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.publishedVersio

Autoimmunity and viral immunity in Addison’s disease

Autoimmune Addison’s disease (AAD) is caused by an immunological destruction of the steroid producing cells of the adrenal cortex. Both genetic and environmental factors are involved in disease development, and while multiple studies have highlighted several genes linked to the disease, far less is known about possible environmental factors and the role played by the adrenal tissue itself. By studying the well-established human adrenocortical carcinoma cell line NCI- H295R as a model of the adrenal cortex, adrenal cells were shown to respond to environmental factors in the form of virus induced type I and III interferons (IFNs) by potentiating chemokine production and by upregulation of MHC class I and the tissue specific enzyme 21-hydroxylase (21OH). This can be important traits in the T cell- mediated adrenal tissue destruction, recruiting potentially 21OH-specific autoreactive T cells to the tissue. In addition, the same IFNs were shown to have a direct cytotoxic effect on the adrenocortical cells. From a large clinical material we have found that AAD patients have elevated levels of the chemokines CXCL10 and CXCL9 in their sera, while producing significantly lower levels of the same chemokines after stimulation of PBMC with type I and II IFNs and the TLR3 ligand poly (I:C). This is also the case when investigating the relative mRNA expression of selected IFN stimulated genes (ISGs) after IFN or poly (I:C) stimulation. Cytomegalovirus (CMV) has been implicated in autoimmune disease development, including AAD. Nevertheless, we found that AAD patients in general have normal humoral and cellular immunity towards CMV, with no differences in CD8+ T cell specific responses. However, the AAD patients were found to have significantly lower levels of total circulating CD8+ T cells. While CMV infections do not appear to be linked to AAD disease development in general, individual patients showed signs supporting CMV as a possible perpetrator. One patient had serological signs suggesting a reactivating CMV infection, while having extremely low levels of CMV specific CD8+ T cells. The same patient was also part of the chemokine study, where she had low chemokine production after IFN stimuli in addition to an upregulation of three ISGs in her peripheral blood. Intriguingly, the daughter of this patient also had AAD and, despite of being anti-CMV IgG positive, had virtually no CMV specific CD8+ T cells. These findings suggest that an inheritable immunological phenotype may increase individuals’ susceptibility to develop AAD, but also impair their ability to control viral infections. Taken together, the work included in this thesis provides important insight into AAD development. We have shown using adrenocortical cells that the adrenal cortex could play a part in its own destruction in response to interferons induced by viruses. Furthermore, while having a normal cellular and humoral immunity towards the common virus CMV, the innate immune system of AAD patients does not appear to function optimally. Thus for individual patients, CMV infection could be a precipitating event in disease development

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

During the last decades antimicrobial resistance has become a global health problem. Metallo-β-lactamases (MBLs) which are broad-spectrum β-lactamases that inactivate virtually all β-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-β-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

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