Results of phase 2 randomized multi-center study to evaluate the safety and efficacy of infusion of memory T cells as adoptive therapy in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia and/or lymphopenia (RELEASE NCT04578210).

BACKGROUND AIMS: There are currently no effective anti-viral treatments for coronavirus disease 2019 (COVID-19)-hospitalized patients with hypoxemia. Lymphopenia is a biomarker of disease severity usually present in patients who are hospitalized. Approaches to increasing lymphocytes exerting an anti-viral effect must be considered to treat these patients. Following our phase 1 study, we performed a phase 2 randomized multicenter clinical trial in which we evaluated the efficacy of the infusion of allogeneic off-the-shelf CD45RA- memory T cells containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells from convalescent donors plus the standard of care (SoC) versus just the SoC treatment. METHODS: Eighty-four patients were enrolled in three Spanish centers. The patients were randomized into the infusion of 1 × 106/kg CD45RA- memory T cells or the SoC. We selected four unvaccinated donors based on the expression of interferon gamma SARS-CoV-2-specific response within the CD45RA- memory T cells and the most frequent human leukocyte antigen typing in the Spanish population. RESULTS: We analyzed data from 81 patients. The primary outcome for recovery, defined as the proportion of participants in each group with normalization of fever, oxygen saturation sustained for at least 24 hours and lymphopenia recovery through day 14 or at discharge, was met for the experimental arm. We also observed faster lymphocyte recovery in the experimental group. We did not observe any treatment-related adverse events. CONCLUSIONS: Adoptive cell therapy with off-the-shelf CD45RA- memory T cells containing SAR-CoV-2-specific T cells is safe, effective and accelerates lymphocyte recovery of patients with COVID-19 pneumonia and/or lymphopenia. TRIAL REGISTRATION: NCT04578210.

NKG2D-CAR memory T cells target pediatric T-cell acute lymphoblastic leukemia in vitro and in vivo but fail to eliminate leukemia initiating cells.

Introduction: Refractory/relapsed pediatric acute leukemia are still clinically challenging and new therapeutic strategies are needed. Interactions between Natural Killer Group 2D (NKG2D) receptor, expressed in cytotoxic immune cells, and its ligands (NKG2DL), which are upregulated in leukemic blasts, are important for anti-leukemia immunosurveillance. Nevertheless, leukemia cells may develop immunoescape strategies as NKG2DL shedding and/or downregulation. Methods: In this report, we analyzed the anti-leukemia activity of NKG2D chimeric antigen receptor (CAR) redirected memory (CD45RA-) T cells in vitro and in a murine model of T-cell acute lymphoblastic leukemia (T-ALL). We also explored in vitro how soluble NKG2DL (sNKG2DL) affected NKG2D-CAR T cells' cytotoxicity and the impact of NKG2D-CAR T cells on Jurkat cells gene expression and in vivo functionality. Results: In vitro, we found NKG2D-CAR T cells targeted leukemia cells and showed resistance to the immunosuppressive effects exerted by sNKG2DL. In vivo, NKG2D-CAR T cells controlled T cell leukemia burden and increased survival of the treated mice but failed to cure the animals. After CAR T cell treatment, Jurkat cells upregulated genes related to proliferation, survival and stemness, and in vivo, they exhibited functional properties of leukemia initiating cells. Discussion: The data here presented suggest, that, in combination with other therapeutic approaches, NKG2D-CAR T cells could be a novel treatment for pediatric T-ALL.

Rationale for combined therapies in severe-to-critical COVID-19 patients.

An unprecedented global social and economic impact as well as a significant number of fatalities have been brought on by the coronavirus disease 2019 (COVID-19), produced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Acute SARS-CoV-2 infection can, in certain situations, cause immunological abnormalities, leading to an anomalous innate and adaptive immune response. While most patients only experience mild symptoms and recover without the need for mechanical ventilation, a substantial percentage of those who are affected develop severe respiratory illness, which can be fatal. The absence of effective therapies when disease progresses to a very severe condition coupled with the incomplete understanding of COVID-19's pathogenesis triggers the need to develop innovative therapeutic approaches for patients at high risk of mortality. As a result, we investigate the potential contribution of promising combinatorial cell therapy to prevent death in critical patients.

Thrombocytopenia requiring platelet transfusions is predictive of retinopathy of prematurity in preterm infants.

BACKGROUND: Recent studies suggest that some hematological parameters and ratios provided by blood count collected on the first day of life may be predictors for the development of retinopathy of prematurity (ROP). The aims of this study were to assess whether hematological parameters and ratios in blood count collected on the first day of life are associated with ROP. METHODS: Retrospective data collection from electronic clinical files of inborn preterm infants with a gestational age of less than 30 weeks delivered from January 2008 to December 2022, in a level III Neonatal Intensive Care Unit (NICU). We collected detailed clinical and demographic data on the mother, pregnancy, mode of delivery, resuscitation, in-hospital neonatal morbidity, pathological examination of the placenta, and hematological parameters and ratios provided by blood count collected on the first day of life. We compared neonates with or without ROP and performed a subgroup analysis on patients with ROP≥2. RESULTS: We included 140 preterm infants with ophthalmic examination; 81(57.9%) developed ROP. The need for platelet transfusions (OR=5.28; 95% CI: 1.31-21.21; P=0.019) and bronchopulmonary dysplasia (BPD) (OR=5.12; 95% CI: 1.14-22.98; P=0.033) were independent risk factors for ROP. In the multivariate analysis, no associations were found between any of the hematological parameters or ratios studied and ROP. CONCLUSIONS: In our study, hematological parameters and ratios on the first day of life were not associated with the development of ROP. However, thrombocytopenia that required platelet transfusions was associated with ROP development and severity.

Inherited human ezrin deficiency impairs adaptive immunity.

BACKGROUND: Inborn errors of immunity (IEI) are a group of monogenic diseases that confer susceptibility to infection, autoimmunity, and cancer. Despite the life-threatening consequences of some IEI, their genetic cause remains unknown in many patients. OBJECTIVE: We investigated a patient with an IEI of unknown genetic etiology. METHODS: Whole-exome sequencing identified a homozygous missense mutation of the gene encoding ezrin (EZR), substituting a threonine for an alanine at position 129. RESULTS: Ezrin is one of the subunits of the ezrin, radixin, and moesin (ERM) complex. The ERM complex links the plasma membrane to the cytoskeleton and is crucial for the assembly of an efficient immune response. The A129T mutation abolishes basal phosphorylation and decreases calcium signaling, leading to complete loss of function. Consistent with the pleiotropic function of ezrin in myriad immune cells, multidimensional immunophenotyping by mass and flow cytometry revealed that in addition to hypogammaglobulinemia, the patient had low frequencies of switched memory B cells, CD4+ and CD8+ T cells, MAIT, γδ T cells, and centralnaive CD4+ cells. CONCLUSIONS: Autosomal-recessive human ezrin deficiency is a newly recognized genetic cause of B-cell deficiency affecting cellular and humoral immunity.

Two Siblings With Recurrent Fevers: The Path to Mevalonate Kinase Deficiency Diagnosis.

Systemic autoinflammatory diseases (SAIDs) are a group of disorders that constitute a rare cause of recurrent fevers. Recurrent fevers are defined as periodic febrile episodes lasting from days to weeks, separated by symptom-free intervals of variable duration. They present multiple etiologies, representing a diagnostic challenge. Mevalonate kinase deficiency (MKD) is a genetic SAID, a rare hereditary recurrent fever syndrome (HRF) caused by pathogenic variants in the mevalonate kinase (MVK) gene. It is characterized by the early onset of periodic fever flares, frequently associated with joint, gastrointestinal, skin, and lymph node involvement. Although elevated serum immunoglobulin D (IgD) levels were previously considered an MKD's hallmark, normal values do not exclude it. High serum immunoglobulin A (IgA) is frequent. An acute-phase response and elevated urinary mevalonic acid (UAV) excretion during flares may aid in the diagnosis. Genetic testing is an essential tool to confirm the diagnosis. The authors report two siblings presenting with early infancy onset of recurrent febrile illness and characteristic associated symptoms, one of which was initially misdiagnosed with periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome. MKD diagnoses were only established at 12 and nine years old, respectively, after the identification of the same two MVKgene variants. The diagnosis in the eldest favored the earlier recognition of MKD in the youngest. Owing to its wide spectrum of manifestations, with many being nonspecific and/or shared with other more frequent entities, a significant proportion of MKD patients present a long delay until its final establishment. These cases illustrate the MKD diagnosis and management's difficulties, reinforcing the importance of a careful clinical history and HRF awareness for its prompt diagnosis and appropriate precocious referral.

Familial CD45RA- T cells to treat severe refractory infections in immunocompromised patients.

Background: Immunocompromised patients are susceptible to high-risk opportunistic infections and malignant diseases. Most antiviral and antifungal drugs are quite toxic, relatively ineffective, and induce resistance in the long term. The transfer of pathogen-specific Cytotoxic T-Lymphocytes has shown a minimal toxicity profile and effectiveness in treating Cytomegalovirus, Adenovirus, Epstein - Barr virus, BK Virus and Aspergillus infections, but this therapy have the main limitations of regulatory issues, high cost, and absence of public cell banks. However, CD45RA- cells containing pathogen-specific memory T-cells involve a less complex manufacturing and regulatory process and are cheaper, feasible, safe, and potentially effective. Methods: We present preliminary data from six immunocompromised patients: four who had severe infectious diseases and two who had EBV lymphoproliferative disease. All of them underwent multiple safe familial CD45RA- T-cell infusions as adoptive passive cell therapy, containing Cytomegalovirus, Epstein - Barr virus, BK virus, and Aspergillus-specific memory T-cells. We also present the method for selecting the best donors for CD45RA- cells in each case and the procedure to isolate and store these cells. Results: The infusions were safe, there was no case of graft-versus host disease, and they showed a clear clinical benefit. The patients treated for BK virus nephritis, Cytomegalovirus encephalitis, Cytomegalovirus reactivation, and disseminated invasive aspergillosis experienced pathogen clearance, complete resolution of symptoms in 4-6 weeks and a lymphocyte increase in 3 of 4 cases after 3-4 months. Donor T cell transient microchimerism was detected in one patient. The two patients treated for EBV lymphoproliferative disease underwent chemotherapy and several infusions of CD45RA- memory T-cells containing EBV cytotoxic lymphocytes. Donor T-cell microchimerism was observed in both patients. The viremia cleared in one of the patients, and in the other, despite the viremia not clearing, hepatic lymphoproliferative disease remained stable and was ultimately cured with EBV-specific Cytotoxic T-Lymphocytes. Conclusion: The use of familial CD45RA- T-cells containing specific Cytotoxic T-lymphocytes is a feasible, safe and potential effective approach for treating severe pathogen infections in immunocompromised patients through a third party donor. Furthermore, this approach might be of universal use with fewer institutional and regulatory barriers.

Donor selection for adoptive cell therapy with CD45RA- memory T cells for patients with coronavirus disease 2019, and dexamethasone and interleukin-15 effects on the phenotype, proliferation and interferon gamma release.

BACKGROUND AIMS: We have previously demonstrated the safety and feasibility of adoptive cell therapy with CD45RA- memory T cells containing severe acute respiratory syndrome coronavirus 2-specific T cells for patients with coronavirus disease 2019 from an unvaccinated donor who was chosen based on human leukocyte antigen compatibility and cellular response. In this study, we examined the durability of cellular and humoral immunity within CD45RA- memory T cells and the effect of dexamethasone, the current standard of care treatment, and interleukin-15, a cytokine critically involved in T-cell maintenance and survival. METHODS: We performed a longitudinal analysis from previously severe acute respiratory syndrome coronavirus 2-infected and infection-naïve individuals covering 21 months from infection and 10 months after full vaccination with the BNT162b2 Pfizer/BioNTech vaccine. RESULTS: We observed that cellular responses are maintained over time. Humoral responses increased after vaccination but were gradually lost. In addition, dexamethasone did not alter cell functionality or proliferation of CD45RA- T cells, and interleukin-15 increased the memory T-cell activation state, regulatory T cell expression, and interferon gamma release. CONCLUSIONS: Our results suggest that the best donors for adoptive cell therapy would be recovered individuals and 2 months after vaccination, although further studies with larger cohorts would be needed to confirm this finding. Dexamethasone did not affect the characteristics of the memory T cells at a concentration used in the clinical practice and IL-15 showed a positive effect on SARS-CoV-2-specific CD45RA- T cells.

c-MET/VEGFR-2 co-localisation impacts on survival following bevacizumab therapy in epithelial ovarian cancer: an exploratory biomarker study of the phase 3 ICON7 trial.

INTRODUCTION: Bevacizumab improves survival outcomes in women diagnosed with epithelial ovarian cancer (EOC). Pre-clinical data showed that the c-MET/VEGFR-2 heterocomplex negates VEGF inhibition through activation of c-MET signalling, leading to a more invasive and metastatic phenotype. We evaluated the clinical significance of c-MET and VEGFR-2 co-localisation and its association with VEGF pathway-related single nucleotide polymorphisms (SNPs) in women participating in the phase 3 trial, ICON7 (ClinicalTrials.gov identifier: NCT00262847). MATERIALS AND METHODS: Patients had FIGO stage I-IIA grade 3/poorly differentiated or clear cell carcinoma or stage IIB-IV epithelial ovarian, primary peritoneal or fallopian tube cancer. Immunofluorescence staining for co-localised c-MET and VEGFR-2 on tissue microarrays and genotyping of germline DNA from peripheral blood leukocytes for VEGFA and VEGFR-2 SNPs was performed. The significance of these biomarkers was assessed against survival. RESULTS: Tissue microarrays from 178 women underwent immunofluorescence staining. Multivariable analysis showed that greater c-MET/VEGFR-2 co-localisation predicted worse OS in patients treated with bevacizumab after adjusting for FIGO stage and debulking surgery outcome (hazard ratio [HR] 1.034, 95% confidence interval [95%CI] 1.010-1.059). Women in the c-MET/VEGFR-2HIGH group treated with bevacizumab demonstrated significantly reduced OS (39.3 versus > 60 months; HR 2.00, 95%CI 1.08-3.72). Germline DNA from 449 women underwent genotyping. In the bevacizumab group, those women with the VEGFR-2 rs2305945 G/G variant had a trend towards shorter PFS compared with G/T or T/T variants (18.3 versus 23.0 months; HR 0.74, 95%CI 0.53-1.03). CONCLUSIONS: In bevacizumab-treated women diagnosed with EOC, high c-MET/VEGFR-2 co-localisation on tumour tissue and the VEGFR-2 rs2305945 G/G variant, which may be biologically related, were associated with worse survival outcomes.

Cancer predisposing syndrome: a retrospective cohort analysis in a pediatric and multidisciplinary genetic cancer counseling unit.

INTRODUCTION: Approximately 10% of pediatric patients with cancer have an inherited, sometimes masked, cancer predisposition syndrome (CPS). Identifying patients with genetic susceptibility to malignant disease is essential for their correct diagnosis and clinical management. MATERIALS AND METHODS: Here, we present the workflow and experience of a multidisciplinary cancer predisposition unit focused on pediatric patients with cancer. RESULTS: Between July 2018 and July 2020, 214 patients were diagnosed with pediatric cancer in our Hospital. Of all, 49 patients were treated at the CPS unit, 48 of whom were recommended a genetic study. Mutational analysis was performed on DNA from peripheral blood samples, with approximately 45% of the patients (n = 22) receiving a confirmed CPS diagnosis, all of whom underwent genetic counseling. These cases represent 20% of all pediatric cancers diagnosed in the same center during this period. Most of the patients were diagnosed with hereditary retinoblastoma; however, we also identified families with Li-Fraumeni syndrome, multiple endocrine neoplasia type 2, hereditary melanoma, hereditary leiomyomatosis, and Gardner syndrome. CONCLUSION: Despite its limitations regarding the type of tumors and number of patients included, this study revealed that implementing a specialized unit focused on children with cancer results in a higher diagnostic rate and better genetic counseling for patients with pediatric cancer predisposition syndromes.

Ruxolitinib does not completely abrogate the functional capabilities of TLR4/9 ligand-activated NK cells.

Introduction: Natural killer (NK) cells are lymphocytes from the innate immune system part of the first defense barrier against infected and transformed cells, representing 5%-15% of peripheral blood lymphocytes. The cytotoxic capacity of NK cells is controlled by a balance between inhibitory and activating NK receptors expressed on their surface, which recognize and interact with the ligands on stressed cells. The cytokines involved in NK cell activation, proliferation, survival, and cytotoxicity are signaled mainly through the Janus kinase and signal transducer and activator of transcription proteins (JAK/STAT) pathway. NK cells are also activated in response to pathogens through Toll-like receptors (TLRs) expressed on their surface. Ruxolitinib is a specific JAK1/2 inhibitor approved for treating myelofibrosis and for steroid-refractory acute and chronic graft-versus-host disease (SR-GvHD). Methods: Purified NK cells from healthy donors were stimulated with two TOLL-like receptor ligands, LPS and CpG, in the presence of different concentrations of Ruxolitinib. Results: This study showed the effects of ruxolitinib on TLR4 and TLR9 ligand-activated NK cells from healthy donors. Ruxolitinib did not completely inhibit STAT3 phosphorylation and had a moderate effect on NK cell cytokine activation via the TLR pathway. Only the highest doses of ruxolitinib led to a decrease in the pro-inflammatory cytokines tumor necrosis factor α, interferon-γ, interleukin-6, and interleukin-1ß. The cytotoxic capacity of stimulated NK cells versus K562, SEM, and MV-4-11 cell lines was reduced by increasing doses of ruxolitinib, but it was not completely abolished and we observed no major changes in degranulation capacity. Phenotypic changes were observed in activated NK cells in the presence of ruxolitinib. In a small cohort of pediatric patients treated with ruxolitinib for SR-GvHD, we observed no decrease in NK cell counts; however, further prospective studies with larger cohorts are necessary to confirm this finding. Discussion: In summary, our results showed that the functional capabilities and phenotype of NK cells activated through TLR4/9 agonists were not completely abolished by the inhibition of the JAK-STAT pathway by ruxolitinib.

Identifying SARS-CoV-2 'memory' NK cells from COVID-19 convalescent donors for adoptive cell therapy.

COVID-19 disease is the manifestation of syndrome coronavirus 2 (SARS-CoV-2) infection, which is causing a worldwide pandemic. This disease can lead to multiple and different symptoms, being lymphopenia associated with severity one of the most persistent. Natural killer cells (NK cells) are part of the innate immune system, being fighting against virus-infected cells one of their key roles. In this study, we determined the phenotype of NK cells after COVID-19 and the main characteristic of SARS-CoV-2-specific-like NK population in the blood of convalescent donors. CD57+ NKG2C+ phenotype in SARS-CoV-2 convalescent donors indicates the presence of 'memory'/activated NK cells as it has been shown for cytomegalovirus infections. Although the existence of this population is donor dependent, its expression may be crucial for the specific response against SARS-CoV-2, so that, it gives us a tool for selecting the best donors to produce off-the-shelf living drug for cell therapy to treat COVID-19 patients under the RELEASE clinical trial (NCT04578210).

Patent ductus arteriosus in preterm newborns: A tertiary hospital experience.

INTRODUCTION: Patent ductus arteriosus (PDA) in preterm infants has been associated with increased mortality and comorbidities. This study aimed to characterize the population of preterm infants diagnosed with PDA and to identify predictive factors of response to medical treatment of PDA. METHODS: An eight-year retrospective observational study was carried out, which included all preterm infants with a gestational age (GA) between 23 and 32 weeks diagnosed with PDA, admitted to the Neonatal Unit of the CHUSJ. Univariate comparative analysis was performed, and models for predicting the effectiveness of PDA treatment with ibuprofen were explored by multivariate logistic regression analysis. RESULTS: 115 cases were included and 34 were excluded, with a final sample of 81 preterm infants with PDA. The univariate analysis revealed significant differences in the closure efficacy via medical treatment with ibuprofen in several variables, and a multivariate logistic regression model was obtained (discriminative capacity 72.2%, sensitivity 98.1%, specificity 57.1%), taking into account the effect of GA, type of delivery, need for diuretics treatment and platelet transfusion. CONCLUSION: This study enabled the population of preterm infants diagnosed with PDA to be characterized and the identification of a predictive model that can help predict the efficacy of medical treatment and thus contribute to optimizing the medical approach to the non-responders.

Facing CAR T Cell Challenges on the Deadliest Paediatric Brain Tumours.

Central nervous system (CNS) tumours comprise 25% of the paediatric cancer diagnoses and are the leading cause of cancer-related death in children. Current treatments for paediatric CNS tumours are far from optimal and fail for those that relapsed or are refractory to treatment. Besides, long-term sequelae in the developing brain make it mandatory to find new innovative approaches. Chimeric antigen receptor T cell (CAR T) therapy has increased survival in patients with B-cell malignancies, but the intrinsic biological characteristics of CNS tumours hamper their success. The location, heterogeneous antigen expression, limited infiltration of T cells into the tumour, the selective trafficking provided by the blood-brain barrier, and the immunosuppressive tumour microenvironment have emerged as the main hurdles that need to be overcome for the success of CAR T cell therapy. In this review, we will focus mainly on the characteristics of the deadliest high-grade CNS paediatric tumours (medulloblastoma, ependymoma, and high-grade gliomas) and the potential of CAR T cell therapy to increase survival and patients' quality of life.