bluuebunny/crossref_metadata_2025 huggingface dataset:
Nedd4-family interacting proteins activate Nedd4-family E3 ubiquitin ligases in T cells to limit pathogenic inflammation. (P1382)
Authors: O'Leary Claire , Riling Chris , Oliver Paula
Published: January 2023

E3 ubiquitin ligases tune signaling pathways in activated T cells to regulate differentiation and cytokine production. Nedd4-family ligases have distinct roles in these processes. These ligases often function with adaptors, which have been shown to aid binding to substrates and/or E2 enzymes. We have found that Nedd4-family interacting protein 1 (Ndfip1) and Ndfip2 promote Nedd4-family E3 ligase catalytic activity by a new mechanism. Our in vitro studies also indicate that Ndfip1 and Ndfip2 have overlapping function. In vivo, Ndfip1 negatively regulates T cell activation and TH2 cytokine production. The in vivo function of Ndfip2 is unknown; therefore, we generated Ndfip2-/- mice. Our studies revealed that, unlike Ndfip1, Ndfip2 is not a prominent negative regulator of T cell activation or TH2 polarization. However, loss of Ndfip2 exacerbated the inflammatory Ndfip1-/- phenotype. This suggested that, like Ndfip1, Ndfip2 limits inflammation. Supporting this, using a T cell transfer model of colitis, we found that transfer of doubly deficient T cells caused more severe disease compared to transfer of Ndfip1-/- T cells. These data indicate that Ndfip2 negatively regulates effector cell function following activation. We have now tested the effects of Ndfip2 deficiency on distinct cellular subsets in models of both acute and chronic inflammatory bowel disease. These studies suggest that Ndfip1 and Ndfip2 are pleiotropic modulators of inflammation.
Replacement of Sϵ with Sμ reveals novel mechanistic insights into rearrangements of the IgH locus and cMyc chromosomal translocations (P1400)
Authors: Senger Kate , Misaghi Shahram , Qu Yan , Sai Tao , Hamidzadeh Kajal , Sun Yonglian , Nguyen Allen , Zhou Meijuan , Yan Donghong , Lin Zhonghua , Soukup Tim , Lorenzo Maria , Garris Christopher , Sebrell Andrew , Xu Min , Caplazi Patrick , Roose-Girma Merone , DeForge Laura , Warming Soren , Lee Wyne , Balazs Mercedesz , Dixit Vishva , Zarrin Ali
Published: January 2023

Preceding antibody constant regions are switch (S) regions varying in length and repeat density that are targets of activation induced cytidine deaminase (AID). We asked how participating S regions influence each other to orchestrate rearrangements at the IgH locus by engineering mice in which the weakest switch, Sϵ, is replaced with prominent recombination hotspot Sμ (SμKI). Inserted Sμ out-competed both endogenous Sγ1 and Sϵ to produce copious polyclonal IgE upon challenge, providing a platform to study IgE biology and therapeutic interventions. Intra-switch recombination (ISR) within endogenous Sμ is also diminished in SμKI mice, although the insertion itself exhibits no ISR. Furthermore, reciprocal translocations between c-myc and endogenous Sμ are dramatically lower in SμKI mice, suggesting the knocked-in switch sequesters critical factors. We propose competition among S regions influences CSR, ISR, and chromosomal translocation.
Human blood-derived raw material: enabling controlled, consistent collection (P4415)
Authors: Ramos Thomas , Busby William , McPherson Gaytha , Burger Scott
Published: January 2023

Human cells are critical raw materials for manufacturing cell therapy products, but often introduce significant variability. Rigorous operational controls and quality systems, however, enable optimal collection of high-quality, consistent cellular material. HemaCare, a long-standing supplier of human-derived blood components, controls apheresis procedures and collection sites under a formal quality system, with GMP-compliant, validated procedures and equipment, and GTP-compliant donor screening and tracking. HemaCare performed 69,658 cellular apheresis collections in the last five years, including patient and normal-donor MNCs, G-CSF-mobilized PBPCs and plateletpheresis products, for use in research, clinical trials, and commercial products. Expanded capabilities include bone marrow, umbilical cord blood, and cord tissue collection, immunomagnetic selection, cryopreservation, and analysis by flow cytometry. HemaCare unmobilized apheresis products showed consistently high MNC purity, with 93.8% of products containing ≥75% MNC, and an average of 85.2% MNC ± 6.6% (mean ± 1 SD). Red blood cell contamination was low, with hematocrit averaging 1.8% ± 0.8%. Approximately 85% of HemaCare donors have donated apheresis products 5 or more times, and this repeat-donor pool also contributes to product consistency, as MNC content of individual donor apheresis products had an average coefficient of variation of 3.5%, compared to a CV of 7.7% for all apheresis products.
Evaluation of the adjuvant properties of chitosan-poly vinyl pyrrolidone nanoparticles (P4423)
Authors: Izaguirre Irma , Tenorio Aldo , Altuzar Victor , Mendoza Claudia , Vivanco Hector
Published: January 2023

Polymeric Nanoparticles (NPs) have recently been shown to possess significant potential as drug or antigen delivery systems. In addition to the carrier function, the adjuvant capacity is also an important and desirable property in the context of vaccination. Here, we evaluated the adjuvant properties for a new NPs formulation based on a two-polymer combination: CS-PVP. The NPs were synthesized by ionic gelation and characterized by TEM, DLS and FTIR. Moreover, biological properties of NPs were addressed in vitro by studying their effect on cell viability and Dendritic Cells (DCs) maturation. 5 x105 bone marrow DCs were stimulated with CS-PVP NPs at different concentrations for 24 hours. At the end of the time, we analyzed the surface expression for costimulatory molecules by flow cytometry. Our results showed an enhanced surface expression of CD40, CD80, CD86 and MHC II on NPs stimulated-DCs at two different concentrations: 250 y 500 μg/mL, confirming the maturation phenotype. Using Human Monocyte-derived Dendritic Cells, we evaluated by ELISA the production of TNF-α and IL-6. 2 x105 Human DCs were stimulated for 24 hours with 300 μg/mL of CS-PVP NPs. Intermediate levels of both TNF-α (260 pg/ml) and IL-6 (75 pg/mL) were detected comparing against LPS. These results clearly indicate that CS-PVP NPs formulation active both murine and human DCs, opening the possibility for using such as a potential adjuvant in vaccination.
In vivo PPAR-gamma activation inhibits MyD88 expression to protect mice from polymicrobial sepsis. (P4425)
Authors: Ferreira Ana , Sisti Flavia , Filgueiras Luciano , Wang Sue , Wang Julia , Cunha Fernando , Alves-Filho Jose , Serezani C. Henrique
Published: January 2023

Sepsis is a disorder characterized by systemic inflammatory response caused by an infectious agent and may progress to multiple organ dysfunction and death. Overwhelming inflammatory response is believed to underlie tissue damage and preceding multiple organ disfunction syndrome. Thus, therapeutic strategies to inhibit excessive inflammation in sepsis is needed. The nuclear receptor peroxisossome proliferating factor (PPAR)-g exhibit anti-inflammatory properties in different immune cells. We hypothesize that prolonged PPAR-g activation decreases systemic inflammatory responses, improving sepsis outcome. We employed a model of polymicrobial sepsis (cecum and ligation puncture-CLP) to evaluate whether prolonged PPAR-g activation inhibits inflammation and improves animal survival. PPAR-g mRNA expression is increased in both peritoneal and lung cells at 6 h after CLP. Our data show that the PPAR-g agonist pioglitazone treatment for 18 h enhances bacterial clearance in the peritoneal cavity; which was associated with enhanced neutrophil migration to the infectious focus. These data correlates with lower expression of the TLR adaptor MyD88 in pioglitazone in vivo and peritoneal macrophages in vitro. Together, our findings suggest that PPAR-g activation impairs TLR responses, by inhibiting expression of MyD88 and preventing overwhelming inflammatory response.
CD56negCD16+ NK cells are mature NK cells generated from CD56+CD16+ NK cells during HIV-1 infection (P4438)
Authors: York Vanessa , Milush Jeffrey , López-Vergès Sandra , Deeks Steven , Martin Jeffrey , Hecht Frederick , Lanier Lewis , Nixon Douglas
Published: January 2023

A subset of CD3negCD56negCD16+ Natural Killer (NK) cells is highly expanded during chronic HIV-1 infection, and the role of this subset in HIV-1 pathogenesis remains unclear. The lack of NK cell lineage-specific markers has complicated the study of minor NK cell subpopulations. Using CD7 as an additional NK cell marker, we found that CD3negCD56negCD16+ cells are a mixed population of CD7+ NK cells and CD7neg myeloid cells. CD7+CD56negCD16+ NK cells are significantly expanded in HIV-1 infection. CD7+CD56negCD16+ NK cells are mature and express KIRs, NKG2A, NKG2C, and natural cytotoxicity receptors similar to CD7+CD56+CD16+ cells. CD7+CD56neg NK cells in healthy donors produced minimal IFNg following K562 target cell or IL-12 plus IL-18 stimulation; however, they did degranulate in response to K562 cells similar to CD7+CD56+ NK cells. HIV-1 infection resulted in reduced IFNg secretion following K562 or cytokine stimulation by both NK cell subsets compared to healthy donors. Decreased granzyme B and perforin expression and increased expression of CD107a in the absence of stimulation, particularly in HIV-1-infected subjects, suggests that CD7+CD56negCD16+ NK cells may have recently engaged target cells. CD7+CD56negCD16+ NK cells have increased expression of the aging-related cyclin-dependent kinase inhibitors p16/INK4a and p21/Waf1. Taken together, CD7+CD56negCD16+ NK cells appear to be mature NK cells generated from chronic target engagement.
Effect of Clonorchis sinensis and N-nitrosodimethylamine on host cell proliferation and modulation of cancer related moleclues (P1356)
Authors: Kim Eun-min , Wi Hae Joo , Bae Young Mee
Published: January 2023

Clonorchis sinensis (C. sinensis) infection is known to cause cholangiocarcinomas in humans, but the mechanism is not clearly understood. In order to verify the role of excretory/secretory products (ESP) from C. sinensis in cell proliferation and the modulation of cancer related molecules, HEK293T cells were cultured with ESP and/or NDMA (N-nitrosodimethylamine), and then the mode of cell cycle and the expression of proteins were analyzed by using FACS, western blotting and confocal analysis. When the ESP of C. sinensis and NDMA were treated, the degree of cell proliferation was upregulated and the proportion of G2/M phase cells were increased by 25%-30% as compared with that of control. The expression pattern of the cell cycle proteins was also changed when both ESP and NDMA were added. Theses indicate that ESP from C. sinensis and NDMA synergistically affected the regulation of cell cycle proteins. In addition, the levels of Cox-2 and connexin 43 (Cx43) which is known as cancer related gap junction protein were upregulated by the addition of ESP and NDMA in vivo. In addition, the level of another gap junction protein, Cx32, which is also known as cancer inhibitor was down- regulated. Our results suggest that exposure to C. sinensis and a small amount of NDMA promote uncontrolled cellular proliferation of the bile duct epithelial cells and the upregulation of cancer related intracellular molecules.
GATA-3 regulation and stability in T helper type 2 cells (P1310)
Authors: Pellet Elsa , Peine Michael , Flossdorf Michael , Löhning Max , Höfer Thomas
Published: January 2023

GATA-3 and T-bet, the key transcription factors of T helper type 1 and type 2 cells, respectively, are specifically upregulated during differentiation of naive Th cells in response to antigen and cytokine signals. GATA-3 expression increases in response to IL-4/STAT6 signaling, whereas T-bet is induced by the IL-12/STAT4 and IFN-y/STAT1 pathways. GATA-3 and T-bet subsequently induce lineage-specific gene expression. Here, we study the regulation of GATA-3 in Th2 cells as well as the interactions between the Th1 and Th2 signaling pathways using time-resolved dose-response experiments and mathematical modeling. We measured the dose response of GATA-3, T-bet and pSTAT1/4/6 to IL-4 and IL-12 in vitro during differentiation and fitted mathematical models to the data. GATA-3 expression was graded and depended on the cytokines’ concentrations. The activation of STAT6 downstream of IL-4 and STAT4 downstream of IL-12 was stronger and longer-lasting with higher cytokine concentrations. We discovered several cross-regulatory mechanisms between the Th1 and Th2 pathways. Furthermore, the different amounts of GATA-3 reached in vitro were stably maintained in vivo for 30 days after transfer into naive mice, suggesting a cell-intrinsic mechanism allowing quantitative memory of GATA-3 expression in resting Th2 cells. Our results provide insight into the molecular mechanisms that underlie the induction and maintenance of Th2 cell differentiation.
Pulmonary hepcidin protects against acute lung injury (P3345)
Authors: Chen QiXing , Song ShengWen , Fang XiangMing
Published: January 2023

Hepcidin is a small cysteine-rich cationic peptide mainly produced by the liver, and is a principle iron regulatory hormone. Hepcidin could modulate acute inflammatory response via interaction with its receptor ferroportin. Recent studies showed that hepcidin was also expressed in tracheal epithelial cells. However, the role of this epithelia-derived hepcidin in lung inflammation remained unknown. Adenovirus-mediated hepcidin specific shRNA and a relative negative control were constructed and were administrated to mice intratracheally. Ten days later, the mice were subjected to cecal ligation and puncture to induce acute lung injury (ALI). The severity of lung injury was scored 24 hours after onset of ALI. The bacterial colony counts, white blood cell counts and total protein content in bronchoalveolar lavage fluid were also measured. Furthermore, the protein levels of ferroportin were analyzed using immunobloting. Adenovirus-mediated hepcidin specific shRNA significantly suppressed tracheal hepcidin levels ten days after administration. Interference of this epithelia-derived hepcidin increased bacterial proliferation, white blood cell counts and total protein content in bronchoalveolar lavage fluid, and thus exacerbated lung injury. However, the ferroportin levels were comparable between the two groups. Tracheal epithelia-derived hepcidin plays an important role in protecting against acute lung injury, which might function via a ferroportin-independent mechanism.
New fluorophore for multiplexing with violet laser excitation (P3289)
Authors: Anderson April , Low Quentin , Bone Bradley , Zhou Wenjun , Dubbels Bradley , Bradford Jolene , Gee Kyle
Published: January 2023

Flow cytometry instruments with solid state violet laser diodes are becoming more common due to their small size, low power requirements, cost, and reliability. The use of violet-excited fluorochromes in multiparametric flow cytometry has been essential in the expansion of multicolor flow cytometry, enabling greater numbers of markers to be detected in one sample. We have developed a novel violet-excited dye, Pacific Green™ dye, which has an excitation/emission maximum of 411 and 500 nm, respectively. This development addresses the need to transfer well-resolved markers off the Blue 488 nm and Red 635 nm excitation lines and onto the Violet 405 nm excitation line, thus enabling the detection of other markers with the 488 nm and 635 nm lasers. Pacific Green™ dye can be used for three color immunophenotyping using violet laser excitation with Pacific Blue™ and Pacific Orange ™ dyes with minimal compensation without 488 nm excitation. Data is shown for human lymphocytes and mouse splenocytes stained with Pacific Green™ direct conjugates. Six color immunophenotyping data is shown using direct conjugates of Pacific Green™, Alexa Fluor® 488, phycoerythrin, PE-Cy®7, Pacific Blue™ and Pacific Orange™ conjugates. Pacific Green™ dye can be used with the far red emitting QDot®605, 655, and 705 conjugates for higher multiplexing with violet laser excitation. Pacific Green™ dye can be paired with fixable blue or yellow-emitting viability stain with violet excitation to exclude dead cells.
Preservations in anhydrous sodium chloride reduces keratinocytes alloantigenicty. (P2223)
Authors: Domaszewska-Szostek Anna , Moscicka-Wesolowska Maria , Szymanska Beata , Olszewski Waldemar Lech
Published: January 2023

A large amount of human keratinocytes (KCs) is needed to cover burned skin areas. There is a need to develop an efficient method of long term preservation of these cells. We checked whether KCs isolated from skin preserved in anhydrous sodium chloride survive and can be successfully transplanted. Fragments of normal human skin were preserved in sodium chloride. After 7 or 30 days preservation they were rehydrated, the epidermis was separated and KCs were isolated. Subsequently, the cells were cultured for 7 or 21 days. Viability of cultured KCs was tested by the Viability/Cytotoxicity Test. KCs were transplanted to the dorsum of SCID mice. Histopathological evaluation revealed that the morphology of KCs was totally preserved. KCs from all epidermal layers could be identified. All grafts were taken by the recipients. Three weeks after transplantation, keratinocytes synthesized keratins 10 and 16, and expressed antigens specific for stem and transient amplifying cells, as well as markers of proliferation. Cells isolated from transplants and cultured in vitro remained viable and produced various enzymes. All graft cells remained HLA I+. Taken together, keratinocytes isolated from human skin and preserved in anhydric sodium chloride present very low alloantigenicity can be successfully transplanted to SCID mice.
Aberrant glycosylation of plasma proteins in HIV-infected patients (P6361)
Authors: Ruhaak L. Renee , Williams Cynthia , Fenton Anne , Nagy Lauren , Hong Qiuting , Dandekar Satya , Lebrilla Carlito
Published: January 2023

Background: Glycosylation is one of the most abundant post-translational modifications, and is involved in protein structure formation and protein-protein interactions. Aberrant glycosylation profiles are often observed on plasma proteins from patients with inflammatory diseases. Increased levels of non-galactosylated glycans have been reported on serum IgGs of HIV-infected patients. We investigated the effects of HIV infection on protein glycosylation by N-glycomic profiling of plasma and plasma IgG. Methods: We obtained plasma samples of 22 HIV infected patients (11 therapy-naïve, 11 receiving anti-retroviral therapy) and 11 HIV-negative controls. First, a nano-LC-TOF-MS strategy was employed for the evaluation of plasma N-glycan profiles in each of the samples. Then, a UPLC-QQQ-MS method was used to evaluate the IgG specific glycosylation patterns. N-glycan peak integrals were used for biostatistical analysis. Results and conclusion: Several neutral, fucosylated and sialylated glycan compositions as well as high mannose type glycans in plasma samples were significantly altered in therapy-naïve HIV infected patients compared to controls. Moreover, galactose-deficient glycans were increased on the IgG in these patients, independent of IgG subclass. Interestingly, these effects were largely reduced in HIV infected patients receiving therapy. These results suggest an important role for protein glycosylation in immune dysfunction that is driven by active HIV infection.
Comparative lipidomics reveals a global sampling of major cellular membrane lipids by human CD1 proteins (P5006)
Authors: Huang Shouxiong , Cheng Tan-Yun , Altman John , Moody D. Branch
Published: January 2023

Human CD1 proteins have differentially shaped grooves and present bacterial or self lipid antigens to T cells, but it is unknown whether the pool of endogenous lipids are differently sampled by each type of CD1 proteins. We used recently established lipidomic approach to analyze the molecules extracted from human CD1a, CD1b, CD1c, and CD1d proteins. In the study, we demonstrated that this highly sensitive and accurate lipidomic approach detected more than one thousand diverse molecules defined by accurate mass retention time values, which represented hundreds of lipid species associated with CD1 proteins. More than fifty percent of these molecules were shared by all four types of CD1 proteins and additional thirty percent shared by two or three CD1 isoforms. Further mass spectrometry analyses of collision-induced dissociation showed that the molecules eluted from all four types of CD1 proteins mainly are lipids from subcellular membranes, including phospholipids of conventional, ether-linked, and lyso forms and sphingolipids. Surprisingly, CD1 isoforms with bigger grooves preferred to lipids with shorter chains, which can be explained by small spacer or scaffold lipids that bind together with antigens. Whereas previous studies argued that one lipid molecule blocks CD1 grooves, analogous to MHC class II-associated CLIP peptide, we conclude that CD1 proteins broadly sample cellular lipids and two small lipids are able to be adapted into a big groove.
MHC II-peptide complex conformation constrained by interactions throughout the peptide binding groove determines HLA-DM susceptibility (P5014)
Authors: Yin Liusong , Trenh Peter , Stern Lawrence
Published: January 2023

HLA-DM (DM) mediates the exchange of peptides loaded onto MHC II. However, the determinants of DM-mediated peptide release remain unclear and controversial. In this study, we synthesized a series of peptides derived from HLA-A2104-117 and measured their kinetic stabilities when bound to HLA-DR1 (DR1) in the absence or presence of DM. As expected from previous work, we found that peptides with non-optimal pocket 1 residues were highly DM susceptible. Surprisingly we found that substitution of the pocket 9 residue can counteract the low binding affinity, low kinetic stability and high DM-susceptibility of peptides containing non-optimal pocket 1 residues. Surface Plasmon Resonance demonstrated that DM bound well to DR1 loaded with a peptide variant with pocket 1 alanine (A1), while no binding was observed for a rescue variant with leucine at pocket 9 (A1L9) or for the wild-type peptide (W1Q9). We determined the crystal structure of DR1-A1L9 to 2.3Å resolution, confirming the expected binding frame with alanine at pocket 1. DR1-A1 appeared to adopt a different conformation which can be better detected and edited by DM, as evidenced by the sodium dodecyl sulfate-resistance and recognition by a conformation-specific monoclonal antibody UL-5A1. Together with the results that multiple substitutions influence DM-susceptibility, our data suggest that conformation of MHC II-peptide complex constrained by interactions throughout the peptide binding site determines DM-susceptibility.
Silicon-based peptide microarrays for epitope mapping of serum autoantibodies from patients with systemic lupus erythematosus with single-amino acid resolution (P4030)
Authors: Haddon David , Jarrell Justin , Liu Chih-Long , Price Jordan , Tangsombatvisit Stephanie , Credo Grace , Xu Guangyu , Varma Madoo , Baechler Emily , Utz Paul
Published: January 2023

We have developed a silicon-based peptide microarray with >5,700 features, corresponding to 891 unique peptides derived from the U1-70 protein, a well-known target of autoantibodies in systemic lupus erythematosus (SLE). U1-70 is a component of the U1-small nuclear ribonucleoprotein (U1-snRNP) complex, which is one of five snRNPs that comprise the spliceosome. The peptides on the microarray represent every possible overlapping peptide, up to 21 amino acids in length, spanning amino acids 110 to 170 within the U1-70 RNA binding domain. The microarray includes regions of U1-70 targeted by SLE patient autoantibodies, although the precise epitopes are not known. Additionally, the region includes a peptide (131-150) that our lab recently identified as a target of autoreactive CD4+ T cells. We have validated the microarray using commercial anti-U1-70 antibodies, and have shown that this array is capable of sensitive and specific determination of their minimal epitopes. We went on to evaluate a cohort of SLE (n=24) patients and identified two minimal reactive epitopes, peptides 116-121 (n=3) and 143-148 (n=1), of their U1-70 autoantibodies. Further analysis of a larger SLE cohort (n=80) by peptide ELISA revealed additional patients with reactivity to peptides 116-121 (1/80) and 143-148 (5/80). Currently we are correlating reactivity to these peptides with patient clinical information in an effort to better understand the development and function of anti-U1-70 autoantibodies in SLE.
Higher cytotoxic capacity in peripheral blood of sarcoidosis patients (P3135)
Authors: Wahlström Jan , Enger Tobias , Lorenz Daniel , Rao Ramana , Forsslund Helena , Rahman Jubayer , Skold Magnus , Kullberg Susanna , Eklund Anders , Brighenti Susanna , Grunewald Johan
Published: January 2023

BACKGROUND. Sarcoidosis is a granulomatous disease of unknown etiology, mainly affecting the lungs, where greatly increased numbers of CD4+ T cells typically are seen. However, CD8+ T cells can sometimes make up the majority of cells recovered by bronchoalveolar lavage (BAL). Previous studies have extensively studied Th1 cytokine-producing T cells and established their role in the pathological process. Little is however known about cytotoxic cells in sarcoidosis. METHODS: Peripheral blood (from 17 sarcoidosis patients and 11 healthy controls) and BAL fluid (from 19 patients and 4 controls) was obtained. Flow cytometric analysis of cell subsets was performed after staining with antibodies against the surface molecules CD3, CD4 and CD8 as well as antibodies against the cytotoxic intracellular mediators perforin, granzyme B and granulysine. The PBMC cytotoxic activity was quantified with the 51Cr release assay. RESULTS: Sarcoidosis patients had a significantly higher percentage of both perforin+ (p < 0.001) and granzyme B+ (p < 0.05) CD8+ T cells in blood. In BAL, however, a tendency in the opposite direction was seen. Higher cytotoxic activity was observed in PBMC from patients with sarcoidosis compared to controls. CONCLUSION: Cytotoxic cells, including CD8+ T cells, may be involved in the propagation or regulation of the inflammatory process in sarcoidosis, but further studies are needed to delineate their role in this disease.
γδ T cells and Th17 collaborate in CD18hypo PL/J psoriasiform dermatitis (P3139)
Authors: Gatzka Martina , Hainzl Heidi , Peters Thorsten , Singh Kamayani , Tasdogan Alpaslan , Wlaschek Meinhard , Scharffetter-Kochanek Karin
Published: January 2023

The contribution of IL-17 to the pathogenesis of psoriasis is substantiated by the therapeutic efficacy of antibodies against the common p40 subunit of IL-12 and IL-23, like Ustekinumab, suppressing Th17 and Th1 signature cytokine production. To dissect the roles of distinct IL-17 producing T cell subsets in psoriasis, we systematically analyzed the occurrence of γδ and CD4+ T cells in blood, lymph nodes and skin of CD18hypo PL/J mice, developing psoriasiform dermatitis at 12-14 weeks of age due to reduced expression of CD18/β2 integrin to 2-16% of wild-type levels, and assessed the effects of blocking different targets within the IL-23/IL-17 axis. Severity of CD18hypo PL/J psoriasiform dermatitis correlated with a loss of skin-resident Vγ5+ T cells and concurrent skin infiltration with inflammatory γδTCRlow cells preceded by increases in Vγ4+ T cells in local lymph nodes. Moreover, inflammatory CD18hypo PL/J γδ T cells enhanced allogeneic CD4+ T cell responses in vitro. Injection of diseased CD18hypo PL/J mice with anti-γδTCR, -IL-17, and -IL-23/p19 antibodies resulted in resolution of skin inflammation and eliminated pathological γδ T cells. These results for the first time demonstrate a critical function of pathological skin-infiltrating γδ T cells in a complex psoriasis model and of wild-type CD18 levels in their suppression.
Increased expression of PRRs and NOS in patients with endometriosis (P3143)
Authors: Park Dong Choon , Yeo Joon Hyung , Yeo Seung Geun
Published: January 2023

Objective: To assess these immune responses, we evaluated the levels of expression of Toll-like receptors (TLR)-1, -2, -4, -5, and -9; nucleotide-binding oligomerization domains (NOD)-1 and -2; interleukins-1β, -6, -8, -10, and -12; interferon-γ; tumor necrosis factor-α; inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS); and immunoglobulins (Igs) in patients with endometriosis. Methods: The levels of TLRs, NODs, cytokines, and NOS mRNAs in peritoneal effusion were assessed by real time polymerase chain reaction, and IgG, IgA and IgM concentrations were measured by enzyme-linked immunosorbent assays (ELISA) in 40 patients with and 40 without endometriosis. Findings from the two groups were compared. Results: We observed expression of all Pattern Recognition Receptors (PRRs), cytokines, and NOS mRNAs and immunoglobulins in the effusion fluid of patients with and without endometriosis. The levels of TLR-2 and -9; NOD-1 and -2; iNOS and eNOS mRNA and CA 125 were significantly higher in the endometriosis than in the non-endometriosis group (p<0.05 each). Moreover, PRRs, cytokine, and NOS showed significant correlations (p<0.05). Conclusions: PRRs, cytokines, and NOS, which act cooperatively in the innate immune response, are closely associated with endometriosis. Increased expression of PRRs and NOS in peritoneal fluid may be associated with endometriosis.
Pathophysiology of cytopenias in an autoimmune lymphoproliferative disease linked to bone marrow fibrosis (P3165)
Authors: Mailloux Adam , Zhang Ling , Moscinski Lynn , Bennett John , Yang Lili , Yoder Sean , Sokol Lubomir , Loughran Thomas , Epling-Burnette Pearlie
Published: January 2023

Large Granular Lymphocyte (LGL) leukemia is associated with unexplained cytopenias, autoimmune features, and CD57+CD8+perforin+ T-cell lympoproliferation. Like many autoimmune disorders, the pathophysiology of cytopenias in LGL leukemia is unknown. Bone marrow (BM) pathology from 24 LGL leukemia patients was retrospectively studied using H&E, reticulin, and trichrome-stained sections, and revealed that BM fibrosis was present in 21 out of 24 patients (88%). Fibrosis, a previously undefined complication, was significantly associated with splenomegaly, the presence of cytopenias, and coexistance of autoimmune diseases. LGL cell numbers in the BM, but not peripheral blood, was associated with fibrosis severity. Colony forming assays in the presence of exogenous bioartificial collagen matrix using healthy bone marrow mononuclear cells (BM-MNCs) revealed a direct inhibitory effect of collagen on progenitor growth. Consistent with microarray analysis, primary mesenchymal stromal cultures (MSCs) from LGL leukemia BM displayed heightened collagen deposition. In co-culture assays, collagen matrix produced by LGL MSCs directly inhibited healthy hematopoietic progenitor growth support. This study demonstrates that excessive collagen matrix deposition by deregulated MSCs may result from the local BM accumulation of activated CD8+ LGL leukemia T-cells. Therefore, BM fibrosis should be explored as a mechanism for secondary cytopenias associated with autoimmune diseases.
Design of less immunogenic therapeutic factor VIII proteins (P3169)
Authors: Pratt Kathleen , James Eddie , Puranik Komal , Kwok WIlliam , Ettinger Ruth
Published: January 2023

Hemophilia A patients lack functional factor VIII (FVIII), which is essential for normal blood clotting. Approximately 1 in 4 patients receiving replacement therapy by infusions of FVIII develop neutralizing antibodies that can cause serious bleeds. Tetramer-guided epitope mapping (TGEM) experiments identified an HLA-DRB1*01:01-restricted T-cell epitope in 2 subjects with missense substitution A2201P. The same epitope was identified using samples from a subject with severe hemophilia. T-cell clones were isolated by single-cell sorting and expansion of tetramer-positive CD4 T cells. Peptide-MHC binding assays using recombinant DR0101 and FVIII peptides with systematic arginine substitutions identified anchor residues F2196, M2199, A2201 and S2204. Recombinant FVIII-C2 domain with substitution F2196A abrogated proliferation of 4 clones. Six FVIII proteins with rational substitutions designed to interfere with epitope presentation (F2196A, F2196K, F2196L, M2199A, M2199W and M2199R) were produced in a BHK expression system. All had pro-coagulant activities, measured by both clotting and chromogenic assays, similar to that of wild-type FVIII. Their immunogenicity is now being evaluated. These experiments provide proof of principle for the design of less immunogenic, functional FVIII proteins targeted to specific subsets of patients.
Diminished B cell response and peripheral B cell abnormalities in patients with SLE are linked to disturbances of the spleen tyrosine kinase (P4056)
Authors: Fleischer Sarah , Daridon Capucine , Dörner Thomas
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a breakdown of self-tolerance, autoantibody production, B cell hyperreactivity and an impaired B cell homeostasis. Recent data suggested that an impaired B cell response could also lead to autoimmunity. In this study, the BCR downstream signaling spleen tyrosine kinase Syk of healthy donors (HD), SLE, rheumatoid arthritis (RA) and primary Sjögren’s syndrome (pSS) patients was analyzed in detail and revealed new insights into peripheral B cell abnormalities based on intracellular characteristics. Interestingly, SLE B cells showed a significantly lower but long-lasting Syk (Y352) phosphorylation after BCR engagement compared to HD (p<0.05). Furthermore, a significant enlarged frequency of Syk(bright) B cell population has been identified within the CD27(-) B cell subset in the blood of SLE patients (p<0.001), but no correlation with the disease activity was observed. Notably, the frequency of Syk(bright) B cells was not increased in RA or pSS patients. Further characterization showed that Syk(bright) cells exhibit an increased basal level of phosphorylated Syk, expressed higher levels of CD19 and CD95 and mainly do not express CD38 as compared to Syk(dim) cells. Thus, SLE patients show a diminished but long-lasting BCR response and exhibit an enlarged unique Syk(bright) B cell subset that could be responsible for the hyperactivity of SLE B cells.
Germinal center B cells but not TFH require T cell-derived IL21 in a model of autoimmune arthritis (P4068)
Authors: Block Katharine , Huang Haochu
Published: January 2023

IL21 is a pluripotent cytokine known to promote germinal center B cell differentiation and is thought be an autocrine factor for TFH differentiation and maintenance. The effects of autocrine IL21 on TFH have been addressed in several mouse models, but its requirement is still disputed. We investigated the importance of IL21 as a T cell autocrine factor in a model of autoimmune arthritis. Adoptive transfer of KRN transgenic CD4+ T cells into appropriate hosts drives germinal center formation and autoantibody production against glucose-6-phosphate isomerase, leading to inflammation and joint destruction. By comparing transfer of T cells deficient in IL21 to transfer of T cells lacking IL21R we were able to distinguish whether T cells themselves require IL21 to induce disease or whether it is required exclusively by other cell types. We found that T cells deficient in IL21 did not induce germinal center formation or autoantibody production, but had normal TFH differentiation. Transfer of T cells lacking IL21R was similar to wild-type transfer in antibody titer, GC B cell frequency, and TFH frequency both early and late in disease. To test whether IL21R signaling was required by autoreactive B cells to induce disease, KRN T cells and IL21R sufficient or deficient B cells were transferred into T and B cell deficient hosts. Mice that received IL21R deficient B cells did not develop arthritis, demonstrating that B cells require IL21 from TFH for a proper germinal center response.
Combination of agonist anti-OX40 therapy with CTLA-4 blockade augments anti-tumor effector CD4 and CD8 T cells (P4318)
Authors: Redmond William , Linch Stefanie , Kasiewicz Melissa
Published: January 2023

Ligation of the TNF receptor family co-stimulatory molecule OX40 (CD134) with an agonist anti-OX40 mAb enhances anti-tumor immunity by augmenting CD4 and CD8 T cell clonal expansion, effector differentiation, as well as turning off the suppressive activity of FoxP3+CD4+ regulatory T cells (Treg). Moreover, the targeted blockade of the checkpoint inhibitor CTLA-4 on CD4 effector and Treg cells enhances anti-tumor immunity by boosting effector T cell responses and alleviating the suppressive capacity of Treg. However, monotherapy with anti-OX40 or anti-CTLA-4 mAb can have limited efficacy, particularly against poorly immunogenic tumors. Therefore, we investigated whether combination immunotherapy could overcome this defect to generate optimal therapeutic responses. We demonstrate that combined anti-OX40/anti-CTLA-4 therapy significantly enhanced tumor regression and survival of TRAMP-C1 prostate or MCA-205 sarcoma tumor-bearing hosts. Mechanistic studies revealed that combined anti-OX40/anti-CTLA-4 therapy elicited CD8 T cell proliferation and differentiation as well as the expansion of effector CD4 T cells producing Th1 and Th2 cytokines, but did not inhibit the expansion or suppressive function of Treg cells. Together, these data demonstrate that combined anti-OX40/anti-CTLA-4 immunotherapy elicits potent anti-tumor immunity and indicate that clinical evaluation of this combination regimen to boost tumor immunotherapy is warranted.
Inhibition of T cell protein tyrosine phosphatase enhances IL-18-dependent hematopoietic stem cell expansion (P6268)
Authors: Bourdeau Annie , Trop Sebastien , Doody Karen , Dumont Daniel , Tremblay Michel
Published: January 2023

The clinical application of hematopoietic progenitor cell-based therapies for the treatment of hematological diseases is hindered by current protocols, which are cumbersome and have limited efficacy to augment the progenitor cell pool. We report that inhibition of T cell protein tyrosine phosphatase (TC-PTP), an enzyme involved in the regulation of cytokine signaling, through gene knockout results in a 9-fold increase in the number of hematopoietic progenitors in murine bone marrow. This effect could be reproduced using a short (48 h) treatment with a pharmacological inhibitor of TC-PTP in murine bone marrow, as well as in human bone marrow peripheral blood and cord blood. We also demonstrate that the ex-vivo use of TC-PTP inhibitor only provides a temporary effect on stem cells and did not alter their capacity to reconstitute all hematopoietic components in vivo. We establish that one of the mechanisms whereby inhibition of TC-PTP mediates its effects involves the IL-18 signaling pathway, leading to increased production of IL-12 and IFN-gamma by progenitor cells. Together, our results reveal a previously unrecognized role for IL-18 in contributing to the augmentation of the stem cell pool, and provide a novel and simple method to rapidly expand progenitor cells from a variety of sources using a pharmacological compound.
Expression and clinical significance of CCR9 in prostate cancer (P6276)
Authors: Singh Shailesh , Singh Rajesh , Gupta Pranav , Lillard, Jr. James , Grizzle William
Published: January 2023

Despite recent advances in diagnosis and treatment, prostate cancer is the second leading cause of cancer-related deaths in US. Precise etiology and molecular target to treat prostate cancer is yet to be defined. In recent years, involvement of chemokines and their corresponding receptors in pathogenesis of prostate cancer has gained tremendous attention. We have previously shown that CC chemokines receptor-9 (CCR9) is significantly expressed in prostate cancer cell lines compared to normal epithelial cells derived from prostate. Furthermore, we have shown higher invasive and migratory potential of prostate cancer cells under the chemotactic gradient of CCL25, the only known natural ligand of CCR9. The aim of this study was to establish the clinical relevance of CCR9 in prostate cancer using tissue micro array (TMA). The TMAs were stained for CCR9 and virtual slides were created using an Aperio ScanScope scanning system to numerically analyze the Immuno-intensity of CCR9. The ScanScope generated true color digital images of each stained sample, which were viewed and analyzed using Aperio ImageScope (version 6.25 software). Our data show higher expression of CCR9 in prostate cancer tissues compared to match benign tissues. Interestingly, CCR9 immuno-intensity was stronger in tissues with Gleason grade 8 and above. This study suggests the potential involvement of CCR9 in pathogenesis of prostate cancer.
Autophagosome-enriched cancer vaccine strategy provides a novel non-genetic method to engineer leukocytes to express molecules that may augment cancer immunotherapy. (P4252)
Authors: Paustian Christopher , Hilton Traci , Van De Ven Rieneke , Haley Daniel , Twitty Chris , Hu Hong-Ming , Fox Bernard , Aung Sandra
Published: January 2023

CD80 expression by APC provides signal 2, a critical element in priming T cell immune responses to antigen. As limited CD80 expression on APC can result in reduced immune responses, strategies that increase CD80 may have benefit. We report here that the autophagosome-enriched vaccine named DRibbles (DRiPs and SLiPs in Blebs), derived from human tumor cells transfected with a plasmid encoding huCD80, are highly efficient at transferring membrane proteins onto human PBMCs. When PBMCs were incubated with a DRibble vaccine derived from a CD80 expressing breast-tumor cell line, a significant increase in surface expression of CD80 was observed on PBMCs within 5 minutes (p<0.001). This effect was time and concentration-dependent and repeatable across multiple donors (N=5) in multiple experiments (N≥16). DRibble vaccine could transfer CD80 onto as high as 60-86.4% of monocytes and 41-86.1% of CD4+ T cells. Transfer of CD80 to CD8 T cells was substantially less effective. Transfer of membrane-associated proteins between leukocyte populations is a well-documented phenomenon, however, this is the first report of transfer of membrane-associated proteins from cell-free autophagosomes. Current efforts are investigating the functional properties associated with increased expression of CD80. Given the critical role of CD80 in augmenting immune responses this provides a novel strategy to further augment the response to DRibble vaccines, which are being explored in clinical trials.
Age related pharmacokinetic and pharmacodynamic response of intranasal rGM-CSF (P4190)
Authors: Resetar Erin , Eichinger Katherine , Anderson Kacey , Empey Kerry
Published: January 2023

Respiratory syncytial virus (RSV) is one of the leading causes of respiratory tract infections in children. Nearly every child will be infected by the age of two, yet there is currently no vaccine or effective treatment for RSV infection. Previous studies have shown that immunotherapy, such as recombinant GM-CSF (rGM-CSF), expedites viral clearance, likely due to increased activation and accumulation of pulmonary antigen-presenting cells (APCs). This study examines the pharmacokinetics and pharmacodynamics of a weight-based, inhaled rGM-CSF dose in infant compared to adult mice. Using a Luminex assay, the concentration of rGM-CSF in digested lung tissue and blood was analyzed over 24 hours. In lung digest, rGM-CSF was rapidly detected in both ages with levels peaking at 1 hour and decreasing to endogenous levels by 8 hours. rGM-CSF levels in the blood peaked at 0.5 hours and decreased by 4 hours in pups, while adult levels peaked at 1 hour and decreased by 8 hours. Whole lung was analyzed by flow cytometry for macrophage activation at 4, 8, 24, and 48 hours post dose. In pups and adults, CD11c+/CD11b+ expression increased, suggesting activation or infiltration of APCs. CD86 and MHC class II expression increased significantly in adults, with a more marked MHCII increase at 48 hours. Using a non-compartmental pharmacokinetic modeling approach, we predict that increasing the dose from 50 to 170ng/g will achieve similar biological outcomes in pup compared to adult mice.
Proliferation promotes functional changes in NK cells during viral infection. (P6054)
Authors: Tarrio Margarite , Biron Christine
Published: January 2023

Following mouse cytomegalovirus (MCMV) challenge, NK cells control viral replication by killing infected cells and producing IFNγ in response to IL-12 induced at day 1.5 after infection. Previous work in our laboratory has demonstrated that under conditions of high viral replication, NK cells make significant amounts of IL-10 and deliver immunoregulatory functions at 3 to 4 days of infection (Lee et al., J. Exp. Med. 206:2235, 2009). The condition is accompanied by extensive NK cell proliferation. To test the hypothesis that proliferation is required for the changing function, culture systems were developed examining NK cell responses to IL-12 with and without division supported by high dose IL-2. Proliferating NK cells were induced to express the IL-10-reporter GFP in response to IL-12. One cell division was sufficient as was entrance into cell cycle measured by incorporation of BrdU. In contrast to intracellular IFNγ, proliferation was required for IL-10-GFP expression. Cell transfer studies into MCMV-infected mice demonstrated that IL-10-GFP levels were elevated with increasing divisions. When evaluated either in vitro or in vivo, decreasing intracellular IFNγ expression correlated with increasing IL-10-GFP expression as divisions progressed. Taken together, the studies reveal a previously unappreciated role for proliferation in shaping NK cell responses to infection. (Supported in part by the National Institutes of Health, USA.)
Lymphatic endothelial cells induce anergy of β-galactosidase specific CD4 T cells by providing antigen to CD11c cells (P1147)
Authors: Rouhani Sherin , Tewalt Eric , Davis Holly , Chen Lieping , Engelhard Victor
Published: January 2023

Lymphatic endothelial cells (LEC) express a variety of peripheral tissue antigens (Ags) and induce deletional tolerance of tyrosinase-specific CD8 T cells. LEC express MHC II molecules, suggesting they may tolerize CD4 T cells. We expressed β-galactosidase (β-gal) selectively in LEC using Lyve-1cre x β-galflox mice to investigate the ability of LEC to tolerize β-gal specific CD4 and CD8 T cells (Bg2 and Bg1, respectively). Using in vitro co-cultures and MHC I-/- bone marrow chimeras, we demonstrated that Bg1 CD8 T cells undergo deletional tolerance after recognizing Ag presented by MHC I molecules on LEC. Peptide-pulsed LEC induce Bg2 CD4 T cell proliferation in vitro, indicating LEC express functional MHC II. However, Bg2 cells proliferate when co-cultured with CD11c cells but not with LEC from β-gal+ mice. When Ag presentation is restricted to LEC in MHC II-/- bone marrow chimeras, Bg2 cells do not proliferate or upregulate CD69, CD25 or CD44. However, Bg2 cells do proliferate when transferred into chimeras with MHC II+ β-galneg bone marrow, demonstrating that hematopoietic cells acquire and present Ag from LEC. Bg2 cells activated in vivo by hematopoietic presentation of LEC-derived Ag do not proliferate in response to subsequent rechallenge, demonstrating that they have become anergic. Thus, LEC show a diminished ability to directly present MHC II Ags, but play an important role in CD4 tolerance by serving as a reservoir of Ag used by hematopoietic cells to induce anergy.