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[Cancer Research 63, 1040-1045, March 1, 2003]
© 2003 American Association for Cancer Research

Immunology

CD4+ T Lymphocytes Play a Critical Role in Antibody Production and Tumor Immunity against Simian Virus 40 Large Tumor Antigen1

Ronald C. Kennedy2, Michael H. Shearer, Allison M. Watts and Robert K. Bright

Department of Microbiology and Immunology, Texas Tech University Health Science Center, Lubbock, Texas 79430


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of CD4+ T lymphocytes in antitumor immunity has been largely attributed to providing signals required for the priming of MHC class I-restricted CD8+ cytotoxic T lymphocytes, and CD8+ cytotoxic T lymphocytes are thought to serve as the predominant mediators of tumor killing in vivo. We decided to evaluate the role of T lymphocyte subsets in tumor immunity induced by recombinant SV40 large tumor antigen (Tag) within an experimental murine pulmonary metastasis model of SV40 Tag-expressing tumors. Studies in BALB/c mice used in vivo depletion of either CD4+ or CD8+ T cells in the induction phase of the immune response to SV40 Tag. These studies indicate that CD4+ T cells but not CD8+ T cells were critical in the production of antibodies to SV40 Tag and in tumor immunity as the result of recombinant SV40 Tag immunization. On the basis of the predominance of the IgG1 isotype in the antibody response to SV40 Tag immunization, Th2 type CD4+ T cells appeared to be involved. SV40 Tag immunization was not as effective in the induction of tumor immunity in therapeutic modalities when compared with the prophylactic setting. Our results suggest that CD4+ T cells, along with antibody responses, play a role in the induction of tumor immunity to a viral-encoded tumor antigen.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SV40 Tag3 is a viral-encoded oncoprotein that represents a tumor-specific antigen and a target for in vivo tumor immunity (reviewed in Ref. 1 ). It has been demonstrated previously that immunization with baculovirus-derived recombinant SV40 Tag induces complete protection from i.p. tumor challenge of BALB/c mice with a syngeneic SV40-transformed cell line designated mKSA (2) . In addition, SV40 Tag immunization is able to induce complete protection from the development of lung tumor foci in an experimental pulmonary metastasis model whereby mKSA cells are inoculated i.v. (3 , 4) . In characterizing the in vitro immune response induced by SV40 Tag immunization it was observed that in BALB/c mice, Tag does not induce detectable levels of Tag-specific CTLs; however, it does induce Tag-specific serum antibodies (5) . In vitro assays using peritoneal exudate cells taken from immunized mice revealed that ADCC was functioning as a mechanism of tumor cell death (5) . Neither complement-dependent cytotoxicity nor NK cells appeared to play a role in tumor cell death. This provided indirect in vitro evidence that antibodies to SV40 Tag were important in the observed tumor immunity. We were interested in additionally discerning the in vivo mechanism of tumor protection induced by SV40 Tag immunization and determining whether humoral immune responses were important. The prevailing belief regarding the immunological rejection of cancer is that T lymphocyte-mediated immunity is essential for the destruction of most solid tumors with CD8+ CTLs representing the major effector cell mediating tumor cell destruction. Because CD8+ CTL responses were not induced by SV40 Tag immunization, we believed that CD4+ T lymphocytes might be a critical mediator of the observed tumor immunity by providing help for the induction of antibodies to SV40 Tag. To evaluate this possibility, we decided to examine the T-cell subsets involved in providing the observed tumor immunity. Therefore, we depleted BALB/c mice of either CD4+ or CD8+ T cell subsets during the course of immunization with SV40 Tag and subsequently challenged the mice i.v. with mKSA tumor cells to evaluate tumor immunity. On the basis of the results of our experiments, it appears that functional CD4+ T cells, but not CD8+ T cells, along with the presence of SV40 Tag-specific antibody are critical for the development of tumor immunity in this model. Additionally, the predominance of the murine IgG1 isotype in the antibody response to SV40 Tag immunization suggests the role of Th2-type CD4+ T cells in the induction of these immune responses. Although SV40 Tag immunization was capable of reducing lung tumor foci and increasing survival time in mice treated therapeutically with existing tumors, it was not as effective as was observed in a prophylactic setting where complete tumor immunity was observed.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Mice.
The SV40-transformed BALB/c mouse kidney fibroblast cell line designated mKSA (6) was used for tumor challenge. Cells were cultured in DMEM with L-glutamine (Life Technologies, Inc., Rockville, MD) and supplemented with 0.1 mM nonessential amino acids, 100 units/ml penicillin, 500 µg/ml streptomycin (Mediatech, Washington, DC), and 10% heat-inactivated fetal bovine serum (BioWhittaker, Walkersville, MD). Cells were incubated in a humidified, 5% CO2 atmosphere at 37°C. Before injection, cells were detached from flasks by 5-min exposure to PBS and 1 mM EDTA (pH 7.5). Cells were washed, resuspended in PBS, counted, and adjusted to the appropriate density with additional PBS before inoculation. Six to 8-week-old female BALB/c mice were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained under standard conditions. Treatment and care of the animals were in accordance with institutional guidelines and the Animal Welfare Assurance Act.

In Vivo Depletion of CD4+ and CD8+ T Cells.
Mabs GK1.5 (rat IgG2b antimurine CD4) and YTS169.4 (rat IgG2b antimurine CD8) were used for in vivo depletion of the CD4+ and CD8+ T-cell subsets, respectively. The rat IgG Mabs were purified by affinity chromatography, and total protein content was quantified by absorbance at 280 nm. Purified rat Mabs and a control rat IgG preparation were administered by i.p. injection of 100 µg of antibody in 0.1 ml PBS, according to the schedule specified in Fig. 1Citation . Groups of Mab-depleted and control immunized mice were sacrificed 12 days after the last immunization with the rat antibodies (corresponding to day 21 or tumor cell challenge, see Fig. 1Citation ), and splenocytes and lymph node preparations were analyzed by flow cytometry for CD4+ (stained with clone RM4–4 conjugated to fluorescence isothiocyanate) and CD8+ (stained with clone 2.43 conjugated to phycoerythrin) T-cell subsets. These are commercially available rat Mabs that detect epitopes distinct from those recognized by the depleting Mabs. Flow cytometry indicated a >95% depletion in the specific CD4+ or CD8+ T-lymphocyte populations when compared with control mice (data not shown). Our flow cytometry methods have been described elsewhere (5 , 7)



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  		Fig. 1. Schedule for SV40 Tag immunization, CD4 depletion, CD8 depletion, and tumor challenge. Mice were depleted of T-cell subsets by injections of 100 µg of Mab GK1.5 (anti-CD4), Mab YTS169.4 (anti-CD8), or rat IgG at -2, -1, 2, 5, 6, and 9 days. Groups of mice were sacrificed at day 21, and CD4 and CD8 T-cell populations analyzed by flow cytometry. SV40 Tag immunization consisted of 1 µg Tag in alum at day 0 and 7. Groups of mice that were challenged with tumor cells received 5 x 105 viable mKSA cells i.v. at day 21. Tumor cell-challenged mice were sacrificed 14 days after challenge, and lungs were removed and analyzed for tumor foci formation.

 
Immunization and Tumor Challenge.
SV40 Tag was prepared using a baculovirus expression vector system (8) . Groups of 5 BALB/c mice were immunized i.p. with an alum precipitate of recombinant SV40 Tag as described previously (2 , 5) . Control groups of 5 mice each received alum alone. Injections of 1 µg of SV40 Tag were administered at 1-week intervals for a total of two injections (Fig. 1)Citation . Serum was obtained from each mouse before immunization (preimmune), and at 7 days after the first and second immunization. Mice were challenged 14 days after the second SV40 Tag injection, with 5 x 105 viable mKSA cells being delivered i.v. in 50 µl of sterile PBS. In the first set of experiments, mice were sacrificed 14 days after challenge, and the number of lung tumor foci was determined using a computer-based image analysis system (9) . In the second set of experiments, 1 mouse from each group was sacrificed 21 days after challenge, and lung tumor foci were examined. Tumor immunity was determined in the remaining 4 mice/group based on survival (3, 4, 5) . To evaluate the effects of SV40 Tag immunization on established tumors, mice were inoculated with 5 x 105 mKSA cells, i.v., and then immunized with recombinant SV40 Tag on days 3, 9, and 15 after tumor challenge. Control groups of mice received alum rather than SV40 Tag in alum. Eighteen days after tumor challenge, 3 mice from each group were euthanized, and lungs were obtained. The remaining 7 mice were used to determine survival time.

ELISA.
Detection of SV40 Tag-specific serum antibody was carried out using ELISA as described previously (2 , 4 , 5) . Briefly, 200 ng of recombinant SV40 Tag in borate-buffered saline was coated onto 96-well microtiter plates overnight at 4°C. Nonspecific binding was blocked by the addition of 200 µl of 10% normal goat serum borate-buffered saline and incubated at 37°C for 1 h. Mouse serum was added at various dilutions and incubated for 1 h at 37°C. Anti-SV40 Tag reactivity was detected using horseradish peroxidase-conjugated goat antimouse IgG Fc-specific reagent (Sigma, St. Louis, MO) diluted 1:1000 in blocking solution. Plates were developed with peroxidase substrate 2,2'-azino-di(3-ethyl-benzthiazoline sulfonic acid) containing 0.01% H2O2. An absorbance at 410 nm (A410 nm) determined to be approximately three times the absorbance values obtained for 1:10 dilutions of the preimmune sera was established as a cutoff for positive reactivity and antibody end point titer determinations. Immune serum was examined using serial 4-fold dilutions, beginning with a dilution of 1:50, and the dilution that resulted in the last absorbance above the cutoff was the end point titer. Anti-SV40 Tag antibodies failed to bind a control recombinant antigen (hepatitis B surface antigen) in similar assays (data not shown). All of the serum samples were run in triplicate and reported as the reciprocal end point titer. These methods have been described in detail elsewhere (4 , 5) . To determine the IgG antibody isotype distribution, immune serum at a 1:100 dilution was added to SV40 Tag-coated microtiter wells in triplicate as described above. IgG1 and IgG2a anti-SV40 Tag responses were determined using sheep antimouse IgG1- and IgG2-specific isotyping reagents (Binding Site, San Diego, CA) that were conjugated with horseradish peroxidase, and the assays were performed as described above. Two murine monoclonal anti-SV40 Tag preparations, PAb 405 (IgG1) and PAb 419 (IgG2a), were used as controls. The IgG2a:IgG1 ratios were determined from the mean absorbance values as has been described previously (10 , 11) .

Determination of Tumor Cell Foci in the Lungs.
The left lung of each animal was removed after euthanization and stained by intratracheal injection of the lobes with 10% India ink. Lungs were then suspended in Fekete’s destaining solution. To remove subjectivity in the counting of tumor cell foci in the lungs of inoculated mice, we have used a computer-assisted method (9) . After destaining 15 min, the number of foci visible on the ventral surface of the lung was quantified using an IS-1000 digital Imaging System (Alpha Innotech Co., San Leandro, CA). Density threshold parameters were defined to ensure that the foci counted consistently fell above a gray scale value of 25 (compared with black lung background). Size threshold parameters were set to count only those foci falling above 4 pixels in diameter on the computer image. SE was determined to demonstrate variability within each group (9) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vivo Depletion and Recombinant SV40 Tag Immunization.
The schedule used for the in vivo depletion of CD4 and CD8 T-cell subsets using rat antimouse CD4 and CD8 Mabs is depicted in Fig. 1Citation . The specific rat Mab for CD4 depletion (GK1.5) and CD8 depletion (YTS169.4) have been used by other investigators previously (12 , 13) , and we used a similar schedule for in vivo depletion. In our initial studies, groups of 5 mice each were depleted with either GK1.5 (CD4), YTS169.4 (CD8), or similarly immunized with purified rat IgG as a control before and after immunization with recombinant SV40 Tag. Serum was obtained before and 7 days after each injection with SV40 Tag, and mice were sacrificed on day 21 (corresponding to tumor cell challenge) and 12 days after the last injection with the rat IgG preparations. Lymph node and splenocytes were obtained and examined by flow cytometry for CD4 and CD8 expression when compared with the control rat IgG immunized group. In each instance, the levels of CD4+ in the GK1.5-treated group and CD8+ in the YTS169.4-treated group were decreased by >95% when compared with the rat IgG immunized group (data not shown). This suggested that the depletion scheme used in Fig. 1Citation would reduce the levels of CD4 and CD8 subpopulations in the treated mice. To evaluate the effects of this in vivo depletion, we also examined the antibody response to SV40 Tag as the result of the recombinant protein immunization. As shown in Table 1Citation , mice treated with either the rat IgG preparation or YTS169.4 (anti-CD8) generated detectable anti-SV40 Tag responses after the second SV40 Tag injection. Mean antibody titers were 3040 (rat IgG-treated) and 1760 (anti-CD8-treated) with similar ranges of antibody responses (800–3200). The group treated with GK1.5 (anti-CD4), along with a control group of mice that received alum alone, failed to produce detectable anti-SV40 Tag responses. These data suggested that the anti-CD4 treatment group of mice were functionally depleted, as no antibody response to SV40 Tag was observed. Alternatively, the anti-CD8 treatment group appeared to possess functioning CD4+ T cells, as SV40 Tag immunization generated antibodies to SV40 Tag. No IgG anti-SV40 Tag responses were observed in any of the groups of mice after the first injection, which is similar to what we have observed previously after a single injection of SV40 Tag.


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  		Table 1 Antibody titers to SV40 Tag in mice treated with anti-CD4, anti-CD8, or rat IgG

 
To compare the antibody responses in the rat IgG control-treated and anti-CD8-treated groups of mice, we examined the binding of sera at various dilutions to SV40 Tag by ELISA. A representative set of binding curves is shown in Fig. 2Citation . It is apparent from the binding curves that the anti-SV40 Tag antibody responses in the rat IgG- and anti-CD8-treated groups of mice were similar with respect to the slope of the binding curve and the titer. No anti-SV40 Tag-specific antibodies were observed in the alum immunized or the anti-CD4-treated groups of mice. Together, these data indicate that our Mab treatment regimen is successful for functionally depleting CD4+ T cells, and that the antibody response to SV40 Tag does not appear to differ in the rat IgG- and anti-CD8-treated groups of mice.



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  		Fig. 2. Representative SV40 Tag-specific antibody binding curves from SV40 Tag-immunized mice treated with anti-CD4 Mab, anti-CD8 Mab, or rat IgG. The mean absorbance (A410 nm) values for the various 4-fold dilutions of groups of 5 mice each are shown; bars, ±SD.

 
Anti-CD4-treated Mice Are Susceptible to Tumor Formation.
In the next set of experiments, we evaluated whether anti-CD4 and anti-CD8 treatment before recombinant SV40 Tag immunization had an effect on tumor immunity. Groups of 5 mice each received anti-CD4, anti-CD8, or rat IgG treatment before and after immunization with SV40 Tag as described in Fig. 1Citation and were challenged i.v. with mKSA tumorigenic cells 14 days after the second SV40 Tag immunization. Tumor immunity was determined 14 days after mKSA challenge by evaluating the presence of lung tumor foci in an experimental model of pulmonary metastasis that has been described previously (3 , 4) . As shown in Table 2Citation , the rat IgG- and anti-CD8-treated groups of mice developed anti-SV40 Tag responses after the second SV40 Tag injection. The alum immunized and anti-CD4-treated groups of mice failed to develop detectable antibodies to SV40 Tag. These data are similar to the data described in Table 1Citation . However, after tumor cell challenge, both the alum immunized and anti-CD4-treated groups of mice developed lung tumor foci in each of the 5 individual mice. The numbers of lung foci ranged from 6 to 12 (average of 10) for the alum immunized and 1 to 9 (average of 5) for the anti-CD4-treated mice. No lung tumor cell foci were observed in any of the rat IgG- and anti-CD8-treated mice indicating that tumor immunity was complete. To additionally examine the level of tumor immunity, additional groups of mice were treated and immunized as described in Fig. 1Citation , and tumor immunity was evaluated in a single mouse 21 days after mKSA tumor cell experimental challenge. The remaining 4 mice/group were evaluated for tumor immunity based on survival time. Representative tumor cell foci present in the lungs from alum-immunized (Fig. 3A)Citation and anti-CD4-treated mice (Fig. 3C)Citation differ from the lack of tumor cell foci in the lungs from rat IgG-treated (Fig. 3B)Citation and anti-CD8-treated (Fig. 3D)Citation mice 21 days after tumor cell challenge. The number of tumor foci quantitated by computer-assisted video image analysis in the alum-immunized mouse was 23 foci versus 44 foci in the anti-CD4-treated mouse. This increase in the number of tumor foci in the anti-CD4-treated mice late in the experimental metastasis model may reflect the fact the CD4+ T cells may exhibit direct antitumor effects. However, this is not observed early in tumor foci development in the lungs, because alum-immunized and anti-CD4-treated mice had similar numbers of tumor foci when lungs were evaluated 14 days after tumor cell challenge (Table 2)Citation . The survival time for the remaining alum- and anti-CD4-treated mice ranged from 20 to 24 days after challenge. The rat IgG- and anti-CD8-treated mice survived >60 days (data not shown). These data clearly point to a critical role that CD4+ T cells have in the induction of antibody responses and in tumor immunity as the result of recombinant SV40 Tag immunization.


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  		Table 2 Antibody titers and development of lung metastases in mice treated with anti-CD4, anti-CD8, or rat IgG

 


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  		Fig. 3. Representative lung tumor cell foci in mice treated with anti-CD4 or anti-CD8 Mabs and immunized with recombinant SV40 Tag. A, tumor cell foci in the lung of a control alum immunized mouse. B, lack of tumor cell foci in the lung of a rat IgG treated and SV40 Tag-immunized mouse. C, tumor cell foci in the lung of an anti-CD4-treated and SV40 Tag-immunized mouse. D, lack of tumor cell foci in the lung of an anti-CD8-treated and SV40 Tag-immunized mouse.

 
Next, we determined the IgG isotype distribution of the antibody response to SV40 Tag in rat IgG- and anti-CD8-treated mice (Table 3)Citation . It is apparent that IgG1 was the predominant antibody response to SV40 Tag immunization in both groups of mice when compared with IgG2a. The IgG2a:IgG1 anti-SV40 Tag ratios ranged from 0.03 to 0.07 in the rat IgG-treated group and 0.02 to 0.08 in the anti-CD8-treated group. These data indicate that the immune response to multiple injections of SV40 Tag is Th2 biased, because IgG1 is the predominant subclass of antibody that is synthesized. Little to no IgG2a anti-SV40 Tag was observed in these mice. The synthesis of IgG2a is associated with a Th1-biased immune response (10 , 11 , 14) .


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  		Table 3 IgG antibody isotypes in mice that developed anti-SV40 tag responses

 
To determine what effects recombinant SV40 Tag immunization had on established tumors, we challenged mice with tumor cells before the initiation of SV40 Tag injections. We selected a tumor cell challenge dose similar to that used in the prophylactic immunization experiments, and the selection of the time point after challenge to obtain lungs from mice was based on the latest time when control group mouse survival became questionable. As shown in Table 4Citation , the control alum-treated mice had a higher number of tumor foci (mean value of 29.33; n = 3) when compared with the SV40 Tag immunized mice (mean value of 6.66; n = 3). Similarly, the survival time was increased in the group of mice that received SV40 Tag (mean survival time of 35 days) when compared with control mice (mean survival time of 21 days). The SV40 Tag immunization was capable of reducing the number of lung tumor foci and increasing the survival time in a therapeutic modality; however, it was not as effective as was observed in a prophylactic setting where complete tumor immunity was observed.


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  		Table 4 Effect of SV40 tag immunization on established tumors

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The role of CD4+ T-helper cells in antitumor immunity has been largely attributed to providing signals required for the priming of MHC class I-restricted CD8+ CTLs. The CD8+ CTLs are thought to serve as the predominant mediators of tumor killing in vivo. Indeed, many studies that involve the induction of antitumor immune responses in vivo target tumor antigen-specific CTL responses (Refs. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , reviewed in Ref. 26 ). In a number of these instances CD4+ T cells were required for the induction of CD8+ CTLs that exhibited antitumor immunity (17 , 20 , 22 , 23) . The involvement of both CD4+ and CD8+ T lymphocytes has been reported in immunity against polyoma virus-induced tumors (27) , whereas CD8+ CTL responses specific for the viral-encoded human papillomavirus type 16 tumor antigen E7 correlated with both tumor immunity and the regression of established tumors (28 , 29) . The antitumor immune responses were dependent on E7-specific CD8+ cells but not CD4+ T cells (28) . Although the majority of vaccination strategies for the treatment of solid malignancies focus on the generation of CD8+ CTL responses, antibody-based therapies have been successful against some cancers in the clinical setting (reviewed in Ref. 30 ). These antibody-based immunotherapies involve passive administration of Mabs specific for CD20 and HER-2/neu for the treatment of non-Hodgkin’s lymphoma and breast cancer, respectively. Thus, it is reasonable to speculate that antibodies induced by tumor vaccination strategies may exhibit antitumor immunity in some systems.

Previous studies from our laboratories suggested that antibodies to SV40 Tag may play a role in tumor immunity resulting from immunization with recombinant SV40 Tag (2 , 4 , 5) . In comparative studies using other SV40 Tag immunization modalities, such as anti-idiotypes or SV40 Tag synthetic peptides where only partial tumor immunity was observed, the presence of tumor immunity appeared to correlate with the levels of antibodies to SV40 Tag (31 , 32) . Additional studies demonstrated recombinant SV40 Tag failed to generate any detectable CD8+ CTL responses, whereas antibodies to SV40 Tag were induced and were capable of mediating ADCC against the mKSA tumor cells in vitro (5) . The role of antibodies that mediated complement-dependent cytotoxicity and direct NK cell activity against mKSA tumor cells as in vitro mechanisms in the observed recombinant SV40 Tag-induced tumor immunity was ruled out. In this present study, the predominance of the IgG1 anti-SV40 Tag suggests a biased for a Th2 type CD4+ T-cell response. The lack of an IgG2a anti-SV40 Tag response being induced by multiple injections of recombinant SV40 Tag suggests that Th1 type CD4+ T-cell responses are not involved in the observed tumor immunity in this model. In murine systems, the IgG1 subclass is most effective at mediating ADCC in the context of CD32 (Fc{gamma}RII) effector cells, whereas the IgG2a subclass is more effective at mediating ADCC using CD64 (Fc{gamma}RI) effector cells (reviewed in Ref. 33 ). Because these two Fc receptors are not expressed on NK cells, these data, along with our previous report (5) , support the possible role of ADCC and macrophage/monocytes effector cells in the complete tumor immunity resulting from recombinant SV40 Tag immunization. Additional studies to evaluate the direct role of macrophages/monocytes as possible effectors cells involved in ADCC in this system are warranted. The potential exists whereby ADCC may play only a partial role in the observed complete tumor immunity, and CD4+ T cells, particularly Th2 type cells, may also have direct effects that complement the antitumor activities of antibodies that can mediate ADCC. This could include the secretion of cytokines and their potential for direct cytotoxic and/or apoptotic events against tumor cells that also impart a necessary component of the observed tumor immunity. Nonetheless, our study clearly supports a role for both CD4+ T cells and antibodies as components of tumor immunity.

It was of interest to note that in a therapeutic setting, immunization with recombinant SV40 Tag had only partial antitumor effects on pre-existing tumors when compared with immunization in a prophylactic modality. These partial effects included less of a tumor burden as assessed by the number of tumor lung foci and an increase in survival time when compared with control groups. This was not a surprising observation, as a number of murine tumor systems have indicated that immunization is only effective either before or early after tumor cell inoculation (34, 35, 36, 37) . Indeed, some potential therapeutic effects were observed with recombinant SV40 Tag immunization after tumor cell challenge. However, because tumor immunity in the therapeutic setting was not complete to examine mechanistic aspects related to the induction of tumor immunity in this system, we selected the prophylactic modality where complete tumor immunity was observed.

In this present report, we analyze the role of T-cell subset involvement in mediating tumor rejection in the murine experimental pulmonary metastasis model after immunization with a tumor-specific antigen. The data described in this present report have implicated an important role for CD4+ T-helper cell function in B-cell priming for antibody production. This is in contrast to reports by another group of investigators who describe the requirement of CD8+ T lymphocytes for recombinant SV40 Tag-induced tumor immunity in their system (37 , 38) . These investigators determined that whereas CD4+ T-helper cells may play a role in protection, their role is to provide signals to activate CD8+ CTL responses and that these effector cells are responsible for the observed recombinant SV40 Tag-induced tumor immunity (38) . It is noteworthy that similar to our studies published previously (4 , 5) , no CD8+ CTL responses were observed in the spleen or draining lymph nodes that would be indicative of systemic tumor immunity. Rather, CD8+ CTL were observed only at the site of the experimental tumor challenge (38) . A number of other important differences between these studies can explain the discrepancies in the results and conclusions of the investigations.

These results are relevant to the treatment of human cancer in both general and specific terms. The use of Mabs as passively administered therapy modalities has been successfully used to treat several types of human cancers, and this directly implicated antibodies in tumor immunity (reviewed in Ref. 30 ). ADCC and specific effector cells that mediate this form of antitumor immunity appear to play a role. Our studies implicate both the need to activate tumor antigen-specific CD4+ T cells and antibodies as the basis for the observed immunity in this murine tumor model. Thus, active immunization strategies should include the targeting of both antibody induction and CD4+ T-cell activation to impart potentially the most complete form of tumor immunity in cancer scenarios where the mechanism(s) of tumor immunity are unknown. In specific terms, SV40 and SV40 Tag have been reported to be involved in a number of human malignancies (reviewed in Refs. 39, 40, 41 ). Of particular relevance to SV40 Tag vaccination strategies is the association of SV40 with malignant pleural mesothelioma (reviewed in Ref. 42 ). This is a tumor of the pleura that originates in the serosal lining and is exceptionally lethal. Current therapies, including surgery, radiation, and chemotherapy, are ineffective at slowing the course of the disease. The median survival from the time of diagnosis is rarely >1 year. Thus, the need for new alternative cancer therapies to treat malignant pleural mesothelioma is important. Within mesothelioma cells, it has been shown that SV40 Tag binds essential cellular tumor suppressor gene products, including p53 and pRb, suggesting that SV40 and SV40 Tag may play roles in cellular transformation (43 , 44) . It was reported recently that SV40 Tag-specific CTLs could be generated from the peripheral blood of malignant pleural mesothelioma patients (45) . These CTLs were capable of recognizing mesothelioma tumor cells that expressed SV40 Tag in an MHC class I-restricted manner, suggesting that SV40 Tag represents an immunological target in humans with mesothelioma. The potential exists whereby recombinant SV40 Tag or components thereof may function as a therapeutic cancer vaccination strategy for treating malignant mesotheliomas expressing SV40 Tag (reviewed in Ref. 46 ). Additionally, asbestos exposure has also been associated with malignant mesothelioma (42) . Individuals who are exposed to both SV40 and asbestos have two associated risk factors for developing mesothelioma (47) . Such high-risk individuals could be vaccinated using SV40 Tag-based strategies before the onset of symptoms, a scenario that may approximate prophylactic immunization. In these treatment scenarios, the generation of antibodies and CD4+ T cells specific for SV40 Tag would be indicative of immunological responsiveness to the vaccination protocol and based on the studies describe herein using murine systems, a preferred outcome associated with tumor immunity.


   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 Supported in part by Grant CA-77351 from the NIH. Back

2 To whom requests for reprints should be addressed, at Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, 3601 4th Street STOP 6591, Lubbock, TX 79430. Phone: (806) 743-2414; Fax: (806) 743-2334; E-mail: ronald.kennedy{at}ttuhsc.edu Back

3 The abbreviations used are: Tag, large tumor antigen; ADCC, antibody-dependent cell-mediated cytotoxic; CTL, cytotoxic T lymphocyte; Mab, monoclonal antibody; NK, natural killer. Back

Received 4/18/02. Accepted 12/27/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bright R. K., Shearer M. H., Pass H. I., Kennedy R. C. Immunotherapy of SV40 induced tumours in mice: a model for vaccine development. Dev. Biol. Stand., 94: 341-355, 1998.[Medline]
  2. Shearer M. H., Bright R. K., Lanford R. E., Kennedy R. C. Immunization of mice with baculovirus derived recombinant SV40 large tumor antigen induces protective tumor immunity to a lethal challenge with SV40 transformed cells. Clin. Exp. Immunol, 91: 266-271, 1993.[Medline]
  3. Watts A. M., Shearer M. H., Pass H. I., Kennedy R. C. Development of an experimental murine pulmonary metastasis model incorporating a viral encoded tumor specific antigen. J. Virol. Methods, 69: 93-102, 1997.[Medline]
  4. Watts A. M., Shearer M. H., Pass H. I., Kennedy R. C. Comparison of SV40 large Tag recombinant protein and DNA immunization in the induction of protective immunity from experimental pulmonary metastasis in a murine model. Cancer Immunol. Immunother., 47: 343-351, 1999.[Medline]
  5. Bright R. K., Shearer M. H., Kennedy R. C. Immunization of BALB/c mice with recombinant simian virus 40 large tumor antigen induces antibody dependent cell mediated cytotoxicity against simian virus 40 transformed cells: an antibody based mechanism for tumor immunity. J. Immunol., 153: 2064-2071, 1994.[Abstract]
  6. Kit S., Kurimura T., Dubs D. R. Transplantable mouse tumor line induced by injection of SV40-transformed mouse kidney cells. Int. J. Cancer, 4: 384-392, 1969.[Medline]
  7. Shearer M. H., Chanh T. C., Dalgleish A. G., Kennedy R. C. Idiotype cascades associated with the CD4-HIV glycoprotein 120 interaction: immunization with anti-idiotypic antibodies induces anti-anti-idiotypic responses with anti-CD4 specificity and in vitro neutralizing activity. AIDS Res. Hum. Retrovir., 16: 77-86, 2000.[Medline]
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