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NEOPLASIA
From the Institute of Haematology, Royal Prince Alfred
Hospital, Camperdown, New South Wales, Australia; Centenary Institute
of Cancer Medicine and Cell Biology, Newtown, New South Wales,
Australia; and the Department of Clinical Chemistry, Turku University
Central Hospital, Turku, Finland.
The occurrence of clonal T cells in multiple myeloma (MM), as
defined by the presence of rearrangements in the T-cell receptor (TCR)- Expanded populations of T cells, as measured by an
increase in the number of cells positively stained by monoclonal
antibodies to variable (V) regions of human T-cell receptor (TCR)
subfamilies, have been detected in the peripheral blood of patients
with multiple myeloma (MM) and smouldering myeloma.1-5
These populations consist mainly of CD8+ cells and persist
for long periods, suggesting that they are the result of chronic
antigenic stimulation.5,6 In contrast, transient antigenic
stimulation is associated with temporary CD8+ T-cell
expansions.7 Although persistent T-cell expansions in MM
may involve up to 25% of total T cells,5 such expansions may not necessarily be clonal. On the basis of Southern blot analysis of the TCR Flow cytometric analysis offers the means to isolate and characterize
such clones in terms of specificity and function. Previous flow
analysis indicated that the expanded T-cell clones, as judged by
specific positivity with anti-TCRV The most sensitive way to demonstrate clonality is by analysis of the
length of the complementarity-determining region 3 (CDR3) of the
TCRV genes, followed by
sequencing.18,19 In the present study, we have used this
technique to demonstrate the genuine monoclonal or oligoclonal nature
of the expanded populations of CD57+CD8+TCRV Patients
Monoclonal antibodies
Cell surface staining and flow sorting Ficoll-separated cells were stained with biotinylated anti-CD8 and incubated on ice for 20 minutes. After 2 washes with phosphate-buffered saline (PBS), a cocktail of FITC-conjugated anti-CD57, PE-conjugated anti-TCRV , and streptavidin
conjugated to Alexa Fluor-594 was added, and the cells were
incubated for a further 20 minutes on ice. Stained cells were washed
once with PBS and remained on ice until sorting by means of a FACStar
Plus Cytometer (Becton Dickinson).
CD57+TCRV+CD8+ and
CD57 TCRV+CD8+
cells were sorted into 2 separate tubes containing RPMI-1640 containing
10% heat-inactivated fetal calf serum (ICN, Costa Mesa, CA), 1 mM
L-glutamine, 100 IU/mL penicillin, and 160 U/mL (160 µg/mL)
gentamicin. The tubes were kept cool during the sort.
CDR3 length analysis Six patients (A through F) with expanded TCRV
subsets were chosen for CDR3 length analysis. They were selected
because their T-cell expansions could be labeled at high fluorescent
intensity with PE-conjugated anti-TCRV antibodies and
could therefore be sorted accurately. The CDR3 length analysis has also
been carried out on 6 age-matched controls. The QuickPrep Micro mRNA
Purification Kit (Amersham Pharmacia Biotech, Piscataway, NJ) with
oligo-deoxythymidine (dT)-cellulose was used to obtain messenger RNA
(mRNA) from approximately 103 to 104 sorted
cells according to the manufacturer's protocol. Complementary DNA
(cDNA) was subsequently prepared by means of the T-primed First-Strand
Kit (Amersham), which uses Moloney murine leukemia virus reverse
transcriptase and an oligo-dT primer to generate full-length
first-strand cDNA from the mRNA template. The cDNA was stored at
 70°C until in vitro amplification of TCR genes was performed with a
5' primer specific for the appropriate TCR subfamily19 and a 32P-labeled 3' primer for
the TCR constant region, which was common to all
rearranged  -chain genes.19 The reverse primer was
radioactively end-labeled in a solution consisting of 27 MBq (0.73 mCi)/mL  32P]adenosine 5'-triphosphate (ATP)
(Amersham), and 2.3 µM T4 polynucleotide kinase
(9.6 × 102 U/mL) (Boehringer Mannheim, Mannheim,
Germany). The polymerase chain reaction (PCR) reaction mix also
contained the following components at the final concentrations listed
here: 2.3 µM forward primer; 0.38 µM deoxy-ATP (dATP),
d-cytidine 5'-triphosphate, d-ribothymidine 5'-triphosphate, and
d-guanosine 5'-triphosphate (Boehringer Mannheim); a 1 ×
PCR-reaction buffer with either 1.5 mM or 3 mM MgCl2
(Boehringer Mannheim); and 0.01 U Taq polymerase (Boehringer Mannheim).
We added 5 µL PCR reaction mix to each well of a Thermo-fast
96-well plate (Advanced Biotechnologies, Epsom, Surrey, United Kingdom)
containing 5 µL cDNA. The mixture was overlaid with 25 µL mineral
oil (ICN). PCR amplification was performed in an Omni-e thermal cycler
(Hybaid, Ashford, United Kingdom) by means of the following
thermocycling protocol: initial denaturation at 95°C for 5 minutes;
followed by 45 cycles of 95°C for 30 seconds, 55°C for 30 seconds,
and 72°C for 1 minute; with a final extension at 72°C for 7 minutes. Polyacrylamide gel electrophoresis was used to distinguish PCR
products, which had been diluted 2-fold with sequencing gel-loading
buffer, denatured for 5 minutes at 95°C in an Omni-e thermal cycler,
and snap-chilled on ice. To allow an estimation of PCR bands sizes,
32P]ATP-labeled molecular weight markers (Number VIII)
(Boehringer Mannheim) were electrophoresed on the same gel. The PCR
products were electrophoresed at 900 V for approximately 3 hours.
Following electrophoresis, gels were transferred to 3-mm
chromatography paper (Whatman International, Maidstone, United
Kingdom), covered in plastic wrap (Huntsman, Castle
Hill, Australia), and directly exposed to X-OMAT AR scientific imaging
film (Eastman Kodak, Rochester, NY) overnight at 70°C, by means of
intensifying screens (DuPont NEN, Denver, CO). Film was developed in a
Curix 60 film processor (Agfa-Gevaert, Munich, Germany).
Sequencing of PCR products of specific TCRV
subfamilies for DNA sequencing, PCR was performed with the use of cDNA
samples with Ready-to-go PCR beads (Amersham). The PCR amplification
conditions were similar to those described above except that no
radioactive label was used. Samples were taken from 3 patients (A, B,
and C) who had been previously shown to have single, dual, and multiple dominant bands, respectively, on polyacrylamide gel electrophoresis. Direct sequencing of the PCR products was carried out by Supermac (Camperdown, Australia). Where direct sequencing failed, the
Original TA Cloning Kit (Invitrogen, Carlsbad, CA) was used to clone
the PCR products in plasmid vectors according to the manufacturer's protocol. The linearized vector supplied in this kit ends with single
3'deoxythymidine (dT) residue that allows PCR inserts to ligate
efficiently with the vector, since the nontemplate-dependent activity
of Taq polymerase adds a single deoxyadenosine (dA) to the 3' ends of
PCR products. A few isolated white transformants were picked
individually and allowed to grow for 12 hours at 37°C in 5 mL
Luria-Bertani medium containing 50 µg/mL ampicillin in a rotary
shaking incubator at 225 rpm. The plasmid DNA was then purified with a
NucleoSpin kit (Macherey-Nagel, Düren, Germany). DNA sequencing
employing dye-terminator chemistry with M13 forward and reverse primers
was performed on the extracted plasmid DNA. PCR products from
TCRV age-matched controls were also direct sequenced (Supermac).
Estimation of T-cell proliferative index Ficoll-separated cells from patients A to F were first labeled with nonconjugated antibodies specific for the TCRV
subfamily known to be expanded in each individual. After a 20-minute
incubation on ice, cells were washed and stained for a further 20 minutes with biotinylated antimouse immunoglobulin (Dako, Carpinteria, CA), except for anti-TCRV1 for which a biotinylated
rabbit anti-rat immunoglobulin (Dako) was used. Cells were then washed and stained with streptavidin conjugated to Alexa-488 (Molecular Probes). Alternatively, to determine the proliferative index in total
CD8 and CD4 populations in MM patients (no. = 8; patient A and
patients J through P) and normal controls (no. = 4; healthy laboratory staff), cells were labeled initially with biotinylated anti-CD8 or anti-CD4 (Pharmingen), followed by streptavidin conjugated to Alexa-488, and then washed with PBS. The supernatant was removed and
the pellet resuspended. While the cells were gently vortexed, 100 µL
DNA-Prep lysing and permeabilizing reagent (Beckman Coulter, Fullerton,
CA) and then 2 mL DNA-Prep Stain (Beckman Coulter) was added,
and the sample was incubated for 15 minutes at room temperature. Cells
were analyzed on a Coulter Epics XL flow cytometer. Multicycle for
Windows (Phoenix Flow Systems, San Diego, CA) was used to analyze the
DNA histograms.
CD95 surface staining Ficoll-separated cells from 12 MM patients (patients A through I and N through P) were examined for expression of CD95 on total CD3+ T cells or TCRV subsets (both expanded
and nonexpanded) by means of 3-color labeling. An aliquot of cells was
stained with biotinylated anti-CD8 to allow the percentage of CD8 or
CD4 cells within each TCRV subset to be calculated
(Table 2). Propidium iodide (PI) was
added to all samples before flow analysis. Cells were analyzed on a
Coulter Epics XL flow cytometer. Normally, more than 99% of the cells
in the lymphocyte gate were viable as assessed by PI. Biotinylated
mouse IgG1 was used as the isotype control. For a 4-color labeling
experiment, 5 MM patients (A through E) were examined for expression of
CD95 on total CD3+ T cells and different
TCRV subsets. For 4-color labeling, Ficoll-separated
cells were stained with biotinylated anti-CD95 (Pharmingen), which was
followed by a cocktail of FITC-conjugated anti-CD57, PE-conjugated
anti-TCRV or anti-CD3, and streptavidin conjugated to
Alexa-594 (Molecular Probes) and APC-conjugated anti-CD4 (Pharmingen).
Stained cells were washed once and then fixed in 1% paraformaldehyde
for at least 30 minutes before analysis by means of a FACStar Plus
cytometer.
Overrepresentation of CD8+ cells within
TCRV + T-cell populations exceeding the
mean + 3 SD of the percentage of V+
cells in peripheral blood T cells of normal controls, were found in
79% of patients with MM (no. = 38; mean age = 62 years), but in
only 19% of normal controls (no. = 17; mean age = 45
years).5 In the present study, we included an age-matched
control group (no. = 27; mean age = 66 years) and found that 63%
(17 of 27 subjects) had at least one T-cell expansion, consistent with
the normal age-dependent increase in T-cell expansions.20
Since the T-cell expansions in MM patients had previously been shown to
express predominantly CD85 whereas those in the normal
control group of younger age did not (data not shown), we decided to
make a more detailed comparison of the frequency and phenotype of
T-cell expansions in patients with MM versus the age-matched controls.
Data from the 27 age-matched controls in this study and those MM
patients from the previous group5 for whom
V analysis had included CD8 status (22 of the 38) were
reanalyzed after gating for either CD8+CD3+ or
CD8CD3+cells (Figure
1). Given the small percentage of
circulating CD4+CD8+ and
CD4CD8 T cells in human blood,
CD8CD3+ cells were composed predominantly of
CD4+ T cells. The
CD8+CD3+TCRV+ and
CD8CD3+TCRV+
percentages were calculated as a percentage either of total
CD3+ cells or of CD8+CD3+ or
CD8CD3+ T cells, respectively. The latter
calculation was included to correct for the low CD4+ T-cell
counts that are characteristic of many patients with MM and that may
mask any CD8 expansions. TCRV+
expansions were defined as those populations exceeding the mean + 3 SD of the percentage in the normal controls.
The new analysis indicated that CD8
CDR3 length analysis suggests that the CD57+ subset of expanded cytotoxic T cells is monoclonal or oligoclonal We have previously shown that the phenotype of the expanded V populations in MM was predominantly CD57+
and CD28, which differs significantly from the
nonexpanded V populations.5 The reactivity
of T cells to specific anti-TCRV antibodies can
represent either a true clonal expansion or a polyclonal response. Therefore, the clonality of sorted
CD57+CD8+TCRV+ T
cells from either MM patients or age-matched controls was compared with
that of
CD57CD8+TCRV+ T
cells by means of TCRV CDR3 length analysis and DNA
sequencing of the variable region of the TCR. Cells were sorted
by their CD57 phenotype because we have recently shown that expanded
cytotoxic T cells contain intracytoplasmic perforin, and its expression is directly associated with the expression of CD57 in expanded V populations.5
PCR was performed with cDNA prepared from the sorted cells, a 5'
primer specific for the appropriate TCRV
Direct DNA sequencing and plasmid cloning confirmed the monoclonality or biclonality of the CD57+ expanded T-cell population TCRV CDR3 PCR products of patients A
(V1+), B (V1+),
and C (V14+) were sequenced to ensure that
the distribution of bands observed was a true reflection of the
clonality of the V+ expansions. These
patients were chosen because they had previously been shown to have
single, dual, and multiple dominant bands, respectively, in CDR3 length
analysis. In every case, PCR products that appeared as a single band
gave unambiguous direct DNA sequencing data (Figure 2A, lanes 1 and 2;
Figure 3A, Gel-A1, A2; Figure 2A, lane 9;
Figure 3C, Gel-C), indicative of a dominant sequence within the PCR
product. On the other hand, none of the
TCRV+CD8+CD57 PCR
products yielded readable sequence data except for patient A, whose
CD57 PCR product indicated a monoclonal population
(Figure 2A, lane 2; Figure 3A, Gel-A2).
Plasmid cloning and sequencing of the cDNA were performed on the PCR
products of the 2 patients (B, C) in whom direct sequencing failed to
provide interpretable sequencing results of the CDR3 regions in
TCRV The monoclonality of the CD57+ subsets of the expanded
CD8+TCRV The majority of the expanded T cells in MM patients are not proliferating We have previously shown that the expanded T-cell clones of patients with MM remain stable over an 18-month period.5 To examine the level of turnover in the expanded T-cell populations, we examined the proliferative index of T cells from the expanded TCRV subsets of patients with MM by measuring the
proportion of cells in the S phase by means of a protocol previously
established to measure cycling plasma cells in our
laboratory.21 The proliferative index of the
TCRV expanded cells from 6 MM patients (patients A
through F) was compared with that of total CD8+ and
CD4+ T cells in MM (no. = 8, patients A, J through P) and
normal controls (no. = 4) (Figure 4).
Only 0.1% to 1.8% of the TCRV expanded cells of MM
patients were in the S phase, a percentage that did not differ
significantly from the CD8+ and CD4+ T cells of
MM patients or normal controls examined (ANOVA, P = .84).
On the other hand, the TCRV expanded T cells and total
CD8+ T cells of these MM patients retained the capacity to
proliferate in a dose-related fashion in response to polyclonal stimuli
such as PHA or concanavalin A. The percentage of cells in S phase
increased within 24 hours of 15 µg/mL PHA stimulation from 1.6% to
11.1% in a dose-related fashion (Figure 4, far right column).
CD57+ T cells express CD95 at lower levels than
their CD57
populations was approximately 2-fold lower than that of their
CD57 counterparts (Figure
5A; Table 2). Because CD95 expressed by the CD8+ subset of the
TCRV+CD57 cells could not be
definitively determined by a 3-color staining protocol, it was unclear
whether the increase in the percentage of CD8+ cells within
particular TCRV populations was responsible for the
decrease in CD95 expression (eg, Table 2, V1 of patient
A and V7 of patient B). To unambiguously examine the
expression of CD95 on expanded CD57+ subsets, 4-color
labeling was used so that the CD95 levels in each of the 4 subsets
(namely, CD4+CD57,
CD4+CD57+, CD8+CD57,
and CD8+CD57+) could be determined. It was
found that there was a 30% reduction of CD95 expression in
CD8+CD57+ cells compared with their
CD8+CD57 counterparts (Figure 5B, column 3 versus 4; P < .0001, Mann-Whitney test). The reduction in
CD95 expression within the CD4+ compartment was even more
marked (Figure 5B, column 1 versus 2; P < .0001,
Mann-Whitney test). This suggests that although CD95 (Fas) may be
involved in the accumulation of CD57+ T cells, an
additional factor, such as chronic antigenic stimulation, could be more
important in the determination of the fate of these cells. The median
CD95 levels in these 4 subsets from patients with MM were similar to
that of the age-matched normal controls (Figure 5C). The number of
CD4+CD57+ cells was low in the age-matched
controls, and in some cases the number was too low to include in the
subset analysis.
There is currently considerable interest in the development of
immunotherapy as a novel treatment option for patients with myeloma and
other malignancies. In myeloma, the unique tumor antigen, the idiotype,
has been adopted as the immunizing agent. The potential value of
immunotherapy in MM is based on a dual rationale. First, it has been
postulated that plateau-phase disease may have an immunoregulatory
basis,22 and second, our group has recently found that
expanded T-cell clones with a cytotoxic phenotype (CD8+,
CD57+, CD28 In the present study, flow cytometric analysis of TCR V In the CDR length analysis study, the extracted mRNA encodes those
TCR Using the Southern blot technique, we previously reported expanded
clonal T cells in 32% of MM patients (no. = 119).9 This is a lower percentage than in the cohort of MM patients in this study,
in which T-cell expansions composing more than 5% of total CD3+ cells were seen in 59% of patients. This discrepancy
could be due to a number of factors: (1) the sensitivity of the
Southern blot technique previously used may have been lower than the
5% limit estimated by van Dongen and Wolvers-Tettero8;
(2) total T-cell counts of patients may have differed in the 2 study
times; and (3) the presence of more than one TCR within the T-cell
expansions, due either to biclonality within the CD57+
subset (as demonstrated in one third of MM patients in this study) or
polyclonality within the CD57 The CD57+ clonal T cells had a low rate of turnover, as
demonstrated by S-phase analysis, and expressed relatively lower levels of the apoptotic marker CD95 than their CD57 After prolonged stimulation and proliferation,
CD8+CD28+ cells tend to lose expression of
CD28.33 Thus, the previous demonstration by our group that
the expanded TCRV In our previous study,5 we showed that the expression of
perforin was directly associated with that of CD57 in the expanded TCRV The identity of possible tumor-associated antigens in myeloma is still unknown. Circulating idiotype remains an attractive candidate owing to its persistence throughout the course of the disease and the fact that it represents a unique tumor antigen. Idiotype is a possible antigen, and anti-idiotypic peptide responses that lead to tumor protection have been demonstrated in artificial transgenic models,40-46 but evidence of anti-idiotype CD4+-dependent or CD8+-dependent responses in MM remains controversial.24-29 Idiotype-specific cytotoxic T lymphocytes in MM capable of lysing autologous primary tumor cells have been recently reported.47 Having shown that monoclonality or oligoclonality is a property of the CD57+CD8+ T-cell subset, we aim to identify whether these CD8+ expanded T-cell clones are really tumor-specific and, if so, which specific tumor antigen they recognize. Some plasma cell-specific antigens, such as HM1.2448 and CD138 (syndecan-1),49,50 have been previously identified and may be potential targets for immunotherapy. Knowledge of the specificity of the CD57+CD8+ T-cell clones may also allow us to design immunization regimens for MM patients who have failed to make spontaneous antitumor responses.
We wish to thank Joseph Webster for expert technical assistance in the flow-sorting work on FACStar Plus and Dr Robert Brink for his kind gift of the Original TA Cloning Kit.
Submitted August 17, 2000; accepted June 26, 2001.
Supported by the Foundation IV fund and the International Myeloma Foundation (D.M.-Y.S.) and the Academy of Finland (M.R.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Daniel M.-Y. Sze, Institute of Haematology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia 2050; e-mail: d.sze{at}centenary.usyd.edu.au.
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© 2001 by The American Society of Hematology.
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Y. Le Priol, D. Puthier, C. Lecureuil, C. Combadiere, P. Debre, C. Nguyen, and B. Combadiere High Cytotoxic and Specific Migratory Potencies of Senescent CD8+CD57+ Cells in HIV-Infected and Uninfected Individuals J. Immunol., October 15, 2006; 177(8): 5145 - 5154. [Abstract] [Full Text] [PDF]
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C. C. Ibegbu, Y.-X. Xu, W. Harris, D. Maggio, J. D. Miller, and A. P. Kourtis Expression of Killer Cell Lectin-Like Receptor G1 on Antigen-Specific Human CD8+ T Lymphocytes during Active, Latent, and Resolved Infection and its Relation with CD57 J. Immunol., May 15, 2005; 174(10): 6088 - 6094. [Abstract] [Full Text] [PDF]
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J. M. Brenchley, N. J. Karandikar, M. R. Betts, D. R. Ambrozak, B. J. Hill, L. E. Crotty, J. P. Casazza, J. Kuruppu, S. A. Migueles, M. Connors, et al. Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8+ T cells Blood, April 1, 2003; 101(7): 2711 - 2720. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
2 = 1.85, P > .1), and the
number was unaffected by being calculated as a percentage of
CD8
