Participation of leukocyte function-associated antigen-1 and NK cells in the homing of thymic CD8+NKT cells to the liver

A distinct CD8+NKT cell population expressing TCRalpha/beta or TCRgamma/delta has been identified in liver and thymus. We wondered whether cell adhesion molecules play a role in the homing of CD8+NKT cells to the liver. The number of liver CD8+NKT cells was markedly reduced in leukocyte function-associated antigen (LFA)-1-/- mice compared with wild-type (WT) mice. The reduction was restricted to the liver only and no measurable alterations were found in other organs. In the liver of SCID mice reconstituted with thymocytes from LFA-1-/- or WT mice, the number of donor-derived CD8+NKT cells was comparable and the vast majority of these cells expressed TCRalpha/beta. In a reciprocal radiation thymocyte reconstitution system with LFA-1-/- and WT mice, LFA-1 expressed on liver cells other than CD8+NKT cells appeared to be required for the homing of thymic CD8+NKT cells to the liver. The accumulation of donor thymocyte-derived CD8+NKT cells in the liver of SCID mice was severely impaired by in vivo depletion of NK cells, but not of Kupffer cells. These results not only indicate that thymus provides a source for CD8+NKT cells expressing TCRalpha/beta in the liver, but also suggest that LFA-1 expressed on NK cells is involved in the homing of thymic CD8+NKT cells to the liver.     

Age-related bias in function of natural killer T cells and granulocytes after stress: reciprocal association of steroid hormones and sympathetic nerves

Stress-associated immune responses were compared between young (8 weeks of age) and old (56 weeks) mice. Since stress suppresses the conventional immune system (i.e. T and B cells) but inversely activates the primordial immune system (i.e. extrathymic T cells, NKT cells, and granulocytes), these parameters were analysed after restraint stress for 24 h. The thymus became atrophic as a function of age, and an age-related increase in the number of lymphocytes was seen in the liver. Although the number of lymphocytes in both the thymus and liver decreased as the result of stress, the magnitude was much more prominent in the thymus. To determine stress-resistant lymphocyte subsets, two-colour immunofluorescence tests were conducted in the liver and spleen. NKT cells were found to be such cells in the liver of young mice. On the other hand, an infiltration of granulocytes due to stress was more prominent in the liver of old mice than in young mice. Liver injury as a result of stress was prominent in young mice. This age-related bias in the function of NKT cells and granulocytes seemed to be associated with a difference in the responses of catecholamines (high in old mice) and corticosterone (high in young mice) after stress. Indeed, an injection of adrenaline mainly induced the infiltration of granulocytes while that of cortisol activated NKT cells. The present results suggest the existence of age-related bias in the function of NKT cells and granulocytes after stress and that such bias might be produced by different responses of sympathetic nerves and steroid hormones between young and old mice.     

The differential effect of stress on natural killer T (NKT) and NK cell function

When C57Bl/6 mice were exposed to restraint stress for 12 h or 24 h, lymphocytopenia was induced in the liver, spleen, and thymus. We examined which types of lymphocytes were sensitive or resistant to such stress by a immunofluorescence test. T cells of thymic origin were sensitive while NKT and NK cells were resistant. In contrast to the increase in the proportion of NK cells, NK activity of liver lymphocytes against YAC-1 targets decreased at 24 h after stress. On the other hand, their NKT cytotoxicity against syngeneic thymocytes increased in parallel with an increase in their proportion. In perforin -/- B6 mice and B6-gld/gld (Fas ligand-) mice, NK cells were found to mediate cytotoxicity through perforin while NKT cells mediated self-reactive cytotoxicity through Fas ligand. These results suggest that stress increases the proportion of both NK and NKT cells, but that NK cytotoxicity is suppressed while self-reactive NKT cytotoxicity is not, due to a diversity of their functional mechanisms.     

Size of the population of CD4+ natural killer T cells in the liver is maintained without supply by the thymus during adult life

Given that there are few natural killer T (NKT) cells in the liver of athymic nude mice and in neonatally thymectomized mice, it is still controversial whether all NKT cells existing in the liver are supplied by the thymus or if some such cells develop in the liver. To determine whether or not NKT cells are consistently supplied from the thymus during adult life, thymectomy was conducted in mice at the age of 8 weeks. Interestingly, the proportion and number of CD4+ NKT cells increased or remained unchanged in the liver after adult thymectomy and this phenomenon continued for up to 6 months after thymectomy. The administration of alpha-galactosylceramide induced severe cytopenia (due to apoptosis) of CD4+ NKT cells in the liver on day 1, but subsequent expansion of these NKT cells occurred in thymectomized mice similar to the case in normal mice. However, in thymectomized mice given lethal irradiation (9.5 Gy) and subsequent bone marrow transfer, the population of CD4+ NKT cells no longer expanded in the liver, although that of CD8+ NKT cells did. These results suggest that thymic CD4+ NKT cells, or their progenitors, may migrate to the liver at a neonatal stage but are not supplied from the thymus in the adult stage under usual conditions. CD8+ NKT cells can be generated in the liver.     

HBV转基因小鼠肝脏NKT细胞功能与PD1、CD28表达分析

目的研究HBV转基因小鼠肝脏中NKT细胞的功能与表面PD1、CD28表达的关系。方法分离小鼠肝脏、脾脏、胸腺和腹膜淋巴结单个核细胞,利用流式细胞检测技术,分别检测其淋巴细胞中NKT细胞的频率,同时检测肝脏NKT细胞PD1、CD28的表达及IFN-γ、IL-4的分泌功能,比较肝脏、脾脏、胸腺和腹膜淋巴结这几个主要免疫组织淋巴细胞中NKT细胞所占的比例,并分析肝脏NKT细胞PD1、CD28的表达与细胞功能的关系。结果与正常同品系小鼠比较,HBV转基因小鼠肝脏、脾脏、胸腺和腹膜淋巴结NKT细胞数量明显减少(P<0.05),与脾脏、胸腺和腹膜淋巴结相比,肝脏淋巴细胞中含有大量的NKT细胞;与正常同品系小鼠比较,HBV转基因小鼠肝脏NKT细胞PD1的表达明显增多(P<0.05),CD28的表达明显减少(P<0.05),肝脏NKT细胞IFN-γ、IL-4的分泌功能明显降低(P<0.05)。结论肝脏中含有大量的NKT细胞,HBV转基因小鼠肝脏NKT细胞的功能存在明显的缺陷,并提示PD1的增加和CD28的降低可能与NKT细胞功能的下调密切相关。     

Simultaneous activation of natural killer T cells and autoantibody production in mice injected with denatured syngeneic liver tissue

Denatured syngeneic liver tissue prepared by mechanical procedures was intraperitoneally injected into adult C57BL/6 mice. In parallel with a decrease in the total number of lymphocytes in the liver, spleen, and thymus from days 1-7 after the injection, the proportion of the CD4+NK1.1+CD3(int) subset of these cells (i.e. natural killer T or NKT cells) increased in the liver. Even the absolute number of these NKT cells increased in the liver on days 14 and 21. In response to the injection of denatured liver tissue, tissue damage was induced in the liver, as shown by elevated levels of serum transaminases and hepatocyte degeneration observed by electron microscopy. Sera obtained on days 7 and 14 contained autoantibodies including anti-DNA antibodies. The proportion of CD1d(high)B cells in the liver was found to decrease on days 1-7. In other words, denatured liver tissue stimulated both NKT cells and certain B cells in the liver. These results suggest that liver lymphocytes might contain not only autoreactive T cells (e.g. CD3(int) or NKT cells) but also some B cells (e.g. B-1 cells) which produce autoantibodies and that the denatured tissue had the potential to stimulate these lymphocytes and to evoke an autoimmune-like state.     

Three day neonatal thymectomy selectively depletes NK1.1+ T cells

Neonatal thymectomy of mice 3 days after birth but not at birth leads to T cell-mediated, organ-specific, autoimmune disease in a strain- dependent manner. The mechanisms that lead to disease in this model remain unknown, but the answer may lie in a deficiency of thymus- dependent cells or factors. One candidate is the relatively rare population of NK1.1 + T cells (NKT cells). Conventional alphabetaTCR+ T cells appear in the thymus from days 17-18 of embryogenesis and start emigrating to the periphery around birth, whereas the development of NKT cells is thought to be delayed until at least 1 week after birth. We have confirmed this to be the case in both (BALB/c x C57BL/6)F1 (autoimmune susceptible) and C57BL/6 (autoimmune resistant) mice. Moreover, examination of T cells (in spleen, lymph nodes, liver and bone marrow) from mice following 3 day neonatal thymectomy revealed a significant reduction in the presence of NKT cells in all tissues. However, the extent of depletion was generally more pronounced in (BALB/c x C57BL/6)F1 than in C57BL/6 mice, and the few remaining NKT cells in C57BL/6 mice were enriched for a CD4-CD8int subset which is absent from the thymus and may represent a distinct lineage of thymus- independent NKT cells. Given mounting evidence of a role for NKT cells in protection from autoimmune disease, it is possible that their specific removal by neonatal thymectomy may contribute to the susceptibility of these mice to autoimmune disease.      

Age-dependent variation in the proportion and number of intestinal lymphocyte subsets, especially natural killer T cells, double-positive CD4+ CD8+ cells and B220+ T cells, in mice

The age-dependent variation in the proportion and number of lymphocyte subsets was examined at various extrathymic sites, including the liver, small intestine, colon and appendix in mice. In comparison with young mice (4 weeks of age), the number of total lymphocytes yielded by all tested organs was greater in adult (9 weeks) and old (40 weeks) mice. The major lymphocyte subset that expanded with age was interleukin-2 receptor (IL-2R) beta+ CD3int cells (50% of them expressed NK1.1) in the liver, whereas it was CD3+ IL-2Rbeta- NK1.1- cells at all intraepithelial sites in the intestine. Although NK1.1+ CD3+ cells were present at intraepithelial sites in the intestine, the proportion of this subset was rather low. The ratio of CD4 to CD8 tended to decrease among natural killer T (NKT) cells and T cells at all intraepithelial sites in the intestine with age. A unique population of double-positive CD4+ CD8+ cells in the small intestine increased in old mice. B220+ T cells were found mainly in the appendix and colon, and the proportion of these T cells decreased in old mice. Conventional NKT cells were very few in Jalpha281-/- and CD1d-/- mice in the liver, while NKT cells which existed in the appendix remained unchanged even in these mice. This was because unconventional CD8+ NKT cells were present in the intestine. The present results suggest that despite the fact that both the liver and intraepithelial sites in the intestine carry many extrathymic T cells, the distribution of lymphocyte subsets and their age-associated variation are site-specific.     

Tissue-specific expansion of NKT and CD5+B cells at the onset of autoimmune disease in (NZBxNZW)F1 mice

Natural killer T (NKT) cells and CD5(+)B cells were searched for in various immune organs of autoimmune prone (NZBxNZW)F(1) (NZB/W F(1)) mice. The number of lymphocytes increased in the liver, spleen, and peritoneal cavity after the onset of disease (at the age of 30 weeks) while the number of thymocytes decreased at that time. Prominent changes of lymphocyte subsets were seen in the liver and peritoneal cavity, namely, expansion of IL-2Rbeta(+)TCRalpha beta(int) cells in the liver and of CD5(+)B220(+) cells in the peritoneal cavity. The majority of TCRalpha beta(int) cells in the liver were NK1.1(+), and CD5(+)B cells in the peritoneal cavity were CD1d(+). Proteinuria became prominent in NZB/W F(1) mice with the progression of disease. In parallel with this progression, the proportion of NKT cells decreased slightly in the liver, but their absolute number remained at a high level in this organ. These NKT cells were CD4(+) and used an invariant chain of Valpha14Jalpha281 for TCRalpha. Reflecting the elevation of CD5(+)B cells, autoantibodies against hepatocyte cytoplasmand denatured DNA were detected in sera. Although NKT cells are known to be immunoregulatory cells in some autoimmune mice, the present results raise the possibility that NKT cells as well as CD5(+)B cells might be associated with the onset of autoimmune diseases in NZB/W F(1) mice. Indeed, NKT cells in F(1) mice had a high potential to induce autoimmune-like inflammationwhen alpha-galactosylceramide was administered or when active NKT cells were transferred into young F(1) mice.     

Positive selection of NKT cells by CD1(+), CD11c(+) non-lymphoid cells residing in the extrathymic organs

Previously, we found that NK1.1(+), TCRalpha beta(+) natural killer T (NKT) cells develop in cytokine-supplemented suspension cultures of fetal liver established from normal, but not from beta2 microglobulin-deficient [beta2m(- / -)] mice, and that recombination-deficient SCID fetal liver can reconstiute NKT cell development in beta2m(- / -) fetal liver cultures. We found here that cells of SCID adult liver, bone marrow, spleen and thymus were able to reconstitute NKT cell development in the former culture system with efficiency comparable to normal thymic cells. The reconstitution of NKT cells was also seen in the bone marrow chimeras that had been administered a combination of beta2m(- / -) and Rag-2(- / -) bone marrow cells. Development of NKT cells was hampered by depletion of CD11c(+) or CD11b(+) cells, but not by removal of B220(+) or Gr-1(+) cells from cultures of normal fetal liver cells. Furthermore CD11c(+), CD11b(+) and / or CD11c(+) CD11b(-) cells (both populations were CD1-dull positive) enriched from Rag-2-deficient fetal livers and pulsed with alpha-galactosylceramide, a possible antigen for NKT cells, were shown to reconstitute the NKT cell development in beta2m(- / -) fetal liver cultures. Collectively, our findings suggest that non-lymphoid cells, presumably CD11c(+), CD11b(+) and / or CD11c(+), CD11b(-) dendritic cells, are involved in the mechanism of positive selection of NKT cells in the thymus and extrathymic organs.     

Regulatory roles of CD1d-restricted NKT cells in the induction of toxic shock-like syndrome in an animal model of fatal ehrlichiosis

CD1d-restricted NKT cells are key players in host defense against various microbial infections. Using a murine model of fatal ehrlichiosis, we investigated the role of CD1d-restricted NKT cells in induction of toxic shock-like syndrome caused by gram-negative, lipopolysaccharide-lacking, monocytotropic Ehrlichia. Our previous studies showed that intraperitoneal infection of wild-type (WT) mice with virulent Ehrlichia (Ixodes ovatus Ehrlichia [IOE]) results in CD8+ T-cell-mediated fatal toxic shock-like syndrome marked by apoptosis of CD4+ T cells, a weak CD4+ Th1 response, overproduction of tumor necrosis factor alpha and interleukin-10, and severe liver injury. Although CD1d-/- mice succumbed to high-dose IOE infection similar to WT mice, they did not develop signs of toxic shock, as shown by elevated bacterial burdens, low serum levels of tumor necrosis factor, normal serum levels of liver enzymes, and the presence of few apoptotic hepatic cells. An absence of NKT cells restored the percentages and absolute numbers of CD4+ and CD8+ T cells and CD11b+ cells in the spleen compared to WT mice and was also associated with decreased expression of Fas on splenic CD4+ lymphocytes and granzyme B in hepatic CD8+ lymphocytes. Furthermore, our data show that NKT cells promote apoptosis of macrophages and up-regulation of the costimulatory molecule CD40 on antigen-presenting cells, including dendritic cells, B cells, and macrophages, which may contribute to the induction of pathogenic T-cell responses. In conclusion, our data suggest that NKT cells mediate Ehrlichia-induced T-cell-mediated toxic shock-like syndrome, most likely via cognate and noncognate interactions with antigen-presenting cells.     

The role of intrahepatic CD8+ T cell trapping and NK1.1+ cells in liver-mediated immune regulation

The liver was previously suggested as a site of lymphocyte clearance. Liver-associated lymphocytes that express NK1.1 marker (NKT LAL) play a role in immune modulation. Aim: To determine the role of the liver and of NKT LAL in determining the CD4+/CD8+ balance during tolerance induction. Methods: Colitis was induced in C57 mice by intracolonic instillation of trinitrobenzenesulfonic acid (TNBS). Immune tolerance was induced via five oral feedings of colitis-extracted proteins (CEP) from TNBS-colitis colonic wall, starting on the day of colitis induction (group A). Control mice were fed with BSA (group B). To determine the role of NKT cells in immune modulation, NK1.1 depletion was performed in nonfed (group C) and fed (group D) mice. To further evaluate the role of NKT cells in this model, mice in group E were tolerized following NKT depletion. To determine the effect of NKT depletion in a tolerized environment, tolerized mice in group F were NKT depleted following tolerance induction. Peripheral and intrahepatic NK1.1+ and CD4+/CD8+ T cells were determined in all groups. Colitis was assessed by standard clinical and histologic scores. Serum cytokines levels were measured by ELISA. Results: Oral tolerance induction led to a marked alleviation of colitis as manifested by a significant improvement of the clinical, macroscopic, and microscopic scores of colitis (group A vs. group B). NK1.1+ depletion without tolerance induction had a favorable effect on colitis (C). Depletion of NKT LAL prevented the ability to induce tolerance (group D). However, induction of tolerance following NK1.1+ depletion, and NK1.1+ depletion following tolerance induction led to a marked improvement of colitis (groups E and F). Tolerance induction led to a significant increase in NKT LAL numbers. The peripheral CD4+/CD8+ ratio increased up to 3-fold in tolerized vs. non-tolerized mice. A similar increase was observed in NKT-depleted healthy mice in groups C, E, and F (P < 0.005). In contrast, NK1.1+ depletion in the presence of antigen in the bowel led to a reverse effect with a significant decrease in the peripheral CD4+/CD8+ ratio. An opposite effect was observed in the intrahepatic CD4+/CD8+. The peripheral/intrahepatic CD4+/CD8+ ratio increased significantly in tolerized and in healthy mice (A, D, E, F, P < 0.005). In contrast, NK1.1+ depleted fed mice in group C manifested a marked decrease in the peripheral/intrahepatic CD4+/CD8+ ratio. Induction of tolerance led to a marked increase in the IL-10/interferon gamma (IFNgamma) and IL-4/IFNgamma ratios. Conclusions: In the experimental colitis model, the liver is an important site for CD8+ accumulation during tolerance induction in a process that is independent of NK1.1+ cells. NK1.1+ cells play a dual role in the pro/anti-inflammatory balance. In the presence of antigen, these lymphocytes may be accountable for keeping an anti-inflammatory lymphocyte balance. However, in the absence of antigen, they may induce a pro-inflammatory shift.     

C57BL/6小鼠不同组织器官中NKT细胞的比较

目的比较C57BL/6小鼠肝脏、肺脏、脾脏和肠系膜淋巴结中NKT细胞的含量、亚型和功能的特点。方法分离正常C57BL/6小鼠肝脏、肺脏、脾脏和肠系膜淋巴结的淋巴细胞,利用细胞表面分子染色的方法,观察不同组织器官中CD3+NK1.1+NKT细胞及其亚型的含量;淋巴细胞经过PMA和离子霉素刺激后,应用细胞内细胞因子染色的方法,通过流式细胞仪观察NKT细胞IFN-γ、IL-4、IL-9和IL-17的产生情况。结果肝脏中NKT细胞的含量为(25.2±12)%,显著高于肺脏、脾脏和肠系膜淋巴结。肝脏、脾脏和肠系膜淋巴结中NKT细胞以CD4+细胞亚群为主,而肺脏中NKT细胞以CD4-CD8-亚群为主,同时肠系膜淋巴结的NKT细胞中存在CD4+CD8+亚群。不同组织器官中NKT细胞IFN-γ、IL-4、IL-9和IL-17产生的能力有差别。结论 C57BL/6小鼠肝脏、肺脏、脾脏和肠系膜淋巴结的NKT细胞在含量、表型和功能方面可能存在明显的差异。     

Comparison of intrahepatic lymphocytes from normal and growth hormone transgenic mice with chronic hepatitis and liver cancer.

Mice expressing an ovine growth hormone-mouse metallothionein promoter fusion gene (METoGH mice) develop chronic hepatitis which becomes progressively more severe over time, hepatocellular adenomas, and eventually carcinoma in the oldest animals. T-lymphocyte expression of activation/memory-associated markers was compared between liver and blood lymphocytes isolated from METoGH and non-transgenic mice at 7, 10 and 12 months of age. The percentage of intrahepatic lymphocytes (IHL) which were CD4+ was markedly diminished in METoGH mice at all times. CD4+ and CD8+ IHL in METoGH mice expressed Ly-6A/6D at increased density, and were CD45RBlo at later time-points. Ly-6C+ and NK1.1+ CD4+ cells, which are common in normal mouse liver, were found at decreased frequency in METoGH livers. Further analysis demonstrated that, as a proportion of total T-cell receptor (TCR) alpha beta cells, NK1.1+ TCR alpha beta int CD4+ cell numbers (NKT cells) were diminished in the livers of METoGH mice. Observations made in METoGH mice support the hypothesis that sustained liver inflammation and hepatocellular injury may be linked to liver cancer. Additionally, it is possible that the relative lack of NKT cells may create an environment permissive for the growth of liver tumours.     

Studies on the Thymus in Chagas'' Disease II. Thymocyte Subset Fluctuations in Trypanosoma cruzi-infected mice: Relationship to Stress

Changes in thymic T-cell subsets in mice acutely infected with Trypanosoma cruzi have been studied in both C3H/HeJ and C57BL/6 mice. The significant decrease in thymocyte number, observed in both mouse strains on day 14 post-infection correlated with a drastic decrease in CD4+CD8+ cell number, whereas the number of CD4-CD8-, CD4+CD8- and CD4-CD8+ cells remained essentially unchanged. The important increase in CD3hi cell frequency confirmed that resistant thymocytes during Chagas' disease development were mostly medullary thymocytes, whereas the thymic cortex was largely depleted, as previously observed on thymus sections. This involution of the thymus could have been due to the increase of circulating glucocorticoid levels observed after infection. However, similar cell modifications were found in infected adrenalectomized mice whose serum corticosterone levels were only slightly augmented. Thus, the thymic alterations appear not to be linked to stress responses, at least those dependent on high levels of circulating glucocorticoids.     

Endoplasmic reticulum stress, hepatocyte CD1d and NKT cell abnormalities in murine fatty livers

The liver regulates lipid homeostasis and is enriched with natural killer T (NKT) cells that respond to lipid antigens. Optimal maturation and activation of NKT cells requires their interaction with lipid antigens that are presented by cluster of differentiation-1 (CD-1) molecules on antigen-presenting cells. Hepatocytes express CD1d and present lipid antigens to NKT cells. Depletion and dysregulation of hepatic NKT cells occurs in mice with fatty livers. Herein, we assess whether reduced CD1d content on steatotic hepatocytes contributes to fatty liver-associated NKT cell abnormalities. We show that despite expressing normal levels of CD1d mRNA, fatty hepatocytes from ob/ob mice have significantly less CD1d on their plasma membranes than normal hepatocytes. This has functional significance because ob/ob hepatocytes are less able to activate CD1d-restricted T-cell responses in vitro, and CD1d-reactive NKT cells are reduced in ob/ob livers. Events in the endoplasmic reticulum (ER) normally regulate CD1d trafficking to plasma membranes. Hepatic steatosis has been associated with ER stress. To determine if ER stress reduces CD-1 accumulation on hepatocytes, we evaluated hepatic ER stress in ob/ob mice and treated cultured hepatocytes and lean mice with tunicamycin to induce ER stress. Lipid accumulation and ER stress occurred in the livers of both ob/ob and tunicamycin-treated mice. Tunicamycin caused dose-dependent decreases in hepatocyte CD1d, inhibited hepatocyte activation of CD1d-restricted T-cell responses, depleted liver populations of CD1d-reactive NKT cells and promoted Th-1 polarization of hepatic cytokine production. In conclusion, ER stress-related decreases in hepatocyte CD1d contribute to NKT cell dysregulation in fatty livers.     

Activation-induced NKT cell hyporesponsiveness protects from alpha-galactosylceramide hepatitis and is independent of active transregulatory factors

NK T (NKT) cells, unique lymphocytes expressing features of NK and T lymphocytes, can specifically be activated with the glycolipid antigen alpha-galactosylceramide (alpha-GalCer). In humans and mice, this activation provokes pronounced cytokine responses. In C57BL/6 mice, alpha-GalCer injection additionally induces NKT-mediated liver injury, representing a model for immune-mediated hepatitis in humans. However, a single alpha-GalCer pretreatment of mice prevented NKT-mediated liver injury, cytokine responses (systemically and locally in the liver), and up-regulation of hepatocellular Fas upon alpha-GalCer rechallenge. As alpha-GalCer is used as a NKT cell-activating agent in clinical trials, an investigation of tolerance induction appears crucial. We demonstrate that alpha-GalCer tolerance does not depend on Kupffer cells, IL-10, Caspase-3-mediated apoptosis, or CD4+CD25+ T regulatory cells (Tregs), which are crucial in other models of immunological tolerance. Amending relevant, earlier approaches of others, we cocultivated highly purified, nontolerized and tolerized liver NKT cells ex vivo and could convincingly exclude the relevance of transdominant NKT Tregs. These results strongly suggest alpha-GalCer-induced tolerance to be exclusively caused by NKT cell intrinsic hyporesponsiveness. Tolerized mice showed specific diminishment of the intrahepatic CD4+ NKT cell subpopulation, with the CD4(-) population largely unaffected, and revealed down-modulation of alpha-GalCer-specific TCR and the NKT costimulator glucocorticoid-induced TNFR-related protein on liver NKT cells, whereas inhibitory Ly49I was increased. In conclusion, alpha-GalCer tolerance could serve as a model for the frequently observed NKT cell hyporesponsiveness in tumor patients and might help to develop strategies for their reactivation. Conversely, approaches to render NKT cells hyporesponsive may constitute new therapeutic strategies for diseases, where aberrant NKT cell activation is causally involved.     

Tissue-specific segregation of TCRgamma delta+ NKT cells according to phenotype TCR repertoire and activation status: parallels with TCR alphabeta+NKT cells

Whereas the majority of NKT cells in the mouse express an alpha beta TCR (NKTalpha beta cells), a small subset of NKT cells express a gamma delta TCR (NKTgamma delta). Here we have systematically analyzed the phenotype, TCR repertoire and activation status of NKTgamma delta cells in the thymus, liver, spleen and bone marrow of normal C57BL/6 mice. Our data indicate that NKTgamma delta cells segregate in a tissue-specific manner according to these parameters. While most NKTgamma delta cells in the thymus and liver have a recently activated CD62L(lo) phenotype and a TCR repertoire that is heavily biased to Vgamma1.1 and Vdelta6.3, the majority of NKTgamma delta cells in the spleen and bone marrow are CD62L(hi) and have a much less biased TCR repertoire. Moreover, expression of NK markers is high on NKTgamma delta cells in spleen and bone marrow but low in thymus and liver. Collectively our results reveal a tissue-specific segregation of NKTgamma delta cells that is strikingly similar to that recently described for CD1d-dependent and Cd1d-independent NKTalpha beta cells. We therefore propose that chronic TCR activation by tissue-specific endogenous ligands is a generic property of NKT cells of both the alpha beta and gamma delta lineages.     

Phenotypic characterization of CD8(+)NKT cells

We describe a novel CD8(+)NKT cell population expressing TCRalpha /beta or TCRgamma /delta. These CD8(+)NKT cells were prominent in the liver, and except for the thymus, virtually absent in other lymphoid organs. CD8(+)NKT cells expressed activation markers and comprised a high proportion of Ly49(+) cells. The development of the majority of CD8(+)NKT cells expressing TCRalpha /beta, but not TCRgamma /delta, depended on classical MHC class I. No CD8(+)NKT cells were detectable in young athymic mice, whereas the cells expressing TCRgamma /delta, but not TCRalpha /beta, appeared randomly in aged athymic mice. CD8(+)NK1(+) TCRalpha /beta cells showed polyclonal TCRVbeta usage and were virtually devoid of TCRValpha14. CD8(+)NK1(+) TCRgamma /delta cells predominantly expressed TCRVgamma1, 2 and 4, and Vdelta4, 5, 6 and 7. CD8(+)NKT cells, in particular those expressing TCRgamma /delta, were a major population in early life. IFN-gamma, but not IL-4, was induced in CD8(+)NKT cells by in vitro stimulation, independent of the TCRalpha /beta or TCRgamma /delta lineage. Hence, these cells represent a unique, though heterogeneous T cell population that shares markers with, but is distinct from, both conventional NKT cells and conventional CD8(+) T cells, and that may play a role in immune regulation.     

Role of CD44 and its v7 isoform in staphylococcal enterotoxin B-induced toxic shock: CD44 deficiency on hepatic mononuclear cells leads to reduced activation-induced apoptosis that results in increased liver damage

Exposure to bacterial superantigens such as staphylococcal enterotoxin B (SEB) leads to the induction of toxic shock syndrome which results in multiorgan failure, including liver damage. In the present study, we investigated the role of CD44 in SEB-induced liver injury. Injection of SEB into d-galactosamine-sensitized CD44 wild-type (WT) mice led to a significant increase in CD44 expression on liver T cells, NK cells, and NKT cells. Administration of SEB to CD44 knockout (KO) mice caused significantly enhanced liver damage which correlated with elevated numbers of T cells, NK cells, NKT cells, and macrophages in the liver and increased production of tumor necrosis factor alpha and gamma interferon compared to CD44 WT mice. Furthermore, liver mononuclear cells from CD44 KO mice were resistant to SEB-induced apoptosis, and cDNA microarray analysis revealed that SEB activation of such cells led to the induction of several antiapoptotic genes and repression of proapoptotic genes. Examination of CD44 isoforms revealed that SEB exposure altered CD44 variant 7 (v7) isoform expression. Interestingly, mice bearing a specific deletion of the CD44v7 exon exhibited increased susceptibility to SEB-induced hepatitis. Finally, treatment of CD44 WT mice with anti-CD44 monoclonal antibodies reduced expression of CD44 in liver mononuclear cells and caused increased susceptibility to SEB-induced liver injury. Together, these data demonstrate that the expression of CD44 and/or CD44v7 on SEB-activated liver mononuclear cells facilitates their rapid apoptosis, thereby preventing severe liver injury in wild-type mice, and suggest that CD44 plays an important role in the regulation and elimination of immune cells in the liver.