Srividya Sundararaman

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 Immunospot® 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)-γ 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-γ 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.

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.

High Reproducibility of ELISPOT Counts from Nine Different Laboratories.

The primary goal of immune monitoring with ELISPOT is to measure the number of T cells, specific for any antigen, accurately and reproducibly between different laboratories. In ELISPOT assays, antigen-specific T cells secrete cytokines, forming spots of different sizes on a membrane with variable background intensities. Due to the subjective nature of judging maximal and minimal spot sizes, different investigators come up with different numbers. This study aims to determine whether statistics-based, automated size-gating can harmonize the number of spot counts calculated between different laboratories. We plated PBMC at four different concentrations, 24 replicates each, in an IFN-γ ELISPOT assay with HCMV pp65 antigen. The ELISPOT plate, and an image file of the plate was counted in nine different laboratories using ImmunoSpot® Analyzers by (A) Basic Count™ relying on subjective counting parameters set by the respective investigators and (B) SmartCount™, an automated counting protocol by the ImmunoSpot® Software that uses statistics-based spot size auto-gating with spot intensity auto-thresholding. The average coefficient of variation (CV) for the mean values between independent laboratories was 26.7% when counting with Basic Count™, and 6.7% when counting with SmartCount™. Our data indicates that SmartCount™ allows harmonization of counting ELISPOT results between different laboratories and investigators.

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.

High-Throughput GLP-Capable Target Cell Visualization Assay for Measuring Cell-Mediated Cytotoxicity

One of the primary effector functions of immune cells is the killing of virus-infected or malignant cells in the body. Natural killer (NK) and CD8 effector T cells are specialized for this function. The gold standard for measuring such cell-mediated cytolysis has been the chromium release assay, in which the leakage of the radioactive isotope from damaged target cells is being detected. Flow cytometry-based single cell analysis of target cells has recently been established as a non-radioactive alternative. Here we introduce a target cell visualization assay (TVA) that applies similar target cell staining approaches as used in flow cytometry but based on single cell computer image analysis. Two versions of TVA are described here. In one, the decrease in numbers of calcein-stained, i.e., viable, target cells is assessed. In the other, the CFSE/PI TVA, the increase in numbers of dead target cells is established in addition. TVA assays are shown to operate with the same sensitivity as standard chromium release assays, and, leaving data audit trails in form of scanned (raw), analyzed, and quality-controlled images, thus meeting requirements for measuring cell-mediated cytolysis in a regulated environment.

A Positive Control for Detection of Functional CD4 T Cells in PBMC: The CPI Pool

Testing of peripheral blood mononuclear cells (PBMC) for immune monitoring purposes requires verification of their functionality. This is of particular concern when the PBMC have been shipped or stored for prolonged periods of time. While the CEF (Cytomegalo-, Epstein-Barr and Flu-virus) peptide pool has become the gold standard for testing CD8 cell functionality, a positive control for CD4 cells is so far lacking. The latter ideally consists of proteins so as to control for the functionality of the antigen processing and presentation compartments, as well. Aiming to generate a positive control for CD4 cells, we first selected 12 protein antigens from infectious/environmental organisms that are ubiquitous: Varicella, Influenza, Parainfluenza, Mumps, Cytomegalovirus, Streptococcus, Mycoplasma, Lactobacillus, Neisseria, Candida, Rubella, and Measles. Of these antigens, three were found to elicited interferon (IFN)-γ-producing CD4 cells in the majority of human test subjects: inactivated cytomegalo-, parainfluenza-, and influenza virions (CPI). While individually none of these three antigens triggered a recall response in all donors, the pool of the three (the ‘CPI pool’), did. One hundred percent of 245 human donors tested were found to be CPI positive, including Caucasians, Asians, and African-Americans. Therefore, the CPI pool appears to be suitable to serve as universal positive control for verifying the functionality of CD4 and of antigen presenting cells.

When Results of T cell Immune Monitoring Match/Do Not Match Clinical Outcomes of Tumor Vaccine Trials: What More Could and Should We Measure?

Activation of cancer-specific T cells by tumor vaccines can lead to the rejection of the tumor. However, monitoring the tumor-specific T cell response continues to be a challenge. The magnitude and cytokine effector lineage of the vaccine-induced T cells, their ability to kill, and the avidity of the T cells for the tumor are among the primary parameters that define the T cell’s potential impact on the tumor. Due to determinant spreading, immunization with a tumor antigen can result in recruitment of T cells with specificity for unrelated antigens of the tumor. The efficacy of this second wave T cell repertoire will depend on clonal sizes, effector lineages, and their avidities for the tumor. Comprehensive immune monitoring should be able to measure all the above parameters of the primary and secondary T cell responses to the tumor. In this chapter, we propose an ELISPOT-based protocol which can help accomplish the above goal with reasonable cost and effort, requiring only 10 ml of patient blood.

Abstract B57: NK cell-mediated cytotoxicity detection can be miniaturized by direct imaging in assay wells using a Terasaki plate format using only 100,000 effector cells (PBMC)

Introduction: Natural Killer (NK) Cell - and Antibody Dependent (ADCC) Cell - Cytotoxicity has traditionally been assessed by the release of radioactive Chromium from target cells following lysis. This assay is laborious, and requires substantial quantities of patient blood to detect minor changes in cell lysis due to the inherent background noise (spontaneous release). We have developed an assay that can visualize individual target cells to detect cytolytic activity without involving radioactivity, via high-throughput imaging in microtiter well format. We also developed a miniaturized version of this assay using Terasaki plates to measure NK activity with one tenth of the blood needed for a classic Chromium Release assay, and with less labor. Methods: The assay we developed, Target cell Visualization Assay (TVA™) images individual fluorescence-labeled target cells. Labelled K562 tumor cells were used as targets, and peripheral blood mononuclear cells (PBMC) as effector cells. In an assay setup similar to Chromium Release Assays (CRA), constant number of labelled target cells was incubated with serially diluted effector cells. Four hours later, the cells were transferred to flat bottom microtiter plates and the number of viable tumor cells was quantitated using a plate reader; CTL ImmunoSpot® Analyzer, S6ULT-00-9000. For the miniaturized version, one tenth of effector and target cells were plated in a Terasaki plate format and imaged directly in the assay wells. Results: Only viable target cells retain the fluorescent dye; it is lost upon cell death. When effector and target cells are mixed in various ratios, the % of target cell lysis is inversely proportional to the number of effector cells in the well. The TVA™ and CRA in a 96-well format provided equivalent results. However, the TVA™ is a non-radioactive system and can be performed with much less labor. The TVA™ assay exhibited high intermediate precision as well as inter-assay repeatability. The assay results obtained in Terasaki plates, miniaturizing the assay tenfold, paralleled those of the 96-well format. Conclusion: The miniaturized TVA™ uses only 100,000 effector cells for eight serial dilutions, for Effector:Target ratios starting from 100:1. We have demonstrated the feasibility of detecting and assessing NK function in a non-radioactive, high-throughput capable system using a fraction of the blood/effector cells as is required by traditional CRA. This is of particular value when access to PBMC is limited, such as pediatric, geriatric and immune deficient populations. The assay readout and analysis is automated by the CTL ImmunoSpot® software providing audit trails for each test condition. Citation Format: Srividya Sundararaman, Kinga Karacsony, Diana Roen, Jaya Ghosh, Paul V. Lehmann. NK cell-mediated cytotoxicity detection can be miniaturized by direct imaging in assay wells using a Terasaki plate format using only 100,000 effector cells (PBMC). [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B57.

Imaging-based, non-radioactive measurements of natural killer cell activity provides comparable results as classic chromium release assays but with fewer cells and less labor

Natural Killer Cell activity has traditionally been assessed by detecting the lysis of tumor cells in The Chromium Release assay. This classic assay relies on radioactive labels, is laborious, and requires substantial quantities of patient blood. Moreover the Chromium Release assay provides low signal to noise ratios. We have developed an assay that can visualize individual target cells to detect cytolytic activity within a high signal to noise range, without involving radioactivity, via high-throughput imaging. We also developed a miniaturized version of this assay to measure NK activity with one tenth of the blood needed for a classic Chromium Release assay, and with less labor.

Log-Normal ELISPOT spot size distribution permits count harmonization among different laboratories

ELISPOT assays are primarily used to detect the number of T cells that respond to a given antigen. With that number being absolute for any given donor sample, ELISPOT counts should be similar between laboratories, if subjectivity in counting is avoided. Due to the differences in spot sizes ranging from microns to millimeters, setting cut offs for minimal and maximal spot sizes will lead to substantial variability between investigators when determined subjectively, irrespective of experience. In contrast, if spot size distributions would follow predictable statistical functions, objective gating decisions could be made using common standards, eliminating subjective calls of the counting process. This study aims to determine if ELISPOT size distribution follows predictable statistical distributions and therefore if ELISPOT counting can be made objective based on statistical principles.