*Purpose: The role of NK cell during antibody-mediated rejection (ABMR) has been previously recognized, but an in-depth characterization of pathways that contribute to such immune response is still not well understood. Here, we aimed to characterize differential phenotypic, functional, and transcriptomic changes of circulating CD56^dimCD16^bright NK cells during ABMR as compared to those occurring during T-cell mediated rejection (TCMR).

*Methods: Cross-sectional phenotypic analysis of circulating NK cells by spectral flow cytometry was implemented at the time of event in 71 kidney transplant recipients: 17 anti-HLA DSA+ mixed ABMR, 17 TCMR, and control patients (17 DSA+ free of rejection, 20 DSA- stable free of rejection). Cytotoxicity assay against T2 lymphoblastic cells in presence of DSA (antibody-dependent cellular cytotoxicity (ADCC)) and stimulation with cg-chain cytokine IL-21 were conducted. Plasma cytokines/chemokines profiles were established with Meso Scale Discovery immunoassays. Single-cell RNA sequencing (10X Genomics) on sorted circulating NK cells and kidney allograft bulk transcriptomic analysis were performed.

*Results: CD56^dimCD16^bright NK cells from ABMR patients showed increased (i) Th1-related transcription factors EOMES/T-bet co-expression, (ii) IL-21 responsiveness potential (IL-21R expression), (iii) CD16a-inducible CD160, CD161/NK1.1, NKG2D cytotoxic and activating receptors. Increased Type-1 inflammatory cytokine release (high IFN-γ/IL-10 and TNF-α/IL-10) was noted during ADCC. Plasma from ABMR patients showed elevated IL-21 levels that correlated with CD160 and CD161 expression, and with plasma TNF-α. IL-21 stimulation enhanced EOMES and CD160 expression, IFN-γ and TNF-α release. During ABMR, circulating NK cell transcriptomic profile involved enhanced FCGR3A and Th1 pathways, as well as transcripts involved in polarization and release of cytotoxic granule granulysin/granzymes. ABMR biopsies, unlike those from TCMR, featured strong involvement of NK cell signaling and FcγR-mediated activation pathways as well as increased FCGR3A, IL21R, TBX21, EOMES, CD160, KLRK1 transcripts suggesting infiltration of such NK cells, and supporting the results obtained on circulating CD56^dimCD16^bright NK cells.

*Conclusions: Significant and unique DSA- and IL-21-triggered Type-1 inflammatory/cytotoxic NK cell changes occur during ABMR and potentially contribute to allograft injury. Early detection of activated, Type-1 inflammatory, cytotoxic IL-21R+ CD56^dimCD16^bright NK cells in the blood of ABMR patients could help for timely therapeutic intervention.

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