bluuebunny/crossref_metadata_2025 huggingface dataset:
Rap1b-GTPase Regulated Cytokine Generation in murine NK Cells (89.19)
Authors: Awasthi Aradhana , Samarakoon Asanga , Chu Haiyan , Wodnicka Magdalena , White Gilbert C. , Malarkannan Subramaniam
Published: January 2023

Rap1 belongs to Ras superfamily that consists of more than hundred GTP-binding proteins involved in a variety of essential cellular processes. Rap1 exists in two closely related isoforms: Rap1a and Rap1b. Although Rap1 isoforms are known to regulate cell growth, differentiation and survival, their precise functions in lymphocytes have not been defined. In this study, using single gene knockout mice we analyzed the role of Rap1 isoforms in the development and effector functions of natural killer (NK) cells. Both Rap1a and Rap1b are expressed in NK cells and the lack of these GTPases did not affect the development and terminal maturation of NK cells. Moreover, NK–mediated cytotoxicity against ‘missing-self’, ‘induced-self’ and ‘allo’ target cells were not altered by the absence of either Rap1a or Rap1b. However, the ability of NK cells to generate inflammatory cytokines in the absence of Rap1b isoform was significantly reduced. Further, NK cells lacking Rap1a generated normal levels of these cytokines demonstrating the functional divergence between Rap1a and Rap1b isoforms. We conclude that Rap1b is a critical regulator of a signalling pathway that governs the cytokine generation in NK cells.
Plasmacytoid Dendritic Cell Receptor BDCA2/FcεRγ Complex Uses BCR-like Signaling Cascade to Modulate Toll-Like Receptor Responses (89.20)
Authors: Cao Wei , Zhang Li , Rosen David B. , Bover Laura , Watanabe Gokuran , Bao Musheng , Lanier Lewis L , Liu Yong-Jun
Published: January 2023

BDCA2 is a C-type lectin uniquely expressed by human plasmacytoid dendritic cells (pDCs). Ligation of this receptor by mAb inhibits Toll-like receptor (TLR)-mediated type I IFN responses by pDCs. However, it remains unclear how BDCA2 signals. We demonstrate here that BDCA2 forms a receptor complex with the transmembrane adapter FcεRIγ. Furthermore, pDCs express a comprehensive signaling machinery similar to that of the B cell receptor (BCR), which is distinct from that involved in T cell receptor (TCR) signaling. BDCA2 crosslinking resulted in the activation of BCR-like signal cascade in pDCs including phosphorylation of Syk and Src family kinases, as well as adapter molecule BLNK, leading to NFAT activation and calcium flux. This potently suppressed the ability of pDCs to produce IFN and other cytokines in response to both TLR7 and TLR9 ligands. Therefore, BDCA2 negatively regulates pDCs’ innate immune functions by associating with FcεRIγ and activating a BCR-like signaling pathway.
Role of leptin signaling in regulating NK cell development in mouse bone marrow (89.21)
Authors: Lo Cherry Kam Chun , Ko King Hung , Lu Liwei
Published: January 2023

Increasing evidence suggests that leptin is involved in regulating immune response by acting as a growth factor on lymphocytes. Previous studies showed reduced number of NK cells in various peripheral organs of leptin-receptor-deficient db/db mice. However, it remains unknown whether leptin-receptor signaling is involved in regulating NK cell development in the bone marrow (BM). In this study, we characterized NK cell development in the BM of db/db mice at a prediabetic stage. Total number of NK cells was moderately reduced in the BM of mutant mice whereas both frequencies and total numbers of NK cell subpopulations were significantly reduced at various differentiation stages. Moreover, BM NK cells from db/db mice displayed markedly increased apoptosis but did not show any defects in cell cycling status and proliferative capacity. Consistently, recombinant leptin could significantly enhance the survival of NK cells of wild-type mice. To verify if NK cell functional activity was impaired in db/db mice, sorting-purified splenic DX5+CD3− NK cells were evaluated for their cytotoxicity and rmIL-15-induced proliferation capacity. Interestingly, db/db NK cells showed unimpaired capacities in their proliferation and cytotoxic function. In conclusion, our findings demonstrate that leptin-receptor deficiency impairs NK cell development in the db/db mouse BM via affecting NK cell survival.

This work was supported by grants from the Research Grants Council of Hong Kong (HKU7423/04M).
A novel role for HMGB1 in TLR9-mediated inflammatory responses to CpG-DNA (89.24)
Authors: Chu Wen-Ming , Ivanov Stanimir , Dragoi Ana-Maria , Wang Xin
Published: January 2023

Microbial CpG-DNA and related synthetic oligonucleotides (CpG-ODN) activate immune cells to produce inflammatory cytokines through Toll-like receptor 9 (TLR9). TLR9 is an intracellular receptor and is mainly expressed in immune cells. It is proposed that TLR9 is initially localized to the endoplasmic reticulum (ER), and upon movement of CpG-DNA into early endosomes redistributes to CpG-DNA-enriched vesicles, in which it is activated and MyD88 is recruited, leading to subsequent immune responses. However, it is not clear how TLR9 is activated by CpG-DNA, and it remains unknown whether CpG-DNA binding proteins are involved in this activation process. We demonstrate that the high mobility group box 1 protein (HMGB1) is involved in this process. We found that CpG-ODN-treated macrophages and DCs quickly secrete HMGB1; moreover, HMGB1 engaged CpG-ODNs and enhanced their immunostimulatory potential in a TLR9-dependent manner. Confocal microscopy analysis revealed that HMGB1 pre-associates with TLR9, the ER marker calnexin, the ERGIC-53 and the Golgi marker GM130 in quiescent cells, and accelerates redistribution of TLR9 from the ER to the early endosomes in response to CpG-DNA. HMGB1-deficient cells exhibited substantially decreased responses to CpG-DNA, but the defect was complemented by extracellular HMGB1. Thus, the DNA-binding protein HMGB1 shuttles in and out of immune cells, and is important for early endosomal translocation and subsequent activation of TLR9 in response to CpG-DNA.
The Role of Membrane Micro-domains on Target Cells in Control on NK Cell Activity (89.29)
Authors: Gross Catharina Christiane , Long Eric O.
Published: January 2023

Natural killer (NK) cells are large granular lymphocytes that comprise 5–15% of peripheral blood mononuclear cells and function through cytokine production or cytotoxicity. The response of NK cells to contact with other cells is regulated through many different receptor-ligand interactions. Lipid rafts are membrane micro-domains, enriched in glycosphingolipids, sphingomyelins, and cholesterol, which influence receptor signaling in lymphocytes. It is not clear yet to what extent distribution of ligands in micro-domains of target cells controls NK cell responses. To investigate the role of membrane micro-domains on target cells, and to avoid the use of inhibitors such as cyclodextrin, cholesterol-auxotroph insect cells were grown in serum-free conditions to eliminate cholesterol-enriched domains. Insect cells transfected with the GPI-linked CD48 and ULBP1 molecules, ligands for 2B4 and NGK2D respectively, are currently being tested for their ability to activate NK cells.
Estrogen Deficiency Induces Bone Loss in Mice Lacking ITAM Adapter Proteins, DAP12 and FcRg (89.31)
Authors: Wu Y. , Torchia J. , Yao W. , Lane N. E. , Lanier L. L. , Nakamura M. C. , Humphrey M. B.
Published: January 2023

DAP12 and or FcRγ ITAM signals are required during normal osteoclast (OC) development in vivo and in vitro. To determine whether ITAM deficient mice are resistant to bone loss induced by estrogen deficiency, we performed ovariectomy (OVX) or SHAM surgery on 10–12 week female DAP12−/−, FcRγ−/−, DAP12−/−FcRγ−/− and control C57BL/6 (B6) mice. MicroCT of the distal femur showed statistically significant losses of trabecular bone in DAP12−/−FcRγ−/−, DAP12−/−, FcRγ−/− and B6 OVX groups compared to SHAM groups, BV/TV dropping from 56% to 35% (p<0.01), 21% to 12% (p<0.01), 12% to 7% (p<0.01), and 6% to 3% (p<0.05) respectively. Bone turnover measured by serum osteocalcin and urinary DPD increased at 4 weeks post-OVX in all four strains. In vitro OC bone resorption assays revealed a 2.7 or 3.6 fold increase in OC resorption from B6 and FcRγ−/− OVX mice that were not seen in DAP12−/− and DAP12−/−FcRγ−/− OVX OC. FSH, reported to stimulate postmenopausal osteoporosis, combined with RANKL could not rescue the in vitro OC defect in DAP12−/− and DAP12−/−FcRγ−/− cultures. TGB-β followed by TNF-α stimulation induced OC differentiation in B6 cells in vitro without RANKL; however, DAP12−/− and DAP12−/−FcRγ−/− OC cultures were not rescued. We conclude that OC differentiation during estrogen deficiency induced osteoporosis bypasses the requirement of ITAM signaling and the unidentified novel effect is microenvironment dependent.
D-3-deoxy-dioctanoyl-PI induces cell death in the human leukemic monocyte lymphoma cell line U-937 by disruption of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (89.33)
Authors: Blair Derek , Pu Mingming , Wang Yanling K. , Chen Wei , Xu Yingju , Miller Scott J. , Roberts Mary F. , Chiles Thomas C.
Published: January 2023

D-3-deoxy-phosphatidylinositol derivatives have a potentially wide array of targets in the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway. As such, these phosphatidylinositol molecules have been shown to have cytotoxic activity against a variety of human cancer cell lines. Here, we demonstrate that the D-3-deoxy-dioctanoyl-PI (D-diC8PI), but not the L-isomer or D-3,5-dideoxy-diC8PI, is able to induce the death of the U-937 human leukemic monocyte lymphoma cell line, even at low concentrations (<50 μM). In order to further investigate the mechanism of the cytotoxicity of D-3-deoxy-diC8PI we examined its effect on the activity of various molecules in the PI3K/Akt pathway. In vitro, low concentrations (<50 μM) of D-3-deoxy-diC8PI have no significant effect on the activity of PI3K or PTEN. However, incubation of U-937 cells with D-3-deoxy-diC8PI at these concentrations results in significant decreases in phosphorylation of key components of the PI3K/Akt signalling pathway, including the transcription initiation factor eIF4e and the translation factor S6 ribosomal protein. These data further delineate the mechanism of action of such cytotoxic D-3-deoxy-PI compounds.
A novel in vivo siRNA delivery technology for dendritic cell-specific gene silencing and immune modulation (89.5)
Authors: Vladau Costin , Zheng Xiufen , Zhang Xusheng , Li Mu , Suzuki Motohiko , Chen Dong , Garcia Bertha , Min Wei-Ping
Published: January 2023

Silencing immune molecules, such as the costimulatory molecule CD40, using siRNA was shown to have therapeutic potential and promise in immune modulation. However, a major barrier to the clinical application of siRNA is the current lack of an effective and cell-specific delivery system. Herein, we present a new method of selectively delivering siRNA to DC in vivo using stealth immunoliposomes (SILs). CD40 siRNA-containing SILs were generated using 4 types of lipids and decorated with surface-bound, DC-specific mAbs. DC-specific binding capacity of SILs was demonstrated by fluorescence microscopy and flow cytometry. Upon treatment with CD40 siRNA-SILs, DC expression of CD40 was successfully suppressed in vitro. Administration of CD40 siRNA-SILs resulted in DC-specific tissue targeting, as evidenced by increased uptake of fluorescence-tagged siRNA. Additionally, DC from mice treated with CD40 siRNA-SILs exhibited CD40-specific gene silencing in vivo. Tolerogenic properties of DC treated with CD40 siRNA-SILs were determined by inhibition of allogeneic T cell proliferation in MLR. Furthermore, a strong in vivo immune modulation was observed in mice treated with CD40 siRNA-SILs. In conclusion, this is the first demonstration of DC-specific siRNA delivery and gene silencing in vivo, which highlights the potential of DC-mediated immune modulation and the feasibility of siRNA-based clinical therapy.
CpG oligodeoxynucleotide and double-stranded RNA synergize inflammatory cytokine gene expressions in chicken monocytes (90.13)
Authors: He Haiqi , Genovese Kenneth J. , Kogut Michael H.
Published: January 2023

Toll-like receptors (TLRs) recognize microbial components and initiate innate immune responses that control microbial infections. We have investigated the innate immune response of chicken monocytes to CpG-motif containing oligodeoxydinucleotide, CpG-ODN, and the analog of viral double-stranded RNA, poly I:C. Our results show ligands poly I:C and CpG-ODN of the TLR3 and TLR9, respectively, synergized the induction of many genes involving inflammatory responses including nitric oxide and cytokines, IL-1β, IFN-α, IFN-β, IFN-γ, IL-18, and IL-12, in chicken monocytes. We also demonstrated the differential responses of some cytokines such as IL-18 and IFN-β to CpG-ODN and poly I:C stimulations, respectively. Those results demonstrate differential roles for TLR3 and TLR9 in signaling immune responses to bacterial and viral infections in chicken monocytes as well as a synergistic interaction between the two signaling pathways. Furthermore, our results suggest synergistic interaction between TLR3 and TLR9 pathways may contribute the “lethal synergism” from bacterial and viral co-infections.
Activation of OX40 down-regulates IL-17 production (91.10)
Authors: Li Jian , Shang XiaoZhou , Li Lily , Schantz Allen , Bracht Michelle , Benson Jacqueline , Elloso Merle , Orlovsky Yevgeniya , Sweet Ray
Published: January 2023

IL-17 is an important cytokine involved in multiple inflammatory diseases partially through the up-regulation of various pro-inflammatory cytokines and chemokines. The regulation of IL-17 production has been well studied, but the role of OX40 on IL-17 regulation has not been fully illustrated. In this study, we investigated the effect of activation of OX40 on IL-17 production. As expected, stimulation of PHA activated human peripheral blood mononuclear cells (PBMCs) with soluble OX40L increased both Th1 (IFNγ) and Th2 (IL-4 and IL-13) cytokine production. However, addition of soluble OX40L significantly inhibited IL-17 production, and this inhibition was abrogated by IFNγ mAb. IL-23 is a major stimulator for IL-17 and IL-23 enhanced IL-17 production was also inhibited by soluble OX40L in purified human CD4 and CD8 T cells. The downregulation of IL-17 by activation of OX40 was also demonstrated in murine splenocytes from OVA TCR transgenic mice. Blockage of OX40/OX40L interaction with either OX40L antibody or mouse OX40/Fc protein enhanced IL-17 production. In addition, agonistic OX40 antibody inhibited antigen induced IL-17 production and antagonistic OX40 antibody enhanced IL-17 production in splenocytes from OVA transgenic mice. Taken together, our results show that activation of OX40 exerts a negative regulation on IL-17 production. The inhibition is probably mediated through the up regulation of IFNγ production.
Preformed CD40 ligand exists in secretory lysosomes of effector and memory CD4 T cells and is recruited to the cell surface upon antigenic stimulation (91.2)
Authors: Koguchi Yoshinobu , Thauland Timothy J , Slifka Mark K , Parker David C
Published: January 2023

CD40 ligand (CD40L) is essential for the establishment of humoral and cellular immunity. Although its functions are well characterized, little is known about the regulation of its surface expression because of CD40-mediated modulation. Intracellular staining shows that most CD40L in resting, effector CD4 T cells is intracellular (iCD40L). We used a surface mobilization assay, in which the detection antibody is included during stimulation to enhance sensitivity, to show that iCD40L comes to the cell surface within 30min upon stimulation in a cycloheximide-insensitive manner. Microscopic analysis demonstrated that iCD40L is co-localized with Fas ligand but not with CTLA-4 in secretory lysosomes. We also found that LCMV-specific effector and memory Th1 cells have iCD40L, which comes to the cell surface upon antigen recognition. Finally, we observed that both naïve and effector/memory CD4 T cells from unmanipulated animals contain iCD40L, while only effector/memory T cells exhibit rapid surface mobilization of iCD40L. Surface mobilization of iCD40L is observed in T cells from CD40-deficient mice, indicating that CD40 is not required for sorting of CD40L to secretory lysosomes. Our results suggest that effector CD4 T cells can provide CD40-mediated stimulation to antigen bearing APCs by delivering iCD40L during transient interactions.

This work was supported by Grant AI29544 from the NIH.
The Wiskott-Aldrich Syndrome protein (WASp) controls Th2, not Th1, effector function in vivo (91.6)
Authors: Morales-Tirado Vanessa Marie , Katzman Shoshana , Fowell Deborah
Published: January 2023

Signals through the TCR and cytokine receptors cooperate to direct uncommitted CD4+ T cells into Th1 and Th2 cells. The signals required for the acquisition of Th effector function are poorly understood. Remodeling of the actin cytoskeleton is essential for the transduction of signals from external stimuli, translating into morphological changes, polarity and reorganization of proteins for the formation and stabilization of the immunological synapse (IS). The Wiskott Aldrich Syndrome protein (WASp) is a key regulator of the actin cytoskeleton, suggested to be the link between TCR signals and cytoskeletal reorganization. However, it is unclear how these initial signaling events modulate cell fate decisions for effector function.

We investigated the role of WASp during the Th cell differentiation by using WASp−/− CD4+ T cells. We identified two distinct WASp-dependent control points in vitro: regulation of cytokine gene transcription and regulation of targeted cytokine secretion. For Th2 development, we observe defects in signals for sustained IL-4 gene transcription. In contrast, Th1 development is WASp-independent. Although WASp−/− cells contain similar levels of intracellular IFN□ when compared to WT cells, we observe a defect in cytokine polarization to the IS for directed secretion. The final secretion of both Th1 and Th2 cytokines is severely impaired. In vivo the major defect appears to be in Th2 function. The outcome of these studies could provide possible explanations for the pathology observed in human WAS-patients, characterized by eczema, thrombocytopenia and immunodeficiency. NIH T32 AI007362
Lung RNA profiling suggests an essential role for micro RNAs in regulating allergic lung disease (91.9)
Authors: polikepahad sumanth , El-Daye Jad , Naghavi Arash , Miller Jonathan , Gunaratne Preethi , Corry David Brian
Published: January 2023

MicroRNAs (miRNAs) are small non-coding RNAs, which negatively regulate gene expression by translational inhibition of target mRNAs. In vertebrates, miRNAs are produced in most organs, including lungs. In the current study, we hypothesized that the expression of miRNAs would correlate inversely with expression of critically important target genes involved in the pathogenesis of allergic lung disease. After intranasal challenge of mice with a potent allergen derived from Aspergillus oryzae on alternating days for 2 weeks, lungs were isolated, high quality RNA was extracted and subjected to microarray analysis in comparison to naïve lungs obtained in parallel. In allergic lungs, ~ 30 known miRNAs were suppressed and ~100 were induced, including many new miRNA candidates uncovered through genome-wide predictions. Bioinformatics analysis revealed that miRNA 199a*, which was suppressed, and miR-144 which was induced in allergic lungs, are candidate regulators of STAT-6, GATA-3 and costimulatory factors, all of which are critical mediators of allergic lung disease. These studies suggest the existence of many new miRNAs and pivotal role for miRNA 199a* in controlling the expression of allergic lung disease.
Germinal center helper T-cells are dual functional regulatory cells with suppressive activity to conventional CD4+ T-cells (92.1)
Authors: Zheng Biao , Marinova Ekaterina , Han Shuhua
Published: January 2023

Germinal center (GC) reaction is a T cell-dependent process in which activated B cells mature to produce high-affinity antibodies and differentiate into memory B cells. The GC microenvironment is almost exclusively reserved for the optimal antigen-specific B cell clonal expansion, selection and maturation, but lack of significant conventional CD4+ T- cell responses. The mechanisms that ensure such a focused B cell response in the GC are not known. Here we report that human CD4+CD57+T-cells, which are the major helper T-cells in GCs, actively suppress the activation of conventional CD4+ T-cells, particularly Th1 cells, via direct contact-dependent mechanism and soluble mediators. Our findings demonstrate that GC T-cells are unique regulatory cells that provide critical help signals for B-cell response but suppress conventional effector T-cells in the same local environment.
Redistribution of Lipid-rafts to T Cell –Antigen Presenting Cell Contact Is Not a Prerequisite for CD4 T Cell Activation (92.13)
Authors: Yang Wen , Xu Yanfei
Published: January 2023

During T cell activation, lipid-rafts were reported to be polarized in the contact area of T cells and antigen presenting cells (APCs) in the literature. Whether such polarization of lipid rafts is a prerequisite for T cell activation is still unclear. To address this question, we studied the distributions of lipid-rafts in naive murine T cell receptor transgenic CD4 T cells upon engaging APCs presenting peptide antigen, under conditions that the T cells were untreated, pretreated with GM1-binding reagent chorela toxin B (ctB), or pretreated with ctB followed by crosslinking with anti-ctB antibodies (ctB+actB). In consistent with findings reported in the literature, lipid-rafts, as indicated by GM1, were concentrated in the contact sites between untreated T cells and APCs in the presence of antigen. In contrast, GM1 was polarized to the opposite end of ctb+actB-treated T cells, the urope. The redistribution of GM1 to the T-APC contact and/or the urope of T cells was less obvious in the ctB-treated-only T cells, but to the latter was significantly enhanced within minutes of adding the actB to the T cells previously engaged with APCs presenting antigen. However, under these conditions with or without GM1-pretreatments, no detectable inhibition on T cell activation was observed as assayed by secretion of IL-2 and T cell proliferation. Results from this study clearly indicated that redistribution of GM1, a lipid-raft marker, to the T-APC contact site is not essential for CD4 T cell activation.
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

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

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

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

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

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

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

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

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>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 < stably transfected < 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

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

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

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

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

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

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<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

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)
Programming for a CD8 T Cell Memory Response Requires IL-12 and/or Type I IFN (95.23)
Authors: Xiao Zhengguo , Jameson Stephen C , Mescher Matthew F
Published: January 2023

Naïve CD8 T cells proliferate in response to Ag and costimulation, but require signals from IL-12 or IFNα/β to make a productive response. Whether these are the only cytokines with this activity, and their relative contributions, are only beginning to be defined. Response to LCMV infection by CD8 cells lacking the IFNα/β receptor (IFNIR−/−) is reduced > 99%, but response to vaccinia virus (VV) remains largely intact (J.Exp.Med. 202, 637; J. Immunol. 176, 4225). We have studied responses of OT-I T cells to OVA-expressing VV and Listeria monocytogenes (LM), using cells that lack IFNIR, IL-12 receptor (IL-12R−/−), or both (DblKO). Clonal expansion of receptor-deficient OT-I cells to LM-OVA or VV-OVA is comparable to that of wild type (wt) cells, but effector functions are compromised, and their numbers decline more rapidly. IL-12R−/− cells exhibit greater defects than do IFN-IR−/− cells, and DblKO cells are the most compromised. Compared to the wt memory population following VV-OVA infection (> day 30), memory cell numbers are reduced by 27% for IFN-IR−/− cells, 96% for IL-12R−/− cells, and >99% for DblKO cells. For LM-OVA, the reduction in memory is 76% for IFN-IR−/− cells, 97% for IL-12R−/− cells, and >99% for DblKO cells. Thus, both IL-12 and IFNα/β contribute to responses to VV and LM, with IL-12 making the greater contribution, and programming for CD8 T cell memory requires signals from one or the other of these cytokines.