Mechanistic Studies in a New Tolerance Trial of Combined Kidney and Bone Marrow Transplantation

Farshid Fathi – Columbia Center for Translational Immunology, Department of Medicine, Columbia University, New York, NY, USA; Benjamin Vermette – Columbia Center for Translational Immunology, Department of Medicine, Columbia University, New York, NY, USA; Yasmeen S. Saad – Columbia Center for Translational Immunology, Department of Medicine, Columbia University, New York, NY, USA; Kevin Breen – Columbia Center for Translational Immunology, Department of Medicine, Columbia University, New York, NY, USA; Morcel Khwajazadeh – Columbia Center for Translational Immunology, Department of Medicine, Columbia University, New York, NY, USA; Vineetha Mohan – Columbia Center for Translational Immunology, Department of Medicine, Columbia University, New York, NY, USA; David Cohen – Department of Medicine, Division of Nephrology, Columbia University, New York, NY, USA; Joshua Weiner – Columbia Center for Translational Immunology, Department of Medicine, Columbia University, New York, NY, USA; Megan Sykes – Columbia Center for Translational Immunology, Department of Medicine, Columbia University, New York, NY, USA

Abstract Text: Tolerance was achieved in a small cohort receiving combined kidney and bone marrow transplantation (CKBMT) with transient mixed chimerism. Cytokine release syndrome causing renal dysfunction led to modification of the regimen by increasing anti-CD2 antibody and adding a short course of MMF. In the previous trial, donor-reactive T cell clones (DRTCCs) were identified via high throughput TCR sequencing and tracked post-transplant. Loss of DRTCCs and expansion of donor-specific Tregs correlated with tolerance.
In the current protocol, high-throughput CDR3-TCRβ sequencing on DNA extracted from FACS-sorted CFSE-low CD4+ and CD8+T cells isolated from pre-transplant mixed lymphocyte reactions (MLRs) and unstimulated PBMCs identified DRTCCs. These were tracked in unstimulated CD4 and CD8 populations at months 4, 12, 24.
The number of circulating CD4 and/or CD8 DRTCCs decreased post-transplant in all patients. When normalized to their pre-transplant total frequencies, all patients except Pt2 who had a higher CD8 relative ratio, showed a relative reduction ( < 1) or a relative ratio around 1 in circulating CD4 and/or CD8 DRTCCs by 4 months post-transplant (Pt1 CD4: 0.03, CD8: 0.09; Pt2 CD4: 0.8, CD8: 2.6; Pt3 CD4: 1.2, CD8: 0.5; Pt5 CD4: 1.4, CD8: 0.5). Transient multilineage donor chimerism was observed in all patients, peaking at 12-70% and decreased to < 1% by 30 days in three patients. Circulating Tregs were enriched (up to 79.5%), then gradually declined to baseline by 7.5-12 months.
Our data indicates an early loss of DRTCCs in CKBMT recipients. Ongoing studies of tolerance and donor-specific Treg sequences will identify mechanistic correlations of outcomes.