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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Mol Ther. Author manuscript; available in PMC 2010 July 1.
Published in final edited form as:
PMCID: PMC2731416
NIHMSID: NIHMS120820

Therapeutic Immunity by Adoptive Tumor-Primed CD4+ T Cell Transfer in Combination with In Vivo GITR Ligation

Abstract

Tumor-primed CD4+ T cells from splenocytes of tumor-rejection mice in combination with in vivo GITR ligation (the combination therapy) elicited effective host CD8+ T cell-dependent therapeutic immunity against a murine breast tumor. GITR ligation in vitro enhanced tumor-primed CD4+ T cell activity and partially abrogated Treg suppressor function. DC from TDLN of tumor-bearing mice treated by the combination therapy stimulated Ag-specific T cells and produced IL-12 ex vivo. Whereas tumor-primed CD4+ T cells or in vivo GITR ligation alone induced a tumor-specific IFN-γ producing cellular response, the combination therapy enhanced and sustained it. Furthermore, the combination therapy in vivo attenuated Treg ability to suppress IL-12 production by DC and IFN-γ production by effectors ex vivo. Importantly, tumor-primed CD4+ CD25 T cells from splenocytes of untreated tumor-bearing mice in combination with in vivo GITR ligation also elicited an effective therapeutic effect in this model. These data suggest that the combination therapy may improve DC function, accentuate tumor-specific T cell responses and attenuate Treg suppressor function, thereby eliciting effective therapeutic immunity.

Keywords: Adoptive tumor-primed CD4+ T cell transfer, in vivo GITR ligation, Therapeutic immunity

INTRODUCTION

CD4+ T cells, the major orchestrators of the immune system, are essential in eliciting tumor-specific neutralizing antibodies and cellular immunity for tumor rejection via multiple mechanisms (12). Adoptively-transferred CD4+ T cells can enhance antitumor activity mediated by adoptively-transferred CD8+ T cells and initiate host CD8+ T cell dependent or independent antitumor immunity in various models (311). A recent study has demonstrated that adoptive tumor-primed CD4+ T cell transfer (CD4 AT) can induce a clinical effective immune response against metastatic melanoma (12). Thus, CD4 AT has clearly emerged as an important approach in combating tumors.

CD4+CD25+Foxp3+ regulatory T cells (Treg) constitutively express glucocorticoid-induced TNF receptor (GITR) (1314). Activated T cells, NK cells, monocytes, macrophages, B cells, mast cells and dendritic cells (DC) also express GITR (15). GITR ligation via agonistic α-GITR mAb has been shown to 1) inhibit Treg-dependent suppression and enhance T cell responses (13, 16), 2) impede established tumors (17), 3) induce tumor immunity against B16 melanoma in a concomitant immunity model (18), 4) promote DNA vaccine-induced CD8+ T cell-dependent tumor protection (19), 5) predominantly act on T effectors (Teff) rather than Treg (20), 6) enhance adenoviral vector vaccine-induced cytotoxic T lymphocytes (CTL) (21), 7) induce CD4+ Teff which are resistant to Treg suppression in a murine CT26 tumor model (22), and 8) reduce the frequency of Treg in the spleen and tumor-draining lymph nodes (TDLN) (23). Otherwise, GITR ligation via GITRL reduced Treg suppressive activity, generated Treg-resistant Teff and promoted CD8+ T cell infiltration (2429). Therefore, in vivo GITR ligation has potential to induce and/or augment tumor-specific immunity (1729).

Either CD4 AT or in vivo GITR ligation alone can induce antitumor immunity in some tumor models. Whether CD4 AT in combination with in vivo GITR ligation via agonistic α-GITR mAb can elicit effective therapeutic antitumor immunity has not yet been documented. In this report, we explored this possibility in a murine breast tumor model. We also examined possible mechanisms underlying the combination strategy.

RESULTS

CD4 AT in combination with in vivo GITR ligation (the combination therapy) elicits therapeutic immunity against a murine breast tumor

The murine breast tumor 4T1.2-Neu (3031) shares many characteristics with many human advanced breast cancers such as aggressive metastasis, inherent resistance to chemotherapy, poor immunogenicity, major histocompatibility complex (MHC) class II negative, production of various immune suppression factors, induction and/or expansion of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg), and expression of “oncoantigen” Her2/Neu (10, 3034). In our previous study, adoptively-transferred tumor-primed CD4+ T cells, which were isolated from α-CD25 mAb-pretreated mice that rejected the tumor, generated an effective host CD8+ T cell-dependent tumor-specific protection (10). They did not do so in a therapeutic setting although the foreign Ag (rat Her2/Neu) is not tolerated in mice (Fig. 1A) (35).

FIGURE 1
The combination therapy elicits effective therapeutic immunity against a murine breast tumor

To explore whether in vivo GITR ligation via α-GITR mAb can improve therapeutic immunity induced by adoptively-transferred tumor-primed CD4+ T cells, 1 d after CD4 AT, α-GITR mAb or rat IgG was administrated. As shown in Fig. 1A, the combination therapy was effective in eliciting a therapeutic response against the breast tumor when compared with α-GITR mAb, tumor-primed CD4+ T cells, and tumor-primed CD4+ T cells in combination with rat IgG (p<0.005). Importantly, tumor-primed CD4+CD25 T cells, which were isolated from untreated tumor-bearing mice (a clinical relevant setting), in combination with in vivo GITR ligation also elicited the effective therapeutic immunity in this tumor model (p<0.0005) (Fig. 1B). The failure of naive CD4+CD25 T cells in the combination therapy suggestsantigen-specificity is necessary (supplemental data Fig. S1). Collectively, this data shows that the combination therapy can elicit effective therapeutic immunity against the breast tumor.

Therapeutic immunity elicited by the combination therapy is host CD8+ T cell-dependent

We next tested whether tumor-primed CD4+ T cells in vitro pretreated by α-GITR mAb are sufficient in eliciting a therapeutic activity. Tumor-primed CD4+ T cells were pretreated with α-GITR mAb in vitro. These pretreated tumor-primed CD4+ T cells did not generate an effective therapeutic effect after adoptive transfer (Fig. 2). This suggests that α-GITR mAb in vivo might also act on other immune cells in combination with adoptively-transferred tumor-primed CD4+ T cells in eliciting therapeutic immunity. To examine whether host CD8+ T cells are required in the therapeutic immunity elicited by the combination therapy, CD8+ T cells were depleted during and after treatment. Depletion of CD8+ T cells abrogated the therapeutic effect elicited by the combination therapy (Fig. 2). This data shows that therapeutic immunity elicited by the combination therapy is host CD8+ T cell-dependent.

FIGURE 2
Therapeutic immunity elicited by the combination therapy is host CD8+ T cell-dependent

GITR ligation in vitro enhances tumor-primed CD4+ T cell activity and partially abrogates Treg suppressor function

Tumor-primed CD4+ T cells from α-CD25 mAb-pretreated mice that rejected the tumor may presumably include tumor-specific CD4+ T cells, non-specific CD4+ T cells and new Treg that have arisen in the hosts as a consequence of Treg homeostasis or of tumor-driven peripheral conversion (36). However, tumor-primed CD4+ T cells, at least under conditions of the experimental protocols, consisted mostly of CD4+CD25 T cells (>98%) expressing CD40L (detectable), CD44 and CD62L (high levels), and responded to tumor-specific stimulation (supplemental data Figs. S2 and S3). GITR is highly expressed on tumor-primed CD4+CD25 T cells or Treg (Fig. 3A–B). To determine whether α-GITR mAb acts on tumor-primed CD4+ T cells, tumor-primed CD4+ T cells were cultured in the presence or absence of α-GITR mAb. α-GITR mAb stimulated IFN-γ production by tumor-primed CD4+ T cells (Fig. 3C). This suggests that GITR ligation in vitro enhances tumor-primed CD4+ T cell activity. DC stimulated tumor-primed CD4+ T cells for IFN-γ production, which was suppressed by Treg (Fig. 3D). To test whether α-GITR mAb can abrogate Treg suppressor function, Treg were preincubated with α-GITR mAb. α-GITR mAb-pretreated Treg decreased their ability to suppress IFN-γ production by DC-stimulated tumor-primed CD4+ T cells in vitro (p<0.05) (Fig. 3D). This suggests that GITR ligation in vitro partially abrogates Treg suppressor function.

FIGURE 3
GITR ligation in vitro enhances tumor-primed CD4+ T cell activity and reduces Treg suppressor function

DC isolated from TDLN of tumor-bearing mice treated by the combination therapy stimulate Ag-specific T cell proliferation and produce IL-12 ex vivo

To determine the impact of the combination therapy on the function of DC in TDLN, tumor-bearing mice were non-treated or treated by tumor-primed CD4+ T cells, α-GITR mAb, tumor-primed CD4+ T cells in combination with rat IgG or the combination therapy. 2 d after last treatment, DC were purified from pooled TDLN, and cultured with OT-II in the presence of OVA-specific MHC class-II peptides. As shown in Fig. 4A, DC isolated from TDLN of tumor-bearing mice treated by the combination therapy effectively stimulated OVA-specific OT-II proliferation ex vivo when compared with non-treatment, tumor-primed CD4+ T cells, α-GITR mAb or tumor-primed CD4+ T cells in combination with rat IgG (p<0.05). Furthermore, as shown in Fig. 4B, DC isolated from TDLN of tumor-bearing mice treated by the combination therapy produced significant IL-12 ex vivo when compared with non-treatment, tumor-primed CD4+ T cells, α-GITR mAb or tumor-primed CD4+ T cells in combination with rat IgG ex vivo (p<0.005). This data suggests that the combination therapy in vivo may improve DC function.

FIGURE 4
DC from TDLN of tumor-bearing mice treated by the combination therapy stimulate Ag-specific T cell proliferation and produce IL-12 ex vivo

The combination therapy enhances and sustains a tumor-specific IFN-γ-producing cellular response

To determine the effect of the combination therapy on cellular immunity, tumor-bearing mice were non-treated or treated by tumor-primed CD4+ T cells, α-GITR mAb, tumor-primed CD4+ T cells in combination with rat IgG or the combination therapy. 12 d later, splenocytes from mice of each group were restimulated with 4T1.2-Neu or CT26. Although α-GITR mAb, tumor-primed CD4+ T cells or tumor-primed CD4+ T cells in combination with rat IgG generated a tumor-specific cellular immune response when compared with non-treatment (p<0.005), the combination therapy markedly augmented it (Fig. 5A) (p<0.0001). 60 d later, splenocytes from surviving mice that were treated by α-GITR mAb or the combination therapy were restimulated with 4T1.2-Neu or CT26. The combination therapy sustained a tumor-specific IFN-γ-producing cellular response when compared with α-GITR mAb (p<0.0001) (Fig. 5B). Notably, an IFN-γ-producing T cell response was specific to 4T1.2-Neu but not CT26 (10, 37, Fig. 4), despite the sharing of a murine virus Ag gp70 (38). This data shows that the combination therapy induces an effective long-term tumor-specific cellular immune response.

FIGURE 5
The combination therapy enhances and sustains a tumor-specific IFN-γ-producing cellular response

The combination therapy in vivo attenuates Treg suppressor function

CD4+CD25+ T cells, which were purified from tumor-bearing mice treated by the combination therapy, expressed Foxp3 and were unable to produce IFN-γ upon ex vivo stimulation (Fig. 6A–B). To investigate the influence of the combination therapy on Treg suppressor function in tumor-bearing mice, we tested whether Treg ability to dampen DC function can be attenuated by the combination therapy. In the presence of LPS, purified splenic DC were cultured alone or with Treg purified from tumor-bearing mice non-treated or treated by tumor-primed CD4+ T cells, α-GITR mAb, tumor-primed CD4+ T cells in combination with rat IgG or the combination therapy. Treg purified from tumor-bearing mice non-treated or treated by tumor-primed CD4+ T cells, α-GITR mAb or tumor-primed CD4+ T cells in combination with rat IgG suppressed splenic DC ability to produce IL-12 (Fig. 6C). In contrast, Treg purified from tumor-bearing mice treated by the combination therapy did not do so (Fig. 6C). We next tested whether Treg ability to inhibit effectors can be decreased by the combination therapy. In the presence of tumor cell stimulation, effectors from tumor-bearing mice treated by the combination therapy were cultured alone or with Treg as described above. As shown in Fig. 6D, Treg purified from tumor-bearing mice treated by the combination therapy did not inhibit effectors. Taken together, this data suggests that the combination therapy might in vivo attenuate Treg suppressor function.

FIGURE 6
The combination therapy in vivo attenuates Treg suppressor function

DISSCUSSION

In this 4T1.2-Neu breast tumor model, both adoptively-transferred tumor-primed CD4+ T cells and in vivo GITR ligation induced a tumor-specific cellular response (Fig. 4A). However, neither elicited an effective therapeutic response against the breast tumor (Fig. 1A). The requirement for CD4+ T cells in the in vivo GITR ligation-induced anti-CT26 tumor immunity raises the possibility that CD4+ T cells work as helper T cells in augmenting effector mechanisms downstream (22). In this study, adoptively-transferred tumor-primed CD4+ T cells in combination with in vivo GITR ligation elicited effective therapeutic immunity against the breast tumor (Figs. 12).

As previously published (20, 22), GITR ligation in vitro enhanced IFN-γ production by tumor-primed CD4+ T cells (Fig. 3C), and in vivo induced a tumor-specific IFN-γ-producing T cell response (Fig. 5A). In this model, GITR ligation in vitro partially reduced Treg suppressor function (Fig. 3D). However, GITR ligation in vivo did not attenuate Treg suppressor function (Fig. 6C–D). The action of agonistic GITR ligation on Treg is controversial (13, 1623). The conflicting observations are likely due to different experimental and/or disease models used in various studies.

α-GITR mAb in vitro dramatically stimulated the production of IFN-γ by tumor-primed CD4+ T cells (Fig. 3C). However, α-GITR mAb-in vitro pretreated tumor-primed CD4+ T cells did not induce therapeutic immunity (Fig. 2). It is possible that IFN-γ produced by α-GITR mAb-stimulated tumor-primed CD4+ T cells may not be sufficient for a desired therapeutic effect. Data also suggest that α-GITR mAb in vivo might act on other immune cells in combination with adoptively-transferred tumor-primed CD4+ T cells in eliciting therapeutic immunity. The requirement of host CD8+ T cells in an effective therapeutic effect elicited by the combination therapy further indicates that adoptively-transferred tumor-primed CD4+ T cells act with α-GITR mAb on host immune components in eliciting host CD8+ T cell-dependent therapeutic immunity.

α-GITR mAb has been shown to target Treg, CD4+ and CD8+ T cells, NK cells and B cells (15). DC are essential in tumor immunity induced by adoptively-transferred tumor-primed CD4+ T cells (10). The data suggests that the combination therapy may modulate DC in TDLN becoming more effective in stimulating Ag-specific T cells and producing IL-12 than tumor-primed CD4+ T cells, α-GITR mAb or tumor-primed CD4+ T cells in combination with rat IgG (Fig. 4). Regarding the mechanisms of the combination therapy-mediated DC targeting in vivo, there are several possibilities: 1) α-GITR mAb might be is first to ‘educate’ tumor-primed CD4+ T cells and/or other immune cells, then these ‘educated’ tumor-primed CD4+ T cells and/or other immune cells may be superior in conditioning DC for inducing effective tumor-specific immunity; 2) α-GITR mAb might be first to ‘educate’ DC since DC express GITR (15, data not shown), then α-GITR mAb-in vivo ‘educated’ DC may be optimal to respond the condition by tumor-primed CD4+ T cells for inducing effective tumor-specific immunity; 3) tumor-primed CD4+ T cells and α-GITR mAb in vivo might simultaneously act on DC for inducing effective tumor-specific immunity. It is also likely that all these defined mechanisms operate together in TDLN. Regardless of the precise mechanisms, this observation suggests that the combination therapy might in vivo modulate DC function for inducing optimal therapeutic immunity.

Considering the fact that DC in TDLN are critical in initiating tumor-specific immunity in tumor-bearing hosts, it was expected that the combination therapy vigorously induces tumor-specific T cell responses. Although tumor-primed CD4+ T cells, α-GITR mAb or tumor-primed CD4+ T cells in combination with rat IgG generated a tumor-specific IFN-γ-producing cellular response (Fig. 5A), the combination therapy was effective in doing so and sustained it (Fig. 5A–B). It therefore seems that the combination therapy may improve DC function (the precise mechanisms needed to be further investigated) for induction of a long-term tumor-specific T cell response.

In this study, Treg depletion in vivo may be necessary to investigate the role of Treg in this combination therapy. However, in this tumor model, mice pretreated with α-CD25 mAb before tumor inoculation effectively rejected the tumor (10). Thus, injection of α-CD25 mAb before tumor inoculation is not feasible in this model. Nor is it feasible to inject α-CD25 mAb after tumor inoculation because injection of α-CD25 mAb depletes CD25+ activated T cells, resulting in the failure of therapeutic immunity (data not shown). Alternatively, we have focused on the specific effects of Treg, which were purified from tumor-bearing mice treated by the combination therapy or controls, on dampening DC function and inhibiting effectors ex vivo. Our data shows that the combination therapy but not α-GITR mAb or CD4 AT alone reduced Treg ability to suppress IL-12 production by DC (Fig. 6C). Furthermore, Treg isolated from tumor-bearing mice treated by the combination therapy were not effective in inhibiting IFN-γ production by effectors (Fig. 6D). Effectors from tumor-bearing mice treated by the combination therapy were suppressed by Treg purified from tumor-bearing mice that were untreated (Fig. 6C–D), suggesting that the combination therapy may not render effectors refractory to suppression by Treg. Thus, it is likely that the combination therapy in vivo attenuates Treg suppressor function rather than making Teff resistant to Treg suppression in tumor-bearing mice. It is now recognized that Treg suppressor function may be compartmentalized (39), whether there are differential effects of the combination therapy on Treg in various lymph compartments needs to be addressed in the future studies.

It is unknown how adoptively-transferred tumor-primed CD4+ T cells and α-GITR αmAb contribute mechanistically to attenuate Treg suppressor function. Adoptively-transferred and/or endogenously-induced tumor-primed CD4+ T cells may provide immune stimulatory signals that cooperate in vivo with α-GITR mAb which directly act on Treg and/or indirectly modulate other immune cells such as DC or both, resulting in attenuation of Treg suppressor function. This is a complicated mechanism and needs to be further investigated in the future studies.

In summary, this study has demonstrated that, in the breast tumor model, neither adoptively-transferred tumor-primed CD4+ T cells nor in vivo GITR ligation alone can in vivo improve DC function, attenuate Treg suppressor function and elicit effective therapeutic immunity, and the combination therapy is crucial for achieving this effect. The success of tumor-primed CD4+ CD25 T cells from untreated tumor-bearing mice (a clinical relevant setting) in this approach suggests a possibility of a practical translation of the combination therapy. However, we acknowledged that the combination therapy should also be tested on more advanced tumors, when neovasculature and stroma are clearly formed, before a clinical application could be considered.

MATERIALS AND MATHODS

Mice, cell lines and antibodies

BALB/c, BALB/c-Tg (DO11.10)10Loh/J mice (female, 6–8 wks) were purchased from Taconic and The Jackson Laboratory, and housed in specific pathogen-free conditions in the University of Pittsburgh animal facility. All animal procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals. Murine breast tumor cell 4T1.2-Neu (30) was maintained in DMEM (IRVINE Scientific) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 2mM glutamine (Invitrogen), antibiotic antimycotic solution (Sigma) and G-418 (500 μg/ml) (Invitrogen). Murine colon carcinoma cell CT26 (ATCC) was cultured in RPMI 1640 (IRVINE Scientific) supplemented with 10% FBS, 2mM glutamine and antibiotic antimycotic solution. α-CD25 mAb (PC61.5, ATCC) or agonistic α-GITR mAb (DTA-1) was purified from culture supernatants of a hybridoma cell line (13) using MAb purification kit (Amersham) or purchased from eBioscience. Purified rat IgG (Sigma) and rat IgG2b (eBioscience) served as isotype controls.

Tumor-primed CD4+ T cells

Tumor-primed CD4+ T cells were isolated from splenocytes of α-CD25 mAb-pretreated mice that rejected the tumor or untreated tumor-bearing mice (10). Briefly, BABL/c mice were injected i.p. with 600μg of α-CD25 mAb. 3 d later, mice were inoculated s.c with 4T1.2-Neu (1×105) in 20μl endotoxin-free 1×PBS (Sigma) at the 4th mammary fat pad. 3–4 wk later, tumor-primed CD4+ T cells were purified from splenocytes of α-CD25 mAb-pretreated mice that rejected the tumor using α-mouse CD4 micro-beads (Miltenyi Biotec). To prepare tumor-primed CD4+CD25 T cells from untreated tumor-bearing mice, BALB/c mice were inoculated s.c with 4T1.2-Neu (1×105) as described above. 14–21 d later, tumor-primed CD4+CD25 T cells were obtained by removal of CD4+CD25+ T cells in splenic CD4+ T cells using mouse CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec). Freshly-purified tumor-primed CD4+ or CD4+CD25 T cells were used in experiments.

Function of agonistic α-GITR mAb on tumor-primed CD4+ T cells and Treg in vitro

DC purified from splenocytes of naive BALB/c mice using α-mouse CD11c micro-beads (Miltenyi Biotec) were loaded with 4T1.2-Neu tumor Ag (10). Treg were purified from splenocytes of naive BALB/c using mouse CD4+CD25+ regulatory T cell isolation kit. Tumor-primed CD4+ T cells or Treg (1×106) were cultured in the presence of agonistic α-GITR mAb or rat IgG2b (100μg/ml) in 200μl RPMI 1640 10%FBS at 37°C, 5% CO2 for 2 d. Tumor Ag-loaded DC (4×105) were cultured with tumor-primed CD4+ T cells (1×106) in the presence or absence of Treg or agonistic α-GITR mAb-pretreated Treg (1×106 Treg were preincubated with 100μg agonistic α-GITR mAb in 200μl 1× PBS on ice for 30 min, then washed for use) (1×106) in 200μl RPMI 1640 10%FBS at 37°C, 5% CO2 for 2 d. The concentration of IFN-γ in the culture supernatants was determined by ELISA (BD Biosciences).

Function of DC isolated from TDLN of tumor-bearing mice non-treated or treated by tumor-primed CD4+ T cells, agonistic α-GITR mAb, tumor-primed CD4+ T cells in combination with rat IgG or the combination therapy

4T1.2-Neu (1×105) were inoculated into BALB/c (3–5mice/group) as described above at d 0. These mice were adoptively transferred i.v. by tumor-primed CD4+ T cells (1×107) and were administrated i.p. with 800μg agonistic α-GITR mAb or rat IgG at d 3 and d 4, respectively. 2 d later, DC were purified from pooled TDLN using α-mouse CD11c micro-beads. Purified DC (5×104) were cultured with OT-II (5×104) purified from naive DO11.10 TCR transgenic mice and OVA MHC class II peptides (OVA323–339, purity>95%, synthesized and HPLC purified in the core facility of University of Pittsburgh) at a final concentration of 1μM in 200μl RPMI 1640 10%FBS at 37°C, 5% CO2 for 3 d. 3H (1μCi per well; Du Pont/New England Nuclear) was added during the last 16–18 h of culture. The concentration of IL-12 in culture supernatants was determined by ELISA (eBioscience, BD Biosciences).

Tumor-specific IFN-γ-producing cellular responses in tumor-bearing mice non-treated or treated by tumor-primed CD4+ T cell, agonistic α-GITR mAb, tumor-primed CD4+ T cells in combination with rat IgG or the combination therapy

Tumor-bearing mice (3mice/group) treated by tumor-primed CD4+ T cells, agonistic α-GITR mAb or the combination therapy were described above. At d 12, splenocytes (1×106) from mice in each group were restimulated in vitro with mitomycin C-treated 4T1.2-Neu or CT26 (1×105) in 200μl RPMI 1640 10%FBS at 37°C, 5% CO2 for 3 d (10). At d 60, splenocytes (1×106) from surviving mice that were treated by the combination therapy or agonistic α-GITR alone were restimulated in vitro as described above. The concentration of IFN-γ in the culture supernatants was determined by ELISA.

Function of Treg isolated from tumor-bearing mice non-treated or treated by tumor-primed CD4+ T cells, agonistic α-GITR mAb, tumor-primed CD4+ T cells in combination with rat IgG or the combination therapy

Tumor-bearing mice (3mice/group) treated by tumor-primed CD4+ T cells, agonistic α-GITR mAb alone or the combination therapy were described above. At d 21, freshly-purified naive splenic DC (4×105) were cultured alone or cultured with Treg (4×105) purified from splenocytes of mice of each group in the presence of LPS (100ng/ml, Sigma) in 200μl RPMI 1640 10%FBS at 37°C, 5% CO2 for 2–3 d. The concentration of IL-12 in the culture supernatants was determined by ELISA. At the same time, splenocytes (1×106) from tumor-bearing mice treated by the combination therapy were restimulated in vitro with mitomycin C-treated 4T1.2-Neu (1×105) in the presence or absence of Treg (1×106) purified from splenocytes of mice of each group as described above in 500μl RPMI1640 10%FBS at 37°C, 5% CO2 for 3 d. The concentration of IFN-γ in the culture supernatants was determined by ELISA. Foxp3 protein expression in CD4+CD25+ T cells of splenocytes from tumor-bearing mice treated by the combination therapy was performed using the mouse specific Foxp3 (FJK-16s) staining kit (eBioscience).

The combination therapy

4T1.2-Neu (1×105) were inoculated s.c. into BALB/c (3–5mice/group) at the 4th mammary fat pad on d 0. These mice were adoptively transferred i.v. by freshly-purified tumor-primed CD4+ T cells which were isolated from splenocytes of α-CD25 mAb-pretreated mice that rejected the tumor or untreated tumor-bearing mice (1×107), and were administrated i.p. with 800μg agonistic α-GITR mAb or rat IgG at d 3 and d 4, respectively. In some experiments, α-CD8 mAb (53-6.7, 200μg/injection) was injected i.p. at d 3, 6 and 9. In other experiments, tumor-primed CD4+ T cells (1×107) were pretreated by agonistic α-GITR mAb in vitro. A primary tumor was observed (~5–8mm mean diameter before tumor rejection, usually at d 10–16 after tumor inoculation) in all mice inoculated with tumor cells, and measured using an electric caliper in the two perpendicular diameters every other day. Mice were sacrificed for humane reasons when the primary tumor reached 10mm in mean diameter, when ulceration, bleeding or both developed, or when mice became ill due to metastatic diseases.

Statistical analysis

Data were statistically analyzed using Student’s t test (Graph Pad Prism version 5). Data from animal survival experiments were statistically analyzed using Log Rank test (Graph Pad Prism version 5). P < 0.05 is considered to be statistically significant.

Supplementary Material

Fig 1

Fig 2

Fig 3

Acknowledgments

We are indebted to C. Donahue (Univ. of Pittsburgh) for her help. This work was supported by a NIH grant R01CA108813 (to Z.Y.).

Footnotes

DISCLOSURES

The authors have no financial conflict of interest.

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