DNA vaccination against the ovarian carcinoma-associated antigen folate receptor alpha (FRalpha) induces cytotoxic T lymphocyte and antibody responses in mice.

Human folate receptor alpha (FRalpha) is a folate-binding protein that is selectively overexpressed in ovarian carcinoma and has been regarded as a suitable target antigen for immunotherapy purposes. To study the possible use of this antigen in DNA vaccination, FRalpha cDNA was ligated into the VR1012 (Vical) expression vector under the transcriptional control of the cytomegalovirus promoter. A total of 100 microg of purified plasmid DNA was injected intramuscularly in BALB/c mice three times at 14-day intervals. At 10 days after the second injection, the sera of the animals (100%) displayed significant antibody titers (by indirect immunofluorescence and fluorescence-activated cell sorter analysis) against syngeneic C26 cells transduced with FRalpha, but not against unmodified C26 cells. Immunoglobulin G2a was the predominant isotype. In addition, specific cytotoxic T lymphocyte activity against FRalpha-transduced C26 cells could be detected in splenocytes from all immunized animals. Coinjection of a plasmid containing interleukin-2 cDNA increased both antibody titers and cytotoxic T lymphocyte activity. Challenge by subcutaneous injection with FRalpha-transduced C26 cells (performed 10 days after the third injection) showed a statistically significant delay in tumor growth. Vaccination with the FRalpha and interleukin-2 cDNA mixture, which was performed after an intravenous injection of FRalpha-transduced cells, enhanced the mean survival time and reduced the number of lung metastases, thus suggesting that such vaccination is effective even against preexisting tumor cells.

Expression of two interleukin-15 mRNA isoforms in human tumors does not correlate with secretion: role of different signal peptides.

Interleukin (IL)-15 is a four-helix bundle cytokine sharing several biological properties with IL-2. By reverse transcriptase-polymerase chain reaction analysis, human cancer cell lines of different histotypes are shown to express two IL-15 amplification products: a 524-bp band corresponding to the IL-15 mRNA found in macrophages, and another of 643 bp corresponding to an alternatively spliced mRNA including a 119-bp alternative exon. IL-15 was undetectable in the supernatant of tumor cell lines expressing either one or both of the mRNA isoforms as evaluated by a bioassay or by ELISA, indicating that IL-15 is not secreted. However, IL-15 could be detected intracellularly in some tumor cells by confocal microscopy analysis. Since the pre-proteins encoded by the two mRNA isoforms differ in the signal peptide sequence, we have analyzed the characteristics of these signal peptides and their possible role in controlling secretion. The two IL-15 cDNA isoforms, expressed in COS-7 cells, induced very low levels of IL-15 secretion. However, substitution of the sequence encoding natural signal peptide(s) with the one from IgV kappa chain in the IL-15 cDNA results in a significantly higher secretion of biologically active IL-15 (15-30-fold) upon cDNA transfection. A poor efficiency of natural signal peptides may represent one of the mechanisms involved in the control of IL-15 secretion.

Reciprocal expression of CD70 and of its receptor, CD27, in human long term-activated T and natural killer (NK) cells: inverse regulation by cytokines and role in induction of cytotoxicity.

By transfection of COS cells with an expression vector containing CD70 cDNA we demonstrate that two previously described MoAbs (ED6 and LD6) recognize CD70. By means of these MoAbs, we show that the surface expression of CD70 inversely correlates with the expression of its receptor, CD27, on activated T and NK cell populations and clones, although a subpopulation of cells expressing low density of both molecules exists. In addition, culture in the presence of IL-4 significantly enhances CD27 and reduces CD70 surface expression in phytohaemagglutinin (PHA)-activated peripheral blood lymphocytes (PBL), while tumour necrosis factor-alpha (TNF-alpha) displays opposite effects, indicating that receptor and ligand are reciprocally regulated by these cytokines. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of CD27 and CD70 mRNA suggests a transcriptional control of CD27 antigen expression in T cell clones. In addition, we show by the use of a re-directed killing assay that in cytotoxic T cell receptor (TCR) alpha/beta+ T cell clones, CD27 molecule may be involved in the regulation of cytolytic functions and may act synergistically with CD2. Finally, CD70 also acts as a signal-transducing molecule in some activated CD70+ TCR gamma/delta+ T or NK cell clones. In conclusion, our data indicate that CD27 and CD70 molecules are differentially expressed and regulated on long term-activated T and NK cells and are involved in the control of cellular functions.