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.].

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. cruzi specific T cell clones from patients with Chagas disease, which enable the acquisition of information on functionality and specificity of individual T cells.

Peptides from common viral and bacterial pathogens can efficiently activate diabetogenic T-cells.

Cross-reactivity between an autoantigen and unknown microbial epitopes has been proposed as a molecular mechanism involved in the development of insulin-dependent diabetes (type 1 diabetes). Type 1 diabetes is an autoimmune disease that occurs in humans and the nonobese diabetic (NOD) mouse. BDC2.5 is an islet-specific CD4+ T-cell clone derived from the NOD mouse whose natural target antigen is unknown. A biometrical analysis of screening data from BDC2.5 T-cells and a positional scanning synthetic combinatorial library (PS-SCL) was used to analyze and rank all peptides in public viral and bacterial protein databases and identify potential molecular mimic sequences with predicted reactivity. Selected sequences were synthesized and tested for stimulatory activity with BDC2.5 T-cells. Active peptides were identified, and some of them were also able to stimulate spontaneously activated T-cells derived from young, pre-diabetic NOD mice, indicating that the reactivity of the BDC2.5 T-cell is directed at numerous mouse peptides. Our results provide evidence for their possible role as T-cell ligands involved in the activation of diabetogenic T-cells.

Correction: Santos, R.S., et al. Improvement of IFNγ ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis. Cells 2015, 4, 1-18.

The authors wish to make the following corrections to this paper [1]: [...].

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.

Identification of novel human leukocyte antigen-A*0201-restricted, cytotoxic T lymphocyte epitopes on CD133 for cancer stem cell immunotherapy.

Targeting cancer stem cells (CSCs) with immunotherapy may be an effective means to prevent recurrences in glioblastoma multiforme (GBM). It is well established that CD133 is expressed in the population of GBM tumor cells representing CSCs. This raises a possibility that CD133 could serve as a potential target for cytotoxic T cells (CTLs) to target glioblastoma cancer stem cells. Two potential human leukocyte antigen (HLA)-A*0201-restricted CD133 epitopes, ILSAFSVYV (CD133-405) and YLQWIEFSI (CD133-753), showed strong binding to HLA-A*0201 molecules. In vitro immunogenicity studies generated peptide-specific CD8(+) CTLs from normal donors. Autologous monocyte-derived dendritic cells pulsed with the CD133-405 or CD133-753 peptides generated CTLs that efficiently recognized the CD133 epitopes presented in T2 HLA-A*0201 cells and specifically lysed CD133+ HLA-A*0201(+) GBM CSCs. These studies demonstrated natural processing and subsequent presentation of these epitopes in GBM CSCs and the ability of CTLs to kill CSCs bearing the antigen. Immunization studies in mice using the mouse homolog CD133 epitopes demonstrated immunogenicity in the absence of autoimmune damage. The results presented in this study support the use of CD133-specific epitope vaccines to target CSCs in glioblastoma and other cancers.

Cytokine production but lack of proliferation in peripheral blood mononuclear cells from chronic Chagas' disease cardiomyopathy patients in response to T. cruzi ribosomal P proteins.

BACKGROUND: Trypanosoma cruzi ribosomal P proteins, P2ß and P0, induce high levels of antibodies in patients with chronic Chagas' disease Cardiomyopathy (CCC). It is well known that these antibodies alter the beating rate of cardiomyocytes and provoke apoptosis by their interaction with ß1-adrenergic and M2-muscarinic cardiac receptors. Based on these findings, we decided to study the cellular immune response to these proteins in CCC patients compared to non-infected individuals. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated proliferation, presence of surface activation markers and cytokine production in peripheral blood mononuclear cells (PBMC) stimulated with P2ß, the C-terminal portion of P0 (CP0) proteins and T. cruzi lysate from CCC patients predominantly infected with TcVI lineage. PBMC from CCC patients cultured with P2ß or CP0 proteins, failed to proliferate and express CD25 and HLA-DR on T cell populations. However, multiplex cytokine assays showed that these antigens triggered higher secretion of IL-10, TNF-α and GM-CSF by PBMC as well as both CD4+ and CD8+ T cells subsets of CCC subjects. Upon T. cruzi lysate stimulation, PBMC from CCC patients not only proliferated but also became activated within the context of Th1 response. Interestingly, T. cruzi lysate was also able to induce the secretion of GM-CSF by CD4+ or CD8+ T cells. CONCLUSIONS/SIGNIFICANCE: Our results showed that although the lack of PBMC proliferation in CCC patients in response to ribosomal P proteins, the detection of IL-10, TNF-α and GM-CSF suggests that specific T cells could have both immunoregulatory and pro-inflammatory potential, which might modulate the immune response in Chagas' disease. Furthermore, it was possible to demonstrate for the first time that GM-CSF was produced by PBMC of CCC patients in response not only to recombinant ribosomal P proteins but also to parasite lysate, suggesting the value of this cytokine to evaluate T cells responses in T. cruzi infection.