Ellen Marks

The Transcription Factor T-bet Regulates Intestinal Inflammation Mediated by Interleukin-7 Receptor(+) Innate Lymphoid Cells

Summary Mice lacking the transcription factor T-bet in the innate immune system develop microbiota-dependent colitis. Here, we show that interleukin-17A (IL-17A)-producing IL-7Rα+ innate lymphoid cells (ILCs) were potent promoters of disease in Tbx21−/−Rag2−/− ulcerative colitis (TRUC) mice. TNF-α produced by CD103−CD11b+ dendritic cells synergized with IL-23 to drive IL-17A production by ILCs, demonstrating a previously unrecognized layer of cellular crosstalk between dendritic cells and ILCs. We have identified Helicobacter typhlonius as a key disease trigger driving excess TNF-α production and promoting colitis in TRUC mice. Crucially, T-bet also suppressed the expression of IL-7R, a key molecule involved in controlling intestinal ILC homeostasis. The importance of IL-7R signaling in TRUC disease was highlighted by the dramatic reduction in intestinal ILCs and attenuated colitis following IL-7R blockade. Taken together, these data demonstrate the mechanism by which T-bet regulates the complex interplay between mucosal dendritic cells, ILCs, and the intestinal microbiota.

Developing in vitro expanded CD45RA+ regulatory T cells as an adoptive cell therapy for Crohn's disease

Background and aim Thymus-derived regulatory T cells (Tregs) mediate dominant peripheral tolerance and treat experimental colitis. Tregs can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. Treg cell therapy is also conceptually attractive for Crohns disease (CD). However, barriers exist to this approach. The stability of Tregs expanded from Crohns blood is unknown. The potential for adoptively transferred Tregs to express interleukin-17 and exacerbate Crohns lesions is of concern. Mucosal T cells are resistant to Treg-mediated suppression in active CD. The capacity for expanded Tregs to home to gut and lymphoid tissue is unknown. Methods To define the optimum population for Treg cell therapy in CD, CD4+CD25+CD127loCD45RA+ and CD4+CD25+CD127loCD45RA− Treg subsets were isolated from patients’ blood and expanded in vitro using a workflow that can be readily transferred to a good manufacturing practice background. Results Tregs can be expanded from the blood of patients with CD to potential target dose within 22–24 days. Expanded CD45RA+ Tregs have an epigenetically stable FOXP3 locus and do not convert to a Th17 phenotype in vitro, in contrast to CD45RA− Tregs. CD45RA+ Tregs highly express α4β7 integrin, CD62L and CC motif receptor 7 (CCR7). CD45RA+ Tregs also home to human small bowel in a C.B-17 severe combined immune deficiency (SCID) xenotransplant model. Importantly, in vitro expansion enhances the suppressive ability of CD45RA+ Tregs. These cells also suppress activation of lamina propria and mesenteric lymph node lymphocytes isolated from inflamed Crohns mucosa. Conclusions CD4+CD25+CD127loCD45RA+ Tregs may be the most appropriate population from which to expand Tregs for autologous Treg therapy for CD, paving the way for future clinical trials.

CCR6-Dependent Positioning of Memory B Cells Is Essential for Their Ability To Mount a Recall Response to Antigen

Chemokine-dependent localization of specific B cell subsets within the immune microarchitecture is essential to ensure successful cognate interactions. Although cognate interactions between T cells and memory B cells (Bmem) are essential for the secondary humoral immune responses, the chemokine response patterns of Bmem cells are largely unknown. In contrast to naive B cells, this study shows that Ag-specific Bmem cells have heightened expression of CCR6 and a selective chemotactic response to the CCR6 ligand, CCL20. Although CCR6 appears be nonessential for the initial clonal expansion and maintenance of Bmem, CCR6 is essential for the ability of Bmem to respond to a recall response to their cognate Ag. This dependency was deemed intrinsic by studies in CCR6-deficient mice and in bone marrow chimeric mice where CCR6 deficiency was limited to the B cell lineage. Finally, the mis-positioning of CCR6-deficient Bmem was revealed by immunohistological analysis with an altered distribution of CCR6-deficient Bmem from the marginal and perifollicular to the follicular/germinal center area.

Cutting Edge: Retinoic Acid Signaling in B Cells Is Essential for Oral Immunization and Microflora Composition

Retinoic acid (RA) is a critical regulator of the intestinal adaptive immune response. However, the intrinsic impact of RA on B cell differentiation in the regulation of gut humoral immunity in vivo has never been directly shown. To address this issue, we have been able to generate a mouse model where B cells specifically express a dominant-negative receptor α for RA. In this study, we show that the silencing of RA signaling in B cells reduces the numbers of IgA+ Ab-secreting cells both in vitro and in vivo, suggesting that RA has a direct effect on IgA plasma cell differentiation. Moreover, the lack of RA signaling in B cells abrogates Ag-specific IgA responses after oral immunization and affects the microbiota composition. In conclusion, these results suggest that RA signaling in B cells through the RA receptor α is important to generate an effective gut humoral response and to maintain a normal microbiota composition.

Diversity of gut microflora is required for the generation of B cell with regulatory properties in a skin graft model

B cells have been reported to promote graft rejection through alloantibody production. However, there is growing evidence that B cells can contribute to the maintenance of tolerance. Here, we used a mouse model of MHC-class I mismatched skin transplantation to investigate the contribution of B cells to graft survival. We demonstrate that adoptive transfer of B cells prolongs skin graft survival but only when the B cells were isolated from mice housed in low sterility “conventional” (CV) facilities and not from mice housed in pathogen free facilities (SPF). However, prolongation of skin graft survival was lost when B cells were isolated from IL-10 deficient mice housed in CV facilities. The suppressive function of B cells isolated from mice housed in CV facilities correlated with an anti-inflammatory environment and with the presence of a different gut microflora compared to mice maintained in SPF facilities. Treatment of mice in the CV facility with antibiotics abrogated the regulatory capacity of B cells. Finally, we identified transitional B cells isolated from CV facilities as possessing the regulatory function. These findings demonstrate that B cells, and in particular transitional B cells, can promote prolongation of graft survival, a function dependent on licensing by gut microflora.

Retinoic Acid Signaling in B Cells Is Required for the Generation of an Effective T-Independent Immune Response.

Retinoic acid (RA) plays an important role in the balance of inflammation and tolerance in T cells. Furthermore, it has been demonstrated that RA facilitates IgA isotype switching in B cells in vivo. However, it is unclear whether RA has a direct effect on T-independent B cell responses in vivo. To address this question, we generated a mouse model where RA signaling is specifically silenced in the B cell lineage. This was achieved through the overexpression of a dominant negative receptor α for RA (dnRARα) in the B cell lineage. In this model, we found a dramatic reduction in marginal zone (MZ) B cells and accumulation of transitional 2 B cells in the spleen. We also observed a reduction in B1 B cells in the peritoneum with a defect in the T-independent B cell response against 2,4,6-trinitrophenyl. This was not a result of inhibited development of B cells in the bone marrow, but likely the result of both defective expression of S1P1 in MZ B cells and a defect in the development of MZ and B1 B cells. This suggests that RARα expression in B cells is important for B cell frequency in the MZ and peritoneum, which is crucial for the generation of T-independent humoral responses.

Transcription factor T-bet regulates intestinal inflammation mediated by innate lymphoid cells with the interleukin-7 receptor

Abstract Interactions between the innate immune system and the intestinal microbiota have an important role in the maintenance of mucosal homoeostasis. Indeed, impaired regulation of innate immune pathways are now considered to be a key driver of gut inflammation and may lead to the emergence of inflammatory bowel disease, such as ulcerative colitis or Crohns disease. Mice lacking the transcription factor T-bet in the innate immune system develop microbiota-dependent colitis that resembles ulcerative colitis. Here, we show that inflammatory bowel disease in this model is mediated by a newly discovered population of innate immune cells termed innate lymphoid cells (ILCs) that selectively produce the inflammatory cytokine interleukin (IL) 17A. Depletion of intestinal ILCs, or blockade of the IL23/IL17 axis significantly attenuated chronic inflammatory bowel disease in these mice. We also show that elevated colonic production of tumour necrosis factor α (TNFα), a key cytokine involved in the pathogenesis of inflammatory bowel disease, was produced by CD103– CD11b+ intestinal dendritic cells (DCs). TNFα synergised with IL23 to drive IL-17A production by ILCs, demonstrating a previously unrecognised layer of cellular crosstalk between DCs and ILCs. Other cytokines implicated in pathogenesis of inflammatory bowel disease, such as TL1A and IL6, also promoted IL-17 production by ILCs, indicating that this novel intestinal innate immune cell population is responsive to other recognised inflammatory signals to drive gut inflammation. By sequencing bacterial rRNA genes we also identify a particular component of the intestinal microbiota of TRUC mice that drives excess TNF-α production and triggers colitis. Lastly, we show that T-bet is a transcriptional repressor of IL-7R expression, a key molecule involved in controlling intestinal ILC homoeostasis. The importance of IL-7R signalling in TRUC disease was highlighted by the dramatic reduction in intestinal ILCs and attenuated colitis following IL-7R blockade. Taken together, these data demonstrate the mechanism by which T-bet regulates the complex interplay between mucosal DCs, ILCs, and the intestinal microbiota. Funding Wellcome Trust.