Reversible NK1.1 surface expression on invariant liver natural killer T cells during Listeria monocytogenes infection

The invariant (i) natural killer (NK)T cells consistently express the Valpha14 chain of the T cell receptor (TCR) and recognize alpha-galactosylceramide (alpha-GalCer) presented by the nonpolymorphic presentation molecule CD1d. Despite their name, the iNKT cells represent a heterogeneous population, which can be divided on the basis of NK1.1 surface expression. Here we show that NK1.1 surface expression on liver iNKT cells in mice fluctuates during Listeria monocytogenes infection. At early stages of listeriosis, iNKT cells expressing NK1.1 were numerically reduced and those lacking NK1.1 were increased. At later time points, the NK1.1(-) iNKT cell population contracted, whereas NK1.1(+) iNKT cells reemerged. Alterations in NK1.1 surface expression on iNKT cells were paralleled by numerical changes of interleukin (IL)-12 producers in the liver and were completely prevented by endogenous IL-12 neutralization, whereas NK1.1 surface alterations on iNKT cells following alpha-GalCer stimulation were not prevented. Adoptive cell transfer experiments revealed that the liver NK1.1(-) iNKT cells from NK1.1(+) cell-depleted L. monocytogenes-infected mice accumulated in the liver of recipient recombination-activating gene-1-deficient mice where they acquired NK1.1 surface expression. Thus, we present first evidence that NK1.1 surface expression on liver iNKT cells is reversible during L. monocytogenes infection, and that different mechanisms underlie stimulation by TCR and IL-12.     

CD1d-independent regulation of NKT cell migration and cytokine production upon Listeria monocytogenes infection

Natural killer T (NKT) cells are a unique T-cell population that is positively selected by CD1d-expressing cells. In this study, we examined the kinetics of conventional CD4+TCRbeta+ and CD4-TCRbeta+ cells along with various NKT cell populations from WT and CD1d KO mice after oral Listeria monocytogenes (Lm) infection at different time points in tissue compartments. We found that CD4+TCRbeta+ cells expressing NK1.1+ (NKT) were constitutively expressed in the lung of both strains of mice, but disappeared after infection. In contrast, CD4-TCRbeta+ NK1.1+ cells migrated to the spleen. Here, we demonstrated that endogenous IL-12 was predominantly expressed in the spleen of CD1d KO mice 2 days after infection, whereas IL-4 was predominantly expressed in the liver of WT mice. Higher levels of IFN-gamma were expressed in MLN of CD1d KO but not in WT mice on day 5. Thus, tissue-specific ligands orchestrate the localization and activation of NKT cells to control immune response to Listeria, which may explain the difference in disease susceptibility.     

Protection from type 1 diabetes by invariant NK T cells requires the activity of CD4+CD25+ regulatory T cells

Invariant NK T (iNKT) cells regulate immune responses, express NK cell markers and an invariant TCR, and recognize lipid Ags in a CD1d-restricted manner. Previously, we reported that activation of iNKT cells by alpha-galactosylceramide (alpha-GalCer) protects against type 1 diabetes (T1D) in NOD mice via an IL-4-dependent mechanism. To further investigate how iNKT cells protect from T1D, we analyzed whether iNKT cells require the presence of another subset(s) of regulatory T cells (Treg), such as CD4+ CD25+ Treg, for this protection. We found that CD4+ CD25+ T cells from NOD.CD1d(-/-) mice deficient in iNKT cell function similarly in vitro to CD4+ CD25+ T cells from wild-type NOD mice and suppress the proliferation of NOD T responder cells upon alpha-GalCer stimulation. Cotransfer of NOD diabetogenic T cells with CD4+ CD25+ Tregs from NOD mice pretreated with alpha-GalCer demonstrated that activated iNKT cells do not influence the ability of T(regs) to inhibit the transfer of T1D. In contrast, protection from T1D mediated by transfer of activated iNKT cells requires the activity of CD4+ CD25+ T cells, because splenocytes pretreated with alpha-GalCer and then inactivated by anti-CD25 of CD25+ cells did not protect from T1D. Similarly, mice inactivated of CD4+ CD25+ T cells before alpha-GalCer treatment were also not protected from T1D. Our data suggest that CD4+ CD25+ T cells retain their function during iNKT cell activation, and that the activity of CD4+ CD25+ Tregs is required for iNKT cells to transfer protection from T1D.     

Essential role of bystander cytotoxic CD122+CD8+ T cells for the antitumor immunity induced in the liver of mice by alpha-galactosylceramide

We recently reported that NK cells and CD8(+) T cells contribute to the antimetastatic effect in the liver induced by alpha-galactosylceramide (alpha-GalCer). In the present study, we further investigated how CD8(+) T cells contribute to the antimetastatic effect induced by alpha-GalCer. The injection of anti-CD8 Ab into mice 3 days before alpha-GalCer injection (2 days before intrasplenic injection of B16 tumors) did not inhibit IFN-gamma production nor did it reduce the NK activity of liver mononuclear cells after alpha-GalCer stimulation. However, it did cause a reduction in the proliferation of liver mononuclear cells and mouse survival time. Furthermore, although the depletion of NK and NKT cells (by anti-NK1.1 Ab) 2 days after alpha-GalCer injection no longer decreased the survival rate of B16 tumor-injected mice, the depletion of CD8(+) T cells did. CD122(+)CD8(+) T cells in the liver increased after alpha-GalCer injection, and antitumor cytotoxicity of CD8(+) T cells in the liver gradually increased until day 6. These CD8(+) T cells exhibited an antitumor cytotoxicity toward not only B16 cells, but also EL-4 cells, and their cytotoxicity significantly decreased by the depletion of CD122(+)CD8(+) T cells. The critical, but bystander role of CD122(+)CD8(+) T cells was further confirmed by adoptive transfer experiments into CD8(+) T cell-depleted mice. Furthermore, it took 14 days after the first intrasplenic B16/alpha-GalCer injection for the mice to generate CD8(+) T cells that can reject s.c. rechallenged B16 cells. These findings suggest that alpha-GalCer activates bystander antitumor CD122(+)CD8(+) T cells following NK cells and further induces an adaptive antitumor immunity due to tumor-specific memory CD8(+) CTLs.     

The role of CD1d in the immune response against Listeria infection

To address the role of CD1d in mucosal immune regulation in bacterial infection, we infected CD1d KO mice with Listeria monocytogenes (Lm). A higher systemic bacterial burden associated with inflammatory lymphocytic infiltrations within the intestine was found in CD1d KO compared with wild type (WT) mice. Lm induced strong IFN-gamma mRNA expression in the liver of WT and the intestine of CD1d KO mice, thus demonstrating the dual, opposing immune activities of IFN-gamma in Lm infection that is dependent on CD1d and/or NKT cells. Analysis of hepatic T cell population demonstrated a reduction of NK1.1(+)TCRbeta+ cells in both mice, followed by recovery only in WT mice. Last, the proportion of alpha4beta1 integrin on lung lymphocytes from CD1d KO was dramatically increased compared with WT mice. Thus, the absence of CD1d resulted in increased susceptibility towards Listeria infection, induced changes in NKT cells, and increased trafficking of alpha4beta1 molecule to inflamed lung.     

Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by alpha-galactosylceramide in mice

The role of mouse liver NK1.1 Ag(+) T (NKT) cells in the antitumor effect of alpha-galactosylceramide (alpha-GalCer) has been unclear. We now show that, whereas alpha-GalCer increased the serum IFN-gamma concentration and alanine aminotransferase activity in NK cell-depleted C57BL/6 (B6) mice and B6-beige/beige mice similarly to its effects in control B6 mice, its enhancement of the antitumor cytotoxicity of liver mononuclear cells (MNCs) was abrogated. Depletion of both NK and NKT cells in B6 mice reduced all these effects of alpha-GALCER: Injection of Abs to IFN-gamma also inhibited the alpha-GalCer-induced increase in antitumor cytotoxicity of MNCS: alpha-GalCer induced the expression of Fas ligand on NKT cells in the liver of B6 mice. Whereas alpha-GalCer did not increase serum alanine aminotransferase activity in B6-lpr/lpr mice and B6-gld/gld mice, it increased the antitumor cytotoxicity of liver MNCS: The alpha-GalCer-induced increase in survival rate apparent in B6 mice injected intrasplenically with B16 tumor cells was abrogated in beige/beige mice, NK cell-depleted B6 mice, and B6 mice treated with Abs to IFN-gamma. Depletion of CD8(+) T cells did not affect the alpha-GalCer-induced antitumor cytotoxicity of liver MNCs but reduced the effect of alpha-GalCer on the survival of B6 mice. Thus, IFN-gamma produced by alpha-GalCer-activated NKT cells increases both the innate antitumor cytotoxicity of NK cells and the adaptive antitumor response of CD8(+) T cells, with consequent inhibition of tumor metastasis to the liver. Moreover, NKT cells mediate alpha-GalCer-induced hepatocyte injury through Fas-Fas ligand signaling.     

Repeated alpha-galactosylceramide administration results in expansion of NK T cells and alleviates inflammatory dermatitis in MRL-lpr/lpr mice

NK T (NKT) cells expressing the invariant Valpha14-Jalpha18 TCR alpha-chain recognize glycolipid Ags such as alpha-galactosylceramide (alpha-GalCer) presented by the MHC class I-like molecule CD1d. Upon activation by alpha-GalCer, invariant NKT cells secrete multiple cytokines and confer protection in certain immune-mediated disorders. Here we have investigated the role of NKT cells in the development of inflammatory dermatitis in MRL-lpr/lpr mice, which shares features with lupus in humans. Our results show that the numbers Sand functions of NKT (TCRbeta(+)CD1d/alpha-GalCer tetramer(+)) cells, particularly of the NK1.1(-) subset, are reduced in MRL-lpr/lpr mice compared with MRL-fas/fas and/or nonautoimmune C3H/Hej and BALB/c mice. Repeated treatments with alpha-GalCer result in the expansion of NKT cells and alleviate dermatitis in MRL-lpr/lpr mice. Our results indicate that NKT cell deficiency can be corrected by repeated alpha-GalCer treatment and that NKT cells may play a protective role in inflammatory dermatitis of lupus-prone mice.     

NK T cells contribute to expansion of CD8(+) T cells and amplification of antiviral immune responses to respiratory syncytial virus

CD1d-deficient mice have normal numbers of T lymphocytes and natural killer cells but lack Valpha14(+) natural killer T cells. Respiratory syncytial virus (RSV) immunopathogenesis was evaluated in 129xC57BL/6, C57BL/6, and BALB/c CD1d(-/-) mice. CD8(+) T lymphocytes were reduced in CD1d(-/-) mice of all strains, as shown by cell surface staining and major histocompatibility complex class I tetramer analysis, and resulted in strain-specific alterations in illness, viral clearance, and gamma interferon (IFN-gamma) production. Transient activation of NK T cells in CD1d(+/+) mice by alpha-GalCer resulted in reduced illness and delayed viral clearance. These data suggest that early IFN-gamma production and efficient induction of CD8(+)-T-cell responses during primary RSV infection require CD1d-dependent events. We also tested the ability of alpha-GalCer as an adjuvant to modulate the type 2 immune responses induced by RSV glycoprotein G or formalin-inactivated RSV immunization. However, immunized CD1-deficient or alpha-GalCer-treated wild-type mice did not exhibit diminished disease following RSV challenge. Rather, some disease parameters, including cytokine production, eosinophilia, and viral clearance, were increased. These findings indicate that CD1d-dependent NK T cells play a role in expansion of CD8(+) T cells and amplification of antiviral responses to RSV.     

NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells

Modification in the function of dendritic cells (DC), such as that achieved by microbial stimuli or T cell help, plays a critical role in determining the quality and size of adaptive responses to Ag. NKT cells bearing an invariant TCR (iNKT cells) restricted by nonpolymorphic CD1d molecules may constitute a readily available source of help for DC. We therefore examined T cell responses to i.v. injection of soluble Ag in the presence or the absence of iNKT cell stimulation with the CD1d-binding glycolipid alpha-galactosylceramide (alpha-GalCer). Considerably enhanced CD4(+) and CD8(+) T cell responses were observed when alpha-GalCer was administered at the same time as or close to OVA injection. This enhancement was dependent on the involvement of iNKT cells and CD1d molecules and required CD40 signaling. Studies in IFN-gammaR(-/-) mice indicated that IFN-gamma was not required for the adjuvant effect of alpha-GalCer. Consistent with this result, enhanced T cell responses were observed using OCH, an analog of alpha-GalCer with a truncated sphingosine chain and a reduced capacity to induce IFN-gamma. Splenic DC from alpha-GalCer-treated animals expressed high levels of costimulatory molecules, suggesting maturation in response to iNKT cell activation. Furthermore, studies with cultured DC indicated that potentiation of T cell responses required presentation of specific peptide and alpha-GalCer by the same DC, implying conditioning of DC by iNKT cells. The iNKT-enhanced T cell responses resisted challenge with OVA-expressing tumors, whereas responses induced in the absence of iNKT stimulation did not. Thus, iNKT cells exert a significant influence on the efficacy of immune responses to soluble Ag by modulating DC function.     

Protective immunosurveillance of the central nervous system by Listeria-specific CD4 and CD8 T cells in systemic listeriosis in the absence of intracerebral Listeria

The invasion of the CNS by pathogens poses a major risk for damage of the highly vulnerable brain. The aim of the present study was to analyze immunological mechanisms that may prevent spread of infections to the CNS. Intraperitoneal application of Listeria monocytogenes to mice induced infection of the spleen, whereas pathogens remained absent from the brain. Interestingly, Listeria-specific CD4 and CD8 T cells homed to the brain and persisted intracerebrally for at least 50 days after both primary and secondary infection. CD4 and CD8 T cells resided in the leptomeninges, in the choroid plexus, and, in low numbers, in the brain parenchyma. CD4 and CD8 T cells isolated from the brain early after infection (day 7) were characterized by an activated phenotype with spontaneous IFN-gamma production, whereas at a later stage of infection (day 28) restimulation with Listeria-specific peptides was required for the induction of IFN-gamma production by CD4 and CD8 T cells. In contrast to splenic T cells, T cells in the brain did not exhibit cytotoxic activity. Adoptively transferred T cells isolated from the brains of Listeria-infected mice reduced the bacterial load in cerebral listeriosis. The frequency of intracerebral Listeria-specific T cells was partially regulated by the time of exposure to Listeria and cross-regulated by CD4 and CD8 T cells. Collectively, these data reveal a novel T cell-mediated pathway of active immunosurveillance of the CNS during bacterial infections.     

The complementarity determining region 2 of BV8S2 (V beta 8.2) contributes to antigen recognition by rat invariant NKT cell TCR

Invariant NKT cells (iNKT cells) are characterized by a semi-invariant TCR comprising an invariant alpha-chain paired with beta-chains with limited BV gene usage which are specific for complexes of CD1d and glycolipid Ags like alpha-galactosylceramide (alpha-GalCer). iNKT cells can be visualized with alpha-GalCer-loaded CD1d tetramers, and the binding of mouse CD1d tetramers to mouse as well as to human iNKT cells suggests a high degree of conservation in recognition of glycolipid Ags between species. Surprisingly, mouse CD1d tetramers failed to stain a discrete cell population among F344/Crl rat liver lymphocytes, although comprised iNKT cells are indicated by IL-4 and IFN-gamma secretion after alpha-GalCer stimulation. The arising hypothesis that rat iNKT TCR recognizes alpha-GalCer only if presented by syngeneic CD1d was then tested with the help of newly generated rat and mouse iNKT TCR-transduced cell lines. Cells expressing mouse iNKT TCR reacted to alpha-GalCer presented by rat or mouse CD1d and efficiently bound alpha-GalCer-loaded mouse CD1d tetramers. In contrast, cells expressing rat iNKT TCR responded only to alpha-GalCer presented by syngeneic CD1d and bound mouse CD1d tetramers only poorly or not at all. Finally, CD1d-dependent alpha-GalCer reactivity and binding of mouse CD1d tetramers was tested for cells expressing iNKT TCR comprising either rat or mouse AV14 (Valpha14) alpha-chains and wild-type or mutated BV8S2 (Vbeta8.2) beta-chains. The results confirmed the need of syngeneic CD1d as restriction element for rat iNKT TCR and identified the CDR2 of BV8S2 as an essential site for ligand recognition by iNKT TCR.     

Conventional alpha beta T cells are sufficient for innate and adaptive immunity against enteric Listeria monocytogenes

We have begun to dissect the cellular requirements for generation of immunity against enteric infection by Listeria monocytogenes using a novel T(-) B(-) NK(-) mouse strain (mice double deficient for the common cytokine receptor gamma-chain (gamma(c)) and the recombinase-activating gene-2 (RAG2/gamma(c) mice). Initial experiments showed that C57BL/6 mice and alymphoid RAG2/gamma(c) mice had similar kinetics of bacterial accumulation in the spleen, liver, and brain early after intragastric L. monocytogenes infection (up to day 3), calling into question the physiologic role of gut-associated lymphoid cells during the passage of this enterobacterium into the host. However, in contrast to C57BL/6 mice, RAG2/gamma(c) mice rapidly succumbed to disseminated infection by day 7. Polyclonal lymph node CD4(+) and CD8(+) alphabeta T cells were able to confer RAG2/gamma(c) mice with long-lasting protection against enteric L. monocytogenes infection in the absence of gammadelta T, NK, and NK-T cells. Moreover, these alphabeta T-reconstituted RAG2/gamma(c) mice produced IFN-gamma at levels comparable to C57BL/6 mice in response to L. monocytogenes both in vitro and in vivo. Protection was IFN-gamma dependent, as RAG2/gamma(c) mice reconstituted with IFN-gamma-deficient alphabeta T cells were unable to control enteric L. monocytogenes infection. Furthermore, alphabeta T cell-reconstituted RAG2/gamma(c) mice were able to mount memory responses when challenged with lethal doses of L. monocytogenes. These data suggest that NK, NK-T, gammadelta T, and B cells are functionally redundant in the immunity against oral L. monocytogenes infection, and that in their absence alphabeta T cells are able to mediate the early IFN-gamma production required for both innate and adaptive immunity.     

A subset of NKT cells that lacks the NK1.1 marker, expresses CD1d molecules, and autopresents the alpha-galactosylceramide antigen

In the present report, we characterize a novel T cell subset that shares with the NKT cell lineage both CD1d-restriction and high reactivity in vivo and in vitro to the alpha-galactosylceramide (alpha-GalCer) glycolipid. These cells preferentially use the canonical Valpha14-Jalpha281 TCR-alpha-chain and Vbeta8 TCR-beta segments, and are stimulated by alpha-GalCer in a CD1d-dependent fashion. However, in contrast to classical NKT cells, they lack the NK1.1 marker and express high surface levels of CD1d molecules. In addition, this NK1.1(-) CD1d(high) T subset, further referred to as CD1d(high) NKT cells, can be distinguished by its unique functional features. Although NK1.1(+) NKT cells require exogenous CD1d-presenting cells to make them responsive to alpha-GalCer, CD1d(high) NKT cells can engage their own surface CD1d in an autocrine and/or paracrine manner. Furthermore, in response to alpha-GalCer, CD1d(high) NKT cells produce high amounts of IL-4 and moderate amounts of IFN-gamma, a cytokine profile more consistent with a Th2-like phenotype rather than the Th0-like phenotype typical of NK1.1(+) NKT cells. Our work reveals a far greater level of complexity within the NKT cell population than previously recognized and provides the first evidence for T cells that can be activated upon TCR ligation by CD1d-restricted recognition of their ligand in the absence of conventional APCs.     

Treatment with alpha-galactosylceramide before Trypanosoma cruzi infection provides protection or induces failure to thrive

Trypanosoma cruzi, a protozoan parasite, chronically infects many mammalian species and triggers a chronic inflammatory disease. Invariant Valpha14 NK T (iNKT) cells are a regulatory subset of T cells that can contribute to protection against pathogens and to control of chronic inflammatory diseases. alpha-Galactosylceramide (alpha-GalCer) is an iNKT cell-specific glycolipid Ag: a single immunization with alpha-GalCer stimulates robust IFN-gamma and IL-4 production by iNKT cells, while multiple immunizations stimulate IL-4 production, but limited IFN-gamma production. We recently demonstrated that iNKT cells help control T. cruzi infection and affect the chronic Ab response. Therefore, alpha-GalCer treatment might be used to increase protection or decrease chronic inflammation during T. cruzi infection. In this report, we show that a single dose of alpha-GalCer before T. cruzi infection decreases parasitemia. This protection is independent of IL-12, but dependent upon iNKT cell IFN-gamma. In addition, alpha-GalCer treatment of the IFN-gamma(-/-) mice exacerbates parasitemia through IL-4 production. Furthermore, a multiple dose regimen of alpha-GalCer before T. cruzi infection does not lower parasitemia and, surprisingly, after parasitemia has resolved, causes poor weight gain. These data demonstrate that during T. cruzi infection glycolipids can be used to manipulate iNKT cell responses and suggest the possibility of developing glycolipid treatments that can increase protection and possibly decrease the chronic inflammatory pathology.     

In vivo administration of anti-asialo-GM1 antibody enhances splenic clearance of Listeria monocytogenes

Resistance of mice to infection by Listeria monocytogenes involves a biphasic response. The first phase consists of the first 48 h after infection, during which there is multiplication of Listeria in the liver and spleen of infected mice. In these nonimmune mice, macrophages and polymorphonuclear leukocytes are the effector cells involved in controlling multiplication. In the second phase, cell-mediated immunity develops, beginning on day 2, during which multiplication of Listeria is prevented by macrophages possessing increased microbicidal activity that is mediated through the action of lymphokines released by immunologically committed T lymphocytes. The purpose of the present study was to define a role for natural killer (NK) cells in natural resistance to Listeria during the first 48 h after infection, prior to the development of specific immunity. Splenic NK cell activity was enhanced following a sublethal intravenous injection of viable Listeria as early as 24 h after injection and remained elevated throughout the nonimmune phase of infection. Interestingly, treatment of mice with anti-asialo-GM1 significantly enhanced the ability of mice to clear Listeria from the spleen relative to infected controls possessing intact NK cell populations. This was evidenced by 23-fold fewer bacteria obtained from the spleens of anti-asialo-GM1-treated mice. In addition, Percoll-enriched NK cell populations obtained from 48-hour Listeria-infected mice do not exhibit in vitro listericidal activity. These observations suggest a regulatory role of NK cells in resistance against Listeria and preclude a role for NK cells in direct cytolysis. Perhaps these cells modulate the immune response to Listeria by down-regulating the activity of the immune cells crucial to listerial resistance.     

Overexpression of IL-15 in vivo increases antigen-driven memory CD8+ T cells following a microbe exposure.

To elucidate potential roles of IL-15 in the maintenance of memory CD8+ T cells, we followed the fate of Ag-specific CD8+ T cells directly visualized with MHC class I tetramers coupled with listeriolysin O (LLO)(91-99) in IL-15 transgenic (Tg) mice after Listeria monocytogenes infection. The numbers of LLO(91-99)-positive memory CD8+ T cells were significantly higher at 3 and 6 wk after infection than those in non-Tg mice. The LLO(91-99)-positive CD8+ T cells produced IFN-gamma in response to LLO(91-99), and an adoptive transfer of CD8+ T cells from IL-15 Tg mice infected with L. monocytogenes conferred a higher level of resistance against L. monocytogenes in normal mice. The CD44+ CD8+ T cells from infected IL-15 Tg mice expressed the higher level of Bcl-2. Transferred CD44+ CD8+ T cells divided more vigorously in naive IL-15 Tg mice than in non-Tg mice. These results suggest that IL-15 plays an important role in long-term maintenance of Ag-specific memory CD8+ T cells following microbial exposure via promotion of cell survival and homeostatic proliferation.     

A cell-type specific CD1d expression program modulates invariant NKT cell development and function

Invariant NK T (iNKT) cells are a distinct subset of T cells that rapidly produce an array of immunoregulatory cytokines upon activation. Cytokines produced by iNKT cells subsequently transactivate other leukocytes and elicit their respective effector functions. In this way, iNKT cells play a central role in coordinating the development of immune responses in a variety of settings. However, the mechanisms governing the quality of the iNKT cell response elicited remain poorly defined. To address whether changes in the CD1d expression pattern could regulate iNKT cell function, we generated a transgenic (Tg) mouse model in which thymocytes and peripheral T cells express high levels of CD1d (Lck-CD1d Tg+ mice). The expression of CD1d by T cells was sufficient to rescue development of iNKT cells in mice deficient of endogenous CD1d. However, the relative proportions of iNKT cell subsets in Lck-CD1d Tg+ mice were distinctly different from those in wild-type mice, suggesting an altered developmental program. Additionally, iNKT cells were hyporesponsive to antigenic stimulation in vivo. Interestingly, Lck-CD1d Tg+ mice develop liver pathology in the absence of any exogenous manipulation. The results of these studies suggest that changes to the CD1d expression program modulate iNKT cell development and function.     

Inducible costimulator protein controls the protective T cell response against Listeria monocytogenes

The inducible costimulator protein (ICOS) was recently identified as a costimulatory molecule for T cells. Here we analyze the role of ICOS for the acquired immune response of mice against the intracellular bacterium Listeria monocytogenes. During oral L. monocytogenes infection, low levels of ICOS expression were detected by extracellular and intracellular Ab staining of Listeria-specific CD4(+) and CD8(+) T cells. Blocking of ICOS signaling with a soluble ICOS-Ig fusion protein markedly impaired the Listeria-specific T cell responses. Compared with control mice, the ICOS-Ig treated mice generated significantly reduced numbers of Listeria-specific CD8(+) T cells in spleen and liver, as determined by tetramer and intracellular cytokine staining. In contrast, the specific CD8(+) T cell response in the intestinal mucosa did not appear to be impaired by the ICOS-Ig treatment. Analysis of the CD4(+) T cell response revealed that ICOS-Ig treatment also affected the specific CD4(+) T cell response. When restimulated with listerial Ag in vitro, reduced numbers of CD4(+) T cells from infected and ICOS-Ig-treated mice responded with IFN-gamma production. The impaired acquired immune response in ICOS-Ig treated mice was accompanied by their increased susceptibility to L. monocytogenes infection. ICOS-Ig treatment drastically enhanced bacterial titers, and a large fraction of mice succumbed to the otherwise sublethal dose of infection. Thus, ICOS costimulation is crucial for protective immunity against the intracellular bacterium L. monocytogenes.