Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells.
ABSTRACT Concomitant tumor immunity describes immune responses in a host with a progressive tumor that rejects the same tumor at a remote site. In this work, concomitant tumor immunity was investigated in mice bearing poorly immunogenic B16 melanoma. Progression of B16 tumors did not spontaneously elicit concomitant immunity. However, depletion of CD4(+) T cells in tumor-bearing mice resulted in CD8(+) T cell-mediated rejection of challenge tumors given on day 6. Concomitant immunity was also elicited by treatment with cyclophosphamide or DTA-1 monoclonal antibody against the glucocorticoid-induced tumor necrosis factor receptor. Immunity elicited by B16 melanoma cross-reacted with a distinct syngeneic melanoma, but not with nonmelanoma tumors. Furthermore, CD8(+) T cells from mice with concomitant immunity specifically responded to major histocompatibility complex class I-restricted epitopes of two melanocyte differentiation antigens. RAG1(-/-) mice adoptively transferred with CD8(+) and CD4(+) T cells lacking the CD4(+)CD25(+) compartment mounted robust concomitant immunity, which was suppressed by readdition of CD4(+)CD25(+) cells. Naturally occurring CD4(+)CD25(+) T cells efficiently suppressed concomitant immunity mediated by previously activated CD8(+) T cells, demonstrating that precursor regulatory T cells in naive hosts give rise to effective suppressors. These results show that regulatory T cells are the major regulators of concomitant tumor immunity against this weakly immunogenic tumor.
- SourceAvailable from: Rostyslav Bilyy[Show abstract] [Hide abstract]
ABSTRACT: Large amounts of dead and dying cells are produced during cancer therapy and allograft rejection. Depending on the death pathway and stimuli involved, dying cells exhibit diverse features, resulting in defined physiological consequences for the host. It is not fully understood how dying and dead cells modulate the immune response of the host. To address this problem, different death stimuli were studied in B16F10 melanoma cells by regulated inducible transgene expression of the pro-apoptotic active forms of caspase-3 (revCasp-3), Bid (tBid), and the Mycobacterium tuberculosis-necrosis inducing toxin (CpnTCTD). The immune outcome elicited for each death stimulus was assessed by evaluating the allograft rejection of melanoma tumors implanted subcutaneously in BALB/c mice immunized with dying cells. Expression of all proteins efficiently killed cells in vitro (>90%) and displayed distinctive morphological and physiological features as assessed by multiparametric flow cytometry analysis. BALB/c mice immunized with allogeneic dying melanoma cells expressing revCasp-3 or CpnTCTD showed strong rejection of the allogeneic challenge. In contrast, mice immunized with cells dying either after expression of tBid or irradiation with UVB did not, suggesting an immunologically silent cell death. Surprisingly, immunogenic cell death induced by expression of revCasp-3 or CpnTCTD correlated with elevated intracellular reactive oxygen species (ROS) levels at the time point of immunization. Conversely, early mitochondrial dysfunction induced by tBid expression or UVB irradiation accounted for the absence of intracellular ROS accumulation at the time point of immunization. Although ROS inhibition in vitro was not sufficient to abrogate the immunogenicity in our allo-immunization model, we suggest that the point of ROS generation and its intracellular accumulation may be an important factor for its role as damage associated molecular pattern in the development of allogeneic responses.Frontiers in Immunology 01/2014; 5(560).
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ABSTRACT: Cancer immunotherapy is a rapidly evolving field that offers a novel paradigm for cancer treatment: therapies focus on enhancing the immune system's innate and adaptive anti-tumor response. Early immunotherapeutics have achieved impressive clinical outcomes and monoclonal antibodies are now integral to therapeutic strategies in a variety of cancers. However, only recently have antibodies targeting innate immune cells entered clinical development. Innate immune effector cells play important roles in generating and maintaining antitumor immunity. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are important innate immune mechanisms for tumor eradication. These cytolytic processes are initiated by the detection of a tumor-targeting antibody and can be augmented by activating co-stimulatory pathways or blocking inhibitory signals on innate immune cells. The combination of FDA-approved monoclonal antibodies with innate effector-targeting antibodies has demonstrated potent preclinical therapeutic synergy and early-phase combinatorial clinical trials are ongoing. Copyright © 2015 Elsevier Ltd. All rights reserved.Current opinion in immunology. 01/2015; 33C:1-8.
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ABSTRACT: The role of the microenvironment in high-grade lymphoma is not well defined. In this report, we employ immunohistochemistry to characterise programmed death-1 (PD-1/CD279) and FoxP3 expression in 70 cases of diffuse large B-cell lymphoma (DLBCL). PD-1 is a surface marker characteristic of follicular helper T-cells whilst FoxP3 is characteristic of Tregs. We demonstrate variable infiltration with CD4(+) T-cells (<10 to >50 % of all lymph node cells) and PD-1(hi) cells (0.1 to 1.5 % of all cells). CD4(+) T-cells can be distributed in clusters or more diffusely and PD-1(hi) cells, but not FoxP3(+) cells, are found in rosettes around lymphoma cells. Cases with high CD4(+) T-cell numbers tended to have higher numbers of both PD-1(hi) and FoxP3(+) cells. Cases with total CD4(+) T-cell, PD-1(hi) and FoxP3(+) numbers above the median associate with better clinical outcome. Overall, we demonstrate that infiltration by CD4(+) T-cells, including both FoxP3(+) and PD-1(hi) subsets, correlates with prognosis in DLBCL. In distinction to previous reported series, patients (91 %) were treated with rituximab-containing regimens, suggesting that the effects of CD4+ T-cell infiltration are maintained in the rituximab era. This work suggests that determinants of total CD4(+) T-cell infiltration, either molecular characteristics of the lymphoma or the patients' immune system, and not individual T-cell subsets, correlate with clinical outcome.Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin 07/2014; · 2.56 Impact Factor
The Journal of Experimental Medicine
J. Exp. Med.
Volume 200, Number 6, September 20, 2004 771–782
The Rockefeller University Press • 0022-1007/2004/09/771/12 $8.00
Concomitant Tumor Immunity to a Poorly Immunogenic
Melanoma Is Prevented by Regulatory T Cells
Mary Jo Turk, José A. Guevara-Patiño, Gabrielle A. Rizzuto,
Manuel E. Engelhorn, and Alan N. Houghton
The Swim Across America Laboratory of Tumor Immunology, Memorial Sloan-Kettering Cancer Center,
New York, NY 10021
Concomitant tumor immunity describes immune responses in a host with a progressive tumor
that rejects the same tumor at a remote site. In this work, concomitant tumor immunity was
investigated in mice bearing poorly immunogenic B16 melanoma. Progression of B16 tumors
did not spontaneously elicit concomitant immunity. However, depletion of CD4
tumor-bearing mice resulted in CD8
T cell–mediated rejection of challenge tumors given on
day 6. Concomitant immunity was also elicited by treatment with cyclophosphamide or DTA-1
monoclonal antibody against the glucocorticoid-induced tumor necrosis factor receptor. Im-
munity elicited by B16 melanoma cross-reacted with a distinct syngeneic melanoma, but not
with nonmelanoma tumors. Furthermore, CD8
specifically responded to major histocompatibility complex class I–restricted epitopes of two
melanocyte differentiation antigens.
T cells lacking the CD4CD25 compartment mounted robust concomitant immunity,
which was suppressed by readdition of CD4
T cells efficiently suppressed concomitant immunity mediated by previously activated CD8
cells, demonstrating that precursor regulatory T cells in naive hosts give rise to effective suppressors.
These results show that regulatory T cells are the major regulators of concomitant tumor immunity
against this weakly immunogenic tumor.
T cells in
T cells from mice with concomitant immunity
mice adoptively transferred with CD8
cells. Naturally occurring CD4
GITR • cyclophosphamide
melanocyte differentiation antigen • cancer immunity • immune suppression •
Concomitant tumor immunity describes the immune re-
sponse in a host bearing a progressive tumor that rejects an
inoculum of the same tumor at a distant site. The phenom-
enon was first reported by Ehrlich (1) and Bashford et al.
(2) in the early 1900’s, although these early observations
could be attributed to alloantigen recognition and trans-
plantation immunity in outbred mice. Subsequently, the
establishment of inbred mouse strains and syngeneic tumor
transplants directly established the relevance of concomi-
tant immunity to tumor immunity. More than 70 yr later,
North et al. conducted a series of notable experiments
demonstrating concomitant immunity in BALB/c mice
bearing the chemically induced Meth A fibrosarcoma (3–7).
Immunity, recognized as rejection of secondary tumors in-
oculated on days 6–9, was mediated by CD8
cells that were detected as the primary tumor grew. Subse-
quently, immunity was down-regulated by CD5
(Ly-12 ) suppressor cells that were induced as the primary
tumor progressed (3, 8).
Although skepticism developed regarding the existence
of suppressor cells, studies in recent years have confirmed a
central role of suppressor cell populations in regulating immu-
nity. Naturally occurring CD4
regulatory T cells) constitutively express the transcription
factor FoxP3 (9, 10), CD25 (11), and glucocorticoid-induced
TNF receptor (GITR; reference 12). They are selected in
the thymus and comprise
5–10% of the peripheral CD4
T cell repertoire in mice (11). Absence of CD4
FoxP3 T cells is associated with severe autoimmunity (9).
suppressor T cells (renamed
The online version of this article contains supplemental material.
Address correspondence to Alan N. Houghton, The Swim Across
America Laboratory of Tumor Immunology, Memorial Sloan-Kettering
Cancer Center, 1275 York Ave., New York, NY 10021. Phone: (212)
639-7595; Fax: (212) 794-4352; email: firstname.lastname@example.org
Abbreviations used in this paper:
ceptor; mAb, monoclonal antibody.
GITR, glucocorticoid-induced TNF re-
Concomitant Immunity to Melanoma
It has been proposed that mechanisms underlying autoim-
munity and tumor immunity are linked (13). In fact, care-
fully timed depletion of CD25
enhance tumor immunity and autoimmunity in both im-
munized and naive mice (14, 15). In addition to naturally
CD25 regulatory T cells, IL-10–induced
Tr1-type cells (16) and NK T cells (17–19) have also been
implicated in suppressor functions.
This is an appropriate time to readdress the role of sup-
pressor T cells in the historical context of concomitant tu-
mor immunity. Although strongly immunogenic, carcino-
gen-induced tumors elicit both concomitant immunity and
immune suppression (3, 20), concomitant immunity has
not been described for hosts bearing poorly immunogenic
tumors of spontaneous origin. The goals of the present pa-
per were twofold: to explore concomitant tumor immunity
in the context of a poorly immunogenic tumor and to in-
vestigate a role for CD4
other suppressor T cell populations in this model. For these
experiments, we have used B16 melanoma, an extensively
studied, poorly immunogenic tumor.
T cells has been shown to
regulatory T cells or
Materials and Methods
Mice and Tumors.
accordance with institutional protocol guidelines at Memorial-
Sloan Kettering Cancer Center (MSKCC) under an approved
) mice (males, 8–10 wk old) were
obtained from The Jackson Laboratory.
donated by M. Exley and S. Balk (Harvard Medical School, Bos-
ton, MA) and bred at MSKCC. All tumors were of C57BL/6 or-
igin. The B16F10 mouse melanoma cell line was originally ob-
tained from I. Fidler (M.D. Anderson Cancer Center, Houston,
TX) and passaged intradermally in mice four times to ensure re-
producible and aggressive intradermal tumor growth (B16). Im-
munization with 5
10 irradiated B16 melanoma cells provides
no tumor protection, and as few as 2
tumors. B16-GMCSF was generated by I. Hara and A. Hough-
ton at MSKCC by retroviral transduction of B16F10 with a ret-
roviral vector encoding the gene for mouse
procedures described previously (22). JBRH melanoma was pro-
vided by P. Livingston (MSKCC, New York, NY), and Lewis
lung carcinoma was acquired from American Type Culture Col-
lection. LiHa fibrosarcoma, a cutaneous tumor induced by di-
methylbenzanthrene treatment of an
was provided by T. Merghoub (MSKCC, New York, NY). Cells
were cultured in RPMI 1640 medium containing 7.5% FBS.
Growth medium for B16-GMCSF cells was supplemented with
1.0 mg/ml G418.
Cells were harvested after limited passage in vitro and were
used only if viability was
96%. Tumors were generated by in-
tradermal inoculation of 10
live cells (except for Lewis lung car-
cinoma, where 2
10 cells were used). For experiments requir-
ing two tumors on the same mouse, primary tumors were given
on the right flank and challenge tumors were given on left flank
6 d later, unless otherwise specified. Growth of secondary tumors
was only monitored for those mice that supported growth of pri-
mary tumors. Tumor diameters were measured approximately
every other day, and mice were killed when one of the tumors
All mouse procedures were performed in
mice (21) were
cells can give rise to
ulcerated, reached a maximum diameter of 1 cm, or when mice
Monoclonal Antibody (mAb) and Drug Treatments.
ments were administered by intraperitoneal injection. Mice were
depleted of CD4
and CD8 T cells by injection with 250
GK1.5 and 2.43 monoclonal antibodies, respectively (bioreactor
supernatants). NK cells were depleted by injection with 500
PK136 mAb to NK1.1. Antibodies were injected on days 4, 10,
and 17 after the primary tumor inoculation (unless otherwise
specified), and flow cytometry was used to confirm
tion of target cells for at least 7 d after injection. For in vivo
GITR stimulation, 1 mg of affinity-purified DTA-1 mAb (DTA-1
hybridoma was a gift from S. Sakaguchi, Kyoto University, Ky-
oto, Japan) was injected at the specified time points. Cyclophos-
phamide (Sigma-Aldrich) in PBS was administered in a single
dose of 150 mg/kg as described previously (23) at specified time
Peptides and ELISPOT Assay.
Genemed Synthesis, Inc. and used at
by HPLC. Peptides from dopachrome tautomerase/tyrosinase-
related protein 2 (DCT): DCT
published data), are restricted by K
(25) is restricted by D
from mouse prostate-specific membrane antigen was mutated at
an anchor residue for optimized strong binding to D
used as an irrelevant peptide control.
Multiscreen-IP plates (Millipore) were coated with 100
antibody (10 mg/ml; clone AN18; Mabtech)
in PBS, incubated overnight at 4
unbound antibody, and blocked with RPMI 1640 plus 7.5% FBS
for 2 h at 37
C. CD8 T cells were harvested from pooled spleen
and inguinal lymph nodes of killed mice (10 mice/group), puri-
fied using anti-CD8 MACS magnetic beads (Miltenyi Biotec),
and plated at a concentration of 2
10 irradiated B16 cells or EL-4 leukemia cells
(American Type Culture Collection) that had been pulsed with
g/ml peptide for 1 h were added to a final volume of 100
l/well. After incubation for 20 h at 37
sively washed with PBS plus 0.05% Tween and incubated for 2 h
C with 100
l/well biotinylated antibody against mouse
(2 mg/ml; clone R4-6A2; Mabtech). Spot development
was performed as described previously (26). Spots were counted
with an automated ELISPOT reader system with KS 4.3 software
(Carl Zeiss MicroImaging, Inc.).
Concomitant Immunity in RAG1
Naive T cell populations were freshly isolated from spleens of
C57BL/6J untreated donor mice. All cells were purified using
magnetic beads, according to the manufacturer’s instruc-
tions (Miltenyi Biotec). CD8
fied by positive selection, and CD4
populations were purified in two steps, by negative and positive
selection (mouse regulatory T cell isolation kit; Miltenyi Biotec).
Cell purity of
90% for all populations was confirmed by flow
cytometry. Purified lymphocytes were transferred intravenously
into five groups of 15
ents received either 6
CD8 T cells mixed with each of the following: 10
CD4CD25 T cells, or 9
1 d after reconstitution,
10 B16 cells in the right flank, followed 6 d later by an
identical inoculum in the left flank.
Adoptive Transfer of Immunity and Suppression.
donor mice were inoculated with 10
Peptides were synthesized by
80% purity, as confirmed
. A nine–amino acid sequence
(24) and DCT
. The gp100/pmel 17 peptide
C, washed with PBS to remove
cells/well. For antigen
C, plates were exten-
Hosts, after Adoptive Transfer.
populations were puri-
CD25 and CD4
recipient mice/group. Recipi-
T cells alone, or 6
mice were challenged with
live B16 cells in the right
Turk et al.
flank followed 6 d later by an identical inoculum in the left flank,
and CD4 depletion on days 4 and 10. 12 d after primary tumor
T cells were isolated from pooled spleen
and tumor-draining lymph nodes. All cells were purified using
MACS magnetic beads, as described before, and adoptive transfer
was conducted with fresh unstimulated cells. CD8
was 93%, as determined by flow cytometry, and
cells were obtained from each immunized donor. For adoptive
transfer of immunity, 10
recipients (10 mice/group). A separate
group of mice received 10
C57BL/6J mice, as a negative control.
CD25 T cells were isolated from spleens of un-
treated C57BL/6J mice, whereas tumor-bearing CD4
and CD4?CD25? T cells were isolated from combined spleen
and tumor-draining lymph nodes of mice bearing day-12 B16 tu-
mors. Purity of CD4?CD25? and CD4?CD25? T cells was 85
and 95%, respectively, as determined by flow cytometry. For
adoptive transfer of suppression, groups of RAG1??? recipients
(10 mice/group) were intravenously injected with 107 immune
CD8? T cells combined with each of the following putative sup-
pressor populations: 8 ? 105 naive CD4?CD25? T cells, 8 ? 105
CD4?CD25? T cells from tumor-bearing mice, or 5 ? 106
CD4?CD25? T cells from tumor-bearing mice. All mice were
challenged with 105 B16 cells 1 d after adoptive transfer, and tu-
mor growth was monitored for 60 d.
To determine significant differences
between different groups of mice with respect to tumor-free sur-
vival, log rank analysis (comparisons pooled over strata) of
Kaplan-Meier data was conducted using SPSS 10.0 software for
Windows. Statistical differences between tumor sizes and groups
of wells in ELISPOT assay were determined by two-tailed Stu-
dent’s t test.
Online Supplemental Material.
primary and secondary tumors in CD1??? and IL-10??? mice.
Online supplemental material is available at http://www.jem.
T cell purity
T cells were injected intrave-
T cells taken from naive
Fig. S1 shows growth of B16
Concomitant Immunity to Poorly Immunogenic B16 Can Be
Elicited by Tumor GM-CSF Expression and/or CD4? T Cell
To determine if concomitant immunity is elic-
ited by a poorly immunogenic tumor, B16 tumors were in-
oculated in the right flanks of C57BL/6 hosts, and 3, 6, 9,
or 12 d later, an identical inoculum was given in the oppo-
site flank. At all time points, mice with progressive primary
tumors supported growth of challenge tumors at rates com-
parable to naive control mice. Out of a total of 156
mice bearing primary tumors, from six independent exper-
iments, 148 supported normal growth of secondary tumors
(Fig. 1 A and not depicted). A similar lack of immunity
against a secondary tumor was observed when primary tu-
mors were ligated or surgically excised, and when mice re-
ceived primary and/or challenge inoculations in the foot-
pad (unpublished data).
Because growth of B16 tumors did not induce concomi-
tant immunity under any of these conditions, we used B16
cells stably transduced with GM-CSF (B16-GMCSF) as a
potential positive control. Immunization of mice with irra-
diated B16 cells transduced with GM-CSF (including our
B16-GMCSF cell line) has been shown to confer protec-
tion against B16, demonstrating that these cells are highly
immunogenic compared with their wild-type counterparts
(references 14, 27 and unpublished data). To determine if
this strongly immunogenic tumor primes concomitant im-
munity to B16, 105 live B16-GMCSF cells were inoculated
in mice, and wild-type B16 challenge tumors were given
6 d later on the opposite flank. As shown in Fig. 1 B, B16-
GMCSF tumors protected ?70% of mice from challenge
tumors (in three independent experiments, a total of 30 out
of 44 mice were protected), reinforcing the conventional
B16 melanoma can be induced by
GM-CSF expression in tumor cells and/
or by CD4? T cell depletion. C57BL/6
mice (10–15/group) were inoculated
with B16 cells (A and C) or B16-
GMCSF cells (B and D) in the right
flank. 6 d later, mice were challenged
with B16 cells in the left flank, and
growth of primary and challenge tumors
was monitored. In each panel, the left
graph represents growth of primary tu-
mors, the middle graph depicts growth
of challenge tumors, and the right graph
shows growth of the challenge tumor in-
oculum given in naive mice. Mice were
either left untreated (A and B) or treated
(C and D) with GK1.5 CD4 depleting
antibody on days 4, 10, and 17 relative
to primary tumor inoculation (arrows).
Significance for each group was deter-
mined by log rank analysis comparing
growth of challenge tumors in tumor-
bearing mice versus naive mice. (A) P ?
0.145; (B) P ? 0.014; (C) P ? 0.001;
(D) P ? 0.0002. Results are shown for
Concomitant immunity to
Concomitant Immunity to Melanoma
belief that immunogenic tumors effectively prime concom-
itant immunity, and showing that a strongly immunogenic
priming tumor can elicit immunity against a weakly immu-
nogenic challenge tumor.
The studies by North et al. showed that growth of im-
munogenic Meth A fibrosarcoma induced potent concom-
itant immunity, followed by loss of immunity around day
12, due to appearance of CD4? suppressor cells (3, 8). Be-
cause no concomitant immunity was observed at any time
point in B16 growth, we hypothesized that a population of
CD4? suppressor cells might be functional at an earlier
stage. To test this possibility, mice bearing primary tumors
were depleted of CD4? cells beginning 4 d after primary
tumor inoculation and continuing throughout tumor growth.
This approach could deplete putative suppressors, along with
helper T cells and other CD4? positive cells, while poten-
tially preserving a window for early T cell help.
In mice bearing wild-type B16 primary tumors, CD4 de-
pletion slightly reduced growth of primary tumors, but
also induced significant concomitant immunity, protecting
?70% of primary tumor-bearing mice from secondary tu-
mors (Fig. 1 C); in a total of seven experiments, 59 out of
89 mice completely rejected secondary tumors, and 9 mice
had secondary tumors that grew to a palpable size and re-
gressed around day 12. CD4 depletion also augmented con-
comitant immunity induced by B16-GMCSF (in two
experiments, 25 out of 30 mice were protected from sec-
ondary tumors), and retarded growth of primary B16-GMCSF
tumors (Fig. 1 D). These data supported the existence of a
CD4? population, present after primary tumor inoculation,
which suppresses concomitant immunity to B16.
CD8? T Cells from Concomitantly Immune Mice Recognize
Melanocyte Differentiation Antigens and Adoptively Transfer Tu-
Because previous studies have shown that
concomitant immunity is a CD8? T cell–dependent phe-
nomenon, we wanted to determine if this convention also
held true for our models. Therefore, upon induction of con-
comitant immunity by tumor inoculation and CD4 deple-
tion, we evaluated the effects of CD8 codepletion. Com-
pared with immune controls, challenge tumors grew rapidly
in CD8-depleted mice, at rates comparable to unimmunized
mice, indicating that concomitant immunity is CD8 depen-
dent (Fig. 2). This was true of mice bearing either B16 or
B16-GMCSF primary tumors. Primary tumor growth rates
also increased in CD8-depleted mice, demonstrating that ef-
fects on primary tumors are also CD8 dependent.
At this point, having established concomitant immunity
in the B16 and B16-GMCSF models, we conducted all
subsequent experiments with wild-type B16 tumors. This
model would allow the identification of relevant immune
responses using unmanipulated tumor cells.
Our original schedule for CD4 depletion was based on
the assumption that CD4 help might be required for early
C57BL/6 mice (5–15/group) were inoculated with B16 cells (A) or B16-
GMCSF cells (B) in the right flank, followed 6 d later by B16 cells in the
left flank. In addition, all mice received CD4-depleting antibody on days
4 and 10 to induce concomitant immunity. In each panel, the left graph
depicts growth of primary tumors and the right graph depicts growth of
challenge tumors in the same mice. The two bottom panels show tumor
outgrowth in mice coinjected with CD8-depleting antibody (arrows).
Significance for each group was determined by log rank analysis compar-
ing growth of challenge tumors in CD8-depleted versus nondepleted
mice. (A) P ? 0.0892; (B) P ? 0.0022.
Concomitant tumor immunity is dependent on CD8? cells.
lacking CD4? T cell help. Mice (15/group) were inoculated with B16
cells in the right flank, followed 6 d later by an identical inoculum in the
left flank. Mice received intraperitoneal injections of CD4-depleting anti-
body on days ?2, 4, 10, and 17 (A, arrows) or days 4, 10, and 17 (B). In
each panel, the left graph represents growth of primary tumors, the middle
graph represents growth of challenge tumors, and the right graph represents
growth of the challenge tumor inoculum given in naive mice.
Priming of concomitant immunity is less effective in mice
Turk et al.
priming of concomitant immunity. To assess this possibil-
ity, the CD4-depleting antibody was also administered be-
ginning 2 d before primary tumor inoculation, to ensure
CD4? T cell absence throughout tumor progression. Al-
though rejection of secondary tumors by day 18 in these
mice (Fig. 3 A) was similar to mice that did not receive the
early CD4? depletion (Fig. 3 B), a substantial proportion of
challenge tumors (7 out of 15) in the early CD4-depleted
mice grew to palpable size and later regressed. These results
show that priming of concomitant immunity is still possi-
ble, but is less efficient in mice deficient in CD4 help at the
time of primary tumor challenge.
We investigated the specificity of concomitant immunity
to B16. Mice bearing primary B16 tumors and depleted of
CD4? T cells were challenged with three different syngeneic
tumor cell lines: Lewis lung carcinoma, LiHa fibrosarcoma,
and JBRH melanoma. It has been shown previously that
Lewis lung carcinoma is subject to immunological rejection
after vaccination (28). CD4 depletion alone did not affect the
growth of these tumors (Fig. 4). Similarly, CD4 depletion
combined with B16 primary tumor growth did not detectably
affect growth of Lewis lung carcinoma or LiHa fibrosarcoma
(Fig. 4, A and B). However, growth of JBRH melanoma was
halted for ?2 wk, consistent with the notion that B16 tumors
elicit immunity to shared melanoma antigens (Fig. 4 C).
This observation led us to study epitope specificity of
CD8? T cells in mice with concomitant immunity. We
currently know of 10 epitopes derived from melanocyte
differentiation antigens that are presented by MHC class I
molecules on B16 cells: 2 from DCT (reference 24 and un-
published data): 1 from gp100 (25), 3 from TYRP-1 (un-
published data), and 4 from tyrosinase (unpublished data).
to B16 is shared with JBRH mela-
noma, but not with Lewis lung carci-
noma or LiHa fibrosarcoma. Mice
were inoculated (left flank) on day 0
with either Lewis lung carcinoma (A),
LiHa fibrosarcoma (B), or JBRH mel-
anoma (C). Mice were either un-
treated; depleted of CD4? T cells on
days ?2, 4, and 11; or depleted of
CD4? T cells on days ?2, 4, and 11
and inoculated with B16 tumors (right
flank) on day ?6 to induce concomi-
tant immunity. Tumor diameter
represents the mean of the greatest
diameters (? SEM) for 15 mice/
group. For JBRH tumors, the differ-
ence between mean sizes in naive
versus B16 tumor-bearing mice was
statistically significant (P ? 0.05; by
two-tailed Student’s t test) at all time
points between days 12 and 20.
ceived (A) inoculation of B16 cells in the right flank followed 6 d later by an identical inoculum in the left flank, (B) inoculation of B16 cells in the right
flank combined with CD4 depletion on days 4 and 10, or (C) inoculation of B16 cells in the right flank followed 6 d later by an identical inoculum in the
left flank and CD4 depletion on days 4 and 10. 12 d after primary tumor inoculation, CD8? T cells from spleen and inguinal lymph nodes were tested by
IFN-? ELISPOT analysis using as targets either B16 cells or peptide-pulsed EL4 lymphoma cells. Lymph node and spleen cells were pooled from groups
of 5–10 mice. Values represent the mean number of spots (n ? 3–4 replicates/point) ? SD. Asterisks depict statistically significant differences (P ? 0.05;
by two-tailed Student’s t test) between T cell responses to target EL4 cells pulsed with relevant versus irrelevant (Irr) peptide; or, for target B16 cells,
differences between responses from naive versus tumor-bearing mice.
CD8? T cells from concomitantly immune mice recognize epitopes from DCT and gp100 melanocyte differentiation antigens. Mice re-
Concomitant Immunity to Melanoma
These epitopes served as a panel to survey CD8? T cell
specificity. For these experiments, CD8? T cells were iso-
lated from lymphoid organs of concomitantly immune
mice and tested by IFN-? ELISPOT analysis using either
B16 cells or EL4 lymphoma cells (the latter pulsed with
each of the 10 epitopes) as targets.
6 d after the primary tumor inoculation in CD4-depleted
mice, no specific CD8? T cells were detected. On day 12,
the mice that did not receive CD4 depletion but bore both
primary and secondary tumors still had no detectable re-
sponse (Fig. 5 A). However, mice that received primary tu-
mors and CD4 depletion had readily detectable CD8? T
cell responses against DCT181 and gp10025 peptides, as well
as B16 cells (Fig. 5 B). Responding T cell numbers were
similar for both epitopes and strongest in the spleen, al-
though significant numbers of DCT181-specific T cells were
also found in tumor-draining and contralateral lymph nodes.
Inoculation of a secondary tumor on day 6 not only boosted
day-12 T cell responses in spleen and tumor-draining lymph
nodes, but also elicited recognition of a third epitope,
DCT363 (Fig. 5 C). In no group were responses detected
against any of the TYRP-1 or tyrosinase peptides.
To determine if this day-12 CD8? T cell population was
sufficient for tumor protection, these immune cells were
adoptively transferred into RAG1??? recipients, which were
challenged with B16 on the following day. Although naive
CD8? T cells afforded no significant protection, immune
CD8? T cells clearly conferred immunity, protecting
?50% of recipient mice from tumor growth (Fig. 6).
Naturally Occurring CD4?CD25? Regulatory T Cells Are
Required for Suppression of Concomitant Immunity in Mice
Bearing Progressive B16 Tumors.
with CD4 depletion indicated the presence of CD4? sup-
Our initial experiments
pressor T cells that block generation of CD8-dependent im-
munity. Nevertheless, the identity of these putative suppres-
sors had not been established. CD4? candidates included
NK T cells, Tr1 regulatory cells, and CD4?CD25? regula-
tory T cells. Significant concomitant immunity was not ob-
served with B16 melanoma in CD1??? mice (12 out of 15
mice grew secondary tumors) or in mice depleted of NK
cells (23 out of 37 mice grew secondary tumors), indicating
that suppression did not require NK T cells (Fig. S1,
available at http://www.jem.org/cgi/content/full/jem.
20041130/DC1, and unpublished data). Concomitant im-
munity was also not observed in IL-10??? mice (12 out of
15 mice grew secondary tumors), showing that suppression
was proceeding in the absence of IL-10 (Fig. S1). Therefore,
transfer immunity to T cell–deficient hosts. Donor C57BL/6 mice were
made concomitantly immune by inoculation of B16 cells in the right flank
followed 6 d later by a secondary inoculation in the left flank and CD4
depletion on days 4 and 10. 12 d after the primary tumor inoculation,
CD8? T cells were harvested from spleens and tumor-draining lymph
nodes of concomitantly immune mice (CD8 immune), or from spleens of
naive C57BL/6 mice (CD8 naive). Cells were purified and adoptively
transferred into RAG1??? recipients, which were challenged with an inoc-
ulum of B16 cells on the following day. Tumor growth was monitored in
adoptively transferred recipients for 60 d. The difference between tumor
incidence in mice receiving naive versus immune CD8? T cells was statisti-
cally significant (P ? 0.013), as determined by log rank analysis.
CD8? T cells from concomitantly immune donors adoptively
Mice (10–15/group) were inoculated with B16 cells in the right flank
followed 6 d later with an identical inoculum in the left flank. In each
panel, the left graph represents growth of primary tumors, the middle
graph depicts growth of challenge tumors, and the right graph shows
growth of the challenge tumor inoculum given to naive mice. 1 mg anti-
GITR antibody (clone DTA-1) was administered intraperitoneally on
days 1 and 7 (A) or days 0 and 7 (B) relative to primary tumor inocula-
tion. (C) Mice received injections of 1 mg rat IgG isotype control anti-
body. Arrows indicate time of antibody administration. The difference
between challenge tumor incidence in mice receiving rat control IgG
versus DTA-1 was statistically significant for both treatment schedules as
determined by log rank analysis. (A) P ? 0.0002; (B) P ? 0.0050.
GITR stimulation induces potent concomitant immunity.
Turk et al.
we undertook a series of experiments to determine if the
suppressors were CD4?CD25? regulatory T cells.
GITR is constitutively expressed on CD4?CD25? sup-
pressor T cells and its ligation by the stimulatory antibody
DTA-1 down-regulates suppressor function in vivo (12).
Consequently, enhancement of concomitant immunity by
DTA-1 would be consistent with a suppressive role for
CD4?CD25? T cells. Mice treated with DTA-1 on days 1
and 7 after primary tumor inoculation demonstrated pro-
gressive growth of primary tumors, while mounting con-
comitant immunity (?85% protection) against challenge
tumors (Fig. 7 A). Similar results were observed when
DTA-1 was administered on days 3 and 9 (unpublished
data). Interestingly, DTA-1 administration on days 0 and 7
(Fig. 7 B) was especially potent, protecting 50% of mice
from primary tumors and 100% of mice from challenge tu-
mors. Although these results suggested that DTA-1 was
blocking the function of suppressor T cells in our model,
alternate mechanisms involving binding to recently acti-
vated CD8? or CD4?CD25? T cells, which also express
GITR, could not be ruled out.
Cyclophosphamide has been implicated in selective tox-
icity to suppressor T cells induced during tumor growth
(23, 29, 30) and, more recently, to CD4?CD25? T cells
(31). Therefore, cyclophosphamide-induced concomitant
immunity would also support involvement of CD4?
CD25? suppressor T cells. The drug was tested at various
early time points, according to the doses used by North et
al. (23), to decrease collateral toxicity to CD8? effectors.
Administration of cyclophosphamide 4 d before inocula-
tion of B16 primary tumors induced concomitant immu-
nity (90% protection) and had no effect on growth of pri-
mary tumors or control tumors in naive mice (Fig. 8 A).
Effects on concomitant immunity were schedule depen-
dent, as treatment on day 2 only induced 40% protection
and treatment on day 0 induced no protection (Fig. 8 B).
To confirm that CD4?CD25? T cells are the suppressors
of concomitant immunity, RAG1??? hosts were partially re-
constituted with naive CD8? T cells mixed with various pu-
rified CD4? populations from naive C57BL/6 mice. Con-
comitant immunity was assessed by inoculation of B16
primary and challenge tumors. As predicted, similar to mice
that had received no cell transfer (Fig. 9 A), mice reconsti-
tuted with CD8? cells combined with the entire CD4?
compartment did not mount immunity (Fig. 9 B), consistent
with the presence of active suppressors. In contrast, mice
that had received a CD4? population depleted of CD25?
cells primed strong immunity (89% protection) against sec-
ondary tumors (Fig. 9 C). Immunity in this group was po-
tent enough that it also affected growth of the primary
tumors: 6/15 primary tumors were rejected and 7/15 dem-
onstrated reduced growth after day 12. Conversely, mice
reconstituted with CD8? T cells and the CD4?CD25?
T cell population had very rapid growth of primary tumors
and required early sacrifice (Fig. 9 D). These results confirm
the suppressive role of CD4?CD25? T cells in priming of
concomitant immunity. Interestingly, mice transferred with
naive CD8? T cells alone were not able to mount concomi-
tant immunity, indicating a requirement for CD4? T cell
help in RAG1??? hosts, in contrast with wild-type C57BL/6
hosts (see CD8? T Cells from Concomitantly Immune
Mice Recognize Melanocyte Differentiation Antigens and
Adoptively Transfer Tumor Immunity).
Having demonstrated that CD4?CD25? T cells could
suppress priming of concomitant immunity, we wished to
determine if this suppressor population required preexpo-
sure to tumor for function. For this experiment, preacti-
vated CD8? effectors were isolated from concomitantly
immune donors and combined with each of three putative
CD4? T cell suppressor populations: CD4?CD25? cells
from naive C57BL/6 donors, CD4?CD25? cells from day-
12 tumor-bearing donors, or CD4?CD25? cells from day-
12 tumor-bearing donors. These individual populations
were cotransferred into RAG1??? recipients, which were
challenged with B16 tumor cells 1 d later. Although we
observed moderate tumor protection (33%) in mice receiv-
ing immune CD8? T cells alone, immunity was com-
nity when administered 4 d before primary tumor inoculation. (A) Mice
(10/group) were treated with cyclophosphamide on day ?4, inoculated
with B16 cells in the right flank on day 0, and reinoculated with B16 in
the left flank on day 6. The left graph represents growth of primary tumors,
the middle graph depicts growth of challenge tumors, and the right graph
shows growth of the challenge tumor inoculum in naive mice that were
treated with cyclophosphamide according to the same schedule. (B) Inci-
dence of B16 secondary tumors (left flank) was measured in mice bearing
day-6 primary tumors and receiving the following: no treatment or cyclo-
phosphamide treatment on days ?4, ?2, or 0 relative to primary tumor
inoculation. The difference between challenge tumor incidence in un-
treated mice and those that received cyclophosphamide on day ?4 was
statistically significant (P ? 0.002) as determined by log rank analysis.
Single-dose cyclophosphamide induces concomitant immu-
Concomitant Immunity to Melanoma
pletely abolished in mice cotransferred with naive CD4?
CD25? T cells, at a suppressor/effector ratio of 1:12, dem-
onstrating that regulatory T cell precursors are naturally
occurring in naive C57BL/6 host (Fig. 10). Interestingly,
CD4?CD25? T cells from tumor-bearing mice did not
suppress with the same efficiency as naive CD4?CD25? T
cells, although this difference did not reach statistical signif-
icance (Fig. 10 A). In addition, CD4?CD25? T cells taken
from tumor-bearing mice did not function as suppressors,
but rather slightly enhanced tumor rejection (Fig. 10, A
Historically, concomitant tumor immunity has been de-
scribed for hosts bearing immunogenic tumors, including
tumors from outbred animals (1, 2, 32), methylcholan-
threne-induced tumors (4, 33–35), and virally transformed
models (36, 37). This response was shown to be complex,
with immunity decaying when primary tumors reached
large sizes (3, 33, 34, 36). The work of North et al. ?20 yr
ago carefully characterized the immune response to Meth
A fibrosarcoma as an equilibrium between tumor-induced
CD8? effector and CD4?CD5? suppressor T cells (3, 8).
Nevertheless, concomitant immunity has not been charac-
terized for poorly immunogenic tumors of spontaneous or-
igin. Resistance to a second inoculum of Madison lung car-
cinoma and Lewis lung carcinoma has been examined (20,
38), but immunological mechanisms were not directly im-
plicated, and it remained unclear if growth of such tumors
could prime host immunity.
In addition to extending this phenomenon to a poorly
immunogenic tumor, the present description of concomi-
tant immunity to B16 melanoma demonstrates possible
fundamental differences between strongly and poorly im-
munogenic tumors. Concomitant immunity to B16 does
not occur as a result of progressive tumor growth alone,
but rather only as a result of progressive tumor growth in
the absence of CD4? suppressor T cells. These suppressors
come from naturally occurring CD4?CD25? regulatory T
cells present de novo in the naive host and are capable of
blocking concomitant immunity at both priming and effec-
tor phases. GM-CSF production by otherwise poorly im-
munogenic tumor cells can induce resistance to a second
tumor inoculum, although this phenomenon is likely to
occur by mechanisms other than simply overcoming sup-
pression, such as enhancing APC recruitment and/or func-
tionality. This supposition is supported by the observation
that depletion of CD4? suppressors further augments the
immune response in mice bearing B16-GMCSF primary
tumors. Growth of primary B16-GMCSF tumors is strongly
dependent on the presence of CD4? T cells, with CD4
depletion resulting in CD8-dependent rejection of even
primary tumors. Therefore, B16-GMCSF may require a
basal level of suppressor T cell function to evade immune
naive T cells from C57BL/6J donor mice as follows: (A) no cells, (B) CD8? and CD4? T cells, (C) CD8? and CD4?CD25? T cells, (D) CD8? and
CD4?CD25? T cells, or (E) CD8? T cells alone. Adoptively transferred mice were inoculated with primary B16 tumors on day 1, followed by challenge
tumors on day 7. In each panel, the left graph depicts growth of primary tumors and the right graph depicts growth of challenge tumors in the same mice.
In D, the experiment was terminated 8 d after tumor challenge because growth of primary tumors was too rapid. Five out of nine tumors had already
appeared by day 9.
CD4?CD25? T cells suppress priming of concomitant immunity. RAG1??? mice were adoptively transferred with various populations of
Turk et al.
Before this paper, concomitant tumor immunity models
had not been characterized with regard to antigen specific-
ity. In the B16 model, we demonstrate that progressive tu-
mor growth elicits immunity to melanocyte differentiation
autoantigens. The number of individual CD8? T cells rec-
ognizing any single epitope was modest compared with
what might be achieved through active vaccination, and
certainly much less than observed after immunity to viruses
or bacteria. However, the gp10025 and DCT181 epitopes
have been described previously as B16 tumor rejection an-
tigens (24, 25, 39, 40), and it is most likely that the entire
population of CD8? T cells recognizing multiple shared
tumor antigens mediates the potent concomitant immunity
that we observe. Furthermore, the presence of cross-reac-
tive immunity to a different melanoma, but not to nonmel-
anomas, supports the argument that rejection antigens in-
clude shared melanocyte differentiation antigens. The fact
that JBRH melanoma is not completely rejected suggests
that immunity is also directed against antigens specific to
B16 and that B16-specific CD8? T cells are required for
complete tumor rejection.
Because these experiments were terminated within ?3
wk due to primary tumor size, we were not able to further
characterize autoimmunity (manifested as hypopigmenta-
tion of coat) or immunological memory. Memory T cell
responses against melanoma differentiation autoantigens
have been characteristically difficult to obtain through vac-
cination (unpublished data). Surgical resection of primary
tumors may facilitate future studies of autoimmunity and
memory by prolonging the lifespan of tumor-bearing hosts.
Postsurgical tumor immunity has been described for highly
immunogenic tumors, in many cases with potency exceed-
ing concomitant immunity (20). This comparison remains
to be defined for a poorly immunogenic tumor.
Unexpectedly, priming of concomitant immunity in im-
mune competent mice was only partially reduced in the
absence of CD4? T cell help. However, this result may re-
flect the regeneration of small numbers of CD4? T cells
between antibody depletions because RAG1??? mice lack-
ing CD4? T cells were not able to mount concomitant im-
munity. Alternatively, local help may be provided by in-
flammation at the tumor site, and the lack of immunity in
RAG1??? hosts may reflect a difference in antigen-present-
ing capabilities between these two strains.
Depletion of CD25? cells by treatment with PC61 mAb
has been shown previously to break immunological unre-
sponsiveness to highly immunogenic and, to a lesser extent,
poorly immunogenic tumors (41, 42). PC61 pretreatment
of naive mice has been reported to significantly reduce
growth of B16 tumors by mechanisms that appeared to in-
volve combinations of NK cells and CD8? T cells (15) or
CD4? and CD8? T cells (41); however, it remains unclear
if relevant CD8? populations were nonspecifically acti-
vated. Our model of concomitant immunity illustrates that,
in addition to CD4?CD25? T cell elimination, progressive
tumor growth is required for generation of tumor-specific
CD8? T cells and tumor immunity.
In our hands, pretreatment of mice with PC61 mAb was
associated with variable growth of primary tumors and
CD4? T cell–mediated rejection of ?20–40% of control
tumors, which made it difficult to assess the role of primary
tumor growth in rejection of secondary tumors (unpub-
lished data). In addition, treatment with PC61 mAb at later
time points was ineffective presumably because of the
depletion of activated CD8? effectors (14). Rather than
effectors of concomitant immunity. CD8 immune T cells were generated
as described in Fig. 6. (A) Incidence of B16 tumors in RAG1??? mice
that had been previously (day ?1) reconstituted with CD8 immune T
cells or CD8 immune T cells mixed with various potential suppressor
populations as follows: naive CD4?CD25? T cells, CD4?CD25? T cells
from day-12 tumor-bearing mice, or CD4?CD25? T cells from day-12
tumor-bearing mice. As determined by log rank analysis, the difference
between tumor incidence in mice receiving immune CD8 cells alone and
those receiving immune CD8 cells combined with naive CD4?CD25? T
cells was statistically significant (P ? 0.0026). However, there was no
statistical difference for mice cotransferred with tumor-bearing CD4?
CD25? T cells (P ? 0.992) or tumor-bearing CD4?CD25? T cells (P ?
0.677). Naive versus tumor-bearing CD4?CD25? T cells did not reach
statistical significance (P ? 0.0729). (B) Tumor growth curves depicting
growth rates of individual tumors in each group of adoptively transferred
CD4?CD25? T cells from naive mice suppress preactivated
Concomitant Immunity to Melanoma
PC61, we used GK1.5 mAb, DTA-1 mAb, and cyclophos-
phamide as tools to block or deplete suppressor T cells. We
were interested in DTA-1 mAb because it has been shown
previously to block CD4?CD25? suppressor T cell func-
tion in vivo (12). However, more recently, in an allogeneic
bone marrow transplant setting, DTA-1 mAb has been
shown to act directly on both effector CD4? and CD8? T
cell populations (43). Therefore, although our data are col-
lectively consistent with the notion that DTA-1 acts to in-
hibit CD4?CD25? suppressor T cells, potential activation
of effector cells is possible, and further experiments are
needed to determine which cell populations are responding
to DTA-1 in the B16 concomitant immunity model.
Cyclophosphamide has been described previously to de-
plete suppressor T cells induced by progressive Meth A tu-
mors and other immunogenic tumors (23, 29, 30). More
recently, a selective reduction in the proportion of CD4?
CD25? T cells has been reported in spleens of cyclophos-
phamide-treated rats (31). We have observed a similar de-
cline in this population in mice on days 4–10 after cyclo-
phosphamide treatment, although other T cell populations
are also affected (unpublished data). Because cyclophospha-
mide had profound effects on concomitant immunity when
administered 4 d before the primary tumor, this evidence
supports the role for cyclophosphamide through a mecha-
nism of selective toxicity against CD4?CD25? T cells.
However, effects on T cell homeostasis are likely, and fur-
ther studies are required to examine the exact mechanism
of cyclophosphamide function.
The Meth A sarcoma has been shown previously to in-
duce a suppressor population beginning after day 9 of
tumor growth (3), and it has been shown recently that
human melanoma can be recognized by a line of CD4?
CD25? regulatory T cells derived from the tumor (44). In
addition to suppressor T cell populations, myeloid cells and
soluble molecules (e.g., IL-10 and TGF-?) have been
shown to contribute to suppression of tumor immunity.
Although our studies do not rule out the possibility that
B16 growth also generates a CD4?CD25? regulatory T
cell population, suppressive CD4?CD25? or CD4?CD25?
T cells could not be transferred from mice bearing day-12
tumors. In fact, CD4?CD25? T cells taken from these
mice appeared to be inefficient suppressors compared with
their naive counterparts. This suggests that tumor growth
primes a population of activated CD4? T cells that partic-
ipates in tumor immunity, implying that CD4?CD25?
T cells isolated from tumor-bearing mice contain a
mixed population with different functions. The fact that
CD4?CD25? T cells from tumor-bearing mice did not
significantly enhance tumor rejection also supports the
conclusion that T helper cells were activated and, there-
fore, were present in the CD4?CD25? population. In any
case, CD4?CD25? T cells from naive mice were potent
suppressors in vivo after adoptive transfer, without requir-
ing preexposure to tumor. The antigen specificity of the
relevant CD4?CD25? T cells within this population re-
mains to be determined, but the finding that regulatory T
cell expansion is driven by presentation of self peptides (45)
presents the intriguing possibility that these cells may rec-
ognize melanocyte differentiation antigens. In this case,
precursor regulatory T cells might not require a priori acti-
vation by tumor because these antigens are already ex-
pressed by normal cutaneous melanocytes in hair follicles.
One fundamental characteristic of concomitant immu-
nity to tumors and pathogens is the observation that
progression of primary tumors or lesions is unaffected by
the developing host immune response. For instance, in
the case of concomitant immunity to Leishmania major,
CD4?CD25? T cells resident in the primary lesion prevent
local immune reactions, while maintaining antigen persis-
tence for priming of systemic immunity (46). In contrast,
concomitant immunity to B16 only proceeds in the ab-
sence of preexisting host suppressor cells and, therefore, the
presence of a local suppressive environment involving
CD4?CD25? T cells only within primary but not chal-
lenge tumors is unlikely. Furthermore, significant numbers
of tumor-reactive, specific CD8? T cells are present in
lymph nodes draining primary tumors, suggesting that pri-
mary tumors can be vulnerable to host immunity under the
right circumstances. Indeed, we have observed decreased
growth of primary tumors in mice with certain types of
concomitant immunity, particularly in those treated with
DTA-1 mAb and in T cell–reconstituted RAG1??? mice
lacking only the CD4?CD25? population. DTA-1 mAb
might activate effector cells in addition to inhibiting
suppressors, whereas in the case of RAG1??? recipients,
emerging T cell homeostasis could skew the response
strongly to tumor immunity. Although participation of lo-
cal CD4? suppressors (such as tumor-associated macro-
phages) cannot be ruled out, our observation that most pri-
mary tumors continue to grow in the face of rejection of
challenge tumors likely reflects the fact that these tumors
are established with stroma and vasculature before CD8
immunity is primed and, therefore, are more formidable
targets for host immunity compared with tumor cells given
on day 6. In summary, these studies illustrate the require-
ments for generating concomitant immunity to a progres-
sive, poorly immunogenic tumor. Our finding that B16 tu-
mors rapidly immunize hosts that lack the CD4?CD25?
regulatory T cell population demonstrates the central role
played by these cells in the blockade of tumor immunity in
this melanoma model. The fact that this concomitant im-
mune response recognizes melanocyte differentiation anti-
gens is consistent with an important role for shared, unal-
tered self antigens in rejection of poorly immunogenic,
The authors thank T. Merghoub, H. Uchi, and M.L. Palomba for
creation of the LiHa tumor cell line; T. Merghoub and M.Y. Tan
for breeding of CD1??? mice; and R. Stan for creating this paper.
Support for this work was provided by the National Institutes of
Health (NIH) grants R01 CA56821, P01 CA33049, CA59350, and
CA47179 (A.N. Houghton). M.J. Turk was supported by NIH
training grant T32 CA09149. M.E. Engelhorn was the recipient of
a fellowship award from the Cancer Research Institute. J.A. Gue-
vara-Patino received support from Mr. W.H. Goodwin, Mrs. A.
Goodwin, the Commonwealth Cancer Foundation for Research,
Turk et al.
and the Experimental Therapeutics Center of MSKCC, and A.N.
Houghton has Damon Runyon/Eli Lilly mentorship support. We
are grateful to Swim Across America, the Louis & Anne Abrons
Foundation, and the Mr. and Mrs. Quentin J. Kennedy Fund.
The authors have no conflicting financial interests.
Submitted: 7 June 2004
Accepted: 10 August 2004
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