High-throughput sequencing of IgG B-cell receptors reveals frequent usage of the rearranged IGHV4-28/IGHJ4 gene in primary immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an acquired form of thrombocytopenia caused by IgG anti-platelet autoantibodies and represents an organ-specific autoimmune disorder. Although the glycoprotein (GP) IIb/IIIa and GPIb/IX have been shown to be targets for autoantibodies, the antigen specificity of autoantibodies is not fully elucidated. To identify the characteristics of IgG B-cell receptor (BCR) repertoires in ITP, we took advantage of adaptor-ligation PCR and high-throughput DNA sequencing methods for analyzing the clone-based repertoires of IgG-expressing peripheral blood B cells. A total of 2,009,943 in-frame and 315,469 unique reads for IGH (immunoglobulin heavy) were obtained from twenty blood samples. Comparison of the IGHV repertoires between patients and controls revealed an increased usage of IGHV4-28 in ITP patients. One hundred eighty-six distinct IGHV4-28-carrying sequences were identified in ITP patients and the majority of these clones used an IGHJ4 segment. The IGHV4-28/IGHJ4-carrying B-cell clones were found in all ITP patients. Oligoclonal expansions of IGHV4-28/IGHJ4-carrying B cells were accompanied by multiple related clones with single amino substitution in the CDR3 region suggesting somatic hypermutation. Taken together, the expansion of IGHV4-28/IGHJ4-carrying IgG-expressing B cells in ITP may be the result of certain antigenic pressure and may provide a clue for the immune pathophysiology of ITP

Anti-hCD20 Antibody Ameliorates Murine PBC

There is considerable interest in expanding B cell-targeted therapies in human autoimmune diseases. However, clinical trials in human primary biliary cholangitis (PBC) using a chimeric antibody against human CD20 (hCD20) have showed limited efficacy. Two potential explanations for these disappointing results are the appearance of anti-drug antibodies (ADAs) and the high frequency of patients with moderate PBC or patients who had failed ursodeoxycholic acid treatment. Here, we studied a novel humanized IgG1 antibody against hCD20 and explored its efficacy in early stage PBC using a well-defined murine model. We developed a unique murine model consisting of dnTGF-bRII mice expressing hCD20 and human Fcg receptors (hFcγRs). Beginning at 4–6 weeks of age, equivalent to stage I/II human PBC, female mice were given weekly injections of an anti-hCD20 antibody (TKM-011) or vehicle control, and monitored for liver histology as well as a broad panel of immunological readouts. After 16 weeks’ treatment, we observed a significant reduction in portal inflammation, a decrease in liver-infiltrating mononuclear cells as well as a reduction in liver CD8+ T cells. Importantly, direct correlations between numbers of liver non-B cells and B cells (r = 0.7426, p = 0.0006) and between numbers of liver memory CD8+ T cells and B cells (r = 0.6423, p = 0.0054) were apparent. Accompanying these changes was a dramatic reduction in anti-mitochondrial antibodies (AMAs), interleukin (IL)-12p40 and IL-5, and elevated levels of the anti-inflammatory chemokine CXCL1/KC. In mice that developed ADAs, clinical improvements were less pronounced. Sustained treatment with B cell-targeted therapies may broadly inhibit effector pathways in PBC, but may need to be administered early in the natural history of PBC

Anti-drug Antibodies Against a Novel Humanized Anti-CD20 Antibody Impair Its Therapeutic Effect on Primary Biliary Cholangitis in Human CD20- and FcγR-Expressing Mice

There is considerable interest in expanding B cell-targeted therapies in human autoimmune diseases. However, clinical trials in human primary biliary cholangitis (PBC) using a chimeric antibody against human CD20 (hCD20) have showed limited efficacy. Two potential explanations for these disappointing results are the appearance of anti-drug antibodies (ADAs) and the high frequency of patients with moderate PBC or patients who had failed ursodeoxycholic acid treatment. Here, we studied a novel humanized IgG1 antibody against hCD20 and explored its efficacy in early stage PBC using a well-defined murine model. We developed a unique murine model consisting of dnTGF-βRII mice expressing hCD20 and human Fcγ receptors (hFcγRs). Beginning at 4–6 weeks of age, equivalent to stage I/II human PBC, female mice were given weekly injections of an anti-hCD20 antibody (TKM-011) or vehicle control, and monitored for liver histology as well as a broad panel of immunological readouts. After 16 weeks' treatment, we observed a significant reduction in portal inflammation, a decrease in liver-infiltrating mononuclear cells as well as a reduction in liver CD8+ T cells. Importantly, direct correlations between numbers of liver non-B cells and B cells (r = 0.7426, p = 0.0006) and between numbers of liver memory CD8+ T cells and B cells (r = 0.6423, p = 0.0054) were apparent. Accompanying these changes was a dramatic reduction in anti-mitochondrial antibodies (AMAs), interleukin (IL)-12p40 and IL-5, and elevated levels of the anti-inflammatory chemokine CXCL1/KC. In mice that developed ADAs, clinical improvements were less pronounced. Sustained treatment with B cell-targeted therapies may broadly inhibit effector pathways in PBC, but may need to be administered early in the natural history of PBC

Clonal hematopoiesis in adult pure red cell aplasia

Idiopathic pure red cell aplasia (PRCA) and secondary PRCA associated with thymoma and large granular lymphocyte leukemia are generally considered to be immune-mediated. The PRCA2004/2006 study showed that poor responses to immunosuppression and anemia relapse were associated with death. PRCA may represent the prodrome to MDS. Thus, clonal hematopoiesis may be responsible for treatment failure. We investigated gene mutations in myeloid neoplasm-associated genes in acquired PRCA. We identified 21 mutations affecting amino acid sequences in 11 of the 38 adult PRCA patients (28.9%) using stringent filtering of the error-prone sequences and SNPs. Four PRCA patients showed 7 driver mutations in TET2, DNMT3A and KDM6A, and 2 PRCA patients carried multiple mutations in TET2. Five PRCA patients had mutations with high VAFs exceeding 0.3. These results suggest that clonal hematopoiesis by stem/progenitor cells might be related to the pathophysiology of chronic PRCA in certain adult patients

Functional characterization of 3 thioredoxin homology domains of ERp72

Folding of secretory proteins is associated with the formation and isomerization of disulfide bonds. ERp72, a protein disulfide isomerase (PDI) family member, possesses 3 thioredoxin homology domains, but the participation of each domain in disulfide-bond formation and isomerization remains to be determined. We analyzed the function of individual domains in the insulin reduction assay system by site-directed mutagenesis with cysteine-to-serine replacement. All domains contributed to apparent steady-state binding (K(m)) and catalysis at saturating substrate concentrations (k(cat)) but in different manners. A mutant ERp72 with mutations in domains 1 and 2 (ERp72-mut-1+2) exhibited reductions in k(cat) of 73.9% when compared with wild type, whereas ERp72-mut-1+3 (mutations in domains 1 and 3) and ERp72-mut-2+3 (mutations in domains 2 and 3) exhibited less substantial reductions in k(cat). ERp72-mut-1+3 and ERp72-mut-2+3 showed elevations in K(m) of 89.9% and 96.2%, respectively, when compared with wild type, whereas ERp72-mut-1+2 exhibited smaller elevations in K(m). These results suggest that domains 1 and 2 make greater contributions to catalyzing efficacy and domain 3 to binding affinity. Domain 2 is involved in binding affinity, in combination with domain 3, in addition to its own contribution to catalyzing efficacy. This assignment of functions to individual domains is similar to that observed in other PDI domains, which is consistent with the high sequence homology between ERp and PDI domains

Correlation between DWI bi-exponential estimates and PET hypoxia imaging activities in head and neck carcinoma

ObjectiveHypoxic tumor has highly malignant property and resistance to radiotherapy and chemotherapy. Assessment of hypoxia is important for selection of treatment method, including hypoxia-targeting therapy, and prediction of treatment effect. 18F-fluoroazomycin arabinoside (FAZA) positron emission tomography (PET) has been reported to be useful for hypoxia imaging, but it will take some time to become prevalent in clinical practice. Under hypoxia, alteration of cell structure leads to change in water diffusion. This change may be detected by diffusion-weighted magnetic resonance imaging (DWI) by bi-exponential model. The purpose of this study was to evaluate the correlation between DWI bi-exponential estimates and FAZA-PET activities and to investigate the usefulness of MRI for hypoxia imaging of head and neck carcinoma.Materials and MethodsEleven patients with head and neck carcinoma (8 primary tumors and 5 lymph nodes without necrosis) were included. They were the subjects that had this MRI protocol in the previous FAZA study [1]. By bi-exponential model, the diffusion coefficients of fast and slow components (Dfast, Dslow) and their fractions were obtained. Apparent diffusion coefficient (ADC) was also obtained at low and high b-value ranges (lowbADC, highbADC). On FAZA-PET images, tumor-muscle ratios (T/M) were measured for comparison between DWI bi-exponential and FAZA-PET estimates using Spearman’s rank correlation coefficient.ResultsThere was a positive correlation between Dslow and T/M (rs=0.63, p=0.022). Dfast, fraction, lowbADC, or highbADC did not correlate with T/M.ConclusionAmong bi-exponential estimates of DWI, only Dslow correlated with FAZA-PET parameter. Dslow might be useful as an indicator of hypoxia.Reference1. Saga, T., et al. ANN 30, 217-224.第44回日本磁気共鳴医学会大

Imaging of Hypoxic Tumor: Correlation between Diffusion-weighted MR Imaging and F-fluoroazomycin Arabinoside Positron Emission Tomography in Head and Neck Carcinoma.

We investigated the usefulness of diffusion-weighted imaging (DWI) for detecting changes in the structure of hypoxic cells by evaluating the correlation between F-fluoroazomycin arabinoside (FAZA) positron emission tomography activity and DWI parameters in head and neck carcinoma. The diffusion coefficient corresponding to the slow compartment of a two-compartment model had a significant positive correlation with FAZA activity (ρ = 0.58, P = 0.016), whereas the diffusional kurtosis from diffusion kurtosis imaging had a significant negative correlation (ρ = -0.62, P = 0.008), which suggests that those DWI parameters might be useful as indicators for changes in cell structure

Anti-drug Antibodies Against a Novel Humanized Anti-CD20 Antibody Impair Its Therapeutic Effect on Primary Biliary Cholangitis in Human CD20- and FcγR-Expressing Mice.

There is considerable interest in expanding B cell-targeted therapies in human autoimmune diseases. However, clinical trials in human primary biliary cholangitis (PBC) using a chimeric antibody against human CD20 (hCD20) have showed limited efficacy. Two potential explanations for these disappointing results are the appearance of anti-drug antibodies (ADAs) and the high frequency of patients with moderate PBC or patients who had failed ursodeoxycholic acid treatment. Here, we studied a novel humanized IgG1 antibody against hCD20 and explored its efficacy in early stage PBC using a well-defined murine model. We developed a unique murine model consisting of dnTGF-βRII mice expressing hCD20 and human Fcγ receptors (hFcγRs). Beginning at 4-6 weeks of age, equivalent to stage I/II human PBC, female mice were given weekly injections of an anti-hCD20 antibody (TKM-011) or vehicle control, and monitored for liver histology as well as a broad panel of immunological readouts. After 16 weeks' treatment, we observed a significant reduction in portal inflammation, a decrease in liver-infiltrating mononuclear cells as well as a reduction in liver CD8+ T cells. Importantly, direct correlations between numbers of liver non-B cells and B cells (r = 0.7426, p = 0.0006) and between numbers of liver memory CD8+ T cells and B cells (r = 0.6423, p = 0.0054) were apparent. Accompanying these changes was a dramatic reduction in anti-mitochondrial antibodies (AMAs), interleukin (IL)-12p40 and IL-5, and elevated levels of the anti-inflammatory chemokine CXCL1/KC. In mice that developed ADAs, clinical improvements were less pronounced. Sustained treatment with B cell-targeted therapies may broadly inhibit effector pathways in PBC, but may need to be administered early in the natural history of PBC