bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of tumor necrosis factor-alpha on in vitro differentiation of natural killer cells (138.13)
Authors: Lee Jiwon , Lee Suk Hyung , Jeong Mira , Choi Inpyo
Published: January 2023

Natural killer (NK) cells are differentiated from hematopoietic stem cells (HSCs) in bone marrow. The differentiation of NK cells is regulated by various factors including soluble growth factors and transcription factors. Here, we demonstrated that tumor necrosis factor-α (TNF-α) is a positive regulator of NK cell differentiation. HSC-derived precursor NK (pNK) cells were further differentiated into mature NK (mNK) cells in the presence of IL-15 in vitro. The potential role of TNF-α on NK cell maturation was evaluated in the presence or absence of IL-15. TNF-α itself induced the expression of NK1.1 and CD122 of mature NK cells and its effects were significantly augmented in the presence of IL-15. IFN-γ production was maximal when NK cells were matured in the combination of IL-15 and TNF-α. Moreover mRNA expression of several transcription factors including T-bet and GATA-3 was increased during TNF-α -mediated in vitro NK cell differentiation. Overall, these data indicate that TNF-α regulates NK differentiation by increasing transcription factors which are crucial for NK maturation.
Severe block in hematopoiesis in the absence of NKAP (138.2)
Authors: Pajerowski Anthony G , Shapiro MIchael J , Shapiro Virginia S
Published: January 2023

During hematopoiesis, proper differentiation of the long-term, multi-potent, self-renewing hematopoietic stem cell (HSC) results in the cellular components of blood. The lineage fate decisions that occur during hematopoiesis result from the coordinated regulation of transcriptional activators, repressors, and chromatin modifiers. We have identified NKAP as a novel regulator of hematopoiesis, that functions at least in part as a transcriptional repressor. In this study we employ a combination of conditional and inducible mouse models of NKAP deletion to explore the role of NKAP in hematopoietic development starting at the HSC stage. Our models allow for both the pan-deletion of NKAP starting in the HSC stage by using a vav-cre transgenic, as well as temporally regulated deletion of NKAP in the hematopietic compartment through the use of the interferon-inducible mx1-cre model system. These complimentary approaches allow us to conclude that NKAP functions in a cell intrinsic manner, as a member of a unique group of transcriptional regulators, and is necessary in the earliest stage of definitive hematopoiesis.
Notch target gene deregulation and leukemogenesis occur independently of RBPJ in Ikaros null thymocytes. (138.23)
Authors: Chari Sheila , Winandy Susan
Published: January 2023

Both Ikaros and Notch are transcriptional regulators that are essential for normal T cell development. Cooperative mutations causing a reduction in Ikaros activity and an increase in Notch activation have been hypothesized to promote T cell leukemia. However, it is not known whether Notch deregulation plays a preferential or obligatory role in the leukemias that arise in Ikaros null (Ik-/-) mice with 100% penetrance. In order to answer this question, we abrogated Notch target gene activation in Ik-/- thymocytes using conditional inactivation of the DNA-binding Notch-target gene activator RBP-J. Through analyses of these mice we have made two important discoveries.

First, thymocytes that lack either Ikaros alone or both Ikaros and RBP-J exhibit high level derepression of Notch target genes. However, this derepression is not observed in thymocytes lacking RBP-J alone, which suggests that Ikaros, but not RBP-J, is required for Notch target gene repression in DP thymocytes. This data represents a major shift in our understanding of regulatory factors involved in Notch target gene repression. Secondly, Ik-/- mice develop clonal T cell populations, a hallmark of leukemia, with similar kinetics in the presence or absence of RBP-J. The appearance of clonal populations in the thymus and spleen of Ik-/- mice lacking RBP-J indicates that T cell transformation in the absence of Ikaros does not require deregulated Notch signaling.
Coordinate Regulation of TCR signaling, Notch, and Gata3 in T Cell Development (138.27)
Authors: Agyemang Amanda , Zhang Xianyu , Laky Karen , Fowlkes B.J.
Published: January 2023

The transcription factors, Notch and Gata3, are both involved in the intrathymic development of CD4 lineage T cells. It has been proposed that Gata3 is critical in CD4 T cell maturation after lineage commitment. To the contrary, we find that disruption of the Gata3 gene re-directs a fraction of MHC II-restricted thymocytes to the CD8 lineage. Moreover, Gata3 binds regulatory regions of the ThPOK (Zbtb7b) gene in vivo and is required for the ThPOK expression that enforces CD4 lineage commitment (Nature Immunol. 9:1122). Collectively, these findings place Gata3 developmentally upstream of the CD4/CD8 lineage decision and raise the question of how Gata3 itself is regulated. Our data and that of others suggest that Gata3 expression is regulated by TCR signaling, but Gata3 can also promote TCR signaling/expression. Therefore, Gata3 may operate in a self-reinforcing feedback loop during positive selection to achieve the levels of TCR signaling required for ThPOK induction and CD4 lineage commitment. In the differentiation of peripheral Th2 cells, Gata3 transcription can be regulated by Notch. We find that Notch and Gata3 promote TCR signal transduction prior to positive selection, as well as the selection/maturation of CD4/CD8 T lineages. Thus, we have designed genetic and expression studies to investigate whether Notch regulates Gata3 in thymocytes.

Intramural Research Program of the National Institute of Allergy and Infectious Diseases.
Characterization of a novel cell surface protein CSP82 on bone marrow cells that induce DP58 (138.32)
Authors: Al-Shaibi Nisreen , Ghosh Swapan K
Published: January 2023

We previously described a novel protein, DP58 that occurs constitutively in murine brain nuclei and as a cytosolic phosphoprotein during bone marrow (BM) differentiation into promyeloid dendritic progenitors (Front. Bioscience 12, 2947, 2007;. BBRC, 321, 26, 2004). DP58 protein is not detectable in immature or mature dendritic cells. In this study, we describe a GPI-linked receptor lactoferrin CSP82 on BM that is involved in induction of cytosolic DP58 in dendritic progenitors. Furthermore, to fully characterize CSP82 receptor, we determined whether iron could be its natural ligand playing important roles in facilitating differentiation of BM cells. Previous reports show that iron has a significant role in the differentiation of macrophages and dendritic cells from haematopoietic stem cells. This report shows that iron (ferrioxamine) acting on CSP82 can facilitate induction of DP58 protein. Moreover, the expression of DP58 protein can be inhibited by desferrioxamine; but when these cells are supplemented with ferrioxamine, DP58 induction is restored. Further support comes from the experiment in which CSP82 expression in BM cells is inhibited by antisense oligos, with consequent failure of iron to induce DP58. Our preliminary results suggest that iron-binding CSP82 receptor on BM cells play important roles in early differentiation of promyeloid dendritic progenitors expressing cytosolic phosphoprotein DP58.
Human cord blood, but not adult bone marrow, gives rise to IL-7-independent B cell production (138.5)
Authors: Payne Kimberly J , Baez Ineavely , Milford Terry-Ann , Benitez Abigail , Galloway Nicholas , Rogerio Jaqueline W , Sahakian Eva , Wall Nathan R , Dovat Sinisa , Parrish Yasmin Khan
Published: January 2023

IL-7 is critical for B cell production in adult mice, however its role in human B lymphopoiesis is controversial. Using a novel, human-only culture model we show that IL-7 increases human B cell production by >60-fold from hematopoietic stem cells (HSCs) in both cord blood (CB) and adult bone marrow (BM). IL-7-induced increases are dose-dependent and specific to CD19+ cells. STAT5 phosphorylation and expression of the Ki-67 proliferation antigen indicate that IL-7 acts directly on CD19+ cells to increase proliferation at the CD34+ and CD34- pro-B cell stages, but not among CD19- B lineage precursors. As compared to CB, adult BM shows a reduction of in vitro generative capacity that is progressively more profound in developmentally sequential B cell precursor populations, resulting in a ~50-fold reduction in IL-7-dependent B lineage generative capacity. Without IL-7, HSCs in CB, but not BM, give rise to a small but consistent population of CD19LO B lineage cells that express EBF and PAX-5 and respond to subsequent IL-7 stimulation. Flt3 ligand, but not TSLP, was required for the IL-7-independent production of human B lineage cells. These data provide evidence that human B cell production from adult BM is dependent on IL-7 and suggest that a mechanism in addition to the "developmental switch" observed in the mouse model may be responsible for IL-7 dependency in adult human B lymphopoiesis. Supported by NIH K01 DK066163 (KJP).
ADAM10 regulates B cell development and immunoglobulin production (138.6)
Authors: Gibb David R , El Shikh Mohey , Kang Dae-Joong , El Sayed Rania , Crawford Howard , Tew John G , Conrad Daniel H
Published: January 2023

Members of the disintegrin and metalloprotease (ADAM) family can regulate lymphocyte development and function by cleaving membrane receptors. Previous studies showed that ADAM10 cleaves the B cell receptors, CD23 and Notch2, in vitro. However, in the absence of in vivo models, the physiologic impact of these events has not been examined. Thus, we generated B cell-specific ADAM10 KO and transgenic (Tg) mice to determine the role of ADAM10 in B cell development and function. Notch signaling regulates the differentiation of common lymphoid progenitors (CLPs) by promoting T cell fate. Accordingly, overexpression of ADAM10 in CLPs completely blocked B cell development. Deletion of ADAM10 from B cells in ADAM10fl/fl, CD19-cre+ mice prevented differentiation of marginal zone B cells, which is dependent on Notch2 signaling. In addition, expression of CD23 on ADAM10null B cells was dramatically elevated while levels of soluble CD23 were reduced in serum. As a result, IgE production by ADAM10fl/fl, CD19-cre+ mice was remarkably suppressed upon immunization, mimicking the response of CD23Tg mice. Finally, production of IgG1 and IgG2b was profoundly inhibited, suggesting a novel role for ADAM10 in class-switching to IgG. These results illustrate the physiologic role of ADAM10 in Notch signaling and CD23 cleavage. Moreover, they identify ADAM10 as a critical regulator of B cell development and immunoglobulin production.

AHA 0815066E, NIAID R01AI18697, NIAID U19AI077435
The interplay of CD23 and ADAM10 and its role in IgE synthesis (139.1)
Authors: mathews joel , Chen Bing-Hung , Saha Nayanendu , Conrad Daniel
Published: January 2023

CD23, the low affinity IgE receptor, is a type II transmembrane protein expressed on hematopoietic cells and contains two important extracellular domains, the IgE binding lectin head, and the coil-coiled stalk, responsible for CD23's trimeric structure. Mouse models have shown that trimetric membrane bound CD23 regulates IgE synthesis, which ability is lost when CD23 is shed. Recently it was shown that the CD23 sheddase is ADAM10, and shedding is controlled by the trimer stability. In this study, we examined the interactions of CD23 and ADAM10. We found that the binding of ADAM10, using either cell lysates containing ADAM10 or rADAM10 (Janes et al. 2005. Cell 123:291-304.), to CD23 is unlike any other protein that interacts with CD23, in that it binds the stalk region, between amino acids 139-157 in a protease independent manner. When CD23 is destabilized, with the anti-stalk antibody, 19G5, ADAM10 binding increased. Stabilizing CD23 with IgE had the opposite affect. These studies will be further examined with surface plasmon resonance.
Autocrine production of cysteinyl leukotrienes activates human mast cells (139.13)
Authors: Kulka Marianna , Catalli Adriana
Published: January 2023

Cysteinyl leukotrienes (CysLT) are potent inflammatory lipid mediators which are derived from 5-lipoxygenase activity. CysLTs mediate some of the pathophysiological responses associated with asthma such as bronchoconstriction, vasodilation and increased microvascular permeability, increased mucus secretion, decreased mucociliary clearance, eosinophil migration, and increased eosinophil survival. We now report that peripheral blood CD34+ progenitor-derived human mast cells and the human mast cell line (LAD2) express both CysLT receptors (CysLT1R and CysLT2R), the G protein-coupled receptors that bind CysLTs. LTC4, LTD4 and LTE4 activated LAD2 to produce MIP-1β and MCP-1 and production of these chemokines was inhibited by the CysLT1R inhibitor, monelukast. LAD2 activated by IgE/anti-IgE produced CysLT (980 + 55 pg/million cells) but LAD2 activated by substance P, a neuropeptide that also binds a G protein-coupled receptor, did not produce CysLT. Montelukast had no effect on SP-mediated LAD2 degranulation. CysLT did not induce human mast cell degranulation but montelukast inhibited FcεRI-mediated human mast cell degranulation by approximately 20% suggesting that autocrine production of CysLT potentiated FcεRI-dependent degranulation. IL-4 (10 ng/mL) in the presence of stem cell factor (SCF; 100 ng/mL) significantly upregulated expression of both CysLT1R and CysLT2R (16% and 32% respectively) and potentiated mast cell responses to LTE4 by up to 25%. Overall, these results show that human mast cell activation and degranulation is subject to autocrine CysLT-induced signaling which can be blocked by CysLT1R inhibitors such as montelukast.

Funded by the Investigator Initiated Studies Program (IISP) from Merck and Company, Inc.
Differential uptake of serum IgE in skin and serosal mast cells(139.14)
Authors: Cheng Laurence E , Wang Zhien , Locksley Richard M
Published: January 2023

Background: Mast cells (MC) are key effector cells in helminth infection and the clinical manifestations of allergic disease. In mice, a subset of MC (Connective Tissue MC) resides at epithelial barriers, such as the skin, or at serosal surfaces, including the peritoneal cavity. These MC exert much of their effector function by virtue of IgE binding to the high affinity Fc epsilon receptor (FcεRI ).

Objective: Although acquisition of serum immunoglobulin E (IgE) by skin MC is central to MC effector function, the mechanisms that govern loading of IgE on skin MC are poorly defined. We hypothesize that different anatomic compartments may exhibit differential serum IgE uptake.

Methods: We used the mouse ear as a source of skin MC. To control the dose and timing of serum IgE, we used a passive, intravenous IgE transfer model with flow cytometric analysis of MC IgE uptake at time points between 1 and 96 hours after infusion. As controls, we examined serum IgE uptake by spleen basophils and peritoneal mast cells (PEC-MC).

Results: We observed differences in IgE uptake by basophils and PEC-MC compared to skin MC. Basophils and PEC-MC displayed uniform loading of IgE that started within 1 hour of IgE exposure. By contrast, skin MC IgE uptake was heterogeneous with only 30% of MC showing significant acquisition of IgE. The timing of uptake was slightly delayed, and the amount of IgE on these cells was also diminished compared to basophils and PEC-MC.

Conclusion and Future Directions: Our data suggest that skin and serosal MC demonstrate distinct patterns of IgE uptake and that a select population of skin MC acquires serum IgE upon exposure. Further work will focus on direct in vivo imaging of MC IgE loading within ear tissue to better understand the underlying reason for decreased skin MC uptake of serum IgE.
Regulation of IL-4 expression by aspirin via a MAPK pathway (139.15)
Authors: Kim Byung Soo , Uhm Tae Gi , Kang Jin Hyun , Park Choon-Sik , Chung Il Yup
Published: January 2023

IL-4, a pleiotropic cytokine produced in Th2 cells, plays a central role in the pathological development of asthma. Aspirin, a widely used nonsteroidal anti-inflammatory drug (NSAID), is reported to have inhibitory effect on IL-4 expression in activated T cells and paradoxically to induce airway hyperreactivity in a subset of asthmatic patients, causing so-called aspirin-intolerant asthma (AIA) by completely unknown mechanism. Here we show the effects of aspirin on IL-4 expression in Jurkat T cells and K562 cells that are shown to be able to express IL-4 mRNA. qPCR analysis revealed that aspirin induced IL-4 mRNA expression in both cell lines. In agreement, aspirin also transactivated IL-4 promoter. Analysis of IL-4 promoter deletion constructs showed that the asprin-mediated IL-4 induction was not attributable to any discrete motif in the IL-4 promoter: all the deletion constructs exhibited the increased reporter activities in response to aspirin. Aspirin was able to activate p38 and ERK1/2, and specific inhibitors for these kinases abolished aspirin-mediated IL-4 induction and IL-4 promoter activity. Aspirin-induced IL-4 expression might implicate airway hyperreactivity found in AIA.
Adenosine receptor A2a inhibits complement-mediated activation of human mast cells by activating Gαs-proteins (139.5)
Authors: Kulka Marianna , Sheen Cecilia , Tancowny Brian P , Schleimer Robert P
Published: January 2023

The complement anaphylatoxin 3a (C3a) and adenosine receptors (AR) are implicated in the inflammatory process in allergic rhinitis although a direct interaction between these pathways has not been demonstrated. To investigate the interaction between these pathways, primary human cultured mast cells (HuMC) and human mast cell line (LAD2) were stimulated with C3a with or without AR agonists and antagonists. The non-selective AR agonist, 5'-N-ethylcarboxamidoadenosine (NECA; 30 uM), inhibited C3a-mediated HuMC and LAD cell degranulation (43 + 4 and 40 + 5 % inhibition respectively compared to control). NECA also blocked C3a-activated production of MCP-1 (57 + 6 %) and C3a-activated chemotaxis (46.7 + 10 %). A selective A2aR agonist, CGS 21680, inhibited C3a-mediated degranulation but the A3R agonist, IB-MECA, had no effect suggesting that inhibition of degranulation was mediated by A2aR. An A2aR antagonist, SCH 58261, blocked the inhibitory effect of NECA but an A2bR and A3R antagonist had no effect. Real-time PCR analysis showed that LAD2 and HuMC expressed mRNA for A2aR, A2bR and A3R but not A1R. Measurements of intracellular cAMP showed that NECA elevated [cAMP]i levels by at least 30% even in C3a-activated cells. Pertussis toxin blocked C3a-activated degranulation. The adenylyl cyclase inhibitor, SQ 22536, had no effect on C3a-activated degranulation but blocked the effect of NECA demonstrating that C3a and NECA mediate their effects through Gαi and Gαs proteins respectively. These results show that adenosine inhibits complement activation of human mast cells through a Gαs-protein pathway.

Funded by a grant from the Ernest S. Bazely Trust and intramural NRC funds
Approaches Against Dust Mite- induced Asthma Through Adaptive T Regulatory Cells (140.12)
Authors: Jin Jin , Hu Yanxin , Liu Qiang , Wang Bin
Published: January 2023

Asthma is a long-lasting lung disease that causes breathing problems. 50 to 80 % of asthma cases may be caused by dust mites and their droppings. This severe reaction is initiated by the allergen-specific T cell responsiveness. Effective antigen-specific treatments or preventions are still lacking. Using a murine asthma model induced by dust mite extract, we first report that co-immunization of DNA and protein vaccines encoding the dust mite specific antigen (Der p1) can prevent allergic responses, including the antigen specific IgE level and infiltration of lymphocytes and mast cells to the site of the allergen challenge. The prevention of Der p1induced asthma is directly related to the induction of a population of Der p1 specific iTreg cells exhibiting a CD4+/CD25-/FoxP3+ phenotype. This subtype of iTreg cells express IL-10 and TGF-beta, but not CTLA-4 and CCR7 which are well-known markers of natural nTreg cells. Adoptive transfer of the iTreg cells is also able to suppress the Der p1-induced asthma, suggesting the inhibition potency of the iTreg cells directly on the asthma causing T cells. The study demonstrates that utility of the co-immunization strategy may present a prophylactive approach clinically and offer us a novel strategy to prevent other allergic and asthma diseases in human and animals.
INTRANASAL CpG THERAPY ATTENUATED EXPERIMENTAL FUNGAL ASTHMA IN A TLR9-DEPENDENT AND INDEPENDENT MANNER (140.4)
Authors: Ramaprakash Hemanth , Hogaboam Cory M.
Published: January 2023

CpG-ODN administration abolishes airway inflammation and remodeling in acute models of allergic airway disease. Herein, we investigated the therapeutic effect of CpG-ODN in a chronic fungal model of asthma. TLR9+/+ and TLR9-/- mice were sensitized to soluble A. fumigatus antigens and challenged with live A. fumigatus conidia. Mice were treated with intraperitoneal (IP) CpG, intranasal (IN) CpG, or left untreated from days 14 to 28-post conidia challenge. All features of allergic airway disease were attenuated in TLR9+/+ mice treated with IN CpG, including airway hyperresponsiveness (AHR), mucus production, and peribronchial fibrosis. TLR9-/- mice treated with IN CpG exhibited attenuated airway remodeling, but not AHR. Whole-lung IL-12 levels were significantly elevated in both TLR9+/+ and TLR9-/- mice receiving IN CpG but not in either group receiving IP CpG. Whole-lung IL-10 levels were significantly elevated in IN CpG-treated TLR9+/+ mice but not in TLR9-/- mice receiving IN CpG. Increased whole-lung transcript and protein levels of the scavenger receptors SR-A and MARCO were observed in TLR9-/- mice compared with TLR9+/+ mice, possibly accounting for the CpG responsiveness in the knockout group. Together, these data show that IN CpG has a therapeutic effect during established fungal asthma, which is TLR9-dependent and independent.

This work was financially supported, in part, by the NIH (grant HL069865 to CMH).
Allergic sensitization in US bakery workers (140.9)
Authors: Biagini Raymond  E. , Page Elena H. , Dowell Chad R. , MacKenzie Barbara  A. , Snawder John E. , Sammons Debbie L.
Published: January 2023

As part of a health hazard evaluation (HHE), the National Institute for Occupational Safety and Health (NIOSH) performed a study at a commercial bakery designed to evaluate allergic sensitization to bakery associated allergens (BAA; alpha-amylase, [AA]; wheat, [W], or flour dust, [FD]). Sera was obtained from 96 employees, dichotomized as having low exposure (N=33) or high (or past) exposure (N=63) to BAA. Workers were evaluated for atopy (AlaTOP®, total IgE), and serum-specific IgE (AA, FD, and W; IMMULITE® 2000). There was no significant difference in the prevalence of atopy between groups by AlaTOP® (47% of high exposure vs. 41% of low exposure, p=0.6) or total IgE (3.9 kU/L [high exposure] compared to 3.7 kU/L [low exposure], p=0.5). At an IMMULITE® specific IgE cutoff of > 0.35 kU/L, there was significantly greater prevalence of positive specific IgE to W. When an IMMULITE® positive cutoff of >0.10 was used, significantly greater prevalence (p<0.05) of specific IgE to FD, AA, and W was observed in the high vs. low exposure groups. Atopics had significantly higher rates of sensitization to W and FD at both the 0.1 and 0.35 kU/L levels, and to AA at the 0.1 level. Exposure to BAA puts workers at a higher risk for sensitization to BAA (specific IgE). As has been shown for other occupational allergens (Annals of Allergy, Asthma, & Immunology, 97:196-202, 2006), using the IMMULITE® at a cutoff of 0.10 kU/L vs. 0.35 kU/L appears to increase the sensitivity of the assay.
Natural Killer cell interactions with dendritic cells and tumor cells are mediated by different NK cell receptors (141.10)
Authors: Wai Lu-En , Martinez Olivia M , Krams Sheri M
Published: January 2023

NK cells infiltrate solid organ allografts early post-transplant. As NK cells can mediate both maturation and killing of dendritic cells (DC), a better understanding of NK-DC interactions after transplant is warranted. This study investigates the interaction of NK cells with DC and tumor cells, and the specific NK cell activation receptors (NKRs) involved. A rat NK cell line was co-cultured with rat bone marrow-derived DC and rat hepatoma McA-RH7777 (McA). Co-culture of NK cells with DC and McA resulted in significant IFNγ production. This IFNγ production required cell contact suggesting that specific NKRs are involved. To specifically address this possibility, the NKRs, NKp30, NKp46, NKG2D, were knocked down using RNAi.

NK cell activation by DC is mediated by NKp46 as knockdown of this receptor markedly decreased IFNγ production. In contrast, NKp30, not NKp46, was responsible for NK cell activation by tumor. Flow cytometry on DC and McA with NKR fusion proteins indicated the presence of putative ligands. Interestingly, individual NKR knockdown did not markedly diminish NK cell killing suggesting NK cells use multiple receptors and/or other mechanisms to kill cells. In conclusion, both DC and tumor activate NK cells but NKp46 and NKp30 mediate these interactions respectively. These data suggest a means to manipulate specific NK cell interactions after transplantation.

Funding: NIH AI44095, and Agency for Science, Technology and Research (Singapore).
Differences in Bcl-2 Expression by T Cell Subsets Alter Their Balance After In Vivo Irradiation to Favor Regulatory NKT Cells and CD4+ CD25+ T Cells in Wild Type But Not p53-/- Mice (141.19)
Authors: Yao Zhenyu , Jones Jennifer Clare , Liu Yinping , Strober Samuel
Published: January 2023

A more nuanced analysis of the changes in the balance of immune cell subsets is needed to understand the impact of radiation on immune function. We show the balance of T cell subsets changes after increasing single doses of total body irradiation(TBI) or after fractionated irradiation of the lymphoid tissues(TLI) of mice. These changes are due to differences in radioresistance and Bcl-2 expression of the NKT, CD4+CD25+ Treg and conventional T cell subsets to favor CD4+Bcl-2hi NKT and CD4+CD25+Bcl-2hi Treg cells. Reduction of the Bcl-2lo mature T cell subsets was at least 100 fold greater than that of the Bcl-2hi subsets. CD4+ NKT cells upregulated Bcl-2 and developed a Th2 bias after irradiation, while conventional T cells failed to do so. GVHD was more severe after bone marrow transplantation in NKT cell deficient than in wild type hosts and the residual NKT cells were 10 fold more abundant in the wild type spleen after the TLI versus TBI. Although the changes in the balance of T cell subsets and increase in Bcl-2hi cells occurred in irradiated wild type mice, these changes did not occur in p53-/- mice. In conclusion, differences in the level of Bcl-2 and associated differences in resistance of T cell subsets to radiation induced p53 dependent apoptosis results in a marked alteration in the balance of T cell subsets favoring NKT cells and CD4+CD25+ T cells that promote transplantation tolerance.
CCL22 protection of islet allografts in diabetes (141.32)
Authors: Montane Joel , Bischoff Loraine , Soukhatcheva Galina , Tan Rusung , Verchere C Bruce
Published: January 2023

CD4+CD25+ T regulatory (Treg) cells prevent spontaneous development of autoimmune diabetes and may delay islet allograft rejection. Tumour cells attract Treg cells to foster immune privilege by producing the chemokine CCL22, which promotes trafficking of Treg cells to the tumour. Therefore, manipulating Treg migration with CCL22 may provide a means of preventing islet transplant rejection in autoimmune diabetes. To drive islet-specific expression of CCL22 we created an adenovirus (Ad-CCL22) in which a mouse CCL22 cDNA is expressed downstream of the CMV promoter. Isolated islets transduced with Ad-CCL22 (10 MOI) produced and secreted high levels of CCL22 in vitro whereas CCL22 production by non-transduced islets was undetectable. To determine whether CCL22 expression prevents islet allograft rejection, donor Balb/c islets were isolated and transduced overnight with Ad-CCL22 (n = 6) or Ad-lacZ (n = 5). Islets (300 per recipient) were then transplanted into the left renal subcapsular space of age-matched female C57/BL6 diabetic recipients. By 5 weeks post-transplant, only 1 of 7 CCL22-expressing islet grafts had been rejected whereas 4 of 5 Ad-LacZ-transduced grafts were rejected. Induced expression of CCL22 by transplanted islets thus delays islet allograft rejection. Manipulation of Tregs cells by CCL-22 in transplanted islets may be a novel therapeutic strategy for diabetes.
Induced Tolerance to Liver Allografts Involves Apoptosis and the Generation of Regulatory T Cells (141.40)
Authors: Fujiki Masato , Esquivel Carlos O , Martinez Olivia M. , Strober Samuel , Krams Sheri M.
Published: January 2023

Post-transplant total lymphoid irradiation (TLI) is a non-myeloablative regimen that has been studied in rodent models for induction of tolerance to allografts. We show in a rat model of orthotopic liver transplantation that 88% of Lewis recipients of DA livers (n=16) survive more than 100 days (and accept donor skin grafts) after receiving post-transplant TLI alone whereas the control group (liver transplantation without host conditioning) rejected their liver allografts within 14 days.

To establish the mechanism of this induced tolerance, lymphocytes were isolated from the livers of groups (n=3-5) of Lewis recipients of Lewis or DA liver grafts, on day 7 post-transplant, and analyzed for apoptosis. Apoptosis of T cells in allografts was increased by TLI treatment (Allo+TLI vs. Allo, CD4+ 41.5 % vs 13.4%, p=0.002, CD8+, 22.5% vs. 9.7% p=0.034). Syngeneic grafts from recipients treated with TLI had minimal apoptosis similar to the levels detected in normal livers.

Further, intragraft CD4+CD25+FoxP3+ cells were increased in the TLI group in the first week post-transplant and remained elevated in the graft and in the spleen. Importantly, adoptive transfer of splenocytes from day >100 liver allograft recipients that received TLI prolonged the survival of donor, but not third-party, heart grafts in naïve recipients. Depletion of CD4+CD25+ cells from transferred splenocytes abrogated this prolongation. We conclude that post-transplant TLI significantly increases apoptosis of infiltrating T cells in the allograft during the first week post-transplant. We suggest that apoptosis of host lymphocytes helps to promote an environment whereby CD4+CD25+FoxP3+ T regulatory cells can develop and accumulate in the graft and periphery, and maintain graft non-responsiveness.

Supported in part by NIH AI044095, AI037683
Pancreatic islets engineered to display on their surface a novel form of FasL protein survive indefinitely by inducing tolerance through phagocytes/CD4+CD25+FoxP3+ Treg cell axis (141.47)
Authors: Zhao Hong , Yolcu Esma S. , Bandura-Morgan Laura , Shirwan Haval
Published: January 2023

Objective: Pancreatic islets engineered to display on their surface a chimeric form of FasL (SA-FasL) protein survive indefinitely in allogeneic recipients. In this study, we tested the role of phagocytes and CD4+CD25+FoxP3+ Treg cells in the induced tolerance.

Methods: To study the function of Treg cells, OT-I transgenic and rag-/- B6 mice were adoptively transferred with 1 x 106 CD4+ Teff cells from naïve B6 together with 2-5x103 Tregs from na ïve, rejected, or tolerant animals one day prior to BALB/c islet Tx. The function of phagocytes in the induced tolerance was investigated using liposome-encapsulated clodronate for depletion.

Results: OT-I mice did not reject BALB/c mice islet grafts (n= 5; >100 days) due to the restricted TCR repertoire for ovalbumin. Adoptive transfer with sorted 1x106 CD4+ Teff cells from naïve B6 mice promptly rejected allogeneic islets (n=5; MST=29.0±9.2 days). Cotransfer of 2-5x103 Tregs from long-term (n=5; >100 days), but not rejecting (n=5; MST=34.4±3.5 days), animals prevented graft rejection. Treatment with clodronate resulted in depletion of phagocytes and acute rejection of SA-FasL-engineered islets (n=5; MST=22.7±10.4 days). In marked contrast, 5/6 mice treated with control liposome accepted their grafts (MST>100 days).

Conclusions: Tolerance to SA-FasL-engineered pancreatic islets is maintained by Treg cells that require phagocytes for induction.

NIH (R21 DK61333, R01 AI47864, R21 HL080108), ADA (1-05-JF-56), and AHA Fellowship 0725348B.
Temporal variability of cellular alloreactivity in kidney transplant candidates (141.8)
Authors: Kramer Kristen L , Siegel Hanni , Lalli Peter , Baldwin William , Fairchild Robert , Poggio Emilio
Published: January 2023

Assessment of preexistent cellular alloimmunity (CA) in transplant candidates aids in understanding individual immunological risk. We tested the extent of CA over time and characterized factors associated with it. Blood samples were obtained every 3 mo from 21 hemodialysis (HD) pts and 5 healthy controls over 1 yr. CA was tested by IFN- ELISPOT using a panel of 6 HLA-typed B cell lines as stimulators (panel of reactive T cells, PRT). For each time point, PRT was calculated and reactivity to >75% of the panel was considered highly reactive. In HD pts, the PRT % increased over baseline with time but was stable in healthy subjects. African-American (AA) pts had significantly higher PRT values at any time point than non-AA, and pts on HD>5 yrs had a trend to higher PRT values. PRT was not different based on gender or age. By logistic regression analysis, race was found to invariably be independently associated with higher PRT values at any time point (OR:1.50, p<0.01), while there was a trend to higher PRT values for pts with longer HD. These data expand upon our previous reports that AA race is a significant risk factor for cellular alloreactivity, potentially accounting for the worse posttransplant outcomes in this population. Serial measurements of preexisting cellular anti-HLA immunity in transplant candidates by PRT is warranted as results can vary in an individual candidate over time.
WIP is critical for T cell responsiveness to IL-2 (33.1)
Authors: Le Bras Severine , Massaad Michel , Suresh Koduru , Kumar Lalit , Oyoshi Michiko , Hartwig John , Geha Raif S
Published: January 2023

The WASP-interacting protein WIP stabilizes WASP, the product of the gene mutated in Wiskott-Aldrich syndrome. WIP-deficient T cells have low WASP levels, limiting the usefulness of WIP KO mice in defining the role of WIP in T cell function. To define this role, we compared WIP/WASP double knockout (DKO) mice to WASP KO mice on DO11.10 background. T cell development was normal in both strains, but peripheral T cell numbers were significantly decreased in DKO mice. WASP KO T cells proliferated and secreted IL-2 normally in response to OVA peptide (OVAp). In contrast, T cells from DKO mice proliferated poorly in response to OVAp in vitro and cutaneous hapten hypersensitivity was deficient in these mice. DKO T cells up-regulated CD25 expression and secreted normal amounts of IL-2 after antigen stimulation, but had defective response to IL-2, evidenced by failure to further up-regulate CD25 expression, phosphorylate STAT5, and induce expression of STAT5 dependent genes. DKO, but not WASP KO, T cells had a disrupted subcortical actin cytoskeleton and impaired actin polymerization following TCR ligation. These results indicate that WIP is essential for IL-2 signaling and responsiveness in T cells, possibly because of its critical role in TCR-triggered actin cytoskeletal reorganization.
IL-2 differentially regulates perforin and FasL mediated cytotoxicity in CD4 T cells (33.10)
Authors: Brown Deborah M. , Harris Leah , Moore Tyler , Swain Susan L. , Condon Shirley A.
Published: January 2023

CD4 T cell effectors can promote survival against lethal influenza virus via perforin mediated cytolytic mechanisms, however; our understanding of how naïve CD4 cells differentiate into class II restricted killers remains obscure. To address this, TCR Tg CD4 cells were activated in vitro and examined for their ability to lyse target cells. We found that cytokine polarized CD4 T cell effectors displayed cytolytic activity with the hierarchy Th0>Th1>Th2>Th17; however, IL-6, TGF-β, IL-10, IL-12 or TNF-α were not required for inducing cytolytic activity in CD4 effectors. Antigen dose had a marked effect on cytotoxicity: low concentrations of peptide induced more potent cytolytic activity than relatively high concentrations. At low peptide concentration, exogenous IL-2 was necessary to drive granzyme B (GrB) expression and perforin mediated lysis. Using IL-2 deficient T cells, we found that IL-2 signaling was required in the first 24 h of culture, but increasing concentrations of IL-2 did not increase cytolytic activity. Studies are underway to determine whether Stat5 signaling is required for induction of cytolytic activity in CD4 T cells. Thus, low antigen dose and early activation signals via IL-2 direct the CD4 T cell response toward effectors with perforin mediated cytolytic potential.
3D cell-matrix contact prevents IFN-γ induced upregulation of endothelial MHC II expression in high glucose environment (33.17)
Authors: Methe Heiko , Kim Jong-Oh
Published: January 2023

Cell matrix interactions are critical to function. Embedding endothelial cells (EC) within 3D matrices (3D-EC) retains biosecretory function and limits cell immunogenicity. Cardiovascular risk factors induce alterations in matrix composition and EC functionality. We examined how glucose levels dictate IFN-γ induced MHC II expression in EC.

EC cultured on polystyrene plates (TC-EC) and 3D-EC were grown to confluence in 0-50 mM glucose with or without 1000 U/ml IFN-γ. Intracellular signaling and MHC II expression were analyzed via PCR, Western Blot, and flow cytometry.

Without glucose phosphorylation of JAK- and STAT-proteins upon IFN-γ stimulation were attenuated 3.5-fold (p<0.001) in 3D-EC compared to TC-EC. This was mirrored by reduced MHC II expression in 3D-EC (PCR: 0.3±0.1 vs. 0.1±0.1 relative units, p<0.005). Suppressor of cytokine signaling protein (SOCS)-1 and -3 expression were 2.5-fold higher in unstimulated 3D-EC (p<0.002). Matrix-embedding amplified upregulation of SOCS by IFN-γ. Low and high glucose medium significantly upregulated IFN-γ signaling pathways and MHC II expression (25 mM: 0.7±0.2, 50 mM: 0.9±0.2 relative units, p<0.0001 vs. no glucose) in TC-EC but had no significant effect on SOCS and MHC II expression (0.2± 0.1 relative units) in 3D-EC.

Glucose amplifies IFN-γ dependent signaling in TC-EC. In contrast, increased SOCS expression in 3D-EC associates with a higher threshold at which IFN-γ induces MHC II, even in states of hyperglycemia. Matrix-embedding presents EC with the near-physiological state of substrate adherence and confluence. Intact cell-matrix contact shields EC from immune recognition and atherosclerotic risk factors like hyperglycemia.
Role of platelet-activating factor in protecting against lipopolysaccharide-induced endotoxic shock (33.3)
Authors: Jeong Young-Il , Jung In Duk , Lee Jun Sik , Lee Chang-Min , Chun Sung Hak , Jeong Soo kyung , Shin Yong Kyoo , Park Yeong-Min
Published: January 2023

AbstractPlatelet-activating factor (PAF) has been long believed to be associated with many pathophysiological processes during septic shock. Here we present novel activities for PAF in the LPS-mediated endotoxic shock. In vivo PAF treatment immediately after LPS challenge markedly improved the survival rate against mortality from endotoxic shock. In addition, this treatment prominently attenuated LPS-induced profound hypotension, excessive polymorphonuclear neutrophil infiltration, severe multiple organ failure, and lymphocyte apoptosis. These protective effects of PAF was correlated with significantly decreases in the production of the inflammatory mediators such as TNF-α, IL-1β, IL-12, and IFN-γ, while increasing production of the anti-inflammatory cytokine IL-10 in vivo and in vitro. In response to LPS, PAF inhibits ERK1/2 and p38 MAPK activation, which is critical for proinflammatory cytokines production. Furthermore, PAF upregulates SOCS1 and SOCS3, which are essential for cross-talk inhibition in LPS-induced cytokine signaling. Taken together, these results suggest that PAF may provide protection against endotoxic shock via a complex mechanism involving modulation of inflammatory and anti-inflammatory mediators, as well as balanced regulation between activation and subsequent deactivation of the signaling pathways in response to LPS.
MAGUK-controlled CARMA1 and BCL-10 turnover propose a new mechanism for NF-κB inactivation in lymphocytes (33.31)
Authors: Moreno-Garcia Miguel E , Sommer Karen , Rawlings David J
Published: January 2023

In lymphocytes, the antigen receptor utilizes a unique scaffolding molecule, CARMA1, to organize an oligomerization cascade that ultimately activates NF-kappaB. Activation of CARMA1 is characterized by conformational changes that expose binding sites to downstream effectors, including BCL10. We found that CARMA1 activation induces its own turnover, and also drives the turnover of BCL10. In B and T cell lines, activated CARMA1 is post-translationally modified by K48-linked polyubiquitin chains, and its degradation is blocked by proteasome inhibitors. CARMA1 ubiquitination was enhanced in vitro by E3 ubiquitin protein ligases cIAP2 and NEDD4 (but not Cbl-b, ITCH, or beta-TrCP). The MAGUK region of CARMA1 plays a unique role in mediating these events as the SH3 and GUK domains are main ubiquitin acceptors, and deletion of a domain (HOOK) located between SH3 and GUK was sufficient to induce E3 recruitment and constitutive ubiquitination. Additionally, we show that CARMA1 promoted parallel ubiquitination and degradation of BCL10 via a TAK1-dependent mechanism. Functionally, we demonstrated that over-expression of cIAP2 reduced the CARMA1-dependent NF-kappaB activation in antigen receptor stimulated lymphocytes. We hypothesize that up-regulation of E3 ligases by NF-kappaB provides a feed-back control mechanism to shut-down NF-kappaB activation by degrading the CARMA1 scaffold and associated downstream effectors including BCL-10.

This work was funded by NIH 5RO1HD37091 and 5RO3TW007322, and CRDF UKB2-2831-06.
Apoptosis signal-regulating kinase (ASK)-1 mediates apoptosis in B cells (34.6)
Authors: Mizuguchi Junichiro , Takada Eiko , Furuhata Masae
Published: January 2023

Engagement of membrane immunoglobulin (mIg) on B cells results in reduction of the mitochondrial membrane potential (ƒ¢ƒμm) and apoptosis, which plays a crucial role in the regulation of immune responses. In this study, we show that the apoptosis signal-regulating kinase 1 (ASK1)-JNK1 signaling pathway participates in mIg-induced apoptosis in normal B cells as well as WEHI-231 B lymphoma cells. Stimulation of WEHI-231 cells with anti-IgM induces phosphorylation and subsequent activation of ASK1, leading to JNK activation. Anti-IgM stimulation produces superoxide anions (O2-), accompanied by loss of ƒ¢ƒμm and an increase in cells with sub-G1 DNA content. Anti-IgM-induced O2- production, loss of ƒ¢ƒμm, and increase in the sub-G1 fraction were all reduced substantially in WEHI-231 cells overexpressing a dominant-negative form of ASK1, compared with control vector alone. The mIg-mediated O2- production, loss of ƒ¢ƒμm, and increase in the sub-G1 fraction were partially abrogated by the reactive oxygen species scavenger N-acetyl-L-cysteine (NAC). Similarly to WEHI-231 B lymphoma cells, the mIg-mediated apoptotic features were moderately compromised in mouse ASK1-deficient B cells. Taken together, these results suggest that mIg engagement induces JNK activation through ASK1 activation, resulting in production of O2- that leads to loss of ƒ¢ƒμm and finally to apoptosis.
Granzyme B is Important in the Control of B Cell Homeostasis (34.7)
Authors: Zhang Yingyu , xu guangwu , Shi Yufang
Published: January 2023

The role of granzyme B in the homeostasis of B cells is unknown. In our study, splenomegaly with increased B number was observed in Granzyme B deficient (gzb-/-) mice over 8 months age along with significantly increased B cells in the thymus. Moreover, gzb-/- mice over 6 months old have considerably longer intestines with accumulation of lymphocytes in the lamina propria. Serum levels of immunoglobulin isotypes dramatically increased in mice over 4 months old. However, no significant increase was observed in serum Th1/Th2 cytokine levels or in vitro T cell functions . Flowcytometry analysis of B cells in the thymus and spleen from gzb-/- mice showed similar characters to B cells from wild type spleen. Bone marrow transplantation of bone marrow from wild type mice to gzb-/- mice partially corrected the abnormalities observed suggesting that the critical change may lie in the hematopoietive system. In vivo BrdU staining uncovered an increased population of BrdU positive CD45- cells in the spleen. In addition, the percentage of BrdU positive B cells also slightly increased indicating possible role of granzyme B in the control of B cell homeostasis possibly through the abnormal function of this CD45- population of cells. Moreover, coculture of freshly isolated or activated CD8+ T cells with B cells did not show increased cell death in the B cells from gzb-/- mice. Our results showed the importance of granzyme B in the control of B cell homeostasis especially in the intestinal mucosa.
Indigenous opportunistic bacteria inhabit mammalian gut-associated lymphoid tissues for mucosal antibody-mediated symbiosis (39.11)
Authors: Obata Takashi , Goto Yoshiyuki , Shibata Naoko , Sato Shintaro , Kunisawa Jun , Kiyono Hiroshi
Published: January 2023

The indigenous bacteria of the gastrointestinal (GI) tract create natural cohabitation niches together with mucosal antibodies (Abs) such as secretory IgA (S-IgA). However, the nature of this symbiosis is still not well understood. Here we report that opportunistic bacteria, mostly Alcaligenes spp., specifically inhabit the gut-associated lymphoid tissues (GALT), namely Peyer's patches (PPs) and isolated lymphoid follicles (ILFs) of naive, specific-pathogen-free (SPF) but not germ-free (GF) mice. Consistent with this result, antigen-specific Ab-producing cells are present in the mucosal (e.g., PPs) but not systemic compartment (e.g., spleen). In the absence of antigen-specific mucosal Abs in PPs, intra-tissue inhabited Alcaligenes were greatly diminished. Thus, Alcaligenes uniquely inhabits PPs by inducing PP antigen-specific Ab production; this creates an optimal symbiotic environment on the interior of PPs.
Distinct roles of dendritic cells and macrophages in B cell class switching (39.12)
Authors: Xu Wei , Dullaers Melissa , Oh Sangkon , Banchereau Jacques
Published: January 2023

Immunoglobulin A (IgA) provides the first line of immune protection at mucosal surface. Dendritic cells (DCs) can induce naïve B cell class switching towards IgA in a T cell -independent manner. We questioned whether resident macrophages, antigen presenting cells in tissue, can also shape protective mucosal immunity. Human DCs (by IL-4 and GM-CSF) and macrophages (by M-CSF) were generated from monocytes of the same donor. Autologous IgD+CD27- naïve B cells were activated by anti-IgM (to mimic antigen engagement with BCR), CpG and IL-2, and cultured with DC or macrophages on L cell-coated plates (to mimic stoma environment). We showed here that both anti-inflammatory CD163+ macrophages and DCs induced naïve B cell proliferation and differentiation towards CD38+CD27+CD20- plasmablasts. However, CD38+ cells induced by macrophages showed more terminally differentiated CD138+ plasma cells than those induced by DCs. Macrophage-induced plasma cells preferentially expressed surface IgA whereas DC-induced plasma cells preferentially expressed surface IgG. ELISA showed that macrophage-stimulated B cells produce more IgA (both IgA1 and IgA2) than DC-stimulated B cells which produce more IgG. Macrophage-primed plasma cells also expressed more CCR10 than those primed by DCs. Both tonsil and colon sections confirmed that CD138+ plasma cells were surrounded prevalently by CD163+ resident macrophages. Our data suggest a previously unrecognized mechanism by which macrophages help B cells to establish protective mucosal immunity.