|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 251-259
Hairy Cell Leukemia-Specific Recognition by Multiple Autologous
HLA-DQ or DP-Restricted T-Cell Clones
By
Lisette van de Corput,
Hanneke C. Kluin-Nelemans,
Michel G.D. Kester,
Roel Willemze, and
J.H. Frederik Falkenburg
From the Laboratory of Experimental Hematology, Department of
Hematology, Leiden University Medical Center, Leiden, The Netherlands.
 |
ABSTRACT |
We studied in patients with hairy cell leukemia (HCL) whether
autoreactive T cells could be isolated with specific reactivity to the
HCL cells. HCL cells were activated via triggering of CD40 on the cell
membrane and used as stimulator cells to generate autologous T-cell
clones. Two types of CD4+BV2+ T-cell clones
with different CDR3 rearrangements and one type of
CD4+BV8S3+ T-cell clone were generated from
the spleen or blood. These clones specifically recognized the
autologous HCL cells, without reactivity to autologous peripheral blood
mononuclear cells (PBMC), phytohemagglutinin blasts, or Epstein-Barr
virus-transformed B cells in a primed lymphocyte test. Blocking and
panel studies using HCL cells from 11 other patients showed that
recognition of the HCL cells by the BV2+ T cells was
restricted by HLA-DQA1*03/DQB1*0301, and the BV8S3+ T
cells were restricted by DPB1*04. The T-cell clones did not recognize
DPB1*04+ or DQ3+ PBMC from healthy donors
or DP/DQ matched malignant cells from patients with other hematologic
malignancies, except for one patient with acute lymphoblastic leukemia.
These HCL-specific T-cell clones may be used for the detection of an
HCL-specific tumor antigen.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
HAIRY CELL LEUKEMIA (HCL), a chronic
B-cell malignancy comprised by mature neoplastic B cells with hairy
protrusions, is characterized by splenomegaly caused by the HCL cells
which home to and accumulate in the spleen. The HCL cells are also
found in the bone marrow, and at later stages of the disease they
appear in the peripheral blood.1 Patients with HCL suffer
from opportunistic infections2 caused by pancytopenia and a
T-cell-related immune deficiency.3,4 Signs of abnormal
T-cell activation and distribution have been found in the HCL spleens
compared with normal spleens.5-7 Splenic T cells from
patients with HCL show spontaneous cytokine gene expression of
interleukin-2 (IL-2), IL-4, granulocyte/macrophage-colony stimulating
factor (GM-CSF), and interferon- (IFN-),5 and a
reversed CD4/CD8 ratio.5 Recently, we demonstrated that T lymphocytes from blood and spleen of most HCL patients are clonally expanded and show a restricted and skewed repertoire of the T-cell receptor- family (TCRBV).8 Besides clonal excess of
certain T cells, several TCRBV families were weakly expressed or even absent.8 In part, these findings may explain the immune
deficiency and lack of tumor surveillance in patients with HCL. We
hypothesized that T cells with specific activity to the HCL cells exist
in vivo, but are in an anergic state9,10 or are probably
suppressed by the tumor cells. It may be possible to expand these T
cells more optimally in vitro by using activated HCL cells, which are triggered via the CD40 antigen to upregulate expression of adhesion and
costimulatory molecules important for the completion of an effective
immune response.11,12 To test this hypothesis, we analyzed
whether T cells capable of recognizing autologous HCL cells could be
isolated from patients.
In this study, we show that multiple leukemia-reactive CD4+
T-cell clones with different TCRBV expression that specifically recognized autologous HCL cells in HLA-DQ and HLA-DP restricted ways
could be generated.
| |
MATERIALS AND METHODS |
Patients and cell samples.
The diagnosis HCL of patient A was confirmed by histology of the spleen
and bone marrow, cytomorphology, and immunophenotyping (reactivity with
monoclonal antibodies [MoAbs] against CD11c, CD19, CD25, CD103, and
expression of monotypic immunoglobulins). In 1991 a therapeutic
splenectomy had been performed, and seven months after splenectomy,
IFN- therapy was started because of granulocytopenia and
thrombocytopenia. After 24 months, he obtained a partial hematologic
remission with a normalization of the blood counts and a reduction of
circulating HCL cells from 90% to 2%. HLA class I and II typing of
patient A was performed by standard serology methods and HLA-DR/DQ and
HLA-DP typing by DNA analysis using sequence-specific primers (SSP) and
sequence-specific oligos (SSO). The HLA type was class I: A2 A28, B44
(B12) B60 (B40), Cw10 Cw5; class II: DRB1*04 DQA1*03 DQB1*0301
(homozygous), DPB1*0101 DPB1*0402.
After informed consent, mononuclear light density cells were isolated
from the spleen by gentle mechanical disruption, washing, and
Ficoll-Isopaque (1.077 g/mL) density gradient centrifugation. Peripheral blood lymphocytes (PBL) were obtained both during active disease and during remission of the disease 2 years after IFN- therapy was started. Peripheral blood mononuclear cells (PBMC) were
isolated by Ficoll density cell separation. The cells were cryopreserved in 10% dimethylsulfoxide and thawed directly before use.
Cryopreserved spleen samples from 11 other HCL patients (B-L) were
used. PBL or bone marrow samples were obtained from patients with
B-cell chronic lymphocytic leukemia (B-CLL, n = 1), B-cell lineage
acute lymphoblastic leukemia (ALL, n = 5), acute myeloid leukemia (AML,
n = 2), chronic myeloid leukemia (CML, n = 4) and non-Hodgkin's
lymphoma (NHL, n = 1). All samples contained 80% to 95% malignant
cells. As controls, PBL or bone marrow samples from 3 healthy donors
were used. HLA class II typing of all samples was performed by standard
serology and SSP/SSO DNA methods.
Activation of HCL cells using the CD40 ligand system.
To activate HCL cells, 3T6 mouse fibroblast cells transfected with
human CD40 ligand (CD40L) were used.13 In a fully
humidified atmosphere containing 5% CO2 for 7 or 8 days, 5 × 105 HCL cells were cocultured with 5 × 104 irradiated (70 Gy) 3T6 cells/well in a
24-well plate in a final volume of 2 mL complete medium. Complete
culture medium consisted of Iscove's modified Dulbecco's medium
(IMDM; Bio-Whittaker, Verviers, Belgium), 5%
heat-inactivated fetal calf serum (FCS), penicillin/streptomycin (20 U/mL and 20 µg/mL, respectively), 10 U/mL nystatin (Sanofi, Maassluis, The Netherlands), 3 U/mL polymyxin B (Pfizer, Capelle a/d
IJssel, The Netherlands), 20 µg/mL kanamycin (Gist
Brocades, Delft, The Netherlands), 5 µg/mL bovine insulin (Sigma
Chemical Co, St Louis, MO), 0.5% human serum albumin (CLB, Amsterdam,
The Netherlands), 45 µg/mL human transferrin (Boehringer, Mannheim, Germany), and 0.01 mmol/l beta-mercaptoethanol (Sigma). To analyze the
expression of adhesion and costimulatory molecules, the HCL cells from
five patients were labeled using the following fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labeled MoAbs: CD19 (leu-12), CD11c (leu-M5), CD11a (LFA-1), CD54 (leu-54),
CD58 (LFA-3), CD80 (B70/B7-1), CD86 (B70/B7-2), pan HLA class I
(W6.32), and HLA-DR from Becton Dickinson (San Jose, CA). CD40 (MoAb
89; Schering Plough, Dardilly, France) and CD72 (MCA 687; Serotec, Oxford, UK) were used as first-step unconjugated MoAbs
followed by a conjugated isotype-specific goat-anti-mouse (GAM)-FITC
antibody. The data are expressed as fluorescence intensity in arbitrary units.
Generation of HCL-reactive T-cell lines.
Two different methods were used to generate HCL-reactive T-cell lines.
Using the first method, the autologous T cells and the HCL cells, both
derived from the spleen, were cultured together in the CD40L system. In
the second method, HCL cells were first activated in the CD40L system,
isolated, irradiated, and added as stimulator cells to autologous PBMC
obtained during hematologic remission.
Spleen-derived T-cell lines.
To generate HCL-reactive T-cell lines, the spleen suspension consisting
of 96% HCL cells and 4% T cells from HCL patient A was added at a
concentration of 5 × 105 cells/well to the CD40L
system. After 7 days of culture and weekly thereafter, the complete
medium was demi-refreshed and IL-2 (Roussel Uclaf, Paris, France) was
added at a final concentration of 100 U/mL. From day 40 on, the cell
cultures were weekly demi-refreshed using RPMI 1640 with 10% pooled
human serum prescreened for human immunodeficiency virus, HBs Ag, and
allo-antibodies (Red Cross Bloodbank Leidsenhage, Leiden, The
Netherlands) and IL-2. T-cell cultures obtained were phenotyped, and
tested for cytotoxicity in a 51Cr-release assay as
described14 and specific proliferation in a
3H-thymidine assay as described below. On day 71, the
T-cell line that showed specific proliferation in the primed lymphocyte
test (see below) was restimulated with autologous HCL cells. The T cells were further expanded by restimulation with autologous HCL cells
once every 2 to 3 weeks and cloned by limiting dilution (see below).
PBL-derived T-cell lines.
To generate HCL-reactive T-cell lines from the blood, HCL cells
activated in the CD40L system were used as irradiated (25 Gy)
stimulator cells, and added (1.5 × 106 cells/well) to
autologous PBMC (5 × 105 cells/well) obtained during
hematologic remission in a 24-well plate. After 7 days of culture using
RPMI 1640 with 10% pooled human serum (2 mL/well) and weekly
thereafter, the cell cultures were restimulated with autologous
activated HCL cells (106 cells/2 mL), demi-refreshed, and
IL-2 was added at a final concentration of 100 U/mL. Eleven wells with
growing T cells from the 24-well plate obtained were tested for
cytotoxicity in a 51Cr-release assay as
described.14
Limiting dilution assay.
T-cell lines were further cloned by limiting dilution. T cells (1 to
0.3 cells/well) were added to autologous activated HCL cells
(104 cells/well) in 96-well microtiter plates containing
RPMI with 10% pooled human serum and IL-2 (100 U/mL). After 3 to 4 weeks, T cells were isolated from wells that showed proliferation and further expanded by restimulation with autologous HCL cells in RPMI
with 10% pooled human serum and IL-2 once every 3 weeks.
Primed lymphocyte test (PLT).
Before use, stimulator cells were thawed and incubated in RPMI with
10% pooled human serum at 37°C overnight. As controls, autologous
PBMC during hematologic remission were used, autologous phytohemagglutinin (PHA) blasts, or Epstein-Barr virus-transformed autologous B cells (EBV-lymphoblastoid cell line [EBV-LCL]). An EBV-LCL was established by in vitro transformation of 107
spleen cells and further cultured in RPMI with 10% human serum. PHA
blasts were generated by culturing PBMC obtained during hematologic remission with PHA (0.8 µg/mL) for 3 days and further culture in
medium containing IL-2 (100 U/mL). Responder T cells (104
cells/well) and irradiated stimulator cells (PHA blasts, PBMC, leukemic
cells at 105 cells/well, or EBV-LCL at 2.5 × 104 cells/well) were cocultured in a 96-well microtiter
plate at a final volume of 150 µL/well in RPMI containing 10% pooled
human serum. After 72 hours, 3H-thymidine (1 µCi/well)
was added to the cultures. After 18 hours, the cells were obtained with
an automatic microharvester, and counts per minute (cpm) were
determined on a liquid scintillation spectrometer. As control for
background 3H-thymidine incorporation, responder and
stimulator cells cultured separately were used. All PLT were performed
in sixfold, from which the mean and standard deviation (SD) were
determined.
To determine the restriction of the recognition of the stimulator cells
by the T cells, blocking studies were performed. Responder T cells were
preincubated with saturating concentrations of CD4 or CD8 MoAbs (RIV6
and FK18, respectively) for 15 minutes,15,16 or stimulator
cells were preincubated with MoAbs against pan-HLA class I (W6.32 or
B9.12.1),17 pan-HLA class II (PdV5.2),18 HLA-DR
(B8.11.2), HLA-DQ (SPVL3), or HLA-DP (B7.21).
Immunophenotyping.
The phenotypes of the generated T-cell lines were analyzed by flow
cytometry (FACscan; Becton Dickinson, Mountain View, CA). Two-color
fluorescence was used with the following FITC- or PE-labeled MoAbs: CD3
(leu-4), CD4 (leu-3a), CD8 (leu-2a), TCR-/ (WT31), TCR- -1
(11F2), CD25 (anti-IL2-R), CD28 (leu-28) from Becton Dickinson, and
CD45RO (UCHL 1; Dako, Glostrup, Denmark). To analyze the expression of
HLA-DQ and HLA-DP on the different stimulator cells, SPVL3 and B7.21
were used as a first-step unconjugated MoAb, respectively, followed by
a conjugated GAM-FITC antibody. Only viable cells were analyzed using
LDS 751.19
To determine the TCRBV protein expression of the T-cell lines and
clones, TCRBV-specific MoAbs were used. All MoAbs have been described
previously.20 For the first screening, pools consisting of
several MoAbs were used. When a pool of MoAbs showed positive staining,
the appropiate individual MoAbs were tested.
TCRBV-polymerase chain reaction (PCR).
Total RNA was isolated with Trizol (GIBCO-BRL, Gaithersburg, MD)
according to the manufacturer's procedure, and if necessary, glycogen
was used as carrier. cDNA was prepared from samples of 1 to 2 µg RNA
each (derived from at least 0.5 to 1 × 106 T cells)
using M-MLV BRL reverse transcriptase (GIBCO-BRL) for 60 minutes at
37°C as described.21 An aliquot of each cDNA reaction was individually amplified using a mixture of TCRBV family-specific primers or a single BV-specific primer and a C primer. All primers have been described previously,8,21-23 except for the
TCRBV8 primer (5 -CCATGATGCGGGGACTGGAGTTGC-3). Each
50-µL PCR reaction contained cDNA in 10 mmol/L TrisHCl, pH 8.4; 50 mmol/L KCl; 1.5 mmol/L MgCl2; 20 µg/mL bovine serum
albumin; 20 pmol of TCRBV family-specific primers and the C primer; 50 µmol/L dNTPs; 20Pm [-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 subsequently 1 minute at 94°C, 56°C, and 72°C. Length of
CDR3 products was determined by denaturing polyacrylamide gel
electrophoresis gels. After electrophoresis, amplified DNAs were
visualized by autoradiography.
The following clone-specific primers for the CDR3 region with a four
nucleotide overlap with the TCRBV and TCRBJ genes were designed:
TCRN4F10, GTCTCCAGACCTAGTGGGC; TCRN1F2, TCCCCCCCCTGGTTTGCAC; TCRNBV8S3, CTTCCCCCTGTCCAGGACC. PCR-amplification was performed using
these specific primers in combination with the TCRBV-specific primers.
Cytokine mRNA expression was analyzed by reverse transcriptase-PCR
using cytokine specific primers as described.5
| |
RESULTS |
Activation of HCL cells using the CD40L system.
At different time intervals during culture, the HCL cells were analyzed
for the expression of several adhesion, costimulatory, and HLA
molecules by flow cytometry. After 1 hour of incubation at 37°C in
medium alone, the HCL cells expressed low levels of CD54 (ICAM-1), CD58
(LFA-3), and CD80 (Table 1). Furthermore, the HCL cells expressed CD40, CD72, and HLA class II, and very high
levels of HLA class I. HCL cells completely lacked the expression of
CD11a (LFA-1) and CD86 (B7.2; Table 1). When HCL cells were cultured in
medium alone no difference in the expression of the above-mentioned
molecules was observed, except for the expression of HLA class I and
II, which significantly increased from 2 days of culture. Expression of
CD54 and CD58 was only slightly upregulated after 6 days of culture. To
induce and upregulate the expression of CD11a, CD54, CD80, and CD86,
the CD40L system was used. Triggering of the CD40 antigen on the HCL
cells by CD40L transfectants highly increased the expression of CD54
and CD80 (Table 1 and Fig 1). The
expression of CD86 was strongly induced. The expression of CD11a could
not be induced by triggering of CD40 on the HCL cells.
View this table:
[in this window]
[in a new window]
|
Table 1.
Comparison of the Expression of Adhesion and
Costimulatory Molecules on HCL Cells (n = 5) When Cultured in
Medium Alone or in the CD40L System
|
|
View larger version (31K):
[in this window]
[in a new window]
| Fig 1.
FACS analysis of HCL cells after 6 days of culture in
medium alone (left), and in the CD40L system (right). The expression of
CD11a, CD54, CD80, and CD86 are shown by a thick black line, and the
isotype control is shown by a thin line.
|
|
Generation and characterization of HCL-reactive T-cell lines and
clones.
Using the activated HCL cells for stimulation, two proliferative T-cell
lines (C6 and D1) generated independently from each other were derived
from the spleen and peripheral blood of patient A, respectively. Both
T-cell lines were TCR+/CD4+ and
specifically proliferated in response to autologous HCL cells as was
measured by 3H-thymidine incorporation after 3 days of
coculture (Fig 2). The T-cell lines showed
similar proliferation in response to autologous HCL cells derived from
the spleen or blood, and were not reactive to autologous PBMC (obtained
during hematologic remission), PHA blasts, or EBV-transformed B cells
(Fig 2). The antigen-specific proliferation of both T-cell lines was
blocked by an MoAb against HLA class II. To exclude positive reactivity
measured in the PLT as a result of immune responses generated to
allotypic human serum components, the different stimulator cells were
incubated in medium with 10% FCS overnight before using in the PLT. No
difference was measured in proliferative responses to autologous cells
incubated in 10% pooled human or 10% FCS serum (data not shown). The
T-cell lines could not lyse the autologous HCL cells.
View larger version (18K):
[in this window]
[in a new window]
| Fig 2.
Proliferation of T-cell lines C6 and D1 in response to
different autologous stimulator cells. Both T-cell lines specifically
recognized the autologous HCL cells in an HLA class II-restricted way,
without reactivity against autologous PBMC, PHA blasts, and EBV-LCL in
the PLT. The PLT was performed in sixfold, and the SD of the means were
lower than 15%.
|
|
To determine how many and which T-cell clones with a certain TCRBV
chain were present in the T-cell lines, a TCRBV-PCR was performed. Both
T-cell lines consisted of several T-cell clones that expressed
different TCRBV families (Fig 3). By
fluorescence-activated cell sorting (FACS) analysis both T-cell lines
consisted of a population of BV2+ T cells and a T-cell
population that could not be determined by the MoAbs available. By
TCRBV-PCR analysis this T-cell population was BV8S3+.
Initially, we cloned T-cell line C6 by limiting dilution. After 4 weeks
of culture, 25 T-cell clones were obtained, of which 15 expressed BV2
and showed two different TCR rearrangement patterns. In addition, 10 T-cell clones were BV8S3+, all expressing the same TCR
rearrangement pattern. T-cell line D1 contained two of these three
different T-cell clones (Fig 3). Two different BV2+ T-cell
clones (4F10 and 1F2) and one BV8S3+ T-cell clone (BV8S3)
were sequenced and showed functionally rearranged TCRB genes with
different CDR3 regions (Table 2). Using
direct PCR with the TCRBV subfamily-specific primers or with the
CDR3-specific primers, the BV2+ and the BV8S3+
T cells could not be detected in the original blood and spleen, with a
sensitivity of 1% for BV2 and 0.01% for BV8. Using a nested PCR with
the TCRBV-specific primers and the C primer in the first step and
the CDR3 specific primers in the second step, only the BV2+
T cells from 1F2 could be detected in the original spleen and blood
(data not shown).
View larger version (49K):
[in this window]
[in a new window]
| Fig 3.
PCR analysis of the TCRBV expression using BV-specific
primers showed that in both T-cell lines BV2+ and
BV8S3+ T-cell clones were present. By limiting dilution,
two different types of BV2+ T-cell clones 4F10 and 1F2,
and one type of BV8S3+ T-cell clone were obtained. PCR
mix A contained primers for TCR BV2, BV6, BV17, BV18, and BV21; mix B
contained primers for TCR BV5S1, BV7, BV8, BV9, BV14, and BV23. Both
BV2+ T-cell clones 4F10 and 1F2 show two bands because of
the difference in running pattern of the single-stranded cDNA fragments
after denaturation.
|
|
HLA-DQ and HLA-DP restriction of recognition by BV2+
T cells and BV8S3+ T cells.
Both BV2+ T-cell clones 4F10 (4,072 ± 313 cpm) and 1F2
(4,569 ± 428 cpm), and the BV8S3+ T-cell clone (6,107 ± 651 cpm) specifically recognized the autologous HCL cells of
patient A. To determine the restriction of recognition of the HCL
cells, HLA class I and II reactive MoAbs were used for blocking
studies. The specific proliferative responses of the three T-cell
clones were completely blocked by CD4 and HLA class II MoAbs, but not
by CD8 and HLA class I MoAbs, showing class II-restricted recognition
(data not shown). The specific proliferative responses of the
BV2+ T-cell clones were blocked by HLA-DQ MoAbs and not by
HLA-DR and HLA-DP MoAbs (Fig 4A and B).
Proliferation of both BV2+ T-cell clones 4F10 and 1F2 in
response to autologous HCL cells and HCL cells of patient B with the
same HLA class II type (DR4 DQ3 DQB1*0301 DP4) was HLA-DQ restricted.
In contrast, the specific proliferative response of the
BV8S3+ T-cell clone was blocked by HLA-DP MoAbs (Fig 4C),
but not by HLA-DR and HLA-DQ MoAbs, showing that recognition of HCL
cells by BV8S3+ T cells was restricted by HLA-DP.
View larger version (32K):
[in this window]
[in a new window]
| Fig 4.
HCL-cell-specific recognition by the T-cell clones was
restricted by HLA class II. The restriction of recognition of the HCL
cells by the T-cell clones was determined by blocking studies. The HCL
cells from patient A (white bar) and B (black bar) were incubated with
saturating concentrations of pan-HLA class II (PdV5.2), HLA-DR
(B8.11.2), HLA-DQ (SPVL3), or HLA-DP (B7.21) MoAbs for 15 minutes
before the responder T cells were added. The specific proliferative
responses of the BV2+ T-cell clones 4F10 (A) and 1F2 (B)
were blocked by an HLA-DQ MoAb. The BV8S3+ T-cell clone
(C) recognized the HCL cells in an HLA-DP-restricted way.
|
|
BV2+ and BV8S3+ T-cell clones
recognize HCL cells from other patients in an HLA class II-restricted
way.
Panel studies were performed using HCL cell samples obtained from 11 other patients (B-L) with compatible or different HLA class II types
(Table 3). FACS analysis showed that all
HCL cell samples expressed high levels of HLA-DQ. Two of eight
HLA-DQ3+ HCL cell samples were DR4+ and
expressed DQB1*0301, three of eight samples were DR5+ and
expressed DQB1*0301 but a different DQ-chain, and three of eight
samples expressed another DQ-chain, such as DQB1*0302 or DQB1*0303.
The BV2+ T-cell clones 1F2 and 4F10 proliferated in
response to both HLA-DR4+ DQ3+
DQB1*0301+ HCL cell samples from patients A and B
(Fig 5A and Table 3), and did not recognize
the HLA-DR5+ DQ3+ DQB1*0301+ HCL
cell samples (E, G, and H). The HLA-DQ3+
DQB1*0302+ and DQB1*0303+ HCL cell samples (C,
D, and F), and the HLA-DQ3 HCL cell samples (I-L)
were also not recognized by both BV2+ T-cell clones.
View larger version (22K):
[in this window]
[in a new window]
View larger version (19K):
[in this window]
[in a new window]
| Fig 5.
The recognition of the HCL cells by the
BV2+ and the BV8S3+ T-cell clones was
restricted by HLA-DQA1*03/DQB1*0301 and HLA-DPB1*04, respectively.
Panel studies were performed to further determine the restriction of
recognition of the HCL cells by the T-cell clones. Proliferation of the
BV2+ T-cell clones 4F10 and 1F2 (A) and the
BV8S3+ T-cell clone (B) in response to HCL cells from
different patients are shown.
|
|
The same panel of HCL cell samples was used to study the
BV8S3+ T-cell clone. The BV8S3+ T-cell clone
proliferated in response to all nine HLA-DPB1*04+ HCL cell
samples tested (Fig 5B). HLA-DPB1*0402+ HCL cells (A, B, C,
and E) were superior to HLA-DPB1*0401+ HCL cells (D, F, G,
and J) as stimulator cells. The BV8S3+ T cells did not
recognize the HCL cells from HLA-DPB1*04 patients.
Reactivity with other hematologic malignancies.
To further determine the specificity of recognition of the T-cell
clones, blood or bone marrow samples from HLA-DR4DQ3+
(DQA1*03 DQB1*0301/0302) and DPB1*0401/0402+ healthy
controls and patients with different hematologic malignancies were used
as stimulator cells. FACS analysis showed that all donor and other
malignant cell samples used expressed HLA-DQ and HLA-DP. Both the
BV2+ and the BV8S3+ T-cell clones showed no
proliferation in response to almost all malignant cell samples from 13 patients with CLL, NHL, CML, AML, ALL, and 3 donor samples
(Fig 6). The cells from 1 patient with ALL
(B) behaved differently because the malignant cells weakly stimulated
both the BV2+ and the BV8S3+ T-cell clones,
although the ALL cells were DQB1*0302+ and
DPB1*0401+. Proliferation of the BV2+ and
BV8S3+ T-cell clones in response to the ALL cells (B) could
be blocked by HLA-DQ and HLA-DP MoAbs, respectively. The
BV8S3+ T cells also showed weak proliferation in response
to bone marrow mononuclear cells from donor A and malignant cells from
ALL patient E, which expressed DPB1*0401/0402.
View larger version (25K):
[in this window]
[in a new window]
View larger version (22K):
[in this window]
[in a new window]
| Fig 6.
Reactivity of the BV2+ and
BV8S3+ T-cell clones with malignant cells from patients
with other hematologic malignancies. Proliferation of the
BV2+ T-cell clones 4F10 and 1F2 (A) and the
BV8S3+ T-cell clone (B) in response to malignant cell
samples and normal cell samples from HLA-DR4DQ3+
DPB1*04+ patients with CLL, NHL, ALL, CML, AML, and
healthy individuals, respectively.
|
|
Characterizations of the T-cell clones 4F10, 1F2, and BV8S3.
Ten days after restimulation with autologous HCL cells, the T-cell
clones were analyzed by flow cytometry. All T-cell clones were
CD4+, CD28+, CD45RO+, and expressed
CD25. The T-cell clones did not express a typical cytokine profile of
Th1 or Th2 T cells because both BV2+ T-cell clones and the
BV8S3+ T-cell clone showed cytokine gene expression of
IL-2, IL-4, IL-5, IFN-, and IL-10.
| |
DISCUSSION |
We isolated autologous HCL-reactive T cells both from the spleen and
blood of a patient with HCL. Patients with HCL frequently show a
restricted TCRBV repertoire,8 which may partly explain the
T-cell-related immune deficiency. In addition, other factors may cause
T-cell dysfunction in vivo, such as IL-10 and tumor necrosis factor-
that are produced by the HCL cells,5,7,24-28 and lack or
impaired expression of CD28 on especially the CD4+ T
cells.29 Beside these factors, the HCL cells themselves
showed impaired expression of adhesion30 or costimulatory
molecules31 necessary for the completion of the antitumor
response.32 Therefore, we used HCL cells activated in the
CD40L system for the generation of the T-cell clones to overcome the
lack of stimulation or induction of anergy by the nonactivated HCL
cells.33-37 We generated two T-cell lines, one from the
spleen and one from the blood, showing specific proliferation in
response to autologous HCL cells. Both T-cell lines consisted of an
identical BV2+ and BV8S3+ T-cell clone.
It is remarkable that the recognition of the HCL cells was restricted
by HLA-DQ and DP, whereas DR-restricted T-cell responses are more
common. Recognition of the HCL cells by both BV2+ T-cell
clones was restricted by HLA-DQA1*03/DQB1*0301. HCL cells expressing
other DQ- or DQ-chains were not recognized. This is consistent
with the data of Kwok et al38 that
DQA1*0301/DQB1*0301 and DQA1*0501/DQB1*0301 dimers show different
specificities for the AYK peptide, a modified poly-alanine peptide. The
effect of polymorphisms of both DQ- and DQ-chains on peptide
binding is expected because both chains contribute residues for the
binding pockets of the class II molecules.39 The
BV8S3+ T-cell clone recognized all nine
HLA-DPB1*04+ HCL cell samples tested, with recognition of
DPB1*0402+ HCL cells being superior to
DPB1*0401+ HCL cells. HLA-DPB1*04 HCL
cell samples and DPB1*04+ cells from most other hematologic
malignancies were not recognized by the BV8S3+ T cells,
indicating recognition of an HCL-specific protein processed and
presented in HLA-DPw4 molecules. Both BV2+ and the
BV8S3+ T-cell clones weakly proliferated in response to ALL
patient B's malignant cells, which expressed DQB1*0302 and DPB1*0401
(Fig 6). Possibly, the ALL cells expressed an antigenic peptide similar to an HCL-associated peptide, which in combination with DQB1*0302 or
DPB1*0401 was inefficiently recognized by the TCR. The
BV8S3+ and BV2+ T cells may have given
increased proliferation in response to the cells of ALL patient B if
the cells had expressed DPB1*0402 and DQB1*0301, respectively.
It is possible that the BV2+ T cells recognize the same
antigen as the BV8S3+ T cells. Several antigenic epitopes
may be derived from one protein. Multiple class II molecules can bind
to a single large denatured or partially cleaved protein at diverse
sites,40-41 after which antigenic fragments may undergo
additional proteolysis.42 It is as yet not known if HCL
cells express proteins which may be related to the origin and
development of the disease. Mutated proteins expressed by tumor cells
can function as antigenic targets to which immune responses can be
generated. Several antigenic peptides have been associated with
autologous tumor-specific T-cell responses. These antigens include
proteins encoded by the MAGE, BAGE, or GAGE families of genes;
tissue-specific differentiation antigens; antigens encoded by genes
that are overexpressed; viral antigens; and antigens as a result of a
mutation.43-45
In other B-cell malignancies such as B-CLL, B-NHL, multiple myeloma,
and in monoclonal gammopathy of undetermined significance, oligoclonal
T-cell expansions have also been found.46-50 From blood of
a CLL patient, a proliferative T-cell clone was generated that recognized autologous B-CLL cells restricted by HLA-DR11.51 Farace et al52 observed in a patient with B-CLL a clonally
expanded BV19+ T-cell subpopulation. After expansion in
vitro, this BV19+ T-cell clone specifically recognized
autologous CLL cells as measured by production of GM-CSF and
IFN-.52 In contrast, in HCL patient A only the
BV2+ T-cell clone 1F2 could be detected in the original
spleen and blood at low levels, whereas the other two clones were
negative. This suggests that the frequency of the T-cell clones was
less than 1 out of 104 T cells in vivo. The T-cell clones
generated in vitro were not from the same TCRBV family as the T cells
clonally expanded in vivo, which expressed predominantly
BV3.20 Previous results showed that 25% of all T cells
expressed BV3, whereas only 3% to 6% expressed BV2 or
BV8.20 Possibly, the increased costimulatory capacity of
the HCL cells cultured in the CD40L system and the culture conditions
preferentially favored the expansion of the CD4+ T-cell
clones. Alternatively, the in vivo-expanded clones may have had too
limited residual capacity left to be further expanded in vitro. Thus,
the observation that different clones were isolated after in vitro
stimulation as compared with the clonal expansion in vivo does not
exclude the possibility that these clones in vivo may also recognize
the HCL cells. Dietrich et al53 already described this
discrepancy for T-cell clones from patients with renal cell carcinoma
and glioblastoma. The T-cell clones overexpressed in vivo disappeared
after restimulation with autologous tumor cells in vitro, whereas T
cells with other V-chains oligoclonally expanded.
In conclusion, autologous HLA-DQ or DP-restricted HCL-specific T-cell
clones could be generated from the spleen and blood of a patient with
HCL. These T-cell clones may be used to provide more insight into the T
cell-HCL cell interaction, especially into the inhibitory and
stimulatory factors which play a remarkable role in this disease.
Furthermore, these HCL-reactive T-cell clones can be used for the
detection of a putative HCL-specific tumor antigen.
| |
ACKNOWLEDGMENT |
We thank Dr F.H.J. Claas for the HLA-typing, and Dr F. Koning for
critically reading the manuscript.
| |
FOOTNOTES |
Submitted May 26, 1998;
accepted September 8, 1998.
Supported by the Dutch Cancer Society (Koningin Wilhelmina Fonds),
grant no. RUL 94-842.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Lisette van de Corput, PhD,
Leiden University Medical Center, Department of
Hematology, Bldg 1 C2-R, PO Box 9600, 2300 RC Leiden, The Netherlands.
| |
REFERENCES |
1.
Cawley JC, Burns GF, Hayhoe FJG:
Hairy Cell Leukemia. Berlin, Germany, Springer-Verlag, 1980, p 85.
2.
Golomb HM, Hadad LJ:
Infectious complications in 127 patients with hairy cell leukemia.
Am J Hematol
16:393, 1984[Medline]
[Order article via Infotrieve]
3.
Kluin-Nelemans JC:
Hairy cell leukemia and its T cell interactions.
Leuk Lymphoma
4:159, 1991
4.
Knight RA, Worman CP, Cawley JC:
Defective autologous and allogeneic mixed lymphocyte reactions in hairy cell leukemia.
Clin Exp Immunol
53:600, 1983[Medline]
[Order article via Infotrieve]
5.
Kluin-Nelemans JC, Kester MGD, Oving I, Cluitmans FHM, Willemze R, Falkenburg JHF:
Abnormally activated T lymphocytes in the spleen of patients with hairy cell leukemia.
Leukemia
8:2095, 1994[Medline]
[Order article via Infotrieve]
6.
Corput van de L, Kester MGD, Falkenburg JHF, Willemze R, Kluin-Nelemans JC:
TCR+ cells expressing V9V2, which normally predominate the blood, are found in the spleens of patients with hairy cell leukemia.
Leukemia
11:106, 1997
7.
Corput van de L, Falkenburg JHF, Kluin-Nelemans JC:
T cell dysfunction in hairy cell leukemia: An updated review.
Leuk Lymphoma
30:31, 1998[Medline]
[Order article via Infotrieve]
8.
Kluin-Nelemans JC, Kester MGD, Melenhorst JJ, Landegent JE, van de Corput L, Willemze R, Falkenburg JHF:
Persistent clonal excess and skewed T-cell repertoire in T cells from patients with hairy cell leukemia.
Blood
87:3795, 1996[Abstract/Free Full Text]
9.
Mueller DL, Jenkins M:
Molecular mechanisms underlying functional T cell unresponsiveness.
Curr Opin Immunol
7:375, 1995[Medline]
[Order article via Infotrieve]
10.
Schwartz RH:
Models of T cell anergy: Is there a common molecular mechanism?
J Exp Med
184:1, 1996[Free Full Text]
11.
Clark EA, Ledbetter JA:
How B and T cells talk to each other.
Nature
367:425, 1994[Medline]
[Order article via Infotrieve]
12.
Sharpe AH:
Analysis of lymphocyte costimulation in vivo using transgenic and `knock out' mice.
Curr Opin Immunol
7:389, 1995[Medline]
[Order article via Infotrieve]
13.
Planken EV, Dijkstra NH, Bakkus MH, Willemze R, Kluin-Nelemans JC:
Proliferation of precursor B-lineage acute lymphoblastic leukemia (ALL) by activating the CD40 antigen.
Br J Haematol
95:319, 1996[Medline]
[Order article via Infotrieve]
14.
Faber MF, Luxemburg-Heijs van SAP, Willemze R, Falkenburg JHF:
Generation of leukemia-reactive cytotoxic lymphocyte clones from the HLA-identical bone marrow donor of a patient with leukemia.
J Exp Med
176:1283, 1992[Abstract/Free Full Text]
15.
Uytdehaag FGCM, Loggen HG, Logtenberg T, Lichtveld RA, van Steenis B, van Asten JAAM, Osterhaus ADME:
Human peripheral blood lymphocytes from recently vaccinated individuals produce both type-specific and intertypic cross-reacting neutralizing antibody on in vitro stimulation with one type of poliovirus.
J Immunol
135:3094, 1985[Abstract]
16.
Roelen DL, Datema G, van Bree FPMJ, Zhang L, van Rood JJ, Claas FHJ:
Evidence that antibody formation against a certain HLA alloantigen is associated not with a quantitative but with a qualitative change in the cytotoxic T cells recognizing the same antigen.
Transplantation
53:899, 1992[Medline]
[Order article via Infotrieve]
17.
Barnstable CJ, Bodmer WF, Brown G, Galfre GG, Milstein C, Williams AF, Ziegler A:
Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis.
Cell
14:9, 1978[Medline]
[Order article via Infotrieve]
18.
Koning F, Schreuder I, Bontrop R, de Vries P, Giphart M, Termijtelen A, Bruning H:
A monoclonal antibody with MT2 specifity.
Dis Markers
2:75, 1984
19.
Terstappen LWMM, Shah VO, Conrad MP, Recktenwald D, Loken MR:
Discriminating between damaged and intact cells in fixed flow cytometric samples.
Cytometry
9:477, 1988[Medline]
[Order article via Infotrieve]
20.
Kluin-Nelemans JC, Kester MGD, van de Corput L, Boor PPC, Landegent JE, van Dongen JJM, Willemze R, Falkenburg JHF:
Correction of abnormal T cell receptor repertoire during interferon- therapy in patients with hairy cell leukemia.
Blood
91:4224, 1998[Abstract/Free Full Text]
21.
Cluitmans FHM, Esendam BHJ, Landegent JE, Willemze R, Falkenburg JHF:
Regulatory effects of T cell lymphokines on cytokine gene expression in monocytes.
Lymphokine Cytokine Res
12:457, 1993[Medline]
[Order article via Infotrieve]
22.
Hawes GE, Struyk L, Elsen van den PJ:
Differential usage of T cell receptor V gene segments in CD4+ and CD8+ subsets of T lymphocytes in monozygotic twins.
J Immunol
150:2033, 1993[Abstract]
23.
Struyk L, Kurnick JT, Hawes GE, Laar van JM, Schipper R, Oksenberg JR, Steinman L, de Vries RR, Breedveld FC, Elsen van den P:
T cell receptor V-gene usage in synovial fluid lymphocytes of patients with chronic arthritis.
Hum Immunol
37:237, 1993[Medline]
[Order article via Infotrieve]
24.
Lindemann A, Ludwig WD, Oster W, Mertelsmann R, Hermann F:
High-level secretion of tumor necrosis factor-alpha contributes to hematopoietic failure in hairy cell leukemia.
Blood
73:880, 1989[Abstract/Free Full Text]
25.
Buck C, Digel W, Schoniger W, Stefanic M, Rachavachar A, Porzsolt F:
Tumor necrosis factor and hairy cell leukemia.
Lancet
2:402, 1988[Medline]
[Order article via Infotrieve]
26.
Waal Malefyt de R, Yssel H, de Vries JE:
Direct effects of IL-10 on subsets of human CD4+ T cell clones and resting T cells. Specific inhibition of IL-2 production and proliferation.
J Immunol
150:4754, 1993[Abstract]
27.
Taga K, Mostowski H, Tosato G:
Human interleukin-10 can directly inhibit T cell growth.
Blood
81:2964, 1993[Abstract/Free Full Text]
28.
Groux H, Bigler M, de Vries JE, Roncarolo MG:
Interleukin-10 induces a long-term antigen-specific anergic state in human CD4+ T cells.
J Exp Med
184:19, 1996[Abstract/Free Full Text]
29.
Corput van de L, Kester MGD, Falkenburg JHF, Willemze R, Kluin-Nelemans JC:
Defective alloresponse of CD4+CD28- T cells in hairy cell leukemia.Keystone Symposia, Cellular Immunology and the Immunotherapy of Cancer-III, Copper Mountain, CO, 1997 (abstr)
30.
Jansen JH, Harst van der D, Wientjens GHM, Kooy-Winkelaar YMC, Brand A, Willemze R, Kluin-Nelemans JC:
Induction of CD11a/leukocyte function antigen-1 and CD54/intercellular adhesion molecule-1 on hairy cell leukemia cells is accompanied by enhanced susceptibilty to T-cell but not lymphokine-activated killer-cell cytotoxicity.
Blood
80:478, 1992[Abstract/Free Full Text]
31.
Trentin L, Zambello R, Sancetta R, Facco M, Cerutti A, Perin A, Siviero M, Basso U, Bortolin M, Agostini C, Semenzato G:
B lymphocytes from patients with chronic lymphoproliferative disorders are equipped with different costimulatory molecules.
Cancer Res
57:4940, 1997[Abstract/Free Full Text]
32.
Cardoso AA, Schulze JL, Boussiotis VA, Freeman GJ, Seamon MJ, Laszlo S, Billet A, Sallan SE, Gribben JG, Nadler LM:
Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy to alloantigen.
Blood
88:41, 1996[Abstract/Free Full Text]
33.
Cardoso AA, Seamon MJ, Afonso HM, Ghia P, Boussiotis VA, Freeman GJ, Gribben JG, Sallan SE, Nadler LM:
Ex vivo generation of human anti-pre-B-leukemia-specific autologous cytolytic T cells.
Blood
90:549, 1997[Abstract/Free Full Text]
34.
Yellin MJ, Sinning J, Covey LR, Sherman W, Lee JJ, Glickman-Nir E, Sippel KC, Rogers J, Cleary AM, Parker M, Chess L, Lederman S:
T lymphocyte T cell-B cell-activating molecule/CD40-L molecules induce normal B cells or chronic lymphocytic leukemia B cells to express CD80 (B7/BB-1) and enhance their costimulatory activity.
J Immunol
153:666, 1994[Abstract]
35.
Ranheim EA, Kipps TJ:
Activated T cells induce expression of B7/BB1 on normal or leukemic B cells through a CD40-dependent signal.
J Exp Med
177:925, 1993[Abstract/Free Full Text]
36.
Kooten van C, Banchereau J:
Functions of CD40 on B cells, dendritic cells and other cells.
Curr Opin Immunol
9:330, 1997[Medline]
[Order article via Infotrieve]
37.
Van Den Hove LE, Van Gool SW, Vandenberghe P, Bakkus M, Thielemans K, Boogaerts MA, Ceuppens JL:
CD40 triggering of chronic lymphocytic leukemia B cells results in efficient alloantigen presentation and cytotoxic T lymphocyte induction by up-regulation of CD80 and CD86 costimulatory molecules.
Leukemia
11:572, 1997[Medline]
[Order article via Infotrieve]
38.
Kwok WW, Nepom GT, Raymond FC:
HLA-DQ polymorphisms are highly selective for peptide binding interactions.
J Immunol
155:2468, 1995[Abstract]
39.
Kwok WW, Domeier ME, Johnson ML, Nepom GT, Koelle DM:
HLA-DQB1 codon 57 is critical for peptide binding and recognition.
J Exp Med
183:1253, 1996[Abstract/Free Full Text]
40.
Castellino F, Zhong G, Germain RN:
Antigen Presentation by MHC class II molecules: Invariant chain function, protein trafficking, and the molecular basis of diverse determinant capture.
Hum Immunol
54:159, 1997[Medline]
[Order article via Infotrieve]
41.
Corradin G, Demotz S:
Peptide-MHC complexes assembled following multiple pathways: An opportunity for the design of vaccines and therapeutic molecules.
Hum Immunol
54:137, 1997[Medline]
[Order article via Infotrieve]
42.
Nelson CA, Vidavsky I, Viner NJ, Gross ML, Unanue ER:
Amino-terminal trimming of peptides for presentation on major histocompatibility complex class II molecules.
Proc Natl Acad Sci USA
94:628, 1997[Abstract/Free Full Text]
43.
Guilloux Y, Lucas S, Brichard VG, Van Pel A, Viret C, De Plaen E, Brasseur F, Lethé B, Jotereau F, Boon T:
A peptide recognized by human cytolytic T lymphocytes on HLA-A2 melanomas is encoded by an intron sequence of the N-acetylglucosaminyltransferase V gene.
J Exp Med
183:1173, 1996[Abstract/Free Full Text]
44.
Boon T, Coulie PG, Van de Eynde B:
Tumor antigens recognized by T cells.
Immunol Today
18:267, 1997[Medline]
[Order article via Infotrieve]
45.
Boon T, Bruggen van der P:
Human tumor antigens recognized by T lymphocytes.
J Exp Med
183:725, 1996[Free Full Text]
46.
Serrano D, Monteiro J, Allen SL, Kolitz J, Schulman P, Lichtman SM, Buchbinder A, Vinciguerra VP, Chiorazzi N, Gregersen PK:
Clonal expansion within the CD4+CD57+ and CD8+CD57+ T cell subsets in chronic lymphocytic leukemia.
J Immunol
158:1482, 1997[Abstract]
47.
Wen T, Mellstedt H, Jondal M:
Presence of clonal T cell populations in chronic B lymphocytic leukemia and smoldering myeloma.
J Exp Med
171:659, 1990[Abstract/Free Full Text]
48.
Shi I, Bonnefoix T, Heuzé-Le Vacon F, Jacob M-C, Leroux D, Gressin R, Sotto M-F, Chaffanjon P, Bensa J-C, Sotto J-J:
Autotumour reactive T-cell clones among tumour-infiltrating T lymphocytes in B-cell non-Hodgkin's lymphomas.
Br J Haematol
90:837, 1995[Medline]
[Order article via Infotrieve]
49.
Moss P, Gillespie G, Frodsham P, Bell J, Reyburn H:
Clonal populations of CD4+ and CD8+ T cells in patients with multiple myeloma and paraproteinemia.
Blood
87:3297, 1996[Abstract/Free Full Text]
50.
Halapi E, Werner Å, Wahlström J, Österborg A, Jeddi-Tehrani M, Yi Q, Janson CH, Wigzell H, Grunewald J, Mellstedt H:
T cell repertoire in patients with multiple myeloma and monoclonal gammopathy of undetermined significance: Clonal CD8+ T cell expansions are found preferentially in patients with low tumor burden.
Eur J Immunol
27:2245, 1997[Medline]
[Order article via Infotrieve]
51.
Sherman W, Liu Z, Inghirami G, Reed EF, Harris FPE, Suciu-Foca NM:
Major histocompatibility complex-restricted recognition of autologous chronic lymphocytic leukemia by tumor-specifc T cells.
Immunol Res
12:338, 1993[Medline]
[Order article via Infotrieve]
52.
Farace F, Orlanducci F, Dietrich P-Y, Gaudin C, Angevin E, Courtier M-H, Bayle C, Hercend T, Triebel F:
T cell repertoire in patients with B chronic lymphocytic leukemia: Evidence for multiple in vivo T cell clonal expansions.
J Immunol
153:4281, 1994[Abstract]
53.
Dietrich P-Y, Walker PR, Schnuriger V, Saas P, Perrin G, Guillard M, Gaudin C, Caignard A:
TCR analysis reveals significant repertoire selection during in vitro lymphocyte culture.
Int Immunol
9:1073, 1997[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
M. Hoogendoorn, J. Olde Wolbers, W. M. Smit, M. R. Schaafsma, I. Jedema, R. M.Y. Barge, R. Willemze, and J.H. F. Falkenburg
Primary Allogeneic T-Cell Responses against Mantle Cell Lymphoma Antigen-Presenting Cells for Adoptive Immunotherapy after Stem Cell Transplantation
Clin. Cancer Res.,
July 15, 2005;
11(14):
5310 - 5318.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. H. Slager, C. E. van der Minne, M. Kruse, D. D. Krueger, M. Griffioen, and S. Osanto
Identification of Multiple HLA-DR-Restricted Epitopes of the Tumor-Associated Antigen CAMEL by CD4+ Th1/Th2 Lymphocytes
J. Immunol.,
April 15, 2004;
172(8):
5095 - 5102.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M.-R. Rezvany, M. Jeddi-Tehrani, H. Wigzell, A. Osterborg, and H. Mellstedt
Leukemia-associated monoclonal and oligoclonal TCR-BV use in patients with B-cell chronic lymphocytic leukemia
Blood,
February 1, 2003;
101(3):
1063 - 1070.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. H. Kessler, N. J. Beekman, S. A. Bres-Vloemans, P. Verdijk, P. A. van Veelen, A. M. Kloosterman-Joosten, D. C.J. Vissers, G. J.A. ten Bosch, M. G.D. Kester, A. Sijts, et al.
Efficient Identification of Novel Hla-A*0201-Presented Cytotoxic T Lymphocyte Epitopes in the Widely Expressed Tumor Antigen Prame by Proteasome-Mediated Digestion Analysis
J. Exp. Med.,
January 1, 2001;
193(1):
73 - 88.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction