EVALUATING THE ENGINEERING OF CYTOMEGALOVIRUS-SPECIFIC CD8+ T CELL CLONES

University Of Washington, Seattle WA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Reactivation of cytomegalovirus (CMV) represents a major cause of morbidity and mortality in immunosuppressed recipients of allogeneic hematopoietic stem cell and solid organ transplant as a consequence of deficient CD8+ T cell immunity. Adoptive immunotherapy with antigen-specific T cell clones has been shown to safely correct quantitative or qualitative deficiencies of T cells that permit progression of viral infection. However, T cell therapy has failed to restore protective T cell immunity in the subset of patients that receive immunosuppressive corticosteroids to treat graft versus host disease or graft rejection, respectively. To overcome this obstacle, we have investigated an innovative strategy for selectively interfering with glucocorticoid receptor (GR) signaling using zinc-finger endonucleases (ZFNs) for targeted deletion of the GR gene in mature T cells. We have developed a macaque model for adoptive transfer of CMV-specific T cell clones and employ this model to investigate the safety and efficacy of immunotherapy with CMV-specific T cells that lack the GR gene (GR-/-) after ZFN targeted deletion. We identified immunocompetent macaques with detectable CMV-specific CD8+ T cell responses in the peripheral blood and used a replication defective Ad5/35 vector to deliver ZFNs that target the GR gene in macaque T cells. CMV-specific GR-/- CD8+ T cell clones were then isolated by limiting dilution cloning. The GR-/- T cell clones exhibited CMV-specific cytolytic reactivity comparable to CMV-specific GRwt clones. However, in contrast to GRwt T cells, the GR-/- T cells were resistant to lymphotoxic effects of corticosteroids in vitro, consistent with disruption of the GR gene and lack of a functional GR protein. We are currently generating autologous GR-/- CMV-specific macaque CD8+ T cell clones from 2 macaques that can be used for adoptive transfer studies to evaluate the safety and efficacy of adoptively transferred CD8+ CMV-specific T cell clones with an edited GR gene.