Cell Reports Medicine

In patients with common variable immune deficiencies, primary vaccination followed by two booster doses is recommended for protection against COVID-19. Seroconversion has been shown in 60% of patients. We have no information on whether serum antibodies reflect the generation of durable immune memory. In a longitudinal study on 47 common variable immune deficiencies patients who received the third and fourth vaccine dose, we show that the measurement of specific antibodies is not sufficient to predict the establishment of immune memory and the ability to respond to antigen re-exposure. Our results indicate that the combination of antibodies and memory B cells responses represents a more reliable read-out of vaccine immune efficacy in vulnerable patients. This analysis may not only identify individuals remaining unprotected after vaccination and unable to respond to additional booster doses, but also address the search for the underlying immune defect and suggest patient-tailored management strategies.

Post-Acute Sequelae of SARS-CoV-2 infection (PASC) syndrome or "Long COVID" represents a widespread health challenge that necessitates the development of novel diagnostic approaches and targeted therapies that can be readily deployed. Immune dysregulation has been reported as one of the hallmarks of PASC, but the extent of PASC immune dysregulation in patients over time remains unclear. We therefore assessed SARS-CoV-2-specific antibody responses, peripheral immune cell profiles, autoantibody profiles and circulating cytokines for up to 6 months in participants with a SARS-CoV-2 infection who either convalesced or developed PASC. Compared to convalescent, PASC participants with a broad range of PASC phenotypes exhibited persistently elevated IgG titers for SARS-CoV-2 Envelope and Nucleocapsid proteins over the 6 months of study duration. In contrast, the IgG responses to Spike protein were significantly lower in the PASC cohort with predominantly IgG1 and IgG3 class-switched bias. Using CyTOF analysis, we show elevated numbers of circulating T follicular helper cells (cTFH) and mucosa-associated invariant T cells (MAIT), which also correlated with high anti-Envelope IgG titers. Persistent immune activation was accompanied by augmented serum cytokine profiles with LIF, IL-11, Eotaxin-3, and HMGB-1 in PASC participants, who also demonstrated significantly higher rates of autoantibodies. These findings highlight the persistence of immune dysregulation in PASC, underscoring the need to explore targeted therapies addressing viral persistence, dysregulated antibody production, and autoimmunity.

COVID-19 convalescent plasma (CCP), a passive polyclonal antibody therapeutic, has exhibited mixed results in the treatment of COVID-19. Given that the therapeutic effect of CCP may extend beyond the ability of SARS-CoV-2-specific antibody binding and neutralization to influence the evolution of the endogenous antibody response, we took a systematic and comprehensive approach to analyze SARS-CoV-2 functional antibody profiles of participants in a randomized controlled trial of CCP treatment of individuals hospitalized with COVID-19 pneumonia where CCP was associated with both decreased mortality and improved clinical severity. Using systems serology, we found that the clinical benefit of CCP is related to a shift towards reduced inflammatory Spike (S) responses and enhanced Nucleocapsid (N) humoral responses. We found CCP had the greatest clinical benefit in participants with low pre-existing anti-SARS-CoV-2 antibody function, rather than S or N antibody levels or participant demographic features. Further, CCP induced immunomodulatory changes to recipient humoral profiles persisted for at least two months, marked by the selective evolution of anti-inflammatory Fc-glycan profiles and persistently expanded nucleocapsid-specific humoral immunity following CCP therapy. Together, our findings identify a novel mechanism of action of CCP, suggest optimal patient characteristics for CCP treatment, identify long-last immunomodulatory effects of CCP, and provide guidance for development of novel N-focused antibody therapeutics for severe COVID-19 hyperinflammation.

Androgen receptor signaling inhibitors (ARSi) are a mainstay for patients with metastatic castration-resistant prostate cancer (mCRPC). However, patient response is heterogeneous and the molecular underpinnings of ARSi resistance are not well elucidated. Here we performed transcriptome analysis of circulating tumor cells (CTCs) and peripheral blood mononuclear cells (PBMC) in the context of a prospective clinical trial of men with mCRPC treated with abiraterone (Abi) or enzalutamide (Enza). CTC RNA-sequencing identified that RB loss and enhanced E2F signaling along with BRCA loss transcriptional networks were associated with intrinsic ARSi resistance, while an inflammatory response signature was significantly associated with acquired resistance. Transcriptomic analysis of matching PBMCs identified enrichment of inflammasome gene signatures indicative of activated innate immunity at progression, with concurrent downregulation of T and NK cells. Importantly, CTC gene signatures had a significant positive association with circulating immune macroenvironment (CIME) signatures. Taken together, these data demonstrate that liquid biopsy transcriptomics can identify molecular pathways associated with clinical ARSi resistance paving the way for treatment optimization in patients with mCRPC.

SARS-CoV-2 infection and COVID-19 vaccines elicit memory T cell responses. Here, we report the development of two new pools of Experimentally-defined T cell epitopes derived from the non-spike Remainder of the SARS-CoV-2 proteome (CD4RE and CD8RE). The combination of T cell responses to these new pools and Spike (S) were used to discriminate four groups of subjects with different SARS-CoV-2 infection and COVID-19 vaccine status: non-infected, non-vaccinated (I-V-); infected and non-vaccinated (I+V-); infected and then vaccinated (I+V+); and non-infected and vaccinated (I-V+). The overall classification accuracy based on 30 subjects/group was 89.2% in the original cohort and 88.5% in a validation cohort of 96 subjects. The T cell classification scheme was applicable to different mRNA vaccines, and different lengths of time post-infection/post-vaccination. T cell responses from breakthrough infections (infected vaccinees, V+I+) were also effectively segregated from the responses of vaccinated subjects using the same classification tool system. When all five groups where combined, for a total of 239 different subjects, the classification scheme performance was 86.6%. We anticipate that a T cell-based immunodiagnostic scheme able to classify subjects based on their vaccination and natural infection history will be an important tool for longitudinal monitoring of vaccination and aid in establishing SARS-CoV-2 correlates of protection.

With an estimated 65 million individuals suffering from Long COVID, validated therapeutic strategies as well as non-invasive biomarkers are direly needed to guide clinical management. We used blood digital transcriptomics in search of viral persistence and Long COVID diagnostic biomarkers in a real-world, general practice-based setting with a long clinical follow-up. We demonstrate systemic SARS-CoV-2 persistence for more than 2 years after acute COVID-19 infection. A 2-gene biomarker, including FYN and SARS-CoV-2 antisense RNA, correctly classifies Long COVID with 93.8% sensitivity and 91.7% specificity. Specific immune transcripts and immunometabolism score correlate to systemic viral load and patient-reported anxiety/depression, providing mechanistic links as well as therapeutic targets to tackle Long COVID.

Post-acute sequelae of SARS-CoV-2 (SARS2) infection (PASC) is a heterogeneous condition, but the main viral drivers are unknown. Here, we use MENSA, Media Enriched with Newly Synthesized Antibodies, secreted exclusively from circulating human plasmablasts, to provide an immune snapshot that defines the underlying viral triggers. We provide proof-of-concept testing that the MENSA technology can capture the new host immune response to accurately diagnose acute primary and breakthrough infections when known SARS2 virus or proteins are present. It is also positive after vaccination when spike proteins elicit an acute immune response. Applying the same principles for long-COVID patients, MENSA is positive for SARS2 in 40% of PASC vs none of the COVID recovered (CR) patients without any sequelae demonstrating ongoing SARS2 viral inflammation only in PASC. Additionally, in PASC patients, MENSAs are also positive for Epstein-Barr Virus (EBV) in 37%, Human Cytomegalovirus (CMV) in 23%, and herpes simplex virus 2 (HSV2) in 15% compared to 17%, 4%, and 4% in CR controls respectively. Combined, a total of 60% of PASC patients have a positive MENSA for SARS2, EBV, CMV, and/or HSV2. MENSA offers a unique antibody snapshot to reveal the underlying viral drivers in long-COVID thus demonstrating the persistence of SARS2 and reactivation of viral herpes in 60% of PASC patients. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=181 HEIGHT=200 SRC="FIGDIR/small/24310017v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1a30be5org.highwire.dtl.DTLVardef@16203ecorg.highwire.dtl.DTLVardef@1ef9448org.highwire.dtl.DTLVardef@1f01045_HPS_FORMAT_FIGEXP M_FIG C_FIG

The gut microbiome (GMB) has been associated with outcomes of immune checkpoint blockade therapy in melanoma, but there is limited consensus on the specific taxa involved, particularly across different geographic regions. We analyzed pre-treatment stool samples from 674 melanoma patients participating in a phase-III trial of adjuvant nivolumab plus ipilimumab versus nivolumab, across three continents and five regions. Longitudinal analysis revealed that GMB was largely unchanged following treatment, offering promise for lasting GMB-based interventions. In region-specific and cross-region meta-analyses, we identified pre-treatment taxonomic markers associated with recurrence, including Eubacterium, Ruminococcus, Firmicutes, and Clostridium. Recurrence prediction by these markers was best achieved across regions by matching participants on GMB compositional similarity between the intra-regional discovery and external validation sets. AUCs for prediction ranged from 0.83-0.94 (depending on the initial discovery region) for patients closely matched on GMB composition (e.g., JSD [≤]0.11). This evidence indicates that taxonomic markers for prediction of recurrence are generalizable across regions, for individuals of similar GMB composition. HighlightsO_LIOverall gut microbiome (GMB) composition is largely unchanged during ICB treatment. C_LIO_LIGMB composition varies by geographic region C_LIO_LIWe identified gut bacterial markers associated with recurrence in region-specific analyses. C_LIO_LIRegion-identified markers are generalizable if GMB composition is taken into account by matching. C_LI

Interferons (IFNs) are antiviral cytokines induced very early after SARS-CoV-2 infection and are crucial for viral clearance, shaping immunity, and preventing the development of severe COVID-19. We previously demonstrated that a single injection of peginterferon-lambda1 (PEG-IFN-{lambda}) accelerated viral clearance in COVID-19 patients. To determine if the rapid viral decline was mediated by enhanced immunity, we assessed in vivo responses to PEG-IFN-{lambda} by single cell RNA sequencing and measured SARS-CoV-2-specific T cell and antibody responses between placebo and PEG-IFN-{lambda}-treated patients. PEG-IFN-{lambda} treatment induced interferon stimulated genes in peripheral immune cells expressing IFNLR1, with plasmacytoid dendritic cells having the greatest response, followed by B cells. PEG-IFN-{lambda} did not significantly affect SARS-CoV-2-specific antibody levels in plasma or the magnitude or functionality of virus-specific T cells. However, we identified a delayed T cell response in older adults, suggesting that PEG-IFN-{lambda} can overcome the delay in adaptive immunity to accelerate viral clearance in patients most at risk for severe disease. Taken together, PEG-IFN-{lambda} offers an early COVID-19 treatment option for outpatients to boost innate antiviral defenses without dampening peripheral SARS-CoV-2 adaptive immunity

DARPins (Designed Ankyrin Repeat Proteins) are a non-antibody based protein scaffold which has undergone extensive clinical characterization. DARPins are small, stable proteins derived from naturally occurring ankyrin repeat proteins and are characterized by high binding affinity and specificity, and a flexible architecture enabling multispecific approaches beyond bi-specific molecules. A total of seven clinical DARPin drug candidates across therapeutic areas spanning ophthalmology (abicipar), virology (ensovibep), and oncology - including solid tumors (MP0250, MP0274, MP0310, MP0317) and hematologic neoplasms (MP0250, MP0533) - have been tested in patients, with the two most advanced reaching the registrational phase of development. Here, we systematically review the published clinical experience with DARPin therapeutics and imaging agents generated over the past decade. The DARPin drug candidates have evolved from relatively simple binders/neutralizers, with impact on specific disease hallmarks, to more sophisticated effector function-enabled molecules that modulate complex tumor-host interactions with therapeutic intent. The accumulated clinical safety, pharmacokinetics, and activity data described herein illustrate the versatility of DARPins to influence complex biological processes and thus help patients with medical conditions that are in some cases difficult to treat with other therapeutic modalities. Following recent breakthroughs in protein structure prediction and de novo protein design, DARPins are poised to deliver an array of unprecedented and exquisitely targeted novel drug candidates eagerly awaited by patients with high unmet medical needs.

The role of antigen-specific T cells responding to antigen is a topic of intense studies, and critical for mechanistic insight of diseases and development of therapeutic strategies. Methods for broad-scale detection of antigen-specific CD4 T cells are lacking, while such methods have demonstrated great value in exploring CD8 T cell response in health and disease. Furthermore, major histocompatibility complex II (MHCII) assays are technically challenging due to high HLA diversity, lower binding affinities, low frequencies of ex vivo antigen-specific CD4 T cells and several bottlenecks in production and peptide exchange of MHCII monomers. Here we use peptide-loaded MHCII (pMHCII) proteins multimerized on a barcode- and fluorophore-labelled dextran backbone to provide a method for the detection of peptide-specific CD4 T cells by using a large display of MHCII-associated peptides. We have established a protocol for MHCII production and peptide-exchange suitable for the generation of large libraries of peptide-MHCII complexes. We validate the use of such pMHCII complexes in the form of barcode-labelled MHCII multimers to detect antigen-specific CD4 T cells. We demonstrate that we can identify antigen specific CD4 T cells, using these DNA barcoded peptide-MHCII multimer. The multimer bound CD4 T cells were selected based on the fluorochrome signal, and the co-attached DNA barcodes were hereafter amplified and used to identify the peptide-MHCII response/binding. In cases where the peptide-specific CD4 T cells frequencies are very low, we expanded the cell population with peptide-pools and in the presence of IL2. The given CD4 T cell populations hereby reach a cell number allowing for the DNA-barcoded pMHCII multimers to detect responses otherwise missed out. Applying this technology, we utilized a panel of 150 peptides derived from human cytomegalo virus (CMV), Epstein barr virus (EBV), Influenza (Flu), SARS CoV 2 and SARS CoV1, Hepatitis B virus (HBV), and Hepatitis C virus (HCV) loaded onto HLA-DRB1*01:01 and DRB1*04:01 to screen peripheral blood mononuclear cells (PBMC). We assessed ex vivo responses in 16 participants with HCV infection, and successfully detected naturally occurring viral-specific CD4 T cells at frequencies as low as 0.004% of total CD4 T cells. The low-frequency responses, identified via the barcode screen, were rigorously validated using individual fluorophore-labelled tetramer staining after a peptide-driven expansion in 15 participants. Furthermore, we assessed the recognition of novel HCV epitopes in 11 additional participants. Through this, we identified a total of 12 distinct HCV epitopes, including 9 that have not been previously utilized in assays to detect CD4 T cells. Overall, this barcoded-multimer platform provides a powerful tool for the large-scale discovery of class II epitopes and the broad profiling of CD4 T cell specificities. This method will allow for in-depth analyses of immune interactions, provide a better understanding of the antigen-driven associations between CD4 and CD8 T cell responses, and help dissect the complexities of CD4 T cell protection in HCV infection.

ImportanceThe persistence of SARS-CoV-2 antibodies may be a predictive correlate of protection for both natural infections and vaccinations. Identifying predictors of robust antibody responses is important to evaluate the risk of re-infection / vaccine failure and may be translatable to vaccine effectiveness. ObjectiveTo 1) determine the durability of anti-SARS-CoV-2 IgG and neutralizing antibodies in subjects who experienced mild and moderate to severe COVID-19, and 2) to evaluate the correlation of age and IgG responses to both endemic human seasonal coronaviruses (HCoVs) and SARS-CoV-2 according to infection outcome. DesignLongitudinal serum samples were collected from PCR-confirmed SARS-CoV-2 positive participants (U.S. active duty service members, dependents and military retirees, including a range of ages and demographics) who sought medical treatment at seven U.S. military hospitals from March 2020 to March 2021 and enrolled in a prospective observational cohort study. ResultsWe observed SARS-CoV-2 seropositivity in 100% of inpatients followed for six months (58/58) to one year (8/8), while we observed seroreversion in 5% (9/192) of outpatients six to ten months after symptom onset, and 18% (2/11) of outpatients followed for one year. Both outpatient and inpatient anti-SARS-CoV-2 binding-IgG responses had a half-life (T1/2) of >1000 days post-symptom onset. The magnitude of neutralizing antibodies (geometric mean titer, inpatients: 378 [246-580, 95% CI] versus outpatients: 83 [59-116, 95% CI]) and durability (inpatients: 65 [43-98, 95% CI] versus outpatients: 33 [26-40, 95% CI]) were associated with COVID-19 severity. Older age was a positive correlate with both higher IgG binding and neutralizing antibody levels when controlling for COVID-19 hospitalization status. We found no significant relationships between HCoV antibody responses and COVID-19 clinical outcomes, or the development of SARS-CoV-2 neutralizing antibodies. Conclusions and RelevanceThis study demonstrates that humoral responses to SARS-CoV-2 infection are robust on longer time-scales, including those arising from milder infections. However, the magnitude and durability of the antibody response after natural infection was lower and more variable in younger participants who did not require hospitalization for COVID-19. These findings support vaccination against SARS-CoV-2 in all suitable populations including those individuals that have recovered from natural infection.

BackgroundGestational diabetes mellitus (GDM) affects 15.6% of pregnancies globally, with Vietnam exhibiting one of the highest prevalences at 21%. Current diagnostic approaches at 24-28 weeks limit early intervention opportunities. We developed a multi-modal machine learning framework integrating cell-free DNA (cfDNA) structural features and genetic information for early GDM prediction at 10-12 weeks of gestation in Vietnamese women. MethodsWe analyzed blood samples from 1,086 pregnant women (435 GDM cases, 651 controls) collected at 9-12 weeks. Two parallel analytical pathways were employed: cfDNA profiling extracting cfDNA-specific features (fragment length, end motifs, GC content, nucleosome patterns), and whole-genome imputation generating predictions for [~]19,000 omics traits. Component scores were developed using TabPFN classifier and integrated via logistic regression into a unified master score. ResultsGenome-wide analysis identified five omics traits with significant GDM associations: HSD11B1, NEK7, COMMD10, KLRC4, and OCEL1. Component score optimization revealed distinct patterns--cfDNA scores peaked at 200 features (AUC=71.53), while genetics-based scores improved with up to 2,000 omics traits (AUC=77.21). The final master score, integrating three components (gbSC2000, gbSCBH, cfSC200), achieved AUCs of 86.82 - 87.19 across validation cohorts with 70% sensitivity and 89% specificity. Addition-deletion analysis confirmed that both cfDNA and genetic components provided essential, non-redundant contributions. ConclusionsThis multi-modal framework demonstrates superior performance compared to single-biomarker approaches, enabling risk stratification from very low (4% GDM prevalence) to very high risk (90% prevalence). At the cutoff 0.4, the model identifies 78% of future GDM cases at 10-12 weeks while maintaining an 18% false-positive rate, potentially enabling early interventions to prevent GDM development and associated complications.

Omicron, the most recent SARS-CoV-2 variant of concern (VOC), harbours multiple mutations in the spike protein that were not observed in previous VOCs. Initial studies suggest Omicron to substantially reduce the neutralizing capability of antibodies induced from vaccines and previous infection. However, its effect on T cell responses remains to be determined. Here, we assess the effect of Omicron mutations on known T cell epitopes and report data suggesting T cell responses to remain broadly robust against this new variant.

Treatment response in Type 2 Diabetes Mellitus (T2DM) is highly heterogeneous and difficult to predict using conventional clinical markers. Here, we apply digital-twin modeling of generalised metabolic fluxes (GMFs) to 60 T2DM patients treated with Dapagliflozin, Metformin, or their combination. GMF profiling revealed distinct post-treatment metabolic signatures, with Dapagliflozin exerting stronger effects on hepatic and renal fluxes, whereas Metformin and combination therapy strongly modulates haemoglobin glycation. Within each treatment arm, GMF-based clustering stratifies responders and non-responders, with baseline creatinine concentration emerging as a key determinant of glycemic benefit. Proteomic analyses corroborated these sub-groups, showing concordant differences in pathways linked to insulin resistance and inflammation. Longitudinal GMFs further captured the waning glycemic durability of Metformin through progressive increases in Glucose[->]HbA1c flux. Together, these results establish GMF digital twins as a mechanistic framework to dissect drug-specific effects, stratify heterogeneous responses, and project therapeutic durability in T2DM, offering a new avenue for precision metabolic medicine.

Glioblastoma (GB) is a fatal disease in which most targeted therapies have clinically failed. However, pharmacological reactivation of tumor suppressors has not been thoroughly studied as yet as a GB therapeutic strategy. Tumor suppressor Protein Phosphatase 2A (PP2A), is inhibited by non-genetic mechanisms in GB, and thus it would be potentially amendable for therapeutic reactivation. Here we demonstrate, that small molecule activators of PP2A (SMAPs), NZ-8-061 and DBK-1154, effectively cross the in vitro model of blood-brain barrier (BBB), and in vivo partition to mouse brain tissue after oral dosing. In vitro, SMAPs exhibit robust cell killing activity against five established GB cell lines, and nine patient-derived primary glioma cell lines. Collectively these cell lines have heterogenous genetic background, kinase inhibitor resistance profile, and stemness properties; and they represent different clinical GB subtypes. Oral dosing of either of the SMAPs significantly reduced growth of infiltrative intracranial GB tumors. DBK-1154, with both higher degree of brain/blood distribution, and more potent in vitro activity against all tested GB cell lines, also significantly increased survival of mice bearing orthotopic GB xenografts. In summary, this report presents a proof-of-principle data for BBB-permeable tumor suppressor reactivation therapy for glioblastoma cells of heterogenous molecular background.

While the public health burden of SARS-CoV-2 infection has lessened due to natural and vaccine-acquired immunity, the emergence of less virulent variants, and antiviral medications, COVID-19 continues to take a significant toll. There are > 10,000 new hospitalizations per week in the U.S., many of whom develop post-acute sequelae of SARS-CoV-2 (PASC), or "long COVID", with long-term health issues and compromised quality of life. Early identification of individuals at high risk of severe COVID-19 is key for monitoring and supporting respiratory status and improving outcomes. Therefore, precision tools for early detection of patients at high risk of severe disease can reduce morbidity and mortality. Here we report an untargeted and longitudinal metabolomic study of plasma derived from adult patients with COVID-19. One-carbon metabolism, a pathway previously shown as critical for viral propagation and disease progression, and a potential target for COVID-19 treatment, scored strongly as differentially abundant in patients with severe COVID-19. A follow-up targeted metabolite profiling revealed that one arm of the one-carbon metabolism pathway, the methionine cycle, is a major driver of the metabolic profile associated with disease severity. The methionine cycle produces S-adenosylmethionine (SAM), the methyl group donor important for methylation of DNA, RNA, and proteins, and its high abundance was reported to correlate with disease severity. Further, genomic data from the profiled patients revealed a genetic contributor to methionine metabolism and identified the C677T allele of the MTHFR gene as a pre-existing predictor of disease trajectory - patients homozygous for the MTHFR C677T have higher incidence of experiencing severe disease. Our results raise the possibility that screening for the common genetic MTHFR variant may be an actionable approach to stratify risk of COVID severity and may inform novel precision COVID-19 treatment strategies.

Background Platinum refractory paediatric germ cell tumours (GCTs) carry a poor prognosis, with five year survival below 30% and no validated molecular stratification tool. The biological mechanisms underlying platinum resistance in this population remain poorly defined, limiting the development of targeted therapeutic strategies and early warning biomarkers. Methods We performed integrated plasma multi-omics profiling in 105 pediatric GCT patients (54 refractory and 51 treatment naive) using data-independent acquisition proteomics, untargeted metabolomics, and exploratory lipidomics. Candidate biomarkers were validated using ELISA and spatial multiplex immunofluorescence. Predictive models were constructed using logistic regression and evaluated by ROC analysis, calibration, and decision-curve analysis. Results Multiomics integration has revealed the coordinated dysregulation of sphingolipid metabolism, extracellular matrix remodeling, and immune checkpoint signaling in refractory diseases. Lipidomic analysis demonstrated a significant depletion of sphingolipid associated species, including lysophosphatidylserine, lysophosphatidylethanolamine, and phosphatidylserine. Proteomic profiling identified the upregulation of LAG3 and HTRA1, which was validated by ELISA. Multiplex immunofluorescence demonstrated the spatial enrichment of exhausted CD8 + LAG3 T cells adjacent to CK-PAN tumor cells in refractory tumors. A plasma biomarker panel integrating LAG3, HTRA1, and AFP showed improved discrimination of refractory disease (AUC = 0.821) compared with AFP alone. Conclusions Our study identified a sphingolipid HTRA1 LAG3 immune evasion axis as a defining molecular feature of refractory pediatric germ cell tumors and proposed a clinically applicable plasma biomarker panel for early risk stratification.

The identification of predictive biomarkers for patient treatment response is urgently needed to increase the probability of success of existing and novel experimental therapies. Single-cell profiling has provided novel biological insights into drug responses in the tumor microenvironment, but its potential for biomarker discovery has not been fully explored for therapeutic purposes. We describe a novel approach to discover predictive response biomarkers from single-cell data from a small patient cohort using the T cell receptor sequence intrinsic to each T cell to match clonotypes between pre- and post-treatment tumor samples. As a result, we have identified a predictive gene expression signature for immune checkpoint blockade and validated its predictive performance using data from three larger clinical studies. Our results demonstrated that applying clonotyping with single-cell genomic profiling is a promising novel approach for biomarker identification that does not require data collected from large patient cohorts. This could increase success rates, reduce clinical trial size, and significantly impact future clinical developments of immunomodulatory therapeutics.

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.