[show abstract][hide abstract] ABSTRACT: Antigen presentation by MHC class I molecules requires degradation of epitope source proteins in the cytosol. Although the preeminent role of the proteasome is clearly established, evidence suggesting a significant role for proteasome-independent generation of class I ligands has been reported repeatedly. However, an enzyme responsible for such a role has not been identified. Recently insulin-degrading enzyme (IDE) was shown to produce an antigenic peptide derived from the tumor antigen MAGE-A3 in an entirely proteasome-independent manner, raising the question of the global impact of IDE in MHC class I antigen processing. Here we report that IDE knockdown in human cell lines, or knockout in two different mouse strains, has no effect on cell surface expression of various MHC class I molecules, including allomorphs such as HLA-A3 and HLA-B27 suggested to be loaded in an at least a partly proteasome-independent manner. Moreover, reduced or absent IDE expression does not affect presentation of five epitopes including epitopes derived from beta amyloid and proinsulin, two preferred IDE substrates. Thus, IDE does not play a major role in MHC class I antigen processing, confirming the dominant and almost exclusive role of the proteasome in cytosolic production of MHC class I ligands.
PLoS ONE 01/2014; 9(2):e88365. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The therapeutic efficacy of anthracyclines relies on antitumor immune responses elicited by dying cancer cells. How chemotherapy-induced cell death leads to efficient antigen presentation to T cells, however, remains a conundrum. We found that intratumoral CD11c(+)CD11b(+)Ly6C(hi) cells, which displayed some characteristics of inflammatory dendritic cells and included granulomonocytic precursors, were crucial for anthracycline-induced anticancer immune responses. ATP released by dying cancer cells recruited myeloid cells into tumors and stimulated the local differentiation of CD11c(+)CD11b(+)Ly6C(hi) cells. Such cells efficiently engulfed tumor antigens in situ and presented them to T lymphocytes, thus vaccinating mice, upon adoptive transfer, against a challenge with cancer cells. Manipulations preventing tumor infiltration by CD11c(+)CD11b(+)Ly6C(hi) cells, such as the local overexpression of ectonucleotidases, the blockade of purinergic receptors, or the neutralization of CD11b, abolished the immune system-dependent antitumor activity of anthracyclines. Our results identify a subset of tumor-infiltrating leukocytes as therapy-relevant antigen-presenting cells.
[show abstract][hide abstract] ABSTRACT: Insulin Degrading Enzyme (IDE) is a protease conserved through evolution with a role in diabetes and Alzheimer's disease. The reason underlying its ubiquitous expression including cells lacking identified IDE substrates remains unknown. Here we show that the fission yeast IDE homologue (Iph1) modulates cellular sensitivity to endoplasmic reticulum (ER) stress in a manner dependent on TORC1 (Target of Rapamycin Complex 1). Reduced sensitivity to tunicamycin was associated with a smaller number of cells undergoing apoptosis. Wild type levels of tunicamycin sensitivity were restored in iph1 null cells when the TORC1 complex was inhibited by rapamycin or by heat inactivation of the Tor2 kinase. Although Iph1 cleaved hallmark IDE substrates including insulin efficiently, its role in the ER stress response was independent of its catalytic activity since expression of inactive Iph1 restored normal sensitivity. Importantly, wild type as well as inactive human IDE complemented gene-invalidated yeast cells when expressed at the genomic locus under the control of iph1(+) promoter. These results suggest that IDE has a previously unknown function unrelated to substrate cleavage, which links sensitivity to ER stress to a pro-survival role of the TORC1 pathway.
PLoS ONE 01/2013; 8(6):e67705. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: In vitro cultures of bone marrow-derived precursors are a convenient method for generating dendritic cells (DC). This method additionally overcomes the problem of low availability of certain DC types, DC heterogeneity, and laborious procedures encountered using ex vivo isolation protocols.Here we describe two standard protocols for in vitro differentiation of steady-state DC equivalents with Fms-like tyrosine kinase 3 ligand (Flt3L) and inflammatory-like DC using granulocyte-macrophages-colony-stimulating factor (GM-CSF). These protocols allow for obtaining up to 2 × 10(8) CD11c(high) inflammatory-like DC and up to 5 × 10(6) equivalents of each CD8+ and CD8- conventional DC and plasmacytoid DC.
Methods in molecular biology (Clifton, N.J.) 01/2013; 960:351-357.
[show abstract][hide abstract] ABSTRACT: Cross-presentation is defined as the ability of certain professional antigen-presenting cells to take up, process and present extracellular antigens on major histocompatibility class I (MHC-I) molecules to CD8+ T cells. The stimulation of naive cytotoxic CD8+ T cells by this process, termed cross-priming, is involved in many different responses, including those to tumors, pathogens, graft tissues, and self-antigens. Dendritic cells (DCs), a heterogeneous cell population, are endowed with the highest cross-priming capacity. Investigation of their cross-presentation capacities, important both for vaccination and for the induction of immune tolerance can be performed by in vivo and in vitro assays. In this chapter we describe the preparation of antigens that can be used to test cross-presentation via pinocytosis, receptor-mediated endocytosis, and phagocytosis.
Methods in molecular biology (Clifton, N.J.) 01/2013; 960:389-400.
[show abstract][hide abstract] ABSTRACT: Peptide epitopes presented by MHC class I molecules are produced through sequential proteolysis, frequently terminating with an aminoterminal trimming step. While the trimming enzymes processing endogenous MHC class I ligands in the endoplasmic reticulum have by now been characterized extensively, we have only recently identified an endosomal enzyme, insulin-regulated aminopeptidase (IRAP) that can trim cross-presented peptides derived from proteins internalized by dendritic cells. Here we summarize the essential features of IRAP as a trimming enzyme, propose an updated model of cellular cross-presentation pathways, and discuss potential additional functions of IRAP and its compartment in dendritic cell biology.
[show abstract][hide abstract] ABSTRACT: Peptides presented by MHC class I molecules are typically produced through antigen degradation by the proteasome followed by trimming by exopeptidases. According to recent results, these include both aminopeptidases and carboxypeptidases in the cytosol and the endoplasmic reticulum. While cytosolic peptidases have a net neutral or destructive effect on MHC ligands, endoplasmic reticulum aminopeptidases are required for efficient class I loading and have a strong effect on the repertoire of peptide/MHC complexes. Cells lacking these enzymes can be eliminated both by NK cells and by CD8+ T cells recognizing complexes formed between an MHC class Ib molecule and a conserved peptide. Cross-presented peptides derived from internalized antigens can be processed by insulin-regulated aminopeptidase, the only endosomal trimming peptidase.
Current opinion in immunology 10/2012; · 10.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Intracellular Toll-like receptors (TLRs) expressed by dendritic cells recognize nucleic acids derived from pathogens and play an important role in the immune responses against the influenza virus (IAV), a single-stranded RNA sensed by different receptors including TLR7. However, the importance of TLR7 processing in the development of anti-viral immune responses is not known. Here we report that asparagine endopeptidase (AEP) deficient mice are unable to generate a strong anti-IAV response, as demonstrated by reduced inflammation, cross presentation of cell-associated antigens and priming of CD8(+) T cells following TLR7-dependent pulmonary infection induced by IAV. Moreover, AEP deficient lung epithelial- or myeloid-cells exhibit impaired TLR7 signaling due to defective processing of this receptor. Indeed, TLR7 requires a proteolytic cleavage by AEP to generate a C-terminal fragment competent for signaling. Thus, AEP activity is critical for TLR7 processing, opening new possibilities for the treatment of influenza and TLR7-dependent inflammatory diseases.
[show abstract][hide abstract] ABSTRACT: Type 1 diabetes results from the destruction of β-cells by an autoimmune T-cell response assisted by antigen-presenting B cells producing autoantibodies. CD8(+) T-cell responses against islet cell antigens, thought to play a central role in diabetes pathogenesis, can be monitored using enzyme-linked immunosorbent spot (ELISpot) assays. However, such assays have been applied to monitoring of adult patients only, leaving aside the large and increasing pediatric patient population. The objective of this study was twofold: 1) to develop a CD8(+) T-cell interferon-γ ELISpot assay for pediatric patients and 2) to determine whether zinc transporter 8 (ZnT8), a recently described target of autoantibodies in a majority of patients, is also recognized by autoreactive CD8(+) T cells. Using DNA immunization of humanized mice, we identified nine HLA-A2-restricted ZnT8 epitopes. Among 36 HLA-A2(+) children with diabetes, 29 responded to ZnT8 epitopes, whereas only 3 of 16 HLA-A2(+) control patients and 0 of 17 HLA-A2(-) control patients responded. Some single ZnT8 epitopes performed as well as the group of epitopes in discriminating between patients and control individuals. Thus, ZnT8 is a major CD8(+) T-cell autoantigen, and ELISpot assays display similar performance in adult and pediatric type 1 diabetes.
[show abstract][hide abstract] ABSTRACT: Dendritic cells (DCs) use cellular pathways collectively referred to as cross-presentation to stimulate CD8(+) T cells with peptide Ags derived from internalized, exogenous Ags. We have recently reported that DCs rely on aminoterminal trimming of cross-presented peptides by insulin-responsive aminopeptidase (IRAP), an enzyme localized in a regulated endosomal storage compartment. Considering a report contending that this role is limited to inflammatory DCs (Segura et al. 2009. Proc. Natl. Acad. Sci. USA 106: 20377-20381), in this study, we examined the role of IRAP in steady-state DC subpopulations. Steady-state conventional DCs (cDCs) and plasmacytoid DCs expressed similar amounts of IRAP. IRAP colocalized with the endosomal markers Rab14 and syntaxin 6, both known to be associated with regulated endosomal storage compartments, in CD8(+) and CD8(-) cDCs-however, to a greater extent in the former population. Likewise, IRAP recruitment to phagosomes was significantly stronger in CD8(+) DCs. IRAP deficiency compromised cross-presentation of soluble and particulate Ag by both CD8(+) and CD8(-) cDCs, again with a stronger effect in the former population. Thus, the requirement of IRAP in cross-presentation extends to steady-state cDCs. Moreover, these data suggest that increased recruitment of an IRAP(+)/Rab14(+) compartment to Ag-containing vesicles contributes to the superior cross-presentation efficacy of CD8(+) cDCs.
The Journal of Immunology 02/2012; 188(4):1840-6. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Production of MHC-I ligands from antigenic proteins generally requires multiple proteolytic events. While the proteolytic steps required for antigen processing in the endogenous pathway are clearly established, persisting gaps of knowledge regarding putative cross-presentation compartments have made it difficult to map the precise proteolytic events required for generation of cross-presented antigens. It is only in the past decade that the importance of aminoterminal trimming as the final step in the endogenous presentation pathway has been recognized and that the corresponding enzymes have been described. This review focuses on the aminoterminal trimming of exogenous cross-presented peptides, with particular emphasis on the identification of insulin responsive aminopeptidase (IRAP) as the principal trimming aminopeptidase in endosomes and phagosomes.
[show abstract][hide abstract] ABSTRACT: Major histocompatibility complex (MHC) class I cross-presentation is thought to involve two pathways, one of which depends on both the TAP transporters and the proteasome and the other on neither. We found that preincubation of TAP-deficient dendritic cells at low temperature increases the density of MHC class I at the surface and fully restores cross-presentation of phagocytosed antigen, but not of soluble antigen internalized through receptors. Restoration of cross-presentation by TAP-deficient cells requires antigen degradation by the proteasome. Thus, TAP might mainly be required for recycling cell surface class I molecules during cross-presentation of phagocytosed antigens. Furthermore, phagosomes-but not endosomes-seem to have a TAP-independent mechanism to import peptides generated by cytosolic proteasome complexes.
[show abstract][hide abstract] ABSTRACT: Major histocompatibility complex (MHC) class I molecules provide the molecular basis for the comprehensive surveillance of an organism by the cytotoxic arm of the adaptive immune system. To exert this function correctly, class I molecules must be loaded with peptide ligands of appropriate length, sequence and affinity that provide a rapidly updated and sufficiently comprehensive picture of the state of the cell. This is accomplished by a sophisticated cellular machinery using a blend of cellular house-keeping proteins and dedicated transporters, chaperones and peptidases. The last 10 years have seen substantial progress in our comprehension of this machinery. It seems now clear that a large proportion of MHC class I ligands are derived from short-lived products of the ribosomal apparatus, many of which correspond to defective proteins. Despite much effort to identify alternative proteolytic pathways, cytosolic production of epitopes still appears to depend almost entirely on the proteasome, while cytosolic aminopeptidases act mainly to limit antigen presentation. In contrast, clear evidence for a critical role of trimming peptidases residing in the endoplasmic reticulum has emerged. These enzymes play a role in responses against pathogens and are associated with autoimmune diseases, most notably ankylosing spondylitis. Much has also been learned about the intricate chaperone interactions in peptide-loading complexes, especially with respect to the structural role of tapasin-ERp57 conjugates and to the editing function of tapasin. In contrast, cross-presentation of exogenous antigens by MHC class I molecules still remains somewhat poorly understood and is likely to attract much research effort for years to come.
[show abstract][hide abstract] ABSTRACT: Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.
[show abstract][hide abstract] ABSTRACT: Peptide ligands presented by MHC class I molecules are produced by intracellular proteolysis, which often involves multiple steps. Initial antigen degradation seems to rely almost invariably on the proteasome, although tripeptidyl peptidase II (TPP II) and insulin-degrading enzyme (IDE) may be able to substitute for the proteasome in rare cases. Recent evidence suggests that the net effect of cytosolic aminopeptidases is destruction of potential class I ligands, although a positive role in selected cases has been documented. This may apply particularly to the trimming of long precursors by TPP II. In contrast, trimming of ligand precursors in the endoplasmic reticulum is essential for the generation of suitable peptides and has a substantial impact on the repertoire of ligands presented. Trimming by the ER aminopeptidase (ERAP) enzymes most likely acts on free precursors and is adapted to the needs of class I molecules by way of a molecular ruler mechanism. Trimming by ERAP enzymes also occurs for cross-presented ligands, which can alternatively be processed in a special endosomal compartment by insulin-regulated aminopeptidase.
Cellular and Molecular Life Sciences CMLS 03/2011; 68(9):1553-67. · 5.62 Impact Factor