Recombinant antibody against Trypanosoma cruzi from patients with chronic Chagas heart disease recognizes mammalian nervous system.

Background: To deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients. Methods: Immunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein target_s orthologues, focusing mainly on post-translational modifications. Findings: A2R1 recognizes a parasite protein of ~50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system. Interpretation: Our findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection

In silico and molecular characterization of Q4D1D9_TRYCC, a novel high-molecular weight antigen from Trypanosoma cruzi

Chagas illness, a potentially life-threatening disease, is an infection caused by thehemoflagellate parasite Trypanosoma cruzi. By phage display technology, involving cDNAisolated from B cells of patients with chronic Chagas disease (CCC), we selected arecombinant monoclonal antibody, named scFv 6B6, which recognized a protein of ~300kDa only expressed in T. cruzi. Although preliminary, data showed that only plasma frompatients with CCC but not those from with Leishmaniasis or non-infected subjects reactedagainst 6B6 antigen. In this work, immunoprecipitation coupled to mass spectrometryrevealed that 6B6 antigen was a hypothetical protein of 323 kDa, named Q4D1D9_TRYCC.According to phylogenetic analysis, this protein is highly conserved throughout evolution inall linages of T. cruzi so far identified but lacks orthologues in other kinetoplastid parasites.In parallel, bioinformatic approaches established that this protein has the most commonlypost-translational modifications (acetylation, glycosylation, among others) identified in T.cruzi and the B cell epitopes predictors mapped that almost the entirely protein sequence isimmunogenic. Structural predictions using RaptorX server allowed us to foretell six structuraldomains of Q4D1D9_TRYCC. Interestingly, we found that this protein presentspyrroloquinoline quinone-dependent alcohol dehydrogenase (PQQ) domains, an enzymefunction that has not been yet documented in Trypanosomatids. We hypothesize that theenzymatic activity of Q4D1D9_TRYCC may be involved in some detoxification processexerted by the parasite during the mammalian host invasion. To date, our finding allowed theidentification of a novel T. cruzi protein as a promising diagnostic candidate for Chagasdisease. Moreover, the inhibition of its enzymatic activity could be tested for thedevelopment of new drugs against T. cruzi.Fil: Ossowski, Micaela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gallardo, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Niborski, Leticia Laura. Institute Curie; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Mariño, Karina Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Santos, Javier. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Potenza, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaXXXII Reunión Anual de la Sociedad Argentina de ProtozoologíaCiudad Autónoma de Buenos AiresArgentinaSociedad Argentina de Protozoologi

Evaluation of in-house ELISA using Trypanosoma cruzi lysate and recombinant antigens for diagnosis of chagas disease and discrimination of its clinical forms

The aim of this work was to investigate the potential usefulness of Trypanosoma cruzi lysate, recombinant protein JL7, and peptides P013, R13, JL18, JL19, and P0b as serological markers for human Chagas disease. We analyzed 228 sera from Brazilian Chagas disease patients classified into four clinical groups and 108 from non-chagasic patients. We defined the diagnostic sensitivity, specificity, and Kappa index measured by enzyme-linked immunosorbent assay (ELISA). As previously described, the highest values of diagnostic parameters were achieved for T. cruzi lysate and JL7; peptide P013 showed high specificity but low sensitivity. The other peptides resulted in lower sensitivity and specificity in our ELISA than T. cruzi lysate and JL7 protein. Antibodies against JL7 protein were mainly detected in sera from patients with severe chagasic cardiomyopathy, compared with those from the indeterminate form, whereas peptides failed to discriminate between the clinical forms of the disease.Fil: Longhi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidade Federal de Goiás; BrasilFil: Brandariz, Silvia B.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidade Federal de Goiás; BrasilFil: Lafon, Sonia Olga. Universidade Federal de Goiás; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Niborski, Leticia Laura. Universidade Federal de Goiás; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Luquetti, Alejandro O.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidade Federal de Goiás; BrasilFil: Levin, Mariano Jorge. Universidade Federal de Goiás; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gomez, Karina Andrea. Universidade Federal de Goiás; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Hnf1b haploinsufficiency differentially affects developmental target genes in a new renal cysts and diabetes mouse model

International audienceHeterozygous mutations in HNF1B cause the complex syndrome Renal Cysts and Diabetes (RCAD), characterized by developmental abnormalities of the kidneys, genital tracts and pancreas, and a variety of renal, pancreas and liver dysfunctions. The pathogenesis underlying this syndrome remains unclear as mice with heterozygous null mutations have no phenotype, while constitutive/conditional Hnf1b-ablation leads to more severe phenotypes.We generated a novel mouse model carrying an identified human mutation at the intron-2 splice donor-site. Unlike heterozygous previously characterized, heterozygous for the splicing mutation exhibited decreased HNF1B protein levels and bilateral renal cysts from embryonic stage E15, originated from glomeruli, early proximal tubules (PT) and intermediate nephron segments, concurrently with a delayed PT differentiation, hydronephrosis and rare genital tract anomalies.Consistently, mRNA-sequencing showed that most down-regulated genes in embryonic kidneys were primarily expressed in early PTs and Henle's Loop and involved in ion/drug transport, organic acid and lipid metabolic processes, while the expression of previously identified targets upon Hnf1b-ablation, including cystic disease genes was weakly or not affected. Postnatal analyses revealed renal abnormalities, ranging from glomerular cysts to hydronephrosis and rarely multicystic dysplasia. Urinary proteomics uncovered a particular profile predictive of progressive decline in kidney function and fibrosis, and displayed common features with a recently reported urine proteome in a RCAD pediatric cohort. Altogether our results show that HNF1B reduced levels lead to developmental disease phenotypes associated with the deregulation of a subset of its targets. They further suggest that this model represents a unique clinical/pathological viable model of the RCAD disease

Tumor invasion in draining lymph nodes is associated with Treg accumulation in breast cancer patients

International audienceTumor-draining lymph node (TDLN) invasion by metastatic cells in breast cancer correlates with poor prognosis and is associated with local immunosuppression, which can be partly mediated by regulatory T cells (Tregs). Here, we study Tregs from matched tumor-invaded and non-invaded TDLNs, and breast tumors. We observe that Treg frequencies increase with nodal invasion, and that Tregs express higher levels of co-inhibitory/stimulatory receptors than effector cells. Also, while Tregs show conserved suppressive function in TDLN and tumor, conventional T cells (Tconvs) in TDLNs proliferate and produce Th1-inflammatory cytokines, but are dysfunctional in the tumor. We describe a common transcriptomic signature shared by Tregs from tumors and nodes, including CD80, which is significantly associated with poor patient survival. TCR RNA-sequencing analysis indicates trafficking between TDLNs and tumors and ongoing Tconv/Treg conversion. Overall, TDLN Tregs are functional and express a distinct pattern of druggable co-receptors, highlighting their potential as targets for cancer immunotherapy

Serological based monitoring of a cohort of patients with chronic Chagas disease treated with benznidazole in a highly endemic area of northern Argentina

This study aimed to evaluate well-documented diagnostic antigens, named B13, 1F8 and JL7 recombinant proteins, as potential markers of seroconversion in treated chagasic patients. Prospective study, involving 203 patients treated with benznidazole, was conducted from endemic areas of northern Argentina. Follow-up was possible in 107 out of them and blood samples were taken for serology and PCR assays before and 2, 3, 6, 12, 24 and 36 months after treatment initiation. Reactivity against Trypanosoma cruzi lysate and recombinant antigens was measured by ELISA. The rate of decrease of antibody titers showed nonlinear kinetics with an abrupt drop within the first three months after initiation of treatment for all studied antigens, followed by a plateau displaying a low decay until the end of follow-up. At this point, anti-B13, anti-1F8 and anti-JL7 titers were relatively close to the cut-off line, while anti-T. cruzi antibodies still remained positive. At baseline, 60.8% (45/74) of analysed patients tested positive for parasite DNA by PCR and during the follow-up period in 34 out of 45 positive samples (75.5%) could not be detected T. cruzi DNA. Our results suggest that these antigens might be useful as early markers for monitoring antiparasitic treatment in chronic Chagas disease

CD8+T cell responsiveness to anti-PD-1 is epigenetically regulated by Suv39h1 in melanomas

International audienceAbstract Tumor-infiltrating CD8 + T cells progressively lose functionality and fail to reject tumors. The underlying mechanism and re-programing induced by checkpoint blockers are incompletely understood. We show here that genetic ablation or pharmacological inhibition of histone lysine methyltransferase Suv39h1 delays tumor growth and potentiates tumor rejection by anti-PD-1. In the absence of Suv39h1, anti-PD-1 induces alternative activation pathways allowing survival and differentiation of IFNγ and Granzyme B producing effector cells that express negative checkpoint molecules, but do not reach final exhaustion. Their transcriptional program correlates with that of melanoma patients responding to immune-checkpoint blockade and identifies the emergence of cytolytic-effector tumor-infiltrating lymphocytes as a biomarker of clinical response. Anti-PD-1 favors chromatin opening in loci linked to T-cell activation, memory and pluripotency, but in the absence of Suv39h1, cells acquire accessibility in cytolytic effector loci. Overall, Suv39h1 inhibition enhances anti-tumor immune responses, alone or combined with anti-PD-1, suggesting that Suv39h1 is an “epigenetic checkpoint” for tumor immunity

Clonally Expanded T Cells Reveal Immunogenicity of Rhabdoid Tumors

International audienceRhabdoid tumors (RTs) are genomically simple pediatric cancers driven by the biallelic inactivation of SMARCB1, leading to SWI/SNF chromatin remodeler complex deficiency. Comprehensive evaluation of the immune infiltrates of human and mice RTs, including immunohistochemistry, bulk RNA sequencing and DNA methylation profiling studies showed a high rate of tumors infiltrated by T and myeloid cells. Single-cell RNA (scRNA) and T cell receptor sequencing highlighted the heterogeneity of these cells and revealed therapeutically targetable exhausted effector and clonally expanded tissue resident memory CD8+ T subpopulations, likely representing tumor-specific cells. Checkpoint blockade therapy in an experimental RT model induced the regression of established tumors and durable immune responses. Finally, we show that one mechanism mediating RTs immunogenicity involves SMARCB1-dependent re-expression of endogenous retroviruses and interferon-signaling activation