P1A as an Oncogene Figure 2. P1A Tg mice developed tumors. A. Thymus weights of P1A+Rag22/2 mice and Rag22/2 mice. Enlarged thymus in P1A+Rag22 mice after 7 months of age compared to P1A+Rag22 mice. B. P1A+Rag22 mice had shorter survival rates compared to P1A2Rag22 groups. Only mice that died due to tumor formation were recorded. p = 0.0004 (Kaplan-Meier analysis). C. Although several P1A+Rag2+ mice died due to tumor formation, differences in the tumor incidences were not statistical significant. p = 0.1157 (Kaplan-Meier analysis). doi:10.1371/journal.pone.0013439.g002 Discussion P1A, the first identified tumor rejection antigen, is presented to cytotoxic T lymphocytes by bound to major histocompatibility complex (MHC) class I molecules on the tumor cell surface [14]. Our data on the expression of P1A in tumor cells and its presence in embryonic stem cells and testis indicated that P1A is an onco- fetal antigen. In testis, it has been shown that P1A expression is restricted to spermatogonia cells which are MHC class I negative cells [5]. In addition, MHC class I molecules are not detected in the undifferentiated ES cells by FACS or immunofluorescence staining in ES cells [15]. Hence, the low to no expression of cell- surface MHC class I on spermatogonia and ES cells suggests that they are immune privilege. Therefore, these normal cells expressing P1A antigen are compromised in the immune system due to low MHC class I expression, but not tumor cells. The phenotypes of P1A Tg mice suggested that onco-fetal antigen P1A was indeed involved in tumorigenesis. First, we observed that P1A Tg mice developed thymic leukemia after 7 months of age, particularly in immunocompromised mice. Second, we demonstrated higher BrdU incorporation in P1A Tg BM cells compared to WT cells in vivo. Third, there was more CFU from P1A Tg BM compared to control in vitro. To our knowledge, this is the first evidence that P1A may promote tumorigenesis, and the first example that onco-fetal antigen can be involved in tumorigenesis. Mice with inactivated Rag2 cannot rearrange lymphocyte antigen receptors and therefore lack peripheral T cells and B cells [13,16]. In thymus, after TCR-b gene rearrangement, the CD4 2 CD82 double negative cells differentiate into CD4+CD8+ double positive thymocytes which are selected to single-positive cells (CD4 or CD8) following TCR-a rearrangement. As a result, T cell development in Rag2-deficient thymus is arrested prior to the CD4+CD8+ stage. The appearance of a thymic tumor at CD4+CD8+ stage suggest that transformation of T cells may have resulted in expression of the CD4 and CD8 molecules. The role for P1A in the process is unknown. Likewise, B cell development in the Rag2-deficient mice is blocked at the pro-B cell stage which could express cell surface marker, B220 [13,16]. The widespread expression of B220 among the tumors is therefore anticipated. However, the occurrence of thymic tumor in all tumor-bearing mice deserves consideration. CD42CD82 double negative thymocytes contain a mixture of lineage-committed progenitors which can reconstitute T, B, NK and dendritic cell lineages [17,18,19]. Our transgene P1A was expressed during hematopoietic progenitor development; therefore, any stage of cells may be the targets of the transformation or immortalization process by P1A activation. The heterogeneity was probably the result of tumor initiation at different stage of progenitor cell development. Hence, P1A induced tumorigenesis is not stage-specific and resulted in various thymic tumor types in mice, such as B cells (B220+), plasmacytoid dendritic cells (B220+CD11c+) and myeloid cells (Gr1+CD11b+). PLoS ONE | www.plosone.org 3 October 2010 | Volume 5 | Issue 10 | e13439