Inbred Strain Characteristics Impact the NKT Cell Repertoire.

NKT cells are primed lymphocytes that rapidly secrete cytokines and can directly kill cancerous cells. Given the critical role NKT cells play in cancer immune surveillance, we sought to investigate the effect of mutations in Brca1, specifically a conditional deletion of exon 11, on type I invariant NKT cell development. We observed a significant reduction in invariant NKT cells in both primary lymphoid and peripheral organs in Brca1 mutant mice compared with wild-type C57BL/6. However, the original Brca1 mutant strain was on a mixed background containing FVB/N. We determined that strain differences, rather than mutations in Brca1, led to the observed loss in NKT cells. Importantly, we found that whereas FVB/N mice lack Vß8, there was a striking increase in the total number of thymic type I CD1d-α-galactosylceramide tetramer positive NKT cells and skewing of the NKT cell population to NKT2 compared with C57BL/6 mice. Collectively, our data demonstrate the profound effect genetics can have on NKT cell subset differentiation.

Efficient preparation of human and mouse CD1d proteins using silkworm baculovirus expression system.

CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and ß2-microglobulin (ß2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with ß2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 µg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.

Sterile activation of invariant natural killer T cells by ER-stressed antigen-presenting cells.

Invariant NKT (iNKT) cells have the unique ability to shape immunity during antitumor immune responses and other forms of sterile and nonsterile inflammation. Recent studies have highlighted a variety of classes of endogenous and pathogen-derived lipid antigens that can trigger iNKT cell activation under sterile and nonsterile conditions. However, the context and mechanisms that drive the presentation of self-lipid antigens in sterile inflammation remain unclear. Here we report that endoplasmic reticulum (ER)-stressed myeloid cells, via signaling events modulated by the protein kinase RNA-like ER kinase (PERK) pathway, increase CD1d-mediated presentation of immunogenic endogenous lipid species, which results in enhanced iNKT cell activation both in vitro and in vivo. In addition, we demonstrate that actin cytoskeletal reorganization during ER stress results in an altered distribution of CD1d on the cell surface, which contributes to enhanced iNKT cell activation. These results define a previously unidentified mechanism that controls iNKT cell activation during sterile inflammation.

CD1d-mediated lipid presentation by CD11c+ cells regulates intestinal homeostasis.

Intestinal homeostasis relies on a continuous dialogue between the commensal bacteria and the immune system. Natural killer T (NKT) cells, which recognize CD1d-restricted microbial lipids and self-lipids, contribute to the regulation of mucosal immunity, yet the mechanisms underlying their functions remain poorly understood. Here, we demonstrate that NKT cells respond to intestinal lipids and CD11c+ cells (including dendritic cells (DCs) and macrophages) are essential to mediate lipid presentation within the gut ultimately controlling intestinal NKT cell homeostasis and activation. Conversely, CD1d and NKT cells participate in the control of the intestinal bacteria composition and compartmentalization, in the regulation of the IgA repertoire and in the induction of regulatory T cells within the gut. These changes in intestinal homeostasis require CD1d expression on DC/macrophage populations as mice with conditional deletion of CD1d on CD11c+ cells exhibit dysbiosis and altered immune homeostasis. These results unveil the importance of CD11c+ cells in controlling lipid-dependent immunity in the intestinal compartment and reveal an NKT cell-DC crosstalk as a key mechanism for the regulation of gut homeostasis.

Administration of α-Galactosylceramide Improves Adenine-Induced Renal Injury.

Natural killer T (NKT) cells are a subset of lymphocytes that reacts to glycolipids presented by CD1d. Invariant NKT cells (iNKT) correspond to >90% of the total population of NKTs and reacts to α-galactosylceramide (αGalCer). αGalCer promotes a complex mixture of Th1 and Th2 cytokines, as interferon (IFN)-γ and interleukin (IL)-4. NKT cells and IFN-γ are known to participate in some models of renal diseases, but further studies are still necessary to elucidate their mechanisms. The aim of our study was to analyze the participation of iNKT cells in an experimental model of tubule-interstitial nephritis. We used 8-wk-old C57BL/6j, Jα18KO and IFN-γKO mice. They were fed a 0.25% adenine diet for 10 d. Both adenine-fed wild-type (WT) and Jα18KO mice exhibited renal dysfunction, but adenine-fed Jα18KO mice presented higher expression of kidney injury molecule-1 (KIM-1), tumor necrosis factor (TNF)-α and type I collagen. To analyze the role of activated iNKT cells in our model, we administered αGalCer in WT mice during adenine ingestion. After αGalCer injection, we observed a significant reduction in serum creatinine, proinflammatory cytokines and renal fibrosis. However, this improvement in renal function was not observed in IFN-γKO mice after αGalCer treatment and adenine feeding, illustrating that this cytokine plays a role in our model. Our findings may suggest that IFN-γ production is one of the factors contributing to improved renal function after αGalCer administration.

Increased Expression of Two Alternative Spliced Variants of CD1d Molecule in Human Gastric Cancer.

BACKGROUND: CD1d presents glycolipid antigens to invariant natural killer T (iNKT) cells. The role of CD1d in the development of peptic ulcer and gastric cancer has not been revealed, yet. OBJECTIVE: To clarify the expression of alternatively spliced variants of CD1d in peptic ulcer and gastric cancer. METHODS: Patients with dyspepsia were selected and divided into three groups of non-ulcer dyspepsia (NUD), peptic ulcer disease (PUD), and gastric cancer (GC), according to their endoscopic and histopathological examinations. H. pylori infection was diagnosed by rapid urease test and histopathology. The expression levels of V2, V4, and V5 spliced variants of CD1d molecule were determined by quantitative Reverse Transcriptase PCR. RESULTS: Relative gene expression levels of V4 were higher in GC patients (n=37) than those in NUD (n=49) and PUD (n=51) groups (p<0.05 and p<0.01, respectively). Moreover, GC patients showed higher expression levels of V5 compared to NUD and PUD groups (p<0.001 and p<0.001, respectively). Positive correlation coefficients were attained between V4 and V5 expression in patients with PUD (r=0.734, p<0.0001) and GC (r=0.423, p<0.01), but not in patients with NUD. Among NUD patients, the expression levels of V4, but not V5, were higher in H. pylori-positive patients than in H. pylori-negative ones (p<0.01). CONCLUSION: Collectively, both membrane-bound (V4) and soluble (V5) isoforms of CD1d were over-expressed in gastric tumor tissues, suggesting that they are involved in anti-tumor immune responses.

TLR9-induced miR-155 and Ets-1 decrease expression of CD1d on B cells in SLE.

B cells present lipid antigens to CD1d-restricted invariant natural killer T (iNKT) cells to maintain autoimmune tolerance, and this process is disrupted in systemic lupus erythematosus (SLE). Inflammation may inhibit CD1d expression to exacerbate the pathology of lupus. However, how inflammation regulates CD1d expression on B cells is unclear in SLE. In the present study, we showed that the surface expression of CD1d on B cells from SLE mice was decreased and that stimulation of inflammatory responses through TLR9 decreased the membrane and total CD1d levels of CD1d on B cells. Moreover, inflammation-related microRNA-155 (miR-155) negatively correlated with the expression of CD1d in B cells. miR-155 directly targeted the 3'-untranslated region (3'-UTR) of CD1d upon TLR9 activation in both humans and mice. The inhibitory effects of miR-155 on CD1d expression in B cells impaired their antigen-presenting capacity to iNKT cells. In addition, Ets-1, a susceptibility gene of SLE, also directly regulated the expression of the CD1d gene at the transcriptional level. These findings provide new insight into the mechanism underlying decreased CD1d expression on B cells in SLE, suggesting that inhibition of inflammation may increase CD1d expression in B cells to ameliorate SLE via modulating iNKT cells.

Tumor-induced CD11b(+) Gr-1(+) myeloid-derived suppressor cells exacerbate immune-mediated hepatitis in mice in a CD40-dependent manner.

Immunosuppressive CD11b(+) Gr-1(+) myeloid-derived suppressor cells (MDSCs) accumulate in the livers of tumor-bearing (TB) mice. We studied hepatic MDSCs in two murine models of immune-mediated hepatitis. Unexpectedly, treatment of TB mice with Concanavalin A (Con A) or α-galactosylceramide resulted in increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels in comparison to tumor-free mice. Adoptive transfer of hepatic MDSCs into naïve mice exacerbated Con A induced liver damage. Hepatic CD11b(+) Gr-1(+) cells revealed a polarized proinflammatory gene signature after Con A treatment. An IFN-γ-dependent upregulation of CD40 on hepatic CD11b(+) Gr-1(+) cells along with an upregulation of CD80, CD86, and CD1d after Con A treatment was observed. Con A treatment resulted in a loss of suppressor function by tumor-induced CD11b(+) Gr-1(+) MDSCs as well as enhanced reactive oxygen species (ROS)-mediated hepatotoxicity. CD40 knockdown in hepatic MDSCs led to increased arginase activity upon Con A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40(-/-) tumor-induced myeloid cells resulted in exacerbation of hepatitis and increased ROS production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor-induced hepatic MDSCs act as proinflammatory immune effector cells capable of killing hepatocytes in a CD40-dependent manner.

Splenic B cells from Hymenolepis diminuta-infected mice ameliorate colitis independent of T cells and via cooperation with macrophages.

Helminth parasites provoke multicellular immune responses in their hosts that can suppress concomitant disease. The gut lumen-dwelling tapeworm Hymenolepis diminuta, unlike other parasites assessed as helminth therapy, causes no host tissue damage while potently suppressing murine colitis. With the goal of harnessing the immunomodulatory capacity of infection with H. diminuta, we assessed the putative generation of anti-colitic regulatory B cells following H. diminuta infection. Splenic CD19(+) B cells isolated from mice infected 7 [HdBc(7(d))] and 14 d (but not 3 d) previously with H. diminuta and transferred to naive mice significantly reduced the severity of dinitrobenzene sulfonic acid (DNBS)-, oxazolone-, and dextran-sodium sulfate-induced colitis. Mechanistic studies with the DNBS model, revealed the anti-colitic HdBc(7(d)) was within the follicular B cell population and its phenotype was not dependent on IL-4 or IL-10. The HdBc(7(d)) were not characterized by increased expression of CD1d, CD5, CD23, or IL-10 production, but did spontaneously, and upon LPS plus anti-CD40 stimulation, produce more TGF-ß than CD19(+) B cells from controls. DNBS-induced colitis in RAG1(-/-) mice was inhibited by administration of HdBc(7(d)), indicating a lack of a requirement for T and B cells in the recipient; however, depletion of macrophages in recipient mice abrogated the anti-colitic effect of HdBc(7(d)). Thus, in response to H. diminuta, a putatively unique splenic CD19(+) B cell with a functional immunoregulatory program is generated that promotes the suppression of colitis dominated by TH1, TH2, or TH1-plus-TH2 events, and may do so via the synthesis of TGF-ß and the generation of, or cooperation with, a regulatory macrophage.

Recombinant murine calreticulin fragment 39-272 expands CD1d(hi)CD5+ IL-10-secreting B cells that modulate experimental autoimmune encephalomyelitis in C57BL/6 mice.

Calreticulin (CRT) is a Ca²âº binding molecular chaperone in the endoplasmic reticulum, but can also accumulate in soluble form in serum and/or synovial fluid of patients with rheumatic disorders. We have recently shown that a prokaryotically expressed recombinant CRT fragment 39-272 (rCRT/39-272) exhibited potent stimulatory activities against macrophages and B cells. However, intraperitoneal (i.p.) administration of rCRT/39-272 effectively suppressed delayed-type hypersensitivity in mice, attributable to production of anti-CRT Abs favoring Th2 cell differentiation. In this study, we further demonstrate that i.p. injection of rCRT/39-272 reduced disease severity in mouse model of experimental autoimmune encephalomyelitis (EAE), by inhibiting autoantigen-specific Th1 differentiation in vivo. Interestingly, the EAE-modulating effect of rCRT/39-272 was attributed to activation/expansion of CD1d(hi)CD5⁺ IL-10-secreting B (B10) cells rather than induction of CRT-specific antibodies in mice. In vitro, rCRT/39-272 could activate and expand murine splenic B10 cells through a Toll like receptor 4 (TLR4)-dependent pathway. The rCRT-activated B10 cells were able to not only enhance Th2 cell differentiation in vitro but also reduce EAE scores of recipient animals in passive transfer experiments. These results revealed soluble CRT, likely released by tissue cells under stress conditions, as a potentially important multi-faced player in immunoregulation and immunopathological responses.

B cell: T cell interactions occur within hepatic granulomas during experimental visceral leishmaniasis.

Hepatic resistance to Leishmania donovani infection in mice is associated with the development of granulomas, in which a variety of lymphoid and non-lymphoid populations accumulate. Although previous studies have identified B cells in hepatic granulomas and functional studies in B cell-deficient mice have suggested a role for B cells in the control of experimental visceral leishmaniasis, little is known about the behaviour of B cells in the granuloma microenvironment. Here, we first compared the hepatic B cell population in infected mice, where ≈60% of B cells are located within granulomas, with that of naïve mice. In infected mice, there was a small increase in mIgM(lo)mIgD(+) mature B2 cells, but no enrichment of B cells with regulatory phenotype or function compared to the naïve hepatic B cell population, as assessed by CD1d and CD5 expression and by IL-10 production. Using 2-photon microscopy to quantify the entire intra-granuloma B cell population, in conjunction with the adoptive transfer of polyclonal and HEL-specific BCR-transgenic B cells isolated from L. donovani-infected mice, we demonstrated that B cells accumulate in granulomas over time in an antigen-independent manner. Intra-vital dynamic imaging was used to demonstrate that within the polyclonal B cell population obtained from L. donovani-infected mice, the frequency of B cells that made multiple long contacts with endogenous T cells was greater than that observed using HEL-specific B cells obtained from the same inflammatory environment. These data indicate, therefore, that a subset of this polyclonal B cell population is capable of making cognate interactions with T cells within this unique environment, and provide the first insights into the dynamics of B cells within an inflammatory site.

Enhancing immunostimulatory function of human embryonic stem cell-derived dendritic cells by CD1d overexpression.

Human embryonic stem cell-derived dendritic cells (hESC-DCs) may potentially provide a platform to generate "off-the-shelf" therapeutic cancer vaccines. To apply hESC-DCs for cancer immunotherapy in a semiallogeneic setting, it is crucial for these cells to "jump-start" adaptive antitumor immunity before their elimination by host alloreaction. In this study, we investigated whether CD1d upregulation in hESC-DCs may exploit invariant NKT (iNKT) cell adjuvant activity and boost antitumor immunity. Using a baculoviral vector carrying the CD1d gene, we produced CD1d-overexpressing hESC-DCs and demonstrated that the upregulated CD1d was functional in presenting α-galactosylceramide for iNKT cell expansion. Pulsed with melanoma Ag recognized by T cell 1 peptide, the CD1d-overexpressing hESC-DCs displayed enhanced capability to prime CD8(+) T cells without relying on α-galactosylceramide loading. Blocking the CD1d with Ab reduced the immunogenicity, suggesting the importance of hESC-DC and iNKT cell interaction in this context. The CD1d-overexpressing hESC-DCs also induced a proinflammatory cytokine profile that may favor the T cell priming. Moreover, a similar immunostimulatory effect was observed when the CD1d upregulation strategy was applied in human monocyte-derived dendritic cells. Therefore, our study suggests that the upregulation of CD1d in hESC-DCs provides a novel strategy to enhance their immunogenicity. This approach holds potential for advancing the application of hESC-DCs into human cancer immunotherapy.

Distinct APCs explain the cytokine bias of α-galactosylceramide variants in vivo.

α-Galactosylceramide represents a new class of vaccine adjuvants and immunomodulators that stimulate NKT cells to secrete Th1 and Th2 cytokines. Synthetic variants with short or unsaturated acyl chains exhibit a striking Th2 bias in vivo but no evidence of defect in TCR signaling or stimulation of NKT cells in vitro. Using cd1d1(fl/fl) mice, we demonstrated that distinct APC types explained the cytokine bias in vivo. Whereas NKT stimulation by α-Galactosylceramide required CD1d expression by dendritic cells (DCs), presentation of the Th2 variants was promiscuous and unaffected by DC-specific ablation of CD1d. This DC-independent stimulation failed to activate the feedback loop between DC IL-12 and NK cell IFN-γ, explaining the Th2 bias. Conversely, forced presentation of the Th2 variants by DC induced high IL-12. Thus, lipid structural variations that do not alter TCR recognition can activate distinct Th1 or Th2 cellular networks by changing APC targeting in vivo.

Mutation of a positively charged cytoplasmic motif within CD1d results in multiple defects in antigen presentation to NKT cells.

CD1d is an MHC class I-like molecule that presents glycolipid Ags to types I and II NKT cells. The YxxI motif in the cytoplasmic tail of CD1d contributes to its intracellular localization to the endolysosomal compartment and is important for Ag presentation to type I NKT cells. In this study, we identified the (327-329)RRR motif in CD1d and showed that it is critical for the control of CD1d intracellular trafficking and Ag presentation. The replacement of the arginines in this motif with alanines resulted in the extensive accumulation of CD1d in lysosomes but did not affect the cell surface expression. The defect in its cellular localization was accompanied by defects in Ag presentation to both type I and type II NKT cells. These results demonstrated that the (327-329)RRR motif of CD1d is required for proper cellular distribution of CD1d and optimal Ag presentation to both type I and type II NKT cells.

Analysis of invariant natural killer T cells in human paracoccidioidomycosis.

Invariant natural killer T (iNKT) cells are capable of recognizing lipid antigens and secreting Th1/Th2 cytokines. Deficiency in iNKT cell number or function has been partially implicated in susceptibility to some infectious diseases, such as tuberculosis. We evaluated iNKT cells in paracoccidioidomycosis, another chronic granulomatous disease endemic in Latin America. iNKT cells were detected using PBS57-loaded tetramer staining and flow cytometry. Circulating iNKT cell numbers were similar among healthy individuals who had previously been cured of paracoccidioidomycosis (susceptible individuals, n = 7) and healthy Paracoccidioides brasiliensis-infected (n = 5) and non-infected individuals (n = 5). iNKT from all three groups expanded similarly upon α-GalCer and a synthetic analog (OCH) stimulation. IFN-γ was the dominant cytokine produced both by ex vivo and by expanded iNKT cells, followed by IL-4 and IL-10, in the three groups. No deficit in the monocyte expression of CD1d was detected. In conclusion, individuals who had developed paracoccidioidomycosis in the past have no impairment in iNKT number, expansion capacity, and cytokine secretion.

Herpes simplex virus 1 glycoprotein B and US3 collaborate to inhibit CD1d antigen presentation and NKT cell function.

Herpes simplex viruses (HSVs) are prevalent human pathogens that establish latency in human neuronal cells and efficiently evade the immune system. It has been a major medical challenge to eradicate them and, despite intensive efforts, an effective vaccine is not available. We previously showed that upon infection of antigen-presenting cells, HSV type 1 (HSV-1) rapidly and efficiently downregulates the major histocompatibility complex class I-like antigen-presenting molecule, CD1d, and potently inhibits its recognition by CD1d-restricted natural killer T (NKT) cells. It suppresses CD1d expression primarily by inhibiting its recycling to the cell surface after endocytosis. We identify here the viral glycoprotein B (gB) as the predominant CD1d-interacting protein. gB initiates the interaction with CD1d in the endoplasmic reticulum and stably associates with it throughout CD1d trafficking. However, an additional HSV-1 component, the serine-threonine kinase US3, is required for optimal CD1d downregulation. US3 expression in infected cells leads to gB enrichment in the trans-Golgi network (TGN) and enhances the relocalization of both gB and CD1d to this compartment, suggesting that following internalization CD1d is translocated from the endocytic pathway to the TGN by its association with gB. Importantly, both US3 and gB are required for efficient inhibition of CD1d antigen presentation and NKT cell activation. In summary, our results suggest that HSV-1 uses gB and US3 to rapidly inhibit NKT cell function in the initial antiviral response.

Antigen-specific cytotoxicity by invariant NKT cells in vivo is CD95/CD178-dependent and is correlated with antigenic potency.

Invariant NKT (iNKT) cells are a unique subset of T lymphocytes that rapidly carry out effector functions following activation with glycolipid Ags, such as the model Ag alpha-galactosylceramide. Numerous studies have investigated the mechanisms leading to Th1 and Th2 cytokine production by iNKT cells, as well as the effects of the copious amounts of cytokines these cells produce. Less is known, however, about the mechanisms of iNKT cell cytotoxicity. In this study, we investigated the effect of Ag availability and strength, as well as the molecules involved in iNKT cytotoxicity. We demonstrate that the iNKT cell cytotoxicity in vivo correlates directly with the amount of CD1d expressed by the targets as well as the TCR affinity for the target glycolipid Ag. iNKT cells from spleen, liver, and thymus were comparable in their cytotoxicity in vitro. Surprisingly, we show that the Ag-specific cytotoxicity of iNKT cells in vivo depended almost exclusively on the interaction of CD95 (Fas) with CD178 (FasL), and that this mechanism can be efficiently used for tumor protection. Therefore, unlike NK cells, which rely mostly on perforin/granzyme-mediated mechanisms, the Ag-specific cytotoxicity of iNKT cells in vivo is largely restricted to the CD95/CD178 pathway.

Secretion of IFN-gamma but not IL-17 by CD1d-restricted NKT cells enhances rejection of skin grafts expressing epithelial cell-derived antigen.

NKT cells are key regulators of autoimmunity, tumor immune surveillance, and the immune response to pathogens. The role of NKT cells in regulating adaptive immunity to cutaneous Ags is largely unknown. This study explores the role of CD1d-restricted NKT cells in cross-priming of CD8 effector T cells to OVA expressed in epithelial keratinocytes (K5mOVA transgenic mouse). In a skin grafting model, we show that NKT cells enhance the rejection of K5mOVA skin grafts by promoting generation of OVA-specific CD8 effector T cells in the skin-draining lymph nodes. This is associated with a decrease in the proportion of both Th17 cells and IL-17-producing NKT cells within the lymph node, thereby inducing a Th1-biased response by increasing the ratio of IFN-gamma to IL-17 production. Administration of a strong agonist ligand (alpha-galactosylceramide) for NKT cells induced higher levels of local IFN-gamma production, enhancing the rate of K5mOVA graft rejection. Thus, NKT cells can promote adaptive immunity to cell-associated Ag expressed in skin by local regulation of IFN-gamma production in secondary lymphoid tissue during cross-priming of effector CD8 T cells.

Human CD1D gene expression is regulated by LEF-1 through distal promoter regulatory elements.

CD1d-expressing cells present lipid Ag to CD1d-restricted NKT cells, which play an important role in immune regulation and tumor rejection. Lymphoid enhancer-binding factor-1 (LEF-1) is one of the regulators of the Wnt signaling pathway, which is a powerful regulator in cellular growth, differentiation, and transformation. There is little evidence connecting Wnt signaling to CD1d expression. In this study, we have identified LEF-1 as a regulator of the expression of the gene encoding the human CD1d molecule (CD1D). We found that LEF-1 binds specifically to the CD1D promoter. Overexpression of LEF-1 in K562 or Jurkat cells suppresses CD1D promoter activity and downregulates endogenous CD1D transcripts, whereas knockdown of LEF-1 using LEF-1-specific small interfering RNA increases CD1D transcripts in K562 and Jurkat cells but there are different levels of surface CD1d on these two cell types. Chromatin immunoprecipitation showed that the endogenous LEF-1 is situated at the CD1D promoter and interacts with histone deacetylase-1 to facilitate the transcriptional repressor activity. Knockdown of LEF-1 using small interfering RNA potentiates an acetylation state of histone H3/H4, supporting the notion that LEF-1 acts as a transcriptional repressor for the CD1D gene. Our finding links LEF-1 to CD1D and suggests a role of Wnt signaling in the regulation of the human CD1D gene.

Tumor necrosis factor-alpha promotes myocarditis in female mice infected with coxsackievirus B3 through upregulation of CD1d on hematopoietic cells.

Coxsackievirus B3 (CVB3) induces cardiac inflammation (myocarditis) in male but not female C57BL/6 mice. Protection of females correlates with reduced expression of TNF-alpha and IL-1beta at both the mRNA and protein levels in the heart. Treatment of females with 300 ng/mouse of recombinant TNF-alpha on days +1 and +3 relative to infection restores myocarditis susceptibility to levels approximating those of infected male mice, showing that TNF-alpha deficiency is central to disease resistance. Female mice express little CD1d on spleen lymphocytes or cardiac myocytes, while females treated with TNF-alpha show increased CD1d expression in both cell populations. TNF-alpha treatment of male or female CD1d knockout (CD1dKO) mice failed to restore myocarditis susceptibility, demonstrating that of the multiple potential TNF-alpha activities, its ability to upregulate this non-classical major histocompatibility complex antigen is its dominant function in myocarditis susceptibility. Bone marrow chimeric mice were produced between female C57BL/6 and C57BL/6 CD1dKO mice so that either hematopoietic or non-hematopoietic cells were CD1d deficient. TNF-alpha treatment of chimeric mice having wild-type (CD1d+) hematopoietic cells restored myocarditis susceptibility, while TNF-alpha treatment of chimeric mice having CD1dKO hematopoietic cells, but CD1d+ myocytes, failed to develop myocarditis, showing that CD1d expression in lymphoid cells controls disease susceptibility.