Polyclonal lymphoid expansion drives paraneoplastic autoimmunity in neuroblastoma.

Neuroblastoma is a lethal childhood solid tumor of developing peripheral nerves. Two percent of children with neuroblastoma develop opsoclonus myoclonus ataxia syndrome (OMAS), a paraneoplastic disease characterized by cerebellar and brainstem-directed autoimmunity but typically with outstanding cancer-related outcomes. We compared tumor transcriptomes and tumor-infiltrating T and B cell repertoires from 38 OMAS subjects with neuroblastoma to 26 non-OMAS-associated neuroblastomas. We found greater B and T cell infiltration in OMAS-associated tumors compared to controls and showed that both were polyclonal expansions. Tertiary lymphoid structures (TLSs) were enriched in OMAS-associated tumors. We identified significant enrichment of the major histocompatibility complex (MHC) class II allele HLA-DOB∗01:01 in OMAS patients. OMAS severity scores were associated with the expression of several candidate autoimmune genes. We propose a model in which polyclonal auto-reactive B lymphocytes act as antigen-presenting cells and drive TLS formation, thereby supporting both sustained polyclonal T cell-mediated anti-tumor immunity and paraneoplastic OMAS neuropathology.

Bacterial Infections and Clinical Outcomes Among Febrile Infants up to 90 Days Old With SARS-CoV-2 Infection: A Multicenter Cohort Study.

We present a large, multicenter, cohort study that aimed to assess bacterial infection rates among febrile infants up to 90 days old presenting to the pediatric emergency department with severe acute respiratory syndrome coronavirus 2 infection during 2021-2022 throughout successive variant waves. Overall, 417 febrile infants were included. Twenty-six infants (6.2%) had bacterial infections. All bacterial infections consisted of urinary tract infections, and there were no invasive bacterial infections. There was no mortality.

Elevated cfDNA after exercise is derived primarily from mature polymorphonuclear neutrophils, with a minor contribution of cardiomyocytes.

Strenuous physical exercise causes a massive elevation in the concentration of circulating cell-free DNA (cfDNA), which correlates with effort intensity and duration. The cellular sources and physiological drivers of this phenomenon are unknown. Using methylation patterns of cfDNA and associated histones, we show that cfDNA in exercise originates mostly in extramedullary polymorphonuclear neutrophils. Strikingly, cardiomyocyte cfDNA concentration increases after a marathon, consistent with elevated troponin levels and indicating low-level, delayed cardiac cell death. Physical impact, low oxygen levels, and elevated core body temperature contribute to neutrophil cfDNA release, while muscle contraction, increased heart rate, ß-adrenergic signaling, or steroid treatment fail to cause elevation of cfDNA. Physical training reduces neutrophil cfDNA release after a standard exercise, revealing an inverse relationship between exercise-induced cfDNA release and training level. We speculate that the release of cfDNA from neutrophils in exercise relates to the activation of neutrophils in the context of exercise-induced muscle damage.

Viral infection reveals hidden sharing of TCR CDR3 sequences between individuals.

The T cell receptor is generated by a process of random and imprecise somatic recombination. The number of possible T cell receptors which this process can produce is enormous, greatly exceeding the number of T cells in an individual. Thus, the likelihood of identical TCRs being observed in multiple individuals (public TCRs) might be expected to be very low. Nevertheless such public TCRs have often been reported. In this study we explore the extent of TCR publicity in the context of acute resolving Lymphocytic choriomeningitis virus (LCMV) infection in mice. We show that the repertoire of effector T cells following LCMV infection contains a population of highly shared TCR sequences. This subset of TCRs has a distribution of naive precursor frequencies, generation probabilities, and physico-chemical CDR3 properties which lie between those of classic public TCRs, which are observed in uninfected repertoires, and the dominant private TCR repertoire. We have named this set of sequences "hidden public" TCRs, since they are only revealed following infection. A similar repertoire of hidden public TCRs can be observed in humans after a first exposure to SARS-COV-2. The presence of hidden public TCRs which rapidly expand following viral infection may therefore be a general feature of adaptive immunity, identifying an additional level of inter-individual sharing in the TCR repertoire which may form an important component of the effector and memory response.

Correction of T-Cell Repertoire and Autoimmune Diabetes in NOD Mice by Non-myeloablative T-Cell Depleted Allogeneic HSCT.

The induction of partial tolerance toward pancreatic autoantigens in the treatment of type 1 diabetes mellitus (T1DM) can be attained by autologous hematopoietic stem cell transplantation (HSCT). However, most patients treated by autologous HSCT eventually relapse. Furthermore, allogeneic HSCT which could potentially provide a durable non-autoimmune T-cell receptor (TCR) repertoire is associated with a substantial risk for transplant-related mortality. We have previously demonstrated an effective approach for attaining engraftment without graft versus host disease (GVHD) of allogeneic T-cell depleted HSCT, following non-myeloablative conditioning, using donor-derived anti-3rd party central memory CD8 veto T cells (Tcm). In the present study, we investigated the ability of this relatively safe transplant modality to eliminate autoimmune T-cell clones in the NOD mouse model which spontaneously develop T1DM. Our results demonstrate that using this approach, marked durable chimerism is attained, without any transplant-related mortality, and with a very high rate of diabetes prevention. TCR sequencing of transplanted mice showed profound changes in the T-cell repertoire and decrease in the prevalence of specific autoimmune T-cell clones directed against pancreatic antigens. This approach could be considered as strategy to treat people destined to develop T1DM but with residual beta cell function, or as a platform for prevention of beta cell destruction after transplantation of allogenic beta cells.

The proteasome regulator PSME4 modulates proteasome activity and antigen diversity to abrogate antitumor immunity in NSCLC.

Immunotherapy revolutionized treatment options in cancer, yet the mechanisms underlying resistance in many patients remain poorly understood. Cellular proteasomes have been implicated in modulating antitumor immunity by regulating antigen processing, antigen presentation, inflammatory signaling and immune cell activation. However, whether and how proteasome complex heterogeneity may affect tumor progression and the response to immunotherapy has not been systematically examined. Here, we show that proteasome complex composition varies substantially across cancers and impacts tumor-immune interactions and the tumor microenvironment. Through profiling of the degradation landscape of patient-derived non-small-cell lung carcinoma samples, we find that the proteasome regulator PSME4 is upregulated in tumors, alters proteasome activity, attenuates presented antigenic diversity and associates with lack of response to immunotherapy. Collectively, our approach affords a paradigm by which proteasome composition heterogeneity and function should be examined across cancer types and targeted in the context of precision oncology.

Modulating the proliferative and cytotoxic properties of patient-derived TIL by a synthetic immune niche of immobilized CCL21 and ICAM1.

A major challenge in developing an effective adoptive cancer immunotherapy is the ex-vivo generation of tumor-reactive cells in sufficient numbers and with enhanced cytotoxic potential. It was recently demonstrated that culturing of activated murine CD8+ T-cells on a "Synthetic Immune Niche" (SIN), consisting of immobilized CCL21 and ICAM-1, enhances T-cell expansion, increases their cytotoxicity against cultured cancer cells and suppresses tumor growth in vivo. In the study reported here, we have tested the effect of the CCL21+ICAM1 SIN, on the expansion and cytotoxic phenotype of Tumor Infiltrating Lymphocytes (TIL) from melanoma patients, following activation with immobilized anti-CD3/CD28 stimulation, or commercial activation beads. The majority of TIL tested, displayed higher expansion when cultured on the coated SIN compared to cells incubated on uncoated substrate and a lower frequency of TIM-3+CD8+ cells after stimulation with anti-CD3/CD28 beads. Comparable enhancement of TIL proliferation was obtained by the CCL21+ICAM1 SIN, in a clinical setting that included a 14-day rapid expansion procedure (REP). Co-incubation of post-REP TIL with matching target cancerous cells demonstrated increased IFNγ secretion beyond baseline in most of the TIL cultures, as well as a significant increase in granzyme B levels following activation on SIN. The SIN did not significantly alter the relative frequency of CD8/CD4 populations, as well as the expression of CD28, CD25, several exhaustion markers and the differentiation status of the expanded cells. These results demonstrate the potential capacity of the CCL21+ICAM1 SIN to reinforce TIL-based immunotherapy for cancer patients.

Quantifying changes in the T cell receptor repertoire during thymic development.

One of the feats of adaptive immunity is its ability to recognize foreign pathogens while sparing the self. During maturation in the thymus, T cells are selected through the binding properties of their antigen-specific T-cell receptor (TCR), through the elimination of both weakly (positive selection) and strongly (negative selection) self-reactive receptors. However, the impact of thymic selection on the TCR repertoire is poorly understood. Here, we use transgenic Nur77-mice expressing a T-cell activation reporter to study the repertoires of thymic T cells at various stages of their development, including cells that do not pass selection. We combine high-throughput repertoire sequencing with statistical inference techniques to characterize the selection of the TCR in these distinct subsets. We find small but significant differences in the TCR repertoire parameters between the maturation stages, which recapitulate known differentiation pathways leading to the CD4+ and CD8+ subtypes. These differences can be simulated by simple models of selection acting linearly on the sequence features. We find no evidence of specific sequences or sequence motifs or features that are suppressed by negative selection. These results favour a collective or statistical model for T-cell self non-self discrimination, where negative selection biases the repertoire away from self recognition, rather than ensuring lack of self-reactivity at the single-cell level.

From pseudo to real-time dynamics of T cell thymic differentiation.

Numerous methods have recently emerged for ordering single cells along developmental trajectories. However, accurate depiction of developmental dynamics can only be achieved after rescaling the trajectory according to the relative time spent at each developmental point. We formulate a model which estimates local cell densities and fluxes, and incorporates cell division and apoptosis rates, to infer the real-time dimension of the developmental trajectory. We validate the model using mathematical simulations and apply it to experimental high dimensional cytometry data obtained from the mouse thymus to construct the true time profile of the thymocyte developmental process. Our method can easily be implemented in any of the existing tools for trajectory inference.

Extrachromosomal DNA Amplification Contributes to Small Cell Lung Cancer Heterogeneity and Is Associated with Worse Outcomes.

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine lung cancer. Oncogenic MYC amplifications drive SCLC heterogeneity, but the genetic mechanisms of MYC amplification and phenotypic plasticity, characterized by neuroendocrine and nonneuroendocrine cell states, are not known. Here, we integrate whole-genome sequencing, long-range optical mapping, single-cell DNA sequencing, and fluorescence in situ hybridization to find extrachromosomal DNA (ecDNA) as a primary source of SCLC oncogene amplifications and driver fusions. ecDNAs bring to proximity enhancer elements and oncogenes, creating SCLC transcription-amplifying units, driving exceptionally high MYC gene dosage. We demonstrate that cell-free nucleosome profiling can noninvasively detect ecDNA amplifications in plasma, facilitating its genome-wide interrogation in SCLC and other cancers. Altogether, our work provides the first comprehensive map of SCLC ecDNA and describes a new mechanism that governs MYC-driven SCLC heterogeneity. ecDNA-enabled transcriptional flexibility may explain the significantly worse survival outcomes of SCLC harboring complex ecDNA amplifications. SIGNIFICANCE: MYC drives SCLC progression, but the genetic basis of MYC-driven SCLC evolution is unknown. Using SCLC as a paradigm, we report how ecDNA amplifications function as MYC-amplifying units, fostering tumor plasticity and a high degree of tumor heterogeneity. This article is highlighted in the In This Issue feature, p. 799.

SLAM-Drop-seq reveals mRNA kinetic rates throughout the cell cycle.

RNA abundance is tightly regulated in eukaryotic cells by modulating the kinetic rates of RNA production, processing, and degradation. To date, little is known about time­dependent kinetic rates during dynamic processes. Here, we present SLAM­Drop­seq, a method that combines RNA metabolic labeling and alkylation of modified nucleotides in methanol­fixed cells with droplet­based sequencing to detect newly synthesized and preexisting mRNAs in single cells. As a first application, we sequenced 7280 HEK293 cells and calculated gene­specific kinetic rates during the cell cycle using the novel package Eskrate. Of the 377 robust­cycling genes that we identified, only a minor fraction is regulated solely by either dynamic transcription or degradation (6 and 4%, respectively). By contrast, the vast majority (89%) exhibit dynamically regulated transcription and degradation rates during the cell cycle. Our study thus shows that temporally regulated mRNA degradation is fundamental for the correct expression of a majority of cycling genes. SLAM­Drop­seq, combined with Eskrate, is a powerful approach to understanding the underlying mRNA kinetics of single­cell gene expression dynamics in continuous biological processes.

Conserved degronome features governing quality control associated proteolysis.

The eukaryotic proteome undergoes constant surveillance by quality control systems that either sequester, refold, or eliminate aberrant proteins by ubiquitin-dependent mechanisms. Ubiquitin-conjugation necessitates the recognition of degradation determinants, termed degrons, by their cognate E3 ubiquitin-protein ligases. To learn about the distinctive properties of quality control degrons, we performed an unbiased peptidome stability screen in yeast. The search identify a large cohort of proteome-derived degrons, some of which exhibited broad E3 ligase specificity. Consequent application of a machine-learning algorithm establishes constraints governing degron potency, including the amino acid composition and secondary structure propensities. According to the set criteria, degrons with transmembrane domain-like characteristics are the most probable sequences to act as degrons. Similar quality control degrons are present in viral and human proteins, suggesting conserved degradation mechanisms. Altogether, the emerging data indicate that transmembrane domain-like degron features have been preserved in evolution as key quality control determinants of protein half-life.

A hierarchy of selection pressures determines the organization of the T cell receptor repertoire.

We systematically examine the receptor repertoire in T cell subsets in young, adult, and LCMV-infected mice. Somatic recombination generates diversity, resulting in the limited overlap between nucleotide sequences of different repertoires even within the same individual. However, statistical features of the repertoire, quantified by the V gene and CDR3 k-mer frequency distributions, are highly conserved. A hierarchy of immunological processes drives the evolution of this structure. Intra-thymic divergence of CD4+ and CD8+ lineages imposes subtle but dominant differences observed across repertoires of all subpopulations in both young and adult mice. Differentiation from naive through memory to effector phenotype imposes an additional gradient of repertoire diversification, which is further influenced by age in a complex and lineage-dependent manner. The distinct repertoire of CD4+ regulatory T cells is more similar to naive cells in young mice and to effectors in adults. Finally, we describe divergent (naive and memory) and convergent (CD8+ effector) evolution of the repertoire following acute infection with LCMV. This study presents a quantitative framework that captures the structure of the repertoire in terms of its fundamental statistical properties and describes how this structure evolves as individual T cells differentiate, migrate and mature in response to antigen exposure.

Triage performance in adolescent patients with SARS-CoV-2 infection in Israel.

OBJECTIVE: The aim of this study was to assess the performance of the Pediatric Canadian Triage and Acuity Scale (PaedCTAS) in adolescent patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: A time-series study was conducted in the Emergency Departments (EDs) of 17 public hospitals during the Delta (B.1.617.2) variant spread in Israel. Data were collected prospectively from June 11, 2021 to August 15, 2021. Multivariate regression analyses were performed to identify independent variables associated with hospital admission and with admission to an Intensive Care Unit (ICU). RESULTS: During the study period, 305 SARS-CoV-2 patients ages 12-18 years presenting to the ED were included, and 267 (87.5%) were unvaccinated. Sixty-seven (22.0%) and 12 (3.9%) patients were admitted to pediatric wards and ICUs, respectively. PaedCTAS level 1-2 and the presence of chronic disease increased the odds of hospital admission (adjusted odds ratio (aOR) 5.74, 95% CI, 2.30-14.35, p < 0.0001), and (aOR 2.9, 95% CI, 1.48-5.67, p < 0.02), respectively. PaedCTAS level 1-2 and respiratory symptoms on presentation to ED increased the odds of ICU admission (aOR 27.79; 95% CI, 3.85-176.91, p < 0.001), and (aOR 26.10; 95% CI, 4.47-172.63, p < 0.0001), respectively. PaedCTAS level 3-5 was found in 217/226 (96%) of the patients who were discharged home from the ED. CONCLUSIONS: The findings suggest that PaedCTAS level 1-2 was the strongest factor associated with hospital and ICU admission. Almost all the patients who were discharged home had PaedCTAS level 3-5. Study findings suggest good performance of the PaedCTAS in this cohort.

Dissemination and Use of Point-of-Care Ultrasound by Pediatricians in Europe: A Research in European Pediatric Emergency Medicine Network Collaborative Survey.

OBJECTIVE: We surveyed the dissemination and use of point-of-care ultrasound (POCUS), physician training levels, and barriers and limitations to use of POCUS among pediatricians and pediatric emergency medicine (PEM) physicians across Europe and Israel. METHODS: A questionnaire was distributed through the PEM section of the European Society for Emergency Medicine and the Research in European Pediatric Emergency Medicine Network. RESULTS: A total of 581 physicians from 22 countries fully completed the questionnaire. Participants were primarily pediatric attending physicians (34.9% [203 of 581]) and PEM attending physicians (28.6% [166 of 581]). Most of the respondents, 58.5% (340 of 581), reported using POCUS in their practice, and 61.9% (359/581) had undergone POCUS training. Point-of-care ultrasound courses represented the most common method of becoming proficient in POCUS. Overall, the Focused Assessment with Sonography in Trauma scan was the mostly taught application, with 76.3% (274 of 359). Resuscitative, diagnostic, and procedural POCUS were rated as very useful or useful by the most of respondents.The lack of qualified personnel to train (76.9% [447 of 581]), and the insufficient time for physicians to learn, POCUS (63.7% [370 of 581]) were identified as the main limitations to POCUS implementation. CONCLUSIONS: The dissemination of pediatric POCUS in the European and Israeli centers we surveyed is limited, and its applications are largely restricted to the Focused Assessment with Sonography in Trauma examination. This is likely related to lack of training programs. In contrast, the potential value of use of POCUS in PEM practice is recognized by the majority of respondents.

Transcription feedback dynamics in the wake of cytoplasmic mRNA degradation shutdown.

In the last decade, multiple studies demonstrated that cells maintain a balance of mRNA production and degradation, but the mechanisms by which cells implement this balance remain unknown. Here, we monitored cells' total and recently-transcribed mRNA profiles immediately following an acute depletion of Xrn1-the main 5'-3' mRNA exonuclease-which was previously implicated in balancing mRNA levels. We captured the detailed dynamics of the adaptation to rapid degradation of Xrn1 and observed a significant accumulation of mRNA, followed by a delayed global reduction in transcription and a gradual return to baseline mRNA levels. We found that this transcriptional response is not unique to Xrn1 depletion; rather, it is induced earlier when upstream factors in the 5'-3' degradation pathway are perturbed. Our data suggest that the mRNA feedback mechanism monitors the accumulation of inputs to the 5'-3' exonucleolytic pathway rather than its outputs.

Pediatric Hospitalizations After School Reopening During the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Alpha (B.1.1.7) Variant Spread: A Multicenter Cross-sectional Study in Israel.

This multicenter, cross-sectional study provides evidence on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-associated emergency department visits and hospitalizations in pediatric wards and intensive care units after school reopening during the SARS-CoV-2 Alpha (B.1.1.7) variant spread in Israel. Study findings suggest that school reopening was not followed by an increase in SARS-CoV-2-related pediatric morbidity.

Obesity screening in the pediatric emergency department - A missed opportunity?

Objectives: There is a low rate of body mass index measurements and obesity screening in primary pediatric care. Pediatric emergency department (PED) visits, with their large volumes and routine weight measurements, provide a unique opportunity to identify and address obesity. The study objectives were to examine the rate of addressing obesity in the PED and to identify its predicting factors. Methods: From electronic medical records of PED visits during 2010-2019, we extracted data on age, gender, weight, time, listed diagnoses, and discharge texts. The primary outcome was a listed diagnosis of "obesity" on discharge letters of children with obesity. Secondary outcomes were addressing weight in the discharge letter and written recommendations for obesity-related treatment. Mixed models were used to test for associations between each of the three outcomes and patient/visit characteristics. Results: There were 150,250 PED visits by 88,253 different children and adolescents. Obesity was found in 10,691 children (12.1%). Among these, listed "obesity" diagnosis was present in only 240 (1.5%) visits. Text addressing overweight/obesity was recorded in 721 (4.4%) visits, and weight-related recommendations were documented in 716 (4.4%) visits. "Obesity" was documented in females more often than in males, in older children, in children with higher weights, and in visits conducted during the mornings. Conclusions: The rate of obesity diagnosis in the PED was extremely low, hence the potential screening ability of the PED in this matter is highly under-utilized. PEDs could increase the recognition of obesity, thus assisting in the global efforts in tackling this disease.

Combined presentation and immunogenicity analysis reveals a recurrent RAS.Q61K neoantigen in melanoma.

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote "off-the-shelf" precision immunotherapies, alleviating limitations of personalized treatments.

Dynamic changes in tRNA modifications and abundance during T cell activation.

The tRNA pool determines the efficiency, throughput, and accuracy of translation. Previous studies have identified dynamic changes in the tRNA (transfer RNA) supply and mRNA (messenger RNA) demand during cancerous proliferation. Yet dynamic changes may also occur during physiologically normal proliferation, and these are less well characterized. We examined the tRNA and mRNA pools of T cells during their vigorous proliferation and differentiation upon triggering their antigen receptor. We observed a global signature of switch in demand for codons at the early proliferation phase of the response, accompanied by corresponding changes in tRNA expression levels. In the later phase, upon differentiation, the response of the tRNA pool relaxed back to the basal level, potentially restraining excessive proliferation. Sequencing of tRNAs allowed us to evaluate their diverse base-modifications. We found that two types of tRNA modifications, wybutosine and ms2t6A, are reduced dramatically during T cell activation. These modifications occur in the anticodon loops of two tRNAs that decode "slippery codons," which are prone to ribosomal frameshifting. Attenuation of these frameshift-protective modifications is expected to increase the potential for proteome-wide frameshifting during T cell proliferation. Indeed, human cell lines deleted of a wybutosine writer showed increased ribosomal frameshifting, as detected with an HIV gag-pol frameshifting site reporter. These results may explain HIV's specific tropism toward proliferating T cells since it requires ribosomal frameshift exactly on the corresponding codon for infection. The changes in tRNA expression and modifications uncover a layer of translation regulation during T cell proliferation and expose a potential tradeoff between cellular growth and translation fidelity.