Isolation of functional autologous collagen-II specific IL-10 producing Tr1 cell clones from rheumatoid arthritis blood Valérie Brun a , Virginie Neveu a , Yves Marie Pers b , Sylvie Fabre b , Brigitte Quatannens a , Hervé Bastian a , Nathalie Clerget-Chossat a , Christian Jorgensen b , Arnaud Foussat a, ⁎ a TxCell, SA, Allée de la Nertière, 06560 Valbonne Sophia-Antipolis, France b Department of physical medicine & therapeutics of osteo-articular diseases, INSERM U844 Hôpital Saint Eloi-Bâtiment INM 80 rue Augustin Fliche 34295 Montpellier cedex 5, France abstract article info Article history: Received 4 February 2011 Received in revised form 1 March 2011 Accepted 2 March 2011 Available online 12 March 2011 Keywords: Regulatory Tr1 cells Immunotherapy Rheumatoid arthritis IL-10 producing regulatory type 1 (Tr1) cells represents a subpopulation of CD4+ regulatory cells able to prevent in vitro bystander T-cell proliferation and to inhibit a wide range of inflammatory diseases in mice. Our aim was to evaluate the frequency and function of joint specific Tr1 cells in the peripheral blood of severe Rheumatoid Arthritis (RA) patients. The collagen II protein was chosen to isolate Tr1 cells specific for a joint antigen. We successfully isolated Tr1 clones from 9 out of 11 RA patients. We showed that cells from patients display the same phenotype and surface marker regulation as previously shown for human Tr1 cells, characterized by expression of markers of regulation (FoxP3, CD25) at the activated but not at the resting state. Importantly, cells from patients showed Tr1 cytokine secretion (IL-10 and IFN-γ) and immunosup- pressive action on bystander T cell proliferation. Based on these results, we demonstrated that collagen II specific Tr1 cells can be isolated from the blood of severe refractory patients and that these cells are not altered in their phenotype and function. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Type 1 regulatory T lymphocytes (Tr1) together with natural Foxp3+ regulatory T (nTreg) cells are the two main regulatory T cell populations believed to have a crucial role in vivo in the tolerance mechanism to self and environmental antigens. Tr1 cell mechanism of action involves mainly Interleukin-10 secretion after antigen specific activation [1–4]. Indeed, adoptive transfer of Tr1 cells has been shown to suppress chronic inflammation in animals in a variety of disease models in an IL-10 dependent manner [3–6]. Interestingly, in vitro studies have demonstrated that immunosuppressive molecules such as vitamin D3 derivatives or steroid compounds are able to induce indirectly Tr1 cells by a tolerogenic action on dendritic cells [4,7,8]. In addition in the human settings, a wide array of immunosuppressive therapeutic strategies such as peptide immunization or TNFα blockade have been shown to induce Tr1 like cells suggesting that this regulatory population may be a key component of their mode of action in patients suffering from chronic inflammatory diseases [9,10]. Correlations have also been made between patient remission and the capacity of dendritic cells to differentiate Tr1 cells, particularly in Rheumatoid Arthritis patients [11]. The role of IL-10 for the control of Rheumatoid Arthritis and other inflammatory diseases has been largely debated. In general, approaches that proposed systemic delivery of IL-10 did not show high levels of efficacy. In contrast local delivery of IL-10 seems to be more encouraging as shown by the efficacy of local gut mucosa IL-10 delivery by use of probiotics in Crohn's Disease [12] or by IL-10 delivery in the inflamed joints after IL-10 gene transfer of joint antigen specific CD4+ T cells in the collagen-induce arthritis model in mice [13]. In this regard, transfer of natural IL-10 producing cells such as Tr1 cells, if targeted to the site of inflammation could be a new competitive strategy to deliver specifically and locally this immuno- suppressive cytokine. Beyond the classical medications used to control arthritic inflam- mation in Rheumatoid Arthritis patients, a lot of new compounds are now tested in clinical trials to try to block inflammation in severe refractory patients that do not respond to methotrexate or anti-TNFα agents. These new strategies involve different types of compounds such as proinflammatory cytokine blocking antibodies (Tocilizumab) or molecules targeting directly B cells or cross talk between immune cells (Abatacept) [14,15]. The main objective beyond inhibiting inflammation in autoimmune diseases is to restore immunogical homeostasis. For this latter purpose, cell therapy based on autologous regulatory cells such as Tr1 cells could be another of these new needed therapeutic strategies [16] using a more disease specific mode of action given by the fine antigen specificity of the cells. In this study, we evaluated a new technology to generate joint-antigen (collagen II) International Immunopharmacology 11 (2011) 1074–1078 ⁎ Corresponding author. Tel.: +33 497 218 300; fax: +33 493 641 580. E-mail address: foussat@txcell.com (A. Foussat). 1567-5769/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2011.03.001 Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp