Crystal Structure of a Superantigen Bound to the High-Affinity, Zinc-Dependent Site on MHC Class II

Abstract CXCL14 is a primordial CXC-type chemokine that induces migration of immature dendritic cells (DCs), tissue resident macrophages, and natural killer cells. It has been reported that CXCL14 plays multiple roles in tumor suppression, exacerbation of autoimmune arthritis, and induction of obesity-associated insulin resistance. However, underlying molecular mechanisms of these phenomena remain to be elucidated. Although CXCL14 binds to the CXCL12 receptor CXCR4 with high affinity, chemoattractive activity of CXCL14 is much weaker than CXCL12. In this study, we newly discovered that CXCL14 specifically binds to CpG DNA and activates Toll-like receptor 9 (TLR9), thereby inducing inflammatory cytokines such as IL-6, IL-12 p40, and TNFa in mouse bone marrow-derived dendritic cells (BMDCs). Cell surface expression levels of MHC class-II and CD86 in BMDCs were also enhanced by the combination of CpG DNA and CXCL14. In this experimental setting, CXCL14 interacted with certain classes of CpG DNA, but not with RNA ligands for TLR3 and TLR8. In addition, this CpG DNA-cooperative activity was not present in CXCL8 and CXCL12, excluding a nonspecific interaction between CpG DNA and an alkaline chemokine. In BMDCs and inguinal lymph node DCs, intracellular transport of a low concentration of CpG DNA was greatly enhanced by the addition of CXCL14. Confocal microscopical analyses revealed that CpG DNA and CXCL14 mainly co-localized in EEA1+ endosome and LAMP1+ lysosomal compartments where TLR9 is present. Furthermore, we demonstrated that CXCL14 binds to CpG DNA in vitro in the neutral pH condition with high affinity (Kd=9.8 nM). This interaction was completely dissociated in pH 6.0, implying that CpG DNA can be released and passed to TLR9 in the endosome and lysosome. Consistent with our hypothesis, induction of IL-12 p40, MHC class-II, and CD86 by the combination of CpG DNA and CXCL14 was not observed in BMDCs derived from TLR9 knockout (KO) mice. Moreover, after systemic administration of CpG DNA, plasma concentration of IL-12 p40 and frequency of MHC-class II+CD11c+CD8+DCs in spleen were significantly decreased in CXCL14-deficient mice when compared to littermate control mice. Taken together, these results demonstrated that CXCL14 serves as a specific carrier for CpG DNA into conventional DCs for activating TLR9-mediated adaptive immune system. This is also the first demonstration of a DNA sensing function of chemokine. The combination of CXCL14 and CpG DNA would be a promising vaccine adjuvant for enhancing immnunosurveillance against pathogens and malignant cancers. Disclosures Tanegashima: Tokyo Metropolitan Institute of Medical Science: Patents & Royalties. Takahashi:Tokyo Metropolitan Institute of Medical Science: Patents & Royalties. Tsuji:Tokushima University: Patents & Royalties. Shigenaga:Tokushima University: Patents & Royalties. Otaka:Tokushima University: Patents & Royalties.

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Epitope Identification and Generation of an Antigen-Specific T-Cell Clone in a Mild Hemophilia A Inhibitor Subject with Missense Genotype R593C.

Blood ◽

10.1182/blood.v110.11.1159.1159 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1159-1159 ◽

Cited By ~ 1

Author(s):

Eddie A. James ◽

Simon D. van Haren ◽

Ruth A. Ettinger ◽

Arthur R. Thompson ◽

William W. Kwok ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Hemophilia A ◽

Cell Clone ◽

Peptide Binding ◽

Class Ii ◽

High Affinity ◽

T Cell Clone ◽

A2 Domain

Abstract The development of inhibitory anti-FVIII antibodies is a major clinical problem in hemophilia A. While less common in mild/moderately severe patients, the relative risk of inhibitor formation is elevated in patients with missense mutations in the FVIII A2 domain, especially those with an R593C genotype. In this study, T-cell responses to 14 FVIII A2 domain peptides with predicted DRB1*1101 MHC binding motifs were investigated using MHC class II tetramer reagents. CD4+ T cells were isolated from a hemophilic subject (DRB1*1101, 1302) with FVIII missense genotype R593C and stimulated with pooled peptides. This subject had developed a longstanding low titer inhibitor after receiving multiple FVIII infusions. Staining with fluorescent, peptide-loaded tetramers revealed that the hemophilic subject, but not an HLA-matched healthy control, had a DRB1*1101-restricted response to peptide A2589–608, which contained the wild-type R593 sequence. MHC class II tetramers with bound A2589–608 were used to sort antigen-specific T cells and then generate a T-cell clone recognizing this high avidity DRB1*1101-restricted epitope. FVIII residues 594–602 (FLPNPAGVQ) comprise a predicted high-affinity binding motif. Peptide binding assays confirmed that A2589–608 bound to recombinant, monomeric DRB1*1101 protein with high affinity. A peptide with the corresponding hemophilic sequence (A2589–608, 593C) bound with affinity that was threefold lower, but in a range that should be sufficient for presentation to T cells. The other 12 A2 peptides had a wide spectrum of binding affinities for the DRB1*1101 protein. Interestingly, prediction algorithms suggest that the peptides containing residue 593 occupy the MHC Class II peptide-binding groove in a register that places this residue just outside the p1 anchor position. Thus we anticipate that residue 593 modulates T-cell recognition of peptide A2589–608. This provides a potential explanation for inhibitor development in mild hemophilia A patients with a FVIII R593C genotype who are HLA-DRB1*1101. Immunogenicity of this FVIII region with respect to other HLA types is under investigation.

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