The risk of COVID-19 in IBD patients is increased by urban living and is not influenced by disease activity or intravenous biologics.

Background: Patients with inflammatory bowel disease (IBD) may have a modified immune response to SARS-CoV-2. The objectives were to evaluate the prevalence of COVID-19 in patients treated with infliximab or vedolizumab, to analyze the factors associated with the infection, the impact of treatments and trough levels. Methods: Patients with IBD treated with intravenous biologics in 14 French centers were included between March and June 2020 and followed-up for 6 months. Blood samples were collected for serologies and trough levels. The analysis of factors associated with COVID-19 was conducted in a matched 1:1 case-control sub-study with positive patients. Results: In total, 1026 patients were included (74.9% infliximab). Over the follow-up period, 420 patients reported the occurrence of COVID-19 symptoms; 342 had been tested of whom 18 were positive. At the end of follow-up, 38 patients had a positive serology. Considering both nasal tests and serologies together, 46 patients (4.5%) had been infected. The risk of COVID-19 was related neither to the use of treatments (whatever the trough levels) nor to disease activity. Infections were more frequent when using public transport or living in flats in urban areas. Conclusions: The prevalence rate of COVID-19 in this IBD population treated with intravenous infliximab or vedolizumab was the same as the one in the French population before the start of the vaccination campaign. The risk was increased by urban living and was not influenced by disease activity or biologics. Sanitary barrier measures remain the best way to protect against SARS-CoV-2 in patients with IBD in biological therapy.

The biological sample collection of the OFSEP French MS registry: An essential tool dedicated to researchers.

Today's medicine strives to be personalized, preventive, predictive and participatory. This implies to have access to multimodal data to better characterize patients groups and to combine clinical and imaging data with high-quality biological samples. Collecting such data is one of the objectives of the Observatoire français de la sclérose en plaques (OFSEP), the French MS registry. On December 2022, the OFSEP biocollection includes 4,888 patients with scientific characteristics and about 90,000 samples. Thanks to its richness, this biocollection open for the scientific community, contributes to address unmet needs in MS through identification of multiomics determinants of MS activity, progression and secondary effects.

The French prospective multisite registry on sudden unexpected infant death (OMIN): rationale and study protocol.

INTRODUCTION: Even after 'back-to-sleep' campaigns, sudden unexpected infant death (SUID) continues to be the leading cause of death for infants 1 month to 1 year old in developed countries, with devastating social, psychological and legal implications for families. To sustainably tackle this problem and decrease the number of SUIDs, a French SUID registry was initiated in 2015 to (1) inform prevention with standardised data, (2) understand the mechanisms leading to SUID and the contribution of the already known or newly suggested risk factors and (3) gather a multidisciplinary group of experts to coordinate and develop innovative and urgent research in the SUID area. METHODS AND ANALYSIS: This observational multisite prospective observatory includes all cases of sudden unexpected deaths in children younger than 2 years occurring in the French territory covered by the 35 participating French referral centres. From these cases, various data concerning sociodemographic conditions, death scene, personal and family medical history, parental behaviours, sleep environment, clinical examinations, biological and imagery investigations and autopsy are systematically collected. These data will be complemented as of 2018 with a biobank of diverse biological samples (blood, hair, urine, faeces and cerebrospinal fluid), with other administrative health-related data (health claim reimbursements and hospital admissions) and socioenvironmental data. Insights from exploratory descriptive statistics and thematic analysis will be combined for the design of targeted strategies to effectively reduce preventable infant deaths. ETHICS AND DISSEMINATION: The French sudden unexpected infant death registry (Observatoire National des Morts Inattendues du Nourrisson registry;OMIN) was approved in 2015 by the French Data Protection Authority in clinical research (Commission Nationale de l'Informatique et des Libertés: number 915273) and by an independent ethics committee (Groupe Nantais d'Ethique dans le Domaine de la Santé: number 2015-01-27). Results will be discussed with associations of families affected by SUID, caregivers, funders of the registry, medical societies and researchers and will be submitted to international peer-reviewed journals and presented at international conferences.

T-cell therapy using a bank of EBV-specific cytotoxic T cells: lessons from a phase I/II feasibility and safety study.

We report herein the results we obtained and the limitations we experienced during the production and use of a bank of Epstein-Barr virus (EBV)-transformed human cytotoxic T lymphocytes (EBV-CTLs). To assess the feasibility and toxicity of this strategy, we selected and stored, in liquid nitrogen, 4 billion EBV-CTLs from each of the 13 selected donors. Subsequently, in a multicenter phase I/II study, 11 patients with EBV-associated lymphoma resistant to conventional treatments received 1-3 doses of 5 million EBV-CTLs/kg with 1-3 and 0-4 compatibilities for human leukocyte antigen (HLA)-I and HLA-II, respectively. Except for one event of fever after injection, no immediate or delayed toxicity, no graft versus host disease, and no graft rejection attributable to CTL infusion were observed. Three patients presented complete remission and 1 partial remission after treatment. Considering the clinical options currently available, and the constrains associated with CTL preparation and implementation, we conclude that CTL banks should consist of a reasonably small number of cell lines with documented specificities. This objective could be more easily achieved if the few homozygous donors for the most frequent HLA alleles of the targeted population could be made available for such a project.

Upregulation of adhesion molecules on leukemia targets improves the efficacy of cytotoxic T cells transduced with chimeric anti-CD19 receptor.

T lymphocytes engineered to express chimeric antigen receptors (CARs) interact directly with cell surface molecules, bypassing MHC antigen presentation dependence. We generated human anti-CD19ζ CAR cytotoxic T lymphocytes and cytokine-induced killer cells and studied their sensitivity to the expression of adhesion molecules for the killing of primary B-lineage acute lymphoblastic leukemia (B-ALL) targets. Despite a very low basal expression of surface adhesion molecules, B-ALL blasts were lysed by the anti-CD19ζ-CAR transduced effectors as expected. We next investigated the regulatory role of adhesion molecules during CAR-mediated cytolysis. The blocking of these accessory molecules strongly limited the chimeric effector's cytotoxicity. Thereafter, B-ALL cells surface adhesion molecule level expression was induced by IFN-γ or by the combined use of CD40L and IL-4 and the cells were submitted to anti-CD19ζ-CAR transduced effectors lysis. Upregulation of adhesion molecules expression by blasts potentiated their killing. The improved cytotoxicity observed was dependent on target surface expression of adhesion molecules, particularly CD54. Taken together, these results indicate that adhesion molecules, and principally CD54, are involved in the efficiency of recognition by effector chimeric ζ. These observations have potential implications for the design of immunotherapy treatment approaches for hematological malignancies and tumors based on the adoption of CAR effector cells.

Viral DNA contamination is responsible for Epstein-Barr virus detection in cytotoxic T lymphocytes stimulated in vitro with Epstein-Barr virus B-lymphoblastoid cell line.

Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (LCLs) are used to prepare human EBV-specific T lymphocytes (EBV-CTL) in vitro. Within an LCL, up to 5-7% the cells release infectious EBV, and this has fostered safety concerns for therapeutic applications because of the exposure of T cells to EBV. The release of infectious viruses can be prevented by ganciclovir, but this drug may seriously affect LCL growth. In the wake of these concerns, the present work was designed to compile safety data on EBV-CTL preparation for the purpose of submission to a regulatory agency. We showed that further to supernatant exclusion, the number of EBV genome copies (EBVc) associated with the EBV-CTL always made up a constant proportion of the EBVc number detected in the culture supernatant. In addition, such was the case whether infectious virus could be produced by the LCL or not, suggesting that the EBV signal detected was due to a DNA contamination rather than an infection. Furthermore, we demonstrated that the number of EBVc associated with the EBV-CTL was highly sensitive to DNAse treatment, and finally that EBVc could no longer be detected after the EBV-CTL had been amplified in the absence of LCL. Consequently, during in vitro EBV-CTL preparation, either T cells cannot be infected or they die rapidly after EBV infection.

Exploration of the lysis mechanisms of leukaemic blasts by chimaeric T-cells.

Adoptive transfer of specific cytotoxic T lymphocytes (CTL) and Cytokine Induced Killer Cells (CIK) following genetic engineering of T-cell receptor zeta hold promising perspective in immunotherapy. In the present work we focused on the mechanisms of anti-tumor action of effectors transduced with an anti-CD19 chimaeric receptor in the context of B-lineage acute lymphoblastic leukemia (B-ALL). Primary B-ALL blasts were efficiently killed by both z-CD19 CTL and z-CD19 CIK effectors. The use of death receptor mediated apoptosis of target cells was excluded since agonists molecules of Fas and TRAIL-receptors failed to induce cell death. Perforin/granzyme pathway was found to be the mechanism of chimaeric effectors mediated killing. Indeed, cytolytic effector molecules perforin as well as granzymes were highly expressed by CTL and CIK. CD19 specific stimulation of transduced effectors was associated with degranulation as attested by CD107 membrane expression and high IFN-gamma and TNF-alpha release. Moreover inhibitors of the perforin-based cytotoxic pathway, Ca(2+)-chelating agent EGTA and Concanamycin A, almost completely abrogated B-ALL blast killing. In conclusion we show that the cytolysis response of z-CD19 chimaeric effectors is predominantly mediated via perforin/granzyme pathway and is independent of death receptors signaling in primary B-ALL.

Effector memory alphabeta T lymphocytes can express FcgammaRIIIa and mediate antibody-dependent cellular cytotoxicity.

Human memory T cells are comprised of distinct populations with different homing potential and effector functions: central memory T cells that mount recall responses to Ags in secondary lymphoid organs, and effector memory T cells that confer immediate protection in peripheral tissues. In the present study we demonstrate that a proportion of effector memory T cells express FcgammaRIIIa (CD16), are perforin positive, and directly mediate Ab-dependent cytotoxicity ex vivo. This particular alphabeta T lymphocyte subset has the morphology of large granular lymphocytes, increases proportionately in vivo during reactive lymphocytosis, and can be detected in vitro among EBV-specific T lymphocytes after stimulation with EBV Ags. Consequently, during a normal immune response, amplification of these effector memory T lymphocytes that are capable of Ab-dependent cytotoxicity may have beneficial or harmful consequences depending on the presence of pathogen- or tissue-specific Abs, respectively.

Antibody-dependent cellular cytotoxicity (ADCC) is mediated by genetically modified antigen-specific human T lymphocytes.

In the context of transplantation, donor and virus-specific T-lymphocyte infusions have demonstrated the dramatic potential of T cells as immune effectors. Unfortunately, most attempts to exploit the T-cell immune system against nonviral malignancies in the syngeneic setting have been disappointing. In contrast, treatments based on monoclonal antibodies (Abs) have been clinically successful and have demonstrated the clinical relevance of several antigens as therapeutic targets and the importance of the antibody-dependent cellular cytotoxicity (ADCC) pathway. In the present study, we considered the possibility of arming specific T cells with a receptor that would enable them to mediate ADCC. After transduction with a CD16/gamma receptor gene, CD4(+) and CD8(+) cytotoxic T lymphocytes displayed stable expression of the CD16 receptor at their surface. In the absence of Ab, CD16/gamma expression did not affect the capacity of specific T lymphocytes to kill their target following "natural" T-cell receptor recognition. When tested against the autologous B-lymphoblastoid cell line (BLCL) coated with anti-CD20 mAb, the newly expressed Fc receptor enabled the T cells to kill the BLCL through ADCC. Adoptive transfer of such newly designed immune effector may be considered to increase antibody efficiency by harnessing the immune potential of T cells.

T cell repertoire and Epstein-Barr virus-specific T cell response in chronic active Epstein-Barr virus infection: a case study.

In a patient with chronic active Epstein-Barr virus infection associated with vasculitis and fulminant CD4+ T cell lymphoproliferative disorder, we probed the peripheral blood mononuclear cells (PBMC) for the presence of an EBV-specific T cell repertoire and tested the possible relationship between the lymphocytic infiltrate and the EBV-specific T cell response. Our results give credence to the presence of an apparently normal EBV-specific memory T cell response after in vitro reactivation of the patient's PBMC with autologous infected B lymphoblastoid cell lines. In keeping with the characterization of the vasculitis, certain T cell subsets were detected after expansion of skin lesion-infiltrating lymphocytes and were found to be infected with EBV. These particular T cell expansions were neither the effectors nor the targets of the in vitro reactivated EBV-specific T cells, thus excluding a simple relationship between EBV, the skin lesions, and the T cell expansions frequently observed in these patients.

Long-term preservation of antibody-dependent cellular cytotoxicity (ADCC) of natural killer cells amplified in vitro from the peripheral blood of breast cancer patients after chemotherapy.

Twenty percent of breast cancer adenocarcinomas overexpress the oncogene c-erb-2 that is recognized by the humanized anti-Her2/neu monoclonal antibody Herceptin. Results from clinical studies suggest that antibody-dependent cellular cytotoxicity (ADCC) is involved in the clinical response of Herceptin-treated patients. The purpose of the current study was to evaluate the possibility of amplifying in vitro the CD3-/CD16+ natural killer (NK) cell subset that mediates ADCC from breast cancer patients after chemotherapy. Peripheral blood mononuclear cells from six breast cancer patients taken 2 months after chemotherapy completion were co-cultured with an autologous irradiated Epstein-Barr virus-transformed B-lymphoblastoid cell line (LCL) in the presence of interleukin-2 (IL-2) for 4-6 weeks. These LCL + IL2 activated cultures (ACs) were tested for ADCC potential, and their CD3/CD16 NK proportion was quantified. Among the ACs, the proportion of CD3-/CD16+ NK cells increased up to 64% over the first 2 weeks of culture and the ACs continued to expand for 1 month thereafter. Control and patient ACs displayed ADCC activity (tested in the presence of Rituximab against the autologous LCL to take into account any possible effect of inhibitory NK receptors) as well as against the MCF-7(Her2/neu) breast cancer cell line in the presence of Herceptin. This ADCC activity was maintained during the entire culture period. In conclusion, chemotherapy in breast cancer patients does not obviate the possibility of amplifying in vitro the NK cell subset that mediates ADCC. Consequently, adoptive transfer of lymphocytes mediating ADCC can be considered using this protocol to test its benefit in patients under Herceptin treatment.

Preparation of genetically homogeneous antigen-specific thymidine kinase positive T-lymphocyte clones for the control of alloreactivity post-bone marrow transplantation.

We have previously proposed the use of HLA-specific T-cell clones transduced with a suicide gene to produce an allogeneic effect that can be controlled after allogeneic bone marrow transplantation. Procedures described so far to obtain specific T-cells transduced with a suicide gene have led to the recovery of heterogeneous polyclonal T-cells with a limited level of purity. We have therefore developed an approach to select specific T-cell clones in which the suicide transgene is inserted at a unique site of the genome, and used it to produce CD(+)-cytotoxic HLA-DP-specific T-cell clones. Immunization was performed by a one-way mixed lymphocyte culture and responder T lymphocytes were transduced at day 16, 6 days after the second stimulation. Transductions were carried out using gibbon ape leukemia virus (GALV)-pseudotyped retroviral particles harboring a bicistronic Thy-1/TK vector produced by TEFLY GA16-pKM4 clone 34 packaging cells. Three to 5 days later, CD90 immunomagnetic selection and cloning were performed on the transduced T cells. Our results demonstrate that this procedure led to the recovery of T-cell clones, the majority of which had the expected specificity and a single site of transgene insertion. Such clonotransgenic T-cell populations represent suitable tools to drive a defined alloreaction that can be controlled after bone marrow transplantation.

Efficient transduction and selection of human T-lymphocytes with bicistronic Thy1/HSV1-TK retroviral vector produced by a human packaging cell line.

BACKGROUND: T-cells expressing the HSV1-TK suicide gene can be used for the control of graft-versus-host disease following allogeneic stem cell transplantation. To develop clinical trials based on such a strategy, we have generated under good manufacturing procedures a novel 'split genome' human packaging cell line (1704 cells). METHODS: To minimize the risk of generating replication-competent retroviruses, pol was truncated to remove sequences overlapping with env. To improve retroviral infection and selection of transduced T-cells, high titers of GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector coding for the CD90 GPI-anchored membrane molecule were produced by 1704 cells. RESULTS: Using 1704 cell supernatant and an optimized transduction protocol, approximately 50% of primary T-cells were transduced and could then be purified (approximately 95%) using clinical-grade immunomagnetic beads directed against CD90. Over 96% of these OKT3/IL-2-activated CD90(+)-selected T-cells were killed by ganciclovir. Cell proliferation and cytokine production of transduced T-cells and HLA-restricted cytotoxicity of transduced T-cell clones were identical to those of their non-transduced counterparts cultured under the same conditions. CONCLUSIONS: GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector allow efficient transduction and rapid selection of human T-cells under conditions applicable for clinical trials using the new human 1704 packaging cell line.