Oxidative Stress is a shared characteristic of ME/CFS and Long COVID.

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.

Premature skewing of T cell receptor clonality and delayed memory expansion in HIV-exposed infants.

While preventing vertical HIV transmission has been very successful, HIV-exposed uninfected infants (iHEU) experience an elevated risk to infections compared to HIV-unexposed and uninfected infants (iHUU). Here we present a longitudinal multimodal analysis of infant immune ontogeny that highlights the impact of HIV/ARV exposure. Using mass cytometry, we show alterations in T cell memory differentiation between iHEU and iHUU being significant from week 15 of life. The altered memory T cell differentiation in iHEU was preceded by lower TCR Vß clonotypic diversity and linked to TCR clonal depletion within the naïve T cell compartment. Compared to iHUU, iHEU had elevated CD56loCD16loPerforin+CD38+CD45RA+FcεRIγ+ NK cells at 1 month postpartum and whose abundance pre-vaccination were predictive of vaccine-induced pertussis and rotavirus antibody responses post 3 months of life. Collectively, HIV/ARV exposure disrupted the trajectory of innate and adaptive immunity from birth which may underlie relative vulnerability to infections in iHEU.

Mtb HLA-E-tetramer-sorted CD8+ T cells have a diverse TCR repertoire.

HLA-E molecules can present self- and pathogen-derived peptides to both natural killer (NK) cells and T cells. T cells that recognize HLA-E peptides via their T cell receptor (TCR) are termed donor-unrestricted T cells due to restricted allelic variation of HLA-E. The composition and repertoire of HLA-E TCRs is not known so far. We performed TCR sequencing on CD8+ T cells from 21 individuals recognizing HLA-E tetramers (TMs) folded with two Mtb-HLA-E-restricted peptides. We sorted HLA-E Mtb TM+ and TM- CD8+ T cells directly ex vivo and performed bulk RNA-sequencing and single-cell TCR sequencing. The identified TCR repertoire was diverse and showed no conservation between and within individuals. TCRs selected from our single-cell TCR sequencing data could be activated upon HLA-E/peptide stimulation, although not robust, reflecting potentially weak interactions between HLA-E peptide complexes and TCRs. Thus, HLA-E-Mtb-specific T cells have a highly diverse TCR repertoire.

Impaired innate and adaptive immune responses to BNT162b2 SARS-CoV-2 vaccination in systemic lupus erythematosus.

Understanding the immune responses to SARS-CoV-2 vaccination is critical to optimizing vaccination strategies for individuals with autoimmune diseases, such as systemic lupus erythematosus (SLE). Here, we comprehensively analyzed innate and adaptive immune responses in 19 patients with SLE receiving a complete 2-dose Pfizer-BioNTech mRNA vaccine (BNT162b2) regimen compared with a control cohort of 56 healthy control (HC) volunteers. Patients with SLE exhibited impaired neutralizing antibody production and antigen-specific CD4+ and CD8+ T cell responses relative to HC. Interestingly, antibody responses were only altered in patients with SLE treated with immunosuppressive therapies, whereas impairment of antigen-specific CD4+ and CD8+ T cell numbers was independent of medication. Patients with SLE also displayed reduced levels of circulating CXC motif chemokine ligands, CXCL9, CXCL10, CXCL11, and IFN-γ after secondary vaccination as well as downregulation of gene expression pathways indicative of compromised innate immune responses. Single-cell RNA-Seq analysis reveals that patients with SLE showed reduced levels of a vaccine-inducible monocyte population characterized by overexpression of IFN-response transcription factors. Thus, although 2 doses of BNT162b2 induced relatively robust immune responses in patients with SLE, our data demonstrate impairment of both innate and adaptive immune responses relative to HC, highlighting a need for population-specific vaccination studies.

HLA-DQB1*05 subtypes and not DRB1*10:01 mediates risk in anti-IgLON5 disease.

Anti-IgLON5 disease is a rare and likely underdiagnosed subtype of autoimmune encephalitis. The disease displays a heterogeneous phenotype that includes sleep, movement, and bulbar-associated dysfunction. Presence of IgLON5-antibodies in CSF/serum, together with a strong association with HLA-DRB1*10:01∼DQB1*05:01, support an autoimmune basis. In this study, a multicentric HLA study of 87 anti-IgLON5 patients revealed a stronger association with HLA-DQ than HLA-DR. Specifically, we identified a predisposing rank-wise association with HLA-DQA1*01:05∼DQB1*05:01, HLA-DQA1*01:01∼DQB1*05:01 and HLA-DQA1*01:04∼DQB1*05:03 in 85% of patients. HLA sequences and binding cores for these three DQ heterodimers were similar, unlike those of linked DRB1 alleles, supporting a causal link to HLA-DQ. This association was further reflected in an increasingly later age of onset across each genotype group, with a delay of up to 11 years, while HLA-DQ-dosage dependent effects were also suggested by reduced risk in the presence of non-predisposing DQ1 alleles. The functional relevance of the observed HLA-DQ molecules was studied with competition binding assays. These proof-of-concept experiments revealed preferential binding of IgLON5 in a post-translationally modified, but not native, state to all three risk-associated HLA-DQ receptors. Further, a deamidated peptide from the Ig2-domain of IgLON5 activated T cells in two patients, compared to one control carrying HLA-DQA1*01:05∼DQB1*05:01. Taken together, these data support a HLA-DQ-mediated T cell response to IgLON5 as a potentially key step in the initiation of autoimmunity in this disease.

Features of acute COVID-19 associated with post-acute sequelae of SARS-CoV-2 phenotypes: results from the IMPACC study.

Post-acute sequelae of SARS-CoV-2 (PASC) is a significant public health concern. We describe Patient Reported Outcomes (PROs) on 590 participants prospectively assessed from hospital admission for COVID-19 through one year after discharge. Modeling identified 4 PRO clusters based on reported deficits (minimal, physical, mental/cognitive, and multidomain), supporting heterogenous clinical presentations in PASC, with sub-phenotypes associated with female sex and distinctive comorbidities. During the acute phase of disease, a higher respiratory SARS-CoV-2 viral burden and lower Receptor Binding Domain and Spike antibody titers were associated with both the physical predominant and the multidomain deficit clusters. A lower frequency of circulating B lymphocytes by mass cytometry (CyTOF) was observed in the multidomain deficit cluster. Circulating fibroblast growth factor 21 (FGF21) was significantly elevated in the mental/cognitive predominant and the multidomain clusters. Future efforts to link PASC to acute anti-viral host responses may help to better target treatment and prevention of PASC.

γδ T cell antigen receptor polyspecificity enables T cell responses to a broad range of immune challenges.

γδ T cells are essential for immune defense and modulating physiological processes. While they have the potential to recognize large numbers of antigens through somatic gene rearrangement, the antigens which trigger most γδ T cell response remain unidentified, and the role of antigen recognition in γδ T cell function is contentious. Here, we show that some γδ T cell receptors (TCRs) exhibit polyspecificity, recognizing multiple ligands of diverse molecular nature. These ligands include haptens, metabolites, neurotransmitters, posttranslational modifications, as well as peptides and proteins of microbial and host origin. Polyspecific γδ T cells are enriched among activated cells in naive mice and the responding population in infection. They express diverse TCR sequences, have different functional potentials, and include the innate-like γδ T cells, such as the major IL-17 responders in various pathological/physiological conditions. We demonstrate that encountering their antigenic microbiome metabolite maintains their homeostasis and functional response, indicating that their ability to recognize multiple ligands is essential for their function. Human γδ T cells with similar polyspecificity also respond to various immune challenges. This study demonstrates that polyspecificity is a prevalent feature of γδ T cell antigen recognition, which enables rapid and robust T cell responses to a wide range of challenges, highlighting a unique function of γδ T cells.

Hypoimmune induced pluripotent stem cells survive long term in fully immunocompetent, allogeneic rhesus macaques.

Genetic engineering of allogeneic cell therapeutics that fully prevents rejection by a recipient's immune system would abolish the requirement for immunosuppressive drugs or encapsulation and support large-scale manufacturing of off-the-shelf cell products. Previously, we generated mouse and human hypoimmune pluripotent (HIP) stem cells by depleting HLA class I and II molecules and overexpressing CD47 (B2M-/-CIITA-/-CD47+). To determine whether this strategy is successful in non-human primates, we engineered rhesus macaque HIP cells and transplanted them intramuscularly into four allogeneic rhesus macaques. The HIP cells survived unrestricted for 16 weeks in fully immunocompetent allogeneic recipients and differentiated into several lineages, whereas allogeneic wild-type cells were vigorously rejected. We also differentiated human HIP cells into endocrinologically active pancreatic islet cells and showed that they survived in immunocompetent, allogeneic diabetic humanized mice for 4 weeks and ameliorated diabetes. HIP-edited primary rhesus macaque islets survived for 40 weeks in an allogeneic rhesus macaque recipient without immunosuppression, whereas unedited islets were quickly rejected.

A Paratope-Enhanced Method to Determine Breadth and Depth TCR Clonal Metrics of the Private Human T-Cell Vaccine Response after SARS-CoV-2 Vaccination.

Quantitative metrics for vaccine-induced T-cell responses are an important need for developing correlates of protection and their use in vaccine-based medical management and population health. Molecular TCR analysis is an appealing strategy but currently requires a targeted methodology involving complex integration of ex vivo data (antigen-specific functional T-cell cytokine responses and TCR molecular responses) that uncover only public antigen-specific metrics. Here, we describe an untargeted private TCR method that measures breadth and depth metrics of the T-cell response to vaccine challenge using a simple pre- and post-vaccine subject sampling, TCR immunoseq analysis, and a bioinformatic approach using self-organizing maps and GLIPH2. Among 515 subjects undergoing SARS-CoV-2 mRNA vaccination, we found that breadth and depth metrics were moderately correlated between the targeted public TCR response and untargeted private TCR response methods. The untargeted private TCR method was sufficiently sensitive to distinguish subgroups of potential clinical significance also observed using public TCR methods (the reduced T-cell vaccine response with age and the paradoxically elevated T-cell vaccine response of patients on anti-TNF immunotherapy). These observations suggest the promise of this untargeted private TCR method to produce T-cell vaccine-response metrics in an antigen-agnostic and individual-autonomous context.

Acute Respiratory Illness Is Associated with Memory T Cell Differentiation and Other Immune Cell Changes in an Age-Associated Manner.

Respiratory viruses such as influenza are encountered multiple times through infection and/or vaccination and thus have the potential to shape immune cell phenotypes over time. In particular, memory T cell compartments may be affected, as both CD4+ and CD8+ T cell responses likely contribute to viral control. In this study, we assessed immune phenotypes using cytometry by time of flight in the peripheral blood of 22 humans with acute respiratory illness and 22 age-matched noninfected controls. In younger infected individuals (1-19 y of age), we found decreased B and NK cell frequencies and a shift toward more effector-like CD4+ and CD8+ T cell phenotypes, compared with young healthy controls. Significant differences between noninfected and infected older individuals (30-74 y of age) were not seen. We also observed a decrease in naive CD4+ T cells and CD27+CD8+ T cells as well as an increase in effector memory CD8+ T cells and NKT cells in noninfected individuals with age. When cell frequencies were regressed against age for infected versus noninfected subjects, significant differences in trends with age were observed for multiple cell types. These included B cells and various subsets of CD4+ and CD8+ T cells. We conclude that acute respiratory illness drives T cell differentiation and decreases circulating B cell frequencies preferentially in young compared with older individuals.

Integrated single-cell chromatin and transcriptomic analyses of human scalp identify gene-regulatory programs and critical cell types for hair and skin diseases.

Genome-wide association studies have identified many loci associated with hair and skin disease, but identification of causal variants requires deciphering of gene-regulatory networks in relevant cell types. We generated matched single-cell chromatin profiles and transcriptomes from scalp tissue from healthy controls and patients with alopecia areata, identifying diverse cell types of the hair follicle niche. By interrogating these datasets at multiple levels of cellular resolution, we infer 50-100% more enhancer-gene links than previous approaches and show that aggregate enhancer accessibility for highly regulated genes predicts expression. We use these gene-regulatory maps to prioritize cell types, genes and causal variants implicated in the pathobiology of androgenetic alopecia (AGA), eczema and other complex traits. AGA genome-wide association studies signals are enriched in dermal papilla regulatory regions, supporting the role of these cells as drivers of AGA pathogenesis. Finally, we train machine learning models to nominate single-nucleotide polymorphisms that affect gene expression through disruption of transcription factor binding, predicting candidate functional single-nucleotide polymorphism for AGA and eczema.

Disrupted memory T cell expansion in HIV-exposed uninfected infants is preceded by premature skewing of T cell receptor clonality.

While preventing vertical HIV transmission has been very successful, the increasing number of HIV-exposed uninfected infants (iHEU) experience an elevated risk to infections compared to HIV-unexposed and uninfected infants (iHUU). Immune developmental differences between iHEU and iHUU remains poorly understood and here we present a longitudinal multimodal analysis of infant immune ontogeny that highlights the impact of HIV/ARV exposure. Using mass cytometry, we show alterations and differences in the emergence of NK cell populations and T cell memory differentiation between iHEU and iHUU. Specific NK cells observed at birth were also predictive of acellular pertussis and rotavirus vaccine-induced IgG and IgA responses, respectively, at 3 and 9 months of life. T cell receptor Vß clonotypic diversity was significantly and persistently lower in iHEU preceding the expansion of T cell memory. Our findings show that HIV/ARV exposure disrupts innate and adaptive immunity from birth which may underlie relative vulnerability to infections.

A structural blueprint for interleukin-21 signal modulation.

Interleukin-21 (IL-21) plays a critical role in generating immunological memory by promoting the germinal center reaction, yet clinical use of IL-21 remains challenging because of its pleiotropy and association with autoimmune disease. To better understand the structural basis of IL-21 signaling, we determine the structure of the IL-21-IL-21R-γc ternary signaling complex by X-ray crystallography and a structure of a dimer of trimeric complexes using cryo-electron microscopy. Guided by the structure, we design analogs of IL-21 by introducing substitutions to the IL-21-γc interface. These IL-21 analogs act as partial agonists that modulate downstream activation of pS6, pSTAT3, and pSTAT1. These analogs exhibit differential activity on T and B cell subsets and modulate antibody production in human tonsil organoids. These results clarify the structural basis of IL-21 signaling and offer a potential strategy for tunable manipulation of humoral immunity.

Organoid modeling of lung-resident immune responses to SARS-CoV-2 infection.

Tissue-resident immunity underlies essential host defenses against pathogens, but analysis in humans has lacked in vitro model systems where epithelial infection and accompanying resident immune cell responses can be observed en bloc. Indeed, human primary epithelial organoid cultures typically omit immune cells, and human tissue resident-memory lymphocytes are conventionally assayed without an epithelial infection component, for instance from peripheral blood, or after extraction from organs. Further, the study of resident immunity in animals can be complicated by interchange between tissue and peripheral immune compartments. To study human tissue-resident infectious immune responses in isolation from secondary lymphoid organs, we generated adult human lung three-dimensional air-liquid interface (ALI) lung organoids from intact tissue fragments that co-preserve epithelial and stromal architecture alongside endogenous lung-resident immune subsets. These included T, B, NK and myeloid cells, with CD69+CD103+ tissue-resident and CCR7- and/or CD45RA- TRM and conservation of T cell receptor repertoires, all corresponding to matched fresh tissue. SARS-CoV-2 vigorously infected organoid lung epithelium, alongside secondary induction of innate cytokine production that was inhibited by antiviral agents. Notably, SARS-CoV-2-infected organoids manifested adaptive virus-specific T cell activation that was specific for seropositive and/or previously infected donor individuals. This holistic non-reconstitutive organoid system demonstrates the sufficiency of lung to autonomously mount adaptive T cell memory responses without a peripheral lymphoid component, and represents an enabling method for the study of human tissue-resident immunity.

Spheromers reveal robust T cell responses to the Pfizer/BioNTech vaccine and attenuated peripheral CD8+ T cell responses post SARS-CoV-2 infection.

T cells are a critical component of the response to SARS-CoV-2, but their kinetics after infection and vaccination are insufficiently understood. Using "spheromer" peptide-MHC multimer reagents, we analyzed healthy subjects receiving two doses of the Pfizer/BioNTech BNT162b2 vaccine. Vaccination resulted in robust spike-specific T cell responses for the dominant CD4+ (HLA-DRB1∗15:01/S191) and CD8+ (HLA-A∗02/S691) T cell epitopes. Antigen-specific CD4+ and CD8+ T cell responses were asynchronous, with the peak CD4+ T cell responses occurring 1 week post the second vaccination (boost), whereas CD8+ T cells peaked 2 weeks later. These peripheral T cell responses were elevated compared with COVID-19 patients. We also found that previous SARS-CoV-2 infection resulted in decreased CD8+ T cell activation and expansion, suggesting that previous infection can influence the T cell response to vaccination.

Entropic analysis of antigen-specific CDR3 domains identifies essential binding motifs shared by CDR3s with different antigen specificities.

Antigen-specific T cell receptor (TCR) sequences can have prognostic, predictive, and therapeutic value, but decoding the specificity of TCR recognition remains challenging. Unlike DNA strands that base pair, TCRs bind to their targets with different orientations and different lengths, which complicates comparisons. We present scanning parametrized by normalized TCR length (SPAN-TCR) to analyze antigen-specific TCR CDR3 sequences and identify patterns driving TCR-pMHC specificity. Using entropic analysis, SPAN-TCR identifies 2-mer motifs that decrease the diversity (entropy) of CDR3s. These motifs are the most common patterns that can predict CDR3 composition, and we identify "essential" motifs that decrease entropy in the same CDR3 α or ß chain containing the 2-mer, and "super-essential" motifs that decrease entropy in both chains. Molecular dynamics analysis further suggests that these motifs may play important roles in binding. We then employ SPAN-TCR to resolve similarities in TCR repertoires against different antigens using public databases of TCR sequences.

NK-like CD8+ γδ T cells are expanded in persistent Mycobacterium tuberculosis infection.

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.