T-cell Mediated Immune Response Research Articles

Identification and characterization of HLA-A*0301 epitopes in HIV-1 gag proteins using a novel approach

Identification of CTL epitopes correlated to immune protection is important for the development of vaccines that enhance T-cell mediated immune responses. The correlation of positively selected amino acids (PS) of HIV-1 with host HLA alleles can identify regions containing potential T-cell epitopes. However, the specific epitopes have to be identified and characterized using overlapping peptides through T-cell functional assays. In this study we used a new approach to identify and characterize potential epitopes in the gag region containing PS mutations that significantly correlated with HLA-A*0301. The iTopia Epitope Discovery System was used to rapidly screen a panel of peptides overlapping the regions containing PS mutations and the peptides identified were assessed for relative affinity and complex stability. The potential epitopes were then validated by interferon gamma (IFN-γ) ELISpot assays with patient PBMCs. Using this approach we identified/confirmed the predicted HLA-A*0301 epitopes in two regions of gag containing PS mutations V7I and K403R, one previously reported and the other novel. Five of the seven peptides that bound to A*0301 contained the K403R mutation and corresponded to the documented LARNCRAPRK-A3 supertype epitope. Two epitope variants, RASVLSGGK and RASILSGGK containing the V7I mutation, were identified using the iTopia Epitope Discovery System, however only the consensus variant (RAK9C) was confirmed using the ELISpot assay and it represents a novel A*0301 epitope.

Abstract 5168: Adenosine Formed by CD73 on Treg Inhibits T-cell Mediated Immune Response via NFkB

CD73-derived adenosine (ADO) acts as potent inhibitor of inflammation and we have recently shown that the CD73−/−/ApoE−/− double mutants show enhanced plaque formation with local accumulation of immune cells. Since regulatory T-cells (Treg) have been recently shown to express CD73 as a novel marker enzyme, the present study explored the potential role of CD73-derived adenosine on NFkB activity and cytokine release in CD4+ cells. We asked: does adenosine physiologically formed by Treg inhibit the immune response of the entire Th-cell population? Does the lack of CD73 alter the expression pattern of various enzymes of the ecto-nucleotidase cascade and ATP/adenosine receptors in Tregs? T-cells, isolated from murine spleens by magnetic micro beads, were stimulated with α CD3/ α CD28 which activates the nuclear translocation of transcriptionfactor NF κ B, known to be involved in the secretion of pro-inflammatory cytokines such as IL-2. Using EMSA we found activated NF κ B in T-cells lacking CD73 to be significantly upregulated (WT: 4.36±0.21, CD73−/−: 6.58±0.75; n=4; p=0.029) which was paralleled by increases in release of the pro-inflammatory cytokines IL-2 and IFN- γ (ELISA). Treatment of T-cells with ADO (25 μ M) caused a massive A2a ADO receptor mediated reduction of IL-2 and IFN-ã release. Similarly, AMP (50 μ M) decreased the IFN-ã release of WT CD4+ cells. Expression analysis revealed that there were no differences in the enzymes and receptors of the ecto-nucleotidase cascade in WT and CD73−/− Tregs. Upon stimulation with ãCD3/ãCD28, however, mRNA levels of the ATP receptor P2X7 - a ligand-gated ion channel that controls CD4+ activation - was significantly reduced. Expression of the ADO transporter CNT2 was also significantly reduced while the expression of the A2b ADO receptor was increased. In summary, our data show that stimulated CD73-deficient T-cells show a proinflammtory phenotype with increased levels of active NF κ B and released proinflammatory cytokines. Thus, CD73-derived ADO is a physiological inhibitor of the immune response relevant to atherogenesis. Downregulation of P2X7 on Tregs may be important to prevent apoptosis and to maintain suppressive function of Tregs by converting extracellular ATP to ADO.

Association of Cytotoxic T Lymphocyte-associated antigen-4 gene haplotype with the susceptibility to gastric cancer

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) was widely accepted as a pivotal molecule in downregulating T-cell mediated immune responses. In this study we investigated the polymorphisms which would impact the CTLA-4 gene expression and function to assess the association with the risk of gastric cancer. 205 gastric cancer patients and 262 healthy controls were included in the case-control study. PCR and restriction fragment length polymorphism (RFLP) methods were performed to identify the +49A/G and promoter -1661A/G polymorphisms. The promoter -1772T/C polymorphism was detected by PCR amplification refractory mutation system (ARMS) technique. A significant difference was observed between case and control groups. The frequency of +49A/G polymorphism AG and -1661A/G polymorphism GG genotype were significantly higher in patients than in controls (OR = 2.15, OR = 1.88, respectively). No significant difference was found in the allelic frequency of -1772T/C polymorphism between cases and controls (P = 0.478). By the haplotype analysis, logistic regression showed the frequency of haplotype A (GAT) and D (AGT) in the case group revealed significant difference compared with in control group (OR = 2.00, P < 0.001; OR = 1.62, P = 0.043, respectively). Our findings implied the genetic variations within CTLA-4 gene would be a critical risk factor to the susceptibility of gastric cancer.

Chronic myeloid leukemia patient manifesting fatal hepatitis B virus reactivation during treatment with imatinib rescued by liver transplantation: case report and literature review

Imatinib mesylate (imatinib) is now a standard treatment for patients with chronic myeloid leukemia (CML). Although imatinib is known to have a potential impact on various infectious organisms by altering the T-cell mediated immune response, only two cases of hepatitis B virus (HBV) reactivation during imatinib treatment have actually been reported. The role of liver transplantation (LT) after fatal HBV reactivation in patients with potentially treatable or curable hematologic malignancy is also unknown. Therefore, this report presents a case of fatal HBV reactivation during imatinib treatment for CML, where the patient is rescued by LT. Following a successful living donor LT, the liver function improves rapidly and the patient remains in complete cytogenetic remission after retreatment with imatinib for 6 months. The present report also covers the role of tyrosine kinase inhibitor in triggering HBV reactivation and a literature review of fulminant hepatic failure in CML patients taking imatinib.

Effect of B7.1 Costimulation on T-Cell Based Immunity against TAP-Negative Cancer Can Be Facilitated by TAP1 Expression

Tumors deficient in expression of the transporter associated with antigen processing (TAP) usually fail to induce T-cell-mediated immunity and are resistant to T-cell lysis. However, we have found that introduction of the B7.1 gene into TAP-negative (TAP−) or TAP1-transfected (TAP1+) murine lung carcinoma CMT.64 cells can augment the capacity of the cells to induce a protective immune response against wild-type tumor cells. Differences in the strength of the protective immune responses were observed between TAP− and TAP1+ B7.1 expressing CMT.64 cells depending on the doses of γ-irradiated cell immunization. While mice immunized with either high or low dose of B7.1-expressing TAP1+ cells rejected TAP− tumors, only high dose immunization with B7.1-expressing TAP− cells resulted in tumor rejection. The induced protective immunity was T-cell dependent as demonstrated by dramatically reduced antitumor immunity in mice depleted of CD8 or CD4 cells. Augmentation of T-cell mediated immune response against TAP− tumor cells was also observed in a virally infected tumor cell system. When mice were immunized with a high dose of γ-irradiated CMT.64 cells infected with vaccinia viruses carrying B7.1 and/or TAP1 genes, we found that the cells co-expressing B7.1 and TAP1, but not those expressing B7.1 alone, induced protective immunity against CMT.64 cells. In addition, inoculation with live tumor cells transfected with several different gene(s) revealed that only B7.1- and TAP1-coexpressing tumor cells significantly decreased tumorigenicity. These results indicate that B7.1-provoked antitumor immunity against TAP− cancer is facilitated by TAP1-expression, and thus both genes should be considered for cancer therapy in the future.

Designing Short Peptides as Viral Vaccines

The genetic diversity across global population (Black, Caucasoid, oriental, Hispanic, mixed race, Pacific Islander, American Indian and Australian aboriginal) are other vital issues in personalized/population based medicine. The human leukocyte antigen (HLA) genes in human chromosome 6 are extensively studied across global population using genetic data and about a 2000 HLA alleles have been named so far at IMGT/HLA database. Extensive HLA polymorphism and peptide diversity are critical issues in HLA-peptide binding estimation for T-cell mediated immune response. The precise evaluation of HLA peptide binding finds application in epitope design for the development of vaccines and diagnostics of diseases associated with CD4+ (example, type 1 diabetes, malaria) and CD8+ (example, several viral infections) T-cellular immunity. HLA class II binding peptides have an extended conformation at the binding groove unlike class I. This increases peptide binding combinations of varying length at the groove having eventual effect in the host immune response to infectious agents. Discussion on the significance of prediction models to large HLA allele coverage representing sampled global population towards the development of diagnostics and vaccines will be presented.

Increased levels of interleukin-8 in human seminal plasma.

The role of cell-mediated immunity in the aetiopathogenesis of male infertility is far from being defined. The cytochemokine interleukin-8 (IL-8) has a key role in T-cell mediated immune responses. The aim of this study was to confirm the presence of IL-8 in human seminal plasma, to show differences between IL-8 concentrations in fertile and infertile subjects, and to show the potential relationship between IL-8 amounts in semen and spermiogram parameters. IL-8 levels were determined in the seminal plasma of 77 men divided as follows: (a) into seven groups according to the aetiological diagnosis of fertility and (b) into two groups on the basis of a normal or abnormal spermiogram. The mean value of IL-8 in the seminal plasma was 31.5 times higher than the upper limit in normal serum. There is a borderline statistical significant difference among the means of the various groups (P < 0.051). The Tukey's HSD test for multiple comparisons indicated no two groups as being significantly different, whereas the less conservative test LSD showed significant differences between the group with infection and groups with normal controls, Klinefelter's syndrome, mumps orchitis, cryptorchidism, or varicocele. There was no significant difference in IL-8 levels between men with normal and those with abnormal spermiograms. Furthermore, there was no correlation between IL-8 levels and the variables of the spermiogram. Even though the conclusions of this study have to be tempered by the sample size, IL-8 concentration in seminal plasma may be considered as a potential marker for the diagnosis of male accessory gland infection.

Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines

BackgroundGluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4+ T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (Triticum aestivum) (AABBDD).ResultsThe effects of deleting individual gluten loci on both the level of T-cell stimulatory epitopes in the gluten proteome and the technological properties of the flour were analyzed using a set of deletion lines of Triticum aestivum cv. Chinese Spring. The reduction of T-cell stimulatory epitopes was analyzed using monoclonal antibodies that recognize T-cell epitopes present in gluten proteins. The deletion lines were technologically tested with respect to dough mixing properties and dough rheology. The results show that removing the α-gliadin locus from the short arm of chromosome 6 of the D-genome (6DS) resulted in a significant decrease in the presence of T-cell stimulatory epitopes but also in a significant loss of technological properties. However, removing the ω-gliadin, γ-gliadin, and LMW-GS loci from the short arm of chromosome 1 of the D-genome (1DS) removed T-cell stimulatory epitopes from the proteome while maintaining technological properties.ConclusionThe consequences of these data are discussed with regard to reducing the load of T-cell stimulatory epitopes in wheat, and to contributing to the design of CD-safe wheat varieties.

A West Nile Virus (WNV) recombinant canarypox virus (ALVAC®) vaccine elicits WNV specific neutralising antibodies and T-cell mediated immune responses in the horse: Absence of inhibiting anti-vector immunity following repeated injections

A West Nile Virus (WNV) recombinant canarypox virus (ALVAC®) vaccine elicits WNV specific neutralising antibodies and T-cell mediated immune responses in the horse: Absence of inhibiting anti-vector immunity following repeated injections

Association of Genetic Variation in Inducible Costimulator Gene With Outcome of Kidney Transplantation

The closely-linked genes of CD28, cytotoxic T-lymphocyte associated antigen 4 (CTLA4), inducible costimulator (ICOS), and programmed cell death 1 on chromosome 2q encode costimulatory molecules, which are regulators of the T-cell activity. The T-cell mediated immune response has a major role in allograft rejection. Hence, the variation in these genes may have an effect on graft survival and the amount of immunosuppression needed, but so far the studies have restricted solely to the CTLA4 gene. We determined 13 single nucleotide polymorphisms in CD28, CTLA4, ICOS, and PPCD1 genes in 678 adult patients who received a kidney from deceased donor. The effect of genetic variation on the outcome of renal transplantation was analyzed. Two markers on the ICOS gene, rs10183087 and rs4404254, were associated with delayed graft function (odds ratio=5.8; P=0.020 and odds ratio=5.8; P=0.019, respectively). Interestingly, the same ICOS variation has been shown to regulate the expression level of ICOS. We also demonstrated an association of the ICOS polymorphism rs10932037 with the graft survival (P=0.026). The present results indicate that potentially functional genetic variation in T-cell costimulatory molecule ICOS has an effect on the outcome of kidney transplantation.

Array CGH Analysis of Chronic Lymphocytic Leukemia Reveals Frequent Cryptic Monoallelic and Biallelic Deletions of Chromosome 22q11.

Bacterial artificial chromosome array-based comparative genomic hybridization (array CGH) was used to detect recurrent genomic imbalances in 187 CLL cases. Detection rates of clinically relevant chromosomal abnormalities involving losses of 11q23, 13q14, 17p13, and whole chromosome 12 gains were consistent with previously reported results. However, submicroscopic deletions of chromosome 22q11 were observed in 28 cases (15%), and the frequency of these deletions was second only to loss of the 13q14 region, the most commonly detected recurrent chromosomal aberration in CLL. The del(22)(q11) occurred as the sole genomic alteration in three cases, but was associated with additional copy number changes in 25 cases, most commonly with the 13q14 deletion, where it was found in 17 of the 28 cases (61%). Twenty-four cases exhibited monoallelic 22q11 losses, four cases showed biallelic losses, and two cases also exhibited concomitant gains of whole chromosome 22.Breakpoint mapping using oligonucleotide-based array CGH analysis showed that the 22q11 deletions ranged in size from 0.34 Mb up to approximately 1 Mb, and all cases included a minimally deleted 340,279 bp region located within the lambda immunoglobulin light chain variable region locus. The minimally deleted region included four protein coding genes, including the closely related zinc finger protein genes ZNF280A and ZNF280B, the gamma-glutamyltransferase light chain 2 (GGTLC2) gene, and the preferentially expressed antigen in melanoma (PRAME) locus. Quantitative real-time PCR expression studies for these four genes revealed that the expression of ZNF280A, ZNF280B, and PRAME mRNA was significantly lower in the 22q11 deletion cases compared to non-deleted cases by both the two sample t-test and the Wilcoxon rank sum test methods. Mean expression of ZNF280A mRNA was 26-fold lower in the del(22) (q11) samples compared to non-deleted samples (t-test, p=0.02612; Wilcoxon rank sum test, p=0.002511). Mean expression of ZNF280B mRNA was decreased by 11-fold in the del(22)(q11) samples compared to non-deleted samples (t-test, p=0.01442; Wilcoxon rank sum test, p=9.768x10−6). Mean expression of PRAME mRNA was 11-fold lower in the del(22)(q11) cases compared to non-deleted cases (t-test, p=0.005517; Wilcoxon rank sum test, p=0.0317). However, mean expression of GGTLC2 mRNA was only 1.6-fold lower in the del(22)(q11) cases compared to non-deleted cases. This change was marginally significant by t-test (p=0.06718) and not significantly different by the Wilcoxon rank sum test (p=0.1329). CLL cases with a biallelic del(22)(q11) exhibited the lowest levels of expression of ZNF280A, ZNF280B, and PRAME mRNA.The ZNF280A and ZNF280B genes are closely related zinc finger proteins which exhibit significant sequence similarity to the Drosophila Suppressor of Hairy-wing (Su[Hw]) protein, a leucine zipper protein that contributes to the regulation of gene expression in the Drosophila genome through the establishment of endogenous insulators (independent transcriptional domains). To date, there is no direct evidence linking these two zinc finger proteins with human cancers. However, it is conceivable that alterations in the copy number of these genes could have a significant effect on gene regulation in CLL, and possibly other human cancers.The PRAME gene encodes a membrane- and cytoplasm-associated protein that was first described as a tumor antigen in melanoma that triggered autologous cytotoxic T-cellmediated immune responses. High levels of PRAME mRNA expression have been described in a number of human malignancies, including CLL. High PRAME expression is present in acute leukemias with a more favorable prognosis such as AML with the t(8;21), AML-M3 with the t(15;17), and childhood B-ALL with or without the t(12;21). These findings have made PRAME an attractive candidate for immunotherapy and a potentially useful marker for minimal residual disease monitoring. Our study is the first to correlate submicroscopic deletions of the PRAME locus with decreased expression of PRAME mRNA in CLL. The lack of reciprocal duplications and the presence of biallelic deletions also point to a possible role for loss of the PRAME gene locus in the pathogenesis of CLL.Further studies are needed to assess the prognostic significance of submicroscopic 22q11 deletions and to determine whether these alterations contribute to the pathogenesis of CLL.

The utility of rhesus monkey (Macaca mulatta) and other non-human primate models for preclinical testing of Leishmania candidate vaccines

Leishmaniasis causes significant morbidity and mortality, constituting an important global health problem for which there are few effective drugs. Given the urgent need to identify a safe and effective Leishmania vaccine to help prevent the two million new cases of human leishmaniasis worldwide each year, all reasonable efforts to achieve this goal should be made. This includes the use of animal models that are as close to leishmanial infection in humans as is practical and feasible. Old world monkey species (macaques, baboons, mandrills etc.) have the closest evolutionary relatedness to humans among the approachable animal models. The Asian rhesus macaques (Macaca mulatta) are quite susceptible to leishmanial infection, develop a human-like disease, exhibit antibodies to Leishmania and parasite-specific T-cell mediated immune responses both in vivo and in vitro, and can be protected effectively by vaccination. Results from macaque vaccine studies could also prove useful in guiding the design of human vaccine trials. This review summarizes our current knowledge on this topic and proposes potential approaches that may result in the more effective use of the macaque model to maximize its potential to help the development of an effective vaccine for human leishmaniasis.

Prophylactic Efficacy of High‐Molecular‐Weight Antigenic Fractions of a Recent Clinical Isolate of Leishmania donovani Against Visceral Leishmaniasis

T-cell mediated immune responses are key determinants to the natural course of infection caused by intracellular parasites such as Leishmania. Thus, T-cell activating proteins of these microbes continue to generate active interest particularly in view of their possible role in the design and development of newer and more effective vaccines. We have recently reported the presence of T-cell immunostimulatory antigens with the high-molecular-weight (MW) fractions (134-64.2 kDa) of whole Leishmania donovani antigen (strain 2001), which stimulated variable amounts of IFN-gamma, IL-12 and IL-10 in exposed immune individuals. The present study was undertaken to further evaluate these high-MW antigenic fractions (MW range >100-60 kDa) for potential protective efficacy. The high-MW region of the parasite was resolved into five antigenic fractions (Prep A-E) using continuous elution gel electrophoresis. Prior to in vivo protection studies in hamsters, these fractions were used to evaluate in vitro cellular responses in eight Leishmania-exposed individuals and treated cured hamsters. The protective efficacy of prep (A + B), C, D and E in combination with BCG was evaluated in inbred hamsters using standard immunization protocol. Proliferative responses were seen in all eight of eight exposed individuals to prep D [median stimulation index (SI): 5.2 (range 3.9-7.1)] and E [median SI: 5.6 (range 4.4-8.2)], five of eight individuals to prep B and prep C and three of eight to prep A [median SI: 0.2 (range 0.1-7.2)]. The median proliferative responses to prep D and prep E were significantly higher than to fraction prep A; (P < 0.05) but not to prep B and prep C. However, prep A-E induced equivalent levels of IFN-gamma, IL-10 and IL-12 cytokines. Fractions D and E also exhibited marked parasite inhibition in spleen (52.5% and 73.7%) and liver (65% and 80.2%) as compared with prep (A + B) (23% in spleen and 24% in liver) and prep C (38% in spleen and 24% in liver). Prep D and prep E vaccinated animals showed higher in vitro stimulatory responses (mean SI: 6.6 and 8.8) and nitric oxide (NO) induction (mean NO levels: 6.4 and 10.7 mug/ml) against whole cell extract as compared with other groups. The protection also correlated with presence of suppressed Leishmania-specific IgG levels in prep D and prep E immunized hamsters. These studies indicate the presence of immunostimulatory and protective molecules in 60-80 kDa region of L. donovani, which may be further exploited for developing a subunit vaccine.

The IL-33/ST2 pathway: therapeutic target and novel biomarker

For many years, the interleukin-1 receptor family member ST2 was an orphan receptor that was studied in the context of inflammatory and autoimmune disease. However, in 2005, a new cytokine--interleukin-33 (IL-33)--was identified as a functional ligand for ST2. IL-33/ST2 signalling is involved in T-cell mediated immune responses, but more recently, an unanticipated role in cardiovascular disease has been demonstrated. IL-33/ST2 not only represents a promising cardiovascular biomarker but also a novel mechanism of intramyocardial fibroblast-cardiomyocyte communication that may prove to be a therapeutic target for the prevention of heart failure.

Influence of hatching asynchrony and within-brood parental investment on size, condition, and immunocompetence in nestling red-billed choughs

Studies of hatching asynchrony have rarely assessed the effect of parental investment strategies on the development of intra-brood hierarchies using indicators of nestling quality. The influence of hatching asynchrony and other variables related to parental investment on immunocompetence was evaluated measuring T-cell mediated immune response in broods of red-billed choughs (Pyrrhocorax pyrrhocorax), a large long-living passerine with a pronounced sexual size dimorphism. The results showed that T-cell mediated immune response depends on parental effects as shown by the differences between broods on hatching asynchrony. Male nestlings were both heavier and larger than female nestlings, but there was no effect of hatching order on these traits, nor on sex differences in immunocompetence. Differential investment strategy in relation to laying order did not favour older offspring or either of the sexes. Successful reproduction in this species might be so unpredictable and infrequent that strategies of parental investment in the brood could have evolved to attempt to maximize the survival of all nestlings by avoiding within-brood hierachies of size, body condition, and immunocompetence. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94, 675–684.

In Vitro Expanded Human CD4+CD25+ Regulatory T Cells are Potent Suppressors of T-Cell-Mediated Xenogeneic Responses

Cellular rejection of xenografts is predominantly mediated by CD4 T cells. Little is known of the effectiveness of CD4CD25 T regulatory (Treg) cells at suppressing this strong T-cell mediated immune response. In this study, we evaluated the activity of fresh Treg cells and expanded Treg cells to suppress the xeno immune response in vitro. Human Treg cells were preferentially expanded by CD3/CD28 expand beads, interleukin (IL)-2, and rapamycin. Human CD4CD25 T cells were stimulated with irradiated porcine peripheral blood mononuclear cells in the presence or absence of fresh or expanded human Treg cells for 5 days before proliferation assay. In a separate experiment, the porcine xenoantigen-stimulated CD4CD25 T cells were separated from Treg cells by transwells and assessed for cytotoxicity of porcine peripheral blood mononuclear cells target cells. Cytokine-producing cells and cytokine release in the cocultures were examined by enzyme-linked immunosorbent spot and enzyme-linked immonosorbent assay, respectively. Human Treg were expanded up to 3,500-fold after 14 days in culture. The addition of fresh Treg suppressed the T-cell mediated xenoimmune response. Compared with fresh Treg cells, expanded Treg cells were more potent at suppressing CD4CD25 T-cell-mediated antiporcine xenogeneic responses. This suppression required cell contact. However, the enhanced suppression by expanded Treg cells was associated with increased secretion of IL-4 and IL-10 when compared with their nonexpanded Treg counterparts. This study shows that expanded human Treg cells were capable of suppressing antiporcine xenogeneic responses in vitro and involve both contact dependent and cytokine mediated mechanisms.

NS1 Specific CD8+ T-Cells with Effector Function and TRBV11 Dominance in a Patient with Parvovirus B19 Associated Inflammatory Cardiomyopathy

BackgroundParvovirus B19 (B19V) is the most commonly detected virus in endomyocardial biopsies (EMBs) from patients with inflammatory cardiomyopathy (DCMi). Despite the importance of T-cells in antiviral defense, little is known about the role of B19V specific T-cells in this entity.Methodology and Principal FindingsAn exceptionally high B19V viral load in EMBs (115,091 viral copies/μg nucleic acids), peripheral blood mononuclear cells (PBMCs) and serum was measured in a DCMi patient at initial presentation, suggesting B19V viremia. The B19V viral load in EMBs had decreased substantially 6 and 12 months afterwards, and was not traceable in PBMCs and the serum at these times. Using pools of overlapping peptides spanning the whole B19V proteome, strong CD8+ T-cell responses were elicited to the 10-amico-acid peptides SALKLAIYKA (19.7% of all CD8+ cells) and QSALKLAIYK (10%) and additional weaker responses to GLCPHCINVG (0.71%) and LLHTDFEQVM (0.06%). Real-time RT-PCR of IFNγ secretion-assay-enriched T-cells responding to the peptides, SALKLAIYKA and GLCPHCINVG, revealed a disproportionately high T-cell receptor Vbeta (TRBV) 11 expression in this population. Furthermore, dominant expression of type-1 (IFNγ, IL2, IL27 and T-bet) and of cytotoxic T-cell markers (Perforin and Granzyme B) was found, whereas gene expression indicating type-2 (IL4, GATA3) and regulatory T-cells (FoxP3) was low.ConclusionsOur results indicate that B19V Ag-specific CD8+ T-cells with effector function are involved in B19V associated DCMi. In particular, a dominant role of TRBV11 and type-1/CTL effector cells in the T-cell mediated antiviral immune response is suggested. The persistence of B19V in the endomyocardium is a likely antigen source for the maintenance of CD8+ T-cell responses to the identified epitopes.

Recent advances in research on hepadnaviral infection in the woodchuck model

The woodchuck model is an excellent animal model to study hepadnaviral infection. The new progresses in this model made possible to examine the T-cell mediated immune responses in acute and chronic hepadnaviral infection. Recently, a new assay for cytotoxic T-cells based on detection of CD107 was established for the woodchuck model. In addition, new immunotherapeutic approaches based on combination of potent antiviral treatment and DNA-protein vaccines were proven to be useful for treatment of chronic hepatitis B.

Dendritic cell acquisition of epitope cargo mediated by simple cationic peptide structures

In this study we evaluate the uptake by murine dendritic cells (DCs) of different synthetic, branched cationic peptide structures with a view to facilitating peptide epitope delivery. The level of cell uptake by fluorescenated peptides was measured by flow cytometry following quenching of extracellular fluorescence with trypan blue. Branched peptides containing either N-terminal arginine or N-terminal lysine residues were able to mediate cell entry but the peptide containing four arginine residues in a branching configuration (R4) was found to be superior not only to other branched peptides in translocating to the cell interior and also to a peptide containing four arginine residues arranged linearly. Fluorescenated R4 was found to be localized within intracellular vesicle-like compartments as well as being distributed throughout the cell cytoplasm. Uptake of R4 utilized an energy-dependent process that appeared to involve phosphatidylinositol-3-kinase and could induce intermediate levels of DC maturation. R4 when conjugated to a T-helper cell and CTL epitope construct was able to induce antigen-specific CD8+ T-cell mediated immune responses in mice when administered in adjuvant as were DCs that were pulsed with this construct and then matured with LPS. Fluorescenated R4 was also found to translocate into the interior of other cell types indicating that it may be useful for the delivery of peptide cargo into other specialized cells.

Cytokines as adjuvants for improving anti-HIV responses

Cytokines as adjuvants for improving anti-HIV responses