bluuebunny/crossref_metadata_2025 huggingface dataset:
TGF-β1 suppresses mast cell functions in vivo. (151.15)
Authors: Fernando Josephine , Ryan John
Published: January 2023

We have previously demonstrated the immunosuppressive effects of TGFβ1 on IgE signaling in human and mouse mast cells in vitro. Here we extend our focus to in vivo models, including mast cell-dependent airway inflammation. TGFβ1 injection into unimmunized mice reduced peritoneal mast cell numbers and their expression of the high affinity IgE receptor, as well as cKit. Further, TGFβ1 delivered as an aerosol to ovalbumin-immunized mice decreased pulmonary eosinophilic inflammation and mucus production. TGFβ1 did not affect Syk, Stat5, Fyn or Akt mRNA expression. However, mast cell Fyn and Stat5 protein levels were diminished by TGFβ1 treatment in vitro, while Akt and Syk were unaffected. Interestingly, we found that mast cells from the 129/SvJ mouse strain were resistant to TGFβ1-mediated suppression. Unlike their C57BL/6 counterparts, 129/SvJ mast cells did not demonstrate reduced IgE receptor expression or IgE-mediated cytokine production after culture with TGFβ1. These data show that TGFβ1 is a potent suppressor of the mast cell response that may be altered by host genetic makeup. We further find that the effects of TGFβ1 include selective reduction in key IgE-mediated signaling molecules.
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

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

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

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

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

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

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

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 (>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

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

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

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

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

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

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

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.
Development of iNKT cells: influence of Y-chromosome genes (153.44)
Authors: Toussaint Leon , Case Laure , Terrizzi Stephanie , Ericson Jeffrey , Benoist Christophe , Teuscher Cory , Brossay Laurent
Published: January 2023

In previous studies, we identified a mouse strain, B6.Ynkt, in which only males were deficient in iNKT cells and demonstrated that the deficiency was linked to Y-chromosome factors. We reasoned that the deficiency could be due to either a consomic effect of the Y-chromosome or due to a spontaneous germ line mutation on the Y- chromosome that occurred during the backcross to B6 background. To test the consomic hypothesis, we examined the iNKT cell frequency and number, in 16 different Y consomic strains. Although there is variation between the different strains tested none of them have a drastic decrease in iNKT cell number and frequency. To test the second hypothesis, we performed µarray on residual iNKT cells and conventional CD4+ T cells from the B6.Ynkt strain. The gene expression profile from these cells was compared to B6 iNKT cells and CD4+ T cells. We found that 4 contiguous genes on the Y-chromosome, were not expressed in the B6.Ynkt iNKT cells but were expressed in wild-type B6 iNKT cells. Importantly, these genes were expressed similarly in conventional CD4+ T cells from both wild-type B6 and B6.Ynkt mice suggesting the phenotype observed was not due to a deletion in this area. Genomic analysis confirmed that these 4 genes are present in both strains of mice. Taken together, our results argue against a consomic effect. Rather the data revealed a possible genetic expression repression of specific genes found contiguously on the Y-chromosome.
A simple and fast method for the enrichment of lymphoid progenitors from mouse bone marrow (153.9)
Authors: Tabatabaei-Zavareh Nooshin , Thomas Terry , Fairhurst Maureen
Published: January 2023

The expression of IL-7Rα in the common lymphoid progenitor (CLP) population marks initiation and/or commitment to the lymphoid lineage. CLPs hold potential for only B, T and NK cell lymphoid lineages and are defined as Lin−IL-7Rα+c-KitloSca-1lo. Study of lymphocyte development largely relies on access to CLPs or other IL-7Rα+ lymphoid progenitors. Fluorescence-activated cell sorting (FACS) commonly used to isolate lymphoid progenitors is costly, time-consuming and possibly detrimental to cell viability. We describe a fast and efficient method for the isolation of lymphoid progenitors from mouse bone marrow (BM). This method is based on immunomagnetic, column-free cell separation technology (EasySepTM). Using this method, lineage positive cells are first depleted by cross-linking them to magnetic particles using biotinylated antibodies. Next, IL-7Rα+ cells are positively selected from the Lin−/lo population. After enrichment, average purity of Lin−IL-7Rα+ lymphoid progenitors as assessed by flow cytometry is 32 ± 10% (n=18). Lin−IL-7Rα+c-KitloSca-1lo cells are enriched 60-fold from 0.08 ± 0.05% in start BM to 4.6 ± 2.5%. Limiting dilution analysis of enriched cells shows increased frequencies of B cell (1:73, n=12), T cell (1:16, n=5) and NK cell (1:65, n=6) progenitors as compared to non-depleted control BM. This system introduces an easy method for the enrichment of lymphoid progenitors with good viability that will enable study of developmental immunology.
TLR-9 agonist protects human monocytic cells against HIV-Vpr-induced apoptosis: a critical role for Calmodulin-dependent protein kinase-II, c-JUN N terminal kinase and anti-apoptotic c-IAP-2 gene (154.11)
Authors: Saxena Mansi , Busca Aurelia , Pandey Sunita , Kumar Ashok
Published: January 2023

Monocytic cells unlike CD4+ T cells survive HIV induced apoptosis and serve as key viral reservoirs. Mechanisms underlying the development of resistance to HIV-induced cytopathic effects are poorly understood. In chronic HIV infections, microbial products translocate from the gut and may activate lymphocytes conferring anti-apoptotic signals. By using Vpr52-96 peptide, an accessory protein of HIV known to cause apoptosis in various cell types, as a model for apoptosis-inducing protein, we demonstrate that Vpr52-96 induced apoptosis in primary monocytes and undifferentiated THP-1 cells. However, monocyte-derived macrophages and PMA-differentiated THP-1 cells exhibited profound resistance to Vpr-induced apoptosis. Interestingly, prior treatment of primary monocytes and undifferentiated THP-1 cells with TLR-9 agonist, CpG, induced resistance to Vpr-mediated apoptosis. This resistance was found to be mediated by extracellular calcium influx, activation of c-Jun N terminal kinase via Calmodulin-dependent kinase II and induction of anti apoptotic cIAP2 gene. Our results also revealed a novel pathway by which Vpr may dysregulate TLR signaling in monocytes by targeting MyD88. Interestingly, CpG protected these cells by blocking the Vpr-induced inhibition of MyD88. Overall, our results suggest that anti-apoptotic cIAP2 gene and calcium activated JNK signaling play a crucial role in conferring resistance induced by CpG against Vpr-mediated apoptosis in monocytic cells.
Basic mechanisms of Friend virus-induced immunomodulation and tumorigenesis (154.34)
Authors: Shen Limei , Balkow Sandra , Bros Matthias , Grabbe Stephan
Published: January 2023

Friend murine leukemia Virus (FV) infection of immunocompetent mice is a well established model to acquire further knowledge about retrovirus-associated mechanisms, enabling the development of preventive and therapeutic treatments against retrovirus-induced diseases. Our own studies show that formation of the immunological synapse between FV-infected DCs and T cells is altered. Those DCs induced a regulatory phenotype and suppressive function in cocultured CD4+ T cells (Treg). In order to identify key molecules that contribute to this Treg induction, expression patterns of cytoplasmatic proteins in uninfected and FV-infected DCs were comparatively analyzed by Protein-mass fingerprinting. By that approach about 600 differentially expressed proteins were identified. Interestingly, many of these are cytoskeletal proteins, and some are known to be involved in DC-T cell interactions (e.g. Myosins1F, 9, and 14). Notably, several MHCII antigens are downregulated in FV-infected DCs as compared with uninfected DCs. Recent studies show that the endocytic capacity of FV-infected DCs was significantly enhanced as compared with control DCs. Our current work is focussed on elucidating the function of signalling proteins which are differentially expressed in FV-infected vs. uninfected DCs. Taken together, our work is aimed to understand how retroviral infection of DCs alters DC-T cell interactions, and at which extent these effects contribute to retrovirus-induced immunosuppression.
Human herpesvirus 6 suppresses T cell proliferation through the induction of infected cell cycle arrest in G2/M phase (154.38)
Authors: Yao Kun , Li Lingyun , Zhou Feng , Chi Jing , Wang Fang , Peng Guangyong , Gu Bin , Xie Fangyi , Qing Jian , Feng Dongju , Lu Shiqiang
Published: January 2023

Human herpesvirus 6 is an important immunosuppressive and immunomodulatory virus which infects immune cells and strongly suppresses the proliferation of infected cells. However, the mechanisms responsible for the regulation and suppression mediated by HHV-6 are still unknown. In this study, we examined the ability of HHV-6A to manipulate cell cycle progression in the infected cells and explored the potential molecular mechanisms. We demonstrated that infection with HHV-6A imposed a growth-inhibitory effect on HSB-2 cells by inducing cell cycle arrest at G2/M phase. We then showed that the activity of Cdc2-cyclinB1 complex was significantly decreased in HHV-6A infected HSB-2 cells. Furthermore, we found that inactivation of Cdc2-cyclinB1 in infected cells was through the inhibitory Tyr15 phosphorylation resulting from the elevated Wee1 expression and inactivated Cdc25C; Meanwhile, the reduction of Cdc2-cyclinB1 activity in infected cells was also partly due to the increased expression of cell cycle regulatory molecule p21 in a p53-dependent manner. In addition, HHV-6A infection activated the DNA damage checkpoint kinases, Chk2 and Chk1. Our data suggest that HHV-6A infection induces G2/M arrest in infected T cells via various regulatory mechanisms. These results demonstrate potential mechanisms involved in immune suppression and modulation mediated by HHV-6 infection, and provide new insights relevant to the development of novel vaccines and immunotherapeutic approaches.
Ectromelia NF-κβ inhibitor 002 is essential for virus pathogenesis (154.4)
Authors: Rubio Daniel , Alcami Antonio , Sigal Luis
Published: January 2023

Ectromelia virus is an orthopoxvirus and the causative agent of mousepox in mice. The virus codifies several proteins to control the host response. One of them is the NF-κβ inhibitor, ECTV 002 which has orthologs in other poxviruses including cowpox, monkeypox and variola viruses. We generated an ectromelia virus that lacks this gene (ECTV Δ002) and its correspondent revertant virus. Both viruses grew normally in vitro; however, when used to infected BALB/C mice in the footpad, only the WT and revertant viruses killed the mice. When using different doses, the LD50s were lower than 1pfu for the WT and revertant viruses but higher than 105 for the ECTV Δ002 indicating an attenuation of at least 5 orders of magnitude. We then analyzed the virus spread by measuring virus titers in different organs. The virus load in the draining lymph node was lower in mice infected with the ECTV Δ002 than with the revertant virus and consequently, the virus titer in peripheral organs like spleen or liver was lower as well. When we compared the immune responses in the draining lymph nodes by flow cytometry, we did not find significant differences between ECTV Δ002 and WT viruses. However, 7 dpi, the mice infected with ECTV Δ002 had elevated numbers of NK, CD8 and CD4 cells in their spleens as compared to mice infected with revertant virus. Altogether our results show that ECTV 002 is essential for ECTV pathogenesis and that the control of the infection occurs early in the draining lymph node.
A cationic lipid/DNA complexes (JVRS-100) combined with influenza A H5N1 vaccine induce long-term antibody responses and protection against H5N1 virus in BALB/c mice (155.31)
Authors: Liu Feng , Hancock Kathy , Katz Jacqueline , Fairman Jeffery , Lu Xiuhua
Published: January 2023

Highly pathogenic avian influenza A H5N1 viruses pose a pandemic threat. An adjuvant is needed due to poor immunogenicity of pre-pandemic H5N1 vaccine in humans. JVRS-100 is a novel adjuvant, composed of cationic liposomes and plasmid DNA. Here, we investigated the long-term immunogenicity and protective efficacy of JVRS-100-adjuvanted H5N1 vaccine in mice. One year after immunization with one dose of vaccine with or without JVRS-100 adjuvant, significantly higher levels of hemagglutination-inhibition antibody and IgG antibody could be detected in serum of mice immunized with adjuvanted vaccine compared to that of mice receiving vaccine only. No or limited waning of antibody response was observed up to one year after immunization. IgG2a clearly predominated in serum of mice that received adjuvanted vaccine, suggesting that JVRS-100 preferentially stimulates a Th1 response. Mice were challenged with a high lethal dose of H5N1 virus 14 months after immunization. Co-administration of JVRS-100 with H5N1 vaccine significantly reduced virus titers in the lungs and brains and completely protected mice from severe disease and death. Our results indicate that the addition of JVRS-100 to H5N1 vaccine enhanced long-term antibody responses and protection against H5N1 virus lethal disease in mice. Therefore, JVRS-100 warrants further investigation as a potential adjuvant for use in human vaccination against avian influenza viruses with pandemic potential.
IRX-2, a novel immunotherapeutic, enhances NK cell function and protects NK cells from TGFβ-mediated inhibition. (156.14)
Authors: Schilling Bastian , Schuler Patrick , Harasymczuk Malgorzata , Egan James , Whiteside Theresa
Published: January 2023

Background: IRX-2 is a primary biologic which has been used for therapy of head and neck cancer with promising clinical results. Since NK cell function is compromised in head and neck cancer (HNC) patients, we tested the effects of IRX-2 on human NK cells in vitro. Methods: PBMC were isolated 10 HNC patients and 10 healthy donors (HD). NK cell phenotype and function were compared before and after culture ± IRX-2 or 50 IU/ml IL-2. NK cell phenotype, signaling, cytotoxicity and cytokine production were studied by flow cytometry. Results: Cytotoxicity was impaired in HNC patients (614 LU20 vs. 2760 LU20, p<0.01). Mean expression of NKG2D, NKp30 and NKp46 was significantly lower in HNC patients than in HD (p<0.05). Patients’ NK cells treated with IRX-2 expressed higher levels of these receptors than those treated with IL-2 (p<0.05) and showed higher cytotoxicity (3000 LU20 vs. 1300 LU20, p<0.01). In the presence of TGF-β, IL-2 did not increase the expression of activating NK cell receptors while IRX-2 still upregulated NKG2D and NKp30 (p<0.05). More importantly, IRX-2 protected NK cells from TGF-β- mediated impairment of cytotoxicity (p<0.05), while IL-2 did not. Conclusions: IRX-2 is more effective in enhancing and protecting NK cell function of HNC patients in vitro than IL-2 alone. Our data suggest that clinical responses previously observed in HNC patients treated with IRX-2 could be, at least in part, attributed to restored NK cell function.
Cytomegalovirus-based cancer vaccines expressing TRP2 induce potent antitumor effect against melanoma in mice (156.20)
Authors: Xu Guangwu , Smith Tameka , Hill Ann
Published: January 2023

Cytomegalovirus (CMV) induces strong and long-lasting immune responses, which make it an attractive candidate for a cancer vaccine vector. In this study, we tested whether a tumor antigen expressed in CMV can induce an anti-tumor effect. We expressed the melanoma tumor antigen, tyrosinase-related protein 2 (TRP2), in murine cytomegalovirus (MCMV). A single prophylactic dose of MCMV-TRP2 induced complete rejection of B16 melanoma cells. In a therapeutic setting, MCMV-TRP2 significantly inhibited the growth of melanoma. In addition, the growth of B16 cells inoculated five months after a single vaccination with MCMV-TRP2 was still inhibited, which indicates the vaccine induced a long-term protection. MCMV-TRP2 induced the production of both TRP2-specific CD8 T cells and antibodies. Depletion of CD4 and CD8 T cells did not compromise the antitumor effect, but MCMV-TRP2-vaccinated B cell deficient (µMT) mice did not reject B16 cells. This indicated that antibody rather than T cells was the main mediator of the antitumor effect induced by MCMV-TRP2. We also made a spread-deficient MCMV-TRP2 vaccine, which showed a similar antitumor effect. In conclusion, our study indicates that TRP2 expressed in MCMV induces a strong and long-lasting anti-melanoma effect through an antibody dependent mechanism. Our findings demonstrated for the first time that CMV is a promising vector for cancer vaccines.
Sequential administration of chemotherapy and tumor lysate pulsed autologous dendritic cells induce stronger anti-tumor activity in treatment of metastatic colorectal cancer: immunologic results and the underlying mechanisms from a phase II trial. (156.3)
Authors: Wu Yanfeng , Wan Tao , Cao Xuetao
Published: January 2023

We conducted a randomized phase II trial to determine whether sequential administration of chemoherapy plus DC vaccine is superior to chemotherapy alone. Our studies indicated that appropriate doses of oxaliplatin(OXA) and/or 5-Fu could enhance HLA-A2 and Fas expression, depressed FasL expression on SW480 cell surface and stimulate higher counts of IFN-γ secretion antigen-specific T cells, decrease the productions of immunosuppression cytokines such as IL-10, TGF-β, and VEGF in supernatants of treated SW480 cells, in peripheral blood of patients with metastatic colorectal cancer(MCRC) and CT26 liver metastasis mice models. The proportions of CD4+CD25+ Treg were observed to reduce in PBMCs from MCRC patients or in splenocytes from CT26 liver metastasis mice, as well as CD11b+Gr-1+ MSCs after the chemotherapy. Sequential antigen-pulsed DC administration could stabilize above-mentioned results afterwards. We also found that the production of Hsp70, a new Th1 polarization adjuvant which is a chaperone important to the DCs’ cross-presentation, significant increased in peripheral blood of MCRC patients after OXA and 5-Fu chemotherapy. The survival time of CT26 liver metastasis mice had been significant prolonged and specific anti-tumor immune response induced by receiving sequential immuno-chemotherapy. We hope that these results may provide new strategies and methods in clinical trials revolving immunotherapy.
Carcinoembryonic antigen-related cell adhesion molecule 1 down regulates Syk coupled neutrophil NLRP3 inflammasome activation through ITIM and the recruitment of SHP-1 (157.12)
Authors: Lu Rongze , Pan Hao , Shively John
Published: January 2023

IL-1β is a critical pro-inflammatory cytokine produced by innate immune cells, and is regulated by the inflammasome. Recently thioredoxin -interacting protein (TXNIP) has been shown to associate with and activate the Nod-like receptor protein 3(NLRP3) inflammosome in a ROS-sensitive manner. We have found LPS induced the NLRP3 inflammasome activation in neutrophils was dependent on ROS production and lysosomal damage, both of which were regulated by spleen tyrosine kinase (Syk).We have demonstrated LPS induced phosphorylation of Syk and formation of a complex platform including Toll -like receptor 4(TLR4), TXNIP and p-Syk, to facilitate ROS production the inflammosome activation. We also found that carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) associated with TLR4 ,TXNIP and p-Syk complex in response to LPS. IL-1β, pro-caspase-1, ROS and lysosomal destabilization are enhanced in neutrophils from ceacam1-/- mice. CEACAM negatively regulates phosphorylation of Syk and the inflammosome activation by recruiting phosphatase SHP-1 through CEACAM1’s (immunoreceptor tyrosine-based inhibition motif) ITIMs. Pharmaceutical inhibition of Syk activation or knock down of Syk attenuated Syk phosphorylation and the inflammosome activation in ceacam1-/- neutrophils. Reconstitution of CEACAM1 restores inflammasome activation to normal level. Therefore, we identify CEACAM1 as a negative regulator of the Syk coupled NLRP3 inflammasome activation.
Cytosolic DNA sensing by AIM2 inflammasome is integral to anti-microbial defense (157.5)
Authors: Rathinam Vijay , Waggoner Stephen , Jiang Zhaozhao , Sharma Shruti , Monks Brian , Waggoner Lisa , Fitzgerald Katherine
Published: January 2023

Cytosolic DNA of microbial and eukaryotic origins elicits strong inflammatory responses characterized by the production of type I interferons and IL-1β/IL-18. The maturation of proforms of IL-1β and IL-18 into bioactive forms is mediated by a multiprotein complex in the cytosol called inflammasomes. Recently, AIM2 (absent in melanoma 2) protein was shown to recognize cytosolic DNA and form an inflammasome complex. To understand the role of AIM2 in innate immunity, we generated AIM2 deficient mice. Using the AIM2-deficient mice, we demonstrate that AIM2 inflammasome is an essential and non-redundant mechanism mediating IL-1β and IL-18 responses to cytosolic DNA from bacterial and viral infections. In contrast, cytosolic DNA-induced activation of NF-κB and IRF-3 are normal in AIM2-/- cells indicating that AIM2 is least likely to play a role in type I interferon production elicited by cytosolic DNA. Importantly, AIM2 deficient mice infected with vaccinia virus (VV) or mouse cytomegalovirus (mCMV) demonstrated a marked reduction in serum IL-18 levels and in number of IFN-γ producing splenic NK cells, which are critical events in the early control of VV and mCMV infection. Correspondingly, AIM2 deficiency hampered the control of virus replication in the infected mice. Overall, we demonstrate that AIM2 inflammasome is an integral component of cytosolic DNA sensing in microbial infections.
NLRC4 inflammasomes in dendritic cells evoke non-cognate effector function by memory CD8 T cells that improve immunity to Salmonella typhimurium (158.1)
Authors: Bedoui Sammy , Andreas Kupz , Guarda Greta , Gebhardt Thomas , Sander Leif , Curtis Roy , Tschopp Jurg , Strugnell Richard
Published: January 2023

Salmonella enterica is the causative agent of typhoid fever, a condition with considerable global human morbidity and mortality. Although it is well established that interferon-γ (IFN-γ) is critical for the control of Salmonella infections, the cellular source of this protective cytokine and its regulation during Salmonella infection remain unclear. CD4 T cells have been suggested as prime producers of IFN-γ during Salmonella infection. However, in contrast to non-lethal Salmonellosis ensuing in CD4 T cell deficient hosts, mice lacking IFN-γ rapidly succumb to the infection, suggesting that cells other than CD4 T cells contribute to IFN-γ-mediated protection against Salmonella infection. Here, we identify a novel mechanism by which Salmonella Typhimurium-derived flagellin triggers IFN-γ secretion by CD62Lhi memory CD8 T cells in an antigen-independent fashion. Our findings not only resolve that this phenomenon depends on the release of IL-18 by dendritic cells (DC) upon flagellin recognition through NLRC4 inflammasomes in vivo, but also demonstrate that non-cognate memory CD8 T cells improve survival following Salmonella Typhimurium infection. Thus, these findings uncover a novel, innate role for memory CD8 T cells in protection against Salmonella and establish a functional link between inflammasome activation in DC and CD8 T cell function in vivo.
Macrophage migration inhibitory factor increases MHC II and CD86 on dendritic cells and favors proinflammatory cytokine production mediated by ERK1/2 phosphorylation in Trypanosoma cruzi infection (158.14)
Authors: Rodríguez-Sosa Miriam , Terrazas César A , Huitron Erick , Vazquez Alicia , Juárez Imelda , Camacho Griselda , Elsa A. Calleja
Published: January 2023

Macrophage migration inhibitory factor (MIF) has been found to be involved in host resistance to several parasitic infections. To determine the mechanisms of MIF-dependent responses to Trypanosoma cruzi (Queretaro strain), we investigated host resistance in MIF-/- mice (BALB/c background) during an intraperitoneal (i.p.) infection. We focused on the potential involvement of MIF on Dendritic Cell (DC) maturation and cytokine production. Following i.p. T. cruzi infection, wild type (WT) mice developed a strong IL-12 response with an adequate maturation of their DC in draining mesenteric lymph node (MLNDC), these mice were resistant to a challenge with 5x103 parasites. In contrast, similarly infected MIF-/- mice mounted a weak IL-12 response, displayed immature MLNDCs in the early phases of infection and rapidly succumbed to a similar challenge. Lack of maturation and IL-12 production of DCs in response to total or soluble T. cruzi antigens was confirmed by in vitro studies, such effects were reversed following treatment with recombinant MIF. Interestingly, whereas DC from WT mice exposed to total T. cruzi antigens had ERK1/2 MAPK phosphorylation, MIF-/- DC did not. These findings demonstrate that MIF-induced early DC maturation and IL-12 production mediate resistance to T. cruzi infection, probably by activation of ERK1/2 MAPK pathway.