bluuebunny/crossref_metadata_2025_split huggingface dataset:
Itk and Btk regulate mast cell responses to lipopolysaccharide: firing or dampening? (151.18)
Authors: Huang Weishan , Morales J. Luis , August Avery 
Published: January 2023

Abstract
LPS induces mast cell derived cytokines such as tumor necrosis factor (TNF) α, to cause septic hypothermia. The non receptor tyrosine kinases Itk and Btk are both expressed as signaling regulators in mast cells, but their roles in mast cell response to LPS are unclear. Here we show that Itk-/- mice exhibited less severe LPS induced hypothermia, with no difference observed in Btk-/- mice, compared to C57BL/6 (WT) mice, strikingly however, Itk/Btk double knockout (DKO) mice suffered exacerbated and prolonged hypothermia. Bone marrow derived mast cells (BMMCs) lacking Itk, Btk or both altered degradation of the NFκB inhibitor IκBα in response to LPS. DKO BMMCs exhibited dramatically reduced degradation of IκBα, but significantly enhanced production of TNFα and other LPS responsive genes, suggesting that Itk and Btk altered NF-κB response to suppress TNFα secretion by BMMCs in response to LPS. Analysis of modified existing computational models for LPS and TNFα stimulation revealed novel NFκB/IκBα signaling dynamics which predict our experimental results in WT BMMCs, suggesting that BMMCs may be a good model to integrate primary and secondary responses against LPS, revealing how stimulation of TLR4 ignites TNFα, which triggers and spreads the flame, activating a regulatory network with negative feedback and feed forward loops. This combination of experimental and computational approach promises to reveal novel roles for Itk and Btk in the regulation of the BMMC response to LPS.
The chymase, mouse mast cell protease 4, degrades TNF, limits inflammation, and promotes survival in a mouse model of sepsis (151.3)
Authors: Piliponsky Adrian , Chen Ching-Cheng , Rios Eon , Abrink Magnus , Pejler Gunnar , Tsai Mindy , Galli Stephen 
Published: January 2023

Abstract
Mouse mast cell protease (mMCP)-4, the mouse counterpart of human mast cell chymase, is thought to have pro-inflammatory effects in innate or adaptive immune responses associated with mast cell activation. However, human chymase can degrade the pro-inflammatory cytokine, TNF, a mediator that can be produced by mast cells and many other cell types. We found that mMCP-4 can reduce levels of mouse mast cell-derived TNF in vitro through degradation of transmembrane and soluble TNF. We then searched for possible effects of interactions between mMCP-4 and TNF in vivo by analyzing the features of a classic model of polymicrobial sepsis, cecal ligation and puncture (CLP), in C57BL/6-mMCP-4-deficient mice versus C57BL/6 wild type mice, and in C57BL/6-KitW-sh/W-sh mice containing adoptively-transferred mast cells that were either wild type or lacked mMCP-4, TNF, or both mediators. We found that mMCP-4 reduced levels of intraperitoneal TNF and numbers of peritoneal neutrophils after CLP, and also significantly enhanced survival in mice subjected to this procedure. Our findings support the conclusion that mMCP-4 can enhance survival after CLP at least in part by limiting detrimental effects of TNF, and suggest that mast cell chymase may represent a physiologically important negative regulator of TNF.
Phenotypic and functional characterization of adaptive regulatory T cells using a novel TCR-transgenic mouse model of EAE (152.29)
Authors: Yadav Mahesh , Louvet Cedric , Davini Dan , Bailey-bucktrout Samantha , Schack David , Head Richard , Ruminski Peter , Weiss David , Kuster Dan , Bluestone Jeffrey 
Published: January 2023

Abstract
A defect in functions of Foxp3+ CD4 T helper cells called regulatory T cells (Tregs) play Tregs results in the onset of autoimmune diseases, however the basis for these Treg defects and specific roles of nTregs and aTregs and their relative contributions in autoimmunity remain unknown. We have developed a novel mouse model (1B3) that allows for the direct study of the aTreg population, with a defined TCR specificity and autoreactive potential. 1B3 mouse when bred to RAG2-/- background developed spontaneous EAE by 2-3 weeks of age. 1B3.RAG-/- mice had significant number of Foxp3+ cells in periphery but there was a complete lack of Treg generation in the thymus. aTregs isolated from 1B3.RAG-/-exhibited CpG demethylation in TSDR and showed suppressive activity in vitro. Microarray analysis demonstrated that compared to nTregs, aTregs displayed significantly lower levels of surface markers PD-1, Nrp-1 and previously described transcription factor Helios. PD-1 and Nrp-1 allowed us to separate Foxp3+ Tregs into Helios+/hi nTregs and Helios-/lo aTregs in WT NOD or B6 mice. This is the first report describing phenotypic markers to distinguish aTregs from nTregs. aTregs were less effective in controlling effector T cells and exhibited unstable Foxp3 expression in vivo. Thus nTregs are critical in maintaining tolerance while aTregs are generated in periphery to control local inflammatory response but may not provide long lasting protection due to less stable Foxp3 expression.
Memory CD4+ T cell-derived secondary effectors are distinct from and functionally superior to primary effectors arising from naïve cells (152.33)
Authors: Strutt Tara , McKinstry K. , Bradley Linda , Swain Susan 
Published: January 2023

Abstract
The properties of secondary (2°) CD4+ T cell effectors generated from memory cells during recall responses are not defined but have been assumed to mirror those of primary (1°) effectors arising from naïve cells. Here we rigorously compare these two effector populations following influenza virus infection using a well-characterized adoptive transfer model employing populations of T cell receptor transgenic CD4 T cells (HNT) recognizing an epitope of the A/PR8 (H1N1) influenza viral hemagglutinin. Both 1° and 2° effectors develop and migrate to infected lungs with similar kinetics, but 2° effectors accumulate in greater number and demonstrate enhanced multifunctional capabilities. We show that the 2° effector response in the lung is a critical component of protection against influenza mediated by memory CD4+ T cells and that upon transfer to unprimed hosts 2° effectors are superior to 1° effectors at mediating protection. We employ transcriptome analysis to further characterize differences between 1° and 2° effectors. Our approach provides novel insight into the regulation of expansion, survival, trafficking, and functions of effector CD4+ T cells during pathogen challenge and may reveal ideal candidates for manipulation with the goal of directing the generation of larger populations of multifunctional and protective effectors in a variety of settings apart from flu infection.
Gastric epithelial cells directly influence Th17 development and activation during H. pylori infection (152.5)
Authors: Beswick Ellen , Pinchuk Iryna , Earley Rachel , Reyes Victor 
Published: January 2023

Abstract
Gastric epithelial cells (GECs) express class II MHC, act as antigen presenting cells (APCs), and influence CD4+ T cell responses during H. pylori (Hp) infection. Hp, as the major global cause of gastroduodenal ulcers and gastric cancer, elicits GEC responses that include production of proinflammatory cytokines and upregulation of class II MHC and costimulatory molecules that provide signals for CD4+ T cell activation. Recently, the proinflammatory CD4+ phenotype, Th17, was detected in the Hp infected gastric mucosa. Since GECs produce inflammatory cytokines and act as APCs during Hp infection, we hypothesized that the GEC response to Hp infection influences development of Th17 cells in the gastric mucosa. In order to examine this, we investigated Hp-induced GEC production of IL-6 and TGF-β, the cytokines required for Th17 development. These cytokines induced naive CD4+ T expression of RORγt and IL-17A in culture with infected GECs. IL-21, which may play a role in Th17 development, was also found in co-cultures of GECs and CD4+ T cells. Surprisingly, B7-H2 blocking on GECs increased Th17 development. Th17 proliferated in culture and class II MHC was crucial in Th17 proliferation as determined by blocking class II MHC on GECs and measuring CFSE labeled CD4+ T cells gated on RORγt by flow cytometry. These observations suggest a novel mechanism where GECs induce Th17 development and proliferation, which is likely a crucial mechanism in inflammation during Hp infection.
Identification of the emergence of clonally multipotent hematopoietic stem and progenitor populations in the mouse embryo (153.10)
Authors: Inlay Matthew , Serwold Thomas , Mosley Adriane , Weissman Irving 
Published: January 2023

Abstract
In the adult hematopoietic system, all mature blood cells derive from the hematopoietic stem cell (HSC). However, HSCs are not the first blood-forming cells to appear during embryogenesis. Rather, erythrocyte-biased progenitors can be identified in the yolk sac (YS) blood islands approximately a week after fertilization. After the establishment of circulation, fully functional HSCs can be identified within the aorta-gonad-mesonephros (AGM) and fetal liver (FL) tissues. The origin and maturation of HSCs in embryonic development remains an open and important question. To help address this issue, we sought to identify and isolate the blood-forming populations within these embryonic tissues at the stages when hematopoietic potential first appears. Using a modified OP9 stromal line that can yield 8 hematopoietic lineages (B, T, NK, DC, granulocyte, macrophage, platelet, and erythrocyte), we identified populations with multipotent potential at the clonal level in all tissues. While these embryonic multipotent cells share many of the surface markers that define adult HSCs, there are also distinct tissue-specific differences. Furthermore, these populations lack the ability to engraft into adult irradiated recipient mice, indicating their development into mature HSCs is incomplete. Our data support a model by which the ability to generate all hematopoietic lineages emerges prior to the ability to engraft.
CXCR4 blockade reverses panleukopenia in patients with WHIM syndrome (153.11)
Authors: Liu Qian , Malech Harry , Murphy Philip , McDermott David 
Published: January 2023

Abstract
WHIM syndrome is a rare congenital immunodeficiency disorder characterized by warts, hypogammaglobulinemia, infections and myelokathexis (neutropenia due to impaired egress from bone marrow); most patients also have severe panleukopenia. The disease is caused by autosomal dominant gain-of-function truncation mutations in the C-terminus of chemokine receptor CXCR4. CXCR4 normally controls neutrophil and hematopoietic stem cell distribution between blood and bone marrow, T cell development and B cell lymphopoiesis. Here we present preliminary data from a Phase 1 study of the safety and efficacy of the FDA-approved CXCR4 antagonist Mozobil as mechanism-based therapy in WHIM syndrome, using a 7-day dose-escalation protocol. To date, we have recruited two unrelated adult patients, both having the most common WHIM mutation, CXCR4 R334X. In both patients, Mozobil increased lymphocytes and monocytes in blood to the normal range, and neutrophils in blood to the safe level (&gt;500 cells/microliter). One patient has now been treated with Mozobil daily for 6 months with no loss of efficacy. No side effects were noted in either patient. Thus, our data provide the first pharmacologic evidence that panleukopenia in WHIM syndrome is due to CXCR4-dependent leukocyte retention in storage sites, and justify continued study of Mozobil as mechanism-based therapy for patients with this disease.
L111 and E605: vital residues in the functionality of the Bruton’s tyrosine kinase (153.18)
Authors: Vargas Alexander , López-Herrera Gabriela , Vences-Catalan Felipe , Maravillas-Montero José , Rojo-Domínguez Antonio , Mogica-Martínez Dolores , Espinosa-Rosales Francisco , Santos-Argumedo Leopoldo 
Published: January 2023

Abstract
X-linked agammaglobulinemia (XLA) is a primary immunodeficiency due to mutations in the gene encoding Bruton’s tyrosine kinase (Btk). Btk is a key protein in the B cell receptor signaling pathway. Functional characterization of the mutated Btk in patients with XLA is important in order to define that they are disease causing mutations. Previously, we identified two novel missense mutations in Btk in XLA patients. Using in silico assays we determined that L111P and E605G produced modifications in the structural conformation, affecting the protein organization and its functionality. The absence of catalytic activity of Btk from the patients was reflected on the lack of the Y223 phosphorylation, lack of calcium release and aberrant Btk redistribution after BCR crosslinking. This work shows for that the L111 and E605 residues are fundamental for the activation and functionality of the Bruton’s tyrosine kinase. Additionaly, in silico analysis can be a useful tool for understanding Btk mutations found in primary immunodeficiency patients.
B cell activation contributes to splenomegaly in PPARγ deficient mice (153.21)
Authors: Liu Ya-Hui , Tsai Yau-Sheng , Tsai Pei-Jane 
Published: January 2023

Abstract
PPARγ is a nuclear transcription factor that modulates diverse biofunctions, including lipid biosynthesis, glucose metabolism and inflammation. However, the specific role of PPARγ in the immune function has not been fully explored. We have previously generated mice with PPARγ gene expression from 25% to 100% normal. We found that mice with 25% expression of PPARγ (PpargC/-) exhibited a two-fold increase in spleen size at their young age and lasted throughout the life. It has been demonstrated PPARγ deficiency enhanced bone formation and relocated hematopoiesis to the spleen. However, micro-CT analysis showed trabecular volume, number, thickness, and bone mineral density were increased in old, but not in young, PpargC/- mice. The population of hematopoietic stem cells (HSCs) and the expression level of transcription factors, crucial for HSC differentiation and self-renewal, in spleen were also increased in old PpargC/- mice. However, young PpargC/- with splenomegaly revealed neither increased bone formation nor relocation of hematopoiesis to the spleen. In vivo BrdU incorporation study demonstrated that B cells were highly proliferated in young PpargC/- mice. Therefore, further studies are needed to examine the role of PPARγ in immune system. These results suggest that splenomegaly in young PpargC/- mice was not likely caused by extramedullary hematopoiesis, but may be contributed by increased activation of B cells, implicating the beneficial role of PPARγ in immunomodulation.
Blk-haploinsufficiency impairs the development, but enhances the functional responses, of marginal zone B cells (153.22)
Authors: Samuelson Elizabeth , Laird Renee , Maue Alexander , Rochford Rosemary , Hayes Sandra 
Published: January 2023

Abstract
Blk was identified two decades ago as a B cell-specific Src family kinase. Surprisingly, despite its restricted expression, Blk was found to have a non-essential role in B cell development and activation. Recent studies, however, have discovered that Blk is expressed in many cell types outside of the B lineage, including IL-17-producing γδ T cells and pancreatic β-cells. The discovery that Blk has a broader expression pattern than previously reported prompted us to perform a more comprehensive analysis of Blk expression patterns in hematopoietic cells. During this analysis, while using B cell subsets as positive controls, we found that Blk is differentially expressed in mature splenic B cell subsets, with marginal zone (MZ) B cells expressing high levels, and follicular (FO) B cells expressing low levels, of Blk. To determine whether this difference in Blk expression levels reflected a differential requirement for Blk in MZ and FO B cell development, we analyzed the effects of reducing Blk levels on splenic B cell development in mice expressing one functional allele of Blk (Blk+/-). We report that reducing Blk levels impaired the generation of MZ B cells but not FO B cells. Moreover, although there were fewer MZ B cells in Blk+/- mice compared to Blk+/+ mice, Blk+/- MZ B cells were hyper-responsive to BCR stimulation, both in vitro and in vivo. Thus, this study has revealed a previously unappreciated role for Blk in the development and activation of MZ B cells.
Transcription factor Fli-1 is involved in regulation of dendritic cell and monocyte development (153.33)
Authors: Suzuki Eiji , Williams Sarah , Karam Eva , Zhang Xian 
Published: January 2023

Abstract
The Fli1 gene is a member of the ets family of transcription factors that is expressed in hematopoietic cells. We have reported that Fli-1 plays an important role in the B cell and megakaryocyte development. C57BL/6 (B6) mutant Fli-1 mice (Fli-1ΔCTA) express a truncated Fli-1 protein that lack the C-terminal transactivation domain Fli-1ΔCTA B6 mice had significant decreased follicular B cells, increased marginal zone B cells and decreased platelets compared with wild-type mice. In this report, we examined the role of Fli-1 in dendritic cell and monocyte development using Fli-1ΔCTA B6 mice. Fli-1ΔCTA and wild-type B6 mice were sacrificed at the ages of 8-12 weeks and dendrtic cell and monocyte populations in blood, spleen and bone marrow were analyzed with Flow cytometery using specific antibodies. Fli-1ΔCTA B6 mice had significantly increased conventional dendritic cells and monocytes in blood compared with wild-type control B6 mice. Also dendritic cell population in the spleen and the common dendtitic cell precursor population in bone marrow are significantly increased compared to wild-type controls. Our results demonstrate that the Fli1 plays an important role in myeloid cell development and the CTA domain in Fli-1 protein is negatively regulates the myeloid cell development. Further molecular mechanisms regarding effects of Fli-1 on myeloid cell differentiation are under investigation.
Sustained LAIR/ITIM expression is linked to C1Q mediated dendritic cell differentiation arrest at the monocyte to DC interface (153.35)
Authors: Santiago-Schwarz Frances , Espaillat Mel Pilar 
Published: January 2023

Abstract
LAIR-1, an immune cell receptor with ITIM motifs, is highly downregulated on DCs arising from monocyte (mono) precursors. Indicative that LAIR/ITIM signaling modulates DC growth, engaging LAIR with anti-LAIR Ab inhibits DC differentiation and sustains the growth of LAIRhi mono-mΦs. Soluble C1q also disrupts DC differentiation to yield mono-mΦs over DCs. Since C1q contains the collagen motif known to be a ubiquitous ligand for LAIR, we predicted that C1q activity on DC growth is linked to LAIR. Studies were conducted during the 3 day mono/DC interface, which denotes a key transition from innate to adaptive immunity. Flow cytometry analysis of human mono/DC precursors cultured with DC growth factors (GM-CSF+IL-4) revealed reduced levels of LAIR by 24h; by 72h, ~50% of cells dimly expressed LAIR. In contrast, when C1q was combined with GM-CSF+IL-4, most cells were LAIRhi at 72h. C1q effects were dose related peaking at 20ug/ml (physiologic). Mono/DC markers studied in parallel with LAIR confirmed DC differentiation arrest with C1q. M-CSF also sustained the growth of LAIRhi mono-mΦs. Our results identify biologic factors regulating LAIR at the critical mono/DC interface and substantiate that C1q interacts with specific ligands on mono precursors to modulate DC differentiation. C1q binds to monos via its globular heads (likely via gC1qR). Thus, Gly-Pro-Hyp motifs on the C1q collagen tail could be available to engage LAIR to promote ITIM signaling and interrupt DC growth.
Eomesodermin and T-bet each specify a distinct natural killer cell lineage (153.36)
Authors: Gordon Scott , Mooney Erin , Reiner Steven 
Published: January 2023

Abstract
Natural killer (NK) cells play a critical role in host defense against tumors and intracellular pathogens. Eomesodermin (Eomes) and T-bet are T-box transcription factors that direct fate and function in cytotoxic lineages, such as CD8+ T cells and NK cells. Eomes and T-bet redundantly regulate differentiation of CD8+ effector T cells. Here we report that Eomes and T-bet play complementary and non-redundant roles in NK cell development. We find that conventional, CD49b-expressing (DX5+) NK cells fail to develop in the absence of Eomes. T-bet, however, is dispensable for the formation of conventional NK cells, even though this lineage expresses both T-bet and Eomes. Eomes-dependent, DX5+ NK cells are absent in neonatal mice. Instead, non-canonical, immature-appearing NK cells, characterized by the expression of the death ligand TRAIL, comprise the early neonatal NK cell repertoire. Non-canonical NK cells express T-bet but not Eomes and are absent in animals lacking T-bet. T-bet-dependent, TRAIL+ NK cells are less prevalent in lymphoid tissue of adult mice but represent a major subset of NK cells in the adult liver. In animals lacking both Eomes and T-bet, we find that neither Eomes-dependent, DX5+ NK cells nor T-bet-dependent, TRAIL+ NK cells develop, and that double mutant mice are devoid of all NK cells. Eomes and T-bet thus coordinate transcriptional programs required for development of two genetically, phenotypically, and functionally distinct NK cell lineages.
TCR signaling drives early growth response 2 gene expression and enforces natural killer T cell lineage commitment (153.40)
Authors: Seiler Michael , Mathew Rebecca , Spooner Chauncey , Barr Kenneth , Singh Harinder , Bendelac Albert 
Published: January 2023

Abstract
The transcription factor PLZF is required to drive lineage diversification of would-be conventional T cells into the natural killer T cell fate. However, the molecular machinery linking extracellular signaling to this fate choice is unknown. Recently, the early growth response 2 (Egr2) transcription factor, a calcineurin-dependent lineage resolution factor, was found to be important for NKT development. Here, we utilize an Egr2/IRES-GFP transgenic mouse model to show that Egr2 is uniquely induced immediately after TCR engagement in NKT cells. Competitive bone marrow chimeras with T cell conditional knockout mice indicate that Egr2 is required for both stage zero to stage 1, and stage 2 to stage 3 transitions of NKT development. Importantly, Egr1/Egr2 double deficient, stage 1 arrested NKT cells fail to express PLZF, in striking contrast to wild type stage 1 NKTs. Gel shift analysis shows in vitro translated Egr2 protein binds to a conserved site within the zbtb16 (PLZF) promoter. ChIP-seq of Egr2-bound chromatin from thymic NKTs confirmed in vivo binding of the zbtb16 promoter. Finally, we show that strong TCR signals are important for Egr2 induction in NKT cells. We propose that Egr2 links the NKT TCR signal with resolution of a mixed lineage state, driving PLZF expression in addition to other critical NKT lineage properties. We also suggest other receptor-dependent cell fates may be controlled by similar induction of resolution factors.