Effects of T-Cell Depletion on Allogeneic Hematopoietic Stem Cell Transplantation Outcomes in AML Patients.

1. Introduction

Cytogenetic risk stratification in acute myelogenous leukemia (AML) allows clinicians to determine which patients are most likely to benefit from allogeneic hematopoietic stem cell transplantation (HCT), with evidence to support a survival advantage in patients with intermediate or high-risk cytogenetics [ 1 ]. In addition to more accurate patient selection based on cytogenetic risk factors, over the past decades, transplantation outcomes have also improved as a result of more accurate patient selection tools, such as the HCT Sorror comorbidity index [ 2 ], improvements in HLA matching techniques and supportive care.

Despite these improvements, graft- versus -host disease (GVHD) remains a leading cause of post-transplant morbidity and mortality. A variety of T-cell depletion (TCD) techniques have been developed and used over the years in an effort to reduce transplant-related mortality (TRM) due to GVHD ( Table 1 ). In this chapter we will focus on outcomes of HCT using T cell depletion for the treatment of AML in recipients of matched or partially mismatched donors (7/8 or 8/8 HLA-matched), with a primary focus on ex vivo CD34+ selection of the graft. Other methods of T-cell depletion will be mentioned for historic context only.

2. T-Cell Depletion Techniques

The goal of T-cell depleting a graft is to reduce GVHD while maintaining the graft- versus -leukemia or lymphoma (GVL) effect. A variety of TCD techniques have been used with mixed results. When reviewing reports utilizing TCD for transplantation it is critical to determine the following: (1) Which technique is being used? (2) Which cell population is being removed ( i.e. , T, B, NK or all non-hematopoietic cells) and to what extent? Different techniques lead to both quantitative and qualitative differences in the cells being depleted with important clinical implications; (3) Is post-transplant GVHD prophylaxis utilized? and (4) What is the graft source and what is the degree of HLA matching? All of these factors significantly affect outcomes and therefore must be considered when interpreting published reports.

Early TCD techniques differed in the use of negative vs. positive selection. Negative selection can be achieved either through physical methods such as counterflow elutriation [ 3 , 4 , 5 ] or soybean lectin agglutination (SBA) and sheep red blood cell (sRBC)-rosette depletion (E-rosetting) [ 6 , 7 , 8 , 9 , 10 ], or immunological methods using monoclonal antibodies [ 5 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. Monoclonal antibodies can be used with or without complement, or conjugated to toxins. Antibodies vary in their specificities, which can be narrow, such as T10B9 targeting the α/β T cell receptor (TCR), or broad, such as combination of antibodies targeting CD2, CD4 and CD8 [ 30 ].

Currently, the most common method for T cell depletion relies on positive selection of CD34+ hematopoietic stem cells from the graft [ 32 , 33 , 34 , 36 , 37 ]. CD34+ selection of peripheral blood stem cells (PBSCs) was performed in initial studies with the ISOLEX 300i magnetic cell selection system (Baxter, Deerfield, IL, USA), followed by E-rosetting [ 32 , 33 , 37 ]. The ISOLEX device is no longer being manufactured, and the most commonly used method in current studies uses immunomagnetic beads with the CliniMACS CD34 Reagent System (Miltenyi Biotech, Gladbach, Germany) for CD34+ selection [ 31 , 34 , 37 ]. The CliniMACS system can also be used to negatively select grafts through depletion of CD3+ and CD19+ cells or depletion of TCRαβ+ T cells [ 35 , 38 , 39 , 40 , 41 ]. The two CD34+ selection methods differ in the degree of TCD; for example, the CliniMACS CD34 Reagent System can achieve a 5-log reduction in T cells, whereas the ISOLEX 300i system achieves a 3.5-log reduction, requiring additional T-cell depletion through E-rosetting.

The cell dose and graft source also impact outcomes. In recipients of T-cell depleted marrow grafts from HLA-identical donors, the risk of GVHD was shown to increase if the graft contained >1 × 10 5 T cells/kg [ 42 ]. Differences between TCD methods can have a significant impact on clinical outcomes, including the risk of graft failure, GVHD and relapse.

3. Outcomes in AML with T-Cell Depletion

One of the primary benefits of allogeneic HCT is derived from the GVL effect, driven by the recognition of tumor cells by donor T cells. Therefore, a concern in the utilization of TCD is the potentially negative impact on relapse resulting from reduced T-cell doses included in the graft. However, it is clear that certain diseases rely more on the GVL effect than others. For example, early studies with TCD in chronic myelogenous leukemia (CML) were associated with a significantly increased risk of relapse. In a retrospective study of 46 patients who received TCD grafts form HLA-identical siblings, relapse at 3-years was significantly higher in the TCD group (62% vs . 24%, p = 0.0003). However, a significant proportion of these patients were then salvaged with donor lymphocyte infusion (DLI), supporting the role of GVL for this disease [ 43 ].

On the other hand, studies utilizing TCD in AML and ALL have shown favorable outcomes in recipients of 7/8 or 8/8 HLA matched related or unrelated donors that are comparable to those seen in unmodified, conventional grafts, calling into question the contribution of GVL in those diseases [ 34 , 36 , 44 , 45 ].

4. TCD in AML—Early Studies

Studies using TCD done in the 1980’s and 1990’s mostly used bone marrow from sibling donors as the graft source and used a variety of techniques (lectin separation, elutriation, E-rosettes, antibodies against T and NK cells, antibodies with or without complement). These techniques were associated with a 1–2 log reduction in T cells in the graft, and patients were often given post-transplant cyclosporine for additional GVHD prophylaxis. These studies were also associated with increased graft failure [ 8 , 46 ]. The addition of antithymocyte globulin (ATG) and thiotepa to the conditioning regimens, which had traditionally included cyclophosphamide and total body irradiation (TBI) or busulfan, decreased the rate of graft failure and eliminated the need for post transplant GVHD prophylaxis. Favorable results were reported in recipients of HLA-identical donors using this approach along with sequential soybean lectin agglutination and sheep red blood cell-rosette depletion. A study by Papadopoulos et al. included 31 patients with AML in CR1 or CR2 and showed a disease-free survival (DFS) at 4 years of 70% with no GVHD or graft rejection [ 9 ]. Similar results were reported by Aversa et al. using the same regimen in 54 patients with acute leukemia, including 2 with HLA-DR mismatched donors [ 10 ]. No GVHD or graft rejection was observed and the event-free survival at 4.9 years was 74% in 30 patients with AML.

These early studies demonstrated the feasibility of TCD, and overcame graft rejection with the utilization of ATG, at the expense of added immunosuppression and ensuing delayed immune recovery.

5. TCD in AML—Contemporary Studies

Contemporary studies have utilized two main approaches of TCD by CD34+ selection described above, either the ISOLEX 300i Magnetic Cell Separator followed by sRBC-rosette depletion or, more recently, the Miltenyi CliniMACS CD34 Reagent System. In 2011, Devine et al. [ 34 ] reported the results of a Blood and Marrow Clinical Trials Network study (BMT CTN 0303) utilizing CD34+ selection with the Miltenyi CliniMACS CD34 Reagent System. The study included 44 patients with AML in CR1 or CR2 (excluding patients with t (15;17), and core binding factor leukemia) who received T cell depleted PBSCT from HLA-identical siblings after conditioning with hyperfractionated TBI (HFTBI), thiotepa, cyclophosphamide and ATG. No additional GVHD prophylaxis was given. All patients engrafted; the incidence of aGVHD (grades II–IV) was 22.7% and extensive cGVHD was 6.8% at 2 years. The relapse rate at three years for patients in CR1 was 17.4%, with a DFS for all patients at 6 months of 82% and a 2-year OS of 60%. Rates of infection were comparable to other studies; however EBV reactivation occurred in 18% of patients leading to 1 death from Epstein-Barr Virus (EBV) post-transplant lymphoproliferative disease (PTLD) [ 36 , 47 , 48 ]. In a second report, the outcomes of patients from the BMT CTN 0303 study were compared to a similar cohort of patients (AML in either CR1 or CR2, PBSCT from HLA-identical siblings) who received a conventional HCT on BMT CTN 0101 study (a study comparing fluconazole with voriconazole as antifungal prophylaxis after HCT) [ 44 ]. There were no differences in leukemia relapse (23% vs. 27% in the TCD and conventional graft groups, respectively) and 2-year OS (65% vs. 59% in the TCD and conventional graft groups, respectively). However rates of GVHD were higher in the conventional graft group (aGVHD 23% vs. 39%, p = 0.07 and cGVHD 19% vs. 50%, p < 0.001).

More recently, a retrospective study compared the use of TCD HCT to conventional grafts in patients with AML in CR1 by examining outcomes of 115 patients who received TCD grafts at Memorial Sloan Kettering Cancer Center (MSKCC) with a cohort of 181 patients at MD Anderson Cancer Center (MDACC) [ 36 ]. A hundred and seven patients in the MSKCC cohort received PBSC grafts, including 85 that were CD34-selected with the ISOLEX 300i Magnetic Cell Separator followed by sRBC-rosette depletion, and 22 with the Miltenyi CliniMACS CD34 Reagent System. Patients at both centers received myeloablative conditioning (MAC) and both cohorts included recipients of matched related, matched unrelated and mismatched donors. Patients at MSKCC were more likely to be recipients of a mismatched graft (27% vs. 14%, p < 0.001). Patients at MSKCC did not receive additional GVHD prophylaxis. Patients at MDACC received tacrolimus and mini-methotrexate for GVHD prophylaxis, and ATG for HLA-mismatched donors. There were no significant differences in the rate of relapse at 3 years between groups (18% vs. 25%, in the TCD vs. conventional grafts, respectively, p = 0.3). However, rates of GVHD were significantly lower in the TCD group (5% vs. 18% for aGVHD, p = 0.005, and 13% vs. 53% for cGVHD, p < 0.001).

Although contemporary studies that compare outcomes of conventional to TCD grafts are retrospective, the results suggest that TCD transplants offer similar DFS and OS with significantly lower rates of GVHD.

As noted above additional TCD approaches are being investigated beyond CD34 selection [ 35 , 38 , 39 , 40 , 41 ]. The potential advantages of negative selection by depletion of CD3+/CD19+ or TCRαβ+ T cells include the presence of additional cells in the graft such as natural killer (NK) cells or TCRγδ+ T cells, which may play a role in relapse or infection prevention. To date, the published studies of these approaches have been in recipients of haplo-identical grafts and there has been limited data on patients with AML. In a study by Bethge et al . [ 35 ], EFS was 35% at one year in 16 patients with AML who received a CD3/CD19 depleted transplant from a haplo-identical donor.

Finally, an alternative GVHD prophylaxis approach that will be compared to CD34-selection in an upcoming phase 3 trial (BMT CTN 1301) relies on the use of post-transplant high dose cyclophosphamide after a T-replete bone marrow graft from a matched donor [ 49 , 50 , 51 ]. In a recently published study using this approach in 138 patients with AML, the 3-year DFS and OS were 43% (95% CI, 35% to 52%) and 53% (95% CI, 45% to 62%), respectively [ 50 ]. DFS (48% vs. 29% at 3 years) and OS (55% vs. 50% at 3 years) were higher in patients with AML in morphologic CR compared to those with active disease. The approach was associated with low rates of grade III to IV acute GVHD (11% at 100 days) and chronic GVHD (13% at 2 years).

6. Impact of T-Cell Depletion on Engraftment and Immune Reconstitution

Hematopoietic stem cell transplantation, regardless of donor source and manipulation, is associated with significant and prolonged immunosuppression and risk of severe and fatal infections [ 48 , 52 ], disease relapse and secondary malignancies [ 20 , 53 ]. The use of TCD, whether in vivo with agents such as alemtuzumab or ATG, or ex vivo with T-cell depletion considerably affects immune recovery. In early studies comparing immune reconstitution in TCD and unmodified grafts the rate of CD3+, CD4+ and CD8+ T cell reconstitution was significantly delayed in TCD recipients, correlating with increased risk of infections, including EBV-PTLD [ 48 , 54 ]. T-cell receptor (TCR) studies using 5′-RACE PCR with deep sequencing have confirmed these findings by showing more rapid recovery of TCR diversity in conventional graft recipients compared to TCD grafts [ 55 ]. Lower T-cell levels result from decreased thymic output, which can be quantified via measurements of T-cell receptor excision circles (TRECs). Studies have shown that older patients and recipients of TCD grafts have lower TRECs than unmodified graft recipients; however this difference abates beyond 9 months [ 56 ].

ATG plays an important role in TCD by reducing the risk of graft rejection. However it is associated with delayed immune recovery and an increased risk of opportunistic infections (OI), with approximately 15% of the patients in early TCD studies dying from OIs [ 9 , 10 ]. Furthermore, a study of immune reconstitution in patients receiving TCD grafts with or without ATG found delayed immune reconstitution with ATG, which was associated with increased OIs [ 54 ]. Although studies of TCD performed without ATG by substituting cyclophosphamide with fludarabine have demonstrated durable engraftment in recipients of matched related donors, there did not appear to be a significant effect on immune recovery or the risk of OIs [ 32 ].

It is important to note however that, in addition to age and TCD, the presence of GVHD also significantly hampers immune reconstitution via direct effects on the thymus [ 57 , 58 , 59 ], as well as the immunosuppressive drugs required for treatment of GVHD [ 60 , 61 , 62 , 63 , 64 ]. Although TCD impacts immune reconstitution leading to higher infection related deaths, GVHD in conventional grafts similarly leads to increased mortality. This is a potential explanation for the similar RFS and OS outcomes observed in patients with AML in the MSKCC/MDACC retrospective study and the BMT CTN study [ 36 , 44 ].

7. Strategies to Enhance Immune Recovery Post HCT

HCT affords a curative treatment option to many patients with otherwise incurable malignancies. However, the benefit of this therapy comes at the risk of significant complications, including infection, GVHD, relapse and secondary malignancies [ 20 , 48 , 52 , 53 , 65 , 66 ]. The rationale for TCD is to mitigate GVHD while preserving the benefit of GVL. TCD and HCT in general, are associated with prolonged immunosuppression. Therefore, strategies to optimize post transplant immunity, enhance GVL, while minimizing infectious complications are needed.

The addition of T cells post transplantation represents one such strategy. In one recent study, 19 pediatric patients (13 transplanted for malignant disease) received CD34+ selected matched unrelated donor (MUD) HCT with CD3+ T cells added back, at a dose of 1.0–2.5 × 10 5 CD3+/kg, and tacrolimus for GVHD prevention [ 67 ]. Rates of aGVHD, cGVHD and extensive cGVHD were 15.8%, 23.3% and 0%, respectively, which are low compared to conventional HCT. All patients on this study had neutrophil engraftment, and infection-related mortality at one year was 5%–6%, showing the feasibility of this approach. This approach has also been used in recipients of CD34-selected haplo-identical grafts [ 68 ]. The same group previously reported low rates of acute and chronic GVHD in a retrospective series of 16 patients who received DLI (up to 6 × 10 4 CD3+/kg) from haplo-identical donors to enhance immune recovery and/or treat infections [ 69 ]. One ongoing trial is evaluating the effect of serial DLI post TCD in patients with advanced multiple myeloma ( NCT01131169 ).

Another strategy to boost immune reconstitution post-transplant is the use of Keratinocyte Growth Factor (KGF). KGF has been shown in pre-clinical models to play an important role in T cell homeostasis and immune recovery, as well as in thymic regeneration after radiation injury [ 70 , 71 , 72 ]. Based on these data, KGF along with sex steroid ablation is being studied in an ongoing phase II clinical trial ( NCT01746849 ).

We recently published the results of a phase I study using recombinant human IL-7 (rhIL-7, CYT107, Cytheris) in recipients of TCD HCT and demonstrated enhanced immune recovery, with significant increases in CD4+ and CD8+ T cells along with increased T cell function, without causing significant GVHD or other serious toxicity [ 73 ].

8. Benefits of TCD HCT in AML

Although reduced intensity conditioning (RIC) regimens allow older patients to undergo HCT, they are associated with higher relapse rates in AML [ 74 , 75 ]. However, the combined toxicity of GVHD prophylaxis that usually includes a calineurin inhibitor (CNI) and methotrexate with myeloablative conditioning (MAC), makes this approach prohibitive in older patients. Unlike conventional grafts, CD34+ selected grafts do not require post-transplant GVHD prophylaxis, and as a result, older patients can be treated with MAC. In addition, patients with renal insufficiency can also successfully undergo transplantation by avoiding the use of CNIs.

The use of CD34+ selected grafts is associated with significant reductions in both acute and chronic GVHD. In addition to the obvious advantage of lowering GVHD, patients without fully matched donors are also able to undergo transplantation, therefore expanding the pool of potential donors.

Finally, CD34+ selected grafts are an ideal platform for post-transplant immunotherapy with adoptive cell therapy targeting both minimal residual disease and viral reactivation by CMV and EBV, among others [ 76 , 77 , 78 , 79 ]. The administration T cells specific for tumor or viral antigens post-transplant has the potential advantage of overcoming any loss of GVL or increased infectious risk associated with TCD without affecting the benefit of reduced GVHD.

9. Conclusions

After three decades of investigation, it is reasonable to consider CD34+ selected allografts for patients with AML in CR1 based on prospective data [ 34 ], and well conducted retrospective studies [ 36 , 44 ]. These contemporary studies are significantly more homogeneous in their methodology and patient inclusion than prior studies, mostly using the CliniMACS CD34 Reagent System for CD34+ selection, and reporting consistent favorable outcomes for patients with AML in CR. The use of CD34+ selected grafts overcomes the morbidity and mortality associated with GVHD, a significant contributor of transplant-related complications, without compromising the benefit of transplantation and affording the same overall survival as conventional transplantation.

TCD represents an important step in graft manipulation, allowing older patients, and those with comorbidities to successfully undergo transplantation. Ongoing research aims to continue to decrease morbidity and mortality associated with transplantation by improving immune reconstitution and the GVL effect.

As mentioned above, an ongoing national phase 3 trial will compare TCD with the CliniMACS CD34 Reagent System to post-transplant cyclophosphamide [ 49 ], and a control arm (tacrolimus and methotrexate) in patients with acute leukemia and MDS who are eligible for a MAC transplant from a matched related or unrelated donor (BMT CTN 1301).

Acknowledgments

This work was supported in part by PO1 CA23766 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Gabriela Soriano Hobbs was supported in part by an American Society of Clinical Oncology Young Investigator Award.

Author Contributions

Gabriela Soriano Hobbs and Miguel-Angel Perales wrote reviewed the literature, wrote the manuscript and edited its contents.

Conflicts of Interest

The authors declare no conflict of interest.

References

1. Koreth J. Schlenk R. Kopecky K.J. Honda S. Sierra J. Djulbegovic B.J. Wadleigh M. DeAngelo D.J. Stone R.M. Sakamaki H. Allogeneic stem cell transplantation for acute myeloid leukemia in first complete remission: Systematic review and meta-analysis of prospective clinical trials JAMA 2009 301 2349 2361 10.1001/jama.2009.813 19509382 PMC3163846

2. Sorror M.L. Maris M.B. Storb R. Baron F. Sandmaier B.M. Maloney D.G. Storer B. Hematopoietic cell transplantation (HCT)-specific comorbidity index: A new tool for risk assessment before allogeneic HCT Blood 2005 106 2912 2919 10.1182/blood-2005-05-2004 15994282 PMC1895304

3. De Witte T. Hoogenhout J. de Pauw B. Holdrinet R. Janssen J. Wessels J. van Daal W. Hustinx T. Haanen C. Depletion of donor lymphocytes by counterflow centrifugation successfully prevents acute graft- versus -host disease in matched allogeneic marrow transplantation Blood 1986 67 1302 1308 3516253

4. Wagner J.E. Donnenberg A.D. Noga S.J. Cremo C.A. Gao I.K. Yin H.J. Vogelsang G.B. Rowley S. Saral R. Santos G.W. Lymphocyte depletion of donor bone marrow by counterflow centrifugal elutriation: Results of a phase I clinical trial Blood 1988 72 1168 1176 3048436

5. Wagner J.E. Thompson J.S. Carter S.L. Kernan N.A. Effect of graft-versus-host disease prophylaxis on 3-year disease-free survival in recipients of unrelated donor bone marrow (T-cell Depletion Trial): A multi-centre, randomised phase II-III trial Lancet 2005 366 733 741 10.1016/S0140-6736(05)66996-6 16125590

6. Reisner Y. Kapoor N. Kirkpatrick D. Pollack M.S. Cunningham-Rundles S. Dupont B. Hodes M.Z. Good R.A. O’Reilly R.J. Transplantation for severe combined immunodeficiency with HLA-A,B,D,DR incompatible parental marrow cells fractionated by soybean agglutinin and sheep red blood cells Blood 1983 61 341 348 6217853

7. Reisner Y. Kapoor N. Kirkpatrick D. Pollack M.S. Dupont B. Good R.A. O’Reilly R.J. Transplantation for acute leukaemia with HLA-A and B nonidentical parental marrow cells fractionated with soybean agglutinin and sheep red blood cells Lancet 1981 2 327 331 10.1016/S0140-6736(81)90647-4 6115110

8. Young J.W. Papadopoulos E.B. Cunningham I. Castro-Malaspina H. Flomenberg N. Carabasi M.H. Gulati S.C. Brochstein J.A. Heller G. Black P. T-cell-depleted allogeneic bone marrow transplantation in adults with acute nonlymphocytic leukemia in first remission Blood 1992 79 3380 3387 1596577

9. Papadopoulos E.B. Carabasi M.H. Castro-Malaspina H. Childs B.H. Mackinnon S. Boulad F. Gillio A.P. Kernan N.A. Small T.N. Szabolcs P. T-cell-depleted allogeneic bone marrow transplantation as postremission therapy for acute myelogenous leukemia: Freedom from relapse in the absence of graft- versus -host disease Blood 1998 91 1083 1090 9446672

10. Aversa F. Terenzi A. Carotti A. Felicini R. Jacucci R. Zei T. Latini P. Aristei C. Santucci A. Martelli M.P. Improved outcome with T-cell-depleted bone marrow transplantation for acute leukemia J. Clin. Oncol. 1999 17 1545 1550 10334542 10.1200/JCO.1999.17.5.1545

11. Prentice H.G. Blacklock H.A. Janossy G. Bradstock K.F. Skeggs D. Goldstein G. Hoffbrand A.V. Use of anti-T-cell monoclonal antibody OKT3 to prevent acute graft- versus -host disease in allogeneic bone-marrow transplantation for acute leukaemia Lancet 1982 1 700 703 10.1016/S0140-6736(82)92619-8 6122004

12. Filipovich A.H. McGlave P.B. Ramsay N.K. Goldstein G. Warkentin P.I. Kesey J.H. Pretreatment of donor bone marrow with monoclonal antibody OKT3 for prevention of acute graft- versus -host disease in allogeneic histocompatible bone-marrow transplantation Lancet 1982 1 1266 1269 10.1016/S0140-6736(82)92840-9 6123018

13. Prentice H.G. Blacklock H.A. Janossy G. Gilmore M.J. Price-Jones L. Tidman N. Trejdosiewicz L.K. Skeggs D.B. Panjwani D. Ball S. Depletion of T lymphocytes in donor marrow prevents significant graft- versus -host disease in matched allogeneic leukaemic marrow transplant recipients Lancet 1984 1 472 476 10.1016/S0140-6736(84)92848-4 6142207

14. Martin P.J. Hansen J.A. Thomas E.D. Preincubation of donor bone marrow cells with a combination of murine monoclonal anti-T-cell antibodies without complement does not prevent graft- versus -host disease after allogeneic marrow transplantation J. Clin. Immunol. 1984 4 18 22 10.1007/BF00915282 6365949

15. Martin P.J. Hansen J.A. Buckner C.D. Sanders J.E. Deeg H.J. Stewart P. Appelbaum F.R. Clift R. Fefer A. Witherspoon R.P. Effects of in vitro depletion of T cells in HLA-identical allogeneic marrow grafts Blood 1985 66 664 672 3896348

16. Herve P. Flesch M. Cahn J.Y. Racadot E. Plouvier E. Lamy B. Rozenbaum A. Noir A. Des Floris R.L. Peters A. Removal of marrow T cells with OKT3-OKT11 monoclonal antibodies and complement to prevent acute graft- versus -host disease. A pilot study in ten patients Transplantation 1985 39 138 143 10.1097/00007890-198502000-00006 3881852

17. Trigg M.E. Billing R. Sondel P.M. Exten R. Hong R. Bozdech M.J. Horowitz S.D. Finlay J.L. Moen R. Longo W. Clinical trial depleting T lymphocytes from donor marrow for matched and mismatched allogeneic bone marrow transplants Cancer Treat. Rep. 1985 69 377 386 3888387

18. Mitsuyasu R.T. Champlin R.E. Gale R.P. Ho W.G. Lenarsky C. Winston D. Selch M. Elashoff R. Giorgi J.V. Wells J. 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 Ann. Intern. Med. 1986 105 20 26 10.7326/0003-4819-105-1-20 3521427

19. Patterson J. Prentice H.G. Brenner M.K. Gilmore M. Janossy G. Ivory K. Skeggs D. Morgan H. Lord J. Blacklock H.A. Graft rejection following HLA matched T-lymphocyte depleted bone marrow transplantation Br. J. Haematol. 1986 63 221 230 10.1111/j.1365-2141.1986.tb05544.x 3521712

20. Maraninchi D. Gluckman E. Blaise D. Guyotat D. Rio B. Pico J.L. Leblond V. Michallet M. Dreyfus F. Ifrah N. Impact of T-cell depletion on outcome of allogeneic bone-marrow transplantation for standard-risk leukaemias Lancet 1987 2 175 178 10.1016/S0140-6736(87)90763-X 2885638

21. Cahn J.Y. Herve P. Flesch M. Plouvier E. Racadot E. Vuillier J. Montcuquet P. Noir A. Rozenbaum A. Leconte des Floris R. Marrow transplantation from HLA non-identical family donors for the treatment of leukaemia: A pilot study of 15 patients using additional immunosuppression and T-cell depletion Br. J. Haematol. 1988 69 345 349 10.1111/j.1365-2141.1988.tb02372.x 3044438

22. Martin P.J. Hansen J.A. Torok-Storb B. Moretti L. Press O. Storb R. Thomas E.D. Weiden P.L. Vitetta E.S. Effects of treating marrow with a CD3-specific immunotoxin for prevention of acute graft- versus -host disease Bone Marrow Transplant. 1988 3 437 444 2973360

23. Laurent G. Maraninchi D. Gluckman E. Vernant J.P. Derocq J.M. Gaspard M.H. Rio B. Michalet M. Reiffers J. Dreyfus F. Donor bone marrow treatment with T101 Fab fragment-ricin A-chain immunotoxin prevents graft- versus -host disease Bone Marrow Transplant. 1989 4 367 371 2789084

24. Filipovich A.H. Vallera D. McGlave P. Polich D. Gajl-Peczalska K. Haake R. Lasky L. Blazar B. Ramsay N.K. Kersey J. T cell depletion with anti-CD5 immunotoxin in histocompatible bone marrow transplantation. The correlation between residual CD5 negative T cells and subsequent graft- versus -host disease Transplantation 1990 50 410 415 10.1097/00007890-199009000-00011 1698319

25. Antin J.H. Bierer B.E. Smith B.R. Ferrara J. Guinan E.C. Sieff C. Golan D.E. Macklis R.M. Tarbell N.J. Lynch E. Selective depletion of bone marrow T lymphocytes with anti-CD5 monoclonal antibodies: Effective prophylaxis for graft- versus -host disease in patients with hematologic malignancies Blood 1991 78 2139 2149 1717080

26. Soiffer R.J. Fairclough D. Robertson M. Alyea E. Anderson K. Freedman A. Bartlett-Pandite L. Fisher D. Schlossman R.L. Stone R. CD6-depleted allogeneic bone marrow transplantation for acute leukemia in first complete remission Blood 1997 89 3039 3047 9108425

27. Soiffer R.J. Freedman A.S. Neuberg D. Fisher D.C. Alyea E.P. Gribben J. Schlossman R.L. Bartlett-Pandite L. Kuhlman C. Murray C. Freeman A. CD6+ T cell-depleted allogeneic bone marrow transplantation for non-Hodgkin’s lymphoma Bone Marrow Transplant. 1998 21 1177 1181 10.1038/sj.bmt.1701271 9674848

28. Alyea E.P. Weller E. Fisher D.C. Freedman A.S. Gribben J.G. Lee S. Schlossman R.L. Stone R.M. Friedberg J. DeAngelo D. Comparable outcome with T-cell-depleted unrelated-donor versus related-donor allogeneic bone marrow transplantation Biol. Blood Marrow Transplant. 2002 8 601 607 10.1053/bbmt.2002.v8.abbmt080601 12463479

29. Lee S.J. Zahrieh D. Alyea E.P. Weller E. Ho V.T. Antin J.H. Soiffer R.J. Comparison of T-cell-depleted and non-T-cell-depleted unrelated donor transplantation for hematologic diseases: Clinical outcomes, quality of life, and costs Blood 2002 100 2697 2702 10.1182/blood-2002-03-0984 12351374

30. Champlin R.E. Passweg J.R. Zhang M.J. Rowlings P.A. Pelz C.J. Atkinson K.A. Barrett A.J. Cahn J.Y. Drobyski W.R. Gale R.P. T-cell depletion of bone marrow transplants for leukemia from donors other than HLA-identical siblings: Advantage of T-cell antibodies with narrow specificities Blood 2000 95 3996 4003 10845940

31. Urbano-Ispizua A. Brunet S. Solano C. Moraleda J.M. Rovira M. Zuazu J. de La Rubia J. Bargay J. Caballero D. Diez-Martin J.L. Allogeneic transplantation of CD34+-selected cells from peripheral blood in patients with myeloid malignancies in early phase: A case control comparison with unmodified peripheral blood transplantation Bone Marrow Transplant. 2001 28 349 354 10.1038/sj.bmt.1703154 11571506

32. Jakubowski A.A. Small T.N. Young J.W. Kernan N.A. Castro-Malaspina H. Hsu K.C. Perales M.A. Collins N. Cisek C. Chiu M. T cell depleted stem-cell transplantation for adults with hematologic malignancies: Sustained engraftment of HLA-matched related donor grafts without the use of antithymocyte globulin Blood 2007 110 4552 4559 10.1182/blood-2007-06-093880 17717135 PMC2234775

33. Jakubowski A.A. Small T.N. Kernan N.A. Castro-Malaspina H. Collins N. Koehne G. Hsu K.C. Perales M.A. Papanicolaou G. van den Brink M.R. T Cell-Depleted Unrelated Donor Stem Cell Transplantation Provides Favorable Disease-Free Survival for Adults with Hematologic Malignancies Biol. Blood Marrow Transplant. 2011 17 1335 1342 10.1016/j.bbmt.2011.01.005 21232623 PMC3094599

34. Devine S.M. Carter S. Soiffer R.J. Pasquini M.C. Hari P.N. Stein A. Lazarus H.M. Linker C. Stadtmauer E.A. Alyea E.P. III Low risk of chronic graft- versus -host disease and relapse associated with T cell-depleted peripheral blood stem cell transplantation for acute myelogenous leukemia in first remission: Results of the blood and marrow transplant clinical trials network protocol 0303 Biol. Blood Marrow Transplant. 2011 17 1343 1351 10.1016/j.bbmt.2011.02.002 21320619 PMC3150599

35. Bethge W.A. Faul C. Bornhauser M. Stuhler G. Beelen D.W. Lang P. Stelljes M. Vogel W. Hagele M. Handgretinger R. Haploidentical allogeneic hematopoietic cell transplantation in adults using CD3/CD19 depletion and reduced intensity conditioning: An update Blood Cells Mol. Dis. 2008 40 13 19 10.1016/j.bcmd.2007.07.001 17869547

36. Bayraktar U.D. de Lima M. Saliba R.M. Maloy M. Castro-Malaspina H.R. Chen J. Rondon G. Chiattone A. Jakubowski A.A. Boulad F. Ex Vivo T Cell Depleted versus Unmodified Allografts in Patients with Acute Myeloid Leukemia in First Complete Remission Biol. Blood Marrow Transplant. 2013 19 898 903 10.1016/j.bbmt.2013.02.018 23467126 PMC4059063

37. Aversa F. Terenzi A. Tabilio A. Falzetti F. Carotti A. Ballanti S. Felicini R. Falcinelli F. Velardi A. Ruggeri L. Full haplotype-mismatched hematopoietic stem-cell transplantation: A phase II study in patients with acute leukemia at high risk of relapse J. Clin. Oncol. 2005 23 3447 3454 10.1200/JCO.2005.09.117 15753458

38. Bertaina A. Merli P. Rutella S. Pagliara D. Bernardo M.E. Masetti R. Pende D. Falco M. Handgretinger R. Moretta F. HLA-haploidentical stem cell transplantation after removal of alphabeta+ T and B cells in children with nonmalignant disorders Blood 2014 124 822 826 10.1182/blood-2014-03-563817 24869942

39. Zecca M. Strocchio L. Pagliara D. Comoli P. Bertaina A. Giorgiani G. Perotti C. Corbella F. Brescia L. Locatelli F. HLA-haploidentical T cell-depleted allogeneic hematopoietic stem cell transplantation in children with Fanconi anemia Biol. Blood Marrow Transplant. 2014 20 571 576 10.1016/j.bbmt.2014.01.015 24462983

40. Lang P. Teltschik H.M. Feuchtinger T. Muller I. Pfeiffer M. Schumm M. Ebinger M. Schwarze C.P. Gruhn B. Schrauder A. Transplantation of CD3/CD19 depleted allografts from haploidentical family donors in paediatric leukaemia Br. J. Haematol. 2014 165 688 698 10.1111/bjh.12810 24588540

41. Gonzalez-Llano O. Rodriguez-Romo L.N. Mancias-Guerra Mdel C. Tarin-Arzaga L. Jaime-Perez J.C. Herrera-Garza J.L. Cantu-Rodriguez O.G. Gutierrez-Aguirre C.H. Garcia-Sepulveda R.D. Garcia-Marin A.Y. Feasibility of an outpatient HLA haploidentical stem cell transplantation program in children using a reduced-intensity conditioning regimen and CD3-CD19 depletion Hematology 2014 19 10 17 10.1179/1607845413Y.0000000088 23601986

42. Kernan N.A. Collins N.H. Juliano L. Cartagena T. Dupont B. O’Reilly R.J. Clonable T lymphocytes in T cell-depleted bone marrow transplants correlate with development of graft-v-host disease Blood 1986 68 770 773 3527302

43. Sehn L.H. Alyea E.P. Weller E. Canning C. Lee S. Ritz J. Antin J.H. Soiffer R.J. Comparative outcomes of T-cell-depleted and non-T-cell-depleted allogeneic bone marrow transplantation for chronic myelogenous leukemia: Impact of donor lymphocyte infusion J. Clin. Oncol. 1999 17 561 568 10080600 10.1200/JCO.1999.17.2.561

44. Pasquini M.C. Devine S. Mendizabal A. Baden L.R. Wingard J.R. Lazarus H.M. Appelbaum F.R. Keever-Taylor C.A. Horowitz M.M. Carter S. Comparative outcomes of donor graft CD34+ selection and immune suppressive therapy as graft- versus -host disease prophylaxis for patients with acute myeloid leukemia in complete remission undergoing HLA-matched sibling allogeneic hematopoietic cell transplantation J. Clin. Oncol. 2012 30 3194 3201 10.1200/JCO.2012.41.7071 22869882 PMC3434978

45. Hobbs G.S. Hilden P. Hamdi A. Goldberg J.D. Poon M. Ledesma C. Devlin S. Rondon G. Papadopoulos E.B. Jakubowski A.A. Outcomes in Patients with Acute Lymphoblastic Leukemia in First or Second Complete Remission Receiving Ex-Vivo T-Cell Depleted or Unmodified Allografts: Comparison of Results At Two Institutions Blood 2013 122 3370 10.1182/blood-2013-06-501452

46. Marmont A.M. Horowitz M.M. Gale R.P. Sobocinski K. Ash R.C. van Bekkum D.W. Champlin R.E. Dicke K.A. Goldman J.M. Good R.A. T-cell depletion of HLA-identical transplants in leukemia Blood 1991 78 2120 2130 1912589

47. Small T.N. Immunologic reconstitution following stem cell transplantation Curr. Opin. Hematol. 1996 3 461 465 10.1097/00062752-199603060-00010 9372118

48. Small T.N. Papadopoulos E.B. Boulad F. Black P. Castro-Malaspina H. Childs B.H. Collins N. Gillio A. George D. Jakubowski A. Comparison of immune reconstitution after unrelated and related T-cell-depleted bone marrow transplantation: Effect of patient age and donor leukocyte infusions Blood 1999 93 467 480 9885208

49. Luznik L. Bolanos-Meade J. Zahurak M. Chen A.R. Smith B.D. Brodsky R. Huff C.A. Borrello I. Matsui W. Powell J.D. High-dose cyclophosphamide as single-agent, short-course prophylaxis of graft- versus -host disease Blood 2010 115 3224 3230 10.1182/blood-2009-11-251595 20124511 PMC2858487

50. Kanakry C.G. Tsai H.L. Bolanos-Meade J. Smith B.D. Gojo I. Kanakry J.A. Kasamon Y.L. Gladstone D.E. Matsui W. Borrello I. Single-agent GVHD prophylaxis with posttransplantation cyclophosphamide after myeloablative, HLA-matched BMT for AML, ALL, and MDS Blood 2014 124 3817 3827 10.1182/blood-2014-07-587477 25316679 PMC4263989

51. Kanakry C.G. O’Donnell P.V. Furlong T. de Lima M.J. Wei W. Medeot M. Mielcarek M. Champlin R.E. Jones R.J. Thall P.F. Multi-institutional study of post-transplantation cyclophosphamide as single-agent graft- versus -host disease prophylaxis after allogeneic bone marrow transplantation using myeloablative busulfan and fludarabine conditioning J. Clin. Oncol. 2014 32 3497 3505 10.1200/JCO.2013.54.0625 25267759 PMC4209101

52. Storek J. Gooley T. Witherspoon R.P. Sullivan K.M. Storb R. Infectious morbidity in long-term survivors of allogeneic marrow transplantation is associated with low CD4 T cell counts Am. J. Hematol. 1997 54 131 138 10.1002/(SICI)1096-8652(199702)54:2<131::AID-AJH6>3.0.CO;2-Y 9034287

53. Curtis R.E. Rowlings P.A. Deeg H.J. Shriner D.A. Socie G. Travis L.B. Horowitz M.M. Witherspoon R.P. Hoover R.N. Sobocinski K.A. Solid cancers after bone marrow transplantation N. Engl. J. Med. 1997 336 897 904 10.1056/NEJM199703273361301 9070469

54. Small T.N. Avigan D. Dupont B. Smith K. Black P. Heller G. Polyak T. O’Reilly R.J. Immune reconstitution following T-cell depleted bone marrow transplantation: Effect of age and posttransplant graft rejection prophylaxis Biol. Blood Marrow Transplant. 1997 3 65 75 9267666

55. Van Heijst J.W. Ceberio I. Lipuma L.B. Samilo D.W. Wasilewski G.D. Gonzales A.M. Nieves J.L. van den Brink M.R. Perales M.A. Pamer E.G. Quantitative assessment of T cell repertoire recovery after hematopoietic stem cell transplantation Nat. Med. 2013 19 372 377 23435170 10.1038/nm.3100 PMC3594333

56. Lewin S.R. Heller G. Zhang L. Rodrigues E. Skulsky E. van den Brink M.R. Small T.N. Kernan N.A. O’Reilly R.J. Ho D.D. Direct evidence for new T-cell generation by patients after either T-cell-depleted or unmodified allogeneic hematopoietic stem cell transplantations Blood 2002 100 2235 2242 12200390

57. Weinberg K. Blazar B.R. Wagner J.E. Agura E. Hill B.J. Smogorzewska M. Koup R.A. Betts M.R. Collins R.H. Douek D.C. Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation Blood 2001 97 1458 1466 10.1182/blood.V97.5.1458 11222394

58. Clave E. Busson M. Douay C. Peffault de Latour R. Berrou J. Rabian C. Carmagnat M. Rocha V. Charron D. Socie G. Acute graft-versus-host disease transiently impairs thymic output in young patients after allogeneic hematopoietic stem cell transplantation Blood 2009 113 6477 6484 10.1182/blood-2008-09-176594 19258596

59. Olkinuora H. von Willebrand E. Kantele J.M. Vainio O. Talvensaari K. Saarinen-Pihkala U. Siitonen S. Vettenranta K. The impact of early viral infections and graft- versus -host disease on immune reconstitution following paediatric stem cell transplantation Scand. J. Immunol. 2011 73 586 593 10.1111/j.1365-3083.2011.02530.x 21323694

60. Perales M.A. Ishill N. Lomazow W.A. Weinstock D.M. Papadopoulos E.B. Dastigir H. Chiu M. Boulad F. Castro-Malaspina H.R. Heller G. Long-term follow-up of patients treated with daclizumab for steroid-refractory acute graft- vs. -host disease Bone Marrow Transplant. 2007 40 481 486 10.1038/sj.bmt.1705762 17618322

61. Willenbacher W. Basara N. Blau I.W. Fauser A.A. Kiehl M.G. Treatment of steroid refractory acute and chronic graft- versus -host disease with daclizumab Br. J. Haematol. 2001 112 820 823 10.1046/j.1365-2141.2001.02582.x 11260089

62. Arai S. Margolis J. Zahurak M. Anders V. Vogelsang G.B. Poor outcome in steroid-refractory graft- versus -host disease with antithymocyte globulin treatment Biol. Blood Marrow Transplant. 2002 8 155 160 10.1053/bbmt.2002.v8.pm11939605 11939605

63. McCaul K.G. Nevill T.J. Barnett M.J. Toze C.L. Currie C.J. Sutherland H.J. Conneally E.A. Shepherd J.D. Nantel S.H. Hogge D.E. Treatment of steroid-resistant acute graft- versus -host disease with rabbit antithymocyte globulin J. Hematother. Stem Cell Res. 2000 9 367 374 10.1089/15258160050079470 10894358

64. Khoury H. Kashyap A. Adkins D.R. Brown R.A. Miller G. Vij R. Westervelt P. Trinkaus K. Goodnough L.T. Hayashi R.J. Treatment of steroid-resistant acute graft- versus -host disease with anti-thymocyte globulin Bone Marrow Transplant. 2001 27 1059 1064 10.1038/sj.bmt.1703032 11438821

65. Storek J. Joseph A. Espino G. Dawson M.A. Douek D.C. Sullivan K.M. Flowers M.E. Martin P. Mathioudakis G. Nash R.A. Immunity of patients surviving 20 to 30 years after allogeneic or syngeneic bone marrow transplantation Blood 2001 98 3505 3512 10.1182/blood.V98.13.3505 11739150

66. Storek J. Witherspoon R.P. Storb R. T cell reconstitution after bone marrow transplantation into adult patients does not resemble T cell development in early life Bone Marrow Transplant. 1995 16 413 425 8535315

67. Geyer M.B. Ricci A.M. Jacobson J.S. Majzner R. Duffy D. Van de Ven C. Ayello J. Bhatia M. Garvin J.H. Jr. George D. T cell depletion utilizing CD34(+) stem cell selection and CD3(+) addback from unrelated adult donors in paediatric allogeneic stem cell transplantation recipients Br. J. Haematol. 2012 157 205 219 10.1111/j.1365-2141.2012.09048.x 22313507

68. Dvorak C.C. Gilman A.L. Horn B. Oon C.Y. Dunn E.A. Baxter-Lowe L.A. Cowan M.J. Haploidentical related-donor hematopoietic cell transplantation in children using megadoses of CliniMACs-selected CD34(+) cells and a fixed CD3(+) dose Bone Marrow Transplant. 2013 48 508 513 10.1038/bmt.2012.186 23178543

69. Dvorak C.C. Gilman A.L. Horn B. Jaroscak J. Dunn E.A. Baxter-Lowe L.A. Cowan M.J. Clinical and immunologic outcomes following haplocompatible donor lymphocyte infusions Bone Marrow Transplant. 2009 44 805 812 10.1038/bmt.2009.87 19421175

70. Alpdogan O. Hubbard V.M. Smith O.M. Patel N. Lu S. Goldberg G.L. Gray D.H. Feinman J. Kochman A.A. Eng J.M. Keratinocyte growth factor (KGF) is required for postnatal thymic regeneration Blood 2006 107 2453 2460 10.1182/blood-2005-07-2831 16304055 PMC1895735

71. Jenq R.R. King C.G. Volk C. Suh D. Smith O.M. Rao U.K. Yim N.L. Holland A.M. Lu S.X. Zakrzewski J.L. Keratinocyte growth factor enhances DNA plasmid tumor vaccine responses after murine allogeneic bone marrow transplantation Blood 2009 113 1574 1580 10.1182/blood-2008-05-155697 19011222 PMC2644085

72. Vadhan-Raj S. Goldberg J.D. Perales M.A. Berger D.P. van den Brink M.R. Clinical applications of palifermin: Amelioration of oral mucositis and other potential indications J. Cell Mol. Med. 2013 17 1371 1384 10.1111/jcmm.12169 24251854 PMC4117550

73. Perales M.A. Goldberg J.D. Yuan J. Koehne G. Lechner L. Papadopoulos E.B. Young J.W. Jakubowski A.A. Zaidi B. Gallardo H. Recombinant human interleukin-7 (CYT107) promotes T-cell recovery after allogeneic stem cell transplantation Blood 2012 120 4882 4891 10.1182/blood-2012-06-437236 23012326 PMC3520625

74. Hegenbart U. Niederwieser D. Sandmaier B.M. Maris M.B. Shizuru J.A. Greinix H. Cordonnier C. Rio B. Gratwohl A. Lange T. Treatment for acute myelogenous leukemia by low-dose, total-body, irradiation-based conditioning and hematopoietic cell transplantation from related and unrelated donors J. Clin. Oncol. 2006 24 444 453 10.1200/JCO.2005.03.1765 16344316

75. Aoudjhane M. Labopin M. Gorin N.C. Shimoni A. Ruutu T. Kolb H.J. Frassoni F. Boiron J.M. Yin J.L. Finke J. Comparative outcome of reduced intensity and myeloablative conditioning regimen in HLA identical sibling allogeneic haematopoietic stem cell transplantation for patients older than 50 years of age with acute myeloblastic leukaemia: A retrospective survey from the Acute Leukemia Working Party (ALWP) of the European group for Blood and Marrow Transplantation (EBMT) Leukemia 2005 19 2304 2312 10.1038/sj.leu.2403967 16193083

76. O’Reilly R.J. Doubrovina E. Trivedi D. Hasan A. Kollen W. Koehne G. Adoptive transfer of antigen-specific T-cells of donor type for immunotherapy of viral infections following allogeneic hematopoietic cell transplants Immunol. Res. 2007 38 237 250 10.1007/s12026-007-0059-2 17917029

77. Koehne G. Doubrovina E. Hasan A. Barker J.N. Castro-Malaspina H. Perales M.A. Jakubowski A. Papadopoulos E. Young J.W. Boulad F. A Phase I Dose Escalation Trial of Donor T Cells Sensitized with Pentadecapeptides of the CMV-pp65 Protein for the Treatment of CMV Infections Following Allogeneic Hematopoietic Stem Cell Transplants Blood 2009 114 2262

78. Doubrovina E. Oflaz-Sozmen B. Prockop S.E. Kernan N.A. Abramson S. Teruya-Feldstein J. Hedvat C. Chou J.F. Heller G. Barker J.N. Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+ lymphomas after allogeneic hematopoietic cell transplantation Blood 2012 119 2644 2656 10.1182/blood-2011-08-371971 22138512 PMC3311278

79. Blyth E. Clancy L. Simms R. Ma C.K. Burgess J. Deo S. Byth K. Dubosq M.C. Shaw P.J. Micklethwaite K.P. Donor-derived CMV-specific T cells reduce the requirement for CMV-directed pharmacotherapy after allogeneic stem cell transplantation Blood 2013 121 3745 3758 10.1182/blood-2012-08-448977 23435462