Research

Projects

Clinical Immunology

Vaccine studies

Vaccine studies provide a unique opportunity to explore human immunology through a timed exposure to a known stimulus. Our vaccine studies include several influenza vaccination studies conducted at Stanford, as well as in collaboration with industry. We also collaborate with the HIV Vaccine Trial Network to analyze samples from subjects around the world enrolled in HIV vaccine trials. For each of these studies, we perform deep immunoprofiling by CyTOF of PBMCs from subjects pre- and post-vaccination. Our goal is to understand the role of NK cells during a vaccine response, whether beneficial or harmful to a positive vaccine outcome. This will shed light on how NK cells respond to vaccines, and whether certain vaccination methods generate a more beneficial NK response. This work is primarily supported by the National Institutes of Health.

COVID-19 clinical immunology and pathogenesis

Dr. Blish directs the safe processing of samples for Stanford’s COVID-19 Biobank. This involves the majority of the lab members processing and coordinating patient specimens (e.g. whole blood) for research use across the campus and across the country. Special protocols are used to preserve cells for functional studies, and to support NIH multi-site studies. Samples come from the hospital and urgent care settings. For the Blish lab, the goal of these studies is to identify pathways that are altered in COVID-19 patients’ immune cells and then test these pathways in BSL2 and BSL3 settings, in order to better understand how patients get sick, and how we can best treat or prevent the disease. (Wilk, Rustagi, Zhao et al. medRxiv 2020)

Microbial Pathogenesis

SARS-CoV-2 virology and pathogenesis

Dr. Blish directs Stanford’s SARS-CoV-2 (the causative agent of COVID-19) biosafety level 3 (BSL3) research program. Lab members complete the NIH BSL3 training program in order to safely perform experiments with infectious SARS-CoV-2 in the one lab space on campus approved for BSL3 work. These experiments involve growing the virus and performing infections in cell culture and primary tissue models, and studying the results by imaging and single-cell techniques. Promising drug candidates, an urgent human health need, are also screened against the virus in relevant infection models. This work is primarily funded by the Bill and Melinda Gates Foundation and the Chan Zuckerberg Biohub.

Innate Immune Cell Biology

Understanding NK cells at the single cell level

Human natural killer (NK) cells were once thought to be a largely homogeneous population of innate immune cells, responsible for rapid identification and killing of virus-infected or cancerous cells. Through research in the Blish lab, it was revealed that NK cells are not as homogeneous as once thought, and that the diversity of the NK repertoire within an individual is astounding. This led to our focus on understanding NK cell biology at the single cell level, using many research methods to understand the diversity and breadth of an individual’s immune response. Our single cell methods include CyTOF, spectral flow cytometry, and single cell RNA-sequencing. This work is primarily supported by the National Institutes of Health, the Burroughs Wellcome Foundation, and the Bill and Melinda Gates Foundation. (Horowitz et al. SciTransMed 2013)

Single-cell immune profiling in acute viral infections

Our primary focus when researching NK cells at the single cell level is understanding the NK response to viral infections. We work with acute and chronic infection cohorts to determine the in vivo NK response to infection, including studies with dengue, influenza, Zika virus, and HIV. In order to understand the biological mechanisms at play when an NK cell responds to a virus-infected cell, we use in vitro co-cultures of primary NK cells and infected target cells to dissect NK cell responses.

Our studies of NK cell responses to viral infection would not be complete without studying the flip side, and focusing on the infected cell itself. Our goal is to understand the pathways by which different target cells, particularly monocytes and T cells, respond to infection and to elucidate the communication pathways between the cells that are infected, bystander cells, and NK cells. This allows us to dissect mechanisms that drive NK recognition, and also reveal mechanisms by which viruses seek to evade NK cell responses.

The dynamic intercellular communication between different immune cell subsets in the setting of viral infections remains poorly understood despite its impact on disease resolution and pathology. Using a combination of single-cell proteomic, transcriptomic, and epigenomic profiling from several cohorts of primary infections, the Blish lab has begun unraveling the complex cell-cell cooperativity networks in the setting of influenza, dengue, HIV, SIV, and SARS-CoV-2 infection. This work is primarily supported by the National Institutes of Health, the Burroughs Wellcome Foundation, and the Bill and Melinda Gates Foundation. (McKechnie et al. Frontiers in cellular and infection microbiology 2019) (Zhao et al. Frontiers in Immunology 2020) (Kronstad et al. JI 2018) (Bayless et al. Cell host & microbe 2016)

Methods development

NK cells are highly resistant to standard transfection techniques, which presents a substantial barrier to understanding the mechanisms of NK cell-target cell recognition. In collaboration with Paul Wender (Stanford Chemistry), we have developed a novel bio-orthogonal transfection technique using Charge-Altering Releasable Transporters (CARTs), a class of biodegradable and dynamic delivery vectors capable of transfecting a wide variety of cargo. We are using CARTs to manipulate NK cells with minimal off-target effects in order to study the contributions of individual NK cell receptors in target cell recognition. This work is primarily supported by the Chan Zuckerberg Biohub and the National Institutes of Health. (Wilk, Benner, et al. bioRxiv 2020)

Publications

George E. and Lucy Becker Professor in Medicine

Publications

Prior vaccination prevents overactivation of innate immune responses during COVID-19 breakthrough infection. Science translational medicine Chan, L., Pinedo, K., Stabile, M. A., Hamlin, R. E., Pienkos, S. M., Ratnasiri, K., Yang, S., Blomkalns, A. L., Nadeau, K. C., Pulendran, B., O'Hara, R., Rogers, A. J., Holmes, S. P., Blish, C. A. 2025; 17 (783): eadq1086 More

Abstract

At this stage in the COVID-19 pandemic, most infections are "breakthrough" infections that occur in individuals with prior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure. To refine long-term vaccine strategies against emerging variants, we examined both innate and adaptive immunity in breakthrough infections. We performed single-cell transcriptomic, proteomic, and functional profiling of primary and breakthrough infections to compare immune responses from unvaccinated and vaccinated individuals during the SARS-CoV-2 Delta wave. Breakthrough infections were characterized by a less activated transcriptomic profile in monocytes and natural killer cells, with induction of pathways limiting monocyte migratory potential and natural killer cell proliferation. Furthermore, we observed a female-specific increase in transcriptomic and proteomic activation of multiple innate immune cell subsets during breakthrough infections. These insights suggest that prior SARS-CoV-2 vaccination prevents overactivation of innate immune responses during breakthrough infections with discernible sex-specific patterns and underscore the potential of harnessing vaccines in mitigating pathologic immune responses resulting from overactivation.

View details for DOI 10.1126/scitranslmed.adq1086

View details for PubMedID 39879318
Sex differences and immune correlates of Long Covid development, symptom persistence, and resolution. Science translational medicine Hamlin, R. E., Pienkos, S. M., Chan, L., Stabile, M. A., Pinedo, K., Rao, M., Grant, P., Bonilla, H., Holubar, M., Singh, U., Jacobson, K. B., Jagannathan, P., Maldonado, Y., Holmes, S. P., Subramanian, A., Blish, C. A. 2024; 16 (773): eadr1032 More

Abstract

Sex differences have been observed in acute coronavirus disease 2019 (COVID-19) and Long Covid (LC) outcomes, with greater disease severity and mortality during acute infection in males and greater proportions of females developing LC. We hypothesized that sex-specific immune dysregulation contributes to LC pathogenesis. To investigate the immunologic underpinnings of LC development and symptom persistence, we performed multiomic analyses on blood samples obtained during acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and 3 and 12 months after infection in a cohort of 45 participants who either developed LC or recovered. Several sex-specific immune pathways were associated with LC. Males who would later develop LC exhibited increases in transforming growth factor-β (TGF-β) signaling during acute infection, whereas females who would go on to develop LC had reduced TGFB1 expression. Females who developed LC demonstrated increased expression of XIST, an RNA gene implicated in autoimmunity, during acute infection compared with females who recovered. Many immune features of LC were also conserved across sexes, such as alterations in monocyte phenotype and activation state. Nuclear factor κB (NF-κB) transcription factors were up-regulated in many cell types at acute and convalescent time points. Those with ongoing LC demonstrated reduced ETS1 expression across lymphocyte subsets and elevated intracellular IL-4 in T cell subsets, suggesting that ETS1 alterations may drive aberrantly elevated T helper cell 2-like responses in LC. Altogether, this study describes multiple innate and adaptive immune correlates of LC, some of which differ by sex, and offers insights toward the pursuit of tailored therapeutics.

View details for DOI 10.1126/scitranslmed.adr1032

View details for PubMedID 39536117
Comparative analysis of cell-cell communication at single-cell resolution. Nature biotechnology Wilk, A. J., Shalek, A. K., Holmes, S., Blish, C. A. 2023 More

Abstract

Inference of cell-cell communication from single-cell RNA sequencing data is a powerful technique to uncover intercellular communication pathways, yet existing methods perform this analysis at the level of the cell type or cluster, discarding single-cell-level information. Here we present Scriabin, a flexible and scalable framework for comparative analysis of cell-cell communication at single-cell resolution that is performed without cell aggregation or downsampling. We use multiple published atlas-scale datasets, genetic perturbation screens and direct experimental validation to show that Scriabin accurately recovers expected cell-cell communication edges and identifies communication networks that can be obscured by agglomerative methods. Additionally, we use spatial transcriptomic data to show that Scriabin can uncover spatial features of interaction from dissociated data alone. Finally, we demonstrate applications to longitudinal datasets to follow communication pathways operating between timepoints. Our approach represents a broadly applicable strategy to reveal the full structure of niche-phenotype relationships in health and disease.

View details for DOI 10.1038/s41587-023-01782-z

View details for PubMedID 37169965

View details for PubMedCentralID 8104132
SARS-CoV-2 escapes direct NK cell killing through Nsp1-mediated downregulation of ligands for NKG2D. Cell reports Lee, M. J., Leong, M. W., Rustagi, A., Beck, A., Zeng, L., Holmes, S., Qi, L. S., Blish, C. A. 2022: 111892 More

Abstract

Natural killer (NK) cells are cytotoxic effector cells that target and lyse virally infected cells; many viruses therefore encode mechanisms to escape such NK cell killing. Here, we interrogate the ability of SARS-CoV-2 to modulate NK cell recognition and lysis of infected cells. We find that NK cells exhibit poor cytotoxic responses against SARS-CoV-2-infected targets, preferentially killing uninfected bystander cells. We demonstrate that this escape is driven by downregulation of ligands for the activating receptor NKG2D (NKG2D-L). Indeed, early in viral infection, prior to NKG2D-L downregulation, NK cells are able to target and kill infected cells; however, this ability is lost as viral proteins are expressed. Finally, we find that SARS-CoV-2 non-structural protein 1 (Nsp1) mediates downregulation of NKG2D-L and that Nsp1 alone is sufficient to confer resistance to NK cell killing. Collectively, our work demonstrates that SARS-CoV-2 evades direct NK cell cytotoxicity and describes a mechanism by which this occurs.

View details for DOI 10.1016/j.celrep.2022.111892

View details for PubMedID 36543165
NKp30 and NKG2D contribute to natural killer cell-mediated recognition of HIV-infected cells. iScience Pi, R., Zhao, N. Q., Bien, A. J., Ranganath, T., Seiler, C., Holmes, S., Marson, A., Nguyen, D. N., Blish, C. A. 2025; 28 (10): 113548 More

Abstract

Natural killer (NK) cells respond rapidly in early HIV-1 infection. HIV-1 prevention and control strategies harnessing NK cells could be enabled by mechanistic understanding of how NK cells recognize HIV-infected T cells. Here, we profiled the phenotype of human primary NK cells responsive to autologous newly HIV-infected CD4 T cells in vitro. We characterized the patterns of NK cell ligand expression on CD4 T cells at baseline and after infection with a panel of transmitted/founder HIV-1 strains to identify key receptor-ligand pairings. CRISPR editing of CD4 T cells to knock out the NKp30 ligand B7-H6, or the NKG2D ligand MICB reduced NK cell responses to HIV-infected cells in some donors. Blockade of NKp30 or NKG2D on NK cells compromised their specificity of killing HIV-infected cells. Collectively, we identified receptor-ligand pairs including NKp30:B7-H6 and NKG2D:MICB that contribute to NK cell recognition of HIV-infected cells.

View details for DOI 10.1016/j.isci.2025.113548

View details for PubMedID 41079618

View details for PubMedCentralID PMC12514549
Circulating endothelial signatures correlate with worse outcomes in COVID-19, respiratory failure and ARDS. Critical care (London, England) Costa Monteiro, A. C., Pickering, H., Sarma, A., Taylor, C. S., Jenkins, M. M., Hsu, F. M., Nadel, B., Levy, O., Baden, L. R., Melamed, E., Ehrlich, L. I., McComsey, G. A., Sekaly, R. P., Cairns, C. B., Haddad, E. K., Shaw, A. C., Hafler, D. A., Montgomery, R. R., Corry, D. B., Kheradmand, F., Atkinson, M. A., Brakenridge, S. C., Higuita, N. I., Metcalf, J. P., Hough, C. L., Messer, W. B., Pulendran, B., Nadeau, K. C., Davis, M. M., Geng, L. N., Fernandez-Sesma, A., Simon, V., Krammer, F., Kraft, M., Bime, C., Calfee, C. S., Erle, D. J., Bosinger, S., Eckalbar, W., Maecker, H., Rahman, A., Guan, L., Peters, B., Kleinstein, S. H., Augustine, A. D., Diray-Arce, J., Becker, P. M., Rouphael, N., Agus, M., Kulkarni, H., Schaenmann, J. M., Salehi-Rad, R., Matthay, M. A., Reed, E. F., Sapru, A. 2025; 29 (1): 432 More

Abstract

Elevated circulating endothelial cells (CECs), released from monolayers after insult, have been implicated in worse outcomes in ARDS and COVID-19, however there is no consensus proteomic phenotype that define CECs. We queried whether a transcriptomic approach would alternatively support the presence of endothelial cells in circulation and correlate with worsening respiratory failure.To test whether elevated endothelial cell signatures (ECS) in circulation plays a role in worse respiratory outcomes, we used unsupervised bulk-transcriptome deconvolution to quantify ECS% in two cohorts. Our pilot analysis included pediatric patients requiring invasive mechanical ventilation (CAF-PINT, NCT01892969). Our validation cohort included adult hospitalized patients with COVID-19 (IMPACC, NCT04378777), testing the association of ECS% to outcomes in patients at risk of acute respiratory failure/ARDS. Primary outcome was 28-day mortality.In CAF-PINT, day 0 ECS% was higher in non-survivors compared to survivors of respiratory failure (2.8%, IQR 2.4-3.4% versus 2.6%, IQR 2.2-3.0% n = 244, p < 0.05, Wilcoxon rank-sum). In IMPACC, baseline ECS% (< 72 h of hospitalization) was higher in COVID-19 non-survivors versus survivors (2.9%, IQR 2.6-3.4%, versus 2.7%, IQR 2.3-3.1%, n = 932, p < 0.001, Wilcoxon rank-sum). Each 1% increase in baseline ECS% was significantly associated with mortality (adjusted OR 1.36, CI 1.03-1.79) by multivariable logistic regression. Increased baseline ECS% was associated with worse respiratory trajectories (2.5%, IQR 2.2-2.8% for trajectory with no oxygen requirements, 2.9%, IQR 2.6-3.4% for the trajectory with fatal outcome by day 28, n = 932, p < 0.001, one-way ANOVA).Quantifying ECS by deconvolution supports a transcriptomics-driven approach towards the non-invasive evaluation of endothelial damage in respiratory outcomes. This is a first step towards elucidating mechanistic components linking endothelial damage to ARDS utilizing non-invasive, circulating transcriptomic data by leveraging a novel deconvolution approach.

View details for DOI 10.1186/s13054-025-05596-0

View details for PubMedID 41088445

View details for PubMedCentralID PMC12522733
Olfactory Dysfunction After SARS-CoV-2 Infection in the RECOVER Adult Cohort. JAMA network open Horwitz, L. I., Becker, J. H., Huang, W., Akintonwa, T., Hornig-Rohan, M. M., Maranga, G., Adams, D. R., Albers, M. W., Ayache, M., Berry, J., Brim, H., Bryan, T. W., Charney, A. W., Clark, R. A., Cortez, M. M., D'Anza, B., Davis, H., Donohue, S. E., Erdmann, N., Flaherman, V., Fong, T. G., Frontera, J. A., Goldberg, M. P., Goldman, J. D., Harkins, M. S., Hodder, S. L., Jacoby, V. L., Jagannathan, P., Jia, X., Kelly, J. D., Krishnan, J. A., Kumar, A., Laiyemo, A. O., Levitan, E. B., Martin, J. N., McCaffrey, K. M., McComsey, G. A., Metz, T. D., Murthy, G., Nguyen, H., Okumura, M., Parry, S., Parthasarathy, S., Patterson, T. F., Peluso, M. J., Sorochinsky, C., Walker, T., Wiegand, S. L., Wiley, Z., Wisnivesky, J., Ashktorab, H., Foulkes, A., Lee-Iannotti, J. K. 2025; 8 (9): e2533815 More

Abstract

Olfactory dysfunction is common after SARS-CoV-2 infection and has been associated with cognitive loss in other conditions. Formal testing is needed to characterize the presence, severity, and patterns of olfactory dysfunction.To characterize long-term olfactory dysfunction after SARS-CoV-2 infection.This prospective cohort study included adults enrolled in the Researching COVID to Enhance Recovery (RECOVER)-Adult study. All those with and a random sample of those without self-reported change or loss in smell or taste were offered olfactory testing, performed at 83 sites in 35 US states and territories. Participants included 2956 enrollees with prior infection (1393 with and 1563 without self-reported change or loss) and 569 without prior infection (9 with and 560 without self-reported change or loss in taste) who underwent olfactory testing a mean (SD) of 671.6 (417.8) days after the index date. Data were collected from October 29, 2021, to June 6, 2025.SARS-CoV-2 infection.Olfactory function, as defined by age- and sex-standardized performance on the University of Pennsylvania Smell Identification Test (UPSIT), a well-validated test comprising 40 unique odors.The study included 3525 participants with a mean (SD) age of 47.6 (15.2) years; of 3520 with data available, 2548 (72.4%) were female or intersex. Among 1393 infected participants with self-reported change or loss, 1111 (79.8%) had hyposmia on the UPSIT, including 321 (23.0%) with severe microsmia or anosmia. Among 1563 infected participants without self-reported change or loss, 1031 (66.0%) had hyposmia, including 128 (8.2%) with severe microsmia or anosmia. Participants with prior infection and self-reported change or loss scored at the 16th age- and sex-standardized UPSIT percentile, compared with the 23rd and 28th percentiles for those without self-reported change or loss with and without prior known infection, respectively. Younger women had scores corresponding to lower mean age- and sex-standardized percentiles. Among participants who self-reported change or loss in smell, those with abnormal UPSIT scores more often reported cognitive problems (742 of 1111 [66.8%]) than those with normal UPSIT scores (179 of 282 [63.5%]).In this cohort study of RECOVER-Adult participants, self-reported change or loss in smell or taste was an accurate signal of verified hyposmia, but a high rate of hyposmia among those with no reported change or loss was also observed. Formal smell testing may be considered in those with prior SARS-CoV-2 infection to diagnose occult hyposmia and counsel patients about risks.

View details for DOI 10.1001/jamanetworkopen.2025.33815

View details for PubMedID 40996759
Single-Cell and Plasma Proteomics Do Not Differentiate Patients With and Without SARS-CoV-2 Antigenemia in Convalescence in a Cohort of 100 Patients. Open forum infectious diseases Pienkos, S., Swank, Z., Hamlin, R. E., Rao, M., Grant, P., Bonilla, H., Jacobson, K., Jagannathan, P., Singh, U., Walt, D. R., Subramanian, A., Blish, C. 2025; 12 (9): ofaf515 More

Abstract

Plasma samples obtained approximately 3 (n = 100) and 12 months (n = 78) after acute SARS-CoV-2 infection were tested for S1, spike, and N antigens. There were no significant differences in plasma proteins or single-cell protein expression levels on immune cells between those with and without plasma antigen detected.

View details for DOI 10.1093/ofid/ofaf515

View details for PubMedID 40926879

View details for PubMedCentralID PMC12415173
Membrane-wide screening identifies potential tissue-specific determinants of SARS-CoV-2 tropism. PLoS pathogens Dinesh, R. K., Wang, C., Qu, Y., Rustagi, A., Cousins, H., Zengel, J., Wang, X., Barnard, T. R., Johnson, W. A., Xu, G., Zhang, T., Magazine, N., Beck, A., Heilbroner, L. M., Peters-Schulze, G., Wilk, A. J., Wang, M., Huang, W., Howitt, B., Carette, J., Altman, R., Blish, C. A., Cong, L. 2025; 21 (7): e1013157 More

Abstract

While SARS-CoV-2 primarily infects the respiratory tract, clinical evidence indicates that cells from diverse cell types and organs are also susceptible to infection. Using the CRISPR activation (CRISPRa) approach, we systematically targeted human membrane proteins in cells with and without overexpression of ACE2, thus identifying unrecognized host factors that may facilitate viral entry. Validation experiments with replication-competent SARS-CoV-2 confirmed the role of newly identified host factors, particularly the endo-lysosomal protease legumain (LGMN) and the potassium channel KCNA6, upon exogenous overexpression. In orthogonal experiments, we show that disruption of endogenous LGMN or KCNA6 decreases viral infection and that inhibitors of candidate factors can reduce viral entry. Additionally, using clinical data, we find possible associations between expression of either LGMN or KCNA6 and SARS-CoV-2 infection in human tissues. Our results identify potentially druggable host factors involved in SARS-CoV-2 entry, and demonstrate the utility of focused, membrane-wide CRISPRa screens in uncovering tissue-specific entry factors of emerging pathogens.

View details for DOI 10.1371/journal.ppat.1013157

View details for PubMedID 40674406
scMetaIntegrator: a meta-analysis approach to paired single-cell differential expression analysis. bioRxiv : the preprint server for biology Ratnasiri, K., Mach, S. N., Blish, C. A., Khatri, P. 2025 More

Abstract

Traditional differential gene expression methods are limited for analysis of single cell RNA-sequencing (scRNA-seq) studies that use paired repeated measures and matched cohort designs. Many existing approaches consider cells as independent samples, leading to high false positive rates while ignoring inherent sampling structures. Although pseudobulk methods address this, they ignore intra-sample expression variability and have higher false negatives rates. We propose a novel meta-analysis approach that accounts for biological replicates and cell variability in paired scRNA-seq data. Using both real and synthetic datasets, we show that our method, single-cell MetaIntegrator (https://github.com/Khatri-Lab/scMetaIntegrator), provides robust effect size estimates and reproducible p-values.

View details for DOI 10.1101/2025.06.04.657898

View details for PubMedID 40501846

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Vaccine studies

COVID-19 clinical immunology and pathogenesis

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Understanding NK cells at the single cell level

Single-cell immune profiling in acute viral infections

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