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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2883-2889
IMMUNOBIOLOGY
Killer inhibitory receptor (CD158b) modulates the lytic activity
of tumor-specific T lymphocytes infiltrating renal cell carcinomas
Nadia Guerra,
Maryvonne Guillard,
Eric Angevin,
Hamid Echchakir,
Bernard Escudier,
Alessandro Moretta,
Salem Chouaib, and
Anne Caignard
From INSERM U487, Institut Fédératif de Recherche
(IFR54), Institut Gustave Roussy, Villejuif, France; and Dipartimento
di Medicina Sperimentale, Universita degli Studi di Genova, Genova,
Italy.
 |
Abstract |
In this study, we showed that renal tumors contain substantial
subsets of CD8+ p58+ T cells. From 1 of
these tumors, T cells were amplified in mixed lymphocytes-tumor cell
cultures and p58+ T cells were selected immunologically.
After expansion, phenotypic and functional features of
p58+ and p58 T cells were examined. The
p58+ T cells expressed p58.2 receptor and corresponded to
CD3+, CD8+, T-cell receptor (TCR)
 / + T cells that were CD56+ and
CD28. Functionally, p58+ T cells showed a
low level of lytic activity against autologous tumor cells that was
dramatically and specifically increased by anti-p58.2 monoclonal
antibody. On the other hand, p58 CD8+ T
cells did not lyse autologous tumor cells and had non-major histocompatibility complex-restricted cytotoxicity against K562 and
Daudi cells. A p58+ cytotoxic T lymphocyte (CTL) clone
(4C7) with the same characteristics as the p58+ T-cell
line was derived. This CTL clone did not lyse autologous normal B cells
but lysed several HLA-A1+ renal tumor cell lines.
Analysis of TCR repertoire diversity showed that the p58+
T-cell line contained 3 TCR rearrangements, whereas the TCR repertoire of p58 T cells was polyclonal. Interestingly, TCR
transcripts of p58+ T cells and of CTL clone 4C7 were
detected as prominent ex vivo in tumor cells but not in peripheral
blood mononuclear cells, suggesting that these cells are antigen
specific and amplified at the tumor site.
(Blood. 2000;95:2883-2889)
© 2000 by The American Society of Hematology.
| |
Introduction |
Renal cell carcinomas (RCC), like melanomas, are
considered potentially immunogenic tumors. In this context, durable
tumoral responses were obtained after interleukin 2 (IL-2) treatment in 15% to 20% of patients with metastasis, with some of these responses resulting from elicitation of T-cell responses.1,2 However, in spite of the presence of a large CD3+ infiltrate, it is
difficult to generate stable tumor-specific cytotoxic T lymphocytes
(CTL) from RCC.3 Furthermore, the rare specific CTL
described in this model had functional alterations that resulted in
marginal lytic potential as well as low proliferation efficiency.4 We previously showed that some renal tumors
contained a large expansion of T-cell clones characterized in situ by
T-cell receptor (TCR) analysis but such cells were counterselected in vitro.5-7 In an attempt to understand why T cells amplified
in situ do not proliferate and efficiently lyse tumor cells in vitro, we examined the possible role of the described natural killer receptors
(NK-R) in the interactions between tumor cells and effector T
lymphocytes infiltrating the tumors (TIL).
The identification of the family of NK-R and the characterization of
related killer inhibitory receptors (KIR) and killer triggering
receptors have led to a better understanding of the mechanisms
regulating target-cell recognition as well as activation of NK and T
cells.8,9 In humans, NK-R belong to 2 distinct molecular
families, which display different specificities for polymorphic
determinants of HLA-I molecules. The first family belongs to the Ig
superfamily and comprises receptors such as HLA-C-specific
p58,10-12 HLA-B-specific p70,13 and
HLA-A-specific p140 that contain 2 (p58) or 3 (p70, p140) Ig domains.
The second family includes type II membrane proteins, represented by
CD94 expressed as an heterodimer associated with NKG2
molecules14,15 and involved in the recognition of HLA-E
molecules.16
Substantial evidence indicates that the interaction between KIR and HLA
molecules results in the delivery of a negative signal by NK cells that
leads to target-cell protection.17 All Ig-type inhibitory
receptors have long cytoplasmic domains with immunoreceptor tyrosine-based inhibition motifs (ITIM) mandatory to the transduction of their inhibitory function18,19 and are denoted KIR2DL
and KIR3DL. Highly homologous NK-R that have similar HLA-I allele specificity but are devoid of intracytoplasmic ITIM and contain a
charged lysine residue in the transmembrane have been
identified20,21 and are denoted KIR2DS and KIR3DS. These
truncated receptors trigger lysis in redirected cytolytic
assays.22 Although the nature of the triggering
receptor-ligand interactions is less defined, these receptors assemble
with an adaptor molecule bearing immunoreceptor tyrosine-based
activation motifs, triggering cell
activation.23,24 Both types of NK-R (Ig type
and lectin type) have also been detected on peripheral T lymphocytes
from healthy donors with a selective expression on minor T-cell
subsets, mostly of the CD8+, CD28,
CD45RO+ memory phenotype.25,26 It is
established that NK-R engagement also leads to inhibition of T-cell
functions, including TCR-mediated cytolytic activity and lymphokine
production.27-29
Current knowledge about the roles of these receptors in the immune
response to tumors is limited because few data are available. However,
identification of a new tumor-associated antigen recognized by a CTL
clone expressing an inhibitory p58 NK receptor was recently reported.30 This CTL clone was unable to lyse primary tumor cells expressing HLA-C molecules but efficiently killed metastatic cells that had lost HLA-C molecule expression. Taking into account the
facts that alteration of expression of HLA molecules is a common
feature of tumor cells and that tumor-specific CTL are poorly
characterized in RCC, we hypothesized that NK receptors might be
involved in the lethargy of the immune response to renal tumors.
In this study, we provide evidence indicating that most RCC contain a
substantial subset of CD8+ T cells expressing p58
receptors. Functional analysis of these in situ-amplified
p58+ T cells revealed that these effectors were potentially
highly cytotoxic and tumor specific but that their lytic activity was dramatically inhibited by the p58 receptor.
| |
Materials and methods |
Isolation and culture of tumor cells
Tumor cells from 9 previously untreated patients with RCC were
studied. The autologous tumor cell lines were derived from the primary
tumor as described previously.31 Briefly, total cell
suspensions were subjected to Ficoll-Hypaque centrifugation to
eliminate the dead cells. Viable cells were cultured in specific culture medium to amplify tumor cells and TIL. For tumor cells, Dulbecco modified Eagle medium/Ham F12 medium supplemented with 10%
fetal calf serum (FCS) and Ultroser G (Gibco BRL, Scotland) was used as
complete medium. Confluent culture dishes were treated with EDTA and
trypsin, and cells were subcultured 1 in 3.
Immunofluorescence analysis
The phenotype of TIL was analyzed by indirect 2-color fluorescence.
Cells (2 × 105) were first incubated for 30 minutes
at 4°C with the unlabeled monoclonal antibodies (mAbs) HP3B1
(anti-CD94, IgG2a), EB6 (anti-p58.1, IgG1), or GL183 (anti-p58.2,
IgG1), which were purchased from Immunotech (Marseille, France) and
correspond to purified mAbs. Culture supernatants of the mAbs 11PB6
(anti-p58.1, IgG1), Z27 (anti-p70, IgG1), Q66 (anti-p140, IgG1), and
Z199 (anti-NKG2-A, IgG2b) were also used. Cells were washed twice with
phosphate-buffered saline (PBS) and then incubated for 20 minutes at
4°C with phosphatidyl ethanolamine (PE)-conjugated goat antimouse
Ig. After a saturation step for 10 minutes with mouse Ig, cells were
incubated with anti-CD8 fluorescein isothiocyanate-conjugated (FITC)
mAb or anti-CD3 FITC mAb (Immunotech). Expression of activation markers
was analyzed by using anti-CD69 PE and anti-CD25 FITC (Immunotech),
anti-CD56, anti-CD28, and anti-CTLA-4 followed by GAMPE. In single-cell
tumor suspensions, analysis was performed on 5000 gated TIL (appearing in FSC/SSC as small lymphocytes comparable to peripheral
blood mononuclear cells (PBMC) after exclusion of dead cells and
debris. Background levels were measured by using isotypic controls.
Analysis was done on a fluorescence-activated cell sorter (Becton
Dickinson, Pont de Claix, France) using Cell Quest software (Becton
Dickinson). Compensation was set up with single stained samples.
Derivation of p58+ and p58 T-cell lines
and CTL
TIL cell lines were obtained from mixed lymphocytes-tumor cell
culture (MLTC). Briefly, from dissociated tumor cell suspensions, 3 × 105 TIL/mL were grown for 2 weeks in medium
(RPMI and 10% human AB serum) supplemented with 30 U/mL IL-2 (Roussel
Uclaff, Romainville, France) in 24-well culture plates. The medium was
renewed 3 times a week. After 2 weeks, the percentage of
CD3+ T cells expressing p58 receptors was determined, and
the p58+ T cells were immunoselected by using anti-p58.2
mAb (GL183) and immunomagnetic beads (Dynal, France). After
immunoselection, p58+ and p58 T-cell
subsets were expanded separately in bulk cultures
(3 × 103 cells/well) on allogeneic feeder cells
(104cells/well) in the presence of autologous tumor cells
(2 × 103 cells/well). Then, p58+ T
cells were cloned by limiting dilution 0.8 to 1 cell/well and using the
feeder-cell conditions described above.
Assay for cytolytic activity
The cytolytic activity of T-cell lines against autologous tumor
cells (patient DM), allogeneic renal tumor cell lines (LM, LR, MT, GF,
and VM), and NK targets K562 and Daudi cells was measured in a 4-hour
chromium 51 (51Cr)-release assay. Tumor cells were used in
amounts of 2 × 103 cells/well, and the ratio of
effector to target (E:T) cells ranged from 20:1 to 2:1. The percentages
of specific lysis were determined as described
previously.31 In some experiments, mAbs 11PB6 (anti-p58.1), GL183 (anti-p58.2), B1.23.2 (anti-HLA-B or C, IgG2a), or UCHT1 (anti-CD3, IgG1) were added at predefined saturating concentrations at
the beginning of the cytolytic assay. Data were expressed as the
percentage of specific lysis at the indicated E:T ratio.
The cytolytic activity of T-cell lines was also assessed in a
CD3-redirected lysis assay using P815 mastocytoma mouse cells. Briefly,
51Cr-labeled P815 cells
(2 × 103) coated with anti-CD3
(1µg/mL) were incubated with serial dilutions of T cells (E:T ratio
ranging from 10:1 to 2:1). CD3-redirected lysis of labeled P815 cells
was modulated by the presence of the anti-NK-R mAbs.
Usage of the TCRBV gene segment
Usage of the TCRBV gene segment was determined by a semiquantitative
polymerase chain reaction (PCR) analysis as described previously.32 Briefly, tumor samples (0.2-0.5 g) squeezed
by a pulverizer (Spex 6700; Spex Industries, Edison, NJ) were
resuspended in 6 mol/L guanidinium thiocyanate buffer. Total RNA was
then purified by cesium chloride gradient centrifugation. For TIL and PBMC (5 × 106 cells), total RNA was extracted with
use of a modified guanidinium thiocyanate-phenol-chloroform method
(Trizol; Eurobio, Les Ulis, France). Complementary DNA (cDNA) was
prepared with a standard method using reverse transcriptase and an
oligo-deoxythymidine primer (Invitrogen, Netherlands). cDNA
amplification was done over 30 to 40 cycles with the 5' sense
primers specific for the 24 BV subfamilies and one 3' antisense
primer specific for BC. The intensity values for the different peaks in
all BV subfamilies were added, and the percentages of each BV subfamily
were evaluated and represented as histograms. Although PCR efficiency
may vary from 1 oligonucleotide pair to another, this method was
previously found to allow intersample comparison.33
CDR3 size analysis of TCRBV transcripts
To study the overexpressed TCRBV transcripts further, a PCR-based
method that determines size-distribution patterns of the V-D-J junction
(CDR3) was used.33 Briefly, BV/BC-amplified products were
copied in 1- to 5-cycle runoff reactions primed with
fluorescence-labeled oligonucleotides (ABI fluorophore Fam) specific
for a BC or the 13 BJ fragments. Runoff products were then subjected to
electrophoresis on an automatic sequencer (ABI; Applied Biosystems,
Foster City, CA) in the presence of fluorescent size markers and
analyzed by automatic fluorescence quantification and size
determination with use of a computer program (Genescan 672; Applied Biosystems).
| |
Results |
Expression of p58 NK receptors by renal TIL
Phenotypic analysis of the TIL population before in vitro culture
was performed by using 2-color immunofluorescence analysis in 9 tumors.
From 7 tumors containing a substantial CD3+ T-cell
population, p58+ T cells were identified and corresponded
to a marked subset of CD8+ T cells (range, 6% to 30%;
Figure 1). From one of these tumors (patient DM: HLA A1, A2, B5, B8, and Cw7-Cw14), a permanent tumor cell
line was established, and phenotypic, functional, and molecular analyses of p58+ T cells were done. Our data indicate
that CD3+ represented 71% of the cells in the TIL gate,
with 40% of CD4 and 38% of CD8 cells. A small subset of p58.2 cells
representing 6% of the CD8+ T cells was detected (Figure
2). Although the percentages of p58.2 cells
were low, these cells constituted a well-defined CD8bright
T-cell subset, since the staining intensity of anti-p58.2 mAb (GL183)
was high. With respect to the lectin-type NK receptors, CD94 was
detected on 7.5% of CD8+ T cells and NKG2-A was detected
on 6% of CD8+ T cells (Figure 2). In addition, 4-color
analysis revealed that about 30% of p58+ T cells expressed
CD94 but not NKG2-A (data not shown). NK receptors were not detected on
CD4+ T cells.
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| Fig 1.
Expression of p58 receptors of T lymphocytes infiltrating
the tumors (TIL) in a series of renal cell carcinomas (RCC).
Non-adherent cells obtained from enzymatically dissociated tumors were
analyzed by cytometry after double-fluorescence staining with
anti-p58.2 GAMPE and fluorescein isothiocyanate-conjugated (FITC)
CD8.
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| Fig 2.
Analysis of natural killer receptors on TIL population
from patient DM before in vitro culture.
Double-immunofluorescence analysis with unlabeled anti-p58.2,
anti-CD94, and anti-NKG2A GAMPE and FITC CD8.
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Differential expression of CD56 and CD28 antigens on
p58+ and p58 T cells
Fourteen days after the MLTC was begun, the TIL cell line was
phenotyped to detect CD3+ p58+ T cells. In
these cultures, anti-p58.2 (GL183) stained 35% of the cells,
indicating that p58+ cells were amplified during MLTC. From
this culture, p58+ cells were immunoselected by using
anti-p58.2 (GL183) and were expanded, as were p58 T cells.
Comparative phenotypic analysis of both subsets was performed after a
10-day expansion. As shown in Figure 3,
p58.2+ cells were CD3/CD8+ T cells,
CD45RO+, CD28,
CTLA-4, and 60% CD56low. In addition,
p58+ T cells were CD25+, CD69+ and
expressed a TCR  / when BMA031 mAb was used (data not shown). As
expected from examination of the HLA-C alleles of patient DM that
correspond to the HLA-C ligand of p58.2, the use of specific anti-p58.1
mAb 11PB6 confirmed the exclusive membrane expression of the p58.2
receptor. Expression of the other Ig-type NK-R was not detected (p70,
p140), and 45% expressed CD94 but not NKG2-A (data not shown). On the
other hand, p58 cells also corresponded to
CD3/CD8+, CD45RO+, CD25+,
CD69+, TCR /+ T cells that were
CD56, CD28+, and
CTLA-4/low (Figure 3) and did not express any
Ig-type NK-R. A low-intensity expression of CD94 was detected on 15%
of the cells, but NKG2-A was not detected. Thus, expression of CD56 and
CD28 distinguished p58+ from p58 T
cells, whereas early activation markers CD25 and CD69 were expressed on
both subsets.
Differential cytotoxic potential and specificity of
p58+ and p58 T cells
When the lytic activity of p58+ and
p58 T cells was tested against autologous tumor
cells, both T-cell subsets showed low levels of cytotoxic activity
(Figure 4). In addition, p58+ T
cells lysed K562 with a low efficiency and did not kill Daudi cells,
suggesting that these cells do not have NK/LAK activity. On the other
hand, p58 T cells efficiently lysed NK cell targets
K562 and NK-resistant Daudi cells, indicating that these cells mainly
mediate non-major histocompatibility complex-restricted killing (eg,
LAK activity) (Figure 4).
To refine the functional analysis, the p58+ T-cell line was
further cloned by limiting dilution. A CTL clone (4C7) that showed a
high proliferative capacity was derived. CTL clone 4C7 expressed p58.2
and CD56 and was CD28. It had a low level of lytic
activity against autologous tumor cells.
Inhibitory effect of the p58.2 receptor on the CD3-mediated
lytic activity of p58+ T cells
To assess whether the lytic activity of p58+ T cells is
modulated by the p58 receptor, cytotoxic assays were performed in the presence of neutralizing anti-p58 mAbs. Lysis of autologous tumor cells
by p58+ T cells and CTL clone 4C7 increased significantly
when anti-p58.2 mAb was added during the cytotoxic assay, whereas the
control, anti-p58.1 mAb 11PB6, had no effect (Figure
5A). Preincubation of target cells with
anti-HLA-B/C-specific mAb (BB1 23.2) induced a similar increase in
lysis of tumor cells. Thus, the p58+ T-cell line and CTL
clone exhibit marked lytic activity against tumor cells when
interaction of p58 and HLA-C is blocked. Further evidence that
p58+ T cells and CTL clone 4C7 mediated a TCR-specific
lysis of tumor cells was that the addition of anti-HLA-B/C to
counteract the p58 receptor in the presence of anti-CD3 mAb resulted in
background-level lytic activity (Figure 5A). Moreover, the production
of IFN by p58+ T cells stimulated by tumor cells
increased significantly after blockage of interaction of HLA-C and p58
(data not shown). With regard to p58 T cells, the
marginal lysis observed was not modified when anti-p58.2 (GL183) was
added to the test (Figure 5A), and anti-CD3 induced a partial
inhibition of p58 T-cell cytotoxicity (Figure 5A).
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| Fig 5.
Anti-p58 monoclonal antibody (mAb) inhibition of the
cytolytic capacities of p58+ T cells.
(A) Lysis of tumor cells by p58+ and p58
T-cell lines and the p58+ cytotoxic T lymphocyte (CTL)
clone 4C7. The cytotoxic activity of effector cells against autologous
RCC was tested in a standard chromium-release assay at the indicated
ratio of effector to target cells. Inhibition of the lytic activity was
tested after preincubation of the cells for 30 minutes with the
indicated mAbs. (B) CD3-redirected lysis of P815 cells induced by
T-cell lines was generated by anti-CD3 mAb (1 µg/mL) in the
presence of anti-NK-R mAbs (2µg/mL).
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To confirm the presence of the modulator effect of the p58.2 receptor
on the TCR-mediated signal, we performed CD3-redirected lysis
experiments with FcR+ murine P815 cells. Both
p58+ and p58 T cells, as well as CTL
clone 4C7, mediated efficient CD3/TCR-triggered lysis of P815 cells but
did not kill P815 in the absence of anti-CD3 mAb. Furthermore, in
p58+ T cells, CD3-redirected lysis of P815 was almost
completely abrogated in the presence of anti-p58.2 mAb, whereas
anti-p58.1 had no significant effect and anti-p70 had no effect at all.
Comparable results were obtained with CTL clone 4C7. In
p58 T cells, CD3-redirected lysis was not modified
by anti-p58 mAb (Figure 5B). It is noteworthy that the 2 T-cell subsets
displayed comparable levels of CD3-redirected lysis, indicating that
the CD3 lytic machinery normally operates in p58+ T cells.
Specific lysis for allogeneic renal tumor cells exhibited by the
p58+ T-cell line and p58+ CTL
To analyze the specificity of lysis mediated by p58+ T
cells, normal autologous cells (phytohemagglutinin (PHA)-blasts and Epstein-Barr virus (EBV) B cells), as well as allogeneic tumor cell
lines, were used as targets. Autologous PHA-blasts cells and
EBV-transformed B cells were not lysed, even in the presence of
anti-p58.2 or anti-HLA-B/C mAbs, indicating that the antigen recognized
by p58+ T cells is not expressed by activated T and B cells
(Table 1).
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Table 1.
Cytotoxic activity of p58+ cytotoxic T
lymphocyte clone 4C7 against autologous normal targets and allogeneic
tumor cell lines
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Five allogeneic renal tumor cell lines (3 HLA-A1+ and 2 HLA-A2+) were tested. As shown on Table 1, p58+
T cells lysed the 3 HLA-A1+ cell lines (LM, GF, and MT) in
the presence of anti-p58.2 or anti-HLA-BC mAbs. Cell lysis of these
allogeneic tumor cells was comparable to the lysis of autologous tumor
cells, whereas the LR and VM tumor cell lines (HLA-A2+)
were not lysed, even in the presence of anti-p58.2 mAb. In addition, normal renal cells from patient LM were susceptible to lysis by CTL
clone 4C7. These data show that antigen expressed by DM tumor cells is
present in other renal tumors and possibly expressed by normal renal
cells and that the DM p58+ T cells exhibited
HLA-A1-restricted lysis.
Expression by p58+ T cells and CTL clone 4C7 of BVBJ
TCR transcripts that are amplified in the tumor
To assess the presence of p58+ T cells in vivo, TCR
transcripts expressed by p58+ T cells were characterized
and their in situ representativeness was analyzed. Analysis of usage of
the BV gene fragment in the p58+ T-cell line revealed that
the TCRBV repertoire was very restricted, with expression of 2 specificities: BV6 (43%) and BV8 (55%). On the other hand, the TCR
repertoire of p58 T cells was polyclonal (Figure
6A and 6B). In tumor, although the TCR
repertoire was diverse, BV6 and BV8 specificities corresponded to 7%
and 11%, respectively, of the BV transcripts (Figure 6C). The
repertoire in PBMC was polyclonal and different from that of tumor
(Figure 6C).
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| Fig 6.
Analysis of T-cell receptor (TCR) BV repertoire expressed
by in vitro-derived p58+ and p58 T-cell
lines and ex vivo samples of tumor and peripheral blood mononuclear
cells (PBMC).
Comparison of relative expression of TCRBV gene segment in a
p58+ TIL cell line (A), a p58 TIL cell
line (B), and a tumor fragment ( ) and PBMC ( ) (C). For each
sample, the spots obtained by Southern blotting of TCRBV gene segments
amplified by polymerase chain reaction (30 cycles) were analyzed by
densitometry, and each BV signal was expressed as a percentage of the
sum of the values of all BV spots.
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We focused on the 2 TCR BV6 and BV8 specificities in the
p58+ T-cell line to refine identification of clonal
transcripts using the 13 different BJ primers. One dominant BV6 clone
was detected, corresponding to a TCR BV6BJ2S1 rearrangement of 274 nt.
The TCRBV8 transcripts contained 2 cDNA clones, 1 prominent
rearrangement, TCR BV8JB2S1 of 214 nt, and 1 minor rearrangement, TCR
BV8BJ2S7 of 242 nt (Figure 7).
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| Fig 7.
Detection of p58+ T cells and CTL clone
4C7-specific TCR rearrangements by CDR3 analysis ex vivo in tumor
cells and PBMC.
RNA from p58+ T cells, tumor, and PBMC were reverse
transcribed and amplified by using a TCRBV6 or TCRBV8 and a BC primer.
Amplified DNA was copied in a runoff reaction by using fluorescent BC
or BJ primers. The amplified products were analyzed on an automated
sequencer. The profiles obtained show the size in nt
(x-axis) and fluorescence intensity (y-axis) of the
amplified products. The absolute FU values are indicated
by asterisks.
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The presence of these rearrangements in vivo was determined by CDR3
size analysis of BV6 and BV8 TCR transcripts in tumor and PBMC (Figure
7). In tumor, the TCR VB6BJ2S1 (274 nt) and BV8BJ2S1 (214 nt)
rearrangements were prominent, whereas the minor BV8BJ2S7 peak was not
clearly detected. In PBMC obtained at the time of nephrectomy, TCR BV6
and VB8 transcripts displayed a bell-shaped pattern consistent with
polyclonality, and the transcripts specific for p58+ were
barely detectable. Interestingly, the cytotoxic CTL clone 4C7 expressed
the TCR BV6J2S1 rearrangement of 274 nt prominently in situ, suggesting
that the selective intratumor amplification of the TCR BV6BJ2S1
transcript corresponds to the dominant p58+ tumor-specific
CTL. This molecular analysis of p58+ tumor-specific T cells
confirms that such cells are amplified at the tumor site and constitute
the main tumor-specific population infiltrating RCC.
| |
Discussion |
The current studies emphasize that NK-R may be an important
component of the regulation of the T-cell response in RCC. First, double-fluorescence analysis of uncultured TIL provided evidence that
most renal tumors contain a substantial subset of p58+ T
cells. Furthermore, the functional study indicated that
p58+ TIL correspond to antigen-specific cytotoxic T
cells, whose lytic and secretory activities were significantly
inhibited by the p58 receptor. Exploiting the high-resolution
properties of the analysis of the length of the CDR3 TCRBV transcripts
and the fact that TCR expression is a stable and conserved
feature of specific antigen recognition, we demonstrated expansion of
the p58+ tumor-specific T-cell clones at the tumor site. On
the other hand, p58 TIL mainly mediated a
non-specific lytic activity and exhibited a polyclonal TCR repertoire.
The tumor-specific T cells we obtained after a single in vitro
stimulation by tumor cells (while avoiding culture skewing as much as
possible) corresponded to few p58+ T-cell clones bearing an
inhibitory p58 receptor and represented 5% to 10% of the
CD8+ TIL subset. Interestingly, CTL clone 4C7 derived from
a p58+ T-cell line had the same phenotypic and functional
characteristics as the T-cell line and expressed the BV6BJ2S1
rearrangement that was prominent in the p58+ T cells and
found to be amplified in the tumor in vivo. These results indicate
that, in RCC, p58+ T cells correspond to tumor-specific T
cells that are primed in vivo, proliferate, and display high lytic
activity in vitro. A recent study using the sensitive tetramer
technology provided evidence of the presence of
CD94/NKG2-A+, Mela A/Mart1-specific CTL in the peripheral
blood of a few patients with melanoma. In these patients, lysis of
melanoma cells was inhibited by this inhibitory NK
receptor.34
Although there is no evidence that CD158b expression by itself provides
antigen specificity, expression of this marker correlates in this model
to high lytic activity, clonal TCRBV gene usage, and in situ expansion
of the corresponding T cells. The expression of inhibitory NK receptors
on tumor-specific T cells would explain the absence of tumor-specific
activity in bulk TIL populations and the low frequency of CTL detected
by conventional cytotoxicity assays of autologous tumor cells in
RCC compared with that observed in melanoma. It might also explain the
skewing usually observed between in situ-amplified and in
vitro-derived T-cell subsets in RCC.6
Few data are available on the mechanisms controlling the induction of
these receptors on T cells. In contrast to the CD94/NKG2 receptor,
which can be induced in vitro under particular T-cell stimulation
conditions,35-37 Ig-type NK-R induction cannot yet be
reproduced in vitro. In our model, expression of p58 was stable and was
not modulated by either interferon- or interleukin 10 after T-cell
stimulation with tumor cells (data not shown). In addition, few data
implicating Ig-type NK-R in the recognition of solid tumors have been
reported. Introduction of an inhibitory p70 receptor (KIR3DL) in a
gp100 melanoma-specific, HLA-A2-restricted CTL resulted in inhibition
of lysis of melanoma cells coexpressing HLA-A2 and HLA-Bw4
allotypes.38 A melanoma-specific p58+ CTL was
obtained from PBMC stimulated by a metastatic cell line that had lost
expression of the complete HLA haplotype. Although this CTL killed
metastatic cells efficiently, its specific activity was inhibited by
primary tumor cells expressing HLA-Cw7. This p58+ CTL
recognized a new antigen, PRAME, which is expressed in a large range of
melanomas, sarcomas, and myeloid leukemias and also detected in normal
testicular, endometrial, and ovarian tissues.30 In the
context of an immune response against tumor, expression or
overexpression of normal tissue-specific antigen presented by tumor
cells with altered HLA molecules in the presence of particular immunoregulatory cytokines may be responsible for amplification of
KIR+ antigen-specific CTL.
In patients with RCC, who frequently present with slow-growing large
tumor burdens, prolonged chronic exposure of T cells to antigen
(overexpressed normal proteins) may lead to activation of potentially
autoreactive T cells. Therefore, expression of NK-R may represent a
mechanism involved in the down-regulation of a deleterious immune
reaction. Alternatively, activation and expansion of p58+
CTL may be a consequence of altered expression of HLA-C molecules at
the surface of tumor cells. Indeed, like many tumors, RCC lose or have
alterations of expression of the HLA molecules with tumor progression,
and several mechanisms may be involved in these
alterations.39,40 Furthermore, it has been shown that HLA-C
molecules bind to the inhibitory NK receptor with extremely fast
association and dissociation rates, and such kinetics may facilitate
the rapid and precise immunosurveillance of cells with absent or
diminished expression of HLA-I molecules.41 In this
context, reduced levels of expression of HLA molecules have been shown
to play a major role in the susceptibility of melanoma cells to
NK-mediated lysis.42 The fact that in vitro-derived tumor
cells were not lysed by p58+ T cells in the absence of
anti-p58 mAb suggests that these cells expressed HLA-C.
This finding supports the hypothesis suggesting that the nature of the
antigen is the main mechanism activating p58+ T cells in
RCC. The p58+ CTL correspond to high-affinity CTL requiring
few interactions of TCR and HLA-I, and an overall (not allele specific)
down-regulated expression of the HLA-I molecule probably affects the
NK-R activity more than the TCR-mediated recognition. Thus, in RCC
infiltrated by p58+ T cells, HLA-C expression may tune the
lytic-activity level of the tumor-specific response.
The nature of the antigens recognized by p58+ T cells is
still highly undetermined. It may be postulated that such antigens derived from self proteins recognized by autoreactive CTL that are
unable to lyse normal cells expressing large amounts of HLA molecules
but are efficiently activated by contact with cells with altered
expression of HLA-I molecules. In our model, although the
antigen recognized by p58+ CTL was not expressed by normal
activated T and B cells, it may correspond to a tissue-specific antigen
expressed by normal and tumoral renal cells. It is interesting that RCC
do not express any of the tumor-specific and tumor-associated antigens
identified so far by tumor-specific CTL in melanoma. In addition, the
antigens recognized by classical CD8+ CTL in RCC that
have been characterized correspond to private antigens and are encoded
by mutated epitopes of the HLA-A2 allele,43 hsp70-2,44 the open reading frame of carboxyl
esterase,45 or the rarely expressed RAGE
gene.46 Thus, the characterization of the antigens
recognized by p58+ CTL may be of major interest.
In summary, we found that substantial subsets of TCR /
CD8+ T cells that expressed NK receptors infiltrated RCC.
Tumor-specific T-cell clones derived from this subset inhibited the
lysis of renal tumor cells, suggesting that NK receptors may modulate
T-cell activities in vivo. Overall, these results point to a new
mechanism in the regulation of T-cell effector functions against human
renal tumors that may have important implications for antitumor
immunity in RCC.
| |
Acknowledgments |
We thank Yann Lécluse for immunofluorescence analyses and Dr Ryad
Tamouza for HLA genotyping of several tumor cell lines.
| |
Footnotes |
Submitted November 1, 1999; accepted December 27, 1999.
Supported by grants awarded by INSERM and the Association for Cancer
Research (ARC 9702 to A.C.).
Reprints: Anne Caignard, Unité INSERM U487 Cytokines et
Immunité Antitumorale, Institut Gustave Roussy, PR1, 39 rue Camille Desmoulins, F-94805 Villejuif, France; e-mail:
caignard{at}igr.fr.
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.
| |
References |
1.
Rosenberg SA, Yang JC, Topalian SL, et al.
Treatment of 283 consecutive patients with metastatic melanoma and renal cell cancer using high dose bolus interleukin 2.
JAMA.
1994;271:907[Abstract/Free Full Text].
2.
Bukowski RM, Olencki T, Wang Q, et al.
Phase II trial of interleukin-2 and interferon-alpha in patients with renal cell carcinoma: clinical results and immunologic correlates responses.
J Immunother.
1997;20:301.
3.
Itoh K, Platsoucas CD, Balch CM.
Autologous tumor specific cytotoxic T lymphocytes in the infiltrate of human metastatic melanomas. Activation by interleukin 2 and autologous tumor cells, and involvement of the T cell receptor.
J Exp Med.
1998;168:1419[Abstract/Free Full Text].
4.
Alexander J, Kudoh S, Melsop K, et al.
T cells infiltrating renal cell carcinoma display a poor proliferative response even though they can produce interleukin 2 and express interleukin 2 receptors.
Cancer Res.
1993;53:1380[Abstract/Free Full Text].
5.
Gaudin C, Dietrich PY, Robache S, et al.
In vivo local expansion of clonal T cell subpopulations in renal cell carcinoma.
Cancer Res.
1995;55:685[Abstract/Free Full Text].
6.
Dietrich PY, Walker PR, Schnuriger V, et al.
TCR analysis reveals significant repertoire selection during in vitro lymphocyte culture.
Int Immunol.
1997;9:1073[Abstract/Free Full Text].
7.
Angevin E, Kremer F, Gaudin C, Hercend T, Triebel F.
Analysis of T-cell immune response in renal cell carcinoma: polarization to type 1-like differentiation pattern, clonal T-cell expansion and tumor-specific cytotoxicity.
Int J Cancer.
1997;72:431[Medline]
[Order article via Infotrieve].
8.
Pende D, Sivori S, Accame L, et al.
HLA-G recognition by human natural killer cells. Involvement of CD94 both as inhibitory and as activating receptor complex.
Eur J Immunol.
1997;8:1875.
9.
Ryan JC, Seaman WE.
Divergent functions of lectin-like receptors on NK cells.
Immunol Rev.
1997;155:79[Medline]
[Order article via Infotrieve].
10.
Moretta A, Tambussi G, Bottino C, et al.
A novel surface antigen expressed by a subset of human CD3CD16+ natural killer cells: role in cell activation and regulation of cytolytic function.
J Exp Med.
1990;171:695[Abstract/Free Full Text].
11.
Ciccone E, Pende D, Viale O, et al.
Involvement of HLA class I alleles in natural killer (NK) cell-specific functions: expression of HLA-Cw3 confers relative protection from lysis by alloreactive NK clones displaying a defined specificity (specificity 2).
J Exp Med.
1992;176:963[Abstract/Free Full Text].
12.
Moretta A, Vitale M, Bottino C, et al.
P58 molecules as putative receptors for major histocompatibility complex (MHC) class I molecules in human natural killer (NK) cells: anti-p58 antibodies reconstitute lysis of MHC class I-protected cells in NK clones displaying different specificities.
J Exp Med.
1993;178:597[Abstract/Free Full Text].
13.
Litwin V, Gumperz JE, Parham P, Phillips JH, Lannier LL.
NKB1: an NK receptor involved in the recognition of polymorphic HLA-B molecules.
J Exp Med.
1994;180:537[Abstract/Free Full Text].
14.
Aramburu J, Balboa MA, Ramirez A, et al.
A novel functional cell surface dimer (Kp43) expressed by natural killer cells and T cell receptor-gamma/delta+ T lymphocytes. Inhibition of the IL-2 dependent proliferation by anti-Kp43 monoclonal antibody.
J Immunol.
1990;144:3238[Abstract].
15.
Chang C, Rodriguez A, Carretero M, Lopez-Botet M, Phillips JH, Lanier LL.
Molecular characterization of human CD94: a type II membrane glycoprotein related to C type lectin superfamily.
Eur J Immunol.
1995;9:2433.
16.
Braud VM, Allan DS, O'Callaghan CA, et al.
HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C.
Nature.
1998;391:795[Medline]
[Order article via Infotrieve].
17.
Moretta A, Bottino C, Vitale M, et al.
Receptors for HLA class I molecules in human natural killer cells.
Annu Rev Immunol.
1996;14:619[Medline]
[Order article via Infotrieve].
18.
Burshtyn DN, Scharenberg AM, Wagtmann N, et al.
Recruitment of tyrosine phosphatase HCP by the killer cell inhibitory receptor.
Immunity.
1996;4:77[Medline]
[Order article via Infotrieve].
19.
Bruhns P, Marchetti P, Fridman WH, et al.
Differential roles of N- and C-terminal immunoreceptor tyrosine-based inhibition motifs during inhibition of cell activation by killer cell inhibitory receptors.
J Immunol.
1999;162:3168[Abstract/Free Full Text].
20.
Moretta A, Sivori S, Vitale M, et al.
Existence of both inhibitory (p58) and activatory (p50) receptors for HLA-C molecules in human natural killer cells.
J Exp Med.
1995;182:875[Abstract/Free Full Text].
21.
Wagtmann N, Biassoni R, Cantoni C, et al.
Molecular clones of the p58 NK cell receptor reveal immunoglobulin related molecules with diversity in both extra and intracellular domains.
Immunity.
1995;2:439[Medline]
[Order article via Infotrieve].
22.
Bottino C, Sivori S, Vitale M, et al.
A novel surface molecule homologous to the p58/p50 family of receptors is selectively expressed on a subset of human natural killer cells and induces both triggering of cell functions and proliferation.
Eur J Immunol.
1996;8:1816.
23.
Olcese L, Cambiaggi A, Semenzato G, Bottino C, Moretta A, Vivier E.
Human killer cell activatory receptors for MHC class I molecules are included in a multimeric complex expressed by natural killer cells.
J Immunol.
1997;198:5083.
24.
Lanier LL, Corliss BC, Wu J, Leong C, Phillips JH.
Immunoreceptor DAP-12 bearing a tyrosine based activation motif is involved in activating NK cells.
Nature.
1998;391:703[Medline]
[Order article via Infotrieve].
25.
Mingari MC, Vitale C, Cambiaggi A, et al.
Cytolytic T lymphocytes displaying natural killer (NK)-like activity: expression of NK related functional receptors for HLA class I molecules (p58 and CD94) and inhibitory effect on the TcR-mediated cell lysis or lymphokine production.
Int Immunol.
1995;7:697[Abstract/Free Full Text].
26.
Speiser DE, Valmori D, Rimoldi D, et al.
CD28-negative cytolytic effector T cells frequently express NK receptors and are present at variable proportions in circulating lymphocytes from healthy donors and melanoma patients.
Eur J Immunol.
1999;6:1990.
27.
Ferrini S, Cambiaggi A, Meazza R, et al.
T cell clones expressing the natural killer cell-related p58 receptor molecule display heterogeneity in phenotypic properties and p58 function.
Eur J Immunol.
1994;10:2294.
28.
Phillips JH, Gumperz JE, Parham P, Lanier LL.
Superantigen dependent cell mediated cytotoxicity inhibited by MHC class I receptors on T lymphocytes.
Science.
1995;268:403[Abstract/Free Full Text].
29.
D'Andrea A, Chang C, Phillips JH, Lanier LL.
Regulation of T cell lymphokine production by killer cell inhibitory receptor recognition of self HLA class I alleles.
J Exp Med.
1996;184:789[Abstract/Free Full Text].
30.
Ikeda H, Lethé B, Lehmann F, et al.
Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor.
Immunity.
1997;6:199[Medline]
[Order article via Infotrieve].
31.
Caignard A, Guillard M, Gaudin C, Escudier B, Triebel F, Dietrich PY.
In situ demonstration of renal cell carcinoma specific T-cell clones.
Int J Cancer.
1996;66:564[Medline]
[Order article via Infotrieve].
32.
Génévé C, Diu A, Nierat J, et al.
An experimentally validated panel of subfamily specific oligonucleotide primers (Valpha 1-w29/Vbeta 1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction.
Eur J Immunol.
1992;22:1261[Medline]
[Order article via Infotrieve].
33.
Pannetier C, Even J, Kourilsky P.
T-cell repertoire diversity and clonal expansions in normal and clinical samples.
Immunol Today.
1995;16:176[Medline]
[Order article via Infotrieve].
34.
Speiser DE, Pittet MJ, Valmori D, et al.
In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes.
J Exp Med.
1999;190:775[Abstract/Free Full Text].
35.
Bertone S, Schiavetti F, Bellomo R, et al.
Transforming growth factor-beta induced expression of CD94/NKG2-A inhibitory receptors in human T lymphocytes.
Eur J Immunol.
1999;29:23[Medline]
[Order article via Infotrieve].
36.
Mingari MC, Ponte M, Bertone S, et al.
HLA class I-specific inhibitory receptors in human T lymphocytes: interleukin 15-induced expression of CD94/NKG2A in superantigen or alloantigen activated CD8+ T cells.
Proc Natl Acad Sci U S A.
1998;95:1172[Abstract/Free Full Text].
37.
Guerra N, Benlhassan K, Carayol G, et al.
Effect of TGF-beta on NK receptor expression by allostimulated CD8+ T lymphocytes.
Eur Cytokine Netw.
1999;10:357[Medline]
[Order article via Infotrieve].
38.
Bakker AB, Phillips JH, Figdor CG, Lanier LL.
Killer cell inhibitory receptors for MHC class I molecules regulate lysis of melanoma cells mediated by NK cells, gamma delta T cells, and antigen-specific CTL.
J Immunol.
1998;160:5239[Abstract/Free Full Text].
39.
Luboldt HJ, Kubens BS, Rubben H, Grosse-Wilde H.
Selective loss of human leukocyte antigen class I allele expression in advanced renal cell carcinoma.
Cancer Res.
1996;56:826[Abstract/Free Full Text].
40.
Seliger B, Hohne A, Knuth A, et al.
Reduced membrane major histocompatibility complex class I density and stability in a subset of human renal cell carcinomas with low TAP and LMP expression.
Clin Cancer Res.
1996;8:1427.
41.
Vales-Gomez M, Reyburn HT, Mandelboim M, Strominger JL.
Kinetics of interaction of HLA-C ligands with natural killer cell inhibitory receptors.
Immunity.
1998;9:337[Medline]
[Order article via Infotrieve].
42.
Pende D, Accame L, Pereti L, et al.
The susceptibility to natural killer cell-mediated lysis of HLA class I-positive melanomas reflects the expression of insufficient amounts of different HLA class I alleles.
Eur J Immunol.
1998;28:2384[Medline]
[Order article via Infotrieve].
43.
Brandle D, Brasseur F, Weynants P, Boon T, Eynde BJVD.
A mutated HLA-A2 molecule recognized by autologous cytotoxic T lymphocytes on a human renal cell carcinoma.
J Exp Med.
1996;183:2501[Abstract/Free Full Text].
44.
Gaudin C, Kremer F, Angevin E, Scott V, Triebel F.
A hsp70-2 mutation recognized by CTL on a human renal cell carcinoma.
J Immunol.
1999;162:1730[Abstract/Free Full Text].
45.
Ronsin C, Chung-Scott V, Poullion I, Aknouche N, Gaudin C, Triebel F.
A non-AUG-defined alternative open reading frame of the intestinal carboxyl esterase mRNA generates an epitope recognized by renal cell carcinoma-reactive tumor infiltrating lymphocytes in situ.
J Immunol.
1999;163:483[Abstract/Free Full Text].
46.
Gaugler B, Brouwenstijn N, Vantomme V, et al.
A new gene coding for an antigen recognized by autologous cytolytic T lymphocytes on human renal cell carcinoma.
Immunogenetics
1996;44:323[Medline]
[Order article via Infotrieve].
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Abstract
Introduction
