bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular interaction between CD99 and CD99-related proteins (94.27)
Authors: Ma Min-Jung , Lee Hae Young , Choi Eun Young
Published: January 2023

Functions of CD99 have been well known to be related to apoptosis and homotypic aggregation of thymocytes, and transendothelial migration of lymphocytes and monotypes. Moreover, CD99- like 2 protein (CD99L2), a protein with homology to CD99, has also been found to be involved in cellular diapedesis. Despite these well known cellular functions, information on the biochemical and molecular characteristics of CD99 and the related proteins is relatively rare. The molecules have been suggested to interact in a hemophilic fashion during the cellular transendothelial migration. We investigated molecular interaction among the mouse CD99-related proteins using bimolecular fluorescence complementation analysis (BiFC). In BiFC analysis, the dimerization-induced fluorescence was detected when CD99 formed homodimers, at the perinuclear region, and the dimerization was formed via extracellular region of the molecules. Not only CD99 formed homodimer, but also did CD99 form dimmers with mouse CD99L2 or with an alternatively-spliced version of CD99L2. But the distribution pattern of the fluorescence emitted when CD99 formed dimers with CD99L2 or with an alteratively-spliced version of CD99L2 was different from the pattern observed when the CD99 formed homodimers, with more dispersed distribution inside of the cells, implying the change in molecular interaction according to the cellular location of CD99. This result suggests that molecular interactions among the CD99-related molecules may control the cellular localization of the CD99.
Endoglycan is a CD34-related marker of marginal zone B cells and is upregulated by TLR ligands (94.7)
Authors: Hirukawa Alison , Merkens Helen , Frohwerk Erin J , Lam Mindy , David Muriel , Furness Sebastian GB , Hughes Michael R , McNagny Kelly M
Published: January 2023

Endoglycan is a CD34-related sialomucin proposed to play a role in selectin-dependent adhesion of leukocytes to vasculature. Here we have investigated the distribution and function of endoglycan by generating a specific monoclonal antibody to an extracellular epitope of the protein. On non-hematopoietic cells, endoglycan is a relatively selective marker of smooth muscle cells of the gut, airway, and vasculature. Surprisingly, endoglycan does not appear to be a marker of vascular endothelia. Within the mature lineages of the hematopoietic system, endoglycan is restricted to a subset of CD4+CD8+ thymocytes and a subset of mature peripheral B cells. In the spleen of naïve mice, the highest levels of endoglycan surface expression is on marginal zone B (MZB) cells with much lower expression on follicular B cells. To further explore the regulation of endoglycan expression we treated splenocytes with a panel of toll-like receptor (TLR) ligands and cytokines. LPS, Pam3CSK4, CpG and IL-1 all enhanced endoglycan surface expression on splenic B cells approximately four-fold within 48 hrs. Interestingly, TLR ligand-dependent upregulation was blocked in the presence of IL-4, IL-5 or TGF-β, suggesting a selective requirement for endoglycan in T cell independent responses.

This work was funded by an operating grant from the Heart and Stroke Foundation of Canada. EJF is a recipient of an NSERC post-graduate scholarship.
Expression analyses of CHST4, a carbohydrate sulfotransferase required for L-selectin ligand biosynthesis, suggests novel role in epithelial cells (95.10)
Authors: Hopkins Deborah A , Allred Sean , Pretorius James , Harrison Janine , Hale Kari , Trueblood Esther , Ramachandran Chidambaram
Published: January 2023

Slowed lymphocyte rolling along high endothelial venules (HEV) is initiated by the L selectin: PNAd interaction, as an essential first step in a cascade of reactions leading to extravasation of lymphocytes across HEV and into the lymphoid tissues. CHST4, a golgi resident carbohydrate sulfotransferase, has been described as an HEV restricted sulfotransferase in part responsible for the sulfation step required for L selectin: PNAd interaction. The MECA79 antibody which recognizes sulfated PNAd provides a useful tool for monitoring sulfotransferase activity. The presence of both CHST4 and the MECA79 sulfated epitope have been described in HEV as well as lymphoid aggregates and tertiary lymphoid tissues at the sites of chronic inflammation. For the studies described, we investigated the expression of CHST4 with real time PCR, In Situ Hybridization (ISH), Immunohistochemistry (IHC) as well as MECA79 IHC. Expression patterns were examined in an extensive collection of normal human tissue panels as well as diseased tissue collected from several inflammatory conditions. Resulting data, in addition to confirming an HEV and lymphoid aggregate restricted expression profile also revealed expression of the gene in epithelial and ductal epithelial cells in a small set of diverse organ tissues. The functional consequences of such expression and the co-localized MECA79 epitope expression are currently unknown.
CD146+ T lymphocytes are increased in both the peripheral circulation and in the synovial fluids of patients with various musculoskeletal diseases and may facilitate T and B interactions (95.11)
Authors: McCoy, Jr J Philip , Tatlici Gulnaz , Samsel Leigh , Raghavachari Nalini , Liu Poching , Liu Delong , Munson Peter J , Gourley Mark
Published: January 2023

The endothelial junction adhesion molecule CD146 appears on activated lymphocytes in vitro and in very low percentages of circulating lymphocytes in healthy individuals. Preliminary evidence suggests that CD146 facilitates adherence of these cells to the endothelium, and perhaps transendothelial migration. 24 synovial fluids (SF) were obtained from 21 patients with various arthritic conditions. Peripheral blood (PB) was obtained from 6 of these patients and PB from 23 healthy individuals. Our data indicate CD4+CD146+ cells accumulate in SFs preferentially compared to PB and these cells display a unique genotypic profile from their CD146- counterparts. Using Affymetrix HG-U133+ PM arrays, comparative data analysis between CD146 positive and negative SF CD4+ cells revealed differential expression of 230 probesets. CD146+ CD4+ cells compared to CD146- CD4+ cells displayed increased expression of numerous genes including collagen type VI, alpha 3, CXCL13, and CD109 and decreased expression CCR7 and cytotoxicity-associated genes such as granzymes h and k, perforin, and CCL5. Both CD109 and CXCL13 have been shown to mediate T cell - B cell interactions and the upregulation of these genes on CD146+ T cells in SF may indicate these cells facilitate such interactions.
IQGAP1 mediates lymphocyte cytoskeletal polarization and peripheral effector functions (95.16)
Authors: Gorman Jacquelyn A , Gomez Timothy S , Billadeau Daniel D
Published: January 2023

IQGAP1 is a scaffolding protein that integrates the actin and microtubule cytoskeletons. Although IQGAP1 has been shown to participate in numerous cellular activities in fibroblasts and epithelial cells including E-cadherin-mediated cell-cell adhesion, cell polarity, motility, proliferation, and MAP kinase signaling, its function in thymocyte development or peripheral T cell effector functions has not yet been investigated. IQGAP1 localizes to the immune synapse in CD4+ and CD8+ T cells suggesting a possible role in T cell activation. Using wild type and IQGAP1-deficient mice, we demonstrate that neither thymocyte development nor peripheral T cells numbers are affected in the absence of IQGAP1. Surprisingly, IQGAP1-deficient CD4+ T cells make significantly more IL-2 compared to wild type cells. Despite this, IQGAP1-deficient T cells show diminished T cell proliferation and increased cell death following TCR and CD28 ligation, which is most likely due to increased activation-induced cell death. Interestingly, IQGAP1-deficient T cells spread abnormally in response to anti-CD3, fail to accumulate F-actin or polarize their microtubule-organizing center (MTOC) toward anti-CD3 coated beads. Consistent with the latter observation, CD8+ T cells do show defective cell-mediated killing. Furthermore, we find that IQGAP1-deficient T cells fail to polarize following integrin stimulation. Taken together, our data identify IQGAP1 as an important mediator of proper cytoskeletal polarization during T cell activation. This work was supported by NIAID grant AI065474 to D.D.B. and NIH -T32-AI07425 pre-doctoral Immunology Training Grant to J.A. Gorman.
Unexpected role of selectin ligands in T cell trafficking to the lung (95.6)
Authors: Onami Thandi M. , Harp John R.
Published: January 2023

Selectin mediated tethering represents one of the earliest steps in T cell extravasation into lymph nodes via HEVs. Fucosyltransferases (FucT) -IV and -VII are required for production of the sialyl Lewis X moiety, which is essential for selectin mediated T cell entry into LNs. We examined T cell trafficking to lymphoid and non-lymphoid compartments such as the lung using FucT -IV and -VII double knockout (FucT DKO) mice. Examination of uninfected FucT DKO mice revealed significantly increased numbers of T cells localized in lungs, with no evidence of induced bronchial associated lymphoid tissue (iBALT). Following LCMV viral infection, we noted similar numbers of antigen specific CD8 T cells, but reduced numbers of total activated CD8 T cells in the lungs compared to wildtype mice. Finally, we transferred bona fide naïve or memory CD8 T cells into recipient mice and show increased trafficking of T cells into lungs of uninfected FucT DKO mice. We conclude from these studies that while selectin mediated entry may play an important role in T cell trafficking to the inflamed lung, in the absence of selectin ligand expression, T cells preferentially migrate to and are retained in the lung under non-pathological conditions. In situations where normal trafficking of T cells to LNs are compromised, tissues such as the lung may become sites for T cell accumulation, and this may play a role in the initiation of T cell mediated lung diseases.

This work was supported by NIH grants AI057719, GM62116 (CFG), and University of Tennessee start up funds
Characterization of a new monoclonal antibody to human IL-17A (96.14)
Authors: Bonnevier Jody L , deMedeiros Ricardo , Cheng Qi , Coley Kristen , Galitz David , Munshi Cyrus , LaBresh Kurt , Herrera Julio , Ni Jessie H.T.
Published: January 2023

Although IL-17A is made by CD4+ TH17 cells and there is an ever-increasing body of literature on these cells, other cells make IL-17A, including CD8+ (Tc17) and gamma-delta T cells, NK cells, and neutrophils. There are many factors that contribute to published findings of the percentage of cells producing IL-17A, including the activation or differentiation conditions, time course of activation, and antibody used for detection. It has been published that high levels of IL-17A are secreted by human PBMCs after anti-CD3/CD28-treatment alone for only 24 hrs. We have developed a new monoclonal Ab (clone 41802) that is specific for human IL-17A. The specificity of this Ab has been rigorously tested. Clone 41802 detects an increase in IL-17A in PMA/ionomycin-treated human PBMCs over resting cells by intracellular flow cytometry. This finding correlated with real-time PCR data in activated vs resting CD4+ PBMCs. This clone also detects IL-17A in a population of activated CD3+ PBMCs that is also positive for IL-22 and IL-23R by flow cytometry. Importantly, clone 41802 detects IL-17A in transfectant cells overexpressing human IL-17A, but not in cells overexpressing human IL-17F. The specificity of clone 41802 was further confirmed by western blot. In conclusion, clone 41802 is specific for human IL-17A. Although a larger percentage of IL-17A+ cells are detected in activated PBMCs than with other commercially available clones, this indicates an interesting biological finding, not a lack of antibody specificity.
Pro-inflammatory adjuvants enhance the cognate helper activity of aged CD4 T cells (96.9)
Authors: Eaton Sheri , Maue Alexander C , Lanthier Paula A , Sweet Kathryn B , Blumerman Seth L
Published: January 2023

The humoral response in aged individuals is significantly reduced compared to young, thus contributing to the reduced efficacy of vaccines commonly seen in older populations. Using an adoptive transfer model, we have shown that age-related intrinsic declines in the function of naïve CD4 T cells from aged donors contribute significantly to reduced humoral responses. This decline leads to reduced B cell expansion and differentiation as well as reduced IgG production. In this study, we show that the helper function of aged CD4 T cells can be enhanced using a Toll-like receptor (TLR) binding adjuvant or an adjuvant containing proinflammatory (PI) cytokines. In addition, we show that the helper function of aged CD4 T cells can be enhanced by adding PI cytokines during in vitro effector generation. We observed enhanced expansion and differentiation of B cells and affinity maturation of IgG. Importantly, we found that PI cytokines also induced significant production of a variety of effector cytokines, including IL-4, IFNg, IL-17, and IL-21, by both young and aged CD4 T cells. The use of PI adjuvants can also improve humoral responses in intact aged animals. We propose that one of the mechanisms involved in the ability of adjuvants to enhance both young and aged T cell responses includes driving multifaceted T cell differentiation and production of multiple cytokines by responding T cells. This work funded by NIH grants AG21054 and AG02160 to LH.
A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses (97.1)
Authors: Yanaba Koichi , Bouaziz Jean-David , Sato Shinichi , Tedder Thomas F
Published: January 2023

B cells mediate multiple functions that influence immune and inflammatory responses. While B cells contribute to disease in most autoimmune mouse models, we have also found that B cells have regulatory functions and inhibit inflammation. Specifically, T cell-mediated inflammation in a mouse model of contact hypersensitivity was exaggerated in CD19-deficient (CD19-/-) mice and wild type mice depleted of CD20+ B cells, while inflammation was substantially reduced in mice with hyperactive B cells due to CD19 overexpression (hCD19Tg). These inflammatory responses were negatively regulated by a unique CD1dhiCD5+ B cell subset that was absent in CD19-/- mice, represented only 1-2% of spleen B220+ cells in wild type mice, but was expanded to ~10% of spleen B220+ cells in hCD19Tg mice. Adoptive transfer of these CD1dhiCD5+ B cells normalized inflammation in wild type mice depleted of CD20+ B cells and in CD19-/- mice. Remarkably, IL-10 production was restricted to this CD1dhiCD5+ B cell subset, with IL-10 production diminished in CD19-/- mice, yet increased in hCD19Tg mice. Thereby, CD1dhiCD5+ B cells represent a unique subset of potent regulatory B cells that we have labeled as "B10 cells" due to their unique production of IL-10 and the fact that other regulatory B cell subsets are likely to exist.
Interleukin-21 mediates unanticipated suppressive effects via its induction of IL-10 (97.5)
Authors: Spolski Rosanne , Leonard Warren J.
Published: January 2023

Interleukin-21 (IL-21) is a type I cytokine produced by CD4+ T cells and NK T cells that regulates the function of T, B, NK, and myeloid cells. IL-21 can augment both humoral and cell-mediated immune responses through its effects on lymphoid differentiation and function, and we and others have demonstrated a critical role for IL-21 in the autoimmune NOD mouse model of diabetes. Elevated levels of IL-21 were previously correlated with development of disease in the BXSB-Yaa mouse model of systemic lupus erythematosus, and interestingly, these mice also had increased serum IL 10. We now demonstrate that IL-21 potently induces IL-10 production by both T and B cells, with increased IL-10 mRNA and protein production in IL-21 transgenic mice but reduced production in IL-21R KO mice. TCR priming in the presence of IL-21 led to the accumulation of cells with IL 10-dependent immunosuppressive activity on both T cell and B cell function. Furthermore, we demonstrate that IL-21 is required for optimal induction of IL-10 by either IL-6 or IL-27. These results identify a novel mechanism for the induction and amplification of IL-10 production in both naive and activated T cells, and moreover identify unexpected immunosuppressive actions for IL-21.
Involvement of a receptor tyrosine kinase in the activation of cAMP and ERK1/2 by a retroviral-derived immunosuppressive peptide (98.13)
Authors: Haraguchi Soichi , Cianciolo George J , Day Noorbibi K
Published: January 2023

Several endogenous and exogenous retroviruses contain an evolutionarily conserved amino acid sequence (putative immunosuppressive domain, ISD) within the ectodomain of their transmembrane envelope proteins. We investigated the physiological and pathological role of ISD using a synthetic biopeptide composed of 17 amino acid CKS-17 homologous to the highly conserved region and showed that it not only exhibited important immune-regulatory functions, but also inhibited the production of IL-2, IL-12, IFN-γ, and TNF-α, while enhancing IL-10. Moreover, CKS-17 activated several intracellular signaling molecules including elevation of cAMP levels and phosphorylation of ERK1/2. Since the activation of the cAMP and ERK1/2 pathways has been shown to favor the development of Th2-type immune responses as well as influence viral replication and infectivity, these findings provided an important molecular mechanism(s) responsible for the observed cytokine patterns and virus-associated pathogenesis. In the present study, we show that AG879 (an inhibitor to TrkA, ErbB2, and FLK-1) completely blocks CKS-17-induced elevation of cAMP levels and phosphorylation of ERK1/2 using a monocytic cell line THP-1. By contrast, other receptor tyrosine kinase inhibitors (AG825, AG1296, AG1478) do not have this effect. We also show that while PI3K inhibitors (wortmannin, Ly294002) reduce the activation of ERK1/2 by CKS-17, these inhibitors have no effect on the elevation of cAMP levels by CKS-17. These results suggest that the action of the ISD may be mediated by an AG879-sensitive receptor tyrosine kinase that induces the phosphorylation of ERK1/2 through, at least, PI3K but elevates cAMP levels independently of PI3K. Supported in part by the Eleanor Naylor Dana Charitable Trust.
Monocytic THP-1 cells stimulated by normal human serum (NHS) release cytokine-bearing plasma membrane-derived vesicles (PMVs), and can be inhibited by methyl-beta-cyclodextrin, calpeptin and Rho-kinase inhibitor, Y-27632 (98.27)
Authors: Ansa-Addo Ephraim A , Cestari Igor dos Santos , Pathak Paras , Ramirez Marcel I , Inal Jameel M.
Published: January 2023

Stimulation of human acute monocytic cells with sublytic complement leads to an increase of intracellular calcium resulting in the release of two forms of microvesicles, one derived from the surface membrane (between 0.1-1 μm), which we term plasma membrane-derived vesicles (PMVs) and exosomes (50-100nm) released by exocytosis. Much work has been documented on the release of PMVs, using various stimuli, and the roles they play in disease, but comparatively little is available on inhibition of PMV release. Although upon stimulation cells release exosomes in addition to PMVs, little has been reported on ways of differentiating between these two microvesicle types. Our aim was therefore to identify inhibitors of PMV release and to effectively differentiate between PMVs and exosomes. Methyl-β -cyclodextrin, an inhibitor of lipid rafts, calpeptin, which inhibits calpain and Rho-kinase inhibitor, Y-27632 were found to inhibit effectively the release of PMVs. Exosomes separated from PMVs by sucrose gradient were confirmed by electron microscopy and compared to PMVs were found to express less phosphatidylserine, but more CD63. Of the various cytokines carried by PMVs and exosomes, macrophage migration inhibition factor was found at a four-fold higher level in PMVs.

Source of Research Support: Royal Society, London.
Distinct patterns of dendritic cell cytokine release stimulated by beta-glucans and Toll-Like receptor agonists (98.33)
Authors: Huang Haibin , Ostroff Gary R. , Levitz Stuart M.
Published: January 2023

β-glucans (βGs) derived from fungal cell walls have potential uses as immunomodulating agents and vaccine adjuvants. Yeast glucan particles (YGPs) are purified Saccharomyces cerevisiae cell walls treated so they are >80% β1,3-D-glucan and free of mannan and proteins. YGPs stimulated secretion of the proinflammatory cytokine TNF-α in wild type murine bone marrow-derived myeloid DCs (BMDCs), but did not stimulate IL-12p70 production. A purified soluble βG, scleroglucan, also stimulated TNF-α in BMDCs. These two βGs failed to stimulate TNF-α in dectin-1 (βG-receptor) knockout BMDCs. Co-stimulation of wild-type BMDCs with βGs and specific TLR ligands resulted in greatly enhanced TNF-α production but decreased IL-12p70 production compared with TLR ligands alone. The up-regulation of TNF-α and down-regulation IL-12p70 required dectin-1, but not IL-10. Similar patterns of cytokine regulation were observed in human monocyte-derived DCs (hDCs) co-stimulated with YGPs and, the TLR4 ligand, LPS. Finally, co-stimulation of BMDCs with the TLR9 ligand, CpG, and YGPs resulted in upregulated secretion of IL-1α and IL-10, downregulated IL-1β, IL-6 and IP-10, and had no significant effects on IL-12p40, KC, MCP-1 or MIP-α, compared with CpG alone. Thus, βGs have disparate effects on cytokine responses following DC stimulation with TLR ligands.
CCR7/CCL19 Mediates T Lymphocyte Expression of EDG-1 (98.8)
Authors: Shannon Laurie A , Byers Melissa A , Fassold Brian C , Roth Megan , Calloway Psachal , Zhang Elizabeth Y , Vines Charlotte M
Published: January 2023

C-C Chemokine Receptor 7 (CCR7) regulates migration of naïve T cells to lymph nodes, via chemotaxis to the ligand, CCL21. Approximately 72 hours after lymph node entry, Endothelial Differentiation Gene 1 (EDG-1) is up-regulated on T cells and mediates lymph node egress. It is unclear how EDG-1 expression is up-regulated. We hypothesized that lymph node egress occurs in response to CCR7 receptor interaction with dendritic cells presenting the second CCR7 ligand, CCL19. To test this hypothesis, we used murine or human T cells, stimulated with CCL19 and found that EDG-1 mRNA was up-regulated at 24, 48, and 72 hours. The cells displayed increased migration to EDG-1 ligand S1P over the same time period confirming surface expression of EDG-1. Extracellular Regulated Kinase 5 (ERK5) can mediate transcription of Kruppel-Like Factor 2 (KLF-2). KLF-2 directly mediates transcription of EDG-1. To determine if the hypothesized ERK5-KLF-2-EDG-1 pathway was active in our system, we assayed murine and human T cells for changes in ERK5 and KLF-2. We found that stimulation with CCL19 led to increased phosphorylation and expression of ERK5 at 24, 48, and 72 hours. Similarly, KLF-2 levels were increased over the same interval. Taken together, our data suggests that after lymph node entry of T cells via CCR7/CCL21, T cells exit the lymph nodes via EDG-1 in response to activation of T cell CCR7 by CCL19 on activated dendritic cells.
Dissecting the antimicrobial activity of CCL28 through protein mutagenesis (98.9)
Authors: Liu Bin , Wilson Eric
Published: January 2023

Some chemokines have been shown act as antimicrobial proteins in vitro, suggesting that they may contribute directly to innate immune protection. Based on the study of defensins and other antimicrobial peptides (AMPs), it has been proposed that the characteristics of cationicity, hydrophobicity and amphipathicity of the AMPs determine the killing activity of these peptides. However, amino acid characteristics necessary for the antimicrobial activity of chemokines have not been elucidated. Using mouse CCL28 we have determined the primary sequence features required for antimicrobial activity of this chemokine. Our data demonstrate, through the use of successive truncations, that the holoprotein (108 amino acids) is necessary for full antimicrobial activity of CCL28. Consecutive truncations resulted in an almost complete loss of activity following the removal of the 24 C terminal amino acids. Charge reversal as well as positive charge to neutral charge mutations, support the hypothesis that positively charged amino acids are essential for the antimicrobial activity of CCL28. Finally, the antimicrobial activity of chimeric proteins in which the N-terminus of CCL5 or CCL27 was fused with the C terminus of CCL28 suggest that although the C terminal region of CCL28 is essential for antimicrobial activity, the interaction of the N and C terminal regions of CCL28 is necessary for optimal antimicrobial activity. These experiments are the first to investigate the role of primary structure in the killing activity of an antimicrobial chemokine.
PDCD4-deficient NOD mice are resistant to type 1 diabetes (99.12)
Authors: Ruan Qingguo , Kameswaran Vasumathi , Chen Youhai H
Published: January 2023

Development of autoimmune diseases is associated with heightened protein translation. However, the mechanism of translational regulation and the roles of translation factors in the pathogenesis of autoimmune diseases are not clear. Programmed cell death 4 (PDCD4) is a translation factor that inhibits cap-dependent translation by binding to eIF4A. It is also a newly identified target of micro-RNA 21 that regulates immune responses. To determine the roles of PDCD4 in type 1 diabetes (T1D), we generated PDCD4-deficient NOD mice. We report here that PDCD4-deficient NOD mice are resistant to T1D, and PDCD4-deficient lymphocytes and macrophages produce increased levels of both anti-inflammatory (such as IL-4 and IL-10) and pro-inflammatory cytokines (such as IL-17, IFN-gamma and TNF-alpha). Additionally, PDCD4-deficient NOD mice are also resistant to high dose streptozotocin-induced diabetes, and over-expression of PDCD4 in islet beta cells induces apoptosis and cell cycle arrest, which appear to be related to reduced AP-1 activation. Thus, PDCD4 may regulate the development of autoimmune diabetes by targeting both inflammatory cells and pancreatic beta cells.
A key role for Fas ligand expressed on B cells in initiation of autoimmune diabetes (99.17)
Authors: xiao zuoxiang , Li Meng , Schneck Jonathan P. , Hamad Abdel Rahim A.
Published: January 2023

Type-1 diabetes (T1D) is an organ specific autoimmune disease that affects children. T, B and dendritic cells infiltrate islets in the early stage of disease leading to insulitis followed by islet destruction and hyperglycemia. Development of T1D in non-obese diabetic (NOD) mice is completely inhibited by gld or lpr mutations of Fas and FasL, respectively, even though mutant mice develop T cell lymphoproliferation and the Fas pathway is not essential for T cell activation. Recently, we have shown heterozygous gld mutation (gld/+) provides complete protection without causing lymphoproliferation. Using NOD-gld/+ mouse as a model, we found that the protection is not due to deletion of autoreactive T cells or switching of T helper polarization. Islet-reactive BDC2.5 TCR transgenic CD4 T cells transferred into NOD-gld/+ mice were efficiently primed in the pancreatic lymph nodes but failed to accumulate in the pancreas. FasL deficiency selectively reduced expression of CD40 and frequency of B cells in the pancreas. Transfer of wild type B cells into NOD-gld/+ mice results in mobilization of endogenous T cells particularly CD8 T cells and of co-transferred BDC2.5 T cells resulting in severe insulitis. These results provide the first evidence that B cells play an essential role in gld-mediated protection from T1D and that expression of functional FasL on B cells is key for initiation insulitis.

Supported by grants from AHA and JDRF.
Allogeneic neonatal blood transfusion ameliorates autoimmune diabetes in unconditioned non-obese diabetic mice by functionally deleting pathogenic T cells (99.19)
Authors: Jayaraman Sundararajan , Ajani Shahnaz , Garza Rebecca , Jayaraman Arathi , Kwon Steve , Prabhakar Bellur , Holterman Mark
Published: January 2023

We show that transfusion of unconditioned, pre-diabetic non-obese diabetic mice with newborn blood containing multiple Sca-1+ stem/progenitor subsets derived from allogeneic diabetes-resistant mice can prevent diabetes. Diabetes protection was characterized by enhanced glucose-induced insulin release and diminished cellular infiltration of islets. Allogeneic neonatal blood transfusion induced only transient hematopoietic microchimerism, which did not correlate with the level of protection obtained. Splenocytes derived from cured mice failed to transfer diabetes into immunodeficient NODscid mice, indicating deletion/inactivation of diabetogenic T cells by transfused neonatal blood cells. Diabetes cure did not result in alloantigen-specific T cell unresponsiveness as assessed by T cell proliferation. Paradoxically, T cell anergy to allogeneic antigens was displayed by a majority of diabetic but not cured mice that received newborn blood cells. These results indicate that deletion/inactivation of diabetogenic T lymphocytes is instrumental in preventing the progression of insulitis to overt diabetes by allogeneic newborn blood cell transplantation.
Double-stranded RNA protects against antigen-induced arthritis via the type I interferon receptor (99.23)
Authors: Magnusson Mattias
Published: January 2023

Viral infections may contribute to different forms of arthritis. Both viral components, e.g. dsRNA and host-derived anti-viral agents, e.g. type I interferons (IFN), can induce arthritis if present in a joint. This joint inflammation is transient and mediated by antigen-independent (innate) immunity. In order to study the impact viral components may have on the development of antigen-dependent arthritis, dsRNA was administered systemically during the activation of antigen-induced arthritis.

The methylated bovine serum (mBSA)-induced arthritis model used consists of two s.c. immunizations with mBSA in Freund's incomplete adjuvant one week apart, followed by intraarticular injection of mBSA alone three weeks after the first immunization. The importance of IFN-signalling in the onset of mBSA-induced arthritis was studied (1) by using mice deficient for the IFN receptor (IFNAR ko, strain 129) and (2) by including the IFN-activator viral dsRNA in the immunizations.

Administration of dsRNA during the immunizations with mBSA inhibited arthritis development and severity in wt, but not in IFNAR ko mice (p<0.05). This protection was accompanied by lower levels of anti-mBSA-specific IgG in serum (p<0.05). This anti-inflammatory effect of IFN-signalling contrasts the potent arthritogenic properties dsRNA and IFN have if present in the joint and underscores the opposing effects IFN signalling may have locally/systemically and on innate/adaptive immunity.

Grant resources for this work includes The King Gustav V 80-years foundation and the Swedish Physicians Society
Smoking and nicotine exposure delays development of arthritis in animal model (99.24)
Authors: Lindblad Sofia Silfverswärd , Mydel Piotr , Jonsson Ing-Marie , Senior Robert M , Tarkowski Andrej , Bokarewa Maria
Published: January 2023

Recent results of epidemiological studies indicate smoking as an environmental risk factor for development of rheumatoid arthritis (RA). The aim of this study was to evaluate the role of smoking and nicotine in the pathogenesis of arthritis using an animal model.

DBA/1 mice were exposed to cigarette smoke for 16 weeks prior to immunization with collagen type II (CII) and through the whole course of experiment. In a separate experiment DBA/1 mice were subjected to nicotine in drinking water from the day of immunization with CII until the end of experiment. Severity of arthritis was evaluated clinically and morphologically and compared to control mice. Levels of specific anti-CII antibodies as well as of autoantibodies (aCCP and RF) and cytokines were evaluated.

Clinical evaluation of arthritis showed a delayed onset of arthritis in smoking mice as compared to non-smoking (p<0.05). Histological index and weight changes were comparable between both groups, however smoking mice were gaining weight faster than non-smoking (p<0.05). Additionally, smoking mice had decreased ability to produce antibodies (aCCP and anti-CII) as compared to non-smoking (p<0.05). Similar results were obtained in the mice exposed to nicotine. Nicotine had a direct anti-inflammatory effect diminishing cytokine production by stimulated spleenocytes in vitro.

Neither smoking nor nicotine exposure aggravates development of CII-induced arthritis in mouse model.
A new mode of TNF-[alpha] inhibition by microRNA (99.26)
Authors: Frenzel Laurent , Semaan Noha , Alsaleh Ghada , Wachsmann Dominique , Gottenberg Jacques-Eric , Sibilia Jean
Published: January 2023

TNF-α, is a major cytokine implicated in rheumatoid arthritis (RA). TNF-α expression is regulated both at the transcriptional and the postranscriptional levels and recent data demonstrated that miRNAs are implicated in TNF-α response in macrophages. LPS-activated synoviocytes fibroblast-like (FLS) isolated from RA patients express TNF-α mRNA but they do not release TNF-α. This inhibition is due to a rapid degradation of TNF-α mRNA. We demonstrated previously that miR-346, which is overexpressed in LPS-activated FLS inhibits Bruton's tyrosine kinase (Btk) expression, which controls TNF-α stability. In fact, transfection of antisense oligonucleotides targeting miR-346 reestablished Btk and TNF-α expression in activated FLS. We also showed that transfection of miR-346 in THP-1 cells, inhibited TNF-α secretion in response to LPS. Tristetraprolin (TTP) is a protein which inhibited TNF-α synthesis by binding to AU rich elements of TNF-α mRNA. We demonstrated in LPS-activated RA FLS, that inhibition of miR-346 decreases TTP expression. In parallel we also showed that inhibition of Btk by LFMA13 or miR-346, increases the expression of TTP in LPS-activated THP-1 cells which results in inhibition of TNF-α synthesis. These results indicate that Btk controls TNF-α synthesis by regulating TTP expression. In conclusion, our findings provide evidence that miR-346 can act as a negative regulator of inflammation.
Select T Cell Receptor Utilization by CD4+ T Cells in a Murine Model of Systemic Lupus Erythematosus is Similar to Human Disease (99.31)
Authors: Trivedi Sapna , Culpepper Schartess , Zang Yun , Martinez Laisel , Greidinger Eric L , Hoffman Robert W
Published: January 2023

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease associated with significant morbidity and mortality. We have identified and characterized U1-70kDa small nuclear ribonucleoprotein (70kDa) autoantigen specific T cells in a murine model of autoimmunity when a subset of mice developed kidney disease similar to lupus nephritis. Mice developed proteinuria and histology showed CD4+ T cells associated with glomerular lesions. CD4+ T cells were freshly isolated from kidney and spleen and cultured with autoantigen to generate short-term T cell lines. Following RNA extraction from cell lines and cDNA preparation, a PCR reaction was performed using primers for the TRBV subgroups. The product was purified and underwent cloning and sequencing. T cell epitope mapping revealed that the majority of T cells were directed against antigenic peptides residing within the RNA binding domain of 70KDa. We also found that TCR beta (TRB) V usage was highly restricted among 70kDa reactive T cells which selectively utilized TCR BV subgroups 1 and 8.3 in the kidney and 1,2, 6, 8.1, 8.2, and 8.3 in the spleen and the lung. The TRB third complementary-determining regions (CDR3) had conserved sequence motifs which were shared across different TRBV subgroups. When compared for homology, the TRBV and CDR3 regions used by both murine and human 70kDa-specific CD4+ T cells were highly similar including those CD4+ T cells found at two sites of inflammation; the kidney and lung.

The NIH and Dept Veterans Affairs provided support for this work.
IL-21 Receptor signaling by B-cells is essential for BXSB-Yaa pathogenesis (99.39)
Authors: Bubier Jason A , Sproule Thomas J , Foreman Oded , Spolski Rosanne , Morse Herbert C , Leonard Warren J , Roopenian Derry C
Published: January 2023

IL-21 is a member of the IL-2 cytokine family that exerts potent biological effects on multiple hemopoietic cell types. It is produced by activated CD4 T cells, while its receptor, IL-21R, is found on cells including NK cells, dendritic, T cells and B cells. To investigate the role of IL-21 signaling in autoimmune disease, we addressed whether this cytokine contributes to the severe systemic lupus erythematosus (SLE)-like syndrome developed by BXSB-Yaa mice. We show that BXSB-Yaa CD4 T cells produce IL-21 but lack discriminative T follicular helper cell markers. We also show that BXSB-Yaa mice completely lacking the IL-21R or Igh6 are remarkably resistant to multiple aspects of the prototypic BXSB-Yaa autoimmune disease. Finally, we show that mixed bone marrow chimeric BXSB-Yaa mice lacking IL-21R specifically in the B-cell compartment are similarly resistant. These results suggest that IL-21 produced by a novel population of BXSB-Yaa CD4 T-cells acts through B-cells to evoke multiple features characteristic of severe SLE. The results indicate that IL-21 signaling through B-cells is an essential step in the pathogenesis of this SLE-like autoimmune disease and potentially others. Funded by an Arthritis Foundation grant to JB.
Bruton’s tyrosine kinase and microRNA miR-346 regulate IL-18 release by LPS-activated rheumatoid fibroblast-like synoviocytes (99.52)
Authors: Wachsmann dominique , Alsaleh Ghada , Semaan Noha , Suffert Guillaume , Pfeffer Sebastien , Frenzel Laurent , Gottenberg Jacques-Eric , Sibilia Jean
Published: January 2023

We reported previously that LPS-activated synoviocytes fibroblast-like (FLS) isolated from rheumatoid arthritis (RA) patients express IL-18 mRNA but they do not release IL-18. As this inhibition was due to a rapid degradation of IL-18 mRNA, our group has carried out a study to identify miRNAs that could play a role in this "anti-inflammatory" response. As assessed by microarray analysis, 15 miRNAs were overexpressed more than two fold in LPS-activated FLS and among them, we identified one miRNA, miR-346 that had partial sequence complementarity within the 3'-UTR region of the IL-18 mRNA. We then used antisense oligonucleotides molecules to block miR-346 activity; transfection of the inhibitor targeting miR-346 reestablished IL-18 release by LPS-activated FLS. Moreover we also showed that transient transfection of miR-346 in THP-1 cells, inhibited IL-18 secretion by these cells in response to LPS. Using a luciferase assay we showed that miR-346 does not target directly IL-18 mRNA. In search for another target that could explain the effect of miR-346 on IL-18, we also demonstrated that miR-346 inhibited Btk expression at the transcriptional level in LPS-activated RA FLS and that inhibition of Btk expression regulated negatively IL-18 release. These results suggest an important role of miR-346 in the control of IL-18 synthesis and indicate that its expression may be critical to prevent an excessive inflammatory response.
Neuropilin-1 is important for proper suppression of inflammation and autoreactivity (99.6)
Authors: Bynoe Margaret S , Solomon Benjamin D , Chae Wookjin
Published: January 2023

Neuropilin-1(Nrp1) is a surface molecule originally identified for its role in neuronal development. Recently, Nrp1 has been implicated in several aspects of immune function including maintenance of the immune synapse and development of regulatory T cells (Treg). In this study we provide the first evidence for a role of Nrp1 in the pathogenesis of an immune mediated disease, experimental autoimmune encephalomyelitis (EAE), which is an animal model for the central nervous system inflammatory disorder multiple sclerosis (MS). Using a cell type specific deletion system, we observed that the lack of Nrp1 on CD4+ T cells results in increased EAE disease severity. These conditional knockout mice exhibit preferential Th17 lineage commitment and decreased Treg numbers and functionality. Conversely, mice receiving CD4 T cells that were retrovirally transduced with Nrp1 and immunized for EAE developed only mild EAE compared to controls. This suggests that Nrp1 is essential for proper maintenance of peripheral tolerance and its absence can result in unchecked autoreactive responses leading to diseases like EAE and MS. This work was supported by National Institutes of Health Grants AI 57854 and AI 072434 (to MSB).
The IFN-γ / IFN-γ Rα axis is involved in immunoediting of breast cancer that leads to the de novo generation of a novel breast cancer stem cell (BCSC)-like tumor variant and subsequent tumor recurrence (100.12)
Authors: Kmieciak Maciej , Knutson Keith , Manjili Masoud
Published: January 2023

Tumor dormancy remains a major hurdle in the treatment of cancers. Recently, the association between cancer stem cells (CSC) and tumor dormancy has been speculated in variety of cancers. Despite conventional therapies of breast cancer, most of the US deaths are attributed to relapse which is likely due to a failure of current treatment regimens to eliminate breast cancer stem cells (BCSC). We reported IFN-γ-induced HER-2/neu antigen loss and tumor relapse. We performed in vitro and in vivo studies using neu positive mammary carcinoma (MMC) tumors and their dominant negative (dn) IFN-γ Rα MMC sub-line. We showed that IFN-γ producing CD8+ T cells induced the de novo generation of a distinct BCSC variant that differed from conventional BCSC. The novel phenotype: i) is not intrinsically quiescent, ii) is reversible by blocking IFN-γ Rα signaling in the tumor, iii) upregulates serine protease inhibitors (SPI-6/PI-9) and MUC-1 that could render the tumors refractory to granzyme/perforin- and antibody-induced apoptosis, respectively, and iv) downregulates HER-2/neu expression upon emerging under the immune pressure. While HER-2/neu+ conventional BCSC have been shown to be sensitive to HER-2/neu-targeted immunotherapy, these novel BCSC-like phenotypes are refractory to such immunotherapy because of HER-2/neu loss. Identification of such tumor immunoediting pathways and mechanisms is expected to improve immunotherapy of breast cancer, by pathway blocking strategies.
IL-17 promotes inflammation associated tumor development (100.14)
Authors: Xu Hui , He Donggou , Li Hui , Yusuf Nabiha , Elmets Craig , Athar Mohammad , Katiyar Santosh , Mountz John
Published: January 2023

The mechanism for inflammation associated tumor development is a central issue for tumor biology and immunology. IL-17 is an important cytokine for inflammatory and autoimmune diseases. Although IL-17 producing cells are detected in cancer patients and tumor bearing mice, the role of IL-17 mediated inflammation in tumor development remains to be determined. In the current studies, we showed that IL-17 receptor-A gene deficient mice (IL-17R-/-) were resistant to chemical induced carcinogenesis in the skin. Moreover, the defect in IL-17R reversed the increased susceptibility of IL-12p35 deficient mice to tumor development. Analysis showed that angiogenesis and infiltration of myeloid cells were inhibited whereas the infiltration of CD8 T cells was increased in the inflamed skin of IL-17R-/- mice. The development of myeloid derived suppressor cells was inhibited in IL-17R-/- mice. Inflammation induced Cox-2 activity and S100A8/A9 proteins were inhibited and inflammation associated epidermal hyperplasia were suppressed in IL-17R-/- mice. Furthermore, the production of tumor promoting inflammatory cytokines IL-1β and TNF-α were reduced. These findings demonstrate that IL-17 signals are required for inflammation associated tumor development. The study provides insights into a novel mechanism by which IL-17 mediates tumor promoting inflammation and induces tumor escape from immune surveillance. It has major implications for targeting IL-17 in prevention and treatment of tumors.
Do stress response genes play a role in maintaining MDSC survival and suppression? (100.17)
Authors: Beury Daniel , Fitzgerald Phillip , Srivastava Minu , Ostrand-Rosenberg Suzanne
Published: January 2023

Myeloid- derived suppressor cells (MDSC) contribute to immune suppression in tumor bearing individuals. Their suppression and survival are increased by inflammation. MDSC production of toxic radicals is the primary mechanism of suppression but the toxic mediators do not affect the MDSC themselves. We are investigating stress response genes that may maintain MDSC survival and facilitate suppression. BALB/c mice were injected with BALB/c derived mammary carcinoma 4T1 or 4T1 constitutively expressing the pro-inflammatory cytokine IL-1β (4T1/IL-1β). Primary tumors were surgically removed after 25 days. At 35 days, spleens were harvested and assayed for the percentage of Gr1+ CD11b+ MDSC. Surgical removal of 4T1 results in a regression of the MDSC population, however MDSC do not regress in mice bearing 4T1/IL-1β tumors, confirming that inflammation increases MDSC survival. Western blots and confocal microscopy of MDSC induced by 4T1 and 4T1/IL-1β tumors localize Nrf2 in the nucleus, demonstrating that Nrf2 is activated in MDSC. Catalase, a protein transcriptionally regulated by Nrf2, when added to a T cell activation assay eliminates MDSC suppressive activity. Addition of catalase to T cells co-cultured with MDSC and peptide causes a synergistic effect, activating T cells to a greater level than peptide alone. Collectively these data suggest that stress genes may play a role in maintaining MDSC survival and suppressive activity.
CD28 costimulates macrophage suppression via increased IFNg production (100.21)
Authors: Riggs James , Kozlowski Megan , Composto Gabriella , Silberman Dan
Published: January 2023

The high macrophage to T cell ratio (Mf:T) characteristic of the tumor microenvironment can be modeled using peritoneal cavity (PerC) cell culture. C57BL/6J PerC Mfs suppress T cell activation by IFNg-triggered expression of iNOS and IDO, enzymes that consume Arg and Trp. Attempts to reverse suppression by CD28 costimulation actually increased suppression and ELISPOT analyses revealed this was due to increased numbers of IFNg-secreting cells (gSC). Comparative analyses showed that C57BL/6 mice had more gSC than B-1 B cell deficient, BALB.xid mice and that these strains both had more gSC than BALB/c mice even though the latter had the greatest increase in gSC following costimulation. Titration experiments revealed that increased PerC cell density reduced the need for costimulation and increased gSC number. Both CD80 and CD86 knockout (KO) mice had fewer gSC than wildtype mice with CD80 KOs having fewer than CD86 KOs. These data reveal a “braking” role for IFNg in T cell responses and suggest that, rather than promote T cell activation, CD28-mediated costimulation could foster suppression of cell-mediated immunity via increased formation of Mf-T cell synapses within tumor microenvironments. Supported by NIH AREA R15- CA136901-01.