CX3CL1⁺ HEV licenses gut-imprinted CX3CR1+ myeloid cells to form an ectopic TLS niche and drive chronic renal allograft dysfunction

Background: Tertiary lymphoid structures (TLS) frequently emerge in chronically rejected renal allografts and strongly correlate with donor-specific antibody production and long-term graft survival. However, the cellular origin of the TLS niche and the mechanisms initiating its maturation remain incompletely defined.
Methods: A multicenter biopsies cohort of chronic allograft dysfunction (CAD) was analyzed to determine the association between TLS maturity and graft outcomes. TLS maturity was quantified based on B/T segregation, PNAd⁺ high endothelial venules (HEV), CD35⁺ follicular dendritic cell networks, GL7/Fas⁺ germinal center (GC) markers, and AID expression. Spatial transcriptomics defined TLS microenvironment dynamics. In syngeneic and allogeneic murine kidney transplantation models (4-16 weeks), single-cell RNA sequencing of intestine, blood, spleen, bone marrow, and graft tissues, combined with CX3CR1^CreER–tdTomato fate mapping, traced myeloid origins. Multispectral immunofluorescence delineated TLS architecture. Functional causality was tested using CX3CL1 blockade, endothelial CX3CL1 or myeloid CX3CR1 deletion, CXCL13 neutralization, conditional myeloid MHCII deletion, and bone marrow chimeras.
Results: TLS maturity independently predicted inferior graft survival and accelerated eGFR decline. Spatial multi-omics revealed progressive accumulation of CX3CR1⁺ myeloid cells surrounding developing GC-like structures, exhibiting enhanced CXCL13 and MHCII programs and tight spatial coupling with CXCR5⁺ B cells, Tfh cells, and HEV networks. Cross-organ single-cell integration and fate mapping demonstrated that intragraft CX3CR1⁺ myeloid cells were predominantly gut-derived. CX3CL1 was highly expressed on HEV-like vessels, and endothelial CX3CL1 or myeloid CX3CR1 deletion markedly reduced myeloid accumulation and TLS formation. Upon entry into the graft, gut-derived CX3CR1⁺ cells underwent environmental reprogramming in a high allo-stimulation, characterized by suppression of mucosal IL-10 programs and amplification of MHCII, CXCL13, and lymphoid-organizing modules. Functionally, these reprogrammed cells promoted GC-B and Tfh interactions, enhanced AID-dependent class switching, supported plasma cell survival via BAFF/APRIL, and amplified donor-specific antibodies, accelerating vascular injury and fibrosis.
Conclusion: We identify a gut–graft axis in which CX3CL1⁺ HEV recruits gut-derived CX3CR1^hi myeloid cells that are environmentally reprogrammed to construct an ectopic mucosal-like TLS niche, driving chronic humoral rejection. Targeting the CX3CL1–CX3CR1 pathway may disrupt intragraft humoral niches and mitigate chronic allograft injury.