Ubiquitination of Major Histocompatibility Complex II Changes Its Immunological Recognition Structure.

Ubiquitination is a process that dictates the lifespan of major histocompatibility complex class II (MHC II)/peptide complexes on antigen-presenting cells. This process is tightly controlled by the levels of ubiquitin ligases, and disruptions in the turnover of MHC II can lead to the improper development of CD4+ T cells within the thymus and hinder the formation of regulatory T cells in the peripheral tissue. To investigate the underlying mechanisms, we utilized dendritic cells lacking the Membrane-associated RING-CH (MARCH) I ubiquitin ligase. We discovered that the overexpression of MARCH I decreases the interaction with LAG-3. Moreover, the MHC II molecules tethered with ubiquitin also showed diminished binding to LAG-3. We employed Diffracted X-ray Blinking (DXB), a technique used for single-molecule X-ray imaging, to observe the protein movements on live cells in real time. Our observations indicated that the normal MHC II molecules moved more rapidly across the cell surface compared to those on the MARCH I-deficient dendritic cells or MHC II KR mutants, which is likely a result of ubiquitination. These findings suggest that the signaling from ubiquitinated MHC II to the T cell receptor differs from the non-ubiquitinated forms. It appears that ubiquitinated MHC II might not be quickly internalized, but rather presents antigens to the T cells, leading to a range of significant immunological responses.

Elucidation of binding mechanism, affinity, and complex structure between mWT1 tumor-associated antigen peptide and HLA-A*24:02.

We have applied our advanced computational and experimental methodologies to investigate the complex structure and binding mechanism of a modified Wilms' Tumor 1 (mWT1) protein epitope to the understudied Asian-dominant allele HLA-A*24:02 (HLA-A24) in aqueous solution. We have applied our developed multicanonical molecular dynamics (McMD)-based dynamic docking method to analyze the binding pathway and mechanism, which we verified by comparing the highest probability structures from simulation with our experimentally solved x-ray crystal structure. Subsequent path sampling MD simulations elucidated the atomic details of the binding process and indicated that first an encounter complex is formed between the N-terminal's positive charge of the 9-residue mWT1 fragment peptide and a cluster of negative residues on the surface of HLA-A24, with the major histocompatibility complex (MHC) molecule preferring a predominantly closed conformation. The peptide first binds to this closed MHC conformation, forming an encounter complex, after which the binding site opens due to increased entropy of the binding site, allowing the peptide to bind to form the native complex structure. Further sequence and structure analyses also suggest that although the peptide loading complex would help with stabilizing the MHC molecule, the binding depends in a large part on the intrinsic affinity between the MHC molecule and the antigen peptide. Finally, our computational tools and analyses can be of great benefit to study the binding mechanism of different MHC types to their antigens, where it could also be useful in the development of higher affinity variant peptides and for personalized medicine.

Aire Controls in Trans the Production of Medullary Thymic Epithelial Cells Expressing Ly-6C/Ly-6G.

Medullary thymic epithelial cells (mTECs), which express a wide range of tissue-restricted Ags (TRAs), contribute to the establishment of self-tolerance by eliminating autoreactive T cells and/or inducing regulatory T cells. Aire controls a diverse set of TRAs within Aire-expressing cells by employing various transcriptional pathways. As Aire has a profound effect on transcriptomes of mTECs, including TRAs not only at the single-cell but also the population level, we suspected that Aire (Aire+ mTECs) might control the cellular composition of the thymic microenvironment. In this study, we confirmed that this is indeed the case by identifying a novel mTEC subset expressing Ly-6 family protein whose production was defective in Aire-deficient thymi. Reaggregated thymic organ culture experiments demonstrated that Aire did not induce the expression of Ly-6C/Ly-6G molecules from mTECs as Aire-dependent TRAs in a cell-intrinsic manner. Instead, Aire+ mTECs functioned in trans to maintain Ly-6C/Ly-6G+ mTECs. Thus, Aire not only controls TRA expression transcriptionally within the cell but also controls the overall composition of mTECs in a cell-extrinsic manner, thereby regulating the transcriptome from mTECs on a global scale.

Single-molecule motions of MHC class II rely on bound peptides.

The major histocompatibility complex (MHC) class II protein can bind peptides of different lengths in the region outside the peptide-binding groove. Peptide-flanking residues (PFRs) contribute to the binding affinity of the peptide for MHC and change the immunogenicity of the peptide/MHC complex with regard to T cell receptor (TCR). The mechanisms underlying these phenomena are currently unknown. The molecular flexibility of the peptide/MHC complex may be an important determinant of the structures recognized by certain T cells. We used single-molecule x-ray analysis (diffracted x-ray tracking (DXT)) and fluorescence anisotropy to investigate these mechanisms. DXT enabled us to monitor the real-time Brownian motion of the peptide/MHC complex and revealed that peptides without PFRs undergo larger rotational motions than peptides with PFRs. Fluorescence anisotropy further revealed that peptides without PFRs exhibit slightly larger motions on the nanosecond timescale. These results demonstrate that peptides without PFRs undergo dynamic motions in the groove of MHC and consequently are able to assume diverse structures that can be recognized by T cells.

Beta3GnT2 (B3GNT2), a major polylactosamine synthase: analysis of B3GNT2-deficient mice.

The polylactosamine structure is a fundamental structure of carbohydrate chains and carries a lot of biofunctional carbohydrate epitopes. To investigate the biological function of polylactosamine chains, here we generated and analyzed knockout mice lacking the gene B3gnt2, which encodes a major polylactosamine synthase. In beta1,3-N-acetylglucosaminyltransferase (B3gnt2) B3gnt2-deficient (B3gnt2-/-) mice, the number of polylactosamine structures was markedly lower than in wild-type mice. Flow cytometry, LEL lectin-blotting, and glycan analysis by metabolic labeling demonstrated that the amount of polylactosamine chains on N-glycans was greatly reduced in the tissues of B3gnt2-/- mice. We examined whether immunological abnormalities were present in B3gnt2-/- mice. We screened polylactosamine-carrying molecules of wild-type mice by lectin microarray analysis and found that polylactosamine was present on CD28 and CD19, two established immune co-stimulatory molecules. Polylactosamine levels on these molecules were lower in B3gnt2-/- mice than in wild-type mice. B3gnt2-/- T cells were more sensitive to the induction of intracellular Ca2+ flux on stimulation with anti-CD3epsilon/CD28 antibodies and proliferated more strongly than wild-type T cells. B3gnt2-/- B cells also showed hyperproliferation on BCR stimulation. These results showed that hyperactivation of lymphocytes occurred due to a lack of polylactosamine on receptor molecules in B3gnt2-/- mice. This finding indicates that polylactosamine has an important role in immunological biofunctions. We can therefore attempt to identify the in vivo biological function of glycans using glycogene-deficient mice.

Lack of lacto/neolacto-glycolipids enhances the formation of glycolipid-enriched microdomains, facilitating B cell activation.

In a previous study, we demonstrated that beta1,3-N-acetylglucosaminyltransferase 5 (B3gnt5) is a lactotriaosylceramide (Lc(3)Cer) synthase that synthesizes a precursor structure for lacto/neolacto-series glycosphingolipids (GSLs) in in vitro experiments. Here, we generated B3gnt5-deficient (B3gnt5(-/-)) mice to investigate the in vivo biological functions of lacto/neolacto-series GSLs. In biochemical analyses, lacto/neolacto-series GSLs were confirmed to be absent and no Lc(3)Cer synthase activity was detected in the tissues of these mice. These results demonstrate that beta3GnT5 is the sole enzyme synthesizing Lc(3)Cer in vivo. Ganglioside GM1, known as a glycosphingolipid-enriched microdomain (GEM) marker, was found to be up-regulated in B3gnt5(-/-) B cells by flow cytometry and fluorescence microscopy. However, no difference in the amount of GM1 was observed by TLC-immunoblotting analysis. The GEM-stained puncta on the surface of B3gnt5(-/-) resting B cells were brighter and larger than those of WT cells. These results suggest that structural alteration of GEM occurs in B3gnt5(-/-) B cells. We next examined whether BCR signaling-related proteins, such as BCR, CD19, and the signaling molecule Lyn, had moved into or out of the GEM fraction. In B3gnt5(-/-) B cells, these molecules were enriched in the GEM fraction or adjacent fraction. Moreover, B3gnt5(-/-) B cells were more sensitive to the induction of intracellular phosphorylation signals on BCR stimulation and proliferated more vigorously than WT B cells. Together, these results suggest that lacto/neolacto-series GSLs play an important role in clustering of GEMs and tether-specific proteins, such as BCR, CD19, and related signaling molecules to the GEMs.

The role of {beta}-TrCP1 and {beta}-TrCP2 in circadian rhythm generation by mediating degradation of clock protein PER2.

The mammalian circadian clock proteins undergo a daily cycle of accumulation followed by phosphorylation and degradation. The mechanism by which clock proteins undergo degradation has not been fully understood. Circadian clock protein PERIOD2 (PER2) is shown to be the potential target of F-box protein beta-TrCP1, a component of ubiquitin E3 ligase. Here, we show that beta-TrCP2 as well as beta-TrCP1 target PER2 protein in vitro. We also identified beta-TrCP binding site (m2) of PER2 being recognized by both beta-TrCP1 and beta-TrCP2. Luciferase-PER2 fusion system revealed that m2 site was responsible for the stability of PER2. The role of beta-TrCP1 and beta-TrCP2 in circadian rhythm generation was analysed by real-time reporter assay revealing that siRNA-mediated suppressions of beta-TrCP1 and/or beta-TrCP2 attenuate circadian oscillations in NIH3T3 cell. beta-TrCP1-deficient mice, however, showed normal period length, light-induced phase-shift response in behaviour and normal expression of PER2, suggesting that beta-TrCP1 is dispensable for the central clock in the suprachiasmatic nucleus. Our study indicates that beta-TrCP1 and beta-TrCP2 were involved in the cell autonomous circadian rhythm generation in culture cells, although the role of beta-TrCP2 in the central clock in the suprachiasmatic nucleus remains to be elucidated.

Polylactosamine on glycoproteins influences basal levels of lymphocyte and macrophage activation.

beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is a polylactosamine synthase that synthesizes a backbone structure of carbohydrate structures onto glycoproteins. Here we generated beta3GnT2-deficient (beta3GnT2(-/-)) mice and showed that polylactosamine on N-glycans was markedly reduced in their immunological tissues. In WT mice, polylactosamine was present on CD28 and CD19, both known immune costimulatory molecules. However, polylactosamine levels on these molecules were reduced in beta3GnT2(-/-) mice. beta3GnT2(-/-) T cells lacking polylactosamine were more sensitive to the induction of intracellular calcium flux on stimulation with anti-CD3epsilon/CD28 and proliferated more strongly than T cells from WT mice. beta3GnT2(-/-) B cells also showed hyperproliferation on BCR stimulation. Macrophages from beta3GnT2(-/-) mice had higher cell surface CD14 levels and enhanced responses to endotoxin. These results indicate that polylactosamine on N-glycans is a putative immune regulatory factor presumably suppressing excessive responses during immune reactions.

Affinity maturation of secreted IgM pentamers on B cells.

We prepared a series of hapten-BSA conjugates with varying ratios of biotin to measure ligand-receptor interactions on B cells by flow cytometry using avidin for detection. Surface plasmon resonance measurements of the interaction with a monoclonal anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody suggested that NP(5)-BSA or NP(7)-BSA harboring 29 or 23 biotin molecules (NP(5)-BSA-bio(29) or NP(7)-BSA-bio(23)) would be suitably sensitive for flow cytometric analysis. By using NP-BSA-bio, we analyzed NP-binding cells in immunized mice. Unexpectedly, 30-40% of spleen cells expressing IgM could bind to NP(5)-BSA or NP(7)-BSA after immunization of mice with NP(40)-chicken gamma-globulin. The proteins binding to NP(7)-BSA-bio(23) on the cell surface were analyzed by immunoprecipitation and western blotting. Surprisingly, most of the proteins binding NP-BSA-bio on the cell surface were not the membrane form of IgM monomer, but a secreted IgM pentamer. It is likely that the IgM pentamer bound through Fc receptors for polymeric IgA or IgM and contributed to antigen binding. Comparison of the binding ratio of NP(0.9)-BSA:NP(5)-BSA between B cells of primary and secondary immunization suggested that the affinity of IgM matured during immunization.

Differential binding properties of B7-H1 and B7-DC to programmed death-1.

Programmed death-1 (PD-1) is a negative regulatory receptor expressed on activated T and B cells. Two ligands for PD-1, B7-H1 (PD-L1) and B7-DC (PD-L2), have been identified, but their binding properties have not been characterized yet. In this study, we generated soluble Ig fusion proteins of these molecules and examined the kinetics and relative affinities of the interactions between B7-H1 or B7-DC and PD-1 by flow cytometry and surface plasmon resonance. The interaction of B7-DC/PD-1 exhibited a 2-6-fold higher affinity and had different association/dissociation kinetics compared with the interaction of B7-H1/PD-1. Our results suggest that the differential binding properties of B7-H1 and B7-DC may be responsible for differential contributions of these two PD-1 ligands to immune responses.

Amelioration of collagen-induced arthritis by blockade of inducible costimulator-B7 homologous protein costimulation.

B7 homologous protein (B7h)/B7-related protein 1 (B7RP-1) is a new member of the B7 family of costimulatory molecules that specifically interacts with inducible costimulator (ICOS) expressed on activated T cells. Collagen type II (CII)-induced arthritis (CIA) is an experimental model of arthritis that has been used to dissect the pathogenesis of human rheumatoid arthritis. In this study, we have investigated the effect of neutralizing anti-B7h mAb on the development and disease progression of CIA. Administration of anti-B7h mAb significantly ameliorated the disease as assessed by clinical arthritis score and histology in the joints, and a beneficial effect was also obtained by a delayed treatment after the onset of disease. Expression of ICOS and B7h was observed in the inflamed synovial tissue as well as in the draining lymph nodes (LNs) and expansion of ICOS(+) T cells in the LN was reduced by the anti-B7h mAb treatment. Expression of mRNA for proinflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6 in the joints was inhibited by the treatment. Proliferative responses and production of IFN-gamma and IL-10 upon restimulation with CII in vitro were significantly inhibited in LN cells from the anti-B7h mAb-treated mice. Serum anti-CII IgG1, IgG2a, and IgG2b levels were also reduced. Our present results showed a beneficial effect of the B7h blockade on CIA through anti-inflammatory actions and inhibition of both Th1- and Th2-mediated immune responses, suggesting that the ICOS-B7h interaction plays an important role in the pathogenesis of CIA and thus the blockade of this pathway may be beneficial for the treatment of human rheumatoid arthritis.