Seroconversion with progress of disease.

<p>CD28 abs measured in one female patient during a period of more than 10 years in association with the course of the disease.</p

Detailed data of melanoma patients.

<p>SSM = superficial spreading melanoma, NMM = nodular malignant melanoma, LMM = Lentigo maligna melanoma, ALM = acrolentiginous melanoma, AMM = amelanotic melanoma, UCM = unclassified melanoma, others = melanoma of the mucosa or uvea.</p

Specificity of the CD28 autoantibody Elisa.

<p>Human recombinant proteins were immobilized and measured by Elisa as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058087#s2" target="_blank">methods</a> part. Sera from 2 patients were used. No unspecific signal was detected. ACT-g: actin-γ, MAZ: myc-ass. zinc finger protein, PC9: pyruvate-carboxylase 9, SLP2: stomatin-like protein 2, ATG13: autophagy related 13 homolog.</p

Competition assay using a mouse CD28 monoclonal antibody and purified human CD28 autoantibodies.

<p>Jurkat cells were incubated with mixtures of mouse monoclonal CD28 antibodies with increasing amounts of purified human autoantibodies (CD28 or G250). Competition was detected for human CD28 autoantibodies while human G250 autoantibodies had no effect. Cell viability was measured by EZ4U assay.</p

CD28 abs and death risk, overall survival and progress-free survival. CD28 abs and death risk

<p>(<b>A</b>). Distribution of patients with or without CD28 abs according to whether patients died from melanoma or not (p = 0.043, chi-square test), n = 230; <b>CD28 abs and overall survival</b> (<b>B</b>). Kaplan-Meier curve showing correlation between overall survival and occurrence of CD28 abs in melanoma patients (p = 0.559, Log-rank test), n = 230; <b>CD28 abs and progress-free survival</b> (<b>C</b>). Kaplan-Meier curve showing correlation between progress-free survival and occurrence of CD28 abs in melanoma patients (p = 0.952, Log-rank test), n = 230.</p

The inhibitory effect of human CD28 autoantibodoes is titer-dependent.

<p>Jurkat cells were incubated with sera derived from patient #64 that had been collected at different time points and that showed different titers of CD28 autoantibodies. Sera derived from a healthy donor (HD) or from a melanoma patients without CD28 autoantibodies were used as control. Cell viability was measured by EZ4U assay.</p

Analysis of recombinant CD28 expression in HEK cells.

<p>(A) Coimmunoprecipitation. Recombinant HEK cells were lysed with lysis buffer, and 200–500 µl of cell lysate was incubated with rabbit αFLAG antibody at 4°C for 2 hours, then 20 µl of protein A agarose slurry (GE Healthcare) was added for another 2 hours. The beads were washed three times with at least 10 volumes of lysis buffer before resolving by SDS-PAGE. Detection was done either with mouse αFLAG or mouse αCD28. As control HEK293-SLP2-FLAG was used. 1: HEK293 lysate, 2: HEK293-CD28-FLAG lysate, HEK293-SLP2-FLAG lysate. (B) Westernblot. Cells were lysed and analysed by immunoblot using αFLAG or αCD28 antibodies. 1: HEK293 lysate, 2: HEK293-CD28-FLAG lysate (C) Elisa. Recombinant CD28 is recognized by a commercial αCD28 mAb. HEK293-CD28-FLAG lysate is coated on NUNC maxisorp via FLAG-tag. Detection was done with 1: αCD30 or 2: αCD28.</p

Purified human CD28 autoantibodies have an effect on Jurkat T cell viability (EZ4U assay).

<p>Jurkat cells were incubated with autoantibodies purified from patients serum by affinity chromatography. Purified CD28 autoantibodies show reduced cell viability while purified G250 autoantibodies have no effect on cell viability when compared with Jurkat cells not incubated with antibodies.</p

Titration of CD28 autoantibodies in the sera of patients and healthy donors measured by Elisa.

<p>Sera from three patients (#4974, #5243, #4690) and two healthy donors were titrated. The curves obtained from the healthy donors cannot be distinguished in this figure.</p

Occurrence of CD28 abs in different diseases.

<p>CD28-antibodies in the serum of healthy blood donors (A), patients with plasmocytoma (B), melanoma (C), psoriasis vulgaris (D) and allergies (E). Line: mean value (MV), dashed line: MV ±3 SD.</p