J.I. Riezu-Boj

Specific and general HLA-DR binding motifs: comparison of algorithms.

Using panels of peptides well characterized for their ability to bind to HLA DR1, DRB1*1101, or DRB1*0401 molecules, algorithms were deduced to predict binding to these molecules. These algorithms consist of blocks of 8 amino acids containing an amino acid anchor (Tyr, Phe, Trp, Leu, Ile, or Val) at position i and different amino acid combinations at positions i+2 to i+7 depending on the class II molecule. The sensitivity (% of correctly predicted binder peptides) and specificity (% of correctly predicted non-binder peptides) of these algorithms, were tested against different independent panels of peptides and compared to other algorithms reported in the literature. Similarly, using a panel of 232 peptides able to bind to one or more HLA molecules as well as 43 non-binder peptides, we deduced a general motif for the prediction of binding to HLA-DR molecules. The sensitivity and specificity of this general motif was dependent on the threshold score used for the predictions. For a score of 0.1, the sensitivity and specificity were 84.7% and 69.8%, respectively. This motif was validated against several panels of binder and non-binder peptides reported in the literature, as well as against 35, 15-mer peptides from hepatitis C virus core protein, that were synthesized and tested in a binding assay against a panel of 19 HLA-DR molecules. The sensitivities and specificities against these panels of peptides were similar to those attained against the panels used to deduce the algorithm. These results show that comparison of binder and non-binder peptides, as well as correcting for the relative abundance of amino acids in proteins, is a useful approach to deduce performing algorithms to predict binding to HLA molecules.

Hepatitis C virus induces the expression of CCL17 and CCL22 chemokines that attract regulatory T cells to the site of infection

BACKGROUND & AIMS The mechanisms by which Foxp3+ T regulatory cells (Treg) accumulate in HCV infected livers are not known. Here, we studied the role of chemokines CCL17 and CCL22 in this process. METHODS Chemokine mRNA levels were determined by qPCR in liver biopsies from 26 HCV chronically infected patients (CHC), 11 patients with treatment-induced sustained virological response (SVR), 16 patients with other liver diseases unrelated to HCV, and 24 normal livers. Double-immunofluorescence Foxp3/CD3 or CD11c/CCL22 was performed in liver sections. Chemokine production by monocyte-derived dendritic cells (MDDC) co-cultured with uninfected or HCV-JFH1 infected Huh7 cells was measured by qPCR and ELISA. Chemotactic activity of culture supernatants was also tested. RESULTS Foxp3+ Treg were increased in CHC livers as compared to controls. Patients with CHC showed elevated intrahepatic levels of CCL17 mRNA compared to normal livers or livers from subjects with SVR or other forms of liver disease. Intrahepatic CCL22 expression was also higher in CHC than in healthy subjects or SVR patients but similar to that observed in other liver diseases. Dendritic cells producing CCL22 could be found inside the hepatic lobule in CHC patients. Contact between MDDC and HCV-JFH1-infected Huh7 cells induced the expression of CCL17 and CCL22 in a process partially dependent on ICAM-1. Transwell experiments showed that upregulation of these chemokines enhanced Treg migration. CONCLUSIONS Contact of HCV-infected cells with dendritic cells induces the production of Treg-attracting chemokines, an effect which may favour liver accumulation of Treg in CHC. Our findings contribute to explain the mechanism by which HCV escapes the immune response and thus reveals novel therapeutic targets.

Effects of IFN-α as a signal-3 cytokine on human naïve and antigen-experienced CD8(+) T cells.

IFN‐α/β link innate and adaptive immune responses by directly acting on naïve CD8+ T cells. This concept unveiled in mice remains unexplored in humans. To investigate that, human CD8+CD45RO− cells were stimulated with beads coated with anti‐CD3 and anti‐CD28 mAb, mimicking Ag (type‐1) and co‐stimulatory (type‐2) signals, in the presence or absence of IFN‐α and their transcriptional profiles were defined by cDNA‐microarrays. We show that IFN‐α provides a strong third signal directly to human CD8+ T cells resulting in regulation of critical genes for their overall activation. This transcriptional effect was substantiated at the protein level and verified by functional assays. Interestingly, the biological effects derived from this stimulation vary depending on the CD8+ T‐cell population. Thus, whereas IFN‐α increases the proliferative capacity of naïve CD8+ T cells, it inhibits or does not affect the proliferation of Ag‐experienced cells, such as memory and effector CTL, including CMV‐specific lymphocytes. Cytolysis and IFN‐γ‐secretion of all these populations are enhanced by IFN‐α‐derived signals, which are critical in naïve CD8+ T cells for acquisition of effector functions. Our findings in human CD8+ T cells are informative to understand and improve IFN‐α‐based therapies for viral and malignant diseases.

Immunogenicity of variable regions of hepatitis C virus proteins: selection and modification of peptide epitopes to assess hepatitis C virus genotypes by ELISA

The immunogenicity of variable regions of hepatitis C virus (HCV) proteins was studied by ELISA by using 543 synthetic peptides from 120 variable regions and 90 sera from HCV-infected patients. Some regions from certain genotypes were less immunogenic, or even non-immunogenic, compared with their equivalents in other genotypes. However, the mean recognition of all peptides from genotypes 1a, 1b and 3 by sera infected with genotypes 1a, 1b and 3, respectively, showed no significant differences, suggesting a similar overall immunogenicity of variable regions from these genotypes. Proteins NS4a, NS4b and NS5a were found to be the most immunogenic. Recognition of individual peptides by the sera of infected patients showed that the humoral response against HCV is patient-dependent. The work shows that 15-mer peptides may encompass several B-cell epitopes. These epitopes may lie in slightly different positions in different genotypes. Thirty-one percent of the 543 peptides were recognized by some of the 35 healthy donors. This may be a reflection of the large number of antigens to which they had been exposed, but it may also reflect a strategy of HCV to respond to immune pressure. After selection and modification, a set of 40 peptides was used to assess genotypes 1a, 1b, 1, 2 and 3 in the sera of HCV-infected patients, with sensitivities of 34.1, 48.5, 68.8, 58.3 and 48.9% and specificities of 100, 99.1, 97.1, 99.5 and 99%, respectively. The overall sensitivity and specificity for the assessment of genotypes 1, 2 and 3 were 64 and 98%, respectively.

CD8 T Cell Priming in the Presence of IFN-α Renders CTLs with Improved Responsiveness to Homeostatic Cytokines and Recall Antigens: Important Traits for Adoptive T Cell Therapy

Previous mouse and human studies have demonstrated that direct IFN-α/β signaling on naive CD8 T cells is critical to support their expansion and acquisition of effector functions. In this study, we show that human naive CD8 T cells primed in the presence of IFN-α possess a heightened ability to respond to homeostatic cytokines and to secondary Ag stimulation, but rather than differentiating to effector or memory CTLs, they preserve nature-like phenotypic features. These are qualities associated with greater efficacy in adoptive immunotherapy. In a mouse model of adoptive transfer, CD8 T cells primed in the presence of IFN-α are able to persist and to mediate a robust recall response even after a long period of naturally driven homeostatic maintenance. The long-lasting persistence of IFN-α–primed CD8 T cells is favored by their enhanced responsiveness to IL-15 and IL-7, as demonstrated in IL-15−/− and IL-7−/− recipient mice. In humans, exposure to IFN-α during in vitro priming of naive HLA-A2+ CD8 T cells with autologous dendritic cells loaded with MART126–35 peptide renders CD8 T cells with an improved capacity to respond to homeostatic cytokines and to specifically lyse MART1-expressing melanoma cells. Furthermore, in a mouse model of melanoma, adoptive transfer of tumor-specific CD8 T cells primed ex vivo in the presence of IFN-α exhibits an improved ability to contain tumor progression. Therefore, exposure to IFN-α during priming of naive CD8 T cells imprints decisive information on the expanded cells that can be exploited to improve the efficacy of adoptive T cell therapy.

Strict Requirement for Vector-Induced Type I Interferon in Efficacious Antitumor Responses to Virally Encoded IL12

Host responses are increasingly considered important for the efficacious response to experimental cancer therapies that employ viral vectors, but little is known about the specific nature of host responses required. In this study, we investigated the role of host type I interferons (IFN-I) in the efficacy of virally delivered therapeutic genes. Specifically, we used a Semliki Forest virus encoding IL12 (SFV-IL12) based on its promise as an RNA viral vector for cancer treatment. Intratumoral injection of SFV-IL12 induced production of IFN-I as detected in serum. IFN-I production was abolished in mice deficient for the IFNβ transcriptional regulator IPS-1 and partially attenuated in mice deficient for the IFNβ signaling protein TRIF. Use of bone marrow chimeric hosts established that both hematopoietic and stromal cells were involved in IFN-I production. Macrophages, plasmacytoid, and conventional dendritic cells were each implicated based on cell depletion experiments. Further, mice deficient in the IFN-I receptor (IFNAR) abolished the therapeutic activity of SFV-IL12, as did a specific antibody-mediated blockade of IFNAR signaling. Reduced efficacy was not caused by an impairment in IL12 expression, because IFNAR-deficient mice expressed the viral IL12 transgene even more strongly than wild-type (WT) hosts. Chimeric host analysis for the IFNAR involvement established a strict requirement in hematopoietic cells. Notably, although tumor-specific CD8 T lymphocytes expanded robustly after intratumoral injection of WT mice with SFV-IL12, this did not occur in mice where IFNAR was inactivated genetically or pharmacologically. Overall, our results argued that the antitumor efficacy of a virally based transgene therapeutic relied strongly on a vector-induced IFN-I response, revealing an unexpected mechanism of action that is relevant to a broad array of current translational products in cancer research.

IFN-α5 Mediates Stronger Tyk2-Stat-Dependent Activation and Higher Expression of 2′,5′-Oligoadenylate Synthetase Than IFN-α2 in Liver Cells

Interferon-α5 (IFN-α5) is the main IFN-α subtype expressed in the liver. Hepatitis C virus (HCV) infection is associated with low IFN-α5 mRNA levels, possibly reflecting an escape mechanism of the virus. In this work, we sought to compare IFN-α2 and IFN-α5 with respect to activation of early cell signaling cascades and induction of antiviral genes in the human hepatoma HepG2 and Huh7 cell lines. We found that the Tyr701 phosphorylation kinetics of Stat1 mediated by IFN stimulation was higher when cells were incubated with IFN-α5 than when using IFN-α2. Similarly, Tyr1054/1055 phosphorylation kinetics of Tyk2 were more intense after exposure to IFN-α5 than when using IFN-α2. Concomitantly, Tyr705 phosphorylation of Stat3 was higher after stimulation with IFN-α5 than with IFN-α2. In parallel to these findings, the mRNA levels of the antiviral IFN-inducible gene 2′,5′-oligoadenylate synthetase were higher in cell samples treated with IFN-α5 than with IFN-α2. These findings suggest that interaction of IFN-α5 ...

Detection of hepatitis C virus antibodies with new recombinant antigens: assessment in chronic liver diseases

A new serological assay to detect antibodies against hepatitis C, based on a recombinant protein (BHC10) which incorporates structural and non-structural viral antigens, was tested in 67 healthy subjects and 409 patients with various forms of liver disease. Results were compared with the current assay based on the recombinant non-structural viral antigen c100 and with the recently introduced second-generation assay, Ortho2. None of the healthy subjects was positive by any of the assays. In patients with chronic non-A, non-B hepatitis the prevalence of anti-BHC10 was 96.8%, higher than anti-c100 (83.3%, p less than 0.001) and similar to Ortho2 (94.3%). False-positive results were less frequently found when BHC10 was used. These findings show that assays incorporating structural and non-structural antigens provide higher sensitivity to detect hepatitis C virus infection and they define an almost exclusive role of hepatitis C virus in the genesis of chronic non-A, non-B hepatitis.

Conventional but Not Plasmacytoid Dendritic Cells Foster the Systemic Virus–Induced Type I IFN Response Needed for Efficient CD8 T Cell Priming

Plasmacytoid dendritic cells (pDCs) are considered to be the principal type-I IFN (IFN-I) source in response to viruses, whereas the contribution of conventional DCs (cDCs) has been underestimated because, on a per-cell basis, they are not considered professional IFN-I–producing cells. We have investigated their respective roles in the IFN-I response required for CTL activation. Using a nonreplicative virus, baculovirus, we show that despite the high IFN-I–producing abilities of pDCs, in vivo cDCs but not pDCs are the pivotal IFN-I producers upon viral injection, as demonstrated by selective pDC or cDC depletion. The pathway involved in the virus-triggered IFN-I response is dependent on TLR9/MyD88 in pDCs and on stimulator of IFN genes (STING) in cDCs. Importantly, STING is the key molecule for the systemic baculovirus-induced IFN-I response required for CTL priming. The supremacy of cDCs over pDCs in fostering the IFN-I response required for CTL activation was also verified in the lymphocytic choriomeningitis virus model, in which IFN-β promoter stimulator 1 plays the role of STING. However, when the TLR-independent virus-triggered IFN-I production is impaired, the pDC-induced IFNs-I have a primary impact on CTL activation, as shown by the detrimental effect of pDC depletion and IFN-I signaling blockade on the residual lymphocytic choriomeningitis virus–triggered CTL response detected in IFN-β promoter stimulator 1−/− mice. Our findings reveal that cDCs play a major role in the TLR-independent virus-triggered IFN-I production required for CTL priming, whereas pDC-induced IFNs-I are dispensable but become relevant when the TLR-independent IFN-I response is impaired.

Gene expression analysis during acute hepatitis C virus infection associates dendritic cell activation with viral clearance.

Viral clearance during acute hepatitis C virus (HCV) infection is associated with the induction of potent antiviral T‐cell responses. Since dendritic cells (DC) are essential in the activation of primary T‐cell responses, gene expression was analyzed in DC from patients during acute HCV infection. By using microarrays, gene expression was compared in resting and activated peripheral blood plasmacytoid (pDC) and myeloid (mDC) DC from acute HCV resolving patients (AR) and from patients who become chronically infected (ANR), as well as in healthy individuals (CTRL) and chronically‐infected patients (CHR). For pDC, a high number of upregulated genes was found in AR patients, irrespective of DC stimulation. However, for mDC, most evident differences were detected after DC stimulation, again corresponding to upregulated genes in AR patients. Divergent behavior of ANR was also observed when analyzing DC from CTRL and CHR, with ANR patients clustering again apart from these groups. These differences corresponded to metabolism‐associated genes and genes belonging to pathways relevant for DC activation and cytokine responses. Thus, upregulation of relevant genes in DC during acute HCV infection may determine viral clearance, suggesting that dysfunctional DC may be responsible for the lack of efficient T‐cell responses which lead to chronic HCV infection. J. Med. Virol. 88:843–851, 2016.