bluuebunny/crossref_metadata_2025 huggingface dataset:
Tissue resident memory T cells are found in the primary and metastatic tumors of breast cancer patients (TUM2P.922)
Authors: Egelston Colt , Lee Peter
Published: January 2023

Tumor tissues are composed of significant numbers of Tumor Infiltrating Lymphocytes (TILs), which may include CD8+ T cells, CD4+ T cells, and B cells. Of the T cell population, it is known that these cells are often functionally suppressed and defective in their ability to kill and proliferate. However, little is known about how T cells traffic into tumor sites. Furthermore, it is unclear how the various T cell memory subtypes play a role in attempting to eradicate tumor cells. Such knowledge is critical for the development of successful cancer immunotherapies. Here we assayed peripheral blood mononuclear cells (PBMCs), tumor tissue, and sentinel lymph nodes (SLNs) of breast cancer patients to phenotype memory T cells. We found higher proportions of EM cells among CD8 TILs as compared to SLN CD8 T cells. Conversely, the CD4 compartment in both TILs and SLNs was dominated by CM CD4 T cells. Further analysis showed that breast cancer patient CD8+ TILs in primary tumors were made up of a significant population of Tissue Resident Memory cells (TRMs) as identified by CD103 expression and high CD69 expression. We also showed that the CD8+ TILs found in a metastatic brain lesion of a breast cancer patient also contained TRMs. Finally we showed that healthy breast tissue from a patient with no clinical signs of breast cancer also contained CD8+ TRMs.
CpG immunization protects against a lethal influenza challenge and generates antigen specific memory T cells (VAC2P.932)
Authors: Vogel Alexander , Condon Shirley , Brown Deborah
Published: January 2023

Despite extensive research, influenza A virus (IAV) remains a major cause of mortality and healthcare expenditure. Emerging pandemics from highly pathogenic IAV strains such as H5N1 and H1N1 highlight the need for universal, cross protective IAV vaccines. Current vaccine formulations generate strain-specific neutralizing antibodies primarily against the outer coat proteins hemagglutinin and neuraminidase. In contrast to these highly mutable proteins, internal proteins of IAV are more conserved and are therefore a favorable target for developing vaccines that induce strong T cell responses in the absence of humoral immunity. Here, we found that intranasal administration of a TLR 9 agonist (CpG) and inactivated x31 (H3N2) protects mice from a heterosubtypic challenge with a lethal dose of PR8 virus (H1N1). Vaccinated mice also showed less weight loss and lower viral titers compared to control mice following a lethal PR8 challenge. Both CD4 and CD8 effector functions were measured five days post challenge with significant increases in the number of Granzyme B positive cells as well as antigen specific IFN-γ cells. Additionally, vaccinated mice had higher memory recall responses following ex vivo restimulation with NP specific peptides as measured by IL-2 and IFN-γ production. Thus, CpG is a potent inducer of antiviral immunity highlighted by robust T cell responses underscoring the potential of this vaccination strategy as a prophylactic measure against pandemic IAV strains.
Enhanced protection for influenza vaccines using a novel rOv-ASP-1 adjuvant aqueous formulation (VAC2P.933)
Authors: Jiang Jiu , Fisher Erin , Hensley Scott , Lustigman Sara , Murasko Donna , Shen Hao
Published: January 2023

Adjuvants have been used to enhance the efficacy of a variety of vaccines; however, no adjuvant is included in current influenza vaccines used in the United States. In this study, we found that a novel adjuvant, rOv-ASP-1, co-administrated with inactivated influenza vaccine using an aqueous formulation, substantially improved the influenza-specific antibody response and protection against lethal infection in a mouse model. rOv-ASP-1 enhanced the magnitude of the specific antibody response after immunization with low doses of influenza vaccine, allowing antigen-sparring by 10-fold. The rOv-ASP-1 formulated vaccine induced a more rapid response and a stronger Th1-associated antibody response compared to vaccine alone and to the vaccine formulated with the adjuvant alum. Importantly, rOv-ASP-1 significantly enhanced cross-reactive antibody responses and protection when challenged with a heterologous influenza virus strain. These results demonstrate that rOv-ASP-1 is an effective adjuvant that: 1) accelerates and enhances the specific antibody response induced by influenza vaccine; 2) allows for antigen sparing; and 3) augments a Th1-biased and cross-reactive antibody response that confers heterologous protection.
Circulating CXCR5+PD-1+ response predicts influenza vaccine antibody responses in young adults but not older adults (VAC2P.936)
Authors: Canaday David , Herati Ramin , Reuter Morgan , Dolfi Douglas , Mansfield Kathleen , Kurupati Raj , Ertl Hildegund , Schmader Kenneth , Kannan Senthil , Betts Michael , Wherry John
Published: January 2023

Although influenza vaccination is recommend for all adults annually, the incidence of vaccine failure, defined as weak or absent increase in neutralizing antibody titers, is increased in the elderly compared to young adults. The T follicular helper subset of CD4+ T cells provide B cell help in germinal centers and are necessary for class-switched antibody responses. Previous studies suggested a role for circulating T follicular helper cells (cTfh) following influenza vaccination in adults, but cTfh have not been studied in elderly adults where weak vaccine responses are often observed. Here, we studied cTfh expressing CXCR5 and Programmed Death 1 (PD-1). cTfh from elderly adults were present at reduced frequency, had decreased in vitro B cell help ability, and greater expression of activation marker inducible costimulator (ICOS) compared to young adults. At seven days after inactivated influenza vaccination, cTfh correlated with influenza vaccine-specific IgM and IgG responses in young adults but not in elderly adults. In sum, we have identified aging-related changes in cTfh which manifested as reduced influenza vaccine responses. Future rational vaccine design efforts should incorporate Tfh measurement as an immune correlate of protection, particularly in the setting of aging.
Vaccination with DHA-derived specialized proresolving mediators increases the antibody-mediated immune response against influenza virus (VAC2P.925)
Authors: Ramon Sesquile , Baker Steven , Sahler Julie , Feldsott Eric , Martínez-Sobrido Luis , Topham David , Serhan Charles , Phipps Richard
Published: January 2023

Influenza viruses remain a critical global health concern. More efficacious vaccines are needed to protect against influenza virus, yet few adjuvants are approved for routine use. Specialized proresolving mediators (SPMs) are powerful endogenous bioactive lipid regulators of inflammation, with great clinical translational properties. We previously identified endogenous levels of the SPM resolvin D1, protectin D1 and 17-hydroxydocosahexaenoic acid (17-HDHA) in the mouse spleen. Here, we investigated the ability of 17-HDHA to enhance the adaptive immune response using a pre-clinical influenza vaccination mouse model. Our findings revealed that mice immunized with influenza H1N1-derived HA protein plus 17-HDHA increased antigen specific antibody titers. 17-HDHA increased the number of plasma cells and HA-specific antibody-secreting cells in the bone marrow, but it did not affect the frequency of GC B cells or Tfh cells in the draining lymph node. Importantly, the 17-HDHA-mediated increased antibody production was more protective against live pH1N1 influenza virus infection in mice. This is the first report on the immuno-stimulatory effects of omega-3-derived SPMs on the humoral immune response. These findings illustrate a previously unknown biological relation between proresolution signals and the adaptive immune system. In addition, 17-HDHA as well as other immuno-stimulatory SPM have the potential to be used as novel endogenous adjuvants.
VacSIM™ - a new delivery method increases influenza vaccine efficacy (VAC2P.928)
Authors: Samli E. Farah , Shollenberger Lisa , Harn Donald
Published: January 2023

Influenza is a global health issue causing considerable economic burden. Seasonal epidemics of this acute viral infection spread rapidly with unpredictable morbidity. Regular vaccination remains the most effective prevention and as such, there is tremendous need for new technologies focused on improving immunogenicity and efficacy of seasonal flu vaccines. VacSIM™, which stands for Vaccine Self-assembling Immune Matrix, represents a new method of vaccine delivery incorporating an inert biopolymer with unique biophysical properties. It can be stored at 4-22°C, as a viscous-liquid and is flexible enough to incorporate various antigens, adjuvants or organisms for vaccine delivery. Post-injection VacSIM™ undergoes a phase shift to a semi-solid gel, forming a temporary depot of concentrated vaccine components. This gel-matrix depot may slow clearance of the vaccine, simultaneously enabling time-released delivery of the vaccine. We believe the latter increases activation of antigen presenting cells. Mice vaccinated with whole inactivated influenza (A/Puerto Rico/08/34) or with recombinant nucleoprotein via VacSIM™, had an increased humoral response, greater protection from lethal influenza challenge and improved viral clearance from lungs. Studies with other candidate vaccine antigens suggest VacSIM™ is capable of enhancing the immunogenicity and efficacy of a wide variety of vaccines.
B7-H1 blockade improves efficacy of regulatory T cell depletion as cancer immunotherapy by reducing Treg regeneration, possibly through tumor B7-H1 effects (VAC3P.950)
Authors: Curiel Tyler , Sareddy Gangadhara , Hurez Vincent , Qin Kunhua , Pandeswara Sri Lakshmi , Vadlamudi Ratna , Murthy Kruthi
Published: January 2023

Immune co-signaling blockade is effective cancer immunotherapy but is not curative alone. Treg depletion has shown potential human anti-cancer efficacy, but is also not curative alone. As B7-H1 immune co-signaling facilitates Treg generation, we hypothesized that adding αB7-H1 to Treg depletion would improve treatment by hindering Treg regeneration. We show here that αB7-H1 plus denileukin diftitox (DD) to deplete Tregs is a synergistic combo that effects complete and durable (>7 mos) tumor regressions in bulky B16 melanoma, whereas neither agent alone does. αB7-H1 efficacy alone or with DD is lost in IFN-γ KO or T cell KO mice, and is mediated in part by tumor-specific CD8+ (but not CD4+) T cells. αB7-H1 is highly effective in B7-H1 KO mice, demonstrating a major role for tumor B7-H1 in treatment efficacy. Reducing tumor B7-H1-mediated Treg generation appears to be a mechanism as predicted. The αB7-H1/DD combo was also effective in ID8 ovarian cancer showing broad application. We engineered B7-H1hi/lo B16 tumors for further immune studies. Surprisingly, B7-H1hi B16 grew faster than B7-H1lo in RAG KO, suggesting B7-H1 as an adaptive immunity-independent tumor growth/survival factor. IFNγ upregulated tumor B7-H1, but αB7-H1 did not prevent IFN-γ-mediated tumor inhibition in vitro. B7-H1hi/lo tumors in B7-H1 KO are being used to define tumor vs host effects of αB7-H1 ± Treg depletion. As αB7-H1 and DD have been tested clinically, this combo is rapidly clinically translated.
Rescue of vaccine T cell memory following sublethal gamma irradiation (VAC3P.952)
Authors: Berkson Julia , McFarland Hugh , Rosenberg Amy
Published: January 2023

Subsequent to sublethal γ radiation exposure, survivors reacquire immune competence, but lose vaccine immunity. Our goal is to rescue CD8+ T cell-mediated memory responses following γ radiation exposure. In a mouse model of Listeria monocytogenes (LM) infection, we found that rapid revaccination (rescue) of immunized mice following irradiation preserves CD8+ T cell mediated immunity. Rescued mice are able to survive a wild type LM challenge infection when the rescue vaccine strain, LM-deltaActA, is given 0-3 days following radiation exposure. A signature of rescue was revealed in gene profiling studies in which a group of cytokines and chemokines were uniquely up regulated in the spleens of LM-rescued animals as compared to mice irradiated without rescue. These included factors important in resistance to LM, such as proinflammatory factors CCL2, TNFα, and IL-6 and bacterocidal factors CXCL9, 10, and 11 as well as anti-apoptotic factors including IL-15 and IER3, which may have a role in rescue of memory CTL. RAW264.7, a macrophage cell line, produced a similar cytokine profile when irradiated and infected with LM, suggesting a probable myeloid source for the majority of these factors. Monocyte activation by the combination of irradiation and LM infection may thus create conditions of rescue by producing cytokines and chemokines that enhance clearance of LM and rescue CD8+ CTL mediating vaccine immunity.
Potency and stability of LAP+FOXP3+ Tregs in control GVHD development (VAC3P.954)
Authors: Tran Dat , Wang Huihui , Pham Anha , Cooper Laurence
Published: January 2023

Intro: We have developed previously a method to isolated bona fide Tregs from expansion cultures based on their selective surface expression of latency-associated peptide (LAP). In this study, we investigated their in vivo potency and stability using a humanized GVHD murine model. Methods: CD25+ cells isolated from PBMC via AutoMACS were stimulated with OKT3 loaded K562 aAPC expressing CD64 and CD86 in media +IL2 and rapamycin. 36h post-restimulation at wk2, the Tregs were re-isolated with antiLAP via AutoMACS. The LAP+ Tregs and LAP- nonTregs were expanded for 2wks. In vitro suppression, phenotype and TSDR demethylation were performed after 4wk expansion. CD25-depleted human PBMC were injected into NSG mice to induce xGVHD and compared with coinjection of LAP+ Tregs or LAP- nonTregs. Results: LAP+ repurification results in >85% LAP+FOXP3+ Tregs, leaving behind FOXP3- and FOXP3+ nonTregs within the LAP- population. After a total of 4wk expansion, LAP+ Tregs were >1 billion, >85% FOXP3, highly suppressive and anergic in vitro, and >95% demethylated TSDR (p<0.05). In GVHD model, PBMC alone resulted in a median survival of 29 days. The LAP+ Tregs increased median survival to 43 days (p<0.01), while the LAP- non-Tregs had a median survival of 28.5 days. Conclusions: LAP+ Tregs are highly purified, stable, and potent based on these preclinical studies. LAP+ Tregs are an ideal immunotherapeutic population for clinical trials to prevent or treat GVHD and other autoimmune diseases.
Endogenous NK cell HMGB1 is central to HDIL-2 induced systemic autophagy (VAC3P.946)
Authors: Liang Xiaoyan , LI Guanqiao , Vernon Philip , Buchser William , Loughran Patricia , Basse Per , Zeh Herbert , Lotze Michael
Published: January 2023

Administration of high dose IL-2 (HDIL-2) has durable antitumor effects in 8-10% of patients with metastatic melanoma and RCC but is associated with substantial toxicity. We explored the underlying mechanisms of this toxicity. Administration of HDIL-2 (600KIU/mouse) significantly enhances serum level of the inflammatory cytokines IFN-γ, IL-6, IL-18 and HMGB1. These increases are associated with increased LC3-II in multiple organs in B6 mice. This is consistent with the notion that HDIL-2 induces systemic autophagy. These responses are lethal in RAG1-/- mice. IL-2 treatment with chloroquine (CQ, 50mg/kg/d) enhances survival in RAG1-/- mice from 20% in the IL-2 alone cohort to 82% in mice receiving CQ+IL-2 (p=0.01). Surprisingly, NK cell depletion provides 100% survival in HDIL-2 treated RAG1-/- mouse (p<0.01). Although no HDIL-2 toxicity was observed in NSG mice, this could be reversed with the adoptive transfer of NK cells. Serum levels of cytokines were significant enhanced (all p<0.05): HMGB1 (41.9±13.5 to 256.6±26.8 ng/ml), RAGE (2.4±0.5 to 15.8±0.5 ng/ml), IFN- γ (0±18 to 420±0 pg/ml), IL-18(205±19.7 to 2318±430 pg/ml). Interestingly, treatment with α-HMGB1 Ab conferred protection. Combinations of IL-2 (200K IU/mouse) and IL-18 (25ug/kg/d X5) induced lethal systemic autophagy in WT mice. This was significantly abrogated in mice with HMGB1 genominically ablated in NKp46+ cells. Thus, NK cell plays a critical role in HDIL-2 toxicity and endogenous HMGB1 may be a key factor.
Induction and protective effect of innate interferons during human astrovirus infection (VIR1P.1003)
Authors: Cline Troy , Huerta Teddy , Freiden Pamela , Fugate Nicole , Schultz-Cherry Stacey
Published: January 2023

Members of the relatively new Astroviridae family are non-enveloped, single-stranded RNA viruses that cause diarrheal disease in a wide range species. Human astroviruses, of which there are eight canonical strains, are a leading cause of gastroenteritis in children <2 years old, the elderly, and immunocompromised patients. Because of the lack of a small animal model of astroviral gastroenteritis, little is known about the host response to infection. The role of type I and type III interferons (IFN), IFN-α/β and IFN-λ respectively, in mediating innate antiviral immune responses through a STAT1-dependent mechanism have been well established. Using human astrovirus type 1 (HAstV-1), the most common serotype isolated from human cases of gastroenteritis, we investigated the innate IFN response to astrovirus in cultured colonic epithelial cells (Caco2). HAstV-1 induced modest levels of IFN-β in Caco2 cells by 24 hours post-infection. Pre-treatment of cells with IFN-β protected Caco2 cells from HAstV-1 infection in a dose-dependent manner. Further, viral titers were significantly elevated in the presence of a neutralizing antibody to IFN-β. These data are in contrast to IFN-λ, which is induced by HAstV-1 to levels approximately 10-fold greater than those of IFN-β. However, IFN-λ pretreatment was not protective. Consistent with this, IFN-λ treatment did not induce STAT1 phosphorylation, suggesting that Caco2 cells are not responsive to IFN-λ.
Identifying the DHHC palmitoyl acyltransferase responsible for activating the antiviral protein IFITM3 (VIR1P.1008)
Authors: McMichael Temet , Hach Jocelyn , Chesarino Nicholas , Yount Jacob
Published: January 2023

Influenza virus poses a significant health threat to the global population. The intrinsic immune factor, interferon inducible transmembrane protein 3 (IFITM3) has been shown to inhibit the infection efficiency of all tested influenza virus subtypes, and understanding its mechanism of action and post-translational regulation may provide new strategies for combating infections. Palmitoylation represents a post-translational modification that positively affects IFITM3 membrane affinity and is necessary for its complete anti-influenza virus activity. Palmitoylation is generally carried out by palmitoyl acyltransferases (PATs) that have conserved DHHC amino acid motifs. Currently, it is not known which PATs are responsible for the palmitoylation of IFITM3. Our preliminary screening, using a PAT overexpression assay and chemical reporters to measure IFITM3 palmitoylation, has implicated four candidate PATs. Quantification of IFITM3 antiviral activity against influenza virus upon PAT overexpression has implicated a single PAT able to increase IFITM3 activity. Confocal imaging of this PAT demonstrated a unique pattern of localization among the other candidates suggesting that the site of IFITM3 palmitoylation is a critical factor in controlling its activity. Identifying the PAT responsible for palmitoylating IFITM3 should promote our understanding of the post-translational regulation, cellular trafficking, and activity of this important antiviral effector.
Inhibition of Langerhans cell maturation by human papillomavirus type 16: a novel role for the annexin A2 heterotetramer in immune suppression (VIR1P.961)
Authors: Kast Wijbe , Woodham Andrew , Raff Adam , Raff Laura , Yan Lisa , Skeate Joseph , Wong Michael , Lin Yvonne , Da Silva Diane
Published: January 2023

Human papillomaviruses (HPV) are associated with several cancers. During its life cycle HPV16 infects the epithelial basal cells through a receptor we recently identified, the annexin A2 heterotetramer (A2t). During infection, HPV16 also interacts with Langerhans cells (LC), the antigen presenting cells of the epithelium, and induces immune suppression mediated by the HPV16 L2 minor capsid protein. Despite the importance of these virus-immune cell interactions, the specific mechanisms of HPV16 entry into LC and HPV16-induced immune suppression remain undefined. An N-terminal peptide of HPV16 L2 has been shown to specifically interact with A2t. Here, we show that incubation of human LC with this peptide blocks binding of HPV16. Inhibiting this interaction with an A2t ligand or by siRNA down-regulation of A2t, significantly decreases HPV16 internalization into LC in an L2-dependent manner. A2t is associated with inhibition of LC maturation as demonstrated through attenuated secretion of Th1-associated cytokines and decreased surface expression of MHC II on LC exposed to A2t. Conversely, small molecule inhibition of A2t prevents HPV16-induced inhibition of LC immune function as indicated by increased secretion of inflammatory cytokines and surface expression of MHC II and CD86 in HPV16 treated LC pre-exposed to A2t inhibitors. These results demonstrate that HPV16 suppresses LC maturation through an interaction with A2t, revealing a novel immune suppressive role for this protein.
Licensed NK-mediated response to murine cytomegalovirus specifically upregulates dendritic cell co-stimulatory ligands required in T cell priming (VIR1P.963)
Authors: Teoh Jeffrey , Stadnisky Michael , Bullock Timothy , Brown Michael
Published: January 2023

Ly49G2+ (G2+) NK cells mediate murine (M)CMV resistance in mice with the self-MHC Dk ligand expressed. A prior study showed that G2+ NK-mediated MCMV resistance resulted in increased numbers of conventional dendritic cells (cDC) and virus-specific CD8 T cells after infection, independent of CD4 T cells. However, the molecular basis underlying licensed NK-mediated enhancement of T-cell immunity is unknown. Here we examined the role of co-stimulatory ligands expressed in splenic cDC after MCMV infection. An increase in the intracellular expression of both CD86 and CD70 was observed by 72-hr, first in the CD8α+ DC and then in the CD11b+ DC. To test functionality, we used CD86 and CD70 mAbs to block co-stimulation before infection. CD86-block had no effect on CD8 T cells. In contrast, CD70-block impaired both the frequency and the numbers of MCMV-specific CD8 T cells without perturbing cDC numbers, cDC maturation, or G2+ NK-mediated viral control. We next examined the importance of CD70-CD27 signaling in CD27-/- mice with the self-ligand Dk expressed. We observed similar deficits in Ag-specific CD8 T cells, despite having intact G2+ NK-mediated MCMV control. Moreover, CD8 T cells in both CD70-blocked and CD27-/- infected mice responded to MCMV peptide re-stimulation with less degranulation and cytokine production. We infer that a licensed G2+ NK-mediated response to MCMV upregulates co-stimulatory molecule expression in cDC that is needed to efficiently prime CD8 T cells.
Targeting Robo4 pathway reduces corneal angiogenesis induced by herpes simplex virus. (VIR1P.967)
Authors: Gimenez Fernanda , Rouse Bary
Published: January 2023

The cornea is a complex tissue that must preserve its transparency to maintain optimal vision. Eye infections such as with herpes simplex virus (HSV) can result in a chronic immuno-inflammatory syndrome and pathological neovascularization that impairs vision. Recent reports demonstrate that angiogenic sprouting shows morphological similarities to axon growth. Moreover there are several key molecules that upon binding to receptors regulate the direction of both capillary and axon guidance. In this study we show that Roundabout 4 (ROBO4) receptor plays a significant role reducing angiogenesis. Using ROBO4 -/- mice we demonstrate that these mice are hyper-susceptible to HSV induced corneal angiogenesis compared to wild type mice. Moreover, providing additional soluble ROBO4 protein by subconjunctival administration significantly diminished the extent of corneal angiogenesis in wild type animals. Finally, by administration of soluble ROBO4 protein to ROBO-/- HSV infected mice it was possible to rescue the wild type phenotype. In conclusion, the administration of soluble ROBO4 protein promotes the activation of the ROBO4 pathway. This could represent a valuable therapeutic tool to control corneal angiogenesis related to HSV induced stromal keratitis.
Defective viral genomes arising in vivo promote the onset of lung antiviral immunity (VIR2P.1023)
Authors: Lopez Carolina , Tapia Karla , Kim Won-keun
Published: January 2023

In infections with viruses well adapted to the host, virus-encoded proteins that delay the cellular response allow the virus to replicate to high titers prior to host intervention. The mechanisms overcoming viral evasion and leading to the production of the primary antiviral cytokine IFNbeta are not well established. We show that the appearance of defective forms of the viral genome may provide the answer. Defective viral genomes, which were until recently considered an epiphenomenon of in vitro virus replication, accumulate in infected cells at a faster rate than standard viral genomes and potently trigger the sustained activation of the transcription factors IRF3 and NF-kB and the production type I IFNs through a mechanism independent of IFN signaling. Remarkably, our work demonstrates that defective viral genomes are generated naturally during respiratory infections in vivo even in mice lacking the type I IFN receptor, and their appearance coincides with the production of cytokines during infections with Sendai virus or influenza virus. Notably, the hallmark antiviral cytokine IFNβ is only expressed in lung epithelial cells containing defective viral genomes, while cells within the lung that contain standard viral genomes alone do not express this cytokine. Together, our data indicate that defective viral genomes generated naturally during viral replication are a primary source of danger signals for the initiation of the host immune response to infection in vivo.
Effect of asthma on susceptibility to influenza A virus infection (VIR2P.1029)
Authors: Furuya Yoichi , Sanfilippo Alan , Metzger Dennis
Published: January 2023

Asthma exacerbations by respiratory infections are a serious health issue and, thus significant effort has been put into understanding the impact of respiratory infections on asthma. However little remains known about how the asthmatic phenotype could influence immune responses against respiratory viral pathogens. Using a mouse model of allergic lung inflammtion, we investigated the susceptibility of asthmatics to influenza A virus challenge. Unexpectedly, asthmatic mice were highly resistant to primary infection with the H1N1 2009 pandemic strain. However, the increased survival rate did not correlate with viral lung titers. Strikingly, asthmatic mice exhibited a significant reduction in cytokine storm, total protein levels and cytotoxicity in the airway; observations that indicate minimal tissue injury. Further, we found that asthmatic mice had significantly increased levels of TGF-β1 and that the heightened resistance of asthmatic mice was abrogated in the absence of TGF-β receptor II. Thus, we conclude that a transient increase in TGF-β expression following an asthma attack is responsible for temporary protection against influenza virus challenge via suppression of lung immunopathology.
The human respiratory syncytial virus nucleoprotein is expressed on the surface of dendritic cells and inhibits immunological synapse assembly by T cells (VIR2P.1018)
Authors: Céspedes Pablo , Bueno Susan , Ramírez Bruno , Depoil David , Sacristan Catarina , Cammer Michael , Dustin Michael , Kalergis Alexis
Published: January 2023

The human Respiratory Syncytial Virus (hRSV) is the major cause of children severe respiratory tract infections worldwide. Both, the poor activation of T cells and the limited generation of memory T cells following disease resolution are considered key processes favoring hRSV epidemics, which are based on re-infections. It is known that hRSV infects Dendritic cells (DCs) dampening their capacity to prime antigen-specific naïve T cells, a process needed to mount a proper antiviral immune response and eliminate hRSV from the airways. Here we show that the hRSV Nucleoprotein (N) is expressed at the surface of a variety of infected cells, including DCs. After estimating the approximate density of N in the surface of DCs and other cells, we evaluated whether these physiological densities could affect the assembly of the immunological synapse (IS) by naïve CD4+ T cells. We observed that N, but not control proteins inhibited the assembly of IS by interfering with TCR-pMHC interactions, which was accompanied with reduced accumulation of TCR and reduced central clustering of pMHC at the T cell-bilayer interface, as well as with reduced phosphorylation of tyrosine residues in the interacting pole of stimulated T cells. Altogether, our findings propose N-hRSV as a key virulence factor responsible for the suboptimal T cell activation observed in hRSV-infected individuals.
Optineurin is dispensable for LPS- and Salmonella typhimurium-induced autophagy (INM3P.412)
Authors: Munitic Ivana , Giardino-Torchia Maria Letizia , Ashwell Jonathan
Published: January 2023

In vitro studies demonstrated that optineurin, a ubiquitin (Ub)-binding protein, becomes an autophagy cargo receptor upon being phosphorylated on Ser177 by TANK binding kinase 1 (TBK1) in response to cytosolic Salmonella typhimurium. Phosphorylated optineurin then regulates autophagy by delivering ubiquinated bacteria to autophagosomal membranes via light chain 3 (LC3), hence restricting their growth. To test the in vivo relevance of these findings, we used a mouse model with C-terminal optineurin truncation, which lacks the Ub-binding region (Optn470T). No difference was found between WT and Optn470T macrophages in LC3 lipidation during LPS-induced autophagy. Similarly, p62+LC3+ aggresome-like structures generated upon LPS or Salmonella typhimurium stimulation in Optn470T macrophages colocalized with lysosomal marker LAMP1 to the same extent as in WT cells, demonstrating no perturbation in autophagosome fusion to lysosomes. Importantly, upon in vivo infection with Salmonella typhimurium, no difference was found in bacterial replication in WT and Optn470T mice. These results argue against the role of optineurin in Salmonella typhimurium infection in vivo.
The involvement of Mycobacterium leprae Hlp-DNA complex in bacterial recognition and airways epithelial activation (INM3P.404)
Authors: Dias Andre , Silva Camila , Silva Carlos Adriano , Santos João Pedro , Pereira Geraldo , Pessolani Maria Cristina
Published: January 2023

Airways are considered the main site of entry of Mycobacterium leprae (ML), the causative agent of leprosy. ML expresses a histone-like protein (Hlp), present in the cell envelope, which has been implicated in the binding of this mycobacterium to respiratory epithelial cells (ECs). So we investigated whether mycobacterial DNA-Hlp complexes are able to increase the human alveolar epithelial cell line A549 responses to ML through recognition by TLR9. ELISA results showed that ML and soluble CpG-Hlp complexes increase IL-8 secretion in ECs. Moreover, immunoblotting and ELISA assays revealed that ML induces the NF-κB nuclear translocation in A549 cells and the IL-8 production was dependent of that translocation. The antagonist of the nucleic acid PRRs also affected the ML-induced IL-8 production by ECs. In culture medium, the M. smegmatis wild-type (wt), but not the mutant strain for the gene hlp, was able to form clumps in the presence of exogenous mycobacterial DNA. Furthermore, the addition of CpG to M. smegmatis wt increased the production of IL-8 by A549 cells. Finally, conditioned medium from ECs pre-treated with ML was able to increase the CD40 and CD86 co-stimulatory molecules expression on plasmacytoid dendritic cells (DCs). Altogether, these observations suggest a potential role for the M. leprae DNA-Hlp complex in the activation of the airways epithelium, and an involvement of DCs in the protection of the host at the initiation of infection.
Interleukin-17 receptor A (IL-17RA) is a key regulator of type 2 inflammatory responses involved in maintaining immune system homeostasis. (IRC7P.431)
Authors: FLOUDAS ACHILLEAS , Saunders Sean , Fallon Padraic
Published: January 2023

IL-17 cytokine family signaling is a key mediator of skin inflammatory disorders including atopic dermatitis and psoriasis. It has previously been shown that deficiency of IL-17 producing cells and especially TCRγδ T cells attenuates the skin inflammatory phenotype observed in murine models of psoriasis. Mice lacking IL-17 receptor A expression, through which IL-17A and other IL-17 family cytokines signal, develop severe and progressive skin inflammation. This spontaneous skin inflammation was characterized by increased epidermal thickness with cellular infiltration, which was accompanied with lymphadenopathy, and type 2 responses, including eosinophilia and increased serum levels of IgE. On a cellular level a significant increase in the numbers of innate lymphoid cell subpopulations was observed. Additionally IL-17RA-/- mice have increased γδ T cells with an activated memory-like phenotype. To further elucidate the role of IL-17 family cytokines in atopic dermatitis (AD) we crossed IL-17RA-/- mice with mice, carrying a mutation previously associated with human AD, that results in a spontaneous atopic dermatitis-like disease. The absence of IL-17RA signaling resulted in a severely accelerated AD like inflammation. This highlights that IL-17RA signaling is required for maintaining homeostasis including the regulation of Th2 responses.
Antigen presentation via MHC class II by lymphatic endothelial cells dampens CD4+ T cell response (IRC7P.436)
Authors: Potin Lambert , Maillat Lea , Dubrot Juan , Duraes Fernanda , Hugues Stéphanie , Swartz Melody
Published: January 2023

Lymphatic endothelial cells (LECs) can directly interact with CD8+ T cells via presentation of peptide-MHCI complexes, resulting in tolerance due to lack of costimulatory molecules and high levels of PD-L1 expression by LECs. Moreover, we and others have recently demonstrated that LECs can present peptide-MHCII complexes (pMHCII) to CD4+ T cells. However, the functional implications of antigen presentation by LECs to CD4+ T cells remain unclear. Here, we explore the tolerogenic potential of pMHCII presentation by LECs to CD4+ T cells using ovalbumin (OVA) as a model antigen. In vitro, LECs isolated from mice lacking MHCII could acquire pMHCII from wildtype dendritic cells (DCs). Subsequent LEC interactions with TCR-transgenic CD4+ T cells resulted in increased levels of CD4+ cell apoptosis, indicating deletion. Moreover, the ability of DCs to induce CD4+ T cell proliferation was decreased when LECs were present in the culture system. To examine the role of endogenous MHCII expression by LECs, we created an inducible mouse model lacking MHCII expression in LECs using a Cre-recombinase approach. In these mice, T cell responses to vaccination effects were enhanced with respect to circulating OVA-specific CD8+ T cells, indicating increased T cell help (CD4). Together, these data suggest that LEC expression of MHCII may serve to dampen or regulate immune responses. Further studies are needed to elucidate the functional role of LEC expression of MHCII in pathological conditions.
GITR sustains IL-2+ CD4 helper T cells to facilitate early control of a chronic viral infection (IRC7P.422)
Authors: Clouthier Derek , Zhou Angela , Wortzman Michael , Luft Olga , Levy Gary , Watts Tania
Published: January 2023

CD4 T cells are critical for control of persistent infections; however, the key signals that regulate CD4 T help during infection remain incompletely defined. Here we show that during lymphocytic choriomeningitis virus (LCMV) clone 13 infection, glucocorticoid-induced TNFR (GITR)-deficient mice exhibit defective CTL responses and viral control. Differences in CTL responses and viral control between GITR+/+ and GITR-/- mice were lost when CD4 T cells were depleted. Furthermore, mixed bone marrow chimeric mice and transfer of LCMV-specific CD4 or CD8 T cells demonstrated that these effects of GITR are CD4 T cell-intrinsic. GITR is dispensable for initial CD4 T cell proliferation and differentiation, but supports the post-priming accumulation of IL-2+ Th1 cells to facilitate CTL expansion and early viral control. In vivo GITR-dependent activation of p65 NF-kB and S6 ribosomal protein was detected at day 3 p.i., a time at which GITR-L is maximally expressed on macrophages. Defects in CD4 T cell accumulation in GITR-deficient T cells were apparent starting at day 5 p.i. Consistently, we pinpoint IL-2-dependent CD4 T cell help for CTLs to between days 4-8 p.i. GITR also increases the ratio of T follicular helper to T follicular regulatory cells, thereby enhancing LCMV-specific IgG production. Together, these findings identify a CD4 T cell-intrinsic role for GITR in sustaining early CD8 and late humoral responses to collectively promote control of chronic LCMV clone 13 infection.
The iron sequestering protein Lipocalin 2 is critical to IL-33-exposed dendritic cell stimulation of Th2 responses and allergic airway disease (IRC7P.424)
Authors: Lott Jeremy , Liu Quan , Matta Benjamin , Mathews Lisa , Pociask Derek , Sperling Anne , Kolls Jay , Turnquist Heth
Published: January 2023

IL-33 is an IL-1 cytokine with emerging pleotropic functions, but ascribed a prominent role in Type 2-mediated responses to pathogens due to its capacity to generate and support T helper type 2 (Th2) cells. Likewise, IL-33 and Th2 cell are viewed as dominant drivers of inflammatory lung disorders, including asthma. Previous studies have revealed that IL-33 stimulates the capacity of dendritic cells (DC) to initiate Th2 responses and, thus instigate allergic airway disease (AAD) in mice. However, the precise mechanisms by which IL-33 promotes DC-mediated Th2 polarization was not known. To answer this question, we preformed microarray analysis on DC after exposure to IL-33. This analysis identified multiple genes involved in iron homeostasis and metabolism upregulated by IL-33. The most dramatically increase gene in DC by IL-33 was Lipocalin 2 (Lcn2), an iron sequestering protein that facilitates DC iron uptake. After IL-33-exposure, DC deficient in Lcn2 displayed a significantly reduced capacity to generate Th2 responses, however, LPS-exposed DC lacking Lcn2 were fully capable of Th1 polarization. Identical finding were observed when DC iron uptake was blocked through chelation during their exposure to IL-33. In a DC-induced model of AAD, blocking DC iron uptake resulted in significantly decreased disease severity. These data define a novel role for IL-33-stimulated iron uptake by Lcn2 in the generation of Th2 responses.
Engineered FVIII-specific human CD4 T cells: does TCR avidity modulate T-helper phenotypes? (IRC7P.425)
Authors: Adair Patrick , Kim Yongchan , Pratt Kathleen , Scott David
Published: January 2023

We recently demonstrated that expanded human CD4 T cells can be transduced to express TCRs specific for a given epitope. These cells proliferate and secrete cytokines in response to their cognate peptide/MHC. Herein, we examined whether TCR- transduced cells could be skewed to different T-helper subsets, or alternatively if previously skewed T cells could be modulated after transduction. Two HLA DR1-restricted TCRs were cloned from Th2 and Th17/Th1-type CD4+ T-cell clones specific for a coagulation factor VIII peptide (pC2). Engineered CD4+ T cells expressing these TCRs exhibited different proliferation kinetics during titration with pC2. All pC2-stimulated cells expressed IFN-g, IL-4, and IL-2 intracellularly but did not express cytokine signatures characteristic of their parental Th2 and Th17/Th1 clones. CD45RA+ and CD45RA- T cells were skewed to Th1, Th2 or Th17 and then transduced with each of the 2 TCRs. The higher-avidity TCR cells that were skewed to Th2 and then stimulated with low [pC2] maintained a Th2 cytokine signature, whereas the same cells stimulated with higher [pC2] expressed lower levels of IL-4 and GATA3. In contrast, Th1, Th17 and Th0-skewed cells transduced with the same TCR expressed their respective cytokines at all pC2 doses. Differences between the low-avidity TCR-engineered cells were less pronounced. These studies will inform our strategies for designing novel T-helper cellular therapies.
Synergistic T cell modulation by innate and adaptive immune signals (IRC8P.446)
Authors: Jun Janice
Published: January 2023

One of the most effective anti-inflammatory agents that has successfully prevented disease in multiple inflammatory disease models is the capsular carbohydrate polysaccharide A (PSA) of the commensal bacteria Bacteroides fragilis. PSA triggers a potent anti-inflammatory response characterized by T cell-dependent IL-10 production, yet little is known about the antigenic mechanism underlying this extraordinarily effective suppressive response. While this effect of PSA is known to require TCR engagement on CD4+ T cells by MHCII presentation of processed PSA by APCs, little else is known about why this engagement, which is common to all conventional T cell antigens, selectively leads to IL-10 production and the inhibition of inflammatory disease. We have taken a reductionist approach to identify the types of antigenic stimuli selectively leading to IL-10 production in T cells and have discovered that innate signals directly on the T cell, independent of the APCs, can modulate IL-10 secretion if the T cell receptor signaling cascade is actively engaged. Moreover, these innate signals leading to IL-10 production are specific for the effector/memory T cell subsets and are not effective within the classically-defined naïve or regulatory T cell populations. This suggests a novel role for T cell-intrinsic innate signals working in conjunction with traditional adaptive signals in inducing a potent IL-10 response with therapeutic ramifications.
Cellular and molecular basis of Ag-specific and Ag-nonspecific T-dependent antibody responses in vivo (IRC8P.447)
Authors: Watanabe Masashi , Hodes Richard
Published: January 2023

Although B7 is essential for T-dependent Ag-specific Ab production, little is known about cell type specific role of B7 for these responses. Utilizing B7cKO mice, the cell type specific roles of B7 costimulation for T-dependent GC and Ab responses were analyzed. Upon NP-OVA/Alum immunization, NP-specific IgG1 production was profoundly defective in mice lacking B7 expression on DC (DC-B7KO) but was intact in mice lacking B7 on B cells (BC-B7KO). Consistent with this, NP-binding GC B cells and Ag-specific CD4+ T cell expansion were not observed in DC-B7KO. Surprisingly, however, GC formation and total numbers of GC B and TFH were undiminished in DC-B7KO; whereas BC-B7KO showed reduction of total number of GC B and TFH despite normal numbers of NP-binding GC B. These results demonstrate that B7 on DC but not B cells is critical for Ag-specific CD4+ T cell expansion and IgG1 production. In contrast, a distinct pathway dependent upon B7 on B cells but not DC supported apparently Ag-nonspecific GC response. Of interest, MHCII on B cells but not on DC was required for Ag-nonspecific GC response, indicating that cognate T-B but not T-DC interaction was responsible for eliciting Ag-nonspecific GC response. Our study revealed a previously unappreciated B7-dependent Ag-nonspecific Ab production pathway and demonstrated distinct cellular and molecular pathways for Ag-specific and Ag-nonspecific components of T-dependent Ab responses.
RGC-32 regulates T lymphocyte cell cycle activation in a phosphatidylinositol 3- kinase and IL-2 -dependent manner (IRC8P.442)
Authors: Rus Horea , Tegla Cosmin , Cudrici Cornelia , Nguyen Vinh , Kruszewski Adam , Mekala Amurgam , Rus Violeta , Badea Tudor
Published: January 2023

We have previously shown that RGC-32 is involved in cell cycle regulation in vitro. To define the in vivo role of RGC-32, we generated RGC-32 knockout mice. To assess the effect of RGC-32 deficiency on cell cycle activation in T cells, we determined the proliferative rates of CD4+ and CD8+ T cells from the spleens of RGC-32-/- mice, as compared to wild-type (WT) mice. After stimulation with anti-CD3/anti-CD28, CD4+ T cells from RGC-32-/- mice displayed a significant increase in [3H]-thymidine incorporation when compared to WT mice. In addition, both CD4+ and CD8+ T cells from RGC-32-/- mice displayed a significant increase in the proportion of proliferating Ki67+ cells, indicating that in T cells, RGC-32 has an inhibitory effect on cell cycle activation induced by T-cell receptor/CD28 engagement. Furthermore, Akt and FOXO1 phosphorylation induced in stimulated CD4+ T-cells from RGC-32-/- mice were significantly higher, indicating that RGC-32 inhibits cell cycle activation by suppressing FOXO1 activation. We also found that IL-2 mRNA and protein expression were significantly increased in RGC-32-/- CD4+ T cells when compared to WT CD4+ T cells. In addition, the effect of RGC-32 on the cell cycle and IL-2 expression was inhibited by pretreatment of the samples with LY294002, indicating a role for phosphatidylinositol 3- kinase (PI3K). Thus, RGC-32 is involved in controlling the cell cycle of T cells in vivo, and this effect is mediated by IL-2 in a PI3K-dependent fashion.
Mechanism of lipid droplet accumulation in mononuclear phagocytes during sepsis (IRM9P.461)
Authors: ROSAS-BALLINA MAURICIO , Guan Xue Li , Schürmann Nura , Schmidt Alexander , Bumann Dirk
Published: January 2023

Lipid droplet formation in phagocytes is associated with various important inflammatory conditions, and their accumulation modulates disease progression. We used proteomics, lipidomics, and cell bioenergetics experiments to unravel the metabolic basis of lipid droplet accumulation in a murine model of Salmonella infection. Lipid droplets accumulated in inflammatory monocytes and neutrophils within spleen inflammatory lesions, and this is recapitulated in vitro by treating monocytes with the pro-inflammatory cytokine IFNγ. Bioenergetic experiments suggested that iNOS-derived nitric oxide blocks the mitochondrial electron transport chain thus inhibiting respiration and oxidation of fatty acids, which instead accumulate within lipid droplets. Surprisingly, metabolite tracing with 13C-labeled substrates revealed that de novo synthesis of fatty acid from glutamine or glucose plays no major role in lipid accumulation, in stark contrast to the otherwise similar metabolism of cancer cells. Instead, the glycerol head group in triacylglycerol and phospholipid entirely derives from glucose, while the fatty acid carbons originate from uptake of external lipids. Our results identify neutrophils and inflammatory monocytes as a depot of neutral lipid during infection, and establish remodeling of imported lipids with exchange of head groups for newly synthesized glycerol as the underlying metabolic mechanism of lipid droplet accumulation upon activation with IFNγ.