Dendritic cells (DC) are professional Ag-capturing and -presenting cells of the immune system. Because of their exceptional capability of activating tumor-specific T cells, cancer vaccination research is now shifting toward the formulation of a clinical human DC vaccine. We developed a short term and serum-free culture protocol for rapid generation of fully mature, viable, and highly stimulatory CD83+ DC. Human monocytes were cultured for 24 h in serum-free AIM-V medium, followed by 24-h maturation by polyriboinosinic polyribocytidylic acid (polyI:C). Short term cultured, polyI:C-maturated DC, far more than immature DC, showed typical mature DC markers and high allogeneic stimulatory capacity and had high autologous stimulatory capacity in an influenza model system using peptide-pulsed DC. Electroporation of mRNA as an Ag-loading strategy in these cells was optimized using mRNA encoding the enhanced green fluorescent protein (EGFP). Monocytes electroporated with EGFP mRNA, followed by short term, serum-free differentiation to mature DC, had a phenotype of DC, and all showed positive EGFP fluorescence. Influenza matrix protein mRNA-electroporated monocytes cultured serum-free and maturated with polyI:C showed high stimulatory capacity in autologous T cell activation experiments. In conclusion, the present short term and serum-free ex vivo DC culture protocol in combination with mRNA electroporation at the monocyte stage imply an important reduction in time and consumables for preparation of Ag-loaded mature DC compared with classical DC culture protocols and might find application in clinical immunotherapy settings.

We developed reverse transcriptase (RT) PCR assays for the detection of mRNA from three spliced genes of human herpesvirus 6 (HHV-6), the immediate-early genes U16/U17 and U89/U90 and the late gene U60/U66. Sequence analysis determined the splicing sites of these genes. The new assays may be instrumental in investigating the association between HHV-6 and disease.Human herpesvirus 6 (HHV-6) is the causative agent of exanthema subitum (10). High HHV-6 viral loads have been demonstrated in the settings of allograft rejection, immunodeficiencies, malignancies, and multiple sclerosis, although an etiological correlation is still uncertain (1). This is primarily due to the lack of sensitive diagnostic tools specific for active infection. PCR detection of viral DNA in plasma has been proposed as a marker of active infection, but its sensitivity is low (9). The aim of the present work was to develop HHV-6 reverse transcriptase (RT)-PCR assays, since the expression of viral mRNA constitutes an unambiguous marker of active infection. To discriminate mRNA from genomic DNA, we focused on the detection of spliced gene expression; here we report on RT-PCR assays for the immediate-early genes U16/ U17 and U89/U90 and the late gene U60/U66, as well as on the splicing patterns of these genes for both HHV-6A and HHV-6B.HHV-6A(GS) strain was propagated in the human T-cell line HSB-2; HHV-6B(PL1) strain was propagated in activated peripheral blood mononuclear cells (PBMC). mRNA was extracted using oligo(dT) 25 -coated magnetic beads (Dynabeads mRNA direct kit; Dynal, Oslo, Norway), and total RNA was extracted using the Qiagen RNeasy minikit (Qiagen, Hilden, Germany). Primers (Table 1) were designed based on the HHV-6(U1102) sequence (4).Poly(A) ϩ RNA was treated with the Access RT-PCR kit (Promega, Madison, Wis.). Except for primers C1bis and C2bis, initial denaturation (94°C for 2 min) was followed by 35 amplification cycles (94°C for 1 min, 50°C for 1 min, and 72°C for 2 min) and by final elongation (72°C for 10 min). For primers C1bis and C2bis, initial denaturation (95°C for 15 min) was followed by 40 amplification cycles (95°C for 20 s, 50°C for 45 s, and 72°C for 30 s) and by final elongation (72°C for 2 min).Total RNA samples were treated (35°C for 15 min) with RNase-free DNase (Ambion, Austin, Tex.) and then subjected to reverse transcription (36°C for 90 min) using Moloney murine leukemia virus RT (Promega). One-tenth of the RT reaction mixture was added to 50 pmol of each primer, 100 M each deoxynucleoside triphosphate, 2.5 mM MgCl 2 , 1ϫ Perkin-Elmer buffer II, and 1.25 U of Taq polymerase (Applied Biosystems, Foster City, Calif). Initial denaturation (95°C for 15 min) was followed by 40 amplification cycles (95°C for 20 s, 60°C [50°C for primers C1bis and C2bis] for 45 s and 72°C for 30 s) and by final elongation (72°C for 2 min).For sequencing, amplimers were purified using the Qiaquick spin extraction kit (Qiagen), amplified, and sequenced with the primers mentioned above (Table 1; Fig. 1). The nucleotide positions of...

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Human immunodeficiency virus type 1 (HIV-1) infection is characterized by dysfunction of HIV-1-specific T-lymphocytes. In order to suppress the virus and delay evolution to AIDS, antigen-loaded antigen-presenting cells (eg. dendritic cells (DC), B-lymphocytes) might be useful to boost and broaden HIV-1-specific T-cell responses. Monocyte-derived DC from untreated HIV-1-infected patients were electroporated with codon-optimized (“humanized”) mRNA coding for consensus HxB-2 (hHXB-2) Gag protein. These DC elicited a strong HIV-1 Gag-specific interferon (IFN)-γ response by an HLA-A2-restricted CD8+ T-cell line. Moreover, hHXB-2 gag mRNA-electroporated DC also triggered IFN-γ secretion by autologous peripheral blood mononuclear cells (PBMC), CD8+ T-cells and CD4+ T-cells from all patients tested. Similar observations were made with CD40-activated cultured autologous B-cells (from HIV-1-seropositive patients) electroporated with hHXB-2 gag mRNA. Gag mRNA-electroporated, but not mock-electroporated, DC or B-cells secreted Gag protein. Next, a novel strategy was developed, using autologous virus sequences. Proviral DNA was amplified by polymerase chain reaction (PCR) from PBMC and viral cDNA was amplified by reverse transcriptase PCR (RT-PCR) from plasma virus. Proviral and viral mRNA were then obtained by in vitro transcription of proviral DNA and plasma viral cDNA, respectively. Significant specific IFN-γ T-cell responses were induced in all patients tested by DC electroporated with patients’ autologous proviral and plasma viral mRNA, coding for Gag or Env. The stimulatory effect was seen on PBMC, CD8+ T-cells and CD4+ T-cells, demonstrating both major histocompatibility complex (MHC) class I and MHC class II antigen presentation. Moreover, a significant interleukin (IL)-2 T-cell response was induced by DC electroporated with hHxB-2 or proviral gag mRNA. Sequence analysis in 4 randomly chosen patients showed that they were infected by 4 different subtypes. In a heteroduplex mobility assay (HMA) up to 50% of the cloned amplified sequences exhibited a differential migration pattern; by sequencing a high degree of variation was demonstrated, particularly between clones derived from proviral DNA and plasma viral cDNA, with mutations in an immunodominant epitope (Gag) or mutations and deletions in non-immunodominant epitopes (Env). The stimulatory effect of autologous DC electroporated with autologous viral sequences opens a major perspective for the development of patient-specific immunotherapy for HIV-1 disease, that might be necessary to control the virus, in view of the major inter-patient and intra-patient sequence variability.