Switch receptor T3/28 improves long-term persistence and antitumor efficacy of CAR-T cells.

BACKGROUND: Chimeric antigen receptor (CAR) T cells have been successfully used in tumor immunotherapy due to their strong antitumor responses, especially in hematological malignancies such as B cell acute lymphoid leukemia. However, on-target off-tumor toxicity and poor persistence severely limit the clinical application of CAR-T cell therapy. METHODS: T-cell immunoglobulin mucin domain molecule 3 (TIM-3) was used to develop a second-generation 41BB CD19 CAR linked with a T3/28 chimera, in which truncated extracellular TIM-3 was fused with the CD28 transmembrane and cytoplasmic domains. The efficacy of T3/28 CAR-T cells was evaluated in vitro and in vivo. RESULTS: We demonstrated that the switch receptor T3/28 preserved the TCM phenotype, improved proliferative capacity, and reduced exhaustion of CAR-T cells, resulting in superior in vitro and in vivo antitumor activity in B lymphoma. Importantly, the switch receptor T3/28 substantially prolonged the persistence of CAR-T cells, and the interleukin-21/Stat3 axis probably contributed to the enhanced cytotoxicity of T3/28 CAR-T cells. CONCLUSION: Overall, the T3/28 chimera significantly prolonged the persistence of CAR-T cells, and T3/28 CAR-T cells possessed potent antitumor activity in mice, shedding new light on potential improvements in adoptive T cell therapies.

The orphan nuclear receptor NR4A3 controls the differentiation of monocyte-derived dendritic cells following microbial stimulation.

In response to microbial stimulation, monocytes can differentiate into macrophages or monocyte-derived dendritic cells (MoDCs) but the molecular requirements guiding these possible fates are poorly understood. In addition, the physiological importance of MoDCs in the host cellular and immune responses to microbes remains elusive. Here, we demonstrate that the nuclear orphan receptor NR4A3 is required for the proper differentiation of MoDCs but not for other types of DCs. Indeed, the generation of DC-SIGN+ MoDCs in response to LPS was severely impaired in Nr4a3-/- mice, which resulted in the inability to mount optimal CD8+ T cell responses to gram-negative bacteria. Transcriptomic analyses revealed that NR4A3 is required to skew monocyte differentiation toward MoDCs, at the expense of macrophages, and allows the acquisition of migratory characteristics required for MoDC function. Altogether, our data identify that the NR4A3 transcription factor is required to guide the fate of monocytes toward MoDCs.

NR4A1 and NR4A3 restrict HSC proliferation via reciprocal regulation of C/EBPα and inflammatory signaling.

Members of the NR4A subfamily of nuclear receptors have complex, overlapping roles during hematopoietic cell development and also function as tumor suppressors of hematologic malignancies. We previously identified NR4A1 and NR4A3 (NR4A1/3) as functionally redundant suppressors of acute myeloid leukemia (AML) development. However, their role in hematopoietic stem cell (HSC) homeostasis remains to be disclosed. Using a conditional Nr4a1/Nr4a3 knockout mouse (CDKO), we show that codepletion of NR4A1/3 promotes acute changes in HSC homeostasis including loss of HSC quiescence, accumulation of oxidative stress, and DNA damage while maintaining stem cell regenerative and differentiation capacity. Molecular profiling of CDKO HSCs revealed widespread upregulation of genetic programs governing cell cycle and inflammation and an aberrant activation of the interferon and NF-κB signaling pathways in the absence of stimuli. Mechanistically, we demonstrate that NR4A1/3 restrict HSC proliferation in part through activation of a C/EBPα-driven antiproliferative network by directly binding to a hematopoietic-specific Cebpa enhancer and activating Cebpa transcription. In addition, NR4A1/3 occupy the regulatory regions of NF-κB-regulated inflammatory cytokines, antagonizing the activation of NF-κB signaling. Taken together, our results reveal a novel coordinate control of HSC quiescence by NR4A1/3 through direct activation of C/EBPα and suppression of activation of NF-κB-driven proliferative inflammatory responses.

The transcription factor NR4A3 controls CD103+ dendritic cell migration.

The transcription factor NR4A3 (also known as NOR-1) is a member of the Nr4a family of nuclear receptors and is expressed in myeloid and lymphoid cells. Here, we have shown that Nr4a3 is essential for the migration of CD103+ dendritic cells (DCs) to lymph nodes (LNs). Nr4a3-deficient mice had very few CD103+ migratory DCs (mDCs) present in LNs, and mixed-chimera studies revealed that this migratory defect was cell intrinsic. We further found that CD103+ DCs from Nr4a3-deficient mice displayed a marked loss of surface expression of the chemokine CCR7. This defect in CCR7 expression was confined to CD103+ DCs, as CCR7 expression on T lymphocytes was unaffected. Moreover, CCR7 was not induced on CD103+ DCs from Nr4a3-deficient mice in response to either administration of the TLR7 agonist R848 or infection with Citrobacter rodentium in vivo. The transcription factor FOXO1 has been shown to regulate CCR7 expression. We found that FOXO1 protein was reduced in Nr4a3-deficient DCs through an AKT-dependent mechanism. Further, we found a requirement for NR4A3 in the maintenance of homeostatic mitochondrial function in CD103+ DCs, although this is likely independent of the NR4A3/FOXO1/CCR7 axis in the regulation of DC migration. Thus, NR4A3 plays an important role in the regulation of CD103+ mDCs by regulating CCR7-dependent cell migration.

Mycobacterium tuberculosis keto-mycolic acid and macrophage nuclear receptor TR4 modulate foamy biogenesis in granulomas: a case of a heterologous and noncanonical ligand-receptor pair.

The cell wall of Mycobacterium tuberculosis is configured of bioactive lipid classes that are essential for virulence and potentially involved in the formation of foamy macrophages (FMs) and granulomas. Our recent work established crosstalk between M. tuberculosis cell wall lipids and the host lipid-sensing nuclear receptor TR4. In this study, we have characterized, identified, and adopted a heterologous ligand keto-mycolic acid from among M. tuberculosis lipid repertoire for the host orphan NR TR4. Crosstalk between cell wall lipids and TR4 was analyzed by transactivation and promoter reporter assays. Mycolic acid (MA) was found to transactivate TR4 significantly compared with other cell wall lipids. Among the MA, the oxygenated form, keto-MA, was responsible for transactivation, and the identity was validated by TR4 binding assays followed by TLC and nuclear magnetic resonance. Isothermal titration calorimetry revealed that keto-MA binding to TR4 is energetically favorable. This keto-MA-TR4 axis seems to be essential to this oxygenated MA induction of FMs and granuloma formation as evaluated by in vitro and in vivo model of granuloma formation. TR4 binding with keto-MA features a unique association of host nuclear receptor with a bacterial lipid and adds to the presently known ligand repertoire beyond dietary lipids. Pharmacologic modulation of this heterologous axis may hold promise as an adjunct therapy to frontline tuberculosis drugs.

Dexamethasone counteracts the immunostimulatory effects of triiodothyronine (T3) on dendritic cells.

Glucocorticoids (GCs) are widely used as anti-inflammatory and immunosuppressive agents. Several studies have indicated the important role of dendritic cells (DCs), highly specialized antigen-presenting and immunomodulatory cells, in GC-mediated suppression of adaptive immune responses. Recently, we demonstrated that triiodothyronine (T3) has potent immunostimulatory effects on bone marrow-derived mouse DCs through a mechanism involving T3 binding to cytosolic thyroid hormone receptor (TR) ß1, rapid and sustained Akt activation and IL-12 production. Here we explored the impact of GCs on T3-mediated DC maturation and function and the intracellular events underlying these effects. Dexamethasone (Dex), a synthetic GC, potently inhibited T3-induced stimulation of DCs by preventing the augmented expression of maturation markers and the enhanced IL-12 secretion through mechanisms involving the GC receptor. These effects were accompanied by increased IL-10 levels following exposure of T3-conditioned DCs to Dex. Accordingly, Dex inhibited the immunostimulatory capacity of T3-matured DCs on naive T-cell proliferation and IFN-γ production while increased IL-10 synthesis by allogeneic T cell cultures. A mechanistic analysis revealed the ability of Dex to dampen T3 responses through modulation of Akt phosphorylation and cytoplasmic-nuclear shuttling of nuclear factor-κB (NF-κB). In addition, Dex decreased TRß1 expression in both immature and T3-maturated DCs through mechanisms involving the GC receptor. Thus GCs, which are increased during the resolution of inflammatory responses, counteract the immunostimulatory effects of T3 on DCs and their ability to polarize adaptive immune responses toward a T helper (Th)-1-type through mechanisms involving, at least in part, NF-κB- and TRß1-dependent pathways. Our data provide an alternative mechanism for the anti-inflammatory effects of GCs with critical implications in immunopathology at the cross-roads of the immune-endocrine circuits.

Interleukin-17-producing gammadelta T cells selectively expand in response to pathogen products and environmental signals.

Gammadelta T cells are an innate source of interleukin-17 (IL-17), preceding the development of the adaptive T helper 17 (Th17) cell response. Here we show that IL-17-producing T cell receptor gammadelta (TCRgammadelta) T cells share characteristic features with Th17 cells, such as expression of chemokine receptor 6 (CCR6), retinoid orphan receptor (RORgammat), aryl hydrocarbon receptor (AhR), and IL-23 receptor. AhR expression in gammadelta T cells was essential for the production of IL-22 but not for optimal IL-17 production. In contrast to Th17 cells, CCR6(+)IL-17-producing gammadelta T cells, but not other gammadelta T cells, express Toll-like receptors TLR1 and TLR2, as well as dectin-1, but not TLR4 and could directly interact with certain pathogens. This process was amplified by IL-23 and resulted in expansion, increased IL-17 production, and recruitment of neutrophils. Thus, innate receptor expression linked with IL-17 production characterizes TCRgammadelta T cells as an efficient first line of defense that can orchestrate an inflammatory response to pathogen-derived as well as environmental signals long before Th17 cells have sensed bacterial invasion.

T helper type 17-related cytokine expression is increased in the bronchial mucosa of stable chronic obstructive pulmonary disease patients.

There are increased numbers of activated T lymphocytes in the bronchial mucosa of stable chronic obstructive pulmonary disease (COPD) patients. T helper type 17 (Th17) cells release interleukin (IL)-17 as their effector cytokine under the control of IL-22 and IL-23. Furthermore, Th17 numbers are increased in some chronic inflammatory conditions. To investigate the expression of interleukin (IL)-17A, IL-17F, IL-21, IL-22 and IL-23 and of retinoic orphan receptor RORC2, a marker of Th17 cells, in bronchial biopsies from patients with stable COPD of different severity compared with age-matched control subjects. The expression of IL-17A, IL-17F, IL-21, IL-22, IL-23 and RORC2 was measured in the bronchial mucosa using immunohistochemistry and/or quantitative polymerase chain reaction. The number of IL-22(+) and IL-23(+) immunoreactive cells is increased in the bronchial epithelium of stable COPD compared with control groups. In addition, the number of IL-17A(+) and IL-22(+) immunoreactive cells is increased in the bronchial submucosa of stable COPD compared with control non-smokers. In all smokers, with and without disease, and in patients with COPD alone, the number of IL-22(+) cells correlated significantly with the number of both CD4(+) and CD8(+) cells in the bronchial mucosa. RORC2 mRNA expression in the bronchial mucosa was not significantly different between smokers with normal lung function and COPD. Further, we report that endothelial cells express high levels of IL-17A and IL-22. Increased expression of the Th17-related cytokines IL-17A, IL-22 and IL-23 in COPD patients may reflect their involvement, and that of specific IL-17-producing cells, in driving the chronic inflammation seen in COPD.

Defects along the T(H)17 differentiation pathway underlie genetically distinct forms of the hyper IgE syndrome.

BACKGROUND: The hyper IgE syndrome (HIES) is characterized by abscesses, eczema, recurrent infections, skeletal and connective tissue abnormalities, elevated serum IgE, and diminished inflammatory responses. It exists as autosomal-dominant and autosomal-recessive forms that manifest common and distinguishing clinical features. A majority of those with autosomal-dominant HIES have heterozygous mutations in signal transducer and activator of transcription (STAT)-3 and impaired T(H)17 differentiation. OBJECTIVE: To elucidate mechanisms underlying different forms of HIES. METHODS: A cohort of 25 Turkish children diagnosed with HIES were examined for STAT3 mutations by DNA sequencing. Activation of STAT3 by IL-6 and IL-21 and STAT1 by IFN-alpha was assessed by intracellular staining with anti-phospho (p)STAT3 and -pSTAT1 antibodies. T(H)17 and T(H)1 cell differentiation was assessed by measuring the production of IL-17 and IFN-gamma, respectively. RESULTS: Six subjects had STAT3 mutations affecting the DNA binding, Src homology 2, and transactivation domains, including 3 novel ones. Mutation-positive but not mutation-negative subjects with HIES exhibited reduced phosphorylation of STAT3 in response to cytokine stimulation, whereas pSTAT1 activation was unaffected. Both patient groups exhibited impaired T(H)17 responses, but whereas STAT3 mutations abrogated early steps in T(H)17 differentiation, the defects in patients with HIES with normal STAT3 affected more distal steps. CONCLUSION: In this cohort of Turkish children with HIES, a majority had normal STAT3, implicating other targets in disease pathogenesis. Impaired T(H)17 responses were evident irrespective of the STAT3 mutation status, indicating that different genetic forms of HIES share a common functional outcome.

Over-expression of forkhead box P3 and its association with receptor activator of nuclear factor-kappa B ligand, interleukin (IL) -17, IL-10 and transforming growth factor-beta during the progression of chronic periodontitis.

AIM: T regulatory (Treg) cells have been detected in periodontitis lesions, and forkhead box P3 (Foxp3) expression has been negatively correlated to receptor activator of nuclear factor-kappa B ligand (RANKL). The aim of this study was to correlate T-helper type 1 (Th1), Th2, Th17 and Treg transcription factor expressions, in gingival tissues from patients undergoing active periodontal tissue destruction, with bone loss-associated cytokines. MATERIALS AND METHODS: In 10 chronic periodontitis patients undergoing disease progression, the mRNA expressions of T-bet, GATA-3, Foxp3, RORC2, interleukin (IL)-1beta, IL-10, IL-17, RANKL, interferon (IFN)-gamma and transforming growth factor (TGF)-beta1 were quantified using real-time reverse transcription-polymerase chain reaction. The levels of these markers were compared between active and inactive periodontal lesions. RESULTS: In active periodontal lesions, Foxp3, T-bet, RANKL, IL-17, IL-1beta and IFN-gamma were significantly over-expressed compared with inactive lesions. The expression of IFN-gamma was the highest within the active periodontal lesions, similar to that of TGF-beta1 within the inactive ones. There was a positive correlation between RANKL and IL-17. Additionally, RANKL and IL-17 were positively correlated with RORC2, but no correlation was detected with Foxp3. CONCLUSIONS: These results lead us to speculate that Foxp3(+) cells that do not have a regulatory function might have a role in the pathogenesis of active periodontal lesions by down-regulating TGF-beta1 and IL-10 synthesis that lead to the over-expression of Th17-associated cytokines RANKL and IL-17.

Effects of leukotriene B4 and prostaglandin E2 on the differentiation of murine Foxp3+ T regulatory cells and Th17 cells.

Leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) are shown to be potent immunoregulatory lipid mediators. Here, we examined the effects of LTB4 and PGE2 on the differentiation of immunosuppressive CD4+CD25+Foxp3+ T regulatory cells (Treg) and pro-inflammatory IL-17-producing cells (Th17) from murine naïve CD4+ T cells. Using MACS-purified murine CD4+CD62L+ naïve T cells, we found that three days later in the presence of TGF-beta1, (28.65+/-6.83)% cells were converted into Treg cells, the mRNA expression of the key transcription factor Foxp3 peaked at 36h. Both LTB4 and PGE2 dose-dependently decreased the percentage of Treg cells and the mRNA expression of Foxp3. When the CD4+CD62L+ T cells were activated under Th17-promoting conditions in the presence of TGF-beta1 plus IL-6, three days later the production of IL-17 was markedly increased and the key transcription factor RORgammat mRNA peaked at 48h. LTB4 dose-dependently increased the secretion of IL-17 and the expression of RORgammat mRNA, whereas PGE2 decreased the secretion of IL-17 and the RORgammat mRNA expression. Our results suggest a distinct mode of immunoregulative action by PGE2 and LTB4, which may further our understanding of the role for lipid inflammatory mediators in the physiopathology of autoimmune diseases such as rheumatoid arthritis.

Identification of IL-17-producing FOXP3+ regulatory T cells in humans.

IL-17-producing CD4(+) T helper (Th17) cells have recently been defined as a unique subset of proinflammatory helper cells whose development depends on signaling initiated by IL-6 and TGF-beta, autocrine activity of IL-21, activation of STAT3, and induction of the orphan nuclear receptor RORgammat. The maintenance, expansion, and further differentiation of the committed Th17 cells depend on IL-1beta and IL-23. IL-17 was originally found produced by circulating human CD45RO(+) memory T cells. A recent study found that human Th17 memory cells selectively express high levels of CCR6. In this study, we report that human peripheral blood and lymphoid tissue contain a significant number of CD4(+)FOXP3(+) T cells that express CCR6 and have the capacity to produce IL-17 upon activation. These cells coexpress FOXP3 and RORgammat transcription factors. The CD4(+)FOXP3(+)CCR6(+) IL-17-producing cells strongly inhibit the proliferation of CD4(+) responder T cells. CD4(+)CD25(high)-derived T-cell clones express FOXP3, RORgammat, and IL-17 and maintain their suppressive function via a cell-cell contact mechanism. We further show that human CD4(+)FOXP3(+)CCR6(-) regulatory T (Treg) cells differentiate into IL-17 producer cells upon T-cell receptor stimulation in the presence of IL-1beta, IL-2, IL-21, IL-23, and human serum. This, together with the finding that human thymus does not contain IL-17-producing Treg cells, suggests that the IL-17(+)FOXP3(+) Treg cells are generated in the periphery. IL-17-producing Treg cells may play critical roles in antimicrobial defense, while controlling autoimmunity and inflammation.

Novel therapeutic targets along the Th17 pathway.

The recent discovery of IL-17-producing CD4(+) Th subset significantly revised the Th1/Th2 dichotomy model proposed by Mosmann and Coffman almost two decades ago. Th17 cells are involved in the pathogenesis of many human autoimmune diseases. Th17 cells, their developmental pathways and their effector functions, therefore, provide novel therapeutic targets.

Differentiation and functional analysis of human T(H)17 cells.

BACKGROUND: T(H)17 cells are of pathologic relevance in autoimmune disorders and presumably also in allergy and asthma. Regulatory T (Treg) cells, in contrast, suppress inflammatory and allergen-driven responses. Despite these disparate functions, both T-cell subsets have been shown to be dependent on TGF-beta for their development. OBJECTIVE: The aim of the study was to analyze the differentiation and function of human T(H)17 cells in comparison with other T(H) cell subsets. METHODS: Naive human CD4(+) T cells were differentiated in vitro, and gene expression was analyzed by means of quantitative real-time PCR, ELISA, and immunofluorescence. The function of T(H) cell subsets was assessed by monitoring the response of primary bronchial epithelial cells in coculture experiments. RESULTS: In vitro differentiated T(H)17 cells differ from Treg and other T(H) cells in their potency to induce IL-6 and IL-1beta expression in primary bronchial epithelial cells. TGF-beta, IL-1beta, IL-6, and IL-23 are necessary during T(H)17 cell differentiation to acquire these functions, including IL-17 production. In contrast, TGF-beta alone is necessary and sufficient to induce the transcription factor RORC2. This transcription factor, previously thought to be specific for T(H)17 cells, is also expressed in Treg cells, CD25(+) cells, cytotoxic T cells, and natural killer T cells. CONCLUSION: This study demonstrates mechanisms of differentiation to human T(H)17 cells, a subset that effectively and uniquely modulates the function of primary bronchial epithelial cells.

Human fetal lymphoid tissue-inducer cells are interleukin 17-producing precursors to RORC+ CD127+ natural killer-like cells.

The human body contains over 500 individual lymph nodes, yet the biology of their formation is poorly understood. Here we identify human lymphoid tissue-inducer cells (LTi cells) as lineage-negative RORC+ CD127+ cells with the functional ability to interact with mesenchymal cells through lymphotoxin and tumor necrosis factor. Human LTi cells were committed natural killer (NK) cell precursors that produced interleukin 17 (IL-17) and IL-22. In vitro, LTi cells gave rise to RORC+ CD127+ NK cells that retained the ability to produce IL-17 and IL-22. Postnatally, similar populations of LTi cell-like cells and RORC+ CD127+ NK cells were present in tonsils, and both secreted IL-17 and IL-22 but no interferon-gamma. Our data indicate that lymph node organogenesis is controlled by an NK cell precursor population with adaptive immune features and demonstrate a previously unappreciated link between the innate and adaptive immune systems.

Regulation and function of proinflammatory TH17 cells.

Naïve CD4(+) helper T (TH) cells, upon activation by antigen-presenting cells (APC), differentiate into different types of effector cells that are characterized by their distinct cytokine production profiles and immune regulatory functions. In addition to TH1 and TH2 cells, a third subset of effector TH cells has recently been described and termed TH17. Since their identification, TH17 cells have emerged as crucial players in infectious, inflammatory, and autoimmune diseases, and cancer. In this review, we summarize the latest discoveries on the cytokine-mediated regulation and transcriptional programming of TH17 cells and their roles in different immune responses and diseases.

Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense.

Natural killer (NK) cells are innate lymphocytes with spontaneous antitumor activity, and they produce interferon-gamma (IFN-gamma) that primes immune responses. Whereas T helper cell subsets differentiate from naive T cells via specific transcription factors, evidence for NK cell diversification is limited. In this report, we characterized intestinal lymphocytes expressing the NK cell natural cytotoxicity receptor NKp46. Gut NKp46+ cells were distinguished from classical NK cells by limited IFN-gamma production and absence of perforin, whereas several subsets expressed the nuclear hormone receptor retinoic acid receptor-related orphan receptor t (RORgammat) and interleukin-22 (IL-22). Intestinal NKp46+IL-22+ cells were generated via a local process that was conditioned by commensal bacteria and required RORgammat. Mice lacking IL-22-producing NKp46+ cells showed heightened susceptibility to the pathogen Citrobacter rodentium, consistent with a role for intestinal NKp46+ cells in immune protection. RORgammat-driven diversification of intestinal NKp46+ cells thereby specifies an innate cellular defense mechanism that operates at mucosal surfaces.

Interleukin 10 suppresses Th17 cytokines secreted by macrophages and T cells.

IL-17 and IL-22 are typical cytokines produced by the Th17 T cell subset, but it is unclear if Th17 cytokines can be produced by other cell types. We demonstrate that IL-10-deficient and IL-10R-deficient macrophages stimulated with lipopolysaccharide produce high levels of IL-17 and IL-22. Addition of exogenous IL-10 to IL-10-deficient macrophages abolished IL-17 production. When IL-10-deficient and IL-10R-deficient splenocytes were cultured under Th17 polarizing conditions, the population of IL-17-producing cells was increased and the cultures produced significantly higher levels of IL-17 and IL-22. The addition of recombinant IL-10 to IL-10-deficient splenocytes significantly decreased the percentage of IL-17-producing CD4(+) T cells. Finally, the mRNA for the Th17 transcription factor retinoic acid-related orphan receptor (ROR)gammat was significantly elevated in IL-10-deficient spleen cells and macrophages. These data demonstrate that Th17 cytokines and RORgammat are also expressed in macrophages and that IL-10 negatively regulates the expression of Th17 cytokines and RORgammat by both macrophages and T cells.

In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORgamma t+ T cells.

The nuclear hormone receptor retinoic acid receptor-related orphan receptor gamma t (RORgamma t) is required for the generation of T helper 17 cells expressing the proinflammatory cytokine interleukin (IL)-17. In vivo, however, less than half of RORgamma t(+) T cells express IL-17. We report here that RORgamma t(+) T alphabeta cells include Foxp3(+) cells that coexist with IL-17-producing RORgamma t(+) T alphabeta cells in all tissues examined. The Foxp3(+) RORgamma t(+) T alphabeta express IL-10 and CCL20, and function as regulatory T cells. Furthermore, the ratio of Foxp3(+) to IL-17-producing RORgamma t(+) T alphabeta cells remains remarkably constant in mice enduring infection and inflammation. This equilibrium is tuned in favor of IL-10 production by Foxp3 and CCL20, and in favor of IL-17 production by IL-6 and IL-23. In the lung and skin, the largest population of RORgamma t(+) T cells express the gammadelta T cell receptor and produce the highest levels of IL-17 independently of IL-6. Thus, potentially antagonistic proinflammatory IL-17-producing and regulatory Foxp3(+) RORgamma t(+) T cells coexist and are tightly controlled, suggesting that a perturbed equilibrium in RORgamma t(+) T cells might lead to decreased immunoreactivity or, in contrast, to pathological inflammation.

Cutting edge: NKT cells constitutively express IL-23 receptor and RORgammat and rapidly produce IL-17 upon receptor ligation in an IL-6-independent fashion.

Th17 cells require IL-6 and TGFbeta for lineage commitment and IL-23 for maintenance. Unexpectedly, naive IL-6(-/-) splenocytes stimulated with anti-CD3 and IL-23 produced normal amounts of IL-17 during the first 24 h of culture. These rapid IL-6-independent IL-17 producers were identified as predominantly DX5(+) TCRbeta(+) NKT cells, and a comparable response could be found using the invariant NKT-specific ligand alpha-galactosylceramide. Human NKT cells also produced IL-17. NKT cells constitutively expressed IL-23R and RORgammat. Ligation of either TCR or IL-23R triggered IL-17 production and both together had a synergistic effect, suggesting independent but convergent pathways. IL-17 production was not restricted to a particular subset of NKT cells but they were NK1.1 negative. Importantly, in vivo administration of alpha-galactosylceramide triggered a rapid IL-17 response in the spleen. These data suggest an important biological role for innate IL-17 production by NKT cells that is rapid and precedes the adaptive IL-17 response.