bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chronic alcohol consumption decreases NK cells in the peripheral lymph nodes and exacerbates B16BL6 melanoma metastasis into the draining lymph nodes (40.5)
Authors: Zhang Hui , Meadows Gary G. 
Published: January 2023

Abstract
Chronic alcohol consumption decreases NK cells in the spleen (SP) and compromises their release from the bone marrow (BM). NK cells in the lymph nodes (LN) are important to T cell priming and polarization. Little is known as to how alcohol consumption affects NK cells in the LN. To address this issue, female C57BL/6 mice were given 20% (w/v) alcohol in drinking water for 3-6 months. The percentage, number, phenotype and functions of NK cells in the peripheral LN were studied. Alcohol consumption decreased the percentage and numbers of NK cells in the LN without altering the degree of NK cell apoptosis. Ly49H+, Ly49D+ and Ly49G2+ NK cells also decreased in the LN. The decrease in these cell populations was more pronounced in the SP, and there were no changes in the BM. The results from normal SP cell transfer experiments indicated a time-dependent decrease in accumulation of NK cells into the LN of alcohol consuming as compared to water drinking mice. Alcohol consumption increased the percentage of CXCR3+CD3- and decreased CD62L+CD3-NK cells in the SP and BM. The percentage of CXCR3+CD3+ T cells and CD62L+CD3+ T cells did not change. Consistent with the decrease of NK cells in the LN, alcohol consumption enhanced melanoma metastasis in the draining LN. The results indicate that decreased NK cells in the LN are associated with decreased CD62L+ NK cells in the SP and BM, which impairs NK cell migration to the LN.
Supported by R01 AA07293.
Peritoneal B cells responding to CD40 ligation resist macrophage-mediated suppression (40.8)
Authors: Swider Adam , Sacavage Steve , Deeney Brian , Kozlowski Megan , Riggs James E. 
Published: January 2023

Abstract
Although cells essential for anti-tumor immunity exist within tumors they often fail to prevent cancer progression. The immunosuppressive tumor microenvironment, notable for a high macrophage to T cell ratio (Mö:T), inhibits T cell activation primarily by Arg and Trp catabolism. We have assessed the impact of high M ö ratios on B cell (Mö:B) activation in a model culture system. LPS and F(ab')2 anti-IgM failed to activate B cells at high Mö:B ratios unless cyclooxygenase was inhibited by indomethacin. In contrast, B cells activated by CD40 ligation were resistant to Mö-mediated suppression. Compared with spleen (SP) B cells, peritoneal cavity (PerC) B cells responding to CD40 ligation were particularly resistant to suppression. PerC B cells from B-1 B cell subset-deficient BALB.xid mice were also resistant to Mö-mediated suppression. The CD40 response, however, was suppressed in cultures where both B (áCD40) and T (áCD3) cells were activated illustrating that suppression by amino acid catabolism overrides the CD40 response of B cells in high Mö:B environments. These results are relevant to the development of strategies designed to reverse the immune suppression characteristic of many tumors. Supported by NIH AREA R15-AI060356-01.
Identification of molecular checkpoints in patients with prostate cancer immunized with PSA146-154 peptide vaccine (41.24)
Authors: Perambakam Supriya , Xie Hui , Edassery Seby , Peace David J 
Published: January 2023

Abstract
The objective of the current study is to delineate genes that may be differentially expressed in responding versus non responding patients in the context of a completed PSA146-154 peptide vaccine protocol. 28 HLA-A2+ prostate cancer patients with high risk, local disease or metastatic, hormone sensitive disease in remission were vaccinated on weeks 1, 4, and 10 with peptide plus GM-CSF or peptide loaded autologous DC. Before and after PBMC were assessed for specific tetramer response and gene expression profiles per Affymetrix whole human genome arrays. Clinical status was evaluated per serial serum PSA. 13 of 27 patients achieved stable serum PSA (1 not evaluable), of whom 9 patients developed specific tetramer responses at six months post first vaccination. Thus the detectable immunity was associated with lower risk of PSA progression (p=0.02). Comparison of array data between responders and non responders indicated that 114 of 54,675 genes were differentially expressed (p&lt;0.001). Notably VEGF-A, LIF, TNFRSF21 and IFNK, pertaining to the cytokine-cytokine receptor interaction pathway were predicted to be impacted (Impact factor=5.19). Taqman gene assays indicated that the expression of VEGF-A relative to GAPDH was greater in non responding compared to responding patients. Hence, VEGF-A is one of the likely candidates to impede vaccine efficacy, possibly by mediating its effects on antigen presenting cells.
Acetal-modified Dextran: An Acid-sensitive, Biodegradable Material for Effective Microparticulate Antigen Delivery (41.53)
Authors: Cohen Joel A , Broaders Kyle E , Beaudette Tristan T , Bachelder Eric M , Fréchet Jean MJ 
Published: January 2023

Abstract
Polymer-based microparticle systems capable of delivering proteins to antigen-presenting cells (APCs) of the immune system have been investigated for the enhancement of vaccines and immunotherapy. The size of the particles in these systems can be controlled to passively target APCs , without being taken up by other cells in the body. We have developed a new concept in acid-sensitive microparticles based on the use of a water-soluble polysaccharide, dextran, that can be rendered insoluble in aqueous solutions by protecting the hydroxyl groups with acid-sensitive acetals. The resulting polymer is soluble in organic solvents used to generate protein loaded particles by a variety of methods in which the modified polymer is condensed around the encapsulated antigen. Once administered, the acetals rapidly hydrolyze under the acidic conditions present in the phagosomes of APCs to provide a solubility switch that enables particle degradation and release of encapsulated antigen in a controlled manner. We have recently demonstrated the ability to control the rate of particle degradation over a range of minutes to several hours at pH 5, with interesting results observed in how degradation rate affects the presentation of model antigens by APCs to T cells in vitro, as well as how degradation rate may affect the processing mechanism of the released antigen within APCs.
Alphavirus replicon particles expressing melanocyte differentiation antigens are effective vaccines for melanoma (41.55)
Authors: Avogadri Francesca , Merghoub Taha , Hirschhorn Daniel , Maughan Maureen , Morris John , Houghton Alan N 
Published: January 2023

Abstract
Melanocyte differentiation antigens are attractive candidates for melanoma immunotherapy, but because they are expressed by malignant cells as well as their normal counterpart eliciting effective immune responses is challenging.
Here we evaluated an alphavirus replicon vector system as a potential melanoma immunotherapy. Use of propagation-defective virus-like replicon particles (VRP) based on an attenuated strain of Venezuelan equine encephalitis (VEE) virus is especially attractive because VRP express heterologous proteins to high levels and target expression to dendritic cells. VRP vaccines have been shown to elicit both humoral and cellular immune responses to the vectored gene products in many animal disease models and in phase I clinical trials.
The efficacy of VRP expressing the melanocyte differentiation antigens tyrosinase, gp100 or TRP-2/DCT was analyzed against challenge with the poorly immunogenic and highly aggressive B16 murine melanoma. TRP-2/DCT-expressing VRP were the most effective in delaying tumor occurrence when compared to the other antigens tested. Although both TRP2/DCT-specific CD8+ T cells and IgG are induced, the data suggest that B-cells may play a more important role in the effector phase of tumor protection.
These studies demonstrate that VRP vaccines present a promising approach for melanoma immunotherapy.
Immunological evaluation of patients with osteosarcoma received adjuvant treatment with specific Transfer Factor. (41.63)
Authors: Rodriguez-Flores Azucena , Castillo-Juarez Paola , Vazquez Dotor , Gonzalez-Guzman R , Rico-Martinez G. , Estrada-Parra Sergio 
Published: January 2023

Abstract
Osteosarcoma is a malignant bone tumor with a tendency to metastasis. Based on the immune response and the degree of progress in primary injury Levin developed a classification for osteosarcoma ranging from Phase I to Phase IV associated with the loss of the immune response of cytotoxic cell type and primarily to the development of metastases. Transfer Factor is a polypeptide with a molecular weight of about 6 kilodalton present in dialyzable leukocyte extract the human capacity for immunoregulation.
The present study is conducting a pilot clinical trial evaluating the different populations and lymphocyte subpopulations in peripheral blood at baseline and post-surgical type, neoadjuvant chemotherapy and specific transfer factor.
So far 16 patients have been assessed, of which it was found that 62.5 percent have decreased the number of lymphocyte cells, T lymphocytes, 44 percent and 68.8 percent total cytotoxic and helper response at baseline compared to healthy individuals. After treatment we observed that 63 percent of patients increased the number of T lymphocytes, 75 percent increased their cytotoxic T lymphocytes and 50 per cent of these cells cooperating. The 87.5% of patients come back to phase I of Levins classification
A novel human-mouse chimeric death receptor (DR5) molecule mediates TRA-8 induced apoptosis in mouse cell lines (41.9)
Authors: Li Jun , Yang Ping-Ar , Hsu Hui-Chen , Mountz John D. 
Published: January 2023

Abstract
DR5 is a pro-apoptotic protein considered as a target for cancer and autoimmune therapy. An anti-human DR5 antibody, TRA-8, has been shown to induce apoptosis in various cells. The purpose of this study is to generate a humanized mouse DR5 molecule that can be used to study the DR5 mediated apoptosis and immune responses using TRA-8 in a mouse.
A chimeric DR5 consisting of a human extracellular domain, a mouse transmembrane and intracellular domain was subcloned downstream of a 1kb, 3kb putative promoters, and a 3 kb first intron of mouse DR5. Transfection was performed by using Lipofectamine and nucleofection. Chimeric DR5 expression was determined by FACS and Western blotting. Cell viability was estimated by the ATPLite.
Maximal cell surface expression was seen with the 1 kb promoter in NIH3T3 and 4T1 cells. Longer promoter with first intron conferred authentic expression. TRA-8 enhanced apoptosis with efficacy varying from 15% (shorter promoter, high DR5 expression, high cell death with/without TRA-8) to 60% (longer promoter plus the first intron, authentic DR5 expression, low cell death without TRA-8, high cell death with TRA-8).
This novel DR5 molecule with regulatory elements confers its authentic expression and enables TRA-8 induced apoptosis in mouse cells. It will lead to the generation of a transgenic mouse for analysis of the TRA-8 apoptotic mechanism, therapeutic response in both autoimmune diseases and cancer.
Multicolor flow cytometry using only Qdot® conjugated primary antibodies (42.12)
Authors: Jaron Shulamit , Godfrey William L. 
Published: January 2023

Abstract
Qdot® nanocrystals allow researchers 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 a multicolor panel consisting solely of Qdot® conjugates. We show the effectiveness of using only Qdot®-conjugated antibodies in a typical 8-color phenotyping panel. Experiments were performed using standard staining protocols with human peripheral blood leucocytes. Analysis was performed on an LSR II flow cytometer using 405 nm and 488 nm lasers equipped with octagon optical pathways. Attention is paid to experimental set-up including the selection of appropriate filters to minimize within-laser and cross-laser compensation. The fluorescence from Qdot® 525, 605, 655, 705, and 800 nanocrystal conjugates were easily isolated from adjacent channels due to the narrow emission profiles of the nanocrystals. More care was needed in the filter selection and compensation between 565 and 585, 625 and 605, and 625 and 655 nanocrystal conjugates. By selecting the right combination of laser/detector and filters, compensation can be minimized. These results demonstrate the ease of performing a multicolor experiment with Qdot® nanocrystal conjugates using no more than two lasers.
Supported by Life Technologies.
Numbers matter: quantitative and dynamic analysis of the formation of a mature immunological synapse using imaging flow cytometry (42.15)
Authors: Ahmed Fariyal , Friend Sherree , George Thaddeus C , Barteneva Natasha , Lieberman Judy 
Published: January 2023

Abstract
Activation of T lymphocytes by antigen-presenting cells (APC) results in the formation of an immunological synapse. Following intercellular contact with the target cell, key signaling and adhesion molecules polarize within minutes to hours to the T cell-APC interface. We use multispectral imaging flow cytometry, a new technology which combines flow cytometry with imaging, to visualize and quantify the recruitment of signaling molecules CD3ε and Lck during the evolution of a mature immune synapse. We use two independent analysis methods to show that the dynamics of Lck and CD3ε recruitment are comparable; however, CD3ε exhibits longer residence times (&gt; 8 min) following Ca++ triggered activation in the synapses of thousands of T cell-macrophage conjugates.
Engineering XID(Btk) site specific Homing Endonucleases for gene repair in hematopoietic stem cells (42.19)
Authors: Wang Yupeng , Jarjour Jordan , Havranek Jim , Baker David , Scharenberg Andrew M. , Rawlings David J. 
Published: January 2023

Abstract
X-linked agammaglobulinemia (XLA), a human immunodeficiency caused by mutations in Btk, represents an ideal candidate target for HSC gene therapy. However, current gene replacement methods carry risks of insertional mutagenesis as well as gene silencing. In contrast, gene repair induced by site specific DNA double strand breaks (DSB) offers a novel means to treat genetic disease with reduced risk and the potential to utilize endogenous gene expression control elements. LAGLIDADG Homing Endonucleases (LHEs) have been shown to efficiently induce site specific DSB and markerless modification of genes without toxicity. Due to the limited number of LHEs currently available, engineering site specific LHEs represents a critical step for enabling technology for DSB-induced gene modification.
In the current study we have begun to generate a site specific variant of the LHE I-AniI, targeted to recognize a unique mutation in the mouse BTK gene in the XID model of human XLA. Our approaches combine: protein computational design with random mutation and selection using yeast library surface display; or alternatively, iterative somatic hyper-mutation and selection within DT40 B cell library targeted to the light chain locus. Using these combinatorial methods we have generated a panel of I-AniI variants with novel DNA binding and cleavage specificity towards the XID site. Preliminary characterization of a subset of these I-AniI variants will be presented. Ultimately, new I-Anil variants and donor DNA repair template will be delivered into HSC derived from XID/Tec-/- mice; and evaluated for capacity to re-engraft and reconstitute B lineage development and function in vivo.
A proinflammatory program of perinatal plasmacytoid dendritic cells activate natural killer (NK) cells to damage the bile duct epithelium in experimental biliary atresia (43.2)
Authors: Saxena Vijay , Shivakumar Pranavkumar , Meithke Alex G , Sabla Gregg E , Chougnet Claire , Bezerra Jorge A 
Published: January 2023

Abstract
Biliary atresia results from an NK cell-mediated inflammatory injury of the bile duct epithelium with onset in the early postnatal period. Based on key immunoregulatory roles of dendritic cells (DC), we hypothesized that early neonatal DC activation triggers an effector immune response that results in biliary atresia. We tested this hypothesis in a mouse model of rotavirus-induced biliary atresia. Following rotavirus challenge on day 1 of life, viral immunogenic protein NSP3 was detected in hepatic PDCA-1+ plasmacytoid DC (pDC) but not CD11c+ conventional DC (cDC) within 3 days of infection. Hepatic pDC increased 3 fold, while non-pDC (CD11b+CD11c+ cells) decreased 2.5 fold. Rotavirus-naïve perinatal hepatic pDC and cDC spontaneously expressed B7.1/2 (2-3 fold higher than adults), with a much higher increased expression in B7.1/2, IFNγ, and IL-12p40 by pDC after rotavirus challenge. IL-15 was spontaneously expressed by naïve perinatal pDC and increased dramatically after rotavirus infection, but in vitro activation of NK cells required co-culture with both pDC and cDC. Most notably, ablation of pDC decreased the liver population of NK cells and prevented the mucosa injury and obstruction of neonatal bile ducts, leading to improved symptoms and long-term survival. Thus early activation of pDC due upon viral exposure regulates neonatal duct injury and obstruction in biliary atresia.
Adoptive transfer of CD4 T cells into T cell deficient Mycobacterium avium infected mice results in a fatal Th1 disease that mimics human Immune Reconstitution Inflammatory Syndrome (IRIS) (43.6)
Authors: Barber Daniel L. , Mayer Katrin D. , Antonelli Lis R. , Sher Alan 
Published: January 2023

Abstract
IRIS is the paradoxical worsening of pathology in HIV patients after initiation of anti-retroviral therapy (ART). Patients with severe CD4 T cell loss and concurrent mycobacteria infection display a high risk of developing IRIS after ART. To study the mechanism(s) underlying the pathogenesis of IRIS, we have developed a mouse model that focuses on a fundamental feature of the clinical scenario: CD4 T cell reconstitution in the context of concurrent mycobacteria infection. Both WT and TCRα-/- mice survive for many months after Mycobacterium avium infection. However, when TCRα-/- mice are infected with M. avium and then inoculated with CD4 T cells from naïve mice several weeks later, they develop a rapid wasting disease and succumb as early as day 12 post-transfer. IFNγ-/- CD4 T cells induce much less disease than WT CD4 T cells, and TNFα neutralization delays its onset. TCR Tg CD4 T cells specific for irrelevant Ag's cause no disease indicating that this is an Ag driven T cell response. Interestingly, both self-Ag and M. avium specific CD4 T cells induce rapid mortality after transfer into TCRα-/- M. avium infected mice. This suggests that (1) the combination of lymphopenia and concurrent infection triggers the reconstituting CD4 T cells to mount dysregulated Ag-driven Th1 responses that lead to fatal wasting and (2) IRIS-inducing CD4 T cells can be specific for either self or mycobacterial antigens.
Work supported by NIAID Intramural Research Program.
Local intrahepatic antigen presentation induces dysfunctional antiviral CD8+ T cell responses (44.19)
Authors: Dolina Joseph S , Lukens John R , Kim Taeg S , Hahn Young S 
Published: January 2023

Abstract
Immune responses to pathogens in the liver are uniquely characterized by a tolerogenic feature as a result of constant exposure to bacterial constituents and food-derived antigens. Little is known about how the immune dysregulation in the liver contributes to persistent viral infection. Specifically, it is important to understand how the site of antigen delivery impacts the quality of CD8+ T cells either trafficked to or generated within the liver. In this study, we show that subcutaneous (SC) adenovirus infection results in the trafficking of CD8+ T cells to the liver with effective antiviral function. In contrast, when antigen is delivered directly to the liver during intravenous (IV) adenovirus infection, intrahepatic CD8+ T cell responses are defective in their ability to produce inflammatory cytokines and lyse target cells. Further, these intrahepatic CD8+ T cells express elevated levels of PD-1, downregulate TCR, and exhibit increased apoptosis. IV adenovirus infection distinctively results in local intrahepatic antigen presentation responsible for limiting the CD8+ T cell immune response. These findings not only have applications for expanding the field of knowledge on chronic liver infections, but they also demonstrate that the delivery route of vaccine can orchestrate the quality of immune mediators present in the liver. NIH Grants DK063222 and Training Fellowship T32 AI007496 supported this work.
An activating receptor's critical role in supporting NK cell proliferation for survival during viral infections (44.2)
Authors: Lee Seung-Hwan , Kim Kwang-Sin , Fodil-Cornu Nassima , Vidal Silvia M , Biron Christine A 
Published: January 2023

Abstract
Activating receptors, including the Ly49H molecule recognizing MCMV, can stimulate NK cell cytotoxicity and proliferation. Ly49H induction of NK cell cytotoxicity contributes to defense against MCMV, but its in vivo importance in stimulating NK cell expansion remains unclear. To define Ly49H's role in supporting cell proliferation during infection, experiments were carried out evaluating NK cell responses in Ly49h-/-, perforin 1 (Prf1)-/- and wild type (wt) B6 mice. NK cell numbers were similar in uninfected mice, and either deficiency alone resulted in high MCMV burdens. In the absence of Ly49h, however, NK cell numbers declined, with remaining populations having an immature phenotype. In the absence of Prf1, NK cells expanded and had higher rates of proliferation, eccentric phenotypes, and Ly49H expression on nearly all cells. Expansion was abolished, with negative consequences for survival, in mice deficient for both Ly49H and Prf1 (Ly49h-/-Prf1-/-), and NK cell depletions in Prf1-/- mice decreased survival during infection. The results prove that Ly49H can drive dramatic NK cell expansion during challenges with MCMV. Moreover, by dissecting functions for proliferation from cytotoxicity, they demonstrate a previously unappreciated critical role for activating receptors in keeping NK cells present during viral infection. Supported by NIH CA41268 and the Canadian Institutes of Health Research MOP-7781.
Poor anti-viral immunity in aged mice is associated with defects in CD8 T cell recall responses (44.5)
Authors: Decman Vilma , DiMenna Lauren J. , Laidlaw Brian J. , Ertl Hildegund C.J. , Wherry E.  John 
Published: January 2023

Abstract
Aging leads to an increased susceptibility to infectious diseases. To investigate the age-related changes in T cell memory we compared anti-viral CD8 T cell immunity in young versus aged mice following systemic (lymphocytic choriomeningitis virus; LCMV) or local (influenza virus) viral infections. The number of memory CD8 T cells formed was similar or only modestly different between aged and young animals, though in some cases changes in immunodominance and cytokine production were apparent. Both influenza virus-specific and LCMV-specific memory CD8 T cells from aged mice, however, had major substantially reduced proliferative capacity upon secondary infection. As a result, aged mice displayed increased morbidity and mortality upon viral rechallenge. The studies described herein demonstrate that the defects intrinsic to aged CD8 T cells are at least partially responsible for diminished immunity of aged mice. Thus, the strategies that accelerate the induction of T cell responses or that overcome proliferative deficiencies may prove beneficial.
These studies were funded by grants from AHA, NIH (HHSN266200500030C) and the Ellison Medical Foundation.