Engineering allorejection-resistant CAR-NKT cells from hematopoietic stem cells for off-the-shelf cancer immunotherapy.

The clinical potential of current FDA-approved chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapy is encumbered by its autologous nature, which presents notable challenges related to manufacturing complexities, heightened costs, and limitations in patient selection. Therefore, there is a growing demand for off-the-shelf universal cell therapies. In this study, we have generated universal CAR-engineered NKT (UCAR-NKT) cells by integrating iNKT TCR engineering and HLA gene editing on hematopoietic stem cells (HSCs), along with an ex vivo, feeder-free HSC differentiation culture. The UCAR-NKT cells are produced with high yield, purity, and robustness, and they display a stable HLA-ablated phenotype that enables resistance to host cell-mediated allorejection. These UCAR-NKT cells exhibit potent antitumor efficacy to blood cancers and solid tumors, both in vitro and in vivo, employing a multifaceted array of tumor-targeting mechanisms. These cells are further capable of altering the tumor microenvironment by selectively depleting immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells. In addition, UCAR-NKT cells demonstrate a favorable safety profile with low risks of graft-versus-host disease and cytokine release syndrome. Collectively, these preclinical studies underscore the feasibility and significant therapeutic potential of UCAR-NKT cell products and lay a foundation for their translational and clinical development.

Activation of cDCs and iNKT cells contributes to triptolide-induced hepatotoxicity via STING signaling pathway and endoplasmic reticulum stress.

Triptolide (TP) exhibits therapeutic potential against multiple diseases. However, its application in clinics is limited by TP-induced hepatoxicity. TP can activate invariant natural killer T (iNKT) cells in the liver, shifting Th1 cytokine bias to Th2 cytokine bias. The damaging role of iNKT cells in TP-induced hepatoxicity has been established, and iNKT cell deficiency can mitigate hepatotoxicity. However, the activation of iNKT cells in vitro by TP requires the presence of antigen-presenting cells. Therefore, we hypothesized that TP could induce dendritic cells (DCs) to activate iNKT cells, thereby leading to hepatotoxicity. The hepatic conventional DCs (cDCs) exhibited immunogenic activities after TP administration, upregulating the expression of CD1d, co-stimulatory molecules, and IL-12. Neutralization with IL-12p40 antibody extenuated TP-induced hepatotoxicity and reduced iNKT cell activation, suggesting that IL-12 could cause liver injury by activating iNKT cells. TP triggered the activation and upregulation of STING signaling pathway and increased endoplasmic reticulum (ER) stress. Downregulation of STING reduced cDC immunogenicity, inhibiting the activation of iNKT cells and hepatic damage. These indicated the regulatory effects of STING pathway on cDCs and iNKT cells, and the important roles it plays in hepatoxicity. ER stress inhibitor, 4-phenylbutyrate (4-PBA), also suppressed iNKT cell activation and liver injury, which might be regulated by the STING signaling pathway. Our results demonstrated the possible mechanisms underlying TP-induced hepatoxicity, where the activation of cDCs and iNKT cells was stimulated by upregulated STING signaling and increased ER stress as a result of TP administration.

Invariant Natural Killer T-cell Dynamics in Human Immunodeficiency Virus-associated Tuberculosis.

BACKGROUND: Tuberculosis (TB) is the leading cause of mortality and morbidity in people living with human immunodeficiency virus (HIV) infection (PLWH). PLWH with TB disease are at risk of the paradoxical TB-associated immune reconstitution inflammatory syndrome (TB-IRIS) when they commence antiretroviral therapy. However, the pathophysiology is incompletely understood and specific therapy is lacking. We investigated the hypothesis that invariant natural killer T (iNKT) cells contribute to innate immune dysfunction associated with TB-IRIS. METHODS: In a cross-sectional study of 101 PLWH and HIV-uninfected South African patients with active TB and controls, iNKT cells were enumerated using α-galactosylceramide-loaded CD1d tetramers and subsequently functionally characterized by flow cytometry. In a second study of 49 people with HIV type 1 (HIV-1) and active TB commencing antiretroviral therapy, iNKT cells in TB-IRIS patients and non-IRIS controls were compared longitudinally. RESULTS: Circulating iNKT cells were reduced in HIV-1 infection, most significantly the CD4+ subset, which was inversely associated with HIV-1 viral load. iNKT cells in HIV-associated TB had increased surface CD107a expression, indicating cytotoxic degranulation. Relatively increased iNKT cell frequency in patients with HIV-1 infection and active TB was associated with development of TB-IRIS following antiretroviral therapy initiation. iNKT cells in TB-IRIS were CD4+CD8- subset depleted and degranulated around the time of TB-IRIS onset. CONCLUSIONS: Reduced iNKT cell CD4+ subsets as a result of HIV-1 infection may skew iNKT cell functionality toward cytotoxicity. Increased CD4- cytotoxic iNKT cells may contribute to immunopathology in TB-IRIS.

Frontiers in alopecia areata pathobiology research.

This current review explores selected and as yet insufficiently investigated frontiers in current alopecia areata (AA) pathobiology research, with an emphasis on potential "new" players in AA pathobiology that deserve more systematic exploration and therapeutic targeting. Indeed, new evidence suggests that CD8+ T cells, which have long been thought to be the central players in AA pathobiology, are not the only drivers of disease. Instead, subsets of natural killer (NK) and so-called "unconventional" T cells (invariant NK T cells, γδ T cells, classic NK cells, and type 1 innate lymphoid cells), all of which can produce large amounts of IFN-γ, might also drive AA pathobiology independent of classical, autoantigen-dependent CD8+ T-cell functions. Another important new frontier is the role of regulatory lymphocyte subsets, such as regulatory T cells, γδ regulatory T cells, NKT10 cells, and perifollicular mast cells, in maintaining physiologic hair follicle immune privilege (IP); the extent to which these functions are defective in patients with AA; and how this IP-protective role could be restored therapeutically in patients with established AA. Broadening our AA research horizon along the lines suggested above promises not only to open the door to innovative and even more effective immunotherapy strategies for AA but will also likely be relevant for other autoimmune disorders in which pathobiology, ectopic MHC class I expression, and IP collapse play an important role.

Soluble factors derived from neuroblastoma cell lines suppress dendritic cell differentiation and activation.

Dendritic cells (DC) play a key role in the initiation of both antitumor immunity and immunological tolerance. It has been demonstrated that exposure to soluble factors produced by tumor cells modulates DC functions and induces tolerogenic DC differentiation. In this study, we investigated the effects of neuroblastoma cell line-derived soluble factors on DC differentiation. Monocytes isolated from healthy volunteers were incubated with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor in the presence of culture supernatants from neuroblastoma cell lines. The culture supernatants from neuroblastoma cell lines, such as NLF and GOTO, partially blocked both downregulation of CD14 and upregulation of CD1a, and dramatically decreased IL-12 and tumor necrosis factor (TNF)-α production from mature DC, while no effect of SH-SY5Y cell supernatant was noted. In addition, IL-6 and IL-10 production from monocytes was increased by the supernatants of NLF and GOTO cells at 24 hours after incubation. Furthermore, we evaluated DC functions through stimulation of invariant natural killer T (iNKT) cells. α-Galactosylceramide-pulsed DC co-cultured with supernatants of NLF cells were unable to sufficiently stimulate iNKT cells. The decreased ability of iNKT cells to produce interferon (IFN)-γ after stimulation with neuroblastoma cell line supernatant-cultured DC was reversed by addition of IL-12. CD40 expression and IL-12 production in NLF-sup-treated DC were increased by addition of exogenous IFN-γ. These results indicate that tolerogenic DC are induced in the neuroblastoma tumor microenvironment and attenuate the antitumor effects of iNKT cells. Interactions between iNKT cells and αGalCer-pulsed DC have the potential to restore the immunosuppression of tolerogenic DC through IFN-γ production.

Deficient invariant natural killer T cells had impaired regulation on osteoclastogenesis in myeloma bone disease.

Recent research showed that invariant natural killer T (iNKT) cells take part in the regulation of osteoclastogenesis. While the role of iNKT cells in myeloma bone disease (MBD) remains unclear. In our study, the quantity of iNKT cells and the levels of cytokines produced by them were measured by flow cytometry. iNKT cells and osteoclasts were induced from peripheral blood mononuclear cells after activation by α-GalCer or RANKL in vitro. Then, gene expressions and the levels of cytokines were determined by RT-PCR and ELISA, respectively. The results showed that the quantity of iNKT and production of IFN-γ by iNKT cells were significantly decreased in newly diagnosed MM (NDMM), and both negatively related with severity of bone disease. Then, the osteoclasts from healthy controls were cultured in vitro and were found to be down-regulated after α-GalCer-stimulated, while there was no significant change with or without α-GalCer in NDMM patients, indicating that the regulation of osteoclastogenesis by iNKT cells was impaired. Furthermore, the inhibition of osteoclastogenesis by iNKT cells was regulated by IFN-γ production, which down-regulated osteoclast-associated genes. In conclusion, the role of α-GalCer-stimulated iNKT cells in regulation of osteoclastogenesis was impaired in MBD, as a result of iNKT cell dysfunction.

Frequency and proliferative response of circulating invariant natural killer T cells in pediatric patients with malignant solid tumors.

BACKGROUND: Invariant natural killer T (iNKT) cells play an important role in tumor immunity, enhancing both innate and acquired immunity. We have previously shown the enhancement of antibody-dependent cellular cytotoxicity against neuroblastoma by activated iNKT cells. As a first step towards clinical application, we studied the frequency and proliferative response of circulating iNKT cells in children with and without cancer. METHODS: Blood samples were collected from 10 patients with pediatric malignant solid tumors and 11 patients with non-neoplastic diseases (control). The frequency of circulating iNKT cells was quantified by flow cytometry. Whole peripheral blood mononuclear cells were then stimulated with α-galactosylceramide (α-GalCer) for 7 days, and the expansion rate of the iNKT-cell fraction was assessed. RESULTS: The frequency of iNKT cells in the patients of the cancer and control group did not differ to a statistically significant extent. The iNKT-cell population increased after α-GalCer stimulation in all cases. The iNKT cells of patients who had undergone intensive chemotherapy also had the potential to expand in vitro. CONCLUSIONS: Unlike adult cancer patients, the numbers of circulating iNKT cells were not decreased in pediatric cancer patients. α-GalCer stimulation induced a proliferative response in all of the patients.

Sphingosine 1-phosphate alleviates Coxsackievirus B3-induced myocarditis by increasing invariant natural killer T cells.

Sphingosine 1-phosphate (S1P), via binding to its specific receptors of S1PR1, participates in the regulation of both innate and adaptive immunity. Recent reports have identified S1P as a messenger mediating inflammation. However, roles of S1P in Coxsackievirus B3 (CVB3)-induced myocarditis were largely unknown. Here, we investigated the effect of S1P treatment on CVB3-induced myocarditis in vivo. We found that CVB3 infection downregulated S1PR1 expression in spleen and decreased the proportion of invariant natural killer T cells (iNKT) in CD3 positive T cells both in spleen and in blood from left ventricle, which accompanied by severe inflammation lesions and more virus capsid protein (VP1) expression in heart tissue. In comparison, S1P supply upregulated iNKT in the spleen and in blood from left ventricle, which represented the strengthening of anti-inflammatory effects. Indeed, inflammation infiltration, VP1 expression and apoptosis in the myocardium was all downregulated. These results demonstrated that S1P supplement could alleviate CVB3-induced myocarditis.

Generation and characterization of CD1d-specific single-domain antibodies with distinct functional features.

Ligation of the CD1d antigen-presenting molecule by monoclonal antibodies (mAbs) can trigger important biological functions. For therapeutic purposes camelid-derived variable domain of heavy-chain-only antibodies (VHH) have multiple advantages over mAbs because they are small, stable and have low immunogenicity. Here, we generated 21 human CD1d-specific VHH by immunizing Lama glama and subsequent phage display. Two clones induced maturation of dendritic cells, one clone induced early apoptosis in CD1d-expressing B lymphoblasts and multiple myeloma cells, and another clone blocked recognition of glycolipid-loaded CD1d by CD1d-restricted invariant natural killer T (iNKT) cells. In contrast to reported CD1d-specific mAbs, these CD1d-specific VHH have the unique characteristic that they induce specific and well-defined biological effects. This feature, combined with the above-indicated general advantages of VHH, make the CD1d-specific VHH generated here unique and useful tools to exploit both CD1d ligation as well as disruption of CD1d-iNKT interactions in the treatment of cancer or inflammatory disorders.

West Nile virus-infected human dendritic cells fail to fully activate invariant natural killer T cells.

West Nile virus (WNV) infection is a mosquito-borne zoonosis with increasing prevalence in the United States. WNV infection begins in the skin, and the virus replicates initially in keratinocytes and dendritic cells (DCs). In the skin and cutaneous lymph nodes, infected DCs are likely to interact with invariant natural killer T cells (iNKTs). Bidirectional interactions between DCs and iNKTs amplify the innate immune response to viral infections, thus controlling viral load and regulating adaptive immunity. iNKTs are stimulated by CD1d-bound lipid antigens or activated indirectly by inflammatory cytokines. We exposed human monocyte-derived DCs to WNV Kunjin and determined their ability to activate isolated blood iNKTs. DCs became infected as judged by synthesis of viral mRNA and Envelope and NS-1 proteins, but did not undergo significant apoptosis. Infected DCs up-regulated the co-stimulatory molecules CD86 and CD40, but showed decreased expression of CD1d. WNV infection induced DC secretion of type I interferon (IFN), but no or minimal interleukin (IL)-12, IL-23, IL-18 or IL-10. Unexpectedly, we found that the WNV-infected DCs stimulated human iNKTs to up-regulate CD69 and produce low amounts of IL-10, but not proinflammatory cytokines such as IFN-γ or tumour necrosis factor (TNF)-α. Both CD1d and IFNAR blockade partially abrogated this iNKT response, suggesting involvement of a T cell receptor (TCR)-CD1d interaction and type I interferon receptor (IFNAR) signalling. Thus, WNV infection interferes with DC-iNKT interactions by preventing the production of proinflammatory cytokines. iNKTs may be a source of IL-10 observed in human flavivirus infections and initiate an anti-inflammatory innate response that limits adaptive immunity and immune pathology upon WNV infection.

Function and expression of CD1d and invariant natural killer T-cell receptor in the cotton rat (Sigmodon hispidus).

The cotton rat (Sigmodon hispidus) belongs to the rodent family of Cricetidae and provides a powerful model to study the pathogenesis of human respiratory viruses and measles virus. Recent studies in other rodent models have suggested a role for invariant natural killer T (iNKT) cells in antiviral immunity and vaccination against respiratory virus infections. Using new experimental tools, we provide the first evidence for a functional CD1d cell molecule (crCD1d) and iNKT T-cell receptor in cotton rats. The crCD1d cDNA sequence was identified and crCD1d transductants showed that monoclonal antibody WTH-2 stains crCD1d as efficiently as mouse or rat CD1d. The expression of crCD1d was clearly weaker for thymocytes and B cells, and higher for T cells, which is different to what is found in murine species. The antigen-presenting capacity of crCD1d was demonstrated with crCD1d-immunoglobulin dimers loaded with the glycolipid PBS57, which bound iNKT T-cell receptors. Evidence for functional cotton rat iNKT cells was provided by detection of interferon-γ and interleukin-4 in cultures of splenocytes stimulated with PBS57 and α-galactosylceramide and by specific staining of about 0·2% of splenocytes with PBS57-loaded crCD1d dimers. Canonical AV14/AJ18 rearrangements were identified and found to contain multiple members of the AV14 (AV11) family. One of them was expressed and found to bind CD1d dimers. In summary, these data provide the first evidence for functional CD1d molecules and iNKT T-cell receptors in cotton rats and provide the tools to analyse them both in the cotton rat model of infectious diseases.

A bidirectional crosstalk between iNKT cells and adipocytes mediated by leptin modulates susceptibility for T cell mediated hepatitis.

BACKGROUND & AIMS: Immunometabolism is an emerging field of clinical investigation due to the obesity epidemic worldwide. A reciprocal involvement of immune mediators in the body energy metabolism has been recognized for years, but is only partially understood. We hypothesized that the adipokine leptin could provide an important modulator of iNKT cells. METHODS: The expression of leptin receptor (LR) on resting and activated iNKT cells was measured by flow cytometry. FACS-sorted hepatic iNKT cells were stimulated with anti-CD3/CD28Ab coated beads in the absence or presence of a neutralizing anti-leptin Ab. Furthermore, we evaluated the outcome of LR blocking nanobody treatment in ConA induced hepatitis and towards metabolic parameters in WT and iNKT cell deficient mice. RESULTS: The LR is expressed on iNKT cells and leptin suppresses iNKT cell proliferation and cytokine production in vitro. LR deficient iNKT cells are hyper-responsive further enforcing the role of leptin as an important inhibitor of iNKT cell function. Consistently, in vivo blockade of LR signaling exacerbated ConA hepatitis in wild-type but not in iNKT cell deficient mice, through both Janus kinase (JAK)2 and mitogen-activated protein kinase (MAPK) dependent mechanisms. Moreover, LR inhibition altered fat pad features and was accompanied by insulin resistance, only in wild-type mice. Curiously, this interaction was strictly dependent on MAPK mediated LR signaling in iNKT cells and uncoupled from the more central effects of leptin. CONCLUSIONS: Our data support a new concept of immune regulation by which leptin protects towards T cell mediated hepatitis via modulation of iNKT cells.

Activation of invariant natural killer T cells in regional lymph nodes as new antigen-specific immunotherapy via induction of interleukin-21 and interferon-γ.

Invariant natural killer T (iNKT) cells play important immunoregulatory functions in allergen-induced airway hyperresponsiveness and inflammation. To clarify the role of iNKT cells in allergic rhinitis (AR), we generated bone marrow-derived dendritic cells (BMDCs), which were pulsed by ovalbumin (OVA) and α-galactosylceramide (OVA/α-GalCer-BMDCs) and administered into the oral submucosa of OVA-sensitized mice before nasal challenge. Nasal symptoms, level of OVA-specific immunoglobulin (IgE), and T helper type 2 (Th2) cytokine production in cervical lymph nodes (CLNs) were significantly ameliorated in wild-type (WT) mice treated with OVA/α-GalCer-BMDCs, but not in WT mice treated with OVA-BMDCs. These anti-allergic effects were not observed in Jα18(-/-) recipients that lack iNKT cells, even after similar treatment with OVA/α-GalCer-BMDCs in an adoptive transfer study with CD4(+) T cells and B cells from OVA-sensitized WT mice. In WT recipients of OVA/α-GalCer-BMDCs, the number of interleukin (IL)-21-producing iNKT cells increased significantly and the Th1/Th2 balance shifted towards the Th1 dominant state. Treatment with anti-IL-21 and anti-interferon (IFN)-γ antibodies abrogated these anti-allergic effects in mice treated with α-GalCer/OVA-BMDCs. These results suggest that activation of iNKT cells in regional lymph nodes induces anti-allergic effects through production of IL-21 or IFN-γ, and that these effects are enhanced by simultaneous stimulation with antigen. Thus, iNKT cells might be a useful target in development of new treatment strategies for AR.

invariant Natural Killer T cell therapy as a novel therapeutic approach in hematological malignancies.

Invariant Natural Killer T cell therapy is an emerging platform of immunotherapy for cancer treatment. This unique cell population is a promising candidate for cell therapy for cancer treatment because of its inherent cytotoxicity against CD1d positive cancers as well as its ability to induce host CD8 T cell cross priming. Substantial evidence supports that iNKT cells can modulate myelomonocytic populations in the tumor microenvironment to ameliorate immune dysregulation to antagonize tumor progression. iNKT cells can also protect from graft-versus-host disease (GVHD) through several mechanisms, including the expansion of regulatory T cells (Treg). Ultimately, iNKT cell-based therapy can retain antitumor activity while providing protection against GVHD simultaneously. Therefore, these biological properties render iNKT cells as a promising "off-the-shelf" therapy for diverse hematological malignancies and possible solid tumors. Further the introduction of a chimeric antigen recetor (CAR) can further target iNKT cells and enhance function. We foresee that improved vector design and other strategies such as combinatorial treatments with small molecules or immune checkpoint inhibitors could improve CAR iNKT in vivo persistence, functionality and leverage anti-tumor activity along with the abatement of iNKT cell dysfunction or exhaustion.

Nanomapping of CD1d-glycolipid complexes on THP1 cells by using simultaneous topography and recognition imaging.

CD1d molecule, a monomorphic major histocompatibility complex class I-like molecule, presents different types of glycolipids to invariant natural killer T (iNKT) cells that play an important role in immunity to infection and tumors, as well as in regulating autoimmunity. Here, we present simultaneous topography and recognition imaging (TREC) analysis to detect density, distribution and localization of single CD1d molecules on THP1 cells that were loaded with different glycolipids. TREC was conducted using magnetically coated atomic force microscopy tips functionalized with a biotinylated iNKT cell receptor (TCR). The recognition map revealed binding sites visible as dark spots, resulting from oscillation amplitude reduction during specific binding between iNKT TCR and the CD1d-glycolipid complex. THP1 cells were pulsed with three different glycolipids (α-GalCer, C20 and OCH12) for 4 and 16 hr. Whereas CD1d-α-GalCer and CD1d-C20:2 complexes on cellular membrane formed smaller microdomains up to ~10 000 nm(2) (dimension area), OCH12 loaded CD1d complexes presented larger clusters with a dimension up to ~30 000 nm(2). Moreover, the smallest size of recognition spots was about 25 nm, corresponding to a single CD1d binding site. TREC successfully revealed the distribution and localization of CD1d-glycolipid complexes on THP1 cell with single molecule resolution under physiological conditions.

Efficacy of Invariant Natural Killer T Cell Infusion Plus Transarterial Embolization vs Transarterial Embolization Alone for Hepatocellular Carcinoma Patients: A Phase 2 Randomized Clinical Trial.

Purpose: Invariant NKT cells (iNKT) are CD1d-restricted T cells with the capacity of antitumor immunity. The safety of autologous iNKT cell treatment in hepatocellular carcinoma (HCC) has been verified. This study aimed to investigate its efficacy in advanced HCC after transarterial chemoembolization (TACE) failure. Patients and methods: This open-label, randomized, controlled, trial enrolled 60 patients with unresectable HCC after TACE failure at three centers. Transarterial embolization (TAE) was used instead of TACE to protect iNKT cell function. Patients were randomly assigned (1:1) to receive TAE therapy with (TAE-iNKT) or without (TAE) biweekly iNKT cell infusion. The primary endpoint was progression-free survival (PFS). Secondary endpoints included overall survival (OS), objective response rate (ORR), disease control rate (DCR), quality of life (QoL), peripheral blood cell count, and safety. Results: Fifty-four patients completed the study. Median PFS was significantly higher in TAE-iNKT patients (5.7 months [95% CI, 4.3-7.0 months]) compared with TAE patients (2.7 months [95% CI, 2.3-3.2 months]; hazard ratio 0.32 [95% CI, 0.16-0.63]; P<0.001). Higher ORR and DCR were observed in TAE-iNKT patients (52% and 85%, respectively) compared with TAE patients (11% and 33%; respectively). Five TAE-iNKT patients and 1 TAE patient achieved completed response. The median time to deterioration in QoL was longer in TAE-iNKT patients (9.2 months [95% CI, 6.0-13.3 months]) compared with TAE patients (3.0 months [95% CI, 2.9-3.0 months]). The mean lymphocytes were higher in the TAE-iNKT group than in the TAE group at 8 (1.48 vs 0.95×109/L, P = 0.007) and 12 (1.49 vs 0.89×109/L, P = 0.001) weeks. Grade 3 adverse events occurred in 1 TAE-iNKT patient (4%) and 5 TAE patients (19%). All the other adverse events were grade 1-2. Conclusion: iNKT cell infusion significantly improved PFS, ORR, DCR, and QoL with manageable toxicity during TAE therapy in patients with HCC. Trial Registration ClinicalTrials.gov Identifier: NCT04011033.

Mettl3-m6A-Creb1 forms an intrinsic regulatory axis in maintaining iNKT cell pool and functional differentiation.

N6-methyladenosine (m6A) methyltransferase Mettl3 is involved in conventional T cell immunity; however, its role in innate immune cells remains largely unknown. Here, we show that Mettl3 intrinsically regulates invariant natural killer T (iNKT) cell development and function in an m6A-dependent manner. Conditional ablation of Mettl3 in CD4+CD8+ double-positive (DP) thymocytes impairs iNKT cell proliferation, differentiation, and cytokine secretion, which synergistically causes defects in B16F10 melanoma resistance. Transcriptomic and epi-transcriptomic analyses reveal that Mettl3 deficiency disturbs the expression of iNKT cell-related genes with altered m6A modification. Strikingly, Mettl3 modulates the stability of the Creb1 transcript, which in turn controls the protein and phosphorylation levels of Creb1. Furthermore, conditional targeting of Creb1 in DP thymocytes results in similar phenotypes of iNKT cells lacking Mettl3. Importantly, ectopic expression of Creb1 largely rectifies such developmental defects in Mettl3-deficient iNKT cells. These findings reveal that the Mettl3-m6A-Creb1 axis plays critical roles in regulating iNKT cells at the post-transcriptional layer.

Advancing cell-based cancer immunotherapy through stem cell engineering.

Advances in cell-based therapy, particularly CAR-T cell therapy, have transformed the treatment of hematological malignancies. Although an important step forward for the field, autologous CAR-T therapies are hindered by high costs, manufacturing challenges, and limited efficacy against solid tumors. With ongoing progress in gene editing and culture techniques, engineered stem cells and their application in cell therapy are poised to address some of these challenges. Here, we review stem cell-based immunotherapy approaches, stem cell sources, gene engineering and manufacturing strategies, therapeutic platforms, and clinical trials, as well as challenges and future directions for the field.

A single-cell analysis of thymopoiesis and thymic iNKT cell development in pigs.

Many aspects of the porcine immune system remain poorly characterized, which poses a barrier to improving swine health and utilizing pigs as preclinical models. Here, we employ single-cell RNA sequencing (scRNA-seq) to create a cell atlas of the early-adolescent pig thymus. Our data show conserved features as well as species-specific differences in cell states and cell types compared with human thymocytes. We also describe several unconventional T cell types with gene expression profiles associated with innate effector functions. This includes a cell census of more than 11,000 differentiating invariant natural killer T (iNKT) cells, which reveals that the functional diversity of pig iNKT cells differs substantially from the iNKT0/1/2/17 subset differentiation paradigm established in mice. Our data characterize key differentiation events in porcine thymopoiesis and iNKT cell maturation and provide important insights into pig T cell development.

SOX chemotherapy with anti-PD-1 and iNKT cell immunotherapies for stage IV gastric adenocarcinoma with liver metastases: A case report.

Gastric cancer (GC) is the fourth most common cancer worldwide, with overall 5-year survival rate of approximate 20%. Although multimodal treatments that combine surgery with chemotherapy and immunotherapy have been shown to improve survival, pathological complete response (pCR) is rare in advanced GC patients with liver metastases. Pre-clinical studies and clinical trials have demonstrated the antitumor efficacy of invariant natural killer T (iNKT) cells in various malignancies, including GC. While multimodal therapy comprised of chemotherapy, anti-programmed cell death-1 (PD-1) therapy, and iNKT cell immunotherapy have not been reported in GC patients. This case report describes the treatment of an early 60s patient diagnosed with advanced stage IVB (T1N1M1) adenocarcinomas of gastric cardia with liver metastases who received multimodal therapy comprised of SOX chemotherapy, anti-programmed cell death-1 (PD-1) therapy, and iNKT cell immunotherapy followed by surgical resection. Dramatic decreases in tumor area were observed in both the primary tumor and metastatic lesions following six cycles of SOX chemotherapy and iNKT cell immunotherapy, and four cycles of anti-PD-1 therapy. This combined treatment resulted in the transformation of a remarkably large, unresectable liver metastases into a resectable tumor, and the patient received total gastrectomy with D2 lymph node dissection and liver metastasectomy. Subsequent pathological examination detected no cancer cells in either the primary site or liver metastatic lesions, supporting the likelihood that this treatment achieved pCR. To our knowledge, this report represents the first case of a metastatic gastric cancer patient displaying pCR after six months of multimodal therapy, thus supporting that a SOX chemotherapy, anti-PD-1 therapy, and iNKT cell immunotherapy combination strategy may be effective for treating, and potentially curing, patients with advanced gastric adenocarcinoma.