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Supplemental Figure 1. Cytokine production by B cells of healthy volunteers with ibrutinib treatment (n = 4). B cells of healthy volunteers were stimulated with CpG for 24 h and treated with DMSO (Control) or ibrutinib (10 μM). With a Legendplex assay, cytokine levels of IL-6, TNF-⍺, IFN-γ and IL-10 were determined in the culture supernatant. Bars represent mean. Error bars indicate SEM. * p

Background: We previously showed that the bacterial lipopeptide Pam3Cys-Ser-Ser, meanwhile established as a toll-like receptor (TLR) 1/2 ligand, acts as a strong adjuvant for the induction of virus specific CD8+ T cells in mice, when covalently coupled to a synthetic peptide.Case presentation: We now designed a new water-soluble synthetic Pam3Cys-derivative, named XS15 and characterized it in vitro by a TLR2 NF-κB luciferase reporter assay. Further, the capacity of XS15 to activate immune cells and stimulate peptide-specific CD8+ T and NK cells by 6-sulfo LacNAc+ monocytes was assessed by flow cytometry as well as cytokine induction using immunoassays. The induction of a functional immune response after vaccination of a volunteer with viral peptides was assessed by ELISpot assay and flow cytometry in peripheral blood cells and infiltrating cells at the vaccination site, as well as by immunohistochemistry and imaging.XS15 induced strong ex vivo CD8+ and TH1 CD4+ responses in a human volunteer upon a single injection of XS15 mixed to uncoupled peptides in a water-in-oil emulsion (Montanide™ ISA51 VG). A granuloma formed locally at the injection site containing highly activated functional CD4+ and CD8+ effector memory T cells. The total number of vaccine peptide-specific functional T cells was experimentally assessed and estimated to be 3.0 × 105 in the granuloma and 20.5 × 106 in peripheral blood.Conclusion: Thus, in one volunteer we show a granuloma forming by peptides combined with an efficient adjuvant in a water-in-oil-emulsion, inducing antigen specific T cells detectable in circulation and at the vaccination site, after one single vaccination only. The ex vivo T cell responses in peripheral blood were detectable for more than one year and could be strongly boosted by a second vaccination. Hence, XS15 is a promising adjuvant candidate for peptide vaccination, in particular for tumor peptide vaccines in a personalized setting.

Overlapping synthetic peptides of human SO protein chain. (PDF 193Â kb)

Clinical data and laboratory parameters before and during UDCA-treatment and availability of serum and peripheral blood mononuclear cells in 53 PSC patients included in the study. (DOCX 30Â kb)

Receiver operating curve (ROC) analysis for IgG-antibodies to the four SO-proteins comparing sera from PSC patients with those from healthy donors, PBC, AIH, alcoholic liver disease (ALD), viral hepatitis, ulcerative colitis (CU), Crohn disease (CD), and collagen disorders. (PDF 257Â kb)

Schematic presentation of the enzyme sulphite oxidase (SO) with its different domains, and the immuno-dominant epitopes recognised by PSC sera (aa = amino acid; mo = molybdenum binding site). (PDF 165 kb)

Activity of IgG-antibodies against the four SO-proteins in sera from untreated PSC-patients without and with IBD as well as in patients with pure IBD as determined by ELISA. Individual values (•) and median (―) are given. p

Uveitis is an intraocular inflammatory disease in which autoimmune reactions have been discussed in playing an important role. Many data in this respect derived from the animal model of "experimental autoimmune uveitis", where several organ-specific autoantigens have been described such as the retinal S-antigen and the interphotoreceptor retinoid-binding protein. However, their diagnostic and pathogenic role in humans has been controversially discussed. In recent studies, the possible relevance of betaB1-crystallin, present in lens and ciliary body, has been outlined. We, therefore, wanted to analyse whether sera from patients with uveitis might contain antibodies to lens proteinsHuman lenses from cadaveric eyes were shock frozen, homogenized, and resuspended. The resulting suspension (human lens protein fraction--HLPF) was analyzed for antigenicity by ELISA and Western blotting with patients' sera. A total of 165 patients with uveitis, 54 patients with scleritis and episcleritis, 56 patients with other eye diseases, and 112 healthy blood donors were studied.Twenty-six (49%) of the 53 patients with anterior uveitis, 17 (32%) of the 53 patients with intermediate uveitis and 7 (22%) of 32 patients with posterior uveitis reacted in the ELISA with the HLPF. Antibodies to lens antigens were detected in one-third of patients with panuveitis and retinal vasculitis. In contrast, only 12% of the healthy blood donors were positive in the ELISA. The number of patients with an autoimmune response to alpha-crystallins in Western blot predominated in all investigated groups.These data indicate that antibodies to lens proteins occur in a high incidence in sera from patients with uveitis. Forty-nine percent of the patients with anterior uveitis and only 12% of healthy controls were positive in the ELISA. In our groups of patients and controls the autoantibodies reacted in the Western blot predominantly with alpha-crystallin. Further studies are required to analyze in more detail the clinical and etiopathogenetic relevance of the antilens antibodies in uveitis.

Additional file 7: Table S1. Induction of cytokine release by XS15. Table S2. Antibody responses to XS15.

Additional file 7: Table S1. Induction of cytokine release by XS15. Table S2. Antibody responses to XS15.

Additional file 1. Supplementary Materials and Methods.

Additional file 8: Fig. S1. Impact of XS15 on slanMo-mediated T-cell polarization. Fig. S2. Impact of XS15 on slanMo-mediated IFNγ expression by WT1 peptide-specific CD8+ T cells. Fig. S3. Ex vivo phenotype of granuloma infiltrating cells (GICs). Fig. S4. Ex vivo assessment of regulatory T cells (Treg) in granuloma infiltrating cells (GICs) and PBMCs. Fig. S5. Ex vivo phenotype of checkpoint receptors in granuloma infiltrating cells (GICs) and PBMCs. Fig. S6. Detection of vaccinated peptides in granuloma tissue by mass spectrometry.

Additional file 8: Fig. S1. Impact of XS15 on slanMo-mediated T-cell polarization. Fig. S2. Impact of XS15 on slanMo-mediated IFNγ expression by WT1 peptide-specific CD8+ T cells. Fig. S3. Ex vivo phenotype of granuloma infiltrating cells (GICs). Fig. S4. Ex vivo assessment of regulatory T cells (Treg) in granuloma infiltrating cells (GICs) and PBMCs. Fig. S5. Ex vivo phenotype of checkpoint receptors in granuloma infiltrating cells (GICs) and PBMCs. Fig. S6. Detection of vaccinated peptides in granuloma tissue by mass spectrometry.

Additional file 1. Supplementary Materials and Methods.