As the amount of data we have relating to cells, properties, surroundings, and interactions increases exponentially, we are motivated to develop pan-system measurements and analyses to paint comprehensive pictures of immune response in health and disease. Relying on massive transcriptomic datasets generated from complex tissues, like melanoma tumors, inflamed human gut, M. tuberculosis (MTB)-induced granulomas, and healthy or SHIV-infected monkey tissues, we have begun to construct social networks of integrated responses to physiological perturbations. The technologies outlined above uniquely enable us to generate foundational datasets (e.g., transcriptomes from interacting cell pairs) for deconvolving and interpreting the potential drivers of observed ensemble behaviors, as well as for identifying which properties we cannot explain, and thus need to study. To date, our lab has generated over 2 million single-cell transcriptomes across multiple tissues, individuals, and species; we are utilizing this data, paired with metadata and additional characteristics, to look for common cellular network motifs, such as division of labor, quorum sensing, persistence, or bet-hedging.

Vaccines remain the most effective tool to prevent infectious diseases. Here, we introduce an in vitro booster vaccination approach that relies on antigen-dependent activation of human memory B cells in culture. This stimulation induces antigen-specific B cell proliferation, differentiation of B cells into plasma cells, and robust antibody secretion after a few days of culture. We validated this strategy using cells from healthy donors to retrieve human antibodies against tetanus toxoid and influenza hemagglutinin (HA) from H1N1 and newly emergent subtypes such as H5N1 and H7N9. Anti-HA antibodies were cross-reactive against multiple subtypes, and some showed neutralizing activity. Although these antibodies may have arisen as a result of previous influenza infection, we also obtained gp120-reactive antibodies from non–HIV-infected donors, indicating that we can generate antibodies without prior antigenic exposure. Overall, our novel approach can be used to rapidly produce therapeutic antibodies and has the potential to assess the immunogenicity of candidate antigens, which could be exploited in future vaccine development.

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.

 

The immune system plays an important role in regulating homeostatic balance across tissues and individuals in the face of changing and challenging environments. Given the pivotal and outsized impact cell subsets (e.g., rare precocious DCs) can have on ensemble dynamics (e.g., global activation of an antiviral response and deactivation of inflammation), we aim to understand the functional consequences of variation in cellular composition across tissues, as well as how different immune cells adapt to changing environmental conditions.

Motivating questions in the lab include:

  1. How can we perform observational and experimental studies to understand the fundamental units of tissues structure and function?
  2. Can we derive basic principles governing homeostatic and pathogenic immune responses within tissues?
  3. What dictates the evolution of clonal antigen-specific T & B cell responses?

To this end, we are several multiple tissues from multiple organisms across common sources of variation. By examining consistent and unique themes that emerge across these systems, we aim to extract basic principles that govern homeostatic and pathogenic immune responses within tissues. Ultimately, we intend to leverage this information to rationally engineer immune responses (e.g., in vaccines and immunotherapies).

Our immune system collaborates with environment- and diet-dependent commensals to establish and maintain homeostasis, and to defend against pathogenic threats (e.g., viruses, bacteria, fungi). We are interested in understanding the nature and impact of these interactions on host tissues, as well as potential avenues to modulate them for therapeutic or prophylactic ends.

Illustrative questions and areas of study include:

  1. How do microbial composition and byproducts influence cellular differentiation and phenotypic diversity within the gut?
  2. How do pathogens (e.g. HIV and TB) impact target cell phenotypes and overall tissue function in the context of acute and systemic infection?
  3. To what degree can therapeutic intervention (e.g. cART for HIV-1) re-establish homeostatic setpoint (i.e. composition and function)?

We have several projects and collaborations (local and international) actively exploring these and related questions in vaccine design that have both inspired, and take advantage of, some of our unique tools to profile thousands of single cells from limited clinical samples anywhere in the world, and develop clinically relevant hypotheses.

A diverse array of mechanisms—including genetic mutations, environmental triggers, and diet—can alter cell function and reduce tissue stability, ultimately leading to malignancy, autoimmunity, or immunodeficiency. By identifying which cells these factors affect and in what ways, we aim to develop targeted therapeutic interventions in areas such as cancer, allergy, and inflammatory bowel disease.

Motivating questions that drive our research include:

  1. How do the coordinated interactions between epithelial and immune populations inform barrier tissue function in the context of homeostasis, inflammation and malignancy?
  2. How can we leverage information across systems to derive a set of unifying principles of cellular ecology in health and disease?

Current projects aim to contrast the cellular microenvironments of healthy, inflamed, and malignant (Tirosh et al., 2016; Patel et al., 2014) tissues to examine inflammation-induced changes and the drivers of malignant transformation, as well as to identify which cells remember prior insult. We are similarly profiling aberrant immune behaviors in immune privileged tissues, such as the nervous systems. As in our host-microbial studies, our goal is to identify common features shared across different immune-related diseases that we can probe further in natural (tissues, models) and engineered (patterned cells and cellular structures, organoids) ensembles.

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+CXCR3+PD-1lo CD4+ T cells. These CXCR3+PD-1lo 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+PD-1lo Tfh-like cells contained higher proportions of stem cell–like memory T cells, and upon antigenic stimulation differentiated into PD-1hi Tfh-like cells in a Notch-dependent manner. Together, these data suggest that CXCR5+CXCR3+PD-1lo 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.

CD8+ T cell recognition of virus-infected cells is characteristically restricted by major histocompatibility complex (MHC) class I, although rare examples of MHC class II restriction have been reported in Cd4-deficient mice and a macaque SIV vaccine trial using a recombinant cytomegalovirus vector. Here, we demonstrate the presence of human leukocyte antigen (HLA) class II-restricted CD8+ T cell responses with antiviral properties in a small subset of HIV-infected individuals. In these individuals, T cell receptor β (TCRβ) analysis revealed that class II-restricted CD8+ T cells underwent clonal expansion and mediated killing of HIV-infected cells. In one case, these cells comprised 12% of circulating CD8+ T cells, and TCRα analysis revealed two distinct co-expressed TCRα chains, with only one contributing to binding of the class II HLA-peptide complex. These data indicate that class II-restricted CD8+ T cell responses can exist in a chronic human viral infection, and may contribute to immune control.

Innate lymphoid cells (ILCs) play a central role in the response to infection by secreting cytokines crucial for immune regulation, tissue homeostasis, and repair. Although dysregulation of these systems is central to pathology, the impact of HIV-1 on ILCs remains unknown. We found that human blood ILCs were severely depleted during acute viremic HIV-1 infection and that ILC numbers did not recover after resolution of peak viremia. ILC numbers were preserved by antiretroviral therapy (ART), but only if initiated during acute infection. Transcriptional profiling during the acute phase revealed upregulation of genes associated with cell death, temporally linked with a strong IFN acutephase response and evidence of gut barrier breakdown. We found no evidence of tissue redistribution in chronic disease and remaining circulating ILCs were activated but not apoptotic. These data provide a potential mechanistic link between acute HIV-1 infection, lymphoid tissue breakdown, and persistent immune dysfunction.

We introduce a microfluidic platform that enables off-chip single-cell RNA-seq after multi-generational lineage tracking under controlled culture conditions. We use this platform to generate whole-transcriptome profiles of primary, activated murine CD8+ T-cell and lymphocytic leukemia cell line lineages. Here we report that both cell types have greater intra- than inter-lineage transcriptional similarity. For CD8+ T-cells, genes with functional annotation relating to lymphocyte differentiation and function—including Granzyme B—are enriched among the genes that demonstrate greater intra-lineage expression level similarity. Analysis of gene expression covariance with matched measurements of time since division reveals cell type-specific transcriptional signatures that correspond with cell cycle progression. We believe that the ability to directly measure the effects of lineage and cell cycle-dependent transcriptional profiles of single cells will be broadly useful to fields where heterogeneous populations of cells display distinct clonal trajectories, including immunology, cancer, and developmental biology.

Extensive cellular heterogeneity exists within specific immune-cell subtypes classified as a single lineage, but its molecular underpinnings are rarely characterized at a genomic scale. Here, we use single-cell RNA-seq to investigate the molecular mechanisms governing heterogeneity and pathogenicity of Th17 cells isolated from the central nervous system (CNS) and lymph nodes (LN) at the peak of autoimmune encephalomyelitis (EAE) or differentiated in vitro under either pathogenic or non-pathogenic polarization conditions. Computational analysis relates a spectrum of cellular states in vivo to in-vitro-differentiated Th17 cells and unveils genes governing pathogenicity and disease susceptibility. Using knockout mice, we validate four new genes: Gpr65PlzpToso, and Cd5l (in a companion paper). Cellular heterogeneity thus informs Th17 function in autoimmunity and can identify targets for selective suppression of pathogenic Th17 cells while potentially sparing non-pathogenic tissue-protective ones.

Epigenetic changes are crucial for the generation of immunological memory. Failure to generate or maintain these changes will result in poor memory responses. Similarly, augmenting or stabilizing the correct epigenetic states offers a potential method of enhancing memory. Yet the transcription factors that regulate these processes are poorly defined. We find that the transcription factor Oct1 and its cofactor OCA-B are selectively required for the in vivo generation of CD4+ memory T cells. More importantly, the memory cells that are formed do not respond properly to antigen reencounter. In vitro, both proteins are required to maintain a poised state at the Il2 target locus in resting but previously stimulated CD4+ T cells. OCA-B is also required for the robust reexpression of multiple other genes including Ifng. ChIPseq identifies ∼50 differentially expressed direct Oct1 and OCA-B targets. We identify an underlying mechanism involving OCA-B recruitment of the histone lysine demethylase Jmjd1a to targets such as Il2Ifng, andZbtb32. The findings pinpoint Oct1 and OCA-B as central mediators of CD4+ T cell memory.

Encounters between immune cells and invading bacteria ultimately determine the course of infection. These interactions are usually measured in populations of cells, masking cell-to-cell variation that may be important for infection outcome. To characterize the gene expression variation that underlies distinct infection outcomes and monitor infection phenotypes, we developed an experimental system that combines single-cell RNA-seq with fluorescent markers. Probing the responses of individual macrophages to invading Salmonella, we find that variation between individual infected host cells is determined by the heterogeneous activity of bacterial factors in individual infecting bacteria. We illustrate how variable PhoPQ activity in the population of invading bacteria drives variable host type I IFN responses by modifying LPS in a subset of bacteria. This work demonstrates a causative link between host and bacterial variability, with cell-to-cell variation between different bacteria being sufficient to drive radically different host immune responses. This co-variation has implications for host-pathogen dynamics in vivo.

The mammalian immune system is tasked with protecting the host against a broad range of threats. Understanding how immune populations leverage cellular diversity to achieve this breadth and flexibility, particularly during dynamic processes such as differentiation and antigenic response, is a core challenge that is well suited for single cell analysis. Recent years have witnessed transformative and intersecting advances in nanofabrication and genomics that enable deep profiling of individual cells, affording exciting opportunities to study heterogeneity in the immune response at an unprecedented scope. In light of these advances, here we review recent work exploring how immune populations generate and leverage cellular heterogeneity at multiple molecular and phenotypic levels. Additionally, we highlight opportunities for single cell technologies to shed light on the causes and consequences of heterogeneity in the immune system.

Recent molecular studies have shown that, even when derived from a seemingly homogenous population, individual cells can exhibit substantial differences in gene expression, protein levels and phenotypic output1–5, with important functional consequences4,5. Existing studies of cellular heterogeneity, however, have typically measured only a few pre-selected RNAs1,2 or proteins5,6 simultaneously, because genomic profiling methods3 could not be applied to single cells until very recently7–10. Here we use single-cell RNA sequencing to investigate heterogeneity in the response of mouse bone-marrow-derived dendritic cells (BMDCs) to lipopolysaccharide. We find extensive, and previously unobserved, bimodal variation in messenger RNA abundance and splicing patterns, which we validate by RNA-fluorescence in situ hybridization for select transcripts. In particular, hundreds of key immune genes are bimodally expressed across cells, surprisingly even for genes that are very highly expressed at the population average. Moreover, splicing patterns demonstrate previously unobserved levels of heterogeneity between cells. Some of the observed bimodality can be attributed to closely related, yet distinct, known maturity states of BMDCs; other portions reflect differences in the usage of key regulatory circuits. For example, we identify a module of 137 highly variable, yet co-regulated, antiviral response genes. Using cells from knockout mice, we show that variability in this module may be propagated through an interferon feedback circuit, involving the transcriptional regulators Stat2 and Irf7. Our study demonstrates the power and promise of single-cell genomics in uncovering functional diversity between cells and in deciphering cell states and circuits.

Individual genetic variation affects gene responsiveness to stimuli, often by influencing complex molecular circuits. Here we combine genomic and intermediate-scale transcriptional profiling with computational methods to identify variants that affect the responsiveness of genes to stimuli (responsiveness quantitative trait loci or reQTLs) and to position these variants in molecular circuit diagrams. We apply this approach to study variation in transcriptional responsiveness to pathogen components in dendritic cells from recombinant inbred mouse strains. We identify reQTLs that correlate with particular stimuli and position them in known pathways. For example, in response to a virus-like stimulus, a trans-acting variant responds as an activator of the antiviral response; using RNA interference, we identify Rgs16 as the likely causal gene. Our approach charts an experimental and analytic path to decipher the mechanisms underlying genetic variation in circuits that control responses to stimuli.

Despite their importance, the molecular circuits that control the differentiation of naive T cells remain largely unknown. Recent studies that reconstructed regulatory networks in mammalian cells have focused on short-term responses and relied on perturbation-based approaches that cannot be readily applied to primary T cells. Here we combine transcriptional profiling at high temporal resolution, novel computational algorithms, and innovative nanowirebased perturbation tools to systematically derive and experimentally validate a model of the dynamic regulatory network that controls the differentiation of mouse TH17 cells, a proinflammatory T-cell subset that has been implicated in the pathogenesis of multiple autoimmune diseases. The TH17 transcriptional network consists of two self-reinforcing, but mutually antagonistic, modules, with 12 novel regulators, the coupled action of which may be essential for maintaining the balance between TH17 and other CD4+ T-cell subsets. Our study identifies and validates 39 regulatory factors, embeds them within a comprehensive temporal network and reveals its organizational principles; it also highlights novel drug targets for controlling TH17 cell differentiation.

Developing a detailed understanding of enzyme function in the context of an intracellular signal transduction pathway requires minimally invasive methods for probing enzyme activity in situ. Here, we describe a new method for monitoring enzyme activity in living cells by sandwiching live cells between two vertical silicon nanowire (NW) arrays. Specifically, we use the first NW array to immobilize the cells and then present enzymatic substrates intracellularly via the second NW array by utilizing the NWs’ ability to penetrate cellular membranes without affecting cells’ viability or function. This strategy, when coupled with fluorescence microscopy and mass spectrometry, enables intracellular examination of protease, phosphatase, and protein kinase activities, demonstrating the assay’s potential in uncovering the physiological roles of various enzymes.

Deciphering the signaling networks that underlie normal and disease processes remains a major challenge. Here, we report the discovery of signaling components involved in the Toll-like receptor (TLR) response of immune dendritic cells (DCs), including a previously unkown pathway shared across mammalian antiviral responses. By combining transcriptional profiling, genetic and small-molecule perturbations, and phosphoproteomics, we uncover 35 signaling regulators, including 16 known regulators, involved in TLR signaling. In particular, we find that Polo-like kinases (Plk) 2 and 4 are essential components of antiviral pathways in vitro and in vivo and activate a signaling branch involving a dozen proteins, among which is Tnfaip2, a gene associated with autoimmune diseases but whose role was unknown. Our study illustrates the power of combining systematic measurements and perturbations to elucidate complex signaling circuits and discover potential therapeutic targets.