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An ISCT Stem Cell Engineering Committee Position Statement on Immune Reconstitution: the importance of predictable and modifiable milestones of immune reconstitution to transplant outcomes

      Abstract

      Allogeneic stem cell transplantation is a potentially curative therapy for some malignant and non-malignant disease. There have been substantial advances since the approaches first introduced in the 1970s, and the development of approaches to transplant with HLA incompatible or alternative donors has improved access to transplant for those without a fully matched donor. However, success is still limited by morbidity and mortality from toxicity and imperfect disease control. Here we review our emerging understanding of how reconstitution of effective immunity after allogeneic transplant can protect from these events and improve outcomes. We provide perspective on milestones of immune reconstitution that are easily measured and modifiable.

      Keywords

      Abbreviations:

      ADCC (Antibody dependent cellular cytotoxicity), CBT (Umbilical cord blood transplant), CLPs (Common lymphoid progenitors), CRFS (Chronic GvHD Relapse Free Survival), DFS (Disease Free Survival), GvHD (Graft versus Host Disease), HCT (Allogeneic hematopoietic stem cell transplant), HPCSs (Hematopoietic progenitor cells), IR (Immune Reconstitution), NRM (Non-relapse mortality), OS (Overall Survival), PTCy (Post-Transplant Cyclophosphamide), TRM (Treatment related mortality), TCD (T cell depleted)

      Introduction

      Integral to the process of allogeneic hematopoietic stem cell transplant (HCT) is a period of profound immune compromise with defects in both innate and adaptive immunity. While restoration of innate immunity typically occurs in the first month after transplant, defects in adaptive immunity persist for longer. These defects arise from the need to both eradicate the recipient immune system to prevent rejection of the non-self HSCs, and to suppress the donor immune system to avoid overwhelming hyper-acute Graft versus Host Disease (GvHD). Critical to successful HCT is re-establishing T cell immunity – especially CD4+ T cell immunity - in the post-transplant period [
      • Elfeky R.
      • et al.
      Immune reconstitution following hematopoietic stem cell transplantation using different stem cell sources.
      ]. The reconstitution of T cell immunity occurs by both homeostatic expansion of populations infused at the time of transplant as well as by de novo thymic dependent T cell production. For over two decades we have understood that the pace of this process is controlled by recipient age, donor/host HLA disparity, intensity of the conditioning regimen, method of GvHD prophylaxis, incidence of GvHD, and graft composition. Reconstitution can take up to 1-2 years, with a significant number of both pediatric and adult patients experiencing even longer lasting deficits [
      • Hakim F.T.
      • et al.
      Age-dependent incidence, time course, and consequences of thymic renewal in adults.
      ,
      • Mackall C.L.
      • et al.
      AGE, THYMOPOIESIS, AND CD4+ T-LYMPHOCYTE REGENERATION AFTER INTENSIVE CHEMOTHERAPY.
      ,
      • de Koning C.
      • Nierkens S.
      • Boelens J.J.
      Strategies before, during, and after hematopoietic cell transplantation to improve T-cell immune reconstitution.
      ,
      • van den Brink M.R.
      • Velardi E.
      • Perales M.A.
      Immune reconstitution following stem cell transplantation.
      ].
      While some of the factors controlling immune reconstitution are not modifiable – e.g. recipient age – others are. Approaches to limit the incidence of GvHD have profound impacts on both the incidence of GvHD and the pace of post-transplant immune reconstitution. Standard approaches for prevention of GvHD employ serotherapy [anti-thymocyte globulin (ATG) and Alemtuzumab] to eliminate T cells by Antibody-dependent Cellular Cytotoxicity (ADCC), calcineurin inhibitors to limit T cell activation [
      • Schreiber S.L.
      • Crabtree G.R.
      The mechanism of action of cyclosporin A and FK506.
      ], and methotrexate and mycophenolate mofetil to limit proliferation [
      • Allison A.C.
      Immunosuppressive drugs: the first 50 years and a glance forward.
      ]. These approaches are effective in limiting the incidence of GvHD in the setting of fully HLA matched HCT and in Cord Blood transplant; but are insufficient to control GvHD arising in the HLA disparate transplant setting. In addition to serotherapy, approaches to limit the incidence of GvHD by eliminating donor T cells either before or after infusion have included in vivo and ex vivo depletion of T cells (reviewed in [
      • Gooptu M.
      • Antin J.H.
      GVHD Prophylaxis 2020.
      ]). There are currently three primary methods for accomplishing T cell depletion: ex vivo positive selection of CD34+ HSCs, ex vivo negative selection of α/β T cells, and in vivo depletion with post-transplant cyclophosphamide (PTCy). At present, all three approaches are in clinical use [
      • Al Malki M.M.
      • et al.
      Proceedings From the Fourth Haploidentical Stem Cell Transplantation Symposium (HAPLO2016), San Diego, California, December 1, 2016.
      ]. However, any technique to limit the number and function of T cells in the post-transplant period also risks delaying immune reconstitution and, depending on the indication for transplant, increasing the risk of rejection and relapse. Several factors influence the incidence of GvHD and the pace of immune reconstitution with these approaches to HCT including the type of cells depleted [i.e., T cells only versus other populations such as B cells and natural killer (NK) cells]. The few multicenter trials comparing T cell depletion (TCD) with conventional approaches to GvHD prophylaxis have predictably demonstrated a decreased incidence of acute and chronic GvHD, but also an increased incidence of infections in the TCD setting such that this has not translated into improved overall survival [
      • Malard F.
      • et al.
      Ex vivo and in vivo T cell-depleted allogeneic stem cell transplantation in patients with acute myeloid leukemia in first complete remission resulted in similar overall survival: on behalf of the ALWP of the EBMT and the MSKCC.
      ,
      • Mitsuyasu R.T.
      • et al.
      Treatment of donor bone marrow with monoclonal anti-T-cell antibody and complement for the prevention of graft-versus-host disease. A prospective, randomized, double-blind trial.
      ]. Most recently, a multi-center BMT CTN trial compared standard calcineurin-inhibitor based GvHD prophylaxis to two different platforms for T cell depletion with preliminary results presented in abstract form (LBA1, TCT 2021) reporting superior chronic GvHD relapse free survival (CRFS) but inferior overall survival (OS) driven by increased treatment related mortality (TRM) in the ex vivo CD34+ selected HCT arm. Across platforms, the post-HCT immune reconstitution has demonstrated a key role in improving non-relapse and in some settings, relapse related outcomes [
      • Ishaqi M.K.
      • et al.
      Early lymphocyte recovery post-allogeneic hematopoietic stem cell transplantation is associated with significant graft-versus-leukemia effect without increase in graft-versus-host disease in pediatric acute lymphoblastic leukemia.
      ,
      • Le Blanc K.
      • et al.
      Lymphocyte Recovery Is a Major Determinant of Outcome after Matched Unrelated Myeloablative Transplantation for Myelogenous Malignancies.
      ,
      • Savani B.N.
      • et al.
      Absolute lymphocyte count on day 30 is a surrogate for robust hematopoietic recovery and strongly predicts outcome after T cell-depleted allogeneic stem cell transplantation.
      ].
      STATEMENT: Successful HCT requires balancing the need for immune suppression to achieve engraftment and control of GvHD while limiting the deleterious effects of delaying immune reconstitution. There is now sufficient support to use established milestones of immune reconstitution as primary endpoints in prospective trials. In addition, milestones or biomarkers of immune reconstitution can be used for risk-based stratification of the management of complications of transplant such as viral reactivations and GvHD and to initiate revaccination.

      Milestones of Immune Reconstitution

      The best demonstration of complete restoration of the adaptive immune system after HCT is response to vaccination with specific antibody production. It has been demonstrated that revaccination based on acquisition of immune milestones is associated with higher rates of vaccine mediated immunity [
      • Haynes A.S.
      • et al.
      An Immune Recovery-Based Revaccination Protocol for Pediatric Hematopoietic Stem Cell Transplant Recipients: Revaccination Outcomes Following Pediatric HSCT.
      ,
      • Pao M.
      • et al.
      Response to pneumococcal (PNCRM7) and haemophilus influenzae conjugate vaccines (HIB) in pediatric and adult recipients of an allogeneic hematopoietic cell transplantation (alloHCT).
      ]. However, most current recommendations are to initiate revaccination post-transplant based on time rather than acquisition of immune milestones, [
      • Carpenter P.A.
      • Englund J.A.
      How I vaccinate blood and marrow transplant recipients.
      ,
      • Cordonnier C.
      • et al.
      2017 ECIL 7 vaccine guidelines.
      ,
      • McMasters M.
      • et al.
      Casting a wider protective net: Anti-infective vaccine strategies for patients with hematologic malignancy and blood and marrow transplantation.
      ] and response to vaccination is not uniformly assessed. The COVID-19 pandemic has provided a new opportunity to evaluate response to vaccination after HCT with to date 233 HCT recipients reported in single center assessments of the efficacy of mRNA COVID-19 directed vaccination, Response was demonstrated in 75% [
      • Ram R.
      • et al.
      Safety and Immunogenicity of the BNT162b2 mRNA COVID-19 Vaccine in Patients after Allogeneic HCT or CD19-based CART therapy-A Single-Center Prospective Cohort Study.
      ] 38.2% [
      • Easdale S.
      • et al.
      Serologic Responses following a Single Dose of SARS-Cov-2 Vaccination in Allogeneic Stem Cell Transplantation Recipients.
      ] and 83% [
      • Le Bourgeois A.
      • et al.
      Safety and Antibody Response After 1 and 2 Doses of BNT162b2 mRNA Vaccine in Recipients of Allogeneic Hematopoietic Stem Cell Transplant.
      ] of vaccinated HCT recipients. In two studies, recipients were vaccinated a median of 32 [
      • Ram R.
      • et al.
      Safety and Immunogenicity of the BNT162b2 mRNA COVID-19 Vaccine in Patients after Allogeneic HCT or CD19-based CART therapy-A Single-Center Prospective Cohort Study.
      ] and 21.8 [
      • Le Bourgeois A.
      • et al.
      Safety and Antibody Response After 1 and 2 Doses of BNT162b2 mRNA Vaccine in Recipients of Allogeneic Hematopoietic Stem Cell Transplant.
      ] months post-HCT and in one 47/55 [
      • Easdale S.
      • et al.
      Serologic Responses following a Single Dose of SARS-Cov-2 Vaccination in Allogeneic Stem Cell Transplantation Recipients.
      ] were vaccinated after 6 months, despite which all three identified a longer time from HCT as predictive of response. Other identified factors predictive of a poor response included being on immune suppression [
      • Easdale S.
      • et al.
      Serologic Responses following a Single Dose of SARS-Cov-2 Vaccination in Allogeneic Stem Cell Transplantation Recipients.
      ,
      • Le Bourgeois A.
      • et al.
      Safety and Antibody Response After 1 and 2 Doses of BNT162b2 mRNA Vaccine in Recipients of Allogeneic Hematopoietic Stem Cell Transplant.
      ] and lymphopenia [
      • Le Bourgeois A.
      • et al.
      Safety and Antibody Response After 1 and 2 Doses of BNT162b2 mRNA Vaccine in Recipients of Allogeneic Hematopoietic Stem Cell Transplant.
      ]. Short of response to vaccination, other milestones of immune reconstitution evaluated at least 6 months after HCT have been demonstrated to be associated with both response to vaccination and improved overall survival, [
      • Haynes A.S.
      • et al.
      An Immune Recovery-Based Revaccination Protocol for Pediatric Hematopoietic Stem Cell Transplant Recipients: Revaccination Outcomes Following Pediatric HSCT.
      ,
      • Forlenza C.J.
      • Small T.N.
      Live (vaccines) from New York.
      ,
      • Goldberg J.D.
      • et al.
      Early recovery of T-cell function predicts improved survival after T-cell depleted allogeneic transplant.
      ,
      • Haddad E.
      • et al.
      SCID genotype and 6-month posttransplant CD4 count predict survival and immune recovery.
      ] but have not been assessed prospectively as primary outcomes. Other investigators have evaluated immune reconstitution as a continuous variable [
      • Goldberg J.D.
      • et al.
      Early recovery of T-cell function predicts improved survival after T-cell depleted allogeneic transplant.
      ,
      • Gooptu M.
      • et al.
      Effect of Sirolimus on Immune Reconstitution Following Myeloablative Allogeneic Stem Cell Transplantation: An Ancillary Analysis of a Randomized Controlled Trial Comparing Tacrolimus/Sirolimus and Tacrolimus/Methotrexate (Blood and Marrow Transplant Clinical Trials Network/BMT CTN 0402).
      ]. The advantage of using specified milestones of immune reconstitution includes that if validated they can 1) be predictors of outcomes 2) be applied across studies and 3) identify individuals at increased risk of mortality in whom alternative either pre-emptive or adoptive approaches to foster better immune reconstitution may be appropriate.
      STATEMENT: Simple validated milestones of immune reconstitution are needed and should be prospectively evaluated as primary outcomes in HCT.

      Absolute Lymphocyte Count as a Milestone of Immune Reconstitution

      The absolute lymphocyte count (ALC) has been evaluated as a simple surrogate for rapid NK recovery by several groups [
      • Kim D.H.
      • et al.
      Clinical impact of early absolute lymphocyte count after allogeneic stem cell transplantation.
      ,
      • Savani B.N.
      • et al.
      Absolute lymphocyte count on day 30 is a surrogate for robust hematopoietic recovery and strongly predicts outcome after T cell-depleted allogeneic stem cell transplantation.
      ,
      • Le Blanc K.
      • et al.
      Lymphocyte recovery is a major determinant of outcome after matched unrelated myeloablative transplantation for myelogenous malignancies.
      ,
      • Tedeschi S.K.
      • et al.
      Early lymphocyte reconstitution is associated with improved transplant outcome after cord blood transplantation.
      ]. A study evaluating ALC at Day 21 found that an ALC ≥ 350/uL was associated with late events including lower incidence of relapse and fungal infections at 1 year and better 3 year OS and Disease Free Survival (DFFS) [
      • Kim D.H.
      • et al.
      Clinical impact of early absolute lymphocyte count after allogeneic stem cell transplantation.
      ]. This effect was subsequently evaluated in the setting of TCD HCT from matched sibling donors in which multivariable analysis identified that at Day 30 an ALC above the median of 450/uL in patients transplanted for AML (but not ALL) was associated with improved survival and less relapse, and for all patients was associated with less NRM [
      • Savani B.N.
      • et al.
      Absolute lymphocyte count on day 30 is a surrogate for robust hematopoietic recovery and strongly predicts outcome after T cell-depleted allogeneic stem cell transplantation.
      ]. While potentially the simplest of milestones to measure, these studies did not identify a specific threshold ALC that would apply across transplant platforms and need to be confirmed in modern era multicenter trials.

      NK cell Reconstitution

      NK cells are the first lymphocytes to recover and typically recover by several weeks to a month after HCT, though recovery of their effector function takes longer. In both HLA disparate TCD and HLA matched T replete HCT early NK cell reconstitution is associated with both better NRM and OS [
      • Savani B.N.
      • et al.
      Rapid natural killer cell recovery determines outcome after T-cell-depleted HLA-identical stem cell transplantation in patients with myeloid leukemias but not with acute lymphoblastic leukemia.
      ,
      • Mancusi A.
      • et al.
      Haploidentical hematopoietic transplantation from KIR ligand-mismatched donors with activating KIRs reduces nonrelapse mortality.
      ,
      • Minculescu L.
      • et al.
      Early Natural Killer Cell Reconstitution Predicts Overall Survival in T Cell-Replete Allogeneic Hematopoietic Stem Cell Transplantation.
      ,
      • Hattori N.
      • et al.
      Status of Natural Killer Cell Recovery in Day 21 Bone Marrow after Allogeneic Hematopoietic Stem Cell Transplantation Predicts Clinical Outcome.
      ].

      Milestones Extrapolated from HIV

      In early studies of individuals infected with HIV, CD4+ lymphopenia was identified as a predictor of opportunistic infections. It was demonstrated that a progressive decline in CD4+ T cell numbers was associated with progression to AIDS and an increasing spectrum of infections. Salient from this work was the initial identification of the risk of pneumocystis pneumonia at a CD4+ count of less than 200 cells/uL [
      Guidelines for prophylaxis against Pneumocystis carinii pneumonia for persons infected with human immunodeficiency virus.
      ]. The use of this threshold for initiation of prophylaxis has persisted and has been extrapolated beyond the realm of HIV. This early work also identified the modifiable risk of other opportunistic infections including candidiasis, cryptococcus, cytomegalovirus (CMV), herpes zoster and M. tuberculosis such that the incidence of each of these infections decreased as the level of CD4+ deficiency decreased [reviewed in [
      • Justiz Vaillant A.A.
      • Naik R.
      HIV-1 Associated Opportunistic Infections, in StatPearls.
      ].
      In 1999, Small et al [
      • Small T.N.
      • et al.
      Comparison of immune reconstitution after unrelated and related T-cell-depleted bone marrow transplantation: effect of patient age and donor leukocyte infusions.
      ] linked the kinetics of T cell reconstitution after TCD HCT to outcomes and demonstrated that life-threatening and fatal opportunistic infections occurred exclusively in patients whose CD4 count was less than 200 cells/uL. The delayed recovery of total and naïve CD4+ T cells is associated with prolonged inversion of the CD4+/CD8+ ratio and delayed recovery of T cell mitogen responses, which places patients at increased risk of opportunistic infections, including CMV, Epstein-Barr virus (EBV), adenovirus, and human herpesvirus-6, as well as poorer OS [
      • Goldberg J.D.
      • et al.
      Early recovery of T-cell function predicts improved survival after T-cell depleted allogeneic transplant.
      ,
      • Huang Y.T.
      • et al.
      Co-Infections by Double-Stranded DNA Viruses after Ex Vivo T Cell-Depleted, CD34(+) Selected Hematopoietic Cell Transplantation.
      ,
      • Novitzky N.
      • et al.
      Immune reconstitution at 6 months following T-cell depleted hematopoietic stem cell transplantation is predictive for treatment outcome.
      ].
      A subsequent study using this CD4+ cutoff of 200 cells/uL at 3 months confirmed a strong correlation with OS, NRM, opportunistic infections and T-cell recovery at 12 months [
      • Berger M.
      • et al.
      Lymphocyte subsets recovery following allogeneic bone marrow transplantation (BMT): CD4+ cell count and transplant-related mortality.
      ]. In a multivariate analysis, together with a higher CD34+ cell transplant dose, rapid recovery of CD4+ T cells at 3 months was a favorable prognostic factor for OS and NRM [
      • Kim D.H.
      • et al.
      Rapid helper T-cell recovery above 200 x 10 6/l at 3 months correlates to successful transplant outcomes after allogeneic stem cell transplantation.
      ].

      Milestones of CD4+ Immune Reconstitution Identified in Retrospective HCT Studies

      Two small retrospective studies [
      • Berger M.
      • et al.
      Lymphocyte subsets recovery following allogeneic bone marrow transplantation (BMT): CD4+ cell count and transplant-related mortality.
      ,
      • Fedele R.
      • et al.
      The impact of early CD4+ lymphocyte recovery on the outcome of patients who undergo allogeneic bone marrow or peripheral blood stem cell transplantation.
      ] identified thresholds for early CD4+ reconstitution that correlated with subsequent outcomes of interest. Berger and colleagues demonstrated that even as early as Day +35 differences in CD4+ T cell number were associated with subsequent outcomes of interest. A CD4+ count above versus below a median of 86/uL was associated with superior TRM due to a lower risk of lethal infections and GvHD. In multivariate analysis MSD and recipient age less than 16 years were significantly associated with a better CD4+ cell recovery. Fedele et al [
      • Fedele R.
      • et al.
      The impact of early CD4+ lymphocyte recovery on the outcome of patients who undergo allogeneic bone marrow or peripheral blood stem cell transplantation.
      ] used a receiver operating characteristic curve to find the cut-off value of early (approximately Day +20) CD4+ cell count to differentiate groups. They found a CD4 threshold of 114/uL associated with improved TRM at 2 years and OS at 5 years. In multivariate analysis stem cell source and donor type were associated with CD4+ reconstitution.
      More recently, in single center and multi-center retrospective studies initially exclusively in recipients of bone marrow or cord blood transplant and now validated in recipients of ex vivo TCD transplant, we have shown that another milestone of early CD4+ T-cell reconstitution predicts survival after HCT [
      • Admiraal R.
      • et al.
      Association between anti-thymocyte globulin exposure and CD4+ immune reconstitution in paediatric haemopoietic cell transplantation: a multicentre, retrospective pharmacodynamic cohort analysis.
      ,
      • van Roessel I.
      • et al.
      Early CD4+ T cell reconstitution as predictor of outcomes after allogeneic hematopoietic cell transplantation.
      ]. We show that a threshold of CD4+ IR of 50 CD4+ T-cells/uL within 100 days after transplantation is associated with decreased NRM, improved EFS and improved OS. A retrospective analysis of a large cohort of adult and pediatric recipients of CD34+ HCT has been performed with similar findings (manuscript under review [
      • Lakkaraja M.
      • et al.
      Anti-Thymocyte Globulin Exposure in CD34+ T Cell Depleted Allogeneic Hematopoietic Cell Transplantation.
      ]).In addition, this same measure of early CD4+ T cell reconstitution can overcome the risk associated with GvHD [
      • de Koning C.
      • et al.
      CD4+ T-cell reconstitution predicts Survival Outcomes after acute Graft-versus-Host-Disease; a dual center validation.
      ] and viral reactivation [
      • Admiraal R.
      • et al.
      Viral reactivations and associated outcomes in the context of immune reconstitution after pediatric hematopoietic cell transplantation.
      ]. This simple, easily replicated milestone is informative regardless of age, indication for transplant and transplant platform and is feasibly applied even in small centers and those with limited resources.
      Other tools for measuring immune reconstitution can add to our understanding of how a diverse T cell repertoire is reestablished post-HCT and is associated with outcomes including infections, GvHD and relapse. Early studies by complementarity determining region 3 (CDR3) spectra-typing demonstrated that skewed T-cell repertoires emerging early after TCD HCT could persist for years [
      • Wu C.J.
      • et al.
      Reconstitution of T-cell receptor repertoire diversity following T-cell depleted allogeneic bone marrow transplantation is related to hematopoietic chimerism.
      ]. T cell repertoire diversity can now be measured by next generation sequencing of V-gene and CDR3 composition and has again demonstrated restricted diversity early post-HCT with more rapid normalization seen after CBT compared to TCD transplant [
      • van Heijst J.W.
      • et al.
      Quantitative assessment of T cell repertoire recovery after hematopoietic stem cell transplantation.
      ] and can link characteristics of donor and recipient repertoire to the repertoire that emerges post-HCT [
      • Yew P.Y.
      • et al.
      Quantitative characterization of T-cell repertoire in allogeneic hematopoietic stem cell transplant recipients.
      ,
      • Mamedov I.Z.
      • et al.
      Quantitative tracking of T cell clones after haematopoietic stem cell transplantation.
      ,
      • Tickotsky-Moskovitz N.
      • et al.
      CDR3 and V genes show distinct reconstitution patterns in T cell repertoire post-allogeneic bone marrow transplantation.
      ]. This type of analysis has shown lower T cell diversity in patients not only with GvHD [
      • Yew P.Y.
      • et al.
      Quantitative characterization of T-cell repertoire in allogeneic hematopoietic stem cell transplant recipients.
      ] but preceding the development of GvHD [
      • Yew P.Y.
      • et al.
      Quantitative characterization of T-cell repertoire in allogeneic hematopoietic stem cell transplant recipients.
      ,
      • Leick M.
      • et al.
      T Cell Clonal Dynamics Determined by High-Resolution TCR-β Sequencing in Recipients after Allogeneic Hematopoietic Cell Transplantation.
      ]
      Measuring thymic output by identifying recent thymic emigrants (RTEs) or T cell receptor excision circles (TREC) can also quantify de novo lymphocyte reconstitution which is associated with reduced TRM and improved OS (recently reviewed by Velardi et al [
      • Velardi E.
      • et al.
      The role of the thymus in allogeneic bone marrow transplantation and the recovery of the peripheral T-cell compartment.
      ]). Measurements of RTEs and TREC have also been used to compare different GvHD prophylaxis regimens [
      • Törlén J.
      • et al.
      Effect of Graft-versus-Host Disease Prophylaxis Regimens on T and B Cell Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation.
      ] as well as finding similar modifiers of de novo IR of older age and ATG exposure, but are not yet easily performed on all recipients of HCT and may have limitations in measuring thymic function after HCT [
      • Gaballa A.
      • et al.
      Evaluating Thymic Function After Human Hematopoietic Stem Cell Transplantation in the Personalized Medicine Era.
      ,
      • Ringhoffer S.
      • et al.
      T-cell reconstitution after allogeneic stem cell transplantation: assessment by measurement of the sjTREC/βTREC ratio and thymic naive T cells.
      ,
      • Mikhael N.L.
      • Elsorady M.
      Clinical significance of T cell receptor excision circle (TREC) quantitation after allogenic HSCT.
      ]. Alternatively, thymic function has been measured by evaluation for early thymic emigrants by flow cytometry for eg CD31 [
      • Janeczko-Czarnecka M.
      • et al.
      Thymic activity in immune recovery after allogeneic hematopoietic stem cell transplantation in children.
      ,
      • Perlingeiro Beltrame M.
      • et al.
      Immune reconstitution in patients with Fanconi anemia after allogeneic bone marrow transplantation.
      ]. These approaches will likely be important in future studies of immune reconstitution after HCT as well as assessing thymic dysfunction in both autoimmune and alloimmune reactions post-HCT.
      Despite the promise of sophisticated measures of thymic activity to aide our assessment of immune reconstitution, they remain expensive and difficult to perform prospectively. One advantage of simple measures off IR such as ALC and early CD4 IR of 50/uL is that they are simple assessments that can be performed as part of standard of care, in real time, prospectively and are predictive of outcomes. Unlike other milestones of immune reconstitution that have not been demonstrated to predict outcomes across different transplant platforms (Table 1), early CD4 IR of 50/uL has been show to predict outcomes including NRM and OS in both pediatric and adult recipients of conventional, cord blood and TCD HCT regardless of the indication for transplant or conditioning regimen. Importantly these simple measures can be used even in regions with limited resources to identify patients at risk of inferior outcome.
      Table 1
      Author (reference)NAge RangeHCT TypesDiseaseMilestoneHow Milestone IdentifiedOutcomes Predicted
      Kim DH (24)8216-54PBSC, BM

      MA + NMA
      Malignant + AAALC 350 Day 21not describedHigher OS, DFS, and infections

      Lower Relapse and CI of CMV
      Savani (25)15710-56TCD

      MA
      Malignant + AAALC > 450 Day 30MedianHigher OS

      Lower NRM, Relapse (myeloid), GvHD and graft failure
      LeBlanc (26)1020.5–58PBSC, BM

      MA
      Malignant

      (Myeloid)
      ALC

      Day 30
      Continuous no thresholdHigher OS and RFS

      Lower TRM
      Tedeschi (27)400.6-64CBT

      MA + NMA
      Malignant

      Non-Malig
      ALC 150 & 100

      Day 30
      Continuous & DichotomousHigher OS (150)

      Lower NRM, graft failure, no association with relapse (100)
      Savani (28)5410-58TCD

      MA
      MalignantNK > 150

      Day 30
      MedianHigher OS

      Lower Relapse, NRM and aGvHD
      Minculescu (30)29816-74PBSC, BM

      MA + NMA
      MalignantNK > 150

      CD4 >200
      Validation StudyHigher OS (NK)

      Lower TRM, and infections including CMV (CD4 & NK)

      No association w/ relapse or GvHD
      Hattori (31)8118-71PBSC, BM, CBT

      MA, NMA
      MalignantBM NK

      Day 21
      MedianHigher OS and RFS

      Lower NRM and Infections
      Small (34)622-69TCD

      MA
      Malignant

      Non-Malig
      CD4 >200 PHANo specified timeLower infections/Lethal infections
      Fedele (39)9911-67PBSC, BM

      MA, RIC
      Malignant +AACD4> 115 Day 20ROC curve of CD4+Higher OS

      Lower TRM, Disease Related Mortality No association with GvHD
      Goldberg (21)3752-68TCD

      MA
      MalignantCD4 & PHA

      Day 180
      Landmark analysisHigher OS and EFS (PHA)

      Univariate Higher OS and EFS (CD4) No assoc.w/relapse
      Novitzky (36)4215-54TCD

      MA
      MalignantCD45RA

      Day 180
      MedianHigher Relapse Free Survival
      Berger (37)7584-72BM

      MA, RIC
      Malignant + AACD4 >84

      Day 35
      MedianLower TRM and lethal infections
      Kim (38)6916-58PBSC, BM

      MA
      MalignantCD4>200

      Day 100
      Prior studiesMultivariate Higher OS, Lower NRM

      Univariate Higher infection & relapse, lower cGvHD
      Admiraal (40)2510.2-23PBSC, BM, CBT

      MA, RIC
      Malignant

      Non-Malig
      CD4 >50

      Day 100
      Prior studiesHigher OS and EFS

      Lower NRM and relapse (myeloid)
      Van Roessel (41)3150.2–26BM, PBSC, CBT, TCD

      MA, RIC
      Malignant

      Non-malig
      CD4 >50

      Day 100
      Validation StudyHigher OS and EFS

      Lower NRM No impact on relapse or aGVHD
      Lakkaraja (42)5540.2-73TCDMalignant Non-maligCD4 >50

      Day 100
      Validation StudyLower NRM
      STATEMENT The milestone of early minimal CD4+ IR of 50 CD4 T cells/uL by day 100 is powerful, simple and predictive of outcomes of interest in pediatric and adult recipients of transplant for malignant and non-malignant disease and across transplant platforms. This milestone is currently being evaluated prospectively as a primary outcome in both single and multi-center trials.

      Predictors of Immune Reconstitution

      Precision dosing

      Across transplant platforms, the precise dosing of Busulfan based on levels and adjustments to achieve targeted exposure has been the standard for more than two decades. This approach has been demonstrated to improve outcomes by reducing the risk of relapse or incomplete stem cell chimerism with underdosing and toxicity with overdosing [
      • Ljungman P.
      • et al.
      High busulfan concentrations are associated with increased transplant-related mortality in allogeneic bone marrow transplant patients.
      ,
      • McCune J.S.
      • Gibbs J.P.
      • Slattery J.T.
      Plasma concentration monitoring of busulfan: does it improve clinical outcome?.
      ]. More recent work has demonstrated that the model used for these calculations has profound effects on outcomes of interest including freedom from infectious complications and emphasizes the importance of personalized dosing [
      • Bartelink I.H.
      • et al.
      Association of busulfan exposure with survival and toxicity after haemopoietic cell transplantation in children and young adults: a multicentre, retrospective cohort analysis.
      ]. Targeted dosing of other agents used in cytoreduction including melphalan [
      • Sweiss K.
      • et al.
      Predictors of increased melphalan exposure correlate with overall survival, nonrelapse mortality, and toxicities in patients undergoing reduced-intensity allogeneic stem cell transplantation with fludarabine and melphalan.
      ] and fludarabine [
      • Langenhorst J.B.
      • et al.
      Fludarabine exposure in the conditioning prior to allogeneic hematopoietic cell transplantation predicts outcomes.
      ] can also improve HCT outcomes with evidence that over-exposure to fludarabine also impairs post-HCT IR [
      • Langenhorst J.B.
      • et al.
      Fludarabine exposure in the conditioning prior to allogeneic hematopoietic cell transplantation predicts outcomes.
      ]. Current studies are assessing the role of targeted peri-HCT exposure to fludarabine and subsequent immune reconstitution.
      A major factor hampering CD4+ IR is exposure to residual anti-thymocyte globulin (ATG) at the time of infusion of the stem cell graft [
      • Admiraal R.
      • et al.
      Association between anti-thymocyte globulin exposure and CD4+ immune reconstitution in paediatric haemopoietic cell transplantation: a multicentre, retrospective pharmacodynamic cohort analysis.
      ,
      • Scordo M.
      • et al.
      Standard Antithymocyte Globulin Dosing Results in Poorer Outcomes in Overexposed Patients after Ex Vivo CD34(+) Selected Allogeneic Hematopoietic Cell Transplantation.
      ]. This residual exposure is related to the dose and timing of ATG administration [
      • Admiraal R.
      • et al.
      Association between anti-thymocyte globulin exposure and CD4+ immune reconstitution in paediatric haemopoietic cell transplantation: a multicentre, retrospective pharmacodynamic cohort analysis.
      ] as clearance is dependent on the size of the target pool at the time of administration. As a result, the effect of lymphodepleting serotherapy can vary by the specific cytoreductive regimen as was demonstrated in a randomized control trial where the ALC at the time of ATG administration varied by conditioning regimen [
      • Soiffer R.J.
      • et al.
      Prospective, Randomized, Double-Blind, Phase III Clinical Trial of Anti-T-Lymphocyte Globulin to Assess Impact on Chronic Graft-Versus-Host Disease-Free Survival in Patients Undergoing HLA-Matched Unrelated Myeloablative Hematopoietic Cell Transplantation.
      ] Exposure to ATG can be controlled by using prospective modeling to individualize ATG dose and timing such that host immune cells are exposed to appropriate ATG levels prior to transplant to minimize the risk of rejection while donor immune cells in the post-HCT period are not over-exposed [
      • Admiraal R.
      • et al.
      Association between anti-thymocyte globulin exposure and CD4+ immune reconstitution in paediatric haemopoietic cell transplantation: a multicentre, retrospective pharmacodynamic cohort analysis.
      ]. Furthermore, pharmacodynamics of ATG may be altered by exposure to growth factors early post transplantation, and recent pre-clinical and clinical studies suggest administration of filgrastim after ATG administration may be detrimental to immune reconstitution [
      • de Koning C.
      • et al.
      Filgrastim enhances T-cell clearance by antithymocyte globulin exposure after unrelated cord blood transplantation.
      ]. Similar studies have demonstrated that alemtuzumab can impair post-HCT immune reconstitution [
      • Bhoopalan S.V.
      • et al.
      Pharmacokinetics of alemtuzumab in pediatric patients undergoing ex vivo T-cell-depleted haploidentical hematopoietic cell transplantation.
      ] and prospective studies are needed to determine whether dosing nomograms can be developed to improve the predictable exposure to this agent.

      Transplant Platform

      The stem cell source and approach to GvHD prophylaxis have significant impact on the quality of post-HCT immune reconstitution. Historically, when compared with conventional peripheral blood stem cell (PBSCT) or bone marrow (BMT) transplant, cord blood transplant (CBT) has been associated with rapid NK and B cell IR, but increased infection risk due to delayed T cell IR [
      • Komanduri K.V.
      • et al.
      Delayed immune reconstitution after cord blood transplantation is characterized by impaired thymopoiesis and late memory T-cell skewing.
      ,
      • Jacobson C.A.
      • et al.
      Immune reconstitution after double umbilical cord blood stem cell transplantation: comparison with unrelated peripheral blood stem cell transplantation.
      ,
      • Ballen K.
      • et al.
      Infection Rates among Acute Leukemia Patients Receiving Alternative Donor Hematopoietic Cell Transplantation.
      ]. However, this difference in IR and infectious complications has not been consistently associated with increased infectious related mortality [
      • Parody R.
      • et al.
      Severe infections after unrelated donor allogeneic hematopoietic stem cell transplantation in adults: comparison of cord blood transplantation with peripheral blood and bone marrow transplantation.
      ]. More recent studies have demonstrated that this delay in T cell IR is strongly influenced by exposure to ATG [
      • Lindemans C.A.
      • et al.
      Impact of thymoglobulin prior to pediatric unrelated umbilical cord blood transplantation on immune reconstitution and clinical outcome.
      ], and thus modifiable. In pediatric and adult CBT recipients no or low exposure to ATG is associated with improved early CD4+ IR [
      • Politikos I.
      • et al.
      Robust CD4+ T-cell recovery in adults transplanted with cord blood and no antithymocyte globulin.
      ,
      • Admiraal R.
      • et al.
      Excellent T-cell reconstitution and survival depend on low ATG exposure after pediatric cord blood transplantation.
      ]. In addition, the exact composition of individual grafts can be associated with outcomes including IR in both conventional and TCD PBSCT/BMT and in CBT (reviewed in [
      • Storek J.
      Immunological reconstitution after hematopoietic cell transplantation - its relation to the contents of the graft.
      ]). Examples include improved early CD4+ IR in recipients of CB units with higher CD3+ composition [
      • Castillo N.
      • et al.
      Cord Blood Units with High CD3(+) Cell Counts Predict Early Lymphocyte Recovery After In Vivo T Cell-Depleted Single Cord Blood Transplantation.
      ] and higher PBSC graft composition of NK cells [
      • Minculescu L.
      • et al.
      Improved Relapse-Free Survival in Patients With High Natural Killer Cell Doses in Grafts and During Early Immune Reconstitution After Allogeneic Stem Cell Transplantation.
      ]. While graft engineering to achieve these beneficial characteristics is not uniformly possible, in the setting of conventional PBSCT the CD34 dose is frequently capped as a result of a retrospective analysis demonstrating inferior outcome in recipients of PBSCT grafts containing high doses of CD3 and CD34 cells [
      • Czerw T.
      • et al.
      High CD3+ and CD34+ peripheral blood stem cell grafts content is associated with increased risk of graft-versus-host disease without beneficial effect on disease control after reduced-intensity conditioning allogeneic transplantation from matched unrelated donors for acute myeloid leukemia - an analysis from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation.
      ]. Importantly, this cannot be extrapolated to in vivo or ex vivo TCD HCT platforms [
      • Garnier A.
      • et al.
      Absence of influence of peripheral blood CD34+ and CD3+ graft cell counts on outcomes after reduced-intensity conditioning transplantation using post-transplant cyclophosphamide.
      ,
      • Handgretinger R.
      • et al.
      Transplantation of megadoses of purified haploidentical stem cells.
      ,
      • Nakamura R.
      • et al.
      Transplant dose of CD34(+) and CD3(+) cells predicts outcome in patients with haematological malignancies undergoing T cell-depleted peripheral blood stem cell transplants with delayed donor lymphocyte add-back.
      ]. In fact, new approaches to ex vivo graft engineering such as α/β T cell and CD3/CD19 depletion allow optimization of the T cell and CD34 graft composition for recipients of ex vivo TCD HCT [
      • Reisner Y.
      • et al.
      Hematopoietic stem cell transplantation across major genetic barriers: tolerance induction by megadose CD34 cells and other veto cells.
      ,
      • Bertaina A.
      • et al.
      Unrelated donor vs HLA-haploidentical α/β T-cell- and B-cell-depleted HSCT in children with acute leukemia.
      ] resulting in reports of improved T cell IR compared to historical approaches to ex vivo TCD relying on CD34+ selection [
      • Arnold D.E.
      • et al.
      Immune Reconstitution Following TCRαβ/CD19-Depleted Hematopoietic Cell Transplantation for Hematologic Malignancy in Pediatric Patients.
      ,
      • de Witte M.A.
      • et al.
      αβ T-cell graft depletion for allogeneic HSCT in adults with hematological malignancies.
      ].
      Post-transplant cyclophosphamide (PTCy) as a method of in vivo TCD is also reported to foster improved IR over historical methods for TCD. The early NK cell reconstitution seen after other TCD and conventional transplants is blunted by the administration of cyclophosphamide [
      • Russo A.
      • et al.
      NK cell recovery after haploidentical HSCT with posttransplant cyclophosphamide: dynamics and clinical implications.
      ]. However, while cyclophosphamide eliminates rapidly dividing cells interfering with early homeostatic expansion, transferred regulatory T cells preferentially survive cyclophosphamide leading to an increased Treg/Tcon ratio [
      • Kanakry C.G.
      • et al.
      Aldehyde dehydrogenase expression drives human regulatory T cell resistance to posttransplantation cyclophosphamide.
      ,
      • Rambaldi B.
      • et al.
      Impaired T- and NK-cell reconstitution after haploidentical HCT with posttransplant cyclophosphamide.
      ]. The cessation of mycophenolate at 6 weeks after PTCy HCT has been identified as a factor allowing for CD8 T cell reconstitution comparable to that after standard conventional BMT [
      • Storek J.
      Immunological reconstitution after hematopoietic cell transplantation - its relation to the contents of the graft.
      ]. In this setting it was also demonstrated that naïve T cells can differentiate directly into memory stem T cells (Tscm) such that the expanded population of Tscm after PTCy HCT originate from naive precursors and contribute to early peripheral reconstitution by differentiating into effectors.[
      • Roberto A.
      • et al.
      Role of naive-derived T memory stem cells in T-cell reconstitution following allogeneic transplantation.
      ,
      • Cieri N.
      • et al.
      Generation of human memory stem T cells after haploidentical T-replete hematopoietic stem cell transplantation.
      ]. Current efforts to improve IR after PTCy HCT include modifying the post-HCT immune suppression [
      • Ruggeri A.
      • et al.
      Timing of Post-Transplantation Cyclophosphamide Administration in Haploidentical Transplantation: A Comparative Study on Behalf of the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation.
      ,
      • Kasamon Y.L.
      • et al.
      Shortened-Duration Tacrolimus after Nonmyeloablative, HLA-Haploidentical Bone Marrow Transplantation.
      ] and the dose and timing of serotherapy to achieve predictable exposure. One can imagine that an improved understanding of the pharmacokinetics of cyclophosphamide and its metabolites could allow targeted dosing based on the number of specific populations of T cells infused with the graft. While the use of PTCy based transplantation is easily applied in countries or regions with limited resources, it is these same areas that do not have the capacity to treat the consequences of poor IR. Simultaneously, it is important that centers with the resources to do so continue to perform in depth monitoring of immune reconstitution and explore ways to improve immune reconstitution in patients treated with this platform.
      STATEMENT: Milestones of immune reconstitution, including CD4+ IR, are modifiable by transplant regimens that include personalized exposure.

      Future perspectives

      Improving the probability of achieving early CD4+ IR after HCT is an important strategy to enhance HCT success. individualizing the conditioning regimens to allow for better and more predictable IR is one approach that could improve both thymic dependent and thymic independent IR across transplant platforms. In addition to already established approaches to individualized dosing of Busulfan and ATG, a similar approach to dosing of other serotherapies and fludarabine has the potential to further improve immune reconstitution.
      The absence of post-transplant immune suppressive medications makes the TCD platforms with CD3/CD19, α/β depletion and CD34+ selection ideal for the study of preemptive strategies such as adoptive transfer of T regs, human T cell progenitors or cytotoxic lymphocytes directed against herpes viruses. Strategies to enhance immune recovery post-transplant represent a critical unmet need in the TCD setting.

      Strategies to Enhance Thymic Dependent post-HCT Immune Reconstitution

      The thymus is the principal lymphoid organ responsible for generating and supplying naïve T cells. After allo-HCT, T-cell differentiation of donor progenitors within the recipient thymus is required in order to generate the naïve RTEs that provide a diverse TCR repertoire with robust response to pathogens. Insufficient recovery in thymopoiesis translates into a predisposition to severe infections. Approaches to enhance thymic regeneration after HCT are being adapted from pre-clinical models including sex-steroid ablation [
      • Velardi E.
      • Dudakov J.A.
      • van den Brink M.R.
      Sex steroid ablation: an immunoregenerative strategy for immunocompromised patients.
      ], infusion of pre-thymic precursors [
      • André I.
      • et al.
      Ex vivo generated human T-lymphoid progenitors as a tool to accelerate immune reconstitution after partially HLA compatible hematopoietic stem cell transplantation or after gene therapy.
      ,
      • Awong G.
      • et al.
      Human proT-cells generated in vitro facilitate hematopoietic stem cell-derived T-lymphopoiesis in vivo and restore thymic architecture.
      ,
      • Zakrzewski J.L.
      • Holland A.M.
      • van den Brink M.R.M.
      Adoptive precursor cell therapy to enhance immune reconstitution after hematopoietic stem cell transplantation.
      ], growth hormone (GH) [
      • French R.A.
      • et al.
      Age-associated loss of bone marrow hematopoietic cells is reversed by GH and accompanies thymic reconstitution.
      ], keratinocyte growth factor (KGF) [
      • Kelly R.M.
      • et al.
      Short-term inhibition of p53 combined with keratinocyte growth factor improves thymic epithelial cell recovery and enhances T-cell reconstitution after murine bone marrow transplantation.
      ], insulin-like growth factor (IGF-1) [
      • Alpdogan O.
      • et al.
      Insulin-like growth factor-I enhances lymphoid and myeloid reconstitution after allogeneic bone marrow transplantation.
      ] and interleukin-7 (IL-7) [
      • Sportès C.
      • et al.
      Administration of rhIL-7 in humans increases in vivo TCR repertoire diversity by preferential expansion of naive T cell subsets.
      ] (reviewed by [
      • Velardi E.
      • et al.
      The role of the thymus in allogeneic bone marrow transplantation and the recovery of the peripheral T-cell compartment.
      ].

      Strategies to Enhance Thymic Independent post-HCT Immune Reconstitution

      Thymic independent T cell immune reconstitution is largely dependent on graft composition and homeostatic expansion of T cells infused at the time of transplant. As a result, targeted exposure of the graft to immune ablating therapies is critical to effective immune reconstitution.
      Similarly, a better understanding of the role that graft composition plays in outcomes of interest will likely contribute to an improved ability to identify patients at risk of poor post-HCT IR. Correlations between outcomes and graft composition will depend on better identification of the frequency of pluripotent CD34+CD90+ hematopoietic stem cells (HSC) within the population of hematopoietic progenitor cells (HPSCs) identified by CD34+ and by improved enumeration of common lymphoid progenitors (CLPs). The different graft composition of these populations will likely explain some of the differences in IR between transplant platforms. Such analyses will be instrumental not only for prediction of clinical outcome, but also for optimization of novel graft engineering strategies.
      Finally, T cell reconstitution can be mediated by infusion of well characterized populations of donor derived cell products. These cell products can be defined doses of bulk T cells (DLI) [
      • Oved J.H.
      • et al.
      CD3(+)/CD19(+) Depleted Matched and Mismatched Unrelated Donor Hematopoietic Stem Cell Transplant with Targeted T Cell Addback Is Associated with Excellent Outcomes in Pediatric Patients with Nonmalignant Hematologic Disorders.
      ,
      • Amrolia P.J.
      • et al.
      Add-back of allodepleted donor T cells to improve immune reconstitution after haplo-identical stem cell transplantation.
      ,
      • Verholen F.
      • et al.
      Hodgkin's lymphoma relapsing after autologous transplantation: allogeneic hematopoietic stem cell transplantation using a strategy of reduced intensity conditioning, and T-cell depletion with T-cell add-back.
      ,
      • Geyer M.B.
      • et al.
      T cell depletion utilizing CD34(+) stem cell selection and CD3(+) addback from unrelated adult donors in paediatric allogeneic stem cell transplantation recipients.
      ,
      • Im H.J.
      • et al.
      Haploidentical HCT using an αβ T-cell-depleted graft with targeted αβ(+) cells by add-back after a reduced intensity preparative regimen containing low-dose TBI.
      ,
      • Hofmann S.
      • et al.
      Donor lymphocyte infusion leads to diversity of specific T cell responses and reduces regulatory T cell frequency in clinical responders.
      ] but the risk of provoking GvHD is high and it is increased by the presence of HLA disparity between the donor and the recipient. Adoptive cell therapies with donor-derived alloreactive-depleted (eg CD45RA-(naïve) or CD4 depleted) T cells [
      • van Balen P.
      • et al.
      CD4 Donor Lymphocyte Infusion Can Cause Conversion of Chimerism Without GVHD by Inducing Immune Responses Targeting Minor Histocompatibility Antigens in HLA Class II.
      ,
      • Müller N.
      • et al.
      Generation of alloreactivity-reduced donor lymphocyte products retaining memory function by fully automatic depletion of CD45RA-positive cells.
      ,
      • Perruccio K.
      • et al.
      Optimizing a photoallodepletion protocol for adoptive immunotherapy after haploidentical SCT.
      ,
      • Dunaikina M.
      • et al.
      Safety and efficacy of the low-dose memory (CD45RA-depleted) donor lymphocyte infusion in recipients of αβ T cell-depleted haploidentical grafts: results of a prospective randomized trial in high-risk childhood leukemia.
      ], T cells transduced with a suicide gene [
      • Vago L.
      • et al.
      T-cell suicide gene therapy prompts thymic renewal in adults after hematopoietic stem cell transplantation.
      ,
      • Zhou X.
      • et al.
      Inducible caspase-9 suicide gene controls adverse effects from alloreplete T cells after haploidentical stem cell transplantation.
      ,
      • Ciceri F.
      • et al.
      Infusion of suicide-gene-engineered donor lymphocytes after family haploidentical haemopoietic stem-cell transplantation for leukaemia (the TK007 trial): a non-randomised phase I-II study.
      ], T-cell clones or lines directed against life-threatening pathogens [
      • Gottlieb D.J.
      • et al.
      Prophylactic antigen-specific T-cells targeting seven viral and fungal pathogens after allogeneic haemopoietic stem cell transplant.
      ,
      • Walter E.A.
      • et al.
      Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.
      ,
      • Leen A.M.
      • et al.
      Cytotoxic T lymphocyte therapy with donor T cells prevents and treats adenovirus and Epstein-Barr virus infections after haploidentical and matched unrelated stem cell transplantation.
      ,
      • Koehne G.
      • et al.
      Immunotherapy with Donor T Cells Sensitized with Overlapping Pentadecapeptides for Treatment of Persistent Cytomegalovirus Infection or Viremia.
      ,
      • Rooney C.M.
      • et al.
      Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients.
      ,
      • Doubrovina E.
      • et al.
      Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+ lymphomas after allogeneic hematopoietic cell transplantation.
      ], and regulatory or conventional T cell [
      • Zorn E.
      • et al.
      Combined CD4+ donor lymphocyte infusion and low-dose recombinant IL-2 expand FOXP3+ regulatory T cells following allogeneic hematopoietic stem cell transplantation.
      ,
      • Maury S.
      • et al.
      CD4+CD25+ regulatory T cell depletion improves the graft-versus-tumor effect of donor lymphocytes after allogeneic hematopoietic stem cell transplantation.
      ,
      • Brunstein C.G.
      • et al.
      Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics.
      ,
      • Brunstein C.G.
      • et al.
      Umbilical cord blood-derived T regulatory cells to prevent GVHD: kinetics, toxicity profile, and clinical effect.
      ,
      • Theil A.
      • et al.
      T cell receptor repertoires after adoptive transfer of expanded allogeneic regulatory T cells.
      ,
      • Di Ianni M.
      • et al.
      Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation.
      ] infusions after -HCT (including after haploidentical HCT) can reduce transplantation-related mortality due to infectious complications. The results reported so far are encouraging, but they have been obtained primarily in single center trials in a limited number of patients. In addition, while donor derived viral specific T cells have been demonstrated to persist long term [
      • Heslop H.E.
      • et al.
      Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients.
      ] this approach may not contribute to broad protection or overall immune reconstitution [
      • Fabrizio V.A.
      • et al.
      Adoptive therapy with CMV-specific cytotoxic T lymphocytes depends on baseline CD4+ immunity to mediate durable responses.
      ]. A comparison of the efficacy of these different cell therapy approaches requires confirmation in larger cohorts of patients, homogeneous in terms of disease and treatment. Most importantly, future efforts focused on standardizing and simplifying the manufacture of these cell-therapy products are needed to make them accessible to a broad number of patients including those in resource poor regions.
      Statement
      Understanding the role of graft composition and control of peri-transplant exposure to immune-ablating therapies will improve the ability to minimize the number of patients at risk for poor post-HCT immune reconstitution. For those at risk, adoptive T-cell therapies can accelerate reconstitution of cellular immunity with enhanced antitumor effects following HCT.
      OVERALL STATEMENT
      Milestones of immune reconstitution that are predictable and modifiable are associated with improved HCT outcomes. A simple, early milestone of CD4+IR (50/uL) by day 100 that is validated across transplant platforms and can be used in regions with limited resources, should be validated prospectively, and used to stratify transplant recipients for interventions to improve CD+4IR or compensate for its absence.

      Conflicts of Interest

      Allistair Abraham: Served on the safety monitoring committee for Sangamo Therapeutics and has no financial interest in the development of gene therapies
      Jaap Jan Boelens: Consulting: Avrobio, BlueRock, Race Oncology, Advanced Clinical, Omeros, Sanofi, Medexus
      Carmem Bonfim: Consulting: Zodiac, Amgen, Novartis
      Sandra Cohen: ExCellThera: royalties, consulting, shares
      Susan Prockop: Receives support for the conduct of clinical trials through MSK from AlloVir, Atara, and Jasper. Inventor of IP related to development of third party viral specific T cells program with all rights assigned to MSK

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