTX) patients are resistant to suppression by CD8⁺CD28⁻ regulatory T cells (T_reg). CD8⁺CD28⁻ T_regs isolated by immunomagnetic bead separation from peripheral blood (PB) samples from (a) healthy controls (HC) (n = 5) or (b) RA(MTX) (n = 3) were co-cultured with autologous responder T cells, two allogeneic RA(MTX) responder T cells or two allogeneic HC responder T cells. All cultures were stimulated with CD3/CD28 beads. Cell proliferation was determined by the uptake of [³H]-thymidine for the last 18 h of culture (mean ± counts per minute). Statistical significance was determined using the Student's paired t-test.