Impaired clearance of herpes simplex virus type 1 from mice lacking CD1d or NKT cells expressing the semivariant V alpha 14-J alpha 281 TCR

Ag-presenting molecule CD1 and CD1-restricted NKT cells are known to contribute to defense against a range of infectious pathogens, including some viruses. CD1-restricted NKT cells, a distinct subpopulation of T cells, have striking and rapid effector functions that contribute to host defense, including rapid production of IFN-gamma and IL-4, and activation of NK cells. Consideration of the important contributions of innate and adaptive immunity to clearance of HSV prompted us to investigate the role of CD1 and of NKT cells expressing the V alpha 14-J alpha 281 TCR in the pathogenesis of HSV infection. To address this issue, we compared infection in wild-type mice with that in CD1 gene knockout (GKO) and J alpha 281 GKO mice. In this study, we report impaired clearance of virus and viral Ags, and more florid acute infection in mice lacking CD1 (and by inference, CD1-restricted T cells), in comparison with parental C57BL6 mice. In J alpha 281 GKO mice there was also impairment of virus clearance, resembling that seen in CD1 GKO mice. These results imply roles for the V alpha 14-J alpha 281 subset of NKT cells and for CD1d in control of HSV infection.     

CD1d-restricted NKT cells: an interstrain comparison

CD1d-restricted Valpha14-Jalpha281 invariant alphabetaTCR(+) (NKT) cells are well defined in the C57BL/6 mouse strain, but they remain poorly characterized in non-NK1.1-expressing strains. Surrogate markers for NKT cells such as alphabetaTCR(+)CD4(-)CD8(-) and DX5(+)CD3(+) have been used in many studies, although their effectiveness in defining this lineage remains to be verified. Here, we compare NKT cells among C57BL/6, NK1.1-congenic BALB/c, and NK1.1-congenic nonobese diabetic mice. NKT cells were identified and compared using a range of approaches: NK1.1 expression, surrogate phenotypes used in previous studies, labeling with CD1d/alpha-galactosylceramide tetramers, and cytokine production. Our results demonstrate that NKT cells and their CD4/CD8-defined subsets are present in all three strains, and confirm that nonobese diabetic mice have a numerical and functional deficiency in these cells. We also highlight the hazards of using surrogate phenotypes, none of which accurately identify NKT cells, and one in particular (DX5(+)CD3(+)) actually excludes these cells. Finally, our results support the concept that NK1.1 expression may not be an ideal marker for CD1d-restricted NKT cells, many of which are NK1.1-negative, especially within the CD4(+) subset and particularly in NK1.1-congenic BALB/c mice.     

NKT cells are critical for the initiation of an inflammatory bowel response against Toxoplasma gondii

We demonstrated in this study the critical role of NKT cells in the lethal ileitis induced in C57BL/6 mice after infection with Toxoplasma gondii. This intestinal inflammation is caused by overproduction of IFN-gamma in the lamina propria. The implication of NKT cells was confirmed by the observation that NKT cell-deficient mice (Jalpha281(-/-)) are more resistant than C57BL/6 mice to the development of lethal ileitis. Jalpha281(-/-) mice failed to overexpress IFN-gamma in the intestine early after infection. This detrimental effect of NKT cells is blocked by treatment with alpha-galactosylceramide, which prevents death in C57BL/6, but not in Jalpha281(-/-), mice. This protective effect is characterized by a shift in cytokine production by NKT cells toward a Th2 profile and correlates with an increased number of mesenteric Foxp3 lymphocytes. Using chimeric mice in which only NKT cells are deficient in the IL-10 gene and mice treated with anti-CD25 mAb, we identified regulatory T cells as the source of the IL-10 required for manifestation of the protective effect of alpha-galactosylceramide treatment. Our results highlight the participation of NKT cells in the parasite clearance by shifting the cytokine profile toward a Th1 pattern and simultaneously to immunopathological manifestation when this Th1 immune response remains uncontrolled.     

Long-term survival of corneal allografts is dependent on intact CD1d-reactive NKT cells

BALB/c mice that tolerate the allogeneic grafts develop allogeneic-specific anterior chamber-associated immune deviation. Because CD1d-reactive NKT cells are essential for anterior chamber-associated immune deviation, we postulated that the survival of C57BL/6 (B6) cornea graft in BALB/c mice was also dependent on CD1d-reactive NKT cells. The B6 corneal graft rejection rate in BALB/c vs Jalpha281 knockout (KO) mice, which lack NKT cells, was measured. While there were no difference in the early phase of rejection, the survival rates at 12 wk after grafting for BALB/c and Jalpha281 KO mice were 50 and 0%, respectively. Because anti-CD1d mAb abrogated the corneal graft survival in the wild-type mice we concluded that CD1d-reactive NKT cells were essential for graft survival. Moreover, allospecific T regulatory (Tr) cells correlated with acceptance of B6 grafts in BALB/c mice, and the adoptive transfer of these allospecific Tr cells to Jalpha281 KO mice allowed a 50% survival rate of B6 cornea grafts. In conclusion, CD1d-reactive NKT cells are required for induction of allospecific Tr cells and are essential for survival of corneal allografts. Mechanisms that contribute to cornea graft acceptance may lead to new therapies for improvement in graft survival in high-risk corneas and other transplanted tissues and grafts.     

NKT cells are critical to initiate an inflammatory response after Pseudomonas aeruginosa ocular infection in susceptible mice

CD4(+) T cells produce IFN-gamma contributing to corneal perforation in C57BL/6 (B6) mice after Pseudomonas aeruginosa infection. To determine the role of NK and NKT cells, infected corneas of B6 mice were dual immunolabeled. Initially, more NKT than NK cells were detected, but as disease progressed, NK cells increased, while NKT cells decreased. Therefore, B6 mice were depleted of NK/NKT cells with anti-asialo GM1 or anti-NK1.1 Ab. Either treatment accelerated time to perforation, increased bacterial load and polymorphonuclear neutrophils, but decreased IFN-gamma and IL-12p40 mRNA expression vs controls. Next, RAG-1 knockout (-/-; no T/NKT cells), B6.TCR Jalpha281(-/-) (NKT cell deficient), alpha-galactosylceramide (alphaGalCer) (anergized NKT cells) injected and IL-12p40(-/-) vs B6 controls were tested. IFN-gamma mRNA was undetectable in RAG-1(-/-)- and alphaGalCer-treated mice at 5 h and was significantly reduced vs controls at 1 day postinfection. It also was reduced significantly in B6.TCR Jalpha281(-/-), alphaGalCer-treated, and IL-12p40(-/-) (activated CD4(+) T cells also reduced) vs control mice at 5 days postinfection. In vitro studies tested whether endotoxin (LPS) stimulated Langerhans cells and macrophages (Mphi; from B6 mice) provided signals to activate NKT cells. LPS up-regulated mRNA expression for IL-12p40, costimulatory molecules CD80 and CD86, NF-kappaB, and CD1d, and addition of rIFN-gamma potentiated Mphi CD1d levels. Together, these data suggest that Langerhans cell/Mphi recognition of microbial LPS regulates IL-12p40 (and CD1d) driven IFN-gamma production by NKT cells, that IFN-gamma is required to optimally activate NK cells to produce IFN-gamma, and that depletion of both NKT/NK cells results in earlier corneal perforation.     

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.     

CD8+ T cells rapidly acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in vivo

NKT cells express both NK cell-associated markers and TCR. Classically, these NK1.1+TCRalphabeta+ cells have been described as being either CD4+CD8- or CD4-CD8-. Most NKT cells interact with the nonclassical MHC class I molecule CD1 through a largely invariant Valpha14-Jalpha281 TCR chain in conjunction with either a Vbeta2, -7, or -8 TCR chain. In the present study, we describe the presence of significant numbers of NK1.1+TCRalphabeta+ cells within lymphokine-activated killer cell cultures from wild-type C57BL/6, CD1d1-/-, and Jalpha281-/- mice that lack classical NKT cells. Unlike classical NKT cells, 50-60% of these NK1.1+TCRalphabeta+ cells express CD8 and have a diverse TCR Vbeta repertoire. Purified NK1.1-CD8alpha+ T cells from the spleens of B6 mice, upon stimulation with IL-2, IL-4, or IL-15 in vitro, rapidly acquire surface expression of NK1.1. Many NK1.1+CD8+ T cells had also acquired expression of Ly-49 receptors and other NK cell-associated molecules. The acquisition of NK1.1 expression on CD8+ T cells was a particular property of the IL-2Rbeta+ subpopulation of the CD8+ T cells. Efficient NK1.1 expression on CD8+ T cells required Lck but not Fyn. The induction of NK1.1 on CD8+ T cells was not just an in vitro phenomenon as we observed a 5-fold increase of NK1.1+CD8+ T cells in the lungs of influenza virus-infected mice. These data suggest that CD8+ T cells can acquire NK1.1 and other NK cell-associated molecules upon appropriate stimulation in vitro and in vivo.     

Immune responses of different mouse strains after challenge with equivalent lethal doses of Toxoplasma gondii

Most immunological studies that utilize different strains of inbred mice following T. gondii infection fail to compensate for differences in host susceptibility to the size of the parasite innoculum. To address this concern, susceptible C57BL/6 and resistant CBA/J mice were orally infected with either an equivalent 50% lethal dose (LD50) of brain cysts of the 76K strain of T. gondii (15 cysts in C57BL/6, 400 cysts in CBA/J) or the same dose of parasites in each mouse strain. C57BL/6 mice receiving 400 cysts (LD50 of CBA/J mice) died post infection, whereas CBA/J mice that received 15 cysts (LD50 of C57BL/6 mice) survived. Parasite loads in the brains and serum Toxoplasma-specific IgG1 titers of LD50-infected C57BL/6 mice were significantly higher than those in LD50- or 15 cysts-infected CBA/J mice, whereas splenocyte proliferation to Toxoplasma antigen and the percentage of CD8 alpha+ T cells were reduced in LD50-infected C57BL/6 mice. In contrast, serum IgG2a and IgM titers, the percentage of gamma delta T cells and IFN-gamma expression of spleen of LD50-infected CBA/J mice were higher than those of either 15 cysts-infected CBA/J mice or LD50-infected C57BL/6 mice. These observations demonstrate that the immune response between LD50-infected C57BL/6 and CBA/J mice was more prominent when compared to C57BL/6 or CBA/J mice receiving the same parasite inoculum. These observations would suggest that caution must be excersized in the planning and interpretation of data when the size of the parasite inoculum has not been adjusted for mouse strain.     

Regulation of NKT cells by Ly49: analysis of primary NKT cells and generation of NKT cell line

TCRalphabeta(+)NK1.1(+) (NKT) cells are known to express various NK cell-associated molecules including the Ly49 family of receptors for MHC class I, but its functional significance has been unclear. Here, we examined the expression of Ly49A, C/I and G2 on various NKT cell populations from normal and MHC class I-deficient C57BL/6 mice as well as their responsiveness to alpha-galactosylceramide (alpha-GalCer), a potent stimulator of CD1d-restricted NKT cells. The frequency and the level of Ly49 expression varied among NKT cells from different tissues, and were regulated by the expression of MHC class I and CD1d in the host. Stimulation of various NKT cells with alpha-GalCer suggested that Ly49 expression inversely correlates with the responsiveness of NKT cells to alpha-GalCer. Moreover, alpha-GalCer presented by normal dendritic cells stimulated purified Ly49(-), but not Ly49(+), splenic NKT cells, whereas MHC class I-deficient dendritic cells presented alpha-GalCer to both Ly49(+) and Ly49(-) NKT cells equally well. Therefore, MHC class I on APCs seems to inhibit activation of NKT cells expressing Ly49. To further characterize CD1d-restricted NKT cells, we generated an alpha-GalCer-responsive NKT cell line from thymocytes. The line could only be generated from Ly49(-)NK1.1(+)CD4(+) thymocytes but not from other NKT cell subsets, and it lost expression of NK1.1 and CD4 during culture. Together, these results indicate the functional significance of Ly49 expression on NKT cells.     

Type II NKT cells stimulate diet-induced obesity by mediating adipose tissue inflammation, steatohepatitis and insulin resistance

The progression of obesity is accompanied by a chronic inflammatory process that involves both innate and acquired immunity. Natural killer T (NKT) cells recognize lipid antigens and are also distributed in adipose tissue. To examine the involvement of NKT cells in the development of obesity, C57BL/6 mice (wild type; WT), and two NKT-cell-deficient strains, Jα18(-/-) mice that lack the type I subset and CD1d(-/-) mice that lack both the type I and II subsets, were fed a high fat diet (HFD). CD1d(-/-) mice gained the least body weight with the least weight in perigonadal and brown adipose tissue as well as in the liver, compared to WT or Jα18(-/-) mice fed an HFD. Histologically, CD1d(-/-) mice had significantly smaller adipocytes and developed significantly milder hepatosteatosis than WT or Jα18(-/-) mice. The number of NK1.1(+)TCRβ(+) cells in adipose tissue increased when WT mice were fed an HFD and were mostly invariant Vα14Jα18-negative. CD11b(+) macrophages (Mφ) were another major subset of cells in adipose tissue infiltrates, and they were divided into F4/80(high) and F4/80(low) cells. The F4/80(low)-Mφ subset in adipose tissue was increased in CD1d(-/-) mice, and this population likely played an anti-inflammatory role. Glucose intolerance and insulin resistance in CD1d(-/-) mice were not aggravated as in WT or Jα18(-/-) mice fed an HFD, likely due to a lower grade of inflammation and adiposity. Collectively, our findings provide evidence that type II NKT cells initiate inflammation in the liver and adipose tissue and exacerbate the course of obesity that leads to insulin resistance.     

Genetic background influences natural killer cell activation during bacterial peritonitis in mice, and is interleukin 12 and interleukin 18 independent

Some mouse strains produce strong pro-inflammatory, T-helper (Th)1 responses (e.g. C57BL/6), or strong anti-inflammatory, Th2 responses (e.g. BALB/c). The exact mechanisms for development of distinct immune responses to infection are not completely understood, although cytokines such as interleukin (IL)-12, IL-18 and IL-4 are known to play roles. Natural killer T (NKT)/natural killer (NK) cells are important regulators of immune responses in infection and non-infection models, and NKT/NK activation is also regulated by IL-12 and IL-18 in many models. We investigated the role of IL-12/IL-18 in NKT/NK activation in murine bacterial peritonitis, as well as differential NKT and NK cell activation in C57BL/6 and BALB/c mice. No differences in NKT or NK cell activation or intracellular interferon (IFN)-gamma were determined between mice given control, anti-IL-12 or anti-IL-18 antibodies or in NKT/NK cell activation in STAT4-/- mice (deficient in IL-12 signaling) or wild type controls. However, there were significant differences in the activation of NKT and NK cells between C57BL/6 mice and BALB/c mice, with NKT/NK cytokine production following Th1 or Th2 lines dependent on strain. This suggests a role for NKT and NK cell activation in the development of Th1 and Th2 responses during bacterial infection independently of IL-12 or IL-18.     

The I-Abm12 mutation, which confers resistance to experimental myasthenia gravis, drastically affects the epitope repertoire of murine CD4+ cells sensitized to nicotinic acetylcholine receptor.

Susceptibility to experimental autoimmune myasthenia gravis (EAMG), which is induced in mice by injection of purified Torpedo nicotinic acetylcholine receptor (TAChR), is influenced by the I-A locus products, which restrict presentation of AChR Th epitopes. The bm12 mutation of the I-Ab molecule in the C57BL/6 strain, which is highly susceptible to EAMG, yields the EAMG resistant mutant B6.C-H-2bm12 (bm12). We investigated here the consequences of the bm 12 mutation on the CD4+ response to the TAChR alpha subunit. Upon immunization with TAChR, CD4+ cells became sensitized to TAChR and anti-AChR antibodies were produced in both bm12 and C57BL/6 strains. Overlapping synthetic peptides, corresponding to the complete sequence of TAChR alpha subunit, were used to identify Th epitopes. CD4+ cells from C57BL/6 mice recognized peptides T alpha 150-169, T alpha 181-200, and T alpha 360-378. CD4+ cells from bm12 mice did not respond to any synthetic sequence. Upon injection of the three C57BL/6 Th epitope peptides, either individually or as a pool, CD4+ cells from C57BL/6 mice recognized each peptide and TAChR. Therefore they recognized epitopes similar or identical to those originated from TAChR processing. CD4+ cells from bm12 mice injected with the same peptides responded to T alpha 360-378 strongly, to a lesser extent to T alpha 181-200, never to peptide T alpha 150-169. Only CD4+ cells sensitized against the T epitope peptide T alpha 181-200 responded to TAChR. We tested if lack of response to T alpha 150-169, and the low response to T alpha 181-200, was due to inability of the I-Abm12 molecule to present the T epitope peptides. bm12 and C57BL/6 APC were used to present the T epitope peptides to specifically sensitized CD4+ cells from C57BL/6 mice. All T epitope peptides were presented by bm12 APC, although T alpha 150-169 was presented less efficiently than by C57BL/6 APC. Resistance to EAMG induced by the bm12 mutation may be due to the change in the epitope repertoire of AChR-specific Th cells, and lack of recognition of otherwise immunodominant Th epitopes. For at least one epitope this might be due to absence of potentially reactive, specific CD4+ clones.     

Oral tolerance to nickel requires CD4+ invariant NKT cells for the infectious spread of tolerance and the induction of specific regulatory T cells

Previously, oral administration of nickel to C57BL/6 wild-type (WT) mice was shown to render both their splenic T cells and APCs (i.e., T cell-depleted spleen cells) capable of transferring nickel tolerance to naive syngeneic recipients. Moreover, sequential adoptive transfer experiments revealed that on transfer of tolerogenic APCs and immunization, the naive T cells of the recipients differentiated into regulatory T (Treg) cells. Here, we demonstrate that after oral nickel treatment Jalpha18(-/-) mice, which lack invariant NKT (iNKT) cells, were not tolerized and failed to generate Treg cells. However, transfer of APCs from those Jalpha18(-/-) mice did tolerize WT recipients. Hence, during oral nickel administration, tolerogenic APCs are generated that require iNKT cell help for the induction of Treg cells. To obtain this help, the tolerogenic APCs must address the iNKT cells in a CD1-restricted manner. When Jalpha18(-/-) mice were used as recipients of cells from orally tolerized WT donors, the WT Treg cells transferred the tolerance, whereas WT APCs failed to do so, although they proved tolerogenic on transfer to WT recipients. However, Jalpha18(-/-) recipients did become susceptible to the tolerogenicity of transferred WT APCs when they were reconstituted with IL-4- and IL-10-producing CD4(+) iNKT cells. We conclude that CD4(+) iNKT cells are required for the induction of oral nickel tolerance and, in particular, for the infectious spread of tolerance from APCs to T cells. Once induced, these Treg cells, however, can act independently of iNKT cells.     

Exacerbated susceptibility to infection-stimulated immunopathology in CD1d-deficient mice

Mice lacking functional CD1d genes were used to study mechanisms of resistance to the protozoan parasite Toxoplasma gondii. Wild-type (WT) BALB/c mice, CD1d-deficient BALB/c mice, and WT C57BL/6 mice all survived an acute oral infection with a low dose of mildly virulent strain ME49 T. gondii cysts. In contrast, most CD1d-deficient C57BL/6 mice died within 2 wk of infection. Despite having parasite burdens that were only slightly higher than WT mice, CD1d-deficient C57BL/6 mice displayed greater weight loss and intestinal pathology. In C57BL/6 mice, CD4(+) cells can cause intestinal pathology during T. gondii infection. Compared with WT mice, infected CD1d-deficient C57BL/6 mice had higher frequencies and numbers of activated (CD44(high)) CD4(+) cells in mesenteric lymph nodes. Depletion of CD4(+) cells from CD1d-deficient mice reduced weight loss and prolonged survival, demonstrating a functional role for CD4(+) cells in their increased susceptibility to T. gondii infection. CD1d-deficient mice are deficient in Valpha14(+) T cells, a major population of NKT cells. Involvement of these cells in resistance to T. gondii was investigated using gene-targeted Jalpha18-deficient C57BL/6 mice, which are deficient in Valpha14(+) T cells. These mice did not succumb to acute infection, but experienced greater weight loss and more deaths than B6 mice during chronic infection, indicating that Valpha14(+) cells contribute to resistance to T. gondii. The data identify CD4(+) cells as a significant component of the marked susceptibility to T. gondii infection observed in CD1d-deficient C57BL/6 mice, and establish T. gondii as a valuable tool for deciphering CD1d-dependent protective mechanisms.     

Influence of the maternal effect on allogenic inhibition of hematopoietic stem cells]

Bone marrow cells (0,5-10(6)) of female mice of CBA or C57BL strains were injected intravenously to lethally irradiated CBA, C57BL/6, (femaleCBA X maleC57BL/6)F1 and (femaleC57BL/6 X maleCBA)F1 mice. Spleen of recipients as assayed for colony count on the 9th day after bone marrow transplantation by the method of Till and McCullouch. Stem cells of CBA mice demonstrated failure of allogenic inhibition in (CBA X C57BL/6)F1 hybrid mice and formed the same number of colonies as in the spleen of syngenic recipients. The level of allogenic inhibition of CBA stem cells transplanted to (C57BL/6 X X CBA)F1 hybrid mice was 50%. Bone marrow cells of C57BL/6 mice formed colonies in spleen of (CBA X C57BL/6)F1 mice at least in 20 times less than in syngenic combination. In the transplantation of bone marrow from C57BL/6 mice to (C57BL/6 X CBA)F1 hybrid mice the allogenic inhibition was less pronounced (77-85%) as compared with the transfer of cells to (CBA X C57BL/6)F1 hybrid mice (95%). The sex of a recipient did not influence the number of formed colonies. The different level of allogenic inhibition of parental stem cells can not be explained by the effect of linkage with sex as the female of reciprocal hybrid mice have identical structure of sex chromosomes (X(CBA)XC57BL/6). The data obtained indicate that the maternal effect affects allogenic inhibition of stem cells in parent--F1 system. It is possible that the maternal influence may be determined by cytoplasmic factors of inheritance which affect the expressivity of recessive genes Hh, controlling the inheritance of specific haematopoietic cell antigens.     

The formation of titan cells in Cryptococcus neoformans depends on the mouse strain and correlates with induction of Th2‐type responses

Cryptococcus neoformans is a pathogenic yeast that can form titan cells in the lungs, which are fungal cells of abnormal enlarged size. Little is known about the factors that trigger titan cells. In particular, it is not known how the host environment influences this transition. In this work, we describe the formation of titan cells in two mouse strains, CD1 and C57BL/6J. We found that the proportion of C. neoformans titan cells was significantly higher in C57BL/6J mice than in CD1. This higher proportion of titan cells was associated with a higher dissemination of the yeasts to the brain. Histology sections demonstrated eosinophilia in infected animals, although it was significantly lower in the CD1 mice which presented infiltration of lymphocytes. Both mouse strains presented infiltration of granulocytes, but the amount of eosinophils was higher in C57BL/6J. CD1 mice showed a significant accumulation of IFN‐γ, TNF‐α and IL17, while C57BL/BL mice had an increase in the anti‐inflammatory cytokine IL‐4. IgM antibodies to the polysaccharide capsule and total IgE were more abundant in the sera from C57BL/6J, confirming that these animals present a Th2‐type response. We conclude that titan cell formation in C. neoformans depends, not only on microbe factors, but also on the host environment.     

Role of CD1d in coxsackievirus B3-induced myocarditis

The myocarditic (H3) variant of Coxsackievirus B3 (CVB3) causes severe myocarditis in BALB/c mice and BALB/c mice lacking the invariant J alpha 281 gene, but minimal disease in BALB/c CD1d(-/-) animals. This indicates that CD1d expression is important in this disease but does not involve the invariant NKT cell often associated with CD1d-restricted immunity. The H3 variant of the virus increases CD1d expression in vitro in neonatal cardiac myocytes whereas a nonmyocarditic (H310A1) variant does not. V gamma 4(+) T cells show increased activation in both H3-infected BALB/c and J alpha 281(-/-) mice compared with CD1d(-/-) animals. The activated BALB/c V gamma 4(+) T cells from H3-infected mice kill H3-infected BALB/c myocytes and cytotoxicity is blocked with anti-CD1d but not with anti-MHC class I (K(d)/D(d)) or class II (IA/IE) mAbs. In contrast, H3 virus-infected CD1d(-/-) myocytes are not killed. These studies demonstrate that CD1d expression is essential for pathogenicity of CVB3-induced myocarditis, that CD1d expression is increased early after infection in vivo in CD1d(+) mice infected with the myocarditic but not with the nonmyocarditic CVB3 variant, and that V gamma 4(+) T cells, which are known to promote myocarditis susceptibility, appear to recognize CD1d expressed by CVB3-infected myocytes.     

Roles of tumor necrosis factor alpha (TNF-alpha) and the p55 TNF receptor in CD1d induction and coxsackievirus B3-induced myocarditis

Giving C57BL/6 mice 10(4) PFU of coxsackievirus B3 (H3 variant) fails to induce myocarditis, but increasing the initial virus inoculum to 10(5) or 10(6) PFU causes significant cardiac disease. Virus titers in the heart were equivalent at days 3 and 7 in mice given all three virus doses, but day 3 titers in the pancreases of mice inoculated with 10(4) PFU were reduced. Tumor necrosis factor alpha (TNF-alpha) concentrations in the heart were increased in all infected mice, but cytokine levels were highest in mice given the larger virus inocula. TNF-alpha(-/-) and p55 TNF receptor-negative (TNFR(-/-)) mice developed minimal myocarditis compared to B6;129 or C57BL/6 control mice. p75 TNFR(-/-) mice were as disease susceptible as C57BL/6 animals. No significant differences in virus titers in heart or pancreas were observed between the groups, but C57BL/6 and p75 TNFR(-/-) animals showed 10-fold more inflammatory cells in the heart than p55 TNFR(-/-) mice, and the cell population was comprised of high concentrations of CD4(+) gamma interferon-positive and Vgamma4(+) cells. Cardiac endothelial cells isolated from C57BL/6 and p75 TNFR(-/-) mice upregulate CD1d, the molecule recognized by Vgamma4(+) cells, but infection of TNF(-/-) or p55 TNFR(-/-) endothelial cells failed to upregulate CD1d. Infection of C57BL/6 endothelial cells with a nonmyocarditic coxsackievirus B3 variant, H310A1, which is a poor inducer of TNF-alpha, failed to elicit CD1d expression, but TNF-alpha treatment of H310A1-infected endothelial cells increased CD1d levels to those seen in H3-infected cells. TNF-alpha treatment of uninfected endothelial cells had only a modest effect on CD1d expression, suggesting that optimal CD1d upregulation requires both infection and TNF-alpha signaling.     

Tumour necrosis factor receptor deficiency alters anxiety-like behavioural and neuroendocrine stress responses of mice

Tumour necrosis factor (TNF)-alpha is known to be involved in anxiety and the regulation of the hypothalamic-pituitary-adrenal axis. To examine the role of its receptors in neuroendocrine immunomodulation, we studied behaviour, corticosterone production and T-cell activation in mice with a C57BL/6J background and deficient for one or both TNF receptors (TNFR1-/-, TNFR2-/-, and TNFR1+2-/-) compared to wildtype C57BL/6J mice with and without psychological stress. Stress was induced by social disruption (SDR), and anxiety-like behaviour was examined using the elevated plus maze (EPM). Anxiety of unstressed TNFR1+2-/- mice was increased compared to C57BL/6J mice as shown by reduced ratios of entries into open arms relatively to total entries. SDR-stressed TNFR1+2-/- mice showed reduced ratios of entries into open arms relatively to total entries, reduced ratios of distances walked in open relatively to distances walked in both arms and reduced time in open arms compared to C57BL/6J mice. Locomotor activity of unstressed and SDR-stressed TNFR1-/- and TNFR2-/- mice was reduced. Serum corticosterone concentrations of control mice do not differ between mouse strains. However, TNFR1+2-/- mice had significantly higher corticosterone concentrations than C57BL/6J mice after SDR. EPM testing significantly increased corticosterone concentrations in all strains. Mitogen-induced activation-marker expression was reduced in TNFR1-/- T-helper cells under control and stress conditions, while activation marker expression of TNFR2-/- and TNFR1+2-/- cells was only slightly affected by stress compared to C57BL/6J T cells. Our study suggests that both TNF receptors contribute to anxiety-like behaviour and corticosterone responses, whereas TNFR1 has a larger impact on T-cell activation.