IL-23 stabilizes an effector Treg cell program in the tumor microenvironment.

Interleukin-23 (IL-23) is a proinflammatory cytokine mainly produced by myeloid cells that promotes tumor growth in various preclinical cancer models and correlates with adverse outcomes. However, as to how IL-23 fuels tumor growth is unclear. Here, we found tumor-associated macrophages to be the main source of IL-23 in mouse and human tumor microenvironments. Among IL-23-sensing cells, we identified a subset of tumor-infiltrating regulatory T (Treg) cells that display a highly suppressive phenotype across mouse and human tumors. The use of three preclinical models of solid cancer in combination with genetic ablation of Il23r in Treg cells revealed that they are responsible for the tumor-promoting effect of IL-23. Mechanistically, we found that IL-23 sensing represents a crucial signal driving the maintenance and stabilization of effector Treg cells involving the transcription factor Foxp3. Our data support that targeting the IL-23/IL-23R axis in cancer may represent a means of eliciting antitumor immunity.

The A946T variant of the RNA sensor IFIH1 mediates an interferon program that limits viral infection but increases the risk for autoimmunity.

The single-nucleotide polymorphism rs1990760 in the gene encoding the cytosolic viral sensor IFIH1 results in an amino-acid change (A946T; IFIH1T946) that is associated with multiple autoimmune diseases. The effect of this polymorphism on both viral sensing and autoimmune pathogenesis remains poorly understood. Here we found that human peripheral blood mononuclear cells (PBMCs) and cell lines expressing the risk variant IFIH1T946 exhibited heightened basal and ligand-triggered production of type I interferons. Consistent with those findings, mice with a knock-in mutation encoding IFIH1T946 displayed enhanced basal expression of type I interferons, survived a lethal viral challenge and exhibited increased penetrance in autoimmune models, including a combinatorial effect with other risk variants. Furthermore, IFIH1T946 mice manifested an embryonic survival defect consistent with enhanced responsiveness to RNA self ligands. Together our data support a model wherein the production of type I interferons driven by an autoimmune risk variant and triggered by ligand functions to protect against viral challenge, which probably accounts for its selection within human populations but provides this advantage at the cost of modestly promoting the risk of autoimmunity.

Trans-presentation of IL-6 by dendritic cells is required for the priming of pathogenic TH17 cells.

The cellular sources of interleukin 6 (IL-6) that are relevant for differentiation of the TH17 subset of helper T cells remain unclear. Here we used a novel strategy for the conditional deletion of distinct IL-6-producing cell types to show that dendritic cells (DCs) positive for the signaling regulator Sirpα were essential for the generation of pathogenic TH17 cells. Using their IL-6 receptor α-chain (IL-6Rα), Sirpα+ DCs trans-presented IL-6 to T cells during the process of cognate interaction. While ambient IL-6 was sufficient to suppress the induction of expression of the transcription factor Foxp3 in T cells, trans-presentation of IL-6 by DC-bound IL-6Rα (called 'IL-6 cluster signaling' here) was needed to prevent premature induction of interferon-γ (IFN-γ) expression in T cells and to generate pathogenic TH17 cells in vivo. Our findings should guide therapeutic approaches for the treatment of TH17-cell-mediated autoimmune diseases.

Trypanosoma cruzi trans-sialidase initiates a program independent of the transcription factors RORγt and Ahr that leads to IL-17 production by activated B cells.

Here we identified B cells as a major source of rapid, innate-like production of interleukin 17 (IL-17) in vivo in response to infection with Trypanosoma cruzi. IL-17(+) B cells had a plasmablast phenotype, outnumbered cells of the TH17 subset of helper T cells and were required for an optimal response to this pathogen. With both mouse and human primary B cells, we found that exposure to parasite-derived trans-sialidase in vitro was sufficient to trigger modification of the cell-surface mucin CD45, which led to signaling dependent on the kinase Btk and production of IL-17A or IL-17F via a transcriptional program independent of the transcription factors RORγt and Ahr. Our combined data suggest that the generation of IL-17(+) B cells may be a previously unappreciated feature of innate immune responses required for pathogen control or IL-17-mediated autoimmunity.

Cutting Edge: Systemic Autoimmunity in Murine STAT3 Gain-of-Function Syndrome Is Characterized by Effector T Cell Expansion in the Absence of Overt Regulatory T Cell Dysfunction.

Germline gain-of-function mutations in the transcriptional factor STAT3 promote early-onset multisystemic autoimmunity. To investigate how increased STAT3 promotes systemic inflammation, we generated a transgenic knock-in strain expressing a pathogenic human mutation STAT3K392R within the endogenous murine locus. As predicted, STAT3K392R mice develop progressive lymphoid hyperplasia and systemic inflammation, mirroring the human disease. However, whereas the prevailing model holds that increased STAT3 activity drives human autoimmunity by dysregulating the balance between regulatory T cells and Th17 cell differentiation, we observed increased Th17 cells in the absence of major defects in regulatory T cell differentiation or function. In addition, STAT3K392R animals exhibited a prominent accumulation of IFN-γ-producing CD4+ and CD8+ T cells. Together, these data provide new insights into this complex human genetic syndrome and highlight the diverse cellular mechanisms by which dysregulated STAT3 activity promotes breaks in immune tolerance.

T-bet represses T(H)17 differentiation by preventing Runx1-mediated activation of the gene encoding RORγt.

Overactive responses by interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) are tightly linked to the development of autoimmunity, yet the factors that negatively regulate the differentiation of this lineage remain unknown. Here we report that the transcription factor T-bet suppressed development of the T(H)17 cell lineage by inhibiting transcription of Rorc (which encodes the transcription factor RORγt). T-bet interacted with the transcription factor Runx1, and this interaction blocked Runx1-mediated transactivation of Rorc. T-bet Tyr304 was required for formation of the T-bet-Runx1 complex, for blockade of Runx1 activity and for inhibition of the T(H)17 differentiation program. Our data reinforce the idea of master regulators that shape immune responses by simultaneously activating one genetic program while silencing the activity of competing regulators in a common progenitor cell.

Cutting Edge: DOCK8 Regulates a Subset of Dendritic Cells That Is Critical for the Development of Experimental Autoimmune Encephalomyelitis.

Dedicator of cytokinesis 8 (DOCK8) is a guanine nucleotide exchange factor with an essential role in cytoskeletal rearrangement, cell migration, and survival of various immune cells. Interestingly, DOCK8-deficient mice are resistant to the development of experimental autoimmune encephalomyelitis (EAE). To understand if EAE resistance in these mice results from an alteration in dendritic cell (DC) functions, we generated mice with conditional deletion of DOCK8 in DCs and observed attenuated EAE in these mice compared with control mice. Additionally, we demonstrated that DOCK8 is important for the existence of splenic conventional DC2 and lymph node migratory DCs and further established that migratory DC, rather than resident DC, are essential for the generation and proliferation of pathogenic T cell populations upon immunization with myelin Ag in adjuvant. Therefore, our data suggest that limiting migratory DCs through DOCK8 deletion and possibly other mechanisms could limit the development of CNS autoimmunity.

Temporal overexpression of IL-22 and Reg3γ differentially impacts the severity of experimental autoimmune encephalomyelitis.

IL-22 is an alpha-helical cytokine which belongs to the IL-10 family of cytokines. IL-22 is produced by RORγt+ innate and adaptive lymphocytes, including ILC3, γδ T, iNKT, Th17 and Th22 cells and some granulocytes. IL-22 receptor is expressed primarily by non-haematopoietic cells. IL-22 is critical for barrier immunity at the mucosal surfaces in the steady state and during infection. Although IL-22 knockout mice were previously shown to develop experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), how temporal IL-22 manipulation in adult mice would affect EAE course has not been studied previously. In this study, we overexpressed IL-22 via hydrodynamic gene delivery or blocked it via neutralizing antibodies in C57BL/6 mice to explore the therapeutic impact of IL-22 modulation on the EAE course. IL-22 overexpression significantly decreased EAE scores and demyelination, and reduced infiltration of IFN-γ+IL-17A+Th17 cells into the central nervous system (CNS). The neutralization of IL-22 did not alter the EAE pathology significantly. We show that IL-22-mediated protection is independent of Reg3γ, an epithelial cell-derived antimicrobial peptide induced by IL-22. Thus, overexpression of Reg3γ significantly exacerbated EAE scores, demyelination and infiltration of IFN-γ+IL-17A+ and IL-17A+GM-CSF+Th17 cells to CNS. We also show that Reg3γ may inhibit IL-2-mediated STAT5 signalling and impair expansion of Treg cells in vivo and in vitro. Finally, Reg3γ overexpression dramatically impacted intestinal microbiota during EAE. Our results provide novel insight into the role of IL-22 and IL-22-induced antimicrobial peptide Reg3γ in the pathogenesis of CNS inflammation in a murine model of MS.

CD4+ CCR6+ T cells, but not γδ T cells, are important for the IL-23R-dependent progression of antigen-induced inflammatory arthritis in mice.

IL-23 plays an important role in the development of arthritis and the IL-23 receptor (IL-23R) is expressed on different types of T cells. However, it is not fully clear which IL-23R+ T cells are critical in driving T cell-mediated synovitis. We demonstrate, using knock-in IL-23R-GFP reporter (IL-23RGFP/+ ) mice, that CD4+ CCR6+ T cells and γδ T cells, but not CD8+ T cells, express the IL-23R(GFP). During early arthritis, IL-23R(GFP)+ CD4+ CCR6+ T cells, but not IL-23R(GFP)+ γδ T cells, were present in the inflamed joints. IL-23RGFP/+ mice were bred as homozygotes to obtain IL-23RGFP/GFP (IL-23R deficient/IL-23R-/- ) mice, which express GFP under the IL-23R promotor. Arthritis progression and joint damage were significantly milder in IL-23R-/- mice, which revealed less IL-17A+ cells in their lymphoid tissues. Surprisingly, IL-23R-/- mice had increased numbers of IL-23R(GFP)+ CD4+ CCR6+ and CCR7+ CD4+ CCR6+ T cells in their spleen compared to WT, and IL-23 suppressed CCR7 expression in vitro. However, IL-23R(GFP)+ CD4+ CCR6+ T cells were present in the synovium of IL-23R-/- mice at day 4. Finally, adoptive transfer experiments revealed that CD4+ CCR6+ T cells and not γδ T cells drive arthritis progression. These data suggest that IL-23R-dependent T cell-mediated synovitis is dependent on CD4+ CCR6+ T cells and not on γδ T cells.

IL-4 inhibits TGF-beta-induced Foxp3+ T cells and, together with TGF-beta, generates IL-9+ IL-10+ Foxp3(-) effector T cells.

Transcription factor Foxp3 is critical for generating regulatory T cells (T(reg) cells). Transforming growth factor-beta (TGF-beta) induces Foxp3 and suppressive T(reg) cells from naive T cells, whereas interleukin 6 (IL-6) inhibits the generation of inducible T(reg) cells. Here we show that IL-4 blocked the generation of TGF-beta-induced Foxp3(+) T(reg) cells and instead induced a population of T helper cells that produced IL-9 and IL-10. The IL-9(+)IL-10(+) T cells demonstrated no regulatory properties despite producing abundant IL-10. Adoptive transfer of IL-9(+)IL-10(+) T cells into recombination-activating gene 1-deficient mice induced colitis and peripheral neuritis, the severity of which was aggravated if the IL-9(+)IL-10(+) T cells were transferred with CD45RB(hi) CD4(+) effector T cells. Thus IL-9(+)IL-10(+) T cells lack suppressive function and constitute a distinct population of helper-effector T cells that promote tissue inflammation.

ILC3 deficiency and generalized ILC abnormalities in DOCK8-deficient patients.

BACKGROUND: Dedicator of cytokinesis 8 (DOCK8) deficiency is the main cause of the autosomal recessive hyper-IgE syndrome (HIES). We previously reported the selective loss of group 3 innate lymphoid cell (ILC) number and function in a Dock8-deficient mouse model. In this study, we sought to test whether DOCK8 is required for the function and maintenance of ILC subsets in humans. METHODS: Peripheral blood ILC1-3 subsets of 16 DOCK8-deficient patients recruited at the pretransplant stage, and seven patients with autosomal dominant (AD) HIES due to STAT3 mutations, were compared with those of healthy controls or post-transplant DOCK8-deficient patients (n = 12) by flow cytometry and real-time qPCR. Sorted total ILCs from DOCK8- or STAT3-mutant patients and healthy controls were assayed for survival, apoptosis, proliferation, and activation by IL-7, IL-23, and IL-12 by cell culture, flow cytometry, and phospho-flow assays. RESULTS: DOCK8-deficient but not STAT3-mutant patients exhibited a profound depletion of ILC3s, and to a lesser extent ILC2s, in their peripheral blood. DOCK8-deficient ILC1-3 subsets had defective proliferation, expressed lower levels of IL-7R, responded less to IL-7, IL-12, or IL-23 cytokines, and were more prone to apoptosis compared with those of healthy controls. CONCLUSION: DOCK8 regulates human ILC3 expansion and survival, and more globally ILC cytokine signaling and proliferation. DOCK8 deficiency leads to loss of ILC3 from peripheral blood. ILC3 deficiency may contribute to the susceptibility of DOCK8-deficient patients to infections.

A dominant function for interleukin 27 in generating interleukin 10-producing anti-inflammatory T cells.

Regulatory T cells (T(reg) cells) expressing the transcription factor Foxp3 are key in maintaining the balance of immune homeostasis. However, distinct induced T regulatory type 1 (Tr1) cells that lack Foxp3 expression also regulate T cell function, mainly by producing the immunosuppressive cytokine interleukin 10 (IL-10). However, the factors required for the induction of IL-10-producing suppressive T cells are not fully understood. Here we demonstrate that dendritic cells modified by T(reg) cells induced the generation of IL-10-producing Tr1 cells. The differentiation of naive CD4+ T cells into IL-10-producing cells was mediated by IL-27 produced by the T(reg) cell-modified dendritic cells, and transforming growth factor-beta amplified the generation of induced IL-10+ Tr1 cells by IL-27. Thus, IL-27 and transforming growth factor-beta promote the generation of IL-10-producing Tr1 cells.

γδ T cells enhance autoimmunity by restraining regulatory T cell responses via an interleukin-23-dependent mechanism.

Mice that lack interleukin-23 (IL-23) are resistant to T cell-mediated autoimmunity. Although IL-23 is a maturation factor for T helper 17 (Th17) cells, a subset of γδ T cells expresses the IL-23 receptor (IL-23R) constitutively. Using IL-23R reporter mice, we showed that γδ T cells were the first cells to respond to IL-23 during experimental autoimmune encephalomyelitis (EAE). Although γδ T cells produced Th17 cell-associated cytokines in response to IL-23, their major function was to prevent the development of regulatory T (Treg) cell responses. IL-23-activated γδ T cells rendered αß effector T cells refractory to the suppressive activity of Treg cells and also prevented the conversion of conventional T cells into Foxp3(+) Treg cells in vivo. Thus, IL-23, which by itself has no direct effect on Treg cells, is able to disarm Treg cell responses and promote antigen-specific effector T cell responses via activating γδ T cells.

Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth.

Approximately 2% of colorectal cancer is linked to pre-existing inflammation known as colitis-associated cancer, but most develops in patients without underlying inflammatory bowel disease. Colorectal cancer often follows a genetic pathway whereby loss of the adenomatous polyposis coli (APC) tumour suppressor and activation of ß-catenin are followed by mutations in K-Ras, PIK3CA and TP53, as the tumour emerges and progresses. Curiously, however, 'inflammatory signature' genes characteristic of colitis-associated cancer are also upregulated in colorectal cancer. Further, like most solid tumours, colorectal cancer exhibits immune/inflammatory infiltrates, referred to as 'tumour-elicited inflammation'. Although infiltrating CD4(+) T(H)1 cells and CD8(+) cytotoxic T cells constitute a positive prognostic sign in colorectal cancer, myeloid cells and T-helper interleukin (IL)-17-producing (T(H)17) cells promote tumorigenesis, and a 'T(H)17 expression signature' in stage I/II colorectal cancer is associated with a drastic decrease in disease-free survival. Despite its pathogenic importance, the mechanisms responsible for the appearance of tumour-elicited inflammation are poorly understood. Many epithelial cancers develop proximally to microbial communities, which are physically separated from immune cells by an epithelial barrier. We investigated mechanisms responsible for tumour-elicited inflammation in a mouse model of colorectal tumorigenesis, which, like human colorectal cancer, exhibits upregulation of IL-23 and IL-17. Here we show that IL-23 signalling promotes tumour growth and progression, and development of a tumoural IL-17 response. IL-23 is mainly produced by tumour-associated myeloid cells that are likely to be activated by microbial products, which penetrate the tumours but not adjacent tissue. Both early and late colorectal neoplasms exhibit defective expression of several barrier proteins. We propose that barrier deterioration induced by colorectal-cancer-initiating genetic lesions results in adenoma invasion by microbial products that trigger tumour-elicited inflammation, which in turn drives tumour growth.

IL-7/IL-7 Receptor Signaling Differentially Affects Effector CD4+ T Cell Subsets Involved in Experimental Autoimmune Encephalomyelitis.

IL-17-producing CD4(+) T (Th17) cells, along with IFN-γ-expressing Th1 cells, represent two major pathogenic T cell subsets in experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). The cytokines and transcription factors involved in the development and effector functions of Th1 and Th17 cells have been largely characterized. Among them, IL-23 is essential for the generation of stable and encephalitogenic Th17 cells and for the development of EAE. The IL-7/IL-7R signaling axis participates in cell survival, and perturbation of this pathway has been associated with enhanced susceptibility to MS. A link between IL-23-driven pathogenic T cells and IL-7/IL-7R signaling has previously been proposed, but has not been formally addressed. In the current study, we showed that Th17 cells from mice with EAE express high levels of IL-7Rα compared with Th1 cells. Using mice that constitutively express IL-7Rα on T cells, we determined that sustained IL-7R expression in IL-23R-deficient mice could not drive pathogenic T cells and the development of EAE. IL-7 inhibited the differentiation of Th17 cells, but promoted IFN-γ and GM-CSF secretion in vitro. In vivo IL-7/anti-IL-7 mAb complexes selectively expanded and enhanced the proliferation of CXCR3-expressing Th1 cells, but did not impact Th17 cells and EAE development in wild-type and IL-23R-deficient mice. Importantly, high IL-7 expression was detected in the CNS during EAE and could drive the plasticity of Th17 cells to IFN-γ-producing T cells. Together, these data address the contribution of IL-23/IL-23R and IL-7/IL-7R signaling in Th17 and Th1 cell dynamics during CNS autoimmunity.

Cutting edge: the pathogenicity of IFN-γ-producing Th17 cells is independent of T-bet.

During the development of experimental autoimmune encephalomyelitis (EAE), the proportion of pathogenic and myelin-specific cells within CNS-infiltrating cytokine-producing Th cells is unknown. Using an IL-17A/IFN-γ double reporter mouse and I-A(b)/myelin oligodendrocyte glycoprotein 38-49 tetramer, we show in this study that IL-17(+)IFN-γ(+) Th cells, which are expanded in the CNS during EAE, are highly enriched in myelin oligodendrocyte glycoprotein-specific T cells. We further demonstrate that IL-23 is essential for the generation and expansion of IFN-γ-producing Th17 cells independently of the Th1-associated transcription factors T-bet, STAT1, and STAT4. Furthermore, Th17 and IL-17(+)IFN-γ(+) Th cells can induce CNS autoimmunity independently of T-bet. Whereas T-bet is crucial for Th1-mediated EAE, it is dispensable for Th17 cell-mediated autoimmunity. Our results suggest the existence of different epigenetic programs that regulate IFN-γ expression in Th1 and Th17 cells.

Colitogenic effector T cells: roles of gut-homing integrin, gut antigen specificity and γδ T cells.

Disturbance of T-cell homeostasis could lead to intestinal inflammation. Naive CD4 T cells undergoing spontaneous proliferation, a robust proliferative response that occurs under severe lymphopenic conditions, differentiate into effector cells producing Th1- and/or Th17-type cytokines and induce a chronic inflammation in the intestine that resembles human inflammatory bowel disease. In this study, we investigated the key properties of CD4 T cells necessary to induce experimental colitis. α4ß7 upregulation was primarily induced by mesenteric lymph node (mLN) resident CD11b(+) dendritic cell subsets via transforming growth factor beta (TGFß)/retinoic acid-dependent mechanism. Interestingly, α4ß7 expression was essential but not sufficient to induce inflammation. In addition to gut-homing specificity, expression of gut Ag specificity was also crucial. T-cell acquisition of the specificity was dramatically enhanced by the presence of γδ T cells, a population previously shown to exacerbate T-cell-mediated colitis. Importantly, interleukin (IL)-23-mediated γδ T cell stimulation was necessary to enhance colitogenicity but not gut antigen reactivity of proliferating CD4 T cells. These findings demonstrate that T-cell colitogenicity is achieved through multiple processes, offering a therapeutic rationale by intervening these pathways.

Overexpression of the Ctla-4 isoform lacking exons 2 and 3 causes autoimmunity.

CTLA-4 is a potent inhibitor of T cell activation, primarily upon binding to its costimulatory ligands (B7.1 and B7.2) expressed on APCs. However, variants of CTLA-4 can also function independently of B7 molecules. 1/4CTLA-4 is a highly conserved isoform encoded by exons 1 and 4 of the Ctla4 gene that lacks the ligand-binding and the transmembrane domains, and as yet, its function is not known. To investigate the function of 1/4CTLA-4, we generated transgenic (Tg) mice overexpressing this variant. Cytokine production by 1/4CTLA-4 Tg T cells was elevated compared with wild type T cells. The frequency of CD44(high) memory T cells in 1/4CTLA-4 Tg mice was increased, and as the mice aged, the frequency further increased. 1/4CTLA-4 Tg mice >1 y old had increased expression of T cell activation markers and developed spontaneous autoimmunity, including elevated production of autoantibodies. In contrast with young 1/4CTLA-4 Tg mice, aged 1/4CTLA-4 Tg mice had elevated frequencies of Foxp3(+) regulatory T cells, but the regulatory T cells from these mice were not able to inhibit colitis development. Collectively, these data suggest that the function of the 1/4CTLA-4 isoform is distinct from that of CTLA-4 in that it enhances T cell activation and promotes autoimmunity rather than inhibiting immune responses.