The role of H2‐O and HLA‐DO in major histocompatibility complex class Il‐restricted antigen processing and presentation

Class II transcriptional activator (CIITA) is a master regulator of MHC class II genes, including DR, DP, and DQ, and MHC class II-associated genes DM and invariant chain. To determine the repertoire of genes that is regulated by CIITA and to identify uncharacterized CIITA-inducible genes, we used representational difference analysis. Representational difference analysis screens for differentially expressed transcripts. All CIITA-induced genes were MHC class II related. We have identified the α subunit, DNα, of the class II processing factor DO as an additional CIITA-inducible gene. Northern analysis confirmed that DNα is induced by IFN-γ in 2fTGH fibrosarcoma cells, and CIITA is necessary for high-level expression in B cells. The β subunit, DOβ, is not inducible in fibrosarcoma cells by IFN-γ or exogenous CIITA expression. Moreover, in contrast to other class II genes, DOβ expression remains high in the absence of CIITA in B cells. The promoters for DNα and DOβ contain the highly conserved WXY motifs, and, like other class II genes, expression of both DNα and DOβ requires RFX. These findings demonstrate that both DNα and DOβ are regulated by RFX. However, DNα is defined for the first time as a CIITA-inducible gene, and DOβ as a MHC class II gene whose expression is independent of CIITA.

Section: Discussionmentioning

confidence: 99%

Identification of Class II Transcriptional Activator-Induced Genes by Representational Difference Analysis: Discoordinate Regulation of the DNα/DOβ Heterodimer

Taxman

Cressman

Ting

2000

“…In order for loading of peptides derived from antigens to occur, MHCII-bound CLIP must be exchanged for self- and foreign-derived peptides. Two MHCII homologues, H2-M (HLA-DM in humans; DM) and H2-O (HLA-DO in humans; DO) control this process (2, 3). H2-M facilitates the exchange of CLIP for antigen-derived peptides, prolongs the half-life of peptide receptive MHCII molecules and also ensures that only high affinity MHCII-peptide complexes are presented at the cell surface (4–8).…”

Section: Introductionmentioning

confidence: 99%

TLR Agonists Downregulate H2-O in CD8α− Dendritic Cells

Porter

Denzin

2011

Peptide loading of MHC class II (MHCII) molecules is catalyzed by the non-classical MHCII-related molecule, H2-M. H2-O, another MHCII-like molecule, associates with H2-M and modulates H2-M function. The MHCII presentation pathway is tightly regulated in dendritic cells (DCs); yet how the key modulators of MHCII presentation, H2-M and H2-O, are affected in different DC subsets in response to maturation is unknown. Here we show that H2-O is markedly downregulated in vivo in mouse CD8α− DCs in response to a broad array of TLR agonists. In contrast, CD8α+ DCs only modestly downregulated H2-O in response to TLR-agonists. H2-M levels were slightly down-modulated in both CD8α− and CD8α+ DCs. As a consequence, H2-M:H2-O ratios significantly increased for CD8α− but not CD8α+ DCs. The TLR-mediated downregulation was DC-specific, as B cells did not show significant H2-O and H2-M downregulation. TLR4 signaling was required to mediate DC H2-O downregulation in response to LPS. Finally, our studies showed that the mechanism of H2-O downregulation was likely due to direct protein degradation of H2-O as well as down regulation of H2-O mRNA levels. The differential H2-O and H2-M modulation after DC maturation support the proposed roles of CD8α− dendritic cells in initiating CD4-restricted immune responses by optimal MHCII presentation and CD8α+ DCs in promoting immune tolerance via presentation of low levels of MHCII-peptide.

“…Peptide loading of class II molecules requires productive interactions with H-2M and H-2O molecules (44,45). Therefore, we determined whether the HLA class II molecules expressed in mouse cells formed stable dimers in SDS, indicating their assembly with peptide Ag.…”

Section: Regulated Expression Of Hla Dr and Dq In Yac-transgenic Micementioning

confidence: 99%

A 320-Kilobase Artificial Chromosome Encoding the Human HLA DR3-DQ2 MHC Haplotype Confers HLA Restriction in Transgenic Mice

Chen

Dudek

Wijburg

et al. 2002

MHC class II haplotypes control the specificity of Th immune responses and susceptibility to many autoimmune diseases. Understanding the role of HLA class II haplotypes in immunity is hampered by the lack of animal models expressing these genes as authentic cis-haplotypes. In this study we describe transgenic expression of the autoimmune prone HLA DR3-DQ2 haplotype from a yeast artificial chromosome (YAC) containing an intact ∼320-kb region from HLA DRA to DQB2. In YAC-transgenic mice HLA DR and DQ gene products were expressed on B cells, macrophages, and dendritic cells, but not on T cells indicating cell-specific regulation. Positive selection of the CD4 compartment by human class II molecules was 67% efficient in YAC-homozygous mice lacking endogenous class II molecules (Aβnull/null) and expressing only murine CD4. A broad range of TCR Vβ families was used in the peripheral T cell repertoire, which was also purged of Vβ5-, Vβ11-, and Vβ12-bearing T cells by endogenous mouse mammary tumor virus-encoded superantigens. Expression of the HLA DR3-DQ2 haplotype on the Aβnull/null background was associated with normal CD8-dependent clearance of virus from influenza-infected mice and development of CD4-dependent protection from otherwise lethal infection with Salmonella typhimurium. HLA DR- and DQ-restricted T cell responses were also elicited following immunization with known T cell determinants presented by these molecules. These findings demonstrate the potential for human MHC class II haplotypes to function efficiently in transgenic mice and should provide valuable tools for developing humanized models of MHC-associated autoimmune diseases.