bluuebunny/crossref_metadata_2025 huggingface dataset:
A Systemic age dependent defect in immune-cell signaling response induced by inflammation (138.2)
Authors: Shen-Orr Shai , Furman David , Kidd Brian , Maecker Holden , Dekker Cornelia , Butte Atul , Davis Mark
Published: January 2023

Immune system function generally degrades with age and is associated with increased risk of infection and disease. Though differences between young and old have been noted in many immune system components, no system wide understanding of how these disparate observation act together exists to date, nor how they relate to genes found to associated with increased longevity, many of which are immune related. Here we characterize the immune system of 29 young and old individuals by concurrently measuring from peripheral blood, immune cell subset frequency, serum cytokines, gene expression and individual cellular responses to cytokine stimuli by pathway specific phospho-protein abundance. We identify age-dependent changes in STAT signaling baseline and in response to stimulation by a panel of 7 different cytokines, particularly prominent in CD8 and CD4 T cells, but also in monocytes and B-cells. The observed differences in cellular responses are not due to adaptation to higher level of cytokine stimuli but rather to an inert inability to mount a full response, many times augmented by a higher base line phosphorylation level in the elderly. We construct an immune network spanning multiple biological layers and identify co-occurring modules which link longevity associated genes with these cellular immune phenotypes. We quantify the contributions of these modules to the observed reduction in cellular response to stimuli in the elderly and suggest a common responsible mechanism.
Long lasting macrophage cytokine suppression by alpha 7 nAChR requires JAK/STAT (138.23)
Authors: Olofsson Peder , Rosas-Ballina Mauricio , Chavan Sangeeta , Parrish William , Hudson LaQueta , Ochani Mahendar , Li Jianhua , Pavlov Valentin , Tracey Kevin
Published: January 2023

The central nervous system regulates innate immunity reflexively, as action potentials transmitted in the vagus nerve suppress cytokine release during endotoxemia. The mechanism of cytokine release by this neural circuit requires signaling through the alpha 7 nicotinic acetylcholine receptor (a7 nAChR), but the duration of this protection is incompletely understood. Electrical vagus nerve stimulation (VNS) in mice significantly inhibited serum TNF levels during endotoxemia, even when endotoxin was given 2, 24 or 48 h after VNS. This inhibition of TNF was abolished in a7 nAChR deficient mice, confirming the mechanism that requires this receptor. One-hour exposure of human macrophages to acetylcholine in vitro reduced endotoxin-induced increases in IKKbeta, IkappaBalpha activity and TNF release for up to 24 h after removal of acetylcholine. Levels of Supressor of Cytokine Signaling 1-3, A20, BCL3, IkappaBNS and IkappaBzeta were not altered in human macrophages 15 min to 8 h after exposure to acetylcholine, but exposure of macrophages to the selective JAK/STAT inhibitor AG490 abolished the cytokine suppressing effect of acetylcholine. Together with previous work indicating that JAK/STAT forms a signaling complex with a7 nAChR, these results suggest that JAK/STAT activation is required for long lasting suppression of macrophage cytokine release after transient exposure to acetylcholine.
Notch signaling regulates human and mouse TH17 development. (139.13)
Authors: Keerthivasan Shilpa , Suleiman Reem , Le Poole Caroline , Nickoloff Brian , Osborne Barbara , Miele Lucio
Published: January 2023

TH17 immune cells, a recently discovered subset of CD4+ T-helper cells, are known to play important role in pathogenesis of several autoimmune diseases, inflammatory disorders and cancers. Although Notch ligand DLL4 was shown to drive TH17 polarization, downstream pathways conducting those signals are unknown. We demonstrated that the Notch signaling pathway has an important role in TH17 polarization. We found that gamma secretase inhibitors (GSI) that inhibit Notch signaling blocked the in-vitro polarization of mouse or human naïve CD4+ T cells towards the TH17 pathway without affecting cell proliferation. Next, we showed that, among various Notch receptors, Notch-1 is upregulated in TH17 polarizing cells and that knocking down Notch-1 by siRNA inhibited TH17 polarization. We further discovered that, in addition to polarizing naïve T cells towards TH17 pathway, Notch-1 is important for maintenance of this phenotype. Furthermore, reporter luciferase assays and chromatin immunoprecipitation (ChIP) assays suggest that the IL-17 as well as RORC promoters are direct transcriptional targets of Notch-1. Finally, in-vivo administration of GSI inhibits TH17 mediated disease progression in mouse experimental autoimmune encephalomyelitis model of multiple sclerosis. Thus our data suggest a key role for Notch signaling, especially Notch-1, in TH17 polarization.
Signaling through the membrane-bound estrogen receptor GPR30 induces IL10 and IL17A expression in Th17 cells (139.2)
Authors: Brunsing Ryan , Hathaway Helen , Prossnitz Eric
Published: January 2023

In 2005, the orphan G protein-coupled receptor GPR30 was shown to bind estrogen and mediate some downstream events associated with estrogen signaling. While work on GPR30-mediated suppression of autoimmune inflammation has been published, no definitive work on GPR30 in T cells has been reported. To address whether signaling through GPR30 can affect CD4+ T cell populations, CD4+CD62Lhi naïve T cells were stimulated in culture with anti-CD3/CD28 antibodies. We observed a dose-dependent increase in the number of IL10+ and IL10+IL17A+ cells in cultures treated with the GR30-directed agonist G-1, as measured by FACS. This effect is lost in cells from GPR30KO mice. Furthermore, the induction of IL10 and IL17A was limited to the RORγt+Foxp3- (Th17) population, while no effects were observed in Foxp3+ cells. Interestingly, the effects of systemic G-1 in the animal model T-cell mediated colitis varied between the peripheral compartment, where IL17A induction was again observed, and the GI mucosa, where a decrease in the number of IL17A+, IFNγ+, and TNFα+ cells was observed. These findings provide insight into the function of the membrane-bound estrogen receptor GPR30 in CD4+ T cell physiology. In addition, these data may help reconcile an apparent contradiction between work suggesting an immunosuppressive role for estrogen in several autoimmune disease models and clinical reality where women suffer from a substantially higher incidence of autoimmune disease.
Regulatory T cell hybridoma reveal novel Foxp3 inducing factors (139.7)
Authors: Sharma Rahul , Ju Angela , Sung Sun-Sang , Ju Shyr
Published: January 2023

It has been difficult to generate cloned regulatory T cells (Tregs) or Treg hybridoma for the studies on antigen specificity and the regulation of Foxp3 by TCR independent factors. Here, we report the first successful generation of cloned, inducible Treg hybridomas (iTreg-hyb) using the CD4+ T cells of TCR transgenic DO11.10 mice bearing the Foxp3-IRES-eGFP knock-in gene. Although, the iTreg-hyb expressed low Foxp3-GFP, strong expression could be induced by either polyclonal (using anti-CD3 mAb) or antigen specific (using OVA[323-339] peptide presented by syngeneic Rag2 deficient spleen cells) TCR stimulation in the presence of Interleukin (IL) -2 and Transforming Growth Factor-β1. Interestingly, CD11c+ cells isolated from the spleens of Rag2 deficient mice alone could induce Foxp3-GFP expression, albeit with 30-fold lower efficiency. The induction of Foxp3-GFP by the latter mechanism was specific as it did not up-regulate the TCR stimulation dependent Treg associated markers. Using the iTreg-hyb, we have identified several Foxp3 inducing factors which include recombinant IL-6, IL-9, IL-27, and a <5-kd soluble factor produced by Rag2 deficient spleen cells. Our study shows that it is feasible to generate iTreg-hyb, which is useful not only to study the role of self-reactivity but also to uncover TCR independent pathways for the induction of Foxp3 expression by cytokines and novel factors.
Critical role of Ral GTPase as a gatekeeper in controlling neutrophil selective degranulation of secondary granules (140.11)
Authors: Chen Celia , Soto Ileana , Zen Ke , Liu Yuan
Published: January 2023

Neutrophil (PMN) inflammatory responses, including cell adhesion, diapedesis and phagocytosis, are dependent on the mobilization and release of various intracellular granules and vesicles. In this study, we observed that inhibition of the Ras family GTPases by damnacanthal and manumycin A resulted in specifically release of PMN secondary granules, but not primary or tertiary granules, in the absence of stimulation. By screening Ras family members for activity changes during PMN activation and degranulation, we identified Ral GTPase to be a critical regulator that selectively controls secondary granule release. In particular, we found that Ral exists as an active GTPase in resting PMN, while quick inactivation correlates with chemoattractant-induced degranulation. Indeed, we demonstrated that over-expression of a constitutively active Ral mutant (Ral23V) in PMN completely blocked secondary granule release. Subcellular fractionation and immunofluorescence labeling further found that Ral is associated with the plasma membrane and redistributed to secondary granules following chemoattractant stimulation. Blocking clathrin-mediated plasma membrane endocytosis, however, inhibited Ral intracellular translocation and PMN degranulation. Thus, these results provide strong evidence demonstrating Ral to be an essential gatekeeper for secondary granules in PMN. Dysregulation of Ral results in impairment of PMN transmigration in response to inflammatory stimuli.
Interaction between mouse CD99 and CD99L2 proteins influences cellular localization of each protein in the cells and cell-cell interaction. (140.14)
Authors: Ma Min-Jung , Lee Hae-Young , Choi Eun-Young
Published: January 2023

CD99 and CD99 like 2 protein (CD99L2) are membrane glycoprotein leukocyte antigens that do not belong to any known protein family. They are expressed at the endothelial cell junction and are known to participate in the diapedesis process. Using mouse CD99 and CD99L2 gene, we show that CD99 interact with CD99L2. To examine molecular interaction between mouse CD99 and mouse CD99L2 and to localize the site of the interaction, we used a bimolecular fluorescence complementation (BiFC) assay via flow cytometry and confocal analysis. The interaction between mouse CD99 and mouse CD99L2 was verified by isothermal tiltration calorimetry (ITC) and coimmunoprecipitation. The interaction between CD99 and CD99L2 proteins is mediated by cytoplasmic domain of CD99. Their interaction enhanced the localization of each protein to the cell surface membrane and also increased cell-cell interaction via homophilic way. The results from this study suggest that the interaction of CD99 and CD99L2 may control the location and functions of CD99 and CD99L2 as cell adhesion molecules. *This study is supported by a fund from national research foundation (2007-2003619), republic of Korea.
Monocyte trafficking to hepatic sites of bacterial infection is chemokine-independent and directed by focal ICAM-1 expression (140.2)
Authors: Shi Chao , Velázquez Peter , Hohl Tobias , Leiner Ingrid , Dustin Michael , Pamer Eric
Published: January 2023

Recruitment of CCR2+Ly6C+ monocytes to sites of infection is essential for efficient clearance of microbial pathogens. While CCR2-mediated signals promote monocyte emigration from bone marrow, the contribution of CCR2 to later stages of monocyte recruitment remains unresolved. Herein, we show that CCR2 deficiency markedly worsens hepatic L. monocytogenes infection because Ly6C+ monocytes are retained in the bone marrow. Intravenously transferred, CCR2-deficient Ly6C+ monocytes traffic normally to hepatic foci of infection and contribute to bacterial clearance. Pertussis toxin treatment of adoptively transferred monocytes does not impair their intrahepatic trafficking, suggesting that chemokine signaling, once CCR2+Ly6C+ monocytes emigrate from the bone marrow, is not required for monocyte localization to sites of bacterial infection in the liver. Expression of ICAM-1 is induced in close proximity to foci of bacterial infection in the liver and blockade of CD11b and CD44 diminishes monocyte localization to these hepatic foci. Our studies demonstrate that Ly6C+ monocyte recruitment from the bloodstream to the L. monocytogenes-infected liver does not require chemokine receptor mediated signals but instead is principally dependent on integrin- and extracellular matrix-mediated monocyte adhesion.
Long-term immune characteristics of cow's milk allergy in a mouse model (141.12)
Authors: Gangur Venu , Gonipeta Babu , Parvataneni Sitaram
Published: January 2023

Specific changes in immune memory underlying the ability to outgrow cow’s milk allergy (CMA) are incompletely understood. We studied the long-term immune characteristics of CMA in an adjuvant-free mouse model we have recently published (Gonipeta et al 2009. J. Dairy Sci. 92:4738-4744). Thus, BALB/c mice were sensitized to cow’s milk whey protein using a transdermal sensitization protocol. Subsequently, exposure to whey protein was withdrawn for 3, 5 or 8 months. Fate of circulating IgE and IgG2a antibodies was monitored. Subsequently, mice were given booster exposures and examined for memory IgE, IgG2a, and spleen cell IL-4 and IFN-γ responses. Following allergen withdrawal, IgE antibody levels began to drop. Whereas, significant memory IgE response to booster exposures was evident after 3 months of allergen withdrawal, a weak or no memory IgE response was noted after 5 or 8 months of allergen withdrawal. Despite booster exposures, spleen cell memory IL-4 responses to whey protein was absent after 3, 5 or 8 months of allergen withdrawal. Milk sensitized mice, upon 8 months of allergen withdrawal, exhibited elevated IFN-γ and IgG2a responses suggesting a shift to a TH1 immune profile. In summary, these data suggest that: (i) upon withdrawal of allergen exposure, whey protein specific memory IgE, and memory IL-4 do not persist for long periods; and (ii) there occurs a shift in immune memory towards elevation of TH1 responses as mice age. Funding: US EPA STAR Grant#R833133
Role of T-cells in diesel particle exacerbation of allergen-induced pulmonary inflammation (141.2)
Authors: Brandt Eric , Lee Gerald , Gibson Aaron , Bass Stacey , Khurana-Hershey Gurjit
Published: January 2023

The mechanisms underlying the adjuvant effect of diesel exhaust particles (DEP) on allergen-induced airways responses are not well understood. We aim to define how T-cells are affected by exposures to DEP and house dust mite (HDM). Balb/c mice were exposed intra-tracheally to DEP, HDM, or both, three times a week for three weeks. Airway inflammation and FACS analysis of lung and draining lymph node T-cells were assessed 24h or 1 week after the last exposure. Exposure to DEP alone generated airway neutrophilia whereas exposure to HDM was characterized by eosinophilia. Coexposure to HDM and DEP resulted in a significant increase in BALF cells over HDM alone. The percentage of CD4+ T-cells expressing the activation marker CD69 were increased in all three groups over saline exposed mice. However, significantly more activated CD4+ T-cells were observed in the lungs and mediastinal lymph nodes of mice coexposed to HDM+DEP. Alterations in Foxp3+ regulatory T-cells were limited following coexposure to HDM and DEP resulting in a favorable ratio of activated T-cells over Tregs. One week after the last exposure, pulmonary inflammation and DEP containing macrophages could still be observed. Interestingly, levels of activated T-cells remain elevated in all three groups compared to saline exposed control mice, but the further increase observed after coexposure was no longer significant. Taken together, these results suggest that DEP exacerbates the early acute allergic responses.
Resveratrol induces myeloid-derived suppressor cells (MDSCs) and down-regulates CXCR3+ T cell expression and NF-kB activity to abrogate chronic colitis in IL-10-/- mice (142.10)
Authors: Singh Udai , Singh Narendra , Singh Balwan , Hofseth Lorne , Price Robert , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Inflammatory bowel disease, a chronic mucosal inflammation, is caused by hyperactivation of effector immune cells, which produce pro-inflammatory cytokines. Macrophages, along with Gr-1+ CD11b+ cells known as myeloid-derived suppressor cells (MDSCs), are known for their immunosuppressive function. Resveratrol is one of the most promising natural molecules for the treatment of chronic inflammatory diseases. In the present study, we investigated the resveratrol-mediated cellular and molecular mechanisms of chronic colitis inhibition in IL-10-/- mice. Resveratrol effectively attenuated various pathological markers of colitis by down-regulating Th1 responses. Resveratrol reversed colitis-associated decreases in body weight and increased levels of serum amyloid A and colon TNF-α, IL-6, RANTES, IL-12, and IL-1β. After resveratrol treatment, the percentage of CD4+ T cells that expressed CXCR3+ was decreased in the spleen, mesenteric lymph nodes (MLNs), and intestinal lamina propria (LP). However, the percentages of MDSCs in LP lymphocytes and spleens were increased after resveratrol treatment as compared with controls. Interestingly, resveratrol treatment after chronic colitis significantly reversed NF-κB activity in the colon. This study demonstrates for the first time that resveratrol protects against chronic colitis in IL-10-/- mice by inducing MDSCs, down-regulating mucosal and systemic effector CD4+T cells that express CXCR3+, and reducing NF-κB activity in the colon LP.
The splicing factor ASF/SF2 regulates the expression of the human T cell receptor CD3 zeta chain (143.11)
Authors: Moulton Vaishali , Tsokos George
Published: January 2023

T cells from patients with systemic lupus erythematosus (SLE) express decreased levels of the T cell receptor associated (TCR)-associated CD3 zeta (ζ) chain, a feature directly linked to their aberrant function. The decrease in CD3ζ protein expression is in part due to decreased levels of the functional wild-type (WT) isoform of the 3` untranslated region (UTR) of CD3ζ mRNA with concomitant increased levels of an unstable alternatively spliced (AS) isoform. In order to identify factors involved in the post transcriptional regulation of CD3ζ, we performed mass spectrometric analysis of Jurkat T cell nuclear proteins “pulled down” by a CD3ζ 3`UTR defined oligonucleotide which identified the splicing protein Alternative Splicing factor / Splicing factor 2 (ASF/SF2). We show for the first time that ASF/SF2 binds specifically to the 3`UTR of CD3ζ and regulates expression of CD3ζ protein by limiting the production of the alternatively spliced isoform. During activation of human T cells, an increase in the WT CD3ζ mRNA is associated with increased expression of ASF/SF2. Finally, we show a significant correlation between ASF/SF2 and CD3ζ protein levels in T cells from SLE patients. These results identify ASF/SF2 as a novel factor in the regulation of CD3ζ expression in human T cells, and a potential molecular mechanism underlying the altered CD3ζ chain expression in SLE.
Non-MHC genes increase susceptibility to autoimmune myocarditis by driving T cells towards a Th17 response (143.14)
Authors: Chen Ping , Ligons Davinna , Baldiviano G. , Talor Monica , Zheng Dongfeng , Barin Jobert , Rose Noel , Cihakova Daniela
Published: January 2023

MHC genes do not fully explain susceptibility to experimental autoimmune myocarditis (EAM) since A.SW and B10.S mice share the same MHC (H-2S) haplotype, yet A.SW is susceptible to EAM while B10.S is resistant. These two inbred mice strains provide an opportunity to study myocarditis susceptibility factors while excluding the effects of MHC-associated genetic components. We induced EAM in A.SW and B10.S mice by immunizing with murine cardiac myosin on day 0 and 7. A.SW displayed severe myocarditis compared to B10.S (p=0.008). The number of CD4+ T cells from heart and myosin specific T cells proliferation were greater in A.SW (p<0.001). A.SW and B10.S had different proportions of T helper (Th) subsets in their hearts after immunization; A.SW had more Th17 cells (7.05±1.25 vs. 2.24±1.05, p=0.003) and B10.S more Th1 cells (8.94±1.26 vs. 2.9±1.06, p=0.001). In support of these in vivo data we found that CD4+ T cells from naïve A.SW have higher potential to differentiate towards the Th17 pathway than CD4+ T cells from naïve B10.S (11.93±3.72 vs. 5.5±1.14, p=0.04). In contrast, B10.S T cells differentiated towards a Th1 phenotype compared with that from naïve A.SW (12.73±0.61 vs. 8.14±0.85 p=0.002). Additionally, A.SW have a lower proportion of regulatory T cell in their hearts (p=0.002) and spleens (p<0.001) compared to B10.S. Our data suggest that the tendency of A.SW differentiate towards a Th17 phenotype contributes to their heightened susceptibility of autoimmune myocarditis.
Role of IDO in B Cell-Mediated Immune Responses (143.2)
Authors: Mandik-Nayak Laura , Pigott Elizabeth , Galanti Rebecca , Ridge Natalie
Published: January 2023

Rheumatoid arthritis (RA) is a chronic and debilitating inflammatory autoimmune disease of unknown etiology. As with other autoimmune disorders, elevated tryptophan catabolism has been detected in RA patients. This is indicative of activation of the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO); however, the role that IDO plays in the disease process is not well understood. The current paradigm for IDO function is immunosuppressive, providing an important inhibitory role to limit effector T cell responses. In contrast, the role of IDO in B cell-mediated responses has not been addressed. The widely held belief that IDO acts solely in a T cell immunosuppressive role has led to the general assumption that inhibition of IDO activity should exacerbate autoimmune disorders. However, using the K/BxN mouse model of RA and 1-methyl-tryptophan (1MT), an inhibitor of IDO, we found that inhibiting IDO activity had the unexpected consequence of ameliorating, rather than exacerbating arthritis symptoms. This alleviation of joint inflammation was not due to an altered T cell response, but rather resulted from a diminished autoreactive B cell response. We have preliminary data demonstrating that 1MT is also able to inhibit B cell responses to immunization with model antigens. These data suggest for the first time an important role for IDO in directing B cell responses to both self and foreign antigens.
Altered B cell Development and Anergy in the Absence of Foxp3 (143.20)
Authors: Leonardo Steven , Josephson Jennifer , Hartog Nicholas , Gauld Steven
Published: January 2023

The importance of regulatory T cells in immune tolerance is illustrated by the human immune dysregulatory disorder IPEX (immune dysregulation, polyendocrinopathy, enteropathy, Xlinked), caused by a lack of regulatory T cells due to decreased or absent expression of Foxp3. Although the majority of work on regulatory T cells has focused on their ability to suppress T cell responses, the development of significant autoantibody titers in patients with IPEX suggests regulatory T cells also contribute to the suppression of autoreactive B cells. Although these observations suggest that regulatory T cells have an important role in B cell biology, no studies have directly determined whether an absence of regulatory T cells impacts B cell development or B cell tolerance. Using a murine model of IPEX, harboring a mutation in the Foxp3 gene derived from a human IPEX patient, we show that B cell development is significantly altered in the absence of regulatory T cells. Furthermore, we identify a loss of B cell anergy as a likely mechanism to explain the production of autoantibodies and loss of tolerance that occurs in the absence of regulatory T cells. Our results suggest that regulatory T cells, by either direct, or indirect mechanisms, modulate B cell development and anergy.
Breakdown of autoreactive tolerance in mice deficient in apoptotic clearance (143.24)
Authors: Shao Wenhai , Kuan Anita , Wang Charlie , Zhen Yuxuan , Eisenberg Robert , Cohen Philip
Published: January 2023

The Mer receptor tyrosine kinase (Mertk) helps in clearance of apoptotic cells through the bridging molecules Gas6 and/or Protein S. Mertk deficient (Mer-KO) mice develop lupus-like autoimmunity with gradual accumulation of apoptotic cells in spleen and thymus. Such Mer-KO mice have two-fold expanded numbers of splenic marginal zone (MZ) B cells, yet, they develop normal response to OVA comparing to B6 control mice. When challenged respectively with type I (NP-LPS) and type II (NP-Ficoll) T-independent antigens, Mer-KO developed greater antibody responses to NP-Ficoll than their B6 counterparts. We further explored the role of the Mertk in regulating autoreactive B cells. Mer-KO mice bearing a DNA-specific immunoglobulin heavy-chain transgene (3H9/Mer-KO) spontaneously produced anti-dsDNA antibodies at the age of 3-5 months. Allotype specific ELISA allowed us to identify the transgene as source of autoantibody in Mer-KO/3H9 mice. We further analyzed MZ and follicular (FO) B cells by ELISPOT. Antibody-secreting cell counts (ASC) of MZ B cells are 70 (3H9/Mer-KO), 40 (Mer-KO), and 20 (3H9), respectively, while ASC counts of FO B cells are all around the baseline level of 20 cells. Taken together, our data show that Mer has a major effect on the development of the marginal zone B-cell compartment. Mer is important in establishing DNA-specific B cell tolerance in 3H9 anti-DNA transgenic mice.
CD5-dependent CK2 activation pathway regulates T cell activation and threshold for anergy (143.27)
Authors: Sestero Christine , McGuire Donald , DeSarno Patrizia , Raman Chander
Published: January 2023

We have reported that a major biological activity of CD5 is to regulate the survival of activated T cells, a property mediated by the CD5-dependent CK2 activation pathway. In order to address the physiological role of the CD5-dependent CK2 activation pathway in T cell activation, the generation of Th effector cells, and autoimmunity, we generated by gene targeting a mouse (CD5ΔCK2BD) that expressed CD5 lacking the ability to bind and activate CK2. CD4 and CD8 T cells from CD5ΔCK2BD mice exhibited enhanced AICD and hyporeactivity to antigen receptor stimulation compared to T cells from CD5WT and CD5-/- mice. The ability to mobilize calcium and upregulate CD69 were greatly enhanced in CD5-/- T cells but not in CD5WT T cells or CD5ΔCK2BD T cells, indicating that the CD5-dependent CK2 activation pathway did not regulate TCR-proximal signaling pathways. CD5-/- mice developed attenuated experimental autoimmune encephalomyelitis (EAE) when immunized with 150 μg MOG, yet the disease in CD5ΔCK2BD mice was comparable to that seen in CD5WT mice. In contrast, immunization with tolerogenic doses of MOG resulted in attenuated EAE in CD5WT mice but not in CD5-/- and CD5ΔCK2BD mice. The results indicated that loss of the CD5-dependent CK2 pathway made T cells refractory to induction of anergy, a prediction that was recapitulated in in vitro recall assays. Our results reveal a novel role for CD5 in setting thresholds for anergy in T cells. Supported by grants from the NIH and NMSS.
Radioresistance of B-1 cells from (NZB/NZW) F1 mice: possible role of microRNAs (143.46)
Authors: Laurindo Maria , Salerno Erica , Popi Ana , Novaes e Brito Ronni , Yuan Yao , Kasar Siddha , Mariano Mario , Raveche Elizabeth
Published: January 2023

The (NZB/NZW)F1 (B/W) mouse spontaneously develops an autoimmune disease comparable with systemic lupus erythematosus in humans. B-1 cells may be an important cell type in the development of autoimmunity and B-1 cells are distinguished from conventional B cells by developmental origin, surface marker expression and functions, including the production of autoantibodies. Here, we present findings that B-1 cells (but not conventional B cells) from the peritoneal cavity of B/W mice are resistant to 9 Gy of radiation in contrast to B-1 cells derived from C57BL/6 mice. Similar to NZB mice which have a point mutation near the mir-15a/16 loci on chromosome 14 associated with decreased expression of microRNAs miR-15a and miR-16, the B/W are hemizygous for this mutation and also have decreased miR-15a/16. We hypothesized that increased expression of targets of miR-16 in B-1 cells from the B/W mice (such as Bcl-2) could be responsible for the radioresistance observed. Transfection of peritoneal cells from B/W with miR-16 sensitizes the B-1 cells to irradiation. Moreover, 1 hour after irradiation, peritoneal cells from the radiosensitive C57BL/6 also demonstrated an increase in miR-16 expression. Our data indicate a possible role of microRNAs in contributing to the radiation response, with decreased miR-16 associated with radioresistance in B-1 cells derived from the B/W strain. The possible implications of these findings in the pathogenesis of autoimmune disease are under investigation.
Thymic self-reactivity selects for natural Th17 cells which can be distinguished by unique markers (143.49)
Authors: Nowyhed Heba , Marks Benjamin , Choi Jin-Young , Craft Joseph
Published: January 2023

CD4 T helper (Th) cells that secrete interleukin (IL)-17 (Th17) complement the function of Th1 and Th2 cells as a third effector lineage, with a distinct role in neutrophil recruitment necessary for extracellular pathogen clearance and the promotion of certain autoimmune conditions. We have previously demonstrated that natural Th17 (nTh17) cells develop within the thymus in a manner similar to that of natural T regulatory (nTreg) cells (Marks et al., Nat. Immunol. 2009), with activation in response to an increase of self-antigen due to tissue destruction and subsequent host protection via IL-22 secretion. In this study, we report microarray analysis of lymph node nTh17 cells, revealing specific patterns of gene expression compared to naïve, Th1, and nTreg cells. We identified 317 genes uniquely over- or under-expressed in nTh17 cells compared to controls. These results give new insights into the differences among naïve, nTreg, nTh17, and Th1 cells. We further identified two markers, IL-17RE and IL-17RC, that are expressed in nTh17 cells and not in the other subsets or in induced Th17 cells. Neither has been previously reported to be expressed in Th17 cells. IL-17RC appears to be functional in nTh17 cells, in that it offers a way for them to regulate the levels of secretion of IL-17A and IL-22. IL-17RC and IL-17RE could potentially be used as specific markers for natural Th17 cells.
Estrogen upregulates IL-17-secreting cells and IL-17 levels in normal and autoimmune mice: IL-27 and IFNγ downregulate IL-17 (143.50)
Authors: Khan Deena , Dai Rujuan , Karpuzoglu Ebru , Ahmed S. Ansar
Published: January 2023

Estrogen, an important immunomodulator, plays critical role in gender related immune disorders including autoimmune diseases by regulating Th1, Th2 and Tregs homeostasis. Despite extensive research, it is still not known whether estrogen modulates recently identified Th17 cell, and its key effector cytokine IL-17A (IL-17). To address this critical gap in literature, in the present study, we determined whether estrogen regulates potent proinflammatory IL-17 in wildtype (C57BL/6) and autoimmune lupus prone (NZB/W) mice. By comprehensive analysis, we report for the first time that estrogen upregulates not only IL-17 levels in the supernatants of activated splenocytes, but also IL-17-specific transcription factor, retinoic acid-related orphan receptor gamma T (RORγT). Addition of IL-17 suppressive, IL-27 or IFNγ, at the time of initiation of culture inhibit IL-17 induction significantly and expression of RORγT+ cells. However, delay in addition of IL-27 or IFNγ decreased IL-17 (albeit less profoundly) but not RORγT suggesting that once the cell is committed to IL-17 secreting cell then the magnitude of inhibitory effect of IL-27 and IFNγ is lowered. These novel studies may have far-reaching implications that could clarify our understanding of the mechanism of estrogen-mediated proinflammatory responses. Future studies are being conducted to study in detail the signaling events, which favor IL-17 induction in estrogen treated mice and inhibition by IL-27 and IFNγ.
Dendritic cells propagate early autoimmunity in the absence of functional Tregs (143.55)
Authors: Hoyer Katrina , Isakson Sara , Abbas Abul
Published: January 2023

BALB/c IL-2-KO mice develop systemic autoimmunity, dying by 4 wks from complications of autoimmune anemia. Disease in these mice is Th1-mediated, and IFNγ production is required for early disease (Blood 2008). We asked whether APC are required for IFNγ production by T cells in the IL-2-KO mouse. Our data show that APC:T cell interactions through B7, but not via B cells, are necessary for IFNγ production in the absence of IL-2, suggesting that dendritic cells (DC) mediate this alteration. Disease in IL-2-KO mice is marked by DC accumulation, activation and elevated cytokine production, including IL-12. Surprising, in the absence of IL-12 T cell activation and disease is unchanged, thus, IL-12 is dispensable for IFNγ induction as a part of the underlying kinetics of autoimmunity. This led us to ask whether DC are necessary for IFNγ induction and autoimmunity in these mice. The depletion of either CD11c-high-DC or plasmacytoid DC significantly augmented the survival of IL-2-KO mice, demonstrating that DC contribute to the propagation of autoimmunity. IL-2-KO DC induced heightened proliferation and cytokine production by naïve T cells compared to WT DC. Also, DC from mice without overt T cell activation still increased the proliferation of naïve T cells, suggesting that enhanced DC function is not dependent on T cell activation. Overall, our data suggest that DC are directly activated in the absence of functional Tregs, and are critical regulators of autoimmune development.
The initiative to Link Animal Models to Human DIsease (LAMHDI) (144.12)
Authors: Anderson David , Buccigrossi Robert , Ellisman Mark , Eppig Janan , Evans-Kavaldjian Haig , Grethe Jeffrey , Hamel Raymond , Kemnitz Joseph , Martone Maryann , Pohland Kimberly , Preuss Nina , Sullivan Maureen , Turner Judith , Wagner Keith , Westerfield Monte , Yurt Hakan
Published: January 2023

We report a new Web-based resource (LAMHDI) integrating information about animal models for human disease research. This NCRR supported resource provides a simple interface to existing animal model information for the biomedical research community. It provides more efficient access to animal model information, better categorizes and organizes existing data, facilitates interactions between researchers and resource providers, and promotes broader and more applicable use of animal models for human disease research. Approach Phase 1: A first-generation prototype is available at www.LAMHDI.org. This portal provides searchable access to existing mouse and zebrafish databases through an integrated search engine, as well as linked access to information about a variety of other species including nonhuman primates and rats. The portal also allows researchers to contribute data on animal models, thereby extending the impact of existing information through direct posting by the research community. Phase 2: The second generation will add extensive new information for additional animal model species and will increase the depth and connectivity of the data through development of semantic datastores. A sophisticated software architecture will support intelligent relational searches across diverse datasets to highlight associations between diseases, animal models, and biologic pathways.
New Protocol for the Enrichment of Untouched Human Dendritic Cells (144.14)
Authors: Cheng Qi , deMedeiros Ricardo , Hoium Brian , Resnik Ernesto , Erickson Christian , Ni Jessie
Published: January 2023

We have developed a new negative selection protocol for the enrichment of untouched human dendritic cells (DCs) from human blood. The protocol can be carried out in less than one hour, after PBMC enrichment, and is characterized as a two-step negative selection procedure, using two different cocktails of biotinylated antibodies that target non-DC lineage markers. DCs enriched with this protocol performed better in T cell proliferation assays, as compared to DCs enriched with a commercial kit. Flow cytometric analysis of the isolated cells show that 90% of the enriched cells are CD14-/CD19-/BDCA1+/BDCA2+/BDCA3+ (total DCs), as compared to less than 10% before cell enrichment. Within these isolated DCs 2-8% are plasmacytoid DCs (BDCA2+/CD45+) and 82-88% are myeloid DCs (20-40% BDCA1+/CD11c+, 20-40% BDCA3+/CD11c+, 15-20% CD16+/CD11c+ and 20-25% CD56+/MHCII+). On average 1 million DCs were isolated per 100 million PBMCs. The advantages of this protocol include: a) better performance in T cell proliferation assays, compared to DCs enriched with a commercially available kit, and b) higher myeloid DC heterogeneity in the enriched DC fraction. The above observations could be the result of the fact that the isolated DCs are not bound to antibodies that could initiate activation cascades, and that CD16+ and CD56+ DCs were absent from the cell population isolated with the commercial kit.
Identification of biologically significant genes using Gene Ontology (GO) and pathways analysis (144.16)
Authors: Golovan Serguei , Husain Mainul
Published: January 2023

Due to the small sample size and high dimensionality, it is not always possible to identify all of the biologically relevant genes from microarray analysis by simply selecting an arbitrary fold change and p-value thresholds. Such concerns are especially relevant in the analysis of immune response. It has been long known that large variability exist in immune responsiveness with the coefficient of variation (CV) as high as 230% reported for antibody response. Similarly, immune related genes were shown to have CV in excess of 100%. As a consequence, some genes involved in immune response will never be detected as differentially expressed as the number of replications required to detect statistically significant effects may be unobtainable even in well-designed experiments. To better understand which biological pathways were affected by allergenic response to ovomucoid treatment in mouse spleen, two gene sets for GO analysis were selected using different selection criteria. First set included differentially expressed genes that were biologically and statistically significant between treatments (>1.5 fold, P<0.05). Genes for the second gene set were selected based only on their biological significance (>1.5 fold). It is demonstrated that not only new biologically significant genes were identified in previously detected GO and pathway categories (p<0.05) but completely new categories (p<0.05) were detected using second gene set.
Characterization of antibodies generated toward small hydrophobic environmental pollutants (144.17)
Authors: Spier Candace , Harris Thomas , Unger Michael , Kaattari Stephen
Published: January 2023

Antibodies are currently being exploited for their use as analytical reagents for the detection of environmental pollutants. They exhibit exquisite sensitivity and specificity such that they can be used to detect very low concentrations of targeted pollutants. One important class of pollutants for which antibodies are being developed is the polycyclic aromatic hydrocarbons (PAHs). Besides being small, PAHs are extremely hydrophobic, which increases the challenge in developing antibodies to these molecules. We have synthesized derivatives of PAHs and heterocycles (dibenzothiophene, benzothiophene, biphenyl, and fluorene) to serve as immunizing and screening haptens. By making modifications to our sera screening methods, some interesting aspects of antibody affinity to small hydrophobic haptens have been brought to our attention. Namely, the overall titer to these molecules is low (i.e., <10,000 L/mol), the site of attachment greatly influences antibody specificity, and the choice of screening hapten can influence the characterization of the antibody. Knowing these outcomes promotes the need to expand screening and immunizing routines, which will increase the success of developing antibodies to other small hydrophobic pollutants. This research has been supported by a Cooperative Institute for Coastal and Estuarine Environmental Technology grant.
An Alternative Method for Intrathymic Injection in Mice (144.8)
Authors: Handon Robin , Mueller Kristen , Schwatzberg Pamela , Hoogstraten-Miller Shelley
Published: January 2023

Intrathymic injection has traditionally been performed using surgical procedures to open the chest cavity for direct visualization and injection into mice thymus. This technique subjects the mice to significant pain and distress, which can alter T cell development and potentially increase mortality. Intrathymic injection can also be performed by blind injection into the thoracic cavity. However, the success rate for this technique is usually low. In order to improve success rates with non-surgical intrathymic injection, our lab has preformed intrathymic injections in isoflurane-anesthetized mice using ultrasound (US)-mediated guidance. The success rate using this technique has been high as the needle and injectate can both be visualized by US as they enter the thymus. Using this technique, we have been able to label thymocytes using either FITC or Biotin to trace their fates. Complications have been very rare and were limited to the accidental laceration of an intrathoracic vessel that required in the euthanasia of one mouse out of approximately 85 injected to date. Moreover, it is important to note, that we were able to see the immediate hemorrhage on US and euthanized the mouse while it was still anesthetized, thereby avoiding any pain or distress. Our results suggest that use of US-mediated guidance is a refinement that can help reduce morbidity, distress and animal usage in experiment involving intrathymic injection.
Promotion of engraftment by donor NK cell infusion and IL-15 in nonmyeloablative allogeneic bone marrow transplantation (145.18)
Authors: Liu Haiyan , Hu Bo , Lin DanDan , Bao Guangming , Liang Yong , He Yan , Wu Yan , Zhao Lixiang , Murphy William
Published: January 2023

Donor NK cells have been shown to be able to promote engraftment during allogeneic bone marrow transplantation (allo-BMT). The biological activity of NK cells can be significantly enhanced by IL-15. We carried out experiments to determine the effects of combining donor NK cell infusion and IL-15 administration in a murine nonmyeloablative allo-BMT model. Mice infused with donor NK cells and treated with IL-15 during BMT resulted in increased donor engraftment compared with either treatment alone, although single treatment could also significantly promote donor engraftment. The counts of donor derived cell subsets were all increased in the spleens of the recipient mice with combinational treatment compared with either treatment alone. The alloreactivity to donor type antigens was significantly reduced in the recipient mice with combinational treatment, which was manifested by decreased proliferation and IL-2 secretion of splenocytes from recipient mice in response to donor type antigens in mixed lymphocyte responses and decreased capacity of the splenocytes killing donor type tumor targets. In conclusion, the combination of donor NK cell infusion and IL-15 administration could promote donor engraftment and development of all donor derived cell subsets, as well as suppress the host allo-responses in a nonmyeloablative allo-BMT model. The results provide a promising way to promote donor engraftment without involving systemic and nonspecific suppression of the immune system.
Pancreatic islets engineered to display on their surface a novel form of FasL protein establish localized tolerance via Treg cells (145.2)
Authors: Zhao Hong , Yolcu Esma , Bandura-Morgan Laura , Shirwan Haval
Published: January 2023

Objective: Islets engineered to display on their surface a chimeric FasL (SA-FasL) protein induce localized tolerance in allogeneic recipients. This study tests if CD4+CD25+FoxP3+ Tregs play a role in the initiation and maintenance of tolerance. Methods: C57BL/6 mice were transplanted with SA-FasL-engineered BALB/c islets. The frequency of Tregs in various tissues were analyzed using flow cytometry and confocal microscopy. The role of Tregs in localized tolerance was tested by; i) in vivo depletion early or late post-Tx or ii) in an adoptive transfer model using sorted Treg cells from naïve, rejected, or tolerant animals with CD4+ T cells from naïve mice. Results: We observed increased numbers of Treg cells only within the islet grafts on day 5 post-Tx and long-term (>100 days) SA-FasL-islet grafts as compared to controls, but not in peripheral lymphoid organs. In vivo depletion of Treg cells early (day 14) or late (day >100) post-Tx resulted in acute graft rejection. Also, cotransfer of sorted 2-5x103 Tregs from LT (n=6; >100 days) graft survivors, but not rejectors (n=4; MST=36±2.3 days) with 1x106 CD4+ T effector cells from naïve C57BL/6 prevented rejection of unmanipulated BALB/c islets in an adoptive transfer model. Importantly, Tregs from long-term graft survivors expressed higher levels of FoxP3 as compared to Tregs from naïve or rejectors mice. Conclusions: Localized tolerance achieved by SA-FasL-engineered islet grafts is initiated and maintained by Treg cells
Liver DC modulate Foxp3 expression in allogeneic CD4+ T cells via IL-27 (145.3)
Authors: Matta Benjamin , Sumpter Tina , Raimondi Giorgio , Thomson Angus
Published: January 2023

Liver dendritic cells (DC) possess immunoregulatory properties that influence the nature of T cell responses. We investigated the impact of IL-27 on CD4+ T cell responses by liver DC. Both plasmacytoid DC (mPDCA-1+) and myeloid DC (CD11c+ mPDCA-1-) were immunobead-purified from livers and spleens of Flt3L-treated C57BL/6 mice. DC phenotype was analyzed by flow cytometry and cytokine levels were quantified by ELISA. Ebi-3 (a component of the IL-27 molecule) protein expression was determined by Western blot. T cell allostimulatory capacity in MLR was quantified by CFSE dilution analysis, and Foxp3+ T cells quantified by intracellular flow cytometric analysis. Liver DC exhibited reduced expression of antigen (Ag)-presenting and costimulatory molecules and induced inferior T cell proliferation compared to splenic DC. They also produced elevated levels of IL-27p28, IL-10, and IL-6, but reduced IL-12p40 levels compared to splenic DC. Liver DC expressed higher levels of Ebi-3 protein compared to spleen DC. Blockade of IL-27 in allogeneic MLR resulted in greater levels of Foxp3+ T cells when liver but not spleen DC were used as stimulators. Thus, liver DC exhibit lower Ag-presenting and costimulatory molecule expression but produce greater levels of immunoregulatory cytokines than splenic DC, which may contribute to their poor CD4+ T cell allostimulatory capacity. Interestingly, IL-27 produced by liver DC may inhibit their ability to induce Foxp3+ regulatory T cells.