CMV-Reactive Gamma/Delta T Cells Expanded From CMV Seronegative Normal Healthy Individuals Lyse CMV-Infected Targets – Potential Adoptive Immunotherapy for Recipients of Stem Cell Transplants From CMV Seronegative Donors.
Authors: Knight Andrea , Mackinnon Stephen , Lowdell Mark W 
Published: October 2019

Abstract 2233

Poster Board II-210

Reactivation of cytomegalovirus (CMV) early post-transplant remains a serious complication in lymphopenic recipients after allogeneic stem cell transplantation (SCT). CMV-specific alpha/beta T cells are the principal effector T cells in CMV infection in normal immunocompetent individuals. The role of gamma/delta T cells in anti-CMV responses is not currently fully understood. The majority of human circulating gamma/delta T cells express a TCR encoded by the Vgamma9 and Vdelta2 gene segments. Minor subset of circulating gamma/delta T cells known as Vdelta2neg cells (mainly Vdelta1 and Vdelta3) are expanded in CMV seropositive normal individuals compared to CMV seronegative donors (p=0.0002). CMV-reactive Vdelta2neg gamma/delta T cell lines were generated from seropositive healthy donors and shown to lyse CMV infected target cells in vitro. We have reported an expansion of the same subset of gamma/delta T cells in recipients of allogeneic SCT associated with CMV reactivation. Vdelta2neg cell lines were generated from a CMV+ patient who received a graft from a CMV+ sibling donor at two separate early time points post-transplant and also shown to be CMV-reactive with the ability to lyse CMV infected targets. Since gamma/delta T cells are part of the innate immune response we investigated whether CMV-reactive gamma/delta T cells can be isolated from CMV seronegative donors. Polyclonal Vdelta2neg gamma/delta T cell lines were raised and expanded from multiple CMV seronegative healthy subjects using feeder cells from seronegative and seropositive donors. Briefly, Vdelta2neg gamma/delta T cells were purified from peripheral blood mononuclear cells from CMV seronegative donors by immunomagnetic sorting and cultured in the presence of irradiated PBMC from two HLA-mismatched normal donors. Gamma/delta T cells were cultured for up to 5 weeks. The degree of gamma/delta T cell expansion ranged from 78-3250 fold and the purity of Vdelta2neg gamma/delta T cell lines averaged 88.3 %. Lines were co-cultured with uninfected MRC5 fibroblasts and CMV infected fibroblasts with TB40E or VHLE clinical CMV strains at different effector/target ratios. Vdelta2neg gamma/delta T cells showed specific cytotoxicity by lysis of CMV infected but not uninfected fibroblasts. The frequency of generation of CMV-reactive Vdelta2neg gamma/delta T cells was not affected by the use of feeder cells from seropositive versus seronegative donors. These results suggest that CMV-reactive Vdelta2neg gamma/delta T cells represent a novel approach for an adoptive immunotherapy for CMV seropositive recipients of grafts from CMV seronegative donors who are lacking protective CMV immunity and are at greater risk of CMV reactivation. CMV-reactive Vdelta2neg gamma/delta T cells generated from CMV seropositive donors could be used adoptively in patients suffering CMV reactivation without a risk of GvHD.

Disclosures:

Lowdell: Cell Medica Ltd: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.