CMVpp65-Specific T Cells Generated from Naïve T Cell Populations Recognize Atypical but Not Canonical Epitopes and May be Protective in Vivo

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 CMVnegs and found that of >20 T-cell lines, all recognized atypical epitopes of pp65. HLA-A2+ cCTL derived from naive T-cells recognized novel pp65 epitopes (e.g. LQT) but not the typical epitope NLV. Analysis of the avidity of naïve donor CTL specific for the atypical CMV epitopes revealed that the avidity was similar to that of CMVpos CTL recognizing typical epitopes(mean: 300pM), but 30x more avid than paired CMVpos CTL recognizing atypical epitopes (P<0.05). 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 less polyclonal (P=0.02). To address the concern that atypical epitopes might not be naturally presented by CMV-infected cells and therefore not expanded in CMVpos donors in response to virus infection, we tested whether cCTL generated using CMV-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 T-Cell receptor (TCR) sequencing we identified TCRs recognizing typical and atypical epitopes. When we searched for these sequences in unmanipulated cord blood units, five different TCRs recognizing atypical epitopes were found in 5/5 CB units whereas of 22 NLV sequences, only 1 sequence was found in 1/5 cord blood units suggesting that T-Cells recognizing atypical epitopes are more prevalent during the primary infection and are later replaced by T-Cells recognizing typical epitopes. 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. Indeed, in a clinical trial of CB-derived virus-specific T-Cells, one patient with active CMV viremia received three doses of virus-specific T-Cells and was ultimately able to clear the virus with detectable CMV-specific T-Cells in the peripheral blood of the patient post-first T-Cell infusion.