Persistence of SARS CoV-2 S1 Protein in CD16+ Monocytes in Post-Acute Sequelae of COVID-19 (PASC) up to 15 Months Post-Infection

The recent COVID-19 pandemic is a treatment challenge in the acute infection stage but the recognition of chronic COVID-19 symptoms termed post-acute sequelae SARS-CoV-2 infection (PASC) may affect up to 30% of all infected individuals. The underlying mechanism and source of this distinct immunologic condition three months or more after initial infection remains elusive. Here, we investigated the presence of SARS-CoV-2 S1 protein in 46 individuals. We analyzed T-cell, B-cell, and monocytic subsets in both severe COVID-19 patients and in patients with post-acute sequelae of COVID-19 (PASC). The levels of both intermediate (CD14+, CD16+) and non-classical monocyte (CD14Lo, CD16+) were significantly elevated in PASC patients up to 15 months post-acute infection compared to healthy controls (P=0.002 and P=0.01, respectively). A statistically significant number of non-classical monocytes contained SARS-CoV-2 S1 protein in both severe (P=0.004) and PASC patients (P=0.02) out to 15 months post-infection. Non-classical monocytes were sorted from PASC patients using flow cytometric sorting and the SARS-CoV-2 S1 protein was confirmed by mass spectrometry. Cells from 4 out of 11 severe COVID-19 patients and 1 out of 26 PASC patients contained ddPCR+ peripheral blood mononuclear cells, however, only fragmented SARS-CoV-2 RNA was found in PASC patients. No full length sequences were identified, and no sequences that could account for the observed S1 protein were identified in any patient. That non-classical monocytes may be a source of inflammation in PASC warrants further study.

Ly6c as a New Marker of Mouse Blood Vessels: Qualitative and Quantitative Analyses on Intact and Ischemic Retinas

Ly6c is an antigen commonly used to differentiate between classical and non-classical monocytes/macrophages. Here we show its potential as a marker of the mouse vasculature, particularly of the retinal vascular plexuses. Ly6c was immunodetected in several tissues of C57BL/6 mice using isolectin IB4 as the control of vasculature staining. In the retina, Ly6c expression was analyzed qualitatively and quantitatively in intact, ischemic, and contralateral retinas from 0 to 30 days after the insult. Ly6c expression was observed in all organs and tissues tested, with a brighter signal and more homogeneous staining than the IB4. In the retinas, Ly6c was well expressed, allowing a detailed study of their anatomy. The three retinal plexuses were morphologically different, and from the superficial to the deep one occupied 15 ± 2, 24 ± 7, and 38 ± 1.4 percent of the retinal surface, respectively. In the injured retinas, there was extravasation of the classically activated monocyte/macrophages (Ly6chigh) and the formation of new vessels in the superficial plexus, increasing the area occupied by it to 25 ± 1%. In the contralateral retinas, the superficial plexus area decreased gradually, reaching significance at 30 days, and Ly6c expression progressively disappeared in the intermediate and deep plexuses. Although the role of Ly6c in vascular endothelial cell function is still not completely understood, we demonstrate here that Ly6c can be used as a new specific marker of the mouse vasculature and to assess, qualitatively and quantitatively, vascular changes in health and disease.

TIM3+ TRBV11-2 T cells and IFNγ signature in patrolling monocytes and CD16+ NK cells delineate MIS-C

In rare instances, pediatric SARS-CoV-2 infection results in a novel immunodysregulation syndrome termed multisystem inflammatory syndrome in children (MIS-C). We compared MIS-C immunopathology with severe COVID-19 in adults. MIS-C does not result in pneumocyte damage but is associated with vascular endotheliitis and gastrointestinal epithelial injury. In MIS-C, the cytokine release syndrome is characterized by IFNγ and not type I interferon. Persistence of patrolling monocytes differentiates MIS-C from severe COVID-19, which is dominated by HLA-DRlo classical monocytes. IFNγ levels correlate with granzyme B production in CD16+ NK cells and TIM3 expression on CD38+/HLA-DR+ T cells. Single-cell TCR profiling reveals a skewed TCRβ repertoire enriched for TRBV11-2 and a superantigenic signature in TIM3+/CD38+/HLA-DR+ T cells. Using NicheNet, we confirm IFNγ as a central cytokine in the communication between TIM3+/CD38+/HLA-DR+ T cells, CD16+ NK cells, and patrolling monocytes. Normalization of IFNγ, loss of TIM3, quiescence of CD16+ NK cells, and contraction of patrolling monocytes upon clinical resolution highlight their potential role in MIS-C immunopathogenesis.

Effects of Age and Social Adversity on Immune Cell Populations in a Non-Human Primate Model of Human Aging

Significant hallmarks of aging are immune function decline and rising cumulative inflammation. These immunosenescent signatures are also found in individuals who experience chronic social adversity, independently of age. However, no studies to date have examined how social adversity alters immune function across the lifespan –data that are essential to identify the molecular routes through which social adversity might lead to increased aging-related disease. Over a two-year period, we investigated how age and social adversity (quantified by low social status) affected immunity. We measured immune cell proportions at baseline and their gene regulation after in vitro stimulation with pathogen molecules that stimulated both Th1 and Th2 immune responses in a population of free-ranging rhesus macaques. We first performed flow cytometry on peripheral whole blood to quantify changes on immune cell proportions across the lifespan (n=235) and in animals of different social statuses (n=141). We found significant decreases in CD20+ B cells and CD3+/CD4+ T cell proportions with age, suggesting diminished antibody production and adaptive immune responses in older individuals. Age-associated increases in CD3+/CD8+, CD3+/CD4+/CD25+ T regulatory cells and CD14-/CD16+/HLA-DR+ non-classical monocytes indicated heightened baseline inflammation in older animals. Social adversity recapitulated the effects of aging in CD14+/CD16-/HLA-DR+ classical monocytes, indicating immune deficits in phagocytosis and pathogen clearance in older and lower status individuals. Using RNA-seq, our stimulations (n=1,320) will allow us to identify molecular immune pathways that are disrupted by age and social adversity, similarities in response between age and adversity, and how the effect of adversity varies across the lifespan.

Immune Cell and Adipose Gene Expression Reprogramming in Juvenile Monkeys Born to Health and Weight-Diverse Mothers

Over 93 million Americans are obese and 66 million suffer from metabolic disease. Roughly 40% of obese people do not have metabolic abnormalities (metabolically health obese [MHO]), while approximately 15% of lean do (metabolically unhealthy lean [MUL]). African green monkeys (AGMs) demonstrate naturally occurring obesity and metabolic syndrome (MetS) without diet manipulation, and MetS criteria are heritable. Age-matched maternal AGMs were classified by adjusted MetS criteria ([n=44]; waist >40cm, fasting glucose (FG) >100 mg/dL, SBP/DBP >135/85mmHg, and HDL-c <50mg/dL) and classified as metabolically healthy lean (MHL), MHO, MUL or metabolically unhealthy obese (MUO). Age, weight and sex-matched pre-pubertal juvenile offspring from these mothers were additionally selected (n=9-11/group; ages=1.1-3.4years) for evaluation. We assessed monocyte subtypes by flow cytometry, and subcutaneous adipose gene expression patterns by RNAseq. Non-classical monocytes were increased in obese and unhealthy mothers (MHO p=0.02, MUL p=0.003, MUO p=0.00002) compared to MHL. MUL and MUO juvenile offspring also had more non-classical monocytes compared to MHL (p=0.05 and p=0.07). Monocyte chemoattractant protein-1 (MCP)-1 was measured in plasma and found to be elevated in MUO juveniles (p=0.02). Patterns of increased cytokine and extracellular matrix gene expression were seen in MUL and MUO juveniles’ adipose (6-7/group), mirroring obese and unhealthy mothers’ adipose gene expression patterns. Maternal health and obesity influence offspring immune cells and adipose gene expression prior to weight gain and metabolic disease onset. Our data underscore maternal monocyte and adipose profiles as inherited phenotypes that present prior to adipose expansion and may be targets to improve intergenerational health trajectories.

Data-Driven Analysis of COVID-19 Reveals Persistent Immune Abnormalities in Convalescent Severe Individuals

Severe SARS-CoV-2 infection can trigger uncontrolled innate and adaptive immune responses, which are commonly associated with lymphopenia and increased neutrophil counts. However, whether the immune abnormalities observed in mild to severely infected patients persist into convalescence remains unclear. Herein, comparisons were drawn between the immune responses of COVID-19 infected and convalescent adults. Strikingly, survivors of severe COVID-19 had decreased proportions of NKT and Vδ2 T cells, and increased proportions of low-density neutrophils, IgA+/CD86+/CD123+ non-classical monocytes and hyperactivated HLADR+CD38+ CD8+ T cells, and elevated levels of pro-inflammatory cytokines such as hepatocyte growth factor and vascular endothelial growth factor A, long after virus clearance. Our study suggests potential immune correlates of “long COVID-19”, and defines key cells and cytokines that delineate true and quasi-convalescent states.

Kinetic Changes of Peripheral Blood Monocyte Subsets and Expression of Co-Stimulatory Molecules during Acute Dengue Virus Infection

Monocytes, one of the main target cells for dengue virus (DENV) infection, contribute to the resolution of viremia and to pathogenesis. We performed a longitudinal study by a detailed phenotypic comparison of classical (CD14++CD16−, non-classical (CD14+CD16++) and intermediate (CD14++CD16+) monocyte subsets in blood samples from dengue fever (DF) to the severe dengue hemorrhagic fever (DHF) and healthy individuals. Various costimulatory molecules of CD40, CD80, CD86 and inducible costimulatory ligand (ICOSL) expressed on these three monocyte subsets were also analyzed. DENV-infected patients showed an increase in the frequency of intermediate monocytes and a decrease in the classical monocytes when compared to healthy individuals. Although these differences did not correlate with disease severity, changes during the early phase of infection gradually returned to normal in the defervescence phase. Moreover, decreased frequency of classical monocytes was associated with a significant up-regulation of co-stimulatory molecules CD40, CD86 and ICOSL. Kinetics of these co-stimulatory molecule-expressing classical monocytes showed different patterns throughout the sampling times of acute DENV infection. Different distribution of monocyte subsets and their co-stimulatory molecules in the peripheral blood during acute infection might exacerbate immune responses like cytokine storms and ADE, and future studies on intracellular molecular pathways utilized by these monocyte linages are warranted.

Inflammasome-Primed Myeloid Cells Maintain a Pro-Tumor Microenvironment in Multiple Myeloma

Background: Multiple myeloma (MM) disease progression is influenced by signals from the bone marrow (BM) microenvironment. Recently, we showed that the MM BM is characterized by inflammatory mesenchymal stromal cells (iMSCs) that transcribe MM survival factors and are predicted to recruit proliferating myeloma cells via CCL2-CCR2 interactions (de Jong et al. Nat Immunol. 2021). iMSCs also transcribed high levels of chemokines that can bind to CXCR1 and 2. Myeloid cells are known to express CXCR1/2, and have been implicated in both pro- and anti-tumor responses in various malignancies. Therefore, we hypothesized that iMSCs attract and influence myeloid populations in the MM BM. Results: Using flow cytometry, we verified expression of CXCR1/2 on myeloid cell populations in the BM of 5 newly diagnosed MM (NDMM) patients. CD15 + neutrophils were the most dominant population expressing these receptors, as 22.4% (± 9.8%) of cells expressed CXCR2 alone, and 72.6% (± 8.0%) expressed both CXCR1 and CXCR2. CD14 + monocytes only expressed CXCR2 (86.9% ± 15.8%). Importantly, less than 1% of myeloma cells expressed these receptors (n = 17 NDMM). As these findings suggested neutrophils and monocytes as potential targets of iMSC-mediated chemotaxis, we set out to identify MM-associated alterations in this population by performing single cell RNA sequencing of the full neutrophilic and monocytic lineages (n = 5 NDMM and 2 controls). In line with our flow cytometric data, CXCR1 transcripts were absent in monocytes, while CXCR2 was transcribed by classical monocytes of both myeloma patients and controls. Interestingly, CXCR1 and CXCR2 transcription was increased in mature neutrophils of MM patients compared to controls. Additionally, both mature classical monocytes as well as mature neutrophils of MM patients had an activated transcriptome as defined by increased transcription of C3AR1, SLPI, and IL6R, the plasma cell supportive factor TNFSF13B (encoding BAFF), and the inflammatory cytokines IL1B and IL18. Transcription of IL1B and IL18 can be regulated by pattern-recognition receptors (PRRs) binding damage-associated molecular patterns (DAMPs) resulting from e.g. matrix breakdown. Transcription of PRRs as TLR1, 2 and 4 was increased in mature neutrophils and classical monocytes of MM patients compared to controls. Secretion of IL-1β and IL-18 relies on the cleavage of pro-forms of these cytokines by the inflammasome, a multiprotein complex that is assembled in response to alarmins. Transcription of inflammasome components PYCARD, NLRP3 and CASP1 was increased in mature neutrophils and classical monocytes of patients with MM. Additionally, protein levels of both IL-18 and IL-1β are increased in BM plasma from MM patients, implicating activated neutrophils and monocytes as a potential sources of these cytokines. Conclusion: In MM, mature neutrophils and classical monocytes are activated and might interact with iMSCs via CXCR1 and/or 2. Moreover, these myeloid cells are inflammasome-primed and are likely to be sources of the increased IL-1β levels in the MM BM. Therefore, myeloid cells and iMSCs may form a feed-forward loop in which myeloid cells contribute to a pro-MM environment by maintaining iMSC and by directly providing BAFF to tumor cells. Disclosures Broyl: Celgene/BMS: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Sonneveld: Janssen: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; SkylineDx: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene/BMS: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.