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The NIAID DAIDS-sponsored External Quality Assurance Program Oversight Laboratory (EQAPOL) manages an interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) external proficiency program. The ELISpot program evaluates the accuracy and variability of results across laboratories. The variability in the program is quantified via the dispersion, which is the ratio of the variance over the mean of the background-corrected spot-forming cells (SFC) replicates obtained under stimulation with different peptide pools (CMV, CEF). This report includes the longitudinal analysis of the ELISpot program cohort composed of 22 laboratories from 2011 to 2022 to assess whether the within-lab variability has improved over time. Random intercept models of the dispersion over time showed a significant decrease in overall dispersion from an average of approximately 1.8 in 2011 to approximately 1.25 in 2022. Out of the 21 sites, 16 sites (4 being statistically significant) had a negative trend for dispersion over time. Our finding of a reduction of overall within-lab variability demonstrates the need for and benefit of proficiency testing programs. • The ELISpot EQAPOL program assessed performance of site-specific assay protocols. • Examined 22 laboratories, with various levels of participation, from 2011 to 2022. • Participation in the ELISpot program was associated with improved site precision. [ABSTRACT FROM AUTHOR]

Purpose Hypersensitivity pneumonitis (HP) is an immunoallergic disease due to chronic exposure to high quantities of different microorganisms such as Mycobacterium immunogenum (Mi), a mycobacterium, and Lichtheimia corymbifera (Lc), a filamentous fungus. It has recently been demonstrated that the protein DLDH (dihydrolipoyl dehydrogenase), is common to these microorganisms. This study aimed to investigate the immune potential of overlapping peptide pools covering the MiDLDH and LcDLDH. Experimental design A selection of 34 peptides, from the MiDLDH and LcDLDH, able to interact with Major Histocompatibility Complex (MHC) 1 and MHC 2, was obtained using three different epitope prediction websites. By means of ELISPOT assays, we compared the frequency of Interferon gamma (IFNγ) secreting peripheral blood mononuclear cells (PBMC) after stimulation with overlapping peptide pools. Tests were performed using cells from 35 healthy blood donors. Results One peptide pool containing five peptides from MiDLDH and able to interact with MHC 2 induced a marked IFNγ specific immune response (Pool F, p < 0.001, Wilcoxon signed-rank test). Conclusion This study demonstrated that peptides from microorganisms involved in HP were able to induce a high IFNγ specific immune response after stimulation of PBMCs from healthy blood donors which could be useful to develop an effective prevention strategy. [ABSTRACT FROM AUTHOR]

To investigate in detail the effect of infection or vaccination on the human immune system, ELISpot assays are used to simultaneously test the immune response to a large number of peptides of interest. Scientists commonly use “peptide pools”, where, instead of an individual peptide, a test well contains a group of peptides. Since the response from a well may be due to any or many of the peptides in the pool, pooled assays usually need to be followed by confirmatory assays of a number of individual peptides. We present a statistical method that enables estimation of individual peptide responses from pool responses using the Expectation Maximization (EM) algorithm for “incomplete data”. We demonstrate the accuracy and precision of these estimates in simulation studies of ELISpot plates with 90 pools of 6 or 7 peptides arranged in three dimensions and three Mock wells for the estimation of background. In analysis of real pooled data from 6 subjects in a HIV-1 vaccine trial, where 199 peptides were arranged in 80 pools if size 9 or 10, our estimates were in very good agreement with the results from individual-peptide confirmatory assays. Compared to the classical approach, we could identify almost all the same peptides with high or moderate response, with less than half the number of confirmatory tests. Our method facilitates efficient use of the information available in pooled ELISpot data to avoid or reduce the need for confirmatory testing. We provide an easy-to-use free online application for implementing the method, where on uploading two spreadsheets with the pool design and pool responses, the user obtains the estimates of the individual peptide responses. [ABSTRACT FROM AUTHOR]

Abstract: Peptide vaccination constitutes a novel immunotherapeutical approach for the treatment of patients with solid tumors, lymphoma and leukemia. Moreover it might be of use in hematooncological patients for the prevention and therapy of infections like cytomegalovirus (CMV) reactivation due to immunosuppression. To meet good manufacturing practice (GMP) criteria, we introduce here a bio-assay to validate peptide vaccines for peptide content and bio-activity. As a paradigm for peptide vaccine preparation the immunogenic CMV peptide 495–503 NLVPMVATV lyophilisate was resolubilized in dimethyl sulfoxide, phosphate buffered saline and admixed with Montanide™. Addition of different amounts of peptide (10–80 µg) to a mixed lymphocyte peptide culture (MLPC) resulted in the generation of interferon (IFN) gamma and granzyme B releasing CD8+ CMV tetramer+ T cells in a dose dependent manner. The combination of FACS and ELISPOT results allowed the definition of the peptide amount in a vaccine preparation. Storage at ±4 °C over 24 h did not result in a significant change of the immunogenicity of the vaccine. In contrast, cryopreservation of the vaccine at −20 °C resulted in a loss of immunogenicity. Quantitation of tumor/viral antigen peptides admixed with adjuvants, such as incomplete Freund''s adjuvant (IFA), is feasible through bio-assays as the modified ELISPOT/FACS assay described here, meeting GMP criteria for multi-center trials. [Copyright &y& Elsevier]

Abstract: An automated method for counting spot-forming units in the ELISpot assay is described that uses a statistical model fit to training data that is based on counts from one or more experts. The method adapts to variable background intensities and provides considerable flexibility with respect to what image features can be used to model expert counts. Point estimates of spot counts are produced together with intervals that reflect the degree of uncertainty in the count. Finally, the approach is completely transparent and “open source” in contrast to methods embedded in current commercial software. An illustrative application to data from a study of the reactivity of T-cells from healthy human subjects to a pool of immunodominant peptides from CMV, EBV and flu is presented. [Copyright &y& Elsevier]

Abstract: To improve the investigation of the role of human memory B lymphocytes following hepatitis B virus (HBV) infection or vaccination, we developed a method to characterize circulating memory B cells specific to hepatitis B surface antigen (HBsAg). Our approach combined: (1) purification of CD19+ cells, (2) CD40–CD40L polyclonal stimulation, and (3) enumeration of memory B cells differentiated into anti-HBs antibody (Ab)-secreting cells (HBs-SCs) by a HBs-ELISPOT assay. In this way, HBs-SCs were detected in 17 HBsAg-vaccinated and nine HBV-immunized subjects including four individuals with serum anti-HBs Ab levels <10 mIU/ml, but not in six controls. IgG+, IgA+ plus IgM+ HBs-SCs, representing 5–1736 cells/106 circulating B cells and 0.02–0.58% of total immunoglobulin-SCs generated by the B cell polyclonal stimulation, were counted by an Ig two-colour ELISPOT assay. In addition, anti-HBs Abs were found in 8/15 supernatants recovered from B cell cultures which contained HBs-SCs, suggesting that the HBs-ELISPOT assay is more reliable in tracking HBsAg-specific memory B cells than ELISA measurement of anti-HBs Abs secreted in supernatants. This new approach could be useful to explore the presence and the longevity of HBsAg-specific memory B cells in vaccinated and immunized subjects, in chronic HBV infection and after liver transplantation for HBV-related disease. [Copyright &y& Elsevier]

Abstract: In the process of metastasis, malignant cells are released from the primary tumor and migrate to specific organs via the lymphatic and blood circulation systems. These circulating tumor cells have been characterized by immunochemistry, the reverse transcription-polymerase chain reaction, and flow cytometry. Using the MCF-7 breast cancer cell line, we have developed a two-color ELISPOT assay to detect cells secreting cathepsin D protease and MUC1 glycoprotein, markers associated with the risk of metastases in breast cancer. The threshold of detection of this ELISPOT assay was one cathepsin D- or MUC1-secreting MCF7 cell per 5 ml of control blood. In 16 patients with breast carcinoma metastases, 1 to 1940 cathepsin D- or MUC1-secreting cells per 2×107 PBMC were enumerated, whereas none were found in 11 controls. Moreover, in six patients 6–60% of MUC1-secreting cells also expressed the CXCR4 chemokine receptor, which is involved in the homing of metastatic breast cancer cells. The ELISPOT assay described here allowed us to enumerate cathepsin D- and/or MUC1-secreting cells in the MCF-7 cell line and in the peripheral blood of patients with disseminated breast cancer. The combination of the ELISPOT assay and CXCR4-positive cell sorting identified subsets of MUC1-secreting cells in the peripheral blood of these patients. [Copyright &y& Elsevier]

Abstract: Although the number of defined T cell epitopes of clinically relevant antigens is constantly increasing, there is still an enormous need to identify further peptides, processed from new antigens or presented by rare HLA molecules, respectively. Here we introduce a novel two-step approach for the rapid identification of T cell epitopes. It was established in the CMV infection model. From the peripheral blood of healthy donors sharing HLA-A1 according to HLA serotyping we isolated CD8+ T lymphocytes and generated dendritic cells (DCs). DCs were electroporated with CMV pp65 mRNA and tested for recognition by autologous CD8+ T lymphocytes in IFN-γ ELISPOT assays. In all 10 CMV-seropositive donors, CMV pp65-specific CD8+ T cells were readily detectable ex vivo. In 7 of them the response was at least in part restricted by HLA-A1.1 as verified in IFN-γ ELISPOT assays with pp65 mRNA-electroporated K562 cells stably transfected with HLA-A*0101 (K562/A*0101). In a subsequent step various 3′-deleted pp65 RNA fragments were rapidly generated by in vitro transcription of plasmid DNA-templates linearized with restriction enzymes at different sites within the pp65-coding sequence. Polyadenylated mRNA fragments were then electroporated into K562/A*0101 cells and tested for recognition by ex vivo CD8+ T cells in IFN-γ ELISPOT assays. We thereby identified a 76 bp-long sequence as target of the HLA-A*0101-associated pp65-specific T cell response. From this region, 10 peptides predicted by current algorithms were synthesized and tested for recognition. Peptide pp65 364–373 (previously identified by a reverse immunology approach by [Hebart, H., Daginik, S., Stevanovic, S., Grigoleit, U., Dobler, A., Baur, M., Rauser, G., Sinzger, C., Jahn, G., Loeffler, J., Kanz, L., Rammensee, H. G., Einsele, H., 2002. Sensitive detection of human cytomegalovirus peptide-specific cytotoxic T-lymphocyte responses by interferon-gamma-enzyme-linked immunospot assay and flow cytometry in healthy individuals and in patients after allogeneic stem cell transplantation, Blood 99, 3830.]) was positively tested and found to be the dominant target epitope of the HLA-A1-restricted anti pp65 T cell response in all donors. We conclude that (i) the use of HLA-transfected K562 cells allows to dissect antigen-specific T cell responses to partial responses associated with defined HLA class I alleles and (ii) transfection of in vitro transcribed RNA fragments allows to identify immunogenic regions of a given antigen. The latter technique bypasses the need of prior cloning and sequencing of cDNA fragments, reduces the number of synthetic peptides to be tested and thus saves both costs and time. [Copyright &y& Elsevier]

Abstract: The ELISPOT assay is a commonly used technique for quantifying the occurrence of T lymphocyte cells secreting a cytokine after stimulation with an antigen or peptide. The assay endpoint, the number of spot-forming cells (SFC) at a specific concentration of effector cells, is typically estimated using either a simple arithmetic mean or the predicted value from a linear regression model. We compare statistical modeling approaches for summarizing these assays using data from the Pediatric AIDS Clinical Trial Group (PACTG) study 299. A simulation study was conducted to compare methods under controlled conditions. Assuming the optimal effector cell concentration is known, we demonstrate that the simple mean is appropriate if assays are conducted at the same concentration for all samples. Normalizing simple means to a summary concentration using results from a range of concentrations is not valid. A random effects or mixed model is superior to the simple mean when a large within-assay (subject) variance relative to between-subject variance exists. When assays are conducted over a range of effector cell concentrations for each individual, the theoretical linearity assumption of the regression model is often violated and can result in biased estimates. In this case, nonlinear models provide more accurate estimation. Collecting data over a range of concentrations allows reassessment of the optimal cell concentration after the data are generated. [Copyright &y& Elsevier]

Most assay systems for the quantification of antigen-specific T-cell responses in infectious, malignant and autoimmune disease depend on the peptide antigen format and are therefore restricted to known epitopes and their presenting HLA molecules. Here we tested in ELISPOT assays the application of in vitro-transcribed clonal mRNA as an alternative antigen format covering all potential epitopes of a given antigen. As model antigens, we chose pp65 of human cytomegalovirus (HCMV) and human tyrosinase (hTyr). Antigen-presenting cells (APC) were K562 cells stably transfected with single HLA class I alleles and autologous dendritic cells (DC). As effectors, we applied in vitro-generated anti-tyrosinase T-cell populations as well as ex vivo-CD8+ lymphocytes from HCMV-seropositive donors. APC electroporated with clonal mRNA were efficient inducers of spot formation by antigen-experienced CD8+ T cells. They were equivalent to peptide-loaded targets. mRNA electroporation did not induce non-specific spot formation. While the use of autologous mRNA-electroporated DC can uncover the complete individual T-cell response towards an antigen, mRNA-electroporated K562 cells stably transfected with single HLA class I alleles help to detect CD8+ T-cell responses restricted by single HLA class I molecules. [Copyright &y& Elsevier]

Several methods are now available to evaluate the frequencies of virus-specific CD8 T cells but require a systematic comparison to help at choosing the best strategy for evaluation. First, we compared the ELISpot-IFNγ assay, intracellular IFNγ staining and HLA class I tetramer-binding assay to quantify the HIV-specific CD8 T cells. Second, we determined the frequency of recognition of HIV antigens and evaluated whether the mode of antigen presentation might influence the results: We compared HIV antigen presentation in the same ELISpot-IFNγ assays by using recombinant vaccinia viruses (rVVs) encoding for HIV-LAI Gag, Pol, Env, Nef, Tat and Vif proteins, or a panel of 49 synthetic 8–11 amino acid length peptides tested either individually or pooled. Third, we compared the antigens recognized by memory CTL analysis using chromium release assay (CRA) on CTL lines and by effector CD8 cell analysis using ELISpot assay. Our results show that: (1) Flow cytometry and ELISpot assay measuring IFNγ production give the same frequency of HIV-specific CD8 T cells; (2) tetramer-binding assay detects more HIV-specific CD8 T cells than other methods; (3) pools of optimal peptides and sum frequencies of individual optimal peptides give similar results in ELISpot assay; (4) ELISpot assays using peptides are more sensitive than those using rVV; and (5) CRA and ELISpot assay when using rVV provide a comparable profile of HIV antigen recognition by memory CTLs (CRA) and effector CTLs (ELISpot) in two thirds cases. These results have important implications for the choice of immunological methods to evaluate CD8 T cells responses to vaccines. [Copyright &y& Elsevier]

To be able to determine the frequencies of helper-type T lymphocyte (Th) precursors specific for viral or tumor-associated antigens, an ELISPOT assay for IL-5 production was developed. The assay reproducibility was first determined using fresh or cryopreserved peripheral blood mononuclear cells (PBMC) obtained from six normal donors and tested for IL-5 production after 48 h stimulation with phytohemagglutinin (PHA). Both inter-assay (within-subject CV=1.6%) and intra-assay (within-subject CV=0.8%) variabilities were found to be acceptable, and the frequencies of IL-5 secreting cells were comparable in cryopreserved and fresh PBMC of the same donors. The presence and frequency of Th precursor cells to viral capsid L1 protein (viral-like particles, VLP-L1) of human papillomavirus type 16 (HPV-16) in PBMC obtained from seven non-immunized donors and two volunteers immunized with VLP-L1 were then evaluated. Using autologous dendritic cells as antigen-presenting cells (APC) for VLP-L1 in direct 48-h ELISPOT assays, the mean frequency of IL-5 secreting CD4+ T cells was found to be <1/105 (negative) in normal donors but was 1/2, 436 and 1/3678 in the two volunteers immunized with VLP-L1. The assay is applicable to monitoring of the frequency of antigen-specific T cells in the peripheral circulation of individuals immunized with HPV-derived antigens. To test the assay utility in the assessment of Th tumor-reactive lymphocytes, we also used it to determine the frequency of the wild-type sequence (wt) p5322–36 peptide-specific, HLA-DR4-restricted T cells in the bulk CD4+ T cell line. This frequency was 1/33. The ELISPOT assay for IL-5 production can be reliably used to measure Th-type responses in a variety of experimental settings. [Copyright &y& Elsevier]