Multiple environmental antigens may trigger autoimmunity in psoriasis through T-cell receptor polyspecificity.

Introduction: Psoriasis is a T-cell mediated autoimmune skin disease. HLA-C*06:02 is the main psoriasis-specific risk gene. Using a Vα3S1/Vß13S1 T-cell receptor (TCR) from a lesional psoriatic CD8+ T-cell clone we had discovered that, as an underlying pathomechanism, HLA-C*06:02 mediates an autoimmune response against melanocytes in psoriasis, and we had identified an epitope from ADAMTS-like protein 5 (ADAMTSL5) as a melanocyte autoantigen. The conditions activating the psoriatic autoimmune response in genetically predisposed individuals throughout life remain incompletely understood. Here, we aimed to identify environmental antigens that might trigger autoimmunity in psoriasis because of TCR polyspecificity. Methods: We screened databases with the peptide recognition motif of the Vα3S1/Vß13S1 TCR for environmental proteins containing peptides activating this TCR. We investigated the immunogenicity of these peptides for psoriasis patients and healthy controls by lymphocyte stimulation experiments and peptide-loaded HLA-C*06:02 tetramers. Results: We identified peptides from wheat, Saccharomyces cerevisiae, microbiota, tobacco, and pathogens that activated both the Vα3S1/Vß13S1 TCR and CD8+ T cells from psoriasis patients. Using fluorescent HLA-C*06:02 tetramers loaded with ADAMTSL5 or wheat peptides, we find that the same CD8+ T cells may recognize both autoantigen and environmental antigens. A wheat-free diet could alleviate psoriasis in several patients. Discussion: Our results show that due to TCR polyspecificity, several environmental antigens corresponding to previously suspected psoriasis risk conditions converge in the reactivity of a pathogenic psoriatic TCR and might thus be able to stimulate the psoriatic autoimmune response against melanocytes. Avoiding the corresponding environmental risk factors could contribute to the management of psoriasis.

Detection and characterization of syndecan-1-associated heparan sulfate 6-O-sulfated motifs overexpressed in multiple myeloma cells using single chain antibody variable fragments.

This study was undertaken to generate human single chain variable antibody fragments (scFvs) reacting specifically against multiple myeloma (MM) cells using the phage display technique. To isolate myeloma-specific scFvs, we used a simple subtractive strategy by adsorbing the Griffin #1 antibody phage library against myeloma cells in the presence of excess decoy biotinylated HL60 cells, and then removing the unwanted decoy cells using streptavidin coated plates. From eleven scFvs that were isolated, two antibodies, D4A4 and D6B10 stained MM cell lines and patient MM cells with higher intensity than normal plasma cells. Both D4A4 and D6B10 scFvs immunoprecipitated syndecan-1 from myeloma cells and recognized sulfated motifs on syndecan-1-associated heparan sulfate (HS) chains. ScFv D4A4 competed with D6B10 for binding to MM cells. However, they differed in their fine specificities. ScFv D6B10 recognized HS 2,6-O-, N-sulfated motifs and, in contrast, binding of scFv D4A4 required N-sulfation combined with either 2-O- or 6-O-sulfation. Increased D6B10 binding on MM cells suggests that their HS chains contain a greater number of 2,6-O-, N-sulfated motifs than normal plasma cells. Since these highly sulfated motifs bind various angiogenic and growth factors and present them to their respective receptors, they could be instrumental for MM cell survival, proliferation and metastasis. Therefore, scFvs D4A4 and D6B10 provide a means to easily monitor changes in sulfation patterns of heparan sulfate during myeloma tumor progression.

TNF-alpha increases the carbohydrate sulfation of CD44: induction of 6-sulfo N-acetyl lactosamine on N- and O-linked glycans.

CD44 and sulfation have both been implicated in leukocyte adhesion. In monocytes, the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) stimulates CD44 sulfation, and this correlates with the induction of CD44-mediated adhesion events. However, little is known about the sulfation of CD44 or its induction by inflammatory cytokines. We determined that TNF-alpha induces the carbohydrate sulfation of CD44. CD44 was established as a major sulfated cell surface protein on myeloid cells. In the SR91 myeloid cell line, the majority of CD44 sulfation was attributed to the glycosaminoglycan chondroitin sulfate. However, TNF-alpha stimulation increased CD44 sulfation two- to threefold, largely attributed to the increased sulfation of N- and O-linked glycans on CD44. Therefore, TNF-alpha induced a decrease in the percentage of CD44 sulfation due to chondroitin sulfate and an increase due to N- and O-linked sulfation. Furthermore, TNF-alpha induced the expression of 6-sulfo N-acetyl lactosamine (LacNAc)/Lewis x on these cells, which was detected by a monoclonal antibody after neuraminidase treatment. This 6-sulfo LacNAc/Lewis x epitope was induced on N-linked and (to a lesser extent) on O-linked glycans present on CD44. This demonstrates that CD44 is modified by sulfated carbohydrates in myeloid cells and that TNF-alpha modifies both the type and amount of carbohydrate sulfation occurring on CD44. In addition, it demonstrates that TNF-alpha can induce the expression of 6-sulfo N-acetyl glucosamine on both N- and O-linked glycans of CD44 in myeloid cells.

Orderly and nonstochastic acquisition of CD94/NKG2 receptors by developing NK cells derived from embryonic stem cells in vitro.

In mice there are two families of MHC class I-specific receptors, namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1(b) and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently, how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study, we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers, but not Ly49 molecules, and their cytotoxicity is inhibited by Qa-1(b) on target cells. Using RT-PCR analysis, we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order, with their order of acquisition being first CD94; subsequently NKG2D, NKG2A, and NKG2E; and finally, NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells, and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells, suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic, orderly, and cumulative.