A novel transgenic mouse model for immunological evaluation of carcinoembryonic antigen–based DNA minigene vaccines (original) (raw)
A CEA minigene encoded by expression vectors is expressed in mammalian cells. To achieve optimal vaccine efficacy, three expression vectors were constructed based on the backbone of pCMV/ER/Myc (Figure 1A). To check gene expression, 293T cells were transfected with either pHI-myc, pHI-CAP1-6D-myc, or pHI-691-myc using Lipofectamine 2000 (Invitrogen). Protein expression was assessed by Western blotting of cell lysates with monoclonal anti–myc Ab (Invitrogen), and single bands with the expected molecular weight of 15 kDa or 16 kDa were detected (Figure 1B). A lysate from cells transfected with pHI-CAP1-6D-myc revealed patterns identical to those of pHI-691-myc (data not shown). The vaccine vectors pHI, pHI-CAP1-6D, and pHI-691 were generated by introducing a stop codon immediately downstream from the peptide coding sequences, so that mature peptides did not contain the myc epitope. Structures were confirmed by DNA sequencing, and empty pCMV vectors were also included for control purposes.
Expression vectors are constructed and verified. (A) Schematic map of vector constructs. Minigenes encoding HIVtat translocation peptide, a spacer, and human CEA epitopes CAP1-6D or CEA691 were assembled by PCR with overlapping oligonucleotides as templates. The PCR fragments generated were cloned into a pCMV vector by using _Bss_HII and _Xho_I restriction sites. (B) Proteins encoded by minigenes were expressed in mammalian cells. This expression was indicated when 293T cells were transfected with either pHI-myc or pHI-691-myc for 24 hours, harvested, lysed, and analyzed by Western blotting with monoclonal antibody against myc.
CEA691-specific, HLA-A2–restricted immunity is induced by a DNA vaccine encoding the pHI-691 CEA minigene. To verify that a specific immune response was induced by the minigene vaccines, vaccination vectors were transfected into doubly attenuated S. typhimurium (dam–; AroA–) by electroporation and used to vaccinate C57-CEA-A2Kb double transgenic mice three times at 2-week intervals. To this end, ELISPOT assays were performed 2 weeks after the last immunization with splenocytes isolated from immunized mice; synthetic peptides (10 ∝g/ml) were applied as stimulators. All experimental groups revealed an increased number of spots when splenocytes were stimulated with the CEA691 peptide, compared with unstimulated splenocytes. Although this increase is minimal in the groups of mice immunized with either the pCMV, pHI, or pHI-CAP1-6D DNA vaccines, it is quite dramatic in the mice vaccinated with pHI-691 (Figure 2A), which suggests the existence of CEA691-specific cells that can be induced to secrete IFN-γ in C57-CEA-A2Kb double transgenic mice.
An HLA-A2–restricted, CEA691-specific response is induced by the pHI-691 DNA minigene vaccine. Groups of C57BL/6-CEA-A2Kb mice (n = 4) were immunized three times at 2-week intervals with attenuated S. typhimurium harboring the vectors indicated. Two weeks after the last immunization, mice were sacrificed and ELISPOT assays performed on splenocytes isolated by using synthetic peptides (10 ∝g/ml) as stimulators (A). The remaining splenocytes were stimulated with irradiated MC-38-CEA-A2Kb cells for 5 days, and ELISPOT assays were then performed using either irradiated unloaded or peptide-loaded T2 cells as stimulators (B). (C) HLA-A2 expression of T2 (left) and B3 (right) cells. B3 is an EBV-transformed cell line generated from a healthy HLA-A2+ individual as described in Methods. Cells were treated with control antibody (thin dashed lines) or anti–HLA-A2 antibody (thick solid lines) and then stained with PE-conjugated goat anti–mouse Ig.
However, in all experimental groups, stimulation of splenocytes with the CAP1-6D peptide failed to significantly increase the number of spots compared with unstimulated splenocytes; that is, CAP1-6D–specific, IFN-γ–secreting cells could not be detected. This finding suggests that CAP1 may not be a dominant Ag epitope in CEA-A2Kb double transgenic C57BL/6J mice, despite the fact that it is one of the more dominant HLA-A2–restricted, CEA-specific epitopes in humans (9, 17). This notion is further supported by the finding that no CAP1-6D–specific, IFN-γ–secreting cells were detected in mice immunized with a vector encoding the full-length CEA gene (data not shown).
To confirm that the CEA691-specific immune response induced was indeed HLA-A2 restricted, splenocytes from vaccinated mice were also cultured in vitro in the presence of irradiated (1,000 Gy) MC-38-CEA-A2Kb cells for 5 days and thereafter used in ELISPOT assays. HLA-A2+ human T2 cells deficient in transporter associated with antigen processing (TAP) were used as stimulators. Such cells were used either unloaded or loaded overnight with 10 ∝g/ml CAP1-6D or CEA691 peptides and irradiated (1,000 Gy). In mice immunized with the pHI-691 DNA minigene vaccine, an increased number of IFN-γ–secreting cells was detected when splenocytes from such mice were stimulated with CEA691-loaded T2 cells. This increase was particularly evident when compared with splenocytes that were stimulated with either unloaded or CAP1-loaded T2 cells (Figure 2B). Similar results were obtained by substituting T2 cells with another HLA-A2+ cell line, B3 (data not shown). B3 is an EBV-transformed human B cell line established in our laboratory from a normal individual, which maintained high levels of HLA-A2 expression (Figure 2C). Because HLA-A2 is the only MHC molecule in common among the stimulating T2 or B3 cells of human origin and the responding CEA-A2Kb transgenic mouse splenocytes, these data suggest that the CEA691-specific reaction observed is indeed HLA-A2 restricted.
When compared with those with no stimulation added to the splenocytes, the addition of irradiated T2 cells by themselves induced more splenocytes to secrete IFN-γ in every experimental group tested. However, this finding is not surprising because of the human origin of T2 cells. This reaction was stronger in the pHI group than in the pCMV vaccine group of mice, which suggests that pHI is an effective immune stimulator. Moreover, stimulation with CEA691-loaded T2 cells failed to induce more cells to secrete IFN-γ in the pCMV, pHI, and pHI-CAP1-6D experimental groups of mice. This failure may be caused by the dominance of the responses induced against T2 cells, which are xenogeneic, except for their HLA-A2 expression.
HLA-A2–restricted, CEA691-specific responder cells are cytotoxic. The fact that the HLA-A2–restricted, CEA691-specific responder cells are cytotoxic was confirmed with standard 51Cr-release assays performed with splenocytes isolated from immunized mice. Target cells included CAP1-6D–loaded or CEA691-loaded T2 cells. CAP1-6D–loaded T2 cells were used as a control because CAP1-6D–specific cells could not be detected in ELISPOT assays. Splenocytes obtained from the pHI-691 experimental group of mice exhibited significantly higher killing of CEA691-loaded T2 cells than did splenocytes from pCMV, pHI, and pHI-CAP1-6D control groups (P < 0.0005, _P_ < 0.0005, and _P_ < 0.005 at an effector/target [E/T] ratio of 100:1, respectively [Figure 3A]). However, when T2 cells loaded with CAP1-6D were used as targets, the cytotoxic activity of splenocytes isolated from the pHI-691–immunized group was less significant compared with that of mice vaccinated with either pHI or pHI-CAP1-6D (_P_ < 0.05 and _P_ > 0.05 at an E/T ratio of 100:1, respectively [Figure 3B]), which indicates the specificity of this cytotoxic activity. As previously mentioned, because both effector mouse splenocytes and human T2 target cells only share HLA-A2, this specific cytotoxicity might be HLA-A2 restricted as well.
HLA-A2–restricted, CEA691-specific T cells induced by the pHI-691 minigene vaccine are cytotoxic. Groups of C57BL/6-CEA-A2Kb mice (n = 4) were immunized three times at 2-week intervals with attenuated S. typhimurium harboring the vectors indicated. Mice were sacrificed 2 weeks after the last immunization, and isolated splenocytes were stimulated with irradiated MC-38-CEA-A2Kb cells for 5 days. Cytotoxicity assays were performed with (A) CEA691-loaded T2 cells or (B) CAP1-6D–loaded T2 cells as targets.
CD8+ T cells mediate HLA-A2–restricted cytotoxic killing. To verify that the induced HLA-A2–restricted, CEA691-specific killing was mediated by CD8+ T cells, freshly isolated splenocytes were fractionated into CD8+ and CD8– subpopulations by magnetic-activated cell sorting (MACS). The purity of the cell fractions is shown in Figure 4A. These fractionated cells were stimulated with irradiated MC-38-CEA-A2Kb in the presence of IL-2 for 5 days, and then standard 51Cr-release assays were performed using CEA691-loaded T2 cells as targets. Killing of target cells was only observed with the CD8+ population, whereas the CD8– population showed hardly any killing at all (Figure 4B). The CD8+ population from the pHI-691 experimental group of mice showed higher cytotoxicity than that of the pHI-CAP1-6D group, which was used as a control group in this experiment (P < 0.005, P < 0.005, and P < 0.05 at E/T ratios of 20:1, 10:1, and 5:1, respectively [Figure 4C]).
DNA minigene–induced cytotoxicity is mediated by CD8+ T cells. Groups of C57BL/6-CEA-A2Kb mice (n = 4) were immunized three times at 2-week intervals with attenuated S. typhimurium harboring either pHI-CAP1-6D or pHI-691 vectors. Mice were sacrificed 2 weeks after the last immunization, and isolated splenocytes were fractionated into CD8+ or CD8– populations. Each cell population was stimulated with irradiated MC-38-CEA-A2Kb cells for 5 days. Thereafter, cytotoxicity assays were performed with CEA691-loaded T2 cells as targets. (A) Purity of each subpopulation after MACS. (B) Cytotoxicity of each cell population at E/T ratio of 20:1. (C) Cytotoxicity of CD8+ populations is shown at different E/T ratios.
When CD8+ cells were enriched by MACS, the percentage of CD8+ T cells increased from approximately 30% in the unfractionated culture to more then 90% in the CD8+ cell culture. However, the CD8+ fraction did not show higher killing rates (Figure 4C) when compared with unfractionated splenocytes (Figure 3A), presumably because of the requirement of CD8– T cells for optimal proliferation in cell culture. One line of evidence supporting this notion is that culture of the CD8+ fraction resulted in lower CD8 expression when compared with the unfractionated cultured splenocytes (data not shown).
Taken together, these data indicate that the pHI-CEA691 minigene vaccine can effectively induce an HLA-A2–restricted, CEA691-specific CTL response in CEA-A2Kb double transgenic mice.
Protective immunity against CEA-A2Kb–transduced colon carcinoma cells. HLA-A2–restricted, CEA691-specific CTLs, induced by the pHI-691 minigene DNA vaccine, recognized and killed CEA-A2Kb doubly transduced MC-38 murine colon carcinoma cells, that is, MC-38-CEA-A2Kb cells, whose surface expression of HLA-A2 and CEA is depicted in Figure 5A (left). This process was demonstrated by isolating splenocytes from mice 2 weeks after the last immunization and stimulating them in vitro with irradiated MC-38-CEA-A2Kb cells for 5 days and then testing them in a standard 51Cr-release assays. As expected, splenocytes from the group of mice immunized with the pHI-691 vaccine showed the most effective cytotoxic killing of MC-38-CEA-A2Kb target cells when compared with pCMV, pHI, and pHI-CAP1-6D control groups (P < 0.0005, _P_ < 0.001, and _P_ < 0.005 at an E/T ratio of 100:1, respectively [Figure 5B]). This enhanced killing ability appeared to be HLA-A2 restricted because the killing percentage of the HLA-A2– MC-38-CEA cells (HLA-A2 and CEA expression shown in Figure 5A [right]) or parental MC-38 target cells did not differ significantly among the experimental groups (_P_ > 0.05 at an E/T ratio of 100:1 [Figure 5C and data not shown]). Taken together, these data suggest that those immune cells that presumably are HLA-A2 restricted and CEA691-specific CTLs, induced by the pHI-691 vaccine, are indeed capable of recognizing and killing CEA-A2Kb doubly transfected MC-38 cells in vitro.
The DNA minigene vaccine pHI-691 induces HLA-A2–restricted killing of MC-38-CEA-A2Kb cells. (A) Surface expression of HLA-A2 and CEA by MC-38-CEA-A2Kb (left) and MC-38-CEA (right). Tumor cells were washed and incubated with isotype control Ab (thin dashed lines), anti–HLA-A2 (thin solid lines), or anti-CEA (heavy solid lines) and stained with PE-conjugated (Fab′)2 of goat anti–mouse Ig Ab. Groups of C57BL/6-CEA-A2Kb mice (n = 4) were immunized three times at 2-week intervals with attenuated S. typhimurium harboring the vectors indicated. Mice were sacrificed 2 weeks after the last immunization, and isolated splenocytes were stimulated with irradiated MC-38-CEA-A2Kb cells for 5 days. Thereafter, cytotoxicity assays were performed with (B) MC-38-CEA-A2Kb or (C) MC-38-CEA as target cells. max, maximum.
To test the specificity of this immune response, splenocytes isolated from pHI-691 vaccinated group of mice were stimulated weekly with irradiated MC-38-CEA-A2Kb cells. These cells, after being restimulated four times, were more than 86% CD8+ as demonstrated by flow cytometry (Figure 6A). These stimulated cells were then harvested and analyzed by 51Cr-release assays against either unloaded or peptide-loaded HLA-A2+ B3 cells. In 51Cr-release assays, the cytotoxic killing efficacy of CEA691-loaded B3 cells was greatly enhanced; that is, a much higher killing percentage was observed at lower E/T ratios, with the specificity still being maintained as indicated by the lower percentage of specific killing of unloaded B3 cells (Figure 6B).
Repetitive stimulation with MC-38-CEA-A2Kb cells results in enriched HLA-A2–restricted, CEA691-specific CTLs. C57BL/6-CEA-A2Kb mice (n = 4) were immunized three times at 2-week intervals with attenuated S. typhimurium harboring the pHI-691 vector. Mice were sacrificed 2 weeks after the last immunization, and isolated splenocytes were stimulated weekly with irradiated MC-38-CEA-A2Kb cells. After four stimulations, cells were harvested and cytotoxicity assays performed. (A) Expression of CD8 on stimulated cells. Stimulated splenocytes were stained with PE-conjugated isotype control antibody (thin dashed line) or anti-CD8 Ab (thick solid line). (B) Cytotoxicity assays performed with either unloaded or CEA691-loaded B3 cells as targets.
The presence of T cells able to kill tumor cells in the circulation of vaccinated animals suggests that this T cell response may also be effective in vivo, that is, protecting mice from a tumor challenge. To test this hypothesis, mice were challenged subcutaneously in the right flank with 5 ∞ 105 MC-38-CEA-A2Kb cells 2 weeks after the last immunization. These animals were sacrificed 28 days later, and tumor weights were assessed. In all groups of experimental animals, tumor size varied considerably within each group. This finding is not too surprising because of the complex genetic background of the doubly transgenic mice and tumor cell lines. However, the average tumor size was still reduced in the pHI-691 group, and the difference reached significance when compared with the pCMV group (P < 0.02 [Figure 7A]). Importantly, 3 of 10 mice rejected the tumor challenge completely, whereas all mice in the pCMV, pHI, and pHI-CAP1-6D experimental groups uniformly developed tumors (Figure 7A). This experiment was repeated with similar results (data not shown). The protection by the minigene vaccine is specific for MC-38-CEA-A2Kb tumor cells as well as HLA-A2 restricted because no protection was observed against parental MC-38-CEA and MC-38 tumor cells (Figure 7B).
DNA minigene vaccine pHI-691 exhibits HLA-A2–restricted antitumor effects in CEA-A2Kb double transgenic mice. Groups of mice (n = 7–10) were immunized three times at 2-week intervals with attenuated S. typhimurium harboring the vectors indicated. Mice were challenged 2 weeks after the last immunization subcutaneously with 5 ∞ 10S5 MC-38-CEA-A2Kb, MC-38, or MC-38-CEA cells. Mice were sacrificed 28 days later and tumor weights assessed. (A) MC-38-CEA-A2Kb tumor weight, the horizontal bar showed the average. (B) Average tumor weight of each group of mice (n = 8) challenged with different tumor cells. Top shows MC-38 tumor size; bottom shows average MC-38-CEA tumor weight. *P < 0.02 compared with pCMV group.