Glatiramer acetate ameliorates inflammatory ... - Wiley Online Library

Eur. J. Immunol. 2013. 43: 125–136

DOI: 10.1002/eji.201242758

Cellular immune response

Glatiramer acetate ameliorates inflammatory bowel disease in mice through the induction of Qa-1-restricted CD8+ regulatory cells Yunliang Yao∗1,2,3 , Wenzheng Han∗4 , Jingjing Liang1,2 , Jian Ji5 , Jianli Wang1 , Harvey Cantor6,7 and Linrong Lu1,2 1

Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, P. R. China Program in Molecular and Cellular Biology, Zhejiang University School of Medicine, Hangzhou, P. R. China 3 Department of Microbiology and Immunology, Medical School of Huzhou Teachers College, Huzhou, P. R. China 4 Xin Yuan Institute of Medicine and Biotechnology, Life Science College, Zhejiang Sci-Tech University, Hangzhou, China 5 College of Animal Sciences, Key Laboratory of Animal Molecular Nutrition of the Ministry of Education, Zhejiang University, Hangzhou, P. R. China 6 Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA 7 Department of Pathology, Harvard Medical School, Boston, MA, USA 2

Inflammatory bowel diseases (IBDs) are complex multifactorial immunological disorders characterized by dysregulated immune reactivity in the intestine. Here, we investigated the contribution of Qa-1-restricted CD8+ Treg cells in regulating experimental IBD in mice. We found that CD8+ T cells induced by T-cell vaccination ameliorated the pathological manifestations of dextran sulfate sodium induced IBD when adoptively transferred into IBD mice. In addition, CD8+ cell suppressive activity was induced by vaccination with glatiramer acetate (GA), an FDA-approved drug for multiple sclerosis (MS). We next showed that GA-induced CD8+ Treg cells worked in a Qa-1-dependent manner and their suppressive activity depends on perforin-mediated cytotoxicity. Finally, we confirmed the role of CD4+ T cells in dextran sulfate sodium induced colitis progression, and clarified that GA-induced CD8+ T cells exerted their therapeutic effects on colitis by targeting pathogenic CD4+ T cells. Our results reveal a new regulatory role of Qa-1-restricted CD8+ Treg cells in IBD and suggest their induction by GA vaccination as a potential therapeutic approach to IBD.

Keywords: CD8+ Treg

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Colitis

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Inflammation r Qa-1

Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction Inflammatory bowel diseases (IBDs) are severe gastrointestinal disorders that include ulcerative colitis and Crohn’s disease. Both Correspondence: Professor Linrong Lu e-mail: [email protected]

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Crohn’s disease and ulcerative colitis patients have activated innate (macrophage, neutrophil) and acquired (T and B cell) immune responses and loss of tolerance to enteric commensal bacteria [1]. Histologically, mucosal accumulation of leukocytes is ∗

These authors contributed equally to this work.

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also a characteristic feature of IBD, and the activation of T cells and monocyte macrophages has been regarded as a crucial factor in its pathogenesis [2, 3]. Although the specific enterobacterial antigens have not yet been characterized, it is generally acknowledged that CD4+ T cells play important roles in experimental mucosal inflammation as effector cells, not only because these cells make up the main cell populations that infiltrate mucosal tissues in all IBD models studied thus far [1, 4], but also because in instances in which they are deleted in vivo, inflammation is ameliorated [5]. Although significant progress has been made on the pathogenesis of IBD in recent years, the immunological treatment of this disease still relies largely on the use of anti-inflammatory drugs and immunosuppressants. The use of immunomodulation carries the risk of promoting cancer and/or infection [6, 7]. Treg cells, which are already used in clinical trials in the transplantation setting, represent a promising strategy for engineering tolerance to self and nonself antigens in inflammatory diseases. Numerous studies have already demonstrated that IBDs can be suppressed by CD4+ Foxp3+ Treg cells in different animal models [8–11]. Given that lymphocytes with immunosuppressive potential have also been identified in the CD8+ population, CD8+ Treg cells might also exert therapeutic effects on IBDs [12–14]. We previously identified a subset of Qa-1-restricted CD8+ T cells responsible for maintaining self-tolerance through the inhibition of autoreactive CD4+ T cells, and they effectively attenuate experimental autoimmune encephalomyelitis (EAE) [15]. Qa-1-restricted CD8+ Treg cells can be induced by both T-cell vaccination (TCV) [16] and activation of CD4+ T cells upon peptide immunization [17]. Since activation of enteroantigen-specific CD4+ T cells is also playing crucial roles in the pathogenesis of IBD, we were interested in evaluating the therapeutic effects of Qa-1-restricted CD8+ T cells on experimental IBD and exploring potential therapeutic approaches toward this disease through the induction of CD8+ suppressor cells. Glatiramer acetate (GA; Copaxone), an FDA-approved drug for the treatment of multiple sclerosis (MS), is a random polymer (average molecular mass 6.4 kD) composed of four amino-acids found in myelin basic protein. Interestingly, a previous study has also demonstrated that GA ameliorates various pathological manifestations of IBD in animal models [18]. It is generally believed that GA exerts its therapeutic effect on MS through the modulation of CD4+ Th1-type responses to a protective Th2 phenotype [19]. And in mouse IBD models, GA treatment is accompanied by reduced expression of proinflammatory cytokines such as TNF-α and IFN-γ and enhanced levels of regulatory anti-inflammatory TGF-β and IL-10 [18]. Further, some studies also suggested that treatment of MS patients with GA can induce upregulation of HLA-E-restricted CD8+ T cells, which in turn exert a regulatory/suppressor function and are capable of modulating in vivo immune responses by directly killing pathogenic CD4+ T cells [20, 21]. Given that Qa-1 (a nonclassical MHC class Ib molecule) is the homologue of HLA-E in mice, which is the key molecule in mediating CD8+ regulatory T-cell activity [22], we then checked whether GA can induce Qa-1-restricted CD8+ Treg cells in mice


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and further tested its therapeutic contributions in experimental IBD in mice. In this work, we demonstrated the treatment of IBD by GAinduced Qa-1-restricted CD8+ Treg cells in two different mouse colitis models. We also showed that the GA-induced CD8+ T cells targeted to pathogenic CD4+ T cells, and their suppressive activity depended on its cytotoxicity mediated by perforin. Our work reveals a new regulatory role of Qa-1-restricted CD8+ Treg cells in IBD and suggests their induction by GA vaccination as a potential therapeutic approach to IBD.

Results TCV ameliorates dextran sulfate sodium (DSS)-induced IBD in mice Qa-1-restricted CD8+ Treg cells can be induced in mice by vaccination with ConA-activated T cells [16]. We first evaluated the therapeutic effect of TCV on DSS-induced IBD. Splenocytes activated in vitro were irradiated and adoptively transferred into recipient C57BL/6 mice on the day of DSS colitis induction. T-cellvaccinated mice showed much milder symptoms than the control mice, as evidenced by a slower rate of weight loss (Fig. 1A) and a lower disease activity index (DAI) (Fig. 1B). Histological examination of the colon also showed less mucosal damage, demonstrating the therapeutic effect of TCV on colitis in this model (Fig. 1C and D).

Therapeutic effect of GA vaccination on DSS-induced IBD We next determined the therapeutic effect of GA on IBD and found that GA vaccination led to less weight loss (Fig. 2A) and a lower DAI (Fig. 2B). Histological analysis showed less severe symptoms in the colon of GA-vaccinated mice, including inflammatory infiltrates, thickened walls, and disruption of mucosal structures, which were more evident in the untreated group (Fig. 2C and D).

Treatment of DSS-induced IBD with GA-induced CD8+ T cells To elucidate the role of CD8+ Treg cells in the GA treatment of IBD, we purified CD8+ T cells from GA-vaccinated mice and adoptively transferred them into recipient mice. Naive CD8+ T cells from PBS-injected mice were given to the control mice. We found that the mice given naive CD8+ T cells suffered extensive weight loss from day 7 after DSS induction. In contrast, the weight loss was significantly retarded in mice given GA-induced CD8+ T cells (Fig. 3A). The lowered DAI (Fig. 3B) and improved histological condition of the colon (Fig. 3C and D) also demonstrated the therapeutic effects of GA-induced CD8+ T cells.

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Figure 1. Evaluation of DSS-induced colitis treated with TCV. C57BL/6 mice were exposed to 2% DSS in drinking water. Activated and irradiated T cells from C57BL/6 mice were injected (5 × 106 cells per mouse i.p.) on the day of disease induction. (A) Relative weight was defined by percentage of initial body weight. (B) Daily disease activity index was calculated and plotted. (C) Representative photomicrographs of histological features of the colon 9 days after DSS induction. Colon tissue samples were stained with H&E and are shown at 20× magnification; scale bar = 50 μm. (D) Histological scores of each group. Data are shown as mean ± SD of five mice per group and are from one experiment representative of three performed. *p < 0.05, Mann–Whitney test.



Figure 2. Therapeutic effect of glatiramer acetate (GA) on DSS colitis. C57BL/6 mice were immunized daily with 500 μg lysozyme or GA subcutaneously for 14 days and then exposed to 2% DSS in the drinking water. (A) Relative weight of the mice and (B) disease activity index are shown. (C) Histological appearance and (D) histological scores 9 days after DSS induction are shown. (C) Colon tissue samples were stained with H&E and are shown at 20× magnification; scale bar = 50 μm. Data are shown as mean ± SD of five mice per group and are from one experiment representative of three performed. *p < 0.05, Mann–Whitney test.

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Figure 3. Treatment of DSS-induced IBD with GA-induced CD8+ T cells. C57BL/6 mice were exposed to 2% DSS in the drinking water. GAinduced or naive CD8+ T cells were injected (i.v. 106 cells per mouse) on the day of disease induction. (A) Relative weight and (B) disease activity index were recorded daily. (C) Microscopic appearance and (D) histological score of the colon 9 days after DSS induction are shown. Colon tissue samples were stained with H&E and are shown at 20× magnification, scale bar = 50 μm. (E) Cytokine mRNA expression on day 9 in the colon of mice with DSS colitis. (A, B, D, and E) Data are shown as mean ± SD of five mice per group and are from one experiment representative of three performed. *p < 0.05, Mann–Whitney test. (F) Migration of GA-induced CD8+ T cells in vivo. Mice with DSS colitis (day 8) were infused with 5 × 106 CFSE-labeled naive or GA-induced CD8+ T cells. Twenty-four hours later, singlecell suspensions of mesenteric lymph nodes (MLNs), intestinal epithelial lymphocytes(IELs), and lamina propria lymphocytes (LPLs) were prepared. The presence of infused CD8+ T cells was identified by flow cytometric analysis of the green CFSE marker. (G) Frequencies of the indicated transferred CD8+ T cells in MLN, IEL, and LPL. Each symbol represents an individual mouse and the bar represents the SD; data shown are representative of three experiments performed. *p < 0.05 and **p < 0.01, Mann– Whitney test.

When we measured the expression of inflammatory cytokines related to the pathogenesis of colitis in colon tissue using real-time RT-PCR, we found that colonic mRNA expression of TNF-α, IFN-γ, IL-6, and IL-17A was also lower in GA CD8+ T-cell-treated mice


with DSS colitis (Fig. 3E). However, the expression of IL-10 and TGF-β did not significantly differ between the two groups. We further traced the migration of GA-induced CD8+ T cells in vivo by injecting 5 × 106 carboxyfluorescein diacetate

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succinimidyl ester (CFSE) labeled CD8+ T cells into C57BL/6 mice with established DSS IBD (day 8). We prepared the mesenteric lymph nodes (MLNs), intestinal epithelial lymphocytes (IELs), and lamina propria lymphocytes (LPLs) from the recipient mice 24 h later and analyzed them for CFSE-labeled cells. We showed that CFSE-labeled GA CD8+ T cells were present in all these tissues of mice with DSS colitis, but not naive CD8+ T cells in the control group (Fig. 3F and G).

Qa-1-restriction of GA-induced CD8+ Treg cells We further tested the Qa-1-restriction of GA-induced CD8+ T cells by using CD8+ T cells from Qa-1-deficient (Qa-1−/− ) mice. CD8+ T cells from both WT and Qa-1−/− mice with or without GA vaccination were adoptively transferred into DSS-fed mice to evaluate their therapeutic effects on IBD colitis. We found that CD8+ T cells purified from GA-vaccinated Qa-1-/- mice lost their therapeutic activity in IBD, which demonstrated that GA-induced CD8+ Treg cells work in a Qa-1-dependent manner (Supporting Information Fig.1A–D).


The Qa1.D227K mouse is a Qa-1 mutant knock-in carrying a Qa-1 amino acid exchange mutation that disrupts Qa-1 binding to the T-cell receptor/CD8 coreceptor, and is deficient in generating Qa-1-restricted CD8+ Treg cells [15]. Similar to the cells from Qa1−/− mice, GA-induced CD8+ T cells from Qa1.D227K mice failed to treat IBD (Fig. 4). On the contrary, CD8+ T cells from another Qa-1 knock-in strain (Qa-1.R72A), which expresses an amino acid exchange mutation (R72A) that fails to bind to CD94/NKG2A but spares Qa-1-dependent peptide presentation to the TCR, were fully capable of ameliorating DSS-induced colitis (Fig. 4). These data further strengthened our hypothesis that Qa-1-restricted CD8+ Treg cells are responsible for the therapeutic effect of GA-induced CD8+ T cells.

Perforin expression contributes to the therapeutic effect Qa-1-restricted CD8+ Treg cells mediate suppression through perforin-dependent cytotoxicity [15, 22]. We next tested the contribution of perforin and Fas ligand to the therapeutic effects of

Figure 4. Qa-1-restriction of GA-induced CD8+ Treg cells. Colitis was induced in C57BL/6 mice by administration of 2% DSS in the drinking water, CD8+ T cells enriched from mesenteric lymph nodes of GA-vaccinated WT, Qa1−/− , and mutant knock-in (B6.Qa-1.D227K and B6.Qa-1.R72A) mice were transferred into recipients (106 cells per mouse i.v.) on the day of disease induction. Mice injected with naive WT CD8+ T cells were used as control. (A) Relative weight, (B) disease activity index, (C) representative photomicrographs, and (D) scores of histological features from colons 9 days after DSS induction are shown. (A, B, and D) Data are shown as mean ± SD of five mice per group and are from one experiment representative of three performed. (C) Colon tissue samples were stained with H&E and are shown at 20× magnification, scale bar = 50 μm. *p < 0.05, Mann– Whitney test.


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Figure 5. Cytotoxic effector pathways of GAinduced CD8+ T cells. CD8+ T cells from mesenteric lymph nodes of GA-vaccinated WT, FasL−/− , or prf1−/− mice were adoptively transferred into host mice fed with 2% DSS in the drinking water. (A) Relative weight and (B) disease activity index were monitored every day. (C) Histological appearance and (D) histological score of colons 9 days after DSS induction are shown. (A, B, and D) Data are shown as mean ± SD of five mice per group and are from one experiment representative of three performed. (C) Colon tissue samples were stained with H&E and are shown at 20× magnification, scale bar = 50 μm. *p < 0.05, Mann–Whitney test.

GA-induced CD8+ T-cells. CD8+ T cells from GA-vaccinated wildtype, perforin-deficient (Prf1−/− ), or Fas ligand deficient (FasL−/− ) mice were transferred into recipients with DSS colitis. The symptoms of IBD were inhibited by FasL−/− but not by Prf1−/− CD8+ T cells (Fig. 5), demonstrating that the perforin-mediated cytotoxicity is essential for the suppressive activity of CD8+ Treg cells, similar to our previous findings in the treatment of EAE [15].

Suppression of CD4+ T-cell activation by GA-induced CD8+ T cells CD8+ Treg cells are characterized by their ability to suppress CD4+ T-cell activation. We tested the suppressive activity of GA-induced CD8+ T cells toward OT2 T cells both in vitro and in vivo. GAinduced CD8+ T cells were titrated into OT2 T cells stimulated with graded doses of OVA peptide. The results showed that GAinduced CD8+ T cells suppressed the activation of OT2 T cells in vitro when stimulated with a low concentration of OVA peptide, as judged by decreased secretion of IFN-γ upon activation (Fig. 6A). We next cotransferred GA-induced CD8+ T cells with OT2 T cells into rag-1−/− mice and immunized the mice with OVA peptide. In vivo activation of OT2 T cells was also substantially suppressed, C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

marked by their decreased ability to produce IFN-γ upon in vitro recall stimulation (Fig. 6B).

GA-induced CD8+ Treg cells target pathogenic CD4+ T cells in DSS-IBD The role of CD4+ T cells in DSS-IBD is controversial. We first analyzed the percentages of IFN-γ (Th1) and IL-4 (Th2) producing T cells in the MLNs of mice with DSS colitis. These mice had significantly higher numbers of Th1 and Th2 cells than control mice, suggesting the activation of CD4+ T cells in DSS-induced colitis (Fig. 7A). Interestingly, treatment with GA CD8+ T cells effectively reduced the number of both Th1 and Th2 cells (Fig. 7A and B). To directly verify the contribution of CD4+ T cells to DSSinduced IBD, we depleted CD4+ T cells in C57BL/6 mice by antiCD4 mAb (GK1.5) injection before the transfer of GA-induced or naive CD8+ T cells (Fig. 7C). We found that depletion of CD4+ T cells reduced the colitis symptoms to an extent similar to treatment with GA CD8+ T cells (Fig. 7D–G). More importantly, GA CD8+ T-cell treatment and CD4+ depletion did not have an additive therapeutic effect on colitis, again suggesting that CD8+

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T cells, they suffered much milder weight loss (90% CD8+ T cells as determined by cell surface staining and flow cytometry. Purified CD8+ T cells (106 ) were injected i.v. into recipient mice.

Histology Large intestinal pieces were washed thoroughly in PBS with 2% FBS and fixed in formalin. Tissues were embedded in paraffin and 5- to 6-μm sections were cut. The sections were stained with hematoxylin/eosin (H&E) and examined under a light microscope. The degree of histological damage and inflammation was graded in a blinded fashion by a veterinary pathologist. The following parameters were scored: (i) amount of inflammation (0, none; 1, mild; 2, moderate; 3, severe; 4, accumulation of inflammatory cells in the gut lumen), (ii) distribution of lesions (0, none; 1, focal; 2, multifocal; 3, nearly diffuse; 4, diffuse), and (iii) depth of inflammation and layers involved (0, none; 1, mucosa only; 2, mucosa and submucosa; 3, limited transmural involvement; 4, transmural). The overall histological score was the sum of the three parameters (maximum score 12).

RNA isolation and real-time PCR for cytokines In vivo CD8+ T-cell tracking Total RNA was isolated from the distal colon using TaKaRa RNAiso Plus (TaKaRa Bio, Tokyo, Japan). cDNA was synthesized from 1 μg total RNA using the Reverse Transcriptase M-MLV (RNase H@) kit (TaKaRa Bio). PCR amplification of cDNA was performed by SYBR Premix Ex TaqTM II (Perfect Real Time) (TaKaRa Bio) on CFX-Touch (Bio-Rad Laboratories, Inc.). To normalize the amount of total RNA in each reaction, β-actin cDNA was used as an internal control. The primers used were as follows: IFN-γ, 5 -ATC TGG AGG AAC TGG CAA AA-3 (forward) and 5 -TGA GCT CAT TGA ATG CTT GG-3 (reverse); IFN-α, 5 -CTG GGA CAG TGA CCT GGA CT3 (forward) and 5 -GCA CCT CAG GGA AGA GTC TG-3 (reverse); IL-4, 5 -CGA AGA ACA CCA CAG AGA GTG AGC T-3 (forward) and 5 -GAC TCA TTC ATG GTG CAG CTT ATC G-3 (reverse); IL-17A, 5 -ATC CCT CAA AGC TCA GCG TGT C-3 (forward) and 5 -GGG TCT TCA TTG CGG TGG AGA G-3 (reverse); TGF-β, 5 TAC AGG GCT TTC GAT TCA GC-3 (forward) and 5 -CGC ACA CAG CAG TTC TTC TC-3 (reverse); IL-10, 5 -CCA AGC CTT ATC C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

CD8+ T cells were labeled with 1 μM CFSE (Molecular Probes) in PBS at 37◦ C for 5 min and washed three times immediately before i.v. injection into C57BL/6 mice (5 × 106 ) with established DSSIBD (day 8). Twenty-four hours later, MLNs, Payer patch, IELs, and LPLs were harvested. The cells were stained with allophycocyaninconjugated anti-CD3 and PE-conjugated anti-CD8 antibody, and analyzed by FACS for CFSE-labeled cells. The IELs and LPLs were prepared as described [33, 34]. Briefly, colon specimens were washed extensively in HBSS without Ca2+ and Mg2+ (Sangon, China), opened longitudinally, and cut into 5-mm pieces. Fragments were floated in medium under constant EDTA (10 mM) for 30 min at 37◦ C. The cell suspension (released IELs) was collected and washed in complete medium. Lymphocytes were separated from the suspension using mouse lymphocyte separation medium (Dakewe, China). For LPLs preparation, the tissue freed of their epithelial cell layers were floated in medium and digested under

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constant shaking for 45 min at 37◦ C by collagenase type VIII (240 U/mL, Sigma) and DNase I (10 mg/mL, Sigma). The cell suspension (released LPLs) was collected and washed in complete medium. Lymphocytes were separated from the suspension using mouse lymphocyte separation medium (Dakewe).

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CD4+ T-cell depletion

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C57BL/6 mice were injected i.v. with anti-CD4 (GK1.5; QuantoBio, China) monoclonal antibody (mAb) at 150 μg per injection at 3 days before and 4 days after DSS-IBD induction.

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Splenocytes from C57BL/6 mice were labeled with anti-CD4PE and anti-CD45RB-FITC. T cells were gated and electronically sorted using BD FACSAria II (BD Biosciences). Sorted donor lymphocytes (4 × 105 ) were resuspended in 200 μL PBS and injected i.v. into Rag1−/− mice. The mice in the treatment group were also i.v. injected with 106 purified CD8+ cells. Then the recipients were weighed weekly and euthanized after 8 weeks. The degree of histological damage and inflammation in the colon was assessed after H&E staining.

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Statistical analysis

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Data are presented as the mean ± SD values. Comparison between two groups was performed by Mann–Whitney test. A value of p < 0.05 was considered significant.

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Acknowledgments: The authors thank Dr. D. Wang and H. Hu for their helpful discussion; Dr. I. Bruce for his critical reading. This work was supported by grants from the National Natural Science Foundation of China (30972724, 31070782 to L.L.), National Basic Research Program of China (973 Program) (2011CB944100 and 2012CB945004 to L.L.), and Zhejiang Provincial Natural Science Foundation of China (R2090202 to L.L.)

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Conflict of interest: The authors declare no financial or commercial conflict of interest.

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Abbreviations: DAI: disease activity index · DSS: dextran sulfate

Bylund-Fellenius, A. C., Dextran sulfate sodium (DSS) induced experi-

sodium · IBD: inflammatory bowel disease · IEL: intestinal epithelial

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TCV: T-cell vaccination

27 Stevceva, L., Pavli, P., Husband, A. J. and Doe, W. F., The inflammatory infiltrate in the acute stage of the dextran sulphate sodium induced colitis: B cell response differs depending on the percentage of DSS used to induce it. BMC Clin. Pathol. 2001. 1: 3.

Full correspondence: Professor Linrong Lu, 866 Yuhang Tang Road, Medical Research Building B819, Hangzhou, 310058, P. R. China Fax: +86-571-88208284 e-mail: [email protected]

28 Dieleman, L. A., Ridwan, B. U., Tennyson, G. S., Beagley, K. W., Bucy, R. P. and Elson, C. O., Dextran sulfate sodium-induced colitis occurs in severe combined immunodeficient mice. Gastroenterology 1994. 107: 1643–1652. 29 Shintani, N., Nakajima, T., Okamoto, T., Kondo, T., Nakamura, N. and Mayumi, T., Involvement of CD4+ T cells in the development of dextran


Received: 21/6/2012 Revised: 17/8/2012 Accepted: 18/9/2012 Accepted article online: 24/9/2012

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