(Miltenyi Biotec, Paris, France). DCs were generated by culturing monocytes for 6 days in RPMI 1640 containing 10% fetal calf serum, interleukin-4 (IL-4; 500 ...
ARTHRITIS & RHEUMATISM Vol. 60, No. 9, September 2009, pp 2848–2854 © 2009, American College of Rheumatology
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secretion of human DC chemokines, and we investigated the function of each of the Treg cell–associated molecules, including CTLA-4, in modulating DC chemokines. Monocytes from peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using CD14 beads (Miltenyi Biotec, Paris, France). DCs were generated by culturing monocytes for 6 days in RPMI 1640 containing 10% fetal calf serum, interleukin-4 (IL-4; 500 IU/106 cells), and granulocyte–macrophage colony-stimulating factor (GM-CSF; 1000 IU/106 cells). To isolate Treg cells, CD4⫹CD45RO⫹ memory T cells were first purified from PBMCs by negative selection (Invitrogen, Cergy-Pontoise, France). CD4⫹CD45RO⫹ CD25high Treg cells were then isolated from purified CD4⫹CD45RO⫹ T cells by positive selection using CD25 beads (Miltenyi Biotec). The flow-through population, depleted of CD25⫹ cells, was used for comparison with Treg cells. DCs were either cultured in medium containing GM-CSF and IL-4 or cocultured with T cell subsets at a 1:5 ratio for 72 hours. As shown in Figure 1A, DCs cultured with Treg cells had significantly reduced levels of secretion of several key chemokines, i.e., CCL3, CCL17, CCL19, and CCL22, which are critically involved in mediating inflammatory responses. (In acute inflammatory conditions, CCL3 mediates the recruitment and activation of polymorphonuclear cells and T cells. CCL17 and CCL22 play a role in mediating both influx of CCR4⫹ cells and allergic inflammation. CCL19 is involved in attracting CCR7⫹ cells.) Whereas, in the cells from 10
DOI 10.1002/art.24784
CD4ⴙCD25ⴙ regulatory T cells modulate human dendritic cell chemokines via multiple mechanisms: comment on the article by Kolar et al To the Editor: We read with interest the recent article by Kolar et al in which the authors report that expression of CTLA-4 maintains the suppressive function of regulatory T cells (Treg cells) (1). Naturally occurring CD4⫹CD25⫹ Treg cells play a critical role in maintaining immune tolerance (2). Deficiency of Treg cells can lead to fatal autoimmune diseases and inflammatory conditions with dysregulated lymphoproliferation (2,3). Kolar and colleagues found that, following blockade of CTLA-4, Treg cells have a reduced capacity to suppress effector T cells. In addition to suppressing effector T cells, Treg cells can target several other cell types, including professional antigen-presenting cells (APCs), such as dendritic cells (DCs) (4). DCs are sentinels within the immune system and play a crucial role in initiating, regulating, and maintaining immune responses. DCs secrete a wide range of chemokines, which direct immune cell migration and regulate adhesive interactions between cells (5). Thus, DC-derived chemokines play an important role in determining the fate of immune responses and are critically involved in the pathogenesis of autoimmune diseases and inflammation. Therefore, we questioned how Treg cells suppress immune responses by modulating the
Figure 1. Regulatory T cells (Treg cells) modulate human dendritic cell (DC) chemokines by multiple mechanisms. A, Modulation of DC chemokines by regulatory T cells. Human DCs were cultured in the presence of granulocyte–macrophage colony-stimulating factor and interleukin-4 (IL-4) as a control (Ctr) or were cocultured with CD4⫹CD45RO⫹CD25⫹ Treg cells (Treg) or CD4⫹CD45RO⫹ memory T cells depleted of Treg cells (RO), at a 1:5 ratio for 72 hours. Chemokines in coculture were quantified in cell-free culture supernatants by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Lille, France). Bars show the mean and SEM levels of chemokine secretion (n ⫽ 10 donors). ⴱ ⫽ P ⬍ 0.05 versus Treg cell–depleted cells, by Mann-Whitney test. B, Effects of antibodies on Treg cell–mediated regulation of DC chemokine secretion. DCs were cocultured with Treg cells for 72 hours in the absence or presence of individual blocking/neutralizing antibodies. Anti–transforming growth factor  (anti-TGF; R&D Systems), anti–IL-10 (R&D Systems), and anti–CTLA-4 (Ancell, Bayport, MN) monoclonal antibodies were used at a concentration of 10 g/ml, and anti–lymphocyte activation gene 3 (anti–LAG-3; Immutep, Chatenay-Malabry, France) was used at a concentration of 20 g/ml. Chemokines in coculture were quantified by ELISA. Bars show the mean and SEM levels of secretion (n ⫽ 2 donors). 2848
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donors, Treg cell–depleted CD4⫹CD45RO⫹ cells induced high amounts of CCL3 (mean ⫾ SEM 82.7 ⫾ 14.7 pg/ml), CCL17 (81.9 ⫾ 34.9 ng/ml), CCL19 (473.5 ⫾ 79.4 pg/ml), and CCL22 (116 ⫾ 27.4 ng/ml), Treg cells significantly inhibited the secretion of all of these chemokines (mean ⫾ SEM 15.7 ⫾ 4 pg/ml, 37.9 ⫾ 13.4 ng/ml, and 70.7 ⫾ 21.4 ng/ml for CCL3, CCL17, and CCL22, respectively, with CCL19 not induced at all). Thus, our results suggest that, in addition to suppressing T cells, Treg cells can diminish the inflammation and immune response by inhibiting the secretion of DC chemokines implicated in the migration of immune cells. We then determined the effects of specific molecules on Treg cells in regulating DC chemokine secretion. We examined the effects of blocking/neutralizing antibodies to CTLA-4, transforming growth factor  (TGF), IL-10, and lymphocyte activation gene 3 (LAG-3), all of which have been previously reported to contribute to Treg cell activity in certain contexts (2,4,6). DCs were cocultured with Treg cells for 72 hours in the presence of individual blocking/neutralizing antibodies, after which secretion of chemokines was analyzed. Notably, CTLA-4, TGF, and LAG-3 blocking/neutralization partially (though nonsignificantly) reversed the Treg cell– mediated suppressive effect on the secretion of DC-derived CCL17 and CCL22 (Figure 1B); no single blocking/neutralizing antibody completely reversed the Treg cell effects. IL-10 neutralization had minimal effect on CCL17 and CCL22 secretion. None of the antibodies affected the secretion of CCL18 (Figure 1B), or of CCL3 and CCL19 (data not shown). Together, the data indicate that multiple mechanisms contribute to the modulation of DC chemokine secretion by Treg cells. Several non–mutually exclusive mechanisms have been proposed to account for the maintenance of immune tolerance by Treg cells (2). Thus, Treg cells modulate the maturation, activation, and functions of both human and murine DCs (7–11). Recent reports suggest that CTLA-4 plays a major role in Treg cell–mediated immunosuppression in mice (10–12). We found that, in addition to CTLA-4, the modulation of DC chemokine secretion by human Treg cells may be partially regulated by TGF, LAG-3, and (to a lesser degree) IL-10. It is possible that each molecule associated with Treg cells has a distinct role in modulating various aspects of DC biology, such as maturation and expression of costimulatory molecules, secretion of cytokines and chemokines, expression of antiinflammatory molecules, and T cell stimulatory functions. Thus, CTLA-4 has a direct role in regulating DC stimulatory capacity (10,11), while the remaining Treg cell accessory molecules regulate other aspects of DC-mediated immune responses, such as secretion of cytokines and chemokines. The cumulative effect of all of these accessory molecules of Treg cells might impart potent tolerogenic properties to DCs. It remains to be determined, however, whether CTLA-4, TGF, IL-10, and LAG-3 represent components of the same pathway or of multiple pathways that converge at a common point. Supported by grants from INSERM, Centre National de la Recherche Scientifique, UPMC-Paris VI, and Agence Nationale de la Recherche (ANR-07JCJC-0100-01). Ms Navarrete, Mr. Meslier, Ms Teyssandier, and Dr. Andre´ are recipients of Ile-de-France, Fondation pour la Recherche Me´dicale, Ministe`re de la Recherche, and ANR-07-RIB-002-02 fellowships, respectively. Dr. Triebel owns stock options in Immutep and holds patents for LAG-3 monoclonal antibodies.
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Ana-Maria Navarrete, MSc Yann Meslier, MSc Maud Teyssandier, MSc Se´bastien Andre ´, PhD Sandrine Delignat, BSc INSERM Unite´ 872 Universite´ Pierre et Marie Curie and Universite´ Paris Descartes Paris, France Fre´de´ric Triebel, MD, PhD Immutep Orsay, France Srini V. Kaveri, DVM, PhD Se´bastien Lacroix-Desmazes, PhD Jagadeesh Bayry, DVM, PhD INSERM Unite´ 872 Universite´ Pierre et Marie Curie and Universite´ Paris Descartes Paris, France 1. Kolar P, Knieke K, Hegel JK, Quandt D, Burmester GR, Hoff H, et al. CTLA-4 (CD152) controls homeostasis and suppressive capacity of regulatory T cells in mice. Arthritis Rheum 2009;60:123–32. 2. Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell 2008;133:775–87. 3. Valencia X, Lipsky PE. CD4⫹CD25⫹FoxP3⫹ regulatory T cells in autoimmune diseases. Nat Clin Pract Rheumatol 2007;3:619–26. 4. Tang Q, Bluestone JA. The Foxp3⫹ regulatory T cell: a jack of all trades, master of regulation. Nat Immunol 2008;9:239–44. 5. Allen SJ, Crown SE, Handel TM. Chemokine: receptor structure, interactions, and antagonism. Annu Rev Immunol 2007;25:787–820. 6. Liang B, Workman C, Lee J, Chew C, Dale BM, Colonna L, et al. Regulatory T cells inhibit dendritic cells by lymphocyte activation gene-3 engagement of MHC class II. J Immunol 2008;180:5916–26. 7. Tang Q, Adams JY, Tooley AJ, Bi M, Fife BT, Serra P, et al. Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice. Nat Immunol 2006;7:83–92. 8. Houot R, Perrot I, Garcia E, Durand I, Lebecque S. Human CD4⫹CD25high regulatory T cells modulate myeloid but not plasmacytoid dendritic cells activation. J Immunol 2006;176: 5293–8. 9. Bayry J, Triebel F, Kaveri SV, Tough DF. Human dendritic cells acquire a semimature phenotype and lymph node homing potential through interaction with CD4⫹CD25⫹ regulatory T cells. J Immunol 2007;178:4184–93. 10. Onishi Y, Fehervari Z, Yamaguchi T, Sakaguchi S. Foxp3⫹ natural regulatory T cells preferentially form aggregates on dendritic cells in vitro and actively inhibit their maturation. Proc Natl Acad Sci U S A 2008;105:10113–8. 11. Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z, et al. CTLA-4 control over Foxp3⫹ regulatory T cell function. Science 2008;322:271–5. 12. Friedline RH, Brown DS, Nguyen H, Kornfeld H, Lee J, Zhang Y, et al. CD4⫹ regulatory T cells require CTLA-4 for the maintenance of systemic tolerance. J Exp Med 2009;206:421–34.
DOI 10.1002/art.24769
Reply To the Editor: Naturally arising CD4⫹CD25⫹ Treg cells appear to be central control elements within immunoregulation. Treg cells can restrict most types of immune responses, ranging from
