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The complexity of host-pathogen interactions often leads to distinct clinical outcomes upon infection with different pathogen strains. In this Review, we explore the interactions between the highly diverse Trypanosoma cruzi population and the human host. At least 30% of the 7 million individuals with Chagas disease will develop a severe cardiopathy that is among the deadliest heart diseases known. The diversity of the T cruzi population also creates major hurdles for therapy and vaccine development. We also discuss the ecoepidemiological and geographical distribution of T cruzi strains, their susceptibility to treatment, their antigenic diversity, and their effect on the immune response and on disease outcome. Furthermore, we highlight the importance of understanding the T cruzi host-pathogen relationship for guiding new approaches towards development of therapies and vaccines for Chagas disease, and how the information gained by studying this relationship can inspire solutions for other host-pathogen interactions.

Double-negative (DN) T cells are present at relatively low frequencies in human peripheral blood, and are characterized as expressing the alpha-beta or gamma-delta T-cell receptor (TCR), but not the CD4 nor the CD8 co-receptors. Despite their low frequencies, these cells are potent producers of cytokines and, thus, are key orchestrators of immune responses. DN T cells were initially associated with induction of peripheral immunological tolerance and immunomodulatory activities related to disease prevention. However, other studies demonstrated that these cells can also display effector functions associated with pathology development. This apparent contradiction highlighted the heterogeneity of the DN T-cell population. Here, we review phenotypic and functional characteristics of DN T cells, emphasizing their role in human diseases. The need for developing biomarkers to facilitate the translation of studies from animal models to humans will also be discussed. Finally, we will examine DN T cells as promising therapeutic targets to prevent or inhibit human disease development.

Background Our goal was to identify genetic risk factors for cutaneous leishmaniasis (CL) caused by Leishmania braziliensis. Methods Genotyping 2066 CL cases and 2046 controls using Illumina HumanCoreExomeBeadChips provided data for 4 498 586 imputed single-nucleotide variants (SNVs). A genome-wide association study (GWAS) using linear mixed models took account of genetic diversity/ethnicity/admixture. Post-GWAS positional, expression quantitative trait locus (eQTL) and chromatin interaction mapping was performed in Functional Mapping and Annotation (FUMA). Transcriptional data were compared between lesions and normal skin, and cytokines measured using flow cytometry and Bioplex assay. Results Positional mapping identified 32 genomic loci associated with CL, none achieving genome-wide significance (P < 5 × 10−8). Lead SNVs at 23 loci occurred at protein coding or noncoding RNA genes, 15 with eQTLs for functionally relevant cells/tissues and/or showing differential expression in lesions. Of these, the 6 most plausible genetic risk loci were SERPINB10 (Pimputed_1000G = 2.67 × 10−6), CRLF3 (Pimputed_1000G = 5.12 × 10−6), STX7 (Pimputed_1000G = 6.06 × 10−6), KRT80 (Pimputed_1000G = 6.58 × 10−6), LAMP3 (Pimputed_1000G = 6.54 × 10−6), and IFNG-AS1 (Pimputed_1000G = 1.32 × 10−5). LAMP3 (Padjusted = 9.25 × 10−12; +6-fold), STX7 (Padjusted = 7.62 × 10−3; +1.3-fold), and CRLF3 (Padjusted = 9.19 × 10−9; +1.97-fold) were expressed more highly in CL biopsies compared to normal skin; KRT80 (Padjusted = 3.07 × 10−8; −3-fold) was lower. Multiple cis-eQTLs across SERPINB10 mapped to chromatin interaction regions of transcriptional/enhancer activity in neutrophils, monocytes, B cells, and hematopoietic stem cells. Those at IFNG-AS1 mapped to transcriptional/enhancer regions in T, natural killer, and B cells. The percentage of peripheral blood CD3+ T cells making antigen-specific interferon-γ differed significantly by IFNG-AS1 genotype. Conclusions This first GWAS for CL identified multiple genetic risk loci including a novel lead to understanding CL pathogenesis through regulation of interferon-γ by IFNG antisense RNA 1., Genome-wide analysis of 2066 cases and 2046 controls, together with genotypic differences in antigen-specific interferon-γ made by CD3+ T cells, identifies IFNG-AS1 as a genetic risk factor for cutaneous leishmaniasis caused by Leishmania braziliensis.

Multifunctional interleukin 10 (IL10)(+)Th1 cells have been implicated in favorable evolution of many infectious diseases, promoting an efficacious immune response while limiting immunopathology. Here, we investigated the presence of multifunctional CD4(+) and CD8(+) T‐cells that expressed interferon gamma (IFNγ), IL10 and tumor necrosis factor (TNF), or its combinations during dengue infection. Peripheral blood mononuclear cells (PBMCs) from outpatients with dengue (mild dengue forms) and hospitalized patients (or patients with dengue with warning signs and severe dengue) were cultured in the presence of envelope (ENV) or NS3 peptide libraries of DENV during critical (hospitalization period) and convalescence phases. The production of IFNγ, IL10 and TNF by CD4(+) and CD8(+) T‐cells was assessed by flow cytometry. Our data show that patients with mild dengue, when compared with patients with dengue with warning signs and severe dengue, presented higher frequencies of multifunctional T‐cells like NS3‐specific IFNγ/IL10‐producing CD4(+) T‐cells in critical phase and NS3‐ and ENV‐specific CD8(+) T‐cells producing IFNγ/IL10. In addition, NS3‐specific CD8(+) T‐cells producing high levels of IFNγ/TNF and IFNγ/TNF/IL10 were also observed in the mild dengue group. We observed that multifunctional T‐cells produced higher levels of cytokines as measured by intracellular content when compared with single producer T‐cells. Importantly, multifunctional CD4(+) and CD8(+) T‐cells producing IFNγ, TNF and IL10 simultaneously displayed positive correlation with platelet levels, suggesting a protective role of this population. The presence of IL10(+)Th1 and IL10(+)Tc1 multifunctional cells was associated with mild dengue presentation, suggesting that these cells play a role in clinical evolution of dengue infection.

Immune checkpoint inhibitors (ICI) have provided new hope for cancer patients, and in particular for patients with tumors that are immunologically active and classified as hot tumors. These tumors express antigenic and tumor microenvironment (TME) characteristics that make them potential candidates for therapy with checkpoint inhibitors that aim to reactivate the immune response such as anti-PD-1 and anti-CTLA-4. Examples of potentially responsive cancers are, melanoma, non-small cell lung cancer and several other metastatic or unresectable tumors with genetic instability: DNA mismatch repair deficiency (dMMR), microsatellite instability-high (MSI-H), or with a high tumor mutational burden (TMB). Immunotherapy using checkpoint inhibitors is typically associated with adverse events (AEs) that are milder than those with chemotherapy. However, a significant percentage of patients develop short-term immune-related AEs (irAEs) which range from mild (~70%) to severe cases (~13%) that can lead to modifications of the checkpoint inhibitor therapy and in some cases, death. While some studies have investigated immune mechanisms behind the development of irAEs, much more research is needed to understand the mechanisms and to develop interventions that could attenuate severe irAEs, while maintaining the anti-tumor response intact. Moreover, studies to identify biomarkers that can predict the likelihood of a patient developing severe irAEs would be of great clinical importance. Here we discuss some of the clinical ramifications of irAEs, potential immune mechanisms behind their development and studies that have investigated potentially useful biomarkers of irAEs development.

The development of immune checkpoint inhibitors initiated a new era of anti-cancer therapies with very promising results. Although effective for many patients, others are primarily refractory to this treatment. Identifying systemic immune cell subpopulations associated with treatment success or failure can reveal mechanisms behind therapeutic failure and offer potential predictive markers of clinical use. Here, for the first time, we performed a prospective non-interventional study and evaluated the pretreatment systemic immunological status of 33 patients with stage IV non-small cell lung cancer treated with anti-PD-1 combined with chemotherapy by flow cytometry. Treatment outcome was evaluated 9 weeks after treatment to define patients as Responders and Non-responders. Patients were reevaluated 6 months after therapy. Responders presented a higher frequency of activated CD4+ CD69+ T lymphocytes and non-classical monocytes before treatment began, which acted as predictive biomarkers for response measured at nine weeks. Conversely, CTLA-4+CD4+ T lymphocytes were more frequent in Non-responders and may contribute to an immunosuppressive milieu impairing the control of the tumor. Patients with durable responses (³ 6 months) also had higher frequency of activated CD4+ T lymphocytes before treatment started, compared with non-responders. In contrast, non-classical monocytes did not predict a durable response. However, patients with a higher frequency of both immune populations had longer median progression free survival. Our results suggest that higher frequency of peripheral activated CD4+CD69+, CD4+CTLA-4+ T cells may be useful biomarkers of therapeutic success or failure in NSCLC and point to mechanisms underlying therapeutic outcome. Studies are underway to further characterize the functional potential and activation status these systemic immune subpopulations. Citation Format: Amanda B Figueiredo, Stephanie M Iasbeke Ferreira, Guilherme FB Evangelista, Iasmim P Santos, Larissa M Kuil, Clara M Cavalcanti, Kátia LP Morais, Nayane AL Galdino, Ananda D Lopes, Helano C Freitas, Jefferson L Gross, Clóvis A Lopes Pinto, Rubens Chojniak, Vladmir C Cordeiro de Lima, Kenneth J Gollob. Increased systemic activated CD4+ T cells and non-classical monocytes are associated with improved immunotherapy response and progression-free survival in non-small cell lung cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A58.

SUMMARYThe poor prognosis of head and neck cancer (HNC) is associated with the presence of metastasis within the lymph nodes (LNs). Herein, the proteome of 140 multisite samples from a 59-HNC patient cohort, including primary and matched LN-negative or -positive tissues, saliva, and blood cells, reveals insights into the biology and potential metastasis biomarkers that may assist in clinical decision making. Protein profiles are strictly associated with immune modulation across datasets, and this provides the basis for investigating immune markers associated with metastasis. The proteome of LN metastatic cells recapitulates the proteome of the primary tumor sites. Conversely, the LN microenvironment proteome highlights the candidate prognostic markers. By integrating prioritized peptide, protein, and transcript levels with machine learning models, we identified a nodal metastasis signature in the blood and saliva. In summary, we present the deepest proteome characterization wiring multiple sampling sites in HNC, thus providing a promising basis for understanding tumoral biology and identifying metastasis-associated signatures.

Chronic Chagas cardiomyopathy (CCC) is one of the deadliest cardiomyopathies known and the most severe manifestation of Chagas disease, which is caused by infection with the parasite Trypanosoma cruzi. Idiopathic dilated cardiomyopathies (IDC) are a diverse group of inflammatory heart diseases that affect the myocardium and are clinically similar to CCC, often causing heart failure and death. While T-cells are critical for mediating cardiac pathology in CCC and IDC, the mechanisms underlying T-cell function in these cardiomyopathies are not well-defined. In this study, we sought to investigate the phenotypic and functional characteristics of T-cell subpopulations in CCC and IDC, aiming to clarify whether the inflammatory response is similar or distinct in these cardiomyopathies. We evaluated the expression of systemic cytokines, determined the sources of the different cytokines, the expression of their receptors, of cytotoxic molecules, and of molecules associated with recruitment to the heart by circulating CD4+, CD8+, and CD4-CD8- T-cells from CCC and IDC patients, using multiparameter flow cytometry combined with conventional and unsupervised machine-learning strategies. We also used an in silico approach to identify the expression of genes that code for key molecules related to T-cell function in hearts of patient with CCC and IDC. Our data demonstrated that CCC patients displayed a more robust systemic inflammatory cytokine production as compared to IDC. While CD8+ T-cells were highly activated in CCC as compared to IDC, CD4+ T-cells were more activated in IDC. In addition to differential expression of functional molecules, these cells also displayed distinct expression of molecules associated with recruitment to the heart. In silico analysis of gene transcripts in the cardiac tissue demonstrated a significant correlation between CD8 and inflammatory, cytotoxic and cardiotropic molecules in CCC transcripts, while no correlation with CD4 was observed. A positive correlation was observed between CD4 and perforin transcripts in hearts from IDC but not CCC, as compared to normal tissue. These data show a clearly distinct systemic and local cellular response in CCC and IDC, despite their similar cardiac impairment, which may contribute to identifying specific immunotherapeutic targets in these diseases.

BackgroundThe ubiquitin-proteasome system (UPS) is the endogenous intracellular mechanism for maintaining protein homeostasis through protein degradation and turnover. Heterobifunctional small molecules are a new class of compounds that form a ternary complex with an E3 ligase and protein of interest leading to ubiquitination and subsequent degradation of the protein of interest in a process known as Targeted Protein Degradation. This new therapeutic modality enables targeting of “undruggable” proteins such as STAT3, a transcription factor activated in immunofibrotic diseases.ObjectivesKymera has developed heterobifunctional molecules that potently and selectively target STAT3 for degradation and elimination by the ubiquitin-proteasome pathway. The aim of these studies was to evaluate the therapeutic potential of pharmacologically removing STAT3 by targeted protein degradation in various human cell types in vitro, and to prevent the development of skin and lung fibrosis in vivo.MethodsDermal fibroblasts obtained from healthy and systemic sclerosis patients activated with TGF-β were analyzed for development of α-smooth muscle actin (α-SMA)-positive stress fibers and for contractility using collagen gel contraction assay. Contraction assay was also performed using human dermal smooth muscle cells. Human aortic endothelial cells (HAECs) were activated with LPS, and their adhesive properties were assessed in the microcapillary system by the ability to bind peripheral blood mononuclear cells (PBMCs) under shear stress. HAECs proliferation was induced with VEGF. THP-1 cells and CD14+ monocytes were activated with IL-6 or LPS, and secreted cytokines were assessed by CBA. PBMCs activated with LPS, IL-6, IL-21, or IL-23 alone (pSTAT3 induction), or with a combination of anti-CD3/CD38 beads and a pro-Th17 cocktail comprised of cytokines and antibodies to evaluate the development of a Th17 and Treg phenotype by flow cytometry. Cytokines were analyzed by ELISA. All cell types were pre-treated with STAT3 degraders 20h prior to experiment start. Intratracheal instillation of bleomycin was used to induce pulmonary fibrosis. Transgenic Tsk-1 mice were used as a model of spontaneous skin fibrosis.ResultsSTAT3 degraders completely ablated STAT3 in all analyzed cell types with DC50 ranging from 0.25-0.8 nM. STAT3 degradation prevented TGF-β-induced formation of α-SMA-positive stress fibers in dermal fibroblasts (IC50 = 0.35nM) and 2 and 10nM degrader completely abrogated their contractility. Similarly, STAT3 degradation reduced the constitutive contractility of dermal smooth muscle cells of 13% (pConclusionSTAT3 degraders that selectively and potently eliminate STAT3 show robust anti-inflammatory and anti-fibrotic potential in vitro and in vivo. Our results suggest that targeted protein degradation is a promising approach to modulate the STAT3 pathway, making it a novel therapeutic opportunity to treating multiple immunofibrotic diseases.Disclosure of InterestsPrzemyslaw Blyszczuk Grant/research support from: Kymera, Gabriela Kania: None declared, Elena Pachera: None declared, Filip Rolski: None declared, Amela Hukara: None declared, Vanessa Tela: None declared, Michele Mayo Employee of: Kymera, Vaishali Dixit Employee of: Kymera, Bin Yang Employee of: Kymera, Jared Gollob Employee of: Kymera, Nello Mainolfi Employee of: Kymera, Anthony Slavin Employee of: Kymera, Cedric Hubeau Employee of: Kymera, Oliver Distler Speakers bureau: Bayer, Boehringer Ingelheim, Janssen, Medscape, Consultant of: Abbvie, Acceleron, Alcimed, Amgen, AnaMar, Arxx, AstraZeneca, Baecon, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, 4P Science, Galapagos, Glenmark, Horizon, Inventiva, Kymera, Lupin, Miltenyi Biotec, Mitsubishi Tanabe, MSD, Novartis, Prometheus, Roivant, Sanofi and Topadur, Grant/research support from: Kymera, Mitsubishi Tanabe, Boehringer Ingelheim.

Cervical cancer (CC) represents a major global health issue, particularly impacting women from resource constrained regions worldwide. Treatment refractoriness to standard chemoradiotheraphy has identified cancer stem cells as critical coordinators behind the biological mechanisms of resistance, contributing to CC recurrence. In this work, we evaluated differential gene expression in cervical cancer stem-like cells (CCSC) as biomarkers related to intrinsic chemoradioresistance in CC. A total of 31 patients with locally advanced CC and referred to Mário Penna Institute (Belo Horizonte, Brazil) from August 2017 to May 2018 were recruited for the study. Fluorescence-activated cell sorting was used to enrich CD34+/CD45- CCSC from tumor biopsies. Transcriptome was performed using ultra-low input RNA sequencing and differentially expressed genes (DEGs) using Log2 fold differences and adjusted p-value < 0.05 were determined. The analysis returned 1050 DEGs when comparing the Non-Responder (NR) (n=10) and Responder (R) (n=21) groups to chemoradiotherapy. These included a wide-ranging pattern of underexpressed coding genes in the NR vs. R patients and a panel of lncRNAs and miRNAs with implications for CC tumorigenesis. A panel of biomarkers was selected using the rank-based AUC (Area Under the ROC Curve) and pAUC (partial AUC) measurements for diagnostic sensitivity and specificity. Genes overlapping between the 21 highest AUC and pAUC loci revealed seven genes with a strong capacity for identifying NR vs. R patients (ILF2, RBM22P2, ACO16722.1, AL360175.1 and AC092354.1), of which four also returned significant survival Hazard Ratios. This study identifies DEG signatures that provide potential biomarkers in CC prognosis and treatment outcome, as well as identifies potential alternative targets for cancer therapy.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer defined by the lack of expression of estrogen receptor, progesterone receptors, and of the human epithelial growth factor receptor 2. Neoadjuvant chemotherapy has proven efficacy in the treatment of TNBC, and a pathological complete response (pCR) is predictive of improved long-term survival. The immune response exerts a vital role in response to neoadjuvant chemotherapy, as indicated by the relation between the percentage of stromal tumor-infiltrating lymphocytes (TILs) in pre-treated tumor tissue samples and the likelihood of achieving pCR. Despite this, the relationship between the systemic immune response and the tumor microenvironment is unclear. In this prospective study, we determined the systemic plasma immune profile of TNBC patients before treatment using a panel of 27 immune mediators and measured the percentage of TILs from the same patients. Patients who demonstrated pCR had significantly higher systemic immune mediators; GM-CSF, FGF-basic, VEGF, IL-2, and IL-5, than the non-responders. Moreover, responders displayed a strong positive correlation between the cytokines IFN-γ and IL-7 with the percentage of TILs, while non-responders had a negative or no correlation. Finally, systemic immune mediator levels before treatment predict pCR (AUC range 0.64 - 0.71), and the combination of immune mediators and TILs improved pCR prediction (AUC 0.71 - 0.82). In conclusion, increased systemic immune mediators reflect increased TILs percentage and act as potential predictive biomarkers of pCR for TNBC patients submitted to neoadjuvant chemotherapy.

DNA mismatch repair deficiency (dMMR) leads to increased mutation load, which in turn may impact anti-tumor immune responses and treatment effectiveness. Currently, there are different mutational signatures described in primary cancers that are associated with dMMR. Whether the somatic and epigenetic changes in MMR genes precede one or more dMMR signatures, and if so by which mechanism remains unknown. To investigate the relationship between these changes and dMMR signatures, we performed a de novo extraction of mutational signatures in a large cohort of 787 gastric cancer patients. We detected three dMMR-related signatures, one of which clearly discriminates tumors with MLH1 gene silencing caused by hyper-methylation within its promoter (AUC = 98%). We then demonstrate that samples with the highest exposures to signature share features related to better prognosis, encompassing clinical and molecular aspects, as well as altered immune infiltrate composition, predictive of a better response to immune checkpoint inhibitors. Overall, our analysis explored the impact of modifications in MMR-related genes on shaping specific mutational signatures and we provide evidence that patient classification based on mutational signature exposure can identify a group of patients with a good prognosis and who are potentially good candidates for immunotherapy.

BackgroundSignal transducer and activator of transcription 3 (STAT3) is a transcription factor that belongs to a class of targets devoid of catalytic function, thus deemed “undruggable” by standard modalities such as small molecule inhibitors or biologics. STAT3 can be activated by various receptor- and non-receptor tyrosine kinases, playing a critical role in activation pathways triggered by cytokines, hormones, and growth factors, making it an attractive target for the treatment of inflammatory diseases.ObjectivesKymera has developed heterobifunctional molecules that selectively target STAT3 for degradation and elimination by the ubiquitin-proteasome pathway. We sought to evaluate the pharmacologic potential of these STAT3 degraders through in vitro and in vivo studies relevant to human autoimmune disease, including murine collagen-induced arthritis.MethodsWe evaluated the impact of STAT3 degraders on the activation of human monocytes, dermal fibroblasts, CD4+ T cells, and PBMC by LPS, IL-6/IL-6R, IL-21, IL-23, as well as anti-CD3/CD28 plus a cocktail of cytokines and antibodies. STAT3 degradation and pSTAT3 inhibition were determined in comparison to a JAK1/2 small molecule inhibitor. Inhibition of cytokines, chemokines, and collagen release, as well as Th17 (CD4+CD25-RORγt+CXCR6+) and Treg (CD4+CD25+CD127lowFOXP3+) expansion were used as in vitro efficacy assays. Finally, STAT3 degraders were tested in vivo, in a mechanistic (IL-6 challenge) as well as a disease model (murine CIA) relevant to rheumatology indications.ResultsSTAT3 degraders showed broad and potent activity in-vitro against TLR receptor and cytokine-induced activation of immune and stromal cells, including soluble mediator release such as MCP-1/CCL2 and Collagen1a1. STAT3 degradation in CD4+ T cells robustly inhibited the development of Th17 cells, abrogating IL-17, IL-22, IL-8/CXCL8, and TNFα production, and increased Treg numbers in a manner superior to JAK1/2 inhibition. In mice injected with IL-6, plasma levels of serum amyloid A were dose-dependently suppressed by STAT3 degradation. In the murine collagen-induced arthritis model, STAT3 degradation resulted in robust, dose-dependent delay of disease onset and decreased disease incidence, clinical scores, local cytokine expression (paws) and histopathological scores, including the complete alleviation of periosteal bone growth.ConclusionThese data demonstrate the broad activity of STAT3 degradation in alleviating autoimmune inflammation in models relevant to human disease. Targeted protein degradation of STAT3 thus represents a novel therapeutic approach to treating autoimmune/autoinflammatory diseases such as rheumatoid arthritis.Disclosure of InterestsCedric Hubeau Shareholder of: Kymera Therapeutics, Employee of: Kymera Therapeutics, Jeffrey Sullivan Shareholder of: Kymera Therapeutics, Employee of: Kymera Therapeutics, Crystal Brown Shareholder of: Kymera Therapeutics, Employee of: Kymera Therapeutics, Michele Mayo Shareholder of: Kymera Therapeutics, Employee of: Kymera Therapeutics, Vaishali Dixit Shareholder of: Kymera Therapeutics, Employee of: Kymera Therapeutics, Bradley Enerson Shareholder of: Kymera Therapeutics, Employee of: Kymera Therapeutics, Haojing Rong Shareholder of: Kymera Therapeutics, Employee of: Kymera Therapeutics, Bin Yang Shareholder of: Kymera Therapeutics, Employee of: Kymera Therapeutics, Chris De Savi Shareholder of: Kymera Therapeutics, Employee of: Kymera Therapeutics, Jared Gollob Shareholder of: Kymera Therapeutics, Employee of: Kymera Therapeutics, Nello Mainolfi Shareholder of: Kymera Therapeutics, Employee of: Kymera Therapeutics, Anthony Slavin Shareholder of: Kymera Therapeutics, Employee of: Kymera Therapeutics.

Cellular prion protein (PrPC ) is widely expressed and displays a variety of well-described functions in the central nervous system (CNS). Mutations of the PRNP gene are known to promote genetic human spongiform encephalopathies, but the components of gain- or loss-of-function mutations to PrPC remain a matter for debate. Among the proteins described to interact with PrPC is Stress-inducible protein 1 (STI1), a co-chaperonin that is secreted from astrocytes and triggers neuroprotection and neuritogenesis through its interaction with PrPC . In this work, we evaluated the impact of different PrPC pathogenic point mutations on signaling pathways induced by the STI1-PrPC interaction. We found that some of the pathogenic mutations evaluated herein induce partial or total disruption of neuritogenesis and neuroprotection mediated by mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase A (PKA) signaling triggered by STI1-PrPC engagement. A pathogenic mutant PrPC that lacked both neuroprotection and neuritogenesis activities fail to promote negative dominance upon wild-type PrPC . Also, a STI1-α7-nicotinic acetylcholine receptor-dependent cellular signaling was present in a PrPC mutant that maintained both neuroprotection and neuritogenesis activities similar to what has been previously observed by wild-type PrPC . These results point to a loss-of-function mechanism underlying the pathogenicity of PrPC mutations.

Background Rheumatic heart disease (RHD) is associated with inflammation that damages cardiac valves, often requiring surgical interventions. The underlying mechanisms involved in the disease progression are not completely understood. This study aimed to evaluate cytokine plasma levels in patients with RHD as possible markers of disease severity. Methods and results Eighty-nine patients with RHD, age of 41 years ±11.5 years, were prospectively enrolled. RHD severity was defined as valve dysfunction that required invasive intervention, either valve repair or replacement. Peripheral blood samples were collected from all patients for cytokine measurements. The patients were followed up to look at adverse clinical events defined as either the need for valve intervention or death. At baseline, 64 (71.9%) patients had previously undergone valve intervention, whereas 25 patients had stable clinical presentation. Patients with severe RHD displayed higher levels of inflammatory cytokines than patients with stable disease. Cluster analysis showed segregation of severe and stable RHD based on IL-6/TNF-α and IL-6/IL-17A, respectively. IL-6 and TNF-α expression were positively correlated in severe but not in stable RHD patients. During a median follow-up of 23 months, 16 patients (18%) had an adverse outcome. IL-10 at baseline (HR 1.24, 95% CI 1.08–1.43, p = 0.003), and IL-4 (HR 1.12, 95% CI 1.01–1.24, p = 0.041) were predictors of events during the follow-up. Conclusions High levels of cytokines are associated with severity of RHD. The co-regulated expression of IL-6 and TNF-α is associated with severe valve dysfunction, whereas high IL-10 and IL-4 levels predicted subsequently adverse outcome.

Summary Cardiomyopathy is the most severe outcome of Chagas disease, causing more than 12 000 deaths/year. Immune cells participate in cardiomyopathy development either by direct tissue destruction, or by driving inflammation. We have shown that CD4–CD8– [double-negative (DN)] T cells are major sources of inflammatory and anti-inflammatory cytokines, associated with the cardiac (CARD) and indeterminate (IND) forms of Chagas disease, respectively. Here, we sought to identify Trypanosoma cruzi-derived components that lead to activation of DN T cells in Chagas patients. Glycolipid (GCL), lipid (LIP) and protein-enriched (PRO) fractions derived from trypomastigote forms of T. cruzi were utilized to stimulate cells from IND and CARD patients to determine DN T cell activation by evaluating CD69 and cytokine expression. We observed that GCL, but not LIP or PRO fractions, induced higher activation of DN T cells, especially T cell receptor (TCR)-γδ DN T, from IND and CARD. GCL led to an increase in tumour necrosis factor (TNF) and interleukin (IL)-10 expression by TCR-γδ DN T cells from IND, while inducing IFN-γ expression by TCR-γδ DN T cells from CARD. This led to an increase in the ratio IFN-γ/IL-10 in TCR-γδ DN T cells from CARD, favouring an inflammatory profile. These results identify GCL as the major T. cruzi component responsible for activation of DN T cells in chronic Chagas disease, associated predominantly with an inflammatory profile in CARD, but not IND. These findings may have implications for designing new strategies of control or prevention of Chagas disease cardiomyopathy by modulating the response to GCL.

BackgroundCutaneous leishmaniasis (CL) caused by Leishmania braziliensis remains an important public health problem in Brazil. The goal of this study was to identify genetic risk factors for CL.MethodsGenome-wide analysis was undertaken using DNAs from 956 CL cases and 868 controls (phase 1) and 1110 CL cases and 1178 controls (phase 2) genotyped using Illumina HumanCoreExome BeadChips. Imputation against 1000G data provided 4,498,586 quality-controlled single nucleotide variants (SNVs) common across phase 1 and phase 2 samples. Linear mixed models in FastLMM were used to take account of genetic diversity/ethnicity/admixture. Cellular cytokines were measured using flow cytometry.ResultsCombined analysis across cohorts found no associations that achieved genome-wide significance, commonly accepted as P-8. Support for variants at wound-healing genes previously studied as candidate genes for CL included SMAD2 (rs115582038/rs75753347; Pimputed_1000G=1.47×10-4). Top novel GWAS hits at P-5 in plausible candidate genes for CL included SERPINB10 (rs62097497;Pimputed_1000G=2.67×10-6), CRLF3 (rs75270613; Pimputed_1000G=5.12×10-6), STX7 (rs144488134;Pimputed_1000G=6.06×10-6), KRT80 (rs10783496 Pimputed_1000G=6.58×10-6), LAMP3 (rs74285558;Pimputed_1000G=6.54×10-6) and IFNG-AS1 (rs4913269;Pimputed_1000G=1.32×10-5). Of these, LAMP3 (Padjusted=9.25×10-12; +6-fold), STX7 (Padjusted=7.62×10-3; +1.3-fold) and CRLF3 (Padjusted=9.19×10-9; +1.97-fold) were all expressed more highly in CL biopsies compared to normal skin, whereas expression of KRT80 (Padjusted=3.07×10-8; −3-fold) was lower. Notably, the percent peripheral blood CD3+ T cells making interferon-γ in response to Leishmania antigen differed significantly by IFNG-AS1 genotype.ConclusionsIn addition to supporting variants in wound-healing genes as genetic risk factors for CL, our GWAS results provide important novel leads to understanding pathogenesis of CL including through the regulation of interferon-γ responses.

We aimed to investigate the association of CD14 -260C/T (rs2569190) polymorphism and Chagas cardiomyopathy and the functional characteristics of CD14+ and CD14- monocytes upon infection with Trypanosoma cruzi. We observed an association between the T- genotype (absence of allele -260T) related to low CD14 expression and the dilated cardiomyopathy type of Chagas disease. Furthermore, we observed that CD14- monocytes showed a more activated profile upon in vitro infection with T. cruzi than CD14+ monocytes. Our findings suggest that T- genotype is associated with susceptibility to develop Chagas dilated cardiomyopathy, likely linked to the T. cruzi-induced inflammatory profile of CD14- monocytes.

Cell death plays a fundamental role in mounting protective and pathogenic immunity. Etosis is a cell death mechanism defined by the release of extracellular traps (ETs), which can foster inflammation, exert microbicidal activity, and is often associated with innate immune cells. Here we demonstrate that human CD8+ T cells release ETs (herein LETs – lymphocyte extracellular traps), which display unique morphology and functional characteristics. CD8+ T cell-derived LETs form strands that connect distant cells and colocalize with CD107a, a cytotoxicity marker. The release of LETs by CD8+ T cells induces death of neighboring cells without intimate cell-cell contact. Importantly, we show that the progression and severity of debilitating and mutilating forms of human leishmaniasis are associated with CD8+ T cell-derived LETs carrying CD107a+ vesicles. We propose that CD8+ T cell-derived LETs deliver cytotoxicity signals to distant target cells causing their death, providing insights into mechanisms of CD8-mediated pathology.

Gastric cancer (GC) is the fifth most common type of cancer worldwide with high incidences in Asia, Central, and South American countries. This patchy distribution means that GC studies are neglected by large research centers from developed countries. The need for further understanding of this complex disease, including the local importance of epidemiological factors and the rich ancestral admixture found in Brazil, stimulated the implementation of the GE4GAC project. GE4GAC aims to embrace epidemiological, clinical, molecular and microbiological data from Brazilian controls and patients with malignant and pre-malignant gastric disease. In this letter, we summarize the main goals of the project, including subject and sample accrual and current findings.

Aims The aim of the study was to evaluate the immune response triggered by the first contact of human monocytes with two T cruzi strains from distinct discrete typing units (DTUs) IV and V, and whether co-infection with these strains leads to changes in monocyte immune profiles, which could in turn influence the subsequent infection outcome. Methods and results We evaluated the influence of in vitro single- and co-infection with AM64 and 3253 strains on immunological characteristics of human monocytes. Single infection of monocytes with AM64 or 3253 induced opposing anti-inflammatory and inflammatory responses, respectively. Co-infection was observed in over 50% of monocytes after 15 hours of culture, but this percentage dropped ten-fold after 72 hours. Co-infection led to high monocyte activation and an increased percentage of both IL-10 and TNF. The decreased percentage of co-infected cells observed after 72 hours was associated with a decreased frequency of TNF-expressing cells. Conclusion Our results show that the exacerbated response observed in co-infection with immune-polarizing strains is associated with a decreased frequency of co-infected cells, suggesting that the activated response favours parasite control. These findings may have implications for designing new Chagas disease preventive strategies.

Mucosal leishmaniasis (ML) is characterized by high production of inflammatory cytokines. Administration of pentoxifylline (PTX), an inhibitor of TNF-alpha, with pentavalent antimony (Sb(v)), has been successfully used as alternative treatment for refractory ML. Our study aims to investigate the in situ cellular response underlying the effectiveness of this therapy, by evaluating the intensity of the inflammatory infiltrate, cellular composition, and expression of cytokines and granzyme A in lesions from ML before and after treatment with Sb(v) alone or in combination with PTX. Our data showed no differences in the intensity of inflammatory infiltrate comparing before and after treatment, and comparing between different treatments. However, although the number and frequency of CD4(+) and CD8(+) cells were not different before and after treatments or comparing different treatments, frequency of CD68(+) cells decreased after treatment with Sb(v) + PTX, but not with Sb(v). This was due to a reduction in CD68(+) TNF-alpha(+) and not in CD68(+) IL-10(+) cells. The frequency of TNF-alpha(+) cells was correlated with the intensity of the inflammatory infiltrate before treatment, but this correlation was lost after treatment with Sb(v) + PTX. Although the total expression of granzyme A did not significantly change after treatments, a clear trend of decrease was observed after treatment with Sb(v) + PTX. Interestingly, patients who took longer to heal, regardless of the treatment, displayed a higher frequency of granzyme A(+) cells. Our data suggest that treatment with Sb(v) + PTX acts in CD68(+) cells reducing the expression of TNF-alpha but not IL-10, resulting in more efficient modulation of the inflammatory response, accelerating the healing process.

Cervical cancer (CC) is a major cause of death and suffering to women globally with 570,000 new cases in 2017. It disproportionately affects those living in resource-constrained countries such as Brazil, with 90% of the deaths from CC happening in low and middle-income countries. Early detection is still the best strategy for improving response to therapy and survival and cases detected in advanced stages show variable response rates to the standard chemoradiation therapy protocols. Both the genetic landscape and the immune status of patients can dramatically affect cancer progression and response to therapy, as well as disease recurrence. Here we performed a comprehensive sequencing analysis using the cancer gene panel - Ion AmpliSeq™ Cancer hotspot Panel V2 CHPv2, as well as determined the immune infiltrate composition of a group of locally advanced CC patients with the goal of identifying genetic and immune characteristics associated with a clinical response to therapy. The expression levels of CD68+ tumor-associated macrophages (TAMs) and CD8+ tumor-infiltrating lymphocytes (TILs), as well as the immune checkpoint molecules PD-1, PD-L1 and PD-L2 in stroma and in tumor regions were analyzed by immunohistochemistry (IHC). The HPV infection status with high-risk strains was also determined. Twenty-one samples from patients with squamous cell carcinoma segregated into responder (11) and non-responder (10) groups according to standard chemoradiation therapy response were studied. Our findings indicate that responder patients showed an increase of an inflammatory tumor microenvironment as indicated by higher numbers of CD8+ and PD-L2+ TILs, as well as higher expression of PD-L1 immunoreactive area, as compared to the non-responder group. Additionally, our results demonstrate a correlation between the number of gene mutations and PD-L2+ TILs in the responder group. The genes PIK3CA and KDR/VEGFR were the most mutated genes, corroborating past findings. Together, these findings indicate an inflammatory tumor microenvironment present in patients that will respond to future chemoradiation treatment as compared to those that will not. This points to possible future predictors of response to therapy in CC patients.

Aims CD8+ T cells are important in mediating protective responses to intracellular pathogens. However, an uncontrolled response may lead to pathology. The role of CD8+ T cells in different clinical manifestations of human leishmaniasis is controversial and poorly understood. We aim to study the response of CD8+ T cells to the first exposure to different strains of Leishmania, seeking to correlate these findings with clinical manifestations of disease. Methods and results We have evaluated the expression of granzyme A, inflammatory and anti-inflammatory cytokines, as well as CTLA-4 by human naive CD8+ T cells exposed to Leishmania braziliensis and two different strains of Leishmania infantum in vitro. We observed that while exposure to L braziliensis induced an inflammatory profile, as measured by the expression of granzyme A, IFN-gamma and IL-17, as well as a higher IFN/IL-10 ratio, exposure to L infantum led to a regulatory profile, as measured by lower IFN/IL-10 ratio and higher expression of CTLA-4. Conclusion These results may help explain why patients with the visceral clinical form present a weaker cellular response and, consequently, a worse outcome of the disease. The use of CTLA-4 checkpoint inhibitors may emerge as a potential immunotherapy to ameliorate the immune response in visceral leishmaniasis patients.

e21569 Background: Neoadjuvant immunotherapy with nivolumab 3mg/kg and ipilimumab 1mg/kg for two cycles(N3+I1) or anti-PD1 for 3-8 weeks for clinical stage III melanoma have shown rates of pCR/near-pCR about 60% or 30%, respectively. The prognosis of this group seems to be excellent so far. On the other hand, patients classified as pathological non-response have a worse outcome and early identification of this group may allow us to tailor treatment before surgery. Methods: We conducted a retrospective analysis of patients with clinical stage III melanoma treated with neoadjuvant immune checkpoint blockade who did baseline and pre-operative FDG-PET/CT. The total number of FGD avid lesions and the percentual difference between the maximum SUV per lesion was calculated. The pathological results were correlated to FGD-PET/CT findings. Results: Between January 2019 and January 2021, nine patients with clinical stage III melanoma were identified. Six patients received Nivolumab 3m/kg and Ipilimumab 1mg/kg for two cycles and 3 received anti-PD1 for 60 days. Baseline FDG-PET/CT showed just one avid lesion in 7 patients, 2 avid lesions in one patient and 3 avid lesions in one patient. All known lesions identified by CT scan were also captured by FGD-PET/CT. After the neoadjuvant treatment, 4 patients achieved pCR/near-pCR. All of them were treated with N3+I1. Two of them had metabolic complete response (including the one with 3 PET- avid lesions) and 2 had reduction on FDG concentration (-31% and – 76%). Three patients had absence of response: 100% of viable tumor cells (VTC), all of them with increase in FDG concentration (+12% to + 307%) and appearance of a new lesion in one case. Two out of them were treated with anti-PD1. One patient had partial response (40% of VTC). Interestingly, this patient developed sarcoidosis-like reaction with increase of SUV in the index lesion (+68%) and appearance of mediastinal lymph-nodes. One patient, who had two lesions, presented a mixed response: complete response in one and 90% of VTC in the other. FDG-PET/CT was able to detect both responses (metabolic complete response and increase of FGD concentration of 17%, respectively). Conclusions: Reduction in FGD-PET/CT concentration with no appearance of new lesion(s) is associated with significant pathological response. An increase of SUV or appearance of new lesion(s) should be carefully interpreted.