Comparative Multi-Donor Study of IFNγ Secretion and Expression by Human PBMCs Using ELISPOT Side-by-Side with ELISA and Flow Cytometry Assays.

ELISPOT, ELISA and flow cytometry techniques are often used to study the function of immune system cells. It is tempting to speculate that these assays can be used interchangeably, providing similar information about the cytokine secreting activity of cells: the higher the number of cytokine-positive cells measured by flow cytometry, the higher the number of cytokine-secreting cells expected to be detected by ELISPOT and the larger the amount of secreted cytokine expected to be measured by ELISA. We have analyzed the expression level and secretion capacity of IFNγ from peripheral blood mononuclear cells isolated from five healthy donors and stimulated by calcium ionomycin mixed with phorbol 12-myristate 13-acetate in a non-specific manner in side-by-side testing using ELISPOT, ELISA and flow cytometry assays. In our study, we observed a general correlation in donors' ranking between ELISPOT and flow cytometry; ELISA values did not correlate with either ELISPOT or flow cytometry. However, a detailed donor-to-donor comparison between ELISPOT and flow cytometry revealed significant discrepancies: donors who have similar numbers of IFNγ-positive cells measured by flow cytometry show 2-3-fold differences in the number of spot-forming cells (SFCs) measured by ELISPOT; and donors who have the same number of SFCs measured by ELISPOT show 30% differences in the number of IFNγ-positive cells measured by flow cytometry. Significant discrepancies between donors were also found when comparing ELISA and ELISPOT techniques: donors who secreted the same amount of IFNγ measured by ELISA show six-fold differences in the number of SFCs measured by ELISPOT; and donors who have 5-7-times less secreted IFNγ measured by ELISA show a two-fold increase in the number of SFCs measured by ELISPOT compared to donors who show a more profound secretion of IFNγ measured by ELISA. The results of our study suggest that there can be a lack of correlation between IFNγ values measured by ELISPOT, ELISA and flow cytometry. The higher number of cytokine-positive cells determined by flow cytometry is not necessarily indicative of a higher number of cytokine-secreting cells when they are analyzed by either ELISPOT or ELISA. Our ELISPOT vs. ELISA comparison demonstrates that the higher number of SFCs observed in ELISPOT does not guarantee that these cells secrete larger amounts of cytokines compared to donors with lower SFC numbers. In addition, our data indicate that ELISPOT, ELISA and flow cytometry should be performed as complementary, rather than stand-alone assays: running these assays in parallel on samples from the same donors may help to better understand the mechanisms underlying the physiology of cytokine-secreting cells.

Equine ELISPOT assay to study secretion of IFNγ and IL-4 from peripheral blood mononuclear cells.

Human and mouse immune system cells are the most frequently used specimens in ELISPOT assays. In an effect to expand the application of ELISPOT assay to other species, we developed matched antibody pairs for ready-to-use kits designed for studying the frequency of equine IFNγ- and IL-4-secreting peripheral blood mononuclear cells (PBMCs). Equine PBMCs were stimulated with either concanavalin A (Con A) or calcium ionomycin mixed with phorbol 12-myristate 13-acetate (CaI + PMA). We found that Con A, in general, had a more profound stimulating effect than CaI + PMA on IL-4 secretion, whereas both stimulatory and inhibitory effects were observed on IFNγ secretion. Our data demonstrate a large dynamic range in IFNγ and IL-4 secretion among different donors, which may reflect animal health and serve as a valuable diagnostic marker.

ELISPOT assay as a tool to study oxidative stress in lymphocytes.

Enzyme-linked immuno spot (ELISPOT) assay is widely used for vaccine development, cancer and AIDS research, and autoimmune disease studies. The output of ELISPOT assay is a formation of colored spots which appear at the sites of cells releasing cytokines, with each individual spot representing a single cytokine-releasing cell. We worked out a protocol to study oxidative stress in human peripheral blood lymphocytes by determining their potency to secrete IFN-gamma, IL-2, IL-4, IL-5, IL-8, and TNF-alpha in response to acute treatment with hydrogen peroxide. We show that hydrogen peroxide-induced oxidative stress can cause a ∼twofold decrease in the number of lymphocytes secreting the TH1 cytokines IFN-gamma and IL-2, as well as chemokines IL-8 and TNF-alpha. However, the number of cells secreting TH2 cytokines IL-4 and IL-5 in hydrogen -peroxide-treated group did not change. It appears that oxidative stress may affect TH1-TH2 cytokine secretion -balance which, in turn, may underlie developments of various pathological conditions. This protocol can be easily modified to study the effects of many other oxidative stress compounds.

ELISPOT assay for neuroscience research: studying TNFα secretion from microglial cells.

The major application of ELISPOT assays is to study secretion of cytokines and chemokines from immune system cells. We adapted this assay to study TNFα secretion from microglial BV2 cells, which are similar in physiology to microglia in the nervous system. Stimulation of BV2 cells with 1 µg/mL LPS resulted in a robust secretion of TNFα. Unlike uniform round spots formed by TNFα secreted by immune system cells, BV2 cells produced spots with short zigzag "tails" indicating that BV2 cells were actively moving during the incubation. In spite of irregular shapes, spots could be easily counted using an ELISPOT reader. Our study has shown the feasibility of employing an ELISPOT assay as a tool for neuroscience research to study the mechanisms underlying protein secretion from microglial cells. In addition, due to its convenient format, ELISPOT can be used for high-throughput screening of the potency of novel drugs to stimulate or inhibit cytokine secretion by microglial cells in the brain.

Combining ELISPOT and ELISA to measure amounts of cytokines secreted by a single cell.

Enzyme-linked immunospot (ELISPOT) assay allows for the determination of the frequency of -cytokine-secreting cells, but does not answer the question of how much cytokine is secreted per cell. In our study, we combined ELISPOT and ELISA assays and developed a protocol to calculate the amount of IFN gamma secreted by each cell. A suspension of human peripheral blood mononuclear cells was split into two pools and cells from one pool were cultured in a regular ELISPOT plate, whereas cells from the other pool were cultured in an uncoated, "blank," ELISPOT plate. After finishing the incubations, the amount of IFN gamma was measured by ELISA in culture media collected from both plates. The "blank" plate served to measure a total amount of secreted IFN gamma, whereas the ELISPOT plate served to measure the amount of unbound (UB) IFN gamma. Subtracting the amount of unbound IFN gamma from its total amount and dividing it by the number of spots in the ELISPOT plate allows for the calculation of the average amount of IFN gamma in a spot formed by a single cell.

Cometin is a novel neurotrophic factor that promotes neurite outgrowth and neuroblast migration in vitro and supports survival of spiral ganglion neurons in vivo.

Neurotrophic factors are secreted proteins responsible for migration, growth and survival of neurons during development, and for maintenance and plasticity of adult neurons. Here we present a novel secreted protein named Cometin which together with Meteorin defines a new evolutionary conserved protein family. During early mouse development, Cometin is found exclusively in the floor plate and from E13.5 also in dorsal root ganglions and inner ear but apparently not in the adult nervous system. In vitro, Cometin promotes neurite outgrowth from dorsal root ganglion cells which can be blocked by inhibition of the Janus or MEK kinases. In this assay, additive effects of Cometin and Meteorin are observed indicating separate receptors. Furthermore, Cometin supports migration of neuroblasts from subventricular zone explants to the same extend as stromal cell derived factor 1a. Given the neurotrophic properties in vitro, combined with the restricted inner ear expression during development, we further investigated Cometin in relation to deafness. In neomycin deafened guinea pigs, two weeks intracochlear infusion of recombinant Cometin supports spiral ganglion neuron survival and function. In contrast to the control group receiving artificial perilymph, Cometin treated animals retain normal electrically-evoked brainstem response which is maintained several weeks after treatment cessation. Neuroprotection is also evident from stereological analysis of the spiral ganglion. Altogether, these studies show that Cometin is a potent new neurotrophic factor with therapeutic potential.

Expansion of a unique CD57⁺NKG2Chi natural killer cell subset during acute human cytomegalovirus infection.

During human CMV infection, there is a preferential expansion of natural killer (NK) cells expressing the activating CD94-NKG2C receptor complex, implicating this receptor in the recognition of CMV-infected cells. We hypothesized that NK cells expanded in response to pathogens will be marked by expression of CD57, a carbohydrate antigen expressed on highly mature cells within the CD56(dim)CD16(+) NK cell compartment. Here we demonstrate the preferential expansion of a unique subset of NK cells coexpressing the activating CD94-NKG2C receptor and CD57 in CMV(+) donors. These CD57(+)NKG2C(hi) NK cells degranulated in response to stimulation through their NKG2C receptor. Furthermore, CD57(+)NKG2C(hi) NK cells preferentially lack expression of the inhibitory NKG2A receptor and the inhibitory KIR3DL1 receptor in individuals expressing its HLA-Bw4 ligand. Moreover, in solid-organ transplant recipients with active CMV infection, the percentage of CD57(+)NKG2C(hi) NK cells in the total NK cell population preferentially increased. During acute CMV infection, the NKG2C(+) NK cells proliferated, became NKG2C(hi), and finally acquired CD57. Thus, we propose that CD57 might provide a marker of "memory" NK cells that have been expanded in response to infection.

ELISpot assay as a tool to study oxidative stress in peripheral blood mononuclear cells.

Enzyme-Linked Immuno Spot (ELISpot) assay is widely used for vaccine development, cancer and AIDS research, and autoimmune disease studies. The output of an ELISpot assay is a formation of colored spots which appear at the sites of cells releasing cytokines, with each individual spot representing a single cytokine-releasing cell.We have shown that hydrogen peroxide-induced oxidative stress was causing ∼twofold decrease in the number of lymphocytes secreting the TH1 cytokines IFN-gamma and IL-2, as well as chemokine IL-8 and cytokine TNF alpha. However, the number of cells secreting TH2 cytokines IL-4 and IL-5 in hydrogen peroxide-treated group did not change. Our ELISpot data indicate that oxidative stress may affect TH1-TH2 cytokine secretion balance which, in turn, may underlie developments of various pathological conditions. We adopted ELISpot assay for studying oxidative stress in human peripheral blood lymphocytes by analyzing the acute effect of hydrogen peroxide treatment on the frequency of cells secreting IFN-gamma, IL-2, IL-4, IL-5, IL-8, and TNF-alpha.

Roundabout 4 is expressed on hematopoietic stem cells and potentially involved in the niche-mediated regulation of the side population phenotype.

Roundabout (Robo) family proteins are immunoglobulin-type cell surface receptors that are expressed predominantly in the nervous system. The fourth member of this family, Robo4, is distinct from the other family members in that it is expressed specifically in endothelial cells. In this study, we examined the expression of Robo4 in hematopoietic stem cells (HSCs) and its possible role in HSC regulation. Robo4 mRNA was specifically expressed in murine HSCs and the immature progenitor cell fraction but not in lineage-positive cells or differentiated progenitors. Moreover, flow cytometry showed a correlation between higher expression of Robo4 and immature phenotypes of hematopoietic cells. Robo4(high) hematopoietic stem/progenitor cells presented higher clonogenic activity or long-term repopulating activity by colony assays or transplantation assays, respectively. A ligand for Robo4, Slit2, is specifically expressed in bone marrow stromal cells, and its expression was induced in osteoblasts in response to myelosuppressive stress. Interestingly, overexpression of Robo4 or Slit2 in HSCs resulted in their decreased residence in the c-Kit(+)Sca-1(+)Lineage(-)-side population fraction. These results indicate that Robo4 is expressed in HSCs, and Robo4/Slit2 signaling may play a role in HSC homeostasis in the bone marrow niche.

RANK Expression as a cell surface marker of human osteoclast precursors in peripheral blood, bone marrow, and giant cell tumors of bone.

UNLABELLED: RANK expression in vivo on hematopoietic subsets including pre-osteoclasts, identified by monoclonal antibodies, has not been described. We describe the lineages that express RANK in bone marrow, peripheral blood, and GCTs. We show that CD14(+)RANK(high) cells constitute a circulating pre-osteoclast pool. INTRODUCTION: The expression of RANK by subsets of hematopoietic cells has not been adequately studied in humans. While attributed to the monocytoid lineage, the phenotype of the pre-osteoclast (pre-OC) with respect to RANK expression in vivo remains unclear. We tested monoclonal antibodies (MAbs) raised against the extracellular domain of recombinant human RANK for reactivity with normal peripheral blood (PB) and bone marrow (BM) mononuclear cells (PBMNCs and BMMNCs, respectively). We also tested reactivity with giant cell tumor cells (GCT), a confirmed source of pre-OC and mature OCs. MATERIALS AND METHODS: Human PBMNCs, BMMNCs, and GCT cells were analyzed for reactivity with anti-RANK MAbs by flow cytometry in combination with hematopoietic lineage restricted markers. GCTs were also analyzed by immunofluorescence. CD14+ monocytoid cells were sorted by fluorescence-activated cell sorting (FACS) based on their relative RANK expression and cultured under OC-forming conditions. RESULTS: RANK+ cells were detected similarly by three independent anti-RANK MAbs. One MAb (80736) immunoprecipitated RANK-RANKL complexes from surface-biotinylated GCT lysates. Using dual-color flow cytometry, RANK was detected on CD14+ (monocytoid), CD19+ (B-lymphoid), CD56+ (NK cell), and glycophorin A+ erythroid progenitors. Minor populations of both CD3+ T lymphocytes and BM CD34+ hematopoietic progenitors also expressed cell surface RANK. In GCTs, RANK expression was identified on mononuclear CD45(+)CD14(+)alphaVbeta3(+)c-Fms+ cells, likely to be committed pre-OC, and on multinucleated CD45(+)alphaVbeta3(+)TRACP(+) OCs. Importantly, sorted CD14(+)RANK(high) PBMNCs treated with recombinant RANKL and macrophage-colony stimulating factor (M-CSF) gave rise to approximately twice the number of osteoclasts than RANK(mid) or RANK(low) cells. CONCLUSIONS: These results suggest that committed monocytoid RANK+ pre-OCs are represented in the marrow and circulate in the periphery, forming a pool of cells capable of responding rapidly to RANKL. The ability to reliably detect committed pre-OC in peripheral blood could have important clinical applications in the management of diseases characterized by abnormal osteoclastic activity.

The cytomegalovirus m155 gene product subverts natural killer cell antiviral protection by disruption of H60-NKG2D interactions.

Natural killer (NK) cells are an important early mediator of host immunity to murine cytomegalovirus (MCMV) infection. However, MCMV has evolved mechanisms to elude recognition and clearance by NK cells. We have identified an MCMV immune evasion protein that impairs NKG2D-mediated NK cell antiviral activity. Infection of BALB/c 3T3 cells with the Smith strain of MCMV resulted in strong down-regulation of H60, a high affinity ligand for NKG2D, from the surface of virus-infected cells. The MCMV m155 protein specifically down-regulated H60 without affecting expression of the other known NKG2D ligands, RAE-1 and MULT-1. Treatment with the proteasome inhibitors lactacystin or epoxomicin reversed m155 down-regulation of H60. An MCMV mutant virus lacking m155 was severely attenuated in BALB/c mice; however, treatment with neutralizing anti-NKG2D monoclonal antibody or with NK-depleting anti-asialo GM1 antisera restored virulence of the mutant virus. Thus, down-regulation of H60 by m155 is a powerful mechanism of inhibiting NKG2D-mediated antiviral function.

NKG2D-mediated natural killer cell protection against cytomegalovirus is impaired by viral gp40 modulation of retinoic acid early inducible 1 gene molecules.

Natural killer (NK) cells play a critical role in the innate immune response against cytomegalovirus (CMV) infections. Although CMV encodes several gene products committed to evasion of adaptive immunity, viral modulation of NK cell activity is only beginning to be appreciated. A previous study demonstrated that the mouse CMV m152-encoded gp40 glycoprotein diminished expression of ligands for the activating NK cell receptor NKG2D on the surface of virus-infected cells. Here we have defined the precise ligands that are affected and have directly implicated NKG2D in immune responses to CMV infection in vitro and in vivo. Murine CMV (MCMV) infection potently induced transcription of all five known retinoic acid early inducible 1 (RAE-1) genes (RAE-1alpha, RAE-1beta, RAE-1delta, RAE-1 epsilon, and RAE-1gamma), but not H-60. gp40 specifically down-regulated the cell surface expression of all RAE-1 proteins, but not H-60, and diminished NK cell interferon gamma production against CMV-infected cells. Consistent with previous findings, a m152 deletion mutant virus (Deltam152) was less virulent in vivo than the wild-type Smith strain of MCMV. Treatment of BALB/c mice with a neutralizing anti-NKG2D antibody before infection increased titers of Deltam152 virus in the spleen and liver to levels seen with wild-type virus. These experiments demonstrate that gp40 impairs NK cell recognition of virus-infected cells through disrupting the RAE-1-NKG2D interaction.