bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunotherapeutic proteins expressed in transgenic soybean seeds (65.1)
Authors: Bost Kenneth , Hudson Laura , Piller Kenneth
Published: January 2023

Soybean seeds expressing transgenic proteins represent a novel, sustainable platform technology which overcomes some of the current limitations for producing recombinant proteins useful as immunotherapeutics. Advantages include low cost of glycoslyated protein production, greenhouse containment, highest protein/biomass ratio, marketable formulations which require no purification from soy, safety, accurate dosing, low cost of protein purification, low-tech sustainability of product lines, reduced risk of contamination, ease of scalability, minimal waste produced, as well as being a green technology. We have successfully expressed a variety of subunit protein antigens, and potential immunomodulatory proteins in transgenic soybean seeds, and are evaluating their usefulness as vaccines and therapeutics. The flexibility of long-term storage of transgenic seeds or powder in the absence of a cold chain and without degradation allows protein expression to occur years in advance of its therapeutic use. Purification of a transgenic protein is simplified by the limited number of soy proteins present in seeds. Furthermore, transgenic protein expression levels of approximately 13 grams per liter of soy powder and 22 kilograms per greenhouse acre represent the highest protein/biomass ratio for the industry, and contribute to low production costs. As protein expression technologies expand and evolve, transgenic soybean seeds represent an emerging technology with significant advantages.
Development of an alternative method for the identification and production of antigen-specific monoclonal antibodies (65.16)
Authors: Byrne-Steele Miranda , Sanders Catherine , Yang Qunying , Pan Wenjing , Wang Chunlin , Han Jian
Published: January 2023

A critical step in the production of monoclonal antibodies (Abs) is the initial identification of the antigen-specific Abs, which is usually performed by multiple rounds of panning in both hybridoma and phage display. We have developed an alternative method that allows for the rapid and direct identification of antigen-specific Abs from peripheral blood via high-throughput immune repertoire sequencing and LC MS/MS peptide matching. Our B-cell repertoire technology combines novel amplicon rescued multiplex PCR (arm-PCR, patent pending) with high-throughput gene sequencing to access the sequence of a broad spectrum of heavy and light chain V-regions. As a proof of concept, we have sequenced the immune repertoire of 2 healthy individuals at various time points after administration of the 2009-2010 seasonal influenza vaccine. Antigen-specific Abs were purified directly from immune peripheral blood serum and identified using LC MS/MS peptide sequencing, exploiting the B-cell repertoire gene sequencing results as a database for identification. During our study, several unique peptides were successfully matched for each individual’s response to both Flu A strains in the vaccine. Future work includes the development of a method to rapidly clone and express these identified Abs in a human in vitro glycoexpression system. The recombinant Abs will then be tested for their ability to bind flu hemagglutinin, demonstrating the utility of our technology towards the production of mAbs.
Acoustic flow cytometry for multicolor immunophenotyping analysis (65.23)
Authors: Zhang Yu-Zhong , Anderson April , Bradford Jolene
Published: January 2023

The Attune® Acoustic Focusing Cytometer utilizes resonant device that focuses cells or particles into a single tight line. This technology enables both longer cell interrogation times and higher throughput, which simultaneously permits better cell interrogation as well as the analysis of a larger numbers of cells. As a result, we were able to accomplish a high throughput of samples with relatively low sample concentration while maintaining a high degree of separation. Human whole blood stained with several IVD and ASR grade pre-mixed cocktails followed by lyse no-wash procedures were analyzed on the Attune® Acoustic Cytometer. The immunophenotyping data indicated that the analyses obtained are both reliable and reproducible. In addition, the 405 nm violet laser in Attune® Acoustic Cytometer allows researchers using Qdot® nanocrystals to add more parameters to their flow cytometry experiments by filling in the spectral landscape around common organic fluorochrome conjugates. The availability of Qdot® nanocrystals in multiple emission wavelengths and ever more Qdot® conjugates, allows the creation of multicolor panel consisting of Qdot® conjugates. We show the effectiveness of using Qdot® -conjugated antibodies in a typical 6-color T-cell phenotyping panel. Experiments were carried out using standard staining protocols with human peripheral blood leucocytes.
Cryopreserved highly purified immunologically important cell subsets as tools to approach immunological studies (65.27)
Authors: Di Pucchio Tiziana , Glover Chondra , Manak Mark , Anekella Bharathi
Published: January 2023

Cell types, including conventional B and T lymphocytes, regulatory T cells, natural killer and NKT cells, monocytes and dendritic cells are a great resource for a number of immunological studies. Cryopreserved cells are particularly useful in clinical studies as they allow the immunologic assays to be performed after conclusion of studies. Moreover, availability of purified functional cell subsets could help identification and dissection of immunological mechanisms occurring during protective immune responses and lead to development of new cell therapies and vaccine strategies. We show that highly purified human blood cell subsets retain their functionality and differentiation capability when frozen by efficient cryopreservation techniques. Our results demonstrate that frozen PBMC, purified monocytes and T cell subsets maintain their viability and recovery. Furthermore, frozen PBMC and T cell subsets maintain similar ability to proliferate and produce IFN-gamma in response to specific peptide antigen stimulation compared to fresh cell subsets. Notably, fresh and frozen purified monocytes are able to differentiate into phenotypical and functional dendritic cells with similar capability to stimulate allo-CD3 proliferation. Our results indicate that cryopreserved cell subsets could be used as a basic tool in cell-based assays for studying cellular phenotype and functions in studies testing the efficacy of new drugs and vaccines that are designed to modulate immune system.
A polychromatic antibody cocktail to investigate phenotypic and functional markers on human myeloid and plasmacytoid dendritic cells (65.3)
Authors: Jain Pooja , Betts Michael , Manuel Sharron , Makedonas George
Published: January 2023

Antigen presentation is a critical feature of adaptive immunity and essential in self versus nonself discrimination as well as in stimulating immune responses against pathogens. Dendritic cells (DCs) are considered to be the most potent antigen-presenting cells and have long been recognized as key regulators of the immune system. Because DCs are such crucial cells of the immune system, an extensive assessment of their quality in infected individuals is critical. Therefore, we have standardized a unique DC cocktail containing 12 different functional DC markers using an LSR II polychromatic flow cytometer. To this point, we have tested our newly standardized cocktail on the fresh, frozen, and cultured peripheral blood mononuclear cell (PBMCs) as well as on monocyte-derived DCs (MDDCs), a widely used surrogate system for primary blood DCs. This study emphasizes the practicality of utilizing frozen versus fresh cells, especially in rare disease states, wherein obtaining fresh material is virtually impossible. Furthermore, we focus on the feasibility of PBMCs compared to whole blood as starting material. To our knowledge this is the first comprehensive DC phenotyping protocol that uses an extensive list of functional markers (>8). Collectively, these investigations possess great potential to enable investigators to perform immune cell monitoring and develop diagnostic and therapeutic strategies.
Tracking activation of caspases in T cell apoptosis (65.41)
Authors: Gillis Katherine , Clor Julie , Tyagarajan Kamala
Published: January 2023

Caspase activation is critical to immune cell apoptosis, with different caspases activated in different pathways of cell death. Thus, Caspase 8 is implicated in CD95-mediated apoptosis, Caspase 9 is crucial to the intrinsic pathway of apoptosis and Caspase 3,7 activation, is observed in multiple pathways of cell death. Caspases also play a role in immune cell inflammation and proliferation. Simplified methods that allow identification of immune cells and specific caspase activity can provide important mechanistic insights in immune cell apoptosis. We present here results from a simplified strategy to assess caspase activity in CD4 and CD8 T cells using T cell specific cocktails and fluorescently labeled caspase inhibitors. Using this approach, caspase activation with multiple compounds such as anisomycin and staurosporine were studied. Our results indicate that anisomycin causes early activation of both Caspase 9 and Caspase 8 followed by slower activation of Caspase 3,7 activity in both CD4 and CD8 T cells. Differential dose response curves were obtained for Caspase levels in CD4 and CD8 T cells on treatment with staurosporine. In some cases, donor specific differences were observed in the timing and relative activation of the different caspases. Simultaneous assessment of caspase activites in different immune sub-populations allows for a deeper understanding of mechanistic pathways of apoptosis, which is important in drug development and understanding of diseases.
A free NCRR resource for finding and using human disease models (65.46)
Authors: Mungall Chris , Haendel M. , Chatr-aryamontri A. , Oughtred R. , Rust J.
Published: January 2023

LAMHDI.org, a free web-based resource, helps researchers identify model systems to investigate disease mechanisms and therapies by bringing together information about diseases and model organisms. The LAMHDI portal allows search of disease models across species (non-human primates, zebrafish, mice, rats, flies, and yeast, with others in the pipeline) using gene orthology and pathway membership as key linkages. New work includes matching specific phenotypes and common pathways, a zebrafish atlas, and a graphical search based on spatial models of the brain. LAMHDI’s collaboration with BioGRID permits the exploration of networks linked to immune response, such as the NFkappaB and Interferon gamma signaling pathways. Interactions involving homologous members of these pathways provide a valuable resource for exploration across animal models. The goal is to facilitate the identification of models for disease research, make better use of existing model organisms and data about them, and provide the ability to discover new relationships between disease, phenotypes and genes that will further our understanding of disease. Companion efforts will speed the discovery and validation of novel drug candidates in areas as diverse as infectious disease, neuroscience, cardiovascular and metabolic disorders, autoimmunity, and cancer.
Single-cell multiplex RT-qPCR analysis of genes related to migration of murine long-term hematopoietic stem cells during development (65.48)
Authors: García-Ojeda Marcos , Hall Dominique , Lu Alison , De Sena Joseph , Al-Kuhlani Mufadhal , Ciriza Jesús
Published: January 2023

Hematopoietic stem cells (HSC) follow a defined pattern of migration in development. By E14.5, HSC are found in the fetal liver (FL) and colonize the fetal bone marrow (FBM) by E17.5, where they remain during adult life. The mechanism governing HSC migration from the FL to the FBM is not clearly understood. We performed single-cell multiplex RT-qPCR and FACS analysis of 8 genes involved in HSC migration. We show that the expression of the chemokine receptor Cxcr4 varies across microenvironments and developmental times, while N-cadherin and the calcium receptor Casr show only gene expression in FBM at E17.5. Moreover, VE-cadherin maintains high expression only during fetal development, while the integrin subunit α5 increases its variability after E14.5. The integrin subunits α4 and αL as well as the selectin ligand Psgl1 did not show differences in their expression in single HSCs irrespective of the microenvironment. Our data demonstrate that the expression pattern of phenotypically identical, single HSCs fluctuates as a function of developmental and anatomical microenvironment, an observation that could be overlooked using whole population gene expression analysis. This is the first gene expression comparison of migration-related molecules in fetal tissues across developmental times, enhancing the understanding of HSC migration during development. These findings may have potential relevance in improving BM engraftment of clinically transplanted HSC.
Optimization of loading conditions for a violet dye for use in cell proliferation studies (65.9)
Authors: Rohrer Jurg , Elia Jeanne , Rabenstein Jacob
Published: January 2023

Fluorescein diacetate (FDA) and its derivatives, such as carboxyfluorescein succinimidyl ester (CFSE) are nonfluorescent molecules that diffuse into cells and are hydrolyzed by intracellular non-specific esterases to become fluorescent by-products. These fluorescent dye molecules accumulate only in live cells with intact cell membranes and active esterases, while dead cells remain non fluorescent. The precise kinetics of membrane transport and intracellular hydrolysis of FDA and its analogs are related to cellular functions. FDA dyes have been shown to have multiple uses which include, in vitro or in vivo cell tracking experiments and cell proliferation. BD Horizon™ Violet Proliferation Dye (VPD450) dye contains both an esterase-cleavable and an amine-reactive succinimidyl ester group. This dye can be used in multicolor flow cytometry applications with GFP or FITC-labeled cells. Optimization of cell and dye concentrations are important considerations in experimental design with all FDA-based proliferation dyes. A comparison study was performed to determine the effects VPD450 dye loading on cell proliferation, apoptosis, and cell signaling.
Targeting PD-1 and Tim-3 pathways to reverse tumor antigen-specific CD4+ T cell dysfunction in patients with advanced melanoma (66.1)
Authors: Fourcade Julien , Sun Zhaojun , Guillaume Philippe , Luescher Immanuel , Sander Cindy , Kirkwood John , Kuchroo Vijay , Zarour Hassane
Published: January 2023

The coexistence of spontaneous tumor antigen-specific immune T cell responses with progressive disease in cancer patients furthers the need to dissect the molecular pathways involved in tumor-induced T cell dysfunction and exhaustion. We have previously observed in patients with advanced melanoma that NY-ESO-1-specific CD8+ T cells upregulate PD-1 and a fraction of PD-1+ NY-ESO-1-specific CD8+ T cells upregulates Tim-3. We have found that Tim-3+PD-1+ NY-ESO-1-specific CD8+ T cells represent highly dysfunctional T cells. Importantly, Tim-3 and PD-1 pathway blockades synergized to enhance immune functions of NY-ESO-1-specific CD8+ T cells upon prolonged antigen stimulation. Whether the upregulation of PD-1 and Tim-3 is associated with tumor antigen-specific CD4+ T cell dysfunction remains unknown. Here, our findings in advanced melanoma patients show that in contrast to virus-specific and total CD4+ T cells, PD-1 and Tim-3 expression is upregulated by NY-ESO-1-specific CD4+ T cells. Interestingly, PD-1 and Tim-3 pathway blockades synergized to restore the numbers of cytokine-producing and proliferating NY-ESO-1-specific CD4+ T cells. Collectively, our findings support the use of Tim-3 and PD-1 pathway blockades to reverse tumor-induced CD8+ and CD4+ T cell exhaustion in patients with advanced melanoma. Our findings have significant implication in terms of novel immunotherapeutic strategies aiming at promoting T cell-mediated tumor regression in patients with advanced cancers.
Carcinoembryonic antigen-related cell adhesion moclecule-1 regulates myeloid-cell-dependent tumor angiogenesis by inhibition of G-CSFR-Bv8 pathway (66.19)
Authors: Lu Rongze , Pan Hao , Shively John
Published: January 2023

Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs) promote tumor angiogenesis and are refractory to VEGF antibodies treatment. Bv8 expressed in MDSCs has recently been shown to modulate MDSCs mobilization and tumor angiogenesis. Carcinoembryonic antigen-related cell adhesion moclecule-1(CEACAM1) is highly expressed on Gr1+ myeloid cells, however the role of CEACAM1 in myeloid cells mediated tumor angiogenesis has not been investigated. We have demonstrated that tumor growth and angiogenesis was significantly enhanced in Ceacam1-/- mice, which was independent on T and B cells . Meanwhile MDSCs infiltration in tumor, spleen and blood was increased. Tumors coinjected with MDSCs from Ceacam1-/- mice exhibited increased angiogenesis compared to coinjection with MDSCs from control mice. Depletion of Gr1+ cells inhibited tumor growth and angiogenesis in control and Ceacam1-/- mice. MDSCs from Ceacam1-/- mice expressed higher Bv8 in vitro and in vivo. Further we have shown that CEACAM1 recruited Src-homology-phosphatase-1 (SHP-1) to attenuate granulocyte colony-stimulating factor receptor (G-CSFR)-Bv8 pathway. Reconsititution of CEACAM1 into Ceacam1-/- bone marrow cells restored tumor growth and angiogenesis to normal level, and mutations of immunoreceptor tyrosine-based inhibitory motifs ITIMs abolished this restoration. Therefore, we identified CEACAM1, as an inhibitory regulator of G-CSFR-Bv8 pathway, negatively regulates myeloid-cell-dependent tumor angiogenesis.
Mannose receptor regulates macrophage polarization in the tumor microenvironment (66.31)
Authors: Dangaj Denarda , Joshi Shree , Zhao Aizhi , Sandaltzopoulos Raphael , Scholler Nathalie
Published: January 2023

Tumor-associated macrophages (TAMs) share properties with M2 macrophages (MΦ),including high IL10 and mannose receptor (MR) expression. MR cross-linking differentiates DCs into APCs inducing T-cell anergy. However,although MR is abundantly expressed on M2 MΦ,its role on TAMs remains poorly understood. To address this question,we isolated 3 novel recombinant antibodies(scFv) against the human mannose binding domain of MR (CRD4-MR),and set up transwell cocultures allowing chemical exchanges between MΦ and ovarian cancer cells. MΦ phenotype was assessed by FACS,qPCR and bead-based FACS. We also challenged wild type,MR+/- and MR-/- mice with ovarian cancer. We first confirmed that MΦ coculture with tumor cells triggers a tumor associated phenotype and showed that 1 anti-CRD4-MR scFv could block the phenotype switch. We further demonstrated that tumor-released mesothelin (MSLN), but not GPI-anchor truncated MSLN,bound to MR-expressing MΦ,and that MSLN binding could be blocked by all 3 anti-CRD4-MR scFvs. Finally, although tumor challenged MR-/- mice did not survive significantly longer than the wild type,MR+/- mice exhibited lower tumor burden and prolonged survival. MR+/- ascites MΦ secreted significantly higher levels of TNF-a consistent with a pro-inflammatory phenotype. Altogether,tumor-released mesothelin binds to MR and blocking of CRD4-MR binding inhibits TAM phenotype and promotes MΦ with proinflammatory proprieties. Blocking MR function could have therapeutic potential.
NF-κB pathway orchestrates the tumor promoting role of monocytes/macrophages in human renal cell carcinoma (66.45)
Authors: Chittezhath Manesh , Dhillon Manprit , Teo YiLing , Chen Jinmiao , Yang Henry , Lum Josephine , Shalova Irina , Kamaraj Revathy , Singh Rajeev , Wong Alvin , Biswas Subhra
Published: January 2023

Growing evidences suggests that monocyte/macrophages play crucial role in cancer progression. Even though studies in transgenic mouse tumor models have demonstrated the role of myelomonocytic cells in various aspects of tumor progression, information on the role of these cells in human cancers is limited. We investigated the role of monocytes/macrophages in the progression of a human renal Cell Carcinoma (RCC). Gene expression studies have shown that RCC associated monocytes (RCC-Mo) under basal condition exhibit a proinflammatory and protumoral phenotype characterized by high expression of genes such as TNFA, IL6, VEGFA, IL8 and MMP9. Surprisingly, when these cells were exposed to inflammatory stimuli, such as Lipid A or TNF-α, they failed to show an up- regulation of proinflammatory cytokines such as TNFA, CCL3, IL6, and IL1B. This observation correlated to the tumor-induced immunosuppression observed in the patients. Signaling studies showed that the inflammatory/protumoral phenotype of TAMs under basal condition was regulated through NF-κB pathway.Further studies demonstrated defective NF-κB signaling was responsible for the “refractory state” of this RCC-Mo to further inflammatory stimuli. In vivo studies in a human RCC mouse model validated our above findings. In conclusion these results are the first to characterize the transcriptomal and functional phenotype of tumor associated monocytes/macrophages in human cancer which suggested their tumor promoting role.
Blockade of cystine transport inhibits glutathione synthesis and survival of inflammation-induced myeloid-derived suppressor cells (66.6)
Authors: Beury Daniel , Fitzgerald Phillip , Ostrand-Rosenberg Suzanne
Published: January 2023

Tumor-induced myeloid derived suppressor cells (MDSC) are responsible for immune suppression in tumor bearing individuals and are a major obstacle to effective immunotherapy. MDSC suppress T cell activation through several mechanisms including their production of reactive oxygen species (ROS). MDSC accumulation, suppressive activity, and ROS production are enhanced by inflammation. However, MDSC production of ROS does not negatively impact MDSC themselves. Since MDSC sequester cystine/cysteine from their local environment, and cysteine is the rate limiting reagent for glutathione (GSH), which is the predominant antioxidant, we hypothesized that inflammation-induced MDSC utilize high GSH production to attenuate ROS-mediated damage. This hypothesis is supported by our data showing that inflammation-induced MDSC upregulate the subunits of the xc- (cystine/glutamate) transporter, resulting in greater uptake of cystine and higher GSH synthesis. Since MDSC only generate cysteine via the uptake of cystine, the xc- inhibitor sulfasalazine (SASP) may decrease MDSC survival and suppressive activity. This concept is supported by our findings that MDSC treated with SASP are depleted of GSH and are less viable and that inflammation further enhances these effects. Because of their ability to reduce MDSC survival and block MDSC-mediated suppression, xc- inhibitors are potential supplements to cancer immunotherapies.
Response of human natural killer cells to monocyte-derived dendritic cells infected with dengue virus (67.10)
Authors: Lim Daniel , Ooi Eng Eong , Li Na , Yeong Lai Han , Wong Soon Boon Justin
Published: January 2023

Natural Killer (NK) cells are involved in innate responses triggered early during viral infections. However, the factors that regulate the response of primary human NK cells to primary human cells infected with dengue virus (DV) remain poorly characterized. In this study, we demonstrate that naïve NK cells co-cultured with DV-infected monocyte-derived dendritic cells (DCs) produce IFN-γ, upregulate cell surface CD107a, and lyse DV-infected DCs. Type I interferons and TNF-α produced by the DV-infected DCs were crucial for this IFN-γ response, since blocking IFNAR combined with neutralization of TNF-α completely abolished the IFN-γ response in these co-cultures. Cytokines previously reported to play important roles in NK cell activation, e.g. IL-12, IL-15, IL-18, IL-21, and IL-27, were not produced in significant amounts by DV-infected DCs. Transwell experiments suggested that membrane-bound ligands were also important for NK cell recognition of DV-infected DCs. However, we failed to detect the upregulation of ligands for NKG2D or DNAM-1 on DV-infected DCs. Ligands for the natural cytotoxicity receptors were determined not to contribute to the IFN-γ response. In summary, our results have uncovered a previously unappreciated role for the synergy between type I interferons and TNF-α in triggering the anti-dengue response of naïve human NK cells.
Cross reactive TCR modulate the antiviral efficacy of protective HLA class I alleles (67.17)
Authors: Chen Huabiao , Ndhlovu Zaza , Porter Lindsay , Trocha Alicja , Fang Justin , Pereyra Florencia , Yu Xu , Allen Todd , Walker Bruce
Published: January 2023

HLA class I alleles B27 and B57 are associated with protection against HIV-1 disease progression. However, the majority of persons expressing these alleles experience progressive infection, and factors modulating the HLA protective effect remain unclear. Here, we performed a detailed analysis of HIV-1-specific CD8 T cell responses in both progressors and controllers expressing HLA B27 or B57 in terms of TCR recruitment, polyfunctionality, proliferation, avidity, differentiation phenotypes, and functional ability to inhibit viral replication. The data show that HLA B27- or B57-restricted CD8 T cells targeting the same epitopes could be clearly differentiated between controllers and progressors based on potency and cross-reactivity of recognition of HIV-1 and viral variants. This in turn was associated with distinct TCR usage. Superior control of replication of HIV-1 and viral variants was observed by cross-reactive TCR-equipped CD8 T cells while highly antiviral efficacy was associated with polyfunctional CD8 T cells. These data indicate that the efficacy of protective alleles is associated with their ability to cross recognize viral variants, and this in turn is related to specific TCR clonotypes that are generated in natural infection. Clonally diverse CD8 T cell responses to a viral epitope increase the likelihood that multiple divergent strains of viruses may be recognized by cross-reactive CD8 T cells, and have potentially important implications for immunogen design.
Role of B cell memory immunity in secondary heterotypic dengue virus infection in mice (67.2)
Authors: Zompi Simona , Santich Brian , Williams Katherine , Beatty P. Robert , Harris Eva
Published: January 2023

While severe dengue disease has been associated with heterotypic secondary (2°) DENV infection, most 2° infections result in asymptomatic or mild disease. The role of the memory B cell response in mediating cross-protection against a 2° heterotypic infection is not well understood. DENV infection of AG129 (interferon-a/b and -g receptor-deficient) mice reproduces features of human disease. Prior infection with DENV1 protects mice against a lethal 2° infection with DENV2 strain D2S10. DENV1-immune mice treated with cyclophosphamide (CP) and infected with D2S10 died on day 7.5-8.5, demonstrating a role for the cellular immune response in protection. In contrast, naïve CP-treated mice experiencing a 1° infection with D2S10, died on day 4, suggesting that pre-formed Ab play a role in protection. Adoptive transfer of immune memory B cells into naïve mice prior to 2° infection delayed mortality but was not sufficient to protect against a lethal infection. Mice depleted of T cells developed sign of disease but recovered after 2° infection. We are further investigating the respective role of B cells by treating mice with anti-CD20 and by comparing the avidity of post-1°, pre-2° infection serum (LPC response) with that of the supernatant obtained after polyclonal B cell stimulation (B cell memory response). Understanding the role of immune memory in protection will be useful for dengue vaccine development and evaluation.
Analysis of extrinsic pathway mediators in CD8+CD38+ T cells of healthy donors stimulated with Mycobacterium tuberculosis antigens (99.20)
Authors: Alvarez-Jimenez Violeta , Serafin-Lopez Jeanet , Estrada-Parra Sergio , Estrada-García Iris Elvira , Sandoval-Montes Claudia
Published: January 2023

CD38 is expressed in human T cells during early stages of activation and differentiation and can be used as a marker of prognosis for HIV. Currently, CD38 has raised interest since it has been found increased in patients with tuberculosis, however when the active phase is solved a decrease of CD38 in CD8 T lymphocytes is observed. Here we evaluated and determined the role of CD38 in human CD8 T lymphocytes stimulated with M. tuberculosis H37Rv soluble extracts (MTSE) and delipidized MTSE (dMTSE) in vitro. For this purpose, PBMC from healthy donors were incubated with PMA-ionomicyne, media, MTSE or dMTSE. After cultured, cells were harvested and stained with monoclonal antibodies conjugated to different fluorochromes. After kinetics experiments were performed and the expression of perforin, granzyme A, IFN-γ and CD38 in T CD8 cells was determined by flow cytometry. Expression of IFN-γ and perforin was statistically higher in T CD8+CD38+ in comparison with T CD8+CD38-. Remarkably, CD8+CD38+ T cells showed an increased cytotoxicity against macrophages pulsed with MTSE. These results suggest that the population of T CD8+ lymphocytes might have an effector functions in Ag specific dependance on CD38. This is the first report linking CTL activity to the CD38 molecule in tuberculosis.
Host immunity and pathogen genotype contribute to disaccharidase impairment following gut infection in vivo (99.23)
Authors: Solaymani-Mohammadi Shahram , Singer Steven
Published: January 2023

Using a mouse model of infection with the GS strain of the human parasite Giardia duodenalis, we examined the role of host immunity and pathogen genotype in inducing disaccharidase deficiency. The levels of sucrase, maltase, and lactase decreased in the small intestines of C57BL/6J mice at the peak of infection. Lack of functional adaptive immunity in SCID mice resulted in increased parasite burdens, failure to eliminate the infection and no impairment of intestinal enzymes. Experiments using beta2microglonbulin knockout animals implicated CD8+ T cells as mediating disaccharidase deficiency. Interestingly, wild-type mice infected with the WB strain of G. duodenalis, did not exhibit decreased disaccharidase levels. These strains are prototypes of two genetically distinct assemblages within the species G. duodenalis, suggesting that the assemblages may be distinct in causing disaccharidase deficiency in human infections. Analysis of in vitro cytokine production by CD4+ T cells in response to pathogen extracts showed similar production of IL-4, IL-10, IL-13, IL-22, TNF-alpha and IFN-gamma following infection with IFN-gamma being the dominant cytokine for both parasite strains. These findings reveal the significance of both host and pathogen factors as key players in determining the outcome of murine infections, and may explain why some human patients exhibit malnutrition syndrome while others do not.
Identification of novel, biologically distinct T cell responses to chronic viral infection by unsupervised analysis of gene expression profiles of hepatitis C-specific CD8+ T cells. (105.12)
Authors: Gupta Prakash , Wolski David , Alexe Gabriela , Lauer Georg , Haining William
Published: January 2023

During the course of a viral infection, CD8+ T cells responding to the virus differentiate into heterogeneous subpopulations of antigen (ag)-specific cells. Characterizing this phenotypic heterogeneity is limited by the number and choice of molecules that can be assayed on tetramer+ CD8+ T cells by flow cytometry. We studied genome-wide expression profiles of tetramer-sorted ag-specific CD8+ T cells from 27 Hepatitis C virus (HCV) infected patients to identify new molecular subtypes of HCV-specific T cell responses. Unsupervised analysis of HCV-specific profiles by a novel clustering algorithm revealed two distinct subclasses of T cell response among the patients. These molecular phenotypes did not simply represent known differentiation states (e.g. exhaustion or memory), but were instead related to critical biological differences: HCV-specific T cells in subclass 2 showed significant upregulation of proliferation, metabolism and RNA processing genes, suggesting increased T cell fitness. Clinically, patients in this subclass controlled viral load significantly better than those in subclass 1. Subclass-specific markers associated with poor outcome were validated on HCV-specific T cells by flow-cytometry. We demonstrate that unsupervised analysis of genome-wide expression profiles of ag-specific CD8 T cells in humans can identify new biological types of T cell response and novel biomarkers of clinical outcome in chronic viral infection.
CD8 T cell memory precursors are found in bone marrow upon activation after acute LCMV infection (105.14)
Authors: Romagnoli Pablo , Premenko-Lanier Mary , Loria Gilbert , Lefrancois Leo , Altman John
Published: January 2023

The amount and quality of CD8 T cell memory generated in response to an acute infection with an intracellular pathogen is influenced by the signals present in the tissues in which CD8 T cells are activated. Here, we investigated if location dictates the phenotype and functional properties of CD8 T cells responding to acute lymphocytic choriomeningitis virus (LCMV) infection in different lymphoid tissues. First, we show that CD8 T cells isolated 3 days post infection (p.i.) from bone marrow (BM) displayed a memory precursor phenotype, CD62LloCD25loLy6Chi different to the phenotype observed in LCMV-specific CD8 T cells from spleen or peripheral lymph nodes (PLN). Second, we show that T-bet, a T-box transcription factor found predominantly in terminally differentiated effector CD8 T cells, is almost absent in LCMV-specific CD8 T cells from BM in contrast to the high T-bet expression found on LCMV-specific CD8 T cells from PLN and spleen. Differences in T-bet are also detected after acute infection with vesicular stomatitis virus and L. monocytogenes. Finally, we find that CD8 T cells from BM recall better upon secondary challenge when compared to CD8 T cells from spleen or PLN. These findings strongly suggest that CD8 T cell memory precursors can arise in BM early after acute infection with an intracellular pathogen and further investigation is needed to define the environmental signals influencing CD8 T cell memory differentiation.
Partial clearance of a hepatotropic virus infection in HLA class I and II double transgenic human immune system mice (105.19)
Authors: Billerbeck Eva , Horwitz Joshua , Labitt Rachael , Budell William , Rice Charles , Ploss Alexander
Published: January 2023

Humanized mice have emerged as a promising model to study human immunity in vivo. While they are susceptible to many human lymphotropic pathogens, immune responses to infection remain functionally impaired in these mice. It has been shown that the expression of HLA class I or II improves human immunity in this model. However, little is known about the extent of functional human immune responses in non-lymphoid tissues, such as in the liver, and the role of HLA expression in this context. In this study, we analyzed human antiviral immune responses in humanized mice during a hepatotropic adenovirus infection. Specifically, we compared human immune responses of conventional humanized NSG (NOD-SCID IL2RγNULL) mice to those of a novel NSG strain transgenic for HLA-A*0201 and a chimeric HLA-DR*0101. Humanized mice were generated by transplantation of human CD34+ hematopoietic stem cells (HSC) into newborn, sublethally irradiated mice. The development of a human immune system was comparable in both mouse strains. However, the expression of HLA transgenes resulted in more efficient clearance of an adenovirus infection from the liver, which correlated with liver-infiltration and activation of T cells and the detection of antigen-specific adaptive immune responses. While additional modifications to the xeno-recipient may further improve human immunity this study provides first evidence for functional antiviral immunity against a hepatotropic pathogen
PD-1, IL-10 and IFN-γ form a network to regulate HIV-specific CD4 T cell dysfunction (105.2)
Authors: Porichis Filippos , Kwon Douglas , Jennifer Zupkosky , Hart Meghan , McMullen Ashley , Freeman Gordon , Kavanagh Daniel , Kaufmann Daniel
Published: January 2023

Blockade of the PD-1 or IL-10 pathways can restore antigen-specific T cell functions in chronic infections and cancer. PD-1 blockade is currently evaluated in oncology trials and is also considered in HIV infection. It is thus crucial to define the immune restoration elicited by these interventions, and to determine whether combined blockade of these molecules can synergistically revive immune function. We investigated 40 subjects at different stages of HIV infection. We measured proliferation and cytokine production by HIV-specific CD4 T cells stimulated with HIV Gag in the presence of blocking antibodies against PD-L1 and/or IL-10Rα, or IFN-γ. We also determined the modulatory impact of IFN-γ on PD-1 and its ligands on different cell subsets. In viremic subjects, combined blockade of PD-L1 and IL-10Rα resulted in a striking synergistic increase in IFN-γ secretion by HIV-specific CD4 T cells in contrast to only a moderate increase in IL-2 and IL-13 secretion, and proliferation. While IL-10Rα blockade preferentially increased IFN-γ, PD-L1 blockade had a balanced effect on IFN-γ, IL-2 and IL-13. Stimulation of HIV-specific CD4 T cells led to strong up-regulation of PD-L1 on monocytes that was further increased by anti-IL-10Rα. Blockade of IFN-γ abrogated PD-L1 induction and restored proliferation of HIV-specific CD4 T cells. These data provide a mechanistic insight on how the interplay between PD-1, IL-10 and IFN-γ regulates Thelper dysfunction in HIV infection.
PD-1 blockage reverses immune dysfunction and hepatitis B viral persistence in a mouse animal model (105.23)
Authors: Tzeng HorngTay , Chen PeiJer , Hsu PingNing
Published: January 2023

Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Recent studies in animal models of viral infection indicate that the interaction between the inhibitory receptor, programmed death (PD)-1, on lymphocytes and its ligand (PD-L1) play a critical role in T-cell exhaustion by inducing T-cell inactivation. We studied T-cell exhaustion and the effects of PD-1 and PD-L1 blockade on intrahepatic infiltrating T-cells in our recently developed mouse model of HBV persistence. In this mouse animal model, we demonstrated that there were increased intrahepatic PD-1-expressing CD8+ T-cells and increased CD4+ FoxP3+ T cells in mice with HBV persistence, but PD-1 upregulation was resolved in mice which had cleared HBV. The Intrahepatic CD8+ T-cells expressed higher levels of PD-1 and lower levels of CD127 in mice with HBV persistence. Blockade of PD-1/PD-L1 interactions increased HBcAg-specific interferon (IFN)-γ production in intrahepatic T lymphocytes. Furthermore, blocking the interaction of PD-1 with PD-L1 by an anti-PD-1 monoclonal antibody (mAb) reversed the exhausted phenotype in intrahepatic T lymphocytes and viral persistence to clearance of HBV in vivo. Our results indicated that PD-1 blockage reverses immune dysfunction and viral persistence of HBV infection in a mouse animal model, suggesting that the anti-PD-1 mAb might be a good therapeutic candidate for chronic HBV infection.
Kynureninase is the conserved self-antigen mimicked by the 2F5 Neutralizing Epitope of HIV-1 gp41 (105.35)
Authors: Yang Guang , Holl T. , Liu Yang , Li Yi , Lu Xiaozhi , Nicely Nathan , Kepler Thomas , Alam S. , Liao Hua-Xin , Cain Derek , Spicer Leonard , VandeBerg John , Haynes Barton , Kelsoe Garnett
Published: January 2023

Accumulating evidence suggests that the generation of neutralizing HIV-1 antibody may be proscribed by immunological tolerance. A significant majority of monoclonal human antibodies, including 2F5, that neutralize multiple clades of HIV-1 are polyreactive and bind avidly to unknown components of mammalian cells. Knock-in mice expressing the VH and VL regions of 2F5 are characterized by a central defect in B-cell development that indicates the presence of a tolerizing autoantigen(s) that is mimicked by the membrane-proximal, 2F5 epitope of HIV-1 gp41. If immunological tolerance does indeed prevent regular expression of 2F5-like neutralizing HIV-1 antibody, then discrete autoantigens that structurally mimic the HIV-1 2F5 epitope must be present in those mammalian species unable to make 2F5-like antibody. We now demonstrate that the 2F5 antibody and its unmutated ancestor avidly bind human kynureninase (KYNU), a conserved enzyme of tryptophan metabolism. The H4 domain of most mammalian KYNU contains the complete 2F5 epitope (ELDKWA) of gp41; a H4 mutation of human KYNU to that (ELEKWA) of laboratory opossums resulted in a fully active enzyme completely unreactive with the 2F5 antibody. Immunization of opossums with HIV-1 gp140 induced extraordinary titers of ELDKWA antibody but little or no response to adjacent gp41 epitopes. Identification of an epitope shared by most mammals and HIV-1 provides direct evidence that immunological tolerance can impair humoral responses to HIV-1.
Epitope specificity appears to be an important determinant of in vivo killing ability of simian immunodeficiency virus (SIV)-specific CD8+ T cells (105.43)
Authors: Pozzi Lu-Ann , Carville Angela , Varner Valerie , Sen Jayita , Knipe David
Published: January 2023

CD8+ cytotoxic T lymphocytes (CTL) are a critical component of antiviral immunity and play an important role in the control of lentiviruses. We have developed an in vivo CTL assay to directly measure the killing capacity of MHC-restricted SIV-specific CTL in rhesus macaques (RM) vaccinated with a recombinant herpes simplex virus prime/ DNA boost SIV vaccine regimen. Tetramer frequencies of Mamu-A*02-restricted Nef YY9-specific CD8+ T-cells were at least 10-fold higher than Mamu-A*02-restricted Gag GY9-specific CD8+ T-cells in individual animals both pre- and post-challenge. Prior to SIV challenge, both CTL populations showed poor and incomplete killing (22-35%) of target cells over 18 hours. Seven weeks post-challenge, there was a marked increase in the CTL killing capacity of both Nef YY9 and Gag GY9-specific CTL with 26-38% killing occurring over the first two hours and up to 100% killing over 18 hours. Surprisingly, Gag GY9-specific CTL consistently showed equivalent or greater in vivo killing when compared to Nef YY9-specific CTL even though the percentages of IFN-γ secreting and degranulating cells upon peptide stimulation were comparable. These data suggest that epitope specificity rather than tetramer frequency determines the ability of SIV-specific CTL to kill infected cells. Taken together, these data may have important implications for the development of a successful HIV vaccine.
Proteome wide mapping of Dengue Virus-specific T cell responses in human donors reveals antigenic hot spots and a dominance of HLA B restricted responses (105.52)
Authors: Weiskopf Daniela , Angelo Michael , Sidney John , Greenbaum Jason , Fernando Anira , Broadwater Anne , Kolla Ravi , De Silva Aruna , de Silva Aravinda , Grey Howard , Shresta Sujan , Peters Bjoern , Sette Alessandro
Published: January 2023

The frequency of dengue virus (DENV) infection has increased dramatically in the recent decades and the lack of a vaccine has led to significant worldwide morbidity and mortality. To improve on the minimal information available regarding HLA-restricted T cell responses resulting from natural infection with DENV, we endeavored to map T cell responses in individuals with resolved DENV infection. To cover a wide range of HLA phenotypes we first performed bioinformatics predictions of MHC binding motifs of all four DENV serotypes to 16 HLA A and 11 HLA B alleles. To validate this approach, we have tested the predicted motifs in an ex vivo ELISPOT assay for their ability to induce an IFNγ response in HLA-matched PBMC of 250 blood donors from Sri Lanka where DENV is hyper-epidemic. This proteome-wide screen has identified a total of 408 epitopes across all 10 DENV proteins (C, M, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Interestingly, the majority of responses were derived from the highly conserved nonstructural proteins: NS3, NS4B and NS5. Additionally, we have observed a striking dominance of HLA B restricted responses in terms of breath as well as magnitude of the DENV specific response detected. Our data provides specific insight and understanding into the nature of HLA-restricted T cell responses, the role of T cells in DENV infection, and future strategies in vaccine design against this globally intensifying viral threat.
Vitamin A deficiency alters antibody forming cell and CD8+ T cell responses following intranasal vaccination with a respiratory virus vaccine (105.9)
Authors: Hurwitz Julia , Surman Sherri , Jones Bart , Sealy Robert , Woodland David , Rudraraju Rajeev
Published: January 2023

Vitamin A deficiency (VAD) has profound effects on immune responses in the gut, but its influence on other mucosal sites is less well understood. Sendai virus (SeV) is a candidate human parainfluenza virus type I (hPIV-1) vaccine, and a candidate vaccine vector for other respiratory viruses. A single I.N. dose of SeV elicits a protective immune response against hPIV-1 within days after vaccination. To define the effect of VAD on responses toward SeV, we monitored antibody forming cells (AFC) and immunodominant CD8+ T cells in mice. VAD inhibited SeV-specific IgA producing AFCs in the upper respiratory tract (URT) and biased the AFC response in favor of IgG-producing cells in the lower respiratory tract (LRT). In contrast, there was a dramatic reduction of CD8+ T cell frequencies in the LRT airways of VAD animals. Responding CD8+ T cells exhibited unusually high CD103 expression. In both VAD and control mice, e-cadherin (the ligand for αE(CD103)β7) was well expressed in URT tissues, but was expressed only weakly in the LRT. Results support a working hypothesis that the high CD103 expression among responding T cells in VAD mice may enhance T cell entrapment by e-cadherin and inhibit cell egress into the LRT airway. Our results highlight the concept that VAD does not exclusively affect immune responses in the gut, but has dramatic influences on immune cell function and residence in the respiratory tract.
Sensing of low-dose soluble antigen via Immunoglobulin E (106.1)
Authors: Platzer Barbara , Baker Kristi , Schopoff Sandy , Turley Shannon , Blumberg Richard , Fiebiger Edda
Published: January 2023

Dendritic cells (DCs) are the most potent antigen presenting cells and continuously encounter exogenous antigens at low dose. We show here that DCs use IgE-mediated antigen uptake to efficiently sense soluble antigen at a dose range that otherwise escapes immune detection. IgE-mediated antigen sampling via Fc-epsilon-RI simultaneously shuttles into the direct MHC class II-restricted presentation and the cross-presentation pathway resulting in the activation of Th2-type CD4+ T cells as well as cytotoxic CD8+ T cells (CTLs). Fc-epsilon-RI stabilizes at the cell surface of DCs after monovalent ligation with IgE. Antigen-mediated crosslinking of the receptor induces internalization into endo/lysosomal compartments. Absence of IL-12 production during IgE-mediated antigen uptake and presentation is critical for shifting the CD4 response towards Th2, but does not affect the generation of CTLs. Presence of IL-12 during IgE-mediated presentation redirects the immune response towards Th1 by suppressing the production of IL-4 and IL-13. Neither basophils nor mast cells can induce primary T cell responses after antigen sampling via IgE. These results establish a uniquely sensitive pathway for the detection of soluble exogenous antigen by DCs with high relevance for IgE-mediated immune responses in humans.