bluuebunny/crossref_metadata_2025 huggingface dataset:
Selenoprotein K interacts with Vav1 in macrophages to inhibit Rac1 activity (172.17)
Authors: Norton Robert , Huang Zhi , Hoffmann Fukun , Hoffmann Peter
Published: January 2023

Selenium is a mineral micronutrient that is essential in a variety of biological systems, including immune responses. Selenium is used for biosynthesis of the amino acid selenocysteine, which is used by selenoproteins to carry out biological functions. In humans, 25 selenoproteins have been identified and Selenoprotein K (SelK) was recently identified as an endoplasmic reticulum transmembrane protein important for calcium flux during the activation of immune cells. Vav1 is a guanine exchange factor that activates Rac1 and leads to actin polymerization and cytoskeletal rearrangement. Implementation of the SH3 hunter software predicted that the SH3-binding domain of SelK associates with one of the two SH3 domains of Vav1. We were able to show that SelK co-immunoprecipitates with Vav1 from mouse bone marrow derived macrophage (BMDM) whole cell lysates. By pull down assay, we saw higher levels of activated, GTP bound Rac1 in KO BMDM’s compared to WT BMDM’s when treated with RANTES. This suggests that SelK could be involved in Rac1 activation. Studies are underway to determine if Vav1 is being sequestered by SelK, preventing it from becoming activated. Macrophage activation might cause SelK to release Vav1, leading ultimately to actin polymerization, cytoskeletal rearrangement and cell motility. Future experiments will determine if this co-immunoprecipitation is abolished when the SH3 binding domain of SelK is deleted or when the SH3 motif of Vav1 is deleted.
Haptoglobin inhibits the pro-inflammatory activity of HMGB1. (172.19)
Authors: Yang Huan , Levine Jacob , Katz David , Lu Ben , Scheinerman Josh , Dancho Meghan , Ochani Mahendar , Li Jianhua , Chavan Sangeeta , Warren Shaw , Tracey Kevin
Published: January 2023

Severe sepsis accounts for over 200,000 deaths in US annually (Angus et al, CCM, 2001). The mechanism involves endogenous molecules that impair organ function. Extracellular hemoglobin is one such factor enhancing tissue damage during sepsis. We designed haptoglobin-coated beads for a perfusion system to remove hemoglobin in an experimental sepsis model. Surprisingly, we observed that in addition to removing hemoglobin, haptoglobin beads also captured large amounts of HMGB1. HMGB1 is a major mediator on the final common pathway to lethality in sepsis (Yang et al, BBA 2009). Addition of haptoglobin suppressed HMGB1-stimulated TNF and IL-8 release in cultured macrophages. Haptoglobin knockout mice subjected to cecal perforation-induced sepsis had significantly higher serum HMGB1 levels and higher mortality compared to wild type animals (75% survival in wild type vs. 34% in haptoglobin knockout mice; n=15/group, P<0.05). Treatment with anti-HMGB1 antibodies significantly reduced the mortality in haptoglobin knockout mice (12% survival in control vs. 56% in animals receiving HMGB1 antibodies; n=16/group, P<0.05). Structure-function analysis revealed that haptoglobin beta subunit alone is sufficient to recapitulate the protective effects observed with haptoglobin. These findings indicate that haptoglobin is an endogenous modulator of HMGB1 that is capable of reducing the toxicity of HMGB1 in sepsis. Supported in part by grants from NIH (RO1GM62508, to KJT and RO1GM098446, to HY).
Particulates induce macrophages to produce lipid mediators via the Syk activation. (172.25)
Authors: Kuroda Etsushi , Ohata Keiichi , Coban Cevayir , Ishii Ken
Published: January 2023

Some particulate such as silica crystal (silica), aluminum salts (alum) and monosodium urate (MSU) can stimulate the innate immune system to induce inflammatory responses. Recently, we found that silica and alum induce macrophages to produce both IL-1β and lipid mediator prostaglandin E2 (PGE2). We also indicated that particulate-induced PGE2 is involved in the production of IgE in vivo. However, the underlying mechanisms involved in the induction of PGE2 by particulate remain to be elucidated. In this study, we show that Syk plays an important role in particulate-induced PGE2 production. Silica and alum induced macrophages to produce IL-1β and PGE2. However, NALP3 inflammasome-deficient macrophages produced higher amounts of PGE2 comparable to WT macrophages. To determine which signaling pathway is involved in the production of PGE2, we analyzed PGE2 production from macrophages treated with various signaling inhibitors. Interestingly, we found a Syk inhibitor markedly suppressed PGE2 production. Similar to inhibitor experiment, knockdown of Syk by siRNA in macrophages significantly reduced both PGE2 and IL-1β production. In conclusion, we have found that particulate silica, alum and MSU stimulate macrophages to produce lipid mediator PGE2 though a pathway that is dependent on Syk but independent of the NALP3 inflammasome.
Bcl-3 is a regulator of dendritic cell functions (172.30)
Authors: Tassi Ilaria , Claudio Estefania , Wang Hongshan , Siebenist Ulrich
Published: January 2023

Bcl-3 is a nuclear member of the family of IκB inhibitors; it can act as an activator or inhibitor of NF-κB activity, depending on the context, and it functions via association with homodimers of the p50 or p52 subunits of NF-κB. In recent years various NF-κB subunits have been implicated in the development and activation of dendritic cells (DCs), which are important effectors in the activation of the innate and adaptive immune system. Here we have explored, for the first time, the role of Bcl-3 in DC-mediated functions. We report that Bcl-3 is required to prime CD4 T cells in vitro. By using an OT-II adoptive transfer model, we were able to demonstrate that Bcl-3 deficient DCs lacked the capacity to prime T cells in vivo. Furthermore, specific ablation of Bcl-3 in DCs in vivo, revealed that Bcl-3 is required for the development of contact hypersensitivity reactions. Mechanistically, Bcl-3 is dispensable for DC maturation and inflammatory cytokine production. In conclusion, we demonstrated for the first time that Bcl-3 expression in DCs is required for the initiation of the adaptive immune response in vitro and in vivo.
CpG ODN treatment in radiation-combined injury (172.31)
Authors: Stoecklein Veit , Osuka Akinori , Lederer James
Published: January 2023

In the scenario of a nuclear event, radiation injury will be accompanied by additional trauma, especially burns. We developed a mouse model to allow us to study the changes in the immune system induced by these injuries. In this study, we tested the potential of using CpG oligodeoxynucleotides (ODN) as treatments in radiation combined injury (RCI). Mice were exposed to 1, 4 and 6 Gy whole body radiation and underwent near simultaneous burn injury. One day after injury, mice were injected with either CpG ODNs or saline. Seven days later, sepsis was induced by cecal ligation and puncture (CLP) and survival was recorded. Spleen immune cell subsets were assessed by FACS and inflammatory cytokine production by splenocytes was determined at 7 days after CLP. To investigate the role of regulatory T cells (Tregs) in this model, mice were Treg depleted after class A CpG treatment. Treatment with class A CpG increased survival after RCI with 1 Gy followed by CLP significantly whereas treatment with class B or C did not. Class A treatment also conferred a survival benefit following RCI with 4 and 6 Gy. Inflammatory cytokine production was decreased with class A treatment but not with class B or C. The effects of class A treatment were not seen in Treg depleted mice. Class A CpG tracking showed increased uptake in Tregs when compared to class B or C. In summary, CpG treatment is effective in enhancing host defense mechanisms following RCI.
Identification and characterization of a protein G-binding intelectin in Japanese lamprey (172.6)
Authors: Li Qingwei , Wu Fenfang , Pang Yue , Xiao Rong , Liu Xin , Xue Zhuang
Published: January 2023

Intelectins are recently identified extracellular animal lectins. They are glycan-binding receptors recognizing glycan epitopes on foreign pathogens and in the host system. In the present study, we report the identification of a new type of serum lectin from Japanese lamprey (Lampetra japonica). Gene expression analysis and Western blots demonstrated that the lamprey intelectin was expressed in blood, intestine, kidney, heart, gill, liver, medulla, and gonad. The recombinant lamprey intelectin protein (Lj-intelectin) appeared as ~34kD, ~44kD, and ~65kD bands on non-reducing SDS-PAGE gels, suggesting that it is consisted of monomers, dimmers and trimers. In addition, direct binding assays demonstrated that Lj-intelectin bind to protein G. Furthermore, Li-intelectin induced by in vivo bacterial stimulation showed agglutination activity against pathogenic Gram-positive and Gram-negative bacteria, indicating that it may play an important role in self-defense against bacteria in the internal circulation system of lamprey. The Lj-intelectin also exhibited a calcium-dependent agglutination to human red blood cells. These results demonstrate that lamprey intelectin plays an important role in immune response.
Antibiotic induced intestinal dysbiosis in infant mice impacts CD8+ T cell anti-viral immune function and control of infection (IRC8P.494)
Authors: Lamouse-Smith Esi , Radens Caleb , Starnbach Michael
Published: January 2023

Infancy and childhood is a critical period for the development of long-term immune function. Normal acquisition of the gastrointestinal (GIT) flora during this period is now appreciated in its role on immune system development and function. Antibiotics cause GIT flora dysbiosis and may alter normal GIT colonization during infancy. Subsequently, immune development and function may also be affected. In this infant mouse model of antibiotic mediated GIT flora dysbiosis, perinatal antibiotic treatment (AT) alters the composition of the GIT flora in mothers and their infants. CD8+ T cells from AT infant mice activated in vitro with cytokines, proliferate, express activation markers, and produce IFN-γ. However, unlike non-antibiotic treated (CTRL) infant mice, AT mice succumb to vaccinia virus infection. AT infant mice survive sub-lethal infection indicating effective primary immune responses to control infection. Interestingly, the expansion of Teff, Tem, and Tcm subsets differs between CTRL and AT sub-lethally infected infant mice. Transfer of OT-I T cells into AT infant mice prior to lethal infection rescues these mice from death. However, expansion of OT-I T cells is limited in AT infant mice and the differential expression of effector and memory cell markers is also recapitulated. These results suggest that dysbiosis during infancy negatively impacts effective adaptive primary immune responses and has potential consequences on memory T cell development.
Defining the minimal signals necessary to generate an encephalitogenic T cell (IRC8P.476)
Authors: Lee Priscilla , Yang Yuhong , Racke Michael , Lovett-Racke Amy
Published: January 2023

While it is generally accepted that Multiple Sclerosis (MS) is a T cell-mediated disease, the factors determining the encephalitogenicity of T cells remains unclear. Myelin-specific T cells generated with antigen presenting cells (APCs) plus myelin peptide are encephalitogenic, whereas T cells generated with anti-CD3/CD28 antibodies are not, suggessting that APCs provide critical signals beyond T cell receptor activation and co-stimulation, contributing to an encephalitogenic phenotype. To recapitulate the signals provided by APCs, naïve myelin-specific CD4+ T cells were activated with anti-CD3/CD28 in the presence of various cytokines. The degree of encephalitogenicity of T cells was examined by the severity of experimental autoimmune encephalomyelitis (EAE). Although no single cytokine was sufficient, the combination of IL-6/IL-23 or IL-12/IL-23 generated highly encephalitogenic T cells. IL-6 and IL-12 induced the initial expression of IL-23R on naïve T cells, and IL-23 further enhanced the expression of its own receptor and the encephalitogenicity. Neutralizing IL-6 or IL-12 with antibodies significantly reduced the severity of EAE, which was induced by APC-generated T cells. We have identified that IL-6/IL-23 or IL-12/IL-23 combination in addition to TCR activation and co-stimulation were the miminal signals necessary to generate encephalitogenic T cells. This provides potential targets that can be manipulated therapeutically, resulting in innovative treatments for MS.
Gut-associated cross-differentiation of T lymphocyte lineages (LYM7P.726)
Authors: Lui Jenbon , Devarajan Priya , Teplicki Sarah , Miska Jason , Chen Zhibin
Published: January 2023

Peripheral regulatory T cells engage in a necessary role in maintaining the volatile balance between the normal flora of the gut and the host immune system. However, the clonal origin of peripheral Treg cells is not well understood and has proven difficult to elucidate in the presence of naturally occurring thymic Treg cells. We hypothesized that the gut-associated environment enables cross differentiation of the CD8 T cell lineage to CD4 Treg cells for the regulation of immune cell homeostasis. Our preliminary examination found evidence for cross-differentiation from the CD8 lineage to CD4 effector and regulatory T cells. Further studies showed that these cross-differentiated CD4 Treg cells were effective in protecting from intestinal inflammatory damage. We found that in transfer experiments mature CD8 T cells were capable of cross-differentiating into CD4 T cells. Results from normal flora depletion studies by broad spectrum antibiotics showed that cross-differentiation of CD8 T cells into CD4 T cells was dependent on the normal flora of the gut. The results that we have obtained suggest that CD8 T cells have the potential to cross-differentiate into CD4 T cells and Treg cells. This pathway may participate in regulating and maintaining the fine balance of tolerance and immunity between normal flora and host immune system.
Nonclassical MHC Ib-restricted CD8+ T cells recognize Mycobacterium tuberculosis-derived protein antigens and contribute to protection against infection (MPF6P.744)
Authors: Shang Shaobin , Bian Yao , Siddiqui Sarah , Zhao Jie , Wang Chyung-Ru
Published: January 2023

MHC Ib-restricted CD8+ T cells have been implicated to contribute to host defense during Mycobacteria tuberculosis (Mtb) infection, but the relative contribution of various MHC Ib-restricted responses has yet to be defined. In this study, we used mice that lack MHC Ia (Kb-/-Db-/-, DKO), MHC Ia/M3 (Kb-/-Db-/-M3-/-, TKO), or β2m (β2m-/-) to study the role of M3-restricted and other MHC Ib-restricted T cells in immunity against Mtb infection. Unlike their role in Listeria infection, we found that M3-restricted CD8+ T cells only represent a small proportion of the CD8+ T cells responding to Mtb infection. This finding highlights the differential role of various MHC Ib-restricted responses in immunity against distinct microbial pathogens. Non-M3, MHC Ib-restricted CD8+ T cells expanded preferentially in the lungs of TKO mice and expressed higher level of KLRG1 compared to MHC Ia-restricted CD8+ T cells. These CD8+ T cells produced IFNγ, TNFα and IL-2 upon stimulation with Mtb-derived protein antigens in a β2m-dependent but TAP-independent manner. TKO mice had similar bacterial loads in tissues as DKO and wildtype mice, but lower bacterial loads than β2m-/- mice. Our findings indicate non-M3, MHC Ib-restricted CD8+ T cells can specifically recognize Mtb protein antigens and contribute to host immune response to Mtb. Targeting these MHC Ib-restricted CD8+ T cells would facilitate the design of better vaccines against Mtb, due to the limited polymorphism of MHC class Ib molecules.
Invariant natural killer T (iNKT) lymphocytes promote the development of intestinal tumors (TUM2P.914)
Authors: Cardell Susanna , Wang Ying , Sedimbi Saikiran , Löfbom Linda , Porcelli Steven
Published: January 2023

CD1d-restricted NKT cells are known as potent regulatory cells of immune responses. Invariant NKT cells, which are activated by the artificial lipid ligand alpha-galactosylceramide, have anti-tumor functions in several murine experimental models, and iNKT cells can naturally protect against spontaneous tumors, as recently described in two different murine models. APCmin/+ mice develop intestinal tumors due to a mutation in the adenomatous polyposis coli gene, mutated in human colorectal cancer. To investigate whether iNKT cells influence tumor formation in APCmin/+ mice, this strain was crossed with Jalpha281-/- mice or CD1d-/- mice that lack iNKT cells or all NKT cells, respectively. We show that absence of iNKT cells decreased the number of tumors/polyps more than 50%, compared with the iNKT cell sufficient APCmin/+ control litter mate mice. This indicates that iNKT cells naturally promote tumor development in APCmin/+ mice. Moreover, we investigated the modulation of tumor development by iNKT cell directed therapy in preclinical studies. This study was performed by systemically activating iNKT cells with aGalCer, its TH2 skewing analogue C20:2 or the TH1 skewing analogue C-glycoside. The results revealed a decreased number of tumors after treatment with aGalCer. In contrast, in particular the TH2 skewing ligand C20:2 promoted tumor development, which suggests that iNKT cells may promote tumors through TH2 cytokines in this model.
Modeling natural killer cell responses against lung cancer (TUM2P.917)
Authors: Schmidt Leah , Jacks Tyler
Published: January 2023

Natural killer (NK) cells are innate lymphocytes that recognize and kill transformed and pathogen-infected cells. Thought to act as a first line of defense in immune surveillance, NK cells secrete inflammatory cytokines that stimulate adaptive immune responses. Thus, the quality of NK cell responses may impact the potency of adaptive responses against cancer and, perhaps, the efficacy of immunotherapy in individual patients. In a Kras and p53-driven model of autochthonous non-small cell lung cancer, we observe dysfunctional phenotypes in infiltrating NK cells, similar to what is seen in human disease. We have adapted this model to study NK cell responses against lung cancer in a targeted manner, by inducing expression of potent model NK cell ligands in developing tumors. Induced expression of the MCMV surface protein, m157, triggers robust lymphocytic infiltration in tumors within 6 days of induction. Infiltrates include NK cells as well as large numbers of T cells and B cells. Recruitment of T cells and B cells upon ligand induction is dependent on NK cells, and on the interaction of m157 with the Ly49H activating receptor expressed on NK cells. This work demonstrates that NK cells residing in or near established tumors can be stimulated by activating ligands, and that this stimulation leads to the recruitment of adaptive immune responses at the site of tumors. Future work will dissect the mechanisms by which activated NK cells recruit T cells and B cells to tumors.
Nk cells induce tumor cell resistance to Nk cell cytolysis (TUM2P.918)
Authors: Basse Per , Fan Ryan , Bailey Lisa , Wynn Jay , Lotze Michael
Published: January 2023

Live cell microscopy of murine NK cells co-cultured with tumor cells revealed substantial lysis of tumor cells by NK cells that diminished dramatically within the first few hours. The kinetics of conjugate formation and NK cell cycling between targets remained unchanged. When NK cells were re-isolated and placed in co-culture with fresh tumor cells, substantial lysis ensued, indicating that the reduction in killing was not due to exhaustion of the NK cells. When tumor cells were isolated from co-cultures and re-mixed with fresh NK cells, normal cycling and conjugate-formation with tumor cells was observed, but this rarely resulted in cytolysis. Tumor cells lost plastic-adherence and underwent replicative senescence. When removed from co-culture, most of the still viable tumor cells regained their normal adherent morphology, growth rate, and sensitivity to NK-mediated lysis. Pre-incubation of tumor cells with cell-free supernatants from NK cell or NK+tumor cell cultures reduced the killing by 60-90% compared to that of non-preincubated tumor cells. The changes in MHC expression correlated poorly with the observed changes in tumor cell resistance to NK cell-mediated cytotoxicity. Pre-incubation of tumor cells with NK cells from IFNγ-KO donors or supernatants of IFNγ-deficient NK cells did not cause MHC upregulation, but induced almost complete resistance in tumor cells to NK cell-mediated cytolysis. Both IFNγ and MHC upregulation are dispensable for the observed protection.
The complement C5a receptor facilitates cancer metastasis by altering T cell responses in metastases-targeted organs (TUM2P.883)
Authors: Sharma Sharad , Chitala Navinkumar , Vadrevu Suryakumari , Sharma Priya , Cleveland Clayton , Riediger Linley , Fairlie David , Gorczyca Wojciech , Almanza Othon , Karbowniczek Magdalena , Markiewski Maciej
Published: January 2023

The role of complement in cancer metastasis has not yet been recognized. In addition, involvement of adaptive immunity in preventing metastasis to the vital organs is unclear. Here we show that the complement anaphylatoxin C5a receptor (C5aR) facilitates metastasis by suppressing effector CD8+ and CD4+ T cell responses in the lungs. Mechanisms of this suppression involve recruitment of immature myeloid cells co-expressing TGF-β and IL-10 to this organ. TGF- β and IL-10 favored generation of inducible T regulatory (iTreg) cells and Th2 responses that rendered CD8+ T cells dysfunctional. Importantly, pharmacological blockade of C5aR or its genetic ablation in C5aR-deficient mice prevented metastases in a syngeneic model of breast cancer. CD8+ T cell depletion abolished beneficial effect of C5aR blockade on metastasis suggesting that presence of CD8+ T cells is required for C5aR blockade-mediated protection from metastasis. These data reveal a surprising and contrasting role of C5aR-signaling in cancer metastasis that enables iTreg cell generation and suppress T cell responses. Furthermore, present study opens a new avenue for developing complement-based immunotherapies to prevent or reduce cancer metastasis.
Immunogenicity of Teffector and Treg epitopes from H7N9 avian-origin influenza: impact of cross-reactivity with the human genome (VAC2P.934)
Authors: Liu Rui , Moise Leonard , Sangare Kotou , Desrosiers Joe , Martin Bill , Groot Annie
Published: January 2023

Avian-origin H7N9 influenza is a novel influenza A virus (IAV) that emerged in humans in China in 2013. Using immunoinformatic tools, we found this virus has lower T cell epitope content than other IAV subtypes overall, and that some epitopes bear homology to human sequences. We hypothesized that these features may make the virus poorly immunogenic. To evaluate the reactivity of H7N9 epitopes, we synthesized four sets of H7N9 sequences with the following features: (i) unique to H7N9, (ii) cross-conserved with other IAV, (iii) cross-conserved with human genome (HG) and other IAV, and (iv) human analogs of influenza epitopes. 91% of sequences bound to at least one of five common HLA II alleles and 61% displayed strong binding. Epitopes that are conserved with other IAV induced higher IFNγ responses; conversely, epitopes that were conserved with human genome (even though equally well conserved with IAV) induced 3 fold lower IFNγ responses, similar to the level induced by H7N9 unique epitopes and to human analogs of influenza epitopes. Stimulation of human PBMCs from healthy donors with HG cross-conserved epitopes increased the frequency of CD4+CD25+FoxP3+ T cell population. Low overall T cell epitope content and the presence of HG cross-conserved H7N9 T cell epitopes that activate Tregs may explain the very low sero-conversion rates to newly developed H7HA unadjuvanted vaccines. Exploration of alternative vaccine development approaches is warranted for this new influenza virus.
Frailty correlates with age and inflammatory markers but not flu vaccine response in an older veteran cohort (VAC2P.937)
Authors: Canaday David , Oswald Douglas , Hoffman Sarah , Robinson Janet , Tumpey Terrence , Pearce Melissa , Banks Richard , Aung Htin , Burant Christopher , Higgins Patricia , Hornick Thomas
Published: January 2023

Frailty is a geriatric syndrome associated broadly with declines in health and function in older adults. Others have reported associations between frailty and changes in immune function including increased markers of inflammation and impaired response to influenza vaccination. In the current study we have studied a cohort of 118 older veterans with a median age of 81. They underwent Fried frailty testing and received seasonal trivalent inactivated influenza vaccine with blood drawn pre- and post-vaccination. Pre-vaccination serum inflammatory markers (IL-6, TNFR1, TNFR2), anti-flu cell mediated immunity, and pre- and post-hemagglutinin inhibition (HAI) titers were determined. As has been previously described, frailty correlated with age (r=0.32 p<0.001) and serum IL-6 (r=0.41 p<0.001). We extend the markers of inflammation to demonstrate that frailty also correlates with TNFR1 (r=0.32 p=0.001) and TNFR2 (r=0.38 p<0.001). Contrary to a prior report we did not find a correlation between frailty and change in HAI titers after vaccination. We have an estimated power from our cohort size to detect a medium effect size of 0.23. It is possible frailty had a negative effect on vaccine response, but our sample size was too small to detect it although there was not even a trend to suggest that. Therefore, we did not find reduced antibody responses after influenza specifically in the frail veterans.
Activation of the COX2/PGE2 axis by immune effector cells promotes the self-limiting nature of type-1 immunity in cancer tissues (VAC3P.939)
Authors: Wong Jeffrey , Obermajer Natasa , Edwards Robert , Bartlett David , Kalinski Pawel
Published: January 2023

Type-1 immune responses, mediated by IFNγ and TNFα-producing CTLs, Th1, and NK cells, are essential for effective anti-tumor immunity. Despite recent advances in the induction and stabilization of these responses by cancer immunotherapies, the clinical success of these approaches remain limited. Here, we report that the activation of type-1 immunity within the human tumor environment initiates IFNγ- and TNFα-dependent counter-regulation, driven by amplification of prostaglandin E2 (PGE2) and the key regulator of PGE2 synthesis, cyclooxygenase 2 (COX2). We demonstrate that activated NK cells and CTLs induce IFNγ/TNFα-mediated over-expression of indoleamine 2,3-dioxygenase (IDO), inducible nitric oxide synthase (iNOS/NOS2), IL-10, and COX2 by tumor-associated myeloid-derived suppressor cells (MDSCs). Importantly, this self-limiting suppressive feedback driven by type-1 immunity could be eliminated not only by neutralization of IFNγ and TNFα, the factors critically required for the anti-tumor activity of immune effector cells, but also by COX2 blockade, which counteracted the IFNγ/TNFα-driven enhancement of all other suppressive factors, amplifying the therapeutic potential of intratumoral type-1 immunity. Our data demonstrate an intrinsic mechanism underlying the self-limiting character of type-1 immunity within the human tumor environment, and provide rationale for targeting the COX2/PGE2 axis to enhance desirable type-1 responses for cancer immunotherapy.
Aberrant differentiation and exhaustion of adenovirus vaccine-elicited CD8 T cells primed in the absence of CD4 T cells (VAC3P.940)
Authors: Provine Nicholas , Larocca Rafael , Penaloza-MacMaster Pablo , Borducchi Erica , Haining W. , Barouch Dan
Published: January 2023

Replication-incompetent recombinant adenovirus (Ad) vectors have garnered considerable interest as candidate vaccine platforms for an array of diseases. However, the efficacy of such vaccines in settings of impaired CD4 T cell function remains poorly understood. We demonstrate that monoclonal antibody-mediated depletion of CD4 T cells prior to Ad immunization of C57BL/6 mice impaired expansion of responding CD8 T cells and led to aberrant differentiation and exhaustion. The absence of CD4 T cells resulted in a failure of antigen-specific CD8 T cells to differentiate into KLRG1hiCD127lo effector cells. This phenotypic defect corresponded to reduced ex vivo cytotoxicity and decreased expression of IFN-γ and TNF-α. Strikingly, CD8 T cells primed in the absence of CD4 T cells expressed elevated levels of the inhibitory molecules PD-1, LAG-3, and Tim-3. Consistent with this, these cells were identified as exhausted using gene expression profiling and gene set enrichment analysis. Blockade of PD-1 signaling by administration of anti-PDL-1 antibody at the time of immunization rescued expansion of antigen-specific CD8 T cells primed in the absence of CD4 T cells. This treatment also rescued differentiation into KLRG1hiCD127lo effector cells, and corrected defects in expression of IFN-γ and TNF-α. Collectively, these data suggest a novel mechanism of CD4 T cell help whereby CD4 T cells prevent the exhaustion of vaccine-elicited CD8 T cells by modulating PD-1 signaling during priming.
Dendritic cell subversion permits viral persistence (VIR1P.959)
Authors: Popkin Daniel
Published: January 2023

Persistent viral infections share the ability to combat the normally protective function of dendritic cells (DC). We and others postulate that DC inhibition is necessary for viruses to persist in spite of an adaptive T cell response (e.g. HIV, HBV, and HCV). However, resolving whether viral subversion of DC (directly or indirectly) actually permits systemic viral persistence in vivo has not been determined. We developed a model system that allows us to inhibit viral replication in specific cell types to answer this question. We used cell-specific genetic deletion of the host site-1 protease (S1P) to determine a requirement for DC infection in viral persistence. The host site-1 protease (S1P) is required for replication of lymphocytic choriomeningitis virus (LCMV) and other hemorrhagic arenaviruses in addition to HCV replication. We deleted S1P in DC (S1PDCnull) to selectively eliminate viral replication in DC. Three weeks post LCMV infection, S1PDCnull mice had NO detectable viremia in contrast to >5 logs of infectious virus present in WT controls. Thus, productive infection of DC was required for viral persistence. This genetic deletion occurs in <1% of the cells LCMV infects. Thus, overall viral loads were equivalent during the 1st week of infection between WT and S1PDCnull mice. However, S1PDCnull mice possessed more polyfunctional T cells which we reason led to a rapid loss of viral persistence.
Using public T cell responses to predict cytomegalovirus infection status in silico (VIR1P.1004)
Authors: Emerson Ryan , Rieder Mark , Carlson Christopher , Gravley Jenna , Hansen John , Robins Harlan
Published: January 2023

The ability of a host’s adaptive immune system to address an incipient infection relies on the previous existence of T cells which have randomly generated an appropriate antigen-specific T cell receptor (TCR). The nature of V(D)J recombination ensures that despite the enormous space of potential TCRs, there are many TCR specificities that are common in the naïve T cell repertoires across individuals. Hence, many pathogenic antigens are known to induce a “public” T cell response, in which the same functional TCR is found to be immunodominant in multiple individuals. Despite historical limitations on sequencing depth and the limited size of investigational cohorts, previous work on public T cell responses has identified many individual examples of public T cell responses to diseases including CMV, EBV, influenza and others. Using high-throughput TCRβ sequencing, we performed deep characterization of the TCR repertoires of 650 healthy subjects (356 CMV- and 294 CMV+) to enable massively parallel discovery of public T cell responses to CMV. We have identified in one experiment hundreds of CMV-associated public T cell responses. Using cross-validation, we demonstrate that taken collectively public T cell responses to CMV contain enough information to predict CMV status in new subjects using TCR sequencing alone, showing for the first time that assaying the host adaptive immune system could be a valid diagnostic strategy in the context of infectious disease.
IFNγ protects neonatal neuronal precursor cells during measles virus infection of the brain (VIR1P.1007)
Authors: Fantetti Kristen , O'Donnell Lauren
Published: January 2023

In the developing brain, self-renewing neural stem/progenitor cells (NSPC) differentiate into neurons or glia. NSPC survival and differentiation can be altered by neurotropic viruses and by the anti-viral immune response. We hypothesize that interferon-gamma (IFNγ), a pro-inflammatory cytokine required for non-cytolytic clearance of many neurotropic viruses, alters NSPC proliferation and cell fate in measles virus (MV)-infected neonates. Two transgenic mouse lines were used: CD46+ express human CD46, a receptor for MV, under the neuron-specific enolase promoter, thereby restricting MV infection to mature neurons; CD46+/IFNγ-KO lack IFNγ. In our model, NSPCs are not infected, allowing us to evaluate the impact of solely the immune response on NSPCs. Flow cytometry and western blot were used to quantify effects on cell number and IFNγ-mediated signaling, respectively. Mice were mock- or MV-infected on postnatal day 2 and sacrificed 3, 7, and 10 days post-infection. Whole brain isolates were double-labeled with BrdU and markers for NSPCs (nestin), early neuronal lineage (CD24), and glial lineage (GFAP, O4). CD24+ cells showed the greatest cell loss at 7 dpi in both genotypes. NSPCs were reduced in CD46+/IFNγ-KO mice at 3 and 7 dpi, but unaffected in CD46+ mice. Thus, IFNγ may protect against NSPC, but not neuronal, loss. Current experiments aim to address the cell fate of NSPCs exposed to the antiviral immune response and define mechanisms of NSPC loss in CD46+/IFNγ-KO neonates.
Altered lipid metabolism influences course of hepatitis C virus infection and hepatocyte cytokine production (VIR1P.1011)
Authors: Narayanan Sowmya , Hoehn Kyle , Kenwood Brandon , Nieh Albert , Tosello-Trampont Annie , Davis Christine , Caldwell Stephen , Hahn Young
Published: January 2023

Hepatitis C virus (HCV) is remarkably efficient in establishing persistent infection leading to the development of chronic inflammatory liver diseases. Lipids play a central role in regulating inflammatory responses and are necessary for the replication and assembly of HCV. Fatty acids are the building blocks of lipids and are synthesized via a rate-limiting step catalyzed by the enzyme acetyl-CoA carboxylase (ACC). To determine the role of lipid synthesis in regulating innate inflammatory responses and HCV biogenesis, we examined the effect of ACC inhibition on HCV replication and cytokine production. Inhibition of ACC decreases intracellular viral RNA in Huh7.5.1 cells infected with JFH-1, a genotype 2a strain of HCV. Surprisingly, ACC inhibition slightly decreases IL-1β transcription and results in comparable production of IL-8, IL-13, and IP-10 in HCV-infected hepatocytes relative to vehicle controls. Further studies using electron and fluorescent microscopy demonstrate a reduction in the number of lipid droplets, which was accompanied by an accumulation of viral protein. These data highlight the paradoxical immune response of HCV-infected hepatocytes, where the continued accumulation of viral antigen fails to stimulate synthesis of inflammatory cytokines. The potential role of lipids in dampening immune responses in HCV-infected hepatocytes may provide an important link between host factors driving infection and suppression of immune responses in chronic infection.
Sialic acid binding preference of influenza A viruses regulates pathological leukocyte recruitment (VIR2P.1012)
Authors: Obar Joshua , Zickovich Julianne , Graham Amy
Published: January 2023

Influenza A virus (IAV), a seasonal respiratory pathogen, has a high capacity to cause pandemic infections resulting in severe lung pathology. Multiple studies using pathological IAV strains have demonstrated the importance of macrophages and neutrophils, but whether the pathological inflammation induced by different IAV isolates is identical has not been directly compared. Here we demonstrate that while both A/WSN/33 (WSN) and A/PR/8/34 (PR8) influenza viruses cause significant immunopathology in mice the inflammatory milieu differs between the two isolates. Specifically, PR8 infection was associated with greater neutrophil recruitment and CCL4 and CXCL2 expression; in contrast, WSN infection was associated mast cell activation, recruitment of a unique Ly6ghigh Ly6chigh monocyte population, and elevated levels of IFNα, G-CSF, and CCL2. One difference between WSN and PR8 is their hemaggultinin molecules preferentially bind to α2,6- and α2,3-linked sialic acids, respectively. Using a recombinant WSN strain whose sialic acid binding preference is switched to α2,3-linkages, we observed minimal mast cell activation and a switch to a more neutrophilic inflammation. Thus, sialic acid binding preference of IAV isolates has clinically important alterations to the inflammatory cascade(s) activated. These findings have important implications for our ability to therapeutically target the host inflammatory response to limit morbidity following influenza virus infection
Respiratory syncytial virus (RSV)-specific cell-mediated immune responses in human peripheral blood (VIR2P.1025)
Authors: Kim Young-In , Harrison Lisa , DeVincenzo John
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections and hospitalization in infants, but no vaccine exists, primarily because the immune response is poorly understood and immune correlates of protection have not been adequately defined. Antiviral lymphocytes, along with secreted immunoglobulins (Ig) are likely the most important components of effective anti-RSV immunity RSV-specific assays to quantify the effective cell-mediated immune response to naturally-occurring RSV infections are imperative. We therefore quantified IFN-γ secreting human peripheral blood mononuclear cells (PBMCs) with and without stimulation by whole RSV from different clades, and with highly purified RSV-peptide pools. PBMCs from donors demonstrated between-donor variable levels of immune response to the RSV peptide pools and the whole virus preparations. Robust IFN-γ T-cell responses were observed at 72 hours in all PBMCs, thus allowing a single time point assay evaluation. Higher responses were seen in the CD4+ population of PBMCs compared to the CD8+ population. Responses of CD8+ PBMCs to whole virus preparations and to mock infection control preparations were similar and responses to RSV peptide pools were not statistically significant from the negative peptide control. IFN-γ based ELISPOTS and FACS assays are promising tools to quantify RSV-specific T-cell-mediated immune responses in human blood.
Differential antiviral immune response in swine model of influenza vaccine-associated enhanced respiratory disease. (VIR2P.1026)
Authors: Loving Crystal , Vincent Amy , Gauger Phillip , Hughes Holly
Published: January 2023

Genetic drift and introduction of human seasonal influenza A virus (IAV) have resulted in six H1 phylogenetic clusters in swine. In humans reports suggest that prior vaccination against seasonal IAV (pre-2009) may result in enhanced disease following infection with pandemic 2009 IAV (pH1N1). Similarly, vaccine-associated enhanced respiratory disease (VAERD) has been reported in pigs that were vaccinated with one antigenic H1 subtype (human-like H1δ1) and subsequently challenged with pH1N1. To investigate the immune response associated with VAERD to delineate a mechanism for enhanced disease pigs were vaccinated with adjuvanted, inactivated H1δ1 and subsequently challenged with pH1N1 (Vx/Ch). Non-vaccinated (NV)/Ch and NV/non-challenged (NC) were included. Vaccination elicited IAV-specific IgG that cross-reacted with pH1N1 but did not neutralize infectivity. Proinflammatory cytokines IL-8, IL-1β and IL-6 were significantly elevated in lungs of Vx/Ch pigs compared to NV/Ch pigs. However, IFNα levels were significantly lower in lungs of Vx/Ch pigs compared to NV/Ch pigs indicating a difference in activation of antiviral immune pathway(s). IFNα mRNA levels were the same in tracheal epithelial cells collected from Vx/Ch and NV/Ch pigs; however, IFNα mRNA levels in alveolar macrophages (AM) from NV/Ch pigs were significantly increased over Vx/Ch pigs. We hypothesize vaccine-potentiated immunopathology through dysregulation of AM innate responses potentiates disease.
The role of T cell leptin signaling in 2009 pandemic H1N1 influenza infection mortality in obese mice (VIR2P.1013)
Authors: Milner J. , Meyers Matthew , Sheridan Patricia , Mancuso Peter , Beck Melinda
Published: January 2023

Obese humans are more susceptible to death from infection with the 2009 pandemic H1N1 influenza virus (pH1N1) compared with healthy weight individuals. To identify explanatory mechanisms, we utilized a mouse model of obesity and pH1N1 infection. Diet-induced obese C57BL/6J male mice and lean control mice were intranasally infected with influenza A/Cal/07/09 (pH1N1). Obese mice exhibited an 80% mortality rate, whereas no lean mice succumbed. During the infection, obese mice exhibited similar levels of lung viral titers but had elevated levels of lung inflammatory cytokines and greater lung airway damage. Further, obese mice had fewer activated lung CD4+Foxp3+CD103+ regulatory T cells. As a potential mechanism for the heightened inflammatory responses in the lungs of obese mice, we addressed the role of leptin, an immunomodulatory hormone chronically elevated in an obese state. Obese mice had more than twice as much leptin in serum and lung airways during the pH1N1 infection compared with lean mice. Given the regulatory role of leptin in T cell activity, we utilized mice lacking functional leptin receptor in T cells (LepRT-/-). LepRT-/- and LepRTfl/fl littermate controls were maintained on a high fat or low fat diet for 15 weeks. Preliminary studies indicate that disruption of leptin signaling in T cells limits pH1N1 mortality and infection severity in obese mice. Therefore, leptin signaling in T cells may be a critical mediator of pH1N1 severity in obese mice.
Deciphering and correcting impaired respiratory virus-specific immunity caused by vitamin A deficiency (VIR2P.1017)
Authors: Penkert Rhiannon , Surman Sherri , Jones Bart , Sealy Robert , Neale Geoffrey , Rudraraju Rajeev , Hurwitz Julia
Published: January 2023

Vitamin A deficiency (VAD) is a leading contributor to pediatric morbidity and mortality worldwide. Recently we have shown that VAD in mice impairs mucosal IgA responses in the upper respiratory tract during both paramyxovirus and influenza virus vaccination, thus impeding a first defense against these pathogens at their point of entry. The key vitamin A metabolite responsible for normal immune function is retinoic acid (RA). Previously, RA was believed to be produced predominantly by gut dendritic cells expressing aldehyde dehydrogenase (ALDH1A), an enzyme required for metabolism of RA from RA precursors. However, we found that epithelial cells of the respiratory tract autonomously produce ALDH1A. These cells also produce IL-6 and GM-CSF and can facilitate IgA production from activated splenocytes in vitro. Further in vitro analyses demonstrated that outcomes of vitamin A supplementation were affected by a variety of factors including concentration and type of vitamin A metabolite, as well as cells targeted. Based on these results, we supplemented a respiratory virus vaccine with a single intranasal dose of retinol, retinyl palmitate, or a combination of IL-6 and GM-CSF in VAD mice, and observed enhanced virus-specific mucosal IgA. In total, our data provide insight into the role of vitamin A in virus-specific immunity, while defining feasible methods for the correction of impaired mucosal immune responses in individuals with VAD.
Comparison of messenger RNA expression profile of antiviral innate immune response genes in peritoneal macrophages from BALB/c and C57BL/6 mice infected with ectromelia virus (INM3P.413)
Authors: Toka Felix , Dolega Patryk , Mielcarska Matylda , Bossowska Magdalena
Published: January 2023

Although a few mechanisms have explained how, for instance ectromelia virus (ECTV), is successful in causing fatal disease in certain strains of mice and how other strains are not susceptible, a global transcription overview of innate immunity genes is not entirely known. Poxviruses have evolved several mechanisms to avoid immune response of an infected host. Here we have studied the innate gene transcriptional response of BALB/c and C57BL/6 mice peritoneal macrophages to infection with ECTV Moscow strain (ECTV-Mos). Indeed, assessment of four categories of antiviral innate immune response receptors, downstream signalling and responsive components revealed generalized down regulation of many genes in both strains of mice. Of the 84 genes assessed in each mouse strain only 3 and 15 were significantly upregulated in BALB/c and C57BL/6 mice, respectively. The remaining genes were more or less down-regulated. Surprisingly, type I interferon genes were substantially upregulated. The observed upregulation strongly correlated with protein expression in the case of cathepsin genes as well as TLR genes. ECTV-Mos inhibits receptors and signalling components of the innate immune response in mice. Results provide an insight into initial factors that presumably lead to an ineffective antiviral immune response of BALB/c and C57BL/6 mice in the light of infection with ECTV.
Regulation of necroptosis as a viral immune antagonistic mechanism (INM3P.361)
Authors: Hartmann Boris , Albrecht Randy , Marjanovic Nada , Sealfon Stuart
Published: January 2023

Regulation of cell death is an important aspect of immune responses to human pathogens. We observed rapid mRNA degradation in human primary dendritic cells (DC) after infection with seasonal but not pandemic H1N1 influenza A viruses. Investigating the mechanism underlying this phenomenon, we found that virus-induced cell death is responsible for cellular RNA degradation. Our data showed that infection with seasonal H1N1 influenza induces caspase independent but RIPK3 dependent necroptosis in primary human DCs as early as 4 hours post infection. Additionally, we found that necroptosis induction is dependent on de novo viral RNA synthesis. To elucidate which viral proteins mediated necroptosis regulation, DC were infected DCs with recombinant viruses encoding segments from a pair of influenza viruses that differed in inducing necroptosis. These DC cell death assays indicated that HA or NA play a pivotal role in necroptosis regulation. Differences in necroptosis induction observed between the seasonal and pandemic influenza strains in DCs, the main antigen presenting cells in antiviral immunity, could have a significant impact on the adaptive immune response and offer a potential explanation for differences observed in clinical outcomes of infection with pandemic or seasonal influenza virus strains.