CD1d Molecules Research Articles

Structural variants of Bacteroides fragilis-derived alpha-galactosylceramides induce unique natural killer T cell responses.

Abstract Natural killer T (NKT) cell is a unique subset of thymus-derived T cells that recognizes glycolipids presented by CD1d molecule expressed on antigen-presenting cells. The most studied agonistic antigen of NKT cells is alpha-galactosylceramide (αGC). It has been shown that structural features of lipid antigens determine distinct NKT cell responses. In order to drive context-dependent NKT cell responses, understanding the structural characteristics of lipid antigens is important. In the previous study, we have shown that human gut symbiont Bacteroides fragilis (B.fragilis) uniquely produces αGCs (BfaGC), which can normalize the overproliferation of neonatal colonic NKT cells in germ-free mice, and chemically-synthesized structural isomers of BfaGCs induce structure-specific NKT cell responses. In this study, to further investigate the essential structural moiety of BfaGC for the unique immunomodulatory activity, we synthesized BfaGC analogues by modifying their chain lengths and functional groups. We have examined how the analogues differently stimulate NKT cells by analyzing in vitro cytokine production, NKT cell proliferation and effect on specific NKT cell subpopulations. We found the critical features and functional groups on the BfaGC structure leading to immunomodulatory responses. It is expected that our findings may advance further development of lipid antigens to fine-tune context-specific NKT cell responses in immunological diseases.

Abstract 3600: Reversal of iNKT cell dysfunction in cholangiocarcinoma

Abstract Invariant natural killer T (iNKT) cells are CD1d-restricted unconventional T cells possessing innate and adaptive immunity with high anti-tumor activities. Yet, the effectiveness of iNKT cell therapy in clinical trials is limited due to iNKT cell dysfunction after exposure to the tumor microenvironment. Aggressive cholangiocarcinoma (CCA) cell lines were used to study iNKT cell reactivity. These CCA cell lines lacked CD1d molecules and responded poorly to iNKT cells. The iNKT cells became dysfunctional after the exposure to CCA by 48 h. In this study, we overcame iNKT cell dysfunction by searching for a small molecule to prevent or reverse the gene sets regulating iNKT cell dysfunction, thus prolonging anti-tumor activity. It was found that P53 and estrogen early response pathway gene sets were enriched in dysfunctional iNKT cells which were reversed by vorinostat, an HDAC inhibitor. Anti-CCA activity of iNKT cells was regained with a subtherapeutic dose of vorinostat as protection. Interestingly, vorinostat could not totally recover the anti-tumor effect of iNKT cells that have already developed into the dysfunctional stage. Significant tumor size reduction was seen in the humanized mouse model after iNKT cell adoptive therapy was given in combination with vorinostat compared to monotherapy with either iNKT cell or vorinostat alone. This study demonstrated that inhibiting the gene sets regulating iNKT cell dysfunction by vorinostat could prevent iNKT cell dysfunction, and prolong its anti-tumor activities. This combination could be a novel treatment option for CD1d-negative CCA patients resistant to standard chemotherapy. Citation Format: Khin Su Su Htwe, Kitipong Soontrapa, Siwanon Jirawatnotai, Sunisa Prasopporn, Pimkanya More-Krong, Somponnat Sampattavanich, Adisak Wongkajornsilp. Reversal of iNKT cell dysfunction in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3600.

Abstract 1332: Allogeneic CD19-directed CAR-iNKT cells and their phenotypic subsets for the treatment of CD19+ hematological malignancies

Abstract Invariant Natural Killer T (iNKT) cells are a unique subset of innate lymphocytes, constituting <1% of human T cells. iNKT cells express a semi-invariant TCR (iTCR) recognizing glycolipids presented by the monomorphic, MHC-like molecule CD1d. Previous studies indicate that due to distinctive TCR constitution and antigen recognition properties, iNKT cells do not induce acute graft-versus-host disease. Based on the CD4 and CD8 expression, mature human iNKT cells can be classified into CD4+CD8-, CD4-CD8- & CD4-CD8+ subsets with overlapping and distinct functions. The functional profiling of CAR19-iNKT cell subsets serves the dual purpose of ensuring their safety and efficacy as a therapeutic intervention. Here we characterized the phenotypic and functional profile of CD4+ and CD4- CAR19-iNKT cells. iNKT cells were isolated with >99% purity from healthy donors' peripheral blood and were transduced with a CD19 CAR-encoding 3rd generation lentivirus. After expansion, the CAR19-iNKT cells were cryopreserved and later analyzed post-thawing. Differential expression of CD27 and PD1 was noted in CAR+CD4+ and CAR+CD4- iNKT cells compared to other markers. For functional profiling, CAR+CD4+ iNKT cells were positively selected, and 24h cytotoxicity assays were performed using several tumor cell lines including SEM, Ramos and C1R-CD1d (+/- α-GalCer). The CAR+CD4- iNKT cells exhibited superior cytotoxicity to multiple tumor cell lines compared to that of CAR+CD4+ iNKT cells from two donors. However, both subsets lysed α-GalCer pulsed C1R-CD1d cells in a comparable manner indicating the potential of these subsets to recognize antigen through the iTCR. Subsequently, we assessed the proliferation ability of the CAR+CD4+ and CAR+CD4- subsets by exposing the cells to three rounds of stimulation, 24h apart, using SEM (CD19+CD1d-) or K562 (CD19-CD1d-) tumor cells with or without IL-15. Proliferation was assessed seven days following the first stimulation. The CAR+CD4+ subset of CAR19-iNKT cells demonstrated faster proliferation than CAR+CD4- cells in the presence of IL-15 after repeated exposure to tumor cells. Ongoing RNA seq analysis of CAR+CD4+/CAR+CD4- cells will further elucidate differences between these subsets. The outcomes of these studies have shown encouraging results, indicating the potential benefit of having diverse subsets among CAR19-iNKT cells for treating CD19+ cancers. The therapeutic potential of CAR+CD4+ iNKT cell subset could be enhanced to match that of CAR+CD4- subset with the use of α-GalCer. In summary, inclusion of both CD4+ and CD4- iNKT cells is critical for the functionality of allogeneic CAR-iNKT cell therapy. Extensive characterization is crucial for clinical translation, but additional research, including preclinical and clinical trials, is essential to determine the safety and effectiveness of CAR19-iNKT cell therapy in real-world applications. Citation Format: Kanagaraju Ponnusamy, Simon Poon, Nicole van der Weerden, Robson Dossa, Michael J. Baker, Mini Bharathan, Anastasios Karadimitris. Allogeneic CD19-directed CAR-iNKT cells and their phenotypic subsets for the treatment of CD19+ hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1332.

Role of Natural Killer T (NKT) Cells in Myeloma Biology and Therapy.

Natural Killer T (NKT) cells are distinct innate lymphocytes that recognize lipid antigens in the context of nonpolymorphic molecule CD1d. Multiple myeloma (MM) is a hematologic malignancy wherein malignant plasma cells express CD1d and are sensitive to lysis by NKT cells. Progressive malignancy in MM is characterized by NKT cell dysfunction. Several studies have tried to harness the anti-tumor properties of NKT cells in MM to mediate tumor regression. NKT cells are also attractive targets for approaches at immune redirection in MM with chimeric-antigen receptor NKT (CAR-NKT) and bispecific antibodies. In addition to the commonly studied invariant-NKT (iNKT) cells, MM patients often also exhibit alterations in type-II NKT cells and their ligands. In patients and mouse models with Gaucher disease (GD), an inherited lipid-storage disorder with markedly increased risk for MM, distinct type-II NKT cells exhibit a T-follicular helper (NKT-TFH) phenotype and provide help to lipid-specific B cells. Chronic immune activation in this setting eventually sets the stage for malignancy, which can be targeted in both mouse models and GD patients by reducing the underlying antigen. NKT cells are thus integrally linked to MM pathogenesis and an attractive target for MM immunotherapy.

Natural Killer T Cell Diversity and Immunotherapy.

Invariant natural killer T cells (iNKTs), a type of unconventional T cells, share features with NK cells and have an invariant T cell receptor (TCR), which recognizes lipid antigens loaded on CD1d molecules, a major histocompatibility complex class I (MHC-I)-like protein. This interaction produces the secretion of a wide array of cytokines by these cells, including interferon gamma (IFN-γ) and interleukin 4 (IL-4), allowing iNKTs to link innate with adaptive responses. Interestingly, molecules that bind CD1d have been identified that enable the modulation of these cells, highlighting their potential pro-inflammatory and immunosuppressive capacities, as required in different clinical settings. In this review, we summarize key features of iNKTs and current understandings of modulatory α-galactosylceramide (α-GalCer) variants, a model iNKT cell activator that can shift the outcome of adaptive immune responses. Furthermore, we discuss advances in the development of strategies that modulate these cells to target pathologies that are considerable healthcare burdens. Finally, we recapitulate findings supporting a role for iNKTs in infectious diseases and tumor immunotherapy.

Varicella Zoster Virus Downregulates Expression of the Nonclassical Antigen Presentation Molecule CD1d.

The nonclassical antigen presentation molecule CD1d presents lipid antigens to invariant natural killer T (iNKT) cells. Activation of these cells triggers a rapid cytokine response providing an interface between innate and adaptive immune responses. The importance of CD1d and iNKT cells in varicella zoster virus (VZV) infection has been emphasized by clinical reports of individuals with CD1d or iNKT cell deficiencies experiencing severe, disseminated varicella postvaccination. Three strains of VZV (VZV-S, rOka, and VZV rOka-66S) were used to infect Jurkat cells. Flow cytometry of VZV- and mock-infected cells assessed the modulatory impact of VZV on CD1d protein. Infected cell supernatant and transwell co-culture experiments explored the role of soluble factors in VZV-mediated immunomodulation. CD1d transcripts were assessed by reverse-transcription polymerase chain reaction. Surface and intracellular flow cytometry demonstrated that CD1d was strikingly downregulated by VZV-S and rOka in both infected and VZV antigen-negative cells compared to mock. CD1d downregulation is cell-contact dependent and CD1d transcripts are targeted by VZV. Mechanistic investigations using rOka-66S (unable to express the viral kinase ORF66) implicate this protein in CD1d modulation in infected cells. VZV implements multiple mechanisms targeting both CD1d transcript and protein. This provides evidence of VZV interaction with and manipulation of the CD1d-iNKT cell axis.

Type 1 invariant natural killer T cells in chronic inflammation and tissue fibrosis.

Chronic tissue inflammation often results in fibrosis characterized by the accumulation of extracellular matrix components remodeling normal tissue architecture and function. Recent studies have suggested common immune mechanisms despite the complexity of the interactions between tissue-specific fibroblasts, macrophages, and distinct immune cell populations that mediate fibrosis in various tissues. Natural killer T (NKT) cells recognizing lipid antigens bound to CD1d molecules have been shown to play an important role in chronic inflammation and fibrosis. Here we review recent data in both experimental models and in humans that suggest a key role of type 1 invariant NKT (iNKT) cell activation in the progression of inflammatory cascades leading to recruitment of neutrophils and activation of the inflammasome, macrophages, fibroblasts, and, ultimately, fibrosis. Emerging evidence suggests that iNKT-associated mechanisms contribute to type 1, type 2 and type 3 immune pathways mediating tissue fibrosis, including idiopathic pulmonary fibrosis (IPF). Thus, targeting a pathway upstream of these immune mechanisms, such as the inhibition of iNKT activation, may be important in modulating various fibrotic conditions.

Do antigen-presenting CD1a, CD1b, CD1c, and CD1d molecules bind different self-lipids?

Humans express four different lipid antigen-presenting molecules, CD1a, CD1b, CD1c, and CD1d, that are differentially expressed on antigen-presenting cells and which recycle through different endosomal compartments. Huang et al. now answer the question on whether the four CD1 isoforms selectively bind certain lipids.

Anti-CD1d treatment suppresses immunogenic maturation of lung dendritic cells dependent on lung invariant natural killer T cells in asthmatic mice

Our previous findings show that invariant natural killer T (iNKT)cells can promote immunogenic maturation of lung dendritic cells (LDCs) to enhance Th2 cell responses in asthma. It has been accepted that recognition of glycolipid antigens presented by CD1d molecules by the T cell receptors of iNKT cells leads to iNKT cell activation. Therefore, we examine the immunoregulatory influences of anti-CD1d treatment on Th2 cell response and immunogenic maturation of LDCs and subsequently explored whether these influences were dependent on lung iNKT cells in asthmatic mice. We discoveredthat in wild-type mice sensitized and challenged with house dust mite or ovalbumin (OVA), anti-CD1d treatment inhibited Th2 cell response and immunogenic maturation of LDCs. LDCs from asthmatic mice with anti-CD1d treatment had a markedly decreased influence on Th2 cell responses in vivo and in vitro. Furthermore, anti-CD1d treatment reduced the abundance and activation of lung iNKT cells in asthmatic mice. Moreover, in asthmatic iNKT cell-deficient Jα18-/- mice, anti-CD1d treatment did not influence Th2 cell responses and immunogenic maturation of LDCs. Meanwhile, the quantity of CD40L+ iNKT cells in asthmatic mice was significant decreased by anti-CD1d treatment. Finally, the inhibition of anti-CD1d treatment on LDC immunogenic maturation and Th2 cell responses in asthmatic mice was reversed by anti-CD40 treatment. Our data suggest that anti-CD1d treatment can suppress Th2 cell responses through inhibiting immunogenic maturation of LDCs dependent on lung iNKT cells, which couldbe partially related to the downregulation of CD40L expression on lung iNKT cells in asthmatic mice.

An immunostimulatory glycolipid that blocks SARS-CoV-2, RSV, and influenza infections in vivo

Prophylactic vaccines for SARS-CoV-2 have lowered the incidence of severe COVID-19, but emergence of viral variants that are antigenically distinct from the vaccine strains are of concern and additional, broadly acting preventive approaches are desirable. Here, we report on a glycolipid termed 7DW8-5 that exploits the host innate immune system to enable rapid control of viral infections in vivo. This glycolipid binds to CD1d on antigen-presenting cells and thereby stimulates NKT cells to release a cascade of cytokines and chemokines. The intranasal administration of 7DW8-5 prior to virus exposure significantly blocked infection by three different authentic variants of SARS-CoV-2, as well as by respiratory syncytial virus and influenza virus, in mice or hamsters. We also found that this protective antiviral effect is both host-directed and mechanism-specific, requiring both the CD1d molecule and interferon-gamma. A chemical compound like 7DW8-5 that is easy to administer and cheap to manufacture may be useful not only in slowing the spread of COVID-19 but also in responding to future pandemics long before vaccines or drugs are developed.

Understanding NKT cells through sulfatide analogues

Abstract The goal of this research is to further investigate NKT cell activation using sulfatide analogues. NKT cells have been shown to play a significant role in mediating inflammatory immune responses and because of this, there has been strong interest in pharmacologically regulating this compartment to fight cancer. NKT cells recognize lipids that are loaded on CD1d molecules on the surface of cells, and it has been shown that structural modifications of these lipids directly affect NKT cell activation and polarization. Our work is focused on the impact of modifications of sulfatide, which is a glycolipid that can be loaded onto CD1d which is known to activate sulfatide-reactive (generally type II) NKT cells when presented by CD1d monomers on plastic. We have numerous sulfatide analogues with modifications (based on previous work with alpha-galactosylceramide analogues) on the acyl chain, sphingosine chain, and changes of beta-anomeric to alpha-anomeric. These analogues have been screened with NKT cell hybridomas, DN32 and XV19, to assess their effect on activation of type I and type II NKT cells, respectively. We observed that alterations in the sphingosine or acyl chain in sulfatides do not always mimic the effects on activity of similar changes in alpha-galactosylceramide analogues and that numerous sulfatide analogues can activate DN32 (not sulfatide-reactive) when presented on CD1d monomer or bone marrow-derived dendritic cells. Our results provide further insight into NKT cell activation and have identified sulfatide analogues that may be applicable to cancer therapy by activating type I NKT cells. NCI, NIH intramural research program (NIA-C-004020), and institutional grants of NIH (R01 GM111849)

α-GalCer administration expands virtual memory CD8 +T cells through IL-4 secretion

Abstract Virtual memory CD8 +T (CD8 +T VM) cells, a unique subset of CD8 +T cells bearing memory phenotypes, exist in naïve and even in germ-free mice. Distinct from the conventional memory T cells, T VMcells are antigen-inexperienced but able to respond quickly in a cognate antigen-independent manner, thus could provide a broad range of innate-like protective effects. Our previous study showed that T VMcells could be expanded by type 2/interleukin-4 (IL-4) responses. Thus, we used α-galactosylceramide (α-GalCer) to selectively activate invariant natural killer T (iNKT) cells and create a type 2/IL-4 response, which in turn induced robust T VMcell expansion. We further demonstrated that CD1d molecule and NKT cells were necessary for α-GalCer-induced T VMcell expansion, but the T VMcell expansion was only partially dependent on IL-4 in this scenario. It remains unclear how IL-4 induces T VMcell expansion, and what other cytokine(s) may contribute to IL-4-independent T VMcell expansion upon α-GalCer treatment. Among multiple cytokines induced by α-GalCer treatment, we found that in addition to IL-4, IL-15 and type I interferons (IFNs), which have been reported to participate in the development and expansion of T VMcells, were elevated in response to α-GalCer treatment in vivo. We further found that IL-4 and IL-15 but not type I IFNs drove T VMcell proliferation and expansion in vitro. Thus. we speculated that one of the factors that involves in IL-4-independent α-GalCer-induced T VMcell expansion may be IL-15. We are currently testing this hypothesis. Taken together, our results suggested that the activation of iNKT cells by α-GalCer expands T VMcell pool through IL-4 production, thus may regulate the ability of T VMcells to provide broad protection. Supported by grants from MOST (109-2320-B-002-074-MY3), NHRI (EX110/111-11033SI) and NTU (111L7829), Taiwan

Perturbation of Invariant Natural Killer T cells by the Clonal Hematopoiesis-related inflammatory environment

Abstract Invariant Natural Killer T (iNKT) cells are T cells characterized by a semi-invariant TCR and NK markers. They respond to glycolipid antigens presented by the major histocompatibility complex (MHC)-like molecule CD1d. Our single-cell transcriptomic profiling of several tissues identified marrow iNKT as a unique subset, possibly modulating marrow homeostasis. However, little is known about their role in hematological malignancies and how the disease-related environment impacts iNKT cells. For example, clonal hematopoiesis of indeterminate potential (CHIP) corresponds to a condition in which mutated hematopoietic cells have an enhanced fitness and expansion, increasing the risk of leukemia and other diseases. Loss of TET2 in CHIP promotes myeloid skewing and inflammation. TET2 is a methycytosine dioxygenase catalyzing DNA demethylation of genes involved in proliferation, differentiation and oncogenesis. However, the impact that TET2 loss has on iNKT lineage output and function is yet to be defined. In our chimeric mouse model of CHIP, only 15% of TET2 mutants differentiate into iNKT. We also observe a decrease of non-transformed iNKT cells in the marrow of our mice, suggesting environmental inhibition. We expect these bystander iNKT cells to be functionally impaired where inflammation leads to their chromatin rewiring potentially contributing to disease. This is addressed by studying CHIP-exposed iNKT transcriptomes and chromatin accessibility. Understanding the molecular basis of the functional reprogramming by the disease-related inflammation will improve clinical outcomes for marrow transplantation and for the development of cellular therapies using iNKT cells.

The TNF superfamily cytokine receptor Death Receptor 3 is a costimulatory molecule for iNKT cells

Abstract Invariant NKT (iNKT) cells are a small population of thymus-generated T lymphocytes with innate-like phenotype and function that play a disproportionally important role in immune regulation and surveillance. iNKT cells are generated upon strong agonistic TCR signaling by glycolipid-loaded CD1d molecules, which differs from conventional T cells that are positively selected by weak TCR engagement with peptide-loaded MHC molecules. Whether iNKT cells are also distinct in their costimulatory requirement, however, has remained largely unresolved. Here, we report that the TNF superfamily receptor death receptor 3 (DR3) plays a previously unappreciated costimulatory role in peripheral iNKT cell activation. We found that the co-injection of agonistic anti-DR3 antibodies significantly augmented the activation and proliferation of mature iNKT cells in α-GalCer injected mice. Moreover, iNKT cells of α-GalCer and anti-DR3 co-injected mice dramatically increased their expression of proinflammatory cytokines, resulting in severe thymic atrophy. The DR3 costimulatory effect was strictly dependent on iNKT cells, because iNKT-deficient Traj18−/−mice were refractory to α-GalCer and anti-DR3 costimulation-induced inflammation. Because DR3 ligation alone did not suffice to activate iNKT cells, and DR3 exerted its effects only in the context of TCR costimulation, these results strongly support DR3 being a new bona fidecostimulatory molecule for iNKT cells. Considering the interest in utilizing iNKT cells in adoptive T cell therapies and as anti-tumor bioreagents, employing DR3 as a tool to boost iNKT cell immunity will open new venues in translational and clinical research. This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

α-GalCer, an α-Candidate in Tumour Suppression

Alpha-Galactosylceramide (α-GalCer, KRN7000) is an exogenous glycolipid ligand that is presented by CD1d molecules in antigen-presenting cells (APCs). It activatesinvariant natural killer T (iNKT) cells, characterized by semi-invariant T cell receptors (TCRs), which often leads to further downstream activation of the immune system. For example, iNKT cells release cytokines that regulate myeloid-derived suppressor cells (MDSCs) to promote tumor suppression. This critical review aims to clarify the observed effects of α-GalCer by examining recent studies, ranging from in vitro experiments with mice to in vivo clinical trials with humans. Within the current literature, α-GalCer has demonstrated beneficial effects toward tumour suppression. Most pre-clinical studies evaluating α-GalCer have seen success in suppressing tumour growth and increasing patient lifespan, although clinical trials yield inconclusive results. For example, the use of α-GalCer comes with severe limitations, including the induction of immune cell anergy amongst other unwanted side effects. Future studies and trials will be necessary to evaluate the full potential of α-GalCer. Nonetheless, α-GalCer may be a promising agent in combating cancer.

Abstract 690: Modification of mRNA-encoded HPV16 antigens to include endolysosomal trafficking domains drives cross-presentation and results in superior in vivo and ex vivo antigen-specific responses

Abstract Many cancer immunotherapies require the strong activation of the T-cell mediated adaptive immune response to elicit their full anti-tumor potential. Antigen processing and presentation is a complex, multistep process that can be significantly enhanced via antigen modification, leading to better antigen-specific immune responses. Using mRNA, we can express exogenous antigens inside the cell and facilitate the subcellular localization of these antigens to enhance antigen processing and presentation. In this work, we introduced endolysosomal trafficking domains from CD1d molecules to drive subcellular localization of antigenic proteins to the late endosomes (LE) to enhance class I and class II antigen loading. CD1d are molecules that naturally traffic between the membrane and the late endosomes, where antigens are processed and loaded to both class II and class I molecules in a TAP-independent manner. We compared the immunogenicity of a novel endolysosomal trafficking domain, human CD1d, coupled to a chimeric HPV16 E6 and E7 (HPV16E6E7-hCD1d) antigen to antigen designs intended to be either a) secreted (Sec-HPV16E6E7), b) retained in the endoplasmic reticulum (ER) by an ER retention signal KDEL (HPV16E6E7-KDEL) or c) unmodified antigen sequence (HPV16E6E7). To ensure intracellular translation and expression, all antigens were encoded and delivered as mRNAs. The immunogenicity of each antigen design was assessed in HPV16 positive cervical intraepithelial neoplasia (CIN) donor derived PBMCs and healthy controls. Using confocal microscopy, we observed that the hCD1d trafficking domain directed the translated E6E7 protein into late endosomes, which was not observed for any of the other antigen designs. Importantly, antigen localization to late endosomes resulted in significantly improved immunogenicity in vitro as well as in vivo. We observed a consistent increase in numbers of activated antigen-specific T cells across multiple CIN donor PBMCs treated with HPV16E6E7-hCD1d compared to the other 3 designs, as measured by Interferon gamma secretion, surface marker expression, and T-cell proliferation. To assess the generalizability of this approach, we also compared immunogenicity in human donor PBMCs using the human cytomegalovirus antigen pp65, and observed similar results. Lastly, the immunogenicity of each antigen design was tested in vivo by intramuscular injection of nanoparticle formulated mRNAs in C56/Bl6 mice, and inclusion of the murine analogue (mCD1d) resulted in significantly increased generation of HPV16-specific T cell responses. Based on these findings, we are developing a novel mRNA-based cancer therapeutic that includes the addition of hCD1d trafficking domain in the HPV16-specific antigen component. Citation Format: Weiqun Liu, Daniel Fernandez, Colin J. McKinlay, Daniel O. Frimannsson, Meredith L. Leong, W. Martin Kast, Samuel Deutsch, Ole Audun W. Haabeth. Modification of mRNA-encoded HPV16 antigens to include endolysosomal trafficking domains drives cross-presentation and results in superior in vivo and ex vivo antigen-specific responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 690.

α-GalCer, an α-Candidate in Tumour Suppression

Alpha-Galactosylceramide (α-GalCer, KRN7000) is an exogenous glycolipid ligand that is presented by CD1d molecules in antigen-presenting cells (APCs). It activatesinvariant natural killer T (iNKT) cells, characterized by semi-invariant T cell receptors (TCRs), which often leads to further downstream activation of the immune system. For example, iNKT cells release cytokines that regulate myeloid-derived suppressor cells (MDSCs) to promote tumor suppression. This critical review aims to clarify the observed effects of α-GalCer by examining recent studies, ranging from in vitro experiments with mice to in vivo clinical trials with humans. Within the current literature, α-GalCer has demonstrated beneficial effects toward tumour suppression. Most pre-clinical studies evaluating α-GalCer have seen success in suppressing tumour growth and increasing patient lifespan, although clinical trials yield inconclusive results. For example, the use of α-GalCer comes with severe limitations, including the induction of immune cell anergy amongst other unwanted side effects. Future studies and trials will be necessary to evaluate the full potential of α-GalCer. Nonetheless, α-GalCer may be a promising agent in combating cancer.

Immunomodulatory Functions of α-GalCer and a Derivative, α-Carba-GalCer.

Certain glycolipids have immunomodulatory potential by activating natural killer T (NKT) cells, a unique T cell subset. Invariant NKT (iNKT) cells recognize α-galactosylceramide (α-GalCer) and synthetic derivatives presented by CD1d molecules and secrete large amounts of cytokines that modulate immune functions. Some iNKT cell ligands induce distinct reactions biased toward either Th1 or Th2 immune responses, while others show mixed responses. We describe the methods for activating iNKT cells by the ligands as represented by α-GalCer using in vitro assays, such as cell-free or co-culture with antigen-presenting cells. In addition, α-GalCer/CD1d multimer can be used to specifically detect iNKT cells using flow cytometry. α-GalCer is also utilized to activate systemic iNKT cells in vivo, which leads to the production of high levels of cytokines in sera. Collectively, α-GalCer and its derivatives activate iNKT cells that modulate immune balance, and we need to understand the characteristics of these ligands for developing their utility.

Primary Mouse Invariant Natural Killer T (iNKT) Cell Purification and Transduction.

Invariant natural killer T (iNKT) cells are a non-conventional T-cell population expressing a conserved semi-invariant T-cell receptor (TCR) that reacts to lipid antigens, such as α-galactosyl ceramide (α-GalCer), presented by the monomorphic molecule CD1d. iNKT cells play a central role in tumor immunosurveillance and represent a powerful tool for anti-cancer treatment, notably because they can be efficiently redirected against hematological or solid malignancies by engineering with tumor-specific chimeric antigen receptors (CARs) or TCRs. However, iNKT cells are rare and require specific ex vivo pre-selection and substantial in vitro expansion to be exploited for adoptive cell therapy (ACT). This protocol describes a robust method to obtain a large number of mouse iNKT cells that can be effectually engineered by retroviral (RV) transduction. A major advantage of this protocol is that it requires neither particular instrumentation nor a high number of mice. iNKT cells are enriched from the spleens of iVα14-Jα18 transgenic mice; the rapid purification protocol yields a highly enriched iNKT cell population that is activated by anti-CD3/CD28 beads, which is more reproducible and less time consuming than using bone marrow-derived dendritic cells loaded with α-GalCer, without risks of expanding contaminant T cells. Forty-eight hours after activation, iNKT cells are transduced with the selected RV by spin inoculation. This protocol allows to obtain, in 15 days, millions of ready-to-use, highly pure, and stably transduced iNKT cells that might be exploited for in vitro assays and ACT experiments in preclinical studies.

Glycolipids from the gut symbiont Bacteroides fragilis are agonists for natural killer T cells and induce their regulatory differentiation.

Natural Killer T (NKT) cells are a lipid-antigen reactive T cell subset that is restricted to the antigen presenting molecule CD1d. They possess diverse functional properties that contribute to inflammatory and regulatory immune responses. The most studied lipid antigen target for these T cells is α-galactosylceramide (αGC). The commensal organism Bacteroides fragilis (B. fragilis) produces several forms of αGC, but conflicting information exists about the influence of these lipids on NKT cells. Herein, we report the total synthesis of a major form of αGC from B. fragilis (Bf αGC), and several analogues thereof. We confirm the T cell receptor (TCR)-mediated recognition of these glycolipids by mouse and human NKT cells. Despite the natural structure of Bf αGC containing lipid branching that limits potency, we demonstrate that Bf αGC drives mouse NKT cells to proliferate and differentiate into producers of the immunoregulatory cytokine, interleukin-10 (IL-10). These Bf αGC-experienced NKT cells display regulatory function by inhibiting the expansion of naïve NKT cells upon subsequent exposure to this antigen. Moreover, this regulatory activity impacts more than just NKT cells, as demonstrated by the NKT cell-mediated inhibition of antigen-stimulated mucosal-associated invariant T (MAIT) cells (a T cell subset restricted to a different antigen presenting molecule, MR1). These findings reveal that B. fragilis-derived NKT cell agonists may have broad immunoregulatory activity, providing insight into the mechanisms influencing immune tolerance to commensal bacteria and highlighting a potential means to manipulate NKT cell function for therapeutic benefit.