Chimeric autoantibody receptor T cells deplete NMDA receptor-specific B cells.

Anti-NMDA receptor (NMDAR) autoantibodies cause NMDAR encephalitis, the most common autoimmune encephalitis, leading to psychosis, seizures, and autonomic dysfunction. Current treatments comprise broad immunosuppression or non-selective antibody removal. We developed NMDAR-specific chimeric autoantibody receptor (NMDAR-CAAR) T cells to selectively eliminate anti-NMDAR B cells and disease-causing autoantibodies. NMDAR-CAARs consist of an extracellular multi-subunit NMDAR autoantigen fused to intracellular 4-1BB/CD3ζ domains. NMDAR-CAAR T cells recognize a large panel of human patient-derived autoantibodies, release effector molecules, proliferate, and selectively kill antigen-specific target cell lines even in the presence of high autoantibody concentrations. In a passive transfer mouse model, NMDAR-CAAR T cells led to depletion of an anti-NMDAR B cell line and sustained reduction of autoantibody levels without notable off-target toxicity. Treatment of patients may reduce side effects, prevent relapses, and improve long-term prognosis. Our preclinical work paves the way for CAAR T cell phase I/II trials in NMDAR encephalitis and further autoantibody-mediated diseases.

An atlas of cells in the human tonsil.

Palatine tonsils are secondary lymphoid organs (SLOs) representing the first line of immunological defense against inhaled or ingested pathogens. We generated an atlas of the human tonsil composed of >556,000 cells profiled across five different data modalities, including single-cell transcriptome, epigenome, proteome, and immune repertoire sequencing, as well as spatial transcriptomics. This census identified 121 cell types and states, defined developmental trajectories, and enabled an understanding of the functional units of the tonsil. Exemplarily, we stratified myeloid slan-like subtypes, established a BCL6 enhancer as locally active in follicle-associated T and B cells, and identified SIX5 as putative transcriptional regulator of plasma cell maturation. Analyses of a validation cohort confirmed the presence, annotation, and markers of tonsillar cell types and provided evidence of age-related compositional shifts. We demonstrate the value of this resource by annotating cells from B cell-derived mantle cell lymphomas, linking transcriptional heterogeneity to normal B cell differentiation states of the human tonsil.

DNA Damage Signaling Instructs Polyploid Macrophage Fate in Granulomas.

Granulomas are immune cell aggregates formed in response to persistent inflammatory stimuli. Granuloma macrophage subsets are diverse and carry varying copy numbers of their genomic information. The molecular programs that control the differentiation of such macrophage populations in response to a chronic stimulus, though critical for disease outcome, have not been defined. Here, we delineate a macrophage differentiation pathway by which a persistent Toll-like receptor (TLR) 2 signal instructs polyploid macrophage fate by inducing replication stress and activating the DNA damage response. Polyploid granuloma-resident macrophages formed via modified cell divisions and mitotic defects and not, as previously thought, by cell-to-cell fusion. TLR2 signaling promoted macrophage polyploidy and suppressed genomic instability by regulating Myc and ATR. We propose that, in the presence of persistent inflammatory stimuli, pathways previously linked to oncogene-initiated carcinogenesis instruct a long-lived granuloma-resident macrophage differentiation program that regulates granulomatous tissue remodeling.

Amplification of autoimmune organ damage by NKp46-activated ILC1s.

In systemic lupus erythematosus, loss of immune tolerance, autoantibody production and immune complex deposition are required but not sufficient for organ damage1. How inflammatory signals are initiated and amplified in the setting of autoimmunity remains elusive. Here we set out to dissect layers and hierarchies of autoimmune kidney inflammation to identify tissue-specific cellular hubs that amplify autoinflammatory responses. Using high-resolution single-cell profiling of kidney immune and parenchymal cells, in combination with antibody blockade and genetic deficiency, we show that tissue-resident NKp46+ innate lymphoid cells (ILCs) are crucial signal amplifiers of disease-associated macrophage expansion and epithelial cell injury in lupus nephritis, downstream of autoantibody production. NKp46 signalling in a distinct subset of group 1 ILCs (ILC1s) instructed an unconventional immune-regulatory transcriptional program, which included the expression of the myeloid cell growth factor CSF2. CSF2 production by NKp46+ ILCs promoted the population expansion of monocyte-derived macrophages. Blockade of the NKp46 receptor (using the antibody clone mNCR1.15; ref. 2) or genetic deficiency of NKp46 abrogated epithelial cell injury. The same cellular and molecular patterns were operative in human lupus nephritis. Our data provide support for the idea that NKp46+ ILC1s promote parenchymal cell injury by granting monocyte-derived macrophages access to epithelial cell niches. NKp46 activation in ILC1s therefore constitutes a previously unrecognized, crucial tissue rheostat that amplifies organ damage in autoimmune hosts, with broad implications for inflammatory pathologies and therapies.

Renal IL-23-Dependent Type 3 Innate Lymphoid Cells Link Crystal-induced Intrarenal Inflammasome Activation with Kidney Fibrosis.

Chronic inflammasome activation in mononuclear phagocytes (MNPs) promotes fibrosis in various tissues, including the kidney. The cellular and molecular links between the inflammasome and fibrosis are unclear. To address this question, we fed mice lacking various immunological mediators an adenine-enriched diet, which causes crystal precipitation in renal tubules, crystal-induced inflammasome activation, and renal fibrosis. We found that kidney fibrosis depended on an intrarenal inflammasome-dependent type 3 immune response driven by its signature transcription factor Rorc (retinoic acid receptor-related orphan receptor C gene), which was partially carried out by type 3 innate lymphoid cells (ILC3s). The role of ILCs in the kidney is less well known than in other organs, especially that of ILC3. In this article, we describe that depletion of ILCs or genetic deficiency for Rorc attenuated kidney inflammation and fibrosis. Among the inflammasome-derived cytokines, only IL-1ß expanded ILC3 and promoted fibrosis, whereas IL-18 caused differentiation of NKp46+ ILC3. Deficiency of the type 3 maintenance cytokine, IL-23, was more protective than IL-1ß inhibition, which may be explained by the downregulation of the IL-1R, but not of the IL-23R, by ILC3 early in the disease, allowing persistent sensing of IL-23. Mechanistically, ILC3s colocalized with renal MNPs in vivo as shown by multiepitope-ligand cartography. Cell culture experiments indicated that renal ILC3s caused renal MNPs to increase TGF-ß production that stimulated fibroblasts to produce collagen. We conclude that ILC3s link inflammasome activation with kidney inflammation and fibrosis and are regulated by IL-1ß and IL-23.

Tissue niches and immunopathology through the lens of spatial tissue profiling techniques.

Spatial organization plays a fundamental role in biology, influencing the function of biological structures at various levels. The immune system, in particular, relies on the orchestrated interactions of immune cells with their microenvironment to mount protective or pathogenic immune responses. The COVID-19 pandemic has underscored the significance of studying immunity within target organs to understand disease progression and severity. To achieve this, multiplex histology and spatial transcriptomics have proven indispensable in providing a spatial context to protein and gene expression patterns. By combining these techniques, researchers gain a more comprehensive understanding of the complex interactions at the cellular and molecular level in distinct tissue niches, key functional units modulating health and disease. In this review, we discuss recent advances in spatial tissue profiling techniques, highlighting their advantages over traditional histopathology studies. The insights gained from these approaches have the potential to revolutionize the diagnosis and treatment of various diseases including cancer, autoimmune disorders, and infectious diseases. However, we also acknowledge their challenges and limitations. Despite these, spatial tissue profiling offers promising opportunities to improve our understanding of how tissue niches direct regional immunity, and their relevance in tissue immunopathology, as a basis for novel therapeutic strategies and personalized medicine.

Interferon-Driven Immune Dysregulation in Common Variable Immunodeficiency-Associated Villous Atrophy and Norovirus Infection.

PURPOSE: About 15% of patients with common variable immunodeficiency (CVID) develop a small intestinal enteropathy, which resembles celiac disease with regard to histopathology but evolves from a distinct, poorly defined pathogenesis that has been linked in some cases to chronic norovirus (NV) infection. Interferon-driven inflammation is a prominent feature of CVID enteropathy, but it remains unknown how NV infection may contribute. METHODS: Duodenal biopsies of CVID patients, stratified according to the presence of villous atrophy (VA), IgA plasma cells (PCs), and chronic NV infection, were investigated by flow cytometry, multi-epitope-ligand cartography, bulk RNA-sequencing, and RT-qPCR of genes of interest. RESULTS: VA development was connected to the lack of intestinal (IgA+) PC, a T helper 1/T helper 17 cell imbalance, and increased recruitment of granzyme+CD8+ T cells and pro-inflammatory macrophages to the affected site. A mixed interferon type I/III and II signature occurred already in the absence of histopathological changes and increased with the severity of the disease and in the absence of (IgA+) PCs. Chronic NV infection exacerbated this signature when compared to stage-matched NV-negative samples. CONCLUSIONS: Our study suggests that increased IFN signaling and T-cell cytotoxicity are present already in mild and are aggravated in severe stages (VA) of CVID enteropathy. NV infection preempts local high IFN-driven inflammation, usually only seen in VA, at milder disease stages. Thus, revealing the impact of different drivers of the pathological mixed IFN type I/III and II signature may allow for more targeted treatment strategies in CVID enteropathy and supports the goal of viral elimination.

Two-Photon Excitation Spectra of Various Fluorescent Proteins within a Broad Excitation Range.

Two-photon excitation fluorescence laser-scanning microscopy is the preferred method for studying dynamic processes in living organ models or even in living organisms. Thanks to near-infrared and infrared excitation, it is possible to penetrate deep into the tissue, reaching areas of interest relevant to life sciences and biomedicine. In those imaging experiments, two-photon excitation spectra are needed to select the optimal laser wavelength to excite as many fluorophores as possible simultaneously in the sample under consideration. The more fluorophores that can be excited, and the more cell populations that can be studied, the better access to their arrangement and interaction can be reached in complex systems such as immunological organs. However, for many fluorophores, the two-photon excitation properties are poorly predicted from the single-photon spectra and are not yet available, in the literature or databases. Here, we present the broad excitation range (760 nm to 1300 nm) of photon-flux-normalized two-photon spectra of several fluorescent proteins in their cellular environment. This includes the following fluorescent proteins spanning from the cyan to the infrared part of the spectrum: mCerulean3, mTurquoise2, mT-Sapphire, Clover, mKusabiraOrange2, mOrange2, LSS-mOrange, mRuby2, mBeRFP, mCardinal, iRFP670, NirFP, and iRFP720.

Teriflunomide Preserves Neuronal Activity and Protects Mitochondria in Brain Slices Exposed to Oxidative Stress.

Teriflunomide (TFN) limits relapses in relapsing-remitting multiple sclerosis (RRMS) by reducing lymphocytic proliferation through the inhibition of the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) and the subsequent modulation of de novo pyrimidine synthesis. Alterations of mitochondrial function as a consequence of oxidative stress have been reported during neuroinflammation. Previously, we showed that TFN prevents alterations of mitochondrial motility caused by oxidative stress in peripheral axons. Here, we aimed to validate TFN effects on mitochondria and neuronal activity in hippocampal brain slices, in which cellular distribution and synaptic circuits are largely preserved. TFN effects on metabolism and neuronal activity were investigated by assessing oxygen partial pressure and local field potential in acute slices. Additionally, we imaged mitochondria in brain slices from the transgenic Thy1-CFP/COX8A)S2Lich/J (mitoCFP) mice using two-photon microscopy. Although TFN could not prevent oxidative stress-related depletion of ATP, it preserved oxygen consumption and neuronal activity in CNS tissue during oxidative stress. Furthermore, TFN prevented mitochondrial shortening and fragmentation of puncta-shaped and network mitochondria during oxidative stress. Regarding motility, TFN accentuated the decrease in mitochondrial displacement and increase in speed observed during oxidative stress. Importantly, these effects were not associated with neuronal viability and did not lead to axonal damage. In conclusion, during conditions of oxidative stress, TFN preserves the functionality of neurons and prevents morphological and motility alterations of mitochondria.

[B lymphocytes and plasma cells as drivers of rheumatic diseases]. / B-Lymphozyten und Plasmazellen als Treiber rheumatischer Erkrankungen.

Various research groups at the German Rheumatism Research Center in Berlin, in close cooperation with the Department of Rheumatology and Clinical Immunology of the Medical Clinic at the Charité, have made important contributions to the significance of B cells and plasma cells in rheumatic diseases, which are relevant not only for rheumatology but for all clinical specialties in which antibody-mediated diseases play a role. In particular, the research addresses impaired B cell homeostasis, the importance of the IgM Fc receptor in the regulation of autoimmunity, the role of long-lived memory plasma cells in maintaining autoimmunity and ensuring its survival in specific niches organized by stromal cells in bone marrow and inflamed tissues. The research results have contributed to a better understanding of the immunological and molecular mechanisms in rheumatic diseases and their treatment. The identification of the long-lived memory plasma cell has led to promising treatment approaches with curative potential in autoimmune diseases.

[The DRFZ-a pioneer in research on the interaction between immune and stromal cells during de- and regeneration of the musculoskeletal system]. / Das DRFZ ­ ein Vorreiter bei der Erforschung des Zusammenspiels von Immun- und Stromazellen bei der De- und Regeneration des Bewegungsapparats.

Rheumatoid arthritis and osteoarthritis are two related chronic diseases of the musculoskeletal system which are particularly pronounced in the region of joints and bones. Their pathogeneses are associated with chronic inflammation, which can disrupt homeostasis in bones and articular cartilage. Degradation products deriving from articular cartilage can contribute to the exacerbation of inflammation in the joint region. Mechanical stimuli and blood vessels also play a central role in both the regulation of bone growth as well as in the regeneration of bone tissue. Not only chronic inflammatory processes but also hormonal changes after menopause or undesired effects of glucocorticoid therapy have an influence on the balance between bone resorption and deposition, by promoting the former and reducing the latter. This results in decreased bone quality and, in some cases, considerable loss of bone or osteoporosis. An in-depth understanding of these processes at the molecular, cellular, and tissue level, as well as of the changes present in chronic inflammatory diseases, has been the focus of research at the German Rheumatism Research Center (Deutsches Rheuma-Forschungszentrum, DRFZ) since its foundation. Based on an improved understanding of these mechanisms, the DRFZ aims to develop improved prevention and treatment strategies with effects even in early disease stages.

Unbiased classification of mosquito blood cells by single-cell genomics and high-content imaging.

Mosquito blood cells are immune cells that help control infection by vector-borne pathogens. Despite their importance, little is known about mosquito blood cell biology beyond morphological and functional criteria used for their classification. Here, we combined the power of single-cell RNA sequencing, high-content imaging flow cytometry, and single-molecule RNA hybridization to analyze a subset of blood cells of the malaria mosquito Anopheles gambiae By demonstrating that blood cells express nearly half of the mosquito transcriptome, our dataset represents an unprecedented view into their transcriptional program. Analyses of differentially expressed genes identified transcriptional signatures of two cell types and provide insights into the current classification of these cells. We further demonstrate the active transfer of a cellular marker between blood cells that may confound their identification. We propose that cell-to-cell exchange may contribute to cellular diversity and functional plasticity seen across biological systems.

Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing Matrix.

Two-photon microscopy enables monitoring cellular dynamics and communication in complex systems, within a genuine environment, such as living tissues and, even, living organisms. Particularly, its application to understand cellular interactions in the immune system has brought unique insights into pathophysiologic processes in vivo. Simultaneous multiplexed imaging is required to understand the dynamic orchestration of the multiple cellular and non-cellular tissue compartments defining immune responses. Here, we present an improvement of our previously developed method, which allowed us to achieve multiplexed dynamic intravital two-photon imaging, by using a synergistic strategy. This strategy combines a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an unmixing algorithm based on the calculation of spectral similarities with previously measured fluorophore fingerprints. The improvement of the similarity spectral unmixing algorithm here described is based on dimensionality reduction of the mixing matrix. We demonstrate its superior performance in the correct pixel-based assignment of probes to tissue compartments labeled by single fluorophores with similar spectral fingerprints, as compared to the full-dimensional similarity spectral unmixing approach.

Single-cell transcriptomes of murine bone marrow stromal cells reveal niche-associated heterogeneity.

Bone marrow (BM) stromal cells are important in the development and maintenance of cells of the immune system. Using single cell RNA sequencing, we here explore the functional and phenotypic heterogeneity of individual transcriptomes of 1167 murine BM mesenchymal stromal cells. These cells exhibit a tremendous heterogeneity of gene expression, which precludes the identification of defined subpopulations. However, according to the expression of 108 genes involved in the communication of stromal cells with hematopoietic cells, we have identified 14 non-overlapping subpopulations, with distinct cytokine or chemokine gene expression signatures. With respect to the maintenance of subsets of immune memory cells by stromal cells, we identified distinct subpopulations expressing Il7, Il15 and Tnfsf13b. Together, this study provides a comprehensive dissection of the BM stromal heterogeneity at the single cell transcriptome level and provides a basis to understand their lifestyle and their role as organizers of niches for the long-term maintenance of immune cells.

Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies.

Bone healing involves the interplay of immune cells, mesenchymal cells, and vasculature over the time course of regeneration. Approaches to quantify the spatiotemporal aspects of bone healing at cellular resolution during long bone healing do not yet exist. Here, a novel technique termed Limbostomy is presented, which combines intravital microendoscopy with an osteotomy. This design allows a modular combination of an internal fixator plate with a gradient refractive index (GRIN) lens at various depths in the bone marrow and can be combined with a surgical osteotomy procedure. The field of view (FOV) covers a significant area of the fracture gap and allows monitoring cellular processes in vivo. The GRIN lens causes intrinsic optical aberrations which have to be corrected. The optical system was characterized and a postprocessing algorithm was developed. It corrects for wave front aberration-induced image plane deformation and for background and noise signals, enabling us to observe subcellular processes. Exemplarily, we quantitatively and qualitatively analyze angiogenesis in bone regeneration. We make use of a transgenic reporter mouse strain with nucleargreen fluorescent protein and membrane-bound tdTomato under the Cadherin-5 promoter. We observe two phases of vascularization. First, rapid vessel sprouting pervades the FOV within 3-4 days after osteotomy. Second, the vessel network continues to be dynamically remodeled until the end of our observation time, 14 days after surgery. Limbostomy opens a unique set of opportunities and allows further insight on spatiotemporal aspects of bone marrow biology, for example, hematopoiesis, analysis of cellular niches, immunological memory, and vascularization in the bone marrow during health and disease. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Coregistered Spectral Optical Coherence Tomography and Two-Photon Microscopy for Multimodal Near-Instantaneous Deep-Tissue Imaging.

Two-photon microscopy (2PM) has brought unique insight into the mechanisms underlying immune system dynamics and function since it enables monitoring of cellular motility and communication in complex systems within their genuine environment-the living organism. However, use of 2PM in clinical settings is limited. In contrast, optical coherence tomography (OCT), a noninvasive label-free diagnostic imaging method, which allows monitoring morphologic changes of large tissue regions in vivo, has found broad application in the clinic. Here we developed a combined multimodal technology to achieve near-instantaneous coregistered OCT, 2PM, and second harmonic generation (SHG) imaging over large volumes (up to 1,000 × 1,000 × 300 µm3 ) of tendons and other tissue compartments in mouse paws, as well as in mouse lymph nodes, spleens, and femurs. Using our multimodal imaging approach, we found differences in macrophage cell shape and motility behavior depending on whether they are located in tendons or in the surrounding tissue compartments of the mouse paw. The cellular shape of tissue-resident macrophages, indicative for their role in tissue, correlated with the supramolecular organization of collagen as revealed by SHG and OCT. Hence, the here-presented approach of coregistered OCT and 2PM has the potential to link specific cellular phenotypes and functions (as revealed by 2PM) to tissue morphology (as highlighted by OCT) and thus, to build a bridge between basic research knowledge and clinical observations. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Systematic Enzyme Mapping of Cellular Metabolism by Phasor-Analyzed Label-Free NAD(P)H Fluorescence Lifetime Imaging.

In the past years, cellular metabolism of the immune system experienced a revival, as it has become clear that it is not merely responsible for the cellular energy supply, but also impacts on many signaling pathways and, thus, on diverse cellular functions. Label-free fluorescence lifetime imaging of the ubiquitous coenzymes NADH and NADPH (NAD(P)H-FLIM) makes it possible to monitor cellular metabolism in living cells and tissues and has already been applied to study metabolic changes both under physiologic and pathologic conditions. However, due to the complex distribution of NAD(P)H-dependent enzymes in cells, whose distribution continuously changes over time, a thorough interpretation of NAD(P)H-FLIM results, in particular, resolving the contribution of various enzymes to the overall metabolic activity, remains challenging. We developed a systematic framework based on angle similarities of the phase vectors and their length to analyze NAD(P)H-FLIM data of cells and tissues based on a generally valid reference system of highly abundant NAD(P)H-dependent enzymes in cells. By using our analysis framework, we retrieve information not only about the overall metabolic activity, i.e., the fraction of free to enzyme-bound NAD(P)H, but also identified the enzymes predominantly active within the sample at a certain time point with subcellular resolution. We verified the performance of the approach by applying NAD(P)H-FLIM on a stromal-like cell line and identified a different group of enzymes that were active in the cell nuclei as compared to the cytoplasm. As the systematic phasor-based analysis framework of label-free NAD(P)H-FLIM can be applied both in vitro and in vivo, it retains the unique power to enable dynamic enzyme-based metabolic investigations, at subcellular resolution, in genuine environments.

Multiplexed fluorescence microscopy reveals heterogeneity among stromal cells in mouse bone marrow sections.

The bone marrow (BM) consists of multiple, structured micro-environmental entities-the so called niches, which contain hematopoietic cells as well as stromal cells. These niches fulfill a variety of functions, such as control of the hematopoietic stem cell pool, differentiation of hematopoietic cells, and maintenance of immunological memory. However, due to the molecular and cellular complexity and a lack of suitable histological multiplexing methods, the composition of the various BM niches is still elusive. In this study, we apply multiepitope-ligand-cartography (MELC) on bone sections from mice. We combine multiplexed immunofluorescence histology data with various object-based segmentation approaches in order to define irregularly shaped, net-like structures of stromal cells. We confirm MELC as a robust histological method and validate our automated segmentation algorithms using flow cytometry and manual evaluation. By means of MELC multiplexing, we reveal heterogeneous expression of leptin receptor (LpR), BP-1, and VCAM-1 in the stromal network. Moreover, we demonstrate by quantification a preferential contact of B cell subsets as well as of plasma cells to processes of CXCL12-expressing stromal cells, compared with stromal somata. In summary, our approach is suitable for spatial analysis of complex tissue structures.

Cell shape characterization and classification with discrete Fourier transforms and self-organizing maps.

Cells in their natural environment often exhibit complex kinetic behavior and radical adjustments of their shapes. This enables them to accommodate to short- and long-term changes in their surroundings under physiological and pathological conditions. Intravital multi-photon microscopy is a powerful tool to record this complex behavior. Traditionally, cell behavior is characterized by tracking the cells' movements, which yields numerous parameters describing the spatiotemporal characteristics of cells. Cells can be classified according to their tracking behavior using all or a subset of these kinetic parameters. This categorization can be supported by the a priori knowledge of experts. While such an approach provides an excellent starting point for analyzing complex intravital imaging data, faster methods are required for automated and unbiased characterization. In addition to their kinetic behavior, the 3D shape of these cells also provide essential clues about the cells' status and functionality. New approaches that include the study of cell shapes as well may also allow the discovery of correlations amongst the track- and shape-describing parameters. In the current study, we examine the applicability of a set of Fourier components produced by Discrete Fourier Transform (DFT) as a tool for more efficient and less biased classification of complex cell shapes. By carrying out a number of 3D-to-2D projections of surface-rendered cells, the applied method reduces the more complex 3D shape characterization to a series of 2D DFTs. The resulting shape factors are used to train a Self-Organizing Map (SOM), which provides an unbiased estimate for the best clustering of the data, thereby characterizing groups of cells according to their shape. We propose and demonstrate that such shape characterization is a powerful addition to, or a replacement for kinetic analysis. This would make it especially useful in situations where live kinetic imaging is less practical or not possible at all. © 2017 International Society for Advancement of Cytometry.