Jennifer Ziegelbauer

A Central Role for Induced Regulatory T Cells in Tolerance Induction in Experimental Colitis

In addition to thymus-derived or natural T regulatory (nTreg) cells, a second subset of induced T regulatory (iTreg) cells arises de novo from conventional CD4+ T cells in the periphery. The function of iTreg cells in tolerance was examined in a CD45RBhighCD4+ T cell transfer model of colitis. In situ-generated iTreg cells were similar to nTreg cells in their capacity to suppress T cell proliferation in vitro and their absence in vivo accelerated bowel disease. Treatment with nTreg cells resolved the colitis, but only when iTreg cells were also present. Although iTreg cells required Foxp3 for suppressive activity and phenotypic stability, their gene expression profile was distinct from the established nTreg “genetic signature,” indicative of developmental and possibly mechanistic differences. These results identified a functional role for iTreg cells in vivo and demonstrated that both iTreg and nTreg cells can act in concert to maintain tolerance.

IL-10 Produced by Induced Regulatory T Cells (iTregs) Controls Colitis and Pathogenic Ex-iTregs during Immunotherapy

“Natural” regulatory T cells (nTregs) that express the transcription factor Foxp3 and produce IL-10 are required for systemic immunological tolerance. “Induced” regulatory T cells (iTregs) are nonredundant and essential for tolerance at mucosal surfaces, yet their mechanisms of suppression and stability are unknown. We investigated the role of iTreg-produced IL-10 and iTreg fate in a treatment model of inflammatory bowel disease. Colitis was induced in Rag1−/− mice by the adoptive transfer of naive CD4+ T cells carrying a nonfunctional Foxp3 allele. At the onset of weight loss, mice were treated with both iTregs and nTregs where one marked subset was selectively IL-10 deficient. Body weight assessment, histological scoring, cytokine analysis, and flow cytometry were used to monitor disease activity. Transcriptional profiling and TCR repertoire analysis were used to track cell fate. When nTregs were present but IL-10 deficient, iTreg-produced IL-10 was necessary and sufficient for the treatment of disease, and vice versa. Invariably, ∼85% of the transferred iTregs lost Foxp3 expression (ex-iTregs) but retained a portion of the iTreg transcriptome, which failed to limit their pathogenic potential upon retransfer. TCR repertoire analysis revealed no clonal relationships between iTregs and ex-iTregs, either within mice or between mice treated with the same cells. These data identify a dynamic IL-10–dependent functional reciprocity between regulatory T cell subsets that maintains mucosal tolerance. The niche supporting stable iTregs is limited and readily saturated, which promotes a large population of ex-iTregs with pathogenic potential during immunotherapy.

The TCR Repertoires of Regulatory and Conventional T Cells Specific for the Same Foreign Antigen Are Distinct

The relationship between the TCR repertoires of natural regulatory T cells (nTregs) and conventional CD4+ T cells (Tconv) capable of responding to the same antigenic epitope is unknown. In this study, we used TCRβ-chain transgenic mice to generate polyclonal nTreg and Tconv populations specific for a foreign Ag. CD4+ T cells from immunized 3.L2β+/− TCRα+/− Foxp3EGFP mice were restimulated in culture to yield nTregs (EGFP+) and Tconv (EGFP−) defined by their antigenic reactivity. Relative to Tconv, nTreg expansion was delayed, although a higher proportion of viable nTregs had divided after 72 h. Spectratype analysis revealed that both the nTreg and Tconv responses were different and characterized by skewed distributions of CDR3 lengths. CDR3 sequences from nTregs displayed a divergent pattern of Jα usage, minimal CDR3 overlap (3.4%), and less diversity than did CDR3 sequences derived from Tconv. These data indicate that foreign Ag-specific nTregs and Tconv are clonally distinct and that foreign Ag-specific nTreg populations are constrained by a limited TCR repertoire.

Affinity-Based Selection of Regulatory T Cells Occurs Independent of Agonist-Mediated Induction of Foxp3 Expression

Natural regulatory T (nTreg) cells recognize self-peptides with high affinity, yet the understanding of how affinity influences their selection in the thymus is incomplete. We use altered peptide ligands in transgenic mice and in organ culture to create thymic environments spanning a broad range of ligand affinity. We demonstrate that the nTreg TCR repertoire is shaped by affinity-based selection, similar to conventional T cells. The effect of each ligand on the two populations is distinct, consistent with early nTreg cell lineage specification. Foxp3 expression is an independent process that does not rely on “high affinity” binding per se, but requires a high-potency agonistic interaction for its induction. The timing of ligand exposure, TGFβ signaling, and the organization of the thymic architecture are also important. The development of nTreg cells is therefore a multistep process in which ligand affinity, potency, and timing of presentation all play a role in determining cell fate.

Alternatively Activated Macrophages Boost Induced Regulatory T and Th17 Cell Responses during Immunotherapy for Colitis

Induced regulatory T (iTreg) and Th17 cells promote mucosal homeostasis. We used a T cell transfer model of colitis to compare the capacity of iTreg and Th17 cells to develop in situ following the transfer of naive CD4+CD45RBhi T cells into Rag1−/− C57BL/6 or BALB/c mice, the prototypical Th1/M1- and Th2/M2-prone strains. We found that the frequency and number of Foxp3+ iTreg cells and Th17 cells were significantly reduced in C57BL/6 mice compared with the BALB/c strain. C57BL/6 mice with colitis were also resistant to natural Treg cell immunotherapy. Pretreatment of C57BL/6 Rag1−/− mice with IL-4 plus IL-13, or with M2a but not M1 macrophages, dramatically increased the generation of iTreg and Th17 cells. Importantly, M2a transfers, either as a pretreatment or in mice with established colitis, allowed successful immunotherapy with natural Treg cells. M2a macrophages also reduced the generation of pathogenic iTreg cells that lost Foxp3 expression, suggesting that they stabilize the expression of Foxp3. Thus, polarized M2a macrophages drive a directionally concordant expansion of the iTreg–Th17 cell axis and can be exploited as a therapeutic adjuvant in cell-transfer immunotherapy to re-establish mucosal tolerance.

Chronic follicular bronchiolitis requires antigen-specific regulatory T cell control to prevent fatal disease progression

To study regulatory T (Treg) cell control of chronic autoimmunity in a lymphoreplete host, we created and characterized a new model of autoimmune lung inflammation that targets the medium and small airways. We generated transgenic mice that express a chimeric membrane protein consisting of hen egg lysozyme and a hemoglobin epitope tag under the control of the Clara cell secretory protein promoter, which largely limited transgene expression to the respiratory bronchioles. When Clara cell secretory protein–membrane hen egg lysozyme/hemoglobin transgenic mice were crossed to N3.L2 TCR transgenic mice that recognize the hemoglobin epitope, the bigenic progeny developed dense, pseudo-follicular lymphocytic peribronchiolar infiltrates that resembled the histological pattern of follicular bronchiolitis. Aggregates of activated IFN-γ– and IL-17A–secreting CD4+ T cells as well as B cells surrounded the airways. Lung pathology was similar in Ifng−/− and Il17a−/− mice, indicating that either cytokine is sufficient to establish chronic disease. A large number of Ag-specific Treg cells accumulated in the lesions, and Treg cell depletion in the affected mice led to an interstitial spread of the disease that ultimately proved fatal. Thus, Treg cells act to restrain autoimmune responses, resulting in an organized and controlled chronic pathological process rather than a progressive disease.

A model of TH17-associated ileal hyperplasia that requires both IL-17A and IFNγ to generate self-tolerance and prevent colitis

Homeostasis in the ileum, which is commonly disrupted in patients with Crohn’s disease, involves ongoing immune responses. To study how homeostatic processes of the ileum impact CD4+T cell responses, we used TCR transgenic tools to breed mice that spontaneously produced CD4+T cells reactive to an antigen expressed in the ileum. At an early age, the ilea of these mice exhibit crypt hyperplasia and accumulate increased numbers of TH17 cells bearing non-transgenic clonotypes. Half of these mice subsequently developed colitis linked to broad mucosal infiltration by TH17 and TH1 cells expressing non-transgenic clonotypes, chronic wasting disease and loss of ileal crypt hyperplasia. By contrast, adult mice with normal growth continued to exhibit TH17-associated ileal crypt hyperplasia and additionally accumulated ileal-reactive Treg cells. Both IL-17A and IFNγ were protective, as their deficiency precluded ileal-reactive Treg accumulation and exacerbated colitic disease. IL-23R blockade prevented progression to colitis, whereas nTreg cell transfers prevented colitic disease, ileal crypt hyperplasia and ileal-reactive Treg accumulation. Thus, our studies identify an IL-17A and IFNγ-dependent homeostatic process that mobilizes ileal-reactive Treg cells and is disrupted by IL-23.

Granzymes are necessary for suppressive function of regulatory T cells

Purpose Regulatory T (Treg) cells are an essential subset of CD4+ T cells that induce and maintain immunological tolerance. Preclinical animal models have demonstrated that adoptive transfer of Treg cells can prevent or cure diabetes, multiple sclerosis (EAE), inflammatory bowel disease, lupus, arthritis, and graft versus host disease. Defects in Treg cell function and number have been described in a number of different human autoimmune diseases including diabetes, multiple sclerosis, rheumatoid arthritis and juvenile idiopathic arthritis. These data suggest that manipulation of Treg cells may be a useful therapeutic intervention. Treg cells are marked by expression of the forkhead/winged helix transcription factor Foxp3, which is essential for their regulatory functions. Treg cells are well known to mediate their immunomodulatory effects through TGF-b, IL-10 and CTLA-4. Recent work has also suggested a role for the granule/exocytosis pathway in Treg cell suppressive function. Granzyme A and granzyme B (gzm A and gzm B, respectively) mRNAs are expressed at high levels in Treg cells, and Treg cells from gzm B–/– mice showed defects in suppression in vitro. To further examine the role of granzymes in the function of Treg cells in vivo, we used a T cell transfer model of colitis. In this model, naive CD45RBhigh CD4+ T cells transferred to RAG–/– mice and undergo homeostatic expansion and activation.This process results in colitis manifested clinically by weight loss and shortened survival. Previous work has shown that a subset of the transferred conventional T cells develop into Foxp3+ iTreg cells and that these iTreg cells are necessary to mitigate colitis when present together with natural Treg cells (nTreg) cells derived in the thymus. Methods CD45RBhigh T cells from mice expressing a Foxp3EGFP fusion protein and additionally deficient in gzm A or gzm B are transferred to RAG-/mice.In these mice, EGFP expression marks iTreg which have developed in situ. Mice were weighed twice weekly and euthanized when moribund or when they have lost more than 1520% of their initial weight. Mesenteric lymph nodes T cells were analyzed for expression of EGFP.

M2 Macrophages Drive the Production of Induced Regulatory T Cells in the Gut: P-180

BACKGROUND: The microbiota is important in shaping the mammalian host’s immune system and is useful in identifying mechanisms of immune maturation. In the limited number of associations between commensal microbes and the immune system that are known to indicate a profound immunomodulatory relationship, the microbes involved have been identified at the genus (or sometimes the species) level; in contrast, little relevant information has been obtained at the microbial molecular level. A notable exception is the relationship between capsular polysaccharide A (PSA) of the gut commensal Bacteroides fragilis and the induction of regulatory T cells (Tregs) that can limit pathologic inflammation both in the gut and in more distant tissues. The ability of PSA to induce secretion of the potent anti-inflammatory cytokine interleukin 10 (IL-10) is vital to the control of inflammation. Restoration of tissue homeostasis by a functional class of dendritic cells (DCs), designated tolerogenic DCs, has been described and is due, at least in part, to generation or enhancement of the function of Tregs. Although PSA affects DCs, it has not been shown whether DCs play a role in the immunoregulatory activities of PSA. METHODS: We treated specific-pathogen-free animals with B fragilis expressing PSA and followed T regs and associated DCs in mesenteric lymph nodes and other tissues in absence of additional inflammatory stimulus. We purified PSA from B fragilis and utilized in vitro in DC-CD4þT cell co-culture assay and in vivo in TNBS-induced colitis and Myelin-PLP induced multiple sclerosis models. Genetically deficient mice, antibody mediated in vivo inhibition and depletion along with adoptive transfer of PSA treated DC subsets were employed. RESULTS: We show that a subset of DCs known as plasmacytoid DCs (PDCs) when exposed to PSA are potent inducers of IL-10 production by CD4þ T cells in vitro. In the murine model of colonic inflammation, PDCs characteristically phenotype PSA mediated protection in a Toll-like receptor 2 (TLR2) dependent way. Interestingly, PDCs are essential for the immunoprotective activities of PSA in this colitis model as observed by antibody mediated PDC depletion and adoptive transfer experiments. TLR2, an immunosensitive receptor of PSA, is induced in PDCs by PSA and, along with ICOSL and CD86, mediates PSA’s immunoregulatory function in vitro and in vivo. Finally, in a murine model of multiple sclerosis wherein PSA was found to be protective, we observed near complete mortality in mice treated with PDC depleting antibody irrespective of PSA treatment, while the isotype control treated mice survived and were protected significantly by PSA treatment in terms of clinical scores. CONCLUSION(S): Our results demonstrate how a prototypical molecule from the commensal microbiota enables a subset of DCs to modify pathologic outcome in the gut and a distant tissue. Understanding functional polarization of PDCs or other DCs by molecules derived from the commensal microbiota and elucidating how these molecules shape immune development may help identify new therapeutic approaches to inflammatory diseases.