bluuebunny/crossref_metadata_2025_split huggingface dataset:
A novel method for high throughput TCR single cell VDJ-pairing with phenotypic analysis
Authors: Byrne-Steele Miranda , Pan Wenjing , Brown Brittany E , Hou Xiaohong , Depinet Mollye , Eisenhower Mary , Weber Daniel , Han Jian 
Published: January 2023

Abstract
Single cell transcriptional analysis reveals a wealth of information that is obscured when analyzing the RNA of a sample en masse. Furthermore, it is important to know the identity of the TCR chain VDJ-rearrangement associated with the phenotypic state. This information can provide direct calculations of clonal frequency in various cell subsets, tracking of specific lymphocytes with treatment, and reveal paired information for both chains of the receptor for downstream Car-T development. Here, we discuss a novel method that identifies paired VDJ-rearrangements for both TCR alpha and beta chains from single cells barcoded via the BD Rhapsody Express single cell system. The phenotype of the cell is obtained using the BD Rhapsody RNA-seq kit, while the receptor information is amplified from the same cDNA using an iRepertoire-derived method incorporating a multiplex mix of primers associated with both the TCR alpha and beta loci. This targeted-seq strategy requires less read depth when compared to template-switch approaches. In one experiment, 3,000 TCR-beta unique CDR3 (uCDR3) were identified from ~7,000 barcoded cells using 1.5 million NGS reads with a 0.83 Pearson correlation upon repeat. Approximately 1,000 TCR alpha-beta paired uCDR3 were also called from the data. Additionally, the BD phenotyping results independently verify the immune repertoire data since the TCR beta chain constant gene overlays with the TCR-beta uCDR3 clonotypes for CD4+ and CD8+ subsets but is largely absent in the CD14+ monocyte population for both analyses. Thus, the developed technology provides the ability to simultaneously pair both alpha and beta chains for thousands of T-cells while obtaining information related to the functional status of each cell.
Development of high-throughput methods used to identify and characterize novel interactions within the human secretome with focus on the Ig and TNF receptor superfamilies
Authors: Garrett-Thomson Sarah C , Almo Steven 
Published: January 2023

Abstract
While it is estimated that roughly 15% of the human genome encodes secreted proteins, many remain “orphans” with no known ligand or are poorly characterized. Even for those with known ligands, new binding partners continue to be identified that fundamentally reshape our understanding of their role in specific biological processes and diseases (i.e. PD-L1:B7-1, ICOS-L:CD28, PD-L2:RGMb). This highlights the need for systematic screening efforts to identify such interactions, broadening our general knowledge while also providing potential new targets for therapeutic intervention. To this end we have developed a set of novel technologies using both cell microarray and high-throughput flow cytometry techniques to screen for novel extracellular protein: protein interactions. Initially we focused on screening for interactions within the Ig and TNF receptor superfamilies, two protein families that are the central underpinning of our immune response but for which much is still unknown. We have generated two expression-validated libraries for cell-based screening, a 366 member Type I expression library of Ig and TNF receptor proteins and a ~3800 member full-length human plasma membrane protein library encompassing much of the human membrane proteome. Through rounds of screening we identified novel interactions for B7-1 (CD80), ICOS-L, TrkA and TrkC. The identified interactions were validated biochemically and are being characterized for their biological significance. Additionally we are using these technologies to characterize the binding interfaces of both known and newly discovered protein complexes as well as to screen for potential off-target interactions of protein-based therapeutics.
Alpha-1-Antitrypsin Ameliorates Diffuse Alveolar Hemorrhage in the Pristane Induced Lupus Model
Authors: Elshikha Ahmed Samir , Abboud Georges , Danos Abigail , Chen Mong-jen , Yuan Ye , Zeumer Leilani , Morel Laurence , Song Sihong 
Published: January 2023

Abstract
Diffuse alveolar hemorrhage (DAH) is a fatal complication of lupus with &gt;50% mortality in humans. DAH can be induced in B6 mice by a single intraperitoneal (i.p.) injection of pristane. Human alpha-1-antitrypsin (hAAT) is an anti-inflammatory and immuno-regulatory protein. In this study, we investigated the protective effect of treatment with hAAT against pristane induced DAH development in B6, as well as in AAT-deficient (AAT-Ko) and hAAT transgenic (hAAT-Tg) mice. First, we characterized activated splenocytes from B6 and hAAT-Tg mice. We found that TNF-α production was significantly lower in the B cells, CD4+ and CD8+ T cells from hAAT-Tg mice as compared to B6 mice. Conversely, the frequency of CD4+CD25+ and CD4+CD25−IL-10+cells was significantly higher in hAAT-Tg than that in B6 mice. To test the effect of hAAT on DAH development, we treated B6 mice with or without hAAT (2mg/mouse/2 d) and induced DAH by a pristane injection. Two weeks after induction, control B6 mice showed severe lung hemorrhage, while hAAT treatment prevented lung hemorrhage. To investigate the mechanisms responsible for protection, we induced DAH in B6, AAT-Ko and hAAT-Tg mice. One week after induction, the frequencies of pro-inflammatory CD11b−Ly6Chi, pDCA-1+CD11clo, CD11c+CD11b+ and neutrophils were significantly lower in the peritoneal exudate cells of hAAT-Tg mice as compared to B6 and AAT-Ko mice. Moreover, the frequency of CD11b+Ly6Clow cells were significantly increased in hAAT-Tg. These results showed for the first time that hAAT has a therapeutic potential for the treatment of DAH by modulating myeloid cell phenotypes.
De novo fatty acid synthesis induced Sjögren’s syndrome by promoting Th17 differentiation
Authors: Luo Jing , Yin Zhinan 
Published: January 2023

Abstract
Primary Sjögren’s syndrome (pSS) is a chronic inflammatory autoimmune characterized by inflammatory infiltration of immune cells within lacrimal and salivary glands (SGs), which leads to the dysfunction of these exocrine glands. Th17 cells and their secreted cytokines play an important role in the pathogenesis of Sjogren’s syndrome, but the mechanism is still unclear. Recent clinical studies have found that metabolic disorders may be closely related to Sjogren’s syndrome. Our studies have also found that FASN, the key enzyme of lipid synthesis and metabolism in peripheral blood of Sjogren’s syndrome patients, is significantly elevated, and mainly expressed in Th17 cells. Fatty acid synthase (FASN) is downstream of ACC, and previous studies have shown that FASN activity influences both cancer and inflammation. However, it remains to be determined whether FASN is a viable target for Sjögren’s syndrome. Here, we demonstrate that FASN is a critical metabolic induced Sjögren’s syndrome by promoting the generation of inflammatory subsets of Th17 cells. In vivo, knocking out of FASN in Th17 cells, leads to reduction of Sjögren’s syndrome. These study demonstrate that FASN-mediated metabolism disorder of fatty acid synthesis may be a new mechanism to regulate Th17 cell function and lead to Sjogren’s syndrome, which can provide new directions and ideas for the treatment of Sjogren’s syndrome.
A rationally engineered DNase1-Fc fusion protein ameliorates autoimmune glomerulonephritis
Authors: Mouchess Maria , Suto Eric , Chan Sara , Dai Bingbing , Tam Christine , Cao Yi , Darwish Martine , He Dongping , Haller Susan , Eastham-Anderson Jeffrey , Franke Yvonne , Blanchette Craig , Lee Wyne , Austin Cary , Lazarus Robert , Yi Tangsheng 
Published: January 2023

Abstract
Loss of B cell tolerance and generation of autoreactive anti-nuclear antibodies are hallmarks of systemic lupus erythematosus and lupus nephritis. The mechanism of anti-nuclear immunoglobulin accumulation and clearance in lupus nephritis pathogenesis remains largely uncharacterized. Here, we show that innate immune activation in the NZB/W F1 mouse model and human lupus nephritis biopsies rapidly reduces expression of DNase1 (also called DNase I) in renal cortex proximal tubular cells. Unexpectedly, kidney DNase1 levels were no longer affected by TLR ligand agonism upon depletion of macrophages, indicating that local macrophage activation is required for the downmodulation of DNase1 in kidney epithelial cells. To overcome the loss of endogenous DNase1, we rationally designed a hyperactive actin-resistant variant of DNase1 with improved catalytic activity and acceptable in vivo pharmacokinetics. The engineered DNase1-Fc fusion protein ameliorates nephritis in a murine model of lupus and reduces immune complex deposition/complement fixation. Our data suggest that the loss of renal DNase1 through TLR signaling or other innate immune activation impairs clearance of autoreactive anti-nuclear immune complex deposits in the kidney to promote nephritis progression. While autoantibodies and autoreactive B cell activation are central to lupus pathogenesis, we hypothesize that renal cortical epithelial cells expressing high levels of DNase1 serve as gatekeepers to prevent detrimental nuclear immune complex deposition. Our findings provide a therapeutic rationale for using an engineered hyperactive actin-resistant DNase1-Fc for lupus nephritis.
Validation of lanthionine synthetase C-like 2 (LANCL2) as a therapeutic target for systemic lupus erythematosus
Authors: Leber Andrew , Hontecillas Raquel , Zoccoli-Rodriguez Victoria , Bassaganya-Riera Josep 
Published: January 2023

Abstract
Lanthionine synthetase C-like 2 (LANCL2) is a membrane receptor with downstream effects on immunity and metabolism including the differentiation of regulatory CD4+ T cells. Its natural ligand, abscisic acid (ABA), has been shown to be decreased in autoimmune and inflammatory disease. Given the implication of the LANCL2 pathway in autoimmune disease and the progression of LANCL2-specific ligands, such as BT-11, into clinical stage development, we assessed the role of LANCL2 in systemic lupus erythematosus (SLE). In a TLR7/8 resiquimod-induced model of SLE, the loss of LANCL2 significantly decreases survival (P = 0.0008) relative to wild-type, resulting in less than 10% survival and a greater than 60% reduction relative to wild-type. Similarly, LANCL2−/− displayed greater serum anti-dsDNA antibodies and higher proteinuria. Within spleens, the loss of LANCL2 resulted in greater IL-6 and TNF, higher proportions of T follicular helper cells and lower ratios of regulatory CD4+ T cells relative to IFNγ producing cells. The greater inflammation was also mirrored in transcriptional changes at the splenic level indicating a greater susceptibility to severe disease. These results merit further exploration into the implication of LANCL2 signaling in human SLE and characterization of the therapeutic efficacy of LANCL2-specific agonists for future SLE development. Thus, LANCL2 is a novel promising therapeutic target for SLE.
Small Molecule Inhibitor suppresses Tfh cell development in germinal center and reduces the severity of inflammation in a collagen-induced arthritis mouse model
Authors: Zhang Xin , Bhatia Nitasha , Migliore Frank , Hellmers Linh , Olayemi Gbemi , Mcleod Cara , Zakem Jerald , Davis William , Quinet Robert 
Published: January 2023

Abstract

Background
Rheumatoidarthritis (RA) is a autoimmune disease characterized by the accumulation of inflammatory cells in the joints, leading to hyperproliferation of synovial cells and tissue destruction. Our previous data showed autoreactive B cells could be induced by T follicular helper (Tfh) cells to undergo clonal expansion with in germinal center (GC), and ultimately differentiate into autoantibody producing plasma cells in RA. Here, we further investigated the role of Tfh cells in RA pathogenesis and the therapeutic effect of a small molecule inhibitor targeting Tfh cells (SMI-Tfh) in collagen-induced arthritis (CIA)mouse model.

Methods
CIA model was induced by immunization with chicken type II collagen in DBA/1 mice. Disease progression was monitored daily by the paw swollenness. Following the onset of clinical arthritis, mice were treated with SMI-Tfh. At the end of the study, blood, spleen, and affected paws were collected. Pathological changes were examined by H&E staining of tissue sections. Immunofluorescent histochemistry (IHC) staining and flow cytometry analysis were used to identify Tfh cells (CD4+CXCR5+ICOS+) in spleen and blood.

Results
Arthritis onset was developed at day 21 and with peak on day 42 after initial immunization. Destruction of articular cartilage, increased inflammatory cells and Tfh infiltration were observed in the synovial tissue. Mice treated with SMI-Tfh had significantly reduced Tfh cells in GC of the spleen (p&lt;0.01) and less arthritis feature (p&lt;0.05) in CIA mouse.

Conclusion
SMI-Tfh selectively inhibits GC-Tfh cells in the spleen and abrogates the severity of inflammatory arthritis. It may serve as a novel therapy by interfering with autoreactive GC-Tfh cells development in RA.
MyD88 and PKD1 play an essential role in the development of spontaneous polyarthritis in IL-1 receptor antagonist-deficient mice
Authors: Yoon Taewon , Cho Hongsik , Stuart John M , Yi Ae-Kyung 
Published: January 2023

Abstract
IL-1 is an important cytokine that is secreted by various cells, including synovial cells, and can induce the production of proinflammatory mediators. Due to the excessive and uncontrolled IL-1 signaling, IL-1R antagonist knockout mice (IL-1rnKO) develop spontaneous polyarthritis that mimics some pathogenic features of human rheumatoid arthritis (RA). MyD88 is a key signaling adaptor molecule for toll-like receptors (TLRs) and IL-1R. We have previously identified protein kinase D1 (PKD1) to play an indispensable role in the MyD88-dependent proinflammatory response. MyD88 and PKD1 may therefore confer regulatory role on arthritis development in IL- 1rnKO mice. To investigate this, we examined the synovial cell, T cell, and B cell responses, and the incidence and severity of arthritis in IL-1rnKO mice that are deficient in MyD88 or PKD1. We found that MyD88 deficiency and PKD1 deficiency inhibited the development of polyarthritis as well as reduced the levels of synovial macrophages in IL-1rnKO. T cells isolated from lymph nodes expressed significantly reduced CCR6, CXCR3, IFNγ, and IL-17 in MyD88-deficient IL-1rnKO mice. BAFF-R and TACI levels in splenic marginal zone precursor cells (MZP) and maturation of MZP to marginal zone B cells were significantly reduced in MyD88-deficient IL- 1rnKO mice. We are currently investigating whether PKD1 also play a regulatory role in T cell and B cell responses during the pathogenic course of arthritis in IL-1rnKO mice. Taking together, our results suggest that MyD88 and PKD1 play a critical role in the pathogenesis of arthritis in IL-1rnKO. Further insights into PKD1 and its role in arthritis development will contribute to better understanding innate signaling in the development of RA.
Targeting Chemokine Receptors CCR6 and CXCR2 in A Murine Model Of IL-36a-Induced Pustular Psoriasis
Authors: Campbell James J , Ebsworth Karen , Ertl Linda , McMahon Jeffrey P , Wang Yu , Yau Simon , Mali Venkat R , Chhina Vicky , Kumamoto Alice , Liu Shirley , Dang Ton , Newland Dale , Charo Israel F , Zhang Penglie , Schall Thomas J , Singh Rajinder 
Published: January 2023

Abstract
Several types of psoriasiform dermatitis are associated with increased IL-36 cytokine activity in the skin. A rare but severe psoriasis-like disorder, generalized pustular psoriasis (GPP), is linked to loss-of-function mutations in the gene encoding IL-36RA, an important negative regulator of IL-36 signaling. To understand the effects of IL-36 dysregulation in a mouse model, we studied skin inflammation induced by intradermal injections of pre-activated IL-36α. This type of inflammation was characterized by markedly increased total skin thickness and epidermal thickness. The inflammation was accompanied by increased concentrations of CCR6 and CXCR2 ligand chemokines in the skin, and large accumulations of CD4 T cells, neutrophils, and inflammatory DC. The accumulated inflammatory subsets in IL-36α-treated skin all expressed either CCR6 or CXCR2 to some extent, prompting the hypothesis that increased CCR6 and CXCR2 ligand concentrations in IL-36α-treated skin might constitute a viable target for relieving inflammation in this model. We found that the orally bioavailable small molecule CCR6/CXCR2 antagonist CCX624 did indeed reverse inflammatory cell accumulation in skin, as well as decrease total skin thickness and epidermal thickness. CCX624 was effective in both prophylactic and therapeutic dosing regimens. CCX624 treatment was more effective than saturating doses of anti-TNFα or anti-IL17RA, which are used in the clinic for treating GPP as first or second line treatments (respectively). These findings put CCR6 and CXCR2 forward as novel targets for a mechanistically distinct therapeutic approach for inflammatory skin diseases involving dysregulated IL-36 signaling, such as GPP.
1,25-Dihydroxyvitamin D3 ameliorates collagen-induced arthritis via suppression of Th17 cells through miR-124 mediated inhibition of IL-6 signaling
Authors: Zheng Song Guo , Zhou Li , Wang Julie 
Published: January 2023

Abstract

Objectives
To explore the molecular mechanisms in which vitamin D (VD) regulates T cells, especially Th17 cells in collagen-induced arthritis (CIA).

Methods
DBA1/J mice induced for CIA were intraperitoneally treated with VD. CIA clinical symptoms and inflammatory responses including Th1/Th17/Tregs percentages were determined and compared. Mouse naïve CD4+ T cells transduced with miR-124 inhibitor or not were polarized to Th17 cells with or without VD. Subsequently, cellular differentiation and IL-6 signaling moleculars were analyzed.

Results
VD treatment significantly delayed CIA onset, decreased incidence and clinical scores of arthritis, downregulated serum IgG levels and ameliorated bone erosion. VD downregulated IL-17A production in CD4+ T cells while increased CD4+Foxp3+Nrp-1+ cells both in draining lymph nodes and synovial fluid in arthritic mice. VD inhibited Th17 cells differentiation in vivo and in vitro and potentially functioning directly on T cells to restrain Th17 cells through limiting IL-6R expression and its downstream signaling including STAT3 phosphorylation, while these effects were blocked when naïve CD4+ T cells were transduced with miR-124 inhibitor.

Conclusions
VD treatment ameliorates CIA via suppression of Th17 cells and enhancement of Tregs. miR-124-mediated inhibition of IL-6 signaling, provides a novel explanation for VD’s role on T cells in CIA mice or RA patients and suggests that VD may have treatment implications in rheumatoid arthritis.
Identifying and engineering TCR specificity against solid tumor neoantigens
Authors: Naradikian Martin Souren , Montero Leslie , Hall Samantha , Thota Rukman , Frentzen Angela , Bahmanof Milad , Sternberg Luise , Lane Jerome , Kosaloglu-Yalcin Zeynep , Lanka Manasa , Miller Aaron , Peters Bjoern , Cohen Ezra , Schoenberger Stephen 
Published: January 2023

Abstract
Neoantigens (NeoAg) offer attractive therapeutic targets for directing a patient’s immune response to the immunogenic subset of mutations expressed exclusively by their cancer cells. Despite the specificity with which NeoAg enable tumor recognition, the majority of approaches for their identification rely on purely predictive methods such as calculating the ability of mutated peptides to bind to a patient’s set of HLA molecules. These methods have met with limited success in revealing natural targets present on tumor cells. We have developed a novel HLA-agnostic functional approach to NeoAg identification which combines genomic sequencing with bioinformatic analysis to nominate mutations for subsequent functional analysis using patient’s own T cells in an effort to identify natural responses generated under physiologic conditions. Using this, we identified a missense mutation (V205I) in the ribosomal protein RPS2 that is recognized by CD8+ T cells from tumor-infiltrating lymphocytes (TIL) of a metastatic HPV16+ Head and Neck Squamous Cell Carcinoma lesion. We then performed adoptive cellular therapy (ACT) using either unseparated TIL or those enriched for RPS2 V205I-specific CD8+ T cells and found the latter to be superior in controlling outgrowth of tumor of a PDX cell line generated from this lesion in NSG mice. Finally, we used single-cell transcriptomics to isolate the genes encoding the RPS2-specific TCR and show that it recognizes the mutated peptide bound to HLA-B*07:02. These results demonstrate that high-affinity NeoAg-specific T cell responses can be identified in cancer patients, that ACT of these cells can control tumor growth, and that the relevant TCR can be isolated for use in TCR engineering-based immunotherapy.
Substitution of alpha aminobutyric acid for cysteine at class I MHC anchor positions can enhance recognition of cancer epitopes
Authors: Sachs Abraham E , Peters Bjoern , Sidney John , Sette Alessandro , Rosenberg Steven A , Robbins Paul F 
Published: January 2023

Abstract
We curated a list of 11 known and validated class I-restricted cancer neoepitopes and tumor antigens that contain cysteine, six of which were restricted by HLA-A*02:01. We synthesized native (Cys-peptide) and alpha aminobutyric acid substituted (AABA-peptide) minimal epitopes and carried out co-culture assays in which responses of relevant T cell populations to titered doses of the native and substituted peptides were compared. The results indicated that T cells reactive with 6 of the 6 HLA-A*02:01 epitopes recognized peptides containing AABA substitutions at putative MHC anchor positions-at significantly lower concentrations than their Cys-pep counterpart. For 4 of the 5 non-HLA-A*02:01 restricted epitopes, T cells recognized AABA-peptides at lower concentrations than their native counterparts, while for the remaining epitope, the Cys-peptide was recognized at lower concentration than its AABA-peptide counterpart. Three of the HLA-A*02:01 restricted peptides, PHLPP1, HIVEP2, and gp100 contained two cysteine residues, one a putative MHC anchor and the second a putative T cell contact position. Substitution of ABBA for the putative T cell contact residue in these peptides appeared to reduce their ability to stimulate relevant cells. Overall, for 9 of the 11 (82%) cysteine-containing peptides evaluated, substitution of AABA for cysteine resulted in 10-fold or greater decrease in the minimum concentration of peptide recognized by T cells. We conclude that substitution of cysteine with AABA at MHC anchor positions can significantly improve the sensitivity of neoantigen screening assays by offsetting impairment that is likely to be due to cysteine oxidation, an effect that was observed for five HLA class I restriction elements.