Single-cell response to perturbations across biological scales : single organ, organ system and phenotypic individuals

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2019Cataloged from PDF version of thesis.Includes bibliographical references.The biological processes that sustain a complex organism require the orchestrated dynamics of complex cellular ensembles. Several vital systems - such as the immune system, the digestive system and more - must process internal and external signals to maintain functional homeostasis in response to perturbations at the systems-level. To further understand how groups of cells collectively respond to perturbations, we have applied single-cell RNA-sequencing and complementary techniques to explore cellular behaviors within complex systems at multiple relevant biological scales: from within a single organ, to an organ system, to across several human individuals with differing genetic backgrounds linked by a shared phenotype. More specifically, at the level of the organ, we have explored acute injury responses in the liver. We have identified and described a new compensatory phase of the liver response to injury, in which surviving hepatocytes upregulate their expression of critical liver function genes to maintain overall organ function. Next, we extended our approach from a focus on an acute injury targeting a single organ to exploring chronic damage resulting from a long-term high fat diet across multiple gastrointestinal and immune compartments. Our analysis revealed molecular pathways and changes in stem gene expression which may contribute to obesity-related disease. Finally, we characterized shared features across multiple unique human donors with a common phenotype, elite control of HIV-1. We identified and validated a subset of highly functional dendritic cells, and developed broadly applicable computational approaches to identify reproducible responses across donors and to nominate candidate targets for rationally modulating the system. Overall, our work demonstrates the utility of single-cell RNA-sequencing for uncovering important cellular phenotypes that inform systems-level responses at any biological scale.by Kellie Elizabeth Kolb.Ph. D.Ph.D. Massachusetts Institute of Technology, Department of Chemistr

Immunological Fingerprints of Controllers Developing Neutralizing HIV-1 Antibodies

The induction of broadly neutralizing antibodies (bnAbs) is highly desired for an effective vaccine against HIV-1. Typically, bnAbs develop in patients with high viremia, but they can also evolve in some untreated HIV-1 controllers with low viral loads. Here, we identify a subgroup of neutralizer-controllers characterized by myeloid DCs (mDCs) with a distinct inflammatory signature and a superior ability to prime T follicular helper (Tfh)-like cells in an STAT4-dependent fashion. This distinct immune profile is associated with a higher frequency of Tfh-like cells in peripheral blood (pTfh) and an enrichment for Tfh-defining genes in circulating CD4+ T cells. Correspondingly, monocytes from this neutralizer controller subgroup upregulate genes encoding for chemotaxis and inflammation, and they secrete high levels of IL-12 in response to TLR stimulation. Our results suggest the existence of multi-compartment immune networks between mDCs, Tfh, and monocytes that may facilitate the development of bnAbs in a subgroup of HIV-1 controllers.NIH (Grants 5U24AI118672, 1U54CA217377, 1R33CA202820, 2U19AI089992, 1R01HL134539, 2RM1HG006193, 2R01HL095791, 2P01AI039671, 1U2CCA23319501, 1R01DA046277 and 1R01AI138546 )Bill and Melinda Gates Foundation (Grants OPP1139972, OPP1137006 and OPP1116944

Circulating CXCR5[superscript +]CXCR3[superscript +]PD-1[superscript lo] Tfh-like cells in HIV-1 controllers with neutralizing antibody breadth

HIV-1–specific broadly neutralizing antibodies (bnAbs) typically develop in individuals with continuous high-level viral replication and increased immune activation, conditions that cannot be reproduced during prophylactic immunization. Understanding mechanisms supporting bnAb development in the absence of high-level viremia may be important for designing bnAb-inducing immunogens. Here, we show that the breadth of neutralizing antibody responses in HIV-1 controllers was associated with a relative enrichment of circulating CXCR5[superscript +]CXCR3[superscript +]PD-1[superscript lo] CD4[superscript +] T cells. These CXCR3[superscript +]PD-1[superscript lo] Tfh-like cells were preferentially induced in vitro by functionally superior dendritic cells from controller neutralizers, and able to secrete IL-21 and support B cells. In addition, these CXCR3[superscript +]PD-1[superscript lo] Tfh-like cells contained higher proportions of stem cell–like memory T cells, and upon antigenic stimulation differentiated into PD-1[superscript hi ]Tfh-like cells in a Notch-dependent manner. Together, these data suggest that CXCR5[superscript +]CXCR3[superscript +]PD-1[superscript lo] cells represent a dendritic cell–primed precursor cell population for PD-1hi Tfh-like cells that may contribute to the generation of bnAbs in the absence of high-level viremia

Mechanisms of Lymphoma Clearance Induced by High-Dose Alkylating Agents

The extraordinary activity of high-dose cyclophosphamide against some high-grade lymphomas was described nearly 60 years ago. Here we address mechanisms that mediate cyclophosphamide activity in bona fide human double-hit lymphoma. We show that antibody resistance within the bone marrow (BM) is not present upon early engraftment but develops during lymphoma progression. This resistance required a high tumor:macrophage ratio, was recapitulated in spleen by partial macrophage depletion, and was overcome by multiple, high-dose alkylating agents. Cyclophosphamide induced endoplasmic reticulum (ER) stress in BM-resident lymphoma cells in vivo that resulted in ATF4-mediated paracrine secretion of VEGFA, massive macrophage infiltration, and clearance of alemtuzumab-opsonized cells. BM macrophages isolated after cyclophosphamide treatment had increased phagocytic capacity that was reversed by VEGFA blockade or SYK inhibition. Single-cell RNA sequencing of these macrophages identified a super-phagocytic subset that expressed CD36/FCGR4. Together, these findings define a novel mechanism through which high-dose alkylating agents promote macrophage-dependent lymphoma clearance. SIGNIFICANCE: mAbs are effective against only a small subset of cancers. Herein, we recapitulate compartment-specific antibody resistance and define an ER stress-dependent mechanism induced by high-dose alkylating agents that promotes phagocytosis of opsonized tumor cells. This approach induces synergistic effects with mAbs and merits testing across additional tumor types

Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq

To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.National Cancer Institute (U.S.) (1U24CA180922)National Cancer Institute (U.S.) (P30-CA14051