Zen Wei

Concurrent Induction of Antitumor Immunity and Autoimmune Thyroiditis in CD4+CD25+ Regulatory T Cell–Depleted Mice

When CD4+ CD25+ regulatory T cells are depleted or inactivated for the purpose of enhancing antitumor immunity, the risk of autoimmune disease may be significantly elevated because these regulatory T cells control both antitumor immunity and autoimmunity. To evaluate the relative benefit and risk of modulating CD4+ CD25+ regulatory T cells, we established a new test system to measure simultaneously the immune reactivity to a tumor-associated antigen, neu, and an unrelated self-antigen, thyroglobulin. BALB/c mice were inoculated with TUBO cells expressing an activated rat neu and treated with anti-CD25 monoclonal antibody to deplete CD25+ cells. The tumors grew, then regressed, and neu-specific antibodies and IFN-gamma-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-gamma-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.

Human ErbB-2 (Her-2) Transgenic Mice: A Model System for Testing Her-2 Based Vaccines

Her-2 transgenic (Tg) mice were generated with wild-type human c-ErbB-2 (Her-2) under the whey acidic protein promoter. They are tolerant to Her-2 and appropriate for testing Her-2 vaccines. The expression of transmembrane ErbB-2 from the whey acidic protein-Her-2 cassette and its up-regulation by insulin and hydrocortisone was verified by in vitro transfection. The transgene cassette was microinjected into fertilized eggs from B6C3 (C3H × C57BL/6) females mated with B6C3 males. Transgene-positive mice were backcrossed onto C57BL/6 mice. Human ErbB-2 was expressed in the secretory mammary epithelia during pregnancy and lactation and expressed constitutively in the Bergman glia cells within the molecular layer of the cerebellum. Overt, neoplastic transformation was not detected in any tissue examined. Tolerance to Her-2 was demonstrated by inoculating mice with a syngenic tumor expressing high levels of human ErbB-2. Tumors grew exclusively in Her-2 Tg mice without inducing an Ab response, while the nontransgenic littermates remained tumor free for 10 mo and mounted a robust anti-ErbB-2 Ab response. When immunized five times with plasmid DNA encoding secErbB-2 and GM-CSF, respectively, ∼33% of the Her-2 Tg mice rejected a lethal challenge of EL-4/E2 tumor cells, whereas all immunized littermates rejected the tumor. Therefore, Her-2 Tg mice express human ErbB-2 in the brain and mammary gland and demonstrated tolerance to ErbB-2 which was partially overcome by DNA vaccination. The breakable tolerance of Her-2 Tg mice resembles that in human and these mice are particularly suited for testing human ErbB-2 based vaccines.

Immunosurveillance of Erbb2 Carcinogenesis in Transgenic Mice Is Concealed by a Dominant Regulatory T-Cell Self-Tolerance

To assess the role of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells in overcoming immunosurveillance of Erbb2 (HER-2/neu) mammary lesions, we studied the effects of their sustained removal in BALB/c female mice made transgenic for the rat Erbb2 (r-Erbb2) oncogene (BALB-neuT mice), which develop multiple mammary carcinomas. During the progression of these lesions, Treg cells expand in the spleen, tumor draining lymph nodes, and tumors. Repeated administration of anti-CD25 antibodies extends tumor-free survival, reduces carcinoma multiplicity, and leads to the manifestation of a natural antibody and CTL-mediated reactivity against r-Erbb2. Loss of Foxp3(+) Treg cells during anti-CD25 treatment remarkably caused the disappearance of Gr1(+) immature myeloid cells, suggesting a cross-talk between these two inhibitory immune cell types. Treg cell expansion associated with r-Erbb2 overexpression may be seen as a physiologic response to dampen the immune reaction elicited by local anomalous overexpression of a self-antigen.

Cure of Mammary Carcinomas in Her-2 Transgenic Mice through Sequential Stimulation of Innate (Neoadjuvant Interleukin-12) and Adaptive (DNA Vaccine Electroporation) Immunity

Purpose: Whereas neoadjuvant therapy is emerging as a treatment option in early primary breast cancer, no data are available on the use of antiangiogenic and immunomodulatory agents in a neoadjuvant setting. In a model of Her-2 spontaneous mammary cancer, we investigated the efficacy of neoadjuvant interleukin 12 (IL-12) followed by “immune-surgery” of the residual tumor. Experimental Design: Female BALB/c mice transgenic for the rat Her-2 oncogene inexorably develop invasive carcinomas in all their mammary glands by the 23rd week of age. Mice with multifocal in situ carcinomas received four weekly i.p. injections of 100 ng IL-12 followed by a 3-week rest. This course was given four times. A few mice additionally received DNA plasmids encoding portions of the Her-2 receptor electroporated through transcutaneous electric pulses. Results: The protection elicited by IL-12 in combination with two DNA vaccine electroporations kept 63% of mice tumor-free. Complete protection of all 1-year-old mice was achieved when IL-12-treated mice received four vaccine electroporations. Pathologic findings, in vitro tests, and the results from immunization of both IFN-γ and immunoglobulin gene knockout transgenic mice and of adoptive transfer experiments all show that IL-12 augments the B- and T-cell response elicited by vaccination and slightly decreases the number of regulatory T cells. In addition, IL-12 strongly inhibits tumor angiogenesis. Conclusions: In Her-2 transgenic mice, IL-12 impairs tumor progression and triggers innate immunity so markedly that DNA vaccination becomes effective at late points in time when it is ineffective on its own.

CD4+ T cell-mediated HER-2/neu-specific tumor rejection in the absence of B cells

HER‐2/neu (HER‐2) is a cell surface proto‐oncogene that is often overexpressed in carcinomas. Passive administration of anti‐HER‐2 antibodies in breast cancer patients has achieved promising results, but less is known about the role of antibodies in active immunization. We asked whether B cells/antibodies are needed for tumor immunity induced by plasmid (HER‐2 and GM‐CSF) immunization. HER‐2 specific tumor immunity relied completely on both CD4+ and CD8+ T cells. IFN‐γ, and to a lesser extent IL‐4, seemed to be crucial cytokines during tumor rejection. Protection was associated with production of anti‐HER‐2 IgG antibodies in B cell competent mice. After immunization, however, B cell‐deficient mice rejected HER‐2‐expressing tumors as efficiently as control littermates. We conclude that T cells are the main effector cells in DNA vaccine induced immunity against HER‐2 and that anti HER‐2 antibodies are not necessary to elicit a protective anti tumor immune response in this model.

Broadening of Epitope Recognition During Immune Rejection of ErbB-2-Positive Tumor Prevents Growth of ErbB-2-Negative Tumor

Tumor heterogeneity is a limiting factor in Ag-specific vaccination. Ag-negative variants may arise after tumor cells bearing the immunizing Ags are destroyed. In situ priming to tumor-associated epitopes distinct from and not cross-reactive with the immunizing Ags may be crucial to the ultimate success of cancer vaccination. Immunization of BALB/c mice with DNA encoding wild-type human ErbB-2 (Her-2/neu, E2) or cytoplasmic ErbB-2 (cytE2), activated primarily CD4 or CD8 T cells, respectively, and both vaccines protected against ErbB-2-positive D2F2/E2 tumors. In ≥50% of protected mice, a second challenge of ErbB-2-negative D2F2 tumor cells was rejected. Recognition of non-ErbB-2, tumor-associated Ags was demonstrated by immune cell proliferation upon stimulation with irradiated D2F2 cells. This broadening of epitope recognition was abolished if CD4 T cells were depleted before D2F2/E2 tumor challenge, demonstrating their critical role in Ag priming. Similarly, mice that rejected D2F2/cytE2 tumor cells, which express only MHC I epitopes of ErbB-2, were not protected from a second challenge with D2F2 cells. Depletion of CD8 T cells abolished protection against D2F2, indicating the activation of D2F2-specific CTL. Therefore, long term protection may be achieved by immunization with dominant Ag(s), followed by a general enhancement of CD4 T cell activity to promote priming to multiple tumor-associated Ags.

Tumor Regression following DNA Vaccination and Regulatory T Cell Depletion in neu Transgenic Mice Leads to an Increased Risk for Autoimmunity

Modulation of the immune system to amplify anti-tumor immunity carries the risk of developing autoimmune diseases, including hypothyroidism, as seen with cancer patients undergoing clinical trials for immunotherapeutic regimens. Although there is a tendency to view autoimmunity as a positive indicator for cancer immunotherapy, some autoimmune manifestations can be life-threatening and necessitate prolonged medical intervention or removal from trial. We have established murine test models to assess such risks by monitoring, simultaneously, the immune reactivity to tumor-associated rat erbB-2 (neu) and another self Ag, mouse thyroglobulin (mTg). We previously reported that in wild-type, thyroiditis-resistant BALB/c mice that underwent regression of neu+ TUBO tumors following regulatory T cell (Treg) depletion, immune responses to rat neu and mTg with resultant autoimmune thyroiditis (EAT) were both enhanced. In this study, we tested the balance between tumor immunity and autoimmunity in neu-transgenic BALB NeuT female mice. First, growth and progression of neu+ tumor were compared in neu tolerant mice treated with either CD25 mAb to deplete Tregs and/or DNA vaccination. Only Treg depletion followed by neu DNA vaccination abrogated tolerance to neu, resulting in complete regression of neu+ tumors, as well as long-term protection from spontaneous tumorigenesis in 58% of mice. The risk of developing EAT was then assessed by incorporated mTg immunization with or without LPS as adjuvant. In mice with induced tumor regression, mTg response was enhanced with modest increases in EAT development. Therefore, tumor regression induced by Treg depletion and DNA vaccination can exacerbate autoimmunity, which warrants close monitoring during immunotherapy.

Quantitative measurement of anti-ErbB-2 antibody by flow cytometry and ELISA.

To establish standard methods for measuring anti-ErbB-2 antibody, a flow cytometric and an enzyme-linked immunosorbent assay (ELISA) were developed and compared. In the flow cytometric assay, the antibody was measured by binding to human breast cancer cell line SKBR3 and the result expressed as mean channel fluorescence (MCF). In ELISA, the antibody was measured by binding to a recombinant, secreted human ErbB-2 containing the N-terminal 505 amino acids of ErbB-2 fused to myc and His tags (secE2/myc/His or secE2) and the result expressed as O.D.(405). A mouse anti-human ErbB-2 mAb 9G6 was used as the standard. Using flow cytometry, MCF of 9G6 binding increased with linearity between 0.6 and 10 microg/ml. Using ELISA, O.D.(405) increased with linearity between 0.015 and 1 microg/ml, indicating greater sensitivity of ELISA. The titer of an immune mouse serum was determined to be 400 and 8000 with flow cytometric and ELISA assay, respectively, consistent with the assay sensitivity. Based on standard curves generated with 9G6, antibody concentration in the same serum sample was calculated to be approximately 230 microg/ml by ELISA and approximately 270 microg/ml by flow cytometry. Therefore, excellent concordance in antibody concentration was obtained with different assays using linear regression and properly diluted samples. This concordance was verified with multiple serum samples.

DNA Vaccination Controls Her-2+ Tumors that Are Refractory to Targeted Therapies

Her-2/neu(+) tumor cells refractory to antibody or receptor tyrosine kinase inhibitors are emerging in treated patients. To investigate if drug resistant tumors can be controlled by active vaccination, gefitinib and antibody sensitivity of four neu(+) BALB/c mouse mammary tumor lines were compared. Significant differences in cell proliferation and Akt phosphorylation were observed. Treatment-induced drug resistance was associated with increased chromosomal aberrations as shown by spectral karyotyping analysis, suggesting changes beyond neu signaling pathways. When mice were immunized with pneuTM encoding the extracellular and transmembrane domains of neu, antibody and T-cell responses were induced, and both drug-sensitive and drug-resistant tumor cells were rejected. In T-cell-depleted mice, drug-sensitive tumors were still rejected by vaccination, but drug-refractory tumors survived in some mice, indicating their resistance to anti-neu antibodies. To further test if T cells alone can mediate tumor rejection, mice were immunized with pcytneu encoding full-length cytoplasmic neu that is rapidly degraded by the proteasome to activate CD8 T cells without inducing antibody response. All test tumors were rejected in pcytneu-immunized mice, regardless of their sensitivity to gefitinib or antibody. Therefore, cytotoxic T lymphocytes activated by the complete repertoire of neu epitopes were effective against all test tumors. These results warrant Her-2 vaccination whether tumor cells are sensitive or resistant to Her-2-targeted drugs or antibody therapy.

Graves’ hyperthyroidism and thyroiditis in HLA-DRB1*0301 (DR3) transgenic mice after immunization with thyrotropin receptor DNA

Familial and twin studies in Caucasians have established that the MHC class II allele HLA‐DRB1*0301 (DR3) is a strong susceptibility gene in Graves’ hyperthyroid disease (GD). To determine if a DR3 transgene could help establish an animal model for GD, we expressed DR3 molecules in class II‐knockout NOD mice (H2Ag7–). DR3+g7– mice were given cardiotoxin prior to immunization on weeks 0, 3 and 6 with plasmid DNA encoding human thyrotropin receptor (TSHR). Two groups of mice were also coimmunized with plasmid DNA for IL‐4 or GM‐CSF. Serial bleeds on weeks 8, 11 and 14 showed that approximately 20% of mice produced thyroid‐stimulating antibodies (Abs), and approximately 25% had elevated T4 levels. In particular, a subset displayed both signs of hyperthyroidism, resulting in approximately 30% with some aspect of GD syndrome. Additional mice had thyroid‐stimulating blocking Abs and/or TSH‐binding inhibitory immunoglobulins, while most mice showed strong labelling of TSHR+ cells by flow cytometry. Interestingly, lymphocytic infiltration with thyroid damage and Abs to mouse thyroglobulin were also noted. Vector controls were uniformly negative. Thus, DR3 transgenic mice can serve as a model for GD, similar to our earlier reports that this allele is permissive for the Hashimotos thyroiditis model induced with human thyroglobulin.