Development of optimized cytotoxicity assays for assessing the antitumor potential of CAR-T cells.

CAR-T cells are T cells expressing a chimeric antigen receptor (CAR) rendering them capable of killing tumor cells after recognition of a target antigen. CD19 CAR-T cells have revolutionized the treatment of hematological malignancies. Their function is typically assessed by cytotoxicity assays using human allogeneic cell lines expressing the target antigen CD19 such as Nalm-6. However, an alloreactive reaction is observed with these cells, leading to a CD19-independent killing. To address this issue, we developed a fluorescence microscopy-based potency assay using murine target cells to provide an optimized cytotoxicity assay with enhanced specificity towards CD19. Murine NIH/3T3 (3T3) fibroblast-derived cell line and EL4 T-cell lymphoma-derived cell line were used as targets (no xenoreactivity was observed after coculture with human T cells). 3T3 and EL4 cells were engineered to express eGFP (enhanced Green Fluorescent Protein) and CD19 or CD22 using retroviral vectors. CD19 CAR-T cells and non-transduced (NT) control T cells were produced from several donors. After 4 h or 24 h, alloreactive cytotoxicity against CD19+ Nalm-6-GFP cells and CD19- Jurkat-GFP cells was observed with NT or CAR-T cells. In the same conditions, CAR-T but not NT cells specifically killed CD19+ but not CD19- 3T3-GFP or EL4-GFP cells. Both microscope- and flow cytometry-based assays revealed as sensitive as impedance-based assay. Using flow cytometry, we could further determine that CAR-T cells had mostly a stem cell-like memory phenotype after contact with EL4 target cells. Therefore, CD19+ 3T3-GFP or EL4-GFP cells and fluorescence microscopy- or flow cytometry-based assays provide convenient, sensitive and specific tools to evaluate CAR-T cell function with no alloreactivity.

The combination of Ara h 2-sIgE and basophil activation test could be an alternative to oral food challenge in cases of suspected peanut allergy.

BACKGROUND: Most children with peanut sensitisation do not have a clinical peanut allergy (PA). Oral food challenge (OFC) is then necessary to diagnose PA and assess the reactive dose of the allergen. However, OFC is laborious to perform, expensive and stressful. We evaluated whether in vitro tests, such as basophil activation test (BAT), allergen-specific IgE (sIgE) and their combination, could be used to replace OFC for the diagnosis of PA in children. METHODS: Ninety-one patients aged 6 months to 18 years with suspected PA were prospectively recruited. These patients then underwent an OFC to assess PA. Whole peanut-sIgE, Ara h 2-sIgE, Ara h 8-sIgE and %CD63+ basophils (CCR3+ /SCClow ) to peanut measured by BAT were investigated for PA diagnosis. RESULTS: Forty-one patients had a positive peanut OFC, and the remaining 50 were only sensitised. All patients with Ara h 2-sIgE >7 kUA /L were allergic to peanut. A threshold of 6% for activated basophils yielded a sensitivity of 95% and a specificity of 54%. All patients with Ara h 2-sIgE ≤7 kUA /L and BAT ≤6% (n = 22) had a negative OFC except for one who presented an oral syndrome due to PR-10 sensitisation. CONCLUSIONS: We have shown that Ara h 2-sIgE >7 kUA/L is a discriminating threshold for the diagnosis of PA. Furthermore, when Ara h 2-sIgE ≤7 kUA/L and BAT ≤6%, patients do not need to adjust their diet and, thus, do not need an OFC.

Radiation therapy improves CAR T cell activity in acute lymphoblastic leukemia.

Autologous T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19 are approved for the treatment of various CD19+ hematological malignancies. While CAR T cells induce objective responses in a majority of patients, relapse frequently occurs upon loss of CD19 expression by neoplastic cells. Radiation therapy (RT) has been successfully employed to circumvent the loss of CAR targets in preclinical models of pancreatic cancer. At least in part, this reflects the ability of RT to elicit death receptor (DR) expression by malignant cells, enabling at least some degree of CAR-independent tumor killing. In a human model of CD19+ acute lymphoblastic leukemia (ALL), we also observed DR upregulation by RT, both in vitro and in vivo. Moreover, low-dose total body irradiation (LD-TBI) delivered to ALL-bearing mice prior to CAR T cell infusion considerably extended the overall survival benefit afforded by CAR T cells alone. Such an improved therapeutic activity was accompanied by a superior expansion of CAR T cells in vivo. These data encourage the initiation of clinical trials combining LD-TBI with CAR T cells in patients with hematological malignancies.

Icos gene disruption in non-obese diabetic mice elicits myositis associated with anti-troponin T3 autoantibodies.

AIMS: Idiopathic inflammatory myopathies (IIM) are autoimmune inflammatory disorders leading to skeletal muscle weakness and disability. The pathophysiology of IIM is poorly understood due to the scarcity of animal disease models. Genetic deletion of Icos or Icosl (inducible T cell co-stimulator/ligand) in non-obese diabetic (NOD) mice leads to muscle disease. Our aim was to characterise Icos-/- NOD myopathy and to search for novel autoantibodies (aAbs) in this model. METHODS: Diabetes, weight, myopathy incidence/clinical score and grip strength were assessed over time. Locomotor activity was analysed with the Catwalk XT gait analysis system. Muscle histology was evaluated in haematoxylin/eosin and Sirius red-stained sections, and immune infiltrates were characterised by immunofluorescence and flow cytometry. 2D gel electrophoresis of muscle protein extracts and mass spectrometry were used to identify novel aAbs. NOD mice were immunised with troponin T3 (TNNT3) in incomplete Freund's adjuvant (IFA) and R848. An addressable laser bead immunoassay (ALBIA) was developed to measure aAb IgG serum levels. RESULTS: Icos-/- NOD mice did not exhibit diabetes but developed spontaneous progressive myositis with decreased muscle strength and altered locomotor activity. Muscle from these mice exhibited myofibre necrosis, myophagocytosis, central nuclei, fibrosis and perimysial and endomysial cell infiltrates with macrophages and T cells. We identified anti-TNNT3 aAbs in diseased mice. Immunisation of NOD mice with murine TNNT3 protein led to myositis development, supporting its pathophysiological role. CONCLUSIONS: These data show that Icos-/- NOD mice represent a spontaneous model of myositis and the discovery of anti-TNNT3 aAb suggests a new autoantigen in this model.

Methods to monitor in vivo expansion and efficacy of CAR-T cells in preclinical models.

The landscape of CAR-T detection and monitoring techniques in preclinical models is rapidly evolving. In this chapter, we will discuss the most widely used methods. The chapter begins with elaborating on the rational of establishing and optimizing protocols for CAR-T monitoring and explaining why this is a crucial step in CAR-T early development. This conceptual basis will be followed by detailed protocols: polymerase chain reaction (PCR), flow cytometry and bioluminescence imaging (BLI). These in vivo methods can be implemented in labs with interest in CAR-T pre-clinical research. It will provide important tools in the process of CAR-T development and offer better understanding of their efficacy, cytotoxicity and survival.

Autologous Myoblasts for the Treatment of Fecal Incontinence: Results of a Phase 2 Randomized Placebo-controlled Study (MIAS).

OBJECTIVE: The aim of this study was to evaluate the efficacy of intrasphincteric injections of autologous myoblasts (AMs) in fecal incontinence (FI) in a controlled study. SUMMARY OF BACKGROUND DATA: Adult stem cell therapy is expected to definitively cure FI by regenerating damaged sphincter. Preclinical data and results of open-label trials suggest that myoblast therapy may represent a noninvasive treatment option. METHODS: We conducted a phase 2 randomized, double-blind, placebo-controlled study of intrasphincteric injections of AM in 24 patients. The study compared outcome after AM (n = 12) or placebo (n = 12) injection using Cleveland Clinic Incontinence (CCI), score at 6 and 12 months. Patients in the placebo group were eligible to receive frozen AM after 1 year. RESULTS: At 6 months, the median CCI score significantly decreased from baseline in both the AM (9 vs 15, P = 0.02) and placebo (10 vs 15, P = 0.01) groups. Hence, no significant difference was found between the 2 groups (primary endpoint) at 6 months. At 12 months, the median CCI score continued to ameliorate in the AM group (6.5 vs 15, P = 0.006), while effect was lost in the placebo group (14 vs 15, P = 0.35). Consequently, there was a higher response rate at 12 months in the treated than the placebo arm (58% vs 8%, P = 0.03). After delayed frozen AM injection in the placebo group, the response rate was 60% (6/10) at 12 months. CONCLUSIONS: Intrasphincteric AM injections in FI patients have shown tolerance, safety, and clinical benefit at 12 months despite a transient placebo effect at 6 months.

Induction of Hematopoietic Microchimerism by Gene-Modified BMT Elicits Antigen-Specific B and T Cell Unresponsiveness toward Gene Therapy Products.

BACKGROUND: Gene therapy is a promising treatment option for hemophilia and other protein deficiencies. However, immune responses against the transgene product represent an obstacle to safe and effective gene therapy, urging for the implementation of tolerization strategies. Induction of a hematopoietic chimerism via bone marrow transplantation (BMT) is a potent means for inducing immunological tolerance in solid organ transplantation. OBJECTIVES: We reasoned, here, that the same viral vector could be used, first, to transduce BM cells for inducing chimerism-associated transgene-specific immune tolerance and, second, for correcting protein deficiencies by vector-mediated systemic production of the deficient coagulation factor. METHODS: Evaluation of strategies to induce B and T cell tolerance was performed using ex vivo gene transfer with lentiviral (LV) vectors encoding coagulation factor IX (FIX) or the SIINFEKL epitope of ovalbumin. Following induction of microchimerism via BMT, animals were challenged with in vivo gene transfer with LV vectors. RESULTS: The experimental approach prevented humoral immune response against FIX, resulting in persistence of therapeutic levels of circulating FIX, after LV-mediated gene transfer in vivo. In an ovalbumin model, we also demonstrated that this approach effectively tolerized the CD8+ T cell compartment in an antigen-specific manner. CONCLUSION: These results provide the proof-of-concept that inducing a microchimerism by gene-modified BMT is a powerful tool to provide transgene-specific B and T cell tolerance in a gene therapy setting.

Autoimmune Myopathies: Where Do We Stand?

Autoimmune diseases (AIDs) as a whole represent a major health concern and remain a medical and scientific challenge. Some of them, such as multiple sclerosis or type 1 diabetes, have been actively investigated for many decades. Autoimmune myopathies (AIMs), also referred to as idiopathic inflammatory myopathies or myositis, represent a group of very severe AID for which we have a more limited pathophysiological knowledge. AIM encompass a group of, individually rare but collectively not so uncommon, diseases characterized by symmetrical proximal muscle weakness, increased serum muscle enzymes such as creatine kinase, myopathic changes on electromyography, and several typical histological patterns on muscle biopsy, including the presence of inflammatory cell infiltrates in muscle tissue. Importantly, some AIMs are strongly related to cancer. Here, we review the current knowledge on the most prevalent forms of AIM and, notably, the diagnostic contribution of autoantibodies.

Diagnostic Value of Antigen-Specific Immunoglobulin E Immunoassays against Ara h 2 and Ara h 8 Peanut Components in Child Food Allergy.

BACKGROUND: Peanut allergy is one of the most severe food allergies in children. The diagnostic gold standard is the oral food challenge (OFC). However, OFC has inherent risks and is time consuming. The measurement of specific immunoglobulin E (sIgE) to peanut components in blood detects peanut sensitization, but the decision point predicting allergy is still unclear. The aim of this study was to determine the diagnostic value of these tests for the evaluation of child peanut allergy. METHODS: In this retrospective study, 81 children were referred for peanut allergy. The diagnosis of peanut allergy was based on the clinical context and a positive OFC. Levels of sIgE against whole peanuts or peanut components (Ara h 2 and Ara h 8) were determined by immunoassay. RESULTS: The Ara h 2 sIgE assay has the best negative predictive value (0.93) and positive predictive value (1) at a cutoff of 0.1 kU/l. Ara h 2 sIgE titers can predict the risk of anaphylaxis (<0.44 kU/l, low risk; >14 kU/l, high risk). The Ara h 8 sIgE assay is not able to discriminate peanut-allergic patients but can be used to evaluate possible cross-reactions to birch pollen with a low risk of anaphylaxis. The best diagnostic strategy is to first determine the Ara h 2 sIgE level and, if negative, evaluate Ara h 8 sIgE. CONCLUSIONS: We propose an algorithm for a better use of peanut component sIgE immunoassays that should improve their diagnostic value and avoid unnecessary OFC.

Selective Vascular Endothelial Protection Reduces Cardiac Dysfunction in Chronic Heart Failure.

BACKGROUND: Chronic heart failure (CHF) induces endothelial dysfunction in part because of decreased nitric oxide (NO(·)) production, but the direct link between endothelial dysfunction and aggravation of CHF is not directly established. We previously reported that increased NO production via inhibition of protein tyrosine phosphatase 1B (PTP1B) is associated with reduced cardiac dysfunction in CHF. Investigation of the role of endothelial PTP1B in these effects may provide direct evidence of the link between endothelial dysfunction and CHF. METHODS AND RESULTS: Endothelial deletion of PTP1B was obtained by crossing LoxP-PTP1B with Tie2-Cre mice. CHF was assessed 4 months after myocardial infarction. In some experiments, to exclude gene extinction in hematopoietic cells, Tie2-Cre/LoxP-PTP1B mice were lethally irradiated and reconstituted with bone marrow from wild-type mice, to obtain mouse with endothelial-specific deletion of PTP1B. Vascular function evaluated ex vivo in mesenteric arteries showed that in wild-type mice, CHF markedly impaired NO-dependent flow-mediated dilatation. CHF-induced endothelial dysfunction was less marked in endoPTP1B(-/-) mice, suggesting restored NO production. Echocardiographic, hemodynamic, and histological evaluations demonstrated that the selectively improved endothelial function was associated with reduced left ventricular dysfunction and remodeling, as well as increased survival, in the absence of signs of stimulated angiogenesis or increased cardiac perfusion. CONCLUSIONS: Prevention of endothelial dysfunction, by endothelial PTP1B deficiency, is sufficient to reduce cardiac dysfunction post myocardial infarction. Our results provide for the first time a direct demonstration that endothelial protection per se reduces CHF and further suggest a causal role for endothelial dysfunction in CHF development.

High risk of cancer in autoimmune necrotizing myopathies: usefulness of myositis specific antibody.

Cancer can occur in patients with inflammatory myopathies. This association is mainly observed in dermatomyositis, and myositis-specific antibodies have allowed us to delineate patients at an increased risk. Malignancy is also reported in patients with necrotizing autoimmune myopathies, but the risk remains elusive. Anti-signal recognition particle or anti-HMGCR antibodies have been specifically associated with necrotizing autoimmune myopathies. We aimed at screening the incidence of cancer in necrotizing autoimmune myopathies. A group of patients (n = 115) with necrotizing autoimmune myopathies with or without myositis-specific antibodies was analysed. Malignancy occurred more frequently in seronegative necrotizing autoimmune myopathies patients and in HMGCR-positive patients compared to anti-signal recognition particle positive patients. Synchronous malignancy was diagnosed in 21.4% and 11.5% of cases, respectively, and incidence of cancer was higher compared to the general population in both groups. No specific type of cancer was predominant. Patients suffering from a synchronous cancer had a decreased median survival time. Cancer screening is necessary in seronegative necrotizing autoimmune myopathies and in HMGCR-positive patients but not in anti-signal recognition particle-positive patients.

Anti-HMGCR autoantibodies in European patients with autoimmune necrotizing myopathies: inconstant exposure to statin.

Necrotizing autoimmune myopathy (NAM) is a group of acquired myopathies characterized by prominent myofiber necrosis with little or no muscle inflammation. Recently, researchers identified autoantibodies (aAb) against 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) in patients with NAM, especially in statin-exposed patients. Here we report what is to our knowledge the first European cohort of patients with NAM.The serum of 206 patients with suspicion of NAM was tested for detection of anti-HMGCR aAb using an addressable laser bead immunoassay. Forty-five patients were found to be anti-HMGCR positive. Their mean age was 48.9 ± 21.9 years and the group was predominantly female (73.3%). Statin exposure was recorded in 44.4% of patients. Almost all patients had a muscular deficit (97.7%), frequently severe (Medical Research Council [MRC] 5 ≤3 in 75.5%). Subacute onset (<6 mo) was noted for most of them (64.4%). Nevertheless, 3 patients (6.6%) had a slowly progressive course over more than 10 years. Except for weight loss (20%), no extramuscular sign was observed. The mean CK level was high (6941 ± 8802 IU/L) and correlated with muscle strength evaluated by manual muscle testing (r = -0.37, p = 0.03). Similarly, anti-HMGCR aAb titers were correlated with muscular strength (r = -0.31; p = 0.03) and CK level (r = 0.45; p = 0.01). Mean duration of treatment was 34.1 ± 40.8 months, and by the end of the study no patient had been able to stop treatment.This study confirms the observation and description of anti-HMGCR aAb associated with NAM. The majority of patients were statin naive and needed prolonged treatments. Some patients had a dystrophic-like presentation. Anti-HMGR aAb titers correlated with CK levels and muscle strength, suggesting their pathogenic role.

Exploring necrotizing autoimmune myopathies with a novel immunoassay for anti-3-hydroxy-3-methyl-glutaryl-CoA reductase autoantibodies.

INTRODUCTION: Necrotizing autoimmune myopathies (NAM) have recently been defined as a distinct group of severe acquired myopathies, characterized by prominent myofiber necrosis without significant muscle inflammation. Because of the lack of appropriate biomarkers, these diseases have been long misdiagnosed as atypical forms of myositis. NAM may be associated to autoantibodies directed against signal recognition particle (SRP) or 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). The objective of this work was to quantify anti-HMGCR autoantibodies in patients with suspicion of NAM through the development of a new addressable laser bead immunoassay (ALBIA). METHODS: Recombinant HMGCR C-domain was bound to fluorescent beads. After incubation with serum, autoantibodies were revealed using class- or subclass-specific anti-human immunoglobulin G (IgG) antibodies. Anti-HMGCR levels were assayed in 150 patients with suspicion of NAM, 142 controls with different inflammatory/autoimmune diseases and 100 healthy donors. Inhibition with free recombinant HMGCR and immunoprecipitation experiments confirmed test specificity. Reproducibility and repeatability were determined from sera with various levels of anti-HMGCR autoantibodies. A multiplex assay (ALBIA-NAM) was also developed to permit the simultaneous quantification of anti-HMGCR and anti-signal recognition particle autoantibodies. RESULTS: No controls scored positive. Of 150 patients with suspicion of NAM, 24% were positive for anti-HMGCR autoantibodies with levels ranging from 24 to 2,656 AU/mL. Anti-HMGCR positivity could be associated to a cytoplasmic pattern in immunofluorescence assay on HEp-2 cells. Anti-HMGCR-positive patients had high creatine kinase (CK) levels (mean 6,630 IU/L) and only 40% of them had been exposed to statins. Multiplex ALBIA-NAM was equally as effective as monoplex anti-HMGCR and anti-SRP ALBIA. CONCLUSIONS: Both monoplex ALBIA-HMGCR and multiplex ALBIA-NAM reliably detect and quantify anti-HMGCR autoantibodies. A positive result allows ascribing patients with a necrotizing myopathy to an autoimmune form. Anti-HMGCR autoantibodies may be found in patients who have not taken statins.

Altered functions of plasmacytoid dendritic cells and reduced cytolytic activity of natural killer cells in patients with chronic HBV infection.

BACKGROUND & AIMS: Hepatitis B virus (HBV) modulates the immune system to escape clearance. Plasmacytoid dendritic cells (pDCs) initiate antiviral immunity and might determine outcomes of HBV infections. Functional defects in pDCs and natural killer (NK) cells have been reported in patients with chronic HBV infection. However, the mechanisms of these immune dysfunctions and the interactions between pDCs and NK cells have not been determined. We investigated features of pDCs from patients with chronic HBV infection and their interactions with NK cells. METHODS: We used flow cytometry and cytokine assays to analyze pDCs from patients with chronic HBV infection (118 aviremic and 67 viremic) and compared them with pDCs from uninfected individuals (controls). We performed coculture assays to analyze the ability of pDCs to activate heterologous NK cells. RESULTS: Circulating and hepatic pDCs from patients with chronic HBV infection had higher levels of activation than pDCs from controls and defective responses to stimulation with Toll-like receptor 9 ligand (TLR9-L), regardless of the patient's viral load. TLR9-L-activated pDCs from viremic patients with HBV did not induce cytolytic activity of NK cells. This altered function of pDCs was associated with reduced expression of OX40L and could be reproduced by incubating control pDCs with plasma from viremic patients with HBV. A high level of interferon-induced protein 10 (IP-10 or CXCL10) and hepatitis B surface and e antigens might induce these defective pDC functions. CONCLUSIONS: HBV escapes antiviral immunity by altering pDC functions, to disrupt interactions between pDC and NK cells. This could reduce immune control of HBV and lead to chronic infection.

Plasmacytoid dendritic cells induce efficient stimulation of antiviral immunity in the context of chronic hepatitis B virus infection.

UNLABELLED: The immune control of hepatitis B virus (HBV) infection is essential for viral clearance. Therefore, restoring functional anti-HBV immunity is a promising immunotherapeutic approach to treatment of chronic infection. Plasmacytoid dendritic cells (pDCs) play a crucial role in triggering antiviral immunity through their ability to capture and process viral antigens and subsequently induce adaptive immune responses. We investigated the potential of pDCs to trigger antiviral cellular immunity against HBV. We used a human leukocyte antigen A (HLA-A)*0201(+) pDC line loaded with HLA-A*0201-restricted peptides derived from hepatitis B core/hepatitis B surface (HBc/HBs) antigens to amplify specific CD8 T cells ex vivo from chronic HBV patients and established a Hepato-HuPBL mouse model to address the therapeutic potential of the strategy in vivo. Stimulation of PBMCs or liver-infiltrating lymphocytes from HLA-A*0201(+) chronic HBV patients by HBc peptide-loaded pDCs elicited up to 23.1% and 76.1% HBV-specific CD8 T cells in 45.8% of cases. The specific T cells from the "responder" group secreted interferon-γ, expressed CD107 upon restimulation, and efficiently lysed HBV antigen-expressing hepatocytes. Circulating hepatitis B e antigen (HBeAg) was found to distinguish the group of patients not responding to the pDC stimulation. The therapeutic efficacy of the pDC vaccine was evaluated in immunodeficient NOD-SCID ß(2) m(-/-) mice reconstituted with HBV patients' PBMCs and xenotransplanted with human HBV-transfected hepatocytes. Vaccination of Hepato-HuPBL mice with the HBc/HBs peptide-loaded pDCs elicited HBV-specific T cells able to specifically lyse the transfected hepatocytes and reduce the systemic viral load. CONCLUSION: pDCs loaded with HBV-derived peptides can elicit functional virus-specific T cells. HBeAg appears to be critical in determining the outcome of immunotherapies in chronic HBV patients. A pDC-based immunotherapeutic approach could be of interest in attempts to restore functional antiviral immunity, which is critical for the control of the virus in chronic HBV patients.