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More than 65 million individuals worldwide are estimated to have Long COVID (LC), a complex multisystemic condition, wherein patients of all ages report fatigue, post-exertional malaise, and other symptoms resembling myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS). With no current treatments or reliable diagnostic markers, there is an urgent need to define the molecular underpinnings of these conditions. By studying bioenergetic characteristics of peripheral blood lymphocytes in over 16 healthy controls, 15 ME/CFS, and 15 LC, we find both ME/CFS and LC donors exhibit signs of elevated oxidative stress, relative to healthy controls, especially in the memory subset. Using a combination of flow cytometry, bulk RNA-seq analysis, mass spectrometry, and systems chemistry analysis, we also observed aberrations in ROS clearance pathways including elevated glutathione levels, decreases in mitochondrial superoxide dismutase levels, and glutathione peroxidase 4 mediated lipid oxidative damage. Critically, these changes in redox pathways show striking sex-specific trends. While females diagnosed with ME/CFS exhibit higher total ROS and mitochondrial calcium levels, males with an ME/CFS diagnosis have normal ROS levels, but larger changes in lipid oxidative damage. Further analyses show that higher ROS levels correlates with hyperproliferation of T cells in females, consistent with the known role of elevated ROS levels in the initiation of proliferation. This hyperproliferation of T cells can be attenuated by metformin, suggesting this FDA-approved drug as a possible treatment, as also suggested by a recent clinical study of LC patients. Thus, we report that both ME/CFS and LC are mechanistically related and could be diagnosed with quantitative blood cell measurements. We also suggest that effective, patient tailored drugs might be discovered using standard lymphocyte stimulation assays.

Here we report preliminary data demonstrating that some patients with myalgic encephalomyelitis/chronic fatiguesyndrome (ME/CFS) may have catalytic autoantibodies that cause the breakdown of myelin basic protein (MBP). We propose that these MBP-degradative antibodies are important to the pathophysiology of ME/CFS, particularly in the occurrence of white matter disease/demyelination. This is supported by magnetic resonance imagining studies that show these findings in patients with ME/CFS and could explain symptoms of nerve pain and muscle weakness. In this work, we performed a series of experiments on patient plasma samples where we isolated and characterized substrate-specific antibodies that digest MBP. We also tested glatiramer acetate (copaxone), an FDA approved immunomodulator to treat multiple sclerosis, and found that it inhibits ME/CFS antibody digestion of MBP. Furthermore, we found that aprotinin, which is a specific serine protease inhibitor, specifically prevents breakdown of MBP while the other classes of protease inhibitors had no effect. This coincides with the published literature describing catalytic antibodies as having serine protease-like activity. Postpandemic research has also provided several reports of demyelination in COVID-19. Because COVID-19 has been described as a trigger for ME/CFS, demyelination could play a bigger role in patient symptoms for those recently diagnosed with ME/CFS. Therefore, by studying proteolytic antibodies in ME/CFS, their target substrates, and inhibitors, a new mechanism of action could lead to better treatment and a possible cure for the disease.

The response of gamma delta (γδ) T cells in the acute versus chronic phases of the same infection is unclear. How γδ T cells function in acute Mycobacterium tuberculosis (Mtb) infection is well characterized, but their response during persistent Mtb infection is not well understood, even though most infections with Mtb manifest as a chronic, clinically asymptomatic state. Here, we analyze peripheral blood γδ T cells from a South African adolescent cohort and show that a unique CD8 + γδ T cell subset with features of "memory inflation" expands in chronic Mtb infection. These cells are hyporesponsive to T cell receptor (TCR)-mediated signaling but, like NK cells, can mount robust CD16-mediated cytotoxic responses. These CD8 + γδ T cells comprise a highly focused TCR repertoire, with clonotypes that are Mycobacterium specific but not phosphoantigen reactive. Using multiparametric single-cell pseudo-time trajectory analysis, we identified the differentiation paths that these CD8 + γδ T cells follow to develop into effectors in this infection state. Last, we found that circulating CD8 + γδ T cells also expand in other chronic inflammatory conditions, including cardiovascular disease and cancer, suggesting that persistent antigenic exposure may drive similar γδ T cell effector programs and differentiation fates.

In this work, we find that CD8 + T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49 + CD8 + regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8 + T cells efficiently eliminated pathogenic gliadin-specific CD4 + T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIR + CD8 + T cells, but not CD4 + regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49 + CD8 + T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8 + T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases.

Previous studies have demonstrated the regulatory functions of Ly49 + CD8 + T cells toward self-reactive CD4 + T cells in mice. Recently, we found KIR + CD8 + T cells are the equivalent of mouse Ly49 + CD8 + T cells in humans. They are increased in patients with autoimmune or infectious diseases as a negative feedback mechanism to suppress the arising pathogenic cells and maintain peripheral tolerance. Here, we describe the methods on how we characterize the KIR + CD8 + T cells from different diseases using single-cell RNA and TCR sequencing., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Previous reports show that Ly49 + CD8 + T cells can suppress autoimmunity in mouse models of autoimmune diseases. Here we find a markedly increased frequency of CD8 + T cells expressing inhibitory Killer cell Immunoglobulin like Receptors (KIR), the human equivalent of the Ly49 family, in the blood and inflamed tissues of various autoimmune diseases. Moreover, KIR + CD8 + T cells can efficiently eliminate pathogenic gliadin-specific CD4 + T cells from Celiac disease (CeD) patients' leukocytes in vitro . Furthermore, we observe elevated levels of KIR + CD8 + T cells, but not CD4 + regulatory T cells, in COVID-19 and influenza-infected patients, and this correlates with disease severity and vasculitis in COVID-19. Expanded KIR + CD8 + T cells from these different diseases display shared phenotypes and similar T cell receptor sequences. These results characterize a regulatory CD8 + T cell subset in humans, broadly active in both autoimmune and infectious diseases, which we hypothesize functions to control self-reactive or otherwise pathogenic T cells., One-Sentence Summary: Here we identified KIR + CD8 + T cells as a regulatory CD8 + T cell subset in humans that suppresses self-reactive or otherwise pathogenic CD4 + T cells.

One in four myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients are estimated to be severely affected by the disease, and these house-bound or bedbound patients are currently understudied. Here, we report a comprehensive examination of the symptoms and clinical laboratory tests of a cohort of severely ill patients and healthy controls. The greatly reduced quality of life of the patients was negatively correlated with clinical depression. The most troublesome symptoms included fatigue (85%), pain (65%), cognitive impairment (50%), orthostatic intolerance (45%), sleep disturbance (35%), post-exertional malaise (30%), and neurosensory disturbance (30%). Sleep profiles and cognitive tests revealed distinctive impairments. Lower morning cortisol level and alterations in its diurnal rhythm were observed in the patients, and antibody and antigen measurements showed no evidence for acute infections by common viral or bacterial pathogens. These results highlight the urgent need of developing molecular diagnostic tests for ME/CFS. In addition, there was a striking similarity in symptoms between long COVID and ME/CFS, suggesting that studies on the mechanism and treatment of ME/CFS may help prevent and treat long COVID and vice versa.

A central feature of the SARS-CoV-2 pandemic is that some individuals become severely ill or die, whereas others have only a mild disease course or are asymptomatic. Here we report development of an improved multimeric αβ T cell staining reagent platform, with each maxi-ferritin "spheromer" displaying 12 peptide-MHC complexes. Spheromers stain specific T cells more efficiently than peptide-MHC tetramers and capture a broader portion of the sequence repertoire for a given peptide-MHC. Analyzing the response in unexposed individuals, we find that T cells recognizing peptides conserved amongst coronaviruses are more abundant and tend to have a "memory" phenotype, compared to those unique to SARS-CoV-2. Significantly, CD8 + T cells with these conserved specificities are much more abundant in COVID-19 patients with mild disease versus those with a more severe illness, suggesting a protective role., (Copyright © 2021, American Association for the Advancement of Science.)

To identify disease-relevant T cell receptors (TCRs) with shared antigen specificity, we analyzed 778,938 TCRβ chain sequences from 178 non-small cell lung cancer patients using the GLIPH2 (grouping of lymphocyte interactions with paratope hotspots 2) algorithm. We identified over 66,000 shared specificity groups, of which 435 were clonally expanded and enriched in tumors compared to adjacent lung. The antigenic epitopes of one such tumor-enriched specificity group were identified using a yeast peptide-HLA A ∗ 02:01 display library. These included a peptide from the epithelial protein TMEM161A, which is overexpressed in tumors and cross-reactive epitopes from Epstein-Barr virus and E. coli. Our findings suggest that this cross-reactivity may underlie the presence of virus-specific T cells in tumor infiltrates and that pathogen cross-reactivity may be a feature of multiple cancers. The approach and analytical pipelines generated in this work, as well as the specificity groups defined here, present a resource for understanding the T cell response in cancer., Competing Interests: Declaration of interests C.L.M. is a founder of, holds equity in, and receives consulting fees from Lyell Immunopharma and receives consulting fees from NeoImmuneTech, Nektar, Apricity, and Roche. J.W.N. reports research support from Genentech/Roche, Merck, Novartis, Boehringer Ingelheim, Exelixis, Takeda Pharmaceuticals, Nektar Therapeutics, Adaptimmune, and GSK and has served in a consulting or advisory role for AstraZeneca, Genentech/Roche, Exelixis Inc., Jounce Therapeutics, Takeda Pharmaceuticals, Eli Lilly and Company, Calithera Biosciences, Amgen, Regeneron Pharmaceuticals, Natera, and Iovance Biotherapeutics. H.A.W. has received research support from Celgene, Clovis Oncology, Genentech/Roche, Arrys Therapeutics, Novartis, Merck, BMS, Exelixis, Lilly, Pfizer, and has participated on the advisory boards of Helsinn, Mirati, Cellworks, Genentech/Roche, Merck, and ITMIG. N.S.L. has received research funding from Intuitive Foundation and Auspex Diagnostics. E.S. is a consultant for Lyell Immunopharma. L.L. is a consultant for Lyell Immunopharma. S.A.F. is consulting for Lonza PerMed and Samsara BioCapital. M.D. reports research funding from Varian Medical Systems and Illumina; ownership interest in CiberMed and Foresight Diagnostics; patent filings related to cancer biomarkers; paid consultancy from Roche, AstraZeneca, BioNTech, Genentech, Novartis, and Gritstone Oncology; and travel/honoraria from Reflexion. K.C.G. is founder of 3T therapeutics. I.I.W. has received honoraria from Genentech/Roche, Bayer, Bristol-Myers Squibb, AstraZeneca/Medimmune, Pfizer, HTG Molecular, Asuragen, Merck, GlaxoSmithKline, Guardant Health, Oncocyte, and MSD. I.I.W. is also supported by Genentech, Oncoplex, HTG Molecular, DepArray, Merck, Bristol-Myers Squibb, Medimmune, Adaptive, Adaptimmune, EMD Serono, Pfizer, Takeda, Amgen, Karus, Johnson & Johnson, Bayer, Iovance, 4D, Novartis, and Akoya. J.Z. reports grants from Merck and Johnson & Johnson, as well as adversary/consulting/Hornoraria fees from Bristol Myers Squibb, AstraZeneca, GenePlus, Innovent, OrigMed, and Roche outside the submitted work. This study was supported in part by a Cancer Prevention Research Institute of Texas Multi-Investigator Research Award (grant number RP160668) and the University of Texas Lung Specialized Programs of Research Excellence grant (grant number P50CA70907). S.-H.C., D.T., C.L.M., and M.M.D have a patent related to this work., (Copyright © 2021. Published by Elsevier Inc.)

Purpose: Skin-related issues have a significant impact on health, activities of daily living, and quality of life among people with spina bifida. Data presented by select clinics that participate in the National Spina Bifida Patient Registry reported that 26% of individuals had a history of pressure injuries with 19% having had one in the past year. The spina bifida community lack direct guidelines on prevention of these and other skin related issues. The Integument (skin) Guidelines focus on prevention, not treatment, of existing problems., Methods: Using a consensus building methodology, the guidelines were written by experts in spina bifida and wound care., Results: The guidelines include age-grouped, evidence-based guidelines written in the context of an understanding of the whole person. They are presented in table format according to the age of the person with spina bifida., Conclusion: These guidelines present a standardized approach to prevention of skin-related issues in spina bifida. Discovering what results in successful minimization of skin-related issues with testing of technology or prevention strategies is the next step in protecting this vulnerable population.

There is not currently a well-established, if any, biological test to diagnose myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). The molecular aberrations observed in numerous studies of ME/CFS blood cells offer the opportunity to develop a diagnostic assay from blood samples. Here we developed a nanoelectronics assay designed as an ultrasensitive assay capable of directly measuring biomolecular interactions in real time, at low cost, and in a multiplex format. To pursue the goal of developing a reliable biomarker for ME/CFS and to demonstrate the utility of our platform for point-of-care diagnostics, we validated the array by testing patients with moderate to severe ME/CFS patients and healthy controls. The ME/CFS samples' response to the hyperosmotic stressor observed as a unique characteristic of the impedance pattern and dramatically different from the response observed among the control samples. We believe the observed robust impedance modulation difference of the samples in response to hyperosmotic stress can potentially provide us with a unique indicator of ME/CFS. Moreover, using supervised machine learning algorithms, we developed a classifier for ME/CFS patients capable of identifying new patients, required for a robust diagnostic tool., Competing Interests: Conflict of interest statement: R.W.D. is Director of the Scientific Advisory Board of the Open Medicine Foundation.

Background: Myalgic encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a poorly understood disease. Amongst others symptoms, the disease is associated with profound fatigue, cognitive dysfunction, sleep abnormalities, and other symptoms that are made worse by physical or mental exertion. While the etiology of the disease is still debated, evidence suggests oxidative damage to immune and hematological systems as one of the pathophysiological mechanisms of the disease. Since red blood cells (RBCs) are well-known scavengers of oxidative stress, and are critical in microvascular perfusion and tissue oxygenation, we hypothesized that RBC deformability is adversely affected in ME/CFS., Methods: We used a custom microfluidic platform and high-speed microscopy to assess the difference in deformability of RBCs obtained from ME/CFS patients and age-matched healthy controls., Results and Conclusion: We observed from various measures of deformability that the RBCs isolated from ME/CFS patients were significantly stiffer than those from healthy controls. Our observations suggest that RBC transport through microcapillaries may explain, at least in part, the ME/CFS phenotype, and promises to be a novel first-pass diagnostic test.

Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5 + ISCs, the most well-defined ISC pool, but Bmi1-GFP + cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP + cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1 + cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP + cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5 + intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5 + ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5 + ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5 + ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.