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The Journal of Immunology, 2003, 171: 2154-2160.
Copyright © 2003 by The American Association of Immunologists

Reconstitution of CD8+ T Cells by Retroviral Transfer of the TCR {alpha}{beta}-Chain Genes Isolated from a Clonally Expanded P815-Infiltrating Lymphocyte1

Hiroyuki Tahara*, Keishi Fujio*, Yasuto Araki*, Keigo Setoguchi*, Yoshikata Misaki*, Toshio Kitamura{dagger} and Kazuhiko Yamamoto2,*

* Department of Allergy and Rheumatology, Graduate School of Medicine, and {dagger} Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gene transfer of TCR {alpha}{beta}-chains into T cells may be a promising strategy for providing valuable T lymphocytes in the treatment of tumors and other immune-mediated disorders. We report in this study the reconstitution of CD8+ T cells by transfer of TCR {alpha}{beta}-chain genes derived from an infiltrating T cell into P815. Analysis of the clonal expansion and V{beta} subfamily usage of CD8+ TIL in the tumor sites demonstrated that T cells using V{beta}10 efficiently infiltrated and expanded clonally. The TCR {alpha}- and {beta}-chain sequences derived from a tumor-infiltrating CD8+/V{beta}10+ single T cell clone (P09-2C clone) were simultaneously determined by the RT-PCR/single-strand conformational polymorphism method and the single-cell PCR method. When P09-2C TCR {alpha}{beta}-chain genes were retrovirally introduced into CD8+ T cells, the reconstituted T cells positively lysed the P815 tumor cells, but not the A20, EL4, or YAC-1 cells, in vitro. In addition, the CTL activity was blocked by the anti-H2Ld mAb. Furthermore, T cells containing both TCR {alpha}- and {beta}-chains, but not TCR {beta}-chain alone, accumulated at the tumor-inoculated site when the reconstituted CD8+ T cells were adoptively transferred to tumor-bearing nude mice. These findings suggest that it is possible to reconstitute functional tumor-specific CD8+ T cells by transfer of TCR {alpha}{beta}-chain genes derived from TIL, and that such T cells might be useful as cytotoxic effector cells or as a vehicle for delivering therapeutic agents.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recently, novel strategies have been introduced for the study of immune responses against various tumors. An increasing number of tumor Ags have been found by screening for gene products differentially expressed in tumors as opposed to normal tissues and by testing for antigenicity (1). These Ags, including the form of peptides, proteins, DNA, RNA, or viral vectors, are currently used in clinical trials. Effective T cell activation depends on the presentation of these Ags by APCs, such as dendritic cells. Vaccination trials using hybrids of tumor Ags and dendritic cells have demonstrated their beneficial effect for some tumors (2, 3, 4), but these strategies do not appear to be reliably sufficient for the real patient treatments.

The CD8+ CTL play a significant role in Ag-specific immune responses to tumors or pathogens by their cytolytic activity. Tumor-reactive CD8+ T lymphocytes can often be isolated from tumors in mice or patients, and these have been used to study Ag specificity (5), frequency of T cell repertoire (6, 7), tumor Ag gene cloning (8, 9), and experimental adoptive immunotherapy (10, 11, 12). Adoptive transfer of tumor-specific CTL has resulted in regression or eradication of murine tumors (10, 13) and reduction of pulmonary metastases (14). In contrast, functional studies of tumor-infiltrating lymphocytes (TIL)3 are difficult because of their weak activity, although many attempts have been made to analyze the immune responses by freshly isolated TIL. It has also been demonstrated that TIL expanded ex vivo and then adoptively transferred to melanoma patients with IL-2 resulted in objective responses in 34% of melanoma patients (15). This observation indicated that TIL may have potent antitumor activities.

It is generally believed that each T cell has a distinct clonotype of TCR that is responsible for the Ag-specific T cell response. The complementarity-determining regions 3 (CDR3) of TCR seem to play key roles in Ag recognition (16). We previously established a method for analyzing T cell clonality by the RT-PCR/single-strand conformational polymorphism (SSCP) method (17). This method detects nucleotide changes of CDR3 of clonally expanded T cells in vivo. Using this method, we have demonstrated that in patients with solid tumors, T cell oligoclonal expansion was found to extend from the original tumor to the draining lymph nodes and to PBLs during metastasis (18). We have also shown that ovarian tumor-infiltrating T cells exhibited clonal expansion and that such T cells were specific for autologous tumors (19), suggesting that the clonally expanded T cells in the tumor site recognize a tumor Ag in vivo. These findings indicate that the information of specific TCR expressed in the tumor site may make it possible to reconstitute functional tumor-specific CTL.

Several studies have been made on the reconstitution of functional T cells by TCR {alpha}{beta}-chain gene transfer, but most of these studies have examined the reconstitution of TCRs into T cell hybridomas and T cell lines (20, 21, 22, 23, 24). We have recently reported a functional reconstitution of peripheral T lymphocytes by retroviral transfer of TCR {alpha}{beta}-chain cDNAs for restoration of immunity (25). Namely, reconstituted CD4+ T cells with OVA-specific TCR strongly responded to OVA peptide in the in vitro culture and induced an Ag-specific delayed-type hypersensitivity in response to the OVA peptide challenge in vivo. Moreover, other groups have demonstrated that peripheral T lymphocytes may be reconstituted by Ag-specific TCR gene transfer (26, 27, 28, 29).

In this study, we analyzed the TIL derived from the P815 tumor site and the TCR {alpha}- and {beta}-chain DNA sequences of clonally expanded CD8+ single TIL by the RT-PCR/SSCP method and the single-cell PCR method. We also examined the cytotoxic response of the TCR {alpha}{beta}-chain-introduced CD8+ T cells to the original tumor in vitro and the recruitment of these T cells to the P815 tumor site in vivo.


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

DBA/2J and nude mice (BALB/c nu/nu) were obtained from Japan SLC (Shizuoka, Japan). All the mice used were female and were used at 7–8 wk of age.

Cytokines and Abs

Recombinant murine IL-2 and IL-12 were obtained from R&D Systems (Minneapolis, MN). Fc block (anti-CD16/CD32), PE anti-CD8, PE anti-V{beta}10, FITC anti-TCR V{beta} chain (H57-597), and FITC anti-V{beta}2, 4, 7, 10, 13, and 14 mAb were obtained from BD PharMingen (San Diego, CA). Anti-H2Kd, anti-H2Dd, anti-H2Ld, and control IgG were obtained from Cedarlane (Ontario, Canada).

Cell culture

Mastocytoma P815 cells (30) were cultured in complete medium consisting of RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.05 mM 2-ME. Splenocytes were cultured in complete medium supplemented with 50 pg/ml murine IL-2, 2 ng/ml murine IL-12, and 10 µg/ml Con A for 48 h before retroviral infection.

Isolation of CD8+ TILs and flow cytometric analysis

DBA/2 mice were injected s.c. with 1 x 106 tumor cells. After 14–18 days, tumors were resected and each was divided into three pieces (Fig. 2A). One piece was minced to yield 1- to 2-mm pieces. To release the tumor cells and TIL, the tumor pieces were incubated in a mixture of 1 mg/ml type IV collagenase (Sigma-Aldrich, St. Louis, MO) and 20 µg/ml DNase (Sigma-Aldrich) in complete medium for 90 min at 37°C. The cell suspension was strained through nylon mesh and washed with PBS. The CD8+ TIL were isolated from the cell suspension by the treatment of Fc block and PE-labeled anti-CD8 mAb, followed by collection with anti-PE-conjugated paramagnetic beads and a MACS LS separation column, according to the manufacturer’s instructions (Miltenyi Biotech, Bergisch Glandbach, Germany). For flow cytometric analysis, tumors were resected from four P815-bearing mice, and the CD8+ TIL were isolated, as described above. The CD8+ TIL batches were mixed and stained with FITC-conjugated anti-TCR V{beta} chain or V{beta} mAbs.



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  		FIGURE 2. Determination of TCR {alpha}- and {beta}-chain CDR3 gene segments derived from a CD8+ single T cell that clonally expanded in the P815 tumor cells. A, Schematic diagrams of the protocol for the single-cell sorting and the single-cell PCR. B, Analysis of TCR {beta}-chain messages expressed in a single TIL (P09-2C clone). The RNA was extracted from T1, T2, and the P09-2C clone. RT-PCR was then performed with a V{beta}10-specific primer and a common C{beta} primer for T1 and T2, and with series of the V{beta}10-specific primers and common C{beta} primers for a P09-2C clone (see Materials and Methods). The PCR products were analyzed by SSCP. C, Nucleotide and predicted amino acid sequence from the CDR3 regions of P09-2C clone TCR {alpha}{beta}-chains. The TCR{beta} bands on an SSCP gel were cut out, amplified, and subsequently sequenced. For DNA sequencing of the P09-2C clone TCR {alpha}-chain, the PCR products from a single cell were used. N, the N sequence in each chain.

 
Oligonucleotides

Reverse-transcription (RT) reactions were performed using RT primers (C{alpha}RT, 5'-AGC TTG TCT GGT TGC TCC AG-3' and C{beta}RT, 5'-TGT GCC AGA AGG TAG CAG AG-3'). The TCR {alpha}-chains were amplified using TCR{alpha} first primer set (AL3N, 5'-TCT TCA GAA TTC TTT TTT TTT TTT TTT TTT TTT TTT-3' and C{alpha} first, 5'-GTT TTG TCA GTG ATG AAC GT-3'), TCR{alpha} second primer set (AL3N and C{alpha} second, 5'-TCG GCA CAT TGA TTT GGG AG-3'), and TCR{alpha} third primer set (AL3N and C{alpha} third, 5'-AAG TCG GTG AAC AGG CAG AG-3'). The TCR {beta}-chains were amplified using TCR{beta} first primer set (V{beta}10–1st, 5'-GCA AGA CTC TAA GAA ATT GC-3' and C{beta} first, 5'-TGG ACT TCC TTG CCA TTC AC-3'), TCR{beta} second primer set (V{beta}10–second, 5'-CTC ATT GTA AAC GAA ACA GT -3' and C{beta} second, 5'-TTC ACC CAC CAG CTC AGC TC-3'), and TCR{beta} third primer set (V{beta}10–third, 5'-AAT CAA GTC TGT AGA GCC GG-3' and C{beta} third, 5'-GGC TCA AAC AAG GAG ACC TTG-3').

Single-cell sorting and RT-PCR

The CD8+ TIL were stained with FITC-conjugated anti-V{beta}10. The CD8+/V{beta}10+ cells were sorted at a ratio of one cell/well using an automatic cell dispensing unit driven by the FACSVantage and Clone-Cyt software (BD Biosciences, Franklin Lakes, NJ). Each cell was sorted into 20 µl of RT reaction mixture (10 nM C{alpha}RT primer, 10 nM C{beta}RT primer, 1x RT reaction buffer, 100 µM each dNTP (Takara, Ohtsu, Japan), 0.5% Nonidet P-40 (Boehringer Mannheim, Mannheim, Germany), and 0.5 U/µl RNasin (Promega, Madison, WI)) in a 96-well microtiter plate. Immediately, 20 U/µl SUPERSCRIPT II (Life Technologies, Gaithersburg, MD) reagent was added to each well, and the plate was held at 37°C for 90 min. After the reaction mixture received heat inactivation for 10 min at 65°C, an equal volume of TdT solution (2x TdTase reaction buffer, 2.5 mM dATP (Amersham Pharmacia Biotech, Piscataway, NJ), and 0.5 U/µl TdT (Life Technologies, Rockville, MD)) was added to each well, and the plate was incubated for 15 min at 37°C (31). From the single-cell RT reaction mixtures, 2 µl cDNA was added to 23 µl of first PCR premix (1.6 pmol/µl each first primer, 200 mM each dNTP, and 0.25 U/µl KOD-plus-Taq polymerase (Toyobo, Osaka, Japan)) and amplified by a 25-cycle program (95°C for 1 min, 52°C for 1 min, 72°C for 2 min). Two microliters of first PCR products were used for the second PCR (30 cycles of 95°C for 1 min, 54°C for 1 min, 72°C for 2 min), using the second PCR premix (1.6 pmol/µl each second primer, 200 mM each dNTP, and 0.25 U/µl Taq polymerase (Promega)). Then 2 µl of the second PCR products was used for further amplification reaction (35 cycles of 95°C for 1 min, 54°C for 1 min, and 72°C for 2 min), using the third PCR premix (1.6 pmol/µl each third primer, 200 mM each dNTP, and 0.25 U/µl Taq polymerase).

DNA sequencing

For DNA sequencing, TCR {beta}-chain bands on a SSCP gel were cut out and amplified, as described previously (18). Amplified cDNA was purified using SUPREC-01 membrane filter (Takara) and cloned into the pGEM T vector (Promega). Plasmid DNA from positive colonies was sequenced with DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) by an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). For the TCR {alpha}-chain, the amplified PCR products from a single cell were sequenced in the same manner as the TCR {beta}-chain.

RNA extraction and cDNA synthesis

Solid tumors were resected from mice that had been injected s.c. with 1 x 106 P815 tumor cells and divided. Total RNA from the tumor pieces was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction method (15) using ISOGEN (Wako, Tokyo, Japan). Total RNA (20 µg) was converted into cDNA with random primers (Life Technologies) and SUPERSCRIPT II reverse transcriptase.

SSCP

The SSCP study was performed, as described previously (17, 18). In brief, the synthesized cDNA was amplified by PCR with a pair of V{beta}1 to V{beta}19 primers and a C{beta} common primer. The amplified DNA was electrophoresed on a nondenaturing 4% polyacrylamide gel. After transfer onto a nylon membrane, the cDNA was hybridized with a biotinylated internal common C{beta} oligonucleotide probe and visualized by subsequent incubations with streptavidin, biotinylated alkaline phosphatase, and a chemiluminescent substrate system (Phototope-Star Chemiluminescent Detection Kit; New England Biolabs, Beverly, MA).

Construction of TCR expression vectors

Because it is difficult to isolate the full-length P09-2C cDNA from a single cell, we first cloned the full-length cDNA encoding the V{alpha}3 and V{beta}10 TCR that has random CDR3 regions. Then each of the random CDR3 genes was exchanged to the P09-2C CDR3 genes using site-directed mutagenesis by PCR (32). The full-length TCR {alpha}- or {beta}-chains were inserted into the retrovirus vector pMX (33). For cell transfer experiments, the full-length TCR {alpha}-chain was inserted into the pMX-IRES (internal ribosomal entry site)/GFP (green fluorescent protein) vector. The expression plasmids were transfected to PLAT-E cells, and the supernatants were collected, as previously described (34). An efficient packaging cell line, PLAT-E, can produce retroviruses with a titer of ~1 x 107/ml (20).

Retrovirus infection to splenocytes

The mixture of both viral supernatants was placed on nontissue culture 24-well plates coated with RetroNectin and centrifuged for 3 h at 700 x g at room temperature. This step was repeated three times, and then splenocytes were placed into the plates and cultured for 24 h.

Cytotoxicity assay

Cytotoxicity was measured with a standard 51Cr release assay. Plasmid-transduced splenocytes were used as effector cells, and the tumor cells were used as targets. The lysis of target cells was determined by the release of 51Cr after 4 h of coincubation with effector cells. In the case of Fig. 4, C and D, data points are represented as {Delta}% specific lysis. {Delta}% specific lysis = (% specific lysis of TCR{alpha}{beta}-introduced T cells) - (% specific lysis of mock-introduced T cells).



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  		FIGURE 4. The cytotoxicity of T cells introduced with P09-2C TCR {alpha}{beta}-chain genes. Splenocytes from DBA/2 mice were retrovirally transduced with P09-2C TCR {alpha}{beta}-chain genes (A) or TCR {beta}-chain gene alone (B), followed by a test of their cytotoxicity against P815 tumor cells. Mock-introduced T cells were used as a control. C, TCR {alpha}{beta} or mock-introduced splenocytes were tested for their cytotoxicity against A20, EL-4, and YAC-1 target cells. {Delta}% specific lysis = (% specific lysis of {alpha}{beta}-introduced T cells) - (% specific lysis of mock-introduced T cells). Each occurrence of lytic activity was evaluated in a 4-h 51Cr release assay at different E:T ratios. D, P815 target cells were incubated with TCR {alpha}{beta} or mock-introduced splenocytes in the absence or presence of optimal concentrations of mAb at a 50:1 E:T ratio. Data points, means of triplicate determinants. Results are representative of three independent experiments.

 
Blocking experiments

Anti-H2Kd, anti-H2Dd, or anti-H2Ld mAb was added to the mixture of effector and target cells in 200 µl at an optimal concentration, according to the manufacturer’s instruction, and then incubated for 4 h at 37°C.

Cell transfer experiments

TCR{alpha}{beta} (pMX-TCR{alpha}-IRES/GFP + pMX-TCR{beta}), TCR{beta} (pMX-IRES/GFP + pMX-TCR{beta}), or mock expression vectors (pMX- IRES/GFP + pMX) were introduced into splenocytes from DBA/2 mice. For cell transfer, CD8+ T cells were isolated from the infected splenocytes by negative selection using MACS LD column. Nude mice were injected s.c. with 1 x 106 tumor cells and i.v. with 2 x 106 infected CD8+ T cells. After 12 days, CD8+ TIL were isolated, stained with PE anti-V{beta}10, and subsequently analyzed by flow cytometry.

Statistical analysis

The statistical significance of the differences was analyzed with Dunnett’s t test or least significant difference multiple comparison test using SPSS software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of P815-infiltrating T cells by SSCP

The clonal expansion of V{beta} subfamilies among infiltrating T cells in the P815 tumor site was initially analyzed by the RT-PCR/SSCP method (17, 18). Because each CDR3 sequence demonstrates unique mobility due to its unique single-strand conformation, identical mobility of the amplified CDR3 region on a gel indicates that the clones are identical (35). Therefore, a single T cell clone makes a band, which means an expanded clone.

Mice were injected s.c. with 1 x 106 tumor cells. After 14–18 days, the tumors were resected and divided, and cDNA was subsequently recovered from them and amplified by PCR with each V{beta} primer and a C{beta} common primer. We analyzed each amplified V{beta} CDR3 region by SSCP and found clonal expansion in several V{beta} chains (data not shown). The clonal expansions of V{beta}1, 2, 4, 7, 10, 13, and 14 were always detected, and multiple identical bands were observed in different samples called tumor 1 (T1) and tumor 2 (T2) of a mouse, indicating that homogenous Ag-specific immune responses existed in the different areas of the tumor site (Fig. 1). In contrast, peripheral (spleen) repertoires exhibited a smear pattern (data not shown), as described previously (17), because the CDR3 regions of T cells are diverse. Moreover, we performed flow cytometric analysis to investigate V{beta} usage of CD8+ TIL. The P815 solid tumors were resected from four mice, and CD8+ TIL were isolated, as described in Materials and Methods. The CD8+ TIL samples were mixed and stained with anti-TCR V{beta} chains, V{beta}2, 4, 7, 10, 13, or 14 mAbs, and subsequently analyzed by flow cytometry. We could not analyze the V{beta}1 usage by flow cytometry because no murine anti-V{beta}1 Ab was available. Accordingly, we found that CD8+/V{beta}10+ T cells infiltrated at high levels in the tumor (Table I). These results suggested that in the case of P815 tumors, CD8+/V{beta}10+ T cells efficiently infiltrated into the tumor and several identical clones of these T cells had expanded into different areas of the tumor site.



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  		FIGURE 1. Clonality analysis of TCR {beta}-chain messages expressed in P815-infiltrating lymphocytes. Tumor-inoculated samples were obtained from P815-bearing DBA/2 mice and divided into two pieces, called T1 and T2. The RNA was extracted from each sample, and RT-PCR was performed with each of the V{beta}-specific primers and a common C{beta} primer. The PCR products were analyzed by SSCP. Representative results obtained from selected V{beta} amplifications are shown.

 

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  		Table I. Comparison of V{beta} usage from CD8+ TILa and spleen cells

 
Isolation of TCR {alpha}- and {beta}-chains derived from a single TIL that expanded into P815 tumors

Because it seemed that CD8+/V{beta}10+ T cells had infiltrated and expanded into the P815 tumors, we attempted to simultaneously isolate TCR {alpha}- and {beta}-chains from clonally expanded TIL. To acquire the TCR {beta}-chain massage derived from a single TIL that had expanded into tumors, we performed a single-cell RT-PCR/SSCP study. As shown in Fig. 2A, the resected tumor inoculation samples were divided, and one piece was used for the single-cell sorting. The CD8+/V{beta}10+ TIL were sorted at a ratio of one cell/well. The subsequently synthesized cDNA was amplified by PCR with series sets of V{beta}10-specific primers and C{beta} common primers. Then we analyzed whether the mobility of the single-cell PCR products on a gel matched that of products from T1 and T2 by SSCP (Fig. 2A). As shown in Fig. 2B, TCR {beta}-chain from clone P09-2C exhibited the same mobility as that from TIL that expanded into both tumor inoculation sites. In addition, to confirm whether the bands represented the identical CDR3 region, we performed sequence analysis. All of the CDR3 regions were identified and found to use the same V{beta}10/D{beta}1/J{beta}2.5 gene segment (Fig. 2C).

Next, we tried to determine the TCR {alpha}-chain of the P09-2C clone. The P09-2C cDNA was added poly(A) using TdT, as described previously (24), and PCR was performed with a poly(T) primer and series of C{alpha} common primers. The amplified cDNA was then purified and cloned into a vector, on which we conducted DNA sequencing analysis. Consequently, the TCR {alpha}-chain of P09-2C clone was found to consist of the V{alpha}3.5/J{alpha}18 gene segment (Fig. 2C). This finding indicates that CDR3 of the TCR {alpha}- and {beta}-chains from clonally expanded TIL could be simultaneously determined by the RT-PCR/SSCP method and the single-cell PCR method.

Reconstituted CD8+ T cells have cytotoxic activity against P815 tumor cells

Full-length cDNAs encoding V{alpha}3.5 or V{beta}10 TCR that has random CDR3 regions were amplified from cDNA derived from TIL by PCR, and each of the random CDR3 genes was exchanged to the P09-2C CDR3 genes. The full-length P09-2C TCR {alpha}- and {beta}-chains were inserted into a retrovirus vector pMX and introduced into splenocytes using recombinant fibronectin fragment CH-296, known as RetroNectin (Fig. 3). Briefly, the supernatants were placed on RetroNectin-coated plates and centrifuged. Using this method, we achieved high efficiency of transduction for T cells. Among CD8+ T cells that were introduced, P09-2C TCR{alpha}{beta}-chain genes, 52.2~75.9% expressed V{beta}10 on the cell surface, while mock-introduced T cells were similar to nonintroduced splenocytes (11.4~13.4% of CD8+ T cells; data not shown). To test whether the P09-2C TCR-introduced T cells were functional, we measured their cytotoxic effect on P815 tumor cells in a standard 51Cr release assay. As shown in Fig. 4A, P09-2C TCR-introduced T cells positively lysed P815 tumor cells ({alpha}{beta} vs mock; p < 0.01). In contrast, mock-introduced T cells failed to lyse the tumor cells. Because T cells that express the P09-2C TCR {beta}-chain alone also failed to lyse target tumor cells (Fig. 4B), the cytotoxic ability of the T cells was found to be TCR {alpha}{beta}-chains clonotype specific. These results indicate that CD8+ T cells reconstituted with TCR {alpha}{beta}-chains derived from TIL are functional and have a clonotype-specific cytotoxic activity. To determine whether the CTL activity generated by gene transfer is P815 specific, we tested for the CTL activity using a variety of additional target cells. As shown in Fig. 4C, P09-2C TCR-introduced T cells showed no CTL activity against A20 tumor cells (H-2d), EL-4 (H-2b), or YAC-1 cells. Furthermore, to determine whether the CTL activity is MHC class I restricted, we performed blocking studies. We found that the anti-P815 CTL activity was blocked significantly with anti-H2Ld mAb (control IgG vs anti-H2Ld; p < 0.05), but not with anti-H2Kd or anti-H2Dd at a 50:1 E:T ratio (Fig. 4D). These findings suggested that the CTL activity is P815 specific and that this activity is MHC class I restricted.



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  		FIGURE 3. Schematic diagrams of the protocol for retrovirus infection to splenocytes. The mixture of both viral supernatants was placed on nontissue culture 24-well plates coated with RetroNectin and centrifuged for 3 h at room temperature. This step was repeated three times. Then splenocytes were placed into the plates and cultured for 24 h.

 
Reconstituted CD8+ T cells accumulate at the P815 tumor site

The P09-2C clone was present and expanded in the P815 tumor site (Fig. 2B), probably due to the Ag recognition by the TCR. Therefore, we examined whether, like the P09-2C clone, the reconstituted T cells accumulated at the P815 tumor site. The pMX-TCR{alpha}-IRES/GFP plasmid was used to visualize the TCR {alpha}-chains because no murine anti-V{alpha}3 Ab was available. TCR {alpha}{beta}-chains, TCR {beta}-chain plus IRES/GFP, or mock expression vectors were introduced into CD8+ T cells. Nude mice were injected s.c. with 1 x 106 tumor cells and i.v. with 2 x 106 infected CD8+ T cells. After 12 days, the CD8+ TIL were isolated, stained with PE anti-V{beta}10, and subsequently analyzed by flow cytometry. The representative result appears in Fig. 5A. Although CD8+/GFP+/V{beta}10+ was detected in both the TCR {alpha}{beta}-chains together and the TCR {beta}-chain plus IRES/GFP, the TCR {alpha}{beta}-chain-introduced CD8+ T cells were found to more efficiently accumulate at the P815 tumor site than the cells introduced with the TCR {beta}-chain alone. The GFP+/V{beta}10+ populations in the CD8+ TIL from each group are summarized in Fig. 5B. This observation suggests that, like the P09-2C clone, TCR {alpha}{beta}-chain-introduced T cells accumulate at the original tumor site, when reconstituted CD8+ T cells are adoptively transferred to tumor-bearing nude mice.



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  		FIGURE 5. Analysis of the reconstituted T cell accumulation at the original tumor site in nude mice. The DBA/2 splenocytes were retrovirally transferred with IRES/GFP (mock), TCR {beta}-chain gene plus IRES/GFP ({beta}), or TCR {alpha}{beta}-chain genes ({alpha}{beta}), and CD8+ cells were isolated from the splenocytes. Nude mice (n = 4) were injected s.c. with 1 x 106 tumor cells and i.v. with 2 x 106 CD8+ gene-transduced T cells. After 12 days, CD8+ TIL were isolated, stained with PE anti-V{beta}10, and subsequently analyzed by flow cytometry. A, Flow cytometric analysis of P815-infiltrating CD8+ T cells. The horizontal axis shows GFP intensity (TCR{alpha}-IRES/GFP or IRES/GFP for the controls), and the vertical axis shows V{beta}10 expressions. Eight individual mice were examined, and representative data are shown. B, The ratio of GFP+/V{beta}10+ TIL in CD8+ TIL from P815-bearing mice. Values represent the mean of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrated that the TCR {alpha}{beta}-chains from clonally expanded TIL can be detected by the combination of the RT-PCR/SSCP method and the single-cell PCR method. Furthermore, when functional tumor-specific CTL were reconstituted by retroviral transfer of TCR {alpha}{beta}-chain genes from clonally expanded TIL, we proved that the TCR {alpha}{beta}-chain-introduced CD8+ T cells lyse the tumor cells in vitro and accumulate at the tumor site in vivo. This is the first approach for simultaneously determining the individual TCR {alpha}- and {beta}-chains of tumor-infiltrating CD8+ T cells and for investigating the immune response of such TCR to the original tumor under both in vitro and in vivo conditions. Several studies have been made on the reconstitution of functional T cells by TCR {alpha}{beta}-chain gene transfer, but most of these studies have involved the reconstitution of TCRs into T cell hybridomas and T cell lines (20, 21, 22, 23, 24). Gene transfer into peripheral T lymphocytes has been achieved in the last few years (25, 26, 27, 28, 29). Furthermore, these studies have used cloned TCR already proven to have a functional specificity to Ag, such as OVA and influenza nucleoprotein (25, 28, 29). In this report, we reconstituted the functional CD8+ T cells by gene transfer of the functionally unknown TCR, which was supposed to recognize an unknown tumor Ag. This kind of approaches would enable us to reconstitute CTL specifically for an individual patient.

Several studies have shown that TIL are able to recognize tumor Ags and in some cases to lyse the malignant cells. These findings provided a basis for the use of in vitro cultured TIL in adoptive immunotherapy (15, 36). However, the efficacy of such cultured TIL is not sufficient for clinical use. For example, even when the freshly isolated TIL exhibited an activated phenotype and displayed strong cytotoxic activity in vitro, the TIL failed to prevent melanoma cell growth in vivo (37). Moreover, even in cases of clones capable of exerting strong antitumor cytotoxicity in vitro, these tumor-specific CTL were not sufficient to suppress tumor growth (38). The discrepancy between in vitro tumor lytic activity and in vivo tumor progression in the presence of such TIL remains to be clarified. Several studies suggest that tumors may escape from immune recognition by a variety of pathways, e.g., release of factors that inhibit the functioning of TIL.

In our previous experiments, we showed that the antigenic stimulation of a T cell population leads to clonal expansion, detected as a band by the RT-PCR/SSCP method (17, 39). Each band corresponds to an accumulated T cell clone in the heterogeneous T cell population (17). We have previously reported that, by this RT-PCR/SSCP method, distinct T cell clonotypes were detected in patients with solid tumors (18) as well as in samples from rheumatoid arthritis or systemic lupus erythematosus (35, 40). In murine P815 solid tumors, the clonal expansion of T cells with V{beta}1, 2, 4, 7, 10, 13, and 14 was repeatedly detected by the SSCP method (Fig. 1). It is likely that these clones react to a tumor Ag. Previously, Levraud et al. (41) reported that T cells with V{beta}1/J{beta}1.2 or V{beta}1/J{beta}2.5 were found in P815-infiltrating lymphocytes, and the V{beta}1/J{beta}1.2 rearrangement was borne by CTL directed against an Ag derived from P1A, a self protein expressed in P815. Likewise, clonally expanded T cells using V{beta}1 were reproducibly detected in our system. However, it was difficult to simultaneously determine the TCR {alpha}- and {beta}-chains derived from a CD8+/V{beta}1+ single TIL because no murine anti-V{beta}1 Ab is available.

In this study, one of the principal problems involved the quantitative assessment of the RT-PCR/SSCP method. The dominant band on an SSCP gel may not reflect the dominant population in TIL because of differences in primer efficiencies among the V{beta}s and in clone numbers of each population, although it is reasonable to suppose that a dominant band in a distinct V{beta} represents the dominant clone in the V{beta} population. Therefore, we analyzed the V{beta} usage of P815-infiltrating CD8+ T cells by flow cytometry. Given that a clone using a certain V{beta} displays clonal expansion on an SSCP gel and that the usage of the V{beta} is predominant in TIL, it seems quite probable that the clone recognizes a P815 tumor Ag. In our system, the usage of V{beta}10 was increased in TIL when compared with that in spleen (Table I). Recently, several investigators studied the V{beta} usage of TIL (42, 43), but few attempts have been made to simultaneously determine the V{alpha} and V{beta} usage of such cells. In this study, we also tried to determine the V{alpha} sequence of clonally expanded TIL by the single-cell PCR method. As shown in Fig. 2A, the V{alpha} sequences were rendered by 5' poly(A) addition and subsequent PCR amplification (31). It has been reported that the frequency of clonally expanded T cells could be analyzed by the single-cell PCR method (44, 45, 46). The efficiency for obtaining sequenceable PCR products from single cells is low (~10–30%) in our system compared with a previous report (46). The difference may be due to less abundant mRNA in TIL, because sequenceable PCR products were obtained from single spleen T cells in our system at the same efficiency as previously reported (46).

When the P09-2C TCR {alpha}{beta}-chain genes were transferred retrovirally to splenocytes, the ability of the transduced cells to secrete IFN-{gamma} was not significant (data not shown). This finding may be due to the relatively weak activity of the CTL. However, the reconstituted CD8+ T cells positively lysed P815 tumor cells and accumulated at the original tumor site. Hence, it is quite likely that the reconstituted CD8+ T cells recognized a P815 tumor Ag. In fact, the MHC molecule presenting P815A and B Ags was shown to be H-2Ld (8), so it is possible that the reconstituted CTL recognized the P815A or B Ag. Stronger activity against the tumor may be achieved by transferring another set of TCR {alpha}{beta}-chains, because CD8+ T cells introduced with the H2-Kb-specific TCR {alpha}{beta}-chain genes strongly lysed the H2-Kb gene-transfected P815 cells (unpublished data).

Although the TCR {alpha}{beta}-chain-introduced CD8+ cells accumulated at the P815 tumor site, such T cells did not reject the original tumor in vivo (data not shown). This finding may be due to the character of P815 tumor cells. In fact, escape variants have been known to emerge from the original P815 tumor cells in vivo (47).

The P09-2C TCR {alpha}{beta}-chain-introduced CD8+ T cells were definitely accumulated at the P815 tumor site. Recently, lymph node T cells transduced with IL-10 (48) and myelin basic protein-specific Th1 clone transduced with TGF{beta}1 (49) have been shown to ameliorate experimental autoimmune encephalomyelitis development. In addition, type II collagen-specific CD4+ T cells or hybridomas with IL-12 antagonist (IL-12p40) significantly inhibited the development of collagen-induced arthritis, and these CD4+ T cells accumulated in the inflamed joints in mice with collagen-induced arthritis (50). Moreover, when virus-specific TCR{alpha}{beta} genes were introduced into peripheral T cells, such T cells expanded upon viral infection of mice and efficiently homed to effector sites (28). These observations suggest that the Ag-specific T cells would be useful as a therapeutic vehicle. Likewise, this approach may be effective in tumors.

The study presented in this work demonstrates that it is possible to reconstitute functional tumor-specific T cells accumulating at a tumor site. We expect that using the reconstituted tumor-specific T cells as a therapeutic vehicle will make possible the development of gene therapy for patients with malignant tumors.


   Acknowledgments
 
We thank Dr. Hideo Yagita for providing P815 tumor cells. We are also grateful to Dr. Manae Kurokawa for valuable discussions.


   Footnotes
 
1 This work was supported by grants from the Ministry of Health, Labor, and Welfare, and the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Back

2 Address correspondence and reprint requests to Dr. Kazuhiko Yamamoto, Department of Allergy and Rheumatology, Graduate School of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail address: yamamoto-tky{at}umin.ac.jp Back

3 Abbreviations used in this paper: TIL, tumor-infiltrating lymphocyte; CDR, complementarity-determining region; GFP, green fluorescent protein; IRES, internal ribosomal entry site; RT, reverse transcription; SSCP, single-strand conformational polymorphism; T1, tumor 1; T2, tumor 2. Back

Received for publication August 2, 2003. Accepted for publication June 12, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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