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  • Single-cell analysis by mass cytometry reveals metabolic states of early-activated CD8(+) T cells during the primary immune response IMMUNITY Levine, L. S., Hiam-Galvez, K. J., Marquez, D. M., Tenvooren, I., Madden, M. Z., Contreras, D. C., Dahunsi, D. O., Irish, J. M., Oluwole, O. O., Rathmell, J. C., Spitzer, M. H. 2021; 54 (4): 829-+


    Memory T cells are thought to rely on oxidative phosphorylation and short-lived effector T cells on glycolysis. Here, we investigated how T cells arrive at these states during an immune response. To understand the metabolic state of rare, early-activated T cells, we adapted mass cytometry to quantify metabolic regulators at single-cell resolution in parallel with cell signaling, proliferation, and effector function. We interrogated CD8+ T cell activation in vitro and in response to Listeria monocytogenes infection in vivo. This approach revealed a distinct metabolic state in early-activated T cells characterized by maximal expression of glycolytic and oxidative metabolic proteins. Cells in this transient state were most abundant 5 days post-infection before rapidly decreasing metabolic protein expression. Analogous findings were observed in chimeric antigen receptor (CAR) T cells interrogated longitudinally in advanced lymphoma patients. Our study demonstrates the utility of single-cell metabolic analysis by mass cytometry to identify metabolic adaptations of immune cell populations in vivo and provides a resource for investigations of metabolic regulation of immune responses across a variety of applications.

    View details for DOI 10.1016/j.immuni.2021.02.018

    View details for Web of Science ID 000640102800020

    View details for PubMedID 33705706

    View details for PubMedCentralID PMC8046726

  • Systemic immunity in cancer NATURE REVIEWS CANCER Hiam-Galvez, K. J., Allen, B. M., Spitzer, M. H. 2021; 21 (6): 345-359


    Immunotherapy has revolutionized cancer treatment, but efficacy remains limited in most clinical settings. Cancer is a systemic disease that induces many functional and compositional changes to the immune system as a whole. Immunity is regulated by interactions of diverse cell lineages across tissues. Therefore, an improved understanding of tumour immunology must assess the systemic immune landscape beyond the tumour microenvironment (TME). Importantly, the peripheral immune system is required to drive effective natural and therapeutically induced antitumour immune responses. In fact, emerging evidence suggests that immunotherapy drives new immune responses rather than the reinvigoration of pre-existing immune responses. However, new immune responses in individuals burdened with tumours are compromised even beyond the TME. Herein, we aim to comprehensively outline the current knowledge of systemic immunity in cancer.

    View details for DOI 10.1038/s41568-021-00347-z

    View details for Web of Science ID 000638496700001

    View details for PubMedID 33837297

    View details for PubMedCentralID PMC8034277

  • Systemic dysfunction and plasticity of the immune macroenvironment in cancer models NATURE MEDICINE Allen, B. M., Hiam, K. J., Burnett, C. E., Venida, A., DeBarge, R., Tenvooren, I., Marquez, D. M., Cho, N., Carmi, Y., Spitzer, M. H. 2020; 26 (7): 1125-+


    Understanding of the factors governing immune responses in cancer remains incomplete, limiting patient benefit. In this study, we used mass cytometry to define the systemic immune landscape in response to tumor development across five tissues in eight mouse tumor models. Systemic immunity was dramatically altered across models and time, with consistent findings in the peripheral blood of patients with breast cancer. Changes in peripheral tissues differed from those in the tumor microenvironment. Mice with tumor-experienced immune systems mounted dampened responses to orthogonal challenges, including reduced T cell activation during viral or bacterial infection. Antigen-presenting cells (APCs) mounted weaker responses in this context, whereas promoting APC activation rescued T cell activity. Systemic immune changes were reversed with surgical tumor resection, and many were prevented by interleukin-1 or granulocyte colony-stimulating factor blockade, revealing remarkable plasticity in the systemic immune state. These results demonstrate that tumor development dynamically reshapes the composition and function of the immune macroenvironment.

    View details for DOI 10.1038/s41591-020-0892-6

    View details for Web of Science ID 000535417400001

    View details for PubMedID 32451499

    View details for PubMedCentralID PMC7384250

  • Divergent clonal differentiation trajectories of T cell exhaustion. Nature immunology Daniel, B., Yost, K. E., Hsiung, S., Sandor, K., Xia, Y., Qi, Y., Hiam-Galvez, K. J., Black, M., J Raposo, C., Shi, Q., Meier, S. L., Belk, J. A., Giles, J. R., Wherry, E. J., Chang, H. Y., Egawa, T., Satpathy, A. T. 2022


    Chronic antigen exposure during viral infection or cancer promotes an exhausted T cell (Tex) state with reduced effector function. However, whether all antigen-specific T cell clones follow the same Tex differentiation trajectory remains unclear. Here, we generate a single-cell multiomic atlas of T cell exhaustion in murine chronic viral infection that redefines Tex phenotypic diversity, including two late-stage Tex subsets with either a terminal exhaustion (Texterm) or a killer cell lectin-like receptor-expressing cytotoxic (TexKLR) phenotype. We use paired single-cell RNA and T cell receptor sequencing to uncover clonal differentiation trajectories of Texterm-biased, TexKLR-biased or divergent clones that acquire both phenotypes. We show that high T cell receptor signaling avidity correlates with Texterm, whereas low avidity correlates with effector-like TexKLR fate. Finally, we identify similar clonal differentiation trajectories in human tumor-infiltrating lymphocytes. These findings reveal clonal heterogeneity in the T cell response to chronic antigen that influences Tex fates and persistence.

    View details for DOI 10.1038/s41590-022-01337-5

    View details for PubMedID 36289450

  • MARCH1 Controls an Exhaustion-like Program of Effector CD4+ T Cells Promoting Allergic Airway Inflammation. ImmunoHorizons Castellanos, C. A., Hiam-Galvez, K. J., Ishido, S., Satpathy, A. T., Shin, J. 2022; 6 (9): 684-692


    Persistent antigenic signaling leads to T cell exhaustion, a dysfunctional state arising in many chronic infections and cancers. Little is known concerning mechanisms limiting exhaustion in immune-stimulatory diseases such as asthma. We report that membrane-associated RING-CH1 (MARCH1), the ubiquitin ligase that mediates surface turnover of MHC class II (MHCII) and CD86 in professional APCs, plays an essential role in restraining an exhaustion-like program of effector CD4+ T cells in a mouse model of asthma. Mice lacking MARCH1 or the ubiquitin acceptor sites of MHCII and CD86 exhibited increased MHCII and CD86 surface expression on lung APCs, and this increase promoted enhanced expression of immune-inhibitory receptors by effector CD4+ T cells and inhibited their proliferation. Remarkably, ablation of MARCH1 in mice with established asthma reduced airway infiltration of eosinophils and Th2 cells. Thus, MARCH1 controls an exhaustion-like program of effector CD4+ T cells during allergic airway inflammation and may serve as a therapeutic target for asthma.

    View details for DOI 10.4049/immunohorizons.2200056

    View details for PubMedID 36100368

  • BCL6-dependent TCF-1+ progenitor cells maintain effector and helper CD4+ Tcell responses to persistent antigen. Immunity Xia, Y., Sandor, K., Pai, J. A., Daniel, B., Raju, S., Wu, R., Hsiung, S., Qi, Y., Yangdon, T., Okamoto, M., Chou, C., Hiam-Galvez, K. J., Schreiber, R. D., Murphy, K. M., Satpathy, A. T., Egawa, T. 2022


    Soon after activation, CD4+ Tcells are segregated into BCL6+ follicular helper (Tfh) and BCL6- effector (Teff) Tcells. Here, we explored how these subsets are maintained during chronic antigen stimulation using the mouse chronic LCMV infection model. Using single cell-transcriptomic and epigenomic analyses, we identified a population of PD-1+ TCF-1+ CD4+ Tcells with memory-like features. TCR clonal tracing and adoptive transfer experiments demonstrated that these cells have self-renewal capacity and continue to give rise to both Teff and Tfh cells, thus functioning as progenitor cells. Conditional deletion experiments showed Bcl6-dependent development of these progenitors, which were essential for sustaining antigen-specific CD4+ Tcell responses to chronic infection. An analogous CD4+ Tcell population developed in draining lymph nodes in response to tumors. Our study reveals the heterogeneity and plasticity of CD4+ Tcells during persistent antigen exposure and highlights their population dynamics through a stable, bipotent intermediate state.

    View details for DOI 10.1016/j.immuni.2022.05.003

    View details for PubMedID 35637103

  • BCL6-dependent TCF-1+progenitor cells maintain effector and helper CD4 T cell responses to persistent antigen Xia, Y., Sandor, K., Pai, J. A., Daniel, B., Raju, S., Wu, R., Hsiung, S., Qi, Y., Yangdon, T., Okamoto, M., Schreiber, R. D., Murphy, K. M., Satpathy, A., Egawa, T. AMER ASSOC IMMUNOLOGISTS. 2022
  • The effect of low-dose IL-2 and Treg adoptive cell therapy in patients with 1 diabetes JCI INSIGHT Dong, S., Hiam-Galvez, K. J., Mowery, C. T., Herold, K. C., Gitelman, S. E., Esensten, J. H., Liu, W., Lares, A. P., Leinbach, A. S., Lee, M., Nguyen, V., Tamaki, S. J., Tamaki, W., Tamaki, C. M., Mehdizadeh, M., Putnam, A. L., Spitzer, M. H., Ye, C., Tang, Q., Bluestone, J. A. 2021; 6 (18)


    BACKGROUNDA previous phase I study showed that the infusion of autologous Tregs expanded ex vivo into patients with recent-onset type 1 diabetes (T1D) had an excellent safety profile. However, the majority of the infused Tregs were undetectable in the peripheral blood 3 months postinfusion (Treg-T1D trial). Therefore, we conducted a phase I study (TILT trial) combining polyclonal Tregs and low-dose IL-2, shown to enhance Treg survival and expansion, and assessed the impact over time on Treg populations and other immune cells.METHODSPatients with T1D were treated with a single infusion of autologous polyclonal Tregs followed by one or two 5-day courses of recombinant human low-dose IL-2 (ld-IL-2). Flow cytometry, cytometry by time of flight, and 10x Genomics single-cell RNA-Seq were used to follow the distinct immune cell populations' phenotypes over time.RESULTSMultiparametric analysis revealed that the combination therapy led to an increase in the number of infused and endogenous Tregs but also resulted in a substantial increase from baseline in a subset of activated NK, mucosal associated invariant T, and clonal CD8+ T cell populations.CONCLUSIONThese data support the hypothesis that ld-IL-2 expands exogenously administered Tregs but also can expand cytotoxic cells. These results have important implications for the use of a combination of ld-IL-2 and Tregs for the treatment of autoimmune diseases with preexisting active immunity.TRIAL NCT01210664 (Treg-T1D trial), NCT02772679 (TILT trial).FUNDINGSean N. Parker Autoimmune Research Laboratory Fund, National Center for Research Resources.

    View details for DOI 10.1172/jci.insight.147474

    View details for Web of Science ID 000709017600001

    View details for PubMedID 34324441

    View details for PubMedCentralID PMC8492314

  • Global absence and targeting of protective immune states in severe COVID-19. Nature Combes, A. J., Courau, T., Kuhn, N. F., Hu, K. H., Ray, A., Chen, W. S., Chew, N. W., Cleary, S. J., Kushnoor, D., Reeder, G. C., Shen, A., Tsui, J., Hiam-Galvez, K. J., Munoz-Sandoval, P., Zhu, W. S., Lee, D. S., Sun, Y., You, R., Magnen, M., Rodriguez, L., Im, K. W., Serwas, N. K., Leligdowicz, A., Zamecnik, C. R., Loudermilk, R. P., Wilson, M. R., Ye, C. J., Fragiadakis, G. K., Looney, M. R., Chan, V., Ward, A., Carrillo, S., UCSF COMET Consortium, Matthay, M., Erle, D. J., Woodruff, P. G., Langelier, C., Kangelaris, K., Hendrickson, C. M., Calfee, C., Rao, A. A., Krummel, M. F., Cathy, C., Zhan, J., Samad, B., Chak, S., Ghale, R., Giberson, J., Gonzalez, A., Jauregui, A., Lee, D., Nguyen, V., Yee, K., Abe-Jones, Y., Pierce, L., Prasad, P., Sinha, P., Beagle, A., Lea, T., Esmalii, A., Sigman, A., Ortiz, G. M., Raffel, K., Jones, C., Liu, K., Eckalbar, W. 2021; 591 (7848): 124-130


    Although infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has pleiotropic and systemic effects in some individuals1-3, many others experience milder symptoms. Here, to gain a more comprehensive understanding of the distinction between severe and mild phenotypes in the pathology of coronavirus disease 2019 (COVID-19) and its origins, we performed a whole-blood-preserving single-cell analysis protocol to integrate contributions from all major immune cell types of the blood-including neutrophils, monocytes, platelets, lymphocytes and the contents of the serum. Patients with mild COVID-19 exhibit a coordinated pattern of expression of interferon-stimulated genes (ISGs)3 across every cell population, whereas these ISG-expressing cells are systemically absent in patients with severe disease. Paradoxically, individuals with severe COVID-19 produce very high titres of anti-SARS-CoV-2 antibodies and have a lower viral load compared to individuals with mild disease. Examination of the serum from patients with severe COVID-19 shows that these patients uniquely produce antibodies that functionally block the production of the ISG-expressing cells associated with mild disease, by activating conserved signalling circuits that dampen cellular responses to interferons. Overzealous antibody responses pit the immune system against itself in many patients with COVID-19, and perhaps also in individuals with other viral infections. Our findings reveal potential targets for immunotherapies in patients with severe COVID-19 to re-engage viral defence.

    View details for DOI 10.1038/s41586-021-03234-7

    View details for PubMedID 33494096

  • Global Absence and Targeting of Protective Immune States in Severe COVID-19. bioRxiv : the preprint server for biology Combes, A. J., Courau, T., Kuhn, N. F., Hu, K. H., Ray, A., Chen, W. S., Cleary, S. J., Chew, N. W., Kushnoor, D., Reeder, G. C., Shen, A., Tsui, J., Hiam-Galvez, K. J., Munoz-Sandoval, P., Zhu, W. S., Lee, D. S., Sun, Y., You, R., Magnen, M., Rodriguez, L., Leligdowicz, A., Zamecnik, C. R., Loudermilk, R. P., Wilson, M. R., Ye, C. J., Fragiadakis, G. K., Looney, M. R., Chan, V., Ward, A., Carrillo, S., Matthay, M., Erle, D. J., Woodruff, P. G., Langelier, C., Kangelaris, K., Hendrickson, C. M., Calfee, C., Rao, A. A., Krummel, M. F. 2020


    While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a whole-blood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferon-stimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and auto-directed antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense.ONE SENTENCE SUMMARY: In severe COVID-19 patients, the immune system fails to generate cells that define mild disease; antibodies in their serum actively prevents the successful production of those cells.

    View details for DOI 10.1101/2020.10.28.359935

    View details for PubMedID 33140050

  • Human pediatric B-cell acute lymphoblastic leukemias can be classified as B-1 or B-2-like based on a minimal transcriptional signature EXPERIMENTAL HEMATOLOGY Fitch, B., Roy, R., Geng, H., Montecino-Rodriguez, E., Bengtsson, H., Gaillard, C., Hiam, K., Casero, D., Olshen, A. B., Dorshkind, K., Kogan, S. C. 2020; 90: 65-+


    The finding that transformed mouse B-1 and B-2 progenitors give rise to B-cell acute lymphoblastic leukemias (B-ALLs) with varied aggressiveness suggests that B-cell lineage might also be a factor in the initiation and progression of pediatric B-ALLs in humans. If this is the case, we hypothesized that human pediatric B-ALLs would share gene expression patterns with mouse B-1 or B-2 progenitors. We tested this premise by deriving a distinct 30-gene B-1 and B-2 progenitor signature that was applied to a microarray data set of human pediatric ALLs. Cluster analysis revealed that CRLF2, E2A-PBX1, ERG, and ETV6-RUNX1 leukemias were B-1-like, whereas BCR-ABL1, hyperdiploid, and MLL leukemias were B-2-like. Examination of the 30-gene signature in two independent data sets of pediatric ALLs supported this result. Our data suggest that common genetic subtypes of human ALL have their origin in the B-1 or B-2 lineage.

    View details for DOI 10.1016/j.exphem.2020.09.184

    View details for Web of Science ID 000583223100008

    View details for PubMedID 32946981

    View details for PubMedCentralID PMC7606616

  • ImmunoGlobe: enabling systems immunology with a manually curated intercellular immune interaction network. BMC bioinformatics Atallah, M. B., Tandon, V., Hiam, K. J., Boyce, H., Hori, M., Atallah, W., Spitzer, M. H., Engleman, E., Mallick, P. 2020; 21 (1): 346


    BACKGROUND: While technological advances have made it possible to profile the immune system at high resolution, translating high-throughput data into knowledge of immune mechanisms has been challenged by the complexity of the interactions underlying immune processes. Tools to explore the immune network are critical for better understanding the multi-layered processes that underlie immune function and dysfunction, but require a standardized network map of immune interactions. To facilitate this we have developed ImmunoGlobe, a manually curated intercellular immune interaction network extracted from Janeway's Immunobiology textbook.RESULTS: ImmunoGlobe is the first graphical representation of the immune interactome, and is comprised of 253 immune system components and 1112 unique immune interactions with detailed functional and characteristic annotations. Analysis of this network shows that it recapitulates known features of the human immune system and can be used uncover novel multi-step immune pathways, examine species-specific differences in immune processes, and predict the response of immune cells to stimuli. ImmunoGlobe is publicly available through a user-friendly interface at and can be downloaded as a computable graph and network table.CONCLUSION: While the fields of proteomics and genomics have long benefited from network analysis tools, no such tool yet exists for immunology. ImmunoGlobe provides a ground truth immune interaction network upon which such tools can be built. These tools will allow us to predict the outcome of complex immune interactions, providing mechanistic insight that allows us to precisely modulate immune responses in health and disease.

    View details for DOI 10.1186/s12859-020-03702-3

    View details for PubMedID 32778050

  • Depletion of microbiome-derived molecules in the host using Clostridium genetics. Science (New York, N.Y.) Guo, C., Allen, B. M., Hiam, K. J., Dodd, D., Van Treuren, W., Higginbottom, S., Nagashima, K., Fischer, C. R., Sonnenburg, J. L., Spitzer, M. H., Fischbach, M. A. 2019; 366 (6471)


    The gut microbiota produce hundreds of molecules that are present at high concentrations in the host circulation. Unraveling the contribution of each molecule to host biology remains difficult. We developed a system for constructing clean deletions in Clostridium spp., the source of many molecules from the gut microbiome. By applying this method to the model commensal organism Clostridium sporogenes, we knocked out genes for 10 C. sporogenes-derived molecules that accumulate in host tissues. In mice colonized by a C. sporogenes for which the production of branched short-chain fatty acids was knocked out, we discovered that these microbial products have immunoglobulin A-modulatory activity.

    View details for DOI 10.1126/science.aav1282

    View details for PubMedID 31831639

  • Identification of Preferred DNA-Binding Sites for the Thermus thermophilus Transcriptional Regulator SbtR by the Combinatorial Approach REPSA. PloS one Van Dyke, M. W., Beyer, M. D., Clay, E., Hiam, K. J., McMurry, J. L., Xie, Y. 2016; 11 (7): e0159408


    One of the first steps towards elucidating the biological function of a putative transcriptional regulator is to ascertain its preferred DNA-binding sequences. This may be rapidly and effectively achieved through the application of a combinatorial approach, one involving very large numbers of randomized oligonucleotides and reiterative selection and amplification steps to enrich for high-affinity nucleic acid-binding sequences. Previously, we had developed the novel combinatorial approach Restriction Endonuclease Protection, Selection and Amplification (REPSA), which relies not on the physical separation of ligand-nucleic acid complexes but instead selects on the basis of ligand-dependent inhibition of enzymatic template inactivation, specifically cleavage by type IIS restriction endonucleases. Thus, no prior knowledge of the ligand is required for REPSA, making it more amenable for discovery purposes. Here we describe using REPSA, massively parallel sequencing, and bioinformatics to identify the preferred DNA-binding sites for the transcriptional regulator SbtR, encoded by the TTHA0167 gene from the model extreme thermophile Thermus thermophilus HB8. From the resulting position weight matrix, we can identify multiple operons potentially regulated by SbtR and postulate a biological role for this protein in regulating extracellular transport processes. Our study provides a proof-of-concept for the application of REPSA for the identification of preferred DNA-binding sites for orphan transcriptional regulators and a first step towards determining their possible biological roles.

    View details for DOI 10.1371/journal.pone.0159408

    View details for PubMedID 27428627

    View details for PubMedCentralID PMC4948773