bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a ferret model of maternal antibody passive transfer to suckling neonates
Authors: Baker Pari , Beverly Christopher , Byrne Jake , Sun Francis , Langel Stephanie
Published: August 2023

Neonates and infants are at increased risk of severe disease from influenza. While placentally-transferred IgG reduces the risk of influenza disease in neonates, maternal antibodies (matAbs) can also impact the ability of infants to mount an immune response post immunization, referred to as matAb interference. It is not fully understood how matAb interference impacts protection against influenza once matAbs have waned and if these effects are influenza lineage-dependent. Understanding preexisting influenza immunity may allow for the development of strategies to overcome preexisting antibody interference in neonatal and adult vaccination. To study this, we developed a ferret model of maternal anti-influenza antibody passive transfer to suckling kits. Pregnant and lactating ferrets were orally and intranasally immunized 2 weeks prepartum and 2 weeks postpartum with an H1N1 hemagglutinin (HA) adenovirus type 5 (Ad5) vaccine. Serum and breast milk from dams and serum from kits were assessed via ELISA. We performed a regression analysis and identified a positive relationship between peak levels of HA-specific IgG antibodies in dam milk and HA-specific IgG antibodies in kit serum at 5–6 weeks of age (P = 0.0001, R2 = 0.47). Additionally, peak concentrations of HA-specific IgG antibodies in dam milk correlated with the levels of HA-specific IgG antibodies in dam serum at the same time point (P = 0.0012, R2 = 0.89). Our data suggest matAbs traffic from dam blood to milk, are ingested by kits and translocate through the neonatal intestine into serum. We will use this model to study the role of influenza lineage-specific matAb passive transfer to offspring and their role in de novo antibody responses in suckling kits.

Supported by the Bill & Melinda Gates Foundation Grant (INV-22595D).
Regulation of fetal tolerance by KIR+ regulatory CD8+ T cells in human pregnancy
Authors: Li Jing , Li Lin , Prunicki Mary , Narasimhan Purnima , Mallajosyula Venkata , Kirosingh Adam , Jagannathan Prasanna , Winn Virginia , Nadeau Kari Christine , Gaw Stephanie , Davis Mark M
Published: August 2023

Immune responses during pregnancy need to be precisely regulated to protect the fetus from microbial infections and to maintain tolerance for the semi-allogeneic fetus. However, the mechanisms regulating fetal tolerance during pregnancy are not well understood. Recently we have identified KIR+CD8+ T cells as a previously unappreciated subset with regulatory functions in humans to suppress harmful self-reactivity. Therefore, we asked whether KIR+CD8+ T cells also play a role in inducing immune tolerance during pregnancy. We first observed an increased frequency of KIR+CD8+ T cells, but not CD4+ regulatory T cells (Tregs), in the peripheral blood of pregnant women at second trimester compared to age-matched nonpregnant females. Interestingly, those with a male fetus had an even higher level of KIR+CD8+ T cells than the ones with a female fetus. In vitro, we found that KIR+CD8+ T cells can suppress CD8+ T cells specific to H-Y antigens (encoded by the Y chromosome) only in mothers with a male fetus. Therefore, the higher induction of KIR+CD8+ T cells in mothers carrying a male fetus may suppress the additional allogenic immune responses triggered by the Y chromosome from the male fetus. Moreover, KIR+CD8+ T cells expand, lose memory features and gain cytotoxic capacity along gestation and their levels are maintained postpartum. In addition, we found the numbers of these suppressive CD8+ T cells correlated with pregnancy disorders, such as spontaneous abortion or pre-eclampsia. Taken together, our findings suggest an important role of KIR+CD8+ T cells in the maintenance of fetal tolerance by suppressing alloreactive fetus-specific T cells, therefore they may be useful as predictive biomarkers or drug targets for human pregnancy disorders.

Supported by Howard Hughes Medical Institute.
ILC2-dependent induction of small intestinal memory and systemic mucosal resilience
Authors: Cortez Victor S , Locksley Richard M
Published: August 2023

The IL-25-dependent ILC2-tuft cell circuit drives altered intestinal physiology in response to chronic helminth and protist infections. We optimized an induction regimen with recombinant IL-25 that resulted in long-lasting alterations of the small intestine, which we termed small intestinal memory. This state was characterized by expanded ILC2s and epithelial tuft cells, small intestinal lengthening, and enhanced resistance to helminth infection. ILC2s were necessary for the establishment of small intestinal memory, as this state was induced in Rag-deficient, but not Rag x gc-deficient mice or mice with deletion of IL-17RB on ILCs. ILC2s also maintained small intestinal memory, as this state was rapidly lost upon ILC2 ablation. Transcriptomic analysis revealed dynamic changes in ILC2 identities from IL-25-treated mice, demonstrative of innate memory. Strikingly, although these alterations were induced by IL-25, the ILC2 memory state was sustained in the absence of IL-25, tuft cells, or multiple alarmins like IL-33, TSLP, or IL-18. Finally, we found that small intestinal memory conferred resistance to bacterial and viral infections at distant mucosal sites and notably impacted the metabolic fitness of mice. The induction of enhanced mucosal resilience accompanied by increased metabolic fitness by IL-25 mimics the positive effects of chronic helminth infestation, revealing a mechanistic pathway that might underpin the widespread prevalence of these infections among vertebrates and that could be therapeutically adapted to improve mucosal health.
You are what you vape: Implications of THC chronic self-administration in changes in the gut microbiome of rats
Authors: Rosado-Franco Jose J , Ellison Alysha , Moore Catherine , Weerts Elise , Williams Dionna
Published: August 2023

Vaping of cannabis and its constituents, such as Δ9-tetrahydrocanabinol (THC), has been promoted as a safer alternative to smoked cannabis. Nevertheless, chronic effect of THC vaping in gut microbiome modulation and its effect of health and or disease is still largely unknown. Hence, this project intends to study the effect of chronic THC vapor self-administration on changes in the gut microbiome of Sprague-Dawley rats. Animals were randomly assigned to either vehicle vapor (100% propylene glycol [PG]) or THC vapor groups and were allowed to self-administer PG or THC vapor puffs in operant chambers for one hour on intermittent days for a total of nine months. At the end of this period, a subset of rats was tested for anxiety-like behavior using the open field test. Afterwards, rats were euthanized with isoflurane overdose and rapid decapitation. Feces, metabolic and immune organs, and serum were collected at the time of necropsy. Fecal DNA was extracted, and relative bacterial gut abundance was determined sequencing 16S hypervariable region V4. Rats exposed to THC vape show anxiety-like behavior in the open field test, when compared with the PG exposed group. This change in behavior is also accompanied by an imbalance in their Firmicutes/Bacteroides ratio, having less Firmicutes and more Bacteroides, specifically members of the Muribaculaceae family. Currently, the effect of this dysbiosis on immune markers expression, such as: CCL2, IL-6, TNF-a, IFNa, IFNb, MX1 and TRAIL, are being studied to determine if there is any disruption on immune homeostasis caused by altered microbiome abundance.

R01DA052859-03S1
RORγt+ Tregs are not the sole mediators of tolerance to systemic vaccination following oral antigen pre-exposure
Authors: Potchen Nicole B , Johnson Andrew M F , Hager Kevin W , Graham Jessica B , Lund Jennifer M , Kublin James G
Published: August 2023

Many promising vaccine candidates fail to elicit protective immune responses against critical human pathogens. Exposure to antigens in the gut leads to a reduction in systemic antibody responses upon subsequent vaccination due to immune tolerance, which could contribute to low efficacy of some vaccines clinically. Tolerogenic mechanisms to avoid inappropriate inflammation are partly achieved by the high abundance of regulatory T cells (Tregs) in the gut. RORγt+ FoxP3+ Tregs are a largely peripherally derived subset that depend on stimulation from microbial antigens for development and could control type-2 intestinal immune responses in some disease models. Currently, it is unclear which Treg subsets control systemic vaccine responses following oral antigen pre-exposure. By utilizing a variety of adjuvants in our tolerance model, we have determined that vaccine-specific IgG1 is suppressed following oral antigen exposure, but not IgG2c, regardless of adjuvant strength or mechanism of action. We have used a RORγt+ Treg conditional knock-out (cKO) mouse model to determine that lack of RORγt+ expression within Tregs leads to similar suppression of vaccine-specific IgG1, but not IgG2c, in groups orally exposed to vaccine antigen compared to WT controls. cKO mice exhibit higher frequencies of IL-33R+ and Gata3+ Tregs at baseline in the gut, which suggests other subsets could compensate for the loss of function of RORγt+ Tregs. Analysis of local and systemic sites by flow cytometry also suggest the T follicular-helper compartment could be altered in RORγt+ Treg cKO mice. Together, we hypothesize that RORγt+ Tregs are not solely responsible for controlling tolerance to oral antigens in a model of tolerance to a systemic vaccine.

Supported by grants from NIH (R01AI127100-01, 5TL1TR002318-05)
Investigating persistent factor VIII-specific IgM in the humoral immune response to factor VIII
Authors: York Elizabeth S , Ito Jasmine , Batsuli Glaivy
Published: August 2023

Hemophilia A is an X-linked bleeding disorder resulting in bleeding events due to insufficient levels of factor VIII (FVIII). The most significant complication in the management of hemophilia A is the formation of polyclonal neutralizing IgG (i.e., inhibitors) which impede FVIII procoagulant activity. In a hemophilia A (FVIII KO) murine model injected with multiple doses of FVIII, FVIII-specific IgM were found to persist despite an established IgG response. In attempt to characterize the role of IgM in FVIII immunity, 18 IgMs were purified from immortal FVIII-specific hybridomas and FVIII binding was confirmed via ELISA. IgMs demonstrated weak binding to FVIII in a static ELISA model with mean optical densities (OD) at 405 nm of 0.33 ± 0.43 compared to anti-FVIII IgG with a mean OD of 1.85 ± 0.02. A novel fluid-phase ELISA, developed in our lab, utilizing N-hydroxysuccinimide (NHS)-activated magnetic beads indirectly conjugated to FVIII increased IgM-FVIII binding with mean OD of 1.15 ± 0.60. IgM binding also demonstrated porcine FVIII cross-reactivity. To evaluate the effect of IgM on antibody titers in vivo, FVIII KO mice received 5 weekly injections of either FVIII or 4 separate immune complexes (IC) of IgM-FVIII. Mice injected with IgM-FVIII IC showed slightly higher median IgG ELISA titers than FVIII alone. These findings suggest a potential role for persistent anti-FVIII IgM in propagating FVIII inhibitor development. Further studies determining the effect of IgM-FVIII IC on complement activation, as well as anti-FVIII IgM epitope specificity and clonality utilizing computational methods of RNA-sequencing, may provide additional mechanistic insight into the role of persistent IgM in the humoral immune response to FVIII.

Hemostasis and Thrombosis Research Society- Student Research Award (EY), NIH K99HL150595 (GB), and Hemophilia of Georgia Clinical Scientist Development Grant (GB).
Differential sensitivity to replication stress of human NK cells
Authors: Guilz Nicole , Ahn Yong-Oon , Mace Emily
Published: August 2023

Natural killer cells are critical components of the innate immune system that kill virally infected or malignant cells. Though rare, natural killer cell deficiency (NKD) occurs when NK cell development and function are impaired and individuals with NKD are susceptible to severe and recurrent viral infections. Several gene deficiencies have been described to result in NKD, including variants in MCM4, GINS1, MCM10 and GINS4, which are components of the CMG helicase. The helicase unwinds DNA during replication and is found in any actively proliferating cell. NK cells are more strongly impacted by mutational deficiencies in helicase proteins than other lymphocytes, though the mechanisms underlying this are not completely understood. NK cells from individuals with NKD as a result of helicase deficiency have increased DNA damage, cell cycle arrest, and replication stress. Here, we induced replication stress during NK or T cell specific activation by chemical methods, using aphidicolin, and genetic methods like siRNA targeting helicase proteins. We found that the CD56bright subset of NK cells accumulates more DNA damage and replication stress during activation than CD56dim NK cells or activated T cells. The drug aphidicolin increases apoptosis of CD56bright NK cells through caspase activation while simultaneously decreasing intracellular perforin expression, a protein important for killing of target cells. This effect is modeled by siRNA mediated knockdown of CMG proteins, thus linking decreased protein expression to replication stress and impaired NK cell function. These findings lend important insight into the mechanisms by which helicase deficiencies selectively affect human NK cell function and cause NK cell deficiency.

NIH R01A137275
CD8+Helios+ T cells: A unique functional T cell subpopulation?
Authors: Jones Madalyn , Shevach Ethan
Published: August 2023

The zinc finger protein, Helios, is a member of the Ikaros transcription factor family and is expressed in CD4+T cells, CD8+T cells and in some NK cells in both mice and humans. While it has been found to have a role in the homeostasis and suppressive function of CD4+Foxp3+T regulatory cells (Treg), its function in CD8+ T cells and NK cells is not well characterized. We have demonstrated that ~10–40% of CD8+ T cells from normal donors express Helios. Expression of Helios in CD8+ appears to be downregulated with TCR stimulation alone but is maintained in the presence of excess IL-2 signaling. Thus far, extensive flow cytometry studies have failed to show a correlation between Helios expression and any other surface or intracellular markers. Mouse CD8+Helios+Ly-49+ T cells have been reported to inhibit the activation of B cells in germinal centers and have been claimed to be the CD8+ counterpart to CD4+Treg. While these studies have been performed in mice, there is very little data to suggest that human CD8+Helios+ T cells exhibit T suppressor function. In humans, the killer-cell immunoglobulin-like receptors (KIRs), a family of transmembrane glycoproteins, are said to be the functional equivalent of the Ly49 proteins. KIR+CD8+T cells have previously been said to highly express Helios and have a function in autoimmune conditions. Data we have obtained from a CITE-seq analysis has shown that Heliosexpression in CD8s is highly correlated with many NK-cell associated markers. This, along with data showing that these cells produce cytotoxic effector molecules, leads us to speculate that these cells have a role in immune suppression via cytotoxic killing of self-activated immune cells. This work was supported by the Intramural Research Program of NIAID, NIH.
GIMAP5 deficiency reveals a human cellular longevity assurance pathway
Authors: Park Ann Y , Gabrielski Justin Q , Lenardo Michael J
Published: August 2023

Studies in model organisms have revealed that lifespan and senescence are genetically controlled, but how cellular longevity assurance pathways are regulated is unknown. Here, we report new insights into cell longevity and senescence through the investigation of a novel disease caused by mutations in GTPase of the immunity-associated protein 5 (GIMAP5). We identified 17 affected individuals in 9 kindreds with lymphopenia, liver disease, splenomegaly, thrombocytopenia, lymphadenopathy, lung infections, and lymphoma. GIMAP5 is highly expressed in T and NK lymphocytes as well as endothelial cells (EC) and is famous as the gene mutated in the Biobreeding diabetic rat and Sphinx lymphopenic mice. Given the strong disease association, the mechanism of GIMAP5 is an important biological and medical question. We found that loss of GIMAP5 leads to the uncontrolled accumulation of long chain ceramides, which is associated with premature senescence and death of lymphocytes, resulting in severe and recurrent infections in patients. We further showed that Gimap5 deficiency in mice increases senescence markers in T cells and ECs of the lung and liver, suggesting that GIMAP5 is involved in the senescence mechanisms affecting multiple tissues. Therefore, our results illustrate the role of the GIMAP5 regulatory pathway in cell longevity and senescence and reveal new molecular targets for increasing the healthy functional lifespan of human cells. This research was supported by the Intramural Research Program of NIAID, NIH.
Thioacetamide-induced liver fibrosis is reduced in humanized immune system mice
Authors: Chen Wen-Yu
Published: August 2023

Fibrosis is defined as a consequence of the abnormal wound-healing response lading to excessive deposition of extracellular matrix components. Chronic liver diseases accompanied with liver fibrosis have caused increased morbidity and mortality worldwide. Carbon tetrachloride (CCl4) and thioacetamide (TAA) are the well-known toxins used to induce liver fibrosis in murine. Compared to CCl4, TAA displays a longer latency between exposures to the drug and less safety concerns with operators. In addition, the translation of murine experimental data to human events often fails due to differences in the immune system of both species, indicating that humanized mice may be a better choice to model human diseases. The effect of TAA in humanized mice is still unknown. In our study, liver fibrosis in wild type mice was induced by escalating doses of TAA. Results showed that liver fibrosis evaluated by Sirius red staining was significantly induced (Metavir score, F2). In addition, serum levels of AST, ALT and ALP were significantly increased after TAA injection. Humanized mice were generated by injection of human hematopoietic stem cells (hHSC) into NOD.Cg-PrkdcscidIl2rgtm1Wjl/YckNarl (Advanced Severe ImmunoDeficiency (ASID)) mice, termed as hHSC-ASID. The escalating doses of TAA were introduced into hHSC-ASID and ASID mice. Although the serum levels of AST, ALT and ALP were increased in both hHSC-ASID and ASID mice following TAA injection, the degree of liver fibrosis was reduced in hHSC-ASID mice than in ASID mice (Metavir score, F1 vs. F2). These results indicated that human immune cells reduced TAA-induced liver fibrosis. It is worthwhile to assess which immune cell(s) participates in this process.
B cell leukemia elicits a suppressive Tr1-like CD4 T cell response that can be reversed with anti-PDL1-blockade
Authors: Venkatesh Hrishi , Tracy Sean I , Hekim Can , Knutson Todd Philip , Farrar Michael A
Published: August 2023

CD4 T cell exhaustion is associated with relapse in B cell leukemia (B-ALL) patients. However, checkpoint blockade is not effective in treating B-ALL. Using a murine model of B-ALL, we demonstrate that leukemia induced CD4 T cells had a unique regulatory-cytotoxic phenotype (Trctx) with preferential expression of exhaustion markers (PD1/TIM3) as well as IL10 and c-Maf, which are markers of immunosuppressive Tr1 cells. Leukemia cells acting as APCs elicited CD4 T cells with a similar Tr1 phenotype that suppressed bystander T cells. This process mimics the mechanism by which hematopoietic stem cells (HSC) enforce an immunosuppressive niche in the bone marrow that protects the HSC compartment. Anti-PDL1 or nilotinib (targeted therapy) treatment alone had a minimal impact on survival; concurrent treatment with anti-PDL1 and nilotinib had a significant synergistic effect that was dependent on CD4 and CD8 T cells. Nilotinib treatment increased markers of helper and regulatory function in the Trctx cells. In contrast, treatment with nilotinib and anti-PDL1 induced expansion of a leukemia-specific CD4 T cell clone with lower expression of Tr1 markers and higher expression of helper molecules that facilitate recruitment and activation of anti-tumor immune cells. Expansion of the helper-cytotoxic (Thctx) CD4 T cell clone was associated with increased GzmB+ CD8 T cells. We propose that leukemia coopts a naturally occurring process designed to protect the HSC niche, thereby inducing Trctx cells that drive escape from immune surveillance. Combining nilotinib and anti-PDL1 alters the balance between immunosuppressive Trctx and immune-activating Thctx, thereby inducing a protective anti-leukemia immune response that prevents relapse.

Spatial Transcriptomics Award from the University of Minnesota, Children’s Cancer Research Fund
Identification and characterization of a dominated T cell population in tumor associated antigen specific CD8+ T cells of tumor microenvironment
Authors: Chuang Ming-Kai , Lee Tsai-Heng , Chia Jean-San
Published: August 2023

The characteristics and roles of tumor associated antigens (TAAs) specific tumor infiltrative T cells (TILs) are not well understood. Alpha-enolase is one TAA of oral squamous cell carcinoma (OSCC) and pancreatic cancer, and are highly expressed in many cancer types by analyzing the TCGA database. We detected α-enolase responsive CD8+ T cells in peripheral blood mononuclear cells (PBMCs) and TILs of OSCC patients. The α-enolase responsive CD8+ T cells significantly increased in TILs than those in PBMCs. We developed two tetramers to identify two different α-enolase peptides specific CD8+ T cells. Two types of tetramer positive CD8+ TILs, total CD8+ TILs, and total CD8+ T cells from PBMC of three OSCC patients were sorted. Single cell RNA sequencing was performed. Unbiased clustering revealed 11 distinct CD8+ T cell clusters. To our surprise, the cell population patterns of both tetramer positive CD8+ TILs were similar, but were much different from total blood CD8+ T cells or total CD8+ TILs. The percentages of relatively exhausted T cells were similar in three TIL groups, but was nearly absent in blood. The blood CD8+ T cells had the highest percentage of effector memory T cells and naïve T cells. A specific cell cluster dominated (>50% population) in the two tetramer positive CD8+ TILs. The expression of important immune related genes in the specific cell cluster were all relatively low. That hinted this T cell subset was more quiescent than other cell clusters, and may have potentials to be activated. Further clarify the roles of the special T cell subset in tumor immunology is warranted.

Supported by grants from National Science and Technology Council, Taiwan (110-2314-B-002-299)
Modeling Immunological Networks in an Educational Setting using Cell Collective
Authors: Taylor Rebekah , Pandey Sumali , Justement Louis B , Helikar Tomas
Published: August 2023

One challenge in the teaching of immunology is the complexity of the subject. Immunology presents a long list of unique cells, signaling molecules, receptor-ligand interactions, regulatory mechanisms, developmental pathways, and outcomes that can feel burdensome to instructors and unapproachable to students. The beauty of this subject, however, is that these unique features interact in networks that parallel those in other biological fields. Visualizing the interactions between tissues, cells, and molecules of the immune system and their outcomes can promote immunological literacy and a broad understanding of biological systems.

Cell Collective (cellcollective.org) is an open-access, approachable software system that allows students and researchers alike to build models and perform simulations of biological processes. Students can use this interactive platform to probe cell-cell interactions, signaling pathways, metabolic networks, etc. while building systems thinking and computational skills, which are critical for success in STEM fields. After building models, real-time simulations can be run to visualize and understand the dynamics of the biological system under conditions of environmental pressure, disease, mutation, etc. Instructors can assess student knowledge by building assessments into modules in a formative or summative manner. Because it is free, user-friendly, and offers a host of pre-built training, educational and experimental modules, Cell Collective is ideally suited for use in all types of educational settings.

Supported by a grant from the NSF (RCN-UBE) 2120806
Smart textbooks and LMS-integrated digital tools incentivize student engagement and facilitate learning in an undergraduate immunology course
Authors: Walline Crystal C
Published: August 2023

During the COVID-19 pandemic, educators were compelled to quickly pivot their face-to-face courses to a hybrid, online, or asynchronous delivery, and to develop learning management (LMS) course websites to seamlessly delivery course content, assignments, assessments, and other instructional tools. Concurrently, textbook publishers are offering LMS-integrated, comprehensive, digital solutions, including “smart” or adaptive learning electronic textbooks. W.W. Norton & Company publisher has recently released digital resources to complement the 5th edition of Peter Parham’s The Immune System, including chapter reading assignments which incorporate adaptive quizzing and gamification using the InQuizitive feature. Here, based on student responses in early questions, subsequent questions are personally curated by learning objectives and difficulty level. Instructors may customize the difficulty level and determine a minimum score to be achieved to earn the assigned points which incentivizes students to read the textbook prior to classroom meetings or discussions. Additionally, instructor resources include Case Studies in Immunology, slides and graphics, animations, activities, primary literature suggestions, and an LMS-integrated test maker. Overall, student feedback was overwhelmingly positive, grades were substantially higher compared to previous semesters, and because more students came to class prepared, classroom time was used more efficiently such that all foundational topics outlined by the AAI Education Ad Hoc Committee were delivered. Therefore, LMS-integrated, adaptive learning textbooks are an essential component for a successful undergraduate immunology course.
Voiceover lectures and gaming; assessing student and faculty perceptions of this pedagogy and determining its effectiveness in teaching immunology and academic policies
Authors: Cusick John K , Zemeida Savannah , Chou Emily , Chang Esther , Shyu Yennie , Abram Alyssa , Gerriets Valerie
Published: August 2023

Our objective was to obtain feedback from students and faculty on the use of pre-recorded voiceover lectures and in-class games to determine the impact of these pedagogies on educational outcomes. To evaluate understanding of academic progression policies, matriculating medical students were given quizzes before and after participating in a review game covering these policies. To evaluate the understanding of immunology content, students were randomly placed into groups in which a quiz was given after 1) attending a traditional lecture, 2) watching a voiceover and participating in a review game, or 3) no exposure to teaching on subject. Likert scales were used to assess impressions from both students and faculty regarding this pedagogy. Survey results indicated that students who participated in a review game scored significantly higher in comparison to matriculating students who learned about academic policies solely through their orientation. Both students and faculty believe that voiceovers combined with review games is a superior educational method than traditional lectures. Although faculty believe these active learning pedagogies are more tedious to create than traditional lectures, faculty expressed an interest in adopting this pedagogy especially if training and IT support are provided. Collectively, our results indicate that faculty and students both strongly prefer the use of active learning pedagogies over traditional lectures, and student comprehension of academic policies increased significantly after participating in a review game. A significantly increased understanding of immunology content through active learning pedagogy versus traditional lectures has not yet been established in this ongoing study.

Supported by grants from the California Northstate University Institute of Teaching and Learning Excellence (ITLE).
Anti-SARS-CoV-2 IgG levels are associated with circulating levels of chemokines CXCL9/CXCL10 and severity disease, but not with RBD-specific B cells 4 months after COVID-19
Authors: Quiroga Romina Andrea , Novoa Sergio Andrés Sanhueza , Cabrera-Garcia Camilo Daniel , Cáceres Bárbara Evelyn Antilef , Munoz Camila Paz , Llerena Faryd , Fraga Marco , Henriquez Mario , Labarca Gonzalo , Nova-Lamperti Estefania , Colombo Alicia , Fernandez Liliana Lampert
Published: August 2023

Background

It has been reported that the humoral response and cytokine secretion is exacerbated in patients with severe COVID-19. However, it is unknown which cytokines are associated with the exacerbated humoral immune response during and after COVID-19. The aim of this study was to determine the association between cytokines and the humoral response of patients who had COVID-19 with different severity degree.

Methods

60 COVID-19 patients were recruited from Víctor-Rios-Ruiz Hospital during acute phase and at 4-months post-infection. Clinical exams were performed to evaluate lung damage after COVID-19. Serum samples were collected to measure IgG levels (Nucleocapsid + Spike) using commercial CLIA assays and cytokines and chemokines by Cytometric Bead Array (CBA). The percentage of B cell subsets and RBD-specific B cells were measured by flow cytometry.

Results

Anti-SARS-CoV-2 IgG levels, but not RBD-specific B cells, were significantly associated with severity and pulmonary sequelae. Moreover, the data revealed that proinflammatory chemokines CXCL9 and CXCL10 were significantly correlated with Anti-SARS-CoV-2 IgG levels. We also observed that these chemokines promote CD40L upregulation and B cell differentiation into plasma cells.

Conclusions

Severe COVID-19 patients produced higher anti-SARS-CoV-2 IgG levels than patients with mild disease, possibly due to an exacerbated immune response, associated with sustained levels of proinflammatory chemokines CXCL9 and CXCL10. This exacerbated response was associated with structural lung damage post-COVID-19.

COVID1005 project Fondecyt Regular 1211480 ACT210085
Elevated C-reactive protein and depression score among post-COVID-19 patients - A cross-sectional study
Authors: Giridharan Vijayasree Vayalanellore , Cardoso Taiane de A , Silva Ritele H , Fernandes Jessica L , Arent Camila O , Amboni Graziela , Borba Laura A , Padilha Alex Paulo Z , Botelho Maria Eduarda M , Maciel Amanda L , Soares Silvio José B , Bagatini Margarete D , Dallagnol Claudia , Brighenti Marta Elisa , Ignácio Zuleide Maria , Quevedo João , Ceretta Luciane B , Morales Rodrigo , Barichello Tatiana , Reus Gislaine Zilli
Published: August 2023

Immune dysfunction after SARS-CoV-2 infection leads to long COVID neurological sequels in COVID-19 survivors. Despite initial respiratory symptoms, neuropsychiatric manifestations have been reported in many COVID-19 patients. We aimed to investigate the psychological symptoms, gut microbiome status, and C-reactive protein levels (CRP) in post-COVID individuals. This cross-sectional study included age and sex-matched individuals with post-COVID (n = 114) or controls (n = 236). We used Mini International Neuropsychiatric Interview (MINI) to diagnose psychiatric disorders. The depressive and anxiety symptoms were assessed by the Hamilton Rating Scale and the stress level by an inventory of stress symptoms and circulating CRP levels measured. In a small cohort (post-COVID = 18 and controls = 46), the gut microbiome was evaluated by 16S rRNA sequencing. Post-COVID individuals exhibited greater severity of depressive symptoms (p = 0.034), higher levels of stress (p = 0.020), and CRP (p = 0.014) as compared to controls. There was no difference in α-diversity but β-diversity (p = 0.001) was significantly different between control and post-COVID groups. Interestingly, post-COVID individuals with depression had greater CRP levels than those with COVID-19 without current major depression disorder (p = 0.023). Post-COVID-19 individuals have demonstrated severe psychological symptoms and altered gut microbiome levels. Although longitudinal studies are needed to study the mental health trajectory of COVID-19 individuals, the levels of CRP may serve as a promising biomarker for the early detection of post-COVID depression.
New Complement Dependent Cytotoxicity Assay and its application to COVID-19 patient sera harboring anti-lymphocyte antibodies
Authors: Calderon Stephanie , Liu Xiangdong , Lisco Andrea , Kellog Anela , Galindo Frances , Memoli Matthew , Rocco Joseph M , Laidlaw Elizabeth , Manion Maura , Sereti Irini , Perez-Diez Ainhoa
Published: August 2023

Complement dependent cytotoxicity (CDC) is one of the ways IgM and IgG antibodies can mediate the lysis of target cells, being IgM the antibody (Ab) isotype with the highest CDC activity. In a cohort of hospitalized COVID-19 patients we identified that 27% carried IgM anti-lymphocyte Ab in their sera. To quantify the sera potential lytic activity against healthy donor (HD) PBMC we developed a flow-based CDC assay with an internal negative control. The assay allows for normalization of the results and therefore inter-experiment data comparison without the need of using counting beads. In short, after CFSE labelling, 2 million PBMC are incubated with either complete media (CM) or with the different experimental conditions (Ab, sera, plasma), then they are washed thoroughly before adding them to equal numbers of unlabeled PBMC (internal negative control). This mixture of hot CFSE+ and cold CFSE-targets is then incubated with rabbit complement for an hour, stained and flow. A “ratio tube” containing the mixture with the CM incubated CFSE cells, is left without rabbit complement at 4 degrees and used to calculate the initial CFSE+/CFSE− ratio. We calculate the percentage of CDC lysis based on the CFSE+/CFSE− ratio in each tube after normalizing it to the ratio tube. Deploying this assay, we found CDC activity against B and T cells in five and two COVID-19 patients’ sera, respectively, out of 23 patients tested. This improved CDC assay shows high reproducibility and allows for quick, reliable, and easy test of multiple conditions.

This work was supported by the Divisions of Intramural Research of NIAID, NIH
Dysregulated immune networks define distinct respiratory phenotypes of post-acute sequelae of COVID-19
Authors: Canderan Glenda , Muehling Lyndsey M , Kadl Alexandra , Sturek Jeffrey , Bonham Catherine , Murphy Deborah , Lima Sierra , Cross Claire , Wilson Jeffrey , Cheon In Su , Sun Jie , Irish Jonathan , Woodfolk Judith A
Published: August 2023

Long-haul respiratory symptoms are common following severe COVID-19 illness; however, the immune mechanisms contributing to post-acute sequelae of COVID-19 (PASC) remain enigmatic. A comprehensive 2-year longitudinal immune assessment was performed in a cohort of PASC patients receiving pulmonary follow-up care (n=110) by analyzing circulating immune cells using spectral flow cytometry, as well as assessment of virus-specific T cell frequencies, serum antibodies, and plasma mediators. Novel unsupervised machine-learning workflows were used to analyze and integrate multiple clinical and immune measures. Our approach identified discrete respiratory phenotypes according to lung physiology measures that defined the degree of lung fibrosis and its severity. Notably, severe respiratory phenotypes were characterized by immune dysregulation of both innate and adaptive cellular components, including lower ILC-3-like cells and higher activated CD4+ and CD8+ T cells in the blood. On the other hand, all respiratory phenotypes displayed a marked loss of naïve CD8+ T cells and unswitched memory B cells compared to healthy individuals. Spike- and nucleoprotein-specific CD4+ and CD8+ T cells were more abundant in severe respiratory phenotypes. Overall, T cell-derived inflammatory cytokines, along with chemokine receptors and their relationships to cognate ligands, pointed to sustained lung-homing of pathogenic effector cells in fibrotic phenotypes. Integration of high-dimensional data defined discrete cellular and molecular immune networks that provide unconventional and novel mechanistic insight into pulmonary complications post-COVID-19.

UO1AI125056 R21 AI160334
NK cell activation provides protection against herpes simplex encephalitis through TLR3/TRIF-dependent crosstalk between epithelial and dendritic cells
Authors: Eo Seong Kug , Choi Jin Young , Byeon Hee Won , Cho Hye Won , Park Seong Ok , Uyangaa Erdenebelig
Published: August 2023

Herpes simplex encephalitis (HSE), which is a fatal disease causing focal or global cerebral dysfunction following infection with herpes simplex virus type 1 (HSV-1). Previous few studies have been administered on the immunopathological networks of HSE in the context of TLR3 and TRIF defects at the cellular and molecular levels. In this work, we tried to decipher the crosstalk between type I IFN (IFN-I)-producing epithelial layer and IL-15-producing dendritic cells (DC) to activate NK cells for the protective role of TLR3/TRIF pathway in HSE progression after vaginal HSV-1 infection. TLR3- and TRIF-knockout mice showed enhanced susceptibility to HSE progression, with high HSV-1 burden in vaginal tract, lymphoid system, and CNS. The increased HSV-1 burden in TLR3- and TRIF-ablated mice did not correlate with increased infiltration of Ly-6C+ monocytes, but it was closely associated with impaired NK cell activation in vaginal tract. In addition, using delicate ex vivo experiments and bone marrow transplantation, TRIF deficiency in tissue-resident cells, such as epithelial cells in vaginal tract, found to cause impaired NK cell activation by means of low IFN-I production, whereas IFN-I receptor in DC play a role in activating NK cells via IL-15 production in response to IFN-I produced from epithelial layer. These results provide new information about IFN-I- and IL-15-mediated crosstalk between epithelial cells and DC at the primary infection site that can suppress HSE progression in a TLR3- and TRIF-dependent manner.

This study was supported by the Basic Science Research Program through National Research Foundation of Korea (NRF) grants funded by the Ministry of Education and the Ministry of Science and ICT (MSIT), Republic of Korea (2021R1A2B5B02001578, 2019R1A6A1A03033084 for Seong Kug Eo, 2020R1I1A1A01054533 for Erdenebileg Uyangaa, and 2020R1I1A2055591 for Jin Young Choi, http://www.nrf.re.kr).
Cardiac dysfunction mediators CXCL-9 and chemotaxis of phagocytes characterized patients with long-term pulmonary dysfunction after COVID-19
Authors: Novoa Sergio Andrés Sanhueza , Nova-Lamperti Estefania , Cabrera-Garcia Camilo Daniel , Quiroga Romina Andrea , Grez Camila Paz Muñoz , Cáceres Bárbara Evelyn Antilef , Labarca Gonzalo
Published: August 2023

Severe COVID-19 is characterized by marked cytokine release and pulmonary dysfunction; however, it is still unknown which mediators support post-COVID-19 pulmonary dysfunction. Our aim was to determine the signaling pathways associated with long-term pulmonary dysfunction in patients with COVID-19. Our cohort of 60 patients with COVID-19 were followed up at 4- and 12-months post infection. Pulmonary sequelae were analyzed by computed tomography (CT), diffusing capacity of carbon monoxide (DLCO), spirometry, 6-minute walk test (6MWT) and handgrip test. Cytokines, chemokines and blood tests parameters were also analyzed. Furthermore, signaling pathways associated with pulmonary dysfunction were identified by serum proteomics analysis using TimsTOF-Pro and ingenuity pathway analysis. Fourteen patients with CT and abnormal DLCO were classified as patients with long-term pulmonary dysfunction (LTPD), who also presented impaired spirometry tests, 6MWT and handgrip strength than patients without sequelae or patients with CT only. We observed that only CXCL-9 was significantly increased in LTPD patients in comparison with other groups. In addition, the main signaling pathways identified by proteomics in LTPD patients were associated with an immunothrombotic state related to cardiac dysfunction, migration of dysfunctional monocytes and reduced IFN-signaling. In summary, patients with LTPD presented a restrictive lung condition, with greater fatigue, lower aerobic capacity and muscle strength 4-months post-COVID-19. Our data suggest that patients with previous cardiac dysfunction who had severe o moderate COVID-19 are more susceptible to develop LTPD.

PROYECTO COVID1005, ACT210085 y Fondecyt Regular 1211480.
Age-associated variations in anti-spike antibody responses in mild SARS-CoV-2 disease
Authors: Parsons Emily , Richard Stephanie , Laing Eric , Fries Anthony , Livezey Jeffrey , Jones Milissa , Lindholm David , Mende Katrin , Rozman Julia , Ganesan Anarudha , Lalani Tahaniyat , Mody Rupal , Bazan Samantha , Saunders David , Colombo Rhonda , Colombo Christopher , Ewers Evan , Larson Derek , Maves Ryan , Berjohn Catherine , Maldonado Carlos , Simons Mark , Tribble David , Agan Brian , Pollett Simon , Burgess Timothy , Malloy Allison
Published: August 2023

Background

SARS-CoV-2 most severely affects older age groups, while children experience minimal disease. We measured elements of adaptive immunity in children and adults with SARS-CoV-2 to define the age-dependent viral and host response.

Methods

Participants were enrolled in the Epidemiology, Immunology, and Clinical Characteristics of Emerging Infectious Diseases with Pandemic Potential (EPICC) study. Eligibility required that individuals present with COVID-19-like symptoms between March 2020 to March 2022. Magnitude of viral RNA was determined by quantitative PCR. SARS-CoV-2-specific antibodies were measured from blood with multiplex microsphere immunoassays.

Results

Serologic responses against the spike protein were measured at early and convalescent time points and compared by age, predominant infecting SARS-CoV-2 variant, and vaccination status. In those with mild disease, the seropositivity rates did not differ based on age. The magnitude of the anti-spike IgG response differed by age at early time points. Only the oldest age group differed at convalescent time points.

Conclusions

The serologic response to SAR-CoV-2 differs with age, implicating host immunity in pathogenesis. Improved understanding of the host immune response has the potential to optimize vaccine design.

Disclaimer:The views expressed are those of the authors and do not reflect the official policy of the Department of the Army, Department of the Navy, the Department of the Air Force, the Department of Defense or the U.S. Government and the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (HJF). The investigators have adhered to the policies for protection of human subjects as prescribed in 45 CFR 46.

This work was supported by the Defense Health Program (grant number HU00012020067) and the National Institute of Allergy and Infectious Diseases (grant number HU00011920111).
Development and Characterization of a novel humanized ACE2 mouse model to study the pathogenesis of viral lung infection by variable SARS-CoV-2 strains
Authors: Choi Chang-Yong , Villano Jason , Pekosz Andy , Jia Hongpeng
Published: August 2023

Despite global efforts to end the pandemic of COVID19 which has claimed millions of lives in the past few years, there is no sign of the ending in foresee near future. One of the obstacles to end the pandemic is lack of proper animal models to better understand the pathogenesis and test preventive and therapeutic strategies. To answer this call, our group created a novel humanized ACE2 mouse line by removing mouse Ace2 exon 1–9 using CRISPR/Cas9 techinque and put full length human ACE2 cDNA into the same loci. So, the human ACE2 expression is driven by mouse Ace2 promoter. Our validation results indicate that the mice express human ACE2 in a pattern similar to that of mouse Ace2 are expressed. Moreover, inoculating young adult mice (6 weeks old) with variable strains of SARS-CoV-2 (SCV2, Washington or Delta or Omicron strain, 5 × 105 PFU/mouse) by nasal instillation resulted in differential phenotypes, including viral loads in lung, trachea and nasal turbinate, weight loss, pro-inflammatory cytokine profile, although no mortality was observed in such age grouped mice. In addition, we detected brain pathological protein related to Alzheimer’s disease, Tau protein after 6 days of the viral lung infection, indicating our mouse model has the potential to be used for preclinical studies of long COVID diseases. In summary, our novel humanized ACE2 mouse line has great potential in COVID19 related research. Our ongoing research focuses on further characterize the age and strain dependent SCV2 infection of the mouse line and understanding the pathogenesis and therapeutic strategies for long COVID diseases.

Supported by grants from NIH (1R01AI148446-01A1)
pDCs contribute to HIV persistence through depleting stem-like CD8 memory T cells
Authors: Li Guangming , Ma Jianping , Yu Haisheng , Tsahouridis Rania , Lou Yaoxian , Su Lishan
Published: August 2023

Plasmacytoid dendritic cells (pDCs) rapidly produce type I interferons (IFN-I) to suppress HIV-1 replication during acute infection. However, persistent activation of pDCs during chronic HIV-1 infection contributes to HIV-1 diseases. The combination antiretroviral therapy (cART) effectively suppresses HIV-1 replication and extends the life span of HIV-infected individuals. However, the persistence of aberrant inflammation and HIV-1 reservoir cells in patients under cART leads to elevated inflammatory diseases and life-long HIV-1 persistence. We report here that pDC-depletion during suppressive cART in humanized mice resolved HIV-associated inflammatory diseases, reversed T cell immune hyper-activation and exhaustion, rescued anti-HIV T cell response. Importantly, pDC-depletion under cART reduced HIV-1 reservoirs in lymphoid tissues, associating with an increase of stem-like CD8 memory T cells (Tslc) in vivo. In vitro culture system, we confirmed that anti-HIV T cell response could also be enhanced by depleting pDC, accompanied with an increase of Tslc number. We conclude that pathogenic pDCs dysregulated anti-HIV T cell response and contributed to viral reservoir persistence via induction of persistent inflammation, immune activation and a consequent exhaustion/depletion of anti-HIV T cells. Depletion of pDCs rescued Tslc, correlated with HIV control in vivo. Targeting pDC may provide a novel strategy for treating HIV/cART-associated inflammatory diseases, controlling HIV-1 reservoirs during cART, approaching a functional cure for HIV infection.