The cellular, immunogenetic, and antigenic factors affecting the breadth of viral antigen variants recognized by human antibody responses are poorly defined. We developed highly multiplexed panels of DNA-tagged SARS-CoV-2 antigens from up to 20 viral variants to label and sort 6,262 antigen-binding circulating B cells from previously naive mRNA vaccinees or infected patients, and from deceased organ donor lymphoid tissues, to enable antigen receptor and transcriptome sequencing. Atypical B cells and a subset of class-switched memory cells with evidence of recent germinal center exposure were enriched for antigen binding. In contrast to atypical B cells, post-germinal center B cells showed progressively increasing variant binding breadth and somatic hypermutation over time. Vaccination, compared with infection, preferentially stimulated B cells expressing antibodies with inherently high antigen-binding breadth. This large-scale analysis reveals key determinants of antigen-binding breadth, critical for understanding responses to viral infection and guiding vaccine development against rapidly mutating viruses.

Tumor-associated macrophages (TAM) exert essential functions during the immune response to cancer. However, investigations of TAM within a native human tumor microenvironment (TME) have been impeded by a lack of appropriate model systems. Here, patient-derived organoids (PDO) from air-liquid interface (ALI)-grown tumor fragments, containing a human TME that encompassed stroma and immune subsets, robustly preserved TAM that were maintained by endogenous CSF-1 and appropriately responded to polarization signals. Antibody blockade of the CD47 regulatory checkpoint in organoids stimulated phagocytosis and remodeled TAM cytokine secretion profiles that were confirmed in anti-CD47 phase I trial patients. Amongst PDO histologies screened, anti-CD47 tumor killing was notable in clear cell renal cell carcinoma (ccRCC) which was associated with increased TAM infiltration. PDO contained diverse previously described TAM subsets; however, anti-CD47 reprogrammed organoid TAM toward an immunosuppressive SPP1+ phenotype, highlighting a negative feedback mechanism. Our findings uncover a resistance circuit engaged by macrophage checkpoint blockade and position ALI PDO as a robust translational platform for dissecting human macrophage biology and informing precision immunotherapy.

Developing cancer therapies that induce specific death of malignant cells is critical for preventing relapse. Highly effective strategies, such as immunotherapy, exemplify this principle. Here, we provide the mechanistic basis for a small-molecule approach that leverages chemically induced proximity (CIP) to kill diffuse large B cell lymphoma, the most common non-Hodgkin lymphoma. We developed lysine acetyltransferase (KAT)-based TCIPs (transcriptional/epigenetic chemical inducers of proximity), or KAT-TCIPs, which redirect p300/CREB-binding protein (CBP) to activate cell-death networks repressed by the oncogenic driver BCL6. Our lead KAT-TCIP reprograms the epigenome to initiate apoptosis. The crystal structure of the chemically induced p300-BCL6 complex reveals how chance protein-protein interactions may be exploited to confer the potency and selectivity of KAT-TCIPs. Thus, oncogenic drivers can be co-opted to activate robust cell death. Consistent with their gain-of-function mechanism, TCIPs recruiting different transcriptional activators—p300, BRD4, or CDK9—produce distinct genomic responses, suggesting specialized therapeutic uses.

Chronic viral infections are ubiquitous in humans, with individuals carrying multiple viruses that can reactivate during physiological stress, including severe illness1. Notably, SARS-CoV-2 infection has been shown to reactivate chronic viruses such as Epstein-Barr virus and cytomegalovirus, yet the full extent, temporal dynamics and immunological impact of viral reactivation in COVID-19 remain incompletely understood2-7. Here, leveraging multi-omic longitudinal data from 1,154 hospitalized patients with COVID-19 from the Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) study, we reveal significant reactivation of Herpesviridae and Anelloviridae during acute COVID-19, with distinct temporal dynamics for different viruses, and demonstrate that reactivation correlates with disease severity, host immune effects and clinical outcomes. Although our results do not establish causation between virus reactivation and clinical outcomes, we highlight the prevalence of chronic viral reactivation during acute COVID-19 and long COVID. Our findings challenge the prevailing view that chronic viral reactivation is primarily a consequence of immunosuppression, demonstrating that reactivations occur frequently in immunocompetent individuals during severe illness and in association with increased systemic inflammation. Additionally, we demonstrate persistence of viral reactivation in convalescence, and report an association of Anelloviridae with long COVID. This study provides immune, transcriptomic and metabolomic signatures of viral reactivation that could inform future strategies to prognosticate and treat acute COVID-19 and long COVID.

Human vaccine responses vary widely, but the determinants remain incompletely defined. Here we analyzed 66 cytokines across four inactivated influenza vaccine (IIV) cohorts over five seasons (n = 581) and identified baseline serum interleukin (IL)-18 and interferon (IFN)-β as correlates of day 28 antibody responses. To test causality, we evaluated 19 cytokines in human tonsil and spleen organoids and found that type I IFNs, IL-21 and IL-12, but not IL-18 or IFNγ, enhanced antibody production. The addition of IFNβ to IIV recapitulated key features of the live-vaccine cytokine program. IL-12 and IL-21 defined a parallel pathway independent of type I IFNs, with IL-12 inducing IL-21 in humans, unlike in mice. Delivery of IL-21 or IFNβ via mRNA lipid nanoparticles in vivo promoted long-lived plasma cell formation. Together, these findings define parallel pathways that regulate vaccine immunity. Our approach unites high-throughput organoid testing and human cohort studies, establishing a human-centric platform to identify adjuvant candidates.

Tissue-resident immunity mediates host defense against pathogens and enables rapid adaptive memory responses. However, the study of tissue-resident immunity is hindered by a singular lack of experimental systems allowing pathogenic epithelial infection amidst the full spectrum of endogenous immune subsets. Particularly in lung, differing notions of transient versus sustained residency of tissue-resident memory T cells (TRM) have questioned the extent to which recall immunity to respiratory pathogens occurs locally or in concert with secondary lymphoid organs. We thus generated long-term adult human distal lung organoids from intact tissue fragments in 3D air-liquid interface (ALI) culture that co-preserved epithelial and stromal architecture alongside endogenous lung-resident immune cells (T, B, NK, myeloid). The organoid T cells exhibited persistent cytokine-assisted maintenance, expressed residency and memory markers, and preserved T cell receptor (TCR) repertoires of cognate fresh tissue. SARS-CoV-2 vigorously infected the organoid lung epithelium, stimulated inflammatory cytokine production, and crucially, induced widespread SARS-CoV-2-specific, tissue-resident T cell responses. Our studies introduce a robust adult human lung organoid experimental system containing a physiologic air interface and diverse resident immune subsets, demonstrate the organ-autonomous sufficiency of lung pathogen memory T cell responses, distinct from secondary lymphoid tissue, and provide a platform to investigate tissue-resident immunity in health and disease.

Cardiovascular disease remains the leading cause of mortality worldwide, yet substantial risk persists beyond traditional clinical and metabolic predictors. The immune system is a key mediator of this residual risk, but clinically scalable metrics of immune state are lacking. Here, we established the clinical and prognostic relevance of IMM-AGE, a system-level metric of immune aging derived from immune cell correlation structure. We developed a transcriptomic gene-ratio signature and optimized reduced-marker flow cytometry panels that accurately preserve IMM-AGE across blood fractions, platforms and cohorts. Applying these clinic-ready implementations across population-based and disease-specific datasets, we show that elevated IMM-AGE is consistently associated with cardiovascular phenotypes and disease. We leverage the UK biobank to show that incorporation of IMM-AGE into the PREVENT 10-year risk equation increase accuracy of risk stratification. We also show that in elderly patients undergoing transcatheter aortic valve replacement, baseline IMM-AGE independently predicted early maladaptive cardiac remodeling and one-year mortality. Finally, in the Baseline Health Study, a large longitudinal cohort, IMM-AGE stratified cardiovascular event risk among individuals with otherwise similar clinical profiles. Together, these findings establish immune aging as a transferable, biologically grounded risk dimension and support IMM-AGE as a practical tool for precision cardiovascular risk assessment.

The post-acute sequelae of SARS-CoV-2 (PASC), also known as long COVID, remain a significant health issue that is incompletely understood. Predicting which acutely infected individuals will develop long COVID is challenging due to the absence of established biomarkers, clear disease mechanisms, or well-defined sub-phenotypes. Machine learning (ML) models may address this gap by leveraging clinical data to enhance diagnostic precision. Clinical data, including antibody titers and viral load measurements collected at the time of hospital admission, are used to predict the likelihood of acute COVID-19 progressing to long COVID. Machine learning models are trained and evaluated for predictive performance. Feature importance analysis is performed to identify the most influential predictors. The machine learning models achieve median AUROC values ranging from 0.64 to 0.66 and AUPRC values between 0.51 and 0.54, demonstrating predictive capabilities. Low antibody titers and high viral loads at hospital admission emerge as the strongest predictors of long COVID outcomes. Comorbidities—such as chronic respiratory, cardiac, and neurologic diseases—and female sex are also identified as significant risk factors. Machine learning models identify patients at risk for developing long COVID based on baseline clinical characteristics. These models guide early interventions, improve patient outcomes, and mitigate the long-term public health impacts of SARS-CoV-2. Long COVID, or post-acute sequelae of SARS-CoV-2, is a prolonged health condition that can occur after acute COVID-19 infection. However, the ability to predict who will develop long COVID remains limited due to the absence of clear tests or biomarkers. We looked at patients’ medical information, including the amount of virus in their body at hospital admission, and how strong their immune response was. Using computer programs that can find hidden patterns in large sets of data, we discovered that people with a weaker immune response, higher amounts of virus, certain long term health problems and women are more likely to develop long COVID. This study highlights that computer-based tools could help doctors identify high-risk patients early and provide care that may prevent long-term complications. Jayavelu, Samaha et al., apply machine learning models on hospital admission data, including antibody titers and viral load, to identify patients at high risk for Long COVID. Low antibody levels, high viral loads, chronic diseases, and female sex are key predictors, supporting early, targeted interventions.

Azithromycin is a widely used antibiotic and was frequently used to treat hospitalized patients during the COVID-19 pandemic. The impact of empiric azithromycin use on the respiratory microbiome in patients with viral respiratory infections is unclear. Here we used longitudinal metatranscriptomics on nasal swabs from a prospective multicentre cohort of 1,164 patients hospitalized for COVID-19. We compared the upper respiratory microbiome, resistome and systemic immune response in patients treated with azithromycin (n = 366) with those who received no antibiotics (n = 474) or other antibiotics (n = 324). We found that azithromycin altered microbiome composition and increased the expression and relative proportion of macrolide/lincosamide/streptogramin (MLS) resistance genes. These changes occurred after 1 day of exposure and persisted for over a week. MLS resistance gene expression was associated with commensals and potential pathogens, while there were no differences in host inflammatory gene expression in blood and airways. This demonstrates that empiric azithromycin treatment impacts the upper respiratory microbiome and resistome without apparent anti-inflammatory benefit.

The establishment of mixed hematopoietic chimerism is a promising way to induce immune tolerance for islet replacement therapy and to treat the underlying autoimmunity in Type 1 diabetes (T1D). Mixed chimerism not only promotes effective thymic negative selection of autoreactive cells but also restores regulatory T cell (Treg) function and peripheral tolerance. In the current study, we determined that a novel class of donor-derived CD8+CD44+CD122+ Tregs (d-CD8+CD122+ Tregs) plays a crucial role in controlling autoimmunity in non-obese diabetic (NOD) mice with induced mixed chimerism. Using adoptive T cell transfer experiments, we showed that d-CD8+CD122+ Tregs abrogate autoimmunity by selectively depleting the exogenously injected diabetogenic T cells in Recombination-Activating Gene deficient NOD mice. These d-CD8+CD122+ Tregs from NOD chimeras show upregulation of Helios, Programmed cell death protein 1, perforin, granzyme-B, CD39, Folate receptor 4, and downregulation of proinflammatory markers like Scart1 and Scart2. Using in vitro assays, we show that d-CD8+CD122+ Tregs respond specifically to a Complementarity-Determining Region-3 peptide sequence derived from T cell receptors of islet antigen-specific autoreactive T cells. Thus, mixed chimerism might be a method to revitalize CD8+CD122+ Tregs which are decreased in number and functionality in NOD mice. Similarly, we found that individuals with T1D have a deficiency in CD8+CD122+ Tregs, suggesting a potential loss of regulatory function accompanies disease onset. Revitalizing CD8+CD122+ Tregs may offer a new therapeutic strategy of restoring immune tolerance in autoimmune diabetes.

Inflammatory bowel disease (IBD), encompassing ulcerative colitis (UC) and Crohn’s disease (CD), is marked by chronic intestinal inflammation and dysregulated immunity. Although UC and CD affect different areas of the gastrointestinal tract, both diseases share aberrant CD4+ memory T cell responses, with HLA-DRB1 as a major genetic risk factor. HLA-DRB1 encodes MHC class II molecules that influence the CD4+ T cell receptor (TCR) repertoire, yet how these genotypes shape TCR specificity in IBD remains unclear. Here, we genotyped HLA-DRB1 and profiled 3.13 million TCRβ sequences from circulating memory CD4+ T cells in 33 IBD patients (20 UC, 13 CD) and 14 healthy controls. Using the GLIPH2 algorithm, we distilled 468,441 candidates based on CDR3 amino acid motifs into 440 high-confidence TCR specificity groups significantly enriched among individuals sharing HLA-DRB1 alleles. Notably, 5 specificity groups were IBD-enriched and were shared between UC and CD, suggesting common antigen targets in both diseases. We also observed increased frequencies of clonally expanded cytotoxic GZMB+PRF1+ memory CD4+ T cells and KIR+CD8+ T cells in a subset of risk-allele carriers with IBD. These findings elucidate distinct, HLA-linked TCR specificity groups in IBD and provide mechanistic insights that may advance antigen discovery and personalized medicine.

Type 1 diabetes (T1D) is caused by T cell-mediated autoimmune destruction of insulin-producing islet β-cells. Treatment with T-cell depleting therapies delays the progression of stage 2 and 3 T1D, but these agents exert broad immunosuppressive effects on T cell populations, including T regulatory cells (Tregs), which are key in promoting immune tolerance. We evaluated non-obese diabetic (NOD) mice and recently diagnosed T1D patients and identified CD38 as a marker for pathogenic T cell populations. Using adoptive T-cell transfer in Recombination Activating Gene 1 knockout NOD mice and in a humanized mouse model of autoimmune diabetes, we demonstrated that CD38-expressing autoreactive T cells drive diabetes pathogenesis. Furthermore, we found that selective depletion of CD38+ cells, using an anti-CD38 monoclonal antibody (mAb), prevents insulitis and diabetes onset without depleting CD4+CD25+ Tregs. Administration of anti-CD38 mAb did not adversely affect islet function and may selectively eliminate immunogenic senescent islet β-cells. These results support the strategy of selectively depleting diabetogenic T cells using an anti-CD38 mAb to treat T1D and restore immune tolerance. Therefore, transient depletion of autoreactive T cells using anti-CD38 mAb may provide a novel strategy to prevent or abrogate β-cell autoimmunity in T1D.

Vaccine effectiveness against malaria is dramatically reduced in malaria-exposed compared to malaria-naïve populations, potentially due to altered immune responses in secondary lymphoid organs following repeated infection. Newly developed human tonsil and spleen organoids, which replicate key features of B and T cell immunity, provide an exciting opportunity to overcome challenges of other models and to improve our understanding of innate-adaptive interactions in lymphoid tissue. The objectives of this study were to use these organoids to investigate the impact of malaria parasites on 1) cells within lymphoid tissues and 2) responses to a heterologous antigen. When we exposed organoids from malaria-naïve donors to Plasmodium falciparum-infected red blood cells (iRBC), we observed that iRBC exposure did not disrupt organoid formation and significantly increased Vδ2 + γδ T cell frequencies in both tonsil and spleen organoids at multiple timepoints. Single-cell RNA/TCR sequencing revealed that iRBC-responsive Vδ2 + T cells in organoids were clonally expanded and exhibited activated, cytotoxic phenotypes with upregulated expression of granzymes, interferon-stimulated genes, and antigen presentation machinery. TCR repertoire analysis demonstrated that malaria exposure drove clonal expansion of cytotoxic Vδ2 + T cells, contrasting with the diverse, smaller clones observed in control conditions. To validate these findings, we analyzed tonsils from Ugandan children with asymptomatic parasitemia and found expanded Vδ2 + T cells with enhanced cytotoxic potential compared to uninfected controls. When we tested whether malaria pre-exposure affected subsequent recall responses to influenza vaccine, malaria pre-exposure or γδ T cell depletion did not significantly alter cellular frequencies or influenza-specific antibody responses in most donors, though modest reductions were observed in some individuals. This work demonstrates the utility of human lymphoid organoids for studying malaria-host interactions and provides novel insights into Vδ2 + T cell biology, including evidence for clonal expansion and cytotoxic differentiation in response to malaria parasites within secondary lymphoid tissues.

A decline in specific antibody responses is a hallmark of human aging, yet the differential contributions of B and T lymphocytes remain unclear. CXCL13 is a chemokine that shapes germinal center (GC) organization, but the regulation of human-specific CXCL13+ T follicular helper (Tfh) cells during aging is not known. Using human tonsil organoids, single-cell RNA sequencing, and CRISPR perturbations, we mapped age-associated changes in Tfh cells, the cell type that provides help to B cells in GCs. Tonsil organoids from older donors generate weaker influenza-specific antibody responses, which we trace to Tfh cell defects rather than B cells. Single-cell profiling revealed a selective loss of mature CXCL13+ GC-Tfh cells accompanied by accumulation of precursor states. Trajectory analysis shows that aging arrests Tfh maturation at the early activated precursor transition, and CRISPR perturbations identify BACH2 and SOX4 as regulators of differentiation reduced with age. These findings reveal a human-specific mechanism of immune aging with implications for strategies to restore humoral immunity.

Despite advances in vaccine and antiviral drug development, the prevention of respiratory viral infection and transmission remains a substantial challenge worldwide. One obvious limitation of these approaches is that they do not provide robust protection at the initial site of infection, which is the respiratory mucosa. Currently, strategies to enhance mucosal immunity against respiratory pathogens remain lacking. Here we engineered mucus-tethering bispecific nanobodies designed to provide the simultaneous neutralization of viruses by binding to their surface proteins and the entrapment of viruses within the mucus by securing them to mucin. Compared with conventional non-mucus-tethering nanobodies, these mucus-tethering bispecific nanobodies demonstrated increased retention in the respiratory tract, provided enhanced protection against influenza viral infection in mice and reduced SARS-CoV-2 transmission in hamsters. Together, our findings represent a promising strategy for enhancing mucosal defences against respiratory viruses by blocking viral entry and limiting onward transmission. Engineered mucus-tethering bispecific nanobodies neutralize and entrap viruses to enhance mucosal immunity, preventing influenza infection and limiting SARS-CoV-2 transmission.

Depletion of pathogenic T and B cells is a pillar of first-line therapies for inflammatory, autoimmune, and transplantation-related immunological diseases. However, concerns about adverse events, safety in immunocompromised patients, and disease relapse from incomplete depletion, limit clinical utility. Here, we exploit the immunosuppressive properties of Transforming growth factor beta (TGF-β), through selective "silencing" of T and B cells by a targeted TGF-β mimic agonist derived from Helminths. CD4 and CD8 T cell-targeted TGF-β agonists effectively silence antigen-stimulated T cell activation and expansion in mice and human spleen organoids. A mouse CD4 T cell-targeted TGF-β agonist silences antigen-specific antibody responses by reprogramming pro-inflammatory Th1 and T follicular helper cells into quiescent or regulatory T cell phenotypes in vivo. A human CD19 B cell-targeted TGF-β agonist silences antibody responses by disrupting differentiation of germinal center B cells into antibody-secreting cells in human spleen organoids. Cell-type-specific targeted TGF-β agonists ameliorate disease activity in multiple mouse models with minimal off-target effects in vivo. Thus, cell-selective TGF-β agonism is a versatile therapeutic strategy for precise silencing of immune functions.

Targeting the HIV-infected reservoir in lymphoid tissues (LT) will be critical to developing a cure for people living with HIV (PLWH). LT explants used to study HIV infection enable the evaluation of human-specific disease progression and treatment response; however, their short lifespan makes it challenging to assess long-term treatment interventions. We therefore established an immune organoid model of HIV infection using human tonsil or spleen cells, demonstrating productive HIV infection and viral integration into CD4+ T cells. Treatment with a protease inhibitor fully suppressed ongoing viral production, with virologic rebound occurring within days of treatment interruption. The transfer of healthy allogeneic NK cells to target the reservoir upon treatment interruption reduced the number of infected cells, intact viral genomes, and production of de novo infectious viral particles. Adoption of this immune organoid platform will accelerate the evaluation of cure-based strategies to eliminate the HIV reservoir in tissues for PLWH.

Abstract Cancer therapies that activate cell death are critical to avoid relapse. Approximately 30% of diffuse large B cell lymphoma (DLBCL) cases, the most common non-Hodgkin lymphoma, fail standard-of-care treatment regimens, highlighting the need for new death-promoting targeted therapies. Here, we introduce a gain-of-function small molecule modality that kills DLBCL cells at sub-nanomolar potency (IC50 = 0.8 nM) through induced proximity. These bivalent compounds, Lysine Acetyltransferase Transcriptional/Epigenetic Chemical Inducers of Proximity (KAT-TCIPs), leverage the endogenous activity of the co-activating KATs E1A Binding Protein p300 (p300) and CREB-Binding Protein (CBP) to drive the transcription of death-promoting genes normally repressed by oncogenes. Specifically, KAT-TCIPs recruit p300/CBP to genomic loci controlled by the master transcriptional repressor BCL6, dysregulated in ∼40% of DLBCL cases, and rapidly reprogram the epigenome to promote BCL6-dependent cell cycle arrest and apoptosis. We report the first X-ray co-crystal structure of a TCIP molecule bound to p300 and BCL6, which guided the optimization of our lead KAT-TCIP, TCIP3. Additional biophysical characterization of TCIP3 revealed its function as a molecular glue that cooperatively seeds ternary complexes on chromatin. This compound exhibits robust preclinical efficacy in vivo. It ablates germinal center B cells, which are naturally enriched for BCL6 expression, in immunized mice (5 mpk bid dosed intraperitoneally) relative to vehicle controls. Additionally, TCIP3 eliminates tumors in DLBCL cell line-derived xenograft models within 11 days at the same dose. Notably, this molecule spares healthy lymphocytes and fibroblasts in cytotoxicity analyses. Collectively, our findings establish KAT-TCIPs as powerful tools for co-opting the malignant function of oncogenic drivers to activate robust cell death, with implications for precision epigenetic therapies. Citation Format: Meredith Nicole Nix, Sai Gourisankar, Sabin Nettles, Kevin Bowman, Haopeng Yang, Brendan G. Dwyer, Roman C. Sarott, Hind Abuzaid, Michael Martinez, Andrey Krokhotin, Lei Chen, Mark M. Davis, Daniel Fernandez, Tinghu Zhang, Michael R. Green, Stephen M. Hinshaw, Nathanael S. Gray, Gerald R. Crabtree. A bivalent molecular glue linking lysine acetyltransferases to oncogene-induced cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3981.