A number of tumor studies have indicated a link between CD4 help and the magnitude and persistence of CTL activity; however, the mechanisms underlying this have been largely unclear. To evaluate and determine the mechanisms by which CD4+ T cells synergize with CD8+ T cells to prevent tumor growth, we used the novel technique of monitoring in vivo CTL by labeling target cells with CFSE. This approach was supported by the direct visualization of CTL using peptide-MHC tetramers to follow tumor-specific T cells. The data presented demonstrate that while cotransfer of Ag-specific CD4+ T cells was not required for the generation of CTLs, because adoptive transfer of CD8+ T cells alone was sufficient, CD4+ T cells were required for the maintenance of CD8+ T cell numbers. Our data suggest that there is a correlation among the number of CD8+ T cells, in vivo CTL function, and IFN-γ production, with no evidence of a partial or nonresponsive phenotype among tetramer-positive cells. We also show that CD4+ T cells are required for CD8+ T cell infiltration of the tumor.

Tumors often fail to elicit effective immune responses. Despite this observation, much effort has gone toward determining the mechanisms that limit the potential efficacy of the anti-tumor immune response with the ultimate goal of overcoming or bypassing these checkpoints using strategic or novel immunotherapies. Generally these therapies are directed toward improving the CD8+ T cell response because most tumors, particularly solid tumors, are MHC class I+ and class II and therefore constitute targets for CD8+ T cells rather than CD4+ T cells. Moreover, CD8+ T cells are specialized for lytic function, and if tumor eradication occurs it is usually associated with an ability to detect tumor-specific CTLs.

However, it is clear that CD4+ T cells can also play an important role in facilitating an anti-tumor response (1). In some experiments in vivo depletion of CD4+ T cells has been shown to facilitate tumor progression; in others the cotransfer of CD4+ T cells improves tumor eradication. Moreover, studies using peptide immunization have demonstrated a requirement for coimmunization with both MHC class I- and class II-restricted peptides to ensure tumor eradication (2). The traditional explanation for these results is that CD4+ T cells provide IL-2 or help to the CD8+ T cells, an interpretation supported by studies in which IL-2 administration facilitates tumor eradication (3). More recently however, CD4+ T cells have been shown to have another role in the induction of CD8+ T cell responses via the modification of dendritic cells (DC),3 which, in turn, induce cytotoxic effector function in CD8+ T cells (4, 5, 6). Viruses or Abs to CD40 can also mediate this “licensing” of APCs by CD4+ T cells. It is also possible that CD4+ T cells are required in later phases of anti-tumor responses, as CD4+ T cells have been implicated in determining the magnitude and persistence of CTL responses in some chronic viral infections and models of autoimmune disease (7, 8, 9). Taken together these types of experiments provide a basis for the hypothesis that CD4+ T cells may play a broader role in anti-tumor responses than simply providing help for CTL induction. However, confirmation of this process, the mechanisms underlying it, and the role and subsequent fate of tumor-specific T cells are unknown.

To dissect out the role of tumor-specific T cells, we have developed a model system in which the influenza hemagglutinin (HA) gene is the model tumor Ag, and tumor-specific T cells are derived from HA-specific TCR transgenic mice. Two lines of TCR transgenic mice, one class I restricted (CL4 mice) (10) and the other class II-restricted (HNT mice) (11), both of which recognize HA in the context of H-2d, have been used. We have previously demonstrated in this model that tumor-specific CD4+ T cells act synergistically with limiting numbers of anti-tumor CD8+ T cells to prevent tumor growth (12). Here we examine the role that CD4+ T cells have in helping anti-tumor CD8+ T cells. Using MHC tetramers to directly identify the HA-specific CD8+ T cells, we show that the presence of CD4+ T cells not only correlates with the maintenance of specific CD8+ T cell numbers, but also with the maintenance of CTL function. In the absence of CD4+ T cells, CD8+ T cells cannot be found within the tumor mass. It is only in the presence of CD4+ T cells that other cells within the tumor exhibit up-regulation of MHC class II and ICAM expression. Together these data demonstrate that CD4+ T cells contribute to three major postlicensing events: maintenance of CD8 numbers, CD8 infiltration of tumors, and modulation of the tumor environment. These three events dramatically increase the efficacy of anti-tumor CTL effectors.

BALB/c (H-2d) mice were obtained from the Animal Resources Center (Canning Vale, Australia) and maintained under standard conditions in the University Department of Medicine animal holding area. The anti-HA transgenic TCR mice (HNT, class II restricted; and CL4, class I restricted) were generated and screened as described previously (12). For all experiments female mice between 6 and 12 wk of age were used.

The derivation and characterization of the AB1 murine MM cell line and the generation of the AB1-HA transfectant (AB1 cells transfected with the influenza HA gene) have been described previously (12, 13). Cell lines were maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 20 mM HEPES, 0.05 mM 2-ME, 60 μg/ml penicillin (CSL, Melbourne, Australia), 50 μg/ml gentamicin (Δ West, Bentley, Australia), and 5% FCS (Life Technologies). AB1-HA transfectants were selected by culture in medium containing the neomycin analogue geneticin (Life Technologies) at a final concentration of 400 μg/ml. The level of HA expression on transfected cells was measured by FACS analysis, using the biotinylated HA-specific mAb H18 (14) that was originally obtained from Dr. Walter Gerhard (The Wistar Institute, Philadelphia, PA).

Detection of in vivo CTL was performed as described previously (15). Erythrocytes were removed from BALB/c spleens by resuspending cells in RBC lysis buffer for 5 min, followed by three washes in PBS and resuspending at 5 × 106 cells/ml. Cells were divided into two populations, one of which was pulsed with 1 μg/ml of CL4 peptide for 90 min at 37°C. Cells were then labeled with CFSE (Molecular Probes, Eugene, OR) for 10 min at room temperature. For CFSE fluorescence intensities, peptide-pulsed cells were labeled at a final concentration of 5 μM (CFSEhigh) and unpulsed cells at 0.5 μM (CFSElow). Cells were washed with FCS four times and then with PBS before i.v. injecting CFSE-labeled cells into recipients. In all experiments cells were recovered 20 h after transfer and analyzed by FACS for fluorescence intensity using CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA).

Recombinant protein was prepared as previously described by Garboczi and colleagues (16). The plasmid encoding Kd (provided by Dr. Jon Yewdell, National Institute of Allergy and Infectious Disease, National Institutes of Health, and Dr. Altman, Emory College of Medicine, Atlanta, GA) was modified to encode a BirA recognition sequence at the C terminus (17). In some experiments the tetramers were further purified by size exclusion chromatography.

For analysis, 5 × 105 lymphocytes were treated with purified anti-mouse CD16/CD32 (Fc-γIII/II receptor, PharMingen, San Diego, CA), then stained with the HA tetramer for 2 h at room temperature. Abs to CD8 coupled with fluorescein were then added for an additional 20 min. Propidium iodide (1 μg/ml) was added in the final wash to exclude nonviable cells. Events were acquired on a FACScan flow cytometer, and the data were analyzed using CellQuest software (Becton Dickinson).

Cells secreting IFN-γ in an Ag-specific manner were detected using a standard ELISPOT assay (18, 19). Ninety-six-well multiscreen HA plates (Millipore, Bedford, MA) were coated with rat anti-mouse IFN-γ Ab (clone R4-6A2; PharMingen) overnight at 4°C, washed with PBS, then incubated with 200 μl of RPMI 1640 medium supplemented with 10% FCS for 1 h at room temperature. Two-fold dilutions of lymph node (LN) cells from animals previously inoculated s.c. with 2 × 105 AB1-HA cells and i.v. with 1 × 107 CL4 LN cells with or without 1 × 107 HNT LN cells were added to wells starting at 106/well in the presence of 4 × 105 gamma-irradiated (1200 rad) syngeneic spleen cells. Cells were incubated for 26 h with or without CL4 peptide stimulation (1 μg/ml, final concentration). Wells were sequentially washed three times each with ddH20, PBS, and PBS containing 0.05% Tween 20 and then incubated for 20 h at 4°C with biotinylated anti-mouse IFN-γ Ab (4 μg/ml, clone XMG1.2, PharMingen). Wells were washed and incubated with peroxidase-labeled anti-biotin Ab (2 μg/ml; Vector, Burlingame, CA) for 20 h at 4°C. Wells were then washed and spots were developed using freshly prepared substrate (3-amino-9-ethyl-carbazole; Sigma, St. Louis, MO) dissolved in dimethyformamide, diluted in 0.1 M sodium acetate (pH 4.8), and filtered, and 0.015% H2O2 (final concentration) was added to give a 0.3 mg/ml solution. After 30 min the substrate solution was discarded, and plates were washed under running water and air-dried. Colored spots were counted using a stereomicroscope.

Surface Ags were detected using the streptavidin-biotin labeling immunoperoxidase staining technique. Tissues from various sites were removed, placed in compound-embedding medium (OCT; Miles, Elkhart, IN), snap-frozen using dry ice, and stored at −80°C. Ten-micrometer sections were cut, collected on poly-l-lysine-coated slides, and allowed to air-dry. Slides were stored at 4°C (for a maximum of 2 days) before staining. Before immunostaining, sections were fixed with cold ethanol (15 min) and blocked with 1% (v/v) H2O2 (5 min) followed by avidin/biotin block (10 min each). Sections were incubated with the appropriate dilutions of rat anti-mouse mAbs against CD8 (53-6.7, Lyt2), CD4 (RM4-5, L3T4), CD54 (3E2, ICAM-1), and isotype controls rat IgG2a,κ (R35-95) and rat IgG2b,κ (A95-1; all from PharMingen) and rat anti-mouse mAb against class II (TIB-120, M5/114.15.2, provided by P. Holt, TVW Telethon Institute for Child Health and Research, Perth, Australia) for 1 h followed by incubation with a biotinylated secondary Ab for 30 min (mouse anti-rat IgG F(ab′)2; Jackson ImmunoResearch, West Grove, PA). Immunostaining was detected by incubating with streptavidin-HRP (Dako, Copenhagen, Denmark) for 30 min and with diaminobenzidine-H2O2 (Sigma) for 5–10 min. Slides were washed three times for 5 min each time in PBS between each incubation step, counterstained with hematoxylin, and mounted in aqueous mounting medium.

Both CD4+ and CD8+ Ag-specific T cells are required for eradication of the AB1-HA MHC class I+, class II tumor (Fig. 1,A) (12). To determine the minimum number of cells that protect from tumor growth, AB1-HA-inoculated mice were adoptively transferred either with 1 × 107 LN cells from HA-specific, MHC class I-restricted TCR transgenic mice (CL4 mice, CD8+ T cells) plus LN cells from HA-specific, MHC class II-restricted TCR transgenic mice (HNT mice, CD4+ T cells; titrated over a range of 104 to 5 × 106 cells; Fig. 1,B) or with 1 × 107 CD4+ T cells and a titration of CD8+ T cells (Fig. 1,C). When <107 cells of either subpopulation were transferred, the level of protection was markedly reduced. This reduction correlated with the number of titrated cells. CD8+ plus CD4+ T cells were then adoptively transferred into BALB/c mice 7 or 14 days after AB1-HA inoculation, when tumor in control mice is not palpable. Delaying the cotransfer of CD4+ plus CD8+ T cells until day 7 or 14 after tumor inoculation resulted in no protection from tumor growth (Fig. 2). In fact, there was no protection even if these cells were transferred as early as 4 days after tumor inoculation (data not shown). To determine whether delaying the administration of either T cell subpopulation would affect tumor growth, BALB/c mice were inoculated with AB1-HA and concomitantly transferred with either CD4+ or CD8+ T cells. At 7 (Fig. 2,A) or 14 (Fig. 2,B) days after tumor inoculation, mice were given the reciprocal population. The efficacy of tumor eradication was reduced in all experimental groups, with no significant difference between delaying transfer of the second reciprocal cell population from day 7 to day 14, although the kinetics of tumor growth were somewhat delayed compared with those in the control animals that received tumor only. Mice that received CD4+ T cells concurrently with tumor inoculation showed a small, but reproducible, degree of protection compared with those given CD8+ T cells at the time of tumor inoculation (40% tumor-free animals compared with 0%; Fig. 2).

FIGURE 1.

Class II-restricted HA-specific TCR transgenic cells act synergistically with limiting anti-tumor CTLs to prevent the growth of the AB1-HA transfectants. BALB/c mice were inoculated s.c. with 2 × 105 AB1-HA tumor cells. Mice were concurrently injected i.v. with A) 1 × 107 LN cells from either HA-specific CL4 mice (CD8+ T cells) or HNT mice (CD4+ T cells) or LN cells from both; B) 1 × 107 LN cells from CL4 mice mixed with dilutions of HNT LN cells; or C) 1 × 107 LN cells from HNT mice mixed with dilutions of CL4 LN cells. Control mice received the AB1-HA tumor cells. This experiment was performed twice with five mice per group, and similar results were obtained.

FIGURE 1.

Class II-restricted HA-specific TCR transgenic cells act synergistically with limiting anti-tumor CTLs to prevent the growth of the AB1-HA transfectants. BALB/c mice were inoculated s.c. with 2 × 105 AB1-HA tumor cells. Mice were concurrently injected i.v. with A) 1 × 107 LN cells from either HA-specific CL4 mice (CD8+ T cells) or HNT mice (CD4+ T cells) or LN cells from both; B) 1 × 107 LN cells from CL4 mice mixed with dilutions of HNT LN cells; or C) 1 × 107 LN cells from HNT mice mixed with dilutions of CL4 LN cells. Control mice received the AB1-HA tumor cells. This experiment was performed twice with five mice per group, and similar results were obtained.

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FIGURE 2.

Cotransfer of tumor-specific CD4+ and CD8+ T cells is only effective for a short period after tumor inoculation. BALB/c mice were inoculated with AB1-HA (T), then 7 (A) and 14 (B) days postinoculation were adoptively transferred with 1 × 107 HA-specific CD8+ (CL4) plus 1 × 107 CD4+ LN cells (HNT; ▴). BALB/c mice were also inoculated with AB1-HA at the same time as either HA-specific CD8+ (•) or CD4+ (▪) LN cells were adoptively transferred i.v. and subsequently on either day 7 (A) or day 14 (B) were given the reciprocal population. Control mice received the AB1-HA tumor cells only (○). Data are representative of three experiments with five animals per group.

FIGURE 2.

Cotransfer of tumor-specific CD4+ and CD8+ T cells is only effective for a short period after tumor inoculation. BALB/c mice were inoculated with AB1-HA (T), then 7 (A) and 14 (B) days postinoculation were adoptively transferred with 1 × 107 HA-specific CD8+ (CL4) plus 1 × 107 CD4+ LN cells (HNT; ▴). BALB/c mice were also inoculated with AB1-HA at the same time as either HA-specific CD8+ (•) or CD4+ (▪) LN cells were adoptively transferred i.v. and subsequently on either day 7 (A) or day 14 (B) were given the reciprocal population. Control mice received the AB1-HA tumor cells only (○). Data are representative of three experiments with five animals per group.

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We have already shown that the cotransfer of tumor-specific CD4+ T cells increases the proliferation of HA peptide-reactive CD8+ T cells recovered from AB1-HA-inoculated animals (12). However, the direct enumeration of these CD8+ T cells was not possible, as there is neither a clonotypic nor a Vα Ab for this transgenic TCR. The Vβ-chain of the transgenic receptor, Vβ8.2, is relatively widely expressed in wild-type animals and therefore cannot be used as a surrogate marker for transferred cells. To overcome this problem, MHC class I tetramers were produced that consisted of the H-2Kd molecule and the HA peptide IYSTVASSL. To demonstrate that the tetramers bind the appropriate receptors, LN cells from CL4 TCR transgenic mice were stained. Tetramer-positive cells represented >80% of the transgenic CD8+ T cells (49% of the total cells analyzed; Fig. 3,A), but did not stain T cells from nontransgenic animals (Fig. 3,B). Examples of tetramer staining in the LN of animals that received adoptively transferred cells are shown in Fig. 3, C–F. Animals that received CD8+ T cells (Fig. 3, C and D), showed consistently fewer tetramer-positive cells than those that received both CD4+ and CD8+ T cells (Fig. 3, E and F). The summary of results from individual animals in which the number of tetramer-positive cells in lymphoid organs was determined 14 and 28 days after tumor inoculation is shown in Fig. 4. Animals in both experimental groups had similar numbers of tetramer-positive cells on day 14, i.e., 0.22 ± 0.02% (SEM) of the total cells for the CD8 only group and 0.27 ± 0.02% of the total cells for the CD8 plus CD4 group (Fig. 4). However, by day 28 the number of tetramer-positive cells in the CD8 plus CD4 group remained similar to day 14 values (0.22 ± 0.02%), but was significantly reduced to 0.10 ± 0.01% of the total cells in animals given CD8 cells only (p = 0.000009). This reduction in tetramer-positive cells in peripheral lymphoid tissue was not the result of tetramer-positive cells trafficking to the tumor (data not shown). Tetramer-positive cells were never detected in animals inoculated with the AB1-HA tumor only (data not shown).

FIGURE 3.

Enumeration of CD8+ CL4 T cells. A, CL4 TCR transgenic mouse; B, a nontransgenic littermate stained with CD8-FITC vs HA-tetramer. Mice were inoculated s.c. with AB1-HA tumor cells and i.v. with either HA-specific CD8+ LN cells (C and D) or a mixture of HA-specific CD8+ and HA-specific CD4+ LN cells (E and F). LN populations were recovered 14 (C and E) and 28 (D and F) days postinoculation. Plots display CD8 and HA-tetramer staining of LN cells, with the percentage of tetramer-positive CD8+ T cells indicated in each plot. Data are for a representative of three mice per group.

FIGURE 3.

Enumeration of CD8+ CL4 T cells. A, CL4 TCR transgenic mouse; B, a nontransgenic littermate stained with CD8-FITC vs HA-tetramer. Mice were inoculated s.c. with AB1-HA tumor cells and i.v. with either HA-specific CD8+ LN cells (C and D) or a mixture of HA-specific CD8+ and HA-specific CD4+ LN cells (E and F). LN populations were recovered 14 (C and E) and 28 (D and F) days postinoculation. Plots display CD8 and HA-tetramer staining of LN cells, with the percentage of tetramer-positive CD8+ T cells indicated in each plot. Data are for a representative of three mice per group.

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FIGURE 4.

CD4+ T cells act to maintain the CD8+ T cell pool. Mice were inoculated with AB1-HA tumor cells, and HA-specific CD8+ LN cells or AB1-HA tumor cells, and a mixture of HA-specific CD8+ and CD4+ LN cells. LN populations were recovered 14 and 28 days postinoculation. These populations were then double stained with CD8-FITC and the HA-tetramer Ab. Plots display the percentage of tetramer-positive CD8+ T cells in each group. The number of tetramer-positive cells was significantly reduced by day 28 in animals given CD8+ LN cells only (p = 0.000009). The addition of CD4+ LN cells significantly increased the number of tetramer-positive CD8+ T cells on day 28 (p = 0.00002). ▴, Mice with palpable tumors.

FIGURE 4.

CD4+ T cells act to maintain the CD8+ T cell pool. Mice were inoculated with AB1-HA tumor cells, and HA-specific CD8+ LN cells or AB1-HA tumor cells, and a mixture of HA-specific CD8+ and CD4+ LN cells. LN populations were recovered 14 and 28 days postinoculation. These populations were then double stained with CD8-FITC and the HA-tetramer Ab. Plots display the percentage of tetramer-positive CD8+ T cells in each group. The number of tetramer-positive cells was significantly reduced by day 28 in animals given CD8+ LN cells only (p = 0.000009). The addition of CD4+ LN cells significantly increased the number of tetramer-positive CD8+ T cells on day 28 (p = 0.00002). ▴, Mice with palpable tumors.

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These experiments demonstrate that the cotransfer of CD4+ T cells is associated with the maintenance of CD8+ T cell numbers. However, we have also shown that a high frequency of effectors is not sufficient to ensure tumor eradication (12). To determine whether these CD8+ T cells could kill specific targets we tested the functional capacity of CD8+ T cells in AB1-HA-inoculated BALB/c mice given CD8+ T cells alone or CD8+ plus CD4+ T cells at the time of tumor inoculation, using an in vivo CTL assay (15). Control animals were inoculated with tumor only. To analyze CTL effector function in vivo, target cells were differentially labeled by pulsing one population of syngeneic spleen cells with CL4 peptide and labeling them with CFSE to a relatively high fluorescence intensity. To control for Ag specificity, unpulsed syngeneic spleen cells were labeled to a lower fluorescence intensity. Identical numbers of both CFSE-labeled populations were injected i.v. into experimental animals. The dynamics of CTL effector function were measured by monitoring the loss of the CFSEhigh peak in spleen cell suspensions obtained from injected mice by flow cytometry 20 h after transfer of the fluorescently labeled targets. Controls for nonspecific elimination of either peak consisted of naive animals injected with both populations of CFSE-labeled cells. An example of one such experiment is shown in Fig. 5, where the r value, i.e., the ratio of the percentage of total cells in the CFSElow (control) peak to that in the CFSEhigh (target) peak represents the relative cytotoxic capacity of the CD8+ T cells. This assay was performed on days 14 and 28 after tumor inoculation. The expression of a HA Ag by the tumor is not sufficient to induce a specific CTL response (Fig. 5, A and B). The variation seen between AB1-HA-inoculated animals on different days was the same as that observed when CSFE-labeled splenocytes were transferred into unmanipulated syngeneic recipients (data not shown). Transferred CD8+ T cells were able to eradicate the peptide-pulsed splenocytes up to 14 days after tumor inoculation (Fig. 5,C), but this capacity was reduced to that seen in control mice by day 28 (Fig. 5,D). In contrast, CTL function was maintained in animals that had received both T cell subpopulations (Fig. 5, E and F). The results from individual animals for days 14 and 28 after tumor inoculation are shown in Fig. 6. Together these results demonstrate that on day 14 there is little discernible difference in the capacity of either experimental group to kill the peptide-pulsed targets (CD8 only: r = 48.6 ± 1.34; CD8+CD4: r = 49.6 ± 1.24; ±SEM). However, by day 28 animals that received only CD8+ T cells at the time of tumor inoculation demonstrated reduced lytic capacity (r = 27.3 ± 2.5) compared with the day 14 results as well as with those exhibited by the CD8 plus CD4 group on day 28 (r = 46.4 ± 1.7).

FIGURE 5.

A representative example of in vivo CTL effector function. Day 14 and 28 effector mice were generated by inoculating mice i.v. with HA-specific CD8+ LN cells, a mixture of HA-specific CD8+ and CD4+ LN cells, or no LN cells. At the same time these mice were inoculated s.c. with 2 × 105 AB1-HA tumor cells. To assess CTL function, syngeneic spleen cells (used as target cells) were differentially labeled with the fluorescent dye CFSE, resulting in two distinct populations exhibiting either high (CFSEhigh) or low (CFSElow) mean fluorescent intensities as determined by FACS analysis. CFSEhigh cells were pulsed with the CL4-specific peptide, whereas CFSElow cells were not pulsed with peptide. CFSEhigh and CFSElow target cells were mixed at a ratio of 1:1 and injected i.v. into all experimental groups. To test for contact-dependent, specific cytotoxic effector function, the next day spleens were removed and made into single-cell suspensions, and the elimination of CL4-pulsed CFSEhigh target cells in spleen cell suspensions derived from individual mice was detected by flow cytometry. The ratio (r) between the percentage of unpulsed cells (CFSElow) and the percentage of pulsed cells (CFSEhigh) was calculated. Data represent an example of the FACS data from three mice per time point per experimental group.

FIGURE 5.

A representative example of in vivo CTL effector function. Day 14 and 28 effector mice were generated by inoculating mice i.v. with HA-specific CD8+ LN cells, a mixture of HA-specific CD8+ and CD4+ LN cells, or no LN cells. At the same time these mice were inoculated s.c. with 2 × 105 AB1-HA tumor cells. To assess CTL function, syngeneic spleen cells (used as target cells) were differentially labeled with the fluorescent dye CFSE, resulting in two distinct populations exhibiting either high (CFSEhigh) or low (CFSElow) mean fluorescent intensities as determined by FACS analysis. CFSEhigh cells were pulsed with the CL4-specific peptide, whereas CFSElow cells were not pulsed with peptide. CFSEhigh and CFSElow target cells were mixed at a ratio of 1:1 and injected i.v. into all experimental groups. To test for contact-dependent, specific cytotoxic effector function, the next day spleens were removed and made into single-cell suspensions, and the elimination of CL4-pulsed CFSEhigh target cells in spleen cell suspensions derived from individual mice was detected by flow cytometry. The ratio (r) between the percentage of unpulsed cells (CFSElow) and the percentage of pulsed cells (CFSEhigh) was calculated. Data represent an example of the FACS data from three mice per time point per experimental group.

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FIGURE 6.

CD4+ T cells maintain the CTL capacity of the CD8+ T cells. Day 14 and 28 effector mice were generated as described in Fig. 5. The results are derived from individual animals from multiple experiments. Mice that received HA-specific CD8+ LN cells only showed a significant reduction in their lytic capacity by day 28 compared with mice that received both CD8+ plus CD4+ LN cells (p = 0.000001). ▴, Mice with palpable tumors.

FIGURE 6.

CD4+ T cells maintain the CTL capacity of the CD8+ T cells. Day 14 and 28 effector mice were generated as described in Fig. 5. The results are derived from individual animals from multiple experiments. Mice that received HA-specific CD8+ LN cells only showed a significant reduction in their lytic capacity by day 28 compared with mice that received both CD8+ plus CD4+ LN cells (p = 0.000001). ▴, Mice with palpable tumors.

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ELISPOT analysis determined that the number of IFN-γ-secreting cells after CL4 peptide stimulation of LN cells from animals that were inoculated 14 days earlier with tumor and that received either CD8+ T cells or both CD8+ and CD4+ T cells was comparable (51 ± 6 and 60 ± 16 IFN-γ-secreting cells, respectively; Fig. 7). However, those animals that received both CD8+ and CD4+ T cells 28 days after tumor inoculation demonstrated a significant increase in the number of IFN-γ-secreting cells (185 ± 43). In contrast, the number of IFN-γ-secreting cells in those animals that had received only CD8+ T cells by day 28 had a decreased number of IFN-γ-secreting cells (18 ± 8).

FIGURE 7.

CD4+ T cells maintain the IFN-γ producing capacity of CD8+ T cells. The same pool of effector cells used to determine the number of tetramer-positive cells was used in an ELISPOT assay to determine the frequency of cells secreting IFN-γ in response to CL4 peptide. Less than 10 (median = 0) spots per 106 cells were observed in the absence of peptide. Error bars indicate the SEM. Mice that received both HA-specific CD8+ and CD4+ LN cells showed a significant increase in the number of cells secreting IFN-γ by day 28 (p = 0.0000006). This experiment was repeated twice with two animals per group.

FIGURE 7.

CD4+ T cells maintain the IFN-γ producing capacity of CD8+ T cells. The same pool of effector cells used to determine the number of tetramer-positive cells was used in an ELISPOT assay to determine the frequency of cells secreting IFN-γ in response to CL4 peptide. Less than 10 (median = 0) spots per 106 cells were observed in the absence of peptide. Error bars indicate the SEM. Mice that received both HA-specific CD8+ and CD4+ LN cells showed a significant increase in the number of cells secreting IFN-γ by day 28 (p = 0.0000006). This experiment was repeated twice with two animals per group.

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BALB/c mice were inoculated with either AB1-HA or the nontransfected parental line AB1 and then injected i.v. with CD8+, CD4+, or CD8+ plus CD4+ T cells. To investigate infiltration of the tumor, the site of inoculation was excised at 5 days after tumor inoculation and examined by immunohistochemistry. This time point was chosen because the adoptive transfer of CD4+ and CD8+ T cells leads to tumor eradication. On day 5 eradication was not complete, and some tumor tissue could still be discerned (Figs. 8 and 9). At later time points tumor tissue could not be obtained reproducibly from animals in this experimental group. Tumors from AB1-HA-inoculated animals that received CD8+ T cells alone showed little if any T cell infiltration (Fig. 8, A and B). The predominant infiltrating cell was the class II macrophage, which compromised up to 50% of the tumor mass (data not shown) (20). Tumors from the parental AB1-inoculated animals showed little if any T cell infiltration regardless of the cells adoptively transferred (data not shown). Tumors from mice that were adoptively transferred with CD4+ T cells only showed CD4+, but not CD8+, T cell infiltration (Fig. 8, C and D). In contrast, there was an intense CD4+ and CD8+ T cell infiltrate within the tumor milieu from mice that had received both cell types (Fig. 8, E and F). CD4+ and CD8+ T cells were present in approximately equal numbers. In addition, when either CD4+ T cells alone or the combination of CD8+ plus CD4+ T cells were transferred, MHC class II+ cells were present throughout the tumor (Fig. 9, C and E). Again, in contrast to mice that received CD8+ T cells only, when either CD4+ T cells or CD8+ and CD4+ T cells were cotransferred, ICAM expression was also detected throughout the tumor (Fig. 9, D and F). Similar experiments were performed in which T cells were adoptively transferred into animals with established tumors. Under these circumstances, tumor regression does not occur. The pattern of infiltrating cells was similar to that seen when tumor inoculation and adoptive transfer of T cells occurred concurrently, that is, only when CD4+ and CD8+ T cells were cotransferred were both cell types found within the tumor (data not shown).

FIGURE 8.

CD8+ T cells do not infiltrate the tumor in the absence of CD4+ T cells. BALB/c mice were inoculated s.c. with 2 × 105 AB1-HA tumor cells. At the same time animals were injected i.v. with 1 × 107 HA-specific CD8+ LN cells (CL4), CD4+ LN cells (HNT), or 1 × 107 each of CD8+ and CD4+ LN cells. Five days after animals were inoculated with tumor and LN cells the injection area was sampled and prepared for immunohistologic analysis. Tumor sections from mice that received CD8+ LN cells (CL4), CD4+ LN cells (HNT), or a combination of both CD8+ and CD4+ LN cells were stained with anti-CD8 (A, C, and E) or anti-CD4 (B, D, and F) mAbs. Infiltrates were observed when either CD4+ LN cells alone or a combination of both CD8+ and CD4+ LN cells were transferred. Original magnification, ×100.

FIGURE 8.

CD8+ T cells do not infiltrate the tumor in the absence of CD4+ T cells. BALB/c mice were inoculated s.c. with 2 × 105 AB1-HA tumor cells. At the same time animals were injected i.v. with 1 × 107 HA-specific CD8+ LN cells (CL4), CD4+ LN cells (HNT), or 1 × 107 each of CD8+ and CD4+ LN cells. Five days after animals were inoculated with tumor and LN cells the injection area was sampled and prepared for immunohistologic analysis. Tumor sections from mice that received CD8+ LN cells (CL4), CD4+ LN cells (HNT), or a combination of both CD8+ and CD4+ LN cells were stained with anti-CD8 (A, C, and E) or anti-CD4 (B, D, and F) mAbs. Infiltrates were observed when either CD4+ LN cells alone or a combination of both CD8+ and CD4+ LN cells were transferred. Original magnification, ×100.

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FIGURE 9.

Class II and ICAM expression was up-regulated in mice that receive either CD4+ and CD8+ T cells or CD4+ T cells alone. As described for Fig. 8, tumor sections from mice that received CD8+ LN cells (CL4), CD4+ LN cells (HNT), or a combination of both CD8+ and CD4+ LN cells were stained with anti-MHC class II (A, C, and E) or anti-ICAM (B, D, and F) mAbs. Original magnification, ×100.

FIGURE 9.

Class II and ICAM expression was up-regulated in mice that receive either CD4+ and CD8+ T cells or CD4+ T cells alone. As described for Fig. 8, tumor sections from mice that received CD8+ LN cells (CL4), CD4+ LN cells (HNT), or a combination of both CD8+ and CD4+ LN cells were stained with anti-MHC class II (A, C, and E) or anti-ICAM (B, D, and F) mAbs. Original magnification, ×100.

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The role of CD4+ T cells in tumor eradication has received little attention in our search to understand how to better enhance the immune response to tumors and has often been overlooked in favor of CD8+ T cells alone. This focus on CD8+ T cells is particularly evident in translating research from the laboratory to the clinic, where, for example, class I peptides alone are administered to cancer patients (21, 22) or where CTL function is used as the only indicator of an anti-tumor response. Perhaps this is because while there is evidence in animal models that CD4+ T cells can be important in elimination of tumors, their full range of functions has yet to be elucidated. CD4+ T cells are important producers of IL-2 and could also contribute to the anti-tumor response by providing the appropriate cytokine milieu that favors the differentiation of cytotoxic effectors (3). More recently, it has been demonstrated that CD4+ T cells license DC, leading to the modulation of DC function via cross-linking of CD40, so that CD8+ T cell recognition of Ag on the DC leads to the induction of CTL function (4, 5, 6). It is probable that CD4+ T cell-dependent rejection of tumors can be attributed to some or all of these mechanisms. In this study using a powerful transfection-transgenic model coupled with MHC tetramer staining of tumor-specific T cells, we demonstrate other important functions of CD4+ T cells at several crucial stages of an effective anti-tumor response.

The observation that upon titration of tumor-specific T cells of either subpopulation the efficacy of tumor eradication is reduced supports the idea that one reason for the failure of tumor eradication is a relative numerical imbalance of tumor cells and T cells. The fact that so many cells are essential to ensure tumor eradication and at such a short period of time after tumor inoculation (<1 wk) probably explains why this tumor is unable to be rejected in syngeneic recipients that bear a competent repertoire for the tumor Ag. Why there is a need for a 1:1 ratio of CD8+:CD4+ T cells is unclear, as CD4+ cells themselves are unable to act as anti-tumor effectors and, as discussed below, this numerical relationship is probably not required for CTL generation. This 1:1 ratio contrasts with the data reported by Kurts et al., who showed in their transgenic model of autoimmunity that relatively few CD4+ T cells facilitated T cell-mediated organ destruction (9). The reasons for these differences have yet to be fully elucidated; however, it is clear that although the requirement for such a relatively large number of adoptively transferred T cells may partly reflect the nature of the model, a s.c. inoculation of relatively fast-growing cells, CD8+ T cells alone are not sufficient to eradicate tumor.

The inadequacy of CD8+ T cells to reject tumors was not due to an inability of the transferred CD8+ T cells to differentiate into effectors, as shown by the in vivo CTL and ELISPOT data. We interpret the generation of CTLs in this system to be consistent with a requirement for CD4+ T cell licensing of DCs. Preliminary data in our laboratory, where mice have been depleted of CD4+ T cells and inoculated with tumor, and then specific CD8+ T cells adoptively transferred, show abrogated CTL activity, demonstrating CD4+ T cell dependency (unpublished observations). Therefore in the experiments described here, when specific CD4+ T cells were not cotransferred, the endogenous repertoire was sufficient to aid in the generation of these effectors. However, the induction of CTLs is clearly not enough to induce tumor eradication as shown here in the adoptive transfer experiments. In fact, a number of studies have demonstrated that CTL activity does not necessarily correlate with the ability to eradicate tumor. In our own system, 50% of transgenic mice expressing an HA-specific, class I-restricted TCR cannot reject the AB1-HA tumor (12). Wick and colleagues showed that anti-Ld TCR transgenic mice were unable to reject an allogenic tumor (23). More recently, Sarma and colleagues have shown that TCR transgenic mice cannot reject tumors expressing the endogenous tumor Ag P1A (24). The question is why, despite demonstrable CTL activity in these studies, CTL activity is not effective against the target tumor in vivo, i.e., why is the CTL effector function restrained?

We show that CD4+ T cells have at least three functions subsequent to licensing events during an effective anti-tumor response. The first of these is maintenance of the CTL pool. Mice that received both CD4+ and CD8+ T cell populations maintained higher numbers of tetramer-positive cells, within a tighter range, over the period of the experiment than those that received CD8+ T cells only. These results are consistent with our previous finding that a greater number of class I-restricted, HA peptide-reactive cells are recoverable from animals adoptively transferred with both CD8+ and CD4+ populations than from CD8+ T cells alone (25). One can envisage that this potentiation of CD8 numbers is particularly necessary when the immune system is responding to a target that has the capacity to multiply or regenerate, as is the case with tumors and viruses. The ability of CD4+ T cells to maintain the CD8 pool has been described in other models, such as the cross-presentation of a transgenic self Ag and chronic viral infection (9, 26). In the transgenic autoimmune model in which OVA is expressed as a model self Ag in the islets of the pancreas, it has been demonstrated that specific CD4+ T cells moderate the loss of CD8+ T cells that occurs due to deletion after cross-presentation of Ag on APCs (9). The exact mechanism by which CD4+ T cells maintain CD8+ T cell numbers in our model is unknown. It does not appear to be related to modulation of the Fas-Fas ligand system (12), which has been implicated in the cross-presentation model described by Heath and colleagues. A recent paper has shown that anti-CTLA4 treatment increases the number of T cells in the draining LNs after Ag stimulation, probably by inhibiting inhibitory signals associated with binding of CTLA4 to B7 molecules (27). It was also shown that this phenomenon is CD4+ T cell independent. The authors hypothesize that this accumulation of CD8+ T cells due to anti-CTLA4 administration leads to the tumor regression observed in some studies (28). Anti-CTLA4 treatment of AB1-HA-inoculated mice also results in tumor regression (A.L.M., R.A.L., B.W.S.R., and B.S., unpublished observations), and it will be interesting to determine in that system whether an accumulation of tumor-specific cells can be observed and thereby support the hypothesis of McCoy and colleagues (27).

The second postlicensing function of CD4+ T cells is the maintenance of CTL function. In our experiments there was a correlation between CD8+ T cell number and CTL function. Both parameters remained high when CD4+ T cells were cotransferred. Although this may not be immediately surprising, and one might argue that if the numbers are higher, then CTL will also be higher, in the literature there are a number of models in which cells may be detected but are apparently nonfunctional. For example, in some viral infections CD4+ T cells are not necessarily required for the induction of primary CTL responses (29, 30), but in the absence of CD4+ T cells in the chronic phase of disease, although specific CD8+ T cells are still present, CTL activity is diminished (26). How CD4+ T cells act to ensure CTL function is not clear, but maintenance of function is important to ensure a pool of reactive cells that have the potential to eradicate their target.

Even so, large numbers of CTL effectors are not sufficient for tumor eradication. Thus, a third and very important postlicensing function of CD4+ T cells is allowing CD8+ T cell infiltration of the tumor. Because CD8+ T cells transferred alone do not traffic to the tumor, one can now offer a reason for why CTL cells can be generated but have no therapeutic effect. Onrust and colleagues have also demonstrated that Ag-specific CTLs cannot enter a tumor despite an inflammatory site, composed of the same CTLs, occurring directly adjacent to the tumor tissue (31). This inability of specific T cells to enter the tumor correlates with a differential expression of endothelial expressed integrins. Of particular interest in these studies was L-selectin expression, which was down-regulated on the vessels at the tumor site, but not at the site of inflammation. L-selectin was initially characterized as a homing receptor for lymphocytes. Previously, Ando and colleagues (32) had shown that CTLs do not necessarily enter tissues or organs that express specific Ag. In their study the target tissues were not sites of tumor growth or inflammation, so one could argue that there was no reason to expect T cell trafficking to a site of Ag expression. However, in these studies it was found that CTLs could enter tissues in which the constituent blood vessels were characterized by discontinuous endothelium and the absence of a basement membrane. These authors proposed that other components of the immune system, such as CD4+ T cells, might act to modify or disrupt the microvasculature and allow CTL entry of otherwise normal vessels during processes such as infection. We have also hypothesized that the absolute requirement for CD4+ T cells in rejecting the AB1-HA tumor is not solely as helper cells, but also as potential modifiers of the tumor milieu (25). In this paper we showed that after activation in the draining LN, CD4+ T cells, which cannot specifically kill the tumor, are not restricted from trafficking to the tumor and, in fact, allow CD8+ T cells to infiltrate the tumor. How do the CD4+ T cells influence the trafficking pattern of CD8+ T cells? CD4+ T cell trafficking is associated with the up-regulation of class II and the induction of ICAM expression within the tumor site, although the mechanism by which CD4+ T cells contribute to this change, either directly or indirectly, has not yet been established. Perhaps the up-regulation of inflammatory-associated molecules coincides with the activation or modification of the tumor vasculature as suggested by Ando and colleagues (32).

Alternatively, rather than CD4+ T cells altering the CD8 trafficking patterns allowing CTLs to enter the tumor, we may be observing the retention and accumulation of CD8+ T cells. A current paradigm is that naive cells remain within the lymphoid tissues, whereas activated cells migrate from LNs and spleen to pass through tissues in search of Ag. CD8+ T cells in tumor-inoculated mice fulfill these criteria for migratory capacity, as they have encountered Ag in the draining LNs (10), become activated, and differentiate into CTL effectors. Our previous studies have demonstrated that these CTL effectors cannot lyse tumor cells in vitro unless the cells have been exposed to IFN-γ (12). This lack of target recognition is not due to low level class I expression or to an intrinsic resistance to lysis (12). Either the 2- to 3-fold increase in surface class I expression that results from IFN-γ treatment is sufficient to increase the number of specific MHC-peptide complexes above the threshold required for recognition or IFN-γ alters the protein processing machinery, allowing more of the peptide to be loaded into the class I molecule. Thus, in the absence of recognition, CD8+ T cells may pass through the tumor and die due to normal attrition following activation or may traffic to and move into the tumor but are killed, either through intrinsic mechanisms or due to tumor-derived factors. In either case the histological picture would be the same, that is little or no observable CD8+ T cell infiltrate. We postulate that the ability of CD4+ T cells to traffic to the tumor allows them to secrete cytokines at the site, modifying all cells, including the tumor cells. Thus, CD4+ T cells enter the tumor site, where they can secrete IFN-γ and “reveal” the epitope required for CTL recognition, which, in turn, leads to tumor cell lysis and the maintenance of an inflammatory loop that ensures tumor eradication. Because the cotransfer of CD4+ T cells leads to a greater number of CD8+ T cells secreting IFN-γ, the CD8+ T cells once in the tumor could also contribute to IFN-γ production. A broader role for CD4+ T cells has recently been described in a vaccination model of tumor eradication where CD4+ T cell infiltration correlated with increased infiltration by other cells, including eosinophils and macrophages that produced both superoxide and NO (33).

In conclusion, our model has enabled us to clarify the role that CD4+ T cells have in anti-tumor immunity. We show that CD4+ T cells are not merely IL-2 factories for CD8+ effectors or potentiators of CTL induction via the licensing of DCs, but that CD4+ T cells also act to promote tumor eradication in at least three additional ways: by maintaining both numbers and cytotoxic capacity of the CD8+ T cells, by enabling CD8+ T cells to traffic to and/or remain within the tumor, and by altering tumor expression of key MHC and accessory molecules. The combination of these three mechanisms increases the chances of a tumor-CD8+ T cell interaction and thereby facilitates tumor eradication. These data have important implications for the design of tumor immunotherapy protocols. Clearly, such strategies must recruit and maintain anti-tumor CD4+ T cells, so therapies using class I epitopes alone will be most effective if agents that boost CD4+ T cell activity can be coadministered.

We thank Drs. L. Sherman and D. Lo for providing breeding stock for TCR transgenic mice, and R. Himbeck for assistance with breeding the TCR transgenic mice. We also thank Dr. W. Gerhard for providing the H18 hybridoma.

1

This work was supported by the National Health and Medical Research Council of Australia (Grant 961329). A.M. is a recipient of the Dora Lush postgraduate scholarship (National Health and Medical Research Council).

3

Abbreviations used in this paper: DC, dendritic cell; HA, hemagglutinin; ELISPOT, enzyme-linked immunospot; MM, murine malignant mesothelioma; LN, lymph node.

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