Blocking CCL8-CCR8–Mediated Early Allograft Inflammation Improves Kidney Transplant Function

Abstract

BackgroundIn kidney transplantation, early allograft inflammation impairs long-term allograft function. However, precise mediators of early kidney allograft inflammation are unclear, making it challenging to design therapeutic interventions.MethodsWe used an allogeneic murine kidney transplant model in which CD45.2 BALB/c kidneys were transplanted to CD45.1 C57BL/6 recipients.ResultsDonor kidney resident macrophages within the allograft expanded rapidly in the first 3 days. During this period, they were also induced to express a high level of Ccl8, which, in turn, promoted recipient monocyte graft infiltration, their differentiation to resident macrophages, and subsequent expression of Ccl8. Enhanced graft infiltration of recipient CCR8+ T cells followed, including CD4, CD8, and γδ T cells. Consequently, blocking CCL8-CCR8 or depleting donor kidney resident macrophages significantly inhibits early allograft immune cell infiltration and promotes superior short-term allograft function.ConclusionsTargeting the CCL8-CCR8 axis is a promising measure to reduce early kidney allograft inflammation.

AXL inhibition suppresses early allograft monocyte-to-macrophage differentiation and prolongs allograft survival

“…In the presence of AXL inhibition by bemcentinib, the resulting Mϕs are not only diminished in number ( Figure 1C ) but also in their ability to present alloantigen ( Figure 1D ), with a subsequent reduction in the activation and proliferation of allospecific T cells ( Figure 2B ). Our monocle psuedotime analysis of a single-cell RNA-Seq data set of murine heart allografts, we observed a similar upregulation of the Axl transcripts during early intragraft MC-to-Mϕ differentiation ( Figure 3E ), concordant with our published findings in a murine allogeneic kidney transplant model ( 1 ). Lastly, transient AXL blockade using bemcentinib treatment in a murine heart transplant model leads to a global reduction in intragraft inflammation and to an extended allograft survival ( Figures 4 – 6 ).…”

“…We have previously demonstrated in a murine allogeneic kidney transplant model that recipient MCs first arrive at the transplanted kidney as Ly6C + F4/80 – cells in a donor Mϕ-dependent, CCL8/CCR8 axis–dependent manner ( 1 , 2 ). In this model, pseudotime analysis using single-cell transcriptomics of the kidney allograft revealed that graft-infiltrating recipient MCs quickly differentiated to inflammatory Mϕs (iMϕs), and this process appeared to correlate with a transient upregulation of the receptor tyrosine kinase AXL in these cells ( 2 ).…”

“…Following staining incubation, cells were fixed with 4% PFA (Santa Cruz Biotechnology Inc.). Cardiac allograft tissue was digested to a single-cell suspension using collagenase type IV (2 mg/mL, Worthington Biochemical Corporation) with mechanical agitation for 30 minutes at 37°C, followed by RBC lysis with ammonium-chloride-potassium lysis buffer, before staining for extracellular surface markers as per above ( 1 ). Prior to staining, cells were incubated for 15 minutes at 4°C with purified anti-mouse Fc shield (anti-CD16/32; 2.4G2, Tonbo Biosciences 70-0161-U100).…”

“…Immediately upon anastomosis and reperfusion, recipient immune cells enter the donor organ via circulation. We have previously shown that the process of early recipient monocyte (MC) trafficking to and retention in the donor transplanted organ is mediated via CCL8/CCR8 interaction and is dependent on donor macrophages residing in the transplanted donor organ ( 1 ). Through bioinformatics analysis, we determined that recipient MCs, once extravasated, differentiate to macrophages (Mϕs), and replace the donor Mϕs as the new tissue resident Mϕs ( 2 ).…”

Standardization and Interpretation of RNA-sequencing for Transplantation

“…Equipped with the ability to interrogate the transcriptome and clonality of graft-infiltrating cells at a single-cell level, we showed in an allogeneic murine kidney transplant model that graft-infiltrating αβ and γδ T cells both express surface CCR8 and that blocking the CCL8-CCR8 axis severely impairs the recruitment of these cells to the kidney allograft. 89 Our data provide evidence that the CCL8-CCR8 axis is a vital linchpin between donor kidney resident macrophages and early immune cell infiltration and therefore represents a promising therapeutic target.…”