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

Project: Research project

Project Details

Description

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
StatusFinished
Effective start/end date8/1/167/31/18

Funding

  • University of Minnesota (N002854801//1U01AI1202463-01)
  • National Institute of Allergy and Infectious Diseases (N002854801//1U01AI1202463-01)

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