Signaling and Function of Interleukin-2 in T Lymphocytes.

The discovery of interleukin-2 (IL-2) changed the molecular understanding of how the immune system is controlled. IL-2 is a pleiotropic cytokine, and dissecting the signaling pathways that allow IL-2 to control the differentiation and homeostasis of both pro- and anti-inflammatory T cells is fundamental to determining the molecular details of immune regulation. The IL-2 receptor couples to JAK tyrosine kinases and activates the STAT5 transcription factors. However, IL-2 does much more than control transcriptional programs; it is a key regulator of T cell metabolic programs. The development of global phosphoproteomic approaches has expanded the understanding of IL-2 signaling further, revealing the diversity of phosphoproteins that may be influenced by IL-2 in T cells. However, it is increasingly clear that within each T cell subset, IL-2 will signal within a framework of other signal transduction networks that together will shape the transcriptional and metabolic programs that determine T cell fate.

Cellular spermine targets JAK signaling to restrain cytokine-mediated autoimmunity.

Prolonged activation of the type I interferon (IFN-I) pathway leads to autoimmune diseases such as systemic lupus erythematosus (SLE). Metabolic regulation of cytokine signaling is critical for cellular homeostasis. Through metabolomics analyses of IFN-ß-activated macrophages and an IFN-stimulated-response-element reporter screening, we identified spermine as a metabolite brake for Janus kinase (JAK) signaling. Spermine directly bound to the FERM and SH2 domains of JAK1 to impair JAK1-cytokine receptor interaction, thus broadly suppressing JAK1 phosphorylation triggered by cytokines IFN-I, IFN-II, interleukin (IL)-2, and IL-6. Peripheral blood mononuclear cells (PBMCs) from individuals with SLE showing decreased spermine concentrations exhibited enhanced IFN-I and lupus gene signatures. Spermine treatment attenuated autoimmune pathogenesis in SLE and psoriasis mice and reduced IFN-I signaling in monocytes from individuals with SLE. We synthesized a spermine derivative (spermine derivative 1 [SD1]) and showed that it had a potent immunosuppressive function. Our findings reveal spermine as a metabolic checkpoint for cellular homeostasis and a potential immunosuppressive molecule for controlling autoimmune disease.

Interleukin-2 signaling in the regulation of T cell biology in autoimmunity and cancer.

Interleukin-2 (IL-2) is a critical cytokine for T cell peripheral tolerance and immunity. Here, we review how IL-2 interaction with the high-affinity IL-2 receptor (IL-2R) supports the development and homeostasis of regulatory T cells and contributes to the differentiation of helper, cytotoxic, and memory T cells. A critical element for each T cell population is the expression of CD25 (Il2rα), which heightens the receptor affinity for IL-2. Signaling through the high-affinity IL-2R also reinvigorates CD8+ exhausted T (Tex) cells in response to checkpoint blockade. We consider the molecular underpinnings reflecting how IL-2R signaling impacts these various T cell subsets and the implications for enhancing IL-2-dependent immunotherapy of autoimmunity, other inflammatory disorders, and cancer.

Single-cell and spatial transcriptomics reveal aberrant lymphoid developmental programs driving granuloma formation.

Granulomas are lumps of immune cells that can form in various organs. Most granulomas appear unstructured, yet they have some resemblance to lymphoid organs. To better understand granuloma formation, we performed single-cell sequencing and spatial transcriptomics on granulomas from patients with sarcoidosis and bioinformatically reconstructed the underlying gene regulatory networks. We discovered an immune stimulatory environment in granulomas that repurposes transcriptional programs associated with lymphoid organ development. Granuloma formation followed characteristic spatial patterns and involved genes linked to immunometabolism, cytokine and chemokine signaling, and extracellular matrix remodeling. Three cell types emerged as key players in granuloma formation: metabolically reprogrammed macrophages, cytokine-producing Th17.1 cells, and fibroblasts with inflammatory and tissue-remodeling phenotypes. Pharmacological inhibition of one of the identified processes attenuated granuloma formation in a sarcoidosis mouse model. We show that human granulomas adopt characteristic aspects of normal lymphoid organ development in aberrant combinations, indicating that granulomas constitute aberrant lymphoid organs.

IFNγ-Dependent Tissue-Immune Homeostasis Is Co-opted in the Tumor Microenvironment.

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.

Ileitis-associated tertiary lymphoid organs arise at lymphatic valves and impede mesenteric lymph flow in response to tumor necrosis factor.

Lymphangitis and the formation of tertiary lymphoid organs (TLOs) in the mesentery are features of Crohn's disease. Here, we examined the genesis of these TLOs and their impact on disease progression. Whole-mount and intravital imaging of the ileum and ileum-draining collecting lymphatic vessels (CLVs) draining to mesenteric lymph nodes from TNFΔARE mice, a model of ileitis, revealed TLO formation at valves of CLVs. TLOs obstructed cellular and molecular outflow from the gut and were sites of lymph leakage and backflow. Tumor necrosis factor (TNF) neutralization begun at early stages of TLO formation restored lymph transport. However, robustly developed, chronic TLOs resisted regression and restoration of flow after TNF neutralization. TNF stimulation of cultured lymphatic endothelial cells reprogrammed responses to oscillatory shear stress, preventing the induction of valve-associated genes. Disrupted transport of immune cells, driven by loss of valve integrity and TLO formation, may contribute to the pathology of Crohn's disease.

Complex Autoinflammatory Syndrome Unveils Fundamental Principles of JAK1 Kinase Transcriptional and Biochemical Function.

Autoinflammatory disease can result from monogenic errors of immunity. We describe a patient with early-onset multi-organ immune dysregulation resulting from a mosaic, gain-of-function mutation (S703I) in JAK1, encoding a kinase essential for signaling downstream of >25 cytokines. By custom single-cell RNA sequencing, we examine mosaicism with single-cell resolution. We find that JAK1 transcription was predominantly restricted to a single allele across different cells, introducing the concept of a mutational "transcriptotype" that differs from the genotype. Functionally, the mutation increases JAK1 activity and transactivates partnering JAKs, independent of its catalytic domain. S703I JAK1 is not only hypermorphic for cytokine signaling but also neomorphic, as it enables signaling cascades not canonically mediated by JAK1. Given these results, the patient was treated with tofacitinib, a JAK inhibitor, leading to the rapid resolution of clinical disease. These findings offer a platform for personalized medicine with the concurrent discovery of fundamental biological principles.

The trajectory of human B-cell function, immune deficiency, and allergy revealed by inborn errors of immunity.

The essential role of B cells is to produce protective immunoglobulins (Ig) that recognize, neutralize, and clear invading pathogens. This results from the integration of signals provided by pathogens or vaccines and the stimulatory microenvironment within sites of immune activation, such as secondary lymphoid tissues, that drive mature B cells to differentiate into memory B cells and antibody (Ab)-secreting plasma cells. In this context, B cells undergo several molecular events including Ig class switching and somatic hypermutation that results in the production of high-affinity Ag-specific Abs of different classes, enabling effective pathogen neutralization and long-lived humoral immunity. However, perturbations to these key signaling pathways underpin immune dyscrasias including immune deficiency and autoimmunity or allergy. Inborn errors of immunity that disrupt critical immune pathways have identified non-redundant requirements for eliciting and maintaining humoral immune memory but concomitantly prevent immune dysregulation. Here, we will discuss our studies on human B cells, and how our investigation of cytokine signaling in B cells have identified fundamental requirements for memory B-cell formation, Ab production as well as regulating Ig class switching in the context of protective versus allergic immune responses.

Myc promotes polyploidy in murine trophoblast cells and suppresses senescence.

The placenta is essential for reproductive success. The murine placenta includes polyploid giant cells that are crucial for its function. Polyploidy occurs broadly in nature but its regulators and significance in the placenta are unknown. We have discovered that many murine placental cell types are polyploid and have identified factors that license polyploidy using single-cell RNA sequencing. Myc is a key regulator of polyploidy and placental development, and is required for multiple rounds of DNA replication, likely via endocycles, in trophoblast giant cells. Furthermore, MYC supports the expression of DNA replication and nucleotide biosynthesis genes along with ribosomal RNA. Increased DNA damage and senescence occur in trophoblast giant cells without Myc, accompanied by senescence in the neighboring maternal decidua. These data reveal Myc is essential for polyploidy to support normal placental development, thereby preventing premature senescence. Our study, combined with available literature, suggests that Myc is an evolutionarily conserved regulator of polyploidy.

Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion.

Gastro-intestinal helminth infections trigger the release of interleukin-33 (IL-33), which induces type-2 helper T cells (Th2 cells) at the site of infection to produce IL-13, thereby contributing to host resistance in a T cell receptor (TCR)-independent manner. Here, we show that, as a prerequisite for IL-33-induced IL-13 secretion, Th2 cells required the expression of the epidermal growth factor receptor (EGFR) and of its ligand, amphiregulin, for the formation of a signaling complex between T1/ST2 (the IL-33R) and EGFR. This shared signaling complex allowed IL-33 to induce the EGFR-mediated activation of the MAP-kinase signaling pathway and consequently the expression of IL-13. Lack of EGFR expression on T cells abrogated IL-13 expression in infected tissues and impaired host resistance. EGFR expression on Th2 cells was TCR-signaling dependent, and therefore, our data reveal a mechanism by which antigen presentation controls the innate effector function of Th2 cells at the site of inflammation.

The spread of interferon-γ in melanomas is highly spatially confined, driving nongenetic variability in tumor cells.

Interferon-γ (IFNγ) is a critical antitumor cytokine that has varied effects on different cell types. The global effect of IFNγ in the tumor depends on which cells it acts upon and the spatial extent of its spread. Reported measurements of IFNγ spread vary dramatically in different contexts, ranging from nearest-neighbor signaling to perfusion throughout the entire tumor. Here, we apply theoretical considerations to experiments both in vitro and in vivo to study the spread of IFNγ in melanomas. We observe spatially confined niches of IFNγ signaling in 3-D mouse melanoma cultures and human tumors that generate cellular heterogeneity in gene expression and alter the susceptibility of affected cells to T cell killing. Widespread IFNγ signaling only occurs when niches overlap due to high local densities of IFNγ-producing T cells. We measured length scales of ~30 to 40 µm for IFNγ spread in B16 mouse melanoma cultures and human primary cutaneous melanoma. Our results are consistent with IFNγ spread being governed by a simple diffusion-consumption model and offer insight into how the spatial organization of T cells contributes to intratumor heterogeneity in inflammatory signaling, gene expression, and immune-mediated clearance. Solid tumors are often viewed as collections of diverse cellular "neighborhoods": Our work provides a general explanation for such nongenetic cellular variability due to confinement in the spread of immune mediators.

Quorum sensing governs collective dendritic cell activation in vivo.

Dendritic cell (DC) activation by viral RNA sensors such as TLR3 and MDA-5 is critical for initiating antiviral immunity. Optimal DC activation is promoted by type I interferon (IFN) signaling which is believed to occur in either autocrine or paracrine fashion. Here, we show that neither autocrine nor paracrine type I IFN signaling can fully account for DC activation by poly(I:C) in vitro and in vivo. By controlling the density of type I IFN-producing cells in vivo, we establish that instead a quorum of type I IFN-producing cells is required for optimal DC activation and that this process proceeds at the level of an entire lymph node. This collective behavior, governed by type I IFN diffusion, is favored by the requirement for prolonged cytokine exposure to achieve DC activation. Furthermore, collective DC activation was found essential for the development of innate and adaptive immunity in lymph nodes. Our results establish how collective rather than cell-autonomous processes can govern the initiation of immune responses.

Altered skin microbiome, inflammation, and JAK/STAT signaling in Southeast Asian ichthyosis patients.

BACKGROUND: Congenital ichthyosis (CI) is a collective group of rare hereditary skin disorders. Patients present with epidermal scaling, fissuring, chronic inflammation, and increased susceptibility to infections. Recently, there is increased interest in the skin microbiome; therefore, we hypothesized that CI patients likely exhibit an abnormal profile of epidermal microbes because of their various underlying skin barrier defects. Among recruited individuals of Southeast Asian ethnicity, we performed skin meta-genomics (i.e., whole-exome sequencing to capture the entire multi-kingdom profile, including fungi, protists, archaea, bacteria, and viruses), comparing 36 CI patients (representing seven subtypes) with that of 15 CI age-and gender-matched controls who had no family history of CI. RESULTS: This case-control study revealed 20 novel and 31 recurrent pathogenic variants. Microbiome meta-analysis showed distinct microbial populations, decreases in commensal microbiota, and higher colonization by pathogenic species associated with CI; these were correlated with increased production of inflammatory cytokines and Th17- and JAK/STAT-signaling pathways in peripheral blood mononuclear cells. In the wounds of CI patients, we identified specific changes in microbiota and alterations in inflammatory pathways, which are likely responsible for impaired wound healing. CONCLUSIONS: Together, this research enhances our understanding of the microbiological, immunological, and molecular properties of CI and should provide critical information for improving therapeutic management of CI patients.

Metabolic Reprogramming Mediated by the mTORC2-IRF4 Signaling Axis Is Essential for Macrophage Alternative Activation.

Macrophage activation status is intrinsically linked to metabolic remodeling. Macrophages stimulated by interleukin 4 (IL-4) to become alternatively (or, M2) activated increase fatty acid oxidation and oxidative phosphorylation; these metabolic changes are critical for M2 activation. Enhanced glucose utilization is also characteristic of the M2 metabolic signature. Here, we found that increased glucose utilization is essential for M2 activation. Increased glucose metabolism in IL-4-stimulated macrophages required the activation of the mTORC2 pathway, and loss of mTORC2 in macrophages suppressed tumor growth and decreased immunity to a parasitic nematode. Macrophage colony stimulating factor (M-CSF) was implicated as a contributing upstream activator of mTORC2 in a pathway that involved PI3K and AKT. mTORC2 operated in parallel with the IL-4Rα-Stat6 pathway to facilitate increased glycolysis during M2 activation via the induction of the transcription factor IRF4. IRF4 expression required both mTORC2 and Stat6 pathways, providing an underlying mechanism to explain how glucose utilization is increased to support M2 activation.

Catch and Release of Cytokines Mediated by Tumor Phosphatidylserine Converts Transient Exposure into Long-Lived Inflammation.

Immune cells constantly survey the host for pathogens or tumors and secrete cytokines to alert surrounding cells of these threats. In vivo, activated immune cells secrete cytokines for several hours, yet an acute immune reaction occurs over days. Given these divergent timescales, we addressed how cytokine-responsive cells translate brief cytokine exposure into phenotypic changes that persist over long timescales. We studied melanoma cell responses to transient exposure to the cytokine interferon γ (IFNγ) by combining a systems-scale analysis of gene expression dynamics with computational modeling and experiments. We discovered that IFNγ is captured by phosphatidylserine (PS) on the surface of viable cells both in vitro and in vivo then slowly released to drive long-term transcription of cytokine-response genes. This mechanism introduces an additional function for PS in dynamically regulating inflammation across diverse cancer and primary cell types and has potential to usher in new immunotherapies targeting PS and inflammatory pathways.

Clara cell 10 (CC10) protein attenuates allergic airway inflammation by modulating lung dendritic cell functions.

Allergic asthma is a complex inflammatory disorder predominantly orchestrated by T helper 2 (Th2) lymphocytes. The anti-inflammatory protein Clara Cell 10-kDa (CC10), also known as secretoglobin family 1A member 1 (SCGB1A1), shows promise in modulating respiratory diseases. However, its precise role in asthma remains unclear. This study examines the potential of CC10 to suppress allergic asthma inflammation, specifically assessing its regulatory effects on Th2 cell responses and dendritic cells (DCs). Lower CC10 levels in asthma were observed and correlated with increased IgE and lymphocytes. Cc10-/- mice exhibited exacerbated allergic airway inflammation marked by increased inflammatory cell infiltration, Th2 cytokines, serum antigen-specific IgE levels, and airway hyperresponsiveness (AHR) in house dust mite (HDM)-induced models. Conversely, recombinant CC10 significantly attenuated these inflammatory responses. Intriguingly, CC10 did not directly inhibit Th cell activation but significantly downregulated the population of CD11b+CD103- DCs subsets in lungs of asthmatic mice and modulated the immune activation functions of DCs through NF-κB signaling pathway. The mixed lymphocyte response assay revealed that DCs mediated the suppressive effect of CC10 on Th2 cell responses. Collectively, CC10 profoundly mitigates Th2-type allergic inflammation in asthma by modulating lung DC phenotype and functions, highlighting its therapeutic potential for inflammatory airway conditions and other related immunological disorders.

Established and emergent roles for Ikaros transcription factors in lymphoid cell development and function.

Ikaros zinc finger transcription factors are important regulators of the gene programs underlying the development of hematopoietic cell lineages. The family consists of five members: Ikaros, Helios, Aiolos, Eos, and Pegasus, which engage in both homo- and heterotypic intrafamilial interactions to exert diverse functional effects. Pioneering studies focused on the role of these factors in early lymphoid development, as their absence resulted in severe defects in lymphocyte populations. More recent work has now begun to define nuanced, stage-specific roles for Ikaros family members in the differentiation and function of mature T, B, and innate lymphoid cell populations including natural killer (NK) cells. The precise transcriptional mechanisms by which these factors function, both independently and collaboratively, is an area of active investigation. However, several key themes appear to be emerging regarding the pathways influenced by Ikaros family members, including the end-to-end regulation of cytokine signaling. Here, we review roles for Ikaros factors in lymphoid cell development, differentiation, and function, including a discussion of the current understanding of the transcriptional mechanisms they employ and considerations for the future study of this important transcription factor family.

Stromal STAT5-Mediated Trophic Activity Regulates Hematopoietic Niche Factors.

Signal transducer and activator of transcription 5 (STAT5a and STAT5b) are intrinsically critical for normal hematopoiesis but are also expressed in stromal cells. Here, STAT5ab knockout (KO) was generated with a variety of bone marrow hematopoietic and stromal Cre transgenic mouse strains. Vav1-Cre/+STAT5abfl/fl, the positive control for loss of multipotent hematopoietic function, surprisingly dysregulated niche factor mRNA expression, and deleted STAT5ab in CD45neg cells. Single-cell transcriptome analysis of bone marrow from Vav1-Cre/+ wild-type or Vav1-Cre/+STAT5abfl/fl mice showed hematopoietic stem cell (HSC) myeloid commitment priming. Nes+ cells were detected in both CD45neg and CD45+ clusters and deletion of STAT5ab with Nes-Cre caused hematopoietic repopulating defects. To follow up on these promiscuous Cre promoter deletions in CD45neg and CD45+ bone marrow cell populations, more stroma-specific Cre strains were generated and demonstrated a reduction in multipotent hematopoietic progenitors. Functional support for niche-supporting activity was assessed using STAT5-deficient mesenchymal stem cells (MSCs). With Lepr-Cre/+STAT5abfl/fl, niche factor mRNAs were downregulated with validation of reduced IGF-1 and CXCL12 proteins. Furthermore, advanced computational analyses revealed a key role for STAT5ab/Cish balance with Cish strongly co-expressed in MSCs and HSCs primed for differentiation. Therefore, STAT5ab-associated gene regulation supports the bone marrow microenvironment.

Cloning of suppressor of cytokine signaling 7 from silkworm (Bombyx mori) and its response to the infection of Bombyx mori nucleopolyhedrovirus.

Suppressors of cytokine signaling (SOCS) play important roles in the regulation of growth, development, and immunity of eukaryotic organisms. SOCS7 is an important member of the SOCS family, but its physiological and pathological functions remain largely unknown in invertebrates including insects. Here, we first report the cloning of a SOCS7 gene from a domesticated silkworm (Bombyx mori), named BmSOCS7. We have characterized BmSOCS7 expression profiles in silkworm varieties susceptible or resistant to the infection of Bombyx mori nucleopolyhedrovirus (BmNPV) using the real-time fluorescence quantitative PCR. BmSOCS7 expresses highly in embryogenesis and lowly in metamorphosis in resistant silkworms but does in opposite contrast in susceptible silkworms. Its expression is at very low level in the fat body of resistant silkworms but is relatively high in the fat body of susceptible ones. BmNPV inoculation induces a transient downregulation and then a general upregulation of BmSOCS7 expression in BmN cells, while it induces a general downregulation in silkworm midgut, fat body and hemolymph with more pronounced effect in resistant silkworms than susceptible ones and more prominent in the fat body and hemolymph than the midgut. Together, our work reveals that downregulation of BmSOCS7 expression may be an important strategy for silkworm anti-BmNPV immune response, and BmSOCS7 may mainly function in the fat body and hemolymph rather than the midgut to participate in BmNPV infection process.

Efficient T cell adoptive transfer in lymphoreplete hosts mediated by transient activation of Stat5 signaling.

Lymphodepleting pre-conditioning is a nearly universal component of T cell adoptive transfer protocols. The side effects of pre-conditioning regimens used in adoptive cell therapy are clinically significant and include pan-cytopenia, immune suppression, and reactive myelopoiesis. We conducted studies to test the hypothesis that the mechanisms underlying effective engraftment are cell autonomous and not dependent on a lymphodepleted host immune status. These studies leveraged mouse models to examine the role of Stat5 signaling during T cell adoptive transfer. We observed that, by transiently expressing a constitutively active mutamer of Stat5b during the process of adoptive transfer, we could completely obviate the need for lymphodepletion prior to adoptive transfer. Using several functional assays, we benchmark the function of the engrafted T cells against T cells transferred after conventional lymphodepletion. These studies identify a cell-autonomous mechanism driven by transient Stat5b signaling with lasting effects on T cell phenotype and function. Furthermore, the results presented suggest that adoptive T cell therapy could be improved by removing lymphodepletion protocols entirely and replacing them with RNA transfection of T cells with transcripts encoding active Stat5.