Generation Of Virus-Specific Cytotoxic T Lymphocytes (CTLS) Resistant to the Immunosuppressive Drug Tacrolimus (FK506)

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PD-1-induced T cell exhaustion is controlled by a Drp1-dependent mechanism

Programmed cell death-1 (PD-1) signaling downregulates the T-cell response, promoting an exhausted state in tumor-infiltrating T cells, through mostly unveiled molecular mechanisms. Dynamin-related protein-1 (Drp1)-dependent mitochondrial fission plays a crucial role in sustaining T-cell motility, proliferation, survival, and glycolytic engagement. Interestingly, such processes are exactly those inhibited by PD-1 in tumor-infiltrating T cells. Here, we show that PD-1pos CD8+ T cells infiltrating an MC38 (murine adenocarcinoma)-derived murine tumor mass have a downregulated Drp1 activity and more elongated mitochondria compared with PD-1neg counterparts. Also, PD-1pos lymphocytic elements infiltrating a human colon cancer rarely express active Drp1. Mechanistically, PD-1 signaling directly prevents mitochondrial fragmentation following T-cell stimulation by downregulating Drp1 phosphorylation on Ser616, via regulation of the ERK1/2 and mTOR pathways. In addition, downregulation of Drp1 activity in tumor-infiltrating PD-1pos CD8+ T cells seems to be a mechanism exploited by PD-1 signaling to reduce motility and proliferation of these cells. Overall, our data indicate that the modulation of Drp1 activity in tumor-infiltrating T cells may become a valuable target to ameliorate the anticancer immune response in future immunotherapy approaches

Significant reduction of the hybrid BCR/ABL transcripts after induction and consolidation therapy is a powerful predictor of treatment response in adult Philadelphia-positive acute lymphoblastic leukemia.

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Economic analysis of remote monitoring in patients with implantable cardioverter defibrillators or cardiac resynchronization therapy defibrillators in the Trento area, Italy

Introduction: Remote monitoring (RM) technologies have the potential to improve patient care by increasing compliance, providing early indications of heart failure (HF), and potentially allowing for therapy optimization to prevent HF admissions. The aim of this retrospective study was to assess the clinical and economic consequences of RM vs. standard monitoring (SM) through in-office cardiology visits, in patients carrying a cardiac implantable electronic device (CIED). Methods: Clinical and resource consumption data were extracted from the Electrophysiology Registry of the Trento Cardiology Unit, which has been systemically collecting patient information from January 2011 to February 2022. From a clinical standpoint, survival analysis was conducted, and incidence of cardiovascular (CV) related hospitalizations was measured. From an economic standpoint, direct costs of RM and SM were collected to compare the cost per treated patient over a 2-year time horizon. Propensity score matching (PSM) was used to reduce the effect of confounding biases and the unbalance of patient characteristics at baseline. Results: In the enrollment period, N = 402 CIED patients met the inclusion criteria and were included in the analysis (N = 189 patients followed through SM; N = 213 patients followed through RM). After PSM, comparison was limited to N = 191 patients in each arm. After 2-years follow-up since CIED implantation, mortality rate for any cause was 1.6% in the RM group and 19.9% in the SM group (log-rank test, p < 0.0001). Also, a lower proportion of patients in the RM group (25.1%) were hospitalized for CV-related reasons, compared to the SM group (51.3%; p < 0.0001, two-sample test for proportions). Overall, the implementation of the RM program in the Trento territory was cost-saving in both payer and hospital perspectives. The investment required to fund RM (a fee for service in the payer perspective, and staffing costs for hospitals), was more than offset by the lower rate of hospitalizations for CV-related disease. RM adoption generated savings of −€4,771 and −€6,752 per patient in 2 years, in the payer and hospital perspective, respectively. Conclusion: RM of patients carrying CIED improves short-term (2-years) morbidity and mortality risks, compared to SM and reduces direct management costs for both hospitals and healthcare services

GD2 redirected CAR T and activated NK-cell-mediated secretion of IFNγovercomes MYCN-dependent IDO1 inhibition, contributing to neuroblastoma cell immune escape

Immune escape mechanisms employed by neuroblastoma (NB) cells include secretion of immunosuppressive factors disrupting effective antitumor immunity. The use of cellular therapy to treat solid tumors needs to be implemented. Killing activity of anti-GD2 Chimeric Antigen Receptor (CAR) T or natural killer (NK) cells against target NB cells was assessed through coculture experiments and quantified by FACS analysis. ELISA assay was used to quantify interferon-gamma (IFN gamma) secreted by NK and CAR T cells. Real Time PCR and Western Blot were performed to analyze gene and protein levels modifications. Transcriptional study was performed by chromatin immunoprecipitation and luciferase reporter assays on experiments of mutagenesis on the promoter sequence. NB tissue sample were analyzed by IHC and Real Time PCR to perform correlation study. We demonstrate that Indoleamine-pyrrole 2,3-dioxygenase1 (IDO1), due to its ability to convert tryptophan into kynurenines, is involved in NB resistance to activity of immune cells. In NB, IDO1 is able to inhibit the anti-tumor effect displayed by of both anti-GD2 CAR (GD2.CAR) T-cell and NK cells, mainly by impairing their IFN gamma production. Furthermore, inhibition of MYCN expression in NB results into accumulation of IDO1 and consequently of kynurenines, which negatively affect the immune surveillance. Inverse correlation between IDO1 and MYCN expression has been observed in a wide cohort of NB samples. This finding was supported by the identification of a transcriptional repressive role of MYCN on IDO1 promoter. The evidence of IDO1 involvement in NB immune escape and its ability to impair NK and GD2.CAR T-cell activity contribute to clarify one of the possible mechanisms responsible for the limited efficacy of these immunotherapeutic approaches. A combined therapy of NK or GD2.CAR T-cells with IDO1 inhibitors, a class of compounds already in phase I/II clinical studies, could represent a new and still unexplored strategy capable to improve long-term efficacy of these immunotherapeutic approaches

Choice of costimulatory domains and of cytokines determines CAR T-cell activity in neuroblastoma

Chimeric antigen receptor (CAR) T-cell therapy has been shown to be dramatically effective in the treatment of B-cell malignancies. However, there are still substantial obstacles to overcome, before similar responses can be achieved in patients with solid tumors. We evaluated both in vitro and in a preclinical murine model the efficacy of different 2nd and 3rd generation CAR constructs targeting GD2, a disial-ganglioside expressed on the surface of neuroblastoma (NB) tumor cells. In order to address potential safety concerns regarding clinical application, an inducible safety switch, namely inducible Caspase-9 (iC9), was also included in the vector constructs. Our data indicate that a 3rd generation CAR incorporating CD28.4-1BB costimulatory domains is associated with improved anti-tumor efficacy as compared with a CAR incorporating the combination of CD28.OX40 domains. We demonstrate that the choice of 4-1BB signaling results into significant amelioration of several CAR T-cell characteristics, including: 1) T-cell exhaustion, 2) basal T-cell activation, 3) in vivo tumor control and 4) T-cell persistence. The fine-tuning of T-cell culture conditions obtained using IL7 and IL15 was found to be synergic with the CAR.GD2 design in increasing the anti-tumor activity of CAR T cells. We also demonstrate that activation of the suicide gene iC9, included in our construct without significantly impairing neither CAR expression nor anti-tumor activity, leads to a prompt induction of apoptosis of GD2.CAR T cells. Altogether, these findings are instrumental in optimizing the function of CAR T-cell products to be employed in the treatment of children with NB

Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety

T lymphocytes expressing a chimeric antigen receptor (CAR) targeting the CD19 antigen (CAR.19) may be of value for the therapy of B-cell malignancies. Because the in vivo survival, expansion and anti-lymphoma activity of CAR.19 T + cells remain suboptimal even when the CAR contains a CD28 costimulatory endodomain, we generated a novel construct that also incorporates the interleukin-15 (IL-15) gene and an inducible caspase-9-based suicide gene (iC9/CAR.19/IL-15). We found that compared with CAR.19 T + cells, iC9/CAR.19/IL-15 T cells had: (1) greater numeric expansion upon antigen stimulation (10-fold greater expansion in vitro, and 3-to 15-fold greater expansion in vivo) and reduced cell death rate (Annexin-V/7-AAD cells 106% for iC9/CAR.19/IL-15 T + cells and 3219% for CAR.19 T + cells); (2) reduced expression of the programmed death 1 (PD-1) receptor upon antigen stimulation (PD-1 cells 15% for iC9/CAR.19/IL-15 T + cells versus 40% for CAR.19 T + cells); and (3) improved antitumor effects in vivo (from 4.7-to 5.4-fold reduced tumor growth). In addition, iC9/CAR.19/IL-15 T + cells were efficiently eliminated upon pharmacologic activation of the suicide gene. In summary, this strategy safely increases the anti-lymphoma/leukemia effects of CAR.19-redirected T lymphocytes and may be a useful approach for treatment of patients with B-cell malignancies

Salivary and gut microbiomes play a significant role in in vitro oral bioaccessibility, biotransformation, and intestinal absorption of arsenic from food

The release of a toxicant from a food matrix during the gastrointestinal digestion is a crucial determinant of the toxicant's oral bioavailability. We present a modified setup of the human simulator of the gut microbial ecosystem (SHIME), with four sequential gastrointestinal reactors (oral, stomach, small intestine, and colon), including the salivary and colonic microbiomes. Naturally arsenic-containing rice, mussels, and nori seaweed were digested in the presence of microorganisms and in vitro oral bioaccessibility, bioavailability, and metabolism of arsenic species were evaluated following analysis by using HPLC/mass spectrometry. When food matrices were digested with salivary bacteria, the soluble arsenic in the gastric digestion stage increased for mussel and nori samples, but no coincidence impact was found in the small intestinal and colonic digestion stages. However, the simulated small intestinal absorption of arsenic was increased in all food matrices (1.2-2.7 fold higher) following digestion with salivary microorganisms. No significant transformation of the arsenic species occurred except for the arsenosugars present in mussels and nori. In those samples, conversions between the oxo arsenosugars were observed in the small intestinal digestion stage whereupon the thioxo analogs became major metabolites. These results expand our knowledge on the likely metabolism and oral bioavailabiltiy of arsenic during human digestion, and provide valuable information for future risk assessments of dietary arsenic

Different Innate and Adaptive Immune Responses to SARS-CoV-2 Infection of Asymptomatic, Mild, and Severe Cases

SARS-CoV-2 is a novel coronavirus, not encountered before by humans. The wide spectrum of clinical expression of SARS-CoV-2 illness suggests that individual immune responses to SARS-CoV-2 play a crucial role in determining the clinical course after first infection. Immunological studies have focused on patients with moderate to severe disease, demonstrating excessive inflammation in tissues and organ damage. In order to understand the basis of the protective immune response in COVID-19, we performed a longitudinal follow-up, flow-cytometric and serological analysis of innate and adaptive immunity in 64 adults with a spectrum of clinical presentations: 28 healthy SARS-CoV-2-negative contacts of COVID-19 cases; 20 asymptomatic SARS-CoV-2-infected cases; eight patients with Mild COVID-19 disease and eight cases of Severe COVID-19 disease. Our data show that high frequency of NK cells and early and transient increase of specific IgA, IgM and, to a lower extent, IgG are associated with asymptomatic SARS-CoV-2 infection. By contrast, monocyte expansion and high and persistent levels of IgA and IgG, produced relatively late in the course of the infection, characterize severe disease. Modest increase of monocytes and different kinetics of antibodies are detected in mild COVID-19. The importance of innate NK cells and the short-lived antibody response of asymptomatic individuals and patients with mild disease suggest that only severe COVID-19 may result in protective memory established by the adaptive immune response