Blood, 1 February 2001, Vol. 97, No. 3, pp. 809-811
BRIEF REPORT
Full hematopoietic engraftment after allogeneic bone marrow
transplantation without cytoreduction in a child with severe combined
immunodeficiency
Ronald J. Rubocki,
Jennifer
R. Parsa,
Michael S. Hershfield,
Warren G. Sanger,
Samuel J. Pirruccello,
Ines Santisteban,
Bruce G. Gordon,
Sarah E. Strandjord,
Phyllis I. Warkentin, and
Peter F. Coccia
From the Departments of Pediatric-Hematology/Oncology
and Pathology and Microbiology, University of Nebraska Medical Center,
Omaha, NE; and the Department of Rheumatology and Immunology, Duke
University Medical Center, Durham, NC.
 |
Abstract |
Bone marrow transplantation (BMT) for severe combined
immunodeficiency (SCID) with human leukocyte antigen (HLA)-identical sibling donors but no pretransplantation cytoreduction results in
T-lymphocyte engraftment and correction of immune dysfunction but not
in full hematopoietic engraftment. A case of a 17-month-old girl with
adenosine deaminase (ADA) deficiency SCID in whom full hematopoietic
engraftment developed after BMT from her HLA-identical sister is
reported. No myeloablative or immunosuppressive therapy or
graft-versus-host disease (GVHD) prophylaxis was given. Mild acute and
chronic GVHD developed, her B- and T-cell functions became
reconstituted, and she is well almost 11 years after BMT. After BMT,
repeated studies demonstrated: (1) Loss of a recipient-specific chromosomal marker in peripheral blood leukocytes (PBLs) and bone marrow, (2) conversion of recipient red blood cell antigens to donor
type, (3) conversion of recipient T-cell, B-cell, and granulocyte lineages to donor origin by DNA analysis, and (4) increased ADA activity and metabolic correction in red blood cells and PBLs.
(Blood. 2001;97:809-811)
© 2001 by The American Society of Hematology.
| |
Introduction |
Severe combined immunodeficiency (SCID) involves a
failure of T cells to proliferate to various stimuli and a failure of B cells to produce specific antibodies.1,2 Adenosine
deaminase (ADA) deficiency, an autosomal recessive genetic defect,
produces a SCID phenotype.3,4 The enzyme defect leads to
an abnormality in purine nucleoside metabolism that interferes with
lymphocyte viability and function. ADA-deficient SCID is characterized
by growth delay, candidiasis, respiratory infections, opportunistic infections, and, without specific therapy, early death.5
Low or absent ADA activity in erythrocytes or other cells is diagnostic.
Bone marrow transplantation (BMT) is effective therapy for SCID. The
profound T-cell dysfunction associated with ADA-deficient SCID prevents
graft rejection and permits BMT without conditioning with an
HLA-matched sibling donor.1,2,6 Success rates are greater
than 90%, with full recovery of T-cell and B-cell
functions.6 Complete T-cell engraftment is routinely
demonstrated, and approximately 50% of patients demonstrate
engraftment of donor B cells.7 Without conditioning, host
hematopoiesis persists with no evidence of myeloid or erythroid
engraftment.5,7-9
| |
Study design |
J.L., a 17-month-old girl, presented with fever, vomiting,
cough, skin ulcers, a history of failure to thrive, recalcitrant thrush, chronic respiratory infections, and recurrent otitis media. Immunizations were current. Family history was negative for
immunodeficiency. Physical examination revealed an ill-appearing white
female with weight and height below the fifth percentile, a paucity of
lymph nodes, and no hepatosplenomegaly.
Laboratory evaluation revealed normal hemoglobin and platelet counts.
The white blood cell count was 1600/cmm (normal, 6000-17 000/cmm) and
consisted of 10% neutrophils, 20% lymphocytes, 65% monocytes, and
4% eosinophils. Liver function test results were normal. Titers were
negative for human immunodeficiency virus, cytomegalovirus, hepatitis B
virus, herpes simplex virus, Epstein-Barr virus, adenovirus,
poliovirus, Toxoplasma, and Chlamydia
trachomatis.
ADA activity was deficient in erythrocytes and in peripheral blood
mononuclear cells; purine nucleoside phosphorylase (PNP) activity was
normal in both cell types (Table 1).
Metabolic findings in erythrocytes were consistent with ADA deficiency,
including elevated deoxyadenosine nucleotide (dAXP) of 0.491 mol/mL
packed cells (normal, less than 0.002 µM/mL), and reduced
S-adenosylhomocysteine hydrolase activity of 0.42 nmol/h/mg protein
(normal, 4.2 ± 1.9).3
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Table 1.
Adenosine deaminase and purine nucleoside phosphorylase
activity in red blood cells and peripheral blood mononuclear cells
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A 3-year-old sister was HLA-identical (A2, A3, B35, B27, Cw2, Cw4,
DRB1*01, and DRB1*04). Without myeloablative or immunosuppressive therapy, the patient was infused with 7.6 × 108
unfractionated nucleated marrow cells per kilogram from her sister. After BMT, the patient engrafted rapidly, remained well, and required no blood products (Figure 1).
Biopsy-proven grade I skin graft-versus-host disease (GVHD) developed
at day 39 after BMT and resolved with topical corticosteroids. Three
months after BMT, she developed vomiting and diarrhea with elevated
absolute neutrophil and lymphocyte counts (Figure 1). Liver function
tests revealed the following values: aspartate
aminotransferase, 270 IU/L (normal, 0-30 IU/L); alanine
aminotransferase, 262 IU/L (normal, 7-56 IU/L); and 
-glutamyl transpeptidase, 123 IU/L (normal, 8-78 IU/L). The illness resolved rapidly, and liver function findings were normal 3 months later.
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| Figure 1.
Graphic representation of hematologic parameters and
engraftment status at selected times after bone marrow transplantation.
Blood components (absolute lymphocyte count [ALC], absolute
neutrophil count [ANC], platelets) and hemoglobin values are shown in
the lower panel. Percentage donor cells analyzed by cytogenetics in the
recipient after transplantation are shown in the top panel. A specific
1qh+ polymorphism was detected in the recipient before BMT. At 1 and 3 months after BMT, analysis of PHA-stimulated PBLs demonstrated mixed
chimerism. Full donor engraftment was found at 6 months, and all
subsequent findings of PBLs and BM (at 1 and 8 years) have remained
100% of donor origin.
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Eleven months after BMT, lichenoid skin lesions developed that were
consistent with chronic GVHD; they completely resolved after 11 months
of oral and topical corticosteroid therapy. A dermatofibrosarcoma
protuberans was completely excised from the patient's left forearm 8 years after BMT. At long-term follow-up, the patient is well, has no
sequelae from BMT, and shows no evidence of immunodeficiency.
Two-color flow cytometric analysis for lymphocyte
phenotyping,10 natural killer cell assays,11
cell proliferation assays,12 and red blood cell antigen
typing13 were performed as described. GW-banding
procedures14 demonstrated a 1qh+ polymorphism in the recipient's phytohemagglutinin (PHA)-stimulated peripheral blood
lymphocytes (PBLs) and unstimulated BM. For DNA studies, T cells and B
cells were isolated with antibody-coated magnetic beads and
granulocytes enriched by Ficoll separation. DNA was extracted from each
fraction, and amplification of short tandem repeats was
performed.15 ADA activity, PNP activity, and dAXP levels
were measured as previously described.16,17 ADA genotype was determined with DNA extracted from fibroblasts cultured from the
dermatofibrosarcoma protuberans through single-strand conformational polymorphism analysis of ADA exons 4 and 5. The procedures used for
exon 5 have been reported.18 For exon 4, a genomic segment (base pair [bp] 24 787-25 481) was first amplified using the
following: primer 1, (+) 5'-GTA TGC AGT TCC AAA GTA GAG CTG; primer 2, (
) 5'-CAG TTA TGA AGT TAG AGC AGG ACC. The product was then subjected to nested polymerase chain reaction of bp 24 900 to 25 261 using the
following: primer 3, (+) 5'-gcg gaa gct tGG ATG TCA TTT GCT CCT G (5'
end-labeled with 32P-dATP); primer 4, () 5'-gcg cga
att cCA TCT TTC TGA GGC CAT G (lowercase denotes nongenomic 5'
extensions). The final exon 4 polymerase chain reaction product was
then sequenced.
| |
Results and discussion |
Allogeneic BMT is effective treatment for various inherited
defects of the pluripotent hematopoietic progenitor cell (HPC). An immunosuppressive and myeloablative preparative regimen is usually
necessary to suppress the host's immune system to prevent rejection of
the infused stem cells. The only exception to the above experience is
in BMT for SCID, when a matched sibling or a T-depleted haploidentical
stem cell donor is available. Because of the host's severe immune
dysfunction, the donor graft is not rejected.2 Only T-cell
progenitors, which have a selective growth advantage over endogenous
host T cells, engraft.5,7 After BMT all erythroid and
myeloid precursors remain of recipient origin.1 After
unconditioned BMT for SCID with an HLA-matched sibling donor, T-cell
function is restored in a few weeks and B-cell function in a few
months.6 Normalization of B-cell function does not
necessarily reflect donor B-cell engraftment.5,9 In many
cases, B-cells remain partially or completely of host origin, but they
function normally in the presence of donor T cells. Natural killer
cells may also be of donor, host, or mixed origin after unconditioned
BMT.5,8
We report a patient with classic ADA-deficient SCID, heteroallelic for
2 previously reported ADA missense mutations, R101Q (exon 4) and R156C
(exon 5).18-20 Before BMT she had lymphopenia, absent B
cells, an inverted CD4/CD8 ratio, absent natural killer activity, and
markedly depressed in vitro mitogen response to pokeweed, concanavalin
A, and phytohemagglutinin. Her bone marrow had normal cellularity with
depressed T- and B-cell numbers for her age. After BMT, all the above
evaluations gradually normalized. In addition, we found multiple
markers confirming complete erythroid and myeloid engraftment.
Recipient and donor were both blood group A Rh-positive. The recipient
(Jka negative, E positive) had complete conversion to donor
erythrocyte antigens (Jka positive, E negative) when
studied at 4 and 26 months after BMT. Anti-B was weakly detected before
BMT and strongly detected 26 months after BMT.
Before BMT, all mitoses in the BM demonstrated the 1qh+ polymorphism.
At 1 and 3 months after BMT, analysis of PHA-stimulated PBLs
demonstrated mixed chimerism. Full donor engraftment was found at 6 months, and all subsequent findings of PBLs and BM (at 1 and 8 years)
have remained of 100% donor origin (Figure 1).
For DNA studies, 9 short tandem repeat markers were analyzed.
Informative loci (D3S1358, FGA, TH01, TPOX, CSF1PO, D13S317, and
D7S820) collectively demonstrated that all cell types (T, B, and
granulocyte-enriched) in the specimen studied were of donor origin
after BMT.
After BMT, ADA activity in erythrocytes and blood mononuclear cells
increased to the level found in the donor (Table 1). Erythrocyte dAXP
level was less than 0.01 nmol/mL packed cells in all samples obtained
after BMT. All the above studies demonstrated complete T, B, erythroid,
and myeloid cell engraftment from 6 months after BMT without any
evidence of mixed chimerism.
After BMT the host HPC ceased to function in our patient. Because no
episode of pancytopenia or marrow aplasia occurred, transient mixed
chimerism of host and donor hematopoiesis must have developed. Over
time the host stem cells ceased to function, and only the donor stem
cells and their progeny persisted. The most appealing speculation for
this observation is the development of donor T-cell-mediated GVHD
reaction that targeted and destroyed the host HPC without other
manifestations of significant GVHD. Approximately 3 months after
transplantation, the child had an episode of vomiting, diarrhea, and
elevated liver enzyme levels. At that time a significant increase in
hemoglobin, platelet, absolute neutrophil, and absolute lymphocyte counts were found that persisted into long-term follow-up (Figure 1).
The patient's cytogenetic studies also changed during this period,
from 50% to 80% donor cells, with 100% donor cells present at 6 months and later (Figure 1).
It has been reported that severe GVHD with pancytopenia and aplasia
develop in infants with SCID after in utero engraftment with maternal
cells.21 In addition, transfusional GVHD in
immunodeficient infants is characterized by the development of severe
GVHD and aplastic anemia, as is transfusional GVHD in immunocompetent
patients after open heart surgery.22,23 In all the above
examples, donor T-lymphocytes mediate host hematopoietic stem cell
destruction and lead to the development of marrow aplasia and severe
pancytopenia. However, unlike our experience, fatal GVHD developed in
all the reported recipients.
An alternative hypothesis is that because ADA-sufficient cells (donor)
have a selective growth advantage over ADA-deficient cells (host),
donor HPC engraftment would occur. However, in all reported cases of
BMT in ADA SCID, host erythropoiesis and myelopoiesis persist.
Our observation is certainly rare. The Buckely et al9
survey of 87 patients with SCID who underwent either HLA-haploidentical or HLA-identical BMT without a preparative regimen or GVHD prophylaxis found no case of complete hematopoietic engraftment, though red cell
typing was not specifically noted. Haddad et al8
recently reported that 2 of 8 patients with SCID with B cells had
evidence of some donor-derived monocytes after HLA-haploidentical,
T-cell-depleted BMT without a preparative regimen. None of the 5 HLA-identical BMT patients had donor-derived monocytes. We suggest that
investigators following up patients with SCID who underwent
transplantation without a preparative regimen look for evidence of
partial or complete myeloid and erythroid engraftment in their
patients. Identification and further study of this phenomenon may lead
to a better understanding of allogeneic tolerance.
| |
Acknowledgments |
We thank Dr F. X. Arredondo-Vega for assistance in performing
ADA genotype analysis and Dr Roger Kobayashi for referring the patient
to us.
| |
Footnotes |
Submitted January 24, 2000; accepted September 28, 2000.
Supported by National Institutes of Health grant DK20902 (M.S.H.) and
by Enzon, Inc.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Peter F. Coccia, Department of
Pediatric-Hematology/Oncology, University of Nebraska Medical Center,
982168 Nebraska Medical Center, Omaha, NE 69198-2168.
| |
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Abstract
Introduction