Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis

Immune monitoring of functional responses is a fundamental parameter to establish correlates of protection in clinical trials evaluating vaccines and therapies to boost antigen-specific responses. The IFNg ELISPOT assay is a well-standardized and validated method for the determination of functional IFNg-producing T-cells in peripheral blood mononuclear cells (PBMC); however, its performance greatly depends on the quality and integrity of the cryopreserved PBMC. Here, we investigate the effect of overnight (ON) resting of the PBMC on the detection of CD8-restricted peptide-specific responses by IFNg ELISPOT. The study used PBMC from healthy donors to evaluate the CD8 T-cell response to five pooled or individual HLA-A2 viral peptides. The results were analyzed using a modification of the existing distribution free resampling (DFR) recommended for the analysis of ELISPOT data to ensure the most rigorous possible standard of significance. The results of the study demonstrate that ON resting of PBMC samples prior to IFNg ELISPOT increases both the magnitude and the statistical significance of the responses. In addition, a comparison of the results with a 13-day preculture of PBMC with the peptides before testing demonstrates that ON resting is sufficient for the efficient evaluation of immune functioning

Methodological approach to the ex vivo expansion and detection of T. cruzi-specific T cells from chronic Chagas disease patients

The discovery of T cell epitopes is essential not only for gaining knowledge about host response to infectious disease but also for the development of immune-intervention strategies. In Chagas disease, given the size and complexity of the Trypanosoma cruzi proteome and its interaction with the host’s immune system, the fine specificity of T cells has not been extensively studied yet, and this is particularly true for the CD4+ T cell compartment. The aim of the present work was to optimize a protocol for the generation of parasite-specific memory T cell lines, representative of their in vivo precursor populations and capable of responding to parasite antigens after long-term culture. Accordingly, peripheral blood mononuclear cells (PBMC) from both chronic asymptomatic and cardiac patients, and from non-infected individuals, underwent different in vitro culture and stimulation conditions. Subsequently, cells were tested for their capacity to respond against T. cruzi lysate by measuring [3H]-thymidine incorporation and interferon-γ and GM-CSF secretion. Results allowed us to adjust initial T. cruzi lysate incubation time as well as the number of expansions with phytohemagglutinin (PHA) and irradiated allogeneic PBMC prior to specificity evaluation. Moreover, our data demonstrated that parasite specific T cells displayed a clear and strong activation by using T. cruzi lysate pulsed, Epstein-Barr virus (EBV)-transformed human B lymphocytes (B-LCL), as autologous antigen presenting cells. Under these culture conditions, we generated a clone from an asymptomatic patient’s memory CD4+ T cells which responded against epimastigote and trypomastigote protein lysate. Our results describe a culture method for isolating T. cruzispecific T cell clones from patients with Chagas disease, which enable the acquisition of information on functionality and specificity of individual T cells

GM-CSF Production Allows the Identification of Immunoprevalent Antigens Recognized by Human CD4+ T Cells Following Smallpox Vaccination

The threat of bioterrorism with smallpox and the broad use of vaccinia vectors for other vaccines have led to the resurgence in the study of vaccinia immunological memory. The importance of the role of CD4+ T cells in the control of vaccinia infection is well known. However, more CD8+ than CD4+ T cell epitopes recognized by human subjects immunized with vaccinia virus have been reported. This could be, in part, due to the fact that most of the studies that have identified human CD4+ specific protein-derived fragments or peptides have used IFN-γ production to evaluate vaccinia specific T cell responses. Based on these findings, we reasoned that analyzing a large panel of cytokines would permit us to generate a more complete analysis of the CD4 T cell responses. The results presented provide clear evidence that TNF-α is an excellent readout of vaccinia specificity and that other cytokines such as GM-CSF can be used to evaluate the reactivity of CD4+ T cells in response to vaccinia antigens. Furthermore, using these cytokines as readout of vaccinia specificity, we present the identification of novel peptides from immunoprevalent vaccinia proteins recognized by CD4+ T cells derived from smallpox vaccinated human subjects. In conclusion, we describe a “T cell–driven” methodology that can be implemented to determine the specificity of the T cell response upon vaccination or infection. Together, the single pathogen in vitro stimulation, the selection of CD4+ T cells specific to the pathogen by limiting dilution, the evaluation of pathogen specificity by detecting multiple cytokines, and the screening of the clones with synthetic combinatorial libraries, constitutes a novel and valuable approach for the elucidation of human CD4+ T cell specificity in response to large pathogens

Novel T cell driven approach leads to the identification of immunoprevalent antigens

The prevention and treatment of infectious diseases is highly dependent on the availability of reliable diagnostic tests and protective or therapeutic vaccines. There also exists an urgent need to develop reliable biomarkers to monitor treatment success and to predict disease progression from asymptomatic to symptomatic disease in several infectious diseases. The elucidation of the disease-relevant antigens that elicit the protective immune responses is critical and required for the development of diagnostics and treatments. Here we present a novel T cell driven approach that permits the direct identification of pathogen epitopes and protein antigens capable of triggering specific T cell responses upon immunization in humans. © 2012

Using Fusion of Response Metrics and Monte Carlo Simulation to Determine Immune Response in Cancer Immunotherapy Patients

Determining immune responders in the post-treatment clinical context of cancer immunotherapy, in which patients are treated with one or more antigens for the purpose of eliciting an immune response against the cancer can be challenging. In general, the effectiveness of threshold-based criteria, such as spot count difference from control in ELISpot, can vary widely, depending on patient population latent response and also on experimental choices that increase background variation such as IVS testing. On the other hand, inferential statistical tests such as mDFR or the binomial test can be impacted by varying numbers of samples per patient and also by general differences in patient population distribution of response. Furthermore, measuring differences between pre- and post-treatment response using either a direct statistical test, or a difference of some kind between independently determined pre- and post-treatment response are options when determining immune responders. The end result is that no single approach is applicable in all cases; this, in turn, can lead to the data itself dictating the definition of immune responder, a non-objective process that is difficult to apply broadly. In this presentation, a novel heuristic for determining immune responders using multiple metrics combined in a fusion scoring approach will be shown. Monte Carlo simulation is then used to put these fusion scores into a clear context for the purpose of assigning responder status to individual patient samples, and hence to each patient . A specific implementation of this approach will be shown using data from a recent phase 2 glioblastoma immunotherapy trial (ICT-107) in which HLA-A2 patients were treated with six synthetic peptides. Patient samples were tested for immune response using both ELISpot and Multimer, and it will be shown how the method was used for both types of data. Promising associations between responders designated in this manner and survival endpoints suggest that this method of designating patients as immune responders captures some of the underlying mechanism of action of this treatment

Correction: Methodological approach to the ex vivo expansion and detection of T. cruzi-specific T cells from chronic Chagas disease patients.

[This corrects the article DOI: 10.1371/journal.pone.0178380.]

Detected frequencies of TCR-Vβ families expressed on T cells from lines RM30.II and RM30.II.84.

<p>Detected frequencies of TCR-Vβ families expressed on T cells from lines RM30.II and RM30.II.84.</p

Effect of stimulus used for initial expansion of antigen specific memory CD4⁺ T cells and their <i>in vitro</i> response against <i>T</i>. <i>cruzi</i> antigens.

<p>Sorted memory CD4⁺ T cells from an asymptomatic Chagas patient (RM30) and a non-infected subject (MM) were stimulated with PHA (B, D) or parasite lysate (C, E) as detailed under Materials and Methods. Five thousand cells from each culture well were challenged between days 27–32 (depending on cell growth) with parasite lysate or culture media only and the antigen-specific response was measured as IFN-γ secretion and proliferation. Ten thousand autologous overnight-primed B-LCL per well were used as APC. SI was calculated for each well as the response against <i>T</i>. <i>cruzi</i> lysate divided by the cells baseline response (media only condition). Statistical analysis of the percentage of positive wells is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178380#pone.0178380.s006" target="_blank">S3 Table</a>. <b>A</b>. Timeline representation of stimulation and challenge protocols. Numbers in brackets next to symbols indicate the day (or range of days) since protocol start at which each step was performed. <b>B, C</b>. Pie chart representations of the wells that showed cell growth after initial stimulation, and different degrees of antigen specific response against <i>T</i>. <i>cruzi</i> lysate. <b>D, E</b>. SI values (scatter plots) and percentage of positive wells (bars) for each readout. Cultures considered positive were those with an SI≥2 (dotted line). Positive and total studied wells are indicated in numbers above the bars.</p

Antigen specific response in selected cultures from subject RM30.

<p>Cell lines were established from <i>T</i>. <i>cruzi</i> specific cultures, based on results from experiment depicted on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178380#pone.0178380.g003" target="_blank">Fig 3</a>. Cells were submitted to 2 PHA expansion cycles prior to specificity evaluation. Five thousand cells from each culture well were challenged with <i>T</i>. <i>cruzi</i> lysate, using 10⁴ autologous overnight-primed B-LCL per well as APC. SI was calculated for each well as the response against <i>T</i>. <i>cruzi</i> lysate divided by the cells baseline response (media only condition), cultures considered positive were those with an SI≥2 (dotted line). Bars show the mean values and standard deviation for three replicates of each measure.</p

Antigen specific response in cultures resulting from limiting dilution assay (LDA) of culture RM30.II.

<p><b>A</b>. Twenty-five thousand cells from each culture well were challenged with <i>T</i>. <i>cruzi</i> lysate, using 5×10⁴ autologous overnight-primed B-LCL per well as APC. Specificity against <i>T</i>. <i>cruzi</i> antigens was assessed for several potentially monoclonal lines by IFN-γ and GM-CSF secretion. N/A: Non applicable. <b>B</b>. Specific response was tested using lysates from different stages in the parasite’s life cycle (epimastigote and trypomastigote/amastigote) for T cell lines RM30.II.84 and .85.</p