*Purpose: Monocytes play an important role in immune defense by initiating adaptive immunity. They are heterogeneous, plastic, and present different subsets: classical (CD14⁺⁺CD16^–), intermediate (CD14⁺CD16⁺), and non-classical (CD14^dimCD16⁺⁺). While recent clinical studies have demonstrated a critical role for monocytes and macrophages to allo-immunity, a comprehensive phenotypic analysis of circulating blood monocyte subsets during rejection is still lacking. Here, we hypothesize that monocytes with distinct inflammatory programs may differently contribute to TCMR or mixed-ABMR after kidney transplantation (KTx).

*Methods: Peripheral blood samples from 48 KTx were analyzed using 22 multi-color panel flow cytometry. Patients were classified as: Stable (without donor-specific antibodies (DSA), TCMR and/or ABMR before or at time of sample); TCMR (without DSA, at the time of a for cause biopsy Banff >1A ); and mixed-ABMR (with DSA , at the time of a biopsy-proven mixed-ABMR). All samples were downsized to same number of monocytes and concatenated for unsupervised analysis by viSNE and FlowSOM, enabling visualization of high dimensional single-cell data. In addition, we performed scRNA-sequencing on bead-sorted monocytes from selected patients (n=3 from each group).

*Results: Our results reveal significant phenotypic heterogeneity of blood circulating monocytes among the 3 groups. Specifically, TCMR patients displayed one unique classical CD14⁺⁺CD16^– cluster that expressed low checkpoint inhibitory receptors (PD1/PDL-1), intermediate levels of CD11b (adhesion), CCR2 (migration), HLA-DR (antigen presentation) and of the scavenger receptor CD36. Conversely, monocytes from patients undergoing mixed-ABMR comprised of two activated, unique intermediate CD14⁺CD16⁺ cell clusters that expressed high CD32, CD86 (both activation markers) and CCR5 (chemokine receptor), but that differed in CX3CR1 (migration) and CD163 (scavenger) expression. None of these clusters were present in stable patients. Moreover, we identified 43 differentially expressed protein coding genes between TCMR and mixed-ABMR patients’ circulating monocytes, including genes related to apoptosis modulation and signaling, Toll-like receptor signaling pathways, and IL1R associated kinases genes.

*Conclusions: In conclusion, our data suggest that different monocyte subsets may contribute TCMR vs mixed-ABMR, suggesting the potential need for distinct therapeutic targeting of monocytes during cellular vs humoral rejection.

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