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The Journal of Immunology, 2007, 179, 5534 -5542
Copyright © 2007 by The American Association of Immunologists, Inc.
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Dual T Cell Receptor Expressing CD8+ T Cells with Tumor- and Self-Specificity Can Inhibit Tumor Growth without Causing Severe Autoimmunity1

Monika Weinhold*,{dagger}, Daniel Sommermeyer{dagger}, Wolfgang Uckert{dagger},{ddagger} and Thomas Blankenstein2,*,{dagger}

* Institute of Immunology, Charité, Hindenburgdamm 30, Berlin, Germany; {dagger} Max-Delbrück-Center for Molecular Medicine, Berlin, Germany; and {ddagger} Institute of Biology, Humboldt University Berlin, Berlin, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The engineering of Ag-specific T cells by expression of TCR genes is a convenient method for adoptive T cell immunotherapy. A potential problem is the TCR gene transfer into self-reactive T cells that survived tolerance mechanisms. We have developed an experimental system with T cells that express two TCRs with defined Ag-specificities, one recognizing a tumor-specific Ag (LCMV-gp33), the other recognizing a self-Ag in the pancreas (OVA). By using tumor cells expressing high and low amounts of Ag and mice expressing high and low levels of self-Ag in the pancreas (RIP-OVA-Hi and RIP-OVA-Lo), we show that 1) tumor rejection requires high amount of tumor Ag, 2) severe autoimmunity requires high amount of self-Ag, and 3) if Ag expression on tumor cells is sufficient and low in the pancreas, successful adoptive T cell therapy can be obtained in the absence of severe autoimmunity. These results are shown with T cells from dual TCR transgenic mice or T cells that were redirected by TCR gene transfer. Our data demonstrate that the approach of adoptively transferring TCR redirected T cells can be effective without severe side effects, even when high numbers of T cells with self-reactivity were transferred.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The generation of Ag-specific T cells by TCR gene transfer for treatment of cancer patients is certainly a method of increasing relevance, since sufficient quantities of desired Ag-specific T cells can be engineered within a reasonable period of time. Moreover, the possibility of TCR gene transfer circumvents the need to isolate Ag-specific T cells from the patient itself. The antitumor reactivity of genetically modified T cells has been certified in mouse models (1, 2, 3, 4). Recently, promising data demonstrated the capacity of TCR-modified human T cells in a first clinical trial (5). Thereby, T cells are generated that do not occur naturally, namely T cells that can express additional, introduced TCR chains. Hence, the use of dual TCR T cells might be associated with concerns. Expression of endogenous and possibly additional mixed TCRs might lead to novel {alpha}beta TCR pairs of undefined specificities and low expression level of the desired TCRs. This might influence the avidity of the T cell, which is a sum of several contributing components, such as TCR/peptide/MHC binding affinity, TCR expression level, costimulatory molecule expression, and the extracellular microenvironment. Additionally, because activation of T cells is essential for retroviral transduction, TCR gene transfer into self-reactive T cells, which had survived tolerance mechanism, might represent an autoimmune hazard.

The main mechanism to reduce the number of self-specific T cells in the periphery is deletional tolerance in the thymus. However, high avidity T cells that recognize peripheral self-Ags that are absent in the thymus, as well as low avidity T cells that were not negatively selected, are still present in the periphery (6, 7, 8, 9, 10, 11, 12, 13, 14). These potentially autoreactive T cells are controlled by peripheral tolerance mechanisms (summarized in Ref. 15). However, a number of studies showed that peripheral tolerance can be broken (6, 7, 8, 12, 14). Several autoimmune diseases are strongly associated with bacterial (16) or viral infections (6, 7, 8, 13). In these studies, molecular mimicry, bystander effects, or release of self-Ag after tissue destruction lead to subsequent activation of T cells, which before ignored the self-Ag. Furthermore, autoimmunity can be induced by the triggering of second TCRs on self-specific T cells (17, 18). Usually, self-specific T cells have no access to most Ag-expressing somatic tissues as long as they are naive, because their circulation is restricted to blood and lymphoid tissue. Moreover, peripheral tissue cells are usually not capable of activating T cells due to the lack of costimulatory molecules. Adoptively transferred redirected T cells, activated in the course of TCR transduction, may become competent to employ the self-specific endogenous TCRs for migration into self-Ag-expressing peripheral tissue. This might bear the risk to induce autoimmunity. Conversely, one experimental approach in tumor immunotherapy is to circumvent self tolerance toward tumor-associated Ags (10, 12, 19). By this means, autoimmunity could be induced (12, 14, 20). However, several studies have shown immune responses against tumors in absence of autoimmunity (19, 21, 22, 23, 24, 25). In general, moderate autoimmune responses, which accompany tumor rejection, could be accepted (26). For example, the incidence of vitiligo in melanoma patients is associated with a better prognosis. Hence, a therapeutic window between induction of tumor rejection and avoidance of severe autoimmunity might exist.

In the present study, we used OVA peptide- (ova257) and Lymphocyte choriomeningitis virus (LCMV)3 glycoprotein peptide (gp33)-specific OT-I/P14 dual TCR T cells, either isolated from dual TCR transgenic (Dtg) mice or generated by TCR gene transfer, to investigate the influence of TCR expression level and Ag amount on the efficacy of the antitumor immune response in vivo. Moreover, OT-I/P14 Dtg T cells provided a model for T cells that express tumor- and self-specific TCRs simultaneously. We analyzed, whether suppression of the growth of gp33+ tumors is accompanied by autoimmunity in mice that express different amounts of OVA as self-Ag in the pancreatic beta-cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

OT-I/Rag1–/– mice (OT-I) (27) express a transgenic TCR (V{alpha}2, Vbeta5) specific for the OVA-derived H-2Kb-restricted peptide ova257–264 (SIINFEKL). P14/Rag1–/– mice (P14) (28) are transgenic for a TCR (V{alpha}2, Vbeta8), which recognizes the LCMV glycoprotein peptide gp33–41 (KAVYNFATM) in a H-2Db-restricted manner. OT-I/P14/Rag1–/– mice (Dtg) (29) express both, the transgenic OT-I and P14 TCRs. Mice were typed for the presence of OT-I TCRbeta and P14 TCRbeta transgenes on peripheral T cells by flow cytometry. Rag1–/– (B6.129S7-Rag1tm1Mom) (30) and C57BL/6 mice were purchased from The Jackson Laboratory. For phenotyping, the absence of B220+ cells in Rag1–/– mice was analyzed by flow cytometry. RIP-OVA-Lo mice (31, 32) express low amounts of OVA under the control of the rat insulin promoter (RIP) in the beta-cells of the pancreas. RIP-mOVA mice (33) (for convenience named RIP-OVA-Hi mice) express the membrane-bound form of OVA under control of the RIP in the beta-cells of the pancreas, the proximal tubular cells of the kidney, and small amounts in the thymus. The presence of the OVA transgene in genomic DNA of RIP-OVA-Lo and RIP-OVA-Hi mice was analyzed by PCR, using OVA-specific sense (5'-CAA GCA CAT CGC AAC CA-3') and anti-sense (5'-GCA ATT GCC TTG TCA GCA T-3') primers. All mice were on H-2b genetic background. RIP-OVA-Hi mice were backcrossed to C57BL/6 genetic background (N8–N10). Studies have been reviewed and approved by a state government review committee.

Peptides

Chicken OVA-derived ova257–264 (SIINFEKL) and LCMV-derived gp33–41 (KAVYNFATM) peptides were purchased from Biosynthan as HPLC-purified peptides. Lyophilized peptides were resuspended in 100 µl DMSO, diluted with PBS to prepare a 1 mM stock solution, sterile filtered, and stored at –20°C.

Cell lines

The OVA- and gp33-transfected H-2b positive B16 melanoma cells (B16-OVA and B16-gp33) have been described (34, 35). The cells were cultured in DMEM (Invitrogen) supplemented with 10% heat-inactivated FCS (Invitrogen), 100 U/ml penicillin/streptomycin (Invitrogen), and 1.5 mg/ml G418 (Invitrogen). In some experiments, cloned B16-gp33 cells (clone 25), whose growth properties did not differ significantly from original B16-gp33 cells, were used. Ecotropic packaging cells GP+E86 (36) were cultured in DMEM with 10% FCS. Ecotropic packaging cells Plat-E (37) were grown in DMEM with 10% FCS containing blasticidin (10 µg/ml) and puromycin (1 µg/ml) (Sigma-Aldrich). MC57-gp33-Lo fibrosarcoma cells (38) were grown in DMEM with 5% FCS and treated with 200 nM 4-hydroxytamoxifen (Sigma-Aldrich) for 4 days to induce high expression of the gp33-GFP fusion protein. These MC57-gp33-Hi cells were cloned, and a clone expressing uniform high GFP level was used.

Construction of retroviral vectors and production of virus supernatants

The P14 TCR{alpha} and TCRbeta chains were amplified by PCR from P14{alpha}2AR and P142beta8AR plasmid templates using the oligonucleotides P14{alpha} sense (5'-TTT GCG GCC GC AGT CTA GGA GGA ATG GAC AAG-3'), P14{alpha} antisense (5'-CCG GAA TTC TCA ACT GGA CCA CAG CCT CAG-3'), P14beta sense (5'-TTT GCG GCC GC CTG AGA GGA AGC ATG TCT AAC-3'), and P14beta antisense (5'-CCG GAA TTC TCA GGA ATT TTT TTT CTT GAC C-3'). The retrovirus vector MP71GPRE (39, 40) was partially digested using EcoRI (Amersham Pharmacia Biotech), to maintain the posttranscriptional regulatory element (PRE) and to excise the GFP gene (G) and subsequently digested with NotI (Amersham Pharmacia Biotech). The P14 TCR{alpha} and TCRbeta-chain fragments, digested with NotI and EcoRI were inserted into MP71PRE to generate MP71-P14{alpha} and MP71-P14beta, respectively. Stable MP71-P14beta vector particle producer cells were generated as described before (41). Transient producer cells were generated by lipofection (Lipofectamin 2000, Invitrogen) of Plat-E packaging cells with the MP71-P14{alpha} plasmid. Virus supernatant from Plat-E cells was produced in 6-well plates and was used freshly after filtration through a 0.45-µm filter.

Transduction of T cells

Spleen cells (6 x 106 cells per well and ml) were in vitro stimulated with 1 µM peptide and 10 U/ml of IL-2 for 24 h and transduced on two consecutive days with 2 ml of each virus supernatant in 6-well nontissue culture plates coated with RetroNectin (Takara Shuzo) as described (42). Virus supernatant was supplemented with protamine sulfate (4 µg/ml; Sigma-Aldrich). For each transduction, the plates were spinoculated for 1 h at 1000 x g at 32°C. The level of transgene expression was measured 4–5 days after first transduction by flow cytometry.

Spleen cell preparation

Single cell suspensions of spleens, isolated from Stg and Dtg mice or T cell-recipients, were treated with lysing buffer, containing 0.12 M NH4Cl, 1 mM KHCO3, and 0.1 mM Na2EDTA to remove RBC. Unless otherwise indicated, 2 x 106 spleen cells/ml were cultured in RPMI 1640 (Invitrogen), 10% FCS, 100 U/ml penicillin/streptomycin, 2 mM glutamine (Invitrogen), 1 mM HEPES Buffer (Invitrogen), 50 µM 2-ME (Sigma-Aldrich), and 1 mM sodium pyruvate (Invitrogen), and stimulated with 1 µM peptide.

Flow cytometry

FITC- or allophycocyanin-labeled anti-CD8, FITC- or PE-labeled anti-B220, PE-labeled anti-Vbeta8, and FITC-labeled anti-Vbeta5 mAb were purchased from BD Biosciences. Allophycocyanin-labeled recombinant MHC tetramers H-2Db/gp33–41 and PE-labeled recombinant MHC tetramers H-2Kb/ova257–264 were purchased from Immunomics Operations. To analyze the expression of OT-I and P14 TCRs, cells were stained for CD8, Vbeta5 (OT-I), and Vbeta8 (P14) or for CD8 and both tetramers. TCR expression was measured using a FACSCalibur flow cytometer (BD Biosciences) and CellQuest Pro software (BD Biosciences). Dead cells were gated out using forward and sideward light scattering parameters.

Tumor challenge and adoptive T cell transfer

Tumor cells were trypsinized, washed twice in Dulbecco’s PBS (Invitrogen), and injected s.c. into the abdominal region. The tumor size was determined as the mean of the largest diameter and the diameter at right angle. Mice were scored as tumor positive when the tumor size reached 0.5 cm and sacrificed when the tumor size reached 1.5 cm. To generate CTL effector cells, spleen cell suspensions were stimulated as described above. After 72 h, the effector cells were washed twice in Dulbecco’s PBS and injected into the tail vein (i.v.) of age- and sex-matched mice.

Diabetes monitoring

The glucose concentration in blood, isolated from the tail vein of mice, was analyzed using the ELITE glucometer (Bayer). Mice with a blood glucose concentration of >13.9 mM on two consecutive measurements were considered as diabetogenic.

Immunohistochemistry

Staining of frozen sections (6 µm) was performed using biotin-conjugated anti-CD8 mAb (Caltag Laboratories) and streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch), followed by incubation with Fast Red Substrate (DakoCytomation). Sections were counterstained with hematoxylin (DakoCytomation) and analyzed with an Olympus CKX41 microscope (Olympus) equipped with LCAch (4x/0,13; 10x/0,25, 20x/0,40; and 40x/0,55) objectives (Olympus). In addition to the objectives, there was an extra enlargement device included in the body of the microscope. Photographs were taken with a Camedia C-5050 Zoom camera (Olympus).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
OT-I/P14 dual TCR T cells are specific for both ova257 and gp33 peptide

OT-I/P14 dual TCR T cells were either isolated from OT-I/P14 Dtg mice (29) or generated by P14 TCR gene transfer into OT-I CD8+ T cells. All TCR transgenic mice used in this study were on Rag1–/– genetic background. Single TCR transgenic (Stg) T cells expressed either the OT-I (Vbeta5+) or the P14 TCRs (Vbeta8+) (Fig. 1A). Dtg mice expressed both beta chains on approximately 89% of the T cells, whereas 10% were only Vbeta5+ (OT-I). Kb/ova257 and Db/gp33 tetramers were bound by 79% of Dtg T cells, confirming that Dtg T cells express both, OT-I and P14 TCRs on the majority of CD8+ T cells. The P14 TCR expression level on Dtg T cells was reduced in comparison to that on P14 T cells (mean fluorescence intensity of 640 vs 1780 (Vbeta8+) and 370 vs 960 (Db/gp33+)), whereas the OT-I TCR expression level was comparable on OT-I and Dtg T cells. To generate OT-I/P14 Dtg T cells by TCR gene transfer, we transduced ova257 peptide in vitro activated OT-I T cells with the combination of P14{alpha} and P14beta TCR chain-encoding retroviral vectors. Transduction of OT-I T cells with P14beta- (as control) or the combination of P14{alpha}- and P14beta-encoding vectors led to expression of P14beta chains in 72 and 67% of the OT-I T cells, respectively (Fig. 1B). P14 Vbeta8 and OT-I Vbeta5 chains were expressed simultaneously on 56% of P14beta- and 57% of P14{alpha}beta-transduced OT-I T cells. After P14{alpha}beta-cotransduction, 30% of the OT-I T cells were Db/gp33 tetramer positive, whereas P14beta-transduced OT-I T cells bound Kb/ova257 tetramers exclusively (Fig. 1B). The level of P14 TCR expression on P14{alpha}beta-transduced OT-I T cells was comparable with that on P14 effector T cells (TE). Thus, OT-I/P14 dual TCR T cells were either isolated from Dtg mice or generated by P14 TCR gene transfer into OT-I T cells. Thereby, simultaneous expression of OT-I and P14 TCRs on dual TCR T cells enables them to bind both, ova257 and gp33 peptides.


Figure 1
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  		FIGURE 1. OT-I/P14 dual TCR T cells are specific for both, ova257 and gp33 peptides. Freshly isolated spleen cells from OT-I, P14, and dual TCR transgenic (Dtg) mice (A) or for 5 days with the indicated peptide in vitro stimulated spleen cells (from OT-I and P14 mice) that had been transduced (td) with MP71-P14beta- and a combination of MP71-P14{alpha}- and MP71-P14beta-vectors, respectively (OT-I) (B), were analyzed for expression of CD8, Vbeta5, and Vbeta8 or for CD8 and binding of Kb/ova257 and Db/gp33 tetramers by flow cytometry. Numbers indicate the percentage of positive cells within the CD8+-population. Shown is one representative example out of at least two experiments.

 
TCR expression level on tumor-specific CD8+ T cells and Ag amount expressed by tumor cells influence suppression of tumor growth

We showed before that OT-I/P14 dual TCR T cells have similar peptide sensitivity to OT-I and P14 single TCR T cells in vitro (29). However, the lower P14 TCR level on Dtg T cells might impair their efficacy in vivo. Therefore, we challenged Rag1–/–, OT-I, P14, and Dtg mice with 1 x 105 B16-OVA or B16-gp33 melanoma cells. B16-OVA tumor cells grew out rapidly in Rag1–/– and P14 mice (Fig. 2A), whereas OT-I and Dtg mice similarly suppressed the outgrowth of B16-OVA cells. B16-gp33 tumor cells grew out rapidly in Rag1–/– and OT-I mice (Fig. 2B), whereas P14 mice rejected the tumor. Growth of B16-gp33 tumors was less efficiently suppressed in Dtg mice, leading to outgrowth of B16-gp33 tumors in 3/4 mice between days 44 and 72. To test, whether the less effective gp33-reactivity of Dtg mice is sufficient to reject tumor cells that express high level of gp33-Ag, we challenged Rag1–/–, OT-I, P14, and Dtg mice with 2 x 106 MC57-gp33-Hi or MC57-gp33-Lo fibrosarcoma cells that express high and low amount of gp33, respectively (38). MC57-gp33-Hi tumor cells grew out rapidly in Rag1–/– and OT-I mice (Fig. 2C), but were rejected in P14 and Dtg mice. In contrast, MC57-gp33-Lo tumor cells grew out in Rag1–/–, P14, and Dtg mice (Fig. 2D). Next, we analyzed the ability of adoptively transferred P14 TCR transduced OT-I TE to suppress growth of gp33-expressing tumor cells in Rag1–/– mice. For this purpose, gp33 peptide in vitro activated P14 or ova257 peptide in vitro activated OT-I T cells which were either nontransduced or had been transduced with P14{alpha}beta-chain genes were injected i.v. into Rag1–/– mice which had been challenged one day earlier with either 2 x 106 MC57-gp33-Hi or 5 x 104 B16-gp33 tumor cells. Equal numbers of Db/gp33-tetramer+ cells (2.2 x 105 within a total of 2.2 x 106; 10% transduced (Fig. 2E) and 2.8 x 106 within a total of 1.2 x 107; 23% transduced (Fig. 2F)) were injected. In Rag1–/– mice that had received OT-I TE, the MC57-gp33-Hi tumor cells grew out rapidly (Fig. 2E), whereas they were rejected after adoptive transfer of P14 and P14{alpha}beta-transduced OT-I TE, respectively. B16-gp33 tumor cells grew out rapidly in mice that had received none or OT-I TE (Fig. 2F), but were rejected in mice that had received P14 TE. The suppression of B16-gp33 tumor growth by P14{alpha}beta-transduced OT-I TE was less efficient. To test, whether adoptively transferred P14{alpha}beta-transduced OT-I T cells persisted in B16-gp33 tumor-bearing Rag1–/– mice (shown in Fig. 2F), spleen cells were analyzed for expression of the corresponding TCR chains. The P14{alpha}beta-transduced OT-I T cells were detected in 4/4 mice analyzed (Fig. 2G, gated on lymphocytes), indicating that P14 TCR transduced OT-I cells had survived. These data indicate that 1) reduced TCR expression on CD8+ T cells is sufficient to reject tumors, 2) at low amount of Ag on tumor cells, reduced TCR expression impairs T cell efficacy, and 3) TCR transduced T cells can reject tumors that express sufficient amount of Ag.


Figure 2
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  		FIGURE 2. TCR expression level on tumor-specific CD8+ T cells and Ag amount expressed by tumor cells influence the suppression of tumor growth. A–D, Rag1–/–, OT-I, P14, and Dtg mice were s.c. challenged with 1 x 105 B16-OVA (A) and B16-gp33 (B) melanoma cells, respectively or with 2 x 106 MC57-gp33-Hi (C) and MC57-gp33-Lo (D) fibrosarcoma cells, respectively. E–F, Spleen cells from OT-I and P14 mice were stimulated in vitro with the indicated peptides for 8 (E) or 6 days (F). P14 or OT-I effector T cells (TE), the latter either non-transduced or transduced with P14{alpha}beta TCR chain genes (as described in Fig. 1), were injected i.v. into Rag1–/– mice that had been s.c. challenged one day earlier with 2 x 106 MC57-gp33-Hi (E) or 5 x 104 B16-gp33 (F) tumor cells. A total of 2.2 x 105 (E) or 2.8 x 106 (F) tetramer+ TE were adoptively transferred. Shown is the percentage of tumor free mice at the indicated time points after tumor challenge and adoptive T cell transfer, respectively. Total numbers (n) of analyzed mice are indicated. G, P14{alpha}beta-transduced OT-I spleen cells from B16-gp33-tumor bearing Rag1–/– mice (shown in F) were isolated between day 37 and 51 after adoptive T cell transfer and analyzed for expression of Vbeta5 and Vbeta8 by flow cytometry (gated on lymphocytes). Spleen cells from Rag1–/– mice that did not receive T cells served as control. A and B, Similar results were obtained in a second experiment. However, in this experiment tumors grew out after a long latency. C, D, and F, Shown are combined results from two independent experiments. E, Shown is one experiment. G, Shown is one representative example out of four animals analyzed.

 
OT-I/P14 Dtg TE cells induce severe autoimmunity in RIP-OVA-Hi mice

To analyze dual TCR T cells that express simultaneously tumor Ag- (gp33-) and self-Ag- (ova257-) specific TCRs on the surface, we combined the tumor model with an autoimmune model. We asked, whether suppression of tumor growth by Dtg T cells is accompanied by autoimmunity in mice that express OVA as self-Ag. We used RIP-OVA-Hi mice (33), which express a high amount of OVA in the pancreatic beta-cells. RIP-OVA-Hi mice are tolerant for the OVA Ag. Destruction of beta-cells causes reduced insulin production and subsequently increased blood glucose (BG) level. T cells of OT-I, P14, and Dtg mice were stimulated in vitro for 72 h in an Ag-specific manner with ova257 or gp33 peptide. TE (1 x 107 cells) were injected i.v. into RIP-OVA-Hi and C57BL/6 littermate mice, which had been challenged 1 day earlier with B16-gp33 tumor cells. Adoptive transfer of OT-I and Dtg TE induced severe diabetes in RIP-OVA-Hi mice (Fig. 3, A and B, respectively). The BG level increased from 8–13 mM to 21- ≥33.3 mM, whereas it maintained with 7–10 mM normal in RIP-OVA-Hi mice that had received P14 TE (Fig. 3A) and in C57BL/6 mice that had received none, OT-I, or Dtg TE (Fig. 3C). Diabetogenic RIP-OVA-Hi mice showed severe symptoms of diabetes, as weakness and emaciation (diabetic wasting) and were therefore sacrificed after two repetitive measurements of BG level of ≥25 mM. The outgrowth of B16-gp33 tumors was suppressed in RIP-OVA-Hi mice that had received P14 TE (Fig. 3A), whereas tumors grew out rapidly in C57BL/6 mice that had received none or OT-I TE (Fig. 3C). The adoptive transfer of gp33 peptide stimulated Dtg T cells suppressed the outgrowth of B16-gp33 tumors in C57BL/6 mice until day 16–100. 1/4 C57BL/6 mice that were treated with Dtg TE (Fig. 3C) and 1/3 RIP-OVA-Hi mice that had received P14 TE (Fig. 3A) were tumor free after a period of 100 days. These results show that OT-I/P14 Dtg T cells that were activated via the tumor Ag- (gp33) or self-Ag- (ova257) specific TCRs induce severe diabetes in mice that express the self-Ag at high levels.


Figure 3
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  		FIGURE 3. OT-I/P14 Dtg TE induce severe autoimmunity in RIP-OVA-Hi mice. Spleen cells of OT-I, P14, and Dtg mice were stimulated in vitro with the indicated peptides for 72 h. TE (1 x 107) were adoptively transferred into RIP-OVA-Hi (A and B) and C57BL/6 littermate mice (C), which had been s.c. challenged one day earlier with 4 x 105 B16-gp33 (clone 25) melanoma cells. Shown is the blood glucose (BG) concentration (open symbols) and the mean tumor diameter (T, filled symbols) at different time points after T cell transfer as combined results from two independent experiments (B) or from one experiment (A and C). Individual tumor growth curves are shown, when tumors grew out with a latency period (A and C). The number (n) of analyzed mice is indicated. The death time point of mice that had to be killed due to tumors ({dagger}T) or due to severe diabetes ({dagger}Dm) is indicated.

 
Suppression of tumor growth through OT-I/P14 Dtg TE cells in RIP-OVA-Lo mice without severe autoimmunity

To examine the influence of self-Ag expression level, we analyzed suppression of B16-gp33 tumor growth by Dtg TE in RIP-OVA-Lo mice (32), which express low amounts of OVA in the pancreatic beta-cells. In RIP-OVA-Lo mice, the OVA-Ag is ignored by the immune system. T cells of OT-I, P14, and Dtg mice were stimulated in vitro for 72 h in an Ag-specific manner with ova257 or gp33 peptide. TE (1 x 107) were injected i.v. into RIP-OVA-Lo and C57BL/6 mice, which had been challenged one day earlier with B16-gp33 tumor cells. The B16-gp33 tumors grew out rapidly in RIP-OVA-Lo and C57BL/6 mice that had received none or OT-I TE, whereas the adoptive transfer of P14 TE suppressed the outgrowth of B16-gp33 tumors (Fig. 4, A and D). The adoptive transfer of Dtg TE suppressed the growth of B16-gp33 tumors in 8/10 RIP-OVA-Lo mice (Fig. 4, B and C). 99 days after adoptive transfer, 3/4 RIP-OVA-Lo mice that had received P14 TE (Fig. 4A) and 2/10 RIP-OVA-Lo mice that had received Dtg TE were tumor free (Fig. 4, B and C). RIP-OVA-Lo mice that had received OT-I TE became diabetogenic eight days after adoptive T cell transfer (Fig. 4A). The BG concentration increased from 6–9 mM to 14–26 mM, whereas the BG level of 6–9 mM changed neither in RIP-OVA-Lo mice that had received none or P14 TE nor in C57BL/6 mice that had received P14 or OT-I TE (Fig. 4D). The inhibition of tumor growth by ova257 and gp33 peptide stimulated Dtg T cells was accompanied by an increase in the BG level from 6–8 to 12–23 mM (Fig. 4, B and C). 8/10 RIP-OVA-Lo mice developed diabetes. However, in contrast to RIP-OVA-Hi mice, RIP-OVA-Lo mice tolerated the obvious mild autoimmunity, and 9/10 survived long-term without symptoms of weakness. The increased BG level in RIP-OVA-Lo mice was in 3/4 Dtg/gp33- and 1/4 Dtg/ova257-recipients transient. The residual four RIP-OVA-Lo mice showed enhanced BG level in intervals. These results demonstrate suppression of B16-gp33 tumor growth by OT-I/P14 Dtg T cells in the absence of severe autoimmunity, if the self-Ag is expressed at low levels.


Figure 4
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  		FIGURE 4. Suppression of tumor growth through OT-I/P14 Dtg TE in RIP-OVA-Lo mice without severe autoimmunity. TE (1 x 107), generated as described in Fig. 3, were adoptively transferred into RIP-OVA-Lo (A–C) and C57BL/6 mice (D), which had been s.c. challenged one day earlier with 5 x 104 B16-gp33 melanoma cells. Shown is the BG concentration (open symbols) and the mean tumor diameter (T, filled symbols) at different time points after T cell transfer as combined results (A and D), or individual mice are shown (B and C). Individual tumor growth curves are shown, if tumors grew out with a latency period (A and D). The number (n) of analyzed mice is indicated. The death time point of mice that had to be killed due to tumors ({dagger}T), due to severe diabetes ({dagger}Dm), or of mice that died for unknown reason ({dagger}?) is indicated. Shown is one representative example from at least two individual experiments with similar results.

 
Stg and Dtg CD8+ TE infiltrate B16-gp33 tumors and Langerhans-islets in an Ag-specific manner

To investigate infiltration of CD8+ donor T cell populations in RIP-OVA-Hi and RIP-OVA-Lo mice, tumor and pancreas tissue was analyzed after adoptive T cell transfer by immune histology. For this purpose, OT-I, P14, and Dtg TE (1 x 107) were adoptively transferred into RIP-OVA-Hi and RIP-OVA-Lo mice, which had been challenged 6 days earlier with 5 x 105 B16-gp33 tumor cells. Tissues were recovered 6–7 days after adoptive T cell transfer. B16-gp33 tumors isolated from RIP-OVA-Hi and RIP-OVA-Lo mice that had received P14 or Dtg TE were infiltrated by CD8+ T cells (Fig. 5A), whereas no CD8+ cells were detected in tumors from OT-I TE-recipients. Langerhans-islets were hardly detected in RIP-OVA-Hi mice that had received OT-I or Dtg TE (Fig. 5B), whereas islets were intact in recipients of P14 TE. This indicates the rapid islet destruction in RIP-OVA-Hi mice by ova257-specific T cells; only few CD8+ T cells were detected in this late phase of diabetes induction. Islets of mice that had received P14 TE did not show CD8+ T cell infiltration. CD8+ T cells were detected in islets of RIP-OVA-Lo mice that had received OT-I or Dtg TE. These results show that adoptively transferred Stg and Dtg TE migrate into pancreas and tumor tissues in an Ag-specific manner. Moreover, RIP-OVA-Hi and RIP-OVA-Lo mice that had received adoptive transfer of OT-I and Dtg TE differ with regard to the degree of islet destruction, showing complete disruption of islets in RIP-OVA-Hi mice.


Figure 5
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  		FIGURE 5. Ag-specific CD8+ T cell infiltration in RIP-OVA-Hi and RIP-OVA-Lo mice. TE (1 x 107), generated as described in Fig. 3, were adoptively transferred into RIP-OVA-Hi and RIP-OVA-Lo mice, which had been s.c. challenged 6 days earlier with 5 x 105 B16-gp33 melanoma cells. Tissues were recovered 6 or 7 days after adoptive T cell transfer. Frozen sections of tumor (A) and pancreas (B) tissue were stained for CD8+ T cells using biotin-conjugated mAb directed against CD8 and streptavidin-conjugated alkaline phosphatase, followed by incubation with DAKO-Fuchsin. Sections were counterstained with hematoxylin. The size bars represent 100 µm. Shown is one representative example out of three experiments.

 
Recurrence of enhanced BG level after re-stimulation of persistent OT-I/P14 Dtg T cells in RIP-OVA-Lo mice

We asked whether adoptively transferred Dtg TE persisted and remained functional in RIP-OVA-Lo mice that had rejected the B16-gp33 tumor and recovered from diabetes. To restimulate the adoptively transferred Dtg T cells in vivo, mice were re-challenged with 2 x 106 MC57-gp33-Hi fibrosarcoma cells s.c. 64 or 161 days after adoptive T cell transfer (indicated by arrows, Fig. 6, A and B). Mice had been diabetogenic for up to 40 days. At the time point of rechallenge, the BG level was with 6–12 mM in a normal range. Six to sixteen days after re-challenge, the BG level increased to 15–22 mM, whereas it remained normal in mice that had received P14 T cells before rechallenge (data not shown). Blood samples, taken 75 and 169 days after adoptive T cell transfer (indicated by arrowheads, Fig. 6, A and B), confirmed the presence of Dtg CD8+ T cells, as shown by simultaneous binding of Vbeta5 and Vbeta8 mAb (Fig. 6C) or by binding to specific peptide/MHC tetramer complexes (Fig. 6D) (gated on CD8+ T cells). The percentage of donor T cells that persisted in RIP-OVA-Lo mice varied for unknown reason (1–57%; Table I). Thereby, traceability of transferred Dtg T cells in 6/14 mice correlated with increase of BG level after rechallenge with MC57-gp33-Hi tumor cells. These data demonstrate that the adoptively transferred Dtg T cells survived and remained functional in vivo, confirmed by recurrence of enhanced BG level after MC57-gp33-Hi-challenge.


Figure 6
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  		FIGURE 6. Recurrence of enhanced BG level in RIP-OVA-Lo mice after restimulation of persistent OT-I/P14 Dtg T cells. RIP-OVA-Lo mice that had rejected the B16-gp33 tumor cells and recovered from diabetes (experimental setting as described in Fig. 4) were rechallenged with 2 x 106 MC57-gp33-Hi fibrosarcoma cells s.c. 64 (A) or 161 (B) days after adoptive T cell transfer (indicated by arrows). Shown are the BG concentration (open symbols) and the mean tumor diameter (T, filled symbols) at different time points after T cell transfer into RIP-OVA-Lo mice as individual results. Numbers (n) of analyzed mice are indicated. C and D, Seventy-five days (Dtg/ova257) or 169 days (Dtg/gp33) after adoptive T cell transfer (indicated by arrowheads in A and B), peripheral blood cells from RIP-OVA-Lo mice (shown in A and B, respectively) were analyzed for expression of CD8, Vbeta5, and Vbeta8 or for CD8 and binding of Kb/ova257 and Db/gp33 tetramers by flow cytometry. Numbers indicate the percentages of positive cells within the CD8+-population. All data are summarized in Table I.

 

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  		Table I. Recurrence of enhanced BG level in RIP-OVA-Lo mice after in vivo re-stimulation of Dtg T cells by MC57-gp33-Hi tumor challengea

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Since polyclonal T cell populations might contain autoreactive clones that survived tolerance mechanisms, gene transfer of a tumor-reactive TCR may generate TE that react to tumor- as well as to self-Ag. In the present work, OT-I/P14 Dtg T cells provided a model to analyze such dual TCR T cells in a combined tumor/autoimmune model. We have shown that even under rigorous experimental conditions with a high frequency of autoreactive T cells, a therapeutic window can be defined, in which the dual TCR T cells reject the tumor cells without causing severe autoimmunity. Critical factors for the therapeutic window are the amount of Ag expressed by tumor vs self-tissue and the TCR expression level on the transferred T cells. Autoimmunity was devastating in mice expressing high amounts of Ag in the pancreas and transient in mice expressing the Ag in low amounts. Since the probability of deletional tolerance is high for self-Ags that are expressed in large amounts, and low amounts of self-Ag cause minor side effects, toxicity mediated by the endogenous, potentially self-reactive, TCRs can be considered as negligible. Even though OT-I/P14 Dtg T cells infiltrated pancreatic islets in RIP-OVA-Lo mice, the increase in BG level was transient in most of the mice. In some of the mice, alternating increase and decrease of BG level was observed that obviously correlated with a persistent antitumor reactivity of the OT-I/P14 Dtg T cells, until after a long, albeit variable, latency, B16-gp33 tumors frequently grew out. From a previous study, we knew that these were in most cases Ag-loss variants (29). We assume that the low Ag expression in the pancreas of RIP-OVA-Lo mice is unable to maintain locally the effector function of the T cells, while growth of gp33-expressing B16 cells is still suppressed at a distant site. Alternatively, under selective pressure by TE, beta-cells in the pancreas can down-regulate Ag expression (43). The regenerative potential of islet tissue may contribute to prevention of overt diabetes in RIP-OVA-Lo mice (44, 45, 46).

OT-I/P14 dual TCR T cells generated by gene transfer or by crossing the two TCR transgenic mouse lines had comparable efficacy. However, the P14 TCR was expressed at lower level on dual compared with single TCR expressing T cells, which may lead to reduced T cell avidity and function (47, 48). Even though the efficacy of transferred T cells varied between different experiments, OT-I/P14 dual TCR T cells were, on the average, clearly less effective to control B16-gp33 tumor growth in comparison to P14 single TCR T cells. On the other hand, the lower P14 TCR expression level on Dtg T cells was sufficient to reject MC57-gp33-Hi tumors in an Ag-specific fashion. Even though other factors likely contributed to the different efficacy of T cells to reject the two tumor cell lines (49), we conclude that the efficacy of tumor-specific T cells varies, depending on TCR expression level on Dtg T cells and Ag amount expressed by tumor cells. Even if TCR expression on dual TCR T cells is lower compared with normal TCR expression, it can be sufficient to reject tumor cells that express large amounts of the Ag.

In RIP-OVA-Lo mice that remained long-term B16-gp33 tumor free and had recovered from diabetes (14/23), the Dtg T cells persisted, with varying frequency, long-term and remained functional (6/14). Triggering of the Dtg T cells via the gp33-specific TCRs induced an increase in the BG level that must have been mediated via the ova257-specific TCRs on Dtg T cells. Thus, the pancreatic beta-cells in RIP-OVA-Lo mice with persistent Dtg T cells still expressed sufficient amount of OVA to be recognized, however, they were ignored, unless the Dtg T cells were restimulated. These data are comparable to other models showing that low amount of Ag is unable to induce or sustain T cell function (6, 10, 19). Restimulation of persisting Dtg T cells induced a shorter increase of BG level than the initial adoptive transfer of the TE. This could be due to rapid rejection of the highly immunogenic MC57-gp33-Hi cells. Alternatively, dual TCR T cells may have selected for reduced OVA expression in the pancreas. In conclusion, adoptively transferred dual TCR T cells can be safe and effective, even if the antitumor TCR is expressed at reduced level, provided the tumor Ag is expressed at sufficient level and the self-Ag at low level.


   Acknowledgments
 
We thank Martina Grabbert and Robert Manteufel for expert technical assistance; Sven Hartmann (Max Delbrtich-Center for Molecular Medicine, Berlin, Germany) for assistance with immunohistochemistry; T. Brocker, H. Lauterbach, and M. Cannarile (Institute of Immunology, Munich, Germany) for RIP-OVA-Lo and RIP-mOVA mice; R. Schwartz and E. Majane (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD) for P14 mice; R. Dutton (Trudeau Institute, Saranac Lake, NY) for B16-OVA cells, H. Pircher (Universitätsklinikum, Freiburg, Germany) for B16-gp33 cells, P14{alpha}2AR, and P142beta8AR plasmids; A. Banks (Columbia University, New York, NY) for GP+E86 cells; T. Kitamura (Institute of Medical Science, Tokyo, Japan) for Plat-E cells; and H. Schreiber (University of Chicago, Chicago, IL) for MC57-gp33 cells. OT-I/Rag1–/– mice (B6-(TG)TCR-OT-I-RAG1 tm1Mom, line # 4175) were obtained through the National Institute of Allergy and Infectious Diseases Exchange Program, National Institutes of Health.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict 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 from the Sixth Framework Programme (ATTACK 18914) and Deutsche Forschungsgemeinschaft (Sonderforschungsbereich TR36). Back

2 Address correspondence and reprint requests to Dr. Thomas Blankenstein, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle Str. 10, Berlin, Germany. E-mail address: tblanke{at}mdc-berlin.de Back

3 Abbreviations used in this paper: Stg, single TCR transgenic; Dtg, dual TCR transgenic; BG, blood glucose; LCMV, Lymphocyte choriomeningitis virus; TE, effector T cells. Back

Received for publication July 20, 2007. Accepted for publication August 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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