Peripheral NKT Cells Research Articles

Deciphering the circulating immunological landscape of thoracic aortic aneurysm: Insights from a two-sample Mendelian randomization study

BackgroundThoracic Aortic Aneurysm (TAA) poses significant health risks due to aortic dilation. Recent evidence suggests a pivotal role for the immune-inflammatory response in the mechanism of aortic aneurysm formation. In this study, we aim to investigate the causal relationship between circulating immune cells and TAA. MethodsThis study employs a two-sample Mendelian Randomization (MR) approach, utilizing genome-wide association study (GWAS) summary statistics for 731 immune cell types and two TAA data from large-scale studies. Causal effects of both peripheral immune cells on TAA and TAA on peripheral immune cells are explored. To ensure more accurate results, we intersected the findings from two TAA data from large-scale studies, excluding results where the direction of the odds ratio (OR) was inconsistent. FindingsThe study identifies specific immune cells associated with TAA. Notably, CD45+ NKT cell (OR: 0.95, 95CI%: 0.90–0.99 in FinnGen study; OR: 0.91, 95CI%: 0.84–0.99 in CHIP + MGI study) and CD45+ HLA-DR + CD8+ T cells (OR: 0.95, 95CI%: 0.90–0.99 in FinnGen study; OR: 0.90, 95CI%: 0.82–0.99 in CHIP + MGI study) demonstrate a protective role against TAA. In addition, CD28+ CD45RA- CD8+ T cells (relative cell counts and absolute cell counts) and HVEM + CM + CD8+ T cells are adversely affected by TAA. InterpretationThe findings indicate that the potential protective influence exerted by specific subsets of peripheral NKT cells and CD8+ T cells in mitigating the development of TAA, while simultaneously highlighting the reciprocal effects of TAA on peripheral Treg cells subsets and T cell subsets. The complex interaction between immune cells and TAA could provide valuable clues for earlier detection and more efficacious treatment strategies for TAA.

Peripheral Blood Lymphocyte Subsets in Factor VIII Inhibitor-Positive Patients with Severe Hemophilia A: A Case-Control Study.

The present study aimed to investigate different peripheral lymphocyte subsets in patients with severe hemophilia A (HA) and factor VIII (FVIII) inhibitor production. For this, age-matched cases of 19 FVIII inhibitor-positive (IP), 21 FVIII inhibitor-negative (IN) and 45 healthy controls were selected for study. Flow cytometry was used to analyze the peripheral lymphocyte subsets, including T, B, natural killer (NK) and NKT cells. The T cell subsets included CD3 + CD4-CD8- [double negative T (DNT)], CD3 + CD4 + CD8+ [double-positive T (DPT)], CD3 + CD4 + CD8- and CD3 + CD4-CD8+ T cells. Pairwise comparisons of absolute lymphocyte subset values were conducted among the three groups. The cut-off value for absolute lymphocyte counts was determined using receiver operating characteristic curve analysis. The results demonstrated that the absolute values of DPT cells in the IN and IP groups were significantly lower than those in the healthy control group (P = 0.007). The DNT values were also lower in severe HA patients with or without inhibitor than those in healthy subjects, but these differences were not statistically significant (P = 0.053). In addition, the absolute value of CD4+ Th cells in the IP group was lower than that in the healthy controls (P = 0.013). Although not statistically significant (P = 0.064), the absolute values of NKT cells were higher in the IN group compared with the IP group, and higher in the IP group compared with the healthy control group. There were no statistically significant differences in total T, B, CD8 + and NK cells among the IN, IP and healthy control groups. The cut-off value for absolute CD4+ Th cells in the IN group was < 598/µl. The decrease in absolute values of CD4+ Th cells in severe HA patients may contribute to the establishment of infused FVIII immune tolerance. If the CD4+ Th value remains > 598/µl, clinicians should be vigilant for possible FVIII inhibitor production, especially on days prior to FVIII exposure.

Disseminated purulent peritonitis outcome affects NKT cell phenotype

The aim of our study was to investigate the main characteristics of peripheral blood NKT cell phenotype in patients with disseminated purulent peritonitis (DPP) in dynamics of postoperative period, depending on the disease outcome. Fifty-two patients with acute surgical diseases and injuries of the abdominal organs complicated by DPP, and 68 healthy individuals in control group, were examined. Blood sampling was performed before surgery (preoperative period), as well as on the day 7, 14 and 21 of postoperative period. All patients with DPP were divided into two groups depending on disease outcome in postoperative period: patients with favorable disease outcome (n = 34); and patients with unfavorable outcome (n = 18). Study of the phenotype of blood NKT lymphocytes was performed by flow cytometry using direct immunofluorescence of whole peripheral blood samples with monoclonal antibodies. The low relative and absolute level of NKT cells was observed in DPP patients regardless of outcome disease in preoperative period. At the same time, the absolute level of NKT cells returned to normal only in patients with favorable DPP outcome and only by day 21 after surgery. Patients with favorable DPP outcome by the end of examination period had normalized quantity of mature NKT-lymphocytes and significantly decreased level of cytotoxic cells which was apparently associated with migration of such cell subsets to site of inflammation. A reduced level of non-classical (expressing CD8 marker) mature and cytokine-producing NKT cells was detected only in patients with favorable DPP outcome in preoperative period which returned to normal by the end of postoperative period. At the same time, patients with unfavorable disease outcome had reduced quantity of NKT cells of these subsets by day 21 of postoperative treatment. Patients with favorable outcome had high level of mature and cytotoxic CD11b+ NKT cells already in the preoperative period, while patients with unfavorable DPP outcome had increased level of cytotoxic CD11b+ NKT cells only by day 21 after surgery. The proportion of NKT cells expressing activation markers (CD28 and CD57) was reduced in patients in preoperative period that returned to normal immediately after surgery with favorable outcome, while it recovered with unfavorable outcome closer to the end of postoperative examination. The defined features of NKT cell phenotype in patients with unfavorable DPP outcome characterize disturbances in subset ratio and mechanisms of functioning of this cell fraction. This determines a need to develop immunotherapeutic methods aimed at stimulating immunoregulatory activity of NKT cells.

Subsets of thymic resident NKT cells show differential requirements for CD28 co-stimulation during antigen activation

Abstract Natural killer T (NKT) cells rapidly respond to antigenic stimulation with cytokine production and direct cytotoxicity. These innate-like characteristics arise from their differentiation into mature effector cells during thymic development. A subset of mature NKT cells remain thymic resident, but their activation and function remain poorly understood. We examined the roles of CD28 and CTLA-4 in driving activation of these thymic resident NKT cells. Unlike previous studies with peripheral NKT cells, the proliferation of thymic NKT cells was significantly impaired when CD28 engagement was blocked - but unaffected with CTLA-4 activation or blockade. Within NKT cell subsets, however, stage 3 NKT cells marked by higher NK1.1 expression were significantly more sensitive to the loss of CD28 signals compared to NK1.1 negative stage 2 NKT cells. In concordance with this, CD28 blockade suppressed all NKT cell cytokines, lowering the ratio of IFN-γ:IL-4 production by NK1.1 positive NKT cells. Intriguingly, the activation-dependent upregulation of the master transcription factor PLZF did not require CD28 co-stimulation in either NK1.1 positive or negative thymic NKT subsets – underlining a dichotomy between requirements for early activation and the subsequent proliferation and effector function of these cells. Collectively, our studies demonstrate the ability of CD28 co-stimulation to fine tune subset-specific responses by thymic resident NKT cells and contextually shape the milieu in this primary lymphoid organ.

Vascular Endothelial Cells Activate Peripheral Natural Killer T Cells and Participate in Regulation of Downstream Immune Cascades in Patients with Sepsis.

BackgroundThis study investigated the effect of supernatant of endothelial cells stimulated by peripheral blood serum from sepsis patients on phenotype and function of peripheral NKT cells.Material/MethodsTwenty-one patients with sepsis and 21 healthy subjects were included. Peripheral blood (5 ml) was collected from all patients and healthy subjects. To isolate peripheral blood mononuclear cells (PBMCs), Ficoll lymphocyte separation solution was used. Flow cytometry was carried out to determine NKT cell ratio, activity, and cytokine secretion. Human umbilical vein endothelial cells were cultured with serum from sepsis patients for 48 h before changing to fresh medium, and supernatant was collected. The supernatant was used to co-culture PBMCs before analyzing NKT activity and cytokines.ResultsThe ratios of CD3-CD56+NK cells and CD3+CD56+NKT cells were increased in peripheral blood from sepsis patients. Surface receptors p30, G2D, and p44 of CD3+CD56+NKT cells were elevated, while inhibitory receptors NKG2A and 158b were decreased. CD4+ NKT cells in peripheral blood from sepsis patients were enhanced. GranB, IFN-γ, IL-4, and IL-17 in NKT cells from sepsis patients were up-regulated. After co-culture with vascular endothelial cells treated with sepsis serum, expression of p30 and G2D in NKT cells was upregulated, and number of TCRVα24-positive cells was increased. In addition, ratio of CD4+NKT cells was increased, and intracellular expression of IL-4 and IFN-γ was elevated.ConclusionsThe study demonstrates that the level of NKT cells in peripheral blood from sepsis patients is increased, and their activity is enhanced. In addition, vascular endothelial cells from sepsis patients can regulate the activity of NKT cells.

Reactive Oxygen Species Regulate the Inflammatory Function of NKT Cells through Promyelocytic Leukemia Zinc Finger.

Reactive oxygen species (ROS) are byproducts of aerobic metabolism and contribute to both physiological and pathological conditions as second messengers. ROS are essential for activation of T cells, but how ROS influence NKT cells is unknown. In the present study, we investigated the role of ROS in NKT cell function. We found that NKT cells, but not CD4 or CD8 T cells, have dramatically high ROS in the spleen and liver of mice but not in the thymus or adipose tissues. Accordingly, ROS-high NKT cells exhibited increased susceptibility and apoptotic cell death with oxidative stress. High ROS in the peripheral NKT cells were primarily produced by NADPH oxidases and not mitochondria. We observed that sorted ROS-high NKT cells were enriched in NKT1 and NKT17 cells, whereas NKT2 cells were dominant in ROS-low cells. Furthermore, treatment of NKT cells with antioxidants led to reduced frequencies of IFN-γ- and IL-17-expressing cells, indicating that ROS play a role in regulating the inflammatory function of NKT cells. The transcription factor promyelocytic leukemia zinc finger (PLZF) seemed to control the ROS levels. NKT cells from adipose tissues that do not express PLZF and those from PLZF haplodeficient mice have low ROS. Conversely, ROS were highly elevated in CD4 T cells from mice ectopically expressing PLZF. Thus, our findings demonstrate that PLZF controls ROS levels, which in turn governs the inflammatory function of NKT cells.

Reactive oxygen species regulate the inflammatory function of NKT cells through Promyelocytic Leukaemia Zinc Finger (PLZF) protein

Abstract Cellular metabolism controlled by the signaling pathways has a crucial role in dictating the outcome of T cell activation and effector function. Reactive oxygen species (ROS) are byproducts of aerobic metabolism and contribute to both physiological and pathological conditions as a second messenger. In T cells, ROS levels increase upon activation and antigen specific responses of T cells are compromised in the absence of ROS production by mitochondria. However, it is not well understood how NKT cells’ functions are controlled by cell metabolism. In the current study, we investigated the role of ROS in NKT cell function. We found that ROS levels were similar among CD4, CD8 and NKT cell subsets in the thymus but NKT cells showed dramatically increased ROS in the spleen and liver but not in adipose tissues. ROS in the peripheral NKT cells were primarily produced by NADPH oxidases not mitochondria. Accordingly, ROS high NKT cells were susceptible to oxidative stress and underwent apoptotic cell death. Furthermore, ROS play an important role regulating the inflammatory function of NKT cells because antioxidant treatment of NKT cells showed reduced frequencies of IFN-γ+ and IL-17+ cells. In line with this, freshly isolated ROS-high NKT cells had more NKT1 and NKT17 cells but less NKT2 than ROS-low cells. Importantly, these characteristics of NKT cells are regulated by PLZF (Promyelocytic Leukaemia Zinc Finger) protein as evidenced by reduced ROS in NKT cells from adipose tissues that do not express PLZF and also those from PLZF haplodeficient mice. Conversely, ROS were highly elevated in CD4 T cells from mice ectopically expressing PLZF. Together, our study revealed for the first time that ROS regulate NKT cell functions through PLZF.

Roquin Paralogs Differentially Regulate Functional NKT Cell Subsets.

NKT cells represent a small subset of glycolipid-recognizing T cells that are heavily implicated in human allergic, autoimmune, and malignant diseases. In the thymus, precursor cells recognize self-glycolipids by virtue of their semi-invariant TCR, which triggers NKT cell lineage commitment and maturation. During their development, NKT cells are polarized into the NKT1, NKT2, and NKT17 subsets, defined through their cytokine-secretion patterns and the expression of key transcription factors. However, we have largely ignored how the differentiation into the NKT cell subsets is regulated. In this article, we describe the mRNA-binding Roquin-1 and -2 proteins as central regulators of murine NKT cell fate decisions. In the thymus, T cell-specific ablation of the Roquin paralogs leads to a dramatic expansion of NKT17 cells, whereas peripheral mature NKT cells are essentially absent. Roquin-1/2-deficient NKT17 cells show exaggerated lineage-specific expression of nearly all NKT17-defining proteins tested. We show through mixed bone marrow chimera experiments that NKT17 polarization is mediated through cell-intrinsic mechanisms early during NKT cell development. In contrast, the loss of peripheral NKT cells is due to cell-extrinsic factors. Surprisingly, Roquin paralog-deficient NKT cells are, in striking contrast to conventional T cells, compromised in their ability to secrete cytokines. Altogether, we show that Roquin paralogs regulate the development and function of NKT cell subsets in the thymus and periphery.

Slamf6 acts as a negative regulator of NKT cell expansion in the presence of a strong agonist

Abstract Signaling lymphocyte activation marker family member 6 (Slamf6) is a cell surface signaling receptor that plays an important role in NKT cell development. Upon activation with the CD1d ligand α-galactosylceramide (αGalCer), NKT cell Slamf6 expression increases dramatically (~10-fold) and it maintains this high level of expression for at least 5 days after activation. The mechanisms through which Slamf6 regulates NKT cell function are largely unknown. To investigate the effect of Slamf6 on peripheral NKT cell populations, we challenged C57BL/6 (B6) or B6.Slamf6−/− mice with the NKT-specific agonist αGalCer. Examination of liver NKT cell numbers 3 days after challenge revealed a 50-fold increase in B6 mice over vehicle-treated controls. In contrast, we observed a 270-fold increase in NKT cells in B6.Slamf6−/− mice versus controls. An analysis of spleen NKT cells yielded similar results. A comparison of in vivo BrdU uptake by NKT cells between B6 and B6.Slamf6−/− mice revealed no significant differences in proliferation. In addition, NKT cell intracellular IFN-γ, IL-4, and TNF production between B6 and B6.Slamf6−/− mice after administration of αGalCer revealed no difference in the production of these cytokines. We next evaluated the Slamf6-specific effect on NKT cell expansion using an in vitro co-culture system. Cross-linking of Slamf6 on purified B6 NKT cells resulted in diminished NKT cell expansion and increased numbers of TUNEL+ NKT cells. Taken together, these data support a model where Slamf6 acts as a negative regulator of NKT cell expansion in the presence of a strong agonist by regulating the death threshold of NKT cells. These data suggest that Slamf6 blockade could be a useful tool to manipulate the expansion of NKT cells in vivo.

CD1d-dependent expansion of NKT follicular helper cells in vivo and in vitro is a product of cellular proliferation and differentiation.

NKT follicular helper cells (NKTfh cells) are a recently discovered functional subset of CD1d-restricted NKT cells. Given the potential for NKTfh cells to promote specific antibody responses and germinal center reactions, there is much interest in determining the conditions under which NKTfh cells proliferate and/or differentiate in vivo and in vitro. We confirm that NKTfh cells expressing the canonical semi-invariant Vα14 TCR were CXCR5(+)/ICOS(+)/PD-1(+)/Bcl6(+) and increased in number following administration of the CD1d-binding glycolipid α-galactosylceramide (α-GC) to C57Bl/6 mice. We show that the α-GC-stimulated increase in NKTfh cells was CD1d-dependent since the effect was diminished by reduced CD1d expression. In vivo and in vitro treatment with α-GC, singly or in combination with IL-2, showed that NKTfh cells increased in number to a greater extent than total NKT cells, but proliferation was near-identical in both populations. Acquisition of the NKTfh phenotype from an adoptively transferred PD-1-depleted cell population was also evident, showing that peripheral NKT cells differentiated into NKTfh cells. Therefore, the α-GC-stimulated, CD1d-dependent increase in peripheral NKTfh cells is a result of cellular proliferation and differentiation. These findings advance our understanding of the immune response following immunization with CD1d-binding glycolipids.

P02.03 * INCREASED COUNTS OF NK AND NKT CELLS ARE ASSOCIATED WITH PROLONGED SURVIVAL IN PRIMARY GLIOBLASTOMA PATIENTS TREATED WITH DENDRITIC CELL IMMUNOTHERAPY IN COMBINATION WITH RADIO- AND CHEMO-THERAPY

Two clinical studies, DENDR1 and DENDR2 including, respectively, the treatment of first diagnosis and recurrent glioblastoma (GB) patients with dendritic cells (DCs) loaded with autologous tumor lysate are currently active at Istituto Neurologico Besta, Milan. Our first results obtained on a group of recurrent GB patients demonstrated that the response of NK cells correlates with significantly prolonged survival. Here we provide results of the interim analysis on 22 patients affected by primary GB. Patients with post-surgery volume ≤10 cc underwent leukapheresis before radiotherapy and chemotherapy with temozolomide (TMZ). Three intradermal injections of mature DC were done before adjuvant chemotherapy. The subsequent 4 injections were performed 17 ± 3 days after adjuvant TMZ. MRI, clinical and immunological follow-up were performed every 2 months. The median age at surgery was 54.5 years (28-69). RT-TMZ induced significant lymphopenia ( 1000 lymphocytes/microl (5/22) before first vaccination had shorter PFS than others (p < 0.005). Peripheral Blood Lymphocytes (PBLs) were analyzed by flow cytometry to identify CD8+ T cells, NK and NKT cells before and after DC vaccines. The ratio of vaccination/baseline frequencies and counts (V/B ratio) of all of the immunological parameters for each patient was calculated, and the median of all of the observations used as the cut off value to separate patients. V/B ratio was correlated with the progression free survival (PFS) of each patient. Increased V/B ratio for NK cells and in particular NKT cells, but not for CD8 T lymphocytes, was significantly associated with prolonged PFS (median PFS 14 vs 8.0 mo, p = 0.01; 15.0 vs 8.0 mo, respectively). Interferon (IFN)-γ in PBLs was significantly higher in patients with PFS12 (p < 0.02), increasing immediately after the second vaccination as evaluated by real time-PCR. No changes in the expression levels of IFN-γ were observed in the other patients. After a median follow up of 14 months (6-27), the median progression-free survival (PFS) was 9 mo, with PFS6 90% (C. I. 0.78-1.029%) and PFS12 42% (C. I: 0.20-0.64) at Kaplan Meier analysis. Median overall survival (OS) was 22 mo with OS 12 70%. (C. I. 0.50-0.9). 2/4 patients with MGMT methylation were in the group of high V/B ratio. Our results show a positive association between increased peripheral NK and NKT cells response and prolonged survival. Further investigations are required on possible interference of radio-chemotherapy on activation of CD8+ T cells.

NKT cells require IL-7, not IL-15, for survival and homeostasis (LYM4P.761)

Abstract NKT cells are thymus-generated T lineage cells with shared phenotypic and functional characteristics of innate NK cells. Both T and NK cells require γc cytokines for their survival and homeostasis with T cells being dependent on IL-7 while NK cells being dependent on IL-15. As NKT cells carry features of both T and NK cells, it has been a long standing question which γc cytokine would drive the survival and homeostasis of NKT cells. In contrast to the current view, here we report that NKT cells survival is not dependent on IL-15 but requires IL-7. Specifically, we found that IL-15-deficient mice have intact numbers of peripheral NKT cells and that overexpression of IL-15 failed to expand NKT cell numbers even as it significantly increased CD8 T cell numbers. On the other hand, using a new in vivo model of IL-7-deficiency selectively in peripheral lymphoid tissues (K7 mice), we demonstrated that IL-7 is a non-redundant survival factor for NKT cells. Furthermore, transgenic IL-7 expression dramatically increased peripheral NKT cell numbers, suggesting that IL-7 availability determines size of the NKT cell pool. Mechanistically, we identified STAT5 as the transcriptional effector molecule downstream of IL-7 signaling to promote survival and maintain NKT cell homeostasis. Collectively, these data provide a new perspective on γc cytokine requirements for NKT cell survival, proposing a new role for IL-7 in NKT cell biology.

In-vivostimulation of macaque natural killer T cells with α-galactosylceramide

Natural killer T cells are a potent mediator of anti-viral immunity in mice, but little is known about the effects of manipulating NKT cells in non-human primates. We evaluated the delivery of the NKT cell ligand, α-galactosylceramide (α-GalCer), in 27 macaques by studying the effects of different dosing (1-100 μg), and delivery modes [directly intravenously (i.v.) or pulsed onto blood or peripheral blood mononuclear cells]. We found that peripheral NKT cells were depleted transiently from the periphery following α-GalCer administration across all delivery modes, particularly in doses of ≥10 μg. Furthermore, NKT cell numbers frequently remained depressed at i.v. α-GalCer doses of >10 μg. Levels of cytokine expression were also not enhanced after α-GalCer delivery to macaques. To evaluate the effects of α-GalCer administration on anti-viral immunity, we administered α-GalCer either together with live attenuated influenza virus infection or prior to simian immunodeficiency virus (SIV) infection of two macaques. There was no clear enhancement of influenza-specific T or B cell immunity following α-GalCer delivery. Further, there was no modulation of pathogenic SIVmac251 infection following α-GalCer delivery to a further two macaques in a pilot study. Accordingly, although macaque peripheral NKT cells are modulated by α-GalCer in vivo, at least for the dosing regimens tested in this study, this does not appear to have a significant impact on anti-viral immunity in macaque models.

Comment on “Induced IL-17–Producing Invariant NKT Cells Require Activation in Presence of TGF-β and IL-1β”

We read with interest the article by Monteiro et al. ([1][1]) reporting that noncommitted peripheral invariant NKT (iNKT) cells could be induced to produce IL-17 when activated in the presence of TGF-β and IL-1β irrespective of CD4 and NK1.1 expression. Our previous papers showed that IL-17–

Response to Comment on “Induced IL-17–Producing Invariant NKT Cells Require Activation in Presence of TGF-β and IL-1β”

The main conclusion of our manuscript was that uncommitted peripheral invariant NKT (iNKT) cells can acquire IL-17 secretion upon activation in the right microenvironment ([1][1]), in addition to the existence of a thymic-committed population of IL-17 secreting iNKT cells as previously reported ([2

Bi(o)communications among peripheral blood fractions: A focus on NK and NKT cell biology in rheumatoid arthritis

Rheumatoid Arthritis (RA) is an autoimmune disease with unknown pathophysiology involving many interwoven signalling cascades. ROS, NK and NKT cells might be crucial in the disease severity of RA of which the role of NK and NKT cells are controversial in literature. However, the role of oxidative stress, its impact on NK and NKT cell immunobiology and disease activity (DAS28) is largely unknown. Therefore, we studied the role of oxidative stress and NK cell subsets in the pathogenesis of RA. The state of oxidative stress in various peripheral blood fractions, percentage NK and NKT cell expression, their altered apoptotic signaling pathways involving mitochondrial membrane potential, FAS associated death domain (FADD) mediated pathways and DNA damage were analyzed. Results indicated a state of profound oxidative stress in the peripheral blood of RA patients where percentage of NK and NKT cell subsets diminished while ROS levels increased. The depolarized mitochondrial membrane potential, FAS, FASL and active caspase-3 positive NK and NKT cell subsets were considerably elevated in patients. The DNA damage, assessed as percentage of DNA in comet tail, was significantly elevated. Findings of the present work indicate increased apoptosis of peripheral NK and NKT cells in the diseased condition. PBMC and RBC are the major sites of enhanced oxidative stress. The state of oxidative stress and altered immunobiology of NK and NKT cells strongly correlated with Disease activity score. The present study strongly supports the protective role of NK cell subsets in the pathogenesis of RA.

Ameliorated ConA-Induced Hepatitis in the Absence of PKC-theta

Severe liver injury that occurs when immune cells mistakenly attack an individual's own liver cells leads to autoimmune hepatitis. In mice, acute hepatitis can be induced by concanavalin A (ConA) treatment, which causes rapid activation of CD1d-positive natural killer (NK) T cells. These activated NKT cells produce large amounts of cytokines, which induce strong inflammation that damages liver tissues. Here we show that PKC-θ−/− mice were resistant to ConA-induced hepatitis due to essential function of PKC-θ in NKT cell development and activation. A dosage of ConA (25 mg/kg) that was lethal to wild-type (WT) mice failed to induce death resulting from liver injury in PKC-θ−/− mice. Correspondingly, ConA-induced production of cytokines such as IFNγ, IL-6, and TNFα, which mediate the inflammation responsible for liver injury, were significantly lower in PKC-θ−/− mice. Peripheral NKT cells had developmental defects at early stages in the thymus in PKC-θ−/− mice, and as a result their frequency and number were greatly reduced. Furthermore, PKC-θ−/− bone marrow adoptively transferred to WT mice displayed similar defects in NKT cell development, suggesting an intrinsic requirement for PKC-θ in NKT cell development. In addition, upon stimulation with NKT cell-specific lipid ligand, peripheral PKC-θ−/− NKT cells produced lower levels of inflammatory cytokines than that of WT NKT cells, suggesting that activation of NKT cells also requires PKC-θ. Our results suggest PKC-θ is an essential molecule required for activation of NKT cell to induce hepatitis, and thus, is a potential drug target for prevention of autoimmune hepatitis.

IL-15 Regulates Homeostasis and Terminal Maturation of NKT Cells

Semi-invariant NKT cells are thymus-derived innate-like lymphocytes that modulate microbial and tumor immunity as well as autoimmune diseases. These immunoregulatory properties of NKT cells are acquired during their development. Much has been learned regarding the molecular and cellular cues that promote NKT cell development, yet how these cells are maintained in the thymus and the periphery and how they acquire functional competence are incompletely understood. We found that IL-15 induced several Bcl-2 family survival factors in thymic and splenic NKT cells in vitro. Yet, IL-15-mediated thymic and peripheral NKT cell survival critically depended on Bcl-x(L) expression. Additionally, IL-15 regulated thymic developmental stage 2 to stage 3 lineage progression and terminal NKT cell differentiation. Global gene expression analyses and validation revealed that IL-15 regulated Tbx21 (T-bet) expression in thymic NKT cells. The loss of IL-15 also resulted in poor expression of key effector molecules such as IFN-γ, granzyme A and C, as well as several NK cell receptors, which are also regulated by T-bet in NKT cells. Taken together, our findings reveal a critical role for IL-15 in NKT cell survival, which is mediated by Bcl-x(L), and effector differentiation, which is consistent with a role of T-bet in regulating terminal maturation.

Skin and Peripheral Lymph Node Invariant NKT Cells Are Mainly Retinoic Acid Receptor-Related Orphan Receptor γt+ and Respond Preferentially under Inflammatory Conditions

Lymph nodes (LNs) have been long considered as comprising few invariant NKT (iNKT) cells, and these cells have not been studied extensively. In this study, we unravel the existence of stable rather than transitional LN-resident NK1.1(-) iNKT cell populations. We found the one resident in peripheral LNs (PLNs) to comprise a major IL-17-producing population and to express the retinoic acid receptor-related orphan receptor (gamma)t (ROR(gamma)t). These cells respond to their ligand alpha-galactosylceramide (alpha-GalCer) in vivo by expanding dramatically in the presence of LPS, providing insight into how this rare population could have an impact in immune responses to infection. PLN-resident ROR(gamma)t(+) NK1.1(-) iNKT cells express concomitantly CCR6, the integrin alpha-chain alpha(E) (CD103), and IL-1R type I (CD121a), indicating that they might play a role in inflamed epithelia. Accordingly, skin epithelia comprise a major ROR(gamma)t(+) CCR6(+)CD103(+)CD121a(+) NK1.1(-) cell population, reflecting iNKT cell composition in PLNs. Importantly, both skin and draining PLN ROR(gamma)t(+) iNKT cells respond preferentially to inflammatory signals and independently of IL-6, indicating that they could play a nonredundant role during inflammation. Overall, our study indicates that ROR(gamma)t(+) iNKT cells could play a major role in the skin during immune responses to infection and autoimmunity.

Peripheral NK and NKT cell in pediatric subjects with chronic HBV infection

To investigate the characteristic of NK cells and NKT cells in HBV infected pediatric subjects for evaluation of their clinical implication. Fresh peripheral blood samples were obtained from 42 HBV-infected pediatric cases and 15 healthy counterparts. NK cells and NKT cells were analyzed by flow cytometry assay. The clinical data such as serum ALT level and HBV viral load was simultaneously recorded from each HBV carrier. HBV-infected children had an obviously increasing percentage of NK cells 12.071% +/- 7.100%, there were significant differences between the children with chronic B hepatitis and the healthy children (P <0.05). As far as percentage of NKT 3.048% +/- 1.937% was concerned, there were not differences. Furthermore the association was not found between serum HBV viral load level and the NK lymphocyte. Our data may provide valuable information of NK and NKT lymphocyte for evaluation of disease progression of HBV infected children NKI cells.