21-Hydroxylase-Specific CD8+ T Cells in Autoimmune Addison's Disease Are Restricted by HLA-A2 and HLA-C7 Molecules.

Objectives: CD8+ T cells targeting 21-hydroxylase (21OH) are presumed to play a central role in the destruction of adrenocortical cells in autoimmune Addison's disease (AAD). Earlier reports have suggested two immunodominant CD8+ T cell epitopes within 21OH: LLNATIAEV (21OH342-350), restricted by HLA-A2, and EPLARLEL (21OH431-438), restricted by HLA-B8. We aimed to characterize polyclonal CD8+ T cell responses to the proposed epitopes in a larger patient cohort with AAD. Methods: Recombinant fluorescent HLA-peptide multimer reagents were used to quantify antigen-specific CD8+ T cells by flow cytometry. Interferon-gamma (IFNγ) Elispot and biochemical assays were used to functionally investigate the 21OH-specific T cells, and to map the exactly defined epitopes of 21OH. Results: We found a significantly higher frequency of HLA-A2 restricted LLNATIAEV-specific cells in patients with AAD than in controls. These cells could also be expanded in vitro in an antigen specific manner and displayed a robust antigen-specific IFNγ production. In contrast, only negligible frequencies of EPLARLEL-specific T cells were detected in both patients and controls with limited IFNγ response. However, significant IFNγ production was observed in response to a longer peptide encompassing EPLARLEL, 21OH430-447, suggesting alternative dominant epitopes. Accordingly, we discovered that the slightly offset ARLELFVVL (21OH434-442) peptide is a novel dominant epitope restricted by HLA-C7 and not by HLA-B8 as initially postulated. Conclusion: We have identified two dominant 21OH epitopes targeted by CD8+ T cells in AAD, restricted by HLA-A2 and HLA-C7, respectively. To our knowledge, this is the first HLA-C7 restricted epitope described for an autoimmune disease.

Analysis of cellular and humoral immune responses against cytomegalovirus in patients with autoimmune Addison's disease.

BACKGROUND: Autoimmune Addison's disease (AAD) is caused by multiple genetic and environmental factors. Variants of genes encoding immunologically important proteins such as the HLA molecules are strongly associated with AAD, but any environmental risk factors have yet to be defined. We hypothesized that primary or reactivating infections with cytomegalovirus (CMV) could represent an environmental risk factor in AAD, and that CMV specific CD8(+) T cell responses may be dysregulated, possibly leading to a suboptimal control of CMV. In particular, the objective was to assess the HLA-B8 restricted CD8(+) T cell response to CMV since this HLA class I variant is a genetic risk factor for AAD. METHODS: To examine the CD8(+) T cell response in detail, we analyzed the HLA-A2 and HLA-B8 restricted responses in AAD patients and healthy controls seropositive for CMV antibodies using HLA multimer technology, IFN-γ ELISpot and a CD107a based degranulation assay. RESULTS: No differences between patients and controls were found in functions or frequencies of CMV-specific T cells, regardless if the analyses were performed ex vivo or after in vitro stimulation and expansion. However, individual patients showed signs of reactivating CMV infection correlating with poor CD8(+) T cell responses to the virus, and a concomitant upregulation of interferon regulated genes in peripheral blood cells. Several recently diagnosed AAD patients also showed serological signs of ongoing primary CMV infection. CONCLUSIONS: CMV infection does not appear to be a major environmental risk factor in AAD, but may represent a precipitating factor in individual patients.

Peripheral Blood Cells from Patients with Autoimmune Addison's Disease Poorly Respond to Interferons In Vitro, Despite Elevated Serum Levels of Interferon-Inducible Chemokines.

Autoimmune Addison's disease (AAD) is a disorder caused by an immunological attack on the adrenal cortex. The interferon (IFN)-inducible chemokine CXCL10 is elevated in serum of AAD patients, suggesting a peripheral IFN signature. However, CXCL10 can also be induced in adrenocortical cells stimulated with IFNs, cytokines, or microbial components. We therefore investigated whether peripheral blood mononuclear cells (PBMCs) from AAD patients display an enhanced propensity to produce CXCL10 and the related chemokine CXCL9, after stimulation with type I or II IFNs or the IFN inducer poly (I:C). Although serum levels of CXCL10 and CXCL9 were significantly elevated in patients compared with controls, IFN stimulated patient PBMC produced significantly less CXCL10/CXCL9 than control PBMC. Low CXCL10 production was not significantly associated with medication, disease duration, or comorbidities, but the low production of poly (I:C)-induced CXCL10 among patients was associated with an AAD risk allele in the phosphatase nonreceptor type 22 (PTPN22) gene. PBMC levels of total STAT1 and -2, and IFN-induced phosphorylated STAT1 and -2, were not significantly different between patients and controls. We conclude that PBMC from patients with AAD are deficient in their response to IFNs, and that the adrenal cortex itself may be responsible for the increased serum levels of CXCL10.

Structural and biochemical characterization of VIM-26 shows that Leu224 has implications for the substrate specificity of VIM metallo-ß-lactamases.

During the last decades antimicrobial resistance has become a global health problem. Metallo-ß-lactamases (MBLs) which are broad-spectrum ß-lactamases that inactivate virtually all ß-lactams including carbapenems, are contributing to this health problem. In this study a novel MBL variant, termed VIM-26, identified in a Klebsiella pneumoniae isolate was studied. VIM-26 belongs to the Verona integron-encoded metallo-ß-lactamase (VIM) family of MBLs and is a His224Leu variant of the well-characterized VIM-1 variant. In this study, we report the kinetic parameters, minimum inhibitory concentrations and crystal structures of a recombinant VIM-26 protein, and compare them to previously published data on VIM-1, VIM-2 and VIM-7. The kinetic parameters and minimum inhibitory concentration determinations show that VIM-26, like VIM-7, has higher penicillinase activity but lower cephalosporinase activity than VIM-1 and VIM-2. The four determined VIM-26 crystal structures revealed mono- and di-zinc forms, where the Zn1 ion has distorted tetrahedral coordination geometry with an additional water molecule (W2) at a distance of 2.6-3.7 Å, which could be important during catalysis. The R2 drug binding site in VIM-26 is more open compared to VIM-2 and VIM-7 and neutrally charged due to Leu224 and Ser228. Thus, the VIM-26 drug binding properties are different from the VIM-2 (Tyr224/Arg228) and VIM-7 (His224/Arg228) structures, indicating a role of these residues in the substrate specificity.

His224 alters the R2 drug binding site and Phe218 influences the catalytic efficiency of the metallo-ß-lactamase VIM-7.

Metallo-ß-lactamases (MBLs) are the causative mechanism for resistance to ß-lactams, including carbapenems, in many Gram-negative pathogenic bacteria. One important family of MBLs is the Verona integron-encoded MBLs (VIM). In this study, the importance of residues Asp120, Phe218, and His224 in the most divergent VIM variant, VIM-7, was investigated to better understand the roles of these residues in VIM enzymes through mutations, enzyme kinetics, crystal structures, thermostability, and docking experiments. The tVIM-7-D120A mutant with a tobacco etch virus (TEV) cleavage site was enzymatically inactive, and its structure showed the presence of only the Zn1 ion. The mutant was less thermostable, with a melting temperature (T(m)) of 48.5°C, compared to 55.3 °C for the wild-type tVIM-7. In the F218Y mutant, a hydrogen bonding cluster was established involving residues Asn70, Asp84, and Arg121. The tVIM-7-F218Y mutant had enhanced activity compared to wild-type tVIM-7, and a slightly higher Tm (57.1 °C) was observed, most likely due to the hydrogen bonding cluster. Furthermore, the introduction of two additional hydrogen bonds adjacent to the active site in the tVIM-7-H224Y mutant gave a higher thermostability (T(m), 62.9 °C) and increased enzymatic activity compared to those of the wild-type tVIM-7. Docking of ceftazidime in to the active site of tVIM-7, tVIM-7-H224Y, and VIM-7-F218Y revealed that the side-chain conformations of residue 224 and Arg228 in the L3 loop and Tyr67 in the L1 loop all influence possible substrate binding conformations. In conclusion, the residue composition of the L3 loop, as shown with the single H224Y mutation, is important for activity particularly toward the positively charged cephalosporins like cefepime and ceftazidime.

Crystal structure of the mobile metallo-ß-lactamase AIM-1 from Pseudomonas aeruginosa: insights into antibiotic binding and the role of Gln157.

Metallo-ß-lactamase (MBL) genes confer resistance to virtually all ß-lactam antibiotics and are rapidly disseminated by mobile genetic elements in Gram-negative bacteria. MBLs belong to three different subgroups, B1, B2, and B3, with the mobile MBLs largely confined to subgroup B1. The B3 MBLs are a divergent subgroup of predominantly chromosomally encoded enzymes. AIM-1 (Adelaide IMipenmase 1) from Pseudomonas aeruginosa was the first B3 MBL to be identified on a readily mobile genetic element. Here we present the crystal structure of AIM-1 and use in silico docking and quantum mechanics and molecular mechanics (QM/MM) calculations, together with site-directed mutagenesis, to investigate its interaction with ß-lactams. AIM-1 adopts the characteristic αß/ßα sandwich fold of MBLs but differs from other B3 enzymes in the conformation of an active site loop (residues 156 to 162) which is involved both in disulfide bond formation and, we suggest, interaction with substrates. The structure, together with docking and QM/MM calculations, indicates that the AIM-1 substrate binding site is narrower and more restricted than those of other B3 MBLs, possibly explaining its higher catalytic efficiency. The location of Gln157 adjacent to the AIM-1 zinc center suggests a role in drug binding that is supported by our in silico studies. However, replacement of this residue by either Asn or Ala resulted in only modest reductions in AIM-1 activity against the majority of ß-lactam substrates, indicating that this function is nonessential. Our study reveals AIM-1 to be a subclass B3 MBL with novel structural and mechanistic features.