Antileukemia multifunctionality of CD4(+) T cells genetically engineered by HLA class I-restricted and WT1-specific T-cell receptor gene transfer.

To develop gene-modified T-cell-based antileukemia adoptive immunotherapy, concomitant administration of CD4(+) and CD8(+) T cells that have been gene modified using identical HLA class I-restricted leukemia antigen-specific T-cell receptor (TCR) gene transfer has not yet been fully investigated. Here, using CD4(+) and CD8(+) T cells that had been gene modified with a retroviral vector expressing HLA-A*24:02-restricted and Wilms' tumor 1 (WT1)-specific TCR-α/ß genes and siRNAs for endogenous TCRs (WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells), we examined the utility of this strategy. WT1-siTCR/CD4(+) T cells sufficiently recognized leukemia cells in an HLA class I-restricted manner and provided target-specific Th1 help for WT1-siTCR/CD8(+) T cells. By using a xenografted mouse model, we found that WT1-siTCR/CD4(+) T cells migrated to leukemia sites and subsequently attracted WT1-siTCR/CD8(+) T cells via chemotaxis. Therapy-oriented experiments revealed effective enhancement of leukemia suppression mediated by concomitant administration of WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells. Importantly, this augmented efficacy in the presence of WT1-siTCR/CD4(+) T cells was correlated with longer survival and enhanced formation of memory T cells by WT1-siTCR/CD8(+) T cells. Collectively, our experimental findings strongly suggest that this strategy would be clinically advantageous for the treatment of human leukemia.

HLA-restricted presentation of WT1 tumor antigen in B-lymphoblastoid cell lines established using a maxi-EBV system.

Lymphoblastoid cell lines (LCLs), which are established by in vitro infection of peripheral B-lymphocytes with Epstein-Barr virus (EBV), are effective antigen-presenting cells. However, the ability of LCLs to present transduced tumor antigens has not yet been evaluated in detail. We report a single-step strategy utilizing a recombinant EBV (maxi-EBV) to convert B-lymphocytes from any individuals into indefinitely growing LCLs expressing a transgene of interest. The strategy was successfully used to establish LCLs expressing Wilms' tumor gene 1 (WT1) tumor antigen (WT1-LCLs), which is an attractive target for cancer immunotherapy. The established WT1-LCLs expressed more abundant WT1 protein than K562 leukemic cells, which are known to overexpress WT1. A WT1-specific cytotoxic T lymphocyte line efficiently lysed the WT1-LCL in a human leukocyte antigen-restricted manner, but poorly lysed control LCL not expressing WT1. These results indicate that the transduced WT1 antigen is processed and presented on the WT1-LCL. This experimental strategy can be applied to establish LCLs expressing other tumor antigens and will find a broad range of applications in the field of cancer immunotherapy.

siRNA-mediated silencing of PD-1 ligands enhances tumor-specific human T-cell effector functions.

Adoptive cell therapy using tumor-specific T cells is a promising strategy for treating patients with malignancy. However, accumulating evidences have demonstrated that optimal function of tumor-reactive T cells is often attenuated by negative regulatory signal(s) delivered through receptors, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death 1 (PD-1), and their cognate ligands. Although systemic blocking of these molecules needs careful attention on the risk of uncontrolled immune activation, selective inhibition of negative signals in tumor-specific T cells by their genetic modification is an attractive approach to overcome immunological suppression in cancer patients. Here, we demonstrate the improved effector functions of tumor-specific CD4(+) and CD8(+) human T cells by small interfering RNA (siRNA) -mediated silencing of PD-1 ligands, PD-L1 or PD-L2. Tumor antigen MAGE-A4-specific human T-cell clones upregulated the expression of PD-1 ligands upon activation. siRNA-mediated knockdown of PD-L1 or -L2 enhanced the interferon-γ production and antigen-specific cytotoxicity of these cells. Peripheral blood mononuclear cells transduced with a retroviral vector encoding MAGE-A4-specific T-cell receptor α/ß chains also increased their effector functions by this modification. These results suggest that siRNA-mediated knockdown of PD-1 ligands is an attractive strategy to inhibit a negative regulatory mechanism of tumor-specific T cells resulting in enhanced efficacy of adoptive T-cell therapy of cancer using genetically modified autologous lymphocytes.

Rapid alphabeta TCR-mediated responses in gammadelta T cells transduced with cancer-specific TCR genes.

Adoptive T-cell transfer of in vitro cultured T cells derived from cancer patients with naturally developed immune responses has met with some success as an immunotherapeutic approach, although only a limited number of patients showed spontaneous immune responses. To find alternative ways, such as cancer-specific T-cell receptor (TCR) gene transfer, in preparation for sufficient numbers of antigen-specific T cells is an important issue in the field of adoptive T-cell therapy. Given the inherent disadvantage of alphabeta TCR transfer to other alphabeta T cells, namely the possible formation of mixed TCR heterodimers with endogenous alpha or beta TCR, we employed gammadelta T cells as a target for retroviral transfer of cancer-specific TCR and examined whether gammadelta T cells were useful as an alternative population for TCR transfer. Although retroviral transduction to gammadelta T cells with TCR alphabeta genes alone, isolated from a MAGE-A4(143-151)-specific alphabeta CD8(+) cytotoxic T lymphocyte (CTL) clone, did not provide sufficient affinity to recognize major histocompatibility (MHC)-peptide complexes due to the lack of CD8 co-receptor, gammadelta T cells co-transduced with TCR alphabeta and CD8 alphabeta genes acquired cytotoxicity against tumor cells and produced cytokines in both alphabeta- and gammadelta-TCR-dependent manners. Furthermore, alphabeta TCR and CD8-transduced gammadelta T cells, stimulated either through alphabeta TCR or gammadelta TCR, rapidly responded to target cells compared with conventional alphabeta T cells, reminiscent of gammadelta T cells. We propose alphabeta TCR-transduced gammadelta T cells as an alternative strategy for adoptive T-cell transfer.

In vivo persistence of genetically modified T cells generated ex vivo using the fibronectin CH296 stimulation method.

Recombinant human fibronectin fragment (FN-CH296, RetroNectin) has been widely used for retroviral gene therapy to enhance gene transfer efficiency. Based on the observation that immobilized FN-CH296 together with anti-CD3 monoclonal antibodies (anti-CD3) enhanced cell proliferation while conserving the naive phenotype of T cells, we used FN-CH296 costimulation to generate engineered T cells. For comparison, human peripheral blood mononuclear cells were stimulated under three kinds of conditions including anti-CD3 only, anti-CD3 and anti-CD28 monoclonal antibodies conjugated with beads (anti-CD3/anti-CD28) and immobilized FN-CH296 together with anti-CD3 (anti-CD3/FN-CH296); all three treatments were followed by retroviral gene transfer. Of all the stimulation methods, the one involving anti-CD3/FN-CH296 produced the most cell expansion with conservation of the naive phenotype. Engineered T cells were transplanted into NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice, and all the mice were killed 14 days later. Transplanted T cells were detected in all the mice; however, mice injected with anti-CD3/FN-CH296-stimulated T cells showed higher transgene expression in organs than mice injected with anti-CD3-stimulated cells. These results demonstrate that the anti-CD3/FN-CH296 stimulation can be an efficient way to generate large numbers of genetically modified T cells that can provide higher and longer lasting levels of transgene expression in vivo and that are suitable for adoptive T-cell transfer therapy.

Long-term phenotypic, functional and genetic stability of cancer-specific T-cell receptor (TCR) alphabeta genes transduced to CD8+ T cells.

In adoptive T-cell transfer as an intervention for malignant diseases, retroviral transfer of T-cell receptor (TCR) genes derived from CD8(+) cytotoxic T-lymphocyte (CTL) clones provides an opportunity to generate a large number of T cells with the same antigen specificity. We cloned the TCR-alphabeta genes from a human leukocyte antigen (HLA)-A(*)2402-restricted CTL clone specific for MAGE-A4(143-151). The TCR-alphabeta genes were transduced to 99.2% of non-TCR expressing SupT1, a human T-cell line, and to 12.7-32.6% of polyclonally activated CD8(+) T cells by retroviral transduction. As expected, TCR-alphabeta gene-modified CD8(+) T cells showed cytotoxic activity and interferon-gamma production in response to peptide-loaded T2-A(*)2402 and tumor cell lines expressing both MAGE-A4 and HLA-A(*)2402. A total of 24 clones were established from TCR-alphabeta gene-transduced peripheral blood mononuclear cells and all clones were functional on a transduced TCR-dependent manner. Four clones were kept in culture over 6 months for analyses in detail. The transduced TCR-alphabeta genes were stably maintained phenotypically, functionally and genetically. Our results indicate that TCR-transduced alphabeta T cells by retroviral transduction represent an efficient and promising strategy for adoptive T-cell transfer for long term.

Application of fluorescently labeled poly(dU) for gene expression profiling on cDNA microarrays.

The membrane filter hybridization technique has been widely used for gene expression profiling. The preparation of sensitive and reliable probes is critical for quantitative analysis in this technique. We report a method in which fluorescently labeled poly(dU) is used to detect poly(A)-containing mRNA that hybridizes to specific gene targets. The probe can be used commonly for every sample, alleviating problems encountered in preparing cDNA probes by reverse transcription, particularly when many samples are to be analyzed. Moreover, the sensitivity is at least comparable to cDNA probes prepared by conventional protocols, and intensities of signals after hybridization are independent of mRNA sizes and solely dependent on copy numbers. This method was also shown to be applicable to DNA chip technology.

Fluorescent labeling of a DNA sequencing primer.

Several oligonucleotides containing one to four fluorescein labels in various positions were synthesized and the fluorescence intensity and thermal stability of the duplex forms with their complementary sequences were measured. Oligomers that contain two fluorescein molecules, one attached at the 5' terminus and the other at an internal phosphate, were hybridized with less stability than that containing fluorescein at only the 5' terminus, but it formed more efficient primers for dideoxy sequencing with an automated sequencer.

Efficient large-scale sequencing of the Escherichia coli genome: implementation of a transposon- and PCR-based strategy for the analysis of ordered lambda phage clones.

We have developed a strategy for efficient sequence analysis of the genome of E. coli K-12 using insertions of a Tn5-derived mini-transposon into overlapping ordered lambda phage clones to provide universal primer-binding sites, and PCR amplification of DNA segments adjacent to the insertions. Transposon-containing clones were selected by blue plaque formation on a dnaBamber lacZamber E. coli strain. Insertion points every 0.5-1 kb were identified by 'analytical PCR' and segments between the transposon inserts and phage arms were amplified by 'preparative PCR' using one biotinylated and one non-biotinylated primer. Single strands of amplified DNA fragments were coupled to Streptoavidin-coated paramagnetic beads (Dynabeads M280) through their biotin tails, purified magnetically, and used as templates for fluorescence-based automatic nucleotide sequencing.

Practical applications in molecular biology of sensitive fluorescence detection by a laser-excited fluorescence image analyzer.

A new kind of fluorescence image analyzer was developed for a variety of uses, especially in molecular biology. Compounds labeled with fluorescent groups on a gel or nitrocellulose membrane are excited with 532 nm of light from a green laser. The fluorescence emitted passes through light-collecting fibers to a photomultiplier. Imaging data converted from the emitted light are analyzed by a microcomputer and stored on a magnetic optical disk. Dideoxy DNA sequencing was done with the same amount of DNA used for autoradiography, and the sequencing ladders obtained from gel scanning were automatically converted to sequence data by the analyzer. When an agarose gel was analyzed after electrophoresis, DNA stained with ethidium bromide was detected by the analyzer with higher sensitivity rather than by the conventional photographic method. Nylon and nitrocellulose membranes could be read by the analyzer, so blot hybridization experiments can be done without radioisotopes. High-quality computer storage of the imaging data from gel electrophoresis and hybridized membranes, including pulsed-field gels, make it possible to quantify image intensity and to construct many kinds of databases.

Aphidicolin inhibits DNA polymerizing activity but not nucleolytic activity of Escherichia coli DNA polymerase II.

We have purified the DNA polymerase II of Escherichia coli from the recombinant strain carrying the plasmid which encodes the polB gene. We confirmed that the purified protein, of molecular weight 90,000, possesses a 3'----5' exonuclease activity in addition to DNA polymerizing activity in a single polypeptide. Its DNA polymerizing activity was sensitive to the drug aphidicoline, which is a specific and direct inhibitor of the alpha-like DNA polymerases including eukaryotic replicative DNA polymerases. Aphidicolin had no detectable effect on the 3'----5' exonuclease activity. The inhibition by aphidicolin on the polymerizing activity of polymerase II was competitive with respect to dNTP and uncompetitive with respect to template DNA. This mode of action is the same as that on eukaryotic DNA polymerase alpha. The apparent Ki value calculated from Lineweaver-Burk plots was 55.6 microM.