Yusuke Mimura

Prognostic potential of FOXP3 expression in non-small cell lung cancer cells combined with tumor-infiltrating regulatory T cells

Expression of the transcription factor FOXP3 characterizes regulatory T cells (Tregs) that engage in the maintenance of immunological self-tolerance and immune homeostasis. Intra-tumoral accumulation of Tregs is associated with unfavorable prognosis in several kinds of cancers. Recently, expression of FOXP3 and its association with prognosis have also been shown in some cancer cells in clinical studies. For non-small cell lung cancer (NSCLC), however, prognostic significance of tumor FOXP3 expression and its relationship with Tregs remain unknown. FOXP3 expression in cancer cells and tumor-infiltrating lymphocytes was examined by immunohistochemical staining of surgical specimens from 87 patients with NSCLC. Prognostic values of the tumor-infiltrating Treg count and tumor FOXP3 expression status were evaluated retrospectively. FOXP3-positive cancer cells were observed in 27 of 87 (31.0%) patients. There was no significant relationship between Treg count and tumor FOXP3 status. Increased Treg counts were associated with worse overall and relapse-free survival whereas the influence of tumor FOXP3 status on survival was not significant. However, when FOXP3-positive cancer cells were present, the relationship between Treg accumulation and worse prognosis was attenuated. In contrast, patients without tumor FOXP3 expression and high Treg count had significantly worse overall and relapse-free survival (hazard ratio: 3.118 and 3.325, p=0.028 and 0.024, respectively) than other groups. These results suggest that tumor FOXP3 expression has a better prognostic potential in NSCLC and that in combination with tumor-infiltrating Treg count the absence of tumor FOXP3 allows the selection of high-risk patients.

Human Follicular Lymphoma Cells Contain Oligomannose Glycans in the Antigen-binding Site of the B-cell Receptor

Expression of surface immunoglobulin appears critical for the growth and survival of B-cell lymphomas. In follicular lymphoma, we found previously that the Ig variable (V) regions in the B-cell receptor express a strikingly high incidence of N-glycosylation sequons, NX(S/T). These potential glycosylation sites are introduced by somatic mutation and are lymphoma-specific, pointing to their involvement in tumor pathogenesis. Analysis of the V region sugars from lymphoma-derived IgG/IgM reveals that they are mostly oligomannose and, remarkably, are located in the antigen-binding site, possibly precluding conventional antigen binding. The Fc region contains complex glycans, confirming that the normal glycan processing pathway is intact. Binding studies indicate that the oligomannose glycans occupying the V regions are accessible to mannose-binding lectin. These findings suggest a potential contribution to lymphoma pathogenesis involving antigen-independent interaction of surface immunoglobulin of the B-cell receptor with mannose-binding molecules of innate immunity in the germinal center.

Association between cytokine removal by polymyxin B hemoperfusion and improved pulmonary oxygenation in patients with acute exacerbation of idiopathic pulmonary fibrosis

Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) is characterized by severe worsening dyspnea of unknown etiology and high mortality without effective treatment. Recently, direct hemoperfusion with polymyxin B (PMX)-immobilized fiber cartridge (PMX-DHP) has been reported to improve pulmonary oxygenation and survival in patients with AE-IPF although its mechanism of action remains unknown. To gain insights into the pathobiology of AE-IPF through the beneficial effects of PMX-DHP, we analyzed the profile of cytokines adsorbed onto PMX-fibers used in 9 AE-IPF patients. In addition, the sera of these AE-IPF patients collected immediately before and after PMX-DHP, 9 stable IPF patients and 8 healthy individuals were also analyzed. The serum levels of cytokines including IL-9, IL-12, IL-17, PDGF and VEGF were significantly decreased immediately after PMX-DHP (P<0.02), and VEGF and IL-12 were most prominently reduced. In addition to PDGF and VEGF, IL-1β, IL-1ra, IL-8, IL-23, FGF basic, GM-CSF, IP-10, RANTES and TGF-β were eluted from used PMX-fibers. Interestingly, improved pulmonary oxygenation after PMX-DHP was correlated well with the quantities of eluted VEGF. These results suggest that adsorption of proinflammatory, profibrotic and proangiogenic cytokines onto PMX-fibers is one of the mechanisms of action of PMX-DHP in AE-IPF. Notably, removal of VEGF by PMX-DHP may contribute to the rapid improvement in oxygenation by suppressing vascular permeability in the lung.

Glycosylation engineering of therapeutic IgG antibodies: challenges for the safety, functionality and efficacy

Glycosylation of the Fc region of IgG has a profound impact on the safety and clinical efficacy of therapeutic antibodies. While the biantennary complex-type oligosaccharide attached to Asn297 of the Fc is essential for antibody effector functions, fucose and outer-arm sugars attached to the core heptasaccharide that generate structural heterogeneity (glycoforms) exhibit unique biological activities. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for the development and quality control of therapeutic antibodies, and glycan profiles of the Fc are recognized as critical quality attributes. In the past decade our understanding of the influence of glycosylation on the structure/function of IgG-Fc has grown rapidly through X-ray crystallographic and nuclear magnetic resonance studies, which provides possibilities for the design of novel antibody therapeutics. Furthermore, the chemoenzymatic glycoengineering approach using endoglycosidase-based glycosynthases may facilitate the development of homogeneous IgG glycoforms with desirable functionality as next-generation therapeutic antibodies. Thus, the Fc glycans are fertile ground for the improvement of the safety, functionality, and efficacy of therapeutic IgG antibodies in the era of precision medicine.

The molecular specificity of IgG-Fc interactions with Fcγ receptors

The molecular specificity and mechanisms for the interaction of immunoglobulins with Fc binding ligands has been a subject of intensive investigation for many years. Significant information has been gained for human IgG-Fc from x-ray crystallographic analysis of IgG-Fc and IgGFc/ligand complexes. The crystal complexes of IgG-Fc with FcRn, SpA, SpG and rheumatoid factor autoantibody ligands revealed binding sites at the CH2/CH3 interface and an IgG-Fc:ligand stoichiometry of 1:2; reflecting the two fold symmetry of the IgG molecule [1]. For FcγR and C 1 q monomeric IgG must necessarily be functionally monovalent since IgG, FcγR bearing cells and Cl co-exist in the blood, in the absence of activation and the consequent inflammatory reactions.

High-Throughput Screening of Glycan-Binding Proteins Using Miniature Pig Kidney N-Glycan-Immobilized Beads

Glycan recognition leading to cell-cell interactions, signaling, and immune responses is mediated by various glycan-binding proteins (GBPs) showing highly diverse ligand specificities. We describe here a rapid glycan immobilization technique via 4-hydrazinobenzoic acid (HBA)-functionalized beads and its application to high-throughput screening of miniature pig kidney N-glycan-binding proteins by using a mass-spectrometric approach. Without any derivatization steps, the purified pig kidney N-glycans were directly immobilized on to HBA-functionalized beads and subsequently used to identify GBPs from human serum. This screening method showed remarkable performance for identifying potential GBPs closely involved in pig-to-human xenograft rejection mediated by human serum, including antibodies, cytokines, complement components, siglec, and CD antigens. Thus, these results demonstrate that the GBP screening method was firmly established by one-step immobilization of the N-glycans on to microsphere and highly sensitive mass-spectrometric analysis.

Folding of an MHC class II-restricted tumor antigen controls its antigenicity via MHC-guided processing

CD4+ and CD8+ T cell responses to endogenous retroviral envelope glycoprotein gp90 generate protective immunity to murine colon carcinoma CT26. A panel of I-Ad-restricted T cell hybridomas recognize gp90 synthesized by CT26 cells but not by other gp90-expressing tumors. Here we report that antigenicity resides in an incompletely folded form of gp90 that is unique to CT26. In contrast to more compact forms of gp90 that are present in other tumors, this open conformer is captured by recycling I-Ad on antigen-presenting cells and is processed intracellularly. Thus, gp90 acquires immunodominance via MHC-guided processing, and the generation of an MHC class II-restricted response can be controlled by the intracellular folding environment of antigen-expressing cells.

Vigorous inflammatory responses in noninfectious pulmonary complication induced by donor lymphocyte infusion

Donor lymphocyte infusion (DLI) is used for treatment of hematologic malignancy relapse or mixed chimerism after allogeneic hematopoietic stem cell transplantation. Although graft‐versus‐host disease is well recognized as one of the adverse effects of DLI, there are limited reports on noninfectious pulmonary complications (NIPCs) after DLI.

Association of immunoglobulin G4 and free light chain with idiopathic pleural effusion

The cause of pleural effusion remains uncertain in approximately 15% of patients despite exhaustive evaluation. As recently described immunoglobulin (Ig)G4‐related disease is a fibroinflammatory disorder that can affect various organs, including the lungs, we investigate whether idiopathic pleural effusion includes IgG4‐associated etiology. Between 2000 and 2012, we collected 830 pleural fluid samples and reviewed 35 patients with pleural effusions undiagnosed after pleural biopsy at Yamaguchi‐Ube Medical Center. Importantly, IgG4 immunostaining revealed infiltration of IgG4‐positive plasma cells in the pleura of 12 patients (34%, IgG4+ group). The median effusion IgG4 level was 41 mg/dl in the IgG4+ group and 27 mg/dl in the IgG4− group (P < 0·01). The light and heavy chains of effusion IgG4 antibodies of patients in the IgG4+ group were heterogeneous by two‐dimensional electrophoresis, indicating the absence of clonality of the IgG4 antibodies. Interestingly, the κ light chains were more heterogeneous than the λ light chains. The measurement of the κ and λ free light chain (FLC) levels in the pleural fluids showed significantly different κ FLC levels (median: 28·0 versus 9·1 mg/dl, P < 0·01) and κ/λ ratios (median: 2·0 versus 1·2, P < 0·001) between the IgG4+ and IgG4− groups. Furthermore, the κ/λ ratios were correlated with the IgG4+/IgG+ plasma cell ratios in the pleura of the IgG4+ group. Taken together, these results demonstrate the involvement of IgG4 in certain idiopathic pleural effusions and provide insights into the diagnosis, pathogenesis and therapeutic opportunities of IgG4‐associated pleural effusion.

Association of immunoglobulin G4 and free light chain with idiopathic pleural effusion: IgG4 in idiopathic pleural effusion

The cause of pleural effusion remains uncertain in approximately 15% of patients despite exhaustive evaluation. As recently described immunoglobulin (Ig)G4‐related disease is a fibroinflammatory disorder that can affect various organs, including the lungs, we investigate whether idiopathic pleural effusion includes IgG4‐associated etiology. Between 2000 and 2012, we collected 830 pleural fluid samples and reviewed 35 patients with pleural effusions undiagnosed after pleural biopsy at Yamaguchi‐Ube Medical Center. Importantly, IgG4 immunostaining revealed infiltration of IgG4‐positive plasma cells in the pleura of 12 patients (34%, IgG4+ group). The median effusion IgG4 level was 41 mg/dl in the IgG4+ group and 27 mg/dl in the IgG4− group (P < 0·01). The light and heavy chains of effusion IgG4 antibodies of patients in the IgG4+ group were heterogeneous by two‐dimensional electrophoresis, indicating the absence of clonality of the IgG4 antibodies. Interestingly, the κ light chains were more heterogeneous than the λ light chains. The measurement of the κ and λ free light chain (FLC) levels in the pleural fluids showed significantly different κ FLC levels (median: 28·0 versus 9·1 mg/dl, P < 0·01) and κ/λ ratios (median: 2·0 versus 1·2, P < 0·001) between the IgG4+ and IgG4− groups. Furthermore, the κ/λ ratios were correlated with the IgG4+/IgG+ plasma cell ratios in the pleura of the IgG4+ group. Taken together, these results demonstrate the involvement of IgG4 in certain idiopathic pleural effusions and provide insights into the diagnosis, pathogenesis and therapeutic opportunities of IgG4‐associated pleural effusion.