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Immunology |
1 Karmanos Cancer Institute and 2 Department of Immunology and Microbiology, School of Medicine, Wayne State University, and 3 Division of Immunopathology, St. John Hospital and Medical Center, Detroit, Michigan
Requests for reprints: Wei-Zen Wei, Karmanos Cancer Institute, Wayne State University, 110 East Warren Avenue, Detroit, MI 48201. Phone: 313-833-0715 Ext 2360; Fax: 313-831-7518; E-mail: weiw{at}karmanos.org.
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Abstract |
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–secreting T cells were induced. The same mice were also exposed to mouse thyroglobulin by chronic i.v. injections. These mice produced thyroglobulin-specific antibody and IFN-–secreting T cells with inflammatory infiltration in the thyroids of some mice. The immune responses to neu or thyroglobulin were greater in mice undergoing TUBO tumor rejection and thyroglobulin injection than in those experiencing either alone. To the best of our knowledge, this is the first experimental system to assess the concurrent induction and possible synergy of immune reactivity to defined tumor and self-antigens following reduction of regulatory T cells. These results illustrate the importance of monitoring immune reactivity to self-antigens during cancer immunotherapy that involves immunomodulating agents, and the pressing need for novel strategies to induce antitumor immunity while minimizing autoimmunity. |
Introduction |
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In this study, rat neu is used as the model tumor-associated antigen. Overexpression of erbB-2 or Her-2/neu in a number of common cancers, such as breast, ovarian, colorectal, prostate, and pancreatic adenocarcinoma (14–17), is correlated with a more aggressive course of disease (18, 19), rendering Her-2 an important target of cancer therapy. The therapeutic effect of anti–Her-2 monoclonal antibody (mAb), Herceptin, in stage IV breast cancer patients further distinguishes this molecule as an exceptional target of immunotherapy and vaccination. Because of self-tolerance, it is difficult to elicit strong immune responses to Her-2, as we showed in Her-2 transgenic mice (20), and Treg depletion may be a plausible strategy to amplify anti–Her-2/neu immunity.
Depletion of CD4+CD25+ cells combined with CTLA-4 blockade has been shown to enhance the efficacy of B16 melanoma cell vaccine with an increase in autoimmune skin depigmentation, demonstrating the concurrent induction of antitumor immunity and autoimmunity directed at common antigens (21). Autoimmunity induced through modulation of regulatory T cells is, however, not restricted to such common antigens. Autoimmune thyroiditis and a spectrum of other autoimmune diseases have been observed in cancer patients receiving melanoma gp100 or Her-2 peptide vaccines with immunomodulating agents (22, 23). In this study, we examined the induction of autoimmunity in the thyroid which does not share common antigens with Her-2.
We have shown that depletion of Treg in CBA/J mice increased their susceptibility to experimental autoimmune thyroiditis (24), the murine model of Hashimoto's thyroiditis. Hashimoto's thyroiditis, the leading cause of hypothyroidism, is characterized by mononuclear cell infiltration and destruction of the thyroid, elevation of thyroid-stimulating hormone, and decrease of thyroid hormones (T3 and T4). The production of autoantibodies (25) and T-cell proliferation to thyroid antigens (26) are indicators of autoreactivity. Susceptibility to thyroiditis is strongly influenced by the haplotype of class II MHC. For example, human HLA-DRB1*0301 (DR3) transgene (27) and murine H2k (CBA/J) confer susceptibility to autoimmune thyroiditis, whereas murine H2d (BALB/c) is associated with resistance (28).
In genetically susceptible mice, experimental autoimmune thyroiditis is induced by injection of mouse thyroglobulin (mTg), usually in the presence of a strong adjuvant (e.g., complete Freund's Adjuvant or lipopolysaccharide; ref. 29), or by repeated injections of mTg for 4 weeks (30). Like Hashimoto's thyroiditis, experimental autoimmune thyroiditis is also characterized by mononuclear cell infiltration, autoantibody production, and T-cell proliferation. Susceptible mice can be tolerized to mTg by short-term elevation of circulating mTg (31). This induced tolerance is abrogated by depletion of CD25+ cells and mice again become susceptible to experimental autoimmune thyroiditis (24).
In this study, we assessed the risk of experimental autoimmune thyroiditis in genetically resistant BALB/c mice undergoing Treg depletion to induce tumor regression. Our results show that in a Treg-deprived environment, tumor cells effectively prime the immune system, resulting in tumor regression and persistent immunologic memory. The same depletion enhanced autoimmunity to mTg in resistant BALB/c mice. Concurrent tumor regression and mTg immunization resulted in further elevation of both antitumor and anti-mTg immunity. This report describes the first test system to analyze simultaneous antitumor and anti-self immunity while reducing immune regulatory mechanisms as a form of cancer therapy.
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Materials and Methods |
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Depletion of CD25+ T cells with anti-CD25 monoclonal antibody, PC61. The hybridoma line PC61 which produces rat anti-mouse CD25 immunoglobulin G1 [IgG1; American Type Culture Collection (ATCC), Manassas, VA] was propagated in severe combined immunodeficient mice. BALB/c mice were injected i.p. with 
0.5 mg of PC61 or normal rat immunoglobulin (or PBS). Depletion of CD25+ cells was verified by flow cytometry. Lymph node cells were prepared and washed with PBS containing 0.1% sodium azide and 2% cosmic calf serum. All samples were treated with Fc receptor blocker (rat antibody to CD16/CD32; PharMingen, San Diego, CA) for 15 minutes on ice, then washed once. Cells were incubated with goat anti-GITR IgG (R&D Systems, Minneapolis, MN) for 20 minutes on ice followed by allophycocyanin-rat anti-CD4 (RM4-5), FITC-rat anti-CD25 (7D4), and phycoerythrin-donkey anti-goat IgG. Controls were stained with FITC-rat immunoglobulin M, allophycocyanin-rat IgG2b, or normal goat IgG with phycoerythrin-donkey anti-goat IgG. mAb RM4-5, 7D4, and their isotype controls were from PharMingen. Normal goat IgG and phycoerythrin-donkey anti-goat IgG were from Jackson ImmunoResearch Lab (West Grove, PA). Single- and double-stained samples were used for instrument setup. Lymph node cells from naïve animals were measured in parallel. Flow cytometric analysis was done with a FACSCalibur (Becton Dickinson, Mountain View, CA).
Tumor growth and measurement. To measure tumor growth, mice were challenged s.c. with 2 x 105 cells in the flank. Tumor growth was monitored by weekly palpation. Tumor diameters were measured in two dimensions and mice were sacrificed when any one dimension reached 15 mm. Tumor volume was calculated as X2Y / 2, where X and Y represent the short and long dimension, respectively, of the tumor. Comparison of tumor-free mice was analyzed with the log-rank test.
Measurement of anti-neu antibodies. Antibody response to rat neu was determined by flow cytometry as we previously described (35). BALB/c 3T3 cells (ATCC) were stably transfected with rat neu to establish 3T3/N cells. The mouse anti-rat neu mAb (IgG2a, clone 7.16.4), which recognizes an extracellular domain of rat neu protein (Oncogene Research Products, Cambridge, MA), was serially diluted and used to stain 3T3/N cells to establish a standard binding curve. FITC-goat anti-mouse IgG was the secondary antibody (Jackson ImmunoResearch). To determine antibody concentration in test sera, serially diluted test sera were used as primary antibody. Antibody concentration was calculated by regression analysis as we previously reported (35). Normal mouse immunoglobulin was used as negative control. The isotype of bound antibody was measured with FITC-goat anti-mouse IgG1 or IgG2a (Caltag, Burlingame, CA) and the results were expressed as mean channel fluorescence. Flow cytometric analysis was done with a FACSCalibur (Becton Dickinson). The results were analyzed by two-tailed Student's t test and are presented as mean ± SD.
Enumeration of cytokine-producing cells by ELISPOT assay. Spleen cells were suspended in RPMI 1640 supplemented with 10% FCS, 2 mmol/L L-glutamine, and 100 units/mL penicillin and 100 µg/mL streptomycin. Three to four hundred thousand cells were added to each well of the 96-well high-throughput screening immunoprecipitation plates (Millipore, Bedford, MA), which were precoated with rat anti-mouse IFN- (IgG1, clone R4-6A2) or rat anti-mouse interleukin 4 (IL-4; IgG1, clone 11B11), and incubated for 24 to 48 hours at 37°C in 5% CO2. Test wells also contained the engineered antigen-presenting cells 3T3/NKB, which were 3T3 cells expressing rat neu, H2-Kd and CD80. The control 3T3/K cells were 3T3 cells expressing H2-Kd. The ratio of spleen cells to 3T3/NKB was 10:1 and 3T3/NKB were added after spleen cells had been plated. After incubation, cells were discarded and biotinylated rat anti–IFN- (IgG1, clone XMG 1.2) or rat anti–IL-4 (IgG1, clone BVD6-24G2) was added. All antibodies were purchased from BD PharMingen. Plates were incubated for 12 hours at 4°C, then washed to remove unbound antibody. Bound antibody was detected by incubating the plates with 0.9 µg/mL avidin-horseradish peroxidase (Sigma) for 2 hours at room temperature. Following washing, the substrate 3-amino-9-ethylcarbazole in 0.1 mol/L acetic acid and 0.003% hydrogen peroxide was added and the plates were incubated for 5 minutes. The substrate was discarded and the plates were washed six times with water. The visualized cytokine spots were enumerated with the ImmunoSpot analyzer (CTL, Cleveland, OH) and the results expressed as the number of cytokine-producing cells per 106 spleen cells. Data were analyzed by Student's t test.
Immunization with mouse thyroglobulin. mTg was prepared from frozen thyroids by fractionation on a Sephadex G-200 column as previously described (36, 37) and diluted in nonpyrogenic saline before use. The presence of lipopolysaccharide was measured by Limulus amoebocyte assay (Associates of Cape Cod, Woods Hole, MA; ref. 30). A 40 µg dose of mTg contained <0.5 ng of lipopolysaccharide.
Mice were injected i.v. with 40 µg mTg, followed in 3 hours with 20 µg Salmonella enteritidis lipopolysaccharide. The injections were repeated in 7 days. Alternatively, 40 µg mTg was injected i.v. on 4 successive days with 3 days of rest (30). This treatment was repeated for 3 more weeks.
Measurement of anti–mouse thyroglobulin antibody. Anti-mTg antibody titers were determined by ELISA as previously described (38). Briefly, Immulon I microtiter plates (Dynatech Laboratories, Inc., Chantilly, VA) were coated with mTg at 1 µg/well and serially diluted test sera were added. After washing, bound antibody was detected with alkaline phosphatase–labeled goat anti-mouse IgG and enzyme substrate.
Measurement of anti–mouse thyroglobulin T-cell response. T-cell proliferation was measured by [3H]thymidine incorporation. Splenocytes were cultured in triplicate in RPMI + 1% normal mouse serum, 2 mmol/L L-glutamine, 5 x 10–5 mol/L ß-mercaptoethanol, 100 units/mL penicillin, and 100 µg/mL streptomycin, in 96-well plates at 6 x 105 cells/well, either with or without 40 µg/mL mTg for 4 days at 37°C, 6% CO2. The cells were pulsed with 1.2 µCi/well of [3H]thymidine and incubated for 18 hours before harvest onto glass fiber filter paper (Tomtec Mach3Man Cell Harvester, LKB Wallac, Gaithersburg, MD). [3H]Thymidine was measured with a Microbeta Plus 1450 liquid scintillation counter (LKB Wallac). Data were analyzed by two-tailed Student's t test.
IFN-– and IL-4–producing cells were enumerated by ELISPOT assay using 40 µg/mL mTg as the antigen. In some experiments, a two-step ELISPOT assay was done to amplify mTg-specific T-cell response. Spleen cells and mTg were incubated in 96-well tissue culture plates for 3 days before the content of the wells was transferred to high-throughput screening immunoprecipitation plates. The high-throughput screening immunoprecipitation plates were incubated for 24 hours. The detection and enumeration of cytokine spots was done as described earlier.
Histologic evaluation of experimental autoimmune thyroiditis. Thyroid specimens were sectioned vertically through both lobes and 50 to 60 histologic sections were prepared from 10 to 15 step levels. The extent of mononuclear cell infiltration was scored based on the pathology index scale of 0 to 4 and presented as percent thyroid infiltration: 0, no infiltration; 0.5, >0% to 10% thyroid infiltration consisting of perivascular foci without follicular destruction; 1.0, >10% to 20% thyroid infiltration with follicular destruction; 2.0, >20% to 40% diffuse thyroid infiltration; 3.0, >40% to 80% thyroid destruction; and 4.0, >80% to 100% thyroid destruction (36). The sections were scored without knowledge of the groups. Statistical differences were analyzed by the nonparametric Mann-Whitney U test.
Statistical analyses. Tumor growth expressed as percent tumor-free mice was analyzed with the log-rank test. Thyroiditis expressed as percent infiltration of thyroid was analyzed using the nonparametric Mann-Whitney U test. The number of IFN-–producing cells and the antibody levels were analyzed by Student's t test.
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Results |
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35 mm3. At week 9, when the tumors were almost completely eliminated, these same mice had 16.7 ± 9 and 34 ± 9 µg/mL of anti-neu IgG, respectively (P = 0.01). Thus, depletion of CD25+ T cells either before or after TUBO cell inoculation resulted in a more prompt and elevated antibody response, with the highest level of anti-neu IgG observed at week 9 in mice receiving anti-CD25 after TUBO cell inoculation (Fig. 2B). The isotypes of antibodies were further analyzed. Figure 2C shows the representative week 5 results from mice treated with 
-CD25 before tumor inoculation. Both IgG1 and IgG2a were elevated, suggesting activation of both T-helper (Th) 1 and Th2 cells.
To measure neu-specific T-cell response, four tumor-free mice which received anti-CD25 after TUBO cell inoculation were sacrificed at week 9. Their spleen cells were isolated and incubated individually with the engineered 3T3/NKB cells. IFN-– and IL-4–producing cells were enumerated by ELISPOT assay after a 2-day culture and similar results were obtained from the four mice. Figure 2D shows the result from a representative animal. There were 36 ± 2 neu-specific IFN-–producing cells per 106 spleen cells. Neu-specific IL-4–producing cells were not detected (not shown), suggesting a more prominent Th1 response. Stimulation with concanavalin A typically resulted in over 1,000 spots per 106 spleen cells (not shown). Incubation with 3T3/K cells did not induce IFN- or IL-4 production.
Immunologic memory to tumor-associated antigen. To assess the strength of immunologic memory to tumor-associated antigens, the group of eight mice which rejected TUBO tumors by anti-CD25 mAb treatment postinoculation were rechallenged with TUBO or D2F2/neu cells at week 14 (week 0 in Fig. 3A). TUBO tumors were rejected by all three mice receiving this cell line as the second challenge (Fig. 3A). The other five mice received D2F2/neu cells, which were generated by transfecting a prolactin-induced BALB/c mammary tumor D2F2 with the wild-type rat neu (Fig. 3B; ref. 39). D2F2/neu cells were rejected by 4 of 5 (80%) mice. Mice which rejected TUBO tumors had 34 ± 9 µg/mL anti-neu antibody just before they received the second tumor challenge. The level of antibody cross-reactive with D2F2 was below 1 µg/mL and remained low after these mice rejected D2F2/neu, suggesting that the second challenge of D2F2/neu cells was rejected primarily through anti-neu immunity.
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–producing T cells, eradication of the growing tumor, as well as immunologic memory to neu and possibly other tumor-associated antigens. Experimental autoimmune thyroiditis induced in resistant BALB/c mice. We have shown that elimination of Treg in mTg-tolerized CBA/J mice resulted in their reversion to the susceptible phenotype (4, 24). To test whether depletion of Treg in BALB/c mice, an experimental autoimmune thyroiditis–resistant mouse strain, would render them susceptible, mice were treated with anti-CD25 mAb twice, 4 days apart. On days 5 and 12 after the second mAb injection, mice received 40 µg of mTg and 20 µg of lipopolysaccharide (29), and they were sacrificed 4 weeks after the second immunization. Anti-mTg antibody was measured by ELISA. Depletion of Treg before mTg and lipopolysaccharide treatment resulted in a significant increase in anti-mTg antibodies (P = 0.003; Fig. 4A), including both IgG1 and IgG2a. To measure T-cell response, splenocytes were cultured with mTg for 4 days and cell proliferation was measured by [3H]thymidine incorporation. A significant increase (P = 0.002) in T-cell response was detected in mice depleted of Treg (Fig. 4B). The incidence and severity of thyroiditis was significantly increased (P = 0.01) with Treg depletion (Fig. 4C). Compared with control mice without anti-CD25 mAb treatment, the incidence of thyroid infiltration was 100% (7 of 7) versus 43% (3 of 7). Follicular destruction which is associated with >10% mononuclear infiltration of the thyroid was detected only in Treg-depleted mice. Therefore, removal of Treg enabled "resistant" BALB/c mice to develop moderate experimental autoimmune thyroiditis.
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100 mm3 and three of four tumors regressed completely. In all seven mice which received anti-CD25 mAb and mTg, TUBO tumors completely regressed. A repeated experiment showed similar results. Therefore, TUBO tumors were rejected following Treg depletion regardless of the presence of mTg.
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–producing cells were detected following tumor regression. Figure 5D shows the result of a representative experiment from four independent analyses. Reactive T cells increased to 45 per 106 spleen cells after the mice were also immunized with mTg. Neu-specific IL-4–producing cells were not detected (not shown). Therefore, injection of mTg in TUBO cell–bearing mice without anti-CD25 mAb treatment did not result in enhanced anti-neu immunity (Fig. 5C and D), demonstrating that exposure to mTg, a self-antigen, per se did not enhance antitumor immunity. Rather, depletion of regulatory T cells resulted in tumor regression with anti-neu immunity, which is further enhanced when anti-mTg reactivity is also triggered with this regimen.
To assess anti-mTg response in Treg-depleted mice which received mTg immunization after TUBO cell inoculation, serum antibody to mTg was measured. In three of seven mice, the antibody level was higher than that in mice which were immunized with mTg after Treg depletion, but were not inoculated with tumor cells, although the difference between these two groups was not statistically significant with this sample size (Fig. 6A). Mice inoculated with TUBO cells and treated with anti-CD25 mAb did not produce anti-mTg antibody. Anti-mTg antibody in Treg-depleted, mTg-immunized mice did not interact with neu when measured by flow cytometry (not shown), showing the absence of cross-reactivity between mTg and neu. mTg-specific T-cell proliferation or cytokine production was not detected at week 9 (not shown). To amplify possible T-cell responses, spleen cells were cultured with mTg for 3 days before they were transferred to ELISPOT plates precoated with anti–IFN- (Fig. 6B) or anti–IL-4 (Fig. 6C). Using this two-step ELISPOT assay, 612 ± 31 IFN-–producing and 79 ± 10 IL-4–producing cells per 106 spleen cells were detected in Treg-depleted mice which received TUBO and mTg. Mice inoculated with TUBO cells and injected with mTg did not develop T-cell response to mTg (not shown). Histologic analysis of the thyroid glands revealed low level of mononuclear cell infiltration, consisting mostly of T lymphocytes and macrophages as we previously reported (36, 40), in 3 of 7 mice (43%; Fig. 6D). Photomicrograph showing an area of thyroid with mononuclear cell infiltration is shown in Fig. 6E. There were no pathologic changes in thyroids of mice which received mTg without experiencing tumor. Therefore, depletion of Treg enhanced autoreactivity to mTg with further elevation after tumor regression, leading to thyroid infiltration.
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Discussion |
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Anti-neu and anti-mTg IgG1 and IgG2a were detected in Treg-depleted mice, suggesting the activation of both Th1 and Th2 cells. When T-cell response was tested, neu-specific IFN-–producing cells, but not IL-4–producing cells, were detected. mTg-specific IFN-– and IL-4–producing T cells were detected in the amplified two-step ELISPOT assay with greater number in IFN-–producing cells. These results show that depletion of Treg enhanced both Th1 and Th2 immunity.
T-cell response to mTg is usually evaluated by [3H]thymidine incorporation because, unlike anti-mTg antibody response, in vitro proliferation generally correlates with thyroid infiltration (36). Compared with T-cell proliferation, ELISPOT assay for IFN-–producing cells is more sensitive in detecting T-cell response. It is possible to further amplify the sensitivity of ELISPOT assay by the two-step incubation. Immune cells were expanded by preculturing with the antigen for 3 days before they were subjected to ELISPOT assay. If we estimate that effector cells divide every 12 to 16 hours on antigen stimulation, the cell number would increase between 16- and 64-fold in 3 days. Every 16 to 64 spots detected in the amplified ELISPOT assay would represent one responding cell in the starting population. The sensitivity of ELISPOT is increased significantly by preincubation, yet the difference between the control and test groups is unequivocal.
Induction of experimental autoimmune thyroiditis in Treg-depleted BALB/c mice indicated that resistance to experimental autoimmune thyroiditis determined by MHC class II was influenced in part by Treg because mononuclear cell infiltration was much less pronounced in resistant BALB/c mice carrying intact Treg (Fig. 4C). After depletion, greater humoral and cellular immunity to mTg was induced by the conventional immunization with mTg and lipopolysaccharide (Fig. 4A and B). When immunized by repeated mTg injection without lipopolysaccharide as adjuvant, a mild response to mTg was induced, but the response was significantly elevated in mice undergoing tumor regression such that more mice developed inflammatory infiltration in their thyroids (Fig. 6D). The mononuclear cell infiltration of 10% in this experiment was insufficient to cause thyroid hormonal changes. Nevertheless, this synergy between anti-neu and anti-mTg responses was both intriguing and alarming. Although the specific mechanisms remain to be delineated, it is possible that repeated injections of the self-antigen mTg in Treg-depleted mice not only stimulated mTg-specific T cells, but that the attendant inflammatory cytokines released systemically may also enhance antitumor response. From the tumor standpoint, in Treg-depleted mice, TUBO tumors grew to palpable size before sufficient immune effectors were generated to commence tumor regression. At the tumor site, tumor cell destruction can be envisioned following immunologic attack. A cascade of immunologic events may lead to elevated reactivity to both mTg and tumor-associated antigens.
In cancer patients, more strenuous effort than one-time depletion may be required to overcome immune regulatory mechanisms before effective antitumor immunity can be induced, with increasing risk of autoimmunity from each new regimen. Our results showed that even genetically resistant BALB/c mice became more susceptible to experimental autoimmune thyroiditis once Treg were removed, particularly during tumor regression. Patients expressing high-risk HLA haplotypes will require close monitoring of their autoreactivity to self-antigens when their immune regulatory mechanisms are modulated. In a pilot study to test Her-2 peptide vaccine with Flt3 ligand administered systemically as an adjuvant, 2 of 15 subjects developed elevated thyroid stimulating hormone with symptoms of grade 2 hypothyroidism, indicating thyroid destruction. Thyroid hormone replacement therapy was required at the conclusion of the study (22). Significantly elevated levels of antibody to thyroglobulin and thyroid peroxidase were detected in one patient, demonstrating autoimmunity to thyroid antigens. In another study, 14 patients with metastatic melanoma received gp100 peptide vaccines along with mAb to CTLA-4, which is expressed on regulatory and activated T cells (23). In six patients, grade III/IV autoimmune manifestations were observed, including dermatitis, enterocolitis, hepatitis, and hypophysitis. The three patients with objective cancer regression all developed severe autoimmune symptoms requiring treatment. Because of the grade III/IV autoimmune toxicity in 
3 patients, accrual intended for 21 patients ceased after 14 patients were enrolled. Therefore, immunomodulating reagents which can amplify antitumor immunity in a profound manner can trigger significant autoimmunity to self-antigens. For patients with genetic predisposition, the risk of autoimmunity may be overwhelming. With the sensitive in vitro assays to monitor immune reactivity to self-antigens, it may be possible to detect the onset of autoimmunity during cancer immunotherapy before clinical symptoms and counter measures may be taken in a timely fashion.
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Acknowledgments |
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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.
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Footnotes |
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Received 3/21/05. Revised 6/ 2/05. Accepted 7/11/05.
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and the costimulatory molecules CD40L and CD28. Cell Immunol 2001;208:52–61.[CrossRef][Medline]
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) or after (
) tumor cell inoculation or in control mice (+). The results are expressed as percentage of tumor-free mice. Inset, expression of neu on TUBO cells was verified by flow cytometry using mouse anti-rat neu mAb (7.16.4, filled histogram) followed by phycoerythrin-anti-mouse immunoglobulin, and compared with isotype controls (open histogram). B, anti-neu IgG levels in mice treated with anti-CD25 before or after TUBO cell inoculation were measured by flow cytometry and calculated by regression analysis. C, IgG1 and IgG2a levels in mice treated with anti-CD25 mAb after TUBO cell inoculation are reported as mean channel fluorescence (MCF). D, T-cell response to neu antigen. Following anti-CD25 mAb treatment and tumor regression, tumor-free mice were sacrificed at week 9 and their spleen cells isolated and cultured with 3T3/K or 3T3/NKB cells. IFN-
) and five mice with D2F2/neu tumor cells (
). Control groups were naïve mice inoculated with D2F2/neu (
) or TUBO (+) cells. B, flow cytometric analysis of D2F2/neu cells (filled histogram). Normal mouse immunoglobulin was used as control (open histogram).
) mTg treatment. Mice in the control group received PBS (
) or mTg (x). B, tumor volume of individual mice in A. C, at week 10, anti-neu antibody was measured by flow cytometry; D, IFN-