Article Figures & Data
Figures
- Fig. 1.
Induction of anti-CEA/OVA IgG serum titers in CEA.Tg mice. A, CEA.Tg mice (two mice/group) were given 20 μg of either CEA (▴), OVA (▪), or HBSS (•) in adjuvant twice at 2-week intervals. Two weeks after the final treatment, mice were bled, and serum was tested for the presence of anti-CEA or anti-OVA IgG antibodies as outlined in “Materials and Methods.” Data are the mean ± SE of the A490 nm value for each group. In some cases, the error bars are covered by the symbol. B, in the same experiment, CEA.Tg mice (four mice/group) were given 107 pfu (100 μl; s.c.) of V-Wyeth (▪) or rV-CEA (•). Unimmunized mice (▴) received an equal volume of HBSS. Data are the mean (SE < 10%) A490 nm values for four mice in the HBSS- and V-Wyeth-treated groups. A490 nm values are shown for the four individual rV-CEA-immunized CEA.Tg mice (average of triplicate determinations). Results in A and B are from a representative experiment; two to three experiments were performed for each immunogen with similar outcomes.
- Fig. 2.
Comparison of anti-CEA IgG responses in CEA.Tg mice and CEA-negative littermates. Groups (four mice/group) of CEA.Tg mice (•) and CEA-negative littermates (▴) were immunized three times (s.c.) with rV-CEA (107 pfu) at 2-week intervals. Serum samples were collected from individual mice 2 weeks after the final immunization and assayed for the presence of anti-CEA IgG antibodies. Results presented are the mean ± SE of the four mice in each group. In some instances, the error bars are covered by the symbol. Data are from a representative experiment that was repeated once with similar results.
- Fig. 3.
Comparison of CEA-specific CD4+ proliferative responses from CEA.Tg mice (A) and CEA-negative (B) littermates. Mice (two to three mice/group) were given 107 pfu of rV-CEA (▪) or V-Wyeth (▴) by tail scarification (10 μl), whereas control mice (four mice/group) were given an equal volume of HBSS (•). Fourteen days later, mice were sacrificed, spleens were pooled, T cells were isolated, and the proliferative responses to soluble CEA were measured by [3H]thymidine incorporation as described in “Materials and Methods.” Data are presented as the mean ± SE from a representative experiment. The experiment was repeated three to four times with similar results. Note that some error bars are covered by the symbol.
- Fig. 4.
Splenic CD4+ proliferation following a prime and boost immunization strategy. Groups of CEA.Tg mice (two to three mice/group) were immunized twice with 20 μg of CEA in adjuvant (▴), 107 pfu of V-Wyeth (▪), or 107 pfu of rV-CEA (▵) as described in “Materials and Methods.” Unimmunized CEA.Tg mice received HBSS (•). At sacrifice, spleens from each group were removed and pooled, T cells were isolated, and the lymphoproliferative assay was carried out. The SI was calculated as follows: [cpm (antigen-stimulated cells)]/[cpm (unstimulated cells)]. Each data point represents the mean ± SE of triplicate determinations from a representative experiment; two to three separate experiments were carried out with similar results.
- Fig. 5.
IFN-γ (▪) and IL-4 (□) production during CEA-mediated CD4+ T-cell proliferation. CEA.Tg mice (two to three mice/group) were immunized with HBSS, CEA, V-Wyeth, or rV-CEA as outlined in the legend of Fig. 4 <$REFLINK> . Spleens were pooled, and T cells were isolated and incubated with freshly isolated, irradiated antigen-presenting cells and 50 μg of CEA for 48 h. Results shown are the mean from triplicate wells (SE ± 15%) from a representative experiment. The experiment was repeated with similar results. No measurable amounts of either IFN-γ and IL-4 were found when splenic T cells from each group were cultured in the absence of CEA. Concanavalin A-stimulated splenic T cells from each group produced approximately 1–2 μg of IFN-γ and 20–30 ng of IL-4/106 cells during the same time period.
- Fig. 6.
Cytolytic activity of CEA526–533-derived CTLs. The cytolytic activity was tested against EL-4 target cells incubated in the presence of 0.2 μg of CEA526–533. CEA.Tg mice (two mice/group) were previously immunized with HBSS (•), CEA in adjuvant (▴), V-Wyeth (▪), or rV-CEA (▵) as outlined previously. Data presented are the mean ± SE from a representative experiment that was repeated once with similar results.
- Fig. 7.
CEA.Tg mice (•) and CEA-negative littermates (▴) were immunized s.c. with 107 pfu of rV-CEA or V-Wyeth three times at 2-week intervals. Two weeks after the third immunization, all mice were challenged with CEA-expressing tumor cells. Tumor volumes (mm3) for individual mice are shown at 35 days after tumor burden. Tumor-free mice are shown as zero tumor volume. Cumulative data from two independent experiments are presented.
Tables
- Table 1
Screening of CEA.Tg micea
Serum CEA (ng/ml) Genotype No. of mice RT-PCRa No. of mice Range Mean ± SE CEA.Tg mice 25 + 25 12.8–133.0 60.8 ± 14.9 CEA-negative littermates 25 Negb 10 Neg Neg Wild-type B6 mice 10 Neg 10 Neg Neg -
a Individual mice were bled between 28–35 days. Approximately 100 μl of whole blood were used for PCR-based detection of the CEA transgene. Serum was collected from the remainder and tested for CEA levels as described in “Materials and Methods.”
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b Neg, <5 ng CEA/ml serum.
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- Table 2
Analyses of immunoglobulin isotypes in immunized CEA.Tg mice and CEA-negative littermatesa
Immunoglobulin analysisa Mice Immunogen No. of mice Serum dilution IgG1 IgG2a IgG2b IgG3 CEA.Tg mice rV-CEA 1 10 ++ ++ ++ Neg 2 10 Neg + + Neg OVA + SDTb 1 50 ++ Neg Neg Neg 2 50 ++ Neg Neg Neg CEA-negative littermates rV-CEA ×4 1000 ++ ++ ++ Neg -
a Wells in 96-well plates were sensitized with the appropriate immunoglobulin subtype as outlined in “Materials and Methods.” Serum samples from immunized CEA.Tg mice were diluted 1:10 to 1:1250, and the assay was carried out as described. Identification of different immunoglobulin subtypes was based on relative A490 nm using the mean (A490 nm) + 3 SD for the appropriate immunoglobulin subtype as cutoff between negative and positive. For example, wells sensitized with IgG1 and incubated in the presence of serum from nonimmunized CEA.Tg mice had a mean A490 nm + 3 SD of 0.115 + 0.026 = 0.146. Therefore, all IgG-sensitized wells in which the A490 nm was >0.15 were scored +. Moreover, wells in which A490 nm exceeded 0.5 were scored as ++. Data represent the results from duplicate determinations.
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b SDT, super DETOX adjuvant.
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- Table 3
Blood chemistries of CEA.Tg mice and CEA-negative littermates immunized with rV-CEAa
CEA.Tg mice CEA-negative littermates Marker Reference (normal) range Untreated (n = 3) V-Wyeth (n = 3) rV-CEA (n = 10) Untreated V-Wyeth (n= 3) rV-CEA (n = 4) ASTb 72 –288 U/liter Normal Normal Normal Not tested Normal Normal ALT 24 –140 U/liter Normal Normal Normal Normal Normal Creatine phosphokinase 0 –800 U/liter Normal Normal ↑ (2/10) Normal Normal Amylase 602 –2311 U/liter Normal ↑ (3/3) ↑ (7/10) ↑ (2/3) ↑ (4/4) BUN 9 –28 mg/dl Normal Normal Normal Normal Normal Creatinine 0.2 –0.7 mg/dl Normal Normal Normal Normal Normal Bilirubin (total) 0.0 –0.9 mg/dl Normal Normal Normal Normal Normal Uric acid 2.2 –4.6 mg/dl Normal Normal Normal Normal Normal Albumin 2.6 –4.6 g/dl Normal Normal Normal Normal Normal Protein (total) 4.0 –6.2 g/dl Normal Normal Normal Normal Normal -
a CEA.Tg mice and CEA-negative littermates were immunized and subsequently challenged with CEA-expressing tumor cells as outlined in Fig. 7. <$REFLINK> Blood chemistries were performed on individual mice at approximately 1 month after tumor challenge.
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b AST, aspartate aminotransferase; ALT, alanine aminotransferase; ↑, elevation in blood markers; (n, n), number of mice with elevated marker, number of mice tested.
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- Table 4
Analyses of normal tissues from untreated and rV-CEA- and V-Wyeth-treated CEA.Tg micea
Treatment Normal tissue Histochemical analysisb CEA expressionc Untreated Intestinal tract Normal + Esophagus/stomach Normal + Kidneys Normal − Liver Normal − V-Wyeth Intestinal tract Normal + Esophagus/stomach Normal + Kidneys Normal ND Liver Normal ND rV-CEA Intestinal tract Normal + Esophagus/stomach Normal + Kidneys Normal − Liver Normal ND -
a Groups of CEA.Tg mice (two mice/group) were treated twice at 2-week intervals with 107 pfu of rV-CEA or V-Wyeth by tail scarification. Two weeks after the second treatment, mice were sacrificed, and spleens were used to generate the data summarized in Fig. 3 <$REFLINK> . Normal tissues were also removed and prepared for immunohistology. Data presented represent the results from both of the CEA.Tg mice in each group.
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b Routine histological examinations were conducted for each specimen on H&E-stained sections.
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c CEA expression as indicated by the presence of dark brown DAB cellular precipitate. ND, not done.
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Volume 59, Issue 3
