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An abstract on the study relating to the facts and artifacts of induced resistance to plant viruses is presented.

Introduction: SARS-CoV-2 pandemic still poses a significant burden on global health and economy, especially for symptoms persisting beyond the acute disease. COVID-19 manifests with various degrees of severity and the identification of early biomarkers capable of stratifying patient based on risk of progression could allow tailored treatments., Methods: We longitudinally analyzed 67 patients, classified according to a WHO ordinal scale as having Mild, Moderate, or Severe COVID-19. Peripheral blood samples were prospectively collected at hospital admission and during a 6-month follow-up after discharge. Several subsets and markers of the innate and adaptive immunity were monitored as putative factors associated with COVID-19 symptoms., Results: More than 50 immunological parameters were associated with disease severity. A decision tree including the main clinical, laboratory, and biological variables at admission identified low NK-cell precursors and CD14 + CD91 + monocytes, and high CD8 + Effector Memory T cell frequencies as the most robust immunological correlates of COVID-19 severity and reduced survival. Moreover, low regulatory B-cell frequency at one month was associated with the susceptibility to develop long COVID at six months, likely due to their immunomodulatory ability., Discussion: These results highlight the profound perturbation of the immune response during COVID-19. The evaluation of specific innate and adaptive immune-cell subsets allows to distinguish between different acute and persistent COVID-19 symptoms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Noviello, De Lorenzo, Chimienti, Maugeri, De Lalla, Siracusano, Lorè, Rancoita, Cugnata, Tassi, Dispinseri, Abbati, Beretta, Ruggiero, Manfredi, Merolla, Cantarelli, Tresoldi, Pastori, Caccia, Sironi, Marzinotto, Saliu, Ghezzi, Lampasona, Vicenzi, Cinque, Manfredi, Scarlatti, Dellabona, Lopalco, Di Serio, Malnati, Ciceri, Rovere-Querini and Bonini.)

Background: In adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells., Methods: We simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCR ED ) and permanently disrupted LAG3 , TIM-3 or 2B4 genes (IR KO ) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCR ED -IR KO and IR competent (TCR ED -IR COMP ) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro . Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models., Results: We show that upon chronic stimulation, TCR ED -IR KO cells are superior to TCR ED -IR COMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo . Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells., Conclusion: These results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses., Competing Interests: CB, ER, ZM, BC, AP, LV, FC, PG, LN and BCC are inventors on different patents on cancer immunotherapy and genetic engineering. CB has been member of Advisory Board and Consultant for Molmed, Intellia, TxCell, Novartis, GSK, Allogene, Kite/Gilead, Miltenyi, Kiadis, Evir, Janssen and received research support from Molmed s.p.a and Intellia Therapeutics. LV received royalties and research support from GEN-DX and research support from Moderna Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Cianciotti, Magnani, Ugolini, Camisa, Merelli, Vavassori, Potenza, Imparato, Manfredi, Abbati, Perani, Spinelli, Shifrut, Ciceri, Vago, Di Micco, Naldini, Genovese, Ruggiero and Bonini.)

To study and then harness the tumor-specific T cell dynamics after allogeneic hematopoietic stem cell transplant, we typed the frequency, phenotype, and function of lymphocytes directed against tumor-associated antigens (TAAs) in 39 consecutive transplanted patients, for 1 year after transplant. We showed that TAA-specific T cells circulated in 90% of patients but display a limited effector function associated to an exhaustion phenotype, particularly in the subgroup of patients deemed to relapse, where exhausted stem cell memory T cells accumulated. Accordingly, cancer-specific cytolytic functions were relevant only when the TAA-specific T cell receptors (TCRs) were transferred into healthy, genome-edited T cells. We then exploited trogocytosis and ligandome-on-chip technology to unveil the specificities of tumor-specific TCRs retrieved from the exhausted T cell pool. Overall, we showed that harnessing circulating TAA-specific and exhausted T cells allow to isolate TCRs against TAAs and previously not described acute myeloid leukemia antigens, potentially relevant for T cell-based cancer immunotherapy.

Introduction: Despite predicted efficacy, immunotherapy in epithelial ovarian cancer (EOC) has limited clinical benefit and the prognosis of patients remains poor. There is thus a strong need for better identifying local immune dynamics and immune-suppressive pathways limiting T-cell mediated anti-tumor immunity., Methods: In this observational study we analyzed by immunohistochemistry, gene expression profiling and flow cytometry the antigenic landscape and immune composition of 48 EOC specimens, with a focus on tumor-infiltrating lymphocytes (TILs)., Results: Activated T cells showing features of partial exhaustion with a CD137 + CD39 + PD-1 + TIM-3 + CD45RA - CD62L - CD95 + surface profile were exclusively present in EOC specimens but not in corresponding peripheral blood or ascitic fluid, indicating that the tumor microenvironment might sustain this peculiar phenotype. Interestingly, while neoplastic cells expressed several tumor-associated antigens possibly able to stimulate tumor-specific TILs, macrophages provided both co-stimulatory and inhibitory signals and were more abundant in TILs-enriched specimens harboring the CD137 + CD39 + PD-1 + TIM-3 + CD45RA - CD62L - CD95 + signature., Conclusion: These data demonstrate that EOC is enriched in CD137 + CD39 + PD-1 + TIM-3 + CD45RA - CD62L - CD95 + T lymphocytes, a phenotype possibly modulated by antigen recognition on neoplastic cells and by a combination of inhibitory and co-stimulatory signals largely provided by infiltrating myeloid cells. Furthermore, we have identified immunosuppressive pathways potentially hampering local immunity which might be targeted by immunotherapeutic approaches., Competing Interests: CBo: received research support from Intellia Therapeutics and is member of advisory boards/consultant/speaker for Molmed, Intellia, TxCell, Novartis, GSK, Allogene, Kite/Gilead, Miltenyi, Kiadis, QuellTX, Janssen. BS is an employee and shareholder of Intellia Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tassi, Bergamini, Wignall, Sant’Angelo, Brunetto, Balestrieri, Redegalli, Potenza, Abbati, Manfredi, Cangi, Magliacane, Scalisi, Ruggiero, Maffia, Trippitelli, Rabaiotti, Cioffi, Bocciolone, Candotti, Candiani, Taccagni, Schultes, Doglioni, Mangili and Bonini.)

Objective: Colorectal tumours are often densely infiltrated by immune cells that have a role in surveillance and modulation of tumour progression but are burdened by immunosuppressive signals, which might vary from primary to metastatic stages. Here, we deployed a multidimensional approach to unravel the T-cell functional landscape in primary colorectal cancers (CRC) and liver metastases, and genome editing tools to develop CRC-specific engineered T cells., Design: We paired high-dimensional flow cytometry, RNA sequencing and immunohistochemistry to describe the functional phenotype of T cells from healthy and neoplastic tissue of patients with primary and metastatic CRC and we applied lentiviral vectors (LV) and CRISPR/Cas9 genome editing technologies to develop CRC-specific cellular products., Results: We found that T cells are mainly localised at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors, which largely differ from primary to metastatic sites. Our data highlighted CD39 as the major driver of exhaustion in both primary and metastatic colorectal tumours. We thus simultaneously redirected T-cell specificity employing a novel T-cell receptor targeting HER-2 and disrupted the endogenous TCR genes (TCR editing (TCR ED )) and the CD39 encoding gene ( ENTPD1 ), thus generating TCR ED ENTPD1 KO HER-2-redirected lymphocytes. We showed that the absence of CD39 confers to HER-2-specific T cells a functional advantage in eliminating HER-2 + patient-derived organoids in vitro and in vivo ., Conclusion: HER-2-specific CD39 disrupted engineered T cells are promising advanced medicinal products for primary and metastatic CRC., Competing Interests: Competing interests: CBo has been member of Advisory Board and Consultant for Intellia, TxCell, Novartis, GSK, Allogene, Kite/Gilead, Kiadis, Evir, Janssen, Genyo, Epsilen and received research support from Intellia Therapeutics. AP, PDB, GC, ER, BCC and CBo are inventors on different patents on cancer immunotherapy and genetic engineering., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: Ulcerative colitis (UC) is a chronic inflammatory disorder of unknown aetiology. Gut virome dysbiosis is fundamental in UC progression, although its role in the early phases of the disease is far from fully understood. Therefore, we sought to investigate the role of a virome-associated protein encoded by the Orthohepadnavirus genus, the hepatitis B virus X protein (HBx), in UC aetiopathogenesis., Design: HBx positivity of UC patient-derived blood and gut mucosa was assessed by RT-PCR and Sanger sequencing and correlated with clinical characteristics by multivariate analysis. Transcriptomics was performed on HBx-overexpressing endoscopic biopsies from healthy donors.C57BL/6 mice underwent intramucosal injections of liposome-conjugated HBx-encoding plasmids or the control, with or without antibiotic treatment. Multidimensional flow cytometry analysis was performed on colonic samples from HBx-treated and control animals. Transepithelial electrical resistance measurement, proliferation assay, chromatin immunoprecipitation assay with sequencing and RNA-sequencing were performed on in vitro models of the gut barrier. HBx-silencing experiments were performed in vitro and in vivo ., Results: HBx was detected in about 45% of patients with UC and found to induce colonic inflammation in mice, while its silencing reverted the colitis phenotype in vivo . HBx acted as a transcriptional regulator in epithelial cells, provoking barrier leakage and altering both innate and adaptive mucosal immunity ex vivo and in vivo ., Conclusion: This study described HBx as a contributor to the UC pathogenesis and provides a new perspective on the virome as a target for tailored treatments., Competing Interests: Competing interests: SD has served as a speaker, consultant and advisory board member for Schering Plough, Abbott (AbbVie) Laboratories, Merck and Co, UCB Pharma, Ferring, Cellerix, Millenium Takeda, Nycomed, Pharmacosmos, Actelion, Alfa Wasserman, Genentech, Grunenthal, Pfizer, AstraZeneca, Novo Nordisk, Vifor and Johnson and Johnson. LP-B has served as consultant for Merck, AbbVie, Janssen, Genentech, Ferring, Tillots, Vifor, Pharmacosmos, Celltrion, Takeda, Biogaran, Boerhinger-lngelheim, Lilly, Pfizer, Jndex Pharmaceuticals, Amgen, Sandoz, Celgene, Biogen, Samsung Bioepis, Alma, Sterna, Nestlé, Enterome, Mylan, HAC-Pharma, Tigenix, and has served as speaker for Merck, AbbVie, Janssen, Genentech, Ferring, Tillots, Vifor, Pharmacosmos, Celltrion, Takeda, Boerhinger-lngelheim, Pfizer, Amgen, Biogen, Samsung Bioepis. VJ has received has received consulting/advisory board fees from AbbVie, Alimentiv Inc (formerly Robarts Clinical Trials), Arena Pharmaceuticals, Asahi Kasei Pharma, Asieris, Bristol Myers Squibb, Celltrion, Eli Lilly, Ferring, Flagship Pioneering, Fresenius Kabi, Galapagos, GlaxoSmithKline, Genentech, Gilead, Janssen, Merck, Mylan, Pandion, Pendopharm, Pfizer, Protagonist, Reistone Biopharma, Roche, Sandoz, Second Genome,Takeda, Teva, Topivert, Vividion; speaker’s fees from, AbbVie, Ferring, Galapagos, Janssen Pfizer Shire, Takeda, Fresenius Kabi. The other authors declare no conflicts of interest., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

After allogeneic hematopoietic stem cell transplantation (HSCT), the emergence of circulating cytomegalovirus (CMV)- specific T cells correlates with protection from CMV reactivation, an important risk factor for non-relapse mortality. However, functional assays measuring CMV-specific cells are time-consuming and often inaccurate at early time-points. We report the results of a prospective single-center, non-interventional study that identified the enumeration of Dextramerpositive CMV-specific lymphocytes as a reliable and early predictor of viral reactivation. We longitudinally monitored 75 consecutive patients for 1 year after allogeneic HSCT (n=630 samples). The presence of ≥0.5 CMV-specific CD8+ cells/mL at day +45 was an independent protective factor from subsequent clinically relevant reactivation in univariate (P<0.01) and multivariate (P<0.05) analyses. Dextramer quantification correlated with functional assays measuring interferon-γ production, and allowed earlier identification of high-risk patients. In mismatched transplants, the comparative analysis of lymphocytes restricted by shared, donor- and host-specific HLA revealed the dominant role of thymic-independent CMV-specific reconstitution. Shared and donor-restricted CMV-specific T cells reconstituted with similar kinetics in recipients of CMV-seropositive donors, while donor-restricted T-cell reconstitution from CMV-seronegative grafts was impaired, indicating that in primary immunological responses the emergence of viral-specific T cells is largely sustained by antigen encounter on host infected cells rather than by cross-priming/presentation by non-infected donor-derived antigen-presenting cells. Multiparametric flow cytometry and high-dimensional analysis showed that shared-restricted CMV-specific lymphocytes display a more differentiated phenotype and increased persistence than donor-restricted counterparts. In this study, monitoring CMV-specific cells by Dextramer assay after allogeneic HSCT shed light on mechanisms of immune reconstitution and enabled risk stratification of patients, which could improve the clinical management of post-transplant CMV reactivations.

We describe a multi-step high-dimensional (HD) flow cytometry workflow for the deep phenotypic characterization of T cells infiltrating metastatic tumor lesions in the liver, particularly derived from colorectal cancer (CRC-LM). First, we applied a novel flow cytometer setting approach based on single positive cells rather than fluorescent beads, resulting in optimal sensitivity when compared with previously published protocols. Second, we set up a 26-color based antibody panel designed to assess the functional state of both conventional T-cell subsets and unconventional invariant natural killer T, mucosal associated invariant T, and gamma delta T (γδT)-cell populations, which are abundant in the liver. Third, the dissociation of the CRC-LM samples was accurately tuned to preserve both the viability and antigenic integrity of the stained cells. This combined procedure permitted the optimal capturing of the phenotypic complexity of T cells infiltrating CRC-LM. Hence, this study provides a robust tool for high-dimensional flow cytometry analysis of complex T-cell populations, which could be adapted to characterize other relevant pathological tissues., (© 2022 Faccani et al.)

The phenotype of infused cells is a major determinant of Adoptive T-cell therapy (ACT) efficacy. Yet, the difficulty in deciphering multiparametric cytometry data limited the fine characterization of cellular products. To allow the analysis of dynamic and complex flow cytometry samples, we developed cytoChain, a novel dataset mining tool and a new analytical workflow. CytoChain was challenged to compare state-of-the-art and innovative culture conditions to generate stem-like memory cells (T SCM ) suitable for ACT. Noticeably, the combination of IL-7/15 and superoxides scavenging sustained the emergence of a previously unidentified nonexhausted Fit-T SCM signature, overlooked by manual gating and endowed with superior expansion potential. CytoChain proficiently traced back this population in independent datasets, and in T-cell receptor engineered lymphocytes. CytoChain flexibility and function were then further validated on a published dataset from circulating T cells in COVID-19 patients. Collectively, our results support the use of cytoChain to identify novel, functionally critical immunophenotypes for ACT and patients immunomonitoring., (© 2021 Wiley-VCH GmbH.)

This artist-led research project involved 10 visual artists producing 10 ambient portraits and a portrait average of a locally familiar Sitter, and 10 ambient portraits and a portrait average of a less locally familiar Sitter. All were then assessed for likeness by more than 150 members of the general public attending an exhibition during Australia's 2018 National Science Week . The results of this study are that portrait averages can be highly shape accurate and tend to be seen as a good likeness by all viewers. However, the portrait average is not necessarily the best likeness. Extending and validating our previous findings regarding the relationship of likeness, familiarity, and shape accuracy (as measured using geometric morphometrics) in portraiture, unfamiliar viewers favouring shape accurate depictions of a Sitter attained statistical significance. Familiar viewers, however, although also tending to view shape accurate depictions a good to very good likeness, were shown to have a stronger preference for portraits that exaggerate a Sitter's facial distinctiveness, including an exaggeration of their head pose, providing such exaggerations are in approximate proportional agreement.

Critical septic patients affected by necrotizing soft tissue infections (NSTI) require an early, aggressive and multidisciplinary treatment. Pharmacokinetic alterations in antibiotic therapy are peculiar in these infections. Clinical pharmacology represents a first step in this setting. We report a case of septic shock due to NSTI in which clinical pharmacology is taken into account.

Uterine adenomyosis usually manifests as diffuse disease involving the myometrium and the endometrial-myometrial junction, but it may also manifest as a focal lesion. It is usually only a few millimeters in diameter but may sometimes be larger. We report the case of a 32-year-old woman with a large isolated mass in the uterine wall. Transvaginal sonography demonstrated the cystic nature of the mass and its characteristic hemorrhagic pattern, whereas CT confirmed its uterine origin. The patient underwent laparoscopic resection of the mass, and pathologic examinations led to the diagnosis of adenomyosis., (Copyright 2004 Wiley Periodicals, Inc. J Clin Ultrasound 32:95-97, 2004)

Background: The evaluation of right ventricular function can provide valuable information in a variety of cardiac and noncardiac conditions. Functional assessment of the right ventricle is difficult because of its anatomy and geometry. The authors describe a method for assessing right ventricular function using gated myocardial perfusion SPECT., Methods: In 20 patients, right and left ventricular ejection fractions (RVEF, LVEF) were determined using gated blood-pool scintigraphy (GBPS) and gated myocardial perfusion SPECT (GSPECT). To avoid contamination with right atrial activity, the two-frame method was adopted for gated blood-pool data when RVEF was measured. In nine patients with normal right ventricles, an index of wall thickening for the right ventricle was derived from the peak systolic and diastolic counts in the free wall., Results: Linear correlation between the two methods adopted for calculation of LVEF and RVEF was good. Bland-Altman analysis revealed good agreement between the two methods with no specific bias. The mean LVEF was 47.9 +/- 12% (GBPS) and 47.3 +/- 12.4 (GSPECT). The mean RVEF was 43.2 +/- 9.6% (GBPS) and 44.2 +/- 8.5% (GSPECT). In both cases, the values were not significantly different. The mean wall motion index was 35%. There was no correlation between the wall thickness index and ejection fraction, but the index was greater in patients with a normal right ventricle compared with those with reduced RVEF., Conclusions: Gated SPECT offers an alternative to GBPS for the functional assessment of the right ventricle. Using GSPECT will allow the simultaneous assessment of both the right and left ventricles.

Radiochromic film is tested for its broad-band response to ultraviolet (UV) B (290-320 nm) and A (320 nm400 nm), visible and infrared radiation produced by a solar simulator and examined for dosimetry in ultraviolet radiation. Results show that MD-55-2 radiochromic film in solar and fluorescent light sources responds almost exclusively to broad-band UVA radiation with negligible colouration from UVB, visible and low level infrared radiation. A second order polynomial function approximates the change in optical density at 660 nm wavelength for film colouration with exposure to UVA from white light fluorescent and solar UV with exposures measured with a dedicated UVA dosimeter. Using a double exposure technique as used in radiation dosimetry where the film is firstly irradiated to a known UV dose, radiochromic film can be used as a quantitative measure of UVA exposure.

Purpose: TI-201 scintigraphy is plagued with poor image quality because of the low-energy photons of TI-201 decay. Traditionally, a narrow 20% window centered on 71-72 keV has been used to improve sensitivity. Recent studies indicate that better imaging may be possible by optimizing the energy window to 34% centered on 77 keV. In this study, energy window optimization (EWO) was applied to gated TI-201 myocardial perfusion SPECT, and myocardial functional parameters were compared for gated TI-201 SPECT and gated Tc-99m sestamibi (Tc-99m MIBI) SPECT., Methods: Count statistics for standard and optimal TI-201 myocardial scintigraphy were noted in 25 patients by assessing the total counts in a mid-ventricular slice of a rest-gated TI-201 myocardial SPECT study. The feasibility of performing functional studies with the application of EWO to TI-201 was assessed using the count statistics of a mid-ventricular slice of an optimized gated TI-201 SPECT study and a gated Tc-99m MIBI SPECT study. The functional parameters (ejection fraction, wall motion, and thickening) of TI-201 with EWO and Tc-99m MIBI were compared in 60 patients who underwent rest-gated TI-201 SPECT followed by poststress gated Tc-99m MIBI SPECT. The left ventricular ejection fraction was calculated using commercially available software, whereas wall thickness and motion were assessed by the consensus of two readers., Results: The application of EWO increased available counts by more than 25%. It also resulted in sufficient counts being available to perform gated TI-201 SPECT without increasing acquisition times or the dose of TI-201. The average ejection fraction was 60.4% for gated TI-201 SPECT and 59.6% for gated Tc-99m MIBI SPECT (not significantly different). Overall, the image quality was rated excellent in 12% for TI-201 and Tc-99m MIBI and good in 50% and 62%, respectively, and poor in 38% and 26%, respectively., Conclusion: The application of EWO to TI-201 SPECT allows myocardial functional parameters to be assessed without having to increase the acquisition times or the administered dose of TI-201.