bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased ERK1/2 activation leads to sustained T cell receptor signaling in rheumatoid arthritis (49.8)
Authors: Singh Karnail , Deshpande Pratima , Pryshchep Sergey , Weyand Cornelia M , Goronzy Jorg J
Published: January 2023

Immune responses to citrullinated neoantigens indicate a tolerance defect in rheumatoid arthritis (RA). We examined the hypothesis that abnormal TCR signaling in RA T cells lowers the activation threshold. T cells from 33 seropositive RA patients and 33 controls were stimulated by CD3/CD28 crosslinking or superantigen-coated dendritic cells. Activation-induced signaling was quantified by PhosFlow, Western blotting, and confocal microscopy. Basal as well as post-stimulation p-ERK was higher in naïve, memory and effector CD4 and CD8 T cells from RA patients compared to controls (p<0.01 to p<0.001). Increased ERK phosphorylation was also observed upon PMA/ionomycin stimulation suggesting a defect distal of early TCR signals. Phosphorylation of ZAP70 and NFk-B was only elevated at late time points, and this increase was completely amenable to MEK1 and ERK inhibitors. Increased ERK responsiveness led to delayed recruitment of SHP-1 to the immunological synapse of RA T cells and lowered the TCR threshold to suboptimal stimulation. Increased ERK responsiveness was also a characteristic finding in the SKG mouse model of RA and preceded clinical symptoms. MEK-1 inhibition with subtherapeutic doses significantly delayed the onset of less severe disease. We propose that increased ERK activity tunes TCR thresholds to impair tolerance mechanism and induce autoimmunity in RA. Supported by RO1-AR41974 & AI 44142.
IC87114, a Selective Inhibitor of PI3Kδ Suppresses Joint Inflammation and Bone Erosion in Collagen-Induced Arthritis in Rat (50.14)
Authors: Puri Kamal D , Steiner Bart H. , Kashishian Adam S , Chen Hao , Gallatin W. Michael , Giese Neill A
Published: January 2023

PI3Kδ plays an essential role in antigen-receptor signaling, proliferation, activation and function of lymphocytes. Mice deficient in PI3Kδ activity show substantial reduction in immunoglobulin levels, partial impairment in chemoattractant-induced neutrophil migration as well as defects in signaling and function of macrophages. Collagen-induced arthritis (CIA), a commonly used model for studying antirheumatic drugs, requires participation of both B and T cells to initiate disease and reproduces many of the pathogenic mechanisms detected in human rheumatoid arthritis. In this study, we have investigated the ability of IC87114, a selective PI3Kδ inhibitor, to reverse the rheumatoid arthritic like state in the CIA model of arthritis. Clinical arthritis was initiated by immunizing animals with collagen followed by a booster dose on day 7. IC87114 or control treatment was initiated when at least one hind paw was significantly inflamed and continued for an 18-day treatment course. IC87114-treatment of arthritic rats reduced the progression and severity of clinical arthritis that was evident within 6 days after initiation of therapy. IC87114-treatment significantly reduced the level of anti-collagen antibodies. Arthritic rats treated with IC87114 had significantly lower radiographic scores compared with control, indicating that treatment with IC87114 was effective in protecting bone integrity. Histopathological evaluation demonstrated that IC87114 was effective in reducing the histological changes induced by rheumatoid arthritis. The effect of IC87114-treatment on these parameters together suggests a therapeutic potential for PI3Kδ-selective compounds to ameliorate rheumatoid arthritis.
NADPH quinone oxidoreductase induction by sulforaphane inhibits synovial hyperplasia and rheumatoid T cell proliferation. (50.15)
Authors: Kim Wan-Uk , Kong Jin-Sun , Yoo Seung-Ah , Yoon Hyung-Ju , Park Yoon-Jung , Cho Chul-Soo
Published: January 2023

Sulforaphane (SFN), derived from cruciferous vegetables, has been previously shown to induce phase II enzyme and to have anti-carcinogenic activity. In this study, we investigated whether SFN regulates synoviocyte hyperplasia and T cell proliferation in rheumatoid arthritis (RA). SFN induced synoviocyte apoptosis by regulating Bcl-2/Bax expression and pAkt activity. In addition, SFN dose-dependently inhibited T cell proliferation and the production of IL-17 and TNF-alpha by RA mononuclear cells stimulated with anti-CD3 antibody, but enhanced the enzyme activities of NADPH quinone oxidoreductase-1 (NQO1), a representative phase II enzyme. SFN inhibition of synoviocyte survival was abrogated by co-treatment with curcumin, an inhibitor of NQO1 or by downregulation of NQO1 transcripts. The intra-peritoneal administration of SFN to mice with type II collagen (CII)-induced arthritis suppressed arthritis severity, anti-CII antibody levels, and T cell proliferative response to CII. The productions of IL-17, TNF-alpha, IL-6, and IFN-gamma from lymph node and spleen cells of these mice also were markedly reduced by SFN treatment. Moreover, in mice with anti-CII antibody-induced arthritis, a passive model of arthritis that is independent of adaptive immunity, SFN treatment reduced arthritis severity and synovial hyperplasia. In summary, NQO1 induction by SFN was found to inhibit synoviocyte proliferation and TH17 cell activation in vitro. Moreover, the anti-arthritic and immune regulatory effects of SFN were confirmed in vivo, which suggests that SFN may offer a possible treatment option for RA.
Targeted ablation of human CD30 positive cells with an anti-CD30-MMAE antibody-drug conjugate (SGN-35) in vivo inhibits antigen-specific secondary immune responses (50.17)
Authors: Oflazoglu Ezogelin , Zeng Weiping , Klussman Kerry , McEarchern Julie A , Law Che-Leung , Grewal Iqbal S , Sievers Eric L , Gerber Hans-Peter
Published: January 2023

CD30 is a member of the tumor necrosis factor receptor superfamily whose normal physiologic expression is restricted primarily to relatively small subpopulations of activated T- and B-cells. In abnormal physiologic states, CD30 has been found on inflammatory cells in patients with a variety of chronic inflammatory diseases such as atopic dermatitis and asthma. In addition, malignant Reed-Sternberg and anaplastic large cell lymphoma cells express particularly high levels of CD30. Cells expressing CD30 have been shown to be activated and produce cytokines during inflammatory responses. SGN-35 is an antibody-drug conjugate that binds to human CD30, ablates CD30+ tumor cells in vitro, and strongly inhibits growth of HL and ALCL tumors in vivo. In the present study, the effects of SGN-35 on antigen specific secondary immune responses were evaluated. Compared to untreated cells, SGN-35 induced a dose-dependent inhibition of T cell proliferation, with maximum inhibition of 70% following antigen stimulation in vitro. In the hu-SCID mouse tetanus toxoid model, antigen-specific immunoglobulin responses in SGN-35 treated mice were much lower compared to untreated controls (p=0.0019). Taken together, these data demonstrate specific targeting of subsets of activated lymphocytes and suggest that SGN-35 may be employed clinically to decrease antigen specific cells and immunoglobulin that damage tissue in autoimmune and inflammatory diseases.
Differential effects of anti-TNF-a mAb and the TNF-R-Fc fusion molecule on fibroblast-like synoviocyte apoptosis in arthritis. (50.19)
Authors: Pattacini Laura , Salvarani Carlo
Published: January 2023

In our study, we tested the hypothesis that different anti-TNF-a drugs had different effects on apoptosis of fibroblast-like synoviocytes (FLS) obtained from rheumatoid arthritis (RA) patients. FLS and peripheral blood mononuclear cells (PBMC) from RA patients were co-cultured and incubated with one of the 3 biologics for 6 days and cell death and signal transduction pathways examined. Whereas all 3 biologics induced an increase in FLS apoptosis, etanercept was the most efficacious, leading to a 3-fold increase in apoptosis induction (p<0.01). All of the biologics activated PTEN-FAK pathway and up-regulated Bax. However, whereas infliximab and adalimumab caused tmTNF dephosphorylation and Bcl-2 upregulation, etanercept had very little effect. The incubation with the molecule MAK195 - the F(ab)'2 fragment of the anti-TNF-a - as well as the cross-linking of etanercept, resulted in reduction of apoptosis induction and upregulation of Bcl-2. In our study, we have shown that the three drugs have a different efficacy in apoptosis induction. Whereas all of them can activate a pro-apoptotic pathway, only the anti-TNF-a antibodies can also induce a reverse signaling, which, in FLS, results in the upregulation of Bcl-2. This finding might be relevant in the choice of the therapy for patients with RA, since etanercept could be more indicated in patients with a particularly aggressive FLS phenotype.
Potent Therapeutic Effect of Apeaz™ Cream in the Mouse Collagen Induced Arthritis Model (50.20)
Authors: nuhaily Samer , Yei Soonpin , Meza Miguel , Incardona Francesca , Esber Heny J , Damaj Bassam B
Published: January 2023

Rheumatoid arthritis (RA) is a chronic autoimmune disease of unknown etiology, characterized by chronic inflammation in the joints and subsequent destruction of the cartilage and bone. OTC NSAIDs are only approved for temporary pain relief and do not qualify as Disease-modifying antirheumatic drugs (DMARDs). Apeaz™ is an OTC cream approved for temporary relief of pain resulting from arthritis. The main ingredient in Apeaz™ is methyl salicylate as anti-inflammatory with glucosamine sulfate, menthol, camphor and methyl sulfonyl ethane as inactive excipients. Because Collagen-induced arthritis (CIA) is a murine experimental disease model sharing a number of clinical, histological and immunologic features with RA, we used it to study the effect of topically applied Apeaz™, once a day, on the pathogenesis of arthritis. The results show that treatment with Apeaz™ has great benefit at the clinical and pathological levels, as the therapeutic effect of Apeaz™ was associated with the downregulation of both inflammatory and autoimmune components of the disease. Treatment with Apeaz™ completely abrogated joint swelling and destruction of cartilage and bone as demonstrated by histopathology and X-ray analysis of joints. In addition, Apeaz™ reduced the levels of inflammatory cytokines in the joints of arthritic mice. Our data suggest that Apeaz™ could be a viable candidate as a DMARD for RA.
Infection with VSV ameliorates autoimmune disease in FcγR2B deficient mice (50.23)
Authors: Kole Hemanta K , Deane Jonathan A , Bolland Silvia
Published: January 2023

Both genetic and environmental factors, including infection by pathogens, affect the development of autoimmune diseases such as lupus. We are investigating the effect of viral infections on the lupus model of FcγR2B-/- mice. We infected FcγR2B-/- mice a single time at 2 months of age with vesicular stomatitis virus (VSV) and observed the development of autoimmune disease for the following 6 months. Infection with VSV significantly reduced autoantibody titers in serum and improved survival of FcγR2B-/- mice. B cell development in the bone marrow was not affected in VSV infected mice whereas memory B cells, and germinal center, plasma, and IgG producing mature B cell numbers were reduced. VSV infected FcγR2B-/- mice had lower number of follicular helper T cells and CD11c+ cells in the spleen. Increased survival in these mice correlated with a reduction of inflammatory cytokines and chemokines in the serum. The fact that a single round of infection with VSV confers long time protection from lupus in this mouse model indicates that the suppressor mechanism underlying the VSV effect on autoimmunity could be an excellent candidate for therapies seeking long term efficacy.
Histone/Protein Deacetylase Inhibitors (HDACi) and DNA Methyltransferase Inhibitors (DNMTi) Enhance Human Treg Function Through Epigenetic Regulation of Foxp3 (50.27)
Authors: Akimova Tatiana , Wang Liqing , Golovina Tatiana N , Mikheeva Tatiana V , Riley James L , Hancock Wayne W
Published: January 2023

Manipulation of CD4+CD25+FOXP3+ Treg cells may be a valuable new approach to therapy for transplantation and autoimmunity. We report the first data involving epigenetic modulation of the suppressive functions of human Tregs. CD4+CD25- T cells and CD4+CD25+ Treg cells from 14 healthy donors were isolated by magnetic beads. CFSE-labeled CD4+CD25- T cells were stimulated with CD3 mAb and irradiated PBMC, co-cultured for 3-4 d with Tregs and varying concentrations of HDACi (sodium butyrate, valproic acid) and/or DNMTi (decitabine, zebularine, RG108), and cell divisions determined by CFSE dilution. Control wells without drugs provided serial suppression data specific for each donor, and wells without Tregs showed maximal cell divisions that were not markedly impaired by drugs. We found that HDACi or DNMTi directly increased expression of Foxp3 and Foxp3-dependent genes and increased the suppressive activity of human Tregs by up to 3-fold (p<0.05). This approach also markedly improved outcomes in parallel studies of murine Tregs in vitro and in vivo, including in murine models of autoimmunity and transplant rejection. We conclude that epigenetic-based therapies using clinically approved drugs may provide the means to usefully upregulate Treg functions in patients with autoimmunity or post-transplantation.
Amelioration of Lupus Nephritis in MRL/lpr mice by Adoptive Transfer of IgM Anti-dsDNA antibodies (50.28)
Authors: Zhao Minglang , Jiang Chuancang , Diaz Marilyn
Published: January 2023

The role of IgM autoantibodies in lupus nephritis (LN) remains controversial. Our previous work with AID-deficient MRL/lpr mice demonstrated that IgM autoantibodies do not contribute to LN. In fact, these mice experienced an abrogation of LN in spite of extremely high levels of IgM autoantibodies, and their survival levels exceeded those of mice with B cells but lacking antibodies. This suggests that IgM autoantibodies may play a protective role in LN. To determine if IgM autoantibodies do in fact protect against LN and to delineate the characteristics of protective autoantibodies, MRL/lpr mice were passively transferred with serum, IgM anti-dsDNA antibodies and IgM non-autoantibodies collected/generated from AID-sufficient and -deficient MRL/lpr mice. MRL/lpr mice treated with the serum from AID-deficient MRL/lpr mice (having higher levels of IgM autoantibodies but no IgG) showed an apparent reduction in the severity of kidney disease, while the mice treated with the serum from AID-sufficient MRL/lpr mice (having both IgM and IgG autoantibodies) seemed to have increased renal impairment. MRL/lpr mice treated with Anti-dsDNA monoclonal IgM either from AID-sufficient or -deficient MRL/lpr mice displayed a reduction in the severity of kidney damage with a delayed onset of proteinuria, significantly less deposition of immune complexes, inflammatory infiltration, and apoptotic cells in the kidneys. In contrast, MRL/lpr mice treated with non-autoreactive monoclonal IgM did not seem to delay the onset of nephritis. Therefore, passive transfer of autoreactive IgM ameliorated renal impairment in MRL/lpr mice, demonstrating a protective role of IgM autoantibodies in the pathogenesis of LN.

* Zhao and Jiang contributed equally to this work
A critical mediator of leukocyte extravasation during inflammatory disease (50.8)
Authors: Kithcart Aaron P , Sielecki Thais , Short A , Mavrikis G , Williams J , Smith K , Shawler T , Gienapp I , Satoskar A , Whitacre Caroline C
Published: January 2023

Multiple sclerosis (MS) is a demyelinating neurological disease affecting more than 400,000 Americans. MS pathology involves activation of autoreactive lymphocytes and their migration across the blood-brain barrier (BBB) into the central nervous system (CNS). MS patients have a higher level of the ubiquitously expressed, proinflammatory cytokine MIF. Mice lacking MIF have fewer leukocytes in the CNS following induction of experimental autoimmune encephalomyelitis (EAE), an animal model for MS. We found using bone marrow chimeras that mice expressing MIF from myeloid cells had significantly more severe disease than MIF KO mice or mice expressing MIF only in non-myeloid cells (CDI±SD; 28.0±17.9 vs. 10.3±17.9 or 4.7±10.4). Using immunohistochemistry, we found more infiltrates in the CNS of mice expressing MIF from myeloid cells. We utilized a small molecule inhibitor of MIF to evaluate whether disruption of MIF activity could inhibit migration of leukocytes. Mice had reduced severity of EAE following inhibitor administration and reduced migration of leukocytes into the CNS. Other groups have shown that blocking MIF reduces expression of adhesion molecules on the BBB. Our data suggests that during inflammatory disease, MIF from myeloid cells is critical for leukocyte migration and an inhibitor of MIF reduces migration and subsequent clinical disease. (Supported by NIH grant AI 064320 and NMSS grant RG3272)
A proteomics study of MHC-associated peptide profiles displayed by macrophages after stimulation in vitro with human low density lipoproteins (78.12)
Authors: Attiyah Sama M , Bermudez-Fajardo Alexandra , Oviedo-Orta Ernesto
Published: January 2023

Recognition of modified self proteins by macrophages usually induces the generation of peptide fragments derived cytoplasmic pool of cells that can be presented by the major histocompatibility complex (MHC). Despite the potential for presentation of a wide range of peptides only a limited number of peptides are displayed by the complexes. This generates peptide patterns that imprint a signature associated with particular cellular or disease processes. The aim of this study was to investigate whether stimulation of macrophages with human low density lipoprotein (LDL) generates peptide patterns on macrophage MHC class I and II in vitro. The study also assessed the effectiveness of peptide elution methods for peptide extraction. To accomplish this we stimulated J774.2 cells with native or oxidised LDL particles and extracted the MHC-associated peptides using citrate-phosphate buffer at pH 3, 4 & 5. Samples were then analysed by LC/MS/MS. MASCOT analysis was carried out to identify the protein source of peptides and IEDB and NetMHCII 1.0 were used to predict the peptide-binding affinity to MHC class I and II complexes. We identified 54 peptides derived from macrophages stimulated with ox-LDL, 33 peptides derived from cells treated with n-LDL and 10 peptides identified from untreated cells. Peptide derived from proteins involved in macrophage DNA and RNA replication, signal transduction, cytoskeleton and lipid metabolism were identified. We found a differential pattern of MHC-associated peptide expression in treated macrophages. These findings contribute to future identification of disease-associated peptides for their potential use in the development of therapeutic and/or diagnostic tools.
Antigen presentation and T cell activation by Coccidioides-Ag2/PRA-cDNA-transfected primary murine dendritic cells (78.21)
Authors: Awasthi Shanjana , Vilekar Prachi
Published: January 2023

The dendritic cells (DCs) have an important role in antigen presentation and regulation of host defense. Immunization with in vitro modified DCs has been shown to activate protective immune responses. Among various approaches used for modifying DCs, the nonviral genetic transfection of DCs is easy and safe as compared to viral-mediated transfection methods. In this study, we investigated antigen presentation and T cell stimulatory ability of nonvirally, gene-transfected primary murine DCs. We isolated primary murine bone marrow derived DCs and transfected isolated DCs with Ag2/PRA-cDNA (protective epitope of Coccidioides, a fungal pathogen) using TransIT-TKO (Mirus Bio, WI)- a nonviral transfection reagent. We found that upto 30-40% of primary murine DCs were transfected with the plasmid DNA carrying Ag2/PRA-cDNA. The transfected DCs remained viable and expressed the transgene (Ag2/PRA-cDNA)-encoded protein. Only moderate changes were observed in the immunophenotype of genetically-transfected DCs. The activation of T cells was studied by flow-cytometry after co-culture of naive splenic T cells with DCs. The T cells co-incubated with Ag2/PRA-cDNA transfected DCs showed upregulation of cell-surface CD25 and CD69 (activation markers). These results suggest that the primary DCs can be efficiently transfected in vitro using a non viral method; the transfected primary DCs can process and present the antigens and stimulate T cells.
Genetic manipulation of BCG leads to altered presentation of CD4 and CD8 epitiopes from Antigen 85B by mouse dendritic cells. (78.22)
Authors: JAGANNATH CHINNASWAMY , Dhandayuthapani Subramanian
Published: January 2023

The partial efficacy of BCG vaccine has led to a search for more effective vaccines. The immuno-dominant antigen-85B (Ag85B) from BCG vaccine is processed in macrophages and dendritic cells (DCs) dependent upon the phagosomal pH and lysosomal delivery. However, BCG avoids lysosomal localization and its phagosomes are near neutral. We found that induction of autophagy enhanced the lysosomal delivery of BCG vaccine in DCs. This in turn increased the MHC-II dependent presentation of Ag85B which was detected using an Ag85B-specific T cell hybridoma. DCs also showed an enhanced presentation of Ag85B when infected with BCG that was engineered to over-express Ag85B. We found that over expressed (OE)-Ag85B triggered autophagy. Furthermore, Ag85B is secreted from BCG vaccine into the cytosol of DCs although BCG is inefficient in inducing CD8 T cells against Ag85B in mice. Since OE-Ag85B enhanced the MHC-II dependent presentation, we examined whether OE-Ag85B could also be routed through the proteasomal pathway. DCs were infected with BCG-OEAg85B and Ag85B presentation monitored with or without the blockade of proteasomes through lactacystin. Lactacystin enhanced the MHC-II dependent presentation of Ag85B suggesting that OE-Ag85B goes through the proteasome as well as lysosome during autophagy. Mice were then immunized with BCG-OEAg85B and the spleens were found to contain an increased number of CD8 T cells specific for an epitope of Ag85B detected using a pentamer-conjugate. We propose therefore that genetic manipulation of BCG may be used to alter the levels of CD4 and CD8 epitope presentation by DCs and alter vaccine efficacy.
Antigen processing and MHC-II presentation by dermal and tumor infiltrating Dendritic Cells (78.26)
Authors: Gerner Michael Y , Mescher Matthew F
Published: January 2023

MHC-II presentation by DC is necessary for both initial CD4 T cell priming and for peripheral effector function. We have previously found that naïve tumor-specific CD4 T cells were ignorant to poorly immunogenic murine tumors, suggesting a possible insufficiency in DC function. Here, we investigated the phenotype, antigen uptake and MHC-II presentation capacity of normal dermal DC (DDC) and tumor infiltrating DC (TIDC) in lymphoid and peripheral sites. We found that tumors were extensively infiltrated by partially mature TIDC that were phenotypically similar to DDC. However, TIDC were inefficient at MHC-II presentation due to poor intrinsic protein uptake capability. This resulted in both inferior initiation of T cell responses in the draining LN and poor peripheral effector cell accumulation. In addition, toll-like receptor stimulation selectively enhanced MHC-II antigen presentation by DDC but not TIDC in the draining LN, and did not affect overall peripheral antigen uptake of either. These results show that TIDC are functionally distinct from normal interstitial DC, indicating that neoplastic tissues can evade effector CD4 T cells through modification of DC competence. NIH AI34824(M.F.M.), CA82596(M.F.M.), NCI 2 T32(M.Y.G.).
Glabridin inhibits dendritic cell maturation and function by blocking the activation of NF-κB/Rel and MAPKs (78.27)
Authors: Kang Jong Soon , Kim Jee Youn , Han Sang-Bae , Lee Kiho , Park Song-Kyu , Kim Hwan Mook
Published: January 2023

Glabridin is known to have anti-inflammatory, antimicrobial and cardiovascular protective activities. The objective of this study was to investigate the effect of glabridin on dendritic cell maturation and function and the molecular mechanisms responsible for this effect. Glabridin dose-dependently suppressed lipopolysaccharide (LPS)-induced expression of maturation markers of dendritic cells, such as CD80, CD86 and MHC-class II molecules, without affecting cell viability. The inhibitory effect of glabridin on functional maturation of dendritic cells was confirmed by enhancement of endocytosis decreased by LPS treatment and suppression of LPS-induced IL-12 production and allogeneic T cell stimulation. The production of IFN-γ and IL-4 in allogeneic T cells was also suppressed by glabridin treatment. Further study demonstrated that LPS-induced activation of NF-κB/Rel and MAPKs, which are known to be important for dendritic cell maturation and functional activation. Collectively, these results demonstrate that glabridin inhibits LPS-induced maturation and functional activation of dendritic cells, at least in part, by blocking NF-κB/Rel and MAPK pathway.
Exogenous antigen enters the major histocompatibility complex (MHC) class I pathway via multiple endocytic compartments with distinct molecular and proteolytic requirements (78.31)
Authors: Belizaire Roger , Unanue Emil
Published: January 2023

Peptides derived from exogenous proteins are displayed by MHC class I via a pathway termed cross-presentation. Despite extensive study, the endocytic compartment(s) that mediate cross-presentation are poorly understood. Immunization of mice with hen egg-white lysozyme (HEL) protein cross-primes CD8 T cells recognizing HEL 23-31 presented by Db. Here we used liposome-encapsulated HEL to examine the cross-presentation capacity of specific subcellular compartments. Liposome-encapsulated HEL targeted to early endosomes, but not late endosomes/lysosomes, entered the MHC class I pathway. Cross-presentation of early endosomal HEL liposomes apparently required the transporter associated with antigen processing (TAP). However, additional experiments indicated no requirement for proteasome-dependent processing or nascent Db. To test the possibility that early endosomal HEL liposomes were presented by endosomal Db independently of TAP, we incubated TAP-deficient antigen presenting cells (APCs) with early endosomal HEL liposomes and a peptide to promote Db surface-stabilization and eventual entry into the endosomal pathway. Addition of Db surface-stabilizing peptide rescued cross-presentation of early endosomal HEL liposomes by TAP-deficient APCs, implying that Db mis-localization accounted for the cross-presentation defect observed previously. Cross-presentation of early endosomal HEL liposomes was inhibited when endolysosomal acidification was blocked by chloroquine. Surprisingly, chloroquine treatment facilitated cross-presentation of both late endosomal/lysosomal HEL liposomes and soluble HEL. Altogether, these data indicate that exogenous protein antigen can enter the MHC class I pathway through multiple routes within the APC.
Targeted delivery of Poly(dG) conjugated protein antigens to antigen presenting cells results in efficient cross-priming in vivo (78.32)
Authors: Toussaint Helene , Erdos Geza , Falo, Jr. Louis D.
Published: January 2023

The development of strategies to efficiently and specifically deliver antigen to antigen presenting cells (APCs) in vivo is a major goal of vaccine design. Internalization of antigen by APCs is a crucial step in immune activation and the mechanism of antigen internalization and trafficking can influence localization in antigen processing compartments and the presentation of antigen to CD4+ or CD8+ T cells. We have designed an APC targeting strategy based on simple and direct coupling of Poly(dG) oligo deoxynucleotides to protein antigens. In comparison to soluble OVA, we find that conjugates of Poly(dG) and OVA (OVA-Poly(dG)) are rapidly internalized by DCs and transverse distinct intracellular processing pathways. OVA-Poly(dG) is both presented to OVA-specific CD4+ T cells (OT-II cells) and efficiently cross-presented to OVA-specific CD8+ T cells (OT-I cells) by DCs in vitro. Importantly, immunization of naïve animals by direct injection of OVA-Poly(dG) results in efficient priming of potent antigen specific CTL responses. Our results suggest that Poly(dG) can target antigen to APCs and serve as a potent adjuvant to induce strong CD8+ T cell-mediated immunity against tumors or viruses. Funding provided by NIH.
A simple one-step method for isolating highly purified plasmacytoid dendritic cells from human peripheral blood (78.33)
Authors: Kokaji Andy I , Holland Siobhan , Fairhurst Maureen A , Thomas Terry E , Guilbault Benoit G
Published: January 2023

Plasmacytoid dendritic cells (pDC) and myeloid DC (mDC) are the two major human dendritic cell populations. Each subset comprises only a small fraction of peripheral blood mononuclear cells (PBMC) and current protocols for their isolation are time consuming, involve multiple steps and often require special equipment. We have developed a rapid and efficient method for the isolation of pDCs from normal blood that yields excellent cell purities and recoveries. Briefly, PBMCs are isolated by Ficoll-Paque PLUS density gradient sedimentation and pDCs are isolated using immuno-magnetic, column-free, negative selection (EasySep®). Our EasySep® technology involves specifically labeling unwanted cells with a cocktail of bi-specific tetrameric antibody complexes and dextran-coated magnetic particles. Using a hand-held magnet, unwanted cells can then be easily removed from the unlabeled pDCs. Flow cytometric assessment of EasySep® isolated pDCs (Lin-, HLA-DR+, BDCA-4+) demonstrate purities of 93.8% ± 3.8% with cell recoveries of 65.6% ± 16.2% (n=9). Using our RoboSep® cell separator, the entire separation procedure can be fully automated with equivalent cell purities and recoveries.

A.I.K is supported by an NSERC Industrial R&D Fellowship
A Threonine-based Targeting Signal in the Human CD1d Cytoplasmic Tail Controls its Functional Expression (78.4)
Authors: Liu Jianyun , Daniel Shaji , Brutkiewicz Randy
Published: January 2023

CD1d molecules are MHC class I-like molecules that present lipids to a unique subpopulation of T cells called natural killer T (NKT) cells. The cytoplasmic tail of human CD1d possesses a tyrosine-based endosomal targeting motif (YXXZ). As such, these molecules traffic through the endocytic pathway, where it is believe that they are loaded with the antigenic lipid that stimulates NKT cells. In the current study, it was found that the T322 residue in the human CD1d tail is a major signal controlling transport to the cell surface and thus its functional expression. Phosphorylation of this residue or removal of the entire cytoplasmic tail negates its ability to regulate CD1d trafficking, resulting in lysosomal targeting and degradation. These results demonstrate an important role of a heretofore unknown signal in the cytoplasmic tail of CD1d that may have relevance to other type I integral membrane proteins that traverse through the endocytic pathway.
RSV-induced expression of TSLP and asthma development (79.13)
Authors: Headley Mark B , Lee Hai-Chon , Ziegler Steven F.
Published: January 2023

Early infection with Respiratory Syncytial Virus (RSV) has long been recognized to predispose certain individuals towards the development of asthma. The immune response to RSV is unique in that it consists of protective Th1 and immunopathologic Th2-type features. Though much is known about the pathology of this disease it is still unclear how RSV infection leads to asthma development. Thymic Stromal Lymphopoietin (TSLP) is a cytokine that has been shown to be both necessary and sufficient for the development of asthma. Mice that express a lung-specific TSLP transgene (SPC-TSLP) develop a spontaneous and progressive asthma-like disease while TSLPRKO mice are highly resistant to disease in an antigen-specific asthma model. In this study we highlight a possible role for TSLP as the link between RSV infection and asthma development. We demonstrate that TSLP overexpression results in sensitization and the development of allergy to environmental antigens. Further, we show for the first time that RSV and its relative, Sendai Virus, lead to increases in TSLP expression by airway epithelial cells both in vitro and in vivo. Taken together these data suggest that TSLP induced by RSV during primary infection may increase susceptibility or directly result in asthma development. Additionally, in this study we show that the RIG-I viral recognition pathway represents a novel pathway leading to TSLP expression.
Relationship between protein allergenicity and glycosylation (79.14)
Authors: Kimber Ian , Almond Rachael , Flanagan Brian F , Dearman Rebecca J
Published: January 2023

Human native and recombinant forms of lactoferrin (NLF and RLF) are available with identical amino acid sequences, but different glycosylation patterns. We have characterised the intrinsic ability of these proteins to induce IgG and IgE antibody responses in BALB/c strain mice and the impact of co-administration. BALB/c strain mice were immunised with various concentrations (w/v) of NLF or RLF alone or in combination by intraperitoneal injection on day 0 and 7. Sera (day 14) were analysed for protein specific IgG and IgE by enzyme-linked immunosorbant assay and passive cutaneous anaphylaxis assay, respectively. RLF alone (1 to 5%) induced detectable IgG responses, but little or no IgE (titer of 1 with 5%). In contrast, NLF (1 to 0.05%) induced vigorous IgG and IgE responses. Forty and 200 times more RLF than NLF was required to induce equivalent IgG and IgE responses, respectively. Co-administration of NLF (0.2%) and RLF (1%) resulted in a significant down-regulation of IgE (titer of 1) in comparison with administration of NLF alone (1/16). This effect was allergen specific as RLF was without effect on IgE responses to the irrelevant allergens ovalbumin and peanut lectin. These results suggest RLF impacts on the induction phase to selectively inhibit IgE responses and that differential glycosylation patterns may impact on antigen uptake, processing and/or presentation to skew towards a type 2 response.
RSV Infection Promotes Cell Cycle Arrest Through TGF-beta Autocrine Regulation; A Critical Stage for Enhanced RSV Replication in Lung Epithelial Cells (79.21)
Authors: Gibbs John D , Ornoff Douglas M , Igo Heather A , Imani Farhad
Published: January 2023

Respiratory syncytial virus (RSV) is a common respiratory viral infection in children, which is associated with exacerbations of asthma, likely due to induction of inflammatory cytokines. We recently reported that treatment of primary human epithelial cells (PHBE) with TGF-β enhanced RSV replication and RSV-induced inflammatory cytokines. Here, we report that TGF-β enhancement of RSV replication is mediated by induction of cell cycle arrest. The data were confirmed by using pharmacologic inhibitors of cell cycle progression such as purvalanol and olomoucine, which enhanced RSV replication. Our data also showed that RSV infection alone resulted in cell cycle arrest in G2/M phase in PHBE cells or G0/G1 phase in A549 cells. Interestingly, RT-PCR and ELISA data revealed that RSV infection induced the expression of TGF-β. Blocking of TGF-β signaling with anti-TGF-β antibody or by using a specific receptor kinase inhibitor resulted in rescue of the RSV-induced cell cycle arrest, suggesting an autocrine mechanism. Collectively, our data demonstrate that RSV regulates cell cycle through TGF-β in order to enhance its replication. These findings provide a plausible mechanistic pathway for the association of RSV with the induction and exacerbations of asthma.
Interleukin-5 producing CD34+ hematopoietic progenitor cells in eosinophilic diseases (80.10)
Authors: Sjöstrand Margareta , Bossios Apostolos , Samitas Konstantinos , Malmhäll Carina , Gaga Mina , Lötvall Jan
Published: January 2023

CD34+ hematopoietic progenitor cells increase in nasal tissue during pollen season and CD34+ cells from human peripheral blood and bone marrow stimulated in vitro produce Interleukin-5 (IL-5). Poor information exists however regarding the role of CD34+ cells in eosinophilic disease.

The aim of this study was to examine whether CD34+ cells produce IL-5 in patients with eosinophilia. Two individuals were examined. The firsts was diagnosed with Churgh-Strauss syndrome with severe asthma, vasculitis and blood eosinophilia (1.9 x 109 L). IL-5 producing CD34+ cells in blood were evaluated by flow cytometry (FACS). Secondly bronchial biopsies were obtained from a patient with moderate persistent asthma and blood eosinophilia (0.3 x 109 L). The bronchial biopsies were immunohistochemical (IC) stained for IL-5 and CD34.

FACS analysis of the patient with Churgh-Strauss syndrome demonstrate that 1, 3 % of the lymphocyte population in the peripheral blood were CD34+ cells and 44% of these cells were positive for IL-5. IC staining of the bronchial biopsies showed IL-5 producing cells and sporadic cells that were positive for IL5/CD34.

IL-5/CD34 + progenitor cells exist in patients with different eosinophilic diseases and may be important when migrating into the local tissue acting as allergy effector cells or by autocrine regulation promoting proliferation of eosinophilic progenitor cells.

Funded by the Swedish HLF.
TGF beta Suppresses Mast Cell Function In Vivo and In Vitro (80.12)
Authors: Fernando Josephine , Ryan John
Published: January 2023

Mast cell activation is a critical component of allergic disease, and may be regulated by cytokines present in the microenvironment. We recently described the suppressive effects of TGFb1 on IgE signaling in mouse and human mast cells. We have furthered this study, demonstrating that TGF injection or aerosolization reduces mast cell IgE receptor expression in vivo, and suppresses eosinophil recruitment in a model of airway hyperresponsiveness. We find that TGFb1 inhibits expression of the key signaling molecules Akt, Fyn, and Stat5 employed by the IgE receptor. In contrast, expression or activation of ERK, JNK, GSK, and p38 was unaffected. Our data support the theory that TGFb1 possesses potent mast cell suppressive activities both in vitro and in vivo.
Repeated fungal exposure leads to eosinophilia and airway remodeling in a murine model of allergic asthma (80.13)
Authors: Samarasinghe Amali E. , Hoselton Scott A. , Schuh Jane M.
Published: January 2023

Allergic asthma is characterized by airway hyperresponsiveness, inflammation, bronchial smooth muscle cell and goblet cell hyperplasia, and subepithelial fibrosis. Differences between human and mouse lung anatomy makes the accurate reproduction of these characteristics challenging to mimic in a mouse model. We hypothesized that repeated allergen exposure would enhance changes observed in the lungs of allergic mice, thereby closely mimicking human disease. C57BL/6 mice were sensitized and challenged with Aspergillus fumigatus antigen and live conidia via airborne delivery respectively over the course of five weeks. Changes in lung architecture, inflammation, antibody, and cytokine profiles were analyzed at days 3, 7, and 14 after the first challenge. An additional group of mice were subjected to a second fungal challenge two weeks after the first, and the same analysis was carried out. While a single challenge resulted in a rapid infiltration and egress of neutrophils at day 3, there were no neutrophils recruited after the second challenge, although eosinophils were recruited more rapidly and in higher quantities. The trichrome stains showed a striking increase in subepithelial collagen deposition and smooth muscle mass immediately following the second fungal exposure. An increase in immunoglobulin (Ig)-E, and a decrease in IgG2a were observed following the second challenge. While cytokines interleukin (IL)-4, IL-5, interferon (IFN)-γ and tumor necrosis factor (TNF)-α peaked at day 14 after the first challenge, the second challenge resulted in peak cytokine levels at day 3. Airborne fungal conidia permits a natural exposure to allergen and mimics characteristics of human asthma with increased eosinophilia, subepithelial collagen, smooth muscle mass, IgE, and cytokines. (2P20RR015566)
Antagonistic Signaling by IL-4 and TGFb Regulates Mast Cell Function (80.5)
Authors: Macey Matthew , Ryan John
Published: January 2023

Mast cells, like all cells of the immune system, are regulated by crosstalk from multiple cytokines, which collectively form a signaling network. Cytokine networking is an important concept for understanding how the immune system is controlled. We hypothesized that the initial presence of pro-inflammatory cytokines in the absence of anti-inflammatory cytokines could desensitize mast cells from anti-inflammatory signals, rendering them more likely to be activated in allergic reactions. Conversely, we hypothesized that the initial presence of anti-inflammatory cytokines in the absence of pro-inflammatory cytokines could shield mast cells from activation by pro-inflammatory cytokines in allergic type responses. In this study, we show that IL-4 and TGFβ1 act as an antagonistic pair in controlling the mast cell response. Mast cell pre-culture with IL-4 decreased TGFβR1 and TGFβR2 RNA and protein levels through a STAT6-dependent mechanism. Further, IL-4 diminished TGFβ1 signaling, blocking SMAD2 phosphorylation. IL-4 also blocked TGFβ1-mediated mast cell death and suppressed TGFβ1-mediated mast cell migration. Mast cell pre-culture with TGFβ1 mirrored this antagonistic effect. TGFβ1 pre-culture reduced IL-4R protein levels, suppressed Il-4-mediated Stat6 phosphorylation, and diminished mast cell migration toward IL-4. When both are present simultaneously, TGFβ1 appears to have a dominating inhibitory effect over IL-4 by blocking proliferation in vitro and recruitment in vivo. These data are important in understanding how cytokine networks can impact mast-cell mediated diseases like asthma. Supported by NIH grants 1R01 AI59638 and U19A1077435.
Basophil-specific and post-translational silencing of a major endoplasmic reticulum heat shock protein gp96 (grp94, HSP90b1) for TLR and integrin (80.7)
Authors: Liu Bei , Li Zihai
Published: January 2023

Basophil has been implicated in anti-parasite defense, allergy and in polarizing TH2 response. However, little is known about its biological significance in vivo, due to lack of basic understanding of basophil developmental/functional cues. As an essential chaperone for multiple Toll-like receptors (TLRs) and integrins in the endoplasmic reticulum (ER), gp96 (grp94, HSP90b1) also participates in general protein homeostasis and in the ER unfolded protein response (UPR) to ensure cell survival during stress. No nucleic cells in mammals have been described to be naturally devoid of gp96. By genetically ablating gp96, we report herein that gp96 is dispensable for basophil development in vivo. Moreover, we found that gp96 was post-translationally cleaved in primary basophils leading to accumulation of a N-terminal ~50 kDa fragment of gp96 and up-regulation of several components of the UPR pathway. The alteration of gp96 was unique to basophils and was not observed in any other cell types including mast cells. Furthermore, we found that deletion of C-terminal domain of gp96 resulted in loss of its chaperone function. Our study revealed a remarkable biochemical event of gp96 silencing in basophils, arguing for the potential importance of shutting down gp96 post-translationally in regulating basophil development and function.
Caspase-mediated apoptosis of myeloid dendritic cells during acute simian immunodeficiency virus infection (81.15)
Authors: Wijewardana Viskam , Brown Kevin N , Liu Xiangdong , Barratt-Boyes Simon M
Published: January 2023

Myeloid dendritic cells (mDC) are depleted from the circulation and their loss inversely correlates with plasma viral load in human immunodeficiency virus (HIV) infection indicating a possible role of this phenomenon in disease pathogenesis. However, the exact mechanism of mDC loss has not been identified. To address this question we studied mDC dynamics in acute simian immunodeficiency virus (SIV) infection of rhesus macaques. Blood mDC from animals that progressed to AIDS were significantly depleted by 12 weeks of infection whereas mDC from monkeys that controlled infection were not. mDC isolated from lymph nodes at 14 days post infection were not significantly infected but did have significant levels of apoptosis. mDC from infected lymph nodes had a 4-fold increase in caspase-3 expression compared to pre-infected lymph nodes. In addition, inhibition of caspase-8, a hierarchical enzyme in the caspase cascade involving the extrinsic pathway of apoptosis, decreased caspase-3 expression by mDC. Expression of BCL-2 proteins did not differ significantly between mDC from uninfected and infected animals, whereas CD95 (Fas) was upregulated in mDC from infected monkeys, indicating a potential role for Fas/FasL in apoptosis. Our studies show that caspase-mediated apoptosis likely plays a role in the loss of mDC in pathogenic SIV infection. Further studies are underway to investigate the initiators of this apoptosis.
The evolution of affinity maturation: Germinal center-like cell aggregates in fish (81.16)
Authors: Magor Brad G , Saunders Holly L , Oko Andrea L
Published: January 2023

Compared with mammals, fish and amphibians have a limited antibody affinity maturation response, an outcome attributed to their lacking distinguishable germinal centres. There is evidence of Ig hypermutation and antigen driven selection, and as such we'd anticipate some sort of germinal center (GC)-like structures, perhaps akin to the varied GC-like cell aggregates observed in different human autoimmune disease. To establish whether fish have GC-like cell aggregates we did in situ hybridization and laser capture microscopy coupled with gene expression analyses on spleen sections of BSA immunized channel catfish. AID expressing cells were found predominantly in tight association with so called melano-macrophage centers or clusters (MMCs). Melano-macrophages have previously been noted for their ability to capture and retain whole antigen and thus had been proposed to be functionally analogous to follicular dendritic cells. Though far smaller than mammalian GCs the fish MMCs also contain CD4 expressing cells and a few flanking plasma cells. However TUNEL assays did not reveal the expected numbers of surrounding apoptotic cells. While there are alternative theories as to the function of these cell aggregates we are proposing that they are primitive germinal centers.

Funded by the National Science and Engineering Research Council - Canada
Genetic diversity of the avian chB6 alloantigen (81.18)
Authors: Funk Phillip E , OLaughlin Peter J
Published: January 2023

Chickens are one of the classic models of vertebrate immunity. We have been interested in the role of the alloantigen chB6 (formerly called BU1) in the development of B cells within the bursa and have presented evidence that chB6 can trigger apoptosis. chB6 is an alloantigen and in the original report of its cloning three alleles were reported, chB6.1 in RPL line 6 flocks, chB6.2 in RPL line 7, and chB6.3 in Light Sussex birds. However, no further search for other chB6 alleles has been reported. Using the chicken EST database we have identified five potential new alleles of chB6, although some of these EST clones are not full length cDNAs.. We have resequenced these EST clones to confirm the base sequence and translation. These results suggest greater genetic diversity of the chB6 locus than previously appreciated. Furthermore, rt-PCR was used to clone the turkey homologue of chB6. The results presented here are the beginnings of a larger study to examine chB6 and can provide novel insights into the evolution of immune related genes in Aves. This work supported by a summer grant from the DePaul College of Liberal Arts and Sciences.