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Adverse effect of donor-specific anti-human leukocyte antigen (HLA) antibodies directed at HLA-DP/-DQ on engraftment in cord blood transplantation

Open AccessPublished:November 02, 2022DOI:https://doi.org/10.1016/j.jcyt.2022.10.005

      Abstract

      Background aims

      While donor-specific anti-human leukocyte antigen (HLA) antibodies (DSAs) in the recipient before transplantation are associated with graft failure in cord-blood transplantation (CBT), effects of DSAs other than against HLA-A, -B or -DRB1 on transplantation outcomes remained poorly understood.

      Methods

      We retrospectively analyzed 567 single-unit CBT recipients to evaluate impact of DSAs against HLA-DP and -DQ on CBT outcomes.

      Results

      Among 143 recipients (25.2%) who had anti-HLA antibodies, nine harbored DSAs against HLA-DP or -DQ. DSAs against HLA-DP or -DQ were associated with a significantly lower neutrophil engraftment rate (55.6% versus 91.8%, P = 0.032) and with a marginally lower platelet engraftment rate (46.7% versus 75.3%, P = 0.128) at day 100 after transplantation, compared with patients without anti-HLA antibodies. Time to neutrophil and platelet engraftment in patients with DSAs for HLA-DP or -DQ was significantly longer than that in patients without anti-HLA antibodies (median, 25 versus 21 days, P = 0.002 in neutrophil; median 61 versus 46 days, P = 0.014 in platelet). Cumulative incidence of bacterial infection at day 100 was significantly greater (88.9% versus 57.1%, P = 0.024), and re-transplant-free survival was marginally lower (55.6% versus 76.8%, P = 0.132) in patients with DSAs against HLA-DP or -DQ, compared with those without anti-HLA antibodies. These findings suggest that DSAs against HLA-DP or -DQ lead to unfavorable engraftment, which may increase risk of bacterial infection, and reduce survival soon after CBT.

      Conclusions

      Our results suggest the importance of evaluating DSAs against HLA-DP and -DQ in recipients before selecting CB units.

      Key Words

      Introduction

      Cord blood transplantation (CBT) has become a valuable alternative graft source for patients who require allogeneic stem cell transplantation but who do not have appropriate related or human leukocyte antigen (HLA)-matched, unrelated donors [
      • Brunstein CG
      • Setubal DC
      • Wagner JE.
      Expanding the role of umbilical cord blood transplantation.
      ]. Graft failure or engraftment delay is one of the major concerns after CBT, leading to early non-relapse mortality [
      • Eapen M
      • Rocha V
      • Sanz G
      • et al.
      Effect of graft source on unrelated donor haemopoietic stem-cell transplantation in adults with acute leukaemia: a retrospective analysis.
      ,
      • Laughlin MJ
      • Eapen M
      • Rubinstein P
      • et al.
      Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukemia.
      ,
      • Takahashi S
      • Ooi J
      • Tomonari A
      • et al.
      Comparative single-institute analysis of cord blood transplantation from unrelated donors with bone marrow or peripheral blood stem-cell transplants from related donors in adult patients with hematologic malignancies after myeloablative conditioning regimen.
      ]. Re-transplantation is often required in patients with graft failure, and overall survival (OS) of these patients is poor [
      • Rondon G
      • Saliba RM
      • Khouri I
      • et al.
      Long-term follow-up of patients who experienced graft failure postallogeneic progenitor cell transplantation. Results of a single institution analysis.
      ]. Therefore, it is essential to reduce the incidence of graft failure.
      Donor-specific anti-HLA antibodies (DSAs) in the recipient are associated with graft failure in allogeneic hematopoietic stem cell transplantation [
      • Spellman S
      • Bray R
      • Rosen-Bronson S
      • et al.
      The detection of donor-directed, HLA-specific alloantibodies in recipients of unrelated hematopoietic cell transplantation is predictive of graft failure.
      ,
      • Takanashi M
      • Fujiwara K
      • Tanaka H
      • Satake M
      • Nakajima K.
      The impact of HLA antibodies on engraftment of unrelated cord blood transplants.
      ,
      • Yoshihara S
      • Maruya E
      • Taniguchi K
      • et al.
      Risk and prevention of graft failure in patients with preexisting donor-specific HLA antibodies undergoing unmanipulated haploidentical SCT.
      ]. In the setting of CBT, the negative impact of DSAs on engraftment appears significant because the majority of patients receive HLA-mismatched units with relatively lower cell counts, compared with those of bone marrow or peripheral-blood stem cells [
      • Ciurea SO
      • Thall PF
      • Wang X
      • et al.
      Donor-specific anti-HLA Abs and graft failure in matched unrelated donor hematopoietic stem cell transplantation.
      ,
      • Ruggeri A
      • Rocha V
      • Masson E
      • et al.
      Impact of donor-specific anti-HLA antibodies on graft failure and survival after reduced intensity conditioning-unrelated cord blood transplantation: a Eurocord, Societe Francophone d'Histocompatibilite et d'Immunogenetique (SFHI) and Societe Francaise de Greffe de Moelle et de Therapie Cellulaire (SFGM-TC) analysis.
      ]. Most earlier studies regarding effects of anti-HLA antibodies on engraftment focused on anti-HLA-A, -B and -DRB1 antibodies in the recipient [
      • Takahashi S
      • Ooi J
      • Tomonari A
      • et al.
      Comparative single-institute analysis of cord blood transplantation from unrelated donors with bone marrow or peripheral blood stem-cell transplants from related donors in adult patients with hematologic malignancies after myeloablative conditioning regimen.
      ,
      • Fuji S
      • Oshima K
      • Ohashi K
      • et al.
      Impact of pretransplant donor-specific anti-HLA antibodies on cord blood transplantation on behalf of the Transplant Complications Working Group of Japan Society for Hematopoietic Cell Transplantation.
      ]. Among DSAs other than against HLA-A, -B or DRB1, DSAs against HLA-DP or -DQ potentially impact engraftment, because HLA-DP and -DQ antigens are expressed on hematopoietic precursor cells [
      • Piacibello W
      • Aglietta M
      • Stacchini A
      • et al.
      Expression of HLA class II determinants by normal and chronic myeloid leukemia progenitors.
      ,
      • Sparrow RL
      • Williams N.
      The pattern of HLA-DR and HLA-DQ antigen expression on clonable subpopulations of human myeloid progenitor cells.
      ]. However, data on effects of DSAs against HLA-DP or -DQ on engraftment after CBT are limited [
      • Ruggeri A
      • Rocha V
      • Masson E
      • et al.
      Impact of donor-specific anti-HLA antibodies on graft failure and survival after reduced intensity conditioning-unrelated cord blood transplantation: a Eurocord, Societe Francophone d'Histocompatibilite et d'Immunogenetique (SFHI) and Societe Francaise de Greffe de Moelle et de Therapie Cellulaire (SFGM-TC) analysis.
      ,
      • Yamamoto H
      • Uchida N
      • Matsuno N
      • et al.
      Anti-HLA antibodies other than against HLA-A, -B, -DRB1 adversely affect engraftment and nonrelapse mortality in HLA-mismatched single cord blood transplantation: possible implications of unrecognized donor-specific antibodies.
      ]. Moreover, given that the disparity in HLA-DP or -DQ between recipients and donors reportedly affects post-transplant outcomes in CBT [
      • Flomenberg N
      • Baxter-Lowe LA
      • Confer D
      • et al.
      Impact of HLA class I and class II high-resolution matching on outcomes of unrelated donor bone marrow transplantation: HLA-C mismatching is associated with a strong adverse effect on transplantation outcome.
      ,
      • Morishima Y
      • Yabe T
      • Matsuo K
      • et al.
      Effects of HLA allele and killer immunoglobulin-like receptor ligand matching on clinical outcome in leukemia patients undergoing transplantation with T-cell-replete marrow from an unrelated donor.
      , the significance of DSAs against these antigens in HLA-mismatched CBT should be determined.
      Therefore, we performed a retrospective cohort study to address the impact of DSAs against HLA-DP and DQ in recipients of CBT on transplantation outcomes, focusing on hematological recovery. This study collected samples of 567 recipient–cord blood (CB) pairs in Japan; typed 16 alleles in the HLA-A, -B, -C, -DRB1, -DPA1, DPB1, -DQA1 and -DQB1 loci and assessed presence of DSAs against each locus to determine effects of DSAs against HLA-DP and -DQ. Our findings provide valuable information on the CB selection algorithm in CBT and should help to improve transplantation outcomes.

      Patients and Methods

      Patients and CBT

      Data on adult patients (age ≥16 years) who underwent their first CBT using CB units provided by the Japanese Red Cross Kinki Cord Blood Bank between 2012 and 2015 were obtained through the Transplant Registry Unified Management Program sponsored by the Japanese Society for Transplantation and Cellular Therapy [
      • Atsuta Y
      • Suzuki R
      • Yoshimi A
      • et al.
      Unification of hematopoietic stem cell transplantation registries in Japan and establishment of the TRUMP System.
      ,
      • Kanda J.
      Scripts for TRUMP data analyses. Part II (HLA-related data): statistical analyses specific for hematopoietic stem cell transplantation.
      ]. Patients who received multiple CB units, lacked data on survival or died within 30 days after transplantation were excluded. This study was approved by the Institutional Review Board of the Japanese Red Cross Kinki Cord Blood Bank and Kyoto University Hospital and was conducted in accordance with the Declaration of Helsinki.

      CB units and anti-HLA antibodies

      CB units were obtained from the Japanese Red Cross Kinki Cord Blood Bank, one of the largest providers of CB units for CBT among the seven public CB banks in Japan. Before selection as a donor source for transplantation, CB units were serologically typed at the HLA-A, -B and DRB1 loci. Anti-HLA antibodies against HLA-A, -B and DRB1 in recipients were screened before transplantation. CD34+ cells were measured using flow cytometry with single-platform assays [
      • Keeney M
      • Chin-Yee I
      • Weir K
      • Popma J
      • Nayar R
      • Sutherland DR.
      Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines. International Society of Hematotherapy and Graft Engineering.
      ]. In principle, CB units were selected from those with no more than two antigen mismatches to recipients, and CB units that contained ≥2 × 107 total nucleated cell (TNC) counts per kilogram of recipient body weights were prioritized for transplantation. For DSAs, CB units with DSAs against HLA-A, -B or DRB1 were not selected in principle, and in exceptional cases with DSAs against these loci, efforts were made to reduce the DSA titers [
      • Ciurea SO
      • Al Malki MM
      • Kongtim P
      • et al.
      Treatment of allosensitized patients receiving allogeneic transplantation.
      ,
      • Yamashita T
      • Ikegame K
      • Kojima H
      • et al.
      Effective desensitization of donor-specific HLA antibodies using platelet transfusion bearing targeted HLA in a case of HLA-mismatched allogeneic stem cell transplantation.
      ].
      After transplantation, we retrospectively tested for HLA-A, -B, -C, -DRB1, -DPA1, -DPB1, -DQA1 and -DQB1 alleles of recipients and CB units and assessed DSAs in recipients. As a result, information on DSAs against HLA-DP or -DQ was unavailable before transplantation. HLA allele typing was performed using polymerase chain reaction sequence-specific oligonucleotide probes using WAKFlow HLA-typing kits (WAKUNAGA Pharmacy Co. Ltd, Hiroshima, Japan). HLA alleles were assigned automatically using WAKFlow Typing software (WAKUNAGA Pharmacy Co. Ltd, Hiroshima, Japan). If ambiguity remained after polymerase chain reaction sequence-specific oligonucleotide probe method, we confirmed the alleles by next-generation sequencing-based HLA typing (Scisco Genetics, Inc., Seattle, WA, USA). Anti-HLA antibodies were tested using LABScreen PRA and LABScreen Single Antigen (One Lambda, Canoga Park, CA, USA) for class I (HLA-A, -B and -C) and class II (HLA-DRB1, -DPA1, -DPB1, -DQA1 and -DQB1) [
      • Takanashi M
      • Fujiwara K
      • Tanaka H
      • Satake M
      • Nakajima K.
      The impact of HLA antibodies on engraftment of unrelated cord blood transplants.
      ]. Median fluorescence intensity (MFI) of anti-HLA antibody against the HLA allele of each recipient was checked, and an MFI of ≥1000 was defined as positive, as previously reported [
      • Fuji S
      • Oshima K
      • Ohashi K
      • et al.
      Impact of pretransplant donor-specific anti-HLA antibodies on cord blood transplantation on behalf of the Transplant Complications Working Group of Japan Society for Hematopoietic Cell Transplantation.
      ,
      • Yamamoto H
      • Uchida N
      • Matsuno N
      • et al.
      Anti-HLA antibodies other than against HLA-A, -B, -DRB1 adversely affect engraftment and nonrelapse mortality in HLA-mismatched single cord blood transplantation: possible implications of unrecognized donor-specific antibodies.
      ,
      • Takanashi M
      • Atsuta Y
      • Fujiwara K
      • et al.
      The impact of anti-HLA antibodies on unrelated cord blood transplantations.
      ].

      Definitions and statistical analysis

      Neutrophil and platelet engraftment were defined as the first three consecutive measures with a neutrophil count ≥0.5 × 109/L and a platelet count ≥50 × 109/L, without platelet transfusion after transplantation. Viral infection included infections with cytomegalovirus, Epstein–Barr virus and human herpesvirus 6, and bacterial infection included all bacterial infections. Fungal infection included candidemia, proven, probable or possible aspergillosis with previously reported criteria [
      • De Pauw B
      • Walsh TJ
      • Donnelly JP
      • et al.
      Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group.
      ], and other proven fungal infections. OS was defined as time from transplantation to death or last contact, where death of any cause was considered an event and re-transplantation was treated as a censor. Re-transplant-free survival was defined as time from transplantation to either re-transplantation or death due to any cause [
      • Bosch DE
      • Swanson PE
      • Yeh MM.
      Centrizonal hepatocyte dropout in allograft liver biopsies: a clinicopathological study.
      ]. Categorical variables and continuous variables were compared between groups with the Fisher exact test and two-tailed, unpaired Student t-test, respectively. Probabilities of engraftment and infection were estimated using the 1 – Kaplan–Meier method, and groups were compared using the log-rank test. In multivariate analysis, effects of DSAs on transplantation outcomes were adjusted by cell doses using the Cox proportional-hazards model, or the Fine–Gray proportional-hazards model. All tests were two-sided, and P-values of <0.05 were considered statistically significant. All analyses were performed with Stata version 17 software (Stata Corp., College Station, TX, USA).

      Results

      Patient characteristics

      A total of 567 patients were analyzed (Table 1). Median patient age was 54 years (range 16–80 years). Among them, 59.3% were male, and 45.1% had acute myeloid leukemia or myelodysplastic syndrome. Most patients had a previous history of transfusion, both of red blood cell and platelet concentrate. Conditioning regimens were composed of myeloablative conditioning in 50.4%, and total body irradiation was used in 79.0%. For graft-versus-host-disease prophylaxis, 59.2% received methotrexate-containing regimens and 30.7% received mycophenolate mofetil–containing regimens. The median TNC and CD34+ cells infused were 2.78 × 107/kg and 0.76 × 105/kg, respectively. HLA–antigen mismatch at HLA-A, -B and DR in host-versus-graft direction was 0/6 (9.5%), 1/6 (33.0%) and 2/6 (56.8%).
      Table 1Patient characteristics of the entire study population (N = 567).
      N = 567
      Age, y
       Median (range)54 (16–80)
      Sex
       Female / male231 (40.7%) / 336 (59.3%)
      PS
       0-1 / ≥2487 (85.9%) / 77 (13.6%)
      HCT-CI
       ≤2 / ≥3462 (81.5%) / 98 (17.3%)
      Disease
       AML/MDS256 (45.1%)
       ALL91 (16.0%)
       ATL34 (6.0%)
       CML16 (2.8%)
       NHL65 (11.5%)
       AA7 (1.2%)
       Other98 (17.3%)
      Disease risk
       Standard / high177 (31.2%) / 390 (68.8%)
      Pre-transplant therapy period, mo
       Median (range)6.9 (0.1-487.7)
      Previous history of RBC transfusion, number
       <20 / ≥20310 (54.7%) / 181 (31.9%)
      Previous history of PC transfusion, number
       <20 / ≥20262 (46.2%) / 229 (40.4%)
      HLA antigen mismatch HVG direction
       0 / 1 / 254 (9.5%) / 187 (33.0%) / 322 (56.8%)
      HLA antibody
       Negative424 (74.8%)
       Positive and DSA negative123 (21.7%)
       Positive and DSA positive20 (3.5%)
      Sex mismatch
       No / M to F / F to M246 (43.4%) / 102 (18.0%) / 132 (23.3%)
      ABO mismatch
       No186 (32.8%)
       Minor / major / both162 (28.6%) / 133 (23.5%) / 85 (15.0%)
      Conditioning regimens
       MAC / RIC286 (50.4%) / 280 (49.4%)
      TBI
       No / yes119 (21.0%) / 448 (79.0%)
      GVHD prophylaxis
       CyA + sMTX142 (25.0%)
       CyA + MMF18 (3.2%)
       Tac + sMTX194 (34.2%)
       Tac + MMF156 (27.5%)
       Other51 (9.0%)
      Number of TNC infused, 107/kg
       Median2.78
      Number of CD34+ cells infused, 105/kg
       Median0.76
      Follow-up period, mo
       Median (range)51.6 (1.5-85.4)
      AA, aplastic anemia; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ATL, adult T-cell leukemia/lymphoma; CML, chronic myeloid leukemia; CyA, cyclosporine A; DSA, donor-specific anti-HLA antibody; F, female; GVHD, graft-versus-host disease; HCT-CI, hematopoietic cell transplantation-specific comorbidity index; HLA, human leukocyte antigen; HVG, host-versus-graft; M, male; MAC, myeloablative conditioning; MDS, myelodysplastic syndrome; MMF, mycophenolate mofetil; NHL, non-Hodgkin lymphoma; PC; platelet concentrate; PS, Eastern Cooperative Oncology Group Performance Status Scale; RBC, red blood cell; RIC, reduced-intensity conditioning; sMTX, short methotrexate; Tac, tacrolimus; TBI, total body irradiation; TNC, total nucleated cells.

      Anti-HLA antibodies

      In total, 143 of 567 patients (25.2%) were positive (Ab-positive group), and the remaining 424 patients (74.8%) were negative (Ab-negative group) for anti-HLA antibodies (Table 2). Among the Ab-positive group, 20 patients had DSAs (DSA-positive group), whereas the remaining 123 patients did not (DSA-negative group). The DSA-positive group included a significantly larger number of female recipients than the DSA-negative group (95.0% versus 52.0%, P < 0.001). Other patient characteristics were comparable between DSA-positive and -negative recipients. Patient characteristics and transplantation outcomes after CBT among patients with DSAs are summarized in supplementary Table 1. Among 20 DSA-positive patients, nine had DSAs against HLA-DP or -DQ (DSA for DP/DQ group), and the remaining 11 patients had DSAs for HLA-A, -B or -DRB1 without DSAs for HLA-DP or -DQ (other DSA group). Those with DSAs for DP/DQ included DSAs against HLA-DPB1 (n = 5) or -DQB1 (n = 4). There were no patients with DSAs against HLA-DPA1 or -DQA1. Of nine patients with DSAs against HLA-DP/DQ, only one had other DSAs. In the remaining eight cases (1.4% of the total cohort), DSAs other than DP/DQ was not detected. The median value of MFI was 9811 (range, 1603–23 306) among DSA for DP/DQ patients, and 2929 (range, 1196–8693) among other patients with DSA, respectively.
      Table 2Characteristics of HLA antibody-positive patients with/without DSAs.
      Ab positive, totalDSA negativeDSA positiveP value
      N = 143N = 123N = 20
      Age, y0.806
       Median545453
       Range21-7221-7233-65
      Sex<0.001
      P < 0.05.
       Female83 (58.0%)64 (52.0%)19 (95.0%)
       Male60 (42.0%)59 (48.0%)1 (5.0%)
      PS0.755
       0-1117 (81.8%)101 (82.1%)16 (80.0%)
       ≥225 (17.5%)21 (17.1%)4 (20.0%)
      HCT-CI1.000
       ≤2110 (76.9%)94 (76.4%)16 (80.0%)
       ≥331 (21.7%)27 (22.0%)4 (20.0%)
      Disease0.344
       AML/MDS66 (46.2%)60 (48.8%)6 (30.0%)
       ALL18 (12.6%)14 (11.4%)4 (20.0%)
       ATL5 (3.5%)5 (4.1%)0 (0.0%)
       CML5 (3.5%)4 (3.3%)1 (5.0%)
       NHL10 (7.0%)7 (5.7%)3 (15.0%)
       AA2 (1.4%)2 (1.6%)0 (0.0%)
       Other37 (25.9%)31 (25.2%)6 (30.0%)
      Disease risk0.405
       Standard36 (25.2%)33 (26.8%)3 (15.0%)
       High107 (74.8%)90 (73.2%)17 (85.0%)
      Pre-transplant therapy period, mo0.804
       Median5.96.24.9
       Range1.1–231.91.1–231.91.4–122.2
      Previous history of RBC transfusion, units0.612
       <2072 (50.3%)60 (48.8%)12 (60.0%)
       ≥2047 (32.9%)41 (33.3%)6 (30.0%)
      Previous history of PC transfusion, units0.802
       <2062 (43.4%)52 (42.3%)10 (50.0%)
       ≥2057 (39.9%)49 (39.8%)8 (40.0%)
      HLA antigen mismatch HVG direction0.813
       015 (10.5%)14 (11.4%)1 (5.0%)
       158 (40.6%)50 (40.7%)8 (40.0%)
       269 (48.3%)58 (47.2%)11 (55.0%)
      Sex mismatch0.518
       No60 (42.0%)51 (41.5%)9 (45.0%)
       M to F40 (28.0%)34 (27.6%)6 (30.0%)
       F to M21 (14.7%)20 (16.3%)1 (5.0%)
      ABO mismatch0.360
       No48 (33.6%)41 (33.3%)7 (35.0%)
       Minor43 (30.1%)37 (30.1%)6 (30.0%)
       Major31 (21.7%)29 (23.6%)2 (10.0%)
       Both21 (14.7%)16 (13.0%)5 (25.0%)
      Conditioning regimens0.638
       MAC71 (49.7%)60 (48.8%)11 (55.0%)
       RIC72 (50.3%)63 (51.2%)9 (45.0%)
      TBI0.159
       No35 (24.5%)33 (26.8%)2 (10.0%)
       Yes108 (75.5%)90 (73.2%)18 (90.0%)
      GVHD prophylaxis0.737
       CyA + sMTX39 (27.3%)34 (27.6%)5 (25.0%)
       CyA + MMF5 (3.5%)4 (3.3%)1 (5.0%)
       Tac + sMTX43 (30.1%)35 (28.5%)8 (40.0%)
       Tac + MMF40 (28.0%)36 (29.3%)4 (20.0%)
       other14 (9.8%)12 (9.8%)2 (10.0%)
      Number of TNC infused, 107/kg0.115
       Median2.742.723.14
       Range0.26–7.150.26–5.402.03–7.15
      Number of CD34+ cells infused, 105/kg0.809
       Median0.790.790.84
       Range0.23–38.30.23–38.30.41–2.17
      Follow-up period, mo0.414
       Median50.149.658.0
       Range2.2–78.212.0–76.22.2–78.2
      AA, aplastic anemia; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ATL, adult T-cell leukemia/lymphoma; CML, chronic myeloid leukemia; CyA, cyclosporine A; DSA, donor-specific anti-HLA antibody; F, female; GVHD, graft-versus-host disease; HCT-CI, hematopoietic cell transplantation-specific comorbidity index; HLA, human leukocyte antigen; HVG, host-versus-graft; M, male; MAC, myeloablative conditioning; MDS, myelodysplastic syndrome; MMF, mycophenolate mofetil; NHL, non-Hodgkin lymphoma; PC; platelet concentrate; PS, Eastern Cooperative Oncology Group Performance Status Scale; RBC, red blood cell; RIC, reduced-intensity conditioning; sMTX, short methotrexate; Tac, tacrolimus; TBI, total body irradiation; TNC, total nucleated cells.
      low asterisk P < 0.05.

      Effect of anti-HLA antibodies on engraftment

      Among the 567 patients, 510 (89.7%) achieved neutrophil engraftment. In the 57 patients who failed to achieve engraftment, 43 proceeded to re-transplantation, eight had early disease progression and six had non-relapse mortality due to bacterial infection (n = 3), respiratory failure (n = 1), hepatic failure (n = 1) and hemophagocytic syndrome (n = 1). Cumulative incidences of neutrophil and platelet engraftment 100 days after transplantation were 90.2% and 73.3%, respectively (Figure 1A,B). Median times to neutrophil and platelet engraftment were 21 days (range, 0–57 days) and 42 days (range, 2–207 days) after transplantation. The presence of HLA antibody itself had no significant effect on neutrophil or platelet engraftment (85.2% versus 91.8%, P= 0.086, in neutrophils; 67.8% versus 75.3%, P= 0.151, in platelets) (Figure 1C,D).
      Figure 1
      Figure 1Cumulative incidence of neutrophil and platelet engraftment stratified according to anti-HLA antibodies. (A) Neutrophil engraftment in the whole cohort. (B) Platelet engraftment in the whole cohort. (C) Engraftment of neutrophils stratified according to the presence of anti-HLA antibodies. (D) Engraftment of platelets stratified according to the presence of anti-HLA antibodies.

      Effect of DSAs against HLA-DP or -DQ on engraftment

      DSAs appeared to be associated with lower cumulative incidence of neutrophil and platelet engraftment (Figure 2A,B), although it was not statistically significant. Among patients positive for DSAs, the neutrophil engraftment rate was significantly lower and the platelet engraftment rate was marginally lower in DSA for DP/DQ patients compared with Ab-negative patients (55.6% versus 91.8%, P= 0.032, in neutrophils; 46.7% versus 75.3%, P= 0.128, in platelets) (Figure 2C,D). Time to neutrophil and platelet engraftment in DSA for patients with DP/DQ was significantly longer than that in patients who were Ab-negative (median, 25 versus 21 days, P= 0.002 in neutrophil; median 61 versus 46 days, P= 0.014 in platelet), suggesting that DSAs against HLA-DP or -DQ are associated with not only engraftment failure but also engraftment delay. In the meantime, neutrophil and platelet engraftment rates in other patients with DSA were comparable with those in patients who were Ab-negative (90.9%, in neutrophils; 57.6%, in platelets). Adverse effects of DSAs against HLA-DP or -DQ on engraftment remained after adjusting for effects of TNC dose, whereas the statistical significance was marginal due to the small sample size (supplementary Figure 1A,B). These findings suggest that adverse impacts of DSAs on engraftment were mainly due to DSAs against HLA-DP or -DQ, not to those against HLA-A, -B, DRB1 or -C.
      Figure 2
      Figure 2Cumulative incidence of neutrophil and platelet engraftment stratified according to the presence of DSAs. (A) Engraftment of neutrophils stratified according to the presence of DSAs. (B) Engraftment of platelets stratified according to the presence of DSAs. (C) Engraftment of neutrophils stratified according to DSA target antigens. (D) Engraftment of platelets stratified according to DSA target antigens. * indicates p < 0.05.

      Effect of DSAs for HLA-DP or -DQ on transplantation clinical courses

      Our results suggest that DSAs against HLA-DP or -DQ are associated with a lower engraftment rate after CBT. Accordingly, we tried to evaluate the influence of DSAs against HLA-DP or -DQ on transplantation outcomes. We analyzed effects of DSAs for HLA-DP or -DQ on infection-related complications. Cumulative incidence of bacterial infection at day 100 after transplantation was significantly greater in patients with DSAs for HLA-DP/DQ compared with patients who were Ab-negative (88.9% versus 57.1%, P= 0.024), whereas cumulative incidence of bacterial infection was comparable in other DSA patients and DSA-negative patients, compared with patients who were Ab-negative (Figure 3A,B). Adverse effects of DSAs against HLA-DP or -DQ on bacterial infection remained after adjusting for effects of TNC dose (supplementary Figure 2A). There were no significant effects of HLA antibodies, including DSAs, on cumulative incidence of fungal or viral infections (Figure 3C–F).
      Figure 3
      Figure 3Cumulative incidence of infection stratified according to the presence of DSAs. Incidence of bacterial infections stratified according to the presence of anti-HLA antibodies (A) and DSA target antigens (B). Incidence of fungal infections stratified according to the presence of anti-HLA antibodies (C) and DSA target antigens (D). Incidence of viral infections stratified according to the presence of anti-HLA antibodies (E) and DSA target antigens (F). * indicates p < 0.05.

      Effect of DSAs for HLA-DP or -DQ on transplantation outcomes

      While anti-HLA antibodies without donor-specificity, or DSAs other than against HLA-DP or -DQ, had little effect on OS, there was a trend toward worse OS in patients with DSAs for HLA-DP or -DQ compared with those without HLA antibodies, although the difference was not statistically significant (64.8% versus 78.1%, P= 0.344) (Figure 4A,B). There were no adverse or advantageous effects of DSAs for HLA-DP or -DQ on cumulative incidence of disease relapse and resultantly disease-free survival early after transplantation (supplementary Figure 3A,B). Then, in order to evaluate effects of DSAs for HLA-DP or -DQ on graft survival, excluding impact of re-transplantation, which increases recipient survival but not graft survival, we compared re-transplant-free survival according to the presence of DSAs. Re-transplant-free survival was poorer in DSA for patients with DP/DQ, with marginal statistical significance compared with those without HLA antibodies (55.6% versus 76.8%, P= 0.132) (Figure 4C,D). The trend toward lower OS and re-transplant-free survival remained after adjusting for effects of cell dose (supplementary Figure 2B,C). These results suggest that adverse effects of DSAs directed at HLA-DP/-DQ on engraftment may potentially reduce survival of recipients, as well as survival of grafts (i.e., continuous hematopoiesis) in CBT.
      Figure 4
      Figure 4OS and re-transplant-free survival soon after transplantation, stratified according to the presence of DSAs. (A) OS stratified according to the presence of anti-HLA antibodies. (B) OS stratified according to DSA target antigens. (C) Re-transplant-free survival stratified according to the presence of anti-HLA antibodies. (D) Re-transplant-free survival stratified according to DSA target antigens.

      Discussion

      This retrospective cohort study analyzed effects of anti-HLA antibodies, especially DSAs against HLA-DP or -DQ on engraftment and transplantation outcomes after single CBT and revealed three major findings: (i) anti-HLA antibodies without donor-specificity had no adverse effects on neutrophil or platelet engraftment after CBT; (ii) DSAs against HLA-DP and -DQ were associated with lower cumulative incidence of neutrophil and platelet engraftment and (iii) DSAs for HLA-DP or -DQ were significantly associated with greater incidence of bacterial infection soon after transplantation, along with a trend toward lower OS and re-transplant-free survival.
      We initially analyzed the effect of anti-HLA antibodies irrespective of donor specificity on engraftment after CBT and found that anti-HLA antibodies themselves had no significant adverse effect on neutrophil or platelet engraftment, and this result was consonant with previous reports [
      • Yamamoto H
      • Uchida N
      • Matsuno N
      • et al.
      Anti-HLA antibodies other than against HLA-A, -B, -DRB1 adversely affect engraftment and nonrelapse mortality in HLA-mismatched single cord blood transplantation: possible implications of unrecognized donor-specific antibodies.
      ]. DSAs appeared associated with lower cumulative incidence of neutrophil and platelet engraftment, although this relationship was not statistically significant (P= 0.084 in neutrophils; P = 0.073 in platelets). Among patients with DSAs against HLA loci other than HLA-DP or -DQ, the incidence of engraftment was comparable with that among patients without anti-HLA antibodies. The absence of adverse effect of these DSAs on engraftment may be attributed to the selection process of CB units before transplantation. In Japan, before selecting a CB unit, recipient serum sample is assessed whether the recipient has DSAs against the candidate CB unit for HLA-A, -B or -DR. In principle, CB units without DSAs against HLA-A, -B or DRB1 were prioritized for transplantation, and CB units with DSAs against HLA-A, -B or DR were selected in exceptional cases with low risk of graft rejection. Indeed, MFI of DSAs for HLA loci other than DP/DQ was relatively lower than against HLA-DP or -DQ (supplementary Table 1). Moreover, in CBT with DSAs against HLA-A, -B or DR, in order to reduce the risk of engraftment failure, efforts to reduce the titer of DSAs by performing plasmapheresis, intravenous γ globulin and/or random platelet transfusion are usually made before transplantation in Japan [
      • Ciurea SO
      • Al Malki MM
      • Kongtim P
      • et al.
      Treatment of allosensitized patients receiving allogeneic transplantation.
      ,
      • Yamashita T
      • Ikegame K
      • Kojima H
      • et al.
      Effective desensitization of donor-specific HLA antibodies using platelet transfusion bearing targeted HLA in a case of HLA-mismatched allogeneic stem cell transplantation.
      ].
      Although a previous report suggested that DSAs other than against HLA-A, -B or -DRB1 can adversely affect engraftment after CBT [
      • Yamamoto H
      • Uchida N
      • Matsuno N
      • et al.
      Anti-HLA antibodies other than against HLA-A, -B, -DRB1 adversely affect engraftment and nonrelapse mortality in HLA-mismatched single cord blood transplantation: possible implications of unrecognized donor-specific antibodies.
      ], direct assessment of whether anti-HLA antibodies are DSAs was not performed due to a lack of allele information on HLA loci for many recipients and CB units other than HLA-A, -B or -DRB1. Therefore, the exact role of DSAs directed at HLA-DP and -DQ remains unclear. Accordingly, we focused on effects of DSAs for HLA-DP and -DQ on engraftment and found that DSAs for HLA-DP or -DQ were significantly associated with lower incidence of engraftment after CBT. MFI values of DSAs for HLA-DP and -DQ were greater in patients with engraftment failure than those who achieved engraftment (supplementary Table 1), suggesting a threshold of MFI value that determines whether engraftment failure occurs. As HLA-DP and -DQ are expressed on hematopoietic precursor cells [
      • Piacibello W
      • Aglietta M
      • Stacchini A
      • et al.
      Expression of HLA class II determinants by normal and chronic myeloid leukemia progenitors.
      ,
      • Sparrow RL
      • Williams N.
      The pattern of HLA-DR and HLA-DQ antigen expression on clonable subpopulations of human myeloid progenitor cells.
      ], it can be speculated that antigen–antibody interactions on the surface of these cells are associated with graft rejection or engraftment delay.
      Then, we assessed the influence of DSAs against HLA-DP or -DQ on transplantation outcome. We showed for the first time that DSAs against HLA-DP or -DQ significantly increased the cumulative incidence of bacterial infection soon after CBT. These findings suggest that engraftment delay induced by DSAs against HLA-DP or DQ increases the risk of infection-related complications. Our observation that there is a trend toward worse OS and reduced re-transplant-free survival in DSA for DP/DQ patients suggests an adverse impact of DSAs for HLA-DP or -DQ on survival of both recipients and grafts after CBT, and warrants validation in a larger study.
      The strength of this study includes stringent evaluation of the significance of DSAs against HLA-DP and -DQ, based on thorough HLA-allele typing of recipients and CB units. However, limitations of the study should be acknowledged. Since this is a retrospective study, including a small sample of DSA-positive patients, findings should be interpreted with caution and should be confirmed in a larger prospective study. Absence of increased engraftment failure in patients with DSAs against HLA-C could be related to the small sample size of this study and should be interpreted cautiously. The significance of DSA-directed HLA-DR3/4/5 on engraftment was not assessed in this study.
      In conclusion, DSAs against HLA-DP or -DQ are associated with lower engraftment and greater incidence of bacterial infection after CBT, whereas anti-HLA antibodies without donor specificity had no adverse effects on engraftment or infectious complications. While our results warrant further investigation due to a small sample of DSA-positive patients in our cohort, our findings suggest the significance of evaluation of DSAs against HLA-DP and -DQ in a recipient before selecting an HLA-mismatched CB unit and provide insights into donor selection algorithms in CBT.

      Declaration of Competing Interest

      The authors have no commercial, proprietary or financial interest in the products or companies described in this article.

      Funding

      This work was supported in part by the Lotte Foundation and the Program for Development of Next-generation Leading Scientists with Global Insight (L-INSIGHT) at Kyoto University, Kyoto, Japan, sponsored by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in Japan to YA.

      Author Contributions

      Conception and design of the study: TJ, YA, KH, HI, and TK. Analysis and interpretation of data: TJ, YA, KH, HI, AO, NM, JM, YK, FA, and TK. Drafting or revising the manuscript: TJ and YA. All authors have approved the final article.

      Acknowledgments

      The authors thank Dr Fumiaki Nakamura for insightful discussions, and all the physicians and data managers at the centers that contributed valuable data on transplantation.

      Appendix. Supplementary materials

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