Lymphocryptovirus-associated post-transplant lymphoproliferative disorders and viral transcript expression in Cynomolgus macaque recipients of porcine neuronal precursors

Post-transplant lymphoproliferative disorders (PTLD) are a heterogeneous group of diseases arising arise as a consequence of the long-term use of immunosuppressive drugs associated with transplantation. Epstein Barr Virus (EBV)-related PTLD represent the large majority of these disorders. The aim is to characterize the PTLD of 8/27 adult immunosuppressed cynomolgus macaques, which received neural precursors from CTLA4Ig-transgenic pigs to treat pharmacologically-induced Parkinson's disease and to investigate whether the macaque-homologue of EBV (macLCV) was present in the lesions. Methods: The lesions and the viral presence have been evaluated applying morphological criteria and immunohistochemical analysis (anti-CD3, anti-CD5, anti-CD20, anti-CD79cyt, Ki-67, EBNA-2). RT-PCR of transcripts EBNA-1 and Latent Membrane Protein-1 (LMP-1) has been performed in neoplastic cells from 5/8 PTLD primates and 2 hyperplastic lymph-nodes from 2 PTLD+ primates (control). In order to exclude the presence of donors-derived Porcine Lymphotropic Herpesvirus (PLHV), DNA was isolated and screened by PCR from PTLD+ and PTLD- recipients’ PBMCs and from donor pigs. Results: 87.5% of PTLD occurred in the nasal cavity (50%), intestinal tract (50%), or both (12.5%); 50% with lymph nodal involvement. The histological diagnosis and immunohistochemical profile is compatible with a high-grade monomorphic PTLD, most frequently diffuse large B-cell lymphoma. Double-labeled immunohistochemistry for Epstein-Barr virus nuclear antigen (EBNA)-2 and CD20 revealed a high percentage of CD20 + neoplastic cells also positive for EBNA-2, while CD3 + cells in the tumors were negative for EBNA2. The consensus-PCR with specificity for EBNA-2 gene revealed that PTLD+ samples had a 92% of sequence identity between Rhesus and cynomolgus viruses. EBNA-1 and EBNA-2 expression was demonstrated in all PTLD+ specimens (no expression in controls). Four different RT-PCR assays failed to detect LMP-1 in all specimens. PLHV was detected in pigs at a prevalence of 44%, but all recipients tested negative. Pan-herpes screening of the macaques and sequencing revealed the presence macLCV in all recipients. A quantitative PCR assay developed to detect macLCV and used to retrospectively screen viral DNA isolated from the serum of the macaques taken pre-transplant, at euthanasia and at intermediate time points post-transplant showed that the 8 PTLD-affected macaques had a detectable viremia within 36–278 days (mean 117 days ±30 days) post-transplant and on average 60 days before the onset of clinical symptoms. There was no detectable viremia in any of the PTLD- macaques at any time point tested. This report identifies the pathological features of PTLD in cynomolgus monkeys in the setting of neuronal transplantation, a specific pattern of viral transcript expression of macLCV-associated PTLD and the presence of an associated viremia.