Molecular determinants of immunogenic cell death elicited by radiation therapy.

Cancer cells undergoing immunogenic cell death (ICD) can initiate adaptive immune responses against dead cell-associated antigens, provided that (1) said antigens are not perfectly covered by central tolerance (antigenicity), (2) cell death occurs along with the emission of immunostimulatory cytokines and damage-associated molecular patterns (DAMPs) that actively engage immune effector mechanisms (adjuvanticity), and (3) the microenvironment of dying cells is permissive for the initiation of adaptive immunity. Finally, ICD-driven immune responses can only operate and exert cytotoxic effector functions if the microenvironment of target cancer cells enables immune cell infiltration and activity. Multiple forms of radiation, including non-ionizing (ultraviolet) and ionizing radiation, elicit bona fide ICD as they increase both the antigenicity and adjuvanticity of dying cancer cells. Here, we review the molecular determinants of ICD as elicited by radiation as we critically discuss strategies to reinforce the immunogenicity of cancer cells succumbing to clinically available radiation strategies.

Immunologically relevant effects of radiation therapy on the tumor microenvironment.

Focal radiation therapy (RT) has been successfully employed to clinically manage multiple types of cancer for more than a century. Besides being preferentially cytotoxic for malignant cells over their nontransformed counterparts, RT elicits numerous microenvironmental alterations that appear to factor into its therapeutic efficacy. Here, we briefly discuss immunostimulatory and immunosuppressive microenvironmental changes elicited by RT and their impact on tumor recognition by the host immune system.

The humoral and cellular response to mRNA SARS-CoV-2 vaccine is influenced by HLA polymorphisms.

Host genetic variability contributes to susceptibility to SARS-CoV-2 infection and COVID-19 evolution and the role of HLA system has not clearly emerged, suggesting the involvement of other factors. Studying response to vaccination with Spyke protein mRNA represents an ideal model to highlight whether the humoral or cellular responses are influenced by HLA. Four hundred and sixteen workers, vaccinated with Comirnaty beginning 2021, were selected within the Azienda Ospedaliera Universitaria "Città della Salute e della Scienza di Torino." The humoral response was determined with the LIAISON® kit, while the analysis of the cellular response was performed with the Quantiferon SARS-CoV-2 assay, for the S1 (receptor-binding domain; Ag1) and S1 and S2 (Ag2) subunits of the Spyke protein. Six HLA loci were typed by next-generation sequencing. Associations between HLA and vaccine response were performed with univariate and multivariate analyses. An association was found between A*03:01, B*40:02 and DPB1*06:01 and high antibody concentration and between A*24:02, B*08:01 and C*07:01 and low humoral responses. The haplotype HLA-A*01:01 ~ B1*08:01 ~ C*07:01 ~ DRB1*03:01 ~ DQB1*02:01 conferred an increased risk of low humoral response. Considering cellular responses, 50% of the vaccinated subjects responded against Ag1 and 59% against Ag2. Carriers of DRB1*15:01 displayed a higher cellular response both to Ag1 and Ag2 compared to the rest of the cohort. Similarly, DRB1*13:02 predisposed to a robust cellular response to Ag1 and Ag2, while DRB1*11:04 showed an opposite trend. Cellular and humoral responses to Comirnaty are influenced by HLA. Humoral response is mainly associated to class I alleles, with A*03:01, previously associated to protection against severe COVID-19, and response to vaccination, standing out. Cellular response predominantly involves class II alleles, with DRB1*15:01 and DPB1*13:01 prevailing. Affinity analysis for Spyke peptides is generally in line with the association results.

Epigenetic escape of immunosurveillance by malignant cell precursors.

Newly formed malignant cells must escape immunosurveillance to generate progressing neoplastic lesions of clinical relevance. Recent data indicate that the immunogenicity of nascent cancer cells, at least in some settings, is dictated by inherent epigenetic mechanisms rather than by immunoediting and the consequent Darwinian selection of poorly immunogenic phenotypes.

Quantitative assessment of mitophagy in irradiated cancer cells.

Mitophagy is a finely regulated mechanism through which eukaryotic cells selectively dispose of supernumerary, permeabilized or otherwise damaged mitochondria through lysosomal degradation. Dysfunctional mitochondria are prone to release potentially cytotoxic factors including reactive oxygen species (ROS) and caspase activators, such as cytochrome c, somatic (CYCS). Thus, proficient mitophagic responses mediate prominent cytoprotective functions. Moreover, the rapid degradation of permeabilized mitochondria limits the release of mitochondrial components that may drive inflammatory reactions, such as mitochondrial DNA (mtDNA) and transcription factor A, mitochondrial (TFAM), implying that mitophagy also mediates potent anti-inflammatory effects. Here, we detail a simple, flow cytometry-assisted protocol for the specific measurement of mitophagic responses as driven by radiation therapy (RT) in mouse hormone receptor (HR)+ mammary carcinoma TS/A cells. With some variations, this method - which relies on the mitochondria-restricted expression of a fluorescent reporter that is sensitive to pH and hence changes excitation wavelength within lysosomes (mt-mKeima) - can be adapted to a variety of human and mouse cancer cell lines and/or straightforwardly implemented on fluorescence microscopy platforms.

Convalescent or standard plasma versus standard of care in the treatment of COVID-19 patients with respiratory impairment: short and long-term effects. A three-arm randomized controlled clinical trial.

BACKGROUND: The efficacy of early treatment with convalescent plasma in patients with COVID-19 is debated. Nothing is known about the potential effect of other plasma components other than anti-SARS-CoV-2 antibodies. METHODS: To determine whether convalescent or standard plasma would improve outcomes for adults in early phase of Covid19 respiratory impairment we designed this randomized, three-arms, clinical trial (PLACO COVID) blinded on interventional arms that was conducted from June 2020 to August 2021. It was a multicentric trial at 19 Italian hospitals. We enrolled 180 hospitalized adult patients with COVID-19 pneumonia within 5 days from the onset of respiratory distress. Patients were randomly assigned in a 1:1:1 ratio to standard of care (n = 60) or standard of care + three units of standard plasma (n = 60) or standard of care + three units of high-titre convalescent plasma (n = 60) administered on days 1, 3, 5 after randomization. Primary outcome was 30-days mortality. Secondary outcomes were: incidence of mechanical ventilation or death at day 30, 6-month mortality, proportion of days with mechanical ventilation on total length of hospital stay, IgG anti-SARS-CoV-2 seroconversion, viral clearance from plasma and respiratory tract samples, and variations in Sequential Organ Failure Assessment score. The trial was analysed according to the intention-to-treat principle. RESULTS: 180 patients (133/180 [73.9%] males, mean age 66.6 years [IQR 57-73]) were enrolled a median of 8 days from onset of symptoms. At enrollment, 88.9% of patients showed moderate/severe respiratory failure. 30-days mortality was 20% in Control arm, 23% in Convalescent (risk ratio [RR] 1.13; 95% confidence interval [CI], 0.61-2.13, P = 0.694) and 25% in Standard plasma (RR 1.23; 95%CI, 0.63-2.37, P = 0.544). Time to viral clearance from respiratory tract was 21 days for Convalescent, 28 for Standard plasma and 23 in Control arm but differences were not statistically significant. No differences for other secondary endpoints were seen in the three arms. Serious adverse events were reported in 1.7%, 3.3% and 5% of patients in Control, Standard and Convalescent plasma arms respectively. CONCLUSIONS: Neither high-titer Convalescent nor Standard plasma improve outcomes of COVID-19 patients with acute respiratory failure. Trial Registration Clinicaltrials.gov Identifier: NCT04428021. First posted: 11/06/2020.

Cytofluorometric assessment of cell cycle progression in irradiated cells.

Radiation therapy (RT) is well known for its capacity to mediate cytostatic and cytotoxic effects upon the accumulation of unrepaired damage to macromolecules, notably DNA. The ability of ionizing radiation to prevent malignant cells from replicating and to cause their demise is indeed an integral component of the anticancer activity of RT. Neoplastic cells are generally more sensitive to the cytostatic and cytotoxic effects of RT than their healthy counterparts as they exhibit increased proliferative rate and limited capacity for DNA repair. This provides a rather comfortable therapeutic window for clinical RT usage, especially with the development of novel, technologically superior RT modalities that minimize the exposure of normal tissues. Thus, while accumulating evidence indicates that cancer control by RT also involves the activation of tumor-targeting immune responses, assessing cell cycle progression in irradiated cells remains a central approach for investigating radiosensitivity in preclinical tumor models. Here, we detail a simple, flow cytometry-assisted method to simultaneously assess cell cycle distribution and active DNA replication in cultured estrogen receptor (ER)+ breast cancer MCF7 cells. With minimal variations, the same technique can be straightforwardly implemented to a large panel of human and mouse cancer cell lines.

Quantification of cytosolic DNA species by immunofluorescence microscopy and automated image analysis.

When employed according to specific doses and fractionation schedules, radiation therapy (RT) elicits potent tumor-targeting immune responses that rely on the secretion of type I interferon (IFN) by irradiated cancer cells. Most often, this is initiated by the ability of RT to promote the cytosolic accumulation of double-stranded DNA (dsDNA) molecules, which are detected by cyclic GMP-AMP synthase (CGAS) to engage the stimulator of interferon response cGAMP interactor 1 (STING1)-dependent transactivation of type I IFN-coding genes via interferon regulatory factor 3 (IRF3). Here, we describe a simple protocol for the quantification of cytosolic dsDNA species by immunofluorescence microscopy coupled to automated image analysis, as enabled by precise sample processing conditions that permeabilize plasma-but not nuclear or inner mitochondrial-membranes. As compared to subcellular fractionation-based techniques, this approach is compatible with assessments in individual cells aimed at gauging inter-cellular heterogeneity, as well as subcellular tests including co-localization studies.

RT-PCR-assisted quantification of type I IFN responses in irradiated cancer cells.

It is now clear that radiation therapy (RT) can be delivered in doses and according to fractionation schedules that actively elicit immunostimulatory effects. While such effects are often sufficient to drive potent anticancer immunity culminating with systemic disease eradication, the immunostimulatory activity of RT stands out as a promising combinatorial partner for bona fide immunotherapeutics including immune checkpoint inhibitors (ICIs). Accumulating preclinical and clinical evidence indicates that the secretion of type I interferon (IFN) by irradiated cancer cells is a sine qua non for RT to initiate ICI-actionable tumor-targeting immune responses. Here, we detail a simple protocol to quantitatively assess type I IFN responses in irradiated mouse hormone receptor (HR)+ TS/A cells by RT-PCR. With minimal variations, the same technique can be straightforwardly adapted to quantify type I IFN-associated transcriptional responses in a variety of human and mouse cancer cells maintained in vitro.

Cytofluorometric assessment of acute cell death responses driven by radiation therapy.

Radiation therapy (RT) is well known for its capacity to mediate cytostatic and cytotoxic effects on malignant cells, largely reflecting the ability of ionizing radiation to cause direct and indirect damage to macromolecules including DNA and lipids. While low-dose RT generally causes limited cytotoxicity in an acute manner (as it imposes insufficient cellular damage to compromise homeostasis, or instead induces the delayed demise of cells that fail to complete mitosis successfully), high RT doses can mediate an acute wave of cell death that begins to manifest shortly (24-72h) after irradiation. Here, we provide two straightforward techniques to assess the acute cytotoxic effects of RT by the flow cytometry-assisted quantification of plasma membrane permeabilization (PMP, a late-stage manifestation of cell death) and either mitochondrial outer membrane permeabilization (MOMP) or phosphatidylserine (PS) externalization (two early-stage signs of cell death) in mouse mammary adenocarcinoma TS/A cells. With minor variations, the same protocols can be straightforwardly adapted to measure acute cell death responses as elicited by RT in a large panel of human and mouse cancer cells lines of different histological derivation.

Type I IFNs promote cancer cell stemness by triggering the epigenetic regulator KDM1B.

Cancer stem cells (CSCs) are a subpopulation of cancer cells endowed with high tumorigenic, chemoresistant and metastatic potential. Nongenetic mechanisms of acquired resistance are increasingly being discovered, but molecular insights into the evolutionary process of CSCs are limited. Here, we show that type I interferons (IFNs-I) function as molecular hubs of resistance during immunogenic chemotherapy, triggering the epigenetic regulator demethylase 1B (KDM1B) to promote an adaptive, yet reversible, transcriptional rewiring of cancer cells towards stemness and immune escape. Accordingly, KDM1B inhibition prevents the appearance of IFN-I-induced CSCs, both in vitro and in vivo. Notably, IFN-I-induced CSCs are heterogeneous in terms of multidrug resistance, plasticity, invasiveness and immunogenicity. Moreover, in breast cancer (BC) patients receiving anthracycline-based chemotherapy, KDM1B positively correlated with CSC signatures. Our study identifies an IFN-I → KDM1B axis as a potent engine of cancer cell reprogramming, supporting KDM1B targeting as an attractive adjunctive to immunogenic drugs to prevent CSC expansion and increase the long-term benefit of therapy.

Cellular Immune Response to BNT162b2 mRNA COVID-19 Vaccine in a Large Cohort of Healthcare Workers in a Tertiary Care University Hospital.

We describe the results of a T-cell immunity evaluation performed after a median elapsed time of 7 months from second-dose BNT162b2 vaccine administration, in a representative sample of 419 subjects from a large cohort of hospital workers. Overall, the Quantiferon SARS-CoV-2 assay detected a responsive pattern in 49.9%, 59.2% and 68.3% of subjects to three different antigenic stimuli from SARS-CoV-2, respectively, with 72.3% of positivity to at least one antigenic stimulus. Potential predictors of cellular response were explored by multivariable analyses; factors associated with positivity to cellular response (to Ag1 antigenic stimulus) were a previous SARS-CoV-2 infection (OR = 4.24, 95% CI 2.34−7.67, p < 0.001), increasing age (per year: OR = 1.03 95% CI 1.01−1.06, p = 0.019 and currently smoking (compared to never smoking) (OR = 1.93, 95% CI 1.11−3.36, p = 0.010). Increasing time interval between vaccine administration and T-cell test was associated with decreasing cellular response (per week of time: OR = 0.94, 95% CI 0.91−0.98, p = 0.003). A blood group A/AB/B (compared to group O) was associated with higher levels of cellular immunity, especially when measured as Ag2 antigenic stimulus. Levels of cellular immunity tended to be lower among subjects that self-reported an autoimmune disorder or an immunodeficiency and among males. Further studies to assess the protective significance of different serological and cellular responses to the vaccine toward the risk of reinfection and the severity of COVID-19 are needed to better understand these findings.

Myeloid-Derived Suppressor Cells and Radiotherapy.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of pathologically activated, mostly immature, myeloid cells that exert robust immunosuppressive functions. MDSCs expand during oncogenesis and have been linked to accelerated disease progression and resistance to treatment in both preclinical tumor models and patients with cancer. Thus, MDSCs stand out as promising targets for the development of novel immunotherapeutic regimens with superior efficacy. Here, we summarize accumulating preclinical and clinical evidence indicating that MDSCs also hamper the efficacy of radiotherapy (RT), as we critically discuss the potential of MDSC-targeting strategies as tools to achieve superior immunotherapeutic tumor control by RT in the clinic.

Does Learning Through Movement Improve Academic Performance in Primary Schoolchildren? A Systematic Review.

Physically active children have greater motor competence and a faster maturation compared with their sedentary peers. Recent research also suggests that physical activity during childhood may also promote cognitive development and therefore improve academic performance. The aim of this study was to understand if physically active academic lessons may improve academic achievement in primary schoolchildren. A systematic review following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines was conducted. The search was performed on the following database: PubMed, Web of Science, Scopus, Education Resources Information Center (ERIC), and PsycINFO (APA). Studies evaluating schoolchildren aged between 3 and 11 years taking part in educational contexts that include physical activity and natural environments evaluating physical fitness and/or educational outcomes were included. A total of 54 studies (for a total sample of 29,460 schoolchildren) were considered eligible and included in the qualitative synthesis. The Effective Public Health Practice Project risk-of-bias assessment revealed a moderate quality of the included studies with only two considered weeks. Despite differences in the retrieved protocols, physically active academic lessons improve the total time engaged in physical activity, motor skills, and/or academic performance. The results of this review suggest that learning through movement is an effective, low-cost, and enjoyable strategy for elementary schoolchildren.

Factors Influencing Level and Persistence of Anti SARS-CoV-2 IgG after BNT162b2 Vaccine: Evidence from a Large Cohort of Healthcare Workers.

We aimed at evaluating quantitative IgG response to BNT162b2 COVID-19 vaccine among health care workers (HCW), and exploring the role of demographic, clinical, and occupational factors as predictors of IgG levels. On May 2021, among 6687 HCW at the largest tertiary care University-Hospital of Northwestern Italy, at a median of 15 weeks (Interquartile range-IQR 13.6−16.0) after second-dose, serological response was present in 99.8%. Seropositivity was >97% in all the subgroups, except those self-reporting immunodeficiency (94.9%). Overall, the median serological IgG value was 990 BAU/mL (IQR 551−1870), with most of subjects with previous SARS-CoV-2 infection or with shorter time lapse (2−8 weeks) between vaccination and serology with values in the highest quintile (>2080). At multivariable analysis, significant predictors of lower values were increasing age, male, current smoking, immunodeficiency, recent occupational contacts, and increasing time lapse from vaccination; conversely, previous infection and recent household contacts were significantly associated with higher IgG levels. Subjects with previous infection kept a very high level (around 2000 BAU/mL) up to 120 days. These results, besides supporting a high serological response up to 4−5 months, suggest predictive factors of faster decay of IgG levels that could be useful in tailoring vaccination strategies.

Methodological Considerations for Movement Education Interventions in Natural Environments for Primary School Children: A Scoping Review.

BACKGROUND: Education is the ideal setting for carrying out projects to improve primary students' capacities. In recent years, interventions in natural environments have been more frequently proposed, but there is still a lack of standardization, making deeper study of the topic necessary. This review aims to report on what previous scientific research has been carried out, and eventually, to propose standard operating procedures for future interventions. METHODS: This is a scoping review that adopted the PRISMA guidelines. Primary school children have been included, and the interventions had to be proposed adopting nature as the primary element of the learning process. RESULTS: A total of 19 studies have been included, and a wide range of methodological differences has been detected regarding the included intervention protocols. CONCLUSIONS: Learning in nature is a feasible intervention that, despite the high heterogeneity of interventions, demonstrates positive outcomes in the learning sphere of children.

Immunogenic cell stress and death.

Dying mammalian cells emit numerous signals that interact with the host to dictate the immunological correlates of cellular stress and death. In the absence of reactive antigenic determinants (which is generally the case for healthy cells), such signals may drive inflammation but cannot engage adaptive immunity. Conversely, when cells exhibit sufficient antigenicity, as in the case of infected or malignant cells, their death can culminate with adaptive immune responses that are executed by cytotoxic T lymphocytes and elicit immunological memory. Suggesting a key role for immunogenic cell death (ICD) in immunosurveillance, both pathogens and cancer cells evolved strategies to prevent the recognition of cell death as immunogenic. Intriguingly, normal cells succumbing to conditions that promote the formation of post-translational neoantigens (for example, oxidative stress) can also drive at least some degree of antigen-specific immunity, pointing to a novel implication of ICD in the etiology of non-infectious, non-malignant disorders linked to autoreactivity.

MDSCs sneak CSCs out of (immuno)surveillance.

Cancer stem cells (CSCs) are known for their superior tumor-initiating and tumor-repopulating potential, partly reflecting their pronounced ability to evade immune recognition. Liu and colleagues recently identified a new aldehyde dehydrogenase (ALDH)-dependent mechanism whereby triple-negative breast CSCs evade immunosurveillance upon recruitment of myeloid-derived suppressor cells.