Special Issue "ELISPOT Research"

Abstract: Tuberculosis is a global health problem. The Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccine has variable efficacy (0–80%) so there is a drive to develop novel vaccines. The cytokine, interferon gamma (IFNγ), is an essential component of the protective response to M. tuberculosis (M. tb) infection and is also produced in response to BCG vaccination. Induction of an IFNγ response is used as a biomarker of successful vaccination in the assessment of new tuberculosis (TB) vaccines. The IFNγ ELISPOT assay provides an important tool for TB research. It is used for both the diagnosis of infection (T.Spot assay), and for the evaluation of the immunogenicity of new TB vaccine candidates in human clinical trials, in the non-human primate (NHP) model of TB infection studies. The ELISPOT assay captures IFNγ produced by peripheral blood mononuclear cells (PBMCs) following specific stimulation, onto a membrane so individual cells can be enumerated and the frequency of responding cells determined. Hence spot forming units (SFU) per 10⁶ cells provide the traditional measure for ELISPOT assays. The discriminatory power of SFU is limited. In some situations, the number of SFU in BCG vaccinated, and unvaccinated, subjects was found to be similar, although the spots were observed to be larger in vaccinated subjects. Spot size potentially provides a measure of the quantity of cytokine produced by individual cells. The AID ELISPOT plate reader software used to determine frequency of spots also has the capability to determine the size of each spot. Consideration of spot size in combination with spot forming units was investigated in our studies of BCG immunogenicity. This additional readout was found to enhance the discriminatory power of the ELISPOT assay, and provide more information on the immune response to BCG vaccination and infection with M.tb.

Abstract: The traditional enzyme-linked immunospot (ELISpot) assay is the gold standard for the enumeration of antigen-specific B cells. Since B cell availability from biological samples is often limited, either because of sample size/volume or the need of performing multiple analyses on the same sample, the implementation of ELISpot assay formats that allow the simultaneous detection of multiple antibody types is desirable. While dual-color ELISpot assays have been described, technical complexities have so far prevented their wide utilization as well as further expansion of their multicolor capability. An attractive solution is to replace the chromogenic reaction of the traditional ELISpot assay with a fluorescent detection system (fluorospot assay). Fluorospot assays using fluorophore-conjugated secondary antibodies in conjunction with fluorescence enhancers, FITC/anti-FITC and biotin/avidin amplification systems and dedicated equipment for spot detection have been developed to enumerate T-cells secreting two or three specific cytokines and, more recently, IgG and IgA antibody-secreting cells (ASCs). We hereby report a method for a multiplex B cell fluorospot assay that utilizes quantum-dot nanocrystals as reporters without further amplification systems or need of dedicated equipment. With this method we simultaneously enumerated HIV-1 gp41 envelope glycoprotein-specific IgG and IgM antibody-secreting cells with sensitivity comparable to that of the traditional ELISpot assay.

Abstract: We have evaluated a novel B-cell FluoroSpot assay for the analysis of antibody responses in healthy individuals vaccinated intramuscularly with Influenza A (H1N1) antigen (Pandemrix^®, GlaxoSmithKline). Using the FluoroSpot assay and an ELISpot assay run in parallel for comparison, we measured the frequency of cells secreting antigen-specific as well as total IgG or IgA antibodies seven days post vaccination. The assays were based on high affinity monoclonal antibodies for capture and detection of human IgG and IgA. Whereas conventional ELISpot analyzes IgG- and IgA-secreting B cells separately, fluorescent detection enabled simultaneous enumeration of B cells secreting IgG or IgA in the same well. The FluoroSpot protocol was also simpler as the assay could be performed without the need for an amplifying detection step. While having all the advantages of a conventional ELISpot assay, including high sensitivity, robustness and ease of performance, the FluoroSpot assay adds further value in reducing costs, time and material.

Abstract: The role of cytomegalovirus (CMV) infection in the pathogenesis and exacerbation of Inflammatory Bowel Disease (IBD) has been unresolved. Typically, the CMV genome remains dormant in infected cells, but a breakdown of immune surveillance can lead to re-activation of viral replication in the gut mucosa, which is not necessarily associated with viremia or changes in antibody titers. We hypothesized that the detection of CMV-specific CD8 effector T cells should permit the distinction between dormant and active CMV infection. As CD8 effector T cells, unlike memory CD8 T cells, have perforin (PFN) and granzyme B (GzB) preformed in their cytoplasmic granules, we employed single cell resolution ELISPOT assays to measure the CMV antigen-triggered release of these molecules by CD8 T cells isolated from subjects with IBD, and age-matched healthy controls. The frequencies of CMV-specific (GzB) and PFN-producing CD8 T cells were increased in IBD patients compared to healthy controls. Furthermore, the increased CMV reactivity was associated with active IBD disease and with longer disease duration. Notably, PCR on serum frequently failed to detect CMV DNA during flares. The data show that during active IBD there is a flare of CD8 T cell activity against CMV in a substantial proportion of IBD patients, suggesting CMV reactivation that serum PCR does not detect. While it remains open whether CMV reactivation is a cause or consequence of IBD, our data suggest that monitoring CMV antigen-specific effector CD8 T cells with GzB and PFN ELISPOT analysis can provide novel insights into the role of CMV infection in IBD. Additionally, our data have implications for the fields of transplantation, HIV, cancer, and autoimmune diseases, in all of which patient care critically depends on sensitive and reliable detection of a reactivation of CMV infection.

Abstract: In recent years we and others have used the ELISPOT assay successfully to identify novel tumor antigens by the characterization of spontaneous HLA class I restricted immune responses against a number of minimal 9–10 amino acid long peptide epitopes. In the present study, we examined the capability of using longer peptides when scrutinizing Peripheral Blood Mononuclear Cells (PMBC) from melanoma patients for spontaneous immunity by means of ELISPOT IFN-γ secretion assay. To this end, we examined PBMC for the presence of specific T-cell responses against long peptides derived from the tumor associated antigen BCL-X(L). The protein product of the larger BCL-X(L) differs from Bcl-X(S) protein by an inserted region (amino acids 126–188). Thus, we scrutinized eight long peptides covering this inserted region for spontaneous immunity. The peptides were overlapping and consisted of 20–23 amino acids. PBMC were pre-stimulated with peptide-pulsed autologous dendritic cells (DC) and subjected to the IFN-γ ELISPOT assay. Four of the BCL-X(L) derived peptides elicited very frequent responses in several patients. Additionally, in all patients responses against more than one of the peptides could be detected. In conclusion several long BCL-X(L) derived peptide epitopes exist, which may be used in anti-cancer immunity. Furthermore, the ELISPOT assay offers an attractive and sensitive method for the characterization of spontaneous immune reactivity against long peptides.

Abstract: Acute graft-versus-host disease (aGVHD) remains a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Infections may coexist and in certain circumstances aggravate aGVHD. It was described that type 1 as well as type 2 cytokines are important mediators of aGVHD. We measured spot-forming cells (SFCs) for interferon (IFN)-γ, interleukin (IL)-4, IL-10, and IL-17 in unstimulated peripheral blood from 80 patients with hematological disorders who underwent allogeneic hematopoietic stem cell transplantation by using the enzyme-linked immunospot (ELISPOT) assay that reflects the ongoing in vivo immune status. A serial monitoring showed that both type 1 and type 2 cytokine SFCs were correlated with aGVHD activity. The numbers of IFN-γ and IL-4 SFCs in patients with grade II-IV aGVHD were significantly higher than those in patients with grade 0 and/or I aGVHD. Elevation of IFN-γ and IL-4 SFCs was significantly correlated with the severity of aGVHD, but not with infection itself, e.g., cytomegalovirus infection. Cytokine SFCs are clinically relevant biomarkers for the diagnostic and therapeutic evaluation of aGVHD and concurrent infection.

Abstract: Mycobacterium tuberculosis (MTB) is a global heath epidemic, its threat amplified by HIV infection and the emergence of multidrug-resistant tuberculosis (MDR-TB). Interferon (IFN)-gamma release assays (IGRAs) have improved the accuracy of detection of MTB exposure in some subject groups as compared to the Tuberculin Skin Test (TST). However, as IFN-gamma is produced by both fully rested and more recently activated populations of memory T cells, it is not surprising that the measurement of this cytokine alone cannot accurately distinguish Latent TB Infected (LTBI) subjects from those with active (infectious) disease. Accurate and rapid diagnosis of infectious individuals would allow medication to be properly allocated and other actions taken to more effectively curtail MTB spread. Analysis of multi-cytokine profiles ex vivo after stimulation of PBMCs from LTBI and active MTB subjects indicate the real possibility of successfully discerning these two disease states within 24 hours of a subject’s blood draw. Due to the unparalleled sensitivity, low cost, and ease of use of Elispot assays, we propose that via a multiplex Elispot platform the accurate distinction of LTBI from active MTB-infected individuals is within reach.

Abstract: Immunologic risk in kidney transplantation is typically minimized by avoiding, or at least limiting, the potential of donor specific humoral responses by testing for the presence of donor-specific antibodies (DSA). Additionally, selecting donor and recipient pairs with the least number of human leukocyte antigen (HLA) mismatches has been shown to play a role in transplant outcome. However, numerous other factors may play a role in the success of transplant outcome and patient health. Specifically, the use of T-cell allospecific ELISPOT assays have helped elucidate the role of pre-formed cellular responses as additional factors in post-transplant outcome. In this review, we will evaluate numerous uses of ELISPOT assays to assess the pre- and post-transplant immunologic risk of rejection episodes, graft survival and even viral susceptibility as well as the utility of ELISPOT assays in monitoring tolerance and withdrawal of immunosuppressive medications following kidney transplantation.

Abstract: 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.

Abstract: Chronic allograft rejection is in part mediated by host T cells that recognize allogeneic antigens on transplanted tissue. One factor that determines the outcome of a T cell response is clonal size, while another is the effector quality. Studies of alloimmune predictors of transplant graft survival have most commonly focused on only one measure of the alloimmune response. Because differing qualities and frequencies of the allospecific T cell response may provide distinctly different information we analyzed the relationship between frequency of soluble antigen and allo-antigen specific memory IFN-g secreting CD4 and CD8 T cells, their ability to secrete IL-2, and their proliferative capacity, while accounting for cognate and bystander proliferation. The results show proliferative responses primarily reflect on IL-2 production by antigen-specific T cells, and that proliferating cells in such assays entail a considerable fraction of bystander cells. On the other hand, proliferation (and IL-2 production) did not reflect on the frequency of IFN-γ producing memory cells, a finding particularly accentuated in the CD8 T cell compartment. These data provide rationale for considering both frequency and effector function of pre-transplant T cell reactivity when analyzing immune predictors of graft rejection.

Abstract: In order to provide better therapy we strive to increase our knowledge of how the immune system behaves and communicates in common pediatric immunological diseases, such as type 1 diabetes, allergic and celiac diseases. However, when dealing with pediatric diseases, where study subjects are almost exclusively children, blood volumes available for immunological studies are limited and as such must be carefully handled and used to their full extent. Single immune markers can easily be detected by a traditional Enzyme Linked Immunosorbent Assay (ELISA), whereas multiple markers can be detected by a fluorochrome (Luminex) or electrochemiluminescence (MSD) technique. These techniques however are sometimes not sensitive enough to detect low levels of secreted immune markers in limited sample sizes. To detect immune markers at the single-cell level, an Enzyme Linked Immuno-spot (ELISPOT) can be used to pin-point elusive immune markers in common pediatric immunological diseases.

Abstract: The aim of this prospective study was to investigate the diagnostic performance of Borrelia (Bb)-induced interferon (IFN)-γ secretion detected by ELISPOT modified to be feasible for clinical laboratories as a supplementary test to the laboratory diagnosis of Lyme neuroborreliosis (LNB) in an endemic setting. Between 2002 and 2004, patients with symptoms of suspected clinical LNB were included in a study conducted on the Åland islands in the Finnish archipelago, which is a hyper-endemic area for Lyme borreliosis (LB). Fourteen patients with confirmed LNB and 103 patients with non-LNB were included, and the numbers of spontaneous and Bb-induced IFN-γ-secreting cells were assayed by the ELISPOT test. The ELISPOT assay showed a weak diagnostic performance with a sensitivity of 36% and a specificity of 82%. The findings in this study show that this ELISPOT-assay modified to be feasible in clinical routine laboratories is not useful as a supplementary diagnostic tool in the laboratory diagnosis of patients with clinically suspected LNB.

Abstract: T cell monitoring is increasingly performed using cryopreserved PBMC. It has been suggested that resting of PBMC after thawing, that is, culturing them overnight in test medium, produces higher antigen-induced spot counts in ELISPOT assays. To evaluate the importance of overnight resting, we systematically tested cryopreserved PBMC from 25 healthy donors. CEF peptides (comprising CMV, EBV and flu antigens) were used to stimulate CD8 cells and mumps antigen to stimulate CD4 cells. The data show that resting significantly increased antigen-elicited T cell responses only for CEF high responder PBMC. The maximal gain observed was doubling of spot counts. For CEF low responders, and for mumps responders of either low- or high reactivity levels, resting had no statistically significant effect on the observed spot counts. Therefore, resting is not a generally applicable approach to improve ELISPOT assay performance, but can be recommended only for clinical subject cohorts and antigens for which it has a proven benefit. Because resting invariably leads to losing about half of the PBMC available for testing, and because doubling the PBMC numbers plated into the assay reliably doubles the antigen-induced spot counts, we suggest the latter approach as a simple and reliable alternative to resting for enhancing the performance of ELISPOT assays. Our data imply that resting is not required if PBMC were cryopreserved and thawed under conditions that minimize apoptosis of the cells. Therefore, this study should draw attention to the need to optimize freezing and thawing conditions for successful T cell work.

Abstract: Experimental allergic encephalomyelitis (EAE) is mediated by neuroantigen-specific pro-inflammatory T cells of the Th1 and Th17 effector class. Th-17 cells can be clearly defined by expression of IL-17, but not IFN-γ, IL-2 or IL-3. Th1 cells do not express IL-17, but it is unclear presently to what extent they co-express the cytokines canonically assigned to Th1 immunity (i.e., IFN-γ, IL-2 and IL-3) and whether CD4 cells producing these cytokines indeed belong to a single Th1 lineage. It is also unclear to what extent the Th1 response in EAE entails polyfunctional T cells that co-express IFN-γ and IL-2. Therefore, we dissected the Th1 cytokine signature of neuroantigen-specific CD4 cells studying at single cell resolution co-expression of IFN-γ, IL-2 and IL-3 using dual color cytokine ELISPOT analysis. Shortly after immunization, in the draining lymph nodes (dLN), the overall cytokine signature of the neuroantigen-specific CD4 cells was highly type 1-polarized, but IFN-γ, IL-2, and IL-3 were each secreted by different CD4 cells in a mutually exclusive manner. This single cell – single cytokine profile was stable through the course of chronic EAE–polyfunctional CD4 cells co-expressing IL-2 and IFN-γ presented less than 5% of the neuroantigen-specific T cells, even in the inflamed CNS itself. The neuroantigen-specific CD4 cells that expressed IFN-γ, IL-2 and IL-3 in a mutually exclusive manner exhibited similar functional avidities and kinetics of cytokine production, but showed different tissue distributions. These data suggest that Th1 cells do not belong to a single lineage, but different Th1 subpopulations jointly mediate Th1 immunity.