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Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASC) to long-lived plasma cells (LLPC). We provide single cell transcriptional resolution of 17,347 BM ASC from 5 healthy adults. Fifteen clusters were identified ranging from newly minted ASC (cluster 1) expressing MKI67 and high MHC Class II that progressed to late clusters 5-8 through intermediate clusters 2-4. Additional clusters included early and late IgM-predominant ASC of likely extra-follicular origin; IFN-responsive; and high mitochondrial activity ASC. Late ASCs were distinguished by differences in G2M checkpoints, MTOR signaling, distinct metabolic pathways, CD38 expression, and utilization of TNF-receptor superfamily members. They mature through two distinct paths differentiated by the degree of TNF signaling through NFKB. This study provides the first single cell resolution atlas and molecular roadmap of LLPC maturation, thereby providing insight into differentiation trajectories and molecular regulation of these essential processes in the human BM microniche. This information enables investigation of the origin of protective and pathogenic antibodies in multiple diseases and development of new strategies targeted to the enhancement or depletion of the corresponding ASC.One Sentence Summary:The single cell transcriptomic atlas of human bone marrow plasma cell heterogeneity shows maturation of class-switched early and late subsets, specific IgM and Interferon-driven clusters, and unique heterogeneity of the late subsets which encompass the long-lived plasma cells.

Background Crohn’s disease is a lifelong disease characterized by chronic inflammation of the gastrointestinal tract. Defining the cellular and transcriptional composition of the mucosa at different stages of disease progression is needed for personalized therapy in Crohn’s. Methods Ileal biopsies were obtained from (1) control subjects (n = 6), (2) treatment-naïve patients (n = 7), and (3) established (n = 14) Crohn’s patients along with remission (n = 3) and refractory (n = 11) treatment groups. The biopsies processed using 10x Genomics single cell 5' yielded 139 906 cells. Gene expression count matrices of all samples were analyzed by reciprocal principal component integration, followed by clustering analysis. Manual annotations of the clusters were performed using canonical gene markers. Cell type proportions, differential expression analysis, and gene ontology enrichment were carried out for each cell type. Results We identified 3 cellular compartments with 9 epithelial, 1 stromal, and 5 immune cell subtypes. We observed differences in the cellular composition between control, treatment-naïve, and established groups, with the significant changes in the epithelial subtypes of the treatment-naïve patients, including microfold, tuft, goblet, enterocyte,s and BEST4+ cells. Surprisingly, fewer changes in the composition of the immune compartment were observed; however, gene expression in the epithelial and immune compartment was different between Crohn’s phenotypes, indicating changes in cellular activity. Conclusions Our study identified cellular and transcriptional signatures associated with treatment-naïve Crohn’s disease that collectively point to dysfunction of the intestinal barrier with an increase in inflammatory cellular activity. Our analysis also highlights the heterogeneity among patients within the same disease phenotype, shining a new light on personalized treatment responses and strategies.

The self-renewal ability is a unique property of fetal-derived innate-like B-1a lymphocytes, which survive and function without being replenished by bone marrow (BM) progenitors. However, the mechanism by which IgM-secreting mature B-1a lymphocytes self-renew is poorly understood. In this study, we showed that Bmi1 was critically involved in this process. Although Bmi1 is considered essential for lymphopoiesis, the number of mature conventional B cells was not altered when Bmi1 was deleted in the B cell lineage. In contrast, the number of peritoneal B-1a cells was significantly reduced. Peritoneal cell transfer assays revealed diminished self-renewal ability of Bmi1-deleted B-1a cells, which was restored by additional deletion of Ink4-Arf, the well-known target of Bmi1. Fetal liver cells with B cell–specific Bmi1 deletion failed to repopulate peritoneal B-1a cells, but not other B-2 lymphocytes after transplantation assays, suggesting that Bmi1 may be involved in the developmental process of B-1 progenitors to mature B-1a cells. Although Bmi1 deletion has also been shown to alter the microenvironment for hematopoietic stem cells, fat-associated lymphoid clusters, the reported niche for B-1a cells, were not impaired in Bmi1−/− mice. RNA expression profiling suggested lysine demethylase 5B (Kdm5b) as another possible target of Bmi1, which was elevated in Bmi1−/− B-1a cells in a stress setting and might repress B-1a cell proliferation. Our work has indicated that Bmi1 plays pivotal roles in self-renewal and maintenance of fetal-derived B-1a cells.

Tissue-resident macrophages (TRMΦ) are important immune sentinels responsible for maintaining tissue and immune homeostasis within their specific niche. Recently, the origins of TRMΦ have undergone intense scrutiny, in which now most TRMΦ are thought to originate early during embryonic development independent of hematopoietic stem cells (HSCs). We previously characterized two distinct subsets of mouse peritoneal cavity macrophages (MΦ) (large and small peritoneal MΦ) whose origins and relationship to both fetal and adult long-term (LT) HSCs have not been fully investigated. In this study, we employ highly purified LT-HSC transplantation and in vivo lineage tracing to show a dual ontogeny for large and small peritoneal MΦ, in which the initial wave of peritoneal MΦ is seeded from yolk sac–derived precursors, which later require LT-HSCs for regeneration. In contrast, transplanted fetal and adult LT-HSCs are not able to regenerate brain-resident microglia. Thus, we demonstrate that LT-HSCs retain the potential to develop into TRMΦ, but their requirement is tissue specific in the peritoneum and brain.

Myeloid cells comprise the majority of immune cells in tumors, where their content and composition is determined by tumor type and driver mutation. While these cells are essential for shaping the tumor microenvironment, promoting tumor growth, and contributing to therapeutic resistance, targeting tumor-associated myeloid cells, including bone-marrow-derived monocytes and neutrophils, has not been successful in the clinics. Monocyte chemoattractant protein (MCP) family, comprising of Ccl2, Ccl7, Ccl8, Ccl12, are essential for monocytes trafficking to the tumor sites. To eliminate monocyte recruitment, we leveraged CRISPR/Cas-9 based gene editing tool to generate a mouse strain that is devoid of all MCP genes, which we termed quadruple MCP knockout (qMCP-/-). Using these mice in combination with genetically engineered mouse models (GEMM) of glioblastoma (GBM), we abolished tumor monocyte infiltration. Due to the functional redundancy of MCP family members, we show that targeting individual MCP genes leads to compensation by other MCPs. In contrast, when all MCPs are genetically deleted and monocyte recruitment is abolished, neutrophil infiltration ensues. Single-cell RNA sequencing revealed that intratumoral neutrophils promoted proneural-to-mesenchymal transition in GBM, and supported tumor aggression by facilitating hypoxia response via TNF production. Remarkably, pharmacologic or genetic interventions that suppress both monocytes and neutrophil infiltration improve the survival of GBM-bearing mice. Taken together, our findings establish that specific subsets of myeloid cells can influence the dynamism of tumor microenvironment, and they emphasize the importance of targeting both monocytes and neutrophils simultaneously for effective GBM immunotherapy.

SummaryIn late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from Wuhan, China spurring the Coronavirus Disease-19 (COVID-19) pandemic that has resulted in over 219 million confirmed cases and nearly 4.6 million deaths worldwide. Intensive research efforts ensued to constrain SARS-CoV-2 and reduce COVID-19 disease burden. Due to the severity of this disease, the US Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level 3 (BSL3) containment laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred and manipulated at lower containment levels (i.e., BSL2), and maintain the fidelity of downstream assays to expedite the development of medical countermeasures (MCMs). We demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with paraformaldehyde solution or other commonly-used branded reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and antibody detection assays, heat inactivation following cDNA amplification of single-cell emulsions for droplet-based single-cell mRNA sequencing applications, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of protocols for viral inactivation of SARS-CoV-2 and COVID-19 patient samples for downstream contemporary immunology assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.

Single-cell RNA sequencing (scRNA-seq) can reveal accurate and sensitive RNA abundance in a single sample, but robust integration of multiple samples remains challenging. Large-scale scRNA-seq data generated by different workflows or laboratories can contain batch-specific systemic variation. Such variation challenges data integration by confounding sample-specific biology with undesirable batch-specific systemic effects. Therefore, there is a need for guidance in selecting computational and experimental approaches to minimize batch-specific impacts on data interpretation and a need to empirically evaluate the sources of systemic variation in a given dataset. To uncover the contributions of experimental variables to systemic variation, we intentionally perturb four potential sources of batch-effect in five human peripheral blood samples. We investigate sequencing replicate, sequencing depth, sample replicate, and the effects of pooling libraries for concurrent sequencing. To quantify the downstream effects of these variables on data interpretation, we introduced a new scoring metric, the Cell Misclassification Statistic (CMS), which identifies losses to cell type fidelity that occur when merging datasets of different batches. CMS reveals an undesirable overcorrection by popular batch-effect correction and data integration methods. We show that optimizing gene expression matrix normalization and merging can reduce the need for batch-effect correction and minimize the risk of overcorrecting true biological differences between samples.

Single-cell transcriptomics enables the definition of diverse human immune cell types across multiple tissues and disease contexts. Still, deeper biological understanding requires comprehensive integration of multiple single-cell omics (transcriptomic, proteomic, and cell-receptor repertoire). To improve the identification of diverse cell types and the accuracy of cell-type classification in multi-omics single-cell datasets, we developed SuPERR-seq, a novel analysis workflow to increase the resolution and accuracy of clustering and allow for the discovery of previously hidden cell subsets. In addition, SuPERR-seq accurately removes cell doublets and prevents widespread cell-type misclassification by incorporating information from cell-surface proteins and immunoglobulin transcript counts. This approach uniquely improves the identification of heterogeneous cell types in the human immune system, including a novel subset of antibody-secreting cells in the bone marrow.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the ensuing COVID-19 pandemic have caused ∼40 million cases and over 648,000 deaths in the United States alone. Troubling disparities in COVID-19-associated mortality emerged early, with nearly 70% of deaths confined to Black/African-American (AA) patients in some areas, yet targeted studies within this demographic are scant. Multi-omics single-cell analyses of immune profiles from airways and matching blood samples of Black/AA patients revealed low viral load, yet pronounced and persistent pulmonary neutrophilia with advanced features of cytokine release syndrome and acute respiratory distress syndrome (ARDS), including exacerbated production of IL-8, IL-1β, IL-6, and CCL3/4 along with elevated levels of neutrophil elastase and myeloperoxidase. Circulating S100A12+/IFITM2+mature neutrophils are recruited via the IL-8/CXCR2 axis, which emerges as a potential therapeutic target to reduce pathogenic neutrophilia and constrain ARDS in severe COVID-19.Graphical AbstractThe lung pathology due to severe COVID-19 is marked by a perpetual pathogenic neutrophilia, leading to acute respiratory distress syndrome (ARDS) even in the absence of viral burden. Circulating mature neutrophils are recruited to the airways via IL-8 (CXCL8)/CXCR2 chemotaxis. Recently migrated neutrophils further differentiate into a transcriptionally active and hyperinflammatory state, with an exacerbated expression of IL-8 (CXCL8), IL-1β (IL1B),CCL3, CCL4, neutrophil elastase (NE), and myeloperoxidase (MPO) activity. Airway neutrophils and recruited inflammatory monocytes further increase their production of IL-8 (CXCL8), perpetuating lung neutrophilia in a feedforward loop. MdCs and T cells produce IL-1β and TNF, driving neutrophils reprogramming and survival.

CD8+ T cells are important antiviral effectors that can potentiate long-lived immunity against COVID-19, but a detailed characterization of these cells has been hampered by technical challenges. We screened 21 well-characterized, longitudinally-sampled convalescent donors that recovered from mild COVID-19 against a collection of SARS-CoV-2 tetramers, and identified one participant with an immunodominant response against Nuc322–331, a peptide that is conserved in all the SARS-CoV-2 variants-of-concern reported to date. We conducted 38-parameter CyTOF phenotyping on tetramer-identified Nuc322–331-specific CD8+ T cells, and on CD4+ and CD8+ T cells recognizing the entire nucleocapsid and spike proteins from SARS-CoV-2, and took 32 serological measurements on longitudinal specimens from this participant. We discovered a coordination of the Nuc322–331-specific CD8+ T response with both the CD4+ T cell and antibody pillars of adaptive immunity. Nuc322–331-specific CD8+ T cells were predominantly central memory T cells, but continually evolved over a ~6-month period of convalescence. We observed a slow and progressive decrease in the activation state and polyfunctionality of the Nuc322–331-specific CD8+ T cells, accompanied by an increase in their lymph-node homing and homeostatic proliferation potential. These results suggest that following a typical case of mild COVID-19, SARS-CoV-2-specific CD8+ T cells not only persist but continuously differentiate in a coordinated fashion well into convalescence, into a state characteristic of long-lived, self-renewing memory.

CD8+ T cells are important antiviral effectors that can potentiate long-lived immunity against COVID-19, but a detailed characterization of these cells has been hampered by technical challenges. We screened 21 well-characterized, longitudinally-sampled convalescent donors that recovered from mild COVID-19 against a collection of SARS-CoV-2 tetramers, and identified one participant with an immunodominant response against Nuc322-331, a peptide that is conserved in all the SARS-CoV-2 variants-of-concern reported to date. We conducted 38- parameter CyTOF phenotyping on tetramer-identified Nuc322-331-specific CD8+ T cells, and on CD4+ and CD8+ T cells recognizing the entire nucleocapsid and spike proteins from SARS- CoV-2, and took 32 serological measurements on longitudinal specimens from this participant. We discovered a coordination of the Nuc322-331-specific CD8+ T response with both the CD4+ T cell and antibody pillars of adaptive immunity. Nuc322-331-specific CD8+ T cells were predominantly central memory T cells, but continually evolved over a ∼6-month period of convalescence. We observed a slow and progressive decrease in the activation state and polyfunctionality of the Nuc322-331-specific CD8+ T cells, accompanied by an increase in their lymph-node homing and homeostatic proliferation potential. These results suggest that following a typical case of mild COVID-19, SARS-CoV-2-specific CD8+ T cells not only persist but continuously differentiate in a coordinated fashion well into convalescence, into a state characteristic of long-lived, self-renewing memory.

We developed a protocol for the preparation of a single-cell suspension of human intestinal (ileal) biopsies, optimized for contemporary multi-omics single-cell assays including high-dimensional flow cytometry and single-cell RNA sequencing (scRNA-seq) assays. The protocol provides a rapid and efficient method of digesting human intestinal biopsies into a single-cell suspension resulting in a high viable cell yield and preservation of cell-surface markers. Notably, the protocol utilizes a Collagenase-I/Benzonase-based 37°C dissociation method, which collects already digested cells at several steps during the process to prevent over-digestion (fractioning technique). We obtained a total cell yield of ~5.8E6 +/- 1.9E6 cells, with a viability of 71% +/- 6% (n=9) (Figures 1A and 1B) from biopsy weights ranging 10-14 mg. By flow cytometry, all major immune cell lineages were detected, as well as a CD45-/EpCAM+ epithelial cell population (20%-60% of total viable singlets; Figure 1C). scRNA-seq analysis (10X Genomics; 5’/VDJ with feature barcoding) showed high-quality viable cells with low mitochondrial gene counts, indicating this protocol exerts minimal stress on the digested cells (Figure 1D).

Here we present an optimized protocol for the single-cell suspension of murine intestines (here we used ileum) optimized for contemporary multi-omics single-cell assays including high-dimensional flow cytometry and single-cell RNA-seq. This protocol provides a rapid and efficient method of digesting mouse ileum into a single-cell suspension with a short processing time, high viable cell yield, and it preserves cell-surface markers throughout the process. Notably, the protocol utilizes a Collagenase-I/Benzonase-based 37°C water bath dissociation method and collects already digested cells at several steps during the process to prevent over-digestion (fractioning technique). Tissues were normalized by length (2.5 cm) and weight (averaged 79.5 mg) and measured for cell yield and viability. We obtained an average total cell yield of ~4.4E6 +/- 1.4E6 cells, and average viability of ~56 +/- 13% (n=7) (Figure 1). By flow cytometry, ~30%-50% of cells were CD45+ immune cells, with the remainder of the cell population being CD45- non-immune cells (i.e., epithelial and stromal cells). To optimize this protocol, several other conditions were tested, including different digestive enzymes, mechanical digestion techniques, the inclusion of an intraepithelial layer (IEL) digestion, and an antioxidant cocktail addition. We summarize our main findings about the optimization of this protocol below: Collagenase-I provided the highest number of total yield and viable cells when compared to Collagenase-IV and Liberase. Enzymatic digestion in a 37°C waterbath and vortexing the cell solutions proved more successful in cell yield and viability than different combinations of the gentleMACS Tissue Dissociator (Myltenyi) intestine digestion programs, including its m_ intestine _1 and 37_m_LPDK programs and variations in their run times. We have also validated a protocol to separate the IEL before the rest of the ileum, and have found it compatible and therefore able to be adopted with the recommended protocol while maintaining a similar yield of viable cells. An IEL separation protocol has been included in a separate section at the end. By using our "fractioning technique", in which cells that are digested early on are removed before digesting the rest of the tissue separately, total cell yield and viability are increased. We tested several antioxidant combinations (Gluthatione, Vitamin C, and Vitamin E) and found higher live cell yield when excluding these antioxidants from the Collagenase-I digestion step. Based on these findings, this murine intestinal digestion protocol is rapid and efficient and can be used for multi-omics single-cell assays such as high-dimensional flow cytometry and single-cell RNA-sequencing.

COVID-19 patients are generally asymptomatic during initial SARS-CoV-2 replication, but may suffer severe immunopathology after the virus has receded and blood monocytes have infiltrated the airways. In the bronchoalveolar lavage fluid from patients with severe COVID-19, lung-infiltrating monocytes expressed high mRNA levels encoding inflammatory mediators, including CXCL8 and IL-1s, and contained SARS-CoV-2 transcripts. To study this process in more depth, we developed a novel organotypic model whereby primary human blood monocytes are transmigrated across a differentiated human lung epithelium infected by SARSCoV-2. Infiltrating monocytes acquired SARS-CoV-2 from the epithelium and upregulated expression and secretion of inflammatory mediators including CXCL8 and IL-1s, mirroring in vivo data. The JAK1/2 inhibitor baricitinib gained emergency use authorization by the FDA for the treatment of COVID-19 originally in combination with the antiviral remdesivir, and recently as a stand-alone treatment. To explore the mechanisms by which baricitinib alone or in combination with remdesivir may result in more favorable disease outcomes, we leveraged this model to characterize viral burden, gene expression and inflammatory mediator secretion by lung epithelial cells and infiltrating monocytes. As expected, remdesivir decreased viral burden in both the epithelium and monocytes, while baricitinib enhanced antiviral signaling and decreased specific inflammatory mediators in monocytes. Combined use of baricitinib and remdesivir enhanced the rate of virus clearance from SARS-CoV-2-positive monocytes. Taken together, baricitinib enhances the antiviral state of monocytes infiltrating the COVID-19 lung, while decreasing the expression of inflammatory mediators, thus limiting the likelihood of a cytokine storm and ensuing acute respiratory distress syndrome (ARDS). Funding: This study was supported by NSF EAGER award 2032273 (to RT, RFS and KZ) and Woodruff Health Science Center COVID-19 CURE award (to RT, RFS, KZ, MB and VS). RFS is also funded in part by NIH grant R01MH116695. We thank Dr. Matt Frieman (University of Maryland) for sharing primer sequences targeting the N-subgenome of SARS-CoV-2. We thank Dalia Gulick and Gregory Gibson (Georgia Tech) for running the monocyte cDNA samples for multiplexed qRT-PCR. SARS-CoV-2, Isolate USA-WA1/2020 was obtained from BEI Resources, Manassas, VA. Declaration of Interests: RFS is the inventor of the use of baricitinib for coronavirus infections and receives royalties from Eli Lilly and Company. His conflict of interest has been reviewed and approved by Emory University. All other authors have declared that no conflict of interest exists.

SUMMARY Convalescing COVID-19 patients mount robust T cell responses against SARS-CoV-2, suggesting an important role for T cells in viral clearance. To date, the phenotypes of SARS-CoV-2-specific T cells remain poorly defined. Using 38-parameter CyTOF, we phenotyped longitudinal specimens of SARS-CoV-2-specific CD4+ and CD8+ T cells from nine individuals who recovered from mild COVID-19. SARS-CoV-2-specific CD4+ T cells were exclusively Th1 cells, and predominantly Tcm with phenotypic features of robust helper function. SARS-CoV-2-specific CD8+ T cells were predominantly Temra cells in a state of less terminal differentiation than most Temra cells. Subsets of SARS-CoV-2-specific T cells express CD127, can homeostatically proliferate, and can persist for over two months. Our results suggest that long-lived and robust T cell immunity is generated following natural SARS-CoV-2 infection, and support an important role for SARS-CoV-2-specific T cells in host control of COVID-19., Graphical Abstract, Highlights • SARS-CoV-2-specific T cells are diverse, express CD127, and can proliferate • SARS-CoV-2-specific CD4+ T cells are IFNγ-producing, lymphoid-homing, Tfh cells • SARS-CoV-2-specific CD8+ T cells are predominantly less-differentiated Temra • Homing features of SARS-CoV-2-specific CD4+ and CD8+ T cells differ

Wide heterogeneity of disease course ranging from asymptomatic spread to respiratory failure and death has become a hallmark of the SARS-CoV-2 pandemic. While this clinical spectrum is well documented, its immunologic underpinnings are less clear. We have therefore, initiated studies of the B cell responses as they would participate in both early effector responses and in the initiation of memory formation. In terms of effector responses, we were particularly interested in the engagement and clinical correlates of the extra-follicular pathway (EF), we recently described in flaring SLE. In this systemic autoimmune disease, the EF pathway is initiated by newly activated naive B cell (aN) leading to large expansion of autoantibody-producing antibody-secreting cells through the generation of an epigenetically primed B cell precursor which are double negative (DN) for naive (IgD) and memory markers (CD27) and lacking expression of CXCR5 and CD21 (DN2). These highly activated D2 cells are also distinguished by high expression of CD11c and T-bet and are TLR7-driven. Both, TLR7-stimulation which is triggered by ssRNA and the central role played by their murine counterparts (typically characterized as Age-Associated B cells), in viral clearance, strongly supported the hypothesis that DN2 cells and the global EF pathway could be prominently engaged in COVID-19 patients. Also of note, EF B cell activation is particularly prominent in SLE patients of African-American ancestry, a population disproportionately represented in severe COVID-19. In this study we find that critically-ill patients with COVID-19 robustly upregulate constituents of the extrafollicular pathway, produce enormous numbers of antibody secreting cells, and lose unique transitional B cell populations that correlate with positive prognosis. This patient cluster associates tightly with biomarkers of poor outcomes and exhibits high rates of mortality. Thus, this B cell phenotype might serve as an immunological marker of severe COVID infection at early stages and could therefore identify a patient subset likely to benefit from targeted immunomodulatory therapy aimed at alleviating disease burden.

Convalescing coronavirus disease 2019 (COVID-19) patients mount robust Tcell responses against SARS-CoV-2, suggesting an important role of Tcells in viral clearance. To date, the phenotypes of SARS-CoV-2-specific Tcells remain poorly defined. Using 38-parameter CyTOF, we phenotyped longitudinal specimens of SARS-CoV-2-specific CD4+ and CD8+ Tcells from nine individuals who recovered from mild COVID-19. SARS-CoV-2-specific CD4+ Tcells were exclusively Th1 cells and predominantly Tcm cells with phenotypic features of robust helper function. SARS-CoV-2-specific CD8+ Tcells were predominantly Temra cells in a state of less terminal differentiation than most Temra cells. Subsets of SARS-CoV-2-specific Tcells express CD127, can proliferate homeostatically, and can persist for over 2months. Our results suggest that long-lived and robust Tcell immunity is generated following natural SARS-CoV-2 infection and support an important role of SARS-CoV-2-specific Tcells in host control of COVID-19.

Disruption of vulnerable atherosclerotic plaques often leads to myocardial infarction and stroke, the leading causes of morbidity and mortality in the United States. A diagnostic method that detects high-risk atherosclerotic plaques at early stages could prevent these sequelae. The abundance of immune cells in the arterial wall, especially inflammatory Ly-6C(hi) monocytes and foamy macrophages, is indicative of plaque‎ inflammation, and may be associated with plaque vulnerability. Hence, we sought to develop a new method that specifically targets these immune cells to offer clinically-relevant diagnostic information about cardiovascular disease. We combine ultra-selective nanoparticle targeting of Ly-6C(hi) monocytes and foamy macrophages with clinically-viable photoacoustic imaging (PAI) in order to precisely and specifically image inflamed plaques ex vivo in a mouse model that mimics human vulnerable plaques histopathologically. Within the plaques, high-dimensional single-cell flow cytometry (13-parameter) showed that our nanoparticles were almost-exclusively taken up by the Ly-6C(hi) monocytes and foamy macrophages that heavily infiltrate plaques. PAI identified inflamed atherosclerotic plaques that display ~6-fold greater signal compared to controls (P

The current paradigm that a single long-term hematopoietic stem cell can regenerate all components of the mammalian immune system has been challenged by recent findings in mice. These findings show that adult tissue-resident macrophages and innate-like lymphocytes develop early in fetal hematopoiesis from progenitors that emerge prior to, and apparently independently of, conventional long-term hematopoietic stem cells. Here, we discuss these recent findings, which show that an early and distinct wave of hematopoiesis occurs for all major hematopoietic lineages. These data provide evidence that fetal hematopoietic progenitors not derived from the bona fide long-term hematopoietic stem cells give rise to tissue-resident immune cells that persist throughout adulthood. We also discuss recent insights into B lymphocyte development and attempt to synthesize seemingly contradictory recent findings on the origins of innate-like B-1a lymphocytes during fetal hematopoiesis.

Summary B cells are key components of cellular and humoral immunity and, like all lymphocytes, are thought to originate and renew from hematopoietic stem cells (HSCs). However, our recent single-HSC transfer studies demonstrate that adult bone marrow HSCs do not regenerate B-1a, a subset of tissue B cells required for protection against pneumonia, influenza, and other infections. Since B-1a are regenerated by transfers of fetal liver, the question arises as to whether B-1a derive from fetal, but not adult, HSCs. Here we show that, similar to adult HSCs, fetal HSCs selectively fail to regenerate B-1a. We also show that, in humanized mice, human fetal liver regenerates tissue B cells that are phenotypically similar to murine B-1a, raising the question of whether human HSC transplantation, the mainstay of such models, is sufficient to regenerate human B-1a. Thus, our studies overtly challenge the current paradigm that HSCs give rise to all components of the immune system., Highlights • Purified LT-HSC transplantation fails to fully regenerate the murine immune system • LT-HSC transplants selectively fail to regenerate B-1a cells • LT-HSC transplantation does not regenerate VH11-encoded natural antibodies • Human fetal liver regenerate peritoneal B cells that resemble murine B-1a, Ghosn and colleagues show that purified HSC transplantation selectively fails to regenerate B-1a, a subset of B cells known to be required for protection against pneumonia, influenza, and other infections. Moreover, HSC transplantation does not restore a key repertoire (VH11) of natural antibodies, raising the question of whether human HSC transplantation is sufficient to fully regenerate the immune system.

The accepted dogma has been that a single long-term hematopoietic stem cell (LT-HSC) can reconstitute all components of the immune system. However, our single-cell transfer studies have shown that highly purified LT-HSCs selectively fail to reconstitute B-1a cells in otherwise fully reconstituted hosts (i.e., LT-HSCs fully reconstitute follicular, marginal zone, and B-1b B cells, but not B-1a cells). These results suggest that B-1a cells are a separate B cell lineage that develops independently of classical LT-HSCs. We provide an evolutionary two-pathway development model (HSC independent versus HSC dependent), and suggest that this lineage separation is employed not only by B cells but by all hematopoietic lineages. Collectively, these findings challenge the current notion that LT-HSCs can reconstitute all components of the immune system and raise key questions about human HSC transplantation. We discuss the implications of these findings in light of our recent studies demonstrating the ability of B-1a cells to elicit antigen-specific responses that differ markedly from those mounted by follicular B cells. These findings have implications for vaccine development, in particular vaccines that may elicit the B-1a repertoire.

Amyloid fibrils composed of peptides as short as six amino acids are therapeutic in experimental autoimmune encephalomyelitis (EAE), reducing paralysis and inflammation, while inducing several pathways of immune suppression. Intraperitoneal injection of fibrils selectively activates B-1a lymphocytes and two populations of resident macrophages (MΦs), increasing IL-10 production, and triggering their exodus from the peritoneum. The importance of IL-10-producing B-1a cells in this effective therapy was established in loss-of-function experiments where neither B-cell-deficient (μMT) nor IL10(-/-) mice with EAE responded to the fibrils. In gain-of-function experiments, B-1a cells, adoptively transferred to μMT mice with EAE, restored their therapeutic efficacy when Amylin 28-33 was administered. Stimulation of adoptively transferred bioluminescent MΦs and B-1a cells by amyloid fibrils resulted in rapid (within 60 min of injection) trafficking of both cell types to draining lymph nodes. Analysis of gene expression indicated that the fibrils activated the CD40/B-cell receptor pathway in B-1a cells and induced a set of immune-suppressive cell-surface proteins, including BTLA, IRF4, and Siglec G. Collectively, these data indicate that the fibrils activate B-1a cells and F4/80(+) MΦs, resulting in their migration to the lymph nodes, where IL-10 and cell-surface receptors associated with immune-suppression limit antigen presentation and T-cell activation. These mechanisms culminate in reduction of paralytic signs of EAE.

Part of the flow/mass cytometry data analysis process is aligning (matching) cell subsets between relevant samples. Current methods address this cluster-matching problem in ways that are either computationally expensive, affected by the curse of dimensionality, or fail when population patterns significantly vary between samples. Here, we introduce a quadratic form (QF)-based cluster matching algorithm (QFMatch) that is computationally efficient and accommodates cases where population locations differ significantly (or even disappear or appear) from sample to sample. We demonstrate the effectiveness of QFMatch by evaluating sample datasets from immunology studies. The algorithm is based on a novel multivariate extension of the quadratic form distance for the comparison of flow cytometry data sets. We show that this QF distance has attractive computational and statistical properties that make it well suited for analysis tasks that involve the comparison of flow/mass cytometry samples.

The question of whether a single hematopoietic stem cell (HSC) gives rise to all of the B-cell subsets [B-1a, B-1b, B-2, and marginal zone (MZ) B cells] in the mouse has been discussed for many years without resolution. Studies here finally demonstrate that individual HSCs sorted from adult bone marrow and transferred to lethally irradiated recipients clearly give rise to B-2, MZ B, and B-1b, but does not detectably reconstitute B-1a cells. These findings place B-2, MZ, and B-1b in a single adult developmental lineage and place B-1a in a separate lineage derived from HSCs that are rare or missing in adults. We discuss these findings with respect to known developmental heterogeneity in other HSC-derived lymphoid, myeloid, and erythroid lineages, and how HSC developmental heterogeneity conforms to the layered model of the evolution of the immune system that we proposed some years ago. In addition, of importance to contemporary medicine, we consider the implications that HSC developmental heterogeneity may have for selecting HSC sources for human transplantation.

Changes in the frequencies of cell subsets that (co)express characteristic biomarkers, or levels of the biomarkers on the subsets, are widely used as indices of drug response, disease prognosis, stem cell reconstitution, etc. However, although the currently available computational “gating” tools accurately reveal subset frequencies and marker expression levels, they fail to enable statistically reliable judgements as to whether these frequencies and expression levels differ significantly between/among subject groups. Here we introduce flow cytometry data analysis pipeline which includes the Earth Mover’s Distance (EMD) metric as solution to this problem. Well known as an informative quantitative measure of differences between distributions, we present three exemplary studies showing that EMD 1) reveals clinically-relevant shifts in two markers on blood basophils responding to an offending allergen; 2) shows that ablative tumor radiation induces significant changes in the murine colon cancer tumor microenvironment; and, 3) ranks immunological differences in mouse peritoneal cavity cells harvested from three genetically distinct mouse strains.

Rationale: Basophils contribute to anaphylaxis and allergies. We examined the utility of assessing basophil-associated surface antigens (CD11b/CD63/CD123/CD203c/CD294) in characterizing and monitoring subjects with nut allergy. Methods: We used flow cytometry to analyze basophils at baseline (without any activation) and after ex vivo stimulation of whole blood by addition of nut or other allergens for 2, 10, and 30 min. We also evaluated whether basophil expression of CD11b/CD63/CD123/CD203c/CD294 was altered in subjects treated with anti-IgE monoclonal antibody (omalizumab) to reduce plasma levels of IgE. Results: We demonstrate that basophil CD203c levels are increased at baseline in subjects with nut allergy compared to healthy controls (13 subjects in each group, p < 0.0001). Furthermore, we confirm that significantly increased expression of CD203c occurs on subject basophils when stimulated with the allergen to which the subject is sensitive and can be detected rapidly (10 min of stimulation, n = 11, p < 0.0008). In 5 subjects with severe peanut allergy, basophil CD203c expression following stimulation with peanut allergen was significantly decreased (p < 0.05) after 4 and 8 weeks of omalizumab treatment but returned toward pretreatment levels after treatment cessation. Conclusions: Subjects with nut allergy show an increase of basophil CD203c levels at baseline and following rapid ex vivo stimulation with nut allergen. Both can be reduced by omalizumab therapy. These results highlight the potential of using basophil CD203c levels for baseline diagnosis and therapeutic monitoring in subjects with nut allergy.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that often leads to joint damage. The etiology of RA remains unclear, but new insights and therapeutic strategies have been facilitated by murine models. In the K/BxN serum transfer (STA) model, arthritis is triggered by autoantibodies against glucose-6-phosphate isomerase. The SKG model has a ZAP70 point mutation that reduces TCR signaling. After zymosan exposure, SKG mice develop arthritis, mediated by autoreactive Th17 cells. Joint inflammation in these models shares many features with RA, including leukocyte invasion, synovitis, and bone resorption. Neutrophils and Ly-6Chi monocytes are known to play critical roles in these models.1–4 Here, we investigated single-walled carbon nanotubes (SWNT) as candidate drug carriers to specifically target pathogenic cells in arthritic joints. Using FACS analysis, we found that, 2h and 4h after IV administration of SWNT, nearly 80% Ly-6Chi monocytes in blood had taken up SWNT in STA and SKG model, respectively. Near-infrared (NIR) photoluminescence whole-animal imaging of the STA model showed that SWNT were first detected in arthritic joints 3h post injection (p.i.) and reached a maximal level 24h p.i., In contrast, in non-arthritic mice, SWNT detected in joints remained low 24h p.i., suggesting SWNT can specifically target cells homing to or present in arthritic joints. In SKG mice, 2d p.i., SWNT were found in Ly-6Chi monocyte and in neutrophils in bone marrow and liver, but not in blood and spleen. FACS analysis indicated specific uptake of SWNT labelled with Cy5.5 in monocytes and neutrophils in human blood. We conclude that SWNT is a potential carrier to target drugs to pathogenic neutrophils and monocytes in arthritic joints.

The current paradigm posits that a single long-term hematopoietic stem cell (LT-HSC) is capable of regenerating all components of the immune system. However, recent studies have overturned this paradigm by showing that tissue-resident macrophages (in brain, skin, and liver) develop from fetal progenitors in the yolk sac that emerge separate from, and prior to, the LT-HSC. Recently, we have identified novel subsets of tissue-resident macrophages in the mouse peritoneum, namely Small and Large Peritoneal Macrophages (SPM and LPM). Here, using Runx1 lineage-tracing assays, we show that, similar to brain microglia, both SPM and LPM emerge at around embryonic day 8 (E8), prior to, and independent of, the conventional LT-HSC. However, after E11, the fetal liver LT-HSC can be called upon to regenerate both SPM and LPM, but not microglia. Single-cell high-dimensional phenotypic and gene expression assays reveal that LT-HSC-derived SPM and LPM are phenotypically and functionally comparable to their E8-derived yolk sac counterpart. After birth, and throughout adulthood, both peritoneum and liver, but not the brain, call upon LT-HSCs in the bone marrow to continuously regenerate their fetal-derived tissue-resident macrophages. These results suggest that the requirement for LT-HSCs to regenerate macrophages is tissue-specific. In rodents, both peritoneum and liver, but not the brain, might be exposed to injuries and other exogenous insults that could require constant regeneration of their tissue-resident macrophages. Collectively, these findings add a new layer to the complex developmental landscape of the myeloid lineage and challenge the current notion that tissue-resident macrophages develop independently of the LT-HSCs.

Background IgD is an enigmatic antibody isotype best known when coexpressed with IgM on naive B cells. However, increased soluble IgD (sIgD) levels and increased IgD + IgM − B-cell populations have been described in the human upper respiratory mucosa. Objective We assessed whether levels of sIgD and IgD + B cell counts are altered in nasal tissue from patients with chronic rhinosinusitis (CRS). We further characterized IgD + B-cell populations and explored clinical and local inflammatory factors associated with tissue sIgD levels. Methods sIgD levels were measured by means of ELISA in nasal tissues, nasal lavage fluid, sera, and supernatants of dissociated nasal tissues. IgD + cells were identified by using immunofluorescence and flow cytometry. Inflammatory mediator levels in tissues were assessed by using real-time PCR and multiplex immunoassays. Bacterial cultures from the middle meatus were performed. Underlying medical history and medicine use were obtained from medical records. Results sIgD levels and numbers of IgD + cells were significantly increased in uncinate tissue (UT) of patients with chronic rhinosinusitis without nasal polyps (CRSsNP) compared with that of control subjects (4-fold, P + cells were densely scattered in the periglandular regions of UT from patients with CRSsNP. We also found that IgD + CD19 + CD38 bright plasmablast numbers were significantly increased in tissues from patients with CRSsNP compared with control tissues ( P ex vivo ( P P Conclusion sIgD levels and IgD + CD19 + CD38 bright plasmablast counts were increased in nasal tissue of patients with CRSsNP. IgD levels were associated with increased IL-2 levels and the presence of pathogenic bacteria. These findings suggest that IgD might contribute to enhancement mucosal immunity or inflammation or respond to bacterial infections in patients with CRS, especially CRSsNP.

Peritoneal cavity (PerC) B-1 cells have long been known to express CD11b, which is coexpressed with CD18 to form the Mac-1/CR3 complement receptor and adhesion molecule. However, although all PerC B-1 cells are commonly believed to express CD11b, we show here that nearly half of the cells in each of the PerC B-1 subsets (B-1a and B-1b) do not express this surface receptor. The CD11b+cells in each B-1 subset are larger and more granular and express higher levels of surface IgM than the CD11b−B-1 cells. In addition, the CD11b+B-1 cells initiate the formation of tightly associated doublets that are present at high frequency in adult PerC. Finally, and most importantly from a developmental standpoint, the CD11b+B-1 cells have a limited reconstitution capability: when sorted and transferred into congenic recipients, they reconstitute their own (CD11b+) B-1 subset but do not reconstitute the CD11b−B-1 subset. In contrast, CD11b−B-1 cells transferred under the same conditions efficiently replenish all components of the PerC B-1 population in appropriate proportions. During ontogeny, CD11b−B-1 cells appear before CD11b+B-1 cells. However, the clear phenotypic differences between the neonatal and adult CD11b B-1 subsets argue that although CD11b−B-1 give rise to CD11b+B-1 in both cases different forces may regulate this transition.

B-1a cells reside in both the peritoneal cavity and the spleen. LPS stimulates splenic B-1a to differentiate to plasma cells producing natural IgM specific for microbial and self antigens. However, there are conflicting views as to whether the B-1a cells divide before this differentiation occurs, and hence how the resident B-1a population is maintained in the spleen. Studies here resolve this dispute in favor of both sides: we show that (some or all) B-1a cells resident in the spleen respond to LPS by differentiating to plasma cells immediately, without dividing; however, we also show that additional B-1a cells immigrate into the spleen after LPS stimulation and divide at least once before differentiating. Importantly, the studies we presently describe reveal the complex cell migration and differentiation events that collectively underlie the rapid production of natural antibodies in response to in vivo LPS stimulation. Thus, the studies present a different view of the roles that B-1a cells play in the early phases of the innate immune response.

Processes that define immunoglobulin repertoires are commonly presumed to be the same for all murine B cells. However, studies here that couple high-dimensional FACS sorting with large-scale quantitative IgH deep-sequencing demonstrate that B-1a IgH repertoire differs dramatically from the follicular and marginal zone B cells repertoires and is defined by distinct mechanisms. We track B-1a cells from their early appearance in neonatal spleen to their long-term residence in adult peritoneum and spleen. We show that de novo B-1a IgH rearrangement mainly occurs during the first few weeks of life, after which their repertoire continues to evolve profoundly, including convergent selection of certain V(D)J rearrangements encoding specific CDR3 peptides in all adults and progressive introduction of hypermutation and class-switching as animals age. This V(D)J selection and AID-mediated diversification operate comparably in germ-free and conventional mice, indicating these unique B-1a repertoire-defining mechanisms are driven by antigens that are not derived from microbiota. DOI: http://dx.doi.org/10.7554/eLife.09083.001, eLife digest Our immune system protects us by recognizing and destroying invading viruses, bacteria and other microbes. B cells are immune cells that produce protective proteins called antibodies to stop infections. These cells are activated by ‘antigens’, which are fragments of molecules from the microbes or from our own cells. When an antigen binds to a B cell, the cell matures, multiplies and produces proteins called antibodies. These antibodies can bind to the antigen, which marks the microbe for attack and removal by other cells in the immune system. Each antibody consists of two ‘heavy chain’ and two ‘light chain’ proteins. B cells are able to produce a large variety of different antibodies due to the rearrangement of the gene segments that encode the heavy and light chains. In mice, there are two kinds of B cells – known as B-1a and B-2 cells – that play different roles in immune responses. B-1a cells have long been known to produce the ‘natural’ antibodies that are present in the blood prior to an infection. On the other hand, B-2 cells produce antibodies that are specifically stimulated by an infection and are better adapted to fighting it. Previous studies have shown that both types of antibodies are required to allow animals to successfully fight the flu virus. Here, Yang, Wang et al. used a technique called fluorescence-activated cell sorting (or FACS) and carried out extensive genomic sequencing to study how the B-1a and B-2 populations rearrange their genes to produce heavy chains. This approach made it possible to separate the different types of B cells and then sequence the gene for the heavy chain within the individual cells. The experiments show that the “repertoire” of heavy chains in the antibodies of the B-1a cells is much less random and more repetitive than that of B-2 populations. Furthermore, Yang, Wang et al. show that B-1a cells produce and maintain their repertoire of heavy chains in a different way to other B-2 populations. B-1a cells develop earlier and the major genetic rearrangements in the gene that encodes the heavy chain occur within the first few weeks of life. Although the gene rearrangements have mostly stopped by adulthood, the B-1a antibody repertoire continues to evolve profoundly as the B-1a cells divide over the life of the animal. On the other hand, the gene rearrangements that make the heavy chains in the B-2 cells continue throughout the life of the animal to produce the wider repertoire of antibodies found in these cells. In addition, the processes that continue to change the antibody reperotire in the B-1a cells during adulthood do not occur in the B-2 populations. Importantly, the these reperotire-changing processes in B-1a cells also occur in mice that have been raised in germ-free conditions, which demonstrates that – unlike other B cells – the repertoire of heavy chains in B-1a cells is not influenced by antigens from microbes. Instead, it is mainly driven by antigens that are expressed by normal cells in the body. These findings open the way to future work aimed at understanding how B-1a cells help to protect us against infection, and their role in autoimmune diseases, where immune cells attack the body’s own healthy cells. DOI: http://dx.doi.org/10.7554/eLife.09083.002

Chromoblastomycosis is a human chronic, often debilitating, suppurative, granulomatus mycosis of the skin and subcutaneous tissues beginning after inoculation trauma. It occurs worldwide, but is more frequently observed in tropical countries such as Brazil. Some studies have focused on fungus-host interaction, showing a predominantly cell-mediated immune response, with the activation of macrophages involved in fungus phagocytosis. Immunization with live conidia produced a high influx of CD4 T cells into the draining lymph node. The sensitized T cells proliferate in vitro when restimulated with specific antigen and preferentially produce IFN- gamma. To better characterize the role played by T cells on the chromoblastomycosis infection we used mice deficient for CD4 and CD8. Data determined by CFU counts associated with decreased DTH and IFN-gamma production of infected mice clearly demonstrated that, during experimental F. pedrosoi infection, absence of CD4(+) cells induces a more severe disease.

Chromoblastomycosis is characterized by the slow development of polymorphic skin lesions (nodules, verrucas, tumores, plaques and scar tissue). Inside the host, infectious propagules adhere to epithelial cells and differentiate into sclerotic forms, which effectively resist destruction by host effector cells and allow onset of chronic disease. A cellular immune response against fungi is essential to control infection. Amongst the cells of the immune system, macrophages play the most important role in controlling fungal growth. In this study, we show that the fungicidal characteristic of macrophages is dependent on the fungal species that causes chromoblastomycosis. We began by observing that the phagocytic index was higher for Fonsecaea pedrosoi and Rhinocladiella aquaspersa compared with that of other fungi. Complement-mediated phagocytosis was more important for Phialophora verrucosa and R. aquaspersa and was inhibited by mannan when F. pedrosoi and R. aquaspersa conidia were phagocytosed by macrophages. We showed that macrophages killed significantly only R. aquaspersa. We also found that the phagocytosis of fungi has functional consequences for macrophages as phagocytosis resulted in down-modulation of MHC-II and CD80 expression as well as in the inhibition of the basal liberation of NO. However, the inhibition of the basal liberation of NO nor the down-modulation of MHC and co-stimulatory molecules were observed in the presence of R. aquaspersa.

Nowadays, one can hardly imagine biology and medicine without flow cytometry to measure CD4 T cell counts in HIV, follow bone marrow transplant patients, characterize leukemias, etc. Similarly, without flow cytometry, there would be a bleak future for stem cell deployment, HIV drug development and full characterization of the cells and cell interactions in the immune system. But while flow instruments have improved markedly, the development of automated tools for processing and analyzing flow data has lagged sorely behind. To address this deficit, we have developed automated flow analysis software technology, provisionally named AutoComp and AutoGate. AutoComp acquires sample and reagent labels from users or flow data files, and uses this information to complete the flow data compensation task. AutoGate replaces the manual subsetting capabilities provided by current analysis packages with newly defined statistical algorithms that automatically and accurately detect, display and delineate subsets in well-labeled and well-recognized formats (histograms, contour and dot plots). Users guide analyses by successively specifying axes (flow parameters) for data subset displays and selecting statistically defined subsets to be used for the next analysis round. Ultimately, this process generates analysis “trees” that can be applied to automatically guide analyses for similar samples. The first AutoComp/AutoGate version is currently in the hands of a small group of users at Stanford, Emory and NIH. When this “early adopter” phase is complete, the authors expect to distribute the software free of charge to .edu, .org and .gov users.

In cancer imaging, nanoparticle biodistribution is typically visualized in living subjects using a €bulka € imaging modalities such as magnetic resonance imaging, computerized tomography and whole-body fluorescence. Accordingly, nanoparticle influx is observed only macroscopically, and the mechanisms by which they target cancer remain elusive. Nanoparticles are assumed to accumulate via several targeting mechanisms, particularly extravasation (leakage into tumour). Here, we show that, in addition to conventional nanoparticle-uptake mechanisms, single-walled carbon nanotubes are almost exclusively taken up by a single immune cell subset, Ly-6C hi monocytes (almost 100% uptake in Ly-6C hi monocytes, below 3% in all other circulating cells), and delivered to the tumour in mice. We also demonstrate that a targeting ligand (RGD) conjugated to nanotubes significantly enhances the number of single-walled carbon nanotube-loaded monocytes reaching the tumour (P < 0.001, day 7 post-injection). The remarkable selectivity of this tumour-targeting mechanism demonstrates an advanced immune-based delivery strategy for enhancing specific tumour delivery with substantial penetration. © 2014 Macmillan Publishers Limited. All rights reserved.

Generation of hypochlorous acid (HOCl), an important microbicidal agent, is considered to be the main function of myeloperoxidase (MPO), an enzyme present in phagocytes. High amounts of MPO are present in neutrophil azurophilic granules, which are mobilized into the phagolysosome vacuole during phagocytosis. MPO is also present in monocytes and macrophages, although to a lesser degree than in neutrophils. In the present study, we investigated the distribution of MPO in murine peritoneal cells using flow cytometry, confocal microscopy (CM) and transmission electron microscopy (TEM). MPO was observed in macrophages, and surprisingly, we detected MPO in B lymphocytes, specifically in B1-a. MPO was present in cytoplasmic granules, vesicles, mitochondria and the nucleus of murine peritoneal cells. Together, these findings suggest that, in addition to its known microbicidal activity, MPO has a myriad of other unanticipated cellular functions.

β-Amyloid 42 (Aβ42) and β-amyloid 40 (Aβ40), major components of senile plaque deposits in Alzheimer's disease, are considered neurotoxic and proinflammatory. In multiple sclerosis, Aβ42 is up-regulated in brain lesions and damaged axons. We found, unexpectedly, that treatment with either Aβ42 or Aβ40 peptides reduced motor paralysis and brain inflammation in four different models of experimental autoimmune encephalomyelitis (EAE) with attenuation of motor paralysis, reduction of inflammatory lesions in the central nervous system (CNS), and suppression of lymphocyte activation. Aβ42 and Aβ40 treatments were effective in reducing ongoing paralysis induced with adoptive transfer of either autoreactive T helper 1 (T(H)1) or T(H)17 cells. High-dimensional 14-parameter flow cytometry of peripheral immune cell populations after in vivo Aβ42 and Aβ40 treatment revealed substantial modulations in the percentage of lymphoid and myeloid subsets during EAE. Major proinflammatory cytokines and chemokines were reduced in the blood after Aβ peptide treatment. Protection conferred by Aβ treatment did not require its delivery to the brain: Adoptive transfer with lymphocytes from donors treated with Aβ42 attenuated EAE in wild-type recipient mice, and Aβ deposition in the brain was not detected in treated EAE mice by immunohistochemical analysis. In contrast to the improvement in EAE with Aβ treatment, EAE was worse in mice with genetic deletion of the amyloid precursor protein. Therefore, in the absence of Aβ, there is exacerbated clinical EAE disease progression. Because Aβ42 and Aβ40 ameliorate experimental autoimmune inflammation targeting the CNS, we might now consider its potential anti-inflammatory role in other neuropathological conditions.

B-1a cells are primarily thought of as natural antibody-producing cells. However, we now show that appropriate antigenic stimulation induces IgM and IgG B-1a antibody responses and long-lived T-independent antigen-specific B-1a memory that differs markedly from canonical B-2 humoral immunity. Thus, we show here that in the absence of inflammation, priming with glycolipid (FtL) from Francisella tularensis live vaccine strain induces splenic FtL-specific B-1a to mount dominant IgM and activation-induced cytidine deaminase-dependent IgG anti-FtL responses that occur within 3–5 d of FtL priming and fade within 1 wk to natural antibody levels that persist indefinitely in the absence of secondary FtL immunization. Equally surprising, FtL priming elicits long-term FtL-specific B-1a memory cells (IgM>>IgG) that migrate rapidly to the peritoneal cavity and persist there indefinitely, ready to respond to appropriately administrated secondary antigenic stimulation. Unlike B-2 responses, primary FtL-specific B-1a responses and establishment of persistent FtL-specific B-1a memory occur readily in the absence of adjuvants, IL-7, T cells, or germinal center support. However, in another marked departure from the mechanisms controlling B-2 memory responses, rechallenge with FtL in an inflammatory context is required to induce B-1a secondary antibody responses. These findings introduce previously unexplored vaccination strategies for pathogens that target the B-1a repertoire.

In the companion article by Yang and colleagues [Yang Y, et al. (2012) Proc Natl Acad Sci USA , 109, 10.1073/pnas.1121631109], we have shown that priming with glycolipid (FtL) from Francisella tularensis live-vaccine strain ( i ) induces FtL-specific B-1a to produce robust primary responses (IgM >>IgG); ( ii ) establishes persistent long-term production of serum IgM and IgG anti-FtL at natural antibody levels; and ( iii ) elicits FtL-specific B-1a memory cells that arise in spleen but rapidly migrate to the peritoneal cavity, where they persist indefinitely but divide only rarely. Here, we show that FtL rechallenge alone induces these PerC B-1a memory cells to divide extensively and to express a unique activation signature. However, FtL rechallenge in the context of a Toll-like receptor 4 agonist-stimulated inflammatory response readily induces these memory cells to migrate to spleen and initiate production of dominant IgM anti-FtL secondary responses. Thus, studies here reveal unique mechanisms that govern B-1a memory development and expression, and introduce B-1a memory as an active participant in immune defenses. In addition, at a practical level, these studies suggest previously unexplored vaccination strategies for pathogen-associated antigens that target the B-1a repertoire.

Nanoparticle targeting efficiency to tumor is poor and not well-understood. Applying intravital microscopy in a mouse-model to interrogate vasculature-targeted nanotubes, we found that monocytes specifically take up nanotubes and are programmed to enter tumor.