iNKT Cell Activation Research Articles

Synthesis of sulfoCy5‐ and sulfoCy7‐αGalCer Probes as Chemical Tools for Investigating the Uptake of Liposomal αGalCer Conjugates by Antigen Presenting Cells

α‐Galactosylceramide (αGalCer) is a synthetic glycolipid that, when loaded into the CD1d receptor of antigen presenting cells, activates iNKT cells to coordinate the generation of both innate and adaptive immune responses. In vaccines, the αGalCer adjuvant is generally delivered in liposomes, but interestingly, little is known about the intracellular fate of αGalCer–antigen conjugates resulting in the absence of clear “design rules” for this type of compounds and their formulations. To unravel the uptake mechanism of liposomal αGalCer conjugates and understand the fate of its components, we synthesized sulfoCy7–αGalCer 1 and sulfoCy5–αGalCer 2. The fluorescent probes were obtained from a linker–functionalized αGalCer, which was then modified with sulfoCy7/sulfoCy5. After formulation in liposomes, the cellular presentation was studied in a murine dendritic cell line using fluorescence imaging and flow cytometry. Imaging indicates that liposomes loaded with sulfoCy5–αGalCer are taken up by DC2.4 cells in a heterogeneous and concentration‐dependent manner. Additionally, fluorescence antibody staining confirms that αGalCer is ultimately presented by CD1d receptors, but importantly is cleaved from sulfoCy5 before reaching the cell surface. These results validate our synthetic probes as useful in mechanistic studies of the uptake of αGalCer–antigen conjugates as shown here for DC2.4 cells.

TIPE2 protein restrains invariant NKT activation and protects against immune-mediated hepatitis in mice.

Concanavalin A (ConA) administration induces a rapid and severe liver injury in mice, and invariant natural killer T (iNKT) cells are recognized to be the key effector cells in this process. However, the underlying regulatory mechanisms are not well defined. We found that iNKT cells constitutively expressed TIPE2 (Tumor necrosis factor-α-induced protein 8-like 2, or TNFAIPL2). Genetic TIPE2 ablation strongly sensitized mice to ConA-induced hepatitis, accompanied with hyperactivation of iNKT cells. Moreover, Tipe2-/- mice were also more susceptible to α-galactosylceramide (αGalCer)-induced liver injury, with elevated serum ALT level and enhanced proinflammatory cytokine production. CD1d signaling blockade or iNKT cell elimination through antibodies reduced the effect of TIPE2 deficiency on liver injury. Mechanistic studies revealed that TIPE2 in iNKT cells functioned as a negative regulator, limiting iNKT cell activity and cytokine production through PIP3- AKT/mTOR pathway. TIPE2-mediated protection from liver injury was further validated by the administration of adeno-associated viruses expressing TIPE2, which effectively ameliorated ConA-induced hepatic injury. However, TIPE2 was dispensable in two other liver injury models, including D-GalN/LPS and APAP-induced hepatitis. Our findings reveal a new role of TIPE2 in the attenuation of iNKT cell-mediated hepatic injury. We propose that TIPE2 serves as an important regulator of immune homeostasis in the liver, and might be exploited for the therapeutic treatment of autoimmune liver diseases.

The iNKT cell ligand α-GalCer prevents murine septic shock by inducing IL10-producing iNKT and B cells.

α-galactosylceramide (α-GalCer), a prototypical agonist of invariant natural killer T (iNKT) cells, stimulates iNKT cells to produce various cytokines such as IFNγ and IL4. Moreover, repeated α-GalCer treatment can cause protective or pathogenic outcomes in various immune-mediated diseases. However, the precise role of α-GalCer-activated iNKT cells in sepsis development remains unclear. To address this issue, we employed a lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced murine sepsis model and two alternative models. Sepsis was induced in wild-type (WT) C57BL/6 (B6) mice by three methods (LPS/D-GalN, α-GalCer/D-GalN, and cecal slurry), and these mice were monitored for survival rates. WT B6 mice were intraperitoneally injected with α-GalCer or OCH (an IL4-biased α-GalCer analog) one week prior to the induction of sepsis. To investigate the effects of α-GalCer-mediated iNKT cell activation on sepsis development, immune responses were analyzed by flow cytometry using splenocytes and liver-infiltrating leukocytes. In addition, a STAT6 inhibitor (AS1517499) and an IL10 inhibitor (AS101) were employed to evaluate the involvement of IL4 or IL10 signaling. Furthermore, we performed B cell adoptive transfers to examine the contribution of α-GalCer-induced regulatory B (Breg) cell populations in sepsis protection. In vivo α-GalCer pretreatment polarized iNKT cells towards IL4- and IL10-producing phenotypes, significantly attenuating LPS/D-GalN-induced septic lethality in WT B6 mice. Furthermore, α-GalCer pretreatment reduced the infiltration of immune cells to the liver and attenuated pro-inflammatory cytokine production. Treatment with a STAT6 inhibitor was unable to modulate disease progression, indicating that IL4 signaling did not significantly affect iNKT cell-mediated protection against sepsis. This finding was confirmed by pretreatment with OCH, which did not alter sepsis outcomes. However, interestingly, prophylactic effects of α-GalCer on sepsis were significantly suppressed by treatment with an IL10 antagonist, suggesting induction of IL10-dependent anti-inflammatory responses. In addition to IL10-producing iNKT cells, IL10-producing B cell populations were significantly increased after α-GalCer pretreatment. Overall, our results identify α-GalCer-mediated induction of IL10 by iNKT and B cells as a promising option for controlling the pathogenesis of postoperative sepsis.

An intranasal cationic liposomal polysaccharide vaccine elicits humoral immune responses against Streptococcus pneumoniae

Diseases caused by S. pneumoniae are the leading cause of child mortality. As antibiotic resistance of S. pneumoniae is rising, vaccination remains the most recommended solution. However, the existing pneumococcal polysaccharides vaccine (Pneumovax® 23) proved only to induce T-independent immunity, and strict cold chain dependence of the protein conjugate vaccine impedes its promotion in developing countries, where infections are most problematic. Affordable and efficient vaccines against pneumococcus are therefore in high demand. Here, we present an intranasal vaccine Lipo+CPS12F&αGC, containing the capsular polysaccharides of S. pneumoniae 12F and the iNKT agonist α-galactosylceramide in cationic liposomes. In BALB/cJRj mice, the vaccine effectively activates iNKT cells and promotes B cells maturation, stimulates affinity-matured IgA and IgG production in both the respiratory tract and systemic blood, and displays sufficient protection both in vivo and in vitro. The designed vaccine is a promising, cost-effective solution against pneumococcus, which can be expanded to cover more serotypes and pathogens.

Inhibiting iNKT Cell Activation: A Promising Strategy Against Pulmonary Fibrosis.

Inhibiting iNKT Cell Activation: A Promising Strategy Against Pulmonary Fibrosis.

Traversing the bench to bedside journey for iNKT cell therapies.

Invariant natural killer T (iNKT) cells are immune cells that harness properties of both the innate and adaptive immune system and exert multiple functions critical for the control of various diseases. Prevention of graft-versus-host disease (GVHD) by iNKT cells has been demonstrated in mouse models and in correlative human studies in which high iNKT cell content in the donor graft is associated with reduced GVHD in the setting of allogeneic hematopoietic stem cell transplants. This suggests that approaches to increase the number of iNKT cells in the setting of an allogeneic transplant may reduce GVHD. iNKT cells can also induce cytolysis of tumor cells, and murine experiments demonstrate that activating iNKT cells in vivo or treating mice with ex vivo expanded iNKT cells can reduce tumor burden. More recently, research has focused on testing anti-tumor efficacy of iNKT cells genetically modified to express a chimeric antigen receptor (CAR) protein (CAR-iNKT) cells to enhance iNKT cell tumor killing. Further, several of these approaches are now being tested in clinical trials, with strong safety signals demonstrated, though efficacy remains to be established following these early phase clinical trials. Here we review the progress in the field relating to role of iNKT cells in GVHD prevention and anti- cancer efficacy. Although the iNKT field is progressing at an exciting rate, there is much to learn regarding iNKT cell subset immunophenotype and functional relationships, optimal ex vivo expansion approaches, ideal treatment protocols, need for cytokine support, and rejection risk of iNKT cells in the allogeneic setting.

Alpha-galactosylceramide pre-treatment attenuates clinical symptoms of LPS-induced acute neuroinflammation by converting pathogenic iNKT cells to anti-inflammatory iNKT10 cells in the brain.

Invariant natural killer T (iNKT) cells play protective or pathogenic roles in a variety of immune and inflammatory diseases. However, whether iNKT cells contribute to the progression of acute neuroinflammation remains unclear. Thus, we addressed this question with a mouse model of lipopolysaccharide (LPS)-induced acute neuroinflammation. For induction of acute neuroinflammation, wild-type (WT) C57BL/6 (B6) mice were injected intraperitoneally (i.p.) with LPS for either three or five consecutive days, and then these mice were analyzed for brain-infiltrating leukocytes or mouse behaviors, respectively. To examine the role of iNKT cell activation in LPS-induced neuroinflammation, mice were injected i.p. with the iNKT cell agonist α-galactosylceramide (α-GalCer) seven days prior to LPS treatment. Immune cells infiltrated into the brain during LPS-induced neuroinflammation were determined by flow cytometry. In addition, LPS-induced clinical behavior symptoms such as depressive-like behavior and memory impairment in mice were evaluated by the open field and Y-maze tests, respectively. We found that iNKT cell-deficient Jα18 mutant mice display delayed disease progression and decreased leukocyte infiltration into the brain compared with WT mice, indicating that iNKT cells contribute to the pathogenesis of LPS-induced neuroinflammation. Since it has been reported that pre-treatment with α-GalCer, an iNKT cell agonist, can convert iNKT cells towards anti-inflammatory phenotypes, we next explored whether pre-activation of iNKT cells with α-GalCer can regulate LPS-induced neuroinflammation. Strikingly, we found that α-GalCer pre-treatment significantly delays the onset of clinical symptoms, including depression-like behavior and memory impairment, while decreasing brain infiltration of pro-inflammatory natural killer cells and neutrophils, in this model of LPS-induced neuroinflammation. Such anti-inflammatory effects of α-GalCer pre-treatment closely correlated with iNKT cell polarization towards IL4- and IL10-producing phenotypes. Furthermore, α-GalCer pre-treatment restored the expression of suppressive markers on brain regulatory T cells during LPS-induced neuroinflammation. Our findings provide strong evidence that α-GalCer-induced pre-activation of iNKT cells expands iNKT10 cells, mitigating depressive-like behaviors and brain infiltration of inflammatory immune cells induced by LPS-induced acute neuroinflammation. Thus, we suggest the prophylactic potential of iNKT cells and α-GalCer against acute neuroinflammation.

Glycolipid antigen recognition by invariant natural killer T cells and its role in homeostasis and antimicrobial responses.

Due to the COVID-19 pandemic, the importance of developing effective vaccines has received more attention than ever before. To maximize the effects of vaccines, it is important to select adjuvants that induce strong and rapid innate and acquired immune responses. Invariant natural killer T (iNKT) cells, which constitute a small population among lymphocytes, bypass the innate and acquired immune systems through the rapid production of cytokines after glycolipid recognition; hence, their activation could be used as a vaccine strategy against emerging infectious diseases. Additionally, the diverse functions of iNKT cells, including enhancing antibody production, are becoming more understood in recent years. In this review, we briefly describe the functional subset of iNKT cells and introduce the glycolipid antigens recognized by them. Furthermore, we also introduce novel vaccine development taking advantages of iNKT cell activation against infectious diseases.

Studying the cellular basis of small bowel enteropathy using high-parameter flow cytometry in mouse models of primary antibody deficiency.

Primary immunodeficiencies are heritable defects in immune system function. Antibody deficiency is the most common form of primary immunodeficiency in humans, can be caused by abnormalities in both the development and activation of B cells, and may result from B-cell-intrinsic defects or defective responses by other cells relevant to humoral immunity. Inflammatory gastrointestinal complications are commonly observed in antibody-deficient patients, but the underlying immune mechanisms driving this are largely undefined. In this study, several mouse strains reflecting a spectrum of primary antibody deficiency (IgA-/-, Aicda-/-, CD19-/- and JH -/-) were used to generate a functional small-bowel-specific cellular atlas using a novel high-parameter flow cytometry approach that allows for the enumeration of 59 unique cell subsets. Using this cellular atlas, we generated a direct and quantifiable estimate of immune dysregulation. This estimate was then used to identify specific immune factors most predictive of the severity of inflammatory disease of the small bowel (small bowel enteropathy). Results from our experiments indicate that the severity of primary antibody deficiency positively correlates with the degree of immune dysregulation that can be expected to develop in an individual. In the SI of mice, immune dysregulation is primarily explained by defective homeostatic responses in T cell and invariant natural killer-like T (iNKT) cell subsets. These defects are strongly correlated with abnormalities in the balance between protein (MHCII-mediated) versus lipid (CD1d-mediated) antigen presentation by intestinal epithelial cells (IECs) and intestinal stem cells (ISCs), respectively. Multivariate statistical approaches can be used to obtain quantifiable estimates of immune dysregulation based on high-parameter flow cytometry readouts of immune function. Using one such estimate, we reveal a previously unrecognized tradeoff between iNKT cell activation and type 1 immunity that underlies disease in the small bowel. The balance between protein/lipid antigen presentation by ISCs may play a crucial role in regulating this balance and thereby suppressing inflammatory disease in the small bowel.

Abstract 4110: Identification of tumor peptides for therapeutic mRNA vaccination in a mouse multiple myeloma model

Abstract Background: Multiple myeloma (MM) is a plasma cell cancer for which immunotherapy holds the promise of durable control. To date, cancer vaccines mainly focused on tumor-associated antigens with survivin eliciting protective T-cell responses in both preclinical and clinical studies. Given the success of mRNA vaccines in medicine, we developed Galsomes, lipid nanoparticles containing mRNA and α-galactosyl ceramide to activate both CD8+ T cells and invariant natural killer (iNK) T cells, both potential anti-MM effectors. Materials and Methods: Mouse 5T33MM cells were studied by PCR and flow cytometry (FCM) for expression of survivin and CD1d, respectively. Intravenous (IV) prime-boost Galsome vaccination was performed with a 7-day interval in C57BL6/KalwRij mice that received an IV injection of 5x106 5T33MM cells 3 days earlier. As a control, mice were injected with saline. After 2 weeks, MM burden was studied by serum electrophoresis and FCM, incl. expression of CD1d and PD-L1 on MM cells. Activation of iNKT cells and survivin-specific T cells was studied by measuring IFN-γ secretion in ELISA and dextramer staining. Immunopeptidomics was used to identify new vaccine candidates for which Galsome vaccination is ongoing. Result: The 5T33MM cells showed expression of survivin and CD1d, making it a suitable model for Galsome vaccination. Two weeks following prime-boost vaccination, the serum M-protein was reduced, though not significantly. In contrast, MM cells were significantly reduced in bone marrow and spleen. Expression of CD1d and PD-L1 on MM cells was high but unchanged by Galsome vaccination. Activation of iNKT cells and survivin-specific T cells following prime vaccination was shown. However, following boost vaccination these cell populations reduced significantly compared to control. We repeated prime-boost vaccination in healthy mice, observing a similar outcome. This suggests that the negative effect of the boost Galsome vaccination was not related to immune dysfunctionality due to MM progression. So, we studied the impact of survivin mRNA in the vaccine, as survivin is also expressed in activated T cells, making them a potential target. We observed that iNKT cells and ovalbumin-specific T cells were more reduced when ovalbumin mRNA was paired with survivin mRNA in the Galsomes. To identify other vaccine candidates, we studied peptides eluted from MHC-I/peptide complexes on 5T33MM cells. We selected 4 vaccine candidates including the MM idiotype based on immunogenicity, pathway classification and abundance. mRNA and Galsomes were prepared, and the efficacy of these Galsomes in 5T33MM mice is currently under evaluation. Conclusion: These data show that Galsome vaccination has the potential to reduce MM cell levels. To reach higher efficacy, careful selection of the vaccine antigen is required. Moreover, combination with other immunotherapies might be needed to reach durable disease control. Citation Format: Arne Van der Vreken, Fabien Thery, Sofie Meulewaeter, Karin Vanderkerken, Elke De Bruyne, Kim De Veirman, Lorenzo Franceschini, Rein Verbeke, Francis Impens, Ine Lentacker, Karine Breckpot, Eline Menu. Identification of tumor peptides for therapeutic mRNA vaccination in a mouse multiple myeloma model [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 4110.

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.

A Humanized Mouse Model Coupled with Computational Analysis Identifies Potent Glycolipid Agonist of Invariant NKT Cells.

Invariant natural killer T (iNKT) cells play an important role in many innate and adaptive immune responses, with potential applications in cancer immunotherapy. The glycolipid KRN7000, an α-galactosylceramide, potently activates iNKT cells but has shown limited anticancer effects in human clinical trials conducted so far. In spite of almost three decades of structure-activity relationship studies, no alternative glycolipid has yet emerged as a superior clinical candidate. One reason for the slow progress in this area is that standard mouse models do not accurately reflect the specific ligand recognition by human iNKT cells and their requirements for activation. Here we evaluated a series of KRN7000 analogues using a recently developed humanized mouse model that expresses a human αTCR chain sequence and human CD1d. In this process, a more stimulatory, previously reported but largely overlooked glycolipid was identified, and its activity was probed and rationalized via molecular simulations.

Motility and tumor infiltration are key aspects of invariant natural killer T cell anti-tumor function

Dysfunction of invariant natural killer T (iNKT) cells contributes to immune resistance of tumors. Most mechanistic studies focus on their static functional status before or after activation, not considering motility as an important characteristic for antigen scanning and thus anti-tumor capability. Here we show via intravital imaging, that impaired motility of iNKT cells and their exclusion from tumors both contribute to the diminished anti-tumor iNKT cell response. Mechanistically, CD1d, expressed on macrophages, interferes with tumor infiltration of iNKT cells and iNKT-DC interactions but does not influence their intratumoral motility. VCAM1, expressed by cancer cells, restricts iNKT cell motility and inhibits their antigen scanning and activation by DCs via reducing CDC42 expression. Blocking VCAM1-CD49d signaling improves motility and activation of intratumoral iNKT cells, and consequently augments their anti-tumor function. Interference with macrophage-iNKT cell interactions further enhances the anti-tumor capability of iNKT cells. Thus, our findings provide a direction to enhance the efficacy of iNKT cell-based immunotherapy via motility regulation.

Overcoming resistance to programmed cell death protein 1 (PD-1) blockade with allogeneic invariant natural killer T-cells (iNKT).

Gastric cancer is the 5th most common malignancy worldwide with only 36% of patients with metastatic disease surviving beyond 5 years. Despite therapeutic improvements with the advent of immune checkpoint inhibitors, most patients with gastric cancer develop disease progression related to tumor resistance. Novel immunotherapeutic approaches, including invariant natural killer (iNKT) cells, are in clinical development and represent potential therapeutic options to overcome resistance. AgenT-797 is an allogeneic human unmodified iNKT derived from healthy donors. Activation of iNKT cells by tumor lipid antigens can trigger direct cytotoxicity and promote indirect anti-tumor immune responses such as recruitment and activation of T cells, NK cells, and dendritic cells through secretion of cytokines and IFNγ. We describe immune modulation leading to durable tumor response in a patient with microsatellite instability-high (MSI-H) advanced gastric adenocarcinoma treated with agent-797 after progression on standard chemotherapy and anti-PD-1 therapy.

Linkage-Editing Pseudo-Glycans: A Reductive α-Fluorovinyl-C-Glycosylation Strategy to Create Glycan Analogs with Altered Biological Activities.

The acetal (O-glycoside) bonds of glycans and glycoconjugates are chemically and biologically vulnerable, and therefore C-glycosides are of interest as more stable analogs. We hypothesized that, if the O-glycoside linkage plays a vital role in glycan function, the biological activities of C-glycoside analogs would vary depending on their substituents. Based on this idea, we adopted a "linkage-editing strategy" for the creation of glycan analogs (pseudo-glycans). We designed three types of pseudo-glycans with CH2 and CHF linkages, which resemble the O-glycoside linkage in terms of bond lengths, angles, and bulkiness, and synthesized them efficiently by means of fluorovinyl C-glycosylation and selective hydrogenation reactions. Application of this strategy to isomaltose (IM), an inducer of amylase expression, and α-GalCer, which activates iNKT cells, resulted in the discovery of CH2-IM, which shows increased amylase production ability, and CHF-α-GalCer, which shows activity opposite that of native α-GalCer, serving as an antagonist of iNKT cells.

T Cell Receptor-Engaging Monoclonal Antibodies Mobilize the Anti-Tumor Functions of Invariant Natural Killer T Cells.

Invariant natural killer T cells (iNKTs) are innate-type T lymphocytes that directly kill tumor cells or tumor-growth promoting immunosuppressive cells such astumor-associated macrophages. Additionally, iNKTs robustly transactivate the antitumor functions of T, B, natural killer, and dendritic cells as well as reinvigorate exhausted immune cells in the tumor microenvironment. As such, iNKTs make excellent candidates for inclusion in anti-cancer cellular therapies. However, to capitalize on the potential benefits of iNKT cell-based approaches, it is imperative that we develop new and clinically viable strategies to enhance their antitumor function. To that end, two novel monoclonal antibodies (mAbs) that selectively bind to the human (NKTT320) or murine (NKT14m) invariant T cell receptor have been recently developed and characterized. Studies using purified human iNKTs (in vitro) and a model of non-human primate (in vivo) reveal that NKTT320 promotes swift, vigorous and sustained iNKT cell activation that is accompanied by robust production of inflammatory mediators and bystander immune cell activation. Furthermore, NKTT320 augments expression of cytotoxic markers and human iNKT cell degranulation. Similarly, NKT14m prompts dramatic murine iNKT cell activation and functional response both in vitro and in vivo. However, antitumor efficacy of a single dose of NKT14m injection in tumor-bearing mice is limited and tumor-model dependent. In contrast, combination treatment of NKT14m with either low dose interleukin (IL)-12 or the chemotherapeutic agent, cyclophosphamide results in a superior antitumor response in vivo. This is evident by activation of both iNKTs and other immune cells, prolonged survival of the tumor-challenged mice, and long-lasting immunity. Collectively, these recent studies justify further development of anti-iTCR mAbs that can be used alone or in conjunction with immunomodulatory agents to enhance iNKT cell antitumor immunity against various cancers.

Diversification of a Novel α-Galactosyl Ceramide Hotspot Boosts the Adjuvant Properties in Parenteral and Mucosal Vaccines.

The development of potent adjuvants is an important step for improving the performance of subunit vaccines. CD1d agonists, such as the prototypical α-galactosyl ceramide (α-GalCer), are of special interest due to their ability to activate iNKT cells and trigger rapid dendritic cell maturation and B-cell activation. Herein, we introduce a novel derivatization hotspot at the α-GalCer skeleton, namely the N-substituent at the amide bond. The multicomponent diversification of this previously unexplored glycolipid chemotype space permitted the introduction of a variety of extra functionalities that can either potentiate the adjuvant properties or serve as handles for further conjugation to antigens toward the development of self-adjuvanting vaccines. This strategy led to the discovery of compounds eliciting enhanced antigen-specific T cell stimulation and a higher antibody response when delivered by either the parenteral or the mucosal route, as compared to a known potent CD1d agonist. Notably, various functionalized α-GalCer analogues showed a more potent adjuvant effect after intranasal immunization than a PEGylated α-GalCer analogue previously optimized for this purpose. Ultimately, this work could open multiple avenues of opportunity for the use of mucosal vaccines against microbial infections.

Diversification of a Novel α‐Galactosyl Ceramide Hotspot Boosts the Adjuvant Properties in Parenteral and Mucosal Vaccines

AbstractThe development of potent adjuvants is an important step for improving the performance of subunit vaccines. CD1d agonists, such as the prototypical α‐galactosyl ceramide (α‐GalCer), are of special interest due to their ability to activate iNKT cells and trigger rapid dendritic cell maturation and B‐cell activation. Herein, we introduce a novel derivatization hotspot at the α‐GalCer skeleton, namely the N‐substituent at the amide bond. The multicomponent diversification of this previously unexplored glycolipid chemotype space permitted the introduction of a variety of extra functionalities that can either potentiate the adjuvant properties or serve as handles for further conjugation to antigens toward the development of self‐adjuvanting vaccines. This strategy led to the discovery of compounds eliciting enhanced antigen‐specific T cell stimulation and a higher antibody response when delivered by either the parenteral or the mucosal route, as compared to a known potent CD1d agonist. Notably, various functionalized α‐GalCer analogues showed a more potent adjuvant effect after intranasal immunization than a PEGylated α‐GalCer analogue previously optimized for this purpose. Ultimately, this work could open multiple avenues of opportunity for the use of mucosal vaccines against microbial infections.

Development of Novel Invariant TCR-Anchored Bispecific Engagers to Enhance Anti-Cancer Activity of iNKT Cells

iNKT are rare, CD1d-restricted, glycolipid-reactive T cells that bridge adaptive and innate immunity. iNKT cells are characterised by an invariant TCRVa24Ja18 chain nearly always pairing with a TCRVb11 chain (iTCR). The ability of allogeneic iNKT to protect from aGVHD makes them ideal candidates for ‘off-the-shelf’ immunotherapy without need for TCR deletion. Indeed, CAR-iNKT show promise in pre-clinical models of blood cancer as well as early clinical development against B cell lymphoma and outperform CAR-T counterparts. Here we aimed to enhance the modularity of adoptive iNKT cell immunotherapy for blood cancers by designing a bispecific iNKT cell engager (biNTe) in which one arm would engage the iTCR while the other arm would engage tumour antigen(s) of interest. As a proof-of-principle we designed and synthesised a mono- and a bi-valent biNTe, one each, against the clinically validated myeloma target BCMA with purities of 90% and 70% respectively. We focused on the bivalent biNTe which we first used to demonstrate its iNKT cell binding specificity. Indeed, in flow-cytometry assays, biNTe followed by fluorescent anti-human Fc Ab, stained purified iNKT and but not T cells while in the presence of the same iTCR-binding mAb used to generate the biNTes, binding of the bivalent biNTe on iNKT cells was abrogated. This shows the iTCR engaging specificity of the bivalent biNTe. To demonstrate BCMA specificity of the other arm of the biNTe, we used it to stain iNKT cells, followed by staining with biotinylated soluble BCMA and fluorescently labelled streptavidin. This, compared to staining without soluble BCMA, showed strong staining of iNKT cells thus validating the BCMA specificity of the bivalent biNTe. Next, we set up co-cultures of pre-expanded iNKT against the BCMA-expressing and -knockout myeloma cell line MM1.S in the presence of varying concentrations of the biNTe. While, as assessed by a flow-cytometric cytotoxicity assay, only background killing of myeloma cells was observed in the absence of biNTe or when BCMA ko cell were used as targets, in the presence of biNTe, BCMA+ MM1S cells were killed, in a dose dependent manner (Figure 1A). Consistent with iNKT specificity of the biNTe similar co-cultures involving T cells showed only background killing of MM1S cells (Figure 1B). The same results were obtained using the BCMA-expressing myeloma cell line H929 and its corresponding BCMA knockout. The overall estimated EC50 for MM1.S and H929 cells were 6.56 and 3.4 nM respectively. Using the EC50 concentrations we show that biNTe very effectively kill and almost eliminate patient-derived bone marrow myeloma plasma cells (n=3). To test whether engagement of the iTCR by biNTe would impact its ability to engage with CD1d, we co-cultured iNKT and MM1.S cells transduced with CD1D in the presence of biNTe and the activating, iNKT-specific, CD1d-binding glycolipid ligand a-galactosylceramide (aGalCer). We found that compared to no biNTe control and at biNTE concentrations lower than the EC50, killing of myeloma targets cells was consistently increased. This suggests that iTCR retains its ability to engage CD1d and the efficacy of future biNTe-based immunotherapy could be enhanced by aGalCer. While in vivo validation experiments are ongoing, we conclude that biNTe constitute a novel therapeutic approach with notable pre-clinical anti-myeloma activity. We envisage that they can be used in conjunction with other therapeutic modalities such as CAR and iTCR to enhance the modularity and the efficacy of iNKT cells as an ‘off -the-shelf’ platform for the treatment of myeloma, other blood or solid tumour cancers.

Conformationally Restricted Analogues of α-Galactosylceramide as Adjuvant in COVID-19 Subunit Vaccine.

iNKT cells are a type of T lymphocyte that recognizes glycolipid antigens presented by CD1d protein. αGC is an agonistic glycolipid that activates iNKT cells and triggers immune modulatory cytokine responses, making it a promising vaccine adjuvant. To find more potent immunostimulating glycolipids, we prepared 4,6-O-galactosyl conformationally restricted analogues of αGC. Mice vaccinated with the SARS-CoV-2 RBD-Fc vaccine adjuvanted with these newly developed glycolipids produced robust anti-RBD antibody responses, comparable to those achieved with αGC. Importantly, we also found that omitting αGC, α-C-GalCer (Th1-type agonist), or C20:2 (Th2-type agonist) from the booster vaccine had negligible impact on antibody and cellular responses, potentially reducing the frequency of adjuvant use required to maintain potent immune responses.