CLEC4F, a member of C-type lectin, was first purified from rat liver extract with high binding affinity to fucose, galactose (Gal), N-acetylgalactosamine (GalNAc), and un-sialylated glucosphingolipids with GalNAc or Gal terminus. However, the biological functions of CLEC4F have not been elucidated. To address this question, we examined the expression and distribution of murine CLEC4F, determined its binding specificity by glycan array, and investigated its function using CLEC4F knockout (Clec4f−/−) mice. We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells. In contrast to F4/80, CLEC4F is detectable in fetal livers at embryonic day 11.5 (E11.5) but not in yolk sac, suggesting the expression of CLEC4F is induced as cells migrate from yolk cells to the liver. Even though CLEC4F is not detectable in tissues outside liver, both residential Kupffer cells and infiltrating mononuclear cells surrounding liver abscesses are CLEC4F-positive upon Listeria monocytogenes (L. monocytogenes) infection. While CLEC4F has strong binding to Gal and GalNAc, terminal fucosylation inhibits CLEC4F recognition to several glycans such as Fucosyl GM1, Globo H, Bb3∼4 and other fucosyl-glycans. Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer) in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT) cells. This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.

BackgroundChronic rhinosinusitis with nasal polyps is associated with local immunoglobulin hyperproduction and the presence of IgE antibodies against Staphylococcus aureus enterotoxins (SAEs). Aspirin-exacerbated respiratory disease is a severe form of chronic rhinosinusitis with nasal polyps in which nearly all patients express anti-SAEs.ObjectivesWe aimed to understand antibodies reactive to SAEs and determine whether they recognize SAEs through their complementarity-determining regions (CDRs) or framework regions.MethodsLabeled staphylococcal enterotoxin (SE) A, SED, and SEE were used to isolate single SAE-specific B cells from the nasal polyps of 3 patients with aspirin-exacerbated respiratory disease by using fluorescence-activated cell sorting. Recombinant antibodies with “matched” heavy and light chains were cloned as IgG1, and those of high affinity for specific SAEs, assayed by means of ELISA and surface plasmon resonance, were recloned as IgE and antigen-binding fragments. IgE activities were tested in basophil degranulation assays.ResultsThirty-seven SAE-specific, IgG- or IgA-expressing B cells were isolated and yielded 6 anti-SAE clones, 2 each for SEA, SED, and SEE. Competition binding assays revealed that the anti-SEE antibodies recognize nonoverlapping epitopes in SEE. Unexpectedly, each anti-SEE mediated SEE-induced basophil degranulation, and IgG1 or antigen-binding fragments of each anti-SEE enhanced degranulation by the other anti-SEE.ConclusionsSEEs can activate basophils by simultaneously binding as antigens in the conventional manner to CDRs and as superantigens to framework regions of anti-SEE IgE in anti-SEE IgE-FcεRI complexes. Anti-SEE IgG1s can enhance the activity of anti-SEE IgEs as conventional antibodies through CDRs or simultaneously as conventional antibodies and as “superantibodies” through CDRs and framework regions to SEEs in SEE–anti-SEE IgE-FcεRI complexes.

The epsilon chain of membrane-bound IgE (mIgE) is expressed predominantly as a "long" isoform, containing an extra segment of 52 amino acid (a.a.) residues, referred to as C epsilon mX, between the CH4 domain and the C-terminal membrane-anchoring transmembrane peptide. C epsilon mX results from an alternative splicing of the epsilon RNA transcript at 156-bp upstream of the splicing acceptor site used by the "short" isoform. Here, based on an analysis of the C epsilon mX genomic DNA sequences of 320 subjects residing in Taiwan, we report that single-nucleotide polymorphisms have been found at two positions, namely, G/T at #46 and A/G at #93 (along the 156 bp of C epsilon mX), with the former creating an amino acid change from Val to Leu at #16 (along the 52 a.a. of C epsilon mX) and the latter resulting in no change (Gly). Among the 640 C epsilon mX sequences identified, the previously known 46G93A allelic form appeared 293 times, the newly discovered 46T93A allelic form (GeneBank accession no. GU208817) 26 times, and the 46G93G allelic form (GU208818) 321 times. No 46T93G allelic form was found. Serum IgE measurements showed that the polymorphisms did not correlate with the levels of serum IgE. The anti-C epsilon mX monoclonal antibody, 4B12, could bind equally well to mIgE.Fc(L)(16V) and mIgE.Fc(L)(16L). While genetic variation of C epsilon mX of broader populations should also be investigated, these newly discovered genetic variants of C epsilon mX in the Taiwanese population do not seem to affect the feasibility of using an anti-C epsilon mX mAb, such as 4B12, to target mIgE-expressing B cells.