ALBERT

Description: Dendritic cells (DC), the professional antigen presenting cells of the immune system, exert important functions both in induction of T cell immunity as well as of tolerance. Previously, it was accepted that the main function of immature DC (iDC) in their in vivo steady state condition is to maintain peripheral tolerance to self-antigens and that these iDC mature upon encounter of so-called danger signals and subsequently promote T cell immunity. However, a growing body of experimental evidence now indicates that traditional DC maturation can no longer be used to distinguish between tolerogenic and immunogenic properties of DC. In this study, we compared the in vitro stimulatory capacity of immature DC (iDC), cytokine cocktail-matured DC (CC-mDC) and poly I:C-matured DC (pIC-mDC) in the absence and presence of antigen. All investigated DC types could induce at least 2 subsets of regulatory T cells. We observed a significant increase in both the number of functionally suppressive transforming growth factor (TGF)-beta+ interleukin (IL)-10+ T cells as well as of CD4+CD25+FOXP3+ T cells within DC/T cell co-cultures as compared to T cell cultures without DC. The induction of these regulatory T cells correlates with in vitro T cell non-responsiveness after co-culture with iDC and CC-mDC, while stimulation with pIC-mDC resulted in reproducible cytomegalovirus pp65 or influenza M1 matrix peptide-specific T cell activation as compared to control cultures in the absence of DC. In addition, the T cell non-responsiveness after stimulation with iDC was shown to be mediated by TGF-beta and IL-10. Moreover, the suppressive capacity of CD4+ T cells activated by iDC and CC-mDC was shown to be transferable when these CD4+ T cells were added to an established T cell response. In contrast, addition of CD4+ T cells stimulated by pIC-mDC made responder T cells refractory to their suppressive activity. In conclusion, we hypothesize that DC have a complementary role in inducing both regulatory T cells and effector T cells, where the final result of antigen-specific T cell activation will depend on the activation state of the DC. This emphasizes the need for proper DC activation when T cell immunity is the desired effect, especially when used in clinical trials.

Description: Leukemic cells exert immunosuppressive effects that interfere with dendritic cell function and hamper effective anti-leukemic immune responses. Recently, Toll-like receptor 3 (TLR3) was characterized in dendritic cells as an intracellular double-stranded (ds)RNA receptor which is triggered by viral infection or incubation with the synthetic dsRNA analogue polyriboinosinic polyribocytidylic acid [poly(I:C)], leading to maturation and activation of dendritic cells. Until now, little was known on the expression of TLR3 in leukemic cells and their responsiveness to dsRNA treatment. We assessed TLR3 expression in primary and transformed acute myeloid leukemia (AML) cells and hypothesized that the immunogenicity of AML cells could be improved by treatment with the synthetic TLR3 agonist poly(I:C), thereby mimicking viral infection of these leukemic cells. In view of this hypothesis, we electroporated or pulsed transformed and primary AML cells with poly(I:C) and analyzed the effect of poly(I:C) loading on TLR3 expression, costimulatory molecules, cytokine production and allogeneic T cell response. We also assessed the uptake of poly(I:C)-loaded leukemic cells by immature dendritic cells and the subsequent effect on dendritic cell activation and maturation status. We observed that primary and transformed AML cells respond to poly(I:C) electroporation by upregulation of TLR3 expression, apoptosis, elevated levels of costimulatory molecules CD80 and CD86 and by production of type I interferons (IFN). Furthermore, poly(I:C)-electroporated AML cells induced interferon-gamma production by allogeneic T cells. Upon phagocytosis of poly(I:C)-electroporated AML cells, dendritic cells showed an increased expression of maturation markers and marked production of proinflammatory cytokines. In contrast, this set of immune effects was absent or suboptimal when AML cells were passively pulsed with poly(I:C), indicating the superiority of transfection over pulsing with poly(I:C). These results demonstrate that poly(I:C) electroporation is a promising novel strategy to increase the immunogenicity of AML cells.

Description: In adoptive cellular immunotherapy, T cells can be genetically engineered to express a novel T-cell receptor (TCR) that recognizes a tumor-associated antigen. However, mispairing between transgene and endogenous TCR chains may result in a reduction of transgene TCR expression and potentially harmful off-target reactivities. Here, we sought to develop a novel clinically safe strategy to promote transgene expression of a Wilms' tumor 1 (WT1)-specific TCR by Dicer-substrate small interfering RNA (DsiRNA)-mediated silencing of the endogenous TCR, using a double electroporation protocol. First, we isolated and cloned an HLA-A*0201-restricted WT1 peptide-specific TCR derived from a leukemia patient who demonstrated clinical benefit after receiving a WT1-targeted DC vaccine. Next, we produced a codon-optimized TCR sequence from the wild-type TCR construct and both TCR mRNAs were generated by in vitro transcription. TCR expression levels were validated by electroporation of TCR-deficient Jurkat J76.7 cells stably transduced with CD8 and an NFAT-driven GFP reporter gene. TCR functionality was confirmed by high expression levels of GFP (70% GFP+cells) upon TCR signaling after co-culture with WT1 peptide-pulsed T2 cells. In order to suppress the translation of endogenous TCR mRNA in CD8+ T cells, DsiRNA duplexes were designed to specifically target the constant regions of wild-type TCR α- and β-chains, but not the codon-optimized TCR. We further developed a double electroporation protocol combining DsiRNA and TCR mRNA transfection in which DsiRNA electroporation was performed 24 hours prior to TCR mRNA electroporation. Our results show more than 2-fold increase in WT1-specific TCR expression by HLA-A2/WT1 tetramer staining after DsiRNA treatment as compared to TCR mRNA electroporation only. This specific TCR expression was maintained at least 5 days after TCR mRNA electroporation in resting peripheral blood CD8+ lymphocytes from healthy donors. The enhanced TCR expression in DsiRNA-transfected CD8+T cells was also correlated with an increase of epitope recognition as shown by interferon (IFN)-γ ELISpot. To determine the killing capacity of DsiRNA/TCR mRNA-transfected CD8+ T cells against epitope-bearing target cells, we performed a flow cytometry-based cytotoxicity assay using WT1 peptide-pulsed T2 cells. Specific cytotoxicity, which was already present in WT1 TCR-transfected cells, was significantly enhanced in TCR mRNA-electroporated T cells following suppression of the endogenous TCR expression by DsiRNA treatment. Accordingly, DsiRNA-treated TCR mRNA transfected CD8+T cells presented higher levels of CD137 and CD69 activation markers and secretion of cytokines (IFN-γ and tumor necrosis factor-α), granzyme B, and perforin upon TCR triggering as compared to the non-DsiRNA treated T cells. In summary, we show a marked enhancement of transgene WT1-specific TCR expression upon silencing of the endogenous TCR using DsiRNA electroporation prior to TCR mRNA electroporation. Importantly, this enhancement in TCR expression was correlated with a significant increase in WT1-specific CD8+ T-cell killing activity, expression of CD69 and CD137 activation markers and cytokine secretion after recognition of WT1 peptide-bearing target cells. These results pave the way for developing a clinically safer strategy for T cell-based adoptive immunotherapy of patients with WT1-expressing malignancies. Disclosures No relevant conflicts of interest to declare.

Description: It has been seen that a significant number of hemophiliacs show a co-existence of mutations related to an increased risk of thrombosis (Foka et al 2003). It has been also suggested that these thrombophilic genetic defects may actually protect hemophiliacs from recurrent bleeding episodes or may even lead to thrombosis in several cases (Makris et al 2003). Aim of the study was to investigate the coagulation activity and the existence of differences between hemophiliacs with and without thrombophilic mutations. Materials and Methods: 65 subjects were studied (58 hemophiliacs 42±15 and 7 normal subjects 45±10 year old). We divided our subjects in 4 groups: 1- hemophiliacs without mutations (HN) 27 patients (pts), 2- hemophiliacs with FVLeiden and/or FIIG20210A mutations (HHet), 15 pts, 3- hemophiliacs with MTHFR mutation (homozygotes, HTT) 16 pts and 4-normal subjects (NS) 7. Prothrombinase generation was evaluated by determination of F1+2 fragments of Prothrombin and the activation of coagulation by the determination of TAT levels (tested with Elisa). Simultaneously, we applied the Hicks and Pitney test to evaluate these results, and the Thrombin Generation test as a confirmatory standard test. The Hicks and Pitney test was applied in an initial mixture of Cephaline and CaCl2 in equal and sufficient quantity of diluted PPP (1/10) incubated in 37°C. Initially, from this mixture, 0,1ml was transferred to a new tube containing 0,1ml substrate normal plasma and 0,1ml CaCl2. This procedure was repeated 10 consecutive times with 1 min interval. In parallel, 0,1ml from each incubation was frozen for measurement of F1+2 and TAT. Concerning the thrombin generation, we applied Endogenous Thrombin Potential (ETP) as described by Hemker et al (2000). Our results are presented graphically with the picture. In these graphs it can be seen that hemophiliacs with no mutations show a gradual prothrombinase generation. On the contrary, HHet show a rapid generation that is also expressed by the concomitant increase in the levels of F1+2 and TAT. A similar response was observed in the group of normal subjects. This observation can be explained as an adjustment mechanism of the coagulation defect, possibly able to prevent the high frequency and severity of bleeding episode in hemophiliacs with mutations. These results were more revelatory using the ETP test. The comparison between the different groups using the t-test distribution curve was as follow: a- Comparison between NS and HHet, p

Description: 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.

Description: Aim: We decide to study the effect of erythropoietin on the platelet function and glycoproteins expression in patients (pts) with myelodysplastic syndrome (MDS). All patients suffered from primary MDS but none of them received any special treatment. From our study pts that suffered from diseases or they were receiving medication that affect the function of platelets were excluded. Furthermore pts with platelet count

Description: 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, including dendritic cells (DC) might be useful to boost and broaden HIV-1-specific T-cell responses. Monocyte-derived DC from 15 untreated (“naive”) and 15 highly active anti-retroviral therapy (HAART)-treated HIV-1-infected patients were electroporated with codon-optimized (“humanized”) mRNA encoding consensus HxB-2 (hHxB-2) Gag protein. These DC were co-cultured for 1 week with autologous peripheral blood leucocytes (PBL). Potential expansion of specific T-cells was measured by comparing ELISPOT responses of PBL before and after co-culture, using a pool of overlapping peptides, spanning the HxB-2 Gag. Expansion of specific PBL after co-culture was noted for T cells producing interferon (IFN)-gamma, interleukin (IL)-2 and perforin (Wilcoxon signed rank test p