|
|
 Previous Article | Table of Contents | Next Article 
Blood, 1 November 2001, Vol. 98, No. 9, pp. 2817-2827
NEOPLASIA
Clonal cytotoxic T cells are expanded in myeloma and reside in
the CD8+CD57+CD28 compartment
Daniel M.-Y. Sze,
Gillian Giesajtis,
Ross D. Brown,
Maria Raitakari,
John Gibson,
Joy Ho,
Alan G. Baxter,
Barbara Fazekas de
St Groth,
Antony Basten, and
Douglas E. Joshua
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.
 |
Abstract |
The occurrence of clonal T cells in multiple myeloma (MM), as
defined by the presence of rearrangements in the T-cell receptor (TCR)- chains detected on Southern blotting, is associated with an
improved prognosis. Recently, with the use of specific
anti-TCR-variable- (anti-TCRV ) antibodies, the
presence in MM patients of expanded populations of T cells expressing
particular V regions was reported. The majority
of these T-cell expansions have the phenotype of cytotoxic T cells
(CD8+CD57+ and perforin positive). Since
V expansions can result from either a true clonal
population or a polyclonal response, the clonality of
CD8+TCRV+ T cells was tested by
TCRV complementarity-determining region 3 length
analysis and DNA sequencing of the variable region of the TCR. In this
report, the CD57+ and CD57 subpopulations
within expanded TCRV+CD8+ cell
populations are compared, and it is demonstrated that the CD57+ subpopulations are generally monoclonal or biclonal,
whereas the corresponding CD57 cells are frequently
polyclonal. The oligoclonality of CD57+ expanded
CD8+ T cells but not their CD57 counterparts
was also observed in age-matched controls, in which the T-cell
expansions were mainly CD8. The
CD8+CD57+ clonal T cells had a low rate of
turnover and expressed relatively lower levels of the apoptotic marker
CD95 than their CD57 counterparts. Taken together, these
findings demonstrate that MM is associated with
CD57+CD8+ T-cell clones, raising the
possibility that the expansion and accumulation of activated clonal
CD8+ T cells in MM may be the result of persistent
stimulation by tumor-associated antigens, combined with a reduced
cellular death rate secondary to reduced expression of the
apoptosis-related molecule CD95.
(Blood. 2001;98:2817-2827)
© 2001 by The American Society of Hematology.
| |
Introduction |
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, which detects monoclonal expansions representing at
least 4% to 5% of the total Ficoll-separated peripheral blood cells,8 our laboratory previously reported on the
prognostic significance of true T-cell clonal populations in
MM.9 We found that MM patients exhibiting expanded T-cell
clones had a better prognosis than those without detectable clonal
bands on Southern blot analysis.9
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 antibodies,
expressed the phenotype of cytotoxic T cells and were predominantly
CD8+, CD57+, CD28, and perforin
positive.5 However, it was not clear from this flow
analysis that the expanded populations of cytotoxic T cells corresponded to the clonal populations that related to survival advantage detected by previous Southern blot experiment.9
The CD57 antigen is normally present on a minority (16%) of
CD8+ T cells,10 but the percentage of
CD8+CD57+CD28 cells has been
shown to be increased in a number of clinical conditions, including
human immunodeficiency virus (HIV)/acquired immunodeficiency
syndrome,11,12 cytomegalovirus (CMV)
infection,13,14 common variable
immunodeficiency,15 and post-bone marrow
transplantation,16 in addition to MM.17
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+ cells
in patients with MM. Moreover, only a small proportion of
CD57+ T cells were undergoing active proliferation, and
they had significantly lower surface expression of CD95 (Fas) than did
their CD57 counterparts, in both expanded and nonexpanded
TCRV subfamilies. Taken together, these findings
demonstrated that MM, a B-cell clonal disease, is associated with
CD8+ T-cell clones, raising the possibility that the
expansion and accumulation of activated clonal CD8+ T cells
in MM may be the result of persistent stimulation by tumor-associated
antigens, combined with a reduced death rate secondary to the reduced
expression of the apoptosis-related molecule CD95.
| |
Patients, materials, and methods |
Patients
After informed consent, peripheral heparinized blood samples
were collected from 16 patients with well-documented myeloma presenting
to our clinic for routine assessment (Table
1). Patients were treated according to
the Australian Leukaemia Study Group myeloma trial protocol,
which involved multiagent therapy (prednisone, cyclophosphamide,
doxorubicin, and bis-chlornitrosourea) followed by stem cell
transplantation if the patient was younger than age 65 years. Seven of
16 patients had had chemotherapy within the previous 6 months before
testing (Table 1). Four patients had autologous stem cell
transplantation. However, none of these transplantations occurred
within 2 years prior to this study (Table 1). The study was approved by
the Central Sydney Area Health Service Human Ethics Committee. Normal
controls used in the CDR3 length analysis experiments and CD95 studies
were 27 blood donors (mean age = 66 years) at the Red Cross Blood
Transfusion Service, Sydney. The normal control samples used in the
proliferation studies were from healthy laboratory staff.
Monoclonal antibodies
The following directly conjugated mouse monoclonal antibodies
were used: anti-CD3, anti-CD4, anti-CD8, and anti-CD57, all fluorescein
isothiocyanate (FITC) labeled (Becton Dickinson, San Jose, CA);
phycoerythrin (PE)-conjugated anti-TCRV 1, 2, 3, 5.1, 5.2, 5.3, 7, 8, 9, 11, 12, 13.1, 13.6, 14, 16, 17, 18, 20, 21.3, 22, and 23 (all antibodies are mouse immunoglobulin [Ig] except
anti-TCRV1 which is a rat IgG1) (Immunotech, Marseille,
France); anti-CD3 (Becton Dickinson); and peridinin chlorophyll protein
(PerCP)-conjugated anti-CD4 and anti-CD8 (Becton Dickinson).
Unconjugated monoclonal antibodies used in the proliferation analysis
were anti-TCRV antibodies as listed above (Immunotech). Biotinylated sheep anti-mouse immunoglobulin and biotinylated rabbit anti-rat immunoglobulin antibodies were used as secondary reagents for the nonconjugated anti-TCRV antibodies. The allophycocyanin (APC)-conjugated anti-CD4 (Pharmingen, San
Diego, CA) was used. The following biotinylated mouse monoclonal
antibodies were used: anti-CD8, anti-CD95, and biotinylated mouse IgG1
isotype control (Pharmingen). Streptavidin conjugated with Alexa
Fluor-488 (Molecular Probes, Eugene, OR) was used in the proliferation
studies to increase the fluorescent signal in the FITC channel to allow the TCRV subsets to be clearly identified. Streptavidin
conjugated with Alexa Fluor-594 (Molecular Probes) was used to
demonstrate the biotinylated primary antibodies in the dual-laser system.
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
CD57TCRV+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
To obtain 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.
View this table:
[in this window]
[in a new window]
|
Table 2.
Median CD95 expression and proportion of CD8+
cells within the CD57+ and CD57 compartments
of individual TCRV subfamilies in 2 patients
|
|
| |
Results |
Overrepresentation of CD8+ cells within
TCRV expansions in MM patients
We have previously reported that T-cell expansions, defined as
those 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.
View larger version (20K):
[in this window]
[in a new window]
| Figure 1.
CD8 expression in T-cell expansions.
T-cell expansions from 27 age-matched donors were compared with 22 patients with MM. The data of patients with MM have been published
previously5 and were reanalyzed and reported again here
for comparison. Cells were gated for either
CD8CD3+ (panels A through D) or
CD8+CD3+ T cells (panels E through H) before
calculation of the percentage of cells expressing each V
as a function of either total CD3+ T cells (panels A
through B and E through F), CD8CD3+ (panels C
and D), or CD8+CD3+ (panels G and H).
Horizontal bars represent 3 SD above the means for the age-matched
control group. #The 21 V families studied (left-right)
were V1, 2, 3, 5.1, 5.2, 5.3, 7, 8, 9, 11, 12, 13.1,
13.6, 14, 16, 17, 18, 20, 21.3, 22, and 23.
|
|
The new analysis indicated that CD8 expansions were
present both in patients with MM and in age-matched controls (Table
3). The slightly reduced frequency in MM
(10 of 22 patients versus 17 of 27 controls) was not statistically
significant ( 2 = 1.85, P > .1), and the
number was unaffected by being calculated as a percentage of
CD8CD3+ cells rather than total
CD3+ cells. In contrast, reanalysis of V
family expression within CD8+ T cells indicated that
CD8+ expansions in MM patients were significantly more
frequent than in age-matched controls (Table
4). A higher percentage of MM patients
had at least one CD8+ T-cell expansion
(2 = 8.6, P < .005), and the total
number of expansions in the MM patients was also significantly higher
(2 = 26.7, P < .005). Thus, in addition
to the CD8+ expansions previously described,5
MM patients showed the CD4+ expansions expected as a
function of age. Thirteen of the 22 MM patients in this analysis (59%)
had CD8+ expansions representing at least 5% of total
CD3+ T cells, which is the estimated detection limit of the
Southern blot technique previously used to demonstrate the prognostic
significance of T-cell expansions in MM.9 In contrast, the
CD8+ T-cell expansions, which represented more than 5% of
total CD3+ T cells, were found in only 15% of
normal age-matched controls. In summary, MM patients have a greater
number of CD8+ expansions, and these constitute a
significantly greater percentage of total T cells than the age-matched
controls.
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 subfamily, and
a 32P-labeled 3' primer from the TCR
constant region. Figure 2A shows the
length of TCRV CDR3 amplified from the
CD8+CD57+ and
CD8+CD57 T cells of 6 patients (patients A
through F) with MM. A single band derived by PCR of the
CD8+TCRV+CD57+ cDNA
was obtained for 4 of the 6 patients examined (Figure 2A, lanes 1, 9, 13, and 15). One third of the patients in this study (patients B and F)
showed 2 bands of equal intensity (Figure 2A, lanes 5 and 17). In the
case of patient C, a second expanded population (V17+) (Figure 2A, lane 11) showed multiple
bands within the CD57+ subset. The CD57
sorted T cells generally contained multiple bands of nearly equal intensity (Figure 2A, lanes 4, 6, 8, 10, 16, and 18). For example, the TCRV1 and V14 amplification products
of the
CD57TCRV+CD8+
sorted T cells of patients B and C, respectively, both consisted of 5 bands each separated by 3 nucleotides (Figure 2A, lanes 6, 10).
However, in some cases, such as patient A, the CD57
sorted cells of an expanded TCRV also contained a single band (Figure 2A, lane 2). Similarly, the CD57 sorted
cells of an expanded TCRV of patient C were oligoclonal (Figure 2A, lane 12). Therefore, although CD57 T cells
were generally polyclonal, they may occasionally be oligoclonal. The
monoclonality and biclonality of the CD57+ subset of
expanded CD8+TCRV+ T cells were
also observed in age-matched controls (Figure 2B; lanes 23, 25, and 27 showed a single band and lanes 19 and 29 showed 2 equally dominant
bands).
View larger version (34K):
[in this window]
[in a new window]
| Figure 2.
TCRV CDR3 length analysis.
The PCR products (approximately 220 base pairs) and molecular markers
were run on a 6% polyacrylamide gel, and the sizes of the radioactive
PCR bands were determined by exposing an x-ray film to the gel.
Expanded T cells were sorted according to their specific surface
phenotype of TCRV+ and CD8+ and
CD57 expression. Bands are lined up side by side for ease of
comparison. The relative position of the PCR products of different
TCRV families to each other is not the same as the
actual gel. *Expanded TCRV populations are underlined.
(A) CDR3 length analysis results from CD8+ T-cell
expansions found in 6 patients with MM, together with CD8 and CD57
expression data. (B) Results of 6 age-matched normal controls for
comparison. **Direct DNA sequencing has been performed on some of the
PCR products and results are summarized.
|
|
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).
View larger version (35K):
[in this window]
[in a new window]
| Figure 3.
TCRV DNA sequences of patients A through C.
Panels A, B, and C show the sequencing results of patients A, B, and C,
respectively. DNA sequences were derived from direct sequencing of the
CD57+ PCR products from patient A (labeled Gel-A1 in panel
A) and patient C (labeled Gel-C in panel C). No readable sequence was
obtained from direct sequencing of the CD57 PCR products,
except in the case of patient A (labeled Gel-A2 in panel A). Plasmids
B-1 to B-6 are 6 independent sequences obtained from 6 cDNA clones
chosen at random from a library containing the CD57+ PCR
product from patient B. Plasmids B-7 to B-11 are the independent
sequences obtained from 5 cDNA clones from the CD57 PCR
product from the patient shown in panel B. Plasmids C-1 to C-8 are 8 independent sequences obtained from 8 cDNA clones chosen at random from
a library containing the CD57 PCR product from patient C
(panel C). The sequences summarized in this Figure have been given the
European Molecular Biology Laboratory (Heidelberg, Germany) nucleotide
sequence database accession numbers: AJ276183 through AJ276184 for
panel A, AJ276185 through AJ276195 for panel B, and AJ276196 through
AJ276204 for panel C. Keys: ... identical nucleotides; ~ ~ ~ absence of an amino acid at the relative position in the CDR3 sequence
alignment.
|
|
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+CD8+CD57+ T
cells. Cloning and sequencing of the PCR products from the CD57 sorted T cells gave an array of sequences,
indicative of a polyclonal population (Figure 3B, plasmids B-7 to B-11;
Figure 3C, plasmids C-1 to C-8). Cloning and sequencing of the
CD57+ PCR product of the expanded CD8+ T-cell
subset of patient B, which gave 2 bands of equal intensity in the CDR3
length polymorphism analysis, revealed 2 distinct sequences, each
present in 3 of the 6 randomly chosen cDNA clones (Figure 3B, plasmids
B-1 to B-6). These 2 TCRV1 clones differed by only 1 amino acid in length, consistent with the CDR3 length polymorphism
analysis (Figure 2A, lane 5). The CD57 sorted cells of
patient B revealed 4 different clones in 5 plasmid preparations, which
differed in length by 1 to 4 amino acids. Furthermore, T-cell clones
plasmids B-7 and B-8, which had the same CDR3 length, possessed unique
sequences and used a different joining- gene segment. The fact that
the dominant clones of
TCRV1+CD8+CD57+ were
not identified in the corresponding CD57 sorted
populations from patient B may have been due to the limited number of
plasmid preparations analyzed. Of the 8 independent sequences obtained
from 8 cDNA clones chosen at random from a library containing the
CD8+TCRV14+CD57
PCR product of patient C, 8 clones that differed by 1 to 4 amino acids
in length were identified (Figure 3C). Once again, the single dominant
clone identified in the CD57+ sorted cells of patient C was
not found in the CD57 subset.
The monoclonality of the CD57+ subsets of the expanded
CD8+TCRV+ T cells in age-matched
controls was shown by direct DNA sequencing of the PCR products
showing a single band (Figure 2B, lanes 23, 25, and 27) or PCR products
with a single dominant band plus other weaker bands (Figure 2B, lane
21). Neither PCR products with double bands nor multiple dominant bands
shown in polyacrylamide gel electrophoresis (Figure 2B, lanes 19, 20, 22, and 26) gave an interpretable direct DNA sequencing result,
indicating nonclonal populations.
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).
View larger version (13K):
[in this window]
[in a new window]
| Figure 4.
Proliferative index of the expanded TCRV
subsets.
The proliferative index measured by the percentage of cells within S
phase of expanded TCRV+ T cells in MM
patients (far left column) and compared with that of the total
CD8+ or total CD4+ T cells in MM patients
(second and third columns from left) and normal controls (NCs, fourth
and fifth columns from left). No significant difference was found among
the groups (one-way analysis of variance [ANOVA],
P = .84). The positive control was cultured
CD8+ cells of patient D at 24 hours after stimulation with
various amounts of phytohemagglutinin (PHA). *Far right column, shown
as squares in order from top to bottom with the following amounts of
PHA added to the 1-mL culture medium: 15 µg, 11.1%; 5 µg, 5.3%;
1.5 µg, 3.7%, and 0 µg, 1.6%.
|
|
CD57+ T cells express CD95 at lower levels than
their CD57 counterparts
We have previously noted that the proportion of T cells expressing
the apoptotic marker Fas (CD95) tends to be lower in expanded populations.5 CD95 expression as determined by
quantitative flow cytometry of the CD57+ V
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.
View larger version (17K):
[in this window]
[in a new window]
| Figure 5.
Median CD95 expression on T-cell subsets with the use of
3- and 4-color labeling.
(A) Median value of CD95 fluorescence with the use of 3-color labeling
on CD57+ and CD57 subsets of various expanded
and nonexpanded TCRV+ subsets from 2 MM
patients (patients A and B) performed on the same day
(P < .0001, Mann-Whitney test, and Table 2). The staining
pattern is representative of the results found in 12 MM patients
examined. (B) Median CD95 expression on T cells of 5 MM patients
(patients A through E) with the use of 4-color labeling. The
CD57+ and CD57 subsets in either the
CD4+ or the CD8+ subpopulations of the
TCRV+ subpopulations are significantly
different (P < .0001, Mann-Whitney test). (C) Median CD95
expression of T cells in 5 age-matched controls. The CD57+
and CD57 subsets in either CD4+ or
CD8+ subpopulations of the TCRV subsets are
significantly different (P = .027 and .002 respectively,
Mann-Whitney test).
|
|
| |
Discussion |
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, and perforin positive) are
present in a majority of MM patients5 and that their
presence is associated with longer survival, consistent with an
antitumor effect.5 We have also recently shown that patients whose myeloma cells carry the CD86 antigen (B7-2+)
have both a poor prognosis and a lower number of expanded T-cell clones, suggesting that an immunological basis exists for the improved
prognosis.23 In the belief that these expanded T-cell clones may possess anti-idiotypic specificity for myeloma cells, most
studies to date have involved a strategy of idiotype immunization with
the patients' own paraproteins. However, so far, only minimal antitumor responses have been reported with the use of either in vitro
or in vivo markers, which raises questions about the true specificity
of the expanded clones and has necessitated further analysis of their
properties.24-29
In the present study, flow cytometric analysis of TCR V
subset distribution within circulating T cells indicated once again
that MM patients and age-matched controls differed in the frequent
occurrence of large CD8+ T-cell expansions in MM. It has
been previously reported that in the healthy elderly both
CD4+ and CD8+ expansions are
seen.20 This observation has been confirmed in the present
study and extended to include MM patients. The oligoclonality, low
proliferative index, and lower CD95 expression of the CD57+
subpopulations was seen in both MM and aged-matched normal control groups. These findings did not appear to be related to previous therapy
administered to MM patients. Although 7 of 16 patients received
chemotherapy within 6 months of this study, no patient had received
chemotherapy within 2 weeks of the day of study, nor did we observe any
difference in the results of CDR3 length analysis, proliferation study,
and CD95 expression between those who received and those who did not
receive chemotherapy. Four patients had autologous stem cell
transplantation at least 2 years before their entry into this study. We
have investigated the effect of transplantation on T-cell populations,
and this will be reported elsewhere. In summary, we found that
CD8+ T-cell expansions appeared shortly after the
transplantation, but are all short-lived and most disappeared within 3 to 6 months after transplantation.
In the CDR length analysis study, the extracted mRNA encodes those
TCR chains that are expressed as proteins on the surface of sorted T cells, and only in-frame rearrangements are represented. Hence, the PCR products from a polyclonal population form a ladder at
3-nucleotide intervals, corresponding to differences of 1 amino acid in
the length of the CDR3 region. Furthermore, the intensity of each band
is proportional to the initial number of TCRV transcripts with that particular CDR3 length. In this report, we
analyzed the CD57+ and CD57 subpopulations
within expanded TCRV+CD8+ cells
and demonstrated that the CD57+ subpopulations are
generally clonal, whereas the corresponding CD57 cells
are frequently polyclonal. In a study of healthy subjects that used a
similar approach based on sorting for CD28+ and
CD28 cells, the CD8+CD28 T
cells were found to be dominated by relatively few
clones.30 However, the expanded T-cell clones in MM
patients may represent up to 25% of total CD3 cells, whereas such
clones in healthy subjects compose only a small percentage of
total T cells30 and have been postulated to reflect a
response to long-standing viral infection.13
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 population, may have led to
an overestimate of the number of T-cell expansions representing more
than 5% of total T cells.
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
counterparts. This finding provides an explanation for the accumulation of CD8+CD57+ T cells and is consistent with a
previous report that they are not susceptible to spontaneous or
activation-induced apoptosis in vitro.16,31 Further
evidence in support of this concept comes from an in vivo study in
mice, which revealed that cells with the highest levels of CD95
expression are preferentially deleted.32
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+CD8+ cells in
patients with myeloma have reduced levels of CD28 is suggestive of
chronic antigenic stimulation.5 In theory, this could have
been due either to a persistent viral infection in a compromised host
or to an ongoing antitumor response. However, no correlation was
demonstrated between the presence of viral infections, such as CMV and
hepatitis, and expanded T-cell clones in a cohort of MM patients
(no. = 40) previously studied (unpublished observations, September
2000), although one patient did have a transient increase in
the TCRV+CD8+ cells associated
with a recurrent herpes infection. In the current study, 7 of 27 age-matched controls showed no evidence of CD8+ or
CD8 T-cell expansion. Although all these donors were
negative for HIV and hepatitis B antibodies, 5 of them had anti-CMV
antibodies. This suggests that CMV infection in the elderly is not
necessarily associated with T-cell expansion. A role for human herpes
virus 8 in the pathogenesis of myeloma remains controversial, and
clonal expansion of T cells is not a feature of patients with Kaposi sarcoma.34-38 Moreover, expanded
TCRV+CD8+ T-cell populations in
patients with MM can persist for many years, consistent with chronic
stimulation by a tumor-associated antigen.5
In our previous study,5 we showed that the expression of
perforin was directly associated with that of CD57 in the expanded TCRV populations as well as in peripheral blood
lymphocytes in general. It was suggested that CD57 is not a direct
marker of cytotoxicity but may be related to a state of
activation. Similar coexpression of perforin and C |
Top
Abstract
Introduction