The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 181, 4363 -4370
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, Z.
Right arrow Articles by You, Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, Z.
Right arrow Articles by You, Z.

Potent Tumor-Specific Protection Ignited by Adoptively Transferred CD4+ T Cells1

Zuqiang Liu*, Hae S. Noh*, Janet Chen*, Jin H. Kim*, Louis D. Falo, Jr.*,{ddagger} and Zhaoyang You2,*,{dagger},{ddagger}

Departments of * Dermatology and {dagger} Immunology, University of Pittsburgh School of Medicine, and {ddagger} University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Administration of anti-CD25 mAb before an aggressive murine breast tumor inoculation provoked effective antitumor immunity. Compared with CD4+ T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4+ T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by dendritic cells (DCs) produced more IFN-{gamma} and IL-2, and less IL-17 in vitro, and ignited protective antitumor immunity in vivo in an adoptive transfer model. Tumor Ag-loaded DCs activated naive CD8+ T cells in the presence of these CD4+ T cells in vitro. Tumor Ag and adoptively transferred CD4+ T cells were both required for inducing a long-term tumor-specific IFN-{gamma}-producing cellular response and potent protective antitumor activity. Although adoptively transferred CD4+ T cells ignited effective tumor-specific antitumor immunity in wild-type mice, they failed to do so in endogenous NK cell-depleted, Gr-1+ cell-depleted, CD40–/–, CD11c+ DC-depleted, B cell–/–, CD8+ T cell-depleted, or IFN-{gamma}–/– mice. Collectively, the data suggest that adoptively transferred CD4+ T cells orchestrate both endogenous innate and adaptive immunity to generate effective tumor-specific long-term protective antitumor immunity. The data also demonstrate the pivotal role of endogenous DCs in the tumor-specific protection ignited by adoptively transferred CD4+ T cells. Thus, these findings highlight the importance of adoptively transferred CD4+ T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD4+ T cells have emerged as an important arm of the immune system in combating tumors (1, 2, 3). CD4+ T cells could mediate tumor rejection by killing tumor cells via TRAIL-, Fas ligand-, or granzyme-perforin-dependent pathways, by up-regulating MHC class I and II expressions on tumor cells, inhibiting tumor cell proliferation, promoting NK cell function, rescuing CTL from activation-induced cell death, inducing angiogenesis inhibitors, and recruiting macrophages/eosinophils via IFN-{gamma}-mediated mechanisms (2, 3, 4). A recent study further showed that CD4+ T cells were powerful in tumor rejection in comparison with CD8+ T cells, especially in collaboration with NK cells (5).

In most cases, CD4+ T cells may orchestrate broad immune components of hosts to elicit a tumor-specific immune attack when considering the following: first, most tumor cells are MHC class II–/–, which cannot be recognized by CD4+ T cells (6); second, the unique ability of CD4+ T cells to regulate other immune cells in vivo (7); lastly, CD4+ T cell help is especially important in the situation of tumor-bearing host lacking intrinsic danger signals (8). CD4+ T cells freshly purified, restimulated, and/or activated in vitro or genetically modified in vitro have been used to investigate their role in antitumor immunity in multiple animal models (9, 10, 11, 12, 13, 14). Accumulated data suggest that adoptively transferred CD4+ T cells may enhance antitumor activity mediated by adoptively transferred CD8+ T cells and/or initiate endogenous CD8+ T cell-dependent or -independent antitumor immunity (15, 16, 17, 18, 19, 20, 21).

In vivo depletion of CD4+CD25+ regulatory T cells (Treg)3 via anti-CD25 mAb enhanced tumor/Ag-specific T cell responses, provoked potent antitumor activity, and augmented tumor vaccine-induced antitumor immunity in various murine tumor models (22). Tumor rejection was reported in naive mice adoptively transferred by CD4+ T cells purified from mice pretreated with anti-CD25 mAb (23). However, the underlying mechanism(s) is unknown.

Her2/Neu is associated with many of advanced human breast tumors and a well-documented tumor-associated oncoantigen as a target of both cellular- and humoral-mediated antitumor immunity (24). The importance of fresh CD4+ T cells was documented in BALB/c mice, showing that foreign Ag Her2-specific CD4+ T cells are essential to reject a tumor-expressing Her2 in BALB/c mice (25). Although the foreign Ag (rat Her2/Neu) is not tolerated in mice (26), murine breast tumor 4T1.2-Neu shares many characteristics with many human advanced breast cancers. This includes aggressive spontaneous metastasis, inherent resistance to chemotherapy, poorly immunogenic with MHC class II–/–, profoundly producing various immune suppression factors and cells, and stably expressing oncoantigen Her2/Neu (27, 28). Thus, 4T1.2-Neu is an aggressive murine breast tumor.

In this study, we tested whether mice with Treg depletion could provoke antitumor immunity against 4T1.2-Neu and examined in vivo antitumor function of CD4+ T cells freshly purified from those mice. We undertook a series of in vitro and in vivo experiments with various endogenous immune cell-depleted and gene knockout mice to dissect the mechanism(s) underlying the effective tumor-specific protection ignited by adoptively transferred CD4+ T cells in this aggressive breast tumor model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

BALB/c and BALB/c-B cell–/– (C.129 (B6)-IgH-Jtm1Dhu) mice were purchased from Taconic Farms. BALB/c-CD40–/– (CNCr.129P2-Cd40tm1Kik/J), BALB/c-IFN-{gamma}–/– (C.129S7 (B6)-Ifngtm1Ts/J), and BALB/c-CD11c-diphtheria toxin (DT) receptor (DTR) transgenic (C.FVB-Tg(Itgax-DTR/EGFP)57Lan/J) mice were purchased from The Jackson Laboratory. All mice were housed in specific pathogen-free conditions in the University of Pittsburgh animal facility. Mice used in experiments were female, age 6–8 wk. All animal procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals.

Tumor cells

Murine breast tumor cell 4T1.2-Neu (28) was maintained in DMEM (Irvine Scientific) supplemented with 10% heat-inactivated FBS (HyClone), 2 mM glutamine (Life Technologies), antibiotic antimycotic solution (Sigma-Aldrich), and G-418 (500 µg/ml) (Invitrogen). Murine breast tumor cell 4T1.2 (27) was maintained in culture medium of 4T1.2-Neu without G-418. Murine colon carcinoma cell CT26 (American Type Culture Collection) was cultured in RPMI 1640 (Irvine Scientific) supplemented with 10% FBS, 2 mM glutamine, and antibiotic antimycotic solution.

Antibodies

Hybridoma cell PC61 (American Type Culture Collection) was cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, and antibiotic antimycotic solution. The culture supernatants were harvested by centrifuging at 4°C, 2000 rpm for 10 min, and stored at –20°C. Anti-CD25 mAb was purified from culture supernatants using mAb purification kit following vendor’s protocol (Amersham). The protein concentration of anti-CD25 mAb was measured using bicinchoninic acid protein assay kit (Pierce). Purified anti-CD25 mAb was stored at –20°C for further use. Anti-CD8 Ab (YTS 169.4.2.1 and YTS 156.7.7) was provided by H. Waldmann (University of Oxford, Oxford, U.K.). Anti-asialoGM1 Ab used to deplete NK cells was purchased from Wako Pharmaceuticals. Anti-Gr-1 Ab (RB6-8C5) used to deplete Gr-1+ cells was purchased from BD Biosciences. Anti-CD40 Ab (FGK45) was provided by S. Schoenberger (La Jolla Institute for Allergy and Immunology, La Jolla, CA).

Generation and characterization of CD4+ T cells

BALB/c mice were injected i.p. with 600 µg of purified anti-CD25 mAb. Three days later, these mice were inoculated s.c. with 4T1.2-Neu (1 x 105) or CT26 (5–13 x 105) in 20 µl of endotoxin-free PBS (Sigma-Aldrich) at the fourth mammary fat pad or at the left flank. Primary tumor (3 mm mean diameter) was observed in all mice on day 3 after tumor inoculation, and measured by electric caliper in two perpendicular diameters every other day. Mice were sacrificed for humane reasons when primary tumor reached 10 mm in mean diameter; when ulceration, bleeding, or both developed; or when mice became ill due to metastatic diseases. Primary tumor was rejected at ~day 21 after tumor inoculation in ~70% of mice that were pretreated by anti-CD25 mAb. Four to 8 days after tumor rejection, CD4+ or CD8+ cells were purified from splenocytes of anti-CD25 mAb-pretreated mice that rejected tumor using anti-mouse CD4 or CD8 microbeads following vendor’s protocols (Miltenyi Biotec). At the same time, CD4+ T cells were purified from splenocytes of anti-CD25 mAb-pretreated mice that did not reject tumor using anti-mouse CD4 microbeads. To characterize CD4+ T cells, purified CD4+ T cells were stained by FITC anti-CD4 (GK1.5) and PE anti-CD25 (7D4) or anti-CD40L (MR1) and related isotype control Abs (eBioscience and BD Biosciences) following standard protocol, and analyzed by flow cytometry. To determine cytokines produced by CD4+ T cells, purified CD4+ T cells (4 x 105) were cocultured with purified naive BALB/c splenic CD11c+ dendritic cells (DCs) loaded with 4T1.2-Neu or CT26 tumor Ag (4 x 105) (29) in 200 µl of RPMI 1640/10% FBS at 37°C, 5% CO2 for 2 days. The concentration of IFN-{gamma}, IL-2, IL-4, IL-5, IL-10, or IL-17 in the culture supernatants was determined by ELISA following vendors’ protocols (eBioscience, BD Biosciences, and Biolegend).

Determination of antitumor immunity ignited by adoptively transferred T cells

Naive BALB/c mice (3–5 mice/group) were adoptively transferred i.v. with freshly purified splenic CD4+ T cells (1 x 107) from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu (or CT26) or that did not reject 4T1.2-Neu, or freshly purified splenic CD8+ T cells (1 x 107) from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu. One day later, those mice were inoculated s.c. with 4T1.2-Neu (1 x 105) or CT26 (5 x 105), as described above. Mice without CD4+ T cell adoptive transfer served as nontreatment control. Mice were monitored, as described above.

Activation of naive CD8+ T cells by DCs in the presence of CD4+ T cells in vitro

CD4+ T cells from splenocytes of anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu were obtained, as described above. CD11c+ DCs or naive CD8+ T cells were purified from splenocytes of naive BALB/c mice using anti-mouse CD11c or CD8 microbeads following vendors’ protocols (Miltenyi Biotec). Purified CD11c+ DCs were loaded with 4T1.2-Neu tumor Ag (29). CD4+ T cells (4 x 105), DCs (4 x 105), and naive CD8+ T cells (4 x 105) were cocultured in 200 µl of RPMI 1640/10% FBS at 37°C, 5% CO2 for 2 days. The concentration of IFN-{gamma} in the culture supernatants was measured by ELISA.

Detection of IFN-{gamma} production from lymphocyte effectors and antitumor activity ignited by adoptively transferred CD4+ T cells in combination with mitomycin C-treated tumor cell immunization

CD4+ T cells (1 x 107) purified from splenocytes of anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu in 200 µl of endotoxin-free PBS were adoptively transferred i.v. into naive BABL/c mice (3–4 mice/group) on day –1. Mice were immunized s.c. by mitomycin C-treated 4T1.2-Neu (1 x 106) (tumor cells were pretreated with 200 µg/ml mitomycin C (Sigma-Aldrich) for 2 h and washed extensively before use) on day 0. Mice with adoptive CD4+ T cell transfer or mitomycin C-treated 4T1.2-Neu immunization alone served as controls. To measure IFN-{gamma} production from lymphocyte effectors, on day 21 or 60 after immunization, mice were sacrificed and splenocytes (1 x 106) were restimulated in vitro with mitomycin C-treated 4T1.2-Neu, 4T1.2, or CT26 (1 x 105) in 1 ml of RPMI 1640/10% FBS at 37°C, 5% CO2 for 3 days. The concentration of IFN-{gamma} in the culture supernatants was determined by ELISA. To evaluate antitumor activity, on day 21 after tumor cell immunization, adoptive CD4+ T cell transfer, or both, mice were inoculated s.c. with 4T1.2-Neu (1 x 105) at the fourth mammary fat pad. Mice were monitored, as described above.

Depletion of endogenous CD8+ T cells, NK cells, Gr-1+ cells, or CD11c+ DCs in vivo

Days –1 and 0 were defined as the day of adoptive CD4+ T cell transfer and tumor inoculation, respectively. To deplete endogenous CD8+ T cells, NK cells, or Gr-1+ cells, anti-CD8 (200 µg/injection), anti-NK1.1 (40 µl/injection), or anti-Gr-1 (200 µg/injection) Ab was injected i.p. into naive BALB/c mice on days –1, +1, +6, and + 9. Normal BALB/c mice without injection of Ab served as control. To transiently deplete endogenous CD11c+ DCs in vivo, BALB/c-CD11c-DTR transgenic mice were injected i.p. once with diphtheria toxin (DT) (Sigma-Aldrich) at 5 ng/g mouse body weight (30). BALB/c-CD11c-DTR transgenic mice without administration of DT served as control. Depletion was confirmed by flow cytometry and resulted in greater than 95% reduction of relevant cell types. Detailed adoptive CD4+ T cell transfer and 4T1.2-Neu challenge experiments in the mice, described above, are shown in related figure legends and tables.

Statistics

Statistical analyses were performed using Student’s t test (GraphPad Prism version 5). Animal survival is presented using Kaplan-Meier survival curves and was statistically analyzed using log rank test (GraphPad Prism version 5). Value of p < 0.05 is considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Injection of anti-CD25 mAb provokes effective antitumor immunity against an aggressive spontaneous metastatic breast tumor 4T1.2-Neu

After 4T1.2-Neu inoculation at the fourth mammary fat pad, primary tumor grew in all mice and was rejected in ~70% of mice that received in vivo administration of anti-CD25 mAb before tumor inoculation (Table I). Injection of anti-CD25 mAb was ineffective in tumor-bearing mice (Table I). Without injection of anti-CD25 mAb, no mice rejected 4T1.2-Neu (Table I). The data suggest that effective antitumor immunity against 4T1.2-Neu can be provoked by in vivo administration of anti-CD25 mAb before tumor inoculation.


View this table:
[in this window]
[in a new window]

  		Table I. In vivo administration of anti-CD25 mAb prior to tumor inoculation provokes potent antitumor immunity against an aggressive spontaneous metastatic murine breast tumor 4T1.2-Neua

 
Cytokine profiles of CD4+ T cells isolated from anti-CD25 mAb-pretreated mice that rejected tumor and that did not reject tumor

Splenic CD4+ T cells were purified from anti-CD25 mAb-pretreated mice that rejected tumor and that did not reject tumor. Both CD4+ T cells stimulated by DCs produced Th1 cytokines (IFN-{gamma} and IL-2) (Fig. 1, A and B), but not Th2 cytokines (IL-4, IL-5, and IL-10) (data not shown). Compared with CD4+ T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4+ T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by DCs produced more IFN-{gamma} and IL-2 in vitro. However, only IFN-{gamma} produced by CD4+ T cells from anti-CD25 mAb-pretreated mice that rejected tumor was tumor specific (Fig. 1A). Compared with CD4+ T cells from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4+ T cells from anti-CD25 mAb-pretreated mice that rejected tumor produced less amount of IL-17 in the absence or presence of DC stimulation in a nonspecific manner (Fig. 1C).


Figure 1
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 1. Protective antitumor immunity is ignited by adoptively transferred CD4+ T cells freshly purified from anti-CD25 mAb-pretreated mice that rejected tumors. CD4+ T cells isolated from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu and that did not reject 4T1.2-Neu were cultured in vitro in the absence or presence of stimulators (4T1.2-Neu or CT26-loaded DCs) for 2 days. The concentration of IFN-{gamma} (A), IL-2 (B), or IL-17 (C) in culture supernatant was determined by ELISA. Data represent three independent experiments. A, CD4+ T cells from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu: 4T1.2-Neu-DCs vs none or CT26-DCs (p < 0.0001); CD4+ T cells from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu vs CD4+ T cells from anti-CD25 mAb-pretreated mice that did not reject 4T1.2-Neu, p = 0.0002 (4T1.2-Neu-DC stimulation). B, CD4+ T cells from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu: 4T1.2-Neu-DCs vs none (p = 0.0027); CD4+ T cells from anti-CD25 mAb-pretreated mice that did not reject 4T1.2-Neu: 4T1.2-Neu-DCs vs none (p < 0.0001); CD4+ T cells from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu vs CD4+ T cells from anti-CD25 mAb-pretreated mice that did not reject 4T1.2-Neu, p = 0.0171 (4T1.2-Neu-DC stimulation). C, CD4+ T cells from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu vs CD4+ T cells from anti-CD25 mAb-pretreated mice that did not reject 4T1.2-Neu, p < 0.0001 (both nonstimulation and DC stimulation). CD4+ or CD8+ T cells purified from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu or CT26 tumor were adoptively transferred i.v. into naive BALB/c mice on day –1. Mice were inoculated s.c. with 4T1.2-Neu (D) at the fourth mammary fat pad or CT26 (E) at the left flank on day 0. Nontreatment mice and mice that were adoptively transferred with CD4+ T cells purified from anti-CD25 mAb-pretreated mice that did not reject 4T1.2-Neu tumor served as controls. In all experiments, primary tumor was observed on day 3 after tumor inoculation and assessed by palpation every other day. Mice that rejected primary tumor demonstrated long-term survival without metastatic tumors. Data represent two to three independent experiments. CD4+ T cells from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu (n = 11) vs nontreatment (n = 7), p < 0.0001; CD8+ T cells from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu (n = 17) vs nontreatment (n = 7), p = 0.0008; CD4+ T cells from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu (n = 11) vs CD4+ T cells from anti-CD25 mAb-pretreated mice that did not reject 4T1.2-Neu (n = 7), p = 0.0002; CD4+ T cells from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu (n = 11) vs CD8+ T cells from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu (n = 17), p = 0.0006; CD4+ T cells from anti-CD25 mAb-pretreated mice that rejected CT26 (n = 5) vs nontreatment (n = 5), p = 0.0025.

 
Adoptively transferred CD4+ T cells ignite potent protective antitumor activity

In 4T1.2-Neu model, CD4+ T cells from anti-CD25 mAb-pretreated mice that did not reject tumor failed to generate protective antitumor activity (Fig. 1D). Although CD4+ or CD8+ T cells from anti-CD25 mAb-pretreated mice that rejected tumor generated protective antitumor activity in an adoptive transfer model (Fig. 1D), the efficacy of antitumor immunity ignited by adoptively transferred CD4+ T cells was much more potent than adoptively transferred CD8+ T cells (Fig. 1D) (p = 0.0006). As reported by others (31, 32), effective antitumor immunity against CT26 can be provoked by in vivo administration of anti-CD25 mAb before tumor inoculation (data not shown). Furthermore, splenic CD4+ T cells purified from anti-CD25 mAb-pretreated mice that rejected CT26 also generated a significant antitumor activity in this adoptive transfer model (Fig. 1E) (p = 0.0025). Taken together, the data suggest that adoptively transferred CD4+ T cells, which were purified from anti-CD25 mAb-pretreated mice that rejected tumors, are powerful to ignite effective protective antitumor immunity.

Tumor Ag and adoptively transferred CD4+ T cells are both required for a tumor-specific IFN-{gamma}-producing cellular response and potent protective antitumor immunity

To test whether CD4+ T cells could promote a tumor-specific IFN-{gamma}-producing cellular response, BALB/c mice were adoptively transferred by CD4+ T cells and subsequently immunized by mitomycin C-treated 4T1.2-Neu. 4T1.2-Neu immunization in the presence of adoptively transferred CD4+ T cells induced, and sustained a long-term IFN-{gamma}-producing cellular response compared with 4T1.2-Neu immunization or adoptive CD4+ T cell transfer alone (Fig. 2A). Restimulation of the splenocytes with an irrelevant CT26 demonstrated that the cellular response was tumor specific (Fig. 2A). In vivo tumor challenge experiments consistently demonstrated that tumor immunization in the presence of adoptive CD4+ T cell transfer induced potent protective antitumor immunity compared with adoptive CD4+ T cell transfer or tumor cell immunization alone (Fig. 2B). The data suggest that tumor Ag and adoptively transferred CD4+ T cells are both required in generating potent protective antitumor immunity.


Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 2. Tumor Ag and adoptively transferred CD4+ T cells are both required to ignite a long-term tumor-specific IFN-{gamma}-producing cellular response and potent protective antitumor immunity. A, CD4+ T cells purified from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu were adoptively transferred i.v. into naive BALB/c mice. One day later, mice were immunized s.c. with mitomycin C-treated 4T1.2-Neu. Mice with injection of mitomycin C-treated 4T1.2-Neu or CD4+ T cells alone served as controls. Twenty-one or 60 days later, mice were sacrificed and splenocytes were restimulated in vitro with mitomycin C-treated 4T1.2-Neu, 4T1.2, or CT26 for 3 days. The concentration of IFN-{gamma} in the culture supernatants was determined by ELISA. One representative of three independent experiments with similar results is presented. B, CD4+ T cells purified from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu were adoptively transferred i.v. into naive BALB/c mice on day –1. Mitomycin C-treated 4T1.2-Neu were injected s.c. on day 0. Twenty-one days later, mice were inoculated s.c. with 4T1.2-Neu at the fourth mammary fat pad. Primary tumor was observed on day 3 after tumor inoculation. Mice that rejected primary tumor demonstrated long-term survival without metastatic tumors. Data represent two independent experiments. CD4+ plus immunization vs CD4+ or immunization alone, p < 0.005.

 
Adoptively transferred CD4+ T cell-ignited antitumor immunity is tumor specific

To confirm the specificity of potent antitumor immunity ignited by adoptively transferred CD4+ T cells, mice were adoptively transferred by CD4+ T cells and challenged by 4T1.2-Neu, a relevant 4T1.2, or an irrelevant CT26. Mice adoptively transferred by CD4+ T cells effectively rejected 4T1.2-Neu, but failed to reject 4T1.2 and CT26 (Table II). The data suggest that adoptively transferred CD4+ T cell-ignited antitumor immunity is tumor specific.


View this table:
[in this window]
[in a new window]

  		Table II. Adoptively transferred CD4+ T cells orchestrate endogenous immune components to ignite potent tumor-specific protective antitumor immunitya

 
Both endogenous innate and adaptive immunity are involved in potent antitumor immunity ignited by adoptively transferred CD4+ T cells

We next examined which immune components play a role in generating antitumor immunity ignited by adoptively transferred CD4+ T cells against this aggressive solid tumor. First, we examined the role of innate immune cells such as NK cells and Gr-1+ cells. In this experimental model, Gr-1+ or NK cell-depleted mice could not effectively reject tumor (Table II). Next, the role of adaptive immune lymphocyte subsets was determined. B cell–/– mice could not effectively generate antitumor activity in mice adoptively transferred by CD4+ T cells (Table II). Depletion of endogenous CD8+ T cells abrogated effective antitumor immunity ignited by adoptively transferred CD4+ T cells (Table II). Finally, we tested the role of effector mechanisms such as IFN-{gamma}. IFN-{gamma}–/– mice adoptively transferred by CD4+ T cells could not effectively reject tumor (Table II). Taken together, the data suggest that adoptively transferred CD4+ T cells orchestrate both innate and adaptive immunity to generate potent tumor-specific immune responses against this aggressive breast tumor.

DCs play a pivotal role in effective antitumor immunity ignited by adoptively transferred CD4+ T cells

We further determined the role of DCs in adoptively transferred CD4+ T cell-ignited antitumor immunity in vitro and in vivo. Although purified splenic CD4+ T cells from anti-CD25 mAb-pretreated mice that rejected tumor or tumor Ag-loaded DCs alone could not activate naive CD8+ T cells, naive CD8+ T cells were activated in the presence of both CD4+ T cells and DCs in vitro (Fig. 3A), indicating that DCs are required in adoptively transferred CD4+ T cell-ignited CD8+ T cell-dependent antitumor immunity. Consistently, CD40–/– mice lost the ability to reject tumor in this experimental model (Fig. 3B), further suggesting that adoptively transferred CD4+ T cells may act on APC such as CD11c+ DCs via CD40 signaling. To confirm their importance, we showed that CD11c+ DCs are required for successful antitumor activity ignited by adoptively transferred CD4+ T cells by using mice carrying a transgene encoding a DTR under the control of the murine CD11c promoter, which allows inducible ablation of CD11c+ DCs in vivo (30). In CD4+ T cell adoptive transfer and tumor challenge experiment, survival was significantly increased in mice without transient depletion of CD11c+ DCs compared with mice with transient depletion of CD11c+ DCs (Fig. 3C). It is worth mentioning that the efficacy of tumor rejection in BALB/c-CD11c-DTR transgenic mice is lower than in BALB/c wild-type (WT) mice (Figs. 1D, 2B, 3B, and 3C, and Table II). It is possible that BALB/c-CD11c-DTR mice somehow resist adoptive CD4+ T cell transfer, to a certain degree, due to the transgenic manipulation. Taken together, the data suggest that DCs are critical in adoptively transferred CD4+ T cell-ignited antitumor activity.


Figure 3
View larger version (8K):
[in this window]
[in a new window]

  		FIGURE 3. DCs play a pivotal role in effective antitumor immunity ignited by adoptively transferred CD4+ T cells. A, CD4+ T cells were obtained from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu. DCs or CD8+ T cells were purified from splenocytes of naive BALB/c mice. Purified CD11c+ DCs were loaded with 4T1.2-Neu. CD4+ T cells, 4T1.2-Neu-loaded DCs, and naive CD8+ T cells were cocultured for 2 days. The concentration of IFN-{gamma} in the culture supernatants was measured by ELISA. One representative of four independent experiments is shown. CD4/DC vs CD4/DC/CD8, p = 0.0170. B, CD4+ T cells (1 x 107) purified from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu were adoptively transferred i.v. into WT or CD40–/– BALB/c mice on day –1. Mice were inoculated s.c. by 4T1.2-Neu (1 x 105) at the fourth mammary fat pad on day 0. Data represent two independent experiments. WT (n = 6) vs CD40–/– (n = 5), p = 0.0213. C, BALB/c CD11c-DTR transgenic mice were injected i.p. with DT. After 24 h, CD4+ T cells (1 x 107) purified from anti-CD25 mAb-pretreated mice that rejected 4T1.2-Neu were adoptively transferred i.v. into transient CD11c+ DC-depleted or nondepleted (control) mice. One day later, mice were inoculated s.c. by 4T1.2-Neu (1 x 105) at the fourth mammary fat pad. Data represent two independent experiments. No depletion (n = 10) vs transient CD11c+ depletion (n = 9), p = 0.0254. In all experiments, primary tumor was observed on day 3 after tumor inoculation. Mice that rejected primary tumor demonstrated long-term survival without metastatic tumors.

 
Adoptively transferred CD4+ T cells ignite tumor-specific memory antitumor immunity

To test whether adoptively transferred CD4+ T cells generate memory antitumor activity, day 40 after initial 4T1.2-Neu rejection, mice were challenged by 4T1.2-Neu, 4T1.2, or CT26. As shown in Table III, mice that rejected initial 4T1.2-Neu also successfully rejected secondary 4T1.2-Neu, but failed to reject 4T1.2 or CT26. These data showed that adoptively transferred CD4+ T cells generated tumor-specific memory immune response against this tumor.


View this table:
[in this window]
[in a new window]

  		Table III. Adoptively transferred CD4+ T cells generate tumor-specific memory antitumor immunitya

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The 4T1.2-Neu is MHC class II–/– (28); therefore, adoptively transferred CD4+ T cells are unlikely to attack this tumor on their own. From this, it was hypothesized that adoptively transferred CD4+ T cells regulate endogenous immune components to generate antitumor immune responses against this tumor.

Tumor cell immunization (providing Ag) alone did not generate a tumor-specific IFN-{gamma}-producing cellular response and failed to trigger antitumor immunity (Fig. 2). However, tumor cell immunization induced a tumor-specific IFN-{gamma}-producing T cell response and potent antitumor activity in the presence of adoptive CD4+ T cell transfer (providing signal(s)) (Fig. 2). Indeed, tumor inoculation (providing Ag) 1 day later after adoptive CD4+ T cell transfer resulted in potent antitumor immunity (Fig. 1D), whereas tumor inoculation 21 days later after adoptive CD4+ T cell transfer (may be unable to provide strong signal(s) due to insufficient adoptively transferred CD4+ T cells at this time point) was dependent on tumor cell immunization to generate potent antitumor activity (Fig. 2B).

It is likely that both signals provided by adoptively transferred CD4+ T cells and Ag provided by tumor cells are required to generate tumor-specific immunity against this tumor. The question becomes which signal(s) is provided by adoptively transferred CD4+ T cells. First, although CD40-independent antitumor responses have been reported (33), CD40 ligation (mimicking activated CD4+ T cells by using functional anti-CD40 mAb) has been shown to generate, enhance, and restore antitumor CTL function in some tumor models (34, 35, 36, 37, 38). In 4T1.2-Neu (data not shown) and relevant 4T1 models (39), injection of functional anti-CD40 mAb did not induce tumor rejection. This observation indicates that adoptively transferred CD4+ T cells not only provide CD40L signaling, but also other undefined signals/factors, to generate antitumor immunity. Second, multiple experiments suggested that IL-2 and IFN-{gamma} benefited antitumor immune responses. Therefore, there is the possibility that in vivo IL-2 and IFN-{gamma} produced by adoptively transferred CD4+ T cells act on CD8+ T cells or APC to initiate help of CD8+ T cells in this model (2, 3). However, recent data showed that IL-2 and IFN-{gamma} also had a role in immune suppression. In this study, CD4+ T cells, which were purified from anti-CD25 mAb-pretreated mice that rejected tumor and that did not reject tumor, produced IL-2 and IFN-{gamma} stimulated by DCs in vitro (Fig. 1, A and B). Although DCs significantly promoted IL-2 production by both CD4+ T cells in a nonspecific manner (Fig. 1B), IL-2 production did not correlate tumor-specific antitumor activity in vivo in the adoptive transfer model (Fig. 1D and Table II). The observation suggests that IL-2 produced by adoptively transferred CD4+ T cells may not be responsible for effective tumor-specific antitumor immunity. CD4+ T cells from anti-CD25 mAb-pretreated mice that rejected tumor produced a high amount of tumor-specific IFN-{gamma} in vitro (Fig. 1A) and generated effective tumor-specific antitumor activity in vivo (Fig. 1D and Table II), indicating that tumor-specific IFN-{gamma} produced by adoptively transferred CD4+ T cells may have a positive impact on effective antitumor immunity. Third, CD4+ T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor were CD4+CD25– (data not shown). It was hypothesized that the lack of Treg results in antitumor activity of adoptively transferred CD4+ T cells. However, CD4+ T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor were also CD4+CD25– (data not shown), which did not generate antitumor activity in the adoptive transfer model. Furthermore, adoptively transferred CD4+CD25– T cells purified from naive BALB/c or tumor-bearing mice could not activate antitumor immunity (data not shown). The data indicate that the lack of Treg in adoptively transferred CD4+ T cells may not be responsible for effective antitumor immunity. Fourth, CD4+ T cells from anti-CD25 mAb-pretreated mice that did not reject tumor enhanced the capacity to produce IL-17 in vitro (Fig. 1C), but failed to generate antitumor activity in vivo (Fig. 1D). In contrast, CD4+ T cells from anti-CD25 mAb-pretreated mice that rejected tumor reduced the capacity to produce IL-17 in vitro (Fig. 1C), but generated effective tumor-specific antitumor activity in vivo (Fig. 1D and Table II). This observation indicates that IL-17 produced by adoptively transferred CD4+ T cells may be involved in the failure of antitumor immunity. This finding is consistent with a recent report that suggested that IL-17 produced by CD8+ T cells directly accounted for promoting 4T1 (original parental cell line of 4T1.2-Neu) tumor growth (40), even though another recent report found that Th17-polarized cells were superior in mediating destruction of advanced B16 melanoma (41). Taken together, adoptively transferred CD4+ T cells serve as the source of signal(s) to induce antitumor immunity in the presence of tumor Ag. However, the theory that signal(s) provided by adoptively transferred CD4+ T cells accounts for the potent antitumor activity needs further investigation, even though IFN-{gamma} (positive) and IL-17 (negative) may be involved in CD4+ T cell-activated antitumor immunity.

Our in vivo data confirmed that adoptively transferred CD4+ T cells induced CD8+ T cell-dependent antitumor response via acting on DCs (Table II and Fig. 3), but their role in igniting antitumor immunity is complicated. First, NK cells and Gr-1+ cells were required for tumor rejection in this model (Table II). Adoptively transferred CD4+ T cells may directly force NK cells to acquire the capacities of antitumor cells or may supply IFN-{gamma} to maintain an endogenous Th1 response (42). NK cells may also directly kill tumor cells, resulting in enhanced tumor Ag capture by resting DCs and subsequently promoting tumor-specific T cell priming. Gr-1 is widely expressed on myeloid populations that can serve as APC. Thus, in vivo administration of anti-Gr-1 Ab may wipe out APC populations that are critical in adoptively transferred CD4+ T cell-activated immunity, resulting in a dampened tumor-specific immune response (Table II). Second, B cells were required in this model (Table II). It was shown that vaccination with activated rat Her2/Neu induces a CD4+ T cell-dependent protective Ab response, which may be unique to BALB/c mice (43). In addition to adoptively transferred CD4+ T cells being able to induce class switching of B cells for production of antitumor Abs (44), they may also activate B cells to generate cellular-mediated antitumor immunity against this tumor because activated B cells can prime both CD4+ and CD8+ T cells (45, 46, 47). Third, endogenous IFN-{gamma} was necessary in adoptively transferred CD4+ T cell-ignited antitumor activity (Table II). IFN-{gamma} not only contributes to antitumor activity, but also reduces antitumor responses through various mechanisms, under different experimental models and/or conditions (48, 49, 50, 51). Previous studies have indicated that IFN-{gamma} was critical in fighting off this tumor and mediated Ig class switch for producing anti-Her2/Neu Abs of the IgG2a and IgG2b subclasses (44, 52), which were reported to play a pivotal role in cancer prevention. Thus, IFN-{gamma} produced by endogenous immune cells is required in tumor rejection in this model. Taken together, it is likely that adoptively transferred CD4+ T cells interact with many different types of endogenous immune components to reject this aggressive breast tumor.

Recent studies showed that CD4+ T cell help was nonspecific in certain cases (53). Alternatively, in this model, the help provided by adoptively transferred CD4+ T cells is tumor specific. Mice adoptively transferred by 4T1.2-Neu-CD4+ T cells rejected 4T1.2-Neu, but not 4T1.2 or CT26 (Table II). Notably, an IFN-{gamma}-producing T cell response enhanced by adoptive 4T1.2-Neu-CD4+ T cell transfer was not specific to CT26 (Fig. 2A) (39), despite the sharing of a murine virus Ag gp70 (54). Interestingly, splenocytes from mice that were immunized by 4T1.2-Neu in the presence of adoptively transferred 4T1.2-Neu-CD4+ T cells responded to 4T1.2 restimulation (Fig. 2A). However, mice adoptively transferred by 4T1.2-Neu-CD4+ T cells failed to reject 4T1.2 (Tables II and III). The data suggest that cellular-mediated immunity alone might be insufficient to reject this tumor. B cells needed in the potent antitumor immunity ignited by adoptively transferred CD4+ T cells suggests that antitumor Ab response might be required in tumor rejection. Either 4T1.2 or 4T1.2-Neu can be the target of cellular-mediated immune responses. However, 4T1.2-Neu provides a well-demonstrated Neu target of Ab-mediated immune responses, which might explain the demonstrated specificity of adoptively transferred CD4+ T cell-ignited antitumor activity.

In summary, our data show that adoptively transferred CD4+ T cells orchestrate broad immune components of hosts to ignite tumor-specific long-term antitumor immunity. This is endogenous CD8+ T cell-dependent tumor rejection. These findings highlight the importance of adoptively transferred CD4+ T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity. Although adoptively transferred CD4+ T cells successfully ignited potent tumor-specific protective antitumor immunity, they failed to eradicate an established 4T1.2-Neu (data not shown). Considerable Treg elevation induced by this tumor may attenuate antitumor immunity ignited by adoptively transferred CD4+ T cells (55) (data not shown). Further investigation of whether combination of adoptive CD4+ T cell transfer and Treg manipulation could eradicate an established 4T1.2-Neu is being actively pursued.


   Acknowledgments
 
We are indebted to S. Schoenberger (La Jolla Institute for Allergy and Immunology) for the anti-CD40 Ab; H. Waldmann (University of Oxford) for the anti-CD8 Ab; P. Morel (University of Pittsburgh) for the hybridoma cell line (PC61); members of Falo’s laboratory for valuable discussions; and the University of Pittsburgh Experimental Animal Facility technicians for animal care. We thank W. J. Storkus (University of Pittsburgh) for critical review of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 This work was supported by a start-up fund from Department of Dermatology of University of Pittsburgh (to Z.Y.) and by National Institutes of Health Grants R01CA108813 (to Z.Y.) and R01CA106662 and P01CA73743 (to L.D.F.). Back

2 Address correspondence and reprint requests to Dr. Zhaoyang You, W1046 Biomedical Sciences Tower, 200 Lothrop Street, Pittsburgh, PA 15213. E-mail address: youz{at}upmc.edu Back

3 Abbreviations used in this paper: Treg, CD4+CD25+ regulatory T cell; DC, dendritic cell; DT, diphtheria toxin; DTR, DT receptor; WT, wild type. Back

Received for publication January 18, 2008. Accepted for publication July 10, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Ostrand-Rosenberg, S.. 2005. CD4+ T lymphocytes: a critical component of antitumor immunity. Cancer Invest. 23: 413-419. [Medline]
  2. Knutson, K. L., M. L. Disis. 2005. Tumor antigen-specific T helper cells in cancer immunity and immunotherapy. Cancer Immunol. Immunother. 54: 721-728. [Medline]
  3. Assudani, D. P., R. B. V. Horton, M. G. Mathieu, S. E. B. McArdle, R. C. Rees. 2007. The role of CD4+ T cell help in cancer immunity and the formulation of novel cancer vaccines. Cancer Immunol. Immunother. 56: 70-80. [Medline]
  4. Kennedy, R., E. Celis. 2006. T helper lymphocytes rescue CTL from activation-induced cell death. J. Immunol. 177: 2862-2872. [Abstract/Free Full Text]
  5. Perez-Diez, A., N. T. Joncker, K. Choi, W. F. N. Chan, C. C. Anderson, O. Lantz, P. Matzinger. 2007. CD4 cells can be more efficient at tumor rejection than CD8 cells. Blood 109: 5346-5354. [Abstract/Free Full Text]
  6. Matoba, K., N. Iizuka, T. Gondo, T. Ishihara, H. Yamada-Okabe, T. Tamesa, N. Takemoto, K. Hashimoto, K. Sakamoto, T. Miyamoto, et al 2005. Tumor HLA-DR expression linked to early intrahepatic recurrence of hepatocellular carcinoma. Int. J. Cancer 115: 231-240. [Medline]
  7. Wang, R. F., S. A. Rosenberg. 1999. Human tumor antigens for cancer vaccine development. Immunol. Rev. 170: 85-100. [Medline]
  8. Matzinger, P.. 1994. Tolerance, danger and the extended family. Annu. Rev. lmmunol. 12: 991-1045.
  9. Antony, P. A., C. A. Piccirillo, A. Akpinarli, S. E. Finkelstein, P. J. Speiss, D. R. Surman, D. C. Palmer, C. C. Chan, C. A. Klebanoff, W. W. Overwijk, et al 2005. CD8+ T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells. J. Immunol. 174: 2591-2601. [Abstract/Free Full Text]
  10. Shu, S., S. A. Rosenberg. 1985. Adoptive immunotherapy of a newly induced sarcoma: immunologic characteristics of effector cells. J. Immunol. 135: 2895-2903. [Abstract]
  11. Greenberg, P. D., M. A. Cheever, A. Fefer. 1981. Eradication of disseminated murine leukemia by chemoimmunotherapy with cyclophosphamide and adoptively transferred immune syngeneic Lyt-1+2– lymphocytes. J. Exp. Med. 154: 952-963. [Abstract/Free Full Text]
  12. Kagamu, H., S. Shu. 1998. Purification of L-selectinlow cells promotes the generation of highly potent CD4 antitumor effector T lymphocytes. J. Immunol. 160: 3444-3452. [Abstract/Free Full Text]
  13. Chamoto, K., T. Tsuji, H. Funamoto, A. Kosaka, J. Matsuzaki, T. Sato, H. Abe, K. Fujio, K. Yamamoto, T. Kitamura, et al 2004. Potentiation of tumor eradication by adoptive immunotherapy with T-cell receptor gene-transduced T-helper type 1 cells. Cancer Res. 64: 386-390. [Abstract/Free Full Text]
  14. Moeller, M., N. M. Haynes, M. H. Kershaw, J. T. Jackson, M. W. L. Teng, S. E. Street, L. Cerutti, S. M. Jane, J. A. Trapani, M. J. Smyth, P. K. Darcy. 2005. Adoptive transfer of gene-engineered CD4+ helper T cells induces potent primary and secondary tumor rejection. Blood 106: 2995-3003. [Abstract/Free Full Text]
  15. Surman, D. R., M. E. Dudley, W. W. Overwijk, N. P. Restifo. 2000. CD4+ T cell control of CD8+ T cell reactivity to a model tumor antigen. J. Immunol. 164: 562-565. [Abstract/Free Full Text]
  16. Marzo, A. L., B. F. Kinnear, R. A. Lake, J. J. Frelinger, E. J. Collins, B. W. S. Robinson, B. Scott. 2000. Tumor-specific CD4+ T cells have a major "post-licensing" role in CTL mediated anti-tumor immunity. J. Immunol. 165: 6047-6055. [Abstract/Free Full Text]
  17. Fallarino, F., U. Grohmann, R. Bianchi, C. Vacca, M. C. Fioretti, P. Puccetti. 2000. Th1 and Th2 cell clones to a poorly immunogenic tumor antigen initiate CD8+ T-cell-dependent tumor eradication in vivo. J. Immunol. 165: 5495-5501. [Abstract/Free Full Text]
  18. Huang, H., F. Li, J. R. Gordon, J. Xiang. 2002. Synergistic enhancement of antitumor immunity with adoptively transferred tumor-specific CD4+ and CD8+ T-cells and intratumoral lymphotactin transgene expression. Cancer Res. 62: 2043-2051. [Abstract/Free Full Text]
  19. Chamoto, K., D. Wakita, Y. Narita, Y. Zhang, D. Noguchi, H. Ohnishi, T. Iguchi, T. Sakai, H. Ikeda, T. Nishimura. 2006. An essential role of antigen-presenting cell/T-helper type 1 cell-cell interactions in draining lymph node during complete eradication of class II-negative tumor tissue by T-helper type 1 cell therapy. Cancer Res. 66: 1809-1817. [Abstract/Free Full Text]
  20. Wang, L.-X., S. Shu, M. L. Disis, G. E. Plautz. 2007. Adoptive transfer of tumor-primed, in vitro activated, CD4+ T-effectors (TE) combined with CD8+ TE provides intratumoral TE proliferation and synergistic antitumor response. Blood 109: 4865-4872. [Abstract/Free Full Text]
  21. Mumberg, D., P. A. Monach, S. Wanderling, M. Philip, A. Y. Toledano, R. D. Schreiber, H. Schreiber. 1999. CD4+ T cells eliminate MHC class II-negative cancer cells in vivo by indirect effects of IFN-{gamma}. Proc. Natl. Acad. Sci. USA 96: 8633-8638. [Abstract/Free Full Text]
  22. Zou, W.. 2006. Regulatory T cells, tumor immunity, and immunotherapy. Nat. Rev. Immunol. 6: 295-307. [Medline]
  23. Simon, A. K., E. Jones, H. Richards, K. Wright, G. Betts, A. Godkin, G. Screaton, A. Gallimore. 2007. Regulatory T cells inhibit Fas ligand-induced innate and adaptive tumor immunity. Eur. J. Immunol. 37: 758-767. [Medline]
  24. Lollini, P. L., F. Cavallo, P. Nanni, G. Forni. 2006. Vaccines for tumor prevention. Nat. Rev. Cancer 6: 204-216. [Medline]
  25. Wang, L., Y. Miyahara, T. Kato, L. Wang, T. Aota, K. Kuribayashi, H. Shiku. 2003. Essential roles of tumor-derived helper T cell epitopes for an effective peptide-based tumor vaccine. Cancer Immun. 3: 16-28. [Medline]
  26. Reilly, R. T., M. B. C. Gottlieb, A. M. Ercolini, J.-P. H. Machiels, C. E. Kane, F. I. Okoye, W. J. Muller, K. H. Dixon, E. M. Jaffee. 2000. Her-2/neu is a tumor rejection target in tolerized Her-2/neu transgenic mice. Cancer Res. 60: 3569-3576. [Abstract/Free Full Text]
  27. Lelekakais, M., J. M. Moseley, T. J. Martin, D. Hards, E. Williams, P. Ho, D. Lowen, J. Javni, F. R. Miller, J. Slavin, R. L. Anderson. 1999. A novel orthotopic model of breast cancer metastasis to bone. Clin. Exp. Metastasis 17: 163-170. [Medline]
  28. Kim, J. H., N. Majumder, H. Lin, J. Chen, L. D. Falo, Jr, Z. You. 2005. Enhanced immunity by NeuEDhsp70 DNA vaccine is needed to combat an aggressive spontaneous metastatic breast cancer. Mol. Ther. 11: 941-949. [Medline]
  29. Hokey, D. A., A. T. Larregina, G. Erdos, S. C. Watkins, L. D. Falo, Jr. 2005. Tumor cell loaded type-1 polarized dendritic cells induce Th1-mediated tumor immunity. Cancer Res. 65: 10059-10067. [Abstract/Free Full Text]
  30. Jung, S., D. Unutmaz, P. Wong, G. I. Sano, K. De los Santos, T. Sparwasser, S. Wu, S. Vuthoori, K. Ko, F. Zavala, et al 2002. In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens. Immunity 17: 211-220. [Medline]
  31. Golgher, D., E. Jones, F. Powrie, T. Elliott, A. Gallimore. 2002. Depletion of CD25+ regulatory cells uncovers immune responses to shared murine tumor rejection antigens. Eur. J. Immunol. 32: 3267-3275. [Medline]
  32. Casares, N., L. Arribillaga, P. Sarobe, J. Dotor, A. Lopez-Diaz de Cerio, I. Melero, J. Prieto, F. Borras-Cuesta, J. J. Lasarte. 2003. CD4+CD25+ regulatory cells inhibit activation of tumor-primed CD4+ T cells with IFN-{gamma}-dependent antigangiogenic activity, as well as long-lasting tumor immunity elicited by peptide vaccination. J. Immunol. 171: 5931-5939. [Abstract/Free Full Text]
  33. Lu, Z., L. Yuan, X. Zhou, E. Sotomayor, H. I. Levitsky, D. M. Pardoll. 2000. CD40-independent pathways of T cell help for priming of CD8+ cytotoxic T lymphocytes. J. Exp. Med. 191: 541-550. [Abstract/Free Full Text]
  34. Sotomayor, E. M., I. Borrello, E. Tubb, F.-M. Rattis, H. Bien, Z. Lu, S. Fein, S. Schoenberger, H. I. Levitsky. 1999. Conversion of tumor-specific CD4+ T-cell tolerance to T-cell priming through in vivo ligation of CD40. Nat. Med. 5: 780-787. [Medline]
  35. Diehl, L., A. T. den Boer, S. P. Schoenberger, E. I. H. van der Voort, T. N. M. Schumacher, C. J. M. Melief, R. Offringa, R. E. M. Toes. 1999. CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficiency. Nat. Med. 5: 774-779. [Medline]
  36. Stumbles, P. A., R. Himbeck, J. A. Frelinger, E. J. Collins, R. A. Lake, W. S. Robinson. 2004. Tumor-specific CTL are constitutively cross-armed in draining lymph nodes and transiently disseminate to mediate tumor regression following systemic CD40 activation. J. Immunol. 173: 5923-5928. [Abstract/Free Full Text]
  37. Van Mierlo, G. J. D., Z. F. H. M. Boonman, H. M. H. Dumortier, A. T. den Boer, M. F. Fransen, J. Nouta, E. I. H. van der Voort, R. Offringa, R. E. M. Toes, C. J. M. Melief. 2004. Activation of dendritic cells that cross-present tumor-derived antigen licenses CD8+ CTL to cause tumor eradication. J. Immunol. 173: 6753-6759. [Abstract/Free Full Text]
  38. Lodge, A., P. Yu, M. B. Nicholl, I. E. Brown, C.-C. A. Jackson, K. Schreiber, S. L. Sugg, H. Schreiber, J. Shilyansky. 2006. CD40 ligation restores cytolytic T lymphocyte response and eliminates fibrosarcoma in the peritoneum of mice lacking CD4+ T cells. Cancer Immunol. Immunother. 55: 1542-1552. [Medline]
  39. Uno, T., K. Takeda, Y. Kojima, H. Yoshizawa, H. Akiba, R. S. Mittler, F. Gejyo, K. Okumura, H. Yagita, M. J. Smyth. 2006. Eradication of established tumors in mice by a combination antibody-based therapy. Nat. Med. 12: 693-698. [Medline]
  40. Nam, J.-S., M. Terabe, M.-J. Kang, H. Chae, N. Voong, Y.-A. Yang, A. Laurence, A. Michalowska, M. Mamura, S. Lonning, et al 2008. Transforming growth factor β subvert the immune system into directly promoting tumor growth through interleukin-17. Cancer Res. 68: 3915-3923. [Abstract/Free Full Text]
  41. Muranski, P., A. Boni, P. A. Antony, L. Cassard, K. R. Irvine, A. Kaiser, C. M. Paulos, D. C. Palmer, C. E. Touloukian, K. Ptak, et al 2008. Tumor-specific Th17-polarized cells eradicate large established melanoma. Blood 112: 362-373. [Abstract/Free Full Text]
  42. Martin-Fontecha, A., L. L. Thomsen, S. Brett, C. Gerard, M. Lipp, A. Lanzavecchia, F. Sallusto. 2004. Induced recruitment of NK cells to lymph nodes provides IFN-{gamma} for Th1 priming. Nat. Immunol. 5: 1260-1265. [Medline]
  43. Rovero, S., A. Amici, E. Di Carlo, R. Bei, P. Nanni, E. Quaglino, P. Porcedda, K. Boggio, A. Smorlesi, P.-L. Lollini, et al 2000. DNA vaccination against rat Her-2/Neu p185 more effectively inhibits carcinogenesis than transplantable carcinoma in transgenic BALB/c mice. J. Immunol. 165: 5133-5142. [Abstract/Free Full Text]
  44. Nanni, P., L. Landuzzi, G. Nicoletti, C. De Giovanni, I. Rossi, S. Croci, A. Astolfi, M. Iezzi, E. Di Carlo, P. Musiani, et al 2004. Immunoprevention of mammary carcinoma in HER-2/Neu transgenic mice is IFN-{gamma} and B cell dependent. J. Immunol. 173: 2288-2296. [Abstract/Free Full Text]
  45. Schultze, J. L., S. Michalak, M. J. Seamon, G. Dranoff, K. Jung, J. Daley, J. C. Delgado, J. G. Gribben, L. M. Nadler. 1997. CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy. J. Clin. Invest. 100: 2757-2765. [Medline]
  46. Lapointe, R., A. Bellemare-Pelletier, F. Housseau, J. Thibodeau, P. Hwu. 2003. CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells. Cancer Res. 63: 2836-2843. [Abstract/Free Full Text]
  47. Crawford, A., M. MacLeod, T. Schumacher, L. Corlett, D. Gray. 2006. Primary T cell expansion and differentiation in vivo requires antigen presentation by B cells. J. Immunol. 176: 3498-3506. [Abstract/Free Full Text]
  48. Berner, V., H. Liu, Q. Zhou, K. L. Alderson, K. Sun, J. M. Weiss, T. C. Back, D. L. Longo, B. R. Blazar, R. H. Wiltrout, et al 2007. IFN-{gamma} mediates CD4+ T cell loss and impairs secondary antitumor responses after successful initial immunotherapy. Nat. Med. 13: 354-360. [Medline]
  49. Fallarino, F., U. Grohmann, K. W. Hwang, C. Orabona, C. Vacca, R. Bianchi, M. L. Belladonna, M. C. Fioretti, M.-L. Alegre, P. Puccetti. 2003. Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 4: 1206-1212. [Medline]
  50. Beatty, G. L., Y. Paterson. 2000. IFN-{gamma} can promote tumor evasion of the immune system in vivo by down-regulating cellular levels of an endogenous tumor antigen. J. Immunol. 165: 5502-5508. [Abstract/Free Full Text]
  51. Wang, Z., J. Hong, W. Sun, G. Xu, N. Li, X. Chen, A. Liu, L. Xu, B. Sun, J. Z. Zhang. 2006. Role of IFN-{gamma} in induction of Foxp3 and conversion of CD4+CD25– T cells to CD4+ Tregs. J. Clin. Invest. 116: 2434-2441. [Medline]
  52. Pulaski, B. A., M. J. Smyth, S. Ostrand-Rosenberg. 2002. Interferon-{gamma}-dependent phagocytic cells are a critical component of innate immunity against metastatic mammary carcinoma. Cancer Res. 62: 4406-4412. [Abstract/Free Full Text]
  53. Bevan, M. J.. 2004. Helping the CD8+ T-cell response. Nat. Rev. Immunol. 4: 595-602. [Medline]
  54. Luznik, L., J. E. Slansky, S. Jalla, I. Borrello, H. I. Levitsky, D. M. Pardoll, E. J. Fuchs. 2003. Successful therapy of metastatic cancer using tumor vaccines in mixed allogeneic bone marrow chimeras. Blood 101: 1645-1652. [Abstract/Free Full Text]
  55. Valzasina, B., S. Piconese, C. Guiducci, M. P. Colombo. 2006. Tumor-induced expansion of regulatory T cells by conversion of CD4+CD25– lymphocytes is thymus and proliferation independent. Cancer Res. 66: 4488-4495. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
Q. Li, S. Teitz-Tennenbaum, E. J. Donald, M. Li, and A. E. Chang
In Vivo Sensitized and In Vitro Activated B Cells Mediate Tumor Regression in Cancer Adoptive Immunotherapy
J. Immunol., September 1, 2009; 183(5): 3195 - 3203.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Z. Liu, J. H. Kim, L. D. Falo Jr., and Z. You
Tumor Regulatory T Cells Potently Abrogate Antitumor Immunity
J. Immunol., May 15, 2009; 182(10): 6160 - 6167.
[Abstract] [Full Text] [PDF]

Home page
 Am Soc Clin Oncol Ed BookHome page
C. Yee
Adoptive T Cell Therapy: Stratagems and Spoils
ASCO Educational Book, January 1, 2009; 2009(1): 511 - 515.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, Z.
Right arrow Articles by You, Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, Z.
Right arrow Articles by You, Z.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS