Jeroen N. Stoop

Therapeutic effect of tolerogenic dendritic cells in established collagen-induced arthritis is associated with a reduction in Th17 responses.

OBJECTIVE Tolerogenic dendritic cells (DCs) are antigen-presenting cells with an immunosuppressive function. They are a promising immunotherapeutic tool for the attenuation of pathogenic T cell responses in autoimmune arthritis. The aims of this study were to determine the therapeutic action of tolerogenic DCs in a type II collagen-induced arthritis model and to investigate their effects on Th17 cells and other T cell subsets in mice with established arthritis. METHODS Tolerogenic DCs were generated by treating bone marrow-derived DCs with dexamethasone and vitamin D(3) during lipopolysaccharide-induced maturation. Mice with established arthritis received 3 intravenous injections of tolerogenic DCs, mature DCs, or saline. Arthritis severity was monitored for up to 4 weeks after treatment. Fluorescence-labeled tolerogenic DCs were used for in vivo trafficking studies. The in vivo effect of tolerogenic DCs on splenic T cell populations was determined by intracellular cytokine staining and flow cytometry. RESULTS Tolerogenic DCs displayed a semi-mature phenotype, produced low levels of inflammatory cytokines, and exhibited low T cell stimulatory capacity. Upon intravenous injection into arthritic mice, tolerogenic DCs migrated to the spleen, liver, lung, feet, and draining lymph nodes. Treatment of arthritic mice with type II collagen-pulsed tolerogenic DCs, but not unpulsed tolerogenic DCs or mature DCs, significantly inhibited disease severity and progression. This improvement coincided with a significant decrease in the number of Th17 cells and an increase in the number of interleukin-10-producing CD4+ T cells, whereas tolerogenic DC treatment had no detectable effect on Th1 cells or interleukin-17-producing γ/δ T cells. CONCLUSION Treatment with type II collagen-pulsed tolerogenic DCs decreases the proportion of Th17 cells in arthritic mice and simultaneously reduces the severity and progression of arthritis.

Low-strength T-cell activation promotes Th17 responses

We show that the strength of T-cell stimulation determines the capability of human CD4(+) T cells to become interleukin-17 (IL-17) producers. CD4(+) T cells received either high- (THi) or low (TLo)-strength stimulation via anti-CD3/CD28 beads or dendritic cells pulsed with superantigen in the presence of pro-Th17 cytokines IL-1β, transforming growth factor β, and IL-23. We found that TLo, but not THi, stimulation profoundly promoted Th17 responses by enhancing both the relative proportion and total number of Th17 cells. Titration of anti-CD3 revealed that low TCR signaling promoted Th17 cells, but only in the presence of anti-CD28. Impaired IL-17 production in THi cells could not be explained by high levels of Foxp3 or transforming growth factor β-latency-associated peptide expressed by THi cells. Nuclear factor of activated T cells was translocated to the nucleus in both THi and TLo cells, but only bound to the proximal region of the IL-17 promoter in TLo cells. The addition of a Ca(2+) ionophore under TLo conditions reversed the pro-Th17 effect, suggesting that high Ca(2+) signaling impairs Th17 development. Although our data do not distinguish between priming of naive T cells versus expansion/differentiation of memory T cells, our results clearly establish an important role for the strength of T-cell activation in regulating Th17 responses.

Developing tolerogenic dendritic cell therapy for rheumatoid arthritis: what can we learn from mouse models?

One of the therapeutic strategies under development for the treatment of rheumatoid arthritis is based on reinstating immune tolerance by vaccination with autologous dendritic cells with potent tolerogenic function. These tolerogenic dendritic cells (TolDC) can be generated ex vivo and have beneficial therapeutic effects in animal models of arthritis. Although experimental animal models have been instrumental in the development of this novel immunotherapeutic tool, several outstanding questions regarding the application of TolDC remain to be addressed. This paper reviews what has been learnt to date from studying the therapeutic potential of TolDC in animal models of arthritis and discusses issues relating to preventive versus curative effects of TolDC, the antigen specificity of TolDC therapy, the route, dose and frequency of TolDC administration and the safety of TolDC treatment. Lessons learnt from animal models will aid the design of clinical trials with TolDC.

Mast cell depletion in the preclinical phase of collagen-induced arthritis reduces clinical outcome by lowering the inflammatory cytokine profile.

BackgroundRheumatoid arthritis (RA) is a multifactorial autoimmune disease, which is characterized by inflammation of synovial joints leading to the destruction of cartilage and bone. Infiltrating mast cells can be found within the inflamed synovial tissue, however their role in disease pathogenesis is unclear. Therefore we have studied the role of mast cells during different phases of experimental arthritis.MethodsWe induced collagen-induced arthritis (CIA), the most frequently used animal model of arthritis, in an inducible mast cell knock-out mouse and determined the effect of mast cell depletion on the development and severity of arthritis.ResultsDepletion of mast cells in established arthritis did not affect clinical outcome. However, depletion of mast cells during the preclinical phase resulted in a significant reduction in arthritis. This reduction coincided with a decrease in circulating CD4+ T cells and inflammatory monocytes but not in the collagen-specific antibody levels. Mast cell depletion resulted in reduced levels of IL-6 and IL-17 in serum. Furthermore, stimulation of splenocytes from mast cell-depleted mice with collagen type II resulted in reduced levels of IL-17 and enhanced production of IL-10.ConclusionsHere we show that mast cells contribute to the preclinical phase of CIA. Depletion of mast cells before disease onset resulted in an altered collagen-specific T cell and cytokine response. These data may suggest that mast cells play a role in the regulation of the adaptive immune response during the development of arthritis.

Abatacept decreases disease activity in the absence of CD4 + T cells in a collagen-induced arthritis model

IntroductionAbatacept is a fusion protein of human cytotoxic T-lymphocyte–associated protein (CTLA)-4 and the Fc portion of human immunoglobulin G1 (IgG1). It is believed to be effective in the treatment of rheumatoid arthritis by inhibiting costimulation of T cells via blocking CD28–B7 interactions as CTLA-4 binds to both B7.1 (CD80) and B7.2 (CD86). However, the interaction of CD28 with B7 molecules is crucial for activation of naive cells, whereas it is unclear whether the action of already activated CD4+ T cells, which are readily present in established disease, also depends on this interaction. The aim of this study was to determine whether the mode of action of abatacept depends solely on its ability to halt T cell activation in established disease.MethodsArthritis was induced in thymectomized male DBA/1 mice by immunisation with bovine collagen type II. The mice were subsequently depleted for CD4+ T cells. Abatacept or control treatment was started when 80 % of the mice showed signs of arthritis. Arthritis severity was monitored by clinical scoring of the paws, and anti-collagen antibody levels over time were determined by enzyme-linked immunosorbent assay.ResultsTreatment with abatacept in the absence of CD4+ T cells resulted in lower disease activity. This was associated with decreasing levels of collagen-specific IgG1 and IgG2a antibodies, whereas the antibody levels in control or CD4+ T cell–depleted mice increased over time.ConclusionsThese results show that abatacept decreased disease activity in the absence of CD4+ T cells, indicating that the mode of action of abatacept in established arthritis does not depend entirely on its effects on CD4+ T cell activation.

The choice of adjuvant determines the cytokine profile of T cells in proteoglycan‐induced arthritis but does not influence disease severity

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation of the synovial joints. Collagen‐induced arthritis (CIA) and proteoglycan‐induced arthritis (PGIA) are mouse models of inflammatory arthritis; CIA is a T helper type 17 (Th17) ‐dependent disease that is induced with antigen in complete Freunds adjuvant, whereas PGIA is Th1‐mediated and is induced using antigen in dimethyldioctadecyl‐ammonium bromide (DDA) as an adjuvant. To investigate whether the type of adjuvant determines the cytokine profile of the pathogenic T cells, we have compared the effect of CFA and DDA on T‐cell responses in a single arthritis model. No differences in incidence or disease severity between aggrecan‐T‐cell receptor transgenic mice immunized with aggrecan in either CFA or DDA were observed. Immunization with CFA resulted in a higher proportion of Th17 cells, whereas DDA induced more Th1 cells. However, the levels of interleukin‐17 (IL‐17) produced by T cells isolated from CFA‐immunized mice after antigen‐specific stimulation were not significantly different from those found in DDA‐immunized mice, indicating that the increased proportion of Th17 cells did not result in significantly higher ex vivo IL‐17 levels. Hence, the choice of adjuvant can affect the overall proportions of Th1 and Th17 cells, without necessarily affecting the level of cytokine production or disease incidence and severity.

Abatacept decreases disease activity in the absence of CD4+ T cells in a collagen-induced arthritis model

Background Abatacept is a fusion protein of human CTLA-4 and the Fc portion of human IgG1. It is believed to be effective in the treatment of rheumatoid arthritis by blocking the co-stimulation of T-cells via blocking CD28-B7 interaction as CTLA-4 binds to both B7.1 (CD80) and B7.2 (CD86). However, the interaction of CD28 with B7 is crucial for the activation of naïve cells, whereas it is unclear whether the action of already activated CD4+ T-cells, which are readily present in established disease, also depend on this interaction. Objectives The aim of this study was to determine whether the mode of action of Abatacept depends on its ability to halt T-cell activation in established disease. Methods Arthritis was induced in thymectomized male DBA/1 mice by immunisation with bovine collagen type II. The mice were subsequently depleted for CD4+ T-cells. Abatacept or control treatment was started when 80% of the mice showed signs of arthritis. Arthritis severity was monitored by clinical scoring of the paws and anti-collagen antibody levels over time were determined by ELISA. Results Treatment with Abatacept in the absence of CD4+ T-cells resulted in lower disease activity. This was associated with decreasing levels of collagen specific IgG1 and IgG2a antibodies while the antibody levels in control- or CD4+ T-cell-depleted mice increased over time. Conclusions These results show that Abatacept decreased disease activity in the absence of CD4+ T-cells indicating that the mode of action of Abatacept in established arthritis does not entirely depend on its effects on CD4+ T-cell activation. Disclosure of Interest None declared

DX5+CD4+ T cells modulate CD4+ T‐cell response via inhibition of IL‐12 production by DCs

DX5+CD4+ T cells have been shown to dampen collagen‐induced arthritis and delayed‐type hypersensitivity reactions in mice. These cells are also potent modulators of T‐helper cell responses through direct effects on CD4+ T cells in an IL‐4 dependent manner. To further characterize this T‐cell population, we studied their effect on DCs and the potential consequences on T‐cell activation. Here, we show that mouse DX5+CD4+ T cells modulate DCs by robustly inhibiting IL‐12 production. This modulation is IL‐10 dependent and does not require cell contact. Furthermore, DX5+CD4+ T cells modulate the surface phenotype of LPS‐matured DCs. DCs modulated by DX5+CD4+ T‐cell supernatant express high levels of the co‐inhibitor molecules PDL‐1 and PDL‐2. OVA‐specific CD4+ T cells primed with DCs exposed to DX5+CD4+ T‐cell supernatant produce less IFN‐γ than CD4+ T cells primed by DCs exposed to either medium or DX5−CD4+ T‐cell supernatant. The addition of IL‐12 to the co‐culture with DX5+ DCs restores IFN‐γ production. When IL‐10 present in the DX5+CD4+ T‐cell supernatant is blocked, DCs re‐establish their ability to produce IL‐12 and to efficiently prime CD4+ T cells. These data show that DX5+CD4+ T cells can indirectly affect the outcome of the T‐cell response by inducing DCs that have poor Th1 stimulatory function.

Reduced TCR-dependent activation through citrullination of a T-cell epitope enhances Th17 development by disruption of the STAT3/5 balance

Citrullination is a post‐translational modification of arginine that commonly occurs in inflammatory tissues. Because T‐cell receptor (TCR) signal quantity and quality can regulate T‐cell differentiation, citrullination within a T‐cell epitope has potential implications for T‐cell effector function. Here, we investigated how citrullination of an immunedominant T‐cell epitope affected Th17 development. Murine naïve CD4+ T cells with a transgenic TCR recognising p89‐103 of the G1 domain of aggrecan (agg) were co‐cultured with syngeneic bone marrow‐derived dendritic cells (BMDC) presenting the native or citrullinated peptides. In the presence of pro‐Th17 cytokines, the peptide citrullinated on residue 93 (R93Cit) significantly enhanced Th17 development whilst impairing the Th2 response, compared to the native peptide. T cells responding to R93Cit produced less IL‐2, expressed lower levels of the IL‐2 receptor subunit CD25, and showed reduced STAT5 phosphorylation, whilst STAT3 activation was unaltered. IL‐2 blockade in native p89‐103‐primed T cells enhanced the phosphorylated STAT3/STAT5 ratio, and concomitantly enhanced Th17 development. Our data illustrate how a post‐translational modification of a TCR contact point may promote Th17 development by altering the balance between STAT5 and STAT3 activation in responding T cells, and provide new insight into how protein citrullination may influence effector Th‐cell development in inflammatory disorders.

Immunosuppressive DX5+ T cells are potent inhibitors of Th-1 responses via modulation of DCs

Backgroundand objectives DX5+CD4+ T cells have been shown to have both a protective- and therapeutic effect on collagen-induced arthritis. This protective effect was associated with an increase in IL-10 production. To understand the mechanisms used by DX5+ T cells to dampen Th1-associated inflammation in CIA, the authors recently studied their immunomodulatory action on CD4+ T cells. These studies revealed that these cells are very effective in modulating Th1-cell responses through direct effects on CD4+ T cells by production of IL-4. In the presence of DX5+ T cells, IFNγ production was inhibited whereas IL-10 secretion was induced in responding CD4+ T cells. To further define additional mechanisms applied by DX5+ T cells to inhibit Th1-immunity, the authors studied the effects of DX5+ T cells on DC function. Materials and methods D011.10 (OVA specific TCR Tg) mice were used for the generation of bone marrow DCs (BMDCs) and for the isolation of CD4+ T cells. BMDCs were cultured with DX5+ or DX5- supernatants for 3 days. LPS was added after 1 day of incubation. The DCs obtained were cultured at 0.4×106/ml with OVA323–339 peptide and OVA specific CD4+ T at 1×106/ml in total volume of 150 µl for 3 days. At day 3, the secretion of cytokines was determined by flow cytometry. 12p70 levels in BMDCs cell culture supernatants were measured by ELISA. Results The authors demonstrate that DX5+ T cells can also indirectly inhibit Th1 responses through modulation of DC. DX5+ T cells robustly inhibit IL-12-production by DC. This effect was dependent on IL-10 produced by DX5+ T cells and does not require cell-cell contact. In addition, DX5+ T cells modulate the surface phenotype of LPS-matured DC as high levels of the “co-inhibitory” molecules PD-L1 and PD-L2 were induced. Importantly, OVA-specific CD4+ T cells primed by DC exposed to DX5+ T cell supernatant produced less IFNγ as compared to their counterparts primed by conventional DC. Addition of IL-12 restored IFNγ production. When IL-10 derived form DX5+ T cells was neutralised, DC re-established their ability to produce IL12 and to efficiently prime Th1 responses. Conclusion These data show that DX5+ T cells cannot only directly inhibit Th1-cell immunity, but also indirectly through the modulation of DC.