The scavenger receptors SRA-1 and SREC-I cooperate with TLR2 in the recognition of the hepatitis C virus non-structural protein 3 by dendritic cells

Backgrounds & AimsThe hepatitis C virus NS3 protein is taken up by myeloid cells in a TLR2-independent manner and activates myeloid cells via TLR2. This study aimed to identify the endocytic receptor(s) involved in the uptake of NS3 by myeloid cells and its relation with TLR2. Methods Inhibitors and transfected cells were used to identify the nature of the NS3-binding receptors expressed by myeloid cells. The cooperation between scavenger receptors (SRs) and TLR2 in the NS3-mediated activation of myeloid cells was evaluated using inhibitors, cells from TLR2−/− mice, and confocal microscopy. The involvement of SRs in NS3 cross-presentation was evaluated in vitro using an NS3-specific human T-cell clone. Results We observed that SRs are the main binding structures for NS3 on myeloid cells and identified the SRs SRA-1 and SREC-I as endocytic receptors for NS3. Moreover, both SRs and TLR2 cooperate in NS3-induced myeloid cell activation. Conclusion This study highlights a central role for SRs in NS3 uptake and cross-presentation, and demonstrates a tightly orchestrated cooperation between signalling and endocytic innate receptors in NS3 recognition

A novel HLA-B18 restricted CD8+ T cell epitope is efficiently cross-presented by dendritic cells from soluble tumor antigen

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8<sup>+</sup>T cell epitope, NY-ESO-1<sub>88–96</sub> (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1<sub>157–165</sub> epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1<sub>88–96</sub> is much more efficiently cross-presented from the soluble form, than NY-ESO-1<sub>157–165</sub>. On the other hand, NY-ESO-1<sub>157–165</sub> is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A<sub>26–35</sub>; whereas NY-ESO-1<sub>88–96</sub> was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1<sub>88–96</sub> is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18+ melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1<sub>88–96</sub> from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8<sup>+</sup>T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed

A large-scale study of the random variability of a coding sequence: a study on the CFTR gene

Coding single nucleotide substitutions (cSNSs) have been studied on hundreds of genes using small samples (ngapproximate to100-150 genes). In the present investigation, a large random European population sample (average ngapproximate to1500) was studied for a single gene, the CFTR ( Cystic Fibrosis Transmembrane conductance Regulator). The nonsynonymous (NS) substitutions exhibited, in accordance with previous reports, a mean probability of being polymorphic (q>0.005), much lower than that of the synonymous ( S) substitutions, but they showed a similar rate of subpolymorphic (q<0.005) variability. This indicates that, in autosomal genes that may have harmful recessive alleles (nonduplicated genes with important functions), genetic drift overwhelms selection in the subpolymorphic range of variability, making disadvantageous alleles behave as neutral. These results imply that the majority of the subpolymorphic nonsynonymous alleles of these genes are selectively negative or even pathogenic

Stage-Specific Inhibition of MHC Class I Presentation by the Epstein-Barr Virus BNLF2a Protein during Virus Lytic Cycle

gamma-herpesvirus Epstein-Barr virus (EBV) persists for life in infected individuals despite the presence of a strong immune response. During the lytic cycle of EBV many viral proteins are expressed, potentially allowing virally infected cells to be recognized and eliminated by CD8+ T cells. We have recently identified an immune evasion protein encoded by EBV, BNLF2a, which is expressed in early phase lytic replication and inhibits peptide- and ATP-binding functions of the transporter associated with antigen processing. Ectopic expression of BNLF2a causes decreased surface MHC class I expression and inhibits the presentation of indicator antigens to CD8+ T cells. Here we sought to examine the influence of BNLF2a when expressed naturally during EBV lytic replication. We generated a BNLF2a-deleted recombinant EBV (ΔBNLF2a) and compared the ability of ΔBNLF2a and wild-type EBV-transformed B cell lines to be recognized by CD8+ T cell clones specific for EBV-encoded immediate early, early and late lytic antigens. Epitopes derived from immediate early and early expressed proteins were better recognized when presented by ΔBNLF2a transformed cells compared to wild-type virus transformants. However, recognition of late antigens by CD8+ T cells remained equally poor when presented by both wild-type and ΔBNLF2a cell targets. Analysis of BNLF2a and target protein expression kinetics showed that although BNLF2a is expressed during early phase replication, it is expressed at a time when there is an upregulation of immediate early proteins and initiation of early protein synthesis. Interestingly, BNLF2a protein expression was found to be lost by late lytic cycle yet ΔBNLF2a-transformed cells in late stage replication downregulated surface MHC class I to a similar extent as wild-type EBV-transformed cells. These data show that BNLF2a-mediated expression is stage-specific, affecting presentation of immediate early and early proteins, and that other evasion mechanisms operate later in the lytic cycle

CCR7 is involved in the migration of neutrophils to lymph nodes

Increasing evidence suggests that neutrophils may participate in the regulation of adaptive immune responses, and can reach draining lymph nodes and cross-prime naive T cells. The aim of this study was to identify the mechanism(s) involved in the migration of neutrophils to the draining lymph nodes. We demonstrate that a subpopulation of human and mouse neutrophils express CCR7. CCR7 is rapidly expressed at the membrane upon stimulation. In vitro, stimulated human neutrophils migrate in response to the CCR7 ligands CCL19 and CCL21. In vivo, injection of complete Freund adjuvant induces a rapid recruitment of neutrophils to the lymph nodes in wild-type mice but not in Ccr7−/− mice. Moreover, intradermally injected interleukin-17–and granulocyte-macrophage colony-stimulating factor–stimulated neutrophils from wild-type mice, but not from Ccr7−/− mice, migrate to the draining lymph nodes. These results identify CCR7 as a chemokine receptor involved in the migration of neutrophils to the lymph nodes

Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay

<p>Abstract</p> <p>Background</p> <p>Hereditary hemochromatosis (HH) is a common genetic disease characterized by excessive iron overload that leads to multi-organ failure. Although the most prevalent genotype in HH is homozygosity for C282Y mutation of the <it>HFE </it>gene, two additional mutations, H63D and S65C, appear to be associated with a milder form of HH. The aim of this study was to develop a high-throughput assay for <it>HFE </it>mutations screening based on TaqMan technology and to determine the frequencies of <it>HFE </it>mutations in the Slovenian population.</p> <p>Methods</p> <p>Altogether, 1282 randomly selected blood donors from different Slovenian regions and 21 HH patients were analyzed for the presence of <it>HFE </it>mutations by an in-house developed real-time PCR assay based on TaqMan technology using shorter non-interfering fluorescent single nucleotide polymorphism (SNP)-specific MGB probes. The assay was validated by RFLP analysis and DNA sequencing.</p> <p>Results</p> <p>The genotyping assay of the H63D, S65C and C282Y mutations in the <it>HFE </it>gene, based on TaqMan technology proved to be fast, reliable, with a high-throughput capability and 100% concordant with genotypes obtained by RFLP and DNA sequencing. The observed frequency of C282Y homozygotes in the group of HH patients was only 48%, others were of the heterogeneous <it>HFE </it>genotype. Among 1282 blood donors tested, the observed H63D, S65C and C282Y allele frequency were 12.8% (95% confidence interval (CI) 11.5 – 14.2%), 1.8% (95% CI 1.4 – 2.5%) and 3.6% (95% CI 3.0 – 4.5%), respectively. Approximately 33% of the tested subjects had at least one of the three HH mutations, and 1% of them were C282Y homozygotes or compound heterozygotes C282Y/H63D or C282Y/S65C, presenting an increased risk for iron overload disease. A significant variation in H63D allele frequency was observed for one of the Slovenian regions.</p> <p>Conclusion</p> <p>The improved real-time PCR assay for H63D, S65C and C282Y mutations detection is accurate, fast, cost-efficient and ready for routine screening and diagnostic procedures. The genotype frequencies in the Slovenian population agree with those reported for the Central European populations although some deviations where observed in comparison with other populations of Slavic origin. Regional distribution of the mutations should be considered when planning population screening.</p

A Novel Strategy to Screen Bacillus Calmette-Guérin Protein Antigen Recognized by γδ TCR

BACKGROUND: Phosphoantigen was originally identified as the main γδ TCR-recognized antigen that could activate γδ T cells to promote immune protection against mycobacterial infection. However, new evidence shows that the γδ T cells activated by phosphoantigen can only provide partial immune protection against mycobacterial infection. In contrast, whole lysates of Mycobacterium could activate immune protection more potently, implying that other γδ TCR-recognized antigens that elicit protective immune responses. To date, only a few distinct mycobacterial antigens recognized by the γδ TCR have been characterized. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we established a new approach to screen epitopes or protein antigens recognized by the γδ TCR using Bacillus Calmette-Guérin- (BCG-) specific γ TCR transfected cells as probes to pan a 12-mer random-peptide phage-displayed library. Through binding assays and functional analysis, we identified a peptide (BP3) that not only binds to the BCG-specific γδ TCR but also effectively activates γδ T cells isolated from human subjects inoculated with BCG. Importantly, the γδ T cells activated by peptide BP3 had a cytotoxic effect on THP-1 cells infected with BCG. Moreover, the oxidative stress response regulatory protein (OXYS), a BCG protein that matches perfectly with peptide BP3 according to bioinformatics analysis, was confirmed as a ligand for the γδ TCR and was found to activate γδ T cells from human subjects inoculated with BCG. CONCLUSIONS/SIGNIFICANCE: In conclusion, our study provides a novel strategy to identify epitopes or protein antigens for the γδ TCR, and provides a potential means to screen mycobacterial vaccines or candidates for adjuvant

CD28null CD4 T-cell expansions in autoimmune disease suggest a link with cytomegalovirus infection

Immunosenescence is thought to contribute to the increase of autoimmune diseases in older people. Immunosenescence is often associated with the presence of an expanded population of CD4 T cells lacking expression of CD28 (CD28null). These highly cytotoxic CD4 T cells were isolated from disease-affected tissues in patients with rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, or other chronic inflammatory diseases and their numbers appeared to be linked to disease severity. However, we recently demonstrated that the common herpes virus, cytomegalovirus (CMV), not ageing, is the major driver of this subset of cytotoxic T cells. In this review, we discuss how CMV might potentiate and exacerbate autoimmune disease through the expansion of CD28null CD4 T cells