bluuebunny/crossref_metadata_2025 huggingface dataset:
Delta-like ligand 4 regulation of effector T cell function during experimental autoimmune encephalomyelitis (96.8)
Authors: Reynolds Nathan , Lukacs Nicholas , Karpus William
Published: January 2023

Notch signaling mediates cell fate decisions through specific ligand-receptor binding. Signaling by Delta-like ligand 4 (DLL4) influences both CD4+ Th1 and Th17-associated cytokine production and contributes to adaptive immunity. Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease of the central nervous system (CNS) that is mediated by autoreactive Th1 and Th17 CD4+ T cells. We examined the contributions of DLL4 in regulating Th1 and Th17 functions during EAE. CNS-infiltrating myeloid dendritic cells, macrophage and resident microglia express high levels of DLL4 during EAE. Blocking DLL4 during antigen-restimulation of primed T cells resulted in decreased inflammatory cytokine production by these T cells. Administration of anti-DLL4 to PLP139-151-primed mice decreased EAE severity. Similarly, pretreatment of PLP139-151-specific T cells with anti-DLL4 reduced adoptive disease transfer. Treatment of mice with anti-DLL4 following adoptive transfer of primed T lymphocytes blocks localization of these T cells to the CNS and prevents disease. These results suggest DLL4-Notch signaling regulates autoimmune T cell effector function and localization to the CNS. (Supported by NMSS RG4146-A-4)
Tannic acid and quercetin display a therapeutic effect on atopic dermatitis via suppression of angiogenesis and Th2 polarization. (97.7)
Authors: Jung Min Kyung , Cho Dae-Ho
Published: January 2023

Atopic dermatitis (AD) is a chronic inflammatory skin disease that is characterized by inflammation, abnormal angiogenesis and elevated Th2 polarization. Tannic acid (TA) and quercetin are well known polypenolic compounds, have documented anti-inflammatory effect. However, little is known about the effects of TA and quercetin on atopic dermatitis and related mechanisms. The present study investigated whether TA and quercetin had a therapeutic effect on atopic dermatitis. In vitro, we found that TA and quercetins reduced pro-angiogenic factor and Th2-inducing cytokine in human keratinocytes. The inhibitory effect was higher in the combined application of the two agents than in the application of one agent alone. To investigate the effect of combined application with TA and quercetin in vivo, an AD-induced Nc/Nga mouse model was used. The group receiving TA and quercetin showed disease improvement with down-regulation of serum IgE, TARC production and angiogenesis level compare to non-treated group. Taken together, these data demonstrate that TA and quercetin have a therapeutic effect on AD by suppressing angiogenesis, Th2 polarization.
Dendritic Cell subsets: Functional analysis and TLR Phenotyping (98.13)
Authors: Rosenberg Jonathan , Chitta Sriram , Singh Gita , Quintel Payton , Lee Hyun-Ku , Singh Sujay
Published: January 2023

In-depth analysis of subtypes of Dendritic Cells (DCs), both myeloid (mDC) and plasmacytoid (pDC) subsets, has been developed through Toll-like Receptor (TLR) Phenotyping by Flow Cytometry. Sets of TLR specific mAbs are combined with cell lineage and DC markers like CD11c and CD123 for mDC and pDC, in a simple assay for consistent DC analysis. DC subsets in peripheral blood differ in origin, phenotype, and function as analyzed by cytokines, chemokines or pro-inflammatory factors produced upon interaction with pathogens. DC response to invading pathogens depends upon expression of a spectrum of receptors, such as TLRs, which are expressed on the cell surface and in intracellular endosomes. TLR signaling in DC subtypes results in production of cytokines, chemokines and pro-inflammatory factors that elicit and control adaptive immune responses. TLR Phenotyping analysis includes development of 4 color Flow Cytometry assay cocktails for TLR expression of DCs to characterize mDC or pDC in human peripheral blood. In combination with activation and functional markers such as cytokines or chemokines the assay system provides a new research tool to identify changes in TLR expression in steady state or pathogen infected conditions and during differentiation of monocytes, DCs, B cells/B cell lines (EBV transformation), or T cells. The approach also provides additional information on TLR signaling and downstream pro-inflammatory mediator production.
LAB/NTAL regulates zymosan-mediated inflammatory responses in Bone Marrow Derived Dendritic Cells (BMDC) (98.14)
Authors: Orr Selinda , Quigley Laura , Roessler Stephen , Hodge Deborah , Razzook Catherine , Cardone Marco , Zhang Weiguo , Trinchieri Giorgio , Lyakh Lyudmila , McVicar Daniel
Published: January 2023

Immune sensing of fungi occurs via multiple pathogen recognition receptors such as Dectin-1/2, mannose receptor and TLR2. The β-glucans and mannans in fungal cell walls activate these receptors. Stimulation of DCs via TLR2 results in the production of IL-10 and little IL-12, while Dectin-1 utilizes a hemi-ITAM (immunoreceptor tyrosine based activation motif) and Syk to facilitate IL-23 production leading to Th17 polarization. We have recently shown that the linker for activation of B cells/Non-T cell activating linker (LAB/NTAL), a known regulator of ITAM-mediated signaling, is expressed in BMDCs. Here we examine whether LAB regulates the response of BMDC to zymosan by affecting Dectin-1 signaling. We find that LAB, Syk, Cbl, MAPK and Akt are rapidly and transiently phosphorylated following zymosan stimulation. Moreover, zymosan stimulated Lat2-/- BMDC produced decreased IL-10 and IL-12p40 and increased TNF levels suggesting the involvement of LAB in regulating BMDC cytokine production. Unexpectedly, direct engagement of Dectin-1 by purified β-glucans failed to elicit LAB phosphorylation. Interestingly, water-soluble components of zymosan induced phosphorylation of FcRγ, Syk, and LAB. Indeed, purified yeast-derived mannans also stimulated phosphorylation of Syk and LAB in BMDC. These data indicate that LAB regulates the BMDC inflammatory response to fungal pathogens not by affecting Dectin-1 signaling but by regulating mannan-derived signals likely through Dectin-2.
The role of surface charge in cationic liposome-induced antigen-presenting cell (APC) activation and viability (98.21)
Authors: Ma Yifan , Xie Xiaofang , Zhuang Yan , Zhou Dongmei , Cai Lintao
Published: January 2023

Cationic liposomes, an effective carrier for drugs and gene delivery, have recently been reported as a potential vaccine adjuvant. Since the immuno-stimulatory effect of cationic liposomes may be related to their surface charge, we hereby investigated the effect of liposome charge on antigen-presenting cell (APC) activation and viability. The charge of cationic liposomes was adjusted by incorporating into DOTAP (a cationic liposome) variable amounts of DOPC (a neutral lipid). DOPC by itself did not affect monocyte activation or DC maturation. The DOTAP/DOPC liposomes with molar ratios of 4:1, 3:2 or 2:3 not only elevated CD86 on monocytes and induced the production TNF-alpha, but also significantly augmented the expression of CD83 and CD86 on both human and mouse dendritic cells (DCs). However, further increasing the proportion of DOPC significantly attenuated monocyte activation and DC maturation induced by DOTAP/DOPC liposomes. Interestingly, although DOTAP had a higher zeta potential than the DOTAP/DOPC liposome (4:1), it was less potent in inducing monocyte activation and TNF-alpha production. Moreover, the treatment of monocytes with DOTAP for 24 hr caused about 40% apoptosis, while the presence of DOPC significantly increased monocyte viability. Hence, an appropriate surface charge is crucial for enhancing the adjuvant effect of cationic liposomes and reducing their potential cytotoxicity.
Molecular mechanisms of stress-induced glucocorticoid resistance in splenic macrophages: A role for p38 MAPK (98.22)
Authors: Stiner LaTonia , Sheridan John
Published: January 2023

Stress has been shown to increase production of glucocorticoids (GC’s), which suppress the inflammatory response. However, the increased levels of circulating GC’s observed following social disruption stress (SDR) fail to suppress cytokine production or induce apoptosis in splenic CD11b+ cells from WT mice, suggesting that these cells are GC resistant. Moreover, CD11b+ cells from IL-1R1 knockout (KO) mice are not GC resistant following SDR. The results of the present study show that following LPS and corticosterone (LPS/cort) stimulation, CD11b+ cells from WT SDR mice displayed an increase in cytoplasmic p38 phosphorylation (p-p38) when compared to controls. The increase in p-p38 was associated with an increase in nuclear translocation of the GC receptor phosphorylated at Ser234 (GR-pS234), within SDR mice compared to controls. Studies conducted with p38 and JNK inhibitors revealed that a reduction in p38 activity led to an increase in JNK activation and that cytoplasmic GR-pS234 was maintained in SDR and control mice. Furthermore, p-p38 was notably absent in KO macrophages from SDR and control mice stimulated with LPS/cort. These data confirm that SDR enhances GR-pS234 and JNK may facilitate this phosphorylation in the absence of p-p38. These data also support a role for p38 in induction of GC resistance.
Expansion, mobilization, and circulation of myeloid derived cells in mammary tumor bearing (TB) mice. (98.27)
Authors: Talmadge James , Younos Ibrahim , Dafferner Alicia , Samson Holly , Westphal Sherry , Fuminori Abe
Published: January 2023

MDSCs are increased in the marrow (BM), lungs, liver, and tumors of TB hosts as compared to non-TB (NTB) mice. In vivo, BrdU labeling of leukocytes from 4T1 mammary TB mice revealed that MDSCs had an increased rate of proliferation in BM, spleen, lungs, and liver. MDSCs are observed in the highest frequency in the blood and spleen, suggesting hematopoietic progenitors mobilized from BM and proliferated following arrest. We assessed the biodistribution of circulating cells using CFSE-labeled spleen cells from TB mice, adoptively transferred to normal mice by iv injection and rapidly arrested in the lungs. In contrast, CFSE-labeled MDSCs injected into 4T1 TB mice transversed the lungs, briefly arrested in the liver, and were retained in BM and tumors. We posit that in 4T1 TB mice, the normal arrest in the pulmonary capillary bed is prevented by increased levels of nitric oxide from activated MDSCs resulting in capillary vasodilation and reduced arrest. In 4T1 TB mice, CD3+ T-cells are decreased, although the MFI of CFSE suggests proliferation. Annexin V staining revealed a high frequency of apoptotic CD3+ T-cells, associated with MDSC accumulation. In summary, MDSCs proliferate in lymphoid and parenchymal organs resulting in T-cell apoptosis possibly associated with activation-induced cell death. Further, although MDSCs proliferate in many organs, they rapidly circulate preferentially homing to the spleen and liver.
SHP-1 is a central regulator of dendritic cell function (98.3)
Authors: Ramachandran Indu , Song Weitao , Levitt Jonathan
Published: January 2023

Dendritic cells (DCs) are specialized antigen presenting cells that initiate T cell responses upon activation by pathogen. Once the pathogen is cleared, DCs regulate excessive T cell responses by turning on intrinsic inhibitory signals, the mechanisms of which are not entirely understood. SHP-1 is an inhibitory phosphatase that can deactivate signaling from a wide range of activating receptors such as antigen, cytokine and chemokine receptors in lymphocytes and macrophages. Despite our vast knowledge of the role of SHP-1 in several immune cell types, the specific role of SHP-1 in DCs remains unclear. To address the role of SHP-1 in DCs, we modulated SHP-1 activity in primary mouse bone-marrow DCs by using RNAi, chemical inhibition and SHP-1 functional mutants. We showed that in DCs, SHP-1 inhibited MAPK, NFκB, and AKT activity and lymph node (LN)-chemotactic receptor CCR7 expression, but had no effect on DC maturation. Consequently, SHP-1 inhibition lead to enhanced DC proinflammatory cytokine production, survival and LN migration. In vivo, vaccination with SHP-1-inhibited DCs lead to the expansion of tumor-specific cytotoxic T cells and reduction in FOXP3+ regulatory T cells resulting in effective anti-tumor immunity against mouse melanoma and prostate tumors. Collectively, these data demonstrate that SHP-1 functions as a central inhibitor of DC signaling and DC-induced T cell responses.
Establishment of central tolerance against blood-borne antigens by thymic Sirpα+ conventional dendritic cells (98.5)
Authors: Baba Tomohisa , Mukaida Naofumi
Published: January 2023

Intrathymic negative selection and differentiation of regulatory T (Treg) cells are a cardinal event in central tolerance system. Thymic dendritic cells (DCs) are presumed to be capable of inducing the apoptosis of autoreactive T progenitor cells and of alternatively differentiating them into Treg cells. In spite of the presence of heterogenous DC subsets in thymus, similarly observed on other lymphoid organs such as lymph nodes and spleen, the role of each intrathymic DC subset in the central tolerance is still unclear. Recently, we demonstrated that mice deficient in a CC chemokine receptor, CCR2, exhibited a selective diminution in CD11c+CD11b+CD8-Sirpα+ conventional DC (cDC) subset in the thymus with an accumulation of T cells with reactivity against serum self-antigen in the periphery. Here, we further revealed that Sirpα+ cDCs in the thymus can selectively capture blood-borne antigens due to their unique intrathymic localization nearby small vessels and inside perivascular regions, and then induce the antigen-specific Treg cells and subsequent clonal deletion to an intravenously injected antigen, depending on the intensity of antigen presentation. Finally, we established a novel way to in vivo expand antigen-specific Treg cells via intrathymic antigen presentation by thymic Sirpα+ cDCs, which can dampen aberrant immune reaction in an antigen-specific manner.
Investigating signaling downstream of SLAM family members CD84 and Ly108 in thymocytes and activated T cells (99.4)
Authors: Ghai Mala , Cannons Jennifer , Schwartzberg Pamela
Published: January 2023

The SLAM family consists of 6 immunoregulatory receptors including CD84 and Ly108. SLAM receptors are expressed on T, B, NKT cells, DCs and macrophages and are self ligands that have intracellular binding sites for the adaptor molecule SAP. SAP recruits and activates the FynT kinase, resulting in signaling leading to cytokine production, cytotoxicity, enhanced cellular interactions and thereby immune regulation. Defective SAP expression is associated with the disease XLP, characterized by dysregulated hyperimmune response to EBV infection and impaired humoral responses. Both CD84 and Ly108 are highly expressed on developing thymocytes and activated T cells, including Tfh cells, which are important for germinal center responses. We have found that CD84 affects GC formation and that CD84 and Ly108 participate in T:B cell interactions. SAP and Ly108 signaling is also implicated in development of innate T cells, like NKTs. We find that Ly108 phosphorylation varies in different genotypes of mice. While thymi of SAP-/- mice, that lack NKT cells, demonstrate reduced pLy108, thymi from Itk-/- mice that develop increased numbers of innate T cells, demonstrate increased pLy108. As development of this increased innate T cell population in Itk-/- mice requires SAP, we hypothesize that SAP-associated Ly108 may be contributing to their development. We are further characterizing biochemical and cellular properties of these receptors, given their roles in these important T cell lineages.
Dec2 promotes Th2 cell differentiation by enhancing IL-2R signaling (99.6)
Authors: Sun Bing , Liu Zhiduo
Published: January 2023

Helper T cell (Th cell) differentiation is precisely regulated by thousands of genes at different stages. In the present study, we demonstrate that Dec2, a transcription factor belonging to the bHLH (basic helix-loop-helix) superfamily, is progressively induced during the course of Th2 differentiation, especially at the late stage. The up-regulated Dec2 can strongly promote Th2 development under Th2-inducing conditions, as evidenced by retrovirus-mediated gene transfer or transgenic manipulation. In addition, an enhancement of Th2 responses is also detectable in Dec2 transgenic mice in vivo. Conversely, RNAi-mediated suppression of endogenous Dec2 could attenuate Th2 differentiation. Finally, we show that the enhanced Th2 development is at least in part due to substantial up-regulation of CD25 expression elicited by Dec2, thereby resulting in hyperresponsiveness to IL-2 stimulation.
Gamma-interferon-inducible lysosomal thiol reductase diminishes cathepsin S expression and function (100.27)
Authors: Phipps-Yonas Hannah , Semik Vikki , Hastings K.
Published: January 2023

MHC class II-restricted processing requires protein degradation in the endosomal pathway for activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates the processing of class II-restricted antigens by reducing protein disulfide bonds. Our previous work demonstrates that reductase active site mutants of GILT have diminished processing of precursor GILT to mature GILT, suggesting that GILT’s reductase active site may decrease the expression or activity of lysosomal proteases responsible for cleavage of GILT’s N- and C-terminal pro-peptides. Cathepsin S (CatS) is capable of processing precursor GILT to its mature form and mediates essential steps in class II-restricted processing, including proteolysis of large polypeptides and invariant chain cleavage. Therefore, we sought to determine whether GILT’s reductase activity regulates CatS expression and function. Our studies show GILT and CatS colocalize within lysosomes of murine B cells. GILT expression acts post-transcriptionally to decrease CatS steady state protein expression. GILT’s reductase active site is necessary for diminished CatS protein levels. However, GILT does not substantially alter the steady state protein expression of other lysosomal proteins, H2-M, H2-O or CatL. In addition, GILT expression decreases proteolysis of a CatS-selective substrate. Thus, GILT’s reductase activity is likely to fine tune class II-restricted antigen processing through decreasing CatS activity.
Distinct spatial localization of resident dendritic cell subsets in lymph nodes (100.30)
Authors: Gerner Michael , Ifrim Ina , Kastenmuller Wolfgang , Germain Ronald
Published: January 2023

Dendritic cells (DCs) are essential for uptake and presentation of antigens (Ags) for T cell activation and can be divided into subsets with varying Ag-presentation capacities. It is not clear whether intrinsic differences or variation in spatial localization and preferential Ag access both contribute to differential Ag-presentation by DC subsets. We thus developed a novel approach of combining multi-parameter (6 or 7 color) confocal microscopy with analytical masking to allow direct in-situ visualization of cellular populations identified by complex phenotypic surface markers. We determined the spatial localization of major resident lymph node (LN) DC subsets and observed preferential positioning of certain populations to distinct LN zones. Some DCs physically associated with lymphatic sinuses (LS) and extended dendrites directly into the LS. While small protein Ags can be directly delivered to resident DCs via LN conduits, particulate Ags are thought to be presented via migratory DCs arriving from peripheral sites. Using 2-photon microscopy, we observed the LS-associated DCs actively sampling the LS for particulate Ags and the formation of T cell clusters in zones proximal to the LS after particulate Ag injection. These results suggest the existence of specialized micro-domains for DC subsets within LN and suggest that LS-associated DC are important for T cell activation to particulate Ags. This research was supported by the Intramural Research Program of the NIH, NIAID.
LTβR signaling in Dendritic Cells induces a Type I IFN response that is required for optimal clonal expansion of CD8+ T cells (100.40)
Authors: Ng Dennis , Summers deLuca Leslie , Gao Yunfei , Wortzman Michael , Watts Tania , Gommerman Jennifer
Published: January 2023

During an acute infection, dendritic cells (DC) are triggered by pattern recognition receptors (PRR) to produce inflammatory molecules that are important for the expansion and function of CD8+ T lymphocytes in order to clear the infection. In contrast, tumours or autoimmune responses against tissue antigens produce very little inflammation, yet the immune response can proceed in the absence of obvious PRR activation. In these situations, DC require CD4+ T cell help to initiate CD8+ T cell-mediated immunity. Here we generated mixed chimeric mice lacking Lymphotoxin-β receptor (LTβR) specifically on the DC, and we found that they exhibit reduced CD8+ T cell expansion in a help-dependent response. In contrast, inhibition of the CD40 signaling only impaired CD8+ T cell interferon-γ (IFN-γ) production. Furthermore, we have identified that LTβR signaling in DC triggers IFN-α/β expression in the absence of any PRR ligand and is critical for antigen specific CD8+ T cell expansion. Therefore, this study demonstrates that different TNF family members provide integrative signals that shape the licensing potential of antigen-presenting DC.
Inhibition of antigen cross-presentation by intravenous immunoglobulins (100.41)
Authors: Bazin Renée , Trepanier Patrick
Published: January 2023

Intravenous immunoglobulins (IVIg) are therapeutic preparations of human IgG used to treat primary and secondary immunodeficiencies. In addition, IVIg exert anti-inflammatory effects in several autoimmune disorders, and the constant increase in their use exposes providers and patients to considerable risks of shortage. Understanding their numerous and complex mechanisms of action is required to develop appropriate substitutes. Our research group recently demonstrated that IVIg inhibited the in vitro and in vivo activation of CD4 T cells following interaction with dendritic cells (DC) (Aubin et al, Blood 2010). In the present work, we investigated whether IVIg could also interfere with the activation of CD8 T cells, which contribute to the persistence and severity of certain autoimmune conditions. C57BL/6 bone marrow-derived DC were treated with IVIg and used to cross-present ovalbumin to fluorescently labelled ova-specific OT-I CD8 T cells. Cell activation was assessed by measuring T cell fluorescence distribution by flow cytometry. Results showed that IVIg-treated DC were significantly less efficient in activating CD8 T cells. The inhibition correlated with a decrease in IL-2 and IFN-y pro-inflammatory cytokine secretion. These results support a novel immunomodulatory mechanism by which IVIg decrease DC capacity to cross-present antigens to CD8 T cells. The molecular mechanisms of this inhibition and its importance in vivo are currently being investigated.
Elf3 plays a role in regulating dendritic cell maturation and function (100.42)
Authors: Kushwah Rahul , Wu Jing , Oliver Jordan , Hu Jim
Published: January 2023

Elf3 belongs to the family of epithelium-specific Ets transcription factors and can be upregulated in non-epithelial cells upon cytokine stimulation. Among Ets family transcription factors, Elf3 is most highly induced during inflammation, yet its function is not well understood. Here we demonstrate an important role of Elf3 in regulating dendritic cell (DC) function. Intraperitoneal injection of ovalbumin (OVA)-Alum to Elf3-/- mice resulted in increased migration of DCs to the peritoneal space along with hypermaturation followed by increase in subsequent migration of DCs to the draining lymph nodes. In vitro cultured Elf3-/- DCs underwent spontaneous maturation in absence of any inflammatory stimuli and upon exposure to inflammatory stimuli underwent hypermaturation compared to Elf3+/+ DCs, indicating a role of Elf3 in regulation DC maturation. Elf3-/- mice had normal T cell development but mounted an exaggerated Th2 response upon OVA exposure. Naïve T cells cultured with Elf3-/- DCs showed impairment in Th1 and Th17 differentiation with an increased propensity for Th2 differentiation. This was mediated by an impairment of IL-6 and IL-12 production by Elf3-/- DCs. The impairment of IL-6 and IL-12 production was due to impairment of IL-6 and IL-12p40 gene induction in Elf3-/- DCs in response to inflammatory stimuli. Impairment of Th1 differentiation promoted Th2 differentiation. Taken together, our findings identify a role of Elf3 in regulating DC maturation and function.
Identification of a novel motif important for the activity of ring-type E3 ligases using MARCH1 as a model (100.52)
Authors: Bourgeois-Daigneault Marie-Claude , Thibodeau Jacques
Published: January 2023

The MARCH family of proteins are RING-CH type E3 ubiquitin ligases responsible for the ubiquitination and endocytosis of cell surface proteins related to immune response. We are particularty interested in MARCH1 and 8, two members of the family that are known to downregulate the cell surface expression of MHC II, CD86 , TfR and CD95. Canonical sorting motifs in the cytoplasmic portions of murine MARCH1 and human MARCH8 were identified and reported by others to be necessary for the function. We hypothesized that such motifs in human MARCH1 are important for it’s function. Methods: We used co-immunoprecipitations and FRET to monitor interaction of MARCH1 with various proteins. Protein expression, function and localization were determined by flow cytometry, confocal microscopy and western blotting. Results: Our results showed the presence of functional sorting motifs in both N- and C-terminal cytoplasmic portions of human MARCH1. However, these signals are not essential for the activity of MARCH1. We used a series of mutants to identify functional regions on MARCH1. Our results point to a novel motif. Interestingly, that motif is found in many other proteins and appears to be important for the function of other RING-type E3 ligases. Conclusions: Our results confirmed the presence of sorting motifs in MARCH1 and highlight important species-specific functional domains. The newly identified domain of MARCH1 will shed light on the structure of RING-type E3 ubiquitin ligases.
Structural basis of HLA-DO inhibition of HLA-DM catalyzed peptide exchange on MHC class II (100.53)
Authors: Guce Abigail , Mortimer Sarah , Mellins Elizabeth , Karlsson Lars , Stern Lawrence
Published: January 2023

HLA-DO (DO) is a non-polymorphic, non-classical MHC class II molecule that localizes to the lysosome with HLA-DM (DM). DO is known to inhibit MHC class II peptide loading mediated by DM. The mechanism of inhibition by DO as well as the interactions between DO and DM remain unknown. Here, we present the three-dimensional structure of the DM and DO complex determined by x-ray crystallography at 3.2 Å resolution which is also the first resolved structure of DO. DO exhibits close structural similarity to the MHC class II molecule, HLA-DR (DR), with an rmsd of 0.8 Å2 despite only 25% sequence homology.The crystal complex shows DO residues mainly at the N-terminal side of the α1 peptide-binding groove interact with DM residues located at a concave surface. Some interactions are also observed involving DO residues from the IgG-like domain . DM concave surface includes an acidic patch where residues in and surrounding this acidic patch and residues in the IgG-like domain interact with DO. Interestingly, the interaction surface on DM to DO is spatially close to DM’s interaction surface for DR that was suggested by molecular modeling and mutagenesis studies. The novel structure of the DM/DO complex presented here provides an insight on the possible mechanism of inhibition of DO on DM as well as the mechanism of how DM catalyzes peptide loading to peptide free DR.
Structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase ERAP1 (100.57)
Authors: Nguyen Tina , Chang Shih-Chung , Evnouchidou Irini , York Ian , Rock Kenneth , Goldberg Alfred , Stratikos Efstratios , Stern Lawrence
Published: January 2023

ERAP1 trims antigen precursors to fit into MHC class I proteins. To perform this function, ERAP1 has unique substrate preferences, trimming long peptides while sparing shorter ones. To identify the structural basis for ERAP1’s unusual properties, we determined the X ray crystal structure of human ERAP1 bound to bestatin. The structure reveals an open conformation with a large interior compartment. An extended groove originating from the enzyme’s catalytic center can accommodate long peptides and has features that explain ERAP1’s broad specificity for antigenic peptide precursors. Structural and biochemical analysis suggest a mechanism for ERAP1’s length-dependent trimming activity, whereby binding of long but not short substrates induces a conformational change with reorientation of a key catalytic residue towards the active site. ERAP1’s unique structural elements suggest how a generic aminopeptidase structure has been adapted for the specialized function of trimming antigenic precursors.
Conformation and solvent-accessibility of an MHC-I alpha-1 domain segment provide insight into the peptide receptive transition state (100.6)
Authors: Mage Michael , Wang Rui , Boyd Lisa , Revilleza Maria , Natarajan Kannan , Hansen Ted , Margulies David
Published: January 2023

MHC class I (MHC-I) molecules bound to antigenic peptides form the molecular structures recognized by MHC-I-restricted CD8+ T lymphocytes. On binding an antigenic peptide, the newly synthesized MHC-I sequesters a previously exposed region of the alpha-1 domain including residues 46-52, is released from tapasin, and proceeds to the cell surface. However, the changes in shape of MHC-I on peptide binding and release from tapasin remain unclear. We have determined four X-ray structures (from 1.64Å to 1.87 Å resolution) of an overlapping family of peptides from the H-2Ld alpha-1 domain (including residues 46-52) bound to the Fab fragment of 64-3-7, a mAb that reacts with peptide-receptive (PR) but not with peptide-loaded (PL) H-2Ld MHC-I. Positional differences of backbone and sidechain atoms of this alpha-1 domain segment, compared to the same segment in PL H-2Ld, indicate a significant conformational change on peptide loading. Among other changes, the side chain of the invariant Trp51 residue, and of Met52, although buried in PL MHCI, form extensive noncovalent bonds with mAb 64-3-7, indicating an external (solvent exposed) location in PR MHC-I. Furthermore, the 3(10) helix present in this segment of PL MHC-I is also present in the free peptide bound to MAb 64-3-7, and hence in the PR molecule. These findings help to delineate and provide insight into the conformational changes in MHC-I that accompany peptide loading. Supported by the intramural research program of the NIAID.
The role of immunoproteasomes in antigen presentation and thymic selection (100.7)
Authors: Kincaid Eleanor , Che Jenny , Welsh Raymond , Karow Margaret , Murphy Andrew , Valenzuela David , Yancopoulo George , Rock Kenneth
Published: January 2023

Proteasomes, multisubunit complexes that degrade cellular proteins, are instrumental in forming epitopes for MHC class I antigen presentation. An alternate set of catalytic proteasome beta subunits are induced by IFN-gamma and expressed constitutively in dendritic cells, T-cells and B-cells. Immunoproteasomes containing these inducible subunits have increased chymotryptic and tryptic activities and decreased caspase-like activity, and are hypothesized to produce more peptides suitable for binding to MHC class I. To date mutant mice lacking one or two of the immunoproteasome subunits have been generated and have revealed some changes in MHC class I antigen presentation. However, the overall role of the immunoproteasome is unclear because mice that completely lack all three subunits have not been produced up until now. We have generated mice that completely lack immunoproteasomes. These animals have reduced presentation of MHC class I-presented peptides, and the peptides that are presented appear to be substantially different than those on cells from wild type mice. Moreover, the phenotype of these animals is distinct from mice lacking any individual immunoproteasome subunit. The loss of immunoproteasomes also affects T cell development in the thymus. The phenotype of these animals will be discussed.
Enhanced IL-9 receptor (IL-9R) expression in CNS resident cells in the pathogenesis of EAE (101.31)
Authors: Ding Xiaoli , Yan Yaping , Li Ke , Zhou Fang , Zhang Guang-xian , Rostami Abdolmohamad
Published: January 2023

IL-9, which was originally identified in mice as a T cell growth factor, has attracted renewed interest because of the identification of its expression by multiple T helper cell subsets, including Th2, Th9, Th17 and regulatory T cells. It has been reported that IL-9 functions through activating its receptor to promote the cross-phosphorylation of JAK1 and JAK3, leading to the activation of STAT1, STAT3 and STAT5 and upregulating the transcription of IL-9-inducible genes. It remains unknown whether IL-9, in addition to being a key mediator of encephalitogenicity of Th17 and Th9 cells, directly affects CNS cells. In this report, we show that IL-9R expression was significantly increased in brain and spinal cord during the development of experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS). The expression pattern increased with disease severity, and expression was highest at disease peak. Immunocytochemical staining showed that IL-9R was expressed by neurons, astrocytes, microglia and oligodentrocyte progenitor cells (OPCs). Interestingly, except for neurons, IL-9R expression in astrocytes, microglia and OPCs was significantly induced during EAE. These results suggest that IL-9 may directly exert its effect on CNS residential cells and therefore that manipulating IL-9 responsiveness in CNS cells will have important implications for treatment of EAE/MS.
β-catenin as a diagnostic marker and therapeutic target for the treatment of Type I diabetes (101.33)
Authors: Zirnheld Arin , Parnell Sarah , Kosiewicz Michele , Alard Pascale
Published: January 2023

We have found that NOD BMDC exhibit overexpression of β-catenin when compared to cells from diabetes-resistant B6g7 mice. We have found that the high levels of β-catenin in NOD BM-DC lead to the excess inflammatory cytokine production, especially that of IL-12, observed in Type I diabetes. The role of β-catenin in the development of diabetes and the mechanism by which it accumulates in NOD BM-DC remains to be determined. We have examined the cellular location and level of expression of the protein, the stages of disease at which it is overexpressed, its rate of degradation, its interactions with E-cadherin, and the impact of a β-catenin inhibitor on disease incidence and blood glucose levels. Our data show that NOD DC exhibit excess β-catenin expression in both whole cell and nuclear fractions, even before disease onset. We have found that NOD β-catenin is less rapidly degraded at early time points than that from B6g7 cells, suggesting that a defect in proteasomal degradation may allow for the accumulation of β-catenin. Additionally, we have found that β-catenin in NOD cells precipitates less E-cadherin, suggesting there may be a defect in the interaction between β-catenin and E-cadherin at the cell membrane. Finally, quercetin, a β-catenin inhibitor, appears to reduce disease development in NOD mice. These data point to the importance of β-catenin as a potential diagnostic marker and therapeutic target for reducing the inflammation that leads to Type I diabetes.
On the role of IL-1β and CNS myeloid cells in EAE pathogenesis (101.34)
Authors: Ronchi Francesca , Reboldi Andrea , Basso Camilla , Lanzavecchia Antonio , Sallusto Federica
Published: January 2023

IL-1β has been shown to play a role in several inflammatory disorders including mouse experimental autoimmune encephalomyelitis (EAE). We found that mice deficient for the apoptosis-associated speck-like protein containing a caspase recruitment domain, or ASC, did not develop EAE following immunization with MOG in CFA and pertussis toxin (PT). Autoreactive T cells were primed in wild-type (WT) and ASC-/- mice but expanded poorly in ASC-/- mice with reduced capacity to produce simultaneously several inflammatory cytokines, such as IL-17, IL-22, IFNγ, and GM-CSF. The induction of these T cells in WT mice but not ASC-/- mice was dependent on the presence of PT. PT was found to induce IL-1β secretion from antigen-presenting cells, such as DCs and microglia. These data suggest that the disease-inducing effect of PT may be due to its ability to induce production of IL-1β which in turn triggers differentiation of pathogenic multifunctional T cells. In addition to its effect in the induction phase, we are testing whether IL-1β, or other inflammasome-related cytokines, may be required in the effector phases of EAE. We found that ASC-/- mice are resistant to passive EAE induction. In addition, bone marrow chimera experiments of mice transplanted with bone marrow cells from WT mice or ASC-/- mice, showed that IL-1β production by microglial cells, CNS-resident myeloid cells and CNS-migrating inflammatory monocytes is important for EAE pathogenesis.
CD40 on T cells interacts with CD154 to promote autoimmune diabetes (101.7)
Authors: Baker Rocky , Mallevaey Thierry , Gapin Laurent , Haskins Kathryn
Published: January 2023

Type 1 diabetes is caused by the autoimmune destruction of pancreatic beta cells mediated by self-reactive CD4 T cells. Our objective in this study was to determine how CD40 on autoreactive CD4 T cells contributes to their pathogenicity. BDC-5.2.9 is a highly diabetogenic CD4 T cell clone and expresses the costimulatory molecule CD40 which can interact with CD154, also expressed on T cells. To investigate signaling through CD40 on T cells, we produced a variant of the diabetogenic T cell clone BDC-5.2.9 expressing a dominant-negative (DN) form of the CD40 molecule. In vitro, we demonstrated that activated T cells express both CD40 and CD154 on their surface and that these two molecules interact when on the same cell. Engagement of CD154 on activated T cells with CD40 on resting T cells resulted in activation of bystander CD40-positive T cells. Transactivation did not occur when clones were transduced with CD40DN, indicating that CD40 engagement on T cells was necessary for transactivation. Furthermore, when the CD40DN variant of BDC-5.2.9 was tested in vivo, none of the recipients developed diabetes. Ex vivo analysis of the pancreatic infiltrate after transfer of BDC-5.2.9 CD40DN revealed an overall reduction of cytokines produced by T cells in addition to decreased activation and function of recruited macrophages. Our data suggest that targeting CD40 expressed on T cells could be a potential avenue for immunotherapy in T1D.
A novel final step in leukocyte extravasation (102.19)
Authors: Hyun Young-Min , Sumagin Ronen , Sarangi Pranita , Overstreet Michael , Baker Christina , Fowell Deborah , Sarelius Ingrid , Kim Minsoo
Published: January 2023

The real-time dynamics of cell surface adhesion receptor reflect leukocyte migration and trafficking in lymphoid and non-lymphoid tissues, providing valuable insight into the evolving events of the immune response. Despite notable advances in studying leukocyte migration in simplified systems in vitro, live imaging of subcellular adhesion complexes expressed at physiological density in intact tissues has been challenging. To this end we generated a knock-in mouse with CD18 fused to CFP. To enable imaging of CD18 dynamics during transendothelial migration of T lymphocytes, monocytes, and neutrophils, we used multiphoton-intravital microscopy and enhanced three dimensional detection of the fluorescent CD18 fusion protein. These in vivo analyses and parallel experiments in vitro show that extravasating leukocytes become extremely elongated before completing transmigration across the endothelium and subsequent retraction of the stretched uropod from the basolateral side of the endothelial cells takes longer than 20 min, occasionally leaving CD18-enriched microparticle at the basement membrane. Experiments with knock-out mice and blocking antibodies reveal that the uropod elongation is mediated by LFA-1. Therefore we propose that uropod elongation/delayed tail retraction is a novel final, rate-limiting step in the leukocyte extravasation cascade, which may be important for precise regulation of leukocyte recruitment and maintaining endothelial barrier function during inflammation.
CCL18-mediated pulmonary infiltration of T lymphocytes is independent of TRAIL but requires DR5 (102.20)
Authors: Luzina Irina , Atamas Sergei , Lockatell Virginia , Rus Violeta
Published: January 2023

Activities of T lymphocytes are centrally regulated by interactions between multiple members of the TNF/TNF-R family. TRAIL is a member of the TNF family with pro-apoptotic activity. The only signaling TRAIL receptor in mice is death receptor (DR) 5. CCL18, a chemokine with selective chemotactic activity on T lymphocytes, is known for its involvement in pulmonary inflammation and fibrosis associated with autoimmune diseases. We measured soluble TRAIL in bronchoalveolar lavage (BAL) fluids. The levels of TRAIL were significantly elevated (p < 0.01, ANOVA) in BAL from 16 patients with scleroderma lung disease (211±110 pg/ml) compared to 16 patients without lung involvement (115±58 pg/ml), or 8 healthy controls (73±41 pg/ml). To determine whether TRAIL is important for CCL18-induced pulmonary infiltration of lymphocytes, recombinant adenovirus-mediated gene delivery of CCL18 was performed in TRAIL KO, DR5 KO, and WT mice. Overexpression of CCL18 caused profound pulmonary lymphocytic infiltration in WT and TRAIL KO mice, compared to control mice infected with AdV-NULL. In contrast, infiltration of T lymphocytes was completely abrogated in CCL18-expressing DR5 KO mice, based on lung histology. Thus, the levels of TRAIL are elevated in the lungs of patients with autoimmunity-associated inflammation and fibrosis, yet TRAIL does not appear to be involved in the CCL18-mediated T lymphocytic infiltration, whereas its receptor DR5 is required for such T cell response to CCL18.
Lipocalin-2 is involved in immune-complex mediated inflammation (102.23)
Authors: Shashidharamurthy Rangaiah , Parkos Charles , Selvaraj Periasamy , Vijay-Kumar Matam
Published: January 2023

Lipocalin-2 (Lcn-2) is an innate immune protein whose expression is upregulated by several orders of magnitude in response to inflammation/injury/infection by variety of cells including neutrophils. However, the role of Lcn-2 induction in IC-induced inflammation is largely unknown. Here in, we have investigated the role of Lcn-2 during IC-mediated inflammation using Lcn-2 knock out (Lcn-2KO) and wild type (WT) mice. IC-mediated inflammation upregulated systemic Lcn-2 in WT mice when compared to naïve mice. The neutrophil chemoattractant KC was significantly elevated in WT, but not in Lcn-2KO mice, in response to IC-induced inflammation. Consistently Lcn-2KO mice showed about 50% reduced IC-mediated inflammation when compared to WT mice. Histopathological analysis of skin biopsy showed reduced inflammatory cell infiltration at inflamed site in Lcn-2KO mice relative to WT mice. Administration of recLcn-2 into Lcn-2KO mice resulted in inflammation similar to that of WT mice in response to IC. Lastly, we observed that neutralization of Lcn-2 via neutralizing mAb significantly reduced inflammation in WT mice. These results suggest that engagement of FcγRs expressed on local inflammatory cells with ICs leads to the induction of Lcn-2. Such IC-induced Lcn-2 expression may be essential for infiltration inflammatory cells at the site of inflammation. Our studies show that therapeutic targeting of Lcn-2 may be useful approach for treating IC-mediated inflammatory/autoimmune diseases
CD8 T cells are rapidly recruited to both immunized and unimmunized skin after vaccinica virus skin scarification persist long-term as nonmigratory resident memory T cells (Trm), and provide rapid, effective, and specific local immunity (102.25)
Authors: Jiang Xiaodong , Wagers Amy , Clark Rachael , Liu Luzheng , Fuhlbrigge Robert , Kupper Thomas
Published: January 2023

Abundant Trm populate normal, non-inflamed skin in both humans and mice, but little is known about their trafficking patterns. We created parabiotic mice by joining wild-type (WT) mice with GFP-transgenic mice. These mice rapidly developed a shared a circulatory system, with equilibration of GFP+ and GFP- T cells in blood and lymphoid tissues (LT), but this never occurred in skin. To test the trafficking of CD8 Trm, we s.s. immunized OT-1 bearing mice with VACV that expressed ovalbumin (VACV-OVA). Five weeks after s.s., we joined these mice parabiotically with naive mice. After 2 weeks, central memory (Tcm) OT-1 cells had equilibrated in blood and LT. However, OT-1 skin Trm could only be detected in the immunized parabiont, even after 24 weeks of parabiosis. When B-cell-deficient (µMT) mice for parabiosis (both immunized and naïve), we found that the skin of the immunized but not the naïve mouse rapidly cleared virus on challenge. These data suggest that CD8+ Trm can persist in previously infected skin and do not readily migrate out of skin. Next we asked whether uninfected skin of immunized mice contained OT-1 Trm. We could find substantial numbers of OT-1 Trm cells in uninfected skin; this number increased further after multiple infections. VACV was rapidly cleared from uninfected skin of s.s. immunized mice after challenge. Our results demonstrate that CD8+ Trm cells persist long term in the skin of s.s. immunized mice and provide rapid and site specific protection.
Conformational switching in ezrin regulates morphological and cytoskeletal changes required for B cell chemotaxis. (102.9)
Authors: Parameswaran Neetha , Matsui Ken , Gupta Neetu
Published: January 2023

B cell chemotaxis occurs in response to specific chemokine gradients and is critical for homeostasis and immune response. The molecular regulation of B cell membrane-actin interactions during migration is poorly understood. In this study we report a role for ezrin, a member of the membrane-cytoskeleton crosslinking Ezrin-Radixin-Moesin (ERM) proteins, in the regulation of the earliest steps of B cell polarization and chemotaxis. We visualized chemokine-induced changes in murine B cell morphology using scanning electron microscopy, and spatiotemporal dynamics of ezrin in B cells using epifluorescence and total internal reflection microscopy. Upon chemokine stimulation ezrin is transiently dephosphorylated to assume an inactive conformation, and localizes to the lamellipodia. B cells expressing a phosphomimetic conformationally active mutant of ezrin, or those in which ezrin dephosphorylation was pharmacologically inhibited displayed impaired microvillar dynamics, morphological polarization and chemotaxis. Our data suggest a two-fold involvement of ezrin in B cell migration whereby it first undergoes chemokine-induced dephosphorylation to facilitate membrane flexibility, followed by relocalization to the actin-rich lamellipodia for dynamic forward protrusion of the cells.