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Genes Into Primary Human T Lymphocytes 1
* Clinical and Tumor Immunology, Department of Medical Oncology, Erasmus Medical Center-Daniel den Hoed, Rotterdam, The Netherlands; and
Department of Tumor Immunology, University Medical Center, Nijmegen, The Netherlands
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when engineered for TCR gene transfer into human T lymphocytes. Retroviral transduction of primary human T lymphocytes with either one of the two sets of TCR constructs enabled T lymphocytes to specifically kill and produce TNF- when triggered by native gp100pos/HLA-A2pos tumor target cells as well as gp100 peptide-loaded HLA-A2pos tumor cells. Peptide titration studies revealed that the cytolytic efficiencies of the T lymphocyte transductants were in the same range as those of the parental CTL clones. Moreover, primary human T lymphocytes expressing either one of the two engineered gp100-specific TCRs show cytolytic activities in response to a large panel of peptide mutants that are identical with those of the parental CTL. The finding that two gp100-specific TCR, derived from two different CTL, can be functionally introduced into primary human T lymphocytes without loss of the Ag reactivity and peptide fine specificity, holds great promise for the application of TCR gene transfer in cancer immunotherapy. |
Introduction |
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Alternatively, adoptive transfer of Ag-specific, MHC-restricted CTL may be successful in the eradication of tumors in patients with metastatic melanoma and in e.g., allogeneic bone marrow transplant recipients with EBV-positive tumors (10, 11, 12, 13). Unfortunately, patients do not always mount a strong and effective in vivo cytotoxic T cell response to their tumors. In fact, isolation of tumor-specific T lymphocytes has only been possible in a fraction of patients, most likely due to the fact that, at least in an immunocompetent setting, the peripheral T cell repertoire is devoid of high-avidity tumor-specific CTL due to thymic selection (14). In addition, these cells only have a limited lifespan, and expansion of such T lymphocytes to therapeutic doses is often not feasible (15, 16). To overcome these problems, T lymphocytes can be harnessed with antitumor/virus specificities via genetic means. Genes encoding for TCR - and -chain can be cloned from the tumor-specific CTL clones, and transfer of these TCR genes into primary human T lymphocytes reprograms T lymphocytes with a specificity against a tumor Ag. In several in vitro studies, primary human T lymphocytes have been retargeted with a tumor or virus specificity by transfer of TCR genes, and were shown to respond specifically toward target cells expressing both the corresponding Ag and the correct HLA molecule (17, 18, 19, 20, 21, 22). Recently, it was also shown that mouse T cells that were redirected by TCR gene transfer efficiently promoted the rejection of Ag-expressing tumors in vivo (23).
To date, very little is known about the preservation of both the cytotoxic potency and peptide fine specificity of CTL following TCR gene transfer, which is crucial to the successful use of TCR genes for immunogene therapy. In this study, we describe the characterization of the TCRs of two anti-gp100/HLA-A2 CTL clones: one obtained from PBL of a healthy donor via stimulation with peptide-pulsed DC (5), and the other obtained from PBL of a melanoma patient via stimulation with autologous melanoma cells (24). These CTL clones showed identical cytotoxic responses against gp100pos/HLA-A2pos target cells, but clearly displayed different fine specificities for gp100 amino acid substitution mutants. The existence of two gene sets each encoding for TCR molecules specific for gp100 peptide but with distinct gp100 peptide fine specificities enabled us to analyze the preservation of both the cytotoxic potency and peptide fine specificity of the physiological TCRs when engineered for TCR gene transfer into human T lymphocytes. Peptide-loaded HLA-A2pos and native gp100pos/HLA-A2pos tumor target cells triggered lysis and TNF- production by TCR-transduced T lymphocytes. The cytolytic efficiencies of the TCR-transduced T lymphocytes were in the same range as those of the parental CTL clones. Most importantly, the gp100 peptide fine specificities of the transduced TCRs were identical with those of the parental CTLs. Taken together, this report shows for the first time that human TCR genes can be functionally transferred to human T lymphocytes without loss of cytolytic efficiency and peptide fine specificity.
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Materials and Methods |
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PBLs from healthy donors were isolated by centrifugation through Ficoll-Isopaque (density = 1.077 g/cm3; Amersham Pharmacia Biotech, Uppsala, Sweden). Transduced primary human T lymphocytes were cultured in RPMI 1640 medium supplemented with 25 mM HEPES, 200 mM L-glutamine, 10% human serum, and 360 IU/ml human rIL-2 (Proleukin; Chiron, Amsterdam, The Netherlands), and stimulated every 2 wk with a mixture of irradiated allogeneic feeder cells, as described elsewhere (25). The melanoma cell lines BLM (HLA-A2pos), BLM transfected with human gp100-encoding cDNA (BLMgp100), and MEL624 (gp100pos/HLA-A2pos) were cultured as described previously (26, 27). The human amphotropic packaging cell line Phoenix, the melanoma cell lines FM3 (gp100pos/HLA-A2pos; a kind gift from J. Zeuthen, Danish Cancer Society, Copenhagen, Denmark) and LB265-MEL, as well as the TAP-deficient T x B cell hybrid T2 cells (HLA-A2pos) were grown in DMEM (Life Technologies, Paisley, U.K.) supplemented with 10% bovine calf serum (HyClone, Logan, UT). The B lymphoblast cell line (B-LCL) BSM (HLA-A2pos) and K562, a chronic myelogenic leukemic cell line, were cultured in RPMI 1640 medium supplemented with 200 mM L-glutamine and 10% bovine calf serum. The B cell lines LG2-EBV and JY-EBV were cultured in IMDM as described previously (28).
Contamination of cell lines and cells with mycoplasm species was excluded by frequent PCR testing of cellular DNA with mycoplasm specific primers (Gen-Probe, San Diego, CA). HLA-A2 expression of melanoma cell lines was checked by flow cytometry and expression of gp100 was checked by staining cytospin preparations with anti-gp100 polyclonal Ab (HMB45).
Peptides and peptide binding assays
Peptides used in this study were: an irrelevant HLA-A2-binding EBV-peptide (GLCTLVAML), gp100280–288 wild-type (wt) peptide YLEPGPVTA, and the gp100 peptide mutants (A1 to A8, and G9, with the A or G referring to an alanine or glycine residue, respectively, and the number corresponding to the position of the amino acid in the gp100 wt peptide). Peptides were synthesized with a free C terminus either by F-moc peptide chemistry using an ABIMED Multiple Synthesizer (Abimed Analyes-Technik, Langenfeld, Germany) or by T-boc chemistry on a Biosearch SAM2 peptide synthesizer (Biosearch Technologies, Novato, CA). All peptide preparations were >90% pure as analyzed by analytical HPLC. Peptides were dissolved in 100% DMSO and stored at -20°C. The HLA-A2 stabilization assay on T2 cells has been described previously (29). Briefly, peptides at various dilutions were incubated with 105 T2 cells for 14 h at 37°C/5% CO2 in serum-free medium in the presence of 3 µg/ml human 2-microglobulin (Sigma-Aldrich, St. Louis, MO) in a total volume of 100 µl. Peptide-mediated stabilization of HLA-A2 molecules at the cell surface of T2 cells was analyzed by flow cytometry using the anti-HLA-A2 mAb BB7.2, and expressed as a fluorescence index: (experimental mean fluorescence/background mean fluorescence) - 1. The background mean fluorescence values were obtained by incubating T2 cells with an HLA-A2 nonbinding peptide (WMAFKERKV) at a similar concentration as the experimental peptide. A second, competition-based HLA-A2 peptide binding assay using JY-EBV cells was performed as described previously (30). In short, HLA molecules on JY-EBV cells were stripped via a mild acid treatment. Stripped cells were subsequently incubated with various concentrations of peptides for 24 h at 4°C in the presence of 150 nM of a fluorescein (FL)-labeled reference peptide (FLPSDC(-FL)FPSV) and 1.5 µg/ml human 2-microglobulin, and analyzed by flow cytometry. The binding capacity of a peptide is expressed as the concentration required to inhibit 50% of the binding of the FL-labeled reference peptide (IC50).
Generation of CTL
CTL-MPD, specific for the HLA-A2-presented gp100280–288 peptide, was cloned via limiting dilution from a bulk anti-gp100 CTL culture that was induced in vitro using gp100 wt peptide-pulsed DC as described previously (5). After several weeks of restimulation, stable clones were obtained. CTL-MPD was expanded weekly: CTL were mixed with LB265-MEL, LG2-EBV, and JY-EBV cells, the latter two pulsed with the gp100280–288 wt peptide, in IMDM supplemented with 3% human serum, 120 IU/ml human rIL-2, and 5 ng/ml human rIL-7 (Genzyme, Cambridge, MA). CTL-296, also specific for the HLA-A2-presented gp100280–288 peptide, was generated from PBL of a melanoma patient (24). The CTL-296 was maintained via coculture of this CTL with gp100 wt peptide-pulsed T2 cells, LG2-EBV and JY-EBV cells in IMDM containing 10% human serum, 200 mM L-glutamine, and 50 IU human rIL-2. The CTL cultures were restimulated weekly.
Analysis of TCR V/V gene usage of CTL-MPD and CTL-296
RNA was isolated from 106 CTL and used for reverse transcriptase reactions performed with Superscript (Life Technologies) according to the manufacturer’s instructions. The TCR -chain variable regions were amplified using a set of sense primers specific for the TCRAV1 to the TCRAV29 segment (kindly provided by Dr. T. Logtenberg, Crucell bv, Leiden, The Netherlands, and Dr. P. van der Elsen, Leiden University Medical Center, Leiden, The Netherlands) in combination with a TCRAC antisense consensus primer. Nested PCR was performed on TCRVA products before gel electrophoresis. The TCR -chain variable regions were amplified using a combination of degenerate TCRBV sense primers (kindly provided by Dr. H. Dolstra, University Medical Center Nijmegen, Nijmegen, The Netherlands) and a TCRBC2 antisense primer (31). The V and V PCR products were run on agarose gels, blotted onto Hybond filters, and hybridized with either a labeled TCRAC or a TCRBC2 probe. Positive PCR products were cloned, and plasmid DNAs from at least five independent colonies were sequenced using both the CTAB sequencing protocol and the T7 sequencing kit (Amersham Pharmacia Biotech).
Cloning of the gp100-specific MPD and 296 TCR genes and transduction of human T lymphocytes
Sequence analysis of the TCR V and V genes used by CTL-MPD and CTL-296 allowed for the design of specific primers to amplify the full-length (fl) TCR and DNAs using CTL-derived cDNA as template DNA. Primers used to amplify the TCR and TCR DNAs without their signal peptide sequences are as follows: fl-MPD TCR -chain: TCRAMPD, 5'-CTC TCC ATG GAG ACT CTC CTG AAA GTG C-3' and HCA, 5'-CTC TCT CGA GGG ATC CTC AGC TGG ACC ACA GCC GCA GC-3'; fl-MPD TCR -chain: TCRBMPD, 5'-CTC TCC ATG GAC TCC TGG ACC TTC TGC TG-3' and HCB, 5'-CTC TCT CGA GGG ATC GCT AGC CTC TGG AAT CCT TTC TC-3'; fl-296 TCR -chain: TCRA296, 5'-CTC TCC ATG GCA TCC ATT CGA GCT GTA TTT-3' and HCA; fl-296 TCR -chain: TCRB296, 5'-CTC TCC ATG GGC CCC CAG CTC CTT GGC TAT G-3' and HCB.
The PCR products (i.e., the TCR cDNAs) were cloned into the retroviral vector pBullet containing a heterologous signal peptide as described elsewhere (21) and checked by sequence analysis. Human T lymphocytes of healthy donors were activated with anti-CD3 mAbs and transduced with retroviruses harboring the TCR and TCR transgenes. The TCR-encoding retroviruses were produced by the packaging cell line Phoenix. The transduction procedure used for primary human T lymphocytes was optimized and described by Lamers et al. (32). In short, 24-well culture plates were coated with human fibronectin fragments CH-296 (Takara Shuzo, Otsu, Japan) and pretreated with retroviral particles by centrifugation at 2400 rpm for 1 h with 2 ml retrovirus-containing supernatant per well. Next, 106 human T cells were centrifuged in 2 ml of fresh virus-supernatant, and cultured for 4–5 h at 37°C/5% CO2. T cells were allowed to recover in normal T cell medium overnight before a second transduction cycle, after which cells were harvested and transferred to T25 culture flasks.
Flow cytometry of TCR-transduced T lymphocytes
TCR-transduced T cells were analyzed for TCR and CD8 expression by flow cytometry using PE-conjugated anti-TCRV8 mAb (i.e., recognizing fl-MPD TCR), anti-TCRV14 mAb (i.e., recognizing fl-296 TCR) (both from Beckman Coulter, Marseille, France) or APC-labeled gp100/HLA-A2 tetramers, and FITC-conjugated anti-CD8 mAb (BD Biosciences, San Jose, CA), respectively. For immunostaining, 0.1–0.5 x 106 transduced cells were washed with ice-cold PBS containing 0.5% BSA and 0.02% EDTA, and incubated with mAbs on ice for 30 min or tetramers at room temperature for 1 h. For immuno double stainings, cells were first incubated with tetramers, washed, and subsequently incubated with anti-TCRV mAb. Upon completion of the immunostainings, cells were washed again, fixed (with 1% paraformaldehyde), and analyzed on a FACSCalibur (BD Biosciences) using CellQuest software.
Cytotoxicity assay
Cytotoxic activity of both gp100/HLA-A2-specific CTL clones and T lymphocytes that were retrovirally transduced with the fl-MPD and fl-296 TCR genes was routinely measured in a standard 5–6 h 51Cr-release assay (33). Target cells were labeled with 100 µCi Na251CrO4/106 cells for at least 1 h at 37°C/5% CO2. T2, BLM, and BSM cells were pulsed with gp100 peptides for 45 min at 37°C/5% CO2 before cocultivation with effector T cells. To correct for NK cell activity, cold target cell inhibition assays were performed by adding nonlabeled K562 cells at the indicated cold target:hot target ratios. In experiments aimed at specific blocking of the cytolytic activities of fl-MPD and fl-296 TCR-transductants, anti-TCRV8 mAb (at 1 µg/ml final), anti-TCRV14 mAb (1 µg/ml), anti-MHC class I mAb (clone W6/32; Sera-Lab, Crawley Down, U.K.) (10 µg/ml), or mouse 
globulin (mIg; Jackson ImmunoResearch Laboratories, West Grove, PA) (10 µg/ml) was added at the onset of the cytotoxicity assay. Cytolytic efficiencies of parental CTL and TCR transductants were assayed by pulsing T2 cells with various amounts of the gp100 wt peptide, whereas peptide fine specificities were determined by loading T2 cells with a series of gp100 peptide mutants (all at 1 µM final) before cytotoxicity assays. Percentage of specific cytolysis, i.e., specific 51Cr-release, was calculated as described previously (34).
TNF- production
To quantify TNF- production by TCR-transduced T lymphocytes after Ag-specific stimulation, 6 x 104 T cells were cultured in the presence of 2 x 104 tumor cells or peptide-pulsed T2 cells for 18 h. As a positive control, TCR-transduced T lymphocytes were stimulated with PHA and PMA. Supernatants were harvested and levels of TNF- were measured by standard ELISA (Central Laboratory for Blood Transfusions, Amsterdam, The Netherlands) according to the manufacturer’s instructions.
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region
We generated an HLA-A2-restricted gp100280–288-specific CTL by stimulating PBL from a healthy donor with autologous peptide-pulsed DC (5). Limiting dilution subsequently yielded the CTL clone MPD which showed a cytotoxic reactivity against peptide-pulsed and native gp100pos/HLA-A2pos melanoma cells (Fig. 1) that was identical with the reactivity pattern of the CTL bulk culture. No lysis of the gp100neg/HLA-A2pos melanoma cell line BLM was observed, whereas BLM cells pulsed with the gp100 wt peptide or transfected with human gp100-encoding cDNA (BLMgp100) were efficiently lysed by CTL-MPD (Fig. 1). In addition, the gp100pos/HLA-A2pos melanoma cell lines MEL624 and FM3 were also lysed by this CTL clone (Fig. 1). PCR-based typing of the TCR genes of CTL-MPD and sequence analysis of cDNA clones revealed that the TCR - and -chains of CTL-MPD, respectively comprise the segments AV29s1/JA49 and BV8s2/D1s1/JB2s1, respectively (Fig. 2). In fact, sequence analysis of 17 different CTL clones showed that they all express the same TCR -chain and thus are derived from one parental CTL (data not shown).
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-chain of another HLA-A2 restricted anti-gp100280–288 CTL clone, i.e., CTL-296/147, which was generated by stimulating PBL from an HLA-A2pos melanoma patient with autologous gp100pos tumor cells (24). The overall cytotoxic response of this CTL appeared to be identical with that of CTL-MPD (Fig. 1). Sequence analysis performed on cDNA clones from this melanoma patient-derived CTL-296 showed that its TCR consists of the segments AV8s1/JAJ and BV14s1/D1s1/JB2s7 (Fig. 2). Although both CTL-MPD and CTL-296 clearly use different V and J gene segments, the TCR complementarity-determining region (CDR)3 of both clones are strikingly homologous at the amino acid level, and in fact identical in size (Fig. 2). The resemblance between the TCR CDR3 regions of both CTL clones is less pronounced. CTL-MPD and CTL-296 differ in their gp100 peptide fine specificity
To determine the cytolytic efficiencies of CTL-MPD and CTL-296, T2 target cells were pulsed with various amounts of the gp100 wt peptide and specific lysis was measured in a 51Cr-release assay. From Fig. 3A, it is clear that the amount of gp100 wt peptide required to obtain 50% of the maximum lysis of peptide-pulsed T2 cells (ED50) was similar for both CTL-MPD and CTL-296 (ED50 = 100 and 50 pM peptide, respectively). A control HLA-A2-binding peptide was not recognized by both CTL, even at higher concentrations (data not shown). These findings show that both CTL display similar cytolytic efficiencies toward gp100 wt peptide-loaded target cells.
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of both CTL-MPD and CTL-296. It is apparent from Fig. 4A that gp100 peptide mutants with substitutions at the anchor positions still initiated a cytotoxic response by both CTL clones, despite their poor binding to HLA-A2. This is in line with the observation that only a few peptide/MHC complexes on target cells are sufficient to trigger CTL responses (38). However,the gp100 peptide with an alanine substitution at position 3 did not trigger lysis by either CTL clone (see Fig. 4A), indicating that the glutamic acid at position 3 of the gp100 peptide is crucial for TCR recognition. Furthermore, no substitutions were allowed at positions 1 and 4 of the gp100 peptide for peptide-mediated lysis by CTL-MPD. In contrast, peptide analogs with substitutions at positions 1 and 4 were still capable of sensitizing T2 cells for lysis by CTL-296 (Fig. 4A). Peptide titration experiments confirmed that the alanine 1 and 4 mutants were as efficient as the gp100 wt peptide with respect to peptide-induced cytolysis by CTL-296 but not CTL-MPD (data not shown). Taken together, the CTL-MPD displays a peptide requirement that is more stringent than that of CTL-296.
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The existence of two gp100-specific CTL clones with distinct gp100 peptide requirements allowed us to investigate the preservation of peptide fine specificity of native TCRs following TCR gene transfer into human T lymphocytes. To this end, we isolated fl TCR cDNAs from both CTL, engineered them for retroviral expression using the vector pBullet (21) and the packaging cell line Phoenix, and transferred them into primary human T lymphocytes. It is important to note that the used retroviral transduction protocol (described in detail in Ref. 32) allows for high transduction efficiencies of primary human T lymphocytes that can then be directly assayed in functional tests and does not necessitate subsequent enrichment or cloning of TCR-transduced T lymphocytes.
Approximately 50% of the human T lymphocytes stably expressed the fl-MPD and fl-296 TCR transgenes, as determined by flow cytometry using anti-TCRV8 and anti-TCRV14 mAbs, respectively (Fig. 5A). Primary human T cell transductants were checked weekly for expression of the introduced TCR chains by flow cytometry, and TCR expression remained stable for a period of at least 3 mo in continuous culture. Expression of CD8 was always >50% (data not shown). In addition, the TCR-transduced human T lymphocytes specifically bound gp100280–288/HLA-A2 tetramers (Fig. 5B), whereas the binding of an irrelevant EBV/HLA-A2 tetramer was negligible. Interestingly, when analyzing the TCR and gp100/HLA-A2 tetramer stainings at the single-cell level, we observed that of T cells expressing the introduced TCR, only 50–60% are able to bind the gp100/HLA-A2 tetramer. Results were confirmed when looking within the CD8-positive fraction of the T cell transductants.
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mAb (CTL-MPD: anti-TCRV8 mAb, and CTL-296: anti-TCRV14 mAb) and anti-MHC class I mAb (Fig. 6B). mIg as a control Ab did not block the cytolytic responses of either TCR transductant (Fig. 6B). In addition, the fl-MPD and fl-296 TCR-transduced human T lymphocytes were also specifically triggered to produce TNF- in response to peptide-loaded and native gp100pos tumor target cells (Table II).
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300 pM for both fl-MPD and fl-296 TCRpos human T lymphocytes (Fig. 3B), which is in the same range as the cytolytic efficiencies of the parental CTL-MPD and CTL-296 clones (ED50 = 100 and 50 pM, respectively; Fig. 3A). T2 cells loaded with a high concentration of an irrelevant HLA-A2-binding EBV peptide (10 µM final) were not lysed by the TCR-transduced human T lymphocytes (data not shown), and mock-transduced human T lymphocytes did not lyse T2 cells loaded with gp100 wt peptide (at 10 µM final) (Fig. 3B). In total, the TCR-transduced human T lymphocytes show specific immune responses to gp100pos/HLA-A2pos target cells, and exhibit similar cytolytic efficiencies as their corresponding parental CTL clones.
Peptide requirement of gp100-specific TCR-transduced primary human T lymphocytes is identical with that of parental CTL
The preservation of not only the cytotoxic potency but also the peptide fine specificity of CTL by TCR gene transfer is crucial to the potential use of TCR genes for immunotherapy. To address this issue, the peptide fine specificities of the fl-MPD and fl-296 TCR transductants were determined in a 51Cr-release assay with T2 cells pulsed with a series of gp100 peptide analogs (as described in the legend to Fig. 4A). The gp100 peptide with an alanine substitution at position 3 did not trigger lysis by either TCR transductant (Fig. 4B). In addition to position 3, no substitutions were allowed at positions 1 and 4 for peptide-mediated lysis by the fl-MPD TCRpos T lymphocytes, whereas these latter peptide analogs were still capable of sensitizing T2 cells for lysis by fl-296 TCRpos T lymphocytes (Fig. 4B). We conclude from these experiments that the TCR transductants show gp100 peptide fine specificities that are identical with those of the parental CTL (compare Fig. 4, A and B).
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Discussion |
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- and -chains using different TCRV and V gene segments. The two CTL clones are both specific for the same gp100280–288 epitope but clearly differ in their response to amino acid substitutions in this gp100 epitope. fl TCR genes of either CTL clone, when engineered for gene transfer into primary human T lymphocytes, completely preserve the observed gp100-specific Ag reactivity and cytolytic efficiency. Moreover, the characterized peptide fine specificities of the two native anti-gp100 TCRs were also functionally exhibited by T lymphocytes transduced with the fl TCRs.
The TCR chains of the gp100-specific CTL-MPD and CTL-296 have strictly homologous CDR3 but not CDR3 regions (see Fig. 2), implying the selection of a specific amino acid sequence in the CDR3 region. The same pattern of sequence and size conservation of the CDR3 regions of the TCR- and -chains was observed in a panel of CTL with the same Ag specificity by Moss and Bell (39). These findings fit the idea that the TCR- and -chains contribute differently to the recognition of the peptide/MHC complex. This notion is further supported by the crystal structure of a TCR bound to a viral peptide/HLA-A2 complex (40). Although several studies show that conservation of sequence and size of CDR3 of CTL is not an absolute determinant of Ag recognition, TCR CDR3 regions do appear to govern peptide fine specificity of CTL (41, 42).
Analysis of the cytotoxic responses of CTL-MPD and CTL-296 showed that both CTL clones have an identical Ag reactivity toward gp100pos/HLA-A2pos melanoma target cells (see Fig. 1). Furthermore, both CTL clones lyse gp100 wt peptide-pulsed target cells with efficiencies in the picomolar range (see Fig. 3A), suggesting that the TCR of both CTLs bind the gp100 peptide/HLA-A2 complex on target cells with similar affinities. Nevertheless, the specificities of CTL-MPD and CTL-296 for gp100 peptides with amino acid substitutions are strikingly different (see Fig. 4A). The CTL responses toward gp100 peptide mutants indicate that the amino acids in the gp100 epitope important for TCR:peptide/MHC interaction of either CTL clone overlap but are not identical. Both parental CTL clones are equally sensitive to a substitution of the glutamic acid at amino acid position 3 of the gp100 peptide for alanine, but only CTL-MPD is sensitive to alanine substitutions at amino acid positions 1 and 4. Because the tested amino acid changes do not affect binding of the gp100 peptide to HLA-A2 (see Table I), a peptide mutant may be incorrectly positioned in the MHC molecule and result in inadequate TCR recognition and CTL response.
Our observation that peptide-pulsed DCs can induce bona fide CTL clones capable of lysing melanoma tumor cells endogenously processing and presenting the tumor Ag in vitro supports a treatment strategy of melanoma patients with DC pulsed with peptides derived from melanoma-associated Ags, which we and others currently employ. The molecular analysis of the TCR- and -chains expressed by gp100-reactive CTLs allows for TCR gene transfer into primary human T lymphocytes, a possible alternative to immunotherapy protocols. The feasibility of transferring fl as well as chimeric two-chain and single-chain TCR genes to impose anantitumor or antivirus specificity onto T cells has been reported by us and others (17, 18, 20, 21, 22, 23, 43, 44, 45). The present study demonstrates for the first time that TCR gene transfer not only preserves peptide specificity, but even peptide fine specificity, which is crucial to the use of TCR genes for immunogene therapy. Important to TCR gene transfer studies, but often unaccounted for, is the efficiency which one can obtain to introduce TCR genes into primary human T lymphocytes. We used a retroviral transduction protocol that results in high levels of surface expression of TCR chains on human T lymphocytes (50% of bulk cultures of transduced T lymphocytes expressed the appropriate TCR -chain, and 35% of these bulk cultures bound gp100280–288/HLA-A2 tetramers; see Fig. 5). This is in contrast to previous reports that show only poor retroviral transduction efficiencies of TCR genes, necessitating antibiotic selection, enrichment for CD8-positive T cells or even cloning to obtain a tumor or virus-specific response of retargeted T cells (17, 18, 19, 44). Parameters that greatly affect retroviral transduction efficiencies, such as transduction-mediating agents and type of retroviral vector, have been carefully optimized by our group and others, allowing for gene transfer into primary human T lymphocytes with high efficiency (21, 34, 46) and at clinical scale meeting the criteria of Good Medical Practice (32).
Retargeted T cells were analyzed functionally for their lytic activity and ability to produce TNF- in response to the same panel of target cells as used to analyze the CTL clones (see Fig. 6A and Table II). The Ag reactivities of the TCR transductants are identical with those of the parental CTL clones (compare Figs. 1 and 6A), and were confirmed by blocking studies with family type-specific anti-TCR V as well as anti-MHC class I Abs (see Fig. 6B). Subsequent peptide titration experiments revealed that the cytolytic efficiencies of the T lymphocyte transductants are in the same picomolar range as those of the parental CTL clones (compare Fig. 3, A and B). Importantly, primary human T lymphocytes positive for either gp100/HLA-A2-specific TCR show cytolytic activities in response to a large panel of peptide mutants that are identical with those of the parental CTL clones (compare Fig. 4, A and B). Moreover, production levels of TNF- of the TCR transductants in response to the gp100 peptide mutants reflect the peptide fine specificity observed in the cytotoxicity assays (data not shown). The observation that TCRs engineered for gene transfer and introduced into primary human T lymphocytes completely preserve the peptide fine specificities of the native TCRs implies that the exogenous TCR genes, in particular their CDR3 regions, do properly fold when expressed on T cell membranes.
The preservation of the peptide fine specificity of parental CTL by transfer of fl TCR genes makes TCR transductants promising tools for immunogene therapy. However, an important issue that needs to be addressed regarding the clinical use of such TCR gene-transduced T lymphocytes is the possible formation of new, potentially autoimmune TCR heterodimers comprising exogenous and endogenous TCR chains. Flow cytometry at the single-cell level shows that following transfer of both TCR and genes only a fraction (i.e., 50–60%) of CD8-positive T cells expressing the introduced TCR -chain are able to bind the gp100/HLA-A2 tetramer. These findings agree with the observation by Stanislawski and colleagues (22) that upon transfer of MDM-2-specific TCR genes into primary human T cells only 30–50% of T cells positive for the introduced TCR were able to bind the MDM-2-specific tetramer, and suggest that the introduced and surface-expressed TCR chains do pair with endogenous TCR -chains. The transfer of chimeric TCR genes linked to genes encoding signal transduction molecules, such as CD3
, has proven advantageous in this respect since it facilitates pairing between two CD3-containing proteins (21, 47), thereby maximizing the expression of the introduced TCR and reducing the risk that alternative TCR complexes are formed. Another advantage of tumor-specific receptors chimerized to CD3 or other signaling molecules is that they bypass TCR-mediated proximal signaling events (48), which are often defective in cancer patients (49, 50). Recently, we have made chimeric genes comprising the V and V domains of the fl-MPD and fl-296 TCR genes coupled to various signaling molecules, such as CD3 and Fc(
)RI, which are currently tested for their efficiency in T cell retargeting.
Taken together, in this report gp100-specific TCR, derived from two different CTL, have been molecularly analyzed and functionally introduced into primary human T lymphocytes without loss of the Ag reactivity and peptide fine specificity. These results confirm the notion that TCR gene transfer holds promise for immunogene therapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Reno Debets, Clinical and Tumor Immunology, Department of Medical Oncology, Erasmus Medical Center-Daniel den Hoed, Groene Hilledijk 301, 3075 EA Rotterdam, The Netherlands. E-mail address: Debets{at}immh.azr.nl
3 Abbreviations used in this paper: DC, dendritic cell; B-LCL, B lymphoblast cell line; CDR, complementarity-determining region; fl, full length; mIg, mouse globulin; wt, wild type; FL, fluorescein.
Received for publication March 1, 2002. Accepted for publication December 2, 2002.
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Z. Sebestyen, E. Schooten, T. Sals, I. Zaldivar, E. San Jose, B. Alarcon, S. Bobisse, A. Rosato, J. Szollosi, J. W. Gratama, et al. Human TCR That Incorporate CD3{zeta} Induce Highly Preferred Pairing between TCR{alpha} and {beta} Chains following Gene Transfer J. Immunol., June 1, 2008; 180(11): 7736 - 7746. [Abstract] [Full Text] [PDF]
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R.-H. Voss, R. A. Willemsen, J. Kuball, M. Grabowski, R. Engel, R. S. Intan, P. Guillaume, P. Romero, C. Huber, and M. Theobald Molecular Design of the C{alpha} Interface Favors Specific Pairing of Introduced TCR{alpha} in Human T Cells J. Immunol., January 1, 2008; 180(1): 391 - 401. [Abstract] [Full Text] [PDF]
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S. Thomas, S.-A. Xue, M. Cesco-Gaspere, E. San Jose, D. P. Hart, V. Wong, R. Debets, B. Alarcon, E. Morris, and H. J. Stauss Targeting the Wilms Tumor Antigen 1 by TCR Gene Transfer: TCR Variants Improve Tetramer Binding but Not the Function of Gene Modified Human T Cells J. Immunol., November 1, 2007; 179(9): 5803 - 5810. [Abstract] [Full Text] [PDF]
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 W Yang, E. Beaudoin, L Lu, R. Du Pasquier, M. Kuroda, R. Willemsen, I. Koralnik, and R. Junghans Chimeric immune receptors (CIRs) specific to JC virus for immunotherapy in progressive multifocal leukoencephalopathy (PML) Int. Immunol., September 1, 2007; 19(9): 1083 - 1093. [Abstract] [Full Text] [PDF]
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 M. H. M. Heemskerk, R. S. Hagedoorn, M. A. W. G. van der Hoorn, L. T. van der Veken, M. Hoogeboom, M. G. D. Kester, R. Willemze, and J. H. F. Falkenburg Efficiency of T-cell receptor expression in dual-specific T cells is controlled by the intrinsic qualities of the TCR chains within the TCR-CD3 complex Blood, January 1, 2007; 109(1): 235 - 243. [Abstract] [Full Text] [PDF]
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R. A. Willemsen, Z. Sebestyen, C. Ronteltap, C. Berrevoets, J. Drexhage, and R. Debets CD8{alpha} Coreceptor to Improve TCR Gene Transfer to Treat Melanoma: Down-Regulation of Tumor-Specific Production of IL-4, IL-5, and IL-10 J. Immunol., July 15, 2006; 177(2): 991 - 998. [Abstract] [Full Text] [PDF]
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N. Schaft, B. Lankiewicz, J. Drexhage, C. Berrevoets, D. J. Moss, V. Levitsky, M. Bonneville, S. P. Lee, A. J. McMichael, J.-W. Gratama, et al. T cell re-targeting to EBV antigens following TCR gene transfer: CD28-containing receptors mediate enhanced antigen-specific IFN{gamma} production Int. Immunol., April 1, 2006; 18(4): 591 - 601. [Abstract] [Full Text] [PDF]
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S.-A. Xue, L. Gao, D. Hart, R. Gillmore, W. Qasim, A. Thrasher, J. Apperley, B. Engels, W. Uckert, E. Morris, et al. Elimination of human leukemia cells in NOD/SCID mice by WT1-TCR gene-transduced human T cells Blood, November 1, 2005; 106(9): 3062 - 3067. [Abstract] [Full Text] [PDF]
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R. A. Willemsen, C. Ronteltap, P. Chames, R. Debets, and R. L. H. Bolhuis T Cell Retargeting with MHC Class I-Restricted Antibodies: The CD28 Costimulatory Domain Enhances Antigen-Specific Cytotoxicity and Cytokine Production J. Immunol., June 15, 2005; 174(12): 7853 - 7858. [Abstract] [Full Text] [PDF]
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 E. C. Morris, A. Tsallios, G. M. Bendle, S.-a. Xue, and H. J. Stauss A critical role of T cell antigen receptor-transduced MHC class I-restricted helper T cells in tumor protection PNAS, May 31, 2005; 102(22): 7934 - 7939. [Abstract] [Full Text] [PDF]
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 M. H.M. Heemskerk, M. Hoogeboom, R. Hagedoorn, M. G.D. Kester, R. Willemze, and J.H. F. Falkenburg Reprogramming of Virus-specific T Cells into Leukemia-reactive T Cells Using T Cell Receptor Gene Transfer J. Exp. Med., April 5, 2004; 199(7): 885 - 894. [Abstract] [Full Text] [PDF]
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M. H. M. Heemskerk, M. Hoogeboom, R. A. de Paus, M. G. D. Kester, M. A. W. G. van der Hoorn, E. Goulmy, R. Willemze, and J. H. F. Falkenburg Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region Blood, November 15, 2003; 102(10): 3530 - 3540. [Abstract] [Full Text] [PDF]
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H. Wu, G. Walters, J. F. Knight, and S. I. Alexander DNA Vaccination Against Specific Pathogenic TCRs Reduces Proteinuria in Active Heymann Nephritis by Inducing Specific Autoantibodies J. Immunol., November 1, 2003; 171(9): 4824 - 4829. [Abstract] [Full Text] [PDF]
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R. A. Morgan, M. E. Dudley, Y. Y. L. Yu, Z. Zheng, P. F. Robbins, M. R. Theoret, J. R. Wunderlich, M. S. Hughes, N. P. Restifo, and S. A. Rosenberg High Efficiency TCR Gene Transfer into Primary Human Lymphocytes Affords Avid Recognition of Melanoma Tumor Antigen Glycoprotein 100 and Does Not Alter the Recognition of Autologous Melanoma Antigens J. Immunol., September 15, 2003; 171(6): 3287 - 3295. [Abstract] [Full Text] [PDF]
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) and CTL-296 (
) were tested in a standard 5-h 51Cr-release assay as described in Materials and Methods. Target cells were the gp100neg/HLA-A2pos melanoma cell line BLM, with or without exogenous gp100 wt peptide (preincubation with peptide for 45 min at 37°C at a final concentration of 1 µM), BLM cells transfected with the hgp100280–288 cDNA (BLMgp100), and the gp100pos/HLA-A2pos melanoma cell lines MEL 642 and FM3. The E:T cell ratio was 10:1.
) were tested in a 6-h 51Cr-release assay. T2 cells, preincubated with various amounts of gp100 wt peptide, were used as target cells. The E:T cell ratio was 15:1. Cold K562 target cells were added to 51Cr-labeled target cells at a ratio of 30:1. The peptide concentration corresponding to the ED50 of both fl-MPD TCR and fl-296 TCR-transductants was 300 pM. The results shown represent fl-TCR transductions of primary human T lymphocytes of one donor. A second donor provided similar data.
) were tested in a 6-h 51Cr-release assay using T2 cells, pulsed with gp100 peptide mutants, as target cells. The E:T cell ratio was 15:1. Cold K562 cells were added to 51Cr-labeled target cells at a ratio of 30:1. The results shown represent fl-TCR transductions of primary human T lymphocytes of one donor. A second donor provided similar data.
