Background: The complex repertoire of immune receptors generated by B cells enables allo-recognition in the context of transplantation across HLA barriers, and is a major contributor for the persistence of unrecognized, sub-clinical lymphocyte immune activation, resulting in acute and chronic immune mediated graft injury. High-throughput DNA sequencing technologies have been advancing at an extremely rapid pace and are transforming our understanding of humoral immune responses.

Methods: DNA sequencing of rearranged immune receptor loci was performed in 99 serial blood samples, collected at 0, 6, 12 and 24 months after transplantation in 36 unsensitized kidney transplant recipients with and without acute rejection and progressive chronic injury. Matched, centrally scored, histopathology compartment scores and graft function were also obtained on all patients at all time points. We used a series of redundant primers to amplify diverse DNA rearrangements, and the resulting mixtures of barcoded amplicons were sequenced using long-read ultra-deep sequencing. Individual DNA molecules were then characterized on the basis of DNA segments that were joined to make functional (or nonfunctional) immune effectors.

Results: Nearly 600,000 sequences were generated across 99 samples resulting in roughly 200,000 unique clones, defined by the VDJ recombination patterns and the CDR3 region sequence. We observed significantly more B-cell sequences and clones present in the acute rejection group in comparison to the other sub-groups (ANOVA p= 0.0006545), with greater expansion also seen in the patients with greater biopsy chronicity at 24 months (ANOVA p=5.016e-05). Importantly, in the acute rejection group B cell sequences and clones were expanded across all time points, inclusive of the pre-transplant sample. Longitudinal analysis tracking clones across the various time points observed similar and related clones across different people.

Conclusions: VDJ sequencing demonstrates expansion of specific B-cell clones prior to transplantation, even in the absence of allo-sensitization. This B cell clonal expansion is highly predictive of acute rejection and accelerated chronic rejection. B cell immune priming is currently unrecognized, and maybe harnessed for immunosuppressive drug selection at engraftment.

CITATION INFORMATION: Sirota M, Sigdel T, Boyd S, Fire A, Sarwal M. VDJ Immune Repertoire Sequencing Predicts Patients at Risk of Alloimmune Injury. Am J Transplant. 2016;16 (suppl 3).

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