The development of de novo donor-specific human leukocyte antigen (HLA) antibodies (dnDSA) is a major risk factor for acute and chronic antibody-mediated rejection (AMR) and graft loss after kidney transplantation (KTx) (1). Single center studies report that acute AMR was more common in KTx recipients with dnDSA vs. without dnDSA (18.8% vs. 0%, respectively; P < 0.001) (2) and probability of graft survival was lower (79% vs. 94%, respectively; P = 0.05) (3). In a case-control study, dnDSA were more common in patients with graft failure vs. controls without graft failure (54% vs. 16%, respectively; P < 0.001), and chronic active AMR was significantly more common in patients with dnDSA compared to those without dnDSA (61% vs. 12%, P < 0.001) (4). The prevalence of dnDSAs is generally between 5% and 10% at 1-year post KTx and slowly increases thereafter to 20% at 5 years (5). Risk factors for dnDSA development include a high number of HLA mismatches (especially DQ mismatches), and inadequate immunosuppression and non-adherence (1). Since dnDSA development has been associated with worse outcomes, it is important to avoid this undesirable alloimmune response and understand the effects of different immunosuppressive agent combinations on dnDSA formation. Although B cells and plasma cells produce antibodies, T cells also play role in the development of dnDSA, and effective T-cell suppression is required to prevent dnDSA formation (6).

Everolimus (EVR), an inhibitor of mammalian target of rapamycin (mTORi), permits reduced calcineurin inhibitor (CNI) exposure after KTx (7). CNIs affect humoral immune response by acting on T cells, whereas mTORis, such as EVR, affect both T cells and B cells (6). Previous studies with CNI-free EVR-based regimens in KTx patients demonstrated increased formation of dnDSA and transplant rejection compared with CNI (8–10). Also, Liefeldt and colleagues reported an increased risk for dnDSA after conversion to CNI-free therapy with EVR, though another reason could be that a high percentage of patients were also steroid-free, and CNI- plus steroid-free regimen may have led to rejection (11). These CNI elimination immunosuppression regimens may result in inadequate immunosuppression, possibly contributing to DSA development and subsequent graft failure.

An alternative strategy using EVR with reduced CNI exposure was investigated in the ATHENA study, a large, randomized trial involving 612 de novo kidney transplant recipients (ClinicalTrials.gov identifier: NCT01843348; EudraCT number: 2011–005238–21) (7). ATHENA compared EVR + tacrolimus (TAC) or EVR + cyclosporine A (CsA) vs. a standard-of-care regimen of mycophenolic acid (MPA) + TAC; all patients received steroids. The 12-month results of the ATHENA study revealed a comparable efficacy of EVR + TAC or EVR + CsA to MPA + TAC and though non-inferiority of renal function with EVR + TAC/CsA was not achieved, an increase in renal function from Month 1 to 12 was comparable in both EVR and MPA groups (7). Given the paucity of data with regards to the development of dnDSA with EVR + reduced CNI-therapy in de novo kidney transplant recipients, this substudy of ATHENA was initiated to evaluate the effect of EVR or MPA in combination with reduced CNI on the formation of HLA antibodies, including dnDSA, and the impact on clinical outcomes over 1 year.

Primary results of the ATHENA study showed that EVR + reduced CNI was efficient and safe in de novo patients (7). The study showed that renal function was comparable between the EVR arms and standard-of-care regimen (MPA + TAC), and that immunosuppressive efficacy was similar in the EVR + TAC and MPA + TAC groups. In addition, both EVR-based arms had significantly fewer cytomegalovirus infections compared with the MPA + TAC regimen. This prospective substudy of ATHENA showed that EVR + reduced CNI was also very effective against formation of dnDSA. More importantly, after 12 months there was no substantial difference between the 3 immunosuppression regimens on the development of dnDSA, suggesting EVR does not increase the risk for dnDSA development.

Overall, the incidence of dnDSA was extremely low under conditions of a controlled clinical trial and dnDSA, at least in this setting, did not appear to adversely affect clinical outcomes. Our findings confirmed that AMR is not necessarily related to the development of dnDSA per se. Although the substudy was designed to investigate the impact of immunosuppressive regimens on the development of dnDSA, it was interesting to observe that the presence of preformed HLA antibodies (MFI ≥ 500 at baseline, independent of mismatch) and critical HLA antibodies (MFI ≥ 500 at baseline, although being a mismatch) had no influence on clinical outcome, irrespective of EVR or MPA exposure.

Previous preclinical studies involving kidney and heart allotransplantation models have demonstrated an inferior immunosuppressive ability with EVR vs. CNI exposure, indicating that CNI-free regimens present an immunological risk (16, 17). Also, clinical studies have suggested that CNI-free and CNI-sparing regimens may be associated with an increased risk of acute rejection (18, 19). Plus, in addition to acute rejection as an indicator of under-immunosuppression, the CNI-sparing regimen with EVR vs. MPA has been said to lead to the development of DSAs and AMR. Yet, on the contrary, other studies investigating CNI-minimizing regimens have not been associated with an increased incidence of acute rejection/immunological risk in de novo KTx patients (20–23), which goes well with the findings of this substudy. Here, we have presented an ideal platform to prospectively analyze the formation of DSAs in the ATHENA study and to determine if immunosuppression is adequate in controlling the emergence of DSAs and AMR.

In line with our findings, results from several randomized, controlled trials in de novo KTx patients have shown that EVR with reduced-exposure CNI is not associated with a higher incidence of dnDSA compared to standard-exposure CNI (24–26). Moreover, no difference in the incidence of acute rejection between groups was reported. In addition, our findings were comparable with another observational study in patients treated with MPA plus TAC, either once or twice daily over 2 years (incidence of DSA: 3.6% and 1.2% and AMR: 4.8% and 2.7%, respectively, with twice- and once-daily TAC) (27). Hence, these findings, in combination with our results, which both exhibited no increased risk for dnDSAs in patients receiving EVR with reduced CNI, strongly oppose results of other studies that suggest dnDSA may be more frequent in patients given EVR in a CNI-free regimen (8–10), likely reflecting inadequate immunosuppression with CNI-free regimens.

The role of induction therapy in preventing de novo DSA is also worth discussion. Studies have reported that induction with basiliximab is associated with lower incidence of de novo DSA in kidney transplant recipients compared with anti-depleting agent, alemtuzumab or rabbit antithymocyte globulin (rATG) (28). Bath NM et al. (2020) also reported that patients who received induction with basiliximab had lower incidence of de novo DSA than alemtuzumab, which could be because of elevated B-cell activating factor levels in patients treated with alemtuzumab (29). In our study, basiliximab was given as induction therapy in all treatment arms, all of which showed an extremely low incidence of DSA over 1 year treatment. Results from the TRANSFORM study in which majority of patients received induction with basiliximab (83%) showed comparable low incidence of de novo DSA on treatment with EVR + reduced CNI vs. MPA + standard CNI over 12 months (10.2% vs. 13.6%, P = 0.508) and 24 months (22.4% vs. 17.7%, P = 0.508) (24, 26). A retrospective analysis of a randomized trial reported comparable incidence of de novo DSA over 12 months in patients who received r-ATG + EVR + TAC [5/78 (6.4%)] vs. basiliximab + EVR + TAC [3/87 (3.4%)] vs. basiliximab + MPS + TAC [5/90 (5.5%)] (25). A multicenter analysis of 24 patients who were enrolled to 2-year, randomized phase-3 study (RAD001A1202 study) and treated with basiliximab + EVR + reduced CsA vs. basiliximab + MMF + standard CsA showed comparable rates of de novo DSA (15.4% vs. 18.3%) over 10 years (30).

The favorable effects observed in this study with the mTORi, EVR-based regimen, can be explained. The mTOR signaling plays an important role in the pathomechanism of HLA antibody-mediated endothelial cell activation and proliferation, which lead to rejection and vasculopathies. The HLA antibodies stimulate the activation of mTOR, as well as the downstream targets extracellular-signal regulated kinase (ERK), S6 kinase (S6K), and S6 ribosomal protein (S6RP) [the latter stimulating intercellular adhesion molecule 1 (ICAM-1) expression and clustering], and favor monocyte adhesion to the endothelium (31). In an in vitro model, mTOR inhibition suppressed Ezrin/Radixin/Moesin (ERM) phosphorylation, ICAM-1 clustering and monocyte adhesion to HLA antibody-activated endothelium (32). Another in vitro study showed that mTOR inhibition prevented endothelial cell proliferation by downregulating interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-β1 (TGF-β1) and von Willebrand factor (vWF) (33). A recently reported cohort study in hypersensitized kidney transplant recipients (calculated panel reactive antibody ≥50%) showed better results in patients treated with EVR vs. MPA, both in combination with CNI and steroids. The risk of BPAR-free survival over 1 year was lower in the EVR group (hazard ratio: 0.32, confidence interval: 0.11–0.90, P = 0.031) with numerically lower incidence of dnDSA (4/33 [12.1%] vs. 6/38 [18.2%], P = 0.408) (34).

The low number of dnDSAs and events at 12 months in the PP population clearly indicate that close follow-up and monitoring of patients following transplantation play a major role in preventing the development of DSAs. In our study, it is possible that we did not observe any difference in dnDSA development between the 3 immunosuppressive regimens because CNIs were included in all study arms. Moreover, non-adherence increases the likelihood of dnDSA production (1). Similar to other controlled studies, the ATHENA trial conditions were likely to support medication adherence, leading to effective immunosuppression. Although there were high rates of study drug discontinuation and withdrawal in the ATHENA study (7), our primary analysis was based on the PP population. Thus, the treatment effect was estimated among adherent patients, which may have led to the observed comparable rate of dnDSA development across the 3 immunosuppressive regimens. However, it should be noted that similarly low rates of dnDSA development were observed across treatment groups in the ITT population, which included both adherent and non-adherent patients.

There are few limitations which are worth consideration. A follow-up to Month 12 after transplantation does not encompass the long-term effects of the treatment regimens on development of DSA or AMR. Further long-term results, ideally 5- or 10-year data, from prospective studies are required to fully understand the risk of these immunosuppressive regimens on dnDSA and AMR. Nevertheless, late graft loss tends to be related to recurrence of previous disease and adherence to the therapy protocol rather than to the initial immunosuppressive protocol (35). Finally, the substudy was not powered to detect statistical significant differences in clinical outcomes. Thus, the lack of differences in dnDSAs between the groups may be due to the small sample size. The strength of the analysis includes the prospective nature of data sampling, and as such, the data are embedded in a broad analysis of primary and secondary endpoints conducted as part of the primary ATHENA study (7). In the ATHENA study, kidneys were allocated within Eurotransplant according to the standard allocation process (36).

The non-HLA antibodies may also play an important role in transplant rejection and patients with both HLA and non-HLA antibodies have been reported to have poor outcomes and lower graft survival (37–39). A separate substudy of ATHENA evaluated the effect of EVR in combination with CNI on the formation of non-HLA antibodies and graft outcome in KTx patients (40). The results showed that EVR + TAC group had a higher incidence of patients negative for angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) antibodies (82.2% and 76.7%, respectively) compared to MPA + TAC group (71.9% and 65.3%, respectively) over Month 12. Similar to findings found for HLA, non-HLA antibodies had no influence on clinical outcomes and no death was reported in patients.

In conclusion, findings from the ATHENA substudy showed that immunosuppression with EVR or MPA in combination with reduced CNI did not increase the risk for dnDSA development. Interestingly, the overall incidence of dnDSA was extremely low and did not directly influence clinical outcome in the first 12 months after KTx. Preformed HLA antibodies (MFI ≥ 500 at baseline, independent of mismatch) and critical HLA antibodies (MFI ≥ 500 at baseline, although being a mismatch) had no influence on clinical outcome, irrespective of EVR or MPA exposure. Clinical outcomes are in line with previous results of ATHENA trial (7).