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.

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.