Jurkat/E6-1 (ATCC TIB-152) cells were cultured in RPMI-1640 medium (Gibco/Invitrogen, Carlsbad, CA, USA), human embryonic kidney cells (HEK293) and human hepatocellular carcinoma cells (BEL-7402; maintained in our laboratory) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco/Invitrogen). All cultures were supplemented with 10% fetal bovine serum (FBS; Gibco/Invitrogen), 100 U/ml streptomycin and 100 U/ml penicillin. The TCRα12 and β7 chains were isolated from TILs of patients (HLA-A²⁺ and AFP⁺) with hepatocellular carcinoma by our laboratory as previously described (19). CD3ζ chains were isolated from peripheral blood mononuclear cells (PBMCs) of a healthy donor (GenBank accession no. NM_000734.3). The pair of fluorescent proteins [enhanced cyan fluorescent protein (ECFP)/enhanced yellow fluorescent protein (EYFP)] was kindly provided by Dr G. Zhang (Guangzhou University of Chinese Medicine, Guangzhou, China). Monoclonal antibodies (mAbs) used for flow cytometry included FITC-conjugated anti-TCRVα12.1 mAb (Pierce Biotechnology, Rockford, IL, USA) and PE-conjugated anti-TCRVβ7.1 (Beckman Coulter, Brea, CA, USA).

The strategy for the tripartite DNA fragment fusion by SOE PCR is shown in Fig. 2A. Variable fragments for generating TCRαζ-ECFP (including TCRα, CD3ζ-a and EYFP) and TCRβζ-EYFP (including TCRβ, CD3ζ-b and ECFP) were amplified using a set of forward primers and reverse primers. The first step of SOE PCR reactions was performed with 100 ng (3 fragments were mixed with an equimolar ratio) of template without primers, 10X Buffer, 2 mM dNTPs, 25 mM MgSO₄, 0.5 U KOD-Plus-Neo Polymerase in a 25 μl reaction volume. The PCR cycling conditions were as follows: initial denaturation at 94°C for 2 min, followed by 5 cycles at 94°C for 30 sec, at 57°C for 30 sec and at 68°C for 1.5 min and completed with a final extension at 68°C for 7 min. This initial PCRs generate overlapping gene segments that are then used as template DNA for the second step of SOE PCR to create a full-length product. Therefore, another 25 μl reaction mixture (containing 10X Buffer, 2 mM dNTPs, 25 mM MgSO₄, 0.5 KOD-Plus-Neo Polymerase and 1 μM forward primer P1/P1′ and reverse primer P6/P6′) was added to the first reaction mixture for the second step of SOE PCR. The reaction conditions were initial denaturation at 94°C for 2 min, followed by 30 cycles at 94°C for 30 sec, at 62°C for 30 sec, and at 68°C for 1.5 min and a final extension at 68°C for 7 min, 2 μl of the amplicons were analyzed by agarose gel electrophoresis (1.0%) (Fig. 2B and C). Primer sequences for the amplification of variable regions and fusion chains are presented in Table I. The wtTCRα-ECFP and wtTCRβ-EYFP fusing sequences were generated using the same methods.

To construct a bicistronic vector expressing both the TCRαζ-ECFP and TCRβζ-EYFP chains, the fusion sequences TCRαζ-ECFP and TCRβζ-EYFP were cloned into the original pIRES2-EGFP vector (Clontech Laboratories, Inc., Palo Alto, CA, USA). The TCRαζ-ECFP fusion gene was inserted into the BstXI/NotI restriction site of the pIRES2-EGFP vector (Clontech Laboratories), located downstream of an internal ribosomal entry site (IRES) sequence of the plasmid to replace the EGFP gene, then the TRBζ-EYFP fusion gene was inserted into the NheI/SalI restriction site, located upstream of IRES. The other bicistronic vector expressing both non-modified wtTCRα-ECFP and wtTCRβ-EYFP was constructed in a manner similar to the above-mentioned procedure. The single vectors expressing TCRα-ECFP or TCRβ-EYFP which were used to remove the spectral bleed-through (SBT) contamination in the FRET images were constructed as previously described (20). The identity of all constructs was verified by direct sequencing.

We used the Ad5F35 chimeric adenoviral vector (21) which contained the Ad35 fiber knob incorporated into an Ad5 capsid as the packaging vector, and the whole wtTCRαβ cassette (TCRβ-IRES-TCRα) and TCRαζβζ cassette (TCRβζ-IRES-TCRαζ) was cloned into the shuttle plasmid (pDC315) (Fig. 4A). Adenoviral particles were produced by co-transfection of the packaging cells, HEK293, with Ad5F35 vector and recombinant pDC315, containing wtTCRαβ or TCRαζβζ. The virus-containing supernatants were filtered through 0.45-μm filters. Adenoviral particle titers were determind by the 50% tissue culture infectious dose (TCID50) method and the supernatants were directly used for the infection of the target Jurkat T cells. For Jurkat T cell transduction, 6-well plates were seeded with Jurkat cells at 1×10⁶ cells/well in 2 ml of RPMI 1640 medium supplemented with 2% FBS, cultured for 35 min, and then transduced with MOI 100 PFU of wtTCRαβ or TCRαζβζ viral supernatants. Following culture for 24 h at 37°C with 5% CO₂, the culture medium was replaced with RPMI 1640 medium supplemented with 10% FBS. After a further 48-h culture period, the cells were analyzed for TCR expression by flow cytometry.

The TCR-transduced Jurkat T cells (5×10⁵) were analyzed for transgene expression by flow cytometry using FITC-conjugated anti-TCRVα12 mAb and PE-conjugated anti-TCRVβ7 mAbs. The cells were incubated with the mAbs on ice for 30 min. Subsequently, the Jurkat cells were washed twice with PBS and centrifuged for 10 min at 1000 × g, and the cell pellet was resuspended and fixed with 2% paraformaldehyde before taking measurements on an Epics XL flow cytometer (Beckman Coulter). The samples were analyzed using EXPO32 software (Beckman Coulter) and are displayed as dotplots.

The recombinant vectors, pIRES-TCRβ-EYFP/TCRα-ECFP and pIRES-TCRβζ-EYFP/TCRαζ-ECFP, were transfected into the Jurkat and BEL-7402 cells using Lipofectamine LTX/PLUS (Invitrogen) according to the manufacturer’s instructions. The cells transfected with pIRES-TCRα-ECFP and pIRES-TCRβ-EYFP were used as controls to remove the SBT contamination in FRET analysis. After 24 h of transfection, the Jurkat cells grown on glass-bottom dishes were washed twice with PBS and immobilized with 0.05% low metling agarose for 15 min and the BEL-7402 cells were washed twice with PBS solution. Confocal images of the cells were acquired using an Olympus FluoView 1000 confocal laser scanning microscope with FV10-ASW 1.7 software (Olympus, Tokyo, Japan). The FRET donor (TCRα-ECFP or TCRαζ-ECFP) was excited with a 458 nm Ar-laser and the acceptor (TCRβ-EYFP or TCRβζ-EYFP) was excited with a 515 nm Ar-laser. The cells were scanned from 475 to 585 nm with a 10 nm step-size and 20 nm band-width to obtain original images.

FRET efficiency was analyzed using the sensitized acceptor emission (SE) method. FRET occurs when 2 fluorophores (donor and acceptor) have sufficiently large spectral overlap which results in the energy of the donor fluorescence transferring to the acceptor. The spectral overlap between donor and acceptor fluorophores also causes FRET signal contamination, termed SBT. It is important to remove SBT in FRET efficiency measurements. The pFRET images were corrected as shown in the equation and as previously described (22): pFRET = uFRET − DSBT − ASBT, where uFRET is uncorrected FRET, ASBT is the acceptor spectral bleed-through signal, and DSBT is the donor spectral bleed-through signal. The final FRET efficiency equation was calculated as E = 1 − I_DA/{I_DA + pFRET × [(ψ_dd/ψ_aa) × (Q_d/Q_a, where I_DA is the intensity of the donor in the presence of the acceptor, ψ_dd and ψ_aa are the collection efficiency in the donor and acceptor channel, and Q_d and Q_a are the quantum yield of the donor and acceptor, respectively (22,23).

Mean FRET efficiency was calculated from multiple (n=6) cell images in each group and 6 random regions of interest (ROI) in each cell image. Statistical analysis was performed using two-way ANOVA with a Bonferroni’s multiple comparisons test using Graphpad Prism 6 software (GraphPad Software Inc., San Diego, CA, USA). Differences with P-values <0.05 were considered statistically significant. Data are expressed as the means ± SD.

We designed a modified TCR in which the extracellular and transmembrane domain of TCRα or β chains were exchanged for CD3ζ chains at a structurally favorable position. To better observe the expression of the modified TCRαζβζ in living cells, we fused the C terminus of TCRαζ and TCRβζ chains to a pair of cyan and yellow fluorescent proteins, ECFP and EYFP, respectively (Fig. 1). The fusion genes, TCRαζ-ECFP and TCRβζ-EYFP, were obtained by SOE PCR (Fig. 2B and C). To compare the relative levels of surface expression with wtTCRαβ, we also fused the C terminus of the wtTCRα and wtTCRβ chains to a pair of ECFP and EYFP fluorescent proteins (Fig. 1). The fusion genes were then cloned into the pIRES2-EGFP vector.

We first examined whether the TCRαζ-ECFP and TCRβζ-EYFP fusion chains are expressed on the surface of the cells. We transfected the recombinant plasmids into Jurkat cells, as well as BEL-7402 cells, and 24 h after transfection, the fluorescence observation by CLSM revealed that the introduced fusion genes, TCRαζ-ECFP and TCRβζ-EYFP, were located at the plasma membrane of the cells and were effectively expressed in T cells and non-T cells (Fig. 3).

We observed a very interesting phenomenon, namely that in the TCRαζβζ-transduced cells, but not in the TCRαβ-transduced cells, microclusters were clearly evident at the plasma membrane and the cilia of the plasma membrane (Fig. 3). This observation indicated that TCRαζβζ expression was stronger in the Jurkat cells and BEL-7402 cells compared to the expression of wtTCRαβ. To confirm this result, we inserted the wtTCRαβ and TCRαζβζ genes separately into the shuttle plasmid, pDC315, to produce Ad5F35 adenovirus (Fig. 4A). Ad5F35 adenovirus which contained the Ad35 fiber knob incorporated into an Ad5 capsid was able to effectively transduce human T cells. The wtTCRαβ and TCRαζβζ adenoviral vectors were introduced into the Jurkat T cells followed by FACS analysis. Double immunofluorescent staining with anti-TCRVα12 mAb^FITC and anti-Vβ7 mAb^PE showed that the Jurkat cells transduced with wtTCRαβ displayed little surface co-expression (up to 10.7%). By contrast, there was a clear increased surface coexpression (up to 21.2%) in the TCRαζβζ-transduced Jurkat cells (Fig. 4B). These data are in accordance with the imaging evidence showing the enhanced surface expression of TCRαζβζ in the cells. These results demonstrated that the FRET reporter, TCRαζ-ECFP/TCRβζ-EYFP, can be effectively used to express TCRαζβζ may thus be used for monitoring the expression and interaction of TCRαζβζ.

To investigate the interaction of TCRαζ and βζ in T cells, we used Jurkat cells (clone E6-1) expressing endogenous TCR as a recipient TCR cell model. We hypothesized that the introduced wtTCRα and β chains or TCRαζ and βζ chains composing heterodimers would result in FRET efficiency between the donor (ECFP) and acceptor (EYFP) fluorescent proteins. Once the introduced TCRαζ and βζ chains paired with endogenous TCRβ and α chains, it would fail to detect FRET between the mispaired TCR (Fig. 5C). We observed that the TCRαζβζ displayed a higher FRET signal compared to wtTCRαβ in Jurkat cells. (Fig. 5A). Six independent cell images and 6 ROI in each cell image were selected for FRET efficiency analysis in each group. The results from statistical analysis showed that the average FRET efficiency between TCRαζ-ECFP and TCRβζ-EYFP (16.7±2.5%) was significantly increased compared to wtTCRα-ECFP and wtTCRβ-EYFP (7.9±1.3%) in the Jurkat cells (P<0.0001) (Fig. 5D). These data suggested that the fusion of wtTCRα12 and β7 with CD3ζ improved pairing.

To better characterize the highly preferred pairing of TCRαζ and βζ, we selected BEL-7402 cells which are deficient in TCR and CD3 molecules as the next recipient cell model to investigate the interaction of TCRαζβζ and endogenous CD3 subunits. We hypothesized that since there was no endogenous TCR in BEL-7402 cells, the pairing of introduced wtTCRα and β or TCRαζ and βζ would not be interfered with and would result in the same FRET signal. Unexpectedly, we observed that TCRαζβζ still displayed a higher FRET signal compared to wtTCRαβ in BEL-7402 cells (Fig. 5B). The average FRET efficiency between wtTCRα and β chains (10.5±1.0%) was significantly reduced compared to the TCRαζ and βζ chains (17.1±2.1%) in BEL-7402 cells (P<0.0001) (Fig. 5D). The results indicated that the assemble of wtTCRα and β chains was impaired in the absence of CD3 subunits, in spite of no endogenous TCR in the BEL-7402 cells. Of note, we found that the average FRET efficiency between TCRαζ-ECFP and TCRβζ-EYFP in the BEL-7402 cells (17.1±2.1%) was not reduced compared to the Jurkat cells (16.7±2.5%) (P>0.05) (Fig. 5D). These results demonstrated that the expression and assembly of TCRαζβζ was not compromised even without the CD3γɛ, Δɛ and ζζ signaling dimers in the BEL-7402 cells.