524. Immune-Monitoring of Mature Human T Cell Reconstitution in Humanized NRG Mice After Cord Blood Stem Cell Transplantation and Engineered Dendritic Cell Prime/Boost: TCR Polyclonality and No GvHD

Abstract

Cord blood (CB) is a major and convenient “off the shelf” source of stem cells for hematopoietic stem cell transplantation (HSCT) when a matched donor is not available. Although relatively lower incidence of graft versus host disease is observed after CB-HSCT than PB-HSCT, delayed T cell immune reconstitution, relapse and viral infections harmfully impact survival after CB-HSCT. We validated genetically reprogrammed CB-derived monocytes capable to differentiate into dendritic cells in vivo as means to improve human immune reconstitution in lymphopenic hosts and protect them against cytomegalovirus (CMV). Monocytes were isolated from the CB CD34− fraction, transduced with an integrase-defective lentiviral vector (co-expressing GM-CSF, IFN-α and the CMV antigen pp65) and administered after CB-HSCT or cryopreserved. Prime/boost administrations of the engineered “SmyleDCpp65” cell vaccine into NOD.Rag1–/–.IL2rγ–/– mice 6-11 weeks after CB-HSCT resulted into higher levels of human T cell development in the thymus and evidence of maturation of memory and effector T cells in peripheral blood and secondary lymphatic tissues. This model allows mechanistic characterization of human CTL, Th and Treg cell reconstitution (by multicolor flow cytometry), homing (bone marrow, thymus, spleen, lymph nodes, liver; by flow cytometry), immune functions (pp65-specific responses by ELISPOT and cytotoxic assays), T cell receptor polyclonality analyses (by deep sequencing), inflammatory responses (array analyses of plasma cytokines and immune globulins). Despite the de novo activation of human T cells, GVHD was not observed. This model demonstrates the prof-of-concept of an adjuvant cell vaccine to be used in conjunction with CB-HSCT in the clinics and offers a practical experimental system to study adaptive human T cell immune reconstitution and antigen-specific responses in vivo. We are currently evaluating further approaches to maximize lentiviral transduction of CB monocytes and co-stimulation of human antibody responses against the cytomegalovirus gB antigen in the CB-HSCT model.