bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetics of MOG-specific CD4+ T cells in experimental autoimmune encephalomyelitis. (P4127)
Authors: Martinez Ryan , Blanchfield Jennifer , Evavold Brian 
Published: January 2023

Abstract
During experimental autoimmune encephalomyelitis (EAE), effector CD4+ T cells are primed in the periphery and migrate to the central nervous system (CNS) to mediate immune damage to myelin. In experiments described here, we investigated the myelin oligodendrocyte glycoprotein (MOG)-specific CD4+ T cell immune response during C57BL/6 EAE before disease onset. The peripheral lymphoid organs, blood and CNS were analyzed using MHC class II tetramer enrichment to determine the precursor frequency, kinetics, and phenotype of MOG-specific CD4+ T cells during priming and migration to the CNS. We found that naïve B6 mice had a precursor frequency of about 450 MOG-specific CD4+ T cells in peripheral lymphoid organs, which expanded to ~30,000 at day 21 after EAE induction. Our lab has previously shown that identification of myelin-specific CD4+ T cells in the CNS is underestimated using MHC class II tetramers, so we attempted to determine if there were more low affinity MOG-specific CD4+ T cells measureable by the sensitive micropipette adhesion frequency assay. We found, that similar to the CNS, MHC class II tetramer enrichment underestimated the amount of low affinity MOG-specific CD4+ T cells in the peripheral lymphoid organs when compared to micropipette binding frequency assay. These results demonstrate that low affinity, self-antigen specific CD4+ T cells are at higher frequency than previously appreciated, and may play an important role in the pathogenesis of EAE.
The role of TNFα pathway and microRNA in regulating autoimmune regulator gene expression and translation (P4101)
Authors: Nagafuchi Seiho , Oda Yoshie , Matsuo Tomohito , Kurisaki Hironori , Katsuta Hitoshi , Nagafuchi Seiho 
Published: January 2023

Abstract
Autoimmune regulator (AIRE) is a responsible gene for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), presenting multiple autoimmune diseases. AIRE gene is expressed in the thymus and lymph nodes. Thus, AIRE gene plays an important role in both the central and peripheral regulation of immune responses. However, there are some patients with APECED, who lack mutations in AIRE gene. Therefore, dysfunction of the regulatory mechanism of AIRE gene may be associated with the pathogenesis of APECED. In this study, we found that nuclear factor-κB (NF-κB) pathway regulated AIRE gene expression during maturation process of dendritic cells under TNFα stimulation, along with the significant elevation of AIRE gene expression. By inhibiting NF-κB pathway, AIRE gene expression was decreased. We also found that miR-220b inhibited the translation of AIRE gene, binding 3’ untranslated region (3’UTR) of the target mRNA. AIRE protein expression was inhibited by overexpressing microRNAs in 293T cells transfected with AIRE gene which have 3’UTR plasmid but not in AIRE open reading frame (ORF) alone. At least, miR-220b was not expressed in the normal thymus which expresses high level of both AIRE gene and AIRE protein, to delete autoreactive T cells. Thus, it was concluded that there are multiple the regulatory mechanisms of AIRE gene expression, and deterioration of those mechanisms may be associated with APECED phenotype.
Critical role of Annexin A1 in protection against pulmonary Mycobacterium tuberculosis infection. (P3320)
Authors: Tzelepis Fanny , Gillard Joshua , Jaworska Joanna , Nishimura Tomoyasu , Verway Mark , Hassani-Ardakani Kymia , Remold Heinz , Vali Hojattolah , Maziar Divangahi 
Published: January 2023

Abstract
Annexin1 is a 37kDa protein with wide spectrum of biological activity. Although there are data emphasizing on anti-inflammatory role of Annexin1 in inflammatory diseases, little is known about its role in pathogenesis of tuberculosis. Recently, it has been described the involvement of Annexin1 in the formation of apoptotic envelope, generation of “eat me” signal and activation of Tcells. As phagocytosis of apoptotic vesicles from Mycobacterium tuberculosis (Mtb) infected macrophages is one of the main sources of antigens for CD8T cells via cross-presentation, we hypothesized that impaired capacity of Annexin1-/-DC in cross-presentation increases host susceptibility to Mtb infection. We found that Annexin1-/-mice were severely susceptible to Mtb infection. High levels of pulmonary bacterial burden and mortality in Annexin1-/-mice were associated with reduced antigen-specific CD8T cell response in the lungs. This reduced response was not due to an intrinsic role of Annexin1 in macrophage apoptosis or T cell activation as generation of chimeric mice in which only T cells were deficient in Annexin1 did not show impairment in Tcell mediated immunity or protection against Mtb infection. Interestingly, both in vitro and in vivo, Annexin1-/-DC demonstrated a significantly reduced capacity to cross-present antigens to CD8Tcells. Together, these data identify Annexin1 as a central player in protective immunity against Mtb infection, by regulating the power of DC in cross-presentation.
IL-10 from T cells, but not macrophages is required for chronic disease in Leishmania mexicana infection (P3328)
Authors: Buxbaum Laurence 
Published: January 2023

Abstract
Chronic cutaneous disease of mice caused by the protozoan parasite Leishmania mexicana requires IL-10 and FcγRIII (an activating IgG receptor). Macrophages are abundant in lesions and readily secrete IL-10 in response to IgG-coated amastigote stage parasites, making macrophages a prime candidate as the critical source of IL-10. However, both direct and indirect evidence demonstrated that macrophage IL-10 is not essential for chronic disease. Specifically, macrophage-granulocyte specific IL-10-deficient mice have chronic disease identical to wild-type mice. We now show that T cell-derived IL-10 is required for chronic disease. CD4-cre IL-10flox/flox mice lack IL-10 from T cells (both CD4+ and CD8+) and heal their L. mexicana lesions. We had previously shown that depletion of CD25+ T cells had no effect on chronic disease, and thus T cells other than Treg cells should be the important source of IL-10. Given that conventional T cells do not express FcγRs, there is likely to be an indirect pathway by which FcγRIII engagement on some other cell induces IL-10 from T cells. Further work is needed to delineate these pathways.
T cell cytokine profile of early tuberculosis infection and disease in Cynomolgus macaque model (P3335)
Authors: Gideon Hannah , Lin Philana , Flynn JoAnne 
Published: January 2023

Abstract
Mycobacterium tuberculosis (Mtb) remains one of the world’s most successful human pathogen. Addressing questions on immunological events soon after Mtb infection in human is challenging. Non-human primate model (Cynomolgus macaques) provide the necessary platform to understand the early immunological events in Mtb infection. We infected 28 Cynomolgus macaques with a low dose of Mtb (Erdman strain, ~25-50 CFU) and monitored clinically for signs and symptoms of tuberculosis up to six months post infection and classified to have either latent TB infection or active disease based on chest X-ray and Mtb culture. Peripheral blood was collected at time-points: pre Mtb infection; and at days: 10, 20, 30, 42, 56, 90, 120, 150 and 180 post infection. PBMC were isolated and multi-parametric intracellular flow cytometry was performed in fresh PBMC to track the early CD4 and CD8 T cell cytokine profiles (CD107a, GMCSF, IFN-g, IL-2, IL-4, IL-6, IL-10 and IL-17) during the course of Mtb infection. Preliminary analysis shows difference in the cytokine response in the early phase of infection between those who developed active disease or latent infection. In addition, we find a spectrum of cytokine response, which overlap between active and latent tuberculosis.
Intrinsic role of MyD88 for anti-fungal Tc17 cell responses (P3338)
Authors: Nanjappa Som , Wuthrich Marcel , Klein Bruce 
Published: January 2023

Abstract
Our previous studies showed that vaccine-induced Tc17 cells are necessary for antifungal defenses to control lethal pneumonia due to experimental blastomycosis. Although dendritic cell cytokines such as IL-6, TGF-β and IL-1 are generally required for differentiation of Th17 cells, the role of intrinsic MyD88 signaling and upstream pattern recognition receptors (PRRs) on CD8 T cells for anti-fungal Tc17 responses has not been studied. In work here, we found that MyD88 expression in CD8 T cells was essential for the generation of Tc17 responses following vaccination. As IL-18, IL-1 and PRRs mediate signaling through MyD88, we investigated the requirement of each of these components for anti-fungal Tc17 responses. We found in vivo that IL-18R was dispensable and that IL-1R1 signaling was insufficient to account for the Tc17 defect observed in MyD88-/- CD8 T cells, suggesting a potential role for PRRs. Our in vitro data also suggested that TLR2 signaling is required for Tc17 differentiation. Interestingly, Tc1 responses were relatively spared in MyD88-/- CD8 T cells. Mechanistically, MyD88-/- Tc17 not Tc1 cells failed to proliferate and levels of Bcl2 and Bcl-xL were unaffected, indicating poor expansion but not death of MyD88-/- Tc17 cells as a cause of poor Tc17 responses. Thus, intrinsic MyD88 and PRR signals in CD8 T cells are crucial for the generation of anti-fungal Tc17 responses. These data may have potential applications for designing fungal vaccines.
IL-12 regulated checkpoints in the CD8+ T cell response to Toxoplasma gondii (P3340)
Authors: Shah Suhagi , Grotenbreg Gijsbert , Rivera Amariliz , Yap George 
Published: January 2023

Abstract
We have previously shown that IL-12 is necessary for the differentiation of KLRG1+ IFN-γ producing CD8+ T effector cells during the immune response to Toxoplasma gondii. However, which key events during the activation, differentiation and mobilization of CD8+ T effector cells are critically regulated by IL-12 have not been identified. By tracking the dynamics of endogenous and adoptively transferred T. gondii-reactive CD8+ T cells, we were able to identify two key IL-12 dependent checkpoints. IL-12 is critical for the early diversification of CD8+ T cells into KLRG1+ IFN-γ-producing T effector cells, which occurs even prior to their initial proliferation. Unexpectedly, acquisition KLGR1 expression was accompanied by an attenuation of CXCR3 expression and this down regulation was IL-12 dependent. We postulate that this IL-12-driven reduction of CXCR3 expression may allow these mature KLRG1+ CD8+ T cells to outmigrate from secondary lymphoid organs. Failure to suppress CXCR3 may result in the retention of KLRG1+ CD8+ T effector cells in secondary lymphoid organs and underlie the deficit in effector cell numbers observed at the site of T. gondii infection in IL-12 p35-/- hosts. Our study reveals that IL-12 implements the early diversification of CTLs by both promoting and attenuating expression of key CD8+ T lymphocyte genes required for their effector function and mobilization.
BCG-treated M2-macrophages enhance IFN-γ production of T cells (P3348)
Authors: Lardone Ricardo , Guo Yi , Plaisier Seema , Singh Babu , Teles Rosane , Modlin Robert , Sieling Peter , Lee Delphine 
Published: January 2023

Abstract
M1- and M2-polarized Macrophage (MΦ) subtypes differentially regulate anti-mycobacterial defense. To examine their roles in human infection, we compared the gene expression profile of M1- vs. M2-MΦ to the expression profile of lesional skin from patients with restricted (tuberculoid, T-lep) vs. disseminated (lepromatous, L-lep) leprosy (a mycobacterial skin infection). Rank-Rank Hypergeometric Overlap (RRHO) is a novel algorithm for comparing full gene expression profiles from two independent microarray experiments that generates the most statistically significant overlapping gene list without requiring strict p-value/fold change cutoffs. RRHO analysis showed significant overlap of genes between M2-MΦ and L-lep lesions (n = 1559, max log hypergeometric p-value = 196), indicating a role for M2-MΦ in immune unresponsiveness. Bacillus Calmette-Guerin (BCG) is given to protect household contacts against leprosy. We hypothesized that BCG treatment may affect the function of M2-MΦ and investigated its effects in vitro. We found that BCG-treated M2-MΦ secreted less IL-10 (p &lt; 0.05), with no effect on IL-12p40, compared to untreated M2-MΦ. In addition, BCG-treated M2-MΦ increased the frequency of IFN-γ producing cells in an antigen non-specific manner (p &lt; 0.05), suggesting an adjuvant effect of BCG. Our data indicates M2-MΦ can be reprogrammed by BCG, resulting in increased IFN-γ production to enhance anti-mycobacterial immunity.
Modeling immune receptor clustering with nano-scale discoidal membrane bilayers (P3350)
Authors: Anikeeva Nadia , Sykulev Yuri , Blanchette Creig , Fisher Nick 
Published: January 2023

Abstract
MHC proteins on antigen-presenting cells form clusters with each other and other cell surface ligands. The changes in the clustering serve as a sensitive mechanism to modulate T cell responses. We propose to mimic MHC membrane patches using nanolipoprotein particles (NLPs). NLPs are nano-scale discoidal lipid bilayers stabilized by apolipoproteins. Nickel-chelating lipids incorporated into the NLP bilayer capture soluble polyhistidine-tagged pMHC. We have studied the interactions of cognate (non-self) and non-cognate (self) pMHC/NLPs with CD8+ CTL. Both self and non-self pMHC/NLPs were bound to the T-cell surface but to different extents. The apparent equilibrium binding constants of both non-self and self pMHC/NLPs were strongly dependent on pMHC density. A 3-fold difference in pMHC density (from 10:1 to 30:1 per NLP) caused 10-fold and 30-fold increases in the apparent affinity, respectively. Surprisingly, the avidity of self and non-self multivalent interactions appears to be similar when the pMHC density on the NLP surface is high. The similar avidity may be explained by a very rapid on-rate of CD8 binding to clustered MHC and marginal difference in the dissociation rates at the high epitope density resulting from very efficient CD8-MHC rebinding. NLP-based model MHC clusters with pre-determined properties could be incorporated onto glass beads coated with a lipid bilayer to engineer artificial APC with the goal of inducing ex vivo long-lived T-cell effectors for adoptive T-cell therapy.
A new and robust method of tethering IgG surrogate antigens on lipid bilayer membranes to facilitate the TIRFM based live cell and single molecule imaging experiments (P3352)
Authors: Liu Wanli , Zhang Shaosen , Xu Liling , Zhao Xingwang , Xu Yinsheng 
Published: January 2023

Abstract
Our understanding of cell-to-cell interactions has been significantly improved in the past years with the help of TIRFM in combination with an antigen presenting system supported by planar lipid bilayer (PLB) membranes. In TIRFM experiments, it is a challenge to uniformly present ligand molecules on the surface of PLB membranes. Here, we introduce a new and robust method of tethering IgG surrogate antigen ligands on the surface of PLB membranes containing nickel salt. In this method, we use a modified D domain from staphylococcal protein A molecule that is fused with an N-terminus polyhistidine tag (H12-D-domain) to tether IgG surrogate antigens on nickel-containing PLB membranes. We systemically assessed the specificity and capability of H12-D-domain construct to capture IgG molecules from different species through live cell and single molecule TIRFM imaging. We find that these IgG surrogate antigens show good lateral mobility and are uniformly distributed on PLB membranes. Not IgM molecules, nor Fab or F(ab’)2 fragments of IgG molecules can be tethered on PLB membranes by H12-D-domain construct. These tethered IgG surrogate antigens strongly induce the formation and accumulation of signaling active antigen receptor microclusters within the immunological synapse in B or T lymphocyte cells. Thus our method provides a new and robust way to tether IgG surrogate antigens or other molecule fused with IgG Fc portion on PLB membranes for TIRFM based molecule imaging experiments.
Blocking ADP-ribosylation of cell surface proteins during cell preparation with a Nanobody against ART2 enhances chemotaxis of T cells during in vitro migration assays. (P3388)
Authors: Moyron-Quiroz Juan , Rissiek Björn , Soto Hortensia , Pahuja Anil , Wiethe Carsten , Zlotnik Albert , Yang Xifeng , Koch-Nolte Friedrich , Ransom John 
Published: January 2023

Abstract
ADP-ribosylation is the enzymatic transfer of the ADP-ribose moiety from NAD+ to a protein, a posttranslational modification that regulates the function of the targeted proteins. Although in normal conditions there is minimal extracellular NAD+, it is released from cells in inflammation and during preparation of primary lymphocytes. ART2 (CD296) is an ADP-ribosyltransferase expressed by most mature T cells, including NKT, and regulatory T cells. A major substrate for ART2 is P2X7, an ion channel that mediates shedding of CD62L, and NAD+-induced cell death (NICD). In this manner, NAD+ released during cell preparation affects the phenotype and survival of cells expressing ART2. In this study, we used a recombinant variable domain of the llama heavy-chain antibody S+16a to block the ADP-ribosyltransferase activity of ART2. The recombinant antibody was administered i.v. to mice and 1-2 h later, the CD4+ T cells from spleen and lymph nodes were isolated. The cell phenotype and functional state was analyzed by flow cytometry and chemotaxis assay respectively. A single dose of S+16a blocked the shedding of CD62L and reduced the number of apoptotic cells about 80%. The higher cell viability resulted in a 50-100% increase in the cell number responding to the chemokines CCL22 and CXCL12. We conclude that S+16a is a useful tool to protect mature T cells from NICD induced by the released NAD+ from cells during purification, thereby enhancing the efficiency of in vitro migration assays.
In silico high throughput pre-clinical determination of monoclonal antibody immunogenicity (P3391)
Authors: Cousens Leslie , Terry Frances , Ardito Matthew , Martin William , De Groot Anne 
Published: January 2023

Abstract
One of the great surprises of the biologics revolution has been the discovery that recombinant human proteins, including humanized and fully-human monoclonal antibodies (MAb), can be immunogenic when administered to immune-competent subjects. Preclinical and clinical evaluations of the immunogenic potential for biologic drugs primarily focus on humoral immune responses; as a result, the critical contribution of T cells to the development of anti-drug antibodies (ADA) has been somewhat overlooked. Using the EpiMatrix T cell epitope mapping system, we have developed an interactive in silico screening and optimization platform that evaluates the overall immunogenic potential of a biologic as well as identifies individual T cell epitope clusters contributing to its immunogenicity. In contrast to other immunogenicity prediction tools, our platform considers the contribution of regulatory T cell epitopes (Tregitopes) to immunogenic potential. Tregitopes are highly conserved T cell epitopes derived from IgG that we and others have shown activate regulatory T cells and promote tolerance induction to associated antigens. Here we demonstrate the correlation of available clinical immunogenicity data with Tregitope-adjusted immunogenicity scoring for twenty approved MAbs. Further, we present a high-throughput platform from which these scores can be used to triage large pools of candidate MAbs during the discovery phase of antibody development.
A longitudinal flow cytometry based study on phenotypic changes of cryopreserved murine T cells using the BD FACSVerse™ system (P3358)
Authors: Wang Yibing , Cai Shang , Feng Weiguo , Jana Rinku 
Published: January 2023

Abstract
Cryopreservation is commonly used to preserve fresh lymphocytes. However, it is not clear whether the phenotype-distinct populations can be cryopreserved equally effectively, nor how cryopreservation contributes to variation in samples analyzed by flow cytometry. Elucidation of the question has been limited by the issue of instrument-related variations over time. Laboratories have been attempting to establish standardization methods to eliminate such variations. The BD FACSVerse™ system is a high-performance flow cytometer designed to address the need for instrument standardization, specifically when conducting longitudinal and cross-site studies. The flow cytometer provides an easy-to-use setup system designed to maintain daily, consistent instrument settings for different applications, including fluorescence compensation. To take advantage of this new technical capability, we conducted a study to compare cryopreserved murine lymphocytes to freshly prepared ones, looking at the variability between the data sets. Freshly prepared murine cells were initially phenotyped and then frozen down. Afterwards, the cryopreserved cells were thawed on various days and examined using the same phenotyping method. We found that cryopreservation does have an impact on different populations of lymphocytes in a longitudinal study, which demonstrated the utility of the BD FACSVerse system’s built-in standardization in detecting phenotypic changes of cell populations.
Master chaperone gp96 for TLRs and integrins regulates canonical Wnt pathway and gut homeostasis (P3278)
Authors: Liu Bei , Staron Matthew , Hong Feng , Sun Shaoli , Li Zihai 
Published: January 2023

Abstract
gp96 is a master protein chaperone for Toll-like receptors and integrins in the endoplasmic reticulum (ER) and plays critical roles in innate immunity (Yang et al., Immunity, 2007 26:215-226). Using a conditional and lineage-specific knockout strategy, we have unexpectedly observed that gp96 is required for maintaining intestinal integrity. Loss of gp96 in adult mice causes a rapid and profound compromise in intestinal homeostasis with gut-intrinsic defect in the proliferation of intestinal crypts, compromise of nuclear beta-catenin translocation, loss of crypt-villus structure and impaired barrier function. Mechanistic study revealed that the observed intestinal pathology in gp96 KO mice is unlikely due to compromise in the gut defense or dysregulated unfolded protein response. Rather, gp96 was found to be essential for maturation of Wnt co-receptor LRP6 and thus canonical Wnt signaling. Our study places Wnt pathway under the direct regulation of a TLR/integrin-master chaperone, suggesting that innate immunity and organ development converge on gp96.