Dissociated expression of natural killer 1.1 and T‐cell receptor by invariant natural killer T cells after interleukin‐12 receptor and T‐cell receptor signalling

Invariant (i) natural killer T (NKT) cells become undetectable after stimulation with α-galactosylceramide (α-GalCer) or interleukin (IL)-12. Although down-modulation of surface T-cell receptor (TCR)/NKR-P1C (NK1.1) expression has been shown convincingly after stimulation with α-GalCer, it is unclear whether this also holds true for IL-12 stimulation. To determine whether failure to detect iNKT cells after IL-12 stimulation is caused by dissociation/internalization of TCR and/or NKR-P1C, or by block of de novo synthesis of these molecules, and to examine the role of IL-12 in the disappearance of iNKT cells after stimulation with α-GalCer, surface (s)/cytoplasmic (c) protein expression, as well as messenger RNA (mRNA) expression of TCR/NKR-P1C by iNKT cells after stimulation with α-GalCer or IL-12, and the influence of IL-12 neutralization on the down-modulation of sTCR/sNKR-P1C expression by iNKT cells after stimulation with α-GalCer were examined. The s/cTCR⁺s/cNKR-P1C⁺ iNKT cells became undetectable after in vivo administration of α-GalCer, which was partially prevented by IL-12 neutralization. Whereas s/cNKR-P1C⁺ iNKT cells became undetectable after in vivo administration of IL-12, s/cTCR⁺ iNKT cells were only marginally affected. mRNA expression of TCR/NKR-P1C remained unaffected by α-GalCer or IL-12 treatment, despite the down-modulation of cTCR and/or cNKR-P1C protein expression. By contrast, cTCR⁺cNKR-P1C⁺ sTCR⁻ sNKR-P1C⁻ iNKT cells and cNKR-P1C⁺ sNKR-P1C⁻ iNKT cells were detectable after in vitro stimulation with α-GalCer and IL-12, respectively. Our results indicate that TCR and NKR-P1C expression by iNKT cells is differentially regulated by signalling through TCR and IL-12R. They also suggest that IL-12 participates, in part, in the disappearance of iNKT cells after stimulation with α-GalCer by down-modulating not only sNKR-P1C, but also sTCR.     

Invariant and noninvariant natural killer T cells exert opposite regulatory functions on the immune response during murine schistosomiasis

CD1d-restricted natural killer T (NKT) cells represent a heterogeneous population of innate memory immune cells expressing both NK and T-cell markers distributed into two major subsets, i.e., invariant NKT (iNKT) cells, which express exclusively an invariant T-cell receptor (TCR) alpha chain (Valpha14Jalpha18 in mice), and non-iNKT cells, which express more diverse TCRs. NKT cells quickly produce Th1- and/or Th2-type cytokines following stimulation with glycolipid antigen (Ag) and, through this property, play potent immunoregulatory roles in autoimmune diseases, cancer, and infection. No study has addressed the role of NKT cells in metazoan parasite infections so far. We show that during murine schistosomiasis, the apparent frequency of both iNKT cells and non-iNKT cells decreased in the spleen as early as 3 weeks postinfection (p.i.) and that both populations expressed a greater amount of the activation marker CD69 at 6 weeks p.i., suggesting an activated phenotype. Two different NKT-cell-deficient mouse models, namely, TCR Jalpha18-/- (exclusively deficient in iNKT cells) and CD1d-/- (deficient in both iNKT and non-iNKT cells) mice, were used to explore the implication of these subsets in infection. We show that whereas both iNKT and non-iNKT cells do not have a major impact on the immune response during the early phase (1 and 4 weeks) of infection, they exert important, although opposite, effects on the immune response during the acute phase of the disease (7 and 12 weeks), after schistosome egg production. Indeed, iNKT cells contribute to Th1 cell differentiation whereas non-iNKT cells might be mostly implicated in Th2 cell differentiation in response to parasite Ag. Our findings suggest, for the first time, that helminths activate both iNKT and non-iNKT cells in vivo, enabling them to differentially influence the Th1/Th2 balance of the immune response.     

Significant involvement of nuclear factor‐κB‐inducing kinase in proper differentiation of αβ and γδ T cells

Nuclear factor‐κB‐inducing kinase (NIK) is known to play a critical role in maintaining proper immune function. This is exemplified in the spontaneous mutant mouse lacking functional NIK, alymphoplasia (aly), which is simultaneously immune‐compromised and autoimmune‐prone. To investigate the role of NIK in αβ T‐cell repertoire formation, we analysed T‐cell development in aly/aly mice bearing a transgenic T‐cell receptor (TCR). Although there were no apparent abnormalities in the mature αβ T cells of non‐transgenic aly/aly mice, the maturation efficiency of idiotype^high+ T cells in the TCR‐transgenic mice was lower in aly/aly mice compared with those found in aly/+ mice, suggesting that the mature αβ T‐cell repertoire could be altered by the absence of functional NIK. In one strain of TCR‐transgenic aly/aly mice with a negatively selecting H‐2 background, the proportion of CD8^low+ idiotype^high+ cells, which are thought to potentially represent the γδ lineage of T cells, was markedly decreased. When the γδ T cells in non‐transgenic aly/aly mice were investigated, the proportion of γδ T cells in the peripheral organs of aly/aly mice was found to be one‐half to one‐fifth of those in aly/+ mice. Analyses of bone marrow chimera mice indicated that NIK in host cells, rather than in donor cells was important for generating a normal number of peripheral γδ T cells. Collectively, these results suggest that NIK could be involved in thymic positive selection of some αβ T cells and that NIK in non‐haematopoietic cells is important for the optimal development and/or maintenance of γδ T cells.     

Differential activation of CD57‐defined natural killer cell subsets during recall responses to vaccine antigens

Natural killer (NK) cells contribute to the effector phase of vaccine-induced adaptive immune responses, secreting cytokines and releasing cytotoxic granules. The proportion of responding NK cells varies between individuals and by vaccine, suggesting that functionally discrete subsets of NK cells with different activation requirements may be involved. Here, we have used responses to individual components of the DTP vaccine [tetanus toxoid (TT), diphtheria toxoid (DT), whole cell inactivated pertussis] to characterize the NK cell subsets involved in interleukin-2-dependent recall responses. Culture with TT, DT or pertussis induced NK cell CD25 expression and interferon-γ production in previously vaccinated individuals. Responses were the most robust against whole cell pertussis, with responses to TT being particularly low. Functional analysis of discrete NK cell subsets revealed that transition from CD56^bright to CD56^dim correlated with increased responsiveness to CD16 cross-linking, whereas increasing CD57 expression correlated with a loss of responsiveness to cytokines. A higher frequency of CD56^dim CD57⁻ NK cells expressed CD25 and interferon-γ following stimulation with vaccine antigen compared with CD56^dim CD57⁺ NK cells and made the largest overall contribution to this response. CD56^dim CD57^int NK cells represent an intermediate functional phenotype in response to vaccine-induced and receptor-mediated stimuli. These findings have implications for the ability of NK cells to contribute to the effector response after vaccination and for vaccine-induced immunity in older individuals.     

mTORC2 regulates multiple aspects of NKT‐cell development and function

Invariant NKT (iNKT) cells bridge innate and adaptive immunity by rapidly secreting cytokines and lysing targets following TCR recognition of lipid antigens. Based on their ability to secrete IFN‐γ, IL‐4 and IL‐17A, iNKT‐cells are classified as NKT‐1, NKT‐2, and NKT‐17 subsets, respectively. The molecular pathways regulating iNKT‐cell fate are not fully defined. Recent studies implicate Rictor, a required component of mTORC2, in the development of select iNKT‐cell subsets, however these reports are conflicting. To resolve these questions, we used Rictor^fl/fl CD4cre⁺ mice and found that Rictor is required for NKT‐17 cell development and normal iNKT‐cell cytolytic function. Conversely, Rictor is not absolutely required for IL‐4 and IFN‐γ production as peripheral iNKT‐cells make copious amounts of these cytokines. Overall iNKT‐cell numbers are dramatically reduced in the absence of Rictor. We provide data indicating Rictor regulates cell survival as well as proliferation of developing and mature iNKT‐cells. Thus, mTORC2 regulates multiple aspects of iNKT‐cell development and function.     

Engagement of glycosylphosphatidylinositol-anchored proteins results in enhanced mouse and human invariant natural killer T cell responses

Invariant natural killer T (iNKT) cells are a small subset of lymphocytes that recognize glycolipid antigens in the context of CD1d and consequently produce large quantities of pro-inflammatory and/or anti-inflammatory cytokines. Several transmembrane glycoproteins have been implicated in the co-stimulation of iNKT cell responses. However, whether glycosylphosphatidylinositol (GPI)-anchored proteins can function in this capacity is not known. Here, we demonstrate that antibody-mediated cross-linking of the prototype mouse GPI-anchored protein Thy-1 (CD90) on the surface of a double-negative (CD4⁻CD8⁻) iNKT cell line leads to cytokine production at both the mRNA and protein levels. In addition, Thy-1 triggering enhanced cytokine secretion by iNKT cells that were concomitantly stimulated with α-galactosylceramide (αGC), consistent with a co-stimulatory role for Thy-1 in iNKT cell activation. This was also evident when a CD4⁺ mouse iNKT cell line or primary hepatic NKT cells were stimulated with αGC and/or anti-Thy-1 antibody. Cross-linking Ly-6A/E, another GPI-anchored protein, could also boost cytokine secretion by αGC-stimulated iNKT cells, suggesting that the observed effects reflect a general property of GPI-anchored proteins. To extend these results from mouse to human cells, we focused on CD55, a GPI-anchored protein that, unlike Thy-1, is expressed on human iNKT cells. Cross-linking CD55 augmented αGC-induced iNKT cell responses as judged by more vigorous proliferation and higher CD69 expression. Collectively, these findings demonstrate for the first time that GPI-anchored proteins are able to co-stimulate CD1d-restricted, glycolipid-reactive iNKT cells in both mice and humans.     

Ex‐vivo analysis of human Natural Killer T cells demonstrates heterogeneity between tissues and within established CD4+ and CD4− subsets

Our understanding of human type 1 natural killer T (NKT) cells has been heavily dependent on studies of cells from peripheral blood. These have identified two functionally distinct subsets defined by expression of CD4, although it is widely believed that this underestimates the true number of subsets. Two recent studies supporting this view have provided more detail about diversity of the human NKT cells, but relied on analysis of NKT cells from human blood that had been expanded in vitro prior to analysis. In this study we extend those findings by assessing the heterogeneity of CD4⁺ and CD4⁻ human NKT cell subsets from peripheral blood, cord blood, thymus and spleen without prior expansion ex vivo, and identifying for the first time cytokines expressed by human NKT cells from spleen and thymus. Our comparative analysis reveals highly heterogeneous expression of surface antigens by CD4⁺ and CD4⁻ NKT cell subsets and identifies several antigens whose differential expression correlates with the cytokine response. Collectively, our findings reveal that the common classification of NKT cells into CD4⁺ and CD4⁻ subsets fails to reflect the diversity of this lineage, and that more studies are needed to establish the functional significance of the antigen expression patterns and tissue residency of human NKT cells.     

NF-κB-inducing kinase contributes to normal development of cortical thymic epithelial cells: its possible role in shaping a proper T-cell repertoire

Nuclear factor (NF)-κB-inducing kinase (NIK) is known to be a critical regulator of multiple aspects of the immune response. Although the role of NIK in the development of medullary thymic epithelial cells (mTECs) has been well documented, the impact of NIK on the differentiation and function of cortical thymic epithelial cells (cTECs) remains ambiguous. To investigate the possible involvement of NIK in cTEC differentiation, we have compared the gene expression and function of cTECs from a NIK-mutant mouse, alymphoplasia (aly/aly) with those of cTECs from wild-type (WT) mice. Flow cytometric analyses revealed that expression levels of MHC class II, but not MHC class I or other TEC markers, were higher in aly/aly cells than in WT cells. Notably, the proportion of MHC class II^hi+ cTECs was elevated in aly/aly mice. We also demonstrated that expression of Ccl5 mRNA in the MHC class II^hi+ subset of aly/aly cTECs was decreased compared with that in WT cells, implying an abnormal pattern of gene expression in aly/aly cTECs. Analyses of bone marrow chimera using aly/aly or aly/+ mice as hosts suggested that Vβ usage and CD5 expression on WT T-cells were altered when they matured in aly/aly thymi. These results collectively indicate that NIK may be involved in controlling the function of cTEC in selecting a proper T-cell repertoire.     

Rapid and reliable generation of invariant natural killer T-cell lines in vitro

Several tools have proved useful in the study of invariant natural killer T (iNKT) cells, including CD1d-deficient mice, Jα281-deficient mice, synthetic lipid antigens and antigen-loaded CD1d tetramers. However, the generation and examination of long-term primary murine iNKT cell lines in vitro has been challenging. Here, we show the rapid generation of iNKT cell lines from splenic iNKT cells of Vα14 T-cell receptor (TCR) transgenic (Tg) mice. These purified iNKT cells were stimulated by bone marrow-derived dendritic cells (BMDCs) loaded with α-galactosylceramide (αGalCer) and cultured with interleukin (IL)-2 and IL-7. iNKT cells proliferated dramatically, and the cell number exhibited a 100-fold increase within 2 weeks and a 10⁵-fold increase in 8 weeks after repeated stimulation with αGalCer. The iNKT cell lines consisted of iNKT cells expressing Vβ chains including Vβ8.1/8.2, Vβ14, Vβ10, Vβ6 and Vβ7, and responded to stimulation with αGalCer presented both by BMDCs and by plate-bound CD1d. In addition, the iNKT cell lines produced interferon (IFN)-γ when activated by lipopolysaccharide (LPS) or CpG oligodeoxynucleotide (ODN)-stimulated BMDCs. Further, we show that iNKT cell lines produced cytokines in response to microbial antigens. In summary, high-yield iNKT cell lines were generated very rapidly and robustly expanded, and these iNKT cells responded to both TCR and cytokine stimulation in vitro. Given the desire to study primary iNKT cells for many purposes, these iNKT cell lines should provide an important tool for the study of iNKT cell subsets, antigen and TCR specificity, activation, inactivation and effector functions.     

Interleukin‐7 supports survival of T‐cell receptor‐β‐expressing CD4− CD8− double‐negative thymocytes

Among the milestones that occur during T-cell development in the thymus is the expression of T-cell receptor-β (TCR-β) and the formation of the pre-TCR complex. Signals emanating from the pre-TCR trigger survival, proliferation and differentiation of T-cell precursors. Although the pre-TCR is essential for these cell outcomes, other receptors, such as Notch and CXCR4, also contribute. Whether interleukin-7 (IL-7) participates in promoting the survival or proliferation of pre-TCR-expressing cells is controversial. We used in vitro and in vivo models of T-cell development to examine the function of IL-7 in TCR-β-expressing thymocytes. Culturing TCR-β-expressing CD4⁻ CD8⁻ double-negative thymocytes in an in vitro model of T-cell development revealed that IL-7 reduced the frequency of CD4⁺ CD8⁺ double-positive thymocytes at the time of harvest. The mechanism for this change in the percentage of double-positive cells was that IL-7 promoted the survival of thymocytes that had not yet differentiated. By preserving the double-negative population, IL-7 reduced the frequency of double-positive thymocytes. Interleukin-7 was not required for proliferation in the in vitro system. To follow this observation, we examined mice lacking CD127 (IL-7Rα). In addition to the known effect of CD127 deficiency on T-cell development before TCR-β expression, CD127 deficiency also impaired the development of TCR-β-expressing double-negative thymocytes. Specifically, we found that Bcl-2 expression and cell cycle progression were reduced in TCR-β-expressing double-negative thymocytes in mice lacking CD127. We conclude that IL-7 continues to function after TCR-β is expressed by promoting the survival of TCR-β-expressing double-negative thymocytes.     

Impaired natural killer cell‐induced antibody‐dependent cell‐mediated cytotoxicity is associated with human immunodeficiency virus‐1 disease progression

This study evaluates the correlation between natural killer (NK) cell function and human immunodeficiency virus (HIV)-1 disease progression in 133 untreated HIV-1 positive Chinese subjects, including 41 former plasma donors (FPDs) and 92 men who have sex with men, and 35 HIV-negative controls. Flow cytometry was used to determine the abundance of NK cell subsets, the expression levels of receptor species, human leucocyte antigen (HLA) genotyping and the antibody-dependent cell-mediated cytotoxicity (ADCC) responses of NK cells. We observed a decreased expression of CD56^dimCD16⁺ NK cell subsets and an increased expression of CD56⁻CD16⁺ with HIV-1 infection. As well, the expression of activating and inhibitory receptors increased significantly in NK cells, but CD16 receptor levels and the NKG2A/NKG2C ratio were down-regulated with HIV-1 infection. ADCC responses were higher in elite controllers than in all other groups, and were correlated inversely with HIV-1 viral load but correlated positively with CD4 count only in FPDs. Furthermore, individuals infected for < 1 year have lower ADCC responses than those infected for > 1 year. We also observed a negative association between ADCC responses and viral load in those who carry the HLA-A*30/B*13/Cw*06 haplotype. The positive correlation between CD16 expression and ADCC responses and a negative correlation trend between CD158a and ADCC responses were also observed (P = 0·058). Our results showed that the ADCC response is associated with patients'' disease status, receptor expression levels, infection time and specific HLA alleles, which indicates that ADCC may offer protective effects against HIV-1 infection.     

Loss of β‐arrestin 2 exacerbates experimental autoimmune encephalomyelitis with reduced number of Foxp3+ CD4+ regulatory T cells

β-Arrestins are well-known regulators and mediators of G protein-coupled receptor signalling, and accumulating evidence reveals that they are functionally involved in inflammation and autoimmune diseases. Of the two β-arrestins, β-arrestin 1 is documented to play regulatory roles in an animal model of multiple sclerosis (MS), whereas the role of β-arrestin 2 is less clear. Here, we show that β-arrestin 2-deficient mice displayed the exacerbated and sustained symptoms of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. At the cellular level, deficiency of β-arrestin 2 led to a decreased number of Foxp3⁺ CD4⁺ regulatory T (Treg) cells in peripheral lymphoid organs of EAE mice. Consistently, our in vitro observations also revealed that loss of β-arrestin 2 impaired the conversion of Foxp3⁻ CD4⁺ T cells into Foxp3⁺ CD4⁺ inducible Treg cells. Taken together, our data suggest that β-arrestin 2 plays a regulatory role in MS, that is opposite to that of β-arrestin 1, in autoimmune diseases such as MS, which is at least partially through regulation of iTreg cell differentiation.     

The NK receptor KLRG1 is dispensable for virus‐induced NK and CD8+ T‐cell differentiation and function in vivo

The killer cell lectin‐like receptor G1 (KLRG1) is expressed by NK and T‐cell subsets and recognizes members of the classical cadherin family. KLRG1 is widely used as a lymphocyte differentiation marker in both humans and mice but the physiological role of KLRG1 in vivo is still unclear. Here, we generated KLRG1‐deficient mice by homologous recombination and used several infection models for their characterization. The results revealed that KLRG1 deficiency did not affect development and function of NK cells examined under various conditions. KLRG1 was also dispensable for normal CD8⁺ T‐cell differentiation and function after viral infections. Thus, KLRG1 is a marker for distinct NK and T‐cell differentiation stages but it does not play a deterministic role in the generation and functional characteristics of these lymphocyte subsets. In addition, we demonstrate that E‐cadherin expressed by K562 target cells inhibited NK‐cell reactivity in transgenic mice over‐expressing KLRG1 but not in KLRG1‐deficient or WT mice. Hence, the inhibitory potential of KLRG1 in mice is rather weak and strong activation signals during viral infections may override the inhibitory signal in vivo.     

CD49a promotes T‐cell‐mediated hepatitis by driving T helper 1 cytokine and interleukin‐17 production

It is becoming increasingly clear that the T-cell-mediated immune response is important in many diseases. In this study, we used concanavalin A (Con A) -induced hepatitis to investigate the role of CD49a in the molecular and cellular mechanism of the T-cell-mediated immune response. We found that CD49a^−/− mice had significantly reduced levels of serum alanine aminotransferase and were protected from Con A-induced hepatitis. CD49a deficiency led to decreased production of interferon-γ (IFN-γ) and interleukin-17A (IL-17A) after Con A injection. Furthermore, we found that hepatic CD4⁺ T cells and invariant natural killer T cells up-regulated CD49a expression, along with enhanced activation after Con A injection, leading to production of inflammatory cytokines by these T cells. Blockade of CD49a in vivo ameliorated Con A-induced hepatitis with reduced production of IFN-γ and IL-17A. Hence, CD49a promoted Con A-induced hepatitis through enhancing inflammatory cytokine production (IFN-γ and IL-17A) by CD4⁺ T and invariant natural killer T cells. The protective effect of CD49a blockade antibody suggested a new target therapeutic molecule for intervention of T-cell-mediated liver injury.     

Interleukin‐12 (IL‐12), but not IL‐23, induces the expression of IL‐7 in microglia and macrophages: implications for multiple sclerosis

Interleukin-12 (IL-12) p70 and IL-23 are bioactive cytokines and their biological functions are becoming clear. Increased expression of IL-7 in the central nervous system as well as in peripheral immune cells is associated with multiple sclerosis and experimental allergic encephalomyelitis. Here, we describe the induction of IL-7 in primary mouse and human microglia, BV-2 microglial cells, mouse peritoneal macrophages and astrocytes by IL-12p70. Interestingly, IL-12 strongly induced the expression of IL-7 whereas IL-23 and other p40 family members remained weak inducers of IL-7 in these cell types. Consistently, IL-12, but not IL-23 and other p40 family members, induced IL-7 promoter-driven luciferase activity in microglial cells. Among various stimuli tested, IL-12 emerged as the most potent stimulus followed by bacterial lipopolysaccharide and HIV-1 gp120 in inducing the activation of IL-7 promoter in microglial cells. Furthermore, increase in IL-7 mRNA expression by over-expression of IL-12p35 subunit, but not p40 and IL-23 p19 subunit, confirm that p35, but not p40 and p19, is responsible for the induction of IL-7. Finally, by using primary microglia from IL-12 receptor β1-deficient (IL-12Rβ1^−/−) and IL-12Rβ2^−/− mice, we demonstrate that IL-12 induces the expression of IL-7 in microglia and macrophages via both IL-12Rβ2 and IL-12Rβ1. These studies delineate a novel biological function of IL-12 that is absent in IL-23 and other p40 family members.     

β2 Integrin deficiency yields unconventional double-negative T cells distinct from mature classical natural killer T cells in mice

Expressed on leucocytes, β₂ integrins (CD11/CD18) are specifically involved in leucocyte function. Using a CD18-deficient (CD18^−/−) mouse model, we here report on their physiological role in lymphocyte differentiation and trafficking. CD18^−/− mice present with a defect in the distribution of lymphocytes with highly reduced numbers of naïve B and T lymphocytes in inguinal and axillary lymph nodes. In contrast, cervical lymph nodes were fourfold enlarged harbouring unconventional T-cell receptor-αβ (TCR-αβ) and TCR-γδ CD3⁺ CD4⁻ CD8⁻ (double-negative; DN) T cells that expanded in situ. Using adoptive transfer experiments, we found that these cells did not home to peripheral lymph nodes of CD18^wt recipients but, like antigen-experienced T or natural killer (NK) T cells, recirculated through non-lymphoid organs. Lacking regulatory functions in vitro, CD18^−/− TCR-αβ DN T cells did not suppress the proliferation of polyclonally activated CD4⁺ or CD8⁺ (single-positive; SP) T cells. Most interestingly, CD18^−/− TCR-αβ DN T cells showed intermediate TCR expression levels, an absent activation through allogeneic major histocompatibility complex and a strong proliferative dependence on interleukin-2, hence, closely resembling NKT cells. However, our data oppose former reports, clearly showing that, because of an absent reactivity with CD1d-αGalCer dimers, these cells are not mature classical NKT cells. Our data indicate that CD18^−/− TCR-αβ DN T cells, like NKT and TCR-γδ T cells, share characteristics of both adaptive and innate immune cells, and may accumulate as a compensatory mechanism to the functional defect of adaptive immunity in CD18^−/− mice.