Sustained release system from PLGA particles co-encapsulated with inactivated influenza virus with natural killer T cell agonist α-galactosylceramide.

Vaccines against influenza and many other infectious diseases require multiple boosters in addition to the primary dose to improve efficacy, but this approach is not ideal for compliance. The multiple doses could potentially be replaced by sustained or pulsatile release of antigens encapsulated in degradable microparticles (MPs). The efficacy of a vaccine is improved by adding an adjuvant, which can be co-delivered from the particles to enhance immunogenicity. Here, we developed degradable poly-lactic-co-glycolic acid (PLGA) (7-17 kDa) MPs capable of sustained release of ultraviolet killed influenza virus (A/PR/8/34) (kPR8) vaccine and the natural killer T (NKT) cell agonist alpha-galactosylceramide (α-GalCer) and tested their effectiveness at providing long-term protection against influenza virus infection in mice. Multiple formulations were developed for encapsulating the virus and adjuvant separately, and in combination. The MPs exhibited sustained release of both the virus and the adjuvant lasting more than a month. Co-encapsulation significantly increased the encapsulation efficiency (EE) of the vaccine but reduced the release duration. On the other hand, co-encapsulation led to a reduction in EE for the α-GalCer and a change in release profile to a higher initial burst followed by a linear release compared to a low initial burst and slower linear release. The α-GalCer also had considerably longer release duration compared to the vaccine. Mice injected with particle formulations co-encapsulating kPR8 and α-GalCer were protected from a lethal influenza virus infection 30 weeks after vaccination. This study demonstrates that PLGA MP based vaccines are promising for providing effective vaccination and possibly for replacing multiple doses with a single injection.

Alpha-galactosylceramide improves the potency of mRNA LNP vaccines against cancer and intracellular bacteria.

Although various types of mRNA-based vaccines have been explored, the optimal conditions for induction of both humoral and cellular immunity remain rather unknown. In this study, mRNA vaccines of nucleoside-modified mRNA in lipoplexes (LPXs) or lipid nanoparticles (LNPs) were evaluated after administration in mice through different routes, assessing mRNA delivery, tolerability and immunogenicity. In addition, we investigated whether mRNA vaccines could benefit from the inclusion of the adjuvant alpha-galactosylceramide (αGC), an invariant Natural Killer T (iNKT) cell ligand. Intramuscular (IM) vaccination with ovalbumin (OVA)-encoding mRNA encapsulated in LNPs adjuvanted with αGC showed the highest antibody- and CD8+ T cell responses. Furthermore, we observed that addition of signal peptides and endocytic sorting signals of either LAMP1 or HLA-B7 in the OVA-encoding mRNA sequence further enhanced CD8+ T cell activation although reducing the induction of IgG antibody responses. Moreover, mRNA LNPs with the ionizable lipidoid C12-200 exhibited higher pro-inflammatory- and reactogenic activity compared to mRNA LNPs with SM-102, correlating with increased T cell activation and antitumor potential. We also observed that αGC could further enhance the cellular immunity of clinically relevant mRNA LNP vaccines, thereby promoting therapeutic antitumor potential. Finally, a Listeria monocytogenes mRNA LNP vaccine supplemented with αGC showed synergistic protective effects against listeriosis, highlighting a key advantage of co-activating iNKT cells in antibacterial mRNA vaccines. Taken together, our study offers multiple insights for optimizing the design of mRNA vaccines for disease applications, such as cancer and intracellular bacterial infections.

Unaltered influenza disease outcomes in swine prophylactically treated with α-galactosylceramide.

Influenza A viruses (IAV) are a major cause of respiratory diseases in pigs. Invariant natural killer T (iNKT) cells are an innate-like T cell subset that contribute significantly to IAV resistance in mice. In the current work, we explored whether expanding and activating iNKT cells with the iNKT cell superagonist α-galactosylceramide (α-GalCer) would change the course of an IAV infection in pigs. In one study, α-GalCer was administered to pigs intramuscularly (i.m.) 9 days before infection, which systemically expanded iNKT cells. In another study, α-GalCer was administered intranasally (i.n.) 2 days before virus infection to activate mucosal iNKT cells. Despite a synergistic increase in iNKT cells when α-GalCer i.m. treated pigs were infected with IAV, neither approach reduced disease signs, lung pathology, or virus replication. Our results indicate that prophylactic use of iNKT cell agonists to prevent IAV infection is ineffective in pigs. This is significant because this type of approach has been considered for humans whose iNKT cell levels and IAV infections are more similar to those of pigs than mice.

Natural Killer T-Cell Agonist α-Galactosylceramide and PD-1 Blockade Synergize to Reduce Tumor Development in a Preclinical Model of Colon Cancer.

Murine and human invariant natural killer T (iNKT) lymphocytes are activated by α-galactosylceramide (α-GalCer) presented on CD1d. α-GalCer was first described as a lipid that had strong anti-metastatic effects in a mouse melanoma model, and it has subsequently been shown to induce efficient iNKT cell dependent tumor immunity in several tumor models. We have shown that α-GalCer treatment leads to a weak reduction of polyp burden in the autochthonous ApcMin/+ mouse model for human colon cancer, however this treatment resulted in upregulation of the inhibitory receptor PD-1 on iNKT cells. While anti-PD-1 treatment can prevent immune-suppression in other cancer types, human colon cancer is generally resistant to this treatment. Here we have used the ApcMin/+ model to investigate whether a combined treatment with α-GalCer and PD-1 blockade results in improved effects on polyp development. We find that PD-1 expression was high on T cells in polyps and lamina propria (LP) of ApcMin/+ mice compared to polyp free Apc+/+ littermates. Anti-PD-1 treatment alone promoted Tbet expression in iNKT cells and CD4 T cells, but did not significantly reduce polyp numbers. However, the combined treatment with anti-PD-1 and α-GalCer had synergistic effects, resulting in highly significant reduction of polyp numbers in the small and large intestine. Addition of PD-1 blockade to α-GalCer treatment prevented loss of iNKT cells that were skewed towards a TH1-like iNKT1 phenotype specifically in polyps. It also resulted in TH1 skewing and increased granzyme B expression of CD4 T cells. Taken together this demonstrates that a combination of immune stimulation targeting iNKT cells and checkpoint blockade may be a promising approach to develop for improved tumor immunotherapy.

Targeted Co-delivery of Tumor Antigen and α-Galactosylceramide to CD141+ Dendritic Cells Induces a Potent Tumor Antigen-Specific Human CD8+ T Cell Response in Human Immune System Mice.

Active co-delivery of tumor antigens (Ag) and α-galactosylceramide (α-GalCer), a potent agonist for invariant Natural Killer T (iNKT) cells, to cross-priming CD8α+ dendritic cells (DCs) was previously shown to promote strong anti-tumor responses in mice. Here, we designed a nanoparticle-based vaccine able to target human CD141+ (BDCA3+) DCs - the equivalent of murine CD8α+ DCs - and deliver both tumor Ag (Melan A) and α-GalCer. This nanovaccine was inoculated into humanized mice that mimic the human immune system (HIS) and possess functional iNKT cells and CD8+ T cells, called HIS-CD8/NKT mice. We found that multiple immunizations of HIS-CD8/NKT mice with the nanovaccine resulted in the activation and/or expansion of human CD141+ DCs and iNKT cells and ultimately elicited a potent Melan-A-specific CD8+ T cell response, as determined by tetramer staining and ELISpot assay. Single-cell proteomics further detailed the highly polyfunctional CD8+ T cells induced by the nanovaccine and revealed their predictive potential for vaccine potency. This finding demonstrates for the first time the unique ability of human iNKT cells to license cross-priming DCs in vivo and adds a new dimension to the current strategy of cancer vaccine development.

A phase I study of loco-regional immunotherapy by transbronchial injection of α-galactosylceramide-pulsed antigen presenting cells in patients with lung cancer.

We conducted a phase I study of the trans-bronchial injection of α-galactosylceramide (αGalCer)-pulsed antigen presenting cells (APCs) to evaluate their safety, immune responses, and anti-tumor activities. Patients with advanced or recurrent non-small cell lung cancer (NSCLC) refractory to standard treatments were eligible. αGalCer-pulsed APCs were administered intratumorally or intranodally by bronchoscopy. Twenty-one patients were enrolled in this study. No severe adverse events related to the cell therapy were observed during this study in any patient. After αGalCer-pulsed APCs were administrated, increased iNKT cell numbers were observed in PBMCs from eight cases, and IFN-γ producing cells were increased in the peripheral blood of 10 cases. Regarding clinical responses, one case exhibited a partial response and eight were classified as stable disease. In the tumor microenvironment, IFN-γ expression was upregulated after treatment in partial response or stable disease cases and TGF-ß was upregulated in progressive disease cases.

Activation of Invariant Natural Killer T Cell Subsets in C57BL/6J Mice by Different Injection Modes of α-galactosylceramide.

Whether different injection modes of α-galactosylceramide (α-GalCer) affect the activation of different subsets of invariant natural killer T (iNKT) cells in different tissues and organs of mice is unclear. This study included healthy control, subcutaneous injection, and intraperitoneal injection groups (n=10 in each group). The subcutaneous and intraperitoneal injection groups were injected with α-Galcer (0.1 mg/kg weight), and then the changes in thymus, spleen, and liver iNKT cell frequencies and subsets were observed. The intraperitoneal injection of α-GalCer could increase the frequency of splenic iNKT cells, but the subcutaneous injection did not affect the frequency. Neither injection had any effect on the frequency of iNKT cells in the thymus and liver. The subcutaneous injection of α-GalCer increased the rate of iNKT2 subsets in the thymus but did not affect the rate of iNKT1 subsets. However, the intraperitoneal injection of α-GalCer did not affect thymus iNKT1 and iNKT2 subsets. Interestingly, the subcutaneous injection of α-GalCer significantly increased the proportion of iNKT1 in the spleen and liver but did not significantly change the proportion of iNKT2. The intraperitoneal injection of α-GalCer significantly increased the rate of iNKT2 in spleen and liver but decreased the rate of iNKT1. Subsets of iNKT1 or iNKT2 cells in the spleen and liver were selectively activated by the subcutaneous or intraperitoneal injection of α-GalCer. It provides a valuable means for treating tumors and certain autoimmune diseases. Further exploration of the activation mechanism may provide new ideas about the development of related vaccines.

Blockade of CD40L inhibits immunogenic maturation of lung dendritic cells: Implications for the role of lung iNKT cells in mouse models of asthma.

Some studies have shown that maturation of dendritic cells (DCs) is modulated directly by pathogen components via pattern recognition receptors such as Toll-like receptors, but also by signal like CD40 ligand (CD40 L or CD154) mediated by activated T cells. Several reports indicate that invariant natural killer T (iNKT) cells up-regulate CD40 L upon stimulation and thereby induce activation and maturation of DCs through crosslink with CD40. Our previous findings indicated that iNKT cells promote Th2 cell responses through the induction of immunogenic maturation of lung DCs (LDCs) in the asthmatic murine, but its mechanism remains unclear. Therefore, we investigated the immunomodulatory effects of blockade of CD40 L using anti-CD40 L treatment on Th2 cell responses and immunogenic maturation of LDCs, and further analyzed whether these influences of blockade of CD40 L were related to lung iNKT cells using iNKT cell-deficient mice and the combination treatment of specific iNKT cell activation with anti-CD40 L treatment in murine models of asthma. Our findings showed that blockade of CD40 L using anti-CD40 L treatment attenuated Th2 cell responses in wild-type (WT) mice, but not in CD1d-deficient mice sensitized and challenged with ovalbumin (OVA) or house dust mite (HDM). Meanwhile, blockade of CD40 L down-regulated immunogenic maturation of LDCs in WT mice, but not in CD1d-deficient mice sensitized and challenged with OVA. Additionally, agonistic anti-CD40 treatment reversed the inhibitory effects of anti-CD40 L treatment on Th2 cell responses and LDC activation in an OVA-induced mouse model of asthma. Furthermore, LDCs from asthmatic mice treated with anti-CD40 L could significantly reduce the influence on Th2 cell responses in vivo and in vitro. Finally, α-Galactosylceramide plus anti-CD40 L treatment stimulated lung iNKT cells, but suppressed Th2 cell responses in the asthmatic mice. Taken together, our data raise an evidence that blockade of CD40 L attenuates Th2 cell responses through the inhibition of immunogenic maturation of LDCs, which may be at least partially related to lung iNKT cells in murine models of asthma.

Fully Synthetic Invariant NKT Cell-Dependent Self-Adjuvanting Antitumor Vaccines Eliciting Potent Immune Response in Mice.

Constructing an effective therapeutic cancer vaccine is very attractive and promising for cancer immunotherapy. However, the poor immunogenicity of tumor antigens and suppression of the immune system in the tumor microenvironment are two major obstacles for developing effective cancer vaccines. Invariant NKT cells (iNKT cells), which are essential bridges between the innate and adaptive immune systems, can be rapidly activated by their agonists and, consequently, evoke whole immune systems. Herein, we conjugated a potent agonist of the iNKT cell, α-galactosylceramide (α-GalCer), with the tumor-associated MUC1 glycopeptide antigens as novel self-adjuvanting cancer vaccines through click chemistry. Immunological studies revealed that the mouse immune system was potently evoked and that high levels of tumor-specific IgG antibodies were elicited by vaccine conjugates without an external adjuvant. The produced antibodies could specifically recognize and bind to antigen-expressing cancer cells and, subsequently, induce cytotoxicity through complement-dependent cytotoxicity. Thus, the insertion of α-GalCer significantly improved the immunogenicity of the MUC1 glycopeptide and induced strong antigen-specific antitumor responses, indicating that α-GalCer is an effective built-in adjuvant for constructing potent chemical synthetic antitumor vaccines.

A Novel Liposome Formulation Carrying Both an Insulin Peptide and a Ligand for Invariant Natural Killer T Cells Induces Accumulation of Regulatory T Cells to Islets in Nonobese Diabetic Mice.

Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of pancreatic ß cells by autoantigen-reactive diabetogenic cells. Antigen-specific therapies using islet autoantigens for restoring immune tolerance have emerged as promising approaches for the treatment of T1D but have been unsuccessful in humans. Herein, we report that RGI-3100-iB, a novel liposomal formulation carrying both α-galactosylceramide (α-GalCer), which is a representative ligand for invariant natural killer T (iNKT) cells, and insulin B chain 9-23 peptide, which is an epitope for CD4+ T cells, could induce the accumulation of regulatory T cells (Tregs) in islets in a peptide-dependent manner, followed by the remarkable prevention of diabetes onset in nonobese diabetic (NOD) mice. While multiple administrations of a monotherapy using either α-GalCer or insulin B peptide in a liposomal formulation was confirmed to delay/prevent T1D in NOD mice, RGI-3100-iB synergistically enhanced the prevention effect of each monotherapy and alleviated insulitis in NOD mice. Immunopathological analysis showed that Foxp3+ Tregs accumulated in the islets in RGI-3100-iB-treated mice. Cotransfer of diabetogenic T cells and splenocytes of NOD mice treated with RGI-3100-iB, but not liposomal α-GalCer encapsulating an unrelated peptide, to NOD-SCID mice resulted in the prevention of diabetes and elevation of Foxp3 mRNA expression in the islets. These data indicate that the migration of insulin B-peptide-specific Tregs to islet of NOD mice that are involved in the suppression of pathogenic T cells related to diabetes onset and progression could be enhanced by the administration of liposomes containing α-GalCer and insulin B peptide.

CD160 serves as a negative regulator of NKT cells in acute hepatic injury.

CD160 and BTLA both bind to herpes virus entry mediator. Although a negative regulatory function of BTLA in natural killer T (NKT) cell activation has been reported, whether CD160 is also involved is unclear. By analyzing CD160-/- mice and mixed bone marrow chimeras, we show that CD160 is not essential for NKT cell development. However, CD160-/- mice exhibit severe liver injury after in vivo challenge with α-galactosylceramide (α-GalCer). Moreover, CD160-/- mice are more susceptible to Concanavalin A challenge, and display elevated serum AST and ALT levels, hyperactivation of NKT cells, and enhanced IFN-γ, TNF, and IL-4 production. Lastly, inhibition of BTLA by anti-BTLA mAb aggravates α-GalCer-induced hepatic injury in CD160-/- mice, suggesting that both CD160 and BTLA serve as non-overlapping negative regulators of NKT cells. Our data thus implicate CD160 as a co-inhibitory receptor that delivers antigen-dependent signals in NKT cells to dampen cytokine production during early innate immune activation.

Transnuclear mice reveal Peyer's patch iNKT cells that regulate B-cell class switching to IgG1.

Tissue-resident iNKT cells maintain tissue homeostasis and peripheral surveillance against pathogens; however, studying these cells is challenging due to their low abundance and poor recovery from tissues. We here show that iNKT transnuclear mice, generated by somatic cell nuclear transfer, have increased tissue resident iNKT cells. We examined expression of PLZF, T-bet, and RORγt, as well as cytokine/chemokine profiles, and found that both monoclonal and polyclonal iNKT cells differentiated into functional subsets that faithfully replicated those seen in wild-type mice. We detected iNKT cells from tissues in which they are rare, including adipose, lung, skin-draining lymph nodes, and a previously undescribed population in Peyer's patches (PP). PP-NKT cells produce the majority of the IL-4 in Peyer's patches and provide indirect help for B-cell class switching to IgG1 in both transnuclear and wild-type mice. Oral vaccination with α-galactosylceramide shows enhanced fecal IgG1 titers in iNKT cell-sufficient mice. Transcriptional profiling reveals a unique signature of PP-NKT cells, characterized by tissue residency. We thus define PP-NKT as potentially important for surveillance for mucosal pathogens.

HPV18 E1 Protein Plus α-Galactosylceramide Elicit in Mice CD8+ T Cell Cross-Reactivity Against Cells Expressing E1 from Diverse Human Papillomavirus Types.

CD8+ T cell immune response plays a critical role in the clearance of human papillomavirus (HPV)-infected cells. During the natural history of HPV infection, the E1 protein, an early-expressed helicase highly conserved among papillomaviruses, is involved in the replication of HPV genomes. We have previously shown, in a murine model, that immunization with HPV18 E1 protein combined with α-galactosylceramide elicits a specific CD8+ T cell response. We further proved those findings by analyzing whether CD8+ T cells from mice immunized with α-galactosylceramide plus HPV18 E1 protein could have a cytotoxic effect on cells expressing the carboxyl-terminal domain from the E1 proteins of other HPV types. Interestingly, CD8+ T cells raised against HPV18 E1 antigen presented cross-reactivity against the E1 protein from HPV53, 33, 16, and 31. Poor cross-reactivity was observed for HPV11, and none for HPV6. This outcome may be relevant for the design of broad-spectrum immune-protective agents against HPV infections.

α-GalCer and iNKT Cell-Based Cancer Immunotherapy: Realizing the Therapeutic Potentials.

NKT cells are CD1d-restricted innate-like T cells expressing both T cell receptor and NK cell markers. The major group of NKT cells in both human and mice is the invariant NKT (iNKT) cells and the best-known function of iNKT cells is their potent anti-tumor function in mice. Since its discovery 25 years ago, the prototype ligand of iNKT cells, α-galactosylceramide (α-GalCer) has been used in over 30 anti-tumor clinical trials with mostly suboptimal outcomes. To realize its therapeutic potential, numerous preclinical models have been developed to optimize the scheme and strategies for α-GalCer-based cancer immunotherapies. Nevertheless, since there is no standard protocol for α-GalCer delivery, we reviewed the preclinical studies with a focus on B16 melanoma model in the goal of identifying the best treatment schemes for α-GalCer treatment. We then reviewed the current progress in developing more clinically relevant mouse models for these preclinical studies, most notably the generation of new mouse models with a humanized CD1d/iNKT cell system. With ever-emerging novel iNKT cell ligands, invention of novel α-GalCer delivery strategies and significantly improved preclinical models for optimizing these new strategies, one can be hopeful that the full potential of anti-tumor potential for α-GalCer will be realized in the not too distant future.

Nano spray dryer for vectorizing α-galactosylceramide in polymeric nanoparticles: A single step process to enhance invariant Natural Killer T lymphocyte responses.

The recognition of α-galactosylceramide (αGC), a high-affinity CD1d antigen, by the invariant Natural Killer T (iNKT) lymphocytes results in potent immunostimulatory responses that have been exploited in advanced cancer patients. Therefore, to improve αGC biological activity, several studies vectorized this agonist in PLGA and/or PEG-based nanoparticles. Despite promising findings, these approaches require several steps, from organic solvent decontamination through extrusion in membrane systems. Using a nano spray dryer, we vectorized αGC into a cationic copolymer (dimethylaminoethyl methacrylate, butyl methacrylate and methyl methacrylate - DBM) in a single step process, free of organic solvent. This methodology allowed the production of stable αGC-vectorized nanoparticles (DBM + αGC) with a more potent biological activity than the free agonist. DBM nanoparticles improved in vivo αGC loading into the CD1d molecule and induced a higher frequency of IFN-γ-expressing iNKT cells. Consequently, mice treated with DBM + αGC presented higher levels of serum IFN-γ than those treated with free agonist. Also, vectorized nanoparticles improved αGC ability to control the growth of murine lung metastatic carcinoma. Thus, this is the first study showing that nano spray dryer technology is a simple and alternative approach to enhance iNKT responses.

Alpha-galactosylceramide enhances mucosal immunity to oral whole-cell cholera vaccines.

Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae (V. cholerae) that results in 3-4 million cases globally with 100,000-150,000 deaths reported annually. Mostly confined to developing nations, current strategies to control the spread of cholera include the provision of safe drinking water and improved sanitation and hygiene, ideally in conjunction with oral vaccination. However, difficulties associated with the costs and logistics of these strategies have hampered their widespread implementation. Specific challenges pertaining to oral cholera vaccines (OCVs) include a lack of safe and effective adjuvants to further enhance gut immune responses, the complex and costly multicomponent vaccine manufacturing, limitations of conventional liquid formulation and the lack of an integrated delivery platform. Herein we describe the use of the orally active adjuvant α-Galactosylceramide (α-GalCer) to strongly enhance intestinal bacterium- and toxin-specific IgA responses to the OCV, Dukoral® in C57BL/6 and BALB/c mice. We further demonstrate the mucosal immunogenicity of a novel multi-antigen, single-component whole-cell killed V. cholerae strain and the enhancement of its immunogenicity by adding α-GalCer. Finally, we report that combining these components and recombinant cholera toxin B subunit in the SmPill® minisphere delivery system induced strong intestinal and systemic antigen-specific antibody responses.

Vaccination with human papillomavirus-18 E1 protein plus α-galactosyl-ceramide induces CD8+ cytotoxic response and impairs the growth of E1-expressing tumors.

CD8+ T cell-mediated immune response plays a major role in the clearance of virus-infected cells, including human papillomavirus (HPV). The effective treatment of women with normal cytology but persistent high risk-HPV infection or with low-grade intraepithelial lesions could take advantage of novel strategies based on vaccination with viral immunological targets with a wide spectrum of cross-protection. The helicase E1, expressed early during viral replication in HPV infection, is among the most conserved papillomavirus proteins, which makes it a good vaccine candidate. In the present study, we examined E1-specific CD8+ T cell and NK immune responses in a mouse model with α-galactosylceramide (α-GalCer) as an adjuvant. We found that mice immunized with E1 combined with α-GalCer elicited an E1-specific CD8+ T and NK cell cytotoxic responses, which correlated with growth inhibition of grafted melanoma B16-F0 cells expressing E1, both in prophylactic and therapeutic protocols.

Tumor-Targeted Chemoimmunotherapy with Immune-Checkpoint Blockade for Enhanced Anti-Melanoma Efficacy.

Chemoimmunotherapy with chemotherapeutics and immunoadjuvant inhibits tumor growth by activating cytotoxic T cells. However, this process also upregulates the expression of PD-1/PD-L1 and consequently leads to immune suppression. To maximize the anti-tumor immune responses and alleviate immunosuppression, PD-L1 antibody was combined with paclitaxel (PTX) and the immunoadjuvant α-galactosylceramide (αGC), which were coencapsulated into pH-sensitive TH peptide-modified liposomes (PTX/αGC/TH-Lip) to treat melanoma and lung metastasis. Compared to treatment with PD-L1 antibody or PTX/αGC/TH-Lip alone, the combination of PD-L1 antibody and PTX/αGC/TH-Lip further elevated the tumor-specific cytotoxic T cell responses and promoted apoptosis in tumor cells, leading to enhanced anti-tumor and anti-metastatic effects. In adoptive therapy, PD-L1 antibody further alleviated immunosuppression and enhanced the anti-tumor effect of CD8+ T cells. The combination of PD-L1 antibody and chemoimmunotherapy PTX/αGC/TH-Lip provides a promising strategy for enhancing treatment for melanoma and lung metastasis.

Deletion of C-C Motif Chemokine Ligand 5 Worsens Invariant Natural Killer T-Cell-Mediated Hepatitis via Compensatory Up-regulation of CXCR2-Related Chemokine Activity.

BACKGROUND & AIMS: Chemokine-mediated immune cell recruitment plays pivotal roles in liver inflammation. C-C motif chemokine ligand 5 (CCL5) has been shown to be responsible for the recruitment of monocytes/macrophages and has been implicated in various liver diseases, including nonalcoholic fatty liver disease, fibrosis, and hepatocellular carcinoma. Previous studies have also shown that inhibition of CCL5 appears to be a promising therapeutic approach for several chronic liver diseases. However, whether blocking CCL5 could benefit immune cell-mediated hepatitis remains largely elusive. METHODS: By adopting a specific agonist, alpha-galactosylceramide (α-Galcer), of invariant natural killer T cells (iNKTs), we investigated the function and mechanism of CCL5 in the iNKT induced murine hepatitis model. RESULTS: We found significantly increased CCL5 expression in α-Galcer-induced hepatitis murine model. Such an increase in CCL5 is mainly enriched in non-parenchymal cells such as macrophages and iNKTs but not in hepatocytes. Surprisingly, CCL5 blockage by genetic deletion of Ccl5 does not affect the α-Galcer-induced iNKT activation but greatly worsens α-Galcer-induced liver injury accompanied by an increased hepatic neutrophil infiltration. Mechanistically, we demonstrated that greater neutrophil accumulation in the liver is responsible for the enhanced liver injury in Ccl5-/- mice. Such an increased hepatic neutrophil infiltration is mainly caused by an enhanced CXCL1-CXCR2 signal in Ccl5-/- mice. Therapeutically, either antibody-mediated neutrophil depletion or a CXCR2 antagonist, SB225002, mediated CXCR2 signaling blockage significantly ameliorated α-Galcer-induced liver injury in Ccl5-/- mice. CONCLUSIONS: Our present study demonstrates that (1) α-Galcer-induced murine hepatitis could greatly induce CCL5 production in macrophages and iNKT cells; (2) loss of CCL5 could enhance CXCL1 expression in hepatocytes and activate CXCL1-CXCR2 axis in neutrophils to augment their hepatic infiltration; and (3) neutrophil depletion or blockage of CXCL1-CXCR2 axis greatly improves α-Galcer-induced liver injury in Ccl5-/- mice. This study suggests that clinical utilization of CCL5 blockage may compensatorily induce the activation of other chemokine pathways to enhance neutrophil recruitment and liver injury in hepatitis.

Graphene oxide polarizes iNKT cells for production of TGFß and attenuates inflammation in an iNKT cell-mediated sepsis model.

Graphene oxide (GO) modulates the functions of antigen-presenting cells including dendritic cells (DCs). Although carbon nanotubes affect expression of the MHC class I-like CD1d molecule, whether GO can influence immune responses of CD1d-dependent invariant natural killer T (iNKT) cells remains unclear. Here, we investigated the impact of GO on inflammatory responses mediated by α-galactosylceramide (α-GalCer), an iNKT cell agonist. We found that in vivo GO treatment substantially inhibited the capacity of α-GalCer to induce the iNKT cell-mediated trans-activation of and cytokine production by innate and innate-like cells, including DCs, macrophages, NK cells, and γδ T cells. Such effects of GO on α-GalCer-induced inflammatory responses closely correlated with iNKT cell polarization towards TGFß production, which also explains the capacity of GO to expand regulatory T cells. Interestingly, the absence of TLR4, a receptor for GO, failed to downregulate, and instead partially enhanced the anti-inflammatory activity of GO against α-GalCer-elicited responses, implying negative effects of TLR4 signaling on the anti-inflammatory properties of GO. By employing an α-GalCer-induced sepsis model, we further demonstrated that GO treatment significantly protected mice from α-GalCer-induced lethality. Taken together, we provide strong evidence that GO holds promise as an adjuvant to modulate iNKT cell responses for immunotherapy.