TScan-II: A genome-scale platform for the de novo identification of CD4+ T cell epitopes.

CD4+ T cells play fundamental roles in orchestrating immune responses and tissue homeostasis. However, our inability to associate peptide human leukocyte antigen class-II (HLA-II) complexes with their cognate T cell receptors (TCRs) in an unbiased manner has hampered our understanding of CD4+ T cell function and role in pathologies. Here, we introduce TScan-II, a highly sensitive genome-scale CD4+ antigen discovery platform. This platform seamlessly integrates the endogenous HLA-II antigen-processing machinery in synthetic antigen-presenting cells and TCR signaling in T cells, enabling the simultaneous screening of multiple HLAs and TCRs. Leveraging genome-scale human, virome, and epitope mutagenesis libraries, TScan-II facilitates de novo antigen discovery and deep exploration of TCR specificity. We demonstrate TScan-II's potential for basic and translational research by identifying a non-canonical antigen for a cancer-reactive CD4+ T cell clone. Additionally, we identified two antigens for clonally expanded CD4+ T cells in Sjögren's disease, which bind distinct HLAs and are expressed in HLA-II-positive ductal cells within affected salivary glands.

The Role of Interferon-γ in Autoimmune Polyendocrine Syndrome Type 1.

BACKGROUND: Autoimmune polyendocrine syndrome type 1 (APS-1) is a life-threatening, autosomal recessive syndrome caused by autoimmune regulator (AIRE) deficiency. In APS-1, self-reactive T cells escape thymic negative selection, infiltrate organs, and drive autoimmune injury. The effector mechanisms governing T-cell-mediated damage in APS-1 remain poorly understood. METHODS: We examined whether APS-1 could be classified as a disease mediated by interferon-γ. We first assessed patients with APS-1 who were participating in a prospective natural history study and evaluated mRNA and protein expression in blood and tissues. We then examined the pathogenic role of interferon-γ using Aire-/-Ifng-/- mice and Aire-/- mice treated with the Janus kinase (JAK) inhibitor ruxolitinib. On the basis of our findings, we used ruxolitinib to treat five patients with APS-1 and assessed clinical, immunologic, histologic, transcriptional, and autoantibody responses. RESULTS: Patients with APS-1 had enhanced interferon-γ responses in blood and in all examined autoimmunity-affected tissues. Aire-/- mice had selectively increased interferon-γ production by T cells and enhanced interferon-γ, phosphorylated signal transducer and activator of transcription 1 (pSTAT1), and CXCL9 signals in multiple organs. Ifng ablation or ruxolitinib-induced JAK-STAT blockade in Aire-/- mice normalized interferon-γ responses and averted T-cell infiltration and damage in organs. Ruxolitinib treatment of five patients with APS-1 led to decreased levels of T-cell-derived interferon-γ, normalized interferon-γ and CXCL9 levels, and remission of alopecia, oral candidiasis, nail dystrophy, gastritis, enteritis, arthritis, Sjögren's-like syndrome, urticaria, and thyroiditis. No serious adverse effects from ruxolitinib were identified in these patients. CONCLUSIONS: Our findings indicate that APS-1, which is caused by AIRE deficiency, is characterized by excessive, multiorgan interferon-γ-mediated responses. JAK inhibition with ruxolitinib in five patients showed promising results. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Parathyroid Hormone Resistance and Autoantibodies to the PTH1 Receptor.

We describe two cases of acquired parathyroid hormone (PTH) resistance consequent to the development of serum PTH type 1 receptor (PTH1R) autoantibodies, which block PTH binding and signaling. Both cases were associated with other autoimmune manifestations, and one case was associated with atypical membranous glomerulonephritis. In vitro binding and signaling assays identified the presence of PTH1R-blocking IgG autoantibodies, which were not present in serum samples from patients with other renal or autoimmune disorders. (Funded by the Intramural Research Programs of the National Institute of Diabetes and Digestive and Kidney Diseases and others.).

Inhibition of JAK-STAT pathway corrects salivary gland inflammation and interferon driven immune activation in Sjögren's disease.

OBJECTIVES: Inflammatory cytokines that signal through the Janus kinases-signal transducer and activator of transcription (JAK-STAT) pathway, especially interferons (IFNs), are implicated in Sjögren's disease (SjD). Although inhibition of JAKs is effective in other autoimmune diseases, a systematic investigation of IFN-JAK-STAT signalling and the effect of JAK inhibitor (JAKi) therapy in SjD-affected human tissues has not been fully investigated. METHODS: Human minor salivary glands (MSGs) and peripheral blood mononuclear cells (PBMCs) were investigated using bulk or single-cell (sc) RNA sequencing (RNAseq), immunofluorescence (IF) microscopy and flow cytometry. Ex vivo culture assays on PBMCs and primary salivary gland epithelial cell (pSGEC) lines were performed to model changes in target tissues before and after JAKi. RESULTS: RNAseq and IF showed activated JAK-STAT pathway in SjD MSGs. Elevated IFN-stimulated gene (ISGs) expression associated with clinical variables (eg, focus scores, anti-SSA positivity). scRNAseq of MSGs exhibited cell type-specific upregulation of JAK-STAT and ISGs; PBMCs showed similar trends, including markedly upregulated ISGs in monocytes. Ex vivo studies showed elevated basal pSTAT levels in SjD MSGs and PBMCs that were corrected with JAKi. SjD-derived pSGECs exhibited higher basal ISG expressions and exaggerated responses to IFN-ß, which were normalised by JAKi without cytotoxicity. CONCLUSIONS: SjD patients' tissues exhibit increased expression of ISGs and activation of the JAK-STAT pathway in a cell type-dependent manner. JAKi normalises this aberrant signalling at the tissue level and in PBMCs, suggesting a putative viable therapy for SjD, targeting both glandular and extraglandular symptoms. Predicated on these data, a phase Ib/IIa randomised controlled trial to treat SjD with tofacitinib was initiated.

Histopathology and SARS-CoV-2 Cellular Localization in Eye Tissues of COVID-19 Autopsies.

Ophthalmic manifestations and tissue tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported in association with coronavirus disease 2019 (COVID-19), but the pathology and cellular localization of SARS-CoV-2 are not well characterized. The objective of this study was to evaluate macroscopic and microscopic changes and investigate cellular localization of SARS-CoV-2 across ocular tissues at autopsy. Ocular tissues were obtained from 25 patients with COVID-19 at autopsy. SARS-CoV-2 nucleocapsid gene RNA was previously quantified by droplet digital PCR from one eye. Herein, contralateral eyes from 21 patients were fixed in formalin and subject to histopathologic examination. Sections of the droplet digital PCR-positive eyes from four other patients were evaluated by in situ hybridization to determine the cellular localization of SARS-CoV-2 spike gene RNA. Histopathologic abnormalities, including cytoid bodies, vascular changes, and retinal edema, with minimal or no inflammation in ocular tissues were observed in all 21 cases evaluated. In situ hybridization localized SARS-CoV-2 RNA to neuronal cells of the retinal inner and outer layers, ganglion cells, corneal epithelia, scleral fibroblasts, and oligodendrocytes of the optic nerve. In conclusion, a range of common histopathologic alterations were identified within ocular tissue, and SARS-CoV-2 RNA was localized to multiple cell types. Further studies will be required to determine whether the alterations observed were caused by SARS-CoV-2 infection, the host immune response, and/or preexisting comorbidities.

Structural and functional analysis of salivary intercalated duct cells reveals a secretory phenotype.

Currently, all salivary ducts (intercalated, striated and collecting) are assumed to function broadly in a similar manner, reclaiming ions that were secreted by the secretory acinar cells while preserving fluid volume and delivering saliva to the oral cavity. Nevertheless, there has been minimal investigation into the structural and functional differences between distinct types of salivary duct cells. Therefore, in this study, the expression profile of proteins involved in stimulus-secretion coupling, as well as the function of the intercalated duct (ID) and striated duct cells, was examined. Particular focus was placed on defining differences between distinct duct cell populations. To accomplish this, immunohistochemistry and in situ hybridization were utilized to examine the localization and expression of proteins involved in reabsorption and secretion of ions and fluid. Further, in vivo calcium imaging was employed to investigate cellular function. Based on the protein expression profile and functional data, marked differences between the IDs and striated ducts were observed. Specifically, the ID cells express proteins native to the secretory acinar cells while lacking proteins specifically expressed in the striated ducts. Further, the ID and striated duct cells display different calcium signalling characteristics, with the IDs responding to a neural stimulus in a manner similar to the acinar cells. Overall, our data suggest that the IDs have a distinct role in the secretory process, separate from the reabsorptive striated ducts. Instead, based on our evidence, the IDs express proteins found in secretory cells, generate calcium signals in a manner similar to acinar cells, and, therefore, are likely secretory cells. KEY POINTS: Current studies examining salivary intercalated duct cells are limited, with minimal documentation of the ion transport machinery and the overall role of the cells in fluid generation. Salivary intercalated duct cells are presumed to function in the same manner as other duct cells, reclaiming ions, maintaining fluid volume and delivering the final saliva to the oral cavity. Here we systematically examine the structure and function of the salivary intercalated duct cells using immunohistochemistry, in situ hybridization and by monitoring in vivo Ca2+ dynamics. Structural data revealed that the intercalated duct cells lack proteins vital for reabsorption and express proteins necessary for secretion. Ca2+ dynamics in the intercalated duct cells were consistent with those observed in secretory cells and resulted from GPCR-mediated IP3 production.

Coexisting autoantibodies against transcription factor Sp4 are associated with decreased cancer risk in patients with dermatomyositis with anti-TIF1γ autoantibodies.

OBJECTIVES: In dermatomyositis (DM), autoantibodies are associated with unique clinical phenotypes. For example, anti-TIF1γ autoantibodies are associated with an increased risk of cancer. The purpose of this study was to discover novel DM autoantibodies. METHODS: Phage ImmunoPrecipitation Sequencing using sera from 43 patients with DM suggested that transcription factor Sp4 is a novel autoantigen; this was confirmed by showing that patient sera immunoprecipitated full-length Sp4 protein. Sera from 371 Johns Hopkins patients with myositis (255 with DM, 28 with antisynthetase syndrome, 40 with immune-mediated necrotising myopathy, 29 with inclusion body myositis and 19 with polymyositis), 80 rheumatological disease controls (25 with Sjogren's syndrome, 25 with systemic lupus erythematosus and 30 with rheumatoid arthritis (RA)) and 200 healthy comparators were screened for anti-SP4 autoantibodies by ELISA. A validation cohort of 46 anti-TIF1γ-positive patient sera from the University of Pittsburgh was also screened for anti-Sp4 autoantibodies. RESULTS: Anti-Sp4 autoantibodies were present in 27 (10.5%) patients with DM and 1 (3.3%) patient with RA but not in other clinical groups. In patients with DM, 96.3% of anti-Sp4 autoantibodies were detected in those with anti-TIF1γ autoantibodies. Among 26 TIF1γ-positive patients with anti-Sp4 autoantibodies, none (0%) had cancer. In contrast, among 35 TIF1γ-positive patients without anti-Sp4 autoantibodies, 5 (14%, p=0.04) had cancer. In the validation cohort, among 15 TIF1γ-positive patients with anti-Sp4 autoantibodies, 2 (13.3%) had cancer. By comparison, among 31 TIF1γ-positive patients without anti-Sp4 autoantibodies, 21 (67.7%, p<0.001) had cancer. CONCLUSIONS: Anti-Sp4 autoantibodies appear to identify a subgroup of anti-TIF1γ-positive DM patients with lower cancer risk.

DDX58(RIG-I)-related disease is associated with tissue-specific interferon pathway activation.

BACKGROUND: Singleton-Merten syndrome (SGMRT) is a rare immunogenetic disorder that variably features juvenile open-angle glaucoma (JOAG), psoriasiform skin rash, aortic calcifications and skeletal and dental dysplasia. Few families have been described and the genotypic and phenotypic spectrum is poorly defined, with variants in DDX58 (DExD/H-box helicase 58) being one of two identified causes, classified as SGMRT2. METHODS: Families underwent deep systemic phenotyping and exome sequencing. Functional characterisation with in vitro luciferase assays and in vivo interferon signature using bulk and single cell RNA sequencing was performed. RESULTS: We have identified a novel DDX58 variant c.1529A>T p.(Glu510Val) that segregates with disease in two families with SGMRT2. Patients in these families have widely variable phenotypic features and different ethnic background, with some being severely affected by systemic features and others solely with glaucoma. JOAG was present in all individuals affected with the syndrome. Furthermore, detailed evaluation of skin rash in one patient revealed sparse inflammatory infiltrates in a unique distribution. Functional analysis showed that the DDX58 variant is a dominant gain-of-function activator of interferon pathways in the absence of exogenous RNA ligands. Single cell RNA sequencing of patient lesional skin revealed a cellular activation of interferon-stimulated gene expression in keratinocytes and fibroblasts but not in neighbouring healthy skin. CONCLUSIONS: These results expand the genotypic spectrum of DDX58-associated disease, provide the first detailed description of ocular and dermatological phenotypes, expand our understanding of the molecular pathogenesis of this condition and provide a platform for testing response to therapy.

Gain-of-function mutations in ALPK1 cause an NF-κB-mediated autoinflammatory disease: functional assessment, clinical phenotyping and disease course of patients with ROSAH syndrome.

OBJECTIVES: To test the hypothesis that ROSAH (retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis and headache) syndrome, caused by dominant mutation in ALPK1, is an autoinflammatory disease. METHODS: This cohort study systematically evaluated 27 patients with ROSAH syndrome for inflammatory features and investigated the effect of ALPK1 mutations on immune signalling. Clinical, immunologic and radiographical examinations were performed, and 10 patients were empirically initiated on anticytokine therapy and monitored. Exome sequencing was used to identify a new pathogenic variant. Cytokine profiling, transcriptomics, immunoblotting and knock-in mice were used to assess the impact of ALPK1 mutations on protein function and immune signalling. RESULTS: The majority of the cohort carried the p.Thr237Met mutation but we also identified a new ROSAH-associated mutation, p.Tyr254Cys.Nearly all patients exhibited at least one feature consistent with inflammation including recurrent fever, headaches with meningeal enhancement and premature basal ganglia/brainstem mineralisation on MRI, deforming arthritis and AA amyloidosis. However, there was significant phenotypic variation, even within families and some adults lacked functional visual deficits. While anti-TNF and anti-IL-1 therapies suppressed systemic inflammation and improved quality of life, anti-IL-6 (tocilizumab) was the only anticytokine therapy that improved intraocular inflammation (two of two patients).Patients' primary samples and in vitro assays with mutated ALPK1 constructs showed immune activation with increased NF-κB signalling, STAT1 phosphorylation and interferon gene expression signature. Knock-in mice with the Alpk1 T237M mutation exhibited subclinical inflammation.Clinical features not conventionally attributed to inflammation were also common in the cohort and included short dental roots, enamel defects and decreased salivary flow. CONCLUSION: ROSAH syndrome is an autoinflammatory disease caused by gain-of-function mutations in ALPK1 and some features of disease are amenable to immunomodulatory therapy.

Lysosome-associated membrane protein 3 misexpression in salivary glands induces a Sjögren's syndrome-like phenotype in mice.

OBJECTIVES: Sjögren's syndrome (SS) is an autoimmune sialadenitis with unknown aetiology. Although extensive research implicated an abnormal immune response associated with lymphocytes, an initiating event mediated by salivary gland epithelial cell (SGEC) abnormalities causing activation is poorly characterised. Transcriptome studies have suggested alternations in lysosomal function are associated with SS, but a cause and effect linkage has not been established. In this study, we demonstrated that altered lysosome activity in SGECs by expression of lysosome-associated membrane protein 3 (LAMP3) can initiate an autoimmune response with autoantibody production and salivary dysfunction similar to SS. METHODS: Retroductal cannulation of the submandibular salivary glands with an adeno-associated virus serotype 2 vector encoding LAMP3 was used to establish a model system. Pilocarpine-stimulated salivary flow and the presence of autoantibodies were assessed at several time points post-cannulation. Salivary glands from the mice were evaluated using RNAseq and histologically. RESULTS: Following LAMP3 expression, saliva flow was significantly decreased and serum anti-Ro/SSA and La/SSB antibodies could be detected in the treated mice. Mechanistically, LAMP3 expression increased apoptosis in SGECs and decreased protein expression related to saliva secretion. Analysis of RNAseq data suggested altered lysosomal function in the transduced SGECs, and that the cellular changes can chemoattract immune cells into the salivary glands. Immune cells were activated via toll-like receptors by damage-associated molecular patterns released from LAMP3-expressing SGECs. CONCLUSIONS: These results show a critical role for lysosomal trafficking in the development of SS and establish a causal relationship between LAMP3 misexpression and the development of SS.