Faculty Bibliography

An IRF8-dependent subset of conventional dendritic cells (cDCs), termed cDC1, effectively cross-primes CD8⁺ T cells and facilitates tumor-specific T cell responses. Etv6 is an ETS family transcription factor that controls hematopoietic stem and progenitor cell (HSPC) function and thrombopoiesis. We report that like HSPCs, cDCs express Etv6, but not its antagonist, ETS1, whereas interferon-producing plasmacytoid dendritic cells (pDCs) express both factors. Deletion of Etv6 in the bone marrow impaired the generation of cDC1-like cells in vitro and abolished the expression of signature marker CD8α on cDC1 in vivo. Moreover, Etv6-deficient primary cDC1 showed a partial reduction of cDC-specific and cDC1-specific gene expression and chromatin signatures and an aberrant up-regulation of pDC-specific signatures. Accordingly, DC-specific Etv6 deletion impaired CD8⁺ T cell cross-priming and the generation of tumor antigen-specific CD8⁺ T cells. Thus, Etv6 optimizes the resolution of cDC1 and pDC expression programs and the functional fitness of cDC1, thereby facilitating T cell cross-priming and tumor-specific responses.

The IRF8-dependent subset of classical dendritic cells (cDCs), termed cDC1, is important for cross-priming cytotoxic T cell responses against pathogens and tumors. Culture of hematopoietic progenitors with DC growth factor FLT3 ligand (FLT3L) yields very few cDC1s (in humans) or only immature "cDC1-like" cells (in the mouse). We report that OP9 stromal cells expressing the Notch ligand Delta-like 1 (OP9-DL1) optimize FLT3L-driven development of cDC1s from murine immortalized progenitors and primary bone marrow cells. Co-culture with OP9-DL1 induced IRF8-dependent cDC1s with a phenotype (CD103⁺ Dec205⁺ CD8α⁺) and expression profile resembling primary splenic cDC1s. OP9-DL1-induced cDC1s showed preferential migration toward CCR7 ligands in vitro and superior T cell cross-priming and antitumor vaccination in vivo. Co-culture with OP9-DL1 also greatly increased the yield of IRF8-dependent CD141⁺ cDC1s from human bone marrow progenitors cultured with FLT3L. Thus, Notch signaling optimizes cDC generation in vitro and yields authentic cDC1s for functional studies and translational applications.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition caused by an aberrant immune response to microbial components of the gastrointestinal tract. Plasmacytoid dendritic cells (pDCs) are innate immune cells specialized in the production of type I interferons and were recently implicated in the pathogenesis of autoimmune disorders such as lupus and scleroderma. While pDCs were shown to infiltrate intestinal mucosa of IBD patients and proposed to participate in intestinal inflammation, their net contribution to the disease remains unclear. We addressed this question by targeting the pDC-specific transcription factor TCF4 (E2-2) in experimental IBD caused by deficiency of Wiskott-Aldrich syndrome protein (WASP) or of interleukin-10 (IL-10). Monoallelic Tcf4 deletion, which was previously shown to abrogate experimental lupus, did not affect autoimmunity manifestations or colitis in WASP-deficient animals. Furthermore, conditional biallelic Tcf4 targeting resulted in a near-complete pDC ablation, yet had no effect on the development of colitis in IL-10-deficient mice. Our results suggest that, in contrast to other inflammatory and autoimmune diseases, pDCs do not play a major role in the pathogenesis of intestinal inflammation during IBD.

Maturation and migration to lymph nodes (LNs) constitutes a central paradigm in conventional dendritic cell (cDC) biology but remains poorly defined in humans. Using our organ donor tissue resource, we analyzed cDC subset distribution, maturation, and migration in mucosal tissues (lungs, intestines), associated lymph nodes (LNs), and other lymphoid sites from 78 individuals ranging from less than 1 year to 93 years of age. The distribution of cDC1 (CD141hiCD13hi) and cDC2 (Sirp-alpha+CD1c+) subsets was a function of tissue site and was conserved between donors. We identified cDC2 as the major mature (HLA-DRhi) subset in LNs with the highest frequency in lung-draining LNs. Mature cDC2 in mucosal-draining LNs expressed tissue-specific markers derived from the paired mucosal site, reflecting their tissue-migratory origin. These distribution and maturation patterns were largely maintained throughout life, with site-specific variations. Our findings provide evidence for localized DC tissue surveillance and reveal a lifelong division of labor between DC subsets, with cDC2 functioning as guardians of the mucosa.

The cell fate decision between interferon-producing plasmacytoid DC (pDC) and antigen-presenting classical DC (cDC) is controlled by the E protein transcription factor TCF4 (E2-2). We report that TCF4 comprises two transcriptional isoforms, both of which are required for optimal pDC development in vitro. The long Tcf4 isoform is expressed specifically in pDCs, and its deletion in mice impaired pDCs development and led to the expansion of non-canonical CD8+ cDCs. The expression of Tcf4 commenced in progenitors and was further upregulated in pDCs, correlating with stage-specific activity of multiple enhancer elements. A conserved enhancer downstream of Tcf4 was required for its upregulation during pDC differentiation, revealing a positive feedback loop. The expression of Tcf4 and the resulting pDC differentiation were selectively sensitive to the inhibition of enhancer-binding BET protein activity. Thus, lineage-specifying function of E proteins is facilitated by lineage-specific isoform expression and by BET-dependent feedback regulation through distal regulatory elements.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive and largely incurable hematologic malignancy originating from plasmacytoid dendritic cells (pDCs). Using RNAi screening, we identified the E-box transcription factor TCF4 as a master regulator of the BPDCN oncogenic program. TCF4 served as a faithful diagnostic marker of BPDCN, and its downregulation caused the loss of the BPDCN-specific gene expression program and apoptosis. High-throughput drug screening revealed that bromodomain and extra-terminal domain inhibitors (BETis) induced BPDCN apoptosis, which was attributable to disruption of a BPDCN-specific transcriptional network controlled by TCF4-dependent super-enhancers. BETis retarded the growth of BPDCN xenografts, supporting their clinical evaluation in this recalcitrant malignancy.

Hematopoietic stem cells (HSCs) sustain long-term reconstitution of hematopoiesis in transplantation recipients, yet their role in the endogenous steady-state hematopoiesis remains unclear. In particular, recent studies suggested that HSCs provide a relatively minor contribution to immune cell development in adults. We directed transgene expression in a fraction of HSCs that maintained reconstituting activity during serial transplantations. Inducible genetic labeling showed that transgene-expressing HSCs gave rise to other phenotypic HSCs, confirming their top position in the differentiation hierarchy. The labeled HSCs rapidly contributed to committed progenitors of all lineages and to mature myeloid cells and lymphocytes, but not to B-1a cells or tissue macrophages. Importantly, labeled HSCs gave rise to more than two-thirds of all myeloid cells and platelets in adult mice, and this contribution could be accelerated by an induced interferon response. Thus, classically defined HSCs maintain immune cell development in the steady state and during systemic cytokine responses.

Antibodies to DNA and chromatin drive autoimmunity in systemic lupus erythematosus (SLE). Null mutations and hypomorphic variants of the secreted deoxyribonuclease DNASE1L3 are linked to familial and sporadic SLE, respectively. We report that DNASE1L3-deficient mice rapidly develop autoantibodies to DNA and chromatin, followed by an SLE-like disease. Circulating DNASE1L3 is produced by dendritic cells and macrophages, and its levels inversely correlate with anti-DNA antibody response. DNASE1L3 is uniquely capable of digesting chromatin in microparticles released from apoptotic cells. Accordingly, DNASE1L3-deficient mice and human patients have elevated DNA levels in plasma, particularly in circulating microparticles. Murine and human autoantibody clones and serum antibodies from human SLE patients bind to DNASE1L3-sensitive chromatin on the surface of microparticles. Thus, extracellular microparticle-associated chromatin is a potential self-antigen normally digested by circulating DNASE1L3. The loss of this tolerance mechanism can contribute to SLE, and its restoration may represent a therapeutic opportunity in the disease.

A common genetic alteration in acute myeloid leukemia is the internal tandem duplication (ITD) in FLT3, the receptor for cytokine FLT3 ligand (FLT3L). Constitutively active FLT3-ITD promotes the expansion of transformed progenitors, but also has pleiotropic effects on hematopoiesis. We analyzed the effect of FLT3-ITD on dendritic cells (DCs), which express FLT3 and can be expanded by FLT3L administration. Pre-leukemic mice with the Flt3ITD knock-in allele manifested an expansion of classical DCs (cDCs) and plasmacytoid DCs. The expansion originated in DC progenitors, was cell intrinsic, and was further enhanced in Flt3ITD/ITD mice. The mutation caused the down-regulation of Flt3 on the surface of DCs and reduced their responsiveness to Flt3L. Both canonical Batf3-dependent CD8+ cDCs and noncanonical CD8+ cDCs were expanded and showed specific alterations in their expression profiles. Flt3ITD mice showed enhanced capacity to support T cell proliferation, including a cell-extrinsic expansion of regulatory T (T reg) cells. Accordingly, these mice restricted alloreactive T cell responses during graft-versus-host reaction, but failed to control autoimmunity without T reg cells. Thus, the FLT3-ITD mutation directly affects DC development, indirectly modulating T cell homeostasis and supporting T reg cell expansion. We hypothesize that this effect of FLT3-ITD might subvert immunosurveillance and promote leukemogenesis in a cell-extrinsic manner.

Plasmacytoid dendritic cells (pDCs) are primary producers of type I interferon (IFN) in response to viruses. The IFN-producing capacity of pDCs is regulated by specific inhibitory receptors, yet none of the known receptors are conserved in evolution. We report that within the human immune system, receptor protein tyrosine phosphatase sigma (PTPRS) is expressed specifically on pDCs. Surface PTPRS was rapidly downregulated after pDC activation, and only PTPRS- pDCs produced IFN-alpha. Antibody-mediated PTPRS crosslinking inhibited pDC activation, whereas PTPRS knockdown enhanced IFN response in a pDC cell line. Similarly, murine Ptprs and the homologous receptor phosphatase Ptprf were specifically co-expressed in murine pDCs. Haplodeficiency or DC-specific deletion of Ptprs on Ptprf-deficient background were associated with enhanced IFN response of pDCs, leukocyte infiltration in the intestine and mild colitis. Thus, PTPRS represents an evolutionarily conserved pDC-specific inhibitory receptor, and is required to prevent spontaneous IFN production and immune-mediated intestinal inflammation.