bluuebunny/crossref_metadata_2025 huggingface dataset:
Elucidating Inflammation-induced Cell Fate Decisions in Primary Human Tregs
Authors: Ho Patrick , Bluestone Jeffrey A , Tang Qizhi
Published: August 2023

Adoptive regulatory T-cell (Treg) therapy is an emerging therapeutic paradigm for promoting immune tolerance in transplant and autoimmune disease settings. Prior investigations demonstrate that murine Tregs can undergo epigenetic reprogramming within chronically inflamed tissue environments, resulting in acquisition of proinflammatory functions and the capacity to exacerbate tissue damage. Despite the ramifications of Treg lineage decommitment for cell therapy applications, inflammation-induced human Treg cell fate decisions remain poorly understood. Here, we present a robust in vitro model of IL6, IL1β, and IL23-driven Treg instability characterized by progressive FOXP3 and HELIOS downregulation, FOXP3 conserved non-coding sequence (CNS)2 enhancer re-methylation, diminished in vitro suppressive function, and elevated proinflammatory cytokine expression. To gain insight into the gene regulatory networks enabling the loss of Treg identity, we generated single-cell transcriptomic and chromatin accessibility profiles of primary human Tregs maintained in the presence or absence of IL6, IL1β, and IL23. Unsupervised clustering revealed a dysfunctional Treg population with an epigenetic signature consistent with murine Treg to “exTreg” conversion, including altered chromatin accessibility at the IFNγ, IL17A, and FOXP3 CNS2 loci. Inference of transcription factor (TF)-associated changes in chromatin accessibility indicated a key role for E26 transformation-specific (ETS) family members. Ongoing experiments aim to identify specific TF modules that can be targeted to better safeguard the function and stability of Treg therapeutics.
Characterization and Neutralization of Pathologic Memory CD4+ T Cells in Spondyloarthritis Synovial Fluid
Authors: Song Woogil , Yi Kijong , Jo Sungsin , Jung Seyoung , Lee Eun Young , Kim Tae-Jong , Lee Jeong Seok
Published: August 2023

Spondyloarthritis (SpA), which has increased T helper 17 (Th17) cell activity, and neutralizing antibodies targeting IL-17 are effective in patients with SpA. However, not all Th17 cells are pathologic; most Th17 cells have physiological roles. Here, we characterize the immune landscape of blood and synovial cells in SpA through single cell to demonstrate the molecular characteristics of pathologic memory CD4+ T cells with Th17 characters. We identified pathologic Th17 (pTh17) cells as polyfunctional IL-17A and IFN-γ-producing memory CD4+ T cells, with clinically supporting evidence for their pathogenic roles at the inflammatory site of SpA. Transcriptome and flow cytometric analysis found that the co-expression of TNFRSF4 and TNFRSF18 is increased in pTh17 cells. Suppression of ligand-receptor interactions via TNFRSF4 and TNFRSF18 effectively decreased clinical arthritis and decreased pTh17 cells in the curdlan-injected SKG mouse model. Our results provide understanding pTh17 cells in SpA and suggest potential therapeutic targets.
Modified Immune Cell (MIC) Therapy disrupts Tertiary Lymphoid Structures in Murine Lupus Nephritis
Authors: Ponath Gerald , Speer Claudius , Arnold Iris , Hidmark Asa , Wang Lei , Kleist Christian , Schmitt Anita , Daniel Volker , Opelz Gerhard , Terness Peter , Schmitt Michael , Zeier Martin , Morath Christian , Schaier Matthias
Published: August 2023

Induction of tolerance can be achieved with Modified Immune Cells (MIC) by infusion of mononuclear cells challenged with mitomycin C (MMC). Although MIC treatment has been successful in several animal models for autoimmunity and in clinical studies of solid organ transplantation, the mechanism of immunosuppression is not fully elucidated.

In lupus nephritis, the glomerular deposition of immune complexes is associated with accumulating immune cells in the kidney. The infiltrating immune cells frequently establish tertiary lymphoid structures (TLS) supporting adaptive autoimmune responses toward locally displayed antigens.

Since TLS display a high persistence to peripheral B cell depletion, the destruction of TLS presents an essential treatment goal in lupus nephritis. In this study, we used lupus nephritis prone NZB/W F1 mice to show the destruction of TLS in the kidney after MIC treatment.

Independent of treatment, >86% of animals displayed dense lymphocytic aggregates proximal to the pelvic wall of the medulla and the arcuate arteries within the cortex of kidneys. Cell type composition of TLS changed drastically after MIC treatment leading to diminished B-cells, a decreased B-cell/T-cell ratio, and a T cell dominated phenotype. Furthermore, a loss of organization of the TLS was observed after MIC treatment. The strict separation of B-cell and T-cell areas was abrogated, and germinal centers were disintegrated. However, regulatory T-cells remained unchanged indicative of a B-cell centric treatment mechanism.

Our data provides a putative in vivo mechanism how MIC treatment inhibits progression of active lupus nephritis by the destruction of tertiary lymphoid structures within the kidney.

Commercial Support - TolerogenixX GmbH
Glucocorticoids redirect naive T cells to the bone marrow in malnourished mice
Authors: Pearson Caroline , Saravanan Rithanya , Marczak Marissa , Washington Aja , Foster Takesha R , Dadzie Nana , Hanes Jacob , Moore Lindsay , Mister Abigail , Goulmamine Syreen , Gibson David , Adams Olivia , Gubbels Bupp Melanie R
Published: August 2023

Glucocorticoids contribute to the daily migration patterns of T cells in well-nourished organisms and are elevated in the malnourished. We examined the effect of malnutrition on T cell migration by comparing the migration patterns of adoptively transferred malnourished and control T cells in the lymphoid organs of malnourished and control recipients. We found that malnourished T cells generally entered lymphoid tissues more efficiently than control T cells, regardless of recipient. Strikingly, the bone marrow of malnourished recipients attracted naïve malnourished T cells, but not control T cells, more efficiently than control bone marrow. In contrast, the spleens of malnourished and control mice attracted similar numbers of naïve T cells. Further experiments revealed that T cells residing in the bone marrow of malnourished mice express higher levels of CCR7 and lower levels of CD11a than control T cells. We also examined the effect of T cell-specific deficiency of the glucocorticoid receptor on T cell migration to the bone marrow in malnourished mice. Indeed, similarly low percentages of glucocorticoid receptor deficient T cells were observed in the bone marrow of malnourished and control mice, indicating that T cell expression of the glucocorticoid receptor is required for T cell migration to the bone marrow. Overall, we have determined that malnutrition modifies both the bone marrow and naïve T cells to promote naïve T cell migration to the bone marrow and that at least the T cell-specific effects are mediated via the glucocorticoid receptor.

NSF-MRI [DBI- 1920116] NSF-RUI [IOS-1951881]
Early Generation and Anatomical Commitment of Stem-like CD8 T cells
Authors: McManus Daniel Thomas , Medina Christopher , Sobierajska Ewelina , Cardenas Maria A , Prokhnevska Nataliya , Hashimoto Masao , Nasti Tahseen , Valanparambil Rajesh , Ross James , Gill Amanda , Scharer Christopher D , Kissick Haydn , Ahmed Rafi
Published: August 2023

Stem-like CD8 T cells sustain the CD8 T cell response during chronic viral infection and cancer, and residency in lymphoid tissues is a key aspect of their biology. We used the chronic LCMV strain, clone 13, to determine when these cells first emerge and commit to positioning in lymphoid sites. Phenotypical and transcriptional profiling of virus-specific CD8 T cells indicated that stem-like and effector fates diverge as early as day 5 after infection. PD-1+ stem-like CD8 T cells sorted from the early (day 5) and established phases (>day 45) of clone 13 infection showed substantial transcriptional overlap indicating that the canonical gene signature of these cells manifests early and is maintained throughout infection. Intravascular labeling and imaging showed that the spatial commitment of these cells to the splenic white pulp also began on day 5. These observations were striking as stem-like CD8 T cells are considered an adaptation to prolonged antigen exposure, yet their fate and anatomical commitments are made before the trajectory of infection is clearly acute or chronic. There were important time-related changes to these cells as well. Early stem-like cells were proliferating and expressed genes involved in the cell cycle, mTOR signaling, and anabolic metabolism. As infection progressed, they upregulated multiple inhibitory molecules and eventually became quiescent. Thus, the stem-like transcriptional and anatomical commitment occurs very early after infection. These data indicate that the CD8 T cell response is equipped for chronic antigen exposure before the trajectory of infection is clearly acute or chronic and further highlight the importance of location for stem-like CD8 T cells.

Supported by grants from NIH (5R01AI030048-29)
TCR signaling dynamically regulates TCF1 expression and effector differentiation
Authors: Favret Natalie , Rudloff Michael W , Zumbo Paul , Betel Doron , Philip Mary
Published: August 2023

T Cell Factor 1, or TCF1, is a transcription factor required for thymic T cell development, memory T cell formation, and maintenance of exhausted/dysfunctional T cell stem/progenitors in tumors and chronic infection. However, TCF1 regulation and function during T cell activation is not well-understood. We analyzed the expression kinetics of Tcf7 mRNA, encoding TCF1 (RNA-SEQ) and TCF1 protein (flow cytometry and western immunoblotting) in CD8 T cells during the initial hours and cell divisions following activation in vivo. Naive CD8 T cells robustly express TCF1. Within 6 hours after activation in L. monocytogenes-infected B6 mice, antigen-specific CD8 T cells downregulated Tcf7 mRNA/TCF1 protein, only to re-express both prior to undergoing the first cell division. We observed a similar pattern of TCF1 kinetics in polyclonal CD8 and CD4 T cells activated with anti-CD3/CD28 antibody and PMA/ionomycin, which activates signaling pathways downstream of the T cell receptor (TCR). We further determined that the magnitude of pre-division TCF1 downregulation correlated with cognate peptide concentration and TCR avidity, but also with higher granzyme B expression during later cell divisions. Thus, our data suggests that the pre-division TCF1 drop is regulated by TCR signaling strength and may determine effector fate. By decoding TCF1 regulation during T cell activation, we could gain insights allowing us to target TCF1 to augment or inhibit effector T cell function in different disease contexts, including cancer, chronic infection, and autoimmunity.

Supported by grants from NHI (T32 GM137793-02, R37 CA263614)
Microbiome protection against Coccidioides growth
Authors: Hoyer Katrina K , Tejeda-Garibay Susana , Zhao Lihong , Hum Nicholas , Loots Gabriela
Published: August 2023

During infection, the host utilizes multiple mechanisms to prevent colonization and dissemination. Protection begins at the surface, often via the commensal community found within our respiratory, skin, and intestinal tract. Microbiota disruption reduces the effectiveness of this protective barrier. Coccidioidomycosis, or Valley fever, is often misdiagnosed as community acquired bacterial pneumonia and treated with antibiotics that alter microbiota density and diversity. Commensal bacteria influence host immune responses to infection, and antibiotic treatment alters host immunity. A soil microbial antagonist related to Bacillus subtilis inhibits in vitro growth of Coccidioides through a clear zone of inhibition between fungi and bacteria. Whether host microbiota also inhibits Coccidioides is unknown. To assess if host microbiota has inhibition capabilities against Coccidioides, we performed two inhibition assays and measured Coccidioides growth area. A 50/50 inhibition assay was performed in which host microbiota and Coccidioides were placed in direct competition on agar plates. A spike in inhibition assay was performed where host microbiota was grown to 80% confluency, to mimic an established in vivo microbiome, before spiking in Coccidioides. Our data indicate that intestinal and respiratory host microbiome species inhibit Coccidioides growth. To assess if host microbiota depletion allows greater Coccidioides growth, in vitro antibiotic disc diffusion assays and in vivo treatments were performed. Our data suggests that host respiratory and intestinal commensals inhibit Coccidioides growth. Antibiotic treatment may kill protective host bacteria that creates a niche for Coccidioides invasion.

Supported by grant VFR-19-633952 from University of California Office of the President.
Development and validation of multiplexed non-human primate ELISA assays
Authors: Christensen Preston B , Nelson Andy B , Tyler Abby B , Berg Kyle B
Published: August 2023

Multiplex ELISA assays are widely used in biomedical research for drug development and other therapeutic interventions. Monitoring of the immune responses of subjects receiving candidate therapeutics is crucial for mitigating adverse reactions and is necessary for the validation of new drug candidates. Non-human primates (NHP) serve as a crucial animal model for development of novel therapeutics due to similarities in immune and organ systems to humans. To support this line of research and maximize data generated from potentially limited samples, new multiplex ELISAs capable of concurrently detecting up to 18 NHP-specific analytes were developed.

The objective of this development was to develop and validate assays for 35 NHP biomarkers. Characterization of assay performance included intra- and inter-assay precision, linearity/parallelism, interference, and assay robustness. Validation and verification testing show functionality and reproducibility of multiplexed NHP panels containing analytes related to cytokine, chemokine, and cancer proteins. Comparisons between stimulated and unstimulated peripheral blood mononuclear cells showed increased concentrations of target analytes.

In conclusion, Quansys Biosciences demonstrated that Q-Plex™ technology is a reliable and accurate tool for measuring NHP specific immunological biomarkers, therefore improving drug research and development processes. This technology will aid in the discovery, diagnosis, and treatment of disease by utilizing NHP models in the discovery process.
The pathogenic anti-PLA2R autoantibodies cooperatively elicit the activation of complement in primary membranous nephropathy
Authors: Wu Tsai-Yi , Tu Kun-Hua , Lin Kung-Wei , Peng Jhan-Jie , Shih Han-Po , Ku Cheng-Lung
Published: August 2023

Primary membranous nephropathy (pMN) is an autoimmune disease associated with autoantibody-mediated immune complexes and the deposition of activated complement proteins on the glomerulus. The phospholipase A2 receptor 1 (PLA2R) has been identified as a major antigen for autoantibodies in 80% of pMN patients. Antibodies of IgG1 and IgG3, but not IgG4, can activate complement. Unexpectedly, in pMN patients, the PLA2R autoantibodies are IgG4 predominance, and those of IgG1 and IgG3 are less abundant in the affected tissues, arguing the role of PLA2R autoantibodies in the pathologies of pMN.

To study the molecular basis of individual PLA2R-reactive IgGs mediating complement activation in pMN, we employed a single-cell capture method and generated 16 anti-PLA2R monoclonal antibodies (mAbs). We characterize mAbs properties including epitope, affinity and gene feature. Then we adopted the CDC assay with PLA2R-podocytes to investigate the complement activation of the mAbs. Our data show that the combination of CTLD1 and any other epitope showed a synergistic effect that enhanced CDC activation, which single epitope anti-PLA2R mAb cannot achieve. Surprisingly, IgG1 mAb can still promote the CDC when pair with IgG4 mAb in synergistic effects. Moreover, polyclonal anti-PLA2R IgGs from pMN patients but not healthy volunteers can induce CDC. Next, we found that a group of patients only have CysR-IgG3, neither CTLD1-IgG1 nor IgG3; when we add CTLD1-IgG1 mAb can enhance CDC activation.

Our data identify an essential role of complement-reactive IgGs, presumably IgG1 and IgG3 synergies between epitopes synergistic effects, and highlight the importance of immune complexes in complement activation by PLA2R autoantibodies, which underlies pMN.
Complement C4 gene copy number variations and polymorphisms, autoantibodies, and clinical manifestations of juvenile dermatomyositis– a multi-center study
Authors: Coss Samantha Lynn , Aziz Rabheh Abdul , Zhou Danlei , Zhou Bi , Miller Katherine , Wu Yee Ling , Ardoin Stacy , Oberle Edward , Driest Kyla , Al Ahmed Ohoud , Patwardhan Anjali , Akoghlanian Shoghik , Sivaraman Vidya , Barbar-Smiley Fatima , Drew Joanne , Spencer Charles , Pachman Lauren , Morgan Gabrielle , Mamyrova Gulnara , Curiel Rodolfo , Jones Olcay , O’Hanlon Terry , Rider Lisa , Miller Frederick , Padyukov Leonid , Lundberg Ingrid , Notarnicola Antonella , Vencovský Jiří , Stibůrková Blanka , Kryštůfková Olga , Yu Chack-Yung
Published: August 2023

Juvenile dermatomyositis (JDM) is an autoimmune myopathy characterized by rash and muscle weakness. Complement C4 gene copy number (GCN) variation is a known risk factor for JDM. JDM patients often develop autoantibodies. We investigated the relationship between C4 GCN, clinical features, and antibody development.

METHODS

Subjects were recruited (n=255) from the US, Sweden, and the Czech Republic. C4 GCN was determined by real time PCR. Comparative analyses were performed via student’s t test or Mann Whitney U test. Correlation was assessed via linear regression or Fisher’s exact test. All studies were IRB approved, and informed consent was obtained.

RESULTS

C4 GCN correlated with muscle pathology and extra-muscular disease at diagnosis. Lower C4A and C4L GCN were associated with a higher number of abnormal muscle enzymes (p=0.0062 and p=0.0029), while the opposite was true for C4S (p=0.024). Higher C4S GCN correlated with lower muscle strength scores (p=0.039). Lower C4L correlated with abnormal MRI (p= 0.042). C4B GCN correlated with dysphagia (p=0.035). Higher GCN of C4S was a risk factor for arthritis (p=0.032), systemic symptoms (p=0.020), and dysphagia (p=0.0078). C4 GCN was associated with myositis-specific (MSA) and myositis-associated (MAA) antibodies. Patients with homozygous C4A deficiency were more likely to test positive for anti-NXP2 (OR 20.0, p=0.011), and C4A GCN was lower in anti-NXP2 positive subjects (p=0.030). A higher C4B GCN correlated with positive MAA (p=0.043).

DISCUSSION

Here, we show that low C4A/C4L GCN and high C4B/C4S GCN correlate with worse muscle disease, extra-muscular pathology, and autoantibodies. Our data suggest that complement C4 may play a key role in the ongoing pathogenesis of JDM.

Supported by grants from NIH (R21 AR070509) and the CureJM Foundation
The Probable Role of Tregs in Improved Heat Tolerance in Heat-acclimated Mice
Authors: Li Jintao , Qiu Minyue , Zhao Lixin , Zhou Yuxin
Published: August 2023

Heatstroke is a life-threatening illness related to systematic inflammation-induced multiple organ dysfunction. The severity of systematic inflammatory response may be related to the changes in immune regulation triggered by heat acclimation. However, the mechanisms of heat acclimation and its effect on immune regulation are unclear. Here, we assessed the differences in immunocyte subsets in the spleen and lymph nodes of heat-acclimated mice. Higher frequencies of CD4+ Foxp3+ regulatory T cells (Tregs) were observed in the heat-acclimated mice. Our results indicate that the improvement in heat tolerance during acute heat stress correlates with the increase in the number of Tregs. These Tregs mitigated the recruitment of neutrophils, inhibited their activation, and suppressed the severity of acute inflammation. The increased differentiation and development of Tregs might stem from an enhanced expression of Foxp3 and PD-L1. Our results strongly suggest that the increase of Tregs has a regulatory effect on neutrophils, and that the PD-1/PD-L1 pathway may be involved in this regulation. The anti-inflammatory effects of Tregs have never been studied in the context of heat stress-induced systemic inflammation. Thus, the results of our study are significant for devising a potential treatment for systemic inflammatory response syndrome and heatstroke.

National Natural Science Foundation of China (grant number 81570497)
Thresholding CITE-Seq-based Antibody Expression Data to Improve Cell Type Identification in scRNA-Seq
Authors: Khan Amir , Oliaeimotlagh Mohammad , Suthahar Sujit Silas Armstrong , Iqneibi Shahad , Nettersheim Felix , Ley Klaus
Published: August 2023

scRNA-seq is widely used to assess immune cell heterogeneity by measuring individual cell transcriptomes. Surface markers have been used for decades to type immune cells by flow and more recently mass cytometry. Expression of surface markers is poorly correlated with the mRNA levels of their encoding transcripts. CITE-Seq provides a systematic approach to analyze surface phenotypes along with cell transcriptomes by using oligonucleotide-tagged antibodies. Like flow cytometry, CITE-seq-based assessment of surface markers is complicated by non-specific binding. Here, we introduce and validate a systematic method of thresholding the levels of protein marker expression to address the issue of non-specific antibody binding. To remove the non-specific signal, a threshold value separating noise from expression for each surface marker is obtained by comparing the antibody signal in a cell type known to not express this surface marker. If no negative cell type is available, we use deconvolution of overlapping normal distributions to find the best separator in density plots (Ridgeline plots) by deconvolution (normalmixEM tool) of overlapping normal distributions. In total, 34 of 49 antibodies tested were reliably thresholded, thus effectively removing the noise from the signal. In a validation experiment testing 188 antibodies at 4 different concentrations, 124 were successfully thresholded, including all 34 mentioned above.

Supported by the National Institutes of Health (NIH) P01 HL136275
Comparative single-cell transcriptome analysis informs early innate immune response in severe human burns
Authors: Dinh Huy , Hou Yiran , Khatri Parth , Rindy Julie , Gao Anqi , Gibson Angela , Huttenlocher Anna
Published: August 2023

Proper wound healing requires a coordinated time-dependent balance of immune pathways to prevent irreversible systemic damage. Given that innate immune cells are first to mobilize to sites of injury, we hypothesize that neutrophils, macrophages, and their interactions are associated with burn severity. To test this, we employed systems immunology and bioinformatics approaches utilizing single-cell transcriptome (scRNA-Seq) in model organisms and large-scale bulk transcriptome of human burns. To this end, we generated the first to-date scRNA-Seq atlas of 6,671 neutrophils and 12,560 macrophages from a time-course zebrafish tail burn model. Our data unveiled transcriptionally distinct subsets that present different dynamics during the wound healing process. We identified the granularity of IL6-IL6R interactions between macrophages and neutrophils, which impair neutrophil recruitment to sterile thermal injury, making the area vulnerable to microbial invasion and subsequent infection. Furthermore, we generated a comprehensive cross-species reference of neutrophils and macrophages in injury models by integrating publicly available mice scRNA-Seq with our zebrafish data. This reference allows us to deconvolute bulk transcriptome data from different published human cohorts and discover signatures of early innate immune cells associated with burn severity in humans. Our discovery demonstrates the power of using comparative bioinformatics approach to better understand the innate immune system during wound healing. In the future, these advances will guide interventional genomics approaches to identify druggable pathways to optimize burn wound healing in human patients.
The Serological Sciences Network (SeroNet); the U.S. government’s effort to boost testing capacity and escalate standardized research on immune responses to SARS-CoV-2
Authors: Roche Nancy V , Hickey Tom , Freund Elaine , Breads Patrick , Hempel Heidi , Berny-Lang Michelle , Klemm Juli , Finstad Samantha , Cherry Jim , Pinto Ligia , Singer Dinah V
Published: August 2023

In April 2020, the National Cancer Institute (NCI) and the Frederick National Laboratory for Cancer Research (FNLCR) built the Serological Sciences Network(SeroNet) on the foundation of the HPV Serology program to develop serology technologies to better understand the immune response to infection and vaccines. SeroNet members (steering committee, investigators, working groups) are funded by NCI (through The Paycheck Protection Program and Healthcare Enhancement Act) to characterize immune responses elicited by SARS-CoV-2 and the modifiers and mechanisms driving these responses, the serological correlates of disease pathogenesis/protection, and the barriers to SARS-CoV-2 serological testing. Studies were conducted by 8 Centers of Excellence and 13 Research Projects, with reference material development and longitudinal vaccine response studies conducted at FNLCR and 4 subcontracted Capacity Building Centers (CBCs). CBC testing capacity has reached >5,000 tests/week and has collected >8000 aliquots of serum/peripheral blood mononuclear cells (PBMC) samples and ~3000 healthy control/immunocompromised patients (serum, PBMC’s, clinical/follow up data) for use by SeroNet. SeroNet has produced 332 papers on correlates of protection and characterization of the immune response. Study level data for 87 papers is found in the Immunology Database and Analysis Portal (ImmPort). SeroNet has produced study design templates, standard operating procedures, common data elements, and well-characterized SARS-CoV-2 serology standards, such as the first US SARS-CoV-2 serology standard, which is calibrated against the WHO International Standard and evaluation panels to facility assay development and validation.

National Cancer Institute
Clonal dynamics of SARS-CoV-2 specific CD4 T cells in lymph nodes after vaccination
Authors: Bartolo Laurent , Pan Yi-Gen , Afroz Sumbul , Su Laura
Published: August 2023

The ongoing COVID-19 pandemic highlight the importance of vaccination. Past studies have surveyed SARS-CoV-2 specific B cell response in the lymph node (LN) of vaccinated patient, but the quality, the dynamics, and the specificity of the T helper response following mRNA vaccination remains incompletely understood. We address this question in healthy individuals receiving mRNA vaccine (BNT162b or mRNA-1273). We collected axillary draining LN using fine needle aspiration following vaccination and analyzed CD4+ T cells by single cell transcriptome, proteome analysis and TCR sequencing. We observed an accumulation T cell having characteristic signature of activated T follicular helper cell (Tfh), through the course of the 6 months, as well as an increase expansion of Tfh clones. Using TCR sequences to track progenies of same T cell over time, we observed that some Tfh cells in the 6 months’ time point were already detectable after the first vaccine dose. We then generated a library of SARS-CoV-2 specific TCR sequences by performing TCR sequencing on cells that proliferated in response to a spike peptide pool. The majority of these TCR sequences mapped to cells with a strong Tfh signature on the transcriptomic level and accumulated after 6 months. In conclusion, we observed an expansion of SARS-CoV-2 specific Tfh clones after the first dose of vaccine, some of which persisted after 6 months. We hypothesize that, in a protective SARS-CoV-2 response, short-lived clones are progressively replaced by long-lasting SARS-CoV-2 specific Tfh clones. We plan to test this hypothesis by analyzing clonal dynamics at other time points and by using aggregate TCR sequences from the publicly available sequences from individuals with mild or severe COVID-19.

NIH R01AO66358
Spike protein vaccine triggers systemic Th2 and Th17 inflammatory responses and pulmonary immunopathology following SARS-CoV-2 breakthrough infection
Authors: Zhang Tianyi , Magazine Nicholas , McGee Michael C , Babawale Pius , Upshaw Sarah , Carossino Mariano , Kousoulas Konstantin G , August Avery , Huang Weishan
Published: August 2023

Vaccines have shown remarkable protection against COVID-19, but vaccination-enhanced respiratory disease (VAERD) following breakthrough infections poses safety concerns. SARS-CoV-2 Spike (SARS-2 S) protein subunit vaccines induced VAERD in hamsters, where aluminum adjuvant primed a Th2-biased immunization, resulting in elevated type 2 pulmonary inflammation in animals with breakthrough infections. To further understand the potential risks and immunopathogenesis of VAERD, we immunized K18-hACE2 mice with SARS-2 S protein in the presence of aluminum and CpG ODN adjuvants and infected them with a lethal dose of SARS-2. The vaccine induced robust antibody and T cell responses, significantly reducing viral titers and increasing host survival. However, following breakthrough infection, vaccinated animals exhibited severe airway immunopathology, with a dramatic increase of eosinophils and CD4+ T cells, and elevated Th2/Th17 cytokines in the lungs. Intracellular flow cytometric analysis showed a systemic Th2/Th17 inflammatory response, prominently in the lungs. Our data show that the aluminum/ODN adjuvants elicit strong protection against COVID-19 but prime a robust Th2/Th17 inflammatory response that may contribute to rapid onset of T cell-mediated pulmonary immunopathology following breakthrough infection. Our results highlight the need for additional studies to elucidate COVID-19-associated VAERD and to further improve vaccine formulations for broad protection and maximal safety.

This research was supported in part by grants from the National Institutes of Health (P20 GM130555-6610 and R01 AI151139).
Memory CD4 T cell recall following Influenza A Virus infection is unaltered by the pregnancy environment
Authors: Malavet Valeria Flores , Prokop Emily , Strutt Tara M
Published: August 2023

The risk of severe influenza A virus (IAV) infection increases during pregnancy. A widely accepted paradigm suggests that this increased severity is resultant from immunity that is shifted towards Th2- versus Th1-immune responses, the latter of which is essential for control of virus. While this concept is accepted for primary immune responses, whether memory T cell responses are similarly shifted during pregnancy is unclear. Using a reductionist murine model, here we explore whether and how adoptively transferred, Th1-polarized IAV-specific memory CD4 T cells responding against IAV are impacted by pregnancy. Following infection, we find that donor cell migration to the lung, surface marker phenotype, T-BET transcription factor expression, multi-cytokine production capacity in terms of IFN-γ, TNF, and IL-2, and ability to control virus in the lung are comparable between non-gravid and gravid hosts. A significant increase in donor CD4 T cell Th2-associated GATA-3 transcription factor expression and cytokine production following IAV infection was not observed in gravid hosts. Enhancement of Th2-associated IL-4, IL-5, and IL-13 cytokine production was only observed in infected hosts when IAV-specific Th2-polarized CD4 T cells already poised to produce these cytokines were adoptively transferred. These findings suggest that in context of an IAV infection during pregnancy, preexisting Th1-polarized IAV-specific memory cells retain their protective recall potential. Furthermore, they support that employment of vaccination strategies that induce universal T cell immunity prior to pregnancy are a potential means to protect the vulnerable pregnant population from severe seasonal as well as pandemic IAV infection.

The Solomon Klotz Excellence in Immunology Award Florida Education Fund McKnight Doctoral Fellowship Supported by grants from the NIH Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) (5R21HD093948-02)
Clonal replacement sustains long-lived germinal centers primed by respiratory viruses
Authors: de Carvalho Renan , Ersching Jonatan , Barbulescu Alexandru , Mesin Luka , Victora Gabriel
Published: August 2023

Germinal centers (GCs) form in secondary lymphoid organs in response to infection and immunization and are the source of affinity-matured B cells. The duration of GC reactions spans a wide range, and long-lasting GCs (LLGCs) are potentially a source of highly mutated B cells. We show that, rather than consisting of continuously evolving B cell clones, LLGCs elicited by influenza virus or SARS-CoV-2 infection in mice are sustained by progressive replacement of founder clones by naïve-derived invader B cells that do not detectably bind viral antigens. Rare founder clones that resist replacement for long periods are enriched in clones with heavily mutated immunoglobulins, including some with very high affinity for antigen, that can be recalled by boosting. Our findings reveal underappreciated aspects of the biology of LLGCs generated by respiratory virus infection and identify clonal replacement as a potential constraint on the development of highly mutated antibodies within these structures (de Carvalho et al, Cell, 2023).

NIH/NIAID grants R01AI119006, R01AI139117, and R01AI157137. Additional support by NIH grant DP1AI144248 (Pioneer award) and the Robertson Foundation.
The Interconnection Between Periodontitis and Inflammatory Bowel Disease
Authors: Tanwar Himanshi , Gnanasekaran Jeba Mercy , Allison Devon , Das Amitabh , Yesupatham Sathish , Dilshad Yasir , Kear Bernice , Wang Sheng , Wang Xiaobei , Chung Man-Kyo , Scott Alison , Rasko David , Ernst Robert , Raufman Jean-Pierre , Math Vivek Thumbigere
Published: August 2023

Objective

Periodontitis, an oral infection affecting ~50% of Americans, is associated with the pathogenesis of inflammatory bowel disease (IBD), however, the contributory mechanisms remain ill defined. Previous GWAS studies have identified SNPs in the Interferon Regulatory Factor 8 (IRF8) gene as risk factors for IBD. The objective of this study was to explore the relationship between periodontitis and IBD using Irf8 knockout (KO) mice.

Methods

Periodontitis was induced in wild-type (WT) and Irf8 KO mice using the well-established oral ligature-induced periodontitis (LIP) model. The effects of LIP on oral bacterial colonization in the mouth and gut were measured by 16S rRNA and shotgun metagenomics sequencing. The influence of LIP on oral bacteria-mediated immune cell infiltration in the colonic mucosa was measured by flow cytometry, RT-qPCR, and ELISA.

Results

LIP promoted expansion of native oral pathogens and accumulation of the same species in the mouth and gut of Irf8 KO mice, which was associated with colitis development in Irf8 KO mice when compared to WT mice. LIP Irf8 KO mice exhibited ~2-fold increased infiltration of Th17 cells and ~2–4-fold decrease infiltration of Th1 and Treg cells in the colonic mucosa, which led to over production of IL17 and decreased production of IFN-γ and IL10. Similarly, IL-1β-producing cells were found to be higher in the colonic mucosa of LIP Irf8 KO vs WT mice. Resolution of periodontitis correlated with reduced intestinal load of oral bacteria and reversal of colitis in Irf8 KO mice.

Conclusion

Our study provides new perspectives on the pathophysiology of IBD. Irf8 deficiency along with dysbiotic oral and gut microbiota promote colitis in mice by activating IL17 and IL-1β signaling pathways.
Cell-autonomous complement C3 maintains prostate epithelial cell homeostasis and prevents malignant transformation
Authors: Kunz Natalia , West Erin E , Yamamoto Hidekazu , Tajmul Md , Loetscher Jonas , Kellerhals Tamara , Kolev Martin , Balmer Maria L , Chandra Ash , Seymour Len , Klos Andreas , Hess Christoph , Afzali Ben , Kazemian Majid , Dasgupta Prokar , Kemper Claudia
Published: August 2023

Intracellularly active complement C3 and C3a emerged as central regulators of normal immune cell function and proliferation via control of mTOR and glycolysis. Because increased glycolysis is a hallmark of many cancer cells, we hypothesized that augmented cell-intrinsic C3 activity may contribute to epithelial cell malignancy.

However, while benign prostate epithelial cells (PECs) harbored expected intracellular C3/C3a storages, cancerous PECs, surprisingly, exhibited loss of C3/C3a. Further, C3/C3a reduction levels in PECs correlated inversely with worsening Gleason scores in patients, silencing of C3 in healthy PECs induced uncontrolled proliferation, and reconstitution of intracellular C3a in prostate cancer cell line DU145 normalized their hyper-proliferation. In line with an unexpected anti-proliferative role for C3a in PECs, C3ar−/− mice developed spontaneous hyperplasia of prostate tubular cells with age, treatment of DU145 tumors, implanted subcutaneously into the flanks of athymic mice, with C3a-expressing adenovirus significantly reduced tumor growth in vivo, and augmented C3 levels are associated with a better prognosis in patients with prostate and other types of epithelial cell cancers. RNA-sequencing of C3 or C3aR-deficient human PECs identified intrinsic C3a as novel controller of several known PEC oncogenes, likely via up-stream impact on the c-MYC pathway.

In sum, while intracellular C3 drives proliferation in immune cells, in (prostate) epithelial cells, cell-autonomous C3/C3a is a negative regulator of growths and, thus, represses oncogenic transformation. Such cell-specific intracellular C3/C3a activities should be taken into consideration when targeting this system therapeutically.
PD1 blockade promotes IFN-γ production by progenitor exhausted PD1-expressing T cells in atherosclerotic plaques
Authors: Mulholland Megan , Yu Yinda , Lin Shiying , Jakobsson Gabriel , Schiopu Alexandru , Björkbacka Harry , Lichtman Andrew H , Gisterå Anton , Engelbertsen Daniel
Published: August 2023

Objective

Interferon-γ (IFN-γ) is a key cytokine regulating the development of atherosclerosis, but little is known regarding the phenotype and regulation of T cells producing IFN-γ in plaques.

Methods

To identify and characterize IFN-γ-producing T cells in the development atherosclerosis, we utilized IFN-γ-YFP and Nur77-GFP reporter mice crossed onto an atherosclerotic background (Apoe−/− IFN-γYFP/YFP; Apoe−/−Nur77GFP/wt), adoptive transfer of transgenic ApoB-reactive T-cells into mice containing humanized ApoB100 (HuBL mice), and treatment with immune checkpoint inhibitors in atherosclerotic mice.

Results

Aortic CD4+ and CD8+ T cells producing IFN-γ were a complex set of cells comprised predominantly of “progenitor exhausted” PD1-intermediate (PD1int) SLAMF6+ IL7R+ T cells and a minor population of “terminally exhausted” PD1hi TOX+ T cells. However, both PD1int and PD1hi plaque T cells were Ki67+ and displayed elevated levels of recent TCR signaling (Nur77-GFP+). ApoB-specific transgenic CD4+ T cells adoptively transferred to a hypercholesterolemic HuBL mice showed sign of early exhaustion, upregulating PD1, TOX, and Tim3. Finally, PD1 blockade in mice with advanced atherosclerosis resulted in increased levels of IFN-γ production of plaque-infiltrating CD4+ and CD8+ T cells.

Conclusion

IFN-γ-producing T cells in the atherosclerotic plaque display markers classically associated with an exhausted T cell phenotype but still produce cytokines, proliferate, and are responsive to anti-PD1 treatment. Our study suggests that PD1 signaling regulates T cell IFN-γ production in plaques and reveals a potential mechanism by which immune checkpoint blockade therapy aggravates cardiovascular disease.

Supported by grants from Swedish Heart-Lung Foundation (#20200522, #20190449) sourced by Daniel Engelbertsen.
The functional role of Helios in CD4+ T cells
Authors: Yang Neil X , Tison Kole , Penna Vinay , Oguz Cihan , Lack Justin , Shevach Ethan M , Thornton Angela M
Published: August 2023

The transcription factor Helios is expressed in the majority of Foxp3+ Regulatory T cells (Treg), where it plays an essential role in Treg function. Loss of Helios in the Treg population induces systemic autoimmunity, marked by CD4 and CD8 hyperproliferation, enhanced germinal center formation, and systemic immune activation. Helios is also expressed in a small subset of conventional CD4+ T cells. However, when Helios is deleted in all CD4 cells, conventional and Treg, by crossing the mice to CD4Cre, this phenotype is abrogated, indicating that Helios must also play a significant role in the function of conventional CD4+ T cells.

In order to examine specific subsets of CD4+ memory cells, we performed single cell transcriptome analysis of CD44hi memory cells from WT mice and mice with the CD4 specific deletion of Helios. Characterization of the CD44hi Helios+ population showed that Helios expression changed dramatically with age, so cells from young (5–6 weeks) and old (5–6 months) mice were included in the analysis. Both longitudinal (young vs old) and cross-sectional (WT vs KO) comparisons were made, with emphasis placed on cross-sectional comparisons at old age. The sequencing results indicated significant transcriptomic and numeric shifts in multiple subpopulations of the analyzed splenocytes, including the almost complete loss of one cluster in both the young and old Helios deficient mice. Taken together, the observed transcriptomic and clustering changes may explain the role Helios plays in the abrogation of autoimmunity in our model and may provide a starting point for us to analyze its function in other biological pathways.

This work was supported by the Intramural Research Program of NIAID, NIH

This work was supported by the Intramural Research Program of NIAID, NIH
Paternal cadmium exposure decreases DMNT2 expression and increases obesity in male offspring
Authors: McCall Jamie L , Martinez Ivan , Barnett John B
Published: August 2023

Prenatal exposure to toxic agents, including cadmium (Cd), can alter offspring immune cell development and function. As Cd does not readily cross the placenta, it is hypothesized that prenatal Cd alters the epigenetic programming of genes including non-coding RNAs (ncRNAs). We found that prenatal Cd exposure increases CD4+ T cell proliferation following activation and that this phenotype is dependent on the upregulation of an oncogenic long non-coding RNA (lncRNA) known as small nucleolar RNA host gene 7 (lncSnhg7). In our previous studies, both parents were exposed. To determine the contribution of paternal exposure, we treated male mice with 10 ppm Cd in their drinking water for 7 days and then mated them with unexposed females. The male offspring of these mice were significantly larger than control mice at one year of age (p<0.001). The offspring had increased fat mass (p=0.04) and reduced lean mass (p=0.03) as compared to controls as well as increased macrophages within the visceral adipose tissue. We hypothesized that sperm epigenetic alterations contribute to the phenotypes observed in the offspring. Cd exposure has been reported to alter sperm DNA methylation patterns in mice. Furthermore, DMNT2, a tRNA methyltransferase, has been reported to mediate intergenerational transmission of paternally acquired metabolic disorders by altering sperm small non-coding RNAs. We measured DNMT1, DNMT2, DNMT3a, and DNMT3b RNA expression in the sperm of Cd-exposed mice. The DNA methyltransferases (1, 3a, and 3b) were unaffected, but DNMT2 was significantly reduced in the sperm of Cd-exposed males. Thus, acute paternal exposure to Cd prior to conception alters DNMT2 expression which may lead to obesity and increased inflammation in the offspring.

Supported by grants from NIH/NIGMS (R01 ES023845, 2U54 GM104942-03, P20 GM103434, U54 GM-104942) and NSF (NSF/1920920, NSF/1761792).
MR1-deficiency in neonatal mice renders protection from necrotizing enterocolitis
Authors: Liu Jianyun , Hunter Chelsea , Manohar Krishna , Mesfin Fikir , Shelley William Christopher , Brokaw John , Markel Troy
Published: August 2023

Necrotizing enterocolitis (NEC) is a life-threatening disease that affects ~ 7% of premature neonates. NEC maintains high morbidity/mortality and there are long term sequelae including short bowel syndrome. The risk factors of developing NEC include prematurity, very low birth weight, microbial dysbiosis and infections. Mucosal-associated invariant T (MAIT) cells are innate T cells that have both innate and adaptive immune cell characteristics and are enriched in mucosal tissues, such as intestines. MR1 is the antigen presenting molecule for MAIT cells. Therefore, MR1-knockout (MR1 KO) mice lack MAIT cells. We hypothesized that MR1/MAIT cells play a key role in the development of NEC. To test the hypothesis, NEC was induced in 5-day old B6 wild type (WT) and MR1 KO mouse pups with gavage formula feeds supplemented with LPS as well as exposure to intermittent hypoxia and hypothermia. Pups were assessed daily for clinical sickness scores. Animals were euthanized on postnatal day 9. Intestine segments were harvested and assessed for histological NEC and cytokines were measured using multiplex assay. Compared to WT mice, we found MR1 KO pups did not develop typical NEC symptoms. When compared to WT mice with NEC, MR1 KO mice undergoing the treatment for NEC, had lower clinical scores (WT=4 [3–4]; MR1 KO=0 [0–0], p<0.05) and intestinal histology scores (WT=1.5 [1–2.375]; MR1 KO=0 [0–0.3745], p<0.05). The cytokine profile of the intestines in MR1 KO NEC mice, when compared to those in WT NEC mice, showed significant differences in IL-6 and IL-10 levels. In conclusion, deletion of MR1 in mice appears to be protective in experimental NEC. Future work will focus on studying the role MR1/MAIT cells in NEC disease development.
TSLP acts on Tregs to limit effector Th2 cell generation during type 2 immune responses
Authors: Gurram Rama Krishna , Oh Jangsuk , Leonard Warren J
Published: August 2023

Thymic stromal lymphopoietin (TSLP) is a type 2 alarmin secreted by epithelial cells to promote type 2 inflammation. After antigen recognition, naïve CD4 T cells differentiate into effector T (Teff) cells, whereas regulatory T (Tregs) cells suppress the immune response to prevent adverse effects caused by inflammation. Therefore, a delicate balance between Teff and Treg activity is required for the homeostasis of immune responses. Here we generated a TSLPR conditional KO mice (Crlf2fl/fl) to investigate the functional role(s) of TSLP on Teff and Tregs. Conditional deletion of TSLPR on T cells (Cd4CreCrlf2fl/fl) resulted in lower numbers of eosinophils and Th2 cells with OVA-induced airway inflammation. In contrast, conditional deletion of TSLPR on Tregs (Foxp3CreCrlf2fl/fl) resulted in elevated numbers of eosinophils and increased IL-5- and IL-13-secreting effector Th2 cells. Furthermore, phenotypic analysis of Foxp3+ Tregs during type 2 immune responses shows that Tregs from Foxp3CreCrlf2fl/fl mice have upregulated levels of GATA3 and secrete IL-13. Together, these data indicate that TSLP not only promotes suppressive activity by Tregs in the generation of Th2 cell-mediated responses but that it also restrains GATA3 production and IL-13 secretion by the Treg cells, indicating a key role for TSLP in shaping an immunosuppressive environment during type 2 immune responses.

This research was supported by the Intramural Research Programs of NHLBI, NIH.