Genetic Regulation of NKT Cell Function

NKT cells are specialized T cells that play important roles in the host immune response to bacteria and viruses. NKT cells produce a wide variety of cytokines and chemokines after being activated by glycolipids such as α-galactosylceramide (αGalCer). Previous work suggested that the ability of NKT cells to be activated by aGalCer mapped to a genetic region encompassing a gene family (Slam genes) that is known to be important in NKT cell development, but the exact gene in this region which regulates NKT cells is unknown. This study utilizes a panel of C57BL/6 (B6) mice containing different regions of chromosome 1 derived from 129X1/SvJ mice (B6.129 congenics) to identify candidate genes regulating NKT cell function by positionally mapping the genes within this locus. We assessed NKT cell function in B6.129c2 (C2), B6.129c3 (C3), and B6.129c4 (C4) mice, which contain 129 intervals ranging from 0.1-1 megabase pairs (Mbp). To assess NKT cell function, we injected mice with αGalCer, which specifically activates NKT cells. Flow cytometry was utilized to determine NKT cell IL-4, TNF, and IFN-g expression on a per cell basis and ELISA assays were conducted to observe the overall magnitude of the NKT cell response. There was a significant reduction in the TNF, IL-4, and IFNγ production in all congenic mice as compared to B6 controls. These data suggested that the NKT cell response to αGalCer mapped to a 0.1 Mbp region on chromosome 1 (the C3 interval), which excluded Slam genes as potential genes regulating these NKT cell functions. Possible candidate genes of interest in this locus are ApoA2, which encodes a protein involved in lipid transport, and Fcer1g, which encodes a protein that has recently been implicated in the development of different NKT cell subsets

Immune evasion of the CD1d/NKT cell axis

Many reviews on the CD1d/NKT cell axis focus on the ability of CD1d-restricted NKT cells to serve as effector cells in a variety of disorders, be they infectious diseases, cancer or autoimmunity. In contrast, here, we discuss the ways that viruses, bacteria and tumor cells can evade the CD1d/NKT cell axis. As a result, these disease states have a better chance to establish a foothold and potentially cause problems for the subsequent adaptive immune response, as the host tries to rid itself of infections or tumors

Kinetics and cellular site of glycolipid loading control

CD1d-restricted natural killer T cells (NKT cells) possess a wide range of effector and regulatory activities that are related to their ability to secrete both T helper 1 (Th1) cell- and Th2 cell-type cytokines. We analyzed presentation of NKT cell activating α galactosylceramide (αGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation. Using a monoclonal antibody specific for αGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokinebiasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins. Complexes formed by association of these Th2 cell-type cytokine-biasing αGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells. These findings help to explain how subtle alterations in glycolipid ligand structure can control the balance of proinflammatory and antiinflammatory activities of NKT cells

Early growth response gene-2 (Egr-2) regulates the development of B and T cells

The study was supported by Arthritis Research UK. Copyright @ 2011 Li et al.BACKGROUND: Understanding of how transcription factors are involved in lymphocyte development still remains a challenge. It has been shown that Egr-2 deficiency results in impaired NKT cell development and defective positive selection of T cells. Here we investigated the development of T, B and NKT cells in Egr-2 transgenic mice and the roles in the regulation of distinct stages of B and T cell development. METHODS AND FINDINGS: The expression of Egr1, 2 and 3 were analysed at different stages of T and B cell development by RT-PCT and results showed that the expression was strictly regulated at different stages. Forced expression of Egr-2 in CD2+ lymphocytes resulted in a severe reduction of CD4+CD8+ (DP) cells in thymus and pro-B cells in bone marrow, which was associated with reduced expression of Notch1 in ISP thymocytes and Pax5 in pro-B cells, suggesting that retraction of Egr-2 at the ISP and pro-B cell stages is important for the activation of lineage differentiation programs. In contrast to reduction of DP and pro-B cells, Egr-2 enhanced the maturation of DP cells into single positive (SP) T and NKT cells in thymus, and immature B cells into mature B cells in bone marrow. CONCLUSIONS: Our results demonstrate that Egr-2 expressed in restricted stages of lymphocyte development plays a dynamic, but similar role for the development of T, NKT and B cells.This article is provided by the Brunel Open Access publishing fund

Transcriptional analysis of natural killer T cell development

Type 1 NKT cells are an innate-like population of T cells that rapidly respond to both cytokine and TCR stimulation by the production of pro-inflammatory and immunoregulatory cytokines. Different from conventional αβT cells, which express a diverse repertoire of TCR sequences that are generated by random rearrangement and are positively selected by thymic epithelium expressing MHC Class I and Class II products, type 1 NKT cells express a highly restricted TCR (Vα14-Jα18 in mice, and the homologous Vα24-Jα18 chain in humans, paired with a restricted range of β chains) and are positively selected by ligating β2 Microglobulin (β2M)/CD1d and SLAM family members expressed on CD4⁺CD8⁺ (double positive – DP) cortical thymocytes. In the periphery of mice, type 1 NKT cells express a 'memory' or 'activated' like surface phenotype (CD62L⁻CD69⁺CD44hi) and the great majority is either CD4⁺CD8⁻ (single positive; SP) or CD4⁻CD8⁻ (double negative; DN). Studies on NKT cell development indicated that they are originated from the same progenitor as conventional T cells, and branched off from the mainstream lineage at DP stage. NKT cells' development is thymus dependent, they develop in fetal thymic organ culture; neonatal thymectomy on the third day of life selectively depletes them. NKT cells are not found in the peripheral tissues of mice until 1-2 weeks after birth. Many efforts have been made to understand the molecular mechanisms that govern their commitment and homeostasis. However, the very low numbers of developing NKT cells, especially DP NKT cells at Stage 0, makes it difficult to analyse transcriptional programs controlling NKT cell development. To further dissect the events surrounding NKT cell lineage commitment and to examine transcriptional factors controlling early NKT cell ontogeny, a mouse model with increased numbers of immature type 1 NKT cells was generated on the SLAM-deficient, NOD background. These mice were found to have greatly increased numbers of CD24⁺NK1.1⁻ DP NKT cells with the characteristics of pre-selection and Stage 0 NKT cells. This provides an opportunity to compare the transcriptional profiles of these very immature CD24⁺NK1.1⁻ DP NKT cells with those of conventional T cells (CD24⁺NK1.1⁻ DP T cells), and those of more mature NKT cell subsets, including CD24⁺NK1.1⁻ CD4⁺ NKT cells and CD24⁺NK1.1⁻ DN NKT cells. Expression levels of a total of 35,556 transcripts of each biological samples of above four different cell populations were obtained by microarray analysis. Principal component analysis indicated that the four populations clearly separated, in order, across principal component 1 from CD24⁺NK1.1⁻ DP T cells, to CD24⁺NK1.1⁻ DP NKT cells, to CD24⁺NK1.1⁻ CD4⁺ NKT cells, and finally to CD24⁺NK1.1⁻ DN NKT cells. Comparison of gene expression between these populations has provided an overall transcriptional profile during TCR validation, positive selection and lineage commitment of NKT cells. These findings have further confirmed phenotypic changes during NKT cell development observed by previous studies and suggest that immature DP NKT cells are pre-selection progenitors of NKT cells. Our transcriptional regulatory network approach mapped TCR validation to the transition from DP T to DP NKT cells, while positive selection and lineage commitment were associated with the transition from DP NKT to CD4 NKT cells. This is the first time that the effects of positive and negative selection have been examined on their actual population – the immature DP NKT cells. We confirm by in vivo experimentation that both positive and negative selection occur at the latter transition, separating for the first time in any T cell population the events associated with TCR validation from those associated with positive selection. NOD.Vα14Tg mice provide a model to study the earliest identifiable stages of NKT cell commitment and differentiation, and to help dissect factors controlling the numbers and function of this important immunoregulatory population

Immuno-modulatory functions of CD1d-restricted natural killer T cells

This thesis focuses on a small population of non-conventional T lymphocytes, called natural killer T (NKT) cells. NKT cells have many unique features; such as recognition of glycolipids presented by CD1d, expression of NK receptors and rapid production of large amounts of cytokines, such as IL-4 and IFN-gamma,?upon activation. This early and robust cytokine burst is believed to give NKT cells the capacity to act as a link between innate and adaptive immunity. NKT cells are involved in a cohort of diverse immunological situations, including immune responses to tumors, pathogens, and autoimmunity, where both protective and detrimental roles have been described. In order to learn more about what genes are important for the characteristic features of NKT cells in general and for NKT cell subset in particular, we performed microarray experiments, thus studying the expression of all genes in two distinct NKT cell subsets compared to a control T cell population. We defined a gene expression profile shared by NKT cells, as well as gene profiles that were unique to the individual subsets. The common NKT cell gene expression profile suggested that NKT cells were predetermined for performing inflammatory and cytotoxic actions in non-lymphoid tissues such as gut and lung, while sharing many similarities with NK cells in activation and function. The subset unique gene profiles implied that while one subset appeared to be firmly associated with Th1-type reactions, the other subset had an enhanced potential for cytotox and for inducing Th2-associated immune responses. The NKT cells involvement in inflammatory mediation was confirmed in our last study where we showed that NKT cells were activated and potently produced IFN-gamma during the early phase of Salmonella-infection. Additionally, the infection was observed to skew the cytokine production profile of the NKT cells toward a protective IFN-gamma-dominated profile

Peripheral blood but not synovial fluid natural killer T cells are biased towards a Th1-like phenotype in rheumatoid arthritis

Natural killer T (NKT) cells have been implicated in the regulatory immune mechanisms that control autoimmunity. However, their precise role in the pathogenesis of rheumatoid arthritis (RA) remains unclear. The frequency, cytokine profile and heterogeneity of NKT cells were studied in peripheral blood mononuclear cells (PBMCs) from 23 RA patients and 22 healthy control individuals, including paired PBMC–synovial fluid samples from seven and paired PBMC–synovial tissue samples from four RA patients. Flow cytometry revealed a decreased frequency of NKT cells in PBMCs from RA patients. NKT cells were present in paired synovial fluid and synovial tissue samples. Based on the reactivity of PBMC-derived NKT cells toward α-galactosylceramide, RA patients could be divided into responders (53.8%) and nonresponders (46.2%). However, NKT cells isolated from synovial fluid from both responders and nonresponders expanded upon stimulation with α-galactosylceramide. Analysis of the cytokine profile of CD4(+ )and CD4(- )PBMC derived NKT cell lines from RA patients revealed a significantly reduced number of IL-4 producing cells. In contrast, synovial fluid derived NKT cell lines exhibited a Th0-like phenotype, which was comparable to that in healthy control individuals. This suggests that synovial fluid NKT cells are functional, even in patients with nonresponding NKT cells in their blood. We conclude that, because the number of Vα24(+)Vβ11(+)CD3(+ )NKT cells is decreased and the cytokine profile of blood-derived NKT cells is biased toward a Th1-like phenotype in RA patients, NKT cells might be functionally related to resistance or progression of RA. Providing a local boost to the regulatory potential of NKT cells might represent a useful candidate therapy for RA

Loss of Effector and Anti-Inflammatory Natural Killer T Lymphocyte Function in Pathogenic Simian Immunodeficiency Virus Infection

Chronic immune activation is a key determinant of AIDS progression in HIV-infected humans and simian immunodeficiency virus (SIV)-infected macaques but is singularly absent in SIV-infected natural hosts. To investigate whether natural killer T (NKT) lymphocytes contribute to the differential modulation of immune activation in AIDS-susceptible and AIDS-resistant hosts, we compared NKT function in macaques and sooty mangabeys in the absence and presence of SIV infection. Cynomolgus macaques had significantly higher frequencies of circulating invariant NKT lymphocytes compared to both rhesus macaques and AIDS-resistant sooty mangabeys. Despite this difference, mangabey NKT lymphocytes were functionally distinct from both macaque species in their ability to secrete significantly more IFN-γ, IL-13, and IL-17 in response to CD1d/α-galactosylceramide stimulation. While NKT number and function remained intact in SIV-infected mangabeys, there was a profound reduction in NKT activation-induced, but not mitogen-induced, secretion of IFN-γ, IL-2, IL-10, and TGF-β in SIV-infected macaques. SIV-infected macaques also showed a selective decline in CD4+ NKT lymphocytes which correlated significantly with an increase in circulating activated memory CD4+ T lymphocytes. Macaques with lower pre-infection NKT frequencies showed a significantly greater CD4+ T lymphocyte decline post SIV infection. The disparate effect of SIV infection on NKT function in mangabeys and macaques could be a manifestation of their differential susceptibility to AIDS. Alternately, these data also raise the possibility that loss of anti-inflammatory NKT function promotes chronic immune activation in pathogenic SIV infection, while intact NKT function helps to protect natural hosts from developing immunodeficiency and aberrant immune activation