Splenic NKG2D confers resilience versus susceptibility in mice after chronic social defeat stress: beneficial effects of (R)-ketamine

The spleen is a large immune organ that plays a key role in the immune system. The precise molecular mechanisms underlying the relationship between the spleen and stress-related psychiatric disorders are unknown. Here we investigated the role of spleen in stress-related psychiatric disorders. FACS analysis was applied to determine the contribution of the spleen to susceptibility and resilience in mice that were subjected to chronic social defeat stress (CSDS). We found a notable increase in splenic volume and weight in CSDS-susceptible mice compared to control (no CSDS) mice and CSDS-resilient mice. The number of granulocytes, but not of T cells and B cells, in the spleen of susceptible mice was higher than in the spleen of both control and resilient mice. Interestingly, NKG2D (natural killer group 2, member D) expression in the spleen of CSDS-susceptible mice was higher than that in control mice and CSDS-resilient mice. In addition, NKG2D expression in the spleen of patients with depression was higher than that in controls. Both increased splenic weight and increased splenic NKG2D expression in CSDS-susceptible mice were ameliorated after a subsequent administration of (R)-ketamine. The present findings indicate a novel role of splenic NKG2D in stress susceptibility versus resilience in mice subjected to CSDS. Furthermore, abnormalities in splenic functions in CSDS-susceptible mice were ameliorated after subsequent injection of (R)-ketamine. Thus, the brain–spleen axis might, at least in part, contribute to the pathogenesis of stress-related psychiatric disorders such as depression.

Gene Editing and mRNA-Based Therapy: Two Complementary Therapeutic Approaches for the Treatment of Patients with Xmen Disease

Introduction 'X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia' (XMEN) disease is a primary immunodeficiency disease caused by loss-of-function mutations in the MAGT1 gene encoding for the magnesium transporter 1. This leads to the absence of expression of the "Natural-Killer Group 2, member D" (NKG2D) receptor in natural killer (NK) and CD8+ T cells, which is essential for their antiviral and antitumoral cytotoxic activity. In consequence, XMEN patients develop chronic EBV infections and EBV-related lymphoproliferative disorders. Allogeneic bone marrow transplant has been associated with significant mortality, and there are no other effective treatments. In that context, we aimed at developing two complementary approaches to treat XMEN patients: 1) Adoptive transfer of XMEN T/NK cells corrected by transient mRNA therapy or longer-lasting gene editing therapy in order to control infections, and 2) Transplantation of gene-edited CD34+ cells in order to permanently restore production of functional immune cells. Material and methods CD34+ cells and PBMCs were collected from XMEN patients and healthy donors (HD) (NIH Protocol 94-I-073). For mRNA therapy, we expanded T cells with anti-CD3/anti-CD28 beads in RPMI + 10% serum supplemented with 200 IU/mL IL2 for 5-7 days and NK cells with 100 IU/mL IL2 and 10 ng/mL IL15 in culture with K562-mb15-41BBL for 10-15 days. Both XMEN T and NK cells were electroporated (EP) with MAGT1 mRNA and cultured for up to 28 days. For gene editing, XMEN CD34+ or stimulated T cells were electroporated with Cas9 mRNA and sgRNA; a rAAV6 donor encoding for the codon-optimized MAGT1 cDNA was added after EP. Two days post-EP, CD34+ cells were differentiated into NK cells for 35 days in vitro. Results MAGT1 mRNA-based therapy. We first showed a restored MAGT1 expression by western blot at 6h and 24h post-EP of the MAGT1 mRNA. In consequence, NKG2D expression analyzed by flow cytometry was restored in expanded CD8+ T and NK cells starting within the 6h post-EP (20-40%), with a peak at 48h (>85%) and a progressive decrease of the expression over time (still 40% and 75%, respectively, of CD8+ T and NK cells of cells at day 14 post-EP respectively). The cytotoxic activity of mRNA-corrected XMEN NK cells was analyzed by culture with K562 target cells at several effector:target (E:T) ratios and shown to be restored at a level similar to HD NK cells (mRNA-treated: 66.7% ±5.8%; HD: 67.8% ±5.9% at E:T 2:1 ratio) compared to untreated cells (49.0% ±7.2%) (Fig 1a). Anticipating the potential use of these cells for repeated infusions as a treatment modality to control infections, we demonstrated that MAGT1 mRNA-corrected CD8+ T and NK cells that have been cryopreserved and thawed exhibit the same NKG2D expression kinetics following thaw and culture. Gene editing therapy. XMEN CD34+ cells electroporated with Cas9 mRNA and a sgRNA targeting exon 1 of MAGT1 gene showed an in vitro average integration rate of the MAGT1 cDNA AAV donor of 35.6% (range: 33.8-41.9%). The NKG2D expression in AAV-treated CD34+-derived NK cells was approximatively of 23% (range: 14.2-27.9%). Interestingly, their cytotoxic activity was similar to the level of NKG2D expression (23.1% ±4.3%), significantly higher than in untreated cells (9.7% ±2.8%) (Fig 1b). Similar rates of targeted integration and NKG2D expression were also obtained in AAV-treated CD8+ T cells. Conclusion For the first time, we demonstrate the efficiency of two approaches for development of potential cell therapy treatments of XMEN patients. MAGT1 mRNA electroporation can restore efficient transient expression of NKG2D in CD8+ T and NK cells, thus fully restoring the cytotoxic activity of NK cells. In addition, cells electroporated with MAGT1 mRNA can be cryopreserved, thus allowing repeated infusions. In parallel, we showed that efficient targeted insertion can be achieved in CD8+ T cells and CD34+ cells by using an AAV donor although the level of NKG2D expression is lower. Optimizations are currently ongoing in order to reach higher levels of correction. Both approaches could be combined in order to propose a new therapeutic strategy for the treatment of XMEN patients: repetitive adoptive transfer of mRNA-corrected autologous T/NK cells for the prevention or control of intractable infections, and transplantation of gene-edited CD34+ cells for the definitive treatment of these patients. Disclosures Meis: CELLSCRIPT, LLC: Employment. Li:MaxCyte, Inc: Employment. Allen:MaxCyte, Inc: Employment. Clark:CELLSCRIPT, LLC: Employment. Dahl:CELLSCRIPT, LLC: Other: Owner and officer.

Potential therapeutic effects of cyanidin-3-O-glucoside on rheumatoid arthritis by relieving inhibition of CD38+ NK cells on Treg cell differentiation

Background CD38+ NK cells are overabundant in rheumatoid arthritis (RA). Cyanidin-3-O-glucoside (C3G) is an inhibitor of CD38. This study investigated the pathogenic role of CD38+ NK cells and the effect of C3G on RA. Methods Rats with bovine type II collagen-induced arthritis (CIA) were injected with C3G. RA synovial fibroblasts (RASFs) or mononuclear cells (MNCs) were cultured with C3G. MNCs were also cocultured with CD38+ NK cells following C3G pretreatment. Results C3G injection significantly alleviated CIA. C3G also significantly increased the level of interleukin (IL)-10 and the regulatory T (Treg) cell proportion, and it decreased the interleukin (IL)-6 and interferon (IFN)-γ levels and CD38+ NK cell proportion in rat peripheral blood and synovial fluid. Additionally, C3G significantly increased RASF apoptosis and decreased RASF proliferation and IL-6 production in the culture medium. Furthermore, C3G stimulated MNCs to increase IL-2 and IL-10 production and the Treg cell proportion, and it caused MNCs to decrease IL-6 and IFN-γ production and the CD38+ NK cell proportion. Although CD38+ NK cells significantly decreased the Treg cell proportion and IL-10 level in MNCs, CD38+ NK cells that had been pretreated with C3G increased the proportion of Treg cells and IL-10 levels and decreased the IL-6 and IFN-γ levels in the coculture. In CD38+ NK cells, C3G significantly increased Sirtuin 6 (Sirt6) expression and the tumor necrosis factor (TNF)-α level, and it decreased natural killer group 2D (NKG2D) expression and the IFN-γ level. However, when CD38+ NK cells were treated with Sirt6 siRNA, C3G did not change the NKG2D expression, the TNF-α level sharply decreased, and the IFN-γ level increased. When MNCs were cocultured with C3G-pretreated CD38+ NK cells in the presence of TNF-α and an anti-IFN-γ antibody, the IL-10+ Treg cell proportion significantly increased. When MNCs were cocultured with C3G-pretreated CD38+ NK cells in the presence of IFN-γ and an anti-TNF-α antibody, the IL-10+ Treg cell proportion sharply decreased. When CIA rats were injected with both C3G and the Sirt6 inhibitor OSS_128167, the rats exhibited joint inflammation and a low Treg cell proportion, but the CD38+ NK proportion was still low. Conclusion C3G has therapeutic effects on CIA and RA. C3G decreased the proportion of CD38+ cells, RASF proliferation, and proinflammatory cytokine secretion, and it increased the Treg cell proportion. C3G also elevated Sirt6 expression to suppress NKG2D expression, increase TNF-α secretion, and decrease IFN-γ secretion in CD38+ NK cells, which stimulates MNCs to differentiate into Treg cells. This study also demonstrates that the inhibition of Treg cell differentiation in MNCs by CD38+ NK cells is a potential cause of the immune imbalance in RA and CIA.

Prolactin Induces IL-2 Associated TRAIL Expression on Natural Killer Cells from Chronic Hepatitis C PatientsIn vivoandIn vitro

Background:Natural killer cells (NKC) are a major component of the innate immune response to HCV, mediating their effects through TRAIL and IFN-γ. However, their function is diminished in chronic HCV patients (HCVp). Prolactin is an immunomodulatory hormone capable of activating NKC.Objective:The study aims to explore if hyperprolactinemia can activate NKC in HCVp.Methods:We treated twelve chronic HCVp (confidence level =95%, power =80%) for 15 days with Levosulpiride plus Cimetidine to induce mild hyperprolactinemia. Before and after treatment, we determined TRAIL and NKG2D expression on peripheral blood NKC, along with cytokine profiles, viral loads and liver function. We also evaluated in vitro effects of prolactin and/or IL-2 on NKC TRAIL or NKG2D expression and IFN-γ levels on cultured blood mononuclear cells from 8 HCVp and 7 healthy controls.Results:The treatment induced mild hyperprolactinemia and increased TRAIL expression on NKC as well as the secretion of IL-1ra, IL-2, PDGF and IFN-γ. Viral loads decreased in six HCVp. IL-2 and TRAIL together explained the viral load decrease. In vitro, prolactin plus IL-2 synergized to increase TRAIL and NKG2D expression on NKC from HCVp but not in controls.Conclusion:Levosulpiride/Cimetidine treatment induced mild hyperprolactinaemia that was associated with NKC activation and Th1-type cytokine profile. Also, an increase in TRAIL and IL-2 was associated with viral load decrease. This treatment could potentially be used to reactivate NKC in HCVp.

Constitutive expression of ULBP-4 on monocytes regulates NK cell NKG2D expression

Key Points The NKG2D ligand ULBP-4 is expressed on healthy monocytes. Monocyte ULBP-4 expression regulates NKG2D expression by NK cells.

Magnesium alterations of graft immune cells (MAGIC).

e18541 Background: Magnesium plays important roles in cellular processes. Allogeneic hematopoietic stem cell transplantation provides potentially curative therapy to patients with a variety of hematologic malignancies. A common abnormality post transplant is hypomagnesaemia. Recent studies identified magnesium transporter 1 deficiency in association with XMEN syndrome. These patients exhibit low intracellular [Mg++] and low T cell and NK cell NKG2D expression. Therefore, we investigated this potential functional link between magnesium deficiency and immune function in patients post transplant. Methods: A pilot study investigated whether detectable changes in T and NK cells were associated with IV magnesium repletion post hematopoietic transplant. Blood was obtained before and after magnesium transfusions. Immunophenotypic analysis of T and NK cell NKG2D levels using commercially available monoclonal antibodies (CD3, CD16, CD56, CD4, CD8, CD314 [NKG2D]) was performed. Samples from patients demonstrating an increase in NKG2D expression were subsequently analyzed for ex vivo NK activity. Here, NK activity and cytotoxicity of Jurkat cells were assayed using standard laboratory techniques and changes > 2 fold were considered significant. A total of 7 patients were enrolled. Results: NKG2D expression on T and NK cell subsets were determined by flow cytometry using commercially available monoclonal antibodies. NK reactivity and killing of K562 were undertaken using standard cytotoxicity assays. Our findings indicate a significant response in five of the seven enrolled patients. NKG2D expression on CD8+ T cells increased by up to 31%. Conclusions: Our results suggest that magnesium infusions post allogeneic transplant are associated with augmented NKG2D expression on immune cells. Additional studies are needed to clarify the mechanisms by which therapeutic magnesium repletion restores the cytotoxicity of NK cells and T lymphocytes. Although the clinical utility of magnesium supplementation remains to be validated, our data indicate that NKG2D is regulated by magnesium post transplant and magnesium may play a key role in immune regulation.