bluuebunny/crossref_metadata_2025_split huggingface dataset:
LUBAC-mediated signals in thymic epithelial cells prevent early thymic atrophy and immunodeficiency
Authors: Jain Reema , Sheridan Julie M. , Heinlein Melanie , Kupresanin Fiona , Hall Cathrine , Rickard James , Bouillet Philippe , Walczak Henning , Strasser Andreas , Silke John , Gray Daniel H.D. 
Published: January 2023

Abstract
Thymic epithelial cells (TECs) govern the differentiation of haematopoietic precursors into functional T cells. Defects in this process can lead to immunodeficiency and autoimmune disease. Distinct signals from members of the TNF receptor superfamily have key roles in TEC proliferation and differentiation, yet it remains unclear how these stimuli are integrated to support thymus function. We find that components of the linear ubiquitin chain assembly complex (LUBAC) that act downstream of TNFR signaling to regulate NFkB activity are essential for TEC homeostasis and thymic function. TEC-specific loss of LUBAC proteins, HOIL-1 or HOIP, induced early thymic atrophy shortly after birth, with severe loss of tissue in 2-month-old adult mice attended by gross defects in peripheral T cell populations. The requirement for HOIL-1 or HOIP extended to all major TEC subsets, including those expressing the autoimmune regulator, AIRE. In contrast, loss of a third LUBAC component in SHARPIN-deficient mice (Shcpdm/cpdm) had a milder impact, reducing TECs in the thymic medulla that require CD40 signals for their maintenance. Ablation of TNF or Caspase 8 and RIPK3 in Shcpdm/cpdm mice failed to rescue these defects, indicating that CD40/SHARPIN signals are not required to prevent TNF-induced cell death. These differential roles for LUBAC components in TEC correlate with their function in linear ubiquitylation and reveal a critical signaling hub in the maintenance of the thymic microenvironment
Two-photon imaging reveals distinct contributions of Aire+ medullary thymic epithelial cells and dendritic cell subsets to central tolerance induction
Authors: Ehrlich Lauren I. , Lancaster Jessica N. , Thyagarajan Hiran Maye 
Published: January 2023

Abstract
Following positive selection, thymocytes migrate into the medulla where they encounter diverse tissue-restricted antigens (TRAs) that induce central tolerance. Both medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) present TRAs to induce thymocyte negative selection and regulatory T cell induction. However, their relative contributions to tolerance remain unresolved, partially because the genetic models that have been used to address this question can interfere with thymocyte-stromal cell crosstalk, potentially resulting in secondary defects in stromal differentiation that independently alter central tolerance. Thus, we developed a 2-photon microscopy approach to directly observe thymocytes as they undergo tolerance induction in response to interactions with DCs versus Aire+ mTECs presenting various forms of a TRA in an intact thymic stromal environment. We find that both mTECs and DCs present transmembrane TRAs with equivalent efficiency to CD8SP and CD4SP thymocytes. However, in the context of a secreted TRA, DCs play a greater role than mTECs in presenting antigen to CD4SP, but not CD8SP thymocytes. Furthermore, distinct DC subsets differentially contribute to TRA presentation to CD4SP versus CD8SP thymocytes, with a major role for Sirpα+DCs in cross-presentation of TRAs. In the context of a polyclonal T cell repertoire responding to endogenous antigens, DCs play a more significant role than mTECs in activating CD4SP cells. In summary, we find that in an intact thymic microenvironment, multiple DC subsets and Aire+ mTECs cooperate to present self-antigens to auto-reactive thymocytes during the establishment of tolerance against diverse medullary TRAs.
Catalase expression mediates redox regulation of autophagy and promiscuous gene expression in thymic stromal cells
Authors: Hester Allison K. , Semwall Manpreet , Xiao Yangming , Almutairi Abdulaziz , Cepeda Sergio , Venables Thomas , Griffith Ann V. 
Published: January 2023

Abstract
T lymphocytes develop in the thymus, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells (TSCs) directs progenitors along a well-characterized differentiation program. However, the biology of stromal cells comprising the lymphopoietic thymic microenvironment remains relatively under-characterized because stromal cells are rare and difficult to isolate. Using a deconvolution technique to study gene expression essentially in situ, we previously identified a deficiency in the peroxide quenching enzyme catalase (CAT) in thymic stromal cells, and found that CAT deficiency results in high reactive oxygen species (ROS) levels in this population, eventually leading to thymic atrophy in aged mice. Here, we find that when catalase deficiency is complemented by overexpression targeted to mitochondria in transgenic mice (mCAT Tg), both ROS levels and stromal function decline in young mice relative to non-transgenic littermates. TSC transcriptome analysis reveals decreased expression of tissue-restricted antigen (TRA) and autophagy pathway genes in mCat Tg mice. Analysis of autophagy flux reporter mice ubiquitously expressing the RFP-EGFP-LC3 fusion transgene also indicates diminished autophagic flux in mCAT Tg mice, particularly in TSCs. Stromal TRA expression and autophagic flux are required for self-antigen presentation, and therefore promote negative selection of potentially auto-reactive T cells. We propose that oxidative stress generated by low catalase levels in stromal cells promotes these key physiological functions in the young, steady state thymus; in contrast however, the resulting accumulated oxidative damage ultimately impairs function in the aged thymus.
Newly generated CD4+ T cells acquire metabolic quiescence after thymic egress.
Authors: Ge Qing , Zhang Shusong 
Published: January 2023

Abstract
Mature naive T cells circulate through the secondary lymphoid organs in an actively enforced quiescent state. Impaired cell survival and cell functions could be found when T cells have defects in quiescence. One of the key features of T cell quiescence is low basal metabolic activity. It remains unclear at which developmental stage that T cells acquire this metabolic quiescence. We compared mitochondria among CD4 single positive (SP) T cells in the thymus, CD4+ recent thymic emigrants (RTEs), and mature naive T cells in the periphery. The results demonstrate that RTEs and naive T cells had reduced mitochondrial content and mitochondrial reactive oxygen species when compared to SP thymocytes. This down-regulation of mitochondria requires T cell egress from the thymus and occurs early after young T cells enter the circulation. Autophagic clearance of mitochondria, but not mitochondria biogenesis or fission/fusion, contributes to mitochondrial down-regulation in RTEs. The enhanced ASK1/MAPKs and reduced mTOR activities in RTEs relative to SP thymocytes may be involved in this mitochondrial reduction. These results indicate that the gain of metabolic quiescence is one of the important maturation processes during SP-RTE transition. Together with functional maturation, it promotes the survival and full responsiveness to activating stimuli in young T cells.
Time of ART initiation in perinatally HIV-infected children impacts on HIV-specific T cell functionality
Authors: Rinaldi Stefano , Cotugno Nicola , Pallikkuth Suresh , Pahwa Rajendra , Palma Paolo , Pahwa Savita 
Published: January 2023

Abstract
Early initiation of antiretroviral therapy (ART) in vertically HIV-infected children provides an opportunity to limit the size of HIV reservoir. However, how the time of ART initiation impacts host HIV-specific immune responses is still poorly understood. In this study, we analyzed HIV-specific CD4 and CD8 T cell functionality in vertically HIV-infected children (age range 2.3–17.5yrs) enrolled at Bambino Gesù Children Hospital under suppressive ART and durable viral control (plasma HIV-RNA&lt;50cp/mL). Children were designated as early treated (ET, n=8, ART initiated at age &lt;6mo) or late treated (LT, n=7, ART initiated at age &gt;1 year) with longer duration of ART in ET (p&lt;0.05). Antigen-specific (CD40L+CD4+/CD107a+CD8+) T cells were evaluated in cryopreserved PBMC by flow cytometry for intracellular cytokines (IL2, IFN g, TNFα, IL21, Perforin, Granzyme B) following 12hr stimulation with GAG PTE peptides. Differences between groups were determined by Mann-Whitney t tests (P≤0.05).
Frequencies of GAG-specific CD8 and CD4 T cells were not different between ET and LT. Boolean analyses revealed that CD8 T cells of LT had a higher frequency of Granzyme B+ cells compared to ET whereas CD4 T cells were qualitatively superior in ET compared to LT and exhibited higher proportions of IL2, IFNg and TNFα producing T cells and enrichment of polyfunctional T cells.
Our results suggest that time of ART initiation in HIV-infected children has a long-term impact on the quality but not the quantity of the host HIV-specific T-cell immune responses. Larger studies are needed to confirm these results and to further evaluate their role in future strategies aiming at functional cure.
Single cell analyses of human antibody responses to Vibrio cholerae
Authors: Kauffman Robert C. , Bhuiyan Taufiqur , Adekunle Oluwaseyi , Ryan Edward , Qadri Firdausi , Harris Jason , Wrammert Jens 
Published: January 2023

Abstract
Cholera is a severe diarrheal disease affecting 2.9 million people annually resulting in an estimated 95,000 deaths. Infection induces long lasting protective immunity, likely mediated by intestinal antibodies. To further understand humoral immunity against cholera, we used single cell expression cloning to generate panels of human monoclonal antibodies (mAbs) from acutely induced plasmablasts isolated from patients in Bangladesh. mAbs largely targeted the dominant antigens cholera toxin and lipopolysaccharide (LPS). Notably, while LPS responses targeted the O-specific polysaccharide moiety, mAbs varied in serotype specificity and functional characteristics. Unexpectedly, despite all patients being infected by the Ogawa serotype, isolated mAbs from one patient preferentially bound to the Inaba serotype, which had been almost undetectable in circulation for 5 years. Inaba selective mAbs were characterized by high levels of somatic hypermutation. These findings suggest that cholera can generate immunologic memory and induce significant somatic hypermutation in response to this polysaccharide antigen. To expand on these findings we generated a panel of isotype and subclass variants from six LPS specific mAbs, representing a range of affinity and serotype specificity. mAbs were expressed as IgG1, IgG2, IgG3, IgG4, pentameric IgM, and the monomeric and dimeric forms of IgA1 and IgA2. This analysis provided detailed insight into how immunoglobulin isotype/subclass impacts functional characteristics such as agglutination and vibriocidal activity. Moreover, using live-cell microscopy and soft-agar migration assays, we provide novel insight into the impact of these antibody variants on bacterial propulsion.
Pathophysiology of rheumatoid arthritis is associated with polymorphisms in Protein Tyrosine Phosphatase Non-receptor type 2 and 22 (PTPN2/22) and susceptibility to Mycobacteria
Authors: Sharp Robert C. , Beg Shazia A. , Naser Saleh A. 
Published: January 2023

Abstract
Single nucleotide polymorphisms (SNPs) in T-cell regulator genes such as Protein Tyrosine Phosphatase Non-receptor type 2 and 22 (PTPN2/22) have been associated with many inflammatory autoimmune disorders including rheumatoid arthritis (RA). Along with these SNPs and an environmental trigger of a mycobacterial infection from Mycobacterium avium subspecies paratuberculosis (MAP), we hypothesis that this will lead to chronic inflammation of the synovial joints of RA. We gathered 132 consented subjects blood samples, where genotyping was done for nine SNPs found in PTPN2/22 using TaqMan™ genotyping. Quantification of the effect of the SNPs on gene expression of PTPN2/22 and IFN-γ expression was determined through real time PCR. T-cell proliferation assays were done to examine the response of phytohematoagglutonin (PHA) and mycobacterial antigens in subject samples by the use of BrdU proliferation assays. Detection of MAP bacterial IS900 DNA was done by nested PCR. Out of the nine SNPs examined, PTPN2:rs478582/PTPN22:rs2476601 were found significant in RA compared to the healthy controls (p-values &lt; 0.05). Gene expression of PTPN2/22 was downregulated in RA overall compared to healthy controls. IFN-γ expression was upregulated in samples with significant SNPs. T-cell proliferation increased when T-cells tested had significant SNPs (p-values &lt; 0.05). IS900 MAP DNA was detected more significantly in RA than in healthy controls, where samples with either significant SNPs had higher presence of MAP (p-values &lt; 0.05). This study suggests that SNPs in PTPN2/22 dysregulates T-cell regulation and increases susceptibility to Mycobacteria, leading to increased proliferation of T-cells and higher amounts of IFN-γ expression in RA.
Differences in the male and female immune response to Cryptococcus neoformans infections&lt;
Authors: Guess Tiffany , McClelland Erin 
Published: January 2023

Abstract

Introduction
Cryptococcus neoformans (Cn) is responsible for an estimated 220,000 cases of cryptococcus, resulting in more than 181,000 deaths each year worldwide. Prevalence of this disease is skewed between males and females. Numerous studies show differences in Cn infection rates, with males having a higher incidence of disease and death (7:3, m:f). Sexual dimorphism in infections is not uncommon. However, these differences in Cn have not been well elucidated. Our goal is to unravel the complexity of sex-specific, host-pathogen interactions by examining the effect of sex on the immune response to Cn and whether sex hormones contribute to those differences. We hypothesize that males have an increased incidence of disease due, at least in part, to differences in immune response during a Cn infection.

Methods
Peripheral blood mononuclear cells were isolated from 40 healthy volunteers and infected with Cn. Physiological levels of testosterone and estrogen were added to subsets of all infections. Infected cells were incubated for seven days after which they were harvested and B- and T-cell responses assessed via flow cytometry. Analytes measured include: CD3, CD4, CD8 (T-cell markers), and CD19 (B-cell marker). Serotyping was done on all samples and correlated with immune response.

Results
Subjects with previous exposure to Cn exhibited an increased CD19 response. Data show lower numbers of all T-cell markers in males during infection. There was no difference in hormonal treatment groups.

Discussion
The sexual dimorphism seen in Cn infections is commonly observed, but understanding what accounts for these differences is unknown. These results could aid in understanding why males have increased disease in Cn infections.
Differences of C-type lectin receptors and their adaptor molecules in the peritoneal fluid of patients with endometriosis and gynecologic cancers
Authors: Park Dong Choon , Kim Young Il , Lee Jin-Woo , Yeo Seung Geun 
Published: January 2023

Abstract

Objective
The immunological differences between endometriosis and malignant gynecologic tumors were analyzed by assessing C-type lectin receptors, which are associated with innate immunity, and immunoglobulin secretion, which is associated with B cell adaptive immunity, in peritoneal fluid of these patients.

Methods
The levels of expression of C-type lectin receptors mRNAs were measured by real time PCR in peritoneal fluid of 35 patients with benign masses, 36 patients with endometriosis, and 35 patients with gynecologic cancers. In addition, the concentrations of IgG, IgA and IgM were measured by ELISA in peritoneal fluid of the same patients.

Results
The level of galectin mRNA was significantly lower, and the level of MR2 and DAP 12 mRNA significantly higher, in the endometriosis than in the control group (p&lt;0.05 each). Compared with the gynecologic cancer group, the level of Bcl 10 mRNA was significantly lower, and the levels of MR1, MR2, Syk, Card 9, Malt 1, Dec 205, Tim 3, and DAP 12 mRNAs significantly higher, in the endometriosis group (p&lt;0.05 each). The level of MR2 & DAP 12 mRNA in the endometriosis group was significantly higher than in the control groups (p&lt;0.05), whereas the level of galectin mRNA in the endometriosis group was similar to that in the gynecologic cancer group (p&lt;0.05). IgA and IgG concentrations in peritoneal fluid were significantly lower in the gynecologic cancer than in control group (p&lt;0.05 each).

Conclusions
C-type lectin receptors and immunoglobulins act cooperatively and are closely associated in the pathogenesis of endometriosis.
Characterization of the Thymic Hypoplasia in Mouse Models of 22q11.2 Deletion Syndrome (DiGeorge Syndrome)
Authors: van Oers Nicolai S. , Du Qiumei , de la Morena M. Teresa , Dozmorov Igor , Khan Shaheen , Cleaver Ondine 
Published: January 2023

Abstract
Patients with 22q11.2 deletion syndrome have variable, multi-system disorders including a thymic hypoplasia, cardiac anomalies, and hypoparathyroidism. Over 90% have a deletion of 2.5 Mb on chromosome 22q11.2, affecting protein coding genes, microRNAs, long noncoding RNAs, and pseudogenes. 50%~70% have some degree of thymic hypoplasia (DiGeorge syndrome), resulting in a T cell lymphopenia. Successful transplantation of thymic tissue in patients with a thymic aplasia suggests stromal tissue abnormalities. The thymic stroma consists of thymic epithelial cells (TEC), mesenchymal cells, and endothelial populations. We are exploring the TEC-mesenchyme-endothelial interactions during thymus organogenesis in the mouse model of 22q11.2 deletion syndrome (Df1/+). Comparative transcriptome analyses of hypoplastic and normal thymic lobes from embryos revealed a unique mesenchymal cells mRNA expression signature, including reduced levels of the PDGFRa. PDGFRaH2B-EGFP heterozygous mice that have lower level of PDGFRa expression are being crossed with the Df1/+ model to ascertain whether the hypoplasia is influenced by this receptor. Additional mouse models with prominent penetrance of thymic hypoplasia are being used to determine how Tbx1 (encoded on 22q11.2) influences pharyngeal pouch mesenchymal-TEC development. The results from the study will likely lead to novel approaches for thymus reconstitution in various clinical settings.
Compound Heterozygous Mutations in Forkhead Box N1 (FOXN1) Lead to a Severe Immunodeficiency but Normal Hair and Nail Development in Patients
Authors: van Oers Nicolai S. , Khan Shaheen , Hunyh Larry K. , Du Qiumei , Padron Grace T. , Molina Erika , Dozmorov Igor , Markert M Louise , de la Morena M. Teresa 
Published: January 2023

Abstract
Patients with mutations in the Forkhead Box N1 (FOXN1) transcription factor are born with a severe T-cell lymphopenia in conjunction with alopecia and nail dystrophy (OMIM # 600838). The T-cell lymphopenia results from the impaired development and/or function of the thymic epithelial cells (TECs). TECs are essential regulators of positive and negative selection of developing thymocytes. We report on 3 independent patients with compound heterozygous mutations in FOXN1. All 3 patients had a severe T cell lymphopenia. However, each had normal hair growth and nail bed formation, suggesting that the compound heterozygous mutations result in clinical presentations distinct from classic cases. To determine how the mutations impact murine Foxn1 function, transcriptional reporter assays and protein expression studies were done. Only one of the mutations affected the transcriptional activity of murine Foxn1, with Western blot analyses indicating that this mutation caused production of a truncated protein. CRISPR/Cas9 technologies were used to create mouse lines with compound heterozygous mutations in Foxn1. The mice are currently being intercrossed. The impact of the compound heterozygous Foxn1 mutations on T cell development in the thymus will be presented. Findings from this study may suggest novel therapeutic strategies at restoring thymopoiesis in different clinical settings.
Skin Microbiota as Antigenic Triggers for Cutaneous T Cell Lymphoma
Authors: Dehner Carina Anja , Ruff William Edwards , Foss Francine , Girardi Michael , Kriegel Martin A. 
Published: January 2023

Abstract

Background
Cutaneous T Cell lymphoma (CTCL) is a debilitating malignancy of lymphocytes that home to the skin, thereby manifesting in a variety of chronic skin lesions. CTCL is thought to arise from a combination of genetic, epigenetic and environmental factors, but specific triggers remain unknown. We propose a model in which adaptive immune responses to particular skin commensals evolve into clonal proliferation of T cells in genetically predisposed patients.

Methods and Results
10 CTCL patients with active skin lesions were sampled at 3 time points (blood, stool and lesional vs non-lesional skin). Skin swabs of SLE patients and healthy donors served as control. Dual-index V1–3 and V4 16S rRNA sequencing was performed on the MiSeq platform and revealed significantly more Firmicutes in patient skin compared to controls. In addition, single bacterial colonies were cultured from 3 subjects. Full-length 16S rRNA sequencing demonstrated unique bacterial strains in lesional compared to non-lesional cultures. Malignant cutaneous T cells from patient biopsies were extracted and cultured in vitro for antigenic challenge with patient-isolated skin commensal candidates. One patient’s T cells showed significant proliferation to lesionally enriched B. safensis, while not responding to non-lesional bacteria.

Discussion
Dissecting cancer cell-microbiota interactions revealed potential skin commensal candidates involved in the pathogenesis of CTCL. Pathobionts enriched in lesional skin might act as antigenic drivers for progression to clonality similar to processes involved in H. pylori-induced MALT lymphoma, a paradigm that may be applicable to skin lymphomagenesis.
Abnormal T follicular helper cell subsets in Chronic Lymphocytic Leukemia
Authors: Marshall Aaron J. , Zhang Christine , Hou Sen , Wu Xun 
Published: January 2023

Abstract
The B cell malignancy Chronic Lymphocytic Leukemia (CLL) is a slow progressing disease of monoclonal B cell expansion and immune dysfunction associated with recurrent infections and second malignancies. CD4+ CXCR5+ T cells, known as follicular helper T cells (Tfh), have specialized properties in promoting B cell activation but their role in CLL is unknown. Here we examine the phenotype, activation status, clinical relevance and potential function of circulating Tfh cell subsets in CLL patients. We find that the percentage of Tfh cells is significantly increased in CLL patients compared to healthy age-matched controls. Tfh frequencies are significantly increased in advanced stage CLL patients compared to early stage disease. CLL patients also exhibited different composition of Tfh1, Tfh2 and Th17 subsets and show differential expression of activation markers compared to healthy controls. Our results indicate that the Tfh1 subset is particularly activated and expanded in CLL patients with more progressive disease. Tfh subset expansion and activation correlated with specific cytokine and chemokine levels and with decline in serum immunoglobulin levels. A large proportion of CD4+ T cells in CLL bone marrow express activated Tfh1 phenotype, suggesting that these cells are present within lymphoid tissues where CLL cells proliferate. Lastly, we found that treatment of CLL patients with the Btk inhibitor Ibrutinib lead to normalization of Tfh populations and reduced Tfh activation markers. Our results indicate that Tfh subset profiles could serve as useful biomarker to evaluate the progression of CLL and suggest selective expansion of Tfh1 cells may help drive CLL proliferation and immune dysfunction.