Contributions of the N-terminal flanking residues of an antigenic peptide from the Japanese cedar pollen allergen Cry j 1 to the T-cell activation by HLA-DP5.

Cry j 1 is a major allergen present in Japanese cedar (Cryptomeria japonica) pollens. Peptides with the core sequence of KVTVAFNQF from Cry j 1 ('pCj1') bind to HLA-DP5 and activate Th2 cells. In this study, we noticed that Ser and Lys at positions -2 and -3, respectively, in the N-terminal flanking (NF) region to pCj1 are conserved well in HLA-DP5-binding allergen peptides. A competitive binding assay showed that the double mutation of Ser(-2) and Lys(-3) to Glu [S(P-2)E/K(P-3)E] in a 13-residue Cry j 1 peptide (NF-pCj1) decreased its affinity for HLA-DP5 by about 2-fold. Similarly, this double mutation reduced, by about 2-fold, the amount of NF-pCj1 presented on the surface of mouse antigen-presenting dendritic cell line 1 (mDC1) cells stably expressing HLA-DP5. We established NF-pCj1-specific and HLA-DP5-restricted CD4+ T-cell clones from HLA-DP5 positive cedar pollinosis (CP) patients, and analyzed their IL-2 production due to the activation of mouse TG40 cells expressing the cloned T-cell receptor by the NF-pCj1-presenting mDC1 cells. The T-cell activation was actually decreased by the S(P-2)E/K(P-3)E mutation, corresponding to the reduction in the peptide presentation by this mutation. In contrast, the affinity of NF-pCj1·HLA-DP5 for the T-cell receptor was not affected by the S(P-2)E/K(P-3)E mutation, as analyzed by surface plasmon resonance. Considering the positional and side-chain differences of these NF residues from previously reported T-cell activating sequences, the mechanisms of enhanced T-cell activation by Ser(-2) and Lys(-3) of NF-pCj1 may be novel.

Modification of the HLA-A*24:02 Peptide Binding Pocket Enhances Cognate Peptide-Binding Capacity and Antigen-Specific T Cell Activation.

The immunogenicity of a T cell Ag is correlated with the ability of its antigenic epitope to bind HLA and be stably presented to T cells. This presents a challenge for the development of effective cancer immunotherapies, as many self-derived tumor-associated epitopes elicit weak T cell responses, in part due to weak binding affinity to HLA. Traditional methods to increase peptide-HLA binding affinity involve modifying the peptide to reflect HLA allele binding preferences. Using a different approach, we sought to analyze whether the immunogenicity of wild-type peptides could be altered through modification of the HLA binding pocket. After analyzing HLA class I peptide binding pocket alignments, we identified an alanine 81 to leucine (A81L) modification within the F binding pocket of HLA-A*24:02 that was found to heighten the ability of artificial APCs to retain and present HLA-A*24:02-restricted peptides, resulting in increased T cell responses while retaining Ag specificity. This modification led to increased peptide exchange efficiencies for enhanced detection of low-avidity T cells and, when expressed on artificial APCs, resulted in greater expansion of Ag-specific T cells from melanoma-derived tumor-infiltrating lymphocytes. Our study provides an example of how modifications to the HLA binding pocket can enhance wild-type cognate peptide presentation to heighten T cell activation.

Usefulness of daily folic acid supplementation during methotrexate treatment of Japanese patients with rheumatoid arthritis.

OBJECTIVES: We investigated the effect of daily folic acid supplementation on methotrexate (MTX) toxicity and efficacy in Japanese patients with rheumatoid arthritis (RA). METHODS: We followed 19 patients treated with MTX who switched from taking weekly 5 mg folic acid supplementation (weekly regimen) to 1.25 mg daily (daily regimen). White blood cell (WBC) and platelet (PLT) counts, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels were collected for 24 weeks following the change. RESULTS: We observed no significant changes in WBC or PLT counts. AST and ALT levels, which had exceeded the upper limits of their normal ranges at the beginning of the study, were improved significantly at weeks 4 and 8, no subsequent deterioration in liver function was found. Further, no significant changes in ESR and CRP levels were observed. CONCLUSION: Our data indicate that supplementing 1.25 mg of folic acid daily rather than 5 mg weekly reduces toxicity caused by MTX without affecting its efficacy.

Methotrexate Enhances Apoptosis of Transmembrane TNF-Expressing Cells Treated With Anti-TNF Agents.

Background: Concomitant use of methotrexate (MTX) improves the clinical efficacy of anti-TNF agents in the treatment of rheumatoid arthritis (RA). We aimed to clarify the cytotoxic effect of MTX on transmembrane TNF (tmTNF)-expressing cells treated with anti-TNF agents. Methods: Jurkat T cells stably expressing tmTNF were used for the following experiments. Cytotoxicity induced by an anti-TNF agent (infliximab, adalimumab, or certolizumab pegol) with concomitant MTX were compared with that by MTX alone or by an anti-TNF agent alone using flow cytometry. Apoptosis-induction mediated by reverse signal through tmTNF, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP) were evaluated. Folic acid and Y-27632, a Rho kinase inhibitor, were used as inhibitors to study intracellular signaling pathway in apoptosis induced by MTX and anti-TNF agents. Results: Apoptosis of tmTNF-expressing cells was significantly increased by the concomitant administration of MTX and an anti-TNF agent, compared with MTX alone or an anti-TNF agent alone. The apoptosis induction by concomitant MTX was most pronounced in infliximab-treatment. Reverse signal transduction, but not CDC or ADCC/ADCP, was responsible for the coordinate effect of MTX and an anti-TNF agent on tmTNF-expressing cells. Folic acid inhibited MTX-mediated apoptosis, while Y-27632 suppressed JNK activation and infliximab-induced apoptosis via revere signal through tmTNF. Conclusion: The apoptotic effect was enhanced by combination of MTX and an anti-TNF agent in tmTNF-expressing cells. The intracellular pathways induced by MTX and anti-TNF agents seem to be independent. These findings might explain at least in part improved the clinical response upon co-therapy of MTX and an anti-TNF agent in RA.

Sudden Respiratory Failure due to Tracheobronchomalacia by Relapsing Polychondritis, Successfully Rescued by Multiple Metallic Stenting and Tracheostomy.

Relapsing polychondritis (RP) is a rare systemic autoimmune disease that affects cartilaginous structures. RP causes tracheobronchomalacia (TBM) by affecting the bronchial cartilage. TBM is a fatal condition characterized by excessive weakening of the walls of the trachea and bronchi. We herein report a case of a 73-year-old man who experienced sudden respiratory failure due to TBM caused by RP. Immunosuppressive treatment did not improve his respiratory failure. Multiple metallic stentings dramatically improved his severe airway symptoms. When the airway condition becomes lethal in RP patients, then metallic stenting can be a useful treatment option.

Identification of a Hashimoto thyroiditis susceptibility locus via a genome-wide comparison with Graves' disease.

BACKGROUND: Hashimoto thyroiditis (HT) and Graves' disease (GD) share some immunological features. Determining the genetic basis that distinguishes HT from GD is key for a better understanding of the differences between these two related diseases. AIM: The aim of this study was to identify a non-HLA susceptibility locus that is specific to either HT or GD. DESIGN: We performed a two-stage genome-wide comparison between HT and GD in Japan. During the discovery stage, we performed a logistic regression analysis adjusting for sex using 727 413 single nucleotide polymorphisms (SNPs) for 265 HT and 261 GD patients. During the replication stage, 35 SNPs were analyzed for 181 HT and 286 GD cases. A combined meta-analysis was performed using the results from these two stages. An SNP showing a genome-wide significant level was further analyzed using 1363 healthy controls to determine the specificity of susceptibility. RESULTS: A genome-wide direct comparison between HT and GD revealed an SNP at the VAV3 locus with genome-wide significant association signals (rs7537605: P(combined) = 3.90 × 10(-8); odds ratio(combined) = 1.77; 95% confidence interval = 1.44-2.17). An association analysis using healthy controls showed that rs7537605 is significantly associated with HT (P = 1.24 × 10(-5); odds ratio = 1.60; 95% confidence interval = 1.30-1.97) but not with GD (P = .50), suggesting that the variant specifically affects susceptibility to HT. CONCLUSION: A genome-wide direct comparison between HT and GD revealed an HT-specific variant within VAV3 in the Japanese. Considering physiological roles of VAV3, such as a guanine nucleotide exchange factor, our finding provides new insight into the molecular mechanism of HT.

Identification of independent susceptible and protective HLA alleles in Japanese autoimmune thyroid disease and their epistasis.

BACKGROUND: Autoimmune thyroid disease (AITD) includes Graves disease (GD) and Hashimoto thyroiditis (HT), which partially share immunological features. Determining the genetic basis that distinguishes GD and HT is a key to understanding the differences between these 2 related diseases. AIM: The aims of this study were to identify HLA antigens that can explain the immunopathological difference between GD and HT and to elucidate epistatic interactions between protective and susceptible HLA alleles, which can delineate the distinct function of HLA in AITD etiology. DESIGN: We genotyped 991 patients with AITD (547 patients with GD and 444 patients with HT) and 481 control subjects at the HLA-A, HLA-C, HLA-B, DRB1, DQB1, and DPB1 loci. A direct comparison of HLA antigen frequencies between GD and HT was performed. We further analyzed an epistatic interaction between the susceptible and protective HLA alleles in the development of GD and HT. RESULTS: We identified 4 and 2 susceptible HLA molecules primarily associated with GD and HT, respectively, HLA-B*35:01, HLA-B*46:01, HLA-DRB1*14:03, and HLA-DPB1*05:01 for GD and HLA-A*02:07 and HLA-DRB4 for HT. In a direct comparison between GD and HT, we identified GD-specific susceptible class II molecules, HLA-DP5 (HLA-DPB1*05:01; Pc = 1.0 × 10(-9)) and HLA-DR14 (HLA-DRB*14:03; Pc = .0018). In contrast, HLA components on 3 common haplotypes in Japanese showed significant protective effects against the development of GD and HT (HLA-A*24:02-C*12:02-B*52:01-DRB1*15:02-DQB1*06:01-DPB1*09:01 and HLA-A*24:02-C*07:02-B*07:02-DRB1*01:01-DQB1*05:01-DPB1*04:02 haplotypes for GD and HLA-A*33:03-C*14:03-B*44:03-DRB1*13:02-DQB1*06:04-DPB1*04:01 haplotype for GD and HT). Interestingly, the representative protective HLA, HLA-DR13 (HLA-DRB1*13:02), was epistatic to susceptible HLA-DP5 in controlling the development of GD. CONCLUSION: We show that HLA exerts a dual function, susceptibility and resistance, in controlling the development of GD and HT. We also show that the protective HLA allele is partially epistatic to the susceptible HLA allele in GD.

Four cases of MPO-ANCA-positive vasculitis with otitis media, and review of the literature.

Otitis media is one of the common organ injuries that appear during the course of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). We experienced four patients with myeloperoxidase (MPO)-ANCA-positive AAV with otitis media. All were elderly Japanese women. MPO-ANCA in our patients was reminiscent of microscopic polyangiitis (MPA), although chest computed tomography (CT) scans revealed characteristics of both granulomatosis with polyangiitis (GPA), showing bronchial lesions and nodule formation, and MPA, showing interstitial changes. Whether our cases should be classified as GPA or MPA is a matter of discussion. We detail their profiles, and review previous literature on MPO-ANCA-positive AAV with otitis media.