MHC-restricted Antigen Presentation Research Articles

Analysis of LC3-Associated Phagocytosis and Antigen Presentation in Macrophages and B Cells.

Canonical autophagy and the non-canonical autophagy pathway LC3-associated phagocytosis (LAP) play crucial roles in the immune system by processing antigens for major histocompatibility complex (MHC) class II restricted presentation to CD4+ T cells. Recent studies offer insight into the relationship between LAP, autophagy, and antigen processing in macrophages and dendritic cells; however their involvement during antigen processing in B cells is less well understood.In this chapter, we describe how to monitor, manipulate, and understand the role of LAP and classical autophagy during MHC-restricted antigen presentation by human monocyte-derived macrophages as well as in B cell lymphoblastoid cell lines (LCLs). It includes an explanation on how to generate LCLs and monocyte-derived macrophages from primary human cells. Then we describe two different approaches to manipulate the autophagy pathways: silencing of the atg4b gene using CRISPR/Cas9 technology and a lentivirus delivery system for specific ATG4B overexpression. We also propose a method for triggering LAP and measuring different ATG proteins using Western blot and immunofluorescence. Finally, we show an approach to investigate MHC class II antigen presentation by an in vitro co-culture assay that uses the measurement of secreted cytokines, released by activated CD4+ T cells, as readout.

Non-canonical functions of autophagy proteins in immunity and infection

The molecular machinery of macroautophagy, a catabolic pathway for cytoplasmic constituent degradation in lysosomes, remodels membranes by lipid phosphorylation and conjugation of LC3 and GABARAP proteins. In recent year it has become clear that these membrane modifications also regulate endo- and exocytosis. Here I will discuss recent evidence of how such non-canonical functions of the macroautophagy machinery with its autophagy related gene (atg) products influences infectious viral particle secretion, inflammation, and MHC restricted antigen presentation. Especially LC3-Associated Phagocytosis and ATG supported exocytosis will be high-lighted during immunity and infection.

Do the two transcription factors form a positive feedback amplifier circuit in their common fight against pathogens?

Do the two transcription factors form a positive feedback amplifier circuit in their common fight against pathogens?

Endocytosis regulation by autophagy proteins in MHC restricted antigen presentation.

The macroautophagy machinery supports membrane remodeling and fusion events that lead to the engulfment of cytoplasmic constituents in autophagosomes and their degradation in lysosomes. The capacity of this machinery to regulate membrane adaptors and influence vesicle fusion with lysosomes seems to be used not only for autophagosomes, but also for endosomes. We summarize recent evidence that two aspects of endocytosis are regulated by parts of the macroautophagy machinery. These are recruitment of adaptors for the internalization of surface receptors and the fusion of phagosomes with lysosomes. Antigen processing for MHC presentation is affected by these alternative functions of the macroautophagy machinery. Primarily extracellular antigen presentation by MHC class II molecules after phagocytosis benefits from this regulation of phagosome maturation. Furthermore, MHC class I molecules are more efficiently internalized in the presence of the core macroautophagy machinery. The identification of these alternative functions of macroautophagy proteins not only complicates the interpretation of their deficiencies in biological processes, but could also be harnessed for the regulation of antigen presentation to T cells.

Peptidoglycan-linked protein A promotes T cell-dependent antibody expansion during Staphylococcus aureus infection

A hallmark of Staphylococcus aureus disease in humans is persistent infections without development of protective immune responses. Infected patients generate VH3 plasmablast expansions and increased VH3 idiotype Ig; however, the mechanisms for staphylococcal modification of immune responses are not known. We report here that S. aureus-infected mice generate VH3 antibody expansions via a mechanism requiring MHC-restricted antigen presentation to CD4(+) T cells and staphylococcal protein A (SpA), a cell wall-anchored surface molecule that binds Fcγ and VH3 variant heavy chains of Ig. VH3 expansion occurred with peptidoglycan-linked SpA from the bacterial envelope but not with recombinant SpA, and optimally required five tandem repeats of its Ig-binding domains. Signaling via receptor-interacting serine/threonine protein kinase 2 (RIPK2) was essential for implementing peptidoglycan-linked SpA superantigen activity. VH3 clan IgG from S. aureus-infected or SpA-treated animals was not pathogen-specific, suggesting that SpA cross-linking of VH3 idiotype B-cell receptors and activation via attached peptidoglycan are the determinants of staphylococcal escape from adaptive immune responses.

Metformin reduces gene expression of adipogenesis and inflammatory cytokines through the modulation of T cell priming in obese-induced Type II diabetic mice (P5063)

Abstract Inflammation is increasingly regarded as a key process underlying metabolic diseases in obese individuals where obese adipose tissue shows features characteristic of active local inflammation. Metformin is a biguanide derivative used in the treatment of T2D and one of the world's most widely prescribed drugs and have been suggested that metformin has anti-inflammatory properties. Here we show that metformin efficiently attenuates the metabolic disorder in DIO mice and suppresses MHC-restricted antigen presentation in dendritic cells. This study was to investigate the therapeutic effect of metformin on diabetic symptoms in DIO mice. Metformin decreased aipogenesis related gene, FAS, SCD-1, GPAT, SREBP1a, but also suppressed gene expression of innate cytokines than control mice. Metformin decreased both class I- and class II-restricted presentation of exogenous OVA in dendritic cells. Metformin also decreased expression of the co-stimulatory molecules, ICAM-1 and B7-1/-2, and MHC class I and II molecules but not phagocytic activity of DCs. Metformin decreased MHC class II-restricted exogenous antigen presentation in peritoneal macrophages in vivo. These results show that metformin attenuates immune responses followed by decreases MHC-restricted presentation of exogenous antigen in DCs via intracellular processing events. Theses finding suggest that modulation of the antigen presentation pathway could provide a novel means for regulating T cell responses in metabolic diseases.

Metformin Suppresses MHC-Restricted Antigen Presentation by Inhibiting Co-Stimulatory Factors and MHC Molecules in APCs

Metformin is widely used for T2D therapy but its cellular mechanism of action is undefined. Recent studies on the mechanism of metformin in T2D have demonstrated involvement of the immune system. Current immunotherapies focus on the potential of immunomodulatory strategies for the treatment of T2D. In this study, we examined the effects of metformin on the antigen-presenting function of antigen-presenting cells (APCs). Metformin decreased both MHC class I and class II-restricted presentation of OVA and suppressed the expression of both MHC molecules and co-stimulatory factors such as CD54, CD80, and CD86 in DCs, but did not affect the phagocytic activity toward exogenous OVA. The class II-restricted OVA presentation-regulating activity of metformin was also confirmed using mice that had been injected with metformin followed by soluble OVA. These results provide an understanding of the mechanisms of the T cell response-regulating activity of metformin through the inhibition of MHC-restricted antigen presentation in relation to its actions on APCs.

Biodegradable polymer, PLGA, is an efficient vehicle for class II MHC-restricted antigen presentation and the induction of DC maturation (106.15)

Abstract The antigen presentation and maturation activities of antigen-encapsulated biodegradable nanoparticles were compared with that of antigen-antibody immune complexes. Dendritic cells (DCs) were allowed to uptake ovalbumin (OVA) via pinocytosis, phagocytosis or endocytosis, and then the levels of MHC-restricted OVA peptide presentation were measured using OVA specific CD4 and CD8 T cells. Poly(D,L-lactic acid-co-glycolic acid, PLGA)-encapsulated OVA (pOVA) and OVA-IgG complexes (OVA-IC) were equally efficient in inducing MHC class I and II-restricted OVA peptide presentation. Comparison of the amount of OVA peptide presentation showed that class II-restricted presentation pathways were robust process compared to class I-restricted presentation pathways. Maturation inducing activities of pOVA and OVA-IC were also examined. pOVA was as efficient as or better compared to OVA-IC in inducing the expression of cell surface molecules such as class II, CD80, CD86, and the secretion of cytokines IL-6, IL-1β and TNF-α. These results show that the biodegradable polymer, PLGA, is an efficient vehicle not only for class II-restricted antigen presentation but also for induction of DC maturation. The present study also shows that in exogenous antigen presentation by DCs, cross presentation is a much weaker phenomenon compared to MHC class II-restricted presentation.

The rs4774 CIITA missense variant is associated with risk of systemic lupus erythematosus

The major histocompatibility complex (MHC) class II transactivator gene (CIITA) encodes an important transcription factor required for human leukocyte antigens (HLA) class II MHC-restricted antigen presentation. MHC genes, including the HLA class II DRB1*03:01 allele, are strongly associated with systemic lupus erythematosus (SLE). Recently the rs4774 CIITA missense variant (+1632G/C) was reported to be associated with susceptibility to multiple sclerosis. In the current study, we investigated CIITA, DRB1*03:01 and risk of SLE using a multi-stage analysis. In stage 1, 9 CIITA variants were tested in 658 cases and 1363 controls (N=2021). In stage 2, rs4774 was tested in 684 cases and 2938 controls (N=3622). We also performed a meta-analysis of the pooled 1342 cases and 4301 controls (N=5643). In stage 1, rs4774(*)C was associated with SLE (odds ratio (OR)=1.24, 95% confidence interval (95% CI)=1.07-1.44, P=4.2 × 10(-3)). Similar results were observed in stage 2 (OR=1.16, 95% CI=1.02-1.33, P=8.5 × 10(-3)) and the meta-analysis of the combined data set (OR=1.20, 95% CI=1.09-1.33, P(meta)=2.5 × 10(-4)). In all three analyses, the strongest evidence for association between rs4774(*)C and SLE was present in individuals who carried at least one copy of DRB1*03:01 (P(meta)=1.9 × 10(-3)). Results support a role for CIITA in SLE, which appears to be stronger in the presence of DRB1*03:01.

Ubiquitin-Dependent Control of Class II MHC Localization Is Dispensable for Antigen Presentation and Antibody Production

Controlled localization of class II MHC molecules is essential for proper class II MHC-restricted antigen presentation and the subsequent initiation of an adaptive immune response. Ubiquitination of class II MHC molecules on cytosolic lysine (K225) of the β-chain has been shown to affect localization of the complex. We generated mice in which the endogenous β-chain locus is replaced with a GFP tagged mutant version that lacks the cytosolic lysine residue (I-A-β-K225R-EGFP). These mice have elevated levels of class II MHC as compared to I-A-β-EGFP mice, and immature bone marrow-derived dendritic cells show redistribution of class II MHC to the cell surface. Nonetheless, in these same cells efficiency of antigen presentation is unaffected in I-A-β-K225R-EGFP mice, as assayed for presentation of ovalbumin to appropriately specific T cells. The I-A-β-K225R-EGFP animals have normal CD4 T cell populations and are capable of generating antigen-specific antibody in response to model antigens and viral infection. We therefore conclude that in our experimental system modulation of trafficking by ubiquitination of residue K225 of the β-chain is not essential for the function of class II MHC products in antigen presentation or antibody production.

Baccatin III, a synthetic precursor of taxol, enhances MHC-restricted antigen presentation in dendritic cells

Baccatin III, a precursor for the semisynthesis of taxol, is widely considered to be an inactive derivative of taxol. Here we show that baccatin III efficiently enhances MHC-restricted antigen presentation in dendritic cells. Baccatin III increased both class I- and class II-restricted presentation of exogenous OVA in bone marrow-derived dendritic cells (BM-DCs). Baccatin III also increased class I-restricted presentation of virus-encoded endogenous OVA in BM-DCs. Baccatin III did not affect the phagocytic activity of BM-DCs. The antigen presentation-enhancing activity of baccatin III was examined further with nanoparticles containing OVA and baccatin III. Inclusion of baccatin III to nanoparticles containing OVA greatly enhanced their capacity to induce class I-restricted OVA peptide presentation in DCs both in vitro and in vivo. Accordingly, nanoparticles containing both OVA and baccatin III were much more efficient in inducing an OVA-specific CTL response in mice compared to those containing OVA only. These results demonstrate that baccatin III exerts immunomodulatory activities in vivo as well as in vitro on the MHC-restricted antigen presentation.

CIITA is not associated with risk of developing rheumatoid arthritis

The major histocompatibility complex (MHC) class II transactivator gene (CIITA) encodes an important transcription factor regulating genes required for human leukocyte antigen (HLA) class II MHC-restricted antigen presentation. MHC genes, particularly HLA class II, are strongly associated with risk of developing rheumatoid arthritis (RA). Given the strong biological relationship between CIITA and HLA class II genes, a comprehensive investigation of CIITA variation in RA was conducted. This study tested 31 CIITA single-nucleotide polymorphisms in 2542 RA cases and 3690 controls (N=6232). All individuals were of European ancestry, as determined by ancestry informative genetic markers. No evidence for association between CIITA variation and RA was observed after a correction for multiple testing was applied. This is the largest study to fully characterize common genetic variation in CIITA, including an assessment of haplotypes. Results exclude even a modest role for common CIITA polymorphisms in susceptibility to RA.

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.

The HCMV membrane glycoprotein US10 selectively targets HLA-G for degradation.

Human cytomegalovirus (HCMV) encodes an endoplasmic reticulum (ER)-resident transmembrane glycoprotein, US10, expressed early in the replicative cycle of HCMV as part of the same cluster that encodes the known immunoevasins US2, US3, US6, and US11. We show that US10 down-regulates cell surface expression of HLA-G, but not that of classical class I MHC molecules. The unique and short cytoplasmic tail of HLA-G (RKKSSD) is essential in its role as a US10 substrate, and a tri-leucine motif in the cytoplasmic tail of US10 is responsible for down-regulation of HLA-G. Both the kinetics of HLA-G degradation and the mechanisms responsible appear to be distinct from those used by the US2 and US11 pathways, suggesting the existence of a third route of protein dislocation from the ER. We show that US10-mediated degradation of HLA-G interferes with HLA-G–mediated NK cell inhibition. Given the role of HLA-G in protecting the fetus from attack by the maternal immune system and in directing the differentiation of human dendritic cells to promote the evolution of regulatory T cells, HCMV likely targets the HLA-G–dependent axis of immune recognition no less efficiently than it interferes with classical class I MHC–restricted antigen presentation.

CIITA variation in the presence of HLA-DRB1*1501 increases risk for multiple sclerosis

The MHC class II transactivator gene (CIITA) is an important transcription factor regulating gene required for HLA class II MHC-restricted antigen presentation. Association with HLA class II variation, particularly HLA-DRB1*1501, has been well-established for multiple sclerosis (MS). In addition, the -168A/G CIITA promoter variant (rs3087456) has been reported to be associated with MS. Thus, a multi-stage investigation of variation within CIITA, DRB1*1501 and MS was undertaken in 6108 individuals. In stage 1, 24 SNPs within CIITA were genotyped in 1320 cases and 1363 controls (n = 2683). Rs4774 (missense +1614G/C; G500A) was associated with MS (P = 4.9 x 10(-3)), particularly in DRB1*1501 +individuals (P = 1 x 10(-4)). No association was observed for the -168A/G promoter variant. In stage 2, rs4774 was genotyped in 973 extended families; rs4774*C was also associated with increased risk for MS in DRB1*1501+ families (P = 2.3 x 10(-2)). In a third analysis, rs4774 was tested in cases and controls (stage 1) combined with one case per family (stage 2) for increased power. Rs4774*C was associated with MS (P = 1 x 10(-3)), particularly in DRB1*1501+ cases and controls (P = 1 x 10(-4)). Results obtained from logistic regression analysis showed evidence for interaction between rs4774*C and DRB1*1501 associated with risk for MS (ratio of ORs = 1.72, 95% CI 1.28-2.32, P = 3 x 10(-4)). Furthermore, rs4774*C was associated with DRB1*1501+ MS when conditioned on the presence (OR = 1.67, 95% CI = 1.19-2.37, P = 1.9 x 10(-3)) and absence (OR = 1.49, 95% CI = 1.15-1.95, P = 2.3 x 10(-3)) of CLEC16A rs6498169*G, a putative MS risk allele adjacent to CIITA. Our results provide strong evidence supporting a role for CIITA variation in MS risk, which appears to depend on the presence of DRB1*1501.

Cordyceps militarisEnhances MHC-restricted Antigen Presentation via the Induced Expression of MHC Molecules and Production of Cytokines

BackgroundCordyceps militarys water extract (CME) has been reported to exert antitumor and immunomodulatory activities in vivo and in vitro. However, the therapeutic mechanism has not yet been elucidated. In this study, we examined the effects of CME on the antigen presenting function of antigen presenting cells (APCs).MethodsDendritic cells (DCs) were cultured in the presence of CME, and then allowed to phagocytose microspheres containing ovalbumin (OVA). After washing and fixing the efficacy of OVA, peptide presentation by DCs were evaluated using CD8 and CD4 T cells. Also, we confirmed the protein levels of proinflammatory cytokines through western blot analysis.ResultsCME enhanced both MHC class I and class II-restricted presentation of OVA in DCs. In addition, the expression of both MHC class I and II molecules was enhanced, but there was no changes in the phagocytic activity of exogenous OVA. Furthermore, CME induced the protein levels of iNOS, COX-2, proinflammatory cytokines, and nuclear p65 in a concentration-dependent manner, as determined by western blot.ConclusionThese results provide an understanding of the mechanism of the immuno-enhancing activity of CME on the induction of MHC-restricted antigen presentation in relation to their actions on APCs.

IL-1β Produced in Response to Islet Autoantigen Presentation Differentiates T-helper-17 Cells at the Expense of Regulatory T cells: Implications for the Timing of Tolerizing Immunotherapy

The effectiveness of tolerizing immunotherapeutic strategies, such as anti-CD40L or dendritic cells (DC), is greater when administered to young NOD mice than at peak insulitis. RelBlo DC, generated in the presence of an NF-kB inhibitor, induce T regulatory (Treg) cells and suppress inflammation in a model of rheumatoid arthritis. IL-1b is over-expressed in humans and mice at risk of T1DM, dysregulates Treg cells, and accelerates diabetes in NOD mice. We investigated the relationship between IL-1b production and the response to RelBlo DC in the pre-diabetic period. We injected RelBlo DC s.c. into 4- or 14-week-old NOD mice, and tracked the incidence of diabetes and effect on Treg cell function. We measured IL-1b production by stimulated splenocytes from mice of different ages and strains, and proliferative and cytokine responses of T effectors to Treg in vitro. Tolerising RelBlo DC significantly inhibited diabetes progression when administered to 4-week-old but not 14 week old mice. IL-1b production by NOD splenocytes was increased from 6 to 16 weeks of age, when MHC-restricted islet antigen presentation to autoreactive T cells occurred. IL-1 promoted Th17 cells and reduced the capacity of Treg cells to suppress effector cells. RelBlo DC exacerbated the IL-1-dependent decline in Treg function and promoted Th17. IL-1b, generated by islet-autoreactive cells in MHC-susceptible mice, accelerates diabetes by differentiating Th17 at the expense of Treg. Tolerizing DC-therapies can regulate islet autoantigen priming and prevent diabetes, but progression past the IL-1b/IL-17 checkpoint signals the need for other strategies.

In vitro generation of murine myeloid dendritic cells from CD34‐positive precursors

Dendritic cells (DCs) link the innate and adaptive immune system. Currently, murine DCs for cell biology investigations are developed from MHC class II-negative bone marrow (BM) precursor cells, non-depleted BM cells or BM monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Here we demonstrate an isolation procedure of functionally intact myeloid CD11c(+) CD11b(+) DCs derived from murine CD34-positive precursors. DCs derived from CD34(+) cells show functional internalization, maturation, cytokine secretion, MHC-restricted antigen presentation, and MHCII retrograde transport of antigens from the lysosomes to the cell surface. In comparison to the established method, the advantages of this isolation procedure are a shorter cultivation period, a superior transfection efficiency, the yield of a purer and more homogeneous population of immature DCs, and less consumption of cell culture medium and GM-CSF. The new isolation procedure and the functional quality of CD34(+) cell-derived murine myeloid DCs make them ideally suited for immunology and cell biology studies.

Vanilloid receptor 1 agonists, capsaicin and resiniferatoxin, induce efficient anti-viral CTL responses by increasing MHC-restricted antigen presentation (78.29)

Abstract DCs, like the sensory neurons, express vanilloid receptor 1 (VR1). Here we demonstrate that VR1 agonists, capsaicin (CP) and resiniferatoxin (RTX), increase MHC-restricted antigen presentation in DCs. Treatment of DCs, that were infected with recombinant vaccinia virus (VV) expressing OVA (VV-OVA), with CP or RTX significantly increased MHC class I-restricted OVA presentation. In accordance with the in vitro results, oral administration of CP into VV-OVA-infected mice significantly enhanced MHC class I-restricted OVA presentation, and consequently elicited potent OVA-specific CTL activity compared to untreated controls. The effects of VR1 agonists on the exogenous antigen presentation were also examined in DCs. CP and RTX enhanced MHC class I-restricted, but not MHC class II-restricted, presentation of exogenous OVA. CP and RTX did not affect phagocytic activity, the expression level of total MHC molecules and co-stimulatory molecules on DC, suggesting that they enhance the intracellular processing events of phagocytosed antigens. These results demonstrate that VR1 agonists preferentially increase MHC class I-restricted antigen processing pathways. The present study also shows that oral consumption of CP, a common component of pungent foods, could enhance the efficacy of DC-based anti-viral and anti-tumor vaccines.

COX‐1 inhibitors block MHC‐restricted exogenous antigen presentation in dendritic cells

T lymphocytes recognize antigens in the form of short peptides presented by MHC molecules. Although MHC‐restricted antigen presentation is an essential process for the initiation T cell responses, it has received little attention as a target of therapeutic immunomodulation. In an effort to search for and characterize MHC‐restricted antigen presentation modulators, we screened several thousands of compounds for their ability to inhibit or enhance exogenous antigen presentation in dendritic cells (DCs). We found that COX‐1 inhibitors, aspirin and ibuprofen, efficiently block MHC‐restricted presentation of an exogenous antigen, ovalbumin (OVA), when added to cultures of DC2.4 cells and BM‐DCs for 18 h or longer. The antigen presentation inhibitory activity of ibuprofen was much more potent than aspirin. The COX‐1 inhibitors, however, did not affect phagocytic activity, the expression level of total MHC molecules and co‐stimulatory molecules in DCs. DCs differentiated from BM cells in the presence of COX‐1 inhibitors were also suppressed in the antigen presentation capability, and this suppressive activity was more prominent when developing DCs were exposed with the COX‐1 inhibitors at the early stage of maturation. These results suggest that prolonged administration of COX‐1 inhibitors may impair antigen presentation capability of DCs.