Novel tumor antigens elicit anti-tumor humoral immune reactions in a subset of patients with polycythemia vera

Abstract

We attempted to determine whether the immune reactions elicited by aberrantly expressed testis antigens contribute to the beneficial responses to interferon (IFN)-alpha therapy and other therapies in patients with polycythemia vera (PV). We screened a human testis cDNA library using SEREX (serological analysis of tumor antigens by screening an expression cDNA library with sera from three patients with PV who had undergone IFN-alpha-induced or other therapeutics-induced remission). We identified two novel PV associated tumor antigens, PV65 (eIF-2alpha) and PV13 (protamine 2). These 2 antigens elicited IgG antibody reactions in a subset of PV patients but not in healthy donors, suggesting that they are authentic tumor antigens. Increased phosphorylation of PV65 in response to stimulation of IFN-alpha, and upregulation of PV13 in tumor cells might enhance their abilities in elicitation of immune reactions in patients. These findings provide new insights into the mechanism underlying the regulation of the self-antigen repertoire in eliciting anti-tumor immune reactions in patients with polycythemia vera, and suggest their potential as the targets of novel immunotherapy.

Figure 1

The novel tumor antigen PV65 (eIF-2α). (A) Schematic representation of the genomic structure, mRNA, and protein structure of tumor antigen PV65 (eIF-2α, GenBank accession number: NM_032025). (B) The expression of PV65 transcripts in normal tissues detected by Northern blot. The lanes N1 to N10 indicate various normal tissues in the order of brain (N1), liver (N2), placenta (N3), small intestine (N4), colon (N5), thymus (N6), spleen (N7), prostate (N8), testis (N9), and ovary (N10), respectively. The hybridization analyses of the normal tissue and tumor cell expression (BD Clontech) with ³²P-labelled specific probes, as indicated, were performed, respectively. The transcript sizes are indicated with kilobases (kb). (C) The expression of PV65 transcripts in tumor cells detected by Northern blot. The lanes T1 to T10 indicate various tumor cells in the order of acute T cell leukemia (Jurkat cells) (T1), Burkitt’s lymphoma (CA46) (T2), breast cancer (MDA-MD-453) (T3), Burkitt’s lymphoma (Namalwa) (T4), epidermal carcinoma (A-431) (T5), uterine carcinoma (MES-SA) (T6), Burkitt’s lymphoma (Raji) (T7), osteosarcoma (MG-63) (T8), histiocytic lymphoma (U-937) (T9), and cervical adenocarcinoma (Hela S3) (T10), respectively. (D) The expression of PV65 transcripts in the granulocytes from PV patients and healthy donors detected by quantitative RT-PCR. The expression levels of PV65 transcripts are expressed as the ΔCT (PV65-18S). Low ΔCT values indicate higher expression of the specific gene. The experiments were repeated for three times. The mean and standard deviation for each group were calculated. (E) Western blot analyses of PV65 protein and phosphorylated PV65 protein in K562 myeloid leukemia cells. The expression levels of PV65 protein and phosphorylated PV65 protein in K562 cells at 0, 2, 4, and 8 h after stimulation by IFN-α were assayed with Western blots using anti-PV65 and anti-phosphorylated PV65, respectively. The Western blot analysis for the house keeping protein control β-actin was also performed using anti-β-actin as protein loading control. (F) The IgG antibody reactions to the tumor antigen PV65 detected by phage plaque assay. The detection rates in each group are presented with the empty column as the positive (on the top) and the solid column as the negative (on the bottom). The experiments were repeated for three times. The representative results are shown. The groups, whose detection rates of the IgG antibody reactions to PV65 are statistically higher than that of healthy donors (the Chi-Square Goodness-of-Fit Test; p < 0.05), are marked with *.

Figure 2

The novel tumor antigen PV13 (protamine 2). (A) Schematic representation of the genomic structure, mRNA, and protein structure of tumor antigen PV13 (protamine 2, GenBank accession number: NM_002762). (B) The expression of PV13 transcripts in normal tissues detected by Northern blot. The lanes N1 to N10 indicate various normal tissues in the order of brain (N1), liver (N2), placenta (N3), small intestine (N4), colon (N5), thymus (N6), spleen (N7), prostate (N8), testis (N9), and ovary (N10), respectively. The hybridization analyses of the normal tissue and tumor cell expression (BD Clontech) with ³²P-labelled specific probes, as indicated, were performed, respectively. The transcript sizes are indicated with kilobases (kb). (C) The expression of PV13 transcripts in tumor cells detected by Northern blot. The lanes T1 to T10 indicate various tumor cells in the order of acute T cell leukemia (Jurkat cells) (T1), Burkitt’s lymphoma (CA46) (T2), breast cancer (MDA-MD-453) (T3), Burkitt’s lymphoma (Namalwa) (T4), epidermal carcinoma (A-431) (T5), uterine carcinoma (MES-SA) (T6), Burkitt’s lymphoma (Raji) (T7), osteosarcoma (MG-63) (T8), histiocytic lymphoma (U-937) (T9), and cervical adenocarcinoma (Hela S3) (T10), respectively. (D) The IgG antibody reactions to the tumor antigen PV13 detected by phage plaque assay. The detection rates in each group are presented with the empty column as the positive (on the top) and the solid column as the negative (on the bottom). The experiments were repeated for three times. The representative results are shown. The groups whose detection rates of the IgG antibody reactions to PV13 are statistically higher than that of healthy donors (the Chi-Square Goodness-of-Fit Test; p < 0.05) are marked with *.