bluuebunny/crossref_metadata_2025 huggingface dataset:
19F-MRI for tracking NK Cells after adoptive transfer (CAM1P.240)
Authors: Bouchlaka Myriam , Gordon Jeremy , Ludwig Kai , Niles David , Bednarz Bryan , Fain Sean , Capitini Christian
Published: January 2023

Natural killer (NK) cells have shown anti-tumor responses against solid tumors. However, it is not known if NK cells traffic directly to tumors and mediate their cytotoxicity, or if they indirectly mediate their anti-tumor effects by stimulating other effector cells. Current methods of tracking immune cells by imaging of 1H contrast agents, such as iron oxide particles, are not ideal as a large part of the 1H signal comes from water in the tissues. Because there are trace amounts of fluorine in the body, we have optimized the non-radioactive isotope 19-fluorine (19F) as a means of tracking human NK cells by magnetic resonance imaging (MRI). In vitro, we show that NK cell viability, their ability to secrete granzyme B or IFN-γ and cytotoxicity against K562 leukemia cells are not affected by 19F labeling and are similar to unlabeled cells. There was also no impact on expression of natural cytotoxicity receptors, NKG2D or DNAM-1. MR Spectroscopy has shown that NK cells highly take up 19F, with up to 10^12 atoms/cell. In vivo, 19F-labeled NK cells injected in the footpad of NOD-SCID-gamma (c)-/- mice were nontoxic and detectable by MRI. Overall, perfluorcarbon 19F labeling of NK cells represents a successful method to track and quantify the number of apparent cells in a region of interest without altering their cytotoxic function. Importantly, 19F imaging will enable us to monitor NK cells after their infusion allowing us to determine how they induce their anti-tumor effects.
Regulation of integrin LFA-1 (CD11a/CD18) during T cell migration and activation (CAM1P.227)
Authors: Capece Tara , Abid Anna , Kim Minsoo
Published: January 2023

T cell entry into a lymph node is dependent upon several events, including activation of chemokine receptor CCR7 and adhesion of integrin LFA1. T cells become highly motile to scan dendritic cells (DCs) for cognate antigen (Ag), utilizing active LFA1 at the leading edge. Ag challenge induces stable T-DC interactions and immunological synapse (IS) formation, where active LFA1 relocates. LFA1 is an adhesive force in migration and IS formation, but the outcome of chemokine and T cell receptor (TCR) signals are different, as the former induce “go” and the latter mediate “stop” signals. We hypothesize the magnitude of chemokine and TCR signals will determine the outcome of LFA1 mediated interactions and thus T cell activation. We generated fluorescent knock-in (KI) mice to visualize LFA1 distribution and activation patterns. We mated these KI mice with OTI TCR transgenic mice to study LFA1 distribution in T cells activated by varying TCR signal strength with altered peptide ligands (APL). We have thus far shown LFA-1 clusters faster and remains clustered longer at the contact site in interactions with cognate Ag compared to those with weaker APLs. We are repeating these experiments in our LFA1 FRET mice, which allow us to visualize the LFA1 activation, to determine if LFA1 activation induces the LFA1 clustering seen in interactions with cognate Ag. We have also incorporated a photoactivatable chemokine receptor, PA-CCR7, to assess the effect of chemokine signal strength on LFA1.
Essential role of elmo1 in dock2-dependent lymphocyte migration (CAM1P.228)
Authors: de la Rosa Gonzalo , Stevenson Catherine , Anderson Christopher , Murphy Patrick , Capece Tara , Kim Minsoo , Elliott Michael
Published: January 2023

Elmo1 and Elmo2 are highly homologous cytoplasmic adapter proteins that interact with Dock family guanine nucleotide exchange factors to promote activation of the small GTPase Rac. In T lymphocytes, Dock2 is essential for CCR7- and CXCR4-dependent Rac activation and chemotaxis, but the role of Elmo proteins in regulating Dock2 function in primary T cells is not known. Here we show that endogenous Elmo1 but not Elmo2 interacts constitutively with Dock2 in mouse and human primary T cells. CD4+ T cells from Elmo1-/- mice were profoundly impaired in polarization, Rac activation and chemotaxis in response to CCR7 and CXCR4 stimulation. Transfection of full-length Elmo1, but not Elmo2 or a Dock2-binding mutant of Elmo1, rescued defective migration of Elmo1-/- T cells. Lymphocytes from Elmo1-/- mice showed a four-fold reduction in total Dock2 protein levels despite normal levels of Dock2 mRNA, suggesting Elmo1 is critical for the maintenance of Dock2 levels in primary lymphocytes. Indeed, Dock2 polyubiquitination was increased in Elmo1-/- T cells, and treatment with proteasome inhibitors partially restored Dock2 levels in Elmo1-/- T cells. Taken together, these findings reveal a previously unknown, non-redundant role for Elmo1 in controlling Dock2 levels and Dock2-dependent T cell migration. This work provides valuable insights into the molecular regulation of Dock2 by Elmo1 that can be used to design improved Dock2-based therapeutic approaches.
CD103 downregulation marks memory-phenotype CD8 T cells and excludes them from the gut (CAM1P.231)
Authors: Ligons Davinna , Sato Noriko , Park Jung-Hyun
Published: January 2023

CD103 is a cell surface integrin whose primary role is the retention of lymphocytes in epithelial tissues where its ligand E-cadherin is expressed. CD103 is found on all newly generated CD8 single positive thymocytes but not on CD4 lineage cells. Because of such lineage specific expression, CD103 is currently considered a lineage specific marker whose expression is proposed to be controlled by Runx3, a runt-family transcription factor involved in CD8 lineage specification. Here we report the surprising finding that Runx3 was neither necessary nor sufficient for CD103 expression on CD8 T cells. Notably, we identified a subpopulation of CD8 T cells that had downregulated CD103 expression, despite expressing high levels of Runx3. Upon correlating CD103 expression with activation and memory markers, we found that CD103-negative CD8 T cells corresponded to memory phenotype cells and that homeostatic cytokines signals terminated CD103 expression on CD8 T cells. Importantly, CD103-negative CD8 T cells were found in every lymphoid organ and other peripheral tissues with the exception of the gut. Indeed, all CD8+ IELs were CD103-positive, and adoptive transfer of CD103-negative CD8 T cells resulted in their recovery from the LN but not the intestinal mucosal tissues. Collectively, these results suggest that CD8 memory-phenotype acquisition is accompanied by CD103 downregulation to control their tissue distribution and to exclude them from the gut.
The development and survival but not function of follicular B cells is dependent on IL-7Rα Tyr449 signaling. (CCR1P.242)
Authors: Abraham Ninan , Patton Daniel , Plumb Adam , Redpath Stephen , Osborne Lisa , Perona-Wright Georgia
Published: January 2023

IL-7 is a critical cytokine for lymphocyte development but its role in B cells is less well characterized. Using a knock-in mouse with a Tyr to Phe mutation at position 449 (IL-7Rα449F/449F mice) within the cytoplasmic domain of IL-7Rα, we evaluated the role of this YxxM motif in spleen B cells. IL-7Rα449F/449F mice had reduced numbers and increased death of follicular B cells compared to WT, but had significantly more follicular cells than IL-7Rα-/-. The death of IL-7Rα449F/449F follicular cells was not due to a failure to respond to BAFF or lower levels of BAFF. Marginal zone B cells were unaffected in IL-7Rα449F/449F mice. A role for TSLP was ruled out, as TSLPR-/- mice had an identical B cell phenotype to wild-type mice. Bone marrow chimeras and the absence of IL-7Rα on B cells suggested that IL-7 did not directly regulate mature B cells, but that an IL-7-responsive cell was influencing B cells. IL-7 was also critical at the checkpoint between the T1 and T2 stages in the spleen. We tested the functional responses of IL-7Rα449F/449F mice and found no difference in antibody responses to T-dependent or T-independent antigens, or to Influenza/A. IL-7 was important for generation of antibody responses to the intestinal worm H. polygyrus and for naive levels of IgA. Our data shows that IL-7 regulates follicular B cell numbers and survival in a cell-extrinsic manner, via a bone-marrow derived cell, but is not critical for antibody production outside the gut.
CCR2-dependent dendritic cell recruitment to CNS tissues during EAE promotes disease progression and inflammatory cytokine production by CNS T cells (CCR1P.243)
Authors: Clarkson Ben , Rayasam Aditya , Walker Alec , Harris Melissa , Sandor Matyas , Fabry Zsuzsanna
Published: January 2023

Dendritic cells (DCs)—while absent from the healthy central nervous system (CNS)—rapidly accumulate within brain and spinal cord tissue during neuroinflammation associated with experimental autoimmune encephalomyelitis (EAE, a mouse model of multiple sclerosis). Yet, while DCs have been appreciated for their role in initiating adaptive immune responses in peripheral lymphoid organ tissues, how DCs infiltrate the CNS and contribute to ongoing neuroinflammation in situ is poorly understood. Therefore we sought to identify key chemokines and chemokine receptors on DCs that govern DC migration across the blood-brain barrier and within the CNS. Here we report that 1) CD11c+ bone marrow-derived (BM)DCs and CNS-infiltrating DCs express CCR2, 2) compared to CCR2+/+ cells, adoptively transferred CCR2-/- BMDCs or DC precursors do not accumulate in the CNS during EAE, despite abundance in blood, 3) CCR2-/- DCs show less accumulation in the inflamed CNS in mixed bone marrow chimeras, when compared to CCR2+/+ DCs, and 4) ablation of CCR2+/+ DCs during EAE clinical onset delays progression and attenuates T cell cytokine production. These data suggest that CCR2-dependent DC recruitment to the CNS during ongoing neuroinflammation plays a crucial role in effector T cell cytokine production and disease progression, and signify that CNS-DCs and circulating DC precursors might be key therapeutic targets for modifying ongoing neuroinflammation in CNS autoimmune diseases.
Demonstrating the immune response of D. rerio using keyhole limpet hemocyanin (EDU1P.249)
Authors: Young Matthew , Criscitiello Michael
Published: January 2023

In this project, immunization of a model aquatic organism is used in the classroom to facilitate understanding of molecular biology, evolution and the vertebrate adaptive immune system. Molecular immunoglobulin and T cell receptor cloning, sequencing and repertoire analysis techniques that I learned in the Criscitiello Comparative Immunogenetics Lab at Texas A&M this summer were applied to analysis of the lymphocyte antigen receptor repertoire in zebrafish. Students at A&M Consolidated High School performed experiments to quantify and qualify various aspects of the immune response of the model organism, D. rerio. Hemocytometry was used to measure leukocyte counts before and after immunization with keyhole limpet hemocyanin. Serum IgM was analyzed using ELISA. Repertoire analysis was performed using PCR and DNA sequencing. The data from these experiments was used in the classroom to discuss the evolution of vertebrate adaptive immunity, immunization techniques, and vaccination design. Furthermore, the repertoire sequence data contributed to an important phylogenetic perspective for ongoing NSF funded studies at A&M on the plasticity of B and T cell receptor immunogenetics. One goal is for high school students to earn co-authorship on journal publications from the Criscitiello lab. This was the first stage in an iterative system of teaching and research that will continue to include reciprocal pedagogical exchange and hands-on experience in comparative molecular immunology.
Integrating immunology into a clinical presentation-based medical school curriculum: lessons learned (EDU1P.251)
Authors: Piskurich Janet , Baatar Dolgor
Published: January 2023

Clearly there is no one best way to study the complex adaptive system that comprises our ability to respond to a wide variety of microorganisms, many of which have developed medically relevant means to evade the immune system. As a teacher or learning facilitator it can be difficult to even decide where to start. Different text books adopt a variety of approaches. A further problem unique to the educational process of medical students is how to provide a relevant framework to enhance their retention of the concepts of basic immunology and their ability to apply these concepts later as they begin to practice medicine. Paul L. Foster School of Medicine, a new medical school located on the Texas Mexico border, utilizes a clinical presentation-based curriculum model. This model has also been recently adopted by several other new medical schools across the country. In this model, students learn clinically-relevant basic science in the context of common patient presentations. Pros and cons of the integration of the basic science of immunology into a clinical presentation-based curriculum as well as lessons learned over the first five years will be addressed.
Pre-clinical studies of inflammation: automating the most common methods of animal randomization and distribution (EDU1P.254)
Authors: Ibsen Eric , Kumer Jeff
Published: January 2023

To more closely emulate clinical research methodology and improve clinical relevance, researchers are increasingly utilizing more clinically-similar group randomization and distribution methods for Inflammation studies in animals. A common pre-clinical approach is paradoxically the least clinically-relevant; a random number generator in a spreadsheet, which is subject to manual and transcription errors and provides no easy way to exclude animals from distribution based on other parameters. Pre-clinical trial software is available which automates the most recommended randomization and distribution methods based on tumor volume or other parameters: deterministic and pair-matched distribution, pure randomization, stratified sampling randomization and randomization across multiple numeric parameters. Subjects may be excluded initially based on recorded clinical observations and comments and then by acceptable value ranges for any numeric parameter. Animals can be randomized iteratively in a rolling or staggered way and can be re-randomized into new groups as required on drug-resistance studies. Proposed randomization results by ANOVA can be displayed instantly to ensure that the group means are acceptably similar. Standardizing and automating methods of randomization and distribution which are similar to this used clinically can potentially improve the ease, clinical relevance, quality and outcome of pre-clinical Inflammation studies relative to spreadsheet-based methods.
CXCR4 antagonist AMD3100 mobilizes leukocytes from bone marrow and thymus to blood in mice (HEM2P.265)
Authors: Liu Qian , Li Zhanzhuo , Gao Ji-Liang , Wan Wuzhou , McDermott David , Murphy Philip
Published: January 2023

AMD3100 is approved for mobilizing hematopoietic stem cells to blood for transplantation in cancer by blocking chemokine receptor CXCR4. It also mobilizes mature leukocytes to blood; however, there is conflicting published evidence regarding the source of mobilized neutrophils (bone marrow vs lung), and the source(s) of other mobilized leukocytes is unknown. Here we used surgical, pharmacologic and histologic approaches to address these questions in mice. AMD3100 10 mg/kg sq mobilized the same leukocyte subsets to blood in mice as in humans, and increased neutrophil and monocyte content in lung and lymph node, while reducing neutrophil, lymphocyte and monocyte content in bone marrow as well as T cell content in thymus. Direct intrathymic labeling showed that AMD3100 mobilized naïve CD4+ and CD8+ T cells to blood. In contrast, splenectomy and experiments with FTY720, which blocks T cell egress from lymph node, suggested that spleen and lymph node were not major sources of AMD3100-mobilized cells. Importantly, AMD3100 did not inhibit neutrophil trafficking to thioglycollate-inflamed peritoneum. These data provide evidence that CXCR4 is a selective compartmentalization factor for both bone marrow (lymphocyte, monocyte, neutrophil) and thymus (naïve T cells), but is not critical for homeostatic trafficking of leukocytes to uninflamed tissues or for innate immune responses to inflamed sites, a profile that is favorable for chronic administration of AMD3100 in patients.
Initiation of splenic white pulp ontogeny (HEM3P.289)
Authors: Neely Harold , Flajnik Martin
Published: January 2023

Secondary lymphoid organs (SLO) provide the structural framework for the co-concentration of antigen and lymphocytes required for an efficient adaptive immune response. The spleen is the primordial SLO that evolved coincidentally with Ig-/TCR:pMHC-based adaptive immunity. B cells are indispensable for the formation and maintenance of SLO microarchitecture; their expression of lymphotoxin α1β2 (LTα1β2) is required for the LTα1β2:CXCL13 positive feedback loop without which SLO cannot properly form. Despite the spleen’s central role in the evolution of adaptive immunity, neither the initiating event nor the B cell subset compulsory for white pulp (WP) formation has been identified. We therefore sought to identify these initiating components of splenic WP ontogeny. We detected CXCL13 but not LTα1β2 protein expression in the vasculature of late embryonic (E17.5 and beyond) spleen, and its expression was independent of rearranging lymphocytes and TNFα. A substantial influx of transitional B cells occurred within 18 hours of birth (E18.5), but these cells were unresponsive to CXCL13 and do not establish a perivascular splenic niche. At P0.5, B cells acquired CXCL13 responsiveness and aggregated around the splenic vasculature, and CXCL13-responsive B cells were enriched for transitional 2 B cells. Thus, CXCL13 is the initiating component of the CXCL13:LTα1β2 positive feedback loop required for WP ontogeny, and CXCL13-responsive transitional B cells are the initiating B cell subset.
Normal stem and progenitor cell sociology within the leukemic microenvironment (HEM3P.300)
Authors: Welner Robert , Amabile Giovanni , Bararia Deepak , Czibere Akos , Yang Henry , Tenen Daniel
Published: January 2023

Specialized bone marrow (BM) microenvironment niches are crucial for hematopoietic stem and progenitor cell (HSC/HPC) maintenance. We are just starting to learn how the integrity of the niche changes with leukemia, however, the impact on normal HSC/HPC behavior and functionality is yet to be addressed. Therefore, we started by studying the kinetics and differentiation of normal HSC/HPC in mice with Chronic Myeloid Leukemia (CML). Although normal hematopoiesis was increasingly suppressed during the disease progression, the leukemic environment imposed distinct effects on HPC predisposing them toward the myeloid lineage, similar to that of the leukemic population. Meanwhile, the leukemic-exposed normal HSC remained functional on transplantation. Analysis of the microenvironment identified several cytokines were dysregulated in the leukemia, and we found IL-6 to be responsible for many of these bystander responses. These results were similarly validated using BM obtained from CML patient samples. Co-culture of CML BM and human CD34+ HPC resulted in selective proliferation and altered differentiation of the normal primitive progenitors compared to mixed cultures using normal BM. Therefore, our results show how hematologic malignancy can influence the bone marrow, in turn causing disturbances in normal blood cell progenitors. Knowledge of such bystander effects could suggest new therapeutic interventions for cancer prevention and novel therapeutic approach for leukemia patients.
Vimentin is a dominant target of in situ humoral immunity in human lupus tubulointerstitial nephritis (HUM1P.314)
Authors: Clark Marcus , Chang Anthony , Ko Kichul , Liarski Vladimir , Kinloch Andrew
Published: January 2023

In lupus nephritis (LuN), severity of tubulointerstitial inflammation (TII) on renal biopsy predicts subsequent renal failure. In severe TII, infiltrating B and T cells have features of tertiary lymphoid neogenesis including in situ antigen-driven clonal B cell selection. To identify the autoantigen(s) driving selection, we laser captured single CD38+ or Ki-67+ B cell mRNA from seven LuN biopsies. Twenty clonally expanded immunoglobulin heavy and light chain variable region pairs were cloned and expressed as antibodies. Seven more were obtained from an eighth biopsy by flow sorting of CD38+ cells. Immunofluorescence indicated that 13 of 27 antibodies had immunoreactivity with cytoplasmic structures. Only 4 reacted with nuclei and none with dsDNA, Sm or RNP. Interrogation of protein arrays with TII antibodies and immunoprecipitations followed by mass spectroscopy identified a limited number of self-antigens with only vimentin common to both approaches. Eleven of 13 cytoplasmic TII antibodies directly bound vimentin, vimentin was highly expressed by tubulointerstitial infiltrating cells and tested TII antibodies preferentially bound inflamed tubulointerstitum. Finally, high-titers of serum anti-vimentin antibodies were strongly associated with severe TII. These finding suggest that in situ adaptive immunity to vimentin, an antigenic feature of inflammation, feeds forward to worsen TII and renal damage.
Peripheral blood (PB) monocytes from subjects with autism spectrum disorders (ASD) and fluctuating behavioral symptoms following immune insults reveal similar innate immune responses to those observed in pediatric acute onset neuropsychiatric disorders (PANS). (HUM1P.325)
Authors: Jyonouchi Harumi , Geng Lee , Davidow Amy
Published: January 2023

ASD is a behaviorally defined syndrome and encompasses markedly heterogeneous subjects. Some ASD subjects reveal markedly fluctuating behavioral symptoms following immune insults and persistent gastrointestinal (GI) symptoms; categorized as the ASD-inflammatory subtype (ASD-IS) (N=20) in this study. Cyotkine production by purified PB monocytes from ASD-IS subjects in response to TLR2/6, TLR4, TLR7/8, and Dectin 1 agonists were assessed in comparison with cells from control ASD subjects with and without non-IgE mediated food allergy (NFA) (ASD/NFA N=16, ASD/non-NFA children; N=18), non-ASD controls (non-ASD/NFA; N=13, normal controls; N=12, non-ASD/PANS N=18). ASD-IS subjects had no siblings diagnosed with ASD and ASD-IS children are noted to have a higher frequency of recurrent sinopulmonary infection than ASD/non-ASD controls; 8/20 ASD-IS children were diagnosed with antibody deficiency syndrome (ADS). ASD-IS PB monocytes produced higher levels of IL-1β, IL-6, and IL-12 with or without stimuli. In addition, decrease in production of IL-10 and sTNFRII with TLR2/6 and TLR7/8 agonists was more striking than normal control monocytes. Monocytes from ASD/NFA, ASD/non-NFA, or non-ASD/NFA subjects did not reveal such tendencies. However, cytokine production by PANS monocytes revealed similar tendencies. Aberrant innate immune responses detected in PB monocytes may be associated with markedly fluctuating neuropsychiatric symptoms observed in ASD-IS and PANS subjects.
The role of CCL5 in T cell recruitment to the aorta (HUM1P.303)
Authors: LI Jie , Ley Klaus
Published: January 2023

T cell adaptive immunity is involved in the pathogenesis of atherosclerosis. Previously our lab found that APCs present antigens to effector T cells (Teff). This causes local T cell activation and cytokine production, which sustain local inflammation. However, the chemokine(s) that drive T cell homing to the atherosclerotic aorta are not known. By multi-color flow cytometry to analyze chemokine receptor expression on T cells from Apoe-/- aorta, we found that more than 40% of CD4 T cells in Apoe-/- aorta express CCR5. Notably, >80% of the CCR5+ T cells in the plaques are IFN-γ+Foxp3+ Tregs. This is a unique CD4 T cell subset in atherosclerosis. They are found only in the aorta and para-aortic lymph nodes of Apoe-/- mice but not in the spleen or other organs, suggesting that the generation of the Th1-like Tregs is associated with local antigen presentation. To test my hypothesis that CCR5 and its ligand CCL5 play an important role in Teff recruitment to the aorta, Teff cells from CD45.1Apoe-/- mice were incubated with explanted Apoe-/- aortas with media alone, CCL5 or CCR5 neutralizing antibody. The CCL5 and CCR5 neutralizing antibody significantly decreased the number of T cells infiltrating into the aorta (53% and 70% respectively vs. media alone). The result is also confirmed by in vivo adoptive transfer experiment using leukocytes from CCR5-/-Apoe-/-mice. CCR5-deficient CD4 T cells displayed dramatic decrease in homing to the aorta compared to the CCR5 competent T cells.
TREML4 expression by myeloid cells may play a role in coronary artery disease (HUM1P.308)
Authors: Boelte Kimberly , Sen Shurjo , Long Priel Debra , Lau Karen , Cardone Marco , Yoshimura Teizo , Barb Jennifer , Joehanes Roby , Accame David , Teer Jamie , Singh Larry , Adams Lila , Zhang XiaoQing , Chowdhury Soma , Johnson Andrew , Green Eric , Mullikin James , Kolodgie Frank , Virmani Renu , O'Donnell Christopher , Munson Peter , Trinchieri Giorgio , Kuhns Douglas , Biesecker Leslie , McVicar Daniel
Published: January 2023

Triggering Receptor Expressed on Myeloid cells (TREM) family receptors modulate responses of a variety of leukocytes. We found that TREM-like 4 (TREML4) mRNA is elevated in the peripheral blood of patients with advanced coronary artery calcification (CAC) and that a single nucleotide polymorphism (SNP), rs2803496, tracks with the level of TREML4 mRNA in these patients. In an effort to elucidate the mechanism through which TREML4 in peripheral blood could affect CAC, we began by determining what cells express it. Monocytes and lymphocytes of donors with this SNP expressed very low levels of TREML4 mRNA whereas their neutrophils (PMNs) were strongly positive. Consistent with its association with CAC, we found variation in the levels of TREML4 mRNA in PMNs that correlated rs2803496, and a second SNP rs2803495. Unlike murine Treml4, which has the typical TREM family structure, human TREML4 has a noncanonical leader, lacks a portion of the transmembrane domain (TM) and has no cytoplasmic tail. Characterization of the Treml4 cDNA suggests that this truncated TM domain is sufficient to anchor the protein in the plasma membrane but that the TREML4 native leader sequence is ineffective. Lastly, immunostaining of human atherosclerotic plaques shows TREML4 in macrophage-rich regions, as well as on the endothelium. Together our findings suggest that TREML4 may play an important role in atherosclerosis and CAC but that it is unlikely to function as a typical, DAP12-coupled TREM.
A defect of differentiation of peripheral blood monocytes into dendritic cells after cardiopulmonary bypass is delayed by 7 days after the initial insult (HUM1P.309)
Authors: Laudanski Krzysztof , Worthen G. Scott
Published: January 2023

Monocytes (MO) can uniquely differentiate in a variety of the offspring cells including dendritic cells (DC). After severe immune insult, like sepsis, ability of MO to differentiate to DC (MO->DC) is significantly diminished immediately after insult and is related to the acquisition of a predominantly macrophage like characteristic of MO thus leaving only few available precursory MO for MO->DC process. Here, we enrolled patients scheduled for non-emergent heart surgery and assessed leukocyte function before surgery (t0), 24hrs (t24h) and 7 days (t7d) after surgery. We hypothesized that immediately after surgery, MO will acquire a persistent macrophage characteristic with subsequent MO->DC defect. MO were isolated and subjected to a DC differentiation&maturation conditions including 5 days in vitro cultures in X-VIVO15 enriched with IL-4 & GM-CSF. The process of MO->DC was significantly diminished in patients but only at the day 7 after the CPB as shown by diminished expression CD83 (%CD83+ [X±SD] t0=24.5±33.81 vs t24hr=8.84±10.71 [p=ns] vs t7d=0.2±0.4 [p<0.05]) or uptake of DQ ovalbumin. Concomitantly, the number of precursory CD11c+ was diminished but only at 7 days samples (%CD11c+ [X±SD] t0=81.5±15.81 vs t24hr=83.3±15.82 [p=ns] vs t7d=61.9±18.32 [p<0.05]). We conclude that surprisingly CPB diminished number of circulating precursory DC but only after 7 days from insult affecting ability of these precursory cells to convert into DC.
Regulation of microRNA expression in the lungs by exposure to air pollution & antigen (HUM2P.340)
Authors: Lucas Benjamin , Chen Wen-Chi , Bleck Bertram , Reibman Joan , Rom William , Narayanan Narayanan , Qu Qingshan , Park Sung-Hyun , Grunig Gabriele
Published: January 2023

Pulmonary Hypertension is characterized by pulmonary arterial remodeling and increased pressure in the pulmonary circulation. It is often associated with inflammation in the lungs and can lead to right heart failure. Our work shows that urban ambient pollution exacerbates the experimental pulmonary hypertension phenotype just like other types of inflammatory lung conditions. We aimed to identify microRNAs (miRNAs) that are differentially expressed in our mouse model. In addition, we examined plasma samples from individuals occupationally exposed to high levels of air pollution or cigarette smoke, and from controls. Our study is the first to show significantly de-regulated expression of three microRNA species (miR-135a, miR-21, miR-204) in the lungs of mice that were exposed to antigen and particulate matter and developed pulmonary hypertension. De-regulated levels of miR-21 and miR-204 have been reported in human pulmonary hypertension and in experimental pulmonary hypertension. MiR-135a is targeting STAT6 and upregulated expression has been reported in experimental asthma. Using human samples, our study showed that plasma levels of miR-21 and miR-135a, but not levels of miR-204, clustered individuals with high dose exposures and individuals with low dose environmental exposures. Current studies are aimed at identifying the cytokines that control these miRNAs’ expression. The long range goal is to identify miRNAs that indicate an at-risk state of the pulmonary vasculature.
Protoarray analysis reveals novel autoantigens targeted by autoantibodies associated with DNA-repair pathway in systemic erythematosus lupus (HUM2P.330)
Authors: Li Quanzhen , Zhou Jinchun , Wu Tianfu , Mohan Chandra
Published: January 2023

Systemic erythematosus lupus (SLE) is an autoimmune disease characterized by presence of autoantibodies (autoAbs) against a broad spectrum of self-antigens. In order to identify novel autoAbs associated with SLE, we utilized a Protoarray bearing 9,500 antigens to screen IgG and IgM autoAbs in sera of SLE patients. 446 IgG and 1218 IgM autoAbs were identified to be significantly elevated in SLE patients compared with healthy controls (p<0.05). Protoarray revealed not only the previously described autoAbs such as antibodies against dsDNA, SSA/SSB, Histone and Sm/RNP, but also uncovered autoAbs against a broad range of novel antigens including 151 nuclear-associated antigens and 150 cytoplasma or membrane-associated antigens. The Protoarray results were further validated by ELISA in a larger cohort of SLE patients and controls. Besides, 65 of the IgG autoAb-targeted proteins were also disregulated in SLE on mRNA level by microarray analysis. Pathway analysis recognized significant enrichment of antigens involved in cell proliferation and DNA repair pathways which were targeted by IgG autoAbs including APEX1, AURKA, CSNK1G1, EIF2C1, HMGB1, IFIT5, MAPKAPK3, PADI4, PRKRA, RALGPS1, UBE2S and VRK1. The elevated IgG autoAbs to APEX1 and other proteins in the pathway may reflect the compromised DNA-repair activity during DNA replication in SLE. In conclusion, identification of novel autoAbs by protoarray may shed light on some of the pathogenic pathways leading to disease in SLE.
Genetic risk alleles associated with autoantibody production and responsible for initial breach in tolerance to self-antigens in normal individuals (HUM2P.331)
Authors: Li Quanzhen , Raj Prithvi , Raman Indu , Yan Mei , Lian Yun , Zhang Bo , Wakeland Edward
Published: January 2023

Antinuclear antibodies (ANA) are serological hallmarks of SLE. Studies have shown that healthy individuals also develop ANA, raising questions on relevance of ANA positivity and autoimmune disease. We screened 2223 healthy controls (HC) and 143 SLE patients for ANA by ELISA and found that 26% HC are ANA positive, with 17% moderate (20-40 AU) and 9% high (>40 AU) ANA levels. To elucidate the autoantibody profiles associated with ANA in HC and SLE, we performed autoantigen arrays to profile over 90 autoAbs and found that ANA positive HC make significant amount of IgG and IgM autoAbs against many antigens with high frequency to non-nuclear antigens such as collagens and phospholipids. We noticed stronger autoAb signatures in females than males. We then genotyped 1583 HC with high and low ANA by Immunochip and did quantitative association test using ANA as dependent variable. Seven genomic loci showed suggestive genomewide association (p=1.00E-05) with ANA. Multiple independent association signals were observed in HLA class I, II and III region and the strongest association mapped to HLA-DR alleles where peak SNP rs9268832 in HLA-DRA gene reached p value 7.63606E-07. The impact of ANA risk alleles on HLA-DR gene expression was studied in B cells and macrophages from healthy donors by RNA-seq and results show that ANA risk haplotype is associated with up regulation of HLA-DRB1 gene which could be associated with initial breach in tolerance to self-antigens in general population.
MS4A4 regulates KIT receptor trafficking, signaling and function in mast cells (HYP3P.343)
Authors: Cruse Glenn , Music Stephen , Bradding Peter , Beaven Michael , Metcalfe Dean
Published: January 2023

Expression of MS4A family proteins influence the cell cycle and may be involved in tumorigenesis. However, the mechanistic basis for these observations remains elusive. We have identified roles for a member of the MS4A family, FcεRIβ (MS4A2), in protein trafficking and signal transduction in human mast cells (Immunity, 38, p906, 2013) and predicted analogous roles for other MS4A proteins. Employing mast cells, we found that down-regulation of MS4A4 reduced KIT expression (the receptor for SCF), and contributed to effector functions. MS4A4 “stabilized” surface KIT by slowing KIT internalization kinetics in response to SCF. Our data suggest that the mechanism for this action may involve trafficking of KIT by MS4A4 from sorting early endosomes to the endocytic recycling center, diverting receptors away from late endosome-dependent ERK kinase signaling and receptor degradation. Thus, in this instance, gene-silencing of MS4A4 reduced recycling pathways resulting in enlarged EEA1+ early endosomes, prolonged ERK signaling, and increased KIT progression to Rab9+ late endosomes. Therefore, MS4A4 appears to promote mast cell maturation by altering the endocytic trafficking and localization of KIT signaling, fine tuning effector functions. Our proposed function of MS4A4 in KIT trafficking is similar to the role that FcεRIβ plays in FcεRI trafficking and raises the question of whether other MS4A gene family members similarly regulate trafficking of receptors and associated molecules.
Complete suppression of IgE-mediated anaphylaxis by antihistamine plus tyrosine kinase antagonists. (HYP3P.400)
Authors: Morris Suzanne , Perkins Charles , Khodoun Marat , Hogan Simon P , Finkelman Fred
Published: January 2023

IgE/FcεRI-mediated mast cell and basophil activation has a central role in food allergy and drug allergy. Although desensitization therapy can suppress these disorders, it can be complicated by anaphylaxis. Pre-treatment with antihistamines and other mediator antagonists can increase the safety of desensitization, but is not completely effective. Because the tyrosine kinase Syk has an important role in FcεRI-dependent mast cell activation and the tyrosine kinase Kit is critical for mast cell survival, we evaluated whether treating mice systemically with a combination of an antihistamine (triprolidine, 200 µg), the Syk inhibitor fostamatinib (80 mg/kg), and the c-Abl/c-Kit inhibitor, imatinib mesylate (1.25 mg), 30 min prior to challenging them intravenously with an anti-IgE mAb, would prevent development of anaphylaxis (measured by hypothermia); mast cell degranulation (measured by increases in serum MMCP1); and/or basophil activation (measured by increases in IL-4 secretion). Pretreatment with this 3-agent cocktail completely suppressed hypothermia and decreased MMCP1 by >80%, but had little effect on IL-4 secretion. Triprolidine and fostamatinib independently partially suppressed hypothermia and fostamatinib, by itself, suppressed MMCP1 by 50-60%. No obvious drug toxicity was noted. These results suggest the possibility of using a combination of mediator and tyrosine kinase antagonists to increase the safety of rapid and conventional desensitization therapy.
n-3 polyunsaturated fatty acids (PUFAs) inhibit mast cell activation by disrupting FcεRI association with lipid rafts (HYP3P.401)
Authors: Wang Xiaofeng , Kulka Marianna
Published: January 2023

INTRODUCTION: n-3 PUFAs can suppress allergic inflammation. We hypothesized that n-3 PUFAs inhibit mast cell (MC) activation through modification of FcεRI association with lipid rafts on plasma membrane. METHODS: Bone marrow-derived MCs (BMMC) were cultivated from C57BL/6 wild-type (WT) and fat-1 transgenic mice which express fatty acid n-3 desaturase and produce endogenous n-3 PUFAs. Exogenous n-3 PUFAs were supplemented to WT BMMC. Degranulation, cysteinyl leukotriene (cys-LT), and tumor necrosis factor (TNF) and chemokine (C-C motif) ligand 2 (CCL2) release were evaluated following FcεRI activation. Lipid rafts were isolated by sucrose gradient centrifugation. FcεRI expression was determined by flow cytometry and western blot analysis. RESULTS: Gas chromatography-mass spectrometry analysis showed increased n-3 PUFA levels in whole cell lysates and lipid raft fractions of fat-1 BMMC compared to WT BMMC and in whole cell lysates of PUFA-supplemented BMMC. n-3 PUFAs inhibited degranulation and cys-LT, TNF and CCL2 production compared to untreated BMMC. n-3 PUFAs did not alter expression of surface or total cell FcεRI. However, n-3 PUFAs suppressed FcεRI localization in lipid rafts of unstimulated cells, and disrupted FcεRI shuttling to rafts in stimulated BMMC. CONCLUSIONS: Our results suggest that n-3 PUFAs inhibit MC activation by disruption of FcεRI localization and shuttling into lipid rafts.
The role of lactic acid on mast cell function (HYP3P.404)
Authors: Spence Andrew , Abebayehu Daniel , Ryan John
Published: January 2023

Lactic acid is produced from pyruvate during anaerobic respiration due to a high demand and low supply of oxygen. Therefore, it stands to reason that hypoxic tissues such as tumors or asthmatic bronchioles would have increased lactic acid production relative to their healthy counterparts. Mast cells are also prevalent in these tissues. The purpose of this research is to determine the effect lactic acid can have on mast cell function. Murine bone marrow derived mast cells were cultured in the presence of various amounts of L-(+)-lactic acid (0-25mM). BMMCs were activated with either IgE+antigen or IL-33. Cytokine and chemokine production was measured using ELISAs. Our results showed that IgE-mediated cytokine/chemokine production increased with the amount of lactic acid, with significantly enhanced MCP-1, IL-6 and TNFα at 12.5mM lactic acid. However, IL-33-induced cytokine/chemokine production was decreased in the presence of lactic acid, with significant suppression of MCP-1 and IL-6 at 12.5mM lactic acid. These data suggest that a hypoxic environment can alter mast cell inflammatory responses in a stimulus-dependent manner, and warrant further study into molecular mechanisms.
Differential effects of cyclosporine A and TGFβ1 on IgE- versus IL-33-mediated mast cell activation (HYP3P.407)
Authors: Chernushevich Oksana , Ndaw Victor , Ryan John
Published: January 2023

Cyclosporine A (CsA), a potent calcineurin antagonist and immunosuppressant, is known to inhibit IgE-mediated mast cell activation. The present research investigates the role of this drug in suppressing IL-33-mediated mast cell activation. IL-33 is a novel cytokine of IL-1 family that elicits mast cells to produce inflammatory cytokines independently of IgE-FcεRI signals. We have found that low concentrations of CsA (10nM) are capable of suppressing IgE-mediated cytokine production by 50-80% in mouse bone marrow-derived mast cells. By comparison, IL-33-mediated secretion of TNF, IL-6, and MCP-1 was unaffected by CsA under these conditions. These results were consistent among mast cells derived from C57BL/6, 129Sv, C3H, and A/J mouse strains. We have previously reported that TGFβ1 inhibits IgE-mediated cytokine production. Our current experiments showed that TGFβ1 similarly antagonizes IL-33-induced cytokine secretion, and that CsA does not enhance this effect. These data demonstrate that mast cell activation by IL-33, thought to be a key regulator of innate immunity, is responsive to TGFβ1-mediated suppression, but relatively resistant to CsA effects. Understanding how mast cells are regulated by innate signals will shed light on many inflammatory diseases.
Dexamethasone suppresses IL-33-mediated mast cell activation (HYP3P.409)
Authors: Paranjape Anuya , Chernushevich Oksana , Ryan John
Published: January 2023

Dexamethasone, a potent synthetic member of the glucocorticoid class of steroid drugs is known for its immunosuppressant and anti-inflammatory effects. The present research investigates the role of this anti-allergic drug in suppressing IL-33-mediated mast cell activation. IL-33 is a novel cytokine of IL-1 family, which promotes mast cells to produce inflammatory cytokines independently of IgE-FcεRI signals. Initial studies have shown that micromolar quantities of dexamethasone suppress the production of TNF-α and IL-6 by IL-33-stimulated mast cells. While confirming these results by intracellular staining, it was observed that dexamethasone not only decreases the fraction of cytokine-positive cells but also causes a drop in cytokine production per cell. Although dexamethasone pretreatment resulted in a significant reduction in the IL-33 receptor subunit T1/ST2, this did not correlate with decreased cytokine production. These results demonstrate the ability of dexamethasone to suppress mast cell activation during innate immune responses, and support further work into the mechanisms by which this occurs.
Mir-34a regulates macrophage polarization by targeting IL-4Ralpa/STAT6 axis (INM2P.423)
Authors: Tundup Smanla , Srivastava Leena , Chandora Kapil , Harn Donald
Published: January 2023

Macrophages perform diverse functions during infection as well as metabolic diseases. M1 macrophages promote inflammation while M2 macrophages suppress inflammation and drive anti-inflammatory responses. Emerging evidence suggests that microRNAs control numerous cellular processes; however their role in controlling M2 macrophage maturation is not well understood. To test if microRNAs play roles in macrophage M2 polarization, we performed microarray analysis of microRNA expression in mouse M2 macrophages generated under both in-vivo (i.p injection of Schistosome eggs or LNFPIII glycan) and in-vitro (incubation with recombinant IL-4) conditions. Among the microRNAs differentially expressed, we found that expression of mir-34a was significantly upregulated in M2 macrophages and that over expression of miR-34a mimics led to decreased M2 maturation measured as Fizz1 expression. Using reporter assays and western blot analysis we found that miR-34a targets 3' UTR of stat6 gene thereby affecting the IL-4Rα-STAT6 signaling cascade. Our findings show a unique role of mir-34a in regulating macrophage polarization via targeting the IL-4Rα-STAT6 axis, suggesting that mir-34a plays an important role in regulation of inflammation and may have therapeutic function for inflammatory and metabolic diseases.
Enhanced phagocytosis of auto antibodies-opsonized blood cells by macrophages from virally infected mice. (INM3P.440)
Authors: Legrain Sarah , Su Dan , Detalle Laurent , Izui Shozo , Verbeek Sjef , Coutelier Jean-Paul
Published: January 2023

Autoimmune haemolytic anemia and idiopathic thrombocytopenic purpura are common immune disorders resulting from the phagocytosis of autoantibodies-opsonized blood cells. It has been reported that viral infection may be associated with autoimmune haemolytic anemia and idiopathic thrombocytopenic purpura in patients and may incriminate IgG and IgM antibody isotypes. Lactate Dehydrogenase-elevating Virus triggers the activation of immune cells although it does not induce usually any pathology in mice. Here we report that LDV infection increases the pathogenicity of IgM anti-platelet mAbs which is mediated by FcαμR. Interestingly, we show that this mechanism results from an enhanced Fc-dependent phagocytosis by macrophages rather than an increased expression of FcαμR. Our results also indicate that the pathogenicity of IgG anti-red blood cells is increased in virally infected mice. To date, three activating FcγR have been identified in mice, binding IgGs with different affinity : FcγRI, FcγRIII and FcγRIV. Since FcγRI has a high affinity for monomeric IgG2a, this receptor is poorly available for the binding of circulating-opsonized IgG2a. However we report an increased expression of each receptor by macrophages in contrast to FcamR whose expression is not regulated. Furthermore, we show the functional involment of these three receptors in the exacerbation of IgG-mediated anemia.
Inflammatory protease, caspase-1, is secreted from monocytes in a stably active form in response to inflammasome activation (INM3P.441)
Authors: Shamaa Obada , Ghonime Mohammed , Srabani Das , Gavrilin Mikhail , Wewers Mark
Published: January 2023

Caspase-1 is an inflammatory protease which is required for activation of the pro-inflammatory cytokines, interleukin-1β (IL-1β) and IL-18, in addition to a role in a form of cell death termed pyroptosis. Caspase-1 is synthesized as a zymogen which requires the assembly of a multi-protein complex, termed the inflammasome, for its activation. This assembly forms in response to pathogen or danger associated molecular patterns, but where and how this assembly happens in the cell is poorly understood. To address this question we compared the activity of the released caspase-1 to the cytosolic form in the human monocyte cell line, THP1. In response to endotoxin (LPS) and ATP, pro-caspase-1 is rapidly processed and released from monocytes in its mature, p20/p10, form. We found that the released form has activity which is stable for over 12h and this activity is inhibited by the caspase-1 specific inhibitor, YVAD-cmk. Interestingly, we were unable to completely immunodeplete the released caspase-1 activity or show its ability to cleave exogenously added proIL-1β. This contrasts to the in-vitro cell-extract model, where concentrating monocytic lysates spontaneously activates caspase-1. This cytosolic caspase-1 cleaves endogenous proIL-18, but rapidly loses function. These results suggest that caspase-1 activation requires multi-level regulation that links assembly of the inflammasome complex and enzymatic activity to its interaction with its target substrates during release.