Coordinated Cellular Neighborhoods Orchestrate Antitumoral Immunity at the Colorectal Cancer Invasive Front.

Antitumoral immunity requires organized, spatially nuanced interactions between components of the immune tumor microenvironment (iTME). Understanding this coordinated behavior in effective versus ineffective tumor control will advance immunotherapies. We re-engineered co-detection by indexing (CODEX) for paraffin-embedded tissue microarrays, enabling simultaneous profiling of 140 tissue regions from 35 advanced-stage colorectal cancer (CRC) patients with 56 protein markers. We identified nine conserved, distinct cellular neighborhoods (CNs)-a collection of components characteristic of the CRC iTME. Enrichment of PD-1+CD4+ T cells only within a granulocyte CN positively correlated with survival in a high-risk patient subset. Coupling of tumor and immune CNs, fragmentation of T cell and macrophage CNs, and disruption of inter-CN communication was associated with inferior outcomes. This study provides a framework for interrogating how complex biological processes, such as antitumoral immunity, occur through concerted actions of cells and spatial domains.

Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics.

Ebola virus (EBOV) causes epidemics with high mortality yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. Here, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cells during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, finding that immature, proliferative monocyte-lineage cells with reduced antigen-presentation capacity replace conventional monocyte subsets, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying intracellular viral RNA, we identify molecular determinants of tropism among circulating immune cells and examine temporal dynamics in viral and host gene expression. Within infected cells, EBOV downregulates STAT1 mRNA and interferon signaling, and it upregulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating pathways the virus manipulates for its replication. This study sheds light on EBOV tropism, replication dynamics, and elicited immune response and provides a framework for characterizing host-virus interactions under maximum containment.

Robust single-cell matching and multimodal analysis using shared and distinct features.

The ability to align individual cellular information from multiple experimental sources is fundamental for a systems-level understanding of biological processes. However, currently available tools are mainly designed for single-cell transcriptomics matching and integration, and generally rely on a large number of shared features across datasets for cell matching. This approach underperforms when applied to single-cell proteomic datasets due to the limited number of parameters simultaneously accessed and lack of shared markers across these experiments. Here, we introduce a cell-matching algorithm, matching with partial overlap (MARIO) that accounts for both shared and distinct features, while consisting of vital filtering steps to avoid suboptimal matching. MARIO accurately matches and integrates data from different single-cell proteomic and multimodal methods, including spatial techniques and has cross-species capabilities. MARIO robustly matched tissue macrophages identified from COVID-19 lung autopsies via codetection by indexing imaging to macrophages recovered from COVID-19 bronchoalveolar lavage fluid by cellular indexing of transcriptomes and epitopes by sequencing, revealing unique immune responses within the lung microenvironment of patients with COVID.

Immunogenicity of rVSVΔG-ZEBOV-GP Ebola vaccination in exposed and potentially exposed persons in the Democratic Republic of the Congo.

Despite more than 300,000 rVSVΔG-ZEBOV-glycoprotein (GP) vaccine doses having been administered during Ebola virus disease (EVD) outbreaks in the Democratic Republic of the Congo (DRC) between 2018 and 2020, seroepidemiologic studies of vaccinated Congolese populations are lacking. This study examines the antibody response at 21 d and 6 mo postvaccination after single-dose rVSVΔG-ZEBOV-GP vaccination among EVD-exposed and potentially exposed populations in the DRC. We conducted a longitudinal cohort study of 608 rVSVΔG-ZEBOV-GP-vaccinated individuals during an EVD outbreak in North Kivu Province, DRC. Participants provided questionnaires and blood samples at three study visits (day 0, visit 1; day 21, visit 2; and month 6, visit 3). Anti-GP immunoglobulin G (IgG) antibody titers were measured in serum by the Filovirus Animal Nonclinical Group anti-Ebola virus GP IgG enzyme-linked immunosorbent assay. Antibody response was defined as an antibody titer that had increased fourfold from visit 1 to visit 2 and was above four times the lower limit of quantification at visit 2; antibody persistence was defined as a similar increase from visit 1 to visit 3. We then examined demographics for associations with follow-up antibody titers using generalized linear mixed models. A majority of the sample, 87.2%, had an antibody response at visit 2, and 95.6% demonstrated antibody persistence at visit 3. Being female and of young age was predictive of a higher antibody titer postvaccination. Antibody response and persistence after Ebola vaccination was robust in this cohort, confirming findings from outside of the DRC.

ADAM17 stabilizes its interacting partner inactive Rhomboid 2 (iRhom2) but not inactive Rhomboid 1 (iRhom1).

The metalloprotease ADAM17 (a disintegrin and metalloprotease 17) is a key regulator of tumor necrosis factor α (TNFα), interleukin 6 receptor (IL-6R), and epidermal growth factor receptor (EGFR) signaling. ADAM17 maturation and function depend on the seven-membrane-spanning inactive rhomboid-like proteins 1 and 2 (iRhom1/2 or Rhbdf1/2). Most studies to date have focused on overexpressed iRhom1 and -2, so only little is known about the properties of the endogenous proteins. Here, we show that endogenous iRhom1 and -2 can be cell surface-biotinylated on mouse embryonic fibroblasts (mEFs), revealing that endogenous iRhom1 and -2 proteins are present on the cell surface and that iRhom2 also is present on the surface of lipopolysaccharide-stimulated primary bone marrow-derived macrophages. Interestingly, very little, if any, iRhom2 was detectable in mEFs or bone marrow-derived macrophages lacking ADAM17, suggesting that iRhom2 is stabilized by ADAM17. By contrast, the levels of iRhom1 were slightly increased in the absence of ADAM17 in mEFs, indicating that its stability does not depend on ADAM17. These findings support a model in which iRhom2 and ADAM17 are obligate binding partners and indicate that iRhom2 stability requires the presence of ADAM17, whereas iRhom1 is stable in the absence of ADAM17.

Performance of BioFire array or QuickVue influenza A + B test versus a validation qPCR assay for detection of influenza A during a volunteer A/California/2009/H1N1 challenge study.

BACKGROUND: Influenza places a significant burden on global health and economics. Individual case management and public health efforts to mitigate the spread of influenza are both strongly impacted by our ability to accurately and efficiently detect influenza viruses in clinical samples. Therefore, it is important to understand the performance characteristics of available assays to detect influenza in a variety of settings. We provide the first report of relative performance between two products marketed to streamline detection of influenza virus in the context of a highly controlled volunteer influenza challenge study. METHODS: Nasopharyngeal swab samples were collected during a controlled A/California/2009/H1N1 influenza challenge study and analyzed for detection of virus shedding using a validated qRT-PCR (qPCR) assay, a sample-to-answer qRT-PCR device (BioMerieux BioFire FilmArray RP), and an immunoassay based rapid test kit (Quidel QuickVue Influenza A + B Test). RESULTS: Relative to qPCR, the sensitivity and specificity of the BioFire assay was 72.1% [63.7-79.5%, 95% confidence interval (CI)] and 93.5% (89.3-96.4%, 95% CI) respectively. For the QuickVue rapid test the sensitivity was 8.5% (4.8-13.7%, 95% CI) and specificity was 99.2% (95.6-100%, 95% CI). CONCLUSION: Relative to qPCR, the BioFire assay had superior performance compared to rapid test in the context of a controlled influenza challenge study.

Functional comparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs. Lymphoprep Tubes.

BACKGROUND: Cryopreserved human peripheral blood mononuclear cells (PBMCs) are a commonly used sample type for a variety of immunological assays. Many factors can affect the quality of PBMCs, and careful consideration and validation of an appropriate PBMC isolation and cryopreservation method is important for well-designed clinical studies. A major point of divergence in PBMC isolation protocols is the collection of blood, either directly into vacutainers pre-filled with density gradient medium or the use of conical tubes containing a porous barrier to separate the density gradient medium from blood. To address potential differences in sample outcome, we isolated, cryopreserved, and compared PBMCs using parallel protocols differing only in the use of one of two common tube types for isolation. METHODS: Whole blood was processed in parallel using both Cell Preparation Tubes™ (CPT, BD Biosciences) and Lymphoprep™ Tubes (Axis-Shield) and assessed for yield and viability prior to cryopreservation. After thawing, samples were further examined by flow cytometry for cell yield, cell viability, frequency of 10 cell subsets, and capacity for stimulation-dependent CD4+ and CD8+ T cell intracellular cytokine production. RESULTS: No significant differences in cell recovery, viability, frequency of immune cell subsets, or T cell functionality between PBMC samples isolated using CPT or Lymphoprep tubes were identified. CONCLUSION: CPT and Lymphoprep tubes are effective and comparable methods for PBMC isolation for immunological studies.

Role of iRhoms 1 and 2 in Endochondral Ossification.

Growth of the axial and appendicular skeleton depends on endochondral ossification, which is controlled by tightly regulated cell-cell interactions in the developing growth plates. Previous studies have uncovered an important role of a disintegrin and metalloprotease 17 (ADAM17) in the normal development of the mineralized zone of hypertrophic chondrocytes during endochondral ossification. ADAM17 regulates EGF-receptor signaling by cleaving EGFR-ligands such as TGFα from their membrane-anchored precursor. The activity of ADAM17 is controlled by two regulatory binding partners, the inactive Rhomboids 1 and 2 (iRhom1, 2), raising questions about their role in endochondral ossification. To address this question, we generated mice lacking iRhom2 (iR2-/-) with floxed alleles of iRhom1 that were specifically deleted in chondrocytes by Col2a1-Cre (iR1∆Ch). The resulting iR2-/-iR1∆Ch mice had retarded bone growth compared to iR2-/- mice, caused by a significantly expanded zone of hypertrophic mineralizing chondrocytes in the growth plate. Primary iR2-/-iR1∆Ch chondrocytes had strongly reduced shedding of TGFα and other ADAM17-dependent EGFR-ligands. The enlarged zone of mineralized hypertrophic chondrocytes in iR2-/-iR1∆Ch mice closely resembled the abnormal growth plate in A17∆Ch mice and was similar to growth plates in Tgfα-/- mice or mice with EGFR mutations. These data support a model in which iRhom1 and 2 regulate bone growth by controlling the ADAM17/TGFα/EGFR signaling axis during endochondral ossification.

The xenoestrogens biphenol-A and nonylphenol differentially regulate metalloprotease-mediated shedding of EGFR ligands.

The xenoestrogens bisphenol-A (BPA) and nonylphenol (NP) are endocrine disruptors used in the plastic polymer industry to manufacture different products for human use. Previous studies have suggested a role of these compounds in the shedding of signaling molecules, such as tumor necrosis factor α (TNF-α). The aim of this work was to evaluate the effect of BPA and NP on the sheddase ADAM17 and its newly discovered regulators iRhom1 and iRhom2 in the release of EGFR-ligands. We report that BPA and NP can stimulate the release of the ADAM17-substrates HB-EGF and TGF-α. In cells lacking ADAM17 (Adam17-/- mEFs) BPA-stimulated release of HB-EGF, but not TGF-α, was strongly reduced, whereas NP-stimulated shedding of HB-EGF and TGF-α was completely abolished. Inactivation of both ADAM17 and the related ADAM10 (Adam10/17-/- mEFs) completely prevented the release of these substrates. In the absence of iRhom1, BPA- or NP-stimulated release of HB-EGF or TGF-α was comparable to wild-type control mEFs, conversely the BPA-induced release of HB-EGF was abolished in iRhom2-/- mEFs. The defect in shedding of HB-EGF in iRhom2-/- mEF cells could be rescued by overexpressing iRhom2. Interestingly, the NP-stimulated release of HB-EGF was not affected by the absence of iRhom2, suggesting that NP could potentially activate both ADAM10 and ADAM17. We tested this hypothesis using betacellulin (BTC), an EGFR-ligand that is a substrate for ADAM10. We found that NP, but not BPA stimulated the release of BTC in Adam17-/- , iRhom2-/- , or iRhom1/2-/- , but not in Adam10/17-/- cells. Taken together, our results suggest that BPA and NP stimulate the release of EGFR-ligands by differentially activating ADAM17 or ADAM10. The identification of specific effects of these endocrine disruptors on ADAM10 and ADAM17 will help to provide a better understanding of their roles in cell signaling and proinflammatory processes, and provide new potential targets for treatment of reproductive or inflammatory diseases such as asthma or breast cancer that are promoted by xenoestrogens.

iRhoms 1 and 2 are essential upstream regulators of ADAM17-dependent EGFR signaling.

The metalloproteinase ADAM17 (a disintegrin and metalloprotease 17) controls EGF receptor (EGFR) signaling by liberating EGFR ligands from their membrane anchor. Consequently, a patient lacking ADAM17 has skin and intestinal barrier defects that are likely caused by lack of EGFR signaling, and Adam17(-/-) mice die perinatally with open eyes, like Egfr(-/-) mice. A hallmark feature of ADAM17-dependent EGFR ligand shedding is that it can be rapidly and posttranslationally activated in a manner that requires its transmembrane domain but not its cytoplasmic domain. This suggests that ADAM17 is regulated by other integral membrane proteins, although much remains to be learned about the underlying mechanism. Recently, inactive Rhomboid 2 (iRhom2), which has seven transmembrane domains, emerged as a molecule that controls the maturation and function of ADAM17 in myeloid cells. However, iRhom2(-/-) mice appear normal, raising questions about how ADAM17 is regulated in other tissues. Here we report that iRhom1/2(-/-) double knockout mice resemble Adam17(-/-) and Egfr(-/-) mice in that they die perinatally with open eyes, misshapen heart valves, and growth plate defects. Mechanistically, we show lack of mature ADAM17 and strongly reduced EGFR phosphorylation in iRhom1/2(-/-) tissues. Finally, we demonstrate that iRhom1 is not essential for mouse development but regulates ADAM17 maturation in the brain, except in microglia, where ADAM17 is controlled by iRhom2. These results provide genetic, cell biological, and biochemical evidence that a principal function of iRhoms1/2 during mouse development is to regulate ADAM17-dependent EGFR signaling, suggesting that iRhoms1/2 could emerge as novel targets for treatment of ADAM17/EGFR-dependent pathologies.

T-cell STAT3 is required for the maintenance of humoral immunity to LCMV.

STAT3 is a critical transcription factor activated downstream of cytokine signaling and is integral for the function of multiple immune cell types. Human mutations in STAT3 cause primary immunodeficiency resulting in impaired control of a variety of infections, including reactivation of latent viruses. In this study, we investigate how T-cell functions of STAT3 contribute to responses to viral infection by inducing chronic lymphocytic choriomeningitis virus (LCMV) infection in mice lacking STAT3 specifically in T cells. Although mice with conditional disruption of STAT3 in T cells were able to mount early responses to viral infection similar to control animals, including expansion of effector T cells, we found generation of T-follicular helper (Tfh) cells to be impaired. As a result, STAT3 T cell deficient mice produced attenuated germinal center reactions, and did not accumulate bone marrow virus specific IgG-secreting cells, resulting in failure to maintain levels of virus-specific IgG or mount neutralizing responses to LCMV in the serum. These effects were associated with reduced control of viral replication and prolonged infection. Our results demonstrate the importance of STAT3 in T cells for the generation of functional long-term humoral immunity to viral infections.

RAIDD Mediates TLR3 and IRF7 Driven Type I Interferon Production.

BACKGROUND/AIMS: Viral infections represent a global health problem with the need for new viral therapies and better understanding of the immune response during infection. The most immediate and potent anti-viral defense mechanism is the production of type I interferon (IFN-I) which are activated rapidly following recognition of viral infection by host pathogen recognition receptors (PRR). The mechanisms of innate cellular signaling downstream of PRR activation remain to be fully understood. In the present study, we demonstrate that CASP2 and RIPK1 domain-containing adaptor with death domain (CRADD/RAIDD) is a critical component in type I IFN production. METHODS: The role of RAIDD during IFN-I production was investigated using western blot, shRNA mediated lentiviral knockdown, immunoprecipitation and IFN-I driven dual luciferase assay. RESULTS: Immunoprecipitation analysis revealed the molecular interaction of RAIDD with interferon regulatory factor 7 (IRF7) and its phosphorylating kinase IKKε. Using an IFN-4α driven dual luciferase analysis in RAIDD deficient cells, type I IFN activation by IKKε and IRF7 was dramatically reduced. Furthermore, deletion of either the caspase recruitment domain (CARD) or death domain (DD) of RAIDD inhibited IKKε and IRF7 mediated interferon-4α activation. CONCLUSION: We have identified that the adaptor molecule RAIDD coordinates IKKε and IRF7 interaction to ensure efficient expression of type I interferon.

Deficiency of the B cell-activating factor receptor results in limited CD169+ macrophage function during viral infection.

UNLABELLED: The B cell-activating factor (BAFF) is critical for B cell development and humoral immunity in mice and humans. While the role of BAFF in B cells has been widely described, its role in innate immunity remains unknown. Using BAFF receptor (BAFFR)-deficient mice, we characterized BAFFR-related innate and adaptive immune functions following infection with vesicular stomatitis virus (VSV) and lymphocytic choriomeningitis virus (LCMV). We identified a critical role for BAFFR signaling in the generation and maintenance of the CD169(+) macrophage compartment. Consequently, Baffr(-) (/) (-) mice exhibited limited induction of innate type I interferon production after viral infection. Lack of BAFFR signaling reduced virus amplification and presentation following viral infection, resulting in highly reduced antiviral adaptive immune responses. As a consequence, BAFFR-deficient mice showed exacerbated and fatal disease after viral infection. Mechanistically, transient lack of B cells in Baffr(-) (/) (-) animals resulted in limited lymphotoxin expression, which is critical for maintenance of CD169(+) cells. In conclusion, BAFFR signaling affects both innate and adaptive immune activation during viral infections. IMPORTANCE: Viruses cause acute and chronic infections in humans resulting in millions of deaths every year. Innate immunity is critical for the outcome of a viral infection. Innate type I interferon production can limit viral replication, while adaptive immune priming by innate immune cells induces pathogen-specific immunity with long-term protection. Here, we show that BAFFR deficiency not only perturbed B cells, but also resulted in limited CD169(+) macrophages. These macrophages are critical in amplifying viral particles to trigger type I interferon production and initiate adaptive immune priming. Consequently, BAFFR deficiency resulted in reduced enforced viral replication, limited type I interferon production, and reduced adaptive immunity compared to BAFFR-competent controls. As a result, BAFFR-deficient mice were predisposed to fatal viral infections. Thus, BAFFR expression is critical for innate immune activation and antiviral immunity.

iRhom2 controls the substrate selectivity of stimulated ADAM17-dependent ectodomain shedding.

Protein ectodomain shedding by ADAM17 (a disintegrin and metalloprotease 17), a principal regulator of EGF-receptor signaling and TNFα release, is rapidly and posttranslationally activated by a variety of signaling pathways, and yet little is known about the underlying mechanism. Here, we report that inactive rhomboid protein 2 (iRhom2), recently identified as essential for the maturation of ADAM17 in hematopoietic cells, is crucial for the rapid activation of the shedding of some, but not all substrates of ADAM17. Mature ADAM17 is present in mouse embryonic fibroblasts (mEFs) lacking iRhom2, and yet ADAM17 is unable to support stimulated shedding of several of its substrates, including heparin-binding EGF and Kit ligand 2 in this context. Stimulated shedding of other ADAM17 substrates, such as TGFα, is not affected in iRhom2(-/-) mEFs but can be strongly reduced by treating iRhom2(-/-) mEFs with siRNA against iRhom1. Activation of heparin-binding EGF or Kit ligand 2 shedding by ADAM17 in iRhom2(-/-) mEFs can be rescued by wild-type iRhom2 but not by iRhom2 lacking its N-terminal cytoplasmic domain. The requirement for the cytoplasmic domain of iRhom2 for stimulated shedding by ADAM17 may help explain why the cytoplasmic domain of ADAM17 is not required for stimulated shedding. The functional relevance of iRhom2 in regulating shedding of EGF receptor (EGFR) ligands is established by a lack of lysophasphatidic acid/ADAM17/EGFR-dependent crosstalk with ERK1/2 in iRhom2(-/-) mEFs, and a significant reduction of FGF7/ADAM17/EGFR-stimulated migration of iRhom2(-/-) keratinocytes. Taken together, these findings uncover functions for iRhom2 in the regulation of EGFR signaling and in controlling the activation and substrate selectivity of ADAM17-dependent shedding events.

Involvement of Toso in activation of monocytes, macrophages, and granulocytes.

Rapid activation of immune responses is necessary for antibacterial defense, but excessive immune activation can result in life-threatening septic shock. Understanding how these processes are balanced may provide novel therapeutic potential in treating inflammatory disease. Fc receptors are crucial for innate immune activation. However, the role of the putative Fc receptor for IgM, known as Toso/Faim3, has to this point been unclear. In this study, we generated Toso-deficient mice and used them to uncover a critical regulatory function of Toso in innate immune activation. Development of innate immune cells was intact in the absence of Toso, but Toso-deficient neutrophils exhibited more reactive oxygen species production and reduced phagocytosis of pathogens compared with controls. Cytokine production was also decreased in Toso(-/-) mice compared with WT animals, rendering them resistant to septic shock induced by lipopolysaccharide. However, Toso(-/-) mice also displayed limited cytokine production after infection with the bacterium Listeria monocytogenes that was correlated with elevated presence of Listeria throughout the body. Accordingly, Toso(-/-) mice succumbed to infections of L. monocytogenes, whereas WT mice successfully eliminated the infection. Taken together, our data reveal Toso to be a unique regulator of innate immune responses during bacterial infection and septic shock.

Smg1 is required for embryogenesis and regulates diverse genes via alternative splicing coupled to nonsense-mediated mRNA decay.

Smg1 is a PI3K-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbor premature termination codons (PTCs) as a result of events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. By using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5. High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from approximately 9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signaling, membrane dynamics, cell death, and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.

Longitudinal clinical phenotyping of post COVID condition in Mexican adults recovering from severe COVID-19: a prospective cohort study.

Introduction: Few studies have evaluated the presence of Post COVID-19 conditions (PCC) in people from Latin America, a region that has been heavily afflicted by the COVID-19 pandemic. In this study, we describe the frequency, co-occurrence, predictors, and duration of 23 symptoms in a cohort of Mexican patients with PCC. Methods: We prospectively enrolled and followed adult patients hospitalized for severe COVID-19 at a tertiary care centre in Mexico City. The incidence of PCC symptoms was determined using questionnaires. Unsupervised clustering of PCC symptom co-occurrence and Kaplan-Meier analyses of symptom persistence were performed. The effect of baseline clinical characteristics was evaluated using Cox regression models and reported with hazard ratios (HR). Results: We found that amongst 192 patients with PCC, respiratory problems were the most prevalent and commonly co-occurred with functional activity impairment. 56% had ≥5 persistent symptoms. Symptom persistence probability at 360 days 0.78. Prior SARS-CoV-2 vaccination and infection during the Delta variant wave were associated with a shorter duration of PCC. Male sex was associated with a shorter duration of functional activity impairment and respiratory symptoms. Hypertension and diabetes were associated with a longer duration of functional impairment. Previous vaccination accelerated PCC recovery. Discussion: In our cohort, PCC symptoms were frequent (particularly respiratory and neurocognitive ones) and persistent. Importantly, prior SARS-CoV-2 vaccination resulted in a shorter duration of PCC.

Variation of Immune Cell Responses in Humans Reveals Sex-Specific Coordinated Signaling Across Cell Types.

Assessing the health and competence of the immune system is central to evaluating vaccination responses, autoimmune conditions, cancer prognosis, and treatment. With an increasing number of studies examining immune dysregulation, there is a growing need for a curated reference of variation in immune parameters in healthy individuals. We used mass cytometry (CyTOF) to profile blood from 86 humans in response to 15 ex vivo immune stimuli. We present reference ranges for cell-specific immune markers and highlight differences that appear across sex and age. We identified modules of immune features that suggest there exists an underlying structure to the immune system based on signaling pathway responses across cell types. We observed increased MAPK signaling in inflammatory pathways in innate immune cells and greater overall coordination of immune cell responses in females. In contrast, males exhibited stronger pSTAT1 and pTBK1 responses. These reference data are publicly available as a resource for immune profiling studies.