bluuebunny/crossref_metadata_2025_split huggingface dataset:
The impact of MHCII-peptide editing by H2-DM on the generation of neonatal regulatory T cells
Authors: Tuncel Jonatan , Yang Siyoung , Fujikado Noriyuki , Benoist Christophe , Mathis Diane J 
Published: January 2023

Abstract
The transcription factor Aire drives the expression of peripheral-tissue antigens (PTAs) in a subset of thymic medullary epithelial cells (MECs). Presentation of PTAs by MHCII on MECs and thymic DCs induces negative selection of self-reactive thymocytes and positive selection of Tregs. Individuals with mutations in Aire develop a combination of autoimmune diseases. In mice, Aire expression during the first weeks of life is sufficient to protect against ‘Aire-less’ disease. During this ‘neonatal window’, a population of long-lived Aire-dependent Tregs is selected that is important for the suppression of self-reactive T-cells. The mechanism involved in the selection of these Tregs is still unknown, as is their function and property to colonize lymphoid and non-lymphoid organs in the adult mouse. We have previously shown that the expression of Aire-induced genes is not dependent on age; however, age affects the expression of H2-DM, a protein that catalyzes the removal of CLIP from the MHCII peptide-groove and which is more abundant in MECs from young mice. This finding suggested that MECs from young and old mice might present different repertoires of PTAs. To probe how the expression of H2-DM by MECs influences the selection of neonatal Tregs, we have now generated mice that are either H2-DM deficient or express reduced levels of this protein. Transfer of neonatal Tregs from these mice to Aire-KO neonatal recipients may provide important insights into the relevance of MHCII-peptide-editing for the selection of specific Treg populations.
The role of MHC class II polymorphisms for DM-mediated peptide editing and autoimmunity
Authors: Haag Sabrina , Pos Wouter , Pyrdol Jason , Dougan Stephanie K , Wucherpfennig Kai W 
Published: January 2023

Abstract
Polymorphisms in MHC class II (MHCII) genes are the strongest risk factors for the development of autoimmune diseases, such as type I diabetes (T1D). T1D is associated with allelic variants of the MHCII molecule HLA-DQ. In the nonobese diabetic (NOD) mouse, the orthologous molecule, I-Ag7, also predisposes mice to spontaneous diabetes development. To investigate the functional impact of natural MHCII polymorphisms on DM binding and T1D we introduced selected mutations in the genome of the NOD mouse by CRISPR/Cas9 genomic editing.
Our approach was to first identify unique polymorphisms in the putative interface of I-Ag7, and then assess the impact of these polymorphisms on DM-mediated peptide editing in vitro. Several polymorphisms increased the interaction between I-Ag7 and DM and augmented peptide binding. The substitution of lysine (K) 40 in I-Aα with glutamic acid (E) had the strongest effect on DM editing and accelerated peptide binding more than 9-fold. Lysine at position α40 is unique to the diabetes associated I-Ag7 molecule and was therefore selected for in vivo evaluation. Homozygous and heterozygous K40E mutant mice showed increased H2-DM protein expression compared to WT littermate controls. Increased H2-DM levels were accompanied by an increase in intra- and extracellular I-Ag7 expression. This suggests that the K40E mutation promotes the interaction between H2-DM and I-Ag7, and likely, represents increased intracellular dimerization and more stable I-Ag7/peptide complexes on the cell surface. Currently, we are evaluating whether increased editing of the MHCII peptide repertoire mediated by the K40E mutation influences spontaneous diabetes development.
The role of IL-10 and FasL in regulating Th1 and Th17 cell responses toward recoverin in autoimmune retinopathy
Authors: Nikoopour Enayat , Ohara Ray , Lin Cheng-mao , Lundy Steven K , Heckenlively John R 
Published: January 2023

Abstract
Autoimmune retinopathy (AIR) is a condition that manifests in acute and progressive vision loss in humans. Antibodies against the retinal protein recoverin are frequently detected in AIR patients. We have established a murine model to better understand the immunopathology of AIR. Mice were immunized subcutaneously with recombinant mouse recoverin (100–200 μg) emulsified in complete Freund’s adjuvant (CFA) and intraperitoneally with pertussis toxin (400 ng). After three weeks, the immunized mice were scanned with optical coherence tomography (OCT) for detection of pathological changes in the eyes. Cells from spleen and draining lymph nodes were restimulated with recoverin (5 μg/ml) for 4 days. Immunization of C57BL/6 and BALB/c mice with mouse recoverin resulted in recoverin-specific T cell responses. Production of IFN-γ and IL-17 were observed in cells harvested from draining lymph nodes and spleen of the immunized mice upon restimulation in vitro with recoverin. Immunization also led to production of high titers of recoverin-specific antibodies (1/400) in the immunized mice. The recoverin-specific Th1 and Th17 cell response was augmented in IL-10 KO mice, whereas humoral response was unaffected. More importantly, the number of lymphocytes infiltrated into the eyes dramatically increased in IL-10 KO mice upon immunization with recoverin. Immunization of Fas ligand deficient GLD mice with recoverin antigen led to a predominant Th17 cell response toward recoverin. These new insights regarding the anti-recoverin immune response have led us to hypothesize that skewing of recoverin-specific T cell response toward inflammatory Th1 and/or Th17 responses can drive the pathogenesis of AIR.
MOG35-55, not NFM15-35, is the critical autoantigen for inducing demyelinating autoimmune disease.
Authors: Blanchfield Jennifer Lori , Evavold Brian D 
Published: January 2023

Abstract
Here we examine functional significance of CD4+ T cell cross-recognition between MOG35-55 (myelin oligodendrocyte glycoprotein) and NFM15-35 (neurofilament medium polypeptide) in demyelinating autoimmune disease. These epitopes are interesting for synergist potential in promoting autoimmunity due to identical amino acids at proposed TCR contacts while displaying variances at MHC. Surprisingly, NFM15-35 peptide is not encephalitogenic in polyclonal models and we proposed to understand why by studying how NFM15-35 effects the known encephalitogenic MOG CD4+ T cell population using peptide:MHC (pMHC) technologies. We found a NFM15-35 challenge was less potent than MOG35-55 in expanding tetramer positive, MOG38-49:I-Ab T cells. Diminished tetramer staining indicated the expanded polyclonal T cells from NFM challenge were of low affinity for MOG38-49, because tetramer enriches for T cells with high affinity for pMHC. We also found that NFM18-30 specific cells in the CNS of mice with MOG35-55 induced EAE predominantly cross-recognized MOG38-49:I-Ab using the 2-dimensional micropipette adhesion frequency assay. These data suggested MOG35-55, not NFM15-35, was the critical encephalitogenic antigen and we confirmed this by showing that demyelinating disease in NFM deficient mice exhibited identical disease course to wild type mice. Interestingly, alterations of NFM15-35 N-terminal amino acids expanded T cells that cross-recognized MOG38-49 tetramer, a phenotype concomitant with restoring encephalitogenic potential in altered NFM15-35 peptides. Overall, we support MOG35-55 as the critical autoantigen for induced demyelinating disease with the contribution of NFM15-35 being dictated by cross-recognition of MOG.
Adoptive transfer of a novel MIF receptor (CD74+) expressing memory T cell subpopulation is sufficient to transfer inflammatory arthritis
Authors: Doherty Edward H , Piecychna Marta , Leng Lin , Bucala Richard 
Published: January 2023

Abstract
Rheumatoid arthritis (RA) is characterized by relapsing disease and progressive autoimmune joint destruction. The mechanism(s) for disease recurrence in previously affected joints is unknown. High expression alleles of the cytokine macrophage migration inhibitory factor (MIF) are associated with severe erosive RA. We report a novel subpopulation of T cells expressing the MIF receptor CD74, which previously has not been considered to be present in T lineage cells. CD74+ T cells comprise 1% of lymph node T cell and exhibit an effector memory phenotype (CD3+ CD74+ CD44+, CD62Llow). Notably, whole genome expression profiling (RNAseq) of CD74+ T cells revealed reduced expression of the immune checkpoint Ctla4 (3.5-fold, p=0.009), and increased expression of Stat4 and Ptpn22 (1.4-fold, p=0.001 and 0.86-fold, p=0.045), three genes that regulate T cell activation and also are linked genetically to RA pathogenesis. In the collagen-induced arthritis (CIA) mouse model of RA, we observed a 2-fold (p=0.023) expansion of CD74+ T cells after disease induction when compared to Mif−/− mice, which show no expansion of CD74+ T cells and reduced disease severity (disease score: WT: 7.8; Mif −/−: 1.4, p&gt;0.001). Notably, adoptive transfer of CD74+ T cells from mice with established CIA into naive mice recapitulated joint inflammation (disease score: 4.3, p=0.001). Disease was not detectable in naïve mice that received CD74-T cells from CIA hosts. This study identifies a novel population of effector memory T cells that are capable of transferring autoimmune joint inflammation. We hypothesize that MIF responsive CD74+ effector memory T cells mediate recurrent joint inflammation and may be amenable to specific targeting by emerging MIF-directed therapies.
The role of programmed death-1 in regulating islet-specific CD4 T cells
Authors: Martinov Tijana , Spanier Justin , Fife Brian T 
Published: January 2023

Abstract
The inhibitory receptor programmed death (PD)-1 and its ligand PD-L1 negatively regulate T cell function. PD-1 blockade is used in the clinic to reinvigorate anti-tumor immunity. However, patients often experience autoimmune-like symptoms, or develop autoimmunity, including type 1 diabetes. We used peptide:MHC Class II tetramers to track islet-specific CD4 T cells in diabetes-prone (NOD) mice and ask how PD-1 regulated this population. We found that insulin-specific cells with the highest affinity for cognate antigen expressed the most PD-1. In turn, PD-1 blockade promoted the accumulation of high-affinity cells in the pancreas, and exacerbated diabetes. Given that 70% of insulin-specific cells are anergic, we investigated whether PD-1 was required for the maintenance of anergic state, using an adoptive cell transfer model. Anergic cells remained functionally blunted in comparison to effector cells after anti-PD-L1 treatment, suggesting that anergy maintenance depends on other signaling pathways. This work highlights how the differentiation status of a T cell may predetermine its susceptibility to PD-1 blockade, and has implications for cancer immunotherapy and autoimmunity.
Membrane-bound Dickkopf-1 in Foxp3+ regulatory T cells suppresses T cell-mediated autoimmune colitis
Authors: Chae WookJin , Hao Liming , Henegariu Octavian , Yilmaz Saliha , Bothwell Alfred L M 
Published: January 2023

Abstract
Regulation of immunological tolerance is central to maintain or restore homeostasis. Herein we show that Foxp3+regulatory T cells (Tregs) utilize Dickkopf-1 (DKK-1) to regulate T cell-mediated tolerance in T cell-mediated autoimmune colitis model. Tregs from DKK-1 hypomorphic doubleridge mice failed to control CD4+T cell proliferation, resulting in CD4 T-cell-mediated colitis. Foxp3+Tregs showed a robust expression of DKK-1 among other Wnt ligands. DKK-1 was markedly expressed in activated Tregs but not in naïve or effector CD4 T cells. Tregs expressed DKK-1 markedly in the cell membrane, co-expressing other well-known Treg markers in vivo. We found that T cell receptor stimulation is important to induce DKK-1 expression in vitro and in vivo. DKK-1 expression was regulated by the MAPK pathway and was dependent on de novo protein synthesis, but not by the canonical Wnt pathway, the mTOR or mevalonate pathway. Taken together, our results highlight that Tregs utilize DKK-1 to regulate immunological tolerance to prevent autoimmunity.
A novel image cytometric analysis method for T cell-mediated cytotoxicity of 3D tumor spheroids
Authors: Chan Leo , Humbert Laure , Cribbes Scott 
Published: January 2023

Abstract
Cell-mediated cytotoxicity assays have been frequently performed to characterize cytotoxic potential of immune cells, antibodies, and drug compounds. Traditionally, these assays are performed using release assays such as Cr51 (radioactivity), Calcein (fluorescence), or LDH (enzymatic). However, release assays have limitations such as the handling of hazardous material and the indirect measurement of cell death. These assays are commonly performed in 2D cultures, which have difficulty identifying qualifying drug candidates for clinical testing, thus there has been an interest of performing cytotoxicity assays in 3D tumor models. Traditional 3D spheroid analysis methods require the use of standard microscopy, which is time-consuming and subjective. In this work, we demonstrate a novel method of analyzing T cell-mediated cytotoxicity on 3D tumor spheroids in the presence of absence of ImmTAC molecules, which can promote higher T cell killing. In this experiment, MDA-MB-453 GFP expressing breast cancer cells are used to form tumor spheroids in an ultra-low attachment plate. The spheroids are then treated with primary T cells at different E:T ratios, and different concentrations of ImmTAC. The results showed a dose response effect of T cell killing with the addition of ImmTAC molecules by measuring spheroid size in GFP fluorescence and viability using propidium iodide. The captured bright-field and fluorescent images also clearly showed the cytotoxicity effect from the combination of T cells and ImmTAC. The ability to screen cytotoxic effects of immune cells, antibody, and drug compounds on 3D tumor spheroids can provide an alternative tumor model for identifying more qualified cancer drug candidates for drug discovery campaigns.
Gene expression profiling of samples of limited RNA amounts by the NGS application whole transcriptome AmpliSeq-RNA on the Ion Torrent platform offers straightforward, fast, and affordable analysis of human T cell subpopulations.
Authors: Raulf Friedrich , Allard Cyril , Delucis-Bronn Corinne , Angevaare Eline , Vedrine Corinne , Boesch Danielle , Brinkmann Volker 
Published: January 2023

Abstract
The diversity of T cell subpopulations demands a growing number of biomarkers to be studied from small samples. To bridge the gap between multicolor flow cytometry or classical qPCR and RNA-Seq, we applied highly multiplexed whole transcriptome AmpliSeq-RNA with 20,803 genes to perform sensitive and specific gene expression profiling (GEP). This study analyzed untouched primary human naïve and memory T cells and compared results to microarrays.

Method
8 T cell subsets were isolated by FACS from 4 healthy donors without restimulation: CD4+ or CD8+ naïve (CCR7+CD45RA+), central memory (CCR7+CD45RA−), effector memory TEM (CCR7−CD45RA−) and TEMRA (CCR7−CD45RA+). AmpliSeq transcriptome human gene expression kit (Thermo Fisher) generated barcoded libraries from total RNA. 8–10 samples multiplexed per Ion 540 chip were sequenced on an Ion S5 System. Samples were also analyzed by Affymetrix HG-U133 Plus 2 genechips.

Results
The resulting Excel matrix of normalized reads [RPM = reads per million reads] had a dynamic range of more than 5 log units. Biological replicates of T cell subsets showed high correlation with r ≥ 0.925. Technical replicates (same libraries) demonstrated high reproducibility with r ≥ 0.995. A high concordance with genechip data were found for expression pattern of analyzed markers. For several very high or low abundant genes the AmpliSeq-RNA data were more sensitive and more specific than the hybridization-based genechip data.

Conclusion
AmpliSeq-RNA (counting PCR products by NGS) is an interesting alternative to perform straightforward GEP. The analysis of phenotypically well-defined T cell subsets without artificial in vitro stimulation allows more precise determination of in vivo relevant functions.
iPair and iPair+: cognate pairing of human TCR alpha and beta V-regions and phenotypic analysis of single cells using NGS.
Authors: Byrne-Steele Miranda , Pan Wenjing , Hou Xiaohong , Brown Brittany E. , Eisenhower Mary , Han Jian 
Published: January 2023

Abstract
Immune repertoire amplification of T and B cell receptors coupled to NGS provides detailed, sequence-level insight into the immune system. However, information related to the cognate pairing of alpha and beta chains is lost once RNA extraction is performed on the entire sample (or bulk sample). Here, we describe a very sensitive method that allows for the amplification of both human TCR alpha and beta chains including additional phenotypic markers from single cells in the same reaction well. We have reduced the RT-PCR chemistry as low as 1.5 μL, thus providing a cost-effective method to analyze single cells from standard FACs instruments. Plate processing procedures are also simplified since RT-PCR of all targets can be performed in the same plate. Under optimal conditions, the amplification success rate is greater than 90%, with successfully identified pairs as high as 75% of those amplified. During the first round of PCR, RT-PCR is performed with nested, multiplex primers covering both the alpha and beta locus (iPair) and 30 phenotypic markers (iPair+). Communal forward and reverse binding sites are included on the 5′ end of the inside primers. Included on the reverse primer is an in-line 6 nt barcode, which serves as a plate identifier so that multiple 96-well plates may be multiplexed in the same sequencing flow cell. After RT-PCR1, the first round PCR1 products are rescued using beads. A second PCR is performed with dual-indexed primers that complete the sequencing adaptors introduced during PCR1 and provide plate positional information for the sequenced products. An analysis tool has also been developed to help visualize pairing across a plate and to identify those pairs that would be likely candidates for further investigation.
Detection, phenotyping, and quantification of antigen-specific T cells using a peptide-MHC dodecamer
Authors: Huang Jun , Davis Mark M. 
Published: January 2023

Abstract
Here we report a peptide-MHC (pMHC) dodecamer as a “next generation” technology that is a significantly more sensitive and versatile alternative to pMHC tetramers for the detection, isolation, and phenotypic analysis of antigen-specific T cells. In particular, dodecamers are able to detect two- to fivefold more antigen-specific T cells in both human and murine CD4+ and CD8+ αβ T-cell compartments compared with the equivalent tetramers. The low-affinity, tetramer-negative, dodecamer-positive T cells showed comparable effector cytokine responses as those of high-affinity, tetramer-positive T cells. Dodecamers are able to detect early stage CD4+CD8+ double-positive thymocytes on which T-cell receptors are 10- to 30-fold less dense than mature T cells. Dodecamers also show utility in the analysis of γδ T cells and in cytometry by time-of-flight applications. This construct has a simple structure with a central scaffold protein linked to four streptavidin molecules, each having three pMHC ligands or other molecules. The dodecamer is straightforward and inexpensive to produce and is compatible with current tetramer technology and commercially available streptavidin conjugates.
The potential application of ctDNA for improved cancer management, treatment and diagnosis within the Pacific
Authors: Sika-Paotonu Dianne , Guilford Parry 
Published: January 2023

Abstract
The burden of cancer among Pacific peoples living in New Zealand and in the Pacific remains a significant health concern. Given the health inequalities that exist for Pacific peoples and the global burden of cancer, it remains a priority to consider the medical situation for cancer patients in the Pacific. It is acknowledged that within resource constrained environments, the diagnosis, treatment and management of cancer patients is fraught with unique challenges and difficulties. A novel cancer detection method known as circulating tumour DNA (ctDNA) monitors small fragments of tumour DNA found in the blood circulation. These fragments can be isolated and quantified to obtain information providing data concerning cancer progression and treatment response. It is anticipated this simple method of detecting and monitoring cancer will enhance the precision of cancer care for patients, and will eventuate into a particularly useful tool within resource constrained environments such as the Pacific. The purpose of this work was to identify the diagnostic, treatment and pharmacologic options currently available for cancer patient within the main and rural Hospitals in Tonga and to explore the potential utility of ctDNA to support the management, treatment and diagnosis of cancer patients. Cancer diagnosis, treatment and management pathways were available in Tonga but options were limited. The potential diagnostic utility of the ctDNA technology was an element of intense interest as cancer presentation is typically late with medical, nursing and support staff operating within resource constrained environments. The potential application of ctDNA for improved support of cancer patients in Tonga remains a highly appealing possibility.
Interferon epsilon protects primary macrophages against HIV infection
Authors: Chang Theresa Li-Yun , Tasker Carley , Subbian Selvakumar , Gao Pan , Couret Jennifer , Levin Carly , Ghanny Saleena , Soteropoulos Patricia , Zhao Xilin , Landau Nathaniel , Lu Wuyuan 
Published: January 2023

Abstract
Interferon epsilon (IFNe) is a unique type I IFN that is not induced by pattern-recognition response elements. IFNɛ is constitutively expressed in mucosal tissues including the female genital mucosa. Although the direct anti-viral activity of IFNe was thought to be weak compared to IFNa, IFNe controls Chlamydia muridarum and herpes simplex virus 2 in mice, possibly through modulation of immune response. We show here that IFNe induces an antiviral state in human macrophages that blocks HIV-1 replication. IFNe had little or no protective effect in activated CD4+ T cells or transformed cell lines unless activated CD4+ T cells were infected with replication competent HIV-1 at a low MOI. The block to HIV infection of macrophages was maximal after 24 h of treatment and was reversible. IFNe acted on early stages of the HIV life cycle including viral entry, reverse transcription and nuclear import. The protection did not appear to operate through known type I IFN-induced HIV host restriction factors such as APOBEC3A and SAMHD1. IFNe-stimulated immune mediators and pathways had the signature of type I IFNs but were distinct from IFNα2. IFNe induced significant phagocytosis and reactive oxygen species, which contributed to the block to HIV replication in macrophages. These findings indicate that IFNe induces an antiviral state in macrophages that is mediated by different factors than those induced by IFNa2. Understanding the mechanism of IFNe-mediated HIV inhibition through immune modulation has implications for prevention.