Abstract
Control of human CMV (HCMV) infection depends on the cytotoxic activity of CD8+ CTLs. The HCMV phosphoprotein (pp)65 is a major CTL target Ag and pp65495–503 is an immunodominant CTL epitope ...in infected HLA-A*0201 individuals. As immunodominance is strongly determined by the surface abundance of the specific epitope, we asked for the components of the cellular Ag processing machinery determining the efficacy of pp65495–503 generation, in particular, for the proteasome, cytosolic peptidases, and endoplasmic reticulum (ER)-resident peptidases. In vitro Ag processing experiments revealed that standard proteasomes and immunoproteasomes generate the minimal 9-mer peptide epitope as well as N-terminal elongated epitope precursors of different lengths. These peptides are largely degraded by the cytosolic peptidases leucine aminopeptidase and tripeptidyl peptidase II, as evidenced by increased pp65495–503 epitope presentation after leucine aminopeptidase and tripeptidyl peptidase II knockdown. Additionally, with prolyl oligopeptidase and aminopeptidase B we identified two new Ag processing machinery components, which by destroying the pp65495–503 epitope limit the availability of the specific peptide pool. In contrast to cytosolic peptidases, silencing of ER aminopeptidases 1 and 2 strongly impaired pp65495–503-specific T cell activation, indicating the importance of ER aminopeptidases in pp65495–503 generation. Thus, cytosolic peptidases primarily interfere with the generation of the pp65495–503 epitope, whereas ER-resident aminopeptidases enhance such generation. As a consequence, our experiments reveal that the combination of cytosolic and ER-resident peptidase activities strongly shape the pool of specific antigenic peptides and thus modulate MHC class I epitope presentation efficiency.
CD8 super(+) T cells are important for immunity against human cytomegalovirus (HCMV). The HCMV-specific CD8 super(+) T-cell response is characterized by the accumulation of terminally differentiated ...effector cells that have downregulated the costimulatory molecules CD27 and CD28. These HCMV-specific CD8 super(+) T cells maintain high levels of cytotoxic molecules such as granzyme B and rapidly produce the inflammatory cytokine IFN- gamma upon activation. Remarkably, HCMV-specific CD8 super(+) T cells are able to persist long term as fully functional effector cells, suggesting a unique differentiation pathway that is distinct from the formation of memory CD8 super(+) T cells after infection with acute viruses. In this review, we aim to highlight the most recent developments in HCMV-specific CD8 super(+) T-cell differentiation, maintenance, tissue distribution, metabolism and function. HCMV also induces the differentiation of effector CD4 super(+) T cells and NK cells, which share characteristics with HCMV-specific CD8 super(+) T cells. We propose that the overlap in differentiation of NK cells, CD4 super(+) and CD8 super(+) T cells after HCMV infection may be regulated by a shared transcriptional machinery. A better understanding of the molecular framework of HCMV-specific CD8 super(+) T-cell responses may benefit vaccine design, as these cells uniquely combine the capacity to rapidly respond to infection with long-term survival.
Human cytomegalovirus (HCMV) causes significant morbidity in the immunocompromised host. Following primary infection, the virus establishes latent infection in progenitor cells of the myeloid ...lineage. These cells exhibit limited viral gene transcription and no evidence of de novo virion production. It is well recognized that differentiation of latently infected myeloid progenitor cells to dendritic or macrophage-like cells permits viral reactivation in vitro. This has been used to support the concept that viral reactivation in HCMV carriers routinely occurs from such terminally differentiated myeloid cells in vivo. However, to date this has not been shown for in vivo-differentiated macrophages. This study is the first to demonstrate that alveolar macrophages from HCMV carriers express immediate early lytic genes and produce infectious virus. This supports the view, until now based on in vitro data, that terminally differentiated myeloid cells in vivo are sites of HCMV reactivation and potential centers of viral dissemination in latently infected individuals with no evidence of virus disease or dissemination.
To discriminate between primary (PI) and remote (RI) human cytomegalovirus (HCMV) infection, several immunological parameters were monitored for a 2-year period in 53 pregnant women with PI, and 33 ...pregnant women experiencing HCMV PI at least 5 years prior. Cytokine (IFN- gamma and IL-2) production by and phenotype (effector/memory CD45RA super(+)) of HCMV-specific CD4 super(+) and CD8 super(+) T-cells as well as the lymphoproliferative responses (LPR) were evaluated, with special reference to the comparison between a group of women transmitting (T) and a group of non-transmitting (NT) the infection to fetus. While HCMV-specific CD4 super(+) T-cells reached at 90 days post-infection (p.i.) values comparable to RI, CD8 super(+) T-cells reached at 60 days p.i. levels significantly higher and persisting throughout the entire follow-up. Instead, IL-2 production and lymphoproliferative responses were lower in PI than RI for the entire follow-up period. Effector memory CD45RA super(+) CD4 super(+) and CD8 super(+) HCMV-specific T-cells increased until 90 days p.i., reaching and maintaining levels higher than RI. The comparison between T and NT women showed that, at 30 days p.i., in NT women there was a significantly higher IL-2 production by HCMV-specific CD4 super(+) T-cells, and at 60 days p.i. a significantly higher frequency of both specific CD4 super(+) and CD8 super(+) CD45RA super(+) T-cells. HCMV T-cell response appears to correlate with virus transmission to fetus and some parameters (CD4 super(+) lymphoproliferation, and frequency of HCMV-specific CD8 super(+) IL2 super(+) T-cells) may help in dating PI during pregnancy. J. Med. Virol. 88:1238-1246, 2016.
T cells bearing gamma delta T cell antigen receptors (TCRs) function in lymphoid stress surveillance. However, the contribution of gamma delta TCRs to such responses is unclear. Here we found that ...the TCR of a human V sub( gamma )4V sub( delta )5 clone directly bound endothelial protein C receptor (EPCR), which allowed gamma delta T cells to recognize both endothelial cells targeted by cytomegalovirus and epithelial tumors. EPCR is a major histocompatibility complex-like molecule that binds lipids analogously to the antigen-presenting molecule CD1d. However, the V sub( gamma )4V sub( delta )5 TCR bound EPCR independently of lipids, in an antibody-like way. Moreover, the recognition of target cells by gamma delta T cells required a multimolecular stress signature composed of EPCR and costimulatory ligand(s). Our results demonstrate how a gamma delta TCR mediates recognition of broadly stressed human cells by engaging a stress-regulated self antigen.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Copper is an essential cofactor for a variety of biochemical processes including oxidative phosphorylation, cellular antioxidant activity, and elimination of free radicals. The copper transporter 1 ...is known to be involved in cellular uptake of copper ions. In this study, we evaluated the utility of human copper transporter 1 (hCTR1) gene as a new reporter gene for super(64)Cu PET imaging. Human breast cancer cells (MDA-MB-231) were infected with a lentiviral vector constitutively expressing the hCTR1 gene under super cytomegalovirus promoter, and positive clones (MDA-MB-231-hCTR1) were selected. The expression of hCTR1 gene in MDA-MB-231-hCTR1 cells was measured by reverse transcription polymerase chain reaction, Western blot, and super(64)Cu uptake assay. To evaluate the cytotoxic effects induced by hCTR1 expression, the dose-dependent cell survival rate after treatment with cisplatin (Cis-diaminedichloroplatinum (II) CDDP) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion. Small-animal PET images were acquired in tumor-bearing mice from 2 to 48 h after an intravenous injection of super(64)Cu. The hCTR1 gene expression in MDA-MB-231-hCTR1 cells was confirmed at the RNA and protein expression and the cellular super(64)Cu uptake level. MTT assay and trypan blue dye exclusion showed that the cell viability of MDA-MB-231-hCTR1 cells decreased more rapidly than that of MDA-MB-231 cells after treatment with CDDP for 96 or 72 h, respectively. Small-animal PET imaging revealed a higher accumulation of super(64)Cu in MDA-MB-231-hCTR1 tumors than in MDA-MB-231 tumors. With respect to the biodistribution data, the percentage injected dose per gram of super(64)Cu in the MDA-MB-231 tumors and MDA-MB-231-hCTR1 tumors at 48 h after super(64)Cu injection was 2.581 plus or minus 0.254 and 5.373 plus or minus 1.098, respectively. An increase in super(64)Cu uptake induced by the expression of hCTR1 gene was demonstrated in vivo and in vitro, suggesting the potential use of hCTR1 gene as a new imaging reporter gene for PET with super(64)CuCl sub(2).
Abstract
Immunodominance hierarchies operating in immune responses to viral Ags limit the diversity of the elicited CD8 T cell responses. We evaluated in I-Ab+/A2-HHD-II and HLA-DR1+/A2-DR1 mice the ...HLA-A*0201–restricted, multispecific CD8 T cell responses to the human CMV tegument phosphoprotein pp65 (pp65) Ag. Vaccination of mice with pp65-encoding DNA elicited high IFN-γ+ CD8 T cell frequencies to the pp65495–503/(e6) epitope and low responses to the pp65320–328/(e3) and pp65522–530/(e8) epitopes. Abrogation of the e6-specific immunity efficiently enhanced e3- and e8-specific T cell responses by a pp65Δ501–503 DNA vaccine. The immunodominant e6-specific (but not the e3- and e8-specific) CD8 T cell response critically depends on CD4 T cell help. Injection of monospecific DNA- or peptide-based vaccines encoding the e3 or e8 (but not the e6) epitope into mice elicited CD8 T cells. Codelivering the antigenic peptides with different heterologous CD4 T cell helper epitopes enhanced e6-specific (but not e3- or e8-specific) CD8 T cell responses. Similarly, homologous CD4 T cell help, located within an overlapping (nested) pp65487–503 domain, facilitated induction of e6-specific CD8 T cell responses by peptide-based vaccination. The position of the e6 epitope within this nested domain is not critical to induce the immunodominant, e6-specific CD8 T cell response to the pp65 Ag. Distant CD4 T cell epitope(s) can thus provide efficient help for establishing pp65-e6 immunodominance in vaccinated mice. These results have practical implications for the design of new T cell-stimulating vaccines.
IMPORTANCE: The Zika virus (ZIKV) has rapidly reached epidemic proportions, especially in northeastern Brazil, and has rapidly spread to other parts of the Americas. A recent increase in the ...prevalence of microcephaly in newborn infants and vision-threatening findings in these infants is likely associated with the rapid spread of ZIKV. OBJECTIVE: To evaluate the ocular findings in infants with microcephaly associated with presumed intrauterine ZIKV infection in Salvador, Bahia, Brazil. DESIGN, SETTING, AND PARTICIPANTS: Case series at a tertiary hospital. Twenty-nine infants with microcephaly (defined by a cephalic circumference of ≤32 cm) with a presumed diagnosis of congenital ZIKV were recruited through an active search and referrals from other hospitals and health unities. The study was conducted between December 1 and December 21, 2015. INTERVENTIONS: All infants and mothers underwent systemic and ophthalmic examinations from December 1 through December 21, 2015, in the Roberto Santos General Hospital, Salvador, Brazil. Anterior segment and retinal, choroidal, and optic nerve abnormalities were documented using a wide-field digital imaging system. The differential diagnosis included toxoplasmosis, rubella, cytomegalovirus, herpes simplex virus, syphilis, and human immunodeficiency virus, which were ruled out through serologic and clinical examinations. MAIN OUTCOMES AND MEASURES: Ocular abnormalities associated with ZIKV. RESULTS: Twenty-three of 29 mothers (79.3%) reported suspected ZIKV infection signs and symptoms during pregnancy, 18 in the first trimester, 4 in the second trimester, and 1 in the third trimester. Of the 29 infants (58 eyes) examined (18 62.1% female), ocular abnormalities were present in 17 eyes (29.3%) of 10 children (34.5%). Bilateral findings were found in 7 of 10 patients presenting with ocular lesions, the most common of which were focal pigment mottling of the retina and chorioretinal atrophy in 11 of the 17 eyes with abnormalities (64.7%), followed by optic nerve abnormalities in 8 eyes (47.1%), bilateral iris coloboma in 1 patient (2 eyes 11.8%), and lens subluxation in 1 eye (5.9%). CONCLUSIONS AND RELEVANCE: Congenital infection due to presumed ZIKV exposure is associated with vision-threatening findings, which include bilateral macular and perimacular lesions as well as optic nerve abnormalities in most cases.
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