Download CD4 § T-Cells from Mice Immunized to

[CANCER RESEARCH 54, 1055-1058, Fcbruary 15, 1994]
CD4 § T-Cells from Mice Immunized to Syngeneic Sarcomas Recognize Distinct,
Non-Shared Tumor Antigens
P e t e r A . C o h e n , 1 P h i l i p J. C o h e n , S t e v e n A . R o s e n b e r g , a n d J a m e s J. M u l ~
Branches of Surgery [P A. C., S. A. R.] and Dermatology [P J. C.], National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20814, and Systemics, Palo Alto,
CA 94304 [J. J. M.]
ABSTRACT
MATERIALS
We have utilized a newly developed culture system to study the properties of antitumor CD4 + T-cells relevant to the rejection of syngeneic
methylcholanthrene sarcomas. Fresh syngeneic dendritic cells prepared
from spleen, then pulsed with crude lysates of methylcholanthrene sarcomas, evoke antigen-specific proliferation by CD4 + but not by CD8 § T-cells
from tumor-immune mice. Unfractionated splenocytes display similar antigen presenting capacity if they are not irradiated before the pulse with
tumor lysate. CD4 + T-cells from mice immunized to individual methylcholanthrene sarcomas proliferate cross-reactively to dendritic cells
pulsed with fresh tumor digests, but not to dendritic cells pulsed with
cultured tumor cells. This apparent shared recognition of sarcoma lysates
was demonstrated to be a result of sensitization to bacterial collagenase
during the immunization procedure. Therefore, the murine CD4 § T-cell
response to tumor immunization is similar to the CD8 § response in that
sensitization occurs predominantly to tumor specific transplantation antigens rather than to shared tumor antigens. Strategies to avoid artefactual tumor cross-recognition by CD4 § T-cells are discussed.
Mice. Female C57BL/6 and BALB/c mice were purchased from Charles
River Laboratories (Wilmington, MA) and National Cancer Institute-Frederick
Cancer Research and Development Center (Frederick, MD). Animals were
used between ages 8 and 12 weeks.
Murine Tumors. All tumors were of C57BL/6 origin; the MC sarcomas
203 and 207 and adenocarcinoma MC38 were maintained in vivo by serial
s.c. transplantation; the sarcomas were serially passed 9 times before reinitiating with early passage cryopreserved tumor mince. WP4 is a cultured clone
from the MC sarcoma 205, subsequently passaged in mice. The Lewis lung
carcinoma was obtained from the Frederick Cell Repository. All tumors were
demonstrated free of viral pathogens and Mycoplasma by the National
Cancer Institute-Frederick Cancer Research and Development Center.
Culture Medium. All assays and cultures were performed in CM consisting of RPMI 1640 (Biofluids, Rockville, MD) supplemented with 10% heatinactivated fetal calf serum (Biofluids), 100 units/ml pencillin, 100 ~g/ml
streptomycin, 50 txg/ml gentamicin sulfate, 2 ~g/ml Fungizone, 0.05 mM
2-mercaptoethanol, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids,
and 1 p~g/ml indomethacin.
Tumor Digestion and Cell Line Derivation. Serially passed s.c. tumors
were aseptically harvested and finely minced, then enzymatically digested in
50 ml of Hanks' balanced salt solution containing DNase type IV (0.1 mg/ml),
collagenase type IV (1 mg/ml), and hyaluronidase type V (2.5 units/ml)
(Sigma, St. Louis, MO), with constant stirring over 2 h at 25~ After nylon
mesh filtering and 2 washes in Hanks' balanced salt solution, the resulting
single cell suspensions were used either for immunizations or as a source of
tumor antigen.
Murine Tumor Immunizations. Syngeneic mice were immunized to
weakly immunogenic MC sarcomas as described previously (1, 2). Naive mice
were first inoculated intradermally with 106 viable syngeneic sarcoma cells
mixed with 100/xg formalin-killed Corynebacterium parvum; all mice developed small tumors which rapidly regressed. Three weeks following the initial
inoculum, all mice received intradermal challenges with 2 • 105 viable tumor
cells (without C. parvum); 10-60% of mice rejected these challenges dependent on a particular sarcoma's immunogenicity. Three weeks following this
challenge, tumor-free mice received 106 viable tumor cells intradermally; mice
which rejected these challenges (generally 100%) were designated hyperimmune, and used 3 weeks after the last tumor challenge as a source of immune
spleens.
Tumor Antigen. Freshly digested tumor cell suspensions were either used
directly as a source of antigen or first cultured for 1 to 4 weeks in CM without
indomethacin. To prepare tumor antigen, freshly digested or cultured tumor
cells were snap-freeze-thawed (in CM without dimethyl sulfoxide) twice in
rapid succession, then refrozen and stored in liquid nitrogen for later use. No
viable tumor cells were detectable in these FT tumor lysates after the final
thaw.
Dendritic Cell Preparation. Splenic DC were freshly prepared from naive C57BL/6 mice following the method of Girolomoni et aL (7). After partial purification with DNase treatment and Percoll gradient fractionation, DC
were resuspended in CM and placed in 25-cm e Falcon 3013 flasks, 3 spleen
equivalents per flask. Nonadherent cells were discarded 2 h later, leaving
partially purified adherent DC in culture. FT syngeneic tumor cell lysates
were immediately added (1 • 107 tumor cell equivalents/flask), and antigen
pulsing allowed to proceed for 18 h. DC were then harvested, having detached from the flasks during overnight incubation. Each group was counted,
irradiated (1000 R) (8), then cocultured with CD4 § T-cells in proliferation
assays.
T Cell Subset Preparation. While DC were in preparation, fresh CD4 +
T-cells were prepared from the spleens of naive or tumor-immune mice.
INTRODUCTION
It has long been recognized that mice vaccinated to w e a k l y i m m u nogenic M C 2 sarcomas can develop specific resistance to subsequent
challenge with the same sarcoma (1). This i m m u n e protection requires
both CD4 § and CD8 § T effector cells (2). D e v e l o p m e n t of crossprotectivity to other syngeneic sarcomas is seldom observed or is
incomplete, indicating that T S T A rather than tumor-associated antigens are primarily responsible for M C sarcoma rejection following
immunization (3). CD8 § T-cells derived from mice i m m u n i z e d to
syngeneic M C sarcomas often fail to show cross-recognition o f other
sarcomas in 51Cr-release lytic assays or cytokine-release assays, and
do not show cross-protection in adoptive therapy experiments (4).
Similarly, CD8 § T-cells g r o w n from tumor digests (tumor infiltrating
l y m p h o c y t e s ) often manifest a similar absence o f cross-recognition
(5). In contrast, CD8 § T-cells cultured from the l y m p h nodes of mice
bearing progressive syngeneic M C sarcomas often show cross-recognition of other sarcomas in vitro, as well as cross-protection in adoptive therapy (4, 6). Technical problems previously limited the study of
t u m o r - i m m u n e CD4 + T-cells in vitro. In the present report, we describe a newly developed culture technique to demonstrate that, similarly to CD8 § T-cells, CD4 § T-cells derived f r o m t u m o r - i m m u n e
mice predominantly recognize unshared tumor antigens. Apparent
cross-reactive antigens on tumors are shown to be artifacts of tumor
digestion in bacterial collagenase, giving rise during tumor vaccination to collagenase sensitization.
Received 8/16/93; accepted 12/14/93.
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.
1 To whom requests for reprints should be addressed, at Surgery Branch, National
Cancer Institute, Building 10, Room 2B46, 9000 Rockville Pike, Bethesda, MD 20892.
3 The abbreviations used are: MC, methylcholanthrene; DC, dendritic cells; CM,
culture medium; FT, freeze-thawed;TSTA, tumor specific transplantation antigens; PHA,
phytohemagglutinin;APC, antigen presenting cells; pl, one-sided p-valve.
AND
METHODS
1055
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer
Research.
MURINE ANTITUMOR ('D4'
('ELLS RE('O(;NIZI- NON-SItARED ANTIGENS
Splenocytes were first depicted of red blood cells with ammonium chloridepotassium lysing buffer. Negative selection immunoaffinity columns (Pierce
Chemical Co., Rockford, IL) were then utilized with a single modification: the
provided anti-CD8 reagent or purified anti-CD4 (GK1.5; American Type Culture Collection, Rockville, MD) was diluted in CM conditioned by M5/114
hybridoma (anti-I-N'~ American Type Culture Collection) to facilitate quantitative depletion of la + cells (9).
Proliferation Assays. Flat-blottomed 96-well microtiter plates (Costar)
were utilized. Each well received 1 • 105 CD4 + T-cells. DC were added to
achieve a final CD4:DC ratio of 20:1 or 40:1. Each well contained a final
volume of 0.2 ml CM. Control groups routinely performed included: CD4 +
T-cells cultured without DC; pulsed irradiated DC cultured without CD4 +
T-cells; each CD4 + T-cell group with 3/xg/ml PHA (Sigma); each CD4 + T-cell
group sham-pulsed (i.e., cultured in CM only); each DC group with fresh
BALB/c (allogeneic) CD4 + T-cells; each DC group with fresh naive C57BL/6
(syngeneic) CD4 + T-cells. Cultures were continued for a total of 5 days, except
for PHA control groups, which received only 3 days' culture. At 18 h prior to
assay completion, tritiated deoxythymidine (1 p,Ci/well) (Amersham, Arlington Heights, IL) was added to each microtiter well. At completion, plates were
harvested with a semiautomated LKB cell harvester, and measured by a LKB/
Wallac 1205 Betaplate liquid scintillation counter (Gaithersburg, MD). Responses were reported as mean cpm -+ SEM from triplicate samples, and
analyzed by Wilcoxon rank sum analysis.
Fluorescence-activated Cell Sorter Analysis. Cells were routinely analyzed on a Becton-Dickinson FACScan or FACStar Plus using conjugated
direct antibodies and subclass matched controls obtained from Becton-Dickinson (Lincoln Park, N J) and Pharmingen (San Diego, CA). Cells were pretreated with unconjugated anti-CD32 (2.4G2) to block FcR binding by conjugated antibodies (Pharmingen).
I
r[ ]
(A) Sham Pulsed DC alone
(B) MC 203 Pulsed DC alone
(C) Naive CD4+ alone
(D) Immune CD4+ alone
(E) AIIogeneic CD4+ alone
(F) Sham Pulsed DC + AIIo CD4+
(G) MC 203 Pulsed DC + AIIo C D 4 +
H
(H) PHA + DC + Naive C D 4 +
(I) PHA + DC + Immune C D 4 +
(J) Sham Pulsed DC + Naive C D 4 +
(K) MC 203 Pulsed DC + Naive C D 4 +
(L) Sham Pulsed DC+ Immune C D 4 +
(M) MC 203 Pulsed DC + Immune CD4+
0
5
10
15
20
25
30
35
Thousands of C P M
Fig. 1. Representative proliferation assay showing mandatory control groups as described in "Results." CD4 + T-cell splenocyte sources included naive C57BL/6 mice
(Naive CD4+), naivc BALB/c mice (Allogeneic CD4 ~), and MC203-immunc C57BL/6
mice (Immune CD4 ' ). CD44 T-cells were cocultured with sham-pulsed or MC203-pulsed
naive C57BL/6 DC (20:1 ratio) for 5 days, cxcept for PHA groups which were cultured
for 3 days. PHA groups received 3 ~g/ml PHA (Sigma) and sham-pulsed DC. All DC
groups were irradiated (1000 R)just prior to coculture; tritiated deoxythymidine was
added 18 h prior to harvest. Bars, +_SEM.
Naive CD4 alone (A)
(B)
Naive CD4 + Naive CD8 (C)
(D)
Naive CD4 + Immune CD8 (E)
RESULTS
Immune CD4 alone (G)
(H)
Immune C04 + Naive CD8 (I)
Control Group Analysis of 2-Step CD4 § T-Cell Proliferation
Assay. A representative experiment illustrating all routine control
groups is s h o w n in Fig. 1. Irradiated D C alone s h o w e d negligible
proliferation at Day 4 in culture, whether or not pulsed with FT
MC-203 t u m o r antigen. C D 4 + T-cells alone also s h o w e d negligible
proliferation. W h e n naive B A L B / c C D 4 + T-cells were cocultured
with each group o f irradiated C 5 7 B L / 6 DC, strong proliferation was
seen; this c o n f i r m e d that each D C group manifests both Class II
expression and costimulatory function. Each CD4 + T-cell group was
also cultured with s h a m - p u l s e d D C and P H A to confirm proliferation
potential. Naive C D 4 + T-cells s h o w e d consistently low level proliferation w h e n cocultured with each irradiated D C group. Finally,
C D 4 + T-cells from mice i m m u n e to the MC-203 t u m o r s h o w e d low
level proliferation to s h a m - p u l s e d irradiated DC. Taken together, these
control groups enabled the demonstration o f significant antigen-specific proliferative effects w h e n MC-203 i m m u n e C D 4 ~ T-cells were
cocultured with M C - 2 0 3 pulsed, irradiated D C (Fig. 1, Group L versus
Group M, p l = 0.0248).
Tumor-immune CD4 § T-Ceils but Not CD8 § T-Cells Show Specific Proliferation to Tumor-pulsed D C . W h e n C D 4 + and C D 8 §
T-cell subgroups were each prepared from both naive and tumori m m u n e mice, only t u m o r - i m m u n e C D 4 + T-cells d e m o n s t r a t e d specific proliferation w h e n cocultured with tumor-pulsed D C c o m p a r e d
to s h a m - p u l s e d D C (Fig. 2, Group G versus Group H, p l = 0.0248).
This proliferation can be blocked by adding anti-Class II or anti-CD4
antibody 12 h after initial coculture (data not shown). Mixing experim e n t s w h i c h c o m p a r e d adding either naive or t u m o r - i m m u n e CD8 +
T-cells to t u m o r - i m m u n e C D 4 + T-cells d e m o n s t r a t e d nonspecific augmentation of proliferation (Fig. 1, Group J versus Group L, pl -0.4136). In other experiments not shown, irradiation of the tumori m m u n e C D 4 + T-cell subset completely abrogated the proliferative
response of either added C D 8 + T-cell group (9).
(J)
]
I
I --J
Immune CD4 + Immune CD8 (K)
(L)
Naive CDS+ alone (M)
(N)
Immune CD8+ alone (0)
(p)
0
10
20
30
40
50
Thousands of CPM
I i SEM
/
~
Sham Pulsed DC
/ E1 MC203-Pulsed DCI
Fig. 2. Representative T-cell subset proliferation assay as described in "Results." CD4 §
and CD8 § T-cell subsets were prepared from naive and MC203-immune C57BL/6 mice,
thcn cocultured with sham-pulsed or MC203-pulsed naive C57BL/6 DC (40:1 ratio) for 5
days. All DC groups were irradiated (1000 R) just prior to coculturc; tritiated deoxythymidine was added 18 h prior to harvest.
Nonirradiated but not Preirradiated Splenocytes Contain the
Antigen-processing Capacity of Partially Purified DC. Several
previously published efforts to expand antitumor C D 4 + T-cells in
vitro had used preirradiated (3000 R) unfractionated splenocytes as a
source of A P C for antitumor C D 4 + T-cells (10, 11). Even though a
dendritic cell subpopulation is present in such unfractionated splenocytes, such A P C have not proved to be an effective way to present
t u m o r lysates to freshly harvested i m m u n e C D 4 § T-cells (10). We
c o m p a r e d the antigen-presenting function of such preirradiated (3000
R) unfractionated splenocytes to our standard preparation of tumorpulsed splenic DC in which DC are irradiated only after the lysatepulse period. In addition, in order to assess the effect of preirradiation
on naive unfractionated splenocytes, the latter were either preirradiated with 3000 R, then pulsed for 8 h with F T t u m o r lysate, or first
pulsed with t u m o r lysate, then irradiated. Each A P C group was then
cocultured with C D 4 § T-cells. As s h o w n in Fig. 3, unfractionated
splenocytes did have the capacity to process and present t u m o r lysate,
but this proved to be a radiosensitive event.
1056
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer
Research.
MURINE ANTITUMOR
CD4 + CELLS
RECOGNIZE
MC-102. We o b s e r v e d similarly selective proliferation by C D 4 + T-
SplC+MC203 (5:1 Pulse)
cells from mice i m m u n e to M C - 2 0 7 , M C - 1 0 5 , and W P 4 w h e n cultured tumor cells were used as stimulators in vitro.
Cross-Reactive Tumor-immune C D 4 § T-Cell P r o l i f e r a t i o n Is
SplC+MC203 (10:1 Pulsed)
Caused by Immunization to Collagenase. D C were pulsed for 18 h
with either collagenase, hyaluronidase, or DNase, or pulsed with FT
fresh tumor digests or cultured tumor cells, then cocultured with
C D 4 § T-cells either from syngeneic mice i m m u n e to M C - 2 0 3 tumor,
or from naive allogeneic ( B A L B / c ) mice. As seen in Fig. 5, D C pulsed
I
SplC+MC203 (60:1 Pulsed) ~]
MC203 alone
with collagenase, but not hyaluronidase or DNase, e v o k e d strong
proliferation from M C - 2 0 3 i m m u n e C D 4 + T-cells, as did all D C
groups pulsed with fresh collagenase-digested tumor preparations. D C
pulsed with cultured tumor cells did not e v o k e strong proliferation
except for the relevant tumor cell line, M C - 2 0 3 . Importantly, all D C
groups e v o k e d strong proliferative responses b y naive B A L B / c C D 4 +
T-cells, indicating adequate antigen-presenting function in all D C
preparations. Additionally, all D C groups e v o k e d negligible proliferative responses from naive C 5 7 B L / 6 C D 4 + T-cells (latter data not
DC, Sham Pulsed
DC + MC203Pulsed ~`:~:~`:~`:`:`:`:`:~`:`:`:~`:i~`:~i`:`:~`:`:~`:`:`:`:`:~iii~`:~`:`:`:`:`:~`:`:ii~`:`:H
0
10
20
30
40
50
60
Thousands of CPM
I T SEM
/
ANTIGENS
geneic M C sarcomas, namely MC-207, M C - 1 0 5 , M C - 2 0 5 , W P 4 , and
SplC, Sham Pulsed~
SplC+MC203 (30:1 Pulsed)
NON-SHARED
Q P u l s e d , then irradiated
/ I l r r a d i a t e d , then pulsed
Fig. 3. Antigen-presenting function of unfractionated splenocytes versus prepared dendritic cells. DC were prepared, pulsed, and irradiated in the standard way described in
"Materials and Methods." Unfractionated splenocytes (SC) were ammonium chloride
lysing buffer-treated, washed, and then resuspended in CM at 5 X 106 SC/ml. At that time
they were either irradiated (3000 R) and then pulsed with MC203 FT cells for 8 h, or first
203-pulsed and then irradiated. The SC:MC203 pulse ratios are shown on the axis labels.
SC and DC groups were each then cocultured with 203-immune CD4 § T-cells. Each
microwell received 1 • 105 CD4 + T-cells and either 5 x 103 pulsed DC, 5 x l0 s pulsed
SC, or 1 X 105 FF tumor cells alone. Tritiated deoxythymidine was added 18 h prior to
harvest. 203-Immune CD4 § T-cell proliferation following coculture with SC, DC, or FT
tumor cells alone is shown.
shown).
DISCUSSION
The absence o f cross-protection to other syngeneic sarcomas after
mice are immunized to particular sarcomas has long suggested that
recognition of T S T A often plays the central role in the rejection o f
these tumors. Such rejections are dependent upon both C D 4 + and
C D 8 + T-effector cells (2). Previous w o r k had demonstrated that antitumor C D 8 § T-cells taken from t u m o r - i m m u n e mice or g r o w n from
tumor digests (tumor-infiltrating l y m p h o c y t e s ) often fail to recognize
shared s a r c o m a antigens in vitro and fail to s h o w cross-reactive therapeutic effects w h e n used in adoptive transfer experiments (1, 3-5).
D u e to technical obstacles, it w a s previously difficult to characterize
the C D 4 § T-cells involved in M C sarcoma rejections. The present
Thousands of C P M
Sham pulsed DC
3LL Digest Pulsed DC
MC 38 Digest Pulsed DC
MC 203 Digest Pulsed DC
MC 207 Digest Pulsed DC
i
3LL Culture Pulsed DC
i
w o r k demonstrates that C D 4 § T-cells taken from t u m o r - i m m u n e mice
also predominantly recognized unshared tumor antigens. It remains to
be determined w h e t h e r both C D 4 § and C D 8 § T-cells recognize epi-
:
MC 38 Culture Pulsed DC
Thousands of C P M
MC 203 Culture Pulsed DC
l
MC 207 Culture Pulsed DC
100
m
80
60
40
Sham Pulsed DC
20
M C - 2 0 7 Immune
0 10 20 30 40 50 60 70
I
I
WP4 Digest Pulsed DC
MC 207 Digest Pulsed DC
MC 203 Digest Pulsed DC
M C - 2 0 3 Immune
Fig. 4. Proliferation of immune CD4 + T-cells when cocultured with DC previously
pulsed with either fresh tumor digests or cultured tumor cells. Naive C57BL/6 DC were
prepared and pulsed with FT tumor cells either freshly digested from tumor mince (Digest)
or grown for 1 week in vitro (Culture) prior to freeze-thaw. Tumors included Lewis lung
carcinoma (3LL), MC38 adenocarcinoma, and MC207 and MC203 sarcomas. Pulsed DC
were then cocultured with either MC207-immune (left) or MC203-immune (right) CD4 §
T-cells for 5 days. Tritiated deoxythymidine was added 18 h prior to harvest. (Stippled
caps, +- SEM.)
WP4 Culture Pulsed DC
3LL Culture Pulsed DC
MC 203 Culture Pulsed DC
"Triple enzyme" Pulsed DC
DNAase Pulsed DC
CD4 § T-Cells from Mice Immunized to Individual MC Sarcomas Proliferate Cross-Reactively to DC Pulsed with Fresh Tumor
Digests, but not to DC Pulsed with Cultured Tumor Cells. As
s h o w n in Fig. 4, C D 4 + T-cells obtained from mice immunized to
M C - 2 0 3 or M C - 2 0 7 s h o w e d low level proliferation to sham-pulsed
DC, but strong proliferation to D C pulsed with any syngeneic tumor
tested, including nonsarcomas, w h e n fresh tumor digest w a s the
source o f tumor antigen. In contrast, w h e n tumor digests w e r e cultured
in vitro for 1 to 3 w e e k s prior to use as a source o f FT antigen, no
cross-reactive proliferation w a s seen. In multiple experiments not
shown, C D 4 + T-cells from M C - 2 0 3 i m m u n e mice consistently failed
to proliferate cross-reactively to D C pulsed with other cultured syn-
HYALURONIDASE Pulsed DC
COLLAGENASE Pulsed DC
160 120
80
40
Natve AIIoaeneie CD4+
0
40
80
120
Immune Svnaeneie CD4+
Fig. 5. Proliferation of 203-immune CD4 + T-cells when cocultured with DC previously pulsed with either Fq" tumor antigen or the enzymes used in tumor digestion. Naive
C57BL/6 DC were prepared and pulsed with FT tumor cells which were either freshly
digested from tumor mince (Digest) or grown for 1 week in vitro (Culture) prior to
freeze-thaw. Tumors included Lewis lung carcinoma (3LL) and MC sarcomas WP4, 207,
and 203. Alternatively, enzymes were added to DC instead of FT tumor antigen: DNase
1/.tg/ml, collagenase l0/xg/ml, hyaluronidase 25 milliunits/ml, or all 3 (Triple enzyme).
These enzyme concentrations reflected a 1:100 dilution of those used during actual tumor
digestions. MC203-immune CD4 § T-cells were cocultured with each DC group for 5
days. Tritiated deoxythymidine was added 18 h prior to harvest. (Stippled caps, +- SEM.)
1057
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer
Research.
MURINE ANTITUMOR ('D4'
('ELLS RE('O(iNIZE NON-SttAREI) ANTIGENS
topes derived from a single TSTA in the case of each M C sarcoma. It
also remains to be determined w h e t h e r CD4 + T-cells from tumorbearing mice recognize shared tumor antigens in the pattern observed
for CD8 + T-cells from tumor-bearing l y m p h nodes (4, 6).
The results presented here underscore the danger that seemingly
shared tumor antigens m a y often represent experimental artifact. Mice
inoculated with tumor cells freshly digested in clostridial collagenase
develop a prominent i m m u n e response to that enzyme. No such response has been observed for other digestive enzymes, which are
bovine rather than bacterial in origin. Therefore, DC preparation
methods w h i c h begin with collagenase digestions are likely to generate profound artifacts in this type o f tumor model. The repeated
washing o f tumor cells following digestion does not fully remove the
collagenase antigenic activity, but culturing digested tumor cells for
several days eliminates the collagenase activity, as demonstrated in
Fig. 5 by the loss o f cross-reactive tumor recognition. In other experiments not shown, w e have demonstrated that t u m o r - i m m u n e
CD4 + T-cells can be expanded in culture to enrich for either tumorrecognizing or collagenase-recognizing subpopulations.
Although tumor cells grown chronically in culture do not carry the
risk o f generating collagenase sensitization w h e n used as a source o f
tumor for vaccines, animals given repeated injections of cultured
tumor cells are apt to develop sensitization to culture m e d i u m proteins. In this instance, CD4 + T-cells taken from mice i m m u n i z e d to
tumor cultured cell lines show an abnormally high proliferative response to "sham-pulsed" dendritic cells prepared in the same culture
m e d i u m ( e . g . , RPMI 1640 with 10% fetal calf serum) used to culture
the tumor lines (data not shown). Our current strategy to prepare
tumor cells for vaccination involves an initial tumor digestion (of
necessity in collagenase) followed by several days' culture in RPMI
1640 supplemented with 0.5 to 1.0% non-heat-deactivated syngeneic
m o u s e serum. This culture period facilitates catabolism of collagenase
antigen, and adds no new foreign proteins to c o n f o u n d vaccinations.
Either Langerhans cells or spleen DC pulsed with crude FT tumor
preparations can be used to present specific tumor antigens to CD4 +
T-cells (9). Fresh unfractionated splenocytes also possess significant
antigen-presenting capacity, but this is impaired by irradiation prior to
the antigen pulse period. Previous investigators studying antitumor
CD4 + T-cells used unfractionated splenocytes as their source of APC;
however, the splenocytes w e r e irradiated prior to antigen pulsing (10,
11). We believe that this partly explains the historical difficulty associated with growing specific antitumor CD4 + T-cells in vitro.
In n u m e r o u s experiments, we have never observed tumor-specific
CD8 + T-cell proliferation to be e v o k e d by tumor lysate-pulsed DC;
this is true w h e t h e r or not recombinant interleukin-2 is added to the
assay (data not shown). This suggests that DC process and present
crude tumor antigen, in v i t r o at least, solely in a Class II context, and
parallels the experience o f others w h o studied DC presentation of
exogenous protein antigens such as ovalbumin (12). Since CD4 +
T-cells can function effectively in v i v o against Class II nonexpressing
tumors, including the M C sarcomas described in the present study (2,
11, 13), it is possible that A P C within solid tumors process and present
exogenous tumor debris in a Class II context to CD4 + T-effector cells,
triggering cytokine release which leads to accumulation and activation
of CD8 + T-cells (14, 15) as well as accessory cells such as tumoricidal
macrophages and lymphokine-activated killer cells (11, 16-18). Such
accessory cells kill tumor targets independent o f tumor cell Class I and
Class II expression (16, 17), and may explain how CD4 + T-cells
achieve effectiveness in v i v o against Class 11 nonexpressing tumors
(11, 13) as well as Class I nonexpressing tumors (17). We are currently
using our culture system to expand specific antitumor CD4 + T-cells
and investigate their m e c h a n i s m o f tumor rejection in v i v o (19).
ACKNOWLEDGMENTS
We would like to thank Stephen I. Katz and S u y u Shu for helpful
discussions, and Mary Custer, Ellen Fitzgerald, and Arnold Mixon for
excellent technical support.
REFERENCES
1. Shu, S., and Rosenberg, S. A. Adoptive immunotherapy of newly induced murine
sarcomas. Cancer Res., 45: 1657-1662, 1985.
2. Chou, T., and Shu, S. Cellular interactions and the role of interleukin 2 in the
expression and induction of immunity against a syngeneic murine sarcoma. J. Immunol., 139: 2103-2109, 1987.
3. Chang, A. E., Perry-Lalley, D. M., and Shu, S. Distinct immunologic specificity of
tumor regression mediated by effector cells isolated from immunized and tumorbearing mice. Cell. Immunol., 120: 419429, 1989.
4. Chou,T., Chang, A. E., and Shu, S. Y. Generation of therapeutic T lymphocytes from
tumor-bearing mice by in vitro sensitization. Culture requirements and characterization of immunologic specificity. J. Immunol., 140: 2453-2461, 1988.
5. Barth, R. J., Jr., Bock, S. N., Mul6, J. J., and Rosenberg, S. A. Unique murine
tumor-associated antigens identified by tumor infiltrating lymphocytes. J. lmmunol.,
144: 1531-1537, 1988.
6. Sakai, K., Chang, A. E., and Shu, S. Effector phenotype and immunologic specificity
of T-cell-mediatedadoptive therapy for a murine tumor that lacks intrinsic immunogenicity. Cell. lmmunol., 129: 241-255, 1990.
7. Girolomoni, G., Simon, J. C., Bergstresser, P. R., and Cruz, P. D. Freshly isolated
spleen dendritic cells and epidermal Langerhans cells undergo similar phenotypic and
functional changes during short term culture. J. Immunol., 145: 2820-2826, 1990.
8. Inaba, K., Metlay, J. P., Crowley, M. T., and Steinman, R. M. Dendritic cells pulsed
with protein antigens in vitro can prime antigen-specific, MHC-restricted T cells in
situ. J. Exp. Med., 172: 631--640, 1990.
9. Cohen, P. J., Cohen, P. A., Rosenberg, S. A., Katz, S. I., and Mul6, J. J. Murine
epidermal Langerhans cells and splenic dendritic cells present tumor-associated antigens to primed T cells. Eur. J. lmmunol., in press, 1994.
10. Matis, L. A., Ruscetti, S. K., Longo, D. L., Jacobson, S., Brown, E. J., Zinn, S., and
Kruisbeek, A. M. Distinct proliferative T cell clonotypes are generated in response to
a murine retrovirus-induced syngeneic T cell leukemia: viral gp70 antigen-specific
MT4+ clones and Lyt-2+ cytolytic clones which recognize a tumor-specific cell
surface antigen. J. lmmunol., 135: 703-713, 1985.
11. Kahn, M., Sugawara, H., McGowan, P., Okuno, K., Nagoya, S., Hellstrom, K. E.,
Hellstrom, I., and Greenberg, P. CD4 + T cell clones specific for she human p97
melanoma-associated antigen can eradicate pulmonary metastases from a murine
tumor expressing the p97 antigen. J. lmmunol., 146: 3235-3241, 1991.
12. Nair, S., Zhou, F., Reddy, R., Huang, L., and Rouse, B. T. Soluble proteins delivered
to dendritic cells via pH-sensitive liposomes induce primary cytotoxic T lymphocyte
responses ill vitro. J. Exp. Med., 175: 609--612, 1992.
13. Nagarkatti, M., Clary, S. R., and Nagarkatti, E S. Characterization of tumor-infiltrating CD4 + T cells as Thl cells based on lymphokine secretion and functional properties. J. Immunol., 144: 4898--4905, 1990.
14. Ksander, E R., Acevedo, J., and Streilein, J. W. Local T helper cell signals by
lymphocytes infiltration intraocular tumors. J. Immunol., 148: 1955-1963, 1992.
15. Thivolet, C., Bendelac, A., Bedossa, P., Bach, J-E, and Carnaud, C. CD8 + T cell
homing to the pancreases in the nonobese diabetic mouse is CD4 + T cell-dependent.
J. lmmunol., 146: 85-88, 1991.
16. Fidler I. J. Recognition and destruction of target cells by tumoricidal macrophages.
lsr. J. Med. Sci., 14: 177-191, 1978.
17. Levitsky, H. I., and Pardoll, D. M. The effect of tumor class I MHC expression on the
generation of two distinct cytolytic anti-tumor responses. J. Cell. Biochem., 17:119,
1993.
18. Mul6, J. J., Ettinghausen, S. E., Spiess, E J., Shu, S., and Rosenberg, S. A. Antitumor
efficacy of lymphokine-activatedkiller cells and recombinant interleukin-2 in vivo:
survival benefit and mechanismsof tumor escape in mice undergoingimmunotherapy.
Cancer Res., 46: 676-683, 1986.
19. Cohen, E A., Kim, H., Fowler, D. H., Gress, R. E., Jakobsen, M. L., Alexander, R. B.,
Mul6, J. J., Carter, C., and Rosenberg, S. A. Use of interleukin-7, interleukin-2 and
3,-interferonto propagate CD4 + T cells in culture with maintained antigen-specificity.
J. Immunother., 14: 242-252, 1993.
1058
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer
Research.
CD4+ T-Cells from Mice Immunized to Syngeneic Sarcomas
Recognize Distinct, Non-Shared Tumor Antigens
Peter A. Cohen, Philip J. Cohen, Steven A. Rosenberg, et al.
Cancer Res 1994;54:1055-1058.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/54/4/1055
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer
Research.