Clonally expanded EOMES+ Tr1-like cells in primary and metastatic tumors are associated with disease progression.

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.

Characterization of human CD4+EOMES+GzmK+ T-cell subsets unveils an uncoupling of suppressive functions from IL-10-producing capacities.

Human CD4+EOMES+ T cells are heterogeneous and contain Th1-cells, Tr1-cells, and CD4+CTL. Tr1- cells and non-classical EOMES+ Th1-cells displayed, respectively, anti- and pro-inflammatory cytokine profiles, but both expressed granzyme-K, produced IFN-γ, and suppressed T-cell proliferation. Diffusion map suggested a progressive CD4+T-cell differentiation from naïve to cytotoxic cells and identified EOMES+Th1-cells as putative Tr1-cell precursors (pre-Tr1).

Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells.

Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.

Tracking the immune response profiles elicited by the BNT162b2 vaccine in COVID-19 unexperienced and experienced individuals.

Multiple vaccines have been approved to control COVID-19 pandemic, with Pfizer/BioNTech (BNT162b2) being widely used. We conducted a longitudinal analysis of the immune response elicited after three doses of the BNT162b2 vaccine in individuals who have previously experienced SARS-CoV-2 infection and in unexperienced ones. We conducted immunological analyses and single-cell transcriptomics of circulating T and B lymphocytes, combined to CITE-seq or LIBRA-seq, and VDJ-seq. We found that antibody levels against SARS-CoV-2 Spike, NTD and RBD from wild-type, delta and omicron VoCs show comparable dynamics in both vaccination groups, with a peak after the second dose, a decline after six months and a restoration after the booster dose. The antibody neutralization activity was maintained, with lower titers against the omicron variant. Spike-specific memory B cell response was sustained over the vaccination schedule. Clonal analysis revealed that Spike-specific B cells were polyclonal, with a partial clone conservation from natural infection to vaccination. Spike-specific T cell responses were oriented towards effector and effector memory phenotypes, with similar trends in unexperienced and experienced individuals. The CD8 T cell compartment showed a higher clonal expansion and persistence than CD4 T cells. The first two vaccinations doses tended to induce new clones rather than promoting expansion of pre-existing clones. However, we identified a fraction of Spike-specific CD8 T cell clones persisting from natural infection that were boosted by vaccination and clones specifically induced by vaccination. Collectively, our observations revealed a moderate effect of the second dose in enhancing the immune responses elicited after the first vaccination. Differently, we found that a third dose was necessary to restore comparable levels of neutralizing antibodies and Spike-specific T and B cell responses in individuals who experienced a natural SARS-CoV-2 infection.

SARS-COV-2 specific t-cells in patients with thyroid disorders related to COVID-19 are enriched in the thyroid and acquire a tissue-resident memory phenotype.

BACKGROUND: SARS-CoV-2 infections have been associated with the onset of thyroid disorders like classic subacute thyroiditis (SAT) or atypical SAT upon severe COVID disease (COV-A-SAT). Little is known about thyroid anti-viral immune responses. OBJECTIVES: To define the role of T-cells in COV-A-SAT. METHODS: T-cells from COV-A-SAT patients were analyzed by multi-dimensional flow cytometry, UMAP and DiffusionMap dimensionality reduction and FlowSOM clustering. T-cells from COVID-naïve healthy donors, patients with autoimmune thyroiditis (ATD) and with SAT following COVID vaccination were analyzed as controls. T-cells were analyzed four and eight months post-infection in peripheral blood and in thyroid specimen obtained by ultrasound-guided fine needle aspiration. SARS-COV2-specific T-cells were identified by cytokine production induced by SARS-COV2-derived peptides and with COVID peptide-loaded HLA multimers after HLA haplotyping. RESULTS: COV-A-SAT was associated with HLA-DRB1*13 and HLA-B*57. COV-A-SAT patients contained activated Th1- and cytotoxic CD4+ and CD8+ effector cells four months post-infection, which acquired a quiescent memory phenotype after eight months. Anti-SARS-CoV-2-specific T-cell responses were readily detectable in peripheral blood four months post-infection, but were reduced after eight months. CD4+ and CD8+ tissue-resident memory cells (TRM) were present in the thyroid, and circulating CXCR3+T-cells identified as their putative precursors. SARS-CoV-2-specific T-cells were enriched in the thyroid, and acquired a TRM phenotype eight months post-infection. CONCLUSIONS: The association of COV-A-SAT with specific HLA haplotypes suggests a genetic predisposition and a key role for T-cells. COV-A-SAT is characterized by a prolonged systemic anti-viral effector T-cell response and the late generation of COVID-specific TRM in the thyroid target tissue.

Maturation signatures of conventional dendritic cell subtypes in COVID-19 suggest direct viral sensing.

Growing evidence suggests that conventional dendritic cells (cDCs) undergo aberrant maturation in COVID-19, which negatively affects T-cell activation. The presence of effector T cells in patients with mild disease and dysfunctional T cells in severely ill patients suggests that adequate T-cell responses limit disease severity. Understanding how cDCs cope with SARS-CoV-2 can help elucidate how protective immune responses are generated. Here, we report that cDC2 subtypes exhibit similar infection-induced gene signatures, with the upregulation of IFN-stimulated genes and IL-6 signaling pathways. Furthermore, comparison of cDCs between patients with severe and mild disease showed severely ill patients to exhibit profound downregulation of genes encoding molecules involved in antigen presentation, such as MHCII, TAP, and costimulatory proteins, whereas we observed the opposite for proinflammatory molecules, such as complement and coagulation factors. Thus, as disease severity increases, cDC2s exhibit enhanced inflammatory properties and lose antigen presentation capacity. Moreover, DC3s showed upregulation of anti-apoptotic genes and accumulated during infection. Direct exposure of cDC2s to the virus in vitro recapitulated the activation profile observed in vivo. Our findings suggest that SARS-CoV-2 interacts directly with cDC2s and implements an efficient immune escape mechanism that correlates with disease severity by downregulating crucial molecules required for T-cell activation.

Tumor Extracellular Matrix Stiffness Promptly Modulates the Phenotype and Gene Expression of Infiltrating T Lymphocytes.

The immune system is a fine modulator of the tumor biology supporting or inhibiting its progression, growth, invasion and conveys the pharmacological treatment effect. Tumors, on their side, have developed escaping mechanisms from the immune system action ranging from the direct secretion of biochemical signals to an indirect reaction, in which the cellular actors of the tumor microenvironment (TME) collaborate to mechanically condition the extracellular matrix (ECM) making it inhospitable to immune cells. TME is composed of several cell lines besides cancer cells, including tumor-associated macrophages, cancer-associated fibroblasts, CD4+ and CD8+ lymphocytes, and innate immunity cells. These populations interface with each other to prepare a conservative response, capable of evading the defense mechanisms implemented by the host's immune system. The presence or absence, in particular, of cytotoxic CD8+ cells in the vicinity of the main tumor mass, is able to predict, respectively, the success or failure of drug therapy. Among various mechanisms of immunescaping, in this study, we characterized the modulation of the phenotypic profile of CD4+ and CD8+ cells in resting and activated states, in response to the mechanical pressure exerted by a three-dimensional in vitro system, able to recapitulate the rheological and stiffness properties of the tumor ECM.

Polycomb Bodies Detection in Murine Fibromuscular Stroma from Skin, Skeletal Muscles, and Aortic Tissues.

The regulation of chromatin structure depends on a dynamic, multiple mechanisms that modulate gene expression and constitute the epigenome. The Polycomb group (PcG) of proteins are epigenetic factors involved in the transcriptional repression. Among their multilevel, chromatin-associated functions, PcG proteins mediate the establishment and maintenance of higher-order structures at target genes, allowing the transmission of transcriptional programs throughout the cell cycle.In the nucleus, PcG proteins localize close to the pericentric heterochromatin forming microscopically foci, called Polycomb bodies. Here, to visualize the tissue-specific PcG distribution in the aorta, dorsal skin and hindlimb muscles, we combine a fluorescence-activated cell sorter (FACS)-based method with an immunofluorescence staining.

Human airway organoids and microplastic fibers: A new exposure model for emerging contaminants.

Three-dimensional (3D) structured organoids are the most advanced in vitro models for studying human health effects, but their application to evaluate the biological effects associated with microplastic exposure was neglected until now. Fibers from synthetic clothes and fabrics are a major source of airborne microplastics, and their release from dryer machines is poorly understood. We quantified and characterized the microplastic fibers (MPFs) released in the exhaust filter of a household dryer and tested their effects on airway organoids (1, 10, and 50 µg mL-1) by optical microscopy, scanning electron microscopy (SEM), confocal microscopy and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). While the presence of MPFs did not inhibit organoid growth, we observed a significant reduction of SCGB1A1 gene expression related to club cell functionality and a polarized cell growth along the fibers. The MPFs did not cause relevant inflammation or oxidative stress but were coated with a cellular layer, resulting in the inclusion of fibers in the organoid. This effect could have long-term implications regarding lung epithelial cells undergoing repair. This exposure study using human airway organoids proved suitability of the model for studying the effects of airborne microplastic contamination on humans and could form the basis for further research regarding the toxicological assessment of emerging contaminants such as micro- or nanoplastics.

Immunosuppressant Treatment in Rheumatic Musculoskeletal Diseases Does Not Inhibit Elicitation of Humoral Response to SARS-CoV-2 Infection and Preserves Effector Immune Cell Populations.

COVID-19 has proven to be particularly serious and life-threatening for patients presenting with pre-existing pathologies. Patients affected by rheumatic musculoskeletal disease (RMD) are likely to have impaired immune responses against SARS-CoV-2 infection due to their compromised immune system and the prolonged use of disease-modifying anti-rheumatic drugs (DMARDs), which include conventional synthetic (cs) DMARDs or biologic and targeted synthetic (b/ts) DMARDs. To provide an integrated analysis of the immune response following SARS-CoV-2 infection in RMD patients treated with different classes of DMARDs we carried out an immunological analysis of the antibody responses toward SARS-CoV-2 nucleocapsid and RBD proteins and an extensive immunophenotypic analysis of the major immune cell populations. We showed that RMD individuals under most DMARD treatments mount a sustained antibody response to the virus, with neutralizing activity. In addition, they displayed a sizable percentage of effector T and B lymphocytes. Among b-DMARDs, we found that anti-TNFα treatments are more favorable drugs to elicit humoral and cellular immune responses as compared to CTLA4-Ig and anti-IL6R inhibitors. This study provides a whole picture of the humoral and cellular immune responses in RMD patients by reassuring the use of DMARD treatments during COVID-19. The study points to TNF-α inhibitors as those DMARDs permitting elicitation of functional antibodies to SARS-CoV-2 and adaptive effector populations available to counteract possible re-infections.

Integrated longitudinal immunophenotypic, transcriptional and repertoire analyses delineate immune responses in COVID-19 patients.

To understand how a protective immune response against SARS-CoV-2 develops over time, we integrated phenotypic, transcriptional and repertoire analyses on PBMCs from mild and severe COVID-19 patients during and after infection, and compared them to healthy donors (HD). A type I IFN-response signature marked all the immune populations from severe patients during the infection. Humoral immunity was dominated by IgG production primarily against the RBD and N proteins, with neutralizing antibody titers increasing post infection and with disease severity. Memory B cells, including an atypical FCRL5+ T-BET+ memory subset, increased during the infection, especially in patients with mild disease. A significant reduction of effector memory, CD8+ T cells frequency characterized patients with severe disease. Despite such impairment, we observed robust clonal expansion of CD8+ T lymphocytes, while CD4+ T cells were less expanded and skewed toward TCM and TH2-like phenotypes. MAIT cells were also expanded, but only in patients with mild disease. Terminally differentiated CD8+ GZMB+ effector cells were clonally expanded both during the infection and post-infection, while CD8+ GZMK+ lymphocytes were more expanded post-infection and represented bona fide memory precursor effector cells. TCR repertoire analysis revealed that only highly proliferating T cell clonotypes, which included SARS-CoV-2-specific cells, were maintained post-infection and shared between the CD8+ GZMB+ and GZMK+ subsets. Overall, this study describes the development of immunity against SARS-CoV-2 and identifies an effector CD8+ T cell population with memory precursor-like features.

Epigenomic landscape of human colorectal cancer unveils an aberrant core of pan-cancer enhancers orchestrated by YAP/TAZ.

Cancer is characterized by pervasive epigenetic alterations with enhancer dysfunction orchestrating the aberrant cancer transcriptional programs and transcriptional dependencies. Here, we epigenetically characterize human colorectal cancer (CRC) using de novo chromatin state discovery on a library of different patient-derived organoids. By exploring this resource, we unveil a tumor-specific deregulated enhancerome that is cancer cell-intrinsic and independent of interpatient heterogeneity. We show that the transcriptional coactivators YAP/TAZ act as key regulators of the conserved CRC gained enhancers. The same YAP/TAZ-bound enhancers display active chromatin profiles across diverse human tumors, highlighting a pan-cancer epigenetic rewiring which at single-cell level distinguishes malignant from normal cell populations. YAP/TAZ inhibition in established tumor organoids causes extensive cell death unveiling their essential role in tumor maintenance. This work indicates a common layer of YAP/TAZ-fueled enhancer reprogramming that is key for the cancer cell state and can be exploited for the development of improved therapeutic avenues.

Cancer-related CD15/FUT4 overexpression decreases benefit to agents targeting EGFR or VEGF acting as a novel RAF-MEK-ERK kinase downstream regulator in metastatic colorectal cancer.

BACKGROUND: Cancer-related immune antigens in the tumor microenvironment could represent an obstacle to agents targeting EGFR "cetuximab" or VEGF "bevacizumab" in metastatic colorectal cancer (mCRC) patients. METHODS: Infiltrating immune cells into tumor tissues, cancer-related expression of immune antigens (CD3, CD8, CD68, CD73, MPO, CD15/FUT4) from 102 mCRC patients receiving first-line Cetuximab or Bevacizumab plus chemotherapy were assessed by immunohistochemistry and validated in an independent tissue microarrays of 140 patients. Genome-wide expression profiles from 436 patients and 60 colon cancer cell lines were investigated using bioinformatics analysis. In vitro kinase assays of target genes activated by chemokines or growth factors were performed. RESULTS: Here, we report that cancer-related CD15/FUT4 is overexpressed in most of mCRCs patients (43 %) and associates with lower intratumoral CD3+ and CD8+ T cells, higher systemic inflammation (NLR at diagnosis >5) and poorer outcomes, in terms of response and progression-free survival than those CD15/FUT4-low or negative ones (adjusted hazard ratio (HR) = 2.92; 95 % CI = 1.86-4.41; P < 0.001). Overexpression of CD15/FUT4 is induced through RAF-MEK-ERK kinase cascade, suppressed by MEK inhibitors and exhibits a close connection with constitutive oncogenic signalling pathways that respond to ERBB3 or FGFR4 activation (P < 0.001). CD15/FUT4-high expressing colon cancer cells with primary resistance to cetuximab or bevacizumab are significantly more sensitive to MEK inhibitors than CD15/FUT4-low counterparts. CONCLUSION: Cancer-related CD15/FUT4 overexpression participates in cetuximab or bevacizumab mechanisms of resistance in mCRC patients. CD15/FUT4 as a potential target of the antitumor immune response requires further evaluation in clinical studies.