Advertisement

Immunotherapy with CD25/CD71-allodepleted T cells to improve T-cell reconstitution after matched unrelated donor hematopoietic stem cell transplant: a randomized trial

Open AccessPublished:October 09, 2022DOI:https://doi.org/10.1016/j.jcyt.2022.08.010

      Abstract

      Background aims

      Delayed immune reconstitution is a major challenge after matched unrelated donor (MUD) stem cell transplant (SCT). In this randomized phase 2 multi-center trial, Adoptive Immunotherapy with CD25/71 allodepleted donor T cells to improve immunity after unrelated donor stem cell transplant (NCT01827579), the authors tested whether allodepleted donor T cells (ADTs) can safely be used to improve immune reconstitution after alemtuzumab-based MUD SCT for hematological malignancies.

      Methods

      Patients received standard of care or up to three escalating doses of ADTs generated through CD25+/CD71+ immunomagnetic depletion. The primary endpoint of the study was circulating CD3+ T-cell count at 4 months post-SCT. Twenty-one patients were treated, 13 in the ADT arm and eight in the control arm.

      Results

      The authors observed a trend toward improved CD3+ T-cell count at 4 months in the ADT arm versus the control arm (230/µL versus 145/µL, P = 0.18), and three ADT patients achieved normal CD3+ T-cell count at 4 months (>700/µL). The rates of significant graft-versus-host disease (GVHD) were comparable in both cohorts, with grade ≥2 acute GVHD in seven of 13 and four of eight patients and chronic GVHD in three of 13 and three of eight patients in the ADT and control arms, respectively.

      Conclusions

      These data suggest that adoptive transfer of ADTs is safe, but that in the MUD setting the benefit in terms of T-cell reconstitution is limited. This approach may be of more use in the context of more rigorous T-cell depletion.

      Key Words

      Introduction

      Graft-versus-host disease (GVHD) after allogeneic haematopoetic stem cell transplant (HSCT or SCT) is mediated by alloreactive donor T cells infused in the graft that recognize host antigens. Alemtuzumab, a monoclonal anti-CD52 antibody, reduces the incidence of GVHD after unrelated donor SCT through in vivo T-cell depletion [
      • Willemsen L
      • Jol-van der Zijde CM
      • Admiraal R
      • Putter H
      • Jansen-Hoogendijk AM
      • Ostaijen-ten Dam MM
      • et al.
      Impact of Serotherapy on Immune Reconstitution and Survival Outcomes After Stem Cell Transplantations in Children: Thymoglobulin Versus Alemtuzumab.
      ,
      • Kottaridis PD
      • Milligan DW
      • Chopra R
      • Chakraverty RK
      • Chakrabarti S
      • Robinson S
      • et al.
      In vivo CAMPATH-1H prevents graft-versus-host disease following nonmyeloablative stem cell transplantation.
      ,
      • Saif MA
      • Borrill R
      • Bigger BW
      • Lee H
      • Logan A
      • Poulton K
      • et al.
      In vivo T-cell depletion using alemtuzumab in family and unrelated donor transplantation for pediatric non-malignant disease achieves engraftment with low incidence of graft vs. host disease.
      ,
      • Kanda J
      • Lopez RD
      • Rizzieri DA.
      Alemtuzumab for the prevention and treatment of graft-versus-host disease.
      ,
      • Veys P
      • Wynn RF
      • Ahn KW
      • Samarasinghe S
      • He W
      • Bonney D
      • et al.
      Impact of immune modulation with in vivo T-cell depletion and myleoablative total body irradiation conditioning on outcomes after unrelated donor transplantation for childhood acute lymphoblastic leukemia.
      ]. However, non-selective T-cell depletion results in profound immunosuppression, leading to high morbidity/mortality from viral infections [
      • Chakrabarti S
      • Mautner V
      • Osman H
      • Collingham KE
      • Fegan CD
      • Klapper PE
      • et al.
      Adenovirus infections following allogeneic stem cell transplantation: Incidence and outcome in relation to graft manipulation, immunosuppression, and immune recovery.
      ,
      • Chakrabarti S
      • Mackinnon S
      • Chopra R
      • Kottaridis PD
      • Peggs K
      • O'Gorman P
      • et al.
      High incidence of cytomegalovirus infection after nonmyeloablative stem cell transplantation: Potential role of Campath-1H in delaying immune reconstitution.
      ,
      • Cupit-Link MC
      • Nageswara Rao A
      • Warad DM
      • Rodriguez V
      • Khan S
      EBV-PTLD, adenovirus, and CMV in pediatric allogeneic transplants with alemtuzumab as part of pretransplant conditioning: A retrospective single center study.
      ].
      To circumvent this, a number of strategies have been developed to improve immune reconstitution after T-cell-depleted transplant. A number of groups have used donor lymphocytes transduced with a safety “suicide” gene that is activated in the event of GVHD [
      • Ciceri F
      • Bordignon C.
      Suicide-gene-transduced donor T-cells for controlled graft-versus-host disease and graft-versus-tumor.
      ,
      • Di Stasi A
      • Tey S-K
      • Dotti G
      • Fujita Y
      • Kennedy-Nasser A
      • Martinez C
      • et al.
      Inducible Apoptosis as a Safety Switch for Adoptive Cell Therapy.
      ], but it would be clearly preferable to prevent rather than switch off GVHD. Ex vivo-generated donor/third-party virus-specific T cells with specificity for one or multiple viruses show promise, but to date their use has largely been restricted to major academic centers, and randomized studies demonstrating efficacy are lacking [
      • Heslop HE
      • Slobod KS
      • Pule MA
      • Hale GA
      • Rousseau A
      • Smith CA
      • et al.
      Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients.
      ,
      • Peggs KS
      • Thomson K
      • Samuel E
      • Dyer G
      • Armoogum J
      • Chakraverty R
      • et al.
      Directly selected cytomegalovirus-reactive donor T cells confer rapid and safe systemic reconstitution of virus-specific immunity following stem cell transplantation.
      ,
      • Mackinnon S
      • Thomson K
      • Verfuerth S
      • Peggs K
      • Lowdell M.
      Adoptive cellular therapy for cytomegalovirus infection following allogeneic stem cell transplantation using virus-specific T cells.
      ,
      • Ottaviano G
      • Chiesa R
      • Feuchtinger T
      • Vickers M
      • Dickinson A
      • Gennery A
      • et al.
      Adoptive T Cell Therapy Strategies for Viral Infections in Patients Receiving Haematopoietic Stem Cell Transplantation.
      ,
      • Leen AM
      • Bollard CM
      • Mendizabal AM
      • Shpall EJ
      • Szabolcs P
      • Antin JH
      • et al.
      Multicenter study of banked third-party virus-specific T cells to treat severe viral infections after hematopoietic stem cell transplantation.
      ,
      • Tzannou I
      • Papadopoulou A
      • Naik S
      • Leung K
      • Martinez CA
      • Ramos CA
      • et al.
      Off-the-shelf virus-specific T cells to treat BK virus, human herpesvirus 6, cytomegalovirus, Epstein-Barr virus, and adenovirus infections after allogeneic hematopoietic stem-cell transplantation.
      ,
      • Roex MCJ
      • van Balen P
      • Germeroth L
      • Hageman L
      • van Egmond E
      • Veld SAJ
      • et al.
      Generation and infusion of multi-antigen-specific T cells to prevent complications early after T-cell depleted allogeneic stem cell transplantation—a phase I/II study.
      ]. Additional approaches for accelerating immune reconstitution [
      • Lucarelli B
      • Merli P
      • Bertaina V
      • Locatelli F.
      Strategies to accelerate immune recovery after allogeneic hematopoietic stem cell transplantation.
      ] include infusion of T cells depleted of either CD8+ [
      • Dodero A
      • Carniti C
      • Raganato A
      • Vendramin A
      • Farina L
      • Spina F
      • et al.
      Haploidentical stem cell transplantation after a reduced-intensity conditioning regimen for the treatment of advanced hematologic malignancies: Posttransplantation CD8-depleted donor lymphocyte infusions contribute to improve T-cell recovery.
      ,
      • Meyer RG
      • Britten CM
      • Wehler D
      • Bender K
      • Hess G
      • Konur A
      • et al.
      Prophylactic transfer of CD8-depleted donor lymphocytes after T-cell-depleted reduced-intensity transplantation.
      ,
      • Orti G
      • Lowdell M
      • Fielding A
      • Samuel E
      • Pang K
      • Kottaridis P
      • et al.
      Phase i study of high-stringency CD8 depletion of donor leukocyte infusions after allogeneic hematopoietic stem cell transplantation.
      ] or naive [
      • Triplett BM
      • Muller B
      • Kang G
      • Li Y
      • Cross SJ
      • Moen J
      • et al.
      Selective T-cell depletion targeting CD45RA reduces viremia and enhances early T-cell recovery compared with CD3-targeted T-cell depletion.
      ,
      • Bleakley M
      • Heimfeld S
      • Loeb KR
      • Jones LA
      • Chaney C
      • Seropian S
      • et al.
      Outcomes of acute leukemia patients transplanted with naive T cell-depleted stem cell grafts.
      ] T cells or induction of T-cell anergy [
      • Davies JK
      • Gribben JG
      • Brennan LL
      • Yuk D
      • Nadler LM
      • Guinan EC
      Outcome of alloanergized haploidentical bone marrow transplantation after ex vivo costimulatory blockade: Results of 2 phase 1 studies.
      ,
      • Davies JK
      • Brennan LL
      • Wingard JR
      • Cogle CR
      • Kapoor N
      • Shah AJ
      • et al.
      Infusion of Alloanergized Donor Lymphocytes after CD34-selected Haploidentical Myeloablative Hematopoietic Stem Cell Transplantation.
      ].
      Selective ex vivo allodepletion represents a logical approach to improving T-cell recovery post-SCT without causing GVHD. This strategy seeks to remove only those donor T cells that are reactive against the host while preserving T-cell responses to pathogens. Donor T cells are co-cultured with host antigen-presenting cells, following which alloreactive T cells can be identified by expression of surface activation markers such as CD25, CD71 or CD69 or retention of photoactive dyes. They can then be depleted by a variety of methods, including immunotoxins, immunomagnetic separation, chemotherapy, flow cytometry sorting and photodynamic elimination [
      • André-Schmutz I
      • Le Deist F
      • Hacein-Bey-Abina S
      • Vitetta E
      • Schindler J
      • Chedeville G
      • et al.
      Immune reconstitution without graft-versus-host disease after haemopoietic stem-cell transplantation: A phase 1/2 study.
      ,
      • Samarasinghe S
      • Mancao C
      • Pule M
      • Nawroly N
      • Karlsson H
      • Brewin J
      • et al.
      Functional characterization of alloreactive T cells identifies CD25 and CD71 as optimal targets for a clinically applicable allodepletion strategy.
      ,

      Amrolia PJ, Muccioli-Casadei G, Yvon E, Huls H, Sili U, Wieder ED et al. Selective depletion of donor alloreactive T cells without loss of antiviral or antileukemic responses. Blood 2003; 102(6): 2292-9.

      ,
      • Mielke S
      • Nunes R
      • Rezvani K
      • Fellowes VS
      • Venne A
      • Solomon SR
      • et al.
      A clinical-scale selective allodepletion approach for the treatment of HLA-mismatched and matched donor-recipient pairs using expanded T lymphocytes as antigen-presenting cells and a TH9402-based photodepletion technique.
      ,
      • Albon SJ
      • Mancao C
      • Gilmour K
      • White G
      • Ricciardelli I
      • Brewin J
      • et al.
      Optimization of methodology for production of CD25/CD71 allodepleted donor t cells for clinical use.
      ,
      • Li Pira G
      • Di Cecca S
      • Montanari M
      • Moretta L
      • Manca F
      Specific removal of alloreactive T-cells to prevent GvHD in hemopoietic stem cell transplantation: rationale, strategies and perspectives.
      ]. Clinical studies of this approach have demonstrated the feasibility and safety of this strategy [
      • André-Schmutz I
      • Le Deist F
      • Hacein-Bey-Abina S
      • Vitetta E
      • Schindler J
      • Chedeville G
      • et al.
      Immune reconstitution without graft-versus-host disease after haemopoietic stem-cell transplantation: A phase 1/2 study.
      ,
      • Solomon SR
      • Mielke S
      • Savani BN
      • Montera A
      • Wisch L
      • Childs R
      • et al.
      Selective depletion of alloreactive donor lymphocytes: A novel method to reduce the severity of graft-versus-host disease in older patients undergoing matched sibling donor stem cell transplantation.
      ,
      • Amrolia PJ
      • Muccioli-Casadei G
      • Huls H
      • Adams S
      • Durett A
      • Gee A
      • et al.
      Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation.
      ,
      • Roy DC
      • Lachance S
      • Cohen S
      • Delisle JS
      • Kiss T
      • Sauvageau G
      • et al.
      Allodepleted T-cell immunotherapy after haploidentical haematopoietic stem cell transplantation without severe acute graft-versus-host disease (GVHD) in the absence of GVHD prophylaxis.
      ,
      • Roy DC
      • Walker I
      • Maertens J
      • Lewalle P
      • Olavarria E
      • Selleslag D
      • et al.
      ATIR101 administered after T-cell-depleted haploidentical HSCT reduces NRM and improves overall survival in acute leukemia.
      ], and the authors’ previous study [
      • Amrolia PJ
      • Muccioli-Casadei G
      • Huls H
      • Adams S
      • Durett A
      • Gee A
      • et al.
      Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation.
      ] demonstrated accelerated T-cell recovery and cytomegalovirus (CMV)/Epstein–Barr virus (EBV)-specific T-cell responses when patients were infused with a higher dose (105/kg) of allodepleted donor T cells (ADTs). However, the majority of these studies were in the haploidentical setting. Unrelated donor bone marrow/peripheral blood SCT is much more frequently used, and the authors wished to determine whether allodepletion could be used to improve immune reconstitution in this setting.
      The authors developed a methodology for selective immunomagnetic depletion of alloreactive T cells upregulating CD25 and CD71 after activation with host dendritic cells (DCs) and showed that ADTs retain anti-viral responses with minimal host alloreactivity in vitro [
      • Albon SJ
      • Mancao C
      • Gilmour K
      • White G
      • Ricciardelli I
      • Brewin J
      • et al.
      Optimization of methodology for production of CD25/CD71 allodepleted donor t cells for clinical use.
      ]. The authors have now tested this approach in a randomized phase 2 multi-center clinical study, Individualized Cancer Therapy (NCT01827579), to determine whether CD25/CD71 ADTs can be safely used to improve immune reconstitution after alemtuzumab-conditioned matched unrelated donor (MUD) SCT for hematological malignancies.

      Methods

      Study population

      The study was open to adult patients undergoing alemtuzumab-conditioned 9/10 or 10/10 HLA MUD peripheral blood SCT for hematological malignancies at three participating sites in the UK.

      Study design

      Eligible patients were randomized 2:1 to an intervention arm (ADT immunotherapy) or standard-of-care MUD peripheral blood SCT. Patients in the ADT cohort received up to three escalating doses of allodepleted T cells post-transplant (0.1 × 106/kg at day 30, 0.3 × 106/kg at day 60 and 1 × 106/kg at day 90) provided they had engrafted and there were no severe intercurrent infections, severe hepatopathy (bilirubin >50 µM or alanine transaminase >200 U/L), oxygen requirement or grade >1 acute GVHD and circulating CD3+ T cells were less than 700/µL (lower limit of normal for adults).
      The study was approved by the UK South Central Oxford A Research Ethics Committee and the Medicines and Healthcare Products Regulatory Agency as well as by the relevant unrelated donor registries (Anthony Nolan in the UK, German Bone Marrow Donor Center in Germany and National Marrow Donor Program in the US). The study was managed by Cancer Research UK and the University College London Cancer Trials Center. Informed consent was obtained from all patients as well as donors.

      Study objectives

      The primary endpoint of the study was circulating CD3+ T-cell count at 4 months post-hematopoietic SCT (HSCT). Secondary endpoints included incidence of grade 2–4 acute or chronic GVHD; time to recovery of normal T cells and CD4+ cells (>700/µL and >300/µL, respectively) and normal T cell receptor (TCR) diversity, as assessed by Vβ spectratyping; in vitro anti-viral responses of circulating peripheral blood mononuclear cells (PBMCs), as assessed by interferon gamma (IFN-γ) ELISpot; and treatment-related mortality (TRM) and disease-free survival (DFS) at 1 year post-HSCT.

      Transplantation

      All patients received alemtuzumab as part of their conditioning. Alemtuzumab dosing and conditioning regimens were not pre-defined by the trial protocol, but rather were in accordance with institutional protocols. Details of the different conditioning regimens are specified in supplementary Table 1.
      Peripheral blood was the stem cell source for all patients, and the graft was not further manipulated beyond ex vivo T-cell depletion with alemtuzumab, as outlined earlier, for patients receiving myeloablative conditioning. GVHD prophylaxis was with cyclosporine alone, which was withdrawn over 4 weeks from day 50, where possible, in both arms. Acute and chronic GVHD was graded according to Seattle and National Institutes of Health criteria, respectively.

      Generation of ADTs

      Generation of ADTs was performed under current Good Manufacturing Practice conditions at Great Ormond Street Hospital for Children, London, UK. Patient-derived mature DCs were generated from CD14-selected PBMCs from autologous apheresis performed 2 weeks prior to transplant as previously described [
      • Samarasinghe S
      • Mancao C
      • Pule M
      • Nawroly N
      • Karlsson H
      • Brewin J
      • et al.
      Functional characterization of alloreactive T cells identifies CD25 and CD71 as optimal targets for a clinically applicable allodepletion strategy.
      ,
      • Albon SJ
      • Mancao C
      • Gilmour K
      • White G
      • Ricciardelli I
      • Brewin J
      • et al.
      Optimization of methodology for production of CD25/CD71 allodepleted donor t cells for clinical use.
      ]. Donor PBMCs were obtained a week prior to transplant via unstimulated leukapheresis or 500-mL blood draw. Host DCs were irradiated at 30 Gy and co-cultured 1:10 with donor PBMCs in serum-free medium for 4 days. Activated allogeneic donor T cells were then incubated with an anti-CD71-biotin antibody followed by CliniMACS anti-biotin and anti-CD25 beads (Miltenyi Biotec Ltd, Woking, UK). CD25/CD71 cells were then removed through immunomagnetic depletion on the CliniMACS Plus instrument (Miltenyi Biotec Ltd). The resulting ADTs were tested for sterility and residual alloreactivity and cryopreserved. Release criteria for the final product included <0.2% CD3+CD25+ and <0.2% CD3+CD71+, as assessed by flow cytometry, and <10% residual proliferation against host cells, as seen in secondary mixed leukocyte reaction.

      Monitoring immune reconstitution

      Immune reconstitution was studied monthly for the first 6 months and then every 2 months until 1 year post-SCT by analyzing PBMC flow cytometrically for lymphocyte subpopulations [
      • Amrolia PJ
      • Muccioli-Casadei G
      • Huls H
      • Adams S
      • Durett A
      • Gee A
      • et al.
      Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation.
      ]. At 4 months, 6 months and 12 months post-SCT, T-cell function was analyzed via measurement of proliferative responses to mitogenic stimulation with phytohemagglutinin (PHA) using 3H-thymidine uptake. Immunoglobulin levels and T-cell Vβ repertoire were assayed by polymerase chain reaction spectratyping. A normal spectratype consisted of five to eight bands per family with a Gaussian size distribution. Anti-viral responses to CMV and EBV peptides were evaluated in batched cryopreserved PBMCs using IFN-γ ELISpot assays. Anti-CMV response was further analyzed with pentamer analysis in patients who experienced CMV reactivation.

      Data analysis

      The study was designed with 80% power and one-sided test of statistical significance of 15%, requiring 16 patients in the experimental arm and eight in the control arm to detect a standardized mean difference ≥0.85 in circulating CD3 count at 4 months post-SCT. A one-sided significance level of 5–20% is common in early phase 2 trials [
      • Rubinstein LV
      • Korn EL
      • Freidlin B
      • Hunsberger S
      • Ivy SP
      • Smith MA.
      Design Issues of Randomized Phase II Trials and a Proposal for Phase II Screening Trials.
      ]. CD3 count at 4 months was considered to be a meaningful time point with regard to immune reconstitution. A large effect size (0.85) was used because CD3 count is a surrogate endpoint, and it has generally been observed in cancer trials that large effects on a surrogate are associated with smaller/more moderate effects on clinical endpoints such as relapse and survival.
      Statistical analysis in this study was predominantly descriptive. Wilcoxon rank-sum test was used to compare median values for lymphocyte recovery at 4 months, as data were not normally distributed. Similar analysis was done to evaluate differences in median CD3, CD4 and CD8 counts between the control and ADT groups at different time points (4 months, 6 months and 12 months post-SCT). The area under the curve for CD3 counts from 0 months to 4 months was calculated using the trapezoid rule. GVHD and toxicity were reported in terms of frequencies and percentages by treatment group based on the worst grade reported during the study. Mortality and DFS were analyzed using Kaplan–Meier curves and Cox regression.

      Results

      Recruitment and patient characteristics

      Between August 2014 and February 2019, a total of 37 patients were recruited and randomized (Figure 1). A total of 16 patients were taken off the trial because of donor refusal (seven), acute GVHD grade >1 (three), death (one), withdrawal of consent (one), leukapheresis labeling error (one), oxygen requirement (one), cytopenia (one) and intercurrent illness preventing continuation in the study (one). Although the study was designed for a total of 24 evaluable patients (16 in the ADT arm and eight in the control arm), the sample size achieved (21 evaluable patients) was slightly less as a result of these issues. Of the 21 evaluable patients, 13 were treated in the ADT arm and eight were treated in the control arm. All 21 patients were evaluable for the primary endpoint (T-cell recovery at 4 months post-transplant), and 16 patients reached the 12-month follow-up. Patients who did not reach this endpoint included three patients who died prior to 12 months post-SCT (two because of disease progression in the treatment arm and one because of TRM in the control arm) and two patients in the ADT arm who withdrew from the trial.
      Fig 1
      Fig. 1CONSORT diagram of ICAT recruitment and outcome summary. CONSORT, Consolidated Standards of Reporting Trials; ICAT, Individualized Cancer Therapy.
      Baseline patient characteristics are shown in Table 1. Given the size of the study, baseline characteristics were not expected to be equally distributed between the two cohorts. More patients in the ADT group had acute myeloid leukemia (nine of 13 [70%] versus two of eight [25%] in the control group) and were in complete remission (nine of 13 [70%] versus three of eight [38%] in the control group). Only one patient in each cohort had progressive disease prior to transplant. Seventeen patients received reduced-intensity conditioning (11 of 13 in the ADT arm and six of eight in the control arm) and two patients in each group underwent myeloablative conditioning, the details of which are provided in supplementary Table 1. All but one patient received conditioning regimens comprising 50–100 mg of intravenous alemtuzumab in fractions; the remaining patient had the graft treated with 20 mg of alemtuzumab in the bag.
      Table 1Baseline patient characteristics.
      ADT (n = 13)Control (n = 8)
      nn
      Age in yearsmedian (range)1354 (36–68)848 (34–62)
      Sex
      Female323%450%
      Male1077%450%
      Diagnosis
      AML969%225%
      DLBCL216%113%
      FL00%113%
      HL00%113%
      MCL00%113%
      CLL18%00%
      ALL00%00%
      CML00%225%
      Myelodysplasia18%00%
      Disease status
      Morphological CR862%338%
      Cytogenetic CR18%00%
      Partial metabolic response00%113%
      Partial response18%113%
      Stable disease18%113%
      Chronic phase00%113%
      Progressive disease18%113%
      Missing18%00%
      Conditioning
      Myeloablative215%225%
      Non-myeloablative1185%675%
      ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; CML, chronic myeloid leukemia; CR, complete remission; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; HL, Hodgkin lymphoma; MCL, mantle cell lymphoma.

      ADT and treatment compliance

      Characteristics of the ADT products released for the 13 infused patients are summarized in supplementary Table 2. Of the 13 patients treated in the ADT arm, five received one dose, four received two doses and four received three doses. With regard to the nine of 13 patients who did not receive the three planned escalating doses, the reasons were as follows: three already had circulating T cells >700/µL, five had acute GVHD grade >1 at the infusion time point and the dose was not available for one. Table 2 shows a summary of treatment compliance, complications and outcomes for each patient.
      Table 2Summary of treatment compliance, complications and outcomes.
      PatientCohortADT dosesReason for stopping ADT treatmentAUC for CD3 from 0 months to 4 months, count/μLAcute GVHD ≥2Chronic GVHDSystemic steroids before month 4CMV serostatus, Donor/RecipientEBV serostatus, Donor/RecipientViral reactivation within first 6 months (maximum copies/mL - month post-SCT)Other significant complicationsRelapse/progressive diseaseStatus at 12 months post-SCTMonths to death post-transplant (cause)
      AADT3Not applicable890Yes, grade 3NoNo+/++/–CMV (18 600 -M6) EBV (1835 - M6) ADV (37 939 - M5)NoneNoCR
      BADT2Unable to manufacture15Yes, grade 2NoYes–/––/+NoNoneNoCR
      CADT1T cells >700 prior to day 605420NoNoNo+/++/+CMV (320 -M1)NoneYes, at 10.2 months post-SCTPD12.1 (relapse/PD)
      DADT1GVHD grade >1 prior to day 601725Yes, grade 3NoNo–/–+/+EBV (106 772 - M5)Device-related infectionYes, at 6.5 months post-SCTPD
      EADT2GVHD grade >1 prior to day 90590NoYes, moderateNo+/+–/+CMV (2244 - M2)NoneYes, at 11 months post-SCTPD
      FADT3NA495NoNoNo–/–+/–EBV (1522 -M6)NoneYes, at 20.2 months post-SCTCR20.9 (relapse/PD)
      GADT2T cells >700 prior to day 902080NoNoNo+/NA+/NACMV (948 - M1)Parainfluenza, device-related infectionNoCR
      HADT2GVHD grade >1 prior to day 90365Yes, grade 2NoNo–/–+/+EBV (22 052 -M6)Death due to pneumonia 14.5 months post-SCTNoCR14.5 (NRM)
      IADT3Not applicable90NoNoNo–/–+/–NoNoneNoNot assessed, no clinical evidence of disease
      JADT1GVHD grade >1 prior to day 60615Yes, grade 2NoNo–/–+/+NoNoneNANA, last follow-before 12 months
      KADT1T cells >700 prior to day 60, unwell, with oxygen requirement and respiratory infection1450Yes, grade 2Yes, mildYes–/–+/+NoIntracranial hemorrhage, chest infectionYes, at 3.4 months post-SCTNA, dead before 12 months5.6 (relapse/PD)
      LADT1GVHD grade >1 prior to day 60305Yes, grade 3NoYes–/––/–NoNoneNoNot assessed, no clinical evidence of disease
      MADT3Not applicable255NoYes, severeYes–/––/+NoNoneNoCR
      NControl920NoYes, mildNo–/–+/+EBV (2680 -M2)NoneYes, at 6.3 months post-SCTPD
      OControl90Yes, grade 3NoYes–/–+/–NoHeart failure (resolved)NoNot assessed, no clinical evidence of disease
      PControl390NoYes, mildNo–/–+/+NoNoneNoNot assessed, no clinical evidence of disease
      QControl345NoNoNo–/–+/–NoNoneYes, at 7.1 months post-SCTPD
      RControl630NoNoNo–/–+/+NoHypertensionNoStable disease
      SControl105Yes, grade 3NoYes–/–+/–NoNoneNoCR
      TControl30Yes, grade 2Yes, mildYesNA/–NA/+NoNRM, death due to viral infection at 8 monthsNoNA, dead before 12 months8.2 (NRM)
      UControl1030Yes, grade 2NoYes+/++/+CMV (2800- M5)NoneNoCR
      ADT: allodepleted T-cell cohort; ADV: adenovirus; AUC, area under the curve; CMV: citomegalovirus; CR, complete remission; EBV: Ebstein-Barr virus; GVHD: graft vs host disease; NA, not applicable/not available; NRM, non-relapse mortality; PD, progressive disease; SCT: stem-cell transplant

      Immune reconstitution

      The primary endpoint of the study was T-cell reconstitution at month 4 post-transplant (Figure 2A). Data were not normally distributed (Figure 2B), and non-parametric tests did not show a significant difference in median T-cell count between both cohorts, with 230/µL (range, 10–4080) in the treatment cohort and 145/µL (range, 10–500) in the control cohort (one-sided P = 0.18) (Figure 2C). Using log-transformed data, the standardized mean difference was 0.43 (one-sided P = 0.17). Median area under the curve for CD3 for months 0–4 was also not significantly different between the two groups (590 µL/4-month ADT group versus 367.5 µL/4-month control group, two-sided P = 0.38). Three patients (all in the ADT arm) had a normal CD3+ T-cell count of more than 700/µL at 4 months post-SCT, skewing the mean for that population. These patients were C (4080/µL), D (1470/µL) and G (1720/µL) (Table 2). In patients C and G, the increase in CD3+ count was predominantly seen in the CD8+ compartment, whereas in patient D, it was observed in CD4+ T cells. All three of these patients experienced GVHD, but none required treatment with systemic steroids. Patients C and G had early low-level CMV reactivation (Table 2).
      Fig 2
      Fig. 2Median CD3 T-cell recovery between ADT and control cohorts at 4 months. (A) Circulating CD3/µL for all 21 patients post-HSCT. (B) Circulating CD3/µL at 4 months post-SCT. (C) Median circulating CD3 for control and ADT groups at different time points after HSCT. Wilcoxon two-sided P > 0.05 at all time points. (D) Time to normal CD3/µL count after HSCT in both cohorts.
      As shown in Figure 2D, time to normal CD3+ T-cell count was somewhat more rapid in the ADT cohort, though this was not statistically significant (P = 0.35). At 4 months post-SCT, three of 13 (23%) ADT patients had a normal T-cell count versus zero of eight (0%) patients in the control arm. At 6 months, four of 11 (36%) evaluable patients in the ADT cohort had a normal T-cell count versus one of eight (13%) patients in the control cohort. Median CD4 and CD8 counts were comparable between the control and ADT groups at all time points (see supplementary Figure 1).
      To assess T-cell functionality, the authors determined the ability of PBMCs derived from patients post-SCT to proliferate in response to the mitogen PHA. Although a minority of patients in the ADT arm appeared to show improved responses, the median PHA stimulation index was comparable between the two cohorts at 4 months, at 3.3 (range, 0.67–73.1) for the ADT group and 2.7 (range, 1.1–8.2) for the control group (Figure 3A). Figure 3B illustrates the diversity of the TCR repertoire as assessed by Vβ spectratyping at 4 months and 6 months post-SCT. Neither the median number of peaks nor the number of Gaussian families for CD4 and CD8 subsets was significantly different between patients in the ADT and control cohorts. In the ADT and control groups, respectively, the median number of peaks for CD4 at 4 months was six (range, 4–7) and five (range, 4–7) and the median number of peaks for CD8 at 6 months was four (range, 2.5–6) and four (range, 1.5–7). With regard to Gaussian families, in the ADT and control arms, respectively, the median number for CD4 at 4 months was 2.5 (range, 0–10) and three (range, 0–13) and the median number for CD8 at 6 months was zero (range, 0–2) and zero (range, 0–4). However, 10 of 11 (91%) evaluable patients in the ADT group achieved near-normal TCR diversity by month 4, with a median number of peaks between six and seven, compared with two of six (33%) evaluable patients in the control group (Fisher exact test P = 0.028). As expected, both B-cell and natural killer cell recovery and IgG and IgM immunoglobulin levels were similar between the two cohorts at 4 months, 6 months and 12 months post-transplant (see supplementary Figure 2).
      Fig 3
      Fig. 3Functional immune reconstitution tests were not different between ADT and control cohorts. (A) PHA SI at month 4 and month 12 for patients in both cohorts. (B) Vβ repertoire at 4 months and 6 months post-SCT represented by median number of peaks (upper panels) and median number of Gaussian families (lower panels) in CD4 and CD8 subsets. SI, stimulation index.

      Virus-specific immunity

      To assess virus-specific immune reconstitution, in vitro T-cell responses to EBV and CMV peptides were assayed by IFN-γ ELISpot. Four of 12 evaluable patients in the ADT arm and one of eight evaluable patients in the control arm were at risk of CMV reactivation by virtue of patient/donor seropositivity (Table 2), and they all experienced CMV reactivation. As shown in Figure 4A, four of 12 patients in the ADT arm had a significant response (>20 spot-forming units/105 cells) to CMV compared with one of eight control patients. In each case, this was associated with CMV reactivation in patients with CMV-seropositive donors. Five patients experienced CMV reactivation, and the relationship between reactivation and CMV-specific IFN-γ response is illustrated in supplementary Figure 3A. In the four patients treated with ADTs, IFN-γ responses were detectable following ADT infusion (at 2–3 months post-SCT) but not before, suggesting that ADT infusion enhanced the T-cell response to virus reactivation.
      Fig 4
      Fig. 4(A) CMV and (B) EBV responses of ADT and control cohort patients post-SCT. At each time point post-SCT, T-cell responses to EBV and CMV peptides were assayed by IFN-γ ELISpot and viral load was measured by peripheral blood PCR. PCR, polymerase chain reaction; SFUs, spot-forming units.
      Of the five patients who experienced CMV reactivation, four were evaluable by CMV-specific pentamer analysis (three patients in the ADT group and one patient in the control group). CMV-specific CD8 responses were detectable in two of these patients by 4 months post-SCT (see supplementary Figure 3), in each case correlating with prior CMV viremia. The remaining eight of 12 ADT patients and seven of eight control patients did not experience CMV reactivation and did not show significant IFN-γ responses to CMV.
      All 21 patients were at risk of EBV reactivation through either patient or donor seropositivity (Table 2). Four patients in the ADT arm and one patient in the control arm experienced EBV reactivation. Seven of 12 evaluable patients in the ADT group had significant (>20 spot-forming units/105 cells) IFN-γ responses to EBV peptides compared with four of eight controls. Once again, these EBV- specific responses were seen mostly in patients with EBV reactivation and tended to occur somewhat later than recovery of CMV-specific responses (Figure 4B).

      GVHD and toxicity

      The incidence of acute and chronic GVHD was comparable in the ADT and control cohorts (Table 3). Overall, acute GVHD 1–4 occurred in 17 of 21 patients. Acute GVHD grade ≥2 occurred in seven of 13 (54%) patients in the ADT group versus four of eight (50%) patients in the control group (P > 0.99). Severe acute GVHD (grade 3) occurred in three of 13 (23%) ADT patients and two of eight (25%) control patients (P > 0.99). No cases of grade 4 acute GVHD were observed. Chronic GVHD affected three patients in each cohort (one mild, one moderate and one severe in the ADT group and three mild in the control group).
      Table 3Incidence of acute and chronic GVHD.
      Type of GVHDReported gradeADT arm, n (%)

      (N = 13)
      Control arm, n (%)

      (N = 8)
      Acute GVHD14 (31)2 (25)
      24 (31)2 (25)
      3–4a3 (23)2 (25)
      Chronic GVHDMild1 (8)3 (38)
      Moderate1 (8)0 (0)
      Severe1 (8)0 (0)
      aNo cases of grade 4 acute GVHD occurred.
      Overall, eight of 21 patients received systemic steroid treatment for acute or chronic GVHD within the first 4 months post- SCT: four of 13 in the ADT arm and four of eight in the control arm. Overall, toxicity was mostly transplant-related; grade 3–4 toxicities are summarized in supplementary Table 3. A total of 11 of 13 (85%) patients in the ADT group experienced at least one grade 3–4 toxicity versus four of eight (50%) patients in the control arm. Cytopenias accounted for most of these events in both arms.

      Chimerism

      Four patients had mixed chimerism at month 1 post-SCT, of whom two were in the ADT group and two were in the control group. Two of these patients converted to full chimerism by month 12. Three additional patients in the ADT arm went on to develop mixed chimerism between 2 months and 3 months post-transplant; all had converted to 100% chimerism by month 12.

      Patient outcomes

      The median follow-up time was 14 months. Seven patients relapsed or progressed: five of 13 in the study arm and two of eight in the control arm. The 1-year overall survival rate was 92% for patients treated with ADTs compared with 88% in the controls, and 1-year DFS rate was 68% in the ADT group versus 63% in the control group. Three patients died during the 12-month follow-up: two of 13 (C and K) in the ADT group as a result of disease progression/relapse and one of eight (T) in the control group as a result of TRM (viral infection). Two further patients, both in the ADT arm, died after 12-month follow-up: patient F as a result of relapse at 20.9 months post-transplant and patient H as a result of pneumonia at 14.5 months post-transplant.

      Discussion

      The authors report the outcome of the first prospective randomized clinical trial assessing the safety and biological efficacy of ADTs in patients undergoing alemtuzumab-conditioned MUD SCT. This approach proved to be feasible and safe, with comparable 1-year overall survival and DFS for the treatment and control groups.
      Importantly, the authors noted no significant increase in GVHD associated with ADT infusion in the study. The overall incidence of significant (grade 2–4) acute GVHD (seven of 13 patients in the ADT group versus four of eight patients in the control group) and chronic GVHD (three of 13 patients in the ADT arm versus three of eight patients in the control arm) did not differ significantly between the two cohorts. It should be noted, however, that the incidence of significant acute GVHD (11 of 21 [52%] patients) in the authors’ cohort is higher than that reported in other studies on unrelated donor HSCT using alemtuzumab [
      • Kottaridis PD
      • Milligan DW
      • Chopra R
      • Chakraverty RK
      • Chakrabarti S
      • Robinson S
      • et al.
      In vivo CAMPATH-1H prevents graft-versus-host disease following nonmyeloablative stem cell transplantation.
      ,
      • Kanda J
      • Lopez RD
      • Rizzieri DA.
      Alemtuzumab for the prevention and treatment of graft-versus-host disease.
      ,
      • Van Besien K
      • Kunavakkam R
      • Rondon G
      • De Lima M
      • Artz A
      • Oran B
      • et al.
      Fludarabine-Melphalan Conditioning for AML and MDS: Alemtuzumab Reduces Acute and Chronic GVHD without Affecting Long-Term Outcomes.
      ,
      • Ali R
      • Ramdial J
      • Algaze S
      • Beitinjaneh A
      The role of anti-thymocyte globulin or alemtuzumab-based serotherapy in the prophylaxis and management of graft-versus-host disease.
      ,
      • Malladi RK
      • Peniket AJ
      • Littlewood TJ
      • Towlson KE
      • Pearce R
      • Yin J
      • et al.
      Alemtuzumab markedly reduces chronic GVHD without affecting overall survival in reduced-intensity conditioning sibling allo-SCT for adults with AML.
      ,
      • Pérez-Simón JA
      • Kottaridis PD
      • Martino R
      • Craddock C
      • Caballero D
      • Chopra R
      • et al.
      Nonmyeloablative transplantation with or without alemtuzumab: Comparison between 2 prospective studies in patients with lymphoproliferative disorders.
      ,
      • Finazzi MC
      • Boschini C
      • Craddock C
      • Rambaldi A
      • Ward J
      • Malladi RK.
      Characteristics of graft-versus-host disease occurring after alemtuzumab-containing allogeneic stem cell transplants: incidence, organ involvement, risk factors and survival.
      ,
      • Shaw BE
      • Apperley JF
      • Russell NH
      • Craddock C
      • Liakopoulou E
      • Potter MN
      • et al.
      Unrelated donor peripheral blood stem cell transplants incorporating pre-transplant in-vivo Alemtuzumab are not associated with any increased risk of significant acute or chronic graft-versus-host disease.
      ]. This may reflect the use of peripheral blood stem cells as the stem cell source together with single-agent cyclosporine as GVHD prophylaxis and early withdrawal of immunosuppression. André-Schmutz et al. [
      • André-Schmutz I
      • Le Deist F
      • Hacein-Bey-Abina S
      • Vitetta E
      • Schindler J
      • Chedeville G
      • et al.
      Immune reconstitution without graft-versus-host disease after haemopoietic stem-cell transplantation: A phase 1/2 study.
      ] found that GVHD was more common if residual alloreactivity exceeded 1%. In the authors’ study, residual alloreactivy measured as the median residual T cell proliferation in an ADT/host cell co-culture in patients receiving ADTs was 0% in both patients who developed significant acute GVHD (n = 7) and those who did not (n = 6). Relapse rates were comparable between the ADT arm and control arm. It should be noted that ADTs are depleted of alloreactive T cells that mediate graft-versus-leukemia responses and would therefore not be predicted to prevent relapse or shift chimerism.
      The primary endpoint of the study was T-cell immune reconstitution at 4 months post-transplant, as this is the period at which patients are most likely to succumb to infection due to insufficient T-cell recovery, particularly in the setting of alemtuzumab, where T-cell reconstitution is typically not seen until 6–8 months post-transplant [
      • Willemsen L
      • Jol-van der Zijde CM
      • Admiraal R
      • Putter H
      • Jansen-Hoogendijk AM
      • Ostaijen-ten Dam MM
      • et al.
      Impact of Serotherapy on Immune Reconstitution and Survival Outcomes After Stem Cell Transplantations in Children: Thymoglobulin Versus Alemtuzumab.
      ,
      • Veys P
      • Wynn RF
      • Ahn KW
      • Samarasinghe S
      • He W
      • Bonney D
      • et al.
      Impact of immune modulation with in vivo T-cell depletion and myleoablative total body irradiation conditioning on outcomes after unrelated donor transplantation for childhood acute lymphoblastic leukemia.
      ,
      • Schmidt-Hieber M
      • Schwarck S
      • Stroux A
      • Ganepola S
      • Reinke P
      • Thiel E
      • et al.
      Immune reconstitution and cytomegalovirus infection after allogeneic stem cell transplantation: The important impact of in vivo T cell depletion.
      ]. In this study, patients treated with ADTs did not experience significant improvement in T-cell reconstitution; however, there were improved T-cell counts and function in a minority of patients in the ADT arm, although this was associated with CMV reactivation in patients C and G and acute GVHD in patient D. The authors and others have previously reported virus reactivation and GVHD as drivers of accelerated T-cell reconstitution after infusion of ADTs [
      • André-Schmutz I
      • Le Deist F
      • Hacein-Bey-Abina S
      • Vitetta E
      • Schindler J
      • Chedeville G
      • et al.
      Immune reconstitution without graft-versus-host disease after haemopoietic stem-cell transplantation: A phase 1/2 study.
      ,
      • Amrolia PJ
      • Muccioli-Casadei G
      • Huls H
      • Adams S
      • Durett A
      • Gee A
      • et al.
      Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation.
      ]. Moreover, although the T-cell repertoire was not significantly more diverse in the ADT group at 4 months post-SCT, almost all patients in this group achieved near-normal TCR diversity compared with only a minority in the control group. These data suggest that ADTs may have an effect on T-cell recovery, at least in some patients. The overall small study size and the fact that slightly fewer patients than planned were recruited on the treatment arm (13 versus 16)—with donor refusal to participate in the study being the main limiting factor—may have contributed to this failure to reach statistical significance for the primary endpoint. The observed effect (standardized mean difference, 0.43) was smaller than the (large) target of 0.85, so the authors’ study may have missed a modest treatment effect.
      Viral infections/reactivations are a leading cause of morbidity and non-relapse mortality after unrelated donor SCT [
      • Hiwarkar P
      • Gaspar HB
      • Gilmour K
      • Jagani M
      • Chiesa R
      • Bennett-Rees N
      • et al.
      Impact of viral reactivations in the era of pre-emptive antiviral drug therapy following allogeneic haematopoietic SCT in paediatric recipients.
      ,
      • Hill JA
      • Mayer BT
      • Xie H
      • Leisenring WM
      • Huang ML
      • Stevens-Ayers T
      • et al.
      The cumulative burden of double-stranded DNA virus detection after allogeneic HCT is associated with increased mortality.
      ,
      • Guenounou S
      • Borel C
      • Bérard E
      • Yon E
      • Fort M
      • Mengelle C
      • et al.
      Prognostic impact of viral reactivations in acute myeloid leukemia patients undergoing allogeneic stem cell transplantation in first complete response.
      ]. Although the authors did not demonstrate significant differences in virus-specific T-cell responses in this study, it is noteworthy that four patients in the ADT arm displayed early CMV-specific responses, suggesting that patients treated with ADTs can respond to CMV at early time points in the context of reactivation and when the graft received is from a CMV-positive donor. The low frequency of CMV reactivation in the control group precluded assessment of whether they could mount CMV-specific T-cell responses at this stage, but previous data suggest that this is unlikely [
      • Cwynarski K
      • Ainsworth J
      • Cobbold M
      • Wagner S
      • Mahendra P
      • Apperley J
      • et al.
      Direct visualization of cytomegalovirus-specific T-cell reconstitution after allogeneic stem cell transplantation.
      ]. This suggests that virus reactivation post-HSCT may trigger an expansion of ADTs.
      There are a number of potential allodepletion methodologies using different host antigen-presenting cells and depletion strategies [
      • Li Pira G
      • Di Cecca S
      • Montanari M
      • Moretta L
      • Manca F
      Specific removal of alloreactive T-cells to prevent GvHD in hemopoietic stem cell transplantation: rationale, strategies and perspectives.
      ]. At present, it is not possible to define which methodology is superior both because of a lack of standardized assays of residual alloreactivity/anti-viral responses and because clinical studies are not easily comparable. The authors utilized stimulation of donor T cells with host DCs followed by immunomagnetic CD25/CD71 depletion. Although the authors’ production methodology is relatively straightforward, it proved logistically challenging to obtain unstimulated donor leukapheresis prior to stem cell harvest. This is a hurdle that is more easily overcome in the haploidentical SCT setting.
      Roy et al. [
      • Roy DC
      • Lachance S
      • Cohen S
      • Delisle JS
      • Kiss T
      • Sauvageau G
      • et al.
      Allodepleted T-cell immunotherapy after haploidentical haematopoietic stem cell transplantation without severe acute graft-versus-host disease (GVHD) in the absence of GVHD prophylaxis.
      ,
      • Roy DC
      • Walker I
      • Maertens J
      • Lewalle P
      • Olavarria E
      • Selleslag D
      • et al.
      ATIR101 administered after T-cell-depleted haploidentical HSCT reduces NRM and improves overall survival in acute leukemia.
      ] recently reported promising results in CD34-selected haploidentical SCT patients receiving ATIR101, an ADT product obtained through photodepletion. Photodepletion seems to spare some regulatory T cells [
      • Mielke S
      • Nunes R
      • Rezvani K
      • Fellowes VS
      • Venne A
      • Solomon SR
      • et al.
      A clinical-scale selective allodepletion approach for the treatment of HLA-mismatched and matched donor-recipient pairs using expanded T lymphocytes as antigen-presenting cells and a TH9402-based photodepletion technique.
      ] that would be depleted with an anti-CD25 approach. In phase 1 and phase 2 clinical trials [
      • Roy DC
      • Lachance S
      • Cohen S
      • Delisle JS
      • Kiss T
      • Sauvageau G
      • et al.
      Allodepleted T-cell immunotherapy after haploidentical haematopoietic stem cell transplantation without severe acute graft-versus-host disease (GVHD) in the absence of GVHD prophylaxis.
      ,
      • Roy DC
      • Walker I
      • Maertens J
      • Lewalle P
      • Olavarria E
      • Selleslag D
      • et al.
      ATIR101 administered after T-cell-depleted haploidentical HSCT reduces NRM and improves overall survival in acute leukemia.
      ], ATIR101 proved to be safe with a low rate of significant acute GVHD (four of 23 [17%] patients), no severe acute GVHD and reduced non-relapse mortality compared with T-cell-depleted haploidentical transplant, resulting in rates of GVHD- and relapse-free survival similar to those of MUD SCT. Unfortunately, however, a phase 3 registration study of this product is no longer being taken forward.

      Conclusions

      To date, adoptive transfer of ADTs has shown great promise in improving immune reconstitution after ex vivo T-cell-depleted haploidentical SCT [
      • André-Schmutz I
      • Le Deist F
      • Hacein-Bey-Abina S
      • Vitetta E
      • Schindler J
      • Chedeville G
      • et al.
      Immune reconstitution without graft-versus-host disease after haemopoietic stem-cell transplantation: A phase 1/2 study.
      ,
      • Amrolia PJ
      • Muccioli-Casadei G
      • Huls H
      • Adams S
      • Durett A
      • Gee A
      • et al.
      Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation.
      ,
      • Roy DC
      • Walker I
      • Maertens J
      • Lewalle P
      • Olavarria E
      • Selleslag D
      • et al.
      ATIR101 administered after T-cell-depleted haploidentical HSCT reduces NRM and improves overall survival in acute leukemia.
      ]. By contrast, in the present randomized study in the unrelated donor setting, the authors were not able to demonstrate accelerated T-cell recovery with ADTs. A number of potential factors may contribute to this difference. In haploidentical SCT, anti-thymocyte globulin is given as serotherapy, whereas in the authors’ unrelated donor cohort, alemtuzumab was used. Alemtuzumab is known to have a longer half-life in vivo than anti-thymocyte globulin and—although pharmacokinetics are highly variable between patients—can remain in the circulation at potentially lympholytic levels at day 30 and up to 2 months post in vivo administration at doses similar to those utilized in the authors’ study [
      • Peggs KS
      • Verfuerth S
      • Pizzey A
      • Khan N
      • Guiver M
      • Moss PA
      • et al.
      Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines.
      ,
      • Ma L
      • Han X
      • Jiang S
      • Meng Q
      • Zhang L
      • Bao H.
      Haploidentical stem cell transplantation vs matched unrelated donor transplantation in adults with hematologic malignancies: a systematic review and meta-analysis.
      ]. Unfortunately, the authors did not measure circulating alemtuzumab levels in the present study, and it is possible that this may have abrogated the beneficial effect of ADTs. It should be noted, however, that the level of alemtuzumab that is truly lytic to T cells in vivo is not clear, and the authors have previously demonstrated expansion of CMV-specific cytotoxic T lymphocytes adoptively transferred as early as day 32 after alemtuzumab-based reduced-intensity conditioning [
      • Peggs KS
      • Verfuerth S
      • Pizzey A
      • Khan N
      • Guiver M
      • Moss PA
      • et al.
      Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines.
      ]. Further ex vivo T-cell depletion in the haploidentical setting results in more profound T-cell depletion than that achieved through alemtuzumab [
      • Ma L
      • Han X
      • Jiang S
      • Meng Q
      • Zhang L
      • Bao H.
      Haploidentical stem cell transplantation vs matched unrelated donor transplantation in adults with hematologic malignancies: a systematic review and meta-analysis.
      ]. T-cell-depleted haploidentical SCT patients therefore have a priori a lower risk of GVHD. However, in MUD transplants, where the graft is less rigorously T-cell-depleted, ADTs are suppressed by cyclosporine and GVHD is more frequent, resulting in the use of steroids, which further suppress ADT expansion. In this study, 11 of 13 patients in the ADT arm were on cyclosporine at the time of their last ADT dose. Moreover, eight of 21 patients (including four of 13 patients in the ADT arm) received early systemic steroids post-transplant compared with two of 16 patients in the authors’ previous study conducted in the haploidentical setting [
      • Amrolia PJ
      • Muccioli-Casadei G
      • Huls H
      • Adams S
      • Durett A
      • Gee A
      • et al.
      Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation.
      ]. Thus, the authors’ data indicate that immunotherapy with ADTs may be best applied in transplant platforms where the graft is rigorously T-cell-depleted (e.g., with CD34 selection). Whether this approach can be effective in improving immune reconstitution after haploidentical HSCT following TCR-αβ/CD19 depletion or post-transplant cyclophosphamide will require further studies.

      Funding

      This work was funded by a grant from the Medical Research Council Developmental Clinical Studies scheme (K007491) and supported by the National Institute for Health Research Biomedical Research Centers at Great Ormond Street Hospital for Children and University College London Hospital. PJA is the recipient of a National Institute for Health and Care Research research professorship (grant no. 514413).

      Author Contributions

      Conception and design of the study: KSP, SJA, PJA, BP, FC, KC, OS, LC, AL, TT, TP, MF, SM and ET. Acquisition of data: KSP, SJA, CI, RR, JC, RW, AG, SG, AC, MS, BF, TT, TP, MF, SM and ET. Analysis and interpretation of data: KSP, PJA, MO, CI, RR, JC, RW, AG, SG, AC, MS, BF, AL, SA and KCG. Drafting or revising the manuscript: MO, PJA, KSP, SJA, BP, FC, KC, OS and LC. All authors have approved the final article.

      Declaration of Competing Interest

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

      Acknowledgments

      The authors thank Prof Claire Harrison (Guy's and St Thomas’ National Health Service Foundation Trust), Dr John Moppett (University Hospitals Bristol and Weston National Health Service Foundation Trust), Prof David Miles (Mount Vernon Cancer Center), Dr Paul Silcocks (University of Liverpool) and Dr Caroline Kelly (Cancer Research UK Clinical Trials Unit, Glasgow, Scotland) for providing study oversight as the independent data monitoring committee (current and former members). The authors also thank all patients and their families as well as participating sites, donor registries and their staff for their support during the study.

      Appendix. Supplementary materials

      References

        • Willemsen L
        • Jol-van der Zijde CM
        • Admiraal R
        • Putter H
        • Jansen-Hoogendijk AM
        • Ostaijen-ten Dam MM
        • et al.
        Impact of Serotherapy on Immune Reconstitution and Survival Outcomes After Stem Cell Transplantations in Children: Thymoglobulin Versus Alemtuzumab.
        Biol Blood Marrow Transplant. 2015; 21: 473-482
        • Kottaridis PD
        • Milligan DW
        • Chopra R
        • Chakraverty RK
        • Chakrabarti S
        • Robinson S
        • et al.
        In vivo CAMPATH-1H prevents graft-versus-host disease following nonmyeloablative stem cell transplantation.
        Blood. 2000; 96: 2419-2425
        • Saif MA
        • Borrill R
        • Bigger BW
        • Lee H
        • Logan A
        • Poulton K
        • et al.
        In vivo T-cell depletion using alemtuzumab in family and unrelated donor transplantation for pediatric non-malignant disease achieves engraftment with low incidence of graft vs. host disease.
        Pediatr Transplant. 2015; 19: 211-218
        • Kanda J
        • Lopez RD
        • Rizzieri DA.
        Alemtuzumab for the prevention and treatment of graft-versus-host disease.
        Int. J. Hematol. 2011; 93: 586-593
        • Veys P
        • Wynn RF
        • Ahn KW
        • Samarasinghe S
        • He W
        • Bonney D
        • et al.
        Impact of immune modulation with in vivo T-cell depletion and myleoablative total body irradiation conditioning on outcomes after unrelated donor transplantation for childhood acute lymphoblastic leukemia.
        Blood. 2012; 119: 6155-6161
        • Chakrabarti S
        • Mautner V
        • Osman H
        • Collingham KE
        • Fegan CD
        • Klapper PE
        • et al.
        Adenovirus infections following allogeneic stem cell transplantation: Incidence and outcome in relation to graft manipulation, immunosuppression, and immune recovery.
        Blood. 2002; 100: 1619-1627
        • Chakrabarti S
        • Mackinnon S
        • Chopra R
        • Kottaridis PD
        • Peggs K
        • O'Gorman P
        • et al.
        High incidence of cytomegalovirus infection after nonmyeloablative stem cell transplantation: Potential role of Campath-1H in delaying immune reconstitution.
        Blood. 2002; 99: 4357-4363
        • Cupit-Link MC
        • Nageswara Rao A
        • Warad DM
        • Rodriguez V
        • Khan S
        EBV-PTLD, adenovirus, and CMV in pediatric allogeneic transplants with alemtuzumab as part of pretransplant conditioning: A retrospective single center study.
        J Pediatr Hematol Oncol. 2018; 40: e473-e478
        • Ciceri F
        • Bordignon C.
        Suicide-gene-transduced donor T-cells for controlled graft-versus-host disease and graft-versus-tumor.
        Int J Hematol. 2002; 76: 305-309
        • Di Stasi A
        • Tey S-K
        • Dotti G
        • Fujita Y
        • Kennedy-Nasser A
        • Martinez C
        • et al.
        Inducible Apoptosis as a Safety Switch for Adoptive Cell Therapy.
        N Engl J Med. 2011; 365: 1673-1683
        • Heslop HE
        • Slobod KS
        • Pule MA
        • Hale GA
        • Rousseau A
        • Smith CA
        • et al.
        Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients.
        Blood. 2010; 115: 925-935
        • Peggs KS
        • Thomson K
        • Samuel E
        • Dyer G
        • Armoogum J
        • Chakraverty R
        • et al.
        Directly selected cytomegalovirus-reactive donor T cells confer rapid and safe systemic reconstitution of virus-specific immunity following stem cell transplantation.
        Clin Infect Dis. 2011; 52: 49-57
        • Mackinnon S
        • Thomson K
        • Verfuerth S
        • Peggs K
        • Lowdell M.
        Adoptive cellular therapy for cytomegalovirus infection following allogeneic stem cell transplantation using virus-specific T cells.
        Blood Cells, Mol Dis. 2008; 40: 63-67
        • Ottaviano G
        • Chiesa R
        • Feuchtinger T
        • Vickers M
        • Dickinson A
        • Gennery A
        • et al.
        Adoptive T Cell Therapy Strategies for Viral Infections in Patients Receiving Haematopoietic Stem Cell Transplantation.
        Cells. 2019; 8: 47
        • Leen AM
        • Bollard CM
        • Mendizabal AM
        • Shpall EJ
        • Szabolcs P
        • Antin JH
        • et al.
        Multicenter study of banked third-party virus-specific T cells to treat severe viral infections after hematopoietic stem cell transplantation.
        Blood. 2013; 121: 5113-5123
        • Tzannou I
        • Papadopoulou A
        • Naik S
        • Leung K
        • Martinez CA
        • Ramos CA
        • et al.
        Off-the-shelf virus-specific T cells to treat BK virus, human herpesvirus 6, cytomegalovirus, Epstein-Barr virus, and adenovirus infections after allogeneic hematopoietic stem-cell transplantation.
        J Clin Oncol. 2017; 35: 3547-3557
        • Roex MCJ
        • van Balen P
        • Germeroth L
        • Hageman L
        • van Egmond E
        • Veld SAJ
        • et al.
        Generation and infusion of multi-antigen-specific T cells to prevent complications early after T-cell depleted allogeneic stem cell transplantation—a phase I/II study.
        Leukemia. 2020; 34: 831-844
        • Lucarelli B
        • Merli P
        • Bertaina V
        • Locatelli F.
        Strategies to accelerate immune recovery after allogeneic hematopoietic stem cell transplantation.
        Expert Rev Clin Immunol. 2016; 12: 343-358
        • Dodero A
        • Carniti C
        • Raganato A
        • Vendramin A
        • Farina L
        • Spina F
        • et al.
        Haploidentical stem cell transplantation after a reduced-intensity conditioning regimen for the treatment of advanced hematologic malignancies: Posttransplantation CD8-depleted donor lymphocyte infusions contribute to improve T-cell recovery.
        Blood. 2009; 113: 4771-4779
        • Meyer RG
        • Britten CM
        • Wehler D
        • Bender K
        • Hess G
        • Konur A
        • et al.
        Prophylactic transfer of CD8-depleted donor lymphocytes after T-cell-depleted reduced-intensity transplantation.
        Blood. 2007; 109: 374-382
        • Orti G
        • Lowdell M
        • Fielding A
        • Samuel E
        • Pang K
        • Kottaridis P
        • et al.
        Phase i study of high-stringency CD8 depletion of donor leukocyte infusions after allogeneic hematopoietic stem cell transplantation.
        Transplantation. 2009; 88: 1312-1318
        • Triplett BM
        • Muller B
        • Kang G
        • Li Y
        • Cross SJ
        • Moen J
        • et al.
        Selective T-cell depletion targeting CD45RA reduces viremia and enhances early T-cell recovery compared with CD3-targeted T-cell depletion.
        Transpl Infect Dis. 2018; 20https://doi.org/10.1111/tid.12823
        • Bleakley M
        • Heimfeld S
        • Loeb KR
        • Jones LA
        • Chaney C
        • Seropian S
        • et al.
        Outcomes of acute leukemia patients transplanted with naive T cell-depleted stem cell grafts.
        J Clin Invest. 2015; 125: 2677-2689
        • Davies JK
        • Gribben JG
        • Brennan LL
        • Yuk D
        • Nadler LM
        • Guinan EC
        Outcome of alloanergized haploidentical bone marrow transplantation after ex vivo costimulatory blockade: Results of 2 phase 1 studies.
        Blood. 2008; 112: 2232-2241
        • Davies JK
        • Brennan LL
        • Wingard JR
        • Cogle CR
        • Kapoor N
        • Shah AJ
        • et al.
        Infusion of Alloanergized Donor Lymphocytes after CD34-selected Haploidentical Myeloablative Hematopoietic Stem Cell Transplantation.
        Clin Cancer Res. 2018; 24: 4098-4109
        • André-Schmutz I
        • Le Deist F
        • Hacein-Bey-Abina S
        • Vitetta E
        • Schindler J
        • Chedeville G
        • et al.
        Immune reconstitution without graft-versus-host disease after haemopoietic stem-cell transplantation: A phase 1/2 study.
        Lancet. 2002; 360: 130-137
        • Samarasinghe S
        • Mancao C
        • Pule M
        • Nawroly N
        • Karlsson H
        • Brewin J
        • et al.
        Functional characterization of alloreactive T cells identifies CD25 and CD71 as optimal targets for a clinically applicable allodepletion strategy.
        Blood. 2010; 115: 396-407
      1. Amrolia PJ, Muccioli-Casadei G, Yvon E, Huls H, Sili U, Wieder ED et al. Selective depletion of donor alloreactive T cells without loss of antiviral or antileukemic responses. Blood 2003; 102(6): 2292-9.

        • Mielke S
        • Nunes R
        • Rezvani K
        • Fellowes VS
        • Venne A
        • Solomon SR
        • et al.
        A clinical-scale selective allodepletion approach for the treatment of HLA-mismatched and matched donor-recipient pairs using expanded T lymphocytes as antigen-presenting cells and a TH9402-based photodepletion technique.
        Blood. 2008; 111: 4392-4402
        • Albon SJ
        • Mancao C
        • Gilmour K
        • White G
        • Ricciardelli I
        • Brewin J
        • et al.
        Optimization of methodology for production of CD25/CD71 allodepleted donor t cells for clinical use.
        Cytotherapy. 2013; 15: 109-121
        • Li Pira G
        • Di Cecca S
        • Montanari M
        • Moretta L
        • Manca F
        Specific removal of alloreactive T-cells to prevent GvHD in hemopoietic stem cell transplantation: rationale, strategies and perspectives.
        Blood Rev. 2016; 30: 297-307
        • Solomon SR
        • Mielke S
        • Savani BN
        • Montera A
        • Wisch L
        • Childs R
        • et al.
        Selective depletion of alloreactive donor lymphocytes: A novel method to reduce the severity of graft-versus-host disease in older patients undergoing matched sibling donor stem cell transplantation.
        Blood. 2005; 106: 1123-1129
        • Amrolia PJ
        • Muccioli-Casadei G
        • Huls H
        • Adams S
        • Durett A
        • Gee A
        • et al.
        Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation.
        Blood. 2006; 108: 1797-1808
        • Roy DC
        • Lachance S
        • Cohen S
        • Delisle JS
        • Kiss T
        • Sauvageau G
        • et al.
        Allodepleted T-cell immunotherapy after haploidentical haematopoietic stem cell transplantation without severe acute graft-versus-host disease (GVHD) in the absence of GVHD prophylaxis.
        Br J Haematol. 2019; 186: 754-766
        • Roy DC
        • Walker I
        • Maertens J
        • Lewalle P
        • Olavarria E
        • Selleslag D
        • et al.
        ATIR101 administered after T-cell-depleted haploidentical HSCT reduces NRM and improves overall survival in acute leukemia.
        Leukemia. 2020; 34: 1907-1923
        • Rubinstein LV
        • Korn EL
        • Freidlin B
        • Hunsberger S
        • Ivy SP
        • Smith MA.
        Design Issues of Randomized Phase II Trials and a Proposal for Phase II Screening Trials.
        J Clin Oncol. 2005; 23: 7199-7206
        • Van Besien K
        • Kunavakkam R
        • Rondon G
        • De Lima M
        • Artz A
        • Oran B
        • et al.
        Fludarabine-Melphalan Conditioning for AML and MDS: Alemtuzumab Reduces Acute and Chronic GVHD without Affecting Long-Term Outcomes.
        Biol Blood Marrow Transplant. 2009; 15: 610-617
        • Ali R
        • Ramdial J
        • Algaze S
        • Beitinjaneh A
        The role of anti-thymocyte globulin or alemtuzumab-based serotherapy in the prophylaxis and management of graft-versus-host disease.
        Biomedicines. 2017; 5: 67
        • Malladi RK
        • Peniket AJ
        • Littlewood TJ
        • Towlson KE
        • Pearce R
        • Yin J
        • et al.
        Alemtuzumab markedly reduces chronic GVHD without affecting overall survival in reduced-intensity conditioning sibling allo-SCT for adults with AML.
        Bone Marrow Transplant. 2009; 43: 709-715
        • Pérez-Simón JA
        • Kottaridis PD
        • Martino R
        • Craddock C
        • Caballero D
        • Chopra R
        • et al.
        Nonmyeloablative transplantation with or without alemtuzumab: Comparison between 2 prospective studies in patients with lymphoproliferative disorders.
        Blood. 2002; 100: 3121-3127
        • Finazzi MC
        • Boschini C
        • Craddock C
        • Rambaldi A
        • Ward J
        • Malladi RK.
        Characteristics of graft-versus-host disease occurring after alemtuzumab-containing allogeneic stem cell transplants: incidence, organ involvement, risk factors and survival.
        Br J Haematol. 2020; 188: 550-559
        • Shaw BE
        • Apperley JF
        • Russell NH
        • Craddock C
        • Liakopoulou E
        • Potter MN
        • et al.
        Unrelated donor peripheral blood stem cell transplants incorporating pre-transplant in-vivo Alemtuzumab are not associated with any increased risk of significant acute or chronic graft-versus-host disease.
        Br J Haematol. 2011; 153: 244-252
        • Schmidt-Hieber M
        • Schwarck S
        • Stroux A
        • Ganepola S
        • Reinke P
        • Thiel E
        • et al.
        Immune reconstitution and cytomegalovirus infection after allogeneic stem cell transplantation: The important impact of in vivo T cell depletion.
        Int J Hematol. 2010; 91: 877-885
        • Hiwarkar P
        • Gaspar HB
        • Gilmour K
        • Jagani M
        • Chiesa R
        • Bennett-Rees N
        • et al.
        Impact of viral reactivations in the era of pre-emptive antiviral drug therapy following allogeneic haematopoietic SCT in paediatric recipients.
        Bone Marrow Transplant. 2013; 48: 803-808
        • Hill JA
        • Mayer BT
        • Xie H
        • Leisenring WM
        • Huang ML
        • Stevens-Ayers T
        • et al.
        The cumulative burden of double-stranded DNA virus detection after allogeneic HCT is associated with increased mortality.
        Blood. 2017; 129: 2316-2325
        • Guenounou S
        • Borel C
        • Bérard E
        • Yon E
        • Fort M
        • Mengelle C
        • et al.
        Prognostic impact of viral reactivations in acute myeloid leukemia patients undergoing allogeneic stem cell transplantation in first complete response.
        Medicine (Baltimore). 2016; 95: e5356
        • Cwynarski K
        • Ainsworth J
        • Cobbold M
        • Wagner S
        • Mahendra P
        • Apperley J
        • et al.
        Direct visualization of cytomegalovirus-specific T-cell reconstitution after allogeneic stem cell transplantation.
        Blood. 2001; 97: 1232-1240
        • Peggs KS
        • Verfuerth S
        • Pizzey A
        • Khan N
        • Guiver M
        • Moss PA
        • et al.
        Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines.
        Lancet. 2003; 362: 1375-1377
        • Ma L
        • Han X
        • Jiang S
        • Meng Q
        • Zhang L
        • Bao H.
        Haploidentical stem cell transplantation vs matched unrelated donor transplantation in adults with hematologic malignancies: a systematic review and meta-analysis.
        Hematology. 2020; 25: 356-365