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Immunology |
1 Karmanos Cancer Institute, Wayne State University, Detroit, Michigan and 2 Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia
Requests for reprints: Wei-Zen Wei, Karmanos Cancer Institute, Wayne State University, 110 E. Warren Avenue, Detroit, Michigan, 48201. Phone: 313-578-4651; Fax: 313-578-4658; E-mail: weiw{at}karmanos.org.
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Abstract |
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producing T-cell response. The same treatment induced modest improvement in B6 Her-2 Tg mice. In BALB Her-2 Tg mice, however, depletion of CD25hi T cells did not further improve antitumor efficacy. Although their Her-2–specific IgG1 and interleukin-5–secreting T cells increased, the levels of IgG2a and IFN-–secreting T cells did not change. These results are the first to show genetic regulation of the response to a cancer vaccine and an unequal effect of removing CD25hi T cells on antitumor immunity. These results warrant individualized treatment plans for patients with heterogeneous genetic backgrounds and possibly differential intrinsic immune reactivity to tumor antigens. [Cancer Res 2009;69(1):212–8] |
Introduction |
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ErbB-2, a member of the erbB receptor tyrosine kinase family, is weakly to moderately expressed in normal adult tissues (3). Human and rat homologues are designated as Her-2 and neu, respectively (4). Transphosphorylation of tyrosine residues in the cytoplasmic domain is triggered when Her-2 is dimerized with any erbB family member. The consequent signal transduction leads to cell proliferation, migration, adhesion, and transformation (5–8). Dysregulated signal transduction from overexpressed or mutated Her-2 leads to tumorigenesis (9). In 20% to 30% of human breast cancers, overexpression of Her-2 is correlated with more aggressive disease, resistance to chemotherapy and hormone therapy, and poor survival (4, 10–12).
Vaccination of wild-type mice with Her-2 DNA induces robust immune response and protects mice from tumor growth (13), with vaccines advancing to clinical trials (14). With the expected immune tolerance to self–Her-2 in humans, several Tg mouse strains expressing rat neu had been used to evaluate Her-2 vaccines (15, 16). But their immune reactivity to Her-2 did not mimic the response in humans because neu Tg mice responded to Her-2 as a foreign, rather than self-antigen (17). We previously showed that antibody (Ab) induced by Her-2 and neu DNA vaccines recognized only cognate antigen, although T-cell responses to Her-2 and neu were crossreactive (17). This disparate crossreactivity may contribute to some of the confusions in the literature.
To evaluate immunoreactivity to Her-2 as a self-antigen, we generated Her-2 Tg mice by expressing the human Her-2 gene under the whey acidic protein (WAP) promoter (18). Her-2 is expressed in mammary epithelia during pregnancy and lactation and constitutively in the Bergman glia cells within the molecular layer of the cerebellum, but overt neoplastic transformation has not been detected in any tissue. In Her-2 Tg mice, Her-2+ tumors grow progressively without inducing Her-2 Ab, showing immune tolerance to Her-2 in these mice (18).
We tested DNA vaccination in Her-2 Tg mice of BALB, B6, and F1 backgrounds and they showed different response profiles. Treatment with CD25 mAb to deplete regulatory T cells (Treg) is beneficial only in mice of B6 or F1 background that have more profound tolerance. These new findings warrant a personalized vaccination regimen for the heterogeneous patient population to achieve clinical benefit.
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Materials and Methods |
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Mice. All animal procedures were conducted in accordance with accredited institution guidelines and the US Public Health Service Policy on Humane Care and Use of Laboratory Animals. B6, BALB, and F1 female mice were purchased from Charles River Laboratory. Heterozygous C57BL/6 Her-2 Tg mice (B6 Her-2 Tg), which expressed the full-length, wild-type human Her-2 under the WAP promoter were generated in our laboratory and have been maintained by breeding with normal B6 mice (18). B6 Her-2 Tg mice were backcrossed onto the BALB/c background >13 generations to establish syngeneic, heterozygous BALB/c Her-2 Tg (BALB Her-2 Tg) mice. F1 Her-2 Tg mice (F1 Her-2 Tg) were generated by crossing BALB Her-2 Tg mice with wild-type B6 mice or vice versa. Transgene-positive mice were identified by PCR as we previously described (19).
Cell lines. The BALB/c mouse mammary tumor line D2F2 was established from a spontaneous tumor that arose in a prolactin-induced hyperplastic alveolar nodule line, D2 (20). The D2F2/E2 cell line was generated by stable transfection of D2F2 cells with the wild-type Her-2 (13, 17). EO771/E2 was generously provided by Dr. Daniel Allendorf (James Graham Brown Cancer Center, Louisville, KY) and was generated by transfecting the C57BL/6 mammary tumor EO771 with wild-type Her-2. BALB/c APCs 3T3/KB expressed H2Kd and B7.1 (CD80), and 3T3/EKB expressed H2Kd, CD80 and Her-2 (17). C57BL/6 APC TC-1 cells (generously provided by Dr. T.C. Wu, The Johns Hopkins University, Baltimore, MD; ref. 21) expressed endogenous H2Kb and B7.1 (17). TC-1/E2 expressed wild-type Her-2. SKOV3, a human ovarian cancer cell line with amplified Her-2 (22), was purchased from American Type Culture Collection (ATCC). The hybridoma line PC61 (ATCC), which produces rat mAb to mouse CD25 was propagated in severe combined immunodeficient mice. All cell lines were maintained as previously described (2, 17).
Analysis of Her-2 expression in cerebellum. Her-2 expression in the cerebellum of Her-2 Tg mice and transgene-negative littermates were analyzed by Western blot as we previously described (19). Briefly, cerebellum tissue from individual animals was finely cut and lysed in tissue lysis buffer containing protease inhibitors. Three milligrams of total protein were used for each immunoprecipitation reaction, and Her-2 protein was immunoprecipitated from the tissue lysates by incubation with trastuzumab (Genentech) and protein A/G-agarose (Santa Cruz Biotechnology). Proteins were eluted in 2x sample buffer and boiled for 5 min before fractionation in 4% to 20% gradient SDS-PAGE. Proteins were electrotransferred to Immobilon-P (Millipore) polyvinylidene difluoride membranes. Membranes were hybridized with mAb 42 (BD Biosciences), which specifically recognizes human Her-2, followed by horseradish peroxidase–conjugated secondary Ab and detected by chemiluminescence using a Kodak Imaging Station.
Immunization. Mice received DNA electrovaccination as we previously described (17). Briefly, 50 µg each of individual or mixtures of plasmid DNA in a total volume of 50 µL were injected into the quadriceps muscle. DNA injection was followed immediately by square wave electroporation over the injection site using a BTX830 (BTX Harvard Apparatus). A tweezer electrode was used to deliver 8 pulses at 100V for 25 ms per pulse. Wild-type mice were immunized twice and Her-2 Tg mice four times at two-week intervals. To deplete CD25hi Treg, indicated groups of Her-2 Tg mice received i.p. 0.25 mg of CD25 mAb, PC61, 10 d before the first electrovaccination. Depletion of CD25hi T cells was verified by flow cytometry (data not shown).
Measurement of Her-2 Ab by flow cytometry. Her-2 Ab levels were determined as previously described (23). Briefly, SKOV3 cells were incubated with serially diluted mouse sera, and phycoerythrin-conjugated goat anti-mouse IgG Fc was the secondary Ab (Jackson ImmunoResearch). Her-2 mouse mAb TA-1 (EMD Chemicals, Inc.) was used to generate standard binding curves. The concentrations of Her-2 IgG in the test sera were calculated by regression analysis (24). Differences in Ab levels were analyzed by two-tail Student's t test. To determine the IgG isotype, SKOV3 cells were incubated with immune sera at 1:150 dilution, followed by FITC-conjugated goat anti-mouse IgG1, IgG2a, or IgG2c (Jackson ImmunoResearch). Flow cytometric analysis was performed with a FACScalibur (BD Biosciences), and the results were expressed as mean channel fluorescence (MCF). Differences in Her-2 Ab isotype levels were analyzed by Student's t test.
Measurement of T-cell response by IFN- and interleukin-5 ELISpot. All ELISpot reagents were purchased from BD Biosciences, unless otherwise specified. Her-2 reactive T cell levels were enumerated by IFN- and interleukin (IL)-5 ELISpot assay as we previously described (17). Briefly, 96-well HTS IP plates (Millipore), were precoated with 2.5 µg/mL rat anti-mouse IFN- (clone R4-6A2) or rat anti-mouse IL-5 (clone TRFK5). Immune spleen cells (SC) were added to the wells in the presence of TC-1, TC-1/E2, 3T3/KB, or 3T3/EKB at APC/lymphocyte ratio of 1:10. After 48 h of incubation, cells were removed, and captured IFN- or IL-5 were detected with biotinylated rat anti-mouse IFN- (clone XMG1.2) or rat anti-mouse IL-5 (clone TRFK4), avidin-HRP and the substrate 3-amino-9-ethylcarbazole. The visualized cytokine spots were enumerated with the ImmunoSpot analyzer (CTL), and the results were expressed as number of cytokine-producing cells per 106 SCs. Data were analyzed using Student's t test.
Tumor growth inhibition assay. Her-2 Tg mice were inoculated s.c. in the flank with 2 x 105 EO771/E2 or D2F2/E2 cells 2 wk after the fourth electrovaccination. Tumor growth was monitored by weekly palpation, and tumor size was measured weekly in two perpendicular dimensions. Mice were sacrificed when any one dimension of the tumor reached 20 mm. Difference in tumor incidence was analyzed by the log-rank test.
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Results |
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T-cell response induced by Her-2 DNA vaccine. To analyze T-cell response, immune SCs from individual mice were prepared 2 weeks after the final vaccination. IFN- and IL-5 producing T cells were enumerated by ELISpot to evaluate Th1/Tc1 versus Th2 responses. B6 immune SC were incubated with TC-1/E2 cells, which were MHC I+, CD80+, and stably transfected with Her-2 to generate TC-1/E2 (17). BALB/c immune SC were incubated with 3T3/EKB cells, which were 3T3 cells stably transfected with Her-2, H2Kd, and CD80. T cells from F1 mice would respond to either TC-1/E2 or 3T3/EKB.
Vaccinated B6 Her-2 Tg mice showed little to no T-cell response (Fig. 4A and B, left
) as we reported (17), whether or not mice received CD25 mAb, demonstrating their profound T-cell tolerance to Her-2. In BALB Her-2 Tg mice, IFN-–producing T cells were induced at 50 ± 49/106 SC, but IL-5 response was not detected (Fig. 4A and B, middle). Depletion of CD25hi T cells before vaccination did not change IFN-–producing T-cell response, but IL-5–producing T cells were significantly amplified (Fig. 4B, middle). This preferential amplification of Th2 over Th1/Tc1 response is consistent with the increase in IgG1 but not IgG2a. These results may suggest the depletion of activated Th1 or selective enhancement of Th2 cells in BALB Her-2 Tg mice by treatment with 0.25 mg CD25 mAb.
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–producing T cells were induced sporadically and only after Treg depletion; 1 of 5 mice responded to TC-1/E2 and 2 of 5 mice responded to 3T3/EKB (Fig. 4A, right). IL-5–producing cells were not induced in any of the test mice, regardless of CD25 mAb treatment or the APC used for stimulation (Fig. 4B, right).
Interestingly, BALB Her-2 Tg, but not B6 Her-2 Tg or F1 Her-2 Tg mice, showed low level anti–Her-2 IFN-–producing T cells (
20 of 106 SC; data not shown) after CD25hi T-cell depletion, without vaccination, suggesting preexistence of Her-2 reactive T cells. Combined treatment with CD25 mAb and DNA vaccination may not result in a net gain of IFN-–producing T cells because these preexisting reactive T cells may be eliminated by CD25 mAb.
In summary, BALB Her-2 Tg mice were the most responsive to vaccination in both the humoral and cellular compartments. Their Th2, but not Th1/Tc1 response, was amplified when CD25hi T cells were removed. B6 Her-2 Tg mice were very tolerant to Her-2, and their T-cell response was not detectable whether or not CD25hi T cells were depleted, although both IgG1 and IgG2c were amplified in CD25 mAb treated mice, indicating T-cell involvement. To determine if treatment with CD25 mAb resulted in comparable Treg depletion in different mouse strains, Supplementary Fig. S1 showed the kinetics of depletion and recovery of Foxp3+ cells in BALB/c and C57BL/6 mice, which were treated with varying doses of CD25 mAb. At 0.25 mg of CD25 mAb, which was the dose used throughout this study, depletion of CD25hiFoxp3+ cells was achieved in both mouse strains. Partial recovery of these cells was observed at week 3 in C57BL/6 but not BALB/c mice. The more sustained depletion of CD25hiFoxp3+ cells in mice of BALB/c background may result in some loss of preexisting effector T cells in BALB Her-2 Tg mice with little gain in the levels of Th1/Tc1 and IgG2a.
Compared with B6 Her-2 Tg mice, F1 Her-2 Tg mice were more responsive. After CD25 mAb treatment, B6 and F1 Her-2 Tg mice produced comparable levels of IgG1 and IgG2c, but F1 mice also produced IgG2a. About 40% of F1 Her-2 Tg mice had detectable IFN- T-cell response. These results showed the influence of genetic background on immune reactivity to Her-2 vaccine. Treatment with CD25 mAb amplified immune reactivity and favors Th1/Tc1 response in F1 Her-2 Tg mice. In BALB Her-2 Tg mice, treatment with CD25 mAb increased Th2 response without a net gain in Th1 response, the latter may be due to partial loss of activated T cells.
Antitumor immunity in Her-2 Tg mice. The efficacy of DNA vaccination on tumor growth inhibition was tested by electrovaccinating mice four times with pE2TM and pGM-CSF before s.c. tumor challenge. B6 Her-2 Tg mice were inoculated with 2 x 105 EO771/E2, which was a B6 mammary tumor line stably transfected with Her-2. BALB and F1 Her-2 Tg mice received 2 x 105 D2F2/E2, which was a BALB/c mammary tumor line stably transfected with Her-2.
All control mice that received blank vector, regardless of their strain or challenging tumor cells, developed palpable tumors in 2 weeks, which grew progressively (Fig. 5A ). DNA vaccination was not protective against E0771/E2 tumor in B6 Her-2 Tg mice (Fig. 5A, left). Vaccination also failed to protect F1 Her-2 Tg mice from D2F2/E2 tumor (Fig. 5A, right). A significant delay of D2F2/E2 tumor growth was observed only in vaccinated BALB Her-2 Tg mice, although 10 of 12 mice eventually succumbed to tumors (Fig. 5A, middle). Therefore, antitumor immunity was poor in B6 and F1 Her-2 Tg mice but was significant in BALB Her-2 Tg mice.
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Immune reactivity to Her-2 vaccine in wild-type mice. The difference in Her-2 reactivity may be due to intrinsic differences in BALB and B6 mice. To test this possibility, age-matched wild-type B6, BALB, and F1 female mice were electrovaccinated twice with pE2TM and pGM-CSF without Treg depletion. B6 mice produced significantly more Her-2 Ab than BALB mice, and Ab levels in F1 mice were not significantly different than those in B6 or BALB mice (Fig. 6A ). Therefore, when Her-2 was presented as a foreign antigen, B6 mice were equally or more responsive than other mouse strains.
To measure T-cell response, SC from individual immunized animals were incubated with the corresponding APCs for 48 hours. IFN-–producing T cells were detected at 590 ± 149/106 and 658 ± 75/106 SC in wild-type B6 and BALB/c mice, respectively. IL-5–secreting T cells were at 179 ± 126/106 and 129 ± 66/106 SC for B6 and BALB mice, respectively (Fig. 6B and C). F1 mice showed 53 ± 63 and 1147 ± 337/106 SC IFN- T cells when stimulated with TC-1/E2 and 3T3/EKB, respectively (Fig. 6B), suggested a preferential activation of H2d restricted Th1/Tc1 cells in F1 mice. This preferential response to BALB APC was not observed in IL-5 response, which may be mediated by APC in the immune SCs after they take up Her-2 during the incubation (Fig. 6C).
Overall, wild-type BALB and B6 mice showed equivalent T-cell response to foreign Her-2 with B6 mice producing higher levels of Ab. The disparate response in B6 and BALB Her-2 Tg mice was not due to intrinsic difference of the mice but may result from differential thymic selection of Her-2–reactive cells during T-cell development.
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Discussion |
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We showed previously that Her-2/neu immunity was under the control of CD25hi Treg and was amplified by Treg depletion (2, 17, 26). In BALB Her-2 Tg, but not B6 or F1 Her-2 Tg mice, low level T-cell reactivity to self–Her-2 could be detected by simply removing CD25hi T cells without vaccination, indicating preexisting anti–Her-2 T cells in these mice (data not shown). When 0.25 mg CD25 mAb were administered before DNA vaccination, their Th2 response was amplified, but their Th1/Tc1 activity indicated by IgG2a and IFN- production did not change. Because CD25 mAb removed Treg as well as activated effector T cells, these results may indicate a loss of preactivated Th1/Tc1 cells in BALB Her-2 Tg mice when mice were treated with CD25 mAb. Because there was no net gain in tumor rejection, the increase in Th2 response was of little benefit. Therefore, in hosts with endogenous Her-2 reactivity such as BALB Her-2 Tg mice, specific measures for Treg depletion is warranted.
In B6 Her-2 Tg mice, immune reactivity to Her-2 was poor and moderate antitumor activity was achieved in Treg-depleted, vaccinated mice. Although cytokine producing T cells were not detected, Th1 and Th2-mediated production of IgG2c and IgG1, respectively, indicated the activation of T cells, which may be further amplified by additional immune modulating regimen.
When vaccinated after CD25 mAb treatment, F1 Her-2 Tg mice showed the most profound increase in tumor growth inhibition, compared with either parental strain. They generated IgG1, IgG2a, and IgG2c as well as some IFN-–producing T cells but no detectable IL-5–producing cells. Although their T-cell response was not as uniform as that in BALB Her-2 Tg mice, the ability to recognize antigenic epitopes presented by both H2b and H2d and the broader spectrum of Ab isotypes, combined with Th1/Tc1 activation may contribute to greater tumor rejection. These results may suggest more effective antitumor immunity in individuals with broader HLA phenotype and greater Th1/Tc1 activation.
Vaccinated mice live a normal life span without overt toxicity or autoimmunity. However, concurrent exposure to vaccine-independent, self-antigens during Treg depletion or tumor regression can increase their risk of autoimmune diseases to other self-antigens as we reported, and this should be closely monitored (2, 14, 26).
We previously showed that D2F2/E2 or E0771/E2, which were transfected with Her-2, but not dependent on Her-2 signaling for survival, were controlled primarily by T cells with lesser contribution from Ab (17). Consistent with this finding, activation of T cells in BALB and F1 Her-2 Tg mice was associated with greater tumor rejection, although contribution from innate immunity could not be ruled out. Because wild-type B6, BALB, and F1 mice all show excellent reactivity to foreign Her-2 and B6 mice actually produce more Ab than BALB mice, there is no intrinsic deficiency in B6 mice to recognize or respond to Her-2. Different Her-2 immune responses in B6 Her-2 Tg and BALB Her-2 Tg may be the culmination of several factors. B6 and BALB/c Her-2 Tg express MHC H2b and H2d, respectively, resulting in the presentation of different Her-2 epitopes and different repertoire of T cells through positive and negative thymic selection. It is well-established that MHC class II haplotype is the primary determining factor in autoimmune diseases (1). B6 mice express only one MHC class II allele, IA, whereas BALB/c mice express both IA and IE. The greater diversity of MHC class II alleles in BALB/c versus B6 mice could result in effector or regulatory CD4 T cells that are of different functional strength. Also, B6 and BALB/c mice produce a different spectra of IgG. B6 mice secrete IgG2c but not IgG2a (Fig. 3). BALB/c mice secrete IgG2a, but not IgG2c, and F1 mice produce both. Such discrepancy in IgG isotypes may result in different functionality. With F1 mice inheriting the genetic traits from both parents, their response decreases in between the two parental strains.
In addition, activation of IL-5–producing Th2 cells was observed only in CD25 mAb–treated BALB Her-2 Tg mice, but their IFN-–producing T cells did not change. BALB/c mice may have preexisting Th1/Tc1 Her-2 reactivity and the observed results may represent combined effect of removing a portion of Treg as well as preexisting effectors. Further analysis of genetic regulation is warranted to bring cancer vaccine to fruition.
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Disclosure of Potential Conflicts of Interest |
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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.
We thank Dr. Jessica Back, K. David Shim, and Andi Cani for their technical contribution to this study.
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Footnotes |
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Received 8/14/08. Revised 10/16/08. Accepted 10/29/08.
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