Ex vivo generation of regulatory T cells: characterization and therapeutic evaluation in a model of chronic colitis.

Naturally occurring regulatory T cells (nTregs; CD4(+)CD25(+)Foxp3(+)) are capable of suppressing the chronic inflammation observed in a variety of different animal models of autoimmune and chronic inflammatory diseases such as inflammatory bowel diseases, diabetes, and arthritis. A major limitation in exploring how and where nTregs exert their suppression in vivo is the relative paucity of these regulatory cells. Although several laboratories have described different methods to expand flow-purified nTregs or convert conventional/naïve T cells (CD4(+)Foxp3(-)) to Foxp3-expressing "induced" Tregs (iTregs; CD4(+)Foxp3(+)) ex vivo, we have found that many of these approaches are encumbered with their own limitations. Therefore, we sought to develop a relatively simple ex vivo method to generate large numbers of Foxp3-expressing iTregs that can be used to evaluate their trafficking properties, suppressive activity, and therapeutic efficacy in a mouse model of chronic gut inflammation in vivo. We present a detailed protocol demonstrating that polyclonal activation of conventional CD4(+) T cells in the presence of IL-2, TGFß, and all trans retinoic acid induces >90% conversion of these T cells to Foxp3-expressing iTregs as well as promotes a three- to fourfold increase in proliferation following a 4-day incubation period in vitro. This protocol enhances modestly the surface expression of the gut-homing adhesion molecule CCR9 but not α(4)ß(7). Furthermore, we provide preliminary data demonstrating that these iTregs are significantly more potent at suppressing T-cell activation in vitro and are equally effective as freshly isolated nTregs at attenuating chronic colitis in vivo. Finally, we report that this protocol has the potential to generate 30-40 million iTregs from one healthy mouse spleen.

Role of the gut-associated and secondary lymphoid tissue in the induction of chronic colitis.

BACKGROUND: It is well known that enteric bacterial antigens drive the development of chronic colitis in a variety of different mouse models of the inflammatory bowel diseases (IBD). The objective of this study was to evaluate the role of gut-associated lymphoid tissue (GALT; Peyer's patches, isolated lymphoid follicles), mesenteric lymph nodes (MLNs) and spleen in the pathogenesis of chronic colitis in mice. METHODS: Surgical as well as genetic approaches were used to generate lymphopenic mice devoid of one or more of these lymphoid tissues. For the first series of studies, we subjected recombinase activating gene-1-deficient mice (RAG(-/-) ) to sham surgery (Sham), mesenteric lymphadenectomy (MLNx), splenectomy (Splx) or both (MLNx/Splx). In a second series of studies we intercrossed lymphotoxinß-deficient (LTß(-/-) ) mice with RAG(-/-) animals to generate LTß(-/-) x RAG(-/-) offspring that were anticipated to contain functional MLNs but be devoid of GALT and most peripheral lymph nodes. Flow purified naïve (CD4(+) CD45RB(high) ) T-cells were adoptively transferred into the different groups of RAG(-/-) recipients to induce chronic colitis. RESULTS: We found that at 3-5 wks following T-cell transfer, all four of the surgically-manipulated RAG(-/-) groups (Sham, MLNx, Splx and MLNx/Splx) developed chronic colitis that was similar in onset and severity. Flow cytometric analysis revealed no differences among the different groups with respect to surface expression of different gut-homing markers nor were there any differences noted in IFN-γ and IL-17 generation by mononuclear cells isolated among these surgically-manipulated mice. Although we anticipated that LTß(-/-) x RAG(-/-) mice would contain functional MLNs but be devoid of GALT and peripheral lymph nodes (PLNs), we found that LTß(-/-) x RAG(-/-) mice were in fact devoid of MLNs as well as GALT and PLNs. Adoptive transfer of CD45RB(high) T-cells into LTß(-/-) x RAG(-/-) mice or their littermate controls (LTß(+/+) x RAG(-/-) ) induced rapid and severe colitis in both groups. CONCLUSIONS: Taken together, our data demonstrate that: a) neither the GALT, MLNs nor PLNs are required for induction of chronic gut inflammation in this model of IBD and b) T-and/or B-cells may be required for the development of MLNs in LTß(-/-) mice.

T cell transfer model of chronic colitis: concepts, considerations, and tricks of the trade.

The inflammatory bowel diseases (Crohn's disease; ulcerative colitis) are idiopathic chronic inflammatory disorders of the intestine and/or colon. A major advancement in our understanding of the pathogenesis of these diseases has been the development of mouse models of chronic gut inflammation. One model that has been instrumental in delineating the immunological mechanisms responsible for the induction as well as regulation of intestinal inflammation is the T cell transfer model of chronic colitis. This paper presents a detailed protocol describing the methods used to induce chronic colitis in mice. Special attention is given to the immunological concepts that explain disease pathogenesis in this model, considerations and potential pitfalls in using this model, and finally different "tricks" that we have learned over the past 12 years that have allowed us to develop a more simplified version of this model of experimental IBD.