A Whole Blood Enzyme-Linked Immunospot Assay for Functional Immune Endotyping of Septic Patients.

Sepsis initiates simultaneous pro- and anti-inflammatory processes, the pattern and intensity of which vary over time. The inability to evaluate the immune status of patients with sepsis in a rapid and quantifiable manner has undoubtedly been a major reason for the failure of many therapeutic trials. Although there has been considerable effort to immunophenotype septic patients, these methods have often not accurately assessed the functional state of host immunity, lack dynamic range, and are more reflective of molecular processes rather than host immunity. In contrast, ELISpot assay measures the number and intensity of cytokine-secreting cells and has excellent dynamic range with rapid turnaround. We investigated the ability of a (to our knowledge) novel whole blood ELISpot assay and compared it with a more traditional ELISpot assay using PBMCs in sepsis. IFN-γ and TNF-α ELISpot assays on whole blood and PBMCs were undertaken in control, critically ill nonseptic, and septic patients. Whole blood ELISpot was easy to perform, and results were generally comparable to PBMC-based ELISpot. However, the whole blood ELISpot assay revealed that nonmonocyte, myeloid populations are a significant source of ex vivo TNF-α production. Septic patients who died had early, profound, and sustained suppression of innate and adaptive immunity. A cohort of septic patients had increased cytokine production compared with controls consistent with either an appropriate or excessive immune response. IL-7 restored ex vivo IFN-γ production in septic patients. The whole blood ELISpot assay offers a significant advance in the ability to immunophenotype patients with sepsis and to guide potential new immunotherapies.

Comparison of whole blood cytokine immunoassays for rapid, functional immune phenotyping in critically ill patients with sepsis.

BACKGROUND: Sepsis is characterized by highly heterogeneous immune responses associated with a spectrum of disease severity. Methods that rapidly and sensitively profile these immune responses can potentially personalize immune-adjuvant therapies for sepsis. We hypothesized that the ELLA microfluidic approach to measure cytokine production from the whole blood of septic and critically ill patients would deliver faster, more precise results than the existing optic-driven ELISpot quantification. We tested our hypothesis by measuring ex vivo-stimulated production of TNF and IFNγ in critically ill and septic patients (n = 22), critically ill and non-septic patients (n = 10), and healthy volunteers (n = 10) through both ELLA and ELISpot immunoassays. Blood samples were subjected to one of three stimulants for 4 h or 18 h durations during days 1, 7-10, and 14 of critical illness. Stimulants for lymphocytes included anti-CD3/anti-CD28 and phorbol 12-myristate 13-acetate (PMA), whereas LPS was used for monocytes. Stimulated TNF and IFNγ concentrations were then associated with 30-day mortality. RESULTS: Both ELISpot and ELLA immunoassays showed substantial agreement in TNF concentrations post 4 h and 18 h LPS stimulation, with concordance correlation coefficients at 0.62 and 0.60, respectively. ELLA had a broad dynamic measurement range and provided accurate TNF and IFNγ readings at both minimal and elevated cytokine concentrations (with mean coefficients of variation between triplicate readings at 2.1 ± 1.4% and 4.9 ± 7.2%, respectively). However, there was no association between the ELLA-determined cytokine concentrations on the first day of critical illness and 30-day mortality rate. In contrast, using the ELISpot for cytokine quantification revealed that non-survivors had reduced baseline TNF levels at 18 h, decreased LPS-induced TNF levels at 18 h, and diminished TNF levels post 4 h/18 h anti-CD3/28 stimulation. CONCLUSIONS: Our study affirms the feasibility of obtaining dependable immune phenotyping data within 6 h of blood collection from critically ill patients, both septic and non-septic, using the ELLA immunoassay. Both ELLA and ELISpot can offer valuable insights into prognosis, therapeutic strategies, and the underlying mechanisms of sepsis development.

Allergen-induced, eotaxin-rich, proangiogenic bone marrow progenitors: a blood-borne cellular envoy for lung eosinophilia.

BACKGROUND: Eosinophilic inflammation is closely related to angiogenesis in asthmatic airway remodeling. In ovalbumin (OVA)-sensitized mice bone marrow-derived, proangiogenic endothelial progenitor cells (EPCs) are rapidly recruited into the lungs after OVA aerosol challenge and promptly followed by mobilization and recruitment of eosinophils. OBJECTIVE: We hypothesized that bone marrow-derived EPCs initiate the recruitment of eosinophils through expression of the eosinophil chemoattractant eotaxin-1. METHODS: EPCs were isolated from an OVA murine model of allergic airway inflammation and from asthmatic patients. Endothelial and smooth muscle cells were isolated from mice. Eotaxin-1 expression was analyzed by means of immunofluorescence, real-time PCR, or ELISA. In vivo recruitment of eosinophils by EPCs was analyzed in mice. RESULTS: Circulating EPCs of asthmatic patients had higher levels of eotaxin-1 compared with those seen in control subjects. In the murine model OVA allergen exposure augmented eotaxin-1 mRNA and protein levels in EPCs. The EPCs from OVA-sensitized and OVA-challenged mice released high levels of eotaxin-1 on contact with lung endothelial cells from sensitized and challenged mice but not from control animals and not on contact with cardiac or hepatic endothelial cells from sensitized and challenged mice. Intranasal administration of the eotaxin-rich media overlying cultures of EPCs caused recruitment into the lungs, confirming functional chemoattractant activity. CONCLUSIONS: Bone marrow-derived EPCs are early responders to environmental allergen exposures and initiate a parallel switch to a proangiogenic and proeosinophilic environment in the lungs of asthmatic patients.

Four Color ImmunoSpot® Assays for Identification of Effector T-Cell Lineages.

Single color IFN-γ ELISPOT assays have evolved as a highly sensitive T cell immune monitoring platform. By detecting individual T cells that secrete IFN-γ in response to antigen exposure, these assays permit the measurement of the frequency of antigen-specific T cells among white blood cells. These assays therefore are well suited to assess clonal expansions, that is, whether a (Th1) T cell response has been induced to an antigen in a test subject. Single color IFN-γ ELISPOT assays are not suited, however, to provide information on the Th2/Th17 quality of the T cell response, nor do they provide insights into the differentiation state of CD8 cells. Recently it has been established that co-expression profiles of IL-2, TNF-α, and granzyme B along with IFN-γ permit to identify CD8 cell subpopulations. Naïve CD8 cells, central CD8 memory cells, CD8 terminal effector cells, polyfunctional CD8 cells, stem-cell like CD8 memory cells, dysfunctional- and senescent CD8 cells all differ in the extent they produce these molecules upon antigen re-encounter. We therefore have developed, and introduce here, a four color T cell ELISPOT assay in which the co-expression levels of IFN-γ, IL-2, TNF-α, and granzyme B can be established for individual antigen-specific CD8 cells, thereby identifying the activation/differentiation state of these cells.

Multiplex ImmunoSpot® Assays for the Study of Functional B Cell Subpopulations.

B cells mediate humoral immunity by producing antibody molecules, but they also participate in innate and acquired immune functions via the secretion of effector molecules such as cytokines, chemokines, and granzyme. B cell subpopulations releasing such effector molecules have been implicated in immunobiology and a number of diseases.Unlike antigen-specific T cells that can be identified by multimer staining, and then counter-stained to define T cell subpopulations, antigen-specific B cells cannot be detected by flow cytometry. Staining antigen-specific B cells with labeled antigen, in large, has been unsuccessful. Instead, antigen-specific B cells can be and are commonly studied by ELISPOT. In the ELISPOT approach, the B cell is identified via the antibody that it secretes being captured on a membrane by the antigen itself. Should it be feasible to measure simultaneously antibody production and the secretion of other secretory B cell products, it would then be possible to identify B cell subpopulations that co-express effector molecules. Here we introduce multiplex ELISPOT assays in which measurements of antibody secretion are combined with the detection of Granzyme B, IL-6, IL-10, IFN-γ, and TNF-α. Such multiplex assays will help define effector B cell subpopulations, as well as the understanding of their role in health and disease.

Multiplexing T- and B-Cell FLUOROSPOT Assays: Experimental Validation of the Multi-Color ImmunoSpot® Software Based on Center of Mass Distance Algorithm.

Over the past decade, ELISPOT has become a highly implemented mainstream assay in immunological research, immune monitoring, and vaccine development. Unique single cell resolution along with high throughput potential sets ELISPOT apart from flow cytometry, ELISA, microarray- and bead-based multiplex assays. The necessity to unambiguously identify individual T and B cells that do, or do not co-express certain analytes, including polyfunctional cytokine producing T cells has stimulated the development of multi-color ELISPOT assays. The success of these assays has also been driven by limited sample/cell availability and resource constraints with reagents and labor. There are few commercially available test kits and instruments available at present for multi-color FLUOROSPOT. Beyond commercial descriptions of competing systems, little is known about their accuracy in experimental settings detecting individual cells that secrete multiple analytes vs. random overlays of spots. Here, we present a theoretical and experimental validation study for three and four color T- and B-cell FLUOROSPOT data analysis. The ImmunoSpot® Fluoro-X™ analysis system we used includes an automatic image acquisition unit that generates individual color images free of spectral overlaps and multi-color spot counting software based on the maximal allowed distance between centers of spots of different colors or Center of Mass Distance (COMD). Using four color B-cell FLUOROSPOT for IgM, IgA, IgG1, IgG3; and three/four color T-cell FLUOROSPOT for IL-2, IFN-γ, TNF-α, and GzB, in serial dilution experiments, we demonstrate the validity and accuracy of Fluoro-X™ multi-color spot counting algorithms. Statistical predictions based on the Poisson spatial distribution, coupled with scrambled image counting, permit objective correction of true multi-color spot counts to exclude randomly overlaid spots.

ELISPOT Assays in 384-Well Format: Up to 30 Data Points with One Million Cells.

Comprehensive immune monitoring requires that frequencies of T cells, producing different cytokines, are measured to establish the magnitude of Th1, Th2, and Th17 components of cell-mediated immunity. Antigen titration provides additional information about the affinity of T cell response. In tumor immunity, it is also advisable to account for determinant spreading by testing multiple epitopes. Efforts for comprehensive immune monitoring would require substantial numbers of PBMC to run the above tests systematically, which in most test cases is limiting. Immune monitoring with ELISPOT assays have been performed, thus far, in a 96-well format. In this study we show that one can increase cell utilization by performing the assay in 384-well plates whose membrane surface area is one third that of 96-well plates. Systematic testing of PBMC for antigen-specific T cell response in the two formats demonstrated that the 384-well assay corresponds to a one-in-three miniaturization of the 96-well assay. The lowest number of cells that can be used in the 384-well format, while allowing for sufficient contact with APC, is 33,000 PBMC/well. Therefore, with one million PBMC typically obtained from 1 mL of blood, a 30 well T cell ELISPOT assay can be performed in a 384-well format.

Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity.

Most humans become infected with human cytomegalovirus (HCMV). Typically, the immune system controls the infection, but the virus persists and can reactivate in states of immunodeficiency. While substantial information is available on the contribution of CD8 T cells and antibodies to anti-HCMV immunity, studies of the TH1, TH2, and TH17 subsets have been limited by the low frequency of HCMV-specific CD4 T cells in peripheral blood mononuclear cell (PBMC). Using the enzyme-linked Immunospotr assay (ELISPOT) that excels in low frequency measurements, we have established these in a sizable cohort of healthy HCMV controllers. Cytokine recall responses were seen in all seropositive donors. Specifically, interferon (IFN)- and/or interleukin (IL)-17 were seen in isolation or with IL-4 in all test subjects. IL-4 recall did not occur in isolation. While the ratios of TH1, TH2, and TH17 cells exhibited substantial variations between different individuals these ratios and the frequencies were relatively stable when tested in samples drawn up to five years apart. IFN- and IL-2 co-expressing polyfunctional cells were seen in most subjects. Around half of the HCMV-specific CD4 cells were in a reversible state of exhaustion. The data provided here established the TH1, TH2, and TH17 characteristic of the CD4 cells that convey immune protection for successful immune surveillance against which reactivity can be compared when the immune surveillance of HCMV fails.

A trial design for evaluation of empiric programming of implantable cardioverter defibrillators to improve patient management.

The delivery of implantable cardioverter defibrillator (ICD) therapy is sophisticated and requires the programming of over 100 settings. Physicians tailor these settings with the intention of optimizing ICD therapeutic efficacy, but the usefulness of this approach has not been studied and is unknown. Empiric programming of settings such as anti-tachycardia pacing (ATP) has been demonstrated to be effective, but an empiric approach to programming all VT/VF detection and therapy settings has not been studied. A single standardized empiric programming regimen was developed based on key strategies with the intention of restricting shock delivery to circumstances when it is the only effective and appropriate therapy. The EMPIRIC trial is a worldwide, multi-center, prospective, one-to-one randomized comparison of empiric to physician tailored programming for VT/VF detection and therapy in a broad group of about 900 dual chamber ICD patients. The trial will provide a better understanding of how particular programming strategies impact the quantity of shocks delivered and facilitate optimization of complex ICD programming.