Cytokine-producing effector B cells regulate type 2 immunity to H. polygyrus.

Immunity to the intestinal parasite Heligomosomoides polygyrus is dependent on the successful generation of T helper 2 (Th2) memory cells. We showed that B cells contribute to immunity against H. polygyrus by producing antibody (Ab) and by promoting expansion and differentiation of primary and memory Th2 cells. We also demonstrated that cytokine-producing effector B cells were essential for effective immunity to H. polygyrus. Tumor necrosis factor alpha production by B cells was necessary for sustained Ab production, whereas interleukin 2 production by B cells was necessary for Th2 cell expansion and differentiation. These results show that B cells mediate protection from pathogens not only by presenting antigen and secreting antibody but also by producing cytokines that regulate the quality and magnitude of humoral and cellular immune responses.

Optimized protocol for mouse footpad immune cell isolation for single-cell RNA sequencing and flow cytometry.

Single-cell RNA sequencing (scRNA-seq) requires the preparation of a highly viable single-cell suspension to get reliable sequencing results. Here, we present a protocol for isolating mouse footpad leukocytes while maintaining high viability. We describe steps for footpad collection, enzymatic tissue dissociation, leukocyte isolation and purification, and cell fixation and preservation. We then detail combinatorial barcoding, library preparation, scRNA-seq, and data analysis. Cells can be used to generate a complete molecular atlas at the single cell level.

Heat Stress Increases Mammary Epithelial Cells and Reduces Viable Immune Cells in Milk of Dairy Cows.

Somatic cells normally found in milk are generally either immune cells such as lymphocytes, monocytes and granulocytes, or mammary epithelial cells. The number and composition of somatic cells in milk can be influenced by a variety of factors, including infection and temperature-humidity index. The objective of this study was to determine the specific effects of heat stress on the cellular composition of the somatic cell population in milk. We used flow cytometry to ascertain the concentration and viability of mammary epithelial cells, T cells, monocyte/macrophage, and granulocytes in milk from cows maintained under heat stressed conditions compared to thermoneutral conditions. We found a significant 10% increase in the natural log concentration of epithelial cells in the milk of heat stressed cows compared to thermoneutral cows (9.3 vs. 8.4 ln(cells/mL, p = 0.02)). We also found a 12% decrease in the log concentration of live CD45+ cells (p = 0.04), and a 17% decrease in the log concentration of live CD45+ granulocytes (p = 0.04). No changes were found in CD3+CD45+ cells or CD14+CD45+ cells, however, we noted an unusual population of CD14+CD45- cells that showed significant increases of 10% (p = 0.03) and 12% (p = 0.01) in the log concentration of total and dead cells, respectively, under heat stressed conditions. These results suggest that heat stress influences the relative populations and viability of some somatic cells populations in milk. Increased losses of secretory epithelial cells into milk could have implications for milk production, and fewer viable immune cells could negatively impact the immunocompetence of dairy cows under heat stress.

Production of interspecies somatic/pluripotent heterokaryons using polyethylene glycol (PEG) and selection by imaging flow cytometry for the study of nuclear reprogramming.

Fusion of somatic cells to embryonic stem cells induces reprogramming of the somatic nucleus and can be used to study the effect of trans-acting factors from the pluripotent cell over the differentiated nucleus. However, fusion only occurs in a small fraction of the cells exposed to fusogenic conditions, hence the need for a protocol that produces high fusion rate with minimal cell damage, coupled with a method capable of identifying and selecting these rare events. Here, we describe a protocol to induce formation of bi-species mouse pluripotent/bovine somatic heterokaryons, as well as same-species homokaryons, using polyethylene glycol (PEG). To identify bi-species fusion products, heterokaryons were labeled using cell type-specific fluorescent antibodies and selected using imaging (Amnis ImageStream Mark II) and traditional (BD FACSAria I) flow cytometry. Heterokaryons selected with this method produced ES cell-like colonies in vitro. This procedure can be combined with downstream applications such as nucleic acid isolation for RT-PCR and RNA-Seq, and used as a tool to study somatic cell nuclear reprogramming.

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts.

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

Protoplast isolation prior to flow cytometry reveals clear patterns of endoreduplication in potato tubers, related species, and some starchy root crops.

BACKGROUND: Endoreduplication, the process of DNA replication in the absence of cell division, is associated with specialized cellular function and increased cell size. Genes controlling endoreduplication in tomato fruit have been shown to affect mature fruit size. An efficient method of estimating endoreduplication is required to study its role in plant organ development. Flow cytometry is often utilized to evaluate endoreduplication, yet some tissues and species, among them the tubers of Solanum tuberosum, remain intractable to routine tissue preparation for flow cytometry. We aimed to develop a method through the use of protoplast extraction preceding flow cytometry, specifically for the assessment of endoreduplication in potato tubers. RESULTS: We present a method for appraising endoreduplication in potato (Solanum tuberosum) tuber tissues. We evaluated this method and observed consistent differences between pith and cortex of tubers and between different cultivars, but no apparent relationship with whole tuber size. Furthermore, we were able to observe distinct patterns of endoreduplication in 16 of 20 wild potato relatives, with mean endoreduplication index (EI) ranging from 0.94 to 2.62 endocycles per cell. The protocol was also applied to a panel of starchy root crop species and, while only two of five yielded reliable flow histograms, the two (sweet potato and turnip) exhibited substantially lower EIs than wild and cultivated potato accessions. CONCLUSIONS: The protocol reported herein has proven effective on tubers of a variety of potato cultivars and related species, as well as storage roots of other starchy crops. This method provides an important tool for the study of potato morphology and development while revealing natural variation for endoreduplication which may have agricultural relevance.

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

Leukocyte adhesion deficiency type I in a mixed-breed dog.

A 6-month-old, neutered male, mixed-breed dog was examined for a 2-month persistent fever, nonhealing dermal metacarpal area wound, and leukocytosis (47.0-198.0 × 10(3)/µl). Serum chemistry findings included hypoalbuminemia, hyperglobulinemia, hyperphosphatemia, and hyperphosphatasemia. Complete blood cell count results revealed a moderate microcytic, hypochromic nonregenerative anemia with a profound leukocytosis (198.5 × 10(3)/µl), characterized by neutrophilia with toxicity and hypersegmentation, and significant band cells. Tick-borne disease titers (genera Anaplasma, Ehrlichia, and Borrelia) were negative, as were polymerase chain reaction for other infectious agents (genera Hepatozoon, Mycobacterium, Mycoplasma; and Canine distemper virus). No agents were identified in a deep dermal biopsy (conventional and special histochemical stains) of the chronic draining, metacarpal region lesion. Cytology of the draining tract revealed numerous mixed bacteria and a surprising lack of neutrophils. Chronic occult blood loss with iron deficiency was considered a possible cause of the anemia. Differentials for the leukon were chronic established inflammation (occult infectious agent), chronic neutrophilic leukemia, paraneoplastic leukocytosis (neoplastic source of granulocyte colony-stimulating factor [CSF] or granulocyte-macrophage CSF), and leukocyte adhesion deficiency (LAD). The possibility of a LAD disorder was further investigated because of the noted hypersegmented neutrophils, absence of neutrophils in the cytology sample, the animal's young age, and persistence of clinical and laboratory signs. Flow cytometry of blood neutrophils showed a 60% reduction in surface expression of the ß2-integrin (CD18) subunit, whereas neutrophil function tests (oxidative burst and phagocytosis) were normal. Genetic testing revealed a homozygous missense mutation in the ß2-integrin subunit gene, previously recognized only in purebred Irish Setters, leading to a diagnosis of LAD type 1 disorder in this mixed-breed dog.

Role of TLRs in Brucella mediated murine DC activation in vitro and clearance of pulmonary infection in vivo.

Brucellosis is worldwide zoonoses affecting 500,000 people annually with no approved human vaccines available. Live attenuated Brucella abortus vaccine strain RB51 protects cattle through CD4 and CD8 T-cell mediated responses. However, limited information is known regarding how Brucella stimulate innate immunity. Although the most critical toll like receptors (TLRs) involved in the recognition of Brucella are TLR2, TLR4 and TLR9, it is important to identify the essential TLRs that induce DC activation/function in response to Brucella, to be able to upregulate both vaccine strain RB51-mediated protection, and clearance of pathogenic strain 2308. Furthermore, in spite of the importance of aerosol transmission of Brucella, no published studies have addressed the role of TLRs in the clearance of strain 2308 or strain RB51 from intranasally infected mice. Therefore, we used a (a) bone marrow derived dendritic cell model in TLRKO and control mice to assess the differential role of pathogenic and vaccine strains to induce DC activation and function in vitro, and (b) respiratory model in TLRKO and control mice to assess the critical roles for TLRs in clearance of strains in vivo. In support of the essential TLRs in clearance and protection, we performed challenge experiments to identify if these critical TLRs (as agonists) could enhance vaccine induced protection against pathogenic strain 2308 in a respiratory model. We determined: vaccine strain RB51 induced significant (p≤0.05) DC activation vs. strain 2308 which was not dependent on a specific TLR; strain RB51 induced TNF-α production was TLR2 and TLR9 dependent, and IL-12 production was TLR2 and TLR4 dependent; TLR4 and TLR2 were critical for clearance of vaccine and pathogenic Brucella strains respectively; and TLR2 (p<0.05), TLR4 (p<0.05) and TLR9 (p=0.075) agonists enhanced vaccine strain RB51-mediated protection against respiratory challenge with strain 2308 in the lung.

Live Brucella abortus rough vaccine strain RB51 stimulates enhanced innate immune response in vitro compared to rough vaccine strain RB51SOD and virulent smooth strain 2308 in murine bone marrow-derived dendritic cells.

Brucella spp. are Gram-negative, coccobacillary, facultative intracellular pathogens. B. abortus strain 2308 is a pathogenic strain affecting cattle and humans. Rough B. abortus strain RB51, which lacks the O-side chain of lipopolysaccharide (LPS), is the live attenuated USDA approved vaccine for cattle in the United States. Strain RB51SOD, which overexpresses Cu-Zn superoxide dismutase (SOD), has been shown to confer better protection than strain RB51 in a murine model. Protection against brucellosis is mediated by a strong CD4+ Th(1) and CD8+ Tc(1) adaptive immune response. In order to stimulate a robust adaptive response, a solid innate immune response, including that mediated by dendritic cells, is essential. As dendritic cells (DCs) are highly susceptible to Brucella infection, it is possible that pathogenic strains could limit the innate and thereby adaptive immune response. By contrast, vaccine strains could limit or bolster the innate and subsequent adaptive immune response. Identifying how Brucella vaccines stimulate innate and adaptive immunity is critical for enhancing vaccine efficacy. The ability of rough vaccine strains RB51 and RB51SOD to stimulate DC function has not been characterized. We report that live rough vaccine strain RB51 induced significantly better (p ≤ 0.05) DC maturation and function compared to either strain RB51SOD or smooth virulent strain 2308, based on costimulatory marker expression and cytokine production.

CD38 induces apoptosis of a murine pro-B leukemic cell line by a tyrosine kinase-dependent but ADP-ribosyl cyclase- and NAD glycohydrolase-independent mechanism.

Cross-linking of CD38 on hematopoietic cells induces activation, proliferation and differentiation of mature T and B cells and mediates apoptosis of myeloid and lymphoid progenitor cells. In addition to acting as a signaling receptor, CD38 is also an enzyme capable of producing several calcium-mobilizing metabolites, including cyclic adenosine diphosphate ribose (cADPR). It has been previously postulated that the calcium-mobilizing metabolites produced by CD38 may regulate its receptor-based activities. To test this hypothesis, we examined whether the enzyme activity of CD38 controls the apoptosis of an anti-CD38-stimulated leukemic B cell. We show that anti-CD38-induced apoptosis of Ba/F3 cells, a murine pro-B cell line, is not affected by blocking the calcium-mobilizing activity of cADPR or by inhibiting intracellular or extracellular calcium mobilization. In addition, we demonstrate that blocking CD38 enzyme activity with 2'-deoxy-2'-fluoro-nicotinamide arabinoside adenine dinucleotide has no effect on apoptosis and that Ba/F3 cells expressing catalytically inactive mutant forms of CD38 still undergo apoptosis upon CD38 cross-linking. Instead, we find that anti-CD38-induced apoptosis is dependent on tyrosine kinase and caspase activation, and that this process appears to be potentiated by the presence of membrane microdomains. Thus, the receptor-mediated functions of CD38 can be separated from its enzyme activity in a murine leukemic cell line, suggesting that CD38 plays multiple, but independent, biologic roles.

CD4 T cell-independent antibody response promotes resolution of primary influenza infection and helps to prevent reinfection.

It is generally believed that the production of influenza-specific IgG in response to viral infection is dependent on CD4 T cells. However, we previously observed that CD40-deficient mice generate influenza-specific IgG during a primary infection, suggesting that influenza infection may elicit IgG responses independently of CD4 T cell help. In the present study, we tested this hypothesis and show that mice lacking CD40 or CD4 T cells produce detectable titers of influenza-specific IgG and recover from influenza infection in a manner similar to that of normal mice. In contrast, mice completely lacking B cells succumb to influenza infection, despite the presence of large numbers of functional influenza-specific CD8 effector cells in the lungs. Consistent with the characteristics of a T-independent Ab response, long-lived influenza-specific plasma cells are not found in the bone marrow of CD40-/- and class II-/- mice, and influenza-specific IgG titers wane within 60 days postinfection. However, despite the short-lived IgG response, CD40-/- and class II-/- mice are completely protected from challenge infection with the same virus administered within 30 days. This protection is mediated primarily by B cells and Ab, as influenza-immune CD40-/- and class II-/- mice were still resistant to challenge infection when T cells were depleted. These data demonstrate that T cell-independent influenza-specific Ab promotes the resolution of primary influenza infection and helps to prevent reinfection.