Surveillance Study of Influenza Occurrence and Immunity in a Wisconsin Cohort During the 2009 Pandemic.

BACKGROUND: Antibody and T-cell immunity to conserved influenza virus antigens can protect animals against infection with diverse influenza strains. Although immunity against conserved antigens occurs in humans, whether such responses provide cross-protection in humans and could be harnessed as the basis for universal influenza vaccines is controversial. The 2009 pandemic provided an opportunity to investigate whether pre-existing cross-reactive immunity affected susceptibility to infection. METHODS: In 2009, we banked sera and peripheral blood mononuclear cells (PBMC) from blood donors, then monitored them for pandemic influenza infection (pH1N1) by polymerase chain reaction or seroconversion. Antibodies to hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix 2 (M2), and HA-pseudotypes were measured in sera. T-cell inteferon-γ enzyme-linked immunospot responses were measured in PBMC. RESULTS: There were 13 infections in 117 evaluable donors. Pre-existing T-cell reactivity to pH1N1 was substantial (of 153 donors tested, 146 had >100 spot-forming cells/106 cells). Antibodies reactive with pH1N1 were common: anti-NP (all donors) and anti-M2 (44% of donors). Pseudotype-neutralizing antibodies to H1 were detected, but not to highly conserved HA epitopes. Unexpectedly, donors with symptomatic pH1N1 infection had sharp rises in HA pseudotype-neutralizing antibodies, not only pH1N1 but also against multiple seasonal H1s. In addition, an exploratory study of a T-cell marker (response to NP418-426) identified probable infection missed by standard criteria. CONCLUSIONS: Although the number of infections was inadequate for conclusions about mechanisms of protection, this study documents the wide variety of pre-existing, cross-reactive, humoral and cellular immune responses to pandemic influenza virus antigens in humans. These responses can be compared with results of other studies and explored in universal influenza vaccine studies.

Immunological monitoring of the tumor immunoenvironment for clinical trials.

Monitoring of immunotherapeutic clinical trials has undergone a considerable change in the last decade resulting in a general agreement that immune monitoring should guide the development of cancer vaccines. The emphasis on immune cell functions and quantitation of antigen-specific T cells have been playing a major role in the attempts to establish meaningful correlations between therapy-induced alterations in immune responses and clinical endpoints. However, one significant unresolved issue in modern immunotherapy is that when a tumor-specific cellular immune response is observed following the course of immunotherapy, it does not always lead to clinically proven cancer regression. This disappointing lack of a correlation between the tumor-specific cytotoxic immune responses and the clinical efficacy of immunotherapy may be explained, among other reasons, by the notion that the analysis of any single immunological parameter is not sufficient to provide clinically feasible information about the complex interactions between different cell subsets in the peripheral blood and immune, tumor, and stromal cells in the tumor milieu. By contrast, a systemic approach is required for improving the quality of a serial monitoring to ensure that it adequately and reliably measures potential changes induced in patients by administered vaccines or immunomodulators. Comprehensive evaluation of the balance between the immunostimulatory and immunosuppressive compartments of the immune system could be critical for a better understanding of how a given immunotherapy works or does not work in a particular clinical trial. New approaches to characterize tumor-infiltrating leukocytes, their phenotypic, biochemical, and genetic characteristics within the tumor microenvironment need to be developed and validated and should complement current monitoring techniques. These immune-monitoring assays for the local tumor immunoenvironment should be developed, validated, and standardized for reliability and consistency in order to establish the overall performance standards.

ELISPOT Assay for Monitoring Cytotoxic T Lymphocytes (CTL) Activity in Cancer Vaccine Clinical Trials.

The profiling and monitoring of immune responses are key elements in the evaluation of the efficacy and development of new biotherapies, and a number of assays have been introduced for analyzing various immune parameters before, during, and after immunotherapy. The choice of immune assays for a given clinical trial depends on the known or suggested immunomodulating mechanisms associated with the tested therapeutic modality. Cell-mediated cytotoxicity represents a key mechanism in the immune response to various pathogens and tumors. Therefore, the selection of monitoring methods for the appropriate assessment of cell-mediated cytotoxicity is thought to be crucial. Assays that can detect both cytotoxic T lymphocytes (CTL) frequency and function, such as the IFN-γ enzyme-linked immunospot assay (ELISPOT) have gained increasing popularity for monitoring clinical trials and in basic research. Results from various clinical trials, including peptide and whole tumor cell vaccination and cytokine treatment, have shown the suitability of the IFN-γ ELISPOT assay for monitoring T cell responses. However, the Granzyme B ELISPOT assay and Perforin ELISPOT assay may represent a more direct analysis of cell-mediated cytotoxicity as compared to the IFN-γ ELISPOT, since Granzyme B and perforin are the key mediators of target cell death via the granule-mediated pathway. In this review we analyze our own data and the data reported by others with regard to the application of various modifications of ELISPOT assays for monitoring CTL activity in clinical vaccine trials.

Application of a flow cytometric cytotoxicity assay for monitoring cancer vaccine trials.

In this study, we evaluated the applicability of a flow cytometry-based cytotoxicity (FC) assay previously developed by our laboratory, for monitoring cancer vaccine trials. The assay simultaneously measures effector cell degranulation and target cell death. Clinically relevant samples consisted of frozen peripheral blood mononuclear cells (PBMC) from vaccinated melanoma patients with known response to the melanoma peptide g209. Both PBMC and 7 day in vitro-stimulated lymphocyte from patient samples were used as effector cells in the FC assay. Activity against the relevant g209 and the control g280 peptide measured in the FC assay was directly compared with results obtained from the Granzyme B enzyme-linked immunosorbent spot assay and the standard 51Cr-release assay run in tandem. The FC assay detected low or no activity when PBMC were used as effector cells. Using cytotoxic T lymphocytes as effector cells, little or no effector cell degranulation or cytotoxicity was measured in the FC assay in prevaccination samples. After vaccination, an increase in both degranulation and target cell death could be determined when target cells were pulsed with g209. No or low reactivity was found against g280 at any time point. Our findings exhibited excellent correlation between CD107a expression and GrB secretion and also Annexin V binding to target cells and specific lysis measured in the 51Cr-release assay. Results obtained from the FC assay were highly reproducible. Therefore, the FC assay may be applied to vaccine trial monitoring and allows the measurement of effector cell degranulation and target cell death simultaneously in a single sample.

New approaches for monitoring CTL activity in clinical trials.

We have developed a modification of the ELISPOT assay that measures Granzyme B (GrB) release from cytotoxic T lymphocytes (CTLs). The GrB ELISPOT assay is a superior alternative to the 51Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-gamma ELISPOT assay, the GrB ELISPOT directly measures the release of a cytolytic protein. We report that the GrB ELISPOT can be utilized to measure ex vivo antigen-specific cytotoxicity of peripheral blood mononuclear cells (PBMCs) from cancer patients vaccinated with a peptide-based cancer vaccine. We compare the reactivity of patients' PBMCs in the GrB ELISPOT, with reactivity in the tetramer, IFN-gamma ELISPOT and chromium (51Cr)-release assays. Differences in immune response over all assays tested were found between patients, and four response patterns were observed. Reactivity in the GrB ELISPOT was more closely associated with cytotoxicity in the 51Cr-release assay than the tetramer or IFN-gamma ELISPOT assays. We also optimized the GrB ELISPOT assay to directly measure immune responses against autologous primary tumor cells in vaccinated cancer patients. A perforin ELISPOT assay was also adapted to evaluate peptide-stimulated reactivity of PMBCs from vaccinated melanoma patients. Modifications of the ELISPOT assay described in this chapter allow a more comprehensive evaluation of low-frequency tumor-specific CTLs and their specific effector functions and can provide a valuable insight into immune responses in cancer vaccine trials.

DNAse treatment following thawing of cryopreserved PBMC is a procedure suitable for lymphocyte functional studies.

Testing freshly isolated PBMC is not practical for immune monitoring analysis in large clinical trials or natural history studies. Thus, cryopreserved PBMC represent a more practical alternative. However, cell clumping is a common problem following thawing of PBMC isolated from blood that was previously transported and stored. Cell clumping leads to loss of cells, and could affect cell function and/or phenotype. The development and validation of procedures that prevent cell clumping and preserve cell function and surface marker expression levels are necessary to allow evaluation of immune function and phenotype in cryopreserved samples from clinical studies. The incorporation of a DNAse treatment step in the standard thawing procedure efficiently avoided clump formation, and did not result in detectable changes in cell viability, expression of standard leukocyte surface markers or two key parameters of immune function, proliferation and cytokine induction in response to a variety of common stimuli. Therefore, this procedure seems suitable for standard immunologic assays.

Application of the granzyme B ELISPOT assay for monitoring cancer vaccine trials.

Granzyme B (GrB) is present in the granules of cytolytic lymphocytes and is a key mediator of cell-mediated target cell death via the granule-mediated pathway. The release of GrB can be used as an indicator of a cytotoxic T lymphocyte response. Herein, we report that the GrB enzyme-linked immunospot assay (ELISPOT) can be used to measure ex vivo antigen-specific cytotoxicity of peripheral blood mononuclear cells from cancer patients vaccinated with a peptide-based cancer vaccine. We compare the reactivity of patients' peripheral blood mononuclear cells in the GrB ELISPOT with reactivity in the tetramer, interferon (IFN)-gamma ELISPOT, and Cr-release assays. Differences in immune response over all assays tested were found between patients and 4 response patterns were observed. Reactivity in the GrB ELISPOT was more closely associated with cytotoxicity in the Cr-release assay than the tetramer or IFN-gamma ELISPOT assays. Moreover, the higher affinity g209-2M peptide (used for vaccination) elicited greater GrB secretion than the native g209 peptide, although this difference was not observed with IFN-gamma secretion. Taken together with the fact that GrB is a specific mediator released by cytotoxic T lymphocytes, these results show that simultaneous use of the GrB ELISPOT assay with other immunologic assays may provide important additional immunologic insight into patient responses to cancer vaccines.

A novel flow cytometric assay for evaluating cell-mediated cytotoxicity.

Comprehensive evaluation of cell-mediated cytotoxicity is very important, especially in the clinical setting, when a surrogate immunologic endpoint that correlates with a clinical outcome needs to be defined. With the objective of simultaneously evaluating target cell death and effector cell frequency, the authors combined the measuring of the expression of the degranulation marker CD107a by effector cells with the apoptosis marker annexin V binding to target cells. Using human cytotoxic T lymphocytes, the authors found a significant incubation time-dependent increase of surface CD107a expression on effector cells due to degranulation. A parallel increase of annexin V binding to target cells due to target cell apoptosis was also found. These two parameters have shown excellent correlation in all effector/target cell systems studied. To find possible connections between effector cell degranulation (CD107a expression), granzyme B secretion, and target cell death (annexin V binding), the GrB ELISPOT assay and flow cytometric assay were performed and excellent cross-correlation was found between all three parameters. The specificity of the assay was also shown. These data show that this novel assay allows measurement of cytolytic cell activation and frequency as well as target cell death in the same sample.

Evaluating the cytotoxicity of innate immune effector cells using the GrB ELISPOT assay.

BACKGROUND: This study assessed the Granzyme B (GrB) ELISPOT as a viable alternative to the 51Cr-release assay for measuring cytotoxic activity of innate immune effector cells. We strategically selected the GrB ELISPOT assay because GrB is a hallmark effector molecule of cell-mediated destruction of target cells. METHODS: We optimized the GrB ELISPOT assay using the human-derived TALL-104 cytotoxic cell line as effectors against K562 target cells. Titration studies were performed to assess whether the ELISPOT assay could accurately enumerate the number of GrB-secreting effector cells. TALL-104 were treated with various secretion inhibitors and utilized in the GrB ELISPOT to determine if GrB measured in the ELISPOT was due to degranulation of effector cells. Additionally, CD107a expression on effector cells after effector-target interaction was utilized to further confirm the mechanism of GrB release by TALL-104 and lymphokine-activated killer (LAK) cells. Direct comparisons between the GrB ELISPOT, the IFN-gamma ELISPOT and the standard 51Cr-release assays were made using human LAK cells. RESULTS: Titration studies demonstrated a strong correlation between the number of TALL-104 and LAK effector cells and the number of GrB spots per well. GrB secretion was detectable within 10 min of effector-target contact with optimal secretion observed at 3-4 h; in contrast, optimal IFN-gamma secretion was not observed until 24 h. The protein secretion inhibitor, brefeldin A, did not inhibit the release of GrB but did abrogate IFN-gamma production by TALL-104 cells. GrB secretion was abrogated by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid tetra(acetoxymethyl) ester), which sequesters intracellular Ca2+, thereby preventing degranulation. The number of effector cells expressing the degranulation associated glycoprotein CD107a increased after interaction with target cells and correlated with the stimulated release of GrB measured in the ELISPOT assay. CONCLUSIONS: Because of its high sensitivity and ability to estimate cytotoxic effector cell frequency, the GrB ELISPOT assay is a viable alternative to the 51Cr-release assay to measure MHC non-restricted cytotoxic activity of innate immune cells. Compared to the IFN-gamma ELISPOT assay, the GrB ELISPOT may be a more direct measure of cytotoxic cell activity. Because GrB is one of the primary effector molecules in natural killer (NK) cell-mediated killing, detection and enumeration of GrB secreting effector cells can provide valuable insight with regards to innate immunological responses.

A modified human ELISPOT assay to detect specific responses to primary tumor cell targets.

BACKGROUND: The desired outcome of cancer vaccination is to induce a potent T cell response which can specifically recognize and eliminate autologous tumor cells in vivo. Accordingly, immunological assays that demonstrate recognition of native tumor cells (tumor-specific) may be more clinically relevant than assays that demonstrate recognition of tumor protein or peptide (antigen-specific). METHODS: Towards this goal, we adapted the IFN-gamma ELISPOT assay to measure immune responses against autologous primary tumor cells in vaccinated cancer patients. As a model system to develop the assay, we utilized peripheral blood mononuclear cells (PBMC) directly isolated from follicular lymphoma patients vaccinated with tumor-derived idiotype protein. RESULTS: After optimizing several variables, we demonstrated that the modified IFN-gamma ELISPOT assay could be used to reliably and reproducibly determine the tumor-reactive T cell frequency in the PBMC of these patients. The precursor frequency of tumor-reactive T cells was significantly higher in the postvaccine PBMC, compared with prevaccine samples in all patients tested. Furthermore, the specificity of these T cells was established by the lack of reactivity against autologous normal B cells. CONCLUSIONS: These results demonstrate the feasibility of quantitating tumor-specific T cell responses when autologous, primary tumor cells are available as targets.

The Granzyme B ELISPOT assay: an alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity.

BACKGROUND: The interferon-gamma (IFN-gamma) ELISPOT assay is one of the most useful techniques for immunological monitoring of cancer vaccine trials and has gained increased application as a measure of specific T cell activation. However, it does not assess cell-mediated cytotoxicity directly as IFN-gamma secretion is not limited to only cytolytic cells. Granzyme B (GrB) is a key mediator of target cell death via the granule-mediated pathway. Therefore, the release of GrB by cytolytic lymphocytes upon effector-target interaction may be a more specific indicator of CTL and NK cytotoxic ability than IFN-gamma secretion. METHODS: We assessed whether the GrB ELISPOT assay is a viable alternative to the 51Cr-release and IFN-gamma ELISPOT assays for measuring antigen-specific CTL cytotoxicity. Direct comparisons between the three assays were made using human CTL cell lines (alphaEN-EBV and alphaJY) and an in vitro stimulated anti-Flu matrix peptide (FMP)-specific CTL. RESULTS: When the GrB ELISPOT was directly compared to the IFN-gamma ELISPOT and 51Cr-release assays, excellent cross-correlation between all three assays was shown. However, measurable IFN-gamma secretion in the ELISPOT assay was observed only after 1 hour of incubation and cytotoxicity assessed via the 51Cr-release assay after 4 hours, whereas GrB secretion was detectable within 10 min of effector-target contact with significant secretion observed after 1 h. Titration studies demonstrated a strong correlation between the number of effector cells and GrB spots per well. Irrelevant targets or antigens did not induce significant GrB secretion. Additionally, GrB secretion was abrogated when CTL cultures were depleted of CD8+ cells. CONCLUSION: Our findings demonstrate that the GrB ELISPOT assay is a superior alternative to the 51Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-gamma ELISPOT assay, the GrB ELISPOT directly measures the release of a cytotolytic protein. Detection of low frequency tumor-specific CTL and their specific effector functions can provide valuable insight with regards to immunological responses.