bluuebunny/crossref_metadata_2025 huggingface dataset:
Human influenza-specific effector and memory CD8 T cells from older adults show signs of terminal differentiation and senescence (104.3)
Authors: Wagar Lisa , McElhaney Janet , Watts Tania
Published: January 2023

Little is known about normal human aging and its effects on the function of T cells specific for acutely infecting pathogens. To address this issue, we conducted a multicolour flow cytometry study of older persons’ responses to influenza virus challenge ex vivo. Young and elderly donors were recruited in the fall of 2008. PBMCs were stained directly ex vivo or stimulated with A/Puerto Rico/8/34 (PR8) for 18 hours and assessed for functional markers. The data show that most donors have pre-existing influenza-specific T cells (measured by IFNγ) that are reactivated by PR8, although older donors had smaller pre-vaccination populations of flu-responsive T cells. Effector cells identified upon restimulation with influenza displayed a more terminally differentiated phenotype in older compared to younger adults. This phenotype did not change post-vaccination with trivalent inactivated vaccine, which lacks significant internal viral proteins and is a poor inducer of CD8 T cell responses. For HLA-A2+ donors, it was determined by tetramer staining that elderly influenza-specific CD8 T cells express higher levels of senescence markers KLRG1 and CD57 compared to young controls. Our study shows that influenza specific effector and memory T cells from older individuals show signs of terminal differentiation and senescence. Thus, as has been shown for persistent life-long infections, memory CD8 T cells to an acutely infecting pathogen show signs of deterioration with age.
Identification of a genomic region within MCMV critical for the export of m4/Kb complexes (105.17)
Authors: O'Neill Gabriela , Lu Xiuju , Hill Ann
Published: January 2023

The murine cytomegalovirus (MCMV) protein m4/gp34 binds to major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER), and this stable m4/gp34-MHC class I complex is then exported to the cell surface. m4/gp34 can impact antigen presentation to CD8 T cells either positively or negatively. However, its main role may be to interact with natural killer (NK) cells. Recent evidence indicates that m4/gp34 in combination with the MHC class I molecule H-2Dk stimulates the activating NK receptor Ly49P. m4/gp34 is retained in the ER unless it binds to MHC class I. The goal of this study was to understand the molecular basis for export of the m4/gp34-MHC class I complex. We found that when m4/gp34 was expressed in a gutless adenovirus vector, it readily formed complexes with MHC class I. However, the complexes were retained in the ER. Superinfection with MCMV lacking m4 restored the export of m4/gp34-Kb complexes. Screening a panel of virus mutants revealed that a region of the MCMV genome containing open reading frames (ORFs) m166-m170 was needed for efficient export of m4/gp-34-Kb complexes. We will present data describing the impact of individual ORFs within this region on the export of m4/gp34-Kb complexes and on the biological function of m4/gp34.
Transition from inflammatory to anti-inflammatory immune responses in deer mice infected with Sin Nombre virus (105.20)
Authors: Schountz Tony , Acuna-Retamar Mariana , Torres Perez Fernando , Kedmi Shira , Prescott Joseph , Peters Staci , Hjelle Brian
Published: January 2023

Hantavirus cardiopulmonary syndrome (HCPS) is characterized by pronounced inflammation that is thought to contribute to its pathogenesis. Deer mice (Peromyscus maniculatus) are the principal reservoir hosts of Sin Nombre virus (SNV), the etiologic agent of the great majority of HCPS cases in North America. Deer mice have life-long apathogenic persistence with periods of viral shedding in excrement. Virus-specific CD4+ T cells from deer mice possess features of regulatory T cells at persistence. We examined the post-infection kinetics of viral load and immune gene expression in deer mice on days 2, 5, 7, 10, 15 and 20. Viral RNA peaked on day 10 in the lungs and day 15 in the heart, with decreased RNA correlating with the appearance of IgG on day 15. In spleens, Ccl3 and Ccl5 expression was elevated on day 2, while Ccl2 expression was elevated on days 7-15. Several inflammatory cytokine genes were elevated on days 5 and 7, including IL-12p35, IL-21 and IL-23, but then declined. TGFβ1 expression occurred early in infection, but markedly declined and reappeared on days 15 and 20. CD4 expression was elevated on day 7 while CD8 and TCRβ expression were elevated on days 7 and 10, perhaps reflecting a stalled antiviral T cell response. These data suggest that early in infection an inflammatory immune response occurs, but it transitions to an anti-inflammatory response. This switch may interfere with viral clearance and contribute to the persistence and ecology of SNV.
Identification of H-2d-restricted, NS3-specific CD4 and CD8 T-cell epitopes in Japanese encephalitis virus infected mice (105.25)
Authors: Pan Chien-Hsiung , Wei Wen-Min , Wu Sze-Hsien , Hu Hui-mei
Published: January 2023

Japanese encephalitis virus (JEV) is the agent to cause the disorder of central nervous system (CNS) or even death. Neutralizing antibodies is believed to play a major role to prevent JEV entry to CNS. The importance of T-cell responses in the control of JEV infection is not clear. In addition, the presence of heterologous suboptimal neutralizing antibodies among between JEV and other flaviviuses may enhance viral infection. A peptide vaccine constituted with both CD4 and CD8 T cell epitopes to elicit a Th1-type response can provide an alternative solution to reduce the impact of heterologous suboptimal neutralizing antibodies. Total 40 and 31 of MHC-I and II peptides were predicted and used in these study. Spleen cells harvested from NS3 DNA vaccinated or JEV-infected BALB/c mice were stimulated and the IFN-gamma production was detected by ELISPOT assay. The mixture of Kd and Ld-restricted peptides showed the stronger ability to stimulate IFN-gamma production than Dd. Four individual MHC-I restricted peptides were identified. To prove the peptide is CD4 or CD8-dependent, different cell populations were depleted by magnetic beads before performing ELISPOT assay, and all 4 peptides showed a CD8-dependent pattern. For MHC-II, a similar strategy was used and three IFN-gamma stimulating Ad-restricted peptides were mapped. A peptide based JEV vaccine based on this study will be used to test the immunogenicity and protection in a mouse model.
Fc gamma receptors are important for protection from Cytomegalovirus infection (105.28)
Authors: Bootz Anna , Winkler Thomas , Mach Michael
Published: January 2023

Cytomegalovirus is a clinically important pathogen. Previously we could show using murine cytomegalovirus (MCMV) that the adoptive transfer of memory B lymphocytes from immune donors into B- and T-cell immunodeficient RAG-/- mice completely protected from MCMV-induced morbidity and mortality. The protection is correlated with production of protective antibodies. Antibodies can act by neutralization of extracellular virus and/or antibody-dependent cellular cytotoxicity (ADCC) via Fc receptor-mediated effector functions. To investigate the role of Fc receptors for protection we adoptively transferred immune serum from infected donor mice into recipients that were deficient for the expression of Fcγ-receptors. First we used mice lacking the γ-chain (FcRγxRAG-/-), which is the accessory chain of the activating murine IgG receptors FcγRI, FcγRIII, and FcγRIV. FcRγxRAG-/- mice developed severe disease with high viral titers and had a significantly shorter survival compared to the RAG-/- control animals. Essentially no difference was seen between mice lacking the FcγRI and RAG-/- animals. Mice lacking the FcγRIII receptor were significantly less susceptible to MCMV infection than FcRγxRAG-/-, indicating that NK cells were not the major effector cell type involved, since FcγRIII is the only activating Fc-receptor on NK cells. Thus, although Fcγ-receptors are essential for antibody mediated protection from MCMV, NK cells seem not to be the crucial cell type involved.
Enhancing MCMV-specific T cell responses by blocking viral replication (105.44)
Authors: Loo Christopher , Snyder Christopher , Hill Ann
Published: January 2023

Cytomegalovirus (CMV) is a β-herpes virus that is benign in the immunocompetent but can be pathogenic and fatal in the immunocompromised host. Our laboratory investigates murine cytomegalovirus (MCMV) as a model for human CMV. A hallmark of both human and murine CMV infection is the development of a strong cytolytic T cell response (CTL) in the acute phase and a large memory T cell pool in the chronic phase. How the virus initiates both acute and chronic responses in the host are not completely understood. In this study, we investigate the acute CD8 T cell response in the spleen when viral replication is blocked early after infection. Our laboratory has developed a recombinant MCMV that includes the thymidine kinase (TK) gene from HSV-1. In vivo, replication of MCMV-TK can be blocked when mice are administered the drug, Famcyclovir (FVR). Our results show MCMV-TK with FVR, is replication-deficient by undetectable plaque forming units in the spleen at day 3 post-infection. Measurement of peripheral inflammatory cytokines and chemokines are also decreased when viral replication is blocked. However, MCMV-TK infected mice, with FVR treatment, have a greater CTL responses at day seven post-infection. Our goal in this project is to identify the key biological mechanism responsible for this enhanced CD8 T cells response, and contribute to the field of optimal strategies for vaccine development.
FoxP3+ CD4+ cells promote CD8 T cell exhaustion during chronic infection (105.5)
Authors: Penaloza-MacMaster Pablo , Araki Koichi , Kamphorst Alice , Iyer Smita , West Erin , Konieczny Bogna , Freeman Gordon , Rudenski Alexander , Ahmed Rafi
Published: January 2023

Understanding the factors that guide T cell exhaustion may provide opportunities to modulate immune responses during chronic infections. We analyzed the contribution of CD4+ FoxP3+ cells (Tregs), to the process of CD8 T cell exhaustion during chronic lymphocytic choriomeningitis virus (LCMV) infection. Tregs have been shown be the master regulators of immune tolerance. However, a possible suppressive role for these cells during chronic infections is still not very well defined, and most studies show correlations, but not direct causations. Normally, LCMV chronically viremic mice experience a pronounced CD8 T cell exhaustion. We showed for the first time that specific ablation of Tregs (in FoxP3DTR mice) after the onset of a chronic viral infection results in a significant rescue of viral-specific CD8 T cell responses. In addition, PD-1 blockade together with Treg depletion resulted in significant reduction of viral loads, greater than PD-L1 blockade alone. This suggests that Tregs and the PD- pathway control CD8 T cell exhaustion by different mechanisms. The CD8 T cell rescue observed after Treg depletion is dependent on B7 signaling, CD4+ cells, and the presence of viral antigen, suggesting a general role for these factors in maintaining CD8 T cell exhaustion during persistent infections. Our data also indicates a general mechanism that may be used by Tregs to suppress self-specific T cell responses during immune homeostasis.
A cationic lipid/DNA adjuvant enhances the efficacy of an inactivated influenza vaccine against matched and heterosubtypic virus challenge in rhesus macaques. (106.15)
Authors: Carroll Timothy , Matzinger Shannon , Lay Marla , Chang Stella , Fairman Jeffery , Miller Christopher
Published: January 2023

The efficacy of current influenza vaccines depends on eliciting type-specific neutralizing antibodies that do not protect against a range of influenza virus subtypes. To determine if the adjuvant JVRS-100 can enhance vaccine-induced protection against matched and heterosubtyic virus challenge, monkeys were immunized with A/Memphis/7/01 (ΔH1N1) or A/Memphis/1/90 (ΔH3N2); with or without JVRS-100 and challenged with a human seasonal influenza isolate, A/Memphis/7/01. Animals immunized with adjuvant had significantly greater anti-influenza antibody titers than the un-adjuvanted groups (p<0.01). Indeed, ΔH1N1+JVRS100 animals had significantly higher H1 neutralizing titers compared to the un-adjuvanted animals (p<0.001). While only ΔH1N1+JVRS-100 animals had significantly lower levels of virus replication (p<0.01) compared to unimmunized control animals after virus challenge, the ΔH3N2+JVRS-100 animals tended to have less virus replication compared to unimmunized controls. Peak influenza virus replication levels inversely correlated with pre-challenge plasma anti-influenza IgG and H1-neutralizing antibody titers. These results demonstrate that JVRS-100 enhances both matched and heterosubtypic immunity in rhesus monkeys.
Murine dialyzed spleen extract decrease the lesion size of experimental localized cutaneous leishmaniasis (106.25)
Authors: Yam Puc Juan Carlos , Becerril-García Miguel Angel , Estrada-García Iris , Estrada-Parra Sergio , Dumonteil Eric , Pérez-Tapia Sonia Mayra , Limón-Flores Alberto Yairh
Published: January 2023

Leishmaniasis affects 12 millions people in the world and localized cutaneous leishmaniasis (LCL) prevails in México. Drugs used to treat leishmaniasis are not effective and they present side effects. Dialyzable Spleen Extracts (DSE) are a mixture of molecules with the with the ability to regulate immune responses. in this work we determine the effect of DSE administration on a experimental leishmaniasis. First an experimental model for LCL was established in BALB/c mice using intradermal immunization of Leishmania mexicana. In this model lesions show a progressive pattern, with inflammation, follow by ulceration and necrosis. Once LCL was established, different DSE concentrations were used to treat it. Intramuscular administration of 0.1pg of DSE daily for 7 days followed by every third day until 45 day, showed a reduction in lesion size, parasite counts and number of infected macrophages. Combined treatment of DSE with tamoxifen (TAM) had a synergistic effect, decreasing further lesion size than DSE or TAM alone. This experimental model has show to be reproducible, and may be used to assess other anti-leishmania drugs, and it also offers a model to describe the immunological mechanisms underlying DSE effects.
A cationic lipid/DNA adjuvant enhances the effectiveness of Fluzone® in immunosenescent elderly rhesus macaques. (106.30)
Authors: Carroll Timothy , Matzinger Shannon , Genesca Meritxell , Lay Marla , Chang Stella , Fairman Jeffery , Miller Christopher
Published: January 2023

The decline in immunity in older adult humans results in a reduction of influenza vaccine efficacy. Elderly macaques (> 18 years old) have similar declines in immunity as indicated by significantly lower levels of circulating naïve CD4+ T cells, naïve CD8+ T cells and B cells and declines in pDC function compared to juvenile monkeys. To determine if the adjuvant activity of JVRS-100 can improve the efficacy of the inactivated influenza vaccine, Fluzone®, elderly monkeys were immunized with Fluzone® with or without JVRS-100 and challenged with a human seasonal influenza isolate, A/Memphis/7/2001 (H1N1). On the day of challenge, Fluzone®+JVRS-100 immunized animals had significantly higher plasma anti-influenza IgG (p<0.04) and IgA (p<0.01) titers than the un-adjuvanted immunized monkeys. After virus challenge, only the Fluzone®+JVRS-100 immunized animals had a significantly lower level of virus replication (p<0.05) relative to the unimmunized control animals. There were significant inverse correlations between peak influenza virus replication levels in the respiratory tract and plasma and tracheal anti-influenza IgG and IgA antibody titers prior to challenge. These results demonstrate that JVRS-100 enhances the immunogenicity and efficacy of a licensed inactivated influenza vaccine in an immunosenescent elderly monkey population.
Immunotherapy with leishmanial antigen versus immunochemotherapy in murine visceral leishmaniasis (106.7)
Authors: Afrin Farhat , Chouhan Garima , Islamuddin Mohammad , Priya Yadav , Ahmad Farnaz , Want Muzamil
Published: January 2023

Despite significant advances in the treatment of visceral leishmaniasis (VL), conventional chemotherapy (CT) has considerable toxicities and relapses. Immunotherapeutic strategies, aimed to amplify Leishmania-specific immune response prior to or in synergy with CT may lower the required dose or duration of treatment and hence toxicity, improving chemotherapy's overall efficacy, reduce emergence of drug resistant strains, and circumvent the problems of treatment in immunocompromised hosts. Effective antileishmanial intervention blending antigen-based therapy may result in therapeutic harness of cellular immune protection. In the present work, we compared the therapeutic efficacy of leishmanial antigen (freeze-thawed, FT and autoclaved, ALD) when administered alone (immunotherapy, IT) or in combination with pentamidine (immunochemotherapy, ICT) in Leishmania donovani infected mice. FT vaccine administered with pentamidine cleared the splenic and hepatic parasite burden, eliciting Leishmania-specific delayed-type hypersensitivity (DTH) responses and elevated IgG2a levels in contrast to chemo- or immuno-therapy alone. Gamma-IFN and interleukin-4 expression in liver of treated mice and hepatic granuloma formation with associated immune cell recruitment as correlates of cell-mediated immunity are being explored. Our findings underscore the chemo-immunotherapeutic potential of FT antigen that may be extended to drug-nonresponsive VL patients and warrant further investigation.
Distinctive innate immune signatures at the injection site predict the adjuvant-mediated boost of adaptive responses towards a flu subunit vaccine. (106.9)
Authors: Cortese Mario , Caproni Elena , Tritto Elaine , Muzzi Alessandro , Monaci Elisabetta , Baudner Barbara , Seubert Anja , O'Hagan Derek , De Gregorio Ennio
Published: January 2023

Vaccine adjuvants activate specific innate immune pathways which boost the adaptive responses to co-administered antigens, but the efficacy of adjuvants is often antigen-dependent. Recently, several human studies have shown that oil-in-water emulsions such as MF59 are superior to alum in enhancing the response to flu subunit vaccines. However, the mechanism of action of flu adjuvanticity is not fully understood. In this study we wanted to identify the innate immune signatures that are associated to adjuvanticity to flu subunit vaccine in the mouse model. First, we immunized mice with seasonal trivalent flu antigens or tetanus toxoid (TT) mixed with MF59, alum or three TLR-dependent adjuvants: CpG (TLR9), resiquimod (TLR7/8) and Pam3CSK4 (TLR2). All adjuvants were similarly efficient in inducing an adaptive immune response to TT. By contrast, MF59 and, to a lesser extent, Pam3CSK4 enhanced the antibody responses to HA. In order to correlate innate immune gene signatures induced by the vaccine adjuvants with the ability to boost flu adaptive responses, we performed microarray analysis with the same set of adjuvants both in vitro in mouse splenocytes and in vivo in mouse muscle and draining lymph nodes. In summary, we show that strong activation of innate immunity in the muscle, but not in draining LNs, and local recruitment of CD11b+ blood cells at injection site by MF59 and TLR2 vaccine adjuvants correlate with their ability to enhance antibody responses to flu antigens.
Rituximab treatment reduces organ-specific T cell effector responses and ameliorates Experimental Autoimmune Encephalomyelitis (107.3)
Authors: Monson Nancy , Cravens Petra , Hussain Rehana , Harp Christopher , Cummings Matthew , de Pilar Martin Maria , Lyons Jeri-Anne , Lovette-Racke Amy , Cross Anne , Racke Michael , Stuve Olaf , Shlomchik Mark , Eagar Todd
Published: January 2023

Recent clinical trials have established B cell depletion by the anti-CD20 chimeric antibody rituximab as a beneficial therapy for patients with relapsing-remitting multiple sclerosis (MS). However, the impact of rituximab on T cell responses remains largely unexplored. In the experimental autoimmune encephalomyelitis (EAE) model of MS in mice that express human CD20, rituximab administration rapidly depleted peripheral B cells. EAE severity was reduced in a therapeutic model and EAE symptoms were not observed in a preventive model. B cell depletion was also associated with diminished Delayed Type Hypersensitivity (DTH) and a reduction in T cell proliferation and IL-17 production during recall immune response experiments. While rituximab is not considered a broad immunosuppressant, our results indicate a role for B cells as a therapeutic cellular target in regulating encephalitogenic T cell responses in specific tissues.
Synergy between TLR3 and IL-18 promotes IFN-y dependent TRAIL expression in human liver NK cells (108.6)
Authors: Orloff Mark , Tu Zhengkun , Hamalainen-Laanaya Heli , Crispe Nicholas
Published: January 2023

Toll-like receptor 3 (TLR3), which recognizes double-stranded RNA (dsRNA), was the first identified antiviral TLR, and because dsRNA is a universal viral molecular pattern, TLR3 has been assumed to have a central role in the host response to viruses. Natural killer (NK) cells are a component of innate immunity against viral infections through their rapid cytotoxic activity and cytokine production. The synthetic dsRNA polyinosinic-polycytidylic acid (poly I:C), a mimic of a common product of viral infections, is known to rapidly up-regulate their in vivo functions; however, intrahepatic NK cells’ ability to respond to dsRNA is still mostly unknown. Our results show that poly I:C activates liver NK cells directly in the context of cytokines found in the liver, i.e: poly I:C plus inflammatory cytokines (IL-18, IL-12, and IL-2) induced NK cell IFN-g production and TRAIL expression, and anti-inflammatory cytokines (TGF-β and IL-10) inhibit NK cell IFN-γ production. Neutralization of IFN-g blocks poly I:C plus inflammatory cytokines-induced NK cell TRAIL expression, suggesting that IFN-g is an autocrine differentiation factor for these cells. A better understanding of the intrahepatic NK cell activation against viral infection may help in the design of therapies and vaccines for the control of viral hepatitis.
CpG ODN induced NK cell IFNγ production is positively regulated by IL-12 producing neutrophils and suppressed by IL-10 producing B cells (108.8)
Authors: Guillon Laura , Haddad Evette , Chow Mindy , Cohen Carla , Takei Fumio
Published: January 2023

CpG-containing oligodeoxynucleotides (CpG ODN) stimulates spleen cells in vitro and induces IFNγ secretion mostly from NK cells, whereas purified NK cells are not stimulated by CpG ODN. IL-12 neutralization inhibits NK cell IFNγ production, and IL-12 knockout mouse spleen cells stimulated with CpG ODN produce very little IFNγ, indicating that IL-12 is required. Depletion of macrophages and DC has no effect, whereas neutrophil depletion dramatically decreases both IL-12 and IFNγ production by spleen cells, indicating that neutrophils are a key source of IL-12. NK cells in B cell-deficient mouse spleen secrete higher levels of IFNγ than wild type spleen cells, and B cell depletion enhances NK cell IFNγ production, indicating that B cells are suppressive. CD5+ B cells secrete IL-10 in response to CpG ODN. The neutralization of IL-10 increases IFNγ and IL-12 production by CpG ODN-stimulated spleen cells, and B cell-deficient splenocytes produce more IL-12 than WT splenocytes. Furthermore, exogenously added IL-10 effectively inhibits CpG ODN-induced IL-12 production by purified neutrophils, and IL-12/IL-18 induced stimulation of purified NK cells. Therefore, CD5+ B cells produce IL-10 and suppress CpG ODN-mediated NK cell IFNγ production by inhibiting neutrophil IL-12 production as well as by directly acting on activated NK cells themselves.
An activating mutation in CD45 generates a novel murine model of hemophagocytic disorders in the context of a MHC Class 1 restricted TCR. (109.11)
Authors: Tibbetts Rachelle , Lam Viola , Cresalia Nicole , DeGraaf Fleur , Goren Nira , Wong Lawrence , Hai Si-Han , Mills Robyn , Roque Alfonso , Arthur Weiss , Hermiston Michelle
Published: January 2023

Hemophagocytic syndromes encompass a spectrum of life-threatening disorders characterized by severe systemic inflammation and mechanistically are not well understood. We inadvertently generated a novel mouse model that shares many characteristics of these hyperinflammatory conditions. Expression of a single activating point mutation (E613R) in the receptor protein tyrosine phosphatase CD45 results in a lupus-like phenotype on a mixed C57Bl/6 (B6)-129/Sv genetic background. However, CD45E613R mice on an inbred B6 background show no stigmata of disease despite inappropriate regulation of signals emanating from CD45’s substrates, the src family kinases. Surprisingly, introduction of a MHC class I restricted T cell receptor specific for ovalbumin (OT1) into B6 CD45E613R mice results in a severe hyperinflammatory disease reminiscent of hemophagocytic syndromes. Disease is maintained in RAG1 deficient CD45E613R/OTI mice implicating myeloid and/or CD8 T cells in disease pathogenesis. Using an adoptive transfer approach, we find CD45E613R/OT1 CD8 T cells are necessary and sufficient for this phenotype. Further analysis of CD45E613R/OT1 T cells indicates that they are inappropriately activated, secrete excessive levels of interferon gamma, and have defective cytolytic function. These intriguing results support further analysis of the role of src family kinase mediated signaling networks in hemophagocytic disorders.
VDUP1 inhibits NF-κB activity via association with HDAC1 and HDAC3 (109.14)
Authors: Park Young-Jun , Suh Hyun-Woo , Jung Haiyoung , Yoon Suk-Ran , Choi Inpyo
Published: January 2023

NF-κB subunits are important regulators of apoptosis or cellular responses triggered by tumor necrosis factor TNF-α. Termination of nuclear NF-κB action is regulated by histone deacetylase (HDAC) corepressor proteins. However, the molecular mechanism that leads to the termination is not fully understood. Here, we examined the functions of VDUP1, a negative regulator of thioredoxin, in TNF-α-mediated NF-κB activation. VDUP1 level was markedly reduced by TNF-α stimulation, concomitant with elevated NF-κB activation. Furthermore, Enforced VDUP1 overexpression suppressed strongly the increased NF-κB activity. In addition, the overexpresion of VDUP1 resulted in the robust suppression of TNF-α-activated NF-κB activity via association with HDAC1 or HDAC3. Collectively, we identified VDUP1 as a new component of the corepressors by demonstrating its ability to repress NF-κB activation, during which HDAC1 and HDAC3 interact with NF-κB p65 to suppress the NF-κB activity
Selenoprotein K knockout mice exhibit deficient calcium flux in immune cells and impaired immune responses (109.23)
Authors: Hoffmann Peter , Huang Zhi , Hoffmann FuKun , Hashimoto Ann , Verma Saguna , Kumar Mukesh , Roe Kelsey
Published: January 2023

Selenoprotein K (Sel K) is a selenium-containing protein for which no function has been identified. We found that Sel K is an endoplasmic reticulum (ER) transmembrane protein expressed at relatively high levels in immune cells and is regulated by dietary selenium. Sel K-/- mice were generated and found to be similar to WT controls regarding growth and fertility. Immune system development was not affected by Sel K deletion, but specific immune cell defects were found in Sel K-/- mice. Receptor-mediated Ca2+ flux was decreased in T cells, neutrophils, and macrophages from Sel K-/- mice compare to controls. Ca2+-dependent functions including T cell proliferation, T cell and neutrophil migration, and Fcγ-receptor-mediated oxidative burst in macrophages were decreased in cells from Sel K-/- mice compared to controls. West Nile virus (WNV) infections were performed and Sel K-/- mice exhibited decreased viral clearance in the periphery and increased viral titers in brain. Furthermore, WNV-infected Sel K-/- mice demonstrated significantly lower survival (2/23; 8.7%) compared to WT controls (10/26; 38.5%). These results establish Sel K as an ER-membrane protein important for promoting effective Ca2+ flux during immune cell activation and provide insight into molecular mechanisms by which dietary selenium enhances immune responses.
MicroRNA regulation of helper T cell cytokine production and proliferation (109.29)
Authors: Steiner David , Thomas Molly , Blelloch Robert , Ansel K.
Published: January 2023

MicroRNAs have been implicated as important regulators of many aspects of immune cell function. We and others have previously shown that deletion of the microRNA biogenesis enzymes Dicer and Drosha from T cells results in abnormal cytokine production and decreased proliferation of CD4+ T cells ex vivo. However, the role of individual miRNAs in this phenotype remains poorly understood. We conducted a screen for miRNA function in primary T cells and report the identification of individual miRNAs that can independently rescue the defects of miRNA-deficient helper T cells. Multiple members of the miR-17 and miR-92 families rescued proliferation while miR-29 rescued the aberrant IFN-γ expression by these cells. Repression of IFN-γ by miR-29 involved the regulation of both T-bet and Eomes, two transcription factors known to induce IFN-γ production. These results represent a novel screening strategy for miRNA function in primary T cells and further demonstrate the impressive regulatory capability of individual miRNAs in the immune system.
Tyrosine phosphorylation of 3BP2 regulates BCR-mediated activation of NFAT. (109.3)
Authors: Sada Kiyonao , Nakashima Kenji , Ogi Kazuhiro , Chihara Kazuyasu
Published: January 2023

Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2, also referred to SH3BP2) regulates immune receptor-mediated signal transduction. In this report, we focused on the molecular mechanism of 3BP2 function in B cell receptor (BCR) signaling. Engagement of BCR induces tyrosine phosphorylation of 3BP2. Genetic analysis demonstrated that Syk is critical for BCR-mediated tyrosine phosphorylation of 3BP2. Mutational analysis of 3BP2 revealed that both Tyr183 and Src homology 2 (SH2) domain are necessary for 3BP2-mediated BCR-induced activation of nuclear factor of activated T cells (NFAT). Point mutation of Tyr183 or Arg486 in the SH2 domain of 3BP2 diminished BCR-mediated tyrosine phosphorylation of 3BP2. Endogenous 3BP2 forms complex with tyrosine phosphorylated cellular signaling molecules. Peptide binding experiments demonstrated that only phosphorylated Tyr183 in 3BP2 could form a complex with the SH2 domain(s) of phospholipase C (PLC)-γ2 and Vav1 from B cell lysates. These interactions were represented by using bacterial GST-PLC-γ2 or -Vav1 SH2 domain(s). Furthermore, pull down and far-western experiments showed that 3BP2-SH2 domain directly binds to B cell linker protein (BLNK) following BCR stimulation. These results demonstrated that 3BP2 induces protein complex with cellular signaling molecules through phosphorylation of Tyr183 and SH2 domain leading to the activation of NFAT in B cells.
Cytoskeletal and microtubule machineries in concert with integrins are required for the secretion of eosinophil-associated RNases (110.13)
Authors: Shamri Revital , Young Kristen , Melo Rossana , Ghiran Ionita , Weller Peter
Published: January 2023

Eosinophils, both as end-stage effector cells and as collaborative interacting components of innate immune responses, have the capacity to secrete varied cytotoxic proteins that are stored in their cytoplasmic secretory granules, termed specific granules. Among them are the eosinophil-associated RNases (EARs): the human eosinophil-derived neurotoxin and eosinophilic cationic protein. The mouse EAR family includes at least 13 members, six of which were found to be present in specific granules. In the last decade several studies, both in vitro and in vivo, have revealed a role for eosinophils and their EARs in anti-viral defense. However, the secretion mechanism of EARs is not fully understood, especially in mouse eosinophils. Therefore, our study aimed to understand the mechanism of EAR secretion following physiological stimulation with chemokines. We found that dynamic polymerization of the actin cytoskeleton and microtubules, as well as functional integrins were critical for CCR3-mediated secretion of EARs. However, inhibition of regulatory components of the cytoskeletal machinery, such as Rac1 and ROCK-I and -II, increased EARs secretion in mouse and human eosinophils, suggesting a negative regulation of these effectors on EARs secretion. Collectively, these data suggest a mechanism by which EARs are secreted from eosinophils under physiological stimulation, findings that are pertinent to host defense against RNA viruses and eosinophil-associated diseases.
The Structure and Function of Acute Phase Proteins: C-Reactive Protein and Serum Amyloid A in Innate Immunity (110.21)
Authors: Lu Jinghua , Marjon Kristopher , Marnell Lorraine , Mold Carolyn , Du Clos Terry , Sun Peter
Published: January 2023

During an acute infection, serum levels of certain proteins will be increased dramatically at the early stage of the infection, which are so-called acute phase proteins (APPs). Among them, C-reactive protein (CRP) and Serum Amyloid A (SAA) are two major APPs in humans and therefore they have long been used as clinical biomarkers to monitor the inflammation and the outcome of therapy. However, the physiological function of CRP and SAA remains to be better characterized. In our study, we have shown that CRP can functionally activate Fc receptors pathways, particularly FcγRs and FcαRI (CD89), resulting in the phagocytosis of microbial pathogens and cytokine secretion, such as IL6, and IL8. The ability of CRP to activate FcγRs and FcαRI defines a novel function for acute phase proteins in inflammatory responses involving neutrophils and macrophages and also highlights the innate aspect of otherwise humoral immunity-associated antibody receptors. Furthermore, SAA was shown to be able to bind Gram-positive bacteria. We have now determined the X-ray structure of SAA at 2.7Å resolution. The natural hexameric SAA structure revealed a dimeric architecture arranged as two trimers. We also discovered that SAA hexamer could undergo a transition to monomers that are able to bind and activate Toll-like receptor 2 and 4. Taken together, our studies highlighted CRP and SAA as the powerful first lines of host immune defense during acute infection.
Systemic decrease in murine collecting vessel lymphatic function during initial stages of inflammation (110.5)
Authors: Aldrich Melissa , Hall Mary , Angermiller Bonnie , Robinson Holly , Sevick-Muraca Eva
Published: January 2023

The effects of inflammation on the lymphatic system are largely unknown, with conflicting reports of enhanced or decreased activity. We imaged murine lymphatic vessels and function using a custom-designed ICCD camera/low-level laser system and an intradermally injected fluorescent dye, indocyanine green, which is rapidly taken up by the lymphatic vasculature. Mice injected with LPS exhibited a drastic, systemic decrease in collecting vessel lymphatic propulsive flow velocity and frequency at 4-24 hours after the inflammatory insult, gradually increasing to normal values by 7 days post-inflammation. IL-6, TNF-alpha, MCP-1, MCP-5, RANTES, and IL-10 levels were increased in plasma at 4-24 hours. Draining lymph nodes retained ICG at 6-7 days post-inflammation, perhaps indicating restriction of afferent and/or efferent lymphatic vessels. This system for non-invasive imaging of the lymphatic system under inflammatory conditions may allow insights into mechanisms of inflammation and its resolution.
Circadian rhythms influence disease severity in a mouse model of sepsis (110.9)
Authors: Walker Wendy , Bozzi Aaron , Fikrig Erol
Published: January 2023

Septic patients have a heightened risk of mortality between the hours of 2 and 6 am. However, the reasons for this are unknown. Circadian rhythms orchestrate many aspects of mammalian physiology, including inflammatory responses. We investigated how circadian rhythms affect the cecal ligation and puncture (CLP) mouse model of sepsis. We found that mice that underwent CLP had a worse sepsis phenotype when the surgery was performed at zeitgeber time (ZT) 19 (2 am) vs. ZT 7 (2 pm): mice exhibited earlier mortality, more severe hypothermia and worse disease score. This was associated with higher levels of serum proinflammatory cytokines and creatine kinase. These data indicate that in the CLP model, the timing of sepsis induction influences the severity of the inflammatory response and the morbidity and mortality that ensue. Our findings imply that it is important to take circadian rhythms into account when treating patients who are at risk of developing sepsis.
Role of BAT3 in modulating the immune response (111.12)
Authors: Grover Ajay , Izzo Angelo
Published: January 2023

HLA-B-associated transcript 3 (BAT3) also known as Scythe or BAG6, is a nuclear protein that has recently been implicated in the control of apoptosis and in NK-DC cell cross-talk. Here, we demonstrate that BAT3 modulates the immune response by regulating the function of macrophages and dendritic cells. BAT3 is secreted by macrophages and dendritic cells in vitro and extracellular BAT3 down-regulates the production of nitric oxide and proinflammatory cytokines in IFN-γ and LPS stimulated macrophages. Extracellular BAT3 also down-regulates the LPS-stimulated maturation of dendritic cells. The immunodominant Mycobacterium tuberculosis antigen ESAT-6 (early secreted antigenic target-6) up-regulates BAT3 expression in macrophages and dendritic cells in vitro. Intracellular BAT3 regulates ESAT-6 induced apoptosis of macrophages by interacting with the anti-apoptotic protein BCL-2 (B-cell lymphoma 2) indicating that BAT3 may play a role in pathogenesis of tuberculosis. Taken together, the data suggest that BAT3 may exert different immunomodulatory regulations depending on whether it is intra-or extracellular.
New primary immunodeficiency with severe paucity of neutrophil granules despite normal granule protein levels (111.17)
Authors: Ismail Deema , Akuete Kwei , Borregaard Niels , Hostoffer Robert , Tcheurekdjian Haig
Published: January 2023

Neutrophil-specific granule deficiency is a rare congenital disorder, which manifests with severe, recurrent infections and is characterized by the absence of neutrophil specific granules and eosinophil granules. We describe a pathologically distinct primary immunodeficiency characterized by the near absence of all neutrophil granules but sparing of eosinophil granules. The subject is an 8 year old male with infections since birth including recurrent abscesses, chronic cellulitis, recurrent sinopulmonary infections, and profound dental decay. Lymphocyte phenotyping, T-cell and B-cell function, and assessment of neutrophil oxidative burst were normal. Leukocytes appear normal on light microscopy, but neutrophils in peripheral blood and bone marrow examined by electron mocroscopy contained few granules of any kind. Mean (standard deviation) number of granules per neutrophil of subject as compared to control were 1.4 (1.1) and 72.6 (31.4), respectively (p=0.0014). Nonetheless, granule proteins in neutrophil cell lysate were present in amounts comparable to controls. Eosinophils were morphologically normal. This is the first description of an immune deficiency due to near absence of neutrophil granules despite normal granule protein levels. This indicates a defect in granule production as opposed to a defect in granule protein production, which has previously been identified in neutrophil-specific granule deficiency.
The pivotal role of glutathione reductase in host defense against Gram-negative bacteria (111.25)
Authors: Yan Jing , Meng Xiaomei , Wancket Lyn , Lintner Katherine , Wu Haiwa , Nelin Leif , Chen Bernadatte , Smith Charles , Rogers Lynette , Liu Yusen
Published: January 2023

Glutathione reductase (Gsr) is an enzyme that reduces glutathione disulfide to the sulfhydryl form, a major cellular antioxidant. Oxidative burst plays an important role in pathogen killing and initiation of inflammation. Proper redox regulation is crucial for balancing effective pathogen elimination and host preservation. We tested the hypothesis that Gsr plays a critical role in host defense by comparing wildtype (WT) and Gsr- mice after E. coli infection. Compared to WT mice, Gsr- mice exhibited substantially higher mortality, associated with greater bacterial burden, cytokine storm, and striking histological abnormalities. Surprisingly, Gsr- mice displayed increased resistance to LPS. While Gsr- mice exhibited defects in phagocyte mobilization, there appeared no substantial defects in either cell signaling or cytokine production in Gsr- macrophages. The role of Gsr in phagocytic oxidative burst was assessed by both flow cytometry and bioluminescence imaging. The oxidative burst in WT neutrophils after PMA stimulation was sustained for more than 30 min, while oxidative burst was very transient in Gsr- cells (<5 min). Likewise, oxidative burst after E. coli infection was also significantly weaker in the Gsr- neutrophils. Thus, Gsr plays a critical role in host defense against bacterial infection by regulating various immune functions, including facilitation of bacterial killing via sustaining oxidative burst in phagocytes, through maintenance of a delicate redox balance.
The role of α-enolase on the monocyte of rheumatoid arthritis via induction of inflammatory cytokines (112.14)
Authors: Bae Seyeon , Kim Hye Min , Kong Joo Myoung , Yu Yeon Sil , Kim Hang-Rae , Hwang Young Il , Kang Jae Seung , Lee Wang Jae
Published: January 2023

α-enolase (ENO1) is expressed on the surface of activated peripheral blood mononuclear cells. Although it plays an important role on the development of B cells in germinal center, there are some findings regarding the roles of ENO1 on the pathogenesis of cancer. Recently, we found that the production of inflammatory mediators from concanavalin A-activated PBMCs was increased by ligation of ENO1with anti-ENO1 mAb. Therefore, we investigate the roles of ENO1 on the regulation of inflammation and chronic inflammatory disease, rheumatoid arthritis (RA). First, we examined the expression of ENO1 on the surface of PBMCs, synovial fluid mononuclear cells (SFMCs) and synovial tissue (ST) derived from RA patients. The surface expression of ENO1 was higher on PBMCs and SFMCs derived from RA patients than those from osteoarthritis (OA) patients or normal samples. And it was mainly expressed on CD14-positive monocyte/macrophages. Interestingly, the expression of ENO1 on cells from RA patients is dependent of the severity of disease. The production of inflammatory mediators was dramatically increased by the stimulation of ENO1 via the activation of p38MAPK and NF-κB. Taken together, our results strongly suggest that ENO1 is a critical factor for the induction of severe inflammation in RA via the induction of disease-related pro-inflammatory cytokine production.
Inhibition of vascular endothelial growth factor receptor reverses type 1 diabetes in nonobese diabetic mice. (112.17)
Authors: Villalta Armando , Lang Jiena , Kabre Beniwende , Kubeck Sam , Arch Robert , Bluestone Jeff
Published: January 2023

Small molecule receptor tyrosine kinase inhibitors (RTKI) represent a novel therapeutic avenue for the treatment of type 1 diabetes (T1D). These potential therapies target multiple kinases raising questions as to the precise target for intervention. In this investigation, we used a pharmacological approach to test whether vascular endothelial growth factor receptors (VEGF-R) participate in the regulation of disease pathogenesis in T1D in nonobese diabetic (NOD) mice. Treatment of new-onset diabetic NOD mice with PF-337210, an RTKI that inhibits the VEGF-R and platelet-derived growth factor receptors (PDGF-R), reversed diabetes. However, PDGF-R selective inhibitors did not, suggesting that VEGF-R are the principal targets mediating diabetes reversal. RTKI treatment of diabetic mice reduced insulitis, preserved islet mass and improved glucose tolerance, but had no effect on insulin resistance. Immunohistochemical studies suggested that the RTKI targeted VEGF-R expressed by the islet vasculature and not by leukocytes, suggesting that these drugs have a predominant effect on inflammatory processes that contribute to the pathogenesis of T1D rather than a direct effect on diabetogenic leukocytes. Collectively, the results show the inhibition of VEGF-R2 signaling can reverse diabetes by dampening inflammatory events that lead to islet dysfunction, and support the use of VEGF-R specific antagonists as novel therapeutics for the treatment of T1D.
Activation of endothelial roundabout receptor 4 (Robo4) reduces the severity of virus induced keratitis (112.2)
Authors: Sharma Shalini , Mulik Sachin , Rouse Barry
Published: January 2023

Anti angiogenic molecules exert a feedback control to restrain pathological angiogenesis, which include physical binding or inhibition of angiogenic signaling in blood vessel endothelial cells. The latter is the case when Slit2 ligand dependent activation of the blood vessel endothelial cell receptor roundabout 4 (Robo4) occurs. In this study, we demonstrate that Robo4 receptors are up regulated following herpes simplex virus (HSV) infection of the eye on the majority of the new blood vessel endothelial cells that occur in the corneal stroma. However, expression levels of the ligand for Robo4 receptors, Slit2, was not significantly increased during the disease process and the knockdown of Slit2 gene expression using lentiviral shRNAs had no effect on the extent of pathological angiogenesis. In contrast, providing additional Slit2 protein by subconjunctival administration resulted in significantly reduced angiogenesis. The Slit2 binding to Robo4 was shown to block the downstream VEGF signaling molecules Arf 6 and Rac 1 and reduced the anti-apoptotic molecule Bcl-xL in blood vessel endothelial cells. Our results indicate that augmenting the host Robo4/Slit2 system could provide a useful therapeutic approach to control pathological angiogenesis associated with HSV induced stromal keratitis (SK).
Interaction of suppressor of cytokine signaling 2 with phosphorylated Pyk2 regulates human NK cell function (113.1)
Authors: Jung Haiyoung , Lee Suk Hyung , Park Young-Jun , Yoon Suk Ran , Choi Inpyo
Published: January 2023

Natural killer cells are large granular lymphocytes that play an important role in innate immunity, contributing to the elimination of tumor or virus-infected cells. Resting NK cells, however, have only minimal cytolytic activity and secrete a low level of IFN-γ. The cytokine IL-15 can promote the expression of effectors in NK cells. In this study, we demonstrate that suppressor of cytokine signaling 2(SOCS2) has a novel role in IL-15-primed human NK cell function. SOCS2 expression was upregulated in NK cells following stimulation with IL-15. During IL-15 mediated NK cell priming, SOCS2 interacted with phosphorylated proline-rich tyrosine kinase 2(Pyk2) at tyrosine 402(p-Pyk2Tyr402) and induced the proteasome-mediated degradation of p-Pyk2Tyr402 via ubiquitination. Knockdown of SOCS2 resulted in the accumulation of p-Pyk2Tyr402 and blocked NK cell effector functions. In addition, NK cell cytolytic activity and IFN-γ production were inhibited by over-expression of the wild-type of Pyk2 but not by the over-expression of tyrosine 402 mutant of Pyk2. These results suggest that SOCS2 regulates human NK cell effector functions via control of phosphorylated Pyk2 depending on IL-15 existence.