Pros and cons for C4d as a biomarker

Abstract

The introduction of C4d in daily clinical practice in the late nineties aroused an ever-increasing interest in the role of antibody-mediated mechanisms in allograft rejection. As a marker of classical complement activation, C4d made it possible to visualize the direct link between anti-donor antibodies and tissue injury at sites of antibody binding in a graft. With the expanding use of C4d worldwide several limitations of C4d were identified. For instance, in ABO-incompatible transplantations C4d is present in the majority of grafts but this seems to point at 'graft accommodation' rather than antibody-mediated rejection. C4d is now increasingly recognized as a potential biomarker in other fields where antibodies can cause tissue damage, such as systemic autoimmune diseases and pregnancy. In all these fields, C4d holds promise to detect patients at risk for the consequences of antibody-mediated disease. Moreover, the emergence of new therapeutics that block complement activation makes C4d a marker with potential to identify patients who may possibly benefit from these drugs. This review provides an overview of the past, present, and future perspectives of C4d as a biomarker, focusing on its use in solid organ transplantation and discussing its possible new roles in autoimmunity and pregnancy.

Figure 1. The complement system and C4d

(a) The classical pathway of complement is initiated via binding of its recognition molecule C1q to immune complex deposits, antibody-antigen binding or charged molecules. When C1q becomes activated, it subsequently activates its natural substrate C4. C4d is a split product of C4 activation, without a biological function. Although C4d is mainly interpreted as a trace of classical pathway activation, it must be kept in mind that C4 can also be derived from the lectin pathway. Mannose-binding lectin (MBL) or ficolins binding to carbohydrate ligands on the surface of a wide variety of pathogens results in activation of the lectin pathway and cleavage of C4. Consequently, C4d may be generated without prior antibody binding. Classical complement activation converges with other pathways at the level of C3 and proceeds into the formation of the membrane attack complex on complement-activating surfaces, causing direct tissue injury by perforation of the cell membrane. In addition, potent anaphylatoxins C3a and C5a are being formed in the process, which elicit the recruitment of other inflammatory cells to the site of activation. (b) C4d as a footprint for antibody-mediated tissue injury. C4b, the larger molecule that C4d is derived from, has an internal thioester in the molecule, giving it the ability to form a covalent bond with target cells. When C4d is cleaved from C4b, the covalent bond between C4d and the tissue remains intact. Covalently bound C4d has a higher chance to remain at the site of complement activation than the antibodies themselves, which dissociate over time. C4d is anchored tightly to the tissue and therefore acts as a footprint of antibody-mediated tissue injury.

Figure 2. Diagnosing acute antibody-mediated rejection

This flowchart shows that the diagnosis of acute antibody-mediated rejection requires the presence of histological features, a positive C4d stain, and the presence of donor-specific antibodies. AMR, antibody-mediated rejection; ATN, acute tubular necrosis; HLA, human lymphocyte antigen; tx, treatment.

Figure 3. C4d staining patterns in different clinical settings

(a) Acute AMR of a kidney graft with typical peritubular capillary staining of C4d on paraffin-embedded tissue. (b) Glomerular C4d in a native kidney biopsy of a patient with lupus nephritis and thrombotic microangiopathy. (c) C4d in a pancreas graft with typical staining of C4d in interacinar capillaries, suggestive for AMR. (d) Placental C4d in a placenta from a patient with antiphospholipid syndrome and an intrauterine fetal death in this pregnancy. C4d is positive at the fetal–maternal interface on the maternal side of the syncytiotrophoblast, suggesting severe antibody-mediated injury leading to impaired placental development, impaired nutrient exchange, intrauterine fetal growth restriction, and finally, fetal death. AMR, antibody-mediated rejection.

Figure 4. Analogy between AMR and antibody-mediated pregnancy loss

In this scheme the analogy between antibody-mediated rejection of a transplanted graft and ‘antibody-mediated pregnancy loss’ is schematically shown. HLA, human lymphocyte antigen.