bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inactivation of GSK3β by Ser389 phosphorylation prevents thymocyte necroptosis and impacts Tcrβ and Tcrα repertoire diversity
Authors: Pereira Felipe , Sheridan Ryan , Riemondy Kent , Thorton Tina , Fang Qian , Barret Brad , Paludo Gabriela , Thompson Claudia , Collins Patrick L , Santiago Mario , Oltz Eugene M , Rincon Mercedes 
Published: August 2023

Abstract
Double negative (DN) thymocytes go through consecutive rounds of Tcrβ and Tcrα gene recombination that involves the generation of DNA double-strand breaks (DSB), the addition of N-nucleotides between coding ends, and DNA repair with the ultimate goal of enhancing TCR repertoire diversity. How DN thymocytes can overcome such genomic instability in order to generate functional Tcrβ and Tcrα genes remains unclear. We performed scRNA-seq analyses of isolated DN3 and DN4 thymocytes to demonstrate a clear trajectory from DN3 through the DN4 stage that is delineated by G1 and G2M cell cycle checkpoints to facilitate DNA repair during recombination of Tcrβ and Tcrα prior to cell proliferation. Inactivation of GSK3β by phosphorylation on Ser389 in response to V(D)J recombination DSB is required to prevent thymocyte death by necroptosis. Failure to inactivate GSK3β through this pathway alters the Tcrβ and Tcrα V segments usage. We also show that inactivation of GSK3β by phosphorylation on Ser389 is required for expression of the cytidine deaminase Apobec3 in DN3 thymocytes to increase N-nucleotide diversity within the Tcrβ N2-region. Thus, G1 and G2M cell cycle checkpoints to ensure proper DNA repair during V(D)J recombination and unique GSK3β-dependent survival pathways to prevent necroptosis during this process are critical to generate a large diversity of pre-selection TCR repertoire.
Double negative (DN) thymocytes go through consecutive rounds of Tcrβ and Tcrα gene recombination that involves the generation of DNA double-strand breaks (DSB), the addition of N-nucleotides between coding ends, and DNA repair with the ultimate goal of enhancing TCR repertoire diversity. How DN thymocytes can overcome such genomic instability in order to generate functional Tcrβ and Tcrα genes remains unclear. We performed scRNA-seq analyses of isolated DN3 and DN4 thymocytes to demonstrate a clear trajectory from DN3 through the DN4 stage that is delineated by G1 and G2M cell cycle checkpoints to facilitate DNA repair during recombination of Tcrβ and Tcrα prior to cell proliferation. Inactivation of GSK3β by phosphorylation on Ser389 in response to V(D)J recombination DSB is required to prevent thymocyte death by necroptosis. Failure to inactivate GSK3β through this pathway alters the Tcrβ and Tcrα V segments usage. We also show that inactivation of GSK3β by phosphorylation on Ser389 is required for expression of the cytidine deaminase Apobec3 in DN3 thymocytes to increase N-nucleotide diversity within the Tcrβ N2-region. Thus, G1 and G2M cell cycle checkpoints to ensure proper DNA repair during V(D)J recombination and unique GSK3β-dependent survival pathways to prevent necroptosis during this process are critical to generate a large diversity of pre-selection TCR repertoire. Supported by NIH R01 AI051454
Defining the Role of HDAC3 Deacetylation and Decrotonylation in T Cells
Authors: Martinez Kodi , Shapiro Michael , Rajcula Matthew , Lee Hyun Se Kim , Hammer Brady , Mai Elissa , Shapiro Virginia 
Published: August 2023

Abstract
Histone acetyltransferases (HATs) and histone deacetylases (HDACs) facilitate addition or removal of various acyl groups from lysine residues, including acetylation (KAc) and crotonylation (KCr). Recently, mutations in p300 and CBP were identified that abrogate their function as an acetyltransferase but not the ability to mediate crotonylation. These mutants can activate transcription independent of their ability to acetylate histones, demonstrating that crotonylation can also lead to transcriptional activation. The Class I deacetylases HDAC1, HDAC2, and HDAC3 have been shown to have both decrotonylase and deacetylase activity, but the relative importance of each function in T cells is not known. Genetic knockout of HDAC3 using CD2 icre mice results in a severe block in T cell development and spontaneous development of severe colitis. Examination of HDAC3 cKO mice has shown an aberrant ratio of Treg:Th17 cells within the gut, driving inflammation and loss of mucosal barrier integrity. Our lab has also determined that HDAC3 is required for at least three aspects of T cell development: CD4+ CD8+ (DP) cell survival, positive selection, and CD4+ lineage development. Recently, a mutation was identified in either HDAC1 or HDAC3 that eradicated deacetylase activity but retained decrotonylase activity (“VRPP”). Through this mutation we were able to generate a novel mouse model with Cre-mediated deletion of endogenous HDAC3 and linked re-expression of HDAC3 VRPP. Strikingly, this mouse does not develop colitis, although T cell development is still impaired. Thus, HDAC3 decrotonylase activity alone is sufficient to impede the IBD phenotype.
Support by grants from National Institute of Allergy and Infectious Diseases (R01 AI150100)
Learning to live with the thymic roller coaster
Authors: Kang Seung Woo , Manley Nancy R 
Published: August 2023

Abstract
The thymus is the primary lymphoid organ responsible for naïve T-cell production, vital for adaptive immunity. Thymus size and T-cell output dynamically change throughout the lifetime. The thymus increases its size and output during the perinatal stage; hits a plateau around postnatal day 10 and maintains homeostasis during the juvenile stage; then starts to undergo involution. The perinatal thymus is unique, for it exponentially increases the T-cell export whilst generating virtual memory-like T-cells (TVM), poised to respond to foreign antigens.
Our questions are how the thymic microenvironment changes from perinatal expansion to juvenile homeostasis and how FOXN1 regulates the transition. FOXN1 is a master transcription factor that is exclusively expressed in thymic epithelial cells (TECs) and works in a highly dosage-sensitive fashion to determine TEC differentiation and proliferation as well as the total thymic microenvironment via crosstalk. In this study, we use the Foxn1-lacZ model that has a normal Foxn1 expression level during development but has 60% decreased Foxn1 expression level around postnatal day 7. Using scRNA-seq of total thymic stroma, we are studying stromal changes from perinatal expansion to juvenile homeostasis and the effect of decreased Foxn1 level on the transition. We have found changes in TEC subsets, endothelium and fibroblasts before and after the transition contributing to the different perinatal thymic microenvironments. Moreover, we have found a decrease in Foxn1 level causes accelerated transition with the premature occurrence of ageing TECs. Successful completion of the project will determine how the thymic stroma changes across perinatal to juvenile and how Foxn1 regulates the transition.
Supported by grans from NIH-NIAID 1P01AI139449-01A1.
Going oversized: Testing the limits of endogenous thymic regeneration and uncovering the mechanisms of thymic hypertrophy
Authors: Acenas Dante Dennis V , Granadier David , Iovino Lorenzo , Cooper Kirsten , deRoos Paul , Evandy Cindy , Hernandez Vanessa , Dudakov Jarrod 
Published: August 2023

Abstract
The thymus is the primary site of T cell development, generating a diverse repertoire of T cells that protects us from various threats. Despite its crucial role in T cell immunity, the thymus undergoes chronic involution that leads to a decline in T cell production and impaired T cell immunity. Although the thymus is highly sensitive to various forms of damage, and everyday insults like stress and infection, it also has a remarkable capacity to regenerate itself. Though this reparative capacity declines with age. We hypothesized that age-related thymic decline may be attributed to repeated damage and regeneration cycles in response to everyday insults. We expected impaired regeneration with each round of damage. However, we found that after three rounds of damage and repair cycles, overall thymic cellularity consistently returned to homeostasis. Moreover, we also found that unlike injury from ionizing radiation, Dexamethasone (Dex) mediated damage led to thymic hypertrophy.
Preliminary studies revealed that type 2 cytokines, previously implicated to promote thymic regeneration, are elevated in the early phases of Dex induced damage, but not during irradiation. Comparing thymic cellularity between damage models, we found that Dex induced damage had faster recovery kinetics and led to thymic hypertrophy 28 days after damage. We hypothesize that type 2 immunity may be a key contributor to hypertrophic thymic regeneration in Dex induced thymic damage. We will test our hypothesis through genetic mouse models, cytokine modulation, and standard immunophenotyping. The positive results of this study will augment our basic knowledge of the endogenous mechanisms of thymic regeneration and inform the creation of thymic boosting therapies.
I am supported in part by PHS NRSA T32GM007270 from NIGMS.
Nucleated red blood cells: unexpected orchestrators of fetal immunity
Authors: Clements Rebecca Lynn , Jurado Kellie Ann 
Published: August 2023

Abstract
Red blood cells (RBCs) are not typically considered active immune mediators. This dogma stems from the fact that RBC precursors discard organelles as they mature, thus losing the ability to alter gene expression in response to stimuli. Intriguingly, nucleated RBCs (nRBCs) circulate in human fetuses and neonates. Due to evolutionary pressure for successful reproduction, circulation of nRBCs during pregnancy is likely important. However, the role of nRBCs in utero remains unknown. To define the role of human nRBCs, we queried single cell RNA-seq data and found that transcriptomics support nRBCs as putative immune mediators.
Unexpectedly, we found that nRBCs express antigen processing & presentation machinery. nRBCs constitutively express MHC II & co-stimulatory molecules, hallmarks of specialized antigen-presenting cells. We further demonstrated that nRBCs internalize and cleave antigens. Currently, we aim to determine if nRBCs present antigens to T cells and to characterize the elicited response.
In addition, we found that nRBCs express pattern recognition receptors capable of detecting pathogens and initiating an antimicrobial response. We demonstrated that nRBCs respond to viral infection through upregulation of antiviral genes, interferons/interferon-stimulated genes, MHC I, and pro-inflammatory cytokines/chemokines. These data suggest a putative functionality for nRBCs in mediating antiviral immunity in utero. We are currently investigating antiviral functions of nRBCs.
Together, our findings shed light on an unexpected orchestrator of fetal immunity. Knowledge of the fetal immune system has potential to help us understand health and disease during pregnancy, as a neonate, and likely every stage of life after.
Supported by grants from NIH/NIDDK 5T32DK007780, NIH/NIGMS K12GM081259, Burroughs Wellcome Fund 2022 Next Gen Pregnancy Initiative Award
Understanding NKT cells through sulfatide analogues
Authors: Hsu Kevin , Olkhanud Purevdorj B , Camara Kaddy , Luvaga Irunga K , Kanyo Emese , Richardson Steward K , Sassu Nicole , Howell Amy , Berzofsky Jay A 
Published: August 2023

Abstract
The goal of this research is to further investigate NKT cell activation using sulfatide analogues. NKT cells have been shown to play a significant role in mediating inflammatory immune responses and because of this, there has been strong interest in pharmacologically regulating this compartment to fight cancer. NKT cells recognize lipids that are loaded on CD1d molecules on the surface of cells, and it has been shown that structural modifications of these lipids directly affect NKT cell activation and polarization. Our work is focused on the impact of modifications of sulfatide, which is a glycolipid that can be loaded onto CD1d which is known to activate sulfatide-reactive (generally type II) NKT cells when presented by CD1d monomers on plastic. We have numerous sulfatide analogues with modifications (based on previous work with alpha-galactosylceramide analogues) on the acyl chain, sphingosine chain, and changes of beta-anomeric to alpha-anomeric. These analogues have been screened with NKT cell hybridomas, DN32 and XV19, to assess their effect on activation of type I and type II NKT cells, respectively. We observed that alterations in the sphingosine or acyl chain in sulfatides do not always mimic the effects on activity of similar changes in alpha-galactosylceramide analogues and that numerous sulfatide analogues can activate DN32 (not sulfatide-reactive) when presented on CD1d monomer or bone marrow-derived dendritic cells. Our results provide further insight into NKT cell activation and have identified sulfatide analogues that may be applicable to cancer therapy by activating type I NKT cells.
NCI, NIH intramural research program (NIA-C-004020), and institutional grants of NIH (R01 GM111849)
Protein-polymer Bioconjugate Nanoparticles To Investigate Innate and Antigen-Specific CD4 T Cell Responses
Authors: Scotland Brianna L , Cottingham Andrea L , Lasola Jackline J , Pearson Ryan M 
Published: August 2023

Abstract
Nanoparticles (NP) comprised of poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid) (PLA) have been shown to be effective delivery systems for peptide and protein antigens (Ags) capable of modulating Ag-specific T cell responses. Encapsulation and surface conjugation are common Ag association methods using NPs. However, these methods are associated with caveats such as imprecise Ag loading, high Ag burst release, and immune recognition. To improve the efficiency of Ag delivery and address these limitations, we prepared ovalbumin (OVA)-PLGA conjugates that were systematically combined with unmodified PLGA to produce NPs (acNP-OVA) with three precise Ag loadings, negligible immune recognition, and a reduced burst release. acNP-OVA was used to elucidate how NP formulation parameters, such as Ag content, influence innate and Ag-specific CD4+ T cell responses. We observed Ag loading-dependent innate immune cell activation (MHCII, CD80/86) and Ag-specific CD4+ T cell proliferation, cytokine secretions, and regulatory T cell induction in vitro. Ag-encapsulated NPs were employed as a control. CD25 expression and T cell expansion were achieved using both Ag delivery methods and observed at low Ag loading. However, high Ag loading was required to induce IFNg and IL-2 cytokine secretions. Notably, nanostring analysis identified inflammation-associated genes, such as S100a9, was significantly downregulated in dendritic cells due to NP treatment. We anticipate this study will aid in designing NP-based immunotherapies for Ag-specific immunomodulation.
Gene Set Enrichment Analysis Reveals That Fucoidan Induces Type I IFN Pathways in BMDC
Authors: Choi Suyoung , Jeon Sol A , Heo Bu Yeon , Kang Ju-Gyeong , Jung Yunju , Thuy Duong Pham Thi , Song Ik-Chan , Kim Jeong-Hwan , Kim Seon-Young , Kwon Jaeyul 
Published: August 2023

Abstract
Fucoidan, a sulfated polysaccharide extracted from brown seaweed, has been proposed to effectively treat and prevent various viral infections. However, the mechanisms behind its antiviral activity are not completely understood. We investigate here the global transcriptional changes in bone marrow-derived dendritic cells (BMDCs) using RNA-Seq technology. Through both analysis of differentially expressed genes (DEG) and gene set enrichment analysis (GSEA), we found that fucoidan-treated BMDCs were enriched in virus-specific response pathways, including that of SARS-CoV-2, as well as pathways associated with nucleic acid-sensing receptors (RLR, TLR, NLR, STING), and type I interferon (IFN) production. We show that these transcriptome changes are driven by well-known regulators of the inflammatory response against viruses, including IRF, NF-κB, and STAT family transcription factors. Furthermore, 435 of the 950 upregulated DEGs are classified as type I IFN-stimulated genes (ISGs). Flow cytometric analysis additionally showed that fucoidan increased MHCII, CD80, and CD40 surface markers in BMDCs, indicative of greater antigen presentation and co-stimulation functionality. Our current study suggests that fucoidan transcriptionally activates PRR signaling, type I IFN production and signaling, ISGs production, and DC maturation, highlighting a potential mechanism of fucoidan-induced antiviral activity.
이 연구는 충남대학교 연구비와 교육부 조성 한국연구재단(NRF-2019R1I1A3A01060331 to J.K.) 또한, 과학기술정보통신부가 지원하는 한국연구재단(NRF)을 통한 브레인풀 프로그램(NRF-2020H1D3A2A02048437, J.G.K.)과 과학기술부(NRF-2021M3H9A1030267, S.Y.K.)의 지원으로 진행됐다.
Extracellular vesicles deliver feto-placental paternal antigens to maternal secondary lymphoid tissues for T cell recognition
Authors: Powell Juliana Schneider , Shufesky William , Larregina Adriana T , Sadovsky Yoel , Morelli Adrian E 
Published: August 2023

Abstract
Pregnancy constitutes an immunological paradox, where despite the competent maternal immune system, the semi-allogeneic fetus evades rejection. The mechanisms at the maternal-fetal interface and mother’s secondary lymphoid tissues (SLTs) that dodge the immunologic attack to the fetus, remain largely unknown. Passage of extracellular vesicles (EVs) is a mechanism of cell-to-cell communication that transfer antigens (Ags), regulatory mediators, and RNAs. Thus, we tested whether, similar to transplantation, feto-placental EVs could transport fetoplacental Ags via blood to maternal SLT immune cells, in this case in a tolerogenic fashion

Results
In female BALB/c mice impregnated with mOVA C57Bl/6 (B6) males, the surrogate paternal Ag OVA accumulated on follicular dendritic cells (FDCs) in maternal SLTs (E17.5). In a mouse model in which EVs released by the fetoplacental unit are labelled with mNeonGreen linked to EV-CD81, mNeonGreen-CD81 was detected by microscopy in punctate areas on FDCs, conventional DCs, B cells, and macrophages in the mother’s spleen (E17.5). IP followed by western blot of EVs from cultures of trophoblast cells from BALB/c females impregnated with OVA B6 males, revealed OVA on the EVs. Administration of these EVs to non-pregnant females induced defective activation and proliferation of OVA-specific T cells in SLTs. In a human translational mouse model, iv injection of CM-DiI EVs from supernatant of primary human trophoblast cells were captured by B cells, macrophages, and cDCs of human origin in the spleen

Conclusion
In the models tested, systemically released feto-placental EVs carry paternal/trophoblast Ags that reach the maternal SLTs, where the Ags are recognized by maternal T cells. NIH R01 AI148690.
The unexpected effect of the protease CPAF from Chlamydia trachomatis is to increase the global synthesis of the host proteins
Authors: Alvarez Carlos , Mora Valentina 
Published: August 2023

Abstract
MHC-I molecules are involved in the predisposition to several immune diseases. Reactive arthritis (ReA) is strongly linked with Chlamydia trachomatis infection and HLA-B*27. However, how the MHC-I can contribute to the pathology is not well understood.
Chlamydia is an intracellular pathogen and one of its main pathogenic factors is CPAF. This enzyme is secreted to the cytosol of the host during infection and there are no data regarding its global effect on the protein degradation and how could affect the ligands presented by MHC-I molecules.
T-REx-293 cells stably transfected with CPAF were induced with doxycycline and its effects on several known substrates were assayed by Western blot. Moreover, its unknown effects on the global proteome were evaluated by label-free quantitation (LFQ) using a Q-Exactive plus mass spectrometer. Additionally, MHC-I complexes were purified by immunoprecipitation and the eluted peptides were analyzed by MS.
CPAF is highly active degrading known substrates such as Vimentin and RFX5, but also showed an unexpected and profound effect on the cellular proteome increasing the expression of many proteins with a wide range of functions, mostly related to transcription and translation pathways.
More than 3000 MHC-I-restricted peptides were identified showing that the peptidome is altered when CPAF is induced, increasing the diversity and altering the physicochemical properties of the peptides.
As conclusion, CPAF deeply alters the host proteome contributing to alter the peptide supply of the antigen processing pathway which affect the composition of the MHC-I-associated immunopeptidome. These data suggest that the generation of “new ligands” could be a new pathogenic mechanism of C. trachomatis in ReA.
Supported by grants Fondecyt 11170969 and Fondequip EQM190142 from ANID, Chile.
Genetic immunization with self-assembling SARS-CoV-2 Spike-HBsAg nanoparticles
Authors: Merriam Jonah S , Liu Catherine , Wang Lingshu , Shi Wei , Yang Eun-Sung , Zhang Yi , Kong Wing-Pui , Tsybovski Yaroslav , Stephens Tyler , Verardi Raffaello , Leung Kwanyee , Stein Cody , Chen Man , Olia Adam , Harris Darcy , Pegu Amarendra , Graham Barney , Kwong Peter , Mascola John , Koup Richard 
Published: August 2023

Abstract
As the COVID-19 pandemic continues to pose a great challenge to global health, more effective and durable vaccines are needed owing to the waning protection offered by the current available vaccines. Here we report highly immunogenic self-assembling hepatitis B surface antigen (HBsAg) nanoparticles displaying the ectodomain of SARS-CoV-2 WA1 S6P, the prefusion spike stabilized by six proline substitutions. Three self-assembling nanoparticle constructs with different linkers named CL1, CL2 and CL3 expressed well-formed nanoparticles in mammalian cells. These purified nanoparticles can bind diverse monoclonal antibodies (mAbs) specific to different domains of SARS-CoV-2 spike. Immunization by DNA electroporation of CL2 and CL3 in mice elicited significantly more potent and durable neutralizing antibody responses against diverse SARS-CoV-2 strains than non-nanoparticle forms of stabilized spikes, including SARS-CoV-2 S2P and S6P ectodomain and full-length S2P with its coding sequence matching mRNA-1273. Our data showed that genetic delivery of HBsAg based nanoparticle vaccines displaying SARS-CoV-2 spike can elicit potent and durable neutralizing antibody responses and highlight their potential as next generation genetic vaccine candidates against SARS-CoV-2.
NIH Intramural Funding