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CD8⁺ T Cells Targeting a Single Immunodominant Epitope are Sufficient for Elimination of Established SV40 T Antigen-Induced Brain Tumors¹

Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA 17033

	Abstract

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Immunotherapy of established solid tumors is rarely achieved, and the mechanisms leading to success remain to be elucidated. We previously showed that extended control of advanced-stage autochthonous brain tumors is achieved following adoptive transfer of naive C57BL/6 splenocytes into sublethally irradiated line SV11 mice expressing the SV40 T Ag (T Ag) oncoprotein, and was associated with in vivo priming of CD8⁺ T cells (T_CD8) specific for the dominant epitope IV (T Ag residues 404–411). Using donor lymphocytes derived from mice that are tolerant to epitope IV or a newly characterized transgenic mouse line expressing an epitope IV-specific TCR, we show that epitope IV-specific T_CD8 are a necessary component of the donor pool and that purified naive epitope IV-specific T_CD8 are sufficient to promote complete and rapid regression of established tumors. While transfer of naive TCR-IV cells alone induced some initial tumor regression, increased survival of tumor-bearing mice required prior conditioning of the host with a sublethal dose of gamma irradiation and was associated with complete tumor eradication. Regression of established tumors was associated with rapid accumulation of TCR-IV T cells within the brain following initial priming against the endogenous T Ag in the peripheral lymphoid organs. Additionally, persistence of functional TCR-IV cells in both the brain and peripheral lymphoid organs was associated with long-term tumor-free survival. Finally, we show that production of IFN-, but not perforin or TNF-, by the donor lymphocytes is critical for control of autochthonous brain tumors.

	Introduction

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Few immunotherapeutic approaches that target the recruitment of tumor-reactive CD8⁺ T cells (T_CD8)⁵ have been effective against well-established tumors either in experimental models or in cancer patients (1, 2). This is despite success in protection from tumor challenge or control of early-stage tumors in animal models. A variety of explanations have been offered to account for this discrepancy, including, but not limited to, the development of T cell tolerance (3), inhibition by regulatory T cells (4), the immunosuppressive nature and physical barrier provided by the tumor stroma (5, 6), and loss of MHC class I or tumor Ag expression by the tumor cells (7, 8). For targeting self/tumor Ags, the T_CD8 response can be limited by both central tolerance during T cell development (9) and peripheral tolerance mediated by APCs expressing tumor Ags in the absence of an inflammatory trigger (3, 10). However, T_CD8 responsive to a number of human tumor Ags have been identified, isolated, and expanded in vitro, a strategy that has been used for adoptive immunotherapy of cancer (11, 12).

Adoptive transfer of ex vivo-expanded patient-derived T_CD8 has shown promising clinical results, particularly when preceded by lymphodepletion (13, 14). These successes may be partially explained by the activation of T cells targeting unique tumor Ags that may be less affected by central tolerance (15, 16). However, the requirements on the responding T cells to achieve effective tumor regression remain to be determined. Several studies have indicated that the phenotype or stage of differentiation of donor lymphocytes may be particularly important to achieve effective control of tumor progression (17, 18, 19). Additionally, sustained T cell responses are associated with tumor regression (20, 21, 22), although rapid tumor elimination may be important to avoid the development of peripheral tolerance or the appearance of Ag escape variants (7, 23, 24). Sensitization of tumors or the associated stroma to the effects of responding T cells is also indicated to be critical to achieve tumor regression (25, 26).

Tumors within the CNS provide a unique challenge to immunotherapy due to more stringent regulation of lymphocyte circulation (27) and the potential for negative side effects induced by T cell effector functions. While naive T cells are typically not detected in the CNS, memory and activated T cells are granted passage through the blood-brain barrier (27). Thus, current models suggest that naive T cells are triggered by specific Ags in the periphery before entering the CNS. APCs, on the other hand, are capable of trafficking out of the CNS to the draining lymph nodes (28), where they may encounter naive Ag-specific T cells. Thus, tumor models are needed that mimic the unique nature of the CNS to identify effective immunotherapies for cancer.

We previously used a mouse model of autochthonous brain cancer to assess adoptive immunotherapeutic approaches toward advanced-stage disease (29). Line SV11 mice express the SV40 T Ag (T Ag) as a transgene from the SV40 enhancer/promoter, leading to T Ag expression in the thymus as well as the choroid plexus tissue within the brain (30). T Ag expression in the choroid plexus promotes the appearance of small papillomas by 35 days of age, and progressive tumor growth results in mortality by 104 days of age. We previously reported that T_CD8 specific for the three dominant H-2^b-restricted determinants, epitopes I (residues 206–215), II/III (residues 223–231), and IV (residues 404–411), are eliminated from SV11 mice during T cell development (31). However, administration of a nonlethal dose of gamma irradiation before adoptive transfer of naive splenocytes from nontransgenic littermates led to robust priming of donor T_CD8 against the immunodominant H-2K^b-restricted epitope IV in mice with both minimal disease (45 days old) (31) and advanced-stage tumors (80 days old) (29). Epitope IV-specific T_CD8 accumulated to high levels within the brains and tumors of these mice and was associated with reduced tumor burden and extension of the lifespan from 107 days to 170 days of age (29). The requirements to achieve this potent effect on tumor progression, however, remain to be determined.

In this report, we investigated components of the donor pool critical for control of advanced-stage tumors in SV11 mice. While the presence of epitope IV-specific T_CD8 within the brain was previously associated with control of tumor progression, the donor pool in that study was composed of total splenocytes (29). Thus, donor-derived cells other than epitope IV-specific T_CD8 could potentially be required to ensure successful immunotherapy. Here we asked whether epitope IV-specific T_CD8 are a required component of the donor pool and whether epitope IV-specific donor T_CD8 alone are sufficient to control tumor progression. Additionally, we assessed which of several T cell effector functions are important for control of these advanced-stage CNS tumors.

	Materials and Methods

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Mice

C57BL/6 (H-2^b) mice (B6), B6.129S7-IFN-g^tm1Ts/J (IFN- knockout (KO)) mice, C57BL/6-Prf1^tm1Sdz/J (perforin KO) mice, and B6;129S6-Tnf^tm1Gkl/J (TNF- KO) mice were purchased from The Jackson Laboratory. The following mice were obtained through the National Institute of Allergy and Infectious Diseases Exchange Program of the National Institutes of Health: 004216 C57BL/6Ji-K^btm1 (Kb KO) and 004217 C57BL/6Ji-D^btm1 (Db KO). All mice were maintained at the Milton S. Hershey Medical Center (Hershey, PA) animal facility under specific pathogen-free conditions. SV11 (H-2^b) mice express full-length SV40 T Ag under the control of the SV40 promoter (32). The SV11 mouse line was maintained by backcrossing transgene positive males to C57BL/6 females for over 50 generations at the Milton S. Hershey Medical Center. All animal studies were performed under protocols approved by the Institutional Animal Care and Use Committee of the Pennsylvania State University College of Medicine.

Derivation of E1-T251–708 T Ag (line 243) transgenic mice

To construct the plasmid E1-T251–708, containing the T Ag 251–708 segment under control of the rat elastase promoter, the plasmid CAV251–708 (33) was digested with MluI and BglII to release the coding sequence for the fusion protein containing the first seven amino acids of β-galactosidase followed by 31 amino acids encoded by the synthetic polylinker of Bluescript SK+ and then T Ag amino acids 251–708. The released segment was inserted between the corresponding sites of the plasmid pGC2E1 (34), containing the rat elastase 1 promoter. The E1-T251–708 (line 243) transgenic mouse line was generated by injecting purified DNA into fertilized mouse embryos recovered at the one-cell stage from superovulated B6D2F1/J mice that had been mated 20 h earlier to C57BL/6J males, as described previously (34). Pups were screened for the presence of the transgene by PCR analysis using three sets of T Ag primer pairs designed to amplify nucleotides 5139–4925, 4292–4055, and 3502–3233 and, as a control for integrity of the DNA sample, a primer set for the p53 gene as described previously (34). Amplification of the T Ag segment containing nucleotides 3502–3233 but not the other T Ag segments indicated presence of the E1-T251–708 transgene. Line 243 mice have been bred to C57BL/6 mice for more than 10 generations.

Immunoprecipitation and Western blot analysis

Protein from T Ag 251–708 plus activated ras-transformed rat embryo fibroblast cell line (T251–708 + ras) or pancreatic tissue from line 243 mice was immunoprecipitated as previously described (35). Immunoprecipitated proteins were separated by SDS-10% PAGE, electrotransferred to nylon membrane, and probed with a mixture of PAb 419 and PAb 901 as previously described (35). Reacting Abs were detected by chemiluminescence as described previously (36) and a digital image of the resulting film was captured and relevant lanes of the gel were cropped and positioned using Photoshop (Adobe).

Cloning of the epitope IV-specific TCR from CTL clone Y-4

TCR sequences for both the - and β-chains were derived from the epitope IV-specific CTL clone Y-4 (37), which was previously shown by flow cytometry to express Vβ9 (38). Total RNA was extracted from clone Y-4 cells with Tri Reagent (Sigma-Aldrich) and used to identify partial cDNA sequences corresponding to the TCR and β combining regions. A partial cDNA clone revealing an in-frame fusion of Vβ9-Jβ2.5 was generated by oligo(dT)-primed reverse transcription with avian myeloblastosis virus (AMV) reverse transcriptase (Promega) followed by PCR amplification with a 3' constant region antisense oligonucleotide primer (5'-CTTGGGTGGAGTCACATTTCT-3') and a 5' Vβ9-specific sense primer (5'-TACAAGCTTGCAAGAGTTGGA-3'); the mixture was phosphorylated using T4 polynucleotide kinase (Promega), rendered blunt ended with T4 DNA polymerase (Promega), and ligated into SmaI-digested, dephosphorylated pUC19. The V15-J12 variable and combining region sequence of the Y-4 TCR was deduced by sequencing a partial cDNA clone generated by 5'-RACE (Invitrogen). Total Y-4 mRNA was reverse transcribed with SuperScript II (Invitrogen) using a 3' constant region antisense primer (5'-AGAGGGTGCTGTCCTGAGAC-3'), extended with TdT, amplified by PCR using an abridged 5' anchor primer (5'-RACE, Invitrogen) and an internal 3' antisense constant region primer oligonucleotide (5'-CGAGGATCTTTTAACTGGTA-3'), and recombinants were isolated following ligation of the mixture into the cloning vector pGEM-T Easy (Promega).

Genomic sequences encoding the recombined variable and joining regions of the and β TCR subunits were amplified from high-molecular mass DNA prepared as described (39) using 3' antisense genomic primers corresponding to intron sequences flanking the respective TCR and TCRβ J regions: J12 (5'-TGCGGCCGCCTATCAGGTACTTACTGGGGCTGACTGATACCGT-3') (40) and Jβ2.6 (5'-ATCGATTTCCCTCCCGGAGATTCCCTAACCGCGGTCTACTCCAAAC-3') (41) were used in combination, respectively, with 5' sense primers corresponding to 5' noncoding sequences of V15 (5'-ACACCCGGGAAATACAAACAGCTTGCATGGCAAGAGA-3') (42) or Vβ9 (5'-TTCCTTTCCTGTCTCGAGCCATCCATGGATCCTAGA-3') (43) to amplify the genomic sequences from Y-4-derived DNA and incorporate appropriate restriction endonuclease cleavage sites at the ends of each product (, 5'-XmaI, 3'-NotI; β, 5'-XhoI, 3'-ClaI). The amplified genomic V(D)J fragments were subcloned into pGEM-T Easy and the nucleotide sequences were verified. The β-chain genomic VJ clone was further modified by oligonucleotide-directed mutagenesis (Altered Sites, Promega) to eliminate a naturally occurring KpnI site within the Vβ9 sequence (mutagenic primer 5'-GATACCATGTACTGGTATCAAAAGAAGCCAAAC-3') and insert a ClaI site 3' to the Jβ2.5 exon (mutagenic primer 5'-CGTGCGCGTTCTCAGA TCGATTGGGCTGCAGTG-3').

The and β TCR chain fragments were excised by restriction digestion and ligated into the appropriately digested TCR or TCRβ expression cassette plasmids (pTcass and pTβcass, respectively, obtained from Dr. Diane Mathis; see Ref. 44). Recombinant cassette vectors containing the appropriate Y-4 TCR - and β-chain fragments were identified by restriction digestion analysis, and the presence of the proper V(D)J inserts were verified by PCR amplification. Plasmids were purified by cesium chloride gradient centrifugation and digested (, SalI; β, KpnI) to liberate fragments bearing the respective subunit expression cassettes, which were isolated from agarose gels using the QIAEXII method (Qiagen) and eluted directly into microinjection buffer. Fragment solutions were stored at –80°C.

Generation of Tag epitope IV-specific TCR transgenic (TCR-IV) mice

Microinjection of fertilized embryos from B6D2F1/J mice with the purified Y-4 TCR - and β-chain expression cassette fragments was performed by the personnel in the Transgenic Core Facility of the M. S. Hershey Medical Center. The presence of the and β transgene(s) in weanling offspring was determined at 4 wk of age by PCR analysis of tail-derived DNA using the following primer pairs: V15 chain sense, 5'-ACACCCGGGAAATACAAACAGCTTGCATGGCAAGAGA-3'; J12 chain antisense, 5'-TGCG GCCGCCTATCAGGTACTTACTGGGGCTGACTGATACCGT-3'; Vβ9 chain sense, 5'-TTCCTTTCCTGTCTCGAGCCATCCATGGATCCTA GA-3'; and Jβ2.5 chain antisense, 5'-CACTGCAGCCCAATCCCGCTGAGAACGCGCACGT-3'. Amplification of the corresponding 725- and 650-bp fragments from genomic DNA was diagnostic for the presence of the transgenes. Expression of the transgene products was confirmed by staining lymphocytes from various lymphoid tissues with TCRVβ9-specific mAb (BD Pharmingen) and Kb/epitope IV tetramer (38). A single founder line was derived and designated as line F2025 and thereafter given the common designation of line TCR-IV mice. TCR-IV mice were backcrossed 10 or more generations to C57BL/6 mice by mating transgene-positive males with C57BL/6J females.

Cell lines and reagents

B6/wild-type (WT)-19 cells are a C57BL/6 mouse embryonic fibroblast line transformed with SV40 strain VA45–54, which express full-length wild-type T Ag, and were maintained as previously described (45). REF/251–708ras cells are a rat embryonic fibroblast line immortalized by coexpression of ras and T Ag 251–708 and have been described previously (33). K^bKO/WT-Tag cells expressing the wild-type T Ag were derived by transfection of C57BL/6Ji-K^btm1 primary mouse kidney cells with plasmid pPVU-0 (46) followed by selection of immortalized clones as previously described (47). Similarly, D^bKO/WT-Tag cells expressing the wild-type T Ag were derived by transfection of C57BL/6Ji-D^btm1 primary mouse kidney cells with plasmid pSelectESV-1 (48) followed by isolation of immortalized clones. CTL clone K-11, specific for T Ag epitope I, was maintained in vitro as previously described (49). All cell lines were maintained in DMEM media containing 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml kanamycin, 2 mM L-glutamine, 10 mM HEPES, and 0.075% (w/v) NaHCO₃ with 5% FCS. For T cell cultures and preparation of lymphocyte suspensions, RPMI 1640 medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 10 mM HEPES, 50 µM 2-ME, and 10% FCS was used.

In vitro culture and cytotoxicity assay

TCR-IV T cells used in cytotoxicity assays were obtained from TCR-IV transgenic mice immunized 10 days prior with B6/WT-19 cells. Briefly, spleen and lymph node cells were cultured with epitope IV peptide-pulsed (10 nM) bone marrow-derived C57BL/6 dendritic cells, obtained as previously described (50). A ⁵¹Cr-release assay was performed with TCR-IV T cells after 4 days of culture. CTL clone K-11 cells were used 3 days postrestimulation. Target cells were labeled with 100 µCi of sodium ⁵¹chromate in 0.5 ml of RPMI 1640 medium for 1.5 h at 37°C, 5% CO₂. Cells were mixed at the indicated ratios in 0.2 ml of RPMI 1640 medium and incubated for 4 h at 37°C, 5% CO₂. After 4 h, cells were pelleted by centrifugation and 0.1 ml of supernatant was counted using a Packard Cobra gamma counter. Percentage specific lysis was determined as previously described (31).

Irradiation, adoptive transfer, and immunization

SV11 mice received 400 rads of whole body gamma irradiation from a ⁶⁰Co source GammaCell 220 irradiator (Nordion International) 24 h before adoptive transfer. Line TCR-IV donor T_CD8 were isolated by autoMACS sorting using CD8⁺ microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. Briefly, donor splenocytes were incubated with CD8⁺ microbeads for 15 min at 4°C and then washed once before autoMACS sorting using the positive selection protocol. Purity of the positive fraction was determined by flow cytometry and generally ranged between 90 and 95%. SV11 mice received 1 x 10⁶ magnetically isolated TCR-IV T_CD8 i.v. in 0.2 ml of HBSS. For adoptive transfers with C57BL/6 or gene knockout donor mice, 5 x 10⁷ RBC-depleted splenocytes were administered i.v. Immunizations were conducted by i.p. injection of 5 x 10⁷ live B6/WT-19 cells.

Lymphocyte isolation

Brains, spleens, and cervical lymph nodes (CLNs) were removed following administration of 150 mg/kg sodium pentobarbital. Single-cell suspensions of lymphocytes were obtained by mechanical disruption and depletion of RBCs as described previously (31). Isolation of lymphocytes from the brain was performed as previously described (29).

Flow cytometric analysis

Isolated lymphocytes were stained with Abs to cell-surface markers and PE-labeled MHC tetramers (1/200 dilution) as previously described (38). MHC tetramers included H-2K^b/T Ag epitope IV, constructed using the C411L variant peptide in which the carboxyl-terminal cysteine residue was replaced with leucine to increase stability (VVYDFLKL), and the unrelated control H-2K^b/HSV glycoprotein B (gB), which was prepared using the HSV gB epitope 498–505 (SSIEFARL). MHC tetramers were prepared as previously described (38). Cells were then fixed with 2% paraformaldehyde in PBS and analyzed using either a FACScan or FACSCalibur flow cytometer (BD Biosciences). Surface expression of MHC molecules on T Ag-transformed cells was detected by staining with anti-K^b-specific mAb Y3 (51) and anti-D^b-specific mAb B22.249 (52) tissue culture supernatants for 30 min at 4°C followed by staining with goat anti-mouse IgG-FITC (Cappel Laboratories) for 30 min at 4°C. Samples were analyzed by flow cytometry using a FACSCalibur. All flow cytometry data were analyzed using FlowJo software (Tree Star).

Intracellular cytokine staining (ICS)

To detect intracellular IFN- production, 2 x 10⁶ freshly isolated lymphocytes from brain, spleen, or CLN were incubated for 5 h with 1 µM of either T Ag epitope IV C411L peptide or HSV gB498–505 peptide and 1 µg/ml brefeldin A as previously described (53). T_CD8 were stained for intracellular IFN- using the Cytofix/Cytoperm kit (BD Pharmingen) according to the manufacturer’s instructions and as previously described (29). Lymphocytes were analyzed by flow cytometry, and the percentage of IFN--producing T_CD8 specific for epitope IV was determined by subtracting the percentage of T_CD8 producing IFN- in the presence of the unrelated HSV gB498–505 peptide.

Tumor histology

Brains were harvested following perfusion of lethally anesthetized mice with 10 ml of PBS followed by 10 ml of 10% buffered formalin. Once removed, brains was stored in formalin for 24–48 h and then transferred to fresh PBS before embedding in paraffin. Five-micrometer sections representing regions throughout the entire brain were cut and collected onto positively charged slides and stained by standard H&E in the Histology Core of the M. S. Hershey Medical Center.

Lifespan analysis

The lifespan of SV11 mice was determined by monitoring the development of hydrocephalus, which is indicative of end-stage choroid plexus tumors (29, 31). Mice were sacrificed when they became moribund or exhibited symptoms of distress. In some cases, lymphocytes were isolated from brains and spleens for ex vivo analysis with MHC tetramers or by ICS. Additionally, tumor histology was performed on representative brain specimens as described above. Survival curves were generated using the Kaplan-Meier method with GraphPad Prism software (GraphPad Software) and significance was determined by log-rank tests. Values of p <0.05 were considered significant.

Statistical analysis

Significance between the frequency and number of responding T cells was determined using Student’s t test with a p value of <0.05 considered to be significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Choroid plexus tumors progress normally in the absence of epitope IV-specific T_CD8

To determine whether epitope IV-specific T_CD8 are required to achieve control of tumor progression in SV11 mice, we utilized a donor lymphocyte population lacking epitope IV-specific T_CD8 precursors. Mice of the line 243, which express the carboxyl-terminal fragment 251–708 of SV40 T Ag from the rat elastase-1 promoter, were derived by standard transgenic procedures. Western blot of pancreatic tissue from line 243 mice confirmed expression of the expected carboxyl-terminal T Ag fragment 251–708 (Fig. 1A). These mice were previously shown to be tolerant to T Ag epitope IV (404–411) and the immunorecessive epitope V (489–497) using standard cytotoxicity assays, which are dependent on in vitro expansion (54). To ensure the tolerant phenotype of line 243 mice by an independent approach and to assess reactivity ex vivo, groups of mice were immunized with wild-type T Ag-transformed cells (B6/WT-19). After 10 days, freshly isolated splenocytes were stimulated briefly with peptides corresponding to the T Ag epitopes and analyzed for intracellular IFN- production by ICS. The results in Fig. 1B show that line 243 mice failed to generate an epitope IV-specific T_CD8 response, but successfully responded to epitopes I and II/III with T_CD8 levels similar to those observed in B6 mice. Analysis of these splenocytes after 5 days in culture further confirmed the absence of epitope IV-specific T_CD8 following immunization (data not shown). These results demonstrate, using a quantitative analysis, that line 243 mice lack detectable precursors specific for epitope IV, but retain the ability to respond toward epitopes I and II/III.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. Epitope IV-specific precursors are required for successful adoptive immunotherapy in SV11 mice. A, Lysates from T Ag 251–708 plus activated ras-transformed rat embryo fibroblasts (REF/251–708ras) or pancreatic tissue from a line 243 mouse were treated with a non-T Ag-specific Ab (N), PAb 419, whose epitope is in the N terminus of T Ag, or PAb 901, whose epitope is in the C terminus of T Ag. The immunoprecipitated proteins were separated by SDS-10% PAGE, electrotransferred to membrane, probed with a mixture of PAb 419 and PAb 901, and detected by chemiluminescence. B, Groups of three line 243 or B6 mice were immunized i.p. with wild-type T Ag-transformed cells (B6/WT-19). Ten days later, splenocytes were cultured with the indicated T Ag or control Flu peptides for 5 h in the presence of brefeldin A before staining for intracellular IFN- production. Samples were gated on CD8+ cells. The percentage of CD8+ cells producing IFN- is indicated. This experiment was repeated twice with similar results. C, Groups of 8–10 80-day-old SV11 mice were irradiated (400 rads) and reconstituted the following day with line 243 or B6 splenocytes. Survival was monitored and plotted using the Kaplan-Meier method. The median age of survival was compared with SV11 mice that received only irradiation. A p-value of <0.05 was considered significant. D, Groups of SV11 mice were treated as in C. Spleen- and brain-infiltrating lymphocytes were isolated from 100-day-old SV11 mice and cultured with the indicated T Ag or control peptides for 6 h before staining for intracellular IFN- production. The data shown represent the response of three and two SV11 mice that received adoptive transfer with line 243 or C57BL/6 spleen cells, respectively.

Epitope IV-specific T_CD8 are sufficient to promote regression of established tumors in irradiated SV11 mice

While the above experiments suggest that T_CD8 specific for epitope IV are a required component of the donor lymphocyte pool, they do not address whether non-T_CD8 in the donor cell pool contribute to control of tumor progression. Thus, we developed an epitope IV-specific TCR transgenic mouse line, designated line TCR-IV, that expresses both the TCR -chain and TCR β-chain derived from an epitope IV-specific cytotoxic T lymphocyte clone. A high proportion (90%) of the CD8⁺ cells from line TCR-IV mice are specific for epitope IV as measured by MHC tetramer analysis, all of which express the expected TCR Vβ9 chain (Fig. 2A). Additionally, TCR-IV transgenic T cells produce IFN- in response to epitope IV peptide following immunization with wild-type T Ag-transformed cells (Fig. 2B), but not in response to peptides corresponding to the other known T Ag epitopes. The ability of TCR-IV T cells to lyse T Ag-expressing target cells also was determined following in vitro stimulation of lymphocytes from B6/WT-19-immunized mice with epitope IV peptide-pulsed dendritic cells for 4 days. The responding cells efficiently lysed B6/WT-19 cells and cells lacking D^b expression, but not cells lacking K^b or that express a T Ag in which epitope IV has been deleted (Fig. 2C). In contrast, CTL clone K-11, specific for epitope I, efficiently lysed B6/WT-19 cells, K^bKO/WT-Tag cells, and epitope IV mutant T Ag-expressing cells, but not T Ag-transformed cells lacking D^b expression. The MHC phenotype of these cells was confirmed by FACS analysis (Fig. 2D). Taken together, the data in Fig. 2 demonstrate that TCR-IV transgenic T cells are both epitope IV specific and H-2K^b restricted and that these mice fail to develop responses to the other T Ag epitopes.

View larger version (31K): [in this window] [in a new window]   FIGURE 2. Characterization of TCR IV transgenic mice. A, Spleen cells from 9- to 10-wk-old naive TCR-IV transgenic mice were co-stained with anti-CD8, anti-TCRVβ9, and the Kb/IV tetramer. The gated region of the dot plot indicates the percentage of CD8+ T cells specific for epitope IV. The histogram shows Vβ9 expression on CD8+ Kb/IV tetramer+ and CD8+ Kb/IV tetramer– cells. Data show a representative mouse out of six mice examined. B, Eight- to 9-wk-old TCR-IV transgenic mice were immunized with 5 x 106 B6/WT-19 cells. After 10 days, spleen cells were stimulated in vitro with the indicated peptides for 6 h at 37°C followed by ICS for IFN- production (left panels) or were stained directly ex vivo with anti-CD8 and the indicated MHC tetramer (right panels). The percentage of CD8+ cells specific for each epitope is indicated. Data are presented for one of four mice tested, which is representative of the group. C, In vitro-stimulated TCR-IV T cells or CTL clone K-11, specific for epitope I, was mixed at the indicated effector-to-target cell ratios with 51Cr-labeled T Ag-transformed cells in a 4-h cytoxicity assay. The percentage specific lysis of target cells is indicated. Data represent the mean of triplicate samples. D, MHC expression was detected on KbKO/WT-Tag, DbKO/WT-Tag, and B6/WT-19 cells using Abs specific for Kb (mAb Y3) and Db (mAb B22.249). Control cells were stained only with the secondary goat anti-mouse IgG-FITC.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Adoptive transfer of TCR-IV transgenic TCD8 dramatically prolongs the survival of SV11 mice due to regression of established tumors. A, SV11 mice (8–10/group) received 1 x 106 magnetically sorted TCR-IV transgenic TCD8 at 80 days of age with or without irradiation (400 rads) administered the previous day. Survival of SV11 mice was monitored and plotted using Kaplan-Meier analysis, and the median age of survival was compared with either SV11 mice that received no treatment (for 400 rads only and TCR-IV only groups) or SV11 mice that received only irradiation (for 400 rads + TCR-IV group). A p-value of <0.05 was considered significant. B, Groups of 80-day-old SV11 mice received adoptive transfers with 1 x 106 magnetically sorted TCR-IV transgenic TCD8 with or without irradiation. At 120 days of age, brains were harvested and the presence of choroid plexus tumors was detected by H&E staining of paraffin-embedded brain sections. The number of mice tested per group is indicated along with the number in which tumors were detected.

We determined whether control of tumor progression in SV11 mice was associated with activation, accumulation, and persistence of TCR-IV cells within the lymphoid organs of mice that received adoptive transfers. First, we determined whether naive TCR-IV T_CD8 were primed following transfer into SV11 mice with or without prior irradiation of the host. CSFE-labeled TCR-IV T_CD8 were transferred into SV11 mice at 80 days of age. At both 3 and 4 days posttransfer, TCR-IV cells recovered from the spleen had undergone multiple rounds of proliferation regardless of whether the host had received prior irradiation (Figs. 4, A and B). TCR-IV cells transferred into control-irradiated transgene-negative mice did not undergo significant proliferation, demonstrating that Ag recognition was required to induce T cell proliferation (Fig. 4A). We note that we were unable to detect a significant population of TCR-IV T cells in nonirradiated transgene-negative mice following transfer of 1 million donor T_CD8 (data not shown).

View larger version (25K): [in this window] [in a new window]   FIGURE 4. TCR-IV transgenic TCD8 accumulate early in the brains of irradiated SV11 mice. A and B, Groups of three transgene positive (SV11+) or transgene negative (SV11–) mice received 1 x 106 magnetically sorted and CFSE-labeled CD8+ TCR-IV transgenic cells with or without irradiation at 80 days of age. Three (A) or 4 (B) days later, spleen cells were harvested and the Kb/IV tetramer+ CD8+ cells were quantitated by flow cytometry (dot plots). The percentage of CD8+ cells that stained with tetramer is indicated as well as the total number of CD8+ Kb/IV tetramer+ cells per spleen. The intensity of CFSE staining, indicated by the mean fluorescence intensity (MFI), and the level of CD62L expression on CD8+ Kb/IV tetramer+ cells are shown in the histograms. This experiment was repeated twice with similar results. C, Groups of three SV11 mice received 1 x 106 magnetically sorted Thy1.1+CD8+ TCR-IV transgenic cells with or without irradiation at 80 days of age. Five days posttransfer, lymphocytes were isolated from the brain and spleen and the Thy1.1+ CD8+ cells quantified by flow cytometry (dot plots). The percentage of CD8+ cells that stained with tetramer is indicated in the spleen, and the total number of CD8+ Kb/IV tetramer+ cells per organ is indicated for both spleen and brain. The levels of CD62L expression on CD8+ Thy1.1+ cells are shown in the histograms. The data are representative of two independent experiments in which similar results were obtained.

We next examined how quickly TCR-IV cells could be detected in the brains of SV11 mice. During initial experiments, we noted that the intensity of MHC tetramer staining was reduced at early time points postadoptive transfer, particularly at day 5, reducing detection of accumulating cells (data not shown). Thus, we used TCR-IV donor mice on the Thy1.1 congenic background to identify the donor CD8⁺ cells independent of MHC tetramer staining. No TCR-IV cells were detected in the brains of SV11 mice before day 5 (data not shown). By day 5, however, Thy1.1⁺ cells represented a large portion of the CD8⁺ cells in both the spleen and brain of irradiated SV11 mice (Fig. 4C). Most of these cells expressed low levels of CD62L, indicative of an effector phenotype. By comparison, TCR-IV T_CD8 were present at 40-fold lower numbers in the spleens of nonirradiated hosts by day 5, with most cells expressing CD62L, and few donor cells detected in the brain. In fact, this level was dramatically decreased compared with that detected at day 4 (compare with Fig. 4B). The frequency of Thy1.1⁺ CD8⁺ cells detected in the CLNs of both groups was similar to that found in the spleens (data not shown). These results demonstrate that donor TCR-IV T_CD8 rapidly accumulate in the brains of irradiated, but not nonirradiated, SV11 mice and that irradiation enhances TCR-IV cell persistence in the peripheral lymphoid organs.

Increased survival of SV11 mice is associated with long-term persistence of TCR-IV cells in the brain

Having demonstrated that established tumors regress completely by 40 days after adoptive transfer in irradiated SV11 mice (Fig. 3B), we determined the extent to which tumors were eliminated from SV11 hosts at an earlier time point. Groups of 80-day-old SV11 mice received either 1) irradiation alone, 2) transfer with TCR-IV cells alone, or 3) irradiation plus transfer with TCR-IV cells. After 10 days, brains were harvested and tumor burden determined by histological analysis. The results demonstrate that tumors had completely resolved in mice that received both irradiation and adoptive transfer (Fig. 5A). This result was consistent with the early accumulation of TCR-IV T_CD8 in the brains of irradiated SV11 mice (Fig. 4A). As shown previously (29), irradiation was capable of reducing, but not eliminating, tumor burden. Unexpectedly, we observed that adoptive transfer with TCR-IV cells alone also reduced tumor burden. For both of these latter groups, however, tumor progression resumes, resulting in only a slight increase in survival (see Fig. 3A). This result suggested that SV11 tumors are initially susceptible to TCR-IV T cells even in the absence of irradiation, despite only low levels of TCR-IV cells detected at early time points postadoptive transfer.

View larger version (38K): [in this window] [in a new window]   FIGURE 5. Long-term control of tumor progression is associated with persistence of TCR-IV cells in the brain. A, Groups of six 80-day-old SV11 mice received either 400 rads (Irradiation only), TCR-IV cells, or both. After 10 days, brains were harvested and the presence of choroid plexus tumors was detected by H&E staining of paraffin-embedded sections. The samples shown are representative of the group. Arrows indicate the presence of small tumors. A representative tumor from an untreated SV11 mouse at 80 days of age is shown to indicate the average size of tumors at the initiation of treatment. B, Groups of three SV11 mice received 1 x 106 magnetically sorted CD8+ TCR-IV transgenic cells with or without irradiation at 80 days of age. Ten days later, the frequency of epitope IV-specific CD8+ cells was determined by staining of spleen- and brain-derived lymphocytes with control or Kb/IV tetramer. Data from representative mice are shown. This experiment was repeated twice with similar results. C, The absolute number of epitope IV-specific CD8+ cells ± the SE in the spleens and brains of the mice shown in B as measured by tetramer staining or by ICS for IFN- production. The data represent three mice per group ± SE. D, Groups of SV11 mice were treated as in B. The frequency of epitope IV-specific CD8+ cells was determined at the indicated ages using tetramer staining and ICS for peptide IV-induced IFN- production. Error bars indicate the SE for three mice per group.

To determine whether complete tumor regression and/or prolonged control of tumors was associated with enhanced persistence of functional epitope IV-specific T_CD8 in SV11 mice, representative mice from irradiated or nonirradiated recipients were sacrificed at increasing times postadoptive transfer. At 110 days of age, 30 days postadoptive transfer, 12% of T_CD8 in the spleen were specific for epitope IV and 8% produced IFN- in mice that received irradiation (Fig. 5D). In contrast, mice that received adoptive transfer alone had only 1% epitope IV-specific T_CD8 in the spleen with little production of IFN- (Fig. 5D). In the brain, a significant reduction in T_CD8 specific for epitope IV occurred in mice that received adoptive transfer alone: 5–6% epitope-IV-specific T_CD8 (Fig. 5D) compared with 63% epitope-IV-specific T_CD8 detected 20 days earlier (Fig. 5B). Conversely, SV11 mice that received irradiation and adoptive transfer had 50% T_CD8 specific for epitope IV in the brain, with 30% producing IFN- at day 110 (Fig. 5D). The frequency of epitope IV-specific T_CD8 in the spleens gradually decreased between days 110 and 175 in mice that received irradiation plus TCR-IV cells, but the levels remained constant in the brains of these mice over the same period. Of note, an 50% decrease was observed between days 140 and 175 in the proportion of IFN--producing epitope IV-specific T_CD8 in the brain. Mice that received only adoptive transfers were not analyzed at later time points due to tumor progression. These results indicate that TCR-IV T_CD8 primed against the endogenous T Ag in SV11 mice following irradiation and adoptive transfer are capable of persisting in both the spleen and at a high frequency in the brain at late time points, correlating with the observed enhanced survival. In contrast, rapid loss of epitope IV-specific T_CD8 from the brains and peripheral lymphoid organs of mice that received adoptive transfer alone was associated with tumor progression.

IFN--competent donor cells are required for control of tumor progression

We next assessed the requirement for known T_CD8 effector molecules in the donor lymphocytes for control of choroid plexus tumor progression. The cytokine IFN- has been strongly linked to control of tumor progression in various transplantable tumor models (55, 56, 57). Other studies have indicated a role for perforin and TNF- in the control of some tumor types (58, 59, 60). To determine which effector mechanisms may be important for adoptive immunotherapy of well-established tumors in SV11 mice, TNF- KO, IFN- KO, and perforin KO mice were used as donors for adoptive transfers. We first determined whether each mouse line could respond to immunization with T Ag. Groups of mice were immunized with B6/WT-19 cells, and 10 days later CD8⁺ splenocytes were stained with Kb/IV and Db/I tetramers to determine the frequency of T Ag-specific T_CD8. Each knockout mouse developed a predominant T_CD8 response to epitope IV and a subdominant response to epitope I, similar to that observed in the control C57BL/6 mice (Fig. 6A). Thus, all three KO strains are capable of responding to epitope IV.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. IFN--competent donor cells are required for efficient control of tumor progression. A, Groups of mice from the indicated strains were immunized with 3 x 107 T Ag transformed cells (B6/WT-19) and 10 days later spleen cells were analyzed for frequency of epitope I- and IV-specific TCD8 by staining with MHC tetramers. Each data point represents two mice. B, Groups of 8–10 80-day-old SV11 mice were irradiated (400 rads) and received 5 x 107 naive splenocytes from TNF- KO, IFN- KO, or perforin (PFP) KO donors. Survival of SV11 mice was monitored and results were plotted using Kaplan-Meier analysis. The median age of survival was compared with SV11 mice that received no treatment (for 400 rads group) or only irradiation (for all groups that received irradiation plus adoptive transfer). A p-value of <0.05 was considered significant. C, Groups of three 80-day-old SV11 mice received 400 rads of irradiation and adoptive transfer with 5 x 107 B6 or IFN- KO splenocytes. The frequency of epitope IV-specific TCD8 was determined by tetramer staining of spleen- and brain-derived lymphocytes at 100 days of age. Data in the upper panels show the frequency of epitope-IV specific cells as a percentage of CD8+ cells, while data in the lower panels show the absolute number of epitope-IV specific CD8+ cells detected per organ. The results represent data pooled from three independent experiments and the error bars indicate SE. No significant differences in the total number of recovered epitope IV-specific CD8+ cells were detected between recipients of B6 or IFN- KO donor cells as determined by Student’s t test (p = 0.19 for spleen, p = 0.66 for brain).

To ensure that epitope IV-specific T_CD8 were primed following transfer of IFN- KO splenocytes into irradiated SV11 mice, brain-infiltrating lymphocytes and splenocytes were analyzed 20 days postadoptive transfer of IFN- KO splenocytes for the presence of tetramer IV⁺ T_CD8. As shown in Fig. 6C, epitope IV-specific T_CD8 were detected in the spleens and brains of mice that received IFN- KO donor cells, ruling out the possibility that tumors progress due to lack of priming or accumulation of epitope IV-specific T_CD8. No significant differences were detected in the frequency or total number of epitope IV-specific T_CD8 recovered from the brains of mice that received IFN- KO or B6 donor cells. These results indicate that IFN--competent donor lymphocytes are required for effective control of tumor progression in SV11 mice and show that the presence of IFN--deficient epitope IV-specific T_CD8 at the tumor site is not sufficient to mediate tumor immunity.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The present investigation demonstrates that T_CD8 targeting the immunodominant epitope IV of SV40 T Ag are sufficient to induce complete regression and long-lasting control of established autochthonous tumors in the CNS. Derivation of TCR-IV transgenic mice allowed us to test the role of epitope IV-specific T_CD8 in the absence of the complex mixture of accessory donor cells present when using B6-derived donor cells (29). This approach also facilitated transfer of significantly higher numbers of Ag-specific naive donor T cells. Despite this increase, successful adoptive immunotherapy of T Ag-induced tumors still required prior conditioning of the host with gamma irradiation. This requirement was not due to a lack of T cell activation following transfer into SV11 mice, but rather was associated with several distinct consequences for TCR-IV cells in irradiated vs nonirradiated SV11 hosts. This included a more rapid differentiation and higher accumulation of TCR-IV cells in the lymphoid organs, increased kinetics of TCR-IV accumulation in the brain, and prolonged high-level persistence in the brain. This enhanced TCR-IV response was associated with a rapid and complete regression of advanced-stage choroid plexus tumors compared with only a transient partial regression in nonirradiated mice.

We found that TCR-IV T cells transferred into nonirradiated SV11 mice failed to down-regulate CD62L within the first 5 days following adoptive transfer and did not accumulate to the levels observed within the lymphoid organs of irradiated recipients. This was despite evidence of Ag recognition, as indicated by T cell proliferation. This finding is reminiscent of the peripheral tolerance observed in other systems in which recognition of cognate transgenic Ags by cross-presentation led to initial proliferation, but was followed by eventual deletion of responding T_CD8 (61, 62). Lack of CD62L down-regulation may indicate that the signal provided by resident APCs presenting T Ag epitope IV was functionally compromised compared with that provided in irradiated SV11 mice, leading to eventual tolerance (63). A similar observation was found following CD8⁺ T cell recognition of self Ags in the pancreas (62), in which T cells proliferated but failed to down-regulate CD62L to the same extent as T cells from immunized mice. In that model, however, T_CD8 failed to acquire effector function due to the lack of CD4⁺ T cell help (64), whereas TCR-IV T cells did eventually acquire the ability to produce IFN- following transfer into nonirradiated SV11 mice. We note that despite our inability to detect TCR-IV cells that had down-regulated CD62L in the peripheral lymphoid organs of nonirradiated SV11 hosts, TCR-IV cells recovered from the brains at day 10 had lost CD62L expression (data not shown). This result suggests that at least a fraction of TCR-IV cells become fully differentiated in nonirradiated SV11 mice.

The kinetics of T cell infiltration into the brain of SV11 mice was found to be accelerated in irradiated mice. This might be directly related to the magnitude of the response that occurred in the peripheral lymphoid organs. In a previous study using T Ag-immune C57BL/6 donors, we demonstrated that the kinetics of activated T_CD8 infiltration into the brains of tumor-bearing SV11 mice was dependent on the initial dose of donor lymphocytes (65). Thus, the higher numbers of TCR-IV cells that accumulate in the spleen and lymph nodes of irradiated SV11 mice may facilitate more rapid infiltration into the CNS. Alternatively, as described above, TCR-IV cells transferred into nonirradiated hosts may require more time to fully differentiate and gain the proper homing receptors in this toleragenic environment. A third possibility is that irradiation itself may have facilitated T cell migration into the CNS. Using a different model of T Ag transgenic mice, Ganss et al. (26) found that activated T Ag-specific CD4⁺ TCR-transgenic T cells efficiently infiltrated insulinomas in irradiated line Rip Tag5 mice, whereas T cells only accumulated at the periphery of the tumor in nonirradiated mice. Therefore, irradiation promoted better access to the tumor, an observation accompanied by remodeling of the tumor vasculature. A similar observation was made in mice bearing transplantable B16 tumors, in which adoptively transferred activated tumor Ag-specific T_CD8 accumulated within irradiated tumors to higher numbers than in nonirradiated tumors (66). In this latter study, T cell infiltration was associated with increased expression of VCAM-1 on the tumor vasculature.

One of the major consequences of irradiation in SV11 mice was increased T cell persistence both at the tumor site and in the peripheral lymphoid tissues, as TCR-IV T_CD8 quickly disappeared from the lymphoid tissues and brains of SV11 mice in the absence of irradiation. This was despite the presence of similar levels of TCR-IV T_CD8 within the brain 10 days following adoptive transfer. This difference in T cell persistence at the tumor site might be explained by radiation-induced changes within the lymphoid organs or in the tumor microenvironment that promote the T cell response rather than inducing tolerance. Alternatively, persistent TCR-IV T cell accumulation within the brain may be dependent on continuous trafficking of T_CD8 from the periphery. Thus, the higher numbers of TCR-IV T_CD8 that accumulate in the spleens and lymph nodes of irradiated SV11 hosts vs nonirradiated hosts may act as a reservoir for long-term maintenance of the local response at the tumor site.

The synergistic effects of radiation on adoptive T cell immunotherapy have been known for some time (67, 68, 69), although the mechanisms responsible for these effects have only recently begun to be elucidated (12). The ability of radiation to enhance priming of tumor-reactive T cells was recently investigated by Lugade et al. (66) using transplantable OVA-transfected B16 melanoma. These authors found that localized ionizing irradiation enhanced the generation of endogenous Ag-specific T_CD8, which corresponded with increased levels of Ag within the draining lymph node. This finding is consistent with studies showing that irradiation induces up-regulation of costimulatory molecule expression on APCs, including DCs, leading to enhanced T cell stimulatory capacity in vitro (70, 71). In the SV11 model, irradiation was not required to trigger T_CD8 against the endogenous T Ag, as TCR-IV T_CD8 in both the spleen and the CLN proliferated following adoptive transfer. An alternative possibility is that irradiation induces a more robust expansion and/or survival of activated T cells in tumor-bearing mice (22). We found previously that direct presentation by T Ag-transformed cells in vivo can enhance the magnitude of the T_CD8 response toward a given epitope (72). Thus, irradiation might result in increased Ag presentation due to enhanced cross-presentation, localization of tumor cells directly presenting T Ag epitopes into the draining lymph nodes (73, 74), or to increased direct presentation by the tumors due to radiation-induced up-regulation of Ag processing (75). Additionally, recent evidence suggests that ligands for TLRs may circulate systemically due to radiation-induced damage, resulting in activation of host APC populations and increased triggering of T cells (76). Whole-body irradiation is known to result in increased homeostatic proliferation of adoptively transferred T cells following Ag encounter due to the induction of lymphopenia (77, 78) and can simultaneously eliminate regulatory T lymphocytes that might inhibit T cell activation or proliferation (78). The combination of increased stimulation plus the loss of homeostatic regulatory mechanisms may culminate in the dramatically enhanced T_CD8 response to self Ags.

An important finding from the present study is that TCR-IV T cells alone were able to mediate an initial regression of advanced-stage tumors, although these tumors quickly recurred such that only a limited effect on the lifespan occurred. This initial response is explained by the finding that a significant population of TCR-IV T cells accumulates in the brains of nonirradiated SV11 mice by day 10 posttransfer, despite the extremely low levels of donor cells detected in the peripheral lymphoid organs. The inability to induce complete regression of tumors by this time point, as achieved in irradiated mice, may be explained by the observed slower kinetics of T cell infiltration. Alternatively, the ability of irradiation alone to debulk the tumor mass may reduce the amount of tumor that must be eliminated by the T cells themselves. Additionally, radiation-induced changes within the tumor microenvironment may sensitize tumors to infiltrating T cells. A recent study by Zhang and colleagues revealed that cross-presentation of tumor-derived Ag by tumor stromal cells is critical for T_CD8-mediated regression of established transplantable tumors and that transfer of Ag to the stromal cells is induced by both local irradiation and systemic chemotherapy (25). Similar events leading to increased Ag presentation by both tumor and stromal cells could explain the difference between the complete regression of established tumors observed in irradiated SV11 mice vs the partial tumor regression observed in mice given only adoptive transfer, despite the initial accumulation of TCR-IV T_CD8 within the brain in both cases. Our results emphasize that in the absence of complete tumor elimination, regrowth of aggressive tumors can quickly overshadow what initially appears as a significant effect.

Although several previous studies have addressed the role of IFN- in control of transplantable tumors, we demonstrate herein that IFN--competent donor cells play a major role in the immune-mediated control of established autochthonous tumors in the CNS. The importance of IFN- in tumor immunity has been shown by in vivo depletion studies (55, 79), as well as with mice bearing targeted deletions of the specific genes involved (80, 81). While our results indicate that IFN- production by donor cells is required for control of choroid plexus tumors, they do not rule out the additional contribution of host-derived IFN- toward tumor regression. Whether host cell-derived as well as donor T cell-derived IFN- is required for control of tumor progression has been previously investigated, but with variable results (60, 82). Additionally, recent studies have shown that host cells must remain responsive to IFN- in order for IFN--competent donor T_CD8 to induce tumor regression (60, 83, 84). This is likely due to triggering of chemokine release or sensitization of the tumor stroma and/or tumor cells to other effector cells. In SV11 mice, we found that equal numbers of IFN--competent and IFN--deficient donor cells accumulated within the brains, suggesting that lack of T cell function rather than activation or accumulation was the major contributing factor toward continued tumor progression. Overall, we can conclude that IFN- production is an important component of the donor lymphocyte population for mediating regression of established autochthonous CNS tumors in SV11 mice.

In conclusion, we have defined a model in which complete and long-lived regression of established tumors is accomplished by irradiation-enhanced adoptive immunotherapy with T_CD8 targeting a single immunodominant epitope. Our results suggest that irradiation influences T cell differentiation, accumulation, and persistence in the tumor-bearing host and might also enhance the sensitivity of tumors to the responding epitope IV-specific T_CD8, resulting in complete tumor regression.

	Acknowledgments

 
We thank Jeremy Haley and Melanie Epler for excellent technical assistance and Nate Schaffer for assistance in the Flow Cytometry Core Facility of The Pennsylvania State University College of Medicine.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflicts of interest.

	Footnotes

 
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.

¹ This study was supported by Grants CA-25000 and CA-24694 from the National Cancer Institute/National Institutes of Health and by American Cancer Society Research Scholar Grant 04-059-01-LIB.

² Current address: Department of Biological Sciences, Messiah College, Grantham, PA 17027.

³ Current address: University of Pennsylvania, Philadelphia, PA 19104.

⁴ Address correspondence and reprint requests to Dr. Todd Schell, Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA 17033. E-mail address: tschell{at}psu.edu

⁵ Abbreviations used in this paper: B6, C57BL/6; CLN, cervical lymph node; gB, glycoprotein B; ICS, intracellular cytokine staining; KO, knockout; T Ag, SV40 T antigen; T_CD8, CD8⁺ T cell; WT, wild-type.

Received for publication March 21, 2008. Accepted for publication July 17, 2008.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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