Imprinted immune abnormalities in liver transplant patients cured of hepatitis C with antiviral drugs.

Chronic HCV infection induces interferon and dysregulates immune responses through inflammation and chronic antigenic stimulation. Antiviral drugs can cure HCV, providing a unique opportunity to examine the immunological restoration that does and does not occur when a chronic viral infection is eradicated. We quantified blood cytokines levels and used mass cytometry to immunophenotype peripheral blood mononuclear cells before and after HCV cure in 2 groups of patients and controls. At baseline, serum interferon α and soluble CD163 (a macrophage product) were elevated in both liver transplant and nonliver transplant patients compared to controls; the frequencies of several peripheral blood mononuclear cell populations differed from controls; and programmed death protein 1-positivity was increased in nearly all T cell subsets. Many abnormalities persisted after HCV cure, including elevated programmed death protein 1 expression on CD4 naïve and central memory T cells, elevated soluble CD163, and expansion of the plasmablast/plasma cell compartment. Several myeloid-lineage subsets, including Ag-presenting dendritic cells, remained dysregulated. In mechanistic studies, interferon α treatment increased programmed death protein 1 on human T cells and increased T cell receptor signaling. The data identify immunological abnormalities that persist after curative HCV treatment. Before cure, high levels of interferon α may stimulate programmed death protein 1 expression on human T cells, causing persistent functional changes.

Circulating myeloid-derived MMP8 in stress susceptibility and depression.

Psychosocial stress has profound effects on the body, including the immune system and the brain1,2. Although a large number of pre-clinical and clinical studies have linked peripheral immune system alterations to stress-related disorders such as major depressive disorder (MDD)3, the underlying mechanisms are not well understood. Here we show that expression of a circulating myeloid cell-specific proteinase, matrix metalloproteinase 8 (MMP8), is increased in the serum of humans with MDD as well as in stress-susceptible mice following chronic social defeat stress (CSDS). In mice, we show that this increase leads to alterations in extracellular space and neurophysiological changes in the nucleus accumbens (NAc), as well as altered social behaviour. Using a combination of mass cytometry and single-cell RNA sequencing, we performed high-dimensional phenotyping of immune cells in circulation and in the brain and demonstrate that peripheral monocytes are strongly affected by stress. In stress-susceptible mice, both circulating monocytes and monocytes that traffic to the brain showed increased Mmp8 expression following chronic social defeat stress. We further demonstrate that circulating MMP8 directly infiltrates the NAc parenchyma and controls the ultrastructure of the extracellular space. Depleting MMP8 prevented stress-induced social avoidance behaviour and alterations in NAc neurophysiology and extracellular space. Collectively, these data establish a mechanism by which peripheral immune factors can affect central nervous system function and behaviour in the context of stress. Targeting specific peripheral immune cell-derived matrix metalloproteinases could constitute novel therapeutic targets for stress-related neuropsychiatric disorders.

Immunophenotypic correlates of sustained MRD negativity in patients with multiple myeloma.

The role of the immune microenvironment in maintaining disease remission in patients with multiple myeloma (MM) is not well understood. In this study, we comprehensively profile the immune system in patients with newly diagnosed MM receiving continuous lenalidomide maintenance therapy with the aim of discovering correlates of long-term treatment response. Leveraging single-cell RNA sequencing and T cell receptor ß sequencing of the peripheral blood and CyTOF mass cytometry of the bone marrow, we longitudinally characterize the immune landscape in 23 patients before and one year after lenalidomide exposure. We compare patients achieving sustained minimal residual disease (MRD) negativity to patients who never achieved or were unable to maintain MRD negativity. We observe that the composition of the immune microenvironment in both the blood and the marrow varied substantially according to both MRD negative status and history of autologous stem cell transplant, supporting the hypothesis that the immune microenvironment influences the depth and duration of treatment response.

Systems immunology-based drug repurposing framework to target inflammation in atherosclerosis.

The development of new immunotherapies to treat the inflammatory mechanisms that sustain atherosclerotic cardiovascular disease (ASCVD) is urgently needed. Herein, we present a path to drug repurposing to identify immunotherapies for ASCVD. The integration of time-of-flight mass cytometry and RNA sequencing identified unique inflammatory signatures in peripheral blood mononuclear cells stimulated with ASCVD plasma. By comparing these inflammatory signatures to large-scale gene expression data from the LINCS L1000 dataset, we identified drugs that could reverse this inflammatory response. Ex vivo screens, using human samples, showed that saracatinib-a phase 2a-ready SRC and ABL inhibitor-reversed the inflammatory responses induced by ASCVD plasma. In Apoe-/- mice, saracatinib reduced atherosclerosis progression by reprogramming reparative macrophages. In a rabbit model of advanced atherosclerosis, saracatinib reduced plaque inflammation measured by [18F] fluorodeoxyglucose positron emission tomography-magnetic resonance imaging. Here we show a systems immunology-driven drug repurposing with a preclinical validation strategy to aid the development of cardiovascular immunotherapies.

Transitional dendritic cells are distinct from conventional DC2 precursors and mediate proinflammatory antiviral responses.

High-dimensional approaches have revealed heterogeneity amongst dendritic cells (DCs), including a population of transitional DCs (tDCs) in mice and humans. However, the origin and relationship of tDCs to other DC subsets has been unclear. Here we show that tDCs are distinct from other well-characterized DCs and conventional DC precursors (pre-cDCs). We demonstrate that tDCs originate from bone marrow progenitors shared with plasmacytoid DCs (pDCs). In the periphery, tDCs contribute to the pool of ESAM+ type 2 DCs (DC2s), and these DC2s have pDC-related developmental features. Different from pre-cDCs, tDCs have less turnover, capture antigen, respond to stimuli and activate antigen-specific naïve T cells, all characteristics of differentiated DCs. Different from pDCs, viral sensing by tDCs results in IL-1ß secretion and fatal immune pathology in a murine coronavirus model. Our findings suggest that tDCs are a distinct pDC-related subset with a DC2 differentiation potential and unique proinflammatory function during viral infections.

Intratumoral dendritic cell-CD4+ T helper cell niches enable CD8+ T cell differentiation following PD-1 blockade in hepatocellular carcinoma.

Despite no apparent defects in T cell priming and recruitment to tumors, a large subset of T cell rich tumors fail to respond to immune checkpoint blockade (ICB). We leveraged a neoadjuvant anti-PD-1 trial in patients with hepatocellular carcinoma (HCC), as well as additional samples collected from patients treated off-label, to explore correlates of response to ICB within T cell-rich tumors. We show that ICB response correlated with the clonal expansion of intratumoral CXCL13+CH25H+IL-21+PD-1+CD4+ T helper cells ("CXCL13+ TH") and Granzyme K+ PD-1+ effector-like CD8+ T cells, whereas terminally exhausted CD39hiTOXhiPD-1hiCD8+ T cells dominated in nonresponders. CD4+ and CD8+ T cell clones that expanded post-treatment were found in pretreatment biopsies. Notably, PD-1+TCF-1+ (Progenitor-exhausted) CD8+ T cells shared clones mainly with effector-like cells in responders or terminally exhausted cells in nonresponders, suggesting that local CD8+ T cell differentiation occurs upon ICB. We found that these Progenitor CD8+ T cells interact with CXCL13+ TH within cellular triads around dendritic cells enriched in maturation and regulatory molecules, or "mregDC". These results suggest that discrete intratumoral niches that include mregDC and CXCL13+ TH control the differentiation of tumor-specific Progenitor exhasuted CD8+ T cells following ICB.

Peripheral immune-derived matrix metalloproteinase promotes stress susceptibility.

Psychosocial stress has profound effects on the body, including the peripheral immune system and the brain1,2. Although a large number of pre-clinical and clinical studies have linked peripheral immune system alterations to stress-related disorders such as major depressive disorder (MDD)3,4,5, the underlying mechanisms are not well understood. Here we show that a peripheral myeloid cell-specific proteinase, matrix metalloproteinase 8 (MMP8), is elevated in serum of subjects with MDD as well as in stress-susceptible (SUS) mice following chronic social defeat stress (CSDS). In mice, we show that this increase leads to alterations in extracellular space and neurophysiological changes in the nucleus accumbens (NAc), thereby altering social behaviour. Using a combination of mass cytometry and single-cell RNA-sequencing, we performed high-dimensional phenotyping of immune cells in circulation and brain and demonstrate that peripheral monocytes are strongly affected by stress. Both peripheral and brain-infiltrating monocytes of SUS mice showed increased Mmp8 expression following CSDS. We further demonstrate that peripheral MMP8 directly infiltrates the NAc parenchyma to control the ultrastructure of the extracellular space. Depleting MMP8 prevented stress-induced social avoidance behaviour and alterations in NAc neurophysiology and extracellular space. Collectively, these data establish a novel mechanism by which peripheral immune factors can affect central nervous system function and behaviour in the context of stress. Targeting specific peripheral immune cell-derived matrix metalloproteinases could constitute novel therapeutic targets for stress-related neuropsychiatric disorders.

Tissue adaptation and clonal segregation of human memory T cells in barrier sites.

T lymphocytes migrate to barrier sites after exposure to pathogens, providing localized immunity and long-term protection. Here, we obtained blood and tissues from human organ donors to examine T cells across major barrier sites (skin, lung, jejunum), associated lymph nodes, lymphoid organs (spleen, bone marrow), and in circulation. By integrating single-cell protein and transcriptome profiling, we demonstrate that human barrier sites contain tissue-resident memory T (TRM) cells that exhibit site-adapted profiles for residency, homing and function distinct from circulating memory T cells. Incorporating T cell receptor and transcriptome analysis, we show that circulating memory T cells are highly expanded, display extensive overlap between sites and exhibit effector and cytolytic functional profiles, while TRM clones exhibit site-specific expansions and distinct functional capacities. Together, our findings indicate that circulating T cells are more disseminated and differentiated, while TRM cells exhibit tissue-specific adaptation and clonal segregation, suggesting that strategies to promote barrier immunity require tissue targeting.

Cross center single-cell RNA sequencing study of the immune microenvironment in rapid progressing multiple myeloma.

Despite advancements in understanding the pathophysiology of Multiple Myeloma (MM), the cause of rapid progressing disease in a subset of patients is still unclear. MM's progression is facilitated by complex interactions with the surrounding bone marrow (BM) cells, forming a microenvironment that supports tumor growth and drug resistance. Understanding the immune microenvironment is key to identifying factors that promote rapid progression of MM. To accomplish this, we performed a multi-center single-cell RNA sequencing (scRNA-seq) study on 102,207 cells from 48 CD138- BM samples collected at the time of disease diagnosis from 18 patients with either rapid progressing (progression-free survival (PFS) < 18 months) or non-progressing (PFS > 4 years) disease. Comparative analysis of data from three centers demonstrated similar transcriptome profiles and cell type distributions, indicating subtle technical variation in scRNA-seq, opening avenues for an expanded multicenter trial. Rapid progressors depicted significantly higher enrichment of GZMK+ and TIGIT+ exhausted CD8+ T-cells (P = 0.022) along with decreased expression of cytolytic markers (PRF1, GZMB, GNLY). We also observed a significantly higher enrichment of M2 tolerogenic macrophages in rapid progressors and activation of pro-proliferative signaling pathways, such as BAFF, CCL, and IL16. On the other hand, non-progressive patients depicted higher enrichment for immature B Cells (i.e., Pre/Pro B cells), with elevated expression for markers of B cell development (IGLL1, SOX4, DNTT). This multi-center study identifies the enrichment of various pro-tumorigenic cell populations and pathways in those with rapid progressing disease and further validates the robustness of scRNA-seq data generated at different study centers.

A shift in lung macrophage composition is associated with COVID-19 severity and recovery.

Although it has been more than 2 years since the start of the coronavirus disease 2019 (COVID-19) pandemic, COVID-19 continues to be a worldwide health crisis. Despite the development of preventive vaccines, therapies to treat COVID-19 and other inflammatory diseases remain a major unmet need in medicine. Our study sought to identify drivers of disease severity and mortality to develop tailored immunotherapy strategies to halt disease progression. We assembled the Mount Sinai COVID-19 Biobank, which was composed of almost 600 hospitalized patients followed longitudinally through the peak of the pandemic in 2020. Moderate disease and survival were associated with a stronger antigen presentation and effector T cell signature. In contrast, severe disease and death were associated with an altered antigen presentation signature, increased numbers of inflammatory immature myeloid cells, and extrafollicular activated B cells that have been previously associated with autoantibody formation. In severely ill patients with COVID-19, lung tissue-resident alveolar macrophages not only were drastically depleted but also had an altered antigen presentation signature, which coincided with an influx of inflammatory monocytes and monocyte-derived macrophages. In addition, we found that the size of the alveolar macrophage pool correlated with patient outcome and that alveolar macrophage numbers and functionality were restored to homeostasis in patients who recovered from COVID-19. These data suggest that local and systemic myeloid cell dysregulation are drivers of COVID-19 severity and modulation of alveolar macrophage numbers and activity in the lung may be a viable therapeutic strategy for the treatment of critical inflammatory lung diseases.

High Dimensional Immune Profiling of Smoldering Multiple Myeloma Distinguishes Distinct Tumor Microenvironments.

BACKGROUND: Multiple myeloma (MM) is a malignancy of plasma cells that arises from premalignant Monoclonal Gammopathy of Undetermined Significance (MGUS) and often progresses through an asymptomatic Smoldering (SMM) phase. Understanding the interactions between abnormal clonal plasma cells and the tumor microenvironment (TME) in the early disease states (MGUS, SMM) may inform risk assessment and therapy. PATIENTS AND METHODS: We performed high dimensional immunologic analysis of bone marrow specimens from 73 subjects with SMM by mass cytometry and T cell receptor sequencing of CD138-depleted bone marrow (BM) mononuclear cells, and proteomics and seromic profiling of BM plasma. Analysis of individual assay data identified self-organizing subgroups of SMM patients. We then applied novel bioinformatic methods to integrate data from pairs, trios, and quartets of assays. RESULTS: Mass cytometry, TCRSeq and proteomics identified three taxa (sing. taxon) of subjects that shared common characteristics across all three assays. Differential levels of BM plasma pleiotropin (PTN) and BM T cells and their productive clonality emerged as strong distinguishing factors among these taxa. CONCLUSION: These results suggest that the continuum from MGUS to MM does not consist of a single pathway in the TME, and that complex interactions between myeloma cells and the TME may ultimately determine progression and inform clinical management.

Spatial Positioning and Matrix Programs of Cancer-Associated Fibroblasts Promote T-cell Exclusion in Human Lung Tumors.

It is currently accepted that cancer-associated fibroblasts (CAF) participate in T-cell exclusion from tumor nests. To unbiasedly test this, we used single-cell RNA sequencing coupled with multiplex imaging on a large cohort of lung tumors. We identified four main CAF populations, two of which are associated with T-cell exclusion: (i) MYH11+αSMA+ CAF, which are present in early-stage tumors and form a single cell layer lining cancer aggregates, and (ii) FAP+αSMA+ CAF, which appear in more advanced tumors and organize in patches within the stroma or in multiple layers around tumor nests. Both populations orchestrate a particular structural tissue organization through dense and aligned fiber deposition compared with T cell-permissive CAF. Yet they produce distinct matrix molecules, including collagen IV (MYH11+αSMA+ CAF) and collagen XI/XII (FAP+αSMA+ CAF). Hereby, we uncovered unique molecular programs of CAF driving T-cell marginalization, whose targeting should increase immunotherapy efficacy in patients bearing T cell-excluded tumors. SIGNIFICANCE: The cellular and molecular programs driving T-cell marginalization in solid tumors remain unclear. Here, we describe two CAF populations associated with T-cell exclusion in human lung tumors. We demonstrate the importance of pairing molecular and spatial analysis of the tumor microenvironment, a prerequisite to developing new strategies targeting T cell-excluding CAF. See related commentary by Sherman, p. 2501. This article is highlighted in the In This Issue feature, p. 2483.

Unraveling function and diversity of bacterial lectins in the human microbiome.

The mechanisms by which commensal organisms affect human physiology remain poorly understood. Lectins are non-enzymatic carbohydrate binding proteins that all organisms employ as part of establishing a niche, evading host-defenses and protecting against pathogens. Although lectins have been extensively studied in plants, bacterial pathogens and human immune cells for their role in disease pathophysiology and as therapeutics, the role of bacterial lectins in the human microbiome is largely unexplored. Here we report on the characterization of a lectin produced by a common human associated bacterium that interacts with myeloid cells in the blood and intestine. In mouse and cell-based models, we demonstrate that this lectin induces distinct immunologic responses in peripheral and intestinal leukocytes and that these responses are specific to monocytes, macrophages and dendritic cells. Our analysis of human microbiota sequencing data reveal thousands of unique sequences that are predicted to encode lectins, many of which are highly prevalent in the human microbiome yet completely uncharacterized. Based on the varied domain architectures of these lectins we predict they will have diverse effects on the human host. The systematic investigation of lectins in the human microbiome should improve our understanding of human health and provide new therapeutic opportunities.

Mass Cytometry Analysis of Whole Blood Response to an Allergen.

Mass cytometry allows for the use of highly multiplexed antibody panels due to the lack of spill-over between channels detected by mass spectrometry. An advantage over fluorescence cytometry is the relative lack of background, which provides excellent resolution for detection of phosphoproteins and quantification of cell signaling. We have applied mass cytometry to the analysis of whole blood staining after ex vivo stimulation with peanut allergen (Tordesillas et al., J Allergy Clin Immunol 138:1741-4.e9, 2016). This allows for high-dimensional analysis of basophil activation, and analysis of the entire composition of the blood compartment in response to allergen exposure. Here, we describe our optimized protocol for activation and staining of whole blood for mass cytometry analysis that is currently in use in multicenter clinical trials. The protocol can be easily adopted to analyze blood leukocytes in other diseases, including asthma.

Neutralizing Anti-Granulocyte Macrophage-Colony Stimulating Factor Autoantibodies Recognize Post-Translational Glycosylations on Granulocyte Macrophage-Colony Stimulating Factor Years Before Diagnosis and Predict Complicated Crohn's Disease.

BACKGROUND & AIMS: Anti-granulocyte macrophage-colony stimulating factor autoantibodies (aGMAbs) are detected in patients with ileal Crohn's disease (CD). Their induction and mode of action during or before disease are not well understood. We aimed to investigate the underlying mechanisms associated with aGMAb induction, from functional orientation to recognized epitopes, for their impact on intestinal immune homeostasis and use as a predictive biomarker for complicated CD. METHODS: We characterized using enzyme-linked immunosorbent assay naturally occurring aGMAbs in longitudinal serum samples from patients archived before the diagnosis of CD (n = 220) as well as from 400 healthy individuals (matched controls) as part of the US Defense Medical Surveillance System. We used biochemical, cellular, and transcriptional analysis to uncover a mechanism that governs the impaired immune balance in CD mucosa after diagnosis. RESULTS: Neutralizing aGMAbs were found to be specific for post-translational glycosylation on granulocyte macrophage-colony stimulating factor (GM-CSF), detectable years before diagnosis, and associated with complicated CD at presentation. Glycosylation of GM-CSF was altered in patients with CD, and aGMAb affected myeloid homeostasis and promoted group 1 innate lymphoid cells. Perturbations in immune homeostasis preceded the diagnosis in the serum of patients with CD presenting with aGMAb and were detectable in the noninflamed CD mucosa. CONCLUSIONS: Anti-GMAbs predict the diagnosis of complicated CD long before the diagnosis of disease, recognize uniquely glycosylated epitopes, and impair myeloid cell and innate lymphoid cell balance associated with altered intestinal immune homeostasis.

Experimental Validation and Evaluation of the Bending Properties of Additively Manufactured Metallic Cellular Scaffold Structures for Bone Tissue Engineering.

The availability of additive manufacturing enables the fabrication of cellular bone tissue engineering scaffolds with a wide range of structural and architectural possibilities. The purpose of bone tissue engineering scaffolds is to repair critical size bone defects due to extreme traumas, tumors, or infections. This research study presented the experimental validation and evaluation of the bending properties of optimized bone scaffolds with an elastic modulus that is equivalent to the young's modulus of the cortical bone. The specimens were manufactured using laser powder bed fusion technology. The morphological properties of the manufactured specimens were evaluated using both dry weighing and Archimedes techniques, and minor variations in the relative densities were observed in comparison with the computer-aided design files. The bending modulus of the cubic and diagonal scaffolds were experimentally investigated using a three-point bending test, and the results were found to agree with the numerical findings. A higher bending modulus was observed in the diagonal scaffold design. The diagonal scaffold was substantially tougher, with considerably higher energy absorption before fracture. The shear modulus of the diagonal scaffold was observed to be significantly higher than the cubic scaffold. Due to bending, the pores at the top side of the diagonal scaffold were heavily compressed compared to the cubic scaffold due to the extensive plastic deformation occurring in diagonal scaffolds and the rapid fracture of struts in the tension side of the cubic scaffold. The failure in struts in tension showed signs of ductility as necking was observed in fractured struts. Moreover, the fractured surface was observed to be rough and dull as opposed to being smooth and bright like in brittle fractures. Dimple fracture was observed using scanning electron microscopy as a result of microvoids emerging in places of high localized plastic deformation. Finally, a comparison of the mechanical properties of the studied BTE scaffolds with the cortical bone properties under longitudinal and transverse loading was investigated. In conclusion, we showed the capabilities of finite element analysis and additive manufacturing in designing and manufacturing promising scaffold designs that can replace bone segments in the human body.

Design and Validation of Additively Manufactured Metallic Cellular Scaffold Structures for Bone Tissue Engineering.

Bone-related defects that cannot heal without significant surgical intervention represent a significant challenge in the orthopedic field. The use of implants for these critical-sized bone defects is being explored to address the limitations of autograft and allograft options. Three-dimensional cellular structures, or bone scaffolds, provide mechanical support and promote bone tissue formation by acting as a template for bone growth. Stress shielding in bones is the reduction in bone density caused by the difference in stiffness between the scaffold and the surrounding bone tissue. This study aimed to reduce the stress shielding and introduce a cellular metal structure to replace defected bone by designing and producing a numerically optimized bone scaffold with an elastic modulus of 15 GPa, which matches the human's cortical bone modulus. Cubic cell and diagonal cell designs were explored. Strut and cell dimensions were numerically optimized to achieve the desired structural modulus. The resulting scaffold designs were produced from stainless steel using laser powder bed fusion (LPBF). Finite element analysis (FEA) models were validated through compression testing of the printed scaffold designs. The structural configuration of the scaffolds was characterized with scanning electron microscopy (SEM). Cellular struts were found to have minimal internal porosity and rough surfaces. Strut dimensions of the printed scaffolds were found to have variations with the optimized computer-aided design (CAD) models. The experimental results, as expected, were slightly less than FEA results due to structural relative density variations in the scaffolds. Failure of the structures was stretch-dominated for the cubic scaffold and bending-dominated for the diagonal scaffold. The torsional and bending stiffnesses were numerically evaluated and showed higher bending and torsional moduli for the diagonal scaffold. The study successfully contributed to minimizing stress shielding in bone tissue engineering. The study also produced an innovative metal cellular structure that can replace large bone segments anywhere in the human body.

Spatial CRISPR genomics identifies regulators of the tumor microenvironment.

While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying extracellular gene functions, particularly in the tissue context. Here, we developed an approach for spatial functional genomics called Perturb-map. We applied Perturb-map to knock out dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Moreover, we paired Perturb-map and spatial transcriptomics for unbiased analysis of CRISPR-edited tumors. We found that in Tgfbr2 knockout tumors, the tumor microenvironment (TME) was converted to a fibro-mucinous state, and T cells excluded, concomitant with upregulated TGFß and TGFß-mediated fibroblast activation, indicating that TGFß-receptor loss on cancer cells increased TGFß bioavailability and its immunosuppressive effects on the TME. These studies establish Perturb-map for functional genomics within the tissue at single-cell resolution with spatial architecture preserved and provide insight into how TGFß responsiveness of cancer cells can affect the TME.

Limited extent and consequences of pancreatic SARS-CoV-2 infection.

Concerns that infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), may cause new-onset diabetes persist in an evolving research landscape, and precise risk assessment is hampered by, at times, conflicting evidence. Here, leveraging comprehensive single-cell analyses of in vitro SARS-CoV-2-infected human pancreatic islets, we demonstrate that productive infection is strictly dependent on the SARS-CoV-2 entry receptor ACE2 and targets practically all pancreatic cell types. Importantly, the infection remains highly circumscribed and largely non-cytopathic and, despite a high viral burden in infected subsets, promotes only modest cellular perturbations and inflammatory responses. Similar experimental outcomes are also observed after islet infection with endemic coronaviruses. Thus, the limits of pancreatic SARS-CoV-2 infection, even under in vitro conditions of enhanced virus exposure, challenge the proposition that in vivo targeting of ß cells by SARS-CoV-2 precipitates new-onset diabetes. Whether restricted pancreatic damage and immunological alterations accrued by COVID-19 increase cumulative diabetes risk, however, remains to be evaluated.