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Keywords:

  • CD4+ regulatory T cells;
  • TGF-β;
  • Autoimmunity;
  • Suppression

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Naturally occurring CD4+CD25+ regulatory T cells (Treg) are potent suppressors of CD4+ and CD8+ T cell responses in vitro and inhibit several organ-specific autoimmune diseases. While most in vitro studies suggest that CD4+CD25+ Treg cells adopt a cytokine-independent but cell contact-dependent mode of T cell regulation, their precise mechanism of suppression in vivo remains largely unknown. Here we examine the functional contribution of Treg cell-derived TGF-β1 and effector T cell responsiveness to TGF-β in CD4+CD25+ T cell-mediated suppression of inflammatory bowel disease (IBD). We show that CD4+CD25+ Treg cells from either TGF-β1+/+ or neonatal TGF-β1–/– mice can suppress the incidence and severity of IBD as well as colonic IFN-γ mRNA expression induced by WT CD4+CD25 effector T cells. Furthermore, TGF-β-resistant Smad3–/– CD4+CD25+ Treg cells are equivalent to WT Treg cells in their capacity to suppress disease induced by either WT or Smad3–/– CD4+CD25 effector T cells. Finally, anti-TGF-β treatment exacerbates the colitogenic potential of CD4+CD25 effector T cells in the absence of CD4+CD25+ Treg cells. Together, these data demonstrate that in certain situations CD4+CD25+ T cells are able to suppress intestinal inflammation by a mechanism not requiring Treg cell-derived TGF-β1 or effector T cell/Treg cell responsiveness to TGF-β via Smad3.

Abbreviations:
AIG:

autoimmmune gastritis

IBD:

inflammatory bowel disease

LP:

lamina propria

Treg:

regulatory T cells

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

CD25 (IL-2Rα chain)-expressing CD4+ T cells, which constitute 5–10% of normal CD4+ T cells, play a critical role in the induction and maintenance of peripheral tolerance 1, 2. These naturally occurring CD4+CD25+ regulatory T (Treg) cells consist of an anergic lymphocyte population with potent immunosuppressive functions in vivo, as the depletion of CD25-expressing CD4+ T cells correlates with increased immunity to tumors, grafts and intracellular pathogens and provokes the induction of multiple inflammatory diseases including autoimmmune gastritis (AIG) and inflammatory bowel disease (IBD) 3, 4.

Currently, the mechanism(s) by which CD4+CD25+ Treg cells mediate suppression in vitro and in vivo is unclear. Following TCR activation, CD4+CD25+ Treg cells suppress the in vitro proliferation and cytokine production of CD4+ and CD8+ T cells in an antigen non-specific, but contact-dependent manner that does not appear to involve modulation of APC function 57. The role of regulatory cytokines in CD4+CD25+ Treg cell control of inflammation in vivo appears to be tissue and/or context-dependent. While CD4+CD25+ Treg cells from IL-4–/– and IL-10–/– mice are as effective as WT CD4+CD25+ T cells in mediating suppression of AIG, CD4+CD25+ Treg cells from IL-10–/– mice cannot control IBD induced by antigen-experienced effector T cells 8, 9. Similarly, IL-10–/– CD4+CD25+ Treg cells, in contrast to WT cells, fail to attenuate effector T cell responses to Leishmania major in resistant C57BL/6 mice 10.

TGF-β1 is a potently suppressive cytokine that plays a critical role in the regulation of immune function, as illustrated by the development of a severe autoimmune-like syndrome in TGF-β1–/– mice 11, 12. Genetic disruption of TGF-β signaling in T cells by overexpression of a dominant-negative TGF-β type II receptor (DNRIITg), conditional deletion of the TGF-β type II receptor, or inactivation of the gene encoding Smad3, alters the sensitivity of T cells to the inhibitory effects of TGF-β and leads to aberrant T cell responses 1315.

The potential role of TGF-β in CD4+CD25+ Treg cell-mediated suppression remains of great interest. Most murine and human in vitro studies conclude that neither secreted nor membrane-bound forms of active or latent TGF-β are responsible for contact-dependent suppression mediated by CD4+CD25+ T cells 1621. DNRIITg and Smad3–/– T cells, which are defective in their responsiveness to TGF-β, remain susceptible to suppression by CD4+CD25+ T cells in vitro16. More importantly, CD4+CD25+ Treg cells isolated from neonatal TGF-β1–/– mice express CTLA-4, GITR, CD103, and Foxp3, are anergic to TCR signals, and display comparable suppressive activity to WT CD4+CD25+ Treg cells in vitro16, 22. Nonetheless, one can infer from some studies that production of TGF-β by CD4+CD25+ Treg cells is required to protect SCID mice from IBD induced by CD4+CD45RBhigh effector T cells, as treatment of recipients of CD4+CD45RBhigh and CD4+CD25+ Treg cells with neutralizing anti-TGF-β antibody reversed suppression 23, 24. Similarly, one recent study reported a requirement for TGF-β in CD4+CD25+ Treg cell-mediated control of CD8+ T cell anti-tumor activity 25. However, the cellular source of the bioactive TGF-β was not determined in these experiments and remains largely unknown 26.

Here, we critically evaluate the requirement for CD4+CD25+ Treg cell-derived TGF-β1 or TGF-β responsiveness in the suppressive function of these cells in protection from IBD induced by transfer of CD4+CD25 T cells into immunodeficient RAG2–/– mice. We demonstrate that CD4+CD25+ Treg cells from neonatal TGF-β1–/– mice are largely non-pathogenic and are nearly equivalent in their capacity to suppress T cell-mediated inflammation in the colon as TGF-β1+/+ CD4+CD25+ Treg cells. Furthermore, CD4+CD25 T cells from Smad3–/– mice display a similar colitogenic potential as WT cells, but are inhibited by either WT or Smad3–/– CD4+CD25+ Treg cells in vivo. Lastly, anti-TGF-β treatment enhances the colitogenic potential of CD4+CD25 T cells in the absence of CD4+CD25+ Treg cells, suggesting a role for TGF-β from non-Treg sources in controlling inflammation. Taken together, we conclude that while TGF-β clearly plays a role in protection against intestinal inflammation, its production by CD4+CD25+ Treg cells is not essential for their suppressive function in at least one form of colitis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Protection from wasting disease and colitis by CD4+CD25+ Treg cells

We used an adoptive T cell transfer model of IBD in immunodeficient C57BL/10 RAG2–/– mice to directly examine the functional contribution of TGF-β in CD4+CD25+ T cell-mediated disease regulation in vivo. Transfer of WT CD4+CD25 effector T cells induced clinical signs of colitis including hunching, diarrhea, and weight loss in RAG2–/– recipients within 4–8 wk post-T cell transfer (Fig. 1a). These clinical symptoms were associated with prominent T cell infiltration of the intestine, and most mice developed moderate to severe colitis as determined by examination of the lamina propria (LP) score (Fig. 1b–c) or the total histology score (Fig. 2a), which accounts for the infiltration of cells into the LP, submucosa, and serosa. Histologic evaluation of the colon in the mice receiving CD4+CD25 effector T cells alone revealed a prominent leukocytic infiltrate and a moderate epithelial cell hyperplasia with occasional ulceration (Fig. 3b; compare to Fig. 3a showing mice that received no cells). However, when syngeneic CD4+CD25+ Treg cells were co-transferred at a 2:1 effector/Treg cell ratio, recipient mice typically preserved their body weight (Fig. 1a) and displayed no clinical symptoms. In addition, the frequency and severity of disease was reduced as indicated by the lower incidence and inflammatory scores (Fig. 1b–c and Fig. 2a) with reduced epithelial cell hyperplasia and rare LP infiltrates (Fig. 3c). Collectively, our results confirm that CD4+CD25+ Treg cells inhibit the colitogenic potential of CD4+CD25 effector T cells.

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Figure 1. CD4+CD25+ Treg cells from TGF-β1–/– mice protect from wasting disease and suppress colonic inflammation in vivo. (A) Body weight measurements: C57BL/10 RAG2–/– mice (n=3–5 per group) received either no cells (-) or were injected with WT CD4+CD25 T cells (3 × 105/mouse) either alone (closed circle, •) or together with WT (closed square, ▪) or TGF-β1–/– (closed triangle, ▴) CD4+CD25+ T cells (1.5 × 105/mouse). Some mice received TGF-β1–/– CD4+CD25+ T cells alone (open triangle, Δ). Body weights, as an indicator of disease development, were monitored weekly. Results shown are from one representative experiment out of three performed. (B) Colonic LP scores: Experimental groups were as described in (A), and the incidence of colitis was analyzed 12–14 wk post-cell transfer. The following grading system was used focusing on cell infiltration into the LP: no colitis (open column, 0–0.5); mild colitis (lightly hatched column, 1.0–1.5); moderate colitis (gray column, 2.0–3.0); and severe colitis (black column, 3.5–4.0). Results shown are pooled from two separate experiments (n=7–9 per group). (C) Cecal LP scores: Experimental groups were as described in (A), and the incidence of typhlitis was analyzed 12–14 wk post-cell transfer. Grading of histology was done as in (B) (n=7–9 per group).

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Figure 2. TGF-β1–/– CD4+CD25+ Treg cells suppress inflammation and IFN-γ mRNA levels in the colon. (A) Total colonic histology scores. Experimental groups were as in Fig. 1, and colonic tissues were harvested 12 wk post-cell transfer and analyzed for total grade of inflammation as described in the Materials and methods. (B) RT-PCR analysis of IFN-γ relative to G3PDH gene expression was performed on total RNA from colonic tissues from the experiment shown in (A). Similar results were obtained in two additional experiments performed.

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Figure 3. Representative photomicrographs of the colons of RAG2–/– mice after transfer of CD4+ effector and Treg cells. RAG2–/– mice received either no cells or were injected with WT CD4+CD25 T cells (3 × 105/mouse) either alone (B) or together with WT (C) or TGF-β1–/– (D) CD4+CD25+ T cells (1.5 × 105/mouse). Panel (E) shows the colon of a mouse receiving TGF-β1–/– CD4+CD25+ T cells alone. Colonic tissues from each experimental group were analyzed 12–14 wk post-cell transfer. H&E-stained sections of ascending colon, approximately 1 cm from the cecal-colonic junction, are shown.

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CD4+CD25+ Treg cells from TGF-β1–/– mice suppress colitis induction

To directly examine whether production of TGF-β by Treg cells is necessary for suppressor function in vivo, we evaluated the disease-protective effect of CD4+CD25+ T cells isolated from TGF-β1–/– mice. As these mice rapidly develop an autoimmune phenotype by 3–5 wk of age, we isolated CD4+CD25+ Treg cells from 3- to 10-day-old mice prior to the onset of visible signs of disease. Thymus and peripheral lymphoid organs of such neonatal mice have a relatively normal frequency of CD4+CD25+ cells with comparable activation profiles to WT animals, suggesting that the CD4+CD25+ Treg cells present in neonatal TGF-β1–/– mice are not derived from a pre-activated effector T cell pool emerging from the autoimmune syndrome (data not shown).

In contrast to mice given colitogenic CD4+CD25 T cells alone, which developed weight loss and colitis (Fig. 1), mice co-injected with TGF-β1–/– CD4+CD25+ Treg cells preserved their body weight and revealed no clinical symptoms of disease (Fig. 1a). When tissue histology from the latter recipients was analyzed, a significant reduction in disease incidence and severity was observed in the colon. Thus, while >85% of the recipients of colitogenic T cells alone developed moderate to severe colitis, >60% of the mice co-injected with TGF-β1–/– Treg cells showed no or mild colitis, clearly demonstrating the potential of these cells to mediate disease protection (Fig. 1b). The same pattern was observed when analyzing the total histology score (Fig. 2a, Fig. 3d; compare to Fig. 3b). Despite the preserved body weight, the absence of clinical signs of disease, and the suppression of inflammation in colonic tissues mediated by TGF-β1–/– Treg cells, no beneficial effect was observed in the cecum of the same animals (Fig. 1c), possibly indicating the importance of other immunoregulatory mechanisms at this site. In our subsequent studies, we therefore focused on the colon to further examine the disease-protective effect of TGF-β1–/– Treg cells in our model. TGF-β1–/– CD4+CD25+ Treg cells were largely non-pathogenic in nature, as the majority of mice receiving these cells alone did not lose weight or develop colitis (Fig. 1a–b, Fig. 2a, and Fig. 3e). However, in some mice (<10% of mice) given TGF-β1–/– CD4+CD25+ T cells alone, a mild or moderate form of colitis was apparent. This mild pathogenicity is likely due to contamination of the CD4+CD25+ Treg cell population from TGF-β1–/– mice with a small number of CD4+CD25+ cells with an activated phenotype.

To further evaluate the disease-protective potential of TGF-β1–/– CD4+CD25+ T cells in vivo, we examined the level of IFN-γ gene expression in colonic tissues (Fig. 2b). All recipients of CD4+CD25 effector T cells alone expressed significant levels of colonic IFN-γ mRNA relative to animals receiving no cells. TGF-β1–/– CD4+CD25+ Treg cells were able to suppress the gene expression of colonic IFN-γ to a degree similar to that of WT Treg cells, again confirming the capacity of TGF-β1–/– CD4+CD25+ Treg cells to control effector T cell functions in vivo (Fig. 2b). Consistent with the mild to moderate colitis observed in a small fraction of mice receiving TGF-β1–/– CD4+CD25+ Treg cells alone, a low level of IFN-γ transcription was present in these mice, further confirming the existence of a possible contamination of activated effector cells amongst TGF-β1–/– CD4+CD25+ T cells. Together, our results demonstrate that TGF-β1–/– CD4+CD25+ Treg cells can inhibit the disease potential of CD4+CD25effector T cells, improve clinical status, and ameliorate intestinal pathology if co-transferred with pathogenic effector cells. These studies further support the conclusion that production of TGF-β1 by CD4+CD25+ Treg cells is not absolutely required for the suppression of inflammation in vivo.

Smad3–/– CD4+ CD25- effector T cells are highly susceptible to suppression mediated by WT or Smad3–/– CD4+ CD25+ Treg cells in vivo

To determine whether TGF-β signaling in the effector T cell population is required for CD4+CD25+ Treg cell-mediated suppression in vivo, we utilized mice in which the gene encoding Smad3 is deleted. We first confirmed that while Smad3–/– CD4+ T cells are as susceptible to suppression by CD4+CD25+ T cells as WT CD4+ T cells in vitro, they are resistant to the suppressive effects of TGF-β1, as they actively transcribe the IL-2 gene and proliferate following TCR stimulation in the presence of exogenous TGF-β1 (Fig. 4a) 27. We next analyzed the role for Smad3 in our colitis model. RAG2–/– recipients of either WT or Smad3–/– CD4+CD25 T cells all developed colitis with a similar kinetics (Fig. 4b–c). Co-transfer of WT CD4+CD25+ T cells resulted in marked suppression of disease in both groups, as demonstrated by the lack of body weight loss and the high degree of protection as seen on histology (Fig. 4b–c). These results suggest that Smad3-dependent TGF-β signaling in effector CD4+ T cells is not required for CD4+CD25+ Treg cell-mediated control of bacterial-driven mucosal inflammation.

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Figure 4. Smad3–/– effector CD4+ T cells are susceptible to suppression mediated by either WT or Smad3–/– CD4+CD25+ Treg cells in vivo. (A) WT or Smad3–/– CD4+ T cells were stimulated with anti-CD3 and irradiated, T-depleted spleen cells either alone or in the presence of exogenous rhTGF-β1 or WT CD4+CD25+ Treg cells. On day 3 of cultures, proliferation was measured by thymidine incorporation and analysis of IL-2 relative to G3PDH gene expression was performed by RT-PCR (right panel). (B) RAG2–/– mice (n=4–5 per group) received either no cells (-) or were injected with WT (▴, ▪, •) or Smad3–/– (▵, □, ○) CD4+CD25 T cells (3 × 105/mouse) to induce disease. In some instances, recipient mice were also co-injected with WT (▪, □) or Smad3–/– (▴,▵) CD4+CD25+ T cells (1.5 × 105/mouse). Body weights were monitored weekly. (C) Colonic tissues from the experiment shown in (A) were harvested 9 wk post-cell transfer and total grade of inflammation determined.

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Some studies have suggested a correlation between Treg function and the expression of a latent, membrane-bound form of TGF-β1 on CD4+CD25+ Treg cells 2830. While these studies promote the view that CD4+CD25+ Treg cells act as carriers of TGF-β1 to activated effector T cells, the alternate view is that surface TGF-β1 may be bound to TGF-β-signaling receptors and initiate a TGF-β signal in Treg cells themselves. However, CD4+CD25+ cells from Smad3–/– mice suppressed colitis to a similar degree as WT CD4+CD25+ Treg cells (Fig. 4b–c). Together, these studies demonstrate that TGF-β signaling via Smad3 is not required in either the effector or Treg cell pool for protection against colitis in this model. Our results, however, do not rule out the possibility of a TGF-β-dependent process that is occurring via a Smad3-independent signaling pathway in effector and/or Treg cells.

In the absence of CD4+CD25+ Treg cells, anti-TGF-β treatment enhances the pathogenicity of CD4+CD25 effector T cells in vivo

Previous studies have shown that CD4+CD45RBlow or CD4+CD25+ Treg cell-mediated suppression of CD4+CD45RBhigh T cell-induced IBD in SCID recipients can be reversed by in vivo treatment with anti-TGF-β mAb 23, 24. The TGF-β mediating inhibition of disease in this model may originate from multiple cellular sources in vivo, including CD4+CD25+ Treg cells, effector T cells, and host lymphoid or non-lymphoid cells 2, 3.

To determine whether non-CD4+CD25+ Treg cell-derived TGF-β plays any role in suppressing the pathogenic potential of effector T cells, 1 × 106 CD4+CD25 T cells were transferred alone into RAG2–/– mice with or without the administration of a neutralizing anti-TGF-β antibody or an isotype control, and the recipients were monitored for the incidence and severity of IBD. While mice receiving control antibody displayed a disease course similar to animals receiving effector T cells alone, mice co-injected with anti-TGF-β demonstrated a modest increase in disease severity (Fig. 5a–b). Thus, in the absence of CD4+CD25+ Treg cells, TGF-β produced by either activated effector T cells or non-T cell sources in the host may modulate the pathogenicity of effector T cells.

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Figure 5. In the absence of CD4+CD25+ Treg cells, anti-TGF-β treatment enhances the pathogenicity of CD4+CD25 effector T cells in vivo. (A) RAG2–/– mice (n=5 per group) received either no cells (□, ▪) or were injected with 1 × 106 CD4+CD25 T cells (○, •, and ). Some RAG2–/– recipient mice were co-injected with either anti-TGF-β (▪,•) or appropriate isotype control mAb () on day –1 and day 1 of cell transfer and weekly thereafter for the duration of the experiment. Body weights were monitored weekly. (B) Colonic tissues were harvested 9 wk post-cell transfer (mice described in A) and the grade of inflammation in the LP determined.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

At present, most studies indicate that CD4+CD25+ Treg cells suppress T cell activation in vitro by inhibiting the production of IL-2 and other cytokines at the mRNA level by an as-yet-unknown cell contact-dependent pathway that does not require the production of the suppressive cytokines IL-4, IL-10, or TGF-β 5, 16, 1821. The mechanism of CD25-mediated suppression in vivo is considerably more complex and may involve cell contact- and/or cytokine-dependent pathways. The most conclusive data on the role of Treg cell-derived suppressor cytokines has been obtained using IL-10–/– CD4+CD25+ T cells that fail both to suppress IBD induced by antigen-experienced effector T cells and to promote the chronic persistence of L. major in resistant C57BL/6 mice 9, 10. TGF-β has also been implicated in IBD, as the disease-protective effect of CD4+CD45RBlow or CD4+CD25+ cells on CD4+CD45RBhigh-induced colitis was abrogated by anti-TGF-β treatment 28, 29. The cellular source and targets of TGF-β in the effector phase of CD4+CD25+ Treg activity has, however, remained elusive.

The goal of the present experiments was to apply the genetic approach we previously utilized to evaluate the role of TGF-β in suppression mediated by CD4+CD25+ T cells in vitro to the in vivo IBD model. CD4+CD25+ Treg cells purified from neonatal TGF-β1–/– mice were potently suppressive and nearly as efficient as WT CD4+CD25+ Treg cells in their capacity to suppress colonic inflammation induced by CD4+CD25 effector T cells, as measured by clinical disease, histology score, and mRNA levels of colonic IFN-γ. Lastly, anti-TGF-β treatment enhanced the colitogenic potential of CD4+CD25 T cells in the absence of CD4+CD25+ Treg cells, suggesting a role for non-Treg cell-derived TGF-β in the control of IBD.

A second aspect of our studies was to determine the target cell for the immunosuppressive effects of TGF-β in our model of IBD. We first explored the possibility that TGF-β acts on the CD4+CD25+ Treg cells themselves, as it has been shown that the production of this cytokine by islet cells during the priming phase of diabetes protects mice from disease and increases the number of intra-islet CD4+CD25+ T cells 27. Furthermore, CD4+CD25+ T cells from mice expressing a dominant negative form of TGF-βRII failed to expand in vivo and to suppress dextran sulfate sodium-induced colitis 31. However, in our colitis model, CD4+CD25+ Treg cells from Smad3–/– mice were as suppressive as WT CD4+CD25+ Treg cells. In addition, TGF-β did not appear to act selectively on the CD4+CD25 effector cells, as CD4+CD25 T cells from Smad3–/– and Smad3+/+ animals were equally colitogenic and susceptible to inhibition by either WT or Smad3–/– CD4+CD25+ Treg cells, consistent with our previously described observation in vitro16. One caveat in the interpretation of these results is that not all the effects of TGF-β in effector T cells are Smad3-dependent 32. Collectively, our study demonstrates that CD4+CD25+ Treg cells can mediate disease inhibition in the complete absence of suppressor-derived TGF-β1 production and functional Smad3-dependent TGF-β responsiveness in both Treg and effector T cells.

Conflicting results in the literature make it difficult to reach a consensus view on the role of TGF-β in CD4+CD25+ Treg cell-mediated immunosuppression. One study reported that activated CD4+CD25+ T cells express an inactive form of TGF-β1 complexed to its latency associated peptide (LAP), a complex hypothesized to account for the enhanced suppressive capacity of activated CD4+CD25+ T cells in vitro and in vivo28, 29. On the other hand, Oida et al. reported that TGF-β-dependent suppression was observed in the LAP+CD4+CD25 T cell subset 33. In addition, some studies have suggested a correlation between Treg effector function and the expression of a latent, membrane-bound form of TGF-β1 on CD4+CD25+ Treg cells isolated from inflamed lymphoid tissues draining target organs undergoing autoimmune attack 30. However, these studies did not conclusively show functional evidence for a direct effect of Treg cell-derived TGF-β1 on responder T cells. Nonetheless, we have been unable to detect significant expression of secreted or cell surface TGF-β1 or LAP on resting or activated CD4+CD25+ T cells isolated from normal or inflamed lymphoid tissues (data not shown). Furthermore, the above studies did not examine the possibility that cell surface TGF-β1, from autocrine or paracrine sources, mediates its effects by actively signaling in CD4+CD25+ Treg cells themselves, possibly maintaining their survival, differentiation, expansion, or suppressive effector mechanism, as suggested by recent reports 22, 31. In this regard, a recent study by Marie et al. has shown that TGF-β1 signaling in Treg cells may promote Foxp3 expression and subsequent Treg function 34. Thus, it is possible that the very slight increase in intestinal inflammation observed in groups receiving TGF-β1–/– compared to WT Treg cells is not exclusively due to contaminating effector T cells, but instead result at least in part from a lack of TGF-β1 signaling in Treg cells and consequential fading of CD4+CD25+ Treg cell function over time. Although CD4+CD25+ T cells from Smad3–/– mice appeared to function as efficiently as WT CD4+CD25+ T cells in our colitis model, it remains possible that TGF-β could play a stimulatory role in the induction of CD4+CD25+ suppressor activity, perhaps in a Smad3-independent fashion 16.

To assess CD4+CD25+ Treg cell function in TGF-β1–/– mice, it is essential to isolate these cells from neonatal asymptomatic mice, as TGF-β1–/– animals develop a multi-organ autoimmune phenotype by 3–5 wk of age 11, 12. In this study, TGF-β1–/– CD4+CD25+ T cells from 3- to 10-day-old neonatal mice were functionally comparable to WT mice, and the expression of activation markers on total CD4+CD25+ T cells from these two strains was comparable (data not shown), confirming our previous observation 16. Furthermore, the TGF-β1–/– CD4+CD25+ T cells were rarely pathogenic in RAG2–/– recipients (Fig. 1, 2). Thus, these findings strongly suggest that the CD4+CD25+ T cells obtained from neonatal TGF-β1–/– mice were not derived from a pre-activated effector T cell pool emerging from the autoimmune phenotype of TGF-β1–/– mice. Our results differ from those of Nakamura et al. 29 who reported that CD4+CD25+ T cells from TGF-β1–/– mice are not effective suppressors of IBD. However, in that study the colitogenic potential of the TGF-β1–/– CD4+CD25+ T cells alone was not examined. Another difference between the two studies is the type of cells used to induce colitis. While we employed CD4+CD25 T cells, which contain some antigen-experienced cells, Nakamura et al. used naive CD4+CD45RBhigh lymphocytes to induce disease. We do not, however, believe this difference to be significant, as in a preliminary experiment using CD4+CD45RBhigh cells to induce colitis, we observed a disease-protective effect by co-transfer of TGF-β1–/– CD4+CD25+ Treg cells (data not shown). Thus, at this point, we do not understand the discrepancies between the two studies, but from our own results it is clear that under certain circumstances TGF-β secretion from CD4+CD25+ cells is not required for control of intestinal inflammation.

One might conclude on the basis of the above evidence that TGF-β does not play any role in disease protection in this model of IBD. We do not believe that this is the case, as we demonstrate that anti-TGF-β treatment of recipients of CD4+CD25 T cells alone led to an enhanced colitogenic potential of these effector T cells in the absence of CD4+CD25+ Treg cells. This result strongly suggests that TGF-β plays a critical role in the suppression of colitis but that the TGF-β is derived from host non-T cells or non-lymphoid cells or even from the effector T cells themselves. Furthermore, these results support the view that tissue/context-dependent factors may influence the mechanism of immune suppression by CD4+CD25+ Treg cells in vivo. In AIG, CD4+CD25+ Treg cells do not need suppressor cytokines and possibly suppress disease by resorting to a contact-dependent mechanism, a finding similar to what is observed in vitro. However, in the milieu of a bacteria-driven inflammation in the intestine, the contact-dependent pathway may not be sufficient to mediate disease protection and must be supplemented by suppressor cytokine production by the Treg cells (e.g. IL-10) and by production of TGF-β by non-Treg cells. It is also possible that the CD4+CD25+ Treg cells may facilitate the induction of TGF-β production by host cells, which subsequently promotes the induction and differentiation of Treg cells from CD4+CD25 T cell precursors 3538.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Mice

C57BL/6 WT mice were obtained from the National Cancer Institute (NCI) (Frederick, MD), and C57BL/10 RAG2–/– were obtained from either The Jackson Laboratory (Bar Harbor, ME) or Taconic Farms (Germantown, NY). TGF-β1–/– and Smad3–/– mice (both on the C57BL/6×Sv129 genetic background) were generated by targeted gene disruption in murine embryonic stem cells by homologous recombination as previously described 39, 40. All mice were bred and maintained in a specific pathogen-free animal facility. Unless specified, donor mice used in these experiments were generally 3–10 days old, while recipient mice were generally 6–8 wk of age.

Cell purification and flow cytometry

CD4+CD25+ T cells were isolated by cell sorting as previously described 7. Briefly, pooled lymph nodes (axillary, inguinal, brachial, and mesenteric) and thymocytes were collected from appropriate neonatal (3- to 10-day-old) or adult mice. Single-cell suspensions were prepared and gently layered on a Lympholyte-M gradient (Cederlane) to remove dead cells. The resulting cell preparation was stained with tricolor-anti-CD4 (Caltag laboratories, Burlingame, CA) and PE-anti-CD25 (PC61 clone, BD-Pharmingen, San Diego, CA) in PBS/2% FCS for 20 min at 4°C, washed, and then resuspended in RPMI 1640 (without phenol red)/10% FCS for FACS sorting. The purity of sorted populations was typically >98%.

Proliferation assays

Proliferation assays were performed by culturing WT or Smad3–/– CD4+ T cells (5 × 104) in 96-well flat-bottom microtiter plates (0.2 mL) in RPMI 1640 (Invitrogen, Burlington, Canada) supplemented with 10% heat-inactivated FCS, penicillin (100 U/ml), streptomycin (100 μg/ml), 2 mM L-glutamine, 10 mM Hepes, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate (all obtained from Biofluids, Rockville, MD), and 50 μM 2-ME (Sigma, St. Louis, MO) with anti-CD3 (0.5 μg/ml) and irradiated, T-depleted spleen cells (2 × 105) either alone or in the presence of exogenous rhTGF-β (5 ng/ml) or WT CD4+ CD25+ Treg cells (5 × 104 for 72 h at 37°C in 7% CO2. Cell cultures were pulsed with 1 μCi [3H]-thymidine for the last 6–12 h. All the data represent the average counts per minute of triplicate determinations.

Adoptive T cell transfer experiments

To induce IBD, C57BL/10 RAG2–/– mice were injected i.v. with 3 × 105 to 4 × 105 WT or Smad3–/– CD4+CD25 T cells either alone or with 1.5 × 105 to 2 × 105 CD4+CD25+ T cells from either WT or TGF-β1–/– mice at a 2:1 ratio. Mice were weighed weekly and typically developed clinical signs of colitis 4–8 wk post-transfer. In some experiments, recipient mice were treated with affinity-purified anti-TGF-β1 (clone 1D11) or appropriate mouse IgG1 isotype control (1.25 mg i.p. weekly) for the entire duration of the experiment.

Pathology and immunohistochemistry

Colons were removed from mice between 9 and 14 wk after T cell reconstitution and fixed in Bouin's fixative, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E). Sections were evaluated in a blinded fashion, and an average score for the whole section was assigned based on a scoring system described previously [41]. Briefly, inflammation was graded from 0 to 4+ for infiltration of the LP, for submucosal inflammation, and for serosal inflammation. Crypt abscesses and ulceration were also graded on a 0–4+ scale. The total score is the sum of scores for these five variables.

Reverse-transcription polymerase chain reaction (RT-PCR)

Evaluation of cytokine gene expression in inflamed colonic tissue or in CD4+ T cell cultures performed as previously described 42. In brief, colonic tissues were harvested and snap-frozen in liquid N2, and total RNA was prepared with the Trizol RNA extraction reagent (Invitrogen). cDNA was subsequently synthesized using Superscript II RT and oligo-dT primers (Invitrogen). All PCR reactions were done using Platinum Supermix cocktail (Invitrogen) under the following cycling conditions: 4 min at 95°C, 30 cycles of 1 min at 95°C, 55°C, and 72°C, and to finish an extension period of 15 min at 72°C. RT-PCR primers for IFN-γ (5′tgc atc ttg gct ttg cag ctc ttc ctc atg gc3′and 5′tgg acc tgt ggg ttg ttg acc tca aac ttg gc3′; 365 bp) and IL-2 (5′ttc aag ctc cac ttc aag ctc tac agc gga ag3′and 5′gac aga agg cta tcc atc tcc tca gaa agt cc3′; 413 bp) were purchased from BD-Clontech. As an internal control, G3PDH RT-PCR primers (5′tga agg tcg gtg tga acg gat ttg gc3′ and 5′cat gta ggc cat gag gtc cac cac3′; 938 bp) were used (BD-Clontech). All PCR reactions were analyzed in the linear phase of amplification, and products were electrophoresed and quantified using Quantity One 4,4,1 software (Biorad). Semi-quantitative analysis of gene expression was achieved by normalizing the IFN-γ densitometric value with the intensity of the G3PDH amplicon for each sample, and results are reported as arbitrary IFN-γ/G3PDH ratios.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

We would like to point out that while the present manuscript was under review, a similar study by Fahlén et al. was published demonstrating that TGF-β1–/– CD4+CD25+ Treg cells retain the ability to suppress colitis (J. Exp. Med. 2005, 201:737–46).We are grateful to the NIAID and McGill Flow Cytometry Facilities for their effort in cell sorting. We are grateful for the expertise of the Department of Pathology, McGill University, for blinded histological analysis of some experiments. We would also like to thank the animal care staff at the NIAID and the McGill Animal Center for their excellent technical assistance. We acknowledge the financial support of the Canadian Institutes for Health Research (CIHR MOP 67211) and the Canadian Foundation for Innovation (CFI). C. A. P. is a recipient of a Canada Research Chair.

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