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. 2010 Feb 19:8:18.
doi: 10.1186/1479-5876-8-18.

Immune signatures in human PBMCs of idiotypic vaccine for HCV-related lymphoproliferative disorders

Luigi Buonaguro  1 , Annacarmen Petrizzo, Marialina Tornesello, Maria Napolitano, Debora Martorelli, Giuseppe Castello, Gerardo Beneduce, Amalia De Renzo, Oreste Perrella, Luca Romagnoli, Vitor Sousa, Valli De Re, Riccardo Dolcetti, Franco M Buonaguro
Affiliations

Immune signatures in human PBMCs of idiotypic vaccine for HCV-related lymphoproliferative disorders

Luigi Buonaguro et al. J Transl Med. .

Abstract

Hepatitis C virus (HCV) is one of the major risk factors for chronic hepatitis, which may progress to cirrhosis and hepatocellular carcinoma, as well as for type II mixed cryoglobulinemia (MC), which may further evolve into an overt B-cell non-Hodgkin's lymphoma (NHL). It has been previously shown that B-cell receptor (BCR) repertoire, expressed by clonal B-cells involved in type II MC as well as in HCV-associated NHL, is constrained to a limited number of variable heavy (VH)- and light (VL)-chain genes. Among these, the VK3-20 light chain idiotype has been selected as a possible target for passive as well as active immunization strategy. In the present study, we describe the results of a multiparametric analysis of the innate and early adaptive immune response after ex vivo stimulation of human immune cells with the VK3-20 protein. This objective has been pursued by implementing high-throughput technologies such as multiparameter flow cytometry and multiplex analysis of cytokines and chemokines.

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Figures

Figure 1
Figure 1
PBMCs were incubated with increasing doses of VK3-20 protein for 16 hrs. The expression of CD83, CD86 and HLADR was analysed by FACScalibur flow cytometer in CD14+ monocytes, CD123+ pDCs and CD11c+ mDCs. Data analysis was carried out with WinMDI2.8 Software. One representative experiment is shown.
Figure 2
Figure 2
Comparative analysis of the expression of surface maturation/activation markers (CD83, CD86, HLADR) performed on stimulated MDDCs, CD123+ pDCs and CD11c+ mDCs.
Figure 3
Figure 3
6-color flow cytometric analysis was performed on VK3-20 protein-stimulated monocytes, mDC/pDC cell populations and immunophenotype analysis of surface maturation/activation marker expression is shown. Values in each quadrant represent the percentage of positive cells.
Figure 4
Figure 4
Basal level expression of surface maturation/activation markers, indicated as Mean Fluorescence Index (MFI), on PBMC-derived monocytes and DC from control and HCV positive (HCV+) subjects. CD14 = CD14+ monocytes; CD123 = CD123+ pDCs; CD11c = CD11c+ mDCs.
Figure 5
Figure 5
Expression of surface maturation/activation markers, indicated as Mean Fluorescence Index (MFI), induced by the indicated concentrations of VK3-20 and LPS in PBMC-derived monocytes and DC from control subjects. CD14 = CD14+ monocytes; CD123 = CD123+ pDC; CD11c = CD11c+ mDC.
Figure 6
Figure 6
Expression of surface maturation/activation markers, indicated as Mean Fluorescence Index (MFI), induced by the indicated concentrations of VK3-20 and LPS in PBMC-derived monocytes and DC from HCV seropositive subjects. CD14 = CD14+ monocytes; CD123 = CD123+ pDC; CD11c = CD11c+ mDC.
Figure 7
Figure 7
Analysis of basal level production of Th1 and Th2 cytokines in supernatants of PBMCs from control and HCV positive (HCV+) subjects.
Figure 8
Figure 8
Analysis of Th1 and Th2 cytokines in supernatants of PBMCs from control subjects induced by the indicated concentrations of VK3-20 and LPS.
Figure 9
Figure 9
Analysis of Th1 and Th2 cytokines in supernatants of PBMCs from HCV seropositive subjects induced by the indicated concentrations of VK3-20 and LPS.
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