BACH2 regulates diversification of regulatory and proinflammatory chromatin states in TH17 cells.

Interleukin-17 (IL-17)-producing helper T (TH17) cells are heterogenous and consist of nonpathogenic TH17 (npTH17) cells that contribute to tissue homeostasis and pathogenic TH17 (pTH17) cells that mediate tissue inflammation. Here, we characterize regulatory pathways underlying TH17 heterogeneity and discover substantial differences in the chromatin landscape of npTH17 and pTH17 cells both in vitro and in vivo. Compared to other CD4+ T cell subsets, npTH17 cells share accessible chromatin configurations with regulatory T cells, whereas pTH17 cells exhibit features of both npTH17 cells and type 1 helper T (TH1) cells. Integrating single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq), we infer self-reinforcing and mutually exclusive regulatory networks controlling different cell states and predicted transcription factors regulating TH17 cell pathogenicity. We validate that BACH2 promotes immunomodulatory npTH17 programs and restrains proinflammatory TH1-like programs in TH17 cells in vitro and in vivo. Furthermore, human genetics implicate BACH2 in multiple sclerosis. Overall, our work identifies regulators of TH17 heterogeneity as potential targets to mitigate autoimmunity.

Dynamics in protein translation sustaining T cell preparedness.

In response to pathogenic threats, naive T cells rapidly transition from a quiescent to an activated state, yet the underlying mechanisms are incompletely understood. Using a pulsed SILAC approach, we investigated the dynamics of mRNA translation kinetics and protein turnover in human naive and activated T cells. Our datasets uncovered that transcription factors maintaining T cell quiescence had constitutively high turnover, which facilitated their depletion following activation. Furthermore, naive T cells maintained a surprisingly large number of idling ribosomes as well as 242 repressed mRNA species and a reservoir of glycolytic enzymes. These components were rapidly engaged following stimulation, promoting an immediate translational and glycolytic switch to ramp up the T cell activation program. Our data elucidate new insights into how T cells maintain a prepared state to mount a rapid immune response, and provide a resource of protein turnover, absolute translation kinetics and protein synthesis rates in T cells ( https://www.immunomics.ch ).

Publisher Correction: An immunoregulatory and tissue-residency program modulated by c-MAF in human TH17 cells.

In the version of this article initially published, in the legend to Fig. 1b, the description of the frequency of TH17-IL-10+ clones was incomplete for the first group; this should read as follows: "...13 experiments with clones isolated from CCR6+CCR4+CXCR3- T cells...". Also, the label along the vertical axis of the bottom right plot in Figure 5b was incomplete; the correct label is 'IFN-γ+ cells (%)'. Finally, in the first sentence of the final paragraph of the final Results subsection, the description of the regions analyzed was incorrect; that sentence should begin: "DNA motif-enrichment analysis of the subset-specific H3K27ac-positive regions...". The errors have been corrected in the HTML and PDF versions of the article.

An immunoregulatory and tissue-residency program modulated by c-MAF in human TH17 cells.

Different types of effector and memory T lymphocytes are induced and maintained in protective or pathological immune responses. Here we characterized two human CD4+ TH17 helper cell subsets that, in the recently activated state, could be distinguished on the basis of their expression of the anti-inflammatory cytokine IL-10. IL-10+ TH17 cells upregulated a variety of genes encoding immunoregulatory molecules, as well as genes whose expression is characteristic of tissue-resident T cells. In contrast, IL-10- TH17 cells maintained a pro-inflammatory gene-expression profile and upregulated the expression of homing receptors that guide recirculation from tissues to blood. Expression of the transcription factor c-MAF was selectively upregulated in IL-10+ TH17 cells, and it was bound to a large set of enhancer-like regions and modulated the immunoregulatory and tissue-residency program. Our results identify c-MAF as a relevant factor that drives two highly divergent post-activation fates of human TH17 cells and provide a framework with which to investigate the role of these cells in physiology and immunopathology.

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.

Clonal composition and persistence of antigen-specific circulating T follicular helper cells.

T follicular helper (TFH ) cells play an essential role in promoting B cell responses and antibody affinity maturation in germinal centers (GC). A subset of memory CD4+ T cells expressing the chemokine receptor CXCR5 has been described in human blood as phenotypically and clonally related to GC TFH cells. However, the antigen specificity and relationship of these circulating TFH (cTFH ) cells with other memory CD4+ T cells remain poorly defined. Combining antigenic stimulation and T cell receptor (TCR) Vß sequencing, we found T cells specific to tetanus toxoid (TT), influenza vaccine (Flu), or Candida albicans (C.alb) in both cTFH and non-cTFH subsets, although with different frequencies and effector functions. Interestingly, cTFH and non-cTFH cells specific for C.alb or TT had a largely overlapping TCR Vß repertoire while the repertoire of Flu-specific cTFH and non-cTFH cells was distinct. Furthermore, Flu-specific but not C.alb-specific PD-1+ cTFH cells had a "GC TFH -like" phenotype, with overexpression of IL21, CXCL13, and BCL6. Longitudinal analysis of serial blood donations showed that Flu-specific cTFH and non-cTFH cells persisted as stable repertoires for years. Collectively, our study provides insights on the relationship of cTFH with non-cTFH cells and on the heterogeneity and persistence of antigen-specific human cTFH cells.

Noncooperative interactions between transcription factors and clustered DNA binding sites enable graded transcriptional responses to environmental inputs.

A paradigm in transcriptional regulation is that graded increases in transcription factor (TF) concentration are translated into on/off transcriptional responses by cooperative TF binding to adjacent sites. Digital transcriptional responses underlie the definition of anatomical boundaries during development. Here we show that NF-kappaB, a TF controlling inflammation and immunity, is conversely an analog transcriptional regulator that uses clustered binding sites noncooperatively. We observed that increasing concentrations of NF-kappaB are translated into gradual increments in gene transcription. We provide a thermodynamic interpretation of the experimental observations by combining quantitative measurements and a minimal physical model of an NF-kappaB-dependent promoter. We demonstrate that NF-kappaB binds independently to adjacent sites to promote additive RNA Pol II recruitment and graded transcriptional outputs. These findings reveal an alternative mode of operation of clustered TF binding sites, which might function in biological conditions where the transcriptional output is proportional to the strength of an environmental input.

SARS-CoV-2: A Glance at the Innate Immune Response Elicited by Infection and Vaccination.

The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to almost seven million deaths worldwide. SARS-CoV-2 causes infection through respiratory transmission and can occur either without any symptoms or with clinical manifestations which can be mild, severe or, in some cases, even fatal. Innate immunity provides the initial defense against the virus by sensing pathogen-associated molecular patterns and triggering signaling pathways that activate the antiviral and inflammatory responses, which limit viral replication and help the identification and removal of infected cells. However, temporally dysregulated and excessive activation of the innate immune response is deleterious for the host and associates with severe COVID-19. In addition to its defensive role, innate immunity is pivotal in priming the adaptive immune response and polarizing its effector function. This capacity is relevant in the context of both SARS-CoV-2 natural infection and COVID-19 vaccination. Here, we provide an overview of the current knowledge of the innate immune responses to SARS-CoV-2 infection and vaccination.

Protocol for the detection of defined T cell clones in a heterogeneous cell population.

Identifying defined T cell clones within a polyclonal population is key to clarifying their phenotype and function. Here, we present a protocol for detecting specified T cell clones in a heterogeneous cell population. We describe steps for stimulating human CD4+ T cells isolated from blood with a protein antigen, sorting antigen-specific cells by fluorescence-activated cell sorting, and detecting among these the presence of predefined T cell clones, based on their T cell receptor (TCR). TCR cDNA is amplified through 5'-RACE (TCR-SMART) and detected by qPCR. For complete details on the use and execution of this protocol, please refer to Notarbartolo et al. (2021).1.

Jmjd3 contributes to the control of gene expression in LPS-activated macrophages.

Jmjd3, a JmjC family histone demethylase, is induced by the transcription factor NF-kB in response to microbial stimuli. Jmjd3 erases H3K27me3, a histone mark associated with transcriptional repression and involved in lineage determination. However, the specific contribution of Jmjd3 induction and H3K27me3 demethylation to inflammatory gene expression remains unknown. Using chromatin immunoprecipitation-sequencing we found that Jmjd3 is preferentially recruited to transcription start sites characterized by high levels of H3K4me3, a marker of gene activity, and RNA polymerase II (Pol_II). Moreover, 70% of lipopolysaccharide (LPS)-inducible genes were found to be Jmjd3 targets. Although most Jmjd3 target genes were unaffected by its deletion, a few hundred genes, including inducible inflammatory genes, showed moderately impaired Pol_II recruitment and transcription. Importantly, most Jmjd3 target genes were not associated with detectable levels of H3K27me3, and transcriptional effects of Jmjd3 absence in the window of time analysed were uncoupled from measurable effects on this histone mark. These data show that Jmjd3 fine-tunes the transcriptional output of LPS-activated macrophages in an H3K27 demethylation-independent manner.

Human T lymphocytes at tumor sites.

CD4+ and CD8+ T lymphocytes mediate most of the adaptive immune response against tumors. Naïve T lymphocytes specific for tumor antigens are primed in lymph nodes by dendritic cells. Upon activation, antigen-specific T cells proliferate and differentiate into effector cells that migrate out of peripheral blood into tumor sites in an attempt to eliminate cancer cells. After accomplishing their function, most effector T cells die in the tissue, while a small fraction of antigen-specific T cells persist as long-lived memory cells, circulating between peripheral blood and lymphoid tissues, to generate enhanced immune responses when re-encountering the same antigen. A subset of memory T cells, called resident memory T (TRM) cells, stably resides in non-lymphoid peripheral tissues and may provide rapid immunity independently of T cells recruited from blood. Being adapted to the tissue microenvironment, TRM cells are potentially endowed with the best features to protect against the reemergence of cancer cells. However, when tumors give clinical manifestation, it means that tumor cells have evaded immune surveillance, including that of TRM cells. Here, we review the current knowledge as to how TRM cells are generated during an immune response and then maintained in non-lymphoid tissues. We then focus on what is known about the role of CD4+ and CD8+ TRM cells in antitumor immunity and their possible contribution to the efficacy of immunotherapy. Finally, we highlight some open questions in the field and discuss how new technologies may help in addressing them.

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.

LINE1 are spliced in non-canonical transcript variants to regulate T cell quiescence and exhaustion.

How gene expression is controlled to preserve human T cell quiescence is poorly understood. Here we show that non-canonical splicing variants containing long interspersed nuclear element 1 (LINE1) enforce naive CD4+ T cell quiescence. LINE1-containing transcripts are derived from CD4+ T cell-specific genes upregulated during T cell activation. In naive CD4+ T cells, LINE1-containing transcripts are regulated by the transcription factor IRF4 and kept at chromatin by nucleolin; these transcripts act in cis, hampering levels of histone 3 (H3) lysine 36 trimethyl (H3K36me3) and stalling gene expression. T cell activation induces LINE1-containing transcript downregulation by the splicing suppressor PTBP1 and promotes expression of the corresponding protein-coding genes by the elongating factor GTF2F1 through mTORC1. Dysfunctional T cells, exhausted in vitro or tumor-infiltrating lymphocytes (TILs), accumulate LINE1-containing transcripts at chromatin. Remarkably, depletion of LINE1-containing transcripts restores TIL effector function. Our study identifies a role for LINE1 elements in maintaining T cell quiescence and suggests that an abundance of LINE1-containing transcripts is critical for T cell effector function and exhaustion.

High Th2 cytokine levels and upper airway inflammation in human inherited T-bet deficiency.

We have described a child suffering from Mendelian susceptibility to mycobacterial disease (MSMD) due to autosomal recessive, complete T-bet deficiency, which impairs IFN-γ production by innate and innate-like adaptive, but not mycobacterial-reactive purely adaptive, lymphocytes. Here, we explore the persistent upper airway inflammation (UAI) and blood eosinophilia of this patient. Unlike wild-type (WT) T-bet, the mutant form of T-bet from this patient did not inhibit the production of Th2 cytokines, including IL-4, IL-5, IL-9, and IL-13, when overexpressed in T helper 2 (Th2) cells. Moreover, Herpesvirus saimiri-immortalized T cells from the patient produced abnormally large amounts of Th2 cytokines, and the patient had markedly high plasma IL-5 and IL-13 concentrations. Finally, the patient's CD4+ αß T cells produced most of the Th2 cytokines in response to chronic stimulation, regardless of their antigen specificities, a phenotype reversed by the expression of WT T-bet. T-bet deficiency thus underlies the excessive production of Th2 cytokines, particularly IL-5 and IL-13, by CD4+ αß T cells, causing blood eosinophilia and UAI. The MSMD of this patient results from defective IFN-γ production by innate and innate-like adaptive lymphocytes, whereas the UAI and eosinophilia result from excessive Th2 cytokine production by adaptive CD4+ αß T lymphocytes.

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.

Transcriptional signature of human pro-inflammatory TH17 cells identifies reduced IL10 gene expression in multiple sclerosis.

We have previously reported the molecular signature of murine pathogenic TH17 cells that induce experimental autoimmune encephalomyelitis (EAE) in animals. Here we show that human peripheral blood IFN-γ+IL-17+ (TH1/17) and IFN-γ-IL-17+ (TH17) CD4+ T cells display distinct transcriptional profiles in high-throughput transcription analyses. Compared to TH17 cells, TH1/17 cells have gene signatures with marked similarity to mouse pathogenic TH17 cells. Assessing 15 representative signature genes in patients with multiple sclerosis, we find that TH1/17 cells have elevated expression of CXCR3 and reduced expression of IFNG, CCL3, CLL4, GZMB, and IL10 compared to healthy controls. Moreover, higher expression of IL10 in TH17 cells is found in clinically stable vs. active patients. Our results define the molecular signature of human pro-inflammatory TH17 cells, which can be used to both identify pathogenic TH17 cells and to measure the effect of treatment on TH17 cells in human autoimmune diseases.

The histone H3 lysine-27 demethylase Jmjd3 links inflammation to inhibition of polycomb-mediated gene silencing.

Epigenetic chromatin marks restrict the ability of differentiated cells to change gene expression programs in response to environmental cues and to transdifferentiate. Polycomb group (PcG) proteins mediate gene silencing and repress transdifferentiation in a manner dependent on histone H3 lysine 27 trimethylation (H3K27me3). However, macrophages migrated into inflamed tissues can transdifferentiate, but it is unknown whether inflammation alters PcG-dependent silencing. Here we show that the JmjC-domain protein Jmjd3 is a H3K27me demethylase expressed in macrophages in response to bacterial products and inflammatory cytokines. Jmjd3 binds PcG target genes and regulates their H3K27me3 levels and transcriptional activity. The discovery of an inducible enzyme that erases a histone mark controlling differentiation and cell identity provides a link between inflammation and reprogramming of the epigenome, which could be the basis for macrophage plasticity and might explain the differentiation abnormalities in chronic inflammation.