bluuebunny/crossref_metadata_2025_split huggingface dataset:
SHP2 protein-tyrosine phosphatase is a key regulator of mast cell signaling and development in mice (36.22)
Authors: Sharma Namit , Everingham Stephanie , Feng Gen-Sheng , Roers Axel , Craig Andrew 
Published: January 2023

Abstract
SH2 domain-containing phosphatase-2 (SHP2; also called PTPN11) is a positive effector of growth signaling pathways downstream of receptor tyrosine kinases and immune receptors. The development of conditional knockout mouse models for SHP2 has resulted in a better understanding of the role of SHP2 in adult tissues and cell types. In bone marrow-derived mast cells, recent studies have implicated SHP2 in functioning as a positive effector of growth signaling via Kit receptor tyrosine kinase, and TNFα production via the high affinity IgE receptor. In this study, we describe the use of transgenic mice expressing Cre recombinase in connective tissue mast cells (CTMCs) to generate a selective SHP2 knockout in CTMCs (MC-SHP2 KO). Compared to control mice lacking Cre, MC-SHP2 KO mice display a drastic reduction in mast cells within the peritoneum and skin. Previous studies have demonstrated the critical importance of Kit Y567 and Y719 signaling to Src family kinases/Gab2 and phosphatidylinositol 3’ kinase (PI3K) pathways for development of peritoneal and skin mast cells in mice. Thus, our results suggest that SHP2 plays a key role in signaling via the Src/Gab2/PI3K axis downstream of Kit during mast cell development in mice. In vitro differentiation assays for CTMCs are being investigated to further explore the molecular mechanisms by which SHP2 regulates mast cell development.
Identification of an IL-7 Responsive CD4+ Lymphoid Tissue Inducer Cell from Adult Human Blood (36.24)
Authors: Bekiaris Vasileios , Greenberg Brian , Sedy John , Ware Carl 
Published: January 2023

Abstract
In mice, the lymphoid tissue inducer (LTi) cell forms a distinct lineage of hematopoietic cells required for the development of lymphoid tissues, and in the adult may be important for regeneration of lymphoid tissues following infection. LTi cells are rare and constitute a phenotypically mixed population of cells that may differentiate into NK-like cells, which has hindered their identification and characterization. Here, we isolated cells from adult human blood with phenotype and functional responses conserved with mouse CD4+ LTi cells. Human LTi-like cells expressed CD4 and IL-7 receptor-α, Rorc and Tnfsf8, signature markers for mouse CD4+ LTi cells, but lacked lineage markers for T and B lymphocytes, NK and myeloid cells. A culture system that allowed survival of human and mouse LTi cells was independent of IL-7, yet human LTi cells retained responsiveness to IL-7 by induction of lymphotoxin (LT)-α and LTβ, the key signaling system for lymphoid organogenesis. Human CD4+ LTi cells, like T cells, expressed the lymphoid tissue homing chemokine receptor, Ccr7, and the coreceptor for HIV, CXCR4. The identification of adult human CD4+ LTi cells should expand the investigation of these cells in human immunobiology and pathogenesis.
What goes up must come down: The consequences of not turning down pre-TCR induced β-catenin (36.29)
Authors: Sharma Archna , Yu Qing , Misra Sen Jyoti 
Published: January 2023

Abstract
Pre-T cell receptor (pre-TCR) signals regulate β-selection during the maturation of CD4- CD8- double negative (DN) thymocytes to the CD4+ CD8+ double positive (DP) stage. T-cell specific deletion of β-catenin, a central mediator of Wnt- β-catenin-TCF/LEF signaling pathway, impairs T cell development at the β-selection checkpoint. We demonstrate that pre-TCR signals specifically stabilize β-catenin in DN thymocytes during β-selection. Pre-TCR induced β-catenin regulates expression of IL-7 receptor (IL-7R), early growth response (Egr) and Ror-γ-T genes that are essential for traversal through β-selection checkpoint. However, expression of pre-TCR induced β-catenin must be transient and must return to background levels for efficient transition to the DP stage. Sustained expression of β-catenin results in the development of IL-7R and Egr expressing pre-DP thymocytes that fail to up-regulate the expression of Ror-γ-T, which is essential for development to the DP stage. The stalled DN cells, expressing β-catenin, exhibit DNA damage, growth arrest, oncogene-induced-senescence (OIS) and p53 dependent apoptosis. In p53-deficient mice, expression of β-catenin still results in DNA damage and OIS, but the thymocytes survive and eventually progress to thymic lymphoma. We suggest that oncogenic β-catenin is a part of normal developmental signaling event but is prevented from transforming developing thymocytes by p53-independent growth arrest and OIS and p53-dependent apoptosis.
Dkk1 mediated inhibition of Wnt Signaling in postnatal mice leads to loss of TEC progenitors and thymic degeneration (36.32)
Authors: Pezzano Mark , Osada Masako , Jardine Logan , Misir Ruth , Andl Thomas , Millar Sarah 
Published: January 2023

Abstract
The molecular mechanisms, which control both the initial development and subsequent maintenance of TEC microenvironments, are poorly defined. Regulation of Wnt signaling has been shown to impact both early thymocyte and thymic epithelial development. Early blocks in thymic organogenesis or death of the mice have prevented analysis of a role of Wnt signaling in the maintenance of TECs in the postnatal thymus. Tetracycline-regulated expression of the Wnt inhibitor DKK1 in cTECs and mTECs of adult mice, results in rapid thymic degeneration characterized by a reduced Foxn1 expression, loss of K5K8DP putative TEC progenitors, decreased proliferation and the development of cystic structures, similar to an aged thymus. Removal of DKK1 from DKK1-involuted mice results in full recovery, suggesting that canonical Wnt signaling is required for the differentiation or proliferation of TEC populations needed for maintenance of properly organized adult thymic epithelial microenvironments. The results of this study demonstrate that canonical Wnt signaling within TECs is required for the maintenance of epithelial microenvironments in the postnatal thymus, possibly through effects on TEC progenitor/stem cell populations. Downstream targets of Wnt signaling, which are responsible for maintenance of these TEC progenitors may provide useful targets for therapies aimed at counteracting age associated thymic involution or the premature thymic degeneration associated bone marrow transplants.
Impaired thymic selection and abnormal antigen-specific T cell responses in Foxn1Δ/Δ mutant mice (36.36)
Authors: Xiao Shiyun , Manley Nancy 
Published: January 2023

Abstract
Foxn1Δ/Δ mutant mice have a specific defect in thymic development, characterized by a block in TEC differentiation at an intermediate progenitor stage, and blocks in thymocyte development at both the DN1 and DP cell stages, resulting in the production of abnormally functioning T cells that develop from a atypical progenitor. To test the effects of deficient TEC on thymic selection, we used Foxn1Δ/Δ;DO11 Tg and Foxn1Δ/Δ;OT1 Tg mice as positive selection and Foxn1Δ/Δ;MHCII I-E mice as negative selection models. An in vivo system of antigen-specific reactivity was used to test the function of peripheral T cells. Our data show that the capacity for positive and negative selection was reduced in Foxn1Δ/Δ mutants compared to Foxn1+/Δ control mice. These defects were due in part to reduction of MHC I, II expression, although the resulting peripheral T cells have a broad TCR Vβ repertoire. In this deficient thymic environment, immature CD4 and CD8 SP thymocytes emigrate from the thymus into the periphery. These T cells had an incompletely activated profile under stimulation of the TCR signal in vitro, and were either hypersensitive or hyporesponsive to antigen-specific stimulation in vivo. These cell-autonomous defects were compounded by the hypocellular peripheral environment caused by low thymic output, showing that a primary defect in the thymic microenvironment can cause indirect effects on the periphery that can in turn exacerbate functional defects in T cells.
Spontaneous development of IL-17-producing γδ T cells in the thymus occurs via a TGFβ1-dependent mechanism (36.37)
Authors: Do Jeongsu , Fink Pamela , Li Lily , Spolski Rosanne , Robinson Janet , Leonard Warren , Letterio John , Min Booki 
Published: January 2023

Abstract
In naive animals γδ T cells are innate sources of IL17. However, mechanism(s) involved in the generation of these cells remains unexplored. We show here that some γδ T cells spontaneously differentiate into IL17+ cells in vivo and IL17-competent γδ T cells were preferentially found in the peripheral LN but not in the mesenteric LN. Ontogeny study revealed that γδ T cell IL17 differentiation mainly occurs in the thymus particularly during the postnatal period, e.g. prior to 7 days of age; ~40% of γδ TCR+ thymocytes in 5-day old mice expressed IL17 upon stimulation. IL17+ γδ TCR thymocytes were mainly found to be CD44highCD25low, ‘DN1 phenotype’ cells. Because γδ TCR is expressed after the DN2/3 stage of the thymic development pathway, this finding strongly suggest that IL17+ CD44highCD25low cells may be the DN4 γδ TCR+ thymocytes that have upregulated CD44 expression and acquired IL17 expression. In deed, in vitro culture of FACS sorted DN4 γδ TCR+ cells with OP9-DL4 stromal cells confirmed this possibility. Using gene-deficient mice, we also found that TGFβ is the primary factor mediating IL17 differentiation of γδ T cells in the thymus. However, other cytokines involved in Th17 differentiation including IL23, IL6, and IL21 played little or no roles as IL17+ γδ T cells were unaffected in these strains of mice. Taken together, our results identify a novel developmental pathway during which IL17-competent γδ T cells arise in the thymus by a TGFβ1-dependent mechanism
Dynamic regulation of microRNAs in the thymus during stress response (36.41)
Authors: Silge Robert , Belkaya Serkan , de la Morena Maite , van Oers Nicolai 
Published: January 2023

Abstract
Immune responses are dramatically affected by stress, with developing thymocytes undergoing marked atrophy following bacterial infections or steroid injections. MicroRNAs (miRs) have been implicated in stress responses in many different organ systems. We performed miR expression profiling to identify miRs affected by lipopolysaccharide (LPS) or dexamethasone induced thymic atropy. Both treatments caused significant reductions in overall thymic cellularity, with the CD4+CD8+ subset primarily affected. Seven and ten distinct miRs were significantly up- and down- regulated, respectively, in the thymic tissue, compared to controls. Synthetic mimics or inhibitors of several of these miRs, including miR-181a, differentially affected T cell development, as assessed in fetal thymic organ culture assays. These findings indicate that many thymic miRs are stress responsive and can influence the efficiency of T cell development.
The effect of forcibly expressed “immunoproteasome” subunit β5i in cortical thymic epithelial cells (36.46)
Authors: Xing Yan , Hogquist Kristin 
Published: January 2023

Abstract
Cortical thymic epithelial cells (cTECs) provide an essential microenvironment for the positive selection of MHC-restricted T cells. Data in recent years suggests that a novel proteasome subunit—β5t, exclusively expressed in cTECs in the thymus, plays a key role in generating the MHC Class I-restricted CD8+ T cell repertoire during thymic selection. In contrast, the “immunoproteasome” subunit β5i is normally expressed in dendritic cells and medullary thymic epithelial cells, and can be upregulated in other MHC Class I positive cells by IFNγ. To study how positive selection is regulated by a unique peptide repertoire expressed in cTEC, we generated K14-β5i transgenic (Tg) mice in which β5i was expressed in cTECs under the control of the human keratin 14 (K14) promoter. Forced expression of β5i in cTECs did not result in a reduced percentage or number of CD8 T cells. However, there was an increase in the percentage and number of CD44high “memory phenotype” CD8 T cells in the periphery of K14- β5i Tg mice, similar to that observed in β5t deficient mice. This suggests that the expression of β5i in cTECs could induce a distinct peptide repertoire and affect the selection of CD8 T cells in thymus. In addition, these mice exhibit hyperkeratosis of the skin. RAG-deficiency did not rescue the skin disease of K14-β5i Tg mice, indicating that it might be promoted by the over-expression of β5i in keratinocytes.
Importance of TCR ITAM multiplicity in the establishment of central tolerance (36.48)
Authors: Hwang SuJin , Song Ki-Duk , Chu Hamlet , Lee Jan , Lesourne Renaud , Li LiQi , Pinkhasov Julia , Jenkins Marc , Love Paul 
Published: January 2023

Abstract
Thymocyte selection is controlled by signaling motifs (ITAMs) contained within the invariant T cell receptor (TCR) subunits. The current model of thymocyte selection posits that differences in the affinity of the TCR for self-peptides results in differences in the TCR signaling response, with low affinity interactions promoting positive selection and high affinity interactions triggering negative selection. We and others previously demonstrated that attenuation of TCR signaling potential (by mutation of the zeta chain ITAMs) leads to the generation of T cells that express auto-reactive TCRs. However, these T cells did not cause autoimmune disease, presumably because TCR/self-peptide interactions do not induce strong effector responses in the absence of zeta ITAMs. This paradigm was recently challenged in a study reporting that retroviral reconstitution of zeta deficient bone marrow with ITAM mutant zeta chains results in fatal autoimmune disease. In the current study, we generated a knock-in (KI) mutation at the zeta locus that results in expression of a zeta chain where each of the ITAM tyrosines is mutated to phenylalanine (6YF). The phenotype of zeta-6YF mice demonstrates that attenuation of TCR signaling potential is not, by itself, sufficient to abrogate central tolerance. In addition, our results confirm that the threshold for T cell activation is determined by both the affinity of the TCR and by the TCR signaling potential
Age related dynamics of committed T cell progenitors in mice (36.57)
Authors: Peiris Tanuja , Ciriza Jesús , Al-Kuhlani Mufadhal , Graham Ashley , García-Ojeda Marcos 
Published: January 2023

Abstract
Immunosenescence is the reduced ability of the body to mediate an adaptive immune response. Multiple factors contribute to immunosenescence, including age-related changes in hematopoietic stem cell (HSC) phenotype, its niche, and the tissues responsible for lymphocyte development, such as the bone marrow and the thymus. Therefore, understanding lymphocyte development in the aged is fundamental to enhance the immune response capacity of the aging immune system. Here, we investigate the age-related dynamics of committed T cell progenitors (CTP), an HSC-derived bone marrow population capable of generating functional T cells via both thymic and extrathymic pathways. Specifically, we examined the CTP’s frequency and absolute numbers in the bone marrow of young (1 month) and old (12 and 18 months) C57BL/6 mice using flow cytometry. Moreover, we compared the capability of young and aged CTP to generate T cells in vitro. Understanding the biology of CTP in relationship to aging could have important clinical applications in the treatment of diseases related to immunodeficiencies and cancer by hematopoietic stem cell transplantation, as CTP-derived T cells protect against viral infections without inducing graft vs. host disease. This work could pave the way to studies related to human CTP populations and their therapeutic potential.
A simple and fast method for the isolation of mouse lymphoid progenitors from bone marrow (36.6)
Authors: Tabatabaei-Zavareh Nooshin , Fairhurst Maureen , Thomas Terry 
Published: January 2023

Abstract
Lymphocytes are derived from hematopoietic stem cells through an important developmental intermediate called a common lymphoid progenitor (CLP). CLPs are defined as Lin−IL-7Rα+c-KitloSca-1lo. They can differentiate into T, B, and natural killer (NK) cells but lack myeloid and erythroid potential. Study of lymphocyte development largely relies on access to this rare cell population. Fluorescence-activated cell sorting (FACS) commonly used to isolate CLPs is costly, time-consuming and perhaps most importantly detrimental to the cell viability and function. We describe a fast and efficient method for the isolation of CLPs from mouse bone marrow (BM). This method is based on immunomagnetic, column-free cell separation technology (EasySep). Using this method, lineage positive cells were first depleted by cross-linking them to magnetic particles using biotinylated antibodies. Next, IL-7Rα+ cells were positively selected from the Lin−/lo population. Purity of Lin−IL-7Rα+c-Kit+ lymphoid progenitors as assessed by flow cytometry ranged from 18-41%. Limiting dilution analysis of the EasySep enriched cells showed increased frequencies of B cell (1:25), T cell (1:6) and NK cell (1:49) progenitors as compared to non-depleted control BM. This system introduces a rapid and easy method for the enrichment of rare CLPs with greater yield than cell sorting and good viability. This will enable research in the field of cellular, molecular and developmental immunology.
TGF-beta is an essential factor for IL-9 production (36.60)
Authors: Wu Xiao-wei , Rabenstein Jacob , Sepulveda Homero , Wei Ai-Li , Hingorani Ravi , Ernst David , Rohrer Jurg 
Published: January 2023

Abstract
Interleukin-9 (IL-9) is a multifunctional cytokine involved in proliferation, differentiation, and effector functions of various cell types that include T cells, B cells, mast cells, eosinophiles neutrophils, and epithelial cells. In previous studies, Th2 cells were shown to produce IL-9. Recent studies suggest that IL-9 can also be produced by so-called Th9 cells. In this study, we report that TGF-beta is an essential factor for in vitro cultured human peripheral blood mononuclear cells (PBMC) into subsets that secrete IL-9. PBMC were activated in the presence of anti-CD3/CD28 antibodies and polarizing cytokines with or without the addition of TGF-beta. Using flow cytometry to detect both intracellular IL-9 expression and soluble IL-9 secretion, we found that IL-9 was only produced in the presence of TGF-beta. Addition of other cytokines, especially IL-4 further increased IL-9 production. Interestingly, a subset of CD4+IL-9+ T cells was seen to also express IL-17, but not IL-4, IL-10 or IL-12. Additional studies will address the plasticity between the Th9 and Th17 CD4 T cell subsets.
Three functional regions in human tonsil distinguished by in situ gene expression (37.1)
Authors: Zhao Zeng , Rabin Ronald 
Published: January 2023

Abstract
We stained tonsil sections for T cell, B cell, and DC markers and found that T cell zones could be subdivided according to distribution of CD303+ plasmacytoid DC into pDC rich and pDC poor regions; pDC rich regions surrounded the high endothelial venules and pDC poor regions were relatively well populated with CD1c+ myeloid DC. We then modified our immunuhistochemistry technique to preserve RNA integrity and used laser capture microdissection (LCM) to dissect three discrete target regions and characterize differential patterns of gene expression among them. Fifty of the 80 genes probed were differentially expressed among the three regions. Thirty genes in the pDC rich region represented T cell activation and thirteen genes in the pDC-poor region included T cell regulation. Not surprisingly, the seven genes most highly expressed in the GC were primarily related to B cell activation. Expression patterns of master transcription factors of adaptive immune function, IFN-g, and subtypes of types I and III IFN validated a model whereby T cells. Three distinctive regions and genomic functions in situ of tonsil were characterized corresponding to distribution of DC subtypes. The gene profile provides basis of immune responses in vivo for administration of surface vaccines.
A pathogenic role of macrophages in mice intradermally infected with methicillin-resistant Staphylococcus aureus (37.18)
Authors: Asai Akira , Tsuda Yasuhiro , Kobayashi Makiko , Herndon David , Suzuki Fujio 
Published: January 2023

Abstract
In this study, intradermal MRSA infection in burn mice was pathogenically analyzed. Burn mice (3rd degree, 25% TBSA flame burn) and normal mice were infected i.d. with 108 CFU/mouse of MRSA, and abscesses formed in the infection sites and mortality rates of these mice were examined. Also, the abscess formation was examined in burn and normal mice after inoculation with F4/80+Mϕ isolated from tissues surrounding the infection sites of normal (N-F4/80 Mϕ) or burn mice (B-F4/80 Mϕ). Finally, these Mϕ were biophysically characterized. In the results, the abscess was formed in normal mice infected with MRSA and all of them survived. However, the abscess was not formed in burn mice infected with MRSA, and all of them died. The abscess was formed and sepsis was not developed in burn mice inoculated with N-F4/80 Mϕ. However, the abscess was not formed and sepsis was developed in normal mice inoculated with B-F4/80 Mϕ. N-F4/80 Mϕ produced IL-12 (but not IL-10) and were characterized as M1Mϕ, whereas B-F4/80 Mϕ produced IL-10 (but not IL-12) and were characterized as M2Mϕ. These results indicate that the abscess formation is a major host defense against MRSA i.d. infection. M1Mϕ at tissues surrounding the infection site play a pivotal role in the abscess formation. An abscess is not formed in burn mice predominated with M2Mϕ, which are inhibitory on the M1Mϕ generation. This study was supported by SHC NA #8840. A.A was supported by The James W. McLaughlin Fellowship Fund.
Mannose-binding lectin deficiency increases susceptibility to influenza A virus infection. (37.20)
Authors: Takahashi Kazue , Chang Wei-Chuan , White Mitchell , Moyo Patience , McClear Sheree , Thiel Steffen , Hartshorn Kevan 
Published: January 2023

Abstract
Influenza A virus (IAV) infection can cause life-threatening condition even in individuals who are otherwise healthy. IAV infects the airway and the lung, where lung-surfactant protein-A (SP-A), SP-D and alveolar macrophages are thought to be primary innate immune protection. SP-A and SP-D belong to the collectin family that also includes a serum protein, mannose-binding lectin (MBL), that shares structural and functional similarities to lung collectins. MBL is a broad-spectrum pattern recognition molecule that plays a key role in first line host defense as an activator of the lectin complement pathway. In this study, we have found that MBL null mice have increased susceptibility to IAV infection and that recombinant MBL improves the infection. The anti-viral mechanisms of MBL include initiation of the lectin complement pathway as well as a thrombin-like activity that requires the cooperation of other serum factors. These results also demonstrate the ability of MBL to modulate leukocyte recruitment and inflammation in infected lungs. These findings reveal that MBL plays a key role in clearing IAV and maintaining lung homeostasis. In addition, our results suggest that MBL deficiency may be a risk factor in IAV infection and that MBL may be useful as a therapy against IAV infection.