bluuebunny/crossref_metadata_2025 huggingface dataset:
Study of peripheral tolerance in ADA SCID patients after different treatments (143.36)
Authors: Brigida Immacolata , Sauer Aisha , Selleri Silvia , Scaramuzza Samantha , Ripamonti Anna , Casiraghi Miriam , Ferrua Francesca , Olek Sven , Roncarolo Maria Grazia , Aiuti Alessandro
Published: January 2023

Adenosine deaminase (ADA)-SCID is characterized by increased purine metabolites and severe combined immunodeficiency. Autoimmune manifestations have been observed in milder forms or after enzyme replacement therapy (ERT), and recently in patients following gene therapy (GT). nTregs have a full enzymatic machinery to generate and sustain high concentrations of extracellular adenosine and may be involved in the pathogenesis of autoimmunity. We analyzed the frequency of CD4+CD25+FOXP3+CD127- in the peripheral blood, the methylation profile of the FoxP3 gene and the suppressive function of nTreg in ADA-SCID patients following ERT, GT or bone marrow transplant (BMT) as compared to controls. We found that the proportion and function of nTregs after GT mirrored that of controls, while nTreg from the ERT group showed a reduced frequency and impaired function. We also investigated the maturation state of B cells in the peripheral blood of ADA-SCID patients. After ERT and early after GT, patients displayed an increased proportion of transitional B cells (CD24highCD38high) and of cells with an incomplete BCR (CD21-CD35-) as compared to BMT and healthy controls. At later follow up patients treated with GT showed a tendency to normalize the phenotype in parallel to an increased frequency of gene corrected cells. Collectively, these data provide a first indication of predisposition to autoimmunity in ADA-SCID and the role of different treatments in the maintenance of peripheral tolerance.
Evidence supporting the existence of a novel histaminergic pathway in the regulation of EAE susceptibility (143.51)
Authors: Saligrama Naresha , Del Rio Roxana , Case Laure , Noubade Rajkumar , Teuscher Cory
Published: January 2023

Histamine (HA) is a key regulator of experimental allergic encephalomyelitis (EAE), the autoimmune disease model of multiple sclerosis (MS). Histidine decarboxylase deficient mice (HDCKO), which are unable to synthesize HA, exhibit more severe EAE and increased IFN-γ production by splenocytes in response to MOG35-55. HA exerts its effects through four different G protein coupled receptors (GPCR): H1, H2, H3 and H4 (H1-4R). Each HA-receptor has been shown to influence EAE pathogenesis. In the mammalian brain there is, however, evidence for the existence of non-GPCR signaling by HA which is picrotoxin-sensitive and mediated by chloride conductance. GABAA receptor subunits can form HA-gated chloride channels in vitro suggesting that an ionotropic HA-receptor might contain known ligand-gated chloride channel subunits. To test the hypothesis that non-GPCR signaling by HA plays a role in immune responses, we generated H1-4RKO mice and studied their susceptibility to EAE. Here we report that in contrast to HDCKO mice, H1-4RKO mice develop less severe EAE compared to WT animals. Furthermore, splenocytes from immunized H1-4RKO mice produce significantly less IFN-γ compared to WT mice. Taken together these data support the existence of a novel HA signaling pathway in regulating EAE susceptibility.
Role of IFI202b in the suppressive ability of CD8+Treg induced in (NZB x NZW) F1 lupus mice (143.56)
Authors: Singh Ram , Dinesh Ravi , Hahn Bevra
Published: January 2023

Tolerance with an artificial peptide (pConsensus, pCons) based on anti-DNA IgG sequences containing MHC class I and class II T cell determinants, causes NZB/NZW F1 female (BWF1) mice to develop CD8+Foxp3+ inhibitory T (Ti) cells which suppress anti-DNA Ig production. CD8+T cells from mice tolerized with pCons expressed Ifi202b more than two-fold higher than cells from untolerized mice, with an increase 1-4 weeks after tolerization and a return toward baseline at 6 weeks. In vitro polyclonal activation significantly increased Ifi202b mRNA expression in tolerized CD8+T cells. Importantly, silencing of Ifi202b abrogated the suppressive capacity of tolerized CD8+T cells. This was associated with decreased expression of Foxp3, and decreased gene and protein expression of TGFb, but not of IL-2, IFN-g, IL-10 or IL-17. These changes were not related with resistance to apoptosis induced in CD8+Treg. Although Ifi202b is induced by interferons, silencing of another IFN-induced gene upregulated in tolerized CD8+T cells, IFNar1, had no effect on the ability of CD8+T cells to suppress autoantibody production. Taken together, these data identify a role for Ifi202b in the suppressive capacity of peptide-induced regulatory CD8+T cells via effects on the expression of Foxp3 and the synthesis of TGFb.
Deimmunization of HLA-DR Epitopes in the C2 Domain of Human FVIII (144.29)
Authors: Song Chang , Martin William , DeGroot Annie , Scott David
Published: January 2023

To reduce immunogenicity of therapeutic proteins, recombinant proteins can be modified to lower HLA binding of epitopes, a method (deimmunization) useful with monoclonal antibodies. Since the immune response to human clotting factor VIIII limits therapeutic efficacy, we reported that immunodominant peptides in the C2 domain of fVIII could be modified to reduce MHCII binding to display less antigenicity in vitro in a hemophilic fVIII KO mouse model (H-2b). We have extended these studies in humanized HLA-DR transgenic mice and identified responses to C2 peptides p2191-2210, p2231-51, p2254-69, p2271-89; and p2310-30 in DR2, DR3, DR4 mice, as well as in DR3 mice crossed to fVIII KO hemophilic mice. DR transgenic mice were immunized with the designated original peptides (ORG) or with rFVIII in CFA, and LN cells tested in T-cell proliferation assays against rFVIII and ORG peptides. While p2191 dominantly stimulates H-2b, other peptides are immunodominant in DR2, DR3 and DR4 mice. A different pattern was revealed (DR3 x fVIII KO) mice depending on whether they also express H-2b. Efforts to test the reactivity/antigencity and immunogenicity of modified (MOD) peptides predicted to have lower class II binding will be reported. These results will be utilized to design and produce a functional full-length modified rFVIII that can be used as a therapeutic protein in hemophilia A (Supported by NIH R43 HL088834-01).
Unique enzymes improve mass spectroscopy of IgG and make it possible to use affinity purification of acid sensitive IgG molecules (144.31)
Authors: Frediksson Sarah , Hedström Martin , Olsson Fredrik , Allhorn Maria , Collin Mattias
Published: January 2023

The human pathogen Streptococcus pyogenes produces various surface proteins in order to avoid the host immune defence systems. Two of these proteins, EndoS and IdeS, have proven to be very useful as tools in antibody engineering. EndoS hydrolyses the chitobiose core of the aspargine linked glycan on the heavy chain of IgG and IdeS is a cysteine protease that cleaves the IgG molecule at a unique site in the hinge region. Here we show, for the first time, that mass spectrometric analysis of antibody fragments are greatly facilitated by combining EndoS and IdeS activities. Utilization of RPC18 LC-ESI-MS generated a well-defined protein envelope with a deconvoluted mass of 24137 Da corresponding to the IgG1 heavy chain cleaved at position Gly236. This opens up new possibilities for utilizing a combination of these enzymes for fast and accurate analysis of IgG:s in a variety of mass spectrometry applications. In addition, a mutated variant of the EndoS enzyme was found to show a high affinity for IgG molecules and has been evaluated in affinity purification of intact, native IgG molecules since it excludes non-native IgG molecules. Furthermore, the affinity purification using the mutant EndoS can be performed at neutral pH which is favourable in purification of acid sensitive IgG clones. The performance and characteristics of Ides, EndoS and the EndoS mutant is highlighted during the presentation with data from several individual studies.
Rare Event detection using Acoustic Cytometry (144.4)
Authors: Anderson April , Bradford Jolene
Published: January 2023

Detection of rare events includes populations comprising less than 5% of the total cells, which involves the detection of stem cells, minimal residual disease and fetomaternal hemorrhage. Analysis of rare cell populations requires the collection of high numbers of events in order to attain a reliable measure of accuracy, leading to long acquisition times. Acoustic cytometry is a new technology which allows dilute samples to be processed quickly. We compared collection times of the Applied Biosystems® Attune™ Acoustic Focusing Cytometer to conventional cytometers. Varying numbers of CD34+ KG-1a cells were spiked into whole blood collected from a normal donor, stained with CD45 & CD34 conjugates, and then collected on traditional and acoustic cytometers. Two hundred thousand live cells were acquired and acquisition time recorded, with results compared across all instruments tested. Rare CD34+ populations were detected on all instruments, giving equivalent results. Acquisition time was significantly reduced using an acoustic cytometer compared to a standard flow cytometer. Acquisition times on the acoustic cytometer can be reduced by up to 5-10 fold compared to traditional flow cytometers, while maintaining comparable results in rare event detection. Utilizing the acoustic focusing cytometer rare event acquisition can be accomplished in one fifth of the time without sacrificing data integrity.
DN-Treg cells induce long-term cardiac allograft survival in fully MHC mismatched recipients but do not eliminate T memory cells (145.13)
Authors: Zhang Zhu-Xu , Lian Dameng , Huang Xuyan , Wang Shuang , Sun Hongtao , Liu Weihua , Min Wei-ping , Garcia Bertha , Jevnikar Anthony
Published: January 2023

Establishment of immune tolerance is a major goal in transplantation. Regulatory T (Treg) cells play an important role in the regulation of immune responses. Our previous studies have shown that TCRαβ+CD3+CD4-CD8-NK1.1- (double negative, DN) Treg cells suppress anti-donor T cell responses and prolong allograft survival in a single MHC-mismatched mouse model. In this study, we tested whether DN-Treg cells can induce long-term graft survival in the stringent BALB/c to C57BL/6 heart transplant model. Adoptive transfer of 107 DN-Treg cells in combination with rapamycin (RAPA) treatment (day 1- 9) induced long-term graft survival (101 vs. 39 days RAPA alone, p<0.01). DN-Treg treated recipients had few graft-infiltrating CD4+ and CD8+ T cells on day 40 compared to massive infiltration in RAPA treated controls. Memory T (Tm) cells (CD3+CD44+) were found to be resistant to DN-Treg cell-mediated suppression in vitro. The effect of DN-Treg on Tm in vivo was tested in B6-Rag1-/- mice. DN-Treg cells along with CD44+ Tm cells modestly prolonged BALB/c skin graft survival in contrast to DN-Treg with CD44- T cells (26 vs 43 days, P<0.001). In conclusion, DN-Treg cells combined with short term immunosuppression, can prolong allograft survival in the absence of tolerance. Furthermore, this may be related to the resistance of Tm cells to DN-Treg cell suppression, suggesting alternative strategies will be needed to control Tm cells and establish tolerance after transplantation.
Establishment of a novel in vitro gut epithelial culture system for the study of T cell mediated gut injury during early GVHD (145.15)
Authors: Matos Jennifer , Dziopa Eugenia , Friedman Thea , Korngold Robert
Published: January 2023

Graft-versus-host disease (GVHD) is the primary complication of blood and marrow transplantion used to treat hematological diseases. Epithelial cells are apoptotic targets during GVHD. Gut epithelium is a key factor in the progression from localized to systemic GVHD, as damage to the intestines allows endotoxin transfer into general circulation, triggering cytokine release, organ damage and systemic inflammation. However, cellular mechanisms that regulate tissue damage in GVHD are poorly understood. Using the established MHC-matched, miHA-mismatched B6→BALB.B irradiation model of GVHD, we generated a novel primary cell culture system for in vitro study of gut epithelial cell injury by extracting intestinal epithelial cells from BALB.B mice for co-culture with B6 T cells. Our cultures are representative in their heterogeneity, containing both cytokeratin 15+ epithelial cells and smooth muscle cells that are easily differentiated from one another for analysis, with no contaminating dendritic cells or fibroblasts. In the skin, CD4+ and CD8+ T cells target cytokeratin 15+ stem cells of the basal membrane. Therefore, our in vitro gut epithelial cells may be targeted, and apoptosis induced, as in vivo. With this apoptotic co-culture model system in place, we will determine which of the perforin/granzyme, TRAIL, or Fas/FasL apoptotic pathways is critical at the early stages of intestinal GVHD, providing insight into potential therapeutic targets for prevention or treatment of GVHD.
Identification and characterization of langerin positive dendritic cells in the normal cornea and in transplantation. (145.27)
Authors: Saban Daniel , Hattori Taakaki , Chauhan Sunil , Dana Reza
Published: January 2023

A novel population of migratory dendritic cells (DCs), distinct from Langerhans cells (LC), was recently found to which also express langerin (CD207+) and reside throughout the body (skin, lung, gut, and liver). Moreover, such CD207+ DCs were shown to be important in generating immunity. Thus, we investigated whether CD207+ DCs exist in the cornea and their possible migration to lymph nodes (LN) in corneal alloimmunity. Methods: C57BL/6 corneas (n=30) were collected and epithelium separated from stroma. Tissue was pooled separately and digested enzymatically for FACS analysis of CD45, CD11c, CD207, CD205, and CD103. Immunohistology was also examined. Furthermore, BALB/c mice were allografted with C57BL/6 corneas and LNs evaluated for corneal CD207+ DCs vs. LCs. Results: Most epithelial and stromal DCs were CD207+ and co-expressed CD205+, but only the stromal subset expressed CD103+. Interestingly, following allotransplantation, the frequency of the stromal DCs (CD11c+ CD8a- CD207+ CD103+) decreased dramatically (by 4-fold) in ipsilateral LNs relative to naïve mice; whereas, the epithelial DCs (CD103-) markedly increased. A similar, but much less dramatic, trend was seen in the contralateral LN. Conclusions: This indicates the presence of CD207+ DCs in the cornea, distinct from LCs of the epithelium. Interestingly, a dramatic and diametrically opposed flux of such stromal DCs vs. LCs to LNs following transplantation suggests potentially differing functions in alloimmunity.
Extracellular purines serve as an innate trigger to allograft rejection (145.39)
Authors: Reichenbach Dawn , Li Qi , Demetris Anthony , Lakkis Fadi
Published: January 2023

Innate immune triggers are well studied in infectious models, yet they remain unclear in solid organ transplantation. Extracellular purines (e.g. ATP, adenosine), which signal through receptors on immune cells, modulate the extent of ischemia reperfusion injury. To test the effect of purinergic signaling on graft rejection, we used a cardiac transplant model in outbred mice in which accelerated graft rejection occurs within 4 days post-transplantation and is highly dependent on innate mediators. Treatment with NECA, an adenosine receptor agonist, 2 hours prior to heterotopic heart transplantation (n=8) completely abrogated accelerated rejection compared to untreated outbred mice (incidence = 28%, n=35). CD39 and CD73 are ectoenzymes responsible for metabolism of extracellular ATP to adenosine. To determine the effect of increased extracellular ATP in the local graft environment, hearts from CD73-deficient B6 mice were transplanted to BALB/c recipients. Accelerated rejection occurred in 24% of grafts from CD73-/- mice (n=17) compared to 2% of grafts from wt inbred controls (n=42). Skin transplants from CD39-/- and CD73-/- mice to BALB/c mice (n=8/group) rejected at the same rate as wt control B6 to BALB/c skin transplants (n=8), suggesting the effect was restricted to vascularized transplants. Our data suggests that modulation of purine metabolism serves as an early trigger of the innate immune response in vascularized organ transplantation.
Role of T cell subsets in promoting intestinal GVHD. (145.4)
Authors: Anthony Bryan , Wang Jiao-Jing , Gaughan Alice , Hadley Gregg
Published: January 2023

The relative contributions of CD4 vs. CD8 T cell subsets in graft-versus-host disease (GVHD) remains poorly defined. Previous studies in this lab have shown that the integrin CD103 is required for accumulation of CD8 T cells in epithelial compartments and concomitant epithelial injury during GVHD in MHC-mismatched transplant models. The goal of the present study was to define the role of the CD103 pathway in promoting GVHD following MHC-matched BMT, the most common clinical scenario. Our data showed no difference in the ability of WT or CD103-/- splenocytes to cause acute GVHD mortality in this transplant scenario. However, depletion of CD4 T cells on day 6 post transplant completely prevented acute GVHD mortality, despite a robust CD8 T cell response. These data suggested that acute gut injury and mortality elicited following BMT are mediated by CD4 T cells rather than CD8 T cells as is commonly assumed. Consistent with this hypothesis, CD4 depletion also prevented acute mortality following MHC-mismatched transplant. FACS analyses of long-surviving hosts in this model revealed that T cells infiltrating the host gut epithelium were initially CD103- but progressively acquired CD103 expression concomitant with progressive epithelial injury. These data indicate that CD103 is preferentially expressed by CD8 T cells at late post-transplant intervals, and that effective blockade of GVHD pathology will likely require combined blockade of the CD4 and CD103 pathways.
Loss of MRP-14 Augments Costimulatory molecule Expression and Exacerbates Cardiac Allograft Rejection (145.47)
Authors: Shimizu Koichi , Folco Eduardo , Rocha Viviane , Simon Daniel , Libby Peter , Mitchell Richard
Published: January 2023

MRP-14 serves as an early serum marker for monitoring acute allograft rejection. Despite functioning as a putative pro-inflammatory mediator, the pathophysiological role of MRP-14 in cardiovascular disease remains unknown. This study used murine cardiac transplantation to investigate the role of MRP-14 in cardiac allograft rejection using MRP-14-deficient mice. Results: In MHC class II allomismatched cardiac transplantation, allograft survival averaged 5.9±2.9 weeks (n=10) in MRP14-/- recipients compared to > 12 weeks (n = 15, p<0.0001) in wild-type (WT) recipients. At 2 week post-transplantation, MRP14-/-recipient allografts had significantly higher parenchymal rejection (PR) scores (2.8±0.8, n=8) than WT recipients (0.8±0.8, n=12,p<0.0001). MRP14-/-recipients had significantly increased allograft accumulation of T cells and macrophages and mRNA levels of IFN-γ, IP-10, Mig, and I-TAC compared to WT recipients. MRP14-/- dendritic cells (DC) stimulated with IFN-γ expressed significantly higher levels of the co-stimulatory molecules CD86 and ICOSL, and mixed leukocyte reaction using allo-EC-primed MRP14-/- DC resulted in significantly higher antigen-presenting function than WT DC. MRP14-/- DC infected with retroviral vector of MRP-8/14 expressed less CD80 and CD86 than those infected with control vector. Conclusion: This study indicates that MRP-14 modulates DC APC function and T cell priming and deficiency of MRP-8/14 complexes exacerbates acute allograft rejection.
The type 2 TNF receptor plays a critical role in the activation of Tregs in vivo. (34.3)
Authors: Housley William , O'Conor Catherine , Clark Robert
Published: January 2023

Recent studies suggest that regulatory T cells (Tregs) exhibit functional and phenotypic plasticity dependent on the local cytokine milieu. Previous reports have shown that TNF-α stimulates Treg proliferation in vitro and that the type 2 TNF receptor (TNFR2) is expressed on a subset of Tregs with increased in vitro function. We now report that TNFR2 is up-regulated on all activated Tregs and is required for normal Treg suppression in vivo and optimal maintenance of Foxp3 in vitro. Using a model of colitis, wild type (WT) or TNFR2-/- Tregs were transferred into RAG-1-/- mice together with WT CD4+CD25- effector T cells (Teff). TNFR2-/- Tregs were significantly less capable of suppressing both IFNg production and colitis. Surprisingly, TNFR2-/- and WT Tregs, activated in vitro prior to transfer, as well as iTregs generated in-vitro from WT or TNFR2-/- Teff, were identical in suppressive capacity in vivo. These results suggest that TNFR2 plays a critical role in the initial activation of Tregs in vivo. To determine if TNFR2 is involved in the stability of Tregs, Foxp3-GFP Tregs were sorted into TNFR2 High and TNFR2 Low populations and stimulated in-vitro with CD3ab and IL-2. After 3 days, the TNFR2 low population contained significantly fewer Foxp3+ cells than the TNFR2 high population. Overall, our results suggest that TNF-α signaling through TNFR2 may play a critical role in the initial activation of Tregs and may be an important factor for maintaining phenotypic stability.
Baicalein inhibits the enhancing effects of cigarette smoke extract (CSE) on inflammatory cytokine production in IL-1beta activated mast cells (35.5)
Authors: Chi David , Wu Fangbai , Lin Ta-Chang , Hall Kenton , Wu Zong , Krishnaswamy Guha
Published: January 2023

Human mast cells play a role in the pathogenesis of inflammatory airway diseases. We have reported that CSE increased IL-6 and IL-8 production in IL-1beta activated human mast cells (HMC-1). Baicalein (BAI) has been shown to have anti-inflammatory properties. This study aimed to examine the effect of BAI on IL-6 and IL-8 production in HMC-1. Two ml of HMC-1 at 1 x 106 cells/ml were cultured either with or without IL-1beta (10 ng/ml), and in the presence or absence of mainstream (Ms) or sidestream (Ss) CSE (0.25 mg/ml or 0.125 mg/ml), and BAI (30 μM). Treated HMC-1 were incubated for 24 hours and culture supernatants were collected and analyzed for the levels of IL-6 and IL-8 by ELISA. Both concentrations of Ms and Ss CSE significantly increased production of both IL-6 (p < 0.0001) and IL-8 (p < 0.0005) in IL-1beta activated HMC-1. BAI (30 μm) decreased IL-6 and IL-8 production in the Ms CSE (0.25 mg/ml) + IL-1beta treated HMC-1 culture from 2615 ± 38 to 251 ± 19 pg/ml (p < 0.0000001) and from 583 ± 37 to 179 ± 7 pg/ml (p < 0.0005), respectively. A similar inhibitory effect of BAI was also observed in the Ss CSE + IL-1beta treated HMC-1 culture. Our results show that BAI inhibits the enhancing effects of CSE on inflammatory cytokine production in IL-1beta activated mast cells. It suggests that BAI may be a novel candidate for treatment of smoking related inflammatory airway disease (Supported by the Ruth R. Harris Endowment and RDC of ETSU).
Direct comparison of Dll1- and Dll4-mediated Notch activation levels shows differential lympho-myeloid lineage commitment outcomes (36.27)
Authors: Mohtashami Mahmood , Shah Divya , Nakase Hiroshi , Kianizad Korosh , Petrie Howard , Zúñiga-Pflücker Juan Carlos
Published: January 2023

In the thymus, Notch signaling is known to be essential for T-lymphopoiesis, with the Notch ligand Delta like 4 (Dll4) being uniquely involved in this process. However, using stromal cell cultures, either Dll4 or Dll1 has been shown to support T-lymphopoiesis. To address which Dll is more effective at inducing hematopoietic progenitor cells to give rise to T-lineage cells in vitro, we generated OP9 cells expressing a series of incrementally discrete and equivalent levels of Dll1 or Dll4. In keeping with previous findings, OP9 cells expressing high levels of either Dll1 or Dll4 gave rise to T-lineage cells with similar efficacy, and prevented the differentiation of B- and myeloid-lineage cells. However, at limiting levels of Dll expression, Dll4 maintained its ability to inhibit B-lineage choice and induce T-lineage commitment and differentiation at lower levels of expression than Dll1. This manifest property of Dll4 is evident despite its lower levels of steady-state surface expression than Dll1 on OP9 cells. The heightened effectiveness of Dll4 over Dll1 also corresponded to the induction of expression of Notch target genes, and inhibition of B- and myeloid-specific transcription factors. Furthermore, OP9 cells expressing levels of Dll4 equivalent to those present in thymic epithelial cells gave rise to T-lineage cells, as expected, but were also permissive for the differentiation of myeloid cells, while still inhibiting the generation of B-lineage cells.
Phenotypic and functional characterization of group 1 CD1-restricted autoreactive T cells in a transgenic mouse model expressing human group 1 CD1 and a CD1b-specific T cell receptor (36.31)
Authors: Li Sha , Choi Hak-Jong , Shanmuganad Sharmila , Wang Chyung-Ru
Published: January 2023

Unlike other MHC molecules, the CD1 family presents self and foreign lipid antigens to multiple T cell subsets. In humans, this family consists of group 1 CD1 molecules CD1a, b and c and group 2 molecule CD1d. While CD1d-restricted NKT cells have been well-studied, our knowledge of the development and function of group 1 CD1-restricted T cells has been limited by the lack of a suitable animal model. We have generated double transgenic mice (hCD1Tg/HJ1Tg) that express the human group 1 CD1 proteins in a pattern similar to that observed in humans in addition to a TCR from a CD1b-restricted autoreactive T cell clone. Like NKT cells, the majority of HJ1Tg T cells that develop in hCD1Tg/HJ1Tg mice express NK markers and exhibit an activated phenotype. In addition, HJ1Tg T cells are highly enriched in the liver. The development of HJ1Tg T cells with the NKT cell phenotype is group 1 CD1-dependent, as NK1.1+ HJ1Tg T cells are absent in HJ1Tg mice that lack the human group 1 CD1 genes. Upon stimulation with group 1 CD1-expressing dendritic cells, HJ1Tg T cells primarily secrete proinflammatory cytokines, including IFN-γ, IL-17A, IL-6 and MCP-1. These cytokine productions can be further enhanced in the presence of TLR agonists, a phenomenon also observed in autoreactive NKT cells. The similarities between the phenotype and functional properties of HJ1Tg T cells and NKT cells suggest that these two autoreactive T cell subsets may play similar immunological roles.
Arrested bone marrow resident T cell committed progenitors in athymic nu/nu mice can give rise to alpha-beta T cells in an OP9DL1 co-culture system. (36.34)
Authors: Chatterjea Devavani , Garcia-Ojeda Marcos , Schwager Zachary
Published: January 2023

Bone marrow-resident committed T cell progenitors (CTP) are arrested at a Thy1.2hiLin-CD2- stage in athymic nu/nu mice. In WT mice, CTP progress to a Thy1.2hiLin-CD2+ intermediate stage that then can produce mature, function single positive T cells through thymic and extrathymic maturation pathways. The nu/nu bone marrow microenvironment, therefore, likely disrupts the T cell competence of these precursors during the transition from hematopoietic stem cell (HSC) to CTP prior to these cells leaving the bone marrow for the thymus or periphery. This defect is currently not characterized. We investigated the developmental potential of the arrested CTP from nu/nu bone marrow in a hyper-Notch signal environment induced by the OP9DL1 co-culture system that is able to support the development of mature single-positive T cells from HSCs, embryonic stem cells (ESC) and double negative thymocytes. We found that the OP9DL1 co-culture microenvironment allows the arrested nu/nu CTP to progress to the Thy1.2+Lin-CD2+ intermediate stage as well as to double positive CD4+CD8+ and single positive CD4 and CD8 αβ T cells. It is likely therefore that Notch signaling is compromised in the bone marrow of nu/nu mice and this dysregulated signaling results in the absence of acquisition of T cell competence by early hematopoietic precursors in the bone marrow of these congenitally athymic mice.
The development of iNKT cells is dependent on a full complement of functional CD3 ζ ITAMs (36.39)
Authors: Eitson Jennifer , Becker Amy , Norgard Michael , van Oers Nicolai
Published: January 2023

Invariant natural killer T (iNKT) cells are involved in the early phase of an immune response and are proposed necessary for reducing the severity of Lyme disease during Borrelia burgdorferi infection. Little is known about the number of functional TCR encoded ITAMs required for iNKT cell selection and expansion. We analyzed iNKT cell development in CD3 ζ transgenic lines with various tyrosine to phenylalanine substitutions (YF) that eliminated some or all phospho-ζ intermediates. iNKT cell numbers were significantly reduced in the thymus, spleen, and livers of the YF lines compared to wild type mice. Interestingly, the extent of cardiac tissue damage and inflammation in response to B. burgdorferi infections was similar in the YF lines compared to wild type controls, and even mice lacking all iNKT cells (Ja18-null animals). The relative expression of two key transcription factors, Egr2 and PLZF, was significantly reduced in the YF lines. This suggests that the magnitude of TCR/ITAM-driven signals controls the expression of two key transcription factors essential for iNKT cell development. Thus, while a full complement of functional CD3 ζ ITAMs is required for iNKT cell development, these cells are not essential for controlling arthritis or carditis following B. burgdorferi infections.
Modeling the Role of Antigen Stimulation in Treg vs. Teff Fate Selection (36.52)
Authors: Sekar John , Miskov-Zivanov Natasa , Turner Michael , Morel Penelope , Faeder James
Published: January 2023

Peripheral naïve T-cells differentiate into regulatory, helper and other effector cells in response to a cognate antigen. The relative numbers produced of each cell type are critical for immune tolerance and affect tumor and immune-related pathologies. Specifically, the differentiation of CD25+ Foxp3+ regulatory T cells (Treg) is dictated by antigen dose with sensitivity and maintenance effects from TGF-β and IL-2. These overlapping external signals are conveyed and regulated by key intracellular players such as PI3K, PTEN, DNMT1 and Akt/mTor, eventually converging on transcription factors that control the induction of Foxp3 expression. To better understand this intricate system, we have constructed a logical model in which each molecule type is treated as an on/off variable, permitting all known intracellular players to be modeled simultaneously without adjustable parameters. Key subsystems will be modeled later at higher resolution using chemical kinetics and these models will be linked to form a comprehensive, detailed model of T cell differentiation. As a preliminary result, the logical model reproduces the experimental observation that low dose antigen favors Treg induction in the absence of exogenous TGF-β or IL-2. Construction of this model has already suggested novel experimental avenues and identified key components requiring theoretical attention. Experiments suggest that a population model with interacting and evolving phenotypes is needed to complete the picture.
Molecular Basis for Loss of VH4-encoded pre-B cells in Mutant Rabbits (36.7)
Authors: Robbins Gregory , Knight Katherine
Published: January 2023

Developing B cells in mutant ali/ali rabbits predominantly utilize VH4 during VDJ gene rearrangement. However, most of the VH4-utilizing B cells encounter a severe developmental block at the pre-B cell stage. In contrast, wildtype rabbit B cells predominantly rearrange VH1, a gene >90% identical to VH4, and develop normally. We seek to determine how these nearly identical VH genes have profoundly different effects on B cell development. We hypothesize that VH4-utilizing cells are blocked at the pre-B stage due to weak interaction of the μ-chain with surrogate light chain (SL). We expressed VH4-utilizing μ-chains in a pro-B cell line that expresses SL and found that VH4-encoded μ-chains exhibit a 2 fold reduction in preBCR surface localization compared to control VH1-encoded μ-chains. Remarkably, a single amino acid substitution in VH4-framework region 2 (FR2) from tyrosine to an evolutionarily conserved tryptophan, found in VH1 and most VH genes of other species, restored surface preBCR expression. We are testing if this tryptophan substitution in FR2 of VH4 is sufficient to rescue pre-B cell development. These studies should identify evolutionarily conserved VH structural regions required for efficient preBCR formation and B cell development.
IFNgamma Induces Expansion of Hematopoietic Progenitor Cells (36.9)
Authors: Zhao Xin , Ren Guangwen , Liang Li , Ai Phillip , Zheng Betty , Tischfield Jay , Shi Yufang , Shao Changshun
Published: January 2023

The balance between type 1 and type 2 helper T (Th1 and Th2) cells is critical for homeostasis of the immune system, since their distinct cytokine production profile directly determines the activation and functions of other immune cells. However, as the end-differentiated lineage cells, how these effector T cells influence the hematopoietic progenitor cells, is largely unknown. Here we showed that Th1 cell products, but not those of Th2 cells, caused a remarkable expansion o f lineage-Sca-1+C-kit+ (LSK) cells. IFNgamma was found to play a major role in this expansion by activating the expression of Sca-1 in lineage-Sca-1-C-kit+ cells, thus converting them into LSK cells. This process was dependent on IFNgammaR1 signaling, but only partially mediated by STAT1 pathway. Furthermore, such IFNgamma-induced LSK cells had a significantly higher colony forming units (CFUs), compared to control lineage-Sca-1-C-kit+ cells and control LSK cells, which might compensate for the loss of progenitor cells caused by the IFNgamma-treatment. Moreover, the IFNgamma-induced expanded LSK cells were verified as functional stem/progenitor cells through the competitive repopulation experiments in vivo. Finally, the IFNgamma-induced hematopoiesis was more biased toward the differentiation of myeloid lineages in vitro and in vivo. Therefore, our findings demonstrated a novel role of IFNgamma in hematopoiesis, thus providing a new insight into the clinical application of IFNgamma.
Comparison of Global Transcriptional Responses of Chicken Following Primary and Secondary Eimeria acervulina Infections (37.13)
Authors: Lillehoj H. , Kim Chul-hong , Kim Duk Kyung , Keeler C.
Published: January 2023

In this stuy, we compared chicken gene transcriptional profiles following primary and secondary infections with Eimeria acervulina using a 9.6K avian intestinal intraepithelial lymphocyte cDNA microarray (AVIELA). Gene Ontology analysis showed that primary infection significantly modulated the levels of mRNAs for genes involved in the metabolism of lipids and carbohydrates as well as those for innate immune-related genes. By contrast, secondary infection increased the levels of transcripts encoded by genes related to humoral immunity and reduced the levels of transcripts for the innate immune-related genes. Because the observed modulation in transcript levels for gene related to energy metabolism and immunity occurred concurrent with the clinical signs of coccidiosis, these results suggest that altered expression of a specific set of host genes induced by Eimeria infection may be responsible, in part, for the observed reduction in body weight gain and inflammatory gut damage that characterizes avian coccidiosis.
Activated human neutrophil control macrophage infection by L. amazonensis (37.14)
Authors: Brodskyn Claudia , Afonso Lilian , Khoury Ricardo , Santos Theo , Clarencio Jorge , de Oliveira Camila , Barral Aldina , Barral-Netto Manoel , Borges Valeria
Published: January 2023

Neutrophil granulocytes are rapidly recruited from the bloodstream to the site of infection via transmigration through the vascular endothelium and they can be activated in this process. Recently, our group showed that phagocytosis of resting apoptotic human neutrophils by L. amazonensis-macrophages induced an increase in the number of intracellular parasites, whereas phagocytosis of necrotic neutrophils by infected macrophages induced killing of parasites in a mechanism dependent on TNF-α and NE. In the present study, we activated the human neutrophils using extracellular matrix proteins and evaluated the effect of viable and apoptotic activated human neutrophils on macrophage infection by Leishmania amazonensis. Fibronectin interaction with neutrophils induced a higher level of cell activation as evidenced by an increased expression of surface markers, such as CD11b as well as a decrease in CD62L and Annexin V. We also detected an increased production of MMP-9 and NE. Phagocytosis of viable and apoptotic fibronectin-activated neutrophils by Leishmania-infected macrophages led to a decrease in parasite burden in a mechanism dependent of NE, MPO and MMP-9. We also observed increase levels of TNF- α , LTB4 and superoxide production in the co-cultures. Given that the events following Leishmania infection involve interactions with neutrophils, our study describes novel mechanisms associated with parasite elimination, based on neutrophil and the infected macrophage collaboration.
Src-family tyrosine kinases regulate alveolar macrophage alternative activation during Pneumocystis murina lung infection (37.28)
Authors: Christmann Benjamin , Nelson Michael , Metz Allison , Steele Chad
Published: January 2023

We have previously shown that mice triple-deficient in the Src-family tyrosine kinases (SFKs) Hck, Fgr and Lyn (SrcTKO mice) have augmented innate lung clearance of P.murina as a result of elevated proinflammatory cytokine (e.g. IL-1β) and chemokine (e.g. CCL2/MCP-1) levels and increased inflammatory cell recruitment. Augmented clearance in SrcTKO mice correlates with a higher ability of alveolar macrophages (AMs) from these mice to kill P.murina in vitro, though this enhancement is not associated with elevated AM inflammatory responses. Since SrcTKO AMs are not hyper-inflammatory yet kill more efficiently, we hypothesized that they have a different activation pattern compared to WT. We show that AMs and whole lungs from P.murina-infected SrcTKO mice express higher mRNA and protein levels of the alternative macrophage activation markers RELM-α and Arg-1 compared to WT. There is surprisingly no difference in the levels of the pro-alternative activation cytokines IL-4 and IL-13 or other cytokines known to modulate AM activation, e.g. IFN-γ or GM-CSF. However, infected SrcTKO mice have higher lung levels of M-CSF, CCL17/TARC and the IL-4/IL-13 receptor, IL4ra. Treatment of WT AMs with IL-4 or IL-13 enhances their ability to kill P.murina, while a blockade of IL4ra or inhibition of phagolysosome maturation inhibits killing. These results indicate that SFKs regulate AM activation during P.murina infection, and that alternatively activated AMs are better equipped to kill P.murina.
Macrophage responses to interleukin-17 are regulated by location and inflammation (37.35)
Authors: Barin Jobert , Baldeviano G. , Talor Monica , Quader Farhan , Wu Lei , Chen Ping , Zheng Dongfeng , Čiháková Daniela , Rose Noel
Published: January 2023

The purpose of this study was to examine how macrophage-lineage cells participate in T helper 17 (Th17)-mediated inflammatory responses. We were interested in how this response may be affected by compartmentalization, and the presence of an inflammatory microenvirionment. We isolated primary murine F4/80+ macrophages (Mϕ) from a broad variety of compartments including spleen, peripheral blood, peritoneum, gut, lung, liver, and CNS. We assayed the expression of the receptors IL17RA and IL17RC on these cells by flow cytometric methods, and observed the highest levels of IL17RA and IL17RC expression on Mϕ associated with mucosal surfaces, particularly gut and lung. In order to examine the influence of inflammatory stimuli on the expression of IL17RA and IL17RC in vitro, bone marrow-derived macrophages, which express low levels of IL17RA and IL17RC were stimulated with combinations of TLR ligands, and proinflammatory cytokines. We observed coordinate expression of both IL17 receptor subunits controlled by TNFα and peptidoglycan signaling. Following encounter with CFA adjuvant in vivo, the expression of both IL17RA and IL17RC was most dramatically increased on Mϕ in the liver. We further wished to determine biological consequences of IL17 signaling, and have begun to identify genes regulated by IL17 in macrophage-lineage cells. Together, these data indicate that macrophages participate in Th17-mediated immunity in a manner regulated by localization and inflammation.
Proinflammatory cytokines and type I interferon are differentially induced by virulent and avirulent mycobacteria in human macrophages (37.36)
Authors: Novikov Aleksey , Thompson Robert , Sher Alan , Feng Carl
Published: January 2023

One third of the world's population is latently infected with Mycobacterium tuberculosis (MTB), a virulent intracellular pathogen that can survive in macrophages even in the presence of a strong adaptive immune response. The molecular mechanisms underlying MTB persistence are not fully understood. Production of type I interferon (IFN) has been linked to the exacerbation of pulmonary tuberculosis (TB) in mice. However, the role of type I IFN in human TB is as yet undefined. In this study, primary macrophages derived from elutriated monocytes of healthy individuals were infected with MTB or M. bovis Bacillus Calmette-Guérin (BCG), an attenuated vaccine strain, and their responses analyzed by qPCR. We observed that compared to BCG, MTB infection induced markedly lower expression of the NF-kB-dependent pro-inflammatory cytokine genes IL-1 and IL-8 at all time-points examined. On the other hand, transcript levels of the type I IFN associated genes CXCL10 and IFIT1 were significantly higher in MTB infected compared to BCG-infected macrophages. These findings suggest that the differential regulation of type I IFN is a determinant of mycobacterial virulence in human macrophages and that MTB may exploit the host type I IFN pathway to subvert anti-microbial programs in these host cells. This work was supported by the Intramural Research Program of the NIAID and by the Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program.
Intracellular colocalization of galectin-3 with Listeria monocytogenes in macrophages and its implications (37.45)
Authors: Weng I-Chun , Chen Huan-Yuan , Hsu Daniel , Liu Fu-Tong
Published: January 2023

Galectin-3 is a β-galactoside-binding animal lectin expressed in epithelial cells and various immune cell types. In macrophages, It has been shown to be a positive regulator for FcγR-mediated phagocytosis and a negative regulator for LPS-mediated inflammation. However, its role in macrophages during infection process remains unclear. Here we investigated the role of galectin-3 in macrophages during the infection of a Gram-positive bacterium, Listeria monocytogenes (LM). Survival of LM in galectin-3-deficient (gal3-/-) bone marrow-derived macrophages (BMM) was impaired compared to that in galectin-3 wild-type (gal3+/+) BMM. This was not due to a difference in the production of bactericidal mediators nitric oxide and superoxide anion, nor a difference in the efficiency of LM escape from phagosomes between gal3+/+ or gal3-/- BMM. Interestingly, by immunofluorescence staining we detected colocalization of intracellular galectin-3 with LM at as early as 15 min after infection. The colocalization proportion peaked at 30 min post-infection, coinciding with the timing of LM escape. In vitro incubation of LM with recombinant galectin-3 was shown to promote LM growth. Based on these findings, we hypothesize that LM utilizes intracellular galectin-3 to improve its own survival and growth after escape into the host cytosol.
LPS stimulates bactericidal activity in the social amoeba Dictyostelium discoideum (37.47)
Authors: Snyder Michelle , Walk Alexander , Callahan Jennifer , Srisawangvong Pat , Leuschner Jessica , Samaroo Dave , Cassilly Daniel
Published: January 2023

Innate immune cells use evolutionarily-conserved pattern-recognition machinery to detect pathogen-associated molecular patterns (PAMPs). The conservation of pattern-recognition machinery allows for its characterization in various model organisms. The social amoeba Dictyostelium discoideum is a unique model system that can exist as single-celled amoebae that phagocytize bacteria for nutritional uptake. Although D. discoideum has been used to study bacterial phagocytosis, it has not been determined whether D. discoideum uses pattern-recognition machinery to detect bacterial PAMPs. Here we show that D. discoideum mounts responses against the bacterial cell wall PAMP, lipopolysaccharide (LPS). LPS treatment of D. discoideum results in more efficient uptake of bacteria and more efficient clearance of phagocytized bacteria. In addition, as shown by qRT-PCR, D. discoideum treated with LPS upregulate expression of putative pattern-recognition and phago-lysosomal genes. Both LPS-enhanced bactericidal activity and gene expression are dependent on MAPK signaling. We currently are determining the mechanisms by which LPS stimulates D. discoideum by studying intracellular trafficking of phagocytized bacteria and by creating D. discoideum cells that overexpress or are deficient for pattern-recognition and phago-lysosomal genes. The characterization of pattern recognition in D. discoideum should provide insight into conserved molecular mechanisms underlying microbial detection.
IL-17 is essential for host defense against cutaneous Staphylococcus aureus infection (37.50)
Authors: Cho John , Pietras Eric , Garcia Nairy , Ramos Romela Irene , Farzam David , Monroe Holly , Magorien Julie , Blauvelt Andrew , Kolls Jay , Cheung Ambrose , Cheng Genhong , Modlin Robert , Miller Lloyd
Published: January 2023

Although neutrophil recruitment is required for immunity against Staphylococcus aureus, a role for T cells has been suggested based on the increased susceptibility of humans who have altered T cell immune responses to S. aureus cutaneous infections. Here, the contribution of T cells to host defense was investigated in a mouse model of S. aureus cutaneous infection. Mice deficient in γδ but not αβ T cells had substantially larger skin lesions with higher bacterial counts and impaired neutrophil recruitment compared with wild-type mice. This neutrophil recruitment was dependent upon epidermal Vγ5+ γδ T cell production of IL-17 but not IL-21 and IL-22. Further, IL-17 induction required IL-1, TLR2 and IL-23 and was critical for host defense, since IL-17R-deficient mice had a similar phenotype as γδ T cell-deficient mice. Importantly, γδ T cell deficient mice treated with a single dose of recombinant IL-17 had lesion sizes and bacterial counts that resembled wild-type mice, demonstrating that IL-17 can restore the impaired immunity in γδ T cell deficient mice. This study defines a novel role for IL-17-producing epidermal γδ T cells in innate immunity against S. aureus.