Faculty Bibliography

Mammalian aging is associated with multiple defects of hematopoiesis, most prominently with the impaired development of T and B lymphocytes. This defect is thought to originate in hematopoietic stem cells (HSCs) of the bone marrow, specifically due to the age-dependent accumulation of HSCs with preferential megakaryocytic and/or myeloid potential ("myeloid bias"). Here, we tested this notion using inducible genetic labeling and tracing of HSCs in unmanipulated animals. We found that the endogenous HSC population in old mice shows reduced differentiation into all lineages including lymphoid, myeloid, and megakaryocytic. Single-cell RNA sequencing and immunophenotyping (CITE-Seq) showed that HSC progeny in old animals comprised balanced lineage spectrum including lymphoid progenitors. Lineage tracing using the aging-induced HSC marker Aldh1a1 confirmed the low contribution of old HSCs across all lineages. Competitive transplantations of total bone marrow cells with genetically marked HSCs revealed that the contribution of old HSCs was reduced, but compensated by other donor cells in myeloid cells but not in lymphocytes. Thus, the HSC population in old animals becomes globally decoupled from hematopoiesis, which cannot be compensated in lymphoid lineages. We propose that this partially compensated decoupling, rather than myeloid bias, is the primary cause of the selective impairment of lymphopoiesis in older mice.

Developmental origins of dendritic cells (DCs) including conventional DCs (cDCs, comprising cDC1 and cDC2 subsets) and plasmacytoid DCs (pDCs) remain unclear. We studied DC development in unmanipulated adult mice using inducible lineage tracing combined with clonal DNA "barcoding" and single-cell transcriptome and phenotype analysis (CITE-seq). Inducible tracing of Cx3cr1⁺ hematopoietic progenitors in the bone marrow showed that they simultaneously produce all DC subsets including pDCs, cDC1s, and cDC2s. Clonal tracing of hematopoietic stem cells (HSCs) and of Cx3cr1⁺ progenitors revealed clone sharing between cDC1s and pDCs, but not between the two cDC subsets or between pDCs and B cells. Accordingly, CITE-seq analyses of differentiating HSCs and Cx3cr1⁺ progenitors identified progressive stages of pDC development including Cx3cr1⁺ Ly-6D⁺ pro-pDCs that were distinct from lymphoid progenitors. These results reveal the shared origin of pDCs and cDCs and suggest a revised scheme of DC development whereby pDCs share clonal relationship with cDC1s.

Antibodies to double-stranded DNA (dsDNA) are prevalent in systemic lupus erythematosus (SLE), particularly in patients with lupus nephritis, yet the nature and regulation of antigenic cell-free DNA (cfDNA) are poorly understood. Null mutations in the secreted DNase DNASE1L3 cause human monogenic SLE with anti-dsDNA autoreactivity. We report that >50% of sporadic SLE patients with nephritis manifested reduced DNASE1L3 activity in circulation, which was associated with neutralizing autoantibodies to DNASE1L3. These patients had normal total plasma cfDNA levels but showed accumulation of cfDNA in circulating microparticles. Microparticle-associated cfDNA contained a higher fraction of longer polynucleosomal cfDNA fragments, which bound autoantibodies with higher affinity than mononucleosomal fragments. Autoantibodies to DNASE1L3-sensitive antigens on microparticles were prevalent in SLE nephritis patients and correlated with the accumulation of cfDNA in microparticles and with disease severity. DNASE1L3-sensitive antigens included DNA-associated proteins such as HMGB1. Our results reveal autoantibody-mediated impairment of DNASE1L3 activity as a common nongenetic mechanism facilitating anti-dsDNA autoreactivity in patients with severe sporadic SLE.

Plasmacytoid dendritic cells (pDCs) can rapidly produce interferons and other soluble factors in response to extracellular viruses or virus mimics such as CpG-containing DNA. pDCs can also recognize live cells infected with certain RNA viruses, but the relevance and functional consequences of such recognition remain unclear. We studied the response of primary DCs to the prototypical persistent DNA virus, human cytomegalovirus (CMV). Human pDCs produced high amounts of type I interferon (IFN-I) when incubated with live CMV-infected fibroblasts but not with free CMV; the response involved integrin-mediated adhesion, transfer of DNA-containing virions to pDCs, and the recognition of DNA through TLR9. Compared with transient polyfunctional responses to CpG or free influenza virus, pDC response to CMV-infected cells was long-lasting, dominated by the production of IFN-I and IFN-III, and lacked diversification into functionally distinct populations. Similarly, pDC activation by influenza-infected lung epithelial cells was highly efficient, prolonged, and dominated by interferon production. Prolonged pDC activation by CMV-infected cells facilitated the activation of natural killer cells critical for CMV control. Last, patients with CMV viremia harbored phenotypically activated pDCs and increased circulating IFN-I and IFN-III. Thus, recognition of live infected cells is a mechanism of virus detection by pDCs that elicits a unique antiviral immune response.

Plasma DNA fragmentomics is an emerging area in cell-free DNA diagnostics and research. In murine models, it has been shown that the extracellular DNase, DNASE1L3, plays a role in the fragmentation of plasma DNA. In humans, DNASE1L3 deficiency causes familial monogenic systemic lupus erythematosus with childhood onset and anti-dsDNA reactivity. In this study, we found that human patients with DNASE1L3 disease-associated gene variations showed aberrations in size and a reduction of a "CC" end motif of plasma DNA. Furthermore, we demonstrated that DNA from DNASE1L3-digested cell nuclei showed a median length of 153 bp with CC motif frequencies resembling plasma DNA from healthy individuals. Adeno-associated virus-based transduction of Dnase1l3 into Dnase1l3-deficient mice restored the end motif profiles to those seen in the plasma DNA of wild-type mice. Our findings demonstrate that DNASE1L3 is an important player in the fragmentation of plasma DNA, which appears to act in a cell-extrinsic manner to regulate plasma DNA size and motif frequency.

The generation of all blood cell lineages (hematopoiesis) is sustained throughout the entire life span of adult mammals. Studies using cell transplantation identified the self-renewing, multipotent hematopoietic stem cells (HSCs) as the source of hematopoiesis in adoptive hosts and delineated a hierarchy of HSC-derived progenitors that ultimately yield mature blood cells. However, much less is known about adult hematopoiesis as it occurs in native hosts, i.e., without transplantation. Here we review recent advances in our understanding of native hematopoiesis, focusing in particular on the application of genetic lineage tracing in mice. The emerging evidence has established HSCs as the major source of native hematopoiesis, helped to define the kinetics of HSC differentiation, and begun exploring native hematopoiesis in stress conditions such as aging and inflammation. Major outstanding questions about native hematopoiesis still remain, such as its clonal composition, the nature of lineage commitment, and the dynamics of the process in humans. Expected final online publication date for the Annual Review of Cell and Developmental Biology, Volume 36 is October 6, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Adult mammalian hematopoietic stem cells (HSCs) reside in the bone marrow (BM) but can be mobilized into blood for use in transplantation. HSCs interact with BM niche cells that produce growth factor c-Kit ligand (Kitl/SCF) and chemokine CXCL12, and were thought to be static and sessile. We used two-photon laser scanning microscopy to visualize genetically labeled HSCs in the BM of live mice for several hours. The majority of HSCs showed a dynamic non-spherical morphology and significant motility, undergoing slow processive motion interrupted by short stretches of confined motion. HSCs moved in the perivascular space and showed intermittent close contacts with SCF-expressing perivascular stromal cells. In contrast, mobilization-inducing blockade of CXCL12 receptor CXCR4 and integrins rapidly abrogated HSC motility and shape dynamics in real time. Our results reveal an unexpectedly dynamic nature of HSC residence in the BM and interaction with the SCF⁺ stromal niche, which is disrupted during HSC mobilization.

Class-switched antibodies to double-stranded DNA (dsDNA) are prevalent and pathogenic in systemic lupus erythematosus (SLE), yet mechanisms of their development remain poorly understood. Humans and mice lacking secreted DNase DNASE1L3 develop rapid anti-dsDNA antibody responses and SLE-like disease. We report that anti-DNA responses in Dnase1l3^-/- mice require CD40L-mediated T cell help, but proceed independently of germinal center formation via short-lived antibody-forming cells (AFCs) localized to extrafollicular regions. Type I interferon (IFN-I) signaling and IFN-I-producing plasmacytoid dendritic cells (pDCs) facilitate the differentiation of DNA-reactive AFCs in vivo and in vitro and are required for downstream manifestations of autoimmunity. Moreover, the endosomal DNA sensor TLR9 promotes anti-dsDNA responses and SLE-like disease in Dnase1l3^-/- mice redundantly with another nucleic acid-sensing receptor, TLR7. These results establish extrafollicular B cell differentiation into short-lived AFCs as a key mechanism of anti-DNA autoreactivity and reveal a major contribution of pDCs, endosomal Toll-like receptors (TLRs), and IFN-I to this pathway.

Conventional type 1 dendritic cells (cDC1s) are inherently resistant to many viruses but, paradoxically, possess fewer acidic phagosomes that enable antigen retention and cross-presentation. We report that palmitoyl-protein thioesterase 1 (PPT1), which catabolizes lipid-modified proteins in neurons, is highly expressed in cDC1s. PPT1-deficient DCs are more susceptible to vesicular stomatitis virus (VSV) infection, and mice with PPT1 deficiency in cDC1s show impaired response to VSV. Conversely, PPT1-deficient cDC1s enhance the priming of naive CD8⁺ T cells into tissue-resident KLRG1⁺ effectors and memory T cells, resulting in rapid clearance of tumors and Listeria monocytogenes Mechanistically, PPT1 protects steady state DCs from viruses by promoting antigen degradation and endosomal acidification via V-ATPase recruitment. After DC activation, immediate down-regulation of PPT1 is likely to facilitate efficient cross-presentation, production of costimulatory molecules and inflammatory cytokines. Thus, PPT1 acts as a molecular rheostat that allows cDC1s to crossprime efficiently without compromising viral resistance. These results suggest potential therapeutics to enhance cDC1-dependent crosspriming.