BACKGROUND: The need for tolerance protocols in organ transplantation is underscored by the morbidity associated with chronic immunosuppressant use and the inability to prevent chronic rejection. Induction of donor chimerism is currently the most promising strategy to achieve renal allograft tolerance in humans. In an ITN-sponsored trial, several CKBMT recipients have tolerated their allograft for several years in the absence of any immunosuppressive medication, with development of donor-specific unresponsiveness in in-vitro assays. Donor chimerism was present for less than 3 weeks in each of these patients, and the precise mechanisms of tolerance are not known. Assessing deletional tolerance has previously been impossible due to the unavailability of markers for the many thousands of T cell clones responding to HLA alloantigens. METHODS: We have implemented a TCR deep sequencing approach to identify and track the alloreactive T cell repertoire between a given donor-recipient pair. In a commercially-available technique (ImmunoSEQ; Adaptive™), CDR3 regions are amplified with primers specific for all 54 known expressed VΒ and all 13 JΒ regions adapted for solid phase PCR, allowing high throughput sequencing of millions of T cell clones as well as detection of rare clones. We hypothesized that high throughput CDR3 sequencing of a CKBMT recipient's donor-responsive T cells in a one-way mixed lymphocyte reaction prior to transplant would allow identification of the TCRs that specifically recognize their organ donor's alloantigens, providing a method of tracking donor-reactive T cells in the post-transplant period. RESULTS: In a study of one tolerant CKBMT patient, we identified thousands of pre-transplant CD4⁺ and CD8⁺ T cell clones that were significantly enriched upon exposure to irradiated donor PBMC. Of the clones that were expanded at least ten-fold and were detectable pre-transplant, only 22/164 (13.4%) CD4⁺ and 10/64 (15.6%) CD8⁺ clones were still detectable at 1.5 years post-transplant. Donor-reactive clones were much more likely to disappear after transplant than non-donor-reactive clones (OR=35 and 5.7 for CD4⁺ and CD8⁺ clones, respectively; p<10^-15 for each). CONCLUSIONS: Tolerance following CKBMT was associated with deletion of donor-reactive T cells in this patient. Further studies are in progress in additional tolerant patients.

Manitpisitkul, W.: Stockholder, Adaptive Biotechnologies, Other, Adaptive Biotechnologies, Consultant. Robins, H.: Stockholder, Adaptive Biotechnologies, Other, Adaptive Biotechnologies, Consultant.

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