OTULIN haploinsufficiency predisposes to environmentally directed inflammation.

Recently, OTULIN haploinsufficiency was linked to enhanced susceptibility to Staphylococcus aureus infections accompanied by local necrosis and systemic inflammation. The pathogenesis observed in haploinsufficient patients differs from the hyperinflammation seen in classical OTULIN-related autoinflammatory syndrome (ORAS) patients and is characterized by increased susceptibility of dermal fibroblasts to S. aureus alpha toxin-inflicted cytotoxic damage. Immunological abnormalities were not observed in OTULIN haploinsufficient patients, suggesting a non-hematopoietic basis. In this research report, we investigated an Otulin+/- mouse model after in vivo provocation with lipopolysaccharide (LPS) to explore the potential role of hematopoietic-driven inflammation in OTULIN haploinsufficiency. We observed a hyperinflammatory signature in LPS-provoked Otulin+/- mice, which was driven by CD64+ monocytes and macrophages. Bone marrow-derived macrophages (BMDMs) of Otulin+/- mice demonstrated higher proinflammatory cytokine secretion after in vitro stimulation with LPS or polyinosinic:polycytidylic acid (Poly(I:C)). Our experiments in full and mixed bone marrow chimeric mice suggest that, in contrast to humans, the observed inflammation was mainly driven by the hematopoietic compartment with cell-extrinsic effects likely contributing to inflammatory outcomes. Using an OTULIN haploinsufficient mouse model, we validated the role of OTULIN in the regulation of environmentally directed inflammation.

Lymph node and tumor-associated PD-L1+ macrophages antagonize dendritic cell vaccines by suppressing CD8+ T cells.

Current immunotherapies provide limited benefits against T cell-depleted tumors, calling for therapeutic innovation. Using multi-omics integration of cancer patient data, we predict a type I interferon (IFN) responseHIGH state of dendritic cell (DC) vaccines, with efficacious clinical impact. However, preclinical DC vaccines recapitulating this state by combining immunogenic cancer cell death with induction of type I IFN responses fail to regress mouse tumors lacking T cell infiltrates. Here, in lymph nodes (LNs), instead of activating CD4+/CD8+ T cells, DCs stimulate immunosuppressive programmed death-ligand 1-positive (PD-L1+) LN-associated macrophages (LAMs). Moreover, DC vaccines also stimulate PD-L1+ tumor-associated macrophages (TAMs). This creates two anatomically distinct niches of PD-L1+ macrophages that suppress CD8+ T cells. Accordingly, a combination of PD-L1 blockade with DC vaccines achieves significant tumor regression by depleting PD-L1+ macrophages, suppressing myeloid inflammation, and de-inhibiting effector/stem-like memory T cells. Importantly, clinical DC vaccines also potentiate T cell-suppressive PD-L1+ TAMs in glioblastoma patients. We propose that a multimodal immunotherapy and vaccination regimen is mandatory to overcome T cell-depleted tumors.

Development of a cellular model to study CCR8 signaling in tumor-infiltrating regulatory T cells.

The human CC chemokine receptor 8 (CCR8) is specifically expressed on tumor-infiltrating regulatory T cells (TITRs) and is a promising drug target for cancer immunotherapy. However, the role of CCR8 signaling in TITR biology and the effectiveness of CCR8 small molecule antagonists as TITR-targeting immunotherapy remain subjects of ongoing debate. In this work, we generated a novel cellular model of TITRs by culturing peripheral blood mononuclear cell-derived regulatory T cells in medium containing tumor cell-conditioned medium, CD3/CD28 activator, interleukin-2 and 1α,25-dihydroxyvitamin D3. This cellular model (named TITR mimics) highly and stably expressed a series of TITR signature molecules, including CCR8, FOXP3, CD30, CD39, CD134, CD137, TIGIT and Tim-3. Moreover, TITR mimics displayed robust in vitro immunosuppressive activity. To unravel the functional role of CCR8 in TITR mimics, a chemotaxis assay was performed showing strong and CCR8-specific migration toward CCL1, the natural chemokine agonist of CCR8. However, either stimulation (with CCL1) or blocking (with the small molecule antagonist NS-15) of CCR8 signaling did not affect the immunosuppressive activity, proliferation and survival of TITR mimics. Collectively, our work provides a method for the generation of TITR mimics in vitro, which can be used to study TITR biology and to evaluate drug candidates targeting TITRs. Furthermore, our findings suggest that CCR8 signaling primarily regulates migration of these cells.

A Metabolic Gene Survey Pinpoints Fucosylation as a Key Pathway Underlying the Suppressive Function of Regulatory T Cells in Cancer.

Forkhead box P3 (Foxp3)-expressing regulatory T cells (Treg) are the guardians of controlled immune reactions and prevent the development of autoimmune diseases. However, in the tumor context, their increased number suppresses antitumor immune responses, indicating the importance of understanding the mechanisms behind their function and stability. Metabolic reprogramming can affect Foxp3 regulation and, therefore, Treg suppressive function and fitness. Here, we performed a metabolic CRISPR/Cas9 screen and pinpointed novel candidate positive and negative metabolic regulators of Foxp3. Among the positive regulators, we revealed that targeting the GDP-fucose transporter Slc35c1, and more broadly fucosylation (Fuco), in Tregs compromises their proliferation and suppressive function both in vitro and in vivo, leading to alteration of the tumor microenvironment and impaired tumor progression and protumoral immune responses. Pharmacologic inhibition of Fuco dampened tumor immunosuppression mostly by targeting Tregs, thus resulting in reduced tumor growth. In order to substantiate these findings in humans, tumoral Tregs from patients with colorectal cancer were clustered on the basis of the expression of Fuco-related genes. FucoLOW Tregs were found to exhibit a more immunogenic profile compared with FucoHIGH Tregs. Furthermore, an enrichment of a FucoLOW signature, mainly derived from Tregs, correlated with better prognosis and response to immune checkpoint blockade in melanoma patients. In conclusion, Slc35c1-dependent Fuco is able to regulate the suppressive function of Tregs, and measuring its expression in Tregs might pave the way towards a useful biomarker model for patients with cancer. See related Spotlight by Silveria and DuPage, p. 1570.

"IL-2 immunotherapy for targeting regulatory T cells in autoimmunity".

FOXP3+ regulatory T cells (Treg) are indispensable for immune homoeostasis and for the prevention of autoimmune diseases. Interleukin-2 (IL-2) signalling is critical in all aspects of Treg biology. Consequences of defective IL-2 signalling are insufficient numbers or dysfunction of Treg and hence autoimmune disorders in human and mouse. The restoration and maintenance of immune homoeostasis remain central therapeutic aims in the field of autoimmunity. Historically, broadly immunosuppressive drugs with serious side-effects have been used for the treatment of autoimmune diseases or prevention of organ-transplant rejection. More recently, ex vivo expanded or in vivo stimulated Treg have been shown to induce effective tolerance in clinical trials supporting the clinical benefit of targeting natural immunosuppressive mechanisms. Given the central role of exogenous IL-2 in Treg homoeostasis, a new and promising focus in drug development are IL-2-based approaches for in vivo targeted expansion of Treg or for enhancement of their suppressive activity. In this review, we summarise the role of IL-2 in Treg biology and consequences of dysfunctional IL-2 signalling pathways. We then examine evidence of efficacy of IL-2-based biological drugs targeting Treg with specific focus on therapeutic candidates in clinical trials and discuss their limitations.

CTLA4-Ig Effectively Controls Clinical Deterioration and Immune Condition in a Murine Model of Foxp3 Deficiency.

PURPOSE: FOXP3 deficiency results in severe multisystem autoimmunity in both mice and humans, driven by the absence of functional regulatory T cells. Patients typically present with early and severe autoimmune polyendocrinopathy, dermatitis, and severe inflammation of the gut, leading to villous atrophy and ultimately malabsorption, wasting, and failure to thrive. In the absence of successful treatment, FOXP3-deficient patients usually die within the first 2 years of life. Hematopoietic stem cell transplantation provides a curative option but first requires adequate control over the inflammatory condition. Due to the rarity of the condition, no clinical trials have been conducted, with widely unstandardized therapeutic approaches. We sought to compare the efficacy of lead therapeutic candidates rapamycin, anti-CD4 antibody, and CTLA4-Ig in controlling the physiological and immunological manifestations of Foxp3 deficiency in mice. METHOD: We generated Foxp3-deficient mice and an appropriate clinical scoring system to enable direct comparison of lead therapeutic candidates rapamycin, nondepleting anti-CD4 antibody, and CTLA4-Ig. RESULTS: We found distinct immunosuppressive profiles induced by each treatment, leading to unique protective combinations over distinct clinical manifestations. CTLA4-Ig provided superior breadth of protective outcomes, including highly efficient protection during the transplantation process. CONCLUSION: These results highlight the mechanistic diversity of pathogenic pathways initiated by regulatory T cell loss and suggest CTLA4-Ig as a potentially superior therapeutic option for FOXP3-deficient patients.

Homozygous DBF4 mutation as a cause of severe congenital neutropenia.

BACKGROUND: Severe congenital neutropenia presents with recurrent infections early in life as a result of arrested granulopoiesis. Multiple genetic defects are known to block granulocyte differentiation; however, a genetic cause remains unknown in approximately 40% of cases. OBJECTIVE: We aimed to characterize a patient with severe congenital neutropenia and syndromic features without a genetic diagnosis. METHODS: Whole exome sequencing results were validated using flow cytometry, Western blotting, coimmunoprecipitation, quantitative PCR, cell cycle and proliferation analysis of lymphocytes and fibroblasts and granulocytic differentiation of primary CD34+ and HL-60 cells. RESULTS: We identified a homozygous missense mutation in DBF4 in a patient with mild extra-uterine growth retardation, facial dysmorphism and severe congenital neutropenia. DBF4 is the regulatory subunit of the CDC7 kinase, together known as DBF4-dependent kinase (DDK), the complex essential for DNA replication initiation. The DBF4 variant demonstrated impaired ability to bind CDC7, resulting in decreased DDK-mediated phosphorylation, defective S-phase entry and progression and impaired differentiation of granulocytes associated with activation of the p53-p21 pathway. The introduction of wild-type DBF4 into patient CD34+ cells rescued the promyelocyte differentiation arrest. CONCLUSION: Hypomorphic DBF4 mutation causes autosomal-recessive severe congenital neutropenia with syndromic features.

Regulatory T-cell stability and functional plasticity in health and disease.

FOXP3-expressing regulatory T cells (Treg ) are indispensable for immune homeostasis and tolerance, and in addition tissue-resident Treg have been found to perform noncanonical, tissue-specific functions. For optimal tolerogenic function during inflammatory disease, Treg are equipped with mechanisms that assure lineage stability. Treg lineage stability is closely linked to the installation and maintenance of a lineage-specific epigenetic landscape, specifically a Treg -specific DNA demethylation pattern. At the same time, for local and directed immune regulation Treg must possess a level of functional plasticity that requires them to partially acquire T helper cell (TH ) transcriptional programs-then referred to as TH -like Treg . Unleashing TH programs in Treg , however, is not without risk and may threaten the epigenetic stability of Treg with consequently pathogenic ex-Treg contributing to (auto-) inflammatory conditions. Here, we review how the Treg -stabilizing epigenetic landscape is installed and maintained, and further discuss the development, necessity and lineage instability risks of TH 1-, TH 2-, TH 17-like Treg and follicular Treg .

Disrupted Ca2+ homeostasis and immunodeficiency in patients with functional IP3 receptor subtype 3 defects.

Calcium signaling is essential for lymphocyte activation, with genetic disruptions of store-operated calcium (Ca2+) entry resulting in severe immunodeficiency. The inositol 1,4,5-trisphosphate receptor (IP3R), a homo- or heterotetramer of the IP3R1-3 isoforms, amplifies lymphocyte signaling by releasing Ca2+ from endoplasmic reticulum stores following antigen stimulation. Although knockout of all IP3R isoforms in mice causes immunodeficiency, the seeming redundancy of the isoforms is thought to explain the absence of variants in human immunodeficiency. In this study, we identified compound heterozygous variants of ITPR3 (a gene encoding IP3R subtype 3) in two unrelated Caucasian patients presenting with immunodeficiency. To determine whether ITPR3 variants act in a nonredundant manner and disrupt human immune responses, we characterized the Ca2+ signaling capacity, the lymphocyte response, and the clinical phenotype of these patients. We observed disrupted Ca2+ signaling in patient-derived fibroblasts and immune cells, with abnormal proliferation and activation responses following T-cell receptor stimulation. Reconstitution of IP3R3 in IP3R knockout cell lines led to the identification of variants as functional hypomorphs that showed reduced ability to discriminate between homeostatic and induced states, validating a genotype-phenotype link. These results demonstrate a functional link between defective endoplasmic reticulum Ca2+ channels and immunodeficiency and identify IP3Rs as diagnostic targets for patients with specific inborn errors of immunity. These results also extend the known cause of Ca2+-associated immunodeficiency from store-operated entry to impaired Ca2+ mobilization from the endoplasmic reticulum, revealing a broad sensitivity of lymphocytes to genetic defects in Ca2+ signaling.

Selective ablation of thymic and peripheral Foxp3+ regulatory T cell development.

Foxp3+ regulatory T (Treg) cells of thymic (tTreg) and peripheral (pTreg) developmental origin are thought to synergistically act to ensure immune homeostasis, with self-reactive tTreg cells primarily constraining autoimmune responses. Here we exploited a Foxp3-dependent reporter with thymus-specific GFP/Cre activity to selectively ablate either tTreg (ΔtTreg) or pTreg (ΔpTreg) cell development, while sparing the respective sister populations. We found that, in contrast to the tTreg cell behavior in ΔpTreg mice, pTreg cells acquired a highly activated suppressor phenotype and replenished the Treg cell pool of ΔtTreg mice on a non-autoimmune C57BL/6 background. Despite the absence of tTreg cells, pTreg cells prevented early mortality and fatal autoimmunity commonly observed in Foxp3-deficient models of complete Treg cell deficiency, and largely maintained immune tolerance even as the ΔtTreg mice aged. However, only two generations of backcrossing to the autoimmune-prone non-obese diabetic (NOD) background were sufficient to cause severe disease lethality associated with different, partially overlapping patterns of organ-specific autoimmunity. This included a particularly severe form of autoimmune diabetes characterized by an early onset and abrogation of the sex bias usually observed in the NOD mouse model of human type 1 diabetes. Genetic association studies further allowed us to define a small set of autoimmune risk loci sufficient to promote ß cell autoimmunity, including genes known to impinge on Treg cell biology. Overall, these studies show an unexpectedly high functional adaptability of pTreg cells, emphasizing their important role as mediators of bystander effects to ensure self-tolerance.

Cancer immunotherapies transition endothelial cells into HEVs that generate TCF1+ T lymphocyte niches through a feed-forward loop.

The lack of T cell infiltrates is a major obstacle to effective immunotherapy in cancer. Conversely, the formation of tumor-associated tertiary-lymphoid-like structures (TA-TLLSs), which are the local site of humoral and cellular immune responses against cancers, is associated with good prognosis, and they have recently been detected in immune checkpoint blockade (ICB)-responding patients. However, how these lymphoid aggregates develop remains poorly understood. By employing single-cell transcriptomics, endothelial fate mapping, and functional multiplex immune profiling, we demonstrate that antiangiogenic immune-modulating therapies evoke transdifferentiation of postcapillary venules into inflamed high-endothelial venules (HEVs) via lymphotoxin/lymphotoxin beta receptor (LT/LTßR) signaling. In turn, tumor HEVs boost intratumoral lymphocyte influx and foster permissive lymphocyte niches for PD1- and PD1+TCF1+ CD8 T cell progenitors that differentiate into GrzB+PD1+ CD8 T effector cells. Tumor-HEVs require continuous CD8 and NK cell-derived signals revealing that tumor HEV maintenance is actively sculpted by the adaptive immune system through a feed-forward loop.

Context-dependent effects of IL-2 rewire immunity into distinct cellular circuits.

Interleukin 2 (IL-2) is a key homeostatic cytokine, with therapeutic applications in both immunogenic and tolerogenic immune modulation. Clinical use has been hampered by pleiotropic functionality and widespread receptor expression, with unexpected adverse events. Here, we developed a novel mouse strain to divert IL-2 production, allowing identification of contextual outcomes. Network analysis identified priority access for Tregs and a competitive fitness cost of IL-2 production among both Tregs and conventional CD4 T cells. CD8 T and NK cells, by contrast, exhibited a preference for autocrine IL-2 production. IL-2 sourced from dendritic cells amplified Tregs, whereas IL-2 produced by B cells induced two context-dependent circuits: dramatic expansion of CD8+ Tregs and ILC2 cells, the latter driving a downstream, IL-5-mediated, eosinophilic circuit. The source-specific effects demonstrate the contextual influence of IL-2 function and potentially explain adverse effects observed during clinical trials. Targeted IL-2 production therefore has the potential to amplify or quench particular circuits in the IL-2 network, based on clinical desirability.

Unstable regulatory T cells, enriched for naïve and Nrp1neg cells, are purged after fate challenge.

Regulatory T cells (Tregs) are indispensable for the control of immune homeostasis and have clinical potential as a cell therapy for treating autoimmunity. Tregs can lose expression of the lineage-defining Foxp3 transcription factor and acquire effector T cell (Teff) characteristics, a process referred to as Treg plasticity. The extent and reversibility of such plasticity during immune responses remain unknown. Here, using a murine genetic fate-mapping system, we show that Treg stability is maintained even during exposure to a complex microbial/antigenic environment. Furthermore, we demonstrate that the observed plasticity of Tregs after adoptive transfer into a lymphopenic environment is a property limited to only a subset of the Treg population, with the nonconverting majority of Tregs being resistant to plasticity upon secondary stability challenge. The unstable Treg fraction is a complex mixture of phenotypically distinct Tregs, enriched for naïve and neuropilin-1-negative Tregs, and includes peripherally induced Tregs and recent thymic emigrant Tregs These results suggest that a "purging" process can be used to purify stable Tregs that are capable of robust fate retention, with potential implications for improving cell transfer therapy.

Necroptosis in Immuno-Oncology and Cancer Immunotherapy.

Immune-checkpoint blockers (ICBs) have revolutionized oncology and firmly established the subfield of immuno-oncology. Despite this renaissance, a subset of cancer patients remain unresponsive to ICBs due to widespread immuno-resistance. To "break" cancer cell-driven immuno-resistance, researchers have long floated the idea of therapeutically facilitating the immunogenicity of cancer cells by disrupting tumor-associated immuno-tolerance via conventional anticancer therapies. It is well appreciated that anticancer therapies causing immunogenic or inflammatory cell death are best positioned to productively activate anticancer immunity. A large proportion of studies have emphasized the importance of immunogenic apoptosis (i.e., immunogenic cell death or ICD); yet, it has also emerged that necroptosis, a programmed necrotic cell death pathway, can also be immunogenic. Emergence of a proficient immune profile for necroptosis has important implications for cancer because resistance to apoptosis is one of the major hallmarks of tumors. Putative immunogenic or inflammatory characteristics driven by necroptosis can be of great impact in immuno-oncology. However, as is typical for a highly complex and multi-factorial disease like cancer, a clear cause versus consensus relationship on the immunobiology of necroptosis in cancer cells has been tough to establish. In this review, we discuss the various aspects of necroptosis immunobiology with specific focus on immuno-oncology and cancer immunotherapy.

Defective Sec61α1 underlies a novel cause of autosomal dominant severe congenital neutropenia.

BACKGROUND: The molecular cause of severe congenital neutropenia (SCN) is unknown in 30% to 50% of patients. SEC61A1 encodes the α-subunit of the Sec61 complex, which governs endoplasmic reticulum protein transport and passive calcium leakage. Recently, mutations in SEC61A1 were reported to be pathogenic in common variable immunodeficiency and glomerulocystic kidney disease. OBJECTIVE: Our aim was to expand the spectrum of SEC61A1-mediated disease to include autosomal dominant SCN. METHODS: Whole exome sequencing findings were validated, and reported mutations were compared by Western blotting, Ca2+ flux assays, differentiation of transduced HL-60 cells, in vitro differentiation of primary CD34 cells, quantitative PCR for unfolded protein response (UPR) genes, and single-cell RNA sequencing on whole bone marrow. RESULTS: We identified a novel de novo missense mutation in SEC61A1 (c.A275G;p.Q92R) in a patient with SCN who was born to nonconsanguineous Belgian parents. The mutation results in diminished protein expression, disturbed protein translocation, and an increase in calcium leakage from the endoplasmic reticulum. In vitro differentiation of CD34+ cells recapitulated the patient's clinical arrest in granulopoiesis. The impact of Q92R-Sec61α1 on neutrophil maturation was validated by using HL-60 cells, in which transduction reduced differentiation into CD11b+CD16+ cells. A potential mechanism for this defect is the uncontrolled initiation of the unfolded protein stress response, with single-cell analysis of primary bone marrow revealing perturbed UPR in myeloid precursors and in vitro differentiation of primary CD34+ cells revealing upregulation of CCAAT/enhancer-binding protein homologous protein and immunoglobulin heavy chain binding protein UPR-response genes. CONCLUSION: Specific mutations in SEC61A1 cause SCN through dysregulation of the UPR.

Phosphatase Regulator NIPP1 Restrains Chemokine-Driven Skin Inflammation.

Nuclear inhibitor of protein phosphatase 1 (NIPP1) is a ubiquitously expressed nuclear protein that regulates functions of protein serine/threonine phosphatase-1 in cell proliferation and lineage specification. The role of NIPP1 in tissue homeostasis is not fully understood. This study shows that the selective deletion of NIPP1 in mouse epidermis resulted in epidermal hyperproliferation, a reduced adherence of basal keratinocytes, and a gradual decrease in the stemness of hair follicle stem cells, culminating in hair loss. This complex phenotype was associated with chronic sterile skin inflammation and could be partially rescued by dexamethasone treatment. NIPP1-deficient keratinocytes massively expressed proinflammatory chemokines and immunomodulatory proteins in a cell-autonomous manner. Chemokines subsequently induced the recruitment and activation of immune cells, in particular conventional dendritic cells and Langerhans cells, accounting for the chronic inflammation phenotype. The data identifies NIPP1 as a key regulator of epidermal homeostasis and as a potential target for the treatment of inflammatory skin diseases.