Generation and sustained expansion of mouse spleen invariant NKT cell lines with preserved cytokine releasing capacity

Invariant Natural Killer T (iNKT) cells are CD1d restricted innate lymphoid cells with an invariant T cell receptor (TCR) alpha chain gene rearrangement (Valpha24-Jalpha18 in human and Valpha14-Jalpha18 in mouse). iNKT cells play a pivotal role in anti-tumor immune responses via cytokine mediated transactivation of various cells which mediate innate and adaptive immune responses. Here we describe, to our knowledge for the first time, the generation of long-term mouse spleen derived iNKT cell lines. We found that dendritic cells (DC) derived from the D1 line, but not Mf4/4 macrophages, loaded with the artificial iNKT cell ligand alpha-Galactosylceramide (alphaGalCer) could be employed to expand iNKT cells in vitro. Furthermore, exogenously added IL-7, but not IL-2 or IL-15 had a pronounced additive effect on iNKT cell expansion. Using this method up to 10(8) iNKT cells could be obtained from one spleen within 12 to 14 weeks, and cell lines could be continued for up to 24 months. Importantly, the iNKT cell lines had retained the capacity to swiftly secrete substantial amounts of both T helper (Th) 1 and Th2 cytokines upon activation. In conclusion we have generated iNKT cell lines with high yields that can be maintained for up to 24 months, by repeated stimulation using alpha-GalCer loaded D1.DC and IL-7. These in vitro expanded iNKT cells preserved the capacity to swiftly produce both Th1 and Th2 type cytokines and are currently being utilized in pre-clinical adoptive transfer models to identify and optimize the characteristics of therapeutically effective iNKT cells in an anti-tumor setting.     

In vitro expanded human invariant natural killer T-cells promote functional activity of natural killer cells

Invariant natural killer T (iNKT) cells play a pivotal role in cancer immunity through trans-activation of effector cells via swift cytokine secretion. In mice, iNKT cell activation by alpha-galactosylceramide (alpha-GC) induces potent NK cell-mediated anti-tumour effects. Here we investigated whether human iNKT cells could enhance NK cell functional activity in vitro. iNKT cell activation by alpha-GC treatment of peripheral blood mononuclear cells (PBMC) was not sufficient to enhance NK cell effector functions. However, addition of in vitro expanded iNKT cells to PBMC enhanced NK cell-mediated cytotoxicity in an alpha-GC-dependent manner. NK cell activation by iNKT cells was primarily mediated by soluble factors, and could be enhanced by the NK cell activating cytokine IL-21. These results suggest that adoptive transfer of ex vivo expanded iNKT cells will enhance NK cell function and is expected to enhance the efficacy of cancer immunotherapy, particularly in combination with IL-21 and alpha-GC.     

The role of natural killer T cells in lung inflammation

Invariant NK T cells (iNKT) bridge the innate and adaptive immune response, being characterized by the ability to use invariant T cell receptors to recognize glycolipid antigens presented by CD1d, leading to an explosive cytokine effector response. As such it has been proposed that iNKT cells perform important roles as both effector and regulatory cells in a wide range of disease settings. These roles have been characterized in experiments depending on the use of iNKT-null mice, due to lack of either CD1d expression or Jalpha18 and the use of CD1d tetramers loaded with the model glycolipid antigen, alpha-galactosylceramide (alphaGalCer). Several studies have examined lung disease, infectious and allergic, in humans and mice. While the lung itself does not carry an exceptionally large population of iNKT cells (compared with, say, the liver), it does appear to be a site at which these cells can exert a profound effect. Several models of bacterial, fungal and viral murine lung infection have been investigated that have sometimes produced conflicting results. Abrogation of iNKT cell function in knockouts is often associated with disease exacerbation, indicating a regulatory role in lung infection. Studies in murine asthma models and in patients have similarly probed the role of iNKT cells in these settings. While there are again somewhat contradictory findings, evidence suggests a likely role for iNKT cells in mediating airway hyper-responsiveness (AHR), but probably not in Th2 polarization or lung eosinophilia. In marginally different models, administration of alphaGalCer has either ameliorated or exacerbated AHR. Different studies of BAL from human asthma patients show variously that there is either a very enlarged population of iNKT cells in the asthmatic lung, or that there is no significant difference from controls. Taken together, there are some observations that argue compellingly for an important role of iNKT cells in the lung, but resolution of some of the contradictory findings may await the development of reagents capable of providing alternative readouts of iNKT activation in these diverse disease settings.     

Ex vivo expanded human CD4+ regulatory NKT cells suppress expansion of tumor antigen-specific CTLs

NKT cells can produce large amounts of both Th1- and Th2-type cytokines and are an important regulatory cell type. To elucidate their role in acquired immunity, we examined the effect of human Valpha24+Vbeta11+ NKT cells or CD1d-specific ligand alpha-galactosylceramide (alphaGalCer) on the in vitro generation of antigen-specific CTLs from PBMCs using autologous MART-1(26-35) peptide-pulsed dendritic cells as stimulators. Flow cytometry using tetramer for MART-1(26-35) peptide revealed that NKT cells have inhibitory effects on CTL generation. Cytokine analysis using cytometric bead array assay and ELISA showed higher IL-4 and IL-10 secretion in the alphaGalCer(+) and/or NKT cell(+) culture setting, whereas IL-13 secretion in the culture was not affected by the presence of alphaGalCer. The CD4+ NKT cell subset seemed to play a major role in this inhibitory effect by secreting large amounts of Th2-type cytokines. Interestingly however, unlike recent reports utilizing mouse models, IL-13 was not a main effector molecule in our human system. Culture with alphaGalCer in the presence of cytokine-neutralizing antibodies for the Th2 cytokines, IL-4, IL-5 and IL-10, resulted in enhanced CTL generation, suggesting the dominant role of Th2 cytokines over Th1 cytokines. Thus, CD4+ NKT cells can work as immunoregulatory T cells that suppress anti-tumor immune response and, therefore, NKT cells or alphaGalCer could be used as therapeutic modalities to modulate systemic immune responses, such as autoimmune diseases. Conversely, the use of NKT cells along with anti-Th2 cytokine-neutralizing antibodies or CD4-negative NKT cell subset could enhance the generation of antigen-specific CTLs for adoptive immunotherapy.     

Activation of invariant natural killer T cells in regional lymph nodes as new antigen‐specific immunotherapy via induction of interleukin‐21 and interferon‐γ

Invariant natural killer T (iNKT) cells play important immunoregulatory functions in allergen‐induced airway hyperresponsiveness and inflammation. To clarify the role of iNKT cells in allergic rhinitis (AR), we generated bone marrow‐derived dendritic cells (BMDCs), which were pulsed by ovalbumin (OVA) and α‐galactosylceramide (OVA/α‐GalCer‐BMDCs) and administered into the oral submucosa of OVA‐sensitized mice before nasal challenge. Nasal symptoms, level of OVA‐specific immunoglobulin (IgE), and T helper type 2 (Th2) cytokine production in cervical lymph nodes (CLNs) were significantly ameliorated in wild‐type (WT) mice treated with OVA/α‐GalCer‐BMDCs, but not in WT mice treated with OVA‐BMDCs. These anti‐allergic effects were not observed in Jα18^–/– recipients that lack iNKT cells, even after similar treatment with OVA/α‐GalCer‐BMDCs in an adoptive transfer study with CD4⁺ T cells and B cells from OVA‐sensitized WT mice. In WT recipients of OVA/α‐GalCer‐BMDCs, the number of interleukin (IL)‐21‐producing iNKT cells increased significantly and the Th1/Th2 balance shifted towards the Th1 dominant state. Treatment with anti‐IL‐21 and anti‐interferon (IFN)‐γ antibodies abrogated these anti‐allergic effects in mice treated with α‐GalCer/OVA‐BMDCs. These results suggest that activation of iNKT cells in regional lymph nodes induces anti‐allergic effects through production of IL‐21 or IFN‐γ, and that these effects are enhanced by simultaneous stimulation with antigen. Thus, iNKT cells might be a useful target in development of new treatment strategies for AR.     

Rapid and reliable generation of invariant natural killer T-cell lines in vitro

Several tools have proved useful in the study of invariant natural killer T (iNKT) cells, including CD1d-deficient mice, Jα281-deficient mice, synthetic lipid antigens and antigen-loaded CD1d tetramers. However, the generation and examination of long-term primary murine iNKT cell lines in vitro has been challenging. Here, we show the rapid generation of iNKT cell lines from splenic iNKT cells of Vα14 T-cell receptor (TCR) transgenic (Tg) mice. These purified iNKT cells were stimulated by bone marrow-derived dendritic cells (BMDCs) loaded with α-galactosylceramide (αGalCer) and cultured with interleukin (IL)-2 and IL-7. iNKT cells proliferated dramatically, and the cell number exhibited a 100-fold increase within 2 weeks and a 10⁵-fold increase in 8 weeks after repeated stimulation with αGalCer. The iNKT cell lines consisted of iNKT cells expressing Vβ chains including Vβ8.1/8.2, Vβ14, Vβ10, Vβ6 and Vβ7, and responded to stimulation with αGalCer presented both by BMDCs and by plate-bound CD1d. In addition, the iNKT cell lines produced interferon (IFN)-γ when activated by lipopolysaccharide (LPS) or CpG oligodeoxynucleotide (ODN)-stimulated BMDCs. Further, we show that iNKT cell lines produced cytokines in response to microbial antigens. In summary, high-yield iNKT cell lines were generated very rapidly and robustly expanded, and these iNKT cells responded to both TCR and cytokine stimulation in vitro. Given the desire to study primary iNKT cells for many purposes, these iNKT cell lines should provide an important tool for the study of iNKT cell subsets, antigen and TCR specificity, activation, inactivation and effector functions.     

Co-inhibitory roles for glucocorticoid-induced TNF receptor in CD1d-dependent natural killer T cells

Invariant natural killer T (iNKT) cells are a special subset of alphabeta T cells with invariant TCR, which recognize alpha-galactosylceramide (alpha-GalCer) presented by CD1d. In addition to signals through the invariant TCR upon stimulation with alpha-GalCer, costimulatory signals, such as signals through CD28 and OX40, are indispensable for full activation of iNKT cells. In this study, we investigated the functions of a well-known costimulatory molecule, glucocorticoid-induced TNF receptor (GITR), on Ag-induced iNKT cell activation. Unexpectedly, engagement of GITR by agonistic mAb DTA-1 suppressed proliferation and cytokine production of iNKT cells upon alpha-GalCer stimulation. In addition, GITR signals in iNKT cells during only the Ag-priming phase was sufficient to inhibit the iNKT cell activation. Consistent with these results, the GITR-deficient iNKT cells showed enhanced proliferation and increased cytokine production upon alpha-GalCer stimulation both in vitro and in vivo. Furthermore, the in vivo administration of alpha-GalCer suppressed tumor metastasis more efficiently in GITR-deficient mice than in wild-type mice. Collectively, GITR plays a co-inhibitory role in Ag-induced iNKT cell activation.     

Genetic proof for the transient nature of the Th17 phenotype

IL-17-producing CD4(+) T cells (Th17) have been classified as a new T helper cell subset. Using an IL-17 fate mapping mouse strain, which genetically fixes the memory of IL-17 expression, we demonstrate that IL-17A/F-expressing T helper cells generated either in vitro or in vivo are not a stable T-cell subset. Upon adoptive transfer of IL-17F-reporter-positive Th17 cells to RAG-deficient or WT animals, encephalitogenic Th17 cells partially lose IL-17 expression and upregulate IFN-γ. Additionally, we show that Th1 cells can convert in vivo to IL-17A/IFN-γ-coexpressing cells in the mesenteric lymph nodes (mLN). Our data classify IL-17A and IL-17F as cytokines produced transiently in response to the local microenvironment, thus showing that IL-17 expression does not define an end-stage T helper cell subset.     

Functional Invariant NKT Cells in Pig Lungs Regulate the Airway Hyperreactivity: A Potential Animal Model

Important roles played by invariant natural killer T (iNKT) cells in asthma pathogenesis have been demonstrated. We identified functional iNKT cells and CD1d molecules in pig lungs. Pig iNKT cells cultured in the presence of α-GalCer proliferated and secreted Th1 and Th2 cytokines. Like in other animal models, direct activation of pig lung iNKT cells using α-GalCer resulted in acute airway hyperreactivity (AHR). Clinically, acute AHR-induced pigs had increased respiratory rate, enhanced mucus secretion in the airways, fever, etc. In addition, we observed petechial hemorrhages, infiltration of CD4⁺ cells, and increased Th2 cytokines in AHR-induced pig lungs. Ex vivo proliferated iNKT cells of asthma induced pigs in the presence of C-glycoside analogs of α-GalCer had predominant Th2 phenotype and secreted more of Th2 cytokine, IL-4. Thus, baby pigs may serve as a useful animal model to study iNKT cell-mediated AHR caused by various environmental and microbial CD1d-specific glycolipid antigens.     

Innate immunity modulates autoimmunity: type 1 interferon-beta treatment in multiple sclerosis promotes growth and function of regulatory invariant natural killer T cells through dendritic cell maturation

Type 1 interferon-beta (T1IFN-beta) is an innate cytokine and the first-choice therapy for multiple sclerosis (MS). It is still unclear how T1IFN-beta, whose main function is to promote innate immunity during infections, plays a beneficial role in autoimmune disease. Here we show that T1IFN-beta promoted the expansion and function of invariant natural killer (iNKT) cells, an innate T-cell subset with strong immune regulatory properties that is able to prevent autoimmune disease in pre-clinical models of MS and type 1 diabetes. Specifically, we observed that T1IFN-beta treatment significantly increased the percentages of Valpha24(+) NKT cells in peripheral blood mononuclear cells of MS patients. Furthermore, iNKT cells of T1IFN-beta-treated individuals showed a dramatically improved secretion of cytokines (interleukins 4 and 5 and interferon-gamma) in response to antigenic stimulation compared to iNKT cells isolated from the same patients before T1IFN-beta treatment. The effect of T1IFN-beta on iNKT cells was mediated through the modulation of myeloid dendritic cells (DCs). In fact, DCs modulated in vivo or in vitro by T1IFN-beta were more efficient antigen-presenting cells for iNKT cells. Such a modulatory effect of T1IFN-beta was associated with up-regulation on DCs of key costimulatory molecules for iNKT (i.e. CD80, CD40 and CD1d). Our data identified the iNKT cell/DC pathway as a new target for the immune regulatory effect of T1IFNs in autoimmune diseases and provide a possible mechanism to explain the clinical efficacy of T1IFN-beta in MS.     

Involvement of CD4(+) Th1 cells in systemic immunity protective against primary and secondary challenges with Trypanosoma cruzi

In general, gamma interferon (IFN-gamma)-producing CD4(+) Th1 cells are important for the immunological control of intracellular pathogens. We previously demonstrated an association between parasite-specific induction of IFN-gamma responses and resistance to the intracellular protozoan Trypanosoma cruzi. To investigate a potential causal relationship between Th1 responses and T. cruzi resistance, we studied the ability of Th1 cells to protect susceptible BALB/c mice against virulent parasite challenges. We developed immunization protocols capable of inducing polarized Th1 and Th2 responses in vivo. Induction of parasite-specific Th1 responses, but not Th2 responses, protected BALB/c mice against virulent T. cruzi challenges. We generated T. cruzi-specific CD4(+) Th1 and Th2 cell lines from BALB/c mice that were activated by infected macrophages to produce their corresponding cytokine response profiles. Th1 cells, but not Th2 cells, induced nitric oxide production and inhibited intracellular parasite replication in T. cruzi-infected macrophages. Despite the ability to inhibit parasite replication in vitro, Th1 cells alone could not adoptively transfer protection against T. cruzi to SCID mice. In addition, despite the fact that the adoptive transfer of CD4(+) T lymphocytes was shown to be necessary for the development of immunity protective against primary T. cruzi infection in our SCID mouse model, protective secondary effector functions could be transferred to SCID mice from memory-immune BALB/c mice in the absence of CD4(+) T lymphocytes. These results indicate that, although CD4(+) Th1 cells can directly inhibit intracellular parasite replication, a more important role for these cells in T. cruzi systemic immunity may be to provide helper activity for the development of other effector functions protective in vivo.     

Alteration of the relative levels of iNKT cell subsets is associated with chronic mycobacterial infections

CD1d-restricted invariant natural killer T cells (iNKT cells) have been identified as an important type of effector and regulatory T cell, but their roles in the chronic infectious diseases caused by Mycobacterium tuberculosis and Mycobacterium leprae remain poorly defined. Here, we studied circulating human iNKT cells in blood samples from tuberculosis (TB) and leprosy patients. We found that the percentages of iNKT cells among total circulating T cells in TB and leprosy patients were not significantly different from those in normal controls. However, both TB and leprosy patients showed a selective reduction of the proinflammatory CD4(-)CD8beta(-) (DN) iNKT cells with a proportionate increase in the CD4(+) iNKT cells. Similar phenotypic alterations in circulating iNKT cells were observed in a mouse model of M. tuberculosis infection. Taken together, these findings indicate that the selective reduction of circulating DN iNKT cells is associated with chronic infections caused by M. tuberculosis and M. leprae.     

A natural protective function of invariant NKT cells in a mouse model of innate-cell-driven lung inflammation

Activation of invariant natural killer T (iNKT) cells by treatment with their α-galactosyl ceramide ligand provides therapeutic benefits in several immune inflammatory settings. Given the artificial nature of this stimulation, the natural regulatory functions of iNKT remain uncertain. Addressing this issue in a mouse model of innate-cell-driven lung inflammation induced by the cytokine/alarmin IL-33 that targets iNKT cells, we found that eosinophil and neutrophil recruitment was markedly increased in treated iNKT cell-deficient (Jα18 KO) mice, as was the local production of eotaxin and keratinocyte chemoattractant chemokines. By contrast, lung inflammation decreased after adoptive transfer of iNKT cells, which restored the WT inflammatory response in Jα18 KO mice. Finally, we established that this natural anti-inflammatory function of iNKT cells depends on their IFN-γ production and on endogenous IL-12. Our study provides the first evidence of a protective role of iNKT cells during lung inflammation that does not require pharmacological TCR engagement.     

Alpha-galactosylceramide-induced iNKT cells suppress experimental allergic asthma in sensitized mice: role of IFN-gamma

Allergic asthma is a multifaceted syndrome consisting of eosinophil-rich airway inflammation, bronchospasm, and airway hyper-responsiveness (AHR). Using a mouse model of allergic asthma, we previously reported that invariant NKT (iNKT) cells increase the severity of this disease. Herein, we demonstrate that a single i.v. injection of alpha-galactosylceramide (alpha-GalCer), 1 h before the first airway allergen challenge of OVA-sensitized mice, abrogates elicitation of AHR, airway eosinophilia, IL-4 and IL-5 production in bronchoalveolar lavage fluid, and specific anti-OVA IgE antibodies. Further, alpha-GalCer administered intranasally also strongly inhibited the major symptoms of asthma in sensitized and challenged mice. Alpha-GalCer treatment induces iNKT cell accumulation in the lungs, and shifts their cytokine profile from pro-asthmatic IL-4 to a protective IFN-gamma production. The role of IFN-gamma from iNKT cells in protection was shown by adoptive transfer of sorted iNKT cells from OVA-sensitized and alpha-GalCer-treated mice which protected immunized recipients from manifesting asthma by an IFN-gamma-dependent pathway. Our findings demonstrate for the first time that alpha-GalCer administered locally inhibits asthma symptoms, even in predisposed asthmatic mice, through an iNKT cell- and IFN-gamma-dependent pathway.     

Altered Invariant Natural Killer T cell Subsets and its Functions in Patients with Oral Squamous Cell Carcinoma

Invariant natural killer T (iNKT) cells are glycolipid‐reactive T lymphocytes that share receptors and function with natural killer (NK) cells and reportedly play a pivotal role in various immune responses. However, iNKT cells are not well characterized in patients with oral squamous cell carcinoma (OSCC). We investigated the populations and functions of circulating iNKT (CD3⁺6B11⁺) cells from thirty‐eight patients with OSCC and twenty‐eight healthy donors by flow cytometry. Circulating iNKT cells were significantly lower (P < 0.01) in patients as compared to those in healthy controls. Further, iNKT subsets revealed a marked decrease in CD4^?CD8^? (double negative, DN) subset with concomitant increase in CD8⁺ subset in patients as compared to healthy controls (P = 0.03 and P < 0.01, respectively), whereas CD4⁺ subset was similarly distributed in both groups. The functional analysis demonstrated that residual iNKT cells from patients had impaired proliferative response to α‐galactosylceramide (α‐GalCer)‐pulsed dendritic cells (DCs) and Th2‐like cytokine profile. However, in vitro activation with α‐GalCer‐pulsed DCs restores IFN‐γ expression and enhances antitumour activity to human cancer cells lines (SCC‐4, KB and MCF7). It appears that the selectively enriched iNKT subsets and modulation of their function by specific ligand/agonist may be useful for cellular therapy in patients with OSCC. Further, reduced levels of iNKT cells and its DN subset may be used as potential prognostic factors for patients with OSCC.     

NOD mice contain an elevated frequency of iNKT17 cells that exacerbate diabetes

Invariant natural killer T (iNKT) cells are a distinct lineage of innate-like T lymphocytes and converging studies in mouse models have demonstrated the protective role of iNKT cells in the development of type 1 diabetes. Recently, a new subset of iNKT cells, producing high levels of the pro-inflammatory cytokine IL-17, has been identified (iNKT17 cells). Since this cytokine has been implicated in several autoimmune diseases, we have analyzed iNKT17 cell frequency, absolute number and phenotypes in the pancreas and lymphoid organs in non-obese diabetic (NOD) mice. The role of iNKT17 cells in the development of diabetes was investigated using transfer experiments. NOD mice exhibit a higher frequency and absolute number of iNKT17 cells in the lymphoid organs as compared with C57BL/6 mice. iNKT17 cells infiltrate the pancreas of NOD mice where they express IL-17 mRNA. Contrary to the protective role of CD4(+) iNKT cells, the CD4(-) iNKT cell population, which contains iNKT17 cells, enhances the incidence of diabetes. Treatment with a blocking anti-IL-17 antibody prevents the exacerbation of the disease. This study reveals that different iNKT cell subsets play distinct roles in the regulation of type 1 diabetes and iNKT17 cells, which are abundant in NOD mice, exacerbate diabetes development.     

Functions of C2D macrophage cells after adoptive transfer

Macrophage function depends on their in situ location. To test this hypothesis, we examined functional changes of the C2D macrophage cell line after adoptive transfer. In vitro, C2D macrophages reside early in the macrophage lineage and show little functional activity. After in vivo i.p. culture, C2D macrophage cells switch their cytokine/chemokine profile from primarily Th2 cytokines produced in vitro to a Th1 profile including MIP-1alpha, IL-6, and TNF-alpha. The in vivo environment also caused C2D macrophage cells to become more phagocytic than their in vitro counterparts. These data indicate that C2D macrophage cells exhibit distinct functions because of in vivo signals that are absent during in vitro culture.     

Flagellin Modulates the Function of Invariant NKT Cells From Patients With Asthma via Dendritic Cells

PurposeInvariant natural killer T (iNKT) cells play a critical role in the pathogenesis of asthma. We previously reported the association between circulating Th2-like iNKT cells and lung function in asthma patients and the suppressive effect of Toll-like receptor 5 ligand flagellin B (FlaB) on asthmatic in a mouse model. Thus, we investigated whether FlaB modulates the function of circulating iNKT cells in asthmatic patients.MethodsPeripheral blood mononuclear cells (PBMCs) were treated with FlaB, and the secreted and intracellular cytokines of iNKT cells were evaluated by using ELISA and flow cytometry, respectively, following stimulation with α-galactosylceramide. Foxp3⁺ iNKT cells were also measured. To determine the effect of FlaB-treated dendritic cells (DCs) on iNKT cells, we co-cultured CD14⁺ monocyte-derived DCs and T cells from patients with house dust mite-sensitive asthma and analyzed intracellular cytokines in iNKT cells.ResultsA reduction of IL-4 and IL-17 production by iNKT cells in PBMCs after FlaB treatment was alleviated following blocking of IL-10 signaling. A decrease in the frequencies of IL-4⁺ and IL-17⁺ iNKT cells by FlaB-treated DCs was reversed after blocking of IL-10 signaling. Simultaneously, an increase in Foxp3⁺ iNKT cells induced by FlaB treatment disappeared after blocking of IL-10.ConclusionsFlaB may inhibit Th2- and Th17-like iNKT cells and induce Foxp3⁺ iNKT cells by DCs via an IL-10-dependent mechanism in asthmatic patients. In patients with a specific asthma phenotype associated with iNKT cells, FlaB may be an effective immunomodulator for iNKT cell-targeted immunotherapy.     

Abnormalities in iNKT cells are associated with impaired ability of monocytes to produce IL‐10 and suppress T‐cell proliferation in sarcoidosis

Sarcoidosis is a multisystem granulomatous disorder characterized by marked T‐cell expansion of T helper 1 (Th1) cells. The cause of T‐cell overactivity is unknown. We hypothesized that interleukin‐10 (IL‐10) production by a yet undefined cell type might be defective, resulting in loss of regulation of T‐cell activity. Focusing on IL‐10‐producing monocytes, we first showed that monocytes isolated from the peripheral blood of corticosteroid‐naïve sarcoidosis patients (n = 51) produced less IL‐10 compared to controls, and were less able to suppress T‐cell proliferation. In addition, monocytic IL‐10 production correlated negatively with disease activity score. As invariant natural killer T (iNKT) cells are known to both interact with monocytes and be reduced in sarcoidosis patients, we then asked whether iNKT‐specific defects might be responsible for this reduced IL‐10 production. We found that greater numbers of circulating iNKT cells was associated with higher IL‐10 production. Moreover, iNKT cells enhanced monocytic IL‐10 production in vitro. Defective IL‐10 production and T‐cell suppression by sarcoidosis monocytes could be restored following their coculture with iNKT cells, in a CD1d‐ and cell contact‐dependent process. We suggest that reduced iNKT‐cell numbers in sarcoidosis may lead to impaired monocytic IL‐10 production and unchecked T‐cell expansion in sarcoidosis. These findings provide fresh insight into the mechanism of sarcoidosis disease, and interaction between iNKT cells and monocytes.