mRNA transfection of DC in the immature or mature state: comparable in vitro priming of Th and cytotoxic T lymphocytes against DC electroporated with tumor cell line-derived mRNA.

BACKGROUND: The use of mRNA in vaccine studies has generally been through loading or transfection of immature DC followed by a maturation step. A recent study has suggested that this strategy may result in inferior priming of cytotoxic T lymphocytes (CTL). Furthermore the study did not address any possible effects on the priming of CD4(+) T-cell responses. METHODS: We compared mRNA transfection of mature DC with that of immature DC, using as a read-out their capacity to prime autologous T cells during one cycle of in vitro stimulation. In this model system we used mRNA from the tumor cell line Jurkat E6. DC transfected at either the immature stage (day 5) or mature stage (day 7) displayed a similar phenotype. RESULTS: Interestingly, no major differences in their ability to prime CD4 and CD8 T-cell responses were observed. As in vitro priming to some extent may reflect the capacity of these DC to prime T cells in vivo after vaccination, these studies support the use of mRNA-transfected mature DC in clinical protocols. DISCUSSION: Transfection of DC at the end of the maturation process represents a logistical improvement in the GMP production of mRNA-transfected DC for clinical protocols.

Development of a clinical grade procedure for generation of mRNA transfected dendritic cells from purified frozen CD34(+) blood progenitor cells.

Enriched CD34(+) peripheral blood progenitor cells (PBPC) are frequently used as stem cell support in cancer patients following high dose therapy. Since precursor dendritic cells (DCs) originate from haematopoietic progenitor cells, purified CD34(+) cells might also serve as starting cells for ex- vivo production of DC. In the present study we developed a clinical grade procedure for ex- vivo production of DC derived from enriched CD34(+) cells. Different concentrations of CD34(+) cells were grown in gas-permeable Teflon bags with different serum-free and serum-containing media supplemented with GM-CSF, IL-4, TNF-alpha, SCF, Flt-3L and INF-alpha. Serum-free CellGroSCGM medium for 7 days followed by CellGroDC medium in 7 days gave the same results as serum-containing medium. After incubation the cultured cells containing immature DCs were concentrated and transfected with tumour mRNA from human prostate cancer cell lines employing a highly efficient electroporation procedure. Thawed transfected DCs were able to elicit primary T-cell responses in vitro against antigens encoded by the prostate cancer mRNA as shown by ELISPOT assay using mock-transfected DCs as control. Our results show that frozen enriched CD34(+) cells can be an alternative and efficient source for production of DCs for therapeutic purpose.

A protocol for generation of clinical grade mRNA-transfected monocyte-derived dendritic cells for cancer vaccines.

With the aim of producing large quantities of mRNA-transfected monocyte-derived dendritic cells (DCs) to be used as cancer vaccines, a new clinical grade procedure has been developed. Peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis were enriched for monocytes by immunomagnetic depletion of CD19+ B cells and CD2+ T cells employing the ISOLEX 300i device. After 5 days of culture of enriched monocytes in gas permeable Teflon bags, using serum-free medium supplemented with granulocyte/macrophage-colony stimulating factor and interleukin-4 (IL-4), immature DCs were generated. Following transfection with mRNA from three human prostate cancer cell lines (DU145, LNCaP and PC-3), employing a newly developed square wave electroporation procedure, the immature DCs were immediately transferred to Teflon bags and matured for 48 h, using serum-free medium supplemented with IL-1alpha, IL-6, tumour necrosis factor-alpha and PGE2. The electroporation procedure efficiently transferred mRNA into the DCs with minor effect on the viability of the cells. The generated matured transfected DCs show high expression of the antigens CD83, CD80, CD86 and human leucocyte antigen-DR. Freezing and thawing of the transfected matured DCs had minor effect on cell viability and the phenotype. From 4 x 109 PBMCs, about 1 x 108 transfected matured DCs are produced. The thawed transfected DCs were able to elicit primary T-cell responses in vitro against antigens encoded by the prostate cancer mRNA as shown by enzyme-linked immunospot assay using mock-transfected DCs as control. Based on these results, clinical trials in cancer patients have been initiated.

Ribosomal protein insufficiency and the minute syndrome in Drosophila: a dose-response relationship.

Minutes comprise > 50 phenotypically similar mutations scattered throughout the genome of Drosophila, many of which are identified as mutations in ribosomal protein (rp) genes. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. By mobilizing a P element inserted in the 5' UTR of M(3)95A, the gene encoding ribosomal protein S3 (RPS3), we have generated two homozygous viable heteroalleles that are partial revertants with respect to the Minute phenotype. Molecular characterization revealed both alleles to be imprecise excisions, leaving 40 and 110 bp, respectively, at the P-element insertion site. The weaker allele (40 bp insert) is associated with a approximately 15% decrease in RPS3 mRNA abundance and displays a moderate Minute phenotype. In the stronger allele (110 bp insert) RPS3 mRNA levels are reduced by approximately 60%, resulting in an extreme Minute phenotype that includes many morphological abnormalities as well as sterility in both males and females due to disruption of early gametogenesis. The results show that there is a correlation between reduced RPS3 mRNA levels and the severity of the Minute phenotype, in which faulty differentiation of somatic tissues and arrest of gametogenesis represent the extreme case. That heteroalleles in M(3)95A can mimic the phenotypic variations that exist between different Minute/rp-gene mutations strongly suggests that all phenotypes primarily are caused by reductions in maximum protein synthesis rates, but that the sensitivity for reduced levels of the individual rp-gene products is different.

The Drosophila ribosomal protein L14-encoding gene, identified by a novel Minute mutation in a dense cluster of previously undescribed genes in cytogenetic region 66D.

The Minute phenotype results from mutations at > 50 loci scattered throughout the genome of Drosophila. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. Here, we report a novel P-element induced Minute mutation, P¿lacW¿ M(3)66D1, that maps to region 66D on chromosome 3L. Flies heterozygous for P¿lacW¿ M(3)66D1 have a strong Minute phenotype. Molecular characterisation of the chromosomal region revealed three previously undescribed Drosophila genes clustered within a 5-kb genomic fragment. Two of the genes have significant sequence homology to genes for the mammalian ribosomal proteins L14 and RD, respectively, and share a joint 240-bp promoter region harbouring the P-element insert. Quantitative Northern blot analyses showed the mutation to affect RPL14 mRNA levels only. Interestingly, the reduction in abundance of RPL14 mRNA is not constitutive, indicating that the promoter function abolished by the inserted P-element is utilised with different efficiencies in different developmental situations. Remobilisation of the P element produced wild-type flies with normal levels of RPL14 mRNA, demonstrating that the mutant phenotype is caused by the insertion. P¿lacW¿ M(3)66D1 joins a growing list of Minute mutations associated with ribosomal protein-haploinsufficiency.

A novel Drosophila Minute locus encodes ribosomal protein S13.

Minutes comprise > 50 phenotypically similar Drosophila mutations believed to affect ribosomal protein genes. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. To further investigate the proposed Minute to ribosomal protein correspondence, loss-of-function Minute mutations were induced by P-element mutagenesis. Here, we report a previously undescribed Minute locus that maps to 32A on chromosome 2L; this Minute allele is named P{lacW}M(2)32A1 and the gene M(2)32A. Flies heterozygous for P{lacW}M(2)32A1 have a medium Minute phenotype. The gene interrupted by the P-element insertion was cloned. Sequence analyses revealed that it encodes the Drosophila homologue of eukaryotic ribosomal protein S13. It is a single-copy gene and the level of RPS13 transcript is reduced to approximately 50% in P(lacW) M(2)32A1 heterozygotes. Both transcript level and phenotype are restored to wild type by remobilizing the P-element, demonstrating that the mutation is caused by insertion of the P-element construct. These results further strengthen the notion that Minutes encode ribosomal proteins and demonstrate that P-element mutagenesis is a fruitful approach to use in these studies.

Evolution of the Group 1 late embryogenesis abundant (Lea) genes: analysis of the Lea B19 gene family in barley.

The highly conserved Group 1 late embryogenesis abundant (Lea) genes are present in the genome of most plants as a gene family. Family members are conserved along the entire coding region, especially within the extremely hydrophilic internal 20 amino acid motif, which may be repeated. Cloning of Lea Group 1 genes from barley resulted in the characterization of four family members named B19.1, B19.1b, B19.3 and B19.4 after the presence of this motif 1, 1, 3 and 4 times in each gene, respectively. We present here the results of comparative and evolutionary analyses of the barley Group 1 Lea gene family (B19). The most important findings resulting from this work are (1) the tandem clustering of B19.3 and B19.4, (2) the spatial conservation of putative regulatory elements between the four B19 gene promoters, (3) the determination of the relative 'age' of the gene family members and (4) the 'chimeric' nature of B19.3 and B19.4, reflecting a cross-over or gene-conversion event in their common ancestor. We also show evidence for the presence of one or two additional expressed B19 genes in the barley genome. Based on our results, we present a model for the evolution of the family in barley, including the 20 amino acid motif. Comparisons of the relatedness between the barley family and all other known Group 1 Lea genes using maximum parsimony (PAUP) analysis provide evidence for the time of divergence between the barley genes containing the internal motif as a single copy and as a repeat. The PAUP analyses also provide evidence for independent duplications of Group 1 genes containing the internal motif as a repeat in both monocots and dicots.

A Drosophila third chromosome Minute locus encodes a ribosomal protein.

Minutes (M) are a group of over 50 phenotypically similar Drosophila mutations widely believed to affect ribosomal protein genes. This report describes the characterization of the P element-induced M(3)95A(Plac92) mutation [allelic to M(3)95A]. This mutation can be reversed by the mobilization of the P element, demonstrating that the mutation is caused by insertion of this transposable element. The gene interrupted by insertion of the P element was cloned by use of inverse polymerase chain reaction. Nucleotide sequence analysis revealed a 70-75% identity to the human and rat ribosomal protein S3 genes, and to the Xenopus ribosomal protein S1a gene. At the amino acid level, the overall identity is approximately 78% for all three species. This is only the second time that a Minute has been demonstrated to encode a ribosomal protein.

Late embryogenesis-abundant genes encoding proteins with different numbers of hydrophilic repeats are regulated differentially by abscisic acid and osmotic stress.

The late embryogenesis-abundant (Lea) genes, which are suggested to act as desiccation protectants during seed desiccation and in water-stressed seedlings, can be induced by abscisic acid (ABA) and various kinds of water-related stress. Using cotton Lea cDNAs as probes it was found that several of the Lea genes are conserved at the mRNA level in dicots and monocots. By screening a barley cDNA library with a cotton Lea D19 cDNA a family of three members was isolated. The putative B19 proteins have strong similarities to the Em protein in wheat and to LEA proteins from several dicots. However, the middle part of the B19 proteins consists of a 20-amino acid motif repeated three and four times in B19.3 and B19.4, respectively, but only once in B19.1. The gene products are strongly hydrophilic, the internal 20-amino acid motif being the most hydrophilic part. This motif is found once in cotton Lea D19 but is repeated twice in cotton Lea D132, indicating that the repeats are universal among monocot and dicot B19-like genes. The B19 genes are regulated similarly during embryo development, but to very different levels. In contrast, they are differentially regulated by ABA and various types of osmotic stress. In immature embryos all three genes are responsive to ABA and mannitol. However, B19.1 is also responsive to salt. Cold stress does not induce B19 mRNAs; only a stabilization of the transcript levels is seen. These results suggest that the responses to salt stress and exogenous ABA operate through different pathways.