High levels of preformed anti-HLA antibodies are the major cause of antibody-mediated rejection (AMR) and are associated with poor graft survival.1 Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are the primary mechanisms responsible for AMR.2 Current desensitization protocols consist primarily of intravenous immune globulin (IVIG), rituximab, and/or plasmapheresis, and the efficacy of these has been reported.3,4 However, incomplete removal of donor-specific antibodies (DSA) or a possible rebound of DSA posttransplant may increase risk for AMR.5-7 Thus, a new or noble agent or therapy is required to further improve desensitization protocols for successful transplantation.
Imlifidase (IdeS) is an immunoglobulin (IgG)-degrading enzyme of Streptococcus pyogenes that cleaves all 4 human subclasses of IgG with a unique degree of specificity on positions 236 and 237 of the lower hinge region of IgG heavy chains8,9 that is critical for interaction with Fcγ receptors or complements, required for ADCC or CDC, respectively. In a phase I clinical trial, Winstedt et al10 showed that a single ascending dose of IdeS rapidly and efficiently cleaved IgG to F(ab’)2 and Fc fragments in healthy humans with the maximum effect at 2–6 hours after 0.12 or 0.24 mg/kg body weight dosing. Intact IgG began to return at 1 week to 2 months postimlifidase, depending on the dose used. They also showed consistent results using a phagocytic activity assay; that is, serum IgG bound fluorescence beads inside phagocytic cells became background at 24 hours in IdeS-treated subjects and the reduced levels remained for at least 7 days. Tradtrantip et al11 have also shown the inhibitory effect of IdeS on ADCC and CDC in in vitro and animal studies.
We have recently reported on the results of 2 independently conducted open-label and phase 1–2 trials assessing the safety and efficacy of IdeS as a desensitization agent in 25 HLA-sensitized (HS) kidney transplant patients.12 In this study, we showed that the concentration of serum IgG including DSA became nearly 0 at 6–24 hours after IdeS treatment, which permitted HLA-incompatible transplantation in 24 of 25 patients. More recently, successful use of IdeS to HS kidney transplant patients have also been reported by others.13,14 Lorant et al13 showed that positive panel reactive antibody became negative and C1q binding to anti-HLA was abolished after IdeS therapy in 8 patients. Lonze et al14 also reported that positive flow crossmatches became negative in 7 HS patients and all patients received successful kidney transplant with functioning renal allografts at a median follow-up of 235 days. These results indicate that IdeS efficiently cleaves IgG including DSA in HS patients, suggesting a possible utility of IdeS as a desensitization agent.
The Allo-CFC test was established in our laboratory to measure anti-HLA antibody-mediated NK cell activation using intracellular cytokine flow cytometry (CFC), and we showed that this NK cell activation as assessed by intracellular IFNγ production was IgG-FcγRIII interaction, similar to ADCC.15,16 One of several modified Allo-CFC assays developed uses normal individual blood as responder NK cells and anti-HLA antibody-coated third-party peripheral blood mononuclear cells (PBMCs) as stimulater cells.17 An in vitro ADCC assay established in our laboratory uses normal individual PBMC as responder NK cells and lymphoma Farage B (FB) cells as target cells incubated with anti-HLA antibody or control sera. The cytotoxicity is measured by flow cytometry.18 Using these 2 in vitro assays, we investigated the effect of IdeS on anti-HLA antibody-mediated NK cell activation and ADCC in vitro, and tested the capacity of anti-HLA antibody-mediated NK cell activation in sera obtained pretreatment and posttreatment from IdeS-treated HS patients, to assess utility of IdeS as a potential treatment for ADCC-mediated AMR.
MATERIALS AND METHODS
Patients, Blood, PBMC, and Sera
This study was approved by the institutional review board at Cedars-Sinai Medical Center (Protocol numbers: 12562, 42267, and 37068). The study was conducted in accordance with the ethical guideline based on federal regulations and the common rule. In addition, Cedars-Sinai Medical Center has a Federalwide Assurance.
Sodium-heparinized peripheral blood samples were collected from non-HS normal individuals and used as responder cells in the Allo-CFC assays. PBMCs used as stimulator cells in the Allo-CFC assays were isolated from 5 healthy adult volunteers and kept in liquid nitrogen tank until the assays were performed as previously published.16 In the ADCC assay, fresh PBMCs were isolated from normal individuals by density-gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) using SepMates-50 (STEMCELL Technologies, Vancouver, BC, Canada) as previously published,19 and used as effector cells. HS and NC sera used in the Allo-CFC and ADCC assays were pooled sera randomly selected from >20 HS patients with panel reactive antibody >50%, and serum obtained from a non-HS normal individual, respectively. Archived serum samples collected preimlifidase and postimlifidase (1 day [D], 1, 3, and 6 months [M] postimlifidase) from 5 IdeS-treated patients were used as HS sera in Allo-CFC-3. IdeS was administered intravenously on the transplant date, usually 4 to 6 hours before transplantation as previously published.12
A modified Allo-CFC was used as previously described with minor modification.17 Briefly, non-HS normal individual whole blood (responder) mixed with anti-CD28/49d and Brefeldin A was incubated with irradiated third-party allo-PBMCs (stimulator) pretreated with anti-HLA antibody negative (NC) or positive (HS) sera overnight. On the following day, cells were stained with fluorochrome-conjugated mouse monoclonal antibodies to CD3 (FITC; Thermo Fisher Scientific, Rockford, IL), CD8 (V450; BD Biosciences, San Jose, CA), CD16 (PerCP-Cy5.5, BD Biosciences), and CD56 (APC; BioLegend, San Diego, CA) followed by lysing red blood cells. After permeabilization, intracellular IFNγ (PE; BD Biosciences) was stained and then cells were fixed with 1% paraformaldehyde followed by flow cytometry analysis. After cell acquisition, lymphocytes first gated by forward/side scatter were further plotted against CD3 and CD8, then CD56+ NK cells were gated from CD3− cells, and IFNγ+ cell% in NK cells was calculated. In the Allo-CFC-3 written below, CD8+ and CD3+/CD8− T cells were gated from CD3+ cells, and then IFNγ+ cell% in each T cell subset was calculated. CD3+/CD8− was considered as CD4+ T cells in this experiment.
To examine the effect of IdeS on the Allo-CFC, 3 experiments were performed (Figure S1, SDC, http://links.lww.com/TP/B833); in the Allo-CFC-1, HS sera-coated PBMCs were incubated with various concentration of IdeS (0, 0.01, 0.1, 1, 10, and 100 μg/mL) at 37°C for 1 hour. The PBMCs were then washed with RPMI 1640 media (1% HEPES Buffer, 1 × Penicillin/Streptomycin/Glutamine, 10% Human serum) followed by centrifugation at 350g 10 minutes, twice, and then used as stimulator cells. In the Allo-CFC-2, HS sera were incubated with IdeS (0, 0.1, 1, 10, and 100 μg/mL) at 37°C for 1 hour and then used to coat PBMCs. In the Allo-CFC-3, archived sera collected preimlifidase, and 1D, 1M, 3M, and 6M postimlifidase from 5 IdeS-treated patients were used for pretreatment of PBMCs, and then the sera-coated PBMCs were used for the assay.
Antibody-Dependent Cell-Mediated Cytotoxicity
The ADCC assay developed in our laboratory as previously described18,20 was used to examine the effect of IdeS on ADCC (Figure S1, SDC, http://links.lww.com/TP/B833). Briefly, the ADCC assay uses freshly isolated normal individual PBMC as effector (E) and anti-HLA antibody-coated lymphoma FB cells (ATCC CRL-2630, Manassas, VA) as target cells (T). In the ADCC assay, FB cells were first incubated with FITC-anti-human CD19 antibody (Thermo Fisher Scientific, Waltham, MA) at 4°C for 20 minutes followed by washing twice with RPMI-1640-based medium at 125 g for 10 minutes, and then the concentration of prelabeled FB cells was readjusted. Normal individual PBMC was mixed with prelabeled FB cells at the E:T ratio of 5:1 (0.1 × 106 PBMC + 0.02 × 106 FB in total volume of 120 µL) together with HS or NC sera, and incubated at 37°C for 1 hour in the 5% CO2 incubator. To examine the effect of IdeS on ADCC, HS sera pretreated with IdeS at various concentration (0, 0.01, 0.1, 1, 10, and 100 µg/mL) was used for the assay.
After washing, cells were resuspended in 100 µL FACS buffer and then 20 µL of 7-AAD (BD Bioscience) was added followed by 10 minutes incubation on ice. After addition of 250 µL of FACS buffer, cells were submitted for flow cytometry analysis. After cell acquision, CD19+ FB first separated from nonstained PBMC were plotted against 7-AAD, and then 7-AAD+ FB cell% in total CD19+ FB cells was enumerated.
Preimlifidase and postimlifidase anti-HLA antibody levels in 5 patients were measured by Luminex at the HLA laboratory of Cedars-Sinai Medical Center. The total anti-HLA antibody levels (class I and class II) were expressed using a relative intensity scale. Score 10, 5, 2, and 0 for MFIs >10 000, 5000–10 000, <5000, and 0, respectively, are given to each detected antibody, and the sum of these are the final score for serum with multiple HLA class I or class II antibodies.
Paired T-test was used to compare the levels against no IdeS condition in Figures 1–3, and against preimlifidase in Figure 4. P < 0.05 were considered significant.
The Effect of Imlifidase on Anti-HLA Antibody-Mediated NK Cell Activation in Allo-CFC Assays
NK cell activation in response to anti-HLA antibody-coated PBMCs was consistently observed in the Allo-CFC assays (Figures 1A and 2A). IFNγ+ cell% in CD56+ NK cells was significantly elevated when blood was incubated with HS compared to NC sera-coated third-party PBMCs in Allo-CFC-1 (10.0 ± 2.8% versus 2.0 ± 0.9%, P = 0.001; Figure 1A) and Allo-CFC-2 (20.5 ± 9.8% versus 3.9 ± 1.7%, P = 0.01; Figure 2A).
When HS sera-coated PBMCs in the Allo-CFC-1 or HS sera used for coating PBMCs in the Allo-CFC-2 were treated with IdeS, elevated IFNγ+ NK cell% significantly decreased in a dose dependent manner (Figures 1B and 2B); In the Allo-CFC-1, the IFNγ+ NK cell% significantly decreased from 10.0 ± 2.8% at 0 μg/mL to 5.3 ± 3.2% (P = 0.003), 4.4 ± 1.8% (P = 0.001), 3.5 ± 1.5% (P = 0.001), and 2.3 ± 1.8% (P = 0.001) at 0.1, 1.0, 10, and 100 μg/mL, respectively (Figure 1B). Similar results were seen in the Allo-CFC-2; 20.5 ± 9.8% at IdeS 0 μg/mL to 6.8 ± 3.6% (P = 0.02), 5.6 ± 3.1% (P = 0.02) at 10 and 100 μg/mL, respectively (Figure 2B). It should be noted that IdeS showed significant reduction of IFNγ+ NK cell% in the Allo-CFC-1 even at 0.1 and 1 μg/mL, 1/100 and 1/10 of the therapeutic concentration, respectively, and the IFNγ+ NK cell% were close to those observed in the control (NC) group (background, dotted line in Figures 1B and 2B) at the maximum therapeutic level, 10 μg/mL, in the Allo-CFC-1 and 2.
The Effect of Imlifidase on ADCC
The percentage of 7-AAD+ FB cells (ADCC) was significantly elevated when HS sera was added to the assay compared to NC serum (10.9 ± 2.8% versus 4.1 ± 1.9%, P = 0.02, Figure 3A). Here, treatment of HS sera with IdeS reduced ADCC in a dose dependent manner (Figure 3B); 7-AAD+ FB cell%, 10.9 ± 2.8% at IdeS 0 μg/mL, became 7.6 ± 2.2% (P = 0.06), 5.1 ± 0.9% (P = 0.02), and 5.3 ± 1.0(P = 0.03) at 1, 10, and 100 μg/mL, respectively. IFNγ+ NK cell% at the maximum therapeutic level, 10 μg/mL, was again close to that observed in the control (NC) group (background, dotted line in Figure 3B).
Anti-HLA Antibody Levels and Capacity to Induce Antibody-Mediated NK Cell and NKT Cell Activation in Imlifidase-treated Patients
Anti-HLA antibody levels were measured pretreatment and posttreatment in IdeS-treated patients by a single antigen bead Luminex assay, and the results were expressed as the score. Anti-HLA antibody score significantly decreased at day 1 (1D) postimlifidase from the preimlifidase level (baseline) (36 ± 51 versus 287 ± 125, P < 0.01) in all patients, and then returned to the baseline by 1 month in 3 patients (Figure 4A). The score in the remaining 2 patients increased at 1–3 months, but did not return to the baseline by 6 months.
We next examined these sera obtained preimlifidase and postimlifidase in the Allo-CFC-3 to determine the kinetics of antibody-mediated NK cell activation in IdeS-treated patients. The mean IFNγ+ NK cell% preimlifidase was 7.8 ± 4.8% and all patients showed significant reduction at 1D postimlifidase (2.2 ± 1.7%, P = 0.03) (Figure 4B). Four of 5 patients returned to preimlifidase levels by 1M. The IFNγ+ NK cell% gradually increased at 1 and 3 months postimlifidase in the remaining 1 patient and returned to the baseline level at 6 months postimlifidase. We also analyzed the results obtained in the Allo-CFC-3 to determine the kinetics of antibody-mediated T cell activation in these patients since it is known that a subset of T cells, NKT cells, have features similar to NK cells including antibody/CD16-mediated cell activation.21 Among 3 normal blood samples (NC1-3) used as responder cells in the Allo-CFC-3, only CD8+ T cells from normal blood NC2 had high levels of CD16+ cells (15.2%), while CD4+ T cells from NC2 and both T cell subsets from NC1 and NC3 blood had minimal CD16+ cells (≤5%) (Figure 4D). As expected, elevated IFNγ+ cell% in CD8+ cells from NC2 blood (9% and 3%) were observed when PBMCsx were pretreated with preimilifidase serum in the Allo-CFC-3, and then those elevated IFNγ+CD8+ cell% significantly decreased at 1D postimlifidase (Figure 4C). The reduced levels went back to preimlifidase levels by 1 month or 6 months postimlifidase, respectively, which was consistent with the kinetics observed in NK cells (Figure 4B). In contrast, IFNγ+ cell% in the remaining CD4+ and/or CD8+ T cells with low levels of CD16+ cells were minimal and did not change preimlifidase and postimlifidase (Figure 4C and D).
IdeS, IgG-degrading enzyme, cleaves human IgG into Fc, and F(ab′)2 fragments,8,20,22 resulting in abrogration of FcγR-mediated cell activation including ADCC. In this study, we showed that IdeS inhibited anti-HLA antibody-mediated NK cell activation in a dose dependent manner in both Allo-CFC-1 and Allo-CFC-2 assays. The former measures NK cell activation in blood stimulated with anti-HLA antibody-precoated PBMCs that were treated with IdeS (Figure 1B), while the latter in blood stimulated with PBMCs that were precoated with IdeS-treated HS sera (Figure 2B) (Figure S1, SDC, http://links.lww.com/TP/B833). This result suggests that IdeS is capable of cleaving soluble anti-HLA IgG in HS sera and IgG bound on the surface of PBMCs, which is consistent with the previous report.23 In the Allo-CFC-1, significant reduction of NK cell activation by IdeS was observed from 0.1 μg/mL, 1/100 of the maximum therapeutic level, while it was from 10 μg/mL in the Allo-CFC-2. This is likely due to less IdeS available per IgG molecule in the Allo-CFC-2 where HS sera containing 10–20 mg/mL IgG were treated with IdeS compared to the Allo-CFC-1 assay where only IgG bound on PBMCs was cleaved by IdeS.
In this study, we also showed the inhibitory effect of IdeS on ADCC where responder PBMCs mixed with target FB cells were incubated with or without IdeS-treated HS sera (Figure 3B and Figure S1, SDC, http://links.lww.com/TP/B833). Significant inihibition was observed from the maximum therapeutic level of IdeS, 10 μg/mL, but not lower than that, which was similar to the Allo-CFC-2 results. This is likely due to less efficient IgG cleavage by IdeS since HS sera were treated with IdeS and then used for the ADCC assay. Inhibitory effect of IdeS on ADCC was also reported by Tradtrantip et al11 using their ADCC assay where neuromyelitis optica IgG (NMO-IgG) (autoantibodies anti-aquaporin 4)-mediated protein AQP4-expressing CHO cell killing by NK cells was measured with or without IdeS treatment.
Interestingly, significant reduction of ADCC by IdeS was observed from 5 to 10 μg/mL of IdeS, which is consistent with our result. In addition, they showed that IdeS cleaved NMO-IgG in mice with experimental NMO, reducing NMO lesions mediated by ADCC and CDC.
Reduction of FcγR-mediated autoimmune inflammation by IdeS has been reported in other animal models. Mihai et al24 have shown that Arthus reaction induced by Fcγ-FcγR interaction was prevented in IdeS-pretreated mice, and they were completely protected from IgG-dependent edema formation and other inflammatory infiltration. Yang et al25 reported that IdeS treatment prevented albuminuria and the recruitment of leukocytes to the glomeruli in a mouse model of anti-glomerular basement membrane disease induced by rabbit anti-mouse glomerular basement membrane IgG (subclass IgG2a). In our previous study26 where the effect of IdeS on CD4+ T cell and CD56+ NK cell response to CMV antigens or mitogen PHA was compared, we showed that anti-CMV antibody/CD16-mediated NK cell response was completely abrogated by IdeS, while T cell receptor-mediated CMV-specific T-cell response and PHA-stimulated T cell and NK cell response did not change by IdeS. This result confirmed that IdeS affects only antibody-mediated cell response, via degradation of IgGs, regardless of cell types.
We finally determined if anti-HLA antibodies were cleaved in patients treated with IdeS and if serum obtained from the IdeS-treated patients showed less capacity for antibody-mediated NK cell activation in vitro. In the previous study,12 we showed significant reduction of total IgG during 2 hours to 7 days postimlifidase, to a lesser degree during 7–28 days, and no significant reduction by 64 days. In this study, we observed similar trend in anti-HLA antibody levels postimlifidase in 5 patients. All 5 patients showed significant reduction of anti-HLA antibody levels at 1 day postimlifidase. The levels returned to the preimlifidase levels at 1–3 months postimlifidase in 3 patients, while levels in the remaining 2 patients also increased, but did not reach the preimlifidase levels by 6 months postimlifidase (Figure 4A). IFNγ+ NK cell% in the Allo-CFC-3, where sera obtained from these 5 IdeS-treated patients were used for the assay, significantly decreased at 1 day and then returned to the preimlifidase levels by 1 month in most patients (Figure 4B). This was expected since NK cell activation, producing IFNγ, in Allo-CFC is primarily dependant on anti-HLA antibody levels. We also analyzed the kinetics of antibody-mediated T cell activation in these IdeS-treated patients since NKT cells express CD1621,27 and are capable of responding to antibody-coated PBMCs in the Allo-CFC-3 via antibody-CD16 interaction. As expected, CD8+ T cells with high levels of CD16+ cells showed similar kinetics to NK cells preimlifidase and postimlifidase treatment as mentioned above, while the remaining T cell subsets with low levels of CD16+ cells have no or minimal response in the Allo-CFC-3 using patients’ sera obtained preimlifidase and postimlifidase. These results suggest that anti-HLA antibody-mediated NK cell and NKT cell activation, and ADCC were likely to be inhibited by IdeS at least at 1 day to possibly 7 day posttreatment in IdeS-treated patients.
As our previous study suggested,12 the use of IVIG + rituximab pretransplant and posttransplant could prevent the rebound of DSA. All 9 patients with preimlifidase DSA in the Swedish group who did not receive IVIG + rituximab showed DSA postimlifidase, while only 3 of 11 (27%) with preimlifidase DSA in the US group who received pre and posttransplant IVIG and rituximab did so. All 5 patients included in this study had anti-HLA antibodies pretransplant as shown in Figure 4A. At day 1 postimlifidase, those levels became nearly undetected in all patients, but returned to the preimlifidase or 70% of the preimlifidase levels by 6 months postimlifidase in all 5 patients. However, DSA detected preimlifidase in 3 of the 5 patients did not reappear up to 6 months postimlifidase, and all 5 patients did not develop AMR during the study period. This result suggests that IVIG + rituximab may have helped to preferentially deplete or modulate DSA-producing B cells, but not non-DSA anti-HLA antibody producing B cells, after IdeS treatment, reducing DSA levels, which may have resulted in preventing ADCC-mediated, and possibly CDC-mediated AMR. In the above previous study,12 we also showed significantly higher C4d+ score in biopsies obtained from patients with AMR in the Swedish group than those in the US group. This may be due in part to IVIG given to the United States patients as IVIG neutralizes complement.28
In conclusion, IdeS inhibited anti-HLA antibody-mediated NK cell activation and ADCC in a dose-dependent manner in vitro, and the maximum therapeutic concentration of IdeS, 10 μg/mL, showed the greatest inhibition. Anti-HLA antibody levels significantly decreased and the capacity of anti-HLA antibody-mediated NK cell and NKT cell activation were also significantly inhibited in sera obtained after IdeS treatment. These suggest that IdeS inhibits IgG-mediated immune activation including ADCC and CDC in IdeS-treated patients. Thus, IdeS could be useful as a treatment for AMR where ADCC and CDC play important roles.
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