Activation Of NKT Cells Research Articles

IFN-γ-dependent regulation of intestinal epithelial homeostasis by NKT cells

Intestinal homeostasis is maintained through the combined functions of epithelial and immune cells that collaborate to preserve the integrity of the intestinal barrier. However, the mechanisms by which immune cell populations regulate intestinal epithelial cell (IEC) homeostasis remain unclear. Here, we use a multi-omics approach to study the immune-epithelial crosstalk and identify CD1d-restricted natural killer T (NKT) cells as key regulators of IEC biology. We find that NKT cells are abundant in the proximal small intestine and show hallmarks of activation at steady state. Subsequently, NKT cells regulate the survival and the transcriptional and cellular composition landscapes of IECs in intestinal organoids, through interferon-γ (IFN-γ) and interleukin-4 secretion. Invivo, lack of NKT cells results in an increase in IEC turnover, while NKT cell activation leads to IFN-γ-dependent epithelial apoptosis. Our findings propose NKT cells as potent producers of cytokines that contribute to the regulation of IEC homeostasis.

IFITM1 aggravates ConA-Induced autoimmune hepatitis by promoting NKT cell activation through increased AMPK-Dependent mitochondrial function

Although interferon-induced transmembrane 1 (IFITM1) is known for its crucial role in antiviral immunity, its involvement in autoimmune hepatitis (AIH) remains largely unexplored. In this study, we observed that IFITM1 expression is markedly upregulated in a Concanavalin A (ConA)-induced AIH model, with particularly high and markedly elevated expression in natural killer T (NKT) cells. To further understand the role of IFITM1, we examined the responses of IFITM1−/− mice in a model of ConA-induced liver injury. In comparison to wild-type mice, IFITM1−/− mice exhibited reduced sensitivity in this model, as evidenced by significantly ameliorated necrosis areas, lower serum aminotransferase levels, a reduced number of intrahepatic NKT cells, and decreased expression of inflammatory factors, such as IL-1β, IL-6, IFN-γ and TNF-α. Notably, by using IFITM1-GFP mice and IFITM1−/− mice, we demonstrated that IFITM1 expression in NKT cells is crucial for their proliferation, proinflammatory cytokine production, and cytotoxic functions. Furthermore, analysis of single-cell RNA sequencingdata revealed that IFITM1 is essential for mitochondrial function, which is mediated by the AMP-activated protein kinase (AMPK) pathway. We also validated the importance of IFITM1 for the AMPK pathway and mitochondrial ATP synthesis in vivo. Together, our findings elucidate that IFITM1 could regulate NKT cell activation and survival by promoting mitochondrial function during AIH.

Lipidomic scanning of self-lipids identifies headless antigens for natural killer T cells

To broadly measure the spectrum of cellular self-antigens for natural killer T cells (NKT), we developed a sensitive lipidomics system to analyze lipids trapped between CD1d and NKT T cell receptors (TCRs). We captured diverse antigen complexes formed in cells from natural endogenous lipids, with or without inducing endoplasmic reticulum (ER) stress. After separating protein complexes with no, low, or high CD1d-TCR interaction, we eluted lipids to establish the spectrum of self-lipids that facilitate this interaction. Although this unbiased approach identified fifteen molecules, they clustered into only two related groups: previously known phospholipid antigens and unexpected neutral lipid antigens. Mass spectrometry studies identified the neutral lipids as ceramides, deoxyceramides, and diacylglycerols, which can be considered headless lipids because they lack polar headgroups that usually form the TCR epitope. The crystal structure of the TCR-ceramide-CD1d complex showed how the missing headgroup allowed the TCR to predominantly contact CD1d, supporting a model of CD1d autoreactivity. Ceramide and related headless antigens mediated physiological TCR binding affinity, weak NKT cell responses, and tetramer binding to polyclonal human and mouse NKT cells. Ceramide and sphingomyelin are oppositely regulated components of the "sphingomyelin cycle" that are altered during apoptosis, transformation, and ER stress. Thus, the unique molecular link of ceramide to NKT cell response, along with the recent identification of sphingomyelin blockers of NKT cell activation, provide two mutually reinforcing links for NKT cell response to sterile cellular stress conditions.

Fusogenic vesicular stomatitis virus combined with natural killer T cell immunotherapy controls metastatic breast cancer

BackgroundMetastatic breast cancer is a leading cause of cancer death in woman. Current treatment options are often associated with adverse side effects and poor outcomes, demonstrating the need for effective new treatments. Immunotherapies can provide durable outcomes in many cancers; however, limited success has been achieved in metastatic triple negative breast cancer. We tested whether combining different immunotherapies can target metastatic triple negative breast cancer in pre-clinical models.MethodsUsing primary and metastatic 4T1 triple negative mammary carcinoma models, we examined the therapeutic effects of oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express reovirus-derived fusion associated small transmembrane proteins p14 (VSV-p14) or p15 (VSV-p15). These viruses were delivered alone or in combination with natural killer T (NKT) cell activation therapy mediated by adoptive transfer of α-galactosylceramide-loaded dendritic cells.ResultsTreatment of primary 4T1 tumors with VSV-p14 or VSV-p15 alone increased immunogenic tumor cell death, attenuated tumor growth, and enhanced immune cell infiltration and activation compared to control oncolytic virus (VSV-GFP) treatments and untreated mice. When combined with NKT cell activation therapy, oncolytic VSV-p14 and VSV-p15 reduced metastatic lung burden to undetectable levels in all mice and generated immune memory as evidenced by enhanced in vitro recall responses (tumor killing and cytokine production) and impaired tumor growth upon rechallenge.ConclusionCombining NKT cell immunotherapy with enhanced oncolytic virotherapy increased anti-tumor immune targeting of lung metastasis and presents a promising treatment strategy for metastatic breast cancer.

Immunotherapy with an antibody against CD1d modulates neuroinflammation in an α-synuclein transgenic model of Lewy body like disease.

The neuroinflammatory process in synucleinopathies of the aging population such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB) involves microglial activation as well as infiltration of the CNS by T cells and natural killer T cells (NKTs). To evaluate the potential of targeting NKT cells to modulate neuroinflammation, we treated α-syn transgenic (tg) mice (e.g.: Thy1 promoter line 61) with an antibody against CD1d, which is a glycoprotein expressed in antigen presenting cells (APCs). CD1d-presented lipid antigens activate NKT cells through the interaction with T cell receptor in NKTs, resulting in the production of cytokines. Thus, we hypothesized that blocking the APC-NKT interaction with an anti-CD1d antibody might reduce neuroinflammation and neurodegeneration in models of DLB/PD. Treatment with the anti-CD1d antibody did not have effects on CD3 (T cells), slightly decreased CD4 and increased CD8 lymphocytes in the mice. Moreover, double labeling studies showed that compared to control (IgG) treated α-syn tg mice, treatment with anti-CD1d decreased numbers of CD3/interferon γ (IFN γ)-positive cells, consistent with NKTs. Further double labeling studies showed that CD1d-positive cells co-localized with the astrocytes marker GFAP and that anti-CD1d antibody reduced this effect. While in control α-syn tg mice CD3 positive cells were near astrocytes, this was modified by the treatment with the CD1d antibody. By qPCR, levels of IFN γ, CCL4, and interleukin-6 were increased in the IgG treated α-syn tg mice. Treatment with CD1d antibody blunted this cytokine response that was associated with reduced astrocytosis and microgliosis in the CNS of the α-syn tg mice treated with CD1d antibody. Flow cytometric analysis of immune cells in α-syn tg mice revealed that CD1d-tet + T cells were also increased in the spleen of α-syn tg mice, which treatment with the CD1d antibody reduced. Reduced neuroinflammation in the anti-CD1d-treated α-syn tg mice was associated with amelioration of neurodegenerative pathology. These results suggest that reducing infiltration of NKT cells with an antibody against CD1d might be a potential therapeutical approach for DLB/PD.

Abstract 6755: NKT activation enhanced the therapeutic efficacy of neoantigen vaccine in hepatocellular carcinoma

Abstract One sentence introduction: This study indicated the potential of combinational therapy of neoantigen vaccine and iNKT activator α-GalCer in Hepatocellular carcinoma. Hepatocellular carcinoma (HCC) is a leading cause of death around the world. Current immune checkpoint blockade therapies only show effectiveness in some cancer patients, so there is an urgent need for more effective immunotherapy strategies. One alternative is the use of neoantigen-based cancer vaccines, which target tumor-specific antigens (neoantigens) on cancer cells and induce strong T-cell responses. Advances in technology now allow for precise identification of tumor neoantigens using bioinformatic analysis or mass spectrometry. While cancer vaccines have demonstrated strong anti-tumor immune responses and therapeutic effectiveness in some cancer types, their success in HCC remains limited. A contributing factor is the suppressive tumor microenvironment (TME) found in advanced HCC. Some studies suggest that combining neoantigen vaccines with anti-PD1/PD-L1 therapies can significantly enhance HCC treatment outcomes. Overcoming the suppressive immune microenvironment is crucial in cancer treatment, and reactivating intratumoral immunity is essential. In our study, we developed an HCC cancer vaccine using transcriptome sequencing and bioinformatics analysis. Our ELISpot assay results showed that this vaccine induced strong tumor-specific immune responses and significantly inhibited tumor growth in early-stage orthotopic HCC mouse models when administered five days after tumor inoculation. However, the effectiveness decreased in advanced tumors when administered ten days after tumor inoculation. We also discovered a significant relationship between the therapeutic effectiveness of the neoantigen vaccine and the activation of intratumoral NKT cells in HCC. Our study suggested that the type-I NKT cell activator, α-Galactosylceramide (α-GalCer), significantly improved the therapeutic outcomes of the cancer neoantigen vaccine in advanced orthotopic HCC mouse models. In some cases, tumors were almost completely eliminated. This indicates the potential of combining iNKT activators with neoantigen vaccines for HCC treatment. To further understand the role of NKT cells in HCC's TME, we plan to investigate intratumoral NKT cell subtypes following neoantigen vaccine therapy. Additionally, we will develop a new delivery system to deliver type I NKT cell activators and type II NKT inhibitors to tumor sites, aiming to reactivate the suppressive TME in HCC after neoantigen vaccine therapy. Citation Format: Renhao Li, Jing-Chu Hu, Li Rong, Yangfan Wu, Yige He, Jiandong Huang. NKT activation enhanced the therapeutic efficacy of neoantigen vaccine in hepatocellular carcinoma [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 6755.

Preclinical evaluation of therapeutic vaccines for chronic hepatitis B that stimulate antiviral activities of T cells and NKT cells

Background & AimsLiver diseases resulting from chronic HBV infection are a significant cause of morbidity and mortality. Vaccines that elicit T-cell responses capable of controlling the virus represent a treatment strategy with potential for long-term effects. Here, we evaluated vaccines that induce the activity of type I natural killer T (NKT) cells to limit viral replication and license stimulation of conventional antiviral T-cells. MethodsVaccines were prepared by conjugating peptide epitopes to an NKT-cell agonist to promote co-delivery to antigen-presenting cells, encouraging NKT-cell licensing and stimulation of T cells. Activity of the conjugate vaccines was assessed in transgenic mice expressing the complete HBV genome, administered intravenously to maximise access to NKT cell-rich tissues. ResultsThe vaccines induced only limited antiviral activity in unmanipulated transgenic hosts, likely attributable to NKT-cell activation as T-cell tolerance to viral antigens is strong. However, in a model of chronic hepatitis B involving transfer of naive HBcAg-specific CD8+ T cells into the transgenic mice, which typically results in specific T-cell dysfunction without virus control, vaccines containing the targeted HBcAg epitope induced prolonged antiviral activity because of qualitatively improved T-cell stimulation. In a step towards a clinical product, vaccines were prepared using synthetic long peptides covering clusters of known HLA-binding epitopes and shown to be immunogenic in HLA transgenic mice. Predictions based on HLA distribution suggest a product containing three selected SLP-based vaccines could give >90 % worldwide coverage, with an average of 3.38 epitopes targeted per individual. ConclusionsThe novel vaccines described show promise for further clinical development as a treatment for chronic hepatitis B. Impact and ImplicationsAlthough there are effective prophylactic vaccines for HBV infection, it is estimated that 350–400 million people worldwide have chronic hepatitis B, putting these individuals at significant risk of life-threatening liver diseases. Therapeutic vaccination aimed at activating or boosting HBV-specific T-cell responses holds potential as a strategy for treating chronic infection, but has so far met with limited success. Here, we show that a glycolipid-peptide conjugate vaccine designed to coordinate activity of type I NKT cells alongside conventional antiviral T cells has antiviral activity in a mouse model of chronic infection. It is anticipated that a product based on a combination of three such conjugates, each prepared using long peptides covering clusters of known HLA-binding epitopes, could be developed further as a treatment for chronic hepatitis B with broad global HLA coverage.

A natural killer T cell nanoagonist-initiated immune cascade for hepatocellular carcinoma synergistic immunotherapy.

Natural killer T (NKT) cell-mediated immunotherapy shows great promise in hepatocellular carcinoma featuring an inherent immunosuppressive microenvironment. However, targeted delivery of NKT cell agonists remains challenging. Here, we developed a hyaluronic acid (HA) modified metal organic framework (zeolitic imidazolate framework-8, ZIF-8) to encapsulate α-galactosylceramide (α-Galcer), a classic NKT cell agonist, and doxorubicin (DOX) for eliminating liver cancer, denoted as α-Galcer/DOX@ZIF-8@HA. In the tumor microenvironment (TME), these pH-responsive nano-frameworks can gradually collapse to release α-Galcer for activating NKT cells and further boosting other immune cells in order to initiate an antitumor immune cascade. Along with DOX, the released α-Galcer enabled efficient NKT cell activation in TME for synergistic immunotherapy and tumor elimination, leading to evident tumor suppression and prolonged animal survival in both subcutaneous and orthotopic liver tumor models. Manipulating NKT cell agonists into functional nano-frameworks in TME may be matched with other advanced managements applied in a wider range of cancer therapies.

Conditional NKT Cell Depletion in Mice Reveals a Negative Feedback Loop That Regulates CTL Cross-Priming.

NKT cells are unconventional T cells whose biological role is incompletely understood. Similar to TH cells, activated NKT cells can cause dendritic cell (DC) maturation, which is required for effective CTL responses. However, it is unclear whether and how NKT cells affect CTLs downstream of the DC maturation phase. This is partially due to the lack of techniques to conditionally deplete NKT cells invivo. To overcome this problem, we have developed two approaches for this purpose in mice: the first is based on mixed bone marrow chimeras where Jα18 knockout and depletable CD90 congenic bone marrow is combined, and the second used PLZFCre × iDTR bone marrow chimeras, which target innate-like T cells. Using these tools, we found that NKT cell depletion at 20 h, that is, after initial DC activation, did not render CTLs helpless, as CD40L signaling by non-NKT cells sufficed. Instead, NKT cell depletion even augmented CD8 T cell expansion and cytotoxicity by mechanisms distinct from reduced STAT6 signaling. These findings revealed a negative feedback loop by which NKT cells control CTL cross-priming downstream of DC maturation. The techniques described in this study expand the toolbox to study NKT cells and other unconventional T cell subsets invivo and uncovered a hidden immunoregulatory mechanism.

CCRG-06. PP2A DEFICIENCY INDUCES CELLULAR SENESCENCE AND SENSITIZES P53 MUTANT TUMORS TO SENOLYTIC THERAPY

Abstract Loss of p53 can promote tumor progression by preventing induction of senescence, a critical tumor-suppressive mechanism that limits tumor proliferation. Promotion of senescence in a p53-independent manner can be a novel strategy to overcome treatment resistance in p53-mutant brain tumors. Protein-Phosphatase-2A (PP2A), a ubiquitous serine/threonine phosphatase, regulates various cellular processes including cell-cycle progression. We hypothesize that PP2A-deficiency can induce tumor senescence independent of p53-mutational-status and sensitize tumors to senolytic therapy. CRISPR-KO of PP2A was generated in multiple p53-mutant tumor cell lines: D425 (human medulloblastoma), SB28 and GL261 (murine glioma). Hallmarks of senescence such as b-galactosidase, p21, and p16 were assessed via flow cytometry, RT-PCR and Western-Blot. Secretion of senescence-associated secretory phenotype (SASP), such as IL8, was assessed through ELISA. b-galactosidase, p21, p16 and IL8 were dramatically increased in PP2A-KO compared to WT at baseline. The effect was further enhanced when combined with radiation therapy (RT), a known inducer of senescence, demonstrating PP2A-deficiency synergizes with RT and significantly increases cellular senescence. Expression of CD1d, a MHC1-like molecule associated with senescence and required for invariant NKT cell-activation (iNKT), was dramatically increased in PP2A-KO in comparison to WT via flow-cytometry. In-vitro, sensitivity to WT and PP2A-KO cells to senolytic treatments, such as ABT-737 and iNKT-cell therapy, was tested using an XCelligence-assay system and flow-cytometry-based cytotoxicity assay. PP2A-deficient cells were sensitized to ABT-737, a BCL-xL inhibitor that preferentially targets and kills senescent cells. Co-culture revealed increased cytotoxicity of iNKT-cells against PP2A-deficient tumors compared to WT. In vivo, D425 WT or PP2A-KO cells were injected intracranially into nude NU/J mice, radiated 2 days post-injection, and monitored till endpoint. At baseline, PP2A-KO mice survived longer than WT, and those with PP2A-KO and RT survived the longest. In conclusion, we report that PP2A-deficiency in tumor cells can induce p53-independent senescence and sensitize brain tumors to senolytic therapy.

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.

Mitochondrial PGAM5-Drp1 signaling regulates the metabolic reprogramming of macrophages and regulates the induction of inflammatory responses.

Macrophages play a critical role in the regulation of inflammation and tissue homeostasis. In addition to their vital functions for cell survival and physiology, mitochondria play a crucial role in innate immunity as a platform for the induction of inflammatory responses by regulating cell signaling and dynamics. Dynamin-related protein 1 (Drp1) plays a role in the induction of inflammatory responses and the subsequent development of various diseases. PGAM5 (phosphoglycerate mutase member 5) is a mitochondrial outer membrane phosphatase that dephosphorylates its substrate, Drp1. Previous studies showed that PGAM5 regulates the phosphorylation of Drp1 for the activation of NKT cells and T cells. However, it is not clear how PGAM5 regulates Drp1 activity for the induction of inflammation in macrophages. Here, we demonstrate that PGAM5 activity regulates the dephosphorylation of Drp1 in macrophages, leading to the induction of proinflammatory responses in macrophages. In TLR signaling, PGAM5 regulates the expression and production of inflammatory cytokines by regulating the activation of downstream signaling pathways, including the NF-κB and MAPK pathways. Upon LPS stimulation, PGAM5 interacts with Drp1 to form a complex, leading to the production of mtROS. Furthermore, PGAM5-Drp1 signaling promotes the polarization of macrophages toward a proinflammatory phenotype. Our study further demonstrates that PGAM5-Drp1 signaling promotes metabolic reprogramming by upregulating glycolysis and mitochondrial metabolism in macrophages. Altogether, PGAM5 signaling is a linker between alterations in Drp1-mediated mitochondrial dynamics and inflammatory responses in macrophages and may be a target for the treatment of inflammatory diseases.

Off-the-shelf CAR–engineered natural killer cells targeting FLT3 enhance killing of acute myeloid leukemia

AbstractThe majority of patients with acute myeloid leukemia (AML) succumb to the disease or its complications, especially among older patients. Natural killer (NK) cells have been shown to have antileukemic activity in patients with AML; however, to our knowledge, primary NK cells armed with a chimeric antigen receptor (CAR) targeting antigens associated with AML as an “off-the-shelf” product for disease control have not been explored. We developed frozen, off-the-shelf allogeneic human NK cells engineered with a CAR recognizing FLT3 and secreting soluble interleukin-15 (IL-15) (FLT3 CAR_sIL-15 NK) to improve in vivo NK cell persistence and T-cell activation. FLT3 CAR_sIL-15 NK cells had higher cytotoxicity and interferon gamma secretion against FLT3+ AML cell lines when compared with activated NK cells lacking an FLT3 CAR or soluble IL-15. Frozen and thawed allogeneic FLT3 CAR_sIL-15 NK cells prolonged survival of both the MOLM-13 AML model as well as an orthotopic patient-derived xenograft AML model when compared with control NK cells. FLT3 CAR_sIL-15 NK cells showed no cytotoxicity against healthy blood mononuclear cells or hematopoietic stem cells. Collectively, our data suggest that FLT3 is an AML-associated antigen that can be targeted by frozen, allogeneic, off-the-shelf FLT3 CAR_sIL-15 NK cells that may provide a novel approach for the treatment of AML.

CD1d-Dependent Natural Killer T Cells Mediate Hypertension and Vascular Injury Through Interleukin-17A.

Background Different T-lymphocyte subsets, including CD1d-dependent natural killer T (NKT) cells, play distinct roles in hypertension, highlighting the importance of identifying key immune cells for its treatment. This study aimed to determine the unknown effects of CD1d-dependent NKT cells on hypertension and vascular injury. Methods and Results Hypertension models were induced in male CD1d knockout (CD1dko), wild-type, and adoptive bone marrow transfer mice by angiotensin II (Ang II) or deoxycorticosterone acetate salt. Blood pressure was measured by the tail-cuff system and radiotelemetry. Vascular injury was assessed by histologic studies or aortic ring assay. Inflammation was detected by flow cytometry, quantitative real-time polymerase chain reaction, or ELISA. Results showed that Ang II infusion significantly reduced CD1d expression and NKT cell numbers in the aorta of mice. CD1dko mice exhibited worsened blood pressure elevation, vascular injury, and inflammatory response induced by Ang II or deoxycorticosterone acetate salt. However, these effects were markedly reversed in wild-type mice treated with NKT cell-specific activator. Adoptive transfer of CD1dko bone marrow cells to wild-type mice also significantly worsened Ang II-induced responses. Mechanistically, CD1dko increased Ang II-induced interleukin-6 production and activated signal transducer and activator of transcription 3 and orphan nuclear receptor γ, subsequently inducing interleukin-17A production. Neutralizing interleukin-17A partially reversed Ang II-induced hypertension and vascular injury in CD1dko mice. In addition, levels of NKT cells were lower in the blood of patients with hypertension (n=57) compared with normotensive individuals (n=87). Conclusions These findings reveal a previously unknown role for CD1d-dependent NKT cells in hypertension and vascular injury, indicating that NKT cell activation could be a promising therapeutic target for hypertension.

#5264 CELLULAR IMMUNITY ACTIVATION IN RENAL TRANSPLANT RECIPIENTS AFTER VACCINATION WITH TOZINAMERAN: DIFFERENCES BETWEEN RESPONDERS AND NON-RESPONDERS

Abstract Background and Aims Response to vaccination is a complex procedure, resulting from the activation of various immune cell subpopulations and comprising stimulation of several interactive pathways. The aim of our study was to assess the differences in cellular subpopulations between Renal Transplant Recipients (RTRs) classified as “responders” and “non-responders” to vaccination with Tozinameran, against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Method Our research was a prospective observational study. Our sample consisted of 39 RTRs on stable immunosuppression, who had a negative history of corona virus disease 2019 (COVID-19) infection and had not achieved to develop protective humoral immunity (i.e., had a negative test for neutralizing antibodies -Nab-) three weeks after the 2nd vaccine dose (T0). We attempted to evaluate immune response 48 hours before (T1) and 3 weeks after (T2) the 3rd Tozinameran dose. Patients with serum Nab levels > 0.3AU/ml (calculated with chemiluminescence immunoassay -CLIA-) and/or enzyme-linked immunosorbent spot (ELISpot) values > 30SFC/5 × 105 PBMCs were regarded as responders. Results were subsequently correlated with variations in cellular subpopulation compositions. Concentrations of CD4+, activated CD4+, CD8+, activated CD8+ T-cells, CD3+CD16+CD56+ (Natural Killer T -NKT-), activated NKT cells, CD3-CD16+CD56+ (Natural Killer -NK-) cells, CD19+, CD19+CD27- (Naïve), CD19+IgD+CD27- (Marginal), CD19+CD24+CD38high (Transitional), CD19+CD27+CD38high (Plasmablasts) and CD19+CD27+ (Memory) B cells were measured with flow cytometry at T1 and T2. We compared the results in the “responders” and the “non-responders” group. Results The number of responders significantly increased from T1 [17/39, responders: Nab(-) - ELISpot(+)] to T2 [34/39, responders: Nab and/or ELISpot (+)] (x2 = 16.2, p < 0.0001). No difference was observed between responders and non-responders regarding age, renal function, calcineurin inhibitor levels, dialysis and transplantation vintage. Compared to non-responders, responders presented increased concentrations of: activated CD8+ [23.6(29) vs 9.49(10)/μL, p: 0.044], activated NKT [4.77(8) vs 1.73(2)/μL, p: 0.001] and NK cells [198.91(161) vs 104.45(103)/μL, p: 0.024] at T1 and elevated levels of: total B cells [87.63(101) vs 26.86(42)/μL, p: 0.01], marginal [10.03(13) vs 1.12(5.9)/μL, p: 0.045] and memory B-cells [28.31(42) vs 10.57(19)/μL, p: 0.048] at T2 (see table). A significant increase of activated NKT and naïve B-cells from T1 to T2 [310(174) vs 241(200)/μL, p = 0.02 and 56.2(67) vs 1082(834)/μL, p<0.0001, respectively] was evident only in the responders’ group. Conclusion As anticipated, responders to vaccination against SARS-CoV-2 exhibited a quite different immune cell activation profile compared to non-responders. Besides, the 3rd Tozinameran vaccine dose substantially improved RTRs’ immune response and induced a prominent stimulation of the B lymphocyte compartment.

CD1d-dependent natural killer T-cells inactivation aggravates sepsis-induced myocardial injury via T lymphocytes infiltration and IL-6 production in mice

Myocardial edema mediated by endothelial dysfunction plays an important role in sepsis-induced cardiomyopathy (SIC); however, its mechanism is unclear. The current study aimed to provide evidence on the cardioprotection of CD1d-dependent natural killer T (NKT) cells and clarify the possible mechanism in a mouse model of sepsis. Wild-type (WT) and CD1d-dependent NKT-cells inactivation (CD1dko) mice were subjected to sepsis induced by intraperitoneal injection of lipopolysaccharide (LPS). The NKT-cells number and CD1d expression were both increased in the hearts and blood of WT mice after LPS treatment. Compared with WT mice, CD1dko mice exhibited remarkably accelerated LPS-induced mortality, cardiac dysfunction, myocardial injury, endothelial apoptosis, microvascular damage, microvascular permeability and cardiac edema. Mechanistically, CD1d deficiency further increased LPS-induced accumulation of T lymphocytes in the myocardium and upregulation of IL-6 protein levels. Administration of an IL-6 neutralizing antibody to CD1dko mice improved cardiac dysfunction, myocardial injury and edema induced by LPS. Our study identified that CD1d-dependent NKT-cells inactivation exacerbated SIC via T lymphocytes infiltration and IL-6 production. Hence, activation of CD1d-dependent NKT cells may be a potential candidate strategy for SIC treatment.

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)

NKT cells contribute to alleviating lung metastasis in adenoid cystic carcinoma

AbstractSalivary adenoid cystic carcinoma (SACC) with a unique MYB‐NFIB fusion has been considered an “immune‐cold” tumor, but the mechanisms behind this remain unclear. In this study, we analyzed the immune status of 29 SACC patients and found that most lung metastases exhibited an immunoinflammatory state, in contrast to the primary SACC tissues. Single‐cell sequencing data showed that anergic T‐cell types were low in primary inflammatory tissues, while inflammatory metastatic lung tissues had elevated levels of anergic CD8+ natural killer T (NKT)‐like cells and low levels of memory T cells. Primary exclusive tissues had high levels of myeloid‐derived suppressor cells (MDSCs) and low levels of activated CD8+ NKT‐like cells. These data support the fact that metastatic SACC cells might induce a stronger immune response in the lung. Additionally, an in vivo experiment showed that a minimally invasive SACC cell line with higher expression of human leukocyte antigens ‐B and ‐C induced NKT cell activation in mice and effectively attenuated the incidence of lung metastases caused by a highly invasive SACC cell line. This suggests that NKT therapy may be active in treating SACC lung metastasis. Conclusively, this study sheds light on the immune microenvironment of SACC and highlights the potential of NKT‐based therapy.

Abstract CT100: A new therapeutic cancer vaccine inducing multifunctional immunity, artificial adjuvant vector cells

Abstract Background: Cancer immunotherapy is an effective way of battling cancer, and is based on artificial stimulation of the immune system to attack cancer cells. Although several strategies show successful results, it remains to prevent the subsequent cancer escape from the immune surveillance. The further success depends on a variety of functional effector cell types involved and their sustenance following activation. For this purpose, simultaneous induction of innate and adaptive immunity should be one of ideal strategies. We focus on DCs, which play a pivotal role in determining the quality and magnitude of innate and adaptive immunity. To effectively utilize the DC in situ, we have developed a system using allogeneic cells as artificial adjuvant vector cells (aAVCs), comprised of a CD1d-NKT ligand complex on the cell surface and containing tumor antigen inside of the cells. This approach induces adjuvant effects by combining NKT cell activation with delivery of tumor antigen to DCs in vivo. In addition to linking innate and adaptive immunity, aAVC therapy can lead to efficient trafficking of specific anti-tumor CD8+ T cells to the tumor site and also the formation of long-term memory T cells. This novel design of aAVC therapy is a platform and allows for a replacement of any cancer antigens as the therapeutic package. After completion of the regulatory science consultation for discussing the pharmaceutical quality and the design of a clinical study with PMDA in Japan, we developed the WT1-expressing aAVC (aAVC-WT1) for the clinical application. Methods: This first-in-human, open label, single-center trial involves a bifurcated 3+3 design with aAVC-WT1 dose escalations (1 × 106, 1 × 107, and 1 × 108 per body). The three patients in each cohort had not received any chemotherapy during more than 2 weeks before the therapy and had received aAVC-WT1 intravenously twice in a 4-week interval. Nine of the ten enrolled patients with relapsed and refractory AML underwent complete analysis per-protocol set and were evaluated in our phase I dose-escalation trial of aAVC-WT1. Results: No dose-limiting toxicities were observed, whereas activation of NKT and/or NK cells was observed in all patients. Five patients experienced objective leukemic regression, which correlated with WT1-specific T-cell responses. Paired single-cell RNA and TCR sequencing demonstrated effector CD8+ T cell clones in the bone marrow. Some bone marrow CD8+ T cells underwent transition from pre-existing precursor exhausted T cells to functional T cells or emerged as newly activated T cells, some of which were maintained long-term. These demonstrate the feasibility and safety of aAVC-WT1 therapy and the capacity of this platform to activate both innate and adaptive immunity in humans. Conclusion: aAVC-WT1 therapy showed an acceptable safety profile at doses tested and encouraging signs of clinical response. These data support further development of aAVC-WT1 for patients with AML. Clinical trial registry number: UMIN 000028083. Citation Format: Shin-ichiro Fujii, Toyotaka Kawamata, Kanako Shimizu, Jun Nakabayashi, Satoru Yamasaki, Tomonori Iyoda, Jun Jun Shinga, Michihiro Hidaka, Masanori Nojima, Fumitaka Nagamura, Arinobu Tojo. A new therapeutic cancer vaccine inducing multifunctional immunity, artificial adjuvant vector cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT100.

Abstract LB220: S095029: a novel clinical-stage Fc-silenced NKG2A-blocking antibody with best-in-class potential

Abstract NKG2A and its ligand HLA-E are increasingly recognized as an important immune checkpoint restraining multiple lymphocyte populations involved in cancer immunity. Initially described as an NK cell inhibitory receptor, NKG2A’s role in regulating T-cell functions has recently come to the fore and is increasingly viewed as a key component of its immune suppressive functions. In addition, clinical studies have also provided proof-of-concept evidence for the superiority of dual NKG2A/PD-L1 blockade over anti-PD-L1 monotherapy. As of now clinical safety and efficacy results are only available for a single NKG2A-blocking antibody. This is in contrast with other immune checkpoint receptors (e.g., PD-1) where multiple blocking agents with different pharmacological properties have been assessed in the clinic, thereby providing a broad assessment of the target therapeutic potential and benefit to patients. Here, we report the generation and preclinical characterization of a novel monoclonal antibody (mAb) disrupting the NKG2A/HLA-E interaction. The Fc-attenuated IgG1 mAb S095029 binds NKG2A with a nM-range affinity and reverses the inhibitory effects of the NKG2A/HLA-E interaction in several experimental models, both in vitro and in vivo. As a single agent S095029 enhanced the killing activity and cytokine secretion of NK and γδ T-cells in co-culture with cancer cell lines of multiple tissue origins. We also show that S095029 increased the antibody-dependent cellular cytotoxicity (ADCC) mediated by Fc-competent mAbs in different antigen systems with high HLA-E expression. Of note, these immune-activating properties of S095029 compared favorably in benchmarking experiments with other clinical-stage anti-NKG2A mAbs. Finally, S095029 combination with PD-1 blocking agents, with or without an additional ADCC mAb component, conferred superior anti-tumor activity compared to treatments without NKG2A blockade. Overall, our in vitro and in vivo data support the clinical development of S095029 in tumor settings with adequate HLA-E expression and suggest potential differentiating characteristics compared to other anti-NKG2A mAbs. S095029 is currently being evaluated in phase 1 dose escalation studies as a single agent or in combination with anti-PD-1 therapy (NCT05162755). Citation Format: Maria Melander, Bruno Laugel. S095029: a novel clinical-stage Fc-silenced NKG2A-blocking antibody with best-in-class potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB220.