NKT Cell Differentiation Research Articles

IL-27 regulates the differentiation of follicular helper NKT cells via metabolic adaptation of mitochondria

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that express an invariant T cell receptor α chain and contribute to bridging innate and acquired immunity with rapid production of large amounts of cytokines after stimulation. Among effecter subsets of iNKT cells, follicular helper NKT (NKTFH) cells are specialized to help B cells. However, the mechanisms of NKTFH cell differentiation remain to be elucidated. In this report, we studied the mechanism of NKTFH cell differentiation induced by pneumococcal surface protein A and α-galactosylceramide (P/A) vaccination. We found that Gr-1+ cells helped iNKT cell proliferation and NKTFH cell differentiation in the spleen by producing interleukin-27 (IL-27) in the early phase after vaccination. The neutralization of IL-27 impaired NKTFH cell differentiation, which resulted in compromised antibody production and diminished protection against Streptococcus pneumoniae infection by the P/A vaccine. Our data indicated that Gr-1+ cell-derived IL-27 stimulated mitochondrial metabolism, meeting the energic demand required for iNKT cells to differentiate into NKTFH cells. Interestingly, Gr-1+ cell-derived IL-27 was induced by iNKT cells via interferon-γ production. Collectively, our findings suggest that optimizing the metabolism of iNKT cells was essential for acquiring specific effector functions, and they provide beneficial knowledge on iNKT cell-mediated vaccination-mediated therapeutic strategies.

PLZF Acetylation Levels Regulate NKT Cell Differentiation.

The transcription factor promyelocytic leukemia zinc finger (PLZF) is encoded by the BTB domain-containing 16 (Zbtb16) gene. Its repressor function regulates specific transcriptional programs. During the development of invariant NKT cells, PLZF is expressed and directs their effector program, but the detailed mechanisms underlying PLZF regulation of multistage NKT cell developmental program are not well understood. This study investigated the role of acetylation-induced PLZF activation on NKT cell development by analyzing mice expressing a mutant form of PLZF mimicking constitutive acetylation (PLZFON) mice. NKT populations in PLZFON mice were reduced in proportion and numbers of cells, and the cells present were blocked at the transition from developmental stage 1 to stage 2. NKT cell subset differentiation was also altered, with T-bet+ NKT1 and RORγt+ NKT17 subsets dramatically reduced and the emergence of a T-bet-RORγt- NKT cell subset with features of cells in early developmental stages rather than mature NKT2 cells. Preliminary analysis of DNA methylation patterns suggested that activated PLZF acts on the DNA methylation signature to regulate NKT cells' entry into the early stages of development while repressing maturation. In wild-type NKT cells, deacetylation of PLZF is possible, allowing subsequent NKT cell differentiation. Interestingly, development of other innate lymphoid and myeloid cells that are dependent on PLZF for their generation is not altered in PLZFON mice, highlighting lineage-specific regulation. Overall, we propose that specific epigenetic control of PLZF through acetylation levels is required to regulate normal NKT cell differentiation.

Top Reads.

Abstract Antibacterial Autoantibodies See article p. 777 Acetylation Regulates NKT Cell Differentiation See article p. 809 Shark MHC I See article p. 824 Vaccines against Opioids See article p. 860 Aiding Antifungal Immunity See article p. 868 NK Cell Adhesome See article p. 950 MicroRNA-Mediated T Cell Regulation See article p. 985

Unexpected Pro-Fibrotic Effect of MIF in Non-Alcoholic Steatohepatitis Is Linked to a Shift in NKT Cell Populations.

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine with anti-fibrotic properties in toxic liver injury models and anti-steatotic functions in non-alcoholic fatty liver disease (NAFLD) attributed to the CD74/AMPK signaling pathway. As NAFLD progression is associated with fibrosis, we studied MIF function during NAFLD-associated liver fibrogenesis in mice and men by molecular, histological and immunological methods in vitro and in vivo. After NASH diet feeding, hepatic Mif expression was strongly induced, an effect which was absent in Mif∆hep mice. In contrast to hepatotoxic fibrosis models, NASH diet-induced fibrogenesis was significantly abrogated in Mif−/− and Mif∆hep mice associated with a reduced accumulation of the pro-fibrotic type-I NKT cell subpopulation. In vitro, MIF skewed the differentiation of NKT cells towards the type-I subtype. In line with the murine results, expression of fibrosis markers strongly correlated with MIF, its receptors, and markers of NKT type-I cells in NASH patients. We conclude that MIF expression is induced during chronic metabolic injury in mice and men with hepatocytes representing the major source. In NAFLD progression, MIF contributes to liver fibrogenesis skewing NKT cell polarization toward a pro-fibrotic phenotype highlighting the complex, context-dependent role of MIF during chronic liver injury.

MiR-183-96-182 Cluster Is Involved in Invariant NKT Cell Development, Maturation, and Effector Function.

The development, differentiation and function of invariant NKT (iNKT) cells require a well-defined set of transcription factors, but how these factors are integrated to each other and the detailed signaling networks remain poorly understood. Using a Dicer-deletion mouse model, our previous studies have demonstrated the critical involvement of microRNAs (miRNAs) in iNKT cell development and function, but the role played by individual miRNAs in iNKT cell development and function is still not clear. In this study, we show the dynamic changes of miRNA 183 cluster (miR-183C) expression during iNKT cell development. Mice with miR-183C deletion showed a defective iNKT cell development, sublineage differentiation, and cytokine secretion function. miRNA target identification assays indicate the involvement of multiple target molecules. Our study not only confirmed the role of miR-183C in iNKT cell development and function but also demonstrated that miR-183C achieved the regulation of iNKT cells through integrated targeting of multiple signaling molecules and pathways.

Eomes transcription factor is required for the development and differentiation of invariant NKT cells

Eomes regulates the differentiation of CD8+ T cells into effector and memory phases. However, its role in invariant (i)NKT cells remains unknown. Here, we show the impact of Eomes on iNKT cells in the thymus and peripheral tissue using conditional knockout (Eomes-cKO) mice. In the thymus, CD1d-tetramer+CD24+CD44−NK1.1−CD69+stage 0 iNKT cells express higher levels of Eomes than the other iNKT stages. We also found that Eomes regulates NKT1 cell differentiation predominantly. Interestingly, the expression of Eomes in the steady state is low, but can be upregulated after TCR stimulation. We also showed epigenetic changes in the Eomes locus after activation. In addition, vaccination of C57BL/6, but not Eomes-cKO mice with iNKT ligand-loaded dendritic cells generated KLRG1+iNKT cells in lung, characterized as effector memory phenotype by transcriptome profiling. Thus, Eomes regulates not only the differentiation of NKT1 cells in the thymus, but also their differentiation into memory-like KLRG1+iNKT cells in the periphery.

NKAP Regulates Invariant NKT Cell Proliferation and Differentiation into ROR-γt-Expressing NKT17 Cells.

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-γ, 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.

CRISPR-Mediated Slamf1Δ/Δ Slamf5Δ/Δ Slamf6Δ/Δ Triple Gene Disruption Reveals NKT Cell Defects but Not T Follicular Helper Cell Defects.

SAP (SH2D1A) is required intrinsically in CD4 T cells to generate germinal center responses and long-term humoral immunity. SAP binds to SLAM family receptors, including SLAM, CD84, and Ly108 to enhance cytokine secretion and sustained T cell:B cell adhesion, which both improve T follicular helper (Tfh) cell aid to germinal center (GC) B cells. To understand the overlapping roles of multiple SLAM family receptors in germinal center responses, Slamf1Δ/Δ Slamf5Δ/Δ Slamf6Δ/Δ triple gene disruption (Slamf1,5,6Δ/Δ) mice were generated using CRISPR-Cas9 gene editing to eliminate expression of SLAM (CD150), CD84, and Ly108, respectively. Gene targeting was highly efficient, with 6 of 6 alleles disrupted in 14 of 23 pups and the majority of alleles disrupted in the remaining pups. NKT cell differentiation in Slamf1,5,6Δ/Δ mice was defective, but not completely absent. The remaining NKT cells exhibited substantially increased 2B4 (SLAMF4) expression. Surprisingly, there were no overt defects in germinal center responses to acute viral infections or protein immunizations in Slamf1,5,6Δ/Δ mice, unlike Sh2d1a-/- mice. Similarly, in the context of a competitive environment, SLAM family receptor expressing GC Tfh cell, GC B cell, and plasma cell responses exhibited no advantages over Slamf1,5,6Δ/Δ cells.

PLZF Controls the Development of Fetal-Derived IL-17+Vγ6+ γδ T Cells.

Expression of promyelocytic leukemia zinc finger (PLZF) protein directs the effector differentiation of invariant NKT (iNKT) cells and IL-4(+) γδ NKT cells. In this study, we show that PLZF is also required for the development and function of IL-17(+) γδ T cells. We observed that PLZF is expressed in fetal-derived invariant Vγ5(+) and Vγ6(+) γδ T cells, which secrete IFN-γ and IL-17, respectively. PLZF deficiency specifically affected the effector differentiation of Vγ6(+) cells, leading to reduced numbers of mature CD27(-)CD44(+) phenotype capable of secreting IL-17. Although PLZF was not required for Vγ5(+) γδ T cells to develop, when these cells were reprogrammed into IL-17-secreting cells in Skint-1 mutant mice, they required PLZF for their effector maturation, similarly to Vγ6(+) γδ T cells. The impaired effector differentiation of PLZF-deficient Vγ6(+) γδ T cells was not due to increased apoptosis and it was related to reduced proliferation of immature CD27(+)CD44(-) Vγ6(+) γδ T cells, which was required for their differentiation into mature CD27(-)CD44(+) IL-17-secreting cells. Thus, the present study identifies that PLZF function is not restricted to NKT or IL-4(+) T cells, but it also controls the development of IL-17(+) γδ T cells.

Notch 1 and Notch 2 synergistically regulate the differentiation and function of invariant NKT cells.

Invariant natural killer T cells are a distinct subset of T cells that exert Janus-like functions. Moreover, Notch signaling is known to have critical roles in the development and functions of T cells. However, it is not known whether Notch signaling contributes to the development or functions of invariant natural killer T cells. Here, we found that CD4-specific gene ablation of Notch 1 and Notch 2 (N1N2(-/-)) increased the number of invariant natural killer T cells in the thymus but decreased them in the liver. N1N2(-/-) mice showed impaired thymic maturation of invariant natural killer T cells from the NK1.1(-)CD44(+) to the NK1.1(+)CD44(+) stage, resulting in accumulation of NK1.1(-)CD44(+) invariant natural killer T cells in the thymus. Upon activation, hepatic invariant natural killer T cells from N1N2(-/-) mice produced lower cytokine levels and increased apoptosis versus wild-type invariant natural killer T cells. Furthermore, Notch 1/Notch 2-deficient, but not wild type, invariant natural killer T cells failed to promote antibody-induced arthritis in CD1d(-/-) mice. Unlike N1N2(-/-) mice, RBP-j(lox) (/) (lox) CD4-Cre mice showed similar percentages and numbers of thymic invariant natural killer T cells to wild-type mice but had defects in their homeostasis, maturation, and cytokine production in the liver. Taken together, our data indicate distinct effects of Notch signaling on invariant natural killer T cells in the thymus and liver, which are at least partly independent of RBP-j in the thymus.

Differential dependence on nuclear factor-κB-inducing kinase among natural killer T-cell subsets in their development.

Natural killer T cells (NKT cells) are comprised of several subsets. However, the possible differences in their developmental mechanisms have not been fully investigated. To evaluate the dependence of some NKT subpopulations on nuclear factor-κB-inducing kinase (NIK) for their generation, we analysed the differentiation of NKT cells, dividing them into subsets in various tissues of alymphoplasia (aly/aly), a mutant mouse strain that lacks functional NIK. The results indicated that the efficient differentiation of both invariant NKT (iNKT) and non-iNKT cells relied on NIK expression in non-haematopoietic cells; however, the dependence of non-iNKT cells was lower than that of iNKT cells. Especially, the differentiation of CD8(+) non-iNKT cells was markedly resistant to the aly mutation. The proportion of two other NKT cell subsets, NK1.1(+) γδ T cells and NK1.1(-) iNKT cells, was also significantly reduced in aly/aly mice, and this defect in their development was reversed in wild-type host mice given aly/aly bone marrow cells. In exerting effector functions, NIK in NKT-αβ cells appeared dispensable, as NIK-deficient NKT-αβ cells could secrete interleukin-4 or interferon-γ and exhibit cytolytic activity at a level comparable to that of aly/+ NKT-αβ cells. Collectively, these results imply that the NIK in thymic stroma may be critically involved in the differentiation of most NKT cell subsets (although the level of NIK dependence may vary among the subsets), and also that NIK in NKT-αβ cells may be dispensable for their effector function.

Invariant NKT cells require autophagy to coordinate proliferation and survival signals during differentiation.

Autophagy regulates cell differentiation, proliferation, and survival in multiple cell types, including cells of the immune system. In this study, we examined the effects of a disruption of autophagy on the differentiation of invariant NKT (iNKT) cells. Using mice with a T lymphocyte-specific deletion of Atg5 or Atg7, two members of the macroautophagic pathway, we observed a profound decrease in the iNKT cell population. The deficit is cell-autonomous, and it acts predominantly to reduce the number of mature cells, as well as the function of peripheral iNKT cells. In the absence of autophagy, there is reduced progression of iNKT cells in the thymus through the cell cycle, as well as increased apoptosis of these cells. Importantly, the reduction in Th1-biased iNKT cells is most pronounced, leading to a selective reduction in iNKT cell-derived IFN-γ. Our findings highlight the unique metabolic and genetic requirements for the differentiation of iNKT cells.

Let-7 microRNAs target the lineage-specific transcription factor PLZF to regulate terminal NKT cell differentiation and effector function.

Lethal-7 (let-7) microRNAs are the most abundant in the genome but their role in developing thymocytes is unclear. We now report that let-7 miRNAs target Zbtb16 mRNA, which encodes the lineage-specific transcription factor PLZF, to post-transcriptionally regulate PLZF expression and NKT cell effector function. Dynamic up-regulation of let-7 miRNAs during NKT thymocyte development down-regulates PLZF expression and directs terminal differentiation into interferon-γ-producing NKT1 cells. Without let-7 up-regulation, NKT thymocytes maintain high PLZF expression and terminally differentiate into IL-4-producing NKT2 and IL-17-producing NKT17 cells. Let-7 up-regulation in developing NKT thymocytes can be signaled by IL-15, vitamin D and retinoic acid. Such miRNA targeting of a lineage-specific transcription factor constitutes a new level of developmental regulation in the thymus.

Mammalian target of rapamycin complex 1 orchestrates invariant NKT cell differentiation and effector function.

Invariant NKT (iNKT) cells play critical roles in bridging innate and adaptive immunity. The Raptor containing mTOR complex 1 (mTORC1) has been well documented to control peripheral CD4 or CD8 T cell effector or memory differentiation. However, the role of mTORC1 in iNKT cell development and function remains largely unknown. By using mice with T cell-restricted deletion of Raptor, we show that mTORC1 is selectively required for iNKT but not for conventional T cell development. Indeed, Raptor-deficient iNKT cells are mostly blocked at thymic stage 1-2, resulting in a dramatic decrease of terminal differentiation into stage 3 and severe reduction of peripheral iNKT cells. Moreover, residual iNKT cells in Raptor knockout mice are impaired in their rapid cytokine production upon αGalcer challenge. Bone marrow chimera studies demonstrate that mTORC1 controls iNKT differentiation in a cell-intrinsic manner. Collectively, our data provide the genetic evidence that iNKT cell development and effector functions are under the control of mTORC1 signaling.

Phenotypic Analyses of T, B, NK and NKT Cells in a Human Fetus with D and C Alloimmune Anemia

Fetal hypoxygenation due to the anemia may be an important factor in stimulating the maturation processes of the immune system in the fetus, determining the switch from fetal to adult form. We performed an immunological evaluation in a fetus in the 29th week of gestation with secondary hypoxemia and RhD and C alloimmune anemia. We found normal relative values for total T, TCD4+ and TCD8+, and low levels of B memory and NK cells. NKT cells were absent. There IL-8 serum levels were higher than those of the mother. Fetal anemia was not capable of activating the humoral immune response and differentiation of memory B cells. Our data suggest that NKT cell differentiation may be related to thymus development in humans.

Hormonal regulation of the thymic stage of differentiation of invariant NKT cells

Hormonal regulation of the thymic stage of differentiation of invariant NKT cells

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.

Pregnancy alters T-cell development in the mouse thymus (115.11)

Abstract The syngeneic and allogeneic mating combinations were used to assess the effect of pregnancy-associated hormones and fetal antigens on different T-cell subsets and their a2V-ATPase (a2V) expression during pregnancy. Our data shows that a2V was significantly (p≤0.05) increased in thymus recovered from normal mating combinations (♀BALB/cX♂BALB/c and ♀CBAX♂BALB/c) when compared with abortion prone mating combinations (♀CBAX♂DBA). The percentage of NK-T cells (CD3+NKp46+ cells) was significantly (p≤0.05) increased in all studied mating combinations on days 12 and 16 of pregnancy when compared to their respective non-pregnant females and pups. The expression of a2V on NK-T cells was also significantly (p≤0.05) increased as their numbers was increased. The numbers of CD3+CD4+CD8- cells were significantly (p≤0.05) increased, while the numbers of CD3-CD4-CD8- and CD3+CD4+CD8+ cells were significantly (p≤0.05) decreased in all the mating combinations when compared to their respective non-pregnant females and pups. This dynamic change in the numbers of T-cells subsets during pregnancy suggests that T-cells development and differentiation is altered in pregnancy. Moreover, a2V may play a crucial role in differentiation of NK-T cells from different T-cells subsets. Future studies to decipher the mechanism of action of a2V in thymic T-cells development and differentiation will lead to valuable knowledge of thymus development as well as its role in maternal fetal allogeneic relationship.

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.

The sequential activity of Gata3 and Thpok is required for the differentiation of CD1d‐restricted CD4+ NKT cells

While most CD4(+) T cells are MHC class II-restricted, a small subset, including the CD1d-restricted 'invariant' NKT (iNKT) cells, are selected on non-classical MHC-I or MHC-I-like molecules. We previously showed that the sequential activity of two zinc finger transcription factors, Gata3 and Thpok, promotes the differentiation of conventional, MHC II-restricted thymocytes into CD4(+) T cells. In the current study, we show that a Gata3-Thpok cascade is required for the differentiation of CD4(+) iNKT cells. Gata3 is required for iNKT cells to express Thpok, whereas Thpok is needed for proper NKT cell differentiation, and notably for NKT cells to maintain CD4 and terminate CD8 expression. These findings identify the sequential activity of Gata3 and Thpok as a hallmark of CD4(+) T-cell differentiation, regardless of MHC restriction.