Multiparametric Analyses of Human PBMCs Loaded Ex Vivo with a Candidate Idiotype Vaccine for HCV-Related Lymphoproliferative Disorders

Annacarmen Petrizzo,Ena Wang,Valli De Re,Luigi Buonaguro,Franco M Buonaguro,Maria Lina Tornesello,Maria Napolitano,Riccardo Dolcetti,Franco M Marincola,Giovanna D'Alessio,Angelo Salomone Megna,Ranjit Ray

doi:10.1371/journal.pone.0044870

Abstract

Hepatitis C virus (HCV) has been identified as one of the major risk factors for type II mixed cryoglobulinemia (MC), during the clinical evolution of chronic hepatitis, which may lead to development of B cell non-Hodgkin's lymphoma (NHL). We have previously shown that the candidate idiotype vaccine, based on the IGKV3-20 light chain protein, is able to induce activation and maturation of circulating antigen presenting cells (APCs) in both HCV-positive and HCV-negative healthy control subjects, with production of Th2-type cytokines. Here, the effect of the recombinant IGKV3-20 protein on human peripheral blood mononuclear cells (PBMCs) from HCV-positive subjects, with known blood levels of cryoglobulins, is shown via gene expression profiling analysis combined to multiparameter flow cytometry and multiplex analyses of cytokines.

Highlights

Hepatitis C virus (HCV) is a Hepacivirus of the Flaviviridae family, mainly involved in hepatic disorders, including chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) [1].HCV has been implicated as one of the major risk factors for type II mixed cryoglobulinemia (MC), an autoimmune disease leading to B cell non-Hodgkin’s lymphoma (NHL) in about 10% of MC patients [2,3,4,5].The most accredited pathogenetic mechanism of MC during HCV chronic infection is the persistent immune stimulation of Bcell compartment by viral proteins (e.g. HCV E2 protein) which drives the expansion of cellular clones resulting in production of cross-reactive autoantibodies, including cryoglobulins [6,7]
The results showed that IGKV3-20-induced expression of activation markers and co-stimulatory molecules in the evaluated circulating antigen presenting cells (APCs), CD14+ monocyte as well as CD123+ plasmacytoid DC or CD11c+ myeloid DC populations, is largely comparable between HCV-positive and control subjects
In order to evaluate the maturation markers induced by recombinant IGKV3-20 protein, the expression of CD40, CD80, CD83, CD86 and HLA-DR was examined in circulating APCs (i.e. CD14+ monocytes, and CD11c+ myeloid dendritic cells myeloid DC (mDC)) by flow cytometry

Summary

Introduction

HCV has been implicated as one of the major risk factors for type II mixed cryoglobulinemia (MC), an autoimmune disease leading to B cell non-Hodgkin’s lymphoma (NHL) in about 10% of MC patients [2,3,4,5]. The most accredited pathogenetic mechanism of MC during HCV chronic infection is the persistent immune stimulation of Bcell compartment by viral proteins (e.g. HCV E2 protein) which drives the expansion of cellular clones resulting in production of cross-reactive autoantibodies, including cryoglobulins [6,7]. The Id can be a suitable target for active and passive immune-therapeutic strategies to eliminate clonal B cells driving the tumor [11]. Each individual patient needs to be characterized, in order to identify the autologous Id expressed by the clonal tumor B cells and to develop the patient-specific vaccine

Methods

Results

Conclusion