Cell-based immunotherapy in settings of allogeneic stem cell transplantation or donor leukocyte infusion has curative potential, especially in hematologic malignancies. However, this approach is severely restricted due to graft-versus-host disease (GvHD). This limitation may be overcome if target antigens are molecularly defined and effector cells are specifically selected. We chose formin-related protein in leukocytes 1 (FMNL1) as a target antigen after intensive investigation of its expression profile at the mRNA and protein levels. Here, we confirm re- IntroductionAlthough it is controversially discussed whether adaptive immune responses in tumor-bearing hosts play a role in controlling growth and recurrence of human tumors, 1,2 T cells can be converted to highly efficient killers of tumors. Donor leukocyte infusions (DLIs) are responsible for a graft-versus-leukemia effect (GvL) after allogeneic stem-cell transplantation (SCT), representing a therapeutic option with curative potential in different diseases at advanced stages. 3 Although GvL responses have also been shown for low-grade lymphomas, improvement of long-term survival after allogeneic transplantation has not been demonstrated in these patients. [4][5][6][7][8] This can be attributed primarily to the high treatmentassociated mortality due to transfer of T cells recognizing allogeneic minor histocompatibility (MHC) antigens, thereby causing potentially life-threatening graft-versus-host disease (GvHD). For broader applicability of this therapeutic option, it is therefore essential to reduce the risk of detrimental GvHD.One approach to gain a tumor-specific effect while significantly reducing the risk of GvHD is to generate allorestricted T cells with specificity for epitopes derived from tumor-associated antigens (TAAs). 9 Such allorestricted peptide-specific T cells may display high avidity toward MHC-presented TAAs, because they have not been negatively selected in the thymus. Moreover, they can be isolated by tetramers and cloned by limiting dilution to identify the specific TCR responsible for tumor-selective killing. 10 The isolation of a TCR with defined specificity for TAAs facilitates genetic TCR transfer into T-cell lines, 11-14 allowing major expansion of tumor-specific T cells.The choice of an appropriate target antigen is of fundamental importance for the success of an antitumor immunotherapeutic approach. So far, there is no universal antigen that serves as a target antigen in a broad range of malignancies. Many tumor-specific antigens are derived from aberrant expression of cell type-specific proteins and are expressed only in a restricted number of tumor types, limiting the tumors that can be treated. However, even the presence of a suitable antigen does not guarantee effective antigen recognition, because tumor cells evade immune recognition by down-regulation of the specific target or by inhibition of MHC class I-restricted antigen presentation. Thus, the level of epitope presentation in the context of classical or nonclassical MHC...

The Epstein-Barr virus (EBV) growth-transforms B-lymphocytes. The virus-encoded nuclear antigen 2 (EBNA2) is essential for transformation and activates gene expression by association with DNA-bound transcription factors such as RBPJkappa (CSL/CBF1). We have previously shown that EBNA2 contains symmetrically dimethylated Arginine (sDMA) residues. Deletion of the RG-repeat results in a reduced ability of the virus to immortalise B-cells. We now show that the RG repeat also contains asymmetrically dimethylated Arginines (aDMA) but neither non-methylated (NMA) Arginines nor citrulline residues. We demonstrate that only aDMA-containing EBNA2 is found in a complex with DNA-bound RBPJkappa in vitro and preferentially associates with the EBNA2-responsive EBV C, LMP1 and LMP2A promoters in vivo. Inhibition of methylation in EBV-infected cells results in reduced expression of the EBNA2-regulated viral gene LMP1, providing additional evidence that methylation is a prerequisite for DNA-binding by EBNA2 via association with the transcription factor RBPJkappa.

We have previously developed two monoclonal antibodies against the Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA1), designated 1H4 and 2B4. Both detect EBNA1 by in situ staining in established EBV-positive tumours, e.g. Hodgkin's lymphoma and nasopharyngeal carcinoma. An association of EBV with other tumours, notably breast carcinomas, has been reported but remains controversial. Using the antibody 2B4, a nuclear protein has been detected in breast carcinomas that were EBV-negative by other methods, suggesting cross-reactivity with a cellular protein. Furthermore, an association of EBV with various other carcinomas has been reported on the basis of 2B4 immunohistochemistry. Here we show that 2B4 also binds to MAGE-4, a cancer testis antigen expressed in a variety of tumour cells, including breast carcinoma, seminoma and EBV-negative cases of Hodgkin's lymphoma. We conclude that the 2B4 antibody is not suitable for the detection of EBV infection but that additional techniques, particularly in situ hybridization for the detection of the EBV-encoded RNAs (EBERs), should be employed to confirm the presence of EBV. Our results add to the evidence indicating that breast cancer is not an EBV-associated disease.

The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.