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.

Carboxylated polyamidoamine dendron-bearing lipid-based assemblies for precise control of intracellular fate of cargo and induction of antigen-specific immune responses.

For the establishment of advanced medicines such as cancer immunotherapy, high performance carriers that precisely deliver biologically active molecules must be developed to target organelles of the cells and to release their contents there. From the viewpoint of antigen delivery, endosomes are important target organelles because they contain immune-response-related receptors and proteins of various types. To obtain carriers for precision endosome delivery, a novel type of polyamidoamine dendron-based lipid having pH-sensitive terminal groups was synthesized for this study. Liposomes were prepared using these pH-sensitive dendron-based lipids and egg yolk phosphatidylcholine. Their pH-responsive properties and performance as an endosome delivery carrier were investigated. pH-Sensitive dendron lipid-based liposomes retained water-soluble molecules at neutral pH but released them under weakly acidic conditions. Particularly, liposomes containing CHexDL-G1U exhibited highly sensitive properties responding to very weakly acidic pH. These dendron lipid-based liposomes released the contents specifically in the endosome. The timing of content release can be controlled by selecting pH-sensitive dendron lipids for liposome preparation. Significant tumor regression was induced in tumor-bearing mice by the administration of CHexDL-G1U-modified liposomes containing the model antigenic protein. Furthermore, CHexDL-G1U-modified liposomes induced WT1 tumor antigenic peptide-specific helper T cell proliferation. The results demonstrate that dendron lipid-based liposomes are useful as a potent vaccine for cancer immunotherapy.

Development of a novel monoclonal antibody that binds to most HLA-A allomorphs in a conformation-dependent yet peptide-promiscuous fashion.

Specificity analyses of peptide binding to human leukocyte antigen (HLA)-A molecules have been hampered due to a lack of proper monoclonal antibodies (mAbs) for certain allomorphs, such as the prevalent HLA-A1 for Caucasians and HLA-A11 for Asians. We developed a mAb that recognizes a conformational epitope common to most HLA-A allomorphs. The mAb, named A-1, does not discriminate peptides by amino acid sequences, making it suitable for measuring peptide binding. A stabilization assay using TAP-deficient cell lines and A-1 was developed to investigate the specificity of peptide binding to HLA-A molecules. Regarding the evolution of HLA-A genes, the A-1 epitope has been conserved among most HLA-A allomorphs but was lost when the HLA-A gene diversified into the HLA-A*32, HLA-A*31, and HLA-A*33 lineages together with HLA-A*29 after bifurcating from the HLA-A*25 and HLA-A*26 branchs. The establishment of A-1 is expected to help researchers investigate the peptide repertoire and develop computational tools to identify cognate peptides. Since no HLA-A locus-specific mAb has been available, A-1 will also be useful for analyzing the locus-specific regulation of the HLA gene expression.

High affinity IgM(+) memory B cells are generated through a germinal center-dependent pathway.

During a T cell-dependent immune response, B cells undergo clonal expansion and selection and the induction of isotype switching and somatic hypermutation (SHM). Although somatically mutated IgM(+) memory B cells have been reported, it has not been established whether they are really high affinity B cells. We tracked (4-hydroxy-3-nitrophenyl) acetyl hapten-specific GC B cells from normal immunized mice based on affinity of their B cell receptor (BCR) and performed BCR sequence analysis. SHM was evident by day 7 postimmunization and increased with time, such that high affinity IgM(+) as well as IgG(+) memory B cells continued to be generated up to day 42. In contrast, class-switch recombination (CSR) was almost completed by day 7 and then the ratio of IgG1(+)/IgM(+) GC B cells remained unchanged. Together these findings suggest that IgM(+) B cells undergo SHM in the GC to generate high affinity IgM(+) memory cells and that this process continues even after CSR is accomplished.

Pulmonary collectins play distinct roles in host defense against Mycobacterium avium.

Pulmonary collectins, surfactant protein A (SP-A) and surfactant protein D (SP-D), play important roles in the innate immunity of the lung. Mycobacterium avium is one of the well-known opportunistic pathogens that can replicate within macrophages. We examined the effects of pulmonary collectins in host defense against M. avium infection achieved via direct interaction between bacteria and collectins. Although both pulmonary collectins bound to M. avium in a Ca(2+)-dependent manner, these collectins revealed distinct ligand-binding specificity and biological activities. SP-A and SP-D bound to a methoxy group containing lipid and lipoarabinomannan, respectively. Binding of SP-D but not SP-A resulted in agglutination of M. avium. A chimeric protein with the carbohydrate recognition domain of SP-D, which chimera revealed a bouquet-like arrangement similar to SP-A, also agglutinated M. avium. The ligand specificity of the carbohydrate recognition domain of SP-D seems to be necessary for agglutination activity. The binding of SP-A strongly inhibited the growth of M. avium in culture media. Although pulmonary collectins did not increase membrane permeability of M. avium, they attenuated the metabolic rate of the bacteria. Observations under a scanning electron microscope revealed that SP-A almost completely covers bacterial surfaces, whereas SP-D binds to certain areas like scattered dots. These observations suggest that a distinct binding pattern of collectins correlates with the difference of their biological activities. Furthermore, the number of bacteria phagocytosed by macrophages was significantly increased in the presence of SP-D. These data indicate that pulmonary collectins play critical roles in host defense against M. avium.

Mutational analysis of Cys(88) of Toll-like receptor 4 highlights the critical role of MD-2 in cell surface receptor expression.

The role of MD-2 in cell surface expression of Toll-like receptor (TLR) 4 has been controversial. The purposes of this study were to characterize the N-glycan of TLR4 and to investigate the roles of MD-2 in N-linked glycosylation and cell surface expression of TLR4. Lectin blot and cell surface biotinylation revealed that TLR4 exhibited the 110 kDa protein with high mannose type N-glycans and the 130 kDa protein with complex type N-glycans and that only the 130 kDa TLR4 with complex type N-glycans was expressed on the cell surface. The cells transfected with a mutant TLR4(C88A) alone expressed only the 110 kDa TLR4 with a high mannose type N-glycan, which did not appear on the cell surface. However, TLR4(C88A) acquired complex type N-glycans and was expressed on the cell surface when MD-2 was co-transfected. The amount of the 130 kDa TLR4(C88A) with complex type N-glycans expressed on the cell surface depended on that of MD-2 transfected. alpha-Mannosidase II inhibitor blocked the processing N-glycans to complex type, but TLR4 with high mannose type appeared on the cell surface, suggesting that TLR4 is destined to locate on the cell surface before processing N-glycans from a high mannose type to a complex type. From these results, we conclude that MD-2 is critical for cell surface expression of TLR4(C88A). This study provides evidence that MD-2 possesses potential ability to play an essential role in cell surface expression of TLR4.

Pulmonary surfactant protein D inhibits lipopolysaccharide (LPS)-induced inflammatory cell responses by altering LPS binding to its receptors.

Pulmonary surfactant protein D (SP-D) is a member of the collectin family that plays an important role in regulating innate immunity of the lung. We examined the mechanisms by which SP-D modulates lipopolysaccharide (LPS)-elicited inflammatory cell responses. SP-D bound to a complex of recombinant soluble forms of Toll-like receptor 4 (TLR4) and MD-2 with high affinity and down-regulated tumor necrosis factor-alpha secretion and NF-kappaB activation elicited by rough and smooth LPS, in alveolar macrophages and TLR4/MD-2-transfected HEK293 cells. Cell surface binding of both serotypes of LPS to TLR4/MD-2-expressing cells was attenuated by SP-D. In addition, SP-D significantly reduced MD-2 binding to both serotypes of LPS. A chimera containing the N-terminal region and the collagenous domain of surfactant protein A, and the coiled-coil neck and lectin domains of SP-D, was a weak inhibitor of LPS-induced cell responses and MD-2 binding to LPS, compared with native SP-D. The collagenase-resistant fragment consisting of the neck plus the carbohydrate recognition domain of SP-D also was a very weak inhibitor of LPS activation. This study demonstrates that SP-D down-regulates LPS-elicited inflammatory responses by altering LPS binding to its receptors and reveals the importance of the correct oligomeric structure of the protein in this process.

The 6-fluoro-8-methoxy quinolone gatifloxacin down-regulates interleukin-8 production in prostate cell line PC-3.

Fluoroquinolones exhibit immunomodulatory effects on monocytes and macrophages, in addition to their bactericidal activities. It remains unknown even whether the quinolones act directly on the prostate. This study was based on the understanding of the molecular mechanisms of the actions of the fluoroquinolones that can be used for the treatment of chronic prostatitis/chronic pelvic pain syndrome. We investigated whether the 6-fluroro-8-methoxy quinolone gatifloxacin (GFLX) affected the production and secretion of interleukin-8 (IL-8) in the prostate cell line PC-3. GFLX decreased the level of IL-8 release from unstimulated PC-3 cells. GFLX also attenuated IL-8 secretion from PC-3 cells stimulated with peptidoglycan, Mycoplasma hominis, phorbol ester, and tumor necrosis factor alpha (TNF-alpha), indicating that GFLX exhibits an anti-inflammatory effect on the prostate cell line. However, GFLX failed to alter activation of the NF-kappaB and AP-1 elicited by these stimulants. GFLX significantly attenuated the expression of IL-8 mRNA in TNF-alpha-stimulated PC-3 cells and down-regulated the transcriptional activity of the 5'-flanking region of the IL-8 gene from -1481 to +44 bp. The deletion construct without the 5'-flanking region from -1481 to -170 bp but not the construct without the region from -1481 to -188 bp reversed the suppressive effect of GFLX on IL-8 promoter activity. These results demonstrate that GFLX suppresses IL-8 expression in the prostate cell line by decreasing the promoter activity of the IL-8 gene.

Toll-like receptor 4 region Glu24-Lys47 is a site for MD-2 binding: importance of CYS29 and CYS40.

Toll-like receptor 4 (TLR4) is a signaling receptor for lipopolysaccharide (LPS), but its interaction with MD-2 is required for efficient responses to LPS. Previous studies with deletion mutants indicate a critical role of the amino-terminal TLR4 region in interaction with MD-2. However, it is uncertain which region in the TLR4 molecule directly binds to MD-2. The purpose of this study was to determine a critical stretch of primary sequence in the TLR4 region that directly binds MD-2 and is critical for LPS signaling. The synthetic TLR4 peptide corresponding to the TLR4 region Glu(24)-Lys(47) directly binds to recombinant soluble MD-2 (sMD-2). The TLR4 peptide inhibited the binding of a recombinant soluble form of the extracellular TLR4 domain (sTLR4) to sMD-2 and significantly attenuated LPS-induced NF-kappaB activation and IL-8 secretion in wild type TLR4-transfected cells. Reduction and S-carboxymethylation of sTLR4 abrogated its association with sMD-2. The TLR4 mutants, TLR4(C29A), TLR4(C40A), and TLR4(C29A,C40A), were neither co-precipitated with MD-2 nor expressed on the cell surface and failed to transmit LPS signaling. These results demonstrate that the TLR4 region Glu(24)-Lys(47) is a site for MD-2 binding and that Cys(29) and Cys(40) within this region are critical residues for MD-2 binding and LPS signaling.

Surfactant protein A directly interacts with TLR4 and MD-2 and regulates inflammatory cellular response. Importance of supratrimeric oligomerization.

The purpose of the current study was to examine the binding of pulmonary surfactant protein A (SP-A) to TLR4 and MD-2, which are critical signaling receptors for lipopolysaccharides (LPSs). The direct binding of SP-A to the recombinant soluble form of extracellular TLR4 domain (sTLR4) and MD-2 was detected using solid-phase binding, immunoprecipitation, and BIAcore. SP-A bound to sTLR4 and MD-2 in a Ca2+-dependent manner, and an anti-SP-A monoclonal antibody whose epitope lies in the region Thr184-Gly194 blocked the SP-A binding to sTLR4 and MD-2, indicating the involvement of the carbohydrate recognition domain (CRD) in the binding. SP-A avidly bound to the deglycosylated forms of sTLR4 and MD-2, suggesting a protein/protein interaction. In addition, SP-A attenuated cell surface binding of smooth LPS and smooth LPS-induced NF-kappaB activation in TLR4/MD-2-expressing cells. To know the role of oligomerization in the interaction of SP-A with TLR4 and MD-2, the collagenase-resistant fragment (CRF), which consisted of CRD plus neck domain of SP-A, was isolated. CRF assembled as a trimer, whereas SP-A assembled as a higher order oligomer. Although CRD was suggested to be involved in the binding, CRF exhibited approximately 600- and 155-fold higher KD for the binding to TLR4 and MD-2, respectively, when compared with SP-A. Consistently significantly higher molar concentrations of CRF were required to inhibit smooth LPS-induced NF-kappaB activation and tumor necrosis factor-alpha secretion. These results demonstrate for the first time the direct interaction between SP-A and TLR4/MD-2 and suggest the importance of supratrimeric oligomerization in the immunomodulatory function of SP-A.

Prostate cell lines secrete IL-8 in response to Mycoplasma hominis through Toll-like receptor 2-mediated mechanism.

BACKGROUND: The roles of Toll-like receptor 2 (TLR2) and Mycoplasma in prostate inflammation remain unclear. We investigated whether Mycoplasma induced inflammatory cytokine secretion through TLR2-mediated mechanism in prostate cancer cell line. METHODS: Cell surface expression of TLR2 on PC-3 cells were examined by flowcytometry. PC-3 cells were stimulated with Mycoplasma hominis (M. hominis), and IL-8 secretion and NF-kappaB activation were examined. RESULTS: PC-3 cells expressed TLR2 mRNA and cell surface TLR2 protein. The membrane fraction of M. hominis induced IL-8 secretion from PC-3 cells and NF-kappaB activation in a concentration-dependent manner. Transient transfection of the dominant negative mutant TLR2(P681H) into PC-3 cells attenuated M. hominis-induced IL-8 secretion and NF-kappaB activation. Antibody against the extracellular TLR2 domain significantly suppressed M. homnis-induced IL-8 secretion from the prostate cell lines including PC-3, PrEC, and transformed myofibroblasts. CONCLUSIONS: These results clearly demonstrate that the prostate cell line can secrete inflammatory cytokine in response to M. hominis through a TLR2-mediated mechanism.

Generation of monoclonal antibodies against chromosomal antigens that have a high sequence similarity between human and mouse.

We raised monoclonal antibodies by immunizing mice with total chromosome proteins extracted from isolated human metaphase chromosomes. The indirect immunofluorescence screening of hybridoma cell lines provided 15 monoclonal antibodies against the chromosomal antigens. The antigen proteins of the mAbs were identified by immunoblotting as core histones or by immunoprecipitation followed by a peptide mass fingerprinting method as nuclear mitotic apparatus protein, heterogeneous nuclear ribonucleoprotein A2/B1, ribosomal protein S4, linker histone and beta-actin. During mitosis, localizations of these proteins on chromosomes were clearly observed using the obtained antibodies. These results indicate that the current strategy is effective for obtaining monoclonal antibodies useful for immunoblotting and/or immunofluorescent staining of human proteins, using the antigens with high homology to mouse proteins.

Lymphocyte-expressed BILL-cadherin/cadherin-17 contributes to the development of B cells at two stages.

The gene encoding BILL-cadherin/cadherin-17, a nonclassical cadherin expressed on B lymphocytes in a stage-and-site-specific manner, was inactivated by targeted disruption of its transmembrane/cytoplasmic portion-encoding parts. BILL-cadherin deficiency caused a threefold proB cell accumulation, as well as a reduction to half of the numbers of immature B cells in bone marrow. In spleen, CD21(hi)CD23(lo) marginal zone B cells were found reduced and the structure of the marginal zone was impaired. In addition, the size and number of germinal center as well as the number of PNA(+) cells were significantly reduced in BILL-cadherin-deficient mice. In the peritoneal cavity of mutant mice IgM(+)Mac-1(+)CD5(+) B1a cell, that express high BILL-cadherin in wild-type mice, was also reduced in number. The IgG1 and IgG3 antibody response to the T-independent antigen, TNP-Ficoll, was impaired in the mutant mice. These results indicate that BILL-cadherin participates in B lymphocyte development at least at two stages, first at the transition of pro/preB-I cells to preB-II cells possibly in association with surrogate light chain in bone marrow, and later at the point of development, accumulation and reactiveness of immature B cells in spleen.

Estimation of the relative affinity of B cell receptor by flow cytometry.

We have developed a simple method using flow cytometry to estimate the relative affinity of B cell receptor (BCR) possessing the hapten-binding activity. Bovine serum albumin (BSA) was conjugated with a hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP) and biotin (NP-BSA-bio). The interaction between NP-BSA-bio and anti-NP monoclonal antibodies (mAb) was studied as a model of the BCR reaction by surface plasmon resonance (SPR) using a biosensor chip immobilized with mAbs through anti-Fc antibody (Ab). The relative affinity of these mAbs was estimated on the basis of resonance units for the binding of NP(0.5)-BSA-bio(21) relative to that of NP(7.4)-BSA-bio(21) expressed as a ratio (NP(0.5)-BSA-bio(21)/NP(7.4)-BSA-bio(21)). In combination with streptavidin (SA)-R-phycoerythrin (PE), we measured the binding of NP-BSA-bio to BCR by flow cytometry and found that a high number of biotin molecules was necessary to improve the sensitivity of detection of the bound NP-BSA-bio without steric hindrance in the NP-BCR interaction. We demonstrated that the ratio of the mean fluorescence intensity (MFI) of NP(0.5)-BSA-bio(21)/NP(7.4)-BSA-bio(21) at a concentration of 10(-8) M could be used as a practical measure of the affinity. This method is expected to be useful for the study of affinity maturation on the cellular level.

VpreB1/VpreB2/lambda 5 triple-deficient mice show impaired B cell development but functional allelic exclusion of the IgH locus.

At the precursor B cell stage during bone marrow B cell development, Ig muH chain associates with surrogate L (SL) chain, which is encoded by the three genes VpreB1, VpreB2, and lambda 5, to form the pre-B cell receptor (pre-BCR). Surface expression of the pre-BCR is believed to signal both proliferation and allelic exclusion of the IgH locus. Mice which lack either VpreB1/VpreB2 or lambda 5 show a lack of precursor B cell expansion but normal IgH allelic exclusion. This would suggest that one of either lambda 5 or VpreB can make a pre-BCR-like complex which is still able to signal allelic exclusion but not proliferation. To investigate this, we established mice lacking all components of the SL chain. These mice showed severely impaired B cell development which was similar to that previously found in mice lacking either lambda 5 or VpreB1/VpreB2. Surprisingly, the IgH locus was still allelically excluded and thus the SL chain appears not to be involved in allelic exclusion.