bluuebunny/crossref_metadata_2025 huggingface dataset:
Salivary gland lymphocyte response to murine cytomegalovirus (INC1P.344)
Authors: Erick Timothy , Reilly Emma , Wands Jack , Brossay Laurent
Published: January 2023

The β-herpes virus MCMV, a homologue of HCMV, is a well-characterized animal model of viral infection that results in a non-replicative, chronic infection of an immune-competent animal. In most organs, MCMV is cleared within days by cytotoxic lymphocytes. However, the virus persists in the submandibular salivary glands (SMG) for several weeks, before establishing latency for the life of the host. NK cells are crucial for the early containment of MCMV before T cells can mount an effector response. Recent work from our laboratory has shown that SMG NK cells are hyporesponsive during MCMV infection. Here we show that SMG NK cells regain normal effector functions against MCMV when adoptively transferred into different tissue environments. This indicates that the hyporesponsive phenotype observed is tissue specific, and not cell-intrinsic. In addition, using E4BP4 deficient animals, we determined that two CD3-NK1.1+ subsets reside within the salivary glands. One subset is derived from the conventional NK cell population and relies on E4BP4 for development, while the other is E4BP4 independent. When the E4BP4 independent NK cells are transferred into different tissue environments, they produce IFN-γ in response to MCMV infection. The SMGs of E4BP4-/- mice also contain a novel population of CD3+NK1.1+NKp46+ cells that produce IFN-γ during MCMV infection. Altogether, our data show that the SMG tissue environment shapes a unique repertoire of NK1.1+ cells with distinctive phenotypes.
Skin inflammatory response to complex hydrocarbon mixtures (INC1P.357)
Authors: Gallucci Randle , Chastain Lerin , Kemp Jesse , Calhoun Kaitlin
Published: January 2023

Petroleum whether as crude oil or as various refined products is known to variably cause skin irritation. For instance, spill remediation workers that are exposed to fresh and weathered oil appeared to suffer a myriad of health effects, including contact dermatitis. However, the relationship between the form of petroleum and its ability to induce skin inflammation has not been examined. To investigate this, a mouse model of seven-day exposure to crude oil (weathered and fresh), JP8 jet fuel (refined) or acetone (control) was utilized, employing C57BL/6 and Balb/c mice. Immunohistochemistry, multiplex protein analysis, and real-time PCR were then conducted on isolated skin samples. Inflammatory cytokine expression showed that oil exposure significantly increased IL-1b, IL-6, CXCL2, CXCL10, CCL2, CCL3, CCL4, and CCL11 levels, the extent of which differed based on the form of petroleum as well as mouse strain. Histopathology from all forms of petroleum showed obvious inflammation in exposed skin, primarily characterized by epidermal thickening and dermal inflammatory cell infiltration which was most pronounced from jet fuel exposure. Classically activated macrophages dominated M2-like as indicated by CD86 versus CD206 expression respectively for all petroleum types. These results suggest that petroleum is indeed a skin irritant, and its form whether fresh, weathered, or refined may influence the severity of the skin inflammation.
Effect of various types of sea salt spa spring water on cytokine expression in primary human keratinocyte under TLR stimulation (INC1P.404)
Authors: Lee Kyung Ho , Kim Su-Jin , Woo So-Youn , Kim Jin-Wou
Published: January 2023

Background: Balneotherapy is widely used for the treatment of inflammatory skin disease, but the mechanisms are only partly understood. We previously reported spa spring water affected the secretion of inflammatory cytokines from keratinocytes (KCs) in vitro and possibly affected the T cell differentiation. Objective: In this study, we evaluated effect on cytokine secretion in primary KCs of three types of sea salt spa spring water from HaeUnDae, Magumsan, and Sokmodo, selected by low, middle and high salt concentrations, respectively. Methods: We isolated mRNA from KCs and evaluated the expression of TLR1 to TLR10 by RT-PCR. Next, we investigated the immune modulatory effect of three sea salt type spring waters on the expression of cytokines (IL-8, TNF-α, IL-1β, IL-6, and GM-CSF) in the primary KCs under TLR1-6 stimulation by ELISA. Results: The treatment of three types of sea salt spa spring waters induced suppression of inflammatory cytokines under TLR stimulation in the primary KCs. Notably, cytokine secretion from primary human KCs to TLR3 ligand was prominent. Decrease of IL-8, TNF-α, IL-1β, IL-6, GM-CSF secretion under TLR stimulation were observed, depending on the type of TLR stimulations. Conclusion: Three different type of sea salt spa spring water treatment affected the secretion of inflammatory cytokines production from KCs under TLR stimulation. These immune parameters and protocols can be used to evaluate immune modulatory effect of spa spring water in Korea.
ATP protects against sepsis through macrophage P2X7 purinergic receptors by enhancing bacterial killing (INC2P.407)
Authors: Hasko George , Csoka Balazs , Németh Zoltan , Törö Gabor , Idzko Marco , Zech Andreas
Published: January 2023

Extracellular ATP binds to and signals through P2X7 receptors (P2X7R)s to modulate immune function in both inflammasome-dependent and independent manners. We show here using P2X7-/- mice as well as pharmacological receptor and channel ligands that ATP release through connexin/pannexin channels and subsequent P2X7R activation are crucial for the control of mortality, bacterial dissemination and inflammation following sepsis induced by cecal ligation and puncture. Our results with P2X7-/- bone-marrow chimeras, adoptive transfer of macrophages, and myeloid-specific P2X7-/- mice indicate that P2X7R signaling on macrophages is required for the protective effect of P2X7Rs. P2X7R signaling protects through enhancing bacterial killing by macrophages, but independently of the inflammasome. In summary, targeting P2X7Rs provides a new opportunity for harnessing an endogenous protective immune mechanism in the therapy of sepsis.
IL-21 is essential for the optimal control of Mycobacterium tuberculosis infection (INC2P.416)
Authors: Cheekatla Satyanarayana , Venkatasubramanian Sambasivan , Tripathi Deepak , Paidipally Padmaja , Welch Elwyn , Tvinnereim Amy , Vankayalapati Ramakrishna
Published: January 2023

IL-21 is a Th17 cytokine and is known for its anti-tumor and anti-viral effects. In the current study using IL-21receptor (IL-21r-/-) and IL-21 knockout (IL-21-/-) mice, we determined the role of IL-21 and IL-21r in Mycobacterium tuberculosis (M. tb) infection. Around 50% of M. tb H37Rv infected IL-21r-/- mice died in six months compared to no deaths in infected control mice. M. tb infected IL-21r-/- mice have enhanced bacterial burden (7.3 ± 1.0 x 106 vs. 0.7 ± 0.08 x 106,p<0.001), reduced infiltration of CD4+ T-cells (0.9 ± 0.01 x 105 vs. 1.4 ± 0.03 x 105, p<0.001), increased frequency of PD-1,2B4,CD160 & EOMES (transcription factor) and reduced frequency of IL-12Rβ positive T-cells in lungs compared to wild type M. tb infected mice. T-cells from M. tb infected IL-21-/- mice proliferated less, produced less IFN- γ in response γ-irradiated M. tb and expressed more EOMES compared to wild type M. tb infected mice, and this was reversed by recombinant IL-21. EOMES siRNA increased IFN- γ production (p=0.007) and IL-12Rβ expression by T-cells of M. tb infected wild type mice. We also found T-cells from M. tb infected IL-21-/- mice enhance production of IFN-α (p=0.01), IL-10 (p=0.01), reduce IL-12 (p=0.02) and IL-1β (p=0.04) expression by M. tb infected macrophages and were unable control bacterial growth in macrophages compared to T-cells from M. tb infected wild type mice. The sums of our findings suggest that IL-21 and IL-21r are essential for optimal control of M. tb in mice
Characterization of phagocytic cell responses to in vitro stimulation with two commensal fungi. (INC2P.422)
Authors: Limon-Tello Jose , Wolf Andrea , Leal Christian
Published: January 2023

The commensal community in the gut is not only comprised of a very diverse bacterial population but also of an equally diverse fungal community whose role in the gut ecosystem is only beginning to be explored. It is therefore important to be able to isolate and grow out these fungal commensal organisms to characterize host immune responses. By employing the use of selective media we successfully cultured two fungal organisms from the gastrointestinal tract of healthy mice; the basidiomycetous yeasts Rhodotorula mucilaginosa and Guehomyces pullulans. Yeast cells from both organisms stained positive for chitin with calcofluor white, and β-1,3 glucans with a mAb against β-1,3 glucan, or recombinant Dectin-1. Phagocytosis by bone marrow derived dendritic cells and macrophages was less efficient when compared to Candida albicans. Syk phosphorylation was visualized by microscopy after 30 minutes of stimulation. TNFα production was decreased in dendritic cells stimulated with both isolates versus Candida albicans however, cytokine production was not Dectin-1 dependent as Dectin-1 knock out cells produced similar levels of cytokine when compared to WT cells. These are preliminary results which indicate that Dectin-1 may be dispensable for the inflammatory response against these fungi and future work will investigate the role other immune receptors play in the host response to these commensal organisms.
Investigation of heat shock protein interaction with individual CD91 ligand binding domains (INM1P.436)
Authors: Sedlacek Abigail , Mizote Yu , Pappan Nikos , Binder Robert
Published: January 2023

Heat Shock Proteins (HSPs) are a structurally diverse class of proteins that respond to cellular stress. Many of these proteins naturally function to chaperone proteins and peptides throughout the cell. Upon release into the extracellular space, HSPs can act to potentiate immune responses against their chaperoned antigens. HSPs bind the receptor CD91on the surface of antigen presenting cells (APCs) which results in internalization of the HSP+antigen and downstream signaling resulting in APC activation. Interestingly, HSPs that bind CD91 do not share structural homology and result in different patterns of cytokine secretion by APCs following activation. CD91 is comprised of a 515kDa alpha chain which is responsible for ligand binding and an 85kDa transmembrane beta chain which is responsible for signal transduction. Within the alpha chain, 4 separate ligand binding domains have been identified. In these studies, we aim to determine which ligand binding domains are responsible for binding individual HSPs and if differences in binding domain usage result in the different patterns of APC activation. To accomplish this, we have constructed individual mini-genes comprised of a single ligand binding domain tethered to the full length beta chain. These mini-genes were then transfected into RAW309Cr.1 macrophage cells which does not express CD91. These cells will be used to assess HSP binding and downstream events to gain a better understanding of HSP-CD91 interactions.
RIG-I Signaling in the Innate Immune Response After Stroke (INM1P.445)
Authors: de Rivero Vaccari Juan Pablo , Brand Frank , de Rivero Vaccari Juan Carlos
Published: January 2023

Stroke is associated with an inflammatory response that contributes to deleterious outcomes. However, the molecular basis in the regulation of inflammation remains poorly understood. Here, we examine the activation of retinoic acid inducible gene-like (RIG) receptors (RLRs) and the involvement of RLR signaling in the regulation of type I IFNs following mid-cerebral artery occlusion (MCAO). In this study we show that in the rat hippocampus RIG-I and IFN-α expression are increased after MCAO. Similarly, we show that in astrocytes in culture, oxygen glucose deprivation (OGD) resulted in increased RIG-I and IFN-α expression. These data are consistent with immunohistochemical analysis of hippocampal sections, indicating that in glial fibrillary acidic protein (GFAP) positive cells there was an increase in RIG-I immunoreactivity after MCAO. Moreover, n-propyl gallate (n-PG) can be used to inhibit RIG-I/IFN-α signaling in astrocytes. In conclusion, these data indicate that RIG-I is involved in the inflammatory response after stroke.
Insulin resistance in macrophages results in a distinct metabolic profile promoting an M2-like phenotype affecting both acute and chronic innate immune responses (INM1P.430)
Authors: Ieronymaki Eleftheria , Theodorakis Emmanuel , Aznaourova Marina , Vaporidi Katerina , Margioris Andrew , Eliopoulos Aristides , Tsatsanis Christos
Published: January 2023

Obesity and insulin resistance affects innate immune responses. In this study, we evaluated the role of insulin resistance on macrophage activation and metabolism and how these changes may affect acute inflammatory responses and sustained responses such as those regulating the gut microbiome. Herein we demonstrate that primary macrophages from mice exposed to high insulin concentration, either in vitro or in vivo in the model of High Fat Diet as well as macrophages from Akt2-/- mice or from mice lacking IGF1R from macrophages, develop insulin resistance as determined by defective glucose uptake in response to insulin. These macrophages obtained M2-like phenotype and acquired a distinct metabolic profile characterized by decreased glycolytic and increased oxidation capacity. Mice bearing insulin resistant macrophages exhibited reduced acute lung injury in response to polymicrobial sepsis, as evaluated using the Cecal Ligation and Puncture model. The intestinal microbiome was similar in Akt2-/-, LysMCre+/IGF1Rfl/fl and HFD-induced obese mice and differed from this of WT mice, suggesting that insulin resistant macrophages impact microbial flora composition. These data suggest that insulin resistance in macrophages promotes an M2-like phenotype and a distinct metabolic program affecting acute inflammatory responses and shaping the gut microbiome irrespective of the diet.
Upregulation of aryl hydrocarbon receptor expression in CD4 T lymphocytes during an immune response in vivo. (INM1P.431)
Authors: Fechner John , Julliard Walker , O'Driscoll Chelsea , Mezrich Joshua
Published: January 2023

Activation of the aryl hydrocarbon receptor (AHR) can impact T-cell differentiation and the maintenance of tissue-resident T cells. AHR expression, measured by RT-PCR or Western blot has been shown to be low in most CD4 T-cell subsets with highest expression found after stimulation of naïve CD4 T cells under Th17 conditions in vitro. The current study measures AHR expression within CD4 T-cell subsets of naïve or skin-grafted mice using flow cytometry. Cells were freshly isolated from the spleen, lymph nodes or skin of either untreated mice or mice that underwent allogeneic skin transplant. AHR expression in cells from untreated mice was highest in IL-17A+ CD4 T cells, followed by FoxP3+ and IFNγ+ cells. Naïve, CD62L+ CD4 T cells had the lowest expression. AHR expression in splenic or lymph node CD4 T cells from skin-grafted mice was similar to naïve mice with 20 - 30 % of FoxP3+ and <10% of FoxP3- being AHR+. However, CD4 T cells within the skin grafts showed elevated AHR expression compared to spleen or LN with 75 - 85 % of FoxP3+ and 40 - 70% of FoxP3- being AHR+. This is comparable to the >90% AHR+ CD4 T cells found when splenocytes were stimulated in vitro with anti-CD3 antibody in the presence of TGFb and IL-6. We conclude that the up-regulation of AHR expression in CD4 T cells in vitro also happens during immune responses in vivo and is not restricted to a single T cell subset, and may be important in T cell differentiation, trafficking, and cytokine production.
Two dimensional affinity range correlates with phenotypic response to lipid antigens in iNKT cells (IRC4P.600)
Authors: Neeld Dennis , Rossjohn Jamie , Evavold Brian
Published: January 2023

Invariant natural killer T cells (iNKT) are a unique group of T cells that possess an invariant T cell receptor (TCR) alpha chain paired with a limited repertoire of TCR beta chains that recognize lipid antigens presented by the MHC-I like molecule, CD1d. The micropipette adhesion frequency assay was used to define 2D affinities of iNKT cells for a panel of lipid:CD1d antigens. Despite having a limited diversity of αβ TCRs, NKT cells possess approximately a 100-fold range of affinities for the lipids tested. Our results show that αGalCer, an INFγ inducing lipid, has an approximately 10-fold higher affinity than OCH, an IL-4 skewing lipid in B6 mice. Moreover, thymic B6 iNKT cells, which predominantly produce Th1 cytokines (IFNγ), possessed a 4-fold higher affinity as compared to iNKT cells from Balb/C mice that favor more of a Th2 (IL-4) response. iNKT cell phenotype is also affected by their location as cells from the liver of Balb/C mice, which are predominantly IFNγ producers have a 2-fold increase in affinity for αGalCer compared to IL-4 predominant thymic iNKT cells. In addition to affinity differences related to phenotype, TCR expression levels of thymic derived iNKT cells reveal that Balb/C mice express twice as much TCR as the B6 mice. Taken together, these results indicate that NKT2 cytokine producing iNKT cells possess lower 2D affinity and higher TCR density compared to their NKT1 producing counterparts.
MDSC suppression of B cell responses in murine retrovirus-induced immunodeficiency: a role for VISTA (IRC4P.603)
Authors: Green Kathy , Wang Li , Noelle Randolph , Green William
Published: January 2023

Inhibition by myeloid derived suppressor cells (MDSC) of T cell responses is well established in tumor microenvironments. We demonstrated (Green et al., 2013) induction of monocytic MDSCs during infection of B6 mice by LP-BM5 retrovirus, which causes profound immunodeficiency. These MDSCs suppressed not only T, but also B cell, responsiveness ex vivo. Whereas MDSC inhibition of stimulated T cell proliferation and IFN-gamma production was ~100%, iNOS/NO dependent, MDSC suppression of B-cell responses was only ~50% dependent on iNOS/NO - as shown by using iNOS inhibitors and iNOS k.o. mice as a source of MDSCs. We then discovered an additional mechanism(s) in MDSC inhibition of (only) B cell responsiveness that involved VISTA, a new negative checkpoint regulator. Using anti-VISTA blocking mAb, or LP-BM5 infected VISTA-/- MDSCs, MDSC inhibition of B cell responses was dependent on MDSC-expressed VISTA — again accounting for ~50% of total MDSC suppression. Combining the use of reagents to block both iNOS/NO and VISTA lead to an additive, if not synergistic, abrogation of MDSC suppression of B cell responsiveness. Consistent with a direct role of VISTA, in the absence of MDSCs, a VISTA-Ig fusion protein also partially inhibited B cell responsiveness. These results were compatible with a role for MDSC in LP-BM5-induced immunodeficiency and highlight involvement of multiple and unique suppressive pathways in the under-studied area of MDSC suppression of B cell responses.
Loss of cell surface CD47 ‘clustering’ formation and the binding avidity to SIRPα during epithelial cell apoptosis (IRC4P.453)
Authors: Lu Zhiyuan
Published: January 2023

CD47, a ‘self’ marker expressed in most cell types, interacts with immunoreceptor SIRPα expressed on phagocyte to initiate inhibitory signaling that prevents healthy cells from being cleared by phagocytes. Previous studies have suggested that cell may lose surface CD47 during cell aging or apoptosis. In the present study, we demonstrated that the majority of CD47 molecules localize in lipid rafts forming ‘punctate’ pattern in healthy cells, whereas in apoptotic cells, the level of cell surface CD47 wasn’t decreased, but CD47 moved out of rafts and redistributed evenly on the plasma membranes. Importantly, clustering of CD47 in the lipid rafts of cell plasma membranes provided a high binding avidity to SIRPα, and inhibited macrophage phagocytosis of healthy cells. In contrast, dispersed CD47 on apoptotic cell surface resulted in a significant reduction of binding avidity to SIRPα, leading to failure of the protection of apoptotic cells from phagocytosis by macrophages. Disruption of lipid rafts with methyl-β-cyclodextrin treatment disrupted CD47 clustering on cell surface, leading to decreased binding avidity of cells to SIRPα therefore being engulfed by macrophages. Thus, our study reveals that CD47 normally localized in lipid rafts in non-apoptotic cells but redistributed evenly on the plasma membranes when apoptosis occurred, the transformation of CD47 greatly reduced the strength of CD47- SIRPα engagement, resulting in the phagocytosis of apoptotic cells by phagocytes.
Crossreactive influenza A(IAV)-Specific CD4 memory cells enhance viral load during lymphocytic choriomeningitis (LCMV) infection (IRC5P.635)
Authors: Selin Liisa , Agarwal Priti , Kraft Anke , Wlodarczyk Myriam
Published: January 2023

IAV is a common human respiratory pathogen with a strong association with heterotypic and heterologous immunity. IAV-immune mice challenged with LCMV develop detrimental effects with both increased virus titers and immunopathology. Crossreactive CD8 T cell responses between these two viruses mediate the enhanced lung pathology. Here, we questioned whether crossreactive CD4 memory cells in IAV-immune mice could play a role in increasing viral titers during the subsequent LCMV infection. Preferential depletion of memory CD4 but not CD8 cells in IAV-immune mice prevented the increase in LCMV titers. In IAV+LCMV mice there was an early increase in CD4 cells with a shift in the CD4/CD8 ratio. Luminex cytokine assays suggested that there was an increase in Th2 cytokines, IL-4 and IL-5, in the spleen of IAV+LCMV mice, which was IAV CD4 memory cell dependent, as IL-4 decreased in the CD4 depleted mice. Depleting IL-4 or IL-5 returned LCMV titers to naïve levels while adding rIL-4 and rIL-5 together to naïve mice increased virus titers. This suggests that the crossreactive heterologous response was more Th2-polarized and that since memory cytokines are produced early they may shift the normally strong Th1-type response to LCMV towards a more Th2-type response, inhibiting appropriate CD8 activation and impeding viral clearance. This shift to stronger Th2-type response would contribute to detrimental effects as LCMV requires a strong CD8 response and perforin to clear.
Post-transcriptional regulation of interferon lambda during viral infection (IRM5P.640)
Authors: Schwerk Johannes , Thomas Kerri , Aarreberg Lauren , Jarret Abigail , Hong MeeAe , Savan Ram
Published: January 2023

Interferon lambda (IFN-λ) has gained increasing attention recently as an important mediator of antiviral protection in hepatitis C virus (HCV) infection. Similar to many cytokines, we have shown that expression of IFNL is regulated on a post-transcriptional level. Interestingly, single nucleotide polymorphisms (SNPs) within the IFNL genes dictate the outcome of acute HCV infection and serve as a prognostic marker for therapy of chronic HCV infection. Previously, we demonstrated that a functional SNP (rs4803217) within the 3'-untranslated region (UTR) of IFNL3 affects transcript stability and correlates with HCV clearance (G/G) or persistence (T/T) in infected patients and this is mediated by miRNAs and AU-rich elements. In our current study we have identified RNA-binding protein(s) (RBPs) that differentially regulate IFNL3 variants. We identified RBP candidates by carrying out mass spectrometry analysis on both variants of IFNL3 mRNA. We found that these candidates exclusively interact with the IFNL3 (T/T) mRNA. Intriguingly, gene expression of one candidate RBP is strongly induced upon stimulation of cells with HCV RNA or viral PAMP. Collectively, our data suggest that IFNL3 mRNA is post-transcriptionally regulated by miRNA and RBP-mediated mechanisms and that HCV potentially hijacks these regulatory mechanisms to suppress host antiviral responses.
The role of STING in T lymphocytes (IRM5P.643)
Authors: Poltorak Alexander , Larkin Bridget , Surpris Guy , Ilyuha Vladimir
Published: January 2023

The cytoplasmic protein Stimulator of Interferon Genes (STING) plays an essential role in sensing intracellular pathogens and initiating type I IFN responses in myeloid cells. STING detects the presence of cytoplasmic DNA presented by various DNA-binding proteins (e.g. IFI16, DDX41, DAI, RNA Pol III); it also directly binds cyclic dinucleotides produced by intracellular bacteria or by the DNA-binding cyclic GAMP synthase (c-GAS). Once activated, STING phosphorylates TBK1 and IRF3 to trigger type I IFN production. To date STING has only been studied in macrophages and dendritic cells, but early reports showed expression of STING in a variety of tissues including the thymus and spleen, prompting us to ask whether STING might have a functional role in T cells, which account for the majority of cells in the thymus and a larger proportion of cells in the spleen than macrophages and dendritic cells. Here, we show that STING can be activated in T cells by the small molecule DMXAA, resulting in IFNβ production and increased expression of numerous interferon stimulated genes (ISG)—the first evidence to our knowledge of type I IFN production by T cells. Furthermore, RNA-sequencing data revealed unanticipated differences between unstimulated wild type and STING-/- T cells, with STING -/- T cells exhibiting increased expression of genes associated with cytotoxic T lymphocytes, indicating that STING-mediated signaling in T cells may regulate the development of particular T cell subsets.
Defining the chromatin landscape of the Il2-Il21 locus in CD4+ T cells (IRM6P.653)
Authors: Pham Duy , Winstead Colleen , Hatton Robin , Weaver Casey
Published: January 2023

IL-2 and IL-21 are pleiotropic cytokines that control key aspects of the immune response including proliferation, cell survival, and differentiation of multiple cell lineages. Despite their co-localization in the genome, there are both overlapping and divergent patterns of expression of these cytokines during T cell development and differentiation, suggesting both coordinate and distinct transcriptional control within the Il2-Il21 locus. Employing the assay for transposase-accessible chromatin using sequencing (ATAC-seq), we demonstrated that the Il2 promoter and distal regulatory elements spanning the Il2-Il21 locus are accessible as early as 30 min following TCR activation while chromatin accessibility of the Il21 promoter requires IL-6 stimulation. Further, CTCF recruitment and chromatin looping analyses defined a boundary element between the Il2 and Il21 genes. In addition, we identified several conserved non-coding elements in the Il2-Il21 intergenic region that have both repressor and enhancer activities on the Il2 or Il21 transcriptional activity. This study provides insight into the chromatin landscape of the Il2-Il21 locus and identifies regulatory elements responsible for the coordinate and differential expression of IL-2 and IL-21.
Regulation of the immunological synapse and T cell function by the c-terminal eps-15 homology domain-containing protein-dependent endocytic recycling. (IRM7P.711)
Authors: Iseka Fany , Goetz Benjamin , Cypher Luke , Bielecki Timothy , Storck Matthew , Band Vimla , Band Hamid
Published: January 2023

The immunological synapse (IS) is a specialized membrane domain with localized display of T-cell receptor (TCR) in contact with a similar display of peptide-loaded MHC on antigen presenting cells (APCs). Aberrant IS function is linked to ineffective tumor killing and autoimmunity. Accumulating evidence indicates a key role of endocytic traffic in the generation and maintenance of the IS. The C-terminal Eps-15 Homology Domain-containing proteins (EHD1-4) are essential regulators of endocytic recycling of surface receptors. Yet, nothing is known about their role in immune cell function. EHD-regulated receptors include the MHC-I and MHC-II proteins, and EHDs regulate vesicle budding at neural synapse. Our hypothesis is that EHD-dependent recycling of TCR and accessory receptors on T cells and MHC-II on APCs is required for the formation and stability of the IS. Indeed, using antigen-specific TCR transgenic CD4+ T-cells, we observe an accumulation of EHD proteins at the interface of T cells and APCs. CD4+ T-cells from floxed EHD1,3,4 mice bearing CD4-Cre and an antigen-specific TCR transgene demonstrate reduced antigen-driven proliferation. Further in vitro and in vivo analyses are being carried out to test the idea that endocytic recycling orchestrated by EHD proteins plays an important functional role in the formation and/or maintenance of IS and in T-cell function.
Reevaluating the biochemical valency of native TCR/CD3 complexes isolated from T cells (IRM7P.708)
Authors: Reed Brendan , Gil Pages Diana , Palmer Ed , Schrum Adam
Published: January 2023

The αβ T cell antigen Receptor (TCR) is a multi-protein complex expressed together with CD3 subunits (γδεζ). The conventional model has it as monovalent with a stochiometry of (αβγδε2ζ2); however, some previously published work reported observation of bivalent complexes with two αβ heterodimers. To reevaluate the valency of the TCR, we have subjected T cell digitonin-solubilized complexes to immunoprecipitation detected by flow cytometry (IP-FCM), size exclusion chromatography, and other methods for detecting complex size and subunit composition. Our results indicate that precisely bivalent TCRs are found among surface-expressed complexes independent of cross-linking by strong antigenic ligands. Their detection can be masked in the presence of Coomassie reagent from Blue-native PAGE conditions, or if antibodies used in detection assays are themselves bivalent. Therefore, bivalent TCR/CD3 complexes can be found under the biochemical conditions that have classically defined the minimally expressed TCR/CD3 unit (digitonin solubilization). Further investigation is required to determine the functional relevance of these bivalent TCR complexes, although greater antigenic binding and signaling is predicted over that of monovalent TCRs.
PLZF expression maps the early stages of ILC1 lineage development (LYM2P.720)
Authors: Constantinides Michael , Gudjonson Herman , McDonald Benjamin , Ishizuka Isabel , Verhoef Philip , Dinner Aaron , Bendelac Albert
Published: January 2023

Among the variety of tissue-resident NK-like populations recently distinguished from classical recirculating cNK cells, liver innate lymphoid cells type I (ILC1) have been shown to represent a distinct lineage that originates from a novel PLZF-expressing ILC precursor (ILCP) strictly committed to the ILC1, ILC2 and ILC3 lineages. Here, using PLZF-reporter mice and cell transfer assays, we studied the developmental progression of ILC1s and demonstrated substantial overlap with stages previously ascribed to the cNK lineage, including pre-pro-NK, pre-NK precursor (pre-NKP), refined NKP (rNKP) and immature NK (iNK). Although they originated from different precursors, the ILC1 and cNK lineages followed a parallel progression at early stages and diverged later at the iNK stage, with a striking predominance of ILC1s over cNKs early in ontogeny. While a limited set of ILC1 genes were dependent on PLZF for expression, characteristically including Il7r, most of these genes were also differentially expressed between ILC1s and cNKs, indicating that PLZF together with other, yet to be defined, factors contribute to the divergence between these lineages.
Homeostatic regulatory T cell trafficking in peripheral lymph nodes (LYM2P.731)
Authors: Tong Alexander , Allen Frederick , Huang Alex
Published: January 2023

Regulatory T cells (Tregs) express the canonical marker Foxp3 and are critical for suppressing the immune response in homeostatic and inflammatory conditions. As widely studied as CD4+Foxp3+ Tregs are in mice, their physiologic trafficking and scanning behavior of antigen-presenting cells in secondary lymphoid organs (SLOs) have yet to be quantitatively analyzed. Our laboratory has previously described the dynamics of naïve T cell trafficking through SLOs and scanning of dendritic cells, revealing that CD4+ T cells and CD8+ T cells have distinct dynamic behaviors in the lymph node (LN). On average, CD4+ T cells spend less time scanning for self-peptide/MHC (self-pMHC), dwell shorter in LNs, and egress faster compared to CD8+ T cells. In this study, we use flow cytometry and 2-photon laser scanning microscopy to quantitate the trafficking kinetics and surveillance strategy of naïve Tregs in C57BL/6 mice LNs and to compare their behavior to conventional T cells. Our data reveal differences in naïve Treg trafficking in mesenteric versus other peripheral LNs. We also show that self-pMHC recognition regulates the scanning behavior and localization of Tregs in peripheral LNs. In addition, we describe a novel imaging platform for high-resolution intravital microscopy of T cell behavior within mesenteric LNs. These results add to mounting evidence that self-pMHC interactions dynamically control physiologic Treg retainment and contact-dependent suppressive function in peripheral LNs.
Regulation of Foxo1 in T cell homeostasis (LYM2P.733)
Authors: Sullivan Jenna , Thomas Kerri , Campbell Daniel
Published: January 2023

The Foxo (forkhead box O) family of transcription factors has been shown to be important for maintaining homeostasis of both effector and regulatory T cells. Interesting Foxo1 is necessary for proper development and function of regulatory T cells (TR) and absence of Foxo1 results in fatal inflammation due to TR dysfunction. During normal conditions phosphorylation of Foxo1 by Akt results in inactivation of the transcription factor and translocation from the nucleus. We have bred mice that contain a floxed non-phosphorylatable Foxo1 allele under control of CD4-cre (CD4cre-Foxo1CA), resulting in constitutive activation of Foxo1 in both CD4 and CD8 T cells. Mice which are unable to properly regulate Foxo1 have reduced body weight, chronic inflammation and develop spontaneous autoimmunity. CD4cre-Foxo1CA displaying symptoms of autoimmunity have tissue damage in the lung along with increases in eosinophils, alveolar macrophages and mucus production. In the periphery CD4cre-Foxo1CA mice have reduced TR, increased activated effector T cells and an altered B cell compartment. Together this data demonstrates that proper regulation of Foxo1 is essential for T cell homeostasis and prevention of autoimmunity.
Constitutively active ezrin desensitizes IL-7 signaling and impairs T cell homeostasis (LYM2P.735)
Authors: Park Jung-Hyun , Luckey Megan , Liu Yin , Shaw Stephen
Published: January 2023

IL-7 signaling results in tethering of the IL-7 receptor to the actin cytoskeleton, which is mediated by the linker molecules ezrin and moesin. To understand the role of cytoskeleton linker molecules in cytokine signaling, we analyzed mice expressing a constitutively active ezrin transgene in T cells. The ezrin transgene was generated by introducing a point mutation at a conserved C-terminal Thr residue (567T->E) that results in a constitutively open conformation of ezrin. Interestingly, in mice expressing the constitutively active ezrin transgene (T567E-Tg), peripheral T cell numbers were significantly reduced and T cell survival was severely impaired. Moreover, T567E-Tg T cells were defect in IL-7 signaling even as they expressed high levels of surface IL-7 receptor proteins. Additionally, ezrin T567E-Tg induced downregulation of CD8 coreceptor expression, suggesting a “coreceptor tuning” effect of activated ezrin on CD8 T cells. Coreceptor tuning is a model of T cell homeostasis which posits that cytokine signals tailor CD8 coreceptor gene expression to the self-specificity of the TCR. In agreement, TCR disengagement restored IL-7 signaling in T567E-Tg CD8 T cells. Thus, constitutively active ezrin desensitizes IL-7 receptor signaling through increased TCR signaling but not through direct interference with the IL-7 receptor. The molecular mechanisms of ezrin activation on TCR signaling and T cell homeostasis will be discussed.
Metabolic damage and premature thymus aging caused by stromal catalase deficiency (LYM2P.722)
Authors: Griffith Ann , Venables Thomas , Shi Jianjun , Farr Andrew , Van Remmen Holly , Szweda Luke , Fallahi Mohammad , Rabinovitch Peter , Petrie Howard
Published: January 2023

T lymphocytes are essential mediators of immunity produced by the thymus in proportion to mass. The thymus atrophies rapidly with age, resulting in diminished new T cell production. Decreased thymic output is compensated by duplication of existing cells, but results in progressive dominance by memory T cells, and decreased ability to respond to new pathogens or vaccines. We find that accelerated thymic atrophy results from stromal deficiency in the reducing enzyme catalase, leading to increased damage by reactive oxygen species (ROS) generated during aerobic metabolism. Genetic complementation of catalase diminished atrophy, as did chemical antioxidants, providing a mechanistic link between antioxidants, metabolism, and normal immune function. Progenitor lymphoblasts represent the primary source of thymic ROS, likely acting on stromal cells in trans. We propose that thymic atrophy represents a conventional aging process that is accelerated by stromal catalase deficiency in the context of an intensely metabolic lymphoid environment.
Affinity for self drives the preferential accumulation of promiscuous CD4+ T cells over the lifespan. (LYM2P.726)
Authors: Deshpande Neha , Way Sing Sing , Kuhns Michael
Published: January 2023

T cells discriminate self from foreign peptides presented in the context of self-major histocompatibility complex (pMHC) molecules via clonotypic T cell receptors (TCRs). CD8+ T cell recognition and responsiveness to foreign pMHC is known to diminish over the lifespan, which is consistent with gradual thymic involution over time. How the affinity of the CD4+ T cell compartment for self-pMHC, and its capacity to bind foreign-pMHC change over the lifespan are basic aspects of T cell biology that remain largely unexplored. Experimentally restricting thymic selection is known to allow degenerate CD4+ T cells to develop. This suggests that they might accumulate in the CD4+ T cell compartment over time. We report that, while old mice (18-22 months) contain fewer CD4+ T cells than adults (8-12 weeks), those that remain have a higher intrinsic affinity for self-pMHC. Old mice also have more cells that bind distinct foreign-pMHCs, either alone or in combination. The numerical increase of these subsets with age directly correlates with their affinity for self-pMHC. However, no relationship was observed between affinity for self-pMHC and responsiveness to foreign-pMHC. These data demonstrate that the CD4+ T cell compartment preferentially accumulates promiscuous constituents with age as a consequence of higher affinity T cell receptor interactions with self-pMHC. These results have important implications for the design of immunotherapeutics targeting CD4+ T cells for long-term efficacy.
Immunoglobulin relative genes were up-regulated in the liver of cystic echinococcosis mouse (MPF2P.757)
Authors: Lv Guodong , Liu Hui , Li Juan , wen Hao , Lin Renyong
Published: January 2023

Cystic echinococcosis is a neglected parasitic disease all around the world, which due to the infection of Echinococcus granulosus larva in the intermediate host, such as human and sheep. The liver is the major battlefield of the host defends against the parasite, and many immune response and metabolism happened here. This article is aimed to study the gene expression of liver participates in this process, by use of C57 as the experimental animal model and microarray technology. The results showed that 77 genes were up-regulated and 17 genes were down-regulated in the liver adjacent to the parasite lesion. The bioinformatics analysis showed that the most up regulated genes were correlated with immune response process, signal transduction pathway and protein degradation, and down regulated genes were related to metabolism and cell cycle. Immunoglobulin related genes were predicted participation in many important pathways (Fc epsilon RI signaling pathway, primary immunodeficiency, allograft rejection). In addition, transform growth factor receptor 2 was found increased expression in the infected C57BL/6 liver tissue by microarray (2.29-fold) and RT-qPCR (21.4-fold). Quantitative RT-PCR analysis of a random selection of 18 genes confirmed the reliability of the microarray data. Our results provide a hint that immunoglobulin relative genes might participate the interplay between the host and parasite.
Targeting regulatory T cells in chronic inflammatory disease. (MPF2P.749)
Authors: Lieske Nora , Tonby Kristian , Kvale Dag , Dyrhol-Riise Anne , Taskén Kjetil
Published: January 2023

Human regulatory T cells (Tregs) are functionally characterized by their ability to suppress effector T cells in addition to having an essential role in maintaining immunological tolerance. Activated Tregs are upregulated in chronic inflammation and studies in human tuberculosis (TB) patients indicate that patients with active disease have increased frequency of Tregs in peripheral blood compared to patients with latent TB. Tregs also play a role in chronic viral infections. In HIV, Tregs control viral replication in early stages of the disease but limit immune activation and pathogen specific immune responses in later stages. The MAPK/ERK signaling pathway has been shown to be involved in the regulation of the FoxP3 transcription factor, the major regulator of Treg suppressive function. We aimed to target activation of disease specific Tregs by inhibition of the MEK signaling pathway based on the hypothesis that this would improve anti-HIV and anti-TB immunity. Upon stimulation of patient PBMCs with TB- and HIV-specific antigens in presence of MEK inhibitors, we observed significantly downregulated levels of FoxP3 in resting Tregs (CD45RA+FoxP3+) and a reduced number of activated Tregs (CD45RA-FoxP3++). Differential effects on production of IFNγ TNFα and IL-2 could be observed. In conclusion, MEK inhibitors modulate disease antigen-specific Treg activation which may have potential application in new treatment approaches where limiting Treg activity would be favourable.
Lung resident memory CD4 T cells promote chronic inflammation in allergic asthma (MUC1P.910)
Authors: Turner Damian , Goldklang Monica , Trischler Jordis , D’Armiento Jeanine , Farber Donna
Published: January 2023

Common household allergens, like infectious agents, are often encountered at epithelial barrier surfaces within the lung and can sometimes cause allergic asthma. Whereas lung resident memory T cells (TRM) protect against recurrent respiratory infection, allergen-induced CD4 TRM could have deleterious effects, initiating chronic inflammation and recurrent asthma symptoms. We sought to investigate whether lung CD4 TRM are generated in asthma and their role in the pathogenesis of allergic asthma. To this end, mice were inoculated intranasally with house dust mite (HDM) extract (a common household allergen) for three weeks to induce the characteristic symptoms of asthma. The sensitized mice were then rested for 4 weeks and the CD4 population within the lung analyzed. Chronic allergen exposure induces TRM-like CD4 T cells with the characteristic high expression of CD69. HDM-induced TRM are long-lived and persist for weeks in the lung parenchyma after cessation of HDM exposure. Lung CD4 TRM cluster around the bronchioles and are rapidly activated upon subsequent exposure to the allergen. This local activation of CD4 TRM was accompanied by activation of dendritic cells within the lung. These data suggest that early activation of allergen-induced lung CD4 TRM might be an initiating step in the cascade of events that are involved in recurrent asthma episodes.
Can human lung fibroblasts present antigen to autologous T cells? (MUC1P.911)
Authors: Hutton Andrew , Staples Dr Karl , Wilkinson Dr Tom , Warner Jane
Published: January 2023

HLF are a major structural cell in the lung, and their ability to influence CD4 T cells in an Ag-specific manner is unknown. Recurrent bacterial infections, a hallmark of COPD, may involve bacterial presence in the mesenchyme and HLF-Tcell interaction. We have investigated the effect of non-typeable H.Influenzae (NTHi) on HLF immune synapse molecule expression and their ability to stimulate T cells. Lung tissue from patients undergoing resection surgery was collagenase digested and CD45-CD90+ HLF were demonstrated to express HLA-DR at baseline. HLF outgrown from this tissue were cultured and stimulated with IFNγ and/or NTHi for 48h. IFNγ upregulated both ICAM-1 5.6-fold (P<0.001, N=23) and HLA-DR 16.2-fold (P<0.001, N=20). Exposure to NTHi increased ICAM-1 6.1-fold (P<0.001, N=6) but not HLA-DR expression. HLF also expressed low levels of CD40 which increased 2.2-fold upon NTHi stimulation, but no detectable CD80 or 86 (N=5). To assess Ag presentation, HLFs were co-cultured with NTHi-specific autologous T cells overnight and cytokine production measured by FACS. Results indicate HLF preconditioned with IFNγ+NTHi could stimulate autologous T cells raised against NTHi Ags to produce IFNγ or IL-17A. Blocking experiments showed this stimulation to be reliant upon fibroblast HLA-DR expression. In conclusion, NTHi can cause HLF to present antigen to and activate autologous T cells. HLF-activated T cells may cause the lung damage associated with bacterial infections in COPD.
Microarray analysis of epithelial-to-mesenchymal transition genes suggests differential epithelial remodeling in human disease vs. mouse models of asthma (MUC1P.912)
Authors: Loffredo Lucas , Berdnikovs Sergejs
Published: January 2023

Epithelial-mesenchymal transition (EMT) is the process by which epithelial cells revert to a less differentiated, mesenchymal phenotype; it occurs in maintenance of tissue homeostasis and is exaggerated in disease. EMT has been implicated in human asthma, although it is not clear whether this process is caused by inflammation or rather contributes to it. In allergen-induced murine models of asthma, EMT would be directly caused by inflammation; however, evidence for EMT in such models is lacking. To assess weight-of-evidence and causality for EMT, we contrasted gene expression between mouse models and human asthma samples, using data from 12 gene expression microarrays (GEO, NCBI). PCA analysis surprisingly discriminated human and mouse studies based on behavior of 8 classical EMT genes and 25 epithelial differentiation genes. Increased expression of Cdh2, Twist1, Twist2, Snai2 and Zeb1 supported existence of EMT in human asthma. Interestingly, the same genes did not change or decreased their expression in mouse models. Moreover, inducers of EMT Sox4, Fgf18 and Fgfr4 decreased and suppressor of EMT Runx3 increased expression in mice, consistent with mesenchymal-epithelial transition associated with repair. The fact that even chronic mouse models failed to recapitulate the EMT pattern seen in patients suggests that allergic inflammation alone is not sufficient to trigger persistent epithelial de-differentiation, implicating a missing component in the causation of EMT in asthma.