In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

IL-12 priming during initial antigen contact increases generation of effector and memory CD8 T cells by changing properties

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IL-15 and its role in rheumatoid arthritis

Background: IL-15 is involved in all phases of rheumatoid arthritis. Recently we have shown that rheumatoid arthritis synovial fibroblasts (RASF) express both IL-15 and functional IL-15 receptor [1]. Objective: The aim of present study was to identify pathways that are regulated by autocrine IL-15 (IL-15R) in RASF. Methods: RASF were transfected with plasmid encoding IL-15R antagonist (CRB-15, Cardion AG) or control constructs. RNA from transient transfectants were used for Microarray analysis. The differential expression of genes obtained by microarray analysis was verified by SYBR Green real-time PCR. The expression of IL-15Rα, cell proliferation and the expression of p16 and p21 were evaluated in stably transfected cells. Results: The IL-15R antagonist produced by transfected RASF blocked the endogenous IL-15/IL-15Rα interaction, which resulted in an inhibition of cell proliferation (45 ± 10%) via an increase of the expression of p16. In addition, we found that inhibition of IL-15Rα induced the expression of mRNA for FGFR-3. Since two isoforms of FGFR-3 have been identified (FGFR-3b and FGFR-3c) [2], we tested the effect of IL-15Rα inhibition on their expression. In contrast to FGFR-3b, the level of mRNA for FGFR-3c was strongly increased in cells transfected with the IL-15R antagonist (4.71 ± 2.5 in transient transfectants and 6.1 ± 1 fold in stable transfectants). FGFR-3c isoform binds specifically FGF-9, but also FGF-2 [2]. Besides FGFR-3, FGF-2 that is abundant in RA joints binds to FGFR-1. In vitro studies revealed that FGFR-1 transmits a potent mitogenic signal, whereas FGFR-3 usually has no stimulatory effect or inhibits cell proliferation. In contrast to FGFR-3c, blocking of IL-15Rα did not change the mRNA expression for FGFR-1 in RASF. Moreover, we checked whether FGF-2 affects the expression of IL-15Rα. Indeed, FGF-2 strongly decreased the spontaneous and tumor necrosis factor alpha-triggered expression of IL-15Rα at the mRNA and protein levels. Conclusion: Our findings raise the possibility of a negative loop between FGF-2/FGFR-3c and IL-15/IL-15R signaling in RASF. Moreover, the activation of RASF by FGFs could depend on the ratio of FGFR-1/FGFR-3 expression, which is controlled by the endogenous IL-15/IL-15R system

Antigen-specific suppression of inflammatory arthritis by dendritic cells

Purpose Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease. We have shown that NF-κB inactivation in dendritic cells (modified DC) converts them into cells that tolerize rather than immunize to specific antigen [1]. Antigen-exposed modified DC prevent priming of immunity, and they suppress previously primed immune responses. Regulatory CD4+ T cells, which can transfer antigen-specific tolerance in an IL-10-dependent fashion, mediate the tolerance. We hypothesized that modified DC exposed to arthritogenic antigen would suppress clinical arthritis after disease onset. Methods Antigen-induced arthritis was induced in C57/Bl6 mice by priming to methylated bovine serum albumin (mBSA) antigen followed by challenge injection of mBSA to one knee. Knee swelling was apparent within 2 days, with peak clinical signs apparent at 5 days. Mice were treated with antigen-exposed modified DC between 2 and 6 days after mBSA challenge to the knee joint. Results Clinical arthritis was suppressed in each group receiving mBSA-exposed modified DC within 4 days compared with mice that received either no DC or keyhole limpet hemocyanin-exposed modified DC. Clinical improvement was associated with mBSA-specific tolerance in mice receiving mBSA-exposed modified DC. Tolerance induction was not impaired by concomitant administration of anti-tumor necrosis factor alpha monoclonal antibody. Subsequent rechallenge with intra-articular IL-1 induced flare of arthritis in all groups, which could be effectively suppressed by a second administration of mBSA-exposed modified DC. Conclusions The data indicate that modified DC induce antigen-specific immune suppression in this model of inflammatory arthritis, even after full clinical expression of the disease. These observations have important implications for antigen-specific therapy of autoimmunity

Autoimmune Disorders

The present edition entitled "Autoimmune disorders - Pathogenetic aspects" aims to present the current available evidence of etiopathogenetic insights of both systemic and organ specific autoimmune disorders, the crossover interactions among autoimmunity, cardiovascular morbidity and malignancy as well as novel findings in the exciting fields of osteoimmunology and immunology of pregnancy