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The Journal of Immunology, 2004, 172: 1347-1354.
Copyright © 2004 by The American Association of Immunologists

Induction of Tumor Regression by Administration of B7-Ig Fusion Proteins: Mediation by Type 2 CD8+ T Cells and Dependence on IL-4 Production1

Nobuya Yamaguchi*, Shin-ichiro Hiraoka*, Takao Mukai*, Noritami Takeuchi*, Xu-Yu Zhou*, Shiro Ono*, Mikihiko Kogo{dagger}, Kyriaki Dunussi-Joannopoulos{ddagger}, Vincent Ling{ddagger}, Stanley Wolf{ddagger} and Hiromi Fujiwara2,*

* Department of Oncology, Osaka University Graduate School of Medicine, Yamada-oka, Suita, Osaka 565-0871, Japan; {dagger} First Department of Oral and Maxillo-Facial Surgery, Osaka University Faculty of Dentistry, Suita, Osaka 565-0871, Japan; and {ddagger} Department of Inflammation, Wyeth/Research Institute, Cambridge, MA 02140


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD28 signals contribute to either type 1 or type 2 T cell differentiation. Here, we show that administration of B7.2-Ig fusion proteins to tumor-bearing mice induces tumor regression by promoting the differentiation of antitumor type 2 CD8+ effector T cells along with IL-4 production. B7.2-Ig-mediated regression was not induced in IL-4-/- and STAT6-/- mice. However, it was elicited in IFN-{gamma}-/- and STAT4-/- mice. By contrast, IL-12-induced tumor regression occurred in IL-4-/- and STAT6-/- mice, but not in IFN-{gamma}-/- and STAT4-/- mice. Moreover, B7.2-Ig treatment was effective in a tumor model not responsive to IL-12. B7.2-Ig administration elicited elevated levels of IL-4 production. Tumor regression was predominantly mediated by CD8+ T cells, although the induction of these effector cells required CD4+ T cells. Tumor regression induced by CD8+ T cells alone was inhibited by neutralizing the IL-4 produced during B7.2-Ig treatment. Thus, these results indicate that stimulation in vivo of CD28 with B7.2-Ig in tumor-bearing mice results in enhanced induction of antitumor type 2 CD8+ T cells (Tc2) leading to Tc2-mediated tumor regression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The CD8+ T cells are believed to be a major antitumor effector cell population. As in the case of CD4+ T cells, CD8+ T lymphocytes can be classified into two distinct effector cell types depending on their cytokine-secreting profiles following an Ag encounter (1, 2). Type 1 CD8+ T cells (Tc1)3 produce IL-2 and IFN-{gamma}, whereas type 2 CD8+ T cells (Tc2) predominantly secrete IL-4, IL-5, and IL-10. Although the patterns for cytokine production are thus different, both populations of CD8+ effector T cells exert a cytolytic function (3, 4). In addition, both type 1 (IFN-{gamma}) (5, 6) and type 2 (IL-4) (7, 8, 9, 10) cytokines have also been demonstrated to contribute to mediating antitumor effects.

The B7/CD28 costimulatory pathway plays an indispensible role in T cell activation (11). Initially, the role of CD28 costimulation was demonstrated in the activation of type 1 CD4+ T cells (Th1) for Th1 cytokine (IL-2/IFN-{gamma}) gene expression (12). Recent studies have shown that CD28 costimulation also promotes Th2 differentiation and the production of Th2 cytokines (13, 14). Therefore, the B7/CD28 costimulation pathway has the potential to costimulate T cells for both Th1 and Th2 differentiation. Because activation of naive CD8+ T cells requires CD28 costimulation (15), probably more CD28 costimulatory activity than for the activation of CD4+ T cells, the B7/CD28 costimulation could also regulate Tc1 and Tc2 differentiation. However, little is known regarding which differentiation pathway of type 1 or type 2 T cells, particularly Tc1 or Tc2, is promoted more efficiently by CD28 costimulation.

An attempt has been made to enhance the induction of antitumor effector T cells by introducing B7 genes into tumor cells and using such transfected cells as tumor vaccines (16, 17, 18, 19). However, considering the host’s APC-mediated cross-priming for CD8 T cell activation (20, 21, 22), it is unlikely that B7-transfected tumor cells can directly stimulate antitumor CD8+ effector T cell precursors. Thus, it remains to be investigated whether the induction of antitumor T cells during interactions with APC-presenting tumor Ags can be enhanced when CD28 molecules on these T cells are additionally stimulated with exogenous CD28 ligands. In such a case, it is also unknown which element(s) of the Th1/Tc1 or Th2/Tc2 responses is enhanced by incorporating the B7/CD28 costimulation pathway.

Here, we show that stimulation in vivo of the CD28 pathway by B7.2-Ig fusion proteins in tumor-bearing mice results in the regression of growing tumors by promoting the activation of anti-tumor type 2 CD8+ T cells. Namely, the following observations were made: this tumor regression 1) was not induced in IL-4-/- or STAT6-/- mice defective in type 2 T cell responses, but occurred in IFN-{gamma}-/- or STAT4-/- mice unable to develop type 1 T cell responses; 2) was predominantly mediated by CD8+ effector T cells; 3) was associated with enhanced levels of IL-4 production following B7.2-Ig treatment; and iv) was prevented by neutralization of such IL-4. These findings demonstrate the distinct and previously undescribed role for the B7/CD28 costimulation in the enhanced induction of type 2 CD8+ T cell-mediated tumor regression.


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

Male BALB/c and female (C57BL/6 x C3H/He) F1 (B6C3F1) mice were purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan). The following gene-knockout mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred in our laboratory: IFN-{gamma}-deficient (IFN-{gamma}-/-) STAT4-/-, IL-4-/-, and STAT6-/- mice.

Tumor cell lines

The following tumor cell lines were used: CSA1M fibrosarcoma (BALB/c origin) and its variant line (23) (designated CSA1M-variant) as well as OV-HM ovarian carcinoma (B6C3F1 origin) (6). The original CSA1M line was described as the CSA1M-parent when compared with the CSA1M-variant. The CSA1M-parent and -variant were previously shown to be IL-12-responsive and IL-12-unresponsive lines, respectively, although they exhibited common antigenicity at comparable levels (23).

Reagents

To prepare B7.2-Ig as well as B7.1-Ig fusion proteins, expression plasmids encoding mouse B7.2 or B7.1 signal and extracellular domains were fused to the Fc region of mouse IgG2a as previously described in details (24). The inducible costimulator (ICOS) ligand (ICOSL)-IgG2a construct was made by linking the DNA element of ICOSL (GenBank accession no. AF199027, nt 205–897) to a genomic DNA segment encoding the hinge CH2-CH3 domain for mouse IgG2a. B7.2-Ig and ICOSL-Ig fusion proteins were collected from culture supernatants of Chinese hamster ovary (CHO) cells transfected with the recombinant plasmids carrying the above fused DNA fragment and purified on a protein A-Sepharose Fast Flow column (Amersham Pharmacia Biotech, Piscataway, NJ). More than 99% of the protein was in the dimeric, nonaggregated form (24). Recombinant mouse IL-12 was provided by Wyeth/Genetics Institute (Cambridge, MA). The following mAbs were purified from ascitic fluids of the relevant hybridoma cells (obtained from American Type Culture Collection, Manassas, VA): anti-CD4 (GK 1.5), anti-CD8 (2.43), and anti-IL-4 (11B11). Normal (control) rat IgG was purchased from Biomeda (Foster City, CA).

Preparation of tumor-bearing mice

Mice were inoculated s.c. with the parental CSA1M (1 x 106/mouse), the variant CSA1M (1 x 106/mouse), or OV-HM (5 x 105/mouse) tumor cells.

Treatment of mice with fusion proteins or rIL-12

Mice were injected i.p. with 50 µg/mouse B7.2-Ig, B7.1-Ig, or ICOSL-Ig three or five times at 4-day intervals or with 0.5 µg/mouse rIL-12 three or nine times every other day.

Measurement of cytokine concentrations

The concentrations of IL-4 and IFN-{gamma} in culture supernatants and plasma were determined using ELISA kits purchased from Genzyme (Cambridge, MA).

Depletion in vivo of CD4+ or CD8+ T cells

To deplete either the CD4 or CD8 subset of T cells, 200 µg/mouse anti-CD8 or anti-CD4 mAb was injected i.p. twice. The efficacy of each T cell subset depletion was confirmed by flow cytometric analysis of spleen cells from mAb-treated mice as previously described (25).

Histological examination

Tumor masses were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with H&E for histological examination.

Staining procedure of immunohistochemical examination

The following reagents were purchased to perform immunohistochemical examinations: biotinylated anti-mouse CD4 and anti-CD8 mAbs (BD PharMingen, San Diego, CA); biotinylated rat IgG (Jackson ImmunoResearch, West Grove, PA); Histofine SA-PO kit and Histofine DAB kit (Nichirei, Tokyo, Japan). Samples were fixed in 2% paraformaldehyde for 6–12 h at 4°C and then washed sequentially with PBS containing 10, 15, and 20% sucrose for 6 h each at 4°C. The samples were embedded in OCT compound and frozen at -80°C. Cryostat sections (5 µm) were cut, air-dried, and then washed three times with PBS. The sections were incubated in PBS containing 10% hydrogen peroxide at room temperature for 30 min for blocking endogenous peroxidase activity before a biotinylated Ab was added. After pre-incubation with 4% BSA solution, the tissues were overlaid with various biotinylated Abs and incubated in a humidified chamber at room temperature for 2 h. After being washed three times, the sections were incubated with peroxidase-conjugated streptavidin solution for 30 min. After an additional three washes, the labeling was visualized with 0.03% 3,3'-diaminobenzidine tetrahydrochloride solution containing 0.1% hydrogen peroxide for several minutes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Systemic adminstration of B7.2-Ig fusion proteins induces complete regression of growing tumors in various tumor models

A previous study (26) established that the B7.2-Ig fusion protein is effective for costimulation in vitro of anti-CD3-triggered T cells by stimulating CD28 molecules. Regarding sensitization in vivo of antitumor T cells in tumor-bearing mice, our earlier studies showed that APC pick up and present tumor-Ags to prime tumor-reactive T cells (5, 27, 28). We examined whether administration of B7.2-Ig fusion proteins to tumor-bearing mice influences tumor growth by further activating antitumor T cells that are being primed. Fifty micrograms of B7.2-Ig was injected i.p. into CSA1M fibrosarcoma-bearing BALB/c mice or OV-HM ovarian carcinoma-bearing B6C3F1 mice three times at 4-day intervals (Fig. 1). Complete regression of growing tumors was induced without exception in both tumor models.



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  		FIGURE 1. Systemic administration of B7.2-Ig fusion proteins induces complete regression of growing tumors. B7.2-Ig fusion proteins (50 µg/mouse/time) were injected i.p. into CSA1M- or OV-HM tumor-bearing mice three times at 4-day intervals. Tumor growth was individually plotted (five mice per group).

 
Our previous studies (6, 29) showed that complete tumor regression is also inducible in the above tumor models by systemic IL-12 treatment. Thus, it may be speculated that these two tumor models are quite unique because tumor regression is readily induced by two different types of immunomodulatory reagents. Therefore, we examined the effect of B7.2-Ig on tumor growth in IL-12-unresponsive tumor models. A representative of such models is a variant form of CSA1M tumor (designated CSA1M-variant) (23). The parental and variant-type of CSA1M tumors were previously found to exhibit the same tumor immunogenicity (23) but to be responsive and unresponsive, respectively, to IL-12 treatment (23, 30). This was confirmed in Fig. 2A; regression of CSA1M-parent tumors was induced by three IL-12 injections, whereas nine IL-12 injections failed to induce tumor regression and did not induce even significant growth modulation in the CSA1M-variant model. The efficacy of B7.2-Ig treatment in these two CSA1M models is shown in Fig. 2B. The results show that B7.2-Ig treatment is effective in an IL-12-unresponsive tumor model, although more injections of B7.2-Ig were required to induce regression in the CSA1M-variant model than in the CSA1M-parent model, probably due to a higher tumor growth rate in the former. Similar therapeutic effects were observed in another IL-12-unresponsive BAMC-1 tumor (chemically induced BALB/c fibrosarcoma) model (data not shown).



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  		FIGURE 2. B7.2-Ig fusion proteins induce tumor regression in a tumor model unresponsive to IL-12 treatment. A, rIL-12 (0.5 µg/mouse/time) was injected i.p. into CSA1M-parent or CSA1M-variant tumor-bearing mice three times (CSA1M-parent) or nine times (CSA1M-variant) every other day. B, B7.2-Ig fusion protein was injected to CSA1M-parent (three times) or CSA1M-variant (five times) tumor-bearing mice. Tumor growth was expressed as the mean ± SE of five mice per group.

 
IL-12 and B7.2-Ig induce morphologically different patterns of tumor regression

Tumor masses from CSA1M-bearing mice that had been treated with two injections of either IL-12 or B7.2-Ig were removed on various days (1–5 days) after the second injection and subjected to histological examination. Fig. 3 shows light micrographs and immunohistochemical staining of tumor masses obtained 2 days after each second treatment. Moderate infiltration of mononuclear cells was seen around and inside the tumor mass harvested from IL-12-treated mice particularly at the peritumoral stromal area (Fig. 3A). This infiltrate included large numbers of CD8+ and CD4+ T cells (Fig. 3, C and D). T cell infiltration increased at later time points (data not shown) forming a massive T cell migration as shown in our previous report (29).



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  		FIGURE 3. Light micrograph and immunohistochemical staining of tumor masses from IL-12- or B7.2-Ig-treated mice. CSA1M tumor masses were harvested 2 days after the second IL-12 or B7.2-Ig injection. A and E, H&E staining, original magnification, x200. Subcutaneous tissue (SC) and peritumoral stroma (PS) are indicated on both groups of tumor sections. Intratumoral necrosis induced only in a tumor from B7.2-Ig-treated mice is also shown. The corresponding area in a tumor from IL-12-treated mice does not contain necrotic tissue but represents tumor parenchyma with infiltrating leukocytes. B–D and F–H, frozen sections of each tumor mass were stained with anti-CD8, anti-CD4, or control IgG. Pictures represent the staining of CD8+ and CD4+ T cells infiltrating peritumoral stromal areas. Original magnification, x400.

 
Light microscopic examination of a tumor mass from B7.2-Ig-treated mice revealed moderate levels of polymorphonuclear and mononuclear cell infiltration at the location corresponding to the site exhibiting T cell infiltration in tumor masses from IL-12-treated mice. It should be noted that although both CD4+ and CD8+ T cells were also seen in a tumor mass from B7.2-Ig-treated mice (Fig. 3, G and H), the numbers of both T cells did not increase at later time points unlike the case of IL-12 treatment. Instead, tumors from B7.2-Ig-treated mice formed an intratumoral necrosis mass beneath peritumoral stromal tissue/s.c. tissue (Fig. 3E). Such a necrotic mass was never seen at this early stage in the IL-12-treated group of tumor masses. Without an increase in the levels of T cell infiltration, the necrosis mass grew along with B7.2-Ig treatment (data not shown), and the tumor mass eventually disappeared. These histological observations suggest that IL-12 and B7.2-Ig treatment induce fundamentally different mechanisms of tumor regression.

B7.2-Ig-induced tumor regression is mediated by antitumor CD8+ T cells but its induction requires CD4+ T cells

The mice that were cured of an established tumor by B7.2-Ig treatment were found to exhibit protection against a rechallenge with the same tumor (data not shown). Tumor regression associated with the generation of memory responses suggests that the therapeutic effect is mediated by T cells. We examined which T cell subset(s) is responsible for the induction of the therapeutic effect. Either the CD4+ or CD8+ T cell subset was eliminated by two injections of anti-CD4 or anti-CD8 mAb. The efficacy of depletion was confirmed by flow cytometric analysis of spleen cells from mAb-treated mice (data not shown). The treatment with these mAbs were performed at two different stages: one before the B7.2-Ig therapeutic stage and the other before the tumor sensitization stage (i.e., before tumor implantation). As summarized in Fig. 4, the results demonstrate that the B7.2-Ig-mediated therapeutic effect is abrogated by depletion of CD8+ T cells at either stage. In contrast to the absolute requirement for CD8+ T cells at the therapeutic stage, CD4+ T cells were not necessarily required at this stage, although the rate of tumor regression was delayed compared with that in the mAb-untreated, therapeutic group. Notably, depletion of CD4+ T cells before the tumor sensitization stage completely abrogated the generation of the CD8 T cell-mediated therapeutic effect. These results indicate that B7.2-Ig-induced tumor regression is mediated largely by CD8+ T cells but the generation of their therapeutic effect requires CD4+ T cells at the stage of tumor sensitization.



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  		FIGURE 4. Effect of CD8 or CD4 T cell depletion on tumor regression induced by B7.2-Ig treatment. CD4+ or CD8+ T cells were depleted by injection of anti-CD8 or anti-CD4 mAb (200 µg/mouse, twice). This was done either before B7.2-Ig treatment (left) or tumor cell implantation (right). B7.2-Ig treatment was performed by three (CSA1M; upper panels) or five (OV-HM; lower panels) rounds of B7.2-Ig injections at 4-day intervals. Tumor growth was expressed as the mean ± SE of five mice per group.

 
The B7.2-Ig-mediated therapeutic effect is generated in the type 2 (Th2/Tc2), but not in type 1 (Th1/Tc1) T cell-inducing conditions

The preceding observation that B7.2-Ig-induced tumor regression occurs in an IL-12-unresponsive tumor model (Fig. 2) implies that the generation of the B7.2-Ig therapeutic effect is independent of antitumor type 1 T cell induction. To directly demonstrate this, we examined the effect of B7.2-Ig treatment in mice lacking various genes that are responsible for type 1 or type 2 T cell development. Fig. 5 shows that B7.2-Ig treatment induces tumor regression in STAT4-/- or IFN-{gamma}-/- mice, although tumor regression is appreciably delayed in IFN-{gamma}-/- mice. We further compared the effects of B7.2-Ig and IL-12 treatment in two sets of mutant strains deficient in type 1 (IFN-{gamma}-/- or STAT4-/-) or type 2 T cell development (IL-4-/- or STAT6-/-). As summarized in Fig. 6, IL-12-treament was not effective in IFN-{gamma}-/- and STAT4-/- mice but induced tumor regression in IL-4-/- and STAT6-/- mice. In contrast, the B7.2-Ig-mediated therapeutic effect was again induced in IFN-{gamma}-/- and STAT4-/- mice but not in IL-4-/- and STAT6-/- mice. As reported previously (31), tumor growth was reduced in STAT6-/- mice. However, B7.2-Ig treatment failed to induce even the regression of tumors with such slow growth rates.



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  		FIGURE 5. B7.2-Ig administration induces tumor regression in STAT4-/- and IFN-{gamma}-/- mice. B7.2-Ig was injected into wild-type (WT), STAT4-/-, or IFN-{gamma}-/- BALB/c mice bearing CSA1M-parent tumors.

 


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  		FIGURE 6. The IL-4/STAT6 signaling is required to induce tumor regression mediated by B7.2-Ig but not by IL-12. Wild-type (WT), IFN-{gamma}-/-, STAT4-/-, IL-4-/-, or STAT6-/- BALB/c mice bearing CSA1M-parent tumors were treated with either B7.2-Ig or IL-12. The protocols for B7.2-Ig (50 µg/mouse/time) and rIL-12 (0.5 µg/mouse/time) injections were the same as those in Fig. 2 (left).

 
B7.2-Ig treatment induces IL-4 production in tumor-bearing mice

We next examined whether administration of B7.2-Ig to tumor-bearing mice induces the production of type 2 cytokines, particularly IL-4. At various times after a single B7.2-Ig injection into normal BALB/c or CSA1M-parent tumor-bearing BALB/c mice, plasma was collected and examined for IL-4 production. Fig. 7 (upper panels) shows that strikingly elevated levels of IL-4 production that peak 24 h after B7.2-Ig injection are induced in tumor-bearing mice. In contrast, a representative type 1 cytokine IFN-{gamma} was not detected in the same plasma preparation (data not shown). Next, CD4+ or CD8+ T cells were depleted before B7.2-Ig injection. As shown in Fig. 7 (lower panels), anti-CD4 or anti-CD8 T cell-depleted mice still produced appreciable amounts of IL-4. Together, the results indicate that B7.2-Ig injections enhance the production of the type 2 cytokine IL-4 when given in tumor-bearing mice and that this IL-4 production is not abrogated by elimination of CD4+ T cells.



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  		FIGURE 7. B7.2-Ig administration induces strikingly enhanced IL-4 production in tumor-bearing mice. A, Normal or CSA1M-parent tumor-bearing mice were not given or given a single B7.2-Ig injection. At various times later, plasma was obtained and assessed for IL-4 production by ELISA (the mean ± SE of four mice per group). B, CSA1M tumor-bearing mice were depleted of either CD4+ or CD8+ T cells by two injections of anti-CD8 or anti-CD4 mAb (200 µg/mouse/time) before a single B7.2-Ig treatment. Twenty four hours later, plasma was harvested. The IL-4 concentration was individually expressed.

 
Administration of B7.1-Ig or ICOSL-Ig fusion protein induces comparable rates of tumor regression to those by B7.2-Ig

CD28 is stimulated with either B7.2 (CD86) or B7.1 (CD80). It is also known that stimulation of the ICOS pathway induces T cell activation for the production of type 2 cytokines including IL-4 (32, 33, 34). We therefore examined whether B7.1-Ig or ICOSL-Ig fusion protein is also capable of influencing tumor growth. Fig. 8 shows that tumor regression is induced by administration of B7.1-Ig or ICOSL-Ig fusion proteins and that the rate of tumor regression by these three fusion proteins is comparable. These results support the correlation between B7.2-Ig-induced tumor regression and IL-4 production.



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  		FIGURE 8. Tumor regression is induced by B7.2-Ig, B7.1-Ig, as well as ICOSL-Ig fusion proteins. B7.2-Ig, B7.1-Ig or ICOSL-Ig fusion proteins (50 µg/mouse/time) were injected i.p. into tumor-bearing mice three times at 4-day intervals. Tumor growth was expressed as the mean ± SE of five mice per group.

 
Tumor regression depends on IL-4 production induced during B7.2-Ig treatment

To directly demonstrate that B7.2-Ig-induced tumor regression depends on IL-4, IL-4 produced following B7.2-Ig treatment was neutralized by injection of anti-IL-4 mAb. Because CD8+ T cells exclusively mediate tumor regression, the experiment was conducted to determine the effect of IL-4 neutralization in CD4 T cell-depleted tumor-bearing mice. As shown in Fig. 9, tumor-bearing mice depleted of CD4+ T cells again exhibited B7.2-Ig-mediated tumor regression although the regression rate was slower as observed in Fig. 4. Neutralization of the IL-4 produced in these CD4+ T cell-depleted mice by B7.2-Ig administration completely prevented tumor regression, indicating that IL-4 is required for the induction of CD8+ T cell-mediated tumor regression following B7.2-Ig treatment.



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  		FIGURE 9. B7.2-Ig-induced, CD8+ T cell-mediated tumor regression depends on IL-4 production. CD4+ T cells were not depleted or depleted by two injections of anti-CD4 mAb (200 µg/mouse/time) before B7.2-Ig treatment (on day -3 and day -1). Half of these anti-CD4-treated mice were then injected with anti-IL-4-neutralizing mAb (1 mg/mouse/time) twice on day -1 and day 3. Tumor growth was expressed as the mean ± SE of five mice per group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Optimal activation of TCR-triggered T cells requires signaling through costimulatory pathways, particularly through a CD28 pathway (11). Ineffective activation of antitumor T cells due to the absence of appropriate costimulatory molecules on tumor cells could provide one explanation for inadequate immune responses to growing tumors. In this context, several investigations have had some success in enhancing the activation of tumor-specific T cells by introducing costimulatory molecules onto the surface of tumor cells: vaccination with tumor cells transfected with B7.1 or B7.2 cDNA protected mice against tumor challenge (35) and induced regression of established tumors in some therapeutic models (16, 17, 18, 19). As a more clinically applicable approach to providing B7/CD28-mediated costimulation, soluble B7.1- or B7.2-Ig fusion proteins were prepared, and their efficacy particularly in combination with a tumor vaccine was shown in several therapeutic tumor models (24). In the present study, after establishing the efficacy of B7.2-Ig as a single reagent in the eradication of growing tumors, we showed that B7.2-Ig-induced tumor regression is mediated by type 2 CD8+ T cells (probably Tc2) and depends on IL-4 production induced during B7.2-Ig treatment. Accordingly, this tumor regression was not induced in IL-4-/- or STAT6-/- mice with a defect in the development of type 2 T cell responses. In contrast, B7.2-Ig treatment was effective in IFN-{gamma}-/- or STAT4-/- mice in which type 1 T cell differentiation is impaired and IL-12 treatment is not successful. Thus, this study demonstrates a novel and previously undescribed role for the B7/CD28 costimulatory pathway in the induction of antitumor T cell activation leading to tumor eradication in vivo.

T cells organize immune responses involved in tumor regression not only by acting as antitumor effectors but also by activating other effector populations such as macrophages. Among functionally and phenotypically heterogeneous T cell subsets, it has been believed that type 1 T cells, both Tc1 and Th1, play a predominant role in inducing the rejection of established tumors. This notion is consistent with recent observations that administration of IL-12 induces tumor regression that is associated with a massive T cell infiltration (6, 29) and is dependent on IFN-{gamma} production of T cells (29, 36). In fact, IL-12 and IFN-{gamma}, which are cytokines required for Th1 development, are critical in the induction of Th1-associated chemokine receptors (CCR5 and CXCR3) on TCR-triggered T cells (37, 38). T cells infiltrating tumors following IL-12 treatment were found to express CCR5 (39) as shown for joint-infiltrating T cells in rheumatoid arthritis (40, 41). Moreover, the blockade of CCR5 with a CCR5 antagonist resulted in the inhibition of IL-12-induced tumor regression along with down-regulation of T cell infiltration (39). Thus, IL-12 induction of tumor regression has been shown to correlate with the dynamism of type 1 inflammatory T cells that are activated by proinflammatory (IL-12) and inflammatory (IFN-{gamma}) cytokines.

The development of type 1 and type 2 T cells is cross-regulated by the other subset of T cells or by cytokines required for the differentiation of such T cells (reviewed in detail in Ref. 42). According to this theory, IL-4 is considered to down-regulate the above-mentioned type 1 T cell-mediated tumor rejection responses. Nevertheless, the positive antitumor effect of IL-4 was first reported in 1989 before the paradigm of Th1/Th2 was established (7). Recently, the role of IL-4-producing Th2 cells themselves in tumor eradication has also been demonstrated in several independent studies (43, 44, 45). These results indicate that IL-4 does not necessarily counteract Th1-mediated antitumor responses but may be capable of exhibiting its own antitumor pathway.

In addition to Th2, Tc2 have also been shown to mediate anti-tumor immune responses (8, 9, 10, 46). Even the above-mentioned Th2-mediated tumor eradication required the presence of CD8+ T cells, although the distinction between Tc1 and Tc2 was not established (43, 44, 45). Because Tc2 exhibit cytolytic activity against target cells via the perforin pathway (3, 4), Tc2 also function as a final antitumor effector T cell population. However, the cytokine requirement for functional development differs between differentiation of Tc1 and Tc2. Like Th2, IL-4 is required for Tc2 development (2). Accordingly, IL-4-/- mice failed to produce the Tc2-mediated antitumor effect that was otherwise observed in WT mice (9). Consistent with this, our present results showed that tumor regression based on the induction of antitumor Tc2 enhanced by B7.2-Ig administration does not occur in IL-4-/- mice and is prevented by anti-IL-4 mAb treatment.

Furthermore, a series of studies of Schüler et al. (9, 10) provided a better understanding for the induction of antitumor Tc2. In their model, there were two requirements for Tc2 generation: one is the help of CD4+ T cells and the other is IL-4 production by CD8+ T cells themselves but not by CD4+ Th. It becomes increasingly evident that the activation of antitumor CD8+ T cell precursors occurs through interactions not directly with tumor cells but with APC that have picked up and processed tumor Ags, as has been described for "cross-priming" or "cross-presentation" (20, 21, 22). However, such APC have to be preactivated by CD4+ T cells via CD40-CD40L interactions before presenting tumor Ags to CD8+ T cell precursors (47, 48, 49, 50). This mechanism provided an explanation for the requirement of CD4+ Th help in the activation of antitumor CD8+ T cells. In the present study, elimination of CD4+ T cells at the antitumor effector phase (the stage after the start of B7.2-Ig treatment) only partially influenced CD8 T cell-mediated tumor regression. In contrast, CD4+ T cell depletion at the stage of tumor sensitization completely inhibited the B7.2-Ig effect mediated by CD8+ T cells at a later stage. These results are compatible with the above-mentioned requirement for CD4+ T cells in the CD8 T cell induction.

Moreover, Schüler et al. reported that the induction of CD8+ effector T cells during interactions with APC depends on two sequential events: IL-4 production of CD8+ T cells (Tc2) and IL-4-induced functional maturation of APC (10). Our present study showed that both IL-4 production and CD8+ T cell-mediated tumor regression were induced in the mice depleted of CD4+ T cells and that neutralization of the IL-4 prevented tumor regression. Thus, these observations indicate that IL-4 is required for B7.2-Ig-induced, CD8 T cell-mediated tumor regression. This seems to be compatible with the requirement for IL-4 in Tc2 induction (10). Because antitumor CD8+ T cells were induced in the conditions defective in type 1 but not in type 2 T cell responses, these CD8+ T cells could represent Tc2. However, it still remains to be determined whether IL-4 was actually produced by developing Tc2 themselves or was provided by cells other than conventional T cells. Further studies will be required to investigate the involvement of other cell types such as NKT cells in the in vivo production of IL-4.

Here, it may be of value to consider the mechanism of type 1 and type 2 CD8+ T cell (Tc1 and Tc2) development via the cross-presentation pathway together with the role of CD4+ Th help (47, 50). Because APC themselves can produce IL-12 and express B7 (CD80/CD86) molecules, they have the potential to induce both Tc1 and Tc2 differentiation. When CD8+ T cells interacting with APC are stimulated with large amounts of exogenous rIL-12, Tc1 differentiation could be selectively enhanced. Alternatively, if they are stimulated with large amounts of exogenous CD28 ligands (B7.2-Ig), polarization toward Tc2 is induced. Although Tc1 induction by B7.2-Ig is not excluded, Tc2 development appears to be predominant considering the analysis of cytokine production in vivo. It is also reported that the CD4+ T cells that contribute to Tc2 induction via cross-presentation of APC are of type 1 (9). Thus, the following scenario is speculated: first, antitumor Th1 cells are primed preferentially in the absence of external manipulating reagents during tumor growth (tumor sensitization stage). Then, these Th1 cells activate APC for the acquisition of the capacity to cross-present processed tumor Ags to CD8+ T cells. Depending on external B7 stimulation, polarization to Tc2 development occurs during cross-presentation of Th1-activated APC.

However, a critical issue may be raised regarding why type 2 T cell (both Tc2 and Th2) generation was efficiently induced by B7.2-Ig treatment. A number of studies (12, 13) using anti-CD28 mAb have indicated that the B7/CD28 costimulation pathway is potentially effective for enhancing both Th1 and Th2 cytokine production. However, a recent study (26) revealed that there was a great difference in the patterns of Th1 vs Th2 cytokine expression between stimulation of CD28 with anti-CD28 mAb and with the ligands of CD28 (B7.1- or B7.2-Ig). Namely, stimulation in vitro of CD4+ T cells with B7.1- or B7.2-Ig leads to much higher levels of Th2 cytokine production than that with anti-CD28. We have also confirmed this and further observed that administration of anti-CD28 mAb instead of B7.2-Ig to tumor-bearing mice induced neither IL-4 production nor tumor regression (our unpublished observations). Thus, it appears that stimulation of TCR-triggered T cells with B7.2-Ig efficiently leads to the induction of type 2 cytokine production.

A biologically important aspect of the present study concerns the comparison with the IL-12-mediated antitumor effect. As mentioned above, IL-12-induced tumor regression relies on the activation of T cells for Th1/Tc1 differentiation including the expression of Th1 cytokines (29, 36) and Th1 chemokine receptors (37, 38). Moreover, a large number of these activated T cells have to migrate into tumor masses to show the efficacy of IL-12 treatment. Our previous study (23) showed that even though exogenous IL-12 can activate T cells, tumor regression is not inducible when tumor masses fail to exhibit the capacity to accept T cells as exemplified by the development of inflammatory tumor-stroma with VCAM-1/ICAM-1-positive vasculature. A CSA1M-variant was a representative of such IL-12-unresponsive tumor models because this tumor failed to develop inflammatory stroma (23) despite possessing a level of tumor antigenicity comparable to that of the parental CSA1M tumor (23, 30). Surprisingly, B7.2-Ig treatment was shown to be effective in the CSA1M-variant model. Considering that many clinical tumors may have a weak capacity to accept T cells due to the poor development of inflammatory stroma tissue, B7.2-Ig treatment could be a much more clinically applicable approach than IL-12 treatment.

Our present results illustrate that B7.2-Ig treatment of tumor-bearing mice elicits tumor regression that associates with enhanced generation of Tc2 and depends on IL-4 production induced during this treatment. This type of tumor regression differs from the regression induced by IL-12 treatment, in terms of not only the cytokine types required for the induction of the respective effector T cells but also the tumor-infiltration pattern of these T cells. The number of tumor-infiltrating T cells increased along with IL-12 treatment, ultimately leading to a massive T cell infiltration (23, 29, 30). In contrast, the levels of T cell infiltration did not largely change throughout B7.2-Ig treatment (Ref. 24 and this study). Although Tc2 have been shown to exhibit cytotoxic function (3, 4), an intratumoral necrosis emerged beneath the CD8+ T cell infiltrate. Therefore, further studies will be required to determine whether tumor-infiltrating type 2 CD8+ T cells actually mediate antitumor effect as type 2 CTLs (Tc2) and if so to investigate how such Tc2 can induce tumor necrosis leading to tumor eradication. In the light of the applicability of B7.2-Ig treatment to a wider range of clinical tumors, the elucidation of the mechanisms underlying Tc2-mediated tumor regression could determine the prospect of this approach.


   Acknowledgments
 
We thank M. Yasuda and C. Fukuda for their secretarial assistance.


   Footnotes
 
1 This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan. Back

2 Address correspondence and reprint requests to Dr. Hiromi Fujiwara, Department of Oncology, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail address: hf{at}ongene.med.osaka-u.ac.jp Back

3 Abbreviations used in this paper: Tc1, type 1 CD8+ T cell; Tc2, type 2 CD8+ T cell; ICOS, inducible constimulator; ICOSL, ICOS ligand; CHO, Chinese hamster ovary. Back

Received for publication August 11, 2003. Accepted for publication November 4, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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