Expression of Inhibitory Receptors TIGIT, TIM-3, and LAG-3 on CD4+ T Cells from Patients with Different Clinical Forms of Chronic Chagas Disease.

T cells are central to the adaptive immune response against Trypanosoma cruzi infection. In chronic Chagas disease (CCD), circulating parasite-specific memory T cells show reduced functionality and increased expression of inhibitory receptors as a result of persistent antigenic stimulation. This phenotype has been linked to progression of cardiac pathology, whereas the presence of polyfunctional T cells shows association with therapeutic success. In this study, we demonstrate that T. cruzi-specific human CD4+ T cells can be identified by their expression of OX40 and CD25 upon in vitro stimulation. We characterized the expression of the inhibitory receptors T cell immunoreceptor with Ig and ITIM domains (TIGIT), T cell Ig and mucin-domain containing-3 (TIM-3), and lymphocyte activation gene 3 (LAG-3) in CD4+ T cells from CCD patients with and without cardiac alterations. Our results show that, independently of their clinical stage, CCD patients present an increased frequency of CD4+ T cells expressing TIGIT in comparison with non-T. cruzi-infected donors. Exposure to parasite Ags increases the expression of TIM-3 in CD4+ T cells from CCD patients, especially in those with cardiac compromise. Upregulation of LAG-3 was also detected in CCD individuals without cardiac manifestations, predominantly within the subpopulation of cells that did not become activated upon stimulation. Further differences were found between groups in the coexpression of these receptors. Blockade of each individual receptor did not affect activation or the production of IFN-γ and IL-10 by CD4+ T cells in response to parasite Ags. Our results suggest a role for TIGIT, TIM-3, and LAG-3 in the modulation of inflammatory phenomena thought to ultimately lead to tissue damage and cardiac pathology.

Passive and active immunity in infants born to mothers with SARS-CoV-2 infection during pregnancy: prospective cohort study.

OBJECTIVE: To investigate maternal immunoglobulins' (IgM, IgG) response to SARS-CoV-2 infection during pregnancy and IgG transplacental transfer, to characterise neonatal antibody response to SARS-CoV-2 infection, and to longitudinally follow actively and passively acquired antibodies in infants. DESIGN: A prospective observational study. SETTING: Public healthcare system in Santa Clara County (California, USA). PARTICIPANTS: Women with symptomatic or asymptomatic SARS-CoV-2 infection during pregnancy and their infants were enrolled between 15 April 2020 and 31 March 2021. OUTCOMES: SARS-CoV-2 serology analyses in the cord and maternal blood at delivery and longitudinally in infant blood between birth and 28 weeks of life. RESULTS: Of 145 mothers who tested positive for SARS-CoV-2 during pregnancy, 86 had symptomatic infections: 78 with mild-moderate symptoms, and 8 with severe-critical symptoms. The seropositivity rates of the mothers at delivery was 65% (95% CI 0.56% to 0.73%) and the cord blood was 58% (95% CI 0.49% to 0.66%). IgG levels significantly correlated between the maternal and cord blood (Rs=0.93, p<0.0001). IgG transplacental transfer ratio was significantly higher when the first maternal positive PCR was 60-180 days before delivery compared with <60 days (1.2 vs 0.6, p<0.0001). Infant IgG seroreversion rates over follow-up periods of 1-4, 5-12, and 13-28 weeks were 8% (4 of 48), 12% (3 of 25), and 38% (5 of 13), respectively. The IgG seropositivity in the infants was positively related to IgG levels in the cord blood and persisted up to 6 months of age. Two newborns showed seroconversion at 2 weeks of age with high levels of IgM and IgG, including one premature infant with confirmed intrapartum infection. CONCLUSIONS: Maternal SARS-CoV-2 IgG is efficiently transferred across the placenta when infections occur more than 2 months before delivery. Maternally derived passive immunity may persist in infants up to 6 months of life. Neonates are capable of mounting a strong antibody response to perinatal SARS-CoV-2 infection.

T Cell Specificity: A Great Challenge in Chagas Disease.

The CD4+ and CD8+ T cell immune response against T. cruzi, the parasite causing Chagas disease, are relevant for both parasite control and disease pathogenesis. Several studies have been focused on their phenotype and functionally, but only a few have drilled down to identify the parasite proteins that are processed and presented to these cells, especially to CD4+ T lymphocytes. Although approximately 10,000 proteins are encoded per haploid T. cruzi genome, fewer than 200 T cell epitopes from 49 T. cruzi proteins have been identified so far. In this context, a detailed knowledge of the specific targets of T cell memory response emerges as a prime tool for the conceptualization and development of prophylactic or therapeutic vaccines, an approach with great potential to prevent and treat this chronic disease. Here, we review the available information about this topic in a comprehensive manner and discuss the future challenges in the field.

Passive and active immunity in infants born to mothers with SARS-CoV-2 infection during pregnancy: Prospective cohort study.

OBJECTIVE: To investigate maternal immunoglobulins' (IgM, IgG) response to SARS-CoV-2 infection during pregnancy and IgG transplacental transfer, to characterize neonatal antibody response to SARS-CoV-2 infection, and to longitudinally follow actively- and passively-acquired SARS-CoV-2 antibodies in infants. DESIGN: A prospective observational study. SETTING: A public healthcare system in Santa Clara County (CA, USA). PARTICIPANTS: Women with SARS-CoV-2 infection during pregnancy and their infants were enrolled between April 15, 2020 and March 31, 2021. OUTCOMES: SARS-CoV-2 serology analyses in the cord and maternal blood at delivery and longitudinally in infant blood between birth and 28 weeks of life. RESULTS: Of 145 mothers who tested positive for SARS-CoV-2 during pregnancy, 86 had symptomatic infections: 78 with mild-moderate symptoms, and eight with severe-critical symptoms. Of the 147 newborns, two infants showed seroconversion at two weeks of age with high levels of IgM and IgG, including one premature infant with confirmed intrapartum infection. The seropositivity rates of the mothers at delivery was 65% (95% CI 0.56-0.73) and the cord blood was 58% (95% CI 0.49-0.66). IgG levels significantly correlated between the maternal and cord blood (Rs= 0.93, p< 0.0001). IgG transplacental transfer ratio was significantly higher when the first maternal positive PCR was 60-180 days before delivery compared to <60 days (1.2 vs. 0.6, p=<0.0001). Infant IgG negative conversion rate over follow-up periods of 1-4, 5-12, and 13-28 weeks were 8% (4/48), 12% (3/25), and 38% (5/13), respectively. The IgG seropositivity in the infants was positively related to IgG levels in the cord blood and persisted up to six months of age. CONCLUSIONS: Maternal SARS-CoV-2 IgG is efficiently transferred across the placenta when infections occur more than two months before delivery. Maternally-derived passive immunity may protect infants up to six months of life. Neonates mount a strong antibody response to perinatal SARS-CoV-2 infection.

Ex vivo characterization of Breg cells in patients with chronic Chagas disease.

Despite the growing importance of the regulatory function of B cells in many infectious diseases, their immunosuppressive role remains elusive in chronic Chagas disease (CCD). Here, we studied the proportion of different B cell subsets and their capacity to secrete IL-10 ex vivo in peripheral blood from patients with or without CCD cardiomyopathy. First, we immunophenotyped peripheral blood mononuclear cells from patients according to the expression of markers CD19, CD24, CD38 and CD27 and we showed an expansion of total B cell and transitional CD24highCD38high B cell subsets in CCD patients with cardiac involvement compared to non-infected donors. Although no differences were observed in the frequency of total IL-10 producing B cells (B10) among the groups, CCD patients with cardiac involvement showed an increased proportion of naïve B10 cells and a tendency to a higher frequency of transitional B10 cells compared to non-infected donors. Our research demonstrates that transitional B cells are greatly expanded in patients with the cardiac form of CCD and these cells retain the ability to secrete IL-10. These findings provide insight into the phenotypic distribution of regulatory B cells in CCD, an important step towards new strategies to prevent cardiomyopathy associated with T. cruzi infection.

In Silico Guided Discovery of Novel Class I and II Trypanosoma cruzi Epitopes Recognized by T Cells from Chagas' Disease Patients.

T cell-mediated immune response plays a crucial role in controlling Trypanosoma cruzi infection and parasite burden, but it is also involved in the clinical onset and progression of chronic Chagas' disease. Therefore, the study of T cells is central to the understanding of the immune response against the parasite and its implications for the infected organism. The complexity of the parasite-host interactions hampers the identification and characterization of T cell-activating epitopes. We approached this issue by combining in silico and in vitro methods to interrogate patients' T cells specificity. Fifty T. cruzi peptides predicted to bind a broad range of class I and II HLA molecules were selected for in vitro screening against PBMC samples from a cohort of chronic Chagas' disease patients, using IFN-γ secretion as a readout. Seven of these peptides were shown to activate this type of T cell response, and four out of these contain class I and II epitopes that, to our knowledge, are first described in this study. The remaining three contain sequences that had been previously demonstrated to induce CD8+ T cell response in Chagas' disease patients, or bind HLA-A*02:01, but are, in this study, demonstrated to engage CD4+ T cells. We also assessed the degree of differentiation of activated T cells and looked into the HLA variants that might restrict the recognition of these peptides in the context of human T. cruzi infection.

Generation of T. cruzi-Specific Primary CD4+ T Cell Lines from Peripheral Blood Mononuclear Cells Isolated from Chagas Disease Patients.

Human CD8+ and CD4+ T cell lines and clones are valuable tools to explore the role of these cells in the context of diseases, especially in cases in which the main underlying actor is the immune response, like Chagas disease. These cell lines and clones provide a good experimental system to address the phenotypic and functional features of specific T cell subpopulations and furthermore settle the framework necessary for analyzing their antigen/peptide specificity.This chapter details a culture method for the establishment of T. cruzi-specific memory T cell lines from mononuclear cells isolated from Chagas disease patients' peripheral blood. The presented protocol comprises (1) enrichment of memory CD4+ T cells, (2) stimulation with parasite lysate, (3) evaluation of specificity, and (4) expansion and maintenance of specific T cell lines.

The Unsolved Jigsaw Puzzle of the Immune Response in Chagas Disease.

Trypanosoma cruzi interacts with the different arms of the innate and adaptive host's immune response in a very complex and flowery manner. The history of host-parasite co-evolution has provided this protozoan with means of resisting, escaping or subverting the mechanisms of immunity and establishing a chronic infection. Despite many decades of research on the subject, the infection remains incurable, and the factors that steer chronic Chagas disease from an asymptomatic state to clinical onset are still unclear. As the relationship between T. cruzi and the host immune system is intricate, so is the amount and diversity of scientific knowledge on the matter. Many of the mechanisms of immunity are fairly well understood, but unveiling the factors that lead each of these to success or failure, within the coordinated response as a whole, requires further research. The intention behind this Review is to compile the available information on the different aspects of the immune response, with an emphasis on those phenomena that have been studied and confirmed in the human host. For ease of comprehension, it has been subdivided in sections that cover the main humoral and cell-mediated components involved therein. However, we also intend to underline that these elements are not independent, but function intimately and concertedly. Here, we summarize years of investigation carried out to unravel the puzzling interplay between the host and the parasite.

Evaluation of the immune response against Trypanosoma cruzi cytosolic tryparedoxin peroxidase in human natural infection.

Trypanosoma cruzi, the aetiological agent of Chagas disease, has a highly efficient detoxification system to deal with the oxidative burst imposed by its host. One of the antioxidant enzymes involved is the cytosolic tryparedoxin peroxidase (c-TXNPx), which catalyses the reduction to hydrogen peroxide, small-chain organic hydroperoxides and peroxynitrite. This enzyme is present in all parasite stages, and its overexpression renders parasites more resistant to the oxidative defences of macrophages, favouring parasite survival. This work addressed the study of the specific humoral and cellular immune response triggered by c-TXNPx in human natural infection. Thus, sera and peripheral blood mononuclear cells (PBMC) were collected from chronically infected asymptomatic and cardiac patients, and non-infected individuals. Results showed that levels of IgG antibodies against c-TXNPx were low in sera from individuals across all groups. B-cell epitope prediction limited immunogenicity to a few, small regions on the c-TXNPx sequence. At a cellular level, PBMC from asymptomatic and cardiac patients proliferated and secreted interferon-γ after c-TXNPx stimulation, compared with mock control. However, only proliferation was higher in asymptomatic patients compared with cardiac and non-infected individuals. Furthermore, asymptomatic patients showed an enhanced frequency of CD19+ CD69+ cells upon exposure to c-TXNPx. Overall, our results show that c-TXNPx fails to induce a strong immune response in natural infection, being measurable only in those patients without any clinical symptoms. The low impact of c-TXNPx in the human immune response could be strategic for parasite survival, as it keeps this crucial antioxidant enzyme activity safe from the mechanisms of adaptive immune response.

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.

Production of the Main Celiac Disease Autoantigen by Transient Expression in Nicotiana benthamiana.

Celiac Disease (CD) is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen, human tissue transglutaminase (TG2), is a challenge for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein. In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and reactivity of plant-produced TG2 in CD screening test was evaluated. First, a subcellular targeting strategy was tested. Cytosolic, secretory, endoplasmic reticulum (C-terminal SEKDEL fusion) and vacuolar (C-terminal KISIA fusion) TG2 versions were transiently expressed in leaves and recombinant protein yields were measured. ER-TG2 and vac-TG2 levels were 9- to 16-fold higher than their cytosolic and secretory counterparts. As second strategy, TG2 variants were co-expressed with a hydrophobic elastin-like polymer (ELP) construct encoding for 36 repeats of the pentapeptide VPGXG in which the guest residue X were V and F in ratio 8:1. Protein bodies (PB) were induced by the ELP, with a consequent two-fold-increase in accumulation of both ER-TG2 and vac-TG2. Subsequently, ER-TG2 and vac-TG2 were produced and purified using immobilized metal ion affinity chromatography. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bind different epitopes proving that plant-produced antigen has immunochemical characteristics similar to those of human TG2. Lastly, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients while they were not recognized by serum from non-celiac controls. These results confirmed the usefulness of plant-produced TG2 to develop screening assays. In conclusion, the combination of subcellular sorting strategy with co-expression with a PB inducing construct was sufficient to increase TG2 protein yields. This type of approach could be extended to other problematic proteins, highlighting the advantages of plant based production platforms.