bluuebunny/crossref_metadata_2025_split huggingface dataset:
Combination of CpG-ODN+anti-4-1BB but not CpG-ODN+anti-OX40 enhances antitumor immune responses in neu mice modulating the inhibitory function of Tregs (40.7)
Authors: Smith Shannon E , Hoelzinger Dominique B , Dominguez Ana Lucia , Lustgarten Joseph 
Published: January 2023

Abstract
Previous studies have established that 4-1BB and OX40, members of the TNFR family, enhance antitumor immune responses. Recent studies from our laboratory indicate that intratumoral injections of CpG-ODN induce antitumor responses in Her-2/neu tolerant mice. However, this treatment alone is not sufficient to completely eradicate tumors. Here we investigate whether the combination of CpG+anti-OX40 or anti-4-1BB enhance the antitumor responses in neu mice. Our results indicate that CpG+anti-4-1BB but not CpG+anti-OX40 eradicate tumor in neu mice. CpG+anti-4-1BB treatment significantly increased the number of CD8 cells within lymphoid organs as compared to control or anti-OX40 treated animals. More importantly, we observed that anti-4-1BB but not anti-OX40 inhibits the suppressive function of Tregs. Microarray analysis of Tregs revealed specific transcriptional differences brought about by treatment with anti-OX40 or anti-4-1BB. 4-1BB modulates differential gene expression in Tregs of over 300 genes that remain unaffected by OX40. Potential gene candidates include interleukin-9 and protein-tyrosine phosphatase receptor-type zeta-1, which are transcriptionally down and up regulated in the presence of anti-4-1BB. These proteins may play a role in Treg function, and could explain the enhanced anti-tumor effect observed in the presence of anti-4-1BB.
The role of TLR2 in the immunostimulatory effect of Polysaccharide krestin (PSK) (41.13)
Authors: Lu Hailing , Yang Yi , Gad Ekram , Disis Mary L. 
Published: January 2023

Abstract
Polysaccharide krestin (PSK) is a mushroom extract that has long been used in Asia as an anti-cancer drug, although the mechanism of action is unclear. Using neu-transgenic
(neu-tg) mice, a model of human HER2 positive breast cancer, we investigated the anti-tumor effect of PSK and its potential mechanisms. We found that oral administration of PSK significantly inhibited the growth of both implanted and spontaneous breast tumors in these mice. In vitro PSK treatment stimulated splenocytes proliferation and secretion of TNFalpha. PSK activated dendritic cells (DC) from neu-tg mice as shown by increased expression of CD86, CD80, and MHCII. The expression of toll-like receptor 2 (TLR2) on DC was also increased. To study the potential activating effect of PSK on TLRs, we used HEK293 cells transfected with different TLRs and incubated them with PSK (0.5 to 1500ug/ml, 16 hour). PSK activated NFkB in HEK cells transfected with TLR2 in a dose-dependent manner, but had no effect on HEK transfected with other TLRs (4, 7, or 8). To further investigate the role of TLR2 in mediating the immunostimulatory effect of PSK, we used splenocytes from TLR2 knockout (k/o) mice and WT mice and treated them with PSK (25 to 400ug/ml, 72 hour). PSK stimulated the secretion of TNFalpha in splenocytes from WT mice dose-dependently but not in splenocytes from k/o mice. These results suggest that TLR2 is critical in mediating the immunostimulatory effect of PSK.
Immune correlates of clinical responses and survival in ovarian cancer patients treated with intraperitoneal interleukin-2 (41.2)
Authors: Vlad Anda M , Budiu Raluca A , Thaller Julia , Edwards Robert P 
Published: January 2023

Abstract
Ovarian cancer is the most lethal gynecologic cancer and new and improved adjuvant therapies are needed. The confinement of disease to the peritoneal cavity has led to the therapeutic use of intraperitoneal (IP) infusion of biologic agents. Interleukin-2 (IL-2) is a T-cell growth factor important in anti-tumor immunity. The aim of our study was to determine the clinical efficacy of IP IL-2 in a phase II trial for platinum-resistant ovarian cancer and to identify immune correlates of response and survival in 31 IL-2 treated patients. Peripheral blood T cells, collected throughout the treatment were phenotyped and cytokines measured by FACS. Sera and IP fluids were assayed by ELISA for IgE and IgG antibodies and by cytometric bead arrays for VEGF and eotaxin. We found significant associations between baseline IFNγ-secreting CD8 T cells and survival (p=0.04) and between number change during treatment of circulating T cells and survival (p=0.05). Peripheral blood eosinophils were markedly increased at the end of treatment (p&lt;0.0001) and associated with increased eotaxin (p=0.03). Although serum VEGF increased with treatment (p=0.025), measurements in the IP fluid suggest a decrease in local levels of VEGF (p=0.053). This study provides evidence for IP IL-2 in platinum-resistant ovarian cancer and identifies several immune correlates of survival. NIH R21 CA74105-02S1, ACS, Chiron Therapeutics, Magee Health Foundation, PA Dept. of Health.
Development of a transgenic mouse with high affinity TCR reactive to human tyrosinase epitope (41.39)
Authors: MEHROTRA SHIKHAR , Li Mingli , Lyons Gretchen E , Spivey Natalie , Palma Telma Martins da , Naga Osama S , Norell Haakan R , Diaz-Montero C. Marcela , Cole David J , Nishimura Michael I 
Published: January 2023

Abstract
Most tumor-associated antigen epitopes recognized on human melanomas by autologous T lymphocytes are derived from nonmutated self-proteins. Given the belief that T cells bearing high affinity T cell receptor (TCR) would be more effective at targeting self antigens expressed by tumors, understanding tolerance with reference to T cells expressing high affinity TCRs against self-tumor antigens is important for optimizing the anti-tumor T cell response. Thus, using a high affinity TCR isolated from a class-I restricted CD4+ T cells from tumor infiltrating lymphocytes of a patient with metastatic melanoma and reactive to human tyrosinase-derived peptide YMDGTMSQV we developed a novel TCR transgenic mice. Our results show that HLA-A2 restricted TCR passed thymic selection in C57BL/6J (H2-Kb) wild type mice and could be tracked in the periphery. Around 25% of the peripheral CD4+ T cells and more than 75% of the peripheral CD8+ T cells from these TCR transgenic mice expressed functional human tyrosinase TCR. Since tyrosinase is a self-antigen and C57BL/6J mice lack HLA-A2/tyrosinase complexes on cells in their thymus -we developed HLA-A2+TCR+ double transgenic mice by breeding our TCR transgenic mouse and HLA-A2 transgenic mouse strains. Surprisingly, more than 90% of TCR transgenic T cells in the HLA-A2+TCR+ double transgenic mice lacked expression of CD4 or CD8 co-receptors and had CD3+CD4-CD8- phenotype. These double transgenic strains also developed spontaneous autoimmune vitiligo. Thus, our novel TCR transgenic mouse model would allow us to undertake various developmental and translational studies.
Dissociated induction of cytotoxicity and DTH by CFA and CpG (41.8)
Authors: Tigno-Aranjuez Justine T , Lehmann Paul V. , Tary-Lehmann Magdalena 
Published: January 2023

Abstract
Vaccinations typically rely on immunization with live virus for eliciting protective CD8+ T cell immunity. There is increasing interest to use subunit vaccination strategies to achieve such responses. Complete Freund's Adjuvant (CFA) and CpG are considered some of the most potent adjuvants for eliciting immunity. Whereas a wealth of information is available on how these adjuvants affect CD4+ T cell responses, their effects on engaging CD8+ T cell immunity are not completely understood. We immunized C57BL/6J mice with the class I restricted peptides Uty or SIINFEKL using these two adjuvants and tested for cytokine secretion, proliferation, in vivo cytotoxicity and delayed-type hypersensitivity (DTH). Our data show that CFA induced CD8+ T cells to proliferate, mediate DTH, and to secrete IFN-gamma, IL-2 and IL-17. Despite these markers of CD8+ T cell activation, CFA failed to induce an early cytotoxic CD8+ T cell response. In contrast, CpG promoted a vigorous cytolytic response without activating substantial cytokine production, proliferation or DTH. These data have implications for CD8+ T cell sub-unit vaccine design in which cytotoxicity versus DTH plays a key role in host defense. Funding for this work was supported by National Institutes of Health Grant AI47756 and by Cellular Technology Ltd. account OSA483072 to M.T.L.
Ex Vivo-Generation of Gut-Homing Regulatory T-Cells: Characterization and Therapeutic Evaluation in Mice With Established Colitis (42.2)
Authors: Grisham Matthew B. , Karlsson Fridrik , Ostanin Dmitry V. , Jackson Sherry , Gray Laura 
Published: January 2023

Abstract
The objective of this study was to develop an ex vivo method to generate large numbers of Foxp3-expressing Tregs that can be used to evaluate their therapeutic efficacy in a T-cell-dependent model of chronic colitis. We found that polyclonal activation of CD4+CD25- Foxp3- T-cells in the presence of small amounts of IL-2, TGFβ and all trans retinoic acid (RA) converts &gt;90% of these T-cells to Foxp3-expressing Tregs (termed iTregs) as well as induces a 4-5-fold increase in proliferation following a 4 day incubation period in vitro. Furthermore, we found that the presence of RA enhances the surface expression of the gut-homing adhesion molecules α4β7 and CCR9. Finally, we observed that these iTregs are significantly more potent at suppressing T-cell activation in vitro and reversing established colitis in vivo. This protocol routinely generates 30-40 million iTregs from one mouse spleen assuming that the spleen yields approximately 10 million CD4+Foxp3- T-cells and that &gt;90% of these T-cells convert to Foxp3-expressing iTregs as well as undergo a 4-fold proliferation during the 4 day incubation period. We conclude that this protocol generates sufficient numbers of iTregs that can be used in vitro and in vivo to assess the therapeutic efficacy and immunological mechanisms responsible for suppression of inflammation in different models of autoimmune disease (Supported by PO143785).
The neutrophil serine protease inhibitor SerpinB1 protects against inflammatory lung injury and morbidity in influenza virus infection (43.10)
Authors: Gong Dapeng , Benarafa Charaf , Hartshorn Kevan L , Remold-O'Donnell Eileen 
Published: January 2023

Abstract
SerpinB1 is an efficient inhibitor of neutrophil serine proteases. SerpinB1-/- mice fail to clear bacterial lung infection with increased inflammation and neutrophil death. Here, we investigated the role of serpinB1 in influenza virus infection, where infiltrating neutrophils and monocytes facilitate virus clearance but can also cause tissue injury. Influenza virus (H3N2 A/Phil/82) infection caused greater and more protracted body weight loss in serpinB1-/- vs. WT mice (20% vs. 15%; nadir on day 4 vs. day 3). Increased morbidity was not associated with defective virus clearance. Cytokines (IFNγ, TNFα, IL-17, IFNα, G-CSF) and chemokines (MIP-1α, KC, MIP-2) were increased in serpinB1-/- mice vs. WT on days 2-7 post-infection but not on day 1. In WT mice, histology indicated large infiltration of neutrophils peaking on day 1 and maximal airway injury on day 2 that resolved on day 3 coincident with the influx of monocytes/macrophages. In serpinB1-/- mice, neutrophils also peaked on day 1; epithelial injury was severe and sustained with accumulation of dead cells on day 2 and 3. Immunophenotyping of lung digests on day 2 and 3 showed delayed recruitment of monocytes, macrophages and DC in serpinB1-/- mice, but increase of activated CD4 (day 2-3) and CD8 (day 3) T cells. Our findings demonstrate that serpinB1 protects against morbidity and inflammatory lung injury associated with influenza infection. Funded by NIH AI072552.
Subcapsular sinus macrophages prevent lethal vesicular stomatitis virus (VSV) infection (43.11)
Authors: Iannacone Matteo , Moseman Elliott Ashley , Tonti Elena , van Rooijen Nico , von Andrian Ulrich H 
Published: January 2023

Abstract
When a pathogen escapes innate host responses at the site of entry, it has the potential to spread systemically by accessing tissue-draining lymph vessels that channel interstitial fluid to the venous circulation. Before reaching the blood, the tissue-derived lymph must first pass through draining lymph nodes (LNs). LN act as filter stations preventing pathogens from reaching the systemic circulation and they are the staging grounds for adaptive immune responses, providing a specialized environment for the presentation of antigens to B and T cells. It is unclear how viruses are cleared from afferent lymph and how antiviral immune responses are generated. We have previously identified a population of CD11b+CD169+ macrophages on the floor of the subcapsular sinus (SCS) and in the medulla of LNs that capture viral particles within minutes after subcutaneous (sc) injection. Using VSV, an acutely cytopathic RNA virus closely related to rabies virus, we show here that these macrophages are critical to prevent viral access to nerve termini located in the SCS. Indeed, mice depleted of SCS macrophages develop an ascending CNS paralysis that leads to death in 50% of the animals 6-10 days after sc infection. Macrophage-depleted mice die despite mounting an increased neutralizing antibody (nAb) response. The notion that nAb are dispensable for protection against a sc infection (the natural route of transmission of this virus) is further demonstrated by the fact that antibody-deficient mice, which die upon intravenous infection, survive a sc VSV infection as long as SCS macrophages are present. While nAb are unnecessary for survival, B cells are required, and their protective role presumably relies on maintenance of LN architecture and normal macrophage phenotype.
The role of IL-17 in the protective immune response to Mycobacterium tuberculosis (44.18)
Authors: Windish Hillarie Plessner , Zheng John , Bertholet Sylvie , Baldwin Susan L , Ireton Gregory C , Vedvick Tom , Reed Steven G , Coler Rhea N 
Published: January 2023

Abstract
Mycobacterium tuberculosis (Mtb) infection kills more than 1.5 million people each year. While BCG prevents disseminated disease in children, a vaccine that confers long lasting protection from tuberculosis remains elusive. Previous work has demonstrated that expression of IL-17 in response to Mtb antigens supported resistance during subsequent infection. This suggests that an effective tuberculosis vaccine should induce IL-17. We hypothesized that a vaccine, comprised of ID83 and a TLR4 agonist/adjuvant protects against tuberculosis, in part by inducing IL-17. We characterized expression of IL-17 during the response to vaccination of C57Bl/6 mice. We found that IL-17 was produced by γδ T cells, Th17 cells, and also CD8 T cells in response to immunization. IL-17 expression was far more robust in the memory response to Mtb antigens, either during re-stimulation with vaccine antigens or during Mtb infection, than in the primary response. In continuing studies, we investigate the role of IL-17 in response to Mtb antigens and its role in protection that results from ID83 vaccination.
This work was supported by NIH Contract N01-AI-25479, and NIH grants AI-44373, AI-067251 and AI-078054.
MyD88 is required for viral-induced B cell activation and intestinal IgA production (44.20)
Authors: Blutt Sarah Elizabeth , Akira Shizuo , Dustin Lynn , Conner Margaret E 
Published: January 2023

Abstract
The role of TLR signaling in the induction of specific antibody responses is controversial and defined primarily using model antigens. Our study objective was to determine whether TLR signaling is required to generate pathogen-specific antibody responses. To assess whether TLR signaling is required for a viral-specific intestinal B cell responses, we examined whether B cell responses were aberrant after rotavirus infection of mice lacking expression of specific adapter proteins used in TLR signaling (MyD88, TIRAP, TRIF). Rotavirus infection of MyD88-/- mice did not result in a significant induction of either B cell activation or intestinal rotavirus-specific IgA. In contrast, mice lacking TIRAP and TRIF expression had wild type levels of both B cell activation and intestinal IgA production. B cell activation was normal in mice with a defect in BCR signaling (xid) or mice expressing an OVA-specific BCR, indicating that rotavirus-induced B cell activation does not require a rotavirus-specific BCR. These findings indicate that BCR-independent signaling through MyD88 plays a critical role in pathogen induced B cell activation and the generation of pathogen-specific intestinal IgA.
This work was supported by NIH AI10604, AI24998, and by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.
TREATMENT WITH A SPHINGOSINE ANALOG DIMINSHES IMMUNOPATHOLOGY FOLLOWING INFLUENZA VIRUS INFECTION (45.2)
Authors: Walsh Kevin Barry , Marsolais David , McGavern Dorian , Hatta Yasuko , Kawaoka Yoshihiro , Rosen Hugh , Oldstone Michael B.A. 
Published: January 2023

Abstract
Highly pathogenic strains of influenza virus can result in high mortality rates due to severe tissue damage caused by direct injury induced by the virus or by the immune response against the virus (immunopathology). Immune-mediated pulmonary tissue damage is a result of cytokine/chemokine release and the antiviral activity by cells of the innate and adaptive immune responses. Here we report that intratracheal treatment with a sphingosine analog, AAL-R, results in the inhibition of dendritic cell (DC) activation and antigen presentation to T cells in the mediastinal lymph nodes and the lung. Diminished DC function restricts virus-specific T cell proliferation causing a subsequent reduction in the accumulation of T cells in the lungs. The immunosuppressive effect involves signaling through S1P receptors on DC, but is independent of S1P1, S1P2 and S1P3. Inhibition of the accumulation of virus-specific T cells in the lungs occurs without impinging the production of anti-viral neutralizing antibodies. AAL-R treatment hinders cytokine/chemokine production which leads to a reduction in polymorphonuclear leukocyte and macrophage infiltration resulting in reduced lung injury. Importantly, local AAL-R administration into the lung was effective in controlling CD8+ T cell accumulation in the lungs when given four days following influenza virus infection. Administration of sphingosine analogs during influenza virus infection offers a potentially powerful therapeutic for alleviating pulmonary immunopathology.
Age-associated defects in Tcell metabolism (46.10)
Authors: Faulkner Matthew F , Durdik Jeannine M 
Published: January 2023

Abstract
Aging at the cellular and molecular level causes a decline in immune function. We are characterizing the responses of naïve T-cells to activation. Activated T-cells of aged mice initiate proliferation as well as their young counterparts, but have increased apoptotic rates. However, the mechanisms behind this post- activation death are not completely understood. We will show alterations in metabolic activities in T-cells of aged mice through differences in the expression of glucose transporter (Glut 1), glucose uptake and lactate production. Additionally, we will correlate these findings with autophagocytic vesicle formation by analyzing microtubule-associated protein -1 light chain 3 (LC3) as well as comparing relative amounts of actively respiring mitochondria using Mitotracker Red CM-H2XRos, a membrane potential dependant dye, and total mitochondrial mass by staining with the mitochondrial-selective fluorescence label Mitotracker Green. Levels of reactive oxygen species will be then quantified by staining with ROS specific dye DCF-DA and correlated to the ratio of mitochondrial mass to membrane potential.
Tracing memory T cell fate using Ox40-cre and novel reporter alleles (46.17)
Authors: Klinger Mark , Chmura Stephen A , Killeen Nigel 
Published: January 2023

Abstract
OX40 is a member of the Tumor Necrosis Factor Receptor Family that is expressed on activated and regulatory T cells. We have used an Ox40-cre allele with new Cre-dependent reporter alleles to discriminate memory T cells that differ in how much OX40 they expressed during immune responses to viruses. The reporters reveal flux in CD4+ memory T cells resulting in a gradual time-dependent enrichment for cells that previously expressed high amounts of OX40. Such flux was much less evident in CD8+ memory T cells. Differences in amounts of prior OX40 expression were nonetheless found to correlate with phenotypic differences in both CD4+ and CD8+ memory T cells. The results make clear the potential for using the Ox40-cre and reporter alleles for tracing the fate of activated T cells and relating it to differences in prior OX40 expression.
ICOS/B7h Bidirectional Signal to Induce Secretion of IL-1β and IL-17 (46.3)
Authors: Chiocchetti Annalisa , Mesturini Riccardo , Nicola Stefania , Dianzani Umberto 
Published: January 2023

Abstract
B7h is constitutively expressed by antigen presenting cells (APC) and binds ICOS expressed by activated T cells. ICOS triggering costimulates T helper (TH) cell activation and modulates their cytokine secretion, whereas the effect of B7h triggering in APC is unknown. This work investigated B7h reverse signalling in dendritic cells (DC). We induced DC maturation with LPS in the presence or absence of a soluble ICOS-Ig fusion protein; then, both cytokine secretion and allostimolatory activity were evaluated. B7h stimulation substantially modulated LPS-induced DC maturation by influencing cytokine secretion. The most striking effect was 80-fold increase of IL-1b secretion, but other changes were 3-fold increase of IL-10 and 2-fold decrease of TNF-a. In IL-1b secretion, B7h triggering acted as a "second signal" by activating caspase-1, required to cleave pro-IL-1b whose synthesis was induced by LPS. B7h triggering on LPS-activated DC also modulated their capacity to activate allogeneic T cells in mixed lymphocyte cultures (MLC) by decreasing the proliferative response and changing cytokines secretion, including 20-fold decrease of IFN-g and 5-fold increase of IL-17. This effect was partly ascribable to the cytokine milieu secreted by DC and particularly to the high IL-1b levels, known to be involved in differetiation of human Th17 cells. However, ICOS triggering too played a role in IL-17 secretion since blocking of the B7h/ICOS interaction inhibited IL-17 secretion in MLC with DC. Moreover, in naive Th cells activated with anti-CD3 antibodies, ICOS stimulation induced IL-17 secretion in the presence of exogenous IL-1b. In this work we showed that B7h-mediated reverse signaling induced IL-1b by DCs which in turn favors IL-17 secretion by Th cells. On the other side, we demonstrated that ICOS costimulation directly favors Th17 differentiation.