Serum antibodies against CD28-- a new potential marker of dismal prognosis in melanoma patients.

BACKGROUND: Autoantibodies against CD28 have been found in patients with autoimmune and atopic diseases. These antibodies may act as superagonists and activate T cells but may also be antagonistic or induce immunosuppressive effects by activating regulatory T cells. Autoimmunity in melanoma patients has been discussed controversially. OBJECTIVE: We investigated 230 melanoma patients for the occurrence of CD28 antibodies and the effect of the latter on overall and progress-free survival. METHODS: We constructed an ELISA assay to measure CD28 serum antibodies. 230 patients with melanoma and a control-group of 625 patients consistent of 212 patients with virus hepatitis b or c, 149 patients with allergies, 78 patients with psoriasis, 46 patients with plasmocytoma and 140 healthy blood donors were investigated for the occurrence of CD28 antibodies. RESULTS: CD28 abs occur at a higher percentage in patients with melanoma and in patients with viral hepatitis than in other groups investigated (p<0.001). Occurrence of CD28 abs is significantly higher in patients receiving interferons independent from the underlying disease (p<0.001). In vitro CD28 serum antibodies have an inhibitory effect on the CD28 receptor as they lead to reduced stimulation of Jurkat cells. Presence of CD28 was correlated with a higher risk of dying from melanoma (p = 0.043), but not with a significantly shortened overall survival or progression-free survival. CONCLUSION: Interferon therapy appears to induce the production of CD28 abs. In light of reports that these CD28 abs induce immunosuppressive Tregs and - as our data show - that they are inhibitors of CD28 receptor mediated stimulation, the continuation of therapies with interferons in melanoma patients developing CD28 antibodies should be critically reconsidered, since our data indicate a worse outcome of patients with CD28 abs

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

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

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

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

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

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

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

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