The Transcriptional Repressor Gfi1 Plays a Critical Role in the Development of NKT1- and NKT2-Type iNKT Cells.
ABSTRACT: Gfi1 plays an important role in the development and maintenance of many hematopoietic linage cells. However, the impact of Gfi1-deficiency on the iNKT cell differentiation remains unclear. We herein demonstrate a critical role of Gfi1 in regulating the development of iNKT cell subsets. In the thymus of T cell-specific Gfi1-deficient mice, iNKT cells normally developed up to stage 2, while the number of stage 3 NK1.1pos iNKT cells was significantly reduced. Furthermore, CD4pos iNKT cells were selectively reduced in the peripheral organs of T cell-specific Gfi1-deficient mice. The ?-GalCer-dependent production of IFN-?and Th2 cytokines, but not IL-17A, was severely reduced in T cell-specific Gfi1-deficient mice. In addition, a reduction of the ?-GalCer-induced anti-tumor activity was observed in Gfi1-deficient mice. These findings demonstrate the important role of Gfi1 in regulating the development and function of NKT1- and NKT2-type iNKT cell subsets.
Project description:Invariant natural killer T (iNKT) cells develop into three subsets (NKT1, NKT2, and NKT17) expressing a distinct transcription factor profile, which regulates cytokine secretion upon activation. iNKT cell development in the thymus is modulated by signaling lymphocytic activation molecule family (SLAMF) receptors. In contrast to other SLAMF members, Ly9 (SLAMF3) is a non-redundant negative regulator of iNKT cell development. Here, we show that Ly9 influences iNKT cell lineage differentiation. Ly9-deficient mice on a BALB/c background contained a significantly expanded population of thymic NKT2 cells, while NKT1 cells were nearly absent in BALB/c.Ly9-/- thymus. Conversely, the number of peripheral NKT1 cells in BALB/c.Ly9-/- mice was comparable to that in wild-type mice, indicating that the homeostasis of the different iNKT cell subsets may have distinct requirements depending on their tissue localization. Importantly, Ly9 absence also promoted NKT2 cell differentiation in the NKT1-skewed C57BL/6 background. Furthermore, treatment of wild-type mice with an agonistic monoclonal antibody directed against Ly9 impaired IL-4 and IFN-? production and reduced by half the number of spleen iNKT cells, with a significant decrease in the proportion of NKT2 cells. Thus, anti-Ly9 targeting could represent a novel therapeutic approach to modulate iNKT cell numbers and activation.
Project description:Invariant natural killer T (iNKT) cells recognize glycolipids as antigens and diversify into NKT1 (IFN-?), NKT2 (IL-4), and NKT17 (IL-17) functional subsets while developing in the thymus. Mechanisms that govern the balance between these functional subsets are poorly understood due, partly, to the lack of distinguishing surface markers. Here we identify the heparan sulfate proteoglycan syndecan-1 (sdc1) as a specific marker of naïve thymic NKT17 cells in mice and show that sdc1 deficiency significantly increases thymic NKT17 cells at the expense of NKT1 cells, leading to impaired iNKT cell-derived IFN-?, both in vitro and in vivo. Using surface expression of sdc1 to identify NKT17 cells, we confirm differential tissue localization and interstrain variability of NKT17 cells, and reveal that NKT17 cells express high levels of TCR-?, preferentially use V?8, and are more highly sensitive to ?-GalCer than to CD3/CD28 stimulation. These findings provide a novel, noninvasive, simple method for identification, and viable sorting of naïve NKT17 cells from unmanipulated mice, and suggest that sdc1 expression negatively regulates homeostasis in iNKT cells. In addition, these findings lay the groundwork for investigating the mechanisms by which sdc1 regulates NKT17 cells.
Project description:Three subsets of invariant natural killer T (iNKT) cells have been identified, NKT1, NKT2, and NKT17, which produce distinct cytokines when stimulated, but little is known about their localization. Here, we have defined the anatomic localization and systemic distribution of these subsets and measured their cytokine production. Thymic NKT2 cells that produced interleukin-4 (IL-4) at steady state were located in the medulla and conditioned medullary thymocytes. NKT2 cells were abundant in the mesenteric lymph node (LN) of BALB/c mice and produced IL-4 in the T cell zone that conditioned other lymphocytes. Intravenous injection of ?-galactosylceramide activated NKT1 cells with vascular access, but not LN or thymic NKT cells, resulting in systemic interferon-? and IL-4 production, while oral ?-galactosylceramide activated NKT2 cells in the mesenteric LN, resulting in local IL-4 release. These findings indicate that the localization of iNKT cells governs their cytokine response both at steady state and upon activation.
Project description:Invariant NKT (iNKT) cells are a unique lineage with characteristics of both adaptive and innate lymphocytes, and they recognize glycolipids presented by an MHC class I-like CD1d molecule. During thymic development, iNKT cells also differentiate into NKT1, NKT2, and NKT17 functional subsets that preferentially produce cytokines IFN-?, IL-4, and IL-17, respectively, upon activation. Newly selected iNKT cells undergo a burst of proliferation, which is defective in mice with a specific deletion of NKAP in the iNKT cell lineage, leading to severe reductions in thymic and peripheral iNKT cell numbers. The decreased cell number is not due to defective homeostasis or increased apoptosis, and it is not rescued by Bcl-xL overexpression. NKAP is also required for differentiation into NKT17 cells, but NKT1 and NKT2 cell development and function are unaffected. This failure in NKT17 development is rescued by transgenic expression of promyelocytic leukemia zinc finger; however, the promyelocytic leukemia zinc finger transgene does not restore iNKT cell numbers or the block in positive selection into the iNKT cell lineage in CD4-cre NKAP conditional knockout mice. Therefore, NKAP regulates multiple steps in iNKT cell development and differentiation.
Project description:Invariant natural killer T cells (iNKT cells) can produce copious amounts of interleukin 4 (IL-4) early during infection. However, indirect evidence suggests they may produce this immunomodulatory cytokine in the steady state. Through intracellular staining for transcription factors, we have defined three subsets of iNKT cells (NKT1, NKT2 and NKT17) that produced distinct cytokines; these represented diverse lineages and not developmental stages, as previously thought. These subsets exhibited substantial interstrain variation in numbers. In several mouse strains, including BALB/c, NKT2 cells were abundant and were stimulated by self ligands to produce IL-4. In those strains, steady-state IL-4 conditioned CD8(+) T cells to become 'memory-like', increased serum concentrations of immunoglobulin E (IgE) and caused dendritic cells to produce chemokines. Thus, iNKT cell-derived IL-4 altered immunological properties under normal steady-state conditions.
Project description:NKT cells are a distinct subset that have developmental requirements that often differ from conventional T cells. Here, we show that NKT-specific deletion of Hdac3 results in a severe reduction in the number of iNKT cells, particularly of NKT1 cells. In addition, there is decreased cytokine production by Hdac3-deficient NKT2 and NKT17 cells. Hdac3-deficient iNKT cells have increased cell death that is not rescued by transgenic expression of Bcl-2 or Bcl-xL. Hdac3-deficient iNKT cells have less Cyto-ID staining and lower LC3A/B expression, indicative of reduced autophagy. Interestingly, Hdac3-deficient iNKT cells also have lower expression of the nutrient receptors GLUT1, CD71 and CD98, which would increase the need for autophagy when nutrients are limiting. Therefore, Hdac3 is required for iNKT cell development and differentiation.
Project description:Peripheral invariant NKT cells (iNKT) and CD8+ tissue-resident memory T cells (TRM) express high levels of the extracellular ATP receptor P2RX7 in mice. High extracellular ATP concentrations or NAD-mediated P2RX7 ribosylation by the enzyme ARTC2.2 can induce P2RX7 pore formation and cell death. Because both ATP and NAD are released during tissue preparation for analysis, cell death through these pathways may compromise the analysis of iNKT and CD8+ TRM Indeed, ARTC2.2 blockade enhanced recovery of viable liver iNKT and TRM The expression of ARTC2.2 and P2RX7 on distinct iNKT subsets and TRM is unclear, however, as is the impact of recovery from other nonlymphoid sites. In this study, we performed a comprehensive analysis of ARTC2.2 and P2RX7 expression in iNKT and CD8+ T cells in diverse tissues, at steady-state and after viral infection. NKT1 cells and CD8+ TRM express high levels of both ARTC2.2 and P2RX7 compared with NKT2, NKT17, and CD8+ circulating memory subsets. Using nanobody-mediated ARTC2.2 antagonism, we showed that ARTC2.2 blockade enhanced NKT1 and TRM recovery from nonlymphoid tissues during cell preparation. Moreover, blockade of this pathway was essential to preserve functionality, viability, and proliferation of both populations. We also showed that short-term direct P2RX7 blockade enhanced recovery of TRM, although to a lesser degree. In summary, our data show that short-term in vivo blockade of the ARTC2.2/P2RX7 axis permits much improved flow cytometry-based phenotyping and enumeration of murine iNKT and TRM from nonlymphoid tissues, and it represents a crucial step for functional studies of these populations.
Project description:T cell immunoglobulin and mucin-4 (TIM-4), mainly expressed on antigen presenting cells, plays a versatile role in immunoregulation. CD1d-restricted invariant natural killer T (iNKT) cells are potent cells involved in the diverse immune responses. It was recently reported that recombinant TIM-4 (rTIM-4) alone enhanced cytokine production in NKT hybridoma, DN32.D3 cells. Hence, we hypothesized that TIM-4 might regulate iNKT cell biology, especially their function of cytokine secretion. For the first time, we identified that TIM-4 was expressed in thymus iNKT cells, and its expression increased upon iNKT cell migration to the secondary lymphoid organs, especially in lymph nodes. Using TIM-4-deficient mice, we found that lack of TIM-4 did not disturb iNKT cell development, maturation, peripheral homeostasis and cytokine secretion. Moreover, TIM-4 deficiency did not alter the polarization of iNKT sublineages, including NKT1, NKT2 and NKT17. Finally, the mixed bone marrow transfer experiments further confirmed normal iNKT cell development and function from TIM-4-deficient bone marrow. In conclusion, our data suggest that TIM-4 is expressed on iNKT cells but dispensable for their development and function.
Project description:The thymus supports multiple ?? T cell lineages that are functionally distinct, but mechanisms that control this multifaceted development are poorly understood. Here we examine medullary thymic epithelial cell (mTEC) heterogeneity and its influence on CD1d-restricted iNKT cells. We find three distinct mTEClow subsets distinguished by surface, intracellular and secreted molecules, and identify LT?R as a cell-autonomous controller of their development. Importantly, this mTEC heterogeneity enables the thymus to differentially control iNKT sublineages possessing distinct effector properties. mTEC expression of LT?R is essential for the development thymic tuft cells which regulate NKT2 via IL-25, while LT?R controls CD104+CCL21+ mTEClow that are capable of IL-15-transpresentation for regulating NKT1 and NKT17. Finally, mTECs regulate both iNKT-mediated activation of thymic dendritic cells, and iNKT availability in extrathymic sites. In conclusion, mTEC specialization controls intrathymic iNKT cell development and function, and determines iNKT pool size in peripheral tissues.
Project description:Various subsets of invariant natural killer T (iNKT) cells with different cytokine productions develop in the mouse thymus, but the factors driving their differentiation remain unclear. Here we show that hypomorphic alleles of Zap70 or chemical inhibition of Zap70 catalysis leads to an increase of IFN-?-producing iNKT cells (NKT1 cells), suggesting that NKT1 cells may require a lower TCR signal threshold. Zap70 mutant mice develop IL-17-dependent arthritis. In a mouse experimental arthritis model, NKT17 cells are increased as the disease progresses, while NKT1 numbers negatively correlates with disease severity, with this protective effect of NKT1 linked to their IFN-? expression. NKT1 cells are also present in the synovial fluid of arthritis patients. Our data therefore suggest that TCR signal strength during thymic differentiation may influence not only IFN-? production, but also the protective function of iNKT cells in arthritis.