Threonine phosphorylation of STAT1 restricts interferon signaling and promotes innate inflammatory responses.

Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically engineered mice expressing phospho-deficient threonine748-to-alanine (T748A) mutant Stat1 are resistant to LPS-induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase-mediated Thr748 phosphorylation of Stat1, which promotes macrophage inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent modular functionality of Stat1 in innate immune responses: IFN phospho-tyrosine dependent and inflammatory phospho-threonine dependent. Better understanding of the Thr748 phosphorylation of Stat1 may uncover advanced pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually.

A sex-biased imbalance between Tfr, Tph, and atypical B cells determines antibody responses in COVID-19 patients.

Sex-biased humoral immune responses to COVID-19 patients have been observed, but the cellular basis for this is not understood. Using single-cell proteomics by mass cytometry, we find disrupted regulation of humoral immunity in COVID-19 patients, with a sex-biased loss of circulating follicular regulatory T cells (cTfr) at a significantly greater rate in male patients. In addition, a male sex-associated cellular network of T-peripheral helper, plasma blasts, proliferating and extrafollicular/atypical CD11c+ memory B cells was strongly positively correlated with neutralizing antibody concentrations and negatively correlated with cTfr frequency. These results suggest that sex-specific differences to the balance of cTfr and a network of extrafollicular antibody production-associated cell types may be a key factor in the altered humoral immune responses between male and female COVID-19 patients.

Identification of a unique subset of tissue-resident memory CD4+ T cells in Crohn's disease.

T cells differentiate into highly diverse subsets and display plasticity depending on the environment. Although lymphocytes are key mediators of inflammation, functional specialization of T cells in inflammatory bowel disease (IBD) has not been effectively described. Here, we performed deep profiling of T cells in the intestinal mucosa of IBD and identified a CD4+ tissue-resident memory T cell (Trm) subset that is increased in Crohn's disease (CD) showing unique inflammatory properties. Functionally and transcriptionally distinct CD4+ Trm subsets are observed in the inflamed gut mucosa, among which a CD-specific CD4+ Trm subset, expressing CD161 and CCR5 along with CD103, displays previously unrecognized pleiotropic signatures of innate and effector activities. These inflammatory features are further enhanced by their spatial proximity to gut epithelial cells. Furthermore, the CD-specific CD4+ Trm subset is the most predominant producer of type 1 inflammatory cytokines upon various stimulations among all CD4+ T cells, suggesting that the accumulation of this T cell subset is a pathological hallmark of CD. Our results provide comprehensive insights into the pathogenesis of IBD, paving the way for decoding of the molecular mechanisms underlying this disease.

Treg-expressed CTLA-4 depletes CD80/CD86 by trogocytosis, releasing free PD-L1 on antigen-presenting cells.

Foxp3-expressing CD4+CD25+ regulatory T cells (Tregs) constitutively and highly express the immune checkpoint receptor cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), whose Treg-specific deficiency causes severe systemic autoimmunity. As a key mechanism of Treg-mediated suppression, Treg-expressed CTLA-4 down-regulates the expression of CD80/CD86 costimulatory molecules on antigen-presenting cells (APCs). Here, we show that Treg-expressed CTLA-4 facilitated Treg-APC conjugation and immune synapse formation. The immune synapses thus formed provided a stable platform whereby Tregs were able to deplete CD80/CD86 molecules on APCs by extracting them via CTLA-4-dependent trogocytosis. The depletion occurred even with Tregs solely expressing a mutant CTLA-4 form lacking the cytoplasmic portion required for its endocytosis. The CTLA-4-dependent trogocytosis of CD80/CD86 also accelerated in vitro and in vivo passive transfer of other membrane proteins and lipid molecules from APCs to Tregs without their significant reduction on the APC surface. Furthermore, CD80 down-regulation or blockade by Treg-expressed membrane CTLA-4 or soluble CTLA-4-immunoglobulin (CTLA-4-Ig), respectively, disrupted cis-CD80/programmed death ligand-1 (PD-L1) heterodimers and increased free PD-L1 on dendritic cells (DCs), expanding a phenotypically distinct population of CD80lo free PD-L1hi DCs. Thus, Tregs are able to inhibit the T cell stimulatory activity of APCs by reducing their CD80/CD86 expression via CTLA-4-dependent trogocytosis. This CD80/CD86 reduction on APCs is able to exert dual suppressive effects on T cell immune responses by limiting CD80/CD86 costimulation to naïve T cells and by increasing free PD-L1 available for the inhibition of programmed death-1 (PD-1)-expressing effector T cells. Blockade of CTLA-4 and PD-1/PD-L1 in combination may therefore synergistically hinder Treg-mediated immune suppression, thereby effectively enhancing immune responses, including tumor immunity.

Mature dendritic cells enriched in regulatory molecules may control regulatory T cells and the prognosis of head and neck cancer.

We previously reported that regulatory T (Treg) cells expressing CTLA-4 on the cell surface are abundant in head and neck squamous cell carcinoma (HNSCC). The role of expanded Treg cells in the tumor microenvironment of HNSCC remains unclear. In this study, we reveal that the tumor microenvironment of HNSCC is characterized by the high expression of genes related to Treg cells, dendritic cells (DCs), and interleukin (IL)-17-related molecules. Increased expression of IL17A, IL17F, or IL23A contributes to a favorable prognosis of HNSCC. In the tumor microenvironment of HNSCC, IL23A and IL12B are expressed in mature dendritic cells enriched in regulatory molecules (mregDCs). The mregDCs in HNSCC are a migratory and mature phenotype; their signature genes strongly correlate with Treg signature genes in HNSCC. We also observed that IL17A was highly expressed in Th17 cells and exhausted CD8+ T cells in HNSCC. These data suggest that mregDCs in HNSCC may contribute to the prognosis by balancing Treg cells and effector T cells that produce IL-17. Targeting mregDCs may be a novel strategy for developing new immune therapies against HNSCC.

Regulatory T cells control antigen-specific expansion of Tfh cell number and humoral immune responses via the coreceptor CTLA-4.

CD4(+)Foxp3-expressing Treg cells, which constitutively express the inhibitory coreceptor CTLA-4, are indispensable for immune homeostasis. We determined the roles of Treg cells and T follicular regulatory (Tfr) cells in the control of humoral immune responses. Depletion of Treg cells, blocking of CTLA-4 or a Treg cell specific reduction in CTLA-4 expression, resulted in an increase in the formation of antigen-specific Tfh cells, germinal center (GC), and plasma and memory B cells after vaccination. In the absence of Treg cell-expressed CTLA-4, large numbers of Tfr cells were present but were unable to restrain Tfh cell and GC formation. Temporary Treg cell depletion during primary immunization was sufficient to enhance secondary immune responses. Treg cells directly inhibited, via CTLA-4, B cell expression of CD80 and CD86, which was essential for Tfh cell formation. Thus, Treg and Tfr cells control Tfh cell and germinal center development, via CTLA-4-dependent regulation of CD80 and CD86 expression.

Alteration of the immune environment in bone marrow from children with recurrent B cell precursor acute lymphoblastic leukemia.

Due to the considerable success of cancer immunotherapy for leukemia, the tumor immune environment has become a focus of intense research; however, there are few reports on the dynamics of the tumor immune environment in leukemia. Here, we analyzed the tumor immune environment in pediatric B cell precursor acute lymphoblastic leukemia by analyzing serial bone marrow samples from nine patients with primary and recurrent disease by mass cytometry using 39 immunophenotype markers, and transcriptome analysis. High-dimensional single-cell mass cytometry analysis elucidated a dynamic shift of T cells from naïve to effector subsets, and clarified that, during relapse, the tumor immune environment comprised a T helper 1-polarized immune profile, together with an increased number of effector regulatory T cells. These results were confirmed in a validation cohort using conventional flow cytometry. Furthermore, RNA transcriptome analysis identified the upregulation of immune-related pathways in B cell precursor acute lymphoblastic leukemia cells during relapse, suggesting interaction with the surrounding environment. In conclusion, a tumor immune environment characterized by a T helper 1-polarized immune profile, with an increased number of effector regulatory T cells, could contribute to the pathophysiology of recurrent B cell precursor acute lymphoblastic leukemia. This information could contribute to the development of effective immunotherapeutic approaches against B cell precursor acute lymphoblastic leukemia relapse.

Differential control of human Treg and effector T cells in tumor immunity by Fc-engineered anti-CTLA-4 antibody.

Anti-CTLA-4 mAb is efficacious in enhancing tumor immunity in humans. CTLA-4 is expressed by conventional T cells upon activation and by naturally occurring FOXP3+CD4+ Treg cells constitutively, raising a question of how anti-CTLA-4 mAb can differentially control these functionally opposing T cell populations in tumor immunity. Here we show that FOXP3high potently suppressive effector Treg cells were abundant in melanoma tissues, expressing CTLA-4 at higher levels than tumor-infiltrating CD8+ T cells. Upon in vitro tumor-antigen stimulation of peripheral blood mononuclear cells from healthy individuals or melanoma patients, Fc-region-modified anti-CTLA-4 mAb with high antibody-dependent cell-mediated cytotoxicity (ADCC) and cellular phagocytosis (ADCP) activity selectively depleted CTLA-4+FOXP3+ Treg cells and consequently expanded tumor-antigen-specific CD8+T cells. Importantly, the expansion occurred only when antigen stimulation was delayed several days from the antibody treatment to spare CTLA-4+ activated effector CD8+T cells from mAb-mediated killing. Similarly, in tumor-bearing mice, high-ADCC/ADCP anti-CTLA-4 mAb treatment with delayed tumor-antigen vaccination significantly prolonged their survival and markedly elevated cytokine production by tumor-infiltrating CD8+ T cells, whereas antibody treatment concurrent with vaccination did not. Anti-CTLA-4 mAb modified to exhibit a lesser or no Fc-binding activity failed to show such timing-dependent in vitro and in vivo immune enhancement. Thus, high ADCC anti-CTLA-4 mAb is able to selectively deplete effector Treg cells and evoke tumor immunity depending on the CTLA-4-expressing status of effector CD8+ T cells. These findings are instrumental in designing cancer immunotherapy with mAbs targeting the molecules commonly expressed by FOXP3+ Treg cells and tumor-reactive effector T cells.

A distinct subpopulation of CD25- T-follicular regulatory cells localizes in the germinal centers.

T-follicular helper (Tfh) cells differentiate through a multistep process, culminating in germinal center (GC) localized GC-Tfh cells that provide support to GC-B cells. T-follicular regulatory (Tfr) cells have critical roles in the control of Tfh cells and GC formation. Although Tfh-cell differentiation is inhibited by IL-2, regulatory T (Treg) cell differentiation and survival depend on it. Here, we describe a CD25- subpopulation within both murine and human PD1+CXCR5+Foxp3+ Tfr cells. It is preferentially located in the GC and can be clearly differentiated from CD25+ non-GC-Tfr, Tfh, and effector Treg (eTreg) cells by the expression of a wide range of molecules. In comparison to CD25+ Tfr and eTreg cells, CD25- Tfr cells partially down-regulate IL-2-dependent canonical Treg features, but retain suppressive function, while simultaneously up-regulating genes associated with Tfh and GC-Tfh cells. We suggest that, similar to Tfh cells, Tfr cells follow a differentiation pathway generating a mature GC-localized subpopulation, CD25- Tfr cells.

Control of Regulatory T Cells by Co-signal Molecules.

Foxp3-expressing regulatory T cells (Tregs) perform a vital function in the maintenance of immune homeostasis. A large part of Treg suppressive function is derived from their ability to control and restrict the availability of co-signal molecules to other T cells. However, Tregs themselves also depend on many of the same co-signals for their own homeostasis, making this a complex system of feedback. In this chapter, we discuss the critical role of co-signaling in Treg cell biology.

COVID-19 relapse associated with SARS-CoV-2 evasion from CD4+ T-cell recognition in an agammaglobulinemia patient.

A 25-year-old patient with a primary immunodeficiency lacking immunoglobulin production experienced a relapse after a 239-day period of persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Viral genetic sequencing demonstrated that SARS-CoV-2 had evolved during the infection period, with at least five mutations associated with host cellular immune recognition. Among them, the T32I mutation in ORF3a was found to evade recognition by CD4+ T cells. The virus found after relapse showed an increased proliferative capacity in vitro. SARS-CoV-2 may have evolved to evade recognition by CD4+ T cells and increased in its proliferative capacity during the persistent infection, likely leading to relapse. These mutations may further affect viral clearance in hosts with similar types of human leukocyte antigens. The early elimination of SARS-CoV-2 in immunocompromised patients is therefore important not only to improve the condition of patients but also to prevent the emergence of mutants that threaten public health.

Scalable, multimodal profiling of chromatin accessibility, gene expression and protein levels in single cells.

Recent technological advances have enabled massively parallel chromatin profiling with scATAC-seq (single-cell assay for transposase accessible chromatin by sequencing). Here we present ATAC with select antigen profiling by sequencing (ASAP-seq), a tool to simultaneously profile accessible chromatin and protein levels. Our approach pairs sparse scATAC-seq data with robust detection of hundreds of cell surface and intracellular protein markers and optional capture of mitochondrial DNA for clonal tracking, capturing three distinct modalities in single cells. ASAP-seq uses a bridging approach that repurposes antibody:oligonucleotide conjugates designed for existing technologies that pair protein measurements with single-cell RNA sequencing. Together with DOGMA-seq, an adaptation of CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) for measuring gene activity across the central dogma of gene regulation, we demonstrate the utility of systematic multi-omic profiling by revealing coordinated and distinct changes in chromatin, RNA and surface proteins during native hematopoietic differentiation and peripheral blood mononuclear cell stimulation and as a combinatorial decoder and reporter of multiplexed perturbations in primary T cells.

The Proportion of Regulatory T Cells in Patients with Rheumatoid Arthritis: A Meta-Analysis.

BACKGROUND: Regulatory T cells (Tregs) have important functions in peripheral immune tolerance. Dysfunction of Tregs is considered to be a pivotal cause of autoimmune diseases, including rheumatoid arthritis (RA). However, previous reports describing the proportion of Tregs among CD4+ T cells in RA patients were controversial because a range of markers are used to identify Tregs with little consensus. To clarify the status of Tregs in RA, we investigated the proportion of Tregs with focusing on the definitions of them. METHODS: We identified the studies reporting the proportion of Tregs in RA patients using PubMed and Google Scholar. We performed a systematic review of them and a meta-analysis to evaluate the proportion of Tregs (FOXP3-positive and/or CD25-positive) among CD4+ T cells in peripheral blood (PB) and synovial fluid (SF) of RA patients and control subjects. RESULTS: A total 31 studies were selected. The proportion of Tregs defined by all definitions among CD4+ T cells in PB was not significantly different between RA patients and control subjects (-0.65, [-1.30, 0.01]). Then we performed sub-analyses based on individual definitions. The proportion of Tregs defined by either CD25 or FOXP3 alone did not differ between RA patients and control subjects. The proportion of Tregs defined by both FOXP3 and CD25 was lower in RA patients than that in control subjects (-2.42 [-3.49, -1.34]). The proportion of Tregs defined by both FOXP3 and CD25 was higher in SF than that in PB among RA patients (3.27 [0.40, 6.14]). CONCLUSION: The status of Tregs varied according to the definition system. The proportion of Tregs defined by stricter and functionally validated methods decreased in PB and increased in SF among RA patients. If the proportion of Tregs differs in RA, accurate and functionally relevant definitions of Tregs are necessary to elucidate their status in RA.

In vivo induction of T-follicular helper cells by modulation of regulatory T cell function.

Regulatory T cells, particularly their follicular resident subset, T-follicular regulatory cells, play a critical role in the maintenance of immune homeostasis and prevention of the expansion of T-follicular helper cells. One of the key mechanisms used by these cells is CTLA-4 dependent suppression of inflammation. In this chapter, we detail methods to increase the formation of T-follicular helper cells following vaccination by either depleting Treg cells, via Foxp3 linked diphtheria toxin receptor, or blocking CTLA-4 function by the use of antibodies.

Transcriptional and Epigenetic Control of Regulatory T Cell Development.

The control of immune responses against self and nonharmful environmental antigens is of critical importance to the immune homeostasis. Regulatory T (Treg) cells are the key players of such immune regulation and their deficiency and dysfunction are associated with various immune disorders, such as autoimmunity and allergy. It is therefore essential to understand the molecular mechanisms that make up Treg cell characteristics; that is, how their unique gene expression profile is regulated at transcriptional and epigenetic levels. In this chapter, we focus on the components of molecular features of Treg cells and discuss how they are introduced during their development.