Cross-validation of the RT-QuIC assay for the antemortem detection of chronic wasting disease in elk.

Chronic wasting disease is a progressively fatal, horizontally transmissible prion disease affecting several members of the cervid species. Conventional diagnosis relies on ELISA or IHC evaluation using tissues collected post-mortem; however, recent research has focused on newly developed amplification techniques using samples collected antemortem. The present study sought to cross-validate the real-time quaking-induced conversion assay (RT-QuIC) evaluation of rectal biopsies collected from an elk herd with endemic CWD, assessing both binary positive/negative test results as well as relative rates of amplification between laboratories. We found that results were correlative in both categories across all laboratories performing RT-QuIC, as well as to conventional IHC performed at a national reference laboratory. A significantly higher number of positive samples were identified using RT-QuIC, with results seemingly unhindered by low follicle counts. These findings support the continued development and implementation of amplification assays in the diagnosis of prion diseases of veterinary importance, targeting not just antemortem sampling strategies, but post-mortem testing approaches as well.

Effect of salmon type and presence/absence of bone on color, sensory characteristics, and consumer acceptability of pureed and chunked infant food products.

Salmon-based infant food (puree) and toddler food (puree plus chunks) were manufactured from pink salmon, with and without bone, and from Sockeye salmon, with and without bone, to contain 45% salmon, 55% water, and 5% starch. Products were retort processed at 118 to 121 degrees C for 55 min in a steam-jacketed still retort. A trained descriptive panel (n = 7) evaluated infant and toddler foods separately. Instrumental color, pH, and water activity were also determined. Infant and toddler foods were also evaluated by a consumer panel (n = 104) of parents for product acceptability. During the manufacturing process (cooking, homogenization, retort processing), salmon infant food from pink salmon lost much of its characteristic pink color while that from sockeye salmon retained a greater amount. Bitterness was more evident in samples with bones. In the toddler food formulation containing chunks, the odor and flavor characteristics were influenced primarily by the type of salmon. The presence of bone affected visual pink color and lightness, and salmon odor only. Consumers scored products made with sockeye salmon as more acceptable despite the fact that they had more off-flavor than products from pink salmon. The appearance and thickness of the pureed infant food was more acceptable than the toddler food with chunks despite the chunky toddler product having more acceptable salmon flavor. This indicates that the color and appearance of the prototypes were the main drivers for liking. Of the total number of parents surveyed, 73% would feed this salmon product to their children.

Effect of inclusion of salmon roe on characteristics of salmon baby food products.

Baby food was formulated from sockeye salmon (puree alone, puree + chunks, puree + pink row, puree + pink row + chunks, puree + red row, puree + red roe + chunks). In the 1st study, physical (pH, instrumental color, water activity) and descriptive sensory (odor, flavor, texture, visual color) characteristics were determined. Samples containing roe were lighter and less red (by approximately 3 to 4 a* units) than formulations without roe regardless of the type of roe added. Visual pink color followed the same trend. Formulations with roe, both pink and sockeye, were almost twice as fibrous as formulations without roe. Salmon flavor was stronger in samples containing roe from sockeye salmon. In the 2nd study, retort processed samples were stored at room temperature for 6 mo. Sweaty odor decreased over storage time. Visual cream-brown color correlated with L*, a*, b*, and chroma values (r =-0.80, 0.75, 0.80, and 0.84, respectively). TBARS values of all samples were < 0.35 mg MDA/kg and declined after month 0 indicating that these products were oxidatively stable. Overall, adding roe to these products lightened them and increased fibrous texture. Samples containing sockeye salmon roe had stronger salmon flavor. Once retort processed, these products were quite stable in terms of color, odor, and TBARS. Potential nutrient contributions of this type of product to the infant diet warrant additional research.

Protein digestibility evaluations of meat and fish substrates using laboratory, avian, and ileally cannulated dog assays.

Meat and fish serve as important protein sources in the companion animal diet; however, limited protein digestibility data are available for assessing protein digestibility differences among good-quality protein sources. Beef loin, pork loin, chicken breast, pollock fillet, and salmon fillet were evaluated for composition, protein digestibility, and AA bioavailability using the immobilized digestive enzyme assay, cecectomized rooster assay, and ileally cannulated dog assay. Pollock contained the greatest amount of CP, total essential AA (TEAA), and total nonessential AA (TNEAA; DM basis; 96.9, 38.6, and 50.3%, respectively). Salmon contained the next greatest amounts (92.8, 36.4, and 44.6%), followed by chicken (90.3, 36.1, 43.2%). Beef had the least CP content (82.7%), but had slightly greater TEAA and TNEAA concentrations (33.9, 42.0%) compared with pork (86.2, 33.6, 41.3%). Immobilized digestive enzyme assay values were greatest for pollock fillet (0.71) and least for chicken breast (0.52). Beef loin, pork loin, and salmon fillet were similar (0.63, 0.62, and 0.64, respectively). Standardized TEAA and TNEAA digestibility coefficients, evaluated using the cecectomized rooster assay, were greatest (P < 0.05) for pollock fillet (90.4 and 89.8%, respectively) and least (P < 0.05) for chicken breast (86.6 and 85.9%, respectively) and salmon fillet (87.8 and 86.4%, respectively). Dogs assigned to a 5 x 5 Latin square design were fed 5 diets, with each test substrate as the major protein source. No significant differences (P > 0.05) were found in ileal digestibility of protein. Values ranged from 88.9% for chicken to 90.5% for pork loin and pollock fillet. Ileal TEAA and TNEAA coefficients were not different among test substrates, with values between 91.7 and 92.7%, and 88.8 and 90.4%, respectively. Total tract CP apparent digestibility values ranged from 94.4 to 94.8%, with no differences noted among treatments. Despite marked differences in composition and predicted and standardized digestibility values, when the protein sources were added to diets at a concentration of approximately 30% (25% of total energy intake), no differences in test protein substrates were noted in either ileal or total tract nutrient digestibility.

Intensive conditioning regimen of etoposide (VP-16), cyclophosphamide and carmustine (VCB) followed by autologous hematopoietic stem cell transplantation for relapsed and refractory Hodgkin's lymphoma.

Several high-dose therapy regimens are used for autologous hematopoietic stem cell transplantation (auto-HSCT) for relapsed and refractory Hodgkin's lymphoma (HL) with variable disease response. An intensified regimen of etoposide (VP-16) 2,400 mg/m(2), cyclophosphamide 7,200 mg/m(2) and carmustine (BCNU) 600 mg/m(2) (VCB) pre-auto-HSCT was developed to overcome disease recurrence. A total of 43 relapsed and refractory HL patients underwent auto-HSCT between January 1992 and December 2004. At day 100 there were 37 (86%) complete responses. A total of 40 patients survived beyond day 100, 14 of whom subsequently relapsed/progressed. At a median follow-up of 4.9 years (range 1.5-11.4 years), 26 patients (60%) are alive and disease free. Five-year actuarial event-free survival (EFS) was 53% (95% CI 35-70%) and median EFS was 5.9 years. Median progression-free and overall survivals have not been reached. EFS was reduced with an increasing number of prognostic factors (Karnofsky performance status, KPS <90, chemotherapy-resistant disease and >or=3 chemotherapy regimens prior to transplant or=2; P=0.049). Grade III-IV regimen-related toxicity was 9% (n=4). The 1-year cumulative incidence of interstitial pneumonitis (IP) was 36%, however only two patients died of IP complications. Disease progression was the most common cause of death (n=10, 23%). Intensive VCB is an effective and well-tolerated preparative regimen for relapsed and refractory HL auto-HSCT.

High prevalence of early-onset osteopenia/osteoporosis after allogeneic stem cell transplantation and improvement after bisphosphonate therapy.

Osteopenia/osteoporosis (O/O) has been associated with allogeneic stem cell transplantation (alloSCT). We retrospectively reviewed 102 patients undergoing a first alloSCT from 2000 to 2005 at our center to evaluate the prevalence of O/O < or =6 and >6 months post-alloSCT. Fifty-six patients did not have a dual energy X-ray absorptiometry (DXA) scan following alloSCT. Approximately half (n=13/27) of those with a first DXA scan < or =6 months post-alloSCT had O/O and a similar rate (n=9/19) was seen in those with a first DXA scan >6 months. There were no significant differences in patient characteristics between the normal and O/O groups. The dual femur (DF) appeared to be more vulnerable to alloSCT-induced bone mineral density (BMD) loss than the lumbar spine (LS), regardless of screening time. O/O patients were treated with bisphosphonates and 41% had a repeat DXA scan post-treatment. No patient developed jaw osteonecrosis and significant BMD improvement was seen at the LS (mean BMD, 1.03+/-0.13 vs 1.08+/-0.12, P=0.004) but not the DF (mean BMD, 0.84+/-0.06 vs 0.85+/-0.08, P=0.29), indicating BMD loss at the DF is more resistant than the LS to antiresorptive therapy. Our results demonstrate that O/O is an early and late complication post-alloSCT and bisphosphonate treatment reverses BMD loss at the LS.

A whole genome association study of neuroticism using DNA pooling.

We describe a multistage approach to identify single nucleotide polymorphisms (SNPs) associated with neuroticism, a personality trait that shares genetic determinants with major depression and anxiety disorders. Whole genome association with 452 574 SNPs was performed on DNA pools from approximately 2000 individuals selected on extremes of neuroticism scores from a cohort of 88 142 people from southwest England. The most significant SNPs were then genotyped on independent samples to replicate findings. We were able to replicate association of one SNP within the PDE4D gene in a second sample collected by our laboratory and in a family-based test in an independent sample; however, the SNP was not significantly associated with neuroticism in two other independent samples. We also observed an enrichment of low P-values in known regions of copy number variations. Simulation indicates that our study had approximately 80% power to identify neuroticism loci in the genome with odds ratio (OR)>2, and approximately 50% power to identify small effects (OR=1.5). Since we failed to find any loci accounting for more than 1% of the variance, the heritability of neuroticism probably arises from many loci each explaining much less than 1%. Our findings argue the need for much larger samples than anticipated in genetic association studies and that the biological basis of emotional disorders is extremely complex.

In situ assays demonstrate that interferon-gamma suppresses infection-stimulated hepatic fibrin deposition by promoting fibrinolysis.

BACKGROUND: Inflammatory cytokines potently impact hemostatic pathways during infection, but the tissue-specific regulation of coagulation and fibrinolysis complicates studies of the underlying mechanisms. METHODS AND RESULTS: Here, we describe assays that quantitatively measuring prothrombinase (PTase), protein C-ase (PCase) and plasminogen activator (PA) activities in situ, thereby facilitating studies of tissue-specific hemostasis. Using these assays, we investigate the mechanisms regulating hepatic fibrin deposition during murine toxoplasmosis and the means by which interferon-gamma (IFN-gamma) suppresses infection-stimulated fibrin deposition. We demonstrate that Toxoplasma infection upregulates hepatic PTase, PCase, and PA activity. Wild type and gene-targeted IFN-gamma-deficient mice exhibit similar levels of infection-stimulated PTase activity. By contrast, IFN-gamma-deficiency is associated with increased PCase activity and reduced PA activity during infection. Parallel analyses of hepatic gene expression reveal that IFN-gamma-deficiency is associated with increased expression of thrombomodulin (TM), a key component of the PCase, increased expression of thrombin-activatable fibrinolysis inhibitor (TAFI), a PC substrate, and reduced expression of urokinase PA (u-PA). CONCLUSIONS: These findings suggest that IFN-gamma suppresses infection-stimulated hepatic fibrin deposition by suppressing TM-mediated activation of TAFI, thereby destabilizing fibrin deposits, and concomitantly increasing hepatic u-PA activity, thereby promoting fibrinolysis. We anticipate that further application of these in situ assays will improve our understanding of tissue-specific hemostasis, its regulation by cytokines, and its dysregulation during coagulopathy.

Beneficial effects of targeting CCR5 in allograft recipients.

BACKGROUND: The chemokine receptor, CCR5, and its three high-affinity ligands, macrophage inflammatory protein- (MIP) 1alpha, MIP-1beta, and regulated on activation normal T cell expressed and secreted (RANTES), are expressed by infiltrating mononuclear cells during the rejection of clinical and experimental organ allografts, although the significance of these molecules in the pathogenesis of rejection has not been established. METHODS: We studied intragraft events in four allograft models. First, we studied cardiac transplants in fully MHC-mismatched mice that were deficient in CCR5 or two of its ligands, MIP-1alpha or RANTES. Second we tested the effects of a neutralizing rat anti-mCCR5 monoclonal antibody on allograft survival. Third we assessed whether a subtherapeutic course of cyclosporine would potentiate enhance survival in CCR5-deficient recipients. Finally, we tested the effect of targeting CCR5 in a class II-mismatched model. RESULTS: Whereas mice deficient in expression of MIP-1alpha or RANTES reject fully MHC-mismatched cardiac allografts normally, CCR5-/- mice, or CCR5+/+ mice treated with a neutralizing mAb to mCCR5, show enhanced allograft survival. MHC class II-disparate mismatched are permanently accepted in CCR5-/- but not CCR5+/+ recipients. Finally, the beneficial effects of targeting of CCR5 are markedly synergistic with the effects of cyclosporine, resulting in permanent engraftment without development of chronic rejection. CONCLUSIONS: We conclude that CCR5 plays a key role in the mechanisms of host T cell and macrophage recruitment and allograft rejection, such that targeting of CCR5 clinically may be of therapeutic significance.

Fibrinogen stimulates macrophage chemokine secretion through toll-like receptor 4.

Extravascular fibrin deposition is an early and persistent hallmark of inflammatory responses. Fibrin is generated from plasma-derived fibrinogen, which escapes the vasculature in response to endothelial cell retraction at sites of inflammation. Our ongoing efforts to define the physiologic functions of extravasated fibrin(ogen) have led to the discovery, reported here, that fibrinogen stimulates macrophage chemokine secretion. Differential mRNA expression analysis and RNase protection assays revealed that macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, MIP-2, and monocyte chemoattractant protein-1 are fibrinogen inducible in the RAW264.7 mouse macrophage-like cell line, and ELISA confirmed that both RAW264.7 cells and primary murine thioglycolate-elicited peritoneal macrophages up-regulate the secretion of monocyte chemoattractant protein-1 >100-fold upon exposure to fibrinogen. Human U937 and THP-1 precursor-1 (THP-1) monocytic cell lines also secreted chemokines in response to fibrinogen, upon activation with IFN-gamma and differentiation with vitamin D(3), respectively. LPS contamination could not account for our observations, as fibrinogen-induced chemokine secretion was sensitive to heat denaturation and was unaffected by the pharmacologic LPS antagonist polymyxin B. Nevertheless, fibrinogen- and LPS-induced chemokine secretion both apparently required expression of functional Toll-like receptor 4, as each was diminished in macrophages derived from C3H/HeJ mice. Thus, innate responses to fibrinogen and bacterial endotoxin may converge at the evolutionarily conserved Toll-like recognition molecules. Our data suggest that extravascular fibrin(ogen) induces macrophage chemokine expression, thereby promoting immune surveillance at sites of inflammation.

CD1d-reactive T-cell activation leads to amelioration of disease caused by diabetogenic encephalomyocarditis virus.

A subset of CD161 (NK1) T cells express an invariant Valpha14Jalpha281 TCR-alpha chain (Valpha(invt) T cells) and produce Th2 and Th1 cytokines rapidly in response to CD1d, but their physiological function(s) remain unclear. We have found that CD1d-reactive T cells mediate to resistance against the acute, cytopathic virus diabetogenic encephalomyocarditis virus (EMCV-D) in relatively Th1-biased, C57BL/6-based backgrounds. We show now that these results generalize to Th2-biased, hypersensitive BALB/c mice. CD1d-KO BALB/c mice were more susceptible to EMCV-D. Furthermore, alpha-galactosylceramide (alpha-GalCer), a CD1d-presented lipid antigen that specifically activates Valpha(invt) T cells, protected wild-type (WT) mice against EMCV-D-induced encephalitis, myocarditis, and diabetes. In contrast, neither CD1d-KO nor Jalpha281-KO mice were protected by alpha-GALCER: Finally, disease in Jalpha281-KO mice was comparable to WT, indicating for the first time equivalent roles for CD1d-reactive Valpha(invt) and noninvariant T cells in resistance to acute viral infection. A model for how CD1d-reactive T cells can initiate immune responses, which synthesizes current results, is presented.

Requirement of the chemokine receptor CXCR3 for acute allograft rejection.

Chemokines provide signals for activation and recruitment of effector cells into sites of inflammation, acting via specific G protein-coupled receptors. However, in vitro data demonstrating the presence of multiple ligands for a given chemokine receptor, and often multiple receptors for a given chemokine, have led to concerns of biologic redundancy. Here we show that acute cardiac allograft rejection is accompanied by progressive intragraft production of the chemokines interferon (IFN)-gamma-inducible protein of 10 kD (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T cell alpha chemoattractant (I-TAC), and by infiltration of activated T cells bearing the corresponding chemokine receptor, CXCR3. We used three in vivo models to demonstrate a role for CXCR3 in the development of transplant rejection. First, CXCR3-deficient (CXCR3(-/)-) mice showed profound resistance to development of acute allograft rejection. Second, CXCR3(-/)- allograft recipients treated with a brief, subtherapeutic course of cyclosporin A maintained their allografts permanently and without evidence of chronic rejection. Third, CXCR(+/+) mice treated with an anti-CXCR3 monoclonal antibody showed prolongation of allograft survival, even if begun after the onset of rejection. Taken in conjunction with our findings of CXCR3 expression in rejecting human cardiac allografts, we conclude that CXCR3 plays a key role in T cell activation, recruitment, and allograft destruction.

Differential effects of cyclosporine A, methylprednisolone, mycophenolate, and rapamycin on CD154 induction and requirement for NFkappaB: implications for tolerance induction.

BACKGROUND: Recent experimental data indicate that the targeting of the costimulatory molecule CD40-ligand (CD154) may well offer an opportunity for tolerance induction in transplant recipients and patients with autoimmune diseases, although the optimal therapeutic strategy for clinical application of CD154 monoclonal antibody (mAb) is unclear. METHODS: We undertook vascularized heterotopic cardiac allograft transplantation in completely MHC-mismatched mice, treated recipients with CD154 mAb plus various immunosuppressive agents, and performed flow cytometric analysis of CD154 expression by T cells activated in vitro in the presence of corresponding immunosuppressive agents. We also tested the extent to which CD154 induction was NFkappaB-dependent by using NFkappaB/p50-deficient mice as allograft recipients and as source of cells for in vitro studies of CD154 induction, and through use of proteasome inhibitors to block IkappaBalpha degradation and NFKB activation in wild-type mice. RESULTS: Concomitant use of cyclosporin A or methylprednisolone, but not rapamycin or mycophenolate, inhibited CD154 mAb-induced allograft survival. The differential effects of these agents on CD154 mAb-induced tolerance correlated with their capacity to inhibit activation-induced CD154 expression on CD4+ T cells. Full expression of CD154 expression was found to require NF-kappaB activation, and CD154 mAb was ineffective in NF-kappaB/p50 deficient allograft recipients or control mice in which NF-kappaB activation was blocked by proteasome inhibition. CONCLUSIONS: Strategies to use CD154 mAb clinically must take into account the effects of immunosuppressive agents on CD154 induction, which seems to be at least partially NF-kappaB dependent. Our data suggest that ligation of surface-expressed CD154 provides an important signal that modulates T cell activation and thereby contributes to the effects of CD154 mAb, in addition to previously recognized actions involving blockade of CD40/CD154-dependent cell activation and activation-induced cell death.

Targeting of the chemokine receptor CCR1 suppresses development of acute and chronic cardiac allograft rejection.

Although mononuclear cell infiltration is a hallmark of cellular rejection of a vascularized allograft, efforts to inhibit rejection by blocking leukocyte-endothelial cell adhesion have proved largely unsuccessful, perhaps in part because of persistent generation of chemokines within rejecting grafts. We now provide, to our knowledge, the first evidence that in vivo blockade of specific chemokine receptors is of therapeutic significance in organ transplantation. Inbred mice with a targeted deletion of the chemokine receptor CCR1 showed significant prolongation of allograft survival in 4 models. First, cardiac allografts across a class II mismatch were rejected by CCR1(+/+) recipients but were accepted permanently by CCR1(-/-) recipients. Second, CCR1(-/-) mice rejected completely class I- and class II-mismatched BALB/c cardiac allografts more slowly than control mice. Third, levels of cyclosporin A that had marginal effects in CCR1(+/+) mice resulted in permanent allograft acceptance in CCR1(-/-) recipients. These latter allografts showed no sign of chronic rejection 50-200 days after transplantation, and transfer of CD4(+) splenic T cells from these mice to naive allograft recipients significantly prolonged allograft survival, whereas cells from CCR1(+/+) mice conferred no such benefit. Finally, both CCR1(+/+) and CCR1(-/-) allograft recipients, when treated with a mAb to CD4, showed permanent engraftment, but these allografts showed florid chronic rejection in the former strain and were normal in CCR1(-/-) mice. We conclude that therapies to block CCR1/ligand interactions may prove useful in preventing acute and chronic rejection clinically.

Stat6-dependent and -independent pathways for IL-4 production.

Stat6 has been shown to have a crucial role in the IL-4-dependent differentiation of Th2 cells. In this report, we explore whether in vitro Th2 differentiation driven by altered costimulatory signals or Ag dose is Stat6 dependent. We find that blocking B7-1 signaling in vitro promotes the differentiation of IL-4-secreting Th2 cells in wild-type but not Stat6-deficient T cell cultures. Additionally, stimulation with peptide Ag doses that normally result in the production of Th2 cells in vitro fails to do so in cultures of Stat6-deficient cells. We also demonstrate that Stat6 is required for the in vitro differentiation of CD8+ T cells into IL-4-secreting cytotoxic T cell type 2 cells. However, IL-4 expression is not absolutely dependent on Stat6. We demonstrate that populations of T cells that do not require IL-4 for their development, such as NK T cells, are still competent to secrete IL-4 in the absence of Stat6. These results demonstrate that Stat6 is required for the differentiation program leading to the generation of Th2 and cytotoxic T cell type 2 cells but not for IL-4 expression in cells that do not undergo differentiation in response to IL-4.

Avian polyomavirus major capsid protein VP1 interacts with the minor capsid proteins and is transported into the cell nucleus but does not assemble into capsid-like particles when expressed in the baculovirus system.

The baculovirus system was used to construct and isolate AcMNPV-VP1, AcMNPV-VP2 and AcMNPV-VP3 recombinant viruses which express the respective avian polyomavirus (APV) structural proteins in Sf9 insect cells. These recombinant AcMNPVs containing APV structural protein genes were utilized to investigate protein-protein interactions between the structural proteins. Immunofluorescence studies utilizing Sf9 cells infected with the AcMNPV-VP1 revealed that the VP1 protein was expressed and localized in the cytoplasm and not transported into the nucleus. When the cells were co-infected with the VP1 and either VP2 or VP3 recombinant viruses, immunofluorescence of the VP1 protein was localized in the nucleus, indicating that the VP1 protein was transported to the nucleus by both the VP2 and VP3 minor proteins. This observation was suggestive of a protein-protein interaction between the expressed proteins. This protein-protein interaction was substantiated by laser scanning confocal microscopy of Sf9 cells that were co-infected with VP1, VP2 and VP3 recombinant viruses. However, the minor proteins could not be co-isolated with VP1 protein by immunoaffinity chromatography using a monoclonal anti-VP1 serum. In addition, capsid-like particles could not be purified either by CsC1 density gradient centrifugation or by immunoaffinity chromatography. VP1 capsomeres were isolated by immunoaffinity chromatography from Sf9 cells infected with AcMNPV-VP1, with or without the minor protein(s), and these capsomeres could assemble in vitro into capsid-like particles. Electron microscopic observation of thin-sectioned Sf9 cells, which were co-infected with VP1, VP2 and VP3 recombinant viruses, demonstrated capsomere-like structures in the nucleus, but capsid-like particles were not observed.

Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.

Selective ability of mouse CD1 to present glycolipids: alpha-galactosylceramide specifically stimulates V alpha 14+ NK T lymphocytes.

Mouse CD1 (mCD1) glycoproteins are known to present peptides, while human CD1 molecules present glycolipids. In mice, mCD1-autoreactive NK T cells play critical roles in various immune responses, through the secretion of high amounts of cytokines. This study was initiated to determine whether glycolipids are involved in the autorecognition of mCD1 by NK T cells. Alpha-galactosylceramide (alpha-GalCer) was the only glycolipid tested capable of eliciting an mCD1-restricted response by splenic T cells. Moreover, splenic T cells derived from mCD1-deficient mice were not stimulated by alpha-GalCer, suggesting that the responsive T cells are selected by mCD1. Using cytoflow techniques, we confirmed that, in response to alpha-GalCer, IFN-gamma-secreting cells displayed an NK T cell phenotype. The predominance of IFN-gamma vs IL-4, however, is determined by the type of mCD1+ APC, suggesting the potential for APC regulation of cytokine production by NK T cells. Among a panel of 10 mCD1-autoreactive T cell hybridomas, only the ones that express the typical V alpha 14 J alpha 281 TCR rearrangement of NK T cells responded to alpha-GalCer. Fixation or treatment of mCD1+ APCs with an inhibitor of endosomal acidification and the use of mCD1 mutants unable to traffic through endosome still allowed alpha-GalCer to stimulate NK T cells. Thus, endosomal trafficking and Ag processing are not required for glycolipid recognition. In summary, alpha-GalCer might be the autologous ligand, or a mimic of a glycolipid ligand, involved in the mCD1-mediated stimulation of NK T cells.