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.

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.

In response to pathogenic threats, naïve T cells rapidly transition from a quiescent to 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 naïve and activated T cells. Our datasets uncovered that transcription factors maintaining T cell quiescence had constitutively high turnover, which facilitated their depletion upon activation. Furthermore, naïve 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 ( www.immunomics.ch ).

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.

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

Different types of effector and memory T lymphocytes are induced and maintained in protective or pathological immune responses. Here we characterized two human CD4 T17 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 T17 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 T17 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 T17 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 T17 cells and provide a framework with which to investigate the role of these cells in physiology and immunopathology.

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.

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.