Human effector T cells derived from central memory cells rather than CD8+T cells modified by tumor-specific TCR gene transfer possess superior traits for adoptive immunotherapy

Cancer Letters 339 (2013) 195–207

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Human effector T cells derived from central memory cells rather than CD8+T cells modified by tumor-specific TCR gene transfer possess superior traits for adoptive immunotherapy Fenglin Wu a,b,c,d, Wenfeng Zhang b, Hongwei Shao a,c,d, Huaben Bo a,b,c,d, Han Shen a,b,c,d, Jiandong Li a, Yichen Liu a, Teng Wang a,c,d, Wenli Ma b,1, Shulin Huang a,c,d,⇑ a

School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China Southern Medical University, Guangzhou, China c Institute of Bio-Pharmaceutical, Guangdong Pharmaceutical University, Guangzhou, China d Guangdong Provincial Key Laboratory of Biotechnology Candidate Drug Research, Guangzhou, China b

a r t i c l e

i n f o

Article history: Received 14 March 2013 Received in revised form 16 May 2013 Accepted 2 June 2013

Keywords: TCM CD8+T cell TCR Transfer Immunotherapy

a b s t r a c t Adoptive cell therapy provides an attractive treatment of cancer, and our expanding capacity to target tumor antigens is driven by genetically engineered human T lymphocytes that express genes encoding tumor-specific T cell receptors (TCRs). The intrinsic properties of cultured T cells used for therapy were reported to have tremendous influences on their persistence and antitumor efficacy in vivo. In this study, we isolated CD8+ central memory T cells from peripheral blood lymphocytes of healthy donors, and then transferred with the gene encoding TCR specific for tumor antigen using recombinant adenovirus vector Ad5F35-TRAV-TRBV. We found effector T cells derived from central memory T cells improved cell viability, maintained certain level of CD62L expression, and reacquired the CD62L+CD44high phenotype of central memory T cells after effector T cells differentiation. We then compared the antitumor reactivity of central memory T cells and CD8+T cells after TCR gene transferred. The results indicated that tumor-specific TCR gene being transferred to central memory T cells effectively increased the specific killing of antigen positive tumor cells and the expression of cytolytic granule protein. Furthermore, TCR gene transferred central memory T cells were more effective than TCR gene transferred CD8+T cells in CTL activity and effector cytokine secretion. These results implicated that isolating central memory T cells rather than CD8+T cells for insertion of gene encoding tumor-specific TCR may provide a superior tumor-reactive T cell population for adoptive transfer. Ó 2013 Published by Elsevier Ireland Ltd.

1. Introduction Immunotherapy provides an attractive treatment of cancer, the advantage of which is that it can enhance or reconstruct the patients’ anti-tumor immunity with weak side effect. Currently, there are mainly two strategies for tumor immunotherapy, namely, ther-

Abbreviations: TCR, T cell receptor; TCM, central memory T cell; TEM, effector memory T cell; TN, naïve T cell; TM, memory T cell; TE, effector T cell; TIL, tumorinfiltrating lymphocyte; CAR, coxsackie/adenovirus receptors; AFP, alpha-fetoprotein; mAb, monoclonal antibody; CTL, cytotoxic lymphocytes; IFN-c, interferongamma; Calcein-AM, calcein–acetoxymethyl ester; IL-2, interlukin-2; LNs, lymph nodes. ⇑ Corresponding author. Address: School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, 280 E. Rd. outside the City of Guangzhou University, Guangzhou 510006, China. Tel.: +86 20 39352199; fax: +86 20 39352201. E-mail address: [email protected] (S. Huang). 1 Co-corresponding author. 0304-3835/$ - see front matter Ó 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.canlet.2013.06.009

apeutic vaccination and adoptive cell therapy [1], and the latter normally refers to the transfusion of autologous or allogenic immune cells into tumor-bearing hosts. The immune cells include Cytotoxic Lymphocytes (CTLs) with tumor-reactive T cells as its major ingredient and tumor-infiltrating lymphocytes (TILs) etc. The transfusion of tumor-reactive CTLs has been employed in treatment of metastatic melanoma, Epstein-Barr virus Induced Lymphoma and other tumors [2,3]. A major restriction of adoptive T cell therapy is that highly avid T cells can only be separated from some particular patients, because tumor lacks of antigenicity [4]. To solve this problem, reactive T cell clones were selected from TILs. Through this approach, researchers identified the genes of T Cell Receptors (TCRs), which can specifically recognize tumor antigen, and then transduced the genes encoding tumor-specific TCRs into mature T cells to acquire TCR gene engineered T cells that can specifically recognize antigen positive tumor cells in vitro and can mediate anti-tumor immunity after being re-injected into the patient. TCR gene

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engineered T cells were first employed in clinical trials treating melanoma, in which MART-1 antigen specific TCR genes were transduced into melanoma patients’ peripheral blood lymphocytes (PBLs). The transfused TCR gene engineered T cell then directly destroyed antigen positive tumor in vivo [5,6]. By this means, complete tumor regression were observed in some cases. Similar results have been achieved in clinical trials in which NY-ESO-1 antigen-specific TCR gene engineered T cells were utilized to treat melanoma and synovial sarcoma patients [7]. A critical point, which determines the antitumor efficacy of TCR gene engineered T cells, is to identify the TCRs that can recognize tumor antigens and select T cell subsets that are suitable for adoptive cell therapy [8]. Nevertheless, the efficacy of TCR gene engineered T cells in vivo is often limited by the failure of cultured T cells, particularly cloned CD8+T cells, to persist in vivo [9,10]. A vital reason for the short-term survival of transfused T cells is that effector phenotypes are acquired after T cells being activated in vitro [11]. Based on differentiations, Resting T cells fall into two categories, naïve T cells and memory T cells, while the latter, according difference of phenotype, homing ability and function, can be divided into effector memory T cells (TEM) and central memory T cells (TCM) [12]. TCM express CD62L and CCR7, which can promote the homing of them to lymph nodes (LNs) and enable them to proliferate rapidly upon re-exposure to the same antigen [13]. Berger’s [14] landmark research modeled on macaques has proved that effector T cells (TE) derived from TCM can persist long-term in vivo, and reacquired phenotypic and functional properties of memory T cells. In this study, we isolated CD8+ TCM from peripheral blood lymphocytes (PBLs) of healthy donors, and pursued the difference of differentiated phenotype between TE cells derived from TCM and CD8+T cells after they were stimulated in vitro. TCM were then transferred with tumor antigen specific TCR genes screened in advance to redirect their antigen specificity. Result indicated that TE cells derived from TCM exhibited improved survival, maintained certain level of expression of CD62L, CCR7 and CD28, higher level of expression of CD44 and CD45RO, reacquired the CD62L+CD44 high phenotype of TCM after effector differentiation. Specific TCR gene transfer effectively promoted the recognition and killing of antigen positive tumor cells, the expression of cytolytic granule protein by TCM. Furthermore, TCR gene transferred TCM were more effective than transferred CD8+T cells in CTL activity and effector cytokine secretion.

2. Materials and methods 2.1. Ethics statement Human tissue was collected in accordance with a protocol approved the Institutional Review Board (IRB) at the Guangdong Pharmaceutical University. All participants signed a written consent.

tured in RPMI 1640 medium or Dulbecco modified Eagle medium(DMEM, Gibco) respectively, supplemented with 10% FBS (Gibco/Invitrogen) and antibiotics (penicillin G 100 IU/ml and Streptomycin 100 lg/ml) at 37 °C under 5% CO2.

2.3. TCM sorting For sorting T cell subset, CD8+T cells were isolated from PBLs of HLA-A2+ donors by magnetic bead negative selection with CD8+T cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. CD62L positive and CD44 positive cells were then separated by fluorescence activated cell sorting (FACS) using a FACSAria sorter (BD Biosciences) after labeling with anti-human CD62L, and antihuman CD44 fluorescent antibodies (BD Biosciences).

2.4. T cell culture and stimulation TCM and CD8+T cells were then cultured in RPMI1640 medium (Gibco/Invitrogen) supplemented with antibiotics, glutamine, and 10% FBS (Gibco/Invitrogen). 1000 U/mL interferon-c(IFN-c; Gammakine, Boehringer Ingelheim) was added on day 0. Primary stimulation was accomplished by soluble anti-CD3 (OKT3, 30 ng/ mL, R&D Systems) and soluble anti-CD28 (1 ng/mL, R&D Systems) and 300 IU/mL recombinant IL-2 (R&D Systems) on day 1, and with 50 IU/mL IL-2 and IL-15 (1 ng/mL) every 3–5 days thereafter for a total of 35 days. Restimulation was performed on day 5, using T2 cell pulsed with 9-amino long HLA-A2+ epitope from AFP218-226 (LLNQHACAV, 1 lM, China-peptide) for 4 h. In vitro growth of CD8+T cell and TCM was measured by counting viable cells using trypan blue dye exclusion.

2.5. Telomere fluorescene in situ hybridization and flow cytometry (Flow-FISH) Telomere length of TE cells from CD8+T cell and TCM was measured on day 14 using a Telomere PNA Kit/FITC for Flow Cytometry (DAKO) as previously described [15]. Relative telomere length (RTL) was determined by comparing test cells with a control cell line (1301 cell line). Due to variation between samples in prestimulation telomere length, telomere length for each subset was normalized to the mean telomere length for each donor (telomere index = telomere length for subset / average telomere length for donor).

2.6. Construction of recombinant TCR adenovirus vector In previous research, we had screened AFP peptide binding specific TCR genes as follows. CD8+T cells which could be specifically bound to AFP epitope peptide loaded T2 cells were isolated from TILs of patients (HLA-A2+, AFP+) with hepatocellular carcinoma. TCR genotypes of monoclonal expression (TCRa12-2,TCRVb7.1) were identified through analysis of multiplex gene expression profile, and confirmed by anti- AFP+ hepatocellular carcinoma response . We then constructed Ad5F35 chimeric adenoviral vector as previously described [16]. Whole encoding sequence for TCR Va12.2 (TRAV) and TCR Vb7.1 (TRBV) were linked by IRES and then cloned into Ad5F35 vector, resulting in recombinant Ad5F35-TRAV-TRBV. The virus preparations were quantitated by OD260 measurements, viral titers were determined by plaque-forming assay using HEK293 cells and the virus was purified as previously described [17].

2.7. T cell transduction Transduction was performed 2 days after initial stimulation using Ad5F35TRAV-TRBV adenovirus supernatant produced in HEK293 packaging cells. TCM and CD8+T cells of different donors were transduced for 12 h at multiplicities of infection (MOI) ranging from 100 to 200. To investigate the ability of Ad5F35 to transduce T cells, transduced TCM and CD8+T cells were analyzed by flow cytometry with FITC-conjugated anti-human TCR Va12 (Endogen) and PE-conjugated anti-human TCR Vb7 antibodies (Immunotech) on 3–14 days after transduction.

2.2. T-cell isolation and cell lines 2.8. Flow cytometry analysis Peripheral blood lymphocytes (PBLs) from HLA-A2 positive healthy individuals were isolated by Ficoll-plaque plus (GE Healthcare) density gradient centrifugation of buffy coats from healthy donors. Mononuclear cells were washed 3 times with PBS, and resuspended in RPMI 1640 medium (Gibco/Invitrogen) supplemented with 10% fetal bovine serum (Gibco/Invitrogen), 2 mM/L-glutamine (Gibco BRL), 100 U/ mL penicillin, 100 lg/mL streptomycin, and incubated at 37 °C under 5% CO2. Human TAP-deficient T2 cell line, Human hepatocellular carcinoma cell line HepG-2 (AFP+, HLA-A2+) and 1301 cell line (subline of the Epstein-Barr virus (EBV) genome negative T-cell leukaemia line CCRF-CEM) were purchased from American Type Culture Collection (Rockville, MD). Other human hepatocellular carcinoma cell line SMMC-7721 (HLA-A2+, AFP+), Bel-7402(HLA-A2 , AFP+), and human breast carcinoma cell line MCF-7 (HLA-A2+, AFP ), human embryonic kidney cell line HEK293 were maintained in our laboratory. All of the cell lines were cul-

Cells were analyzed by flow cytometry after labeling with fluorescent mAbs against following targets: CD8, CD62L, CD44, CD45RO, CD28, CCR7 (all from eBioscience). After incubation with fluorescent mAbs for 25 mins, cells were washed twice with staining buffer (PBS plus 3% FBS) and centrifuged for 10 min at 1000 g, the cell pellet was resuspended in 0.5 mL PBS for measuring. For intracellular staining, in order to inhibit cytokines secretion and accumulate protein within T cells, brefeldin A (eBioscience) was added at 1.4 lM 6 h before the end of incubation, cells were permeabilized using Cytofix/Cytoperm (BD Biosciences) and performed according to the manufacturer’s protocol, then stained with fluorescent mAbs specific for perforin or granzyme B (eBioscience) . Isotype-matched mAbs served as controls. Samples were analyzed on Epics-XL flow cytometer (Beckman Coulter Inc.), and analyzed by EXPO32 v1.2 software.

F. Wu et al. / Cancer Letters 339 (2013) 195–207 2.9. Cytotoxicity assays One days after restimulation (3 days after transduction), Cytotoxicity was evaluated by the calcein release assay as previously described [18]. Briefly, target cells (HepG2, SMMC-7721, Bel-7402 and MCF-7) were labeled for 30 min at 37 °C with 2 lM calcein–acetoxymethyl ester (Calcein-AM, Dojindo). After washing twice with PBS, labeled target cells were distributed in U-bottom 96-well plates at 1  104 cells per well. Effector cell derived from TCR gene transferred or untransferred TCM and CD8+T cell of different donors were harvested, washed, and added at different effector-to-target (E/T) ratios ranging from 30:1 to 1:1. For antibody blocking experiments, target cells were incubated for 1 h at 4 °C with 10 lg/ml anti-human HLAA2 mAb (MBL, Clone BB7.2) before seeding in 96-well plates. After coculture for 4 h, cells were sedimented by centrifugation, 100 lL of supernatant were collected, calcein release was determined using a fluorescence microplate reader (Thermo Fisher Scientific Inc.) with excitation at 485 nm and emission at 535 nm. The percentage of specific Calcein release was calculated using the following formula: percent specific lysis [(test release minus spontaneous release) divided by (maximal release minus spontaneous release)] times 100. Spontaneous release represented calcein release from target cells in complete medium alone, and maximal release was the calcein release from target cells lysed in medium plus 1% Triton X-100, each measured in at least five replicate wells. 2.10. Cytokine production assays For cytokine production assays, 1 days after restimulation, effector cell from TCR gene transferred or untransferred TCM and CD8+T cell of different donors were cocultured overnight with target cells (HepG2, SMMC-7721, Bel-7402 and MCF-7) at 30:1(E/T). IFN-c and IL-2 contents in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA, R&D Systems). 2.11. Statistical analysis Data were expressed as the mean ± standard deviation (S.D). Statistical significance was evaluated using a repeated measures by one-way Analysis of Variance (ANOVA) with a Bonferroni multiple comparisons posttest. p-Values less than 0.05 were considered to be significant. Prism GraphPad 5 software (GraphPad Software Inc.) was used for these analyses.

3. Results 3.1. Isolation and culture of TCM To detect the distribution of TCM in peripheral blood T cells of healthy people, untouched cytotoxic CD8+T cells were isolated from PBLs of HLA-A2+ donors and stained by anti-human CD62L, CD44 fluorescent antibody, and flow cytometry was adopted to analyze the frequencies of T cell subsets. CD62L is highly expressed in TN and TCM, so it was used to exclude TE cells and TEM. While CD44, as phenotypic mark, was expressed at low level in TN, which could be used to distinguish TN and TCM from each other. CD8+T cells were sorted from PBLs of healthy donors (Fig. 1A). Analysis of frequencies of CD62L+CD44+TCM, CD62L-CD44+TE/TEM cells and CD62L+CD44-TN cells in CD8+T cells (Fig. 1B, representative of nine independent samples) showed that, in human peripheral blood, the frequency of TCM was 13–35% of CD8+T cells, which was variable among different donors. CD62L+CD44+TCM were sorted through fluorescence activated cell sorting (FACS). In comparison with unsorted CD8+T cells, isolation efficiency reached 97% or higher (Fig. 1C and D). We chose 7 samples from HLA-A2+ healthy donors identified by flow cytometry for further stimulation, to obtain TE cells respectively from CD8+T cells and TCM. To determine absolute T cell number of TE cells from CD8+T cell and TCM during 35 days culture period. Cell viability was assessed every 3–4 days by trypan blue dye exclusion (Fig. 1E). We observed that TE cells from each subset displayed rapid expansion after stimulation, and significantly greater number of cells derived from TCM compared with that from CD8+T cells. After reaching the peak on day 14, number of TE cells from TCM displayed slower decline than that from CD8+T cells. Since telomere length was reported to correlate with persistence of transferred T cells in vivo, we also measured relative the

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telomere length of TE cells from CD8+T cell and TCM using FISH and flow cytometry (flow-FISH). Although relative telomere length of TE derived from TCM was longer than that from CD8+T cells, the difference was not significant (Fig. 1F). 3.2. Phenotypic changes of TE cells derived from TCM and CD8+T cells To monitor phenotypic changes of TE cells derived from TCM and CD8+T cells, we observed the expression of CD62L and CD44 within 30 days after stimulation (the culture in vitro lasted 35 days). It is demonstrated that after being stimulated by McAb anti costimulatory molecular, cytokines and antigen, the frequency of CD62L+ cell in TE cells derived from CD8+T cells gradually decreased to the extent that cannot be detected. On the contrary, the frequency of CD62L+ cell in TE cells derived from TCM maintained certain level during the culture period (>10% by the 35th day of culture) (Fig. 2A). At the early stage of stimulation, the expression of CD44 in TE cells either from CD8+T cells or TCM both kept high level (>85%). As TE cells differentiated from expansion phase to contraction phase, the frequency of CD44+ cells in TE cells derived from CD8+T cells gradually decreased, reaching 20% at the end of culture period. In contrast, the frequency of CD44+ cells in TE cells derived from TCM constantly kept relatively high, accounting for 85% during the whole culture period. This phenomenon suggests that CD44+TE cells derived from TCM survive longer than those derived from CD8+T cells (Fig. 2B). Especially in mid-late period of culture (21 days later), cell clusters with high CD44 expression appeared in TE cells from TCM of different donors, meanwhile, all CD62L positive cells were CD44high, i.e., CD62L+CD44high cluster (Fig. 2C). We also compared the expression of other commonly used markers of TCM in TE cells derived from TCM and CD8+T cells. The expression of CD45RO in TE cells derived from TCM was higher than that in TE cells derived from CD8+T cells during the culture period (Fig. 2D). Similar to the trend of CD62L+ T cells, the frequency of CD 28+cells and CCR7+ cells in TE cells derived from TCM maintained certain level during the culture period (>15%), whereas the frequency of CD 28+cells and CCR7+ cells in TE cells derived from CD8+T cells gradually decreased to lower extent (<10%) (Fig. 2E and F). 3.3. Ad vector-mediated TCR gene transfer into TCM Ad5F35 adeno virus vector was generated through homologous recombination (Fig. 3A). The fibers on serotype 5 were replaced with that from serotype 35 target CD46, a ubiquitously expressed complement regulatory protein in almost all human cells [19], resulting in effective promotion of T cells being transduced by adeno virus with exogenous TCR genes. After transduction, with anti-human TCRa12, TCRVb7 fluorescent antibody labeling, the relative efficiency of transgene expression in TCM and CD8+T cells from different donors was detected by flow cytometry. It is shown that the frequency of TCRa12+TCRVb7+ cells is 30–36% of T cells 3 days after transduction, which significantly higher than that of untransduced control groups. (Fig. 3B and C). This demonstrates that TCM and CD8+T cell were efficiently transduced. Notablely, the frequency of TCRa12+TCRVb7+ cells declined gradually by more than 70% over 14 days after transduction in all samples regardless of their origin. 3.4. Promoting CTL effect of TCM by tumor antigen specific TCR gene transfer To figure out the effect of TCR gene transfer upon antigen specificity of TCM, human hepatocellular carcinoma cell lines HepG2(HLA-A2+, AFP+), SMMC-7721 (HLA-A2+, AFP+), Bel-7402(HLA-

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Fig. 1. Identification and isolation of CD62L+CD44+ TCM from PBLs of healthy donors. (A) Sorted CD8+ T cells were stained with anti-human CD8 fluorescent antibody and analyzed by flow cytometry after magnetic bead selection. The frequenecy of CD8+ T cells is shown. (B) Sorted CD8+T cells were stained with anti-human CD62L and CD44 fluorescent antibodies and analyzed by flow cytometry. Gate frequencies are displayed. (C) Unsorted CD8+ T cells (left) and sorted TCM (right) were stained with anti-human CD62L fluorescent antibody and analyzed by flow cytometry. The frequency of CD62L+ T cells is shown. (D) Unsorted CD8+ T cells (left) and sorted TCM (right) were stained with anti-human CD44 fluorescent antibody and analyzed by flow cytometry. The frequency of CD44+ T cells is shown. For (A–D), all figures shown are representative of at least three independent experiments. The dotted line indicates results from staining with isotype-matched antibodies served as controls. (E) In vitro growth of CD8+ T cells and TCM was measured by counting viable cells. Each measured in at least five replicate wells. Error bars represent S.D. and groups are compared using one-way ANOVA with a Bonferroni multiple comparisons posttest. p-Values less than 0.05 were considered to be significant. (F) Results of fluorescence in situ hybridization using flow cytometry (flow FISH) of TE cells derived from CD8+ T cells and TCM from healthy individuals on day 14 in culture. TE cells derived from CD8+T cells (left) and TCM (middle) were analyzed after hybridization with or without FITC-labeled PNA (shaded and open histograms, respectively. Figures shown are representative of four independent experiments). Grouped column scatter (right) shows telomere length of CD8+ T cells (open circle) and TCM (filled rectangle) relative to average length for each donor. Four individual donors are represented.

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Fig. 2. Phenotypic changes of TE cells derived from CD8+T cells and TCM in culture. (A) TE cells derived from CD8+T cells (upper) and TCM (below), were stained with anti-human CD62L fluorescent antibody and analyzed by flow cytometry at different time point in culture as indicated. The frequency of CD62L+ T cells is shown. (B) TE cells derived from CD8+T cells (upper) and TCM (below), were stained with anti-human CD44 fluorescent antibody and analyzed by flow cytometry at different time point in culture as indicated. The frequency of CD44+ T cells is shown. (C) In mid-late period of culture (21 days), TE cells derived from CD8+T cells (left, representative of three donors) and TCM (donor1-3), were stained with anti-human CD62L and anti-human CD44 fluorescent antibodies and analyzed by flow cytometry. Cell clusters with high expression of CD44 appear in TCM-derived TE, meanwhile, all CD62L positive cells are CD44high, i.e., CD62L+CD44high cluster. The frequency of CD62L+CD44high cells is shown. (D) TE cells derived from CD8+T cells (upper) and TCM (below), were stained with anti-human CD45RO fluorescent antibody and analyzed by flow cytometry at different time point in culture as indicated. The frequency of CD45RO+ T cells is shown. (E) TE cells derived from CD8+T cells (upper) and TCM (below), were stained with anti-human CD28 fluorescent antibody and analyzed by flow cytometry at different time point in culture as indicated. The frequency of CD28+ T cells is shown. (F) TE cells derived from CD8+T cells (upper) and TCM (below), were stained with anti-human CCR7 fluorescent antibody and analyzed by flow cytometry at different time point in culture as indicated. The frequency of CCR7+ T cells is shown. For (A–F), All figures shown are representative of at least three independent experiments. The dotted line indicates results from staining with isotype-matched antibodies served as controls.

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Fig. 2 (continued)

A2 , AFP+), and human breast carcinoma cell line MCF-7(HLA-A2+, AFP ) were employed as target cells which were marked by Calcein-AM. The present study evaluated cytotoxic effect of TCR gene transferred TCM, untransferred TCM, TCR gene transferred CD8+T cells and untransferred CD8+T cells by detecting free Calcein-AM released by dead target cells, with 5 parallel samples in each group. 3 independent experiments with T cells from different donors were performed. As shown in Fig. 4A, TCR gene transfer enhanced the ability of T cells to lyse HLA-A2+ AFP+ target cells. The percent specific lysis of TCR gene transferred TCM is significant higher than that of untransferred TCM. Difference in level of lysis is also observed be-

tween TCR gene transferred CD8+T cells and untransferred CD8+T cells. Remarkably, TCR gene transferred TCM have stronger ability to lyse target cells than transferred CD8+T cells at 30:1 effector/target cell (E:T) (P < 0.001). There are no significant differences in level of specific lysis between TCR gene transferred T cells and untransferred control groups in the CTL assays using Bel-7402 and MCF-7 as target cells. To investigate whether TCR gene transferred T cells killing target tumor cells in an HLA-A2-restricted manner, we performed antibody blocking experiments. As shown in Fig. 4B, in the HLAA2 blocking experiments, after blocking the HLA-A2 sites on the

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surface of HepG-2 and SMMC-7721 with HLA-A2 mAb, the specific lysis effects of TCR gene transferred T cells could be significantly eliminated. These results indicate that TCR gene transferred CTL recognized target tumor cells in an HLA-A2-restricted manner. Perforin and granzyme B are proteins thought to play a relevant role in cell-mediated cytotoxicity. 6 h after coculture with target cells at 30:1 (E:T), We performed intracellular staining with antihuman perforin and granzyme B fluorescent antibodies and detected the expression of perforin and granzyme B on TE cells of different groups. As shown in Fig. 5, the frequency of perforin+ cells and granzyme B+ cells in TCR gene transferred TCM is higher than that in untransferred TCM. 3.5. Influence upon cytokines secretion by TCR gene transfer To further analyze antitumor mechanisms of TCR gene transfer T cells, 24 h after cocultured with target cells at 30:1 (E:T), production of IL-2 and IFN-c of different effector cells were detected through ELISA assays. As Fig. 6A shows, after coculture with HLA-A2+ AFP+ target cells, TCR gene transferred CD8+T cell produced greater quantities of IFN-c than untransferred CD8+T cell group, while difference in capacity of secret IFN-c between TCR gene transferred TCM group and that of untransferred TCM is more significant (P < 0.001). Furthermore, Content of IFN-c secretion in TCR gene transferred TCM is higher than that of TCR gene transferred CD8+T cell (P < 0.01). Unlike the results of IFN-c secretion, whether TCM were modified by specific TCR gene or not, they were capable of producing lesser IL-2 than CD8+T cell. We also observed that antigen specific TCR gene modification had no significant effect on the capacity of secret IL-2 of T cells. Taking together, these results indicate that IFN-c is a crucial effector cytokine to mediate the antitumor response, and IL-2 appears to mark differentiation of effective cells. 4. Discussion The key to long-term therapeutic effect is that gene engineered T cells, after being transfused in vivo, can persist their survival and immune function. At clinical transfusion of T cells, the persistence and antitumor efficacy of transfused T cells were strengthened by elimination of regulatory immune cells through depletion of host lymphocytes and application of cytokine IL-2 after cell transfer, etc [20]. It was found, however, that the intrinsic property of T cells, especially the differentiation state, had tremendous influences upon their fate after transfusion [9–11]. To guarantee sufficient effector cells for transfusion, T cells usually need plentiful expansion in vitro by stimulation and activation. After encountering antigen in vivo, T cells proliferated quickly via being activated by TCR, costimulatory signals and cytokine, and generate large amounts of TE cells through programmed differentiation [21]. After antigen elimination, the vast majority (90–95%) of TE cells died in the contraction phase [22], and only few memory T cells could survive long with specificity of antigen recognition. To obtain enough TCM, CD62L and CD44 were used as sorting labels to identify and isolate the TCM of human peripheral blood CD8+T cells. CD8+ TCM express CD62L which promote CD8+ TCM migration into LNs [14]. While CD44 is originally described as a homing receptor of lymphocytes, which is upregulated in response to antigenic stimuli [23]. It was revealed that the peripheral blood of healthy individuals had some TCM which mainly derived from the differentiation of TN cells after they being exposed to antigen in vivo [24]. The intrinsic property of TCM determined their ability of self-renewal and re-differentiation [14]. By dynamic monitoring of phenotype markers, we found that there were significant differences between CD8+T cells and TCM

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during the differentiation process after stimulation in vitro. After effector differentiation, the number of TE cells derived from CD8+T cells declined rapidly after 14 days of culture, the expression of CD62L on TE cells from CD8+T cells decreased gradually to disappearance. Then the expression of CD44 on TE cells from CD8+T cells decreased, along with the differentiation and death of TE cells. Remarkably, the number of TE cells derived from TCM exhibited improved survival, although there was no significant difference in relative telomere length between TE cells derived from TCM and TE derived from CD8+T cells.TE cells from TCM constantly kept a certain frequency of CD62L+ cells, revealing that CD62L, as TCM specificity trait, did not disappear during the whole process of effector differentiation [8]. Meanwhile, the frequency of CD44+ cells of TE cells from TCM kept a relatively high level, and a subset of high CD44 expression gradually appeared after 21 days of culture. A noteworthy phenomenon was that all CD62L positive cells were CD44high. This indicated that some of the TE cells derived from TCM differentiation could reacquire the CD62L+CD44high TCM phenotypic characteristics to sustain their self-renewal and immune function. TE cells derived from TCM also expressed high level of CD45RO which indicated T cell activation. Similar to CD62L, the continuous expression of CCR7 in TE cells derived from TCM enables T cells to migrate to LNs and quickly proliferate when the cells are reexposed to antigen. To further determine whether antigen specificity of TCM could be redirected via TCR gene transfer, we transferred tumor antigen specific TCR genes which were screened previously to CD8+T cells and TCM, respectively. Ad5 is a commonly used viral adenoviral vector in gene therapy, however, T cells have low expression of coxsackie/adenovirus receptors (CAR) and aV integrins which are required for uptake of Ad5 vectors [25], results in low transduction rate of T cells using Ad5 vectors [26]. In this study, chimeric adenoviral vector (Ad5F35) was generated which contained the Ad35 fiber knob incorporated into an Ad5 capsid by homologous recombination. This chimeric Ad5F35 vector is CAR independent, targets CD46 that expressed almost in all human cells, and transduces human T cells effectively [27]. We demonstrated this chimeric fiber-modified Ad5F35 adenoviral vector is able to efficiently transduce human T cell. But the expression level of exogenous TCR genes decreased to 20% over 10–14 days after transduction in vitro. The stability of the expression may be improved by retroviral or lenti viral gene transfer. Specific TCR gene modification could effectively improve the killing ability of T cells to antigen positive tumor cells by an HLA-A2 restrict manner. For TCR gene transferred CD8+T cells and TCM, their cytotoxic activities were both higher than that of untransferred control group, and their expressions of perforin and granzyme B rose along. These results demonstrated that, for both CD8+T cells and TCM, TCR gene transfer could redirect T cells to recognize antigen positive target cells and exert CTL activity, through expressing antigen-specific TCR. It was noteworthy that, compared with TCR gene transferred CD8+T cells, transferred TCM had stronger CTL activity, which might be due to differentiation of CD8+T cells. CD8+T cells can acquire Th-1, Th-2, Treg and Th17 cells effector functions, and form mix cell clusters composed of different subsets which impact their reactivity of antitumor [28,29]. TCR gene modification also improved the ability of T cells to secrete IFN-c. The secretion of IFN-c by TCR gene transferred TCM was higher than that of TCR gene transferred CD8+T cell. After the activation of T cells, IFN-c was a key cytokine of mediating anti-tumor immune response. Although IL-2 production of TCR gene transferred TCM is lower than IFN-c, it has been shown in other work that overexpression of IL-2 by adoptively transferred T cells did not increase their in vivo persistence or clinical effectiveness [30].

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Fig. 3. Recombinating construction and transduction of TCR adenovirus vectors. (A) A chimeric adenoviral vector (Ad5F35) was generated which contained Ad35 fiber knob incorporated into an Ad5 capsid using homologous recombination in HEK 293 cells, whole encoding sequence for TCR Va12.2 (TRAV) and TCR Vb7.1 (TRBV) were linked by IRES and then cloned into Ad5F35 vector. (B) Untransduced TCM (left) and transduced TCM (donor 1,2) were stained with anti-human TCR Va12, Vb7 fluorescent antibodies and analyzed by flow cytometry at different time point after transduction. The frequency of TCR Va12+ TCR Vb7+ T cells is shown. (C) Untransduced CD8+ T cells (left) and transduced CD8+ T cells (donor 1,2) were stained with anti-human TCR Va12, Vb7 fluorescent antibodies and analyzed by flow cytometry at different time point after transduction. The frequency of TCR Va12+ TCR Vb7+ T cells is shown. For (B and C), all figures shown are representative of at least three independent experiments.

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Fig. 4. Promoting CTL effect of TCM by tumor antigen specific TCR gene transfer. (A) Specific lysis of CD8+T cells (open triangles) , TCM (filled inverted triangles) and TCRtransferred CD8+ T cells (open circles), TCR-transferred TCM (filled diamonds) were examined at different effector-to-target ratio (E/T ratio) from 3:1 to 30:1 (i-axis) with target cells as follows: Human hepatocellular carcinoma cell lines HepG-2 (HLA-A2+,AFP+), SMMC-7721 (HLA-A2+, AFP+), Bel-7402 (HLA-A2-, AFP+), and human breast carcinoma cell line MCF-7 (HLA-A2+, AFP-). Target tumor cells were labeled with Calcein-AM before coculture. Percent specific lysis (Y-axis) was calculated using the following formula: percent specific lysis = [(test release-spontaneous release)/ (maximum release –spontaneousre lease)]  100. Spontaneous release represented calcein release from target cells in complete medium alone, and maximal release was the calcein release from target cells lysed in medium plus 1% Triton X-100. (B) Anti-HLA-A2 mAb blocked the peptide-specific CTL reaction on target tumor cell lines. Specific lysis of TCR-transferred CD8+T cells and TCR-transferred TCM were examined at different E/T from 3:1 to 30:1 (X-axis). Target cells HepG-2 and SMMC-7721 were incubated with (filled triangles) or without anti-HLA-A2 antibody (filled squares) for 1 h at 4 °C. Label of target cells and calculation of percent specific lysis (Y-axis) are same with (A). For (A and B), each measured in at least five replicate wells. error bars represent S.D. and groups are compared using one-way ANOVA with a Bonferroni multiple comparisons posttest. p-Values less than 0.05 were considered to be significant. Data shown are representative of at least three independent experiments.

In summary, CD62L+CD44+TCM were identified and isolated from human CD8+T cells. During the process of effector differentiation in vitro, TE cells from TCM exhibited improved survival, constantly kept certain level of expression of CD62L, and reacquired the phenotype of TCM. Tumor-specific TCR genes were transduced by recombinant adedovirus Ad5F35-TRAV-TRBV into TCM. TCR gene transfer effectively enhanced the ability of TCM to recognize and kill antigen positive tumor cells. TCR gene transferred TCM were more effective than TCR gene transferred CD8+T cells in CTL activity,

IFN-c secretion. These results revealed that, compared with CD8+T cells, isolated TCM modified by TCR gene may provide a tumor-reactive T cell population with improved viability and effector function for adoptive T cell therapy. A recent study from Hinrichs et al. [31] compared functional and phenotypic characteristics of human T cells derived from TCR gene modified TN, TCM, and TEM subsets. Their finding is consistent with our data about effector fuction of TCM. But considering the terminal differentiation of TE derived from TCM, they suggested TN may be the superior subset

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Fig. 5. Promoting cytolytic granule protein expression of TCM by tumor antigen specific TCR gene transfer. (A) CD8+T cells, TCM, and TCR-transferred CD8+T cells, TCRtransferred TCM were intracellular stained with anti-human granzyme B fluorescent antibody and analyzed by cytokine flow cytometry 6 h after coculture at 30:1(E/T) with target cell HepG-2. The frequency of granzyme B+T cells is shown. (B) CD8+T cells, TCM, and TCR-transferred CD8+T cells, TCR-transferred TCM were intracellular stained with anti-human granzyme B fluorescent antibody and analyzed by cytokine flow cytometry 6 h after coculture at 30:1(E/T) with target cell SMMC-7721. The frequency of granzyme B+ T cells is shown. (C) CD8+ T cells, TCM, and TCR-transferred CD8+ T cells, TCR-transferred TCM were intracellular stained with anti-human perforin fluorescent antibody and analyzed by cytokine flow cytometry 6 h after coculture at 30:1(E/T) with target cell HepG-2. The frequency of perforin+ T cells is shown. (D) CD8+T cells, TCM, and TCR-transferred CD8+ T cells, TCR-transferred TCM were intracellular stained with anti-human perforin fluorescent antibody and analyzed by cytokine flow cytometry 6 h after coculture at 30:1(E/T) with target cell SMMC-7721. The frequency of perforin+T cells is shown. All figures shown are representative of at least three independent experiments. The dotted line indicates results from staining with isotype-matched antibodies served as controls.

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Fig. 5 (continued)

for adoptive. Since the most important advantage of TE derived from TCR gene modified TCM is to migrate to TM niches and re-acquire phenotypic properties of TM in vivo, further study especially in primate model relevant to human translation may be required to arrive at the final conclusion. Applications of TCM in adoptive T cell therapy may comprise: (1) Screen tumor-specific TCR in TCM isolated from tumor patients.

However, the resemblance between tumor antigen and self antigen should be taken into account, since the T cells that can recognize tumor antigen are rendered tolerant in vivo [32]. (2) In consideration of tumor patients’ immune dysfunction, TCM from HLAmatched healthy donors can be isolated and transferred with tumor-specific TCR genes, then expanded and transfused into the patients [33].

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Fig. 6. Influencing cytokines secretion of TCM by TCR gene transfer. (A) IFN-ccontents (Y-axis) in supernatant of culture medium of CD8+T cells (white column), TCM (grey column), and TCR-transferred CD8+T cells (striped column), TCR-transferred TCM (black column) were detected through ELISA assays, after 24 h coculture at 30:1(E/T) with target cell as follows: HepG-2, SMMC-7402, Bel-7402 and MCF-7. (B) IL-2 contents (Y-axis) in supernatant of culture medium of CD8+T cells (white column), TCM (grey column), and TCR-transferred CD8+T cells s (striped column),TCR-transferred TCM (black column), were detected through ELISA assays, after 24 h coculture at 30:1(E/T) with target cell as follows: HepG-2, SMMC-7402, Bel-7402 and MCF-7. For (A and B), each measured in at least five replicate wells. error bars represent SD, and groups are compared using one-way ANOVA with a Bonferroni multiple comparisons posttest. p-Values less than 0.05 were considered to be significant.(*p < 0.05, **p < 0.01, ***p < 0.001). Data shown are representative of at least three independent experiments.

Conflict of Interest None of the authors has any financial or other interest with regard to the submitted manuscript that might be constructed as a conflict of interest. Acknowledgments This work was supported by the National Major Projects of Science and Technology of China (No. 2009ZX09103-708), National Natural Science Foundation of China (No. 31100664), Natural Science Foundation of Guangdong Province China (No. 10151022401000024), and Science and Technology Planning Project of Guangdong Province, China (No. 2004B31201001, No. 2012A061400021). References [1] C.H. June, T. Adoptive, Cell therapy for cancer in the clinic, J. Clin. Invest. 117 (2007) 1466–1476. [2] E.M. Dudley, J.R. Wunderlich, J.C. Yang, R.M. Sherry, S.L. Topalian, N.P. Restifo, R.E. Royal, U. Kammula, D.E. White, S.A. Mavroukakis, L.J. Rogers, G.J. Gracia, S.A. Jones, D.P. Mangianmeli, M.M. Pelletier, J. Gea-Banacloche, M.R. Robinson, D.M. Berman, A.C. Filie, A. Abati, S.A. Rosenberg, Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma, J. Clin. Oncol. 23 (2005) 2346–2357.

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