NKT Cell Development Research Articles

Identification of gene targets of PLZF in NKT cells (P1289)

Abstract The transcription factor PLZF (promyelocytic leukemia zinc finger) is required for the development of NKT cell effector functions. Studies described here investigate the gene targets of PLZF in NKT cells. The aims are: 1) to determine the gene expression in wild type and PLZF deficient NKT cells, and 2) to define direct genetic targets of PLZF. A hurdle to carrying out gene expression analysis has been the need to use CD1d tetramers to identify invariant NKT cells. Crosslinking of the TCR, as a result of binding the tetramer, can cause changes in signaling and gene expression. Therefore, for aim 1, we used a BAC eGFP/PLZF reporter mouse system. In this mouse, PLZF expressing cells are labeled with eGFP, which obviates the need for CD1d tetramer. Importantly, GFP expression is retained in NKT cells that develop in PLZF deficient mice. Therefore, “untouched” NKT cells can be collected from PLZF expressing and deficient mice. RNA was isolated from these cells and subjected to microarray analysis. Microarray analysis indicated many gene changes, of which only some will be direct targets of PLZF. Aim 2 will be to carry out Chromatin Immunoprecipitation assays followed by high-throughput sequencing. We will utilize an NKT cell hybridoma cell line that expresses PLZF and has retained the capacity to produce IL-4 and IFN-γ. By combining the data, we expect to define genes that are regulated by PLZF and are necessary for the development of NKT cells.

The MicroRNA miR-181 Is a Critical Cellular Metabolic Rheostat Essential for NKT Cell Ontogenesis and Lymphocyte Development and Homeostasis

Regulation of metabolic pathways in the immune system provides a mechanism to actively control cellular function, growth, proliferation, and survival. Here, we report that miR-181 is a nonredundant determinant of cellular metabolism and is essential for supporting the biosynthetic demands of early NKT cell development. As a result, miR-181-deficient mice showed a complete absence of mature NKT cells in the thymus and periphery. Mechanistically, miR-181 modulated expression of the phosphatase PTEN to control PI3K signaling, which was a primary stimulus for anabolic metabolism in immune cells. Thus miR-181-deficient mice also showed severe defects in lymphoid development and Tcell homeostasis associated with impaired PI3K signaling. These results uncover miR-181 as essential for NKT cell development and establish this family of miRNAs as central regulators of PI3K signaling and global metabolic fitness during development and homeostasis.

LKB1 Mediates the Development of Conventional and Innate T Cells via AMP-Dependent Kinase Autonomous Pathways

The present study has examined the role of the serine/threonine kinase LKB1 in the survival and differentiation of CD4/8 double positive thymocytes. LKB1-null DPs can respond to signals from the mature α/β T-cell-antigen receptor and initiate positive selection. However, in the absence of LKB1, thymocytes fail to mature to conventional single positive cells causing severe lymphopenia in the peripheral lymphoid tissues. LKB1 thus appears to be dispensable for positive selection but important for the maturation of positively selected thymocytes. LKB1 also strikingly prevented the development of invariant Vα14 NKT cells and innate TCR αβ gut lymphocytes. Previous studies with gain of function mutants have suggested that the role of LKB1 in T cell development is mediated by its substrate the AMP-activated protein kinase (AMPK). The present study now analyses the impact of AMPK deletion in DP thymocytes and shows that the role of LKB1 during the development of both conventional and innate T cells is mediated by AMPK-independent pathways.

NKT cells: their development, mechanisms and effects of action

NKT cells are a heterogeneous subset of T lymphocytes that share surface markers and functional characteristics with both conventional T lymphocytes and natural killer cells. Most NKT cells express a semi-invariant T cell receptor that reacts with glycolipid antigens presented by the CD1d molecule on the surface of antigen-presenting cells. NKT cells can modulate the immune response against infectious agents, autoantigens, tumors, tissue grafts and allergens. NKT cells mediate the activities through their ability to express pro- and anti-inflammatory cytokines that influence the type and magnitude of the immune response. The manuscript summarizes current views on development of NKT cells as well as mechanisms and effects of their action.

SAP gene transfer restores cellular and humoral immune function in a murine model of X-linked lymphoproliferative disease

AbstractX-linked lymphoproliferative disease (XLP1) arises from mutations in the gene encoding SLAM-associated protein (SAP) and leads to abnormalities of NKT-cell development, NK-cell cytotoxicity, and T-dependent humoral function. Curative treatment is limited to allogeneic hematopoietic stem cell (HSC) transplantation. We tested whether HSC gene therapy could correct the multilineage defects seen in SAP−/− mice. SAP−/− murine HSCs were transduced with lentiviral vectors containing either SAP or reporter gene before transplantation into irradiated recipients. NKT-cell development was significantly higher and NK-cell cytotoxicity restored to wild-type levels in mice receiving the SAP vector in comparison to control mice. Baseline immunoglobulin levels were significantly increased and T-dependent humoral responses to NP-CGG, including germinal center formation, were restored in SAP-transduced mice. We demonstrate for the first time that HSC gene transfer corrects the cellular and humoral defects in SAP−/− mice providing proof of concept for gene therapy in XLP1.

Cutting Edge: Ly9 (CD229), a SLAM Family Receptor, Negatively Regulates the Development of Thymic Innate Memory-like CD8+ T and Invariant NKT Cells

Signaling lymphocytic activation molecule family receptors and the specific adapter signaling lymphocytic activation molecule-associated protein modulate the development of innate-like lymphocytes. In this study, we show that the thymus of Ly9-deficient mice contains an expanded population of CD8 single-positive cells with the characteristic phenotype of innate memory-like CD8(+) T cells. Moreover, the proportion of these innate CD8(+) T cells increased dramatically postinfection with mouse CMV. Gene expression profiling of Ly9-deficient mice thymi showed a significant upregulation of IL-4 and promyelocytic leukemia zinc finger. Analyses of Ly9(-/-)IL4ra(-/-) double-deficient mice revealed that IL-4 was needed to generate the thymic innate CD8(+) T cell subset. Furthermore, increased numbers of invariant NKT cells were detected in Ly9-deficient thymi. In wild-type mice, IL-4 levels induced by α-galactosylceramide injection could be inhibited by a mAb against Ly9. Thus, Ly9 plays a unique role as an inhibitory cell surface receptor regulating the size of the thymic innate CD8(+) T cell pool and the development of invariant NKT cells.

Regulation of Lipid Signaling by Diacylglycerol Kinases during T Cell Development and Function.

Diacylglycerol (DAG) and phosphatidic acid (PA) are bioactive lipids synthesized when the T cell receptor binds to a cognate peptide-MHC complex. DAG triggers signaling by recruiting Ras guanyl-releasing protein 1, PKCθ, and other effectors, whereas PA binds to effector molecules that include mechanistic target of rapamycin, Src homology region 2 domain-containing phosphatase 1, and Raf1. While DAG-mediated pathways have been shown to play vital roles in T cell development and function, the importance of PA-mediated signals remains less clear. The diacylglycerol kinase (DGK) family of enzymes phosphorylates DAG to produce PA, serving as a molecular switch that regulates the relative levels of these critical second messengers. Two DGK isoforms, α and ζ, are predominantly expressed in T lineage cells and play an important role in conventional αβ T cell development. In mature T cells, the activity of these DGK isoforms aids in the maintenance of self-tolerance by preventing T cell hyper-activation and promoting T cell anergy. In this review, we discuss the roles of DAG-mediated pathways, PA-effectors, and DGKs in T cell development and function. We also highlight recent work that has uncovered previously unappreciated roles for DGK activity, for instance in invariant NKT cell development, anti-tumor and anti-viral CD8 responses, and the directional secretion of soluble effectors.

ZBTB7B (Th-POK) Regulates the Development of IL-17–Producing CD1d-Restricted Mouse NKT Cells

CD1d-dependent NKT cells represent a heterogeneous family of effector T cells including CD4(+)CD8(-) and CD4(-)CD8(-) subsets that respond to glycolipid Ags with rapid and potent cytokine production. NKT cell development is regulated by a unique combination of factors, however very little is known about factors that control the development of NKT subsets. In this study, we analyze a novel mouse strain (helpless) with a mis-sense mutation in the BTB-POZ domain of ZBTB7B and demonstrate that this mutation has dramatic, intrinsic effects on development of NKT cell subsets. Although NKT cell numbers are similar in Zbtb7b mutant mice, these cells are hyperproliferative and most lack CD4 and instead express CD8. Moreover, the majority of ZBTB7B mutant NKT cells in the thymus are retinoic acid-related orphan receptor γt positive, and a high frequency produce IL-17 while very few produce IFN-γ or other cytokines, sharply contrasting the profile of normal NKT cells. Mice heterozygous for the helpless mutation also have reduced numbers of CD4(+) NKT cells and increased production of IL-17 without an increase in CD8(+) cells, suggesting that ZBTB7B acts at multiple stages of NKT cell development. These results reveal ZBTB7B as a critical factor genetically predetermining the balance of effector subsets within the NKT cell population.

SAP Is Required for the Development of Innate Phenotype in H2-M3–Restricted CD8+ T Cells

H2-M3--restricted T cells have a preactivated surface phenotype, rapidly expand, and produce cytokines upon stimulation, and, as such, are classified as innate T cells. Unlike most innate T cells, M3-restricted T cells also express CD8αβ coreceptors and a diverse TCR repertoire: hallmarks of conventional MHC Ia-restricted CD8(+) T cells. Although invariant NKT cells are also innate T cells, they are selected exclusively on hematopoietic cells (HC), whereas M3-restricted T cells can be selected on either hematopoietic or thymic epithelial cells. Moreover, their phenotypes differ depending on what cells mediate their selection. Although there is a clear correlation between selection on HC and development of innate phenotype, the underlying mechanism remains unclear. Signaling lymphocyte activation molecule-associated protein (SAP) is required for the development of invariant NKT cells and mediates signals from signaling lymphocyte activation molecule receptors that are exclusively expressed on HC. Based on their dual selection pathway, M3-restricted T cells present a unique model for studying the development of innate T cell phenotype. Using both polyclonal and transgenic mouse models, we demonstrate that although M3-restricted T cells are capable of developing in the absence of SAP, SAP is required for HC-mediated selection, development of preactivated phenotype, and heightened effector functions of M3-restricted T cells. These findings are significant because they directly demonstrate the need for SAP in HC-mediated acquisition of innate T cell phenotype and suggest that, due to their SAP-dependent HC-mediated selection, M3-restricted T cells develop a preactivated phenotype and an intrinsic ability to proliferate faster upon stimulation, allowing for an important role in the early response to infection.

Tandem repeats modify the structure of the canine CD1D gene

Among the CD1 proteins that present lipid antigens to T cells, CD1d is the only one that stimulates a population of T cells with an invariant T-cell receptor known as NKT cells. Sequencing of a 722 nucleotide gap in the dog (Canis lupus familiaris) genome revealed that the canine CD1D gene lacks a sequence homologous to exon 2 of human CD1D, coding for the start codon and signal peptide. Also, the canine CD1D gene contains three different short tandem repeats that disrupt the expected gene structure. Because canine CD1D cDNA lacks sequences homologous to human exon 2 and 3, the functionality of canine CD1d protein may be affected, and this could have consequences for the development and activation of canine NKT cells.

Globosides but Not Isoglobosides Can Impact the Development of Invariant NKT Cells and Their Interaction with Dendritic Cells

Recognition of endogenous lipid Ag(s) on CD1d is required for the development of invariant NKT (iNKT) cells. Isoglobotrihexosylceramide (iGb3) has been implicated as this endogenous selecting ligand and recently suggested to control overstimulation and deletion of iNKT cells in α-galactosidase A-deficient (αGalA(-/-)) mice (human Fabry disease), which accumulate isoglobosides and globosides. However, the presence and function of iGb3 in murine thymus remained controversial. In this study, we generate a globotrihexosylceramide (Gb3)-synthase-deficient (Gb3S(-/-)) mouse and show that in thymi of αGalA(-/-)/Gb3S(-/-) double-knockout mice, which store isoglobosides but no globosides, minute amounts of iGb3 can be detected by HPLC. Furthermore, we demonstrate that iGb3 deficiency does not only fail to impact selection of iNKT cells, in terms of frequency and absolute numbers, but also does not alter the distribution of the TCR CDR 3 of iNKT cells. Analyzing multiple gene-targeted mouse strains, we demonstrate that globoside, rather than iGb3, storage is the major cause for reduced iNKT cell frequencies and defective Ag presentation in αGalA(-/-) mice. Finally, we show that correction of globoside storage in αGalA(-/-) mice by crossing them with Gb3S(-/-) normalizes iNKT cell frequencies and dendritic cell (DC) function. We conclude that, although detectable in murine thymus in αGalA(-/-)/Gb3S(-/-) mice, iGb3 does not influence either the development of iNKT cells or their interaction with peripheral DCs. Moreover, in αGalA(-/-) mice, it is the Gb3 storage that is responsible for the decreased iNKT cell numbers and impeded Ag presentation on DCs.

Suppressing autoimmunity by TGF-β: not just through Treg cells

Transforming growth factor-β (TGF-β) is a pleiotropic factor. It regulates many aspects of T-cell biology including thymocyte development, T cell differentiation and effector T cell function.1 Previous work has shown that the loss of TGF-β signaling in the T-cell lineage leads to severe lymphoproliferative autoimmunity.2,3 Until now, it has been unclear to what extent the autoimmunity observed in those mice is driven by TGF–unresponsive conventional T cells, as TGF- is also crucial for the maintenance and function of regulatory T cells (Treg). A recent study published in Nature Immunology revealed a novel role of TGF-β signaling in non-regulatory naive T cells. It was found that TGF-β controls lymphopenia-driven T-cell proliferation in neonatal mice, thereby preventing the development of autoimmunity.4

The Receptor Ly108 Functions as a SAP Adaptor-Dependent On-Off Switch for T Cell Help to B Cells and NKT Cell Development

Humans and mice deficient in the adaptor protein SAP (Sh2d1a) have a major defect in humoral immunity, resulting from a lack of Tcell help for B cells. The role of SAP in this process is incompletely understood. We found that deletion of receptor Ly108 (Slamf6) in CD4(+) Tcells reversed the Sh2d1a(-/-) phenotype, eliminating the SAP requirement for germinal centers. This potent negative signaling byLy108 required immunotyrosine switch motifs (ITSMs) and SHP-1 recruitment, resulting in high amounts of SHP-1 at the Tcell:B cell synapse, limiting Tcell:B cell adhesion. Ly108-negative signaling was important not only in CD4(+) Tcells; we found that NKT cell differentiation was substantially restored in Slamf6(-/-)Sh2d1a(-/-) mice. The ability of SAP to regulate both positive and negative signals in Tcells can explain the severity of SAP deficiency and highlights the importance of SAP and SHP-1 competition for Ly108 ITSM binding as a rheostat for the magnitude of Tcell help to B cells.

MicroRNA miR-155 regulates NKT cell development and function by targeting ITK (115.6)

Abstract Invariant Natural Killer T (iNKT) cells are potent regulators of diverse immune responses. Our previous study indicated that lack of miRNAs following the deletion of Dicer, a miRNAs-processing enzyme, affect iNKT cell development and function. However, the roles of specific miRNA in iNKT cell development and function has not been reported. To identify which miRNAs are involved in NKT cell development, we first performed a genome-wide miRNA expression profiling during NKT cell development and found that miR-155 was highly expressed in early iNKT cell development, followed by a sharp decrease in late developmental stage in thymus. To test the role of miR-155 in iNKT cells, two miR155 mutant mouse models had been used. With a conventional miR-155 deficient mouse model, we did not find remarkable defects of iNKT cell development and function. However, overexpression of miR-155 in bone marrow or thymus results in iNKT cell developmental defect in the thymus without dramatic effects on other immune cells. Furthermore, miR-155 overexpressed iNKT cells display profound defects in their cytokine production. Bone marrow from miR-155 overexpressed mice poorly reconstituted iNKT cells, suggesting a cell intrinsic effect. Finally, we have identified Itk (Inducible T cell Kinase) as a direct target of miR-155 in the regulation of iNKT cell development and function. Together, our data suggest that dynamic regulation of miR-155 expression is required for iNKT cell development and function.

Endogenous mouse mammary tumor viruses influence the development of thymic αβ NKT cells and innate CD8+ T cells (111.26)

Abstract Mounting evidence has demonstrated the presence of a population of thymic CD8+ T cells that share phenotypic and functional similarity to “innate-like” or “memory-like” T cells. Innate CD8+ T cells have increased expression of CD44, CD122, and Eomesodermin (Eomes), as well as the capacity to rapidly produce interferon (IFN)-γ upon stimulation. In addition to several gene-deficient models, including KLF2-, Itk-, and Id3-deficient mice, innate CD8+ T cells have also been documented in naïve BALB/c mice. The development of such cells is dependent on the production of interleukin (IL)-4 from an expanded population of αβ or γδ NKT cells expressing the transcription factor promyelocytic leukemia zinc finger (PLZF). Interestingly, this innate phenotype among CD8+ T cells is lost in congenic BALB/c mice lacking all endogenous mouse mammary tumor proviruses (BALB/Mtv-null). In the absence of endogenous Mtv proviruses, the expression of CD44, CD122, CXCR3, and Eomes, as well as the production of IFN-γ, was greatly reduced among thymic CD8+ T cells. The loss of this innate CD8+ T cell population in BALB/Mtv-null mice correlated with a reduction in PLZF+, IL-4 secreting αβ NKT cells within the thymus compared to wild type BALB/c. Our findings suggest a potential Mtv-mediated role in modulation of thymic αβ NKT cell development that subsequently influences the generation of innate CD8+ T cells.

BTB-POZ transcription factors recruit the E3 ubiquitin ligase Cullin 3 to initiate lymphoid effector programs (111.38)

Abstract The differentiation of several T and B cell effector programs in the immune system is directed by signature transcription factors that induce rapid epigenetic remodeling. We report that PLZF, the BTB-POZ transcription factor directing the innate-like effector program of NKT thymocytes was prominently associated with cullin 3 (Cul3), an E3 ubiquitin ligase previously shown to use BTB domain-containing proteins as adaptors for substrate binding. PLZF induced the transport of Cul3 from the cytosol to the nucleus where the two proteins were associated within a chromatin associated/modifier protein complex. Furthermore, PLZF expression in thymocytes resulted in selective changes of ubiquitination of multiple components of this complex. Cul3 was also found associated with another BTB-POZ transcription factor, Bcl6, which directs the B cell germinal center program. Conditional deletion in mice demonstrated an absolute, cell-intrinsic requirement of Cul3 for the development of NKT cells and germinal center B cell responses. We conclude that distinct lineage-specific BTB-POZ transcription factors recruit Cul3 to alter the ubiquitination pattern of their associated chromatin associated/modifier complex. We propose that this novel function is essential to direct the differentiation of several T and B lymphocyte effector programs, and may also be involved in the oncogenic role of PLZF and Bcl6 in leukemias and lymphomas.

MicroRNA miR-150 Is Involved in Vα14 Invariant NKT Cell Development and Function

CD1d-restricted Vα14 invariant NKT (iNKT) cells play an important role in the regulation of diverse immune responses. MicroRNA-mediated RNA interference is emerging as a crucial regulatory mechanism in the control of iNKT cell differentiation and function. Yet, roles of specific microRNAs in the development and function of iNKT cells remain to be further addressed. In this study, we identified the gradually increased expression of microRNA-150 (miR-150) during the maturation of iNKT cells in thymus. Using miR-150 knockout (KO) mice, we found that miR-150 deletion resulted in an interruption of iNKT cell final maturation in both thymus and periphery. Upon activation, iNKT cells from miR-150KO mice showed significantly increased IFN-γ production compared with wild-type iNKT cells. Bone marrow-transferring experiments demonstrated the cell-intrinsic characteristics of iNKT cell maturation and functional defects in mice lacking miR-150. Furthermore, miR-150 target c-Myb was significantly upregulated in miR-150KO iNKT cells, which potentially contribute to iNKT cell defects in miR-150KO mice. Our data define a specific role of miR-150 in the development and function of iNKT cells.

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.

Critical Roles of RasGRP1 for Invariant NKT Cell Development

The invariant NKT (iNKT) cell lineage contains CD4(+) and CD4(-) subsets. The mechanisms that control such subset differentiation and iNKT cell maturation in general have not been fully understood. RasGRP1, a guanine nucleotide exchange factor for TCR-induced activation of the Ras-ERK1/2 pathway, is critical for conventional αβ T cell development but dispensable for generating regulatory T cells. Its role in iNKT cells has been unknown. In this study, we report severe decreases of iNKT cells in RasGRP1(-/-) mice through cell intrinsic mechanisms. In the remaining iNKT cells in RasGRP1(-/-) mice, there is a selective absence of the CD4(+) subset. Furthermore, RasGRP1(-/-) iNKT cells are defective in TCR-induced proliferation in vitro. These observations establish that RasGRP1 is not only important for early iNKT cell development but also for the generation/maintenance of the CD4(+) iNKT cells. Our data provide genetic evidence that the CD4(+) and CD4(-) iNKT cells are distinct sublineages with differential signaling requirements for their development.