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The Journal of Immunology, 2005, 175: 6551-6559.
Copyright © 2005 by The American Association of Immunologists

The Cooperation between Two CD4 T Cells Induces Tumor Protective Immunity in MUC.1 Transgenic Mice1

Mara Gerloni, Paola Castiglioni and Maurizio Zanetti2

Laboratory of Immunology, Department of Medicine and Cancer Center, University of California-San Diego, La Jolla, CA 92093


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunity and tumor protection in mice transgenic for human MUC.1, a glycoprotein expressed in the majority of cancers of epithelial origin in humans, were induced by vaccination with B lymphocytes genetically programmed to activate MUC.1-specific CD4 T cells. Their activation required a functional cooperation between two Th cells, one specific for a self (MUC.1) and the other for a nonself T cell determinant. The immunological switch provided by Th-Th cooperation was sufficient to induce MUC.1-specific CD4 and CD8 T cell responses in MUC.1-transgenic mice, and protect them permanently from tumor growth. CD4 T cells specific for MUC.1 lacked cytolytic function, but produced IFN-{gamma} upon restimulation with Ag. We conclude that immunity against tumor self-Ags and tumor protection can be regulated exploiting an inherent property of the immune system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tumors use various strategies to evade immune surveillance and resist immunotherapy. For tumor Ags that are true self-Ags, these factors include an intrinsic poor immunogenicity, immune tolerance/anergy, down-regulation of the MHC molecules, ignorance, secretion of suppressive cytokines, suppressor-regulatory T cells, and neutralization of immune T cells by Fas ligand expression. Some of these factors depend on tumor-host interactions, because tumors are composed of neoplastic cells intimately intermixed with nonneoplastic cells of the host, whereas others reflect more directly acquired functional defects of the immune system. Activation of T cells requires two signals (Ag and costimulation) where lack of costimulation prevents activation and promotes anergy (1). Therefore, in principle, the ability to generate immunity against tumor Ags rests on a simple immunological principle enabling the host to overcome the poor immunogenicity of tumor Ags as well as self-tolerance. Promising new approaches focused on the manipulation of costimulatory molecules have shown that CTLA-4 blockade or OX40 ligation may enhance priming of antitumor T cell responses in nontolerant mice (2, 3) or break peripheral self-tolerance in nontumor model systems (4, 5). In contrast, with the exception of peptide analogues (6, 7), little has been proposed with respect to modifications of the Ag that could strengthen signal 1 and also heighten signal 2.

A response to otherwise subimmunogenic determinants can be induced by triggering a functional cooperation between two CD4 T help cells, Th-Th cooperation or help for helpers (8). As demonstrated previously, this phenomenon requires associative recognition of Ag (9) and is consistent with a three-cell model in which the T cell response to a dominant determinant results in the activation of the APC via up-regulation of costimulatory molecules, which, in turn, enables the presentation of and the response to, a subimmunogenic determinant by a second CD4 T cell (8).

In this report we show that T cell immunity to MUC.1, an Ag expressed in the majority of tumors of epithelial origin, can be induced by injection of syngeneic B lymphocytes as APCs genetically programmed (10) to trigger Th-Th cooperation in vivo. Specifically, we demonstrate that mice transgenic (Tg)3 for the human MUC.1 Ag (11) that are tolerant to MUC.1 and unable to reject MUC.1-expressing tumors (11, 12, 13) respond to the immunization with Tg B lymphocytes mounting durable tumor-protective T cell immunity. The key findings of the study can be summarized as follows: 1) CD4 T cell immunity can be induced by active immunization against a single CD4 T cell determinant of a tumor self-Ag by gauging the composition of the immunogen to enable Th-Th cooperation; 2) genetically programmed B lymphocytes are effective APCs and induce long-lasting tumor immunity; and 3) antiself CD4 T cells induced by Th-Th cooperation jump-start the activation of Ag-specific CD8 T cells specific for the same tumor Ag. These data are relevant to understanding the role of CD4 T cells in tumor immune surveillance and in the generation of protective tumor immunity.


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

The MUC.1-Tg mice colony was established at University of California-San Diego from founders obtained from Dr. S. Gendler (Mayo Clinic, Scottsdale, AZ). MUC.1-Tg-positive mice were identified by PCR analysis as previously described (11). C57BL/6 mice (H-2b) were purchased from Jackson ImmunoResearch Laboratories. All experimental animals were housed at the University of California-San Diego animal facility under standard pathogen-free conditions.

Plasmid DNA, synthetic peptides, and tumor cell lines

Plasmid {gamma}1NV2VTSA3, {gamma}1VTSA3, and {gamma}1NV2NA3 carry chimeric H chain genes under the control of a B cell promoter and are formed by the joining of a human {gamma}1 constant (C) region gene with a rearranged murine variable region gene engineered in the CDRs to code for heterologous Th cell determinants as previously described (8). The superscript numbers identify the CDR in which a given peptide is expressed, e.g., {gamma}1NV2VTSA3 codes for the -NVDP- (NV) peptide in CDR2 and the -VTSA- peptide in CDR3. Each plasmid also carries the enhanced GFP gene inserted at the C terminus of the {gamma}1 C region. Plasmid pSVneo is the original plasmid forming the backbone of the pNeo {gamma}1 vector without the human {gamma}1 C region (14) and was one of the controls used in the protection experiments shown in Fig. 3. Plasmid DNAs were purified using a Megaprep kit (Qiagen) and were stored at –20°C until use. Synthetic peptides VTSAPDTRPAP (-VTSA-), TSAPDTRPA (-VTSA-; 9-mer) NANP NVDP NANP (-NVDP-), and OVA323–339 (ISQAVHAAHAEINEAGR) were synthesized at the Peptide Chemistry Facility (California Institute of Technology). B16-MUC.1 and B16Neo murine (C57BL/6) melanoma tumor lines transfected with a MUC.1 cDNA or control expression vector (11) were gifts from Dr. S. Gendler. These cell lines were maintained in DMEM with 10% FBS, penicillin (50 U/ml), and streptomycin (50 µg/ml), supplemented with 300 µg/ml G418. Tumor cells were treated overnight at 37°C with 200 ng/ml IFN-{gamma} before use in the CTL assay.



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  		FIGURE 3. Development of protective, tumor-specific immunity in vivo in MUC.1-Tg mice after Th-Th cooperation. Top panel, MUC.1-Tg mice were immunized on days 0 and 21 with 5 x 103 Tg B lymphocytes as indicated. Four days after the booster injection, mice were challenged with either 2 x 104 B16-MUC.1 or B16neo tumor cells, and tumor growth was evaluated over time. The number of mice is indicated in parentheses. Survival curves were constructed using the Kaplan-Meier method. Data are cumulative for three independent experiments. Significance (p < 0.00005) was calculated using Fisher’s exact test, based on binomial distribution, where {alpha} is reduced according to the Bonferroni criterion (0.05/number of tests). Bottom panel, Rechallenge experiment. Mice that survived the first tumor challenge were subject to a second challenge with either 2 x 104 B16-MUC.1 or B16neo tumor cells 70 days later. Survival was calculated as described above. The number of mice used is indicated in parentheses. The procedures used are in accordance with National Institutes of Health regulations on laboratory animal welfare and approved institutional protocols.

 
Spontaneous transgenesis of B lymphocytes

This was performed according to the procedure described previously (10). Briefly, spleen cells (4 x 106) in 200 µl of PBS without Ca2+ and Mg2+ (CellGro) were incubated with 25 µg of plasmid DNA for 1 h at 37°C together with 5 µg of PMACS Kk plasmid (Miltenyi Biotec), which codes for a truncated mouse H-2 Kk molecule as a selectable cell surface marker. The cells were then washed and incubated in RPMI 1640 medium (Irvine Scientific) supplemented with HEPES buffer, glutamine, and 10% FCS at 37°C in 5% CO2 atmosphere overnight. The next day, the cells were harvested, and Tg cells were enriched by positive selection on a column MS+/RS+ mounted on the magnetic field of a MACS separator (Miltenyi Biotec). Tg cells were enumerated by flow cytometry on a FACSCalibur (BD Biosciences).

In vivo immunization and tumor challenge experiments

MUC.1-Tg mice were immunized by tail vein injection with a suspension of 5 x 103 Tg B lymphocytes. In the tumor protection experiments, the injection of Tg B lymphocytes was repeated on day 21. Four days after the second immunization, mice were challenged with either 2 x 104 B16-MUC.1 or B16neo tumor cells inoculated s.c. between the scapulae. Mice were inspected daily, and tumor growth was recorded. Mice were killed when tumors reached 2 cm in diameter. In the rechallenge experiment, MUC.1-Tg mice that survived the first challenge were left to rest for 70 days and then rechallenged with either 2 x 104 B16-MUC.1 or B16neo tumor cells. The procedures used are in accordance with National Institutes of Health regulation on laboratory animal welfare according to approved institutional protocols.

Processing of VTSA from full-length MUC.1

To demonstrate processing of the -VTSA- peptide from full-length MUC.1, the following experimental designed was used.

Preparation of dendritic cells (DC) pulsed with apoptotic B16-MUC.1 cells. Bone marrow-derived DC were cultured with recombinant GM-CSF and IL-4 (1000 U/ml; BD Pharmingen) for 6 days, followed by overnight incubation with apoptotic B16-MUC.1 or B16neo tumor cells at a 1:5 (DC to tumor cell) ratio. Apoptosis was induced by mitomycin-C treatment (100 µg/ml for 30 min at 37°C), followed by culture at 3–5 x 105 cells/ml for 48 h in RPMI 1640 containing 0.1% FCS. As a positive control, on day 7 of culture, DC were pulsed with the -VTSA- peptide (13-mer) for 1 h at 3°C. DC were irradiated (3000 rad), washed, and added to the CD4 T cell culture.

Preparation of immune CD4 T cells. Mice immunized by injection of 5000 B lymphocytes Tg for {gamma}1NV2VTSA3 twice on days 0 and 21 were killed 48 h after the booster injection. Spleens were pooled, and CD4 T cells were negatively selected using the StemSep kit (StemCell Technologies) comprising a mixture of biotinylated Abs to the following surface Ags: CD11b, CD45R, CD8, TER119, and Ly-6G. The recovered CD4 T cells were then cultured in a 96-well plate (2 x 105 cell/well) with bone marrow-derived DC at a ratio of 40:1.

Determining Ag presentation. The ability of immune CD4 T cells to be restimulated by MUC.1-pulsed DC was assessed by [3H]thymidine incorporation after 72 h of culture. Culture supernatants were harvested 40 h after initial seeding, and IFN-{gamma} in the culture supernatants was detected by ELISA.

Generation and analysis of a CD4 T cell line

A CD4 T cell line specific for -VTSA- was generated in MUC.1-Tg mice immunized twice with {gamma}1NV2VTSA3-Tg lymphocytes (5 x 103). Four days after the second immunization, mice were killed, and spleens were harvested. CD4 T cells were negatively enriched from pooled spleen cells using anti-CD8 and anti-CD19 magnetic beads following the manufacturer’s instructions (Miltenyi Biotec). Enriched CD4 T cells were restimulated weekly with irradiated (100 Gy) bone marrow-derived DC pulsed with the -VTSA- peptide at a responder-to-stimulator ratio of 10:1. IL-2 (Chiron) was added to a final concentration of 6 U/ml. After 2 wk, CD4 T cells were restimulated with -VTSA- peptide (10 µg/ml) or control OVA323–339 peptide (1 µg/ml) for 6 h at 37°C. Intracellular IFN-{gamma} detection was performed using the commercially available Cytofix/Cytoperm kit (BD Pharmingen) according to the manufacturer’s directions. CD4+/IFN-{gamma}-producing T cells were identified by cell surface staining using the FITC-conjugated Ab anti-CD4 (clone L3T4) and PE-conjugated Ab anti-IFN-{gamma} (clone XMG1.2; BD Pharmingen).

T cell assays

Proliferation assays were performed as previously described (8). Briefly, spleen cells were harvested on day 14 and cultured for 3 days at 37°C in the presence of -VTSA- or -NVDP- peptides (5 µg/ml). Tests were run in triplicate. Results are expressed as cpm or as the stimulation index, calculated as the ratio of (cpm of cells cultured in the presence of synthetic peptide)/(cpm of cells cultured in the absence of peptide). CD8 CTL were detected in a conventional 51Cr release assay (15). Briefly, 21 days after immunization, spleen cells were isolated and restimulated in vitro using the -VTSA- peptide (5 µg/ml) for 6 days. Cultured cells were incubated at the indicated E:T cell ratio with B16-MUC.1 cells, B16neo cells, EL-4 cells pulsed with -VTSA- peptide, or EL-4 cells pulsed with the 9-mer -VTSA- peptide as target cells. CD4 CTL assay was performed after five rounds of in vitro restimulation. Cultured cells were incubated at the indicated E:T cell ratios with B16-MUC.1 cells, B16neo cells, and B6-2 cells pulsed with either the -VTSA- or the OVA323–339 peptide as targets. Results are expressed as the percentage of specific lysis and were determined as follows: [(experimental cpm – spontaneous cpm)/(maximum cpm – spontaneous cpm)] x 100.

Detection of cytokines

IL-2, IL-4, and IFN-{gamma} were detected in culture supernatants harvested 40 h after initial seeding using the OptEIA mouse cytokine sets (BD Pharmingen).

Statistical analysis

Survival curves were constructed using the Kaplan-Meier method. Significance was calculated by Fisher’s exact test, based on binomial distribution, where {alpha} is reduced according to the Bonferroni criterion (0.05/number of tests).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Tg B lymphocytes trigger Th-Th cooperation in vivo

First we established that Ag-presenting B lymphocytes Tg for plasmid {gamma}1NV2VTSA3 could generate an anti-MUC.1 CD4 T cell response attributable to Th-Th cooperation in vivo. To this end, C57BL/6 mice were immunized by i.v. injection of primary syngeneic B lymphocytes rendered Tg for {gamma}1NV2VTSA3. This codes for the subimmunogenic MUC.1 determinant VTSAPDTRPAP (-VTSA-) and a dominant Th cell determinant NANPNVDPNANP (-NVDP-) from a malaria parasite Ag (8). In this approach to immunization, B lymphocytes serve as APCs for both Ag synthesis and presentation (16) and can be considered the sole APC because contaminant DC represent <0.2%. The participation of host APCs in CD4 T cell priming is ruled out by experiments showing that Tg B lymphocytes from MHC class II knockout mice fail to immunize (10). Previously, we also showed that after spontaneous transgenesis ex vivo, naive B lymphocytes up-regulate costimulatory molecules and become de facto functional APCs (16).

C57BL/6 mice immunized with B lymphocytes Tg for {gamma}1NV2VTSA3 generated a vigorous (≥30 stimulation index) response against both the -NVDP- and -VTSA- determinants (Fig. 1A). Immunization with lymphocytes Tg for -VTSA- only did not generate a detectable response, proving that, as demonstrated previously using direct DNA immunization (8) and -VTSA- peptide in IFA (our unpublished observations), -VTSA- per se in unable to induce a CD4 T cell response. Because immunity was generated using B lymphocytes Tg for the dual-epitope transgene {gamma}1NV2VTSA3, the effects observed in vivo are consistent with those described for Th-Th cooperation (8). Furthermore, the experiment demonstrates that a key requirement in Th-Th cooperation is that the two Th determinants (e.g., -NVDP- and -VTSA-) need to be presented by the same APC (linked recognition of Ag) to enable the antiself immunity.



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  		FIGURE 1. Activation of Th-Th cooperation by immunization with Tg B lymphocytes. A, CD4 T cell response induced via Th-Th cooperation in C57BL/6 mice. Mice were immunized by single injection of 5 x 103 spleen B lymphocytes Tg for {gamma}1NV2VTSA3. B lymphocytes Tg for plasmid {gamma}1VTSA3 served as the control. Values represent the mean stimulation index of four mice per group ± SD. B, Titration of the two CD4 T cell responses. C57BL/6 mice were immunized by a single injection of B lymphocytes Tg for {gamma}1NV2VTSA3. Two mice per group received an injection of decreasing amounts (10,000 to 1) of Tg B lymphocytes. Mice were killed on day 14, and spleen CD4 T cells were restimulated in vitro with the -VTSA- ({blacktriangleup}) or the -NVDP- ({square}) peptide. Tests were run in triplicate, and proliferative responses were expressed as cpm. The data shown are from a representative experiment of two independent experiments performed, yielding a similar dose-response profile.

 
The responses against the two Th cell determinants followed a similar dose-response curve (Fig. 1B), suggesting that the thresholds of activation for the two responses are similar and that T cell activation by the nonself-determinant dictates the threshold of T cell activation against the subimmunogenic MUC.1 determinant.

B lymphocytes genetically programmed to trigger Th-Th cooperation immunize MUC.1-Tg mice

Next, we determined whether MUC.1-specific CD4 T cell immunity could be induced in mice Tg for the human MUC.1 Ag (11). MUC.1-Tg mice are reportedly tolerant to MUC.1 and unable to reject MUC.1-expressing tumors (11, 12, 13). In these mice, Abs against MUC.1 do not provide immunity to tumor (12). MUC.1-Tg mice given a single injection of {gamma}1NV2VTSA3-Tg lymphocytes developed a CD4 T cell proliferative response against -VTSA- (Fig. 2). No response was generated by injecting lymphocytes Tg for plasmid control {gamma}1VTSA3, suggesting that the CD4 T cell response against -VTSA- after immunization with lymphocytes Tg for {gamma}1NV2VTSA3 is due to the "help for helpers" effect of Th-Th cooperation. Additional experiments were performed to ensure that MUC.1-Tg mice are unable to respond against -VTSA- or MUC.1 using a variety of immunization conditions. As shown in Table I, the synthetic peptide -VTSA- in immunological adjuvant with or without helper peptide -NVDP-, MUC.1+ B16 tumor cells, or B lymphocytes Tg for {gamma}1VTSA3, all failed to induce a detectable CD4 or CD8 T cell response to the -VTSA- peptide. Thus, the CD4 T cell response against -VTSA- in MUC.1-Tg mice requires Th-Th cooperation, which is based on linked recognition of endogenously processed Ag. A helper effect by epitopes of the backbone H chain transgene was similarly ruled out. Consistent with previously published data on the type of cytokines produced after immunization with Tg B lymphocytes (10), we found that after restimulation in vitro with the -VTSA- peptide, only cultures from mice immunized with the dual-determinant transgene produced IL-2, IFN-{gamma}, and IL-4 (Table II), making this a Th0 response.



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  		FIGURE 2. Th-Th cooperation breaks CD4 T cell tolerance to the MUC.1 Ag. Groups of four MUC.1-Tg or C57BL/6 mice were immunized by single injection of 5 x 103 Tg B lymphocytes as indicated. B lymphocytes Tg for plasmid {gamma}1VTSA3 served as the control. The data shown are from a representative experiment of two independent experiments performed. Results are expressed as stimulation indexes.

 

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  		Table I. MUC.1 Tg mice are tolerant to MUC.1

 

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  		Table II. Cytokines produced by T cells in response to VTSA

 
Despite the fact that CD8 T cell tolerance to MUC.1 has been broken in monkeys after DNA vaccination (17), and in MUC.1-Tg mice immunized with armed DC (18, 19, 20), the induction of CD4 T cells specific for a MUC.1 peptide in MUC.1-Tg mice has not been shown previously.

Active CD4 T cell immunity to MUC.1 protects against tumor

The possibility of generating specific anti-MUC.1 immunity in MUC.1-Tg mice prompted us to investigate whether this was sufficient to protect from MUC.1-expressing tumors in vivo. We used transplantable murine B16 (H-2b) melanoma cells transfected with the human MUC.1 cDNA (12). MUC.1-Tg mice injected with 2 x 104 B16-MUC.1 tumor cells s.c. develop tumors in 100% of mice in ~20 days. In a pilot experiment we found that only two of eight MUC.1-Tg mice were tumor protected if immunized by a single injection of Tg lymphocytes, suggesting that priming per se is insufficient to establish protective immunity (data not shown). Therefore, protection experiments were conducted in mice immunized twice at a 3-wk interval. Four days after the second injection, mice were challenged with B16-MUC.1 tumor cells or B16neo cells as a control. As expected, none of the 12 mice immunized with B lymphocytes Tg for -VTSA- only and challenged with B16-MUC.1 tumor cells were protected. In contrast, all 23 mice vaccinated with B lymphocytes Tg for {gamma}1NV2VTSA3 and challenged with B16-MUC.1 cells survived tumor free (Fig. 3, top panel). Mice challenged with B16neo cells were not protected, suggesting that protection is Ag specific. Protection was also not seen in mice immunized with B lymphocytes Tg for control plasmids and subsequently challenged with B16-MUC.1 tumor cells.

The longevity of protection was assessed in protected mice allowed to rest for 70 days after the initial tumor challenge and then subsequently rechallenged with either 2 x 104 B16-MUC.1 or B16neo cells. We reasoned that if protection was Ag specific, only mice rechallenged with B16-MUC.1 cells would be protected. In line with this prediction, all mice rechallenged with B16-MUC.1 cells survived tumor free for the duration of the observation period (160 days), whereas mice rechallenged with B16neo cells died, as did naive mice challenged with the B16-MUC.1 cells (Fig. 3, bottom panel). Taken together, these results demonstrate unambiguously that protective immunity against a tumor self-Ag enabled by Th-Th cooperation is long lasting. It is noteworthy that the absence of protection against the B16neo challenge implies that no epitope spreading had occurred as a result of the first tumor injection.

Generation of CTL during priming of CD4 T cells against MUC.1

Because Th-Th cooperation specifically expands CD4 T cells reactive with -VTSA- (8), we assumed that CD4 T cells may be involved in the mechanism of protection. However, although there exist only a few studies on CD4 T cell-mediated tumor protection (21, 22, 23, 24, 25), the vast majority of reports emphasized the role of CD8 CTL. Therefore, we considered the possibility that MUC.1-specific CTL were also induced as a result of the immunization. MUC.1-Tg mice immunized with {gamma}1NV2VTSA3-Tg lymphocytes developed specific CTL that lysed B16-MUC.1, but not B16neo, cells in a 4-h 51Cr release assay before (Fig. 4, a–c) and after (Fig. 4, d and e) booster. CTL from MUC.1-Tg mice also lysed EL-4 (H-2b) T lymphoma cells pulsed with the 9-mer peptide TSAPDTRPA, but not the 11-mer VTSAPDTRPAP, demonstrating that CTL are specific for a peptide embedded in the 11-mer structure expressed in the transgene. The protective T cell response was originated in response to immunization and not after contact with tumor cells, because the induction of CTL preceded tumor implantation (Fig. 4, upper panels). Therefore, CTL priming probably results from processing of the 11-mer -VTSA- peptide, which generated a 9-mer peptide recognized by CD8 T cells. Examples of both CD4 and CD8 epitopes within the same peptide structure have been reported for self (26, 27) and nonself (28) Ags. Finally, while the characteristics and specificity of killing rule out lysis by NK cells, a role for cross-priming by DCs is highly unlikely, because Tg B lymphocytes induce both CD4 and CD8 T cell responses in mice lacking functional DCs (10).



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  		FIGURE 4. Induction of MUC.1-specific CTL responses after Th-Th cooperation. a–c, MUC.1-Tg mice were immunized once with 5 x 103 B lymphocytes Tg for {gamma}1NV2VTSA3 (a), {gamma}1VTSA3 (b), or pSVneo (c). The data are presented as the percentage of specific lysis ± SD of four mice per group. Similar results were obtained in two additional experiments. d and e, MUC.1-Tg mice were immunized twice with 5 x 103 B lymphocytes Tg for {gamma}1NV2VTSA3 (d) or {gamma}1VTSA3 (e). Spleen cells were isolated on day 4 after the second immunization and tested as described above. The data are presented as the percentage of specific lysis ± SD of four mice per group. Similar results were obtained in two additional experiments. In both priming and booster experiments, four different target cells were used in each instance as indicated.

 
CD4 T lymphocytes against MUC.1 are noncytolytic, but produce IFN-{gamma}

Next, we investigated the role of CD4 T lymphocytes in tumor cell lysis as a potential mechanism of protection in vivo. In the first experiment we purified CD4 T cells from MUC.1-Tg mice immunized with {gamma}1NV2VTSA3-Tg lymphocytes using anti-CD4 magnetic beads, restimulated them in culture for 5 days, and then tested them in a 51Cr release assay against B16-MUC.1 target cells. CD4 T cells did not kill even at the highest E:T cell ratio (Fig. 5A, left panel), whereas whole spleen T cells from the same mice caused marked lysis (Fig. 5A, right panel). In a second experiment we generated a VTSA-specific CD4 T cell line again from MUC.1-Tg mice immunized with {gamma}1NV2VTSA3-Tg lymphocytes. Cultured CD4 T cells produced IFN-{gamma} after in vitro restimulation with the -VTSA- peptide (Fig. 5B, upper panels), but not ex vivo after priming (Fig. 5B, lower panels). The CD4 T cell line was unable to lyse B6-2 cells, a class II+ nonsecreting murine B cell hybridoma (H-2d,b), pulsed with the 9-mer -VTSA- or B16-MUC.1 tumor cells (Fig. 5C), proving conclusively that VTSA-specific CD4 T lymphocytes lack lytic properties, but secrete IFN-{gamma}.



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  		FIGURE 5. Functional characteristics of anti-VTSA CD4 T cells. A, Purified CD4 T cells from MUC.1-Tg mice immunized once with B lymphocytes Tg for {gamma}1NV2VTSA3 were tested in 51Cr release assay against B16 MUC.1 target cells (left panel). Results refer to the percentage of lysis by CD4 T cells from three individual mice tested at two E:T cell ratios. Lysis by whole spleen lymphocytes from the same mice (right panel) was used as a control. Each column corresponds to a single mouse. B, Specific activation of CD4 T cells. IFN-{gamma}-specific CD4 T cells were detected after 2-wk culture in the case of the CD4 T cell line (upper panels) or ex vivo in the case of spleen lymphocytes 21 days after priming (lower panels). In both instances, T cells were restimulated for 6 h in vitro with the -VTSA- peptide (right panels) or the OVA323–339 peptide, used as control (left panels). Results are expressed as the percentage of IFN-{gamma} -producing CD4 T cells. C, Cytotoxic assay using CD4 T cells. The cytotoxic activity of CD4 T cell line was tested in 5-wk cultures in a conventional 51Cr release assay. Cultured cells were incubated at the indicated E:T cell ratios with B16-MUC.1 cells, B16neo cells, B6-2 cells pulsed with the 9-mer -VTSA- peptide, or B6-2 cells pulsed with control OVA323–339 peptide, as target cells. Results are expressed as the percentage of specific lysis.

 
Processing and presentation of VTSA from full-length MUC.1

To ensure that the -VTSA- sequence is processed and presented from full-length MUC.1, an experiment was performed to assess the ability of DC pulsed with apoptotic B16 MUC.1 cells to restimulate CD4 T cells from MUC.1-Tg mice immunized with {gamma}1NV2VTSA3-Tg lymphocytes. We reasoned that restimulation of VTSA-reactive CD4 T cells would constitute proof that the -VTSA- sequence is processed and presented from full-length MUC.1. As shown in Fig. 6, DC pulsed with apoptotic B16 MUC.1 cells restimulated CD4 T cells generated in vivo. Both T cell proliferation (left panel) and production of IFN-{gamma} (right panel) were in a range comparable to that in DC pulsed with previously processed VTSA peptide, used as a positive control.



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  		FIGURE 6. CD4 T cells induced by immunization against VTSA are restimulated by processed MUC.1 Ag. CD4 T cells from immunized mice were cultured with DC pulsed with either apoptotic tumor cells or the 11-mer -VTSA- peptide. Left panel, The specific proliferative response is shown. Results are expressed as the mean cpm ± SD of triplicate cultures. Right panel, IFN-{gamma} production of the same cultures. Supernatants were collected after 40 h of culture. Experiments were performed using a DC to CD4 T cell ratio of 1:40.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We demonstrate that Th-Th cooperation is an effective way to elicit protective T cell immunity against a bona fide tumor self-Ag. There are two aspects to the significance of these findings. One is that CD4 T cell immunity against a tumor self-Ag and tumor protection were induced strictly by active immunization in vivo. The second is that this was accomplished by enlisting the response of tumor (self)-specific CD4 T cells through the help provided by CD4 T cells specific for a nonself-determinant. This mechanism is consistent with predictions based on associative recognition of linked T cell determinants (29) and validates the basic tenet of the two-signal model for T cell activation (9, 30). We had previously demonstrated that the simultaneous injection of plasmids coding for -VTSA- and -NVDP- in different sites of the same organ did not generate immunity to MUC.1 (8), ruling out that -NVDP- may serve merely as adjuvant. In this study we demonstrate that immunization of MUC.1-Tg mice with 1) the synthetic peptide -VTSA- in immunological adjuvant with or without helper peptide -NVDP-, 2) MUC.1+ B16 tumor cells, or 3) B lymphocytes Tg for {gamma}1VTSA3 all failed to induce a detectable CD4 or CD8 T cell response against -VTSA- (Table I).

CD4 T cells are at the center stage of immune regulation and play important roles in providing help to B cells (31), in CD8 T cell priming (32), and in the generation of durable memory CD8 T cell responses (33, 34, 35) with enhanced protection against viruses (15) and tumors (36). CD4 help can also prevent CD8 T cell tolerance induction in a system in which both CD4 and CD8 T cells express Tg TCR (37). In this study we show that CD4 T cells regulate the induction of a CD4 T cell response against a tumor self-Ag. To the best of our knowledge this is the first direct demonstration of the induction of CD4 T cell immunity against a tumor self-Ag by active immunization. The data imply that the immunological switch provided by two interacting CD4 T cells in vivo (one against a nonself and the other against a self Th cell determinant) is powerful enough to initiate a response against a tumor self-Ag. They also underscore the importance of linked recognition of endogenously processed Ag as a key requirement in Th-Th cooperation.

In our model, activation of VTSA-specific CD4 T cells is the result of Th-Th cooperation, where the response to a self-determinant requires licensing of the Ag-presenting B cell by activated CD4 T cells specific for a nonself-determinant, a three-cell interaction dependent on CD40 and OX40 (10). In previous studies in C57BL/6 mice, we demonstrated that this mechanism was sufficient to overcome the poor immunogenicity of the VTSA determinant (10). In this study we show that the same mechanism enables the induction of specific T cell immunity in MUC.1-Tg mice. The VTSA peptide is a bona fide subimmunogenic Th cell determinant, because it becomes demonstrably immunogenic by changing the molecular environment (i.e., by creating the conditions for Th-Th cooperation) while keeping constant the flanking residues, hence excluding defects in processing of -VTSA- in B lymphocytes harboring the {gamma}1VTSA3. Together with the fact that the -VTSA- sequence is processed from full-length MUC.1 (Fig. 6), we conclude that Th-Th cooperation was key to overcome the subimmunogenicity of the -VTSA- determinant in both wild-type mice and MUC.1-Tg mice. Furthermore, the activation of CD8 T cells against the 9-mer -VTSA- peptide fits within the framework of a model in which activation of CD8 T cells by Ag-presenting B lymphocytes is Th dependent (38). Collectively, our data and interpretation of the phenomenon suggest that the immunological switch that hallmarks functionally Th-Th cooperation is the property of the APC (a B lymphocyte) where inducible costimulation plays a critical role.

We believe that Th-Th cooperation as an immunological switch enables immunity against -VTSA- in MUC.1-Tg mice through the same tempo and requirements for CD40 and OX40 as in wild-type C57BL/6 mice (8). What remains to be clarified is which barrier Th-Th cooperation needed to overcome in MUC.1-Tg mice. For instance, MUC.1-Tg mice possess CD8 T cell precursors for MUC.1 at comparable frequencies as C57BL/6 mice (12), arguing that in these mice there exists a residual T cell repertoire as observed in other Tg mice models (39). In the present study MUC.1-Tg mice were found to respond promptly to immunization via Th-Th cooperation, suggesting the existence of CD4 T cell precursors that can be expanded by immunization. Thus, the refractoriness of MUC.1-Tg mice to respond to MUC.1 and to reject MUC.1+ tumors may be due to two concurrent factors: 1) an intrinsic poor immunogenicity of the MUC.1 Ag, an oncofetal glycoprotein; and 2) central or peripheral tolerance. As demonstrated in this study, Th-Th cooperation, but not immunization with synthetic peptide in immunological adjuvant or with tumor cells, was per se sufficient to induce MUC.1-specific T cell immunity and tumor protection in MUC.1-Tg mice. Taken together, the present findings argue for an effect of Th-Th cooperation in correcting the subimmunogenicity of the -VTSA- determinant and raise the possibility that it may be effective also in settings where unresponsiveness is compounded by a tolerant state. An alternative interpretation would be that the -VTSA- determinant is not processed from full-length MUC.1, precluding its presentation in thymus during the establishment of tolerance. In favor of this view is the fact that the -VTSA- peptide is in all likelihood a poor binder to the MHC class II cleft because it is relatively short, a fact possibly reflected in its lack of immunogenicity when emulsified in IFA. However, as demonstrated in this study, -VTSA- is processed and presented in Tg B lymphocytes and in DC pulsed with apoptotic B16-MUC.1 cells (Fig. 6), making this alternative interpretation unlikely.

Although we are aware of no precedent for immunity against a defined Th cell determinant of a tumor self-Ag by active immunization alone, there exists evidence that memory CD4 T cells generated in wild-type C57BL/6 mice adoptively transferred into MUC.1-Tg mice provide specific tumor immunity and increase survival (12). In our study, protected MUC.1-Tg mice developed both Ag-specific CD4 and CD8 T cell responses. We favor a scenario where activation of the first CD4 T cell against the nonself-determinant is a prerequisite for the subsequent induction of anti-MUC.1 CD4 T cells and thereafter of CTL via intraepitope (11-mer -> 9-mer) processing. Anti-MUC.1 CTL have been shown to be tumor protective in adoptive transfer experiments (40). Thus, we propose that protection conferred by the antitumor immune response triggered by Th-Th cooperation may be due to the combined effects of anti-MUC.1 CD4 T cells producing IFN-{gamma} and CD8 T cells with conventional cytotoxic activity. The effect of IFN-{gamma} may be mediated by the tumoricidal activity of inducible NO synthase and NO produced by activated macrophages (41, 42, 43) or through the inhibition of angiogenesis (44). A role for CD4 T cells and IFN-{gamma} in rejection of B16-MUC.1 tumors in C57BL/6 mice has been suggested (45). Future studies will need to address these issues and ascertain the relative importance of CD4 and CD8 T lymphocytes in the mechanism(s) of protection by adoptive transfer and selective in vivo depletion of T cell populations.

The induction of a tumor-specific CD4 T cell response via Th-Th cooperation may also be an important source of IL-2 helping the local antitumor effect of specific CD8 T cells concomitantly induced. It is becoming apparent that CD8 T lymphocytes of subjects immunized with synthetic peptides of tumor Ags fail to cause tumor regression, being, by and large, in a state of anergy that can be reversed in vitro by exogenous IL-2 (46). Similarly, tumor regression by adoptive antitumor T cell therapy combined with active vaccination requires the in vivo administration of IL-2 (47). Therefore, IL-2 produced locally during Th-Th activation may positively affect the functional status of antitumor CTL.

The results and conclusions of the study presented raise an interesting question about the possible role of Th-Th cooperation as a mechanism to generate or amplify autoimmunity. Although it has been known for a long time that immunization with allogeneic or xenogeneic tissues induces autoreactive B and T cell responses and, in some instances, organ-specific autoimmune diseases, no direct molecular evidence exists for an involvement of Th-Th cooperation. A plausible argument would be that if the initiation of an autoimmune response reflects both the manner in which self-Ags are presented to the immune system and the immune status of Ag-specific T and B cells, Th-Th cooperation could easily link the response to a cross-reacting heterologous Ag (seemingly functioning as a nonself-determinant) to that against the a self-determinant. Thus, Th-Th cooperation could be responsible for the activation of autoreactive CD4 T cells and together with epitope spreading (48) could represent the molecular substrate for the de novo generation or amplification of autoreactive T cell responses in vivo. In this scenario the rate-limiting factor would be the availability, or accessibility, of a nonself-determinant that must, according to the postulate of Th-Th cooperation (49), be presented in linked association with a self-determinant. Ultimately, the frequency with which Th-Th cooperation could lead to autoimmunity will depend on the accumulation of insertional or deletional mutations in the genome of the individual that give origin to a nonself-determinant, the association with predisposing MHC genes (50), and the escape from control by autoimmune regulatory genes such autoimmune regulator (51).

In summary, this study demonstrates that CD4 T cell immunity to a tumor self-Ag can be induced strictly by activating the immunological switch provided by the cooperation between two Th cells. This finding strengthens the importance of cell-cell cooperation within the dynamics of immune regulation and the ability of the immune system to generate antiself responses. They also point to new ways in which the immune response against tumor Ags can be manipulated and protective antitumor immunity induced.


   Acknowledgments
 
We thank Drs. M. Cohn and J. Hernandez for critically reading the manuscript and for their helpful suggestions, and we are indebted to Dr. S. Gendler for originally providing the colony of MUC.1-Tg mice and B16-MUC.1 cells.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
M. Gerloni is an employee of Cosmo Bioscience, which is developing a vaccine based on the use of transgenic lymphocytes.


   Footnotes
 
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 This work was supported by National Institutes of Health Grants RO1CA77427 and R01CA92119. Back

2 Address correspondence and reprint requests to Dr. Maurizio Zanetti, University of California-San Diego, 9500 Gilman Drive, No. 0837, La Jolla, CA 92093-0837. E-mail address: mzanetti{at}ucsd.edu Back

3 Abbreviations used in this paper: Tg, transgenic; DC, dendritic cell. Back

Received for publication February 2, 2005. Accepted for publication September 6, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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