bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation of C4 Binding Protein Deficient Mice: Evaluation of Putative Biological Functions (134.92)
Authors: Soimo Kipruto , Morales John E , Mueller-Ortiz Stacey , Nonaka Maumi , Nonaka Masura , Blom Ana M , Wetsel Rick A
Published: January 2023

The complement C4 binding protein (C4BP) has been suggested to modulate the antibody response to T dependent antigens by binding CD40 on B cells and to confer serum resistance when bound to certain bacteria, including M. catarrhalis. To date, all functions attributed to C4BP have been delineated in vitro. To evaluate functions of C4BP in vivo, we have generated C4bBP deficient mice by targeted disruption of mouse C4BP alpha gene. As expected, the C4bBP-/- serum exhibited significant increased hemolytic activity, and C3 deposition on M. catarrhalis was significantly increased. C4BP-/- mice and C4BP+/+ mice were injected i.p. with M catarrhalis to determine if elevated C3 deposition in C4BP-/- sera would correspond to increased killing of M. catarrhalis in vivo. Contrary to our expectation, the absence of C4BP did not impact the clearance (CFUs) of M.catarrhalis. To investigate if the absence of C4BP would impair the immune response to T-dependent Ags, mice were immunized with DNP-OVA. In contrast to the prediction of Brodeur et al (Immunity 2003), C4bBP-/- mice produced DNP specific IgG1 and IgG2a antibodies at similar levels as did C4bBP+/+ mice. Collectively, these studies provide evidence for the importance of C4BP in regulating complement activation and that C3 deposition on M. catarrhalis is impaired by binding C4BP. However, the absence of C4BP did not cause increased killing of M. catarrhalis in vivo, suggesting that other mechanisms of bacterial clearance compensate for the absence of C4BP. Moreover, C4BP as a major modulator of the antibody response was not supported in these studies, suggesting species dependent differences in C4BP biological functions.
TLR2 and TLR4 differentially regulate DC to stimulate Th17-associated cytokines in response to mycoplasma superantigen (135.30)
Authors: Mu Hong-Hua , Hasebe Akira , Cole Barry
Published: January 2023

Mycoplasma arthritidis induces toxic shock, arthritis and skin necrosis in mice. M. arthritidis mitogen (MAM), a potent superantigen (SAg) secreted by M. arthritidis, contributes to some of these symptoms in certain mouse strains. We previously demonstrated that C3H mouse strains differing in expression of TLR4, differ in immune reactivity to MAM due to differential engagement of TLR2 and TLR4. Herein, we demonstrated that in TLR4-defect C3H mice, following injection of MAM SAg, the splenic DCs were activated to express significantly higher CD8α surface markers than expressed in wild-type mice, and produced remarkably elevated IL-12p40, TNFα and IL-23. In vivo injection of anti-IL-23 antibody, or anti-CD8α antibody greatly suppressed IL-17 in splenic CD4+ cell cultures in comparison with untreated groups. In contrast, in wild-type C3H mice the interaction of MAM with both TLR4 and TLR2 led to reduction of the CD8α+ DC subset; as a consequence the IL-23 is down-regulated, but IL-10 are greatly up-regulated. The results suggest that TLRs control Th17 cell-associated cytokine development in response to MAM, and the CD8α+ DC subset is a major cell type responsible for IL-23 production. DC function that is regulated by discrete TLRs and CD8α molecules in response to MAM SAg might largely influence the outcome of subsequent adaptive immune responses in murine inflammatory diseases caused by M. arthritidis.
Calcium hydroxide attenuates inflammatory potential of lipoteichoic acid from Enterococcus faecalis (135.33)
Authors: Baik Jung Eun , Ryu Young Hee , Han Ji Young , Im Jintaek , Yang Jae Seung , Yun Cheol-Heui , Han Seung Hyun
Published: January 2023

Enterococcus faecalis is a pathogenic gram-positive bacterium closely related with refractory apical periodontitis. Since lipoteichoic acid (LTA) is a major virulence factor of gram-positive bacteria, we prepared highly pure LTA from E. faecalis and investigated its inflammatory responses in a murine macrophage-like cell line, RAW 264.7. Upon exposure to E. faecalis LTA, RAW 264.7 cells substantially produced tumor necrosis factor-α (TNF-α) in concentration- and time-dependent manner. Notably, the LTA was able to stimulate Toll-like receptor 2 (TLR2), but not TLR4. Concomitantly, LTA enhanced the DNA-binding activity of a transcription factor, NF-κB, which plays an important role in the transcriptional activation of genes encoding inflammatory mediators. However, induction of TNF-α production was not observed in the cells exposed to LTA pre-treated with calcium hydroxide, which is widely used endodontic medicament for eliminating viable bacteria and inactivating virulence factors. Furthermore, calcium hydroxide-treated LTA was not able to stimulate TLR2. These results suggest that E. faecalis LTA could stimulate inflammatory responses through TLR2 and calcium hydroxide could detoxify LTA, resulting in attenuation of the inflammatory responses. Supported by the Korea Research Foundation Grant (KRF-2007-314-E00186).
The immune adaptor MPYS/STING/MITA signals via multiple differentially regulated pathways (135.60)
Authors: jin lei , xu liang-guo , cambier john c
Published: January 2023

MPYS, a.k.a. STING and MITA, is a newly described signaling adaptor that functions in both innate and adaptive immunity. MPYS transduces apoptotic death signals when aggregated on the B cell surface, but also plays an obligate role in the intracellular sensing of B-DNA and infectious agents. The basis of MPYS signal transduction is unclear. We report that MPYS signals by two distinct pathways. It associates via its juxtamembrane Box 1 and phosphotyrosine motifs with Tyk2 and STAT1, and utilizes these effectors in ISRE activation. Activation of IFNbeta occurs via MPYS association with and activation of TBK1 and IRF3, and is independent of VISA. MPYS (but not VISA) activation of IFNbeta expression is sensitive to LPS-induced TLR4-independent inhibition, but is positively cooperative with TLR signals. Thus MPYS activates multiple, uniquely regulated signaling pathways to mediate resistance to infection and regulation of adaptive immunity.
TLT2 Ligation Potentiates Neutrophil Migration and the Respiratory Burst Response (135.62)
Authors: Halpert Matthew M , King R. Glenn , Justement Louis B.
Published: January 2023

TREM-like Transcript 2 (TLT2) is a transmembrane receptor expressed on B cells, macrophages and neutrophils in the mouse and human. As of yet, an endogenous ligand has not been identified and the function of TLT2 remains to be elucidated. Because neutrophils are a primary mediator of the host innate immune response to bacterial infection, the analysis of factors that control neutrophil function is of great interest. Preliminary evidence suggests that TLT2 potentiates multiple neutrophil functions, including the respiratory burst and chemotaxis. Using a chemiluminescence system, it was shown that TLT2 ligation increases the primary phase of the respiratory burst by 20-25% in response to the bacterial peptide FMLP, although there is a slight inhibitory delay during the initial phase of the response. Transwell assays have been used to determine the effect that α-TLT2 mAb has on murine neutrophil chemotaxis. In vitro data demonstrate that TLT2 ligation increases migration 1.5-2.5 times in response to different chemotactic stimuli, such as FMLP, IL-8, and MIP-2. Importantly, α-TLT2 mAb treatment potentiates chemotaxis in response to agonists that signal via G-protein coupled receptors, but not in response to agonists that signal via tyrosine kinase-dependent pathways (e.g. G-CSF). These data suggest that TLT2 ligation effectively potentiates multiple neutrophil functional responses.
C-Cbl Regulates Neutrophil Integrin-dependent Respiratory Burst (135.67)
Authors: Peterson Erik J , Zou Liangxing , Ellingson Sonja
Published: January 2023

Signaling through integrin receptors is required for optimal reactive oxygen species (ROS) production by polymorphonuclear neutrophils (PMN). The E3 ubiquitin ligase and adapter protein C-Cbl has been implicated in integrin-mediated adhesion, and is a substrate for integrin-stimulated protein tyrosine kinases. We hypothesized that c-Cbl regulates integrin-driven PMN functions. We observed tyrosine phosphorylation of c-Cbl after beta-2 integrin cross-linking in the NB4 myeloid cell line. To address the requirement for c-Cbl in primary PMN signaling, we utilized c-Cbl deficient mice. We found that while c-Cbl is dispensable for PMN maturation, signaling through both immunoreceptors and integrins on PMNs is modulated by c-Cbl. Upon stimulation of c-Cbl-deficient bone marrow-derived PMNs with beta-2 integrin ligands (poly-RGD or fibrinogen plus TNF-alpha), we observed an 80% decrease in ROS generation compared to stimulated control cells. Phorbol ester-driven ROS generation is unaffected by c-Cbl deficiency. We next addressed the biochemical mechanism of c-Cbl action, investigating MAPK, PI3K, and Ca2+dependent pathways downstream of integrins. We observed no defect in either ERK or Akt phosphorylation after integrin engagement in c-Cbl deficient PMNs; furthermore, we observed that poly-RGD stimulated ROS production is unimpaired in PMNs lacking the PI3K p110gamma subunit. However, c-Cbl-null PMNs displayed decreased phospholipase C phosphorylation. Together, these data strongly suggest that c-Cbl selectively regulates Ca2+driven signaling downstream of beta2 integrins and upstream of ROS generation in PMNs.

Supported by the Minnesota Medical Foundation
Novel functional roles for JAML and CAR in epithelial γδ T cell-specific costimulation (135.68)
Authors: Witherden Deborah A , Verdino Petra , Rieder Stephanie E , Mills Robyn E , Teyton Luc , Fischer Wolfgang H , Wilson Ian A , Havran Wendy L
Published: January 2023

γδ T cells represent a major T cell population in epithelial tissues and provide a crucial first line defence against epithelial insults, yet the molecular events surrounding their activation remain poorly defined. Here we identify a γδ T cell-specific costimulatory molecule, Junctional Adhesion Molecule-Like protein (JAML). Costimulation through JAML leads to cellular proliferation, cytokine and growth factor production. Inhibition of JAML costimulation leads to diminished γδ T cell activation and delayed wound closure akin to that seen in the absence of γδ T cells. Our results identify JAML as a crucial component of epithelial γδ T cell activation. Furthermore, we show that interaction between JAML and its ligand, Coxsackie and Adenovirus receptor (CAR), extends beyond pure cell adhesion, demonstrating an important role for CAR as a signaling receptor for the immune system. This has broader implications for its proposed role in tumor migration and growth.
A critical role of IRAK1 in the differential regulation of Th17 and T regulatory cells (135.8)
Authors: Maitra Urmila , Li Liwu
Published: January 2023

ObjectiveIRAK-1 has been linked to several human inflammatory diseases including autoimmune diseases. Loss of IRAK1 confers resistance to EAE, however, the exact mechanism is not understood. Given the recent discovery of Th17 lineage and its link to diverse autoimmune diseases, we tested the hypothesis that IRAK1 may contribute to the progression of autoimmune diseases via modulating the differentiation of Th17 and T regulatory cells.

MethodsNaïve CD4+ T cells were isolated from splenocytes of wild type (WT) and IRAK1-/- mice. In addition to anti-CD3 and anti-CD28, CD4+ T cells were stimulated with TGF β or TGF β plus IL6. Target proteins and mRNAs were harvested and analyzed via Western blot and RT-PCR. ChIP assays were used to demonstrate in vivo binding of transcription factors to the promoter region of corresponding genes.

ResultsIn response to TGF β and IL6, loss of IRAK1 reduced IL-17A and RORγt expression in naïve CD4+ T cells contrary to WT. Also, IRAK1 knockout impaired TGFβ and IL-6 induced STAT3 Ser727 phosphorylation. ChIP assays indicated reduced in vivo STAT3 binding to the promoter of IL-17 in IRAK1-/- cells. In contrast, the levels of Foxp3 and NFAT were highly induced in IRAK1-/- cells treated with TCR agonists and TGFβ, indicating higher percentage of Treg cells. In vivo binding assays with Smad3 antibodies demonstrated increased binding to the Foxp3 promoter in IRAK1-/- cells.

ConclusionOur data supports a novel role of IRAK1 in the regulation of Th17 and Treg differentiation. These findings may have significant implications in the pathogenesis of autoimmune diseases.

This work is supported by NIH grant R01 AI64414.
Promyelocytic leukemia protein specifically augments the activation of NFAT (136.11)
Authors: Lo Yu-Hsun , Wu Chia-Chen , Shih Hsiu-Ming , Lai Ming-Zong
Published: January 2023

Promyelocytic leukemia (PML) protein is a tumor suppressor with complicated action mechanisms not yet fully understood. Nuclear factor of activated T cells (NFAT) is an important factor for T cell functions. In this study, we found that PML specifically enhanced the transcription activation of NFAT. In PML-null mouse embryonic fibroblasts, no transcription activity of NFAT could be detected. There was a selective requirement of PML isoform in NFAT activation: PML-I and PML-VI, but not PML-IV, increased NFAT transactivation. PML specifically promoted the expression of many, but not all, NFAT targeted genes, including TNF ƒÑ, TRAIL and Cbl-b. A specific binding of PML to NFATc was found by GST pull down assay and immuno-precipitation. The interaction of PML with NFATc in vivo was further confirmed by ChIP and DNA affinity precipitation assay (DAPA) analysis. Knockdown of PML was associated with reduced nuclear presence of NFAT. The unexpected coupling of PML with NFAT reveals a novel mechanism underlying the diverse physiological functions of PML.
Improved peripheral T cell expression of T-cell receptor (TCR)-α transgenes in TCRα/Dad1 locus-like "two gene" reporter systems (136.14)
Authors: Ortiz Benjamin D , Gomos-Klein Janette , Knirr Stefan , Andino Blanca , Harrow Faith , Erhard Karl , Kovalovsky Damian , Sant'Angelo Derek
Published: January 2023

The mouse T-cell receptor (TCR)-a; locus control region (LCR) resides in between the Ca; exons and the Dad1 gene. We reported the "single cell level" activity of the TCRa LCR in the context of linked heterologous single reporter transgenes randomly integrated into the genome of mice. We found that the LCR supports the normal pattern of TCRa expression in the thymus. However, abnormal ectopic expression and T cell transcript reductions were observed in the periphery. These data indicated that important cis-acting elements (not associated with the LCR) are required for strict cell type-specificity, and peripheral T cell maintenance of TCRa expression. Here we describe data from experiments with transgenic mice bearing constructs in which the LCR is flanked by two transcription units (as it is in the genome by TCRa [5'] and Dad1 [3']). In the first system, two heterologous reporter genes flank the LCR. The other system is an 80-kb TCRa/Dad1 Bacterial Artificial Chromosome (BAC) fragment in which a Va promoter driven reporter is inserted upstream of the Ca exons, followed by the TCRa LCR and the Dad1 gene locus. We find that peripheral T cells maintain expression of the TCRa reporter gene in the BAC construct. Surprisingly, in both "two-gene" reporter systems, ectopic, non-T cell expression of the TCRa reporter is virtually eliminated. The data suggest that important cis-acting elements maintaining normal TCRa expression patterns in peripheral T cells reside in the Dad 1 gene locus. Supported by NIH grants AI053050 (to BDO), RR03037 (to the Hunter College Gene Center) and UL1RR024996 (to the Clinical and Translation Science Center at Weill Cornell Medical College).
microRNA expression profiling in Multiple Sclerosis and EAE (136.21)
Authors: Escobar Sabine S , Kennedy Bryan , Kerkof Keith , Peiser Leanne , Arnett Heather A
Published: January 2023

microRNAs (miRNAs) are small endogenous non-coding RNAs that are emerging as key regulators of protein expression; however, little is known about the expression or roles of miRNAs in multiple sclerosis. We isolated and profiled miRNAs from normal and multiple sclerosis donors and have identified a number of miRNAs that are regulated in MS white matter and plaque tissue, as well as some which appear to have altered expression in MS grey matter tissue. Using miRNA and mRNA profiling in parallel, we have been able to identify miRNA families and predicted target networks with relevance to MS. As points of comparison to the MS tissue samples, we also extensively profiled various human immune cell subsets, as well as spinal cord samples from mice with experimental autoimmune encephalomyelitis (EAE), an animal model with similarities to MS, for miRNA expression. miRNA signatures of the whole blood human immune cell populations have enabled identification of cell-type specific miRNAs that can be used to predict the composition of the inflammatory infiltrate in the MS tissue. Identification of the regulation of miRNAs in MS tissue may lead us to a greater understanding of the pathophysiology of the disease, and potentially provide novel targets for therapeutic intervention.
Great genotypic and phenotypic diversities associated with copy-number variations (CNVs) of complement C4 and RP-C4-CYP21-TNX (RCCX) modules in Asian-Indian and European Americans (136.29)
Authors: Yu Chack-Yung , Saxena Kapil , Kitzmiller Kathryn J. , Wu Yee Ling , Zhou Bi , Esack Nazreen , Hiremath Leena , Chung Erwin K. , Yang Yan
Published: January 2023

Inter-individual CNVs probably afford human populations the flexibility to respond to a variety of environmental challenges, but also lead to differential disease predispositions. We investigated gene CNVs for complement C4 and steroid 21-hydroxylase from the RCCX modules located in the MHC among healthy Asian-Indian Americans (AIA) and compared them to European Americans. A combination of definitive techniques that yielded cross-confirmatory results was used. The medium gene copy-numbers for C4 and its isotypes, acidic C4A and basic C4B, were 4, 2 and 2, respectively, but their frequencies were only 53-56%. The distribution patterns for total C4 and C4A are skewed towards the high copy-number side. For example, the frequency of AIA-subjects with three copies of C4A (30.7%) was 3.92-fold of those with a single copy (7.83%). The haplotype with a single short C4B gene and the absence of C4A, which is in linkage- disequilibrium with HLA DRB1*0301 in Europeans and a strong risk factor for autoimmune diseases, has a frequency of 0.012 in AIA but 0.106 among healthy European Americans (p=6.6x10-8). The copy-number and the size of C4 genes strongly determine the plasma C4 concentrations. Parallel variations in copy-numbers of CYP21A and TNXA with total C4 were observed. The high copy-numbers of C4 and the high frequency of RCCX recombinants offer important insights to the prevalence of autoimmune and genetic diseases.
HLA genetic association analysis using the sequence feature variant type approach (136.31)
Authors: Marthandan Nishanth , Karp David R , Arnett Frank , Waller Matthew J , Guidry Paula , Feolo Michael , Marsh Steven GE , Scheuermann Richard H
Published: January 2023

Immunogeneticists have found significant associations between specific HLA alleles and a variety of medical conditions, including autoimmune diseases. Current disease association studies treat each HLA allele as a single complete unit, which does not illuminate the parts of the molecule associated with disease. We have developed a novel approach for genetic association analysis in which HLA genes and proteins are broken down into smaller sequence features. Sequence features are defined based on structural or functional information, and can be overlapping and continuous or discontinuous in the linear sequence. The extent of sequence variation is then assessed for each HLA sequence feature to define all variant types found in the human population. This allows for the independent analysis of disease association with any sequence feature variant type (SFVT). We tested this approach in the analysis of systemic sclerosis patients using HLA-DRB1 and HLA-DQB1 typing data. We identified a region of the HLA-DRB1 protein centered around peptide-binding pocket 7 that appears to be associated with disease risk; aromatic amino acids found in HLA-DRB1*1104 at positions 26, 28, 37 and 67 were associated with increased risk. The SFVT approach is a novel method for identifying the molecular determinants of disease association in highly polymorphic genes like the HLA.

For DAIT-DISC; supported by NIAID N01AI40076
Effector CD4+ T cells expressing IFNγ or IL-17 in pancreatic infiltrates of young NOD mice (137.23)
Authors: Li Cheng-Rui (Michael) , Bradley Linda M.
Published: January 2023

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing pancreatic islet β cells. Previous studies established that the Th1 subset of CD4+ T effector/memory cells plays a central role in the induction of the aoutimmune attack against islet β cells. Using the nonobese diabetes (NOD) mouse model, our recent studies suggested that the Th17 subset CD4+ T effector cells may also play a role in T1D. In an effort to profile the kinetics of effector/memory CD4+ T cell development, we found significant percentages of CD4+ T cells that express effector/memory phenotype (CD44hi CD62L-) in peripheral lymph nodes and spleens of young female NOD mice (2 weeks old). Importantly, we were able to identify islet infiltrating CD4+ T cells in 2-week-old NOD mice, a time point when insulitis cannot be readily detected by histology. Furthermore, intracellular cytokine staining shows that significant proportions of these early infiltrating CD4+ T cells express either IFNγ or IL-17, signature cytokines of Th1 and Th17 subsets of effector/memory cells, respectively. On going efforts are being directed to elucidating how this early effector/memory population of CD4+ T cells develop and to testing the pathogenic potentials of these cells in the NOD model of T1D.
A novel protective role for the Mer receptor tyrosine kinase in glomerular injury in nephrotoxic nephritis (137.32)
Authors: Shao Wen-Hai , Rosenbaum Joshua , McGaha Tracy L. , Cohen Philip L.
Published: January 2023

The Mer receptor tyrosine kinase plays an important role in clearance of apoptotic cells and in modulation of innate immune responses. Mice with a Mer kinase domain deletion (Mer-KO) develop progressive lupus-like autoimmunity. Gas6, the ligand for Mer, has been detected in glomeruli. We had previously noted that normal kidneys express high levels of Mer in glomerular tissue and set out to determine its anatomical location and its function in renal tissue. We hypothesized that glomerular expression of Mer might modulate experimental immunopathology. We thus compared the susceptibility of WT and Mer-KO mice to nephrotoxic anti-glomerular basement membrane-induced murine nephrotoxic nephritis by infusing mice with 7.5 ml/kg nephrotoxic sera. Mer-KO but not WT B6 recipients showed increased proteinuria and blood urine nitrogen by day 3. Glomeruli were hyperplastic and later became necrotic. PAS-positive staining was evident in Mer-KO capillary spaces as well within tubules, consistent with massive protein leakage. This early pathological change was associated with a lower survival rate (40%) in Mer-KO mice compared to WT (100%) at day 15. Altogether, data suggest that Mer acts to preserve the kidney from immune-related inflammation, either through its role as a down regulator of cytokine production or by promoting phagocytosis of apoptotic cells. We are further investigating the role of Mer in nephrotoxic nephritis. Supported by NIDCR and the US Department of Veterans Affairs.
The Dot1L histone H3 methyltransferase is essential for early Mammalian development and hematopoiesis (138.15)
Authors: Fields Patrick E. , Feng Yi , Yang Yanping , Ortega Manoela M. , Copeland Jessica , Vivian Jay L.
Published: January 2023

Histone methylation of H3 is catalyzed by at least two families of proteins, the PRMT family and the SET family. Recent studies have found a new histone H3 methyltransferase without a SET domain named Dot1L (disrupter of telomere silencing 1-like). To investigate the function of Dot1L, a Dot1L deficient mouse line was generated by injection of murine embryonic stem (ES) cells containing a gene trap into C57BL/6 female blastocysts (clone #RRR032; Bay Genomics, San Francisco, CA). Fibroblasts from Dot1L-homozygous mutant embryos were completely defective in their ability to methylate lysine 79 of histone H3. All knockout embryos died by embryonic day 13. The knockout embryos showed significant reductions in blood and blood vessel formation. Further, colony assays revealed a defect primarily in early erythropoiesis. The early erythroid precursor cells were defective in their ability to respond to erythropoietin, and the cells were more susceptible to early apoptosis after stimulation. We conclude that Dot1L plays an important role in early hematopoiesis, primarily affecting early erythroid precursor cells.
MyD88 expression in stem cells regulates hematopoietic reconstitution following bone marrow transplantation (138.16)
Authors: Rahman Adeeb H , Turka Laurence A
Published: January 2023

Myeloid differentiation protein 88 (MyD88) is an adaptor protein that is required for signaling through most Toll-like receptors (TLRs) as well as the IL-1R family. While these pathways are primarily considered in cells of the innate immune system, TLRs are also expressed by hematopoietic stem cells (HSCs) and progenitor cells. We sought to examine the potential importance of MyD88 during hematopoietic development. We find that bone marrow from WT and Myd88-/- C57BL/6 mice contains comparable numbers of HSCs, which give rise to differentiated progenitors with equivalent efficiency in vitro. However, following a competitive mixed bone marrow transplant (BMT) into lethally irradiated recipients, WT HSCs display a significant advantage over Myd88-/- HSCs, resulting in a stable predominance of WT-derived cells in the reconstituted myeloid and lymphoid compartments. This skewed ratio of mature WT to Myd88-/- cells in the periphery of reconstituted mice is apparent in all developmental stages including the earliest long term HSCs. Interestingly, we find that WT HSCs do not exhibit this advantage over Myd88-/- HSCs following competitive BMT into non-irradiated recipients. Overall, our data suggest that factors associated with irradiation, such as inflammation, act on HSCs in a MyD88-dependent fashion to promote long-term engraftment of transplanted bone marrow.
Nuclear factor TOX is required for lymph node organogenesis and NK cell development (138.29)
Authors: Aliahmad Parinaz , Kaye Jonathan
Published: January 2023

Thymocyte selection-associated HMG-box protein (TOX) is a nuclear DNA-binding factor that is expressed at discrete stages of T cell development in the thymus. We have previously reported that development of CD4 T cells, NKT cells and T regulatory cells is severely inhibited in TOX-deficient mice, as a result of a block in the completion of thymic positive selection. In contrast, CD8 T cells, γδT cells, and B cells develop in these animals. Here we document that there are other severe abnormalities in development of the immune system in the absence of TOX, including inhibition of the development of NK cells and loss of lymph nodes (LN). TOX-deficient bone marrow precursor cells fail to develop into NK cells under appropriate conditions in vitro, suggesting an intrinsic defect in hematopoiesis. Peripheral lymph nodes are undetectable in TOX-deficient mice, while the number and size of Peyer's patches are severely reduced. Bone marrow chimeras demonstrate that loss of LNs is not due to aberrant migration of lymphoid populations. Indeed, TOX-deficient mice have few phenotypically defined lymphoid tissue inducer cells, suggesting a developmental defect in organogenesis. We are currently investigating the mechanisms surrounding these phenotypes.
c-Myb is crucial for B-lymphocyte development (138.30)
Authors: Bender Timothy P , Allman David M , Rajewsky Klaus , Fahl Shawn P
Published: January 2023

c-Myb is abundantly expressed in lymphocyte progenitors yet little is known about c-Myb function during lymphocyte development due to the embryonic lethality of Myb null mutations. We have targeted the murine Myb locus with loxP sites for conditional deletion and crossed Mybff mice to the Mb1Cre and CD19Cre strains to ablate c-Myb expression during different stages of B cell development. Mb1Cre mediated deletion begins in pre-pro-B cells and is complete by the pro-B cell stage. We detect a no decrease in the number pre-pro-B cells but <1% the normal number of CD19+ B-lineage cells at any subsequent stage of development, demonstrating that c-Myb is essential for the development past the point of commitment to the B cell lineage. In Mybf/f CD19Cre mice, B-lineage cells are reduced in number by 80% and blocked in transition from pro-B to pre-B cells, resulting in about 10% the normal number of IgM+ bone marrow B cells. c-Myb deficient B cells are hyporesponsive to IL-7 in methylcellulose cloning assays and in vitro proliferation assays, suggesting that at least part of the defect in pro-B to pre-B cell transition is due to a defect in the IL-7 signaling pathway.
IgE Receptor Activates Stat5 via a Fyn-kinase Pathway (139.10)
Authors: Barnstein Brian , Ryan John
Published: January 2023

We have recently found that the transcription factor Stat5 is rapidly activated by IgE crosslinkage, and that its expression is critical to IgE-mediated degranulation, LTB4 production, cytokine secretion, and survival signals. Stat5KO mast cells induced cytokine mRNAs normally following IgE crosslinkage, but these mRNAs were not sustained over time, and were degraded at twice the rate observed in wild type cells. We find that Stat5 is associated with the IgE receptor after antigen-mediated activation. Moreover, cholesterol depletion or Fyn kinase deletion prevents Stat5 tyrosine phosphorylation in response to IgE crosslinkage. In contrast, the Lyn, Syk, MAPK, and PI-3 kinase pathways do not appear to regulate Stat5 tyrosine phosphorylation via the IgE receptor. We also find that Stat5 is serine phosphorylated during IgE signaling. These data suggest that Stat5 is activated through a lipid raft- and Fyn-dependent pathway. Our work demonstrates that Stat5 is a critical factor in IgE-induced mast cell activation that could provide a new clinical target for mast cell-associated inflammatory diseases. Supported by NIH grants 1R01 AI59638 and U19A1077435.
High Resolution Imaging Methods to Probe FcεRI Membrane Organization & Behavior (139.6)
Authors: Wilson Bridget S , Lidke Diane , Lidke Keith , Andrews Nicholas , Murphy Robert , Oliver Janet M
Published: January 2023

We have applied a combination of high resolution microscopy approaches, together with lipid mass spec analysis, to study the membrane organization of high affinity IgE receptors (FcεRI), their signaling partners and the local lipid environment. Using immunogold labeling and transmission electron microscopy (TEM) imaging of fixed membrane sheets, we find resting receptors in singlets and small clusters. After crosslinking, clusters increase dramatically in size and recruit specific downstream signaling molecules to form primary signaling domains. Atomic Force Microscopy (AFM) studies provide maps of topographical features of the cytoplasmic face of the membrane and demonstrate the importance of cholesterol to the height of these features. Consistent with this, lipid mass spec analysis of isolated FcεRI membrane patches show that 50% of the lipid surrounding receptors is cholesterol. Recently, we have generated novel quantum dot (QD) based probes to study the dynamics of early events in FcεRI signaling. Monovalent IgE-QD complexes are used to track the lateral motion of individual FcεRI in resting and activated states. These studies document transient co-confinement of resting IgE-bound receptors, as well as the immobilization of receptors after extensive crosslinking. Mathematical models show that clusters form when receptors rapidly exchange between domains or confinement zones, consistent with experimental results. The motion of QD-IgE has also been tracked in live cells transfected with GFP-actin using total internal reflection microscopy (TIRF), providing direct evidence that actin corrals provide one mechanism for defining confinement zones. Supported by NIH RO1 AI051575 (BW), NIH RO1 GM49814 (JM0) and Sandia Labs (LDRD).
DISTINCT ROLES OF IL-10 IN REGULATION OF PEANUT ALLERGY (140.10)
Authors: Lotoski Larisa C , Simons F Estelle R , Liem Joel , Chooniesdass Rishma , Becker Allan B , HayGlass Kent T
Published: January 2023

Objective: To evaluate the role of IL-10 in regulation of peanut-driven cytokine production in peanut allergic, sensitized, and clinically tolerant human populations.

Methods: Eighteen clinically peanut allergic, 8 sensitized, and 29 peanut non-allergic individuals between 6-45y were studied. PBMC were stimulated with peanut Ag alone, and in the presence of rIL-10 or anti-IL-10R neutralising Ab in short-term primary cultures. Peanut-driven Type 1, Type 2, and regulatory cytokine response profiles were quantified by ELISA.

Results: IL-10 production in response to peanut-specific stimulation was higher in peanut allergic than non-allergic individuals. Exogenous rIL-10 abrogates peanut-driven cytokine production in peanut allergics. Blocking endogenous IL-10 function has no impact on recall Type 2 cytokine responses in non-allergics. Among peanut allergics, anti-IL-10R treatment during Ag stimulation enhanced both Th2 and Th1 recall responses.

Conclusion: IL-10 and the IL-10R play no detectable role in preventing initiation of peanut specific food allergy, but may be important in limiting the intensity of peanut specific Th2 and pro-inflammatory responses in peanut allergic individuals.
Syk, a novel therapeutic target for PTLD, drives Epstein Barr Virus B cell lymphoma growth and survival through the PI3K/Akt pathway (141.23)
Authors: Hatton Olivia L , Lambert Stacie , Vaysberg Maria , Krams Sheri M , Esquivel Carlos O , Martinez Olivia M
Published: January 2023

Post-transplant lymphoproliferative disorder (PTLD)-associated Epstein Barr Virus (EBV)+ B cell lymphomas are a serious, and often fatal, complication of solid organ and bone marrow transplantation. In EBV+ PTLD, latent membrane protein 2a (LMP2a), a mimic of the B cell receptor, provides a constitutive survival signal to latently infected cells. This signal is transmitted through the sequestering and activation of Lyn and Syk and subsequent activation of downstream survival pathways. We hypothesized that inhibition of Syk activity would diminish growth of EBV+ B cell lymphoma lines derived from patients with PTLD. Treatment with R406, a novel Syk inhibitor, decreased proliferation and induced apoptosis of PTLD lines. While the NFkB and p38 pathways were unaffected after PTLD lines were treated with R406, both the Akt and Erk pathways were inhibited. Akt is constitutively active in these cells, and treatment with R406 led to decreased Akt-dependent autocrine IL-10 secretion. These data indicate that Syk mediates its effects on EBV+ PTLD growth and survival through activation of the PI3K/Akt pathway. Syk inhibitors, such as R406, may serve as an effective therapeutic strategy for EBV+ PTLD.
Paracrine Generation of NO by Bystander Human CD8 T Cells Augments Allogeneic Responses by Inhibiting Cytokine Deprivation-Induced Cell Death (141.3)
Authors: Choy Jonathan C. , Pober Jordan S.
Published: January 2023

We previously reported that NO, generated by iNOS expressed in bystander human CD8 T cells, augments the accumulation of allogeneically activated human CD8 T cells in vitro and in vivo. Here we report that iNOS-derived NO does not affect T cell proliferation but rather inhibits cell death of activated human CD8 T cells after activation by allogeneic endothelial cells in culture. Furthermore, exogenous NO does not affect activation-induced cell death of human CD8 T cells but specifically reduces death of activated T cells due to cytokine deprivation. This effect does not involve cGMP signaling, and NO does not affect the protein expression levels of Bcl-2, Bcl-xL, Mcl-1, Bim, or PUMA, which are Bcl-2-related proteins previously implicated as regulators of cytokine deprivation-induced cell death. However, NO inhibits the activity of caspases activated as a consequence of cytokine deprivation in activated T cells. This protective effect correlated with S-nitrosylation of caspases and was phenocopied by ZVAD-FMK and LEDH-FMK, pharmacological inhibitors of caspases. Our findings suggest that NO augments the accumulation of allo-activated human T cells principally by inhibiting cytokine deprivation-induced cell death through S-nitrosylation of caspases.
Cyclosporine A modifies immune-mediating and therapeutic efficacy of allogeneic perivascular porcine endothelial cell implants (141.51)
Authors: Methe Heiko , Edelman Elazer R
Published: January 2023

Non-syngeneic endothelial cell embedded within 3D-matrices (MEEC) when placed in the vascular adventitia control vascular repair processes. Host immunity directed against engrafted allogeneic tissue is a predominant Th1-driven immune response and a major impediment for graft survival. We aimed to identify if cyclosporine A (CsA) would enhance immune compatibility and therapeutic efficacy of allogeneic MEEC.

Pigs (n=4/group) underwent balloon injury of both carotid arteries and received a 12 days course of CsA (group 1), perivascular implants of porcine MEEC (group 2), combination of CsA and MEEC (group 3) or were left untreated (group 4). Host immune reactivity was analyzed after 28 and 90 days in 2 pigs/group respectively.

No immune reactivity against MEEC was observed in treatment groups 1 and 4. MEEC treatment alone (group 2) induced formation of IgG1 antibodies specific for the allogeneic EC and differentiation of host splenocytes into Th2 but not Th1 cytokine-producing cells. CsA-therapy reduced the frequency of IgG1 antibodies and Th2-cytokine producing splenocytes upon MEEC treatment (group 3). Compared with groups 1 and 4 treatment with MEEC (group 2) significantly inhibited luminal occlusion 28 and 90 days after balloon injury. CsA-treatment reduced the ability of MEEC to inhibit luminal occlusion.

MEEC are powerful regulators of vascular injury. MEEC failed to induce a significant Th1-driven allo-immune response but induced differentiation of splenocytes into Th2-cytokine producing cells. This induction and their therapeutic efficacy were alleviated by a 12 day course of CsA. Adventitial MEEC implants -even of allogeneic sources and without immunosuppressive treatment- may be useful in decreasing luminal narrowing in a clinical setting.
The role of Drak2 in T-cells during an alloresponse (141.7)
Authors: Weist Brian M , Walsh Craig M
Published: January 2023

Drak2 is a serine threonine kinase that has previously been shown to negatively regulate activation of T cells through the T cell receptor. Additionally, T cells lacking Drak2 are hypersensitive to low levels of stimulation, but are surprisingly resistant to autoimmunity in an EAE model. Since it has been hypothesized that Drak2 deficient T-cells die after activation, I tested the ability of these T cells to respond to allogeneic stimulation, which would mimic transplantation from an allogeneic donor. Using in vitro mixed lymphocyte reactions, Drak2-/- CD8+ T-cells responded less to allogeneic stimulation compared to wildtype T-cells when analyzed by CFSE dilution. Additionally, allogeneic tumors were used to determine the role of Drak2 during an in vivo allogeneic transplant. Contrary to in vitro results, Drak2-/- mice were able to clear the allogeneic tumor in vivo. Subsequently, T cells isolated from Drak2-/- mice which had cleared an allogeneic tumor displayed proliferation defects when re-challenged in vitro. In the future adoptive transfers of Drak2-/- T-cells into mice lacking B, T, and NK cells will be done to address the specific role of Drak2 in T cell mediated allorejection.
Mast cell-mediated antigen presentation regulates CD8 T cell effector functions (33.15)
Authors: Bulfone-Paus Silvia , Stelekati Erietta , Bahri Rajia , D'Orlando Orietta , Orinska Zane
Published: January 2023

Mast cells (MCs) - long-living cells at sites of host-environment interphase, are important players of the innate immunity and central regulatory cells in adaptive immunity. We have previously described that MCs can detect viral infections and that TLR3-activated MCs contribute to antiviral host defence. MCs can regulate CD8 T cell- dependent autoimmune responses and CD8 T cell chemotaxis. Using three model antigens, we demonstrate that MCs induce antigen-specific CD8 T cell activation and proliferation. This requires direct cell contact and MHC class I-dependent antigen cross-presentation by MCs and induces the secretion of IL-2, IFN-γ and MIP-1α by CD8 T cells. Furthermore, MCs regulate antigen-specific CD8 T cell cytotoxicity by increasing granzyme B expression and promoting CD8 T cell degranulation. MCs also upregulate their expression of costimulatory molecules, and release the T cell proliferation inducer osteopontin upon direct contact. In vivo, adoptive transfer of antigen-pulsed MCs induces MHC class I-dependent, antigen-specific CD8 T cell proliferation. Furthermore, we characterised the bidirectional crosstalk between MCs and CD8 T cells and identified a new pathway of MC activation induced by MC interaction with CD8 T cells. Specifically, a number of genes coding for proteins involved in the IFN-mediated antiviral response are upregulated upon MC-CD8 T cell interaction.
Suppression of TLR4 signaling activated CTL response by scavenger receptor SRA/CD204 (33.18)
Authors: Wang Xiangyang , Yi Huanfa , Yu Xiaofei , Subjeck John
Published: January 2023

Scavenger receptor A (SRA)/CD204 is known to participate in the pathogenesis of atherosclerosis and the pattern recognition of pathogen infection. However, its role in adaptive immune responses has not been well defined. Here we report that the lack of SRA/CD204 promotes TLR4 agonist augmented antigen-specific immunity, as indicated by increased CD8+ T-cell proliferation, enhanced cytolytic activity of T-effector cells, and improved control of tumor growth. Dendritic cells (DCs) deficient in SRA/CD204 display more effective immunostimulatory activities upon TLR4 engagement than those from WT counterparts. Biochemical analysis indicates that SRA/CD204 suppresses TLR4 signaling stimulated activation of transcription factor NF-kappaB and subsequent production of pro-inflammatory cytokines. Moreover, silencing of SRA/CD204 by RNA interference improves the ability of DCs to prime antigen-specific CD8 T-cells, suggesting that antigen presenting cell (APC), especially DC, plays a major role in SRA/CD204-mediated immune modulation. Our findings reveal a previously unrecognized role for SRA/CD204, a non-TLR pattern recognition receptor, as a physiological negative regulator of TLR4 signaling-elicited immune consequences.
Dasatinib potential for anti-inflammatory efficacy while avoiding suppression of T cell activation in preclinical models (33.8)
Authors: Schieven Gary L , Zhang Rosemary , Pitt Sidney , McIntyre Kim , Gillooly Kathleen , Brock Barry , Smykla Richard , Townsend Robert , Saltercid Luisa , Lee Francis
Published: January 2023

Dasatinib potently inhibits Src-family kinases in addition to BCR-Abl and blocks T cell responses in vitro, suggesting potential utility in the treatment of autoimmune disease. A linear relationship was observed between serum concentration and in vitro T cell proliferation IC50 values. Modeling the human PK profile, delayed dasatinib treatment of T cells after T cell stimulation in vitro led to a time dependent decrease in potency as measured by both IC50 and Emax values. Comparison of serum adjusted IC50 values from these studies to the human PK profile suggests that dasatinib at the approved 100 mg QD dose would not be able to achieve a blockade of T cell activation on a daily basis. A similar pattern was observed in preclinical in vivo models. Dasatinib was found to be completely protective in a mouse model of cardiac allograft rejection at a dose of 25 mg BID, whereas a dose of 15 mg BID was not protective. By contrast, dasatinib at a dose of 3 mg/kg BID was highly efficacious in the mouse collagen induced arthritis model. Taken together, these results suggest that dasatinib may be able to provide anti-inflammatory effects while avoiding suppression of T cell activation at clinically relevant doses.
Direct CD8:CD4 T cell interactions improve memory recall of CD8 T cells (33.9)
Authors: Romagnoli Pablo Alberto , Premenko-Lanier Mary F , Loria Gilbert David , Altman John David
Published: January 2023

Protection against intracellular pathogens is conferred by CD8 T cells, which require CD4 T cell help to develop into effective memory CD8 T cells. It is believed that CD4 help is provided via licensing of APCs or IL-2 secretion to optimally stimulate CD8 T cells. We propose a new model where T:T interactions also play a role in CD8 T cell memory formation.

Here, we show that endogenous CD8 T cells, detected by tetramer enrichment, can acquire MHC class II when responding to infection. Next, we demonstrate that CD11c+ APCs are the main source of this MHC class II. Finally, we show that MHC class II bearing activated CD8 T cells can directly interact with experienced CD4 T cells impacting memory recall responses of CD8 T cells in vivo.

These observations suggest that program differentiation of memory CD8 T cells involves a novel direct interaction between CD8 and CD4 T cells. Furthermore, this model may also facilitate the understanding of immune regulation exerted on CD8 T cells depending on the profile of the interacting CD4 T cell population.

Support for this Research comes from the National Institutes of Health, Grant: 1RO1AI042373-04, PI: John D. Altman.
In vitro exposure to Atrazine does not alter activation, but prevents proliferation of primary murine B cells, T cells, or macrophages (34.10)
Authors: Wetzel Scott A , Browning Diana
Published: January 2023

Atrazine (ATR) is one of the most widely applied herbicides in the United States. The US Environmental Protection Agency reported in 2001 that Atrazine and its primary metabolite are the most common ground water contaminants in the US. Atrazine, an estrogen disrupting compound, has been linked to abnormalities in reproduction and as well as development of human cancer. Previous studies from other labs have also implicated the immune system as a target of ATR toxicity. In this study we examined the impact of ATR on the in vitro activation and expansion of mitogen-activated primary murine B cells and primary murine TCR transgenic T cells in an antigen-driven response. In the presence of ATR we observed only minimal changes in the expression of activation markers on macrophages, B cells and T cells. While there was not an increase in apoptosis induction in the presence of ATR, there was significant inhibition of proliferation in all three cell types. Interestingly, the effect is more pronounced on cells derived from male animals, consistent with the estrogen disrupting function of ATR. Thus, it appears that ATR may allow activation of B and T lymphocytes and macrophages while at the same time inhibiting proliferation of these cells. Studies examining potential ATR-induced changes in the effector functions of these cells are underway.

Funded By NIH COBRE 2P20RR017670 Subproject 5