In vitro expansion of Ag-specific T cells by HLA-A*0201-transfected K562 cells for immune monitoring.

BACKGROUND: Development of a practical and sensitive assay for evaluating immune responses against cancer Ag has been a challenge for immune monitoring of patients. We have established a reproducible method using peptide-pulsed K562-A*0201 cells as APC to expand Ag-specific T cells in vitro. This method may be applied for monitoring T-cell responses in cancer immunotherapy clinical trials. METHODS: Autologous PBMC from HLA-A*0201+ healthy donors and patients with melanoma were stimulated with peptide-pulsed K562-A*0201 cells under varying conditions. We investigated (1) different culture conditions, including the requirements for serum and cytokines for expansion of CD8+ T lymphocytes; (2) a range of peptide concentrations for Ag loading; (3) phenotypic characterization of responding T cells; and (4) APC:responder ratios and their effects on T-cell expansion. We validated these conditions by ELISPOT and intracellular cytokine staining (ICS) assays using peptides from influenza, Epslein-Barr Virus (EBV) and tyrosinase. RESULTS: Conditions for optimal T-cell expansion using K562-A*0201 APC included input of 2 x 10(6) PBMC, a 10 microg/mL peptide concentration to pulse K562-A*0201 cells, a 1:30 APC:responder T-cell ratio and culture in 10% autologous plasma supplemented with IL-2 and IL-15. In these conditions, Ag-specific T cells expanded >100-fold over a 10-day culture period (peak at day 12). DISCUSSION: This bulk culture method is simple and reliable for expanding human Ag-specific T cells using peptide-pulsed K562-A*0201 cells. This HLA-matched APC line can be adapted to other HLA haplotypes, and has advantages for monitoring clinical trials of immunotherapy with limited availability of autologous APC and PBMC from patients.

Stable in vivo expression of glucose-6-phosphate dehydrogenase (G6PD) and rescue of G6PD deficiency in stem cells by gene transfer.

Many mutations of the housekeeping gene encoding glucose-6-phosphate dehydrogenase (G6PD) cause G6PD deficiency in humans. Some underlie severe forms of chronic nonspherocytic hemolytic anemia (CNSHA) for which there is no definitive treatment. By using retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein that harbor the human G6PD (hG6PD) complementary DNA, stable and lifelong expression of hG6PD was obtained in all the hematopoietic tissues of 16 primary bone marrow transplant (BMT) recipient mice and 14 secondary BMT recipients. These findings demonstrate the integration of a functional gene in totipotent stem cells. The average total G6PD in peripheral blood cells of these transplanted mice, measured as enzyme activity, was twice that of untransplanted control mice. This allowed the inference that the amount of G6PD produced by the transduced gene must be therapeutically effective. With the same vectors both the cloning efficiency and the ability to form embryoid bodies were restored in embryonic stem cells, in which the G6PD gene had been inactivated by targeted homologous recombination, thus effectively rescuing their defective phenotype. Finally, expression of normal human G6PD in hG6PD-deficient primary hematopoietic cells and in human hematopoietic cells engrafted in nonobese diabetic/severe combined immunodeficient mice was obtained. This approach could cure severe CNSHA caused by G6PD deficiency.

Retroviral-mediated gene transfer in primary murine and human T-lymphocytes.

Recombinant retroviruses are efficient vectors for introducing genes into many mammalian cell types. They are useful in the context of clinical as well as experimental applications, owing to the ability to generate high-titer and helper-free viral stocks. Retroviral vectors are especially appropriate for the transduction of primary lymphocytes, because gene transfer is stable and mediated by nonimmunogenic vectors. Stable integration in chromosomes of cells undergoing clonal expansion ensures that the foreign genetic material will be faithfully transmitted to the cells' progeny. However, oncoretroviral vectors derived from murine leukemia viruses (MLV) require target cell division to integrate. Here we review factors that determine retroviral-mediated gene transfer efficiency in primary T-lymphocytes, in particular, T-cell activation status, viral receptor expression, and culture conditions.

Rapid selection of antigen-specific T lymphocytes by retroviral transduction.

Infusions of donor peripheral blood T cells can induce durable remissions of Epstein-Barr virus (EBV) lymphomas complicating marrow grafts, but they contain alloreactive T cells capable of inducing graft-versus-host disease. EBV-specific T-cell lines or clones avoid this problem but require 30 to 40 days of culture to establish. To accelerate the generation of EBV-specific T cells, we tested whether retroviral vectors, which only integrate in dividing cells, could be used to transduce and select antigen-reactive T cells early after sensitization to autologous EBV-transformed B cells. T cells were transduced with a dicistronic retroviral vector, NIT, which encodes low-affinity nerve growth factor receptor as an immunoselectable marker and herpes simplex virus thymidine kinase as a suicide gene, at different time points after sensitization. EBV-specific cytotoxic T lymphocyte precursor (CTLp) frequencies in purified NIT(+) T-cell fractions transduced on day 8 of culture were comparable to those of EBV-specific T-cell lines cultured for 30 days or more. Alloreactive CTLp frequencies were markedly reduced in the NIT(+) fraction relative to the untransduced T-cell population. NIT(+) fractions transduced on day 8 possessed more CD4(+) T cells than the cell lines at day 30 and exhibited the same selective pattern of reactivity against immunodominant antigens presented by specific HLA alleles. In contrast, T cells transduced with NIT 5 days after stimulation with mitogen and interleukin-2 were relatively depleted of T cells specific for autologous EBV-transformed cells. Thus, retroviral vectors may be used for rapid selection of viral antigen-reactive T cells depleted of alloreactive T cells.

Adenovirus facilitated infection of human cells with ecotropic retrovirus.

Retroviral infection is restricted by the expression of a viral receptor on the surface of the target cell. Retrovirus-mediated gene transfer is therefore not possible in cells that fail to express sufficient levels of the appropriate receptor, representing one major obstacle to the use of recombinant retroviruses in experimental and clinical applications. In this study, we utilized an adenoviral vector to express transiently the receptor for the ecotropic murine leukemia virus in a panel of human cell lines. Following adenoviral infection, the susceptibility to ecotropic retroviral particles of A549, HeLa, RC39 and Meso 33 cells, derived from human lung epithelium, cervical epithelium, kidney and mesothelium, respectively, was measured on a single-cell basis by the detection of a cell surface marker encoded by the recombinant retrovirus. The marker, termed NTP, was found in 10-30%, 25-50% and 50-90% of cells infected at 5, 50 and 250 adenovirus multiplicity of infection, respectively. Southern blot analysis demonstrated the integration of intact retroviral DNA. The integrated vector copy number increased with the adenoviral multiplicity of infection, suggesting that retrovirus infection is proportional to receptor expression by the target cell, albeit not in a linear fashion. Susceptibility to ecotropic retroviral infection was maintained undiminished for at least 3 days, indicating the persistent expression of ecotropic receptor by the adenovirus-transduced cells in that time period and the lack of a major cellular defense triggered by adenovirus infection against the subsequent retroviral infection. Thus, the infection of human cells of various tissues with a recombinant adenovirus expressing the ecotropic murine leukemia virus receptor generates a window of susceptibility where a high retroviral infection rate can be achieved. Increased efficiency of retroviral infection obtained in this fashion is amenable to specific regulation via the controlled expression of the adenovirus-encoded retroviral receptor.

Retrovirally transduced human dendritic cells express a normal phenotype and potent T-cell stimulatory capacity.

Dendritic cells are attractive candidates for vaccine-based immunotherapy because of their potential to function as natural adjuvants for poorly immunogenic proteins derived from tumors or microbes. In this study, we evaluated the feasibility and consequences of introducing foreign genetic material by retroviral vectors into dendritic cell progenitors. Proliferating human bone marrow and cord blood CD34+ cells were infected by retroviral vectors encoding the murine CD2 surface antigen. Mean transduction efficiency in dendritic cells was 11.5% from bone marrow and 21.2% from cord blood progenitors. Transduced or untransduced dendritic cell progeny expressed comparable levels of HLA-DR, CD83, CD1a, CD80, CD86, S100, and p55 antigens. Granulocytes, macrophages, and dendritic cells were equally represented among the transduced and mock-transduced cells, thus showing no apparent alteration in the differentiation of transduced CD34+ precursors. The T-cell stimulatory capacity of retrovirally modified and purified mCD2-positive allogeneic or nominal antigen-pulsed autologous dendritic cells was comparable with that of untransduced dendritic cells. Human CD34+ dendritic cell progenitors can therefore be efficiently transduced using retroviral vectors and can differentiate into potent immunostimulatory dendritic cells without compromising their T-cell stimulatory capacity or the expression of critical costimulatory molecules and phenotypic markers. These results support ongoing efforts to develop genetically modified dendritic cells for immunotherapy.

Recombinant retroviruses pseudotyped with the vesicular stomatitis virus G glycoprotein mediate both stable gene transfer and pseudotransduction in human peripheral blood lymphocytes.

It is essential for the study of T-cell function and the improvement of adoptive cell therapies to efficiently generate large populations of human primary T cells that reliably express foreign genes. This goal is achieved by using recombinant retroviruses pseudotyped with either the gibbon ape leukemia virus (GaLV) envelope or the vesicular stomatitis virus G (VSV-G) glycoprotein. We show here that both retroviral particles mediate stable gene transfer in CD4+ and in CD8+ peripheral blood lymphocytes cultured under optimized conditions. However, VSV-G-pseudotyped virions may cause transduction artifacts that must be carefully excluded. The VSV-G virions require 10- to 100-fold higher concentrations of infectious particles to achieve levels of gene transfer comparable to GaLV-virions. Nonetheless, the physical stability of VSV-G-coated particles enables the concentration of viral stocks to 10(9) infectious particles per milliliter or more.

The internal ribosomal entry site of the encephalomyocarditis virus enables reliable coexpression of two transgenes in human primary T lymphocytes.

It is essential for the improvement of adoptive cell therapies to generate efficiently large populations of human primary T cells that reliably express a suicide gene conferring drug sensitivity, such as herpes simplex virus thymidine kinase (HSVtk). We show here that an optimized dicistronic vector containing the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) is functional in human primary. T lymphocytes that bear on average one integrated vector copy per cell. We demonstrate reliable coexpression of the marker NTP, an inactive mutant of the human low-affinity nerve growth factor receptor and HSVtk. In the dicistronic vector NIT, NTP is expressed as a cap-dependent marker and HSVtk as a nonselectable IRES-dependent gene. Cell-surface expression of NTP is sufficient to allow for the efficient and rapid enrichment of the transduced cells to high purity. Of these purified lymphocytes, 97 +/- 4% and 92 +/- 6% are selectively eliminated when cultured in the presence of 1.0 or 0.1 microM ganciclovic respectively, establishing that the EMCV IRES ensures efficient and sufficient expression of two genes in human primary T cells.

Inhibition of HIV-1 in CEM cells by a potent TAR decoy.

TAR decoys are short RNA oligonucleotides, corresponding to the HIV TAR sequence, which inhibit HIV expression and replication by blocking the binding of the HIV regulatory protein Tat to the authentic TAR region. In previous studies, TAR decoys expressed from a tRNA polIII promoter were moderately effective at inhibiting HIV in isolated human T cell lines and less effective at inhibiting HIV in peripheral blood CD4+ T cells. In this study, a series of modifications was introduced into the tRNA expression cassette in order to improve their effectiveness. These modifications included the addition of sequences which are predicted to have stem-loop secondary structures and addition of a wild-type tRNA processing site. TAR decoy RNA expressed in CEM cells from modified tRNA-based expression cassettes yielded five- to 20-fold more TAR transcripts than unmodified tRNA-based expression cassettes. HIV replication, as measured by a flow cytometric method to quantify intracellular viral p24 expression, was significantly reduced in polyclonal populations of CEM cells expressing a modified tRNA-TAR transcript that contains a wild-type tRNA processing site and stem-loops 5' and 3' to the TAR sequence. Similar modifications to the tRNA expression cassette also increased the intracellular concentration of a random test oligonucleotide, indicating that this improved expression system may also be useful for antisense and ribozyme based gene inhibition strategies.

Inhibition of human immunodeficiency virus type 1 in human T cells by a potent Rev response element decoy consisting of the 13-nucleotide minimal Rev-binding domain.

Intracellular immunization is an anti-viral gene therapy strategy based on the introduction of DNA templates into cells to stably express genetic elements which inhibit viral gene expression and replication. We have recently developed an intracellular immunization strategy for human immunodeficiency virus (HIV) infection that uses RNA decoys. RNA decoys are short RNA oligonucleotides corresponding to the HIV trans activation response element (TAR) or Rev response element (RRE) sequences, which function by inhibiting the binding of the HIV regulatory proteins Tat and Rev to the authentic HIV RNA TAR and RRE regions, respectively. In this report we describe the characterization of potent RRE decoys containing the minimal 13-nucleotide primary Rev binding domain of the RRE. Using an improved tRNA cassette to express high levels of RRE transcripts in CEM cells, we found that this new generation of minimal RRE decoys were more potent inhibitors of HIV in isolated cell lines than previously described TAR or RRE decoys. CEM cells expressing RRE decoys exhibited diminished Rev function in cotransfection assays, confirming the specificity of inhibition of HIV by RRE decoys and indicating that the 13-nucleotide minimal Rev binding domain defined by using in vitro binding studies also binds Rev in vivo. Significant differences in the degree of HIV inhibition between individual CEM cell lines transduced with RRE decoy vectors which were not due to sequence alterations in the tRNA-RRE DNA template, differences in RRE decoy expression level, or endogenous variations in the resistance of CEM clonal cell lines to HIV were observed. In order to evaluate the efficacy of RRE decoys in a more realistic fashion than by comparison of individual clonal cell lines, polyclonal populations of transduced CEM cells were infected with HIV. By using a novel flow cytometric method for quantitating intracellular p24 expression, one version of the RRE decoys tested in this study was found to be capable of durably protecting polyclonal populations of CEM cells from HIV.

Overexpression of RRE-derived sequences inhibits HIV-1 replication in CEM cells.

Overexpression of sequences corresponding to the major Rev-binding site in the Rev response element of human immunodeficiency virus type 1 (HIV-1) (RRE decoys) was used to render cells resistant to HIV-1 replication. This was accomplished by the use of a chimeric tRNA-RRE transcription unit in a double-copy murine retroviral vector to express high levels of HIV-1 RRE-containing transcripts in CEM SS cells. Replication of HIV-1 was inhibited more than 90% in cells expressing chimeric tRNA-RRE transcripts, as determined by in situ immunofluorescence analysis and a p24 antigen ELISA test. Analysis of RNA from HIV-1-infected cells suggests that expression of RRE-containing sequences in CEM SS cells inhibits HIV-1 replication by interfering with Rev function, presumably by competing for Rev binding to its physiological target. The use of a subfragment of RRE as decoy RNA reduces the likelihood that essential cellular factors will be sequestered in cells expressing the decoy RNA. Thus, use of RRE-based decoy RNA to inhibit HIV-1 replication may represent a safer alternative to the use of TAR decoy RNA.

Analysis of trans-acting response decoy RNA-mediated inhibition of human immunodeficiency virus type 1 transactivation.

Overexpression of trans-acting response element (TAR)-containing sequences (TAR decoys) in CEM SS cells renders cells resistant to human immunodeficiency type 1 (HIV-1) replication. Mutagenesis of TAR was used to investigate the molecular mechanism underlying the observed inhibition. A nucleotide change which disrupts the stem structure of TAR or sequence alterations in the loop abolish the ability of the corresponding TAR decoy RNAs to inhibit HIV replication. A compensatory mutation which restores the stem structure also restores TAR decoy RNA function. Synthesis of viral RNA is drastically reduced in cells expressing a functional TAR decoy RNA, but it is unaffected in cells expressing a mutant form of TAR decoy RNA. It is therefore concluded that overexpression of TAR-containing sequences in CEM SS cells interferes with the process of Tat-mediated transactivation of viral gene expression. However, the phenotype of several mutations suggests that TAR decoy RNA does not inhibit HIV-1 gene expression by simply sequestering Tat but rather does so by sequestering a transactivation protein complex, implying that transactivation requires the cooperative binding of both Tat and a loop-binding cellular factor(s) to TAR. Expression of wild-type or mutant forms of TAR had no discernible effects on cell viability, thus reducing concerns about using TAR decoy RNAs as part of an intracellular immunization protocol for the treatment of AIDS.

Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication.

Overexpression of TAR-containing sequences (TAR decoys) was used to render cells resistant to HIV replication. A chimeric tRNA(meti)-TAR transcription unit contained in a double copy murine retroviral vector was used to express high levels of HIV-1 TAR-containing transcripts in CEM SS cells. Replication of HIV-1 was inhibited over 99% in cells expressing chimeric tRNA-TAR transcripts, but an amphotropic murine retrovirus replicated normally in these cells. Expression of TAR sequences in CEM SS cells had no adverse effects on cell viability, indicating that essential cellular factors are not being sequestered in these cells. TAR decoy RNA-mediated HIV inhibition may also be effective against natural HIV isolates in spite of their hypervariable nature, as suggested by the fact that replication of SIVmac was also inhibited in cells expressing HIV-1 TAR decoys.