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.

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.

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.

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.

A robust pipeline with high replication rate for detection of somatic variants in the adaptive immune system as a source of common genetic variation in autoimmune disease.

The role of somatic variants in diseases beyond cancer is increasingly being recognized, with potential roles in autoinflammatory and autoimmune diseases. However, as mutation rates and allele fractions are lower, studies in these diseases are substantially less tolerant of false positives, and bio-informatics algorithms require high replication rates. We developed a pipeline combining two variant callers, MuTect2 and VarScan2, with technical filtering and prioritization. Our pipeline detects somatic variants with allele fractions as low as 0.5% and achieves a replication rate of >55%. Validation in an independent data set demonstrates excellent performance (sensitivity > 57%, specificity > 98%, replication rate > 80%). We applied this pipeline to the autoimmune disease multiple sclerosis (MS) as a proof-of-principle. We demonstrate that 60% of MS patients carry 2-10 exonic somatic variants in their peripheral blood T and B cells, with the vast majority (80%) occurring in T cells and variants persisting over time. Synonymous variants significantly co-occur with non-synonymous variants. Systematic characterization indicates somatic variants are enriched for being novel or very rare in public databases of germline variants and trend towards being more damaging and conserved, as reflected by higher phred-scaled combined annotation-dependent depletion (CADD) and genomic evolutionary rate profiling (GERP) scores. Our pipeline and proof-of-principle now warrant further investigation of common somatic genetic variation on top of inherited genetic variation in the context of autoimmune disease, where it may offer subtle survival advantages to immune cells and contribute to the capacity of these cells to participate in the autoimmune reaction.

Abnormal differentiation of B cells and megakaryocytes in patients with Roifman syndrome.

BACKGROUND: Roifman syndrome is a rare inherited disorder characterized by spondyloepiphyseal dysplasia, growth retardation, cognitive delay, hypogammaglobulinemia, and, in some patients, thrombocytopenia. Compound heterozygous variants in the small nuclear RNA gene RNU4ATAC, which is necessary for U12-type intron splicing, were identified recently as driving Roifman syndrome. OBJECTIVE: We studied 3 patients from 2 unrelated kindreds harboring compound heterozygous or homozygous stem II variants in RNU4ATAC to gain insight into the mechanisms behind this disorder. METHODS: We systematically profiled the immunologic and hematologic compartments of the 3 patients with Roifman syndrome and performed RNA sequencing to unravel important splicing defects in both cell lineages. RESULTS: The patients exhibited a dramatic reduction in B-cell numbers, with differentiation halted at the transitional B-cell stage. Despite abundant B-cell activating factor availability, development past this B-cell activating factor-dependent stage was crippled, with disturbed minor splicing of the critical mitogen-activated protein kinase 1 signaling component. In the hematologic compartment patients with Roifman syndrome demonstrated defects in megakaryocyte differentiation, with inadequate generation of proplatelets. Platelets from patients with Roifman syndrome were rounder, with increased tubulin and actin levels, and contained increased α-granule and dense granule markers. Significant minor intron retention in 354 megakaryocyte genes was observed, including DIAPH1 and HPS1, genes known to regulate platelet and dense granule formation, respectively. CONCLUSION: Together, our results provide novel molecular and cellular data toward understanding the immunologic and hematologic features of Roifman syndrome.

Hematopoietic Stem Cell Niches Produce Lineage-Instructive Signals to Control Multipotent Progenitor Differentiation.

Hematopoietic stem cells (HSCs) self-renew in bone marrow niches formed by mesenchymal progenitors and endothelial cells expressing the chemokine CXCL12, but whether a separate niche instructs multipotent progenitor (MPP) differentiation remains unclear. We show that MPPs resided in HSC niches, where they encountered lineage-instructive differentiation signals. Conditional deletion of the chemokine receptor CXCR4 in MPPs reduced differentiation into common lymphoid progenitors (CLPs), which decreased lymphopoiesis. CXCR4 was required for CLP positioning near Interleukin-7+ (IL-7) cells and for optimal IL-7 receptor signaling. IL-7+ cells expressed CXCL12 and the cytokine SCF, were mesenchymal progenitors capable of differentiation into osteoblasts and adipocytes, and comprised a minor subset of sinusoidal endothelial cells. Conditional Il7 deletion in mesenchymal progenitors reduced B-lineage committed CLPs, while conditional Cxcl12 or Scf deletion from IL-7+ cells reduced HSC and MPP numbers. Thus, HSC maintenance and multilineage differentiation are distinct cell lineage decisions that are both controlled by HSC niches.

The thymoprotective function of leptin is indirectly mediated via suppression of obesity.

Leptin is an adipokine that regulates metabolism and plays an important role as a neuroendocrine hormone. Leptin mediates these functions via the leptin receptor, and deficiency in either leptin or its receptor leads to obesity in humans and mice. Leptin has far reaching effects on the immune system, as observed in obese mice, which display decreased thymic function and increased inflammatory responses. With expression of the leptin receptor on T cells and supporting thymic epithelium, aberrant signalling through the leptin receptor has been thought to be the direct cause of thymic involution in obese mice. Here, we demonstrate that the absence of leptin receptor on either thymic epithelial cells or T cells does not lead to the loss of thymic function, demonstrating that the thymoprotective effect of leptin is mediated by obesity suppression rather than direct signalling to the cellular components of the thymus.

Fundamental properties of unperturbed haematopoiesis from stem cells in vivo.

Haematopoietic stem cells (HSCs) are widely studied by HSC transplantation into immune- and blood-cell-depleted recipients. Single HSCs can rebuild the system after transplantation. Chromosomal marking, viral integration and barcoding of transplanted HSCs suggest that very low numbers of HSCs perpetuate a continuous stream of differentiating cells. However, the numbers of productive HSCs during normal haematopoiesis, and the flux of differentiating progeny remain unknown. Here we devise a mouse model allowing inducible genetic labelling of the most primitive Tie2(+) HSCs in bone marrow, and quantify label progression along haematopoietic development by limiting dilution analysis and data-driven modelling. During maintenance of the haematopoietic system, at least 30% or ∼5,000 HSCs are productive in the adult mouse after label induction. However, the time to approach equilibrium between labelled HSCs and their progeny is surprisingly long, a time scale that would exceed the mouse's life. Indeed, we find that adult haematopoiesis is largely sustained by previously designated 'short-term' stem cells downstream of HSCs that nearly fully self-renew, and receive rare but polyclonal HSC input. By contrast, in fetal and early postnatal life, HSCs are rapidly used to establish the immune and blood system. In the adult mouse, 5-fluoruracil-induced leukopenia enhances the output of HSCs and of downstream compartments, thus accelerating haematopoietic flux. Label tracing also identifies a strong lineage bias in adult mice, with several-hundred-fold larger myeloid than lymphoid output, which is only marginally accentuated with age. Finally, we show that transplantation imposes severe constraints on HSC engraftment, consistent with the previously observed oligoclonal HSC activity under these conditions. Thus, we uncover fundamental differences between the normal maintenance of the haematopoietic system, its regulation by challenge, and its re-establishment after transplantation. HSC fate mapping and its linked modelling provide a quantitative framework for studying in situ the regulation of haematopoiesis in health and disease.

Premature thymic involution is independent of structural plasticity of the thymic stroma.

The thymus is the organ devoted to T-cell production. The thymus undergoes multiple rounds of atrophy and redevelopment before degenerating with age in a process known as involution. This process is poorly understood, despite the influence the phenomenon has on peripheral T-cell numbers. Here we have investigated the FVB/N mouse strain, which displays premature thymic involution. We find multiple architectural and cellular features that precede thymic involution, including disruption of the epithelial-endothelial relationship and a progressive loss of pro-T cells. The architectural features, reminiscent of the human thymus, are intrinsic to the nonhematopoietic compartment and are neither necessary nor sufficient for thymic involution. By contrast, the loss of pro-T cells is intrinsic to the hematopoietic compartment, and is sufficient to drive premature involution. These results identify pro-T-cell loss as the main driver of premature thymic involution, and highlight the plasticity of the thymic stroma, capable of maintaining function across diverse interstrain architectures.

The thymic microenvironment differentially regulates development and trafficking of invariant NKT cell sublineages.

The regulatory role of the thymic microenvironment during trafficking and differentiation of the invariant NKT (iNKT) cell lineage remains poorly understood. In this study, we show that fractalkine receptor expression marks emigrating subpopulations of the NKT1, NKT2, and NKT17 sublineages in the thymus and peripheral organs of naive mice. Moreover, NKT1 sublineage cells can be subdivided into two subsets, namely NKT1(a) and NKT1(b), which exhibit distinct developmental and tissue-specific distribution profiles. More specifically, development and trafficking of the NKT1(a) subset are selectively dependent upon lymphotoxin (LT)α1ß2-LTß receptor-dependent differentiation of thymic stroma, whereas the NKT1(b), NKT2, and NKT17 sublineages are not. Furthermore, we identify a potential cellular source for LTα1ß2 during thymic organogenesis, marked by expression of IL-7Rα, which promotes differentiation of the NKT1(a) subset in a noncell-autonomous manner. Collectively, we propose a mechanism by which thymic differentiation and retention of the NKT1 sublineage are developmentally coupled to LTα1ß2-LTß receptor-dependent thymic organogenesis.

Mast cell chymase degrades the alarmins heat shock protein 70, biglycan, HMGB1, and interleukin-33 (IL-33) and limits danger-induced inflammation.

During infection and tissue damage, virulence factors and alarmins are pro-inflammatory and induce activation of various immune cells including macrophages and mast cells (MCs). Activated MCs instantly release preformed inflammatory mediators, including several proteases. The chymase mouse mast cell protease (MCPT)-4 is thought to be pro-inflammatory, whereas human chymase also degrades pro-inflammatory cytokines, suggesting that chymase instead limits inflammation. Here we explored the contribution of MCPT4 and human chymase to the control of danger-induced inflammation. We found that protein extracts from wild type (WT), carboxypeptidase A3-, and MCPT6-deficient mice and MCs and recombinant human chymase efficiently degrade the Trichinella spiralis virulence factor heat shock protein 70 (Hsp70) as well as endogenous Hsp70. MC-(W(sash))-, serglycin-, NDST2-, and MCPT4-deficient extracts lacked this capacity, indicating that chymase is responsible for the degradation. Chymase, but not MC tryptase, also degraded other alarmins, i.e. biglycan, HMGB1, and IL-33, a degradation that was efficiently blocked by the chymase inhibitor chymostatin. IL-7, IL-22, GM-CSF, and CCL2 were resistant to chymase degradation. MCPT4-deficient conditions ex vivo and in vivo showed no reduction in added Hsp70 and only minor reduction of IL-33. Peritoneal challenge with Hsp70 resulted in increased neutrophil recruitment and TNF-α levels in the MCPT4-deficient mice, whereas IL-6 and CCL2 levels were similar to the levels found in WT mice. The rapid and MC chymase-specific degradation of virulence factors and alarmins may depend on the presence of accessible extended recognition cleavage sites in target substrates and suggests a protective and regulatory role of MC chymase during danger-induced inflammation.

Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3⁺ regulatory T cells.

Foxp3⁺ regulatory T (Treg) cells are a crucial immunosuppressive population of CD4⁺ T cells, yet the homeostatic processes and survival programs that maintain the Treg cell pool are poorly understood. Here we report that peripheral Treg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2-dependent and costimulation-dependent process. By contrast, excess Treg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-xL and Bcl-2 were dispensable for survival of Treg cells, whereas Mcl-1 was critical for survival of Treg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which Treg cells maintain homeostasis via critical survival pathways.

Thymus-autonomous T cell development in the absence of progenitor import.

Thymus function is thought to depend on a steady supply of T cell progenitors from the bone marrow. The notion that the thymus lacks progenitors with self-renewal capacity is based on thymus transplantation experiments in which host-derived thymocytes replaced thymus-resident cells within 4 wk. Thymus grafting into T cell-deficient mice resulted in a wave of T cell export from the thymus, followed by colonization of the thymus by host-derived progenitors, and cessation of T cell development. Compound Rag2(-/-)γ(c)(-/-)Kit(W/Wv) mutants lack competitive hematopoietic stem cells (HSCs) and are devoid of T cell progenitors. In this study, using this strain as recipients for wild-type thymus grafts, we noticed thymus-autonomous T cell development lasting several months. However, we found no evidence for export of donor HSCs from thymus to bone marrow. A diverse T cell antigen receptor repertoire in progenitor-deprived thymus grafts implied that many thymocytes were capable of self-renewal. Although the process was most efficient in Rag2(-/-)γ(c)(-/-)Kit(W/Wv) hosts, γ(c)-mediated signals alone played a key role in the competition between thymus-resident and bone marrow-derived progenitors. Hence, the turnover of each generation of thymocytes is not only based on short life span but is also driven via expulsion of resident thymocytes by fresh progenitors entering the thymus.

Mast cell chymase reduces the toxicity of Gila monster venom, scorpion venom, and vasoactive intestinal polypeptide in mice.

Mast cell degranulation is important in the pathogenesis of anaphylaxis and allergic disorders. Many animal venoms contain components that can induce mast cell degranulation, and this has been thought to contribute to the pathology and mortality caused by envenomation. However, we recently reported evidence that mast cells can enhance the resistance of mice to the venoms of certain snakes and that mouse mast cell-derived carboxypeptidase A3 (CPA3) can contribute to this effect. Here, we investigated whether mast cells can enhance resistance to the venom of the Gila monster, a toxic component of that venom (helodermin), and the structurally similar mammalian peptide, vasoactive intestinal polypeptide (VIP). Using 2 types of mast cell-deficient mice, as well as mice selectively lacking CPA3 activity or the chymase mouse mast cell protease-4 (MCPT4), we found that mast cells and MCPT4, which can degrade helodermin, can enhance host resistance to the toxicity of Gila monster venom. Mast cells and MCPT4 also can limit the toxicity associated with high concentrations of VIP and can reduce the morbidity and mortality induced by venoms from 2 species of scorpions. Our findings support the notion that mast cells can enhance innate defense by degradation of diverse animal toxins and that release of MCPT4, in addition to CPA3, can contribute to this mast cell function.