Download T-Cell Subset Analysis of Lewis Lung Carcinoma

[CANCER RESEARCH 52, 6507-6515, December 1, 1992]
T-Cell Subset Analysis of Lewis Lung Carcinoma Tumor Rejection: Heterogeneity
of Effectors and Evidence for Negative Regulatory Lymphocytes Correlating
with Metastasis
C o h a v a G e l b e r , L e a E i s e n b a c h , M i c h a e l F e l d m a n , a n d R o b e r t S. G o o d e n o w
Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, California 94305 [C. G.]; Baxter Healthcare Corporation, Santa Ana,
California 92705 [R. S. GJ; and Department of Cell Biology, the Weizmann Institute, P.O. Box 76100, Rehovot, Israel [L. E., M. F.]
ABSTRACT
We have analyzed the phenotypes of the T-cell subsets generated in
response to Lewis lung carcinoma clones in C57BL/6J recipients. The
metastatic derivative, which expresses low levels of H - 2 K b gene, predominantly elicited CD8, V~8, and VB9 § T-cells. The nonmetastatic
clone expressing high levels of H-K b gene triggered a more heterogeneous response of V~-5, -6, -8, -9, and -11 CD8 § T-cells. Comparison
of the T-cell receptor (TCR) expression of the T-cells infiltrating the
tumor site with the lymphocytes in the periphery of tum0r-bearing animals revealed a pattern of homing of CD4 § T-cells bearing V~-5, -6,
and -11 TCR chains and CD8 § T-cells bearing V~-5, -6, -9, and -11.
Depletion of V~5 or V~6 + T-cells correlated with accelerated tumor
growth, implying their protective role as tumor-specific effectors and
consistent with the cytotoxicity of T-cells with this TCR phenotype.
V/~ll TCR expression in the tumor-infiltrating lymphocytes increased
with the tumor size. Depletion of Vt/11 § T-cells enhanced resistance to
primary tumor growth and conferred protection from metastasis in recipients cleared of V~5 and VB6 T-cell subsets. Those results suggest
that tumor-specific effectors as well as negative regulator T-cells home,
infiltrate, and coexist in the tumor site.
INTRODUCTION
A considerable body of evidence supports the concept that
tumor-bearing hosts can mount effective cellular immunity that
can be manipulated for clinical benefit in treating neoplastic
diseases, especially solid tumors such as melanoma, lung,
breast, renal, or colon carcinoma (1-12). Despite the capacity
to generate such cellular defenses, however, adoptive immunotherapy has proved to be only partially successful in achieving
significant tumor regressions, underscoring the need to further
elucidate the basic nature of tumor immunity for maximizing
its potential for limiting tumor progression and metastases.
In many respects the underlying issues that challenge cancer
immunotherapy are central to immunology. For example, the
recent characterization of tumor antigens as normal self-cellular peptides (13-15) predicts that their immune recognition by
the tumor-bearing host requires a break in immunological tolerance or the peripheral anergy of lymphocytes responsive to
these antigens. As a result, the small number of potentially
immunogenic self-peptides presented by tumors may not stimulate a vigorous immune response. Furthermore, the ability to
elicit and sustain effective antitumor immunity might be hampered by factors in the host that serve to down-regulate selfreactivity.
Just as the heterogeneity of pathogenic lymphocytes is critical to strategies for ameliorating autoimmunity through T-cell
ablation, the identification of distinct lymphocyte subsets with
the greatest tumoricidal effect could prove essential in devising
Received 5/29/92; accepted 9/24/92.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
new forms of immunotherapy for cancer. The critical questions
are whether there are (a) antigens common to tumors of similar
origin and (b) similarities in the T-cell responses to these tumor
antigens between HLA-matched patients.
In the case of melanoma and other malignancies (16-29), 1
there is strong evidence for the existence of tumor-associated
antigens common to the neoplasms. A number of investigators
have shown that T-cells from different patients can cross-react
with allogeneic tumor lines as long as they express HLA-A2
gene products (23). 1 This raises the possibility that within
groups of melanoma patients with the HLA-A2 haplotype, the
cellular immune responses to tumor antigens may display
shared patterns of TCR 2 V gene usage. Support for this notion
stems from the observed TCR V gene usage reported for MHCrestricted T-cell responses to defined antigens such as myoglobin and cytochrome c (30-33).
In an attempt to elucidate the heterogeneity and phenotype of
cellular effectors generated in an animal tumor model for which
cellular immunity clearly regulates tumor growth, we characterized the T-cell response against the C57BL/6 3LL transplanted into syngeneic recipients. High metastatic, low immunogenic clones of 3LL express low levels of H - 2 K b MHC
antigens. These cells metastasize in syngeneic C57BL/6J or
recombinant strains of mice sharing H-21Y" (34), whereas transfection of syngeneic (35) or allogeneic (36) H - 2 K genes abrogates the metastasis of the transplanted tumor in H - 2 K / H - 2 D
compatible recipients. Since the transfected cells are potent
inducers of H-2K-restricted and alloreactive H-2K CTL (35,
36), 3LL metastasis appears to be controlled by MHC-restricted CTL responses.
We therefore used the nonmetastatic H-2K-positive clone of
the 3LL tumor to characterize the heterogeneity of the anti3LL T-cell response reflected in the T-cell receptor V gene
usage of the effectors generated in syngeneic recipient animals.
To further evaluate the role of the distinct T-cell subsets identified, we also used in vivo T-cell depletion by using the TCR
VB-specific monoclonal antibodies to demonstrate the effects of
specific lymphocyte depletion on primary as well as metastatic
growth.
MATERIALS AND M E T H O D S
Mice. C57BL/6J mice, 8-12 weeks old, were obtained from The
Jackson Laboratory. For tumor injection or T-cell depletion, groups of
at least 6 animals were used for each experiment and for every time
point or measurement.
Y. Kawakami, unpublished data.
2 The abbreviations used are: TCR, T-cell receptor; MHC, major histocompatibility complex; 3LL, Lewis lung carcinoma; CTL, cytotoxic lymphocytes; PBS,
phosphate-buffered saline; i.f.p., intrafootpad; MLTC, mixed lymphocyte tumor
culture; FACS, fluorescence-activated cell sorter; TIL, tumor-infiltrating lymphocytes; PBL, peripheral blood lymphocytes.
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T-CELL SUBSET ANALYS1S OF 3LL TUMOR REJECTION
Tumor Cells. The D122, a highly metastatic derivative, and A9, a
low metastatic subclone, were both derived from the original C57BL/6J
3LL cell line (34). A9, D122, and various D 122-transfected cells were
maintained in Dulbecco's modified Eagle's medium containing 10%
fetal calf serum, glutamine, combined antibiotics, sodium pyruvate, and
nonessential amino acids. H-2K-transfected clones were selected and
maintained in 400 #g/ml geneticin (G418 sulfate; Gibco) as previously
described (35, 36).
Antibodies. Anti-CD4 (L3T4) and anti-CD8 (Lyt 2.1) fluoresceinated or phycoerythrin-conjugated monoclonal antibodies were purchased from Becton Dickinson (San Jose, CA). Hybridomas producing
monoclonal antibodies directed against determinants expressed on
TCR V/~ families were generously provided by various investigators:
V~3 (KJ25, hamster), V/~8 (F23.1, mouse IgG2a), and V~517a (KJ23,
mouse) from Kappler and Marrack; VB5 (MR9-4, mouse IgG), Vfl6
(RR 4-7, rat IgG~), V~9 (MR 10-2, mouse), V~I 1 (RR3-15, rat IgGK),
and V~13 (MR 12-4, mouse) from O. Kanagawa; and V~14 (14.2, rat
IgMK) from D. Raulet.
Tumor Development and Metastatic Assays. Single cell suspensions
of 1-2 x 105 cells in 50 #1 PBS were inoculated i.f.p, into 12- to
15-week-old mice of the various strains. When the tumor diameter
reached 8-9 mm, the tumor-bearing leg was amputated. Amputation
was performed by using a 3-0 silk tie, which is ligated beyond the
iliofemoral joint of the hind leg to prevent bleeding. Mice were sacrificed 30 days postamputation, and their lungs were assayed for metastatic load by measuring lung weight and the number of nodules visualized by fixation in Bouian solution.
In Vitro Cytotoxicity. Mice were immunized with a single dose of
106 viable tumor cells into the pinna of the ear. Spleens were removed
at 9-10 days, and the cells were restimulated in vitro for 5 days on
monolayers of (5000 rads) irradiated cells treated with mitomycin C (80
#g/ml) in RPMI-10% fetal calf serum. Viable lymphocytes were separated by centrifugation with the use of Lympholyte-M (Cederlane, Ontario, Canada), admixed at different ratios with 1000 [75Se]selenomethionine-labeled (Amersham) target cells (37), in U-shaped microtiter
wells, and incubated at 37~ in 5% CO2 for 16 h. Cultures were terminated by centrifugation at 300 • g for 10 min at 4~ prior to counting.
The supernatants (100 ~1) were counted in a Beckman gamma counter.
Percentage of specific lysis was calculated as
release - spontaneous release
Maximal release - spontaneous release
E x p e r i m e n t a l 75Se
x
100
Secondary MLTC. Lymphocytes from primary cultures were
washed and restimulated with fresh irradiated and mitomycin C-treated
tumor cells. Five days later the ceils were used for cytotoxicity assay
and/or TCR V~ chain analysis (FACS).
Preparation of TIL. Excised tumors were minced and treated with
the following enzymes: col|agenase type V (Sigma) at 2 mg/ml, DNase
(Sigma) at 0.2 mg/ml, and hyaluronidase (Sigma) at 0.2 mg/ml in
Hanks' balanced salt solution buffer for 2-3 h at room temperature.
The lymphocytes were separated on Lympholyte-M (Cederlane),
washed, and then depleted of B-lymphocytes by using magnetic beads
coated with goat anti-mouse immunoglobulin (Dynal). The cells were
then stained with CD4 and CD8 fluoresceinated or phycoerythrin-conjugated antibodies for FACS analysis or sorting.
In Vivo Depletion of T-Cell Subsets. C57BL/6J mice (6-10/group)
received weekly i.p. injections of 300 #g of the protein A or protein G
(Pharmacia, Uppsala, Sweden)-purified antibodies starting at least 12
days prior to tumor transplantation. Two mice selected randomly from
each group were bled 24 and 48 h after the first injection and every 21
days thereafter. The PBL were stained with TCR V~-specific monoclonal antibodies to monitor the extent of specific T-cell depletion by
FACS analysis. Depletion of the relevant T-cells was observed as early
as 48 h and as late as 5 days following injection. Repopulation of the
depleted T-cells started no sooner than 2 weeks after the last injection
of antibody. We thus determined that weekly injection successfully
maintains the depletion of the relevant T-ceU subsets.
Immunofluorescence Staining. A sample of 2 x 106 cells in 100 #l
PBS was incubated for 30 min at 0~ with 10 #g/ml of the various
purified monoclonal antibodies. After 2 washings with PBS plus 0.02%
sodium azide and 1% fetal calf serum, the cells were incubated under
the same conditions with 1 gl of fluoresceinated or phycoerythrinconjugated goat anti-mouse immunoglobulin (Becton Dickinson). After
2 further washings the cells were analyzed on the FACS STAR. For
preparative sorting, the washes were done in PBS without azide and
under sterile conditions.
RESULTS
T C R Diversity of 3LL Tumor-specific T-Lymphocytes. To
determine the heterogeneity of the T-cell response induced
against the 3LL tumor, we generated M L T C reactions of
C 5 7 B L / 6 J spleen cells against the metastatic (D122) and nonmetastatic (A9) 3LL t u m o r clones. Splenocytes from
C 5 7 B L / 6 J mice immunized with A9 or D122 cells were resensitized in vitro with the matched t u m o r cells. T-cells were analyzed by FACS for TCR-V/~ expression by using a panel of
monoclonal antibodies to stain the CD8-positive subpopulation. The metastatic derivative D122, that expresses low levels
of the M H C class I H - 2 K ~ gene, predominantly elicited CD8-,
V~8-, and V/39-positive T-cells; whereas the nonmetastatic A9
clone, expressing high levels o f H - K b gene, triggered a more
heterogeneous response of V~5, V/~6, V/~8, and V/~ll CD8positive lymphocytes (Table 1). To compare primary versus
secondary T-cell responses generated in the M L T C , we also
Table 1 Expression of TCR V~ domain on CD8-positive T-cells generated in MLTC reactions against 3LL tumor clones
Mixed lymphocytetumor cultures were prepared with splenocytesfrom 3-6 immunizedC57Bl/6J mice and stimulated in vitro with 3LL tumor clones and H-2K
(class I MHC) transfectants (35, 36). CD8-positive T-cells were sorted out from MLTC preparations and stained with the different TCR-VB-specificmonoclonal
antibodies. Staining and flow cytometric analysis were carried out as described in "Materials and Methods." Standard deviationsare shown.
A9
D122
A9 2nd M L T C
A9-K bm~
D 122-K bmi
D 122-K d
D122-K k
V~3
V~5
V~6
VB8
V~9
VB11
V~13
V~14
V~17
2.60*
0.13"*
0.00
0.04
2.60
0.18
0.68
0.03
3.06
0.15
I. 12
0.06
0.79
0.04
9.10
0.46
0.60
0.05
16.67
1.25
2.75
0.64
15.35
0.77
17.77
0.80
16.18
0.81
7.70
0.39
0.90
1.73
8.05
0.61
7.80
0.39
6.68
0.33
3.80
0.19
8.01
0.40
6.20
0.34
11.10
0.67
2.43
0.19
10.77
0.54
10.34
0.52
10.45
0.52
12.94
0.65
2.90
0.15
9.60
0.11
2.89
0.20
5.47
0.27
5.23
0.26
1.78
0.09
5.51
0.28
4.40
0.22
1.60
0.14
9.64
0.72
5.36
0.27
5.69
0.28
4.24
0.21
7.42
0.37
3.60
0.18
2.20
0.15
2.78
0.21
1.78
0.09
3.39
0.17
2.51
0.13
3.71
0.19
1.90
0.09
1.60
0.05
2.00
0.02
0.73
0.04
0.77
0.04
0.29
0.02
0.93
0.05
0.40
0.02
0.67
0.002
0.90
0.07
0.06
0.003
0.03
0.02
0.05
0.03
0.20
0.01
* % of stained CD8 positiveT cells.
** S.D.
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T-CELL SUBSET ANALYSIS OF 3LL T U M O R REJECTION
analyzed the T-cell heterogeneity of secondary cultures. A sec- Thus, it would appear that the V~I 1-positive T-cells specifically
ondary MLTC of C57BL/6J spleen cells reacted against A9 home to the site of the A9 tumor in recipient syngeneic animals.
tumor cells revealed further enrichment of V~35, V~6, and
The results depicted in Fig. 2 also show the levels of V/33
V/311-CD8 positive T-cells (Table 1). The T-cell receptor rep- TCR expression in TIL as compared to circulating blood lymertoire of C57BL/6J MLTC-derived lymphocytes proliferating phocytes for animals bearing 8-mm tumors: 5% CD4 and 2%
in response to the 3LL tumor clones expressing allogeneic CD8 in TIL versus 0% for both CD4 and CD8 in PBL. Since the
H-2K restriction elements (H-2K bin1, H-2K a, and H-2K k) dem- mean expression of V~33 TCR on CD4- and CD8-positive Tonstrated a similar pattern of the T-cell heterogeneity induced lymphocytes in normal PBL of C57BL/6J mice averages 3%
by the 3LL A9 tumor clone expressing the normal syngeneic (38), the proportion of these T-cells may actually be specifically
H-2K b in which V~/5, V~6, V~/8, V~/9, and V~I 1 TCR predom- down-regulated or depleted in tumor-bearing mice. In contrast,
inated (Table 1). Thus, the immunogenic 3LL tumor cells of the the 30-35% CD8 V/38-positive T-cells found in the PBL of
nonmetastatic phenotype expressing the H-2K b antigen elicit a normal or tumor-bearing mice appears to be markedly undermore heterogeneous T-cell response than the metastatic H-2K b- represented in the TIL population since there were 0% CD4
negative D122 tumor as reflected in the TCR V~/gene usage of V/38 and only 2.8% for the CD8 V138-positive T-cells detected in
the MLTC-derived lymphocytes. The expression of foreign the footpads of tumor-bearing animals. The absence of this V~8
H-2K alleles of class I MHC on the cell surface of the 3LL subpopulation in TIL might be due to their failure to home to
tumor clones (A9 and D122) generated a similar although dis- the tumor site.
V~I 1 T-Cells in TIL Increase with Tumor Size. To establish
tinct degree of T-cell heterogeneity, reflecting the characteristic
immunogenicity of the tumor-bearing foreign MHC gene prod- the relevance of the observed V~I 1 T-cell infiltration on the
nonmetastatic growth of A9, two groups of C57BL/6J mice
ucts.
were
given injections i.f.p, of A9 tumor cells within an interval
Specificity or Reactivity of Various V~/T-Cell Subsets. To
of
8
days.
When the tumors reached a size of 5 mm (22 days
correlate the T-cell phenotypes with biological function or cyposttumor
transplantation) or 8 mm (30 days posttumor transtotoxicity against the tumor, the MLTC-derived double-stained
plantation),
the TIL were harvested and prepared as described
CD8 and V~ T-cells were fractionated and used in a 16-h
cytotoxicity assay against A9 (see "Materials and Methods"). (see "Materials and Methods"). The infiltrating T-cells were
As depicted in Fig. 1, the bulk anti-A9 CTL were capable of analyzed with anti-V135, -V136, or -V~I 1 monoclonal antibodies
lysing the respective tumor targets to 63% at an effector:target together with CD4 or CD8 reagents. As shown in Fig. 3, CD4
ratio of 5-500:1; whereas as few as 5000 bulk CD8 sorted V/35-positive T-cells were represented at equivalent frequencies
T-cells (effector:target ratio of 1:1) could mediate target cell in PBL as compared to TIL harvested from animals bearing 5lysis up to 80%. Thus, removal of CD4-positive T-cells from and 8-mm tumors. However, the CD8 Vt35-positive T-cells apMLTC reactions (comprising 45-55% of the population) in- peared to actually decrease during tumor progression from
12.5% in the TIL harvested from animals bearing 5-mm tumors
creased tumor cell lysis in vitro as much as 100-fold, regardless
down to 6.3% in TIL harvested from animals bearing 8-mm
of the degree of CD8 enrichment. The different CD8-positive
tumors. In contrast, the percentage of CD4 and CD8 V~36fractionated T-cell subsets (V~3, V~/6, V{/8, V~/9, V~/11) showed
positive infiltrating lymphocytes did not appear to vary as a
low levels of target cell killing at the effector:target ratio tested
function of the tumor size. There were 14% CD4-positive T(1:1). It is intriguing that although a variety of T-cell V~ phecells in PBL as compared to 20 and 21% CD4 lymphocytes in
notypes are stimulated in the MLTC, only the V~5 subpopula5- and 8-mm TIL, respectively. Twenty % V~6 CD8 T-cells
tion appears lytic for the tumor in an in vitro assay, denoting
could be detected in PBL as compared to 5.0 and 20.8% V~6
that the homing of different subsets of lymphocytes to the tuCD8-positive lymphocytes in 5- and 8-mm TIL, respectively.
mor site does not always correlate with functional lytic activity
CD4-positive VI311 T-cells in TIL harvested from 5-mm tuand may be due to second signals such as cytokines.
mors represented an average of 25% of the infiltrating lymphoTCR Heterogeneity of A9 TIL. The T-cell response gener- cytes as compared to 7% of the T-cells in the periphery of the
ated to 3LL at the site of inoculation was analyzed in syngeneic same animals. At 8 mm, the V1311 CD4 T-cells represented
recipients. The 3LL tumor was found to be infiltrated with close to 50% of the TIL population. The same pattern was also
different types of hematopoietic cells, primarily granulocytes, observed for CD8-positive V/311 T-cells, i.e., this subset inmacrophages, B- and T-lymphocytes (data not shown), as has creased from 12% in the periphery to 17% in 5-mm tumors and
been reported for many other malignancies studied to date (37). to 33% in 8-mm tumors. Thus, V~I 1 T-cells appear to respond
To examine the lymphocyte fractions, tumors excised from to 3LL tumor antigens, specifically home to the tumor site,
30-35 mice were digested with enzymes (see "Materials and infiltrate, and expand to large numbers concomitantly with inMethods") and then depleted of B-lymphocytes by using mag- creased tumor mass.
netic beads coated with goat anti-mouse immunoglobulin. CD4
In Vivo Depletion of Different T-Cell Subsets. To evaluate
and CD8 T-cells, which usually did not exceed 1% of the total the possible function of the various T-cell subsets observed in
number of cells, were sorted on the FACS and then stained with response to challenge with the 3LL tumor, we used the TCRthe various V~-specific T-cell reagents.
V~-specific monoclonal antibodies to differentially eliminate
The frequency of the V~6 CD8-positive T-cells in the TIL specific T-cell subsets from the periphery of recipient animals.
reflected their level in the PBL of normal and tumor-bearing To examine the consequences of this treatment on the primary
animals, whereas V/35 CD8-positive T-cells were underrepre- tumor growth and lung metastasis, C57BL/6J mice received
sented in the TIL as compared to the lymphocytes in the pe- weekly i.p. injections of 300 ug of the purified antibodies startriphery (Fig. 2). CD4 or CD8 V~I 1-positive T-cells were de- ing 12 days prior to tumor transplantation (see "Materials and
posited in the largest numbers at the tumor site comprising up Methods"). Two mice from each group were randomly selected
to 32% of total CD8 or 50% of the total CD4 lymphocytes. for bleeding every 21 days and the PBL were stained with TCR
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T-CELL SUBSET ANALYSIS OF 3LL TUMOR REJECTION
V~-specific monoclonal antibodies to monitor the status of specific T-cell depletion. As shown in Fig. 4.4, depletion of CD8positive lymphocytes resulted in an accelerated rate of primary
Bulk CTL
tumor growth. Conversely, significant retardation of A9 tumor
growth was observed in mice depleted of (a) V/311, (b) the '.:- 70
combination of V/~5 plus V~6 plus V/~I 1, or (c) CD4 T-cells in
....----0-agreement with the role of these T-cell subsets as possible negative regulators suggested from the patterns of T-cell infiltra~
60
tion. The rate of A9 tumor growth in V/~3-, V/55-, V~6-, V~8-,
or V~9-depleted mice generally corresponded to that of the
control animals (PBS-treated groups; Fig. 4B), showing little or
~. 50
no effect on tumor progression.
Parallel T-cell depletion studies were performed with the
H-2Kb-negative, metastatic derivative of 3LL, D122. Mice depleted of the discrete subsets of T-cells prior to transplantation
O0
200
300
400
500
600
of the D122 tumor cells caused retardation of primary tumor
cells
(xtO00)
growth in recipients depleted of V~9 which represented 9.6% of
CD8 in MLTC (see Table 1); whereas acceleration of primary
tumor growth in the first 30 days of the experiment was observed in animals depleted of V~5 or V/~6 T-cells (Fig. 4C).
Therefore, in spite of the different metastatic phenotypes of the
A9 and D 122 3LL clones, both appear to share antigenic specificities that induce the stimulation of V~5 and V~6 T-cell
80
~
Vb5
p
effectors as well as possible negative regulators (predominantly
~,
Vb6
/
V~9 for D122 and V~I 1 for A9 tumor).
- - vb3
/
Tumor Progression and Metastasis. The A9 derivative of := ,o -- -- A
- m - vb9
/
3LL (expressing H-2K b) has been shown to be immunogenic ~.
60
I
Vb8
/
/
and to elicit a vigorous cytotoxic immune response in immu5o
/
Sorted CD8 positive
nocompetent recipients to account for its nonmetastatic phe- ~,
ets
notype (34, 36). In immunocompromised mice, such as T-cell~.
4o
deficient (nu/nu) but not natural killer-deficient (Bg/Bg) mice,
A9 demonstrates an aggressive phenotype including metastasis
3o
to the lungs, although the number of lung metastases is consid2o
ered very low as compared to D122 in nu/nu mice (34, 35; Fig.
5). Thus, T-cells are the major effectors central to the cellular
10
anti-3LL response controlling growth in transplanted recipients. To demonstrate the efficacy of the effectors characterized
0
0
1
2
3
4
5
6
in vitro to control tumor growth in vivo, we depleted the various
ceils (xl000)
T-cell subsets with the V/~ monoclonals to measure the effect on
Fig. 1. Reactivity of various T-cell subsets in killing the 3LL-derived clone A9.
tumor growth. To test the effect of T-cell depletion on metasCytotoxicity assays were used to measure reactivity of MLTC-derived CTL,
tasis, we monitored the incidence of lung metastases among sorted
subsets (CD8 + V3-TCR) as compared to heterogeneous population with
T-cell-depleted animals following amputation of the limb bear- bulk preparation containing CD4 + CD8 T-cells as described in "Materials and
Methods." The results shown represent the average of triplicates.
ing the primary transplanted tumor.
At 30-45 days following amputation, A9 appeared to be
moderately metastatic in mice depleted of T-ceU subsets in the the A9 tumor presumably by reducing resistance to the tumor
following descending order: CD8 > V/35 > V~6 > VB8 > V~9 mediated by these effectors. Alternatively, elimination of the
lymphocytes (Fig. 5). Thus, the consequence of elimination of V~5 or V~6 T-cells in combination with V/311-positive T-cells
these T-ceU subsets demonstrates an essential role for the CD8 leaves the animals completely susceptible to A9 tumor progresVB5 and V#6 (and to a lesser extent the V158 and VB9)-specific sion. As a result, the Vfll l-positive T-cells may negatively regT-cell response against the A9 tumor. In contrast, removal of ulate T-cell responses controlling tumor growth in such a way
peripheral CD4, V/~3, V/~I 1, or V/~17 T-cells (used as a negative that their elimination enables the host immune system to
control in VB17a-negative C57BL/6 mice) did not promote the mount an adequate tumor-specific T-cell response to restrain
malignancy of the A9 tumor (Fig. 5). Interestingly, lung metas- and limit A9 primary as well as metastatic growth. In other
tasis did not occur with the combined depletion of V~5 plus words, the V~I 1 subpopulation actually promotes or enhances
VB6 plus V~I 1 T-cells; whereas clearance of either the VB5 or the metastatic potential of 3LL by decreasing the effectiveness
VB6 individual lymphocyte subsets alone resulted in increased of V/35/6 CTL.
tumor malignancy. This suggests that the VB 11-positive subset
Timing of VBI1 T-Cell Depletion and Tumor Retardation.
may exert a negative effect on the 3LL-specific T-cell response To study the effect of the timing of depletion on tumor growth,
to the A9 tumor, specifically the V~5 and VB6 CTL which C57BL/6J mice were depleted of V/511 T-cells at various time
presumably control tumor growth in the syngeneic host. There- points relative to tumor transplantation (i.e., 12 days prior to
fore, elimination of either one of the VB5 or V/~6 T-cells in the inoculation as well as 2 and 14 days following tumor transplanpresence of VBI 1 lymphocytes increases the dissemination of tation). At 14 days post-i.f.p, transplantation the tumor is still
6510
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T-CELL
sot
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SUBSET
ANALYSIS
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CD4
3o
9
PBL
[]
TIL
9
3
5
6
8
9
11
V[3 specific antibodies
.,.-..
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O
uo
C.d
0
3
5
6
8
9
9
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growth of a subsequent tumor challenge (35, 36). In the present
study, we have found that differential expression of class I
M H C on the cell surface of the 3LL tumor clones (A9 and
D122) leads to characteristic tumor immunogenicity (34-36)
exemplified by distinguishable TCR differences in response
triggered by the 3LL tumor-specific antigens in association
with H - 2 K b class I MHC restriction element. D 122, the metastatic derivative of 3LL which expresses low levels of the M H C
class I H - 2 K b gene, predominantly elicited CD8-, V~8-, and
V~9-positive T-cells; whereas A9, the nonmetastatic clone expressing high levels of H - K t' gene, triggered a more heterogenous response of V~5, V~6, V~8, V/39, V~I 1, and V/311-CD8positive T-cells (see Table 1). Interestingly, the T-cell receptor
repertoire of C57BL/6J MLTC-derived T-cells proliferating in
response to the 3LL tumor clones expressing allogeneic H-2K
restriction elements (H-2K bml, H-2K d, and H-2K k) demonstrated a similar pattern of the T-cell heterogeneity induced by
the 3LL tumor clone expressing the normal syngeneic H-2K b
(A9; Table 1) suggesting presentation of the same peptides.
Characterization of the T-cell response generated to 3LL
tumor in vivo also revealed restricted heterogeneity in the T-cell
subsets responding to A9 and D 122 by using the available TCRV~-specific monoclonal antibodies. Comparison of the TCR
expression of the T-cells infiltrating the tumor site with the
lymphocytes in the periphery of tumor-bearing animals revealed a pattern of homing of CD4-positive T-cells bearing
V~5, VB6, and V ~ l l TCR chains and CD8-positive T-cells
11
CD4
V[~ specific antibodies
Fig. 2. Profile of TCR V~ gene usage in PBL and TIL from C57BL/6J recipients bearing A9 tumor. Peripheral blood lymphocytes and tumor-infiltrating
lymphoeytesfrom severalC57BL/6J micebearing 8-mm transplanted A9 tumors
were excised, homogenized, and used for floweytometryanalysisas described in
"Materials and Methods." Standard deviations were less than 1%.
1
1
1
5O
!,o
30
invisible. Significant retardation of A9 tumor growth could be
achieved with V ~ l l antibody treatment before (12 days) or "~
shortly after (2 days) tumor injection but not at 14 days posttumor injection (Fig. 6). Thus, the intervention through Vfll 1
depletion at this stage is ineffective, even though the tumor is
still undetectable by caliper measurement. Therefore, removing
V/311-positive T-cells at that point in time cannot reverse their
putative negative effect on the efficacy of the CTL responses.
Whether this reflects the fact that the negative influence is not
irreversible needs to be addressed with additional experimentax:l
tion on the mechanism for the observed negative correlation
between V~I 1 depletion and enhanced host resistance to 3LL
growth and metastasis.
ZO,
~----II
TIL 5mm
TIL 8mm
PBL
4o]
CD;R
O
v
DISCUSSION
1
m
1
!
v~6
I
vm
Many murine and human tumors show marked reduction of
cell surface expression of M H C class I antigens (reviewed in
t~
Ref. 41). The suppression of class I antigens has been correlated
O
~g
with the reduced immunogenicity of tumors in syngeneic recipients and consequently with increased tumorigenicity or malig0
nancy of the neoplastic cells. Our previous studies of the sponTIL 5mm
TIL 8mm
PBL
taneous C57BL/6 3LL carcinoma showed that low-metastatic
Fig. 3. VBI1 T-cell infiltration increased with tumor size. CD4- and CD8clones that expressed H - 2 K ~' and H - 2 D ~ antigens could elicit a
sorted T-cells from multiple C57BL/6J animals bearing 5-mm (TIL) and 8-mm
syngeneic, tumor-specific CTL response, and that immuniza- A9 tumor (PBL and TIL) were stained for VB5,V~6, and V/~I1 TCR expression
tion of mice with H - 2 K b - p o s i t i v e tumor cells retarded the as described in "Materials and Methods."
6511
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Research.
T-CELL SUBSET
ANALYSIS OF
3LL T U M O R
REJECTION
lO
'7
9
~
d/p
B
E
--'0"-
7
unlreated
6-
Z
=
f
-~c " " "
/
x"
5o
o
days
post
tumor
transplantation
10
g
8
-
- "0-
-
unlreated
~
J
7
6
5
4
3
21
9
0
c'
,
~
~,
#
9
9
,
9 "" 1~2
days
~" 7
E
-
- "O- ~.
post
tumor
transplantation
unlreated
Vb5
Yb9
,..,.,
u
s
E
4
2
20
22
24
26
Days
28
post
30
tumor
32
34
3G
38
40
42
transplantation
Fig. 4. In vivo depletion of various T-cell subsets. Clearance of various T-cell subsets was achieved with weekly i.p. injection of 300 gg purified anti-CD4, CD8 and
TCR-Vfl-specific monoclonal antibodies as described in "Materials and Methods." Depletion of the various T-cells was monitored by FACS analysis of peripheral blood
lymphocytes. Tumor transplantation was done 12 days after the first antibody injection in at least 6 animals. A9 tumor growth following depletion of (A) CD4, CD8,
VSI 1, or V135 + Vfl6 + V~I I; or (B) V133, V135, Vt36, VflS, and V~9 T-cell subsets; (C) D122 tumor growth following depletion of Vt35, V~6, V89, and Vlgll T-cell
subsets.
6512
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T-(_'ELL SUBSET ANALYSIS OF 3LL T U M O R REJECTION
nu/nu
CD8
VI35
V~3
V[38
treatment group
V[59 V[311 VI317 V1~5+6+11 VI33
CD4 Control
(depleted T cell subset)
Fig. 5. Metastaticphenotypeof A9 tumorfollowingdepletionof variousT-cell
subsets. Micewere sacrificed30 daysposttumorleg amputation (as describedin
"Materials and Methods") and the lungs were examined for the presence of
metastases. The results shown representthe averageof 6 animals.
9.0
8.0
7.0
6.0
5.0
19
21
25
days post ttunor transplantation
30
Fig. 6. Kinetics of V~l I T-cell depletion in its effect on A9 tumor growth.
Tumor growth was monitored in mice which received i.p. injections of V~I 1specific monoclonal antibodies 12 days prior to tumor challenge of 2 versus 14
days following A9 tumor inoculation. The results represent the average of growth
measured in 6 animals.
vitro assay (Fig. 1). V~6 § CD8 T-cells failed to exhibit CTL
activity (Fig. 1), suggesting that the V/~6+ T-cells (CD4 and/or
CD8) may function as positive regulators via lymphokine secretion. Depletion of CD8-positive T-cell population resulted
in accelerated tumor growth, although with a slower rate than
expected, suggesting that withdrawal of suppressor CD8 § Tceils compensated for the deficiency of CD8 § cytotoxic T-cells.
Depletion of T-cell subsets bearing VB11 TCR enhanced resistance to tumor growth and abrogated metastasis in recipients
despite the clearance of V#5 and V~6 T-cell subsets. This may
be due to a shift in TCR usage similar to that observed for
sperm whale myoglobulin, 3 insulin-dependent diabetes mellitus
(43) and for bovine insulin (44) in recipients that lack the preferred TCR subset as a result of clonal deletion (tolerance),
recombination, or specific antibody treatment. In this study, it
appears that only in the absence of the CD4 V~I 1 subset does
removal of both VB5 and VB6 T-cells affect the metastasis of
A9, indicating that Vfll 1 T-ceUs presumably facilitate A9 tumor growth, possibly in limiting these T-cell effectors from
adequately restricting 3LL tumor progression. In the absence of
VB11-positive T-cells, however, other TCR subsets may substitute for the depleted V~5 and VB6 antitumor T-cells to provide
additional tumor immunity.
Correlation of the primary structural features of the TCR
with its fine antigenic specificity has demonstrated that T-cells
sharing reactivity for the same antigen often exhibit limited
heterogeneity of rearranged V and J gene segments (44, 45).
Because MHC polymorphism is limited relative to the enormous diversity of antigenic peptides, T C R genes may have
evolved to position the highly diversed junctional residues
within the CDR3 of the V/D/J~ junctions for maximal contact
with the antigen bound in the MHC peptide groove (46). PCR
analysis of the TCR expression on cytotoxic TIL clones grown
from A9 and D122 tumors 4 has revealed a high frequency of
TCR bearing the V/~5 element (i.e., 80% of the clones primed
with a V~5-specific primer). Moreover, each one of the clones
used D1 together with a different Jfl segment. Taken together,
our results suggest that the T-cell response against 3LL is limited in its heterogeneity to a few V~ families, and that the
different J segments may contribute additional diversity to the
recognition of 3LL tumor antigens. This is consistent with
T-cell recognition of different tumor-specific peptides in the
context of a single, Le., H - 2 K b restriction element.
Given current attempts to derive adoptive cellular immunotherapies for malignancies such as melanoma, the effect of CD4
VBI 1 lymphocytes on the ability of animals to reject 3LL is
noteworthy from the standpoint of strategies to enhance tumor
immunity by using ex vivo approaches. If suppressive T-cells
home to tumor sites within the body to negatively regulate
effector function, then lymphocyte fractionation may prove
critical to the efficacy of TIL and related therapies. Since the
3LL system represents a useful animal model for tumor progression, our characterization of the T-cells involved in controlling 3LL growth may afford unique opportunities to assess the
efficacy of selected subsets in adoptive immunotherapy. In turn,
this information may yield insights important to the development of improved forms of cellular immunotherapy for human
neoplastic diseases.
bearing V#5, V/~6, VB9, and V/~ll characteristic for the response to the transplanted A9 tumor.
To establish the relevance of the observed V/~I 1 T-cell infiltration on the nonmetastatic growth of A9, two groups of
C57BL/6J mice were given injections i.f.p, of A9 tumor cells
within an interval of 8 days. Comparing the level of V~I 1positive T-cells in the PBL (of mice bearing 8-mm tumor) and
the TIL of 5- versus 8-mm tumor-bearing animals, the V~I 1
CD4-positive T-cells showed a --~7-fold increase in numbers
with increased tumor mass. The VEIl CD8-positive T-cells
showed only .--2-fold increase relative to V~I 1 CD8-positive
T-cells in comparing PBL versus TIL of 5-mm tumor-bearing
animals. Thus, VBI 1-positive T-cells appear to home to the
3LL tumor site, infiltrate, proliferate, and multiply in response
to increased tumor mass.
Depletion of T-cell subsets bearing VB5 or V/~6 TCR correlated with accelerated tumor growth, implying that these T-cell
3 G. Rubberti, A. G a u r , C. G. F a t h m a n , a n d A. L i v i n g s t o n . T-cell r e c e p t o r
subsets play a protective role as tumor-specific cytotoxic effecr e p e r t o i r e influences V ~ e l e m e n t usage in r e s p o n s e to m y o g l o b i n , s u b m i t t e d f o r
tors. The protective function of V/35+ CD8 T-cells can be at- publication.
tributed to their tumoricidal activity as demonstrated in an in
4 G e l b e r et al., u n p u b l i s h e d data.
6513
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Research.
T-CELL SUBSET ANALYSIS OF 3LL TUMOR REJECTION
SUMMARY
We have analyzed the phenotypes of the T-cell subsets generated in response to transplantation of the Lewis lung carcinoma into syngeneic C57BL recipients. The A9 and D 122 3LL
tumor clones differing in their expression of H - 2 K t' elicited
immune responses distinguishable in their profile of T-cell receptor V gene usage. D122, the metastatic derivative of 3LL
which expresses low levels of the MHC class I H - 2 K ~" gene,
predominantly elicited CD8-, V~8-, and V~9-positive T-cells;
whereas A9, the nonmetastatic clone expressing high levels of
H - K t' gene, triggered a more heterogenous response of V~5-,
V~6-, V~8-, V~9-, V~ll-, and V/311-CD8-positive T-cells.
Characterization of the T-cell response generated to 3LL tumor
in vivo also revealed restricted heterogeneity in the T-cell subsets responding to A9 and D122. Comparison of the TCR
expression of the T-cells infiltrating the tumor site with the
lymphocytes in the periphery of tumor-bearing animals revealed a pattern of homing of CD4-positive T-cells bearing
V~5, V/~6, and V~ll TCR chains and CD8-positive T-cells
bearing V~5, V~6, V~9, and V~ll characteristic for the response to the transplanted A9 tumor. V~ll-positive T-cells
appear to home to the 3LL tumor site, infiltrate and proliferate
in response to increased tumor mass.
Depletion of T-cell subsets bearing TCR V~5 or V~6 correlated with accelerated tumor growth, implying that these T-cell
subsets play a protective role as tumor-specific effectors consistent with the cytotoxicity of T-cells with this TCR phenotype.
On the other hand, depletion of T-cell subsets bearing TCR
V~I 1 enhanced resistance to tumor growth and abrogated metastasis in recipients cleared of V~5 and V~6 T-cell subsets. The
effect of CD4 V~I 1 lymphocytes on the ability of animals to
reject 3LL is noteworthy from the standpoint of adoptive immunotherapy. If suppressive T-cells home to tumor sites within
the body to negatively regulate effector function, then lymphocyte fractionation may prove critical to the efficacy of TIL and
related therapies.
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Research.
T-Cell Subset Analysis of Lewis Lung Carcinoma Tumor
Rejection: Heterogeneity of Effectors and Evidence for
Negative Regulatory Lymphocytes Correlating with
Metastasis
Cohava Gelber, Lea Eisenbach, Michael Feldman, et al.
Cancer Res 1992;52:6507-6515.
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