Generation of CD8+ Viral Specific T Cells from EBV and CMV Naive Individuals.

Adoptive T cell immunotherapy (ATCI) with viral specific T cells, as exemplified by ATCI against Epstein-Bar virus (EBV) and Cytomegalovirus (CMV) with viral specific T cells generated from virus-experienced individuals, is efficacious against viral reactivation in immuno-compromised hosts. EBV-seronegative solid organ transplant recipients and CMV-seropositive stem cell transplant patients receiving CMV-seronegative grafts are at high risk of EBV-driven lymphoproliferation and CMV reactivation, respectively. However, due to the absence of virus-specific memory T cells, ex vivo techniques for generating virus-specific CD8+ CTL from virus-naive individuals remain to be developed for reproducibility and efficiency. To extend ATCI to the above patients, we are developing novel ex vivo systems to expand virus specific CD8+ CTL from seronegative individuals. We designed a two step stimulation for the naive T cells to develop into specific T cells. The first step, “de-naiviation”, involves non-specific but high-affinity stimulation of CD45RO/CD25/CD56/CD14 depleted peripheral blood mononuclear cells with anti-CD3 and -CD28 antibodies. The “de-naiviated” T cells were then antigen-specifically stimulated by antigen presenting cells expressing both EBV and CMV antigens. Peripheral blood mononuclear cells from an EBV/CMV seronegative individual were depleted for two rounds with micro-beads conjugated with antibodies against CD45RO, CD25, CD14 and CD56. The resultant cells were a homogenous population of cells mostly CD45RO−/CD45RA+/CD3+/CD25−, the phenotype for naïve T cells. After a period of expansion stimulated by a cocktail containing anti-CD3 (OKT3) and -CD28, 90% of the RO-T cells became RO+/RA−, the phenotype of memory T cells. Nearly all the CD4+ cells and most the CD8 cells became CD25+, suggesting recent activation. Then the “de-naiviated” T cells were stimulated with autologous EBV immortalized B lymphoblastoid cells transduced and expressing the CMV pp65 (CMVpp65) antigen. After three rounds of stimulation, the T cells were screened for specific production of interferon-gamma (IFNg) by ELISA. Clones were isolated from the primed T cells, and FACS analysis showed that the T cell clones produced IFNg in response to EBV BLCL expressing CMVpp65. These T cells also antigen-specificly expressed IL2 and GMCSF. Interestingly, while the cells were predominantly CD8+/CD3+, some of the cells were also positive for CD56, suggesting newly differentiated effector T cells. Work is ongoing to further characterize the T cell clones for antigen specificity, functionality and differentiation status. The novel approach we are developing has the potential to generate EBV and CMV specific CD8+ CTL from virus naive individuals for adoptive T cell immunotherapy against viral infections.