There is strong evidence that anti-HLA antibodies contribute to the development of CR, the leading cause of renal allograft failure.1, 2, 4, 5, 10, 14, 15, 17, 18, 23, 24, 25, 26, 27, 28, 29
While it is hoped that post-transplant alloantibody surveillance might facilitate early diagnosis, there are few guidelines regarding how monitoring should be performed and results interpreted. This report summarizes our experience in attempting to develop a utilitarian and clinically informative monitoring paradigm by correlating clinical outcome with longitudinal changes in FCXM and solid-phase assay reactivity occurring during the first post-transplant year. We intentionally excluded patients with preoperatively negative FCXM who typically experience excellent graft survival and minimal CR.6, 7, 12, 16
We also excluded nine patients with preoperatively positive FCXM because of non-HLA antibodies and a small subgroup of group I (n
=3) with slower antibody elimination. Instead, we focused our attention upon patients with preoperatively positive FCXM because of preformed anti-HLA antibody who, at this center, have poorer graft survival.6, 27
Changes in specificity techniques throughout the years made original specificity data incomparable. To standardize the data, we reassayed patient serum for anti-HLA specificities using one lot of single antigen beads on a Luminex platform and also retested donor HLA for minor histocompatibility antigens not determined originally. Consistent with a recent report, the enhanced sensitivity of single antigen bead testing allowed identification of DSA and non-DSA in 100% of study patients.30
Temporal changes in alloantibody levels were consistent whether measured using FCXM or solid-phase assay. Two post-transplant profiles emerged and showed different clinical outcomes. Patients showing complete elimination of alloantibody enjoyed excellent graft survival and few complications. In contrast, patients failing to eliminate alloantibody had poorer outcomes and higher incidence of CR. Following delineation of specific groups, pretransplant antibody characteristics were compared with the hope that some distinguishing parameters would predict post-transplant outcome. However, similar to previous reports, pretransplant characteristics proved inadequate indicators of post-transplant developments.8, 10, 13
Before transplant, groups were equivalent in terms of demographics, donor characteristics, alloantibody levels, and specificities. Alloantibodies against class I predominated in all groups. However, group II did show a greater total amount of non-DSA against class II than group I.
It has always been puzzling why only a fraction of presensitized patients actually develop CR.6, 7, 8, 9, 10, 16, 19, 20
Our surveillance study provides a partial explanation. The majority of patients (65%) showed rapid reduction of both T- and B-FCXM levels within 6 months of transplant and complete elimination within 12 months. These results were mimicked using solid-phase testing that showed that DSAs and non-DSAs against class I and II were simultaneously eliminated. Elimination of circulating alloantibody transformed this group into a FCXM-negative population that typically has excellent long-term graft survival and minimal CR. The cause of antibody elimination is unknown. However, one possible mechanism could be downregulation of antibody synthesis after transplantation. Considering the short half-life of immunoglobulin G, complete depletion of circulating alloantibody within 6–12 months is entirely possible if synthesis stopped soon after transplantation. Simultaneous depletion of DSA and non-DSA suggests that the effect is global and not specific to DSAs. Alternatively, alloantibody binding to the graft could artificially lower circulating levels below detection thresholds of assays. However, as this would not explain the simultaneous elimination of non-DSAs unless they also bound to the graft, we believe the first possibility is the more likely mechanism.
The second post-transplant profile identified a group that failed to eliminate FCXM, DSA, or non-DSA reactivity against class I and II within the first postoperative year. Persistence of circulating alloantibody predisposed this group for a higher risk of AMR and CR and ultimately poorer graft survival. The critical question is why is the alloantibody not eliminated among group II as occurred among group I? Unfortunately, we do not have an answer. Group II did not demonstrate any unique antibody characteristics before transplant. However, group II demonstrated several unique post-transplant differences that either individually or collectively may predispose this group for CR. First, only group II failed to eliminate alloantibody, which suggests that activation of downregulatory pathways are inhibited in this group. Second, only group II was unable to eliminate DSAs directed against class I and II. Numerous studies show that circulating DSA against either class I or II are deleterious to graft survival. Some studies have suggested that anti-class II DSAs may more aggressively promote CR than anti-class I.10, 15
Thus, the perseverance of anti-class II among group II may enhance the chances of developing CR. Third, only group II was unable to eliminate non-DSA directed against class I and II and maintained significantly high levels of non-DSA against class I and II. The precise role of non-DSAs in allograft survival is unclear but there is growing evidence that they may contribute to allograft destruction by crossreactive recognition of shared epitopes on the allograft. If this occurred among group II, then the high cumulative burden of non-DSA levels combined with sustained DSA levels may escalate the probability of CR.
Interestingly, despite alloantibody persistence, nearly half of group II remained AMR and CR free and had excellent graft outcome. This is consistent with the historic inability to show 100% causal relationship between detection of circulating alloantibody and active disease. Because pretransplant alloantibody characteristics were equivalent between these subgroups (as well as group I), they were useless in forecasting post-transplant antibody profiles or clinical outcomes. Our data suggest that (1) alloantibody persistence is not an automatic trigger for AMR and (2) AMR without antibody persistence is not an automatic trigger for CR (group I patients with AMR have not yet demonstrated CR). Our interpretation is that alloantibody persistence during the first year heightens the risk of AMR. However, alloantibody alone is an insufficient trigger for AMR. The extended period of alloantibody persistence before AMR detection is similar to the lag reported in other studies between de novo alloantibody appearance and rejection. We suggest that perhaps certain minimum conditions must be met in order to initiate alloantibody-mediated tissue damage. Perhaps alloantibody must be composed of complement fixing isotypes at some minimal level in order to show lytic activity. Similarly, perhaps donor HLA expression must exist at some minimal density in order to be an adequate target. Confounding variables such as ongoing immune activating events (like cellular rejection or infection) or nonimmune-mediated allograft damage may boost inadequate alloantibody/donor antigen presentation and trigger rejection. We suggest that if certain minimal conditions are not met, then AMR does not occur, and maintaining high levels of DSA and non-DSA appears clinically irrelevant at least for the short term. On the other hand, AMR with persistent alloantibody must create a smoldering rejection that is difficult to completely eradicate and upon rebound leads to chronic disease. It is hoped that early detection would facilitate AMR reversal. As this study was a blinded protocol in which alloantibody tests were not reported, medical intervention was based upon clinical dysfunction and thus the earliest intervention was not possible.
The heightened sensitivity of the FCXM relative to cytotoxic crossmatching has always generated controversy concerning its relevance and concerns that some reactions are falsely positive. In this study, false-positive FCXMs are unlikely as we only included patients for analysis when the preoperatively positive FCXM reactions were validated by identifiable DSA. Similar to a recent report, we found that use of single antigen bead testing by luminometry detected DSA in virtually all patients with positive FCXM.30
Our goal was to develop a post-transplant monitoring protocol that was clinically informative and manageable from a laboratory perspective. We focused on the patient group most problematic to this center; the presensitized patients with preoperatively positive FCXM. We identified two distinct post-transplant alloantibody profiles that exhibited different clinical outcomes. Identification of patients at greater risk of CR should facilitate earlier diagnosis.
The study results allow us to consider strategies to alter the post-transplant course of patients with persistently positive FCXM and DSA. One strategy would be to avoid transplanting patients with positive FCXM and high levels of DSA. However, complete avoidance would deny transplants to the majority of patients who will develop a group I profile that unfortunately cannot be predicted pretransplant. If transplantation proceeds, preemptive immunomodulation (intravenous immunoglobulin, plasmapharesis, rituximab, Velcade) at the time of transplant might promote antibody depletion in patients who would develop a group II pattern. Perhaps drug tapering protocols should be reconsidered in patients with group II profile. We feel longitudinal monitoring is more effective than testing only when there is evidence of graft dysfunction. Implementing protocol biopsies with C4d staining would be informative. Antibody persistence or elevation coupled with rising serum creatinine or histological changes in the graft should be treated aggressively. Patients with persistent alloantibody who experience AMR would likely benefit from antibody-depleting therapies (perhaps Velcade and rituximab) to reduce or eliminate alloantibody burden that might minimize AMR recurrence. Last, alloantibody monitoring should continue beyond 1 year for patients with persistent antibody.