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.

The long intergenic noncoding RNA landscape of human lymphocytes highlights the regulation of T cell differentiation by linc-MAF-4.

Long noncoding RNAs are emerging as important regulators of cellular functions, but little is known of their role in the human immune system. Here we investigated long intergenic noncoding RNAs (lincRNAs) in 13 subsets of T lymphocytes and B lymphocytes by next-generation sequencing-based RNA sequencing (RNA-seq analysis) and de novo transcriptome reconstruction. We identified over 500 previously unknown lincRNAs and described lincRNA signatures. Expression of linc-MAF-4, a chromatin-associated lincRNA specific to the TH1 subset of helper T cells, was inversely correlated with expression of MAF, a TH2-associated transcription factor. Downregulation of linc-MAF-4 skewed T cell differentiation toward the TH2 phenotype. We identified a long-distance interaction between the genomic regions of the gene encoding linc-MAF-4 and MAF, where linc-MAF-4 associated with the chromatin modifiers LSD1 and EZH2; this suggested that linc-MAF-4 regulated MAF transcription through the recruitment of chromatin modifiers. Our results demonstrate a key role for lincRNA in T lymphocyte differentiation.

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.

Regulatory T-cell-derived interleukin-15 shapes cytotoxic T cell memory.

It is well known that regulatory T-cells (Tregs) are required to prevent autoimmunity, but they may also have some less-well understood immune-stimulatory effects. In particular, in CD8+ T-cell responses Tregs select high-affinity clones upon priming and promote memory by inhibiting inflammation-dependent generation of short-lived effector cells. In the current issue of the European Journal of Immunology [Eur. J. Immunol. 2023. 53: 2149400], Madi et al. report the surprising finding that human and murine FOXP3+ Tregs are a physiologically relevant source of IL-15, a homeostatic cytokine that promotes antigen-independent maintenance of CD8+ memory T-cells. In mice that lack IL-15 selectively in FOXP3+ Tregs the authors show that the composition of the CD8+ T-cell memory pool is altered in the absence of Treg-derived IL-15, since a subset of terminally effector memory cells is drastically reduced. Otherwise Treg-derived IL-15 is dispensable for antiviral immune responses and the generation of anti-viral CD8+ memory T-cells. These findings add to our understanding of the multifaceted role of Tregs in immune responses, and how IL-15 derived from different cellular sources maintains anti-viral T-cell memory.

Eomesodermin-expressing type 1 regulatory (EOMES+ Tr1)-like T cells: Basic biology and role in immune-mediated diseases.

Type 1 regulatory (Tr1) T cells are currently defined all T cells with regulatory functions that lack FOXP3 expression and produce IL-10. Tr1 cells are heterogeneous, and the different reported properties of Tr1-cell populations have caused some confusion in the field. Moreover, understanding the role of Tr1 cells in immune-mediated diseases has been hampered by the lack of a lineage-defining transcription factor. Several independent studies indicated recently that the transcription factor Eomesodermin (EOMES) could act as a lineage-defining transcription factor in a population of IL-10 and IFN-γ co-producing Tr1-like cells, since EOMES directly induces IFN-γ and cytotoxicity, enhances IL-10, and antagonizes alternative T-cell fates. Here, we review the known properties of EOMES+ Tr1-like cells. They share several key characteristics with other Tr1 cells (i.e., "Tr1-like"), namely high IL-10 production, cytotoxicity, and suppressive capabilities. Notably, they also share some features with FOXP3+ Tregs, like downregulation of IL-7R and CD40L. In addition, they possess several unique, EOMES-dependent features, that is, expression of GzmK and IFN-γ, and downregulation of type-17 cytokines. Published evidence indicates that EOMES+ Tr1-like cells play key roles in graft-versus-host disease, colitis, systemic autoimmunity and in tumors. Thus, EOMES+ Tr1-like cells are key players of the adaptive immune system that are involved in several different immune-mediated diseases.

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.

Distinct microRNA signatures in human lymphocyte subsets and enforcement of the naive state in CD4+ T cells by the microRNA miR-125b.

MicroRNAs are small noncoding RNAs that regulate gene expression post-transcriptionally. Here we applied microRNA profiling to 17 human lymphocyte subsets to identify microRNA signatures that were distinct among various subsets and different from those of mouse lymphocytes. One of the signature microRNAs of naive CD4+ T cells, miR-125b, regulated the expression of genes encoding molecules involved in T cell differentiation, including IFNG, IL2RB, IL10RA and PRDM1. The expression of synthetic miR-125b and lentiviral vectors encoding the precursor to miR-125b in naive lymphocytes inhibited differentiation to effector cells. Our data provide an 'atlas' of microRNA expression in human lymphocytes, define subset-specific signatures and their target genes and indicate that the naive state of T cells is enforced by microRNA.

Ex vivo microRNA and gene expression profiling of human Tr1-like cells suggests a role for miR-92a and -125a in the regulation of EOMES and IL-10R.

Ex vivo gene expression and miRNA profiling of Eomes+ Tr1-like cells suggested that they represent a differentiation stage that is intermediate between Th1-cells and cytotoxic CD4+ T-cells. Several microRNAs were downregulated in Eomes+ Tr1-like cells that might inhibit Tr1-cell differentiation. In particular, miR-92a targeted Eomes, while miR-125a inhibited IFN-g and IL-10R expression.

Th17 cells transdifferentiate into regulatory T cells during resolution of inflammation.

Inflammation is a beneficial host response to infection but can contribute to inflammatory disease if unregulated. The Th17 lineage of T helper (Th) cells can cause severe human inflammatory diseases. These cells exhibit both instability (they can cease to express their signature cytokine, IL-17A) and plasticity (they can start expressing cytokines typical of other lineages) upon in vitro re-stimulation. However, technical limitations have prevented the transcriptional profiling of pre- and post-conversion Th17 cells ex vivo during immune responses. Thus, it is unknown whether Th17 cell plasticity merely reflects change in expression of a few cytokines, or if Th17 cells physiologically undergo global genetic reprogramming driving their conversion from one T helper cell type to another, a process known as transdifferentiation. Furthermore, although Th17 cell instability/plasticity has been associated with pathogenicity, it is unknown whether this could present a therapeutic opportunity, whereby formerly pathogenic Th17 cells could adopt an anti-inflammatory fate. Here we used two new fate-mapping mouse models to track Th17 cells during immune responses to show that CD4(+) T cells that formerly expressed IL-17A go on to acquire an anti-inflammatory phenotype. The transdifferentiation of Th17 into regulatory T cells was illustrated by a change in their signature transcriptional profile and the acquisition of potent regulatory capacity. Comparisons of the transcriptional profiles of pre- and post-conversion Th17 cells also revealed a role for canonical TGF-ß signalling and consequently for the aryl hydrocarbon receptor (AhR) in conversion. Thus, Th17 cells transdifferentiate into regulatory cells, and contribute to the resolution of inflammation. Our data suggest that Th17 cell instability and plasticity is a therapeutic opportunity for inflammatory diseases.

Eomesodermin controls a unique differentiation program in human IL-10 and IFN-γ coproducing regulatory T cells.

Whether human IL-10-producing regulatory T cells ("Tr1") represent a distinct differentiation lineage or an unstable activation stage remains a key unsolved issue. Here, we report that Eomesodermin (Eomes) acted as a lineage-defining transcription factor in human IFN-γ/IL-10 coproducing Tr1-like cells. In vivo occurring Tr1-like cells expressed Eomes, and were clearly distinct from all other CD4+ T-cell subsets, including conventional cytotoxic CD4+ T cells. They expressed Granzyme (Gzm) K, but had lost CD40L and IL-7R expression. Eomes antagonized the Th17 fate, and directly controlled IFN-γ and GzmK expression. However, Eomes binding to the IL-10 promoter was not detectable in human CD4+ T cells, presumably because critical Tbox binding sites of the mouse were not conserved. A precommitment to a Tr1-like fate, i.e. concominant induction of Eomes, GzmK, and IFN-γ, was promoted by IL-4 and IL-12-secreting myeloid dendritic cells. Consistently, Th1 effector memory cells contained precommitted Eomes+ GzmK+ T cells. Stimulation with T-cell receptor (TCR) agonists and IL-27 promoted the generation of Tr1-like effector cells by inducing switching from CD40L to IL-10. Importantly, CD4+ Eomes+ T-cell subsets were present in lymphoid and nonlymphoid tissues, and their frequencies varied systemically in patients with inflammatory bowel disease and graft-versus-host disease. We propose that Eomes+ Tr1-like cells are effector cells of a unique GzmK-expressing CD4+ T-cell subset.

The Enigmatic Role of Viruses in Multiple Sclerosis: Molecular Mimicry or Disturbed Immune Surveillance?

Multiple sclerosis (MS) is a T cell driven autoimmune disease of the central nervous system (CNS). Despite its association with Epstein-Barr Virus (EBV), how viral infections promote MS remains unclear. However, there is increasing evidence that the CNS is continuously surveyed by virus-specific T cells, which protect against reactivating neurotropic viruses. Here, we discuss how viral infections could lead to the breakdown of self-tolerance in genetically predisposed individuals, and how the reactivations of viruses in the CNS could induce the recruitment of both autoaggressive and virus-specific T cell subsets, causing relapses and progressive disability. A disturbed immune surveillance in MS would explain several experimental findings, and has important implications for prognosis and therapy.

Novel biomarkers for primary biliary cholangitis to improve diagnosis and understand underlying regulatory mechanisms.

BACKGROUND AND AIMS: Primary biliary cholangitis is an autoimmune biliary disease characterized by injury of bile ducts, eventually leading to cirrhosis and death. In most cases, anti-mitochondrial antibodies and persistently elevated serum alkaline phosphatase are the basis for the serological diagnosis. Anti-nuclear antibodies are also useful and may indicate a more aggressive diseases course. In patients in which anti-mitochondrial antibodies are not detected, an accurate diagnosis requires liver histology. This study aims at identifying specific biomarkers for the serological diagnosis of primary biliary cholangitis. METHODS: Sera from patients affected by primary biliary cholangitis, primary sclerosing cholangitis, hepatitis C virus (with and without cryoglobulinemia), hepatocarcinoma and healthy donors were tested on a protein array representing 1658 human proteins. The most reactive autoantigens were confirmed by DELFIA analysis on expanded cohorts of the same mentioned serum classes, and on autoimmune hepatitis sera, using anti-PDC-E2 as reference biomarker. RESULTS: Two autoantigens, SPATA31A3 and GARP, showed high reactivity with primary biliary cholangitis sera, containing or not anti-mitochondrial antibodies. Their combination with PDC-E2 allowed to discriminate primary biliary cholangitis from all tested control classes with high sensitivity and specificity. We found that GARP expression is upregulated upon exposure to biliary salts in human cholangiocytes, an event involving EGFR and insulin pathways. GARP expression was also detected in biliary duct cells of PBC patients. CONCLUSIONS: This study highlighted SPATA31A3 and GARP as new biomarkers for primary biliary cholangitis and unravelled molecular stimuli underlying GARP expression in human cholangiocytes.

Intestinal IFN-γ-producing type 1 regulatory T cells coexpress CCR5 and programmed cell death protein 1 and downregulate IL-10 in the inflamed guts of patients with inflammatory bowel disease.

BACKGROUND: IL-10 is an anti-inflammatory cytokine required for intestinal immune homeostasis. It mediates suppression of T-cell responses by type 1 regulatory T (TR1) cells but is also produced by CD25+ regulatory T (Treg) cells. OBJECTIVE: We aimed to identify and characterize human intestinal TR1 cells and to investigate whether they are a relevant cellular source of IL-10 in patients with inflammatory bowel diseases (IBDs). METHODS: CD4+ T cells isolated from the intestinal lamina propria of human subjects and mice were analyzed for phenotype, cytokine production, and suppressive capacities. Intracellular IL-10 expression by CD4+ T-cell subsets in the inflamed guts of patients with IBD (Crohn disease or ulcerative colitis) was compared with that in cells from noninflamed control subjects. Finally, the effects of proinflammatory cytokines on T-cell IL-10 expression were analyzed, and IL-1ß and IL-23 responsiveness was assessed. RESULTS: Intestinal TR1 cells could be identified by coexpression of CCR5 and programmed cell death protein 1 (PD-1) in human subjects and mice. CCR5+PD-1+ TR1 cells expressed IFN-γ and efficiently suppressed T-cell proliferation and transfer colitis. Intestinal IFN-γ+ TR1 cells, but not IL-7 receptor-positive TH cells or CD25+ Treg cells, showed lower IL-10 expression in patients with IBDs. TR1 cells were responsive to IL-23, and IFN-γ+ TR1 cells downregulated IL-10 with IL-1ß and IL-23. Conversely, CD25+ Treg cells expressed higher levels of IL-1 receptor but showed stable IL-10 expression. CONCLUSIONS: We provide the first ex vivo characterization of human intestinal TR1 cells. Selective downregulation of IL-10 by IFN-γ+ TR1 cells in response to proinflammatory cytokines is likely to drive excessive intestinal inflammation in patients with IBDs.

Extracellular MicroRNA Signature of Human Helper T Cell Subsets in Health and Autoimmunity.

Upon T cell receptor stimulation, CD4+ T helper (Th) lymphocytes release extracellular vesicles (EVs) containing microRNAs. However, no data are available on whether human CD4+ T cell subsets release EVs containing different pattern of microRNAs. The present work aimed at filling this gap by assessing the microRNA content in EVs released upon in vitro T cell receptor stimulation of Th1, Th17, and T regulatory (Treg) cells. Our results indicate that EVs released by Treg cells are significantly different compared with those released by the other subsets. In particular, miR-146a-5p, miR-150-5p, and miR-21-5p are enriched, whereas miR-106a-5p, miR-155-5p, and miR-19a-3p are depleted in Treg-derived EVs. The in vitro identified EV-associated microRNA signature was increased in serum of autoimmune patients with psoriasis and returned to healthy levels upon effective treatment with etanercept, a biological drug targeting the TNF pathway and suppressing inflammation. Moreover, Gene Set Enrichment Analysis showed an over-representation of genes relevant for T cell activation, such as CD40L, IRAK1, IRAK2, STAT1, and c-Myb in the list of validated targets of Treg-derived EV miRNAs. At functional level, Treg-derived (but not Th1/Th17-derived) EVs inhibited CD4+ T cell proliferation and suppressed two relevant targets of miR-146a-5p: STAT1 and IRAK2. In conclusion, our work identified the miRNAs specifically released by different human CD4+ T cell subsets and started to unveil the potential use of their quantity in human serum to mark the pathological elicitation of these cells in vivo and their biological effect in cell to cell communication during the adaptive immune response.

Maintenance of memory CD8 T cells: Divided over division.

Once generated during an infection, memory CD8+ T cells can provide long-lasting protection against reinfection with an intracellular pathogen, but the longevity of this defense depends on the ability of these pathogen-specific memory cells to be maintained. It is generally believed that the bone marrow plays an important role in this respect, where memory CD8 T cells receive reinvigorating signals from cytokines that induce homeostatic proliferation. However, in the current issue of the European Journal of Immunology, Siracusa et al. (Eur. J. Immunol. 2017. 47: 1900-1905) argue against this dogma, as they provide evidence that CD8 memory T cells in murine bone marrow are not proliferating, but largely quiescent, which protects them from elimination by the cytostatic drug Cyclophosphamide. Interestingly, this is in sharp contrast to the proliferating cell counterparts in the spleen, which are eliminated by this treatment. Here, we will discuss the impact of these results, how they relate to opposing findings by others in the field, and what the relevance of these findings is for humans and clinical applications.

Successful sequential therapy with rituximab and belimumab in patients with active systemic lupus erythematosus: a case series.

OBJECTIVES: B cells play an important role in the initiation and progression of systemic lupus erythematosus (SLE). Accordingly, B cell-targeted therapy has been suggested as a new rational approach for treating lupus. Belimumab, a human monoclonal antibody directed against B lymphocyte stimulator (BLyS), was reported as the first biological treatment effective in reducing mild-to-moderate SLE disease activity by using different scoring systems and endpoints. Conversely clinical trials with rituximab, a chimeric monoclonal antibody directed against the CD20 expressed by B cells, have failed to achieve primary endpoints in spite of a number of reports showing its beneficial effects. Anecdotal reports have described the sequential use of rituximab and belimumab as a more effective treatment than using the individual drugs alone, without compromising safety. METHODS: We report a case series of three patients with active SLE refractory to conventional therapies, who underwent treatment with rituximab followed by belimumab as maintenance therapy. RESULTS: We observed a beneficial effect after sequential treatment with rituximab and belimumab. All patients achieved long-standing remission and could reduce or discontinue corticosteroids. Concomitantly, after rituximab administration we observed a rise in BLyS levels, which were dramatically reduced after belimumab introduction. CONCLUSIONS: The modulation of plasma BLyS kinetics in patients undergoing sequential treatment with rituximab and belimumab may represent a possible rationale behind the effectiveness of this combined therapy.