bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamics and functionality of CD8 T cell immunity within the female reproductive tract (161.4)
Authors: Schenkel Jason , Casey Kerry , Fraser Kathryn , Vezys Vaiva , Masopust David
Published: January 2023

The female reproductive tract (FRT) is a heterogenous mucosal tissue that is exposed to commensal flora, undergoes periodic remodeling, and supports fertilization, yet serves as a major site of microbial infection. Due to its unique physiology and vulnerability to microbes, it is important to have a better understanding of the dynamics of the adaptive immune response within the FRT. We paired flow cytometric analyses with immunofluorescence microscopy in order to characterize the kinetics, localization, phenotype and function of antigen specific CD8 T cells present in the FRT during a systemic infection. Effector CD8 T cells migrated into the vagina, cervix, uterine horns and ovaries, where they localized within the lamina propria, epithelium, or unexpectedly, in large lymphoid aggregates where they were associated with CD11c/MHCII+ cells. After pathogen clearance, maintenance of memory CD8 T cells varied greatly based on location within the FRT. Memory CD8 T cell phenotype was also heterogenous; only a subset retained high levels of granzyme B after pathogen clearance. The cellularity of the lymphoid aggregates changed dramatically throughout the response. We are now exploiting local challenge models to visualize the role of these structures in CD8 T cell migration, antigen trafficking and presentation, and supporting in situ recall responses. These studies should be highly relevant for understanding protective cell-mediated immunity within the female genital mucosa.
NSG-hu mice generate antigen specific human immune responses after gp96 vaccination (161.9)
Authors: Gonzalez Louis , Strbo Natasa , Podack Eckhard
Published: January 2023

NOD.SCID il-2r γc-/- (NSG) KO mice have no functional adaptive immune system and readily accept human xenografts. We have generated NSG mice with human adaptive immunity (NSG-hu) by transplanting CD34+ hematopoietic stem cells isolated from fetal hepatic tissue. These early progenitors give rise to all members of the human adaptive and innate lineages and can be found localized to immunologically relevant compartments (spleen, thymus, mLN, IEL and LPL). Human engraftment appears stable over time and reaches a high proportion of human CD45+ cells when compared to murine CD45+ cells in peripheral blood (54%±24%). Higher proportions of human CD45+ cells were detected in specific tissues (spleen, PEC, IEL and mLN) (82%±29%). We have also found that by using ACK2, an antagonistic anti-mouse stem cell antibody, to further ablate murine hematopoietic stem cells, human engraftment can be boosted (81%±6%). Previously, our lab has developed a cell secreted heat shock protein gp96 based vaccine expressing HIV gag (293gp96HIVgag). NSG-hu vaccinated with 293gp96HIVgag i.p. generates HIV gag-pentamer positive CD8 T cells in the gut. In addition, an interferon gamma ELIPOST response is also observed in the mucosa when stimulated with relevant peptide but not in spleen. We conclude that NSG-hu mice are able to establish and maintain stable hematopoiesis from CD34+ precursor cells. Furthermore, NSG-hu mice are capable of mounting a human HIV-specific immune response after gp96 vaccination.
Glycyrrhizin (GL) improves the resistance of humanized NSG chimeras intradermally infected with Pseudomonas aeruginosa (162.12)
Authors: Chin Jihyang , Lee Jong , Cruz Sharon , Yoshida Shohei , Kobayashi Makiko , Herndon David , Suzuki Fujio
Published: January 2023

In murine studies, GL is shown to be inhibitory on P. aeruginosa burn wound infection. Impaired β-defensin production in tissues surrounding the burn area is restored in mice treated with GL. As a translational study, the effect of GL on HBD-1 production in humanized chimeras was examined. The chimeras were X-irradiated NSG mice grafted with the unburned patient skin (10 x 10 mm: skin-chimeras) and inoculated i.d. with 1~10 x 105 cells/mouse of IMC isolated from patients syngeneic or allogeneic to skin donors (skin/IMC-chimeras). These chimeras were treated i.p. with GL (Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan) and anti-mouse skin-AMP rabbit IgG. Two days after grafting, tissues excised from grafted sites were homogenized and the amounts of HBD-1 in the homogenates were measured. In the results, 19 to 86 ng/ml of HBD-1 were detected in the grafted site tissues of skin-chimeras. The peptide production was inhibited by 77~84% in skin/IMC-chimeras. However, HBD-1 (17~90 ng/ml) was detected in the grafted site skin homogenates of skin/IMC-chimeras treated with 10 mg/kg of GL. P. aeruginosa grew in kidneys of skin/IMC-chimeras infected with 100 CFU/mouse of P. aeruginosa, but the pathogen was not detected in kidneys of the same chimeras treated with GL. These results indicate that HBD-1 is produced at grafted site tissues of skin/IMC-chimeras treated with GL, and sepsis stemming from P. aeruginosa grafted site-infection does not develop in these chimeras.
Glycyrrhizin restores impaired antimicrobial peptide production by human skin tissues cultured with immature myeloid suppressor cells (162.13)
Authors: Yoshida Shohei , Chin Jihyang , Lee Jong , Kobayashi Makiko , Herndon David , Suzuki Fujio
Published: January 2023

We have previously demonstrated that CD34+ CD31+ immature myeloid cells (IMC) appearing in burn patients suppress antimicrobial peptide production by cultured human skin keratinocytes. In the present study, the effect of glycyrrhizin (GL) on the peptide production by unburned patient skin (skin removed for the auto-grafting surgery) transwell-cultured with CD34+ CD31+ IMC was examined. Using a lineage cell depletion kit and flow cytometry, CD34+ CD31+ IMC were isolated from peripheral blood of burn patients. Unburned patient skin tissues (4 x 4 mm) were transwell-cultured with various numbers of IMC for 1 to 7 days in the presence or absence of GL (Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan). Culture fluids obtained were assayed for human β-defensin-1 (HBD-1) by ELISA. In the results, 1-5 ng/ml of HBD-1 were produced by unburned patient skin tissues 2 to 7 days after cultivation. When skin tissues were transwell-cultured with 1 x 104 cells/ml of IMC syngeneic to the skin donors, 86% of HBD-1 production was suppressed. However, HBD-1 was produced by unburned patient skin tissues transwell-cultured with syngeneic IMC and GL. Similarly, HBD-1 was produced by unburned skin tissues transwell-cultured with allogeneic IMC and GL. These results indicate that GL is inhibitory on the IMC suppressor cell activity in HBD-1 production by unburned patient skin tissues.
Dynamics of an innate myeloid cell response to acute CNS viral infection (162.16)
Authors: Nayak Debasis , Zinselmeyer Bernd , McGavern Dorian
Published: January 2023

Viral infections of central nervous system (CNS) often trigger an inflammatory response that gives rise to a wide range of pathological outcomes. The CNS is equipped with an elaborate network of innate immune sentinels that routinely serve as first responders to these infections. The meninges and perivascular spaces are inhabited by specialized macrophages, and the brain parenchyma contains microglia - the most abundant CNS myeloid cell. To gain novel insights into how myeloid cells respond to an acute CNS viral infection, we utilized genomics and two-photon imaging approaches to study a pure innate response to LCMV infection. This was accomplished by monitoring temporal gene expression patterns and myeloid cell dynamics in the brains of CX3CR1-GFP mice lacking LCMV-specific T cells. Two-photon imaging studies revealed that CNS myeloid sentinels responded significantly to acute infection by sequestering DsRed-labeled virus. Microglia but not peripheral myeloid cells proliferated following acute infection and responded directly to viral antigen by reducing process complexity, suggesting a localized response to the presence of virus. However, at the genomic level, acute infection triggered massive changes in CNS gene expression with the majority of genes being linked to innate viral control and the type I interferon system. These data suggest that localized detection of virus by CNS myeloid sentinels can translate into engagement of an extensive anti-viral program.
Chronic morphine administration induces tolerance to suppressive morphine effects in two canonical LPS-induced innate immunity responses in mice. (162.20)
Authors: Molina Martínez L. , Cruz S. , González Espinosa C.
Published: January 2023

The immunosuppressive effects of acute morphine administration are well described. However, little is known about chronic morphine treatment actions using different administration schedules. The aim of this work was to study the effects of various morphine administration schemes on peritoneal Tumor Necrosis Factor-α (TNF-α) release and serum corticosterone levels after i.p. injection of bacterial lipopolysaccharide (LPS) in mice. Our results show that morphine per se (0.1-10 mg/kg) had no effects on TNF-α levels. Morphine inhibited LPS-induced TNF-α release but this effect occurred only at high doses (3.1 y 10 mg/kg) and was not dose dependent. Serum corticosterone levels increased after a single LPS challenge and morphine dose-dependently prevented this effect. After repeated administration, tolerance developed to the inhibitory effects of morphine on peritoneal TNF-α release and serum corticosterone levels in response to LPS. These data suggest that morphine inhibitory effects on innate immunity depend on the opioid administration schedule and duration treatment.
Toll-like receptor 2 activation induces lung endothelial permeability and coagulopathy (162.6)
Authors: Xu Fengyun , Hellman Judith
Published: January 2023

Toll-like receptors (TLRs) recognize components of microbes and initiate host responses in sepsis. TLR2, the receptor for microbial lipoproteins, is important in the inflammatory effects of bacteria and fungi. Endothelial cell (EC) activation, coagulopathy, and vascular leak contribute to sepsis-induced lung injury. We previously found that bacteria shed TLR2 agonists into the blood in sepsis models, that TLR2 activation increases EC expression of inflammatory markers and EC PMN trafficking, modulates EC expression of coagulation factors, increases EC monolayer permeability, and causes lung inflammation and decreased arterial blood oxygenation in mice. We hypothesized that TLR2 activation by the lipopeptide, Pam3Cys, causes lung vascular leak and coagulopathy. Treatment of human lung microvascular EC with Pam3Cys led to increased plasminogen activator inhibitor 1 levels in culture supernatants, and increased monolayer permeability to albumin (p<0.05). Intratracheally administered Pam3Cys also significantly (p<0.05) increased albumin levels in lung homogenates and bronchoalveolar lavage fluid, and increased lung fibrin deposition. The alterations that we observed in lung microvascular EC function closely resemble those that occur in patients with sepsis. Our studies here suggest that TLR2 activation contributes to sepsis-induced EC dysfunction, coagulopathy, and respiratory failure, and suggest that EC TLR2 pathways could potentially be targeted for sepsis interventions.
Resistance to Yersinia pestis infection decreases rapidly and substantially with age in B10.T(6R) mice (162.9)
Authors: Lambert Nathaniel , Nilles Matthew , Bradley David
Published: January 2023

We have previously demonstrated that 2-3 month old, female B10.T(6R) mice are highly resistant to a delta pgm strain of Yersinia pestis and by 5 months of age these mice are highly susceptible to Y. pestis infection. In the current study we further characterize this observation in female B10.T(6R) mice. The LD50 of Y. pestis was ~14000 CFU/ml in young (2-3 month) mice while only ~60 CFU/ml in middle aged (>5 month) mice. Significantly elevated bacterial burden was found in both the spleen and liver of middle aged mice while a significant infiltration of immune cells was observed in resistant young mice. The cellular infiltrate in susceptible middle aged was comparable to susceptible young C57BL6/J mice. Sera and spleens from Y. pestis-infected middle aged B10.T(6R) contained elevated levels of KC, MCP-1 (CCL2), MIP-1a (CCL3), G-CSF, IL-6 and Eotaxin. In many cases, cytokine/chemokine levels in middle aged mice increased in an infectious dose-dependent manner and were comparable to Y. pestis infection in C57BL6/J mice with both experimental groups being significantly increased when compared to infected young B10.T(6R) mice with the exception of IL1b that was only increased in both serum and spleen samples from young mice. These findings suggest that IL1b may be critical to prevent Y. pestis infection and may provide insight into defense mechanism(s) necessary to develop resistance to the plague.
Lineage-specific IKKβ deficiency reveals a role for intestinal epithelial cells in lung immune response to ingested antigens (163.10)
Authors: Bonnegarde-Bernard Astrid , Jee Junbae , Steiner Haley , Fial Micheal , Davis Ian , Boyaka Prosper
Published: January 2023

The sites where immune responses are initiated are believed to play a key role at shaping subsequent responses at distant mucosal sites. However, little is known about the relative contribution of intestinal epithelial cells (IEC) to lung reactivity to foreign antigens and the development of allergic and non-allergic asthma. It has been demonstrated that nuclear factor κB (NF-κB) activation by IκB kinase (IKK)β deletion in macrophages increases pro-inflammatory cytokines responses. To address the potential regulatory effect of IEC for lung reactivity, control C57BL/6 mice and mice deficient in IKKβ in IEC (IKKβΔIEC) or in macrophage (IKKβΔMac) were orally sensitized by administration of ovalbumin and cholera toxin as adjuvant. All groups developed similar levels of antigen-specific IgE responses, but IgG2a responses were enhanced in IKKβΔIEC and, even more in IKKβΔMac. Subsequent nasal challenges of C57BL/6 mice with ovalbumin induced lung inflammation and increased lung resistance. Interestingly, both lung inflammation and lung resistance were lower in IKKβΔIEC mice and further reduced in IKKβΔMac mice. Taken together, these results show that localized alteration of NF-κB signaling in IECs have broader effects on immune responses to ingested antigen, which were similar, although of lower magnitude, than those seen when NF-κB signaling is altered on macrophages. They also reveal that IECs can shape immune responses at distant mucosal sites of the airways.
Mast cell-deficient mice show attenuated thermal pain responses (163.18)
Authors: Chatterjea Devavani , Wetzel Abigail , Mack Madison , Allen Juliann
Published: January 2023

Pain is a cardinal component of inflammation and a significant public health issue. Inflammatory cytokines such as TNF-alpha, IL-1beta, CXCL1/IL-8, as well as neutrophil influx have been implicated in the potentiation of peripheral inflammatory pain in models of thermal and mechanical hindpaw hypernociception in rodents.. Degranulation of mast cells has been shown to be important for phosphorylation of Erk in nociceptive neurons in a rat model of migraine-like stimulation. Mechanical bladder pain in a model of interstitial cystitis was abrogated in mast cell deficient mice. We show that degranulation of plantar mast cells caused by direct secretagogue treatment or passive antibody sensitization and challenge, causes reduction in paw withdrawal latencies as well as significant edema, neutrophil influx, and upregulation of local TNF-alpha and IL-1beta levels, as well as histamine release in the hindpaws of mice in a model of thermal pain. Mast cell-deficient C57BL/6 Wsh/Wsh mice show no edema, histamine release or pain response indicated by reduction in paw withdrawal latencies. This suggests that while pain is a complex and multifactorial physiology, tissue sentinel mast cells may be a critical initiator of inflammatory pain in both protective and allergic/pathological contexts.
Antibodies against GlcNAc modulate the immune response to Aspergillus fumigatus in the lung (163.19)
Authors: Stefanov Emily , Kin Nicholas , Kearney John
Published: January 2023

The incidence of childhood asthma has increased dramatically over the past few years. The hygiene hypothesis postulates that this increase is due in part to overly sanitized environments encountered during childhood. A lack of early microbial exposure is thought to facilitate allergic responses to normally innocuous antigens later in life. Chitin, a biopolymer of N-acetyl glucosamine (GlcNAc), is produced by many allergen-bearing organisms including: fungi, the exoskeleton of insects, crabs, shrimp and parasitic nematodes. Epidemiological studies have shown a negative correlation between Streptococcus pyogenes infection and the development of asthma later in life. S. pyogenes induces antibodies against GlcNAc moieties on its cell wall polysaccharide. We hypothesized that antibodies against GlcNAc, introduced passively, or induced by neonatal immunization of mice with Group A Streptococci, modulate the immune response in the lung to both chitin and Aspergillus fumigatus (AF), a chitin-bearing fungi and common asthma-associated allergen. We show that these antibodies reduce the influx of lymphocytes into the lung after intratracheal challenge with chitin or AF. We also find a dramatic decrease in pro-inflammatory cytokines in the lung when these antibodies are present. These findings are important in understanding allergic asthma and discovering potential ways to prevent its development.
Pathogenetic role of BATF in a murine model of allergic asthma (163.20)
Authors: Übel Caroline , Hildner Kai , Lehr Hans , Rieker Ralf , Murphy Kenneth , Finotto Susetta
Published: January 2023

The transcription factor Basic leucine zipper transcription factor (BATF) is a member of the AP-1 family of transcription factors which upregulate gene expression of genes containing the TPA-DNA response element. However, BATF is thought to inhibit AP-1 activity. BATF has recently been described to be crucial for Th17 differentiation. BATF deficient mice lack this subset of T helper cells which is known to play a key role in inducing airway hyperresponsiveness in allergic asthma. In our study, we analyzed BATF deficient mice in a murine model of allergic asthma. We could show for the first time that BATF deficient mice show decreased lung Th2 cytokine production, reduced airway hyperresponsiveness, serum IgE and IgG1 levels after allergen challenge compared to wild-type littermates. The number of eosinophils was also diminished in the airways of BATF(-/-) compared to wild-type mice. We consider this phenotype to be primarily caused by the lack of IL-17A-producing Th17 cells. These results underline the importance of Th17 and the respective IL-17 cytokines for inducing and maintaining allergic diseases such as asthma. As there is a high similarity between human and murine BATF molecules, targeting this transcription factor could lead to the development of new treatment strategies against human asthma.
Bee venom ameliorates lupus nephritis in New Zealand Black/White mice. (164.15)
Authors: Lee Hyojung , Shin Minkyu , Hong Moochang , Bae Hyunsu
Published: January 2023

Introduction: Bee venom (BV) therapy has been used to treat inflammatory diseases including rheumatoid arthritis in humans and in experimental animals. This study was conducted to examine the therapeutic effect of BV on established lupus nephritis in New Zealand Black/White (NZB/W) F1 female mice. Methods: Beginning at 18 weeks of age, mice were given a subcutaneous injection of either BV (3mg/kg body wt) or an equal volume of saline once a week until the end of the study. Results: The proteinuria level and the serum levels of total IgG, IgG2a and anti-dsDNA IgG antibodies in mice treated with BV were lower than those in the saline-treated mice. Concomitant with serum antibody levels, IgG2a immune complex deposition in the glomeruli was reduced in BV treated mice. Furthermore, renal proinflammatory cytokines were also decreased after BV treatment. Interestingly, CD4+CD25+ Regulatory T cells and CD4+Foxp3 positive T cells were significantly increased in the BV treated mice. Conclusions: Collectively, the administration of BV that has immune modulating effects represents an applicable treatment of lupus nephritis in NZB/W F1 mice.
TLR7 and TLR9 9 agonists enhance the exogenous IFNb-1a’s induction of immunomodulatory cytokine secretion via reconstituting a deficient IFNAR/TLR7 signaling in patients with relapsing remitting multiple sclerosis (164.16)
Authors: Tao Yazhong , Zhang Xin , Troiani Luigi , Markovic-Plese Silva
Published: January 2023

Objective: To explore possibilities to enhance IFNb’s therapeutic effects in RR MS patients. Results: B-cells derived from RR MS patients express a decreased IFNAR1, IFNAR2 and TLR7 gene expression, associated with increased IL-23 and decreased IL-27, IL-10 and IL-4 secretion, in comparison to healthy controls HCs. In-vitro administered IFNb-1a only partially reversed the B-cells’ pathogenic cytokine pattern. TLR9 ligand CpG enhanced exogenous IFNb-1a’s immuomodulatory effects on B-cells by significantly up-regulating IFNb-1, IRF7 and MyD88 gene expression, and IL-27, IL-10 secretion, while combination of TLR7 ligand Loxoribin (Lox) and IFNb-1a induced IL-4 production and endogenous IFN-b1 gene expression. In CD4+ T-cells, TLR7 and TLR9 ligand combinations with IFNb-1a induced a significantly higher IRF7 and MyD88 gene expression in comparison to the untreated CD4 cells. CpG and IFNb-1a combination induced TLR7 and TRAF6, and LOX and IFN-b1a inhibited SIGIRR gene expression. Both combinations with IFNb inhibited the secretion of IL-17A, IL-17F, IL-22 and IL-9, but induced IL-4, TGF-b1 and IL-10, while only CpG and IFNb-1a combination inhibited IL-21 secretion. Both combinations induced endogenous IFNb gene expression in CD4+ cells. Conclusion: TLR7 and TLR9 agonists enhance the in-vitro-mediated IFNb-1a immunomodulatory effects in B-cells and CD4 cells derived from RR MS patients, implicating their therapeutic potential in combination with IFNb-1a.
CD8+ T-cells are required for the action of glatiramer acetate therapy for autoimmune demyelinating disease (164.19)
Authors: Tyler Andrew , Mendoza Jason , Ortega Sterling , Firan Mihail , Karandikar Nitin
Published: January 2023

Multiple sclerosis (MS) is a chronic disabling disease characterized by immune-mediated destruction of central nervous system (CNS) myelin. Murine experimental autoimmune encephalomyelitis (EAE) is a well characterized animal model of MS. The FDA-approved drug glatiramer acetate (GA, Copaxone®), discovered through EAE studies, ameliorates the symptoms of MS without immunosuppression, although its mechanism is still poorly understood. Our studies have suggested that CD8+ T-cells may be involved in exerting the therapeutic effects of the drug. We have shown that in response to drug treatment, the GA-specific subset of CD8+ T-cells greatly expands in MS patients and possesses the ability to suppress/kill CD4+ T-cells in vitro. We now demonstrate that GA also induces a robust CD8+ T-cell response in the mouse model. Importantly, GA treatment is ineffective in ameliorating EAE in mice deficient in CD8+ T-cells or MHC Class I, indicating that pathways involving only CD4+ T-cells and APCs bearing MHC Class II are insufficient to mediate protection. GA-induced CD8+ T-cells transferred into wild-type or CD8-deficient mice restore disease suppression. Induction of GA-induced regulatory CD8+ T-cells requires indoleamine 2,3-dioxygenase (IDO) and their function is dependent on IFNγ, perforin, and MHC Class I. These studies demonstrate an essential in vivo role for cytotoxic/suppressor CD8+ T-cells during GA therapy and identify their potential use as adoptive immunotherapeutic agents.
Baicalein promotes PPAR-β-mediated alternative activation of microglia in alleviating experimental autoimmune encephalomyelitis (164.4)
Authors: Xu Jingwei , Zhang Yanyun
Published: January 2023

Microglia, when induced to an alternatively activated state, play important roles in cellular immunity and tissue repair. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), alternatively activated microglia (aaMG) exhibit a reduced inflammatory response, confined phagocytosis, and promoted functional recovery of myelin, which together can alleviate EAE. Here we investigated the effect of baicalein (BA), a potent neuroprotective compound, on EAE. BA administration significantly attenuated EAE severity by reducing both inflammatory infiltration and demyelination in the central nervous system. The inhibitory effect of BA was not caused by impaired T cell responses in the periphery. Interestingly, we observed that BA significantly increased the expression of alternative activation-associated genes in microglia. Furthermore, we showed that induction of aaMG by BA is attributable to increased peroxisome proliferator-activated receptor (PPAR)-β transcriptional activity through activation of the PI3K signal. Importantly, BA-induced aaMG showed significant preventive effects in EAE mice. Taken together, these data demonstrate that BA has a potent inductive effect on aaMG through PPAR-β expression in microglia that mediated by PI3K activation, which led to reduced disease severity during EAE disease process. This provides novel insights into a therapeutic modality in MS.
Inhibition of activated protein C dramatically attenuated experimental autoimmune encephalomyelitis (164.9)
Authors: Alabanza Leah , Bynoe Margaret
Published: January 2023

Activated Protein C (APC) is an anticoagulant that is involved in the complex two-way interactions between the coagulation and immune systems. APC is widely known to be anti-inflammatory; most notably it has been clinically developed for treatment of severe sepsis. The anti-inflammatory properties of APC include conferring endothelial barrier protection, thus limiting the extravasation of immune cells into tissues. Given that the major pathological component of multiple sclerosis and its mouse model, experimental autoimmune encephalomyelitis (EAE), is the extravasation of autoreactive immune cells in the central nervous system (CNS) due to blood brain barrier (BBB) dysfunction, we investigated whether APC can affect the progression of EAE. We saw that blocking endogenous APC during EAE resulted in considerable immune cell infiltration in the CNS. Interestingly, however, these mice exhibited mild to no outward signs of EAE as assessed by paralysis severity. The anti-APC treated mice also had significantly higher percentage of CD4+Foxp3+ cells both in the CNS and in the periphery compared to controls. Moreover, the cytokine profile of leukocytes from the anti-APC mice was skewed to T helper-2, which is known to be protective in EAE. These data show that lowered APC levels during EAE can lead to divergent effects on EAE pathogenesis, specifically leading to increased BBB dysfunction while simultaneously inducing immunological conditions that are non-pathogenic in EAE.
Administration of a vaccine composed of dendritic cells pulsed with premalignant oral lesion lysate stimulates Th1 and Th17 immunity in mice bearing carcinogen-induced premalignant oral lesions. (165.13)
Authors: De Costa Anna-Maria , Walker David , Schuyler Corinne , Young Rita
Published: January 2023

The use of dendritic cell (DC) vaccines as treatment for malignancy is complicated by the immune suppression often exhibited in the presence of solid carcinomas such as head and neck squamous cell carcinoma (HNSCC). The goal of this study is to determine if an immune response can be elicited by administering a DC vaccine during the premalignant stages of HNSCC, prior to development of immune suppression. Mice treated with the carcinogen 4NQO in drinking water develop premalignant oral lesions which progress to HNSCC. As previous studies demonstrated that premalignant lesions and HNSCC overexpress common tumor antigens, BM-derived DCs were pulsed with premalignant lesion lysate (DCpm) and administered to 4NQO-treated mice exhibiting premalignant lesions. Lymphocytes were then isolated and tested for response to antigenic challenge. The levels of Th1, Th17, and to a lesser extent, Th2 cytokines, were increased in supernatant of unchallenged lymphocytes from DCpm-vaccinated mice compared to controls. In response to premalignant and HNSCC challenge, lymphocytes from DCpm-vaccinated mice released greatly increased levels of Th1 cytokines than control lymphocytes, while levels of Th2 cytokines released by control lymphocytes increased to, or near to, levels released from DCpm-vaccinated mouse lymphocytes. This suggests that DCpm vaccination of 4NQO-treated mice stimulates the development of Th1 immunity that may be specific to premalignant and HNSCC antigens.
Exposure to abnormal self antigens during non-malignant events may provide immunological defense against tumors (165.15)
Authors: Iheagwara Uzoma , Beatty P. , Chan Su-Wan B. , Finn O.
Published: January 2023

Antibodies and T cells specific for several tumor associated antigens exist not only in cancer patients but also in healthy individuals. We hypothesize that non-malignant events such as viral infections elicit abnormal expression of self antigens on infected cells and specific immune memory for those antigens. Abnormal expression of the same antigens on tumor cells triggers specific immune responses and provides anti-tumor immunosurveillance. We are testing this hypothesis by examining changes in expression of known tumor associated antigens MUC1 and cyclin B1 during influenza virus infection of MUC1 transgenic mice. We found increased MUC1 and cyclin B1 expression in acutely infected lung tissue collected three days post infection. Furthermore, MUC1 became hypoglycosylated, the form found on cancer cells. Importantly, we found increased MUC1-specific and cyclin B1-specific antibodies in serum from mice post-infection compared to pre-infection. These antigens are likely to be representative of a much larger number of normal cell molecules that undergo changes in expression and induce an immune response important for cancer immunosurveillance. To address this, post-infection serum was blotted against tumor lysate separated by SDS-PAGE and compared to serum from uninfected mice. We found a marked increase in reactivity with several tumor proteins. In future experiments we will correlate immunity to specific antigens with cancer immunosurveillance.
Regulatory mechanism of Axl-mediated lymphotoxin expression in cancer cells (165.24)
Authors: Song Min-Ji , Lee Eun-Hee , Kang Hyung-Sik
Published: January 2023

We investigated the regulatory mechanism of Axl receptor tyrosine kinase-mediated lymphotoxin (LT) expression. LT expression was markedly downregulated in various cancer cells that over-expressed Axl, but it was increased by siRNA of Axl. Axl-/- mice showed upregulated LT expression in different tissues compared to wild type mice. Blockade of LT signaling resulted in the suppression of apoptosis in these cancer cells, which was further promoted by Axl silencing. Upon triggering of Axl signaling, the activation of PI3K/AKT signaling was induced, but the transcriptional activation of NF-κB was inhibited. These data suggested that Axl suppressed LT expression through NF-κB transcriptional inactivation via PI3K/AKT signaling pathway in cancer cells.
Progressive abrogation of early anti-tumor immunity by dendritic cells provides opportunities for novel diagnostic interventions (165.25)
Authors: Scarlett Uciane , Rauwerdink Adam , Rutkowski Melanie , Martinez Diana , Fields Jennifer , Gonzales Jorge , Fiering Steve , Conejo-Garcia Jose
Published: January 2023

We have generated an inducible genetic model of ovarian carcinoma that leads to terminal disease and recapitulates the immune microenvironment of human tumors. Our model progresses through a phase of dynamic equilibrium that keeps tumor expansion in check for the first 4 weeks, followed by a phase of exponential growth from ~D40-D60 (evasion phase). Our inducible system reveals that dendritic cells, with enhanced endocytic pathways, accumulate at primordial ovarian cancer lesions and drive early anti-tumor immune responses. The immunostimulatory phenotype of tumor-associated dendritic cells is progressively impaired as the disease advances, so that the evasion phase coincides with the exponential accumulation of immunosuppressive dendritic cells. Taking advantage of the spontaneous homing of dendritic cells (with enhanced endocytic activity) to solid tumor masses, we have been able to detect magnetic nanoparticle-derived signals specifically at tumor locations before they become macroscopically visible. We have shown that inflammatory leukocytes function is progressively impaired at tumor locations, which unveils a strategy for early diagnosis.
The effects of 1,25-dihydroxy vitamin D3 on tumor-associated immunosuppression in squamous cell carcinoma of the head and neck. (165.29)
Authors: Walker David , Reeves Travis , De Costa Anna-Maria , Schuyler Corinne , Young M. Rita
Published: January 2023

Head and neck squamous cell carcinoma (HNSCC) evades the host immune system via several mechanisms. In HNSCC patients, 1,25-dihydroxy vitamin D3 (vitamin D3) promotes differentiation of CD34+ progenitor cells into antigen-presenting cells. However, the downstream immune effects of vitamin D3 have not been well-characterized. This is particularly true with respect to systemic versus intra-tumoral effects. The objective of this study is to evaluate the role vitamin D3 in modulating tumor-associated immunosuppression. To compare the peripheral and local immune status of vitamin D3 treatment, we analyzed immune mediator levels in plasma and tissue samples from normal controls, HNSCC patients, and HNSCC patients treated with vitamin D3. Vitamin D3 treatment results in a systemic increase in Th2-associated cytokines, IL-6 (p-value = 0.005), IL-8 (p-value = 0.01), and IL-10 (p-value = 0.005) as compared to untreated HNSCC patients. However, this trend is reversed in tumor homogenates, suggesting systemic markers may not accurately reflect the immunologic status of the tumor environment. Furthermore, inflammatory systemic cytokines IL-2 (p-value = 0.04) and IFN-γ (p-value = 0.05) are increased compared to tumor controls, with a concomitant increase in the pro-tumor mediators GM-CSF (p-value = 0.02 ) and VEGF (p-value = 0.02). These findings are surprising, as treatment with vitamin D3 fails to induce a systemic Th1 response thought to be responsible for anti-tumor activity.
Antigen immunoselection as a mechanism of cancer immunoediting (165.3)
Authors: Matsushita Hirokazu , Vesely Matthew , Uppaluri Ravindra , Schreiber Robert
Published: January 2023

Cancer immunoediting is the process wherein the immune system not only protects against tumor development but also promotes outgrowth of tumors with reduced immunogenicity. Although we and others identified several key immune components that participate in this process, we know very little about the targets of cancer immunoediting. In this study we used a highly immunogenic methylcholanthrene induced sarcoma cell line (d42m1) to ask whether tumor antigens can be immunoediting targets. Like most unedited MCA sarcomas, d42m1 cells form tumors when transplanted into immunodeficient mice but are rejected in naive syngeneic wild type (WT) mice. We identified two antigens of d42m1 by expression cloning: a point mutant of spectrin-β2 and a point mutant of M-phase phosphoprotein 8 (MPP8). In WT mice, d42m1 occasionally produces escape variants lacking mutant spectrin-β2 but maintaining expression of mutant MPP8 that can form tumors in naive WT mice. Enforced expression of mutant but not WT spectin-β2 into escape variant cell lines converted them into regressors. Analysis of the parental d42m1 cell line revealed that only 80% of d42m1 clones expressed mutant spectrin-β2 whereas 100% expressed mutant MPP8. Only spectrin-β2 expressing d42m1 clones were rejected in WT mice. Thus, mutant spectrin-β2 is not only the major rejection antigen of d42m1 sarcoma cells but is the target of a cancer immunoediting immunoselection process that facilitates tumor escape from immune control.
Resveratrol-dependent sensitization of prostate cancer cells (165.33)
Authors: Mishra Manoj , Eroglu Erdal , Singh Udai , Singh Shree
Published: January 2023

Prostate Cancer (PCa) is the most common non-skin malignancy and commonly diagnosed cancer, and second most cancer responsible for death in men in United States. Prior studies in animal models of PCa as well as in human, demonstrated that the prostate produces several growth factors. Addition or blocking of these growth factors can alter the PCa cell proliferation and other important functions. Resveratrol (RES), a polyphenolic compound found in the skin of red fruits, exhibits anti-inflammatory, anti-oxidative, anti-cancer, and anti-proliferative characteristics. In this study, the role RES has been investigated on mouse prostate cancer (PCa) cell lines, derived from transgenic adenocarcinoma of mouse prostate (TRAMP), in C57/B6 mice. Interestingly, TRAMP-C1 and TRAMP-C2 form tumors while TRAMP-C3 fails to form tumors. TRAMP cells were treated with different concentration of resveratrol and at different time points analyzed for intensity of sensitization, cytokines, growth factors as well as apoptotic genes. The results show that TRAMP C1 and C2 were resistant to RES compared to TRAMP C3 suggesting that TRAMP-C3 cells may up regulate “kill me signal” that help in sensitization of these cells compared to TRAMP C1 and C2. This data also demonstrates that RES mode of action is specific for each cell line in activating program cell death that may utilize unique sets of genes during apoptotic pathways.
Tumor growth is attenuated in eosinophil-deficient mouse models (165.41)
Authors: Jacobsen Elizabeth , Taranova Anna , Ochkur Sergei , Protheroe Cheryl , Condjella Rachel , Colbert Dana , Zellner Katie , McGarry Michael , Lee James , Lee Nancy
Published: January 2023

Eosinophil infiltration into tumors has been associated with positive and negative growth human cancers and in mouse models of tumorigenesis. Our previous studies have shown that eosinophils infiltrate tumors and differentially accumulate in the necrotic and capsule regions of tumors. Using various eosinophil-deficient mouse models, we demonstrate that in the absence of eosinophils tumor growth is attenuated. Methods: Tumor growth kinetics were assessed using a B16F10 melanoma subcutaneous cell injection model. Eosinophil chemotaxis was assessed using an ex vivo transwell insert assay system. We compared tumor growth in wild type animals vs. various eosinophil-deficient mouse models that are transgenic line of mice (PHIL) and MBP-/-/EPO-/- double knockout mice that are deficient in eosinophil-specific proteins (major basic protein (MBP) and eosinophil peroxide (EPO)) resulting in a blockade of eosinophilpoiesis. Conclusions: The migration of eosinophils was inhibited through blocking the sphingosine-1-phosphate pathway, suggesting recruitment and accumulation is primarily an inflammatory response. Tumor growth was decreased by ~40-60% in PHIL and in MBP-/-/EPO-/- double knockout mice relative to tumors in wild type animals. In addition, EPO-/- mice, and not MBP-/-mice, replicate this ~40% reduction in tumor size implicating a significant role for EPO in tumor growth kinetics. These data suggest eosinophils respond to inflammatory mediators and mediate augment tumor growth.
Live-imaging of antigen presenting cells in spontaneous breast cancer (165.44)
Authors: Engelhardt John , Krummel Matthew
Published: January 2023

The immune response to tumors is thought to be inactivated, but the subsets of cells that ingest tumor to present to incoming immune cells have remained enigmatic. We generated a genetically encoded fluorescent mouse model of breast cancer to track antigen flow from tumors into immune cells, and defined a subset of tumorAPC (tAPC) that ingest tumor debris in the steady-state, act as a major repository of tumor-derived antigens, and present these antigens to tumor-reactive T cells. Using intravital live-imaging we show that this population represents a unique spatial subset of slow-moving marginating myeloid cells that interact closely with the tumor margin. We further show that these cells nucleate clusters of tumor-specific T cells, but appear to ‘anergize’ them. This study highlights a major route of immunosuppression in the tumor microenvironment and suggests immunotherapies based on modulating the function of tAPC.
Activation of p53 and NF-kB degradation by SOCS1 to promote cancer cell apoptosis and to suppress implanted tumor growth (165.47)
Authors: Lee Choong-Eun
Published: January 2023

The suppressors of cytokine signaling (SOCS), play multiple roles in cell survival, differentiation and apoptosis in diverse cell systems. We have investigated mechanisms of tumor-suppressive functions of SOCS using HCT116 colon cancer cells (HCT116:p53wt and HCT116:p53-/-) by employing over-expression or knock-down of SOCS isoforms. Our data show that SOCS1 but not SOCS3 promoted γ-irradiation-induced apoptosis by up-regulating p53 activation and Bax expression. SOCS1 also modulated cell cycle arrest induced by γ-irradiation resulting in increase in G1 arrest which was associated with up-regulation of p21. In addition, SOCS1 suppressed the radiation-induced survival pathway including Erk, NF-kB and Bcl2 expression. The modulatory effects of SOCS1 on cell cycle changes correlated well with the negative regulation of Jak3/STAT3 and Erk activation, and the transfection of siSTAT3 as well as treatment of inhibitors of Jaks and Erk induced effects similar to SOCS1 over-expression. In addition, the SOCS1-mediated NF-kB degradation appears important in counteracting the survival response triggered upon apoptoptic stimuli. Furthermore, SOCS1-transfected HCT116/SOCS1 tumor cells implanted in nude mice exhibited reduced tumor sizes as compared to HCT116/mock cells upon γ-irradiation. The results indicate the promotion of radiosensitivity by SOCS1, which may be useful for the development of a therapeutic regimen to overcome radioresistance often found in certain colorectal cancers.
Hypoxia inducible factor-α expression correlates with thymosin β4 overexpression related to lymph node metastasis in breast cancer (165.5)
Authors: Yoon Sun Young , Kim Joo Heon , Cho Daeho
Published: January 2023

Two hypoxia inducible factors (HIFs), HIF-1α and HIF-2α, are induced by hypoxia, and high expression of these proteins is correlated with angiogenesis and distant metastasis. Thymosin β4 (Tβ4) is frequently highly expressed in cancer, and this over-expression correlates with malignant progression. The objective of this study was to investigate the clinical correlation of HIF-α with Tβ4 and the intracellular functional roles of Tβ4 on HIF-α activation. We showed that high expression levels of HIF-1α and HIF-2α strongly correlated with Tβ4 expression (P<0.0001), and over-expression of Tβ4 correlated significantly with lymph node metastasis (P<0.05) in patients with breast cancer. Additionally, we demonstrated that hypoxia up-regulates intracellular Tβ4 protein, which then affects HIF-α activity, which is a key in regulating vascular epidermal growth factor (VEGF) expression. We confirmed that hypoxia-induced intracellular Tβ4 and HIF-α activity were reduced by inhibiting Tβ4 expression using Tβ4 shRNA lentivirus. VEGF-A was also down regulated, but VEGF-C and VEGF-D expressions were not affected. These findings suggest that over-expression of Tβ4 is strongly associated with HIF-1α and HIF-2α expression and is also clinicopathologically involved in lymph node metastatic potential of breast cancer through the modulation of HIF-α activation and induction of VEGF-A. Ultimately, these results highlight Tβ4 as a potentially therapeutic target in malignant cancers.
Neural stem/progenitor cells are targets for natural killer cell-mediated killing (169.40)
Authors: Phillips Lori , Gould Elizabeth , Palmer Theo , Krams Sheri , Martinez Olivia
Published: January 2023

Neural Progenitor Cells (NPC) are multipotent progenitors that can differentiate into neurons. NPC in the hippocampus undergo neurogenesis throughout adult life, but the regenerative capacity of this endogenous pool appears inadequate in a variety of CNS diseases. Transplantation of NPC may repopulate or repair damaged areas of the CNS, however, the immune response to NPC allografts is not well characterized. Primary murine NPC display low levels of MHC class I and no class II as determined by flow cytometry. NPC also express intermediate levels of RAE-1, a ligand for the NK cell activation receptor NKG2D. Primary NK cells isolated from BALB/c (H-2d) or C57BL/6 (H-2b) mice can lyse both syngeneic and allogeneic NPC. NK cell killing is significantly inhibited when MHC class I is upregulated on syngeneic NPC targets following IFN-γ exposure (64% and 47% less killing by BALB/c and C57BL/6 NK cell effectors, respectively.) Additionally, NKG2D-/- NK cell effectors are significantly diminished in their ability to target NPC (56% and 68% less killing for matched and mismatched targets, respectively.) This indicates that MHC class I and the NKG2D ligand, RAE-1, play critical roles in NK cell recognition of NPC and suggests that host NK cells that traffic to the NPC graft site after transplantation can recognize and kill transplanted NPC. Thus, strategies to deplete NK cells or decrease NKG2D ligand expression on NPC grafts may lead to improved graft survival and differentiation.
CD11cHI macrophages are specialized APCs in the early human placenta (169.45)
Authors: Houser Brandy , Guerin Leigh , Wu Vernon , DeJong Annemiek , Moody D. Branch , Strominger Jack
Published: January 2023

The human maternal-fetal interface is a unique environment by which there is a necessary establishment of maternal tolerance to fetal antigens as well as uterine spiral artery remodeling and placental growth. Decidual macrophages (dMΦs) comprise approximately 20% of all leukocytes at this interface. Previously we identified two distinct populations based upon CD11c and have been termed, CD11cHI and CD11cLO MΦs (J Immunol. 2011; 186(4)). Based upon microarray analysis, CD11cHI and CD11cLO dMΦs were shown to have significantly divergent functions. Using a DQ-BSA assay we demonstrate that CD11cHI cells process exogenous protein antigens efficiently while processing by CD11cLO cells is very low. Furthermore, several CD1 molecules are expressed on the surface of CD11cHI dMΦs including CD1a, CD1c and CD1d. CD1 molecules are non-polymorphic glycolipid-presenting molecules that have no known function at the maternal-fetal interface. In order to determine functional relevance, we utilized established CD1-specific T cell clones and known ligands for CD1a and CD1c [CD8.2, dideoxymycobactin (DDM); CD8.1, mannosyl-β1-phosphomycoketide (MPM)] respectively. CD11cHI dMΦs, but not CD11cLO dMΦs, were found to specifically stimulate these T cell clones. Thus, these data suggest that the CD11cHI dMΦs may function in two different antigen-presenting systems at the maternal-fetal interface during early human pregnancy, peptide presentation and glycolipid presentation.