Distinct and Non-Overlapping Receptor Repertoires Expanded by DNA Vaccination in Wild-Type and HER-2 Transgenic BALB/c Mice This information is current as of September 25, 2021. Simona Rolla, Chiara Nicoló, Silvia Malinarich, Massimiliano Orsini, Guido Forni, Federica Cavallo and Francesco Ria J Immunol 2006; 177:7626-7633; ; doi: 10.4049/jimmunol.177.11.7626 Downloaded from http://www.jimmunol.org/content/177/11/7626

References This article cites 49 articles, 23 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/177/11/7626.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Distinct and Non-Overlapping T Cell Receptor Repertoires Expanded by DNA Vaccination in Wild-Type and HER-2 Transgenic BALB/c Mice1

Simona Rolla,* Chiara Nicolo´,† Silvia Malinarich,* Massimiliano Orsini,† Guido Forni,‡ Federica Cavallo,2* and Francesco Ria†

Central tolerance to tumor-associated Ags is an immune-escape mechanism that significantly limits the TCR repertoires available for tumor eradication. The repertoires expanded in wild-type BALB/c and rat-HER-2/neu (rHER-2) transgenic BALB-neuT mice following DNA immunization against rHER-2 were compared by spectratyping the variable (V)␤ and the joining (J)␤ CDR 3. Following immunization, BALB/c mice raised a strong response. Every mouse used one or more CD8؉ T cell rearrangements of the V␤9-J␤1.2 segments characterized by distinct length of the CDR3 and specific for 63-71 or 1206-1214 rHER-2 peptides. In Downloaded from addition, two CD4؉ T cell rearrangements recurred in >50% of mice. Instead, BALB-neuT mice displayed a limited response to rHER-2. Their repertoire is smaller and uses different rearrangements confined to CD4؉ T cells. Thus, central tolerance in .BALB-neuT mice acts by silencing the BALB/c mice self-reactive repertoire and reducing the size of the CD8؉ T cell component CD8؉ and CD4؉ T cells from both wild-type and transgenic mice home to tumors. This definition of the T cell repertoires available is critical to the designing of immunological maneuvers able to elicit an effective immune reaction against HER-2-driven carcinogenesis. The Journal of Immunology, 2006, 177: 7626–7633. http://www.jimmunol.org/

umor-prone mice transgenic for provide a sig- not suitable for evaluation of this critical issue in antitumor vac- nificant complementary addition to the data stemming cination, because their TCR repertoire may be dramatically differ- T from tumors engrafted in syngeneic mice. The slowness ent from that of cancer-prone transgenic mice and of patients with of neoplastic progression, its relationship with the surrounding tis- neoplastic lesions overexpressing the target TAA. sues, the natural occurrence of invasion and metastasis, and the Central tolerance to an overexpressed self-TAA involves intra- presence of a long-lasting interaction between the tumor and the thymic deletion of T cells bearing a TCR able to bind them with host immune system are the appealing features of these transgenic high affinity (3). In the thymus, an elaborated system permits the by guest on September 25, 2021 3 models (1). As these transgenic mice and their wild-type (WT) presentation of most self-epitopes by various kinds of APCs (4). counterparts differ in their expression of a single gene, they pro- Failure of this mechanism results in extended autoreactivity (5–7). vide an unique opportunity to assess how overexpression of an A large body of observations confirms that a residual TCR reper- protein product influences the TCR repertoire (2). This toire exists for overexpressed self-Ag in the periphery and encom- comparison acquires crucial importance when evaluating the re- passes “low affinity” TCRs specific for dominant epitopes, and/or sults of vaccination against tumor-associated Ags (TAA). Identi- TCRs characterized by “high affinity” for subdominant or cryptic fication of TAA provides new targets for antitumor immunity. However, most currently defined TAA are products of self-genes epitopes (8–10). Thus the natural tolerance induced by an Ag dif- overexpressed by neoplastic lesions. This poses a significant chal- ferentially expressed during early life may account for very dis- lenge because effective vaccine strategies should be able to by-pass similar peripheral TCR repertoires. self-tolerance. Mice engrafted with a TAA-expressing tumor are Several lines of mice transgenic for the rat-HER-2/neu (rHER-2) oncogene overexpress the transgenic rHER-2 at different times of their life and develop carcinomas whose progression mir- *Dipartimento di Scienze Cliniche e Biologiche, University of Torino, Torino, Italy; rors that of their human counterparts (11, 12). The protein product †Institute of General Pathology, Catholic University Sacro Cuore, Rome, Italy; and ‡Molecular Biotechnology Center, University of Torino, Torino, Italy coded by HER-2 is a member of the epidermal re- Received for publication March 16, 2006. Accepted for publication August 30, 2006. ceptor family endowed with a potent activity. Re- The costs of publication of this article were defrayed in part by the payment of page ceptor tyrosine kinases are central components of cell signaling charges. This article must therefore be hereby marked advertisement in accordance networks and play crucial roles in physiological processes, such as with 18 U.S.C. Section 1734 solely to indicate this fact. embryogenesis, cell proliferation, and apoptosis. Malfunction of 1 This work was supported by grants from Italian Association for Cancer Research, HER-2 is a leading cause of major human diseases, and correlates the Italian Ministries for the Universities and Health, National Multiple Sclerosis Society (Grant RG 3339-A-1), the University of Torino, Compagnia di San Paolo, with more aggressive tumor growth, greater invasiveness, enhanced Torino, and the Center of Excellence on Aging, University of Chieti, Italy. metastatic potential, and increased resistance to therapy (13). Proba- 2 Address correspondence and reprint requests to Dr. Federica Cavallo, Department of bly the most aggressive and consistent model of rHER-2 mammary Clinical and Biological Sciences, Ospedale san Luigi Gonzaga, 10043 Orbassano, Italy. E-mail address: [email protected]. carcinogenesis is provided by female BALB/c mice transgenic for the 3 Abbreviations used in this paper: WT, wild type; rHER-2, rat HER-2/neu; BALB- transforming rHER-2 oncogene under the transcriptional control of a neuT mice, rHER-2 transgenic BALB/c mice; MMTV, mouse mammary tumor virus; long-terminal repeat sequence from mouse mammary tumor virus neu EC-TM , extracellular and transmembrane domains of the protein product of (MMTV) (BALB-neuT mice, ͗http://cancermodels.nci.nih.gov/ rHER-2; RSI, rate of stimulation index; V, variable; J, joining; CDR, complementa- rity-determining region; TAA, tumor-associated Ag. mmhcc/index.jsp͘). rHER-2 protein overexpression and the onset of

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 7627 epithelial nodular side buds stemming from mammary ducts are al- dance with European Union and Institutional guidelines. Female BALB- ϩ ready evident in all 4-wk-old mice, and a palpable invasive carcinoma neuT mice consistently display a palpable invasive rHER-2 carcinoma in is displayed in all their mammary glands around the 33rd week (12, all their mammary glands around the 33rd wk of age (12, 14). 14). The gene expression signatures of this carcinoma progression mRNA extraction from the thymus and RT-PCR neatly model the progression of human breast tumors (15). Total RNA was extracted and purified from thymus of BALB/c and BALB- Comprehension of the features of the T cell repertoire available neuT mice at 2 days and 4 weeks of age using TRIzol reagent (Invitrogen for a rHER-2-specific immune response in BALB-neuT mice will Life Technologies). cDNA was synthesized with Supercript first strand provide information for a more rational design of immunological cDNA System (Invitrogen Life Technologies) following the manufactur- Ј Ј maneuvers to reverse tolerance and allow residual T cells to mount er’s instruction. PCR was performed by 5 - and 3 -specific rHER-2 primers ϩ ATTCATCATTGCAACTGTAGA and AAGCACCTTCACCCTTCC an effective immune response to rHER-2 tumors (2). Although it TTA, respectively (3). Primers that amplify GAPDH (BD Clontech) were has been shown that rHER-2 is expressed in the thymus of FVB used as standard. mice made transgenic for the rHER-2 proto-oncogene (3), infor- DNA expression vectors and vaccination mation about expression of the transforming rHER-2 in the thymus of BALB-neuT mice is lacking. However, whereas simple immu- EC-TMneu plasmid was produced and used as previously described (22). A nization regimens trigger a strong immune reaction leading to the total of 25 ␮g of plasmid in 20 ␮l of 0.9% NaCl with 6 mg/ml polyglu- ϩ tamate (Sigma-Aldrich) were injected into the tibial muscle of anesthetized rejection of large established rHER-2 tumors in WT BALB/c mice. Electric pulses were applied by two electrodes placed on the shaved mice (16, 17), only combined and repeated immunizations can skin covered with a conducting gel. Two square-wave 25 ms, 375 V/cm elicit an immune response strong enough to delay carcinoma onset pulses were generated by a T820 electroporator (BTX) (23). Each vacci- in BALB-neuT mice (18–20). This raises the question of whether nation course consisted of two plasmid administrations with an interval of Downloaded from expression of the oncogene in the thymus actually silences the 14 days. immune response or facilitates its regulation through peripheral Cell lines and tumors mechanisms (17). In the latter eventuality, the TCR repertoires 3T3-NKB cells are BALB/c 3T3 fibroblast (3T3 BALB) transfected with expanded by rHER-2 in BALB/c and BALB-neuT mice should pCMV rHER-2, pRSVneo-Kd, pEXV3-murine B7.1 and pcDNA3.1-Zeo overlap, that remaining in BALB-neuT mice being only reduced in (24) kindly provided by W-Z Wei (Karmanos Cancer Center, Detroit, MI). its width and/or ability to respond. These cells were cultured in medium supplemented with G418 and Zeocin http://www.jimmunol.org/ ϩ We now compare the T cell responses elicited by DNA immu- (Sigma-Aldrich). TUBO cells are a rHER-2 cloned cell line established from a carcinoma that arose spontaneously in a BALB-neuT mouse (22). nization against the extracellular and transmembrane domains of Cells were cultured in DMEM (BioWhittaker Europe) with 20% FBS (In- neu rHER-2 protein product (EC-TM ) in BALB/c mice and trans- vitrogen Life Technologies). Mice were challenged s.c. in the right flank genic BALB-neuT mice. By means of a modified CDR 3-␤ spec- with 1 ϫ 105 TUBO cells (22). Tumors were measured with calipers in the tratyping technique, the bulk T cell population was divided into two perpendicular diameters. When they reached a 4-mm mean diameter, ϳ2400 groups in function of the rearrangement of their variable mice were immunized twice with an interval of 7 days. Tumors were ex- ␤ ␤ cised 21 days after vaccination. BALB-neuT mice were immunized at (V) and the joining (J) gene segments, and the length of the 10–12 wk of age as previously described (23) and mammary glands were TCR’s CDR3 was measured. This dissection of an Ag-specific excised 10 weeks later. TCR repertoire reveals Ag-driven expansions and makes it possi- by guest on September 25, 2021 Synthetic peptides ble to follow Ag-specific T cells (21). Present data show that in every BALB/c mouse immunized to Peptides 63-71 (TYVPANASL) (25), 400-407 (VFETLEEI), 425-432 ϩ (VFQNLRII), and 1206-1214 (ASPPHPSPA) of the rHER-2 sequence are xenogenic rHER-2, responding CD8 cells display one or more d ͗ ␤ ϩ predicted to bind the H-2K glycoproteins with high affinity ( http:// public TCR CDR3- arrangements. CD8 cells carrying these re- www.syfpeithi.de/͘). Peptides (95% pure by HPLC homogeneity and mass arrangements are specifically triggered by 63-71 or 1206-1214 spectrometry weight) were purchased from INBIOS Srl (Biotech Products), rHER-2 peptides. Moreover, two rearrangements specific for dissolved in PBS at 1 mg/ml and stored at Ϫ20°C until use. ϩ Ͼ rHER-2 and carried by CD4 T cells recur in 50% of treated TCR repertoire analysis BALB/c mice. We also show that in BALB-neuT mice rHER-2 is expressed in the thymus. Consequently, the anti-rHER-2 TCR rep- Repertoire analysis was performed using a modification of a previously ␤ described protocol (26). Popliteal and inguinal LN cells were collected ertoire is smaller than that of BALB/c mice, and uses distinct V from mice and cultured for 3 days with or without mitomycin C (Sigma- and J␤ genes. Public and semiprivate rearrangements are distinct Aldrich) treated 3T3-NKB cells (27) or 10 ␮M peptides. Culture medium ϩ from those used by BALB/c mice and confined to CD4 T cells. was RPMI 1640 (Invitrogen Life Technologies), supplemented with 2 mM Once BALB/c as well as residual BALB-neuT repertoires are ac- L-glutamine, 50 ␮M 2-ME, 50 ␮g/ml gentamicin (Sigma-Aldrich), and tivated, rHER-2-specific T cells equally home to tumors. 10% FBS (Invitrogen Life Technologies). Total RNA was isolated from the recovered LN cells with the RNeasy Mini Kit (Qiagen) according to the Thus central tolerance in BALB-neuT mice appears to act by ϩ manufacturer’s instructions. cDNA was synthesized using an oligo-dT mainly silencing the CD8 T cell component of the TCR reper- primer (dT15) (Invitrogen Life Technologies). cDNA was subjected to toire, whereas a non-overlapping CD4ϩ repertoire is generated and PCR amplification using a common constant (C)␤ primer (CACTGATGT escapes tolerance induction. Natural tolerance to HER-2, as well as TCTGTGTGACA) in combination with the following V␤ primers: 1, CT GAATGCCCAGACAGCTCCAAGC; 2, TCACTGATACGGAGCTGAG the pathologic and gene expression analogy between HER-2 car- ϩ GC; 3.1, CCTTGCAGCCTAGAAATTCATG; 4, GCCTCAAGTCGCT cinogenesis in BALB-neuT mice and patients with HER-2 tu- TCCAACCTC; 5.1, CATTATRGATAAAATGGAGAGAGAT; 5.2, mors (14, 15), may lead to similar modulation of the TCR reper- AAGGTGGAGAGAGACAAAGGATTC; 5.3, AGAAAGGAAACCTGC toire. Present results thus suggest that vaccines designed to first CTGGTT; 6, CTCTCACTGTGACATCTGCCC; 7, TACAGGGTCTC trigger CD4ϩ T cell reactivity would be the most rational attempt ACGGAAGAAGC; 8.1, CATTACTCATATGTCGCTGAC; 8.2, CAT TATTCATATGGTGCTGGC; 8.3, TGCTGGCAACCTTCGAATAGGA; to circumvent tolerance and elicit a protective immune reaction 9, TCTCTCTACATTGGCTCTGCAGGC; 10, ATCAAGTCTGTAGA against HER-2-driven carcinogenesis. GCCGGAGGA; 11, GCACTCAACTCTGAAGATCCAGAGC; 12, GAT GGTGGGGCTTTCAAGGATC; 13, AGGCCTAAAGGAACTAACTC Materials and Methods CCAC; 14, ACGACCAATTCATCCTAAGCAC; 15, CCCATCAGT Mice CATCCCAACTTATCC; 16, CACTCTGAAAATCCAACCCAC; 17, AGTGTTCCTCGAACTCACAG; 18, CAGCCGGCCAAACCTAACAT Specific pathogen-free virgin WT BALB/c and rHER-2 transgenic BALB- TCTC; 19, CTGCTAAGAAACCATGTACCA; 20, TCTGCAGCCTGG neuT (H-2d) females (Charles River Laboratories) were treated in accor- GAATCAGAA. The nomenclature of Arden et al. (28) was adopted. Using 7628 TCR REPERTOIRES IN WT AND TRANSGENIC BALB/c MICE

2 ␮l of PCR product as a template, run-off reactions were performed with collected 14 days after the last immunization. LN cells (5 ϫ 106) the following internal fluorescent J␤: 1.1, ACTGTGAGTCTGGTTCCTT were cultured with 5 ϫ 105 rHER-2ϩ 3T3-NKB cells for 3 days. TACC; 1.2, AAAGCCTGGTCCCTGAGCCGAAG; 1.3, CTTCCTTCT The 288 V␤-J␤ primer combinations were used to perform the first CCAAAATAGAGC; 1.4, GACAGCTTGGTTCCATGACCG; 1.5, GAGTCCCCTCTCCAAAAAGCG; 1.6, TCACAGTGAGCCGGGTGC CDR3 length fragment analysis on a pool of cDNA from four CTGC; 2.1, GTGAGTCGTGTTCCTGGTCCGAAG; 2.2, CCAGCACT BALB/c or BALB-neuT mice. Each CDR3-␤ profile can be de- GTCAGCTTTGAGC; 2.3, GTTCCTGAGCCAAAATACAGCG; 2.4, GT picted as a function of the CDR3 length. Each peak represents a GCCCGCACCAAAGTACAAG; 2.5, GTGCCTGGCCCAAAGTACT 3-bp difference in the product of recombination corresponding to GG; 2.7, CTAAAACCGTGAGCCTGGTGC. The run-off products were denatured in formamide and analyzed on an Applied Biosystem 3100 one amino acid residue. In non-vaccinated control BALB/c and Prism using Gene-scan 2.0 software (Applied Biosystem). Data are re- BALB-neuT mice, most peak patterns display a Gaussian distri- ported as rate of stimulation index (RSI): normalized peak area from stim- bution. After in vitro coculture with 3T3-NKB cells, Ag-driven ulated cells/normalized peak area of non-stimulated cells. T cells carrying perturbation of this distribution was observed in 13 spectra from a TCR rearrangement are considered expanded in a rHER-2-driven manner immunized BALB/c mice and 9 spectra from immunized BALB- when RSI is Ͼ2 (26). neuT mice (Fig. 2A). The BALB-neuT mice repertoire is thus less Immunoaffinity separation of CD4ϩ and CD8ϩ T cells than that of the BALB/c mice. We next examined the distribution ␤ ␤ LN cells pooled from four immunized mice were cultured in vitro for 3 of V (Fig. 2B) and J (Fig. 2C) usage. The repertoires used by the days with or without 3T3-NKB cells. CD8ϩ and CD4ϩ populations were responding do not overlap. BALB/c mice preferentially use V␤ enriched from stimulated cells by immunoaffinity magnetic sorting using genes belonging to families 5, 18, and 20, especially family 5 MACS beads conjugated with anti-CD8 or anti-CD4 Ab (Miltenyi Biotec) (present in 5/13 spectra), whereas BALB-neuT mice do not display according to the manufacturer’s instructions. Total, negatively selected, any bias in their V␤ usage. No obvious bias in J␤ usage is ob- Downloaded from and positively selected cells were collected, and total RNA was isolated for TCR repertoire analysis. served in BALB/c mice, whereas BALB-neuT mice preferentially use J␤ of the 2nd (J␤2) family and therefore the second diversity Results region. This observation suggests that amino acid residues encoded Transgenic BALB-neuT mice express rHER-2 in the thymus by this region play a role in rHER-2 recognition in BALB-neuT mice. Interestingly, whereas T cells using TCRs obtained through It has been shown that FVB mice transgenic for rHER-2 proto- V␤8 gene family recombination constitute up to 30% of the total http://www.jimmunol.org/ oncogene under the MMTV long-terminal repeat promoter express in BALB/c mice and thus form a substantial component of any the mRNA of the transgene in their thymus (3). BALB-neuT mice Ag-specific T cell response displayed by this strain (29), the re- are transgenic for the rHER-2 transforming oncogene under the sponse to EC-TMneu immunization in both BALB/c and BALB- same promoter as the FVB proto-oncogene. To assess whether the neuT mice does not use this family. functional tolerance of BALB-neuT mice is associated with thymic expression of rHER-2, we examined the presence of mRNA en- TCR repertoires expanded in BALB/c and BALB-neuT are coding for rHER-2 in thymuses from 2-day- and 4-week-old mutually exclusive BALB-neuT and control BALB/c mice. A significant amount of The majority of Ag-specific TCR rearrangements are private for specific mRNA was detectable in the thymuses from 2-day-old by guest on September 25, 2021 each mouse. However, we (30) and others (21, 31) have used the BALB-neuT mice. The intensity of the band further increases in immunoscope technique to detect public or semiprivate TCR re- 4-week-old thymuses. As expected, age-matched BALB/c mice arrangements in several Ag-specific responses. TCRs using the are negative at both time points (Fig. 1). same V␤-J␤ rearrangement are considered public when expanded BALB/c and BALB-neuT mice expand distinct TCR repertoires in all mice, semiprivate when used by almost 50% of mice. The following EC-TMneu plasmid electroporation expansion of each rearrangement was evaluated as RSI. To deter- mine whether shared TCRs are also present in the response to We previously reported that vaccination with pcDNA3 vector cod- neu neu EC-TM plasmid immunization, usage of candidate rearrange- ing for the EC-TM domains elicits a strong anti-rHER-2 im- ments chosen on peak expansion with a RSI greater than 4 (not mune response in WT BALB/c mice and a significant but much shown) was individually evaluated on cDNA from 18 BALB/c and less effective response in transgenic BALB-neuT mice (16, 22). To 16 BALB-neuT mice. Public and semiprivate rHER-2-specific re- assess the repertoires expanded by vaccination, both BALB/c and neu neu arrangements are detected in both groups (Fig. 3). All EC-TM BALB-neuT mice received EC-TM plasmids electroporated plasmid immunized BALB/c mice show an Ag-driven expansion into the tibial muscle at weeks 10 and 12 (23). LN cells were of T cells carrying one or more rearrangements involving V␤9- J␤1.2 and identified by 98-, 101-, or 104-bp length. In addition, Ͼ50% of mice display Ag-driven expansions of rearrangements V␤18-J␤1.6 and V␤20-J␤2.7 (138 bp and 113 bp) (Fig. 3, first row). By contrast, none of the BALB-neuT mice uses any of these rearrangements. Instead they use TCRs carrying a rearrangement of V␤13-J␤2.3 genes of 121-bp length, and one derived from V␤11-J␤2.7 genes (either 120 or 123 bp) (Fig. 3, second row). Conversely, none of the BALB/c mice uses these V␤-J␤ rearrangements. To assess the specificity of these public and semiprivate rear- FIGURE 1. rHER-2 transgene is expressed in the thymus of BALB- rangements, LN cells from EC-TMneu immunized BALB-neuT neuT mice. PCR for rHER-2 (upper line) and GAPDH (positive control, and BALB/c mice (five for each strain) were cocultured with 3T3 lower line) was performed on cDNA from BALB-neuT and BALB/c thy- muses collected from 2-day and 4-week-old mice as described in Materials BALB cells. Results showed that none of these rearrangements and Methods. rHER-2 expression was detected in thymus samples of was expanded by 3T3 BALB cells, which lack rHER-2 (Fig. 3, BALB-neuT mice and in rHER-2 overexpressing TUBO cells whereas no third and fourth rows). Moreover, LN cells from BALB-neuT and signal was observed in the corresponding thymus of WT BALB/c mice and BALB/c mice immunized with empty plasmid failed to expand any in rHER-2Ϫ TSA cells. of these rearrangements after coculture with 3T3-NKB cells (eight The Journal of Immunology 7629 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 FIGURE 2. Different TCR repertoires are triggered by EC-TMneu electroporation in WT BALB/c and transgenic BALB-neuT mice. Immunoscope analysis was performed on cDNA pools obtained from LN cells of four BALB/c or BALB-neuT immunized mice and restimulated with 3T3-NKB cells. cDNA were amplified by PCR with a combination of all 24 V␤ primers and a common C␤ primer; after a run-off reaction with 12 J␤ fluorescent primers, products were analyzed in a DNA sequencer. A–C, values of BALB/c are shown in gray whereas those of BALB-neuT mice are shown in black. A, Filled squares indicate V␤-J␤ rearrangements with rHER-2-specific expansion; B, distribution of V␤ subfamily usage. The sum of identified rearrangement mice is equal to 100%, and the frequencies of the 24 V␤ subfamily are calculated as relative percentages compared with this total sum. C, Distribution of J␤ subfamily usage. Percentages are calculated as above. mice per group, not shown). Taken together these results support looked to see whether it was expressed by CD4ϩ or CD8ϩ T cells. the notion that the identified CDR3-␤ regions belong to TCRs that LN cells recovered from 3 days’ coculture with 3T3-NKB were specifically recognize rHER-2-derived epitopes. separated into CD4ϩ and CD8ϩ T cells by immunoaffinity and Overall, we examined the use of eight rearrangements specific then analyzed by immunoscope to look for shared rearrangements. for EC-TMneu plasmid in 16 ϩ 18 mice. These data show that TCR It was found that T cells carrying the public V␤9-J␤1.2 repertoires repertoires used by BALB/c and transgenic BALB-neuT mice are are EC-TMneu plasmid-driven CD8ϩ T cells (Table I). Their distinct and do not overlap. This is in agreement with the result of epitope specificity was determined by assessing the ability of pep- our CDR3 length fragment analysis on a pool of cDNA deriving tides of rHER-2 predicted to bind to H-2Kd glycoproteins with from four BALB/c or four BALB-neuT mice (Fig. 2). high affinity [(p63-71 (TYVPANASL), p400-407 (VFETLEEI), The TCR repertoire residual after tolerance induction is deemed p425-432 (VFQNLRII), p1206-1214 (ASPPHPSPA)] to expand in to sustain low avidity responses (32). This result may rest on a vitro CD8ϩ cells carrying V␤9-J␤1.2 rearrangements. The selective depletion of high-affinity TCRs and/or a shift of speci- BALB/c mouse immunodominant peptide p63-71 (25) selectively ficity toward epitopes displaying low affinity for the restriction expands (RSI ϭ 6) cells carrying the 98 and 104 V␤9-J␤1.2 re- element. When the ability of cells from immunized BALB/c and arrangements (Fig. 4A). Intriguingly, T cells carrying the V␤9- BALB-neuT mice to expand in response to graded numbers of J␤1.2 rearrangement of 101 bases length are expanded by peptide 3T3-NKB cells was compared, it was evident that most rearrange- p1206-1214 (RSI ϭ 2) (Fig. 4C). This peptide belongs to the in- ments belong to T cells that expand effectively at comparable low tracellular domain of rHER-2 and is not encoded by the EC-TMneu doses of stimulating Ag (data not shown). plasmid used for immunization. Nevertheless, the response di-

ϩ rected against it is consistently observed (Fig. 3, and S. Rolla, BALB/c public CD8 TCR repertoires are specific for two unpublished data). This observation suggests that an early spread epitopes of rHER-2 of the immune response to other epitopes of HER-2 occurs, pos- Because all EC-TMneu plasmid immunized BALB/c mice show an sibly through presentation of self-peptides and priming of poten- expansion of T cells carrying the V␤9-J␤1.2 rearrangement, we tially self-reactive T cells. LN cells from immunized BALB-neuT 7630 TCR REPERTOIRES IN WT AND TRANSGENIC BALB/c MICE Downloaded from http://www.jimmunol.org/

FIGURE 3. Different public and semiprivate TCR repertoires triggered by EC-TMneu immunization in BALB/c and BALB-neuT mice. V␤-J␤ junction (CDR3) distribution profiles of recurrent TCR rearrangements in BALB/c and in BALB-neuT mice immunized with EC-TMneu are represented. These profiles are expressed as fluorescence intensity on y-axis and V␤-J␤ size on x-axis in base pairs. Each peak differs from the previous and the following 3 by guest on September 25, 2021 bp. Dotted vertical lines show the bases length of the peaks. Recurrent rearrangements in individually evaluated mice: BALB/c (18 mice, first row): V␤9-J␤1.2, 100%; V␤18-J␤1.6, 55%, V␤20-J␤2.7, 66%; BALB-neuT (16 mice, second row): V␤11-J␤2.7, 100%, V␤13-J␤2.3, 100%. This immunoscope analysis was performed on candidate public repertoires chosen on peaks with an RSI Ͼ4. As control of specificity, expansion of the same rearrangements were tested in LN cells from BALB/c (5 mice, third row) and BALB-neuT mice (5 mice, fourth row) immunized with EC-TMneu and re-stimulated with 3T3 BALB cells that do not express rHER-2 (N.T., not tested)

mice fail to expand any V␤9-J␤1.2 rearrangement, even when peptides for I-Ad and I-Ed restriction elements, but no expansion of stimulated in vitro with the peptides (Fig. 4, B and D). T cells carrying BALB-neuT rearrangements was observed (un- published data). BALB/c mice expand semiprivate CD4ϩ T cell repertoires Immunized BALB/c mice expand T cells expressing two addi- tional semiprivate TCR rearrangements. In ϳ55% of these mice, Table I. Expression of the identified TCR rearrangements on CD8ϩ or ϩ expanded T cells expressed the 138-bp V␤18-J␤1.6 rearrange- CD4 T cells and their localization at the tumor site ment, whereas 66% expressed the 113-bp V␤20-J␤2.7 rearrange- ment (Fig. 3). Both rearrangements are carried by CD4ϩ T cells Recurrent TCR Coreceptor Presence at the Mice Rearrangements Expressiona Tumor Site (Table I). Unfortunately, no sustained expansion of T cells carry- ing either rearrangement was observed following stimulation of BALB/c V␤9-J␤1.2 (98) CD8ϩ Yesb neu V␤9-J␤1.2 (101) CD8ϩ No LN cells from EC-TM plasmid immunized mice with four pep- ϩ d,e V␤9-J␤1.2 (104) CD8 Yes tides selected on the basis of predicted affinity for the I-A re- ϩ V␤18-J␤1.6 (138) CD4 Yes striction elements (unpublished data). V␤20-J␤2.7 (113) CD4ϩ Yes BALB-neuT V␤11-J␤2.7 (120) CD4ϩ Yesc Expanded BALB-neuT public TCR repertoires are expressed by V␤11-J␤2.7 (123) CD4ϩ Yes CD4ϩ T cells V␤13-J␤2.3 (121) CD4ϩ Yes All immunized BALB-neuT mice expand T cells expressing the a Expression of TCR rearrangements in LN cells separated by immunoaffinity ϩ ϩ 121-bp V␤13-J␤2.3 and these expanded T cells frequently carry magnetic sorting following restimulation in CD8 and in CD4 enriched cells. b Expression of the BALB/c TCR rearrangement by T lymphocytes infiltrating the 120-bp or (preferably) the 123-bp V␤11-J␤2.7 rearrangement. TUBO cells transplanted into immunized BALB/c mice detecting using V␤-J␤ CDR3 These rearrangements characterize a T cell repertoire used solely spectratyping. ϩ c Expression or absence of the BALB-neuT TCR rearrangement by T lymphocyte by BALB-neuT mice and carried by CD4 cells (Table I). LN infiltrating rHER-2ϩ neoplastic lesion in the mammary gland of immunized BALB- cells were then restimulated with the previously predicted four neuT mice. The Journal of Immunology 7631

Starting from the 4th week of age, BALB-neuT mice overex- press rHER-2 protein in the mammary gland and undergo a dev- astating expansion of mammary lesions leading to multiple carci- nomas (12, 14). Early EC-TMneu plasmid electroporation elicits a protective immune response that controls the progression of initial mammary lesions. Induction of this response fully rests on CD4ϩ cells (34). Protection is due to IFN-␥-producing T cells and a high production of anti-rHER-2 IgG2a Abs (19, 33), whereas a signif- icant CD8ϩ T cell-mediated cytotoxic response was never found (22, 23, 27). The observation that WT BALB/c mice respond to EC-TMneu plasmid electroporation with a larger repertoire than BALB-neuT is not surprising, because rHER-2 is a protein with several xeno- FIGURE 4. Peptides predicted to bind H-2Kd stimulate CD8ϩ cells car- geneic determinants in BALB/c mice (22, 25). By contrast, the rying V␤9-J␤1.2 rearrangements in BALB/c mice, but not in BALB-neuT mechanisms leading all BALB-neuT mice to expand a set of TCRs mice. The immunodominant peptide p63-71 selectively expands (RSI ϭ 6) specific for rHER-2 never used by BALB/c mice are less obvious. the cells carrying the V␤9-J␤1.2 rearrangement of 98 and 104 bases length, Two mechanisms may be postulated. Recruitment of TCR reper- ϩ whereas intracellular p1206-1214 peptide expands (RSI ϭ 2) CD8 cells toire is critically determined by both a competition between T cells carrying the V␤9-J␤1.2 rearrangement of 101 bases length. Empty profiles: to access the APCs (35, 36) and precursor frequency (37, 38). Downloaded from CDR3 distribution in stimulated LN cells from BALB/c or BALB-neuT Thus, it may be suggested that the TCRs belonging to the reper- mice; shaded profiles: unperturbed Gaussian CDR3 distribution in non- toire activated by immunization in BALB/c mice (namely those stimulated LN cells from BALB/c or BALB-neuT mice (controls). Dotted ␤ ␤ ␤ ␤ vertical lines show the peak of 101 bases length. The profiles are expressed carrying the 138-bp V 18-J 1.6, 113-bp V 18-J 1.6, and 113-bp ␤ ␤ as indicated in Fig. 2. V 20-J 2.7 rearrangements) have a high precursor frequency that prevents activation of other, low frequency T cells. Once this rep- ertoire is deleted by induction of tolerance in BALB-neuT mice, a http://www.jimmunol.org/ CD8ϩ and CD4ϩ T cells infiltrating tumor during rejection cryptic repertoire (exemplified by V␤13-J␤2.3 recombination of express the identified TCR repertories 121 bp and V␤11-J␤2.7 rearrangement of 120 or 123 bp) can ϩ access the APCs to be primed. Rejection of rHER-2 tumors following EC-TMneu plasmid im- T cell maturation in the thymus also involves positive selection munization in BALB/c mice is mainly mediated by Abs and CTL of cells bearing TCRs that interact with self-MHC molecules. As (16), and almost completely Ab-mediated in BALB-neuT mice ϩ in negative selection, promotion of proliferation also depends on (33). Whereas in both cases histochemistry shows that CD4 and CD8ϩ cells infiltrate the tumors (22, 23), direct evidence of presentation of self-epitopes, which shape the overall TCR reper- rHER-2 specificity of the T cells within the tumor mass is lacking. toire that reaches the periphery (39). Thus, the presence of the by guest on September 25, 2021 To assess the expression of the TCR rearrangements by T cells rHER-2 protein in the thymus may allow the positive selection of infiltrating tumors during immuno-mediated rejection, BALB/c distinct TCRs with respect to mouse HER-2. Here we show that neu mice were challenged with TUBO cells and immunized with EC- following EC-TM plasmid electroporation, the immune re- ␤ ␤ TMneu plasmid electroporation when the growing tumor reached sponse of these mice rests on the expansion of V 11-J 2.7 and ␤ ␤ ϩ ϩ 4-mm mean diameter. Tumors were excised 21 days after vacci- V 13-J 2.3 rHER-2-specific CD4 cells, whereas no CD8 pub- nation, total RNA and cDNA were prepared, and immunoscope lic T cells can be detected. Differences in thymic selection of CD8ϩ and CD4ϩ cells may be responsible for this selective ex- analysis of the TCR repertoire identified was performed. Results ϩ ϩ ϩ show that infiltrating T cells express V␤9-J␤1.2, V␤18-J␤1.6, and pansion of self-reactive CD4 cells. CD8 and CD4 cells have V␤20-J␤2.7 rearrangements. Intriguingly, among CD8ϩ T cells different sensitivity to epitope density on the surface of APC, be- ϩ carrying the V␤9-J␤1.2, only those specific for the immunodom- cause CD8 cells can be activated by a 100-fold lower number of ϩ inant p63-71 peptide (i.e., T cells carrying the 98- and 104-bp peptide/MHC complexes with respect to CD4 cells (40–42). V␤9-J␤1.2) are detected within the tumor mass. Therefore, This sensitivity may result in distinct outcomes of negative selec- whereas CD8ϩ cells specific for rHER-2 are directly involved in tion in the thymus, despite equivalent presentation of self-Ag de- the eradication of rHER-2ϩ cells, self-reactive CD8ϩ T cells do rived epitopes. Although it is well established that autoreactive not participate. Moreover, following EC-TMneu plasmid electro- thymocytes are subject to deletion during their development, not ϩ poration, CD4ϩ T cells expressing the V␤11-J␤2.7 and V␤13- all autoreactive CD4 T cells are eliminated from the peripheral T ϩ ϩ J␤2.3 public rearrangements infiltrate mammary lesions in both cell repertoire (43). The generation of CD4 CD25 T cells with 22- and 42-week-old BALB-neuT mice (Table I). immunoregulatory properties is one of the ways in which poten- tially autoreactive CD4ϩ cells are controlled (44). Generation of Discussion these regulatory cells is typically accompanied by the development Ϫ In the present study, we used V␤-J␤ spectratyping to compare the of clonally related CD25 T cells bearing an identical TCR spec- ϩ Ϫ TCR repertoire used by WT BALB/c mice and rHER-2 transgenic ificity (45). These CD4 CD25 cells are both more proliferative ϩ BALB-neuT mice in response to EC-TMneu plasmid electropora- than their CD25 counterparts and unable to suppress T cell pro- ϩ tion. BALB-neuT mice express rHER-2 in the thymus and respond liferation. They may be the CD4 cells expanded after EC-TMneu to electroporation by expanding a smaller repertoire and using dif- electroporation in BALB-neuT mice. Expression of rHER-2 in the ferent V␤ and J␤ regions compared with WT BALB/c mice. thymus of BALB-neuT mice and subsequent positive selection are BALB/c mice expand public CD8ϩ and CD4ϩ cell repertoires, the reasons why these TCR rearrangements are not found in whereas that of BALB-neuT mice comprises CD4ϩ cells only. By BALB/c mice. Indeed, anti-rHER-2 reactive CD4ϩ T cell reper- contrast, no differences in ability to infiltrate rHER-2ϩ tumors toires of BALB-neuT and BALB/c mice are mutually exclusive, were displayed by these repertoires. rather than one being a subgroup of the other. 7632 TCR REPERTOIRES IN WT AND TRANSGENIC BALB/c MICE

The rHER-2 specific CD4ϩ T cell repertoire of BALB-neuT Disclosures mice is potentially still able to prevent tumor development. How- The authors have no financial conflict of interest. ever, T cells expressing a self-reactive repertoire need a strong “stimulus” to become effective (23). Presence of regulatory mech- References anisms that down-regulate the immune response to TAA in the 1. Ostrand-Rosenberg, S. 2004. Animal models of tumor immunity, immunotherapy periphery has been recognized in rHER-2 transgenic mice and hu- and cancer vaccines. Curr. Opin. Immunol. 16: 143–150. 2. Ercolini, A. M., B. H. Ladle, E. A. Manning, L. W. Pfannenstiel, man patients (46, 47). These mechanisms include negative T reg- T. D. Armstrong, J. P. Machiels, J. G. Bieler, L. A. Emens, R. T. Reilly, and ulatory cells (2), immature myeloid cells (48), regulation of endo- E. M. Jaffee. 2005. 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