Foxp3+ Regulatory T Cells Control Humoral Autoimmunity by Suppressing the Development of Long-Lived Plasma Cells This information is current as Eunkyeong Jang, Wang Sik Cho, Mi-La Cho, Hyun-Joo of September 30, 2021. Park, Hye-Joa Oh, Sang Mee Kang, Doo-Jin Paik and Jeehee Youn J Immunol 2011; 186:1546-1553; Prepublished online 5 January 2011; doi: 10.4049/jimmunol.1002942 Downloaded from http://www.jimmunol.org/content/186/3/1546 References This article cites 41 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/186/3/1546.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Foxp3+ Regulatory T Cells Control Humoral Autoimmunity by Suppressing the Development of Long-Lived Plasma Cells Eunkyeong Jang,* Wang Sik Cho,† Mi-La Cho,‡ Hyun-Joo Park,* Hye-Joa Oh,‡ Sang Mee Kang,* Doo-Jin Paik,† and Jeehee Youn*,† Foxp3+ regulatory T cells (Tregs) are crucial for maintaining T cell tolerance, but their role in humoral autoimmunity remains unclear. To address this, we combined a model of autoantibody-dependent arthritis (K/BxN) with Foxp3 mutant scurfy mice to generate Treg-deficient K/BxN mice, referred to as K/BxNsf mice. The disease symptoms of K/BxNsf mice were exacerbated, and this coincided with increases in extrafollicular Th cells, follicular Th cells, and germinal centers. Surprisingly, the K/BxNsf mice exhibited an abnormal accumulation of mature plasma cells in their spleens and a corresponding loss of bone marrow plasma cells. The plasma cells were unresponsive to the bone marrow homing chemokine CXCL12, despite normal expression of the chemokine receptor CXCR4. Importantly, they were long-lived and less susceptible to the cytotoxic action of cyclophosphamide. Downloaded from They also expressed less FcgRIIb and were less apoptotic in response to autoantigen–autoantibody immune complexes. This suggests that Tregs control plasma cell susceptibility to cell death induced by engagement of FcgRIIb with immune complexes. Direct cytotoxic effects of Tregs also contribute to the death of plasma cells. Thus, our results reveal that Tregs suppress the emergence of long-lived splenic plasma cells by affecting plasma cell-autonomous mechanisms as well as T cell help, thereby avoiding the persistence of humoral autoimmunity. The Journal of Immunology, 2011, 186: 1546–1553. http://www.jimmunol.org/ ystemic autoimmune diseases, such as systemic lupus cursors that emerge from these reactions are dividing, rapidly erythematosus (SLE) and rheumatoid arthritis, are char- turning over, Ab-secreting plasmablasts, which in turn differenti- S acterized by activation of autoreactive T and B cells (1, 2). ate into nondividing PCs (6). Most PCs either die within 3–4 d in This activation represents a functional loss of self-tolerance and these organs, which apparently provide only few survival niches leads to autoantibody production. Autoantibodies trigger com- for the cells, or leave the organs in search of survival niches plement- and FcR-mediated inflammatory responses in the target provided mainly by the bone marrow (BM). After arriving at the tissue, which in turn promote tissue destruction, more extensive BM, PCs terminally differentiate to end-stage Ab-secreting cells loss of self-tolerance, and ultimately symptoms of autoimmunity and attain a long life span of months or even years. Therefore, by guest on September 30, 2021 (3). Therefore, persistent production of pathogenic autoantibodies although a small proportion of long-lived PCs persist in the spleen is central to the chronic, destructive clinical manifestations of sys- and LNs, most long-lived PCs reside in the BM. These PCs are not temic autoimmune diseases. eliminated by treatments targeting B lineage cells, such as irra- The persistence of autoantibodies in autoimmune disorders can diation, prednisone, cyclophosphamide, and anti-CD20 Abs (7– be attributed either to a continuous supply of short-lived plasma- 10). Thus, along with their persistent Ab production, the resistance blasts or to the activity of long-lived plasma cells (PCs). Both of of these PCs to cytostatic treatment contributes to the difficulty of these cell populations develop as a result of Ag-specific, cognate resolving long-lived PC-mediated diseases. Despite the patho- interactions with extrafollicular T helper (TEFH) cells at extra- logical significance of long-lived PCs, little is known about how follicular B cell foci or with follicular T helper (TFH) cells within they develop and persist in vivo in autoimmune states. germinal centers (GCs) formed in peripheral lymphoid organs, Several of the steps generating peripheral autoimmunity can be such as spleen and lymph nodes (LNs) (4, 5). The early PC pre- censored by CD4+Foxp3+ regulatory T cells (Tregs). These cells were originally identified by their capacity to suppress the pro- *Department of Biomedical Sciences, College of Medicine, Hanyang University, liferation and activation of other T cells (11). However, recent Seoul 133-791, Korea; †Department of Anatomy and Cell Biology, College of Med- studies have extended their targets to diverse immune cells in- icine, Hanyang University, Seoul 133-791, Korea; and ‡Research Institute of Medical Science, The Catholic University of Korea, Seoul 137-701, Korea cluding B cells. Tregs have been shown to suppress directly the Received for publication September 2, 2010. Accepted for publication November 30, activation of B cells, in addition to their indirect effect through 2010. suppressing the activity of TFH cells (12, 13). Their suppressive This work was supported by a National Research Foundation grant funded by the activities include perforin/granzyme-dependent cytotoxicity tar- Korean government (MEST) (2008-0058736 by Mid-career Researcher Program and geting B cells as well as T cells (14–16). In agreement with these 2009-0081790). in vitro studies, the titer of autoantibodies in autoimmune subjects Address correspondence and reprint requests to Dr. Jeehee Youn, Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, 17 is inversely correlated with the activity of Tregs. For instance, Haengdang-dong, Sungdong-gu, Seoul 133-791, Korea. E-mail address: jhyoun@ depletion and transfer of Tregs in autoimmune animals lead to in- hanyang.ac.kr creased and decreased autoantibody production, respectively (17, Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; BM, bone mar- 18). Reversal of the numerical deficit of Tregs in SLE reduces titers row; dLN, joint-draining lymph node; GC, germinal center; GPI, glucose-6- phosphate isomerase; LN, lymph node; PC, plasma cell; PSGL-1, P-selectin glyco- of autoantibodies in vivo (19). However, how Tregs affect the ac- protein ligand 1; SLE, systemic lupus erythematosus; TEFH, extrafollicular helper T; tivity of autoantibody-producing PCs in vivo remains unknown. T , follicular helper T; Treg, regulatory T cell. FH We undertook the current study to investigate whether Tregs al- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 ter the physiology of PCs, using a murine autoantibody-mediated www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002942 The Journal of Immunology 1547 disease model named K/BxN. The spontaneous development of purified using TRIzol reagent (Life Technologies). The level of FcgRIIb severe arthritis in this model depends on the production of auto- mRNA was measured by quantitative RT-PCR using an iCycler thermo- antibodies to glucose-6-phosphate isomerase (GPI) (20). Pre- cycler (Bio-Rad). Relative amounts of FcgRIIb transcripts were normal- ized to the amounts of b2-microglobulin transcript. The primer sequences viously, we found that despite the loss of self-tolerance, func- used were as follows: FcgRIIb forward, 59-GGA AGA CAC GGT GAC tionally intact Foxp3+ Tregs exist in the K/BxN mice (21). This ACT GA-39 ;FcgRIIb reverse, 59-TGC TCC ATT TGA CAC CGA TA-39; prompted us to consider the in vivo role of Tregs in autoantibody b2-microglobulin forward, 59-TGA CCAGCT TGT ATG CTA TC-39; b2- production. We show in this study that in addition to enhancing the microglobulin reverse, 59-CAG TGT GAG CCA GGA TAT AG-39. development of Th cell subsets, Treg deficiency results in changes BrdU incorporation assays intrinsic to PCs—as evidenced by their aberrant localization, mat- Mice were fed drinking water containing 0.8 mg/ml BrdU (Sigma-Aldrich) uration, and life span—which favor the persistence of humoral for 14–28 d. The water was protected from light and changed every 2 d. immunity. Thus, our results demonstrating PC regulation by Tregs After staining for B220 and CD138, splenocytes and dLN cells extracted provide novel insight into how Tregs limit humoral memory. from the mice were fixed, permeabilized, treated with 50 Kunitz units DNase (Sigma-Aldrich), and then stained with FITC-conjugated anti-BrdU mAb according to the manufacturer’s instructions (BD Bioscience). BrdU Materials and Methods incorporation in B220loCD138+ cells was determined by FACS analysis. Mice Cell culture A cross between KRN TCR-transgenic mice on a C57BL/6 background To determine whether Tregs are cytotoxic to PCs, coculture experiments (K/B) and NOD mice generated arthritic transgenic progeny (K/BxN) and were carried out.