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.

Detecting all Immunoglobulin Classes and Subclasses in a Multiplex 7 Color ImmunoSpot® Assay.

Antibody molecules in peripheral blood have a relatively short half-life of roughly 20 days, and therefore their persistence in the serum depends on continuous replenishment by plasma cells. Serum antibody titers are thus indirect and unreliable indicators of immunological memory. In contrast, memory B cells persist in peripheral blood for decades, and enumerating these cells provides direct evidence of having developed an immune response to a given antigen. ELISPOT is an ideal research tool for enumerating antigen-specific memory B cells. Traditionally, B cell ELISPOT assays have been performed for detecting a single class of immunoglobulin (Ig), using a single colorimetric substrate. For comprehensive monitoring of B cell memory, however, all immunoglobulin classes and subclasses need to be assessed. Thus, seven single color assays would need to be performed to measure the numbers of antigen-specific B cells producing IgM, IgA, IgE, IgG1, IgG2, IgG3, and IgG4. We report here the development of a multiplex seven color B cell ImmunoSpot® assay in which the number of antigen-specific B cells can be established simultaneously for all major antibody classes and subclasses, requiring the PBMC, antigen, and labor corresponding to a single color assay.

B Cells and B Cell Blasts Withstand Cryopreservation While Retaining Their Functionality for Producing Antibody.

In individuals who have once developed humoral immunity to an infectious/foreign antigen, the antibodies present in their body can mediate instant protection when the antigen re-enters. Such antigen-specific antibodies can be readily detected in the serum. Long term humoral immunity is, however, also critically dependent on the ability of memory B cells to engage in a secondary antibody response upon re-exposure to the antigen. Antibody molecules in the body are short lived, having a half-life of weeks, while memory B cells have a life span of decades. Therefore, the presence of serum antibodies is not always a reliable indicator of B cell memory and comprehensive monitoring of humoral immunity requires that both serum antibodies and memory B cells be assessed. The prevailing view is that resting memory B cells and B cell blasts in peripheral blood mononuclear cells (PBMC) cannot be cryopreserved without losing their antibody secreting function, and regulated high throughput immune monitoring of B cell immunity is therefore confined to-and largely limited by-the need to test freshly isolated PBMC. Using optimized protocols for freezing and thawing of PBMC, and four color ImmunoSpot® analysis for the simultaneous detection of all immunoglobulin classes/subclasses we show here that both resting memory B cells and B cell blasts retain their ability to secrete antibody after thawing, and thus demonstrate the feasibility of B cell immune monitoring using cryopreserved PBMC.

Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify.

It is essential to identify donors who have not been infected with human cytomegalovirus (HCMV) in order to avoid transmission of HCMV to recipients of blood transfusions or organ transplants. In the present study, we tested the reliability of seronegativity as an indicator for the lack of HCMV exposure in healthy human blood donors. Eighty-two HCMV seronegative individuals were identified, and their peripheral blood mononuclear cells (PBMC) were tested in ImmunoSpot® assays for the presence of HCMV-specific T- and B-memory lymphocytes. Eighty-two percent (67 of 82) of these HCMV seronegative individuals featured at least one memory cell that was lineage specific for HCMV, with the majority of these subjects possessing CD4+ and CD8+ T cells, as well as B cells, providing three independent lines of evidence for having developed immunity to HCMV. Only 15 of these 82 donors (18%) showed neither T- nor B-cell memory to HCMV, consistent with immunological naïveté to the virus. The data suggest that measurements of serum antibodies frequently fail to reveal HCMV exposure in humans, which may be better identified by direct detection of HCMV-specific memory lymphocytes.

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.

Normal Distribution of CD8+ T-Cell-Derived ELISPOT Counts within Replicates Justifies the Reliance on Parametric Statistics for Identifying Positive Responses.

Accurate assessment of positive ELISPOT responses for low frequencies of antigen-specific T-cells is controversial. In particular, it is still unknown whether ELISPOT counts within replicate wells follow a theoretical distribution function, and thus whether high power parametric statistics can be used to discriminate between positive and negative wells. We studied experimental distributions of spot counts for up to 120 replicate wells of IFN-γ production by CD8+ T-cell responding to EBV LMP2A (426 - 434) peptide in human PBMC. The cells were tested in serial dilutions covering a wide range of average spot counts per condition, from just a few to hundreds of spots per well. Statistical analysis of the data using diagnostic Q-Q plots and the Shapiro-Wilk normality test showed that in the entire dynamic range of ELISPOT spot counts within replicate wells followed a normal distribution. This result implies that the Student t-Test and ANOVA are suited to identify positive responses. We also show experimentally that borderline responses can be reliably detected by involving more replicate wells, plating higher numbers of PBMC, addition of IL-7, or a combination of these. Furthermore, we have experimentally verified that the number of replicates needed for detection of weak responses can be calculated using parametric statistics.

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.

Serial Measurements of Apoptotic Cell Numbers Provide Better Acceptance Criterion for PBMC Quality than a Single Measurement Prior to the T Cell Assay.

As soon as Peripheral Blood Mononuclear Cells (PBMC) are isolated from whole blood, some cells begin dying. The rate of apoptotic cell death is increased when PBMC are shipped, cryopreserved, or stored under suboptimal conditions. Apoptotic cells secrete cytokines that suppress inflammation while promoting phagocytosis. Increased numbers of apoptotic cells in PBMC may modulate T cell functions in antigen-triggered T cell assays. We assessed the effect of apoptotic bystander cells on a T cell ELISPOT assay by selectively inducing B cell apoptosis using α-CD20 mAbs. The presence of large numbers of apoptotic B cells did not affect T cell functionality. In contrast, when PBMC were stored under unfavorable conditions, leading to damage and apoptosis in the T cells as well as bystander cells, T cell functionality was greatly impaired. We observed that measuring the number of apoptotic cells before plating the PBMC into an ELISPOT assay did not reflect the extent of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay did. Our data suggest that measuring the numbers of apoptotic cells prior to and post T cell assays may provide more stringent PBMC quality acceptance criteria than measurements done only prior to the start of the assay.

Optimal thawing of cryopreserved peripheral blood mononuclear cells for use in high-throughput human immune monitoring studies.

Cryopreserved peripheral blood mononuclear cells (PBMC) constitute an important component of immune monitoring studies as they allow for efficient batch- testing of samples as well as for the validation and extension of original studies in the future. In this study, we systematically test the permutations of PBMC thawing practices commonly employed in the field and identify conditions that are high and low risk for the viability of PBMC and their functionality in downstream ELISPOT assays. The study identifies the addition of ice-chilled washing media to thawed cells at the same temperature as being a high risk practice, as it yields significantly lower viability and functionality of recovered PBMC when compared to warming the cryovials to 37 °C and adding a warm washing medium. We found thawed PBMC in cryovials could be kept up to 30 minutes at 37 °C in the presence of DMSO before commencement of washing, which surprisingly identifies exposure to DMSO as a low risk step during the thawing process. This latter finding is of considerable practical relevance since it permits batch-thawing of PBMC in high-throughput immune monitoring environments.

Effect of multiple activation stimuli on the generation of Th1-polarizing dendritic cells.

It was originally reported that only a small fraction of total matured dendritic cells (DCs) produced interleukin (IL)-12, but it has never been determined whether different combinations of activating signals now shown to maximize secreted IL-12 do so through increasing output by the same IL-12 producers, or by recruiting additional cytokine-secreting cells. We therefore tested all combinations of bacterial lipopolysaccharide (LPS) (TLR4 ligand), R848 (TLR8 ligand), interferon (IFN)-γ, and CD40L for activating human monocyte-derived dendritic cells (DC), and determined by intracellular flow cytometry that enhanced IL-12 secretion was accomplished in large part by markedly increasing the proportion of cells producing IL-12, with the triple and quadruple combinations recruiting the most DC. This optimization requirement for multiple signals was not reflected in differential Toll-like receptor (TLR) expression by the cells. Interestingly, DCs activated with single TLR ligands plus IFN-γ were capable of responding with a second burst of IL-12 upon later CD40L stimulation, whereas DCs activated with R848 plus LPS were not, despite the trend of the latter for superior polarization of naive T cells toward IFN-γ-secreting Th1. These results have implications for the biology of IL-12-secreting DCs and choice of activation regimen for prospective use in DC-based immunotherapy.