bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin-12 initiates an immune response that leads to tumour rejection (B151)
Authors: Nelles Megan Elizabeth , Labbe Alain , Walia Jagdeep , Jia Lintao , Furlonger Caren , Nonaka Takahiro , Medin Jeffrey A. , Paige Christopher J.
Published: January 2023

Immunotherapies are developed based on the notion that cancer cells can be targeted and eliminated by an appropriately stimulated immune system. Integral to both the innate and acquired immune systems, cytokines are ideal candidates for use in immunotherapy. We have developed two systems of interleukin-12 (IL-12) therapy, in a murine model, that elicit protective immune responses against the acute lymphoblastic leukemia (ALL) cell line, 70Z/3. The first system involves direct injection of IL-12, and the second is a tumour cell-mediated approach. Initially we found that direct delivery of low doses of IL-12 is sufficient to elicit a long-term protective immune response against an established tumour burden, mediated by both CD4+ and CD8+ T cells. Based on this knowledge, we created a lentiviral vector expressing murine IL-12, and transduced 70Z/3 cells to obtain IL-12 secreting tumor cells. We demonstrated that anti-tumour immunity in the second model is long lasting but is primarily dependent on the CD4+ T cell subset alone. Our results highlight that the mode of IL-12 delivery has a distinct impact on the immune response that is initiated and can lead to tumour clearance by way of disparate mechanisms. Furthermore, we demonstrate some of the underlying conditions within the tumour microenvironment that may have resulted in these differential outcomes.
Induction of active autoimmunity to the murine CD20 extracellular domain protects mice from tumor challenge in a model of B cell lymphoma. (B158)
Authors: Oscherwitz Jon , Gribbin Thomas , Cease Kemp Bailey
Published: January 2023

CD20 is a mostly B-cell restricted differentiation protein with a 40 amino acid extracellular domain (ECD) that has proven to be a clinically effective target for passive antibody therapy of B cell malignancies. Though there is interest in developing a mouse model to study the induction of active immunity to CD20, the CD20 ECD has significant secondary and tertiary structure which make it difficult to target with peptides. Our lab designs and studies the immunogenicity of recombinant immunogens which display peptides as tandemly repeated sequences. To target the CD20 ECD, we constructed and expressed a recombinant immunogen displaying six tandemly repeated copies of the N terminus of the murine CD20 ECD. Immunization of two inbred and one F1 strain of mice, and outbred rabbits, all led to the induction of antibodies reactive with the CD20 C-terminal peptide sequences by ELISA and more importantly, with native CD20 on the cell surface of the murine B cell lymphomas, A20 and 38-C13, by flow cytometry. The CD20-specific rabbit serum was also effective at immunoprecipitating CD20 from A20 cell lysate. Lastly, two independent experiments demonstrated that immunization of mice with the CD20 immunogen led to significant protection from a lethal challenge with the 38-C13 murine B cell lymphoma. Long term follow up of survivor mice demonstrated significant diminution of their auto antibody responses and no gross abnormalities.
Expansion of Functional Polyclonal Human T-regulatory Cell Lines Using Artificial Antigen Presenting Cells and Rapamycin. (B159)
Authors: Golovina Tatiana , Mikheeva Tatiana , Shan Xiaochuan , Suhoski Megan , Riley James , June Carl
Published: January 2023

T-regulatory CD4+CD25+ T lymphocytes play a central role in regulating immune response and preventing autoimmunity. There is a broad spectrum of potential clinical applications of expanded T-regs including but not limited to treatments of graft versus host disease, transplant rejection, and type I diabetes.

K562 cell line with stable expression of lentivirus-introduced CD64 and CD86 markers (KT64/86) and loaded with anti-CD3 antibodies was used to expand human CD4+CD25 high T lymphocytes. Though in several occasions stimulation of CD4+CD25high cells with KT64/86 only resulted in production of functional T-regulatory cells, only when antibiotic rapamycin was added to the cultures, functional Tregs were produced in 100% of stimulations. These cells were also active in vivo in reversing GVHD in NOG mice. Total CD4 lymphocytes grown in the presence of rapamycin though suppressive in vitro were not able to reverse GVHD in mice.

Substitution of CD86 with CD80 resulted in a less potent Tregs. Addition of certain members of TNF receptor family such as CD70, OX40 ligand or 4-1BB ligand to KT64/86 cells, had different effects on expansion of Treg cell lines. While CD70 addition did not show any impact, 4-1BB and OX-40 ligands in most cases stimulated growth of T-regulatory cell lines but at the same time in most cases also attenuated their activity.

The project is supported by JDRF Collaborative Center for Cell Therapy grant to C.J.
Mechanisms Study on Tolerogenic Dendritic Cells Induced by Apoptotic Cells (B161)
Authors: Ren Guangwen , Das Gobardhan , Zhang Liying , Roberts Arthur I , Shi Yufang
Published: January 2023

Apoptosis plays a pivotal function in maintaining the homeostasis of the immune system. Our previous experiments have demonstrated that i.v. administration of apoptotic cells of donor origin significantly prolonged the survival of allograft. However, the mechanism was unknown. In this report, we found that dendritic cells (DCs) play an important role in apoptotic cells-mediated immunosuppression. DCs fed with apoptotic lymphocytes (DCaps) down-regulated expression of MHC class II and costimulatory molecules CD80, CD86, as well as producing less IL-6, IL-12 and TNFalpha, but more IL-10. When DCaps were presented with antigen and then injected into the footpad of syngenic mice, they induced a much weaker immune response in draining lymph nodes compared to normal DCs. However, DCaps exhibited much less suppressive effect in ex vivo stimulation for CD4+ T cells in the presence of anti-CD3 when compared with the in vivo model. Surprisingly, the mixture of both normal DCs and DCaps could strikingly suppress T cells proliferation ex vivo. Further studies on the mechanisms of the helper role of normal DCs toward DCaps to get the immunosuppressive effect will strengthen the understanding of apoptotic cells-mediated immune tolerance.
Expression of TLR2 and TLR4 in Preterm Baboon Lung (B165)
Authors: Awasthi Shanjana , Cropper Jodie
Published: January 2023

Respiratory infections can cause significant morbidity and mortality in prematurely delivered babies. The increased susceptibility of preterm babies to infection is related to immaturity of the immune system. The Toll-like receptors (TLR) are expressed by antigen-presenting cells and play a key role in recognition of pathogens and regulation of immune response. However, their role in preterm babies is not well-understood. In this study, we investigated the expression of TLR2 and TLR4 proteins in lung tissues of fetal (delivered at 125, 140, 175 days of gestation age, dGA; complete term 185 days) and naturally-infected, prematurely delivered (125dGA ventilated with PRN O2) baboons. The expression of TLR2 and TLR4 was studied at protein level by Western blotting using anti-human-TLR2 and TLR4 antibodies. We found that the expression of TLR2 and TLR4 protein increased with the gestational age; the expression was found low in lung tissues of 125dGA baboon which increased to adult level at 175dGA. But in the lung tissues of naturally-infected preterm baboons, the protein levels of both TLR2 and TLR4 were significantly increased as compared to non-infected controls. Our results suggest that the expression of TLR2 and TLR4 is developmentally regulated and it may play a significant role in regulation of immune responses during infection in preterm babies.

Research Support by American Lung Association
NK Cells Lyse T Regulatory Cells in Human Infection with An Intracellular Pathogen (B167)
Authors: Roy S , Barnes P , Garg A , Wu S , Weis S , Rajavelu P , Cosman D , Vankayalapati R
Published: January 2023

Previously we found that regulatory T cells (CD4+CD25+Foxp3+ cells, Tregs) expand in response to M.tuberculosis (M. TB) through mechanism that depend on prostaglandin E2 production. In the current study, we determined the role of NK cells in regulating Tregs expansion in human M. TB infection. M. TB whole cell lysate (TB lysate) resulted in regulatory T cell expansion (15.33±1.02% vs 1.5±0.22%, P<0.001). Addition of monokine-activated NK cells (IL-12, IL-15 and IL-18) markedly inhibited Treg expansion (6.2±1.2% vs 15±1.5%, P<0.001) but freshly isolated NK cells had no effect. NK cells activated with TB lysate-stimulated monocytes also reduced Tregs expansion (13.2±0.3% to 3.4±1.4%, P<0.001) confirming the physiological relevance of this effect. Reduced Tregs expansion was not due to inhibition of PGE2 production or because of IFN γ. Monokine activated NK cells lysed TB lysate-expanded Tregs, but not freashly isolated Tregs (% specific lysis of 21±3% vs 1±1% P<0.001), nor T regs depleted T cells. Anti-NKG2D and anti-NKp46 reduced the % specific lysis of expanded Tregs from 22±3% to 7±3% and 8±3%, respectively (P<0.001) but abs to CD16, DNAM-1, and 2B4 had no effect. NKG2D ligand, ULBP1 expression was markedly upregulated on expanded T regs, compared to T regs depleted cells ( 31±5% vs 2.1 ±0.4%, P=0.002) and freshly isolated Tregs (4.6±1.1%). Anti -ULBP1 significantly inhibited the NK mediated lysis of expanded Tregs (specific lysis of 27±4% vs 9±2%, P<0.001) but abs to ULBP2, ULBP3 and MICA/B had no effect. These finding suggest that NK cells reduce the frequency of M.TB-expanded T regs by direct lysis.
Purine nucleosides downregulate innate immune function and arrest apoptosis in teleost fish leucocytes (B168)
Authors: Salinas Irene , Esteban Maria Angeles , Meseguer Jose
Published: January 2023

Adenosine and other purines are naturally present in the cell environment and exert a general protective role of organs and tissues following injury or stress. Moreover, adenosine is a key regulator of immune responses in higher vertebrates; inhibiting phagocytosis, regulating cytokine secretion and generation of oxygen metabolites. It also affects proliferation, survival and apoptosis of different cell types. We aimed to study purinergic nucleotides are able to i) downregulate in vitro innate immune responses and ii) modulate apoptosis in teleost fish leucocytes.

Experiment 1:

Head kidney leucocytes (HKL) from 6 specimens were incubated or with A or CHA (0.1, 1, 10 100μM) for 30, 120 or 360 min in 96-well plates. Phagocytosis, respiratory burst, peroxidase content and natural cytotoxic activity were measured. CHA significantly decreased phagocytic activity and respiratory burst at the highest assayed concentration.

Experiment 2:

106 HKL were dispensed into 48-well culture plates. 250μl of A or CHA (200μM) or sRPMI (negative control) or 1μg/ml staurosporine (positive control) were added to each well in triplicate. Apoptosis after 30min, 24h or 48h was assessed by a double staining flow cytometry assay using fluorescein diacetate and propidium iodide. Both A and CHA reduced apoptosis in HKL’s cultures after 24h. By analysing the different subpopulations, it was observed that only apoptosis of lymphocytes was significantly inhibited.

Conclusions:

teleost innate immune functions are regulated by purine nucleosides.apoptosis was not induced but arrested by A and CHA in seabream HKL cultures after 24h.
The roles of CARD9 in immune responses against intracellular bacterial infection (B170)
Authors: Zhang Yongliang , Hsu Yen-Michael S. , Dong Chen , Lin Xin
Published: January 2023

CARD9 is an adaptor molecule containing an N-terminal Caspase Recruitment Domain (CARD) and a C-terminal Coiled-coil domain. Previous study showed that it activated NF-κB transcription factor when overexpressed in cell line. In order to understand the role of CARD9 in immune responses, we created and analyzed CARD9 deficient mice. We found that p38 activation was selectively defected in CARD9 deficient macrophages in responses to bacterial and viral mimetics-induced signaling pathways. Macrophages from the CARD9 deficient mice produced significantly decreased amount of proinflammatory cytokines in response to components of intracellular microbial pathogens. Furthermore, in response to Listeria moncytogenes infection, CARD9 deficient mice were impaired in innate immunity and failed to activate anti-bacterial clearance. Together, our data demonstrated that CARD9 plays an essential and selective role in immune responses against intracellular bacterial infection.
Tobacco Smoke Exposure Disrupts CCL20 Production and Antimicrobial Activity by Respiratory Epithelial Cells. (B173)
Authors: Crane-Godreau Mardi A , Maccani Matthew A. , Eszterhas Susan K , Fiering Steven N.
Published: January 2023

The mechanism whereby tobacco smoke (TS) exposure increases the risk of infection is unknown. We tested the hypothesis that exposure to TS suppresses production of respiratory tract CCL20, a defensin-like antimicrobial peptide characterized primarily as a chemokine. When nasal washes from human subjects exposed to TS within the last two years were compared to nasal washes from individuals not exposed for more than 20 years, the latter had approximately six times more CCL20 than the former. In studies of polarized human bronchial epithelial cells (Beas-2b) grown in antibiotic-free media, CCL20 was released preferentially to the apical surface of the cell cultures. When cells were treated with tobacco smoke extract (TSE), CCL20 mRNA and secreted protein levels decreased by approximately half. In bacterial survival assays conducted with apically-collected conditioned media, media collected from TSE-treated cells killed significantly fewer CFU than media from non-TSE-treated cells; assays conducted with media treated with anti-CCL20 antibody resulted in abolition of its bacteriocidal activity. Statistically similar results were obtained in assays using rhCCL20 instead of apical media. These results demonstrate that TS exposure disrupts important antimicrobial activity and signaling by respiratory epithelial cells, a condition which may lead to increased susceptibility to infection.
Functional Implication of BAFF Synthesis and Release in Gmix-stimulated Microglia (B174)
Authors: Kim Kwang Soo , Jou Ilo , Park Sang Myun
Published: January 2023

BAFF (B-cell activating factor) is a recently identified member of TNF ligand superfamily that plays a critical role in B cell differentiation, survival, and regulation of immunoglobulin production. It exerts its effect by binding to three receptors: BAFF-R (BAFF receptor), TACI (transmembrane activator and CAML interactor), and BCMA (B-cell maturation antigen) which was known to be primarily expressed in B-lineage cells.

In this present study, we examined whether or not BAFF is also expressed in microglia and the synthesis and release of BAFF is regulated by stimuli. BAFF is expressed and released in primary rat microglia as well as BV-2 cells, mouse microglial cell line and the expression and release of BAFF is increased by Gmix-stimuli in microglia. The expression and release of BAFF is regulated by JAK-STAT, especially STAT1 and STAT3-dependent signaling pathway. Interestingly, SP600125, and SB203580, inhibitors of p38 and JNK, respectively, did not inhibit BAFF expression, but inhibit the release of BAFF by soluble form. It suggested that p38 and JNK signaling pathway regulate the release, not the synthesis of BAFF in microglia.

In BV-2 cells, only BAFF-R is expressed in cytoplasm and cell-surface. However, in primary rat microglia of microglia, BAFF-R and TACI are expressed in cytoplasm and cell-surface. Recombinant BAFF increases cytokines release, especially, IL-6 and IL-10 in primary rat microglia as well as BV-2 cells. It was suggested that BAFF, secreted by microglia may play important roles in CNS inflammation through regulating microglia as well as infiltrated B cells.
Opposing roles of PGE2 and LXA4 in the regulation of necrosis of Mycobacterium tuberculosis infected macrophages (B175)
Authors: Chen Minjian , Remold Heinz G.
Published: January 2023

The innate immune response against attenuated Mycobacterium tuberculosis (Mtb) can include induction of apoptosis of the host macrophage (Mφ) that limits intracellular growth by sequestering bacilli in apoptotic bodies, facilitates efficient pathogen removal and enhances antigen presentation. In infection with virulent H37Rv a completely different mechanism, necrosis, dominates the outcome of cell death. Necrosis serves as a microbial virulence mechanism allowing dissemination of the pathogen. Initiation of necrosis depends on the induction of mitochondrial inner membrane perturbation causing significant mitochondrial transmembrane potential (Δψm) loss. Here we report that attenuated H37Ra induces in Mφ the production of the prostanoids PGE2, PGD2, PGF2α, and TXA2. PGE2 suppresses Δψm loss in H37Rv-infeted Mφ and rescues the infected Mφ from necrosis. In contrast, virulent H37Rv induces the production of LXA4, which triggers Mφ necrosis by blocking prostanoid synthesis leading to Δψm loss. The production of the above mentioned prostanoids is not induced in H37Rv-infected Mφ. PGE2 acts through the engagement of the PGE2 receptor EP-2, which induces cAMP leading to PI3K and PKA activation. Thus, eicosanoids play a crucial role in the regulation of necrosis of Mtb-infected Mφ and are of critical importance in determining the outcome of an Mtb infection

(Supported by NIH grant AI50216).
Differential role for IL-1 dependent on anthrax infection route (B176)
Authors: Loving Crystal Lynn , Chaplin David D , Iwakura Yoichiro , Merkel Tod J
Published: January 2023

Use of B. anthracis as a bioweapon in 2001 has resulted in an increased emphasis on understanding mechanisms of pathogenesis after spore inhalation. Using a murine aerosol challenge model, our lab is focused on identifying host cells and cytokines involved in early control of disease progression. MyD88−/− mice are susceptible to inhalational B. anthracis, but TLR2, TLR4, TLR9, and TLR2/9 knockout mice are not. As MyD88 is also involved in IL-1R and IL-18R signaling, we investigated the susceptibility of these knockout mice and found that IL-1R−/− mice succumb to aerosol challenge, but IL-18R−/− mice do not. Yet, when challenged by the intraperitoneal (i.p.) route both IL-1R−/− and IL-18R−/− mice exhibit enhanced susceptibility to spore challenge. IL-1α and IL-1β signal via IL-1R, but IL-1β appears to be dispensable for protection, as mice deficient in caspase-1, which cleaves pro-IL-1β into bioactive IL-1β, do not succumb to disease. However, caspase-1−/− mice challenged by the i.p. route succumb to infection. It appears that IL-1α or IL-1β are sufficient for providing protection during inhalational disease, as mice deficient in either molecule alone were resistant to aerosol challenge. Yet, IL-1β is critical for survival after i.p. challenge as IL-1α−/−, but not IL-1β−/−, mice survive i.p. challenge. Overall, these results indicate differential role for IL-1 depending on route of B. anthracis spore challenge.
Mast cells enable heightened humoral immunity during infection (B178)
Authors: Shelburne Christopher P. , John Ashley St. , McLachlan James B. , Staats Herman F. , Abraham Soman N.
Published: January 2023

Dendritic cell (DC) accumulation in T-cell zones of secondary lymph organs is a critical event in the maximization of the primary immune response. Here, we demonstrate that mast cell (MC) control of elevated DC trafficking during infection enables the primary adaptive humoral immune response to proceed with heightened intensity. Elevated DC accumulation in draining lymph nodes (DLNs) was found to be the product of continual and incremental recruitment of DCs into the infected tissue site prior to their egress to DLNs. MCs contribute to this pattern of DC trafficking by the release of TNF, which was found to coordinately activate local blood vessel endothelium and DLNs to increase their expression of CD62E and CCL21, respectively. Blockade of either protein interfered with DC accumulation in DLNs, as well as the intensity of the humoral response to bacterial challenge. Thus, MCs enable the humoral immune response to proceed with enhanced intensity, which may be an important consideration in rationale vaccine design.

Supported by grants DK50814 and AI50021 from the National Institutes of Health.
Immunomodulating Activities of the TLR7 Selective Agonist 851, a Novel Drug Candidate for Treatment of High-risk Human Papillomavirus Infection (B182)
Authors: Tomai Mark Allen , Pederson Linda , Vasilakos John , Gorski Kevin , Reddy Laxma , O’Sullivan Gerry , Frank Rodney , Prestegard Terri , Kieper William
Published: January 2023

TLR agonists have been used in treating diseases including infections, cancer and as vaccine adjuvants. Aldara™ (imiquimod 5% cream) is the first TLR7 agonist approved in the US, and is indicated for the treatment of genital warts caused by human papillomavirus (HPV). Aldara was not well-tolerated when delivered vaginally; therefore a 2nd generation analog, 851, that retained the ability to stimulate cytokine production upon vaginal application was selected for development to treat high-risk cervical HPV infection. In human PBMC and purified plasmacytoid DC cultures, 851 stimulated a >10 fold increase in type I IFN, and indirectly activated human NK cells to express CD69 (8-fold) at concentrations of ≥10μM. In the rat, intravaginal application of 851 gel (0.01% and 0.1% w/v) led to local tissue production of TNF-α and MCP-1 without evidence of systemic cytokine induction. Increases in vaginal levels of TNF-α (9-fold) and MCP-1 (81-fold) were seen with the top strength of 851 tested (0.1%). Vaginal application of 851 results in the influx of NK cells, T cells and macrophages and an efflux of DC from the vaginal tissue. The ability of 851 to induce local cytokine production leading to influx and efflux of various immune cell populations could result in elimination of virally-infected cells from the cervix and vaginal tract including cells infected with high-risk HPV that are known to lead to cervical cancer.
Impaired pro-inflammatory cytokine production and Th1 biased ability of DCs exposed to Taenia antigens (B183)
Authors: Terrazas Luis I , Terrazas César A , Rivera-Montoya Irma , Rodríguez-Sosa Miriam
Published: January 2023

Helminth infectionsinduce Th2-biased immune responses. Mechanisms involved in this phenomenon are notyet clearly defined, but antigen-presenting cells (APC) playan important role in this process. Dendritic Cells (DC) recognize motifs that are conserved between pathogens mainly through Toll-like receptors(TLR) family, which is the main pathway known to be involved in maturating and inducing inflammatory cytokines in DCs. Most of intracellular pathogens drive Th1 type responses largely through these TLRs. By contrast, the result of the interaction between DCs and helminth parasites is less clear. To dissect mechanisms that lead to immune modulation in experimental cysticercosis, we have used DC exposed to Taenia excreted/secreted glycoproteins and examined their role as APCs, as well as in the modulation of DC responses to pro-inflammatory stimuli. We found that Taenia antigens had the ability to downmodulate DC production of TNF-α and IL-12 in response to several inflammatory molecules such as LPS, CpG and Toxoplasma Ags. Taenia-exposed DC displayed increased ability to suppress IFN-γ production but maintained IL-4 production on CD4+ DO11.10 cells in response to OVA in a STAT6-independent manner. Our results reflect the potential ability of T. crassiceps antigens to modulate DC-inflammatory responses to non-related pro-inflammatory molecules.

Work supported by PAPIIT IN208706
Vaccine for Cancer and Infectious Diseases (B194)
Authors: Deisseroth Albert B. , Tang Yucheng , Akbulut Hakan , Maynard Jonathan , Pedersen Line
Published: January 2023

In order to overcome defects in the immune response in older individuals, we have designed an adenoviral vector (Ad-sig-TAA/ecdCD40L) for the in vivo activation and tumor antigen loading of dendritic cells (DCs). This adenoviral vector encodes a fusion protein composed of an aminoterminal tumor associated antigen (TAA) fragment fused to the extracellular domain (ecd) of the CD40 ligand (CD40L) at the carboxyl terminal end. Two sc injections of this vector have broken tolerance to specific tumor associated antigens in two separate TAA.Tg transgenic mouse models (mice transgenic for rat Her-2-Neu (rH2N) and human MUC-1 (hMUC-1)). The immunoprotection extends for over a year and is independent of CD4 cells. We showed that the sc injection of the hMUC-1/ecdCD40L protein following the sc injection of the Ad-sig-TAA/ecdCD40L vector (this vector prime-protein boost is called VPP) increases the levels of both the cellular and humoral immune response over that achievable with just the vector injections alone in the hMUC-1.Tg mice. The antibodies from hMUC-1 VPP vaccinated mice bind to biopsy specimens of human cancers of the breast and prostate. Importantly, the VPP vaccination can induce an immune response even in 18 month old mice which completely suppresses tumor growth in old (18 month old) mice. The Ad-sig-rH2N/ecdCD40L vaccination suppresses the evolution of spontaneous breast cancer in the rH2N.Tg mouse model. The Ad-sig-HA/ecdCD40L vector prime-HA/ecdCD40L protein boost which is directed to the H5N1 avian influenza hemagglutinin (HA) antigen induces neutralizing antibodies against the H5N1 486 avian influenza virus at 1/4000 dilution of the serum.
Novel method for deconstructing humoral responses (B198)
Authors: Barrington Robert A , Feist Frank , Mann Wolfgang , Ketman Ken , Fu Wei , Steen Robert
Published: January 2023

Measurement of antibody production is a critical feature in vaccine design. However, the production and persistence of memory B (Bmem) cells may be more informative for long-term humoral protection against pathogens. In addition to the frequency of available Bmem cells, the heterogeneity of Bmem cells may influence protective responses. To determine heterogeneity, production and persistence of memory B cells with time after encountering pathogens, we implemented the AmpliGrid platform developed by Advalytix AG in concert with 6-parameter polychromatic flow cytometry to deconstruct B cell subpopulations during ongoing immune responses to a model protein antigen. Flow cytometry analysis and cell sorting was performed on a 3-laser FACSAria system. Antigen-binding B cells participating in germinal center (GC) and post-GC responses were sorted directly onto AmpliGrid chips. Subsequent single cell analysis using on-chip 1 ul RT-PCR and sequencing reactions revealed that responding B cells expressing diverse IgH genes can be subdivided into those being selected versus those being eliminated. After the initial response, we find evidence of memory B cells persisting during the subsequent 4–5 months following antigen encounter. This preliminary work suggests it will be of value in the evaluation of experimental vaccines.
Immunodeficiency and accumulation of B1-like B cells in transgenic mice overexpressing catalytically inactive RAG1 (B2)
Authors: Swanson Patrick C. , Anderson Dirk K. , Hassaballa Ashraf E. , Parks Kevin W. , Perry Greg A.
Published: January 2023

Antigen receptor genes are assembled by V(D)J recombination, which is mediated by two lymphoid cell-specific proteins called RAG1 and RAG2. In B cells, primary V(D)J rearrangements yield B cell receptors displaying diverse antigenic specificity. Recent evidence suggests that certain populations of peripheral CD5+ B cells may express the RAG proteins in order to initiate secondary V(D)J rearrangements to “edit” receptor specificity away from autoreactivity or “revise” receptor specificity to further expand an antigen-specific B cell repertoire. In principle, if a means were identified to block this process, B cell maturation and/or function may be impaired. To explore this idea, we generated transgenic mice overexpressing catalytically inactive full-length RAG1 in the periphery. In two founder lines, transgenic mice exhibit a polyclonal expansion of B1-like B cells (B220loCD19+CD5+), pronounced hypogammaglobulinemia, and an impaired humoral immune response. Interestingly, B1-like B cells fail to accumulate in mice expressing both catalytically inactive RAG1 and a heavy chain transgene specific for dsDNA (3H9H56R.KI), but bi-transgenic mice still exhibit hypogammaglobulinemia and may also be susceptible to bacterial infection.

This work was supported by the Health Future Foundation, and the State of Nebraska LB506 and LB692 Research Programs.
Profiling the Mammalian Cell Surface Glycome (B201)
Authors: Li Yu , Tao Sheng-Ce , Qian Jiang , Schnaar Ron , Goldstein Irwin , Schneck Jonathan , Zhu Heng
Published: January 2023

Glycosylation is one of the most complex posttranslational modifications with an extremely high level of diversity that has made it refractory to detailed analysis. To facilitate studies in glycosylation, we have developed a comprehensive lectin microarray for use as a high-throughput analysis of investigating cell surface glycosylation in mammalian cells. Using this approach, we have established a "GlycoCode" and hierarchical organization of 23 mammalian cell lines based on the profile of their surface glycan structures. We also show the potential use of this approach as a sensitive analysis of cell development and differentiation by probing immune system cells. Lectin microarrays represent a novel approach to high-throughput analysis of cell heterogeneity; the results of which can be used to studying diverse cell processes including cell development and differentiation, cell-cell communication, pathogen-host recognition, cell surface biomarker identification, and to aid in development of novel therapeutics.
Real-time PCR array for simultaneous evaluation of multiple cytokine mRNA expression (B204)
Authors: Arikawa Emi , You Min , Wang Jie , Lo Jing-yi , Yu Sean , Yang Jingping
Published: January 2023

Cytokine quantification is an important element in studies of inflammation and immune responses. Quantitative RT-PCR, a rapid and sensitive assay, is the preferred method to quantify cytokine mRNA levels because they are often expressed at low levels. The RT2Profiler™ PCR Array combines the reliable performance of SYBR® Green based RT-qPCR with multi-gene profiling capabilities to simultaneously analyze the expression of a panel of genes from the same pathway. Using PCR Arrays, we have monitored the mRNA levels of 84 different cytokines in human peripheral blood mononuclear cells (PBMC) in response to treatment with 50ng/mL PMA and 1μg/mL ionomycin for up to 24 h. The results identify 23 up-regulated and 6 down-regulated genes (with >5 fold-change & p<0.005) in the stimulated cells when compared to the resting cells at 6 h. At 24 h, the effects of PMA-ionomycin on genes such as BMPs, CSFs, IFN-γ, IL-1β, IL-6, IL-11, TGF-β and TNF are continuously observed, while the effects on other genes such as IL-2, 3, 5, 9, 10, 13, 17 and 22 diminish 24 h after stimulation. To validate these results, the protein level of eight selected cytokines secreted by the PBMC was measured using a multiplex ELISA array. Our data show that changes in cytokine mRNA levels detected by PCR Arrays accurately predict changes in protein levels measured by ELISA. Hence, the PCR Array offers a simple, reliable and sensitive tool for multiple cytokine profiling.
Antitumor effect of resveratrol and potential for combination with immunotherapy (B205)
Authors: Soto Brenda Liz , Hank Jaquelyn A , Polans Arthur S , Sondel Paul M
Published: January 2023

Resveratrol (3, 4′, 5 – trihydroxy-trans-stilbene) is a natural compound currently being investigated for possible health promoting activities. It has antitumor effects on various types of cancer cells in vitro and in some animal models. Clement et al (Blood, 92:3, 996–1002, 1998) demonstrated that trans-resveratrol (t-RV) induced apoptotic cell death in HL60 leukemia cells and T47D breast cancer cells in vitro at doses which were minimally toxic to peripheral blood lymphocytes (PBMC). Additional studies on the effect of t-RV on the immune system have found t-RV to be immunosuppressive in vitro in a time and dose-dependent manner, but not suppressive in vivo. We are currently examining the ability of t-RV to be combined with immunotherapy to treat cancer. Using proliferative and cytotoxicity assays we are defining a dose in vitro and a regimen in vivo that does not suppress the immune system and has additive or synergistic anti-tumor effects when combined with immunotherapy. Our data suggest that t-RV inhibits the proliferation of K562, a natural killer sensitive target, and M21, a human melanoma cell line, in a dose dependent fashion. In addition, t-RV inhibits the ability of PHA to stimulate proliferation of PBMC. Furthermore, t-RV suppresses proliferation of cells already activated with PHA. These responses were dose dependent. We are examining the effects of t-RV on target and effector cells in cell mediated and antibody dependent cytotoxicity assays. These investigations may shed light on ways of combining t-RV with immunotherapy to treat cancer.
Assessing the regulation of T cell function by distinct cytokine combinations using GuavaFlo, a cytometry-based system for multidimensional cellular analysis. (B206)
Authors: Gillis Katherine Anne , Fishwild Dianne M , Lefebvre Ray , Zieske Lynn R , Snyder-Cappione Jennifer E. , Cappione Amedeo J
Published: January 2023

Precise regulation of effector function is critical for mounting a potent, yet specific, immune response to a given antigenic challenge. Distinct cytokine combinations present within a given microenvironment can induce changes in the phenotype and effector functions of immune cells that differ dramatically from the effects exerted by each cytokine alone. Using a multiparametric analysis approach, we investigated the phenotypic and functional changes of all combinations of 6 cytokines (IL-2, IL-4, IL-7, IL-12, TNF-α, and TGF-β) on human T-cell isolates derived. Cultures were assessed for changes in maturation/activation state, as well as determination of their individual proliferative capacity and viability. Cultures were subject to cytokine production profiling through intracellular staining. Samples were acquired using Guava’s EasyCyte Plus system and integrated GuavaFlo software, an analysis platform designed specifically to simplify and expedite the assessment of plate level flow cytometry data sets. We found that unique combinations of cytokines had profound and highly varied effects on lymphocyte fate and function in vitro. Demonstrations of synergy, dominance, and substitution amongst multiple cytokines in our experimental system may reflect actual paradigms underlying such processes as the differentiation of T cell subsets into effector and resting memory phenotypes in vivo.
Live Cell Imaging of Human Mast Cells on a Pico-well LiveCell Array at Single Cell Resolution (B207)
Authors: Maliakal James , Tagen Michael , Kempuraj Duraisamy
Published: January 2023

Mast cells are important effecter cells in allergic and inflammatory responses. When the mast cells are activated they undergo regulated secretion of intracellular granules by exocytosis. This includes inflammatory cytokines such as IL-6, TNF-α and other chemokines. In this study we examined mast cell activation using a microscope slide based pico-well array at a single cell resolution.

The human umbilical cord blood derived mast cells were cultured in the mast cell medium. The cells were counted and 2uL of cell suspension were added to the pico-wells of the array (Livecell array, NUNC). The cells in the array were covered with a sliding cover slip allowing each cell to settle into an individual well. The cover slip also allowed for repeated treatment and washing of reagents via capillary flow. The cells were treated with substance P (SP) to induce activation directly on the array. Live cell imaging showed cells were distributed evenly on the array as one cell per well. After activation, the cells secreted granules which are clearly visible in the pico-wells surrounding the cells. Additionally, LAD2 cells were activated with compound 48/80 (0.1ug/ml). Use of anti-CD63 PE permitted single cell investigation of degranulation. The ability to culture, treat and image non-adherent mast cells in the array allowed us to study single cell responses in a mast cell population, and permits fast screening of potential inhibitors.
The neurotrans mechanism of electronic-acupuncture in DNP-KLH immunized mice. (B21)
Authors: Lee Youngseop , Lee Heekyung , Min Ji-young , Oh Banggul , Lee Mi-Kyung , Hong Moo-Chang , Bae Hyunsu , Kim Yangseok , Shin Min-Kyu
Published: January 2023

Our previous study demonstrated that successive electroacupuncture (EA) at the ST36 acupoint reduces IgE production in BALB/c mice immunized with 2,4-dinitrophenylated keyhole limpet protein (DNP-KLH) by suppression of the Th2 cell lineage development. Here we report that pretreatment of phentolamine (¥á-adrenergic receptor antagonist, 10 mg/kg, i.p.) completely blocks the EA-induced suppression of antigen-specific and total IgE levels in serum, and IL-4 production in anti-CD3 mAb-activated splenocytes in DNP-KLH immunized mice. And pretreatment of Y-25130 (5HT3 receptor antagonist, 3 mg/kg, i.p.) partly blocks EA-induced suppression effect shown above. These results suggest that ¥á-adrenergic receptor play an important role in mediating the suppressive effects of EA on IgE production and Th2 cell response in DNP-KLH immunized mice, and 5HT3 receptor play as a minor factor.

This work was supported by the Brain Korea 21 project in 2007
Simultaneous Determination of Phenotypic Expression and Electronic Volume in Mouse Cells by the Cell Lab Quanta® SC-MPL Analyzer. (B210)
Authors: Cabana Elisa R , Daly Maria , Cheetham Mark , Shankey Vincent T , Thomas Richard , Krishan Awtar
Published: January 2023

The Cell Lab Quanta® SC analyzer, a unique platform with two excitation light sources; a 488 nm diode laser and a HBO arc lamp, can simultaneously measure Electronic Volume (EV), side scatter (SS) and three color fluorescence. This system has an innovative multiplate loader (MPL), which can undertake automatic sample preparation and high throughput analysis.

We have used this instrument to analyze cells from various murine tissues with the objective to describe and identify changes in cellular volume and morphological complexity during development, activation and differentiation. Immunophenotypic analysis of cells isolated from bone marrow, peripheral blood, spleen, thymus and lymph nodes of C57 BL/6 mice was done with conventional combinations of MABs conjugated with FITC, PE and PE cy-5. The MPL was used for sample preparation in 96 well plates.

EV, which is not used at present as a relevant parameter in flow cytometry, is a more precise measure of cell size than the conventional Forward Scatter (FS). EV can be calibrated with NIST traceable beads or appropriate biological calibrators with known Mean Corpuscular Volume.

We conclude that this new automated platform expands the possibilities for researchers by providing means for identifying and differentiating between different cell types on the basis of EV/SS and also by improving the throughput with rapid sample analysis.
Nicotine affects antigen-specific immune responses both in vitro and in vivo (B211)
Authors: Nouri-Shirazi Mahyar , Guinet Elisabeth , Zeng Menghua
Published: January 2023

There is increasing evidence that nicotine, an immunomodulatory component of cigarette smoke, affects immune surveillance and increases the progression of diseases. We recently reported on the effects of nicotine on human dendritic cells (DCs) and proposed a possible mechanism that links cigarette smoke to higher incidences of respiratory tract infection and asthma. To establish the causal relationship between nicotine-induced DC alterations and immunomodulation in vivo, we examined the effects of nicotine on 1) the Th-promoting capacity of mouse bone marrow DCs (BMDC), and 2) the development of cellular and humoral immune responses to antigen challenge in vivo. We report that nicotine promotes the development of mouse BMDC precursors into DCs that preferentially support the proliferation and differentiation of ovalbumin (OVA)-specific naïve T cells into effector memory cells that produce less IFN-γ and more IL-4. Further analysis revealed that these DCs indeed have a reduced capacity for antigen uptake, produce less Th1-promoting cytokine, IL-12, and display enhanced expression of Jagged, a Notch family ligand associated with producing Th2-type cells. Of particular interest, we found that injection of soluble OVA plus LPS induces clonal expansion of adoptively transferred OVA-specific CD4+ T cells in both immunized control and nicotine-exposed mice. However, the frequency of IFN-γ secreting T cells within OVA-specific expanded T cells as well as serum anti-OVA IgG2a were reduced in nicotine-exposed mice. Our in vitro and in vivo data suggests that nicotine weakens host immune defense mechanisms by affecting the development of DC-dependent Th1 cells. Philip Morris USA Inc. and Philip Morris International.
Ropporin is a novel cancer testis antigen for multiple myeloma (B212)
Authors: Chiriva Maurizio , Ferrari Raffaele , Yu Yuefei , Kelly Aaron , D’Cunha Nicholas , Cobos Everardo
Published: January 2023

Multiple myeloma is a fatal hematological malignancy. Despite the recent improvements in standard treatments, immunotherapy promises to be more effective due to its major specificity and its less co-related side effects. Immunotherapy’s success depends on the discovery of novel tumor associated antigens. Ropporin is a testis-specific protein, localized in the principal and end piece of sperm flagella. Our aim was to evaluate its presence in a human normal tissue panel and in a pool of multiple myeloma patients. No expression was detectable in the normal tissues, while positive band signals were seen in 52.6% of patients after PCR, and in 47.3% of patients after immunocytochemistry. We established for the first time that Ropporin is expressed at the transcriptional and at protein level in multiple myeloma. Our data strongly suggest that Ropporin is a novel cancer testis antigen and, possibly, a suitable immunological target in multiple myeloma.
Contribution of heavy-chain and conventional IgGs to the alpaca immune system during fetal, neonatal, and adult life (B215)
Authors: Daley Lisa Patryce , Purdy Stephen R , Davis William C , Appleton Judith A
Published: January 2023

Besides conventional immunoglobulins, camelids produce antibodies that do not incorporate light chains into their structures. Because of their considerable potential for biotechnological and therapeutic applications, these so-called heavy-chain (HC) antibodies have incited great interest in the biomedical community. Comparatively little is known about their immunological functions or the B cells that produce them. Using alpacas, we set out to clarify the life histories of the B cells that synthesize HC or conventional IgGs, and determine the contribution of these isotypes to maternal immunity that is conferred on neonates via colostrum. While conducting these studies, we confirmed the presence of a second conventional IgG isotype and showed that it is synthesized by cells that localize to the marginal zones of the spleen in adult alpacas. Also, we documented for the first time a third HC isotype, most closely related to the previously defined IgG3. Cells staining positive for this HC isotype were detected in ileal Peyer’s patches; conversely, none of the other four HC isotypes were detected at this site. In aggregate, HC isotypes comprised 38% of serum IgG in adult alpacas. All five isotypes were present in colostrum and were passively transferred to crias. The faster decline in concentrations of HC isotypes in cria blood suggests that they have a shorter half-life than the conventional IgGs.
Toll Like Receptor (TLR)- 3 mediated chemokine release in human submandibular gland cell line (B218)
Authors: Li Jingchao , Bae Ge-Hyun , Choi Se-Young , Lee Sung Joong , Oh Seog Bae , Kim Joong-Soo , Park Kyungpyo
Published: January 2023

The recent epidemiologic studies suggest that viral infection can be one of causative factors to induce an exocrinopathy. However, the event leading to the initiation of the disease in relation with viral infection is still unknown. In this experiment, we examined whether TLR3 is expressed in human submandibular gland (HSG) cell lines and involve in chemokine release using real-time PCR, ELISA and chemotaxis experiments. We found TLR3 mRNA was expressed in this cell line. TLR3 stimulation with poly (I: C) increased mRNA expression levels of IP-10, I-TAC, and RANTES in a concentration-dependent manner. Application of neutralization antibody of TLR3 inhibited the increase of mRNA levels, suggesting it’s specifically mediated by TLR3. We confirmed the increase of chemokine concentrations in extracellular medium of HSG treated by poly (I: C). In the chemotaxis experiment, the under layer of culture media including released chemokines from poly (I: C) treated HSG cells induced T-lymphocyte migration from the upper layer of boyden chamber. Our results suggest that TLR3 functionally expressed in salivary glands and plays a role in the initiation of inflammation.
Generation and use of monoclonal antibodies as leucocyte-markers to study the immune function in teleost fish (B219)
Authors: Jose Meseguer , Angeles Esteban M , Alberto Cuesta
Published: January 2023

Fish possess the first full immune system in the vertebrate evolution. To date, leucocyte abundance, distribution and function has attempted to elucidate how similar or different are the fish and mammalian immune system. Fish possess lymphocytes, monocyte-macrophages and granulocytes with main differences in the tissue origin, distribution and functioning. Lack of proper molecular markers, such as commercial specific antibodies, for fish leucocytes is still hampering the success in this field. Considering this, we immunized mice with gilthead seabream (Sparus aurata L.) leucocytes and generated monoclonal antibodies following standard protocols. We chose 3 clones (H4, D2 and C1) and characterised their specificity for seabream leucocytes by means of flow cytometry, immunocytochemistry, magnetic-activated cell sorting (MACS) and gene expression by RT-PCR. Functional studies with the isolated leucocyte populations were also conducted to ascertain their implication in phagocytosis, cell-mediated cytotoxicity or oxygen-free radical production. Generation of monoclonal antibodies is a promising field in order to get good markers for further characterisation of the fish immune system.

Work partially funded by European (QLK2-CT-2002-00722), national (AGL2005-07454-C02-01) and regional (02958/PI/05) projects.