It is thought that tumor rejection by CD8+ T-cell effectors is primarily mediated by direct killing. We show that rejection of different tumors (fibrosarcoma, ras-transformed fibroblasts, colon carcinoma, and plasmacytoma) by CD8+ T cells is always preceded by inhibition of tumor-induced angiogenesis. Angiostasis and tumor rejection were observed in perforin but not in IFN-γ-deficient mice. Furthermore, adoptive transfer of tumor-specific CD8+ T cells from IFN-γ-competent mice inhibited angiogenesis of lung metastases in comparison to those from IFN-γ gene-deficient mice. Taken together with our previous findings, we conclude that IFN-γ-dependent antiangiogenesis is a general mechanism involved in tumor rejection by CD4+ and CD8+ T-cell effectors.

CTL killing is the classical assay to measure effector functions of CD8+ T cells in vitro(1). It correlates often, but not always, with the antitumor efficacy of T cells in vivo(2, 3). CTL can use various effector molecules for killing, e.g., pfp3/granzymeB, Fas-ligand (CD95-L) or tumor necrosis factor (1). Although pfp-mediated lysis appears to be the prime mechanism for tumor cell killing both in vitro and in vivo(4), the relative contribution of different effector molecules during tumor rejection seems to depend on the tumor model (4, 5, 6, 7). Even if direct killing mechanisms by CD8+ T-cell effectors contribute to tumor rejection, it still leaves open the question whether they are effective only in conjunction with other mechanisms.

Several groups showed that IFN-γ and IFNγR are essential for tumor rejection (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18). Tumors transfected to secrete IFN-γ are rejected (8, 9). Blocking endogenous IFN-γ by neutralizing antibodies inhibits tumor rejection (10). IFN-γ- and IFNγR-gene deficient mice are impaired in rejecting tumors (7, 11, 14, 17). The efficacy of CD8+ T cells to mediate tumor rejection upon adoptive transfer correlates with IFN-γ production (16) but not lytic activity (3, 5, 6). The mechanism, however, by which IFN-γ exerts tumoricidal activity, is still not completely resolved. Although some studies proposed a direct action of IFN-γ on the tumor cells (10, 12), others proposed that IFN-γ-mediated tumor rejection did not require the tumor to respond to IFN-γ (13, 14). We previously analyzed the mechanism by which CD4+ T cells as effectors mediate tumor rejection. It turned out that it required IFNγR expression on nonhematopoietic but not hematopoietic or tumor cells, most likely on cells within the tumor stroma, and involved inhibition of angiogenesis (14). It is well known that a growing tumor requires new blood vessels (19). We argued that inhibition of angiogenesis by CD4+ T-cell-derived IFN-γ is an effective way to prevent rapid tumor burden allowing other, perhaps direct-killing, mechanisms to eliminate residual tumor cells (14). Subsequently, we showed that impaired tumor blood vessel formation was not because of tumor cell killing and reduced angiogenic factors provided by the tumor, because IFNγR-mediated blood vessel destruction preceded tumor cell death (17). In the current study, we show in several tumor models that tumor rejection by CD8+ T-cell effectors critically depends on their ability to produce IFN-γ to inhibit tumor angiogenesis.

Mice.

C57BL/6 and BALB/c mice were purchased from Charles River (Sulzfeld, Germany). DBA1 mice were obtained from Bomholtgard, Ry, Denmark. pfp and IFN-γ-deficient mice back-crossed onto the C57BL/6 background (F19 and N8 + 1F15, respectively) as well as the control C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Sex and age-matched mice were used in all experiments. P14 TCR-Tg and TCR-Tg IFN-γ−/− mice have been described previously (7).

Cells.

MCA205 fibrosarcoma (20) and B16F10 melanoma cells (kindly provided by Isaiah Fidler) are of C57BL/6 origin. B16F10-GM-CSF cells were established by infection of B16F10 cells with a GM-CSF-specific retrovirus (kindly provided by Wolfram Ostertag) and upon G418 selection, 1 × 106 cells produced 15 ng/ml/48 h of mouse GM-CSF. J558L plasmacytoma (21) and CT26 colon carcinoma cells (22) are of BALB/c origin. B16gp33 cells express the LCMV glycoprotein peptide gp33 and are recognized by Thy1.1+ P14 TCR-Tg CD8+ T cells in association with H-2Db(7). NIHpEJcl3 cells are Ras-transformed mouse NIH3T3 fibroblasts of DBA1 origin (23). All cell lines were cultured in RPMI 1640 supplemented with 10% FCS, 100 units/ml penicillin, and 100 μg/ml streptomycin.

In Vivo Experiments.

Exponentially growing tumor cells were harvested, washed with D-PBS and s.c. injected in 0.2 ml of D-PBS into the left abdominal region of mice in numbers as indicated. Tumor growth was monitored two to three times/week. Mice bearing a tumor larger than 10 mm in diameter were recorded as tumor positive. To generate protective immunity, C57BL/6 mice as well as pfp−/−, and IFN-γ−/− mice were immunized with 1 × 106 irradiated (100 Gy) MCA205 tumor cells. Two weeks later, mice were contralaterally challenged with living tumor cells as indicated. BALB/c mice were injected with 1 × 106 J558L cells, and 11 days later when tumors had grown to a mean size of 1 cm in diameter, they were treated i.p. with 15 mg/kg cyclophosphamide. This treatment leads to T-cell-mediated tumor rejection and effective protective tumor immunity in this model (17). Tumor-free mice were used for challenge 2 months later. BALB/c mice were also immunized with 1 × 106-irradiated CT26 tumor cells and 2 weeks later challenged. DBA1 mice were immunized with 1 × 106-irradiated NIHpEJcl3 cells and 2 weeks later challenged. To neutralize the IFN-γ activity in vivo, immunized pfp−/− mice were i.p. injected with 500 μg of purified XMG6 (rat antimouse IFN-γ mAb) in 0.5 ml D-PBS 2 h before challenge. As control, immunized mice with no mAb treatment were used.

Adoptive transfer experiments with P14 TCR-Tg cells were performed as described previously (7). In brief, lung metastases were induced by i.v. injection of 106 B16 or B16GP33 tumor cells. After 14 days, 1 × 107in vitro activated TCR-Tg cells were adoptively transferred (i.v.) into tumor-bearing C57BL/6 or IFN-γ−/− mice. For in vitro activation, spleen cells (5 × 106/ml) from TCR-Tg mice were cultured in the presence of GP33 peptide (10−7m) in 24-well plates for 3 days.

Depletion of CD4+ and CD8+ Cells.

Two days before challenge, immunized mice were depleted of CD4+ or CD8+ cells by i.p. injection of 400 μg of rat antimouse mAb GK1.5 (anti-CD4) or 2.43 (anti-CD8) in 0.5 ml D-PBS. Depletion of the respective T-cell subpopulation was controlled by flow cytometric analysis of peripheral blood cells using phycoerythrin-labeled anti-CD4 (RM4-5) and anti-CD8 mAbs (53-6.7; BD PharMingen). Complete depletion of the respective T-cell subset lasted for at least 3 weeks.

Immunohistochemistry.

C57BL/6 mice were left untreated or immunized twice in 2 weeks interval with 1 × 106-irradiated MCA205 tumor cells. Seven days after the second immunization, mice were challenged with 1 × 106-living MCA205 cells. DBA/1 and BALB/c mice were left untreated or immunized twice with 1 × 106-irradiated NIHpECl3 and CT26 tumor cells, respectively, and challenged 7 days later with 1 × 106 cells of the same tumor line. BALB/c mice were left untreated or immunized by injection of 5 × 106 living J558L cells and treated at day 11 with 15 mg/kg cyclophosphamide. Two months after tumors had been completely rejected, mice were challenged with 2 × 107 J558L cells. Two days before challenge, immunized mice received i.p. injections of 0.5 ml of D-PBS, anti-CD4, or anti-CD8 mAb. Tumors were isolated 4 and 6 days after the challenge. Preparation of cryostat tissue sections and alkaline phosphatase immunostaining were done as described previously (24). The mAbs used here were anti-CD31 mAb (MEC 13.3; BD PharMingen, Hamburg, Germany) and biotinylated anti-Thy1 mAb (BD PharMingen). As secondary reagents, the alkaline phosphatase-conjugated goat antirat IgG or streptoavidin, respectively, was used (Jackson Immunoresearch Laboratories, Inc., West Grove, IL). All sections were counterstained with Mayer’s hematoxylin (Chroma Gesellschaft GmbH, Münster, Germany). Tissue sections of three to five mice/group were evaluated.

Tumor Immunity often Requires CD8+ T Cells in the Effector Phase.

Previously, we demonstrated that CD4+ T-cell-mediated tumor rejection involved IFNγR-dependent antiangiogenesis. The aim of this study was to analyze the mechanism by which CD8+ T cells as effectors reject a challenge tumor. Several tumor models were used for which it was known that CD8+ T cells are the main effectors (DBA/1 ras-transformed fibroblast cell line NIHpEJC13 and BALB/c colon carcinoma CT26; Refs. 23, 25). Additionally, in two other models, the relative contribution of CD4+ and CD8+ T cells in the effector phase of tumor rejection was determined (C57BL/6 fibrosarcoma MCA205 and BALB/c plasmacytoma J558L). The four tumor lines were used to immunize mice of the tumor’s origin so that after subsequent challenge with the same tumor between 50 and 100% of the mice rejected the tumor (Table 1). Nonimmunized mice usually developed a tumor. Mice immunized with MCA205 or J558L cells were depleted of CD4+ or CD8+ T cells starting 2 days before challenge. In the MCA205 tumor model, depletion of either CD4+ or CD8+ T cells led to a significant decrease of protection (12.5 and 0% tumor-free mice versus 50% in control mice). In the J558L tumor model, only CD8+ but not CD4+ T cells were found to be crucial in the effector phase. One hundred percent of the CD8+ T cell depleted, but none of the CD4+ T-cell-depleted or control mice developed a tumor (Table 1). In the CT26 and NIHpEJ13 tumor models, it has been shown that depletion of CD8+ but not CD4+ T cells at the time of challenge abrogated tumor rejection (23, 25).

Protective Immunity always Correlates with Inhibition of Tumor-induced Angiogenesis.

Tumor-induced angiogenesis was investigated in the four tumor models by staining of tissue sections with anti-CD31 mAb, a marker on endothelial cells. A clear correlation between tumor rejection and inhibition of tumor-induced angiogenesis after immunization was observed. Tumors isolated at day 6 from naïve mice always contained a substantial amount of blood vessels, evenly distributed within the tumor tissue (Fig. 1,a, c, e, and g). In contrast, MCA205, CT26, J558L, and NIHpEJC13 tumors isolated from immunized mice were almost devoid of endothelial cells, and few CD31+ cells occurred at the rim of the tumor adjacent to neighboring tissue (Fig. 1,b, d, f, and h). The decrease of blood vessel density in the tumor was accompanied by the appearance of necrotic areas in the center of the tumor. In the J558L tumor model, depletion of CD8+ but not CD4+ T cells in immunized mice restored blood vessel formation in the tumor (Fig. 1, i and j) and tumor growth (Table 1). Immunization, tumor rejection, and impaired angiogenesis correlated with infiltration of T cells into the tumor (data not shown). Tumor rejection was always preceded by inhibition of tumor-induced angiogenesis, regardless of whether CD4+ and CD8+ T cells (MCA205) or exclusively CD8+ T cells were required as effectors during tumor rejection (CT26, J558L, and NIHpEJC13). Taken together with our previous results (14), this shows that CD4+ and CD8+ T cells mediate tumor rejection by partially overlapping mechanisms.

Tumor Immunity Can Be Generated in pfp-deficient Mice.

pfp-mediated cytotoxicity is the major effector mechanism of CD8+ T cells to kill target cells (1). To analyze the importance of pfp in the generation of tumor immunity, pfp−/− and control C57BL/6 mice were immunized with irradiated MCA205 cells and challenged 2 weeks later with 1 × 105 MCA205 cells. As shown in Fig. 2, MCA205 tumors grew with similar kinetics in naïve pfp−/− and control mice. Sixty percent of the immunized control mice rejected the challenge tumor. Surprisingly, 70% of the pfp−/− mice also rejected the tumor. Similar results were obtained when 2 × 104 MCA205 cells were used for challenge (data not shown). pfp-independent tumor immunity was also seen in the B16 melanoma model. Mice were immunized with 1 × 106-irradiated B16-GM-CSF cells and 2 weeks later challenged with 1 × 105 B16F10 cells. Again, all naïve mice developed a tumor within 30 days, 50% of immunized control, and 55% of immunized pfp−/− mice were tumor free during an 80-day observation period (data not shown).

Tumor Immunity Cannot Be Generated in the Absence of IFN-γ.

Because tumor immunity against MCA205 and B16F10 tumors did not require pfp, we asked whether IFN-γ is required for tumor immunity. IFN-γ−/− mice were immunized with irradiated MCA205 cells and challenged 2 weeks later. As shown in Fig. 3, although the immunization protected 80% of the control mice against the challenge tumor, no protective immunity could be generated in the IFN-γ-deficient mice.

Subsequently, the role of IFN-γ in generation of tumor immunity in pfp−/− mice was analyzed. The pfp−/− mice were immunized and challenged with MCA205 cells. Two h before challenge, mice were injected with neutralizing anti-IFN-γ mAb. Two of eight of the control mice but all of nine of the antibody-treated mice developed a tumor (Fig. 4). Of note, the inhibition of tumor induced angiogenesis after immunization was observed in the absence of pfp (Fig. 5, a and b) but not IFN-γ (Fig. 5, c and d). The protective immunity correlated again with impaired angiogenesis in tumors. In the B16 model, Hung et al.(11) have already shown that IFN-γ-deficient mice did not reject a challenge tumor after immunization with B16-GM-CSF cells. Taken together, tumor immunity can be generated in pfp but not in IFN-γ-deficient mice and is associated with IFN-γ-dependent inhibition of angiogenesis. It is therefore unlikely that the impaired angiogenesis is because of pfp-mediated tumor cell killing, which results in reduced tumor-derived angiogenic factors.

Adoptive Transfer of Tumor-specific CD8+ T Cells Inhibits Angiogenesis of Lung Metastases in an IFN-γ-dependent Fashion.

Several reasons led us to use an adoptive T-cell transfer model. (a) In the MCA205 tumor model CD4+ T-cell contribution could not be excluded (Table 1). (b) We wanted to ask whether IFN-γ produced in an antigen-specific fashion by CD8+ T cells is necessary for antiangiogenesis. (c) We wanted to analyze whether IFN-γ produced by CD8+ T cells inhibits angiogenesis in other tumor models such as experimental lung metastases. We used B16 cells transfected to express the lymphocytic choriomeningitis virus (LCMV) glycoprotein peptide gp33 and TCR-Tg CD8+ T cells, which recognize gp33 in association with H-2Db. Previously, it has been shown that adoptive transfer of gp33-specific TCR-Tg effector cells resulted in elimination of B16-gp33 pulmonary metastases that was dependent on IFN-γ but not pfp (7). Experimental B16gp33 lung metastases were established, and 14 days later, mice were treated with activated TCR-Tg cells. The adoptively transferred CD8+ T cells accumulated in an antigen-specific manner in lung metastases, as shown by staining for Thy1.1+ donor cells in Thy 1.1 recipients (Fig. 6, a and b). Significantly more transferred cells were present in B16gp33 compared with control B16 tumors. After T-cell transfer, decreased CD31+ cells were detected in B16gp33 tumors from wild-type mice when compared with transfer of IFN-γ-deficient TCR-Tg cells into IFN-γ-deficient mice (Fig. 6c–f). The reason why the transferred T cells from IFN-γ-deficient mice fail to inhibit angiogenesis is not entirely clear. Activation and cytolytic activity of T cells from IFN-γ-deficient mice was comparable with that of wild-type T cells (data not shown), but IFN-γ-deficiency could, for example, impair expansion or homing abilities of the transferred T cells. Taken together, we conclude that the adoptively transferred CD8+ effector cells inhibit tumor-induced angiogenesis in lung metastases in an antigen-specific and IFN-γ-dependent manner.

Our data suggest that IFN-γ-mediated antiangiogenesis is the primary mechanism of CD8+ T-cell effectors to mediate tumor rejection. CD4+ and CD8+ T-cell effectors reject tumors by partially overlapping mechanisms. Whether IFN-γ exerts this effect directly or indirectly is not known. It is likely that other factors with antiangiogenic property induced by IFN-γ are involved (26, 27). Parallel studies showed that in contrast to the T-cell priming phase (28), CD8+ T-cell effectors recognize the antigen directly on the tumor cells but that the IFN-γ necessary for tumor rejection acts on host cells, most likely on tumor stroma cells (29). We do not want to exclude direct killing mechanisms necessary for complete tumor rejection. We rather propose that IFN-γ-mediated antiangiogenesis prevents rapid tumor burden allowing other, perhaps direct killing mechanisms, to eliminate residual tumor cells. This could explain why therapeutic vaccination against established tumors usually fails (30). T cells arrive at the tumor site too late and are confronted with a vascularized tumor and the initial mechanism for tumor rejection is not effective.

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

Supported by Deutsche Forschungsgemeinschaft (SFB 506), the Deutsche Krebshilfe, Dr. Mildred-Scheel-Stiftung, e.V., and the Bundesministerium für Bildung und Forschung Grant 01KV9911.

3

The abbreviations used are: pfp, perforin; IFNγR, IFN-γ receptor; TCR-Tg, T-cell receptor transgenic; GM-CSF, granulocyte macrophage colony-stimulating factor; mAb, monoclonal antibody; LCMV, lymphocytic choriomeningitis virus.

Fig. 1.

Protective immunity correlates with inhibition of tumor-induced angiogenesis. Mice were left untreated or immunized twice and 2 weeks later s.c. challenged as described in “Materials and Methods.” The majority of immunized mice rejected the challenge tumor, the majority of naïve mice did not reject the challenge tumor (Table 1). a and b, naïve and immunized C57BL/6 mice were s.c. injected with 1 × 106 MCA205 cells. c and d, naïve and immunized DBA1 mice were s.c. injected with 1 × 106 NIHpEJcl3 cells. e and f, naïve and immunized BALB/c mice were s.c. injected with 1 × 106 CT26 cells. g and h, naïve and immunized BALB/c mice were s.c. injected with 2 × 107 J558L cells. i and j, 2 days before J558L challenge, BALB/c immunized mice (as in g and h) were injected i.p. with 0.5 ml of D-PBS, anti-CD4, or anti-CD8 mAb. At day 6 after challenge, tumor sections were stained with anti-CD31 mAb for endothelial cells. Shown are representative stainings of tumor sections from three to five mice/group.

Fig. 1.

Protective immunity correlates with inhibition of tumor-induced angiogenesis. Mice were left untreated or immunized twice and 2 weeks later s.c. challenged as described in “Materials and Methods.” The majority of immunized mice rejected the challenge tumor, the majority of naïve mice did not reject the challenge tumor (Table 1). a and b, naïve and immunized C57BL/6 mice were s.c. injected with 1 × 106 MCA205 cells. c and d, naïve and immunized DBA1 mice were s.c. injected with 1 × 106 NIHpEJcl3 cells. e and f, naïve and immunized BALB/c mice were s.c. injected with 1 × 106 CT26 cells. g and h, naïve and immunized BALB/c mice were s.c. injected with 2 × 107 J558L cells. i and j, 2 days before J558L challenge, BALB/c immunized mice (as in g and h) were injected i.p. with 0.5 ml of D-PBS, anti-CD4, or anti-CD8 mAb. At day 6 after challenge, tumor sections were stained with anti-CD31 mAb for endothelial cells. Shown are representative stainings of tumor sections from three to five mice/group.

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Fig. 2.

Tumor immunity can be generated in pfp-deficient mice. Groups of pfp−/− (triangle) or as control, C57BL/6 mice (circle) were left untreated (▵ or ○) or immunized with 1 × 106-irradiated MCA205 cells (▴ or •). Two weeks later, mice were s.c. challenged contralaterally with 1 × 105 MCA205 cells. Tumor growth was observed every 2–3 days for 80 days. Mice that had a tumor of ≥1 cm in diameter were recorded as tumor positive. The tumor-free mice at the end of the experiment had completely rejected the tumor. Each group contained 10–14 mice. Similar results were obtained in a second experiment with 2 × 104 MCA205 challenge cells.

Fig. 2.

Tumor immunity can be generated in pfp-deficient mice. Groups of pfp−/− (triangle) or as control, C57BL/6 mice (circle) were left untreated (▵ or ○) or immunized with 1 × 106-irradiated MCA205 cells (▴ or •). Two weeks later, mice were s.c. challenged contralaterally with 1 × 105 MCA205 cells. Tumor growth was observed every 2–3 days for 80 days. Mice that had a tumor of ≥1 cm in diameter were recorded as tumor positive. The tumor-free mice at the end of the experiment had completely rejected the tumor. Each group contained 10–14 mice. Similar results were obtained in a second experiment with 2 × 104 MCA205 challenge cells.

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Fig. 3.

Tumor immunity cannot be generated in IFN-γ-deficient mice. Groups of IFN-γ−/− (triangle) or as control, C57BL/6 mice (circle) were left untreated (▵ or ○) or immunized with 1 × 106-irradiated MCA205 cells (▴ or •). Two weeks later, mice were s.c. challenged with 1 × 105 MCA205 cells. Tumor growth was observed every 2–3 days for 60 days. Each group contained 10 mice. Shown is one of three experiments with similar results.

Fig. 3.

Tumor immunity cannot be generated in IFN-γ-deficient mice. Groups of IFN-γ−/− (triangle) or as control, C57BL/6 mice (circle) were left untreated (▵ or ○) or immunized with 1 × 106-irradiated MCA205 cells (▴ or •). Two weeks later, mice were s.c. challenged with 1 × 105 MCA205 cells. Tumor growth was observed every 2–3 days for 60 days. Each group contained 10 mice. Shown is one of three experiments with similar results.

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Fig. 4.

Neutralization of IFN-γ activity leads to elimination of the protective immunity generated in pfp−/− mice. The pfp−/− mice were left untreated (○, n = 6) or immunized with 1 × 106-irradiated MCA205 cells (•, n = 8). Two weeks later, mice were s.c. challenged with 1 × 105 MCA205 cells. Another group of the immunized mice was i.p. injected with 500 μg of anti-IFN-γ mAb 2 h before the tumor challenge (▴, n = 9). Tumor growth was observed every 2–3 days for 80 days.

Fig. 4.

Neutralization of IFN-γ activity leads to elimination of the protective immunity generated in pfp−/− mice. The pfp−/− mice were left untreated (○, n = 6) or immunized with 1 × 106-irradiated MCA205 cells (•, n = 8). Two weeks later, mice were s.c. challenged with 1 × 105 MCA205 cells. Another group of the immunized mice was i.p. injected with 500 μg of anti-IFN-γ mAb 2 h before the tumor challenge (▴, n = 9). Tumor growth was observed every 2–3 days for 80 days.

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Fig. 5.

Angiogenesis is inhibited in immunized pfp but not IFN-γ-deficient mice. Tumor sections of naïve (top) or immunized (bottom) pfp−/− mice (a or b) and IFN-γ−/− mice (c or d) were prepared and stained with anti-CD31 mAb as described in Fig. 1. Three to 5 mice were analyzed in each group. Shown are representative stainings of tumors at day 6 after challenge.

Fig. 5.

Angiogenesis is inhibited in immunized pfp but not IFN-γ-deficient mice. Tumor sections of naïve (top) or immunized (bottom) pfp−/− mice (a or b) and IFN-γ−/− mice (c or d) were prepared and stained with anti-CD31 mAb as described in Fig. 1. Three to 5 mice were analyzed in each group. Shown are representative stainings of tumors at day 6 after challenge.

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Fig. 6.

Adoptive transfer of tumor specific T cells leads to antiangiogenesis in the tumor. a and b, accumulation of TCR-Tg T cells specifically in B16GP33 tumors. Experimental lung metastases of B16 (a) and B16GP33 tumors (b) were established by i.v. injection of 1 × 106 tumor cells. Fourteen days later, mice were i.v. injected with 1 × 107in vitro antigen-activated spleen cells isolated from Thy1.1+ P14 TCR-Tg mice. Four days after adoptive transfer, tissue sections of lungs were stained with donor T-cell-specific mAb, anti-Thy1.1. c–f, effect of TCR-Tg cells on tumor-induced angiogenesis. Tissue sections of lung metastases of B16GP33 tumors before (c and e) and 4 days after adoptive T-cell transfer (d and f) were stained with anti-CD31 mAb for endothelial cells. In c and d, in vitro activated TCR-Tg cells were transferred into B16GP33 tumor-bearing wild-type C57BL/6 mice, whereas in e and f, in vitro-activated IFN-γ-deficient TCR-Tg cells were transferred into tumor-bearing IFN-γ-deficient mice.

Fig. 6.

Adoptive transfer of tumor specific T cells leads to antiangiogenesis in the tumor. a and b, accumulation of TCR-Tg T cells specifically in B16GP33 tumors. Experimental lung metastases of B16 (a) and B16GP33 tumors (b) were established by i.v. injection of 1 × 106 tumor cells. Fourteen days later, mice were i.v. injected with 1 × 107in vitro antigen-activated spleen cells isolated from Thy1.1+ P14 TCR-Tg mice. Four days after adoptive transfer, tissue sections of lungs were stained with donor T-cell-specific mAb, anti-Thy1.1. c–f, effect of TCR-Tg cells on tumor-induced angiogenesis. Tissue sections of lung metastases of B16GP33 tumors before (c and e) and 4 days after adoptive T-cell transfer (d and f) were stained with anti-CD31 mAb for endothelial cells. In c and d, in vitro activated TCR-Tg cells were transferred into B16GP33 tumor-bearing wild-type C57BL/6 mice, whereas in e and f, in vitro-activated IFN-γ-deficient TCR-Tg cells were transferred into tumor-bearing IFN-γ-deficient mice.

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Table 1

Tumor immunity often requires CD8+ T cells in the effector phase

Mice, either naïve, immunized, or immunized and treated with anti-CD4 or anti-CD8 mAb, were challenged with the indicated number of tumor cells. Immunization with the same tumor and T-cell subset depletion at the time of challenge was done as described in “Materials and Methods.” Tumor growth was monitored for at least 60 days. Shown are numbers and percentages (in parentheses) of tumor free mice/total number of mice in the group.

MouseChallenged withNaiveImmunized
TumorCell numberControlαCD4 mAbαCD8 mAb
C57BL/6 MCA205 1 × 105 0/10 (0%) 4/8 (50%) 1/8 (13%) 0/7 (0%) 
BALB/c J558L 5 × 106 0/10 (0%) 9/9 (100%) 5/5 (100%) 0/6 (0%) 
BALB/c CT26 1 × 105 1/12 (8%) 9/10 (90%) Rejection* No rejection* 
DBA/1 NIHpEJcl3 1 × 105 1/10 (10%) 9/10 (90%) Rejection* No rejection* 
MouseChallenged withNaiveImmunized
TumorCell numberControlαCD4 mAbαCD8 mAb
C57BL/6 MCA205 1 × 105 0/10 (0%) 4/8 (50%) 1/8 (13%) 0/7 (0%) 
BALB/c J558L 5 × 106 0/10 (0%) 9/9 (100%) 5/5 (100%) 0/6 (0%) 
BALB/c CT26 1 × 105 1/12 (8%) 9/10 (90%) Rejection* No rejection* 
DBA/1 NIHpEJcl3 1 × 105 1/10 (10%) 9/10 (90%) Rejection* No rejection* 
*

For CT26 or NIHpEJcl3 tumor models, it has been shown that CD8+ but not CD4+ T-cell depletion at the time of challenge abrogated tumor rejection (23, 24).

We thank Christel Westen, Tanja Specowius, Angelika Gärtner and Marion Rösch, for excellent technical assistance.

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