*Purpose: Transplant (Tx) rejection currently requires an invasive biopsy for diagnosis. We have tested if serial quantitation of donor reactive T cell clones (DRTC) can serve as an alternative, noninvasive approach.

*Methods: Eighty kidney Tx (KTx) recipients were enrolled and their DRTC repertoire was identified by performing a pre-Tx donor-specific MLR, flow-sorting of recipient CFSE-diluting CD4 and CD8 responding cells and subsequent T-cell immunosequencing using the immunoSEQ^® Assay. Then, in the post-tx period, the pre-identified DRTCs were tracked in KTx biopsies (protocol biopsy at 3, 12 months and for-cause) as well as in blood and urine (3, 6, 12 months and for-cause), again by immunosequencing.

*Results: The pre-transplant donor reactive flow sorted CD4 and CD8 samples identified an average of 8,672 (380 – 21,550) CD4 and 3,100 (267 – 9,476) CD8 clones as DRTC (rearrangements needed to be present in at least 5 cells to be counted). Six KTx recipients developed biopsy proven rejection; 15 had other for-cause biopsies, and 42 were stable. In the 6 rejecting patients, the rejecting biopsies contained amplified presence of DRTC when compared to 3- or 12-month protocol biopsies from the stable subjects (combined CD4 and CD8 DTRC are shown in Table 1) indicating a direct relationship between rejection and increased frequency of DRTC (p < 0.01). When the DRTCs were tracked in the peripheral blood, the rejecting patients had marginally increased DRTC at rejection when compared to the stable subjects at 3 or 12 months (stable subjects did not have direct comparable samples as they did not have rejection). More strikingly, in the urine pellets, rejecting subjects had 5-46 DRTC at rejection as opposed to 5±2 and 6 ±3 In stable subjects at 3 and 12 months respectively (p < 0.01), suggesting that rejection can be diagnosed non-invasively in urine samples. Furthermore, the amplified presence of DRTC could be identified in biopsy and urine prior to (i.e. at 3 months) the diagnosis of rejection which occurred after 3 months in 5/6 patients, thus demonstrating that monitoring for DRTC might predict an upcoming rejection episode. In addition, successful treatment and resolution of the rejection reduced the number of DRTC subsequently identified at 12 months in biopsy, blood and urine.

*Conclusions: These results suggest that monitoring for DRTCs can be used as a potential diagnostic and/or predictive tool and that this can be achieved non-invasively through analysis of blood or urine..

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