Dual Chimeric Antigen Receptor T Cells Targeting CD38 and SLAMF7 with Independent Signaling Demonstrate Preclinical Efficacy and Safety in Multiple Myeloma.

Chimeric antigen receptor (CAR) T-cell therapy for multiple myeloma targeting B-cell maturation antigen (BCMA) induces high overall response rates. However, relapse still occurs and novel strategies for targeting multiple myeloma cells using CAR T-cell therapy are needed. SLAMF7 (also known as CS1) and CD38 on tumor plasma cells represent potential alternative targets for CAR T-cell therapy in multiple myeloma, but their expression on activated T cells and other hematopoietic cells raises concerns about the efficacy and safety of such treatments. Here, we used CRISPR/Cas9 deletion of the CD38 gene in T cells and developed DCAR, a double CAR system targeting CD38 and CS1 through activation and costimulation receptors, respectively. Inactivation of CD38 enhanced the anti-multiple myeloma activity of DCAR T in vitro. Edited DCAR T cells showed strong in vitro and in vivo responses specifically against target cells expressing both CD38 and CS1. Furthermore, we provide evidence that, unlike anti-CD38 CAR T-cell therapy, which elicited a rapid immune reaction against hematopoietic cells in a humanized mouse model, DCAR T cells showed no signs of toxicity. Thus, DCAR T cells could provide a safe and efficient alternative to anti-BCMA CAR T-cell therapy to treat patients with multiple myeloma.

Arginine reduces bacterial invasion in rats with head injury: an in vivo evaluation by bioluminescence.

OBJECTIVES: The benefit of arginine in intensive care unit patients with severe sepsis is still controversial. An excessive supply of arginine could lead to an overproduction of nitric oxide and could be responsible for septic shock and multiorgan failure. However, this claim is not supported by any experimental or clinical data. We set out to determine whether an enteral supply of arginine would modulate bacterial invasion in rats with head injury. METHODS: Male Sprague-Dawley rats with head injury were randomized into two groups. Group 1 included rats with head injury fed a standard enteral nutrition (Sondalis HP, n = 10) and group 2 included rats with head injury fed the standard enteral nutrition plus arginine (4 g/kg/d, n = 11). Two days after head injury, the rats received a single enteral bolus of luminescent Escherichia coli Xen 14. Bacterial proliferation was evaluated in vivo at time + 2 hrs and time + 6 hrs after E. coli challenge. Four days after head injury, blood was sampled for arginine and fibrinogen assay. Muscles, intestine, spleen, and thymus were removed and weighed. RESULTS: There was no mortality in either group. The luminescence signal was similar in the two groups at time +2 hrs (group 1: 414 [5-823] vs. group 2: 496 [0.1-993] (median value[min-max]; not significant) and was significantly lower at time +6 hrs in group 2 (group 1: 71 [0-142] vs. group 2: 8.5 [0-17]; p = .026). Arginine treatment did not improve any nutritional parameters. CONCLUSIONS: Arginine was not responsible for mortality in rats with head injury with infectious complications and reduced the intensity of bacterial invasion.

The Sec61 translocon is a therapeutic vulnerability in multiple myeloma.

Multiple myeloma (MM) is an incurable malignancy characterized by the uncontrolled expansion of plasma cells in the bone marrow. While proteasome inhibitors like bortezomib efficiently halt MM progression, drug resistance inevitably develop, and novel therapeutic approaches are needed. Here, we used a recently discovered Sec61 inhibitor, mycolactone, to assess the interest of disrupting MM proteostasis via protein translocation blockade. In human MM cell lines, mycolactone caused rapid defects in secretion of immunoglobulins and expression of pro-survival interleukin (IL)-6 receptor and CD40, whose activation stimulates IL-6 production. Mycolactone also triggered pro-apoptotic endoplasmic reticulum stress responses synergizing with bortezomib for induction of MM cell death and overriding acquired resistance to the proteasome inhibitor. Notably, the mycolactone-bortezomib combination rapidly killed patient-derived MM cells ex vivo, but not normal mononuclear cells. In immunodeficient mice engrafted with MM cells, it demonstrated superior therapeutic efficacy over single drug treatments, without inducing toxic side effects. Collectively, these findings establish Sec61 blockers as novel anti-MM agents and reveal the interest of targeting both the translocon and the proteasome in proteostasis-addicted tumors.

CAR-T cells derived from multiple myeloma patients at diagnosis have improved cytotoxic functions compared to those produced at relapse or following daratumumab treatment.

Chimeric antigen receptor T cells (CAR-T) have provided promising results in multiple myeloma (MM). However, many patients still relapse, pointing toward the need of improving this therapy. Here, we analyzed peripheral blood T cells from MM patients at different stages of the disease and investigated their phenotype and capacity to generate functional CAR-T directed against CS1 or B Cell Maturation antigen. We found a decrease in naive T cells and elevated frequencies of exhaustion markers in T cells from treated MM patients. Interestingly, individuals treated with daratumumab display elevated ratios of central memory T cells. CAR-T derived from patients at relapse show reduced in vitro expansion and cytotoxic capacities in response to MM cells compared to those produced at diagnosis. Of note, CAR-T from daratumumab treated patients display intermediate defects. Reduced anti-myeloma activity of CAR T cells from treated patients was also observed in a mouse model. Our findings suggest that T cell defects in MM patients, specifically during relapse, have a major impact on their capacity to generate efficient therapeutic CAR-T. Selecting naive or central memory T cell subsets to generate therapeutic T cells could improve the CAR-T therapy for MM.

The long non-coding RNA CRNDE regulates growth of multiple myeloma cells via an effect on IL6 signalling.

Multiple myeloma (MM) is a currently incurable malignancy of antibody-secreting plasma cells. Long non-coding RNAs (lncRNAs) have been recognised as an important class of regulatory molecules which are increasingly implicated in tumorigenesis. While recent studies have demonstrated changes in expression of lncRNAs in MM, the functional significance and molecular pathways downstream of these changes remain poorly characterised. In this study, we have performed CRISPR-mediated deletion of the locus encoding the lncRNA Colorectal Neoplasia Differentially Expressed (CRNDE), a known oncogenic lncRNA that is overexpressed in plasma cells of MM patients and is a marker of poor prognosis. We found that CRISPR-mediated deletion of the CRNDE locus in MM cells decreases proliferation and adhesion properties, increases sensitivity to Dexamethasone and reduces tumour growth in an in vivo xenograft model. Transcriptomic profiling in CRNDE-deleted MM cells demonstrated that CRNDE activates expression of a number of genes previously implicated in the aetiology of MM, including IL6R. We further demonstrate that deletion of the CRNDE locus diminishes IL6 signalling and proliferative responses in MM cells. Altogether this study reveals the IL6 signalling pathway as a novel mechanism by which CRNDE impacts upon MM cell growth and disease progression.

Head injury profoundly affects gut microbiota homeostasis: Results of a pilot study.

OBJECTIVES: Head injury (HI) induces a hypercatabolic state, dysimmunity, and septic complications that increase morbidity and mortality. Although compromised immune function is usually incriminated in infection occurrence, gut dysbiosis could also be involved in this phenomenon and, to our knowledge, has never been considered. To assess if HI could affect microbiota, we explored the impact of HI on intestinal microbiota in a rodent model of fluid percussion. METHODS: Nineteen rats were randomly assigned to two groups: Healthy rats fed ad libitum (n = 7) and HI rats (n = 12), which received standard enteral nutrition for 4 d. Four days after HI, rats were euthanized and cecal contents were sampled. Cecal microbiota was assessed using real-time quantitative polymerase chain reaction. RESULTS: HI significantly decreased the cecal content of strict anaerobic groups, Bacteroides/Prevotella group (HI 8.9 versus healthy controls 9.3 median log10 colony forming units [CFU]/g, P = 0.007), Clostridium cluster XIVab (HI 7.9 versus healthy controls 8.9 median log10 CFU/g, P = 0.002), Lactobacillus/Leuconostoc group (HI 8.5 versus healthy controls 9.4 median log10 CFU/g, P = 0.044), and Bifidobacterium sp. (HI 3.0 versus healthy controls 8.2 median log10 CFU/g, P < 0.001). In contrast, colonization by Escherichia coli was dramatically increased (HI 10.5 versus healthy controls 7.0 median log10 CFU/g, P < 0.001). CONCLUSIONS: HI profoundly modified the gut microbiota homeostasis and thus could contribute to infection in head trauma patients. These preliminary results open a new field of research in the management of patients with HI.

Impact of qualitative and quantitative variations in nitrogen supply on catch-up growth in food-deprived-refed young rats.

BACKGROUND: Optimization of the refeeding strategy for the management of malnutrition in early life may enable to improve the quality of catch-up growth. While some data suggest better assimilation of peptides rather than whole proteins the evidence are scarce. OBJECTIVE: To compare the nutritional properties of peptides, partially hydrolyzed proteins or whole proteins in food-deprived/refed young rats. METHODS: Male SD rats (n = 109, 60-70 g) were food-deprived for 48 h and refed for 2-13 days. According to the set of experiments, refeeding was performed at 90% or 100% basal spontaneous intake or ad libitum with either peptide-, partially hydrolyzed protein- or whole protein-containing pediatric enteral nutrition formulas. Body weight, caloric intake and nitrogen balance were measured daily, intestinal trophicity was measured after two-day refeeding, and body composition was determined at the end of the refeeding period. RESULTS: A 2-day food deprivation in young rats led to significant body and organ weight losses, alterations of gut morphology, and decreased plasma citrulline, a marker of gut function. A cautious 2-day renutrition at 90% pre-deprivation level did not restore nutritional status whatever the form of nitrogen supply. Ad libitum feeding was shown to be feasible with improved nitrogen efficiency. After 13 days, compared to chow diet, body weight gain was the lowest with peptide- and whole protein-containing diets, and significantly improved with partially hydrolyzed proteins with limited improvement in body lean mass (+8%, NS). Additional experiments indicated that in this model it will be necessary to significantly increase nitrogen supply in order to restore initial body weight and lean body mass. CONCLUSIONS: Our results show benefits of ad libitum refeeding on catch-up growth. Our data suggest that partially hydrolyzed proteins may be beneficial in terms of body weight gain but that probably their effectiveness may be improved with higher nitrogen supply.

Age-Associated Decrease of the Histone Methyltransferase SUV39H1 in HSC Perturbs Heterochromatin and B Lymphoid Differentiation.

The capacity of hematopoietic stem cells (HSC) to generate B lymphocytes declines with age, contributing to impaired immune function in the elderly. Here we show that the histone methyltransferase SUV39H1 plays an important role in human B lymphoid differentiation and that expression of SUV39H1 decreases with age in both human and mouse HSC, leading to a global reduction in H3K9 trimethylation and perturbed heterochromatin function. Further, we demonstrate that SUV39H1 is a target of microRNA miR-125b, a known regulator of HSC function, and that expression of miR-125b increases with age in human HSC. Overexpression of miR-125b and inhibition of SUV39H1 in young HSC induced loss of B cell potential. Conversely, both inhibition of miR-125 and enforced expression of SUV39H1 improved the capacity of HSC from elderly individuals to generate B cells. Our findings highlight the importance of heterochromatin regulation in HSC aging and B lymphopoiesis.

Evaluation of a new concept of immune-enhancing diet in a model of head-injured rat with infectious complications: A proof of concept study.

Immune-enhancing diet (IED) utilization in critically ill septic patients is still debated. A new concept of IED has been proposed combining extra glutamine sequentially with either antioxidants or other amino acids, in order to match patient requirements according to their response to injury. We evaluated whether this new IED elicits a more favorable response to stress when compared with two existing IEDs both enriched in arginine but with different levels of anti-oxidants, in a validated rat model combining head injury (HI) and infectious complications. Forty-eight HI rats were randomized into four groups (n = 11-13 per group) to receive, for 4 days, standard enteral nutrition (S), one of the two existing IEDs (IED1, IED2), or the new IED (IED3; providing glutamine and antioxidants for two days and glutamine and specific amino acids for two days). Two days after HI, the rats received an enteral bolus of luminescent Escherichia coli Xen14 to induce infection, and bacterial dissemination was evaluated. Body weight (BW) was recorded daily. Four days after HI, animals were euthanized; blood was sampled; organs were weighed; cumulated nitrogen balance (CNB) and nitrogen efficiency were determined. IED3 was more efficient than IED1 and IED2 in improving BW recovery from D3 (D3 vs. D1, p < 0.05) after HI. It significantly improved CNB and net protein utilization (IED3 vs. S, IED1, IED2, p < 0.05). An IED with sequential administration of anti-oxidants and glutamine may be better suited to meeting nutritional requirements in severe catabolic states.

In vivo bioluminescent imaging of a new model of infectious complications in head-injury rats.

Infectious complications are responsible for 10-25% of mortality in head-injured patients. In the present work we developed a model of infectious complications in head-injury rats using Escherichia coli (E. coli) with a stable copy of the lux operon, and monitored the infection in vivo by optical imaging. Rats were randomized into three groups: AL (healthy rats), HI (head-injury rats), and HI-EC (HI rats+single enteral bolus of E. coli, 1.3×10(9)/rat given 2 days after HI). Infection was evaluated with a camera at 2 and 6 h after E. coli challenge. Blood and organs were sampled to assess biological parameters. HI was associated with body weight loss, muscle atrophy, and plasma amino acid disturbances, in particular glutamine depletion (AL 919±37 versus HI 647±25 and HI-EC 717±20 µmol/L; p<0.05). In the HI-EC rats, the luminescence signal was observed at T+2 (mean [range]: 34,778 cpm [1617-2,918,810]), and was significantly decreased at T+6 (0 cpm [0-847,922]; p<0.05). Bacterial challenge was associated with a specific body weight loss and a decrease in gastrocnemius protein content, in alanine (AL 512±41 versus HI-EC 395±29 µmol/L; p<0.05), and in sulfur plasma amino acids. In conclusion, we propose a controlled model of HI with infectious complications characterized by specific metabolic alterations. Combined with the in vivo monitoring of the infection by bioluminescence, this model offers a valuable tool to evaluate specific strategies for HI patients.

TPL-2-mediated activation of MAPK downstream of TLR4 signaling is coupled to arginine availability.

The innate immune response is influenced by the nutrient status of the host. Mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1 (ERK1) and ERK2, are activated after the stimulation of macrophages with bacterial lipopolysaccharide (LPS) and are necessary for the optimal production of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). We uncovered a role for the extracellular nutrient arginine in the activation of ERK1/2 in LPS-stimulated macrophages. Arginine facilitated the activation of MAPKs by preventing the dephosphorylation and inactivation of the MAPK kinase kinase tumor-promoting locus 2 (TPL-2). Starvation of mice decreased the concentration of arginine in the plasma and impaired the activation of ERK1/2 by LPS. Supplementation of starved mice with arginine promoted the subsequent activation of ERK1/2 and the production of TNF-alpha in response to LPS. Thus, arginine is critical for two aspects of the innate immune response in macrophages: It is the precursor used in the generation of the antimicrobial mediator nitric oxide, and it facilitates MAPK activation and consequently cytokine production.