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Immunology |
Department of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, Italy [P. C., C. C., E. Q., M. G., G. F.]; Center for Experimental Research and Medical Studies, S. Giovanni Battista Hospital, 10126 Turin, Italy [P. C., C. C., M. G., G. F.]; Laboratory of Tumor Immunology, Faculté de Pharmacie, 92296 Chatenay-Malabry, France [F. T.]; Department of Oncology and Neurosciences, "G. D’Annunzio" University, 66013 Chieti, Italy [M. I., E. D. C., P. M.]; Cancer Research Section, Department of Experimental Pathology, University of Bologna, 40126 Bologna, Italy [P-L. L.]; Molecular, Cellular and Animal Biology Department, University of Camerino, 62032 Camerino, Italy [A. A.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and interleukin 4, and a marked expansion of both CD8+/CD11b+/CD28+ effector and CD8+/CD11b+/CD28- memory effector T-cell populations were induced in immunized mice. This combined vaccination also elicited a quicker and higher antibody response to r-p185neu, as well as an early antibody isotype switch. These data suggest that the appropriate costimulation provided by mLAG-3Ig enables DNA vaccination to establish an effective protection, probably by enhancing cross-presentation of the DNA coded antigen. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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HER-2/neu oncogene encodes a tyrosine kinase growth factor receptor (p185neu) homologous to other members of epidermal growth factor receptor family (3) . Its overexpression is frequent in human epithelial tumors and correlates with particular aggressiveness (4) . In the rat, a single point mutation replacing the valine residue at position 664 in the TM3 of r-p185neu with glutamic acid favors r-p185neu homo- and heterodimerization that converts the rat Her-2/neu proto-oncogene into a dominant transforming oncogene (5) .
BALB/c virgin female mice transgenic for the transforming rat HER-2/neu oncogene (referred to as BALB-neuT mice) provide one of the most aggressive models of rat HER-2/neu multifocal mammary carcinogenesis (6 , 7) . In 3-week-old BALB-neuT mice, r-p185neu is markedly overexpressed on the surface of the cells of the rudimental mammary gland (6) . At 6 weeks, the r-p185neu cells give rise to a widespread mammary atypical hyperplasia. Multiple microscopic masses somewhat equivalent to multiple carcinomas in situ are evident in all 10 mammary glands around week 10. These enlarge and converge in a rapidly growing, invasive, and metastasizing carcinoma that becomes palpable in all 10 glands between the 25th and 30th week of age (6, 7, 8) . In these mice, repeated vaccination with plasmids coding for distinct portions of r-p185neu alone (9 , 10) or in combination with the immunomodulatory 163–171 nonapeptide of IL-1ß (11) delayed tumor onset and reduced tumor multiplicity. However, this early protective response eventually declines as time progresses, and none or very few mice are tumor free at 1 year of age (Ref. 11 ; data not shown). This study reports the efficacy of DNA vaccination combined with the administration of soluble mouse LAG-3 (lymphocyte activation gene-3/CD223) generated by fusing the ECD of murine LAG-3 to a murine IgG2a Fc portion [mLAG-3Ig (12 , 13) ] in lengthening the protection offered by anti-r-p185neu DNA vaccination in BALB-neuT mice.
LAG-3 is a type I transmembrane protein associated with the T cell receptor-CD3 complex and binds MHC class II molecules in a manner similar to CD4. It is expressed in all subsets of T and natural killer cells after activation (14) , and its expression is up-regulated by IL-2 and IL-12 (15) . Soluble mLAG-3Ig engages MHC class II glycoprotein more efficaciously than CD4 (16) . The manner in which class II molecules of DCs are loaded with peptides and engaged may have consequences for the immune responses they induce (17) . The presence of LAG-3 on CD4 T cells binding certain forms of class II-associated peptides may more efficiently license DCs for class I peptide presentation to CD8 T cells (17) . For this reason, soluble mLAG-3Ig has been used as vaccine adjuvant for both conventional (18) and tumor antigens (13) .
The additive effect stemming from combining the vaccination with DNA plasmids coding for the extracellular domain and TM of r-p185neu (referred to as p185 plasmids) with mLAG-3Ig keeps most 1-year-old BALB-neuT mice tumor free and markedly extends their disease-free survival and reduces tumor multiplicity when multiple in situ carcinomas are already present.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells.
N202.1A (r-p185neu positive) and N202.1E (r-p185neu negative) cell clones were derived from mammary carcinomas of a FVB-neuN #202 mouse (H-2q) transgenic for the rat HER-2/neu proto-oncogene (19)
, whereas TUBO (r-p185neu positive) cell clones were obtained from a mammary carcinoma of a BALB-neuT mouse (H-2d; Ref. 9
). F1-F (H-2d) is a r-p185neu-negative skin fibroblast line that spontaneously transformed after the 15th in vitro passage (9)
. Cells were cultured in DMEM (BioWittaker, Walkerville, MD) supplemented with 20% (or 10% for F1-F) fetal bovine serum (Life Technologies, Inc., Milan, Italy) at 37°C in a humidified 5% CO2 atmosphere.
p185 Plasmids.
The pCMV vector was derived from the pcDNA3 plasmid (Invitrogen, San Diego, CA) by deleting the SV40 promoter, neomycin resistance gene, and SV40 poly(A). The sequences for the ECD and TM of transforming r-p185neu were generated from the PCR product using the primers 3'-CGCAAGCTTCATCATGGAGCTGGC-5' and 3'-CGGAATTCGGGCTGGCTCTCTGCTC-5' and the primers 3'-CGCAAGCTTCATGGAGCTGGC-5' and 3'-ATGAATTCTTTCCGCATCGTGTACTTCTTCCGG-5', respectively, as described previously (9
, 20)
. PCR products of the expected size were isolated by agarose gel electrophoresis, digested with HindIII and EcoRI, and cloned into the multiple cloning site of the pCMV plasmid to obtain the p185 plasmid used in this work. Escherichia coli strain DH5
was transformed with p185 or the empty plasmid (pcDNA3) and then grown in Luria-Bertani medium (Sigma, St. Louis, MO; Ref. 20
). Large-scale preparation of p185 plasmids was carried out by alkaline lysis, using Endofree Quiagen Plasmid-Giga kits (Qiagen, Chatsworth, CA). DNA was then precipitated, suspended in sterile saline at 1 mg/ml, and stored in aliquots at -20°C for subsequent use in immunization protocols.
mLAG-3Ig.
Recombinant mLAG-3Ig molecules were generated by fusing the ECD of murine LAG-3 to a murine IgG2a Fc portion (13
, 16)
to form a recombinant protein that was then produced in Chinese hamster ovary cells and purified on protein A columns (Ares Advanced Technology, Randolph, MA). The total protein purity was >95% soluble LAG-3Ig by Coomassie Blue SDS-PAGE densitometry. Potential contamination of the purified protein with bacterial endotoxin (lipopolysaccharide) was determined by using the chromogenic Limulus amebocyte lysate assay (BioWittaker). A calibration curve based on enzymatic activity versus lipopolysaccharide was constructed to determine endotoxin units in the test sample, and values of <1 EU/mg were obtained for mLAG-3Ig.
Vaccination Schedules.
At the specified time points, BALB-neuT mice received a single injection of 100 µg of p185 plasmids in 0.1 ml of sterile saline solution (0.9% NaCl; SALF, Bergamo, Italy) in the exposed left quadriceps through a 28-gauge needle syringe, followed a few seconds later by 0.1 ml of DPBS (Sigma) alone (DPBS controls) or containing 1 µg of either mLAG-3Ig or a nonspecific isotype-matched purified control mouse Ig (mIgG2a; PharMingen, San Diego, CA). Subsequent boosts with the same combinations were performed contralaterally.
Whole Mount Analysis, Histology, and Immunohistochemistry.
Whole mount preparations were performed as reported by Medina (21)
. Briefly, the skin of euthanized BALB-neuT mice was fixed overnight in 10% buffered formalin. The mammary fat pads were scored into quarters and gently scraped from the skin. The quarters were immersed in acetone overnight and then rehydrated and stained in ferric hematoxylin (Sigma), dehydrated in increasing concentrations of alcohol, cleared with histo-lemon, and stored in methyl salilcylate (Sigma). Digital photos were acquired with a Nikon Coolpix 995 (Nital SpA, Turin, Italy) mounted on a stereoscopic microscope (MZ6; Leica Microsystems, Milan, Italy). Whole mounts were then embedded in paraffin, sectioned at 4 µm, and stained with H&E. For immunohistochemistry, these sections were incubated for 30 min with anti-r-p185neu antibodies (C-18-G; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-PCNA antibodies (Ylem, Rome, Italy). After washing, they were overlaid with biotinylated goat antirat or antirabbit immunoglobulin (Vector Laboratories, Burlingame, CA) for 30 min. Unbound antibodies were removed, and the slides were incubated with avidin-biotin complex/alkaline phosphatase (Dako, Milan, Italy).
Cytotoxic Activity.
A single-cell suspension of SPCs obtained from the spleen of each of five mice from each treatment group was washed in DPBS and resuspended in RPMI 1640 (BioWittaker) supplemented with 10% fetal bovine serum as described previously (22)
. SPCs (2 x 107) from each suspension were cultured with 5 x 105 Mit-C (Sigma)-treated stimulator TUBO cells (22)
for 6 days at 37°C. Recovered lymphocytes were admixed with 5 x 103 [3H]dThd-labeled TUBO or F1-F target cells at various E:T ratios in round-bottomed 96-well microtiter plates in triplicate. The percentage of specific lysis was then evaluated after 48 h and recorded, and the data were expressed as L.U. (L.U.20/107) calculated according to the equation of Pross et al., as previously described in detail (22)
.
Cytokine Production.
The amount of IFN- and IL-4 released by 2 x 106 SPCs/ml cultured for 2 days at 37°C in RPMI 1640 supplemented with 10% fetal bovine serum in the absence (fresh SPCs) or presence of 1 µg/ml anti-CD3 and anti-CD28 mAb [PharMingen (stimulated SPCs)] was assayed by ELISA (IFN-, PharMingen; IL-4, R&D System Inc., Minneapolis, MN).
Flow Cytometry.
SPCs obtained at progressive time points from five mice from each treatment group were incubated individually or pooled at first with rat anti-CD8 antibody (53.6.72 hybridoma, anti-Lyt 2; America Type Culture Collection, Manassas, VA) and then with anti-r-Ig-coated immunomagnetic beads (Miltenyi Biotec, Calderara di Reno, Italy). Recovered CD8+ cells (>96% purity) were stained with anti-CD28-FITC (Cedarlane Laboratories, Celbio, Pero, Italy) and anti-CD11b-phycoeritrine (PharMingen) antibodies. Two-color analysis of both surface molecules was performed by FACScan (Becton Dickinson, Mountain View, CA). The cells were gated on living cells by cell size and granularity. Data analyzed through CellQuest (Becton Dickinson) software were displayed as bivariate dot plots.
Antibody Response.
At the specified time points, sera were collected from mice in each group, and their binding to r-p185neu-positive N202.1A and r-p185neu-negative N202.1E cells was determined by flow cytometry after 1:200 dilution (9)
. The antibody titer is expressed as sbp x 10-3/ml, calculated as previously described in detail (9
, 22)
. Isotype determinations were carried out by an indirect immunofluorescence procedure on cells preincubated with mouse sera diluted 1:20 by adding a secondary biotin-conjugated mAb directed against mouse IgG1, IgG2a, IgG2b, IgG3, and IgM, all from CALTAG Laboratories (Burlingame, CA). FITC-streptavidin (Dako) was used as the last step.
Statistics.
Differences in tumor incidence were evaluated with the Mantel-Haenszel log-rank test; differences in tumor multiplicity, L.U., IFN- and IL-4 production by SPCs, and antibody titer were evaluated with Student’s t test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, left panel). Only a slight, temporary reduction in carcinoma multiplicity was found (Fig. 1
, right panel). By contrast, in mice vaccinated with p185 plasmids plus either control mIgG2a or mLAG-3Ig, the first appearance was greatly delayed, and carcinoma multiplicity was markedly reduced (Fig. 1
, right panel). Around week 40, whereas carcinomas had become palpable in several glands of the great majority of mice vaccinated with p185 plasmids plus control mIgG2a, almost 70% of those vaccinated with p185 plasmids plus mLAG-3Ig were tumor free and remained so until week 52, when the experiment was ended (Fig. 1
, left panel). The mean tumor multiplicity in this group was about 2 and was significantly lower than that of mice vaccinated with p185 plasmids plus control mIgG2a (Fig. 1
, right panel).
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, top row), whereas they were dramatically and similarly reduced at these times in mice that received p185 plasmids plus either control mIgG2a or mLAG-3Ig. However, by week 52, carcinoma masses of different size and invasiveness were evident in the few surviving mice immunized with plasmids plus control mIgG2a (Fig. 2
, middle row), whereas those that received mLAG-3Ig were almost all free from both palpable masses (Fig. 1)
and side buds (Fig. 2
, bottom row). This inhibition of progression was evident at week 8 and 17 and became more evident by week 52.
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and foci of atypical hyperplasia and neoplastic side buds surrounded by a scanty reactive necrotic and hemorrhagic cell infiltrate were evident in mice vaccinated with p185 plasmids plus control mIgG2a (Fig. 3A)
. Lobular carcinomas and foci of atypical hyperplasia were also found in two of six survivor mice vaccinated with p185 plasmids plus mLAG-3Ig (Fig. 3D)
, although a few of their glands displayed a pronounced reactive cell infiltrate in close contact with neoplastic and hyperplastic epithelial cells, in which the expression of r-p185neu was markedly reduced and mostly confined to the cytoplasm (Fig. 3E)
. This limited expression was associated with reduced PCNA positivity (Fig. 3F)
. By contrast, all of the glands of the other four survivors were formed of ducts and ductules lined with a single layer of epithelial cells without r-p185neu and PCNA expression (Fig. 3, G-I
).
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, SPCs obtained 10 and 28 weeks after the last boost with p185 plasmids plus mLAG-3Ig displayed a significant r-p185neu-specific cytotoxicity (Fig. 4)
. At 10 weeks after the last boost (mice were 17 weeks of age), the cytotoxic response was much stronger in mice immunized with p185 plasmids plus mLAG-3Ig than in mice immunized with p185 plasmid plus mIgG2a. Twenty-eight weeks after the last boost (mice were 35 weeks of age), the cytotoxic response was almost nil in mice immunized with p185 plasmid plus control IgG2a. By contrast, a low but significant cytotoxicity was still evident in mice immunized with plasmids plus mLAG-3Ig (Fig. 4)
. The cytotoxicity of SPCs from mice treated with DPBS or empty plasmids plus mLAG-3Ig was always marginal (data not shown).
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and IL-4 released were found 1 week and 10 weeks after the last boost (Fig. 5)
. However, 28 weeks after the last boost, stimulated SPCs from mice that received p185 plasmids plus mLAG-3Ig released greater amounts of both cytokines. A marked amount of IL-4 was also released at this time by fresh SPCs from mice immunized with p185 plasmids plus mLAG-3Ig, whereas at all time points the titers released by fresh SPCs from mice treated with DPBS or empty plasmids plus mLAG-3Ig were marginal, and those released by stimulated cells were no higher than 100 units/ml IFN- and 150 pg/ml IL-4 (data not shown).
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. This difference was already evident 1 week after the last boost and doubled after 10 weeks. This increase may be correlated with the higher reactivity of mice immunized with p185 plasmids plus mLAG-3Ig because their effector T cells are still able to proliferate and produce IFN- after stimulation (23)
. Furthermore, 10 weeks after the last boost, mice vaccinated with p185 plasmids plus mLAG-3Ig presented a markedly expanded population of CD8+/CD11b+/CD28- cells. These are thought to comprise memory effector cells that kill target cells and migrate to inflammatory sites but no longer proliferate (23
, 24)
. No expansion of these two T-cell populations was found in SPCs from mice treateds with DPBS or empty plasmids plus mLAG-3Ig (data not shown).
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. One week after the last boost, whereas the response in mice immunized with p185 plasmids plus control mIgG2a was mostly due to IgM, a T helper cell switch signal was suggested by the presence of anti-r-p185neu IgG of all subclasses in the sera of mice immunized with p185 plasmids plus mLAG-3Ig (Fig. 7)
. The quick appearance and persistent high production of IgG1 in these sera pointed to the influence of IL-4 in the B-cell isotype switch (25)
. The dominance of IgG1, in fact, was still evident 28 weeks after the last boost with p185 plasmids plus mLAG-3Ig (Fig. 7)
.
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. At this stage, vaccination with plasmids plus control mIgG2a was no longer able to delay the appearance of the first tumor or reduce tumor multiplicity (Fig. 8, C and D)
. By contrast, the time at which one tumor was palpable was markedly extended to week 36 in all mice vaccinated with p185 plasmids plus mLAG-3Ig compared with around week 21 in the other treatment groups. Tumor multiplicity, too, was strongly reduced from week 21 until week 52, when the experiment ended.
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and IL-4 release, and higher titers of anti-r-p185neu antibodies were found in mice vaccinated in the presence of mLAG-3Ig (Fig. 9)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In BALB-neuT mice, the target transforming oncogene is embedded in the genome (5) . This builds up a dynamic relationship between the oncogenic rat HER-2/neu signals and the inhibitory potential of the immune reactions activated by p185 plasmid vaccination. In the absence of the continuous onset of r-p185neu neoplastic cells, the strong and sustained inhibition of carcinogenesis that follows vaccination with p185 plasmids plus mLAG-3Ig would seem to coincide with a definitive cure. These marked preventive effects of DNA vaccination were lost when it was applied in older mice with already evident neoplastic lesions similar to multiple hyperplastic foci and carcinoma in situ. Even here, however, p185 plasmids plus mLAG-3Ig enhanced the immune response and significantly extended tumor latency.
These findings show that DNA vaccination is an efficient way to prevent the progression of early lesions but is much less effective when begun in mice with advanced lesions (1) . The critical part played by mLAG-3Ig in allowing p185 plasmid vaccination to elicit enduring protection is not surprising. Addition of mLAG-3Ig as adjuvant in vaccination with a particulate or soluble protein antigen effectively stimulates the priming of cytotoxic T lymphocytes, activates the proliferative response of spleen T cells along with their Th1-type cytokine production, and induces much higher antibody titers than a conventional adjuvant (18) . Enhancement of these immunological activities and the induction of antitumor immune responses and tumor regression (13 , 18) appear to rest on the ability of the LAG-3-induced MHC class II signal to activate macrophages and immature DCs. Cross-linking of certain MHC class II molecules expressed on their membrane and probably contained in lipid rafts (17) contributes to both their activation and the commitment of T cells toward Th1/T killer activity (26 , 27) .
Activation of CD4 and CD8 T-cell functions after DNA vaccination rests on cross-presentation of antigenic peptides released by transfected myocytes through bone marrow-derived macrophages and DCs (28
, 29)
. The protection against carcinogenesis associated with the presence of mLAG-3Ig correlates with a marked enhancement of both the cellular and the humoral immune response (18)
. In effect, mLAG-3Ig appears to enhance a mixed Th2-Th1 immune response, rather than a Th1 response only. In immunized mice, it induced a persistent cellular cytotoxicity, a sustained release of IFN-, expansion of both CD8+/CD11b+/CD28+ effector and CD8+/CD11b+/CD28- effector memory T-cell populations (23
, 24)
, and a faster shift toward Th1-type IgG2a. However, mLAG-3Ig also induced a shift toward IgG1, enhanced IgG2a production, and persistently enhanced the titer of anti-r-p185neu antibody. These latter activities may be directly related to the much higher IL-4 production observed in the spleen (25)
.
The anti-r-p185neu antibodies in the sera of immunized mice may induce a functional block of the r-p185neu receptor (30) , down-regulate its expression on the cell membrane (30 , 31) , impede its ability to form the homo- or heterodimers that spontaneously transduce proliferative signals to the cells (31 , 32) , and block its ability to bind ligands (33) , as has been observed with anti-Her-2/neu mAb. They also significantly suppress the growth of transplantable p185neu-positive tumors (34 , 35) and the onset of mammary carcinomas in HER-2/neu transgenic mice (31) and delay tumor growth in patients with HER-2/neu-positive tumors (36) . The morphological features of the inhibited mammary cell proliferation associated with marked membrane down-modulation of r-p185neu and diminished nuclear positivity of PCNA, characterizing the regression of both preneoplastic lesions and incipient carcinomas, point to direct inhibition by these antibodies.
The success of DNA vaccination in the inhibition of r-p185neu-positive carcinomas is apparently associated with a high and sustained antibody response to a growth factor receptor, whose down-regulation slows the preneoplastic cell proliferation and tumor development. This inhibition mechanism is different from immunological destruction of the malignant cells (7, 8, 9)
. However, a massive tumor cell killing also takes place because these antibody isotypes activate polymorphonuclear leukocytes and other cells to mediate antibody-dependent cell cytotoxicity (35
, 37
, 38)
and complement-dependent cytotoxicity and inhibit the growth of r-p185neu-positive tumors in vivo (35)
. Moreover, in immunized mice, cytotoxic T cells as well as T cells releasing IFN- and mediating intratumor delayed-type hypersensitivity reactions also appear to play an important effector and regulatory role in the inhibition of BALB-neuT mice mammary carcinogenesis (7, 8, 9
, 39)
.
The association of mLAG-3Ig with DNA vaccination halted the progression of multifocal carcinogenesis in all 10 mammary glands of BALB-neuT mice for a long period. mLAG-3Ig may thus be supposed to provide a costimulatory signal that allows vaccination to establish longer and more effective protection, probably by enhancing the cross-presentation of the DNA-coded protein (27) . The effect of mLAG-3Ig administration is to sustain the persistence of a responsive status instead of increasing an actively ongoing immune reaction. The result of its coadministration becomes more and more significant as the mice age. A similar form of combined vaccination could be considered as a noninvasive option in the management of patients diagnosed with early HER-2/neu (c-erbB-2)-expressing neoplastic lesions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Italian Association for Cancer Research, Italian Ministry for the Universities and Scientific and Technological Research (40% grants) and grant from "Compagnia di S. Paolo", Italy. 
2 To whom requests for reprints should be addressed, at Dipartimento di Scienze Cliniche e Biologiche, Ospedale S. Luigi Gonzaga, 10043 Orbassano (TO), Italy. Phone: 39-011-670-81-19; Fax: 39-011-903-86-39; E-mail: guido.forni{at}unito.it
3 The abbreviations used are: TM, transmembrane domain; DC, dendritic cell; ECD, extracellular domain; SPC, spleen cell; PCNA, proliferating cell nuclear antigen, IL, interleukin; dThd, thymidine; DPBS, Dulbecco’s PBS; Mit-C, mitomycin C; mAb, monoclonal antibody; sbp, serum binding potential; L.U., lytic unit(s).
Received 9/12/02. Accepted 3/12/03.
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