CMV Specific T Cells for Adoptive Transfer Exhibit Multiple Effector Functions Associated with Protective Immunity Including the Concurrent Production of Cytokines and Cytolytic Activity
Authors: Clancy Leighton E , Micklethwaite Kenneth P , Blyth Emily , Sandher Upinder , Gottlieb David J 
Published: October 2019

Abstract
Background: Cytomegalovirus (CMV) reactivation post allogeneic haemopoietic stem cell transplant (HSCT) causes significant morbidity. Adoptive transfer of ex-vivo generated CMV specific T cells has the potential to restore immunity, prevent CMV reactivation and circumvent the need for pharmacotherapy. Donor derived CMV specific T cells were generated for prophylactic infusion into haemopoietic stem cell transplant recipients as part of a phase I/II clinical trial aiming to reduce the incidence of CMV reactivation. T cells were expanded by co-culturing with dendritic cells transfected with Ad5F35pp65, a recombinant adenovirus promoting the presentation of epitopes derived from the immunodominant CMV antigen pp65. The aim of this study was to determine if ex vivo expanded CMV specific T cells have the capacity to produce different cytokines and chemokines associated with protective immunity.
Results: Ex vivo expanded Ad5F35pp65 stimulated cultures were primarily CD3+ T cells (median 92%) with a predominance of CD8+ (14–90%) over CD4+ (2.5–57%) cells. An assay measuring antigen specific production of interferon-γ (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor (TNF) and macrophage inflammatory protein 1β (MIP-1β) by intracellular cytokine flow cytometry was established to quantify the frequency of T cells with specificity towards CMV or adenovirus and to assess the quality of responses reflected by the simultaneous production of multiple cytokines. Ad5F35pp65 stimulated cultures were greatly enriched for CMV specific T cells producing cytokines in response to pp65 (mean 59%, 19.1–90%) compared to the starting PBMC population (0.5–1.5%). Responses directed towards the adenovirus hexon protein were also detected accounting for 0.65 to 9% of T cells. CMV specific CD8 T cells predominantly produced IFN-γ and MIP-1β followed by TNF with means of 61.3%, 59% and 44% respectively. The majority of CMV specific CD8+ T cells produced both IFN-γ and MIP-1β (80–96%) with a substantial proportion of these also producing TNF (72%, 44–88.5%). IL-2 producing CD8+ T cells were less frequent, ranging from 0.5–40%. However, IL-2 producers consistently exhibited the highest level of functionality with the production of all four cytokines. Furthermore, IFN-γ producing CD8+ T cells mobilized CD107, a marker of degranulation and cytotoxic activity. In the majority of cases, fewer CD4+ T cells exhibited specificity towards CMV pp65 (30%, 4–49.5%) however greater than 80% co-produced IFN-γ, MIP-1β and TNF. Furthermore, a high proportion of CD4+ T cells also produced IL-2 (53.4%). Adenovirus specific T cell responses were detected in all cultures but were mainly confined to the CD4+ population. Finally, we examined CMV specific responses in the starting donor PBMC population. Cytokine production profiles of CMV specific CD4 and CD8 T cells closely resembled those of ex vivo generated cultures suggesting the uniform expansion of CMV specific functional subsets. In conclusion, these results demonstrate that ex vivo expanded CMV specific T cells intended for adoptive transfer perform several functions associated with protective immunity in vivo, including the capacity to kill infected cells and the simultaneous production of multiple cytokines.