Allo-Reactive Potential of Multi-Virus Specific Cytotoxic T Cell Lines Recognizing Adenovirus Hexon, Cytomegalovirus pp65 and Epstein Barr Virus.
Authors: Yao Xin , Zhu Fenlu , Xu Huiqing , Keever-Taylor Carolyn A. 
Published: October 2019

Abstract
Abstract 5121

Introduction
Cytomegalovirus (CMV), adenovirus (Ad), and Epstein-Barr virus (EBV) are major pathogens after allogeneic hematopoietic stem cell transplant. The current antiviral therapy is associated with significant myelosuppression, impaired hematopoietic reconstitution and consequently, higher rates of other infections. Adoptive immunotherapy utilizing allogeneic donor-derived virus specific cytotoxic T cell lines (CTL) has been shown to effectively treat refractory viral infections. Although such lines carry the theoretical risk of allo-reactivity and the resultant graft versus host disease, this complication has not been widely reported in recipients of single virus specific cell lines. To improve the efficiency of this therapeutic modality we have developed an approach to generate a single cell line that recognizes all three of these clinically relevant viruses while also having greatly reduced allo-reactive potential.

Methods
Peripheral blood mononuclear cells (PBMC) from 3 healthy CMV seropositive donors were primed 3 times with autologous monocyte-derived dendritic cells pulsed with overlapping pools of pentadecapeptides spanning the antigen coding region of highly conserved regions of the Ad hexon protein and/or pools spanning the entire CMV pp65 protein. The lines were expanded for minimally one additional week using peptide loaded EBV transformed B cells (BLCL) as antigen presenting cells (APC). CMV, Ad and EBV anti-viral activity was monitored weekly by chromium release assay and the specificity of the lines was controlled by varying the peptide pools used for subsequent priming. The IFN-gamma ELISPOT assay was used to identify single peptides and to determine HLA restriction. The frequency and subset of single and pool peptide-reactive cells was determined using IFN-gamma intracellular cytokine production by flow cytometry. Fresh PBMC and established lines were additionally primed with fully allogeneic stimulators and response measured using intracellular cytokine production.

Results
Each of the 3 lines had robust proliferation and CTL activity against CMV and Ad peptide-pulsed targets. The lines consisted of 44.5-82.4% CD3+CD8+cells and 18.1-56.7% CD3+CD4+cells. CMV induced IFN-gamma response in 4.3-76.6% of CD3+CD8+cells and 0.4-47.5% of CD3+CD4+cells. Ad induced 1.4-43.9% of CD3+CD8+cells and 6.9-45.7% of CD3+CD4+cells to produce IFN-gamma. The 3 lines recognized 2-4 single CMV peptides and 2-6 single Ad peptides. The reactivity against these viral peptides was HLA restricted. At the time of testing none of the lines showed a strong EBV response. However, EBV reactivity could be detected with 1-2 additional primings, indicating that these precursors were present. Compared to fresh PBMC from the same donors, there was a 34.0±5.6 fold decrease in the frequency of alloreactive CD3+CD8+cells demonstrated in the intracellular cytokine assay.

Conclusions
Generation of multi-viral specific CTL in a single culture is feasible so long as antigen stimulation is controlled and this approach will greatly reduce the time, cost and number of cells required to restore viral immunity for patients receiving allogeneic stem cell transplant. The risk of allo-reactivity in these lines is greatly reduced compared to fresh PBMC. This makes adoptive cell therapy more practical, easier to implement and safer.

Disclosures
No relevant conflicts of interest to declare.