Proteome-based analysis of serologically defined tumor-associated antigens in cutaneous lymphoma

Abstract

Information on specificities of serological responses against tumor cells in cutaneous lymphoma patients is relatively restricted. To advance the knowledge of serological immune responses against and to assess the scope of tumor antigenicity of cutaneous lymphoma, 1- and 2-dimensional Western blot analyses with sera from patients were combined with proteomics-based protein identification. Testing sera from 87 cutaneous lymphoma patients by 1-dimensional Western blot analysis, 64 cases of seroreactivity against lymphoma cells were found. The positive responses were relatively weak, restricted to few antigens in each case, and heterogeneous. To identify the antigens, proteins of the mycosis fungoides cell line MyLa and primary tumor cells were separated by 2-dimensional gel electrophoresis, Western-blotted and probed with heterogeneous and autologous patient sera. The antigens were identified from silver-stained replica gels by MALDI-TOF mass spectrometry. 14 different antigens were assigned and identified with this proteome-serological approach. Only one, vimentin, had been reported before, the other 13 are new antigens for cutaneous lymphomas.

Figure 1. Pattern of seroreactivities of cutaneous lymphoma patients against the mycosis fungoides cell line MyLa.

Total protein extract of the tumor cells were separated by SDS-PAGE, blotted onto nitrocellulose and probed with the sera of the patients or of healthy control donors. The patients were diagnosed for mycosis fungoides (A male, B female), lymphomatoid papulosis (C), pleomorphic cutaneous T cell lymphoma (D), follicle center cell lymphoma (E), other cutaneous T cell lymphoma (F): CD30+ large cell lymphoma (sera 4, 65 and 84), cytotoxic cutaneous T cell lymphomas (sera 32 and 52), small cell to medium size cell T cell lymphoma (serum 56), CD8+ epidermotropic cytotoxic cutaneous T cell lymphoma (serum 78), parapsoriasis (serum 83) and Sezary syndrome (serum) 18) and other B cell lymphomas (G): diffuse large cell B cell lymphoma (sera 17 and 48), large cell B cell lymphoma (serum 24), mantle cell lymphoma (sera 23, 45 and 71), leukemia (serum 82) and one not specified B cell lymphoma (serum 29), not specified cutaneous lymphomas (H). The lanes marked with an arrow were rated as seropositive. As controls, sera of healthy donors were used (I). The numbers atop of each lane represent the numbers of the sera used and correspond to the patient numbering in Table S1. This numbering is used throughout this report.

Figure 2. Seroreactivities of patients with cutaneous lymphoma against the mycosis fungoides cell line MyLa.

Total protein extracts of MyLa cells were separated by 2-dimensional electrophoresis with an isoelectric focusing pH range of 3–10 in the first and SDS-PAGE in the second dimension, blotted onto nitrocellulose membranes and probed with the sera of the patients as listed in Table 1. Serum dilutions were 1/200. The blots A and B were probed with 8 different sera each, blots C and D with single sera. Antigens were detected in the boxed regions of the gels. The antigen signals were very weak although high concentrations of the sera were used which caused a relatively high background of the blots. To enhance the visibility of the spots, the contrast of the image areas around the spots was intensified. For every Western blot analysis A through D, the original is shown on the left and the regions with the enhanced spots for the regions indicated with Roman numerals on the right. A silver-stained gel of the MyLa proteome corresponding to the Western blots A–D is shown in E. The antigen spots that could be assigned to protein spots in the silver-stained gel are indicated with arrows and numbered.

Figure 3. Identification of MyLa-associated antigens detected by sera of cutaneous lymphoma patients.

The protein spots that could be assigned to antigen spots were picked from the gels and treated with trypsin. The resulting fragments were analyzed by mass spectrometry to identify the antigens. Seven different antigens were identified by peptide mass fingerprint analysis as BiP (spot and spectrum 3), HSP60 (spot and spectrum 5), HSP71 (spot and spectrum 6), HSP60 (spot and spectrum 7), β-tubulin (spot and spectrum 8), the β-subunit of the mt-ATP synthase (spot and spectrum 9) and TIP47, a mannose 6 phosphate receptor-binding protein (spot and spectrum 15). The peptide mass fingerprint spectra are numbered according to the antigen spots in Figure 2. The mass peaks marked with an asterisk match the theoretical spectrum of the assigned proteins. The statistics of the fingerprint analyses is summarized in Table 2.

Figure 4. Seroreactivities of patients with mycosis fungoides against autologous tumor cells.

Protein extracts of tumor cells of patient 85 (panels A–C) and patient 86 (panels D–F) were separated by 2-dimensional electrophoresis with a pH range of 3–10 for isoelectric focusing in the first and SDS-PAGE in the second dimension. The proteins were blotted onto nitrocellulose and probed with the respective sera (panels A and D) or visualized by silver staining in replica gels (panels C and F). Since the antigen reactivities are weak and their visibility further reduced by the high background which is due to the high serum concentrations that had to be used for antigen detection, the antigen spots were enhanced by image processing (panels B and E). Antigen spots that could be assigned to protein spots in the silver-stained gels are indicated with arrows and numbered.

Figure 5. Identification of tumor-associated antigen defined by autologous sera.

The protein spots assigned to antigens were picked and treated with trypsin. The peptide mass fingerprints of the resulting protein fragments were determined by mass spectrometry. Twenty-two of the assigned antigens were identified as aconitase (spot and spectrum 1), β-tubulin (spot and spectrum 2), coronin (spot and spectrum 3), glutamate dehydrogenase (spot and spectrum 4), keratin 16 (spot and spectrum 5), lamin A (spot and spectrum 6), lamin C (spot and spectrum 7), lamin B1 (spots and spectrum 8) and vimentin (spots and spectrum 9). Lamin B1 was identified in two and vimentin in 13 different spots. Only one representative spectrum is shown each. The mass peaks marked with an asterisk correspond to peptide masses which were matched to the theoretical spectrum of the identified protein. The numbers of the spectra corresponds to the numbers of the spots in Figure 4.