The exact composition of leukocyte infiltration during kidney allograft rejection is difficult to comprehend and visualize on the same biopsy slide. Using an innovative technology of multiplex immunofluorescence (mIF), we were able to detect simultaneously NK cells, macrophages, and T cells and to determine their intra‐ or extravascular localization using an endothelial marker. Twenty antibody‐mediated rejection (ABMR), 20 T cell–mediated rejection (TCMR), and five normal biopsies were labeled, with automatic leukocyte quantification and localization. This method was compared to a classic NKp46 immunohistochemistry (IHC) with manual quantification and to mRNA quantification. mIF automatic quantification was strongly correlated to IHC (r = .91, P < .001) and to mRNA expression levels (r > .46, P < .021). T cells and macrophages were the 2 predominant populations involved in rejection (48.0 ± 4.4% and 49.3 ± 4.4%, respectively, in ABMR; 51.8 ± 6.0% and 45.3 ± 5.8% in TCMR). NK cells constituted a rare population in both ABMR (2.7 ± 0.7%) and TCMR (2.9 ± 0.6%). The intravascular compartment was mainly composed of T cells, including during ABMR, in peritubular and glomerular capillaries. However, NK cell and macrophage densities were significantly higher during ABMR in glomerular and peritubular capillaries. To conclude, this study demonstrates the feasibility and utility of mIF imaging to study and better understand the kidney allograft rejection process.

Rejection remains the main cause of premature graft loss after kidney transplantation, despite the use of potent immunosuppression. This highlights the need to better understand the composition and the interactions of the alloreactive inflammatory infiltrate. Here we performed droplet-based single-cell RNA sequencing of 35,152 transcriptomes from 16 kidney transplant biopsies and generated cell-type specific gene expression signatures for deconvolution of bulk tissue. A specific association was identified between recipient-derived FCGR3A+ monocytes, FCGR3A+ NK cells and the severity of intragraft inflammation. Activated FCGR3A+ monocytes overexpressed CD47 and LILR genes and increased paracrine signaling pathways promoting T cell infiltration. FCGR3A+ NK cells overexpressed FCRL3, suggesting that antibody-dependent cytotoxic activity is a central mechanism of NK cell mediated graft injury. To unravel the spatial distribution of immune cells in the allograft, multiplexed immunohistochemistry using 38 markers was applied on 18 independent biopsy slides. In antibody-mediated rejection, FcγRIII+ NK and FcγRIII+ nonclassical monocytes specifically infiltrated the glomerular area. These results highlight the central involvement of innate immune cells in the pathogenesis of allograft rejection and indicate several potential therapeutic targets to improve allograft longevity.