bluuebunny/crossref_metadata_2025_split huggingface dataset:
Open Discovery in an Undergraduate Immunology Course Lab: Testing Anti-Inflammatory Drugs
Authors: Brennan Catherine A , Roshandell Mobina 
Published: January 2023

Abstract
I will present the design and outcomes from a course laboratory module in which undergraduates apply techniques learned earlier in the semester (cell culture, spectrophotometry, standard curves, ELISA) towards the testing of an anti-inflammatory drug of their choice. Students rationalize their dose and assay selections, calculate all dilutions, perform the assays, and analyse and interpret all results. Student-directed course research experiences have been shown to solidify student understanding of advanced scientific concepts. Additionally, I have observed that this exercise, in which students analyse the function of medications used by themselves or loved ones, leads to enthusiastic engagement with and mastery of complex concepts.
Jedi Mindset: Discussing Effective Learning Strategies with Students
Authors: Alard Pascale 
Published: January 2023

Abstract
Students’ ability to retrieve acquired knowledge long-term is critical for every discipline. The research on learning shows undergraduate students use ineffective learning strategies, misjudging passive vs active ways of learning, and prioritizing grade over long-term learning, while they could achieve both by switching to effective learning strategies. Moreover, first generation graduates may be at a disadvantage as they have less opportunity to learn from family member experience. Sixty percent of students who attend medical, dental or graduate school continue to use ineffective strategies, e.g., re-reading.
Our goal was to discuss learning strategies with our incoming graduate students to encourage them to switch to effective ones. We held four consecutive weekly sessions before courses started to discuss the book by Dr. Saundra McGuire entitled, “Teach yourself how to learn”. Incoming graduate students read a few chapters for each session and discussed what they had read with facilitators and senior students, who shared their own experience about the program and provided advice. The final session revisited the various concepts with short podcasts highlighting the different points. A majority of our students were willing to modify their study habits and will be surveyed regularly to assess whether they continue to use effective learning strategies. More importantly, students who switched to effective learning strategies understood concepts better and scored higher on exams, including comprehensive exams. Our study suggests that regularly discussing effective learning strategies with graduate students is easy to implement, and may be beneficial to their understanding of material, performance and long-term retrieval.
Supported by None
Dntt expression reveals developmental hierarchy and lineage specification of hematopoietic progenitors.
Authors: Tussiwand Roxane , Klein Fabian , Roux Julien , Cvijetic Grozdan , Tsapogas Panagiotis 
Published: January 2023

Abstract
Intrinsic and extrinsic cues determine developmental trajectories of hematopoietic stem cells (HSCs) towards erythroid, myeloid and lymphoid lineages. To dissect early lymphoid specification events, we generated two transgenic mouse models reporting and tracing the expression of terminal deoxynucleotidyl transferase (TdT). Surprisingly, transient induction of TdT was detected on a newly identified multipotent progenitor (MPP) subset which lacked self-renewal capacity but maintained multilineage differentiation potential. TdT induction by MPPs reflected a transcriptionally dynamic but uncommitted stage characterized by low expression of lineage-associated genes. Further, analysis using single-cell CITE-Seq indicated that multipotency is associated with expression of Endothelial Cell-Selective Adhesion Molecule (ESAM). Developmental progression along all lineages is defined by downregulation of ESAM and by stable TdT expression within the lymphoid trajectory. Collectively, we propose a new developmental hierarchy of early hematopoietic progenitors for which we provide their transcriptional profile as well as their phenotypic and functional properties at single cell resolution.
Supported by NIH intramural funding
A lineage specific role for PD-1 in agonist selection in the thymus
Authors: Caruso Breanna P , Moran Amy 
Published: January 2023

Abstract
Effective peripheral T cell responses rely upon a diverse T cell repertoire, which is generated during the process of thymic selection. Signaling through the T cell receptor is critical in determining the lineage fate of developing thymocytes; however, it has been established that costimulatory molecules and cytokines can also contribute to fate decisions. Interestingly, coinhibitory molecules, such as programmed cell death protein 1 (PD-1), and their ligands are also expressed in the thymus. Expression of these molecules early in T cell development could regulate lineage fate decisions; however, their potential role in modulating T cell selection is not well-understood. We demonstrate that PD-1 is differentially expressed throughout thymocyte development. Additionally, we show that PD-1’s ligands, PD-L1 and PD-L2, are expressed by thymic epithelial cells. To elucidate the role of PD-1 signaling in regulating T cell selection, we examined mice deficient in PD-1 or PD-L1. Disrupting PD-1 signaling results in an increase in regulatory T cells (Tregs), but not other agonistly selected cell populations. This effect is dose-dependent as mice heterozygous for PD-1 or PD-L1 display an intermediate phenotype. Interestingly, our data suggest that PD-1’s regulation of Treg development is not through its inhibition of T cell receptor signaling, but rather through regulation of IL-2 cytokine production. These data demonstrate that PD-1 signaling restricts Treg development in the thymus, which broadens our understanding of the role of inhibitory proteins to include regulation of T cell development.
Supported by grants from NSF (GRFP AVPRS0015) and NIH (F31 CA261058-01).
Using in vivo barcoding to capture T versus B cell fate commitment in the thymus.
Authors: Abdullah Leena , Emiliani Francesco E , McKenna Aaron , Huang Yina H 
Published: January 2023

Abstract
B cells are a rare subset in the thymus, an environment that supports T cell development and inhibits commitment to other immune cell lineages. Yet thymic B cells play an important role in the negative selection of autoreactive T cells. The origin of thymic B cells from either committed B cells or early thymocyte progenitors remains uncertain. We hypothesize that early thymic progenitor cells are a heterogenous population that can give rise to both T cells and B cells. In our study we combined the CRISPR/Cas9-dependent lineage recorder GESTALT with single cell RNAseq to capture cell lineage and type information to follow progressive cell fate decisions and to identify thymic B cell progenitors and transcriptional regulators permitting B cell differentiation in the thymus. Additionally, to evaluate the functional importance of thymic B cells in shaping the T cell repertoire, we performed TCR-seq on conventional CD4 T cells and Tregs. In the absence of B cells, amino acid usage within either TCR alpha or beta CDR3 regions remained unchanged. However, the dominant TCR alpha CDR3 length from the most abundant thymic Treg clones was reduced compared to wild type. Whether this repertoire shift impacts self-tolerance to B cell or AIRE-dependent antigens, both of which are expressed by thymic B cells, remains to be determined. Together, our data directly assesses lineage relationships leading to thymic T and B cell fate commitment and deepen our understanding of intra-thymic development and function of B cells that are required for central tolerance.
Supported by AI089805 Burroughs-Wellcome Fellowship
The Transcriptional Cofactor Ski in Thymic Epithelial Cells Regulates Peripheral T Cell Responses
Authors: Chiu Honyin , Ziegler Steven F 
Published: January 2023

Abstract
The thymus is an important site for the establishment and maintenance of an appropriate immune response through positive and negative selection of developing T cells. Cortical and medullary thymic epithelial cells (TECs), termed cTECs and mTECs, respectively, interact with developing thymocytes to mediate this selection process. Developing thymocytes also support the maturation and proliferation of mTECs by secreting transforming growth factor-beta (TGF-β) superfamily cytokines. Our lab has previously shown that deletion of TGF-β signaling in TECs enhanced negative selection and functional maturation of thymocytes, and increased the production of regulatory T cells. To investigate whether the proto-oncogene product Ski (Sloan-Kettering Institute), a negative regulator of TGF-β signaling, is associated with thymocyte development in homeostatic condition and inflammatory conditions, we generated mice that deleted Ski specifically in TECs (Foxn1Cre Skifl/fl mice). We found that the mTEC population was decreased as predicted, however we did not find any differences in thymocyte development between the Foxn1Cre Skifl/fl mice and control mice. We also found that CD4 T cells purified from Ski deleted mice showed a defect in proliferation, and Th1 and Th17 differentiation in vitro, while regulatory T cell differentiation was normal. Lastly, we found that these mice were significantly protected from developing experimental autoimmune encephalomyelitis (EAE) disease compared to the control mice. Overall, our findings suggest that Ski signaling in TECs regulates peripheral T cell self-reactivity responses and further study may provide evidence for targeting Ski in T cell driven autoimmune diseases such as multiple sclerosis.
Supported by grants from NIH grant R01-AI136475 (to SZ) and AAI Intersect fellowship (to HC)
Loss of Zfp335 triggers cGAS/STING-dependent apoptosis of post-β selection pre-T cells
Authors: Ratiu Jeremy J , Barclay William , Wang Qun , Shinohara Mari L , Zhuang Yuan 
Published: January 2023

Abstract
Production of a diverse peripheral T cell compartment requires massive expansion of the bone marrow progenitors that seed the thymus. There are two main phases of expansion during T cell development, following T lineage commitment at the DN2 stage and following successful rearrangement and selection for functional TCRβ chains in DN3 thymocytes, which promotes development of DN4 cells to the DP stage. Signals driving expansion of DN2 thymocytes are well studied, however, factors regulating the proliferation and survival of DN4 cells remain poorly understood. Here, we uncover an unexpected link between the transcription factor Zfp335 and control of cGAS/STING-dependent cell death in post-β-selection DN4 thymocytes. Zfp335 controls survival by sustaining expression of Ankle2 and regulating phosphorylation of Baf which functions to promote nuclear envelope integrity and suppress cGAS/STING-dependent cell death. Together, this study identifies Zfp335 as a key transcription factor controlling the survival of proliferating post-β-selection thymocytes and demonstrates a key role for the cGAS/STING pathway driving apoptosis of developing T cells.
Supported by grants from NIH R01-GM059638, P01-AI102853, RO1-AI099100, RO1-NS120417, and RO1-AI160737
The role of skin-derived IFN-kappa in the development of systemic autoimmunity
Authors: Martens Jacob William Shelton , Liu Jianhua , Maz Mitra , Kahlenberg J Michelle 
Published: January 2023

Abstract
Aberrant type I interferon expression and downstream interferon-stimulated gene signatures are central to the immune dysregulation observed in lupus. Recent evidence has suggested that elevated interferon kappa expression in the skin originating from keratinocytes may precede the development of systemic autoimmunity. Using a transgene mouse model overexpressing IFN-κ specifically in keratinocytes, we observe the development of anti-nuclear antibodies and immune complex deposition in the kidneys in the absence of environmental triggers. We also observe immune cell infiltration in the kidneys including activated CD4 T cells, activated cDC2-like dendritic cells, and expansion of IgD-CD27-double negative B cells. A subset of DN B cells has recently been shown to be increased in the blood of lupus patients with active disease and nephritis. This DN B cell subset was shown to be hyperresponsive to TLR7 signaling and showed transcriptional markers indicative of an inclination to differentiate into antibody-secreting plasmablasts. Further studies on the IFN-κ induced DN B cells in our model are needed to confirm phenotype and function of these cells. Future studies also hope to address the role of individual immune cell types in propagating or responding to the skin IFN-κ signal. Elucidating the mechanisms of early immune dysregulation resulting from this heightened IFN signaling would provide promising clinical relevance to treating patients early in their disease course, potentially prior to detrimental systemic manifestations.
Supported by NIAMS(R01 AR071384-05) NIH(T32 AI007413-29)
Association between anti-P. gingivalis antibodies and anti-citrullinated vimentin antibodies in the patients with rheumatoid arthritis.
Authors: Shindo Satoru , Nakamura Shin , Rawas-Qalaji Mohamad , Potempa Jan , Arvikar Sheila , Ouhara Kazuhisa , Shiba Hideki , Kawai Toshihisa 
Published: January 2023

Abstract

Objectives
Anti-citrullinated-protein auto-antibodies (ACPA), especially that react to citrullinated vimentin (CV), are implicated in the pathogenesis of Rheumatoid arthritis (RA). P. gingivalis (Pg), a key pathogen of periodontitis, can citrullinate the host proteins by production of Pg-derived peptidylarginine deiminase (PPAD). This study evaluated the relationship among antibodies (Abs) that react cyclic citrullinated protein (CCP), CV and periodontal pathogens in the serum of RA patients or control subjects.

Methods
Blood serum of healthy subjects (n=10) and RA patients (n=26) were obtained from Precision for Medicine. Six distinctive peptide sequences of Vimentin (#1–#6) and corresponding CV (#1’–#6’) peptides were synthesized. ELISA was employed to detect IgG Ab titers for CCP, intact vimentin and CV peptides. Fixed periodontal pathogens, Pg 33277, F. nucleatum, P. intermedia, A. actinomycetemcomitans (Aa) Y4, S. mitis, S. gordonii, A. odontolyticus, and C. gingivalis were also used as IgG ELISA antigens.

Results
The levels of anti-CCP Ab were higher in RA patients with anti-Pg Ab than those without anti-Pg Ab. Since elevated anti-Pg Ab is hallmark of periodontitis, it is conceivable that RA patients with anti-Pg Ab would have periodontitis. However, there was no association between anti-CCP Ab and the Abs reactive to the other bacteria. Very interestingly, RA patients with anti-Pg Ab, but not those without anti-Pg Ab, showed significantly elevated Ab response to #6’ CV peptide compared to intact #6 vimentin peptide, among six peptide sequences tested.

Conclusions
Our findings indicated that periodontitis may be associated with the induction of ACPA via PPAD-mediated citrullination of host proteins.
Supported by NIH NIDCR grants, DE-027851, DE-028715 and DE-331851
IgA and FcαR are critical components of plasmacytoid DC response to autoantibody-containing immune complexes in SLE.
Authors: Waterman Hayley R , Duffort Matthew , Posso Sylvia , Buckner Jane H , Hamerman Jessica A 
Published: January 2023

Abstract
Systemic lupus erythematosus (SLE) is a heterogenous autoimmune disease characterized by the presence of circulating autoreactive anti-nuclear antibodies (ANAs). ANAs form immune complexes (ICs) upon binding to nuclear antigens and these ANA-ICs promote a feedforward loop by enhancing immune responses in cells that recognize ANA-ICs via Fc receptors (FcRs). Plasmacytoid dendritic cells (pDCs) are FcR expressing cells that promote SLE pathology. ANA-ICs deliver nucleic acids to endosomal TLR7 and TLR9. These TLRs induce type I IFN secretion and immune activation upon nucleic acid recognition. Type I IFNs, including IFNα, and interferon stimulated genes are correlated with SLE disease activity. IgG isotype facilitated ANA-IC internalization via FcgRIIA has been the most studied antibody-mediated route for IFNα production in pDCs. The importance of IgA isotype ANAs remains under investigated despite these autoantibodies being present in half of SLE patients. We show here the novel result that human pDCs express the IgA-specific FcαR (CD89) by flow cytometry, that pDCs from SLE patients express increased FcαR compared to pDCs from healthy controls, and that increased pDC FcαR expression in SLE correlates with hallmark IFNα gene set. Additionally, we found that IgA autoantibodies were a critical component of pDC ANA-IC activation when these ANA-IC were generated with serum from IgA autoantibody positive SLE donors complexed with smRNP nuclear antigen. Therefore, our study shows that IgA-containing immune complexes and FαRI are important contributors to pDC type I IFN production in SLE.
Supported by LRA Lupus Mechanisms and Targets Award (JAH), R21AI154841 (JAH), and ITHS TL1 training grant (HRW).
Small molecule inhibition of spleen tyrosine kinase mitigates gut-liver axis inflammation and histopathology in a novel model of IBD-associated liver disease.
Authors: Jewell Mark L , Xue Yue , Huang Hsin-I , Lamagna Chrystelle , Hammer Gianna E 
Published: January 2023

Abstract
In light of recent studies identifying gain-of-function variants of spleen tyrosine kinase (Syk) as a cause of multi-organ inflammatory disease, Syk inhibition has become an attractive pharmacological target. Therapeutic potential of Syk inhibition may extend beyond that of gain-of-function mutations, since Syk activity initiates downstream responses to several pattern-recognition receptors, which are collectively thought to trigger pathology in several inflammatory diseases, including inflammatory bowel disease (IBD). We previously identified the IBD-linked gene Tnfaip3 (A20) as a suppressor of Syk-dependent inflammatory signals in dendritic cells (DCs) and report here that small molecule inhibition of Syk with FDA-approved drug Fostamatinib mitigates the small intestinal inflammation that develops spontaneously in mice with DC-specific loss of A20 (A20cko mice). In our investigation of IBD in these mice we surprisingly found that A20cko mice also had striking inflammation of the liver that shared many biochemical and histological features characteristic of IBD-associated liver disease, including extensive bridging fibrosis. Importantly, Syk inhibition normalized liver biochemistries and significantly reduced intrahepatic inflammation and fibrosis. Mechanistically, plasma proteomics and liver immunophenotyping pinpointed the key impact of Syk inhibition on restricting chemokine expression linked to recruitment of pro-inflammatory monocytes into in the liver. Collectively this work identifies the interplay of Syk and A20 signaling as drivers of inflammation along the gut-liver axis and suggests Syk is a valid target for pharmacological intervention for IBD and IBD-associated liver disease.
Supported by R01AI145930-01A1
CB2 engagement enhances group 2 innate lymphoid cell expansion and induction of airway hyperreactivity
Authors: Hurrell Benjamin P , Helou Doumet Goerges , Shafiei-Jahani Pedram , Howard Emily Diane , Painter Jacob Dean , Quach Christine , Akbari Omid 
Published: January 2023

Abstract
Cannabinoids modulate the activation of immune cells and physiological processes in the lungs. Group 2 innate lymphoid cells (ILC2)s are central players in type-2 asthma, but how cannabinoids modulate ILC2 activation remains to be elucidated. Using a combination of cannabinoid receptor (CB)2 KO mice, a CB2 antagonist and agonist, we here provide evidence that CB2 signaling in ILC2s is important for the development of ILC2-driven airway inflammation in both mice and human. We show that both naïve and activated murine pulmonary ILC2s express CB2. CB2 signaling did not affect ILC2 homeostasis at steady state, but strikingly stimulated ILC2 proliferation and function upon activation using various models of airway inflammation including IL-33, IL-25 and Alternaria alternata. As a result, ILC2s lacking CB2 induced lower lung inflammation, as we made similar observations using a CB2 antagonist. Conversely, CB2 agonism remarkably exacerbated ILC2-driven airway hyperreactivity and lung inflammation. Mechanistically, transcriptomic and protein analysis revealed that CB2 signaling induced CREB phosphorylation in ILC2s. Human ILC2s expressed CB2, as CB2 antagonism and agonism showed opposing effects on ILC2 effector function and development of airway hyperreactivity in humanized mice. Collectively, our results define CB2 signaling in ILC2s as an important modulator of airway inflammation. Furthermore, our findings highlight the stimulatory capacity of cannabinoids on ILC2s and offer new therapeutic avenues, including the use of substances or pathways able to modulate CB2 and capable of alleviating lung function in patients with lung inflammation.
The role of Cul5 in Group 2 innate lymphoid cells (ILC2s)
Authors: Guha Ipsita , Dar Asif Amin , Kumar Binod , Porter Nadia , Oliver Paula M 
Published: January 2023

Abstract
Over the past few years we have witnessed a change in the study of allergic inflammation from a view that holds T helper type 2 cells at the centre of immune orchestration to one that appreciates the significant contribution made by innate lymphoid type 2 cells (ILC2s). ILC2 cells are considered to be the innate counterpart of Th2 cells and these two evolutionarily related cells cooperate to protect the host against helminth infection and, when dysregulated, in the pathogenesis of allergic diseases. We recently determined that the E3 ubiquitin ligase Cul5 limits the differentiation and pathogenicity of Th2 cells and protects against allergic airway remodelling in mice. Here we show that Cul5 also regulates ILC2 cells. Specifically, following IL-33 treatment, mice lacking Cul5 in all hematopoietic cells contained increased numbers of ILC2 cells in their lungs and this corelated with significant Type 2 inflammation. Additionally, in the absence of Cul5, ILC2 cells were more likely to produce IL-9. Similar results were also observed in mice lacking Cul5 specifically in ILC2 cells (Cul5fl/fl-R5/+), supporting that Cul5 restricts the numbers and function of ILC2 cells. Ex vivo and following in vitro culture, Cul5-deficient ILC2 cells exhibited distinct features that would likely impact their responsiveness to environmental cues such as cytokines. Here we will detail how Cul5 impacts the differentiation and function of ILC2 cells and how this may alter the outcome of type 2 immune responses.