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Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2588-2600
Lymphoid Reconstitution After Autologous PBSC Transplantation
With FACS-Sorted CD34+ Hematopoietic Progenitors
By
Catherine Bomberger,
Meeta Singh-Jairam,
Glenn Rodey,
Anastasia Guerriero,
Andrew M. Yeager,
William H. Fleming,
H. Kent Holland, and
Edmund K. Waller
From the Stem Cell Biology Program, Emory University, Department of
Pathology, Emory University, Division of Hematology and Oncology,
Atlanta, GA.
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ABSTRACT |
T-cell and B-cell reconstitution was studied in nine patients who
received fluorescence activated cell sorter (FACS)-sorted autologous
CD34+ hematopoietic progenitor cells (HPC). The mean
numbers of T cells (CD3+), B cells
(CD19+) and CD34+ HPC administered to
each patient were .004, .002, and 1.8 × 106 cells/kg,
respectively. After high-dose myeloablative chemotherapy (busulfan,
cyclophosphamide, etoposide) CD34+ HPC were infused and
lymphoid reconstitution was monitored using flow cytometry and reverse
transcriptase-polymerase chain reaction (RT-PCR) amplification of VDJ
T-cell receptor (TcR) sequences. Restoration of normal numbers of
peripheral blood T cells and B cells among recipients of FACS-sorted
CD34+ HPC was delayed compared to recipients of
non-T-cell-depleted PBSC autografts. In both patient groups, the
circulating T cells were primarily CD4 ,
CD8+,  TcR+, and
CD45RO+, CD45RA during the first 2 months
after transplant. Subsequent increases in the frequency of
CD45RA+ CD45RO T cells occurred at 2 to 3 months after transplant, suggesting maturation of CD34+
hematopoietic progenitors to "naive" T cells. Analysis of the TcR
repertoire after hematopoietic reconstitution demonstrated decreased
diversity of V TcR expression associated with global decreases in
the absolute number of total peripheral blood T cells and most V
TcR+ subsets. Three of nine recipients of FACS-sorted
CD34+ HPC demonstrated significant increases in the
percentage of  + peripheral T cells and
CD5+ B cells at 3 to 9 weeks after transplantation, and
all patients had transient oligoclonal expansions of T cells expressing
specific V TcR. Transplantation with highly purified
CD34+ HPC results in reduced diversity of the peripheral
T-cell repertoire during the early post-transplant period compared with
patients receiving unmanipulated or MoAb-depleted transplants.
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INTRODUCTION |
QUANTITATIVE AND qualitative differences
in the kinetics of reconstitution for the various blood cell lineages
characterize the period of hematopoietic recovery that follows
myeloablative chemoradiotherapy and bone marrow/peripheral blood
progenitor cell transplantation. Reconstitution of the myelomonocytic,
megakaryocytic, and erythroid lineages depends only on achieving
adequate numbers of granulocytes, monocytes, platelets, and red blood
cells in the circulating blood, and functional cells in these lineages develop immediately following maturation of undifferentiated
CD34+ hematopoietic progenitor cells present in the
transplant graft. However, the contribution of pluripotent stem cells
to lymphoid engraftment after bone marrow transplantation
(BMT) and peripheral blood stem cell transplantation (PBSCT) is less
clear.1 Qualitative aspects of lymphocyte function, which
depend on the presence of antigen specific T cells and B cells within a
diverse immune repertoire, may be more important than the simple
quantity of circulating lymphocytes in the blood and in lymphoid
organs. In patients receiving non-T-cell-depleted allogeneic
transplants, recovery of donor-derived lymphocytes is rapid although
the restoration of a fully functional immune system capable of
responding to antigenic challenge takes 6 months to 1 year after
BMT.2-4 Functional reconstitution of the lymphoid
compartment depends either on the presence of adequate numbers of
antigen-specific T cells and B cells in the transplant graft or the
maturation of lymphoid progenitors and perhaps pluripotent stem cells
into antigen-specific T cells and B cells within the thymic and bone
marrow microenvironments post-BMT/PBSCT.
Whereas removal of mature lymphoid cells from autologous bone marrow
and PBSC autografts has been shown to increase the chance of
relapse-free survival for patients with T-cell or B-cell malignancies, a potentially deleterious consequence is a transient, severe combined immunodeficiency (SCID) after BMT.2,5-8 Although the number of T lymphocytes increases rapidly among recipients of T-cell-depleted autologous BMT, the cytotoxic and proliferative functions of T cells
remain significantly impaired for up to 1 year after BMT.4 Studies comparing patients who received T-cell-depleted autologous BMT
with patients receiving T-cell-replete autologous BMT or allogeneic BMT showed that all three groups of patients had a similar pattern of
immune reconstitution after BMT-an initial increase in the numbers of
circulating natural killer (NK) cells, T cells, and monocytes in
addition to the delayed restoration of normal numbers of B cells in the
peripheral blood over a 6- to 12-month period after
BMT.2-4,9-11 These data suggest that the immunodeficiency seen after transplantation is caused by qualitative defects in cellular
and humoral immunity that are not simply explained by deficits in the
numbers of circulating lymphocytes.2,3 The cause of this
qualitative post-BMT T-cell dysfunction among autologous BMT recipients
who do not receive immunosuppressive drugs is unclear. One possible
explanation may be the decreased diversity of the T-cell repertoire
after BMT.
Phenotypic analysis of the peripheral T-cell compartment using
monoclonal antibodies to specific TcR-V regions in patients receiving
unmanipulated autologous and allogeneic BMT, has shown that one or more
V-specific subsets were overrepresented at some time point after
BMT.12 Studies comparing the peripheral T-cell repertoire
of normal individuals and BMT recipients using reverse transcriptionase-polymerase chain reaction (RT-PCR) of the TcR region (CDR3 size spectratyping) found that the circulating T-cell repertoire in normal controls was both complex and stable over time,
whereas post-BMT patients showed contraction of some TcR V families,
as well as oligoclonal expansions of others resulting in an overall
decreased complexity of the peripheral T-cell
compartment.10,13,14 Although the complexity of the T-cell
repertoire has been studied in depth in the peripheral blood from
normal subjects and after conventional BMT, similar studies in patients
receiving significantly T-cell-depleted autologous BMT have not been
reported. We recently completed a clinical study of patients
transplanted with highly purified fluorescence-activated cell sorter
(FACS)-sorted autologous CD34+ peripheral blood stem
cells.15 In the present study we analyzed the kinetics of
T-cell, B-cell, and NK cell reconstitution in this same group of
patients using multiparameter flow cytometry and RT-PCR spectratyping.
Our hypothesis was that transplantation with CD34+
FACS-sorted autologous PBSC might result in delayed immune
reconstitution of the peripheral B-cell and T-cell compartments as a
result of inefficient de novo differentiation of CD34+
hematopoietic precursors into B cells and T cells in adults. We found
that reconstitution of normal numbers of circulating peripheral blood T
cells and B cells was severely delayed for up to 1 year among
recipients of CD34+ FACS-sorted autologous PBSC
transplants. Phenotypic and genotypic analysis of lymphoid subsets
demonstrated initial posttransplant expansions of a limited number of
different T-cell clones, followed by the appearance of naive-type T
cells, subsequently contributing to the reconstitution of a more
diverse peripheral blood lymphoid compartment.
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MATERIALS AND METHODS |
Patient characteristics.
Nine patients with a median age of 41 years (range, 22 to 58) and
poor-risk non-Hodgkin's lymphoma (NHL) who received FACS-sorted autologous CD34+ hematopoietic stem cells (HSCs) have
recently been described.15 Blood samples for phenotypic
analysis of immune reconstitution were obtained weekly during the
patient's initial hospitalization and then every 2 to 6 months, while
being followed in the outpatient BMT clinic. Peripheral blood samples
from a control group of 11 patients with NHL, Hodgkin's disease (HD)
and multiple myeloma (MM) with median age of 40 (range, 28 to 60) who
underwent the same preparative regimen but received unmanipulated PBSC
transplants were studied for the numbers of circulating T cells, B
cells, and NK cells post-transplant. In addition, a larger control
group, including an additional 9 patients (total of 20) patients were studied for the kinetics of lymphocyte reconstitution posttransplant.
Isolation of CD34+ HSC and transplant regimen.
Patients received 4 g/m2 cyclophosphamide on day 30,
followed by granulocyte-colony-stimulating factor (G-CSF) at 20 µg/kg for 10 days. One or two large-volume aphereses were performed to
collect a PBSC product of at least 6 × 106
CD34+ cells/kg. The CD34+ cells were enriched
by immunoaffinity purification using the CEPRATE device (Cell-Pro,
Bothell, WA); the CD34+ cell-enriched fraction was then
further purified by high-speed FACS sorting using a FACS Vantage cell
sorted (Becton Dickinson, San Jose, CA) and
cryopreserved.15 The pretransplant conditioning regimen
consisted of busulfan, 4 mg/kg orally, ×4 days; cyclophosphamide, 60 mg/kg/d intravenously × 2 days; and etoposide, 10 mg/kg/d
intravenously ×3 days. On day 0, the cryopreserved CD34+
purified cells were thawed and infused. Patients received G-CSF (Amgen,
Thousand Oaks, CA) 10 µg/kg subcutaneously beginning on day
0, and received standard transplantation supportive care therapy.
Isolation of mononuclear cells and determination of peripheral blood
and lymphocyte counts.
Pretransplant samples were obtained from the CD34 PBSC
fraction after immunoaffinity column purification of the apheresis product. At 21, 35, 45, 60, 180, and 365 days after transplantation, 20 ml of EDTA or Na heparin anticoagulated blood was obtained. The
absolute number of mononuclear cells in each sample was determined by
adding the number of lymphocytes and monocytes per microliter, as
determined by a complete blood count performed using an Abbot Cell Dyne
2. The mononuclear cell fraction of the peripheral blood was isolated by centrifugation over a step gradient of Ficoll-Hypaque (density 1.077 g/mL) and washed twice by dilution with Hank's balanced
salt solution (HBSS) plus 3% fetal bovine serum (FBS) and 5 mmol/L
EDTA, followed by centrifugation at 500g for 5 minutes.
FACS analysis.
For flow-cytometric analysis, isolated MNCs were stained with
combinations of monoclonal antibodies directly conjugated to FITC, PE,
and PerCP against the following antigens: CD3, CD4, CD5, CD8, CD16,
CD19, CD45, CD45RO, CD45RA, CD56, pan  , pan  , and specific
V , V , V , and V TcR antibodies (Table 1). In this study,
100-µL cell solution aliquots containing 1 × 105-1 × 106 cells were incubated at 4°C with saturating
concentrations of up to three different monoclonal antibodies (MoAbs)
for 30 minutes, washed, and then fixed in a final concentration of 2%
paraformaldehyde. The stained and fixed samples were then analyzed
using a FACS Caliber flow cytometer (Becton Dickinson). List mode files
of 20,000 events were acquired with a light-scatter gate, which
included lymphocytes and monocytes. The numbers of each phenotypically defined T-, B-, or NK cell type in the peripheral blood per microliter was determined by multiplying the numbers of lymphocytes per microliter by the percentage of nucleated cells with that phenotype that fell
within the lymphocyte gate, as determined by flow cytometry.
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Table 1.
Lymphocytes, Monocytes, and CD34+ Cells in
Unmanipulated Apheresis Products and After CD34+ FACS
Isolation
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Molecular analysis.
Pretransplant samples were initially obtained from the apheresis column
unabsorbed fraction generated in the first step of the
CD34+ selection protocol. Follow-up samples of 20 mL of
EDTA-anticoagulated peripheral blood were obtained from the patients at
various time points after BMT. Isolated MNCs were washed with
phosphate-buffered saline (PBS) plus 3% albumin, and their RNA was
extracted using RNAzol, phenol-chloroform extraction (Tel-Test,
Friendswood, TX). cDNA was synthesized using random hexamer primers
and reverse transcriptase (Promega, Madison, WI). Bidirectional
amplification of the CDR3 region for each of 25 V TcR gene segments
was performed using a two-step PCR to produce gene segments varying in
length from 250 to 500 bp.10,16 The first step consisted of
the addition of a constant region primer, OTCB3, along with a specific
V primer (see Table 3), followed by 45 cycles of PCR.16
This was followed by a three-cycle PCR run-off reaction with a
fluorescently labeled internal probe (6-FAM-labeled TcRBC) and
subsequent separation on a 6% polyacrylamide gel (19 : 1 acrylamide/bis). Fluorescent spectrometry was used to detect the
resultant fluorescently labeled bands containing V TcR sequences.
The fluorescently labeled PCR products were analyzed using Genescan
Analysis 2.02 software (Perkin-Elmer, Norwalk, CT) to produce
histograms representing the V spectratype with individual peaks
separated by 3 bps. In some instances, when patients had very low
lymphocyte counts during the first 1 to 2 months after transplantation
and from some apheresis products, insufficient numbers of lymphocytes
were obtained for successful extraction of RNA, PCR amplification of
TcR CDR3 sequences, and spectratyping.
The overall complexity within a V TcR family and among the different
V TcR families was determined by counting the numbers of different
peaks and determining their relative amplitude on the spectratype
histogram. A novel mathematical formula representing the complexity of
each V TcR family (V complexity) was determined by first
calculating the ratio of the sum of the heights of the major peaks
(MPHs) to the sum of all the peak heights (TPHs) and then dividing this
ratio by the number of major peaks present (no. MP), that is,
(MPHs/TPHs)/no. MP. Major peaks were defined as those peaks on the
spectratype histogram whose amplitude was at least 10% of the TPH. The
V complexity numbers calculated for each V TcR family were added
together to yield an overall complexity score that represented the
complexity of the V TcR repertoire at that point in time.
Oligoclonal representation of a particular V TcR was defined as when
one peak represented greater than 60% of the total peak height (peak
height .6 × TPH) or when two major peaks were identified, each
of which represented greater than 40% of the total peak height
(combined peak height .8 × TPH).
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RESULTS |
The mean numbers of mononuclear and CD34+ cells present in
the graft after CD34+ selection and prior to
cryopreservation were 3.7 ± 4.6 × 103 CD3+
T cells/kg, 1.1 ± 1.2 × 103 CD14+
monocytes/kg, 2.4 ± 5.4 × 103 CD19+ B
cells/kg and 1.8 ± 0.77 × 106/kg CD34+
hematopoietic progenitors. These values represented a significant depletion of lymphocytes and monocytes compared with the unmanipulated apheresis products that had been collected from these patients and were
significantly lower than the numbers of monocytes and lymphocytes in
the nonsorted autologous PBSC grafts obtained from 1 to 3 aphereses of
a control group of patients (Table 1). All patients who received FACS sorted CD34+ autologous PBSC
showed successful myeloid engraftment with a median time to achieve an
absolute neutrophil count (ANC) of 500/µL and 1000/µL of 11 (range,
10 to 14) and 12 (range, 11 to 19) days, respectively. To compare
lymphocyte engraftment between patients receiving CD34+
FACS-sorted cells and the control population, we calculated the median
time to an absolute lymphocyte count of 500/µL for each patient in
both cohorts, reasoning that patients with fewer than 500 total
lymphocytes (and less than 200 CD4+ cells/µL) would be at
increased risk of opportunistic infections.17 The median
time to recovery of more than 500 lymphocytes/µL was 30 days (range,
15 to 290) for the patients who received FACS-sorted CD34+
PBSC and 20 days (range, 10 to 170) for the control population (Fig
1). The mean number of circulating
lymphocytes was significantly higher (P < .05) for
nonsorted control group at all time points between day +5 and day +50
post-transplant, and at day +150 and day +200 post-transplant (Fig 1).

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| Fig 1.
Lymphocyte recovery in patients transplanted with either
nonsorted autologous BM/PBSC or with FACS-sorted CD34+
autologous HPC. Mean lymphocyte counts (±SD) in nine patients after
treatment with marrow lethal therapy and infusion of the highly
purified CD34+ HPC ( ). The mean numbers (±SD) of
lymphocytes in the blood of 20 patients receiving unmanipulated PBSC
autografts ( ). The median time to obtaining an ALC > 500/µL
among recipients of standard PBSC autografts was 20 days (range, 10 to
170) and was 30 days (range, 15 to 290) for the patients who received
FACS sorted CD34+ HPC. The mean lymphocyte count in a
separate population of 10 normal donor subjects was 1,800 cells/µL
with a standard deviation of 400 cells/µL. Significant differences
between the mean values in the two groups of patients are indicated:
*P < .05; #P < .001.
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Overall, T-cell reconstitution was delayed among recipients of
FACS-sorted CD34+ autologous PBSC with mean values (±SD)
of peripheral blood CD34+ T cells/µL of 276 ± 425/µL
at 1 month after BMT, 346 ± 516/µL at 2 months after BMT, 296 ± 224/µL at 6 months and 624 ± 207/µL at 1 year after BMT (Fig
2). For the nonsorted control group, the
corresponding mean values (±SD) of CD3+ T cells/µL were
498 ± 179/µL at 1 month, 874 ± 905/µL at 2 months, 441 ± 181/µL at 6 months, and 551 ± 140/µL at 12 months after BMT (Fig
2, P = NS). Three patients showed significant increases in
the frequency of  T cells during the first 2 months
posttransplant with the percentages of T cells expressing the 
TcR increasing from 2%, 4%, and 5% of the peripheral T cells in the
pretransplant blood samples to maximum values of 31%, 10%, and 14%,
respectively, in PB samples obtained 3 to 5 weeks posttransplant,
followed by subsequent declines into the normal range (data not shown).
Increased numbers of circulating  T cells were not seen among
recipients of nonsorted autografts (data not shown). Following
transplantation, inversion of the CD4/CD8 ratio of peripheral blood T
cells was seen among all nine recipients of FACS-sorted HPC, as well as recipients of unmanipulated PBSC autografts until 6 months after BMT
(Fig 3). The low CD4/CD8 ratio was mainly
the result of extremely low numbers (<100/µL) of CD3+,
CD4+ T cells with relatively normal numbers of
CD3+, CD8+ T cells. Recipients of FACS-sorted
CD34+ HPC had significantly (P < .05) lower
mean numbers (±SD) of circulating CD3+, CD4+
T cells/µL at 1 month, 2 months, and 6 months after BMT
(37 ± 70/µL; 31 ± 45/µL, and 101 ± 25/µL,
respectively), compared with the mean numbers of CD3+,
CD4+ T cells/µL present in the blood of recipients of
nonsorted PBSC autografts at the same time points after BMT
(137 ± 47/µL, 189 ± 241/µL, 195 ± 80/µL,
respectively). CD3+, CD8+ T cells/µL were
slightly lower among recipients of sorted CD34+ autologous
HPC compared with recipients of PBSC autografts, but the differences
did not reach statistical significance (data not shown). Two patients
who received FACS-sorted HPC developed a normal CD4/CD8 ratio at 6 months (ratios, 1.7 and 3.1), whereas a third patient had a normal
CD4/CD8 ratio by 1 year after BMT (ratio, 1.3). One recipient of
FACS-sorted HPC had an early and transient increase of the CD4/CD8
ratio to 7.3 at 3 weeks after FACS-sorted HPC infusion, when the total
number of T cells was <100/µL.

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| Fig 2.
Numbers of peripheral blood T cells in patients
undergoing transplantation with autologous PBSC. Mean T-cell count per
microliter in nine patients after treatment with marrow lethal therapy
and infusion of the highly purified CD34+ HPC ( ). Mean
T-cell count per microliter in the blood of 11 patients receiving
unmanipulated PBSC autografts ( ).
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| Fig 3.
FACS analysis of CD4 and CD8 T-cell subsets in patients
after autologous PBSC transplantation. Mean ratio of
CD3+CD4+ helper T lymphocytes to
CD3+CD8+ suppressor T lymphocytes during
the posttransplant period in nine patients transplanted with
FACS-sorted CD34+ HPC ( ). Mean CD4/CD8 ratio of T
cells in the blood of 11 patients receiving unmanipulated PBSC
autografts ( ).
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CD19+ B-cell recovery was delayed in both groups of
patients, with B-cell counts typically <25 cells/µL until 2 months
after BMT (Fig 4). Significant B-cell
reconstitution did not occur until 6 to 12 months after BMT in both
groups. The mean numbers (±SD) of peripheral blood B cells were 141 ± 30/µL at 6 months after BMT and 259 ± 114/µL at 1 year after
BMT among recipients of FACS-sorted CD34+ HPC transplants
and 329 ± 365/µL at 6 months and 419 ± 456/µL at 12 months
after BMT among recipients of unmanipulated PBSC autografts
(P = NS). During the first 2 months after transplantation with CD34+ sorted PBSCs, CD19+/CD5+
B cells were significantly overrepresented, comprising a median of 36%
(range, 0.4 to 85%) of the peripheral B-cell compartment at 3 weeks
and a median of 65% (range, 25 to 99%) of B cells at 5 weeks after
BMT (Fig 5A). This subset of B cells
persisted at high percentages for up to 1 year post-transplant in some
patients.

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| Fig 4.
Numbers of peripheral blood B cells in patients
undergoing transplantation with autologous PBSC. Mean number of B cells
per microliter (CD19+) in the peripheral blood of eight
patients transplanted with FACS-sorted CD34+ HPC ( ).
Number of B cells per microliter in the blood of 11 patients receiving
unmanipulated PBSC autografts ( ).
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| Fig 5.
FACS analysis of CD5+CD19+ B
lymphocytes and CD45RO and CD45RA T-cell subsets. (A) Percentage of
immature B cells expressing the CD5 T-cell lineage antigen in the
peripheral blood of eight patients transplanted with FACS-sorted
CD34+ HPC. (B) Percentage of T cells in the peripheral
blood of eight patients transplanted with FACS-sorted
CD34+ HPC with the CD45RA+,
CD45RO phenotype (naive T cells). (C) Percentage of
CD45RO , CD45RA (memory) T cells.
Horizontal dashed line, mean value of 10 normal subjects; shaded area,
95% confidence interval.
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The mean number of circulating NK cells
(CD16+/CD56+) reached the blood level of
normal subjects during the first 2 months post-transplant with mean
values (±SD) of 232 ± 151/µL among recipients of FACS-sorted CD34+ HPC, compared with 213 ± 97/µL among recipients
of nonsorted PBSC autografts (P = NS) (Fig
6). Circulating numbers of NK cells decreased in both populations at 3 months post transplant with mean
values of 58 ± 48/µL among recipients of FACS-sorted
CD34+ HPC and 90/µL among recipients of unmanipulated
PBSC autografts with subsequent rises to the normal range by 1 year
after BMT (Fig 6) (P = NS).
CD3+/CD56+ NK-like T cells followed a similar
pattern as that of NK cells showing an early rise at around week 5 after BMT, followed by a decline to lower levels at 6 to 12 months
after BMT (data not shown).

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| Fig 6.
Numbers of peripheral blood NK cells in patients
undergoing transplantation with autologous PBSC. Mean number of NK
cells/µL (CD16/CD56+) in the peripheral blood of
eight patients transplanted with FACS-sorted CD34+ HPC is
shown as open circles ( ). Number of NK cells/µL in the blood of 11 patients receiving unmanipulated PBSC autografts are shown in the open
triangle ( ).
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We used the pattern of expression of the CD45 isoforms CD45RO and
CD45RA as a surrogate marker to determine the developmental stage of T
cells. CD45RA single positive T cells are recent thymic emigrants that
have yet to encounter antigen and are considered "naive," whereas
CD45RO single positive T cells or "memory" T cells have emigrated
from the thymus and encountered antigen in a peripheral lymphoid
organ.18,19 We compared the fraction of CD3+ T
cells that expressed solely CD45RO surface antigen (memory T cells)
with the fraction that expressed solely CD45RA surface antigen and the
fraction expressing both antigens. Before transplantation, a median of
9% (range, 4 to 24%) of blood T cells expressed only the CD45RA
isoform (Fig 5B). After transplantation, the percentage of CD45RA
single positive cells decreased to 4% (range, 1% to 8%) at 3 weeks,
then slowly increased to 10% (range, 3% to 16%) at 5 weeks, 20%
(range, 9% to 26%) at 6 months and 24% (range, 18% to 43%) at 1 year (Fig 5B). Before transplantation, a median of 79% (range, 56 to
84) were single positive for CD45RO surface antigen. Three weeks after
transplantation a median of 81% (range, 69 to 93) of peripheral T
cells were single positive for CD45RO; this had decreased to a median
of 71% (range, 34 to 89) by 5 weeks, and this continued to decrease to
64% (range, 50% to 73%) at 6 months and 50% (range, 42% to 59%)
by 1 year (Fig 5C). The number of CD3+ T cells expressing
both CD45RA and CD45RO (double positive T cells) increased
proportionally with the number of CD45RA single positive T cells
between 3 weeks and 1 year posttransplant (data not shown).
T-cell repertoire reconstitution, flow cytometric analysis.
Using flow cytometric analysis, we measured the frequency of
CD3+ peripheral blood lymphocytes expressing a given TcR-V
region using the MoAb panel shown in Table
2. Before transplantation, all nine
patients studied demonstrated a relatively diverse TcR repertoire, with
the majority of individual TcR-Vs expressed on 2% to 5% of peripheral
T cells, and no significantly overrepresented TcR-V, similar to the
peripheral repertoire of normal individuals (Table
3). After transplantation with FACS-sorted
CD34+ autologous HPC, all nine patients studied showed
significant overexpression of at least one TcR at one or more time
points, as defined by a frequency of an individual TcR-V more than the mean value for normal control patients +2 × SD (Table
4). Six of these nine patients demonstrated
at least one significantly overexpressed TcR-V within the first 2 months with relative underexpression of the remaining repertoire (less
than 2%), as well as complete disappearance of some TcR-Vs. By
contrast, only one of five recipients of nonsorted autografts had a
significantly overexpressed TcR-V within the first 2 months after BMT.
Three of the eight evaluable patients showed significantly
overrepresented subsets of TcR-Vs between 3-5 months after
transplantation, and six of the eight evaluable patients showed
expansions of various T-cell subsets at 6 to 12 months after BMT.
Figure 7 shows the percentage of T cells
expressing different TcR-Vs using a panel of 25 MoAb in patient no. 2 before transplantation and at days 22, 37, 100, 180, and 350 after
transplantation. There were transient increases in the percentages of
peripheral T cells bearing V 9 and V 2 at 3 weeks after
transplantation and a gradual increase in the percentage of T cells
bearing V 3 TcR to 26% at 3 months after BMT with a subsequent
decline to 19% at 6 months, while the normal range for the fraction of
T cells expressing V 3 TcR was 4.5% ± 3.3%. In addition to the
transient overexpression of various TcR-Vs in the post-transplant
period, significantly decreased diversity of the remaining T-cell
receptor repertoire was present with relative underrepresentation of
the remaining TcR-V subsets during the early post-transplant period.
The percentage of peripheral T cells detected by one of our panels of
V specific antibodies decreased from an average of 56% of 
TcR+ T cells before transplantation to 33% at 3 weeks
after transplantation. By 3 months, the peripheral T-cell receptor
diversity had expanded to the point that our antibody panel detected
85% of circulating  TcR+ T cells which remained
stable to 1 year after BMT. Although the T-cell receptor repertoire
stabilized by 6 to 12 months after BMT, the overall pattern of TcR
expression at 1 year after BMT was significantly different compared
with the pretransplant pattern with some TcR-V subsets present at much
lower levels, while others remained significantly overrepresented (Fig
8). This pattern of overrepresentation of
one or more V TcR subsets with relative underexpression of the
remaining subsets was seen in all but one of the nine patients studied
(Table 4).

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| Fig 7.
Analysis of circulating V TcR repertoire after
transplantation with FACS-sorted autologous CD34+ HPC
versus a healthy donor. (A) Percentage of CD3+ peripheral
T cells that express a given V TcR at 3 weeks, 5 weeks, 3 months, 6 months, and 1 year after transplantation for patient #2. (B) Percentage
of CD3+ peripheral blood T cells that express the same
V TcRs over time in a healthy donor. The frequencies of V 9 and
V 2 were identical and are shown using the same symbol.
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| Fig 8.
FACS analysis of circulating V-TcR repertoire in four
patients before and at 1 year after autologous HPC transplantation. Filled bars represent the percentage of peripheral CD3+ T
lymphocytes expressing a given V-TcR before transplantation, and
hatched bars represent the percentage expressing the same V-TcR at 1 year posttransplant.
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CDR3 spectratyping results.
A spectratype histogram for each V TcR family was generated for each
peripheral blood sample studied. By analyzing the number of distinct
peaks present among the PCR products for each TcR-V specific family, we
determined whether the majority of TcR-V cDNA products had the same
nucleotide length and were contained in a single peak or distributed
among a few peaks, corresponding to an oligoclonal population, or
distributed equally among many different peaks, corresponding to a
polyclonal population of T cells. Of the eight patients analyzed by
spectratyping, 7 showed oligoclonal expansions of at least one V TcR
family with one or two peaks predominating on at least one occasion
after transplantation. Table 5 shows the
specific oligoclonal expansions seen at the various time points in all
the patients. The amplitude of the peaks in each PCR-amplified product
corresponded to the relative quantity of different sized CDR3 mRNA
species within a V TcR family, and the number of peaks was a
measurement of the T-cell V TcR family repertoire complexity. Figure
9 shows five representative V -TcR
histograms from patient no. 2 before transplant, and at 3 weeks, 3 months, 6 months, and 1 year after transplantation, with the
corresponding percentage of T cells expressing the V TcR detected by
flow cytometry shown in the upper right corner of each box. Before
transplantation 9.2% of peripheral T cells expressed V 3 with a
single peak comprising 64% of the total peak amplitude, whereas at 3 weeks after BMT, 4 peaks were present, indicating increased V 3 complexity. At 3 months posttransplant when V 3+ T cells constituted
27.8% of all circulating T cells, the V 3 spectratype contained
only two predominant peaks, which comprised more than 88% of the total
peak height. This pattern remained relatively stable for 1 year
posttransplant. By contrast, V 8 was expressed on a relatively
constant fraction of T cells before and after transplant (2.8% to
5.2%). The spectratype of V 8 showed a complex pattern before
transplantation, a single peak at 3 weeks after transplantation,
followed by the development of a complex polyclonal pattern of V 8 expression at 3 months posttransplantation. V 12 was present on
2.4% of T cells before transplantation and spectratype analysis showed
a complex pattern of heterogeneous expression. After transplantation,
the frequency of TcR V 12+ cells detected by flow
cytometry declined to less than .5% with the corresponding appearance
of a single peak on the V 12 spectratype by 1 year posttransplant
that represented 66% of the overall peak height. The V 13 spectratype showed a complex polyclonal pretransplant pattern with the
subsequent appearance of two peaks at 6 months after transplantation.
The V 16 spectratype showed a polyclonal pattern before
transplantation, with the subsequent appearance of a single peak that
persisted at 1 year after transplantation.

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| Fig 9.
Spectratypes of the T-cell repertoire during the first
year after autologous transplantation with FACS-purified
CD34+ HPC. PBLs were analyzed using CDR3 spectratyping
and multiparameter flow cytometry to ascertain the V TcR repertoire
from an individual patient at the time points shown. Histograms of the
relative sizes of the PCR-amplified CDR3 region for five representative
V TcR gene products are shown. Numbers in the upper right corner of each box represent the percentage of cells expressing a specific V
TcR. The scale of the y-axis varies in each small box according to the
maximal peak amplitude, which is proportional to the quantity of RNA
bearing the specific V TcR. The x-axis represents the nucleotide
length of the PCR-amplified TcR gene products.
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An overall V TcR complexity score, the sum of the individual
complexity values for each individual TcR V family was generated for
each recipient of FACS-sorted CD34+ PBSC transplants and
for each nonsorted control patient before and at several time points
after transplantation (Fig 10). We had predicted that individual patients' V spectratypes would decline after transplantation because of the limited number of different mature
T-cell clones present in the autograft. The median of the V TcR
complexity scores for six healthy patients was 175 (range, 141 to 191).
The median of the pretransplant overall complexity scores for five
patients (three recipients of CD34+ FACS-sorted PBSC, two
nonsorted controls) was 105 (range, 83 to 106). At 1-2 months
post-transplant the median complexity score for the transplanted
patients was 67 (range, 31 to 93), significantly less than normal
control and pretransplant values. At 3 to 5 months after BMT, the
median complexity score had increased to 95 (range, 82 to 109), and at
6 to 12 months the median complexity score was 97 (range, 73 to 112).
While technical limitations of the RT-PCR reaction precluded obtaining
V spectratypes of all samples, there was no substantial difference
between the calculated post-transplant complexity scores for patients
receiving FACS-sorted CD34+ autologous PBSC (open circles)
compared to patients receiving unmanipulated autologous PBSC (filled
circles, Fig 10). However, both the pretransplant values and the
post-transplant complexity scores of all patients studied were
substantially lower than values obtained from untreated normal donors.

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| Fig 10.
TcR spectratyping complexity score sums over time.
Complexity scores were generated from CDR3 spectratype analysis at
various time points after transplantation. Individual complexity scores were generated for each V TcR, which correlates to the number of
peaks and their relative peak heights generated by autofluorescence of
electrophoretic gels after RT-PCR of cDNA extracted from PBLs. Individual scores were summed to give an overall complexity score for
that patient's peripheral TcR repertoire at that point in time. Open
circles represent scores for the patients who received FACS-sorted
CD34+ HPC transplants. Broken line represents the median
values for all eight patients analyzed. Closed circles represent the
control patient values. Shaded area and dashed line indicate the mean and standard deviation of seven normal healthy donors.
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DISCUSSION |
Myeloid, erythroid, and megakaryocytic reconstitution after BMT have
been shown to be directly related to the number of CD34+
hematopoietic progenitors present in the graft but, the contribution of
CD34+ hematopoietic progenitor cells to lymphoid
reconstitution after BMT is less clear.1,20,21 Quantitative
recovery of peripheral lymphocytes was delayed among recipients of
FACS-sorted CD34+ HPC compared to conventional PBSC
autografts (Fig 1). The restoration of a fully functional immune system
has been shown to be significantly delayed after autologous
BMT,2-4 a likely consequence of quantitative and
qualitative defects in cellular immunity. Previous studies have
hypothesized that quantitative increases in the number of circulating T
cells in the first 2 months after BMT was mainly a result of the
proliferation of mature T cells present in the graft,22
resulting in a peripheral T-cell compartment lacking sufficient
diversity and complexity to adequately recognize and respond to the
variety of antigenic challenges.10 Our study of nine
patients transplanted with highly purified CD34+ PBSC
progenitors afforded an opportunity to examine lymphoid reconstitution
among patients who received negligible numbers of mature T cells and B
cells in their autograft. The CD34+ column selection
followed by high-speed FACS sorting resulted in a 4.2-log depletion of
T cells and a 4.6-log depletion of B cells with the average number of T
cells and B cells in the stem cell autograft reduced to less than
4,000/kg, and 2,500/kg, respectively.15 We anticipated that
patients who had received a limited number of T cells and B cells in
their CD34+ FACS-sorted autograft would demonstrate a less
diverse and less complex peripheral T-cell compartment, compared with
that of patients receiving conventional PBSC transplants. Although
there were minimal quantitative and temporal differences in the
reappearance of peripheral blood T cells and B cells among patients
receiving FACS-sorted CD34+ HPC transplants compared with a
group of patients that received nonsorted PBSC autografts, there were
significant qualitative differences in the phenotype of circulating
lymphocytes after BMT between these two cohorts of patients.
Several of our findings support the hypothesis that early (<2 months
after BMT) lymphoid engraftment among recipients of FACS-sorted CD34+ HPC was derived from expansions of limited numbers of
mature, post-thymic T cells, whereas T cells appearing in the
circulation at more than 2 months after BMT were derived from
hematopoietic stem cells that differentiated during the post-transplant
period. The patterns of CD45RA/CD45RO and CD4/CD8 expression on T cells were used as surrogate markers for their developmental status of T
cells. Pretransplant analysis of CD45 isoforms in our patient population showed that 70% to 96% of circulating T cells expressed the CD45RO isoform alone, 4% to 30% expressed the CD45RA isoform, and
an average of 12% of all peripheral T cells expressed both isoforms.
After transplantation, the fraction of peripheral blood T cells that
were CD45RO single positive was initially more than 80%. During the
first year after BMT, the frequency of CD45RA single positive T cells
gradually increased. These results suggest that (1) expansion of
CD45RO+ mature "memory" T cells present in the PBSC
graft or that survived myeloablative chemotherapy are responsible for
early recovery of the peripheral T-cell compartment, and (2) the
subsequent appearance of peripheral blood T cells expressing CD45RA and
lacking CD45RO was the result of de novo maturation of
CD34+ progenitor cells to "naive" CD45RA single
positive T cells in the residual thymus or at an extrathymic site(s) of
T-cell development.
Analysis of patients receiving T-cell-depleted marrow as well as
patients receiving unmanipulated marrow in previous studies has shown
that the numbers of circulating CD8+ ("suppressor") T
lymphocytes recover more rapidly than the numbers of CD4+
("helper") T lymphocytes, however; the CD8 "overshoot"
tends to be higher in recipients of untreated marrow
transplants.4,23-25 Inverted CD4/CD8 ratios persisted as
long as 2 years after engraftment, mainly attributable to persistent
low numbers of CD3+, CD4+ T cells as
CD3+, CD8+ T cells approached normal at around
30 days after BMT.9,23-25 None of the recipients of
FACS-sorted CD34+ HPC or unmanipulated PBSC autografts had
normal CD4/CD8 ratios at 2 months after BMT. Overall, recipients of
FACS-sorted CD34+ HPC had lower numbers of
circulating T cells and particularly CD4+ T cells, compared
to recipients of unmanipulated PBSC autografts. Three recipients of
FACS-sorted HPC in our present study had attained normal CD8 levels
(mean, 479/µL ± 169) at 1 year after BMT; however, five of the
nine study patients had decreased numbers of CD8 cells (mean,
86/µL ± 106) at 4 to 6 weeks after BMT that persisted until 6 months after BMT (mean, 181/µL ± 192). The persistent distortion of the CD4/CD8 ratio among recipients of FACS-sorted CD34+
HPC, coupled with the appearance of CD45RA+ "naive" T
cells suggests abnormal thymopoiesis in these patients, producing
predominantly CD8+ T-cell formation in an extrathymic site
that favors CD8+ over CD4+ T cells.
Supporting the hypothesis of abnormal thymopoiesis, or
extrathymic T-cell development in these patients is our observation that six of the nine patients studied demonstrated persistently depressed numbers of circulating T cells between 6 months to 1 year
after BMT, and three patients showed a marked elevation in the number
of circulating  + T cells in their peripheral blood at
1 to 2 months after BMT. Lamb et al26 reported that an
increased frequency of  + T cells after allogeneic
partially matched related donor bone marrow transplantation is
associated with a decreased incidence of leukemia relapse. The clinical
significance of the higher frequency of  + T cells
among some of the patients in the current study is unknown, as the
number of patients and their follow-up evaluation are limited. The
origin of the  + T cells in the immediate
post-transplant period is unclear, but they may represent either de
novo T-cell maturation from undifferentiated hematopoietic progenitors
in an extrathymic site or expansion of small numbers of  T cells
(eg, those contained in the intestinal epithelium) that survived the
myeloablative regime. We did not use - or -specific
oligonucleotide primers to study the complexity of  T cells, but
FACS analysis showed uniform expression of 9 and 2 on the
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