C4d is a degradation fragment of the complement protein C4b, generated when the classical complement pathway is activated. In ABO-compatible kidney transplantation, peritubular capillary C4d deposition follows the binding of anti-HLA donor-specific antibodies and is recognized as a histological hallmark of antibody-mediated injury and poor prognosis. In ABO-incompatible grafts, however, the picture is strikingly different. C4d appears in the peritubular capillaries of more than half of recipients, yet most of these grafts function well for years. This paradox has been attributed to endothelial accommodation, a state in which membrane-bound complement regulatory proteins such as CD46, CD55, and CD59 restrain complement-mediated injury on the graft endothelium despite ongoing antibody binding. The clinical significance of C4d deposition in this setting has remained unclear, and most previous studies evaluated it at only a single time point.

The Tokyo team, led by Yu Kijima and colleagues, took a different approach. Because a single determination may not reflect a sustained process, they defined C4d positivity by its persistence across two sequential post-transplant protocol biopsies, obtained at 3 months and 1 year after surgery. Between 2015 and 2022, their center performed 254 ABO-incompatible kidney transplantations, and 69 recipients with paired protocol biopsies formed the study population. Fifty-four recipients, or 78.3 percent, were classified as persistently C4d-positive, meaning C4d deposition was detectable in both biopsies, while 15 recipients were classified as C4d-negative or nonpersistent. The researchers then compared pre-transplant background characteristics, cellular infiltration, chronic Banff lesions according to the Banff 2019 classification, graft function, and graft survival between the two groups.

The immunosuppressive regimen itself is worth noting for its technical sophistication. Patients received a triple-drug protocol consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone, starting seven days before transplantation, with tacrolimus doses adjusted to trough levels of 8 to 12 nanograms per milliliter during the first month. Induction with the anti-IL-2 receptor antibody basiliximab was performed in all recipients, and all patients received low-dose rituximab for B-cell depletion, which has replaced splenectomy at the center since 2004. Before surgery, recipients underwent three or four sessions of double-filtration plasmapheresis to remove anti-A and anti-B antibodies until titers fell to 1:32 or below. Recipients who received intravenous immunoglobulin at induction were excluded from the analysis because IVIG may inactivate activated complement and thereby influence C4d deposition.

The biopsy protocol was equally rigorous. Renal allograft biopsies were obtained at time zero as a back-table biopsy, at 3 months, and at 1 year, with an adequate specimen defined as one containing seven or more glomeruli and at least one arterial cross-section. C4d staining was performed by indirect immunofluorescence on cryostat sections using a mouse monoclonal anti-human C4d antibody, scored from 0 to 3 according to Banff criteria ranging from no staining to diffuse staining of more than half of the peritubular capillaries. The persistent C4d-positive group included recipients with deposition at both time points, even when the staining score was a reproducible low-grade 1 at both visits. As a sensitivity analysis, the team also repeated all comparisons using a stricter, Banff-concordant definition requiring a C4d score of at least 2 at both time points, which yielded 47 positive and 22 negative recipients and did not change the overall pattern of findings.

The results were largely reassuring. Baseline anti-A and anti-B antibody titers, ABO donor-recipient combinations, donor age, and donor relationship did not differ between the groups, although donor sex did, with a higher proportion of male donors in the C4d-positive group, a finding the authors attribute cautiously to chance given the number of comparisons performed. Microvascular inflammation, defined as a combined glomerulitis plus peritubular capillaritis score of 2 or more, was observed in 6 of 54 persistently C4d-positive recipients, or 11.1 percent, versus 1 of 15, or 6.7 percent, in the negative group. Chronic changes, defined as an interstitial fibrosis score of at least 1, appeared in 27 of 54, or 50.0 percent, versus 6 of 15, or 40.0 percent. None of these differences reached statistical significance, and cellular infiltration scores for interstitial inflammation, tubulitis, glomerulitis, and peritubular capillaritis were similarly comparable at both time points.

Graft function and survival told the same story at the group level. Serum creatinine at 1 year did not differ significantly between the groups, nor did estimated glomerular filtration rate, which measured a median of 45.5 versus 43.6 milliliters per minute per 1.73 square meters. Kaplan-Meier analysis showed no significant difference in graft survival, with a log-rank P value of 0.191, and this conclusion held in a landmark analysis with the time origin at the 1-year biopsy, designed to address the guarantee-time bias inherent in requiring a 1-year biopsy for group assignment. Biopsy-proven rejection was infrequent in both groups: nine episodes were identified among the 69 recipients, seven in the C4d-positive group and two in the negative group, including cases of borderline change, active and chronic active antibody-mediated rejection, and chronic active T-cell-mediated rejection.

Yet one finding refuses to be dismissed. All four graft losses in the analyzed cohort occurred in the persistent C4d-positive group, occurring at 1,849, 1,926, 2,231, and 3,208 days after transplantation. Three of the four were ultimately attributable to chronic active antibody-mediated rejection, and the fourth to BK virus nephropathy. Critically, these cases were not homogeneous. One recipient had biopsy-confirmed chronic active antibody-mediated rejection with a microvascular inflammation score of 3 at the very 1-year protocol biopsy used to define C4d persistence, meaning early persistent C4d was accompanied by concurrent histological injury. In the remaining two rejection cases and the BK virus case, no microvascular inflammation or overt rejection was present on either the 3-month or 1-year biopsies, and the chronic active antibody-mediated rejection was diagnosed later, at 1.9 years post-transplant in one case and at an undetermined later time in the other. This heterogeneity suggests that persistent C4d at 3 months and 1 year does not reliably distinguish recipients who will remain stable from those who will later develop overt humoral injury.

A parallel signal emerged in the antibody data, though with important caveats. Post-transplant donor-specific antibody testing was available for 47 of the 69 recipients, and de novo donor-specific antibodies were detected in 11 of 38 evaluable recipients, or 28.9 percent, in the C4d-positive group, compared with 1 of 9, or 11.1 percent, in the negative group. This difference did not reach statistical significance, and the authors emphasize that it is exploratory at best: testing was not performed at uniform time points, measurements spanned roughly 4 months to 3 years after transplantation, and the 22 recipients without available data had significantly shorter follow-up. Whether persistent C4d deposition is unrelated to subsequent HLA sensitization or reflects low-grade humoral activity that could contribute to later sensitization simply cannot be determined from this retrospective cohort, and the absence of molecular transcript profiling further limited the ability to detect subclinical humoral activity.

The authors are admirably candid about the limits of their work. The study was retrospective and single-center, and the comparator group of only 15 patients left it substantially underpowered for low-frequency endpoints such as rejection and graft loss, meaning the null results represent a failure to detect an association rather than evidence of equivalence. The cohort was also highly selected: only 69 of 254 recipients qualified, with exclusions for insufficient pathological data, prior treated rejection, thrombotic microangiopathy, IVIG induction, retransplantation, and recurrent glomerular disease effectively removing the higher-risk population. Anti-A/B titers were available only before transplantation, so the researchers could not determine whether persistent C4d reflected ongoing antibody exposure with successful accommodation or merely residual complement footprints. Taken together, the findings are consistent with accommodation but hypothesis-generating rather than definitive, and the authors call for confirmation in larger, prospective cohorts with systematic de novo donor-specific antibody surveillance and molecular diagnostics. For now, the message for clinicians is nuanced: persistent C4d in an ABO-incompatible graft is not by itself a cause for alarm, but the small cluster of late losses in the positive group suggests that long-term immunological surveillance, rather than either reassurance or immediate treatment escalation based on early biopsies alone, may be the wisest course.

Subject of Research: Persistent C4d deposition on sequential protocol biopsies in ABO-incompatible kidney transplantation

Article Title: Clinical significance of persistent C4d deposition on sequential protocol biopsies in ABO-incompatible kidney transplantation

Article References: Kijima, Y., Ishida, H., Yagisawa, T., Hirai, T., Unagami, K., Shimizu, T., & Takagi, T. (2026). Clinical significance of persistent C4d deposition on sequential protocol biopsies in ABO-incompatible kidney transplantation. Frontiers in Nephrology, 6, Article 1940137. https://doi.org/10.3389/fneph.2026.1940137

Image Credits: AI Generated

DOI: 10.3389/fneph.2026.1940137

Keywords: ABO-incompatible kidney transplantation, C4d, accommodation, antibody-mediated rejection, protocol biopsy, microvascular inflammation, Banff classification, donor-specific antibodies, complement, graft survival, kidney allograft, transplant immunology