Mass cytometry by time-of-flight (CyTOF) permits the simultaneous assessment of over 40 functional and phenotypic parameters at the single-cell level. This novel tool holds tremendous potential in enabling high-dimensional, quantitative analysis of complex diseases and biological systems such as kidney rejection. Here, we are reporting our high-throughput 34-marker panel analyzing the ploidy, immunophenotype, frequency of cell subsets, and expression levels of peripheral blood of 28 out of 60 patients recruited while undergoing a kidney transplant biopsy for evaluation of acute rejection. Correlational bioinformatic analyses were performed using the spanning-tree progression analysis of density-normalized events (SPADE) and visualized t-distributed stochastic neighbor embedding (viSNE) algorithms. By gating on a broad parent population of CD45⁺ lymphocytes, the SPADE and viSNE algorithms allowed a non-biased approach to mapping the uncharted heterogeneity of kidney transplantation. First, we were able to detect known immune signatures of rejection, such as increased granzyme B (GrB) expression among CD8 T and NK cells in patients diagnosed with cellular rejection compared to non-rejectors, in addition to an increase in T_H1 and T_H17 T cells. Interestingly, we also observed an increase in T_H1 and T_H17-like T_Regs during rejection. Furthermore, we saw a change in different cell populations including a subpopulation of B cells that express CCR6. Aberrations in CCR6 have previously been linked to transplant-related infections and chronic graft-versus-host disease but not rejection. In addition, we identified a subpopulation of APCs co-expressing HLA-DR and CD39, that was absent in the peripheral blood of healthy volunteers, however, present after transplantation, and significantly reduced during rejection (39% vs. 17%, non-rejectors vs. rejectors, *P<0.05). This population is thought to desensitize dendritic cells and neutralize T cell responses by CD39-catalyzed conversion of pro-inflammatory ATP to immunoregulatory adenosine. Analysis of the remaining 32 patients by CyTOF is currently underway to further characterize the changes seen after kidney transplantation and to allow flow sorting of cell subtypes for more in-depth ex vivo functional assays.

CITATION INFORMATION: Eskandari S., Al Dulaijan B., Assaker J., Riella L., Kurdi A., Azzi J. Elucidating Novel Signatures of Allograft Rejection through Cytometry by Time of Flight (CyTOF) in Kidney Transplant Recipients Am J Transplant. 2017;17 (suppl 3).

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