bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of interactions in a soluble tapasin/HLA-B*0801 complex provides a mechanism for peptide selection (93.10)
Authors: Bouvier Marlene , Chen Mingnan
Published: January 2023

The cell-surface presentation of peptides by class I molecules is the culmination of a complex series of events that take place in the endoplasmic reticulum and involves specialized proteins, including tapasin (TPN). Although it is uncontested that TPN plays a critical role in class I maturation, a mechanistic understanding of TPN is lacking.

We examined interactions in a soluble TPN/class I complex using real-time fluorescence measurements. Results show that TPN increases the association and dissociation rates of some, but not all, peptides. TPN acts as a chaperone by increasing the concentration of peptide-deficient class I molecules in solution. We also show that a TPN-assisted mechanism of peptide selection relies on disruption of conserved hydrogen bonds at the C-terminal end of the groove; peptide sequence-dependent interactions along the entire length of the groove also play an important role.

Overall, our results suggest that the presence of TPN in the class I assembly complex ensures that bound candidate peptides that cannot meet a certain minimum energy threshold, as needed to conformationally disengage TPN, are excluded from the repertoire. We propose that the net outcome of a TPN-assisted mechanism of peptide selection is to maximize at any point in time, and according to the pool of peptides available, the diversity of minimally stable class I/peptide molecules on the cell surface.

Funded by NIH-AI45070
Survival of antigenic epitope requires class II MHC capture prior to lysosomal proteolysis (93.21)
Authors: Kim Ae , Hartman Isamu , Sadegh-Nasseri Scheherazade
Published: January 2023

The paradigmatic model for the generation of peptide determinants for class II major histocompatibility complex molecules (MHC II) is that proteolytic fragmentation of antigen precedes the capture of the resulting peptides by MHC II (cut/trim first, bind later). However, circumstantial evidence exists to support an alternate model in which MHC II binding of antigenic epitope occurs prior to the proteolytic fragmentation of that antigen (bind first, cut/trim later). To distinguish between these two models, we analyzed the interaction of HLA-DR1 with two protein antigens, type II collagen and influenza hemagglutinin. Here, using a novel cell?free antigen processing system composed solely of purified soluble protein components, HLA-DR1, two cathepsins, and HLA-DM, combined with mass spectrometric identification of the bound peptides, we demonstrate for both antigens that;

the protein antigens bind MHC class II prior to digestion by lysosomal proteases,the immunodominant epitope of the antigens are degraded by lysosomal proteases if not bound to MHC class II, andthe epitopes, when bound to MHCII, are protected from proteolytic degradation.

These findings conclusively support the “bind first, cut/trim later” model and have important implications in understanding the sequence of events in antigen processing and the correlation between antigen structure and immunodominance.
IL-4 and IL-13 induce type I collagen gene activity via the JNK signaling pathway in human fibroblasts. (94.28)
Authors: Bhogal Rashpal Kaur , Stoica Cristina , McGaha Tracy L , Bona Constantin
Published: January 2023

Little is understood of the molecular mechanisms by which fibrotic diseases such as in Scleroderma, are manifested by interleukins (IL) 4 and 13. Studies support a role for the JNK signaling pathway via transcription factors c-Jun and ATF2, being involved in activating the collagen promoter and translation of the collagen gene. To examine the role of JNK signaling in IL-4/IL-13-induced collagen gene activation, we transfected human fibroblasts with a luciferase reporter plasmid under control of the α2(I) collagen promoter to assess JNK signaling on type I collagen promoter activity. We reveal a critical role for JNK 1 in IL-4/IL-13-induced collagen promoter activity subsequent to ectopic expression of c-Jun and ATF2. In contrast, introduction of a dominant negative (dn) mutant reduces basal and cytokine-induced promoter activity. We demonstrate that the JNK signaling pathway leading to the activation of the collagen gene in fibroblasts is effective not only for TGF-β signaling but also for IL-4/IL-13 signaling.
Mechanisms of processing and secretion of IL-1β in fish (94.30)
Authors: Lopez-Castejon Gloria , Roca Francisco J. , Sepulcre M Pilar , Meseguer José , Mulero Victoriano
Published: January 2023

IL-1β is a potent pro-inflammatory cytokine which is translated as a leaderless 33 kDa precursor protein (proIL-1β) that is processed into its 17 kDa active form by the IL-1β-converting enzyme (ICE, caspase-1). Several stimuli have been reported to activate caspase-1, including the engagement of P2X7 receptors by extracellular ATP and infection with invasive bacteria. While the recognition site for caspase-1 is well conserved in mammals, most of the non-mammalian IL-1β genes cloned so far lack this conserved site. In addition, we have previously shown that extracellular ATP fails to induce the processing and release of this cytokine in the bony fish gilthead seabream. In this study, we report that the stimulation of seabream leukocytes with flagellin or bacterial DNA leads to the processing of proIL-1β into a 20 kDa form, although it largely remains intracellular. However, the infection of leukocytes with salmonella results in the efficient and specific release of the mature form. Interestingly, the processing of IL-1β in this species is not prevented by a specific inhibitor of caspase-1 or a pan-caspase inhibitor. Whatever the case, our results suggest that the processing and release of IL-1β in early vertebrates is mediated by cell death upon bacterial infection by a caspase-1 independent mechanism, while a more sophisticated mechanism operated by activation of ATP-gated P2X7 receptors appeared later in evolution.

This work was supported by the Spanish Ministry of Education and Science (grant BIO2005-05078 to V.M. and fellowship to G.L.-C.).
Function of the fractalkine receptor during autoimmune inflammation (96.15)
Authors: Cardona Astrid E , Sasse Margaret E , Cardona Sandra M , Mazutani Makiko , Hamann Isabell
Published: January 2023

Fractalkine a neuronal derived chemokine, undergoes proteolytic cleavage and is released as a soluble molecule. In the CNS, fractalkine binds CX3CR1 on microglia, promoting microglial survival and tonically inhibiting microglial neurotoxicity. CX3CR1 is also expressed by circulating monocytes, NK cells and a small population of T cells. Cx3cr1−/ − mice exhibited severe EAE, and CX3CR1 was required for the accumulation of regulatory NK in the brain. In order to distinguish the role of CX3CR1 on microglia and NK cells during EAE, we generated bone marrow radiation chimeras. Both KO→WT and WT→KO exhibited a severe clinical EAE. In KO→WT mice, disease was associated with an unusually severe and non-remitting form of EAE with a high proportion of the mice exhibiting ascending paralysis that was sustained up to 45–50 days post-immunization. These results suggest that the EAE phenotype of Cx3cr1−/ − mice is primarily determined by its function on circulating cells, likely CD3−NK cells rather than microglia.
ADAM17 deficiency by mature neutrophils has differential effects on L-selectin shedding (97.14)
Authors: LI YING , HERRERA AMY , WALCHECK BRUCE
Published: January 2023

L-selectin directs neutrophils to sites of inflammation, and upon their activation, surface expression of the receptor is rapidly downregulated by ectodomain shedding. ADAM17 (also known as Tumor necrosis factor-alpha-converting enzyme or TACE) is a sheddase of L-selectin; however, Adam17 gene-targeting (ADAM17−/−) in mice is perinatal lethal and its role in L-selectin shedding by mature leukocytes has not been determined. This issue was addressed here by generating radiation-chimeric mice reconstituted with ADAM17−/−fetal liver progenitor cells. ADAM17-deficient neutrophils, monocytes and lymphocytes failed to shed L-selectin in response to PMA, as did neutrophils infiltrating the inflamed peritoneum. In addition, the lack of functional ADAM17 resulted in significantly increased levels of L-selectin surface expression by leukocytes in peripheral blood, suggesting the sheddase is also involved in the constitutive cleavage of L-selectin. Interestingly, it seems that ADAM17 is not required in all manners of L-selectin turnover. Plasma levels of soluble L-selectin were similar between ADAM17−/−chimeric and control mice, as was the shedding of L-selectin by neutrophils undergoing apoptosis. The latter process, however, was diminished by a metalloprotease inhibitor (TAPI-0), indicating the involvement of a metalloprotease sheddase other than ADAM17. Our data is the first to demonstrate that L-selectin’s surface density on neutrophils is regulated by ADAM17, but homeostatic cleavage of L-selectin is not.

This work is supported by grant HL61613 (B.W.) from the National Institutes of Health.
PARP-1 and iNOS relationship in allergen-induced lung inflammation: dependence of iNOS expression on PARP-1 and its dispensability for eosinophil recruitment post-IL-5 production (98.1)
Authors: Boulares Hamid , Suzuki Yasuhiro , Naura Amarjit , Oumouna Mustapha , Datta Rahul , Hans Chetan
Published: January 2023

The inducible nitric oxide synthase (iNOS) has been suggested to play an important role in the pathogenesis of asthma by contributing to eosinophilia and the ensuing oxidative damage after allergen exposure. IL-5 was shown to induce iNOS expression and that a reduction in iNOS expression reduces airway recruitment of eosinophils in response to allergen exposure. We recently showed that PARP-1 also plays a critical role in the pathogenesis of the disease by promoting eosinophil recruitment by participating in the cascade leading to IL-5 production. Here, we investigated the interrelationship between PARP-1 and iNOS in inducing eosinophilia. Our results show that iNOS gene deletion reduces eosinophil recruitment but to lesser degree as compared to PARP-1 gene deletion, suggesting a partial role for this protein in airway inflammation. PARP-1 gene deletion severely reduced iNOS expression in the airways of ovalbumin (allergen)-challenged mice and consequent nitration of target proteins and this phenomenon was closely associated with an impairment of p65 NF-κB nuclear translocation and phosphorylation of its inhibitor, I-κBα. IL-5-intranasal administration restored the eosinophilia in ovalbumin-challenged PARP-1−/− mice but did not reverse iNOS expression, suggesting that iNOS may be dispensable for eosinophil recruitment after IL-5 production. Altogether, our results further support the role of PARP-1 in the process of eosinophil recruitment during allergen-induced lung inflammation and provide evidence for a potential specific relationship between PARP-1 and iNOS.

Support: NIH-HL072889 and 1P20RR18766 to HB.
Rgs13 acts as a nuclear repressor of CREB in B lymphocytes (B10)
Authors: Xie Zhihui , Johnson Eric N , Druey Kirk M
Published: January 2023

The autonomic nervous system profoundly impacts immune functions, particularly during stress. Norepinephrine stimulates Gαs-coupled β2-adrenergic receptors on B lymphocytes, leading to cAMP response element binding protein (CREB)-mediated transcription of a variety of genes including the B-cell specific Oct 2 coactivator (OCA-B). Regulator of G protein signaling 13 (Rgs13) is a member of a large protein family that attenuates G-protein-mediated signal transduction by acting as GTPase-accelerating-proteins (GAPs) for Gαi and Gαq but not Gαs. Here, we show that Rgs13, which is enriched in germinal center B lymphocytes, is a direct transcriptional repressor of CREB in B cells. OCA-B expression is increased in B cells from Rgs13−/− mice both in the basal state and after exposure to the β2-agonist terbutaline in vitro. β2-adrenergic receptor stimulation induces nuclear translocation of Rgs13 and binding to activated pCREB in a complex with CREB binding protein (CBP/p300). This interaction impairs CREB binding to DNA and transcription of a CRE reporter gene. Rgs13 −/− B lymphocytes exhibit increased basal and agonist-stimulated pCREB-DNA binding. We propose that Rgs13 acts as a direct nuclear inhibitor of CREB-driven transcription in B lymphocytes independent of its GAP activity. As a result, Rgs13 may mediate important aspects of neural regulation of humoral immune responses.
Fcγ receptor activation triggers monocytes to differentiate into CD1b+ immature dendritic cells (B138)
Authors: TANAKA Motoyuki , Krutzik Stephan R , Sieling Peter A , Rea Thomas H , Modlin Robert L
Published: January 2023

Antibodies are potent inducers of inflammation and Fcγ receptors (FcγRs) are considered as important immune-regulators linked to infection immunity, cancer immunity and auto-immunity. We previously reported that CD1b+ dendritic cells (DC) are differentially distributed in leprosy lesions and contribute to the in pathophysiology of the polarized clinical spectrum of the disease. Here we demonstrate that activation of FcγRs on human blood monocytes triggers monocyte differentiation into CD1b+ cells. The CD1b+ population increased in a dose-dependent manner, and the differentiation was blocked by the addition of an anti-CD64 monoclonal antibody. Furthermore, Fcγ receptor ligation triggered transcription of GM-CSF mRNA as measured by real time PCR and CD1b+ cell differentiation was suppressed by the addition of an anti-GM-CSF antibody. Finally, FcγR-induced CD1b+ cells exhibited an immature dendritic cell (DC)-like phenotype. These findings predict that the stimulation of FcγRs induces GM-CSF production that in turn triggers monocyte differentiation into a CD1b+ immature dendritic cell population. In conclusion, these results provide insight into the cross-talk between the humoral and innate immune response as well as new strategies for therapeutic intervention in infectious diseases and autoimmune diseases.
Low incidence of anti-Gal α 1-3Gal and anti-porcine antigens antibodies in xenotransplanted type-1 diabetic patients after 6 years of follow-up (B160)
Authors: Esquivel-Perez Rita , Valdes Rafael A , Ayala-Sumuano Jorge-Tonatiuh
Published: January 2023

We have reported the clinical follow-up of diabetic patients xenotransplanted with islet cells. Here, we report the antibody response elicited against the xenograft after 6 years. Samples were screened by ELISA for anti-Gal IgM, IgG and IgG subclasses. Anti-pig antigen antibodies were tittered by means hemolytic antibody assay. Anti-Gal IgM and IgG titers of pretransplant sera were 90 and 115 respectively with no statistical differences in comparison with sera titers from healthy subjects. After first transplant the mean for anti-Gal IgG was 918 and increased with second and third transplants. After that the levels diminished to basal line. IgM titers were no different. IgG subclasses titers were not different, except for IgG2. Hemolytic anti-pig pretransplant antibodies mean was 320 however at this time the mean is 190. There is a classical humoral response against Gal antigen after each transplant with high levels of IgG but with very lower levels of IgM. However, these levels go backward 6 weeks after each transplant. During this assay we have observed an upsurge of IgG2 over the other subclasses. Low anti-pig levels elicited suggest an immune accommodation induced by this procedure.
Efficient Antibody Selection and Quantitation Using Microcapillary Cytometry (B209)
Authors: Mergia Alemayehu , Fishwild Dianne , Kenney John , Tyagarajan Kamala
Published: January 2023

Optimal selection of antibodies for therapy or research utilizes information from relative affinity measurements, specificity, functional studies and antibody isotype. All of the above are further dependant on accurate quantitative levels of antibodies utilized in the experiments so that a proper comparison of clones may be performed. Guava® RapidQuant™ IgG assays are novel bead-based assays that use microcapillary cytometry to provide quantitation of mouse or human IgG. In this study, mice were immunized with human CD80 and supernatant from each resulting hybridoma was subjected to multiple analyses on the Guava EasyCyte™ platform. The antigen specificity was obtained using a cell-based Guava Express™ assay and quantitative levels of IgG in supernatant were determined using the Guava RapidQuant Mouse IgG Assay. Antibody isotype and relative affinity were also ascertained. From these analyses, top ranking clones from 455 tested were identified based on specificity and productivity data. This study demonstrates the utility of microcapillary cytometry in combination with bead-based and cell-based assays to obtain multiple layers of information in the ranking and selection of the best clones.
A study on the screening and classification of herb medicines that have an influence on interleukin-4 (B216)
Authors: Min Ji-young , Lee Youngseop , Lee Heekyung , Jeon Sung Bae , Shin Min-Kyu , Kim Yangseok , Bae Hyunsu , Hong Moo-Chang
Published: January 2023

This study has examined the effects of 204 herbs, which are used in Traditional Korean Medicine, on the immune system. After the separation of splenocytes of BALB/c mice, two concentrations (1 §¶/§¢, 10 §¶/§¢) of herb medicine were given, and then the inducement of IL-4 was examined and measured using a sandwich ELISA. The amount of IL-4 each herb induced was analyzed, and the herb medicines were classified using two methods. One was the classification method of Traditional Korean Medicine, and the other was according to botanical phylogenetic systematics.

There were significant results for the analysis according to the botanical phylogenetic classification (Order). When there was a low concentration of the herb medicines, IL-4 was decreased by Ranunculales and Rosales, and increased by Malvales. When there was a high concentration of the herb medicines, IL-4 was decreased by Liliales, Ranunculales, Asterales, Fabales, Lamiales, Rosales, and Rutlaes, and increased by Malvales. The results showed the characteristics of individual herb medicines that have an affect on the inducement of IL-4 in splenocytes of a BALB/c mouse. In relation to the immune system, there was significant meaning to the functions of drugs for dispersing actions of wind and cold, drugs for dispersing actions of wind and heat, drugs for reducing intense internal heat, and drugs for eliminating heat and dampness, and also classifying the herb medicines according to botanical phylogenetics (Order).

This work was supported by the Brain Korea 21 project in 2007
The role of PKC-Theta in CD8 T cell activation (B28)
Authors: Valenzuela Javier O. , Wang Xingyu , Hussain Sofia , Beato Francisca , Liang Yaming , Yu Xue-Zhong , Beg Amer A.
Published: January 2023

Protein kinase C- Theta (PKC𝛉) is known to be a key mediator of NF-κB activation downstream of TCR signaling. However, recent in vivo studies with PKC𝛉−/− mice have generated conflicting results regarding its role in T cell activation. To elucidate the role of PKC𝛉 in the activation of CD8 T cells, we generated ovalbumin (ova)-specific PKC𝛉−/− OT1 mice. Stimulation of PKC𝛉−/− OT1 CD8 T cells with microspheres coupled to H2Kb-ova demonstrated a critical function for PKC𝛉 in TCR-induced NF-κB activation and proliferation in vitro. In contrast, ova-induced proliferation and CTL generation in vivo was virtually the same as WT cells. In addition, PKC𝛉−/− CD8 T cells stimulated in vitro with ova-pulsed DC proliferated vigorously and to a similar extent as WT cells. Remarkably, comparable levels of NF-κB were activated in WT and PKC𝛉−/− T cells after stimulation with DC. Proliferation of PKC𝛉−/− cells is severely reduced when DC deficient for B7-1/2 are used to stimulate the cells. Costimulation provided by CD27L also plays a role, as demonstrated by the use of a specific blocking antibody. In contrast to the response against antigen, GVHD responses are severely impaired in the absence of PKC𝛉. These results suggest that impaired TCR/antigen-induced NF-κB activation in PKC𝛉−/− CD8 T cells does not have a significant impact on T cell proliferation when other non-TCR signaling pathways are able to induce NF-κB and T cell activation.
Genetic variation in PTPN22 corresponds to altered signaling in B lymphocytes (B38)
Authors: Arechiga Adrian Fernely , Buckner Jane Hoyt
Published: January 2023

PTPN22 is a gene encoding the protein tyrosine phosphatase Lyp. A missense mutation in the gene, changing residue 1858 from a Cytosine to a Thymidine (1858C/T), is associated with the development of multiple autoimmune disorders. Although studies have demonstrated that Lyp has an inhibitory effect on T cell receptor mediated signaling pathways, the mechanism by which the 1858T variant contributes to the development of autoimmunity remains elusive. In this study, we examine the impact of the 1858T variant on B lymphocyte function. B lymphocytes from healthy subjects expressing the variant allele were compared to those of healthy subjects without the variant. When B cells were stimulated via the B-cell receptor, we observed impaired BCR signal transduction among subjects possessing the variant allele. This was characterized by diminished Calcium mobilization and altered tyrosine phosphorylation in key signaling proteins. These findings demonstrate that the PTPN22 1858T variant alters B cell as well as T cell responses and implicates B cells in the mechanism by which the PTPN221858T variant contributes to autoimmunity.

This work is supported by a JDRF grant to The Center for Translational Research at Benaroya Research Institute.
Cytokines Maintain Lymphocyte Homeostasis through the Activity of Cdc25A (B56)
Authors: Kittipatarin Christina , Khaled Annette
Published: January 2023

Homeostasis of the peripheral immune system is maintained through signals transduced by cytokines like interleukin-7 (IL-7). In the absence of IL-7 cells undergo apoptosis, while excess IL-7 leads to proliferation. The mechanisms by which IL-7 regulates these processes are poorly understood. We observed that expressing an anti-apoptotic protein (BCL-2) or inhibiting a pro-apoptotic protein (BAX) prevented cell death but did not support growth. Using cytokine-dependent lymphocytes, we found that the G1-to-S phase progression was not solely dependent on the synthesis of cyclin dependent kinases (cdks) and cyclins or the inactivation of inhibitors. Rather it was the removal of inhibitory phosphorylations on cdks by a phosphatase, Cdc25A, which primarily transduced cytokine-mediated growth signals. Cytokine deprivation lead to cell cycle arrest by inducing the activity of p38 MAPK, which destabilized Cdc25A. Inhibiting p38 or expressing a mutant Cdc25A, in which p38 target sites were mutated, promoted cell cycling independent of a cytokine signal. Cells expressing the Cdc25A mutant, in the absence of cytokines, actively synthesized DNA, expressed hyperphosphorylated Rb protein, maintained glucose uptake and did not undergo apoptosis, even in the presence of p27. Cdc25A is a novel transducer of cytokine-mediated growth signals and regulation of its activity is a means by which IL-7 controls proliferation.
Aberrant Regulation of Hematopoiesis by T cells in BAZF-Deficient Mice (B7)
Authors: Dent Alexander L , Broxmeyer Hal E. , Cooper Scott H , Sehra Sarita
Published: January 2023

The BAZF (BCL6b) protein is highly similar to the BCL6 transcriptional repressor. While BCL6 has been characterized extensively, relatively little is known about the normal function of BAZF. In order to understand the physiological role of BAZF, we created BAZF-deficient mice. Unlike BCL6-deficient mice, BAZF-deficient mice are healthy and normal in size. However BAZF-deficient mice have a hematopoietic progenitor phenotype that is almost identical to that of BCL6-deficient mice. Compared to wild-type mice, both BAZF-deficient and BCL6-deficient mice have greatly reduced numbers of cycling hematopoietic progenitor cells (HPC) in the bone marrow and greatly increased numbers of cycling HPC in the spleen. In contrast to HPC from wild-type mice, HPC from BAZF-deficient and BCL6-deficient mice are resistant to chemokine-induced myelo-suppression and do not show a synergistic growth response to GM-CSF plus Stem Cell Factor. Depletion of CD8 T cells in BAZF-deficient mice reverses several of the hematopoietic defects in these mice. Since both BAZF- and BCL6-deficient mice have defects in CD8 T cell differentiation, we hypothesize that both BCL6 and BAZF regulate HPC homeostasis by an indirect pathway involving CD8 T cells.
Distinct Processing Patterns Involving Flanking Residues of a GAD65 Peptide Can Be Accountable for the Difference in Diabetes Susceptibility Between NOD and NOR Mice (B77)
Authors: Dai Yang D. , Jensen Kent P. , Marrero Idania , Li Ningli , Quinn Anthony , Sercarz Eli
Published: January 2023

An immunogenic peptide in the GAD65 molecule, p524–543, activates two distinct populations of T cells, which apparently play opposite roles in the development of diabetes in NOD mice. In this study, we compared the fine specificity of these two T-cell repertoires using a nested set of truncated peptides that cover the p524–543 region. Surprisingly, we found that all clones apparently required the same minimum core sequence, around p530–539, for optimal activation. It was the flanking residues surrounding this core that played the crucial role in selecting T cells to activate. Flanking residues on the N-terminal side of the core led to the selection of regulatory cells and a largely Vbeta 12 T-cell repertoire, whereas flanking residues on the C-terminal side led to selection of effector cells and a largely Vbeta 4 T-cell repertoire. We then compared the repertoire in NOD with that in MHC-matched but diabetes-resistant NOR mice. NOD mice exhibited an enriched response to a C-terminal peptide, whereas NOR mice favored a response requiring N-terminal flanking residues. These data suggest that processing or trimming of the flanking residues of diabetes-associated determinants is a decisive event in the selection of islet-infiltrating T cells in NOD mice.
Development of Altered Peptide Ligands for Immunotherapy of Experimental Collagen-induced Arthritis (B96)
Authors: Marshall Dana Robin , Zhou Fang , He Xiaowen , Kayes Tim , Stuart John M
Published: January 2023

Collagen-induced arthritis is an autoimmune disease that results from the immunization of susceptible mouse strains with type II collagen. Amino acid residues 260–274 in the bovine collagen II alpha chain (bCIIá) represent the major MHC Class II epitope for the human DR1 haplotype. Amino acid residues 267Q and 270K are predicted TCR contact residues. Replacement of either of these amino acids with a glycine does not alter the ability of the peptides to bind to soluble DR1 but does substantially decrease their ability to induce proliferation of lymphocytes from bCIIá-immunized B10.M.DR1 mice while still allowing them to interfere with bCIIá-induced proliferation. The altered peptides elicit moderate decreases in disease incidence when co-immunized with bCIIá or injected intraperitoneally prior to the onset of clinical signs of disease.
The immune function of aging T cells (35.6)
Authors: Mirza Noweeda N , Hoelzinger Dominique B , Dominguez Ana L , Lustgarten Joseph
Published: January 2023

Age related changes compromise T cell mediated immunity. Previous findings suggest that the aged immune T cell repertoire can be exploited to mount effective anti-tumor responses to immunogenic tumors. However, antitumor immune responses against tumor self-antigens are significantly dysregulated in the old; hence, the salient differences in the biology and function of T cells between young and old, should be elucidated. These differences fall into three categories: (i) there are lower surface levels of T cell receptor (TCR) on old CD4 and CD8 T cells, (ii) the TCR complex is cycled differently in aged T cells and (iii) the TCR complex may be disassociated in older animals. Therefore these preliminary findings support the hypothesis that aging causes a decline in T cell function. Data analyses from a T cell activation time-course microarray study show that there are large differences in the gene expression profiles of old and young naive cells in CD4 (2022 genes) and CD8 (3666 genes) subsets. These differing genetic signatures point to potentially dysregulated proteins in the old immune system. Further investigation of selected gene products using appropriate mouse models, may provide valuable preclinical information with significant therapeutic potential which could have the opportunity of being translated into clinical trials.

This work was sponsored in part by a grant from AFAR.
Gads regulates the signaling threshold for CD8+ T cell activation (35.9)
Authors: Zhang Elizabeth Yan , Yankee Thomas  M.
Published: January 2023

Gads is an important regulator in T cell receptor (TCR)-mediated Ca2+ mobilization. We investigated the role of Gads in peripheral CD8+ T cell proliferation, using the MHC Class I-restricted ovalbumin-specific TCR transgenic mouse line (OT-I) on either the Gads+/+ or Gads-/- genetic background. We found that the Gads deficiency impaired TCR-mediated proliferation both in vitro and in vivo. This defect was most evident when low doses of cognate antigen (SIINFEKL) or altered peptide ligands (APLs) with lower affinity for the OT-I TCR were used as stimuli. However, the requirement for Gads in the proliferation of CD8+ T cells could be overcome by stimulating cells with high doses of SIINFEKL or APLs. The defect of Gads-/- CD8+ T cells in proliferation was caused by both impaired survival and by delayed entry into the cell cycle. We conclude that Gads regulates the signaling threshold through the TCR by promoting survival and cell cycle entry of CD8+ T cells. This research is supported by NIH grant P20 RR016443.
Modulation of autoimmune diabetes by antibodies specific for N-acetyl-D-glucosamine (167.10)
Authors: Dizon Brian , Greenspan Neil , Kearney John
Published: January 2023

Type I diabetes (T1D) is an autoimmune disease in which β cells are destroyed by the immune system. Childhood exposure to Group A Streptococci (GAS) is negatively correlated with T1D. Additionally, mice and humans produce N-acetyl-D-glucosamine (GlcNAc) specific antibodies in response to GAS infection. Since GlcNAc-specific antibodies exhibit autoreactivity to β cells, we hypothesized that GlcNAc-specific B cells and their secreted antibodies modulate the development of autoimmune diabetes. When neonatal NOD mice were immunized with GAS or control bacteria and monitored for hyperglycemia and glycosuria, NOD mice immunized with GAS were protected from spontaneous autoimmune diabetes. Moreover, passive transfer of sera from NOD mice immunized with GAS protected WT NOD mice from diabetes, demonstrating a protective role for GlcNAc-specific antibodies in vivo. Purified GlcNAc-specific IgM prevented activation and production of TNFα, IL-2, and IFNγ by diabetogenic T cells in vitro. Finally, immunization with GAS inhibited diabetogenic CD4 T cell activation in pancreatic lymph nodes in vivo. These findings indicate that B cells specific for O-GlcNAc, when activated by microorganism-associated antigens during neonatal development, protect against autoimmune diabetes by a mechanism dependent on secretory autoantibodies. These data identify a potential vaccine-based therapy for T1D in which protective autoantibodies are generated upon exposure to GlcNAc-expressing microorganisms.
HMGB1-PAMP complex promote the transformation of SFs to RASFs (171.19)
Authors: Qin Yanghua , Chen Yan , Wang Weiwei , Wang Zhiwei , Tang Gusheng , Dai Shengming , Shen Qian
Published: January 2023

The specific pathogenesis mechanism of rheumatoid arthritis (RA) remains unclear, current views believe the transformation of normal synovial fibroblasts (SFs) to RASFs is crucial for the pathogenesis of RA. Our previous studies showed that HMGB1-PAMP complex directly induced experimental arthritis in DBA/1 mice; pathology showed synovial thickening, and SFs excessive proliferation, suggesting that HMGB1-PAMP complex might induce the formation of RASFs. In current study, osteoarthritis synovial fibroblasts (OASFs) isolated from tissue obtained during reconstructive surgery and cocultured with HMGB1-PAMP complex in vitro for 5-8 generations to induce the transformation of normal SFs to RA-like SF (tOASFs). Then the pathogenicity of tOASFs was evaluated both in vitro and in vivo. Our results showed that tOASFs attached to, invaded into and degraded co-implanted cartilage in SCID mouse model. Histochemitry showed tOASFs excessive proliferation, expression MMPs. In vitro, cell cycle analysis showed more tOASFs pass through the G1/S checkpoint and move to S or G2 phage. Flow cytometry and confocal microscope showed significant reduced apoptosis and enhanced autophagy in tOASFs compared to OASFs, respectively. We further showed up-regulated expression of certain receptors (TLR2, TLR4, and RAGE) and adhesion molecules (ICAM-1 and VCAM-1) in tOASFs. In conclusion, we proved that HMGB1-PAMP complex promoted the formation of RASFs.
Regulation of dendritic cell function by Notch receptors and ligands (172.40)
Authors: Krawczyk Connie , Pearce Edward , Borjian Alborz , Young Stephanie , Mahbuba Raya
Published: January 2023

Cells of the innate immune system, such as Dendritic Cells (DCs), are the first responders and play a key role in creating pathogen-specific microenvironments through the generation of secreted mediators including cytokine, chemokines and lipids, and through modification of cell phenotype. T cells respond to the different activation phenotypes of DCs and in turn differentiate accordingly into distinct effector types, designated as lineages. Differentiation down different lineages is driven by “signal 3” or differentiation cues provided by the microenvironment. We have previously shown that signal 3 provided by DCs is a powerful regulator of T cell function not only at the time of activation, but during differentiation and recall responses. Notch ligand expression on DCs has been shown to be differentially regulated in response to various stimuli such as bacteria, worms, and allergens. Because Notch ligands show distinct and correlative expression patters in response to stimulation with various activators it is enticing to speculate they play a role in inducing or propagating differential immune responses. However their role remains controversial. Notch receptors are also expressed on DCs and their role in regulating DC function is just emerging. We will present evidence that Notch receptors participate in the activation of DCs. Furthermore, we propose that Notch ligands may indirectly influence Th differentiation through direct modification of DC function.
Identification, characterization and inhibition of protein interactions in immune responses (172.42)
Authors: Hindie Valerie , Ruggiero Barbara , Tafelmeyer Petra , Rain Jean-Christophe , Formstecher Etienne
Published: January 2023

Protein interaction mapping provides molecular basis to protein functions and signaling pathways. We have developed a high-throughput yeast two-hybrid technology that allows the exhaustive screening of highly complex, domain-based cDNA libraries relevant to the immune system (e.g. leukocytes and activated mononuclear cells, thymocytes CD4+/CD8+ and T-cells). These libraries are screened to saturation thanks to an optimized mating procedure, allowing to test on average 100 million interactions per screen. This corresponds to a 10-fold coverage of the libraries, and ensures exhaustive and reproducible results. Moreover, multiple independent fragments are isolated for each interactant, enabling the immediate delineation of a minimal interacting domain and the computation of a confidence score. A database containing over five thousand cDNA screens realized with this technique permits a rigorous calculation of relative prey connectivity and understanding of true/false positives and sticky proteins. We have developed additional methods to probe and characterize protein interactions in vitro with a medium to high throughput, using Surface Plasmon Resonance and a miniaturized time-resolved FRET assay which has been used for drug discovery projects targeting protein-protein interactions. Here, we will present results that illustrate the strength and benefits of our protein interaction screening and validation technologies to decipher the molecular components of immune responses.
Hybrid Vγ-Cβ TCR chains support T cell development and an immunocompetent T cell repertoire (LYM7P.713)
Authors: Bowen Steven , Sharrow Susan , Hodes Richard
Published: January 2023

Developing T cells undergo VDJ recombination at four T cell receptor (TCR) loci (γ, δ, β and α) to generate a highly diverse γδ or αβ TCR. During the CD4-CD8- (DN) stage of development, simultaneous recombination at the TCRγ, TCRδ and TCRβ loci can result in unusual interchromosomal trans rearrangements between these loci, particularly between TCRβ and TCRγ, creating hybrid TCR genes encoded by two different chromosomes. We have found that in-frame trans rearrangements between the Vγ2 gene and a variety of Dβ and Jβ genes are transcribed, spliced and expressed on the surface of a small population of peripheral T cells. Vγ2-Cβ hybrid TCR chains are capable of mediating β-selection and require the expression of TCRα for subsequent developmental transitions. However, the relatively low frequency of these cells has made analysis of their function difficult. Here we report that transgenic expression of a Vγ2-Jβ2.4 trans rearrangement is capable of driving T cell development in the thymus through the CD4/CD8 double positive (DP) and single positive (SP) stages as well as maintaining a robust population of CD4+ and CD8+ T cells in the periphery. These mice are also capable of mounting a T-dependent IgG1 response to peptide immunization. These data suggest that non-conventional Vγ-Cβ TCR chains are sufficient to support the development of an immunocompetent T cell repertoire.
Tumoral expression of IL-33 promotes Anti-tumor Immune Responses (TUM2P.912)
Authors: Wang Xuefeng , Gao Xin , Zhao Xin , Yang Qianting , Qi Yuan , Zang Shayang , Zhang Xueguang , Lu Binfeng
Published: January 2023

One major approach of cancer immune therapy is to convert the tumor immunosuppressive microenvironment to one that favors antitumor immune responses. We have recently demonstrated that Interleukin 33 (IL-33) promotes effector functions of CD8 T cells, suggesting a potential function in antitumor immunity. Here we showed that overexpression of IL-33 in two tumor cell lines, 4T1 and B16, potently inhibited tumor growth in vivo. CD45+, CD8+ T cells, NK cells, IFN-γ+ CD8+ T cells and IFN-γ+ NK cells were greatly increased in the IL-33-expressing tumors when compared with those in the control tumors. We further demonstrated that the antitumor effect of IL-33 was dependent on NK cells and CD8 T cells. In contrast, MDSC were greatly decreased in the IL-33-expressing tumors when compared to those in the control tumors. Our results indicate that the intratumoral delivery of IL-33 can be a new strategy of tumor immunotherapy by promoting an immunogenic microenvironment.
Induction of anti-leukemic responses using cellular immunotherapy (TUM2P.921)
Authors: Fast Loren , Reagan John , Nevola Martha , Mulder Arend , Claas F. , Quesenberry Peter
Published: January 2023

Previous work has shown that blood cancers can be treated through stimulation of the cancer patient’s immune system. In initial studies, it was observed that 14/26 patients with refractory hematological malignancies responded (9 major) to the infusion of large numbers (1 -2 x 108 CD3+ cells/kg) of haploidentical white blood cells following low levels of total body irradiation (100cGy). A cytokine storm quickly developed with a median time of 14 hours and resulted in high fevers that were allowed to persist for 48 hours, if possible, and then controlled with corticosteroids if needed. However the potential role of anti-leukemic effector cells in these responses was not determined. Three patients have been enrolled into a re-opened cellular immunotherapy clinical trial in which haploidentical white blood cells are infused with no prior chemotherapy or radiation. Blood samples were obtained from the recipient approximately 1, 24, 48, 72 and 168 hours after infusion and analyzed for the persistence and activity of donor/recipient cells as well as levels of cytokines and other factors in the plasma. Thus far, the results demonstrate elimination of donor cells by day 7, rapid upregulation of PD-1 ligands on leukemic cells and prolonged plasma levels of IL-10. Evaluation of the trends obtained with increased number of patients enrolled in the clinical trials will provide insights into new approaches to enhance the anti-leukemic responses in these patients.
Design of T cell epitope-driven IAV vaccines for swine using immunoinformatics tools (VAC2P.929)
Authors: Liu Rui , Gutierrez Andres , Loving Crystal , Vincent Amy , Moise Leonard , Terry Frances , Martin Bill , Groot Annie
Published: January 2023

Immunoinformatics tools have accelerated human vaccine design, but have not been applied extensively to animal vaccines. iVAX is a comprehensive toolkit to design genome-derived epitope-driven vaccines (GD-EDV) that uses EpiMatrix for epitope-mapping, among other algorithms. EpiMatrix was designed for use with human HLA. Using the pocket profile method, we created new T cell epitope-prediction matrices (PigMatrix) for porcine MHC (SLA). Integration of PigMatrix into iVAX now makes it possible to design GD-EDV for swine pathogens. We analyzed proteins from five influenza A viruses (IAV) representative of those prevalent in North American swine for highly conserved T cell epitopes. IFN-γ ELISpot responses to eleven 9mer epitopes, predicted to bind to class I SLA-1*0401, were measured using samples from pigs vaccinated with adjuvanted, whole-inactivated IAV. Together, these epitopes represented more than 50% of the response observed for whole virus re-stimulation. In further studies, we developed matrices for class I and II SLA alleles and predicted 28 conserved class I and 20 class II T cell epitopes from seven common IAV genomes. These epitopes were concatenated into two multiepitope IAV DNA vaccines for efficacy testing. These early results demonstrate the potential for immunoinformatics tools to develop rapid responses to infections affecting animals used for food. The new enhanced tools will facilitate the design of GD-EDV for swine diseases lacking effective vaccines.
Epigenetic regulation in CD91+ cells following immunization with heat shock proteins (VAC3P.948)
Authors: Kinner Lauren , Binder Robert
Published: January 2023

The immunogenic HSPs are intracellular chaperones of peptides. When HSPs are released from cells they interact with the receptor CD91 on the surface of antigen presenting cells. This allows for cross-presentation of the chaperoned peptides and initiation of various signaling pathways. We have shown that low or high dose gp96 immunization of mice leads to priming of TH1 and Treg responses respectively. Although the mechanisms driving this switch remain largely unknown, these responses have been harnessed for immunotherapy of cancer and autoimmune disease. Our preliminary studies have indicated changes in accumulation and/or recruitment of multiple immune cell subsets in the draining lymph nodes and spleens of mice following immunization with gp96. We propose that such regulation is controlled by DNA methylation states in CD91+ cells. Using next generation sequencing, we have identified these modifications following gp96 immunization. Methyl-seq analysis of CD91+ cells derived from mice immunized with low or high doses of gp96 reveals differential methylation across at least 80 promoter regions, including genes involved in chemotaxis, signaling, and cytokine response. Further exploration will determine which, if any, of these regions are associated with gene silencing. Importantly, these studies show an early requirement for epigenetic changes in response to gp96 and will provide further understanding of the APC-T cell interaction following gp96 immunization and in general.
Genetic background influences the NK recruitment and Th1 polarization in response to TLR agonists (VAC3P.955)
Authors: Nouri-Shirazi Mahyar , Zeng Menghua , Bible Brittany , Tamjidi Saba
Published: January 2023

In response to vaccines, DCs do not act in isolation but potentiate their efficiency by interacting with NK cells. The recognition of PAMPs by TLRs triggers DC maturation which is essential for their migration and T-cell priming. Maturing DCs release IL-12 which regulates the function of NK cells and drives the Th1 cells differentiation. In turn, NK cells which are activated by IL-12 and PAMPs through their own TLRs, provide IFN-γ necessary for enhancing stable IL-12 production by DCs and maintaining Th1 cell polarization. Studies using Balb/c strain showed that TLR agonist R848 induces NK cell recruitment into lymph nodes and augments Th1 polarization through unknown mechanisms. In this study we tested whether the genetic background will influence the NK cell recruitment and subsequent Th1 polarization in response to vaccine formulated with a panel of TLR agonists and alhydrogel. We found that immunization with OVA protein mixed with TLR agonist R848 among adjuvants tested, increased up to 3-fold recruitment of IFN-γ producing CD27+CD11b+ NK cell expressing CXCR3 and CD62L in lymph nodes and spleen of Balb/c but to a much lesser extent in B6 mice. Interestingly, however, the increased in NK cell recruitment had limited impact on Th1 polarization in Balb/c as compared to Th1-prone B6 mice. These data suggest that for optimal Th1 polarization, adjuvant or adjuvant combination should be selected based on their ability to both recruit and maximize DC-NK bidirectional signaling.
Expression of triggering receptor expressed on myeloid cells-1 (TREM-1) is up-regulated following West Nile virus infection (WNV): implication for a role in innate immune responses (VIR1P.968)
Authors: Verma Saguna , Roe Kelsey , Orillo Beverly , Nelson Jacob , Gibot Sebastien
Published: January 2023

Activation of TREM-1 signaling via adaptor protein DAP12 is important for inflammation and activation of antigen presenting cells, however little is known about its role in viral infections. Innate immune responses are essential for the control of Flaviviruses including WNV, which has emerged as a significant cause of viral encephalitis in humans. Here, we investigated the effect of Flavivirus infection on the expression of TREMs and its potential role in production of cytokines. We show that expression of TREM-1 was markedly increased in dengue virus-infected THP-1 cells, which correlated with peak viral titers. Similarly, WNV infection significantly increased the mRNA levels of TREM-1 in mouse embryonic fibroblasts (MEFs), BMDM and BMDC. In vivo characterization of TREMs in mice demonstrated significant up-regulation in the transcripts of TREM-1, -3 and -4 in the peritoneal cavity cells and brain at day 3 and 8 after WNV infection respectively. Interestingly, serum levels of soluble-TREM-1 also increased significantly at days 2-3 after infection. Further, activation of TREM-1 using an agonist antibody increased mRNA of WNV-induced cytokines such as IFN-α, TNF-α and IL-6 in MEFs, which decreased following blocking of TREM-1. Collectively, our results for the first time document response of TREMs to Flavivirus infection and indicate a novel role of TREM-1 in modulating inflammatory response to WNV. Further studies are ongoing to define role of TREM-1 in WNV disease outcome.