INTRODUCTION

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CD8⁺ cytotoxic T lymphocytes (CTL) are potent mediators of antigen-specific tumor cell destruction. Therefore, most attempts to generate an immune response against tumor cells have focused on the induction of CTL, for example, by immunizing with peptides corresponding to major histocompatibility complex (MHC) class I-restricted epitopes. However, as this strategy fails to also stimulate T helper cells, which respond to MHC class II-restricted epitopes, the supportive role of these cells in tumor-specific immune responses may be neglected.

The importance of T helper cells for the outcome of immune responses against infectious pathogens has been recognized for quite some time. Recently, some groups (reviewed in reference 35) have presented evidence that the contribution of T helper cells also may be crucial in immune responses against tumors.

To ascertain more about the involvement of T helper cells in immune responses against tumors, we investigated the rejection of a well-characterized virus-induced tumor, the simian virus 40 (SV40) large-T-antigen (TAg)-expressing mKSA tumor, by BALB/c mice immunized with recombinant TAg. This particular system was chosen for the following reasons. (i) BALB/c mice immunized with TAg readily reject mKSA tumor cell inocula of 10⁶ cells, corresponding to about 10,000 times the 50% lethal dose of these tumor cells in naive mice (54). (ii) BALB/c mice are considered to be low responders or nonresponders with respect to the generation of TAg-specific CTL (1, 2, 16, 17, 36, 41, 42, 46). This weak immune reaction resembles the immune responses against nonviral tumor-specific antigens. (iii) Tumor-associated lymphocytes (TAL) can be isolated very conveniently from BALB/c mice injected intraperitoneally (i.p.) with mKSA cells by peritoneal lavage. These cells can be tested in primary in vitro cytotoxicity assays as well as for cytokine secretion without the need for prolonged in vitro cultivation. Thus, their measured activity approximates that in vivo as closely as possible. (iv) From the site of tumor cell inoculation, TAg-specific CD8⁺ CTL which lyse TAg-expressing target cells in primary in vitro assays can be recovered (54). (v) Although these TAg-specific, MHC class I-restricted CTL appear to be the actual effector cells in eliminating mKSA tumor cells, previous experiments demonstrated that at least at some point in the immune response CD4⁺ T cells are also needed for protective immunity (54).

The requirement of CD4⁺ lymphocytes for mKSA cell rejection could be explained by the following, not mutually exclusive scenarios. First, CD8⁺ CTL activity might be too weak or too slow to completely eradicate mKSA tumor cells, and an antibody response that requires CD4⁺ T helper cells might provide the complementary part in tumor rejection. The possibility of such a combined cell-mediated and humoral anti-mKSA tumor response is supported by the report of Bright and coworkers (2), who proposed an antibody-dependent cell-mediated cytoxicity mechanism for mKSA tumor rejection by BALB/c mice. Second, CD4⁺ cells might provide help to CD8⁺ cells. This interpretation is supported by several studies of adoptive immunization in which CTL were effective against tumors only when coadministered either with T helper cells or with one of the major help-providing lymphokines of these cells, interleukin 2 (IL-2) (18, 27, 38, 39, 53). Third, CD4⁺ lymphocytes could lyse tumor cells via soluble factors (47) or via membrane-bound mechanisms (20, 21). Fourth, they could recruit and activate macrophages or other leukocytes to exert tumoricidal activity (10, 11, 15, 32, 45, 52).

Our results show that there is an absolute requirement for both CD4⁺ and CD8⁺ cells during the entire course of mKSA tumor rejection. During this time, the major function of CD4⁺ T lymphocytes is to enable CD8⁺ cells to proliferate and to mature into fully active effector CTL. However, despite the support of T helper cells, the expansion of TAg-specific CTL is so limited that ex vivo lytically active CTL can be isolated from the tumor site only, while both the spleen and the regional lymph nodes are devoid of them.

	MATERIALS AND METHODS

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Mice and tumor cells. Specific-pathogen-free female BALB/cAnNCrlBR mice were purchased from Charles River Wiga, Sulzfeld, Germany. The mice were kept strictly under barrier conditions and were used when 8 to 12 weeks old. SV40-transformed mKSA (22) tumor cells, originating from BALB/c mice, and SV40-transformed BALB/c fibroblasts (BALB/c-SV40 fibroblasts) were routinely maintained in Dulbecco modified Eagle medium supplemented with 5% fetal calf serum (FCS).

Production and purification of recombinant TAg and production of SV40. TAg was produced by infecting Sf 158 cells with recombinant baculovirus coding for full-length SV40 TAg (24), kindly provided by Ellen Fanning. TAg was purified from cell lysates by immunoaffinity chromatography using PAb108 (19) coupled to CNBr-Sepharose. The purified protein was dialyzed against phosphate-buffered saline (PBS)-40% glycerin and stored at 70°C. SV40 was harvested from cultures of TC7 African green monkey kidney cells infected with SV40.

Immunization of mice with recombinant TAg or with SV40. Ten micrograms of purified TAg in PBS was injected twice i.p. at a 1-week interval (days 14 and 7). Seven days after the second immunization (day 0), the animals were challenged by i.p. injection of 10⁶ viable mKSA cells. Alternatively, mice were immunized with SV40 by injection of 50 µl of SV40 suspension (virus titer, about 10⁸ PFU/ml) into the left hind footpad.

Depleting mice of CD4⁺ or CD8⁺ T lymphocytes. Groups of mice were treated intravenously (i.v.) with either a mixture of 250 µg each of rat monoclonal antibodies (Mab) YTS191.1 and YTA3.1 (37) or 500 µg of rat MAb YTS169.4 (9) to deplete CD4⁺ or CD8⁺ T lymphocytes, respectively. As a control, purified rat immunoglobulin G (IgG) (Sigma, Deisenhofen, Germany) was administered in some experiments. The time of the antibody injection relative to tumor challenge is given in Results. MAb were produced, purified, used, and efficacy controlled as described previously (13).

Magnetic cell sorting. The magnetic cell sorting method has been described elsewhere (28). TAg-immunized or naive mice were inoculated i.p. with 10⁶ mKSA tumor cells. Peritoneal exudate cells were recovered by rinsing the peritoneal cavities with cold PBS, and single-cell suspensions of spleens or mesenteric lymph nodes were prepared from the same animals. These cell preparations were incubated with CD4- or CD8-specific MAb attached to magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) and subsequently passed through MiniMACS columns in a magnetic field (Miltenyi). Flow cytometry revealed >95% purity of the CD4⁺ or CD8⁺ T-lymphocyte preparation.

In vitro restimulation of TAg-specific CTL. Spleen cells (5 × 10⁷) were cocultured with 5 × 10⁶ irradiated (10 Gy) BALB/c-SV40 fibroblasts in 20 ml of complete medium (RPMI 1640, 10% FCS, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, 100 U of penicillin/ml, 100 µg of streptomycin/ml) supplemented with 10% concanavalin A supernatant in upright TC-25 culture flasks for 6 days. The TAg-specific cytotoxicity of these cells was tested with standard chromium release assays.

Chromium release assays. CTL activity was measured as described by Brunner and colleagues (5) with modifications (13). Briefly, ⁵¹Cr-labeled mKSA or fetal BALB/c-SV40 fibroblast target cells were incubated at 37°C with effector cells at various ratios; control target cells were TAg-negative MethA cells (H-2^d) (34) or TAg-positive SV40-transformed fetal C57BL/6J fibroblasts (H-2^b). After 4 h, the release of ⁵¹Cr was measured and percent lysis was calculated as [(experimental counts per minute  spontaneous counts per minute)/(maximal counts per minute  spontaneous counts per minute)] × 100.

Measurement of cytokines. CD4⁺ or CD8⁺ T cells were prepared from peritoneal exudate cells or splenic single-cell suspensions by magnetic cell sorting. Purified cells were kept for 24 h at a density of 10⁶/ml in RPMI 1640-5% FCS in flat-bottom microtiter plates (200 µl/well) before supernatants were harvested (49). The supernatants were tested for their contents of IL-2, gamma interferon (IFN-), or tumor necrosis factor alpha (TNF-) by a commercially available enzyme-linked immunosorbent assay (R&D Systems, Wiesbaden, Germany).

	RESULTS

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Detection of TAg-specific CD8⁺ CTL in TAg-immunized BALB/c mice. CD8⁺ TAL collected from the site of tumor cell inoculation by peritoneal lavage of BALB/c mice immunized twice with 10 µg of recombinant TAg and challenged i.p. with mKSA tumor cells lysed TAg-expressing, MHC class I-matched target cells in primary ex vivo cytotoxicity assays (Fig. 1) (54). However, CD8⁺ T cells prepared from spleens (Fig. 1) (54) or mesenteric lymph nodes (data not shown) from the same mice did not exhibit this cytotoxic activity. This finding indicates that only a limited number of TAg-specific CTL is activated in BALB/c mice and that these few cells are apparently recruited quantitatively to the site of the tumor.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   TAg-specific cytotoxicity mediated by freshly prepared CD8+ cells or by spleen cell bulk cultures prepared from mice immunized with different protocols. TAL or spleen cells were recovered from mice pretreated as follows: twice immunized with TAg, challenged with mKSA cells, CD8+ TAL prepared from the peritoneal cavity 8 days after challenge (filled diamonds; the reduced effector/target ratio is due to the poor yield of CD8+ TAL from this group of five mice); TAg immunized, challenged with mKSA cells, CD8+ spleen cells prepared on day 8 (open diamonds); TAg immunized, challenged with mKSA cells, restimulated in the presence of TAg-expressing cells 4 weeks after challenge (filled circles); SV40 immunized (once), restimulated in the presence of TAg-expressing cells 2 weeks after immunization (filled squares); TAg immunized, restimulated 4 weeks after immunization for three weekly cycles (inverted filled triangles); TAg immunized, restimulated once 4 weeks after immunization (filled triangles); and naive, stimulated once in the presence of TAg-expressing cells (open circles). Lytic activity against MHC-matched, non-TAg-expressing MethA cells or against MHC-mismatched, TAg-expressing SV40-transformed C57BL/6 fibroblasts was below 7% (data not shown).

Both CD4⁺ and CD8⁺ cells are required during the entire course of mKSA tumor rejection. It was previously reported that protective immunity in TAg-immunized BALB/c mice is dependent not only on CD8⁺ but also on CD4⁺ T lymphocytes (54). In order to find out at what time during the course of the immune response T helper cells were needed, groups of mice were depleted of either CD4⁺, CD8⁺, or both T-cell subsets by administering i.v. a single dose of the appropriate MAb at various times. The development of clinical signs of tumor progression (inactivity, ruffled fur, hunched posture, projection of the flanks due to ascites, or development of subcutaneous tumor nodes at the injection site) and the time of tumor-related death of these mice were monitored. Effective antitumor protection was defined as mice staying free of mKSA for life rather than some prolongation of survival compared to that of naive controls.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Mortality of mice after depletion of T-lymphocyte subsets prior to immunization with TAg. Groups of five mice were injected i.v. with a combination of each CD4- and CD8-specific MAb, CD4-specific MAb, or CD8-specific MAb on days 15 and 8; 500 µg of each MAb was injected. A control group received no MAb treatment. One day after T-cell depletion (i.e., days 14 and 7), mice were immunized by i.p. injection of 10 µg of TAg; five control mice received neither TAg nor MAb. On day 0, all mice were challenged by i.p. inoculation of 106 mKSA cells. Development of tumors and death of the mice were monitored daily. Open circles and error bars indicate means and standard errors.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Dependence of course of mortality on T-cell subset depletion 1 day before or 3, 6, or 10 days after mKSA cell challenge. Mice immunized by two i.p. injections of 10 µg of TAg on days 14 and 7 were depleted of CD4+, CD8+ or both T-lymphocyte populations. Depletion was achieved by i.v. injection of 500 µg of the respective MAb as a single dose on day 1, +3, +6, or +10 relative to challenge with 106 mKSA cells on day 0. Tumor development and death of mice were monitored daily.

Efficacy and duration of CD4⁺ or CD8⁺ T-cell depletion by a single MAb injection. To exclude the possibility that T cells that escaped depletion by a single treatment with 500 µg of MAb might be responsible for the survival of several T-cell-depleted mice (Fig. 3), we thouroghly investigated the efficacy and duration of the MAb treatment. We especially addressed concerns that in immunized and tumor cell-challenged mice, depletion of T-cell subsets might be incomplete due to the expansion of the respective T-cell population or due to the down-modulation of CD4 or CD8 coreceptors on activated T cells (6, 51). In Table 1, we show that regardless of whether the respective MAb was injected on the day of challenge or 4 or 7 days after challenge, CD4⁺ as well as CD8⁺ T cells were completely eliminated from the spleens. Moreover, the recovery of the T-cell compartment was so slow that the respective T-cell subset could not be detected up to 18 days after administration of the MAb. The data suggest that the minute numbers of T cells that escaped depletion most probably were not responsible for the survival of MAb-treated mice.

CD4⁺ T lymphocytes are not directly cytolytic for mKSA cells. As CD4⁺ T cells were as important for the protective TAg-specific immune response of BALB/c mice as CD8⁺ T cells, we considered the possibility that CD4⁺ T lymphocytes might directly attack tumor cells either by contact-mediated lytic mechanisms (12, 21, 25, 29, 40) or by the secretion of cytostatic or cytotoxic lymphokines (47). However, the specific recognition of target cells by CD4⁺ T cells usually requires the expression of MHC class II molecules on the target cells. As mKSA cells do not express MHC class II molecules (R. Schirmbeck, personal communication; our unpublished data), direct cytotoxic activity of CD4⁺ T cells against mKSA cells was rather unlikely. Nevertheless, we tested whether CD4⁺ T cells freshly prepared from immune mice on day 4, 6, or 8 after tumor challenge were able to lyse mKSA cells. Although IFN- does not induce or upregulate the expression of MHC class II molecules in mKSA cells (R. Schirmbeck, personal communication), we used native as well as IFN--preincubated mKSA cells as target cells in these assays. In standard 4-h chromium release assays, we could never detect any specific destruction of native or IFN--treated mKSA cells. In addition, overnight cytolytic assays also did not reveal any specific CD4⁺ cell-mediated lysis of mKSA cells (data not shown).

The development of CD8⁺ T-lymphocyte-mediated TAg-specific cytotoxicity is critically dependent on CD4⁺ T helper cells. Since CD4⁺ T lymphocytes are not directly cytolytic for mKSA cells, we assumed that these cells might act as helper cells for the generation of TAg-specific CD8⁺ CTL.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Primary cytotoxicity of CD8+ peritoneal exudate cells from TAg-immunized mice after depletion of CD4+ cells at different times during immunization or maturation. Mice were immunized on days 14 and 7 by i.p. injection of 10 µg of TAg and challenged on day 0 with 106 mKSA cells injected i.p. Depletion of CD4+ cells was achieved by treating groups of five mice on days 15 and 8, day 1, day +2, or day +5 with an i.v. injection of CD4-specific MAb. On day 8, the cytotoxic activity of CD8+ peritoneal exudate cells was determined with a standard 4-h chromium release assay using mKSA cells as targets. Variable effector/target ratios are due to various yields of CD8+ TAL in the groups.

View larger version (14K): [in this window] [in a new window]   FIG. 5.   Primary cytotoxicity of CD8+ TAL after depletion of CD4+ cells during the effector phase. Mice were immunized and challenged with tumor as described in the legend to Fig. 4. CD4+ T cells were depleted on day 7 or 9. The cytotoxicity of CD8+ peritoneal exudate cells was determined on day 10 after tumor challenge. TAL from mice not treated with MAb were included as positive controls.

CD4⁺ T cells freshly isolated from TAg-immunized, mKSA-challenged mice secrete large amounts of IL-2 and IFN-. CD4⁺ T cells mediate their helper functions via the secretion of cytokines. Therefore, we compared the ex vivo capacities of CD8⁺ and CD4⁺ cells to secrete cytokines that are known to support cell-mediated immunity. We determined the amounts of IL-2, IFN-, and TNF- in the supernatants of either CD4⁺ or CD8⁺ cells freshly prepared from spleens or peritoneal exudate lymphocytes (PEL) of TAg-immunized mice at different times after mKSA cell inoculation.

	DISCUSSION

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CD4⁺ T lymphocytes are often essential for humoral and cellular immune responses (18, 27, 39, 53), but only recently has the importance of CD4⁺ T helper cells in tumor-specific immune responses attracted some scientific interest (21, 32, 35, 48). The previous neglect of these cells is due at least in part to methodological problems. For example, in most experimental systems, it is not possible to determine the ex vivo activity of freshly prepared CD4⁺ or CD8⁺ tumor-infitrating lymphocytes (TIL) due to rather small quantities of these cells and difficulties in isolating them from the tumor tissue. Therefore, TIL usually can be studied only following prolonged in vitro cultivation.

TAg-immunized BALB/c mice challenged i.p. with TAg-expressing mKSA tumor cells provide a unique opportunity to analyze the in vivo and ex vivo activities of both CD4⁺ and CD8⁺ T lymphocytes during the entire course of antigen-specific tumor rejection, because T lymphocytes can easily be enriched from peritoneal exudate cells (54). However, since these lymphocytes are not derived from a solid tumor, we define them as TAL rather than as TIL. Studying such freshly isolated cells should provide data approximating the functional state of these lymphocytes in vivo as closely as possible. These data, in conjunction with in vivo experiments, should allow a detailed understanding of the mechanisms of antigen-specific immune responses against tumor cells in vivo.

Studying the contributions of CD4⁺ and CD8⁺ T lymphocytes to TAg-specific tumor elimination in BALB/c mice revealed two major findings. First, TAg-specific CD8⁺ CTL are absolutely dependent on antigen-specific CD4⁺ T helper cells not only during induction and maturation but also during the late effector phase. Second, despite the support of T helper lymphocytes, the expansion of specific CTL appears to be very limited, and the small numbers of TAg-specific CTL performing ex vivo detectable target cell lysis appear to migrate quantitatively to the tumor site so that ex vivo active CTL can be recovered neither from the spleen nor from the regional lymph nodes.

Dependence of CTL on T helper cells. The possibility of measuring primary TAg-specific CTL activity in this system enabled us to disclose the dependence of CD8⁺ lytic effector cells on CD4⁺ T helper cells. The measurement of the lytic activity of CD8⁺ T lymphocytes subsequent to the depletion of CD4⁺ lymphocytes revealed that CD4⁺ T cells are absolutely required for the generation of lytically active TAg-specific CD8⁺ CTL, because elimination of CD4⁺ cells at any time point between 1 day prior to TAg injection and 5 days after tumor challenge completely prevented the emergence of CTL. Moreover, withdrawal of T helper cells at day 7 after tumor challenge stopped the augmentation of the already acquired low level of CTL-mediated lysis present at the moment of T helper cell depletion. However, depletion of T helper cells at day 9 did not significantly alter the lytic activity measurable on day 10 compared to the results for immune controls.

Accumulation of lytically active antigen-specific CTL at the tumor site. The critical dependence of TAg-specific CTL in BALB/c mice on CD4⁺ cells and its consequences might explain why BALB/c mice, in contrast to C57BL/6 mice, have been denoted as low responders or even nonresponders with respect to their ability to mount an SV40 TAg-specific CTL response (1, 2, 4, 16, 23, 33, 42, 46). Beyond confirming this assessment in a previous study (54) and in this report (Fig. 1), as far as the detection of TAg-specific CTL by restimulation in vitro of cells from secondary lymphoid organs is concerned, we found that for the judgment of weak tumor-specific immune responses, topological aspects must be considered. Thus, we failed to detect primary ex vivo active TAg-specific CTL in the spleens or regional lymph nodes of immune mice rejecting i.p.-injected mKSA cells, while we easily detected lytically active CD8⁺ TAL in the peritoneal cavities of the same mice. Apparently, despite the help of CD4⁺ cells, only small numbers of TAg-specific CTL are generated, and these cells are quantitatively recruited to the tumor site.