Zoltan G. Laszik

Spatial transcriptomics allows the unprecedented examination of gene expression levels at the resolution of spatially-situated single cells in a high-throughput manner. As the technology is adopted more broadly, studies frequently collect data from multiple tissue samples, which leads to unique challenges that traditional spatial statistical methods are not equipped to handle. In particular, factors that differ across samples, such as different coordinate systems, different numbers and types of cells, different underlying tissue architectures, among others, preclude the application of traditional methods to our new setting. In this work, we propose a novel method, TESSERA, based on a spatial generalized linear model, for analyzing multi-sample spatial transcriptomics count data. Importantly, we provide a mathematical and computational framework for efficient and scalable model fitting and statistical inference to accompany the specification of our model. Our method for fitting the model enables the estimation of a common set of fixed effects across samples. This allows us to address a variety of differential expression questions, such as identification of which genes are differentially expressed between conditions (e.g., diseases, treatments), while accounting for spatial correlation between cells within a sample. We benchmark our proposed method on simulated data and apply it to a spatial transcriptomics dataset of human kidney samples. We find that our method provides a hitherto nonexistent extension to the multi-sample setting while remaining competitive with or outperforming existing algorithms in the single-sample setting.

The mechanisms of postacute medical conditions and unexplained symptoms after SARS-CoV-2 infection [Long Covid (LC)] are incompletely understood. There is growing evidence that viral persistence, immune dysregulation, and T cell dysfunction may play major roles. We performed whole-body positron emission tomography imaging in a well-characterized cohort of 24 participants at time points ranging from 27 to 910 days after acute SARS-CoV-2 infection using the radiopharmaceutical agent [ ¹⁸ F]F-AraG, a selective tracer that allows for anatomical quantitation of activated T lymphocytes. Tracer uptake in the postacute COVID-19 group, which included those with and without continuing symptoms, was higher compared with prepandemic controls in many regions, including the brain stem, spinal cord, bone marrow, nasopharyngeal and hilar lymphoid tissue, cardiopulmonary tissues, and gut wall. T cell activation in the spinal cord and gut wall was associated with the presence of LC symptoms. In addition, tracer uptake in lung tissue was higher in those with persistent pulmonary symptoms specifically. Increased T cell activation in these tissues was also observed in many individuals without LC. Given the high [ ¹⁸ F]F-AraG uptake detected in the gut, we obtained colorectal tissue for in situ hybridization of SARS-CoV-2 RNA and immunohistochemical studies in a subset of five participants with LC symptoms. We identified intracellular SARS-CoV-2 single-stranded spike protein–encoding RNA in rectosigmoid lamina propria tissue in all five participants and double-stranded spike protein–encoding RNA in three participants up to 676 days after initial COVID-19, suggesting that tissue viral persistence could be associated with long-term immunologic perturbations.

Key Points: Non-full house (NFH) membranous lupus nephritis (MLN) is a minor subset of all MLN cases. Patients with NFH MLN tend to be older when diagnosed with systemic lupus erythematosus, undergo first renal biopsy at an older age, and have fewer extrarenal systemic manifestations. Lower load of C3 glomerular deposits seen in NFH MLN biopsies suggests attenuation of complement-mediated injury, which may have wider systemic implications. Background: Renal involvement in systemic lupus erythematosus (SLE) is a key predictor of morbidity and mortality. Immunofluorescence (IF) staining of glomeruli is typically positive for IgG, IgA, IgM, C3, and C1q—the full house (FH) pattern. However, a subset of patients with membranous lupus nephritis (MLN) have a Non-FH (NFH) IF pattern more typical of idiopathic membranous nephropathy. Methods: From a multi-institutional cohort of 113 MLN cases, we identified 29 NFH MLN biopsies. NFH MLN was defined by IF criteria: ≥1+ glomerular capillary loop IgG staining and<1+ IgA, IgM, and C1q. FH MLN was defined as ≥1+ staining for all five antibodies. Intermediate (Int) cases did not meet criteria for FH or NFH. We compared the pathological and clinical characteristics and outcomes among patients with FH, NFH, and Int IF patterns on kidney biopsy. Results: NFH MLN represents a subset of MLN biopsies (13.4%). Compared with patients with FH MLN, patients with NFH MLN were older at SLE diagnosis (29 versus 22.5 years), had a longer time to initial kidney biopsy (8 versus 3.16 years), and had fewer SLE manifestations (2.5 versus 3.36 involved systems). NFH MLN biopsies showed lower C3 IF intensity (1.16+ versus 2.38+). Int biopsies had findings intermediate between those of NFH and FH groups. Conclusions: NFH IF pattern defines a small subset of MLN biopsies and appears to be associated with milder clinical manifestations and slower disease progression. Less robust C3 deposition in NFH MLN may suggest a pathophysiology distinct from that of FH MLN.

Kidney Precision Medicine Project (KPMP) is building a spatially specified human kidney tissue atlas in health and disease with single-cell resolution. Here, we describe the construction of an integrated reference map of cells, pathways, and genes using unaffected regions of nephrectomy tissues and undiseased human biopsies from 56 adult subjects. We use single-cell/nucleus transcriptomics, subsegmental laser microdissection transcriptomics and proteomics, near-single-cell proteomics, 3D and CODEX imaging, and spatial metabolomics to hierarchically identify genes, pathways, and cells. Integrated data from these different technologies coherently identify cell types/subtypes within different nephron segments and the interstitium. These profiles describe cell-level functional organization of the kidney following its physiological functions and link cell subtypes to genes, proteins, metabolites, and pathways. They further show that messenger RNA levels along the nephron are congruent with the subsegmental physiological activity. This reference atlas provides a framework for the classification of kidney disease when multiple molecular mechanisms underlie convergent clinical phenotypes.

Belatacept offers superior long‐term outcome relative to calcineurin inhibitor (CNI)‐based immunosuppression. However, the higher frequency of early T cell–mediated rejection (TCMR) in belatacept‐treated patients hampered the widespread adoption of costimulation blockade. Here, we applied gene expression analysis and whole‐slide inflammatory cell quantification to assess the impact of belatacept on intragraft immune signature. We studied formalin‐fixed, paraffin‐embedded renal biopsies from 92 patients stratified by histopathologic diagnosis (TCMR, borderline changes, or normal) and immunosuppression regimen (belatacept, CNI). An interaction model was built to explore maintenance treatment‐dependent expression level changes of immune response‐related genes across diagnostic categories of normal, borderline changes, and TCMR. Ninety‐one percent of genes overexpressed in TCMR showed significant correlation with whole section inflammatory load. There were 27 genes that had a positive association with belatacept treatment. These were mostly related to myeloid cells and innate immunity. Genes negatively associated with costimulation blockade (n = 14) could be linked to B‐cell differentiation and proliferation. We concluded that expression levels of genes characteristic of TCMR are strongly interconnected with quantitative changes of the biopsy inflammatory load. Our results might suggest differential involvement of the innate immune system, and an altered B‐cell engagement during TCMR in belatacept‐treated patients relative to CNI‐treated referents.

A bstract: Kidney Precision Medicine Project (KPMP) is building a spatially-specified human tissue atlas at the single-cell resolution with molecular details of the kidney in health and disease. Here, we describe the construction of an integrated reference tissue map of cells, pathways and genes using unaffected regions of nephrectomy tissues and undiseased human biopsies from 55 subjects. We use single-cell and -nucleus transcriptomics, subsegmental laser microdissection bulk transcriptomics and proteomics, near-single-cell proteomics, 3-D nondestructive and CODEX imaging, and spatial metabolomics data to hierarchically identify genes, pathways and cells. Integrated data from these different technologies coherently describe cell types/subtypes within different nephron segments and interstitium. These spatial profiles identify cell-level functional organization of the kidney tissue as indicative of their physiological functions and map different cell subtypes to genes, proteins, metabolites and pathways. Comparison of transcellular sodium reabsorption along the nephron to levels of mRNAs encoding the different sodium transporter genes indicate that mRNA levels are largely congruent with physiological activity.This reference atlas provides an initial framework for molecular classification of kidney disease when multiple molecular mechanisms underlie convergent clinical phenotypes.

The elusive nature of assessing immunological processes in situ in organ transplantation is one of the major impediments to improve diagnostics and treatment. Here, we present a proof-of-concept study using multiplexed in situ hybridization (ISH) (RNAscope) to detect low-abundance cytokines in formalin-fixed paraffin-embedded (FFPE) human transplant kidney biopsies in combination with immunofluorescence (IF) for cell phenotyping. We show that a multiplex IF and ISH (mIFISH) assay is feasible to identify the cellular source of cytokines and chemokines (tumor necrosis factor-α, interferon-γ, and CXCL9) in FFPE transplant kidney biopsies and that quantification of the mRNA and protein signal is also possible at single-cell resolution in the context of tissue complexity. Furthermore, the mIFISH assay allows precise quantitative assessment of tubulitis, one of the key morphological correlates of alloimmune injury. Simultaneous in situ identification and quantification of multiple cellular phenotypes and mRNA expression of proinflammatory cytokines in FFPE tissues offer a novel insight into the biology of alloimmune injury in kidney transplantation and may contribute to improved diagnostic accuracy and patient care.