Donor-specific T Cell Activation and Expansion Following a Pig-to-human Decedent Kidney Transplant

Abstract Text:

Introduction: Pig-to-human decedent transplantation provides a unique window into human anti-pig xenoresponses. We analyzed donor-specific T cell immunity in the context of a long-term (61-day) pig-to-human decedent thymokidney transplant.
Materials and

Methods: Blood and kidney biopsy samples were obtained pre-transplantation and at POD14, 28, 33 and 49. To identify donor-reactive T cell clones (DRTCCs), we performed direct and indirect mixed lymphocyte reactions pre-transplant and on POD 28. We FACS sorted proliferating CFSElow CD4 and CD8 T cells, extracted DNA and performed high-throughput TCRβ CDR3 sequencing (Immunoseq, Adaptive). Donor-reactive T cell clones (DRTCCs) were identified by expansion compared to sequenced unstimulated T cells. Kidney biopsies underwent digestion and lymphocyte isolation followed by single-cell RNA sequencing with TCR sequencing (10X).

Results: Mild AMR on POD33 was successfully reversed. No pathological evidence for cell-mediated rejection was observed up to Day 61, when the experiment was terminated. DRTCCs were expanded in PBMCs at POD 33 and 49, when CD8 DRTCCs formed >15% of the total CD8 repertoires. In rejection-free biopsies (POD14 and 28), scRNA sequencing did not reveal activated T cells or NK cells. At POD33, scRNA sequencing revealed activated T cells and NK cells containing effector gene transcripts, including GZMB, PRF1, granulysin, IFN-γ, CCL4 and CCL5. Activated antigen-presenting cells with upregulated proinflammatory genes were also evident. DRTCCs were detected among graft-infiltrating T cells; transcriptomic analysis is in progress.

Conclusion: Our results suggest a role for activated T cells and NK cells in apparently mild antibody-mediated porcine xenograft rejection in humans.