Invariant NKT Cells Research Articles

First-in-human clinical trial of the NKT cell-stimulatory glycolipid OCH in multiple sclerosis

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system that causes the damage to the myelin sheath as well as axonal degeneration. Individuals with MS appear to have changes in the numbers and functions of T-cell subsets, leading to an immunological imbalance accompanied by enhanced autoreactivity. In previous preclinical studies, (2 S,3 S,4R)-1-O-(α-D-Galactopyranosyl)-N-tetracosanoyl-2-amino-1,3,4-nonanetriol (OCH), a synthetic analog of α-galactosylceramide stimulatory for invariant NKT (iNKT) cells, has shown therapeutic or disease-preventive immunoregulatory effects in autoimmune disease models such as experimental autoimmune encephalomyelitis (EAE). This study is the first-in-human study of oral OCH to evaluate the pharmacokinetics and to examine the effects on immune cells as well as related gene expression profiles. Fifteen healthy volunteers and 13 MS patients who met the study criteria were enrolled. They were divided into five cohorts and received oral administration of various doses of granulated powder of OCH (0.3-30 mg), once per week for 4 or 13 weeks. Plasma OCH concentrations were measured by high-performance liquid chromatography. Frequencies of lymphocyte subsets in peripheral blood were evaluated by flow cytometry, and microarray analysis was performed to determine OCH-induced changes in gene expression. Oral OCH was well tolerated, and its bioavailability was found to be sufficient. Six hours after a single dose of OCH, increased frequencies of Foxp3+ regulatory T-cells were observed in some cohorts of healthy subjects and MS patients. Furthermore, gene expression analysis demonstrated an upregulation of several immunoregulatory genes and downregulation of pro-inflammatory genes following OCH administration. This study has demonstrated immunomodulatory effects of the iNKT cell-stimulatory drug OCH in human. Safety profiles together with the presumed anti-inflammatory effects of oral OCH encouraged us to conduct a phase II trial.

Preclinical Development of Non-Edited CD7 CAR-Modified Invariant NKT Cell Therapy for T-Cell Malignancies and Acute Myeloid Leukemia

The major hurdle in developing chimeric antigen receptor T (CAR-T) cell therapy for T-cell malignancies is CAR-T cell fratricide. This refers to the self-killing of the CAR-T cells due to shared expression of targetable antigens (e.g., CD7) between CAR-T and malignant target T cells. This CAR-T fratricide creates difficulties in producing sufficient numbers of CAR-T cells for infusion and can limit survival of the cells in vivo. Nuclease (e.g., CRISPR)-mediated genomic disruption of CD7 or T cell receptor alpha chain expression provides an alternative approach to enabling expansion of CD7 CAR transduced T cells from allogeneic healthy donors. However, the safety and clinical implications of CRISPR-based genome editing remain unclear. Thus, other approaches to development of allogeneic, non-edited CD7 CAR-T cell therapy for relapsed and refractory T-cell malignancies is warranted. We unexpectedly found that human invariant natural killer T (iNKT) cells in normal blood donors displayed on average approximately 56% CD7 positivity (range 38 - 74%, n = 9 donors). Therefore, we hypothesize that CD7- subsets in iNKT cells expressing CD7 CAR can partially avoid fratricide without nuclease-mediated genome editing to delete CD7 and be expanded for adoptive cell therapy of CD7+ malignancies. By leveraging our proprietary 6000-10000-fold iNKT cell expansion platform, lentiviral transduced CD7 CAR iNKT (CD7 CAR-iNKT) cells can be expanded to clinical scale (n=2) (Fig. 1A). Based on CD19 CAR- and CD7 CAR-iNKT expansion rate (n=5), 100-500 doses (assuming 1-3 infusions per patient) of CAR-iNKT cells can be manufactured from a single donor-derived leukocyte concentrate. CD7 CAR-iNKT cells (n=5, 64-98% CAR+) specifically killed luciferase-expressing CD7+ T-cell acute lymphoblastic leukemia (T-ALL) (HSB2, Jurkat, MOLT13) and CD7+ AML (KG1) at E/T ratios ranging from 20:1 to 2.2:1 compared to mock iNKT cells and CD19 CAR-iNKT cells (p < 0.0001). Control CD19 CAR-iNKT cells (n=3) specifically killed CD19+ B-cell tumor cells but not CD19- T-ALL and AML cells (p < 0.0001). Mock iNKT cells showed minimal cytotoxicity to all target cells except for CD1d+ Jurkat. CD7 CAR-iNKT cells (n=5) also produced significant amounts of IFN-γ in response to CD7+ T-ALL (HSB2, Jurkat, MOLT13) and CD7+ AML (KG1, Kasumi-6) compared to mock (n=5, p < 0.001) and CD19 CAR-iNKT cells (p < 0.05 except for Jurkat, p < 0.01). CD19 CAR-iNKT cells (n=3) produced significant amounts of IFN-γ to CD19+ B-cell leukemia and lymphoma cell lines (Nalm6, Daudi, Raji) compared to mock and CD7 CAR-iNKT cells (p < 0.0001). As expected, CD7 CAR-iNKT cells significantly eliminated almost all CD7+ cells (mean ± SD 0.4% ± 0.1, n=4) compared to mock (61.6% ± 24.4, n=4) and CD19 or ROR1 CAR-iNKT cells (69.9% ± 24.4, n=3) (p < 0.0001). Furthermore, expanded CD7 CAR-iNKT cells were >95% CD3+TCRVα24-Jα18+ and specifically responded to a-galactosylceramide or 7DW8-5 lipid antigens presented by CD1d molecules. The anti-leukemic effect of CD7 CAR-modified iNKT cells was assessed in HSB2 T-ALL and KG1 AML xenograft models (n=5 per group). Three i.v. infusions of thawed CD7 CAR-iNKT cells (5x106 cells/mouse) at days 0, 3, and 6 into leukemia (5x105/mouse, i.v. at day -2)-bearing mice significantly suppressed T-ALL growth by bioluminescent imaging analysis at days 3 (p < 0.0001), 6 (p=0.0001-0.0005), 10 (p=0.0012-0.0024) and 17 (p < 0.0001) compared to PBS and mock controls (Fig. 1B). A significant survival advantage was observed after CD7 CAR-iNKT cell therapy compared to PBS and mock controls (p = 0.0009, Fig. 1C). Two i.v. infusions of thawed CD7 CAR-iNKT cells into pre-established AML mice significantly suppressed AML growth at days 3 (p = 0.0096), 6 (p < 0.0001), 10 (p = 0.0026), 17 (p < 0.0001), 24 (p < 0.0001), 31 (p < 0.0001), 38 (p < 0.0001) and 45 (p = 0.0003) compared to mock control (Fig. 1D). CD7 CAR-iNKT cell therapy also significantly prolonged the animal survival (p = 0.0069, Fig. 1E). In conclusion, these results demonstrate that CD7 CAR-iNKT cells without genome editing can be manufactured to therapeutic doses and mount anti-CD7+ tumor responses in vitro and in vivo. Our CD7 CAR-iNKT cells can be used as allogeneic, off-the-shelf products to treat all subtypes of T-ALL and 30% of CD7+ AML patients. In addition, our CD7 CAR-iNKT cells are likely more effective in 70% CD7+CD1d+ T-ALL patients as they provide dual target specificity and reduce the likelihood of relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The role of regenerative invariant NKT cells in cancer immunotherapy for head and neck cancer

The role of regenerative invariant NKT cells in cancer immunotherapy for head and neck cancer

Invariant NKT cells metabolically adapt to the acute myeloid leukaemia environment

Acute myeloid leukaemia (AML) creates an immunosuppressive environment to conventional T cells through Arginase 2 (ARG2)-induced arginine depletion. We identify that AML blasts release the acute phase protein serum amyloid A (SAA), which acts in an autocrine manner to upregulate ARG2 expression and activity, and promote AML blast viability. Following in vitro cross-talk invariant natural killer T (iNKT) cells become activated, upregulate mitochondrial capacity, and release IFN-γ. iNKT retain their ability to proliferate and be activated despite the low arginine AML environment, due to the upregulation of Large Neutral Amino Acid Transporter-1 (LAT-1) and Argininosuccinate Synthetase 1 (ASS)-dependent amino acid pathways, resulting in AML cell death. T cell proliferation is restored in vitro and in vivo. The capacity of iNKT cells to restore antigen-specific T cell immunity was similarly demonstrated against myeloid-derived suppressor cells (MDSCs) in wild-type and Jα18−/− syngeneic lymphoma-bearing models in vivo. Thus, stimulation of iNKT cell activity has the potential as an immunotherapy against AML or as an adjunct to boost antigen-specific T cell immunotherapies in haematological or solid cancers.

T-bet+ B cells accumulate in adipose tissue and exacerbate metabolic disorder during obesity

Obesity is accompanied by inflammation in adipose tissue, impaired glucose tolerance, and changes in adipose leukocyte populations. These studies of adipose tissue from humans and mice revealed that increased frequencies of T-bet+ B cells in adipose tissue depend on invariant NKT cells and correlate with weight gain during obesity. Transfer of B cells enriched for T-bet+ cells exacerbates metabolic disorder in obesity, while ablation of Tbx21 specifically in B cells reduces serum IgG2c levels, inflammatory cytokines, and inflammatory macrophages in adipose tissue, ameliorating metabolic symptoms. Furthermore, transfer of serum or purified IgG from HFD mice restores metabolic disease in T-bet+ B cell-deficient mice, confirming T-bet+ B cell-derived IgG as a key mediator of inflammation during obesity. Together, these findings reveal an important pathological role for T-bet+ B cells that should inform future immunotherapy design in type 2 diabetes and other inflammatory conditions.

Invariant NKT cells dictate antitumor immunity elicited by a bispecific antibody cotargeting CD3 and BCMA

AbstractCD3-engaging bispecific antibodies (BsAbs) have emerged as powerful therapeutic approaches by their ability to redirect T cells to eliminate tumor cells in a major histocompatibility complex–independent manner. However, how we can potentiate the efficacy of BsAbs remains largely unknown. To address this question, we investigated immunological mechanisms of action of a BsAb cotargeting CD3 and B-cell maturation antigen (BCMA) in syngeneic preclinical myeloma models. Treatment with the CD3/BCMA BsAb stimulated multiple CD3-expressing T-cell subsets and natural killer (NK) cells in the myeloma bone marrow (BM), highlighting its broad immunostimulatory effect. Notably, the BsAb-mediated immunostimulatory and antitumor effects were abrogated in mice lacking invariant NKT (iNKT) cells. Mechanistically, activation of iNKT cells and interleukin-12 production from dendritic cells (DCs) were crucial upstream events for triggering effective antitumor immunity by the BsAb. Myeloma progression was associated with a reduced number of BM iNKT cells. Importantly, the therapeutic efficacy of a single dose of CD3/BCMA BsAb was remarkably augmented by restoring iNKT cell activity, using adoptive transfer of α-galactosylceramide-loaded DCs. Together, these results reveal iNKT cells as critical players in the antitumor activity of CD3 engaging BsAbs and have important translational implications.

Genomic and Transcriptional Mechanisms Governing Innate-like T Lymphocyte Development.

Innate-like lymphocytes are a subset of lymphoid cells that function as a first line of defense against microbial infection. These cells are activated by proinflammatory cytokines or broadly expressed receptors and are able to rapidly perform their effector functions owing to a uniquely primed chromatin state that is acquired as a part of their developmental program. These cells function in many organs to protect against disease, but they release cytokines and cytotoxic mediators that can also lead to severe tissue pathologies. Therefore, harnessing the capabilities of these cells for therapeutic interventions will require a deep understanding of how these cells develop and regulate their effector functions. In this review we discuss recent advances in the identification of the transcription factors and the genomic regions that guide the development and function of invariant NKT cells and we highlight related mechanisms in other innate-like lymphocytes.

Re-programming mouse liver-resident invariant natural killer T cells for suppressing hepatic and diabetogenic autoimmunity

Invariant NKT (iNKT) cells comprise a heterogeneous group of non-circulating, tissue-resident T lymphocytes that recognize glycolipids, including alpha-galactosylceramide (αGalCer), in the context of CD1d, but whether peripheral iNKT cell subsets are terminally differentiated remains unclear. Here we show that mouse and human liver-resident αGalCer/CD1d-binding iNKTs largely correspond to a novel Zbtb16+Tbx21+Gata3+MaflowRorc– subset that exhibits profound transcriptional, phenotypic and functional plasticity. Repetitive in vivo encounters of these liver iNKT (LiNKT) cells with intravenously delivered αGalCer/CD1d-coated nanoparticles (NP) trigger their differentiation into immunoregulatory, IL-10+IL-21-producing Zbtb16highMafhighTbx21+Gata3+Rorc– cells, termed LiNKTR1, expressing a T regulatory type 1 (TR1)-like transcriptional signature. This response is LiNKT-specific, since neither lung nor splenic tissue-resident iNKT cells from αGalCer/CD1d-NP-treated mice produce IL-10 or IL-21. Additionally, these LiNKTR1 cells suppress autoantigen presentation, and recognize CD1d expressed on conventional B cells to induce IL-10+IL-35-producing regulatory B (Breg) cells, leading to the suppression of liver and pancreas autoimmunity. Our results thus suggest that LiNKT cells are plastic for further functional diversification, with such plasticity potentially targetable for suppressing tissue-specific inflammatory phenomena.

Gender-dependent frequency of unconventional T cells in a healthy adult Caucasian population: A combinational study of invariant NKT cells, γδ T cells, and mucosa-associated invariant T cells.

This study tested the hypothesis of gender bias in frequency of unconventional T cells. Unconventional T cells exist as minor subsets of T cells in peripheral blood. Despite their low number, they play a crucial role in various immune-mediated diseases such as inflammation, autoimmunity, allergy, and cancer. Gender-based frequency of these cells altogether on large number of healthy individuals are unestablished creating hurdles to manifest association with various immune-mediated pathologic conditions. In this study, we used a multicolor flow cytometric panel to identify iNKT cells, γδ T cells, and MAIT cells altogether in the peripheral blood samples of 93 healthy adult males and 109 healthy adult females from the Caucasian population. The results revealed iNKT cell median value (% T cells) in females was higher: 0.114% ranging from 0.011 to 3.84%, than males: 0.076% (p value 0.0292), ranging from 0.007 to 0.816% and found to be negatively correlated with age in females (p value 0.0047). However, γδ T cell median value in males was higher: 2.52% ranging from 0.31 to 16.09%, than females: 1.79% (p value 0.0155), ranging from 0.078 to 12.49% and each gender was negatively correlated with age (male p value 0.0003 and female p value 0.0007). MAIT cell median values were 3.04% ranging from 0.11 to 10.75% in males and 2.67% ranging from 0.2 to 18.36% in females. MAIT cells did not show any statistically significant difference between genders and found to be negatively correlated with age (p value<0.0001). Our results could be used for further gender-wise investigations of various pathologic conditions such as cancer and their prognosis, autoimmune diseases, allergies, and their pathogenicity.

Adipocyte CD1d Gene Transfer Induces T Cell Expansion and Adipocyte Inflammation in CD1d Knockout Mice.

CD1d, a lipid Ag-presenting molecule for invariant NKT (iNKT) cells, is abundantly expressed on adipocytes and regulates adipose homeostasis through iNKT cells. CD1d gene expression was restored in visceral adipose tissue adipocytes of CD1d knockout (KO) mice to investigate the interactions between adipocytes and immune cells within adipose tissue. We developed an adipocyte-specific targeting recombinant adeno-associated viral vector, with minimal off-target transgene expression in the liver, to rescue CD1d gene expression in visceral adipose tissue adipocytes of CD1d KO mice, followed by assessment of immune cell alternations in adipose tissue and elucidation of the underlying mechanisms of alteration. We report that adeno-associated virus-mediated gene transfer of CD1d to adipocytes in CD1d KO mice fails to rescue iNKT cells but leads to massive and selective expansion of T cells within adipose tissue, particularly CD8+ T effector cells, that is associated with adipocyte NLRP3 inflammasome activation, dysregulation of adipocyte functional genes, and upregulation of apoptotic pathway proteins. An NLRP3 inhibitor has no effect on T cell phenotypes whereas depletion of CD8+ T cells significantly attenuates inflammasome activation and abolishes the dysregulation of adipocyte functional genes induced by adipocyte CD1d. In contrast, adipocyte overexpression of CD1d fails to induce T cell activation in wild-type mice or in invariant TCR α-chain Jα18 KO mice that have a normal lymphocyte repertoire except for iNKT cells. Our studies uncover an adipocyte CD1d → CD8+ T cell → adipocyte inflammasome cascade, in which CD8+ T cells function as a key mediator of adipocyte inflammation likely induced by an allogeneic response against the CD1d molecule.

High Dimensionality Reduction and Immune Phenotyping of Natural Killer and Invariant Natural Killer Cells in Latent Tuberculosis-Diabetes Comorbidity.

Natural killer (NK) and invariant NKT (iNKT) cells are unique innate lymphocytes that coordinate diverse immune responses and display antimycobacterial potential. However, the role of NK and iNKT cells expressing cytokines, cytotoxic, and immune markers in latent tuberculosis (LTB), diabetes mellitus (DM), or preDM (PDM) and nonDM (NDM) comorbidities is not known. Thus, we have studied the unstimulated (UNS), Mycobacterium tuberculosis (Mtb [PPD, WCL]), and mitogen (P/I)-stimulated NK and iNKT cells expressing Type 1 (IFNγ, TNFα, and IL-2), Type 17 (IL-17A, IL-17F, and IL-22) cytokines, cytotoxic (perforin, granzyme B, and granulysin) and immune (GMCSF, PD-1, and CD69) markers in LTB comorbidities by dimensionality reduction and flow cytometry. Our results suggest that LTB DM and PDM individuals express diverse NK and iNKT cell immune clusters compared to LTB NDM individuals. In UNS condition, frequencies of NK and iNKT cells expressing markers are not significantly different. After Mtb antigen stimulation, NK cell expressing [Type 1 (IFNγ, TNFα, and IL-2), GMCSF in PPD and IFNγ in WCL), Type 17 [(IL-17A), PD-1 in PPD), (IL-17A, IL-17F, and IL-22), PD-1 in WCL], and cytotoxic (perforin, granzyme B in PPD, and WCL)] marker frequencies were significantly reduced in LTB DM and/or PDM individuals compared to LTB NDM individuals. Similarly, iNKT cells expressing [Type 1 (IFNγ, IL-2), GMCSF in PPD), TNFα, GMCSF in WCL), Type 17 (IL-17A), PD-1 in PPD, IL-17F in WCL) cytokines were increased and cytotoxic or immune (perforin, granzyme B, granulysin), CD69 in PPD, perforin and CD69 in WCL] marker frequencies were significantly diminished in LTB DM and/or PDM compared to LTB NDM individuals. Finally, NK and iNKT cell frequencies did not exhibit significant differences upon positive control antigen stimulation between the study population. Therefore, altered NK cell and iNKT cells expressing cytokines, cytotoxic, and immune markers are characteristic features in LTB PDM/DM comorbidities.

A phase 1 study of ABX196 in combination with nivolumab in patients with previously treated hepatocellular carcinoma (HCC).

429 Background: ABX196, a synthetically modified α-galactosylceramide (α-GalCer), activates invariant NKT (iNKT) cells and produced anti-tumor activity in Hepa 106 xenograft HCC models. Pre-administration of anti-PD1 antibodies prevented α-GalCer-induced iNKT cell anergy and may also enhance iNKT cell-induced T cell response. We report on a phase 1 study evaluating the combination of ABX196 plus nivolumab (N) in HCC patients previously treated with at least 1 line of prior systemic therapy. Methods: In a 3+3 dose escalation design, intramuscular ABX196 was dosed at 0.1, 0.2, or 0.4 µg 120 mins after N infusion on day 1 of every other 28-day cycle. N (240mg) was administered intravenously on day 1 and 15 of each cycle. Key objectives were to assess safety, MTD, and signs of clinical benefit. Study endpoints included incidence and severity of AEs, laboratory parameters, DLTs, ORR, and PFS. Results: 10 patients (8 males, 2 females) were enrolled: median age, 66y (49-76y); median # of prior systemic therapies, 2 (1-3), including 9 patients with prior immune checkpoint inhibitor (ICI) therapy; median # of ABX196 doses, 2.5 (1-8). There were 76 AEs (95% G1/G2) and 1 non-treatment related SAE. Common non-serious AEs included diarrhea (6), malaise/fatigue (6), AST/ALT increase (6), and only 1 injection site reaction. Maximum administered dose was 0.4 μg; MTD not reached. Clinical benefit was observed in 5 patients (50%) including 1 patient with a PR (ORR 10%) and 4 patients with SD (40%). Of these 5 patients, 3 had viral hepatitis. Median PFS for all patients was 113.5 days (49-450 days), but for those with clinical benefit it was 276 days (172-450 days). Conclusions: ABX196 plus N was very well tolerated without any DLTs or treatment emergent SAEs. In this small but heavily pre-treated HCC population, ABX196 plus N demonstrated promising signals of clinical benefit, including in patients with previous ICI therapy. These results support further clinical development of ABX196 in the front-line HCC setting. Clinical trial information: NCT03897543. [Table: see text]

Recent Development in NKT-Based Immunotherapy of Glioblastoma: From Bench to Bedside.

Glioblastoma multiforme (GBM) is an aggressive and dismal disease with a median overall survival of around 15 months and a 5-year survival rate of 7.2%. Owing to genetic mutations, drug resistance, disruption to the blood–brain barrier (BBB)/blood–brain tumor barrier (BBTB), and the complexity of the immunosuppressive environment, the therapeutic approaches to GBM represent still major challenges. Conventional therapies, including surgery, radiotherapy, and standard chemotherapy with temozolomide, have not resulted in satisfactory improvements in the overall survival of GBM patients. Among cancer immunotherapeutic approaches, we propose that adjuvant NKT immunotherapy with invariant NKT (iNKT) and cytokine-induced killer (CIK) cells may improve the clinical scenario of this devastating disease. Considering this, herein, we discuss the current strategies of NKT therapy for GBM based primarily on in vitro/in vivo experiments, clinical trials, and the combinatorial approaches with future therapeutic potential.

Use of a Clostridioides difficile Murine Immunization and Challenge Model to Evaluate Single and Combination Vaccine Adjuvants Consisting of Alum and NKT Cell-Activating Ligands.

Adjuvant combinations may enhance or broaden the expression of immune responses to vaccine antigens. Information on whether established Alum type adjuvants can be combined with experimental CD1d ligand adjuvants is currently lacking. In this study, we used a murine Clostridioides difficile immunization and challenge model to evaluate Alum (Alhydrogel™), α-galactosylceramide (α-GC), and one of its analogs 7DW8-5 singly and in combination as vaccine adjuvants. We observed that the Alum/α-GC combination caused modest enhancement of vaccine antigen-specific IgG1 and IgG2b responses, and a broadening to include IgG2c that did not significantly impact overall protection. Similar observations were made using the Alum/7DW8-5 combination. Examination of the impact of adjuvants on NKT cells revealed expansion of invariant NKT (iNKT) cells with modest expansion of their iNKTfh subset and little effect on diverse NKT (dNKT) cells. Side effects of the adjuvants was determined and revealed transient hepatotoxicity when Alum/α-GC was used in combination but not singly. In summary these results showed that the Alum/α-GC or the Alum/7DW8-5 combination could exert distinct effects on the NKT cell compartment and on isotype switch to produce Th1-driven IgG subclasses in addition to Alum/Th2-driven subclasses. While Alum alone was efficacious in stimulating IgG-mediated protection, and α-GC offered no apparent additional benefit in the C. difficile challenge model, the work herein reveals immune response features that could be optimized and harnessed in other vaccine contexts.

Conventional type 1 dendritic cells protect against age-related adipose tissue dysfunction and obesity

Conventional dendritic cells (cDCs) scan and integrate environmental cues in almost every tissue, including exogenous metabolic signals. While cDCs are critical in maintaining immune balance, their role in preserving energy homeostasis is unclear. Here, we showed that Batf3-deficient mice lacking conventional type 1 DCs (cDC1s) had increased body weight and adiposity during aging. This led to impaired energy expenditure and glucose tolerance, insulin resistance, dyslipidemia, and liver steatosis. cDC1 deficiency caused adipose tissue inflammation that was preceded by a paucity of NK1.1+ invariant NKT (iNKT) cells. Accordingly, among antigen-presenting cells, cDC1s exhibited notable induction of IFN-γ production by iNKT cells, which plays a metabolically protective role in lean adipose tissue. Flt3L treatment, which expands the dendritic cell (DC) compartment, mitigated diet-induced obesity and hyperlipidemia in a Batf3-dependent manner. This effect was partially mediated by NK1.1+ cells. These results reveal a new critical role for the cDC1-iNKT cell axis in the regulation of adipose tissue homeostasis.

Protein abundance of the cytokine receptor γc controls the thymic generation of innate-like T cells.

Innate-like T (iT) cells comprise a population of immunoregulatory T cells whose effector function is imposed during their development in the thymus to provide protective immunity prior to antigen encounter. The molecular mechanism that drives the generation of iT cells remains unclear. Here, we report that the cytokine receptor γc plays a previously unappreciated role for thymic iT cells by controlling their cellular abundance, lineage commitment, and subset differentiation. As such, γc overexpression on thymocytes dramatically altered iT cell generation in the thymus, as it skewed the subset composition of invariant NKT (iNKT) cells and promoted the generation of IFNγ-producing innate CD8 T cells. Mechanistically, we found that the γc-STAT6 axis drives the differentiation of IL-4-producing iNKT cells, which in turn induced the generation of innate CD8 T cells. Collectively, these results reveal a cytokine-driven circuity of thymic iT cell differentiation that is controlled by the abundance of γc proteins.

Invariant NKT Cells Promote the Development of Highly Cytotoxic Multipotent CXCR3+CCR4+CD8+ T Cells That Mediate Rapid Hepatocyte Allograft Rejection.

Hepatocyte transplant represents a treatment for metabolic disorders but is limited by immunogenicity. Our prior work identified the critical role of CD8+ T cells, with or without CD4+ T cell help, in mediating hepatocyte rejection. In this study, we evaluated the influence of invariant NKT (iNKT) cells, uniquely abundant in the liver, upon CD8-mediated immune responses in the presence and absence of CD4+ T cells. To investigate this, C57BL/6 (wild-type) and iNKT-deficient Jα18 knockout mice (cohorts CD4 depleted) were transplanted with allogeneic hepatocytes. Recipients were evaluated for alloprimed CD8+ T cell subset composition, allocytotoxicity, and hepatocyte rejection. We found that CD8-mediated allocytotoxicity was significantly decreased in iNKT-deficient recipients and was restored by adoptive transfer of iNKT cells. In the absence of both iNKT cells and CD4+ T cells, CD8-mediated allocytotoxicity and hepatocyte rejection was abrogated. iNKT cells enhance the proportion of a novel subset of multipotent, alloprimed CXCR3+CCR4+CD8+ cytolytic T cells that develop after hepatocyte transplant and are abundant in the liver. Alloprimed CXCR3+CCR4+CD8+ T cells express cytotoxic effector molecules (perforin/granzyme and Fas ligand) and are distinguished from alloprimed CXCR3+CCR4-CD8+ T cells by a higher proportion of cells expressing TNF-α and IFN-γ. Furthermore, alloprimed CXCR3+CCR4+CD8+ T cells mediate higher allocytotoxicity and more rapid allograft rejection. Our data demonstrate the important role of iNKT cells in promoting the development of highly cytotoxic, multipotent CXCR3+CCR4+CD8+ T cells that mediate rapid rejection of allogeneic hepatocytes engrafted in the liver. Targeting iNKT cells may be an efficacious therapy to prevent rejection of intrahepatic cellular transplants.

Thymus-Derived CD4+CD8+ Cells Reside in Mediastinal Adipose Tissue and the Aortic Arch.

Double-positive CD4+CD8αβ+ (DP) cells are thought to reside as T cell progenitors exclusively within the thymus. We recently discovered an unexpected CD4+ and CD8αβ+ immune cell population in healthy and atherosclerotic mice by single-cell RNA sequencing. Transcriptomically, these cells resembled thymic DPs. Flow cytometry and three-dimensional whole-mount imaging confirmed DPs in thymus, mediastinal adipose tissue, and aortic adventitia, but nowhere else. Deep transcriptional profiling revealed differences between DP cells isolated from the three locations. All DPs were dependent on RAG2 expression and the presence of the thymus. Mediastinal adipose tissue DPs resided in close vicinity to invariant NKT cells, which they could activate in vitro. Thymus transplantation failed to reconstitute extrathymic DPs, and frequencies of extrathymic DPs were unaltered by pharmacologic inhibition of S1P1, suggesting that their migration may be locally confined. Our results define two new, transcriptionally distinct subsets of extrathymic DPs that may play a role in aortic vascular homeostasis.

CD138 expression is a molecular signature but not a developmental requirement for RORγt+ NKT17 cells.

Invariant NKT (iNKT) cells are potent immunomodulatory cells that acquire effector function during their development in the thymus. IL-17–producing iNKT cells are commonly referred to as NKT17 cells, and they are unique among iNKT cells to express the heparan sulfate proteoglycan CD138 and the transcription factor RORγt. Whether and how CD138 and RORγt contribute to NKT17 cell differentiation, and whether there is an interplay between RORγt and CD138 expression to control iNKT lineage fate, remain mostly unknown. Here, we showed that CD138 expression was only associated with and not required for the differentiation and IL-17 production of NKT17 cells. Consequently, CD138-deficient mice still generated robust numbers of IL-17–producing RORγt+ NKT17 cells. Moreover, forced expression of RORγt significantly promoted the generation of thymic NKT17 cells, but did not induce CD138 expression on non-NKT17 cells. These results indicated that NKT17 cell generation and IL-17 production were driven by RORγt, employing mechanisms that were independent of CD138. Therefore, our study effectively dissociated CD138 expression from the RORγt-driven molecular pathway of NKT17 cell differentiation.

Description of invariant NKT cells.

Recently discovered subpopulations belonging to cell groups called “invariant” NKT, also referred to as type I NKT or classical NKT cells, appear to have a unique impact on the tumor microenvironment. Considering the characteristics of secreted cytokines, production of chemokines and the capability of cell cytotoxic activity, these cells have a significant function in being immune to bacteria, parasites, viruses, autoimmune and tumor diseases. The profile of secreted cytokine is probably decisive in reference to the distinct functions of the subpopulation, some of them exhibit immunosuppressive activity inhibiting an anticancer immune response, and the other stimulate the organisms for eradicating the tumor. Currently, it has been discovered six fundamental, functionally distinct subpopulations of iNKT cells (NKT1, NKT2, NKT10, NKT17, NKTreg, NKTFH).