Tissue effector signatures define distinct mechanistic phenotypes of microvascular inflammation and predict kidney allograft outcomes beyond DSA and C4d
Overview
Tissue Effector Signatures Define Distinct Mechanistic Phenotypes of Microvascular Inflammation and Predict Kidney Allograft Outcomes Beyond DSA and C4d
B. Handan Özdemir, Alev Ok Atılgan, Aydan A. Özdemir, Mehmet Haberal
Background
Microvascular inflammation (MVI) is a key feature of antibody-mediated rejection (ABMR). However, classification based solely on donor-specific antibodies (DSA) and C4d deposition does not fully capture the biological heterogeneity of MVI. Importantly, MVI can occur in the absence of detectable DSA or C4d, highlighting the need for tissue-based approaches to better define the underlying effector mechanisms and clinical outcomes.
Methods
Totally 166 renal biopsies with MVI were analyzed. Immunostaining was performed to characterize immune effector pathways and endothelial injury, including markers of complement activation (C4d, C5b-9), cytotoxic effector activity (granzyme B, perforin), macrophage infiltration (CD68), NK-cell activation and ADCC signaling (CD16, NKp46), endothelial activation (HLA-DR), inflammatory signaling (TNF-α, CD44, CD47), fibrotic remodeling (TGF-β), and nitric oxide regulation (eNOS, iNOS).
Cases were stratified into mechanistic phenotypes based on DSA, C4d deposition, and tissue effector signatures: Group 1 (DSA⁺/C4d⁺ complement-dominant ABMR), Group 2 (DSA⁺/C4d⁻ ADCC/NK-dominant ABMR), and Group 3 (DSA⁻/C4d⁻ MVI), further subdivided into innate-high (3A) and intermediate innate activation (3B).
Outcomes included time from the index biopsy to the development of interstitial fibrosis (IF), transplant glomerulopathy (TG), and graft loss within 10 years of follow-up.
Results
Phenotype distribution was 36.7% in Group 1, 33.7% in Group 2, 16.9% in Group 3A, and 12.7% in Group 3B. Group 1 showed strong complement deposition with relatively lower cytotoxic and innate inflammatory signaling than other groups (p<0.001). Group 2 showed the most marked cytotoxic effector activity, characterized by increased activation of CD16/NKp46-positive NK cells, granzyme B/perforin expression, macrophage infiltration, elevated TNF-α/CD44 signaling, and reduced CD47 expression (p<0.001). Histologically, this group showed the highest MVI burden and greater interstitial inflammation (p<0.01). Group 3A showed partially overlapping innate effector signatures, whereas Group 3B showed mild inflammatory activity. Innate-dominant phenotypes showed reduced eNOS and increased iNOS expression, consistent with iNOS-mediated endothelial injury, and were associated with earlier IF and TG progression and poorer graft survival (p<0.001). Group 2 also showed the strongest association with persistent MVI and PTC destruction, whereas Group 1 lesions more often showed fluctuating or resolving MVI (p<0.01).
Conclusion:
MVI in renal allografts reflects biologically distinct injury pathways beyond conventional DSA/C4d classification (Figure 1). C4d-negative ADCC/NK-dominant MVI emerges as the most aggressive phenotype, characterized by enhanced cytotoxic signaling, nitric oxide dysregulation, and accelerated chronic graft injury. Tissue-based effector signatures may improve prognostic stratification and support mechanism-targeted therapeutic strategies.
Figure 1
Authors
Affiliations
Abstract
Background: Microvascular inflammation (MVI) is a key feature of antibody-mediated rejection (ABMR). However, classification based solely on donor-specific antibodies (DSA) and C4d deposition does not fully capture the biological heterogeneity of MVI. Importantly, MVI can occur in the absence of detectable DSA or C4d, highlighting the need for tissue-based approaches to better define the underlying effector mechanisms and clinical outcomes.
Methods: Totally 166 renal biopsies with MVI were analyzed. Immunostaining was performed to characterize immune effector pathways and endothelial injury, including markers of complement activation (C4d, C5b-9), cytotoxic effector activity (granzyme B, perforin), macrophage infiltration (CD68), NK-cell activation and ADCC signaling (CD16, NKp46), endothelial activation (HLA-DR), inflammatory signaling (TNF-α, CD44, CD47), fibrotic remodeling (TGF-β), and nitric oxide regulation (eNOS, iNOS).
Cases were stratified into mechanistic phenotypes based on DSA, C4d deposition, and tissue effector signatures: Group 1 (DSA⁺/C4d⁺ complement-dominant ABMR), Group 2 (DSA⁺/C4d⁻ ADCC/NK-dominant ABMR), and Group 3 (DSA⁻/C4d⁻ MVI), further subdivided into innate-high (3A) and intermediate innate activation (3B).
Outcomes included time from the index biopsy to the development of interstitial fibrosis (IF), transplant glomerulopathy (TG), and graft loss within 10 years of follow-up.
Results: Phenotype distribution was 36.7% in Group 1, 33.7% in Group 2, 16.9% in Group 3A, and 12.7% in Group 3B. Group 1 showed strong complement deposition with relatively lower cytotoxic and innate inflammatory signaling than other groups (p<0.001). Group 2 showed the most marked cytotoxic effector activity, characterized by increased activation of CD16/NKp46-positive NK cells, granzyme B/perforin expression, macrophage infiltration, elevated TNF-α/CD44 signaling, and reduced CD47 expression (p<0.001). Histologically, this group showed the highest MVI burden and greater interstitial inflammation (p<0.01). Group 3A showed partially overlapping innate effector signatures, whereas Group 3B showed mild inflammatory activity. Innate-dominant phenotypes showed reduced eNOS and increased iNOS expression, consistent with iNOS-mediated endothelial injury, and were associated with earlier IF and TG progression and poorer graft survival (p<0.001). Group 2 also showed the strongest association with persistent MVI and PTC destruction, whereas Group 1 lesions more often showed fluctuating or resolving MVI (p<0.01).
Conclusion: MVI in renal allografts reflects biologically distinct injury pathways beyond conventional DSA/C4d classification (Figure 1). C4d-negative ADCC/NK-dominant MVI emerges as the most aggressive phenotype, characterized by enhanced cytotoxic signaling, nitric oxide dysregulation, and accelerated chronic graft injury. Tissue-based effector signatures may improve prognostic stratification and support mechanism-targeted therapeutic strategies.