Identification and characterization of peripheral T-cell lymphoma-associated SEREX antigens

Abstract

Peripheral T-cell lymphomas (PTCL) are generally less common and pursue a more aggressive clinical course than B-cell lymphomas, with the T-cell phenotype itself being a poor prognostic factor in adult non-Hodgkin lymphoma (NHL). With notable exceptions such as ALK(+) anaplastic large cell lymphoma (ALCL, ALK+), the molecular abnormalities in PTCL remain poorly characterised. We had previously identified circulating antibodies to ALK in patients with ALCL, ALK(+). Thus, as a strategy to identify potential antigens associated with the pathogenesis of PTCL, not otherwise specified (PTCL, NOS), we screened a testis cDNA library with sera from four PTCL, NOS patients using the SEREX (serological analysis of recombinant cDNA expression libraries) technique. We identified nine PTCL, NOS-associated antigens whose immunological reactivity was further investigated using sera from 52 B- and T-cell lymphoma patients and 17 normal controls. The centrosomal protein CEP250 was specifically recognised by patients sera and showed increased protein expression in cell lines derived from T-cell versus B-cell malignancies. TCEB3, BECN1, and two previously uncharacterised proteins, c14orf93 and ZBTB44, were preferentially recognised by patients' sera. Transcripts for all nine genes were identified in 39 cancer cell lines and the five genes encoding preferentially lymphoma-recognised antigens were widely expressed in normal tissues and mononuclear cell subsets. In summary, this study identifies novel molecules that are immunologically recognised in vivo by patients with PTCL, NOS. Future studies are needed to determine whether these tumor antigens play a role in the pathogenesis of PTCL.

Figure 1. Immunoreactivity of the PTCL, NOS-associated antigens with sera from patients with PTCL, NOS (n = 9), T-NHL (n = 15), B-NHL (n = 32) or healthy controls (n = 17).

Figure 2. RT-PCR analysis of mRNA expression levels of genes encoding PTCL, NOS-associated antigens in cancer cell lines.

−, no reverse transcriptase negative control; +, positive control testis cDNA. TBP was included as a positive control for the quality of the cDNA.

Figure 3. Quantitative real time PCR analysis of genes encoding PTCL, NOS-associated antigens in cancer cell lines.

Black bars represent T-cell lines, pale grey are Classical Hodgkin Lyphoma (CHL), white are myeloma and dark grey are B-NHL. Expression was normalised to fractionated normal CD19⁺ B cells for B-cell derived cell lines or fractionated normal CD3⁺ T cells for T-cell derived cell lines.

Figure 4. RT-PCR analysis of PTCL, NOS SEREX antigens in fractionated peripheral blood cells.

1, resting CD4⁺T cells; 2, activated CD4⁺ T cells; 3, resting CD8⁺ T cells; 4, activated CD8⁺ T cells; 5, resting CD19⁺ B cells; 6, activated CD19⁺ B cells; 7, resting CD14⁺ monocytes; 8, activated monocytes; 9, mononuclear B and T cells; 10, positive control placenta tissue cDNA; 11, no reverse transcriptase negative control; 12, positive control Thiel cDNA.

Figure 5. Western blot analysis of antibodies to PTCL, NOS SEREX antigens against selected whole cell RIPA extracts.

T-ALL, T cell acute lymphoblastic leukaemia; ALCL, ALK+; CTCL, cutaneous T cell lymphoma; FL, follicular lymphoma; GCB, germinal centre-derived diffuse large B cell lymphoma; ABC, activated B cell-derived diffuse large B cell lymphoma; BL, Burkitt lymphoma; MCL, mantle cell lymphoma; HL, Hodgkin lymphoma; CML, chronic myelogenous leukaemia.