Evaluation of analytical performance of AQUIOS CL flow cytometer and method comparison with bead-based flow cytometry methods.

OBJECTIVES: Given that method validation is mandatory for compliance with the International Organization for Standardization (ISO) 15,189 standard requirements, we evaluated the analytical performance of the AQUIOS CL system (Beckman Coulter) and compared it with two bead-based flow cytometry (FCM) protocols (BD FACSCAntoTM-II and Beckman Coulter DxFLEX). There are no comparative literature data on standardized protocols for counting lymphocyte subsets on the new-generation cytometer DxFLEX. METHODS: We evaluated the AQUIOS CL's performance with regard to accuracy, linearity and stability by using dedicated control cell samples and patient samples. We also compared the lymphocyte counts measured on the AQUIOS CL (n=69 samples) with those measured on the BD FACSCAntoTM-II and DxFLEX FCM systems. For 61 samples, FCM results were compared with those measured on the XN-3000 Sysmex hematology analyzer. RESULTS: AQUIOS CL showed acceptable performance - even outside the manufacturer's quantification ranges- and strong correlations with bead-based FCM methods. The FCM techniques and the XN-3000 gave similar absolute lymphocyte counts, although values in samples with intense lymphocytosis (B cell lymphoma/leukemia) were underestimated. CONCLUSIONS: The AQUIOS CL flow cytometer is a time-saving, single-platform system with good performance, especially when the manufacturer's instructions for use are followed. However, AQUIOS CL's possible limitations and pitfalls impose validation of a bead-based FCM method for immunophenotyping verification or as a back-up system. Although the DxFLEX flow cytometer is more time-consuming to use, it can provide standardized lymphocyte subset counts in case of aberrant results on AQUIOS CL or in the event of equipment failure.

mTOR Inhibition Impairs the Activation and Function of Belatacept-Resistant CD4+CD57+ T Cells In Vivo and In Vitro.

Calcineurin inhibitors have improved graft survival in solid-organ transplantation but their use is limited by toxicity, requiring a switch to another immunosuppressor in some cases. Belatacept is one option that has been shown to improve graft and patient survival despite being associated with a higher risk of acute cellular rejection. This risk of acute cellular rejection is correlated with the presence of belatacept-resistant T cells. We performed a transcriptomic analysis of in vitro-activated cells to identify pathways affected by belatacept in belatacept-sensitive cells (CD4+CD57-) but not in belatacept-resistant CD4+CD57+ T cells. mTOR was significantly downregulated in belatacept-sensitive but not belatacept-resistant T cells. The inhibition of mTOR strongly decreases the activation and cytotoxicity of CD4+CD57+ cells. In humans, the use of a combination of mTOR inhibitor and belatacept prevents graft rejection and decreases the expression of activation markers on CD4 and CD8 T cells. mTOR inhibition decreases the functioning of belatacept-resistant CD4+CD57+ T cells in vitro and in vivo. It could potentially be used in association with belatacept to prevent acute cellular rejection in cases of calcineurin intolerance.

Advances in CAR-T Cell Genetic Engineering Strategies to Overcome Hurdles in Solid Tumors Treatment.

During this last decade, adoptive transfer of T lymphocytes genetically modified to express chimeric antigen receptors (CARs) emerged as a valuable therapeutic strategy in hematological cancers. However, this immunotherapy has demonstrated limited efficacy in solid tumors. The main obstacle encountered by CAR-T cells in solid malignancies is the immunosuppressive tumor microenvironment (TME). The TME impedes tumor trafficking and penetration of T lymphocytes and installs an immunosuppressive milieu by producing suppressive soluble factors and by overexpressing negative immune checkpoints. In order to overcome these hurdles, new CAR-T cells engineering strategies were designed, to potentiate tumor recognition and infiltration and anti-cancer activity in the hostile TME. In this review, we provide an overview of the major mechanisms used by tumor cells to evade immune defenses and we critically expose the most optimistic engineering strategies to make CAR-T cell therapy a solid option for solid tumors.

Flare of a mixed cryoglobulinaemic vasculitis after obinutuzumab infusion.

OBJECTIVES: Obinutuzumab (OBZ) is a new humanised type II anti-CD20 monoclonal antibody (mAb) approved in onco-haematology. Its use as an alternative to rituximab (RTX) in case of immunisation in autoimmune diseases has not been fully assessed yet. Here we report the case of a patient suffering from a refractory cryoglobulinaemic vasculitis (CV) associated to Sjögren's syndrome (SS) and treated with OBZ. METHODS: Since the patient was immunised against RTX, she was treated with OBZ at relapse. Three days after the infusion of OBZ, she presented a vasculitis flare. Rheumatoid factor level, complement level and cryoprecipitation were evaluated on consecutive serum samples of the patients and after RTX and OBZ addition in vitro. RESULTS: No evidence for cross-reactivity between anti-RTX Abs and OBZ was found. However, we could observe in vitro that cryoprecipitation was worsened by the simultaneous presence of anti-RTX Abs and RTX. We suggest that the flare of CV after OBZ infusion could be linked to a large release of immune complexes following B cells lysis induced by OBZ. CONCLUSIONS: Based on our report, we think that the use of OBZ needs to be carefully discussed in patients with mixed CV.

Engineering Next-Generation CAR-T Cells for Better Toxicity Management.

Immunoadoptive therapy with genetically modified T lymphocytes expressing chimeric antigen receptors (CARs) has revolutionized the treatment of patients with hematologic cancers. Although clinical outcomes in B-cell malignancies are impressive, researchers are seeking to enhance the activity, persistence, and also safety of CAR-T cell therapy-notably with a view to mitigating potentially serious or even life-threatening adverse events like on-target/off-tumor toxicity and (in particular) cytokine release syndrome. A variety of safety strategies have been developed by replacing or adding various components (such as OFF- and ON-switch CARs) or by combining multi-antigen-targeting OR-, AND- and NOT-gate CAR-T cells. This research has laid the foundations for a whole new generation of therapeutic CAR-T cells. Here, we review the most promising CAR-T cell safety strategies and the corresponding preclinical and clinical studies.

Erythropoietin is a major regulator of thrombopoiesis in thrombopoietin-dependent and -independent contexts.

Thrombopoietin (TPO), through activation of its cognate receptor Mpl, is the major regulator of platelet production. However, residual platelets observed in TPO- and Mpl-loss-of-function (LOF) mice suggest the existence of an additional factor to TPO in platelet production. As erythropoietin (EPO) exhibited both in vitro megakaryocytic potential, in association with other early-acting cytokines, and in vivo platelet activation activity, we sought to investigate its role in this setting. Here, we used multiple LOF models to decipher the reciprocal role of EPO and TPO in the regulation of platelet production in TPO-LOF and Mpl-LOF mice and of platelet size heterogeneity in wild-type mice. We first identified EPO as the major thrombopoietic factor in the absence of the TPO-Mpl pathway. Based on the study of several mouse models we found that the EPO-EPO receptor pathway acts on late-stage megakaryopoiesis and is responsible for large-sized platelet production, while the TPO-Mpl pathway promotes small-sized platelet production. On the basis of our data, EPO might be used for thrombocytopenia supportive therapy in congenital amegakaryocytopoiesis. Furthermore, as a distribution skewed toward large platelets is an independent risk factor and a poor prognosis indicator in atherothrombosis, the characterization of EPO's role in the production of large-sized platelets, if confirmed in humans, may open new perspectives in the understanding of the role of EPO-induced platelets in atherothrombosis.

Anti-Angiotensin type 1 receptor antibodies in chronic graft-versus-host disease.

BACKGROUND: Activating anti-angiotensin type 1 receptor antibodies (AT1R-AA) have been described in patients with systemic scleroderma, an auto-immune disorder with clinical fibrotic features. Chronic graft-versus-host disease (cGvHD) after hematopoietic stem cell transplantation may have clinical fibrotic features, whose pathogenesis may be similar with systemic sclerosis. OBJECTIVE: To evaluate the presence of AT1R-AA and their association with clinical and biological symptoms in cGvHD patients. MATERIAL AND METHODS: Sera from 87 patients including 45 extensive cGvHD and 42 hematopoietic stem cell transplantation patients without cGvHD were retrospectively analyzed for the presence of AT1R-AA using an enzymatic immunoassay. RESULTS: The frequency of AT1R-AA was significantly increased (odds ratio [OR]=3.4, P=0.04) in the cGvHD group (24.4%) compared with the non-cGvHD group (7.1%). In the cGvHD group the positivity of AT1R-AA was significantly associated with: i/ the presence of antinuclear antibodies (OR=5.9, P=0.04) ii/ a more severe global and organ-specific cGvHD scoring (P<0.05), iii/ the presence of active skin or mucosal erosions (OR=19.2, P<0.01). There was no difference between the number and the types of organs involved by the cGvHD between the AT1R-AA-positive versus AT1R-AA-negative subgroups. CONCLUSION: This preliminary study suggests a potential role and prognostic value of AT1R-AA in cGvHD.