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Keywords:

  • Foxp3+ regulatory T cells;
  • γδ T cells;
  • IL-17;
  • Mucosal inflammation

Abstract

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

CD4+CD25+Foxp3+ regulatory T (TREG) cells are critical mediators of peripheral immune tolerance, and abrogation of their function provokes a variety of autoimmune and inflammatory states including inflammatory bowel disease. In this study, we investigate the functional dynamics of TREG-cell responses in a CD4+ T-cell-induced model of intestinal inflammation in αβ T-cell-deficient (TCR-β−/−) hosts to gain insights into the mechanism and cellular targets of suppression in vivo. We show that CD4+ T effector cell transfer into T-cell-deficient mice rapidly induces mucosal inflammation and colitis development, which is associated with prominent Th1 and Th17 responses. Interestingly, we unveil a prominent role for resident γδ T cells in mucosal inflammation as they promote Th1 and particularly Th17 responses in the early phase of inflammation, thus exacerbating colitis development. We further demonstrate that CD4+CD25+Foxp3+ TREG cells readily inhibit these responses and mediate disease protection, which correlates with their accumulation in the draining LN and lamina propria. Moreover, TREG cells can directly suppress γδ T-cell expansion and cytokine production in vitro and in vivo, suggesting a pathogenic role of γδ T cells in intestinal inflammation. Thus, functional alterations in TREG cells provoke dysregulated CD4+ and γδ T-cell responses to commensal antigens in the intestine.


Introduction

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

The gastrointestinal tract represents a major site where immune tolerance mechanisms assure a homeostatic equilibrium between the mucosal immune system and commensal microorganisms 1, 2. Given the permanent co-existence of harmless and pathogenic bacteria that constantly trigger local immune responses, the intestinal mucosa must maintain tolerance in these sites. A disturbance in immune homeostasis of the human gut may provoke inflammatory bowel diseases (IBDs) like Crohn's disease (CD) and ulcerative colitis, both characterized by an abnormal accumulation of activated lymphocytes in the gut resulting in chronic intestinal inflammation 1–5. CD4+Foxp3+ TREG cells are widely recognized as dominant mediators responsible for the control of peripheral tolerance 6–10. Functional abrogation of these cells results in over-activation and uncontrolled inflammatory responses towards tissue-derived antigens and commensal bacteria, leading to the development of various chronic inflammatory disorders 10–13.

Our current understanding of the role of Foxp3+ TREG cells in the prevention of IBD development is largely derived from mouse models where intestinal inflammation is induced by adoptive transfer of CD4+ T effector (TEFF) cells into lymphocyte-deficient nude, SCID or RAG−/− hosts 14. Collectively, these studies show that CD4+Foxp3+ TREG cells prevent colitis development or even cure established disease by restraining pathogenic CD4+ T-cell and DC immune responses 15–18. However, other cellular targets of suppression in vivo remain ill-defined. Recently, increasing evidence points to a significant multi-faceted role for non-CD4+ lymphocytes, including γδ T cells, in the maintenance of intestinal homeostasis 19–21. More specifically, it has been shown that γδ T cells readily accumulate in inflamed tissues of IBD patients 22–25, although, in murine studies, γδ T cells have been shown to either potently reduce 26–28 or exacerbate inflammation 29–33. Some studies also identify γδ T cells as a source of rapidly activated T cells with Th17-like effector properties providing the first line of defense against pathogens 34–36. A significant role of IL-17-producing γδ T cells in the exacerbation of the autoimmunity has also been observed in the mouse models of experimental autoimmune encephalomyelitis 37 and collagen-induced arthritis 30. While some recent studies suggest that TREG cells can suppress some aspects of human or mouse γδ T-cell functions 32, 38–40, the dynamics and impact of this regulation on γδ T-cell function throughout IBD development is ill-defined.

In this study, we investigate the functional dynamics of Foxp3+ TREG cells in the control of γδ T-cell responses in a mouse CD4+ TEFF cell transfer model of intestinal inflammation in αβ T-cell-deficient TCR-β−/− C57BL/6 (B6) mice. We show that transfer of CD4+ TEFF cells rapidly induces colitis development, which is associated with prominent Th1- and Th17-cell responses, a process readily inhibited by CD4+CD25+Foxp3+ TREG cells in the draining LN and the site of intestinal inflammation. Interestingly, we identify gut-residing γδ T cells as key players in mucosal inflammation as they promote an acute wave of Th1- and, particularly, Th17-like responses in the early phase of inflammation, thus exacerbating colitis development, indicating a pathogenic role of γδ T cells in intestinal inflammation. We further show that CD4+CD25+Foxp3+ TREG cells directly suppress γδ T-cell expansion and cytokine production in vitro, and can potently inhibit these responses in vivo and mediate disease protection.

Results

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

equation imageequation image cells prevent the accumulation of colitogenic CD4equation image Tequation image cells in mesenteric sites

Murine models of T-cell-induced colitis have largely used lymphocyte-deficient SCID, RAG−/− and nude recipient mice 18, 41, 42. In order to study the dynamics of TEFF and TREG-cell responses during mucosal inflammation, we established a new mouse model of T-cell-induced colitis in B6 TCR-β−/− mice that are genetically autoimmune-resistant, and harbor a normal adaptive immune system with the exception of αβ T cells.

In this model, colitis was induced in TCR-β−/− recipient mice by the transfer of colitogenic CD4+CD25 (>98% Foxp3) TEFF cells from WT B6 mice, and suppressed by the co-transfer of WT B6 CD4+CD25+ (>95% Foxp3+) TREG cells. By 2–3 wk after T-cell transfer, all recipients of TEFF cells developed clinical signs of colitis, including diarrhea and weight loss, in contrast to the mice reconstituted with TEFF and TREG subsets (Fig. 1A). Although un-reconstituted TCR-β−/− mice spontaneously develop a well-accepted, low level, bacterial-induced mucosal inflammation 41, 43, histological analysis of colonic tissues of recipient mice showed a prominent transmural infiltration of mononuclear cells in the intestinal mucosa and lamina propria (LP) (Fig. 1B and C). Co-transfer of CD4+CD25+ TREG cells significantly suppressed intestinal inflammation and restored normal tissue architecture (Fig. 1B and C).

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Figure 1. Foxp3+ TREG cells inhibit colitis by preventing the accumulation and proliferation of CD4+ TEFF cells in mesenteric sites of TCR-β−/− mice. B6 TCR-β−/− mice received either total CD4+ (TEFF+TREG) or CD4+CD25 (TEFF) cells (1.3×106 each) isolated from GFP-Tg mice. (A) Individual body weight of recipients, as an indicator of colitis development, was monitored and compared with the body weight prior to T-cell transfer. (B and C) Colonic tissues were harvested 21 days post T-cell transfer, stained with H&E and analyzed by microscopy (200× magnification). (D–G) Seven and 21 days post adoptive transfer, (D and E) the frequency and (F and G) proliferation of donor GFP+CD4+ T cells were assessed in LNs and LP. (E) Fold increase of donor CD4+ T-cell accumulation or (G) proliferation between colitic and protected mice is shown at 7 and 21 days post T-cell transfer. Results from one representative experiment out of five (with n≥4 mice per group) are shown as mean±SEM (Student's t test, *p<0.01 and **p<0.01).

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Moreover, flow cytometric analysis of non-draining peripheral (per-) and draining mesenteric (mes-) LNs as well as LP 3 wk post T-cell transfer shows a progressive increase in donor TEFF-cell frequency, particularly in LP of colitic mice (Fig. 1D and E), suggesting a mucosa-specific accumulation/expansion of pathogenic CD4+ TEFF cells in TCR-β−/− recipient mice (Fig. 1D). This corresponded to a prominent increase in TEFF cell absolute numbers in colitic mice compared with healthy controls (a two-fold increase on day 7 and six- to eight-fold increase on day 21) (Fig. 1E), suggesting a dysregulated expansion of donor TEFF cells in the absence of TREG cells.

In order to examine kinetics of lymphocyte proliferation in TCR-β−/− recipient mice, cycling cells from secondary lymphoid tissues and LP were determined by intracellular Ki-67 expression at different time points during disease progression. Our results show a progressive increase in frequencies and absolute numbers of cycling lymphocytes in colitic mice (Fig. 1F), which was significantly decreased in all lymphoid organs examined, as well as in the LP, upon TREG-cell co-transfer (Fig. 1F and G). More importantly, the reduced absolute numbers of donor TEFF cells in mesLN compared with LP (Fig. 1G) suggests that TREG cells hamper the expansion and accumulation of pathogenic cells in the site of tissue inflammation.

Foxp3equation image Tequation image cells inhibit the differentiation and expansion of intestinal Th1 and Th17 CD4equation image Tequation image cells

Studies show that a prominent role for Th1, and in particular Th17, polarized immune responses in autoimmunity and IBD-like disorders in humans and in mouse models 44, 45. In particular, IL-17-secreting T cells are found in lesions of patients with CD 4, 22, 25, and genome-wide association studies of CD and ulcerative colitis patients indicate the importance of Th17-promoting factors, including IL-23, in IBD 46, 47.

We then sought to characterize the inflammatory nature of the mucosal inflammation. We observed a significant increase in IFN-γ IL-1β, IL-12 and IL-6 mRNA expression in colons of mice reconstituted with CD4+CD25 TEFF cells alone, while CD4+CD25+ TREG cell-mediated protection from colitis correlated with higher levels of IL-4 and IL-10 mRNA expression (Fig. 2A). Moreover, we found a marked increase in frequencies and absolute numbers of IFN-γ- and IL-17-producing lymphocytes in secondary lymphoid tissues and LP of colitic mice (Fig. 2B–E), indicating that TREG cells potently suppress the priming and expansion of these cells in protected mice. Interestingly, our results reveal a temporal difference in the emergence of IFN-γ- and IL-17-producing cells. While IFN-γ was highly expressed in the absence of TREG cells in both perLN and mesLN (Fig. 2B), IL-17 secretion was more specific to the intestinal tissue (Fig. 2B and C). This is consistent with previous studies pointing to the mucosa as a privileged site for Th17-cell development due to elevated secretion of specific polarizing mediators such as IL-6 and TGF-β1 25. Moreover, while the frequency of IFN-γ-secreting CD4+ TEFF cells (≈40% of CD4+ T cells) in the inflammatory site remained unchanged during colitis development, the frequency of IL-17+ donor CD4+ TEFF cells steadily dropped from 35% at day 7 to 20% at day 21 (Fig. 2D and E), suggesting a role for different signals in the initial and progressive phases of T-cell-induced colitis in TCR-β−/− mice. Thus, TREG cells prevent accumulation of Th17 cells more effectively than Th1 cells particularly in intestinal sites (Fig. 2D and E).

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Figure 2. Foxp3+ TREG cells inhibit the differentiation and expansion of Th1 and Th17 CD4+ TEFF cells in intestinal tissue of αβ T-cell-deficient mice. Total CD4+ (TEFF+TREG) or CD4+CD25 (TEFF) cells (1.3×106 each) were transferred into TCRβ−/− recipients. (A) After 3 wk, total colonic RNA was analyzed by RT-PCR for gene expression of the indicated cytokines relative to G3PDH expression. Relative cytokine production from one representative experiment out of three is shown and was compared with the basal levels of expression in protected mice. (B and C) Lymphocytes from LNs and LP were harvested 21 days post T-cell transfer, and (C) representative FACS profiles or (B) the frequencies and absolute numbers of total IFN-γ- or IL-17-producing cells are shown. The kinetics of (D) IFN-γ or (E) IL-17 production by donor CD4+ T cells in mesLN and LP at 7, 14 and 21 days post adoptive T-cell transfer are shown. Results from one representative experiment out of three (with n=4 mice per group) are shown as mean±SEM (Student's t test, *p<0.01 and **p<0.01).

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Foxp3equation image Tequation image cells control IFN-γ and IL-17 production by γδ T cells during mucosal inflammation

Upon further analysis of pro-inflammatory cytokine production, we found that CD3+CD4 γδ TCR+ cells accounted for approximately 50% of total IFN-γ-producing cells (Fig. 3A). The kinetic analysis of cytokine production revealed that resident γδ T cells were the predominant cytokine-producers in the mesLN and LP of TCR-β−/− recipient mice during the early phase of intestinal inflammation (Fig. 3B and C). We observed that γδ T cells from TCR-β−/− recipient mice reconstituted with CD4+CD25 TEFF cells alone produced either IFN-γ or IL-17 (15 and 5% respectively) (Fig. 3D and E) throughout colitis development, and this represented over 80% of total IFN-γ- and IL-17-producing cells 4 days post CD4+ T-cell transfer (Fig. 3B and C). At a later stage of intestinal inflammation, the balance of cytokine expression between γδ and αβ T cells tipped in favor of αβ T cells, as 70–80% of IFN-γ-/IL-17-secreting cells in the LP originated from donor CD4+ TEFF pool (Fig. 3B and C). In all instances, co-transfer of CD4+CD25+ TREG cells potently inhibited the priming, differentiation and accumulation of IFN-γ-/IL-17-producing CD4+ and γδ T cells in mesLN and LP (Fig. 3D and E). It is noteworthy to mention that, although some recent studies suggest functional differences in peripheral (non-mesenteric) γδ T cells between WT and TCR-β−/− mice 48, the cytokine profile of mesenteric γδ T cells isolated from TCR-β−/− mice was similar to the cytokine profile of WT mesenteric γδT cells in our experiments (data not shown).

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Figure 3. Foxp3+ TREG cells control the expansion and production of IFN-γ and IL-17 by γδ T cells during TEFF cell-induced mucosal inflammation. Total CD4+ (TEFF+TREG) or CD4+CD25 (TEFF) cells (1.3×106 each) were transferred into TCRβ−/− mice, and the frequency of IFN-γ- or IL-17-producing CD3+ lymphocytes in mesLN and LP was analyzed at the indicated time points. (A) Representative FACS profiles and the relative proportion of host γδ T cells (i.e. CD3+CD4) or donor CD4+ (CD3+CD4+) T cells secreting (B) IFN-γ or (C) IL-17 throughout colitis development is shown. Frequencies and absolute numbers of (D) IFN-γ or (E) IL-17 producing γδ T cells (CD3+CD4) from one representative experiment out of three (with n=3–4 mice per group) are shown as mean±SEM (Student's t test, *p<0.01 and **p<0.01).

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Increased Th17 response and accelerated T-cell induced colitis development in TCR-β−/− mice

While CD4+ T cells are the primary mediators of disease in our model, it has been suggested that B cells largely play an important regulatory role as the onset of colitis is delayed in immunodeficient recipients 19, 49–51. As the role of γδ T cells in colitis development is unknown in our system, we compared the onset and severity of T-cell-induced intestinal inflammation between TCR-β−/− (lacking only αβT cells) and RAG2−/− (lacking all lymphocyte lineages) mice. To this end, both host strains were reconstituted with WT CD4+CD25 TEFF cells, and the onset of colitis as well as cytokine profile was compared. By 10 days post TEFF cells transfer, TCR-β−/− recipient mice rapidly began to show clinical signs of colitis development and lost 30% of their initial body weight within 3 wk (Fig. 4A). In contrast, RAG2−/− recipient mice showed a delayed onset of colitis and less severe body weight loss (>20%) by 3–5 wk post T-cell transfer (Fig. 4A).

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Figure 4. Increased Th17 and accelerated T-cell-induced colitis development in TCR-β−/− compared with RAG−/− mice. CD4+CD25 TEFF cells (0.75×106) were transferred into TCR-β−/− or RAG−/− mice, and (A) the loss of body weight as an indicator of colitis development, is shown for each group. (B) Four weeks post T-cell transfer, colonic tissues were harvested, stained with H&E and analyzed by microscopy (40× and 200× magnifications). Arrowheads indicate cluster formation within the mononuclear cell infiltrate. (C) The absolute numbers and frequencies of (E) IFN-γ- or (F) IL-17- producing donor CD4+ T cells in mesLN and LP from one representative experiment out of three (with n=4 mice per group) are shown as mean±SEM. (D) Representative FACS profiles of cytokine secreting cells are shown (Student's t test, *p<0.001).

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Histological analysis of colonic tissues of TCR-β−/− and RAG2−/− recipient mice 30 days post TEFF cell transfer revealed similar levels of global, intestinal inflammation. However, we observed some differences in the cellular architecture of the inflamed, colonic tissues of TCR-β−/− and RAG2−/− mice. More specifically, mononuclear infiltrates in the LP of TCR-β−/− mice tended to form clusters that were completely absent in the LP of RAG2−/− recipients, although their significance is unclear (Fig. 4B). Moreover, we did not detect a significant change in the frequency, absolute number or phenotype of B cells during colitis development (Supporting Information Fig. 1). While these observations do not exclude a possible role for B cells in this process, they also do not exclude a potential contribution for resident γδ T cells during T-cell-induced immune pathology in the gut.

Flow cytometric analysis of draining mesLN of colitic mice showed a two-fold increase in accumulation of donor CD4+ TEFF cells in TCR-β−/− compared with RAG2−/− recipient mice; however, CD4+ TEFF cells accumulated at a similar rate in the LP of either recipients (Fig. 4C). Interestingly, when we examined frequencies of IFN-γ- and IL-17-secreting donor CD4+ T cells, we observed that RAG2−/− recipient mice harbored significantly fewer IL-17+ TEFF cells compared with TCR-β−/− mice, despite a slightly more elevated frequency in IFN-γ-secreting TEFF cells. Over 50% of donor CD4+ T cells isolated from mesLN and LP of RAG2−/− recipients secreted IFN-γ, and only 10% were positive for IL-17, which is three times less compared with TCR-β−/− recipient mice (Fig. 4D and E). Thus, γδ T cells resident in mesenteric sites of TCR-β−/− mice fuel Th17 responses and actively participate in intestinal inflammation.

Foxp3equation image Tequation image cells control proliferation of CD4equation image and γδ T cells during mucosal inflammation

Our results show that TREG cells potently inhibit the expansion and accumulation of pro-inflammatory cytokine secreting donor CD4+ TEFF and host γδ T cells in T-cell-induced intestinal inflammation in TCR-β−/− mice. Interestingly, by 21 days post CD4+ TEFF cell transfer, co-transfer of TREG cells resulted in a two-fold reduction in the proportion of γδ T cells in mesLN compared with colitic mice receiving only TEFF cells (Fig. 5A and B). Furthermore, this decrease was more profound in the LP and reached an eight-fold reduction in the proportion of γδ T cells (Fig. 5B), suggesting that TREG cells impair the accumulation of γδ T cells in the inflamed gut.

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Figure 5. Foxp3+ TREG cells control proliferation of donor CD4+ and host γδ T cells during TEFF cell-induced mucosal inflammation. Total CD4+ (TEFF+TREG) or CD4+CD25 (TEFF) cells (1.3×106 each) were transferred into TCRβ−/− recipients, and (A and B) the accumulation and (C and D) proliferating capacity of host γδ T cells were assessed in mesLN and LP by FACS 21 days post adoptive transfer. Frequencies and absolute numbers of (A) total or (C) cycling CD3+γδ TCR+ T cells are shown. Fold decrease of host γδ T-cell accumulation (B) and proliferation (D) between protected and colitic mice is shown. Results from one representative experiment out of three (with n=4 mice per group) are shown as mean±SEM (Student's t test, *p<0.01 and **p<0.01).

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To examine the proliferation of donor and host T cells in the presence and absence of TREG cells, the proportion of cycling cells was determined by intracellular Ki-67 expression. Co-transfer of TREG cells significantly decreased the frequency and absolute numbers of cycling donor CD4+ TEFF and resident γδ T-cell populations in lymphoid organs as well as in the LP in recipient TCR-β−/− mice (Fig. 5C and D). Thus, TREG-cell transfer suppresses the expansion and accumulation of resident γδ T cells in the inflamed colon during development of T-cell-induced colitis.

Foxp3equation image Tequation image cells directly suppress γδ T-cell functions in vitro and in vivo

In order to show a direct inhibitory effect of TREG cells on γδ T cells, we performed an in vitro suppression assay where anti-CD3 pre-activated FACS sorted responder populations were co-cultured with titrated numbers of freshly isolated CD4+CD25+ TREG cells. At the highest 1:1 TREG to T responder ratio, TREG cells inhibited γδ T-cell proliferation by 75%, with a similar effect on control CD4+CD25 T responder cells (Fig. 6A). Our results also show that TREG cells suppressed γδ T cells proliferation in vitro in a dose-dependent manner (Fig. 6A).

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Figure 6. Foxp3+ TREG cells directly suppress γδ T-cell proliferation and inflammatory cytokine production in vitro and in vivo. (A) FACS-sorted γδ TCR+ or CD4+CD25 responder T cells were pre-activated for 12 h with soluble anti-CD3 (1 μg/mL), IL-2 (100 U/mL) and irradiated total splenocytes (APCs). FACS-sorted CD4+CD25+ TREG cells were then co-cultured at various TRESPONDER to TREG ratios in fresh medium. Percent proliferation at each ratio, assessed by 3H-thymidine incorporation, was normalized to responder T-cell proliferation. Data are presented as mean±SD of triplicate wells. (B–D) TCR-β−/− recipient mice received TREG cells (0.3×106), and the frequency and cytokine producing potential of resident γδ TCR+ cells were analyzed 2 wk post transfer. Absolute numbers of (B) total and (D) IFN-γ- or IL-17- producing γδ TCR+ cells as well as (C) representative FACS profiles are shown. (E–G) RAG2−/− recipient mice were reconstituted with in vivo-activated sorted γδ T cells (0.7×106) either alone or in combination with TREG (0.3×106). The frequency of total (E) and IFN-γ- or IL-17-producing (F and G) γδ TCR+ cells was assessed in mesLN 2 wk later. Results from one representative experiment out of two (with n= 4–5 mice per group) are shown as mean±SEM (Student's t test, *p<0.01 and **p<0.01).

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To determine whether TREG cells are able to directly inhibit γδ T-cell responses in vivo independently of CD4+ TEFF cells, we first adoptively transferred CD4+CD25+ TREG cells alone in TCR-β−/− recipient mice, and assessed γδ T-cell responses. Administration of TREG cells significantly reduced the accumulation of γδ T cells in both mesLN and LP of recipient mice (Fig. 6B). Moreover, 14 days post TREG-cell transfer, recipient mice showed a significant decrease in the proportion of resident IFN-γ- and IL-17-producing γδ T cells compared with control non-reconstituted mice (Fig. 6C and D). Furthermore, we also adoptively transferred RAG2−/− recipient mice with γδ T cells in the presence or absence of TREG cells. Our results show that although the expansion of donor γδ T cells was unchanged by TREG cell co-administration (Fig. 6E), the secretion of IFN-γ and IL-17 by γδ T cells was significantly inhibited (Fig. 6F and G). We observed a two- and four-fold decrease in the frequency of IFN-γ- and IL-17-secreting γδ T cells in the presence of TREG cells (Fig. 6G).

Overall, we show that TREG cells, in addition to controlling donor CD4+ TEFF cell functions, are also able to directly suppress γδ T cells in vitro as well as significantly dampen the inflammatory response of resident γδ T cells in our in vivo model of T-cell-induced colitis. While TREG cells readily suppressed CD4+ TEFF cells, we make the novel observation that TREG cells are particularly capable of restraining the expansion and effector differentiation of resident pro-inflammatory γδ T cells in the mesLN and intestinal tissue.

Discussion

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

In our study, we investigated the dynamics of TREG and pathogenic T-cell responses in a T-cell-adoptive transfer model of intestinal inflammation in an attempt to gain insights into the mechanisms and cellular targets of TREG cell-mediated suppression in vivo. We show that CD4+CD25+Foxp3+ TREG cells suppress the mucosal inflammation induced by colitogenic CD4+CD25Foxp3 TEFF cells and reduce the pathogenic potential of donor αβ and resident γδ TEFF cells in the intestinal microenvironment of αβ T-cell-deficient TCR-β−/− mice.

We show that γδ T cells are active contributors to the global inflammatory environment in T-cell-induced colitis. Resident γδ T cells actively proliferate, differentiate into Th1- or Th17-like cells and migrate to the mucosal tissue, where they continue to expand and secrete IFN-γ and IL-17. Previous reports have shown that γδ T cells, among other mucosa-residing innate and memory cells, produce a basal level of IL-17 and IL-22, which play an important role in maintenance of a constitutive level of antimicrobial proteins implicated in mucosa surveillance 52, 53 as well as the tonus of endothelial junctions 54. Our results demonstrate that within the first days post CD4+ TEFF cell transfer, γδ T cells produce the majority of IL-17 and IFN-γ. This early cytokine secretion probably acts as an amplification factor for the development of colitogenic CD4+ Th17 cells, given that in RAG2−/− mice, lacking both αβ and γδ T cells, we observed reduced IL-17 expression in CD4+ TEFF cells. IL-17 secreted by γδ T cells may directly act on CD4+ T cells, since in vitro stimulation with IL-17A and IL-23 upregulates IL-17A/F mRNA expression in CD4+ T cells 37, or indirectly, by conditioning resident APCs. Moreover, this early IL-17 production may also act directly on APCs, such as macrophages and subsets of DCs, which are known to express IL-17R more abundantly than T cells, and provoke APC production of IL-23, IL-1, IL-6 and TGF-β1 37, 55, which are crucial factors for pathogenic Th17-cell development. Consistently, IL-17 secretion is significantly more elevated in mucosal tissues, where we detected an elevated level of IL-1β and IL-6 mRNA expression.

Importantly, our results show that CD4+CD25+Foxp3+ TREG cells directly suppress the proliferation and differentiation of γδ T cells in vitro and in vivo. Moreover, we show that in the context of mucosal inflammation, TREG cells restrain the proliferation of resident γδ T cells more strongly than donor CD4+CD25 TEFF cells, although a similar potency in TREG cell-mediated suppression of both populations is observed in vitro. This finding is consistent with a recent study showing that TREG cells inhibit γδ T-cell proliferation in vitro 32, 40. It is possible that the more potent TREG-cell suppression of IL-17 secretion compared with IFN-γ secretion seen in the mucosal tissue occurs as a result of a more profound inhibition of γδ T-cell expansion in situ. Whether this happens due to a greater susceptibility of γδ T cells to direct TREG cell-mediated suppression or indirect inhibition mediated by TREG cell-conditioned APCs requires further investigation. Interestingly, in contrast to γδ T cells, a significant fraction (around 30%) of CD4+ TEFF cells found in mucosa-associated tissues co-expressed IFN-γ and IL-17, an observation reminiscent of recent human studies showing the existence of IFN-γ/IL-17 dual producing CD4+ T cells in colonic biopsies of CD patients 25. Furthermore, our results demonstrate that both CD4+ and γδ T cells from mucosal tissues of recipient mice are more activated as they display a higher proliferation rate and secrete more pro-inflammatory cytokines compared to cells from LNs. Although TREG cells are not able to completely inhibit priming of the pro-inflammatory TEFF cells in the mucosa-draining lymphoid tissues (mesLN), the dramatic reduction in absolute numbers of LP-infiltrating lymphocytes suggests that TREG cells regulate the influx and/or expansion of activated αβ and γδ TEFF-cell subsets in the site of tissue inflammation. These results are consistent with a recent study by Park et al., which identifies IL-10 as a potential mediator in Foxp3+ TREG cell-mediated suppression of γδ T cells 32.

Overall, we show that Foxp3+ TREG cells control colitogenic responses in our T-cell-induced model of intestinal inflammation by inhibiting the expansion and Th1/Th17 differentiation of CD4+ TEFF cells, as well as γδ TEFF cells, in mesLN and directly within the target organ. We show that resident γδ T cells are an early, innate-like source of IL-17 and that γδ T cells amplify Th17 responses and exacerbate colitis development. Moreover, we also demonstrate that Foxp3+ TREG cells also suppress the expansion and cytokine-producing potential of resident γδ T cells at an early stage of colitis development. These findings will increase our understanding of TREG cell-mediated control of bacterially driven mucosal inflammation and may enable us to design novel approaches to potentiate TREG-cell function and consequential tolerance induction in various chronic inflammatory disorders.

Materials and methods

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

Mice

WT, TCR-β−/− and RAG2−/− B6 mice were obtained from Taconic Laboratories, while GFP transgenic B6 (pUbi-GFPtg) mice were provided by Dr. Schaefer 56. All mice were generally used at 6–10 wk of age. Mice were housed and bred under specific pathogen-free conditions according to institutional guidelines at McGill University (animal use protocol ♯4715).

Purification of T-cell subsets

For in vivo adoptive transfer, CD4+CD25+ (TREG), CD4+CD25 (TEFF), CD4+ (total) and γδ TCR+ T-cell subsets from appropriate mice were purified from a pool of splenocytes and LN cells using the autoMACS cell sorter (Miltenyi Biotec) according to the manufacturer's protocol. Briefly, CD4+CD25+ T-cell fraction (∼90% purity) was obtained by positive selection for CD25. The remaining cells were used to obtain CD4+CD25 TEFF fraction (>93% purity) by positive selection for CD4. CD4+ and γδ TCR+ T-cell subsets (>93 and > 90% purity, respectively) were obtained by positive selection for CD4 or γδ TCR. For in vitro suppression assays, T-cell subsets were isolated using a FACSAria Cell Sorter with a purity > 98%. CD4+CD25 TEFF or CD4+CD25+ TREG cells were sorted from WT B6. CD3+γδ TCR+ T cells were sorted form TCR-β−/− mice.

Adoptive T-cell transfers

MACS purified CD4+CD25 TEFF (1.3×106), a mixture of CD4+CD25+ TREG (0.2×106) and CD4+CD25 TEFF (1.3×106) T cells, and (0.7×106) γδ T cells from GFP-Tg or WT donor mice were intravenously transferred into TCR-β−/− or RAG2−/− recipient mice. Individual body weight, as an indicator of disease incidence, was monitored and compared with body weight at the start point.

Histological analysis of colonic tissues

Colonic tissues were collected from recipient mice and either directly mounted in optimum cutting temperature compound or fixed in 10% paraformaldehyde followed by paraffin embedding. Sections of 10 μm for frozen and 6 μm for paraffin embedded tissues were made, subjected to hematoxylin/eosin staining and analyzed by a pathologist giving the score from 0–4 based on previously described criteria 57, 58.

Lymphocyte isolation from LP

In order to isolate lymphocytes from LP, a modified protocol from 59 was used. Briefly, colonic tissues from recipient mice were isolated, washed with PBS and cut into pieces. Epithelial and subepithelial cells as well as intraepithelial lymphocytes were removed by EDTA incubation. The remaining LP were incubated twice for 25 min at 37°C in RPMI medium containing DNAse (5 mg), collagenase A (25 mg), collagenase D (25 mg), dispase I (0.3 g) and penicillin/streptomycin (100 U/mL). Lymphocytes were then collected, passed though the cell strainer and resuspended in medium.

Antibodies and flow cytometry

Single-cell suspensions prepared from different organs of recipient mice were stained and analyzed on FACSCalibur or FACSCanto (Becton Dickinson, Mountain View, CA) using FlowJo software (Tree Star). For surface phenotyping of lymphocyte populations, the following fluorochrome-conjugated or biotinylated mAbs were used: anti-CD4 (RM4-5), anti-CD25 (PC61), anti-CD3 (145-2C11) and anti-γδ TCR (GL-3) (eBioscience or BD Bioscience). For determination of intracellular cytokine production, cells were restimulated with PMA (20 ng/mL), ionomycin (1 nM) for 4 h at 37°C in the presence of BD GolgiStop (1:1000 dilution). Cells were then stained for surface antigens, fixed/permeabilized with Fix/Perm solution (eBioscience) and stained with anti-IFN-γ (XMG1.2), anti-IL-17A (TC11-18H10.1 or eBio17B7), anti-IL-10 (JES5-16E3), anti-IL-2 (JES6-5H4) (purchased from eBioscience or BD Bioscience). In order to determine cellular proliferation in vivo, cells were stained intracellularly with anti-Ki-67 (B56) (BD Bioscience), as described above.

Quantitative real-time PCR

Colons were collected in RNAlater (Qiagen, Mississauga, ON) and frozen at −20°C until use. RNA was extracted following the TRIzol protocol (Invitrogen, Burlington, ON). Total RNA was reverse-transcribed using the cDNA Archive Kit (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) (1 PCR cycle, 95°C, 10 min; 40 PCR cycles, 60°C, 1 min, 95°C, 15 s). cDNA (10 ng total RNA) was amplified in a reaction mix containing TaqMan Universal PCR Master Mix (Applied Biosystems) and corresponding TaqMan Gene Expression Assays (Applied Biosystems). Signals were analyzed by the ABI Prism Sequence Detection System software version 2.2 (Applied Biosystems). The comparative Ct method for relative quantification was used, whereby all threshold cycles were normalized to the expression of 18s rRNA. Cytokine expression is represented as a fold-change relative to control non-diseased mice adoptively transferred with total CD4+ T cells.

In vitro suppression assays

For suppression assay, FACS-sorted γδ TCR+ or CD4+CD25 T cells (50×103) were plated in 96-well, flat-bottomed microtiter plates (0.2 mL) with 200×103 irradiated total splenocytes and activated with soluble anti-CD3 (1 μg/mL) and IL-2 (100 U/mL). After 12 h, 75% of the medium was subtracted from each well, and FACS-sorted CD4+CD25+ TREG cells were added with fresh medium to the co-culture at various ratios. Cells were cultured for a total of 72 h at 37°C and pulsed for the last 12 h with 0.5 uCi of 3H-thymidine to determine the extent of proliferation.

Statistical analysis

Analyses were performed with a Student's t test. Values of p<0.05 were considered significant.

Acknowledgements

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

We acknowledge the financial support of the Canadian Institutes for Health Research (MOP 67211 and MOP 84037 to C.A.P). The authors thank Marie-Hélène Lacombe from the RI-MUHC Immunophenotyping Platform for FACS Sorting and Genny Fortin for the help with RT-PCR. C.A.P. holds the Canada Research Chair.

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

References

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

Supporting Information

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

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