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Keywords:

  • CD8+ Treg;
  • Colitis;
  • Inflammation;
  • Qa-1

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. Reference
  10. Supporting Information

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. Reference
  10. Supporting Information

Inflammatory bowel diseases (IBDs) are severe gastrointestinal disorders that include ulcerative colitis and Crohn's disease. Both 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 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 and further tested its therapeutic contributions in experimental IBD in mice.

In this work, we demonstrated the treatment of IBD by GA-induced 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. Reference
  10. Supporting Information

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-cell-vaccinated 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).

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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.

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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).

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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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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.

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.

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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. GA-induced 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, single-cell 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.

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We further traced the migration of GA-induced CD8+ T cells in vivo by injecting 5 × 106 carboxyfluorescein diacetate 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 Qa-1−/− 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.

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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, Qa-1−/−, 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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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 GA-induced CD8+ T-cells. CD8+ T cells from GA-vaccinated wild-type, 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].

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Figure 5. Cytotoxic effector pathways of GA-induced 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.

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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. GA-induced CD8+ T cells were titrated into OT2 T cells stimulated with graded doses of OVA peptide. The results showed that GA-induced 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, marked by their decreased ability to produce IFN-γ upon in vitro recall stimulation (Fig. 6B).

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Figure 6. Suppressive activity of GA-induced CD8+ T cells against CD4+ T-cell activation. (A) Naive or GA-induced CD8+ T cells (105) were co-cultured with splenocytes from OT2 mice (4 × 105) in 96-well plates with graded doses of ovalbumin (OVA) peptide. IFN-γ concentrations in the supernatant were measured by ELISA. (B) GA-induced CD8+ T cells were cotransferred into Rag-1−/− mice (106 cells per mouse) with CD4+ T cells purified from OT2 mice (106 cells per mouse). The recipient mice were then immunized with 200 μg OVA peptide emulsified in CFA. The lymph node cells were harvested after 12 days and stimulated with graded doses of OVA peptide; IFN-γ concentrations in the supernatant were measured by ELISA. 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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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).

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Figure 7. Role of CD4+ T cells in DSS-induced colitis. (A) Flow cytometric detection of intracellular IFN-γ and IL-4 production by CD4+ T cells. Lymphocytes from mesenteric lymph nodes stimulated with PMA/ionomycin were stained with anti-CD4 and cytokine-specific antibodies (IFN-γ-PE and IL-4-FITC) and analyzed by flow cytometry. The expression of IFN-γ and IL-4 on CD4+ T cells is shown. (B) Frequencies of the indicated cytokine-secreting CD4+ T cells are shown. Each symbol represents an individual mouse and the error bars indicate the SD. *p < 0.05 and **p < 0.01, Mann–Whitney test. (C) C57BL/6 mice with CD4+ T cells deleted by anti-CD4 mAb were given GA-induced or naive CD8+ T cells (106). The depletion efficiency was evaluated by flow cytometry 5 days after the last injection. (D) Body weight changes and (E) disease activity index were determined daily. Nine days after DSS induction, the colon tissues were stained with H&E, and the (F) scores and (G) histological appearance were analyzed. (B, D, E, and F) Data are shown as mean ± SD of five mice per group and are from one experiment representative of three performed. *p < 0.05 and **p < 0.01, Mann–Whitney test. (G) Colon tissue samples were stained with H&E and are shown at 20× magnification, scale bar = 50 μm.

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To directly verify the contribution of CD4+ T cells to DSS-induced IBD, we depleted CD4+ T cells in C57BL/6 mice by anti-CD4 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+ Treg cells ameliorate the symptoms by targeting pathogenic CD4+ T cells.

To further validate the suppressive activity of GA-induced CD8+ T cells on pathogenic CD4+ T cells in colitis, we established a CD4+CD45RBhigh T-cell transfer model of colitis that is dependent on CD4+ T cells for disease. We first transferred sorted CD4+CD45RBhigh T cells (Fig. 8A) into Rag1-/- mice and monitored their weight for 8 weeks, after which the mice were euthanized and the colon subjected to histological analysis. We found that mice injected with CD4+CD45RBhigh plus naive CD8+ T cells lost more than 25% of their weight during this period. On the contrary, when the mice were cotransferred with GA-induced CD8+ T cells, they suffered much milder weight loss (<15%) (Fig. 8B). Similar to what we found in the DSS-colitis model, CD8+ T cells from GA-vaccinated R72A mice retained their therapeutic effect while the cells from Qa-1−/− and D227K mice lost this activity; this also complied with the result in DSS-induced IBD (Fig4B). Taken together, these data further strengthen our hypothesis that GA-induced Qa-1-restricted CD8+ Treg-cell treats colitis by targeting pathogenic CD4+ T cells.

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Figure 8. GA-induced CD8+ T cells prevent development of transfer model of colitis. (A) CD4+CD45RBhigh cells were sorted from C57BL/6 mice and injected i.v. into Rag1−/−-deficient C57BL/6 mice (4 × 105 cells per mouse). The mice in the treatment group were also i.v. injected with 106 purified CD8+ cells as indicated. (B) Weight change of animals was monitored every week for 8 weeks. Data are shown as mean ± SD of five mice per group and are from one experiment representative of three performed. (C) Microscopic sections of colon were stained with H&E and examined for signs of colitis at week 8, images are shown at 20× magnification and scale bar = 50 μm. *p < 0.05 and **p < 0.01, Mann–Whitney test.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. Reference
  10. Supporting Information

IBDs are diseases that cause inflammation of the intestines, in which overactivated CD4+ T cells play important roles in the pathogenesis of the diseases [1, 4]. This makes Treg cells popular candidates for the treatment of this disease. However, there is very little evidence that Foxp3+ Treg cells are dysfunctional in IBD, since Treg cells isolated from the intestinal mucosal of IBD patients are suppressive in vitro [23]. On the contrary, deficiency in CD8+ Treg cells has been reported in the lamina propria of patients with IBD [24]. Thus, CD8+ Treg cells might provide an additional potential target for cellular therapy of IBD.

In this study, we showed that TCV, which is a classical way to induce Qa-1-restricted CD8+ Treg cells, alleviates DSS-colitis efficiently. However, TCV causes a strong inflammatory response, which hardly makes it a viable approach for treatment. We then further focused on GA, an FDA-approved drug for MS. GA treatment ameliorates various pathological manifestations in several IBD animal models [18], and work in human MS patients has suggested that GA vaccination induces HLA-E-restricted CD8+ Treg cells [20].

Here, we employed the DSS-induced colitis model and demonstrated that Qa-1-restricted CD8+ Treg cells were indeed responsible for the therapeutic effects of GA on IBD. The suppression of CD4+ T-cell activation in vivo and in vitro also supported this conclusion. For the first time, we provided direct genetic evidence that Qa-1 was responsible for the GA-induced CD8+ regulatory activity by using Qa-1-deficient mice. Further work with mutant knock-in mice proved that the induction and activation of the Qa-1-restricted CD8+ Treg cells depends on the interactions between Qa-1 and TCR but not CD94/NKG2A. Our work in mice here not only demonstrates that GA vaccination can induce Qa-1-restricted CD8+ Treg cells in mice, but also their therapeutic effect on colitis. Analysis using Qa-1-deficient and knock-in mice also provided genetic evidence for the requirement of Qa-1 in GA-induced CD8+ regulatory activity. Cytotoxicity experiments illustrate that the suppression of GA-induced CD8+ Treg cells required perforin but not FasL, further elucidating the mechanism of this treatment.

Although Qa-1-restricted CD8+ Treg cells are proved to suppress the overactivation of pathogenic CD4+ T cells in IBD, the alleviation is statistically significant but not huge. This is due to the nature of this DSS-induced disease model, both the activation of innate cells and adaptive immune responses contribute to the pathogenesis of the disease. The primary target of the CD8+ regulatory cells is the adaptive immune response mediated by CD4+ T cells, which limits the therapeutic effect to a certain extent to the late stage of the disease. Our work also re-evaluated the contribution of CD4+ T cells in the DSS colitis model. Although CD4+ T lymphocytes are known to play an important role in intestinal inflammatory immune responses, the effect of CD4+ T cells in DSS colitis is controversial [25]; most studies support the DSS model to be independent of CD4+ T cells [26-28]. However some studies have shown that pathogenic CD4+ T cells may participate in the process described by model [29-31]. In our work, we show that DSS colitis is associated with the activation and differentiation of CD4+ T cells in MLNs. More importantly, depletion of CD4+ T cells in C57BL/6 mice led to less severe disease that mimicked the therapeutic effect of CD8+ Treg cells. Our data thus not only demonstrated the pathogenic role of CD4+ T cells in DSS colitis but also proved that GA-induced CD8+ Treg cells exert their therapeutic effect by targeting pathogenic CD4+ T cells. The suppressive activity of these CD8+ T cells against CD4+ T cells was further confirmed in a CD4+CD45RBhigh T-cell transfer model of colitis.

In sum, our work reveals a novel immunologic regulatory role for Qa-1-restricted CD8+ Treg cells in IBD and provides new insights into the immune interplay underlying the disease.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. Reference
  10. Supporting Information

Animals

Female mice at 8–10 weeks of age were used. C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animals Co., Ltd (Shanghai, China). Rag-1 deficient (Rag1−/−), FasL-deficient (FasL−/−), perforin-deficient (prf1−/−), and OT2 TCR (T-cell receptor) transgenic C57BL/6 mice were from Jackson Laboratory (Bar Harbor, ME). The Qa-1-deficient (Qa-1−/−) and Qa-1 knock-in mice (B6.Qa-1 D227K and B6.Qa-1 R72A) had been previously generated [15]. All mice were co-housed for more than 4 weeks in the pathogen-free facility at Zhejiang University Laboratory Animal Center. The experiment protocols were approved by the Review Committee of Zhejiang University School of Medicine, and done in compliance with the institutional guidelines.

Antibodies

FITC-conjugated anti-mouse CD4 (RM4–5) and PE-conjugated anti-mouse CD8a (53–6.7) were from BD Biosciences (San Jose, CA, USA). PE-conjugated anti-mouse CD4 (RM4–5) was from eBioscience (San Diego, CA, USA). Allophycocyanin-conjugated anti-mouse CD3 (145–2C11), FITC-conjugated anti-mouse CD45RB (C363–16A), and allophycocyanin-conjugated anti-mouse CD8a (53–6.7) were from BioLegend (San Diego, CA, USA).

Induction and assessment of DSS IBD

C57BL/6 mice were randomized into groups of five, with comparable average body weight in each group. Acute colitis was induced by giving 2% wt/v DSS( MW 36 000–50 000; MP Biomedicals, LLC, Eschwege, Germany) in the drinking water for 9 days [32]. Body weight, rectal bleeding, stool consistency, and survival were monitored daily after DSS administration. The daily DAI was calculated by grading on a scale 0–4 for the following parameters: change in weight (0, <1%; 1, 1–5%; 2, 5–10%; 3, 10–20%; and 4, >20%), intestinal bleeding (0, negative; 4, positive), and stool consistency (0, normal; 2, loose stools; 4, diarrhea). The combined scores were then divided by 3 to obtain the final DAI.

T-cell vaccination

Purified CD4+ T cells from the spleen were stimulated in vitro using 1 μg/mL Con A (Sigma, St. Louis, MO, USA) in RPMI 1640 medium containing 10% FCS at 37°C. Forty hours later, cells resuspended in PBS were irradiated at 3000 rad and injected i.v. into mice. Control mice were injected with 200 μL PBS.

GA vaccination

GA (Copaxone, Copolymer 1) consists of acetate salts of synthetic polypeptides containing four amino acids: l-alanine, l-glutamate, l-lysine, and l-tyrosine. GA from batch 242 990 599, with an average molecular weight of 7300 kDa was from Sigma. GA was dissolved in PBS and administered daily by s.c. injection (500 μg/day) for 2 weeks.

CD8+ T-cell enrichment and adoptive transfer

Splenocytes and MLNs were harvested from control and immunized mice (2 weeks after GA administration). CD8+ T cells were enriched by negative bead selection using CD8+ T Lymphocyte Enrichment Set-DM (BD Biosciences), according to the manufacturer's guidelines. The enriched cell population contained >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.

RNA isolation and real-time PCR for cytokines

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 Hˉ) 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 CT-3′ (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 GGA AAT GA-3′ (forward) and 5′-TTT TCA CAG GGG AGA AAT CG-3′ (reverse); β-actin, 5′-AAC AGT CCG CCT AGA AGC AC-3′ (forward) and 5′-CGT TGA CAT CCG TAA AGA CC-3′ (reverse).

Cytokine analysis by flow cytometry

Lymphocytes from the MLNs of C57BL/6 mice were harvested and cultured in RPMI 1640 in 96-well plates and activated with phorbol 12-myristate 13-acetate (PMA; 25 ng/mL; Sigma) and ionomycin (1 μg/mL; eBioscience) for 2 h at 37°C and 5% CO2. The cultures were further incubated with brefeldin A (0.5 mg/mL; eBioscience), a transport inhibitor that prevents cytokine release from cells for 4 h. The cells were fixed and permeabilized with Fix/Perm A/B (Caltag Laboratories, Burlington, ON, USA), and the expression of intracellular cytokines was assessed by flow cytometry.

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).

In vivo CD8+ T-cell tracking

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 DSS-IBD (day 8). Twenty-four hours later, MLNs, Payer patch, IELs, and LPLs were harvested. The cells were stained with allophycocyanin-conjugated 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 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).

CD4+ T-cell depletion

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.

Induction of CD4+CD45RBhigh colitis

Splenocytes from C57BL/6 mice were labeled with anti-CD4-PE 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.

Statistical analysis

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. Reference
  10. Supporting Information

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.)

Reference

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. Reference
  10. Supporting Information
  • 1
    Sartor, R. B., Mechanisms of disease: pathogenesis of Crohn's disease and ulcerative colitis. Nat. Clin. Pract. Gastroenterol. Hepatol. 2006. 3: 390407.
  • 2
    Schreiber, S., Monocytes or T cells in Crohn's disease: does IL-16 allow both to play at that game? Gut 2001. 49: 747748.
  • 3
    Boirivant, M., Marini, M., Di Felice, G., Pronio, A. M., Montesani, C., Tersigni, R. and Strober, W., Lamina propria T cells in Crohn's disease and other gastrointestinal inflammation show defective CD2 pathway-induced apoptosis. Gastroenterology 1999. 116: 557565.
  • 4
    Strober, W., Fuss, I. J. and Blumberg, R. S., The immunology of mucosal models of inflammation. Annu. Rev. Immunol. 2002. 20: 495549.
  • 5
    Okamoto, S., Watanabe, M., Yamazaki, M., Yajima, T., Hayashi, T., Ishii, H., Mukai, M. et al., A synthetic mimetic of CD4 is able to suppress disease in a rodent model of immune colitis. Eur. J. Immunol. 1999. 29: 355366.
  • 6
    Kim, T. I., Lee, Y. C., Lee, K. H., Han, J. H., Chon, C. Y., Moon, Y. M., Kang, J. K. et al., Effects of nonsteroidal anti-inflammatory drugs on Helicobacter pylori-infected gastric mucosae of mice: apoptosis, cell proliferation, and inflammatory activity. Infect. Immun. 2001. 69: 50565063.
  • 7
    Wu, C. Y., Wu, M. S., Kuo, K. N., Wang, C. B., Chen, Y. J. and Lin, J. T., Effective reduction of gastric cancer risk with regular use of nonsteroidal anti-inflammatory drugs in Helicobacter pylori-infected patients. J. Clin. Oncol. 2010. 28: 29522957.
  • 8
    Sakaguchi, S., Naturally arising CD4+ Treg cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 2004. 22: 531562.
  • 9
    Wan, Y. Y. and Flavell, R. A., TGF-beta and regulatory T-cell in immunity and autoimmunity. J. Clin. Immunol. 2008. 28: 647659.
  • 10
    Powrie, F., Leach, M. W., Mauze, S., Caddle, L. B. and Coffman, R. L., Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C.B-17 scid mice. Int. Immunol. 1993. 5: 14611471.
  • 11
    Mottet, C., Uhlig, H. H. and Powrie, F., Cutting edge: cure of colitis by CD4+CD25+ regulatory T-cells. J. Immunol. 2003. 170: 39393943.
  • 12
    Poussier, P., Edouard, P., Lee, C., Binnie, M. and Julius, M., Thymus-independent development and negative selection of T cells expressing T-cell receptor alpha/beta in the intestinal epithelium: evidence for distinct circulation patterns of gut- and thymus-derived T lymphocytes. J. Exp. Med. 1992. 176: 187199.
  • 13
    Menager-Marcq, I., Pomie, C., Romagnoli, P., and van Meerwijk, J. P., CD8+CD28- Treg lymphocyte prevent experimental inflammatory bowel disease in mice. Gastroenterology 2006. 131: 17751785.
  • 14
    Das, G., Augustine, M. M., Das, J., Bottomly, K., Ray, P. and Ray, A., An important regulatory role for CD4+CD8 alpha alpha T cells in the intestinal epithelial layer in the prevention of inflammatory bowel disease. Proc. Natl. Acad. Sic. USA 2003. 100: 53245329.
  • 15
    Lu, L., Kim, H. J., Werneck, M. B. and Cantor, H., Regulation of CD8+ regulatory T-cells: interruption of the NKG2A-Qa-1 interaction allows robust suppressive activity and resolution of autoimmune disease. Proc. Natl. Acad. Sci. USA 2008. 105: 1942019425.
  • 16
    Jiang, H., Kashleva, H., Xu, L. X., Forman, J., Flaherty, L., Pernis, B., Braunstein, N. S. et al., T-cell vaccination induces T-cell receptor Vbeta-specific Qa-1-restricted regulatory CD8(+) T-cells. Proc. Natl. Acad. Sic. USA 1998. 95: 45334537.
  • 17
    Wu, Y., Zheng, Z., Jiang, Y., Chess, L. and Jiang, H., The specificity of T-cell regulation that enables self-nonself discrimination in the periphery. Proc. Natl. Acad. Sci. USA 2009. 106: 534539.
  • 18
    Aharoni, R., Kayhan, B., Brenner, O., Domev, H., Labunskay, G. and Arnon, R., Immunomodulatory therapeutic effect of glatiramer acetate on several murine models of inflammatory bowel disease. J. Pharmacol. Exp. Ther. 2006. 318: 6878.
  • 19
    Schrempf, W. and Ziemssen, T., Glatiramer acetate: mechanisms of action in multiple sclerosis. Autoimmun. Rev. 2007. 6: 469475.
  • 20
    Dressel, A., Vogelgesang, A., Brinkmeier, H., Mader, M. and Weber, F., Glatiramer acetate-specific human CD8(+) T-cells: increased IL-4 production in multiple sclerosis is reduced by glatiramer acetate treatment. J. Neuroimmunol. 2006. 181: 133140.
  • 21
    Tennakoon, D. K., Mehta, R. S., Ortega, S. B., Bhoj, V., Racke, M. K. and Karandikar, N. J., Therapeutic induction of regulatory, cytotoxic CD8+ T cells in multiple sclerosis. J. Immunol. 2006. 176: 71197129.
  • 22
    Hu, D., Ikizawa, K., Lu, L., Sanchirico, M. E., Shinohara, M. L. and Cantor, H., Analysis of regulatory CD8 T cells in Qa-1-deficient mice. Nat. Immunol. 2004. 5: 516523.
  • 23
    Holmen, N., Lundgren, A., Lundin, S., Bergin, A. M., Rudin, A., Sjovall, H. and Ohman, L., Functional CD4+CD25high Treg cells are enriched in the colonic mucosa of patients with active ulcerative colitis and increase with disease activity. Inflamm. Bowel Dis. 2006. 12: 447456.
  • 24
    Brimnes, J., Allez, M., Dotan, I., Shao, L., Nakazawa, A. and Mayer, L., Defects in CD8+ Treg cells in the lamina propria of patients with inflammatory bowel disease. J. Immunol. 2005. 174: 58145822.
  • 25
    Perse, M. and Cerar, A., Dextran sodium sulphate colitis mouse model: traps and tricks. J. Biomed. Biotechnol. 2012. 2012: 718617.
  • 26
    Axelsson, L. G., Landstrom, E., Goldschmidt, T. J., Gronberg, A. and Bylund-Fellenius, A. C., Dextran sulfate sodium (DSS) induced experimental colitis in immunodeficient mice: effects in CD4(+) -cell depleted, athymic and NK-cell depleted SCID mice. Inflamm. Res. 1996. 45: 181191.
  • 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.
  • 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: 16431652.
  • 29
    Shintani, N., Nakajima, T., Okamoto, T., Kondo, T., Nakamura, N. and Mayumi, T., Involvement of CD4+ T cells in the development of dextran sulfate sodium-induced experimental colitis and suppressive effect of IgG on their action. Gen. Pharmacol. 1998. 31: 477481.
  • 30
    Kim, T. W., Seo, J. N., Suh, Y. H., Park, H. J., Kim, J. H., Kim, J. Y. and Oh, K. I., Involvement of lymphocytes in dextran sulfate sodium-induced experimental colitis. World J. Gastroenterol. 2006. 12: 302305.
  • 31
    Yanaba, K., Asano, Y., Tada, Y., Sugaya, M., Kadono, T. and Sato, S., Proteasome inhibitor bortezomib ameliorates intestinal injury in mice. PLoS One 2012. 7: e34587.
  • 32
    Wirtz, S., Neufert, C., Weigmann, B. and Neurath, M. F., Chemically induced mouse models of intestinal inflammation. Nat. Protoc. 2007. 2: 541546.
  • 33
    Uematsu, S., Fujimoto, K., Jang, M. H., Yang, B. G., Jung, Y. J., Nishiyama, M. and Sato, S. et al., Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5. Nat. Immunol. 2008. 9: 769776.
  • 34
    Rudolphi, A., Bonhagen, K. and Reimann, J., Polyclonal expansion of adoptively transferred CD4 +alpha beta T cells in the colonic lamina propria of scid mice with colitis. 1996 Eur. J. Immunol. 26: 11561163.
Abbreviations
DAI

disease activity index

DSS

dextran sulfate sodium

IBD

inflammatory bowel disease

IEL

intestinal epithelial lymphocyte

GA

glatiramer acetate

LPL

lamina propria lymphocyte

TCV

T-cell vaccination

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. Reference
  10. Supporting Information

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eji2464-sup-0001-FigureS1.pdf343KSupplementary Fig. 1 Qa-1-restriction pp y g Q of GA-induced CD8+ regulatory T 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 and Qa-1-/- mice were transferred into recipients (i.v. 106 cells per mouse) on the day of disease induction. Mice injected with na・e WT or Qa-1-/- 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. Error bars indicate the SD. Similar results were observed in two independent experiments (with 5 mice per group). (Mann-Whitney test,*p < 0.05)

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