Miller - Alloantigen Delivery Via ECDI-Fixed Cells for Tolerance to Monkey Islet Grafts

Aim 1: To manufacture ADEC products meeting prospectively defined release criteria for evaluation as tolerogens in islet allotransplantation in RM: Workscope for Northwestern University: • Flow cytometry phenotyping of pre-ECDI-treated splenocytes and PBLs from NHP donors Aim 3: To examine the effects of the immunotherapeutic protocol on mechanisms underlying the induction, maintenance, and/or loss of donor-specific tolerance to islet allografts in RM: Workscope for Northwestern University: Aim 3a - Innate immunity. • Cytokine and chemokine innate immunity by ELISA (UMN or NWU, to be determined). Aim 3b - Adaptive direct and indirect alloimmunity. Workscope for Northwestern University: • Monitoring by flow cytometry half-life of infused ECDI-fixed vaccine cells (stained with fluorescent dye) in circulating recipient PBLs post-vaccination • Donor-specific antibodies by flow cytometry • Longitudinal flow cytometric analyses of circulating phenotypes including regulatory, effector, and memory T- and B-cells. • Anti-donor T cell proliferation by CFSE with flow cytometry • Cytokine analyses by ELISPOT • Analyses of the phenotype of graft-infiltrating cells obtained from liver, splenocytes and lymph nodes by flow cytometry, at sacrifice • Analyses of the phenotype of graft-infiltrating cells obtained from liver, splenocytes and lymph nodes by immunohistochemistry, at sacrifice. • Trans-vivo Delayed Type Hypersensitivity assay in mice Aim 3c – preexisting, heterologous, memory alloimmunity Workscope for Northwestern University: • Pre-existing, heterologous, memory alloimmunity and newly formed memory post- transplant by flow cytometry. Please also see Aim 3b above. Aim 3d – induction and function of lymphocytes with regulatory phenotypes Workscope for Northwestern University (to be updated in consultation with PIs): • Quantification of circulating and tissue T, B, NKT cells including cells with regulatory phenotypes by flow cytometry for characterization of cytokine producing cell patterns • Determination of donor specificity of the observed regulation by RT-PCR. • Homing characteristics of regulatory cells by immunohistochemistry on fixed paraffin sections of liver at sacrifice