Detection of the Complement Degradation Product C4d in Renal Allografts: Diagnostic and Therapeutic Implications VOLKER NICKELEIT,* MATTHIAS ZEILER,* FRED GUDAT,* GILBERT THIEL, † and M. J. MIHATSCH* *Institute for Pathology and † Division of Nephrology, Kantonsspital, University of Basel, Basel, Switzerland. Abstract. The immunohistochemical detection of the comple- ment degradation product C4d, a component of the classical complement pathway, offers a new and currently poorly de- fined tool in the evaluation of renal allograft biopsies. Our retrospective study aims at determining the diagnostic and clinical significance of C4d accumulation in kidney trans- plants, employing immunofluorescence microscopy. We ana- lyzed 398 diagnostic allograft biopsies (n = 265 patients with 1 to 5 biopsies obtained 7 to 7165 d posttransplantation [tx]) and correlated the detection of C4d with 18 histologic changes, panel-reactive antibody titers, response to treatment, and out- come. One hundred twenty-five native kidney and baseline tx biopsies served as controls. Linear deposition of C4d along peritubular capillaries was only found in a subgroup (30%) of allografts post-tx, mainly during the early time-course (median, 38 d post-tx; range, 7 to 5646 d). There was no significant association with infections. C4d staining could change from negative to positive and vice versa within days to weeks. The accumulation of C4d was most tightly linked to a morphologic subtype of rejection, transplant glomerulitis (P  0.0001). In addition, tubular MHC class II expression was correlated with C4d deposition (P  0.0001). Both features are signs of “acute active rejection.” In comparison with C4d-negative controls, 43% of C4d-positive patients showed increased (10%) panel- reactive antibody titers (versus 19% in the negative group; P = 0.001). C4d positivity was frequently associated with higher serum creatinine levels at time of biopsy (compared with C4d-negative group; P  0.01). More C4d-positive patients were treated with polyclonal antithymocyte globulins (ATG) or monoclonal anti-CD 3 antibodies (OKT3) (P  0.0001). Out- come did not significantly differ between C4d-positive and C4d-negative groups. In conclusion, the detection of C4d iden- tifies a humoral alloresponse in a subgroup of kidney trans- plants, which is often associated with signs of cellular rejec- tion, i.e. tx glomerulitis. Allograft dysfunction in C4d-positive rejection episodes is often more pronounced. We provide first evidence that C4d-positive rejection might benefit from inten- sive therapy, potentially preventing the previously reported high graft failure rate. In addition, we show that a subgroup of C4d-positive cases may not require any immediate therapeutic intervention. The presence of C4d is clinically relevant and should be reported in the histologic diagnosis. The criterion for establishing a diagnosis of acute renal allo- graft rejection is the histologic evaluation of a graft biopsy (1). Acute rejection is morphologically characterized by the pres- ence of mononuclear cells in the interstitial compartment, tubulitis, transplant endarteritis, or glomerulitis. The cellular inflammatory component is the most striking feature; there- fore, acute rejection is sometimes also referred to as acute cellular rejection (2). However, a humoral response to various donor antigens is serologically well documented, the clinical significance of which is only incompletely understood (3,4). The lack of detectable immunoglobulins or activated comple- ment factors in renal allograft biopsies during acute cellular rejection episodes might in part be explained by their rapid shedding from endothelial cell surfaces. Consequently, in the histologic evaluation of kidney biopsies, humoral components during acute rejection episodes are most likely underestimated (5,6). A new perspective opened up when Feucht et al. (3,7–10) pioneered work on C4d, a complement split product that can be detected in renal allografts. C4d is a degradation product of the complement factor C4, which is typically activated during the classical complement cascade. The classical path- way of complement activation is characteristically initiated by conformational changes in Ig molecules after binding to specific antigens (11,12). After cleavage of most C4 do- mains during activation and breakdown, only the alpha 2 domain (representing the split product C4d) remains as the most long-lived portion of the C4 molecule near the site of C4 activation. The alpha 2 domain covalently binds to endothelial surfaces and basement membranes via a proteo- lytically exposed thioester group, making it a stable mole- cule (in contrast to other cleaved domains) (3). The alpha 2 domain can easily be detected by immunohistochemistry (7–9). Thus, the accumulation of C4d can be regarded as a footprint of a humoral response (3,6,13). Its detection offers Dr. Volker Nickeleit’s current affiliation: Department of Pathology, Nephro- pathology Laboratory, University of North Carolina, Chapel Hill, North Carolina. Received August 8. 2001. Accepted October 9, 2001. Correspondence to Dr. M. J. Mihatsch, Instite for Pathology, Schoenbein- strasse 40, CH-4003 Basel, Switzerland. Phone: +41-61-265-2872; Fax +41- 61-265-3194; E-mail: mjmihatsch@uhbs.ch 1046-6673/1301-0242 Journal of the American Society of Nephrology Copyright © 2001 by the American Society of Nephrology J Am Soc Nephrol 13: 242–251, 2002