Cytometry by time of flight identifies distinct signatures in patients with systemic sclerosis, systemic lupus erythematosus and Sjögrens syndrome.

Systemic sclerosis (SSc), systemic lupus erythematosus (SLE) and primary Sjögrens syndrome (pSS) are clinically distinct systemic autoimmune diseases (SADs) that share molecular pathways. We quantified the frequency of circulating immune-cells in 169 patients with these SADs and 44 healty controls (HC) using mass-cytometry and assessed the diagnostic value of these results. Alterations in the frequency of immune-cell subsets were present in all SADs compared to HC. Most alterations, including a decrease of CD56hi NK-cells in SSc and IgM+ Bcells in pSS, were disease specific; only a reduced frequency of plasmacytoid dendritic cells was common between all SADs Strikingly, hierarchical clustering of SSc patients identified 4 clusters associated with different clinical phenotypes, and 9 of the 12 cell subset-alterations in SSc were also present during the preclinical-phase of the disease. Additionally, we found a strong association between the use of prednisone and alterations in B-cell subsets. Although differences in immune-cell frequencies between these SADs are apparent, the discriminative value thereof is too low for diagnostic purposes. Within each disease, mass cytometry analyses revealed distinct patterns between endophenotypes. Given the lack of tools enabling early diagnosis of SSc, our results justify further research into the value of cellular phenotyping as a diagnostic aid.

Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10.

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.

Somatic Gene Editing of GUCY2D by AAV-CRISPR/Cas9 Alters Retinal Structure and Function in Mouse and Macaque.

Mutations in GUCY2D, the gene encoding retinal guanylate cyclase-1 (retGC1), are the leading cause of autosomal dominant cone-rod dystrophy (CORD6). Significant progress toward clinical application of gene replacement therapy for Leber congenital amaurosis (LCA) due to recessive mutations in GUCY2D (LCA1) has been made, but a different approach is needed to treat CORD6 where gain of function mutations cause dysfunction and dystrophy. The CRISPR/Cas9 gene editing system efficiently disrupts genes at desired loci, enabling complete gene knockout or homology directed repair. Here, adeno-associated virus (AAV)-delivered CRISPR/Cas9 was used specifically to edit/disrupt this gene's early coding sequence in mouse and macaque photoreceptors in vivo, thereby knocking out retGC1 expression and demonstrably altering retinal function and structure. Neither preexisting nor induced Cas9-specific T-cell responses resulted in ocular inflammation in macaques, nor did it limit GUCY2D editing. The results show, for the first time, the ability to perform somatic gene editing in primates using AAV-CRISPR/Cas9 and demonstrate the viability this approach for treating inherited retinal diseases in general and CORD6 in particular.

RNA-Seq and CyTOF immuno-profiling of regenerating lacrimal glands identifies a novel subset of cells expressing muscle-related proteins.

The purpose of the present studies was to use CyTOF and RNA-Seq technologies to identify cells and genes involved in lacrimal gland repair that could be targeted to treat diseases of lacrimal gland dysfunction. Lacrimal glands of female BALB/c mice were experimentally injured by intra-glandular injection of interleukin 1 alpha (IL-1α). The lacrimal glands were harvested at various time points following injury (1 to 14 days) and used to either prepare single cell suspensions for CyTOF immuno-phenotyping analyses or to extract RNA for gene expression studies using RNA-Seq. CyTOF immuno-phenotyping identified monocytes and neutrophils as the major infiltrating populations 1 and 2 days post injury. Clustering of significantly differentially expressed genes identified 13 distinct molecular signatures: 3 associated with immune/inflammatory processes included genes up-regulated at days 1-2 and 3 associated with reparative processes with genes up-regulated primarily between days 4 and 5. Finally, clustering identified 65 genes which were specifically up-regulated 2 days post injury which was enriched for muscle specific genes. The expression of select muscle-related proteins was confirmed by immunohistochemistry which identified a subset of cells expressing these proteins. Double staining experiments showed that these cells are distinct from the myoepithelial cells. We conclude that experimentally induced injury to the lacrimal gland leads to massive infiltration by neutrophils and monocytes which resolved after 3 days. RNAseq and immunohistochemistry identified a group of cells, other than myoepithelial cells, that express muscle-related proteins that could play an important role in lacrimal gland repair.

Clinical relevance of RORγ positive and negative subsets of CD161+CD4+ T cells in primary Sjögren's syndrome.

OBJECTIVE: The relevance of the Th17 pathway in primary SS (pSS) is unclear. Published studies have relied on restimulating circulating CD161+ T cells in vitro for quantitation of IL-17-producing cells. While CD161 marks all IL-17+ T cells, it is also expressed by other Th subsets. The aim of this study was to directly analyse retinoic acid receptor-related orphan nuclear receptor (ROR)-γ expressing and non-expressing subsets of CD161+ T cells to determine the relevance of the Th17 pathway in pSS. METHODS: We quantitated the frequencies of both CD161- and RORγ-expressing T cells by comparative flow cytometry in peripheral blood mononuclear cells from a well-stratified cohort of pSS patients and control subjects. We also analysed the expression of antigen D-related HLA (HLA-DR) and CD161 in labial salivary glands from nine subjects undergoing a diagnostic biopsy. RESULTS: While the frequencies of both RORγ+ and RORγ- subsets of CD161+ CD4+ T cells were increased in peripheral blood from pSS patients, the increase in the RORγ+ subset positively correlated with humoral manifestations of the disease (anti-SSA/SSB autoantibodies and hypergammaglobulinaemia), but not with disease activity, and vice versa for the RORγ- subset. An increased frequency of HLA-DR+ CD161+CD4+ T cells was observed in labial salivary gland biopsies from pSS patients, suggesting chronic activation of CD161+CD4+ T cells in the target tissue of the disease. CONCLUSION: In addition to pointing to CD161 as a marker of a pathogenic subset of CD4+ T cells in pSS patients, our data indicate that even though the RORγ+ (Th17) CD161+ subset might contribute to humoral manifestations of the disease, the RORγ- (non-Th17) CD161+ subset is the one associated with disease activity in pSS patients.

Identification of Novel CD4+ T Cell Subsets in the Target Tissue of Sjögren's Syndrome and Their Differential Regulation by the Lymphotoxin/LIGHT Signaling Axis.

Despite being one of the most common rheumatologic diseases, there is still no disease-modifying drug for primary Sjögren's syndrome (pSS). Advancing our knowledge of the target tissue has been limited by the low dimensionality of histology techniques and the small size of human salivary gland biopsies. In this study, we took advantage of a molecularly validated mouse model of pSS to characterize tissue-infiltrating CD4+ T cells and their regulation by the lymphotoxin/LIGHT signaling axis. Novel cell subsets were identified by combining highly dimensional flow and mass cytometry with transcriptomic analyses. Pharmacologic modulation of the LTßR signaling pathway was achieved by treating mice with LTßR-Ig, a therapeutic intervention currently being tested in pSS patients (Baminercept trial NCT01552681). Using these approaches, we identified two novel CD4+ T cell subsets characterized by high levels of PD1: Prdm1+ effector regulatory T cells expressing immunoregulatory factors, such as Il10, Areg, Fgl2, and Itgb8, and Il21+ effector conventional T cells expressing a pathogenic transcriptional signature. Mirroring these observations in mice, large numbers of CD4+PD1+ T cells were detected in salivary glands from Sjögren's patients but not in normal salivary glands or kidney biopsies from lupus nephritis patients. Unexpectedly, LTßR-Ig selectively halted the recruitment of PD1- naive, but not PD1+, effector T cells to the target tissue, leaving the cells with pathogenic potential unaffected. Altogether, this study revealed new cellular players in pSS pathogenesis, their transcriptional signatures, and differential dependency on the lymphotoxin/LIGHT signaling axis that help to interpret the negative results of the Baminercept trial and will guide future therapeutic interventions.

BAFF overexpression increases lymphocytic infiltration in Sjögren's target tissue, but only inefficiently promotes ectopic B-cell differentiation.

B-cell activating factor (BAFF) levels are increased in rheumatoid arthritis, lupus and primary Sjögren's syndrome (pSS). However, BAFF contribution to pathogenesis is not completely understood. In pSS, immune infiltration of the salivary and lacrimal glands leads to xerostomia and xerophtalmia. Glandular B cell hyperactivation, differentiation into germinal center (GC)-like structures and plasma cell accumulation are histopathological hallmarks that were attributed to increased BAFF. Here, we experimentally tested this hypothesis by overexpressing BAFF in a mouse model of pSS. BAFF overexpression enhanced lymphocytic infiltration and MHCII expression on B cells. Increased BAFF also induced B cell differentiation into GC B cells within the autoimmune target tissue. However, even in these conditions, GC B cells only accounted for <1% of glandular B cells, demonstrating that BAFF is not efficiently promoting ectopic GC formation in pSS and warranting further investigation of therapeutics targeting both BAFF and the related TNF-family member APRIL.

Cytometry by time-of-flight immunophenotyping identifies a blood Sjögren's signature correlating with disease activity and glandular inflammation.

BACKGROUND: Mass cytometry has recently emerged as a promising tool for clinical research. However, few studies have demonstrated its benefit for patient stratification and biomarker identification. Primary Sjögren's syndrome (pSS) is a prototype of chronic autoimmune disease, the pathogenesis of which remains unclear and for which treatment does not exist. OBJECTIVE: This observational case-control study was designed to discover new cellular biomarkers and therapeutic targets in patients with pSS. METHODS: Forty-nine patients with pSS and 45 control subjects were enrolled for clinical evaluation and mass cytometry quantification of 34 protein markers in blood. For a third of these subjects, matched labial salivary gland biopsy specimens were also analyzed by mass cytometry and immunohistochemistry. RESULTS: In salivary gland biopsy specimens from patients with pSS, we identified a high number of activated CD8(+) T cells, terminally differentiated plasma cells, and activated epithelial cells, pointing to new pathogenic mechanisms for future clinical intervention. In blood, we identified a 6-cell disease signature defined by decreased numbers of CD4 and memory B lymphocytes, decreased plasmacytoid dendritic cell numbers, and increased representation of activated CD4 and CD8 T cells and plasmablasts. These blood cellular components correlated with clinical parameters and, when taken together, clustered patients into subsets with distinct disease activity and glandular inflammation. CONCLUSION: This first application of mass cytometry to a well-stratified clinical cohort and small biopsy tissues establishes the benefits of such an approach for the discovery of new biomarkers and therapeutic targets. Similar high-dimensional immunophenotyping strategies could be implemented in longitudinal and interventional clinical settings in this and other disease areas.

Peptide-MHC-I from Endogenous Antigen Outnumber Those from Exogenous Antigen, Irrespective of APC Phenotype or Activation.

Naïve anti-viral CD8+ T cells (TCD8+) are activated by the presence of peptide-MHC Class I complexes (pMHC-I) on the surface of professional antigen presenting cells (pAPC). Increasing the number of pMHC-I in vivo can increase the number of responding TCD8+. Antigen can be presented directly or indirectly (cross presentation) from virus-infected and uninfected cells, respectively. Here we determined the relative importance of these two antigen presenting pathways in mousepox, a natural disease of the mouse caused by the poxvirus, ectromelia (ECTV). We demonstrated that ECTV infected several pAPC types (macrophages, B cells, and dendritic cells (DC), including DC subsets), which directly presented pMHC-I to naïve TCD8+ with similar efficiencies in vitro. We also provided evidence that these same cell-types presented antigen in vivo, as they form contacts with antigen-specific TCD8+. Importantly, the number of pMHC-I on infected pAPC (direct presentation) vastly outnumbered those on uninfected cells (cross presentation), where presentation only occurred in a specialized subset of DC. In addition, prior maturation of DC failed to enhance antigen presentation, but markedly inhibited ECTV infection of DC. These results suggest that direct antigen presentation is the dominant pathway in mice during mousepox. In a broader context, these findings indicate that if a virus infects a pAPC then the presentation by that cell is likely to dominate over cross presentation as the most effective mode of generating large quantities of pMHC-I is on the surface of pAPC that endogenously express antigens. Recent trends in vaccine design have focused upon the introduction of exogenous antigens into the MHC Class I processing pathway (cross presentation) in specific pAPC populations. However, use of a pantropic viral vector that targets pAPC to express antigen endogenously likely represents a more effective vaccine strategy than the targeting of exogenous antigen to a limiting pAPC subpopulation.