Characterization of the developmental landscape of murine RORγt+ iNKT cells.

Invariant natural killer T (iNKT) cells expressing the retinoic acid receptor-related orphan receptor γt (RORγt) and producing IL-17 represent a minor subset of CD1d-restricted iNKT cells (iNKT17) in C57BL/6J (B6) mice. We aimed in this study to define the reasons for their low distribution and the sequence of events accompanying their normal thymic development. We found that RORγt+ iNKT cells have higher proliferation potential and a greater propensity to apoptosis than RORγt- iNKT cells. These cells do not likely reside in the thymus indicating that thymus emigration, and higher apoptosis potential, could contribute to RORγt+ iNKT cell reduced thymic distribution. Ontogeny studies suggest that mature HSAlow RORγt+ iNKT cells might develop through developmental stages defined by a differential expression of CCR6 and CD138 during which RORγt expression and IL-17 production capabilities are progressively acquired. Finally, we found that RORγt+ iNKT cells perceive a strong TCR signal that could contribute to their entry into a specific 'Th17 like' developmental program influencing their survival and migration. Overall, our study proposes a hypothetical thymic developmental sequence for iNKT17 cells, which could be of great use to study molecular mechanisms regulating this developmental program.

IL-17A Is Critical for CD8+ T Effector Response in Airway Epithelial Injury After Transplantation.

BACKGROUND: Airway epithelium is the primary target of trachea and lung transplant rejection, the degree of epithelial injury is closely correlated with obliterative bronchiolitis development. In this study, we investigated the cellular and molecular mechanisms of IL-17A-mediated airway epithelial injury after transplantation. METHODS: Murine orthotopic allogeneic trachea or lung transplants were implemented in wild type or RORγt mice. Recipients received anti-IL-17A or anti-IFNγ for cytokine neutralization, anti-CD8 for CD8 T-cell depletion, or STAT3 inhibitor to suppress type 17 CD4+/CD8+ T cell development. Airway injury and graft inflammatory cell infiltration were examined by histopathology and immunohistochemistry. Gene expression of IL-17A, IFNγ, perforin, granzyme B, and chemokines in grafts was quantitated by real-time RT-PCR. RESULTS: IL-17A and IFNγ were rapidly expressed and associated with epithelial injury and CD8 T-cell accumulation after allotransplantation. Depletion of CD8 T cells prevented airway epithelial injury. Neutralization of IL-17A or devoid of IL-17A production by RORγt deficiency improved airway epithelial integrity of the trachea allografts. Anti-IL-17A reduced the expression of CXCL9, CXCL10, CXCL11, and CCL20, and abolished CD8 T-cell accumulation in the trachea allografts. Inhibition of STAT3 activation significantly reduced IL-17A expression in both trachea and lung allografts; however, it increased IFNγ expression and cytotoxic activities, which resulted in the failure of airway protection. CONCLUSIONS: Our data reveal the critical role of IL-17A in mediating CD8 T effector response that causes airway epithelial injury and lung allograft rejection, and indicate that inhibition of STAT3 signals could drive CD8 T cells from Tc17 toward Tc1 development.

Reversing SKI-SMAD4-mediated suppression is essential for TH17 cell differentiation.

T helper 17 (TH17) cells are critically involved in host defence, inflammation, and autoimmunity. Transforming growth factor ß (TGFß) is instrumental in TH17 cell differentiation by cooperating with interleukin-6 (refs 6, 7). Yet, the mechanism by which TGFß enables TH17 cell differentiation remains elusive. Here we reveal that TGFß enables TH17 cell differentiation by reversing SKI-SMAD4-mediated suppression of the expression of the retinoic acid receptor (RAR)-related orphan receptor γt (RORγt). We found that, unlike wild-type T cells, SMAD4-deficient T cells differentiate into TH17 cells in the absence of TGFß signalling in a RORγt-dependent manner. Ectopic SMAD4 expression suppresses RORγt expression and TH17 cell differentiation of SMAD4-deficient T cells. However, TGFß neutralizes SMAD4-mediated suppression without affecting SMAD4 binding to the Rorc locus. Proteomic analysis revealed that SMAD4 interacts with SKI, a transcriptional repressor that is degraded upon TGFß stimulation. SKI controls histone acetylation and deacetylation of the Rorc locus and TH17 cell differentiation via SMAD4: ectopic SKI expression inhibits H3K9 acetylation of the Rorc locus, Rorc expression, and TH17 cell differentiation in a SMAD4-dependent manner. Therefore, TGFß-induced disruption of SKI reverses SKI-SMAD4-mediated suppression of RORγt to enable TH17 cell differentiation. This study reveals a critical mechanism by which TGFß controls TH17 cell differentiation and uncovers the SKI-SMAD4 axis as a potential therapeutic target for treating TH17-related diseases.

Tbet or Continued RORγt Expression Is Not Required for Th17-Associated Immunopathology.

The discovery of Th17 cell plasticity, in which CD4(+) IL-17-producing Th17 cells give rise to IL-17/IFN-γ double-producing cells and Th1-like IFNγ(+) ex-Th17 lymphocytes, has raised questions regarding which of these cell types contribute to immunopathology during inflammatory diseases. In this study, we show using Helicobacter hepaticus-induced intestinal inflammation that IL-17A(Cre)- or Rag1(Cre)-mediated deletion of Tbx21 has no effect on the generation of IL-17/IFN-γ double-producing cells, but leads to a marked absence of Th1-like IFNγ(+) ex-Th17 cells. Despite the lack of Th1-like ex-Th17 cells, the degree of H. hepaticus-triggered intestinal inflammation in mice in which Tbx21 was excised in IL-17-producing or Rag1-expressing cells is indistinguishable from that observed in control mice. In stark contrast, using experimental autoimmune encephalomyelitis, we show that IL-17A(Cre)-mediated deletion of Tbx21 prevents the conversion of Th17 cells to IL-17A/IFN-γ double-producing cells as well as Th1-like IFN-γ(+) ex-Th17 cells. However, IL-17A(Cre)-mediated deletion of Tbx21 has only limited effects on disease course in this model and is not compensated by Ag-specific Th1 cells. IL-17A(Cre)-mediated deletion of Rorc reveals that RORγt is essential for the maintenance of the Th17 cell lineage, but not immunopathology during experimental autoimmune encephalomyelitis. These results show that neither the single Th17 subset, nor its progeny, is solely responsible for immunopathology or autoimmunity.

Epidermal Notch1 recruits RORγ(+) group 3 innate lymphoid cells to orchestrate normal skin repair.

Notch has a well-defined role in controlling cell fate decisions in the embryo and the adult epidermis and immune systems, yet emerging evidence suggests Notch also directs non-cell-autonomous signalling in adult tissues. Here, we show that Notch1 works as a damage response signal. Epidermal Notch induces recruitment of immune cell subsets including RORγ(+) ILC3s into wounded dermis; RORγ(+) ILC3s are potent sources of IL17F in wounds and control immunological and epidermal cell responses. Mice deficient for RORγ(+) ILC3s heal wounds poorly resulting from delayed epidermal proliferation and macrophage recruitment in a CCL3-dependent process. Notch1 upregulates TNFα and the ILC3 recruitment chemokines CCL20 and CXCL13. TNFα, as a Notch1 effector, directs ILC3 localization and rates of wound healing. Altogether these findings suggest that Notch is a key stress/injury signal in skin epithelium driving innate immune cell recruitment and normal skin tissue repair.

Dysregulated development of IL-17- and IL-21-expressing follicular helper T cells and increased germinal center formation in the absence of RORγt.

Interleukin 17-producing helper T (Th17) cells have been widely defined by the lineage transcription factor retinoid-related orphan receptor (ROR)γt. Pathophysiologically, these cells play a crucial role in autoimmune diseases and have been linked to dysregulated germinal center (GC) reactions and autoantibody production. In this study, we used gene expression and flow cytometric analyses for the characterization of Rorγt(-/-) and Rorγt(-/-)Il21(RFP/+) mice to demonstrate a previously unknown transcriptional flexibility in the development of IL-17-producing Th-cell subsets. We found an accumulation of follicular Th (Tfh) cells by 5.2-fold, spontaneous 13-fold higher GC formation, decreased frequency of follicular Foxp3(+) T-regulatory (Treg) cells (50%), and a 3.4-fold increase in the number of proliferating follicular B cells in RORγt-deficient vs. wild-type mice. Dysregulated B-cell responses were associated with enhanced production of IL-17 (6.4-fold), IL-21 (2.2-fold), and B-cell-activating factor (BAFF) (2-fold) and were partially rescued by adoptive transfer of Treg cells. In an unexpected finding, we detected RORγt-independent IL-17 expression in ICOS(+)CXCR5(+)Tfh and in ICOS(+)CXCR5(-)Th cells. Based on the observed high Irf4 and Batf gene expression, we suggest that CD4(+) T-cell transcription factors other than RORγt can cooperatively induce differentiation of IL-17-producing Th cells, including Th17-like Tfh-cell subsets. We conclude that the occurrence of aberrant Tfh and follicular Treg cells support spontaneous GC formation and dysregulated B-cell responses in RORγt-deficient mice.

Interleukin-23-Independent IL-17 Production Regulates Intestinal Epithelial Permeability.

Whether interleukin-17A (IL-17A) has pathogenic and/or protective roles in the gut mucosa is controversial and few studies have analyzed specific cell populations for protective functions within the inflamed colonic tissue. Here we have provided evidence for IL-17A-dependent regulation of the tight junction protein occludin during epithelial injury that limits excessive permeability and maintains barrier integrity. Analysis of epithelial cells showed that in the absence of signaling via the IL-17 receptor adaptor protein Act-1, the protective effect of IL-17A was abrogated and inflammation was enhanced. We have demonstrated that after acute intestinal injury, IL-23R(+) γδ T cells in the colonic lamina propria were the primary producers of early, gut-protective IL-17A, and this production of IL-17A was IL-23 independent, leaving protective IL-17 intact in the absence of IL-23. These results suggest that IL-17-producing γδ T cells are important for the maintenance and protection of epithelial barriers in the intestinal mucosa.

Runx3 specifies lineage commitment of innate lymphoid cells.

Subsets of innate lymphoid cells (ILCs) reside in the mucosa and regulate immune responses to external pathogens. While ILCs can be phenotypically classified into ILC1, ILC2 and ILC3 subsets, the transcriptional control of commitment to each ILC lineage is incompletely understood. Here we report that the transcription factor Runx3 was essential for the normal development of ILC1 and ILC3 cells but not of ILC2 cells. Runx3 controlled the survival of ILC1 cells but not of ILC3 cells. Runx3 was required for expression of the transcription factor RORγt and its downstream target, the transcription factor AHR, in ILC3 cells. The absence of Runx3 in ILCs exacerbated infection with Citrobacter rodentium. Therefore, our data establish Runx3 as a key transcription factor in the lineage-specific differentiation of ILC1 and ILC3 cells.

Group 3 innate lymphoid cells accumulate and exhibit disease-induced activation in the meninges in EAE.

Innate lymphoid cells are immune cells that reside in tissues that interface with the external environment and contribute to the first line defense against pathogens. However, they also have roles in promoting chronic inflammation. Here we demonstrate that group 3 ILCs, (ILC3s - CD45+Lin-IL-7Rα+RORγt+), are normal residents of the meninges and exhibit disease-induced accumulation and activation in EAE. In addition to production of the pro-inflammatory cytokines IL-17 and GM-CSF, ILC3s constitutively express CD30L and OX40L, molecules required for memory T cell survival. We show that disease-induced trafficking of transferred wild type T cells to the meninges is impaired in ILC3-deficient Rorc-/- mice. Furthermore, lymphoid tissue inducer cells, a c-kit+ ILC3 subset that promotes ectopic lymphoid follicle development, a hallmark of many autoimmune diseases, are reduced in the meninges of EAE-resistant c-kit mutant Kit(W/Wv) mice. We propose that ILC3s sustain neuroinflammation by supporting T cell survival and reactivation in the meninges.

Conversion of Th17 into IL-17A(neg) regulatory T cells: a novel mechanism in prolonged allograft survival promoted by mesenchymal stem cell-supported minimized immunosuppressive therapy.

The ultimate goal in transplantation medicine is the promotion of operational tolerance. Although Th cells of the Th17 type have been predominantly associated with rejection of allogeneic solid organ grafts, regulatory T (T(reg)) cells appear to foster operational tolerance. Induced T(reg) and Th17 cells have a higher lineage plasticity than has been recognized thus far. We found that when mesenchymal stem cells (MSCs) were used to induce long-term acceptance of allogeneic heart grafts in mice, the induction of T(reg) cells was preceded by development of a CD11b(hi)Gr1(int) myeloid-derived immunosuppressive cell-mediated Th17 response. Substantial suppression of Foxp3(+) T(reg) cell generation from retinoic acid receptor-related orphan receptor γ(-/-) T cells by MSCs revealed that retinoic acid receptor-related orphan receptor γ is a common factor in the differentiation of T(reg) and Th17 cells. Immunosuppressant mycophenolate mofetil treatment of enriched IL-17A(+) cells from MSC-primed allograft mouse recipients resulted in a reduction of IL-17A production and an increase in the Foxp3(+) T(reg) cell fraction. Furthermore, identification of IL-17A(+) Foxp3(+) double-positive and ex-IL-17-producing IL-17A(neg)Foxp3(+) T cells strongly argues for direct conversion of Th17 cells into T(reg) cells as the underlying mechanism of immune regulation in MSC-mediated allograft survival. The Th17 into T(reg) conversion identified in this study constitutes an important immunological mechanism by which MSC-induced myeloid-derived immunosuppressive cells mediate operational transplant tolerance. The possibility to create T(reg) cell-regulated operational tolerance in the absence of complete immune suppression provides strong clinical implications for cell therapy-assisted minimization protocols.

Retinoic acid-related orphan receptor γ (RORγ): a novel participant in the diurnal regulation of hepatic gluconeogenesis and insulin sensitivity.

The hepatic circadian clock plays a key role in the daily regulation of glucose metabolism, but the precise molecular mechanisms that coordinate these two biological processes are not fully understood. In this study, we identify a novel connection between the regulation of RORγ by the clock machinery and the diurnal regulation of glucose metabolic networks. We demonstrate that particularly at daytime, mice deficient in RORγ exhibit improved insulin sensitivity and glucose tolerance due to reduced hepatic gluconeogenesis. This is associated with a reduced peak expression of several glucose metabolic genes critical in the control of gluconeogenesis and glycolysis. Genome-wide cistromic profiling, promoter and mutation analysis support the concept that RORγ regulates the transcription of several glucose metabolic genes directly by binding ROREs in their promoter regulatory region. Similar observations were made in liver-specific RORγ-deficient mice suggesting that the changes in glucose homeostasis were directly related to the loss of hepatic RORγ expression. Altogether, our study shows that RORγ regulates several glucose metabolic genes downstream of the hepatic clock and identifies a novel metabolic function for RORγ in the diurnal regulation of hepatic gluconeogenesis and insulin sensitivity. The inhibition of the activation of several metabolic gene promoters by an RORγ antagonist suggests that antagonists may provide a novel strategy in the management of metabolic diseases, including type 2 diabetes.

Decreased RORC-dependent silencing of prostaglandin receptor EP2 induces autoimmune Th17 cells.

Prostaglandin E2 (PGE2) promotes Th17 expansion while otherwise inhibiting other CD4+ T cell subsets. Here, we identified a PGE2-dependent pathway that induces pathogenic Th17 cells in autoimmune disease and is regulated by the transcription factor RORC. Compared with other CD4+ cell types from healthy subjects, there is a surprising lack of the prostaglandin receptor EP2 on Th17 cells; therefore, we examined the hypothesis that RORγt, which is highly expressed in Th17 cells, mediates EP2 downregulation. Chromatin immunoprecipitation followed by DNA sequencing revealed that RORγt binds directly to Ptger2 (the gene encoding EP2 receptor) in Th17 cells isolated from WT mice. In Th17 cells isolated from humans, RORC repressed EP2 by directly silencing PTGER2 transcription, and knock down of RORC restored EP2 expression in Th17 cells. Compared with Th17 cells from healthy individuals, Th17 cells from patients with MS exhibited reduced RORC binding to the PTGER2 promoter region, resulting in higher EP2 levels and increased expression of IFN-γ and GM-CSF. Finally, overexpression of EP2 in Th17 cells from healthy individuals induced a specific program of inflammatory gene transcription that produced a pathogenic Th17 cell phenotype. These findings reveal that RORC directly regulates the effects of PGE2 on Th17 cells, and dysfunction of this pathway induces a pathogenic Th17 cell phenotype.

Double deficiency for RORγt and T-bet drives Th2-mediated allograft rejection in mice.

Although Th1, Th2, and Th17 cells are thought to be major effector cells in adaptive alloimmune responses, their respective contribution to allograft rejection remains unclear. To precisely address this, we used mice genetically modified for the Th1 and Th17 hallmark transcription factors T-bet and RORγt, respectively, which allowed us to study the alloreactive role of each subset in an experimental transplant setting. We found that in a fully mismatched heterotopic mouse heart transplantation model, T cells deficient for T-bet (prone to Th17 differentiation) versus RORγt (prone to Th1 differentiation) rejected allografts at a more accelerated rate, indicating a predominance of Th17- over Th1-driven alloimmunity. Importantly, T cells doubly deficient for both T-bet and RORγt differentiated into alloreactive GATA-3-expressing Th2 cells, which promptly induced allograft rejection characterized by a Th2-type intragraft expression profile and eosinophilic infiltration. Mechanistically, Th2-mediated allograft rejection was contingent on IL-4, as its neutralization significantly prolonged allograft survival by reducing intragraft expression of Th2 effector molecules and eosinophilic allograft infiltration. Moreover, under IL-4 neutralizing conditions, alloreactive double-deficient T cells upregulated Eomesodermin (Eomes) and IFN-γ, but not GATA-3. Thus, in the absence of T-bet and RORγt, Eomes may salvage Th1-mediated alloimmunity that underlies IL-4 neutralization-resistant allograft rejection. We summarize that, whereas Th17 cells predictably promote allograft rejection, IL-4-producing GATA-3(+) Th2 cells, which are generally thought to protect allogeneic transplants, may actually be potent facilitators of organ transplant rejection in the absence of T-bet and RORγt. Moreover, Eomes may rescue Th1-mediated allograft rejection in the absence of IL-4, T-bet, and RORγt.

Expression of RORγt marks a pathogenic regulatory T cell subset in human colon cancer.

The role of regulatory T cells (T(regs)) in human colon cancer (CC) remains controversial: high densities of tumor-infiltrating T(regs) can correlate with better or worse clinical outcomes depending on the study. In mouse models of cancer, T(regs) have been reported to suppress inflammation and protect the host, suppress T cells and protect the tumor, or even have direct cancer-promoting attributes. These different effects may result from the presence of different T(reg) subsets. We report the preferential expansion of a T(reg) subset in human CC with potent T cell-suppressive, but compromised anti-inflammatory, properties; these cells are distinguished from T(regs) present in healthy donors by their coexpression of Foxp3 and RORγt. T(regs) with similar attributes were found to be expanded in mouse models of hereditary polyposis. Indeed, ablation of the RORγt gene in Foxp3(+) cells in polyp-prone mice stabilized T(reg) anti-inflammatory functions, suppressed inflammation, improved polyp-specific immune surveillance, and severely attenuated polyposis. Ablation of interleukin-6 (IL-6), IL-23, IL-17, or tumor necrosis factor-α in polyp-prone mice reduced polyp number but not to the same extent as loss of RORγt. Surprisingly, loss of IL-17A had a dual effect: IL-17A-deficient mice had fewer polyps but continued to have RORγt(+) T(regs) and developed invasive cancer. Thus, we conclude that RORγt has a central role in determining the balance between protective and pathogenic T(regs) in CC and that T(reg) subtype regulates inflammation, potency of immune surveillance, and severity of disease outcome.

Cutting edge: lymphoid tissue inducer cells maintain memory CD4 T cells within secondary lymphoid tissue.

Phylogeny shows that CD4 T cell memory and lymph nodes coevolved in placental mammals. In ontogeny, retinoic acid orphan receptor (ROR)γ-dependent lymphoid tissue inducer (LTi) cells program the development of mammalian lymph nodes. In this study, we show that although primary CD4 T cell expansion is normal in RORγ-deficient mice, the persistence of memory CD4 T cells is RORγ-dependent. Furthermore, using bone marrow chimeric mice we demonstrate that LTi cells are the key RORγ-expressing cell type sufficient for memory CD4 T cell survival in the absence of persistent Ag. This effect was specific for CD4 T cells, as memory CD8 T cells survived equally well in the presence or absence of LTi cells. These data demonstrate a novel role for LTi cells, archetypal members of the innate lymphoid cell family, in supporting memory CD4 T cell survival in vivo.

Robust tumor immunity to melanoma mediated by interleukin-9-producing T cells.

Interleukin-9 (IL-9) is a T cell cytokine that acts through a γC-family receptor on target cells and is associated with inflammation and allergy. We determined that T cells from mice deficient in the T helper type 17 (T(H)17) pathway genes encoding retinoid-related orphan receptor γ (ROR-γ) and IL-23 receptor (IL-23R) produced abundant IL-9, and we found substantial growth inhibition of B16F10 melanoma in these mice. IL-9-blocking antibodies reversed this tumor growth inhibition and enhanced tumor growth in wild-type (WT) mice. Il9r(-/-) mice showed accelerated tumor growth, and administration of recombinant IL-9 (rIL-9) to tumor-bearing WT and Rag1(-/-) mice inhibited melanoma as well as lung carcinoma growth. Adoptive transfer of tumor-antigen-specific T(H)9 cells into both WT and Rag1(-/-) mice suppressed melanoma growth; this effect was abrogated by treatment with neutralizing antibodies to IL-9. Exogenous rIL-9 inhibited tumor growth in Rag1(-/-) mice but not in mast-cell-deficient mice, suggesting that the targets of IL-9 in this setting include mast cells but not T or B cells. In addition, we found higher numbers of T(H)9 cells in normal human skin and blood compared to metastatic lesions of subjects with progressive stage IV melanoma. These results suggest a role for IL-9 in tumor immunity and offer insight into potential therapeutic strategies.

Restricted microbiota and absence of cognate TCR antigen leads to an unbalanced generation of Th17 cells.

Retinoic acid-related orphan receptor (ROR)γt(+) TCRαß(+) cells expressing IL-17, termed Th17 cells, are most abundant in the intestinal lamina propria. Symbiotic microbiota are required for the generation of Th17 cells, but the requirement for microbiota-derived Ag is not documented. In this study, we show that normal numbers of Th17 cells develop in the intestine of mice that express a single TCR in the absence of cognate Ag, whereas the microbiota remains essential for their development. However, such mice, or mice monocolonized with the Th17-inducing segmented filamentous bacteria, fail to induce normal numbers of Foxp3(+) RORγt(+) T cells, the regulatory counterpart of IL-17(+)RORγt(+) T cells. These results demonstrate that a complex microbiota and cognate Ag are required to generate a properly regulated set of RORγt(+) T cells and Th17 cells.

Microbiota-induced tertiary lymphoid tissues aggravate inflammatory disease in the absence of RORgamma t and LTi cells.

The programmed development of lymph nodes and Peyer's patches during ontogeny requires lymphoid tissue inducer (LTi) cells that express the nuclear hormone receptor RORγt. After birth, LTi cells in the intestine cluster into cryptopatches, the precursors of isolated lymphoid follicles (ILFs), which are induced to form by symbiotic bacteria and maintain intestinal homeostasis. We show that in RORγt-deficient mice, which lack LTi cells, programmed lymphoid tissues, ILFs, and Th17 cells, bacterial containment requires the generation of large numbers of tertiary lymphoid tissues (tLTs) through the activity of B cells. However, upon epithelial damage, these mice develop severe intestinal inflammation characterized by extensive recruitment of neutrophils and IgG(+) B cells, high expression of activation-induced deaminase in tLTs, and wasting disease. The pathology was prevented by antibiotic treatment or inhibition of lymphoid tissue formation and was significantly decreased by treatment with intravenous immunoglobulin G (IVIG). Our data show that intestinal immunodeficiency, such as an absence in RORγt-mediated proinflammatory immunity, can be compensated by increased lymphoid tissue genesis. However, this comes at a high cost for the host and can lead to a deregulated B cell response and aggravated inflammatory pathology.

T helper17 cells are sufficient but not necessary to induce acute graft-versus-host disease.

T helper (Th)1 cells were considered responsible for the induction of graft-versus-host disease (GVHD), but recently the concept has been challenged. Th17 cells play a critical role in mediating autoimmune diseases, but their role in the pathogenesis of GVHD remains unclear. Herein we compare the ability of in vitro generated Th1 and Th17 cells from C57BL/6 mice to induce GVHD in lethally irradiated BALB/c recipients. Allogeneic Th17 cells had superior expansion and infiltration capabilities in GVHD target organs, which correlated with their increased pathogenicity when compared with naïve or Th1 controls. Th17 cells caused no pathology in the syngeneic recipients, indicating that antigen-activation was required for their pathogenicity. Polarized Th17 cells could not maintain their phenotype in vivo as they produced a significant amount of interferon (IFN)-gamma after being transplanted into allogeneic recipients; however, IFN-gamma was not required for Th17 cell-induced GVHD. Further, we evaluated the pathogenesis of Th17 cells in GVHD by using polyclonal nonprimed CD4T cells in a clinically relevant allogeneic bone marrow transplantation (BMT) setting. We found that disruption of Th17-differentiation alone by targeting RORgammat (Th17-specific transcription factor) had no significant effect on GVHD development. We conclude that Th17 cells are sufficient but not necessary to induce GVHD.

Isolation of NK cells and NK-like cells from the intestinal lamina propria.

Being exposed to food products, pathogens and harmless commensal bacteria, the mucosal immune system faces a constant challenge. Therefore, maintenance of a homeostatic balance is required to achieve tolerance to harmless bacteria and their products and to induce potent immunity to infection with pathogenic bacteria. Until recently, the literature on mucosal natural killer (NK) cells residing in the intestinal lamina propria was scarce and phenotype and function of gut mucosal NK cells did not receive much attention. Recently, data have become available identifying two distinct subsets of mucosal NKp46(+) lymphocytes based on the expression of the orphan transcription factor RORgammat. In many ways, the RORgammat(-) subset resembled "classical" NK cells in that it was developmentally dependent on IL-15 but not on RORgammat and displayed NK cell function (e.g., cell-mediated cytotoxicity, IFN-gamma production). In contrast, the RORgammat(+) subset developed independent of IL-15 but required RORgammat, suggesting that this subset may be related to lymphoid tissue inducer (LTi) cells. Interestingly, these RORgammat(+) NKp46(+) NK-LTi cells constitutively produced large amounts of IL-22, a cytokine regulating antimicrobial protection and regeneration of epithelial cells. In this chapter, we provide experimental procedures to isolate "classical" NK cells from the intestinal lamina propria as well as the newly described lymphoid tissue inducer-like (LTi-like) cells producing IL-22 and co-expressing NK cell receptors.