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.     

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.     

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.     

Cooperative action of CD8 T lymphocytes and natural killer cells controls tumour growth under conditions of restricted T‐cell receptor diversity

In mice expressing a transgenic T-cell receptor (TCR; TCRP1A) of DBA/2 origin with reactivity towards a cancer-germline antigen P1A, the number of TCRP1A CD8⁺ T cells in lymphoid organs is lower in DBA/2 than in B10.D2 or B10.D2(× DBA/2)F₁ mice. This reduction results from haemopoietic cell autonomous differences in the differentiation of the major histocompatibility complex class I-restricted TCRP1A thymocytes controlled by DBA/2 versus B10.D2-encoded genes. We report here that the lower number of TCRP1A CD8⁺ T cells in DBA/2 mice correlated with their poor resistance to P1A-expressing mastocytoma solid tumours. Functional potency of CD8⁺ cytolytic T lymphocytes (CTL) from the above strains was not compromised, but their number after expansion appeared to be influenced by their genetic background. Intriguingly, non-transgenic DBA/2 mice resisted P1A⁺ tumours more efficiently despite poor representation of P1A-specific CTL. This was partly the result of their more heterogeneous TCR repertoire, including reactivity to non-P1A tumour antigens because mice that had rejected a P1A⁺ tumour became resistant to a P1A⁻ variant of the tumour. Such ‘cross-resistance’ did not develop in the TCRP1A transgenic mice. Nonetheless, reconstitution of RAGº^/º mice with TCRP1A CD8⁺ T cells, with or without CD4⁺ T cells, or exclusive representation of TCRP1A CD8⁺ T cells in RAGº^/º TCRP1A transgenic mice efficiently resisted the growth of P1A-expressing tumours. Natural killer cells present at a higher number in RAGº^/º mice also contributed to tumour resistance, in part through an NKG2D-dependent mechanism. Hence, in the absence of a polyclonal T-cell repertoire, precursor frequencies of natural killer cells and tumour-specific CTL affect tumour resistance.     

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.     

Defective induction of the proteasome associated with T‐cell receptor signaling underlies T‐cell senescence

The proteasome degradation machinery is essential for a variety of cellular processes including senescence and T‐cell immunity. Decreased proteasome activity is associated with the aging process; however, the regulation of the proteasome in CD4⁺ T cells in relation to aging is unclear. Here, we show that defects in the induction of the proteasome in CD4⁺ T cells upon T‐cell receptor (TCR) stimulation underlie T‐cell senescence. Proteasome dysfunction promotes senescence‐associated phenotypes, including defective proliferation, cytokine production and increased levels of PD‐1⁺ CD44^High CD4⁺ T cells. Proteasome induction by TCR signaling via MEK‐, IKK‐ and calcineurin‐dependent pathways is attenuated with age and decreased in PD‐1⁺ CD44^High CD4⁺ T cells, the proportion of which increases with age. Our results indicate that defective induction of the proteasome is a hallmark of CD4⁺ T‐cell senescence.     

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.     

CD40‐mediated signalling influences trafficking,T‐cell receptor expression,and T‐cell pathogenesis,in the NOD model of type 1 diabetes

CD40 plays a critical role in the pathogenesis of type 1 diabetes (T1D). The mechanism of action, however, is undetermined, probably because CD40 expression has been grossly underestimated. CD40 is expressed on numerous cell types that now include T cells and pancreatic β cells. CD40⁺ CD4⁺ cells [T helper type 40 (TH40)] prove highly pathogenic in NOD mice and in translational human T1D studies. We generated BDC2.5.CD40^−/− and re‐derived NOD.CD154^−/− mice to better understand the CD40 mechanism of action. Fully functional CD40 expression is required not only for T1D development but also for insulitis. In NOD mice, TH40 cell expansion in pancreatic lymph nodes occurs before insulitis and demonstrates an activated phenotype compared with conventional CD4⁺ cells, apparently regardless of antigen specificity. TH40 T‐cell receptor (TCR) usage demonstrates increases in several Vα and Vβ species, particularly Vα3.2⁺ that arise early and are sustained throughout disease development. TH40 cells isolated from diabetic pancreas demonstrate a relatively broad TCR repertoire rather than restricted clonal expansions. The expansion of the Vα/Vβ species associated with diabetes depends upon CD40 signalling; NOD.CD154^−/− mice do not expand the same TCR species. Finally, CD40‐mediated signals significantly increase pro‐inflammatory Th1‐ and Th17‐associated cytokines whereas CD28 co‐stimulus alternatively promotes regulatory cytokines.     

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.     

Different immunosuppressive mechanisms in multi‐drug‐resistant tuberculosis and non‐tuberculous mycobacteria patients

Previous studies have demonstrated that cells from both multi-drug-resistant tuberculosis (MDR-TB) and non-tuberculous mycobacteria (NTM) patients respond poorly to mycobacterial antigens in vitro. In the present study, we compared the in vitro response of cells isolated from sensitive TB (NR-TB)-, MDR-TB- and NTM-infected patients. Analysis of T cell phenotype ex vivo revealed that both MDR-TB and NTM patients present an increased percentage of CD4⁺CD25^+- forkhead box protein 3 (FoxP3)⁺ and CD4⁺CD25⁺CD127⁻ regulatory T (T_reg) cells when compared to NR-TB. Increased numbers of T_reg cells and interleukin (IL)-10 serum levels were detected in MDR-TB, whereas elevated serum transforming growth factor (TGF)-β was found in the NTM group. Cells of MDR-TB patients stimulated with early secretory antigenic target (ESAT)-6, but not purified protein derivative (PPD), showed a lower frequency of CD4⁺/interferon (IFN)-γ⁺ T cells and enhanced CD4⁺CD25⁺FoxP3⁺, CD4⁺CD25⁺CD127⁻ and CD4⁺CD25⁺IL-10⁺ T cell population. In addition, increased IL-10 secretion was observed in cultured MDR-TB cells following ESAT-6 stimulation, but not in NR-TB or NTM patients. In vitro blockade of IL-10 or IL-10Rα decreased the CD4⁺CD25⁺FoxP3⁺ frequencies induced by ESAT-6 in MDR-TB, suggesting a role of IL-10 on impaired IFN-γ responses seen in MDR-TB. Depletion of CD4⁺CD25⁺ T lymphocytes restored the capacity of MDR-TB T cells to respond to ESAT-6 in vitro, which suggests a potential role for T_reg/T regulatory 1 cells in the pathogenesis of MDR-TB. Together, our results indicate that although the similarities in chronicity, NTM- and MDR-TB-impaired antigenic responses involve different mechanisms.     

‘Default’ generated neonatal regulatory T cells are hypomethylated at conserved non‐coding sequence 2 and promote long‐term cardiac allograft survival

Regulatory T (Treg) cells play an important role in the maintenance of immune self-tolerance and homeostasis. We previously reported that neonatal CD4⁺ T cells have an intrinsic ‘default’ mechanism to become Treg (neoTreg) cells in response to T-cell receptor (TCR) stimulation. However, the underlying mechanisms are unclear and the effects of neoTreg cells on regulating immune responses remain unknown. Due to their involvement in Foxp3 regulation, we examined the role of DNA methyltransferase 1 (DNMT1) and DNMT3b during the induction of neoTreg cells in the Foxp3^gfp mice. The function of neoTreg cells was assessed in an acute allograft rejection model established in RAG2^−/− mice with allograft cardiac transplantation and transferred with syngeneic CD4⁺ effector T cells. Following ex vivo TCR stimulation, the DNMT activity was increased threefold in adult CD4⁺ T cells, but not significantly increased in neonatal cells. However, adoptively transferred neoTreg cells significantly prolonged cardiac allograft survival (mean survival time 47 days, P < 0·001) and maintained Foxp3 expression similar to natural Treg cells. The neoTreg cells were hypomethylated at the conserved non-coding DNA sequence 2 locus of Foxp3 compared with adult Treg cells. The DNMT antagonist 5-aza-2′-deoxycytidine (5-Aza) induced increased Foxp3 expression in mature CD4⁺ T cells. 5-Aza-inducible Treg cells combined with continuous 5-Aza treatment prolonged graft survival. These results indicate that the ‘default’ pathway of neoTreg cell differentiation is associated with reduced DNMT1 and DNMT3b response to TCR stimulus. The neoTreg cells may be a strategy to alleviate acute allograft rejection.     

TLR4, 5, and 9 Agonists Inhibit Murine Airway Invariant Natural Killer T Cells in an IL-12-Dependent Manner

Purpose

Invariant natural killer T (iNKT) cells may play an important role in the pathogenesis of asthma in mice and humans. Thus, an agent that modulates the function of iNKT cells may have therapeutic potential to control asthma. We hypothesized that lipopolysaccharide (LPS)-, flagellin-, or CpG-induced changes in the cytokine milieu may modify and even inhibit the function of airway iNKT cells in asthma.

Methods

Because increased α-galactosylceramide (GalCer)-induced airway hyperreactivity (AHR) reflects the presence of airway iNKT cells, α-GalCer-induced AHR, as well as inflammatory cells and cytokines in bronchoalveolar lavage (BAL) fluid, were determined 24 hours after in vivo treatment with LPS, flagellin, or CpG in naïve BALB/c mice. Intracellular IL-4 and IFN-γ were measured in spleen iNKT cells after in vitro treatment with LPS, flagellin, or CpG. A role for IL-12 following the treatments was determined.

Results

Intranasal administration of LPS, flagellin, or CpG reduced development of α-GalCer-induced AHR, eosinophilic airway inflammation, and Th1 and Th2 cytokine responses in BAL fluid, while producing IL-12 in BAL fluid. Intraperitoneal administration of IL-12 mAb blocked the suppressive effect of LPS, flagellin, or CpG. In vitro treatment with LPS, flagellin, or CpG reduced production of IL-4 and IFN-γ from α-GalCer-stimulated spleen iNKT cells; these effects were ameliorated by addition of anti-IL-12 mAb.

Conclusions

TLR4, 5, and 9 agonists may suppress the function of airway and spleen iNKT cells via IL-12-dependent mechanisms. Anergy of iNKT cells by IL-12 might play a role in suppression by these TLR agonists.     

Invariant natural killer T‐cell development and function with loss of microRNA‐155

Invariant natural killer T (iNKT) cells are adaptive T cells with innate‐like characteristics including rapid cytokine production and a proliferative response to stimulation. Development of these cells in the thymus is dependent on expression of the microRNA (miRNA) processing enzyme Dicer, indicating that iNKT cells probably have distinct miRNA requirements for gene regulation during development. The miRNA miR‐155 has previously been shown to have numerous roles in T cells, including regulation of proliferation and differentiation, and positive modulation of interferon‐γ expression. We examined the role of miR‐155 in the development and function of iNKT cells. Using germline‐deficient miR‐155 mice, we showed that loss of miR‐155 resulted in unchanged iNKT cell frequency and cell number. Although miR‐155 was up‐regulated in iNKT cells upon activation with α‐galactosylceramide, loss of miR‐155 did not affect cytokine production or proliferation by iNKT cells. Hence, cytokine production occurs in iNKT cells independently of miR‐155 expression.     

Protease‐activated receptor 2 signalling promotes dendritic cell antigen transport and T‐cell activation in vivo

Deficiency of protease-activated receptor-2 (PAR2) modulates inflammation in several models of inflammatory and autoimmune disease, although the underlying mechanism(s) are not understood. PAR2 is expressed on endothelial and immune cells, and is implicated in dendritic cell (DC) differentiation. We investigated in vivo the impact of PAR2 activation on DCs and T cells in PAR2 wild-type (WT) and knockout (KO) mice using a specific PAR2 agonist peptide (AP2). PAR2 activation significantly increased the frequency of mature CD11c^high DCs in draining lymph nodes 24 hr after AP2 administration. Furthermore, these DCs exhibited increased expression of major histocompatibility complex (MHC) class II and CD86. A significant increase in activated (CD44⁺ CD62⁻) CD4⁺ and CD8⁺ T-cell frequencies was also observed in draining lymph nodes 48 hr after AP2 injection. No detectable change in DC or T-cell activation profiles was observed in the spleen. The influence of PAR2 signalling on antigen transport to draining lymph nodes was assessed in the context of delayed-type hypersensitivity. PAR2 WT mice that were sensitized by skin-painting with fluorescein isothiocyanate (FITC) to induce delayed-type hypersensitivity possessed elevated proportion of FITC⁺ DCs in draining lymph nodes 24 hr after FITC painting when compared with PAR2 KO mice (0·95% versus 0·47% of total lymph node cells). Collectively, these results demonstrate that PAR2 signalling promotes DC trafficking to the lymph nodes and subsequent T-cell activation, and thus provides an explanation for the pro-inflammatory effect of PAR2 in animal models of inflammation.     

Exogenous interleukin‐33 targets myeloid‐derived suppressor cells and generates periphery‐induced Foxp3+ regulatory T cells in skin‐transplanted mice

Interleukin-33 (IL-33) has been a focus of study because of its variety of functions shaping CD4⁺ T-cell biology. In the present work, we evaluated the modulatory effect of IL-33 on suppressor cells in an in vivo transplantation model. C57BL/6 wild-type mice were grafted with syngeneic or allogeneic skin transplants and treated with exogenous IL-33 daily. After 10 days of treatment, we analysed draining lymph node cellularity and found in allogeneic animals an increment in myeloid-derived suppressor cells, which co-express MHC-II, and become enriched upon IL-33 treatment. In line with this observation, inducible nitric oxide synthase and arginase 1 expression were also increased in allogeneic animals upon IL-33 administration. In addition, IL-33 treatment up-regulated the number of Foxp3⁺ regulatory T (Treg) cells in the allogeneic group, complementing the healthier integrity of the allografts and the increased allograft survival. Moreover, we demonstrate that IL-33 promotes CD4⁺ T-cell expansion and conversion of CD4⁺ Foxp3⁻ T cells into CD4⁺ Foxp3⁺ Treg cells in the periphery. Lastly, the cytokine pattern of ex vivo-stimulated draining lymph nodes indicates that IL-33 dampens interferon-γ and IL-17 production, stimulating IL-10 secretion. Altogether, our work complements previous studies on the immune-modulatory activity of IL-33, showing that this cytokine affects myeloid-derived suppressor cells at the cell number and gene expression levels. More importantly, our research demonstrates for the first time that IL-33 allows for in vivo Foxp3⁺ Treg cell conversion and favours an anti-inflammatory or tolerogenic state by skewing cytokine production. Therefore, our data suggest a potential use of IL-33 to prevent allograft rejection, bringing new therapeutics to the transplantation field.