bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tissue engineering human thymus from iPSCs to support human T cell development
Authors: Fan Yong , McCormick Catherine , Liu Wen , Zeleniak Ann , Wiegand Connor , Krishnamurthy Ravi , Guan Haonan , Montanari Kaylee , Muraskin Nickia , Banerjee Ipsita 
Published: August 2023

Abstract
Thymus is the primary immune organ responsible for the generation of diverse and self-tolerant T cells that are essential for fighting off invading pathogens while maintaining immune homeostasis. Loss of thymus function, due to either genetic mutations or environmental insults, can lead to prolonged immunodeficiency that can be life-threatening. Rejuvenating the function of the thymus is challenging as the organ undergoes irreversible involution at young age, during which thymic epithelial cells (TECs) – the thymic stromal cells key to the education and selection of developing T cells – are progressively replaced by infiltrating adipocytes. Regeneration of thymus from the patient’s own stem cells will have tremendous clinical benefits in treating immunodeficiency and immunodysregulatory disorders.
We reported here the tissue-engineering of functional artificial thymus from inducible pluripotent stem cells (iPSCs). Taking advantage of the 3D alginate encapsulation technology, we developed a novel protocol to induce the differentiation and maturation of human iPSCs into TECs (iPSC-TECs) at high efficiency. To bioengineer the thymus, iPSC-TECs were injected into decellularized murine thymus scaffolds, in conjunction with T cell progenitors derived from hematopoietic stem/progenitor cells (HSPCs) harvested from the umbilical cord blood. When transplanted under the kidney capsules of hematopoietic humanized mice (aka immunodeficient mice engrafted with HSPCs), iPSC-TEC thymus can support developed a diverse population of human T cells that could mediate adaptive immune responses. Our findings suggest that functional human thymus can be generated from patient-derived iPSCs for regenerative medicine.
Supported in part by the NIH (R01 AI123392 and R21 AI126335), NSF (CBET 1804728), and Highmark Health Award (A023948-HIGHMARK-LANNI-FAN).
Chemotherapy-Induced Thymic Involution: An Ultrastructural Study
Authors: Lagou Maria K , Nishku Xheni , Churaman Joseph , Cummins Leslie , Macaluso Frank P , Karagiannis George S 
Published: August 2023

Abstract
Endogenous thymic repair is quite complex, demanding coordinated thymocyte and thymic stromal responses for T cell development and establishment of central tolerance. While the importance of thymic stroma, notably cortical (cTEC) and medullary (mTEC) thymic epithelial cells, has been shown in gain- and loss-of-function studies, endogenous thymic epithelial repair has never been resolved ultrastructurally. Using Transmission Electron Microscopy (TEM), we analyzed morphometrically thymi in mice receiving Cyclophosphamide (CTX). In vehicle-treated mice, the cortex was populous with thymocytes neighboring cTEC, which extended long cytoplasmic processes into a dendritic meshwork, called “cytoreticulum”. In CTX-treated mice though, thymocytes were scarcely met in the cortex and TEC appeared more circular/ellipsoid. The cytoreticulum was evidently collapsed, increasing total contact surface area among cTEC/mTEC subsets. Despite that thymic macrophages primarily mediate clearance of thymocytes failing positive/negative selection, those in CTX-treated thymi were rich in secondary lysosomes and many were found phagocytosing TEC. The surviving TEC in CTX-treated mice had increased autophagolysosomes per surface area unit of TEC cytoplasm compared to vehicle-treated ones. Analysis via intensity thresholding revealed higher electron density of enclosed particles, consistent with the presence of partly-digested amorphous material, indicating membranous organelle self-digestion (i.e., stress macroautophagy). These data propose an emerging hypothesis that survival of thymic epithelium following cytotoxic insult is mediated via a collective stress response involving macroautophagy activation in TEC.
Funded by: 1)AAI careers in immunology Fellowship - trainee 2)new investigator’s start up funds (PI)
Age-associated changes to lymph node architecture shape T cell responses
Authors: Lancaster Jessica N , Da Silva Ildefonso Alves , Kwok Tina , Fisher Jacob S 
Published: August 2023

Abstract
Aging dramatically impacts T cells, contributing to diminished and delayed responses to infection. While the development and maintenance of lymphocytes certainly undergo intrinsic changes with age, the contribution of cell-extrinsic forces, such as the aging tissue microenvironment, has been difficult to uncouple. Macroscopic changes to the lymph nodes have been well described, but the impact of aging on lymph node stromal subsets is less understood. Here, we visualized naïve T cell migration in the aged murine lymph node using live-cell 2-photon microscopy and determined that T cell motility was impaired in regions of increased fibrotic deposition. Using fluorescent reporters for Ccl19 and Cxcl12, homeostatic chemokines important for stromal:lymphocyte crosstalk, we also found that the distribution of stromal cells expressing these chemokines was altered with age, and that migrating naïve T cells were excluded from regions of chemokine expression. Flow cytometric analyses of fibroblastic reticular cells (FRCs) revealed decreased proportions of FRCs within the mesenteric lymph nodes and decreased putative FRC progenitors in the skin-draining lymph nodes. FRC progenitors at both sites had reduced surface expression of lymphotoxin-beta receptor, an important receptor for FRC differentiation and maintenance. We hypothesize that age-associated changes to FRC maturation contribute to increased lymph node fibrosis and altered localization of homeostatic factors. Current studies have indicated that in vitro cultured lymph node fibroblasts are sensitive to TNF-alpha and respond by increasing expression of alpha-smooth muscle actin, suggesting a mechanism by which aging remodels the lymph node microenvironment.
Influence of SLAM/SAP signaling on γδ T cell TCR signal strength during thymic development
Authors: Hilton Brianna M , Savard Remi , Dienz Oliver , Boyson Jonathan E 
Published: August 2023

Abstract
Innate-like T cells are unusual T cells that are enriched in mucosal tissues, where they constitute a prominent source of pro-inflammatory cytokines like IL-17 and IFN-γ. During thymic development, mouse γδ T cells commit to either γδT1, γδT2, or γδT17 phenotypes through mechanisms that remain unclear. Recent observations from our laboratory have suggested a role for the SLAM/SAP signaling pathway in γδ T cell thymic developmental programming. Here, we investigated the influence of the SLAM/SAP signaling pathway on γδ TCR signal strength during thymic development. Using Nur77GFP expression as a readout of TCR signal strength, we observed significantly greater Nur77GFP expression in B6.Nur77GFP.SAP−/− thymic γδ T cells compared their B6.Nur77GFP counterparts, suggesting that SAP inhibited TCR signal strength. Consistent with these findings, we observed significantly greater Erk phosphorylation in CD3-stimulated B6.SAP−/− thymic γδ T cells. In vitro stimulation of thymic γδ T cells with anti-TCRδ and anti-SLAM family receptors revealed that SLAMF6 co-stimulation resulted in decreased Erk phosphorylation, but increased Nur77GFP expression. Surprisingly, the effects of SLAMF6 co-stimulation on γδ TCR signaling were SAP-independent. Together, these data suggest an inhibitory role for SAP in γδ TCR signaling during thymic developmental programming.
Neural crest-derived mesenchymal cells support thymic reconstitution after lethal irradiation
Authors: Tetteh Doris Narki , Hikosaka-Kuniishi Mari , Yamazaki Hidetoshi 
Published: August 2023

Abstract
The thymus is composed of thymocytes and stromal cells including thymic epithelial and mesenchymal cells. Thymic mesenchymal cells are originated from Neural Crest (NC) cells and the mesoderm. Although it is known that NC-derived (NCd) cells contribute to thymus organogenesis and thymus development, their specific role in T cell regeneration is unclear.
To evaluate the regenerative ability of NCd mesenchymal cells in the thymus, we used lethally irradiated mice to examine the effect of ionizing irradiation (IR) on the thymus size, thymocytes and the thymic stromal cells (thymic epithelial cells (TECs) and thymic mesenchymal cells). Additionally, we ablated NC-derived mesenchymal cells via in vitro diphtheria toxin (DT) administration in double transgenic mice (Wnt1-Cre/+; RosaYFP/DTR mice) that express NC-specific Cre and Cre-driven DT receptor (DTR) and transplanted the thymus depleted of NC-derived mesenchyme under the renal capsules of non-irradiated C57BL/6 (CD45.1) mice.
IR reduced the size of the thymus, the absolute numbers and percentages of thymocytes, primarily (CD4+ CD8+) T cells and TECs. Moreover, IR reduced the number of thymic mesenchymal cells but not that of NC-derived mesenchymal cells. NC-derived mesenchymal cells upregulated several hematopoietic factors such as Flt3l, Periostin, Scf, and Bmp4 after irradiation and supported thymic reconstitution.
Taken together, our findings indicate that NC-derived mesenchymal cells are not only radio-resistant, but also produce molecules that may support early T-cell development for thymic reconstitution after irradiation .
Toll-like receptor expression on young and old hematopoietic cell types
Authors: Lowe Sara Kristen , Elberfeld Lauren , Bhowmick Debajit , Ratliff Michelle 
Published: August 2023

Abstract
Hematopoiesis is an essential process that produces white and red blood cells within the body. As the body ages, we have observed defects in the hematopoietic process including an increase in the number of stem cells, skewing of white blood cells down the myeloid lineage, and a decrease in the production of new B and T cells. Since hematopoietic aging skews the production of professional immune cells, the elderly population has a disadvantage in recognizing and clearing infections compared to young individuals. Immune cells use a common bacterial and viral recognition system, which includes toll-like receptors (TLRs), to identify common pathogen-associated molecular patterns (PAMPs) that bacteria and viruses produce. Upon exposure, immune cells will activate to complete two functions: to direct myeloid differentiation for acute responses and to recognize and eliminate the pathogens via the TNFα or type 1 interferon (IFN-1) signaling pathway.
Deficiencies in aging hematopoiesis, IFN-1 expression, and the TLR responses associated with IFN-1 in immune cells have been described in both mice and humans. The expression of these TLRs and any age-associated changes in that expression, as well as downstream signaling, are not as well established. Our goal is to identify which hematopoietic cells express the four TLRs associated with IFN-1 (TLR-3, −4, −7, −9) and if aging leads to changes in receptor expression and/or downstream signaling in human immune cells. Using flow cytometry, we evaluated receptor expression and the downstream effects of TLR engagement. The data reveals age-associated changes in IFN-1-related innate immune signaling pathways.
Supported by grants from NIH (R00 AG055717)
TLR2/6 agonist treatment after total body irradiation in mice and nonhuman primates induces proliferation of hematopoietic progenitor cells
Authors: Brickey Willie J , Caudell David , Olson John , Huang Guannan , Li Sirui , Macintyre Andrew N , Yang Shuangshuang , Hsu Martin , Chao Nelson J , Cline J Mark , Ting Jenny P 
Published: August 2023

Abstract
Exposure to high doses of radiation, whether intentional or accidental, may cause injury or death. Therapeutics are urgently needed for mitigating injuries caused by radiation exposure. Previously, we identified a synthetic lipopeptide agonist of TLR2/6, namely fibroblast-stimulating lipopeptide (FSL-1), that prolonged survival of mice exposed to lethal ionizing radiation and mitigated hematopoietic acute radiation syndrome. Here, this agent was tested in several mouse models as well as in nonhuman primates given total body irradiation followed 24 hours later with a single subcutaneous injection of FSL-1. These studies revealed the mitigation of radiation-induced loss of platelets and red blood cells, as well as the promotion of bone marrow progenitor cell recovery. These beneficial hematopoietic outcomes were accompanied with a lack of toxicity, showcasing the safe, efficacious application of a single dose of FSL-1 for mitigating hematopoietic dysfunction resulting from radiation. By investigating the mechanism of action of FSL-1 in response to radiation, we confirmed that FSL-1 radio-mitigation activity was mediated through a TLR2/MyD88-dependent process. In FSL-1 treated irradiated mice, we found enhanced numbers of proliferating Ki67+ bone marrow cells. Finally, examination of cellular pathways in mice and nonhuman primate bone marrow cells indicated FSL-1 induced the activation of NF-kB and MAPK signaling. These data suggest that FSL-1 promotes hematopoietic progenitor cell proliferation through the activation of cell survival and activation pathways downstream of TLR2/6 stimulation, and thus may serve to mitigate the injurious consequences of radiation.
Supported by grants from NIH (AI067798) and DOD Congressionally Directed Medical Research Program grant W81XWH-15-1-0574 (JMC).
Characterizing the mucosal-associated invariant T cell response to pulmonary infection
Authors: Melamed Jonathan , Constantinides Michael G 
Published: August 2023

Abstract
Mucosal-associated invariant T (MAIT) cells are an effector population of innate-like T cells that comprise up to 5% of T cells within human lungs. Rather than recognizing peptides like conventional T cells, MAIT cells utilize their semi-invariant T cell receptors to identify small molecules, such as intermediates of riboflavin synthesis, presented by MHC-class 1 related protein (MR1). Because this biosynthetic pathway is highly conserved among bacteria and fungi, MAIT cells promote immunity to a wide array of pulmonary pathogens, such as Legionella and Francisella species. However, due to the low level of MR1 expression in several major cell lineages, the populations that present microbial metabolites to MAIT cells during infection remain unknown, preventing optimization of MAIT cell vaccines. Utilizing the F. tularensis live vaccine strain to induce pneumonic tularemia, we find that MAIT cells are the predominant effector T cell population, accounting for more than 20% of IFN-γ producing T cells. Infection of MR1f/f conditional knockout mice following broad deletion of MR1 with the tamoxifen-inducible Rosa26-creERT2 allele revealed that MR1-mediated presentation is necessary for the MAIT cell response to F. tularensis. Use of MR1f/f strains that express Cre recombinase under control of cell-specific promoters has allowed us to determine the relative contributions towards MAIT cell activation from dendritic cells, macrophages, and neutrophils. This work defines the antigen presenting cells that are critical for MAIT cell activation during an intracellular bacterial infection and identifies cellular targets to enhance MAIT cell responses.
Supported by NIAID grants K22 AI146217 and R21 AI171697
Signs of Dendritic Cell Activation in the Lungs of Mice Exposed to Aerosols of Flavored Vaping Liquid
Authors: Tremblay Felix , Pineault Marie , Thériault Pierre-Olivier , Milad Nadia , Lechasseur Ariane , Routhier Joanie , Beaulieu Marie-Josée , Aubin Sophie , Côté Andréanne , Morissette Mathieu C 
Published: August 2023

Abstract

Rationale
Vaping products contain numerous flavor compounds that are known immunological sensitizers. Sensitization to various antigens can be responsible for the onset of serious respiratory diseases, including hypersensivity pneumonitis. However, respiratory tract sensitization and pathological consequences from exposure to aerosols of flavored vaping liquids need to be thoroughly investigated.

Methods
Female BALB/c mice were exposed to laboratory made nicotine-free vaping liquid containing a mixture of 1% citral (lemon), 1% cinnamaldehyde (cinnamon), 1% dihydrocoumarin (coconut) and 1% vanillin (vanilla) solubilized in propylene glycol and glycerol (50:50). Mice were exposed 2h/day for 4 days to vaping or room air. Bronchoalveolar lavage was performed to assess total and differential cell counts, and flow cytometry was performed on pulmonary tissue and lung-draining lymph nodes to assess immune cell populations.

Results
After 4 days of daily exposure, we found a significant increase in bronchoalveolar lavage immune cells in mice exposed to aerosols from the flavored vaping liquid. We also observed a significant increase in pulmonary conventional dendritic cells (cDCs) as well as their expression of the activation markers CD40, CD86 and MHC-II. This activation is mainly observed on type 2 cDCs. In the lung-draining lymph nodes, flavored vaping liquid lead to a significant increase in total immune cells (CD45+ cells).

Conclusions
Pulmonary conventional dendritic cells activation and the increase in total immune cells in lung-draining lymph nodes caused by exposure to aerosols from flavored vaping liquid suggest the early initiation of immunological sensitization processes in the lungs.

Fonds de recherche du Québec – Santé, Ministère de la Santé et des Services sociaux, Fondation de l’Institut de cardiologie et de pneumologie de Québec, Réseau de recherche en santé respiratoire du Québec.
The FcμR regulates IgM-BCR internalization
Authors: Blandino Rebecca A , Lou Zheng , Cook Sarah , Sciammas Roger , Baumgarth Nicole 
Published: August 2023

Abstract
The FcμR is a cell surface receptor that can bind to both secreted and membrane-bound IgM. In B cells it co-localizes with IgM-BCR in Golgi vesicles, where it restrains surface BCR-IgM expression. B cell-expressed FcmR is required also for maximal IgG response development after influenza A virus infection, but the mechanisms are unknown. To explore the effects of FcmR expression we generated FcmR-deficient and sufficient BCR-”SwHEL” transgenic C57BL/6 mice. The mice, containing about 20% HEL-specific B cells were immunized either directly with hen egg lysozyme (HEL) or with HEL-ovalbumin (OVA), or we created bone marrow irradiation chimeras in which about 2% of naïve B cells expressed the SwHEL-BCR. Consistent with the influenza infection studies, IgG responses following subcutaneous HEL-OVA immunization were significantly diminished in both systems when B cells lacked FcmR−/− expression compared to controls. Confocal and STED microscopy studies on B cells from these mice were done to quantify IgM-BCR internalization following in vitro exposure to HEL-bodipy. FcmR−/− SwHEL B cells did not show IgM “capping” and retained high amounts of antigen on the cell surface compared to the HEL− FcmR+/+ cells. This reduction in internalization resulted in reduced MHCII-peptide loading, as shown via flow cytometric quantification of surface pMHCII expression using the YAE-antibody. Ongoing studies are defining the subcellular compartments of antigen-processing in the presence and absence of the FcmR. We conclude that the FcmR acts as a non-redundant chaperone that optimizes IgM-BCR antigen internalization and processing.
U19AI109962 R01AI148652
HLA-DO promotes Treg differentiation by altering antigen presentation
Authors: Welsh Robin A , Song Nianbin , Antonescu Corina , Florea Liliana , Sadegh-Nasseri Scheherazade 
Published: August 2023

Abstract
HLA-DO in human (H2-O in mice) (DO) is a highly conserved non-classical MHC class II accessory molecule. Unlike the main Class II peptide editor HLA-DM, expression of DO is restricted to the thymic medulla, B cells and certain dendritic cell subsets. DO forms a stable complex with HLA-DM and requires DM for trafficking to MIIC. Using biochemical experiments, our lab has previously discovered that DO has differential effects on editing peptides of different sequences. DO enhances binding of DM-resistant peptides, and reduces the binding of DM-sensitive peptides to the HLA-DR1 molecules. Changes in the quantities of either DM-resistant of DM-sensitive peptides could therefore lead to alterations in both thymic negative selection, and peripheral activation of CD4 T cells. Using single-cell RNA-sequencing (scRNA-seq) of peripheral CD4 T cells from unimmunized DO knock-out (KO) mice we found an increased frequency of “activated” CD4 T cells, in support of our biochemical model. This increased frequency corresponds with a decrease in “naïve” CD4 T cells in both scRNA-seq and FACs analysis. Interestingly, scRNA-seq analysis confirmed our previous findings of elevated regulatory T cells (Treg) levels in DO-KO mice. Genetic analysis showed that unimmunized DO-KO Tregs also have a more activated phenotype. These findings suggests that absence of DO generates a more stimulatory in vivo cellular environment for CD4 T cells, likely due to altered antigen presentation.
Research supported by grants from NIH (R01 AI130210, R01 AI121174 and R37 AI060040)
Loss of HLA-DO function impairs the development of memory CD4 T cells
Authors: Song Nianbin , Welsh Robin A , Sadegh-Nasseri Scheherazade 
Published: August 2023

Abstract
DO is an accessory protein that binds DM for trafficking to MIIC and has peptide editing functions. DO is mainly expressed in thymic medulla and B cells. Using biochemical experiments, our lab has discovered that DO has differential effects on editing peptides of different sequences. DO increases binding of DM-resistant peptides, and reduces the binding of DM-sensitive peptides to the HLA-DR1 molecules. In a separate line of work, we have established that reduced densities of antigen presentation by B cells during the contraction phase of an infection, induces quiescence in antigen experienced CD4 T cells as they differentiate into memory T cells. This quiescent phenotype helps memory CD4 T cell survival and promotes effective memory responses to secondary Ag challenge. Based on our mechanistic understanding of DO function, it would be expected that in immunized DO-KO mice, presentation of altered densities of pMHC by B cells would lead to faulty development of memory CD4 T cells. We tracked the development of DR1 restricted H5N1 flu specific memory CD4 T cells and B cells post flu vaccine immunization in DR1+/DO-KO mice and found that immunized DR1+/DO-KO mice had fewer CD4 memory T cells and memory B cells than DR1+/DO-WT mice. By in vitro and in vivo recall experiments, we found that memory responses elicited in DR1+/DO-KO mice were compromised. Using a second model antigen, OVA, we confirmed our findings, i.e., altered memory development and secondary responses in DO-KO mice. These results demonstrate that in the absence of DO, the presentation of cognate foreign antigens in the DO-KO mice is altered and can hamper the proper development of memory cells. These findings suggest a justification for the late appearance of DO in warm blooded mammals.
R37 AI060040 R01 AI121174 R01 AI130210