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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4224-4231
Correction of Abnormal T-Cell Receptor Repertoire During
Interferon- Therapy in Patients With Hairy Cell Leukemia
By
Hanneke C. Kluin-Nelemans,
Michel G.D. Kester,
Lisette van deCorput,
Patrick P.C. Boor,
Jim E. Landegent,
Jacques J.M. van Dongen,
Roel Willemze, and
J.H. Frederik Falkenburg
From the Laboratory of Experimental Hematology, Department of
Hematology, Leiden University Medical Center, Leiden; and the
Department of Immunology, Erasmus University Rotterdam, Rotterdam, The
Netherlands.
 |
ABSTRACT |
Patients with the B-cell malignancy hairy cell leukemia (HCL)
exhibit a skewed T-cell repertoire with oligoclonal expression or
absence of many members of the T-cell receptor (TCR) BV gene families.
To evaluate whether interferon- (IFN-) therapy would not only
restore normal hematopoiesis, but also the abnormal T-cell repertoire,
we studied T lymphocytes from a cohort of HCL patients treated by
IFN- in the past, at initiation, and at several intervals up to 6 years of IFN- treatment. The junctional regions from 22 TCRBV gene
families were analyzed after polymerase chain reaction amplification of
cDNA (RT-PCR) using family specific primers. In all seven patients
improvement of the skewed T-cell repertoire was not seen until 2 years
of treatment. It consisted of disappearance of oligoclonal
subpopulations and (polyclonal) reappearance of absent TCRBV gene
families. The RT-PCR results were correlated with the TCRBV protein
expression using TCRBV-specific monoclonal antibodies. T lymphocytes
from four patients with active HCL contained large expansions of
particular TCRBV-expressing cells (up to 25% of the CD3+
cells; 600 to 700/µL whole blood), which decreased during IFN- therapy in both patients tested. Finally, restoration of the TCR repertoire matched normalization of the functional immune repertoire as
measured by proliferative, helper, and cytotoxic T-lymphocyte precursor
frequencies against major histocompatibility complex-unrelated individuals. In conclusion, oligoclonal bands of TCRBV gene families found by RT-PCR correspond with a dramatic increase in circulating T
lymphocytes expressing the same TCRBV family. Moreover, IFN- can
restore the skewed T-cell repertoire and suppress persistent T-cell
clones upon treatment of the accompanying malignancy.
| |
INTRODUCTION |
RECENTLY WE described that T cells from
most patients with hairy cell leukemia (HCL) exhibit a marked skewed
repertoire of the T-cell receptor (TCR) genes.1 In
individual patients, different oligoclonal patterns were observed after
polymerase chain reaction (PCR) amplification of the junctional regions
of the rearranged TCRG and TCRB genes.1 These abnormalities
can in part explain the abnormal cellular host defense and extreme
sensitivity to opportunistic infections of these
patients.2-8 We wondered whether the TCR repertoire would
normalize with treatment of the disease. Most patients respond with
partial remissions upon interferon- (IFN-) therapy with
normalization of blood parameters8-10 and gradual
restoration of T-cell immunity.11-13 Although newer therapy regimens with 2-deoxycoformycin and 2-chlorodeoxyadenosine are followed
by more complete remissions than with IFN-,14-16 both cytostatic drugs are characterized by a longstanding T-cell cytopenia with persistently decreased CD4+ T-cell
counts.15,17-19 Therefore, to evaluate the role of
treatment on the T-cell repertoire in patients with HCL, we studied a
cohort of patients solely treated by IFN-. Here we show that indeed many T-cell abnormalities disappeared with time. Analysis of the TCRBV
repertoire at the protein level with TCRBV-specific monoclonal antibodies (MoAbs) matched the results obtained by reverse
transcriptase (RT)-PCR analysis of TCRBV transcripts
before and during IFN- therapy. Moreover, these MoAbs
made it possible to study specific subsets of TCRBV family-expressing T
cells by multicolor immunofluorescence stainings. Finally, to confirm
that the phenotypical improvement also reflected immunological
improvement, we measured T-cell reactivity before and during recovery,
focusing on helper, proliferative, and cytotoxic alloreactive T-cell
precursor frequencies against major histocompatibility complex
(MHC)-unrelated individuals.20,21
| |
MATERIALS AND METHODS |
Cell isolation.
Peripheral blood (PB) cells, collected during the last decade, from
seven patients with HCL treated by IFN- and who had undergone splenectomy were studied. The age of the patients during
the present analysis varied from 29 to 61 years, with a median of 41 years (see Table 1). The diagnosis was
confirmed by histology of the spleen and bone marrow (BM),
cytomorphology, and immunophenotyping (reactivity with MoAbs against
CD11c, CD19, CD25, CD103, and expression of monotypic Igs). Patient
characteristics are summarized in Table 1. The cell samples tested were
obtained before initiation and treatment with IFN-. The majority of
patients was treated with initial doses of 1.5 to 3 million
international units (MIU) IFN-2b, three times a week,
followed by a further reduction of the dose when at 1 year of therapy a
hematologic remission of the HCL was obtained. In most patients the
IFN- therapy was continued at ultra-low doses (about 1 MIU/wk22). Patient G (61 years old and known with HCL for
12 years) was analyzed while receiving his second course of
IFN-. Blood was collected at regular intervals during follow-up. Mononuclear cells were isolated by Ficoll-Isopaque (Pharmacia, Uppsala, Sweden) (1.077 g/mL) density gradient
centrifugation. In most cases the cells were cryopreserved in liquid
nitrogen in 10% dimethyl sulfoxide until further study.
TCRBV-PCR.
Total RNA was isolated by the guanidium isothiocyanate method as
described23 or with Trizol (GIBCO-BRL, Gaithersburg, MD), according to the manufacturer's procedure. cDNA was prepared from samples of 2 µg RNA each (derived from at least 2 × 106 T cells) using Moloney murine leukemia
virus BRL RT (GIBCO-BRL) for 60 minutes at
37°C as described.23,24 One fiftieth of each cDNA
reaction was individually amplified using a TCRBV family-specific primer and a C primer. All primers have been described
previously.1,25,26 Each 50-µL PCR reaction contained cDNA
in 10 mmol/L Tris HCl, pH 8.4, 50 mmol/L KCl, 1.5 mmol/L
MgCl2, 20 µg/mL bovine serum albumin, 20 pmol of a TCRBV
family-specific primer and the C primer, 50 µmol/L dNTPs, 20 Pm
[-32P]dCTP (3,000 Ci/mmol/L; Amersham,
Arlington Heights, IL), and 1.25 U Taq DNA polymerase.
The amplification was started with a denaturation step of 4 minutes at
94°C, followed by 25 to 30 cycles, each cycle consisting of 1 minute at 94°C, 55°C, and 72°C. The number of V-C
amplification cycles required for the individual cDNA samples was first
determined by amplifying the C region of the TCR cDNA for 20 to 30 cycles and visualization on an ethidium bromide-stained agarose gel.
The cycle number at which the constant-region amplicons were clearly
visible was chosen for amplifying the TCRBV repertoire. Clonality
within each family was determined by denaturing polyacrylamide gel
electrophoresis (dPAGE) and single-strand conformation polymorphism
(SSCP) analysis. After electrophoresis, amplified DNAs were visualized
by autoradiography.
Scanning of cDNA-TCR products.
In addition to visual analysis, the dPAGE and SSCP gels were exposed to
a Storage Phosphor Screen, secured with a Phosphor Imager 455 SI and
analyzed by ImageQuant NT software (all from Molecular Dynamics,
Sunnyvale, CA). A selection of 1,500 images was scored by three
different persons, of whom two were not aware of the clinical data. The
scores for the three were concordant in greater than 90% of all
images; the inter-observer variability focusing on the absence or
presence of oligoclonal patterns varied between 1% and 10% per
patient and per TCRBV family. The images usually visualized with a
virtual Y-axis were also expressed using a Y-axis representing the
relative use of total expression. To this end the counts for each pixel
in a graph were added, the integral surface per graph was calculated,
and the amount of radioactivity related to the total amount of all
families in a single experiment.
Flow cytometry analysis of TCRBV protein expression in T-cell
subsets.
Out of an extensive (n = 50) panel of TCRBV-specific MoAbs of
the TCRBV Workshop, the following were used: BV2: E2.2E7.2 and MPB2/D; BV3: CH92, 8F10, and 5E4; BV5.1: IMMU157; BV5.2/5.3: 4H11; BV6.1: CRI304.3; BV6.7: OT145; BV7.1: 3G5; BV8.1/8.2: 56C5.2; BV9.1:
FIN9; BV11.1/11.2: C21; BV12.2: VER2.32; BV13.1/13.2: BAM13; BV13.6: JU74.3; BV14; CAS1.1.3; BV16: TAMAYA1.2; BV17: C1 and E17.5F3;
BV18: BA62.6; BV20: ELL1.4; BV21.3: IG125; BV22(22.1): IMMU546; BV23:
AF23.27,28 These antibodies were kindly provided by
Immunotech (Marseille, France), T Cell Diagnostics (Cambridge, MA), and
T Cell Sciences (Cambridge, MA). Reactivity of the TCRBV MoAbs was
assessed by fluorescein isothiocyanate-labeled goat-anti-mouse antibodies. After blocking with normal mouse serum, cells were further
stained with phycoerythrin (PE)-conjugated MoAbs and PE-Cyanine 5 or
PerCP-conjugated MoAbs of the following clusters: CD3 (Leu-4), CD4
(Leu-3a), CD8 (Leu-2a), anti-TCR-/ (WT31 or BMA031),
anti-TCR- / (11F2) from Becton Dickinson (Mountain View, CA).
Fluorescence was assessed by flow cytometry (FACScan; Becton Dickinson)
using CellQuest software (Becton Dickinson). Appropriate
controls were included to rule out aspecific fluorescence. Only viable
cells were analyzed using LDS.29
Cloning and sequencing experiments.
From one patient with 13% TCRBV2+ cells, the
CD8+BV2+ T-cell subset was sorted using a
FACStar (Becton Dickinson). After amplification by RT-PCR, the PCR
product was cloned into the pCR3.1 vector with a TA cloning system
(Invitrogen, Leek, The Netherlands) and sequenced with the T7
sequencing kit (Pharmacia Biotech, Uppsala, Sweden) using the vector
primers.
T-cell precursor frequencies in mixed lymphocyte cultures.
Cytotoxic and helper T-lymphocyte precursor frequencies (CTLp and HTLp)
were analyzed as described.20,21 In short, responder T
cells from the patient at various time points of IFN- treatment were
twofold diluted across the wells of two 96-well plates from 40,000 cells per well until a concentration of 625 cells per well was
reached. To determine whether contaminating HCL cells in
the responder cell populations could possibly suppress the
proliferation of these cells, responder cells from active disease were
tested with and without depletion of HCL cells. In addition, as a
control, the same HCL cells recovered during these depletion steps were added to responder T-cell suspensions obtained after several years of
IFN- treatment. All of these cocultures were done with frozen PB
mononuclear cells, and always simultaneously realized in a single
experiment. Irradiated stimulator lymphocytes obtained from healthy
donors, which differed at four or more HLA class I and II antigens from
the responder cells, were added at 50,000 cells per well and cultured
for 3 days. For the HTLp analysis, 80 µL of supernatant
was obtained and interleukin-2 (IL-2) release was measured using an
IL-2-dependent murine CTLL-2 cell line.21 Subsequently,
IL-2 was added (120 U/mL) and the cultures were continued until day 10. IL-2 plus phytohemagglutinin (PHA)-stimulated target cells were labeled
with europium (Eu) chelated to diethylenetriaminopentaacetate (DTPA)
and added at a concentration of 5,000 cells per well to each responder
cell concentration. After 4 hours of incubation, released Eu was
measured in the supernatant using a time-resolved fluorometer, and
expressed in counts per second. A well was scored positive if the
counts exceeded the mean ± 3 × SD of the wells with
stimulator cells and corresponding target cells only.
CTLp frequencies were calculated for that responder cell concentration resulting in 37% of the tested wells remaining negative. As controls, identically treated autologous target cells were used.
Proliferative T-lymphocyte precursor frequencies (PTLp) were
identically set up as described above. However, instead of IL-2 release, 3H-thymidine incorporation was measured at day 6 of culture.
All T-precursor frequencies were expressed per 106 T cells.
| |
RESULTS |
In four of seven patients IFN- induced a hematologic response with
normalization of hemoglobin levels, white blood cell and thrombocyte
counts, disappearance of circulating hairy cells, and reappearance of
monocytes. In the remaining patients, the blood counts normalized but a
few circulating HCL cells were still detectable (see Table 1).
RT-PCR of TCRBV families before and during IFN- therapy.
The seven patients were studied before the start of IFN- and during
therapy for a period of 315 to 1,646 days. Some of the results in six
patients obtained before the start of IFN- have been described
before.1 RT-PCR analysis with a panel of 24 primers of
junctional regions of TCRB transcripts from 22 well-established TCRBV
families analyzed on dPAGE gels disclosed oligoclonal and polyclonal
(ie, regular ladder patterns) configurations for the individual TCRBV
families in all seven patients. Markedly abnormal patterns were found
with clonal bands in many different BV families. In addition, several
gaps in the TCRB repertoire were present with BV families missing or
showing abnormal weak signals compared with normal age-matched
controls. For each family the junctional region size distribution
patterns were recorded using a Phosphor Imager, allowing comparison of
the samples during treatment (Fig 1). SSCP
gel analysis of the TCRBV repertoire confirmed the clonal excess
patterns seen on the dPAGE gels. Follow-up during IFN- therapy
disclosed a remarkable improvement of the skewed TCRBV repertoire in
all patients, which became more clear only after 2 or more years of
therapy. To illustrate the slow rate of this response, a detailed
overview of seven abnormal BV families in one patient followed for 3.3 years is given in Fig 1. Not only did oligoclonal populations
disappear, but also a reappearance of polyclonal patterns was observed.
The latter is illustrated for several BV families in two patients in
Fig 2, which clearly shows the restoration
of some of the affected BV families, both quantitatively and
qualitatively. The combined results of the RT-PCR analyses of 22 TCRBV
gene families in the seven patients at the start of IFN- therapy and
after long-term follow-up are summarized in
Fig 3.
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| Fig 1.
Junctional region size distribution patterns of RT-PCR
products from various TCRBV subfamilies from patient A. Various time points are shown. The first blood sample was taken at the start of
IFN- (day 0). To compare different samples in time, Phosphor Imager
scans obtained from the radioactive labeled products on dPAGE gels are
shown. The right part of the figure shows the pattern of a healthy
individual.
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| Fig 2.
Follow-up of junctional region size distribution patterns
of RT-PCR products from TCRBV families in patients A and G showing very
weak signals at initiation of IFN- therapy. In a semi-quantitative method the recovery of polyclonal T cells within a particular TCRBV
gene family is shown. The y-axis represents the relative use of total
expression calculated by adding the counts obtained from each single
pixel within the graph.
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| Fig 3.
Distribution of 22 well-established TCRBV gene families
from seven HCL patients, analyzed before and during IFN- therapy. The X-axis shows the time points in days. Patient B was analyzed 221 days before IFN was started. The Y-axis gives the absolute number of
TCRBV families assessed from 1-23 with TCRBV20 not analyzed, and with
TCRBV5 and TCRBV13 shown as 5a, 5b, 13a, and 13b. The large BV5 was
split into 5a (BV5S1) and 5b (BV5S2-6); amplification of BV13 resulted
in two patterns on the gels, which were analyzed separately. PCR
pattern: ( ), Polyclonal; ( ), absent/weak; ( ), oligoclonal.
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Analysis of TCRBV-expressing blood T lymphocytes by flow cytometry.
To match the findings at the RNA level with TCRBV protein expression,
we determined in four patients with active disease the percentages of
CD3+ T cells in the blood that expressed particular TCRBV
families. For this purpose we used a panel of 25 MoAbs covering about
65% of the TCRBV repertoire in healthy individuals.27,28
The results were compared with a panel of five normal donors, as shown
in Fig 4. In all four patients large
T-lymphocyte expansions (percentual and numerical) expressing a single
BV family were seen. In two of four patients tested, more than 22% and
25% of the CD3+ T lymphocytes expressed a single TCRBV
family. Detailed flow cytometric analysis showed that in most cases the
expanded TCRBV families concerned the CD8+ T lymphocytes,
although "clonal" CD4+ populations were also seen
(Table 2). We calculated the absolute numbers of circulating TCRBV+ cells to illustrate the
presence of large expansions of single BV+ expressing T
cells (Table 3). For example,
patient A with 40% of the CD8+ cells being
BV3+ harbored a total of 617 × 106
BV3+ cells/µL in the blood during active disease. An
excess of BV-family expression was almost always concordant with clonal
populations found by RT-PCR.
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| Fig 4.
TCRBV repertoire of four HCL patients during active
disease. The results are expressed as percentage of CD3+
cells. For specific MoAbs see Materials and Methods. The results obtained for BV2, BV3, and BV17 were confirmed by other MoAbs detecting
the same TCRBV phenotype. For comparison, the TCRBV repertoire obtained
from five healthy donors is given as mean ± SD for each BV family
(gray bars, lower part of each subfigure).
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Table 2.
Phenotype of CD3+ T Cells and Elevated
TCRBV+ Fractions in Three Patients Before and in Two
Patients Also During IFN- Therapy
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Table 3.
TCRBV Repertoire During IFN- Therapy Calculated
as Absolute Numbers TCRBV+ Cells per Microliter of
Blood
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To analyze whether the excessive expression of some BV families
represented true clonal expansions of individual members, we sorted the
CD8+BV2+ cells from patient G. cDNA obtained
from the sorted cells was amplified with TCRBV2 and TCRBC primers. The
TCRBV RT-PCR products were cloned and 30 colonies were subjected to
dPAGE and SSCP analysis. These analyses showed that 28 of the 30 fragments comigrated under various electrophoresis conditions,
indicating their clonal origin. Sequencing of 1 of the 28 fragments
showed a functionally rearranged TCRB gene consisting of the
TCRBV2S1D2S1J2S7C2 gene segments with a junctional region amino
acid sequence of (BV2S1)CSA_IPLNGTD_EQY(BJ2S7).
Follow-up of TCRBV expansions during therapy with IFN-.
In two patients the TCRBV domain expression before and during IFN-
therapy was measured. Results are shown in Table 2. Patient A was
analyzed after 3 years and showed a complete hematologic response with
normalization of the leukocyte differential (disappearance of
circulating hairy cells, reappearance of monocytes). The very high
percentage of BV3+ cells decreased from
24.6% to 7.5% (normal, 3.3% ± 1.2%). By RT-PCR a faint TCRBV
band was still visible 3 years after the start of IFN- (Fig 3).
Also, the increased percentages of BV17+ (10%; normal,
5.4% ± 1.3%) and BV18+ (6.8%; normal, 1.2% ± 0.6%) normalized during IFN-. This normalization was also seen for
the absolute BV17+ and BV18+ cell counts (Table
3). In contrast, patient B, who was assessed after 1.3 years as far as
these experiments are concerned, showed only minor improvements. The
leukocyte differential still yielded 1% hairy cells, 27%
granulocytes, and only 1% monocytes. In this patient both the
increased percentage of BV2+ (12.8%; normal, 6.9% ± 0.5%) and BV20+ (7.2%; normal, 2.5% ± 1.0%)
remained elevated, and the RT-PCR analysis also showed persistent
clonality.
Functional immunologic studies.
To analyze whether the improvement of the TCRBV repertoire correlated
with a functional immunologic improvement, we assessed the
alloreactivity to HLA-nonidentical donors before and during IFN-
therapy. The results from the analysis of helper (HTLp), proliferative
(PTLp), and cytotoxic (CTLp) T-cell precursor frequencies before and
during IFN- are shown in Table 4. In all
three cases HTLp, PTLp, and CTLp frequencies were markedly abnormal at
start of IFN-. Patients A and B were considered good responders upon IFN- treatment and showed in parallel a good
restoration of the immunologic T-cell functions. Patient G, without a
complete hematologic remission and with a persistently abnormal TCRBV
repertoire during 1.3 years of treatment, continued to
have impaired T-cell responses.
HCL cells present in the cultures might have suppressed the functional
reactivity of the responder cells collected during active disease.
Therefore, control experiments were performed and equal numbers of HCL
cells were added to the cultures performed with responder T cells
obtained during remission of the disease. The outcome was not different
from the results shown in Table 4 (data not shown).
| |
DISCUSSION |
The mechanism of acquired T-cell immune dysfunction in the absence of
T-cell lymphocytopenia observed in patients with hematologic malignancies is poorly understood. Untreated patients with HCL have a
T-cell dysfunction, amongst others characterized by severe opportunistic infections.2,7 Furthermore, defective K,
natural killer, and lymphokine-activated killer (LAK)
cell functions30,31 are found, as well as a remarkable
decrease in the percentage and number of circulating
(CD4+CD45R0+) memory T-helper
cells.32
Recently we showed that the T-cell immune deficiency in HCL is
associated with a marked restriction in the TCRBV repertoire with
oligoclonal T-lymphocyte subsets and large gaps in the use of BV
families.1 Patients with active disease and not yet
treated, who were monitored several times for more than 1 year, showed a further progression of these abnormalities.1
Clonal T-cell populations have also been found in other B-cell
malignancies, eg, in chronic lymphocytic leukemia33,34 and multiple myeloma.33,35 Such oligoclonal T cells may play a role in antitumor surveillance. Remarkably, in the studies by Wen et
al,33 only patients with early disease (stage
0 chronic lymphocytic leukemia and smoldering myeloma) harbored
circulating clonal T cells. Moss et al35 found, by
immunopheno- typing, oligoclonal CD4+ and CD8+
T cells in the blood of patients with benign paraproteinemia and
multiple myeloma. The highest percentage of clonal
expansions was seen in a patient with presumably benign IgM
paraproteinemia. Farace et al34 succeeded in the expansion
of a BV19+ T-cell clone that specifically recognized
autologous CLL cells. We analyzed patients with B-cell malignancies
other than HCL1 and confirmed the occurrence of
oligoclonality in some patients with chronic lymphocytic leukemia,
non-Hodgkin's lymphoma, or prolymphocytic leukemia, but the TCRBV
abnormalities in these cases were much less pronounced than in HCL.
Here we show for the first time that a skewed TCRBV repertoire can
normalize upon treatment of the associated malignancy. IFN- induced
a remarkable restoration of the T-cell abnormalities, which took much
more time (at least 2 to 3 years) than was needed for hematopoietic
recovery. This is not unexpected given the long period necessary for a
full recovery of the T-cell repertoire such as is seen after BM
transplantation.36,37 We observed not only a gradual
disappearance of the clonal expansions, but also a polyclonal
reappearance of those BV families that were very low or even absent at
the start of IFN- therapy. Although many of the oligoclonal
T-lymphocyte populations could not be detected after several years, in
some patients with a hematologic remission persistent T-cell clones
could be recognized by RT-PCR. This might be comparable to the recent
observation by Callan et al,38 who showed large clonal
expansions in patients with infectious mononucleosis; analysis 6 months
after recovery did not yield an expansion in any of the eight patients.
However, the investigators could not exclude the persistence of small
clones, because they used the immunophenotyping technique instead of
RT-PCR analysis.38 Similar to acute Epstein-Barr virus
infections, the clonal expansions found in HCL may reflect T-cell
responses to an HCL-specific antigen which obviously needs further
determination.
We correlated the restrictions in the TCRBV repertoire assessed at the
genotypic and immunophenotypic levels with functional tests. We
measured a restoration of the T-cell function by a combined assay in
which HTLp, PTLp, and CTLp frequencies were assessed.
In conclusion, patients with HCL harbor a skewed TCRBV repertoire
consisting of large clonal expansions as well as gaps in many BV
families. Induction of a hematologic remission by IFN- is followed
by a restoration of the T-cell abnormalities after a time period of at
least several years. Functional immunological studies confirm the
severe T-cell immune deficiency and also show improvement in parallel
to the genotypical and phenotypical normalization of the TCRBV
repertoire. These observations give more insight into the mechanisms of
acquired T-cell immune deficiency in relation to malignant diseases.
| |
FOOTNOTES |
Submitted July 14, 1997;
accepted December 31, 1997.
Supported by Grant No. RUL 94-842 from the Dutch Cancer Society
(Koningin Wilhelmina Fonds).
Address reprint requests to J.C. Kluin-Nelemans, MD, PhD,
Leiden University Medical Center, Department of Hematology, Building 1 E1-Q, PO Box 9600, 2300 RC Leiden, The Netherlands.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank A. van de Marel for his help with cell sorting and
are grateful to Immunotech, T Cell Diagnostics, and T Cell Sciences for
providing the TCRBV antibodies.
| |
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Abstract
Introduction
Abstract