Delineation of DNA and mRNA COVID-19 vaccine-induced immune responses in preclinical animal models.

Nucleic acid vaccines are designed based on genetic sequences (DNA or mRNA) of a target antigen to be expressed in vivo to drive a host immune response. In response to the COVID-19 pandemic, mRNA and DNA vaccines based on the SARS-CoV-2 Spike antigen were developed. Surprisingly, head-to-head characterizations of the immune responses elicited by each vaccine type has not been performed to date. Here, we have employed a range of preclinical animal models including the hamster, guinea pig, rabbit, and mouse to compare and delineate the immune response raised by DNA, administered intradermally (ID) with electroporation (EP) and mRNA vaccines (BNT162b2 or mRNA-1273), administered intramuscularly (IM), expressing the SARS-CoV-2 WT spike antigen. The results revealed clear differences in the quality and magnitude of the immune response between the two vaccine platforms. The DNA vaccine immune response was characterized by strong T cell responses, while the mRNA vaccine elicited robust humoral responses. The results may assist in guiding the disease target each vaccine type may be best matched against and suggest mechanisms to further enhance the breadth of each platform's immune response.

DNA-delivered antibody cocktail exhibits improved pharmacokinetics and confers prophylactic protection against SARS-CoV-2.

Monoclonal antibody therapy has played an important role against SARS-CoV-2. Strategies to deliver functional, antibody-based therapeutics with improved in vivo durability are needed to supplement current efforts and reach underserved populations. Here, we compare recombinant mAbs COV2-2196 and COV2-2130, which compromise clinical cocktail Tixagevimab/Cilgavimab, with optimized nucleic acid-launched forms. Functional profiling of in vivo-expressed, DNA-encoded monoclonal antibodies (DMAbs) demonstrated similar specificity, broad antiviral potency and equivalent protective efficacy in multiple animal challenge models of SARS-CoV-2 prophylaxis compared to protein delivery. In PK studies, DNA-delivery drove significant serum antibody titers that were better maintained compared to protein administration. Furthermore, cryo-EM studies performed on serum-derived DMAbs provide the first high-resolution visualization of in vivo-launched antibodies, revealing new interactions that may promote cooperative binding to trimeric antigen and broad activity against VoC including Omicron lineages. These data support the further study of DMAb technology in the development and delivery of valuable biologics.

Prime-boost vaccination regimens with INO-4800 and INO-4802 augment and broaden immune responses against SARS-CoV-2 in nonhuman primates.

The enhanced transmissibility and immune evasion associated with emerging SARS-CoV-2 variants demands the development of next-generation vaccines capable of inducing superior protection amid a shifting pandemic landscape. Since a portion of the global population harbors some level of immunity from vaccines based on the original Wuhan-Hu-1 SARS-CoV-2 sequence or natural infection, an important question going forward is whether this immunity can be boosted by next-generation vaccines that target emerging variants while simultaneously maintaining long-term protection against existing strains. Here, we evaluated the immunogenicity of INO-4800, our synthetic DNA vaccine candidate for COVID-19 currently in clinical evaluation, and INO-4802, a next-generation DNA vaccine designed to broadly target emerging SARS-CoV-2 variants, as booster vaccines in nonhuman primates. Rhesus macaques primed over one year prior with the first-generation INO-4800 vaccine were boosted with either INO-4800 or INO-4802 in homologous or heterologous prime-boost regimens. Both boosting schedules led to an expansion of T cells and antibody responses which were characterized by improved neutralizing and ACE2 blocking activity across wild-type SARS-CoV-2 as well as multiple variants of concern. These data illustrate the durability of immunity following vaccination with INO-4800 and additionally support the use of either INO-4800 or INO-4802 in prime-boost regimens.

Nucleic acid delivery of immune-focused SARS-CoV-2 nanoparticles drives rapid and potent immunogenicity capable of single-dose protection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines may target epitopes that reduce durability or increase the potential for escape from vaccine-induced immunity. Using synthetic vaccinology, we have developed rationally immune-focused SARS-CoV-2 Spike-based vaccines. Glycans can be employed to alter antibody responses to infection and vaccines. Utilizing computational modeling and in vitro screening, we have incorporated glycans into the receptor-binding domain (RBD) and assessed antigenic profiles. We demonstrate that glycan-coated RBD immunogens elicit stronger neutralizing antibodies and have engineered seven multivalent configurations. Advanced DNA delivery of engineered nanoparticle vaccines rapidly elicits potent neutralizing antibodies in guinea pigs, hamsters, and multiple mouse models, including human ACE2 and human antibody repertoire transgenics. RBD nanoparticles induce high levels of cross-neutralizing antibodies against variants of concern with durable titers beyond 6 months. Single, low-dose immunization protects against a lethal SARS-CoV-2 challenge. Single-dose coronavirus vaccines via DNA-launched nanoparticles provide a platform for rapid clinical translation of potent and durable coronavirus vaccines.

Class Ib MHC-Mediated Immune Interactions Play a Critical Role in Maintaining Mucosal Homeostasis in the Mammalian Large Intestine.

Lymphocytes within the intestinal epithelial layer (IEL) in mammals have unique composition compared with their counterparts in the lamina propria. Little is known about the role of some of the key colonic IEL subsets, such as TCRαß+CD8+ T cells, in inflammation. We have recently described liver-enriched innate-like TCRαß+CD8αα regulatory T cells, partly controlled by the non-classical MHC molecule, Qa-1b, that upon adoptive transfer protect from T cell-induced colitis. In this study, we found that TCRαß+CD8αα T cells are reduced among the colonic IEL during inflammation, and that their activation with an agonistic peptide leads to significant Qa-1b-dependent protection in an acute model of colitis. Cellular expression of Qa-1b during inflammation and corresponding dependency in peptide-mediated protection suggest that Batf3-dependent CD103+CD11b- type 1 conventional dendritic cells control the protective function of TCRαß+CD8αα T cells in the colonic epithelium. In the colitis model, expression of the potential barrier-protective gene, Muc2, is enhanced upon administration of a Qa-1b agonistic peptide. Notably, in steady state, the mucin metabolizing Akkermansia muciniphila was found in significantly lower abundance amid a dramatic change in overall microbiome and metabolome, increased IL-6 in explant culture, and enhanced sensitivity to dextran sulfate sodium in Qa-1b deficiency. Finally, in patients with inflammatory bowel disease, we found upregulation of HLA-E, a Qa-1b analog with inflammation and biologic non-response, in silico, suggesting the importance of this regulatory mechanism across species.

Intradermal-delivered DNA vaccine induces durable immunity mediating a reduction in viral load in a rhesus macaque SARS-CoV-2 challenge model.

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health and social and economic infrastructures. Here, we assess the immunogenicity and anamnestic protective efficacy in rhesus macaques of an intradermal (i.d.)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800, currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and induced spike antigen and RBD binding antibodies with ADCP and ADCD activity. Sera from the animals neutralized both the D614 and G614 SARS-CoV-2 pseudotype viruses. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T cell and neutralizing antibody responses. These responses were associated with lower viral loads in the lung. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system, which are likely important for providing durable protection against COVID-19 disease.

Safety and immunogenicity of INO-4800 DNA vaccine against SARS-CoV-2: A preliminary report of an open-label, Phase 1 clinical trial.

BACKGROUND: A vaccine against SARS-CoV-2 is of high urgency. Here the safety and immunogenicity induced by a DNA vaccine (INO-4800) targeting the full length spike antigen of SARS-CoV-2 are described. METHODS: INO-4800 was evaluated in two groups of 20 participants, receiving either 1.0 mg or 2.0 mg of vaccine intradermally followed by CELLECTRA® EP at 0 and 4 weeks. Thirty-nine subjects completed both doses; one subject in the 2.0 mg group discontinued trial participation prior to receiving the second dose. ClinicalTrials.gov identifier: NCT04336410. FINDINGS: The median age was 34.5, 55% (22/40) were men and 82.5% (33/40) white. Through week 8, only 6 related Grade 1 adverse events in 5 subjects were observed. None of these increased in frequency with the second administration. No serious adverse events were reported. All 38 subjects evaluable for immunogenicity had cellular and/or humoral immune responses following the second dose of INO-4800. By week 6, 95% (36/38) of the participants seroconverted based on their responses by generating binding (ELISA) and/or neutralizing antibodies (PRNT IC50), with responder geometric mean binding antibody titers of 655.5 [95% CI (255.6, 1681.0)] and 994.2 [95% CI (395.3, 2500.3)] in the 1.0 mg and 2.0 mg groups, respectively. For neutralizing antibody, 78% (14/18) and 84% (16/19) generated a response with corresponding geometric mean titers of 102.3 [95% CI (37.4, 280.3)] and 63.5 [95% CI (39.6, 101.8)], in the respective groups. By week 8, 74% (14/19) and 100% (19/19) of subjects generated T cell responses by IFN-É£ ELISpot assay with the median SFU per 106 PBMC of 46 [95% CI (21.1, 142.2)] and 71 [95% CI (32.2, 194.4)] in the 1.0 mg and 2.0 mg groups, respectively. Flow cytometry demonstrated a T cell response, dominated by CD8+ T cells co-producing IFN-É£ and TNF-α, without increase in IL-4. INTERPRETATION: INO-4800 demonstrated excellent safety and tolerability and was immunogenic in 100% (38/38) of the vaccinated subjects by eliciting either or both humoral or cellular immune responses. FUNDING: Coalition for Epidemic Preparedness Innovations (CEPI).

Immunogenicity of a DNA vaccine candidate for COVID-19.

The coronavirus family member, SARS-CoV-2 has been identified as the causal agent for the pandemic viral pneumonia disease, COVID-19. At this time, no vaccine is available to control further dissemination of the disease. We have previously engineered a synthetic DNA vaccine targeting the MERS coronavirus Spike (S) protein, the major surface antigen of coronaviruses, which is currently in clinical study. Here we build on this prior experience to generate a synthetic DNA-based vaccine candidate targeting SARS-CoV-2 S protein. The engineered construct, INO-4800, results in robust expression of the S protein in vitro. Following immunization of mice and guinea pigs with INO-4800 we measure antigen-specific T cell responses, functional antibodies which neutralize the SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and biodistribution of SARS-CoV-2 targeting antibodies to the lungs. This preliminary dataset identifies INO-4800 as a potential COVID-19 vaccine candidate, supporting further translational study.

Differential Activation of Unconventional T Cells, Including iNKT Cells, in Alcohol-Related Liver Disease.

BACKGROUND: Liver is enriched in several innate-like unconventional T cells, but their role in alcohol-related liver disease (ALD) is not fully understood. Studies in several acute alcohol feeding models but not in chronic alcoholic steatohepatitis (ASH) model have shown that invariant natural killer T (iNKT) cells play a pathogenic role in ALD. Here, we investigated the activation of iNKT cells in an intragastric (iG) infusion model of chronic ASH as well as the frequency and cytokine phenotype of 3 different unconventional T cells: iNKT, mucosal-associated invariant T (MAIT), and CD8+ CD161hi Vα7.2- cells in peripheral blood of ALD patients. METHODS: Hepatic iNKT cells were investigated using the iG model of chronic ASH that combines feeding of high-cholesterol/high-fat diet (HCFD) with intragastric feeding of ethanol diet (HCFD + iG Alc). Human iNKT, MAIT, and CD8+ CD161hi Vα7.2- cells were examined by flow cytometry in peripheral blood of patients with severe alcoholic hepatitis (SAH) and chronic alcoholics (ChA) and compared with healthy controls. RESULTS: In the iG model of chronic ASH, IFNγ+ iNKT cells accumulate in their livers compared with pair-fed control mice and activated hepatic iNKT cells show high expression of Fas and FasL. Notably, IFNγ+ iNKT cells are also significantly increased in peripheral blood of ChA patients compared with SAH patients. MAIT cells are significantly reduced in all ALD patients, but CD8+ CD161hi Vα7.2- cells are increased in SAH patients. Although MAIT and CD8+ CD161hi Vα7.2- cells displayed a similar cytokine production profile, the production of IFNγ and TNFα is significantly increased in SAH patients, while significant IL-17A production is found in ChA patients. CONCLUSIONS: We found that the 3 unconventional T cells are activated in ALD patients showing interesting differences in their frequency and cytokine production profile between SAH and ChA patients. In the iG murine model of chronic ASH, iNKT cells are also activated secreting proinflammatory cytokines suggesting their involvement in liver disease.

Blockade of IL-17 signaling reverses alcohol-induced liver injury and excessive alcohol drinking in mice.

Chronic alcohol abuse has a detrimental effect on the brain and liver. There is no effective treatment for these patients, and the mechanism underlying alcohol addiction and consequent alcohol-induced damage of the liver/brain axis remains unresolved. We compared experimental models of alcoholic liver disease (ALD) and alcohol dependence in mice and demonstrated that genetic ablation of IL-17 receptor A (IL-17ra-/-) or pharmacological blockade of IL-17 signaling effectively suppressed the increased voluntary alcohol drinking in alcohol-dependent mice and blocked alcohol-induced hepatocellular and neurological damage. The level of circulating IL-17A positively correlated with the alcohol use in excessive drinkers and was further increased in patients with ALD as compared with healthy individuals. Our data suggest that IL-17A is a common mediator of excessive alcohol consumption and alcohol-induced liver/brain injury, and targeting IL-17A may provide a novel strategy for treatment of alcohol-induced pathology.

Distinct PLZF+CD8αα+ Unconventional T Cells Enriched in Liver Use a Cytotoxic Mechanism to Limit Autoimmunity.

Hepatic immune system is uniquely challenged to mount a controlled effector response to pathogens while maintaining tolerance to diet and microbial Ags. We have identified a novel population of innate-like, unconventional CD8αα+TCRαß+ T cells in naive mice and in human peripheral blood, called CD8αα Tunc, capable of controlling effector T cell responses. They are NK1.1+ (CD161+ in human), express NK-inhibitory receptors, and express the promyelocytic leukemia zinc finger (PLZF) transcription factor that distinguishes them from conventional CD8+ T cells. These cells display a cytotoxic phenotype and use a perforin-dependent mechanism to control Ag-induced or T cell-mediated autoimmune diseases. CD8αα Tunc are dependent upon IL-15/IL-2Rß signaling and PLZF for their development and/or survival. They are Foxp3-negative and their regulatory activity is associated with a functionally distinct Qa-1b-dependent population coexpressing CD11c and CD244. A polyclonal TCR repertoire, an activated/memory phenotype, and the presence of CD8αα Tunc in NKT- and in MAIT-deficient as well as in germ-free mice indicates that these cells recognize diverse self-protein Ags. Our studies reveal a distinct population of unconventional CD8+ T cells within the natural immune repertoire capable of controlling autoimmunity and also providing a new target for therapeutic intervention.

Intestinal iNKT cells migrate to liver and contribute to hepatocyte apoptosis during alcoholic liver disease.

We investigated the migration of intestinal immune cells to the liver and their contribution to alcoholic liver disease. In mice fed ethanol, we found that an increased number of invariant natural killer T (iNKT) cells, which respond to the antigen presented by CD1d, migrated from mesenteric lymph nodes to the liver. iNKT cells react to lipid antigens, so we studied their activities in mice with intestinal epithelial cell-specific deletion of Pparg (PpargΔIEC) as a model for altering intestinal lipidomic profiles. Levels of CD1d increased in intestines of ethanol-fed PpargΔIEC mice, and in cell-tracking experiments, more iNKT cells migrated to the liver, compared with mice without disruption of Pparg. Livers of PpargΔIEC mice had increased markers of apoptosis and liver injury after ethanol feeding. iNKT cells isolated from livers of ethanol-fed PpargΔIEC mice induced apoptosis of cultured hepatocytes. An inhibitor of iNKT cells reduced ethanol-induced liver injury in PpargΔIEC mice. Duodenal tissues from patients with alcohol-use disorder have been found to have increased levels of CD1d compared with tissues from patients without alcohol overuse. Ethanol use, therefore, activates iNKT cells in the intestine to migrate to liver, where they-along with the resident hepatic iNKT cells-contribute to hepatocyte death and injury. NEW & NOTEWORTHY In this article, we studied migration of intestinal immune cells into the liver in response to ethanol-induced liver disease. We found that chronic ethanol feeding induces expression of CD1d by enterocytes, which activate invariant natural killer T (iNKT) cells in mesenteric lymph nodes; activation is further increased with loss of peroxisome proliferator-activated receptor gamma gene and altered lipid profiles. The activated iNKT cells migrate into the liver, where they promote hepatocyte apoptosis. Patients with alcohol use disorder have increased expression of CD1d in the small intestine. Strategies to block these processes might be developed to treat alcoholic liver disease.

Differential Activation of Hepatic Invariant NKT Cell Subsets Plays a Key Role in Progression of Nonalcoholic Steatohepatitis.

Innate immune mechanisms play an important role in inflammatory chronic liver diseases. In this study, we investigated the role of type I or invariant NKT (iNKT) cell subsets in the progression of nonalcoholic steatohepatitis (NASH). We used α-galactosylceramide/CD1d tetramers and clonotypic mAb together with intracytoplasmic cytokine staining to analyze iNKT cells in choline-deficient l-amino acid-defined (CDAA)-induced murine NASH model and in human PBMCs, respectively. Cytokine secretion of hepatic iNKT cells in CDAA-fed C57BL/6 mice altered from predominantly IL-17+ to IFN-γ+ and IL-4+ during NASH progression along with the downmodulation of TCR and NK1.1 expression. Importantly, steatosis, steatohepatitis, and fibrosis were dependent upon the presence of iNKT cells. Hepatic stellate cell activation and infiltration of neutrophils, Kupffer cells, and CD8+ T cells as well as expression of key proinflammatory and fibrogenic genes were significantly blunted in Jα18-/- mice and in C57BL/6 mice treated with an iNKT-inhibitory RAR-γ agonist. Gut microbial diversity was significantly impacted in Jα18-/- and in CDAA diet-fed mice. An increased frequency of CXCR3+IFN-γ+T-bet+ and IL-17A+ iNKT cells was found in PBMC from NASH patients in comparison with nonalcoholic fatty liver patients or healthy controls. Consistent with their in vivo activation, iNKT cells from NASH patients remained hyporesponsive to ex-vivo stimulation with α-galactosylceramide. Accumulation of plasmacytoid dendritic cells in both mice and NASH patients suggest their role in activation of iNKT cells. In summary, our findings indicate that the differential activation of iNKT cells play a key role in mediating diet-induced hepatic steatosis and fibrosis in mice and its potential involvement in NASH progression in humans.

Complex Network of NKT Cell Subsets Controls Immune Homeostasis in Liver and Gut.

The liver-gut immune axis is enriched in several innate immune cells, including innate-like unconventional and adaptive T cells that are thought to be involved in the maintenance of tolerance to gut-derived antigens and, at the same time, enable effective immunity against microbes. Two subsets of lipid-reactive CD1d-restricted natural killer T (NKT) cells, invariant NKT (iNKT) and type II NKT cells present in both mice and humans. NKT cells play an important role in regulation of inflammation in the liver and gut due to their innate-like properties of rapid secretion of a myriad of pro-inflammatory and anti-inflammatory cytokines and their ability to influence other innate cells as well as adaptive T and B cells. Notably, a bi-directional interactive network between NKT cells and gut commensal microbiota plays a crucial role in this process. Here, we briefly review recent studies related to the cross-regulation of both NKT cell subsets and how their interactions with other immune cells and parenchymal cells, including hepatocytes and enterocytes, control inflammatory diseases in the liver, such as alcoholic and non-alcoholic steatohepatitis, as well as inflammation in the gut. Overwhelming experimental data suggest that while iNKT cells are pathogenic, type II NKT cells are protective in the liver. Since CD1d-dependent pathways are highly conserved from mice to humans, a detailed cellular and molecular understanding of these immune regulatory pathways will have major implications for the development of novel therapeutics against inflammatory diseases of liver and gut.

Invariant natural killer T cells contribute to chronic-plus-binge ethanol-mediated liver injury by promoting hepatic neutrophil infiltration.

Neutrophil infiltration is a hallmark of alcoholic steatohepatitis; however, the underlying mechanisms remain unclear. We previously reported that chronic-plus-binge ethanol feeding synergistically induces hepatic recruitment of neutrophils, which contributes to liver injury. In this paper, we investigated the roles of invariant natural killer T (iNKT) cells in chronic-plus-binge ethanol feeding-induced hepatic neutrophil infiltration and liver injury. Wild-type and two strains of iNKT cell-deficient mice (CD1d- and Jα18-deficient mice) were subjected to chronic-plus-binge ethanol feeding. Liver injury and inflammation were examined. Chronic-plus-binge ethanol feeding synergistically increased the number of hepatic iNKT cells and induced their activation, compared with chronic feeding or binge alone. iNKT cell-deficient mice were protected from chronic-plus-binge ethanol-induced hepatic neutrophil infiltration and liver injury. Moreover, chronic-plus-binge ethanol feeding markedly upregulated the hepatic expression of several genes associated with inflammation and neutrophil recruitment in wild-type mice, but induction of these genes was abrogated in iNKT cell-deficient mice. Importantly, several cytokines and chemokines (e.g., MIP-2, MIP-1, IL-4, IL-6 and osteopontin) involved in neutrophil infiltration were upregulated in hepatic NKT cells isolated from chronic-plus-binge ethanol-fed mice compared to pair-fed mice. Finally, treatment with CD1d blocking antibody, which blocks iNKT cell activation, partially prevented chronic-plus-binge ethanol-induced liver injury and inflammation. Chronic-plus-binge ethanol feeding activates hepatic iNKT cells, which play a critical role in the development of early alcoholic liver injury, in part by releasing mediators that recruit neutrophils to the liver, and thus, iNKT cells represent a potential therapeutic target for the treatment of alcoholic liver disease.

Inhibition of type I natural killer T cells by retinoids or following sulfatide-mediated activation of type II natural killer T cells attenuates alcoholic liver disease in mice.

UNLABELLED: Innate immune mechanisms leading to liver injury subsequent to chronic alcohol ingestion are poorly understood. Natural killer T (NKT) cells, enriched in the liver and comprised of at least two distinct subsets, type I and II, recognize different lipid antigens presented by CD1d molecules. We have investigated whether differential activation of NKT cell subsets orchestrates inflammatory events leading to alcoholic liver disease (ALD). We found that after chronic plus binge feeding of Lieber-DeCarli liquid diet in male C57BL/6 mice, type I, but not type II, NKT cells are activated, leading to recruitment of inflammatory Gr-1(high) CD11b(+) cells into the liver. A central finding is that liver injury after alcohol feeding is dependent upon type I NKT cells. Thus, liver injury is significantly inhibited in Jα18(-/-) mice deficient in type I NKT cells as well as after their inactivation by sulfatide-mediated activation of type II NKT cells. Furthermore, we have identified a novel pathway involving all-trans retinoic acid (ATRA) and its receptor (RARγ) signaling that inhibits type I NKT cells and, consequently, ALD. A semiquantitative polymerase chain reaction analysis of hepatic gene expression of some of the key proinflammatory molecules shared in human disease indicated that their up-regulation in ALD is dependent upon type I NKT cells. CONCLUSIONS: Type I, but not type II, NKT cells become activated after alcohol feeding. Type I NKT cell-induced inflammation and neutrophil recruitment results in liver tissue damage whereas type II NKT cells protect from injury in ALD. Inhibition of type I NKT cells by retinoids or by sulfatide prevents ALD. Given that the CD1d pathway is highly conserved between mice and humans, NKT cell subsets might be targeted for potential therapeutic intervention in ALD.

Recognition of lysophosphatidylcholine by type II NKT cells and protection from an inflammatory liver disease.

Lipids presented by the MHC class I-like molecule, CD1d, are recognized by NK T (NKT) cells, which can be broadly categorized into two subsets. The well-characterized type I NKT cells express a semi-invariant TCR and can recognize both α- and ß-linked glycolipids, whereas type II NKT cells are less well studied, express a relatively diverse TCR repertoire, and recognize ß-linked lipids. Recent structural studies have shown a distinct mode of recognition of a self-glycolipid sulfatide bound to CD1d by a type II NKT TCR. To further characterize Ag recognition by these cells, we have used the structural data and screened other small molecules able to bind to CD1d and activate type II NKT cells. Using plate-bound CD1d and APC-based Ag presentation assay, we found that phospholipids such as lysophosphatidylcholine (LPC) can stimulate the sulfatide-reactive type II NKT hybridoma Hy19.3 in a CD1d-dependent manner. Using plasmon resonance studies, we found that this type II NKT TCR binds with CD1d-bound LPC with micromolar affinities similar to that for sulfatide. Furthermore, LPC-mediated activation of type II NKT cells leads to anergy induction in type I NKT cells and affords protection from Con A-induced hepatitis. These data indicate that, in addition to self-glycolipids, self-lysophospholipids are also recognized by type II NKT cells. Because lysophospholipids are involved during inflammation, our findings have implications for not only understanding activation of type II NKT cells in physiological settings, but also for the development of immune intervention in inflammatory diseases.

Dendritic cells and anergic type I NKT cells play a crucial role in sulfatide-mediated immune regulation in experimental autoimmune encephalomyelitis.

CD1d-restricted NKT cells can be divided into two groups: type I NKT cells use a semi-invariant TCR, whereas type II express a relatively diverse set of TCRs. A major subset of type II NKT cells recognizes myelin-derived sulfatides and is selectively enriched in the CNS tissue during experimental autoimmune encephalomyelitis (EAE). We have shown that activation of sulfatide-reactive type II NKT cells by sulfatide prevents induction of EAE. In this article, we have addressed the mechanism of regulation, as well as whether a single immunodominant form of synthetic sulfatide can treat ongoing chronic and relapsing EAE in SJL/J mice. We have shown that the activation of sulfatide-reactive type II NKT cells leads to a significant reduction in the frequency and effector function of myelin proteolipid proteins 139-151/I-A(s)-tetramer(+) cells in lymphoid and CNS tissues. In addition, type I NKT cells and dendritic cells (DCs) in the periphery, as well as CNS-resident microglia, are inactivated after sulfatide administration, and mice deficient in type I NKT cells are not protected from disease. Moreover, tolerized DCs from sulfatide-treated animals can adoptively transfer protection into naive mice. Treatment of SJL/J mice with a synthetic cis-tetracosenoyl sulfatide, but not α-galactosylceramide, reverses ongoing chronic and relapsing EAE. Our data highlight a novel immune-regulatory pathway involving NKT subset interactions leading to inactivation of type I NKT cells, DCs, and microglial cells in suppression of autoimmunity. Because CD1 molecules are nonpolymorphic, the sulfatide-mediated immune-regulatory pathway can be targeted for development of non-HLA-dependent therapeutic approaches to T cell-mediated autoimmune diseases.

Two cases of successful pregnancies after hysteroscopic removal of endometrioid adenocarcinoma grade I, stage IA, in young women with Lynch syndrome.

We present two cases of endometrioid adenocarcinoma grade I, FIGO IA (staging according to the International Federation of Gynecology and Obstetrics) in young women, diagnosed within endometrial polyps. Both patients underwent repeated hysteroscopies and multiple biopsies after initial treatment to medroxyprogesterone one 400 mg daily or the insertion of IUD-LND (intrauterine device-levonorgestrel) for three months. In both of them, all histological samples were negative. Both of them decided that they would try to conceive. The first patient conceived spontaneously and the second patient after IVF (in vitro fertilisation) treatment. They both gave birth to full-term infants. Hysterectomy was recommended to both of our patients, and was carried out. Both of the patients fulfilled both Amsterdam II and revised Bethesda criteria for hereditary non-polyposis colorectal cancer (HNPCC).

Study on the phenotype and the function of CD8αα+TCRαβ+regulatory T cells / 中华微生物学和免疫学杂志

Objective To investigate the phenotype and the immunoregulatory function of CD8αα+TCRαβ+regulatory T cells in peripheral blood samples from mice.Methods The distribution profile and the phenotype of CD8αα+TCRαβ+regulatory T cells in C57BL/6 mice were detected by flow cytometry.The cytokines released by CD8αα+TCRαβ+regulatory T cells upon the stimulation with anti-CD3 antibody were analyzed by cytometric bead array.The in vitro immunosuppressive activity of CD8αα+TCRαβ+regulatory T cells on activated CD4+T cells was analyzed by using flow cytometry and carboxyfluorescein succinimidyl ester ( CFSE ) .An adoptive cell transfer assay was set up to evaluate the immunoprotective effects of CD8αα+TCRαβ+ regulatory T cells in a mouse model of experimental autoimmune encephalomyelitis ( EAE) .Results CD8αα+TCRαβ+regulatory T cells were detected in liver, spleen and peripheral blood samples collected from na?ve C57BL/6 mice.Compared with CD8αβ+TCRαβ+regulatory T cells, CD8αα+TCRαβ+regulatory T cells showed a memory-activated phenotype of CD25+CD122high CD44high CD62Llow CD69high NK1.1+DX5+.CD8αα+TCRαβ+regulatory T cells could produce IL-2 after 24 hours stimulation with anti-CD3 antibody, followed by producing IFN-γ, TNF-α, IL-4, IL-17A and traces of IL-6 and IL-10. In vitro, CD8αα+TCRαβ+regulatory T cells specifically suppressed the proliferation of activated CD4+T cells ( P<0.01 ).Moreover, they could delay the onset of EAE in mice and reduce clinical score (P<0.01).Conclusion CD8αα+TCRαβ+regulatory T cells were a unique population with immunoregula-tory function, which could be used as a potential therapeutic target in the treatment of autoimmune disease.