Constitutive Flt3 signaling impacts conventional dendritic cell function.

The development of dendritic cells (DCs) depends on signaling via the FMS-like tyrosine kinase 3 (Flt3) receptor. How Flt3 signaling impacts terminally differentiated DC function is unknown. This is important given the increasing interest in exploiting Flt3 for vaccination and tumor immunotherapy. Here, we examined DCs in mice harboring constitutively activated Flt3 (Flt3-ITD). Flt3ITD/ITD mice possessed expanded splenic DC subsets including plasmacytoid DC, conventional DC (cDC)1, cDC2, double positive (DP) cDC1 (CD11c+ CD8+ CD11b- CD103+ CD86+), noncanonical (NC) cDC1 (CD11c+ CD8+ CD11b- CD103- CD86-) and single positive (SP) cDC1 (CD11c+ CD8+ CD11b- CD103- CD86+). Outcomes of constitutive Flt3 signaling differed depending on the cDC subset examined. In comparison with wild type (WT) DCs, all Flt3ITD/ITD cDCs displayed an altered surface phenotype with changes in costimulatory molecules, major histocompatibility complex class I (MHC I) and II (MHC II). Cytokine secretion patterns, antigen uptake, antigen proteolysis and antigen presenting function differed between WT and Flt3ITD/ITD subsets, particularly cDC2. In summary, Flt3 signaling impacts the function of terminally differentiated cDCs with important consequences for antigen presentation.

Microbial Metabolites in the Maturation and Activation of Dendritic Cells and Their Relevance for Respiratory Immunity.

The respiratory tract is a gateway for viruses and bacteria from the external environment to invade the human body. Critical to the protection against these invaders are dendritic cells (DCs) - a group of highly specialized myeloid cells that monitors the lung microenvironment and relays contextual and antigenic information to T cells. Following the recognition of danger signals and/or pathogen molecular associated patterns in the lungs, DCs undergo activation. This process arms DCs with the unique ability to induce the proliferation and differentiation of T cells responding to matching antigen in complex with MHC molecules. Depending on how DCs interact with T cells, the ensuing T cell response can be tolerogenic or immunogenic and as such, the susceptibility and severity of respiratory infections is influenced by the signals DCs receive, integrate, and then convey to T cells. It is becoming increasingly clear that these facets of DC biology are heavily influenced by the cellular components and metabolites produced by the lung and gut microbiota. In this review, we discuss the roles of different DC subsets in respiratory infections and outline how microbial metabolites impact the development, propensity for activation and subsequent activation of DCs. In particular, we highlight these concepts in the context of respiratory immunity.

Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86.

The MARCH E3 ubiquitin (Ub) ligase MARCH1 regulates trafficking of major histocompatibility complex class II (MHC II) and CD86, molecules of critical importance to immunity. Here we show, using a genome-wide CRISPR knockout screen, that ubiquitin-like protein 3 (UBL3) is a necessary component of ubiquitination-mediated trafficking of these molecules in mice and in humans. Ubl3-deficient mice have elevated MHC II and CD86 expression on the surface of professional and atypical antigen presenting cells. UBL3 also regulates MHC II and CD86 in human dendritic cells (DCs) and macrophages. UBL3 impacts ubiquitination of MARCH1 substrates, a mechanism that requires UBL3 plasma membrane anchoring via prenylation. Loss of UBL3 alters adaptive immunity with impaired development of thymic regulatory T cells, loss of conventional type 1 DCs, increased number of trogocytic marginal zone B cells, and defective in vivo MHC II and MHC I antigen presentation. In summary, we identify UBL3 as a conserved, critical factor in MARCH1-mediated ubiquitination with important roles in immune responses.

MHC Class II Ubiquitination Regulates Dendritic Cell Function and Immunity.

MHC class II (MHC II) Ag presentation by dendritic cells (DCs) is critical for CD4+ T cell immunity. Cell surface levels of MHC II loaded with peptide is controlled by ubiquitination. In this study, we have examined how MHC II ubiquitination impacts immunity using MHC IIKRKI/KI mice expressing mutant MHC II molecules that are unable to be ubiquitinated. Numbers of conventional DC (cDC) 1, cDC2 and plasmacytoid DCs were significantly reduced in MHC IIKRKI/KI spleen, with the remaining MHC IIKRKI/KI DCs expressing an altered surface phenotype. Whereas Ag uptake, endosomal pH, and cathepsin protease activity were unaltered, MHC IIKRKI/KI cDC1 produced increased inflammatory cytokines and possessed defects in Ag proteolysis. Immunization of MHC IIKRKI/KI mice identified impairments in MHC II and MHC class I presentation of soluble, cell-associated and/or DC-targeted OVA via mAb specific for DC surface receptor Clec9A (anti-Clec9A-OVA mAb). Reduced T cell responses and impaired CTL killing was observed in MHC IIKRKI/KI mice following immunization with cell-associated and anti-Clec9A-OVA. Immunization of MHC IIKRKI/KI mice failed to elicit follicular Th cell responses and generated barely detectable Ab to anti-Clec9A mAb-targeted Ag. In summary, MHC II ubiquitination in DCs impacts the homeostasis, phenotype, cytokine production, and Ag proteolysis by DCs with consequences for Ag presentation and T cell and Ab-mediated immunity.

Dendritic cell Flt3 - regulation, roles and repercussions for immunotherapy.

Dendritic cells (DCs) are essential for initiating immune responses. Depending on the environment, the type of DC and the way in which they interact with T cells, these immune responses can be beneficial or detrimental. DCs can be exploited as cellular vectors for vaccines against infection and cancer. The development and maintenance of DCs is dependent on the FMS-like tyrosine kinase 3 (Flt3)/Flt3 ligand (Flt3L) signaling cascade. Flt3 is also one of the most commonly mutated genes in acute myeloid leukemia and as such represents an attractive drug target. In this review, Flt3 is discussed with a particular focus on DCs. We detail the lifecycle of Flt3, from transcription to degradation, and interrogate recent studies as to how this pathway can be manipulated for immunotherapy, vaccination and treatment of autoimmune disease.

Physiological substrates and ontogeny-specific expression of the ubiquitin ligases MARCH1 and MARCH8.

MARCH1 and MARCH8 are ubiquitin ligases that control the expression and trafficking of critical immunoreceptors. Understanding of their function is hampered by three major knowledge gaps: (i) it is unclear which cell types utilize these ligases; (ii) their level of redundancy is unknown; and (iii) most of their putative substrates have been described in cell lines, often overexpressing MARCH1 or MARCH8, and it is unclear which substrates are regulated by either ligase in vivo. Here we address these questions by systematically analyzing the immune cell repertoire of MARCH1- or MARCH8-deficient mice, and applying unbiased proteomic profiling of the plasma membrane of primary cells to identify MARCH1 and MARCH8 substrates. Only CD86 and MHC II were unequivocally identified as immunoreceptors regulated by MARCH1 and MARCH8, but each ligase carried out its function in different tissues. MARCH1 regulated MHC II and CD86 in professional and "atypical" antigen presenting cells of hematopoietic origin, including neutrophils, eosinophils and monocytes. MARCH8 only operated in non-hematopoietic cells, such as thymic and alveolar epithelial cells. Our results establish the tissue-specific functions of MARCH1 and MARCH8 in regulation of immune receptor expression and reveal that the range of cells constitutively endowed with antigen-presentation capacity is wider than generally appreciated.

Ubiquitination of MHC Class II Is Required for Development of Regulatory but Not Conventional CD4+ T Cells.

MHC class II (MHC II) displays peptides at the cell surface, a process critical for CD4+ T cell development and priming. Ubiquitination is a mechanism that dictates surface MHC II with the attachment of a polyubiquitin chain to peptide-loaded MHC II, promoting its traffic away from the plasma membrane. In this study, we have examined how MHC II ubiquitination impacts the composition and function of both conventional CD4+ T cell and regulatory T cell (Treg) compartments. Responses were examined in two models of altered MHC II ubiquitination: MHCIIKRKI /KI mice that express a mutant MHC II unable to be ubiquitinated or mice that lack membrane-associated RING-CH 8 (MARCH8), the E3 ubiquitin ligase responsible for MHC II ubiquitination specifically in thymic epithelial cells. Conventional CD4+ T cell populations in thymus, blood, and spleen of MHCIIKRKI/KI and March8 -/- mice were largely unaltered. In MLRs, March8 -/-, but not MHCIIKRKI/KI, CD4+ T cells had reduced reactivity to both self- and allogeneic MHC II. Thymic Treg were significantly reduced in MHCIIKRKI/KI mice, but not March8 -/- mice, whereas splenic Treg were unaffected. Neither scenario provoked autoimmunity, with no evidence of immunohistopathology and normal levels of autoantibody. In summary, MHC II ubiquitination in specific APC types does not have a major impact on the conventional CD4+ T cell compartment but is important for Treg development.

MARCH1-mediated ubiquitination of MHC II impacts the MHC I antigen presentation pathway.

Major histocompatibility complex class II (MHC II) expression and turn-over are regulated via its ubiquitination by the membrane associated RING-CH 1 (MARCH1) E3 ligase. Unexpectedly, we show that MHC II ubiquitination also impacts MHC I. Lack of MARCH1 in B cells and dendritic cells (DCs) resulted in a significant reduction in surface MHC I expression. This decrease was not directly caused by changes in MARCH1 ubiquitination of MHC I but indirectly by altered MHC II trafficking in the absence of its ubiquitination. Deletion of MHC II in March1-/- cells restored normal MHC I surface expression and replacement of wild type MHC II by a variant that could not be ubiquitinated caused a reduction in MHC I expression. Furthermore, these cells displayed inefficient presentation of peptide and protein antigen via MHC I to CD8+ T cells. In summary, we describe an unexpected intersection between MHC I and MHC II such that the surface expression of both molecules are indirectly and directly regulated by MARCH1 ubiquitination, respectively.