bluuebunny/crossref_metadata_2025 huggingface dataset:
MojoSort: a versatile nanoparticle for magnetic isolation of cells with high purity, yield, and preserved functionality (TECH2P.926)
Authors: Zhang Hong , Tam Miguel , Gohel Dhanesh , Zhao Xinfang , Koury Jeffery , Wiethe Carsten , Yang Xifeng , Ransom John , Monell Craig , Jiang Nan , Lay Gene
Published: January 2023

The isolation of specific cells from a complex population is a technical challenge that researchers face very often. Several strategies have emerged over the years, including gradient centrifugation, Fluorescent Activated Cell Sorting and Magnetic Activated Cell Sorting. Magnetic labeling is the most common approach, as it constitutes a fast, reliable, and convenient method to obtain populations with high purity and yield, and the cells are suitable for downstream applications. Here we present a new type of magnetic nanoparticles that may be used with current separation methods. To illustrate the ease and utility of the system, while maintaining functionality of the isolated cells, bone marrow cells were isolated from C57BL/6 mice using magnetic particles, and characterized by flow cytometry. The cells were then differentiated towards a macrophage-like phenotype in vitro. After culture, the cells were stimulated with LPS and CpG and the activated phenotype was compared to non-stimulated cells. Surface markers analyzed include CD80, CD86 and MHC II. The supernatant was collected and screened for the presence of cytokines, such as IFN-b, GM-CSF, IL-1b, IL-12, among others. The isolated cells show a differential activation phenotype, as well as a defined cytokine profile, in accordance with the stimulation used when compared to non-stimulated cells. Thus, we validated the high quality performance of BioLegend’s new low-cost magnetic cell separation system.
A new protein crosslinking method for labeling and modifying antibodies (TECH2P.758)
Authors: Liao Jinfang , Diwu Jack , Zhao Qin , Guo Haitao , Han Xing
Published: January 2023

The modification and labeling of a protein with a tag macromolecule is an important task in biological research. There are a few methods available for crosslinking two macromolecules. The direct crosslinking of two macromolecules suffer extremely low yield and difficult purification. A common method is to use a small crosslinker (such as SMCC) in numerous bioconjugations, but SMCC-modified protein is extremely unstable and often self-reactive during storage since proteins often contain both amine and thiol groups that cause significant amount of homo-crosslinking. We have recently developed an effective crosslinking method to selectively link two macromolecules. The new crosslinking method uses two unique crosslinkers that readily react upon mixing. Specifically, a pair of crosslinkers is used, including Buccutite FOL and Buccutite MTA. A protein (e.g., antibody) is first labeled with Buccutite FOL, and the tag macromolecule is labeled with Buccutite MTA. The two resulted Buccutite-modified conjugates can be readily purified by a desalting column or dialysis. Buccutite FOL-labeled protein and Buccutite MTA-tag macromolecule are mixed under extremely mild neutral conditions to give the desired protein-tag macromolecule conjugate. The Buccutite bioconjugation system is run at neutral pH, and completed within 1 hour. The conjugation can be run at low concentration, thus eliminating the pretreatment step for some diluted proteins. No catalyst is required.
Development of detection tools for health monitoring in new world and old world camelid (TECH2P.762)
Authors: Holzloehner Pamela , Schliebs Erik , Maier Natalia , Naumann Friederike , Füner Jonas , Micheel Burkhard , Hanack Katja
Published: January 2023

Keeping and breeding of new and old world camelids in Europe is a very complex process influenced by altered climate conditions in comparison to the natural habitat in desert ecosystems. Especially the veterinary care for these animals is lacking specific detection systems for general health monitoring, diagnosis of certain diseases or vaccination. In order to build up efficient tools for detecting important parameters in this field highly affine monoclonal antibodies were developed reacting against camelid immunoglobulins. The humoral immune response of New and Old World Camelids is characterized by generating conventional as well as heavy chain only antibodies which is a unique feature in mammals. To investigate the immunological status of Camelids after vaccination murine monoclonal antibodies were produced by hybridoma technology, characterized and used for assay development. We were able to generate monoclonal mouse antibodies that bind (I) to all subtypes, (II) only the conventional subtype IgG1 and (III) all heavy chain subtypes. All generated monoclonal antibodies showed no cross reactivity with immunoglobulins from other species. Specific monoclonal antibodies were used for assay development to monitor vaccination success against e.g. Coronavirus, Rotavirus and Clostridium. Further the detection systems were extended for disease control as e.g. erysipelas. With these tools it is now possible to develop efficient diagnostic systems for health monitoring in Camelids.
Performance qualification of a 13-color intracellular cytokine staining assay for the assessment of T cell responses in human PBMCs (TECH3P.932)
Authors: Graves Andrew , Henson Dawn , Padilla Marcelino , Schaeffer Jacqueline , Hokey David
Published: January 2023

One challenge to tuberculosis vaccine research is the lack of a correlate of immunity, which researchers are attempting to address through use of more high-throughput, high-data assays for assessing trial specimens. Multiparameter intracellular cytokine staining (ICS) assays have proven to be one of the most cost-effective tools available. Previously, we reported the development of a 13-color ICS panel for use in assessing T cell responses from human PBMCs. In an effort to better understand the metrics of the assay performance, a qualification study under GCLP conditions was executed. The parameters assessed in this performance qualification included specificity, sensitivity, selectivity, linearity, and intermediate precision. The lower limits of quantification for cytokine detection were shown to be under 0.03%. Using Fisher’s exact test to determine positivity, the false positive and false negative rates were shown to be 0% in this performance qualification. Furthermore, assessment of intermediate precision suggested that the CD3, CD4, CD8, CCR7, and CD45RO markers each showed a percent coefficient of variation (%CV) of 25% or under, while intracellular and functional markers were generally under a 30% CV as measured via responses to Staphylococcal enterotoxin B. The results of this performance qualification study illustrate the capabilities of this panel for assessing clinical trial responses and suggest it is a reliable assay for the assessment of T cell responses.
Incorporation of BD HorizonTM BV421 and BD HorizonTM BV480 immunofluorescence reagents improves immunofluorescence microscopy (TECH3P.933)
Authors: Rohrer Jurg , Wright Adam , Martin Jody
Published: January 2023

New fluorophores offered by BD Biosciences, BD HorizonTM BV421 and BD HorizonTM BV480, have unique spectra, are very bright, photostable, and work well in immunofluorescence microscopy. With narrow excitation and emission spectra these 2 fluorophores are compatible with most fluorescent protein models (eGFP, YFP, RFP, and tdTomato). Additionally, they are compatible with the standard green, yellow, red, and far-red immunofluorescence imaging reagents, (Alexa Fluor® 488, Alexa Fluor® 568, and Alexa Fluor® 647). These new fluorophores enable robust 5-color imaging. The workflow is easy and the dyes are functional (with simple optimization) on most fluorescence microscopes. No spectral unmixing is required for analysis. Because BV421 and BV480 are brighter than standard imaging fluorophores, there is the potential to lower the limit of detection with improved resolution. Side by side experiments with standard fluorophores on an epifluorescence microscope demonstrate improved resolution, via increased signal to noise, using both BV421 and BV480. Brighter fluorophores improve ease of use during microscope focusing and data acquisition by decreasing exposure time. In addition to enhanced sensitivity, these fluorophores enable easy and routine 5 color staining. Examples from human and mouse model systems using directly conjugated primary antibodies, show how these dyes enable simple multi-parameter imaging thereby permitting correlation of more parameters per sample
Tolerogenic synthetic nanoparticles for the prevention of anti-drug antibodies against biologic therapies (TECH3P.936)
Authors: Ferrari Joseph , LaMothe Robert , Kolte Pallavi , Griset Aaron , O'Neil Conlin , Browning Erica , Fu Fen-ni , Altreuter David , Johnston Lloyd , Farokhzad Omid , Langer Robert , von Andrian Ulrich , Maldonado Roberto , Kishimoto Takashi
Published: January 2023

The development of anti-drug antibodies (ADAs) is a common cause for treatment failure and adverse events, such as hypersensitivity reactions, associated with biologic therapies. Therefore, prevention of ADAs in an antigen-specific manner would be highly desirable in order to improve the safety and efficacy of marketed products. Here we describe the use of SVP: polymeric, synthetic, biodegradable nanoparticles carrying a tolerogenic immunomodulator, rapamycin, to induce durable, antigen-specific immune tolerance. In mice, intravenous or subcutaneous co-injections of SVP with free antigen results in robust CD4+ T cell and B cell tolerance (i.e. the inhibition of their activation over multiple challenges), an increase in regulatory cells and the inhibition of antigen-specific hypersensitivity reactions. Only encapsulated rapamycin, not the free form, could induce immunological tolerance to a variety of antigens. Co-injections of SVP and antigen in both rats and cynomologous monkeys also results in B cell tolerance. In mice that spontaneously develop rheumatoid arthritis (RA), we show that treatment with SVP and adalimumab prevents the formation of anti-adalimumab ADAs therefore normalizing adalimumab pharmacokinetics and improving the clinical and histological manifestations of RA. SVP therapy represents a novel antigen-specific approach for the prevention of ADAs against biologic therapies, as well as the treatment of allergies and autoimmune diseases.
MHC-mismatched mixed chimerism augments thymic regulatory T cell production and prevents relapse of EAE (THER7P.960)
Authors: Wu Li-Min , Li Nainong , Zhang Mingfeng , Xue Shengli , Riggs Arthur , Zeng Defu
Published: January 2023

Multiple sclerosis (MS) is an autoimmune inflammatory disease in central nervous system with demyelination, axon damage, and paralysis. There is no yet curative therapy. Induction of MHC-mismatched mixed chimerism is proposed to be a potential cure for autoimmune diseases, but this has not yet been tested in animal models of MS, experimental autoimmune encephalomyelitis (EAE). Here, we report that MHC-mismatched mixed chimerism with C57BL/6 (H-2b) donor in SJL/J (H-2s) EAE recipients eliminates symptoms and prevents relapse. This cure is demonstrated by not only disappearance of clinical signs but also reversal of autoimmunity and elimination of infiltration of T, B and macrophage cells in the spinal cord, as judged by HE, immunofluorescent staining and flow cytometry analysis. We also see regeneration of myelin as judged by fast-blue staining and prevention of axon damage as judged by electronic microcopy. The reversal of autoimmunity is associated with a marked reduction of autoreactivity of CD4+ T cells and significant increase of Foxp3+ regulatory T percentage among host-type CD4+ T cells in the spleen and lymph nodes. We have also found a marked increase of Foxp3+ regulatory T percentage among host-type CD4+ and CD4+CD8+ thymocytes in the thymus. Thymectomy leads to loss of prevention of EAE relapse by the mixed chimerism. These results indicate that induction of MHC-mismatched mixed chimerism can augment thymic production of Foxp3+ regulatory T cells and cure EAE.
Targeting epidermal fatty acid binding protein for treatment of experimental autoimmune encephalomyelitis (THER7P.947)
Authors: Rao Enyu , Singh Puja , Li Yan , Zhang Yuwen , Chi Young-In , Suttles Jill , Li Bing
Published: January 2023

Multiple sclerosis (MS) is an autoimmune disease in which dysregulated immune cells attack myelin in the central nervous system (CNS), leading to irreversible neuronal degeneration. Our previous studies have demonstrated that epidermal fatty acid binding protein (E-FABP) widely expressed in immune cells, in particular in dendritic cells (DCs) and T lymphocytes, fuels their overactive immune responses in the mouse model of experimental autoimmune encephalomyelitis (EAE). In the present study, we conducted an intensive computational docking analysis to identify novel E-FABP inhibitors for regulation of immune cell functions and for treatment of EAE. We demonstrate that compound [2-(4-acetylphenoxy)-9,10-dimethoxy-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one; designated as EI-03] binded lipid binding pocket of E-FABP and enhanced the expression of peroxisome proliferator-activating receptor (PPAR) γ. Further in vitro experiments showed that EI-03 regulated DC functions by inhibition of TNFα production while promoting IL-10 secretion. Moreover, EI-03 treatment counterregulated T cell balance by decreasing effector T cell differentiation (e.g. Th17, Th1) but increasing regulatory T cell development. Most importantly, mice treated with this newly identified compound exhibited reduced clinical symptoms of EAE in different mouse models. Taken together, we have identified a new compound which displays a potential therapeutic benefit for treatment of MS by targeting E-FABP.
Targeting the anti-tumor function of B cells in non-small cell lung cancer patient tumors (TUM9P.1008)
Authors: Bruno Tullia , Moore Brandon , Munson Daniel , Kern Jeffrey , Slansky Jill
Published: January 2023

Understanding the function of tumor infiltrating lymphocytes (TILs) in NSCLC patient tumors will contribute to the development of rationally designed treatments and an increase in the current 18% survival rate. We hypothesize that TIL-Bs help generate potent, long-term immune responses against cancer. We used un-manipulated, primary human B cells from fresh tumor and tumor adjacent lung tissue to determine the phenotype and function of TIL-Bs in NSCLC patients. The total number of B cells at the site of the tumor versus the tumor-adjacent tissue was increased compared to other immune subsets. In analyzing the markers of activation and exhaustion, we observed a spectrum of activation of TIL-Bs. Further, we observed that TIL-Bs present autologous tumor antigens to CD4 TILs in a subset of NSCLC patients. These data suggest that some patients have functional TIL-Bs and some patients have nonfunctional TIL-Bs; TIL-Bs that could not present tumor-specific antigens had higher expression of B cell exhaustion markers. Lastly, we are combining blockade of the immune inhibitory PD-1:PD-L1 pathway in our antigen presentation assays because PD-1 expression is increased on CD4 TILs in our NSCLC patient tumors. Results from this study will advance the understanding of the anti-tumor function of TIL-Bs in solid tumors and will develop a model of B cell exhaustion in human cancer. Ultimately, we will predict which TIL-B functions to target in future NSCLC immunotherapies.
PTPROt maintains T cell immunity in the microenvironment of hepatocellular carcinom (TUM9P.1002)
Authors: Sun Beicheng
Published: January 2023

Background & Aims: Intratumoral T cells play a central role in anti-tumor immunity, and the balance between T effector cells (Teff) and regulatory T cells (Treg) affects the prognosis of cancer patients. However, educated by tumor microenvironment, T cells frequently fail in their responsibility. In this study, we aimed to investigate the role of truncated isoform of protein tyrosine phosphatase receptor - type O (PTPROt) in T cell mediated anti-tumor immunity. Methods: We recruited 70 hepatocellular carcinoma (HCC) patients and 30 healthy volunteers for clinical investigation, and analyzed cellular tumor immunity by using ptpro-/- C57BL/6 mice and NOD/SCID mice. Results: PTPROt expression was significantly down-regulated in human HCC infiltrating T cells due to the hypoxia microenvironment (***P<0.001); PTPROt expression highly correlated with the intratumoral Teff/Treg ratio (**P<0.01) and clinicopathologic characteristics (*P<0.05). Moreover, PTPROt deficiency attenuated T cell mediated anti-tumor immunity and remarkably promoted mouse HCC growth (***P<0.001). Mechanistically, deletion of PTPROt decreased Teff quantity and quality through phosphorylation of lymphocyte specific tyrosine kinase (Lck), but increased Treg differentiation through phosphorylation of signal transducer and activator of transcription (STAT) 5. Conclusions: In support of the Teff/Treg homeostasis, PTPROt serves as an important tumor suppressor in HCC microenvironment.
Intestinal microbiota alters omental tumor growth (TUM9P.1004)
Authors: Meza-Perez Selene , Silva-Sanchez Aaron , Garcia-Hernandez Maria de la Luz , Mudunuru Uma , Rangel-Moreno Javier , Simpler Scott , Sinclair Stacey , Weaver Casey , Schoeb Trenton , Lord Edith , Randall Troy
Published: January 2023

The omentum is an adipose tissue that contains milky spots (MS). The MS are similar to secondary lymphoid organs and can generate B and T cell immune responses to peritoneal antigens. Additionally, omentum CD4+CD25+FoxP3+ T regulatory cells (Tregs) display an activated phenotype CD44hiCD62Llow. Moreover, the omentum also collects metastasizing tumor cells in the peritoneal cavity and tumors growing in the omentum are associated with poor clinical prognosis. Here we show that intestinal microbiota affects Tregs activation profile and impairs tumor growth in omentum. Our transplantable tumor model in wild type (WT) mice shows that peritoneal tumors grow progressively in the omentum and peritoneal cavity in WT mice, whereas in germ free (GF) mice tumors do not grow. Omental tumor growth in WT mice is associated with an increase in PD-1+ Tregs, while tumor-specific CD8+ T are reduced. However, in GF mice numbers of PD1+ Tregs remain unaltered but tumor specific CD8+ T cells are still present. After co-housed GF mice with WT mice, ex-GF mice showed tumor growth and restore the increase in PD-1+ Tregs with low number of specific CD8+ T cells. The induction of tolerance requires PD-1+ Tregs. These data suggest that intestinal microbiota has a role in omental Tr activation and in the tolerance to peritoneal tumors.
MicroRNA-422a downregulates DNAM-1 expression in NK cells and impairs DNAM-1-mediated cytotoxicity (TUM10P.1033)
Authors: zhu min , Zhang Wei , Sang Hai-wei , Fu Xiang-ning
Published: January 2023

Natural killer (NK) cells play a crucial role in innate immunity against cancer. Activation of NK cells is regulated by a balance between activating and inhibitory signals from cell surface receptors. Among these receptors, DNAM-1 and its ligands ( PVR and Nectin-2) play a vital role in NK cell-mediated recognition and lysis of tumor cells. microRNAs (miRNAs) are key regulators of gene expression that may be involved in NK function of immune surveillance against cancer. Several miRNA have been identified as modulators of NK activating receptor and its ligands expression, such as NKG2D and MICA. However, little is known about DNAM-1 gene regulation by miRNA. Based on bioinformatics, we identified miR-422a with putative docking sites in the 3'UTR of DNAM-1. Furthermore, we investigated the potential interaction between the 3'UTR of DNAM-1 gene and miR-422a as well as its functional role in the regulation of DNAM-1 expression and cytotoxicity in NK cells both in vitro and in vivo. We found that TGF-β1 but not L-kynurenine upregulated miR-422a expression in NK cells, resulting in markedly inhibiting NK cytotoxicity against lung cancer cells, which was mediated by suppression of DNAM-1. Conversely, miR-422a downregulation significantly increased DNAM-1 expression in NK cells, resulting in more efficient DNAM-1-mediated cytotoxicity. Our findings indicate that miR‑422a is a novel regulator for DNAM-1. Therapeutic control of miR-422a may provide new strategies against lung cancer.
CXCL10 within the tumor microenvironment induces gemcitabine resistance in pancreatic cancer cells (TUM10P.1037)
Authors: Delitto Daniel , Black Brian , Sorensen Heather , Knowlton Andrea , Thomas Ryan , Behrns Kevin , Moldawer Lyle , Trevino Jose , Wallet Shannon , Hughes Steven
Published: January 2023

The systemic treatment of pancreatic cancer (PC) is hindered by the rapid development of chemoresistance to current cytotoxic therapies. Mechanisms governing the development of chemoresistance remain poorly characterized, particularly with respect to contributions from the tumor microenvironment. Here, intratumoral soluble mediator concentrations from resected PC specimens (n=26) as well as supernatants from co-cultures of PC cell lines and primary tumor-associated pancreatic stellate cells (PSCs) (n=16) were evaluated using a panel of 43 growth factors, chemokines and cytokines. CXCL10 levels were significantly increased in PC specimens as well as upon co-culture of PC cells with tumor-associated PSCs. In addition, high intratumoral CXCL10 concentrations correlated with reduced overall survival (HR 6.9; P = .006). Thus, the effect of CXCL10 on viability, proliferation, and apoptosis of PC cell lines was evaluated with and without gemcitabine treatment. While CXCL10 treatment had a small effect on the viability of PC cell lines, it significantly increased viability of these cells in the presence of gemcitabine. In addition, gemcitabine treatment induced the expression of the CXCL10 receptor, CXCR3. Thus, paracrine CXCL10 signaling between stromal, PC and immune cells within the pancreatic cancer microenvironment the may be responsible for chemoresistance to gemcitabine.
VEGF neutralization alters the innate immune cell compartment and enhances intratumoral T cell activation (TUM10P.1040)
Authors: Walsh Kevin , de Almeida Patricia , Mak Judy , Hernandez Genevive
Published: January 2023

Although targeting the vascular endothelial growth factor (VEGF) pathway has been used in clinic as an anti-angiogenic therapy to combat cancer for almost a decade, the precise mechanisms of action have yet to be fully defined. Here we demonstrate in a panel of syngeneic tumor models that in addition to it’s anti-angiogenic activity, VEGF blockade augmented inflammation within tumors. Enhanced inflammation coincided with altered myeloid cell composition and modulation of dendritic cell activation. Modifications within the innate immune cell compartment mediated by anti-VEGF treatment correlated with enhanced functional capacity of tumor infiltrating T cells. This was characterized by increased cytokine production of total as well as antigen-specific T cells and decreased surface expression of negative immune regulators. VEGF receptors are detected on both innate immune cells and T cells suggesting that VEGF neutralization has a direct effect on these compartments. Based on these data, VEGF neutralization increased tumor inflammation by modulation of the innate immune cell compartment as well as promotion of anti-tumor T cell responses. Therefore, therapeutics that target the VEGF pathway have potential to work in concert with immunotherapeutics.
Acute myeloid leukemia transcription profile based on natural killer cell status at diagnosis correlates with clinical outcome (TUM10P.1047)
Authors: Toubert Antoine , Khaznadar Zena , Boissel Nicolas , Agaugue Sophie , Henry Guylaine , Cheok Meyling , Vignon Marguerite , Geromin Daniela , Cayuela Jean Michel , Lachuer Joel , Preudhomme Claude , Dombret Herve , Dulphy Nicolas
Published: January 2023

Acute myeloid leukemia (AML) is a heterogeneous group of malignancies sensitive to natural killer (NK) cell anti-tumor responses. However, NK cells are frequently defective in AML. Here, we found in an exploratory cohort (n = 46) that a different expression of activating receptors (NCR) on NK-cells identified a subgroup of patients with reduced expression of DNAM-1, NKG2D, NCRs and perforin especially in CD56bright NK-cells. Patients with a deficient NK-cell profile had a significantly higher risk of relapse (P = 0.03) independently of cytogenetic classification in multivariate analysis. We hypothesized that the transcription patterns in AML cells could give a clue on the NK/AML crosstalk. Analysis of the gene expression in AML blasts following classification according to their NK-cell status showed extensive differences in immune-related and oncogenic pathways. A favorable NK status was associated with pro-inflammatory AML transcription patterns whereas patients with defective NK cells showed a profound gene expression decrease in AML blasts for cytokine and chemokine signaling, antigen processing and adhesion molecule pathways. A set of 388 leukemic classifier genes defined in the exploratory cohort was independently validated in a multicentric cohort of 194 AML patients (ALFA-0701). In total, these data evidenced the prognostic value of NK-cell responses at diagnosis of AML and the relationships between NK-cells and AML independently of known cytogenetic status.
Surviving the odds: targeting PIM2 in plasma cell survival (TUM10P.1051)
Authors: Nair Jayakumar , Baldino Carmen , Caserta Justin , Carlson Louise , Fetterly Gerald , Lee Kelvin
Published: January 2023

Plasma cells (PC) depend on interactions with bone marrow stroma and the resultant growth factors for their long lived survival. Very little is known about the molecular mediators of these survival signals. One such molecule that has recently come of interest is PIM2. PC malignancies such as multiple myeloma (MM) preferentially express PIM2 over its counterparts PIM1 or PIM3 downstream of the PI3K/AKT/mTOR pathway essential for differentiation and survival of PC. In this study, we explored an experimental drug JP_11646 (Jasco Pharmaceuticals LLC) that has a high potency towards PIM kinases, more specifically towards PIM2. Using MM cell lines as a model for long term PC survival, we have shown for the first time that inhibiting PIM2 deregulates survival pathways that involve IL-6, DC protection of MM and CD28 activation by deregulating key survival factors such as NFκB, decreased phosphorylation and inactivation of the PIM2 substrate and pro-apoptotic protein BAD, lower levels of the inactive phosphorylated translational repressor 4EBP1, besides affecting transcription and translation of PIM2 itself. This was recapitulated when PIM2 was silenced with siRNA. Through northerns and protein assays, we also explore the transcriptional and translational dimensions involved in regulating PIM2 in plasma cells. An understanding of the role of PIM2 in MM biology will help to understand how plasma cells survive long term and maintain humoral immunity.
Plant-derived viral-like nanoparticle immunotherapy suppresses development of metastatic lung cancer (TUM10P.1058)
Authors: Lizotte Patrick , Wen Amy , Steinmetz Nicole , Fiering Steven
Published: January 2023

Conventional chemo- and radiotherapy are largely ineffective for metastatic melanoma and emerging immune checkpoint inhibitors, while promising, remain ineffective in the majority of patients and carry associated autoimmune side effects. We hypothesized that treatment of lung tumor-bearing mice with a viral-like nanoparticle could modulate the lung immune environment and prevent the development of B16F10 metastatic-like lesions. We report here that inhalation of a self-assembling viral-like particle derived from the cowpea mosaic virus (CPMV) suppresses the development of tumors in the lungs of intravenous challenged mice. The disparity in tumor burden between CPMV- and PBS-treated mice was pronounced, and the effect was immune-mediated as it was not seen in Ifn-γ-/-, Il-12-/-, and NOD/scid/Il2Rγ-/- mice. CPMV nanoparticles were rapidly taken up by antigen-presenting cells that had trafficked to the lung following treatment and resulted in their activation and cytokine/chemokine production. CPMV particles are manufactured in plants and purified of nucleic acids and therefore are nonreplicative and free of endotoxin and other pattern recognition receptor agonists. CPMV nanoparticles are highly scalable, stable, nontoxic, and modifiable with drugs and antigens. These properties, combined with their inherent immunogenicity and significant efficacy against a poorly immunogenic tumor, present CPMV as an attractive novel immunotherapy against cancer metastatic to the lung.
Engineering anti-HIV-1 antibodies to enhance activity and increase half-life (VAC11P.1104)
Authors: Sievers Stuart , Patel Sonal , Klein Florian , Bennett Kathleen , Nussenzweig Michel , Bjorkman Pamela
Published: January 2023

HIV/AIDS remains one of the most serious threats to global public health. Although anti-HIV drugs have been effective among the wealthiest populations, new methods to prevent infections are needed to control HIV-1 infections globally. Strategies to combat HIV-1 benefit from structural knowledge of how antibodies recognize HIV envelope proteins and how the immune system eliminates viruses. Until recently, only a small number of broadly neutralizing antibodies against HIV-1 had been characterized, and the immunological basis for their breadth and potency remains poorly understood. However, it was recently demonstrated that antibodies could be engineered to greatly enhance their breadth and potency. Unfortunately, these and other engineering efforts can result in a decrease in antibody half-life in various animal models. This decrease in half-life correlates with polyreactivity, an increase in reactivity to a variety of antigens. In order to make better targets for passive delivery therapies, we are using a variety of computational and structure-based techniques. We have constructed several mutations in regions that have been predicted to have high aggregation propensities, and have shown that these novel reagents have reduced polyreactivity and longer in vivo half-lives, yet maintain potency in neutralization assays. Further characterization will help further our understanding of the relationship between antibody potency, polyreactivity, and half-life.
Characterization of activated B cells, a novel antigen-specific B cell population that emerges in blood after infection and vaccination in humans (VAC11P.1062)
Authors: Ellebedy Ali , Ahmed Rafi
Published: January 2023

Plasma cells (PCs) and memory B cells (MBCs) provide a remarkably stable humoral immune memory. In humans, antigen-specific antibody secreting cells (ASCs) and MBCs are detected in peripheral blood shortly after infection and vaccination. While ASCs can be distinguished by surface staining, the phenotype of recently generated MBCs is unknown. Here, we defined the phenotype of the latter cells, and termed them activated B cells (ABCs). We confirmed the specificity of ABCs after influenza vaccination and infection by ELISPOT (after in vitro differentiation) and by HA-specific staining. Similarly to ASCs, ABCs actively proliferate, up-regulate CD27 and down-regulate CD21. In contrast to ASCs, ABCs do not spontaneously secrete antibodies and up-regulate CD20 expression. ABCs represent a distinct lineage from ASCs as they maintained Pax5 expression and showed only a slight increase in IRF4 expression compared to naive B cells. Using this characterization, we were able to clearly distinguish ABCs and ASCs in peripheral blood of two Ebola virus-infected patients who were being cared for at Emory University Hospital. Defining ABCs is critical for addressing basic questions regarding the fundamental differences between the memory and plasma cell compartments in humans and because ABCs could be used for procuring therapeutic monoclonal Abs that may be different in terms of their protective potential from those derived from ASCs.
Identification and characterization of monoclonal antibodies suitable for development of influenza A vaccine seed virus (VAC11P.1101)
Authors: He Yu , Bucher Doris
Published: January 2023

The seed viruses for influenza A vaccine production are high-yield (hy) reassortant viruses generated in embryonated chicken eggs by reassorting wild type (wt) viruses with a hy donor virus, A/Puerto Rico/8/1934 (PR8). Currently, reassortants with wt hemagglutinin (HA) and neuraminidase (NA) are selected for growth by using polyclonal antibodies (pAbs) against PR8. The efficiency and reproducibility of reassortment will be greatly enhanced with monoclonal antibodies (mAbs) which not only provide high specificity against PR8 but could also be prepared as defined reagents with guaranteed activity in unlimited quantities. In this study, from a panel of hybridoma clones prepared against PR8 surface glycoproteins, we found four HA specific mAbs with complete neutralization of PR8 virus in an in vitro plaque inhibition assay, which are designated as mAb-1B3, mAb-1H6, mAb-2A6, and mAb-2D11. All four mAbs have been shown highly specific in both in vitro and in ovo neutralization activity to PR8. Furthermore the epitopes of all four mAbs were mapped to the area around the receptor binding site (RBS) at the membrane-distal tip of HA. Their epitopes are composed of four regions of HA residues 158-172, 183-197, 208-222, and 253-267 (H1 numbering with signal sequence). The HA residues 158-172 and 253-267 are two common regions for all four mAbs, while residues 183-197 is specific for mAb-1H6 and mAb-2A6, and residues 208-222 is specific for mAb-2D11.
Anti-CCR4 antibody enhances anti-tumor immunity by modulating tumor-infiltrating Tregs in an ovarian cancer xenograft animal model (VAC12P.1121)
Authors: Chang DeKuan , Sun Jiusong , Zhu Quan , Drapkin Ronny , Matulonis Ursula , Marasco Wayne
Published: January 2023

Recent experimental and clinical data demonstrate that regulatory T cells (Tregs) are recruited to the tumor site where they can suppress tumor-specific immunity. Increasing evidence indicates that Treg recruitment to the tumor is mediated by the production of chemokine CCL22, a ligand of chemokine receptor CCR4, by tumor cells and tumor resident macrophages. Therefore, blocking Treg migration and function through CCL22/CCR4 axis may have therapeutic value in cancer treatment. With a mouse model bearing CCL22-secreting ovarian xenograft and tumor-specific T cells, we assessed the therapeutic effect of human anti-CCR4 antibody mAb2-3 to block or modulate immunological axes involved in tumor immune evasion. CCR4 expression was demonstrated at a high level on functional CD4+CD25+ Tregs and T cell proliferation was restored by removal of CCR4+ Tregs in vitro. In vivo examinations showed isotyping of mAb2-3 had marked effects on its mode of action: mAb2-3 IgG1 results in Treg depletion whereas IgG4 blocks in vivo chemotactic migration to CCL22-secreting ovarian cancer (OVC) cells without Treg depletion. The mAb2-3-treated mice bearing OVC xenograft and tumor-primed T cells showed the ability to inhibit Treg recruitment and resulted in potent anti-tumor immunity. The results revealed that depletion of Tregs and blockade of Treg recruitment through mAb2-3 IgG1 and IgG4, respectively, could reverse Treg suppression and improve cellular immunity against tumors.
Adenovirus 5 vaccination elicits IL-27, which impairs CD4 and CD8 T cell functionality (VAC12P.1113)
Authors: Larocca Rafael , Provine Nicholas , Iampietro M. , Borducchi Erica , Penaloza-MacMaster Pablo , Bricault Christine , Blass Eryn , Parenteau Lily , Blackmore Stephen , Stephenson Kathryn , Barouch Dan
Published: January 2023

In pre-clinical studies Ad26 has shown increased protective efficacy compared to Ad5. We hypothesize this is due, in part, to differences in phenotype and function of the elicited CD4 and CD8 T cell responses. Thus, our goal is to elucidate the phenotypic differences of the CD4 and CD8 T cell repertoire following Ad5 or Ad26 vaccination and the underlying mechanism. B6 mice vaccinated with Ad5 upregulate PD1 and Tim3 (p<0.01) expression on both CD4 and CD8 T cells 10 days post-immunization. On day 60, high PD1 and low CD226 expression (P<0.01) was observed following only Ad5 immunization. Ad5, but not Ad26, induced a population of regulatory CD4 T cells that co-produce IFN-γ and IL-10 (p<0.01). In Ad5 vaccinated non-human primates we also observed elevated expression of PD-1 and production of IL-10 (p<0.01), and less IL-2 expression by CD4 T cells. Mouse DCs stimulated in vitro with Ad5 showed a four-fold increase on Il27 mRNA compared to Ad26 stimulated cells. Immunization of IL-27ra KO mice with Ad5 resulted in downregulation of IL-10 (p<0.01), and up-regulation of IL-2 and CD226 (p<0.01) by CD4 T cells compared to WT. CD8 T cells from KO mice exhibited increased TNF-α and IL-2 polyfunctionality (p<0.01). These data demonstrate that Ad5 vaccination induces greater IL-27 production that drives inhibitory molecule expression and dysfunctional cytokine secretion by CD4 and CD8 T cells. These data suggest a mechanistic explanation for one reason why Ad5 is a suboptimal vector.
Denileukin diftitox reduces EL4 lymphoma growth by depleting regulatory T cells, which can be improved by αICOS (VAC12P.1116)
Authors: Liu Yang , Padron Alvaro , Drerup Justin , Dao Vinh , Liu Aijie , Hurez Vincent , Curiel Tyler
Published: January 2023

Immunotherapy shows promise in carcinoma treatments. As less is known about effects on lymphomas, we tested EL4 T cell lymphoma. We found that EL4 cells were CD25-CTLA4-B7H1loPD1hi by flow cytometry. Immune checkpoint blockade with αCTLA4, αB7H1, or αPD1 antibodies had no effect on tumor growth in EL4-challenged mice. αCD25 worked as prophylaxis (before EL4 challenge) but not therapy, despite depleting regulatory T cells (Tregs). In Foxp3DTR mice, specific Treg depletion reduced EL4 tumor growth, suggesting Treg depletion as a good treatment strategy. Denileukin diftitox (DD) is a relatively-specific Treg depletion drug with clinical benefits in mouse and human cancer. DD (5 μg/4 days starting 4 days after EL4 challenge) significantly delayed EL4 tumor growth and increased survival (P=.002) in WT mice. DD protection was lost in βδ TCR KO mice lacking all T cells, suggesting a T cell dependent treatment mechanism. In EL4-challenged WT, DD depleted Tregs > αCD25 but significantly increased ICOS by 2-fold on remaining Tregs > than on CD4+ or CD8+ T cells in tumor draining lymph nodes. Nearly all Tregs within tumors were ICOS+. ICOS+ Tregs were mostly CD44+ with hi Ki-67, suggesting activation that reduced DD treatment effects. In support, αICOS (100 μg/4 days starting 1 day after DD) enhanced DD-mediated EL4 tumor growth inhibition, but did not improve overall survival. DD-mediated Treg depletion plus αICOS and/or other adjuncts could lead to effective lymphoma immunotherapy.
Tumor pericyte-associated antigens DLK1 and DLK2 as targets for cancer immunotherapy (VAC13P.1131)
Authors: Fabian Kellsye , Storkus Walter
Published: January 2023

The vasculature of solid tumors is essential for their progressive growth and is abnormal structurally and functionally. Notably, pericytes and vascular endothelial cells (VEC) in tumors exhibit altered epigenetic programming and express antigens that allow for their differential recognition (vs. vascular cells in normal tissues) by therapeutic T cells. Therefore, targeting immune reactivity against these antigens via specific vaccination may be a viable therapeutic approach against solid tumors. We show that pericytes isolated from human and murine renal cell carcinoma (RCC) and melanoma overexpress the antigens Delta-like homologue 1 (DLK1) and its homologue DLK2. Using recombinant lentiviruses and adenoviruses encoding full-length murine DLK1 and DLK2 proteins, we show that coordinate genetic vaccination against these two antigens leads to superior therapeutic inhibition of RENCA and B16 tumor growth in vivo when compared to tumor-bearing mice vaccinated against either DLK1 or DLK2 alone. The coordinate vaccination protocol also resulted in the activation of DLK1- and DLK2-specific CD8+ T cells, recruitment of T cells into the tumor microenvironment, loss of DLK1 and DLK2 expressing pericytes and structural normalization of tumor-associated blood vessels. We conclude that specific vaccination against (oncofetal) DLK1 and/or DLK2 may represent an effective strategy for the treatment of vascularized, solid forms of cancer (including RCC and melanoma, among others).
Interferon epsilon protects primary human macrophages against HIV-1 infection by blocking early stages of the viral life cycle (VIR9P.1152)
Authors: Tasker Carley , Chang Theresa
Published: January 2023

Interferon epsilon (IFNε), a type-I IFN, is hormonally regulated and plays a protective role against several viral and bacterial pathogens. In this study, we examined the effect of IFNε on HIV infection and its underlying mechanism. IFNε at 10ng/mL inhibited HIV infection of human monocyte derived macrophages (MDMs) but not activated CD4+ T cells. Pretreatment of MDMs with IFNε protected MDMs against infection by replication competent X4, R5, and X4R5 dual-tropic viruses and HIV transmitted/founder viruses. We further delineated the underlying mechanism of the anti-HIV activity of IFNε and found IFNε down-regulated cell surface expression of the co-receptor CCR5 but not CD4 or the co-receptor CXCR4. Although IFNε treatment affected HIV entry, it also blocked infection by HIV reporter virus pseudotyped with VSV envelope, which enters cells independently of CD4 and HIV co-receptors, indicating that IFNε treatment inhibited HIV infection after viral entry. Quantitative PCR analysis of HIV reverse transcribed (RT) DNA products revealed IFNε blocked the step of early RT. IFNε induced expression of the HIV host restriction factor APOBEC3A in MDMs, however siRNA knockdown of A3A determined it may not play a significant role in IFNε mediated HIV inhibition. Taken together, IFNε protects MDMs against HIV infection through multiple mechanisms by down-regulation of CCR5 expression and by inhibiting the synthesis of HIV reverse transcribed products.
Phosphorylation of STAT1 and NF-κB by human plasmacytoid dendritic cells during HIV-infection (VIR9P.1154)
Authors: Serei Virian , Dai Jihong , Fitzgerald-Bocarsly Patricia
Published: January 2023

Plasmacytoid dendritic cells (pDC) are critical for anti-viral immunity. TLR7 and TLR9 allow pDC to recognize viruses and initiate signals leading to IRF7 phosphorylation, which is responsible for IFN-α production, as well as NF-κB phosphorylation, which is responsible for TNF-α and IL-6 induction. During HIV-infection, lower pDC numbers are found, along with impaired IFN-α production. Chronic immune activation is also found, with patients paradoxically displaying an IFN-signature, indicating the presence of IFN-α, and higher serum TNF-α and IL-6. To see if pDC are affected in vivo by IFN-α or TNF-α, we compared baseline p-STAT1 and p-NF-κB in pDC in HIV-patients vs healthy controls. At baseline, HIV-patient vs. healthy pDC showed a trend towards elevated p-STAT1, while p-NF-κB levels were similar. HIV-patient pDC phosphorylated STAT1 and NF-κB less than healthy controls in response to IFN-α and TNF-α, respectively. In response to viruses, HIV-patient pDC phosphorylated STAT1 more than healthy controls. Despite normal production of TNF-α and IL-6, HIV-patient pDC were unable to phosphorylate NF-κB in response to viruses, suggesting that other mechanisms exist for TNF-α and IL-6 induction. In conclusion, impaired NF-κB phosphorylation by HIV-patient pDC suggest that they are immunologically exhausted and this can lead to a number of downstream effects on protein expression. In contrast, enhanced p-STAT1 in response to viruses can drive the IFN-signature seen in HIV-patients.
Regulatory CD4 T cells differentially modulate HIV-1 infection of polarized M1 and M2 macrophages (VIR9P.1156)
Authors: Robinson Tanya , Zhang Mingce , Lowe Robert , Cron Randy
Published: January 2023

Macrophages are long-lived, widely distributed and relatively resistant to HIV-1 cytotoxicity. Effector macrophages are functionally polarized into M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes. M1 macrophages suppress CCR5 (R5) HIV-1 replication, while M2 macrophages promote it. Recently, regulatory CD4 T cells (Tregs) were shown to inhibit HIV-1 infection of co-cultured CD4 T cells, but their effect on macrophage infection is unknown. Using an in vitro model of polarization, we generated M1 (GM-CSF+IFN-γ+LPS) and M2 (M-CSF+IL-4) macrophages with predicted phenotypes and infected them with R5 HIV-1 reporter viruses. We consistently found higher levels of HIV-1 (GFP and luciferase reporter viruses) infection of the M2 cells. Next, we co-cultured Tregs with HIV-1 infected polarized M1 and M2 MDM at a 1:1 ratio. Treg co-cultures yielded decreased HIV-1 infection of M1 but increased infection of M2 macrophages. Surprisingly, our results showed that Tregs mediate divergent effects on HIV-1 infection of M1 and M2 MDM depending on their polarization into a M1 or M2 phenotype. We are currently exploring whether Treg-mediated effects on HIV-1 infection of MDM are cytokine dependent and/or cell contact dependent using a transwell system and inhibitory cytokine antibodies. The research will provide increased knowledge of the role Tregs play in HIV-1, as well as a new understanding of how the M1 to M2 macrophage switch contributes to HIV-1/AIDS pathogenesis.
PSGL-1, an immune checkpoint regulator that promotes T cell exhaustion in chronic viral infection and cancer (VIR10P.1166)
Authors: Tinoco Roberto , Carrette Florent , Barraza Monique , Magana Jonathan , Swain Susan , Bradley Linda
Published: January 2023

Chronic viruses, including HIV and hepatitis B and C, can survive in humans by inducing T cell dysfunction. Using a murine chronic virus that models human infections, we investigated the function of the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1) that is upregulated on responding T cells. PSGL-1-deficient mice unexpectedly cleared the virus due to dramatic increases in the intrinsic survival of multifunctional effector T cells that had downregulated PD-1 and other inhibitory receptors. Notably, this response resulted in immunopathology requiring CD4+ T cells. Mechanistically, PSGL-1 ligation on exhausted CD8+ T cells sustained PD-1 expression and diminished their survival during TCR stimulation. In a model of malignant melanoma where T cell dysfunction also occurs, PSGL-1 deficiency led to PD-1 downregulation, improved T cell responses and tumor control. Thus, PSGL-1 not only plays a fundamental role in balancing viral control and immunopathology, but also functions as a checkpoint that regulates T cell function in the tumor microenvironment.
CD11c-expressing B cells from aged and autoimmune mice localize at T cell B cell border in spleen and present antigen more efficiently than Follicular B cells (BA3P.104)
Authors: Rubtsov Anatoly , Rubtsova Kira , Kappler John , Marrack Philippa
Published: January 2023

Besides secretion of antigen-specific antibodies, B cells may play an important role in the generation of immune responses by efficiently presenting antigen to T cells. We and others recently described a subpopulation of CD11c+ B cells (Age/autoimmune associated B cells, ABCs) which appear with age, during virus infections and at the onset of some autoimmune diseases and which participate in autoimmune responses by secreting autoantibodies. Here we assessed the ability of these cells to present antigen and activate antigen-specific T cells. We demonstrated that ABCs present antigen to T cells, in vitro and in vivo, better than follicular B cells (FO cells) do. Our data indicate that ABCs express higher levels of the chemokine receptor, CCR7, and have higher responsiveness to CCL21 and CCL19 than FO cells and are localized at T/B cell border in spleen. Using multiphoton microscopy we have shown that in vivo, CD11c+ B cells form significantly more stable interactions with T cells than Follicular B cells. Together these data identify a previously undescribed role for ABCs as potent antigen-presenting cells and suggest another potential mechanism by which these cells can influence immune responses and/or the development of autoimmunity.