Papers: Graduate Student, Stanford University by Jonathan M Irish
There is compelling evidence from transgenic mouse studies and analysis of mutations in human car... more There is compelling evidence from transgenic mouse studies and analysis of mutations in human carcinomas indicating that the TGF-β signal transduction pathway is tumor suppressive. We have shown that overexpression of TGF-β1 in mammary epithelial cells suppresses the development of carcinomas and that expression of a dominant negative type II TGF-β receptor (DNIIR) in mammary epithelial cells under control of the MMTV promoter/enhancer increases the incidence of mammary carcinomas. Studies of human tumors have demonstrated inactivating mutations in human tumors of genes encoding proteins involved in TGF-β signal transduction, including DPC4/Smad4, Smad2, and the type II TGF-β receptor (TβRII). There is also evidence that TGF-β can enhance the progression of tumors. This hypothesis is being tested in genetically modified mice. To attain complete loss of TβRII, we have generated mice with loxP sites flanking exon 2 of Tgfbr2 and crossed them with mice expressing Cre recombinase under control of the MMTV promoter/enhancer to obtain Tgfbr2 mgKO mice. These mice show lobuloalveolar hyperplasia. Mice are being followed for mammary tumor development. Tgfbr2 mgKO mice that also express polyoma virus middle T antigen under control of the MMTV promoter (MMTV-PyVmT) develop mammary tumors with a significantly shorter latency than MMTV-PyVmT mice and show a marked increase in pulmonary metastases. Our data do not support the hypothesis that TGF-β signaling in mammary carcinoma cells is important for invasion and metastasis, at least in this model system. The importance of stromal-epithelial interactions in mammary gland development and tumorigenesis is well established. These interactions probably involve autocrine and paracrine action of multiple growth factors, including members of the TGF-β family, which are expressed in both stroma and epithelium. Again, to accomplish complete knockout of the type II TGF-β receptor gene in mammary stromal cells, FSP1-Cre and Tgfbr2 flox/flox mice were crossed to attain Tgfbr2 fspKO mice. The Despite over a decade of scrutiny and over 20 published reports from various countries, the degree to which ATM mutations lead to breast References 1. Gatti RA, Tward A, Concannon P: Cancer risk in ATM heterozygotes: a model of phenotypic and mechanistic differences between missense and truncating mutations. Mol Biol Metab 1999, 68:419-423. 2. Spring K, Ahangari F, Scott SP, Waring P, Purdie DM, Chen PC, Hourigan K, et al.: Mice heterozygous for mutation in Atm, the gene involved in ataxia-telangiectasia, have heightened susceptibility to cancer. Nat Genet 2002, 32:185-190. 3. Scott SP, Bendix R, Chen P, Clark R, Dork T, Lavin MF: Missense mutations but not allelic variants alter the function of ATM by dominant interference in patients with breast cancer. Proc Natl Acad Sci USA 2002, 99:925-930. 4. Concannon P: ATM heterozygosity and cancer risk. Nat Genet 2002, 32:89-90. 5. Chenevix-Trench G, Spurdle AB, Gatei M, Kelly H, Marsh A, Chen X, Donn K, et al.: Dominant negative ATM mutations in breast cancer families.
Altered growth factor responses in phospho-protein-driven signaling networks are crucial to cance... more Altered growth factor responses in phospho-protein-driven signaling networks are crucial to cancer cell survival and pathology. Profiles of cancer cell signaling networks might therefore identify mechanisms by which such cells interpret environmental cues for continued growth. Using multiparameter flow cytometry, we monitored phospho-protein responses to environmental cues in acute myeloid leukemia at the single cell level. By exposing cancer cell signaling networks to potentiating inputs, rather than relying upon the basal levels of protein phosphorylation alone, we could discern unique cancer network profiles that correlated with genetics and disease outcome. Strikingly, individual cancers manifested multiple cell subsets with unique network profiles, reflecting cancer heterogeneity at the level of signaling response. The results revealed a dramatic remodeling of signaling networks in cancer cells. Thus, single cell measurements of phospho-protein responses reveal shifts in signaling potential of a phospho-protein network, allowing for categorizing of cell network phenotypes by multidimensional molecular profiles of signaling.
Papers: Postdoc, Stanford University by Jonathan M Irish
Blood, 2007
Loss or mutation of the TP53 tumor suppressor gene is not commonly observed in acute myeloid leuk... more Loss or mutation of the TP53 tumor suppressor gene is not commonly observed in acute myeloid leukemia (AML), suggesting that there is an alternate route for cell transformation. We investigated the hypothesis that previously observed Bcl-2 family member overexpression suppresses wild-type p53 activity in AML. We demonstrate that wild-type p53 protein is expressed in primary leukemic blasts from patients with de novo AML using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and phospho-specific flow cytometry. We found that p53 was heterogeneously expressed and phosphorylated in AML patient samples and could accumulate following DNA damage. Overexpression of antiapoptosis protein Bcl-2 in AML cells was directly correlated with p53 expression and phosphorylation on serine residues 15, 46, and 392. Within those patients with the highest levels of Bcl-2 expression, we identified a mutation in FLT3 that duplicated phosphorylation site Y591. The presence of this mutation correlated with greater than normal Bcl-2 expression and with previously observed profiles of potentiated STAT and MAPK signaling. These results support the hypothesis that Flt3-mediated signaling in AML enables accumulation of Bcl-2 and maintains a downstream block to p53 pathway apoptosis. Bcl-2 inhibition might therefore improve the efficacy of existing AML therapies by inactivating this suppression of wild-type p53 activity. (Blood.
Human tumors contain populations of both cancerous and host immune cells whose malignant signalin... more Human tumors contain populations of both cancerous and host immune cells whose malignant signaling interactions may define each patient's disease trajectory. We used multiplexed phospho-flow cytometry to profile single cells within human follicular lymphoma tumors and discovered a subpopulation of lymphoma cells with impaired B cell antigen receptor (BCR) signaling. The abundance of BCR-insensitive cells in each tumor negatively correlated with overall patient survival. These lymphoma negative prognostic (LNP) cells increased as tumors relapsed following chemotherapy. Loss of antigen receptor expression did not explain the absence of BCR signaling in LNP tumor cells, and other signaling responses were intact in these cells. Furthermore, BCR signaling responses could be reactivated in LNP cells, indicating that BCR signaling is not missing but rather specifically suppressed. LNP cells were also associated with changes to signaling interactions in the tumor microenvironment. Lower IL-7 signaling in tumor infiltrating T cells was observed in tumors with high LNP cell counts. The strength of signaling through T cell mediator of B cell function CD40 also stratified patient survival, particularly for those whose tumors contained few LNP cells. Thus, analysis of cell-cell interactions in heterogeneous primary tumors using signaling network profiles can identify and mechanistically define new populations of rare and clinically significant cells. Both the existence of these LNP cells and their aberrant signaling profiles provide targets for new therapies for follicular lymphoma.
Papers: Assistant Professor, Vanderbilt University by Jonathan M Irish
The plasticity of AML drives poor clinical outcomes and confounds its longitudinal detection. How... more The plasticity of AML drives poor clinical outcomes and confounds its longitudinal detection. However, the immediate impact of treatment on the leukemic and non-leukemic cells of the bone marrow and blood remains relatively understudied. Here, we conducted a pilot study of high dimensional longitudinal monitoring of immunophenotype in AML. To characterize changes in cell phenotype before, during, and immediately after induction treatment, we developed a 27-antibody panel for mass cytometry focused on surface diagnostic markers and applied it to 46 samples of blood or bone marrow tissue collected over time from 5 AML patients. Central goals were to determine whether changes in AML phenotype would be captured effectively by cytomic tools and to implement methods for describing the evolving phenotypes of AML cell subsets. Mass cytometry data were analyzed using established computational techniques. Within this pilot study, longitudinal immune monitoring with mass cytometry revealed fundamental changes in leukemia phenotypes that occurred over time during and after induction in the refractory disease setting. Persisting AML blasts became more phenotypically distinct from stem and progenitor cells due to expression of novel marker patterns that differed from pre-treatment AML cells and from all cell types observed in healthy bone marrow. This pilot study of single cell immune monitoring in AML represents a powerful tool for precision characterization and targeting of resistant disease.
• FL TILs have reduced cytokine signaling.
Mutations in early follicular lymphoma progenitors are associated with suppressed antigen presentation
Proceedings of the National Academy of Sciences of the United States of America, Jan 23, 2015
Follicular lymphoma (FL) is incurable with conventional therapies and has a clinical course typif... more Follicular lymphoma (FL) is incurable with conventional therapies and has a clinical course typified by multiple relapses after therapy. These tumors are genetically characterized by B-cell leukemia/lymphoma 2 (BCL2) translocation and mutation of genes involved in chromatin modification. By analyzing purified tumor cells, we identified additional novel recurrently mutated genes and confirmed mutations of one or more chromatin modifier genes within 96% of FL tumors and two or more in 76% of tumors. We defined the hierarchy of somatic mutations arising during tumor evolution by analyzing the phylogenetic relationship of somatic mutations across the coding genomes of 59 sequentially acquired biopsies from 22 patients. Among all somatically mutated genes, CREBBP mutations were most significantly enriched within the earliest inferable progenitor. These mutations were associated with a signature of decreased antigen presentation characterized by reduced transcript and protein abundance of...
Blood, 2013
Follicular lymphoma (FL) is currently incurable using conventional chemotherapy or immunotherapy ... more Follicular lymphoma (FL) is currently incurable using conventional chemotherapy or immunotherapy regimes, compelling new strategies. Advances in high-throughput sequencing technologies that can reveal oncogenic pathways have stimulated interest in tailoring therapies toward actionable somatic mutations. However, for mutation-directed therapies to be most effective, the mutations must be uniformly present in evolved tumor cells as well as in the self-renewing tumor-cell precursors. Here, we show striking intratumoral clonal diversity within FL tumors in the representation of mutations in the majority of genes as revealed by whole exome sequencing of subpopulations. This diversity captures a clonal hierarchy, resolved using immunoglobulin somatic mutations and IGH-BCL2 translocations as a frame of reference and by comparing diagnosis and relapse tumor pairs, allowing us to distinguish early versus late genetic eventsduring lymphomagenesis. We provide evidence that IGH-BCL2 translocations and CREBBP mutations are early events, whereas MLL2 and TNFRSF14 mutations probably represent late events during disease evolution. These observations provide insight into which of the genetic lesions represent suitable candidates for targeted therapies.
Single cell mass cytometry is revolutionizing our ability to quantitatively characterize cellular... more Single cell mass cytometry is revolutionizing our ability to quantitatively characterize cellular biomarkers and signaling networks. Mass cytometry experiments routinely measure 25-35 features of each cell in primary human tissue samples. The relative ease with which a novice user can generate a large amount of high quality data and the novelty of the approach have created a need for example protocols, analysis strategies, and datasets. In this chapter, we present detailed protocols for two mass cytometry experiments designed as training tools. The first protocol describes detection of 26 features on the surface of human peripheral blood mononuclear cells. In the second protocol, a mass cytometry signaling network profile measures 25 node states comprised of five key signaling effectors (AKT, ERK1/2, STAT1, STAT5, and p38) quantified under five conditions (Basal, FLT3L, SCF, IL-3, and IFNγ). This chapter compares manual and unsupervised data analysis approaches, including bivariate plots, heatmaps, histogram overlays, SPADE, and viSNE. Data files in this chapter have been shared online using Cytobank ( http://www.cytobank.org/irishlab/ ).
High-content single-cell biology and machine-learning tools are powering a new era of 'systems im... more High-content single-cell biology and machine-learning tools are powering a new era of 'systems immunology'. Routine mass-cytometry experiments now measure more than 30 features of each of millions of cells, and comprehensive maps of cell identity can be derived from a single cytometry tube1, 2. The application of computational tools has substantially augmented the ability to visualize high-dimensional mass-cytometry data and model results3, 4, 5, 6. These modern tools have revealed that traditional analyses can overlook cells whose phenotypes are not well understood ('Cyto Incognito', Fig. 1). In contrast to expert-driven manual analyses, computational approaches aim to comprehensively describe cellular phenotypes with minimal bias. With the increasing capacity of single-cell cytometry, both canonical markers and novel markers can be measured simultaneously and act together to robustly distinguish cell identity in highly heterogeneous tissues. Mass-cytometry panels are designed so that each cell will have multiple well-characterized functional markers that unambiguously confirm its identity. Machine-learning approaches then arrange cells into maps or organize cells along progressions on the basis of similar features (Fig. 1). In this issue of Nature Immunology, Becher and colleagues use mass cytometry to create a modern reference map of the healthy mouse myeloid system across eight tissues and reveal the role of the receptor for the growth factor GM-CSF (encoded by Csf2rb) in the development of various cell populations, including tissue-resident natural killer cells, nonlymphoid dendritic cells (DCs), alveolar macrophages and eosinophils7.
The flood of high-dimensional data resulting from mass cytometry experiments that measure more th... more The flood of high-dimensional data resulting from mass cytometry experiments that measure more than 40 features of individual cells has stimulated creation of new single cell computational biology tools. These tools draw on advances in the field of machine learning to capture multi-parametric relationships and reveal cells that are easily overlooked in traditional analysis. Here, we introduce a workflow for high dimensional mass cytometry data that emphasizes unsupervised approaches and visualizes data in both single cell and population level views. This workflow includes three central components that are common across mass cytometry analysis approaches: (1) distinguishing initial populations, (2) revealing cell subsets, and (3) characterizing subset features. In the implementation described here, viSNE, SPADE, and heatmaps were used sequentially to comprehensively characterize and compare healthy and malignant human tissue samples. The use of multiple methods helps provide a comprehensive view of results, and the largely unsupervised workflow facilitates automation and helps researchers avoid missing cell populations with unusual or unexpected phenotypes. Together, these methods develop a framework for future machine learning of cell identity.
Differences in the quality of BCR signaling control key steps of B cell maturation and differenti... more Differences in the quality of BCR signaling control key steps of B cell maturation and differentiation. Endogenously produced H2O2 is thought to fine tune the level of BCR signaling by reversibly inhibiting phosphatases. However, relatively little is known about how B cells at different stages sense and respond to such redox cues. In this study, we used phospho-specific flow cytometry and high-dimensional mass cytometry (CyTOF) to compare BCR signaling responses in mature human tonsillar B cells undergoing germinal center (GC) reactions. GC B cells, in contrast to mature naive B cells, memory B cells, and plasmablasts, were hypersensitive to a range of H2O2 concentrations and responded by phosphorylating SYK and other membrane-proximal BCR effectors in the absence of BCR engagement. These findings reveal that stage-specific redox responses distinguish human GC B cells.
Papers by Jonathan M Irish
Dissecting Complex Cellular Systems with High Dimensional Single Cell Mass Cytometry
The Human Innate Immunity Handbook, 2016
Systems immune monitoring in cancer therapy
European journal of cancer (Oxford, England : 1990), Jan 4, 2016
Treatments that successfully modulate anti-cancer immunity have significantly improved outcomes f... more Treatments that successfully modulate anti-cancer immunity have significantly improved outcomes for advanced stage malignancies and sparked intense study of the cellular mechanisms governing therapy response and resistance. These responses are governed by an evolving milieu of cancer and immune cell subpopulations that can be a rich source of biomarkers and biological insight, but it is only recently that research tools have developed to comprehensively characterize this level of cellular complexity. Mass cytometry is particularly well suited to tracking cells in complex tissues because >35 measurements can be made on each of hundreds of thousands of cells per sample, allowing all cells detected in a sample to be characterized for cell type, signalling activity, and functional outcome. This review focuses on mass cytometry as an example of systems level characterization of cancer and immune cells in human tissues, including blood, bone marrow, lymph nodes, and primary tumours. Th...
The use of fluorescently-tagged apoptolidins in cellular uptake and response studies
The Journal of antibiotics, Jan 9, 2016
The apoptolidins are glycomacrolide microbial metabolites reported to be selectively cytotoxic ag... more The apoptolidins are glycomacrolide microbial metabolites reported to be selectively cytotoxic against tumor cells. Using fluorescently tagged active derivatives we demonstrate selective uptake of these four tagged glycomacrolides in cancer cells over healthy human blood cells. We also demonstrate the utility of these five fluorescently tagged glycomacrolides in fluorescent flow cytometry to monitor cellular uptake of the six glycomacrolides and cellular response.The Journal of Antibiotics advance online publication, 9 March 2016; doi:10.1038/ja.2016.22.
Melanoma-specific MHC-II expression represents a tumour-autonomous phenotype and predicts response to anti-PD-1/PD-L1 therapy
Nature Communications, 2016
Anti-PD-1 therapy yields objective clinical responses in 30-40% of advanced melanoma patients. Si... more Anti-PD-1 therapy yields objective clinical responses in 30-40% of advanced melanoma patients. Since most patients do not respond, predictive biomarkers to guide treatment selection are needed. We hypothesize that MHC-I/II expression is required for tumour antigen presentation and may predict anti-PD-1 therapy response. In this study, across 60 melanoma cell lines, we find bimodal expression patterns of MHC-II, while MHC-I expression was ubiquitous. A unique subset of melanomas are capable of expressing MHC-II under basal or IFNγ-stimulated conditions. Using pathway analysis, we show that MHC-II(+) cell lines demonstrate signatures of 'PD-1 signalling', 'allograft rejection' and 'T-cell receptor signalling', among others. In two independent cohorts of anti-PD-1-treated melanoma patients, MHC-II positivity on tumour cells is associated with therapeutic response, progression-free and overall survival, as well as CD4(+) and CD8(+) tumour infiltrate. MHC-II(+) tumours can be identified by melanoma-specific immunohistochemistry using commercially available antibodies for HLA-DR to improve anti-PD-1 patient selection.
Multiparameter analysis of stimulated human peripheral blood mononuclear cells: A comparison of mass and fluorescence cytometry
Cytometry. Part A : the journal of the International Society for Analytical Cytology, Jan 24, 2015
Mass and fluorescence cytometry are quantitative single cell flow cytometry approaches that are p... more Mass and fluorescence cytometry are quantitative single cell flow cytometry approaches that are powerful tools for characterizing diverse tissues and cellular systems. Here mass cytometry was directly compared with fluorescence cytometry by studying phenotypes of healthy human peripheral blood mononuclear cells (PBMC) in the context of superantigen stimulation. One mass cytometry panel and five fluorescence cytometry panels were used to measure 20 well-established lymphocyte markers of memory and activation. Comparable frequencies of both common and rare cell subpopulations were observed with fluorescence and mass cytometry using biaxial gating. The unsupervised high-dimensional analysis tool viSNE was then used to analyze data sets generated from both mass and fluorescence cytometry. viSNE analysis effectively characterized PBMC using eight features per cell and identified similar frequencies of activated CD4+ T cells with both technologies. These results suggest combinations of un...
Blood, 2009
Despite the success of passive immunotherapy with monoclonal antibodies (mAbs), many lymphoma pat... more Despite the success of passive immunotherapy with monoclonal antibodies (mAbs), many lymphoma patients eventually relapse. Induction of an adaptive immune response may elicit active and longlasting antitumor immunity, thereby preventing or delaying recurrence. Immunomodulating mAbs directed against immune cell targets can be used to enhance the immune response to achieve efficient antitumor immunity. Anti-CD137 agonistic mAb has demonstrated antitumor effi-cacy in various tumor models and has now entered clinical trials for the treatment of solid tumors. Here, we investigate the therapeutic potential of anti-CD137 mAb in lymphoma. We found that human primary lymphoma tumors are infiltrated with CD137 ؉ T cells. We therefore hypothesized that lymphoma would be susceptible to treatment with anti-CD137 agonistic mAb. Using a mouse model, we demonstrate that anti-CD137 therapy has potent antilymphoma activity in vivo. The antitumor effect of anti-CD137 therapy was mediated by both natural killer (NK) and CD8 T cells and induced long-lasting immunity. Moreover, the antitumor activity of anti-CD137 mAb could be further enhanced by depletion of regulatory T cell (T regs ). These results support the evaluation of anti-CD137 therapy in clinical trials for patients with lymphoma. (Blood. 2009;114:3431-3438)
Mutations in early follicular lymphoma progenitors are associated with suppressed antigen presentation
Proceedings of the National Academy of Sciences of the United States of America, Jan 23, 2015
Follicular lymphoma (FL) is incurable with conventional therapies and has a clinical course typif... more Follicular lymphoma (FL) is incurable with conventional therapies and has a clinical course typified by multiple relapses after therapy. These tumors are genetically characterized by B-cell leukemia/lymphoma 2 (BCL2) translocation and mutation of genes involved in chromatin modification. By analyzing purified tumor cells, we identified additional novel recurrently mutated genes and confirmed mutations of one or more chromatin modifier genes within 96% of FL tumors and two or more in 76% of tumors. We defined the hierarchy of somatic mutations arising during tumor evolution by analyzing the phylogenetic relationship of somatic mutations across the coding genomes of 59 sequentially acquired biopsies from 22 patients. Among all somatically mutated genes, CREBBP mutations were most significantly enriched within the earliest inferable progenitor. These mutations were associated with a signature of decreased antigen presentation characterized by reduced transcript and protein abundance of...
Blood, 2013
• Analysis of coding genomes of FL tumor subpopulations reveals striking clonal diversity at diag... more • Analysis of coding genomes of FL tumor subpopulations reveals striking clonal diversity at diagnosis and progression. • Within a hierarchy of somatic evolution of FL coding genomes, many recurrent mutations are subclonal at diagnosis. Follicular lymphoma (FL) is currently incurable using conventional chemotherapy or immunotherapy regimes, compelling new strategies. Advances in high-throughput sequencing technologies that can reveal oncogenic pathways have stimulated interest in tailoring therapies toward actionable somatic mutations. However, for mutation-directed therapies to be most effective, the mutations must be uniformly present in evolved tumor cells as well as in the self-renewing tumor-cell precursors. Here, we show striking intratumoral clonal diversity within FL tumors in the representation of mutations in the majority of genes as revealed by whole exome sequencing of subpopulations. This diversity captures a clonal hierarchy, resolved using immunoglobulin somatic mutations and IGH-BCL2 translocations as a frame of reference and by comparing diagnosis and relapse tumor pairs, allowing us to distinguish early versus late genetic eventsduring lymphomagenesis. We provide evidence that IGH-BCL2 translocations and CREBBP mutations are early events, whereas MLL2 and TNFRSF14 mutations probably represent late events during disease evolution. These observations provide insight into which of the genetic lesions represent suitable candidates for targeted therapies. (Blood. 2013;121(9):1604-1611)
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Papers: Graduate Student, Stanford University by Jonathan M Irish
Papers: Postdoc, Stanford University by Jonathan M Irish
Papers: Assistant Professor, Vanderbilt University by Jonathan M Irish
Papers by Jonathan M Irish