Molecular Characteristics Of IGVH And Antigen Selection In EBV Infected B-NHL Cell Lines

B cell non-Hodgkin lymphoma (B-NHL) occupies about70-80%lymphoma. The pathogenetic machnism of lymphoma is extremely complexed and has been a hotspot for researchers now. Epstein-Barr virus (EBV) is a y-1herpes virus and relates to a variety of tumors. There are higher infection rates of EBV in the patients with Burkitt lymphoma (BL) or the subtype of diffuse large B-cell lymphoma (DLBCL)-senile EBV positive DLBCL. EBV integrates its own genome into the genome of host cell, and expresses its own gene products to cause abnormalities of the host cell directly and indirectly. The highest correlated gene which associated with many human tumors is the p53gene so far. As a tumor suppressor gene, wild type of p53gene often causes the abnormality of P53protein functionally by mutations or deletions and induces tumorigenesis. The occurrence rate of p53mutations was5-25%in malignant lymphoma. DLBCL patients carrying p53mutations have the lower rate of complete remission and overall survival significantly.Immunoglobulin heavy chain (IGH) genes are normally expressed in mature B cells. The vriable region of IGH (IGVH) gene consists of three framework regions (FRs) and three complementarity determining regions (CDRs). In the germinal center (GC) after recognition for the encountered antigens, B cells will occur high-frequency point mutations mainly focus on CDR of IGVH gene so called somatic hypermutation (SHM), and produce high affinity IG molecules to attend immunological reaction. This process is vulnerable to appear variance by the interference of internal and external factors (such as individual genetic defection, viral infection, etc.), which constitute an important risk factor for lymphoma. Our previous study also found that patients with different types of lymphoma carried biased IGVH gene fragment, and it was associated with clinical prognosis in some lymphomas. Here we would like to know, whether EBV works as a certain antigen and plays an important role in the pathgenesis and progression of lymphoma? What are the possible mechanisms?So, we selected8cell lines of B-NHL which came from BL, DLBCL and MCL types of lymphoma respectively. By using PCR, in situ hybridization (ISH) and immunohistochemical staining (IHC), we detected the gene of LMP1, LMP2, EBER and their protein expressions. We found5cell lines of B-NHL (Raji, Daudi, Namalwa, OCI-ly10and OCI-ly19) were EBV infected. All mutations of p53genes were point mutations and mainly focused on the exon7. There were3hot spots and4Loop-L3mutations of p53gene in cell lines of B-NHL carrying EBV, which indicated a poor prognosis. At the same time, we used PCR-based GeneScan and sequencing to analysis the molecular characteristics of IGVH gene, such as rearrangement types, mutation status, gene segment usages and amino acid sequence of CDR3. We found that7cell lines were clonal rearrangement except Pfeiffer and6cell lines carried mutant IGVH genes except Jeko-1. The mutations of IGVH genes were mainly point mutations and nucleotide substitutions, with2.4%to21.2%mutational frequency, and the average was10.7%. Furthermore Daudi and OCI-lyl9cell lines which carrying EBV showed antigen selection (R/S<1.5). We also found that7cell lines carried biased usage of VH3(3/7) and VH4(3/7) gene families, with the main useage of VH3-21(2/7) and VH4-4(2/7). And for D and J fragments of IGVH gene, D3and J4fragments were used frequently. Furthermore, two EBV positive cell lines Raji and OCI-ly19carrying VH3-21had the same nucleotide and amino acid sequence of CDR3region. And the other two EBV positive cell lines Namalwa and OCI-ly10using VH4-4also had the same complementarity determining regions.In conclusion, our study found that8cell lines of B-NHL carried mutated p53gene which mainly focus on exon7, and EBV positive cell lines of B-NHL contained3hot spots and4Loop-L3mutations of p53gene which indicated a poor prognosis. Furthermore, EBV positive cell lines of B-NHL carried the mutant IGVH gene with biased usage of VH3-21and VH4-4. Evidences of antigen selection were also found in two cell lines of Daudi and OCI-ly19carrying EBV. The same complementarity determining regions of VH3-21or VH4-4suggested that a certain antigen (probably EBV) play the role in antigen recognition and driving which may associate with the beginning and progress of lymphoma. Take together, our finds provided clues and evidences for the molecular mechanism and target treatment in lymphoma.