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Aims: Olfactory receptor 2 (Olfr2) has been identified in a minimum of 30% of vascular macrophages, and its depletion was shown to reduce atherosclerosis progression. Mononuclear phagocytes, including monocytes and macrophages within the vessel wall, are major players in atherosclerosis. Single-cell RNA sequencing studies revealed that atherosclerotic artery walls encompass several monocytes and vascular macrophages, defining at least nine distinct subsets potentially serving diverse functions in disease progression. This study investigates the functional phenotype and ontogeny of Olfr2-expressing vascular macrophages in atherosclerosis., Methods and Results: Olfr2+ macrophages rapidly increase in Apoe-/- mice's aorta when fed a Western diet (WD). Mass cytometry showed that Olfr2+ cells are clustered within the CD64 high population and enriched for CD11c and Ccr2 markers. Olfr2+ macrophages express many pro-inflammatory cytokines, including Il1b, Il6, Il12, and Il23, and chemokines, including Ccl5, Cx3cl1, Cxcl9, and Ccl22. By extracting differentially expressed genes from bulk RNA sequencing (RNA-seq) of Olfr2+ vs. Olfr2- macrophages, we defined a signature that significantly mapped to single-cell data of plaque myeloid cells, including monocytes, subendothelial MacAir, and Trem2Gpnmb foamy macrophages. By adoptive transfer experiments, we identified that Olfr2 competent monocytes from CD45.1Apoe-/-Olfr2+/+ mice transferred into CD45.2Apoe-/-Olfr2-/- recipient mice fed WD for 12 weeks, accumulate in the atherosclerotic aorta wall already at 72 h, and differentiate in macrophages. Olfr2+ macrophages showed significantly increased BrdU incorporation compared to Olfr2- macrophages. Flow cytometry confirmed that at least 50% of aortic Olfr2+ macrophages are positive for BODIPY staining and have increased expression of both tumour necrosis factor and interleukin 6 compared to Olfr2- macrophages. Gene set enrichment analysis of the Olfr2+ macrophage signature revealed a similar enrichment pattern in human atherosclerotic plaques, particularly within foamy/TREM2hi-Mφ and monocytes., Conclusions: In summary, we conclude that Olfr2+ macrophages in the aorta originate from monocytes and can accumulate at the early stages of disease progression. These cells can undergo differentiation into MacAir and Trem2Gpnmb foamy macrophages, exhibiting proliferative and pro-inflammatory potentials. This dynamic behaviour positions them as key influencers in shaping the myeloid landscape within the atherosclerotic plaque., Competing Interests: Conflict of interest: M.O. and K.L. are named as co-inventors on pending patents held by the La Jolla Institute for Immunology relating to cardiovascular diagnostics and therapeutics and might have the right to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics. None of the other authors have any competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Competing Interests: None.

Immune related adverse events (irAEs) after immune checkpoint blockade (ICB) therapy occur in a significant proportion of cancer patients. To date, the circulating mediators of ICB-irAEs remain poorly understood. Using non-targeted mass spectrometry, here we identify the circulating bio-active lipid linoleoyl-lysophosphatidylcholine (LPC 18:2) as a modulator of ICB-irAEs. In three independent human studies of ICB treatment for solid tumor, loss of circulating LPC 18:2 preceded the development of severe irAEs across multiple organ systems. In both healthy humans and severe ICB-irAE patients, low LPC 18:2 was found to correlate with high blood neutrophilia. Reduced LPC 18:2 biosynthesis was confirmed in preclinical ICB-irAE models, and LPC 18:2 supplementation in vivo suppressed neutrophilia and tissue inflammation without impacting ICB anti-tumor response. Results indicate that circulating LPC 18:2 suppresses human ICB-irAEs, and LPC 18:2 supplementation may improve ICB outcomes by preventing severe inflammation while maintaining anti-tumor immunity.

Background: Both cancer and cardiovascular disease (CVD) are the leading causes of death worldwide. Although our previous study detected a relationship between CVD and cancer incidence, limited evidence is available regarding the relationship between CVD, cardiovascular risk factors, and cancer mortality., Methods and Results: A prospective cohort study using data from the continuous NHANES (National Health and Nutrition Examination Survey, 1999-2016) merged with Medicare and National Death Index mortality data, through December 31, 2018. We included individuals with no history of cancer at baseline. The primary exposure was CVD at baseline. We also conducted a comprehensive risk factor analysis as secondary exposure. The main outcome was cancer mortality data collected from Medicare and National Death Index. We included 44 591 adult individuals representing 1 738 423 317 individuals (52% female, 67% non-Hispanic White, and 9% Hispanic). Competing risk modeling showed a significantly higher risk of cancer mortality in individuals with CVD (adjusted hazard ratio [aHR], 1.37 [95% CI 1.07-1.76], P =0.01) after adjusting for age, sex, and race and ethnicity. Notably, cancer mortality increased with aging (aHR, 1.08 [95% CI 1.05-1.11], P <0.0001), current smoking status (aHR, 6.78 [95% CI, 3.43-13.42], P <0.0001), and obesity (aHR, 2.32 [95% CI, 1.13-4.79], P =0.02). Finally, a significant interaction ( P =0.034) was found where those with CVD and obesity showed higher cancer mortality than those with normal body mass index (aHR, 1.73 [95% CI, 1.03-2.91], P =0.04)., Conclusions: Our study highlights the close relationship between cardiovascular health and cancer mortality. Our findings suggest that obesity may play a significant role in cancer mortality among individuals with CVD. These findings emphasize the need for a more proactive approach in managing the shared risk factors for CVD and cancer.

Introduction: This study aimed to investigate the relationship between risk factors of cancer among individuals with existing cardiovascular disease (CVD)., Methods: The analysis included 438 and 2100 CVD patients aged 65+ from NHANES-III and Continuous datasets, respectively. Competing risk models with subdistribution hazards ratio (aHR) were used to identify risk factors., Results: Females in NHANES-III had lower cancer risk (aHR 0.39, P  = 0.001) compared to males. Poor physical activity was associated with increased cancer risk in both datasets (aHR 2.59 in NHANES-III, aHR 1.59 in Continuous). In NHANES-Continuous, age (aHR 1.07, P  < 0.001) and current smoking (aHR 2.55, P  = 0.001) also showed a significant association with developing cancer. No other factors investigated showed significant associations., Discussion: This study highlights the interplay between traditional risk factors and the elevated risk of cancer in CVD patients. Further research with larger samples and wider age ranges is needed to solidify these findings and inform intervention strategies., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Avirup Guha reports financial support was provided by the 10.13039/100000968American Heart Association. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Monocytes are associated with human cardiovascular disease progression. Monocytes are segregated into three major subsets: classical (cMo), intermediate (iMo), and nonclassical (nMo). Recent studies have identified heterogeneity within each of these main monocyte classes, yet the extent to which these subsets contribute to heart disease progression is not known. Peripheral blood mononuclear cells (PBMC) were obtained from 61 human subjects within the Coronary Assessment of Virginia (CAVA) Cohort. Coronary atherosclerosis severity was quantified using the Gensini Score (GS). We employed high-dimensional single-cell transcriptome and protein methods to define how human monocytes differ in subjects with low to severe coronary artery disease. We analyzed 487 immune-related genes and 49 surface proteins at the single-cell level using Antibody-Seq (Ab-Seq). We identified six subsets of myeloid cells (cMo, iMo, nMo, plasmacytoid DC, classical DC, and DC3) at the single-cell level based on surface proteins, and we associated these subsets with coronary artery disease (CAD) incidence based on Gensini score (GS) in each subject. Only frequencies of iMo were associated with high CAD (GS > 32), adj.p = 0.024. Spearman correlation analysis with GS from each subject revealed a positive correlation with iMo frequencies (r = 0.314, p = 0.014) and further showed a robust sex-dependent positive correlation in female subjects (r = 0.663, p = 0.004). cMo frequencies did not correlate with CAD severity. Key gene pathways differed in iMo among low and high CAD subjects and between males and females. Further single-cell analysis of iMo revealed three iMo subsets in human PBMC, distinguished by the expression of HLA-DR, CXCR3, and CD206. We found that the frequency of immunoregulatory iMo&#95;HLA-DR + CXCR3 + CD206 + was associated with CAD severity ( adj.p = 0.006). The immunoregulatory iMo subset positively correlated with GS in both females (r = 0.660, p = 0.004) and males (r = 0.315, p = 0.037). Cell interaction analyses identified strong interactions of iMo with CD4 + effector/memory T cells and Tregs from the same subjects. This study shows the importance of iMo in CAD progression and suggests that iMo may have important functional roles in modulating CAD risk, particularly among females.

Chimeric antigen receptor T cells (CAR-Ts) have remarkable efficacy in liquid tumors, but limited responses in solid tumors. We conducted a Phase I trial (NCT02107963) of GD2 CAR-Ts (GD2-CAR.OX40.28.z.iC9), demonstrating feasibility and safety of administration in children and young adults with osteosarcoma and neuroblastoma. Since CAR-T efficacy requires adequate CAR-T expansion, patients were grouped into good or poor expanders across dose levels. Patient samples were evaluated by multi-dimensional proteomic, transcriptomic, and epigenetic analyses. T cell assessments identified naive T cells in pre-treatment apheresis associated with good expansion, and exhausted T cells in CAR-T products with poor expansion. Myeloid cell assessment identified CXCR3 + monocytes in pre-treatment apheresis associated with good expansion. Longitudinal analysis of post-treatment samples identified increased CXCR3 - classical monocytes in all groups as CAR-T numbers waned. Together, our data uncover mediators of CAR-T biology and correlates of expansion that could be utilized to advance immunotherapies for solid tumor patients., Competing Interests: Declaration of interests C.J.W. receives research funding from Pharmacyclics and hold equity in BioNTech, Inc. F.M. is a cofounder of and has equity in Harbinger Health, has equity in Zephyr AI, and serves as a consultant for Harbinger Health, Zephyr AI, and Red Cell Partners and Exscientia. F.M. declares that none of these relationships are directly or indirectly related to the content of this manuscript. E.S. consults for and holds equity in Lyell Immunopharma and consults for Lepton Pharmaceuticals and Galaria. M.S.M. is currently employed at Normunity and holds stock in AstraZeneca; her contributions to this work were made prior to these industry positions which are not relevant to the content of this manuscript. C.L.M. is an inventor on numerous patents and patents pending related to CAR-T cell therapies. C.L.M. holds equity in and receives research funding from Lyell Immunopharma and holds equity in and consults for CARGO Therapeutics and Link Cell Therapies. C.L.M. consults for Immatics, Mammoth, Ensoma, and Red Tree Venture Capital., (Published by Elsevier Inc.)

IgMs that inactivate oxidation-specific epitopes (IgM OSE ), which are secondary products of lipid peroxidization, protect against inflammatory diseases, including diet-induced atherosclerosis. However, the human B cell subtype that produces IgM OSE remains unknown. In this study, we used single-cell mass cytometry and adoptive transfer of B cell subtypes to NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice to identify B 27+IgM+CD24hi cells as the major producers of IgM OSE in humans. Notably, these cells have characteristics of human circulatory marginal zone B (MZB) cells, which are known to be atheoroprotective IgM producers in mice. CD24 antibody treatment to reduce MZB cells and IgM in a hyperlipidemic humanized mouse model provides the evidence that MZB cells protect against vascular inflammation. Consistent with these findings, the frequency of B 27+IgM+CD24hi cells (MZB) in patients inversely correlates with coronary artery disease severity., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Purpose: Due to their abundance in the blood, low RNA content, and short lifespan, neutrophils have been classically considered to be one homogenous pool. However, recent work has found that mature neutrophils and neutrophil progenitors are composed of unique subsets exhibiting context-dependent functions. In this study, we ask if neutrophil heterogeneity is associated with melanoma incidence and/or disease stage., Experimental Design: Using mass cytometry, we profiled melanoma patient blood for unique cell surface markers among neutrophils. Markers were tested for their predictiveness using flow cytometry data and random forest machine learning., Results: We identified CD79b + neutrophils (CD3 - CD56 - CD19 - Siglec8 - CD203c - CD86 Lo CD66b + CD79b + ) that are normally restricted to the bone marrow in healthy humans but appear in the blood of subjects with early-stage melanoma. Further, we found CD79b + neutrophils present in tumors of subjects with head and neck cancer. AI-mediated machine learning analysis of neutrophils from subjects with melanoma confirmed that CD79b expression among peripheral blood neutrophils is highly important in identifying melanoma incidence. We noted that CD79b + neutrophils possessed a neutrophilic appearance but have transcriptional and surface-marker phenotypes reminiscent of B cells. Compared to remaining blood neutrophils, CD79b + neutrophils are primed for NETosis, express higher levels of antigen presentation-related proteins, and have an increased capacity for phagocytosis., Conclusion: Our work suggests that CD79b + neutrophils are associated with early-stage melanoma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AR declared a shared affiliation with the authors YZ and CO to the handling editor at the time of review., (Copyright © 2023 Meyer, Dinh, Alimadadi, Araujo, Chatterjee, Gutierrez, Zhu, Hunter, Liang, Seumois, Kiosses, Catz, Vijayanand, Ottensmeier and Hedrick.)

Coronary artery disease (CAD) is a major cause of death worldwide. The role of CD8+ T cells in CAD is unknown. Recent studies suggest a breakdown of tolerance in atherosclerosis, resulting in active T cell receptor (TCR) engagement with self-antigens. We hypothesized that TCR engagement would leave characteristic gene expression signatures. In a single cell RNA-sequencing analysis of CD8+ T cells from 30 patients with CAD and 30 controls we found significant enrichment of TCR signaling pathways in CAD+ subjects, suggesting recent TCR engagement. We also found significant enrichment of cytotoxic and exhaustion pathways in CAD cases compared to controls. Highly significant upregulation of TCR signaling in CAD indicates that CD8 T cells reactive to atherosclerosis antigens are prominent in the blood of CAD cases compared to controls., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Iqneibi, Saigusa, Khan, Oliaeimotlagh, Armstrong Suthahar, Kumar, Alimadadi, Durant, Ghosheh, McNamara, Hedrick and Ley.)

CD8 + T cells drive anti-cancer immunity in response to antigen-presenting cells such as dendritic cells and subpopulations of monocytes and macrophages. While CD14 + classical monocytes modulate CD8 + T cell responses, the contributions of CD16 + nonclassical monocytes to this process remain unclear. Herein we explored the role of nonclassical monocytes in CD8 + T cell activation by utilizing E2-deficient (E2 -/- ) mice that lack nonclassical monocytes. During early metastatic seeding, modeled by B16F10-OVA cancer cells injected into E2 -/- mice, we noted lower CD8 + effector memory and effector T cell frequencies within the lungs as well as in lung-draining mediastinal lymph nodes in the E2 -/- mice. Analysis of the myeloid compartment revealed that these changes were associated with depletion of MHC-II lo Ly6C lo nonclassical monocytes within these tissues, with little change in other monocyte or macrophage populations. Additionally, nonclassical monocytes preferentially trafficked to primary tumor sites in the lungs, rather than to the lung-draining lymph nodes, and did not cross-present antigen to CD8 + T cells. Examination of the lung microenvironment in E2 -/- mice revealed reduced CCL21 expression in endothelial cells, which is chemokine involved in T cell trafficking. Our results highlight the previously unappreciated importance of nonclassical monocytes in shaping the tumor microenvironment via CCL21 production and CD8 + T cell recruitment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Padgett, Marcovecchio, Olingy, Araujo, Steel, Dinh, Alimadadi, Zhu, Meyer, Kiosses, Thomas and Hedrick.)

Introduction: Previous studies suggest that monocytes are an important contributor to tuberculosis (TB)-specific immune signatures in blood., Methods: Here, we carried out comprehensive single-cell profiling of monocytes in paired blood samples of active TB (ATB) patients at diagnosis and mid-treatment, and healthy controls., Results: At diagnosis, ATB patients displayed increased monocyte-to-lymphocyte ratio, increased frequency of CD14+CD16- and intermediate CD14+CD16+ monocytes, and upregulation of interferon signaling genes that significantly overlapped with previously reported blood TB signatures in both CD14+ subsets. In this cohort, we identified additional transcriptomic and functional changes in intermediate CD14+CD16+ monocytes, such as the upregulation of inflammatory and MHC-II genes, and increased capacity to activate T cells, reflecting overall increased activation in this population. Single-cell transcriptomics revealed that distinct subsets of intermediate CD14+CD16+ monocytes were responsible for each gene signature, indicating significant functional heterogeneity within this population. Finally, we observed that changes in CD14+ monocytes were transient, as they were no longer observed in the same ATB patients mid-treatment, suggesting they are associated with disease resolution., Discussion: Together, our study demonstrates for the first time that both intermediate and classical monocytes individually contribute to blood immune signatures of ATB and identifies novel subsets and associated gene signatures that may hold disease relevance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hillman, Khan, Singhania, Dubelko, Soldevila, Tippalagama, DeSilva, Gunasena, Perera, Scriba, Ontong, Fisher, Luabeya, Taplitz, Seumois, Vijayanand, Hedrick, Peters and Burel.)

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in severe immune dysfunction, hospitalization, and death. Many patients also develop long-COVID-19, experiencing symptoms months after infection. Although significant progress has been made in understanding the immune response to acute SARS-CoV-2 infection, gaps remain in our knowledge of how innate immunity influences disease kinetics and severity. We hypothesized that cytometry by time-of-flight analysis of PBMCs from healthy and infected subjects would identify novel cell surface markers and innate immune cell subsets associated with COVID-19 severity. In this pursuit, we identified monocyte and dendritic cell subsets that changed in frequency during acute SARS-CoV-2 infection and correlated with clinical parameters of disease severity. Subsets of nonclassical monocytes decreased in frequency in hospitalized subjects, yet increased in the most severe patients and positively correlated with clinical values associated with worse disease severity. CD9, CD163, PDL1, and PDL2 expression significantly increased in hospitalized subjects, and CD9 and 6-Sulfo LacNac emerged as the markers that best distinguished monocyte subsets amongst all subjects. CD9 + monocytes remained elevated, whereas nonclassical monocytes remained decreased, in the blood of hospitalized subjects at 3-4 months postinfection. Finally, we found that CD9 + monocytes functionally released more IL-8 and MCP-1 after LPS stimulation. This study identifies new monocyte subsets present in the blood of COVID-19 patients that correlate with disease severity, and links CD9 + monocytes to COVID-19 progression., (©2022 Society for Leukocyte Biology.)

Dysregulated secretion in neutrophil leukocytes associates with human inflammatory disease. The exocytosis response to triggering stimuli is sequential; gelatinase granules modulate the initiation of the innate immune response, followed by the release of pro-inflammatory azurophilic granules, requiring stronger stimulation. Exocytosis requires actin depolymerization which is actively counteracted under non-stimulatory conditions. Here we show that the actin nucleator, WASH, is necessary to maintain azurophilic granules in their refractory state by granule actin entrapment and interference with the Rab27a-JFC1 exocytic machinery. On the contrary, gelatinase granules of WASH-deficient neutrophil leukocytes are characterized by decreased Rac1, shortened granule-associated actin comets and impaired exocytosis. Rac1 activation restores exocytosis of these granules. In vivo, WASH deficiency induces exacerbated azurophilic granule exocytosis, inflammation, and decreased survival. WASH deficiency thus differentially impacts neutrophil granule subtypes, impairing exocytosis of granules that mediate the initiation of the neutrophil innate response while exacerbating pro-inflammatory granule secretion., (© 2022. The Author(s).)

Background: Cryopreserved peripheral blood mononuclear cells (PBMCs) are frequently collected and provide disease- and treatment-relevant data in clinical studies. Here, we developed combined protein (40 antibodies) and transcript single-cell (sc)RNA sequencing (scRNA-seq) in PBMCs., Results: Among 31 participants in the Women's Interagency HIV Study (WIHS), we sequenced 41,611 cells. Using Boolean gating followed by Seurat UMAPs (tool for visualizing high-dimensional data) and Louvain clustering, we identified 50 subsets among CD4+ T, CD8+ T, B, NK cells, and monocytes. This resolution was superior to flow cytometry, mass cytometry, or scRNA-seq without antibodies. Combined protein and transcript scRNA-seq allowed for the assessment of disease-related changes in transcriptomes and cell type proportions. As a proof-of-concept, we showed such differences between healthy and matched individuals living with HIV with and without cardiovascular disease., Conclusions: In conclusion, combined protein and transcript scRNA sequencing is a suitable and powerful method for clinical investigations using PBMCs., (© 2022. The Author(s).)

Despite the decades-old knowledge that males and people with diabetes mellitus (DM) are at increased risk for coronary artery disease (CAD), the reasons for this association are only partially understood. Among the immune cells involved, recent evidence supports a critical role of T cells as drivers and modifiers of CAD. CD4+ T cells are commonly found in atherosclerotic plaques. We aimed to understand the relationship of CAD with sex and DM by single-cell RNA (scRNA-Seq) and antibody sequencing (CITE-Seq) of CD4+ T cells. Peripheral blood mononuclear cells (PBMCs) of 61 men and women who underwent cardiac catheterization were interrogated by scRNA-Seq combined with 49 surface markers (CITE-Seq). CAD severity was quantified using Gensini scores, with scores above 30 considered CAD+ and below 6 considered CAD-. Four pairs of groups were matched for clinical and demographic parameters. To test how sex and DM changed cell proportions and gene expression, we compared matched groups of men and women, as well as diabetic and non-diabetic subjects. We analyzed 41,782 single CD4+ T cell transcriptomes for sex differences in 16 women and 45 men with and without coronary artery disease and with and without DM. We identified 16 clusters in CD4+ T cells. The proportion of cells in CD4+ effector memory cluster 8 (CD4T8, CCR2+ Em) was significantly decreased in CAD+, especially among DM+ participants. This same cluster, CD4T8, was significantly decreased in female participants, along with two other CD4+ T cell clusters. In CD4+ T cells, 31 genes showed significant and coordinated upregulation in both CAD and DM. The DM gene signature was partially additive to the CAD gene signature. We conclude that (1) CAD and DM are clearly reflected in PBMC transcriptomes, and (2) significant differences exist between women and men and (3) between subjects with DM and non-DM.

Atherosclerosis is accompanied by a CD4 T cell response to apolipoprotein B (APOB). Major Histocompatibility Complex (MHC)-II tetramers can be used to isolate antigen-specific CD4 T cells by flow sorting. Here, we produce, validate and use an MHC-II tetramer, DRB1*07:01 APOB-p18, to sort APOB-p18-specific cells from peripheral blood mononuclear cell samples from 8 DRB1*07:01+ women with and without subclinical cardiovascular disease (sCVD). Single cell RNA sequencing showed that transcriptomes of tetramer+ cells were between regulatory and memory T cells in healthy women and moved closer to memory T cells in women with sCVD. TCR sequencing of tetramer+ cells showed clonal expansion and V and J segment usage similar to those found in regulatory T cells. These findings suggest that APOB-specific regulatory T cells may switch to a more memory-like phenotype in women with atherosclerosis. Mouse studies showed that such switched cells promote atherosclerosis., Competing Interests: DISCLOSURES There are no conflicts of interest.

Non-small cell lung carcinoma (NSCLC) is the leading cause of cancer-related deaths globally. Immune checkpoint blockade (ICB) has transformed cancer medicine, with anti-programmed cell death protein 1 (anti-PD-1) therapy now well-utilized for treating NSCLC. Still, not all patients with NSCLC respond positively to anti-PD-1 therapy, and some patients acquire resistance to treatment. There remains an urgent need to find markers predictive of anti-PD-1 responsiveness. To this end, we performed mass cytometry on peripheral blood mononuclear cells from 26 patients with NSCLC during anti-PD-1 treatment. Patients who responded to anti-PD-1 ICB displayed significantly higher levels of antigen-presenting myeloid cells, including CD9 + nonclassical monocytes, and CD33 hi classical monocytes. Using matched pre-post treatment samples, we found that the baseline pre-treatment frequencies of CD33 hi monocytes predicted patient responsiveness to anti-PD-1 therapy. Moreover, some of these classical and nonclassical monocyte subsets were associated with reduced immunosuppression by T regulatory (CD4 + FOXP3 + CD25 + ) cells in the same patients. Our use of machine learning corroborated the association of specific monocyte markers with responsiveness to ICB. Our work provides a high-dimensional profile of monocytes in NSCLC and links CD33 expression on monocytes with anti-PD-1 effectiveness in patients with NSCLC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Olingy, Alimadadi, Araujo, Barry, Gutierrez, Werbin, Arriola, Patel, Ottensmeier, Dinh and Hedrick.)

Neutrophils are the most abundant myeloid cells in human blood and are emerging as important regulators of cancer. However, their functional importance has often been overlooked on the basis that they are short-lived, terminally differentiated and non-proliferative. Recent studies of their prominent roles in cancer have led to a paradigm shift in our appreciation of neutrophil functional diversity. This Review describes how neutrophil diversification, which in some contexts can lead to opposing functions, is generated within the tumour microenvironment as well as systemically. We compare neutrophil heterogeneity in cancer and in other pathophysiological contexts to provide an updated overview of our current knowledge of the functions of neutrophils in cancer., (© 2021. Springer Nature Limited.)

Cystic fibrosis (CF) is an inherited life-threatening disease accompanied by repeated lung infections and multiorgan inflammation that affects tens of thousands of people worldwide. The causative gene, cystic fibrosis transmembrane conductance regulator (CFTR), is mutated in CF patients. CFTR functions in epithelial cells have traditionally been thought to cause the disease symptoms. Recent work has shown an additional defect: monocytes from CF patients show a deficiency in integrin activation and adhesion. Because monocytes play critical roles in controlling infections, defective monocyte function may contribute to CF progression. In this study, we demonstrate that monocytes from CFTR ΔF508 mice (CF mice) show defective adhesion under flow. Transplanting CF mice with wild-type (WT) bone marrow after sublethal irradiation replaced most (60-80%) CF monocytes with WT monocytes, significantly improved survival, and reduced inflammation. WT/CF mixed bone marrow chimeras directly demonstrated defective CF monocyte recruitment to the bronchoalveolar lavage and the intestinal lamina propria in vivo. WT mice reconstituted with CF bone marrow also show lethality, suggesting that the CF defect in monocytes is not only necessary but also sufficient to cause disease. We also show that monocyte-specific knockout of CFTR retards weight gains and exacerbates dextran sulfate sodium-induced colitis. Our findings show that providing WT monocytes by bone marrow transfer rescues mortality in CF mice, suggesting that similar approaches may mitigate disease in CF patients., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Atherosclerosis is characterized by the abundant infiltration of immune cells starting at early stages and progressing to late stages of the disease. The study and characterization of immune cells infiltrating and residing in the aorta has being tackled by several methodologies such as flow cytometry and mass cytometry (CyTOF). Flow cytometry has been primarily used to address the aortic leukocyte composition; however, only a limited number of markers can be analyzed simultaneously. CyTOF started to overcome these limitations by employing rare element-tagged antibodies and combines mass spectrometry with the ease and precision of flow cytometry. CyTOF currently allows for the simultaneous measurement of more than 40 cellular parameters at single-cell resolution.In this chapter, we describe the methodology used to isolate single immune cells from mouse aortas, followed by protocols for flow cytometry and CyTOF for aortic immune cell characterization., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The tumor microenvironment is a highly complex ecosystem of diverse cell types, which shape cancer biology and impact the responsiveness to therapy. Here, we analyze the microenvironment of esophageal squamous cell carcinoma (ESCC) using single-cell transcriptome sequencing in 62,161 cells from blood, adjacent nonmalignant and matched tumor samples from 11 ESCC patients. We uncover heterogeneity in most cell types of the ESCC stroma, particularly in the fibroblast and immune cell compartments. We identify a tumor-specific subset of CST1 + myofibroblasts with prognostic values and potential biological significance. CST1 + myofibroblasts are also highly tumor-specific in other cancer types. Additionally, a subset of antigen-presenting fibroblasts is revealed and validated. Analyses of myeloid and T lymphoid lineages highlight the immunosuppressive nature of the ESCC microenvironment, and identify cancer-specific expression of immune checkpoint inhibitors. This work establishes a rich resource of stromal cell types of the ESCC microenvironment for further understanding of ESCC biology., (© 2021. The Author(s).)

Despite the important function of neutrophils in the eradication of infections and induction of inflammation, the molecular mechanisms regulating the activation and termination of the neutrophil immune response is not well understood. Here, the function of the small GTPase from the RGK family, Gem, is characterized as a negative regulator of the NADPH oxidase through autophagy regulation. Gem knockout (Gem KO) neutrophils show increased NADPH oxidase activation and increased production of extracellular and intracellular reactive oxygen species (ROS). Enhanced ROS production in Gem KO neutrophils was associated with increased NADPH oxidase complex-assembly as determined by quantitative super-resolution microscopy, but normal exocytosis of gelatinase and azurophilic granules. Gem-deficiency was associated with increased basal autophagosomes and autolysosome numbers but decreased autophagic flux under phorbol ester-induced conditions. Neutrophil stimulation triggered the localization of the NADPH oxidase subunits p22 phox and p47 phox at LC3-positive structures suggesting that the assembled NADPH oxidase complex is recruited to autophagosomes, which was significantly increased in Gem KO neutrophils. Prevention of new autophagosome formation by treatment with SAR405 increased ROS production while induction of autophagy by Torin-1 decreased ROS production in Gem KO neutrophils, and also in wild-type neutrophils, suggesting that macroautophagy contributes to the termination of NADPH oxidase activity. Autophagy inhibition decreased NETs formation independently of enhanced ROS production. NETs production, which was significantly increased in Gem-deficient neutrophils, was decreased by inhibition of both autophagy and calmodulin, a known GEM interactor. Intracellular ROS production was increased in Gem KO neutrophils challenged with live Gram-negative bacteria Pseudomonas aeruginosa or Salmonella Typhimurium, but phagocytosis was not affected in Gem-deficient cells. In vivo analysis in a model of Salmonella Typhimurium infection indicates that Gem-deficiency provides a genetic advantage manifested as a moderate increased in survival to infections. Altogether, the data suggest that Gem-deficiency leads to the enhancement of the neutrophil innate immune response by increasing NADPH oxidase assembly and NETs production and that macroautophagy differentially regulates ROS and NETs in neutrophils., (©2021 Society for Leukocyte Biology.)

Objective: CD4 T cells are important regulators of atherosclerotic progression. The metabolic profile of CD4 T cells controls their signaling and function, but how atherosclerosis affects T-cell metabolism is unknown. Here, we sought to determine the impact of atherosclerosis on CD4 T-cell metabolism and the contribution of such metabolic alterations to atheroprogression., Approach and Results: Using PCR arrays, we profiled the expression of metabolism genes in CD4 T cells from atherosclerotic apolipoprotein-E knockout mice fed a Western diet. These cells exhibited dysregulated expression of genes critically involved in glycolysis and fatty acid degradation, compared with those from animals fed a standard laboratory diet. We examined how T-cell metabolism was changed in either Western diet–fed apolipoprotein-E knockout mice or samples from patients with cardiovascular disease by measuring glucose uptake, activation, and proliferation in CD4 T cells. We found that naive CD4 T cells from Western diet–fed apolipoprotein-E knockout mice failed to uptake glucose and displayed impaired proliferation and activation, compared with CD4 T cells from standard laboratory diet–fed animals. Similarly, we observed that naive CD4 T-cell frequencies were reduced in the circulation of human subjects with high cardiovascular disease compared with low cardiovascular disease. Naive T cells from high cardiovascular disease subjects also showed reduced proliferative capacity., Conclusions: These results highlight the dysfunction that occurs in CD4 T-cell metabolism and immune responses during atherosclerosis. Targeting metabolic pathways within naive CD4 T cells could thus yield novel therapeutic approaches for improving CD4 T-cell responses against atheroprogression.

Objective: Cardiovascular disease (CVD) remains a significant global health concern with a high degree of mortality. While CD4 + T cells have been extensively studied in CVD, the importance of CD8 + T cells in this disease, despite their abundance and increased activation in human atherosclerotic plaques, remains largely unknown. Thus, the objective of this study was to compare peripheral T-cell signatures between humans with a high (severe) risk of CVD (including myocardial infarction or stroke) and those with a low risk of CVD. Approach and Results: Using mass cytometry, we uncovered a naive CD8 + T (T N ) cell population expressing CD95 (termed CD95 + CD8 + stem cell memory T [CD8 T SCM ] cells) that was enriched in patients with high compared with low CVD. This T-cell subset enrichment within individuals with high CVD was a relative increase and resulted from the loss of CD95 lo cells within the T N compartment. We found that CD8 T SCM cells positively correlated with CVD risk in humans, while CD8 + T N cells were inversely correlated. Atherosclerotic apolipoprotein E-deficient (ApoE -/- ) mice also displayed respective 7- and 2-fold increases in CD8 + T SCM frequencies within the peripheral blood and aorta-draining paraaortic lymph nodes compared with C57BL/6J mice. CD8 + T SCM cells were 1.7-fold increased in aortas from western diet fed ApoE -/- mice compared with normal laboratory diet-fed ApoE -/- mice. Importantly, transfer of T SCM cells into immune-deficient Rag.Ldlr recipient mice that lacked T cells increased atherosclerosis, illustrating the importance of these cells in atherogenesis., Conclusions: CD8 + T SCM cells are increased in humans with high CVD. As these T SCM cells promote atherosclerosis, targeting them may attenuate atherosclerotic plaque progression.

Background: Understanding neutrophil heterogeneity and its relationship to disease progression has become a recent focus of cancer research. Indeed, several studies have identified neutrophil subpopulations associated with protumoral or antitumoral functions. However, this work has been hindered by a lack of widely accepted markers with which to define neutrophil subpopulations., Methods: To identify markers of neutrophil heterogeneity in cancer, we used single-cell cytometry by time-of-flight (CyTOF) coupled with high-dimensional analysis on blood samples from treatment-naïve patients with melanoma., Results: Our efforts allowed us to identify seven blood neutrophil clusters, including two previously identified individual populations. Interrogation of these neutrophil subpopulations revealed a positive trend between specific clusters and disease stage. Finally, we recapitulated these seven blood neutrophil populations via flow cytometry and found that they exhibited diverse capacities for phagocytosis and reactive oxygen species production in vitro., Conclusions: Our data provide a refined consensus on neutrophil heterogeneity markers, enabling a prospective functional evaluation in patients with solid tumors., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Neutrophils are the most abundant peripheral immune cells and thus, are continually replenished by bone marrow-derived progenitors. Still, how newly identified neutrophil subsets fit into the bone marrow neutrophil lineage remains unclear. Here, we use mass cytometry to show that two recently defined human neutrophil progenitor populations contain a homogeneous progenitor subset we term "early neutrophil progenitors" (eNePs) (Lin - CD66b + CD117 + CD71 + ). Surface marker- and RNA-expression analyses, together with in vitro colony formation and in vivo adoptive humanized mouse transfers, indicate that eNePs are the earliest human neutrophil progenitors. Furthermore, we identified CD71 as a marker associated with the earliest neutrophil developmental stages. Expression of CD71 marks proliferating neutrophils, which were expanded in the blood of melanoma patients and detectable in blood and tumors from lung cancer patients. In summary, we establish CD117 + CD71 + eNeP as the inceptive human neutrophil progenitor and propose a refined model of the neutrophil developmental lineage in bone marrow., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mechanisms linking immune sensing of DNA danger signals in the extracellular environment to innate pathways in the cytosol are poorly understood. Here, we identify a previously unidentified immune-metabolic axis by which cells respond to purine nucleosides and trigger a type I interferon-β (IFN-β) response. We find that depletion of ADA2, an ectoenzyme that catabolizes extracellular dAdo to dIno, or supplementation of dAdo or dIno stimulates IFN-β. Under conditions of reduced ADA2 enzyme activity, dAdo is transported into cells and undergoes catabolysis by the cytosolic isoenzyme ADA1, driving intracellular accumulation of dIno. dIno is a functional immunometabolite that interferes with the cellular methionine cycle by inhibiting SAM synthetase activity. Inhibition of SAM-dependent transmethylation drives epigenomic hypomethylation and overexpression of immune-stimulatory endogenous retroviral elements that engage cytosolic dsRNA sensors and induce IFN-β. We uncovered a previously unknown cellular signaling pathway that responds to extracellular DNA-derived metabolites, coupling nucleoside catabolism by adenosine deaminases to cellular IFN-β production., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The diverse leukocyte infiltrate in atherosclerotic mouse aortas was recently analyzed in 9 single-cell RNA sequencing and 2 mass cytometry studies. In a comprehensive meta-analysis, we confirm 4 known macrophage subsets-resident, inflammatory, interferon-inducible cell, and Trem2 (triggering receptor expressed on myeloid cells-2) foamy macrophages-and identify a new macrophage subset resembling cavity macrophages. We also find that monocytes, neutrophils, dendritic cells, natural killer cells, innate lymphoid cells-2, and CD (cluster of differentiation)-8 T cells form prominent and separate immune cell populations in atherosclerotic aortas. Many CD4 T cells express IL (interleukin)-17 and the chemokine receptor CXCR (C-X-C chemokine receptor)-6. A small number of regulatory T cells and T helper 1 cells is also identified. Immature and naive T cells are present in both healthy and atherosclerotic aortas. Our meta-analysis overcomes limitations of individual studies that, because of their experimental approach, over- or underrepresent certain cell populations. Mass cytometry studies demonstrate that cell surface phenotype provides valuable information beyond the cell transcriptomes. The present analysis helps resolve some long-standing controversies in the field. First, Trem2 + foamy macrophages are not proinflammatory but interferon-inducible cell and inflammatory macrophages are. Second, about half of all foam cells are smooth muscle cell-derived, retaining smooth muscle cell transcripts rather than transdifferentiating to macrophages. Third, Pf4 , which had been considered specific for platelets and megakaryocytes, is also prominently expressed in the main population of resident vascular macrophages. Fourth, a new type of resident macrophage shares transcripts with cavity macrophages. Finally, the discovery of a prominent innate lymphoid cell-2 cluster links the single-cell RNA sequencing work to recent flow cytometry data suggesting a strong atheroprotective role of innate lymphoid cells-2. This resolves apparent discrepancies regarding the role of T helper 2 cells in atherosclerosis based on studies that predated the discovery of innate lymphoid cells-2 cells.

Monocytes and monocyte-derived cells, including Mϕs and dendritic cells, exhibit a diverse array of phenotypic states that are dictated by their surrounding microenvironment. These cells direct T cell activation and function via cues that range from being immunosuppressive to immunostimulatory. Solid tumors and atherosclerotic plaques represent two pathological niches with distinct immune microenvironments. While monocytes and their progeny possess a phenotypic spectrum found within both disease contexts, most within tumors are pro-tumoral and support evasion of host immune responses by tumor cells. In contrast, monocyte-derived cells within atherosclerotic plaques are usually pro-atherogenic, pro-inflammatory, and predominantly directed against self-antigens. Consequently, cancer immunotherapies strive to enhance the immune response against tumor antigens, whereas atherosclerosis treatments seek to dampen the immune response against lipid antigens. Insights into monocyte-T cell interactions within these niches could thus inform therapeutic strategies for two immunologically distinct diseases. Here, we review monocyte diversity, interactions between monocytes and T cells within tumor and plaque microenvironments, how certain therapies have leveraged these interactions, and novel strategies to assay such associations., (©2020 Society for Leukocyte Biology.)

Background: The role of tumor-associated macrophages (TAMs) in determining the outcome between the antitumor effects of the adaptive immune system and the tumor's anti-immunity stratagems, is controversial. Macrophages modulate their activities and phenotypes by integration of signals in the tumor microenvironment. Depending on how macrophages are activated, they may adopt so-called M1-like, antitumor or M2-like, protumor profiles. In many solid tumors, a dominance of M2-like macrophages is associated with poor outcomes but in some tumor types, strong M1-like profiles are linked to better outcomes. We aimed to investigate the interrelationship of these TAM populations to establish how they modulate the efficacy of the adaptive immune system in early lung cancer., Methods: Macrophages from matched lung (non-tumor-associated macrophages (NTAMs)) and tumor samples (TAMs) from resected lung cancers were assessed by bulk and single-cell transcriptomic analysis. Protein expression of genes characteristic of M1-like (chemokine (C-X-C motif) ligand 9) or M2-like (matrix metallopeptidase 12) functions was confirmed by confocal microscopy. Immunohistochemistry related the distribution of TAM transcriptomic signatures to density of CD8 + tissue-resident memory T cells (T RM ) in tumors and survival data from an independent cohort of 393 patients with lung cancer., Results: TAMs have significantly different transcriptomic profiles from NTAMs with >1000 differentially expressed genes. TAMs displayed a strong M2-like signature with no significant variation between patients. However, single-cell RNA-sequencing supported by immuno-stained cells revealed that additionally, in 25% of patients the M2-like TAMs also co-expressed a strong/hot M1-like signature (M1 hot ). Importantly, there was a strong association between the density of M1 hot TAMs and T RM cells in tumors that was in turn linked to better survival. Our data suggest a mechanism by which M1 hot TAMs may recruit T RM cells via CXCL9 expression and sustain them by making available more of the essential fatty acids on which T RM depend., Conclusions: We showed that in early lung cancer, expression of M1-like and M2-like gene signatures are not mutually exclusive since the same TAMs can simultaneously display both gene-expression profiles. The presence of M1 hot TAMs was associated with a strong T RM tumor-infiltrate and better outcomes. Thus, therapeutic approaches to re-program TAMs to an M1 hot phenotype are likely to augment the adaptive antitumor responses., Competing Interests: Competing interests: PV reports grants and personal fees from Pfizer, from null, outside the submitted work., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Nonclassical monocytes maintain vascular homeostasis by patrolling the vascular endothelium, responding to inflammatory signals, and scavenging cellular debris. Nonclassical monocytes also prevent metastatic tumor cells from seeding new tissues, but whether the patrolling function of nonclassical monocytes is required for this process is unknown. To answer this question, we utilized an inducible-knockout mouse that exhibits loss of the integrin-adaptor protein Kindlin-3 specifically in nonclassical monocytes. We show that Kindlin-3-deficient nonclassical monocytes are unable to patrol the vascular endothelium in either the lungs or periphery. We also find that Kindlin-3-deficient nonclassical monocytes cannot firmly adhere to, and instead "slip" along, the vascular endothelium. Loss of patrolling activity by nonclassical monocytes was phenocopied by ablation of LFA-1, an integrin-binding partner of Kindlin-3. When B16F10 murine melanoma tumor cells were introduced into Kindlin-3-deficient mice, nonclassical monocytes showed defective patrolling towards tumor cells and failure to ingest tumor particles in vivo. Consequently, we observed a significant, 4-fold increase in lung tumor metastases in mice possessing Kindlin-3-deficient nonclassical monocytes. Thus, we conclude that the patrolling function of nonclassical monocytes is mediated by Kindlin-3 and essential for these cells to maintain vascular endothelial homeostasis and prevent tumor metastasis to the lung., (©2020 Society for Leukocyte Biology.)

While the ontogeny and recruitment of the intestinal monocyte/macrophage lineage has been studied extensively, their precise localization and function has been overlooked. Here we show by imaging the murine small and large intestines in steady-state that intestinal CX3CR1 + macrophages form an interdigitated network intimately adherent to the entire mucosal lamina propria vasculature. The macrophages form contacts with each other, which are disrupted in the absence of microbiome, monocyte recruitment (Ccr2 -/- ), or monocyte conversion (Nr4a1 -/- ). In dysbiosis, gaps exist between the perivascular macrophages correlating with increased bacterial translocation from the lamina propria into the bloodstream. The recruitment of monocytes and conversion to macrophages during intestinal injury is also dependent upon CCR2, Nr4a1 and the microbiome. These findings demonstrate a relationship between microbiome and the maturation of lamina propria perivascular macrophages into a tight anatomical barrier that might function to prevent bacterial translocation. These cells are also critical for emergency vascular repair.

Macrophages (MΦ) play a critical role in tumor growth, immunosuppression, and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in MΦs that promote tumor immunosuppression will provide therapeutic benefit. PI3Kγ has been recently established by our group and others as a novel immuno-oncology target. Herein, we report that an MΦ Syk-PI3K axis drives polarization of immunosuppressive MΦs that establish an immunosuppressive tumor microenvironment in in vivo syngeneic tumor models. Genetic or pharmacologic inhibition of Syk and/or PI3Kγ in MΦs promotes a proinflammatory MΦ phenotype, restores CD8 + T-cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Assay for transposase-accessible Chromatin using Sequencing (ATAC-seq) analyses on the bone marrow-derived macrophages (BMDM) show that inhibition of Syk kinase promotes activation and binding of NF-κB motif in Syk MC-KO BMDMs, thus stimulating immunostimulatory transcriptional programming in MΦs to suppress tumor growth. Finally, we have developed in silico the "first-in-class" dual Syk/PI3K inhibitor, SRX3207, for the combinatorial inhibition of Syk and PI3K in one small molecule. This chemotype demonstrates efficacy in multiple tumor models and represents a novel combinatorial approach to activate antitumor immunity., (©2020 American Association for Cancer Research.)

The role of nonclassical, patrolling monocytes in lung tumor metastasis and their functional relationships with other immune cells remain poorly defined. Contributing to these gaps in knowledge is a lack of cellular specificity in commonly used approaches for depleting nonclassical monocytes. To circumvent these limitations and study the role of patrolling monocytes in melanoma metastasis to lungs, we generated C57BL/6J mice in which the Nr4a1 superenhancer E2 subdomain is ablated ( E2 -/- mice). E2 -/- mice lack nonclassical patrolling monocytes but preserve classical monocyte and macrophage numbers and functions. Interestingly, NK cell recruitment and activation were impaired, and metastatic burden was increased in E2 -/- mice. E2 -/- mice displayed unchanged "educated" (CD11b + CD27 + ) and "terminally differentiated" (CD11b + CD27 - ) NK cell frequencies. These perturbations were accompanied by reduced expression of stimulatory receptor Ly49D on educated NK cells and increased expression of inhibitory receptor NKG2A/CD94 on terminally differentiated NK cells. Thus, our work demonstrates that patrolling monocytes play a critical role in preventing lung tumor metastasis via NK cell recruitment and activation., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Neuropilin 1 (Nrp1) is a type I transmembrane protein that plays important roles in axonal guidance, neuronal development, and angiogenesis. Nrp1 also helps migrate thymus-derived regulatory T cells to vascular endothelial growth factor (VEGF)-producing tumors. However, little is known about the role of Nrp1 on CD4 T cells in atherosclerosis. In ApoE -/- mice fed a Western diet for 15 wk, we found a 2-fold increase in Nrp1 + Foxp3 - CD4 T cells in their spleens, periaortic lymph nodes, and aortas, compared with chow-fed mice. Nrp1 + Foxp3 - CD4 T cells had higher proliferation potential, expressed higher levels of the memory marker CD44, and produced more IFN-γ when compared with Nrp1 - CD4 T cells. Treatment of CD4 T cells with oxLDL increased Nrp1 expression. Furthermore, atherosclerosis-susceptible mice selectively deficient for Nrp1 expression on T cells developed less atherosclerosis than their Nrp1-sufficient counterparts. Mechanistically, we found that CD4 T cells that express Nrp1 have an increased capacity to migrate to the aorta and periaortic lymph nodes compared to Nrp1 - T cells, suggesting that the expression of Nrp1 facilitates the recruitment of CD4 T cells into the aorta where they can be pathogenic. Thus, we have identified a novel role of Nrp1 on CD4 T cells in atherosclerosis. These results suggest that manipulation of Nrp1 expression on T cells can affect the outcome of atherosclerosis and lower disease incidence., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Monocytes are innate immune cells of the mononuclear phagocyte system that have emerged as important regulators of cancer development and progression. Our understanding of monocytes has advanced from viewing these cells as a homogenous population to a heterogeneous system of cells that display diverse responses to different stimuli. During cancer, different monocyte subsets perform functions that contribute to both pro- and antitumoral immunity, including phagocytosis, secretion of tumoricidal mediators, promotion of angiogenesis, remodeling of the extracellular matrix, recruitment of lymphocytes, and differentiation into tumor-associated macrophages and dendritic cells. The ability of cancer to evade immune recognition and clearance requires protumoral signals to outweigh ongoing attempts by the host immune system to prevent tumor growth. This review discusses current understanding of monocyte heterogeneity during homeostasis, highlights monocyte functions in cancer progression, and describes monocyte-targeted therapeutic strategies for cancer treatment., (©2019 The Authors. Society for Leukocyte Biology Published by Wiley Periodicals, Inc.)

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Monocytes are innate blood cells that maintain vascular homeostasis and are early responders to pathogens in acute infections. There are three well-characterized classes of monocytes: classical (CD14 + CD16 - in humans and Ly6C hi in mice), intermediate (CD14 + CD16 + in humans and Ly6C + Treml4 + in mice), and nonclassical (CD14 - CD16 + in humans and Ly6C lo in mice). Classical monocytes are critical for the initial inflammatory response. Classical monocytes can differentiate into macrophages in tissue and can contribute to chronic disease. Nonclassical monocytes have been widely viewed as anti-inflammatory, as they maintain vascular homeostasis. They are a first line of defense in recognition and clearance of pathogens. However, their roles in chronic disease are less clear. They have been shown to be protective as well as positively associated with disease burden. This review focuses on the state of the monocyte biology field and the functions of monocytes, particularly nonclassical monocytes, in health and disease.

Objective- Three distinct human monocyte subsets have been identified based on the surface marker expression of CD14 and CD16. We hypothesized that monocytes were likely more heterogeneous in composition. Approach and Results- We used the high dimensionality of mass cytometry together with the FlowSOM clustering algorithm to accurately identify and define monocyte subsets in blood of healthy human subjects and those with coronary artery disease (CAD). To study the behavior and functionality of the newly defined monocyte subsets, we performed RNA sequencing, transwell migration, and efferocytosis assays. Here, we identify 8 human monocyte subsets based on their surface marker phenotype. We found that 3 of these subsets fall within the CD16 + nonclassical monocyte population and 4 subsets belong to the CD14 + classical monocytes, illustrating significant monocyte heterogeneity in humans. As nonclassical monocytes are important in modulating atherosclerosis in mice, we studied the functions of our 3 newly identified nonclassical monocytes in subjects with CAD. We found a marked expansion of a Slan + CXCR6 + nonclassical monocyte subset in CAD subjects, which was positively correlated with CAD severity. This nonclassical subset can migrate towards CXCL16 and shows an increased efferocytosis capacity, indicating it may play an atheroprotective role. Conclusions- Our data demonstrate that human nonclassical monocytes are a heterogeneous population, existing of several subsets with functional differences. These subsets have changed frequencies in the setting of severe CAD. Understanding how these newly identified subsets modulate CAD will be important for CAD-based therapies that target myeloid cells.

Neutrophils are the first line of defense against bacteria and fungi and help combat parasites and viruses. They are necessary for mammalian life, and their failure to recover after myeloablation is fatal. Neutrophils are short-lived, effective killing machines. Their life span is significantly extended under infectious and inflammatory conditions. Neutrophils take their cues directly from the infectious organism, from tissue macrophages and other elements of the immune system. Here, we review how neutrophils traffic to sites of infection or tissue injury, how they trap and kill bacteria, how they shape innate and adaptive immune responses, and the pathophysiology of monogenic neutrophil disorders., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

In this Letter, affiliation number 1 was originally missing from the HTML; the affiliations were missing for author Ming-Yow Hung in the HTML; and the Fig. 4 legend erroneously referred to panels a-h, instead of a-g. These errors have been corrected online.

Neutrophils are short-lived cells that play important roles in both health and disease. Neutrophils and monocytes originate from the granulocyte monocyte progenitor (GMP) in bone marrow; however, unipotent neutrophil progenitors are not well defined. Here, we use cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) methodologies to identify a committed unipotent early-stage neutrophil progenitor (NeP) in adult mouse bone marrow. Importantly, we found a similar unipotent NeP (hNeP) in human bone marrow. Both NeP and hNeP generate only neutrophils. NeP and hNeP both significantly increase tumor growth when transferred into murine cancer models, including a humanized mouse model. hNeP are present in the blood of treatment-naive melanoma patients but not of healthy subjects. hNeP can be readily identified by flow cytometry and could be used as a biomarker for early cancer discovery. Understanding the biology of hNeP should allow the development of new therapeutic targets for neutrophil-related diseases, including cancer., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Rationale: Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is poorly understood., Objective: We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single-cell RNA-sequencing and mass cytometry (cytometry by time of flight) to define an atlas of the immune cell landscape in atherosclerosis., Methods and Results: Using single-cell RNA-sequencing of aortic leukocytes from chow diet- and Western diet-fed Apoe -/- and Ldlr -/- mice, we detected 11 principal leukocyte clusters with distinct phenotypic and spatial characteristics while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on the single-cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic Apoe -/- and Ldlr -/- mice. We confirmed the phenotypic diversity of these clusters with a novel mass cytometry 35-marker panel with metal-labeled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of single-cell RNA-sequencing, mass cytometry, and fluorescence-activated cell sorting, we detected 3 principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Leukocyte cluster gene signatures revealed leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients., Conclusions: The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunologic mechanisms and cell-type-specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis., (© 2018 American Heart Association, Inc.)

Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes 1 . Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL 2-4 . Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr -/- background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr -/- mice, Ldlr -/- E06-scFv mice had 57-28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis.

The clinical successes in immunotherapy have been both astounding and at the same time unsatisfactory. Countless patients with varied tumor types have seen pronounced clinical response with immunotherapeutic intervention; however, many more patients have experienced minimal or no clinical benefit when provided the same treatment. As technology has advanced, so has the understanding of the complexity and diversity of the immune context of the tumor microenvironment and its influence on response to therapy. It has been possible to identify different subclasses of immune environment that have an influence on tumor initiation and response and therapy; by parsing the unique classes and subclasses of tumor immune microenvironment (TIME) that exist within a patient's tumor, the ability to predict and guide immunotherapeutic responsiveness will improve, and new therapeutic targets will be revealed.

Regulatory T (Treg) cells contribute to the anti-inflammatory response during atherogenesis. Here we show that during atherogenesis Treg cells lose Foxp3 expression and their immunosuppressive function, leading to the conversion of a fraction of these cells into T follicular helper (Tfh) cells. We show that Tfh cells are pro-atherogenic and that their depletion reduces atherosclerosis. Mechanistically, the conversion of Treg cells to Tfh cells correlates with reduced expression of IL-2Rα and pSTAT5 levels and increased expression of IL-6Rα. In vitro, incubation of naive T cells with oxLDL prevents their differentiation into Treg cells. Furthermore, injection of lipid-free Apolipoprotein AI (ApoAI) into ApoE -/- mice reduces intracellular cholesterol levels in Treg cells and prevents their conversion into Tfh cells. Together our results suggest that ApoAI, the main protein in high-density lipoprotein particles, modulates the cellular fate of Treg cells and thus influences the immune response during atherosclerosis.