Combined donor kidney plus facilitating cell enriched hematopoietic stem cell (FCRx) transplants (Tx), after low intensity conditioning with fludarabine, 200-cGy total body irradiation and cyclophosphamide were initiated in a phase IIb tolerance trial. Recipients were maintained on immunosuppression with tacrolimus and mycophenolate mofetil post-Tx. Twelve of the 19 patients with >24 months follow-up developed robust chimerism, with rapid return of CD4+ and CD8+ T central and effector memory cell populations, and diverse PBMC TCR repertoire. In the current study, we evaluate the extent of TCR repertoire development in CD8 and CD4 cell subsets as a measure of immune-competence in chimeric and non-chimeric patients.

PBMC drawn after 2-year post-Tx were fractionated into CD8+ and CD4+ cell subsets using Miltenyi Treg isolation kit. DNA was isolated using Qiagen QIAamp DNA micro kit. TCR sequences were obtained using high throughput Illumina HiSeq platform by Adaptive biotechnologies.

We previously reported a 97% novel post-Tx TCR repertoire versus pre-Tx in both chimeric and non-chimeric patients. Current analysis revealed that there is only a statistically insignificant reduction in clone richness within CD8+, CD4+CD25-, and CD4+CD25+ cells in the chimeric recipients as compared to those of the donor.

Additionally, we found a large pool of stable overlapping clones (55/1000 top clones in CD8+ cells; 30/1000 in CD4+CD25+ cells) in chimeric samples and the donor, suggesting a robust immune reconstitution with donor cells at post-tx. These specific clones were not present in post-Tx samples of non-chimeric patients, which instead had overlapping clones with recipient pre-tx.

We conclude that patients receiving HLA mismatched kidneys and FCRx exhibit robust immune reconstitution within CD8+ and CD4+ subsets by 2 years post-Tx, chimeric with the donor and non-chimeric with the recipient.

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