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Int. J. Cancer: 71, 178–182 (1997)
r 1997 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
IDENTIFICATION OF AN ENRICHED CD41 CD8a11 CD8b1 T-CELL SUBSET
AMONG TUMOR-INFILTRATING LYMPHOCYTES IN HUMAN RENAL
CELL CARCINOMA
Ludwig E. VAN DEN HOVE1, Stefaan W. VAN GOOL1,2, Hein VAN POPPEL3, Luc BAERT3, Lieve COOREVITS1, Boudewijn VAN DAMME4,
Paola DAL CIN5, Herman VAN DEN BERGHE5 and Jan L. CEUPPENS1*
1Laboratory of Experimental Immunology, Faculty of Medicine, Catholic University of Leuven, Leuven, Belgium
2Division of Pediatrics, Faculty of Medicine, Catholic University of Leuven, Leuven, Belgium
3Division of Urology, Faculty of Medicine, Catholic University of Leuven, Leuven, Belgium
4Division of Pathology, Faculty of Medicine, Catholic University of Leuven, Leuven, Belgium
5Center for Human Genetics, Faculty of Medicine, Catholic University of Leuven, Leuven, Belgium
Three-color immunofluorescence flow-cytometric analysis
of freshly isolated tumor-infiltrating lymphocytes (TIL) from
patients with primary renal cell carcinoma (RCC) revealed a
unique, not previously described TIL subset with a CD31
CD41 CD8a11 CD8b1 phenotype. This subset represented at
least 5% of CD31 TIL in 15 of 21 patients with clear cell RCC,
whereas it was not or only marginally represented in patients
with papillary RCC or sarcomatoid RCC. In one-third of the
patients with clear cell RCC, more than 20% of CD31 TIL and
in one patient more than half of the CD31 TIL displayed this
phenotype. The occurrence of this subset was not associated
with pathological stage, tumor diameter, nuclear grade,
cytogenetic abnormalities or vascular invasion in this patient
cohort. When present, the CD31 CD41 CD8a11 CD8b1
subset was detected in similar proportions in tumor tissue
and tumor capsula, and it was also detected in adjacent
non-tumoral renal tissue, albeit in much lower proportions.
Despite strong cell surface expression of various activation
markers (CD69, CD54 and HLA-DR), CD31 CD41 CD8a11
CD8b1 cells displayed no ex vivo cytolytic activity in an
anti-CD3-redirected cytotoxicity assay. In contrast with CD31
CD41 CD82 cells from the same tumor sample, they were
markedly deficient in IL-2Ra up-regulation following antiCD3 triggering. The possibility that these cells represent
either anergic cells or a highly specialized effector population
with a discrete, as yet undescribed function is discussed. Int. J.
Cancer 71:178–182, 1997.
r 1997 Wiley-Liss, Inc.
Renal cell carcinoma (RCC) is one example of human cancer in
which the immune system is thought to play a role in the control of
malignant cell growth. The basis for this belief originated from
observations that metastases may regress following nephrectomy,
which has been interpreted to be the consequence of an immune
response (reviewed in de Riese et al., 1991). Furthermore, about
15–20% of patients with advanced RCC respond to systemic
interleukin-2 (IL-2) administration, and durable (.60 months)
responses with complete disappearance of all measurable disease
have been achieved in some patients with metastatic disease
(Whittington and Faulds, 1993). Finally, RCC tumors are infiltrated
by a large mononuclear infiltrate mainly composed of T lymphocytes (tumor-infiltrating lymphocytes, TIL), which have been
considered to be part of the host immune response against the
tumor and to be enriched in T cells with tumor specificity
(Mitropoulos et al., 1994; Gaudin et al., 1995). The observations of
defects in CD3-z-chain expression in CD31 TIL of some patients
(Finke et al., 1993; Farace et al., 1994; Tartour et al., 1995), the
defective expression of IL-2Ra (CD25) on in vitro stimulated
CD31 TIL of some patients (Alexander et al., 1993), the heterogeneity in TIL proliferative responses (Alexander et al., 1993; Tartour
et al., 1995), the failure to grow TIL from some tumor samples
(Whiteside and Parmiani, 1994) and the variable results with TIL
therapy in clinical trials (Pierce et al., 1995) suggest that at least
some TIL subsets are functionally impaired. In the present report,
we describe a unique, previously uncharacterized CD31 TIL subset
in human RCC that expresses CD4, CD8a and CD8b chains and
that is markedly deficient in IL-2Ra expression following stimulation.
MATERIAL AND METHODS
Tissue specimens
Peripheral blood and tumoral (tumor stroma and tumor capsula)
and adjacent non-tumoral renal tissues were obtained from 27
patients during surgery for a primary RCC (Table I). Tumor
specimens were examined for pathological tumor and nodal stage
(TNM classification; Spiessl et al., 1992), largest tumor diameter,
nuclear grade (I–IV according to Fuhrman et al., 1982), histologic
cell type (clear cell, papillary, granular cell, chromophobe cell,
sarcomatoid or collecting duct according to Murphy et al., 1994)
and presence or absence of vascular invasion. The latter was
demonstrated to be the single most significant prognostic factor
after radical surgery (Van Poppel et al., 1995). Thymic tissue was
obtained from a 29-week-old fetus during autopsy.
Isolation of lymphocytes from renal and thymic tissue
and from peripheral blood
Single-cell suspensions from renal and thymic tissue specimens
were prepared by mincing the tissues with scissors and scalpel into
tiny pieces, which were passed through a metal mesh. Enrichment
for viable lymphocytes was done by centrifugation of the singlecell suspensions or peripheral blood over a Lymphoprep gradient
(Nycomed, Oslo, Norway). For purification of CD41 and CD81
TIL subsets and peripheral blood CD81 T cells, magnetic immunoselection on isolated lymphocytes with anti-CD4- and anti-CD8coated Dynabeads (Dynal, Oslo, Norway), respectively, was used.
Beads were applied with a bead-to-target ratio of 3:1.
Immunofluorescence studies
Three-color analyses were performed by direct and combined
indirect/direct immunofluorescence with FITC (fluorescein isothiocyanate)-, PE (phycoerythrin)- and PerCP (peridinin chlorophyll
protein)-conjugated and unconjugated monoclonal antibodies
(MAbs). PerCP-, PE- or FITC-labeled mouse MAbs anti-CD3,
anti-CD4, anti-CD8a, anti-CD25, anti-CD28, anti-CD45, antiCD45RA, anti-CD54, anti-CD56, anti-CD57, anti-CD62L, antiCD69, anti-HLA-DR, anti-T-cell receptor a/b, anti-T-cell receptor
g/d, the appropriate mouse immunoglobulin isotype controls and
rat-anti-mouse IgG1 were all from Becton Dickinson (Mountain
Contract grant sponsor: Nationaal Fonds voor Wetenschappelijk Onderzoek, contract grant number G.3123.94; Contract grant sponsor: Algemene
Spaar, Lijfrentekas, Brussels, Belgium.
*Correspondence to: Laboratory of Experimental Immunology, University Hospital Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium. Fax:
32/16346035.
Received 28 September 1996; accepted 27 December 1996
‘‘DOUBLE-POSITIVE’’ TUMOR-INFILTRATING LYMPHOCYTES
TABLE I – RELATIVE NUMBER OF
Patient1
Sex2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
CD31CD41CD8a11
% CD31CD41CD8a11
lymphocytes
179
TIL AND HISTOPATHOLOGIC, CYTOGENETIC AND CLINICAL FINDINGS IN 27 PATIENTS WITH PRIMARY RCC
pTN
stage3
Maximal
tumor
diameter
(cm)
Nuclear
grade4
Histologic
cell
type5
Venous
invasion
Follow-up6
(months)
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Papillary
No
No
No
No
Yes
No
No
Yes
Yes
Yes
No
No
No
Yes
No
Yes
No
No
NED (11)
NED (18)
NED (16)
NED (18)
NED (5)
NED (16)
NED (18)
NED (13)
NED (20)
NED (14)
NED (8)
NED (8)
NED (8)
AMD (18)
NED (12)
NED (18)
AMD (16)
NED (16)
Papillary
No
NED (18)
Sarcomatoid
Clear
Clear
Papillary
Yes
No
No
No
CRD (14)
NED (15)
NED (16)
NED (18)
II
II
Clear
Papillary
Yes
No
NED (21)
NED (18)
IV
III
Clear
Papillary
Yes
No
CRD (4)
NED (7)
Within
CD31 TIL
Within
total TIL
M
F
M
M
M
M
M
M
M
M
M
F
M
M
F
M
M
M
54
32
32
30
25
24
22
22
16
14
13
6
6
5
5
3
3
2
31
30
27
25
20
17
14
13
12
10
7
4
4
3
1
2
2
2
pT2N0
pT2N0
pT2N0
pT2N0
pT2N0
pT2N0
pT2N0
pT2N0
pT3bN0
pT3bN0
pT2N0
pT3aN0
pT3aN0
pT2N0
pT2N0
pT3bN0
pT3aN1
pT2N0
3.5
6
6.5
6
8
3
4
10
10
7.5
6
7.5
5
8
9
13.5
5
6
I
III
IV
II
III
II
III
III
II
II
II
III
III
III
III
III
III
II
19
M
2
1
pT2N0
5
II
20
21
22
23
M
M
F
M
2
1
1
1
1
1
1
1
pT2Nx
pT2N0
pT2N0
pT2N0
4
10.5
3
5
III
III
II
III
24
25
M
M
0
0
0
0
pT3aN1
pT2N0
8
5
26
27
F
F
0
0
0
0
pT4N1
pT2N0
14
3.5
Tumor
karyotype7
[number of cells]
46,XY [15]
46,XX [15]
46,XY [10]
Failure
45,X,2Y,del(3)(p21p25) [7]
50,XY,17,112,119,120 [20]
Failure
Failure
Failure
Failure
46,XY [20]
Not assessed
46,XY [15]
Failure
Not assessed
Failure
46,XY [17]
47,XY,17/46,X,2Y,17/46,XY
[2/2/18]
48,X,2Y,17,17,der(11)t(3;
11)(q11;p15),117 [20]
Failure
46,XY [10]
47,XX,17/46,XX [5/15]
90–103,XXY,2Y,21,der(9;
12)(q10;
q10)32,1mar1,1mar2/
46,XY [25/5]
Not assessed
47,X,2Y,12,17,17,28,der(13;15)
(q10;q10),215,der(15;
17)(q10;q10),116 [20]
46,XX,del(3)(p14p23) [3]
46,XX [15]
1The 27 patients were ordered according to the relative number of CD31CD41CD8a11 lymphocytes within total CD31 TIL.–2M, male; F,
female.–3Pathological tumor and node stages were determined on the basis of criteria stipulated by the UICC (Spiessl et al., 1992).–4Nuclear grade
was determined on the basis of criteria according to Fuhrman et al. (1982).–5Histologic cell type was classified according to the method of the
AFIP (Murphy et al., 1994).–6AMD, alive with metastatic disease; CRD, cancer-related death; NED, no evidence of disease.–7Tumor karyotype
was determined by conventional cytogenetic analysis on G-banded metaphase cells obtained from short-term culture.
View, CA). Normal mouse serum was from DAKO (Glostrup,
Denmark). Anti-CD1a was from Immuno Quality Products (Groningen, The Netherlands). In the case of direct immunofluorescence, 5 3 105 cells were incubated with the MAb for 15 min, red
cells were lysed with FACS lysing solution (Becton Dickinson) and
cells were fixed in a 0.5% paraformaldehyde/PBS solution. In the
case of combined indirect/direct immunofluorescence, 5 3 105
cells were first incubated with unconjugated anti-CD8b (IgG1;
Immunotech, Marseille, France) for 15 min. After 2 washings, cells
were incubated with rat-anti-mouse-IgG1-PE for 15 min, washed
twice and incubated with normal mouse serum to block the free
antigen-binding sites of the rat-anti-mouse antibodies. After 15 min
incubation, anti-CD8a-FITC and anti-CD4-PerCP MAbs were
added, after which the procedure of direct immunofluorescence
was followed. Three-color immunofluorescence analyses were
performed on a FACScan flow cytometer (Becton Dickinson).
Functional assays
Ex vivo cytotoxic activity of the cells was tested in a 4 hr target
cell lysis assay immediately after isolation, using murine P815
mastocytoma cells as (NK-resistant) targets and OKT3 (2 µg/ml) as
anti-CD3 bridging MAb, as previously described (van Gool et al.,
1993). This redirected cytotoxicity assay evaluates all T-cell
cytotoxic activity, irrespective of antigen specificity (Leeuwenberg
et al., 1985).
For T-cell stimulation, 2.5 3 105 immunomagnetically purified
CD41 TIL were cultured in 1 ml complete medium in 24-well
flat-bottomed tissue culture plates in the presence of anti-CD3
MAb UCHT1. This anti-CD3 MAb was shown to have a strong
stimulatory capacity in the absence of accessory signals (Verwilghen et al., 1991). Culture plates were coated by incubating the
wells with UCHT1 (5 µg/ml) in 300 µl PBS for 4 hr at 37°C and
then washed 3 times for 3 min with PBS. After 48 hr of stimulation,
cells were collected for immunophenotyping.
RESULTS
CD31 CD41 CD8a11 T cells accumulate in the tumor tissue
of patients with RCC
Freshly isolated TIL from RCC were studied for CD3, CD4 and
CD8a expression by 3-color flow-cytometric analysis. As shown in
Figure 1 for 3 representative patients, an aberrant CD41 CD8a11
T-cell population could clearly be identified in some tumor tissues
(Fig. 1a,b). The CD41 CD8a11 T-cell population is different from
the CD31 CD41 CD8adim population, which could also be detected
in many of the tumor samples (3.7% 6 3.2% of total TIL; .5% of
total TIL in 8 of the 27 tumor samples) but which could clearly be
separated in the dot plot from the CD41 CD8a11 T cells (Fig. 1b).
For comparison, thymocytes which contain abundant CD4 and
CD8a double-positive cells are also shown in Figure 1. Table I
gives an overview of all patients with RCC who have been studied.
In approximately one-third (8/27) of the patients, the CD41
CD8a11 T-cell subset represented more than 20% of CD31 TIL
and in one patient more than half of CD31 TIL. Significant
180
VAN DEN HOVE ET AL.
FIGURE 1 – CD4 and CD8a cell surface antigen expression on
freshly isolated TIL of 3 patients with clear cell RCC (a–c) and on
freshly isolated thymocytes (d). TIL were isolated from RCC tumor
tissue stained with anti-CD4-FITC, anti-CD8a-PE and anti-CD3PerCP; and analyzed on a FACS. FACS dot plots show the results of
CD4 and CD8a expression for the gated CD31 population. Thymocytes were isolated from the thymus of a 29-week-old fetus and
analyzed similarly.
proportions ($5%) of CD31 CD41 CD8a11 TIL were detected in
15 of 21 patients with clear cell RCC, whereas this subset was only
marginally present (,3%) in all 5 patients with papillary RCC and
in the one patient with sarcomatoid RCC, suggesting a possible
relation with the histological tumor type. Furthermore, the occurrence of this subset was not clearly associated with pathological
stage, tumor diameter, nuclear grade, cytogenetic abnormalities or
vascular invasion in this patient cohort (Table I). The CD41
CD8a11 T-cell subset was detected in similar proportions in tumor
tissue (6.5% 6 8.8% of total TIL) and tumor capsula (6.2% 6 7.9%)
and in the adjacent normal renal tissue, albeit in a lower proportion
(1.7% 6 2.3%). CD41 CD8a11 T cells were detectable in the
peripheral blood of some of the 27 patients as a tiny population
(0.4% 6 0.4% of total lymphocytes), which did not correlate with
the presence of its counterpart in the tumor (not shown).
CD31 CD41 CD8a11 TIL co-express CD8b but not CD1a and
strongly express early and late activation antigens
To further investigate the potential origin and in vivo significance
of the CD31 CD41 CD8a11 TIL, additional 3-color immunofluorescence analysis was performed on TIL from 3 patients containing
high proportions of CD41 CD8a11 T cells. This revealed that the
CD41 CD8a11 TIL subset expressed the T-cell receptor a/b (Fig.
2b) but not the T-cell receptor g/d (not shown); CD8b (Fig. 2d);
and strongly expressed CD69 (mean 6 SD 5 97% 6 2%), CD54
(99% 6 1%), HLA-DR (99% 6 1%) and CD28 (76% 6 10%) as
well. Only a minor fraction of the cells were positive for CD45RA
(15% 6 12%), CD25 (9% 6 7%), CD62L (18% 6 17%), CD
(16 1 56) (25% 6 9%), CD57 (33% 6 12%) and CD1a
(6% 6 2%).
CD31 CD41 CD8a11 CD8b1 TIL of patients with RCC display
no ex vivo cytolytic activity in an anti-CD3-redirected
cytotoxicity assay against murine P815 cells
To examine the function of the CD31 CD41 CD8a11 CD8b1
TIL subset, we first assessed the ex vivo cytolytic activity of
FIGURE 2 – Identification of CD31 CD41 CD8a11 CD8b1 cells
among TIL of RCC patient 5 by 3-color immunofluorescence. Freshly
isolated TIL were stained with anti-T-cell receptor a/b-FITC, antiCD8a-PE and anti-CD4-PerCP (a, b) or anti-CD8a-FITC, antiCD8b-PE and anti-CD4-PerCP (c, d) and analyzed on a FACS. FACS
fluorescence dot plots (a, c) show the expression levels (fluorescence
intensities on a 4-log scale) of CD4/CD8a cell surface antigens. CD41
CD8a11 cells were gated and analyzed for expression of T-cell
receptor a/b (b) and CD8b (d) in the 3rd color. FACS histograms (b, d)
show the relative cell number vs. the fluorescence intensity (on a 4-log
scale). Dotted line shows staining of control mouse IgG.
immunomagnetically purified CD31 CD8a1 TIL of 2 patients with
high proportions of CD31 CD41 CD8a11 CD8b1 TIL in 51Crrelease assays and compared this with the ex vivo cytolytic activity
of immunomagnetically purified CD31 CD8a1 T cells from the
peripheral blood of 3 healthy donors. FcgR-expressing mouse
mastocytoma P815 cells and anti-CD3 MAb were used to detect
cytotoxic activity regardless of antigen specificity of the cytotoxic
effector cells (%SRP815). As can be seen in Figure 3, the CD31
CD8a11 TIL of both patients failed to lyse P815 cells in the
anti-CD3-redirected cytotoxicity assay, whereas the freshly isolated peripheral blood CD31 CD8a1 T cells of 3 healthy donors
demonstrated a strong cytolytic activity in this assay. No significant
LAK activity (evaluated by 51Cr-release in the absence of anti-CD3
MAb) was detected in any of the experiments (data not shown).
Anti-CD3-stimulated CD31 CD41 CD8a11 CD8b1 TIL
demonstrate a defective IL-2Ra up-regulation
when compared with CD31 CD41 CD8a2 TIL
Subsequently, we investigated the up-regulation of the IL-2Ra
chain (IL-2Ra, CD25) on anti-CD3-triggered CD31 CD41 CD8a11
CD8b1 and CD31 CD41 CD82 TIL. As shown in Figure 4, CD31
CD41 CD8a11 CD8b1 TIL demonstrated a strongly reduced
capacity to up-regulate IL-2Ra when compared with CD31 CD41
CD8a2 TIL. In both experiments, we also noted that the expression
level (mean fluorescence intensity) of IL-2Ra among the positive
cells was higher on CD31 CD41 CD8a2 (mean 6 SD 5
1,143 6 304) than on CD31 CD41 CD8a11 CD8b1 (305 6 65)
TIL (Fig. 4b,c). The addition of IL-2 (10 U/ml) to the anti-CD3stimulated CD41 TIL did not significantly enhance IL-2Ra expression on any subset (data not shown).
‘‘DOUBLE-POSITIVE’’ TUMOR-INFILTRATING LYMPHOCYTES
FIGURE 3 – Ex vivo cytotoxic activity of CD31 CD8a1 T cells
isolated from tumor tissue of 2 patients with primary RCC and from
peripheral blood of 3 healthy donors. CD31 CD8a1 T cells were
purified with Dynabeads (.95% pure). Purified CD31 CD8a1 TIL of
patients 1 and 2 contained 55% and 35% of CD31 CD41 CD8a11
CD8b1 T cells, respectively. Murine P815 cells and anti-CD3 MAbs
were used to detect cytotoxic activity regardless of antigen specificity
(%SRP815). Results are expressed as mean of triplicate experiments.
FIGURE 4 – Induction of IL-2Ra (CD25) on CD41 TIL of RCC
patient 3. CD41 TIL were isolated from RCC tumor tissue and
stimulated for 48 hr with immobilized anti-CD3. Cells were then
stained with anti-CD4-FITC, anti-CD25-PE and anti-CD8a-PerCP and
analyzed on a FACS. The FACS fluorescence dot plot shows the
expression (fluorescence intensities on a 4-log scale) of CD4 and CD8a
cell surface antigens (a). The CD41 CD8a11 and CD41 CD8a2
populations were further analyzed for CD25 expression. FACS histograms (b, c) show the relative cell number vs. the fluorescence intensity
of CD25 staining (on a 4-log scale). Dotted line shows staining with
control mouse IgG. Similar results were obtained with CD41 TIL of
another patient.
DISCUSSION
We report the identification of an enriched CD41 CD8a11
CD8b1 T-cell subset in the tumor tissue of patients with RCC. In
nearly one-third (8/27) of the patients, this subset comprised more
than 20% of total CD31 TIL, and in one patient more than half of
CD31 TIL displayed this phenotype.
The presence of a substantial population of CD41 CD8a11
CD8b1 T cells outside of the thymus was surprising since
peripheral T cells with this phenotype are very rarely detected.
Persistent expansions of CD41 CD8a11 T cells in the peripheral
181
blood are extremely rare (Tonutti et al., 1994). A minor subset
(3–10%) of double-positive CD31 CD41 CD8a1 intra-epithelial
lymphocytes has been described in the small intestine of normal
mice (Mosley et al., 1990; Lefrançois, 1991). Relevant to this,
peripheral CD41 CD82 T cells have been shown to co-express the
CD8a chain when they migrate into the small intestinal epithelium
(Reimann and Rudolphi, 1995), and CD41 CD82 T-cell clones
have been shown to co-express CD8a when cultured in the
presence of IL-4 (Paliard et al., 1988; Hori et al., 1991). The CD41
CD8a11 CD8b1 TIL subset is, however, not an intratumoral
homologue of the intra-epithelial double-positive T cells or the
IL-4-stimulated T-cell clones because the latter do not express the
CD8b chain (Lefrançois, 1991; Hori et al., 1991). Up-regulation of
CD8a chains on CD41 cells, however, may explain the emergence
of a distinct CD31 CD41 CD8adim CD8b2 TIL subset observed in
some patients (Fig. 1b,c). The local production of IL-4 in RCC as
supported by some reports (Schoof et al., 1993; Maeurer et al.,
1995) may explain the induction of these CD8adim-positive CD41
TIL.
The CD41 CD8a11 CD8b1 T cells in RCC tumor tissue may
have several origins. First, they may derive from peripheral blood
double-positive cells (which are probably immature thymic emigrants). In the thymus, CD31 CD41 CD8a1 cells represent
intermediates in T-cell differentiation, which are derived from
CD42 CD82 precursors and which further differentiate into mature
CD31 CD41 CD8a2 or CD31 CD42 CD8a1 T cells (Kisielow and
von Boehmer, 1995). However, this TIL subset did not express the
CD1a cell surface marker of its counterpart in the thymus and
displayed a different CD8a expression pattern from that of
double-positive thymocytes (compare b with d in Fig. 1). If these
cells are thymic emigrants, we have to assume that they lost CD1a
expression upon migration into the tumor. A second (more likely)
possibility is that the CD31 CD41 CD8a11 CD8b1 TIL arise in
situ by further differentiation of mature single-positive CD41 T
cells, which up-regulate CD8a and ultimately also express CD8b.
Third, CD42 CD81 single-positive T cells which acquire CD4
expression also have been described (e.g., following infection with
human lymphotropic herpes virus 6 [HHV-6]; Lusso et al., 1991).
The possibility that the CD31 CD41 CD8a11 CD8b1 TIL subset
originated from CD31 CD42 CD8a11 CD8b1 cells which upregulated CD4 thus should also be explored. Irrespective of their
differentiation pathway, the aberrant T-cell phenotype most likely
results from interaction with the tumor micro-environment, with
either epithelial cell surface molecules or secreted molecules.
With respect to their function, the expression of both CD8
isoforms together with CD4 suggests a functional capacity to
interact with both MHC class I and class II molecules, broadening
their immunological competence and contributing to high-affinity
attachment to target tissues upon recognition of foreign antigen by
the T-cell receptor. However, in contrast to freshly collected
peripheral blood CD31 CD8a1 T cells from controls, freshly
collected CD31 CD8a1 TIL containing high proportions of CD31
CD41 CD8a11 CD8b1 TIL (55% and 35%, respectively) demonstrated no anti-CD3-redirected cytotoxic activity against P815
cells. This strongly suggests that the CD31 CD41 CD8a11 CD8b1
TIL may not fulfill a cytotoxic effector function in vivo. Furthermore, CD31 CD41 CD8a11 CD8b1 TIL demonstrated a markedly
high expression of CD69, CD54 and HLA-DR activation markers
but were nearly negative for CD25. This combination of early and
late activation markers together with a low expression of CD25 is
indicative of a particular in vivo activation state that cannot be
mimicked by in vitro activation of T cells via the T-cell receptor/
CD3 complex (Iannone et al., 1994). Furthermore, in contrast with
CD31 CD41 CD8a2 TIL from the same tumor sample, CD31
CD41 CD8a11 CD8b1 TIL were incapable of expressing IL-2Ra
in response to anti-CD3 triggering, suggesting a decreased capacity
for IL-2-dependent clonal expansion. A marked defect in IL-2Ra
expression of some TIL samples was reported in the study of
Alexander et al. (1993), and our results suggest that the enriched
182
VAN DEN HOVE ET AL.
CD31 CD41 CD8a11 CD8b1 TIL subset may importantly
contribute. Finally, we have preliminary data which suggest that
these cells also have a lower capacity to produce cytokines.
Defective cytotoxic activity and lack of IL-2Ra expression upon
anti-CD3 triggering collectively raise the possibility that CD31
CD41 CD8a11 CD8b1 TIL represent T cells in an anergic state, as
has been suggested to occur in the tumor micro-environment as a
consequence of incomplete stimulation by tumor cells lacking
B7-co-stimulatory molecules (for review, see Chen et al., 1993).
Alternatively, we cannot exclude the possibility that this subset
represents a highly specialized effector population with a discrete,
as yet unidentified function.
Our characterization of true ‘‘double-positive’’ T lymphocytes
among RCC TIL expressing CD4 and both CD8 chains may
stimulate further research to reveal their origin, antigenic specificity and potential in vivo significance. An elucidation of their exact
function may provide a better understanding of host-tumor interactions at tumor sites and may be of important clinical interest
regarding the potential use of TIL in adoptive cellular immunotherapy.
ACKNOWLEDGEMENTS
The authors thank Dr. P. Beverley, Imperial Research Cancer
Fund, London, UK, for generously providing the anti-CD3 MAb
UCHT1, and Mrs. V. Boets, Miss K. Brugmans and Miss M. Adé
for providing skilled technical assistance in the preparation of renal
tissue samples. This work was supported by a grant from the
Nationaal Fonds voor Wetenschappelijk Onderzoek (G.3123.94)
and by a grant for cancer research from the Algemene Spaar—en
Lijfrentekas, Brussels, Belgium. This text presents research results
of the Belgian program on Interuniversity Poles of Attraction
initiated by the Belgian State Prime Minister’s Office, Science
Policy Programming. The scientific responsibility is assumed by its
authors. S.V.G. is a postdoctoral researcher of the Fonds voor
Wetenschappelijk Onderzoek Vlaanderen, Brussels.
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