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Duncan M. Morgan
MIT Chemical Engineering
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publications
TCR sequencing paired with massively parallel 3′ RNA-seq reveals clonotypic T cell signatures
Published in Nature Immunology, 2019
High-throughput 3′ single-cell RNA-sequencing (scRNA-seq) allows cost-effective, detailed characterization of individual immune cells from tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including variable sequences of T cell antigen receptors (TCRs) that confer antigen specificity. Here, we present a strategy that enables simultaneous analysis of TCR sequences and corresponding full transcriptomes from 3′-barcoded scRNA-seq samples. This approach is compatible with common 3′ scRNA-seq methods, and adaptable to processed samples post hoc. We applied the technique to identify transcriptional signatures associated with T cells sharing common TCRs from immunized mice and from patients with food allergy. We observed preferential phenotypes among subsets of expanded clonotypes, including type 2 helper CD4+ T cell (TH2) states associated with food allergy. These results demonstrate the utility of our method when studying diseases in which clonotype-driven responses are critical to understanding the underlying biology.
Recommended citation: Tu, A. A., Gierahn, T. M., Monian, B., Morgan, D. M., Mehta, N. K., Ruiter, B., Shreffler, W. G., Shalek, A. K., Love, J. C. "TCR sequencing paired with massively parallel 3′ RNA-seq reveals clonotypic T cell signatures." Nature Immunology. 20 1692-1699 (2019). https://doi.org/10.1038/s41590-019-0544-5
Clonally expanded, GPR15-expressing pathogenic effector Th2 cells are associated with eosinophilic esophagitis
Published in Science Immunology, 2021
Eosinophilic esophagitis (EoE) is an allergic disorder characterized by the recruitment of eosinophils to the esophagus, resulting in chronic inflammation. We sought to understand the cellular populations present in tissue biopsies of patients with EoE and to determine how these populations are altered between active disease and remission. To this end, we analyzed cells obtained from esophageal biopsies, duodenal biopsies, and peripheral blood of patients with EoE diagnosed with active disease or remission with single-cell RNA and T cell receptor (TCR) sequencing. Pathogenic effector TH2 (peTH2) cells present in the esophageal biopsies of patients with active disease expressed distinct gene signatures associated with the synthesis of eicosanoids. The esophageal tissue–resident peTH2 population also exhibited clonal expansion, suggesting antigen-specific activation. Peripheral CRTH2+CD161− and CRTH2+CD161+ memory CD4+ T cells were enriched for either a conventional TH2 phenotype or a peTH2 phenotype, respectively. These cells also exhibited substantial clonal expansion and convergence of TCR sequences, suggesting that they are expanded in response to a defined set of antigens. The esophagus-homing receptor GPR15 was upregulated by peripheral peTH2 clonotypes that were also detected in the esophagus. Finally, GPR15+ peTH2 cells were enriched among milk-reactive CD4+ T cells in patients with milk-triggered disease, suggesting that these cells are an expanded, food antigen–specific population with enhanced esophagus homing potential.
Recommended citation: Morgan, D. M., Ruiter, B., Smith, N. P., Tu, A. A., Monian, B., Stone, B. E., Virk-Hundal, N., Yuan, Q., Shreffler, W. G., Love, J. C. "Clonally expanded, GPR15-expressing pathogenic effector Th2 cells are associated with eosinophilic esophagitis." Science Immunology, 6, eabi5586 (2021). https://doi.org/10.1126/sciimmunol.abi5586
Lack of CD8+ T cell effector differentiation during priming mediates checkpoint blockade resistance in non–small cell lung cancer
Published in Science Immunology, 2021
In non–small cell lung cancer (NSCLC), response to immune checkpoint blockade (ICB) is associated with pro-grammed cell death ligand 1 expression that is induced by interferon-g–producing, tumor-infiltrating CD8+ T cells. However, not all tumors with a CD8+ T cell infiltrate respond to ICB, and little is known about the mechanisms governing ICB resistance in T cell–infiltrated NSCLC. We used an orthotopic NSCLC mouse model to study ICB- refractory CD8+ T cell responses. Single-cell RNA sequencing of the NSCLC mouse tumors revealed that lung cancer– specific tumor-infiltrating CD8+ T cells exhibited clonal expansion but lacked expression of genes associated with effector and exhausted T cell responses, indicating that they underwent a differentiation program distinct from conventional T cell exhaustion. This lung cancer–specific T cell dysfunction program was established early during priming in the mediastinal lymph node and was characterized by robust proliferation but a failed up-regulation of effector and exhausted T cell characteristics. Intriguingly, CD8+ T cells from patients with NSCLC expressed an analogous gene expression program, which appeared distinct from conventional T cell exhaustion. Administra-tion of recombinant interleukin-2 (IL-2) and IL-12 was sufficient to restore effector T cell differentiation and induce control of KP lung tumors. These findings imply that a CD8+ T cell differentiation trajectory, activated during T cell priming in the mediastinal lymph node, limits the response of CD8+ T cells to ICB and thereby may contribute to failure of ICB in a subset T cell–infiltrated NSCLC.
Recommended citation: Horton, B. L., Morgan, D. M., Momin, N., Zagorulya, M., Torres-Meija, E., Bhandakar, V., Wittrup, K. D., Love, J. C., Spranger, S. (2021). "Lack of CD8+ T cell effector differentiation during priming mediates checkpoint blockade resistance in non–small cell lung cancer." Science Immunology . 6 abi8800 (2021). https://doi.org/10.1126/sciimmunol.abi8800
Peanut oral immunotherapy differentially suppresses clonally distinct subsets of T helper cells
Published in The Journal of Clinical Investigation, 2022
Food allergy affects an estimated 8% of children in the United States. Oral immunotherapy (OIT) is a recently approved treatment, with outcomes ranging from sustained tolerance to food allergens to no apparent benefit. The immunological underpinnings that influence clinical outcomes of OIT remain largely unresolved. Using single-cell RNA-Seq and paired T cell receptor α/β (TCRα/β) sequencing, we assessed the transcriptomes of CD154+ and CD137+ peanut-reactive T helper (Th) cells from 12 patients with peanut allergy longitudinally throughout OIT. We observed expanded populations of cells expressing Th1, Th2, and Th17 signatures that further separated into 6 clonally distinct subsets. Four of these subsets demonstrated a convergence of TCR sequences, suggesting antigen-driven T cell fates. Over the course of OIT, we observed suppression of Th2 and Th1 gene signatures in effector clonotypes but not T follicular helper–like (Tfh-like) clonotypes. Positive outcomes were associated with stronger suppression of Th2 signatures in Th2A-like cells, while treatment failure was associated with the expression of baseline inflammatory gene signatures that were present in Th1 and Th17 cell populations and unmodulated by OIT. These results demonstrate that differential clinical responses to OIT are associated with both preexisting characteristics of peanut-reactive CD4+ T cells and suppression of a subset of Th2 cells.
Recommended citation: Monian, B, Tu, A. A., Ruiter, B., Morgan, D. M., Petrossian, P. M., Smith, N. P., Gierahn, T., M., Ginder, J., Shreffler, W. G., Love, J. C. "Peanut oral immunotherapy differentially suppresses clonally distinct subsets of T helper cells" The Journal of Clinical Investigation. 132(2):e150634 (2022). https://www.jci.org/articles/view/150634
WAT3R: Recovery of T-Cell Receptor Variable Regions from 3` Single-Cell RNA Sequencing.
Published in bioRxiv, 2022
Diversity of the T-cell receptor (TCR) repertoire is central to adaptive immunity. The TCR is composed of α and β chains, encoded by the TRA and TRB genes, of which the variable regions determine antigen specificity. To generate novel biological insights into the complex functioning of immune cells, combined capture of variable regions and single-cell transcriptomes provides a compelling approach. Recent developments enable the enrichment of TRA and TRB variable regions from widely used technologies for 3’-biased single-cell RNA-sequencing (scRNA-seq). However, a comprehensive computational pipeline to process TCR-enriched data from 3’ scRNA-seq is not available. Here we present an analysis pipeline to process TCR variable regions enriched from 3’ scRNA-seq cDNA. The tool reports TRA and TRB nucleotide and amino acid sequences linked to cell barcodes, enabling the reconstruction of T-cell clonotypes with associated transcriptomes. We demonstrate the software using peripheral blood mononuclear cells (PBMCs) from a healthy donor and detect TCR sequences in a high proportion of single T-cells. Detection of TCR sequences is negligible in non-T-cell populations, demonstrating specificity. Finally, we show that TCR clones are larger in CD8 Memory T-cells than other T-cell types, indicating an association between T-cell clonotypes and differentiation states.
Recommended citation: Ainciburu, M., Morgan, D. M., DePasquale, E. A. K., Love, J., C., Propser, F., van Galen, P. "WAT3R: Recovery of T-Cell Receptor Variable Regions from 3` Single-Cell RNA Sequencing." bioRxiv. https://doi.org/10.1101/2022.01.26.477886
Mitochondrial variant enrichment from high-throughput single-cell RNA sequencing resolves clonal populations
Published in , 2022
The combination of single-cell transcriptomics with mitochondrial DNA (mtDNA) variant detection can be used to establish lineage relationships in primary human cells, but current methods are not scalable to interrogate complex tissues. Here, we combine common 3′ single-cell RNA-sequencing protocols with mitochondrial transcriptome enrichment to increase coverage by more than 50-fold, enabling high-confidence mutation detection. The method successfully identifies skewed immune-cell expansions in primary human clonal hematopoiesis.
Recommended citation: Miller, T. E., Lareau, C. A., Verga, J. A., DePasquale, E. A. K., Liu, V., Ssozi, D., Sandor, K., Yin, Y., Ludwig, L. S., El Farran, C. A., Morgan, D. M., Satpathy, A. T., Griffin, G. K., Lane, A. A., Love, J. C., Bernstein, B. E., Sankaran, V. G., van Galen, P. "Mitochondrial variant enrichment from high-throughput single-cell RNA sequencing resolves clonal populations" In Press.
talks
Single-Cell RNA Sequencing Identifies Metabolic Characteristics of T Cells in Eosinophilic Esophagitis
Published:
Eosinophilic esophagitis (EoE) is a tissue-specific allergic disorder characterized by the recruitment of eosinophils to the esophagus of affected patients. Treatment of EoE relies on antigen avoidance through the use of elimination diets, as the mechanisms of disease are not fully understood and interventional treatments are often ineffective. To understand the tissue ecosystem present in EoE, we used single-cell RNA sequencing to profile 24,968 single cells from biopsies of the esophagus and duodenum of nine patients with active EoE or with EoE in remission achieved by antigen avoidance. Analysis of the T cells recovered from these patients revealed T cell populations including CD4+ T helper cells, FOXP3+ T regulatory cells, CD8+ tissue resident memory cells, and GATA3+ effector-like Th2 cells. Th2 cells were enriched in the esophageal tissue of patients with active disease, expressed common mediators of Th2 including IL13 and IL5 as well as markers of pathogenic Th2 cells such as IL1RL1 and IL17RB. Paired alpha/beta TCR sequencing revealed that this population exhibited clonal expansion and contained an exclusive set of clones not detected in other T cell populations. Pathway analysis of the Th2 subset revealed an upregulation of genes associated with arachidonic acid metabolism and prostaglandin D2 (PGD2) synthesis, suggesting that pathogenic T cells contribute to the production of PGD2 in EoE, promote the recruitment of eosinophils. Thus, we identified a pathway upregulated in a clonal population associated with EoE, providing insight into the mechanisms of EoE and informing future interventional treatments.
teaching
Teaching experience 1
Undergraduate course, University 1, Department, 2014
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Teaching experience 2
Workshop, University 1, Department, 2015
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