The Clinical and Pathologic Phenotype of Antibody-Mediated Vascular Rejection Diagnosed Using Arterial C4d Immunoperoxidase

Abstract

Introduction: The diagnosis of antibody-mediated vascular rejection (AM-VR) should be reliable and accurate. We hypothesized that arterial C4d (C4d_art) immunoperoxidase deposition represents endothelial interaction with antibody.

Methods: From 3309 consecutive, kidney transplant biopsies from a single center, 100 vascular rejection (VR) cases were compared against rejection without arteritis (n = 540) and normal controls (n = 1108). The clinical utility of C4d_art for diagnosis and classification of AM-VR was evaluated against an independent reference test.

Results: C4d_art occurred in 20.4% of acute, 11.0% of subclinical, and 46% of VR episodes. Semiquantitative C4d_art score significantly correlated with immunodominant donor-specific antibodies (DSAs) (rho = 0.500, P < 0.001), peritubular capillary C4d (C4d_ptc), microvascular inflammation, and Banff v scores. Banff v3 arteritis suggested AM-VR. Addition of C4d_art to Banff antibody-mediated rejection (AMR) schema increased diagnostic sensitivity for AM-VR from 57.9% to 93.0%, accuracy 74.0% to 92.0%, and specificity 95.4% to 90.2% versus Banff 2019 (using C4d_ptc). Death-censored graft failure was associated with C4d_art AM-VR criteria using Cox regression (adjusted hazard ratio [HR] 4.310, 95% CI 1.322-14.052, P = 0.015). VR was then etiologically classified into AM-VR (n = 57, including 36 mixed VR) or "pure" (TCM-VR, n = 43). AM-VR occurred within all post-transplant periods, characterized by greater total, interstitial, and microvascular inflammation, arterial and peritubular C4d, DSA levels, and graft failure rates compared with TCM-VR. Mixed VR kidneys had the greatest inflammatory burden and graft loss (P < 0.001).

Conclusion: C4d_art is a suggestive biomarker of the humoral alloresponse toward muscular arteries. Inclusion of C4d_art into the Banff schema improved its diagnostic performance for detection of AM-VR and etiologic classification of arteritis.

Keywords: C4d; antibody; kidney transplantation; vascular rejection.

Graphical abstract

Figure 1

Study flow diagram. Numbers are biopsy samples, except for graft outcome which used the first biopsy in a unique kidney for actuarial survival (i.e., not total kidney number). Absent C4d was treated as indeterminant. ABOi, ABO-incompatible; aHUS, (atypical) hemolytic uremic syndrome; NIL, no significant histology; TMA, thrombotic microangiopathy; VR, vascular rejection; v0REJ, rejection without arteritis.

Figure 2

Arterial C4d in arteries. (a) Normal artery without C4d staining (C4d_art 0). (b and c) Incomplete noncircumferential C4d in endothelial/subendothelial and intimal layers of muscular arteries with variable involvement of the IEL (C4d_art 1). (d, e, f) Small, medium, and large muscular arteries with circumferential arterial C4d staining (C4d_art 2) of moderate intensity. (h) Antibody-mediated VR with fibrinoid necrosis (Banff v3 lesion) (g,h) with circumferential C4d staining of multiple arteries (C4d_art 3, arrowed g) at post-transplant day 8 with strong class 2 donor-specific antibody (MFI 20,360). Immunoperoxidase from formalin-fixed paraffin-embedded tissue with rabbit polyclonal anti-C4d antibody and hematoxylin and eosin stains are illustrated. C4d_art, C4d staining of arterial endothelium and intima; IEL, internal elastic lamina; MFI, median fluorescence intensity; VR, vascular rejection.

Figure 3

Graft survival by AM-VR classifier. (a) Kaplan-Meier death-censored graft survival of VR (from first index biopsy) of 91 unique kidney transplants dichotomized by presence of C4d_art reported by routine pathologists (P = 0.056 vs. negative). (b) Actuarial graft loss of AM-VR (including mixed AM-VR) classified by Banff 2019 (using C4d_ptc, P = 0.068) or expanded Banff 2019 (using both C4d_ptc and C4d_art, P = 0.037) versus TCM-VR assessed by routine pathologists. (c) Actuarial graft loss of AM-VR classified by expanded Banff 2019 with re-evaluation of C4d_art by the reference pathologist (P = 0.011 vs. pure TCM-VR). AM-VR, antibody-mediated vascular rejection; C4d_art, C4d staining of arterial endothelium and intima; C4d_glom, endothelial C4d staining of glomerular capillaries; C4d_ptc, C4d staining of peritubular capillaries; TCM-VR, T-cell–mediated vascular rejection; VR, vascular rejection.

Figure 4

Arterial wall C4d staining in VR and outcomes. (a) Superficial endothelial/subendothelial C4d staining correlated with C4d in intimal layers extending to IEL, P < 0.001, n = 258 muscular arteries) in VR samples (n = 65) of mixed etiologies. (b) Endothelial/subendothelial C4d was common in AM-VR compared with T-cell–mediated vascular rejection (TCM-VR, P < 0.001). (c) Death-censored graft survival of VR (from first index biopsy only) of 91 unique kidney transplants etiologically into TCM-VR (n = 38), pure AM-VR (n = 19), and mixed VR (AM-VR with TCMR, n = 34). Key: mean ± SEM. Key: ∗P < 0.05 versus TCM-VR. (d) Death-censored graft survival of 834 first biopsies from unique kidney transplants classified by Banff 2019 AMR with and without C4d_art (text for numbers) versus a negative control group without AMR or C4d_art. Key: ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 for each combination. AM-VR, antibody-mediated vascular rejection; C4d_art, C4d staining of arterial endothelium and intima; IEL, internal elastic lamina; MVI, microvascular inflammation; TCMR, T-cell–mediated rejection; TCM-VR, T-cell–mediated vascular rejection; VR, vascular rejection.

Figure 5

VR classified by pathophysiology. VR was etiologically classified into AM-VR, TCM-VR, or mixed VR and evaluated against histologic and clinical markers. (a) Prevalence of total inflammation (Banff ti category) and MVI by etiology. (b) Median fluorescence intensity of the immunodominant DSA was greater in AM-VR and mixed VR compared with TCM-VR. (c) Prevalence of arterial and peritubular capillary C4d against etiologic phenotypes. (d) Incidence of VR by time post-transplant and etiologic case-mix. (e) Acute histologic inflammation scores by etiologic VR diagnosis. (f) Renal function by VR phenotypes. Mean ± SEM. Key: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. AM-VR, antibody-mediated vascular rejection; C4d_art, C4d staining of arterial endothelium and intima; DSA, donor-specific antibody; IEL, internal elastic lamina; MVI, microvascular inflammation; TCM-VR, T-cell–mediated vascular rejection; VR, vascular rejection.