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
Rewiring STAT signaling from the cell surface with Trikine immunotherapeutics. Science (New York, N.Y.) Rodriguez, G. E., Zhao, Y., Nishiga, Y., Peprah, F., Shen, J., Abhiraman, G. C., Ogishi, M., Zhang, C., Saco, J., Waghray, D., Serasanambati, M., Torres, L., Simone, B. W., Su, L., Wilson, S. C., Yang, A., Sun, Q., Picton, L., Saxton, R. A., Bhandarkar, V., Lee, M. J., Andrews, E., Jiang, H., Obenaus, M., Yen, M., Atajanova, T., Blish, C. A., Spranger, S., Wherry, E. J., Kirane, A., Ribas, A., Raulet, D. H., Kalbasi, A., Dougan, S. K., Dougan, M., Sage, J., Garcia, K. C. 2026: eadx9954 More

Abstract

Cytokines dimerize two receptor chains to activate Janus kinases and STAT transcription factors that regulate immune cells but have therapeutic liabilities. We engineered "Trikines" to compel cis formation of three-chain cytokine receptor complexes at the cell surface that induce bespoke STAT transcriptional signaling programs. Trikines co-activated pSTAT5 and pSTAT3 signatures distinct from natural cytokines, by assembling trimeric combinations of Interleukin-2 (IL-2), Interleukin-10 (IL-10), and Interleukin-21 (IL-21) receptors. In pre-clinical models, an IL-2-based-Trikine restrained terminal differentiation of T cells, promoted stemness, and enhanced durability of tumor control without observable toxicity. An IL-10-based Trikine induced immune infiltration into poorly immunogenic tumors, showing efficacy in pre-clinical models of small cell lung cancer and pancreatic cancer. Trikines obviate the need for cell engineering to customize STAT signatures and may hold potential for immunotherapy.

View details for DOI 10.1126/science.adx9954

View details for PubMedID 41712697
Interferon stimulation and NKG2D expression drive enhanced natural killer cell antibody-dependent cellular cytotoxicity against viral infections. Journal of leukocyte biology Chan, L., Pinedo, K., Yang, S., Blomkalns, A. L., Nadeau, K. C., Rogers, A. J., Blish, C. A. 2026 More

Abstract

Natural killer (NK) cell antibody-dependent cellular cytotoxicity (ADCC) contributes to effective antiviral immunity, yet the relative contribution of NK cell-intrinsic factors and antibodies in mediating these responses remain poorly understood. Here, we combined functional ADCC assays with single-cell transcriptomics of peripheral NK cells from COVID-19 participants. Our analysis revealed distinct transcriptional programs between participants with different ADCC response levels: NK cells from participants with lower ADCC responses upregulated proliferation pathways, while those with high ADCC responses showed enhanced expression of interferon-stimulated genes and NKG2D. Blocking NKG2D significantly reduced NK cell ADCC degranulation and cytokine responses. Paradoxically, greater interferon-mediated NK cell activation was associated with reduced proficiency of participants' antibodies to mediate ADCC, suggesting a regulatory checkpoint mechanism. These findings enhance our understanding of the molecular determinants of ADCC responses and provide novel insights into leveraging these responses for more effective vaccination and therapeutic strategies.

View details for DOI 10.1093/jleuko/qiag019

View details for PubMedID 41634919
Machine learning models predict long COVID outcomes based on baseline clinical and immunologic factors. Communications medicine Doni Jayavelu, N., Samaha, H., Wimalasena, S. T., Hoch, A., Gygi, J. P., Gabernet, G., Ozonoff, A., Liu, S., Milliren, C. E., Levy, O., Baden, L. R., Melamed, E., Ehrlich, L. I., McComsey, G. A., Sekaly, R. P., Cairns, C. B., Haddad, E. K., Schaenman, J., Shaw, A. C., Hafler, D. A., Montgomery, R. R., Corry, D. B., Kheradmand, F., Atkinson, M. A., Brakenridge, S. C., Agudelo Higuit, N. I., Metcalf, J. P., Hough, C. L., Messer, W. B., Pulendran, B., Nadeau, K. C., Davis, M. M., Gen, L. N., Fernandez Sesma, A., Simon, V., Krammer, F., Kraft, M., Bime, C., Calfee, C. S., Erle, D. J., Langelier, C. R., Guan, L., Maecker, H. T., Peters, B., Kleinstein, S. H., Reed, E. F., Augustine, A. D., Diray-Arce, J., Becker, P. M., Rouphael, N., Altman, M. C. 2026; 6 (1): 1 More

Abstract

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.

View details for DOI 10.1038/s43856-025-01230-w

View details for PubMedID 41484172

View details for PubMedCentralID PMC12764860
Interferon-mediated NK cell activation increases cytolytic activity against T follicular helper cells and limits antibody response to SARS-CoV-2. Nature immunology de Los Rios Kobara, I., Jayewickreme, R., Lee, M. J., Wilk, A. J., Blomkalns, A. L., Nadeau, K. C., Yang, S., Rogers, A. J., Blish, C. A. 2025 More

Abstract

Natural killer (NK) cells are innate lymphocytes known for their ability to kill infected or malignant cells, but they have an overlooked role in regulating antibody responses. In mice, NK cells can kill T follicular helper (TFH) cells, decreasing somatic hypermutation and antibody titers. Although human NK cell activation correlates with poor vaccine response, the mechanisms of NK cell regulation of adaptive immunity in humans are poorly understood. Here we found that, in ancestral severe acute respiratory syndrome coronavirus 2 infection, individuals with the broadest neutralization profile had fewer NK cells that expressed inhibitory and immaturity markers, whereas NK cells from narrow neutralizers were highly activated and expressed interferon-stimulated genes. ISG-mediated activation in NK cells from healthy donors increased cytotoxicity toward induced TFH-like cells via NKG2D and NKp30. This work reveals that NK cell activation and dysregulated inflammation play a role in poor antibody response to severe acute respiratory syndrome coronavirus 2 and opens exciting avenues for designing improved vaccines and adjuvants to target emerging pathogens.

View details for DOI 10.1038/s41590-025-02341-1

View details for PubMedID 41272165

View details for PubMedCentralID 7169933
Dysregulated regulatory T cell response in immunosuppressed individuals is associated with increased inflammation in COVID-19 breakthrough infections Chan, L., Pinedo, K., Stabile, M., Hamlin, R., Pienkos, S., Yang, S., Blomkalns, A. L., Nadeau, K., O'hara, R., Rogers, A. J., Pulendran, B., Agbor-Enoh, S., Blish, C. A. OXFORD UNIV PRESS. 2025 More

View details for DOI 10.1093/jimmun/vkaf283.490

View details for Web of Science ID 001627214000001
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

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Vaccine studies

COVID-19 clinical immunology and pathogenesis

SARS-CoV-2 virology and pathogenesis

Understanding NK cells at the single cell level

Single-cell immune profiling in acute viral infections

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