Extending the Option of CMV-Specific T Cells from the CMV-Seronegative Donor

Adoptive transfer of CMVpp65-specific T-cells(cCTL) from CMV-seropositive(CMVpos) donors effectively restores antiviral-immunity after stem cell transplantation. However, the naïve T-cells in cord blood(CB) and adult CMV-seronegative(CMVneg) donors require different culture systems to generate cCTL. With a novel GMP-applicable protocol we have reliably generated cCTL from CB and found that 15/15 CB T-cell lines recognized atypical epitopes of pp65. We then expanded cCTL from CMVneg donors in a GMP-applicable way by isolating naive T-cells and stimulating them with pp65-Pepmix-pulsed antigen-presenting cells supplemented with IL2,IL7,IL12, and IL15. cCTL expanded from 8/11 CMVneg donors were primarily CD8+ T-cells(mean 71%). cCTL secreted IFN-γ in response to pp65-peptides(mean 224 spots) as measured in Elispot assays and lysed pp65-pulsed target cells. cCTL derived from naive T-cells recognized only novel and atypical pp65 epitopes (e.g. LQTGIHVR,MLNIPSINV) but not the typical epitope NLVPMVATV as confirmed by ELISPOT and Pentamer. Analysis of the avidity of naïve donor CTL specific for the atypical CMV epitopes revealed that the ½-maximum effective concentration was similar (mean:600pM) to CMVpos CTL recognizing typical epitopes(mean: 300pM), and more avid than CMVpos CTL recognizing atypical epitopes(mean:4nM). TCR sequencing performed on T-cells specific for typical(CMVpos) and atypical(CMVpos,CMVneg, and CB) epitopes revealed that CMVpos donor cCTL recognizing typical epitopes were markedly less polyclonal. To address the concern that atypical epitopes might not be naturally presented by CMV-infected cells and therefore not recognized by in vitro generated CTL, we tested whether cCTL generated using CMV AD169-infected fibroblasts would recognize the same epitopes. CMVpos cCTL recognized typical epitopes while CB/CMVneg cCTL recognized only atypical epitopes, suggesting that the epitopes are naturally processed/presented by APCs. Using deep TCR sequencing from the peripheral blood of CBT recipients, the atypical epitope MLN was found in a similar number of HLA-A2+ patients (6/9) than the typical epitope (NLV) (5/9). These results reveal major, previously-unreported differences in the naïve and memory CMV specific T-cell repertoire but suggest that atypical epitopes may indeed be important. Hence, we demonstrated that atypical epitopes are naturally presented by CMV-infected cells and are now evaluating the clinical efficacy of these CTL in recipients of CBT.