bluuebunny/crossref_metadata_2025 huggingface dataset:
Lymphoid organ specificity in the maintenance of secondary lymphoid tissues by TNF produced by B and T cells (95.26)
Authors: Tumanov Alexei , Grivennikov Sergei I , Kruglov Andrei A , Kuprash Dmitry V , Nedospasov Sergei A
Published: January 2023

Secondary lymphoid organs play an important role in generation of efficient immune responses. Using a genetic approach we revealed distinct contribution of TNF produced by B and T cells in homeostasis of secondary lymphoid organs. In spleen, the development of follicular dendritic cells (FDC) and B cell follicles was controlled primarily by TNF produced by B cells while TNF from T cells provided a distinct complementary signal, similarly to contributions reported earlier for the surface lymphotoxin (LT). Organized marginal zone structure was dependent on TNF from both B-, T- and other cell types. In contrast, in Peyer’s patches, the lymphoid microarchitecture was exclusively dependent on TNF produced by B cells. Furthermore, we show that TNF signals from both B and T cells acted in concert for the expression of TNF dependent genes in lymph nodes, generation of FDC and B cell follicles, and in mounting an efficient humoral immune response to thymus-dependent antigen. Finally, we found that soluble form of TNF provided by B cells was critical for the maintenance of secondary lymphoid organs. Thus, the organization of different secondary lymphoid organs is maintained by distinct contribution of TNF produced by B and T cells, on one hand, and distinct role of soluble versus membrane TNF form, on the other hand.
Immunorestorative effect of intranasal granulocyte/macrophage colony stimulating factor (GM-CSF) on alcoholic lung macrophages is mediated through local release of zinc (96.30)
Authors: Joshi Pratibha C , Guidot David M
Published: January 2023

Our recent studies have shown that chronic alcohol ingestion dampens alveolar macrophage immune function by decreasing GM-CSF receptor expression, and that these chronic defects can be reversed with rGM-CSF treatment in vivo (JI 2005). However, the mechanisms by which alcohol and GM-CSF modulate macrophage GM-CSF receptors are not clear. Zinc is an essential micronutrient that is implicated in many immunostimulatory functions. Since alcoholics are known to be deficient in zinc, we hypothesized that zinc may be responsible for the functional integrity of alveolar macrophages. To explore the impact of zinc deficiency on normal alveolar macrophage immune function, we treated these cells in vitro with or without a zinc chelator and then added zinc acetate before evaluating phagocytosis by flow cytometry. In parallel, we added 0.2% zinc acetate to the liquid diet of alcohol-fed rats. Finally, we measured zinc levels in the bronchoalveolar lavage from these animals using a zinc specific dye FluoZin-3. Zinc chelator treated alveolar macrophages showed a decrease in phagocytosis that was restored by zinc suggesting an impact of zinc deficiency on macrophage function. In in vivo model, zinc upregulated the membrane GM-CSF receptors on alveolar macrophages similar to that reported earlier with the in vivo GM-CSF treatment. The decreased levels of zinc in the lavage of alcohol-fed rats were significantly increased by in vivo zinc and intranasal GM-CSF. These data suggest a novel mechanism of zinc release in the lungs through which GM-CSF restores the immune function of the hyporesponsive alveolar macrophages from alcohol-fed animals.

Supported by P50 AA013757 and 2R01 AA011660-05A1
Gp100 is Chemotactic for Mononuclear Cells and Utilizes Chemokine Receptors (96.4)
Authors: Howard OM Zack , Buzas Krisztina , Dong Huifang , Oppenheim Joost J
Published: January 2023

Based on the observations that some patients vaccinated with melanoma antigens develop autoimmune vitiligo, and other cancer patients develop autoantibodies to tumor antigens, we tested two, melanoma tumor antigens gp100 and, NY-ESO-1 to determine if tumor antigens, like autoimmune antigens, are chemoattractants. We consistently observed a chemotactic response by human iDC, monocytes and T cells to nM concentrations of gp100, but not at all to NY-ESO-1. Using desensitization studies and receptor transfected HEK 293 cells, we showed that human gp100 interacts with human CCR2, the receptor for CCL2 (MCP1), and thereby accounted for its capacity to attract CCR2 expressing monocytes, T cells and iDC, but not mDC. Pathophysiological doses of CCL2 did not activate iDC, however, gp100 at concentrations of 2–10 mg/ml induced low levels of CD40, CD80, MHC Class II and functional CCR7. High levels of IL6, IL10, MDC, TARC and functionally active TGF-b were induced by gp100 stimulation. Based on CCR2 expression, we predict that gp100 can recruit innate, adaptive and regulatory immune cells to sites of tumor induced tissue damage. Consequently, the net effect of the tumor antigen stimulus can potentially result in inflammation, tumor immunity or tolerance depending on how it is presented to these cell types.

*Funded in part by DHHS #NO1-CO-12400
Ligand binding of leukocyte integrin Very Late Antigen-4 induces exposure of sulfhydryl groups and is subject to redox modulation by reactive oxygen species (97.10)
Authors: Liu Si-Yen , Tsai Ming-Yi , Chuang Kuo-Pin , Shieh Chi-Chang
Published: January 2023

Activation of leukocyte integrins is important for selective recruitment of cells into inflammatory tissues. Our previous studies showed that the binding between the integrin Very Late Antigen-4 (VLA-4) and Vascular Cell Adhesion Molecule-1 (VCAM-1) is modulated by reactive oxygen species. In this study we investigated the molecular nature of redox modulation on the activation states of VLA-4. We found that ligand binding of VLA-4 induced exposure of sulfhydryl groups on the α4 peptide. Moreover, low concentration (10μM), but not higher concentrations (≥100μM), of hydrogen peroxide enhanced the ligand binding ability of VLA-4 to VCAM-1. This activating effect may be dependent on S-glutathionylation of α4 subunit. Increased concentrations (up to15μM) glutathione in the medium enhanced the H2O2 induced activation of VLA-4. The redox regulation of the VLA-4 involves outside-in signaling and cytoskeleton rearrangement. In conclusion, our data indicate that ligand binding of VLA-4 involves redox modulation. Redox-sensitive site(s) thus may play a role in regulating the activation of VLA-4 in inflammatory tissues.
The adhesion molecules CD44 and hyaluronan, not physical trapping, are responsible for neutrophil sequestration in inflamed liver sinusoids (97.5)
Authors: McAvoy Erin , McDonald Braedon , Kubes Paul
Published: January 2023

Adhesion molecules important for neutrophil recruitment in many organs during endotoxemia are not involved in recruitment into the sinusoids of the liver. Using a whole body dual radiolabel adhesion molecules screen, we observed that hyaluronan (HA), a CD44 ligand, is disproportionately expressed in the liver (versus other organs) under both basal and endotoxemic conditions. Using intravital microscopy, we observed that blocking CD44-HA interactions reduced neutrophil adhesion in sinusoids but had no effect on rolling or adhesion in post-sinusoidal venules (mediated by α4 and β2 integrins). Inhibition of CD44-HA interactions, but not integrins, reduced the entry of neutrophils into the liver parenchyma and significantly improved LPS-induced hepatic injury. Anti-CD44 antibody rapidly detached adhering neutrophils in sinusoids and improved sinusoidal perfusion in endotoxemic mice, revealing CD44 as a potential therapeutic target in LPS-associated liver disease. In conclusion, the identification of CD44-HA as partners for neutrophil adhesion in sinusoids, disproves the hypothesis that physical trapping is entirely responsible for sequestration of neutrophils in sinusoids.

This work was funded by Alberta Heritage Foundation for Medical Research (AHFMR), and the Canadian Institutes of Health Research (CIHR).
Non-hematopoietic NADPH Oxidase Regulation of Lung Eosinophilia and Airway Hyperresponsiveness in Experimentally-Induced Asthma (98.3)
Authors: Abdala-Valencia Hiam , Earwood Julie , Babcock George , Garvy Beth , Wills-Karp Marsha , Cook-Mills Joan
Published: January 2023

Infiltration of eosinophils into the lung in experimental asthma is dependent on the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. Ligation of VCAM-1 activates endothelial cell NADPH oxidase which is required for VCAM-1-dependent leukocyte migration in vitro. To examine whether endothelial-derived NADPH oxidase modulates eosinophil recruitment in vivo, mice deficient in NADPH oxidase (CYBB mice) were irradiated and received wild type hematopoietic cells to generate chimeric CYBB mice. In response to OVA challenge, the chimeric CYBB mice had increased numbers of eosinophils bound to the endothelium as well as reduced eosinophilia in the lung tissue and bronchoalveolar lavage. This occurred independent of changes in VCAM-1 expression, cytokine/chemokine levels (IL-5, IL-10, IL-13, IFNã, or eotaxin), or numbers of T cells, neutrophils or mononuclear cells in the lavage fluids or lung tissue of OVA-challenged mice. Importantly, the OVA-challenged chimeric CYBB mice had reduced airway hyperresponsiveness (AHR). The AHR in OVA-challenged chimeric CYBB mice was restored by bypassing the endothelium with intratracheal administration of eosinophils. These data suggest that VCAM-1 induction of NADPH oxidase in the endothelium is necessary for the eosinophil recruitment during allergic inflammation.

(supported by NIH HL069428, J.M.C-M.)
T-Cell Induced Colitis Is Associated With Enhanced Myelopoiesis, PMN Infiltration into Secondary Lymphoid Tissue and Modulation of T-Cell Activation (98.6)
Authors: Ostanin Dmitry V. , D’Souza Dwain , Hoffman Jason , Gray Laura , Pruett Stephen , Grisham Matthew B.
Published: January 2023

The objective of this study was to determine how the adoptive transfer of naïve T-cells into lymphopenic recipients affects myelopoiesis during the development of chronic colitis. We found that adoptive transfer of CD4+CD45RBhigh T-cells (5x105 cells) into RAG-1−/− recipients induced chronic colitis that was preceded by a dramatic leukocytosis/myelopoiesis in which total circulating leukocytes increased 5–6 fold compared to unmanipulated RAG-1−/− mice with PMNs representing >85% of the total blood leukocytes. In addition, we found that serum levels of the granulopoietic cytokines IL-17 and G-CSF increased by 4- and 9-fold, respectively compared to the healthy CD45RBlow→RAG-1−/− controls. Corresponding to these dramatic increases in systemic PMN production in the CD45RBhigh→ RAG-1−/− mice, were large and significant increases (30–50-fold) in the presence of granulocytes (Gr-1+/Mac-1+) in the spleen, MLNs and colonic lamina propria. Co-culture of irradiated leukocytes (5 x 103 cells) obtained from the MLNs or spleen (>60% PMNs) of colitic mice with CD4+ T-cells (105 cells) enhanced CD/CD28-induced T-cell proliferation by 2–3 fold. We conclude that the T-cell induced myelopoiesis and infiltration of PMNs into the secondary lymphoid tissue of lymphopenic recipients may enhance T-cell activation thereby modulating the onset and/or severity of colonic inflammation

(Supported DK64023).
In vitro generated antigen specific CD4+CD25+Foxp3+ regulatory T cells control the severity of virus induced immunoinflammatory lesion (99.3)
Authors: sehrawat sharvan , Suvas Susmit , Rouse Barry T
Published: January 2023

A previous model of stromal keratitis (SK) developed in our laboratory was established in which several TCR transgenic (Tg) mice back crossed to SCID or RAG KO were shown to develop typical SK lesions upon ocular infection with HSV. The lesions were judged to be the consequence of a bystander activation of T cells that in turn orchestrated tissue damage. Most studies were done with the CD4 TCR Tg crossed with DO11.10 RAG KO mice which recognize the OVA323-339 peptide. In current studies we evaluated if CD4+CD25+Foxp3+ OVA-specific Tregs were capable of modulating the bystander tissue damage caused by HSV infection. To this end, we generated CD4+CD25+Foxp3+ regulatory T cells with OVA-specificity by in-vitro culturing splenocytes from DO11.10 TCR Tg RAG KO mice for five days in presence of a cocktail TGFβ1(5ng/ml), OVA323-339 peptide (0.62uM) and rIL-2 (25U). A total of 1x106 CD4+CD25+Foxp3+ cells were adoptively transferred i.v. in DO11.10 TCR Tg Rag KO one day prior to ocular infection with 5x105 pfu of HSV I KOS. The SK lesion and angiogenesis were scored every alternate day beginning at day 4. The lesion severity (p=0.05) and the angiogenesis (p=0.005) were significantly reduced in treated mice as compared to untreated mice. The implications of these observations in terms of the necessary specificity characteristics of Treg to control tissue damage will be discussed.
Studying lineage relationships of T cells by cellular barcoding (99.7)
Authors: van Heijst Jeroen W.J. , Schepers Koen , Swart Erwin , Gerlach Carmen , Kerkhoven Ron M. , Arens Ramon , Schumacher Ton N.M.
Published: January 2023

Antigen-specific T cell responses are composed of phenotypically and functionally distinct subsets. However, it is often unclear how these subsets arise from a pool of naive T cells. To address such issues, we have developed a novel technique that allows the study of lineage relationships of hematopoietic cells. In this technique, progenitor cells are labeled with a unique stretch of DNA. Upon cell division, all offspring of a labeled progenitor will inherit this ‘barcode’. By analyzing the barcodes present in separate cell populations, it can be determined whether these populations are derived from a common progenitor.

To demonstrate the value of this technique, we analyzed kinship of CD8+ T cells accumulating at subcutaneous tumor and lung effector sites. Even though distinct T cells are primed in the draining lymph node of each site, the offspring of these cells is found back at both effector sites. This indicates that CD8+ T cells are endowed with the capacity to migrate to distinct effector sites, independent of the site of priming in this model.

In a parallel project, we have used cellular barcoding to determine the number of T cell progenitors recruited into the immune response under conditions of varying antigen dose. We find that at increasing antigen dose both the recruitment of progenitor cells as well as their rate of proliferation are increased, and thus both factors contribute to the size of the CD8+ T cell response.
Reduction of contact sensitivity responses to FITC, but not DNFB in mice lacking the tyrosine kinase, Itk (B103)
Authors: Ralph Kerry L. M. , Raymond Ernie L. , Sellati Rosemarie , Haynes Nancy , Thibodeau Michael S. , Nabozny Gerald H.
Published: January 2023

Itk (inducible T cell kinase) is a member of the Tec kinase family of nonreceptor protein tyrosine kinases that are expressed in T cells, NK cells, and mast cells. Defects in Itk protein lead to impaired T cell activation and effector function. Although mice lacking Itk are impaired in their ability to generate protective Th1 and Th2 responses in vivo, LCMV clearance requiring CD8+ T cells appear to be intact. Contact sensitivity (CS) is a type IV hypersensitivity reaction resulting from secondary skin exposure to a reactive hapten. Murine models of CS using chemical haptens such as DNFB are shown to be predominantly CD8+ T cell mediated with CD4+ T cells acting as modulators. Recently, data from CS models using FITC suggest a dominant role of Th2-type CD4+ T cells. The purpose of our studies was to determine whether Itk plays a role in the development of contact sensitivity responses to FITC or DNFB using Itk knockout mice. Indeed mice lacking Itk have significantly reduced ear swelling and leukocyte infiltration following FITC ear challenge compared to WT mice, which correlates with the lack of FITC-specific cytokine secretion from primed Itk KO LNC in vitro. In contrast, Itk−/− mice were able to generate ear swelling responses comparable to WT mice following DNFB challenge. Together, these data further support the effector role of CD4+ T cells in FITC-CS and the inability of Itk−/− mice to generate effective Th2 responses in vivo.
IL-17 is selectively induced by epicutaneous sensitization and mediates neutrophil recruitment to the lungs following antigen challenge (B104)
Authors: He Rui , Oyoshi Michiko , Geha Raif
Published: January 2023

IL-17 producing effector CD4+ T cells (TH17) have been recently identified to be a new lineage of T helper cells, and thought to be important contributors to tissue inflammation. We here show that IL-17 is selectively induced by epicutaneous (EC) sensitization with ovalbumin (OVA) compared to intraperitoneal (i.p.) immunization. This is evidenced by increased serum IL-17 and increased IL-17 production by OVA stimulated cells from draining lymph nodes and spleen in EC sensitized mice. Significantly higher expression of IL-17 and CXCL-2 and infiltration of neutrophils was found in the BAL fluid and lungs of EC-sensitized mice compared with i.p-immunized mice following inhaled OVA challenge. Blockade of IL-17 prior to inhaled OVA challenge reduced CXCL-2 expression, airway inflammation and responsiveness to methacholine in EC sensitized mice. FITC+ dendritic cells (DCs) collected from draining lymph nodes 24 hrs after painting the skin with FITC, but not DCs from spleen, induced IL-17 secretion by TCR-OVA CD4+ T cells. This was inhibited by intradermal injection of anti- TGFβ neutralizing antibody. These results indicate that IL-17 is selectively induced by EC sensitization, in part due to the presence of TGFβ in the skin, and suggest that IL-17 might play an important role in airway hyper-reactivity and neutrophil infiltration in the lungs when skin is the initial site of antigen sensitization.
Defining the role of BmIL-5 in tropical pulmonary eosinophilia (B110)
Authors: Kwon Sung (Steve) , Gnanasekar Munirathinam , Balakrishnan Anand Setty , Ramaswamy Kalyanasundaram
Published: January 2023

This study describes characterization of a novel pathogen-derived human IL-5 receptor binding molecule designated BmIL-5. This molecule was identified in our laboratory by screening a phage displayed cDNA expression library of the infective larvae of the human lymphatic filarial parasite, Brugia malayi. Soluble human IL-5 receptor (huIL-5R) was used as the bait. This screening procedure yielded a single clone and sequence analyses of the insert from this clone showed a novel protein designated as “Brugia malayi-derived IL-5-like molecule” (BmIL-5). Recombinant BmIL-5 was then expressed and purified. ELISA-based binding assays confirmed binding of rBmIL-5 to huIL-5R. Functional significance of this binding was then evaluated using a human eosinophil cell line, named Hs 454.T. Initial studies showed that BmIL-5 binds to eosinophil surface. To test whether this binding initiates any signaling events, we evaluated STAT1 phosphorylation status in the eosinophil. We also performed a series of experiments to identify the receptor binding region of BmIL-5 by peptide mapping. In these studies, we designed overlapping 20 mer peptides and evaluated the binding pattern of these peptides to huIL-5R. These studies identified peptide 1 and peptide 11 as the domains binding to huIL-5R. An interesting observation was that, both these peptides interfered with the binding of huIL-5 to huIL-5R in vitro. Thus, we believe that BmIL-5 interferes with IL-5 function by preventing IL-5 binding to IL-5R. Our findings demonstrate a novel method of how pathogens potentially manipulate the human immune system.
TSLP-TSLPR interactions regulate CD4+ T cell-dependent immunity and inflammation in the intestine (B112)
Authors: Taylor Betsy C. , Zaph Colby , Troy Amy , Berman-Booty Lisa , Du Yurong , Artis David
Published: January 2023

Thymic stromal lymphopoeitin (TSLP) is an IL-7-like cytokine expressed by epithelial cells that has been implicated in promoting Th2 cytokine-dependent inflammation in the skin and lung. However, the role of IEC-derived TSLP in regulating immunity and/or inflammation in the intestine has not been tested. Following exposure to the intestinal-dwelling nematode pathogen Trichuris, IECs isolated from genetically resistant C57BL/6 mice exhibit an upregulation in expression of TSLP mRNA. To address the functional role of TSLP-TSLPR interactions in the intestine, WT and TSLPR−/− mice were infected with Trichuris. While WT mice exhibited expression of Trichuris-specific Th2 cytokines in the mesenteric LN (mLN) and cleared infection, mLN cells isolated from TSLPR−/− mice secreted increased IFNγ over WT controls and these mice harbored persistent infection. Further, infected TSLPR−/− mice developed infection-induced intestinal inflammation. In vitro studies suggest IEC-derived TSLP can influence DC function. Consistent with this, DC isolated from the gut-associated lymphoid tissue of TSLPR−/− mice exhibited more IL-12/23p40 than WT DC. In addition, conditioning of WT BMDC with rTSLP inhibited LPS induced IL-12/23p40 in vitro. Taken together, these studies implicate a role for TSLP-TSLPR interactions in IEC-DC cross-talk in the intestine that limits the expression of proinflammatory cytokines.
Stimulation of IL-19 Expression by IL-13 and IL-17A in Human Airway Epithelium (B114)
Authors: Huang Fei , Thai Philip , Wachi Shinichiro , Wu Reen
Published: January 2023

Increasing evidences have shown that IL-19 is up-regulated in chronic inflammatory diseases, such as psoriasis, and asthma. Its significance is further highlighted for its ability to enhance Th2 cytokines expression in T cells, and inflammatory cytokines in monocytes. We have recently found that IL-17A and IL-13 are able to stimulate the expression of IL-19 secretion in both primary airway epithelium and human bronchial epithelium-1 (HBE1) cell line. Additionally, IL-17A, a proinflammatory cytokine which is crucial to many autoimmune diseases and bacterial infection, and IL-13, a Th2 cytokine which is predominated in allergic responses, can synergistically stimulate IL-19 expression. A similar synergism on IL-19 stimulation was observed when cells were treated with IL-4 and IL-17A. The synergism was not observed when IL-17A was co-treated with other proinflammatory, Th1, or immunosuppressive cytokines, such as TNF-α, IL-2, IFN-gamma, IL-10, etc. The increased expression of IL-19 is regulated by IL-13, and IL-17-induced STAT6, and NF-κB transcriptional activation respectively. In conclusion, the synergism of IL-13 and IL-17 on IL-19 production highly suggested that the airway mucosal immunity is regulated through an IL-13/IL-17A/IL-19 /Th2 cytokine axis. This axis needs to be further characterized and verified in allergic/Th2 dysregulated diseases.
Evualation of biocompatibility of epoxy resin-based, zinc oxide eugenol-based, calcium hydroxide-based root canal sealers by in situ proinflammatory cytokine production. (B118)
Authors: SOLIS-SOTO JUAN MANUEL , QUINTANILLA-RODRIGUEZ LIZETH EDITH
Published: January 2023

Histopathological studies have demonstrated that root canal sealers induce mild to severe inflammatory reactions. Proinflammatory cytokines are one of the main causative agents of inflammation. In situ production of these cytokines can be an indicative of citotoxicity. In the present work it is studied the in situ expression of proinflammatory cytokines as a biocompatibility marker for dental sealers of different composition. After inoculating the dorsal dermis of CD-1 mice with AH Plus (epoxy resin based), Tubliseal (Zinc-oxide eugenol based) and Sealapex (calcium hydroxide based), the tissue was prepared for immunocytochemical studies to analyze presence of IL-1beta, IL-6, IL-12, TNF-alpha and Interferon-gamma. AH Plus induced the strongest reaction (p<0.01), mainly in the acute inflammation, with immunoreactivity to IL-1beta, TNF-alpha and IL-12 in several cells in the inflammatory tissue and in the dermis. Few cells of the epidermis were immunoreactive to IL-1beta. No significative difference was found in the amount of immunoreactive cells after inoculation with Tubliseal and Sealapex (p<0.05), only a few cells were immunoreactive to IL-12, IL-1beta and TNF-alpha. Proinflammatory cytokines are an important key in the pathogenesis of the inflammation caused by root canal sealers. Calcium hydroxide based dental sealers showed better in situ biocompatibility.

This work was supported by grants from PROMEP (SEP) 103.5/05/2485 and PAICYT SA1420-06 (UANL).
Neonatal influenza infection induces persistent airway dysfunction (B119)
Authors: Cormier Stephania , You Dahui , Ripple Michael , Simms Tiffany
Published: January 2023

Infection with respiratory syncytial virus (RSV) and influenza is common during infancy. Interestingly, neonatal infection with RSV predisposes to persistent airway hyperreactivity (AHR), pulmonary inflammation, and Th2-skewed immune responses upon reinfection as an adult. A major difference in the host immune response to infection with influenza (Th1 dominant response) and RSV (Th2 response) exists. The present research sought to determine whether neonatal infection with a strong Th1 immune stimulator, such as influenza, could elicit an initial Th1 response and what the subsequent response to reinfection would be. Seven day old mice were infected with influenza (H1N1, 100 TCID50/mouse) or RSV (10e6 TCID50/mouse). Bronchoalveolar lavage fluid (BALF) was isolated to measure cytokines and cellularity at 10 d.p.i. (H1N1) or 6 d.p.i. (RSV). Pulmonary function, lung histology, and BALF cellularity and cytokine production were measured in adult mice (10–12 wks). In the acute phase of infection, H1N1 caused a Th1 response in the lung, characterized by INF-g production and neutrophil recruitment; while RSV led to a Th2 response characterized by IL-13 production. Both viruses elicited unresolved lung dysfunction and pulmonary inflammation characterized by elevations in alveolar macrophages. These data highlight the importance of delaying respiratory viral infections until the immune system matures.

This publication was made possible by NIH grant number P20 RR020159 from the LSU/Tulane COBRE-CEIDR Program of the National Center for Research Resources. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH.
Activated CD8 T cells can stimulate CD4 T cells by direct interaction (B12)
Authors: Romagnoli Pablo Alberto , Altman John D
Published: January 2023

Protective immunity against viruses is characterized by the generation of effective CD8 T cell memory in the presence of helper CD4 T cells. CD4 T cells impact in the programming of CD8 T cells by licensing antigen presenting cells (APCs) involved in priming. It is also known that CD4 T cells are necessary for activated CD8 T cells to differentiate into memory. However, it is not well understood how help is delivered at this second stage due to:

low probability of having CD4, CD8 T cells and APCs together; andabsence of accepted models where CD4 interact directly with CD8 T cells.

Here we propose a model of CD8:CD4 interaction where MHC class II molecules, transferred onto CD8 T cells by trogocytosis, are able to present antigen directly to CD4 T cells. Our results show that only antigen-specific CD8 T cells received MHC class II molecules transfer from APCs . These MHC class II positive activated CD8 T cells are able to prime naïve and restimulate experienced CD4 T cells to release great amounts of helper cytokines like IL2, IFNγ and TNFα and induce proliferation of antigen specific CD4 T cells in vivo.

These observations suggest a novel functional interaction between CD8 and CD4 T cells that might explain how help is delivered in the differentiation phase of memory CD8 T cells. Furthermore, this model would not only contemplate the occurrence of direct CD4 T cell help for CD8 T cells, but also the possiblity of an interface for immune regulation depending on the profile of CD4 T cells contacting the activated CD8 T cell.

Support for this Research comes from the National Institutes of Health, Grant: 1RO1AI042373-04, PI: John D. Altman.
Cellular neuropathologic effects of hemozoin, a toxin produced by Plasmodium falciparum, the causative agent of human malaria (B121)
Authors: Martiney James A , Harkins Caroline , Berman Joan W , Eugenin Eliseo A
Published: January 2023

Malaria, caused by an intracellular protozoan parasite of the genus Plasmodium, is responsible for 2–3 million deaths and 300–500 acute illnesses annually. A major morbidity factor is cerebral malaria, characterized by enhanced blood-brain barrier permeability, leukocyte infiltration and/or activation, and neuronal dropout. The pathogenesis of cerebral malaria remains unknown. Hemozoin, a by-product of intraerythrocytic parasite mediated hemoglobin catabolism, is released when the parasite lyses the host erythrocyte. This potential toxin is found in the brain parenchyma of cerebral malaria cases. The effects, if any, of hemozoin on human brain cells has not been studied in detail. Our data demonstrates that astrocytes actively phagocytize hemozoin, resulting in a time and concentration dependent toxicity and loss of viability. Further analysis demonstrated a secondary loss of neurons in astrocyte-neuron co-cultures. Morphologic characteristics of apoptosis such as membrane blebbing were observed in astrocytes exposed to hemozoin. Apoptosis was subsequently corroborated via Western blot analyses and TUNEL confocal microscopy. Taken together, our results suggests that hemozoin can induce significant pathogenic responses in CNS cells that can contribute to the development of cerebral malaria.

This work was supported by the NIH Centers for AIDS Research Grant AI-051519 and particularly by a KO1 grant from the National Institute of Mental Health to E.A.E. (MH076679).
Diverse and Distinctive Dendritic Cell Subtypes in the Respiratory Tract (B129)
Authors: Kim Taeg S , Braciale Thomas J
Published: January 2023

Dendritic cells (DCs), relatively rare subpopulations of cells widely distributed in organized lymphoid organs and periphery tissues, represent the most efficient antigen presenting cells capable of inducing robust pathogen-specific CD8+ and CD4+ T immunity in vivo. Upon pulmonary microbial infections, DCs residing in the respiratory tract (RDCs) acquire pathogen-derived Ags in the lung, leave the organ and enter the regional lymph nodes. The emigrated RDCs take a critical part in the induction of an adaptive cellular immune response. However, the nature of migratory RDCs is poorly understood, which is in part due to a tenuous characterization of RDC subtypes. We combined phenotypic analysis with functional assays to define a cellular composition of DCs in the respiratory tract of naïve mice. In the ends, we defined at least six subpopulations of CD11c+ cells including alveolar macrophages. Among the RDC subtypes, CD103+ and CD11b med-hi MHC II hi DC subsets feature greater CD86 expression and phagocytic/pinocytic activity than the rest. Our study shows the complexity of DC network in the respiratory tract, implying that the diverse subtypes of RDC may display a specialized role in establishing a protective host immune response upon pulmonary infection. Future study aims at probing a functional specialization among RDC subtypes in a respiratory viral infection model.

Supported by USPHS Grants AI-15608, HL-33391, AI-37293.
Endothelial cell apoptosis induced by Dengue virus (B133)
Authors: Yen Yu-Ting , Chen Hsuen-Chin , Lin Yang-Ding , Wu-Hsieh Betty
Published: January 2023

Vascular leakage and hemorrhagic diathesis are life-threatening complications that occur to dengue hemorrhagic patients. However, the pathogenic mechanism of hemorrhage is not well understood. It has been shown that endothelial cells are permissive to dengue virus (DV) infection in vitro and DV antigen is found in endothelial cells of autopsy specimens collected from patients who died of DHF. Therefore, the study of interaction between DV and endothelial cells will shed light on the mechanism of dengue hemorrhage.

We established a dengue hemorrhage mouse model in immunocompetent C57BL/6 mice by injecting DV serotype 2 strain 16681 intradermally. While high dose DV inoculation induced hemorrhage in all of the mice, 1/3 of those injected with 4–8 x 107 PFU developed hemorrhage in the subcutaneous tissues by day 3 of infection. Importantly, hemorrhage was accompanied by severe thrombocytopenia and vascular leakage. Double immunofluorescence staining revealed that DV targeted both macrophages and endothelial cells in the subcutaneous tissues. In vitro studies showed that human umbilical cord vein endothelial cells (HUVEC) and microvascular endothelial cell line (HMEC) were susceptible to DV and TNF-α enhanced DV-induced apoptosis. In addition, DV infection induced endothelial cell NOS and ROS production and endothelial cell apoptosis, which were also enhanced by TNF-α. These findings demonstrate that dengue virus and TNF-α-induced endothelial cell apoptosis is mediated by ROS and NO.
Interleukin-15: Multiple roles in controlling HIV infection in chimpanzees (B143)
Authors: Rodriguez Annette Rose , Hodara Vida L , Morrow LaShayla , Sanchez Melissa , Knudsvig Tracey , Murthy Krishna K
Published: January 2023

We have recently shown that Interleukin-15 (IL-15) plays a crucial role in eliciting interferon-gamma (IFN-γ) production from CD3−CD8+ NK cells in HIV infected chimpanzees (Pan troglodytes). CD3−CD8+ IFN-γ+ NK cells were consistently higher than CD3+CD8+ IFN-γ+ T cells and CD3−CD56+ IFN-γ+ NK cells by flow cytometry analysis. Accordingly, these findings prompted further investigation into the role of IL-15 during HIV infection. Analysis of fresh chimpanzee leukocytes revealed that a rare subpopulation of T cells express surface IL-15. HIV infected chimpanzees were shown express surface IL-15 on < 1 % to 3 % of T cells. Multiparameter flow cytometry analysis utilizing a whole blood system demonstrated that the cells were CD3+CD4+CD16+IL15+ and CD3+CD8+CD16+IL15+ T cells. These populations were shown to be CD25 negative, however, in vitro stimulation of PBMCs with interleukin-15 resulted in upregulation CD4+CD25+ T cells. Additional blood specimens from HIV infected chimpanzees examined during routine physicals also exhibited CD3−CD8+ IFN-γ+ NK cells > CD3+CD8+ IFN-γ+ T cells (p< 0.009, n=10). Research has shown that CD8+ lymphocytes play a substantial role in controlling HIV replication and declining CD4 cells leads to an increase in susceptibility to pathogens. During this study IL-15 was also shown to induce IFN-γ and TNF-α expression from this CD3−CD8+ NK cells. These findings provide additional insights on the influence of IL-15 and chimpanzee resistance mechanisms during HIV infection.
Ablation of Cbl-b Provides Protection Against Transplanted and Spontaneous Tumors (B146)
Authors: Chiang Jeffrey Y. , Jang Ihn Kyung , Hodes Richard , Gu Hua
Published: January 2023

A significant challenge to efforts aimed at inducing effective anti-tumor immune response is that CD8+ T cells, which play a prominent role in these responses, may be unable to respond to tumors that lack costimulatory signals and that are protected by an immune suppressive environment such as that mediated by TGF-β produced by tumor cells themselves or by infiltrating regulatory T cells (Treg), often resulting in tolerance or anergy of tumor-specific T cells. Here, we show that the in vitro activation of Cbl-b-deficient (Cbl-b−/−) CD8+ T cells does not depend on CD28 costimulation and is resistant to TGF-β suppression. In vivo studies further demonstrate that Cbl-b−/− mice, but not wildtype controls, efficiently reject inoculated E.G7 and EL4 lymphomas that do not express B7 ligands, and that introduction of Cbl−/− mutation into tumor-prone ataxia telangiectasia mutated (ATM)-deficient mice markedly reduces the incidence of spontaneous thymic lymphomas. Immunohistological study shows that E.G7 tumors from Cbl-b−/− but not wildtype mice contain massively infiltrating CD8+ T cells. Adoptive transfer of purified Cbl-b−/− but not wildtype CD8+ T cells into E.G7 tumor-bearing mice leads to efficient eradication of established tumors. Thus, our data indicate that ablation of Cbl-b can be an efficient strategy for eliciting immune responses against both inoculated and spontaneous tumors.
aAPC immunization activates low affinity T cells for treatment of tumors (B147)
Authors: Zoso Alessia , De Santo Carmela , Marigo Ilaria , Bennet Jonathon Drew , Zanovello Paola , Schneck Jonathan P. , Bronte Vincenzo , Oelke Mathias
Published: January 2023

The development of effective anti-tumor immune responses is normally constrained by low affinity tumor specific CTL which are unable to eradicate the tumor. Strategies to rescue anti-tumor activity of low affinity melanoma-specific CTL in vivo might improve immunotherapy effectiveness. To boost the in vivo efficiency of low affinity CTL clone we immunized mice with artificial Antigen Presenting Cells (aAPC), made by covalent coupling of pepMHC-Ig dimer and B7.1-Ig molecules to magnetic beads, in a lung metastatic tumor model. Systemic administration of aAPC resulted in the complete tumor eradication and was associated with proliferation of adoptively transferred low affinity CTL clones. In vitro studies showed that aAPC, in contrast to tumor cells effectively stimulated cytokine release and TCR down-regulation, explaining the efficient in vivo activation of low affinity CTL. Thus, aAPC represent a novel adjuvant for cancer immunotherapy.
Rat dendritic cells induce tumor cell death via a NO-dependent pathway (B152)
Authors: NICOLAS Alexandra , CATHELIN Dominique , FRASZCZAK Jennifer , BONNOTTE Bernard
Published: January 2023

Dendritic cells play an essential role in anti-tumor immune response as they are powerful antigen-presenting cells for activation of specific antitumor T lymphocytes. Recently, several reports reveal that dendritic cells may also act as potent cytotoxic cells against tumor cells. We demonstrated here that LPS-activated rat bone-marrow-derived dendritic cells (BMDCs) are cytotoxic against rat, mouse and human tumor cells. These LPS-activated BMDCs exhibit a mature DC phenotype, produce high amounts of IL-12, IL-6, TNF-a, and retain their T lymphocytes stimulation and phagocytic properties. The tumor cell death induced by these BMDCs involves high release of nitric oxide (NO). BMDC tumoricidal capacity is strongly reduced by NMMA, an inhibitor of the inducible NO synthase (iNOS). In addition, iNOS expression and high amounts of intracellular NO were detected in LPS-activated BMDCs cocultured in the presence of tumor cells. In line with these results, we demonstrated that intratumoral injection of LPS activates NO secretion by tumor-infiltrating DCs which impedes tumor progression.
Immunosurveillance of lung melanoma metastasis in EBI-3 deficient mice by IKDCs- induced CD8+ T cells (B154)
Authors: Sauer Kerstin Annika , Maxeiner Joachim Heinrich , Scholtes Petra , Karwot Roman , Lehr Hans Anton , Galle R. P. , Blumberg Richard Steven , Finotto Susetta
Published: January 2023

Epstein-Barr virus-induced gene 3 codes for a soluble type 1 receptor homologous to the p40 subunit of IL-12 that is expressed by antigen presenting cells following activation. Adoptive intravenous injection of the B16-F10 cell line resulted in a significant reduction of lung tumour metastasis in EBI-3 (−/−) recipient mice compared to wild type. Consistently, we found that EBI-3 (−/−) mice have decreased number of VCAM-1+ endothelial cells. The immunological finding accompanying this therapeutic effect was the orchestrate priming and T cell selection performed by a newly described Dendritic Cell (DC) subset called Interferon-γ producing killer Dendritic Cells (IKDC) which induced the CD8+ T cell response in the lung of EBI-3 deficient mice including Fas and TNF-α programmed tumour cell death. Finally, adoptive transfer of EBI-3 (−/−) DCs primed EBI-3 (−/−) CD8+ T cells into wild type mice decreased the number and size of lung tumour metastasis in recipient mice. Taken together, these data demonstrate that EBI-3 deficiency reduced lung tumour metastasis both because they decrease adhesion of tumour cells to lung vessels and they activate T cell mediated tumour cell apoptosis in the lung.
LAB/NTAL Regulates DAP12 Signaling and Macrophage Differentiation (B163)
Authors: Whittaker Gillian Claire , Quigley Laura , Zhang Weigou , McVicar Daniel
Published: January 2023

Recent studies have highlighted a significant, yet controversial role for DAP12 in the suppression of Toll receptor (TLR)-mediated inflammatory cytokine production by macrophages and plasmacytoid DC. Here we report that the Linker for Activation of T cells (LAT) and the Linker for Activation of B Cells (LAB) are critically regulated during myeloid development such that macrophages express LAB but little if any LAT. Consistent with LAB as a primary adaptor in macrophages, we readily observed its DAP12-inducible phosphorylation. Analogous to its role in Mast cells, we found that LAB also attenuates DAP12 signaling, however, the increased signaling in LAB−/− MΦ was not the result of increased utilization of LAT. Instead, LAB nucleated a DAP12-induced complex containing the E3 ligase c-Cbl and the p85 regulatory domain of PI3K. In the absence of LAB, DAP12-induced MAPK activation was ablated whereas c-Cbl was hyperphosphorylated resulting in enhanced p85/c-Cbl interactions. MΦ of DAP12 or TREM2−/− mice exhibit global increases in TLR-induced cytokine production, yet LPS stimulated LAB−/− BMM_ demonstrated increased IL-10 and decreased IL-12 p40 production. Together these data suggest that the utilization of LAB by TREM-2/DAP12 facilitates Erk1/2 activation while suppressing proximal signaling so as to yield macrophages with the appropriate balance of pro- and anti-inflammatory responses.
Hepcidin is synergistically induced in mouse macrophages by mycobacteria and IFN-γ, and is present in the mycobacterial phagosome (B166)
Authors: Sow Fatoumata Ba , Schlesinger Larry , Satoskar Abhay , Zwilling Bruce , Lafuse William
Published: January 2023

In mammals, infection results in the production of inflammatory cytokines and acute-phase reactants in the liver, including the synthesis of the antimicrobial peptide, hepcidin. Hepcidin controls extracellular iron by regulating its intestinal absorption and release from macrophages. It acts by binding to and mediating the degradation of the sole iron export protein ferroportin 1.

Here, we investigated the expression of hepcidin in mouse macrophages infected with the intracellular pathogens Mycobacterium avium and Mycobacterium tuberculosis H37Rv. We show that stimulation with mycobacteria and IFN-γ synergistically induce hepcidin mRNA in mouse macrophages. The induction of hepcidin mRNA by M. tuberculosis was found to be dependent on TLR2, TLR4, and STAT1. IL-6 and IL-1β, which induce hepcidin expression in liver hepatocytes, did not induce hepcidin mRNA expression in RAW264.7 cells. Iron loading, which induces hepcidin mRNA in the liver, inhibited hepcidin mRNA expression induced by IFN-γ and M. avium. These results implicate differential expression of hepcidin in macrophages and hepatocytes.

Immunofluorescence studies show that hepcidin is present in the M. avium- and M. tuberculosis-containing phagosomes. These results suggest that hepcidin production by infected macrophages is an IFN-γ induced host defense mechanism against mycobacteria.

This work was supported by grant NIH-RO1DK-57667
Innate immunity activation in mouse dendritic cells infected by Baculovirus (B188)
Authors: Hashimoto Kahoko , Suzuki Tomoyuki , Sakai Ryo , Miyazawa Yuki , Saito Ryo , Yamamoto Hiroshi , Nakayama Toshinori , Miyano-Kurosaki Naoko , Takaku Hiroshi
Published: January 2023

Baculovirus is an insect virus of double stranded, which has been used as a powerful tool for producing of recombinant proteins. In most cases, as mammalian cells have not been infected by the baculovirus, the infection can induce the activation in innate immunity system. For this reason, we investigated how baculovirus induce the innate immunity in the mouse bone marrow derived dendritic cells (BMDC). Among the lymphoid cells and tissues, baculovirus could activate BMDC and also macrophage cell line; RAW264.7 much more than T cells, B cells or NK cells. By the baculovirus infection, BMDC increased gene expression in many inflammatory cytokine genes, with increasing TLR genes. These results related the production of cytokines such as IL-12, IL6. IL-1 beta. These data could support our previous data, in which baculovirus infection could prevent the melanoma cells metastasis, and could recover the hepatocyte inflammation in vivo mouse models.
The Role of Beta Cell Antigen Presentation in Dendritic Cell-Based Type 1 Diabetes Immunotherapy (B189)
Authors: Xia Chang-Qing , Qiu Yushi , Peng Ruihua , Lo-Dauer Jeannette , Clare-Salzler Michael J
Published: January 2023

Dendritic cells (DCs) are promising cells for type 1 diabetes (T1D) immunotherapy. Most studies have been focusing on how to modulate DCs with tolerogenicity, but little attention has been directed towards the antigenic epitopes presented by DCs. In this study, we evaluated the roles of beta cell antigens presented by immature DCs (iDCs) in T1D prevention. At first, we established that beta cell antigen pulsing was required for iDCs to prevent T1D by using iDC pulsed with whole beta cell (NIT1) lysates. We further evaluated the capacity of different beta cell-derived antigenic peptides (Insulin B9-23, GAD78-97, GAD217-236, proInsulin C3-A19) in priming NOD splenic T cells in vitro. We discovered that GAD217-236 predominantly stimulated Th2 cells. Consistent with the in vitro results, 4 weekly intravenous injections of GAD217-236-pulsed iDCs significantly prevented diabetes in younger mice. Moreover, the same results were achieved when older NOD mice were treated. In the T cell recall experiments, it was noted that the splenocytes from the mice treated with GAD217-236-plused iDCs, in contrast to PBS-treated mice, produced significantly higher IL-4 and IL-10. Of interest, four beta cell antigenic peptides mentioned above stimulated the similar cytokine-producing pattern, suggesting that the immune response induced by GAD217-236-pulsed iDCs spreads to other beta cell antigens through epitope spreading.
Evaluation of cellular immune responses induced in mice by alphavirus replicon particle vaccines for SARS-CoV (B190)
Authors: Reap Elizabeth A , Norberg Pamela K , Watson Aubrey D , Cecil Chad , Wagner Jordan , Kamrud Kurt I , Smith Jonathan
Published: January 2023

An RNA replicon vector system derived from an attenuated strain of VEE virus was used to produce virus-like replicon particles (VRP) expressing SARS-CoV genes. We previously showed in mouse studies that complete protection was conferred by SARS S VRP and partial protection with SARS M VRP. In this study, two assays for cellular immunity were used to characterize the T cell phenotypes and cytokine profiles induced after SARS VRP vaccination. Following SARS M, N, S or E VRP immunization of BALB/c mice, splenocytes were harvested for antigen specific ICS and ELISPOTs. The SARS S VRP induced both CD4 and CD8 responses with the latter being the dominant component as measured by ICS for IFN-γ or TNF-α. Polyfunctional CD8 cells secreting both IFN-γ and TNF-α were also detected. Following SARS N VRP immunization, T cells secreting IFN-γ or TNF-α were readily detected with cells secreting both predominately identified as CD4 cells. Th1 cytokine secretion was not detected in cells from SARS M VRP inoculated mice. SARS E VRP elicited a robust CD8 response as shown by cytokine expression in cells stimulated with a 9mer peptide. IL-4 was not detectable after any SARS VRP vaccination. The IFN-γ ELISPOT results paralleled those from ICS, indicating that the SARS VRP vaccines induced Th1 biased responses, although the cytokines elicited differed depending on the SARS gene expressed.

This study was supported by NIH grant #UC1-AI62582