Content uploaded by Harry D Bear
Author content
All content in this area was uploaded by Harry D Bear on Feb 17, 2015
Content may be subject to copyright.
1988;48:1422-1427. Published online March 1, 1988.Cancer Res
Harry D. Bear, Brian M. Susskind, Karen A. Close, et al.
from Tumor-bearing Host and Immune Spleens Generationin VitroT-Lymphocytes and Requirements for Their
Phenotype of Syngeneic Tumor-specific Cytotoxic
Updated Version http://cancerres.aacrjournals.org/content/48/6/1422
Access the most recent version of this article at:
Citing Articles http://cancerres.aacrjournals.org/content/48/6/1422#related-urls
This article has been cited by 3 HighWire-hosted articles. Access the articles at:
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and .pubs@aacr.orgDepartment at
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions .permissions@aacr.orgDepartment at
To request permission to re-use all or part of this article, contact the AACR Publications
American Association for Cancer Research Copyright © 1988 on July 10, 2011cancerres.aacrjournals.orgDownloaded from
[CANCER RESEARCH 48, 1422-1427, March 15, 1988)
Phenotype of Syngeneic Tumor-specific Cytotoxic T-Lymphocytes and
Requirements for Their in Vitro Generation from Tumor-bearing
Host and Immune Spleens1
Harry D. Bear,2 Brian M. Susskind, Karen A. Close, and Sandra K. Barrett
Departments of Surgery and Microbiology and Immunology and the Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University,
Richmond, Virginia
ABSTRACT
Cells required for the in vitro generation of syngeneic cytotoxic 1-
lymphocytes (CTL) against the P815 mastocytoma in the DBA/2 mouse
strain were investigated. For both immune and tumor-bearing host spleen
cells, CTL effector cells were eliminated by treatment with anti-Thyl.2,
ami-1 yH.I. or anti-Lyt2.1 and C", but were resistant to anti-L3T4
(GK1.5). Thus, CTL effectors (and their precursors) were Lytl*2%
L3T4-.
However, P81S-specific CTL could not be generated in the absence of
L3T4* cells, whose function could be replaced with exogenous interleu-
kin-2 (11-2). When monoclonal antibodies against I.3T4 were added to
mixed leukocyte tumor cultures, CTL generation was markedly inhibited.
Depletion of accessory cells also led to a marked reduction in CTL
generation, which could be restored to control levels by adding adherent
cells from normal spleens or with exogenous IL-2, but not with IL-1.
Thus, accessory cells are apparently required to present the tumor
antigens of this la-negative tumor to T-helper cells.
INTRODUCTION
Several studies have demonstrated that adoptive transfer of
specifically sensitized lymphocytes, either with or without in
vitro resensitization, can cause tumor regression in the adoptive
host (1-3). In the course of trying to develop strategies for
adoptive immun«»therapyof tumors using autologous lymphoid
cells, a number of T-cell subpopulations have been defined with
different antitumor functions. As numerous animal models and
in vitro correlates of antitumor immunity have been developed,
a number of apparent conflicts have arisen regarding the phe-
notypes and/or functions of the T-cells which can mediate host
resistance to tumors. Previous studies of antitumor effector
cells have relied on the use of lymphocytes from immunized
tumor-resistant animals, but experiments to delineate the de
tails of the in vitro responses of cells from animals bearing
progressive tumors have not been described. In order to exploit
the immune response to cancers clinically, the responses of
lymphocytes from TBHs3 must be understood.
In some models, development of Lytl~2+ CTL appears to be
the critical factor in tumor destruction (4-9), while for other
tumors Lytl+2~ cells, which act as helper cells or produce
delayed-type hypersensitivity responses apparently mediate
host resistance (10-13). Recently, Greenberg (14) showed that
in the Friend leukemia (FBL-3) model some CTL are Lytl~2+
Received 7/16/87; revised 12/8/87; accepted 12/15/87.
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.
1This investigation was supported by IS I'I IS grant number CA 42443-01,
awarded by the National Cancer Institute, Department of Health and Human
Services. '
2Recipient of a Clinical Oncology Career Development Award from the
American Cancer Society. To whom requests for reprints should be addressed, at
Division of Surgical Oncology, P.O. Box 11, Medical College of Virginia, Rich
mond, VA 23298.
3The abbreviations used are: TBH, tumor-bearing host; CTL, cytotoxic T-
lymphocyte(s); HBSS, Hanks' balance salt solution; IL, interleukin; MLTC,
mixed leukocyte tumor culture; TH, T-helper cell(s); i.d., intradermal(ly); Ts,
suppressor T (cells); Con A, concanavalin A.
and others are Lytl+2+. Adoptive transfer of CTL could cure
animals with disseminated tumor, but only if IL-2 was given to
support their proliferation in vivo (14). In the same system it
was shown that adoptive transfer of noncytotoxic Lytl+2~ cells
from immune mice also could induce complete remissions
without adding IL-2 and without generation of Lyt2+ CTL (11).
Shu and Rosenberg (15) have described a sarcoma model in
which cytotoxic effector cells from immune mice boosted in
vitro could mediate tumor regression if IL-2 was provided,
whereas adoptive transfer of fresh, noncultured Lytl+2* im
mune lymphocytes, which were not cytotoxic, induced tumor
regression without added IL-2.
Recently, we have described an in vitro model of syngeneic
CTL generation from tumor antigen-primed splenocytes
against the P815 mastocytoma and showed that this response
is regulated by Ts cells from "late" TBH (16). In the course of
characterizing the mechanisms of this suppression, we noted
that the in vitro CTL response of immune spleen cells was more
susceptible to suppression than the response of "early" TBH
spleen cells. As a step towards determining the reasons for this
difference and working out the detailed mechanisms of Ts
function, we set out to determine the cell phenotypes and
interactions involved in the in vitro CTL response to syngeneic
tumor for both TBH and immune spleen cells. In this report,
we describe the details of this response and the cells involved.
This represents the first description of such data for the in vitro
response to the P815 tumor, or for any tumor using lympho
cytes from syngeneic TBH mice.
MATERIALS AND METHODS
Virus-free DBA/2NCrIBR mice were obtained from Charles River
Laboratories, Wilmington, MA. Mice were housed in a virus-free
environment, given food and water ad libitum, and screened regularly
for pathogenic viruses using standard serological tests. Sprague-Dawley
rats were also obtained from Charles River. Care of animals was in
compliance with institutional and NIH guidelines.
Tumors. The P815 mastocytoma (//-2^, was obtained from Dr.
Vincent J. Merluzzi at the Sloan Kettering Institute, Rye, NY. P815
was passaged as asdics in syngeneic DBA/2 mice. All tumor lines were
also cryopreserved in RPMI 1640 (Hazelton Research Products, Den
ver, PA) plus 20% fetal calf serum and 10% dimethyl sulfoxide over
liquid N2. For each experiment, frozen P815 tumor cells were thawed,
cultured in vitro for 24-48 h, washed, and resuspended in HBSS. Five
million cells were then injected i.p. into DBA/2 mice, and after one i.p.
passage, tumor asdics was harvested 1 wk later for use to inject TBHs,
to immunize mice, or for in vitro experiments.
Monoclonal Antibodies. Monoclonal anti-Lytl.l antibody (18-2.3
ascites) was kindly provided by Dr. F. W. Shen, Memorial Sloan
Kettering Cancer Center, New York, NY and was used at a dilution of
1:1000. Hybridoma cell lines secreting monoclonal anti-Thy 1.2
(30.H12, ATCC TIB107), anti-Lyt2.1 (116-13.1, ATCC HB129), anti-
L3T4 (GK1.5, ATCC TIB207), anti-I-Ad (MK-D6, ATCC HB3) were
obtained from the American Type Culture Collection (Rockville, MD).
Ascites containing monoclonal rat IgG antibody against human C3
1422
American Association for Cancer Research Copyright © 1988 on July 10, 2011cancerres.aacrjournals.orgDownloaded from
T„AND ACCESSORY CELLS IN TUMOR-SPECIFIC CTL GENERATION
(F3A6) was kindly provided by Dr. Shaun Ruddy at Medical College
of Virginia, Richmond, VA.
Antibodies for cell depletion experiments were prepared after reclon-
ing the hybridomas by limiting dilution and screening for production
of cytotoxic antibody against DBA/2 thymocytes. Positive clones were
expanded and inoculated i.p. into pristane-treated nude mice at 5 x K)'1
cells/mouse (17). Ascites and serum were obtained from these mice 10-
20 days later and their cytotoxic titers determined before use. These
preparations were diluted 1:100 (anti-Thy 1.2) or 1:50 (anti-Lyt2.1 and
anti-L3T4) for use with C' as described below.
Inoculation of TBHs and Immunization. PSIS cells from ascites were
washed in HBSS and injected i.d. on the abdomen at a dose of 10"
cells/mouse in 0.05 ml for TBHs. For immunization, IO6 P815 cells
were inoculated i.d. mixed with 100 tig of formalin-killed Corynebac-
terium parvum (Burroughs Wellcome, Research Triangle, NC) in a total
volume of 0.05 ml.
In Vitro Generation and Assay of ('Ils. The detailed methods for in
vitro generation of and assay of cytotoxic cells have been described (16).
Briefly, spleen cell suspensions were prepared in HBSS, and erythro-
cytes were lysed by a 5-min incubation in Gey's hemolytic medium
(18). After washing, viable cells were resuspended in complete medium
composed of RPM1 1640, penicillin (100 units/ml), streptomycin (100
Mg/ml), 2 mM L-glutamine, 10 mM 4-(2-hydroxyethyl)-l-piperazineeth-
anesulfonic acid buffer, 1 mM sodium pyruvate, 1% minimal essential
medium nonessential amino acids (Gibco, Grand Island, NY), 5 x 10
M mercaptoethanol, and 10% fetal calf serum (Hyclone, Logan, UT)
MLTCs were set up in 24-well culture plates (Costar, Cambridge, MA),
with 1.5 x IO7responder spleen cells and 5 x IO5mitomycin C-treated
P815 cells in 2.5 ml in each well. After 5 days at 37°Cin a humidified
atmosphere of 5% CO2:air, cells from MLTCs were harvested, washed
3 times, resuspended in complete medium, and assayed for cytotoxicity
against "Cr-labeled P815 target cells (16). All tests were performed in
triplicate. Spontaneous release was determined by incubating target
cells with medium alone and maximum release by incubating in 10%
Triton-X in distilled H2O. The spontaneous and maximum releases
were determined from 6 replicates of each. The percentage specific
release was calculated as
% specific release =100
cpm with effector cells - cpm spontaneous release
cpm maximum release —cpm spontaneous release
The SEs for triplicate assays were always 10% or less and are not
shown. Maximum release was generally 2500-4000 cpm; spontaneous
release was 20-30% of maximum release for 18-h and 10-15% for 6-h
assays.
Elimination and Enrichment of Lymphocyte Populations. For pheno-
typic characterization and fractionation of lymphocytes using antibody
and C', cells were incubated at 4°Cfor 45 min with an appropriate
dilution of the particular antibody in plastic culture tubes. The cells
were then centrifugea, supernatant fluid was removed, and the cells
were resuspended in an optimal dilution (usually 1:12) of rabbit C'
(Lowtox, Accurate Chemical Co., Westbury, NY). Antibody and C'
were diluted for use in cytotoxicity medium (Accurate Chemical Co.).
After incubation with C' for 45 min at 37°C,the cells were washed,
and viability was determined by trypan blue exclusion. The remaining
viable cells were then used in the experiments described. It should be
noted that one treatment of CTL with anti-Lytl.l antibody had little
effect on the response; 2 treatments were required to eliminate CTL or
CTL precursors. When 2 cycles of treatment with antibody and C' were
performed, dead cells were removed after the first round of treatment
by centrifugation through Lympholyte-M (Accurate Chemical Co.).
Adherent accessory cells were depleted by fractionation of spleen
cells over nylon wool columns, as described (19). The effluent cells
were further depleted of adherent cells by incubating 3 x IO7cells in 5
ml of complete medium in 100-mm plastic Petri dishes (Falcon, Ox-
nard, CA) at 37"C for 2 h. In some experiments, the nonadherent cells
were then treated with monoclonal anti-I-Ad and C'. For these experi
ments, P815 stimulator cells from ascites were also depleted of adherent
cells before mitomycin C treatment. As a source of adherent accessory
cells in reconstitution experiments, 1.5 x IO7normal spleen cells were
incubated at 37°Cfor 2 h in each well of the 24-well plates in which
the MLTC were to be set up. Nonadherent cells were removed by
vigorous washing, and then the accessory cell-depleted responder cells
and stimulator cells were added to these wells.
Interleukin Preparations and Assays. Rat T-cell growth factor-con
taining supernatants were prepared by incubating Sprague-Dawley rat
spleen cells with 2 ^g/ml Con A (Calbiochem-Behring, San Diego, CA)
and harvesting supernatants at 48 h. These were stored at —80°C,and
residual Con A was neutralized before use by addition of 10 mg/ml
methyl-D-mannoside (Sigma, St. Louis, MO). This material contained
80 units/ml of IL-2 activity, when a unit is defined by 50% maximal
stimulation of CTLL-2 proliferation (20). IL-2 levels were assayed by
stimulation of the IL-2-dependent cell line CTLL-2, as previously
described (20). Briefly, after washing in HBSS, 5-10 x IO3 CTLL-2
cells in 100 /¿Iwere plated in each well of a 96-well plate, in which
serial dilutions of materials to be assayed were already present in 100
fi\ (assayed in triplicate). Plates were incubated for 24 h at 37°Cin 5%
CO2 and humidified air. Each well was then pulsed with 1 ^ Ci [methyl-
3H]thymidine, incubated for 6 h, and harvested onto glass-fiber filter
paper for counting in a liquid scintillation counter. SEs for triplicate
assays were 10% or less and are not shown. Human recombinant IL-2
was obtained from Cetus Corporation, Emeryville, CA. Amounts of
this material used are expressed in terms of the manufacturer's units,
which we found to be the equivalent of approximately 2 units in the
CTLL assay.
11,1 was produced from lipopolysaccharide stimulated macrophages
by the method of Hoffmann et al. (21). Briefly, peritoneal exúdatecells
were collected from AKR mice, injected 7 days previously with 1 mg
C. parvum i.p., and incubated at 37°Cin 24-well tissue culture plates
at 2.5 x 10' cells/ml, 1 ml/well, in tissue culture medium containing
10 /ig/ml lipopolysaccharide (Escherichia coli 055:B5; Difco Labora
tories, Detroit, MI). Nonadherent cells were removed after 6 h by
washing wells with warm HBSS. Adherent cells were lysed by the
addition of 1 ml/well of distilled, deionized water and a cycle of freezing
and thawing. Cell lysates were filtered (pore size, 0.45) and stored at
-20°C.
IL-1 activity was determined by stimulation of the IL-1 responsive
D10.G41 helper T-cell line (22) (ATCC TIB224). Twenty thousand
D l O.G4.1 cells were cultured in flat-bottomed microtiter plates in tissue
culture medium containing 2.5 g/ml Con A and serial dilutions of the
IL-1 preparation for 72 h at 37°C.Cultures were pulsed with 1 fid of
[3H]thymidine for the last 6 h of incubation, then harvested and proc
essed as described above for the IL-2 assay. The IL-1 preparation
contained 450 units/ml of activity (20).
RESULTS
In Vitro CTL Response to P815. Spleen cells from either
P815-immune or syngeneic "early" TBH mice generated CTL
activity during the MLTC (Fig. 1). We have previously shown
that this cytotoxicity is antigen specific (16). At no time after
Immune,
unstlmulated Early TBH, Early TBH,
unstlm. +P8l5mc
Fig. 1. In vitro CTL responses of immune and early TBH spleen cells, with or
without PSISmc stimulator cells. Results were similar at eflectortarget ratio = 5
and 40. •effectortarget ratio = 10; D, effectortarget ratio = 20. PSISmc,
mitomycin C-treated P815 cells.
1423
American Association for Cancer Research Copyright © 1988 on July 10, 2011cancerres.aacrjournals.orgDownloaded from
TH AND ACCESSORY CELLS IN TUMOR-SPECIFIC CTL GENERATION
III
a»
<
u
u
oc
o
ILI
O.
CO
100
90
80
70
60
50
40
30
20
10
O
-10
-20
•C* »Ion«
•ntl Thy 12 * C'
10 20 40
E:T RATIO 80
Fig. 2. Phenotype of CTL from early TBH spleens. Early TBH (10 days)
spleen cells were stimulated 5 days in MLTC. They were then harvested and
treated twice with antibody and C' or C' alone. E:T, effectortarget ratio.
tumor cell inoculation (Days 1-21) could spleen cells from
either immune or TBH mice be shown to have any cytotoxic
activity against P815 when tested directly after removal from
the animal. In vitro restimulation wjth tumor cells was required
for CTL to be activated. Spleen cells from normal mice did not
produce any CTL activity in vitro, whether stimulated with
mitomycin C-treated P815 cells or not.
Phenotype of the CTL Effectors. Treatment of cells derived
from early TBH spleens after a 5-day MLTC with anti-Thyl.2,
anti-Lytl.l, or ant i•I.y12.1 antibody and C' eliminated their
cytotoxic activity (Fig. 2). Initial experiments had suggested
that CTL effectors were predominantly Lytl~2+ and that a
minor population of Lytl+2+ CTL might also be present. By
using 2 cycles of depletion and removal of dead cells between
treatments, however, all of the anti-PS 15 CTL effectors could
be depleted with either anti-Lytl or anti-Lyt2 antibody. More
over, this response could not be reconstituted by mixing Lytl"
and Lyt2" cells.
The inability to restore the response by mixing Lytl" and
Lyt2~ cells indicates that both markers are present on the same
cells, and thus the antitumor CTL in our system are Lytl+2+.
Treatment of CTL with anti-L3T4 and C', on the other hand,
had no significant effect on CTL activity (Fig. 2). The results
of similar experiments were identical for spleen cells from
immune mice (data not shown).
Phcnutype(s) of Cells Required for in Vitro CTL Response.
When L3T4+ cells were depleted on Day 0 rather than Day 5,
before establishing the MLTC, the CTL responses of both
immune and early TBH cells were essentially eliminated (Table
1). Anti-Lytl.l or anti-Lyt2.1 antibody and C' treatment also
abrogated the response, and remixing I,yt 1 and I,\2 cells did
not restore it (data not shown). In contrast, when L3T4~ and
Lyt2~ cells were combined, normal levels of CTL could be
generated. Thus, it appears that Lytl*2+, L3T4~ CTL precur
sors and Lyt2~, L3T4+ (TH) cells interact in the secondary in
vitro response to syngeneic P815 tumor cells for both immu
nized and TBH mice. The importance of L3T4* TH cells in this
response was further demonstrated by the addition of anti-
L3T4 monoclonal antibody to the MLTC (Table 2). This has
been shown by others to block the activation of major histocom-
patibility complex Class 11-dependent THcells (23,24), and was
able to inhibit P815-induced CTL generation in vitro. Results
shown are for TBH spleen cells, and were similar for immune
spleen cells.
Role of Interleukin-2. Unresponsive L3T4-depleted spleen
Table 1 Phenotypes of cells required for anti-PSIS CTL generation in vitro
Spleen cells from syngeneic immune mice or early TBHs (10 days after i.d.
inoculation of 10' P81S cells) were treated with antibody and C' or C' alone
prior to in vitro stimulation with mitomycin C-treated P815 cells in MLTCs. All
treatments were carried out twice, with removal of dead cells by density gradient
centrifugation. After 5 days, cells were harvested and assayed for cytotoxicity
against P81S targets.
%, specific release
(effectontarget =40)°Before
culture, treatment of cells
X2withC'
alone
Anti-Thyl.2 + C'
Alili I Ml I +C'
Anti-Lyt2.1 + C'
Anti-L3T4 + C'
Lyt2- and L3T4~ cells mixed (1:1)Immune
mice49
-5-4
-6-25
67Tumor-bearing
hosts64
-4
-6I
367
* Results were similar for other effectortarget ratios from 5-80.
Table 2 Inhibition of anti-PSIS CTL generation by addition ofanti-L3T4
antibody to MLTC
Spleen cells from early TBH mice (10 days after i.d. inoculation of P815) were
stimulated with mitomycin C-treated P815 cells in MLTC. Varying concentra
tions of ascites containing monoclonal rat IgG antibody against anti-L3T4
antibody (GK1.5) or monoclonal rat IgG antibody against human C3 (F3A6)
were added to MLTC medium on Day 0. After 5 days, cells were harvested and
assayed for cytotoxicity against P815 target cells.
% specific release, at
effectortarget ratio equal to
Antibody added to
early TBH spleencellsExperiment
1NoneAnti-L3T4
(4%)°Anti-L3T4(l%)Experiment
2NoneAnti-L3T4(I%)Anti-human
C3 (I %)544714251531107522194928652090413469477840102636010352101
°Percentages in parentheses, final concentration (vol/vol) of ascites in MLTC
on Day 0.
Table 3 Restoration by interleukin-2 of CTL response of To-depleted cells
Spleen cells from early TBH mice (10 days after i.d. inoculation of P815 cells)
were treated with C' alone or monoclonal anti-L3T4 antibody and C'. Cells were
treated twice, and dead cells were removed by density gradient centrifugation.
Cells were then stimulated with mitomycin C-treated P815 cells in MLTC. In
some groups either crude T-cell growth factor (supernatants from Con A-stimu-
lated rat spleen cells) or recombinant II. 2 was added to MLTC medium on Day
0. After 5 days in culture, cells were harvested and assayed for cytotoxicity against
P815 target cells.
Responder cells
treatedwithC'
alone
Anti-L3T4 + C'
Anti-L3T4 + C'
Anti-L3T4 + C'Added
tocultureT-cell
growth factor (20%)°
Recombinant IL-2 (10 units ml)"%
specific release, at
effector:target ratio
equalto5
10 20 408014
28 35 44 80
-2 1 5 10 11
26 52 67 84 75
32 61 68 71 108
°Final concentration in MLTC expressed as percentage of vol/vol for T-cell
growth factor and in manufacturer's units for rIL-2. In each case, this represents
16-20 units IL-2/ml as determined by CTLL-2 assay.
cells could be made responsive by the addition of 10-20 units/
ml of exogenous IL-2, as either crude supernatant or purified
recombinant material (Table 3). These doses were comparable
to the levels we have been able to measure in MLTC superna
tants and did not induce significant killing activity by normal
spleen cells nor by immune cells in the absence of stimulating
tumor cells (data not shown). Moreover, treatment of respond
ing cells on Day 0 with anti-L3T4 and C' completely abrogated
measurable IL-2 production in MLTCs (Fig. 3); C' alone or
anti-Lyt2.1 and C' had no effect.
Role of Accessory Cells. Since L3T4* cells apparently recog-
1424
American Association for Cancer Research Copyright © 1988 on July 10, 2011cancerres.aacrjournals.orgDownloaded from
TH AND ACCESSORY CELLS IN TUMOR-SPECIFIC CTL GENERATION
r ss-,
o :
¿ 20
UJ
¡ «H
10
5
O
o.
o
1.2 1.4 1.8 1.16
SUPERNATANT DILUTION
Fig. 3. IL-2 production in MLTC dependence on presence of L3T4* cells.
P81S-immune spleen cells were treated twice with ( " alone or antibody and ( ".
and the remaining viable cells were then set up in MLTCs. Supernatant fluids
were harvested on Days I or 2 of culture, filtered, and assayed for IL-2 activity.
Results are for Day 2 and were similar for Day I . Background incorporation of
[JH]thymidine (medium alone) was 3372 cpm.
110
uj 100
£ 90
uj 80
70
60
50
40
30H
20
10
UJ
oc
u
u.
ü
UJ
a
co
s«
10 20
E.T RATIO
40
Fig. 4. Role of accessory cells in tumor-specific CTL generation. Spleen cells
from early TBHs (11 days) were depleted of adherent cells or left untreated before
addition to MLTCs. Adherent cells from normal spleens were added to some
wells. PSISmc, mitomycin C-treated P815 cells; N.S., normal spleen.
Table 4 Replacement of accessory cells by exogenous IL-2 but not IL-1
Spleen cells from early TBH mice (10 days after i.d. inoculation of P815 cells)
either untreated or depleted of accessory cells, were stimulated with mitomycin
C-treated P815 cells in MLTC. In some groups either recombinant IL-2 or crude
IL-1 was added to MLTC medium on Day 0. After 5 days in culture, cells were
harvested and assayed for cytotoxicity against P815 target cells.
% specific release,
at effectortarget
ratio equal to
cellsNone
(unstimulated)
None
Accessory cells depleted"
Accessory cells depleted"Added
tocultureRecombinant
1L-210-5
35
-3
8320-S
49-4
8740-5
63
-1
92
Accessory cells depleted" (10 units/ml)*
Crude IL-1 (2%)" 8
" Early TBH spleen cells were depleted of accessory cells by incubation on
nylon wool columns and plastic dishes. Nonadherent cells were then treated with
monoclonal unii I A1'antibody (MK-D6) and ( ". The remaining viable cells were
then plated in MLTC at numbers equal to untreated cells.
4 Final concentrations in MLTC; 2% IL-1 is 9 units/ml.
nize foreign antigen only in the context of self-Class II major
histocompatibility complex determinants (23, 24), and since
P815 cells are Ia~ (25), the requirement for L3T4* cells implies
that tumor antigen must be presented by la* accessory cells
such as macrophages for the CTL response to proceed. In order
to test the accessory cell requirement, responder spleen cells
were depleted of adherent cells prior to stimulation in MLTCs.
As shown in Fig. 4, removal of adherent cells reduced the CTL
response to the level of the unstimulated control. This response
could be fully restored by the addition of plastic-adherent cells
from normal spleens. If, in fact, the accessory cells are required
for antigen presentation to I,, cells, then the need for these
cells should be able to be "bypassed" by providing the TH
product, IL-2. As shown in Table 4, this was indeed the case;
exogenous recombinant IL-2 restored the CTL response to
early TBH spleen cells after removal of accessory' cells. More
over, providing exogenous IL-1, a monocyte product, did not
restore the response of accessory cell-depleted responder cells.
DISCUSSION
In the experiments reported here, details of the cell interac
tions involved in the in vitro generation of tumor-specific CTL
from syngeneic immune or TBH animals against the P815
tumor have been delineated. While similar experiments have
been reported in other syngeneic tumor systems using lympho
cytes from immune, tumor-resistant animals, they have not
been reported for the P815 tumor, nor for any tumor-specific
response by lymphocytes from TBHs with progressively grow
ing tumors.
Although no active CTL could be detected in the spleens of
early TBH or immune animals, in vitro stimulation of spleno-
cytes from either source with specific tumor antigen resulted in
marked CTL activity. We have shown that primed CTL pre
cursors from early TBH and immune animals and the cytotoxic
effectors generated in MLTC are all Lytl+2+, L3T4"T cells and
that CTL activation requires the presence of Lyt2", L3T4*
cells. The function of the latter T-cells could be replaced with
crude or recombinant IL-2, and CTL development could be
inhibited by antibody against L3T4.
Thus, the secondary in vitro CTL response to the P815
mastocytoma involves Lytl+2+, L3T4" CTL precursors and
Lyt2~, L3T4+ TH cells. The fact that L3T4+ cells could be
replaced by recombinant IL-2 suggests that other TH cell factors
(e.g., CTL-differentiation factor) (26, 27) may not be required
in this system. We cannot rule out, however, that L3T4-de-
pleted cells might produce such factors in the presence of IL-2.
Accessory cells were also required for the CTL response, as
might be predicted from the Class II restriction on antigen
recognition by L3T4+ cells (23, 24), the fact that P815 cells are
Ia~ (25), and the ability to block the anti-PS 15 MLTC with
anti-L3T4 antibody. Removal of accessory cells markedly re
duced or eliminated the CTL response, and the response could
be restored to control levels by addition of plastic-adherent cells
from normal spleens or with exogenous recombinant IL-2. II
1, on the other hand, could not bypass the requirement for
accessory cells. These results suggest that accessory cells are
required to process tumor antigen and present it to L3T4+ TH
cells.
Anti-PS 15 CTL effectors generated in vitro and their precur
sors were found by us to be Lytl+2% L3T4~. This agrees with
the findings in several other tumor models (7, 8, 28-30).
Although other investigators have found some or all CTL
effectors specific for syngeneic tumors to be Lytl~2+ (6,14, 15,
31), similar to the descriptions of "classical" allospecific CTL
(32, 33), most, if not all, mature functional T-lymphocytes have
not been shown to bear some Lytl determinants (14, 34, 35).
1425
American Association for Cancer Research Copyright © 1988 on July 10, 2011cancerres.aacrjournals.orgDownloaded from
TH AND ACCESSORY CELLS IN TUMOR-SPECIFIC CTL GENERATION
Thus, Lytl phenotype is actually a quantitative rather than a
qualitative feature of T-cells. Our finding that 2 treatments with
anti-Lytl.l antibody were required to eliminate CTL suggests
a low density of Lytl on these cells and may explain why some
of the studies cited above suggested that CTL were Lytl~2+.
We found that L3T4* TH cells are required for the syngeneic
anti-PS 15 CTL response, apparently to produce IL-2. Whether
or not there are Lyt2*, L3T4~, Class I-restricted TH cells which
respond to the P815 tumor, as has been shown for alloantigen
(36-38), is not known. However, if they do exist, they are
insufficient for the secondary in vitro CTL response of spleen
cells to this syngeneic tumor, since only background levels of
CTL responsiveness and no IL-2 production were seen in the
absence of L3T4* cells. In contrast to these results, Kern et al.
(39) found that for the FBL-3 leukemia, treatment of responder
cells with anti-L3T4 and C' only partially inhibited CTL gen
eration, suggesting that L3T4~, Lyt2+ TH cell may provide
some help in that response. This difference between our results
and those of Kern et al. (39) may be simply methodological,
perhaps resulting from our use of 2 cycles of treatment with
antibody and C'. Alternatively, it may be that the cellular
interactions involved in the response to a virally induced tumor
(FBL-3) are different from the response to a chemically induced
tumor (P815). There is precedent for differential requirements
in responses to cellular antigens. For example, Mizuochi et al.
(37) have shown that the primary CTL response to Class I
alloantigen involves both L3T4+ and Lyt2+ TMcells, which both
produce IL-2. In contrast, only L3T4+ TH cells participate in
the response to trinitrophenylated-self, and no involvement of
Lyt2+ TH cells could be demonstrated (38). Thus, it may be that
FBL-3 tumor antigen(s) is analogous to Class I alloantigen,
while the P815 tumor antigen(s) is analogous to trinitrophen
ylated-self. Aside from our findings suggesting that help for the
CTL response to the syngeneic P815 tumor is provided exclu
sively by L3T4+ cells, our results with TBH and immune cells
are otherwise in agreement with those in the FBL-3-immune
model (39). For both tumors CTL are Lyt2+, and to some
extent Lytl+; TH cells, IL-2, and macrophages are required for
the CTL response to both tumors.
The relationship of these in vitro observations to in vivo
antitumor effects is unclear. Our results are consistent with the
observation that in the FBL-3 model exogenous IL-2 must be
given along with CTL in vivo to induce tumor regression (14).
In fact, it is interesting to note that the finding that Lyt2+ cells
alone could not mediate FBL-3 tumor regression in vivo con
flicts somewhat with the suggestion that Lyt2+ TH cells may
play a role in the in vitro CTL response to FBL-3 tumor cells
(14). In contrast, the finding by North's group that adoptive
transfer of Lytl~2+ precursors of cytotoxic cells alone is suffi
cient for the destruction of several tumors (including P815) in
vivo in T-cell deficient hosts (4, 5, 40) raises some question as
to whether, in fact, TH cells are always required for in vivo CTL
generation. As we have shown, however, primed TH cells which
support CTL generation in vitro are present in early TBHs.
Therefore, when primed CTL precursors are transferred to
TBHs treated with irradiation or cyclophosphamide to inacti
vate suppressor cells, resistant TH cells may already be present
in the adoptive host.
Further delineation of the functions of cells required for in
vitro responses and active in adoptive immunotherapy for dif
ferent tumor models is needed. CTL precursors and effectors
in the DBA/2 anti-PS 15 MLTC are shown here to be Lytl+2+.
Generation of CTL activity in this syngeneic mouse-tumor
combination also requires L3T4+ TH cells, adherent accessory
cells, and IL-2 (the product of TH cells). We have also shown
that the CTL response of TBH lymphocytes is similar to that
of cells from immune mice. Now that the components of this
in vitro, tumor-specific CTL response have been delineated, we
are proceeding with experiments to determine which of these
are the targets of Ts cells and which may be defective in late
TBHs.
REFERENCES
10.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Fefer, A., and Goldstein, A. L. (eds.). The Potential Role of T Cells in Cancer
Therapy. New York: Raven Press, 1982.
Mokyr, M. B., and Dray, S. In vitro immunization as a method for generating
cytotoxic cells potentially useful in adoptive immunotherapy. Methods Can
cer Res., 19: 385-417, 1982.
Greenberg, P. D., Cheever, M. A., and Fefer, A. Therapy of established
tumors by adoptive transfer of T lymphocytes. In: R. B. Herberman (ed.),
Basic and Clinical Tumor Immunology, pp. 301-335. Boston: Martinus
Nijhoff Publishers, 1983.
North, R. J., and Bursuker, I. Generation and decay of the immune response
to a progressive fibrosarcoma. I. Lytl*2" suppressor T cells down regulate
the generation of Lytl~2* effector T cells. J. Exp. Med., ¡59:1295-1311,
1984.
North, R. J., and Dye, E. S. Lyl*2" suppressor T cells down-regulate the
generation of Lyl~2* effector T cells during progressive growth of the P815
mastocytoma. Immunology, 54:47-56, 1985.
Mills, C. D., and North, R. J. Expression of passively transferred immunity
against an established tumor depends on generation of cytolytic T cells in
recipients. Inhibition by suppressor T cells. J. Exp. Med., 157: 1448-1460,
1983.
Rosenstein, M., Eberlein, T. J., and Rosenberg, S. A. Adoptive immunother
apy of established syngeneic solid tumors: Role of T lymphoid subpopula
tions. J. Immunol., 132: 2117-2122, 1984.
Evans, R. Phenotypes associated with tumor rejection mediated by cyclo
phosphamide and syngeneic tumor-sensitized T lymphocytes: potential mech
anisms of action. Int. J. Cancer, 33: 381-388, 1984.
Yamasaki, T., Handa, H., Yamashita, J., Watanabe, Y., Namba, Y., and
Hanaoka, M. Specific adoptive immunotherapy with tumor-specific cytotoxic
T-lymphocyte clone for murine malignant gliomas. Cancer Res., 44: 1776-
1783, 1984.
Bhan, A. K., Perry, L. L., Cantor, H., McCluskey, R. T., Benacerraf, B., and
Green, M. I. The role of T cell subsets in the rejection of a methylcholan-
threne-induced sarcoma (SI509a) in syngeneic mice. Am. J. Pathol., 702:
20-27, 1981.
Greenberg, P. D., Kern, D. E., and Cheever, M. A. Therapy of disseminated
murine leukemia with cyclophosphamide and immune Lyt-l*,2~ T cells.
Tumor eradication does not require participation of cytotoxic T cells. J. Exp.
Med., 161: 1122-1134, 1985.
Fernandez-Cruz, E., Worda, B. A., and Feldman, J. D. Elimination of
syngeneic sarcomas in rats by a subset of T lymphocytes. J. Exp. Med., 152:
823-841, 1980.
Shu, S., Hunter, J. T., Rapp, H. J., and Fonseca, L. S. Adoptive immunity
to a syngeneic guinea pig hepatoma: characteristics of effectors and quanti
tative analysis of tumor rejection. In: A. Fefer and A. Goldstein (eds.), The
Potential Role of T Cells in Cancer Therapy, pp. 79-91. New York: Raven
Press, 1982.
Greenberg, P. D. Therapy of murine leukemia with cyclophosphamide and
immune Lyt-2* cells: cytolytic T cells can mediate eradication of disseminated
leukemia. J. Immunol., 136: 1917-1922, 1986.
Shu, S., and Rosenberg, S. A. Adoptive immunotherapy of a newly induced
sarcoma: immunologie characteristics of effector cells. J. Immunol., 135:
2895-2903, 1985.
Bear, H. D. Tumor-specific suppressor T-cells which inhibit the in vitro
generation of cytolytic T-cells from immune and early tumor-bearing host
spleens. Cancer Res., 46: 1805-1812, 1986.
McKearn, T. J. Method for growing hybridomas in rats or mice. In: R. H.
Kenne«, T. J. McKearn, K. B. Bechtol (eds.). Monoclonal Antibodies.
Hybridomas: A New Dimension in Biological Analyses, pp. 403-404. New
York: Plenum Press, 1980.
Zola, H., and Brooks, D. Techniques for the production and characterization
of monoclonal hybridoma antibodies. In: J. G. R. Hurrell (ed.), Monoclonal
Hybridoma Antibodies. Techniques and Applications, pp. 1-57. Boca Raton,
FL: CRC Press, 1982.
Julius, M. H., Simpson, E., and Herzenberg, L. A. A rapid method for the
isolation of functional thymus-derived murine lymphocytes. Eur. J. Immu
nol., J: 645-649, 1973.
Gillis, S., Fern, M. M., Ou, W., and Smith, K. A. T cell growth factor:
parameters of production and a qualitative microassay for activity. J. Im
munol., 120: 2027-2032, 1978.
Hoffman, M. K., Koenig, S., Mittler, R. S., Oettgen, H. F., Ralph, P.,
Galanois, C., and Hammerling, U. Macrophage factor controlling differen
tiation of B cells. J. Immunol., 122:497-502, 1979.
Kaye, J., Gillis, S., Mizel, S. G., Shevach, E. M., Malek, T. R., Dinarello, C.
A., Cachman, L. B., and Janeway, C. A., Jr. Growth of a clone helper T cell
1426
American Association for Cancer Research Copyright © 1988 on July 10, 2011cancerres.aacrjournals.orgDownloaded from
TH AND ACCESSORY CELLS IN TUMOR-SPECIFIC CTL GENERATION
line induced by a monoclonal antibody specific for the antigen receptor:
interleukin 1 is required for the expression of receptors for interleukin 2. J.
Immunol., 133: 1339-1345, 1984.
23. Dialynas, D. B., Wilde, D. B., Marrack, P., Pierres, A., Wall, K. A., Harran,
W., Otten, G., Loken, M. R., Pierres, M., Kappler. J., and Fitch, F. W.
Characterization of the murine antigenic determinant, designated L3T4a,
recognized by monoclonal antibody GK1.5: expression of L3T4a by func
tional T cell clones appears to correlate primarily with Class H MHC antigen
reactivity. Immunol. Rev., 74: 29-56, 1983.
24. Marrack, P., Endres, R., Shimonkevitz, R.. Zlotnik, A., Dialynas, D.. Fitch,
D., and Kappler, J. The major histocompatibility complex-restricted antigen-
receptor on T cells. II. Role of the L3T4 product. J. Exp. Med., ¡58:1077-
1091, 1983.
25. McKenzie, I. F. C., and Potter, T. Murine lymphocyte surface antigens. Adv.
Immunol., 27: 179-338, 1979.
26. Raulet, D. II., and Bevan, M. J. A differentiation factor required for the
expression of cytotoxic T-cell function. Nature (Lond.), 296:754-757,1982.
27. Conzelmann, A., Corthesy, P., Cianfriglia, M., Silva, A., and Nabholz, M.
Hybrids between rat lymphoma and mouse T cells with inducible cytolytic
activity. Nature (Lond.), 29«:170-172, 1982.
28. Shiku, H., Toshitada, T., Bean, M. A., Old, L. J., and Oettgen, H. F. Ly
phenotype of cytotoxic T cells for syngeneic tumor. J. Exp. Med., 144: 11la
ll 20, 1976.
29. Lala, P. K., and McKenzie, I. F. C. An analysis of T lymphocyte subsets in
tumor-transplanted mice on the basis of Lyt antigenic markers and functions.
Immunology, 47: 663-677, 1982.
30. Dye, E. S. The antimetastatic function of concomitant antitumor immunity.
I. Host Ly-1 ' 2' effector T cells prevent the enumeration of metastatic tumor
cells in a biological assay. J. Immunol., 136:1504-1509, 1986.
31. Maier, T., Levy, J. G., and Kilburn, D. G. The Lyt phenotype of cells involved
in the cytotoxic response to syngeneic tumor and of tumor-specific suppressor
cells. Cell. Immunol., 56: 392-399, 1980.
32. Cantor, 11.,and Boyse, E. A. Functional subclasses of T lymphocytes bearing
different Ly antigens. I. The generation of functionally distinct T-cell sub
classes is a differentiative process independent of antigen. J. Exp. Med., 141:
1376-1389, 1975.
33. Cantor, H., and Boyse, E. A. Lymphocytes as models for the study of
mammalian cellular differentiation. Immunol. Rev., 33: 105-124, 1977.
34. Ledbetter, J. A., Rouse, R. V., Micklem, H. S., and Herzenberg, L. A. T cell
subsets defined by expression of Lyt 1,2,3 and Thy-1 antigens. Two-param
eter immunofluorescence and cytotoxicity analysis with monoclonal antibod
ies modifies current views. J. Exp. Med., 152: 280-295, 1980.
35. Nakayama, E., Shiku, H., Stocken, H., Oettgen, E., and Old, L. J. Cytotoxic
T cells: Lyt phenotype and blocking of killing activity by Lyt antisera. Proc.
Nati. Acad. Sci. USA, 76: 1977-1981, 1979.
36. Sprent, J., and Schaefer, M. Properties of purified T cell subsets. I. In vitro
responses to class I vs. class II H-2 alloantigens. J. Exp. Med., 162: 2068-
2088, 1985.
37. Mizuochi, T., Ono, S., Malek, T. R., and Singer, A. Characterization of two
distinct primary T cell populations that secrete interleukin 2 upon recognition
of class I or class II major histocompatibility antigens. J. Exp. Med., 163:
603-619, 1986.
38. Mizuochi, T. H., Golding, H., Rosenberg, A. S., Glimcher, L. H., Malek, T.
R., and Singer, A. Both L3T4* and Lyt-2* helper T cells initiate cytotoxic T
lymphocyte responses against allogeneic major histocompatibility antigens
but not against trinitrophenylated self. J. Exp. Med., 162:427-443, 1985.
39. Kern, D. E., Klarnet, J. P., Jensen, M. C. V., and Greenberg, P. D. Require
ment for recognition of class II molecules and processed tumor antigen for
optimal generation of syngeneic tumor-specific class I-restricted CTL. J.
Immunol., 136:4303-4310, 1986.
40. North, R. J. The therapeutic significance of concomitant anti-tumor immu
nity. I. Lytl~2* T cells from mice with a progressive tumor can cause
regression of an established tumor in irradiated recipients. Cancer Immunol.,
18: 69-74, 1984.
1427
American Association for Cancer Research Copyright © 1988 on July 10, 2011cancerres.aacrjournals.orgDownloaded from
