bluuebunny/crossref_metadata_2025_split huggingface dataset:
Secretion of IL-12p40, but not IL-12 or IL-23, from immature dendritic cells interacting with naive T cells (92.14)
Authors: Douglas Taneka , Bartley Jeanette , Sayles Leslie , Swier Kevin 
Published: January 2023

Abstract
IL-12 is a 70 kD heterodimeric cytokine that consists of two subunits, p40 and p35. The p40 subunit can also form p80 homodimers, or it can associate with p19 to form IL-23. IL-12 is known to be induced in dendritic cells (DCs) by pathogens, through toll-like receptors (TLRs), and by activated T cells, through CD40-CD40L engagement. To investigate the response of antigen presenting cells (APCs) to naïve T cells, we cocultured DCs and OVA peptide with CD4+ CD62L+ OVA-specific T cells. Naïve T cells induced immature DCs to secrete p40, but not IL-12, during antigen presentation, in sharp contrast to the nearly exclusive secretion of IL-12 heterodimers by DCs presenting to CD62L− T cells. Maximal secretion of p40 occurred within 24 hr after T cells were added to the APCs. During this time period, CD40 and CD40L expression increased on DCs and T cells, respectively, but no significant difference was seen in the expression of CD40L on CD62L+ and CD62L− T cells. IL-23 induction was measured by real-time RT-PCR and by ELISA. IL-23 was not induced by naïve T cells unless the DCs were activated. Western blot analysis will reveal whether the excess p40 induced by naïve T cells is secreted primarily as monomers or homodimers. Future experiments will also address the functional significance of excess p40 secretion during the encounter of immature APCs with naïve T cells.
(Supported by NIH grant S06-GM08043 to K.S.)
Mycobacteria-induced Gr-1+ subsets from distinct myeloid lineages have opposite effects on T cell expansion. (92.16)
Authors: van der Veen Roel 
Published: January 2023

Abstract
Similar to the regulation of vasodilation, the balance between nitric oxide (NO) and superoxide (O2−) regulates expansion of activated T cells in mice. Reduction of suppressive NO levels by O2− is essential for T cell expansion and development of autoimmunity. In mice primed with heat-killed Mycobacterium, a splenocyte population positive for Gr-1 (Ly-6G/C) is the exclusive source of both immunoregulatory free radicals. Distinct Gr-1+ cell sub-populations were separated according to Ly-6G expression. In culture with activated T cells, predominantly monocytic Ly-6G Gr-1+ cells produced T cell inhibitory NO, but no O2−. However, mostly granulocytic Ly-6G+ cells produced O2− simultaneously, but had no measurable effect on proliferation. Recombination of the two purified Gr-1+ subpopulations restored controlled regulation of T cell proliferation through NO and O2− interaction. Co-culture of p47phox−/− and iNOS−/− Gr-1+ cells confirmed this inter-cellular interaction. These data suggest that bacterial products induce development of distinct Gr-1+ myeloid lineages, which, upon stimulation by activated T cells, interact via their respective free radical products to modulate T cell expansion.
Essential role of heat shock protein 90 in direct and cross-presentation (93.13)
Authors: Garg Manish , Callahan Margaret K , Srivastava Pramod K 
Published: January 2023

Abstract
Direct antigen presentation involves the generation of peptides by proteasomes, ferrying of peptides from cytosol to endoplasmic reticulum and finally, formation and transport of peptide-MHC I complexes. Chaperones hsp90, hsp70, gp96 and TriC have previously been shown to associate with MHC class I epitopes and their precursors. Here, we have treated cells with hsp90 inhibitors Radicicol, 17-AAG or geldanamycin and observe that (i) treatment of cells with such inhibitors leads, in a dose-and time- dependent fashion, to a decrease in the levels of peptide-bound but not total cell surface MHC I; (ii) Radicicol does not affect transport of non-MHC I, control proteins to the cell surface; (iii) Radicicol does not impair MHC I synthesis; and (iv) the reduction in surface MHC I expression caused by Radicicol, is restored by pulsing cells with exogenous peptides. These results indicate an essential role for hsp90 in peptide-transport during endogenous (direct) presentation.
During indirect or cross-presentation, antigen presenting cells acquire, process and present exogenous antigen onto the MHC I. Our previous studies (Binder and Srivastava, 2005, Nature Immunology 6, 593) have shown an essential role for HSP-chaperoned peptides in this process. In that study, cell lysates replete with or depleted of one or several HSP-peptide complexes were used to study cross-priming. We have now examined cross-priming by intact cells instead of cell lysates and we observe that hsp90 plays a unique, essential and non-redundant role in cross-priming as well. Data that bear on the mechanism of post-proteasomal transport of peptides, shall be presented.
Recognition of Melanoma-Associated Antigens gp100 or Mart1/Melan-A in the Context of HLA-A2 by Soluble Single Chain T-Cell Receptor Multimers (93.15)
Authors: Zhu Xiaoyun , Liu Bai , Han Kaiping , Lee Hyung-il , You Lijing , Rhode Peter R. , Morgan Richard A. , Wong Hing C. 
Published: January 2023

Abstract
Melanoma-associated antigens gp100 and Mart1/Melan-A are specifically recognized by HLA-A2-restricted CD8+ CTLs derived from melanoma patients and are currently being evaluated in antigen-based vaccines for the treatment of melanoma patients. In this study, TCR genes isolated from the CTL clones recognizing gp100209-217/HLA-A2 and Mart126-35/HLA-A2 were used to generate soluble single chain TCR multimeric reagents, referred to as gp100scTCR and Mart1scTCR multimers. Like original CTL clones, both gp100scTCR and Mart1scTCR multimers recognize T2 cells pulsed with either native or analogue peptides. Gp100scTCR multimer recognize T2 cells pulsed with up to 50 nM of gp100209-217 or gp100209-217T210M and Mart1scTCR multimers recognize T2 cells pulsed with as low as 10 nM of Mart126-35 or Mart126-35A27L. Moreover, gp100scTCR multimer recognizes native peptide/HLA-A2 displayed on the surface of gp100+/HLA-A2+ melanoma cell lines 526 Mel and 624 Mel but not gp100+/HLA-A2− melanoma cell lines 888 Mel and 938 Mel. Thus, these tumor antigen-specific TCR multimers have potential uses as quality control reagents of dendritic cell-based vaccines and a variety of other research applications.
Multiple thermodynamic mechanisms contribute to the binding of pepMHC ligands by wild type and high-affinity TCRs (93.16)
Authors: Jones Lindsay L , Bankovich Alexander J , Colf Leremy A , Garcia K Christopher , Kranz David M 
Published: January 2023

Abstract
To better understand the mechanisms that govern TCR-pMHC binding and the role that different regions of the TCR play in affinity and antigen specificity, we have studied the TCR from mouse CTL clone 2C, which recognizes QL9/Ld as a strong agonist. High affinity mutations of the 2C TCR were previously generated in various CDRs (1β, 2α, 3α and 3β) by in vitro directed evolution. Using a single-chain version of the TCR and a stabilized Ldprotein, we performed isothermal titration calorimetry (ITC) on these mutants, which bind with high affinity to QL9/Ld. Unexpectedly, our results showed that the wild-type interaction was favorable entropically. In contrast, all high affinity mutants except one (2C-1β) bound in an enthalpically driven manner. In addition, we measured the binding and kinetics of these mutants for both QL9/Ld and position 5 peptide variant Y5/Ld using SPR. While other mutants had higher affinity for the Y5 peptide variant than for QL9, the 2C-1β mutant had a lower affinity for Y5. Thus it appears that multiple thermodynamic mechanisms are used to achieve high affinity in this system. In addition, some high-affinity TCR mutants could achieve high affinity while exhibiting enhanced fine specificity.
Supported by grants from the NIH.
Viral and Self HLA Class I Peptides Mark the Surface of Influenza Infected Cells (93.2)
Authors: Wahl Angela Raquel , Schafer Fredda , Bardet Wilfried , Buchli Rico , Samikkannu Malaroviyam , Fleshman Annette , Gonzalez Melva , Air Gillian , Hildebrand William 
Published: January 2023

Abstract
The influenza virus attacks epithelial cells of the lower and upper respiratory tracts. Following infection, the virus alters the proteome of the infected cell and triggers anti-viral cellular immune responses. The infected cell’s altered proteome is reflected at the cell surface by the HLA (Human Leukocyte Antigen) class I molecule. Class I HLA sample and display endogenously loaded peptides at the surface of all nucleated cells so that CTL can distinguish infected from uninfected cells. To identify class I ligands that mark infected cells, we collect soluble HLA B*0702 and A*0201 molecules from influenza infected and uninfected cells. Peptide ligands and their class I carriers are size separated, uninfected and infected peptide pools are fractionated by RP-HPLC, and peptides are comparatively mapped via mass spectrometry. Thus far, we have identified B*0702 peptides derived from the Influenza A/PR/8 hemagglutinin and nucleoprotein molecules and multiple B*0702 and A*0201 host-derived peptides that are either uniquely expressed or up-regulated on the surface of influenza infected cells. The identification of viral peptides unique to the class I of influenza infected cells indicates a route for eliciting CTL via vaccination whilst changes in class I HLA presented self peptide epitopes indicate “danger” signals that demark the infected cell.
Peptide-loading enhancement for antigen presenting cells (93.6)
Authors: Liu Bai , You Lijing , Han Kaiping , Lee Hyung-il , Rhode Peter , Henrickson Sarah , von Andrian Ulrich H , Wong Hing C 
Published: January 2023

Abstract
Exogenous β2-microglobulin has been shown to enhance the loading of antigenic peptides into MHC class I complexes of antigen presenting cells (APCs). In this study, we employed the multimeric soluble singe-chain T cell receptors and flow cytometry analysis to confirm the activity of exogenous β2 -microglobulin in enhancing the loading of various peptides onto APCs. To our surprise, we found that the enhancement of peptide loading on APCs was restricted to a fraction of the peptides tested. However, a chemical component, commonly used in biological research, exhibits similar peptide loading enhancement activity as that of exogenous β2-microglobulin. The peptide loading enhancement profiles of exogenous β2-microglobulin and the chemical are different and seem to be peptide-dependent. Although the underling mechanism of the chemical for peptide loading enhancement is unknown, the chemical is also found to significantly enhance the endogenous peptide presentation of the gp33 protein of lymphocytic choriomeningitis virus in 5T33 mouse myeloma cells. Thus, a mixture of β2-microglobulin and the chemical could have broad utility in increasing antigen presentation in a variety of experimental applications including vaccine-based approaches.
Characterization of C3adesArg/acylation stimulating protein (ASP) binding to C5L2 transfected cells and 3T3-L1 preadipocytes (94.17)
Authors: Cianflone Katherine , Cui Wei , Lapointe Marc , Kalant David , Maslowska Magdalena 
Published: January 2023

Abstract
C5L2 is a recently identified receptor for C5a and C3a/C3adesArg (ASP). C5a/C5adesArg binds C5L2 with high affinity with no functional activation. By contrast, some studies demonstrate C3a/ASP binding/activation of C5L2; others do not. We evaluated the influence of diverse ASP-C5L2 binding methods used.
Orphan receptors (n&gt;30) were screened with 125I-ASP (20oC & 4oC) or fluorescently labeled ASP (Fl-ASP, 37oC). Only C5L2 transfected cells were positive. Cell-associated Fl-ASP increased markedly from transiently-transfected &lt; stably transfected &lt; Fl-ASP sorted HEK-C5L2 for both hC5L2 and mC5L2. Similar results were obtained with transfected C5L2-CHO and C5L2-SW872 cells. Non-transfected cells ± Fl-ASP demonstrated background fluorescence only.
Recombinant ASP prepared under non-denaturing conditions demonstrated 10X greater bioactivity vs. proteolytically-derived plasma ASP (maximal TG synthetic activity 100–300 nM vs. 5 uM).
In adherent HEK-C5L2 (F-ASP sorted) and 3T3-L1 cells, blocking with 10% FCS, protamine sulfate or ovalbumin prevented 125I-ASP non-specific binding (NSB), while BSA blocking increased it. Optimal specific binding (Kd 66±14 nM) was obtained at 20oC (vs.4oC) in PBS or HAG-CM buffer. By contrast, cells in suspension demonstrated only non-specific binding. ASP-C5L2 binding has distinct characteristics that lead to phosphorylation, internalization and increased TG.
Funding: CIHR
Regulation of IL-15 trans-presentation to NK cells (94.18)
Authors: Vielkind Susina , Long Eric 
Published: January 2023

Abstract
NK cell activation is determined by a balance of signals delivered by multiple activating and inhibitory NK receptors that engage specific ligands on target cells. The cytokine IL-15 is essential for the development, survival, proliferation, and effector function of NK cells. Trans-presentation of IL-15 by IL-15Rα expressing cells raises the possibility that stimulation by IL-15 could be regulated by other receptor-ligand interactions.
To test the contribution of individual receptor - ligand interactions, the evolutionary distant Drosophila insect cell line SC2 was transfected with the IL-15R alpha chain. Expression of IL-15Rα on SC2 cells was sufficient to induce proliferation of resting NK cells. Proliferation was not enhanced by co-expression of ICAM-1. On the other hand, trans-presentation of IL-15 enhanced the binding of NK cells to ICAM-1. To test if IL-15 trans-presentation is under negative regulation by inhibitory receptors for MHC class I, IL-15Rα was co-expressed with HLA-C or HLA-E on SC2 cells, and on the MHC class I-negative human cell lines 721.221 and K562. Binding to ICAM-1 by freshly isolated NK cells, and their proliferation in response to trans-presented IL-15 is being evaluated in subsets of NK cells expressing different inhibitory receptors, using multi-color flow cytometry.
Stimulation by Toll-like receptor 5 modulates osteoclast differentiation through STAT1/IFN-β (94.20)
Authors: Lee Zang Hee , Ha Hyunil , Lee Jong-Ho , Kim Ha-Neui , Kwak Han Bok 
Published: January 2023

Abstract
Stimulation of Toll-like receptors (TLRs) has been shown to differently modulate osteoclast differentiation, depending on experimental condition. However, the molecular mechanism by which stimulation by TLRs modulates osteoclast differentiation still remains to be elucidated. In the present study, we examined the effect of flagellin, a specific microbial ligand of TLR5, on receptor activator of NF-κB ligand (RANKL)-stimulated osteoclastogenesis. Flagellin suppressed RANKL induction of c-Fos protein expression in bone marrow-derived macrophages, without affecting c-Fos mRNA expression. Ectopic overexpression of c-Fos and an active form of NFATc1 reversed flagellin-induced anti-osteoclastogenic effect. The inhibitory effect of flagellin was mediated by IFN-β production. Flagellin stimulated IFN-β expression and release in BMMs and IFN-β-neutralizing antibody prevented flagellin-induced c-Fos down-regulation and anti-osteoclastogenic effect. IFN-β gene induction by flagellin, LPS, or RANKL was dependent on STAT1 activation. Treatment with flagellin or RANKL stimulated STAT1 activation, and STAT1-deficiency or a JAK2 inhibitor AG490 dramatically prevented IFN-β induction in response to flagellin or RANKL. Taken together, these results demonstrate that IFN-β acts as a critical modulator for osteoclastogenesis in response to TLR5 activation.
Fish lymphocytes regulate the inflammatory response via the production of chemokines (94.24)
Authors: Roca Francisco Jose , Sepulcre Pilar , Lopez-Castejon Gloria , Sarropoulou Elena , Kotoulas Georgios , Meseguer Jose , Mulero Victoriano 
Published: January 2023

Abstract
Macrophages provide the first line of defense against infections through the recognition of different pathogen-associated molecular patterns (PAMPs). Upon activation, they produce pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα), which regulate not only innate but also adaptive immunity. In the bony fish gilthead seabream, we have shown that TNFα produced by activated macrophages induces the recruitment and activation of professional phagocytic granulocytes when injected intraperitoneally, but it hardly activates these cells in vitro. In this study, we report that seabream TNFα is able to up-regulate the expression of E-selectin in endothelial cells, leading to a concomitant increased adhesion of activated granulocytes and lymphocytes. More importantly, although all fish immune cell types analyzed, i.e. granulocytes, macrophages and lymphocytes, produce the CCL4-like chemokine in response to different PAMPs, only lymphocytes and endothelial cells are able to produce this chemokine in response to TNFα. These results demonstrate that TNFα in fish is mainly involved in the recruitment of leukocytes to the inflammatory foci rather than in their activation and suggest a role for fish lymphocytes in the regulation of early immune responses to infection.
This work was supported by the Spanish Ministry of Education and Science (grant BIO2005-05078) and the Sixth Framework Programme of the European Union (grant SSP8-CT-2003-501984).
The functional activity of human IL-17A, IL-17F and IL-17A/IL-17F cytokines is dependent on the receptors IL-17R and IL-17RC (94.31)
Authors: Wright Jill F , Bennett Frann , Li Bilian , Brooks Jonathan , Luxenberg Deborah P , Whitters Matthew J , Tomkinson Kathleen N , Fitz Lori J , Wolfman Neil M , Collins Mary , Carreno Beatriz M , Dunussi-Joannopoulos Kyri , Chatterjee-Kishore Moitreyee 
Published: January 2023

Abstract
IL-17A and IL-17F are members of the IL-17 cytokine family and have been detected in tissue samples from patients with autoimmune disease. Recent data has shown that these cytokines are secreted by a new CD4+ T cell lineage known as Th17 cells. We have recently demonstrated that activated human CD4+ T cells also produce the IL-17A/IL-17F heterodimer with in vitro functional activity. Both IL-17R and IL-17RC are required for activity of IL-17A and IL-17F. However, the question remains whether IL-17A/IL-17F also utilizes the same receptors for activity or whether it signals through different receptor chains. ELISA and BiaCore-based binding experiments were performed to determine if IL-17A/IL-17F could bind to IL-17R and IL-17RC. We find that IL-17A, IL-17F and IL-17A/IL-17F each bind to IL-17R but with different affinities, whereas they bind to IL-17RC with comparable affinity. Using cell based assays, we have evaluated the functional activity of IL-17A, IL-17F and IL-17A/IL-17F in the presence of soluble receptors, antibodies to the receptors and IL-17R and IL-17RC siRNA. We find that the activity of IL-17A, IL-17F and IL-17A/IL-17F is decreased when the expression of IL-17R is reduced, whereas the activity of the cytokines decreases to a lesser extent when the expression of IL-17RC is reduced. In conclusion, our results suggest that IL-17A, IL-17F and IL-17A/IL-17F each bind and signal through the same receptor complex.
The paradoxical pro- and anti-inflammatory activities of IFN-γ reflect negative feedback loop regulation and apoptosis. (94.5)
Authors: Dalton Dyana K. , Li Xujian 
Published: January 2023

Abstract
During the shut-down of immune responses most activated CD4+ T cells undergo apoptosis. Conflicting studies have cast doubt on the essential roles for TNF-related death molecules in T cell apoptosis. Recent studies have emphasized the roles of pro-apoptotic Bcl-2-related proteins, Bim and Bax, which are thought to be induced by growth factor withdrawal after antigen clearance. Yet, how the Bcl-2-related genes are induced in activated T cells, especially during chronic infections, is not understood. We show new evidence for a coordinated IFN-γ-dependent pattern of expression of pro-apoptotic Bcl-2-related and TNF-related death molecules, caspases, and Apaf-1 during CD4+ T cell activation by BCG. Several IFN-γ-induced molecules, including TNF-family molecules, cause damage to the mitochondria and promote Bim- and Bax-mediated apoptosis. This extends the known roles for IFN-γ in promoting apoptosis of activated CD4+T cells, and reveals previously unknown connections between Bcl-2-related and TNF-family molecules in the process of T cell apoptosis. We propose that CD4+ T cells are sensitized to undergo apoptosis after secreting IFN-γ: reflecting a negative feedback loop to limit further IFN-γ production.
Support: R01 NS043355 and NMSS RG 3820
Severely reduced B cell TACI expression in newborn mice lead to impaired BAFF or APRIL induced immunoglobulin secretion (95.13)
Authors: Akkoyunlu Mustafa , Kanswal Sunita , Katsenelson Nora 
Published: January 2023

Abstract
In this study, we investigated the expression and the functions of the TNF family cytokines BAFF and APRIL, and their receptors (BAFF-R, BCMA and TACI) in newborn mice. We found that while macrophage BAFF and APRIL expressions were increased, B cell TACI expression was dramatically reduced (p&lt;0.001) in newborns. Since TACI is shown to be important in BAFF/APRIL mediated IgA and IgG isotype switch and secretion, we analyzed the effect of BAFF and APRIL on newborn B cells and showed that both BAFF and APRIL failed to induce IgA or IgG secretion. We have previously shown that (manuscript in revision) the CpG-ODNs up-regulate the expression of TACI on adult mouse B cells and augment BAFF/APRIL mediated IgA/IgG secretion. We therefore studied the effect of CpG-ODNs on the expression of TACI and found that as with adult B cells, newborn mice also manifested increased TACI expression (p ≤ 0.0001) after stimulation with CpG-ODN. Moreover, CpG-ODN pretreatment of B cells rendered them susceptible to BAFF or APRIL mediated IgA/IgG secretion. Similarly, in in vivo experiments, CpG injected newborn mice showed increased TACI expression and ex vivo stimulation of B cells from these mice with BAFF or APRIL yielded increased IgA/IgG secretion. Given the pivotal role of TACI in mediating antibody responses to polysaccharide vaccines, low TACI expression could be a possible contributing factor to the susceptibility of newborns to infections with encapsulated bacteria.
IL-12/23 Signals Delivered by Dendritic Cells Predominantly Within the First 48 Hours of T cell Priming Dictates the Functional Capacity of CTLs (95.14)
Authors: Henry Curtis J. , Grayson Jason M. , Westcott Marlena M. , Brzoza Kristina L. , Hiltbold Elizabeth M. 
Published: January 2023

Abstract
Interleukin-12 is critical in establishing CTL-mediated protective immunity against various pathogens. Our initial studies focusing on the role of IL-12 in dictating CTL function used anti-IL-12p40 antibodies, which neutralize both IL-12 (p35/p40 subunits) and IL-23 (p19/p40 subunits). By monitoring interferon gamma (IFN-γ) production as a measure of CTL differentiation, we determined that IL-12p40 signals were required predominantly during the first 48 hours of priming. However, with these studies we could not assess the individual contributions of IL-12 and IL-23.
Therefore, our goal was to evaluate the individual contributions of IL-12 and IL-23 in regulating CTL primary and secondary responses. Wild-type, IL-12p35−/−, or IL-12p40−/− DCs infected with Listeria monocytogenes were used to prime CTLs; IFN-γ and granzyme B were used as measures of CTL differentiation. CTLs primed by IL-12p35 and p40−/− DCs exhibited reduced IFN-γ production; the greatest reduction occurred in CTLs primed by IL-12p40−/− DCs, indicating that IL-23 played a minor role in CTL priming. In addition, mice immunized with IL-12p35−/− DCs exhibited a reduced ability to clear Listeria. This deficiency correlated with fewer CTLs producing IFN-γ upon secondary antigenic exposure. Thus, the magnitude of primary and secondary CTL responses is influenced more by the activity of IL-12 than IL-23 during priming.
(NIH AI057770)