LEF1 Drives a Central Memory Program and Supports Antitumor Activity of Natural Killer T Cells.

Vα24-invariant natural killer T cells (NKT) possess innate antitumor properties that can be exploited for cancer immunotherapy. We have shown previously that the CD62L+ central memory-like subset of these cells drives the in vivo antitumor activity of NKTs, but molecular mediators of NKT central memory differentiation remain unknown. Here, we demonstrate that relative to CD62L- cells, CD62L+ NKTs express a higher level of the gene encoding the Wnt/ß-catenin transcription factor lymphoid enhancer binding factor 1 (LEF1) and maintain active Wnt/ß-catenin signaling. CRISPR/Cas9-mediated LEF1 knockout reduced CD62L+ frequency after antigenic stimulation, whereas Wnt/ß-catenin activator Wnt3a ligand increased CD62L+ frequency. LEF1 overexpression promoted NKT expansion and limited exhaustion following serial tumor challenge and was sufficient to induce a central memory-like transcriptional program in NKTs. In mice, NKTs expressing a GD2-specific chimeric-antigen receptor (CAR) with LEF1 demonstrated superior control of neuroblastoma xenograft tumors compared with control CAR-NKTs. These results identify LEF1 as a transcriptional activator of the NKT central memory program and advance development of NKT cell-based immunotherapy. See related Spotlight by Van Kaer, p. 144.

Anti-GD2 CAR-NKT cells in patients with relapsed or refractory neuroblastoma: an interim analysis.

Vα24-invariant natural killer T (NKT) cells have shown potent anti-tumor properties in murine tumor models and have been linked to favorable outcomes in patients with cancer. However, low numbers of these cells in humans have hindered their clinical applications. Here we report interim results from all three patients enrolled on dose level 1 in a phase 1 dose-escalation trial of autologous NKT cells engineered to co-express a GD2-specific chimeric antigen receptor (CAR) with interleukin-15 in children with relapsed or resistant neuroblastoma (NCT03294954). Primary and secondary objectives were to assess safety and anti-tumor responses, respectively, with immune response evaluation as an additional objective. We ex vivo expanded highly pure NKT cells (mean ± s.d., 94.7 ± 3.8%) and treated patients with 3 × 106 CAR-NKT cells per square meter of body surface area after lymphodepleting conditioning with cyclophosphamide/fludarabine (Cy/Flu). Cy/Flu conditioning was the probable cause for grade 3-4 hematologic adverse events, as they occurred before CAR-NKT cell infusion, and no dose-limiting toxicities were observed. CAR-NKT cells expanded in vivo, localized to tumors and, in one patient, induced an objective response with regression of bone metastatic lesions. These initial results suggest that CAR-NKT cells can be expanded to clinical scale and safely applied to treat patients with cancer.

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.

Double-J ureteric stenting in pregnancy: A single-centre experience from Iraq.

OBJECTIVES: To evaluate the safety and effectiveness of ureteric stenting with a JJ stent in pregnant women, to relieve renal obstruction and intractable flank pain. PATIENTS AND METHODS: All pregnant patients presenting with intractable flank pain, with or without complications, to a tertiary national teaching hospital in Kurdistan/Iraq, and necessitating ureteric stenting with a JJ stent, were prospectively assessed for this study between March 2008 and March 2010. RESULTS: In all, 30 pregnant patients presented with intractable flank pain necessitating JJ ureteric stenting during the 25 months. Intractable flank pain (23 patients, 77%) was the most common indication for ureteric stenting, followed by flank pain with clinical sepsis (six, 20%). All pregnant women had hydronephrosis on ultrasonography (US), and 12 (40%) had evidence of coexisting renal stones on US. All ureteric stents were inserted successfully. The mean (range) indwelling time was 47.4 (3-224) days. Radiologically, 14 (47%) and 15 (50%) had complete resolution of the hydronephrosis on follow-up US in late pregnancy and in the early postnatal period, respectively. Two-thirds of patients had a clinical improvement immediately (15, 50%) and soon after (five, 17%) surgery. Stent encrustation (three, 10%), stent migration (three, 10%) and stent irritation (five, 17%) were reported as complications. The post-natal evaluation confirmed that half the patients had urinary calculus disease. CONCLUSION: Ureteric stenting during pregnancy can be safe, with no intraoperative imaging and even under local anaesthesia. It provides good symptom relief and has a low complication rate. We therefore advocate it as a first-line treatment in pregnant women with therapy-resistant flank pain.