Interleukin 2, but not other common gamma chain-binding cytokines, can reverse the defect in generation of CD4 effector T cells from naive T cells of aged mice.

Development of effectors from naive CD4 cells occurs in two stages. The early stage involves activation and limited proliferation in response to T cell receptor (TCR) stimulation by antigen and costimulatory antigen presenting cells, whereas the later stage involves proliferation and differentiation in response to growth factors. Using a TCR-transgenic (Tg(+)) model, we have examined the effect of aging on effector generation and studied the ability of gamma(c) signaling cytokines to reverse this effect. Our results indicate that responding naive CD4 cells from aged mice, compared with cells from young mice, make less interleukin (IL)-2, expand poorly between days 3 to 5, and give rise to fewer effectors with a less activated phenotype and reduced ability to produce cytokines. When exogenous IL-2 or other gamma(c) signaling cytokines are added during effector generation, the Tg(+) cells from both young and aged mice proliferate vigorously. However, IL-4, IL-7, and IL-15 all fail to restore efficient effector production. Only effectors from aged mice generated in the presence of IL-2 are able to produce IL-2 in amounts equivalent to those produced by effectors generated from young mice, suggesting that the effect of aging on IL-2 production is reversible only in the presence of exogenous IL-2.     

Inhibition of T helper cell type 2 cell differentiation and immunoglobulin E response by ligand-activated Valpha14 natural killer T cells.

Murine Valpha14 natural killer T (NKT) cells are thought to play a crucial role in various immune responses, including infectious, allergic, and autoimmune diseases. Because Valpha14 NKT cells produce large amounts of both interleukin (IL)-4 and interferon (IFN)-gamma upon in vivo stimulation with a specific ligand, alpha-galactosylceramide (alpha-GalCer), or after treatment with anti-CD3 antibody, a regulatory role on helper T (Th) cell differentiation has been proposed for these cells. However, the identity of the cytokine produced by Valpha14 NKT cells that play a dominant role on the Th cell differentiation still remains controversial. Here, we demonstrate by using Valpha14 NKT-deficient mice that Valpha14 NKT cells are dispensable for the induction of antigen-specific immunoglobulin (Ig)E responses induced by ovalbumin immunization or Nippostrongylus brasiliensis infection. However, upon in vivo activation with alpha-GalCer, Valpha14 NKT cells are found to suppress antigen-specific IgE production. The suppression appeared to be IgE specific, and was not detected in either Valpha14 NKT- or IFN-gamma-deficient mice. Consistent with these results, we also found that ligand-activated Valpha14 NKT cells inhibited Th2 cell differentiation in an in vitro induction culture system. Thus, it is likely that activated Valpha14 NKT cells exert a potent inhibitory effect on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-gamma. In marked contrast, our studies have revealed that IL-4 produced by Valpha14 NKT cells has only a minor effect on Th2 cell differentiation.     

Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice

C57BL/6 mice genetically deficient in interleukin 15 (IL-15(-/-) mice) were generated by gene targeting. IL-15(-/-) mice displayed marked reductions in numbers of thymic and peripheral natural killer (NK) T cells, memory phenotype CD8(+) T cells, and distinct subpopulations of intestinal intraepithelial lymphocytes (IELs). The reduction but not absence of these populations in IL-15(-/-) mice likely reflects an important role for IL-15 for expansion and/or survival of these cells. IL-15(-/-) mice lacked NK cells, as assessed by both immunophenotyping and functional criteria, indicating an obligate role for IL-15 in the development and functional maturation of NK cells. Specific defects associated with IL-15 deficiency were reversed by in vivo administration of exogenous IL-15. Despite their immunological defects, IL-15(-/-) mice remained healthy when maintained under specific pathogen-free conditions. However, IL-15(-/-) mice are likely to have compromised host defense responses to various pathogens, as they were unable to mount a protective response to challenge with vaccinia virus. These data reveal critical roles for IL-15 in the development of specific lymphoid lineages. Moreover, the ability to rescue lymphoid defects in IL-15(-/-) mice by IL-15 administration represents a powerful means by which to further elucidate the biological roles of this cytokine.     

The activated type 1-polarized CD8(+) T cell population isolated from an effector site contains cells with flexible cytokine profiles.

The capacity of activated T cells to alter their cytokine expression profiles after migration into an effector site has not previously been defined. We addressed this issue by paired daughter analysis of a type 1-polarized CD8(+) effector T cell population freshly isolated from lung parenchyma of influenza virus-infected mice. Single T cells were activated to divide in vitro; individual daughter cells were then micromanipulated into secondary cultures with and without added IL-4 to assess their potential to express type 2 cytokine genes. The resultant subclones were analyzed for type 1 and 2 cytokine mRNAs at day 6-7. When the most activated (CD44(high)CD11a(high)) CD8(+) subpopulation from infected lung was compared with naive or resting (CD44(low)CD11a(low)) CD8(+) cells from infected lung and from normal lymph nodes (LNs), both clonogenicity and plasticity of the cytokine response were highest in the LN population and lowest in the activated lung population, correlating inversely with effector function. Multipotential cells were nevertheless detected among clonogenic CD44(high)CD11a(high) lung cells at 30-50% of the frequency in normal LNs. The data indicate that activated CD8(+) T cells can retain the ability to proliferate and express new cytokine genes in response to local stimuli after recruitment to an effector site.     

CD1-mediated gamma/delta T cell maturation of dendritic cells

The vascular endothelial growth factor (VEGF) receptor fetal liver kinase 1 (flk1; VEGFR-2, KDR) is an endothelial cell-specific receptor tyrosine kinase that mediates physiological and pathological angiogenesis. We hypothesized that an active immunotherapy approach targeting flk1 may inhibit tumor angiogenesis and metastasis. To test this hypothesis, we first evaluated whether immune responses to flk1 could be elicited in mice by immunization with dendritic cells pulsed with a soluble flk1 protein (DC-flk1). This immunization generated flk1-specific neutralizing antibody and CD8+ cytotoxic T cell responses, breaking tolerance to self-flk1 antigen. Tumor-induced angiogenesis was suppressed in immunized mice as measured in an alginate bead assay. Development of pulmonary metastases was strongly inhibited in DC-flk1-immunized mice challenged with B16 melanoma or Lewis lung carcinoma cells. DC-flk1 immunization also significantly prolonged the survival of mice challenged with Lewis lung tumors. Thus, an active immunization strategy that targets an angiogenesis-related antigen on endothelium can inhibit angiogenesis and may be a useful approach for treating angiogenesis-related diseases.     

A comparative study on roles of natural killer T cells in two diet-induced non-alcoholic steatohepatitis-related fibrosis in mice

BackgroundImmune responses are important in the progression of non-alcoholic fatty liver disease (NAFLD). Natural killer T (NKT) cells are main components of the innate immune system that modulate immunity. However, the role of NKT cells in NAFLD remains controversial.ObjectiveWe aimed to investigate the role of NKT cells in non-alcoholic steatohepatitis (NASH)-related fibrosis in fast food diet (FFD)- and methionine choline-deficient (MCD) diet-induced mouse models.MethodsHepatic NKT cells were analysed in wild-type (WT) and CD1d-/- mice fed FFD or MCD diets. Hepatic pathology, cytokine profiles and liver fibrosis were evaluated. Furthermore, the effect of chronic administration of α-galactosylceramide (α-GalCer) on liver fibrosis was investigated in both FFD- and MCD-treated mice.ResultsFFD induced a significant depletion of hepatic NKT cells, thus leading to mild to moderate NASH and early-stage fibrosis, while mice fed MCD diets developed severe liver inflammation and progressive fibrosis without a significant change in hepatic NKT cell abundance. FFD induced a similar liver fibrogenic response in CD1d-/- and WT mice, while MCD induced a higher hepatic mRNA expression of Col1α1 and TIMP1 as well as relative fibrosis density in CD1d-/- mice than WT mice (31.8 vs. 16.3, p = .039; 40.0 vs. 22.6, p = .019; 2.24 vs. 1.59, p = .036). Chronic administration of α-GalCer induced a higher hepatic mRNA expression of TIMP1 in MCD-treated mice than controls (36.7 vs. 14.9, p = .005).ConclusionNKT cells have protective roles in NAFLD as the disease progresses. During diet-induced steatosis, mild to moderate NASH and the early stage of fibrosis, hepatic NKT cells are relatively depleted, leading to a proinflammatory status. In severe NASH and the advanced stage of liver fibrosis, NKT cells play a role in inhibiting the NASH-related fibrogenic response. Chronic administration of α-GalCer induces NKT cell anergy and tolerance, which may play a role in promoting the liver fibrogenic response.     

Alteration of interleukin 4 production results in the inhibition of T helper type 2 cell-dominated inflammatory bowel disease in T cell receptor alpha chain-deficient mice.

T cell receptor alpha chain-deficient (TCR-alpha(-/-)) mice are known to spontaneously develop inflammatory bowel disease (IBD). The colitis that develops in these mice is associated with increased numbers of T helper cell (Th)2-type CD4(+)TCR-betabeta (CD4(+)betabeta) T cells producing predominantly interleukin (IL)-4. To investigate the role of these Th2-type CD4(+)betabeta T cells, we treated TCR-alpha(-/-) mice with anti-IL-4 monoclonal antibody (mAb). Approximately 60% of TCR-alpha(-/-) mice, including those treated with mock Ab and those left untreated, spontaneously developed IBD. However, anti-IL-4 mAb-treated mice exhibited no clinical or histological signs of IBD, and their levels of mucosal and systemic Ab responses were lower than those of mock Ab-treated mice. Although TCR-alpha(-/-) mice treated with either specific or mock Ab developed CD4(+)betabeta T cells, only those treated with anti-IL-4 mAb showed a decrease in Th2-type cytokine production at the level of mRNA and protein and an increase in interferon gamma-specific expression. These findings suggest that IL-4-producing Th2-type CD4(+)betabeta T cells play a major immunopathological role in the induction of IBD in TCR-alpha(-/-) mice, a role that anti-IL-4 mAb inhibits by causing Th2-type CD4(+)betabeta T cells to shift to the Th1 type.     

Development of CD8alpha/alpha and CD8alpha/beta T cells in major histocompatibility complex class I-deficient mice.

Peripheral CD8(+) T cells mainly use CD8alpha/beta, and their development is mainly dependent on the major histocompatibility complex (MHC) class I proteins K(b) and D(b) in H-2(b) mice. In this report, we have shown that the development of CD8alpha/beta TCR-alpha/beta cells in lymphoid organs as well as in intestinal intraepithelial lymphocytes (iIELs) is dependent on the MHC class I K(b) and D(b) proteins. In contrast, TCR-alpha/beta CD8alpha/alpha cells are found mainly in iIELs, and their numbers are unaffected in K(b)D(b) double knockout mice. Most of the TCR-gamma/delta cells in the iIELs also bear CD8alpha/alpha, and they are also unaffected in K(b)D(b) -/- mice. In beta2-microglobulin (beta2m)-deficient mice, all of the TCR-alpha/beta CD8alpha/alpha and CD8alpha/beta T cells disappear, but TCR-gamma/delta cells are unaffected by the absence of beta2m.     

Neonatal tumor necrosis factor alpha promotes diabetes in nonobese diabetic mice by CD154-independent antigen presentation to CD8(+) T cells

Neonatal islet-specific expression of tumor necrosis factor (TNF)-alpha in nonobese diabetic mice promotes diabetes by provoking islet-infiltrating antigen-presenting cells to present islet peptides to autoreactive T cells. Here we show that TNF-alpha promotes autoaggression of both effector CD4(+) and CD8(+) T cells. Whereas CD8(+) T cells are critical for diabetes progression, CD4(+) T cells play a lesser role. TNF-alpha-mediated diabetes development was not dependent on CD154-CD40 signals or activated CD4(+) T cells. Instead, it appears that TNF-alpha can promote cross-presentation of islet antigen to CD8(+) T cells using a unique CD40-CD154-independent pathway. These data provide new insights into the mechanisms by which inflammatory stimuli can bypass CD154-CD40 immune regulatory signals and cause activation of autoreactive T cells.     

Anergy and cytokine-mediated suppression as distinct superantigen-induced tolerance mechanisms in vivo.

Recombinant-activating gene 2 (RAG-2-/-) T cell receptor-transgenic mice repeatedly injected with the superantigen staphylococcal enterotoxin A entered a tolerant state in which splenic CD4+ T cells produced little interleukin (IL)-2, interferon gamma, or IL-4. This state resulted from a combination of both clonal anergy and cytokine-mediated immunosuppression. The anergy persisted for at least 3 wk and could be distinguished from the suppression by a decrease in IL-2 production per cell, a block in the activation of early response kinases, and a failure to be reversed with anti-transforming growth factor (TGF)-beta. Full suppression lasted for only 1 wk and involved both IL-10 and TGF-beta, but required additional unknown molecules for optimal effect. These experiments show that complex in vivo interactions of multiple peripheral tolerance mechanisms can now be dissected at both the cellular and molecular levels.