HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van der Veken, L. T. Articles by Heemskerk, M. H. M. Search for Related Content PubMed PubMed Citation Articles by van der Veken, L. T. Articles by Heemskerk, M. H. M.

β T Cell Receptor Transfer to T Cells Generates Functional Effector Cells without Mixed TCR Dimers In Vivo¹

Lars T. van der Veken^2,*, Miriam Coccoris^2,, Erwin Swart, J. H. Frederik Falkenburg^*, Ton N. Schumacher and Mirjam H. M. Heemskerk^3,*

^* Laboratory of Experimental Hematology, Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands; and Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The successful application of T cell-based immunotherapeutic applications depends on the availability of large numbers of T cells with the desired Ag specificity and phenotypic characteristics. Engineering of TCR-transferred T lymphocytes is an attractive strategy to obtain sufficient T cells with an Ag specificity of choice. However, the introduction of additional TCR chains into T cells leads to the generation of T cells with unknown specificity, due to the formation of mixed dimers between the endogenous and introduced TCR chains. The formation of such potentially autoaggressive T cells may be prevented by using T cells as recipient cells, but the in vivo activity of such TCR-engineered T cells has not been established. In the present study, we have investigated the in vivo functionality of TCR-transduced T cells, in particular their Ag specific proliferative capacity, Ag specific reactivity, in vivo persistence, and their capacity to mount recall responses. The results demonstrate that β TCR engineering of T cells forms a feasible strategy to generate Ag-specific effector T cells that do not express mixed TCR dimers. In view of increasing concerns on the potential autoimmune consequences of mixed TCR dimer formation, the testing of β TCR engineered T cells in clinical trials seems warranted.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Several lines of evidence suggest that T cell-based adoptive immunotherapy forms an attractive approach for the treatment of various malignancies. Infusion of donor lymphocytes for the treatment of hematological malignancies has been shown to result in 20–80% complete remissions depending on the nature of the leukemia (1, 2, 3, 4, 5). Successful adoptive immunotherapy based on adoptive transfer of ex vivo-expanded tumor infiltrating lymphocytes in conditioned recipients has been demonstrated for metastatic melanoma (6, 7).

Although promising, the complex isolation methods of T cells with the desired Ag specificity and laborious and time-consuming tissue culture procedures are likely to prevent the widespread application of these approaches. We and others have demonstrated that these difficulties can be overcome by an alternative strategy, namely the transfer of T cell specificity by genetic introduction of TCR - and β-chains into T lymphocytes (8, 9, 10, 11, 12, 13, 14, 15). Because T cell specificity is exclusively determined by the TCR, genetic transfer of TCR chains to T cells generates T cells with the desired Ag specificity. The potential in vivo efficacy of TCR-transferred T cells has been demonstrated in mouse models (14, 15, 16, 17). As shown in these various murine models, TCR-transferred T cells can be activated by Ag in vivo, home to effector sites, and can break tolerance against defined self Ags. Importantly, TCR gene transfer can largely halt tumor progression in a spontaneous prostate carcinoma model, under conditions in which vaccination is without effect (18). Recently, in a clinical phase 1 study, autologous peripheral blood-derived lymphocytes transduced with a melanoma Ag recognized by T cells 1-specific TCR were reinfused in patients with metastatic melanoma after lymphodepleting chemotherapy (19). Persistence of the TCR modified T cells was observed for >2 mo and two of fifteen patients showed objective tumor regression illustrating that, although optimization is required, TCR gene transfer is feasible in a clinical setting.

A potential complication in TCR gene transfer is the formation of mixed TCR dimers that occurs when introduced TCR - and β-chains pair with the endogenous TCR - and β-chains (20). As the specificity of these mixed TCR dimers is unknown, the formation of unwanted autoreactivities cannot be excluded (12, 21). Importantly, while no evidence for mixed dimer induced autoimmunity was observed in earlier murine experiments and a first phase I trial (14, 15, 16, 17, 19), more recent data in mouse model systems indicate that under conditions of aggressive conditioning, severe mixed dimer-dependent autoimmune pathology can occur (G. Bendle, personal communication).

Prior work has illustrated that the and TCR chains that are expressed by T cells cannot form heterodimers with and β TCR chains (22, 23). We previously demonstrated that the redirection of T cells instead of β T cells allows TCR gene transfer without the formation of mixed TCR dimers (24). Specifically, in vitro experiments showed that after retroviral transfer of MHC class I- or MHC class II-restricted TCRs fully functional, redirected human effector cells were obtained capable of lysing leukemic cells in an Ag specific manner. TCR gene transfer into T cells requires either the cotransfer of the CD4 or CD8 coreceptor, or the use of coreceptor-independent TCRs, as the majority of T cells lack the expression of these coreceptors (25, 26). However, as demonstrated previously, the joint introduction of TCR and coreceptor genes can be achieved readily (16, 24).

A substantially greater concern is formed by the lack of information on the in vivo capacities of TCR-modified T cells. As compared with β T cells, relatively little is known on the in vivo survival and effector functions of T cells. Furthermore, although T cells can be detected in peripheral blood and spleen, this T cell subset mainly resides in epithelial sites and this could affect the capacity of β TCR-modified T cells to react to Ag (27, 28, 29, 30, 31).

To address these issues, we investigated the in vivo function of β TCR-modified T cells in a mouse model. Our data demonstrate that β TCR gene transfer into T cells can be used as a safe method to generate functional Ag specific effector cells for adoptive immunotherapy.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice and T cells

C57BL/6 (B6), B6 Ly5.1⁺, B6 Ly5.1/2⁺, B6 Ly5.2⁺, and RIP-OVA^high mice (32) were obtained from the animal department of the Netherlands Cancer Institute. All animal experiments were conducted in accordance with institutional and national guidelines and were approved by the Experimental Animal Committee of the Netherlands Cancer Institute (DEC).

To isolate spleen derived T cells from B6 Ly5.1⁺, B6 Ly5.1/2⁺, or B6 Ly5.2⁺ mice, splenocytes were stained with PE-conjugated anti-TCR specific mAbs (BD Biosciences), anti-PE Microbeads (Miltenyi Biotec) were added and T cells were enriched by AutoMACS (Miltenyi Biotec) according to the manufacturer’s protocol (>50% purity). Subsequently, the recovered cells were further purified using a FACSVantage (BD Biosciences) fluorescence-activated cell sorter. Purity of the thus obtained T cell population was >99%.

Construction of the retroviral vectors and production of the retroviral supernatant

The pMX vector encoding the TCR - and TCR β chains of the OT-I TCR separated by an internal ribosome entry site and the pMX vector encoding the murine CD8 and CD8β chains separated by an internal ribosome entry site have been described previously (16, 17). Retroviral supernatants were obtained by transfection of Phoenix-E packaging cells with the indicated retroviral vectors in combination with pCLEco (33), using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals). Retroviral supernatants were obtained 48 h after transfection and used for transduction of purified T cells.

Retroviral transfer of the OT-I β TCR and coreceptors to T cells

Purified T cells were stimulated in 24-well plates for 24 h in RPMI 1640 medium (Life Technologies) supplemented with 8% FCS (BioWhittaker), penicillin (100 U/ml), and streptomycin (100 µg/ml; Boehringer Mannheim) in the presence of irradiated autologous feeder cells, Con A (2 µg/ml; Calbiochem) and IL-7 (5 ng/ml; Santa Cruz Biotechnology).

Non-tissue culture-treated 24-well plates (Becton Dickinson) were coated with 0.5 ml of 30 µg/ml recombinant human fibronectin fragment CH-296 (RetroNectin; Takara) at room temperature for 2 h. The CH-296 solution was removed and replaced with 0.5 ml of 2% BSA (Sigma- Aldrich) in PBS for 30 min at room temperature. The T cells were plated on RetroNectin coated plates (0.3 x 10⁶ cells/well) in 0.5 ml of retroviral supernatant and cultured at 37°C for 24 h. For in vivo assays, the cells were washed once in HBSS (Life Technologies), resuspended in HBSS, and injected in mice i.v.

Influenza A and recombinant vaccinia virus

For live influenza infections, anesthetized mice were infected by intranasal administration of 50 µl of HBSS (Life Technologies) containing 200 PFU of influenza A/WSN/33 (WSN)-OVA(I) (34) virus that expresses the OVA_257–264 epitope (WSN-OVA), or 0.5 hemagglutinating units of influenza A/HK/2/68 virus. For recombinant vaccinia infections, 2 x 10⁷ PFU of vaccinia recombinant for GFP-OVA_257–264 (rVV-OVA) were injected i.p.

Peptides

The H-2K^b binding peptides OVA_257–264 (SIINFEKL) and SV40 large T_404–411 (VVYDFLKC), the H-2D^b binding peptides NT_366–374 (influenza A/HK/2/68: ASNENMDAM), and PR_366–374 (influenza A/PR/8/34: ASNENMETM) were synthesized by standard 9H-fluoren-9-ylmethoxycarbonyl chemistry.

Flow cytometric analysis

For analysis of T cell responses, peripheral blood was drawn at the indicated time points. Erythrocytes were removed by incubation in erylysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA (pH 7.4)) on ice for 15 min. Cells were stained with the indicated Abs for 10–15 min at room temperature. The following mAbs: anti-TCR FITC, anti-TCR PE, anti-TCRVβ5.1 FITC, anti-TCRV2 PE, anti-Ly5.1 PECy5, anti-Ly5.2 PECy5, and anti-CD8 allophycocyanin were obtained from BD Pharmingen, anti-CD8β PE was obtained from Caltag Laboratories. Before analysis, propidium iodide (1 µg/ml; Sigma-Aldrich) was added to select for propidium iodide-negative (living) cells.

Intracellular IFN- staining

For intracellular IFN- staining, cells were incubated in the presence of either 0.1 µg/ml OVA or 1 µg/ml SV40 peptide for 4 h at 37°C, in the presence of GolgiPlug (1 µl/ml; BD Biosciences). Subsequently, cells were surface stained with FITC-conjugated anti-Ly5.2 (BD Biosciences) and PE-conjugated anti-CD8 (Caltag Laboratories) mAbs for 15 min on ice. After washing, cells were incubated in Cytofix/Cytoperm solution (BD Biosciences) for 20 min on ice, washed, and stained for intracellular IFN- with allophycocyanin-conjugated anti-IFN- mAb (BD Biosciences) on ice for 20 min.

In vivo cytotoxicity assay

Splenocytes were prepared from RIP-OVA^high mice and divided into two groups. Cells were labeled with 0.5 or 5 µmol/L CFSE (Molecular Probes) for 20 min at 37°C. Cells were washed, kept on ice, and subsequently the CFSE^high cells were pulsed with 0.1 µmol/L OVA_257–264 peptide for 1 h at 37°C. Five million cells from the CFSE^low and the CFSE^high peptide-pulsed cells were mixed together in equal proportions and injected i.v. into mice that had received T cells transduced with the OT-I TCR and CD8 coreceptor, and had been infected with either WSN-OVA or influenza A/HK/2/68. Spleens were removed 5 h later and single-cell suspensions were generated for FACS analysis. The percentage of target cell killing was determined as: 100 – ((percentage of peptide-pulsed targets in WSN-OVA infected recipients/percentage of unpulsed targets in WSN-OVA infected recipients)/(percentage of peptide-pulsed targets in influenza A/HK/2/68 infected recipients/unpulsed targets in influenza A/HK/2/68 infected recipients) x 100).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Functional analysis of βTCR transduced T cells

To investigate the in vivo functionality of T cells that have been redirected by the introduction of an β TCR, spleen derived T cells were isolated by immunomagnetic bead isolation and subsequent FACS sorting (>99% purity). Purified T cells were then stimulated with ConA and autologous irradiated feeder cells in the presence of IL-7, and after 24 h of stimulation retrovirally transduced with the OVA specific OT-I TCR. All TCR transductions were performed in combination with a retroviral vector encoding the CD8β coreceptor, as most T cells lack expression of this coreceptor. The OT-I CD8 transduced T cell population was infused i.v. (1 x 10⁵ cells per mouse), and the mice then received an intranasal infection with either a recombinant influenza A strain that expresses the OVA257–264 epitope (WSN-OVA) or a control influenza A strain (A/HK/2/68). Subsequently, the frequency of OT-I⁺ CD8⁺ T cells in peripheral blood was determined by combined TCR, TCR V2, Ly5.2, and CD8 staining at various time points post infection. In mice that were infected with WSN-OVA a significant accumulation of the OT-I modified T cells was observed, with peak immune responses at day 9 post adoptive transfer (Fig. 1A, middle panels). In contrast, in control mice that were infected with influenza A/HK/2/68 no significant numbers of OT-I modified T cells could be detected (Fig. 1A, top panel). The TCR⁺ TCR V2⁺ CD8⁺ cell population that was detected in mice infected with WSN-OVA expressed the Ly5.2 marker present on the donor cells, and was hence fully donor-derived. Furthermore, the vast majority of Ly5.2⁺ cells present in these mice expressed the TCR, indicating that there was little if any contribution of the small amount (<1%) of cotransferred β T cells (Fig. 1A, lower panel). These data demonstrate that T cells that are engineered to express an MHC class I-restricted TCR are capable of Ag-specific survival or proliferation upon interaction with Ag-expressing APC in vivo.

View larger version (39K): [in this window] [in a new window]   FIGURE 1. Ag-specific expansion and cytokine production of T cells transduced with an βTCR. Purified T cells from Ly5.2+ mice were transduced with the OT-I TCR in combination with CD8β and infused i.v. into Ly5.1+ mice. Subsequently, the mice received an intranasal infection with either WSN-OVA or influenza A/HK/2/68. A, Representative FACS profiles at day 9 gated on CD8+ cells from mice that received OT-I plus CD8 transduced T cells i.v. and which were subsequently infected with either influenza A/HK/2/68 (upper panels) (n = 4) or WSN-OVA (middle panels) (n = 4). In addition, flow cytometry profiles at day 9 are shown gated on Ly5.2+ CD8+ cells of the WSN-OVA infected mice (lower panel). B, Representative flow cytometry profiles of intracellular IFN- staining of peripheral blood derived lymphocytes from Ly5.1+ mice at the peak of the response (day 9) that received Ly5.2+ OT-I plus CD8 transduced T cells, and were subsequently infected with either influenza A/HK/2/68 (data not shown) (n = 4) or WSN-OVA (n = 4). Peripheral blood-derived lymphocytes were ex vivo stimulated with the OVA257–264 peptide or SV40 large T404–411 control peptide, and subsequently, Ag specific intracellular IFN- production was determined by flow cytometry. C, Representative flow cytometry profiles of intracellular IFN- staining of total splenocytes derived from Ly5.1+ mice at the peak of the response (day 9) that received Ly5.2+ OT-I + CD8 transduced T cells, and were subsequently infected with either influenza A/HK/2/68 (n = 4) or WSN-OVA (n = 4). Splenocytes were in vitro stimulated with SV40 control peptide (data not shown) or OVA peptide, cultured for 14 days, restimulated with OVA or SV40 control peptide and, subsequently, Ag-specific intracellular IFN- production was determined by flow cytometry. βTCR transduced T cells proliferated in vivo and produced cytokines upon Ag-specific stimulation.

In addition, total splenocytes of A/HK/2/68-infected and WSN-OVA-infected mice were stimulated with OVA peptide and cultured for 14 days. Subsequently, the cultured splenocytes were restimulated with either SV40 or OVA peptide and intracellular IFN- production was determined (Fig. 1C). No Ly5.2⁺ cells were detected in influenza A/HK/2/68 infected mice, indicating that efficient recovery of β TCR transduced T cells requires in vivo Ag encounter. In contrast, substantial numbers of the adoptively transferred Ly5.2⁺ OT-I CD8 transduced T cells were found in cultures from WSN-OVA infected mice and a large fraction of these cells produced IFN- after stimulation with OVA peptide. Collectively, these data indicate that T cells modified with an β TCR exhibit the potential to undergo Ag-driven expansion and display effector functions following in vivo activation.

Self-Ag-specific reactivity of βTCR transduced T cells

To investigate whether OT-I CD8 transduced T cells can expand upon Ag specific stimulation in a situation where the endogenous T cell repertoire is tolerant toward the OVA Ag, OT-I CD8 transduced T cells were infused in RIP-OVA^high mice. In these mice, OVA is expressed in pancreatic β cells and no endogenous OVA-specific cytotoxic and helper T cell responses are observed (35, 36). Following infusion of OT-I CD8 transduced T cells (5 x 10⁴ per mouse), the mice were infected either by intranasal application of influenza A/HK/2/68 or WSN-OVA, or by i.p. injection of a recombinant vaccinia virus expressing GFP-OVA_257–264 (rVV-OVA). At various time points, the percentage of OT-I CD8 transduced T cells in peripheral blood was monitored (Fig. 2). In mice that received OT-I CD8-transduced T cells and were subsequently infected with either WSN-OVA or rVV-OVA, a substantial increase in βTCR transduced T cell number was observed, with a peak response of 8% of total CD8⁺ T cells in the latter group. Similar to the kinetics observed upon Ag-induced proliferation of TCR-engineered β T cells (14, 17), expansion was followed by a rapid contraction at the time of Ag clearance. As expected, no expansion of the V2⁺ TCR⁺ CD8⁺ T cell population was observed in mice that were infected with influenza A/HK/2/68. Thus, βTCR-engineered T cells can respond to in vivo Ag encounter in a situation where the endogenous T cell repertoire is tolerant toward the Ag of interest.

View larger version (7K): [in this window] [in a new window]   FIGURE 2. Self-Ag-specific reactivity of βTCR transduced T cells. Purified T cells were transduced with the OT-I TCR in combination with CD8β and infused i.v. into RIP-OVAhigh mice which were subsequently infected intranasally with either influenza A/HK/2/68 (n = 3) or WSN-OVA (n = 3), or were infected i.p. with rVV-OVA (n = 4). The percentages of V2+ TCR+ cells of total CD8+ cells in peripheral blood at different time points after infection are shown. Data represent group averages ± SD. OT-I TCR-transduced T cells expanded in vivo in an Ag specific manner in the absence of an endogenous OVA-specific T cell response.

Analysis of intestinal sites for the presence of βTCR-engineered T cells

A significant percentage of the CD8⁺ lymphocytes present within the intestinal epithelium consists of T cells (27, 28, 29, 30, 31). To investigate whether βTCR-transduced spleen-derived T cells would preferentially home to intestinal epithelial sites upon i.v. infusion, the number of βTCR transduced T cells in peripheral blood was compared with the number of βTCR-transduced T cells in the intra epithelial lymphocyte (IEL)⁴ and lamina propria-resident (LP) lymphocyte fractions. RIP-OVA^high mice received either OT-I CD8 transduced T cells, CD8 transduced T cells (2 x 10⁵ per mouse), or no T cells and were subsequently infected with WSN-OVA. As unmodified T cells lack expression of CD8β (26), expression of CD8β was used to distinguish endogenous and adoptively transferred T cells. The percentage of CD8β⁺ T cells in blood was monitored at various time points after the infection (Fig. 3A). Only in peripheral blood of mice that received T cells transduced with the OT-I TCR in combination with the CD8 coreceptor CD8β⁺ T cells were detected. At the peak of the response (day 12) blood, IEL and LP lymphocyte fractions were analyzed by flow cytometric analysis. Although in peripheral blood percentages of total T cells up to 5% were observed, the percentage of total T cells in the IEL and LP reached levels up to 36 and 14%, respectively. In peripheral blood 7.5% of the T cells consisted of OT-I CD8 transduced T cells (Fig. 3B). Analysis of IEL and LP derived T cells of the mice that received either OT-I CD8 transduced T cells, or CD8 transduced T cells or no T cells showed that no significant numbers of OT-I CD8 transduced T cells could be detected in the intestine of the mice (Fig. 3C). This indicates that the OT-I CD8 transduced T cells do not preferentially home to intestinal epithelial sites upon i.v. infusion.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. Analysis of intestinal sites for the presence of TCR-transduced T cells. RIP-OVAhigh mice received an adoptive transfer of purified T cells transduced with CD8β (mock) (n = 3), T cells transduced with the OT-I TCR + CD8β (n = 3), or no cells (n = 3) and were subsequently infected with WSN-OVA. A, The percentages of TCR V2+ TCR+ cells of total CD8β+ cells in peripheral blood at different time points after infection are shown. Data represent group averages ± SD. B, At the peak of the response (day 12) cells derived from blood, lamina propria (LP) and intra epithelial lymphocyte (IEL) fractions were analyzed by flow cytometry. The total number of T cells in blood, IEL and LP fractions varied from 5 to 36%, respectively (representative histograms of OT-I TCR plus CD8β group shown). C, Representative flow cytometry profiles are shown of gated TCR+ cells from blood, IEL, and LP fractions of RIP-OVAhigh mice that received T cells transduced with CD8β (mock), T cells transduced with the OT-I TCR plus CD8β, or no cells and were subsequently infected with WSN-OVA. OT-I TCR plus CD8β transduced T cells were found mainly in peripheral blood and no βTCR transduced T cells were found in the intestinal epithelial sites.

In vivo persistence of βTCR-transduced T cells

To investigate whether infused OT-I CD8-transduced T cells are able to persist in vivo and have the capacity to mount a recall response RIP-OVA^high mice received an i.v. infusion of OT-I CD8 transduced T cells (4 x 10⁵) and subsequently an intranasal infection with WSN-OVA or influenza A/HK/2/68. At various time points, the percentage of OT-I CD8 transduced T cells in blood was monitored. Mice that received OT-I CD8 transduced T cells and subsequently a WSN-OVA infection showed an increase in βTCR-transduced T cell number from day 7 with a peak of the response at day 10 and subsequent contraction (Fig. 4). To test whether the Ag clearance preceding the observed contraction of the immune response was accompanied by cytolytic activity of the OT-I CD8 transduced T cells, an in vivo cytotoxicity experiment was performed. Influenza A/HK/2/68 or WSN-OVA infected RIP-OVA^high mice infused with OT-I CD8 transduced T cells (4 x 10⁵ per mouse) received CFSE^low labeled cells and OVA peptide pulsed CFSE^high labeled cells i.v. The percentages of direct Ag specific target cell lysis of OVA peptide pulsed cells after 5 h varied from 18.5 to 24% (Fig. 5). Thus, OT-I CD8 transduced T cells are capable of Ag specific cytolytic activity in vivo. To test in vivo persistence and the capacity of the OT-I CD8 transduced T cell to mount a recall response, the mice that initially received an WSN-OVA infection were challenged by i.p. injection of rVV-OVA after 12 wk. Upon rVV-OVA challenge a strong increase in the number of OT-I CD8 transduced T cells was observed in the peripheral blood of these mice leading to a recall response both in magnitude and kinetics comparable to the observed primary response (Fig. 4). Thus, βTCR-modified T cells are capable of prolonged in vivo persistence and the βTCR modified T cells can mount recall responses.

View larger version (11K): [in this window] [in a new window]   FIGURE 4. In vivo persistence of βTCR transduced T cells. RIP-OVAhigh mice received an i.v. infusion of OT-I CD8 transduced T cells and were subsequently infected with influenza A/HK/2/68 (n = 3) (•) or WSN-OVA (n = 3) (). At various time points the percentage of OT-I+ CD8+ T cells in blood was analyzed. At 12 wk after the primary response the mice that initially received a WSN-OVA infection (n = 3) were challenged with an intraperitoneal injection of rVV-OVA. Data represent group averages ± SD. βTCR transduced T cells persisted in vivo and generated T cell memory.

View larger version (12K): [in this window] [in a new window]   FIGURE 5. Cytolytic activity of βTCR transduced T cells. RIP-OVAhigh mice received an i.v. infusion of OT-I CD8 transduced T cells and were subsequently infected with influenza A/HK/2/68 (n = 1) or WSN-OVA (n = 3). CFSElow labeled unpulsed splenocytes and CFSEhigh labeled OVA peptide pulsed splenocytes were injected at a 1:1 ratio around the peak (day 11) of the primary response. Flow cytometric analysis of spleen cells of influenza A/HK/2/68 (upper panel) or WSN-OVA (lower panel) was performed 5 h after transfer of CFSE-labeled cells. Percentages of CFSEhigh and CFSElow cells are indicated as well as percentages Ag specific cytotoxicity. The percentage Ag specific cytotoxicity was determined as follows: 100 – [(% peptide pulsed in WSN-OVA infected/% unpulsed in WSN-OVA infected)/(% peptide pulsed in influenza A/HK/2/68 infected/% unpulsed in influenza A/HK/2/68 infected) x 100]. OT-I CD8-transduced T cells were capable of Ag specific cytolytic activity in vivo.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

The currently most established strategy for TCR gene transfer, in which TCR genes are introduced into β T cells results in the formation of mixed TCR dimers due to pairing of endogenous and introduced TCR and TCRβ-chains (20). The specificity of the mixed TCR dimers is unknown and may be harmful, and therefore avoidance of the formation of mixed TCR dimers is desired. We propose TCR gene transfer into T cells instead of β T cells, which prevents the formation of mixed TCR dimers because TCR chains cannot pair with βTCR chains (22, 23).

As compared with earlier experiments in which the in vivo function of TCR modified βT cells was investigated in murine models, TCR-modified T cells appear to have a somewhat lower proliferative capacity and in vivo effector function (17, 37). This may in part be explained by the relatively low cell numbers used in this study. The T cells isolated from spleen tissue underwent a stringent sorting procedure to render a >99% pure population, which unfortunately decreased the number of cells available for infusion. Furthermore, this selection process may also have affected the viability of the T cells, reducing the proliferative capacity and therefore the effectiveness of the cells. This is in contrast to previous studies with TCR-transduced β T cells, in which no sorting procedures were required (14, 17). Importantly, it seems plausible that high numbers of TCR-modified T cells can be generated with substantially greater ease in the human setting (see below).

Conflicting data exist on the capacity of T cells to persist in vivo, to form long term immunological memory after an initial infection, and to subsequently mount a rapid recall response upon reinfection. Although some murine studies showed a lack of effective immunity of T cells to rechallenge (38, 39, 40, 41), adoptive transfer of mouse T cell lines to naive recipients has been demonstrated to successfully restrict a malaria infection (42, 43). Furthermore, in a nonhuman primate model, T cells showed a memory type response with rapid T cell expansion after a rechallenge, leading to clearance of detectable bacteremia (44). Recently, long term expansion of T cells in CMV-seropositive individuals followed by quicker responses to rechallenge in graft recipients by T cells with an effector/memory phenotype was observed (45). In the present study, mice that had initially undergone WSN-OVA infection showed a strong increase in the number of βTCR transduced T cells after rechallenge with rVV-OVA. The magnitude and kinetics of the secondary T cell response of the βTCR-transduced T cells were comparable to those observed in the primary response, indicating that βTCR transduced T cells are able to persist long-term in vivo and can mount recall responses.

For successful clinical application of βTCR engineered T cells some criteria have to be met. Sufficient numbers of T cells have to be available by easy isolation methods, isolated T cells must have the capacity not only to survive but also to proliferate in vitro and it must be possible to induce sufficient activation of the isolated T cells to enable retroviral transduction of the βTCR and the relevant coreceptor. Human T cells can be easily isolated from peripheral blood because percentages of T cells up to 10% of PBMC can be found (46, 47). It has been shown possible to expand isolated T cells in vitro without deterioration of their effector functions (48). Recently, two studies demonstrated the feasibility of clinical application of adoptive immunotherapy of unmodified T cells (49, 50). Up to 8 x 10⁹ infused in vitro cultured T cells were well tolerated by patients and in vivo antitumor reactivity was observed. The feasibility of βTCR and coreceptor transfer to T cells was demonstrated in our previous study in which βTCR engineered T cells were found to proliferate vigorously in vitro, to produce cytokines in an Ag specific manner and to exert Ag specific cytotoxicity against leukemic cells (24). These data together with the data from the present study provide evidence that T cells equipped with an βTCR can function specifically both in vitro and in vivo and that these effector cells may offer a safe alternative for the use of β T cells in immunotherapy (19).

In summary, we show in this study that βTCR transduced T cells proliferate in vivo and produce cytokines in an Ag-dependent fashion. In addition, these gene-modified T cells persisted in vivo and were capable of mounting recall responses. The combination of functional reactivity and lack of mixed dimer formation makes βTCR modified T cells an attractive cell population for future clinical studies.

	Acknowledgments

 
We thank Reinier van der Linden, Menno A. W. G. van der Hoorn, and Maarten van de Keur for expert technical assistance.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ L.V. was supported by grant 2001-2490 from the Dutch Cancer Society. M.C. was supported by the European Union 6th Framework program (ATTACK).

Lars T. van der Veken designed research, performed research, analyzed data, and wrote the paper; Miriam Coccoris designed research, performed research, analyzed data, and wrote the paper; Erwin Swart performed research; J. H. Frederik Falkenburg designed research and wrote the paper; Ton N. Schumacher designed research and wrote the paper; and Mirjam H. M. Heemskerk designed research, analyzed data, and wrote the paper.

² L.V. and M.C. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Mirjam H. M. Heemskerk, Laboratory of Experimental Hematology, Department of Hematology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. E-mail address: MHMHeemskerk{at}LUMC.nl

⁴ Abbreviations used in this paper: IEL, intra epithelial lymphocyte; LP, lamina propria.

Received for publication August 26, 2008. Accepted for publication November 2, 2008.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Kolb, H. J., J. Mittermuller, C. Clemm, E. Holler, G. Ledderose, G. Brehm, M. Heim, W. Wilmanns. 1990. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 76: 2462-2465. [Abstract/Free Full Text]
2. Porter, D. L., M. S. Roth, C. McGarigle, J. L. Ferrara, J. H. Antin. 1994. Induction of graft-versus-host disease as immunotherapy for relapsed chronic myeloid leukemia. N. Engl. J. Med. 330: 100-106. [Abstract/Free Full Text]
3. Collins, R. H., Jr, O. Shpilberg, W. R. Drobyski, D. L. Porter, S. Giralt, R. Champlin, S. A. Goodman, S. N. Wolff, W. Hu, C. Verfaillie, et al 1997. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J. Clin. Oncol. 15: 433-444. [Abstract/Free Full Text]
4. Kolb, H. J., A. Schattenberg, J. M. Goldman, B. Hertenstein, N. Jacobsen, W. Arcese, P. Ljungman, A. Ferrant, L. Verdonck, D. Niederwieser, et al 1995. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood 86: 2041-2050. [Abstract/Free Full Text]
5. Levine, J. E., T. Braun, S. L. Penza, P. Beatty, K. Cornetta, R. Martino, W. R. Drobyski, A. J. Barrett, D. L. Porter, S. Giralt, et al 2002. Prospective trial of chemotherapy and donor leukocyte infusions for relapse of advanced myeloid malignancies after allogeneic stem-cell transplantation. J. Clin. Oncol. 20: 405-412. [Abstract/Free Full Text]
6. Dudley, M. E., J. R. Wunderlich, P. F. Robbins, J. C. Yang, P. Hwu, D. J. Schwartzentruber, S. L. Topalian, R. Sherry, N. P. Restifo, A. M. Hubicki, et al 2002. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 298: 850-854. [Abstract/Free Full Text]
7. Dudley, M. E., 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, et al 2005. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J. Clin. Oncol. 23: 2346-2357. [Abstract/Free Full Text]
8. Clay, T. M., M. C. Custer, J. Sachs, P. Hwu, S. A. Rosenberg, M. I. Nishimura. 1999. Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity. J. Immunol. 163: 507-513. [Abstract/Free Full Text]
9. Cooper, L. J., M. Kalos, D. A. Lewinsohn, S. R. Riddell, P. D. Greenberg. 2000. Transfer of specificity for human immunodeficiency virus type 1 into primary human T lymphocytes by introduction of T-cell receptor genes. J. Virol. 74: 8207-8212. [Abstract/Free Full Text]
10. Stanislawski, T., R. H. Voss, C. Lotz, E. Sadovnikova, R. A. Willemsen, J. Kuball, T. Ruppert, R. L. Bolhuis, C. J. Melief, C. Huber, et al 2001. Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer. Nat. Immunol. 2: 962-970. [Medline]
11. Heemskerk, M. H., R. A. de Paus, E. G. Lurvink, F. Koning, A. Mulder, R. Willemze, J. J. van Rood, J. H. Falkenburg. 2001. Dual HLA class I and class II restricted recognition of alloreactive T lymphocytes mediated by a single T cell receptor complex. Proc. Natl. Acad. Sci. USA 98: 6806-6811. [Abstract/Free Full Text]
12. Heemskerk, M. H., M. Hoogeboom, R. Hagedoorn, M. G. Kester, R. Willemze, J. H. Falkenburg. 2004. Reprogramming of virus-specific T cells into leukemia-reactive T cells using T cell receptor gene transfer. J. Exp. Med. 199: 885-894. [Abstract/Free Full Text]
13. Schumacher, T. N.. 2002. T-cell-receptor gene therapy. Nat. Rev. Immunol. 2: 512-519. [Medline]
14. Kessels, H. W., M. C. Wolkers, d. B. van, M. A. van der Valk, T. N. Schumacher. 2001. Immunotherapy through TCR gene transfer. Nat. Immunol. 2: 957-961. [Medline]
15. Morris, E. C., A. Tsallios, G. M. Bendle, S. A. Xue, H. J. Stauss. 2005. A critical role of T cell antigen receptor-transduced MHC class I-restricted helper T cells in tumor protection. Proc. Natl. Acad. Sci. USA 102: 7934-7939. [Abstract/Free Full Text]
16. Kessels, H. W., K. Schepers, M. D. van den Boom, D. J. Topham, T. N. Schumacher. 2006. Generation of T cell help through a MHC class I-restricted TCR. J. Immunol. 177: 976-982. [Abstract/Free Full Text]
17. de Witte, M. A., M. Coccoris, M. C. Wolkers, M. D. van den Boom, E. M. Mesman, J.-Y. Song, M. van der Valk, J. B. A. G. Haanen, T. N. M. Schumacher. 2006. Targeting self-antigens through allogeneic TCR gene transfer. Blood 108: 870-877. [Abstract/Free Full Text]
18. de Witte, M. A., G. M. Bendle, M. D. van den Boom, M. Coccoris, T. D. Schell, S. S. Tevethia, H. van Tinteren, E. M. Mesman, J. Y. Song, T. N. Schumacher. 2008. TCR gene therapy of spontaneous prostate carcinoma requires in vivo T cell activation. J. Immunol. 181: 2563-2571. [Abstract/Free Full Text]
19. Morgan, R. A., M. E. Dudley, J. R. Wunderlich, M. S. Hughes, J. C. Yang, R. M. Sherry, R. E. Royal, S. L. Topalian, U. S. Kammula, N. P. Restifo, et al 2006. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314: 126-129. [Abstract/Free Full Text]
20. Heemskerk, M. H., R. S. Hagedoorn, M. A. van der Hoorn, L. T. van der Veken, M. Hoogeboom, M. G. Kester, R. Willemze, J. H. Falkenburg. 2007. 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 109: 235-243. [Abstract/Free Full Text]
21. Kessels, H. W., M. C. Wolkers, T. N. Schumacher. 2002. Adoptive transfer of T-cell immunity. Trends Immunol. 23: 264-269. [Medline]
22. Saito, T., F. Hochstenbach, S. Marusic-Galesic, A. M. Kruisbeek, M. Brenner, R. N. Germain. 1988. Surface expression of only and/or β T cell receptor heterodimers by cells with four (, β, , ) functional receptor chains. J. Exp. Med. 168: 1003-1020. [Abstract/Free Full Text]
23. Koning, F., W. L. Maloy, D. Cohen, J. E. Coligan. 1987. Independent association of T cell receptor β and -chains with CD3 in the same cell. J. Exp. Med. 166: 595-600. [Abstract/Free Full Text]
24. van der Veken, L. T., R. S. Hagedoorn, M. M. van Loenen, R. Willemze, J. H. Falkenburg, M. H. Heemskerk. 2006. β T-cell receptor engineered T cells mediate effective antileukemic reactivity. Cancer Res. 66: 3331-3337. [Abstract/Free Full Text]
25. Groh, V., S. Porcelli, M. Fabbi, L. L. Lanier, L. J. Picker, T. Anderson, R. A. Warnke, A. K. Bhan, J. L. Strominger, M. B. Brenner. 1989. Human lymphocytes bearing T cell receptor are phenotypically diverse and evenly distributed throughout the lymphoid system. J. Exp. Med. 169: 1277-1294. [Abstract/Free Full Text]
26. Janeway, C. A., Jr. 1992. The T cell receptor as a multicomponent signalling machine: CD4/CD8 coreceptors and CD45 in T cell activation. Annu. Rev. Immunol. 10: 645-674. [Medline]
27. Stingl, G., F. Koning, H. Yamada, W. M. Yokoyama, E. Tschachler, J. A. Bluestone, G. Steiner, L. E. Samelson, A. M. Lew, J. E. Coligan, et al 1987. Thy-1⁺ dendritic epidermal cells express T3 antigen and the T-cell receptor -chain. Proc. Natl. Acad. Sci. USA 84: 4586-4590. [Abstract/Free Full Text]
28. Asarnow, D. M., W. A. Kuziel, M. Bonyhadi, R. E. Tigelaar, P. W. Tucker, J. P. Allison. 1988. Limited diversity of antigen receptor genes of Thy-1⁺ dendritic epidermal cells. Cell 55: 837-847. [Medline]
29. Goodman, T., L. Lefrancois. 1988. Expression of the T-cell receptor on intestinal CD8⁺ intraepithelial lymphocytes. Nature 333: 855-858. [Medline]
30. Kyes, S., E. Carew, S. R. Carding, C. A. Janeway, Jr, A. Hayday. 1989. Diversity in T-cell receptor gene usage in intestinal epithelium. Proc. Natl. Acad. Sci. USA 86: 5527-5531. [Abstract/Free Full Text]
31. Itohara, S., A. G. Farr, J. J. Lafaille, M. Bonneville, Y. Takagaki, W. Haas, S. Tonegawa. 1990. Homing of a thymocyte subset with homogeneous T-cell receptors to mucosal epithelia. Nature 343: 754-757. [Medline]
32. Kurts, C., J. F. Miller, R. M. Subramaniam, F. R. Carbone, W. R. Heath. 1998. Major histocompatibility complex class I-restricted cross-presentation is biased towards high dose antigens and those released during cellular destruction. J. Exp. Med. 188: 409-414. [Abstract/Free Full Text]
33. Naviaux, R. K., E. Costanzi, M. Haas, I. M. Verma. 1996. The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J. Virol. 70: 5701-5705. [Abstract/Free Full Text]
34. Topham, D. J., M. R. Castrucci, F. S. Wingo, G. T. Belz, P. C. Doherty. 2001. The role of antigen in the localization of naive, acutely activated, and memory CD8⁺ T cells to the lung during influenza pneumonia. J. Immunol. 167: 6983-6990. [Abstract/Free Full Text]
35. Steinaa, L., P. B. Rasmussen, A. Gautam, S. Mouritsen. 2008. Breaking B-cell tolerance and CTL tolerance in three OVA-transgenic mouse strains expressing different levels of OVA. Scand. J. Immunol. 67: 113-120. [Medline]
36. Steinaa, L., P. B. Rasmussen, J. Rygaard, S. Mouritsen, A. Gautam. 2007. Generation of autoreactive CTL by tumour vaccines containing foreign T helper epitopes. Scand. J. Immunol. 65: 240-248. [Medline]
37. Coccoris, M., E. Swart, M. A. de Witte, J. W. van Heijst, J. B. Haanen, K. Schepers, T. N. Schumacher. 2008. Long-term functionality of TCR-transduced T cells in vivo. J. Immunol. 180: 6536-6543. [Abstract/Free Full Text]
38. Skeen, M. J., H. K. Ziegler. 1993. Induction of murine peritoneal T cells and their role in resistance to bacterial infection. J. Exp. Med. 178: 971-984. [Abstract/Free Full Text]
39. Welsh, R. M., M. Y. Lin, B. L. Lohman, S. M. Varga, C. C. Zarozinski, L. K. Selin. 1997. β and T-cell networks and their roles in natural resistance to viral infections. Immunol. Rev. 159: 79-93. [Medline]
40. Jones-Carson, J., A. Vazquez-Torres, H. C. van der Heyde, T. Warner, R. D. Wagner, E. Balish. 1995. T cell-induced nitric oxide production enhances resistance to mucosal candidiasis. Nat. Med. 1: 552-557. [Medline]
41. Roberts, S. J., A. L. Smith, A. B. West, L. Wen, R. C. Findly, M. J. Owen, A. C. Hayday. 1996. T-cell β⁺ and ⁺ deficient mice display abnormal but distinct phenotypes toward a natural, widespread infection of the intestinal epithelium. Proc. Natl. Acad. Sci. USA 93: 11774-11779. [Abstract/Free Full Text]
42. Tsuji, M., P. Mombaerts, L. Lefrancois, R. S. Nussenzweig, F. Zavala, S. Tonegawa. 1994. T cells contribute to immunity against the liver stages of malaria in β T-cell-deficient mice. Proc. Natl. Acad. Sci. USA 91: 345-349. [Abstract/Free Full Text]
43. Tsuji, M., C. L. Eyster, R. L. O'Brien, W. K. Born, M. Bapna, M. Reichel, R. S. Nussenzweig, F. Zavala. 1996. Phenotypic and functional properties of murine T cell clones derived from malaria immunized, β T cell-deficient mice. Int. Immunol. 8: 359-366. [Abstract/Free Full Text]
44. Shen, Y., D. Zhou, L. Qiu, X. Lai, M. Simon, L. Shen, Z. Kou, Q. Wang, L. Jiang, J. Estep, et al 2002. Adaptive immune response of V2V2⁺ T cells during mycobacterial infections. Science 295: 2255-2258. [Abstract/Free Full Text]
45. Pitard, V., D. Roumanes, X. Lafarge, L. Couzi, I. Garrigue, M. E. Lafon, P. Merville, J. F. Moreau, J. Dechanet-Merville. 2008. Long-term expansion of effector/memory V2- T cells is a specific blood signature of CMV infection. Blood 112: 1317-1324. [Abstract/Free Full Text]
46. Hayday, A. C.. 2000. cells: a right time and a right place for a conserved third way of protection. Annu. Rev. Immunol. 18: 975-1026. [Medline]
47. Porcelli, S., M. B. Brenner, H. Band. 1991. Biology of the human T-cell receptor. Immunol. Rev. 120: 137-183. [Medline]
48. Burjanadze, M., M. Condomines, T. Reme, P. Quittet, P. Latry, C. Lugagne, F. Romagne, Y. Morel, J. F. Rossi, B. Klein, Z. Y. Lu. 2007. In vitro expansion of T cells with anti-myeloma cell activity by Phosphostim and IL-2 in patients with multiple myeloma. Br. J. Haematol. 139: 206-216. [Medline]
49. Kobayashi, H., Y. Tanaka, J. Yagi, Y. Osaka, H. Nakazawa, T. Uchiyama, N. Minato, H. Toma. 2007. Safety profile and anti-tumor effects of adoptive immunotherapy using T cells against advanced renal cell carcinoma: a pilot study. Cancer Immunol. Immunother. 56: 469-476. [Medline]
50. Bennouna, J., E. Bompas, E. M. Neidhardt, F. Rolland, I. Philip, C. Galea, S. Salot, S. Saiagh, M. Audrain, M. Rimbert, et al 2008. Phase-I study of Inna cell , an autologous cell-therapy product highly enriched in 92 T lymphocytes, in combination with IL-2, in patients with metastatic renal cell carcinoma. Cancer Immunol. Immunother. 57: 1599-1609. [Medline]

This article has been cited by other articles:

				M. H.M. Heemskerk T-cell receptor gene transfer for the treatment of leukemia and other tumors Haematologica, January 1, 2010; 95(1): 15 - 19. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van der Veken, L. T. Articles by Heemskerk, M. H. M. Search for Related Content PubMed PubMed Citation Articles by van der Veken, L. T. Articles by Heemskerk, M. H. M.