The multivariate and multiparametric analysis described in the present study shows that the basal and VK3-20-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 (pDC) or CD11c+ myeloid DC (mDC) populations, is largely comparable between HCV-seropositive and control subjects. Overall, the markers show a trend of increased expression in all circulating cells, although the most evident and consistent pattern is observed in the CD123+ pDC and/or CD11c+ mDC cells. No significant difference was observed between results obtained in human monocyte-derived dendritic cells (MDDCs) and circulating APCs, confirming previous results from us and other groups [22, 25, 28, 29].
The overall expression pattern suggests maturation/activation induced by VK3-20, although for some specific markers and in some patients the trend does not reach statistical significance. This observation suggests that the HCV seropositivity status does not significantly impair the immune activation status and the responsiveness of circulating APC populations to the VK3-20 immunogenic stimulus. Results obtained in parallel with lipopolysaccharide (LPS) used as a positive activation factor, confirm the responsiveness of circulating APCs from both groups analyzed in the present study. Nonetheless, some HCV+ individuals show a complete lack of maturation induced by VK3-20 in circulating APCs, strongly suggesting the need for individual evaluations to identify possible impairments in response to this immunogen.
The present results confirm and extend data from others showing a normal expression of surface molecules involved in antigen-specific T-cell activation on immature and mature DCs from HIV-1-infected and hepatitis C virus (HCV)-HIV-coinfected individuals [30–32]. Furthermore, monocyte-derived DCs from either HCV-infected or HCV-HIV-coinfected subjects have been previously shown to stimulate a mixed leukocyte reaction in purified, allogeneic CD4+ T cells comparable to that with DCs derived from healthy donors [33–35].
The average basal level of the Th2 (TNF-α, IL-6, IL-4, and IL-10) cytokines is significantly higher (p < 0.02) in HCV-seropositive compared to control subjects. On the contrary, Th1 cytokine levels are equivalent in the two groups. These results suggest a Th2 polarization induced by an established HCV infection, as previously extensively reported [36–39].
VK3-20 induced a significantly higher production of the analysed Th2 cytokines in PBMCs from HCV-seropositive and control subjects, with the highest levels observed in the samples treated with the highest concentration of VK3-20 (15 μg/ml) (p < 0.05). Furthermore, the levels of Th2 cytokines induced in the HCV+ samples were significantly higher than those identified in the control samples (p < 0.01), suggesting the persistence of a prevalent Th2 status. No increase in the production of Th1 cytokines (IL-2 and IFN-γ) was observed (p < 0.4) in the control as well as HCV+ group. In particular, the production of IFN-γ is known to be inhibited by IL-10 , with a sequential detrimental effect on the IL-12-mediated induction of IFN-γ production by NK and T cells [41–43]. Therefore, the high levels of IL-10 and TNF-α induced by VK3-20 could explain the lack of increased production of IFN-γ in both groups. The observed discrepancy between the VK3-20 concentration necessary for the maximal induction of activation markers (1.5 μg/ml) and the one necessary for the maximal induction of cytokine expression (15 μg/ml) may suggest a different pathway of activation involved in the two independent biological effects, which need further investigation.
The similar response observed in HCV-seropositive subjects, regardless of the diagnosis of type II MC or NHL, would suggest the absence of an in vivo priming for the VK3-20. In this regard, the expression of VK3-20 in the clonal B-cell populations of these subjects is currently under evaluation.
The impairment of basal and antigen-induced production of Th1-polarizing cytokines for HCV-seropositive individuals is in concordance with our previous observations on PBMCs from HIV infected subjects exposed ex vivo to a VLP-based HIV vaccine model [25, 44].
The overall results here described represent a proof-of-concept and confirm the possibility of screening donor susceptibility to an antigen treatment using circulating APCs, CD14+ monocytes as well as CD123+ plasmacytoid DC (pDC) or CD11c+ myeloid DC (mDC) populations, without the need of purification and ex vivo selection of DCs, simplifying the identification of "responsive" vaccinees and providing mechanisms of eventual failures in individuals enrolled in clinical trials. When necessary, additional and more detailed studies on fractionated cell types would allow identification and a better characterization of the individual cells involved in mediating the in vivo response. In conclusion, our results indicate that circulating APCs from HCV-seropositive patients can be functional in active autologous immunotherapy strategies. In particular, the results strongly suggest the induction of the innate and early adaptive immune response by the protein corresponding to the VK3-20 light chain of the idiotype most frequently identified on B cell clones sustaining the HCV-associated type II MC and NHL. Therefore, its use as preventive as well as therapeutic vaccination strategy appears to be feasible and potentially effective. However, specific Th1-driving adjuvant strategies might be necessary to obtain the sought efficient therapeutic effect.