Cytotoxic T Lymphocytes (CTL) Specific for CMV, Adenovirus, and EBV Can Be Generated From Naive T Cells for Adoptive Immunotherapy.
Authors: Hanley Patrick , Savoldo Barbara , Cruz Conrad Russell Young , Leen Ann M. , Molldrem Jeffrey J. , Liu Hao , Gee Adrian P. , Heslop Helen E. , Dotti Gianpietro , Rodgers John , Rooney Cliona M , Brenner Malcolm K. , Shpall Elizabeth J. , Bollard Catherine M. 
Published: October 2019

Abstract
Abstract 504

Allogeneic stem cell transplantation is the treatment of choice for patients with high-risk hematologic malignancies. Umbilical cord blood (CB) has emerged as an important source of stem cells for allotransplant patients lacking human leukocyte antigen (HLA)-matched donors–a significant problem for minorities. T cells in UCB grafts are, however, virus-naïve, leading to higher mortality rates due to infections with CMV, EBV, adenovirus (Ad) and other viruses. Peripheral Blood Stem Cell Transplant (PBSCT) from CMV-seronegative (CMVneg) donors to CMV-seropositive (CMVpos) recipients produces a similarly high incidence of CMV infection since donor T cells are naïve to this virus. Adoptive immunotherapy with peripheral blood (PB)-derived CMV/Ad-specific CTL generated from CMVpos donors effectively prevents CMV clinical disease after PBSCT, but this option has not been feasible when the donor cells are naive, irrespective of whether they are sourced from CB or CMV-PBS, since CTL generation from these donors has been unsuccessful. We have now overcome this problem and can routinely generate CMV, Ad and EBV-specific CTLs from CB and CMV specific CTLs from seronegative PBSC. We used an Ad5f35vector carrying the CMVpp65 transgene to transduce CB-derived or PB-derived dendritic cells and stimulate virus-specific CTL in the presence of IL-7, IL-12 and IL-15. This was followed by 2 stimulations with autologous EBV-lymphoblastoid cell lines (LCL) transduced with the same vector. The 9 CB-derived CTL lines we made in this way contained a mean of 87% (range 81-94) CD8+ and 26% (range 12-40) CD4+T cells, and exhibited significant cytotoxicity in 51Cr release assays against CMVpp65, Adhexon, and LCL targets. In IFN-γ ELISPOT assays there was a mean of 209 (range 45-694), 74 (range 0-128), and 157 (range 23-291) SFC following incubation with CMVpp65, Adhexon peptides and LCL, respectively. In addition, we generated CMVpp65, Adhexon, and LCL-specific responses from the peripheral blood of 4 CMVneg adult donors, which produced a mean of 92 (range 50-126), 163 (range 69-293), and 62 (range 37-86) SFC to CMVpp65 respectively. Neither CB- nor CMVneg-derived CTL responded to irrelevant peptides. Of note, the virus-specific T cells that we expanded from both CB and CMVneg donors derived only from T cells with a naive phenotype (CD45RA+/CCR7+). Moreover, both CB and CMVneg-derived CTL recognized ‘unconventional‘ CMV pp65 epitopes, as identified by overlapping pp65 peptide pools and confirmed by IFN-γ ELISPOT as well as multimer analysis (Table 1). In HLA-A2+ subjects, these naive-derived CTLs did not recognize conventional HLA-A2-associated CMV pp65 epitopes such as NLV, suggesting an inherent difference between naïve and memory T cell responses to CMV. In summary, virus-specific responses T cell responses can be obtained even from CB and virus-naive adult donors and may allow prevention and treatment of viral disease in the recipients of these allografts.

Disclosures:
No relevant conflicts of interest to declare.