Natural killer T cell immunotherapy combined with oncolytic vesicular stomatitis virus or reovirus treatments differentially increases survival in mouse models of ovarian and breast cancer metastasis.

BACKGROUND: Oncolytic viruses reduce tumor burden in animal models and have generated promising results in clinical trials. However, it is likely that oncolytic viruses will be more effective when used in combination with other therapies. Current therapeutic approaches, including chemotherapeutics, come with dose-limiting toxicities. Another option is to combine oncolytic viruses with immunotherapeutic approaches. METHODS: Using experimental models of metastatic 4T1 breast cancer and ID8 ovarian peritoneal carcinomatosis, we examined natural killer T (NKT) cell-based immunotherapy in combination with recombinant oncolytic vesicular stomatitis virus (VSV) or reovirus. 4T1 mammary carcinoma cells or ID8 ovarian cancer cells were injected into syngeneic mice. Tumor-bearing mice were treated with VSV or reovirus followed by activation of NKT cells via the intravenous administration of autologous dendritic cells loaded with the glycolipid antigen α-galactosylceramide. The effects of VSV and reovirus on immunogenic cell death (ICD), cell viability and immunogenicity were tested in vitro. RESULTS: VSV or reovirus treatments followed by NKT cell activation mediated greater survival in the ID8 model than individual therapies. The regimen was less effective when the treatment order was reversed, delivering virus treatments after NKT cell activation. In the 4T1 model, VSV combined with NKT cell activation increased overall survival and decreased metastatic burden better than individual treatments. In contrast, reovirus was not effective on its own or in combination with NKT cell activation. In vitro, VSV killed a panel of tumor lines better than reovirus. VSV infection also elicited greater increases in mRNA transcripts for proinflammatory cytokines, chemokines, and antigen presentation machinery compared with reovirus. Oncolytic VSV also induced the key hallmarks of ICD (calreticulin mobilization, plus release of ATP and HMGB1), while reovirus only mobilized calreticulin. CONCLUSION: Taken together, these results demonstrate that oncolytic VSV and NKT cell immunotherapy can be effectively combined to decrease tumor burden in models of metastatic breast and ovarian cancers. Oncolytic VSV and reovirus induced differential responses in our models which may relate to differences in virus activity or tumor susceptibility.

IL-21 arming potentiates the anti-tumor activity of an oncolytic vaccinia virus in monotherapy and combination therapy.

BACKGROUND: Oncolytic viruses (OVs) have shown promise in containing cancer progression in both animal models and clinical trials. How to further improve the efficacy of OVs are intensively explored. Arming OVs with immunoregulatory molecules has emerged as an important means to enhance their oncolytic activities majorly based on the mechanism of reverting the immunosuppressive nature of tumor environment. In this study, we aimed to identify the optimal combination of different OVs and immunomodulatory molecules for solid tumor treatment as well as the underlying mechanism, and subsequently evaluated its potential synergy with other immunotherapies. METHODS: Panels of oncolytic viruses and cells stably expressing immunoregulatory molecules were separately evaluated for treating solid tumors in mouse model. A tumor-targeted replicating vaccinia virus Tian Tan strain with deletion of TK gene (TTVΔTK) was armed rationally with IL-21 to create rTTVΔTK-IL21 through recombination. CAR-T cells and iNKT cells were generated from human peripheral blood mononuclear cells. The impact of rTTVΔTK-IL21 on tumor-infiltrating lymphocytes was assessed by flow cytometry, and its therapeutic efficacy as monotherapy or in combination with CAR-T and iNKT therapy was assessed in mouse tumor models. RESULTS: IL-21 and TTV was respectively identified as most potent immunomodulatory molecule and oncolytic virus for solid tumor suppression in mouse models. A novel recombinant oncolytic virus that resulted from their combination, namely rTTVΔTK-mIL21, led to significant tumor regression in mice, even for noninjected distant tumor. Mechanistically, rTTV∆TK-mIL21 induced a selective enrichment of immune effector cells over Treg cells and engage a systemic response of therapeutic effect. Moreover, its human form showed a notable synergy with CAR-T or iNKT therapy for tumor treatment when coupled in humanized mice. CONCLUSION: With a strong potency of shaping tumor microenvironment toward favoring TIL activities, rTTVΔTK-IL21 represents a new opportunity worthy of further exploration in clinical settings for solid tumor control, particularly in combinatorial strategies with other immunotherapies. ONE SENTENCE SUMMARY: IL21-armed recombinant oncolytic vaccinia virus has potent anti-tumor activities as monotherapy and in combination with other immunotherapies.

CD1d expression in glioblastoma is a promising target for NKT cell-based cancer immunotherapy.

Glioblastoma is the most common and aggressive type of brain tumor with high recurrence and fatality rates. Although various therapeutic strategies have been explored, there is currently no effective treatment for glioblastoma. Recently, the number of immunotherapeutic strategies has been tested for malignant brain tumors. Invariant natural killer T (iNKT) cells play an important role in anti-tumor immunity. To address if iNKT cells can target glioblastoma to exert anti-tumor activity, we assessed the expression of CD1d, an antigen-presenting molecule for iNKT cells, on glioblastoma cells. Glioblastoma cells from 10 of 15 patients expressed CD1d, and CD1d-positive glioblastoma cells pulsed with glycolipid ligand induced iNKT cell-mediated cytotoxicity in vitro. Although CD1d expression was low on glioblastoma stem-like cells, retinoic acid, which is the most common differentiating agent, upregulated CD1d expression in these cells and induced iNKT cell-mediated cytotoxicity. Moreover, intracranial administration of human iNKT cells induced tumor regression of CD1d-positive glioblastoma in orthotopic xenografts in NOD/Shi-scid IL-2RγKO (NOG) mice. Thus, CD1d expression represents a novel target for NKT cell-based immunotherapy for glioblastoma patients.

Using agonists for iNKT cells in cancer therapy.

The capacity of α-galactosylceramide (α-GalCer) to act as an anti-cancer agent in mice through the specific stimulation of type I NKT (iNKT) cells has prompted extensive investigation to translate this finding to the clinic. However, low frequencies of iNKT cells in cancer patients and their hypo-responsiveness to repeated stimulation have been seen as barriers to its efficacy. Currently the most promising clinical application of α-GalCer, or its derivatives, is as stimuli for ex vivo expansion of iNKT cells for adoptive therapy, although some encouraging clinical results have recently been reported using α-GalCer pulsed onto large numbers of antigen presenting cells (APCs). In on-going preclinical studies, attempts to improve efficacy of injected iNKT cell agonists have focussed on optimising presentation in vivo, through encapsulation in particulate vectors, making structural changes that help binding to the presenting molecule CD1d, or injecting agonists covalently attached to recombinant CD1d. Variations on these same approaches are being used to enhance the APC-licencing function of iNKT cells in vivo to induce adaptive immune responses to associated tumour antigens. Looking ahead, a unique capacity of in vivo-activated iNKT cells to facilitate formation of resident memory CD8+ T cells is a new observation that could find a role in cancer therapy.

Killing efficiency affected by mutually modulated PD-1 and PD-L1 expression via NKT-hepatoma cell interactions.

Aim: To explore the expression of programmed death-1 (PD-1) or programmed death ligand 1 (PD-L1), natural killer T (NKT) and hepatoma cells in coculture system, and the influence of abolishing PD-1 on antitumor efficiency. Materials & methods: CRISPR/Cas9 technology, flow cytometry, ELISA, CCK-8 assay and mouse models were performed to investigate the interactions between PD-1/PD-L1 expression on NKT and hepatoma cells, respectively. Results: The NKT and hepatoma cells mutually affected the expression of PD-1/PD-L1. The killing effect was positively correlated with NKT-mediated PD-L1 expression on hepatoma cells. Conclusion: Hepatoma cells in different genetic background responded differently to NKT-induced PD-L1 stimulation, and those cells with lower PD-L1 expression fail to PD-1 blocking intervention. Additionally, the killing effect was more time-efficient with PD-1 knockout than with monoclonal antibody blockade.

Enhancing Neuroblastoma Immunotherapies by Engaging iNKT and NK Cells.

Neuroblastoma (NB) is the most common extracranial solid tumor in children and, in the high-risk group, has a 5-year mortality rate of ~50%. The high mortality rate and significant treatment-related morbidities associated with current standard of care therapies belie the critical need for more tolerable and effective treatments for this disease. While the monoclonal antibody dinutuximab has demonstrated the potential for immunotherapy to improve overall NB outcomes, the 5-year overall survival of high-risk patients has not yet substantially changed. The frequency and type of invariant natural killer T cells (iNKTs) and natural killer cells (NKs) has been associated with improved outcomes in several solid and liquid malignancies, including NB. Indeed, iNKTs and NKs inhibit tumor associated macrophages (TAMs) and myeloid derived suppressor cells (MDSCs), kill cancer stem cells (CSCs) and neuroblasts, and robustly secrete cytokines to recruit additional immune effectors. These capabilities, and promising pre-clinical and early clinical data suggest that iNKT- and NK-based therapies may hold promise as both stand-alone and combination treatments for NB. In this review we will summarize the biologic features of iNKTs and NKs that confer advantages for NB immunotherapy, discuss the barriers imposed by the NB tumor microenvironment, and examine the current state of such therapies in pre-clinical models and clinical trials.

Can we prevent or treat graft-versus-host disease with cellular-therapy?

Acute and chronic graft-versus-host disease (GvHD) are the most important causes of treatment-related morbidity and mortality after allogeneic hematopoietic cell transplants for various diseases. Corticosteroids are an effective therapy in only about one-half of affected individuals and new therapy options are needed. We discuss novel strategies to treat GvHD using cellular-therapy including adoptive transfer of regulatory T-cells (Tregs), mesenchymal stromal cells (MSCs), cells derived from placental tissues, invariant natural killer T-cells (iNKTs), and myeloid-derived suppressor cells (MDSCs).These strategies may be more selective than drugs in modulating GvHD pathophysiology, and may be safer and more effective than conventional pharmacologic therapies. Additionally, these therapies have not been observed to substantially compromise the graft-versus-tumor effect associated with allotransplants. Many of these strategies are effective in animal models but substantial data in humans are lacking.

Immune Networks and Therapeutic Targeting of iNKT Cells in Cancer.

One of the primary goals in tumor immunotherapy is to reset the immune system from tolerogenic to immunogenic - a process in which invariant natural killer T (iNKT) cells are implicated. iNKT cells develop in the thymus and perform immunosurveillance against tumor cells peripherally. When optimally stimulated, iNKT cells differentiate and display more efficient immune functions. Some cells survive and act as effector memory cells. We discuss the putative roles of iNKT cells in antitumor immunity, and posit that it may be possible to develop novel therapeutic strategies to treat cancers using iNKT cells. In particular, we highlight the challenge of uniquely energizing iNKT cell-licensed dendritic cells to serve as effective immunoadjuvants for both arms of the immune system, thus coupling immunological networks.

Development of Hematopoietic Stem Cell-Engineered Invariant Natural Killer T Cell Therapy for Cancer.

Invariant natural killer T (iNKT) cells are potent immune cells for targeting cancer; however, their clinical application has been hindered by their low numbers in cancer patients. Here, we developed a proof-of-concept for hematopoietic stem cell-engineered iNKT (HSC-iNKT) cell therapy with the potential to provide therapeutic levels of iNKT cells for a patient's lifetime. Using a human HSC engrafted mouse model and a human iNKT TCR gene engineering approach, we demonstrated the efficient and long-term generation of HSC-iNKT cells in vivo. These HSC-iNKT cells closely resembled endogenous human iNKT cells, could deploy multiple mechanisms to attack tumor cells, and effectively suppressed tumor growth in vivo in multiple human tumor xenograft mouse models. Preclinical safety studies showed no toxicity or tumorigenicity of the HSC-iNKT cell therapy. Collectively, these results demonstrated the feasibility, safety, and cancer therapy potential of the proposed HSC-iNKT cell therapy and laid a foundation for future clinical development.

NKT Cells Coexpressing a GD2-Specific Chimeric Antigen Receptor and IL15 Show Enhanced In Vivo Persistence and Antitumor Activity against Neuroblastoma.

PURPOSE: Vα24-invariant natural killer T cells (NKT) are attractive carriers for chimeric antigen receptors (CAR) due to their inherent antitumor properties and preferential localization to tumor sites. However, limited persistence of CAR-NKTs in tumor-bearing mice is associated with tumor recurrence. Here, we evaluated whether coexpression of the NKT homeostatic cytokine IL15 with a CAR enhances the in vivo persistence and therapeutic efficacy of CAR-NKTs. EXPERIMENTAL DESIGN: Human primary NKTs were ex vivo expanded and transduced with CAR constructs containing an optimized GD2-specific single-chain variable fragment and either the CD28 or 4-1BB costimulatory endodomain, each with or without IL15 (GD2.CAR or GD2.CAR.15). Constructs that mediated robust CAR-NKT cell expansion were selected for further functional evaluation in vitro and in xenogeneic mouse models of neuroblastoma. RESULTS: Coexpression of IL15 with either costimulatory domain increased CAR-NKT absolute numbers. However, constructs containing 4-1BB induced excessive activation-induced cell death and reduced numeric expansion of NKTs compared with respective CD28-based constructs. Further evaluation of CD28-based GD2.CAR and GD2.CAR.15 showed that coexpression of IL15 led to reduced expression levels of exhaustion markers in NKTs and increased multiround in vitro tumor cell killing. Following transfer into mice bearing neuroblastoma xenografts, GD2.CAR.15 NKTs demonstrated enhanced in vivo persistence, increased localization to tumor sites, and improved tumor control compared with GD2.CAR NKTs. Importantly, GD2.CAR.15 NKTs did not produce significant toxicity as determined by histopathologic analysis. CONCLUSIONS: Our results informed selection of the CD28-based GD2.CAR.15 construct for clinical testing and led to initiation of a first-in-human CAR-NKT cell clinical trial (NCT03294954).

IL-21 Selectively Protects CD62L+ NKT Cells and Enhances Their Effector Functions for Adoptive Immunotherapy.

T cells expressing CD19-specific chimeric Ag receptors (CARs) produce high remission rates in B cell lymphoma, but frequent disease recurrence and challenges in generating sufficient numbers of autologous CAR T cells necessitate the development of alternative therapeutic effectors. Vα24-invariant NKTs have intrinsic antitumor properties and are not alloreactive, allowing for off-the-shelf use of CAR-NKTs from healthy donors. We recently reported that CD62L+ NKTs persist longer and have more potent antilymphoma activity than CD62L- cells. However, the conditions governing preservation of CD62L+ cells during NKT cell expansion remain largely unknown. In this study, we demonstrate that IL-21 preserves this crucial central memory-like NKT subset and enhances its antitumor effector functionality. We found that following antigenic stimulation with α-galactosylceramide, CD62L+ NKTs both expressed IL-21R and secreted IL-21, each at significantly higher levels than CD62L- cells. Although IL-21 alone failed to expand stimulated NKTs, combined IL-2/IL-21 treatment produced more NKTs and increased the frequency of CD62L+ cells versus IL-2 alone. Gene expression analysis comparing CD62L+ and CD62L- cells treated with IL-2 alone or IL-2/IL-21 revealed that the latter condition downregulated the proapoptotic protein BIM selectively in CD62L+ NKTs, protecting them from activation-induced cell death. Moreover, IL-2/IL-21-expanded NKTs upregulated granzyme B expression and produced more TH1 cytokines, leading to enhanced in vitro cytotoxicity of nontransduced and anti-CD19-CAR-transduced NKTs against CD1d+ and CD19+ lymphoma cells, respectively. Further, IL-2/IL-21-expanded CAR-NKTs dramatically increased the survival of lymphoma-bearing NSG mice compared with IL-2-expanded CAR-NKTs. These findings have immediate translational implications for the development of NKT cell-based immunotherapies targeting lymphoma and other malignancies.

iNKT cells ameliorate human autoimmunity: Lessons from alopecia areata.

Alopecia areata (AA) is understood to be a CD8+/NKG2D+ T cell-dependent autoimmune disease. Here, we demonstrate that human AA pathogenesis of is also affected by iNKT10 cells, an unconventional T cell subtype whose number is significantly increased in AA compared to healthy human skin. AA lesions can be rapidly induced in healthy human scalp skin xenotransplants on Beige-SCID mice by intradermal injections of autologous healthy-donor PBMCs pre-activated with IL-2. We show that in this in vivo model, the development of AA lesions is prevented by recognized the iNKT cell activator, α-galactosylceramide (α-GalCer), which stimulates iNKT cells to expand and produce IL-10. Moreover, in pre-established humanized mouse AA lesions, hair regrowth is promoted by α-GalCer treatment through a process requiring both effector-memory iNKT cells, which can interact directly with CD8+/NKG2D+ T cells, and IL-10. This provides the first in vivo evidence in a humanized model of autoimmune disease that iNKT10 cells are key disease-protective lymphocytes. Since these regulatory NKT cells can both prevent the development of AA lesions and promote hair re-growth in established AA lesions, targeting iNKT10 cells may have preventive and therapeutic potential also in other autoimmune disorders related to AA.

Novel Approaches to Exploiting Invariant NKT Cells in Cancer Immunotherapy.

iNKT cells are a subset of innate-like T cells that utilize an invariant TCR alpha chain complexed with a limited repertoire of TCR beta chains to recognize specific lipid antigens presented by CD1d molecules. Because iNKT cells have an invariant TCR, they can be easily identified and targeted in both humans and mice via standard reagents, making this a population of T cells that has been well characterized. iNKT cells are some of the first cells to respond during an infection. By making different types of cytokines in response to different infection stimuli, iNKT cells help determine what kind of immune response then develops. It has been shown that iNKT cells are some of the first cells to respond during infection with a pathogen and the type of cytokines that iNKT cells make help determine the type of immune response that develops in various situations. Indeed, along with immunity to pathogens, pre-clinical mouse studies have clearly demonstrated that iNKT cells play a critical role in tumor immunosurveillance. They can mediate anti-tumor immunity by direct recognition of tumor cells that express CD1d, and/or via targeting CD1d found on cells within the tumor microenvironment. Multiple groups are now working on manipulating iNKT cells for clinical benefit within the context of cancer and have demonstrated that targeting iNKT cells can have a therapeutic benefit in patients. In this review, we briefly introduce iNKT cells, then discuss preclinical data on roles of iNKT cells and clinical trials that have targeted iNKT cells in cancer patients. We finally discuss how future trials could be modified to further increase the efficacy of iNKT cell therapies, in particular CAR-iNKT and rTCR-iNKT cells.

Development of Anti-Human Mesothelin-Targeted Chimeric Antigen Receptor Messenger RNA-Transfected Peripheral Blood Lymphocytes for Ovarian Cancer Therapy.

CD19-targeted chimeric antigen receptor (CAR) engineered T/natural killer (NK)-cell therapies can result in durable clinical responses in B-cell malignancies. However, CAR-based immunotherapies have been much less successful in solid cancers, in part due to "on-target off-tumor" toxicity related to expression of target tumor antigens on normal tissue. Based on preliminary observations of safety and clinical activity in proof-of-concept clinical trials, tumor antigen-specific messenger RNA (mRNA) CAR transfection into selected, activated, and expanded T/NK cells may permit prospective control of "on-target off-tumor" toxicity. To develop a commercial product for solid tumors, mesothelin was selected as an antigen target based on its association with poor prognosis and overexpression in multiple solid cancers. It was hypothesized that selecting, activating, and expanding cells ex vivo prior to mRNA CAR transfection would not be necessary, thus simplifying the complexity and cost of manufacturing. Now, the development of anti-human mesothelin mRNA CAR transfected peripheral blood lymphocytes (CARMA-hMeso) is reported, demonstrating the manufacture and cryopreservation of multiple cell aliquots for repeat administrations from a single human leukapheresis. A rapid, automated, closed system for cGMP-compliant transfection of mRNA CAR in up to 20 × 109 peripheral blood lymphocytes was developed. Here we show that CARMA-hMeso cells recognize and lyse tumor cells in a mesothelin-specific manner. Expression of CAR was detectable over approximately 7 days in vitro, with a progressive decline of CAR expression that appears to correlate with in vitro cell expansion. In a murine ovarian cancer model, a single intraperitoneal injection of CARMA-hMeso resulted in the dose-dependent inhibition of tumor growth and improved survival of mice. Furthermore, repeat weekly intraperitoneal administrations of the optimal CARMA-hMeso dose further prolonged disease control and survival. No significant off-target toxicities were observed. These data support further investigation of CARMA-hMeso as a potential treatment for ovarian cancer and other mesothelin-expressing cancers.

Innate immune cells for immunotherapy of autoimmune and cancer disorders.

Modulation of the immune system has been widely targeted for the treatment of several immune-related diseases, such as autoimmune disorders and cancer, due to its crucial role in these pathologies. Current available therapies focus mainly on symptomatic treatment and are often associated with undesirable secondary effects. For several years, remission of disease and subsequently recovery of immune homeostasis has been a major goal for immunotherapy. Most current immunotherapeutic strategies are aimed to inhibit or potentiate directly the adaptive immune response by modulating antibody production and B cell memory, as well as the effector potential and memory of T cells. Although these immunomodulatory approaches have shown some success in the clinic with promising therapeutic potential, they have some limitations related to their effectiveness in disease models and clinical trials, as well as elevated costs. In the recent years, a renewed interest has emerged on targeting innate immune cells for immunotherapy, due to their high plasticity and ability to exert a potent and extremely rapid response, which can influence the outcome of the adaptive immune response. In this review, we discuss the immunomodulatory potential of several innate immune cells, as well as they use for immunotherapy, especially in autoimmunity and cancer.

Invariant natural killer T cells ameliorate murine chronic GVHD by expanding donor regulatory T cells.

Chronic graft-versus-host-disease (cGVHD) can cause multiorgan system disease, typically with autoimmune-like features, resulting in high mortality and morbidity caused by treatment limitations. Invariant natural killer T cells (iNKTs), a small population characterized by expression of a semi-invariant T-cell receptor, rapidly produce copious amounts of diverse cytokines on activation that exert potent immune regulatory function. Here, we show that iNKTs are significantly reduced in a cGVHD murine model that recapitulates several aspects of autoimmunity and organ fibrosis observed in patients with cGVHD. Low iNKT infused doses effectively prevented and, importantly, reversed established cGVHD, as did third-party iNKTs. iNKTs suppressed the autoimmune response by reducing the germinal center (GC) reaction, which was associated with an increase in total Tregs and follicular Tregs (Tfr) that control the GC reaction, along with pathogenic antibody production. Treg depletion during iNKT infusions completely abolished iNKT efficacy in treating cGVHD. iNKT cell interleukin 4 production and GC migration were critical to cGVHD reversal. In vivo stimulation of iNKT cells by α-galactosyl-ceramide was effective in both preventing and treating cGVHD. Together, this study demonstrates iNKT deficiency in cGVHD mice and highlights the key role of iNKTs in regulating cGVHD pathogenesis and as a potentially novel prophylactic and therapeutic option for patients with cGVHD.

Adoptive Transfer of Invariant NKT Cells as Immunotherapy for Advanced Melanoma: A Phase I Clinical Trial.

Purpose: Invariant NKT cells (iNKT) are innate-like CD1d-restricted T cells with immunoregulatory activity in diseases including cancer. iNKT from advanced cancer patients can have reversible defects including IFNγ production, and iNKT IFNγ production may stratify for survival. Previous clinical trials using iNKT cell activating ligand α-galactosylceramide have shown clinical responses. Therefore, a phase I clinical trial was performed of autologous in vitro expanded iNKT cells in stage IIIB-IV melanoma.Experimental Design: Residual iNKT cells [<0.05% of patient peripheral blood mononuclear cell (PBMC)] were purified from autologous leukapheresis product using an antibody against the iNKT cell receptor linked to magnetic microbeads. iNKT cells were then expanded with CD3 mAb and IL2 in vitro to obtain up to approximately 109 cells.Results: Expanded iNKT cells produced IFNγ, but limited or undetectable IL4 or IL10. Three iNKT infusions each were completed on 9 patients, and produced only grade 1-2 toxicities. The 4th patient onward received systemic GM-CSF with their second and third infusions. Increased numbers of iNKT cells were seen in PBMCs after some infusions, particularly when GM-CSF was also given. IFNγ responses to α-galactosylceramide were increased in PBMCs from some patients after infusions, and delayed-type hypersensitivity responses to Candida increased in 5 of 8 evaluated patients. Three patients have died, three were progression-free at 53, 60, and 65 months, three received further treatment and were alive at 61, 81, and 85 months. There was no clear correlation between outcome and immune parameters.Conclusions: Autologous in vitro expanded iNKT cells are a feasible and safe therapy, producing Th1-like responses with antitumor potential. Clin Cancer Res; 23(14); 3510-9. ©2017 AACR.

Extrinsic allospecific signals of hematopoietic origin dictate iNKT cell lineage-fate decisions during development.

Invariant NKT (iNKT) cells are critical to the maintenance of tolerance toward alloantigens encountered during postnatal life pointing to the existence of a process for self-education. However, the impact of developmentally encountered alloantigens in shaping the phenotype and function of iNKT cells has not been described. To better understand this process, the current report examined naïve iNKT cells as they matured in an allogeneic environment. Following the prenatal transfer of fetal hematopoietic cells between age-matched allogeneic murine fetuses, cell-extrinsic signals appeared to dictate allospecific patterns of Ly49 receptor expression and lineage diversity in developing iNKT cells. Regulation for this process arose from cells of hematopoietic origin requiring only rare exposure to facilitate broad changes in developing iNKT cells. These findings highlight surprisingly asymmetric allospecific alterations in iNKT cells as they develop and mature in an allogeneic environment and establish a new paradigm for study of the self-education of iNKT cells.

CD1d-dependent expansion of NKT follicular helper cells in vivo and in vitro is a product of cellular proliferation and differentiation.

NKT follicular helper cells (NKTfh cells) are a recently discovered functional subset of CD1d-restricted NKT cells. Given the potential for NKTfh cells to promote specific antibody responses and germinal center reactions, there is much interest in determining the conditions under which NKTfh cells proliferate and/or differentiate in vivo and in vitro. We confirm that NKTfh cells expressing the canonical semi-invariant Vα14 TCR were CXCR5(+)/ICOS(+)/PD-1(+)/Bcl6(+) and increased in number following administration of the CD1d-binding glycolipid α-galactosylceramide (α-GC) to C57Bl/6 mice. We show that the α-GC-stimulated increase in NKTfh cells was CD1d-dependent since the effect was diminished by reduced CD1d expression. In vivo and in vitro treatment with α-GC, singly or in combination with IL-2, showed that NKTfh cells increased in number to a greater extent than total NKT cells, but proliferation was near-identical in both populations. Acquisition of the NKTfh phenotype from an adoptively transferred PD-1-depleted cell population was also evident, showing that peripheral NKT cells differentiated into NKTfh cells. Therefore, the α-GC-stimulated, CD1d-dependent increase in peripheral NKTfh cells is a result of cellular proliferation and differentiation. These findings advance our understanding of the immune response following immunization with CD1d-binding glycolipids.