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Accelerated humoral renal allograft rejection due to HLA-C14 mediated allosensitization to HLA-Bw6
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Accelerated humoral renal allograft rejection due to HLA-C14 mediated allosensitization to HLA-Bw6 ⁎
Stephen P. Persauda, , Brian Duffyb, Donna L. Phelanb, Thalachallour Mohanakumarc, ⁎ Rowena Delos Santosd, Joseph P. Gaute, Chang Liua, a
Division of Laboratory and Genomic Medicine, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA Department of Laboratories, Barnes-Jewish Hospital, St. Louis, MO 63110, USA c Norton Thoracic Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ 85013, USA d Division of Nephrology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA e Division of Anatomic and Molecular Pathology, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA b
A R T I C L E I N F O
A B S T R A C T
Keywords: HLA Alloimmunization Transplantation Kidney Crossmatch
Objectives: To investigate immunological mechanisms underlying accelerated antibody-mediated rejection (AMR) of a living-related renal allograft in a patient with no detectable antibodies to donor human leukocyte antigens (HLA) in pre-transplant sera. Methods: Pre- and post-transplant HLA antibody specificities were determined by single-antigen bead assay, and crossmatching was performed by flow cytometry- and complement-dependent cytotoxicity-based methods. Intermediate- and high-resolution HLA typing were performed by molecular methods. Results: Pre-transplant patient serum reacted weakly against Bw6-positive beads; cytotoxicity and flow crossmatches were negative. The patient was mismatched for the donor antigens B62 and C10 (Bw6-positive). Following transplantation, strong antibody responses against B62, C10, and all Bw6-positive beads were detected. This reactivity was initially masked by complement interference, but became apparent at 1:20 dilution. High-resolution typing suggested that the anti-C16 antibody reactivity detected was an allele-specific response to donor C∗16:01 (Bw6-positive) but not recipient C∗16:02 (Bw6-negative). Alloimmunization likely occurred during pregnancy, during which HLA-C14 (Bw6-positive) was the only mismatched paternal HLA Class I allele. Conclusions: Sensitization to HLA-Bw6 via exposure to paternal HLA-C14 during pregnancy likely predisposed this patient to AMR. The case demonstrates the immunogenicity of HLA-C14-associated Bw6 epitopes in vivo and the clinical significance of low-level antibodies to HLA-Bw6.
1. Introduction Due largely to improved immunosuppressive therapy in recent years, better short-term renal allograft survival and decreased rates of acute rejection have been reported [1]. However, long-term graft survival has proven less substantial than anticipated [2]. Antibody-mediated rejection (AMR) has been increasingly appreciated as a significant obstacle to improvement of long-term kidney transplantation outcomes [3]. While hyperacute rejection secondary to preformed donor specific antibodies (DSA) has become rare over recent years, acute and chronic AMR remain significant risk factors associated with allograft loss [4]. Antibodies targeting donor human leukocyte antigens (HLA) are of
particular concern in the pathogenesis of acute and chronic AMR. Binding of donor HLA in the allograft by DSA can lead to complementmediated and antibody-dependent cellular cytotoxicity, causing endothelial damage and thrombosis that compromises allograft function and survival [5]. Both pre-existing [6,7] and de novo [8] DSA antibodies have been associated with poorer long-term survival of renal allografts. For this reason, screening for anti-HLA in addition to crossmatching has become the standard of care in the evaluation of kidney transplant patients. Herein, we present a case of a multiparous woman with no prior history of transfusion or transplant who underwent living-related kidney transplantation. Pre-transplant antibody screening was
Abbreviations: AMR, antibody mediated rejection; AHG, anti-human globulin; ATG, anti-thymocyte globulin; CDCXM, complement-dependent cytotoxicity crossmatch; CMV, cytomegalovirus; DSA, donor specific antibody; DTT, dithiothreitol; EBV, Epstein-Barr Virus; EDTA, ethylenediaminetetraacetic acid; FITC, fluorescein isothiocyanate; FL, fluorescence channel; FCXM, flow cytometric crossmatch; HLA, human leukocyte antigen; MCS, median channel shift; MFI, median fluorescence intensity; PE, phycoerythrin; PerCP, peridinin chlorophyll protein; POD, post-operative day; PRA, panel reactive antibody ⁎ Corresponding authors at: 660 S. Euclid Ave, Campus Box 8118, St. Louis, MO 63110, USA. E-mail addresses: [email protected] (S.P. Persaud), [email protected] (C. Liu). http://dx.doi.org/10.1016/j.humimm.2017.09.004 Received 13 June 2017; Received in revised form 25 August 2017; Accepted 28 September 2017 0198-8859/ © 2017 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Persaud, S.P., Human Immunology (2017), http://dx.doi.org/10.1016/j.humimm.2017.09.004
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cell suspensions were analyzed using a FC500 flow cytometer (Beckman Coulter, Brea, CA). For analysis, lymphocytes were gated using forward and side scatter parameters followed by gating of CD3+ T cells (FL4 channel) and CD19+ B cells (FL2 channel). Within these subpopulations, bound human IgG was expressed on a logarithmic FL1 channel and expressed as the median channel number. The median channel shift (MCS) between patient samples and non-sensitized controls was obtained by subtraction of the median channel numbers; MCS cutoff values of 40 and 80 were used for T and B cells, respectively.
consistent with a weak antibody against the Bw6 epitope, but cytotoxicity and flow crossmatches were negative. The patient presented one week post-operatively with serologic and histopathological evidence of acute AMR with development of antibodies to HLA. We discuss the immunologic basis for antibody development leading to rapid antibody-mediated renal allograft rejection. 2. Methods 2.1. HLA typing
2.6. Protein structure image generation DNA was extracted from EDTA-anticoagulated whole blood using the EZ1 DNA Blood kit from Qiagen (Hilden, Germany). Intermediateresolution HLA typing was performed using the LabType SSO kit (One Lambda, Canoga Park, CA) per the manufacturer’s manual on the Luminex 200 instrument (Luminex, Austin, TX). High-resolution HLA typing was performed using the SeCore HLA sequencing kit (Life Technologies, Brown Deer, WI) per the manufacturer’s manual. Intermediate-resolution typing and serogroup assignment are performed in routine clinical practice at our center; high-resolution typing is performed for epitope identification.
The ribbon diagram of HLA-B∗15:01 (Protein Data Bank ID 1XR9) [9] was generated using Swiss-PdbViewer version 4.1 (www.expasy. org/spdbv) [10]. 2.7. Statistics The probability P of the allograft recipient being exposed to her husband’s HLA-C14 allele during pregnancy, given that her husband is heterozygous at the HLA-C locus, was calculated as per Formula (1) below:
2.2. Panel reactive antibody (PRA) assay
P (C14 exposure in utero)=1−(0. 5)n;
Patient sera was analyzed using the LABScreen PRA kit (One Lambda, Canoga Park, CA) per the manufacturer’s instructions using the Luminex platform (Luminex, Austin, TX). The cutoff for positivity was MFI 750 per the manufacturer’s recommendation.
(1)
where n is the number of pregnancies, and 0.5 reflects the probability the fetus inherits the paternal C14 allele. The frequencies of the 80 N eplet in various populations in the United States were obtained from the Allele Frequency Net Database [11], and were determined based on haplotype frequencies.
2.3. HLA antibody identification
3. Case report
Patient serum was analyzed for antibodies to HLA with or without dilution as indicated using the LABScreen single-antigen bead kit (One Lambda, Canoga Park, CA). The assay was performed using the Luminex platform (Luminex, Austin, TX) per the user’s manual except that 25 μL of beads was used per assay. The MFI cutoff for a positive assay was based on experience from our center showing that MFI values ranging from 2000 to 3000 predict positivity by complement-dependent cytotoxicity crossmatching; within this range, 2000 MFI was chosen as the cutoff to maximize sensitivity. Samples with results suspicious for interference by complement were retested with dilution at 1:20. We do not routinely monitor post-transplant DSA per protocol at our institution. However, DSA monitoring in selected high-risk cases (i.e., patient with positive pre-transplant virtual or flow crossmatch) can be initiated at the discretion of the treating physician.
A 36-year-old gravida 4 para 4 Caucasian female with a history of chronic kidney disease stage 4 secondary to reflux nephropathy diagnosed at age 5 presented for evaluation for kidney transplantation. Her past medical history was notable for hypertension, dyslipidemia, anemia and secondary hyperparathyroidism. She had no prior history of transplantation or blood transfusions. Living-related kidney transplant was planned with her brother as donor. The patient and donor were blood type O negative, and initial intermediate-resolution HLA typing identified mismatches at the B, C, DRB1 and DQB1 loci (Table 1). Panelreactive antibody assay was positive at 18% for Class I HLA and negative for Class II HLA. EBV serology was positive in both donor and patient and CMV serology positive only in the patient. The transplant procedure was complicated by significant blood loss, which required transfusion of 2 units of leukocyte-reduced red blood cells (RBCs) intraoperatively, but otherwise the patient tolerated the procedure well. For induction immunosuppression, the patient received
2.4. Complement-dependent cytotoxicity crossmatch (CDCXM) CDCXM was performed by the four-wash Amos technique, with and without anti-human globulin (AHG) augmentation. Donor cells were incubated with recipient sera for 40 min at 37 °C. AHG and rabbit complement were added and cells incubated for 1 h at 37 °C. Nonviable cells were labeled by ethidium bromide uptake. Cell death of greater than or equal to 20% above background was defined as a positive crossmatch.
Table 1 HLA typing results for kidney allograft recipient and select relatives. HLA Locus
2.5. Flow cytometric crossmatch (FCXM)
Subject
A
B
C
DRB1
DRB3/4/5
DQB1
Patient (recipient)
A2
B51
C2
DR11
DR52
DQ7
C16:02 (C16)* C10 C16:01 (C16)* C2
DR15
DR51
DQ6
DR4 DR15
DR53 DR51
DQ8 DQ6
DR1
DR52
DQ2
C14
DR17
A26
4 x 105 leukocytes from peripheral blood or lymph node were incubated with 40 μL patient serum for 30 min at 4 °C, and subsequently washed three times to remove unbound antibody. Lymphocytes were not treated with pronase. Samples were stained for 15 min at 4 °C with anti-CD3 PerCP (clone SK7; BD Biosciences, San Jose, CA) and CD19 PE (clone SJ25C1; BD Biosciences, San Jose, CA) to label T and B lymphocytes, respectively, and goat anti-human IgG F(ab′)2 FITC (polyclonal; Sigma Aldrich, St. Louis, MO) to detect cell-bound DSA. Stained
Brother (donor)
A2 A26
B51 B62
Husband of patient
A2
B51:01 (B51)* B51:07 (B51)*
* Serogroup assignment is listed in parentheses.
2
DQ5
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Fig. 1. Renal biopsy shows findings supporting a diagnosis of acute antibody mediated rejection. (A) Hemorrhagic cortical necrosis is widespread (H & E; 200x). (B & C) Glomerular and arteriolar fibrin thrombi are identified (Masson’s trichrome, 400× and 600×). (D) Immunohistochemistry for C4d shows diffuse linear peritubular capillary staining (200×).
consistent with a low-level antibody against Bw6, and six of the seven positive Class I antigen beads were coated with Bw6-associated antigens (Table 2). The median fluorescence intensity of the antibody was reduced at 1:20 dilution, although the pattern of reactivity was largely unaffected (Fig. 2). Consistently, no reactivity was detected against donor B- or T-lymphocytes by complement-dependent cytotoxicity crossmatch (CDCXM) using four-wash Amos or AHG methodologies, or flow cytometry crossmatch (FCXM). At the time of presentation with suspected graft rejection, however, HLA antibody screening demonstrated antibodies against 36 HLA Class I antigen beads (mean MFI 5005, range 2005-12,575), including DSA at low MFI against C10 (Table 2, Fig. 2). Serum dilution to 1:20 revealed strong reactivity against 59 HLA Class I antigen beads at markedly increased MFIs (mean MFI 15,130, range 2187–22,454). Strong DSA against the mismatched HLA Class I alleles, B62 and C10, were revealed by the serum dilution analysis, but not against the mismatched HLA Class II alleles (Fig. 3). The identification of DSA supported the diagnosis of acute AMR. Notably, the serum dilution analysis also unmasked a strong pattern of reactivity to all beads with Bw6-associated HLA-B and HLA-C antigens (C1, C7, C8, C9, C10, C12, C14 and C16), which were clustered together in a range of high MFI values > 15,000 (Fig. 2, Table 2). Given that the pre-transplant reactivity against mismatched donor antigens and Bw6 was much lower, the rapid increase in the strength of DSAs and the pace of the allograft rejection suggested an anamnestic response against HLA Class I rather than de novo sensitization. This implies an occult alloimmunization with grave clinical consequences that could not be reliably predicted or monitored in a timely manner.
75 mg anti-thymocyte globulin (ATG) and 450 mg methylprednisolone intraoperatively, then received 125 mg ATG once daily and 80 mg prednisone once daily on post-operative days (POD) 1 and 2. Prednisone was reduced to 20 mg daily on POD 3 and tapered thereafter. Mycophenolic acid was started at 720 mg twice daily and tacrolimus started at 2 mg twice daily on POD 1. Tacrolimus was subsequently increased to 5 mg twice daily in order to achieve therapeutic concentrations (7–10 ng/dL), at which point mycophenolic acid was reduced to 360 mg twice daily. Graft function was immediately satisfactory, with serum creatinine at POD 4 of 0.76 mg/dL, and so the patient was discharged to home. The patient presented again on POD 7 with pain at the allograft site and facial and upper body flushing. At this time, serum creatinine was 3.6 mg/dL, tacrolimus trough concentration was within the therapeutic range at 7.4 ng/mL, and a renal ultrasound suggested compromise of the renal artery supplying the allograft. Biopsy of the allograft showed diffuse hemorrhagic cortical necrosis, and glomerular, arteriolar and arterial thromboses (Fig. 1). C4d immunohistochemistry demonstrated diffuse peritubular capillary C4d positivity. These histopathologic findings strongly suggested an acute antibody-mediated rejection (AMR) and prompted removal of the allograft. The post-nephrectomy allograft specimen had a mottled blue gross appearance with petechial hemorrhages most apparent at the corticomedullary junction. Microscopically, extensive arteritis and vascular thrombosis were present along with diffuse cortical necrosis. These findings corroborated the initial diagnostic impression of acute AMR.
3.1. Laboratory investigation for AMR
3.2. Possible mechanism of alloimmunization
Based on the initial intermediate-resolution HLA typing, the donor antigens that were mismatched and thus could have been recognized as foreign by the recipient immune system included B62, C10, DR4 and DQ8. Pre-transplant HLA antibody screening revealed a pattern
HLA alloimmunization may occur as a consequence of transplantation, transfusion and pregnancy [12]. We investigated the likely 3
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Table 2 Summary of pre- and post-transplant HLA antibody screen results. Pre-transplant, no serum dilution
Post-transplant, no serum dilution
Post-transplant, 1:20 diluted serum
HLA-A Positive beads (%) MFI Mean ± SD of positive beads MFI range of positive beads
0/31 (0%) 0 0
8/31 (26%) 3190 ± 2103 2005-8344
8/31 (26%) 4546 ± 1774 2187-7583
HLA-Bw4 Positive beads (%) MFI Mean ± SD of positive beads MFI range of positive beads
1/19 (5%) 2414 2414
11/19 (58%) 5491 ± 2684 2071-9103
9/19 (47%) 8793 ± 4076 3396-16229
HLA-Bw6 Positive beads (%) MFI Mean ± SD of positive beads MFI range of positive beads
6/31 (19%) 2855 ± 739 2011-4018
7/31 (23%) 3425 ± 1469 2256-6514
31/31 (100%) 18787 ± 1440 15116-21992
HLA-C Positive beads (%) MFI Mean ± SD of positive beads MFI range of positive beads
0/16 (0%) 0 0
10/16 (63%) 6339 ± 3881 2242-12575
11/16 (69%) 17603 ± 7504 2503-22454
All beads Positive beads (%) MFI Mean ± SD of positive beads MFI range of positive beads
7/97 (7%) 2792 ± 695 2011-4018
36/97 (37%) 5005 ± 3101 2005-12575
59/97 (61%) 15130 ± 6532 2187-22454
minimal number of leukocytes in filtered RBC units, the short time between transfusion and rejection, and the magnitude of the antibody response. However, while de novo immunization by these RBC units was
sensitizing event that initially primed the patient’s immune system to mount such a destructive memory response. The intraoperative RBC transfusions were unlikely the initial immunizing event given the
Fig. 2. Post-transplant Luminex HLA antibody assay performed using diluted recipient serum reveals high-MFI reactivity against all Bw6-positve beads. Histograms show antibody screen results for the pre-transplant serum without dilution (A), pre-transplant serum with 1:20 dilution (B), post-transplant serum without dilution (C), and post-transplant serum with 1:20 dilution. The y-axis shows the MFI values of single antigen beads to which patient serum reacted, with the identity of the antigen listed below each bar. Bw6-associated antigens are circled. Bold horizontal bars indicate the 2000 MFI cutoff for positivity of the assay. All samples were analyzed during a single run that was performed post-transplant, and the pretransplant data shown are repeats of the original assay using the same sample. Please note that in these repeats, reactivity to only one Bw6-associated antigen was identified in the pretransplant serum as opposed to the 6 antigens as detected in the original assay. The overall pattern of reactivity between the original assay and the repeat is otherwise unchanged.
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Fig. 3. Dilution of recipient post-transplant serum in HLA antibody screen demonstrates strong DSA against B62 and C10. Plots show the relationship between serum dilution and median fluorescence intensity (MFI) for each of the specified donor antigens. Notably, dilution of the pretransplant serum did not reveal positivity for DSA.
containing this epitope could be recognized as non-self during pregnancy, leading to allosensitization and a potent memory antibody response upon exposure to the Bw6-positive allograft.
unlikely, it remained possible that residual leukocytes could bear antigen contributing to the rejection response. In the absence of prior transplantation or transfusion, the patient’s multiple pregnancies were most likely the cause of immunization against allogeneic HLA. We therefore examined the HLA antigens expressed by her husband, to which she may have responded during her pregnancies. HLA typing of the patient’s husband demonstrated a single HLA Class I mismatch at HLA-C, with the patient typing as HLA-C16 and her husband as HLA-C14 (Table 1). How, then, could reactivity to HLA-C14 during pregnancy prime the patient’s immune system to respond to B62 and C10 after transplantation? The weak reactivity to several HLA Bw6associated antigens pre-transplant, taken together with the strong posttransplant reactivity against all Luminex beads with HLA-Bw6 antigens, suggested a mechanism whereby recognition of this common public epitope led to cross-reactivity to distinct HLA antigens encountered during pregnancy and transplantation. The Bw6 epitope is comprised of solvent-exposed amino acid residues spanning positions 77-83 within the α1-helix of most HLA-B and some HLA-C heavy chains, including HLA-C14 (Fig. 4A) [13,14]. The Bw6 peptide contains the 80 N epitope (also referred to as eplet 79RN and TerEp #25), as defined by the HLA Epitope Database [15,16]. Amino acid differences at positions 77 and 80-83 distinguish the Bw6 epitope from the mutually exclusive Bw4 epitope (Fig. 4B) [17]. As the Bw6 epitope is present in the HLA-C14 antigen expressed by the patient’s husband as well as the B62 and C10 antigens expressed by the donor, this epitope could have primed and restimulated antibody responses during pregnancy and transplantation, respectively. This epitope is present in all HLA-C antigen beads to which the patient’s serum reacted strongly (C1, C7, C8, C9, C10, C12, C14, and C16) and absent in the HLA-C antigen beads to which her serum reacted weakly (C2, C4, C5, C6, C15, C17, and C18). The latter group of HLA-C antigens have two amino acid substitutions at positions 77 (Ser to Asn) and 80 (Asn to Lys), and thus do not bear the Bw6 epitope (Fig. 4B). An unexplained issue thus far is that both the patient and the donor were typed as positive for HLA-C16, yet her antibody response included strong reactivity to the C∗16:01 Luminex bead. Reactivity to C16 would be unexpected in this patient if it were a self-antigen. To resolve this issue, high-resolution typing was performed, which demonstrated that the patient expresses the HLA-C∗16:02 allele, whereas her brother expresses HLA-C∗16:01. While both alleles are assigned to the C16 serogroup, C∗16:01 contains a canonical Bw6 epitope whereas C∗16:02 does not. Notably, C∗16:02 differs by the same amino acid substitutions at positions 77 and 80 as the other HLA-C antigens to which the patient weakly responded. The absence of the Bw6 epitope in the patient’s HLA-B and C alleles suggests that the paternal HLA-C14 allele
4. Discussion Herein, we have described a kidney transplant recipient who became alloimmunized against the Bw6 epitope upon encountering paternal HLA-C14 antigens during pregnancy; this set the stage for a rapid anamnestic response upon exposure to the Bw6 epitope in the renal allograft, ultimately leading to AMR and graft loss. Our hypothesis that the recognition of a common epitope of a HLA-C allele could lead to broad recognition of HLA-B alleles is consistent with the findings of several previous reports [18–20]. Notably in our case, high-resolution typing demonstrated the patient expressed an HLA-C16 allele that lacks the Bw6 epitope, which accounted for her susceptibility to Bw6 immunization via exposure to paternal HLA-C14. While the children of this patient were not available for HLA typing to confirm the patient’s exposure to HLA-C14 during her pregnancies, the high probability of encountering HLA-C14 during pregnancy (approximately 94% over four pregnancies, given that the patient’s husband was heterozygous for HLA-C14) and the absence of prior transplantation or transfusion makes this the most likely mechanism by which the patient was initially sensitized. Although HLA-C is considered a low-expression locus, the expression level of HLA-C14 was reported to be the highest among selected HLA-C antigens examined by flow cytometry [21]. This case provided evidence supporting the immunogenicity of the HLA-C14 antigen in vivo. Given the high prevalence of Bw6-associated antigens, it remains possible that Bw6 epitope-positive leukocytes in the two RBC units transfused intraoperatively were an additional source of antigen that could have contributed to the patient’s anamnestic response and rejection. As HLA typing results for the two RBC units given to the patient were not available, this possibility cannot be completely excluded. Although leukoreduced RBC units remain a strong risk factor for alloimmunization in renal transplant patients [22,23], the pattern consistent with a weak anti-Bw6 was present in this case before the patient received her intraoperative RBC transfusions. A recent study described two antibodies with specificities to the 80NRG eplet (Bw6-associated) derived from female patients sensitized to paternal HLA antigens during pregnancy [24]. For both antibodies, HLA-B antigens containing the Bw6 epitope (B∗07:02 and B∗08:01) were implicated as the most likely immunizing agents. Similar to our study, pan-reactivity to Bw6-associated antigens was observed, and the patients who produced these antibodies expressed HLA antigens 5
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Fig. 4. The Bw6 motif is a linear epitope present in most HLA-B and several HLA-C antigens. (A) Ribbon diagram of the HLA-B*15:01 (serogroup B62) peptide binding pocket, comprised of the α1 and α2 domains of the Class I HLA-B heavy chain (Protein Data Bank ID 1XR9). The Bw6 motif (residues 77–83, dark gray) lies within the C-terminal end of the α1 helix; residues within this motif are labeled and their sidechains displayed in stick format. (B) Alignment of the amino acid sequence for the Bw4 and Bw6 motifs and select HLA-C antigens at positions 77 through 83. Residues differing from the Bw6 motif are underlined.
(C∗02:02 and C∗06:02) lacking the 80NRG eplet. This supports the notion that patients whose HLA antigens have these amino acid substitutions are susceptible to immunization against the Bw6 epitope. As the 80 N eplet residing within the Bw6 epitope is highly prevalent in several diverse populations in the United States (93-99%) [11], sensitization to this epitope represents a significant barrier to identifying compatible grafts for transplantation. Thus, accurate interpretation of the antibody pattern is essential. Although solid-phase assays have greatly improved the sensitivity and specificity of HLA antibody detection [25], our case illustrates two pertinent issues related to the use of these assays in the laboratory detection of relevant HLA antibodies and risk assessment for AMR in transplant patients. First, the broad, strong reactivity to HLA Class I demonstrated on the post-transplant HLA antibody screen was an important distinguishing feature of the patient’s aggressive graft rejection course, yet the magnitude of this reactivity was only evident upon serum dilution. Such a prozone-like effect leading to false negative results has been described previously for solid-phase, single-antigen bead assays, and has been attributed to interference with the antigen beads by proteins of the complement cascade, particularly C1 [26]. In addition to serum dilution, mitigation of this effect has been demonstrated by treatments that destabilize the C1 complement complex, such as heat inactivation to 56 °C, and treatment with the divalent cation chelator EDTA or the disulfide-reducing dithiothreitol (DTT). Second, the patient’s pre-transplant serum was positive for a few low-MFI, non-DSA Class I antibodies to HLA-B. However, analysis of all single antigen beads (Fig. 2) demonstrated a broader anti-Bw6 reactivity, involving many Bw6 antigen beads with MFI below the positive cutoff. Thus, assessment of allosensitization in transplant candidates requires consideration of not only those single antigen beads above a certain MFI threshold, but rather the overall pattern of reactivity. In this case, the anti-Bw6 reactivity in the pre-transplant patient serum was relatively weak, given the pattern on dilution study and negative crossmatches. In a patient with truly low-MFI anti-Bw6 reactivity, it remains difficult to predict the probability of a fulminant memory response seen in this case. This theoretical risk would have to be weighed against the high cPRA associated with listing Bw6 as an unacceptable group. A study of HLA alloimmunization in potential blood donors demonstrated nearly a quarter of women with a history of pregnancy had antibodies to HLA [27], with a correlation between the prevalence of alloimmunization and the number of prior pregnancies. Such a high seroprevalence of anti-HLA would suggest that the opportunity for cross-reactivity to public epitopes leading to AMR would be a common occurrence among multiparous patients. However, the majority of sensitized kidney transplant recipients in one study, even those with DSA, did not experience AMR at 5 years post-transplant [28]; notably,
though, patients sensitized against non-DSA had lower rates of 5-year graft rejection than DSA-sensitized patients. Another study demonstrated that among DSA-positive kidney transplant recipients, the DSA MFI for patients with and without acute rejection overlapped within a low range of less than 6000 [29]. These results suggest that even with improvement of methods to detect antibodies to HLA, their interpretation remains controversial. 5. Conclusions We investigated a case of rapidly progressive antibody-mediated renal allograft rejection in a patient with a weak, crossmatch-negative antibody against Bw6. Based on the workup performed by the histocompatibility laboratory at our institution, we concluded that recognition of the public Bw6 epitope in the paternal HLA-C14 allele during pregnancy most likely primed the patient’s immune system against this epitope. This led to rapid renal allograft rejection upon exposure to donor HLA alleles also carrying the Bw6 epitope. This case highlights the immunogenicity of C14-associated Bw6 epitopes in vivo and the clinical significance of low-level anti-Bw6 antibodies in the setting of a negative crossmatch. Whether or not to cross the transplantation barrier imposed by Bw6 in a situation like this remains a personalized decision. For cases in which this barrier is crossed, an important pursuit for future investigation will be to better characterize factors that influence whether a sensitized individual will mount an alloantibody response that leads to AMR. Sources of funding None Disclaimers None References [1] H.U. Meier-Kriesche, J.D. Schold, T.R. Srinivas, B. Kaplan, Lack of improvement in renal allograft survival despite a marked decrease in acute rejection rates over the most recent era, Am. J. Transplant. 4 (3) (2004) 378–383. [2] S.A. Lodhi, K.E. Lamb, H.U. Meier-Kriesche, Solid organ allograft survival improvement in the United States: the long-term does not mirror the dramatic shortterm success, Am. J. Transplant. 11 (6) (2011) 1226–1235. [3] A. Djamali, D.B. Kaufman, T.M. Ellis, W. Zhong, A. Matas, M. Samaniego, Diagnosis and management of antibody-mediated rejection: current status and novel approaches, Am. J. Transplant. 14 (2) (2014) 255–271. [4] J. Sellares, D.G. de Freitas, M. Mengel, J. Reeve, G. Einecke, B. Sis, et al., Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonadherence, Am. J. Transplant. 12 (2) (2012) 388–399.
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[18] R.J. Duquesnoy, M. Marrari, Detection of antibodies against HLA-C epitopes in patients with rejected kidney transplants, Transpl. Immunol. 24 (3) (2011) 164–171. [19] J. Lomago, L. Jelenik, D. Zern, J. Howe, J. Martell, A. Zeevi, et al., How did a patient who types for HLA-B*4403 develop antibodies that react with HLA-B*4402? Hum. Immunol. 71 (2) (2010) 176–178. [20] J.A. Trapani, S. Mizuno, S.H. Kang, S.Y. Yang, B. Dupont, Molecular mapping of a new public HLA class I epitope shared by all HLA-B and HLA-C antigens and defined by a monoclonal antibody, Immunogenetics 29 (1) (1989) 25–32. [21] R. Apps, Y. Qi, J.M. Carlson, H. Chen, X. Gao, R. Thomas, et al., Influence of HLA-C expression level on HIV control, Science 340 (6128) (2013) 87–91. [22] M.S. Leffell, D. Kim, R.M. Vega, A.A. Zachary, J. Petersen, J.M. Hart, et al., Red blood cell transfusions and the risk of allosensitization in patients awaiting primary kidney transplantation, Transplantation 97 (5) (2014) 525–533. [23] M. Karpinski, D. Pochinco, I. Dembinski, W. Laidlaw, J. Zacharias, P. Nickerson, Leukocyte reduction of red blood cell transfusions does not decrease allosensitization rates in potential kidney transplant candidates, J. Am. Soc. Nephrol. 15 (3) (2004) 818–824. [24] R.J. Duquesnoy, The antibody response to an HLA mismatch: a model for nonselfself discrimination in relation to HLA epitope immunogenicity, Int. J. Immunogenet. 39 (1) (2012) 1–9. [25] A. Konvalinka, K. Tinckam, Utility of HLA antibody testing in kidney transplantation, J. Am. Soc. Nephrol. 26 (7) (2015) 1489–1502. [26] M. Schnaidt, C. Weinstock, M. Jurisic, B. Schmid-Horch, A. Ender, D. Wernet, HLA antibody specification using single-antigen beads–a technical solution for the prozone effect, Transplantation 92 (5) (2011) 510–515. [27] D.J. Triulzi, S. Kleinman, R.M. Kakaiya, M.P. Busch, P.J. Norris, W.R. Steele, et al., The effect of previous pregnancy and transfusion on HLA alloimmunization in blood donors: implications for a transfusion-related acute lung injury risk reduction strategy, Transfusion 49 (9) (2009) 1825–1835. [28] T.B. Dunn, H. Noreen, K. Gillingham, D. Maurer, O.G. Ozturk, T.L. Pruett, et al., Revisiting traditional risk factors for rejection and graft loss after kidney transplantation, Am. J. Transplant. 11 (10) (2011) 2132–2143. [29] V. Aubert, J.P. Venetz, G. Pantaleo, M. Pascual, Low levels of human leukocyte antigen donor-specific antibodies detected by solid phase assay before transplantation are frequently clinically irrelevant, Hum. Immunol. 70 (8) (2009) 580–583.
[5] C. Puttarajappa, R. Shapiro, H.P. Tan, Antibody-mediated rejection in kidney transplantation: a review, J. Transplant. 2012 (2012) 193724. [6] C. Lefaucheur, C. Antoine, C. Suberbielle, D. Glotz, Mastering the risk of HLA antibodies in kidney transplantation: an algorithm based on pretransplant single-antigen flow bead techniques, Am. J. Transplant. 11 (8) (2011) 1592–1598. [7] N. Singh, A. Djamali, D. Lorentzen, J.D. Pirsch, G. Leverson, N. Neidlinger, et al., Pretransplant donor-specific antibodies detected by single-antigen bead flow cytometry are associated with inferior kidney transplant outcomes, Transplantation 90 (10) (2010) 1079–1084. [8] C. Wiebe, I.W. Gibson, T.D. Blydt-Hansen, M. Karpinski, J. Ho, L.J. Storsley, et al., Evolution and clinical pathologic correlations of de novo donor-specific HLA antibody post kidney transplant, Am. J. Transplant. 12 (5) (2012) 1157–1167. [9] G. Roder, T. Blicher, S. Justesen, B. Johannesen, O. Kristensen, J. Kastrup, et al., Crystal structures of two peptide-HLA-B*1501 complexes; structural characterization of the HLA-B62 supertype, Acta Crystallogr. D Biol. Crystallogr. 62 (Pt 11) (2006) 1300–1310. [10] N. Guex, M.C. Peitsch, SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling, Electrophoresis 18 (15) (1997) 2714–2723. [11] The Allele Frequency Net Database. Accessed April 22, 2017, http:// allelefrequencies.net/hlaepitopes/hlaepitopes.asp?epitope_type=1&showall=1& epitope_name=80N&epitope_locus=&epitope_pos=&epitope_st_pos=&epitope_ end_pos=&hla_a_01=&hla_a_02=&hla_b_01=&hla_b_02=&hla_c_01=&hla_c_ 02=&hla_selection=&hla_pop_selection=&population=&pop_country=&pop_ region=&pop_ethnic=&pop_sample_size_pattern=equal&pop_sample_size=& epitope_order=order_1. [12] M. Crespo, S. Heidt, D. Redondo, J. Pascual, Monitoring B cell subsets and alloreactivity in kidney transplantation, Transplant. Rev. 29 (2) (2015) 45–52. [13] C.T. Lutz, A. Al-Attar, J.R. May, C.D. Jennings, Alloantibody to a Bw4 epitope in a Bw4+B*27: 05 patient, Transplantation 98 (8) (2014) 853–856. [14] S.M. Rizvi, N. Salam, J. Geng, Y. Qi, J.H. Bream, P. Duggal, et al., Distinct assembly profiles of HLA-B molecules, J. Immunol. 192 (11) (2014) 4967–4976. [15] HLA – Epitope Registry, Accessed February 9, 2017, http://www.epregistry.com. br/index/databases/database/ABC/. [16] R.J. Duquesnoy, M. Marrari, Correlations between Terasaki's HLA class I epitopes and HLAMatchmaker-defined eplets on HLA-A, -B and -C antigens, Tissue Antigens 74 (2) (2009) 117–133. [17] C.T. Lutz, Human leukocyte antigen Bw4 and Bw6 epitopes recognized by antibodies and natural killer cells, Curr. Opin. Organ Transplant 19 (4) (2014) 436–441.
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Human Immunology journal homepage: www.elsevier.com/locate/humimm
Accelerated humoral renal allograft rejection due to HLA-C14 mediated allosensitization to HLA-Bw6 ⁎
Stephen P. Persauda, , Brian Duffyb, Donna L. Phelanb, Thalachallour Mohanakumarc, ⁎ Rowena Delos Santosd, Joseph P. Gaute, Chang Liua, a
Division of Laboratory and Genomic Medicine, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA Department of Laboratories, Barnes-Jewish Hospital, St. Louis, MO 63110, USA c Norton Thoracic Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ 85013, USA d Division of Nephrology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA e Division of Anatomic and Molecular Pathology, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA b
A R T I C L E I N F O
A B S T R A C T
Keywords: HLA Alloimmunization Transplantation Kidney Crossmatch
Objectives: To investigate immunological mechanisms underlying accelerated antibody-mediated rejection (AMR) of a living-related renal allograft in a patient with no detectable antibodies to donor human leukocyte antigens (HLA) in pre-transplant sera. Methods: Pre- and post-transplant HLA antibody specificities were determined by single-antigen bead assay, and crossmatching was performed by flow cytometry- and complement-dependent cytotoxicity-based methods. Intermediate- and high-resolution HLA typing were performed by molecular methods. Results: Pre-transplant patient serum reacted weakly against Bw6-positive beads; cytotoxicity and flow crossmatches were negative. The patient was mismatched for the donor antigens B62 and C10 (Bw6-positive). Following transplantation, strong antibody responses against B62, C10, and all Bw6-positive beads were detected. This reactivity was initially masked by complement interference, but became apparent at 1:20 dilution. High-resolution typing suggested that the anti-C16 antibody reactivity detected was an allele-specific response to donor C∗16:01 (Bw6-positive) but not recipient C∗16:02 (Bw6-negative). Alloimmunization likely occurred during pregnancy, during which HLA-C14 (Bw6-positive) was the only mismatched paternal HLA Class I allele. Conclusions: Sensitization to HLA-Bw6 via exposure to paternal HLA-C14 during pregnancy likely predisposed this patient to AMR. The case demonstrates the immunogenicity of HLA-C14-associated Bw6 epitopes in vivo and the clinical significance of low-level antibodies to HLA-Bw6.
1. Introduction Due largely to improved immunosuppressive therapy in recent years, better short-term renal allograft survival and decreased rates of acute rejection have been reported [1]. However, long-term graft survival has proven less substantial than anticipated [2]. Antibody-mediated rejection (AMR) has been increasingly appreciated as a significant obstacle to improvement of long-term kidney transplantation outcomes [3]. While hyperacute rejection secondary to preformed donor specific antibodies (DSA) has become rare over recent years, acute and chronic AMR remain significant risk factors associated with allograft loss [4]. Antibodies targeting donor human leukocyte antigens (HLA) are of
particular concern in the pathogenesis of acute and chronic AMR. Binding of donor HLA in the allograft by DSA can lead to complementmediated and antibody-dependent cellular cytotoxicity, causing endothelial damage and thrombosis that compromises allograft function and survival [5]. Both pre-existing [6,7] and de novo [8] DSA antibodies have been associated with poorer long-term survival of renal allografts. For this reason, screening for anti-HLA in addition to crossmatching has become the standard of care in the evaluation of kidney transplant patients. Herein, we present a case of a multiparous woman with no prior history of transfusion or transplant who underwent living-related kidney transplantation. Pre-transplant antibody screening was
Abbreviations: AMR, antibody mediated rejection; AHG, anti-human globulin; ATG, anti-thymocyte globulin; CDCXM, complement-dependent cytotoxicity crossmatch; CMV, cytomegalovirus; DSA, donor specific antibody; DTT, dithiothreitol; EBV, Epstein-Barr Virus; EDTA, ethylenediaminetetraacetic acid; FITC, fluorescein isothiocyanate; FL, fluorescence channel; FCXM, flow cytometric crossmatch; HLA, human leukocyte antigen; MCS, median channel shift; MFI, median fluorescence intensity; PE, phycoerythrin; PerCP, peridinin chlorophyll protein; POD, post-operative day; PRA, panel reactive antibody ⁎ Corresponding authors at: 660 S. Euclid Ave, Campus Box 8118, St. Louis, MO 63110, USA. E-mail addresses: [email protected] (S.P. Persaud), [email protected] (C. Liu). http://dx.doi.org/10.1016/j.humimm.2017.09.004 Received 13 June 2017; Received in revised form 25 August 2017; Accepted 28 September 2017 0198-8859/ © 2017 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Persaud, S.P., Human Immunology (2017), http://dx.doi.org/10.1016/j.humimm.2017.09.004
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cell suspensions were analyzed using a FC500 flow cytometer (Beckman Coulter, Brea, CA). For analysis, lymphocytes were gated using forward and side scatter parameters followed by gating of CD3+ T cells (FL4 channel) and CD19+ B cells (FL2 channel). Within these subpopulations, bound human IgG was expressed on a logarithmic FL1 channel and expressed as the median channel number. The median channel shift (MCS) between patient samples and non-sensitized controls was obtained by subtraction of the median channel numbers; MCS cutoff values of 40 and 80 were used for T and B cells, respectively.
consistent with a weak antibody against the Bw6 epitope, but cytotoxicity and flow crossmatches were negative. The patient presented one week post-operatively with serologic and histopathological evidence of acute AMR with development of antibodies to HLA. We discuss the immunologic basis for antibody development leading to rapid antibody-mediated renal allograft rejection. 2. Methods 2.1. HLA typing
2.6. Protein structure image generation DNA was extracted from EDTA-anticoagulated whole blood using the EZ1 DNA Blood kit from Qiagen (Hilden, Germany). Intermediateresolution HLA typing was performed using the LabType SSO kit (One Lambda, Canoga Park, CA) per the manufacturer’s manual on the Luminex 200 instrument (Luminex, Austin, TX). High-resolution HLA typing was performed using the SeCore HLA sequencing kit (Life Technologies, Brown Deer, WI) per the manufacturer’s manual. Intermediate-resolution typing and serogroup assignment are performed in routine clinical practice at our center; high-resolution typing is performed for epitope identification.
The ribbon diagram of HLA-B∗15:01 (Protein Data Bank ID 1XR9) [9] was generated using Swiss-PdbViewer version 4.1 (www.expasy. org/spdbv) [10]. 2.7. Statistics The probability P of the allograft recipient being exposed to her husband’s HLA-C14 allele during pregnancy, given that her husband is heterozygous at the HLA-C locus, was calculated as per Formula (1) below:
2.2. Panel reactive antibody (PRA) assay
P (C14 exposure in utero)=1−(0. 5)n;
Patient sera was analyzed using the LABScreen PRA kit (One Lambda, Canoga Park, CA) per the manufacturer’s instructions using the Luminex platform (Luminex, Austin, TX). The cutoff for positivity was MFI 750 per the manufacturer’s recommendation.
(1)
where n is the number of pregnancies, and 0.5 reflects the probability the fetus inherits the paternal C14 allele. The frequencies of the 80 N eplet in various populations in the United States were obtained from the Allele Frequency Net Database [11], and were determined based on haplotype frequencies.
2.3. HLA antibody identification
3. Case report
Patient serum was analyzed for antibodies to HLA with or without dilution as indicated using the LABScreen single-antigen bead kit (One Lambda, Canoga Park, CA). The assay was performed using the Luminex platform (Luminex, Austin, TX) per the user’s manual except that 25 μL of beads was used per assay. The MFI cutoff for a positive assay was based on experience from our center showing that MFI values ranging from 2000 to 3000 predict positivity by complement-dependent cytotoxicity crossmatching; within this range, 2000 MFI was chosen as the cutoff to maximize sensitivity. Samples with results suspicious for interference by complement were retested with dilution at 1:20. We do not routinely monitor post-transplant DSA per protocol at our institution. However, DSA monitoring in selected high-risk cases (i.e., patient with positive pre-transplant virtual or flow crossmatch) can be initiated at the discretion of the treating physician.
A 36-year-old gravida 4 para 4 Caucasian female with a history of chronic kidney disease stage 4 secondary to reflux nephropathy diagnosed at age 5 presented for evaluation for kidney transplantation. Her past medical history was notable for hypertension, dyslipidemia, anemia and secondary hyperparathyroidism. She had no prior history of transplantation or blood transfusions. Living-related kidney transplant was planned with her brother as donor. The patient and donor were blood type O negative, and initial intermediate-resolution HLA typing identified mismatches at the B, C, DRB1 and DQB1 loci (Table 1). Panelreactive antibody assay was positive at 18% for Class I HLA and negative for Class II HLA. EBV serology was positive in both donor and patient and CMV serology positive only in the patient. The transplant procedure was complicated by significant blood loss, which required transfusion of 2 units of leukocyte-reduced red blood cells (RBCs) intraoperatively, but otherwise the patient tolerated the procedure well. For induction immunosuppression, the patient received
2.4. Complement-dependent cytotoxicity crossmatch (CDCXM) CDCXM was performed by the four-wash Amos technique, with and without anti-human globulin (AHG) augmentation. Donor cells were incubated with recipient sera for 40 min at 37 °C. AHG and rabbit complement were added and cells incubated for 1 h at 37 °C. Nonviable cells were labeled by ethidium bromide uptake. Cell death of greater than or equal to 20% above background was defined as a positive crossmatch.
Table 1 HLA typing results for kidney allograft recipient and select relatives. HLA Locus
2.5. Flow cytometric crossmatch (FCXM)
Subject
A
B
C
DRB1
DRB3/4/5
DQB1
Patient (recipient)
A2
B51
C2
DR11
DR52
DQ7
C16:02 (C16)* C10 C16:01 (C16)* C2
DR15
DR51
DQ6
DR4 DR15
DR53 DR51
DQ8 DQ6
DR1
DR52
DQ2
C14
DR17
A26
4 x 105 leukocytes from peripheral blood or lymph node were incubated with 40 μL patient serum for 30 min at 4 °C, and subsequently washed three times to remove unbound antibody. Lymphocytes were not treated with pronase. Samples were stained for 15 min at 4 °C with anti-CD3 PerCP (clone SK7; BD Biosciences, San Jose, CA) and CD19 PE (clone SJ25C1; BD Biosciences, San Jose, CA) to label T and B lymphocytes, respectively, and goat anti-human IgG F(ab′)2 FITC (polyclonal; Sigma Aldrich, St. Louis, MO) to detect cell-bound DSA. Stained
Brother (donor)
A2 A26
B51 B62
Husband of patient
A2
B51:01 (B51)* B51:07 (B51)*
* Serogroup assignment is listed in parentheses.
2
DQ5
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Fig. 1. Renal biopsy shows findings supporting a diagnosis of acute antibody mediated rejection. (A) Hemorrhagic cortical necrosis is widespread (H & E; 200x). (B & C) Glomerular and arteriolar fibrin thrombi are identified (Masson’s trichrome, 400× and 600×). (D) Immunohistochemistry for C4d shows diffuse linear peritubular capillary staining (200×).
consistent with a low-level antibody against Bw6, and six of the seven positive Class I antigen beads were coated with Bw6-associated antigens (Table 2). The median fluorescence intensity of the antibody was reduced at 1:20 dilution, although the pattern of reactivity was largely unaffected (Fig. 2). Consistently, no reactivity was detected against donor B- or T-lymphocytes by complement-dependent cytotoxicity crossmatch (CDCXM) using four-wash Amos or AHG methodologies, or flow cytometry crossmatch (FCXM). At the time of presentation with suspected graft rejection, however, HLA antibody screening demonstrated antibodies against 36 HLA Class I antigen beads (mean MFI 5005, range 2005-12,575), including DSA at low MFI against C10 (Table 2, Fig. 2). Serum dilution to 1:20 revealed strong reactivity against 59 HLA Class I antigen beads at markedly increased MFIs (mean MFI 15,130, range 2187–22,454). Strong DSA against the mismatched HLA Class I alleles, B62 and C10, were revealed by the serum dilution analysis, but not against the mismatched HLA Class II alleles (Fig. 3). The identification of DSA supported the diagnosis of acute AMR. Notably, the serum dilution analysis also unmasked a strong pattern of reactivity to all beads with Bw6-associated HLA-B and HLA-C antigens (C1, C7, C8, C9, C10, C12, C14 and C16), which were clustered together in a range of high MFI values > 15,000 (Fig. 2, Table 2). Given that the pre-transplant reactivity against mismatched donor antigens and Bw6 was much lower, the rapid increase in the strength of DSAs and the pace of the allograft rejection suggested an anamnestic response against HLA Class I rather than de novo sensitization. This implies an occult alloimmunization with grave clinical consequences that could not be reliably predicted or monitored in a timely manner.
75 mg anti-thymocyte globulin (ATG) and 450 mg methylprednisolone intraoperatively, then received 125 mg ATG once daily and 80 mg prednisone once daily on post-operative days (POD) 1 and 2. Prednisone was reduced to 20 mg daily on POD 3 and tapered thereafter. Mycophenolic acid was started at 720 mg twice daily and tacrolimus started at 2 mg twice daily on POD 1. Tacrolimus was subsequently increased to 5 mg twice daily in order to achieve therapeutic concentrations (7–10 ng/dL), at which point mycophenolic acid was reduced to 360 mg twice daily. Graft function was immediately satisfactory, with serum creatinine at POD 4 of 0.76 mg/dL, and so the patient was discharged to home. The patient presented again on POD 7 with pain at the allograft site and facial and upper body flushing. At this time, serum creatinine was 3.6 mg/dL, tacrolimus trough concentration was within the therapeutic range at 7.4 ng/mL, and a renal ultrasound suggested compromise of the renal artery supplying the allograft. Biopsy of the allograft showed diffuse hemorrhagic cortical necrosis, and glomerular, arteriolar and arterial thromboses (Fig. 1). C4d immunohistochemistry demonstrated diffuse peritubular capillary C4d positivity. These histopathologic findings strongly suggested an acute antibody-mediated rejection (AMR) and prompted removal of the allograft. The post-nephrectomy allograft specimen had a mottled blue gross appearance with petechial hemorrhages most apparent at the corticomedullary junction. Microscopically, extensive arteritis and vascular thrombosis were present along with diffuse cortical necrosis. These findings corroborated the initial diagnostic impression of acute AMR.
3.1. Laboratory investigation for AMR
3.2. Possible mechanism of alloimmunization
Based on the initial intermediate-resolution HLA typing, the donor antigens that were mismatched and thus could have been recognized as foreign by the recipient immune system included B62, C10, DR4 and DQ8. Pre-transplant HLA antibody screening revealed a pattern
HLA alloimmunization may occur as a consequence of transplantation, transfusion and pregnancy [12]. We investigated the likely 3
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Table 2 Summary of pre- and post-transplant HLA antibody screen results. Pre-transplant, no serum dilution
Post-transplant, no serum dilution
Post-transplant, 1:20 diluted serum
HLA-A Positive beads (%) MFI Mean ± SD of positive beads MFI range of positive beads
0/31 (0%) 0 0
8/31 (26%) 3190 ± 2103 2005-8344
8/31 (26%) 4546 ± 1774 2187-7583
HLA-Bw4 Positive beads (%) MFI Mean ± SD of positive beads MFI range of positive beads
1/19 (5%) 2414 2414
11/19 (58%) 5491 ± 2684 2071-9103
9/19 (47%) 8793 ± 4076 3396-16229
HLA-Bw6 Positive beads (%) MFI Mean ± SD of positive beads MFI range of positive beads
6/31 (19%) 2855 ± 739 2011-4018
7/31 (23%) 3425 ± 1469 2256-6514
31/31 (100%) 18787 ± 1440 15116-21992
HLA-C Positive beads (%) MFI Mean ± SD of positive beads MFI range of positive beads
0/16 (0%) 0 0
10/16 (63%) 6339 ± 3881 2242-12575
11/16 (69%) 17603 ± 7504 2503-22454
All beads Positive beads (%) MFI Mean ± SD of positive beads MFI range of positive beads
7/97 (7%) 2792 ± 695 2011-4018
36/97 (37%) 5005 ± 3101 2005-12575
59/97 (61%) 15130 ± 6532 2187-22454
minimal number of leukocytes in filtered RBC units, the short time between transfusion and rejection, and the magnitude of the antibody response. However, while de novo immunization by these RBC units was
sensitizing event that initially primed the patient’s immune system to mount such a destructive memory response. The intraoperative RBC transfusions were unlikely the initial immunizing event given the
Fig. 2. Post-transplant Luminex HLA antibody assay performed using diluted recipient serum reveals high-MFI reactivity against all Bw6-positve beads. Histograms show antibody screen results for the pre-transplant serum without dilution (A), pre-transplant serum with 1:20 dilution (B), post-transplant serum without dilution (C), and post-transplant serum with 1:20 dilution. The y-axis shows the MFI values of single antigen beads to which patient serum reacted, with the identity of the antigen listed below each bar. Bw6-associated antigens are circled. Bold horizontal bars indicate the 2000 MFI cutoff for positivity of the assay. All samples were analyzed during a single run that was performed post-transplant, and the pretransplant data shown are repeats of the original assay using the same sample. Please note that in these repeats, reactivity to only one Bw6-associated antigen was identified in the pretransplant serum as opposed to the 6 antigens as detected in the original assay. The overall pattern of reactivity between the original assay and the repeat is otherwise unchanged.
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Fig. 3. Dilution of recipient post-transplant serum in HLA antibody screen demonstrates strong DSA against B62 and C10. Plots show the relationship between serum dilution and median fluorescence intensity (MFI) for each of the specified donor antigens. Notably, dilution of the pretransplant serum did not reveal positivity for DSA.
containing this epitope could be recognized as non-self during pregnancy, leading to allosensitization and a potent memory antibody response upon exposure to the Bw6-positive allograft.
unlikely, it remained possible that residual leukocytes could bear antigen contributing to the rejection response. In the absence of prior transplantation or transfusion, the patient’s multiple pregnancies were most likely the cause of immunization against allogeneic HLA. We therefore examined the HLA antigens expressed by her husband, to which she may have responded during her pregnancies. HLA typing of the patient’s husband demonstrated a single HLA Class I mismatch at HLA-C, with the patient typing as HLA-C16 and her husband as HLA-C14 (Table 1). How, then, could reactivity to HLA-C14 during pregnancy prime the patient’s immune system to respond to B62 and C10 after transplantation? The weak reactivity to several HLA Bw6associated antigens pre-transplant, taken together with the strong posttransplant reactivity against all Luminex beads with HLA-Bw6 antigens, suggested a mechanism whereby recognition of this common public epitope led to cross-reactivity to distinct HLA antigens encountered during pregnancy and transplantation. The Bw6 epitope is comprised of solvent-exposed amino acid residues spanning positions 77-83 within the α1-helix of most HLA-B and some HLA-C heavy chains, including HLA-C14 (Fig. 4A) [13,14]. The Bw6 peptide contains the 80 N epitope (also referred to as eplet 79RN and TerEp #25), as defined by the HLA Epitope Database [15,16]. Amino acid differences at positions 77 and 80-83 distinguish the Bw6 epitope from the mutually exclusive Bw4 epitope (Fig. 4B) [17]. As the Bw6 epitope is present in the HLA-C14 antigen expressed by the patient’s husband as well as the B62 and C10 antigens expressed by the donor, this epitope could have primed and restimulated antibody responses during pregnancy and transplantation, respectively. This epitope is present in all HLA-C antigen beads to which the patient’s serum reacted strongly (C1, C7, C8, C9, C10, C12, C14, and C16) and absent in the HLA-C antigen beads to which her serum reacted weakly (C2, C4, C5, C6, C15, C17, and C18). The latter group of HLA-C antigens have two amino acid substitutions at positions 77 (Ser to Asn) and 80 (Asn to Lys), and thus do not bear the Bw6 epitope (Fig. 4B). An unexplained issue thus far is that both the patient and the donor were typed as positive for HLA-C16, yet her antibody response included strong reactivity to the C∗16:01 Luminex bead. Reactivity to C16 would be unexpected in this patient if it were a self-antigen. To resolve this issue, high-resolution typing was performed, which demonstrated that the patient expresses the HLA-C∗16:02 allele, whereas her brother expresses HLA-C∗16:01. While both alleles are assigned to the C16 serogroup, C∗16:01 contains a canonical Bw6 epitope whereas C∗16:02 does not. Notably, C∗16:02 differs by the same amino acid substitutions at positions 77 and 80 as the other HLA-C antigens to which the patient weakly responded. The absence of the Bw6 epitope in the patient’s HLA-B and C alleles suggests that the paternal HLA-C14 allele
4. Discussion Herein, we have described a kidney transplant recipient who became alloimmunized against the Bw6 epitope upon encountering paternal HLA-C14 antigens during pregnancy; this set the stage for a rapid anamnestic response upon exposure to the Bw6 epitope in the renal allograft, ultimately leading to AMR and graft loss. Our hypothesis that the recognition of a common epitope of a HLA-C allele could lead to broad recognition of HLA-B alleles is consistent with the findings of several previous reports [18–20]. Notably in our case, high-resolution typing demonstrated the patient expressed an HLA-C16 allele that lacks the Bw6 epitope, which accounted for her susceptibility to Bw6 immunization via exposure to paternal HLA-C14. While the children of this patient were not available for HLA typing to confirm the patient’s exposure to HLA-C14 during her pregnancies, the high probability of encountering HLA-C14 during pregnancy (approximately 94% over four pregnancies, given that the patient’s husband was heterozygous for HLA-C14) and the absence of prior transplantation or transfusion makes this the most likely mechanism by which the patient was initially sensitized. Although HLA-C is considered a low-expression locus, the expression level of HLA-C14 was reported to be the highest among selected HLA-C antigens examined by flow cytometry [21]. This case provided evidence supporting the immunogenicity of the HLA-C14 antigen in vivo. Given the high prevalence of Bw6-associated antigens, it remains possible that Bw6 epitope-positive leukocytes in the two RBC units transfused intraoperatively were an additional source of antigen that could have contributed to the patient’s anamnestic response and rejection. As HLA typing results for the two RBC units given to the patient were not available, this possibility cannot be completely excluded. Although leukoreduced RBC units remain a strong risk factor for alloimmunization in renal transplant patients [22,23], the pattern consistent with a weak anti-Bw6 was present in this case before the patient received her intraoperative RBC transfusions. A recent study described two antibodies with specificities to the 80NRG eplet (Bw6-associated) derived from female patients sensitized to paternal HLA antigens during pregnancy [24]. For both antibodies, HLA-B antigens containing the Bw6 epitope (B∗07:02 and B∗08:01) were implicated as the most likely immunizing agents. Similar to our study, pan-reactivity to Bw6-associated antigens was observed, and the patients who produced these antibodies expressed HLA antigens 5
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Fig. 4. The Bw6 motif is a linear epitope present in most HLA-B and several HLA-C antigens. (A) Ribbon diagram of the HLA-B*15:01 (serogroup B62) peptide binding pocket, comprised of the α1 and α2 domains of the Class I HLA-B heavy chain (Protein Data Bank ID 1XR9). The Bw6 motif (residues 77–83, dark gray) lies within the C-terminal end of the α1 helix; residues within this motif are labeled and their sidechains displayed in stick format. (B) Alignment of the amino acid sequence for the Bw4 and Bw6 motifs and select HLA-C antigens at positions 77 through 83. Residues differing from the Bw6 motif are underlined.
(C∗02:02 and C∗06:02) lacking the 80NRG eplet. This supports the notion that patients whose HLA antigens have these amino acid substitutions are susceptible to immunization against the Bw6 epitope. As the 80 N eplet residing within the Bw6 epitope is highly prevalent in several diverse populations in the United States (93-99%) [11], sensitization to this epitope represents a significant barrier to identifying compatible grafts for transplantation. Thus, accurate interpretation of the antibody pattern is essential. Although solid-phase assays have greatly improved the sensitivity and specificity of HLA antibody detection [25], our case illustrates two pertinent issues related to the use of these assays in the laboratory detection of relevant HLA antibodies and risk assessment for AMR in transplant patients. First, the broad, strong reactivity to HLA Class I demonstrated on the post-transplant HLA antibody screen was an important distinguishing feature of the patient’s aggressive graft rejection course, yet the magnitude of this reactivity was only evident upon serum dilution. Such a prozone-like effect leading to false negative results has been described previously for solid-phase, single-antigen bead assays, and has been attributed to interference with the antigen beads by proteins of the complement cascade, particularly C1 [26]. In addition to serum dilution, mitigation of this effect has been demonstrated by treatments that destabilize the C1 complement complex, such as heat inactivation to 56 °C, and treatment with the divalent cation chelator EDTA or the disulfide-reducing dithiothreitol (DTT). Second, the patient’s pre-transplant serum was positive for a few low-MFI, non-DSA Class I antibodies to HLA-B. However, analysis of all single antigen beads (Fig. 2) demonstrated a broader anti-Bw6 reactivity, involving many Bw6 antigen beads with MFI below the positive cutoff. Thus, assessment of allosensitization in transplant candidates requires consideration of not only those single antigen beads above a certain MFI threshold, but rather the overall pattern of reactivity. In this case, the anti-Bw6 reactivity in the pre-transplant patient serum was relatively weak, given the pattern on dilution study and negative crossmatches. In a patient with truly low-MFI anti-Bw6 reactivity, it remains difficult to predict the probability of a fulminant memory response seen in this case. This theoretical risk would have to be weighed against the high cPRA associated with listing Bw6 as an unacceptable group. A study of HLA alloimmunization in potential blood donors demonstrated nearly a quarter of women with a history of pregnancy had antibodies to HLA [27], with a correlation between the prevalence of alloimmunization and the number of prior pregnancies. Such a high seroprevalence of anti-HLA would suggest that the opportunity for cross-reactivity to public epitopes leading to AMR would be a common occurrence among multiparous patients. However, the majority of sensitized kidney transplant recipients in one study, even those with DSA, did not experience AMR at 5 years post-transplant [28]; notably,
though, patients sensitized against non-DSA had lower rates of 5-year graft rejection than DSA-sensitized patients. Another study demonstrated that among DSA-positive kidney transplant recipients, the DSA MFI for patients with and without acute rejection overlapped within a low range of less than 6000 [29]. These results suggest that even with improvement of methods to detect antibodies to HLA, their interpretation remains controversial. 5. Conclusions We investigated a case of rapidly progressive antibody-mediated renal allograft rejection in a patient with a weak, crossmatch-negative antibody against Bw6. Based on the workup performed by the histocompatibility laboratory at our institution, we concluded that recognition of the public Bw6 epitope in the paternal HLA-C14 allele during pregnancy most likely primed the patient’s immune system against this epitope. This led to rapid renal allograft rejection upon exposure to donor HLA alleles also carrying the Bw6 epitope. This case highlights the immunogenicity of C14-associated Bw6 epitopes in vivo and the clinical significance of low-level anti-Bw6 antibodies in the setting of a negative crossmatch. Whether or not to cross the transplantation barrier imposed by Bw6 in a situation like this remains a personalized decision. For cases in which this barrier is crossed, an important pursuit for future investigation will be to better characterize factors that influence whether a sensitized individual will mount an alloantibody response that leads to AMR. Sources of funding None Disclaimers None References [1] H.U. Meier-Kriesche, J.D. Schold, T.R. Srinivas, B. Kaplan, Lack of improvement in renal allograft survival despite a marked decrease in acute rejection rates over the most recent era, Am. J. Transplant. 4 (3) (2004) 378–383. [2] S.A. Lodhi, K.E. Lamb, H.U. Meier-Kriesche, Solid organ allograft survival improvement in the United States: the long-term does not mirror the dramatic shortterm success, Am. J. Transplant. 11 (6) (2011) 1226–1235. [3] A. Djamali, D.B. Kaufman, T.M. Ellis, W. Zhong, A. Matas, M. Samaniego, Diagnosis and management of antibody-mediated rejection: current status and novel approaches, Am. J. Transplant. 14 (2) (2014) 255–271. [4] J. Sellares, D.G. de Freitas, M. Mengel, J. Reeve, G. Einecke, B. Sis, et al., Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonadherence, Am. J. Transplant. 12 (2) (2012) 388–399.
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