*Purpose: There is an unmet need for reliable minimally-invasive diagnostic biomarkers for immunological allograft monitoring and detection of acute kidney transplant rejection. Here, targeted proteomic analysis was applied to compare 92 proteins in sera of belatacept-treated patients who had a biopsy-proven, acute T cell-mediated rejection (aTCMR) with patients without an aTCMR.

*Methods: Serum samples were collected from kidney transplant recipients who participated in a prospective, randomized-controlled trial between 2013 and 2015. Proximity extension immunoassay (PEA) was used to measure 92 inflammation-related protein concentrations in pre-rejection (day 30 after transplantation) and rejection sera of 11 patients with an aTCMR and 9 patients without an aTCMR. PEA uses two matched oligonucleotide‐labelled antibody probes for each protein and PCR to measure normalized protein expression values.

*Results: Five proteins (CD5, CD8A, NCR1, TNFRSF4 and TNFRSF9) were expressed significantly higher in samples of patients with an aTCMR compared with samples of patients without an aTCMR (adjusted p-value<1.14E-02) and had a good predictive capacity for an aTCMR (area under the curve of a receiver operator curve ranged from 0.83 to 0.91 [p<0.014]). The pathways most enriched among these 5 proteins are related to T cell activation, T cell proliferation, and NK cell-mediated immune responses. Non-hierarchical clustering analysis showed distinct clustering of samples of patient with an aTCMR and of samples of patients without aTCMR. This clustering was not seen in pre-rejection samples. In pre-rejection samples, IFN-γ was expressed at a significantly lower level (NPX value median -0.15, IQR -0.27 - 0.04) than in samples of patients without rejection (median 0.13, IQR -0.07 - 0.15, adjusted p-value=3.67E-03).

*Conclusions: Targeted proteomic analysis with PEA was used for the first time in kidney transplant patients and detected aTCMR in sera of belatacept-treated patients. PEA appears to be a promising minimally-invasive technique to diagnose an aTCMR.

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