Umbilical Cord Blood Transplantation in Severe T-Cell ... - Springer Link

Journal of Clinical Immunology, Vol. 20, No. 6, 2000

Umbilical Cord Blood Transplantation in Severe T-Cell Immunodeficiency Disorders: Two-Year Experience ALAN P. KNUTSEN,1,3 and DONNA A. WALL2

strated that cord blood stem cell transplantation resulted in consistent and stable T-, B- and natural killer (NK) cell development. The kinetics of development were such that T-cell development occurred between 60 to 100 days. Initial T-cell engraftment consisted predominantly of CD45RO⫹, CD3⫹, and CD4⫹ T cells, and at 12 to 24 months changed to CD45RA⫹, CD3⫹, and CD4⫹ T cells, indicating de novo maturation of T cells. NK cell development occurred at approximately 180 days. B cells engrafted early, and study of functional B-cell antibody responses revealed that five of six patients in whom intravenous immune globulin has been discontinued have low detectable antibody responses to tetanus and diphtheria toxoid immunizations at 18 to 24 months posttransplantation. Unrelated umbilical donor cord blood is an alternative source of stem cells for transplantation in children with severe T-cell immune deficiency disorders when a suitable HLA-matched donor is not available and when a T-depleted haploidentical preparation is not beneficial. Benefits of UCB include rapid and reliable recovery of immune function, low risk of GvHD, and low viral transmission rate.

Accepted: May 30, 2000

Hematopoietic stem cell transplantation is the treatment of choice for severe primary T-cell immunodeficiencies. When an HLA-identical sibling as the donor is not available, an alternative donor stem cell source is needed. In primary T-cell immunodeficiencies, T-cell-depleted HLA-haploidentical bone marrow transplantation has been particularly successful in reconstituting the immune system in many but not all of the severe T-cell immune deficiency disorders. This study reports the use of umbilical cord blood (UCB) stem cell transplantation in severe T-cell immune deficiency. Umbilical cord blood was evaluated as a stem cell source for immune reconstitution in children with severe primary T-cell immunodeficiency disorders, such as severe combined immunodeficiency syndrome (SCID), reticular dysgenesis, thymic dysplasia, combined immunodeficiency disease (CID), and Wiskott–Aldrich syndrome (WAS) when a matched sibling donor was unavailable. From 1/96 through 5/98, eight children received unrelated cord blood stem cell transplantation following a preparative regimen for the treatment of combined immunodeficiency diseases. The patients ranged in age from 2 weeks to 8 years. The cord blood units were 3/6 HLA antigen matches in two children, 4/6 in four children, and 5/6 in two child, with molecular HLA-DR mismatch in three of the children. The average time for neutrophil engraftment (absolute neutrophil count ⬎500/mm3) was 12 days (range 10 –15 days) and the average time for platelet engraftment (platelet count ⬎20,000/mm3) was 36 days (range 24 –50 days). A patient with reticular dysgenesis failed to engraft following her first transplant, but fully engrafted after a second unrelated donor cord blood transplantation. Five of six patients exhibited grade I graft-versus-host disease (GvHD), while one child had grade IV skin and gut GvHD. Immunologic reconstitution demon-

KEY WORDS:Umbilical cord blood transplantation; severe combined immunodeficiency; combine immunodeficiency; graft-versus-host disease.

INTRODUCTION

Immune reconstitution using HLA-matched bone marrow donor is the treatment of choice for severe primary T-cell immunodeficiencies, including severe combined immunodeficiency syndrome (SCID), reticular dysgenesis, thymic dysplasia (Nezelof’s syndrome), combined immunodeficiency disease (CID), and Wiskott–Aldrich syndrome (WAS) (1–5). When this is unavailable, an alternative stem cell source is needed, such as T-celldepleted HLA-haploidentical bone marrow. In primary T-cell immunodeficiencies, this donor source has been particularly successful in reconstituting the T-cell immune system in many of the SCID types (1, 6 – 8). However, there may be increased resistance to engraft-

1

Divisions of Allergy/Immunology, Department of Pediatrics, St. Louis University Health Sciences Center and Cardinal Glennon Children’s Hospital, St. Louis, Missouri 63110. 2 Hematology/Oncology/Bone Marrow Transplantation, Department of Pediatrics, St. Louis University Health Sciences Center and Cardinal Glennon Children’s Hospital, St. Louis, Missouri 63110. 3 To whom correspondence should be addressed at Allergy/ Immunology, Pediatric Research Institute, St. Louis University Health Sciences Center, 3662 Park Avenue, St. Louis, Missouri 63110.

466 0271-9142/00/1100-0466$18.00/0 © 2000 Plenum Publishing Corporation

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UMBILICAL CORD BLOOD TRANSPLANTATION

ment or nonengraftment in some of the T-cell defects, such as adenosine deaminase deficiency, SCID with elevated natural killer (NK) cells, and thymic dysplasia and induction of Epstein–Barr virus (EBV)-induced lymphoproliferative disease in WAS. In addition, there is typically a prolonged period for T-cell engraftment to occur, up to 3 to 6 months, and then weak T-cell function and B-cell antibody function frequently remains abnormal. Graft failure and the prolonged time to engraftment is a particular problem if the infant is already infected prior to the time of transplant. Recently, umbilical cord blood transplantation (UCB) has been evaluated as an alternative stem cell source when a genotypic HLA-matched donor was not available in other clinical settings (9 –14). We previously reported the kinetics of T, B, and NK cell engraftment in the first year post-UCB transplantation in six patients with severe T-cell immunodeficiency disorders (15). In the present study, we report the results of eight patients who received unrelated umbilical cord blood transplantation for severe primary T-cell immunodeficiency disorders and development of T-, N-, and B-cell function in the first 2 years posttransplantation.

METHODS

Patients Patients presenting to the Stem Cell Transplant Program at Cardinal Glennon Children’s Hospital with severe T-cell immunodeficiency were considered for unrelated donor cord blood transplantation if they did not have a HLA-matched donor available. HLA matching between umbilical cord blood donor and patient was based on serological class I HLA typing and highresolution molecular class II typing. Priority was given for HLA-DR matching at the high-resolution molecular level. Donor–recipient pair was considered matched for class I HLA if they were identical at the level of serological determination, and for class II HLA if they were identical at the level of high-resolution molecular typing. Other HLA alleles were not evaluated. This study was approved by the Institutional Review Board at Saint Louis University Health Sciences Center. Preparative Regimen and Graft-versus-Host Disease Prophylaxis The transplant regimen used was part of a collaboration with Dr. Joanne Kurtzberg (Duke Cord Blood Collaborative Group). Pretransplant conditioning for all


patients was busulfan 1 mg/kg (1.25 mg/kg if ⬍2 years old) orally every 6 hours on days -9 through -6. The dosage of busulfan was adjusted based on first-dose kinetics (steady-state level 400 – 600 ng/ml). This was followed by cyclophosphamide 50 mg/kg intravenously on days -5 through -2 and antithymocyte globulin (ATG) 30 mg/kg intravenously on days -3 through -1. Umbilical cord blood infusion was on day 0. One child failed to engraft and had early trilineage autologous recovery of hematopoiesis. She subsequently was treated with a salvage preparative regimen with fractionated total body irradiation (1000 cGy), cyclophosphamide, and ATG, followed by a second infusion of unrelated donor cord blood from a second unrelated donor. Prophylaxis for acute graft-versus-host disease (GvHD) included continuous infusion of cyclosporine A beginning on day -2, targeting whole blood levels to be 250 to 350 ng/ml, and methylprednisolone 10 mg/kg per day on days 5–7, 5 mg/kg per day, on days 8 –10, and 3 mg/kg per day on days 11–13, followed by a 10% weekly reduction taper. The patients were evaluated daily during hospitalization for acute GvHD and at least weekly following hospital discharge for the first 100 days after transplantation. The diagnosis of acute GvHD was made by clinical evaluation. Corticosteroids were generally discontinued by day ⫹60 posttransplantation and cyclosporine A was discontinued between day ⫹100 day to ⫹365, depending on clinical evidence of GvHD.

Preparation of Umbilical Cord Blood Previous reports detailed the collection and processing of umbilical cord blood (16). Cryopreserved donor cord blood units were obtained from the New York Placental Blood Program or the St. Louis Cord Blood Bank. All units used were seronegative for cytomegalovirus (CMV), EBV, hepatitis viruses, and human immunodeficiency virus (HIV) types 1 and 2. Hemoglobin electrophoresis was performed on units at risk for sickle-cell disease and thalassemia. The thawing procedure used was developed by Rubinstein et al. (16). Briefly, the umbilical cord blood was placed in a sterile bag and thawed in a 38°C water bath with gentle agitation. After thawing, an equal volume of dextran/albumin solution was added over 10 minutes, centrifuged at 250 g for 10 minutes at 10°C, and the supernatant removed. The buffy coat was resuspended in dextran/albumin and immediately infused into the patient over 5 to 10 minutes.

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Supportive Care All the patients were hospitalized in single rooms with high-efficiency particulate air filtration systems. Prophylactic acyclovir was used if the patient was Herpes simplex virus (HSV) or CMV positive prior to treatment. Fluconazole was used for prevention of fungal infections. Patients were given intravenous immune globulin (IVIG) 500 mg/kg intravenously weekly through day 100 posttransplant and then monthly for the first year. Granulocyte colony stimulating factor (GCSF) at 10 ␮g/kg per day subcutaneously was given from day of transplant until the absolute neutrophil count (ANC) ⬎2000/mm3 was attained. Patient 2 received irradiated granulocyte infusions during the neutrophenic nadir to assist management of necrotizing Pseudomonas pneumonitis that was present prior to transplantation. Engraftment Hematologic recovery was defined as achieving an ANC ⬎ 500/mm3 (first of 2 consecutive days) and a platelet count ⬎20,000/mm3 (first of 7 consecutive days without the need for transfusion). Donor cell engraftment was assessed by restriction fragment length polymorphism (RFLP), quantitating donor-recipient chimerism. Immunologic Studies T, NK, and B cell enumeration and lymphocyte stimulation studies were performed as previously described (15). Cytofluorographic analyses of lymphocyte subpopulations were determined by reacting murine monoclonal antibodies conjugated either with fluorescein (FITC) or phycoerythrin (PE) and then analyzed by flow cytometry (FACScan; Becton Dickinson, San Jose, CA). Lymphoproliferative responses were performed to phytohemagglutinin (PHA) (Difco, Detroit, MI), concanavalin A (Con A) (Sigma, St. Louis, MO), pokeweed mitogen (PWM) (Gibco BRL, Grand Island, NY), Candida albicans (Greer Laboratories, Lenoir, NC), tetanus toxoid (Connaught Laboratories, Willowdale, Ontario Canada), and to alloantigens (mixed B-cell pool). NK activity was assayed by using peripheral blood mononuclear cells, isolated from heparinized venous blood by Ficoll-Hypaque density gradient sedimentation, as effector cells against 51Cr-labeled K562 cell line target at an effector:target ratio of 100:1 (15). Normal controls consisted of healthy adult laboratory personnel volunteers. Serum IgG, IgA, and IgM quantification was performed by Clinical Laboratory in the Department of Pathology using nepholometry (15). Anti-diphtheria and anti-

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tetanus antibody titers were measured by tanned cell hemagglutination (15). Assay for adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activity in red blood cells were performed by M. S. Hershfield at Duke Medical Center (3). Studies for identification of common cytokine receptor gamma chain (␥c) and Janus kinase 3 (Jak3) deficiencies were performed by J. M. Puck at the National Institutes of Health and by J. Roberts at Duke Medical Center, respectively, by Western blot and DNA sequencing analyses on EBV transformed B-cell lines (3).

RESULTS

Patient Characteristics Eight patients received unrelated donor umbilical cord blood transplants for primary T-cell immunodeficiency disorders at Cardinal Glennon Children’s Hospital of Saint Louis University Health Sciences Center from 1/96 through 5/98 (Table I). Patients CG36 to CG74 were previously reported following their first year posttransplantation (15). Primary severe T-cell immunodeficiency disorders were diagnosed as one patient with Jak3 deficiency SCID, two with autosomal recessive form of SCID, one with Nezelof’s syndrome, one with reticular dysgenesis, two with CID, and one with WAS. Four of the patients were ill at the time of transplantation, including Pseudomonas necrotizing pneumonitis that required lobectomy (CG52), bacterial and fungal pneumonitis and bronchiectasis (CG71), RSV pneumonia (CG74), and CMV and PCP pneumonia (CG86). The results of pretransplant immunologic evaluation are presented in Table II. Significantly decreased CD3⫹ T-cell percentages, numbers, and lymphoproliferative responses to mitogens (PHA, Con A, PWM), antigens (Candida albicans, tetanus toxoid), and alloantigen stimulations were present in the patients with SCID and Nezelof’s syndrome. The patients with CID (CG71 and CG86) had normal percentage and number of CD3⫹ T cells; patient CG71 had decreased percentages and numbers of CD4⫹ T cells with markedly decreased T-cell function. CD56⫹ NK cells were elevated in the patients with autosomal recessive (AR) SCID (CG 67, CG 74) and reticular dysgenesis (CG64); however, only one of the AR SCID patients had increased NK cytolytic function. Hematopoietic Engraftment All patients had documentation of 100% donor chimerism (following second transplantation in the child


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Table I. Pretransplantation Morbidity in Patients with Severe Primary T-Cell Immunodeficiency Disorders Who Underwent UCB Transplantationa Patient

Diagnosis

Sex (M/F)

Age at diagnosis/ transplantation

CG36

Nezelof’s syndrome

F

15/15 mo

CG52 CG64 CG67 CG71 CG74

SCID, Jak3 deficiency Reticular dysgenesis SCID, AR CID, CD4 lymphopenia SCID, AR

F F F M F

5/10 mo 2 wk/1, 3 mo NB/2 wk 5/6 yr 5/6 mo

CG86 CG88

CID WAS

F M

3/7 mo 1/8 yr

Pretransplantation morbidity

Outcome

FTT Pseudomonas necrotizing pneumonia, lobectomy LLL Bronchiectasis, bacterial, fungal RSV pneumonia, GvHD CMV pneumonitis, PCP

A/W A/W A/W A/W Died A/W Chronic interstitial pneumonitis A/W

a

Abbreviations: RSV, respiratory syncytial virus; CMV, cytomegalovirus; PCP, Pneumocystis carinii pneumonia; FTT, failure to thrive; GvHD, graft-versus-host disease; SCID, severe combined immunodeficiency; Jak3, Janus kinase 3; AR, autosomal recessive; CID, combined immunodeficiency; WAS, Wiskott–Aldrich syndrome; A/W, alive and well; left lower lobe (LLL).

with reticular dysgenesis) determined by RFLP analysis once peripheral blood neutrophil and lymphocyte numbers recovered, usually at 1 month posttransplantation. All patients in the group reached an ANC ⬎500/mm3; the mean time to engraftment was 12 ⫾ 1 days (range 11

to 16 days) (Table III). The patient with reticular dysgenesis (CG64) rejected her initial UCB transplantation with early trilineage recovery of autologous cells. She was prepared again as described and engrafted with the second cord blood infusion. The mean time to platelet

Table II. Initial Immunologic Studies of Patients with Severe Primary T-Cell Immunodeficiency Disorders Prior to UCB Transplantationa Study 3

Lymphocytes/mm CD3⫹ cells/mm3 % CD4⫹ cells/mm3 % CD8⫹ cells/mm3 % CD20⫹ cells/mm3 % CD56⫹ cells/mm3 % PHA, cpm % NR Con A, cpm % NR PWM, cpm % NR Candida albicans, cpm SI Tetanus toxoid, cpm SI MLC, cpm % NR NK, % lysis IgG, mg/dl IgA, mg/dl IgM, mg/dl IgE, IU/ml ␣-HiB (PRP), ␮g/ml ␣-Diphtheria toxoid ␣-Tetanus toxoid

CG36

CG52

CG64

CG67

CG71

CG74

1598 304 19 96 6 128 8 927 58 288 18 14,315 5.1 17,014 9.0 3867 3.0 2922 1.6 1943 1.4 5773 4.2 1.5 303 175 55 8 ⬍0.5 0 0

610 0 0 12 2 6 1 567 93 73 12 ⫺944 2.0 ⫺218 1.0 ⫺1815 0.8 191 1.1 1863 2.0 1929 1.2 — 56 ⬍7 34 15 ⬍0.5 0 81

574 75 13 34 6 98 17 80 14 264 46 3020 1.0 ⫺386 0.1 ⫺3973 3.3 — — — — ⫺227 0.2 0 1150 ⬍7 ⬍25 ⬍5 — — —

312 0 0 9 3 144 46 16 5 275 88 179 0.1 287 0.1 450 0.1 — — — — 1997 1.2 0 950 ⬍7 ⬍5 ⬍5 — — —

1638 901 55 278 17 606 37 491 30 246 22 30,813 13.7 16,531 7.8 26,096 11.2 7245 1.7 8126 1.8 ⫺1237 1.6 27.5 237 41 44 108 ⬍0.5 0 27

1120 101 9 134 12 45 4 0 0 594 53 18,819 5.9 43,035 18.3 367 0.2 ⫺2194 0.8 ⫺2592 0.7 2290 1.7 72.2 ⬍46 ⬍7 ⬍5 ⬍5 ⬍0.5 3 27

a

CG86 8316 6486 78 4075 49 2245 27 1331 16 416 5 138,735 75.5 36,013 50.9 37,993 67.1 615 1.1 681 1.1 35,553 9.9 14.1 439 13 286 13 ⬍0.3 0 81

CG88 1320 977 74 845 64 607 46 119 9 158 12 167,508 91.2 40,830 57.7 56,236 99.3 2,334 2.7 2,663 2.7 8,945 12.2 — 1750 93 18 3300 ⬍0.5 3 27

Normal values 3800 –9900 2400 – 6900 50 –77 1400 –5100 33–58 600 –2200 13–26 700 –2500 13–35 200 –1200 2–13 257,076 ⫻/⫼ 2.6 ⱖ50 163,503 ⫻/⫼ 3.1 ⱖ50 116,647 ⫻/⫼ 2.9 ⱖ50 16,846 ⱖ3 17,482 ⱖ3 119,893 ⫻/⫼ 1.1 ⱖ30 41 ⫾ 3 268 –717 12–31 18 –191 3–15 ⬎1.0 ⬎2,000 ⬎10,000

Normal lymphocyte population for 9- to 15-month-old patients is expressed as the range for 5th and 95th percentiles. Normal lymphoproliferative responses are expressed as mean ⫾ SE in adult control mean (22). Normal immunoglobulin serum concentrations for 7- to 9-month-old patients are expressed as the range for 5th and 95th percentiles. Abbreviations: PHA, phytohemagglutinin; Con A, concanavalin A; PWM, pokeweed mitogen; MLC, mixed lymphocyte culture to B-cell alloantigens; NR, normal response; SI, stimulation index.


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Table III. Histocompatibility Matching and Cell Dose of UCB, GvHD, and Engraftment in Patients with Severe Primary T-Cell Immunodeficiency Disorders Who Underwent UCB Transplantationa Study

CG36

CG52

CG64

CG67

CG71

CG74

CG86

CG88

Average

Degree of HLA match (n ⫽ 6) Location of mismatch UCB transplantation cell dose (⫻108 nucleated cells/kg) GvHD, grade Skin GI Engraftment, days ANC (⬎500 cells/mm3) Platelets (⬎20,000 cells/mm3) CD3 (⬎1000 cells/mm3)

4 A,B

4 A,B

3 A,B,DRmol

5 A

4 A,DR

3 A,B,B

6 —

4 A,A

— —

1.3 I ⫹ 15 42 239

2.0 I ⫹ 10 50 218

1.4 I ⫹ 11 38 246

1.1 I ⫹ 12 34 101

1.2 IV ⫹ ⫹ 12 24 —

1.6 I ⫹ 12 28 189

1.1 I ⫹ 10 28 54

1.0 I ⫹ 16 49 347

1.3 ⫾ 0.1 —

12 ⫾ 1 37 ⫾ 4 98 ⫾ 2

Abbreviations: DRmol, molecular HLA-DR; GI, gastrointestinal; ANC, absolute neutrophil count; GvHD, graft-versus-host disease. Mean ⫾ SE.

a

engraftment was 36 ⫾ 4 days (range 24 to 50 days) (Table III). GvHD As seen in Table III, the degree of acute GvHD among the patients in the series ranged from grade I in five patients to grade IV in one patient. Two patients had limited GvHD, consisting of mild skin rash and diarrhea that resolved with corticosteroid pulse during the cyclosporine taper. Patient CG71, who had a serological HLA-DR mismatch, had progression of GvHD while on steroids but responded to antithymocyte globulin. Patient CG71 eventually died of infectious complications. None of the surviving patients have developed chronic GvHD with follow up at 12 to 30 months. Patient CG86, who had CMV pneumonitis pretransplantation, has developed chronic fibrosing interstitial pneumonitis. Lymphoid Development The kinetics of development of the T-, B- and NKlymphocyte populations are presented in Fig. 1. The mean absolute lymphocyte count (ALC) began to increase at approximately 2 months predominantly due to increases of CD3⫹ T cells and CD20⫹ B cells. Thereafter, CD3⫹ T cells steadily increased between 2 to 9 months, 3487 ⫾ 1488 cells/mm3, and remained stable thereafter in an age-adjusted normal range. CD4⫹ and CD8⫹ T cells increased proportionally beginning at 3 months. CD4⫹ T cells increased to 1721 ⫾ 589 cells/ mm3 at 9 months. Examination of the kinetics of T-cell development for the individual patients demonstrated a similar pattern and rate of development in all the patients, beginning at 100 days and steadily increasing. Lymphoproliferative responses to mitogens and alloan-

tigens were detectable as early as 60 days preceding the marked increase of T cells and steadily increased to normal (defined as ⬎50% normal response) by 6 months and remained stable, as seen in Fig. 2. At 1 year posttransplantation, the children were immunized. Tetanus toxoid-specific lymphoproliferative responses were present in six of six patients at 18 months posttransplantation, SI 21 ⫾ 16. Development of naı¨ve T cells was measured by expression of CD3⫹CD45RA⫹ and CD4⫹CD45RA⫹ T cells as an indication of de novo thymopoiesis. As seen in Fig. 3, CD45RO⫹CD3⫹ and CD4⫹ T cells predominated at 2 to 6 months posttransplantation. However, between 12 to 24 months posttransplantation, CD45RA⫹CD3⫹ and CD4⫹ T cells predominated compared to CD45RO⫹ T cells (2786 ⫾ 648 vs. 642 ⫾ 72 and 1379 ⫾ 453 vs. 450 ⫾ 73, respectively, at 24 months). Development of NK cells occurred consistently by 6 months, both for CD56⫹ NK cell phenotype (Fig. 1) and NK cytolytic function as determined by percent lysis of K562 cells as seen in Fig. 4. As seen in Fig. 1, donor B cell engraftment occurred by 3 months posttransplantation. Antibody responses to diphtheria and tetanus toxoid immunizations were then determined beginning at 1 year posttransplantation. As seen in Fig. 5, serum IgG levels remained within a normal range for age after IVIG was discontinued at 1 year posttransplantation. Antibody responses to diphtheria and tetanus toxoids were present in six of six patients at 18 months posttransplantation and increased following booster immunizations at 24 months. DISCUSSION

In immune reconstitution of patients with severe T-cell diseases, the ideal stem cell source is an unma-


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Fig. 1. Development of percentages (A) and absolute numbers (B) of CD3⫹, CD4⫹, and CD8⫹ T-, CD20⫹ B-, and CD56⫹ NK cells for patients with severe T-cell immunodeficiency disorders following UCB transplantation. For each patient (with exception of patient CG71 who had grade IV GvHD), CD3⫹ T cells appeared at approximately 100 days and steadily increased thereafter. CD4⫹ and CD8⫹ T cells increased proportionally. CD20⫹ B cells appeared by 3 months and CD56⫹ NK cells at 6 months posttransplantation.

nipulated genotypic HLA matched sibling bone marrow. Umbilical cord blood is recognized as a source of CD34⫹ hematopoietic progenitor cells that can result in durable engraftment with potentially lower degrees of GvHD and lower viral transmission than is expected from unrelated donor bone marrow (16 –21). Also, po-


tentially advantageous is the ability to infuse T cells with the graft, which may result in earlier immune recovery. In evaluating the relative strength of cord blood compared to other stem this study formally evaluated the tempo and quality of immune recovery post transplant. The initial T-cell engraftment was memory CD45RO⫹ T

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Fig. 2. Development of lymphoproliferative responses to PHA, Con A, PWM, alloantigens, and tetanus toxoid for patients with severe T-cell immunodeficiency disorders reported as percent %NR for mitogens and alloantigens and as SI for tetanus toxoid (A) and as cpm (B) following UCB transplantation. Lymphoproliferative responses were detectable to mitogens and alloantigens as early as 60 days, preceding the marked increase of T cell, and steadily increasing to normal (defined as ⬎50%NR) by 180 days and remained stable thereafter. Engraftment of NK cytotoxic function as measured by percent lysis of K562 cells occurred consistently by 180 days (with exception of patient CG71 who had grade IV GvHD).

cells. However, naı¨ve T cell, indicative of stem cell engraftment and thymopoiesis, predominated at 1 year posttransplantation. In the present study, infants with a variety of severe T-cell immunodeficiency disorders were reconstituted

with a minimum cell dose of 1 ⫻ 108 cord blood cells/kg with a resulting neutrophil recovery time that is the same as seen for bone marrow. The engraftment time is similar to that reported for CD34 selected stem cell infusions in the unrelated donor peripheral stem cell transplant set-


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Fig. 3. Development of numbers (A) and percentages (B) of CD45RA⫹CD3⫹ and CDCD45RA⫹CD4⫹ T cells for patients with severe T-cell immunodeficiency disorders following UCB transplantation. Following UCB transplantation CD45RA⫹ CD3⫹ and CD4⫹ T cells predominated for the first 6 months. Between 1 to 2 years, CD45RO⫹ CD3⫹ and CD4⫹ T cells predominated.

ting (21). Platelet recovery is delayed in comparison to marrow and peripheral stem cell transplants, as previously reported (4). Trials directly comparing the alternative donor stem cell sources are needed in order to define the optimal stem cell source for this group of children. In this study, early appearance of T-cell immunocompetence was observed due to engraftment of memory T cells. In addition, umbilical cord blood transplantation resulted in a stable long-term T-cell engraftment with


normal lymphoproliferative responses to mitogens and alloantigens. In addition, donor NK cell number and function appeared consistently at approximately 6 months. T and NK cell development correlated with the clinical observation of few late infections. The kinetics of donor T and NK cell development is similar to that observed with T-depleted haploidentical bone marrow transplantation in patients with SCID regardless of preparative regimen used in these studies (1– 8). In addition,

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Fig. 4. Development of NK cell cytotoxic function as measured by percent lysis of K562 cells for patients with severe T-cell immunodeficiency disorders following UCB transplantation. Development of NK cell cytotoxic function occurred consistently by 180 days (with exception of patient CG71 who had grade IV GvHD).

the patients developed antigen specific T-cell responses. Furthermore, since all of the hematopoietic cells were donor origin and long-term T cell engraftment suggested de novo T-cell maturation, full T-cell and antigen presenting cell (APC) cooperation would be hypothesized.

Donor cord blood B-cell engraftment was observed with UCB transplantation, whereas with T-depleted haploidentical bone marrow transplantation functional humoral immunity develops in approximately 50% of the patients who retain host B cells probably due to intrinsic

Fig. 5. Serum immunoglobulin concentrations in patients with severe T-cell immunodeficiency disorders following UCB transplantation. IVIG was discontinued at 1 year posttransplantation. Serum IgG levels remained within a normal rage for age thereafter.


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B cell defects (1). In T-cell-depleted bone marrow transplantation, cytoreduction treatment improves engraftment of donor B cells and the antibody responses. Evaluation of B cell function in these patients demonstrated engraftment of donor B cells and that tetanus and diphtheria toxoid antibody responses have appeared in six of six patients at 18 to 24 months posttransplantation. In our series, all the patients exhibited some degree of acute GvHD, but the severity of GvHD was typically grade I, mild, and resolved with increased corticosteroid treatment and did not evolve into significant chronic GvHD. The occurrence of grade I GvHD did not correlate with the degree of HLA class I mismatch. However, grade IV GvHD did occur in the only patient with a serological HLA-DR mismatch and this patient died of infectious complications. This is a contrast to HLAmatched related and unrelated donor bone marrow transplantation, for which this is a clear correlation between the degree of mismatch between the donor and recipient and the degree of GvHD (9, 18). The relative immaturity of cord blood T cells and/or their ability to become tolerant to host HLA antigens has been postulated as the reasons for the reduced risk and severity of GvHD from that which would be expected given the degree of HLA mismatch. One important issue in the management of children with severe T-cell disorders is whether to utilize a preparative regimen prior to stem cell infusion for immunosuppression to prevent rejection and/or myeloablation to make “space” to allow donor T-, B-, and monocytic cell engraftment. Indeed, this is an important question for all of the hematopoietic stem cell options and for umbilical cord blood. O’Reilly et al. (1) and Fischer et al. (19) reported graft failures in 30% and 50% of the patients who received T-depleted bone marrow graft transplantation without prior cytoreduction in the United States and Europe, respectively. In both murine models and in human SCID patients, normal to high NK cell activity has a higher incidence of graft failure or delayed immunologic reconstitution and is perhaps a foremost cause for graft rejection. The nature of the preparative regimen and the relative importance of immunosuppression versus ablation has not as yet been fully defined and may be dependent on the nature of the hematopoietic stem cell graft. Furthermore, children with severe T-cell diseases may present with serious infections and cannot eliminate the infection prior to transplantation. This was observed in this cohort of patients, e.g., necrotizing Pseudomonas pneumonia, respiratory synctral virus (RSV) pneumonia, and chronic bronchiectatic lung disease. In these children, dependable early recovery of immunocompetent function was important.


This is not necessarily so when T-cell-depleted haploidentical bone marrow transplantation is performed (1, 2, 19). The pretransplantation preparative regimen used in this study was a conventional regimen that is used in young children undergoing hematopoietic stem cell transplantation for malignant and nonmalignant hematopoietic disorders. It was chosen because it has known myeloablative and immunosuppressive properties. Our goal was to ensure that there would be adequate immunosuppression and hematopoietic space to ensure trilineage engraftment of progenitor cells and at the same time infusion of immunocompetent mature T cells. The role of a myeloablation in the preparative regimen in transplantation for SCID remains controversial. Other groups have obtained stable immune reconstitution using umbilical cord blood transplantation in patients with thymic dysplasia and SCID without ablative therapy (20). However, there are some subsets of severe T-cell immunodeficiency disorders that require immunosuppression preparative regimen, such as reticular dysgenesis, CID, thymic dysplasia, and WAS. Some groups also would use a preparative regimen in SCID with high NK function (1). Further studies will be necessary to determine whether this is a true or only theoretical advantage. In addition, our patients received posttransplantation GvHD prophylaxis of cyclosporine and corticosteroids, which would affect the function of mature T cells in the umbilical cord preparation. As the GvHD prophylaxis has been shortened in these patients with subsequent experience of using UCB, it is unclear how much GvHD prophylaxis is needed. In summary, in unrelated donor transplant for children with primary immunodeficiency disorders, it is transplantation-related mortality that is the greatest risk for these children, including graft rejection, preexisting and acquired opportunistic infections, GvHD, and toxicity from the myeloablative preparative regimen. The major morbidity in this series of patients was related to the transplant preparative regimen with remarkably few infectious problems. We conclude that in the unrelated donor stem cell transplant setting, umbilical cord blood should be considered as an alternative source for children with severe combined immunodeficiency syndromes.

ACKNOWLEDGMENTS

We acknowledge Drs. Pablo Rubinstein and Joanne Kurtzberg for their support of this study and the field of cord blood transplantation. We also acknowledge the efforts of the stem cell transplant team at Cardinal Glennon Children’s Hospital who made this study possible.

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