Endogenous Antigen Presentation Research Articles

Incidental CD8 T cell reactivity against caspase-cleaved apoptotic self-antigens from ubiquitously expressed proteins in islets from prediabetic human leucocyte antigen-A2 transgenic non-obese diabetic mice

Apoptosis is known as a major mechanism which contributes to beta cell decay in type 1 diabetes. Commitment to this pathway generally involves caspase-mediated protein cleavage and was found to induce cross-presentation of a specific antigen repertoire under certain inflammatory conditions. We aimed to assess the significance of the CD8 T cell population reactive against such caspase-cleaved apoptotic self-antigens in pancreatic islets of prediabetic human leucocyte antigen (HLA)-A2 transgenic non-obese diabetic chimeric monochain transgene construct (NOD.HHD) mice. We have reproduced a unique peptide library consisting of human CD8 T cell-derived apoptosis-specific antigens, all of which belong to structural proteins expressed ubiquitously in human islets. Pancreatic islets from prediabetic NOD.HHD mice, harbouring humanized major histocompatibilty complex (MHC) class I, were isolated and handpicked at various ages, and islet-infiltrating CD8 T cells were expanded in vitro and used as responders in an interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay. Human T2 cells were used as antigen-presenting cells (APC) to avoid endogenous antigen presentation. Analogous to the interindividual variability found with peptides from known islet autoantigens such as islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) and insulin, some mice showed variable, low-degree CD8 T cell reactivity against caspase-cleaved self-antigens. Because reactivity was predominantly minor and often undetectable, we conclude that beta cell apoptosis does not routinely provoke the development of dominant cytotoxic T lymphocyte (CTL) reactive against caspase-cleaved self-antigens in the NOD.HHD model.

Abstract 2738: Insights into survival from ovarian cancer revealed by gene set analysis

Abstract Epithelial ovarian cancer (EOC) is the 6th most commonly diagnosed cancer among women in the world, accounting for 4% of all female cancers. On a worldwide basis, an estimated 204,000 new cases are diagnosed and 125,000 women die of EOC annually, with 5-year survival rate at 45%. Metastasis and its complications are the usual cause of EOC-related deaths. Thus, factors related to tumor aggressiveness, response to therapy, and the underlying health state of the patient often predict survival. Candidate gene and genome-wide association studies (GWAS) for survival from EOC have discovered only a few the genetic variants, accounting for only a small proportion of the heritability. One explanation for the missing heritability is that the common analysis approach _ assessing the effect of each single nucleotide polymorphism (SNP) individually _ is not well suited to the detection of small effects of multiple SNPs. Gene set analysis (GSA) overcomes this limitation by assessing the overall evidence of association of a phenotype with all measured variation in a set of genes or pathway. Therefore, we conducted a GSA using data from an EOC GWAS, using the Illumina 610-quad SNP array, to determine novel gene sets (GSs) associated with overall survival (OS) from EOC (N cases = 1057, N deaths = 495). All subjects were non-Hispanic, non-Jewish Caucasians, with sample and SNP call rates >95%. Subjects with ambiguous gender, unresolved identical genotypes, and less than 80% European ancestry were also excluded. SNPs were mapped to genes within 20 kb, with genes then mapped to GSs defined by the following: KEGG, Gene Ontology (biological, cellular, molecular) and PharmGKB. GSA was completed in which the association of each gene with OS was assessed using a principal component gene-level test (adjusting for study site, age and population sub-structure based on the first eigenvector from EigenStrat), followed by the aggregation of the gene-level p-values using the Gamma method with a soft truncation threshold of 0.15. Empirical p-values were determined using permutation methods. Of the 2,573 GSs assessed, 30 GSs had a p-value < 0.01. The top GSs were related to: mismatch repair (p = 3.0E-4), platelet formation (p=0.001), leukocyte adhesive activation (p=0.002), antigen processing and presentation of endogenous antigens (p = 0.002) or peptide antigens (p = 0.002), platinum pathway (p = 0.003), aging and lifespan (p = 0.004, p = 0.007), immune system development (p = 0.004) and somatic diversification (p=0.007). These results illustrate the usefulness of GSA for the development of novel hypotheses that can be followed-up with additional clinical or functional studies. Moreover, application of GSA to survival from EOC allowed the detection of significant GSs (and the corresponding genes/SNPs within these GSs) that impact OS that were not detected by assessing each SNP individually, where the smallest p-value from single SNP analysis of the GWAS for OS was 1.3E-05. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2738. doi:10.1158/1538-7445.AM2011-2738

Immunomodulatory Effects of Hypocrellin A on MHC-restricted Antigen Processing

Hypocrellin A has gained much attention in recent years due to its light-induced antitumor, antifungal and antiviral activities. Here we report that hypocrellin A exerts immunomodulatory effects on MHC-restricted presentation of antigen. Hypocrellin A inhibited class II-MHC restricted presentation of exogenous antigen, but not class I MHC-restricted presentation of exogenous antigen, in dendritic cells. Hypocrellin A also inhibited the cytosolic pathway of endogenous antigen presentation. However, hypocrellin A did not inhibit the expression of class I and class II MHC molecules on dendritic cells (DCs), the phagocytic activity of DCs, or the H-2Kb-restricted presentation of a synthetic peptide, SIINFEKL. These results show that hypocrellin A differentially modulates the MHC-restricted antigen presentation pathways.

Rapid generation of maturationally synchronized human dendritic cells: contribution to the clinical efficacy of extracorporeal photochemotherapy

AbstractExtracorporeal photochemotherapy (ECP) is widely used to treat cutaneous T-cell lymphoma, graft-versus-host disease, and allografted organ rejection. Its clinical and experimental efficacy in cancer immunotherapy and autoreactive disorders suggests a novel mechanism. This study reveals that ECP induces a high percentage of processed monocytes to enter the antigen-presenting dendritic cell (DC) differentiation pathway, within a single day, without added cytokines, as determined by enhanced expression of relevant genes. The resulting DCs are capable of processing and presentation of exogenous and endogenous antigen and are largely maturationally synchronized, as assessed by the level of expression of costimulatory surface molecules. Principal component analysis of the ECP-induced monocyte transcriptome reveals that activation or suppression of more than 1100 genes produces a reproducible distinctive molecular signature, common to ECP-processed monocytes from normal subjects, and those from patients. Because ECP induces normal monocytes to enter the DC differentiation pathway, this phenomenon is independent of disease state. The efficiency with which ECP stimulates new functional DCs supports the possibility that these cells participate prominently in the clinical successes of the treatment. Appropriately modified by future advances, ECP may potentially offer a general source of therapeutic DCs.

Surface Expression of PR1 Peptide on Thymic Dendritic Cells Does Not Eliminate PR1-Specific CD8+ Cells In Umbilical Cord Blood

AbstractAbstract 959Positive and negative selection of developing thymocytes is mediated primarily by cortical and medullary epithelial cells (CEC and MEC), respectively, in the thymus. Tolerance to peripheral tissue antigens (PTA) not normally expressed in the thymus can result when PTA expression is induced by AIRE in MEC or when corticomedullary dendritic cells (DC) present endogenous antigens, both mechanisms contributing to deletion of potentially auto-reactive T cells. However, in some instances, negative selection may be incomplete, leading to the release of autoreactive cells into the periphery. Indeed, T cells specific for PR1, the HLA-A2-restricted self-peptide derived from proteinase 3 (P3) and neutrophil elastase (NE), are present in peripheral blood of healthy adults, albeit at extremely low frequency of fewer than 0.0005% of CD8+ T cells. This suggests there is strong thymic central tolerance to PR1. However, PR1-CTL can increase from 0.1 to 2% of peripheral blood CD8 T cells in CML patients treated with interferon or stem cell transplant, and similar levels can be achieved after PR1 peptide vaccination, which suggests that under some circumstances this central tolerance can be reversed. Because of the increasing use of umbilical cord blood (UCB) as an alternative donor source and because incomplete graft-versus-leukemia (GVL) immunity after UCB transplant contributes to relapse, we sought to determine whether PR1-CTL can be expanded from UCB. We hypothesized that PR1-CTL frequency should be low in UCB due to central tolerance. Surprisingly, we found PR1-CTL at a frequency ranging from 0.007 to 0.345% (mean 0.117%) of CD8+ cells in 57 HLA-A2+ cord blood units, similar to what is observed in immunologically responsive vaccine patients and 100- to 1000-fold higher than in healthy adults. The PR1-CTL were predominantly CCR7+CD45+CD28+ and did not efficiently expand ex vivo following peptide stimulation and low dose IL-2, which was consistent with a naive T cell subset. Therefore, this data suggests that central tolerance to PR1 is incomplete. To study whether PR1 is expressed in human thymus, we used the PR1/HLA-A2-specific antibody 8F4 to study PR1 expression. Thymic CEC and MEC expressed no P3, NE, or PR1 by flow cytometry or immunofluorescence imaging of sectioned fetal thymus, which is consistent with previous reports showing absence of P3 or NE induction by AIRE in MEC. Interestingly, by flow cytometric analysis, we found that PR1 is expressed on the surface of thymic dendritic cells (DC) exclusively. This selective expression of PR1 was further confirmed via immunoflourescent staining of sectioned fetal thymus, and the PR1-expressing DC were localized to the corticomedullary junction and medulla. Thus it appears that PR1 expression by DC in the thymus is insufficient for complete central tolerance to PR1. Furthermore, because we have previously shown that PR1-overexpressing CML cells can induce apoptosis of high affinity PR1-CTL, it is possible that peripheral tolerance mechanisms are most critical for preventing autoreactivity to PR1 in humans. These observations suggest possible strategies to overcome tolerance to PR1 by modifying DC uptake and cross-presentation of soluble P3 and NE, when selective autoimmunity may be desirable for leukemia patients or detrimental for patients with vasculitis such as Wegener's granulomatosis. Disclosures:No relevant conflicts of interest to declare.

Expression profiles of genes within a subregion of chicken major histocompatibility complex B in spleen after Marek’s disease virus infection

Major histocompatibility complex has previously been shown to influence the resistance of chicken to Marek's disease virus (MDV). However, little is known about expression of other genes in the MHC-I and II pathway after MDV infection. This study aimed at investigating 8 immune-related genes in the MHC core region that affects host responses to MDV. Spleens of infected and age-matched uninfected chickens were removed at 4, 7, 14, 21, and 28 d postinfection for gene expression detection using real-time PCR. Different expression patterns of MHC-I and II pathway genes were observed in the spleen. In the MHC-I pathway, the expression of transporter of antigen protein 1 (TAP1), transporter of antigen protein 2 (TAP2), and transporter of antigen protein-binding protein (TAPBP) genes was significantly increased in the spleen of MDV-infected than that of uninfected chickens. It indicated that host antivirus responses were generated to enhance antigen presentation. However, MHC-II pathway genes showed contrary trends. Classical MHC-II β chain major gene (BLB2) and nonclassical class II genes [DM α chain gene (DMA), DM β chain gene-1 (DMB1), and DM β chain gene-2 (DMB2)] had consistent lower transcripts in spleens of MDV-infected than that of uninfected chickens, which reflected that MDV interfered with multiple components of the MHC-II pathway. Overall, expression of most genes in the MHC core region was altered; moreover, the genes in endogenous and exogenous antigen presentation pathways had different expression patterns in the spleen after MDV infection.

Dietary fatty acids modulate antigen presentation to hepatic NKT cells in nonalcoholic fatty liver disease

Dietary fatty acids are major contributors to the development and progression of insulin resistance and nonalcoholic fatty liver disease (NAFLD). Dietary fatty acids also alter hepatic NKT cells that are activated by antigens presented by CD1d. In the current study, we examine the mechanism of dietary fatty acid induced hepatic NKT cell deficiency and its causal relationship to insulin resistance and NAFLD. We discover that dietary saturated fatty acids (SFA) or monounsaturated fatty acids (MUFA), but not polyunsaturated fatty acids (PUFA), cause hepatic NKT cell depletion with increased apoptosis. Dietary SFA or MUFA also impair hepatocyte presentation of endogenous, but not exogenous, antigen to NKT cells, indicating alterations of the endogenous antigen processing or presenting pathway. In vitro treatment of normal hepatocytes with fatty acids also demonstrates impaired ability of CD1d to present endogenous antigen by dietary fatty acids. Furthermore, dietary SFA and MUFA activate the NFkappaB signaling pathway and lead to insulin resistance and hepatic steatosis. In conclusion, both dietary SFA and MUFA alter endogenous antigen presentation to hepatic NKT cells and contribute to NKT cell depletion, leading to further activation of inflammatory signaling, insulin resistance, and hepatic steatosis.

CD8+ T Cell Priming by Dendritic Cell Vaccines Requires Antigen Transfer to Endogenous Antigen Presenting Cells

BackgroundImmunotherapeutic strategies to stimulate anti-tumor immunity are promising approaches for cancer treatment. A major barrier to their success is the immunosuppressive microenvironment of tumors, which inhibits the functions of endogenous dendritic cells (DCs) that are necessary for the generation of anti-tumor CD8+ T cells. To overcome this problem, autologous DCs are generated ex vivo, loaded with tumor antigens, and activated in this non-suppressive environment before administration to patients. However, DC-based vaccines rarely induce tumor regression.Methodology/Principal FindingsWe examined the fate and function of these DCs following their injection using murine models, in order to better understand their interaction with the host immune system. Contrary to previous assumptions, we show that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells.Conclusions/SignificanceThis reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches should focus on creating DC vaccines that are more effective at directly priming T cells, or abrogating the tumor induced suppression of endogenous DCs.

LAMP‐2‐deficient human B cells exhibit altered MHC class II presentation of exogenous antigens

Major histocompatibility complex (MHC) class II molecules present antigenic peptides derived from engulfed exogenous proteins to CD4(+) T cells. Exogenous antigens are processed in mature endosomes and lysosomes where acidic proteases reside and peptide-binding to class II alleles is favoured. Hence, maintenance of the microenvironment within these organelles is probably central to efficient MHC class II-mediated antigen presentation. Lysosome-associated membrane proteins such as LAMP-2 reside in mature endosomes and lysosomes, yet their role in exogenous antigen presentation pathways remains untested. In this study, human B cells lacking LAMP-2 were examined for changes in MHC class II-restricted antigen presentation. MHC class II presentation of exogenous antigen and peptides to CD4(+) T cells was impaired in the LAMP-2-deficient B cells. Peptide-binding to MHC class II on LAMP-2-deficient B cells was reduced at physiological pH compared with wild-type cells. However, peptide-binding and class II-restricted antigen presentation were restored by incubation of LAMP-2-negative B cells at acidic pH, suggesting that efficient loading of exogenous epitopes by MHC class II molecules is dependent upon LAMP-2 expression in B cells. Interestingly, class II presentation of an epitope derived from an endogenous transmembrane protein was detected using LAMP-2-deficient B cells. Consequently, LAMP-2 may control the repertoire of peptides displayed by MHC class II molecules on B cells and influence the balance between endogenous and exogenous antigen presentation.

Abstract SY03-03: Modulating innate and adaptive immunity through the manipulation of dendritic cells

Abstract Dendritic cells (DC) are potent antigen presenting cells used in clinical trials to treat cancer and infectious diseases such as HIV. We have performed two types of clinical trials to evaluate their immunogenicity in vivo. First, we compared DC to conventional vaccine adjuvants. HLA-A2+ resected melanoma patients were enrolled into a randomized clinical trial to compare vaccines comprised of six HLA-A2-restricted peptide antigens and a foreign protein, KLH, either pulsed onto cytokine-matured, monocyte-derived DCs or emulsified in the mineral oil adjuvant Montanide ISA 51. The frequency, magnitude and quality of antigen-specific cellular immune responses in peripheral blood were found to be greater in patients receiving the Montanide vaccine. Furthermore, the primed cells were functionally superior, indicating a need to improve current DC-based vaccine approaches. Preclinical studies in mice were undertaken to evaluate the mechanism by which DC prime T cells. Contrary to previous assumptions, we found that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells. This reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches will focus on creating DC vaccines that are more effective at directly priming T cells, by altering maturation factors, or abrogating the tumor induced suppression of endogenous DCs In a second approach, we used TLR agonists targeting TLR 7, 8 and/or 9 to prime immunity to melanoma associated antigens in a series of clinical trials. They include imiquimod, resiquimod and CpG, agonists for TLR7 and/or TLR 8 and TLR9 respectively. These adjuvants when combined with the cancer testis antigen NY-ESO-1, induced integrated immune responses which were characterized by the induction of both CD4+ and CD8+ T cell immunity as well as antigen-specific antibodies in patients with resected melanoma. Strategies to optimize these various approaches to induce immunity in cancer and other chronic diseases will be discussed. Citation Format: Nina Bhardwaj. Modulating innate and adaptive immunity through the manipulation of dendritic cells [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY03-03

Drug-Mediated and Cellular Immunotherapy in Multiple Myeloma

Multiple myeloma is an immunologically relevant disease, which subverts and suppresses immunity, but that may also be amenable to immunological control. Novel drug and cell-based therapies provide an opportunity for the design of antimyeloma immunotherapy. Reversing the immunosuppression associated myeloma remains a substantial challenge. The minimal residual disease setting achieved by autologous stem cell transplant or highly efficacious induction therapy may reverse this immunoparesis and provide a setting for induction of antimyeloma T-cell responses. Adoptive cytotoxic T-lymphocyte/NK therapy and comprehensive treatment with immunomodulatory drug therapy represent means by which antimyeloma immune responses may be promoted. In addition, apoptosis-inducing therapies may prime endogenous antigen presentation via immunogenic cell death, which again may be enhanced by the addition of immunomodulatory drug therapy.

Roles of autophagy in lymphocytes: reflections and directions.

Recent studies have revealed that autophagy, a fundamental intracellular process, plays many different roles in lymphocyte development and function. Autophagy regulates naive T-lymphocyte homeostasis, specifically by regulating mitochondrial quality and turnover, and is necessary for the proliferation of mature T cells. Autophagy also acts as a cellular death pathway in lymphocytes, both upon prolonged cytokine withdrawal and during acute antigen-receptor stimulation if improperly regulated. Furthermore, during HIV infection, hyperinduction of autophagy leads to massive T-cell death in uninfected CD4(+) T cells, and is rescued by inhibiting autophagic initiation. Constitutively high levels of autophagy in thymic epithelial cells are necessary for optimal processing and presentation of endogenous antigens, and required for proper positive and negative selection of developing thymocytes. Autophagy also promotes the survival of B lymphocytes, as well as the development of early B-cell progenitors. In B cells, autophagy is an alternative death pathway, as antigen-receptor stimulation in the absence of costimulation induces a potent autophagic death. Thus, autophagy plays a complex role in lymphocytes and is regulated during their lifespan to ensure a healthy immune system.

Unique Biological Properties and Application Potentials of CD34(superscript +)CD38(superscript -) Stem Cells from Various Sources

Objective: Somatic CD34(superscript +) CD38(superscript -) stem cells can differentiate into cells of hematopoietic and endothelial lineages and have been clinically used to treat diseases. These stem cells can be obtained from cord blood (CB), bone marrow or granulocyte-macrophage colony-stimulating factor-mobilized peripheral blood. Unmasking genes differentially expressed in hematopoietic stem cells (HSCs) from different anatomic locations can improve our understanding of their basic biological features and help in clinical decision making when applying different HSCs. Materials and Methods: We performed microarray analysis on human CD34(superscript +) CD38(superscript -) HSCs isolated from CB, bone marrow and peripheral blood. Systems biology and advanced bioinformatics tools were used to better understand the biological modules and genetic networks accompanying each HSC subtype. Results: We identified HSC genes differentially expressed in various HSCs and found them to be involved in critical biological processes such as cell cycle regulation, cell motility, and endogenous antigen presentation. Among these three HSC types, HSCs from CB expressed the fewest rejection and immune response-associated genes, thereby showing the best potential as a transplantation source. Analysis of HSC-enriched genes using systems biology tools revealed a complex genetic network functioning in different CD34(superscript +) CD38(superscript -) cells, in which several genes act as hubs, such as MYC in CB HSCs and hepatic growth factor in bone marrow HSCs, to maintain the stability or connectivity of the whole network. Conclusion: This study provides the foundation for a more detailed understanding of CD34(superscript +) CD38(superscript -) HSCs from different sources, and reveals the potentials of different HSCs for different clinical applications.

Unique Biological Properties and Application Potentials of CD34 + CD38 − Stem Cells From Various Sources

Somatic CD34+ CD38- stem cells can differentiate into cells of hematopoietic and endothelial lineages and have been clinically used to treat diseases. These stem cells can be obtained from cord blood (CB), bone marrow or granulocyte-macrophage colony-stimulating factor-mobilized peripheral blood. Unmasking genes differentially expressed in hematopoietic stem cells (HSCs) from different anatomic locations can improve our understanding of their basic biological features and help in clinical decision making when applying different HSCs. We performed microarray analysis on human CD34+ CD38- HSCs isolated from CB, bone marrow and peripheral blood. Systems biology and advanced bioinformatics tools were used to better understand the biological modules and genetic networks accompanying each HSC subtype. We identified HSC genes differentially expressed in various HSCs and found them to be involved in critical biological processes such as cell cycle regulation, cell motility, and endogenous antigen presentation. Among these three HSC types, HSCs from CB expressed the fewest rejection and immune response-associated genes, thereby showing the best potential as a transplantation source. Analysis of HSC-enriched genes using systems biology tools revealed a complex genetic network functioning in different CD34+ CD38- cells, in which several genes act as hubs, such as MYC in CB HSCs and hepatic growth factor in bone marrow HSCs, to maintain the stability or connectivity of the whole network. This study provides the foundation for a more detailed understanding of CD34+ CD38- HSCs from different sources, and reveals the potentials of different HSCs for different clinical applications.

Keratinocytes Function as Accessory Cells for Presentation of Endogenous Antigen Expressed in the Epidermis

The precise contribution(s) of skin dendritic cells (DCs) to immune responses in the skin has not been well delineated. We developed an intradermal (i.d.) injection model in which CD8+ T (OT-I) cells that express ovalbumin (OVA) peptide-specific TCRs (Valpha2/Vbeta5) are delivered directly to the dermis of transgenic (Tg) mice expressing OVA in the epidermis. After i.d. injection, these mice reliably develop skin graft-versus-host disease (GVHD) by day 7. To determine the relative contribution of Langerhans cells (LCs) to the ensuing GVHD-like reaction, we generated K14-OVA x Langerin-diphtheria-toxin-receptor (Langerin-DTR) Tg mice to allow conditional ablation of LCs in the epidermis. To delineate the role of dermal DCs (dDCs) in the reaction, we also generated K14-OVA Tg chimeras using beta(2)-microglobulin-deficient (beta(2)m) congenic donor bone marrow cells. Dermal DCs in these mice cannot present OVA to autoreactive T cells (OT-I cells), whereas the LCs are antigen presentation-competent. Unexpectedly, OT-I cell injection into diphtheria toxin (DT)-treated beta(2)m --> K14-OVA x Langerin-DTR Tg mice resulted in skin GVHD. Thus, in vivo, both LC and dDC appear to be dispensable for the induction of keratinocyte-directed, CD8-mediated effector immune responses. Furthermore and surprisingly, OVA-expressing epidermal cells depleted of LCs that could not initiate allogeneic epidermal lymphocyte reactions activated naive OT-I cells in vitro. These results indicate that keratinocytes may function as accessory cells competent to prime naive skin-reactive T cells.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://network.nature.com/group/jidclub.

Lentiviral Vector-Based Prime/Boost Vaccination against AIDS: Pilot Study Shows Protection against Simian Immunodeficiency Virus SIVmac251 Challenge in Macaques

AIDS vaccination has a pressing need for more potent vaccination vectors capable of eliciting strong, diversified, and long-lasting cellular immune responses against human immunodeficiency virus (HIV). Lentiviral vectors have demonstrated efficiency not only as gene delivery vehicles for gene therapy applications but also as vaccination tools. This is likely due to their ability to transduce nondividing cells, including dendritic cells, enabling sustained endogenous antigen presentation and thus the induction of high proportions of specific cytotoxic T cells and long-lasting memory T cells. We show in a first proof-of-concept pilot study that a prime/boost vaccination strategy using lentiviral vectors pseudotyped with a glycoprotein G from two non-cross-reactive vesicular stomatitis virus serotypes elicited robust and broad cellular immune responses against the vector-encoded antigen, simian immunodeficiency virus (SIV) GAG, in cynomolgus macaques. Vaccination conferred strong protection against a massive intrarectal challenge with SIVmac251, as evidenced both by the reduction of viremia at the peak of acute infection (a mean of over 2 log(10) fold reduction) and by the full preservation of the CD28(+) CD95(+) memory CD4(+) T cells during the acute phase, a strong correlate of protection against pathogenesis. Although vaccinees continued to display lower viremia than control macaques during the early chronic phase, these differences were not statistically significant by day 50 postchallenge. A not-optimized SIV GAG antigen was chosen to show the strong potential of the lentiviral vector system for vaccination. Given that a stronger protection can be anticipated from a modern HIV-1 antigen design, gene transfer vectors derived from HIV-1 appear as promising candidates for vaccination against HIV-1 infection.

Autophagy is essential for ER homeostasis and calcium mobilization in T lymphocytes (35.13)

Abstract The evolutionarily conserved intracellular bulk degradation pathway, macroautophagy, is involved in eliminating intracellular pathogens, endogenous antigen presentation in MHC-II pathway and T lymphocytes homeostasis. However, the underlying mechanisms by which autophagy regulates T lymphocyte function remain unknown. Here we used autophagy related protein 7 (Atg7) conditional knockout mice to investigate these mechanisms. Comparing to wild type T cells, Atg7-deficient T cells displayed impaired calcium influx upon TCR triggering or lower concentration of ionomycin stimulation. However, the proximal signaling of TCR in Atg7-deficient T cells was intact. The calcium influx defect in Atg7-deficient T cells may be related to an abnormal expansion of endoplasmic reticulum (ER) and higher intracellular calcium stores. We found that higher amount of ionomycin or the ER sarco/endoplasmic reticulum Ca2+ATPase (SERCA) pump inhibitor, thapsigargin, could rescue the calcium influx defect in Atg7-deficient T lymphocytes. Atg7-deficient T cells failed to efficiently proliferate and were blocked at S phase when stimulated by TCR or PMA plus ionomycin respectively. In contrast, Atg7-deficient T cells were fully activated and capable of secreting IL-2. Our results suggest that an important role for autophagy in T cells is to maintain ER homeostasis.

Autophagy enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV-1 infection

Viral proteins are usually processed by the 'classical' major histocompatibility complex (MHC) class I presentation pathway. Here we showed that although macrophages infected with herpes simplex virus type 1 (HSV-1) initially stimulated CD8(+) T cells by this pathway, a second pathway involving a vacuolar compartment was triggered later during infection. Morphological and functional analyses indicated that distinct forms of autophagy facilitated the presentation of HSV-1 antigens on MHC class I molecules. One form of autophagy involved a previously unknown type of autophagosome that originated from the nuclear envelope. Whereas interferon-gamma stimulated classical MHC class I presentation, fever-like hyperthermia and the pyrogenic cytokine interleukin 1beta activated autophagy and the vacuolar processing of viral peptides. Viral peptides in autophagosomes were further processed by the proteasome, which suggests a complex interaction between the vacuolar and MHC class I presentation pathways.

Endogenous antigen presenting cell-derived IL-10 inhibits T lymphocyte responses to commensal enteric bacteria

Interleukin-10 deficient (IL-10−/−) mice develop chronic T cell-mediated colitis when colonized with normal commensal bacteria, but germ-free (GF) IL-10−/− mice remain disease-free. Antigen presenting cells (APC) secrete regulatory cytokines that help determine T lymphocyte activation or tolerance. CD4 + T cells from the mesenteric lymph nodes of inflamed IL-10−/− mice secrete more IFN-γ and IL-17 when cultured with cecal bacterial lysate-pulsed splenic APC from IL-10−/− mice than when cultured with normal control APC. GF IL-10−/− APC induce similar IFN-γ and IL-17 responses; therefore, the functional difference between normal and IL-10 deficient APC is inherent to the lack of IL-10 and not secondary to inflammation. Bacterial lysate-pulsed normal APC cultured with CD4 + cells from colitic IL-10−/− mice or with exogenous IFN-γ secrete higher amounts of IL-10 compared to the same APC cultured with naïve T cells. APC enriched for CD11c + cells are potent activators of IFN-γ and IL-17 production by CD4 + cells from IL-10−/− mice. These APC also produce IL-12/IL-23 p40 and IL-10. Recombinant IL-10 suppressed and anti-IL-10 receptor antibody increased IFN-γ, IL-17 and IL-12/IL-23 p40 production in bacterial lysate-pulsed APC and plus CD4 + T cell co-cultures. Taken together, our results show that endogenous IL-10 produced by APC inhibits responses to commensal bacteria and influences the ability of APC to stimulate IFN-γ-producing effector lymphocytes, which reciprocally, induce IL-10 production by APC. Cytokines produced by APC are an important determinant of pathogenic versus protective mucosal immune responses to colonic bacterial stimulation.

Endogenous antigen presenting cell-derived IL-10 inhibits T lymphocyte responses to commensal enteric bacteria

Endogenous antigen presenting cell-derived IL-10 inhibits T lymphocyte responses to commensal enteric bacteria