Characterizing porcine invariant natural killer T cells: A comparative study with NK cells and T cells

CD1d-restricted invariant natural killer T (iNKT) cells are innate-like T cells that share phenotypic characteristics of both NK and conventional T cells (Tconv). Although iNKT cells have been well characterized in mice and humans, functional CD1d and CD1d-restricted iNKT cells are not universally expressed in mammals. Swine express iNKT cells that can be detected using α-galactosylceramide (α-GalCer)-loaded CD1d tetramers. In the present study, we characterized iNKT cells from the blood, spleen, lymph node, lung and liver of commercial mixed-breed pigs, and compared their phenotype to NK cells and Tconv. The principal findings are that pig iNKT cells are CD8α and CD44 positive and CD11b and Nkp46 negative. Most are also negative for the CD4 co-receptor, which is used to distinguish functionally distinct mouse and human iNKT cells subsets. The frequency of IFN-γ-producing CD8α^bright iNKT cells was 3–4-fold higher than CD8α^dull iNKT cells, suggesting that CD8α expression identifies iNKT cells with a unique functional role in immune responses. Finally, large variability was detected among pigs in interactions between iNKT cells and monocytes when iNKT cells were activated with α-GalCer, which raises a cautionary note about manipulating iNKT cells for immunotherapy. Collectively, our study provides important phenotypic and functional information about porcine iNKT cells that will be useful for understanding how iNKT cells contribute to immune responses in swine, with potential implications for human health.     

Direct identification of rat iNKT cells reveals remarkable similarities to human iNKT cells and a profound deficiency in LEW rats

iNKT cells are a particular lymphocyte population with potent immunomodulatory capa‐city; by promoting or suppressing immune responses against infections, tumors, and autoimmunity, iNKT cells are a promising target for immunotherapy. The hallmark of iNKT cells is the expression of a semiinvariant TCR (with an invariant α‐chain comprising AV14 and AJ18 gene segments), which recognizes glycolipids presented by CD1d. Here, we identified iNKT cells for the first time in the rat using rat CD1d‐dimers and PLZF staining. Importantly, in terms of frequencies (1.05% ± 0.52 SD of all intrahepatic αβ T cells), coreceptor expression and in vitro expansion features, iNKT cells from F344 inbred rats more closely resemble human iNKT cells than their mouse counterparts. In contrast, in LEW inbred rats, which are often used as models for organ‐specific autoimmune diseases, iNKT cell numbers are near or below the detection limit. Interestingly, the usage of members of the rat AV14 gene family differed between F344 and LEW inbred rats. In conclusion, the similarities between F344 rat and human iNKT cells and the nearly absent iNKT cells in LEW rats make the rat a promising animal model for the study of iNKT cell‐based therapies and of iNKT‐cell biology.     

Activation of murine invariant NKT cells promotes susceptibility to candidiasis by IL‐10 induced modulation of phagocyte antifungal activity

Invariant NKT (iNKT) cells play an important role in a variety of antimicrobial immune responses due to their ability to produce high levels of immune‐modulating cytokines. Here, we investigated the role of iNKT cells in host defense against candidiasis using Jα18‐deficient mice (Jα18^?/?), which lack iNKT cells. Jα18^?/? mice were more resistant to the development of lethal candidiasis than wild‐type (WT) mice. In contrast, treatment of WT mice with the iNKT cell activating ligand α‐galactosylceramide markedly enhanced their mortality after infection with Candida albicans. Serum IL‐10 levels were significantly elevated in WT mice in response to infection with C. albicans. Futhermore, IL‐10 production increased after in vitro coculture of peritoneal macrophages with iNKT cells and C. albicans. The numbers of peritoneal macrophages, the production of IL‐1β and IL‐18, and caspase‐1 activity were also significantly elevated in Jα18^?/? mice after infection with C. albicans. The adoptive transfer of iNKT cells or exogenous administration of IL‐10 into Jα18^?/? reversed susceptibility to candidiasis to the level of WT mice. These results suggest that activation of iNKT cells increases the initial severity of C. albicans infection, most likely mediated by IL‐10 induced modulation of macrophage antifungal activity.     

The hypervariable region 4 (HV4) and position 93 of the α chain modulate CD1d‐glycolipid binding of iNKT TCRs

TCRs of invariant NKT (iNKT) cells bind α‐galactosylceramide (αGC) loaded CD1d in a highly conserved fashion and show a characteristic TCR gene usage: An “invariant” α chain with a canonical AV14/AJ18 rearrangement in mice (AV24/AJ18 in humans) is paired with β chains containing characteristic Vβ segments. In the rat, a multimember AV14 gene family increases the variability within this system. This study characterizes CD1d binding of rat AV14 gene segments in TCR transductants as well as CD1d binding and iNKT TCR expression of expanded polyclonal F344 rat iNKT populations. It defines an important role of position 93 at the V‐J transition for TCR avidity and species cross‐reactivity of the rat iNKT TCR. Furthermore, for the first time we identified variability within the fourth hypervariable loop (HV4) of the α chain as a modulator of CD1d:αGC binding in rat and mouse. Additionally, we confirmed the importance of the CDR2β for CD1d:αGC binding, but also show that the CDR3β may even have opposite effects on binding depending on the pairing α chain. Altogether, we characterized naturally occurring sources of variability for the iNKT TCR and speculate that they rather level than increase the largely germline encoded differences of iNKT TCR ligand avidity.     

Characterization of regulatory T cells in obese omental adipose tissue in humans

Obesity‐associated visceral adipose tissue (AT) inflammation promotes insulin resistance and type 2 diabetes (T2D). In mice, lean visceral AT is populated with anti‐inflammatory cells, notably regulatory T cells (Tregs) expressing the IL‐33 receptor ST2. Conversely, obese AT contains fewer Tregs and more proinflammatory cells. In humans, however, there is limited evidence for a similar pattern of obesity‐associated immunomodulation. We used flow cytometry and mRNA quantification to characterize human omental AT in 29 obese subjects, 18 of whom had T2D. Patients with T2D had increased proportions of inflammatory cells, including M1 macrophages, with positive correlations to body mass index. In contrast, Treg frequencies negatively correlated to body mass index but were comparable between T2D and non‐T2D individuals. Compared to human thymic Tregs, omental AT Tregs expressed similar levels of FOXP3, CD25, IKZF2, and CTLA4, but higher levels of PPARG, CCR4, PRDM1, and CXCL2. ST2, however, was not detectable on omental AT Tregs from lean or obese subjects. This is the first comprehensive investigation into how omental AT immunity changes with obesity and T2D in humans, revealing important similarities and differences to paradigms in mice. These data increase our understanding of how pathways of immune regulation could be targeted to ameliorate AT inflammation in humans.     

Gastrointestinal stromal tumors and KIT-positive mesenchymal cells in the omentum

Gastrointestinal stromal tumor (GIST) is currently considered to be derived from the interstitial cells of Cajal (ICC). To test the hypothesis that omental mesenchymal tumor is also a type of GIST, we evaluated the expression of specific molecules in GIST, and c-kit gene mutation in omental mesenchymal tumors, and we identified a possible counterpart of ICC in the omentum. Immunohistochemically, all of the omental mesenchymal tumors (n = 5) were positive for both KIT and CD34, and three of the five tumors were also positive for an embryonic form of smooth-muscle myosin heavy chain (SMemb). Polymerase chain reaction-single-strand conformational polymorphism analysis (PCR-SSCP) and direct sequencing revealed mutations in c-kit gene exon 11 in all five tumors. As for the ICC counterparts in the omentum, there were some KIT-positive mesenchymal cells resembling ICC at the surface of the omentum. Double fluorescence immunostaining, using anti-KIT polyclonal antibodies and monoclonal antibodies against other molecules, demonstrated that KIT-, CD34- and SMemb-positive cells were present just beneath the mesothelial cells of the omentum. These results show that omental mesenchymal tumor corresponds to GIST of the omentum, and that KIT-positive bipolar mesenchymal cells may be a counterpart of ICC in the gastrointestinal tract. Identification of a new type of KIT-positive mesenchymal cell in the omentum may lead to the discovery of a new physiological role for this organ.     

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.     

Skewed Invariant Natural Killer T (iNKT) Cells,Impaired iNKT:B Cell Help and Decreased SAP Expression in Blood Lymphocytes from Patients with Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is a syndrome with predominantly defective B cell function. However, abnormalities in the number and function of other lymphocyte subpopulations in peripheral blood (PB) have been described in most patients. We have analysed the distribution of iNKT cell subpopulations in the PB of CVID patients and the ability of these cells to provide in vitro cognate B cell help. The total of iNKT cells was reduced in the PB of CVID patients, especially CD4+, CD4‐/CD8‐ and CCR5+/CXCR3+. These findings were associated with an enrichment of memory‐like and a tendency towards a reduction in TNF‐α‐expressing effector iNKT cells in the peripheral blood mononuclear cells (PBMC) of CVID patients. Moreover, an accumulation of follicular helper iNKT cells in the PB of CVID patients was demonstrated. CVID αGalCer‐pulsed iNKT cells are not able to induce autologous B cell proliferation although they do induce proliferation to healthy donor B cells. Interestingly, autologous and heterologous co‐cultures did not differ in the amount of immunoglobulin secreted by B cells in vitro. Finally, reduced intracellular SAP expression in iNKT cells and other lymphocytes in the blood from CVID patients was observed. These results provide further insights into the immunological mechanisms underlying the iNKT cell defects and the potential targets to improve B cell help in CVID.     

Leishmania donovani‐induced expression of signal regulatory protein α on Kupffer cells enhances hepatic invariant NKT‐cell activation

Signal regulatory protein α (SIRPα) and its cognate ligand CD47 have been documented to have a broad range of cellular functions in development and immunity. Here, we investigated the role of SIRPα–CD47 signalling in invariant NKT (iNKT) cell responses. We found that CD47 was required for the optimal production of IFN‐γ from splenic iNKT cells following exposure to the αGalCer analogue PBS‐57 and in vivo infection of mice with Leishmania donovani. Surprisingly, although SIRPα was undetectable in the liver of uninfected mice, the hepatic iNKT‐cell response to infection was also impaired in CD47^?/? mice. However, we found that SIRPα was rapidly induced on Kupffer cells following L. donovani infection, via a mechanism involving G‐protein‐coupled receptors. Thus, we describe a novel amplification pathway affecting cytokine production by hepatic iNKT cells, which may facilitate the breakdown of hepatic tolerance after infection.     

The A2aR adenosine receptor controls cytokine production in iNKT cells

The purine nucleoside adenosine is an important anti‐inflammatory molecule, inhibiting a variety of immune cells by adenosine receptor‐mediated mechanisms. Invariant NKT (iNKT) cells recognize glycolipids presented on CD1d molecules and produce vigorous amounts of cytokines upon activation, hence regulating immune reactions. The mechanisms polarizing their cytokine pattern are elusive. Previous studies demonstrated that adenosine can suppress IFN‐γ production by iNKT cells. We describe the expression of all four known adenosine receptors A1R, A2aR, A2bR and A3R on mouse iNKT cells. We show that IL‐4 production in primary mouse iNKT cells and a human iNKT line is efficiently inhibited by A2aR blockade with an inverse relation to IL‐4. These data are supported by A2aR‐deficient mice, which exhibit largely decreased levels of IL‐4, IL‐10 and TGF‐β concomitantly with an increase of IFN‐γ upon α‐galactosylceramide administration in vivo. While A2aR inhibits other lymphocyte populations, A2aR is required for the secretion of IL‐4 and IL‐10 by iNKT cells. These data suggest adenosine:A2aR‐mediated mechanisms can control the cytokine secretion pattern of iNKT cells.     

Human CD4+ invariant NKT cells are involved in antibacterial immunity against Brucella suis through CD1d‐dependent but CD4‐independent mechanisms

Human invariant NKT (iNKT) cells are a unique subset of T cells, which recognize glycolipids presented by the CD1d. Among the iNKT cells, several functionally distinct subsets have been characterized according to CD4 and/or CD8 co‐receptor expression. The current study is focussed on the CD4⁺ iNKT cell subset and its role in an anti‐infectious response. We have examined the role of CD4⁺ iNKT cells on the intracellular Brucella suis growth. Our results indicate that CD4⁺ iNKT cells impair the intramacrophagic growth of Brucella. This inhibition is due to a combination of soluble and contact‐dependent mechanisms: IFN‐γ is weakly involved while cytotoxic activities such as the induction of the Fas pathway and the release of lytic granules are major mechanisms. The impairment of Brucella growth by CD4⁺ iNKT cells requires an interaction with CD1d on macrophage surface. Also, we have shown that although CD4 regulates several biological responses of CD4⁺ iNKT cells, it is not involved in their antibacterial activity. Here, we have shown for the first time that the CD4⁺ iNKT cell population has antibacterial activity and thus, participates directly in the elimination of bacteria and/or in the control of bacterial growth by killing infected cells.     

Murine CD8+ Invariant Natural Killer T Cells are Negatively Selected by CD1d Expressed on Thymic Epithelial Cells and Dendritic Cells

Background: CD1d-dependent invariant natural killer (iNKT) cells are found as either CD4 single positive (SP) or CD4/CD8 double negative (DN) cells in mice. The size of the CD8⁺ iNKT population is extremely small. It is known that CD1d expression on developing thymocytes is sufficient for iNKT development and co-receptor choice, which is driven by Th-POK expression. This study aimed to examine the factors involved in the CD4/CD8 co-receptor choice of iNKT cells in addition to Th-POK-driven silencing of CD8 expression. Methods: In this study, we compared iNKT cells of wild-type (WT) mice with those of transgenic mice in which CD1d expression is restricted to developing thymocytes by the proximal Lck (pLCK) promoter. CD8 positive iNKT cell population were analyzed by flow cytometry. Results: We found that there was a substantial population of CD8⁺ iNKT cells in the thymus and spleen of transgenic mice, and these cells are negatively selected in between Stage 2 and Stage 3 of their developmental program by the CD1d expressed on Thymic epithelial cell (TEC) and Dendritic cells in WT mice. Conclusion: We conclude that TEC expression of CD1d in the murine thymus contributed to co-receptor choice of iNKT cells, in addition to Th-POK-driven silencing of CD8. Therefore, mostly CD4 SP and DN iNKT cells are produced under normal physiological conditions in mice.     

Suppression of murine tumour growth through CD8+ cytotoxic T lymphocytes via activated DEC‐205+ dendritic cells by sequential administration of α‐galactosylceramide in vivo

Cancer immunity is mediated through the effective priming and activation of tumour‐specific class I MHC molecule‐restricted CD8⁺ cytotoxic T lymphocytes (CTLs). DEC‐205⁺ dendritic cells (DCs) can cross‐present the epitope(s) of captured tumour antigens associated with class I MHC molecules alongside co‐stimulatory molecules to prime and activate tumour‐specific CD8⁺ CTLs. Immunosuppressive tolerogenic DCs with reduced co‐stimulatory molecules may be a cause of impaired CTL induction. Hepa1‐6‐1 cells were established from the mouse hepatoma cell line Hepa1‐6; these cells grow continuously after subcutaneous implantation into syngeneic C57BL/6 (B6) mice and do not prime CD8⁺ CTLs. In this study, we show that the growth of ongoing tumours was suppressed by activated CD8⁺ CTLs with tumour‐specific cytotoxicity through the administration of the glycolipid α‐galactosylceramide (α‐GalCer), which is a compound known to stimulate invariant natural killer T (iNKT) cells and selectively activate DEC‐205⁺ DCs. Moreover, we demonstrated that sequential repetitive intraperitoneal inoculation with α‐GalCer every 48 hr appeared to convert tolerogenic DEC‐205⁺ DCs into immunogenic DCs with a higher expression of co‐stimulatory molecules and a stronger cross‐presentation capacity, which primed CTL precursors and induced tumour‐specific CD8⁺ CTLs within the tumour environment without activating iNKT cells. These findings provide a new basis for cancer immunotherapy to convert tolerogenic DEC‐205⁺ DCs within tumours into immunogenic DCs through the sequential administration of an immuno‐potent lipid/glycolipid, and then activated immunogenic DCs with sufficient expression of co‐stimulatory molecules prime and activate tumour‐specific CD8⁺ CTLs within the tumour to control tumour growth.     

Invariant natural killer T (iNKT) cell exhaustion in sarcoidosis

Invariant natural killer T (iNKT) cells are integral components of immune responses during many chronic diseases, yet their surface phenotypes, subset distribution, and polyfunctional capacity in this environment are largely unknown. Therefore, using flow cytometry, we determined iNKT cell phenotypic and functional characteristics in subjects with chronic inflammatory disease sarcoidosis and matched controls. We found that sarcoidosis subjects displayed lower iNKT‐cell frequencies, which correlated with lung fibrosis, C‐reactive protein levels, and other measures of clinical disease. The CD4^?CD8^? (double negative, DN) iNKT‐cell population was selectively lower in diseased individuals and the remaining DN iNKT cells exhibited higher frequencies of the activation markers CD69 and CD56. Functionally, both total IFN‐γ⁺ and the dual‐functional IFN‐γ⁺ TNF‐α⁺ iNKT cells were decreased in sarcoidosis subjects and these functional defects correlated with total iNKT‐cell circulating frequencies. As the loss of polyfunctionality can reflect functional exhaustion, we measured the surface antigens programmed death‐1 receptor and CD57 and found that levels inversely correlated with dual‐functional iNKT‐cell percentages. These findings reveal that, similar to traditional T cells, iNKT cells may also undergo functional exhaustion, and that circulating iNKT‐cell frequencies reflect these defects. Programmed death‐1 receptor antagonists may therefore be attractive therapeutic candidates for sarcoidosis and other iNKT‐cell‐mediated chronic diseases.