bluuebunny/crossref_metadata_2025 huggingface dataset:
IFN-γ exerts antiviral activity against polyomavirus infection (42.14)
Authors: Wilson Jarad , Lin Eugene , Pack Christopher , Hadley Annette , Lukacher Aron
Published: January 2023

CD8 T cells use both cytolytic and noncytolytic effector mechanisms to eliminate infectious pathogens. Using the mouse polyoma virus (MPyV) system, we have previously shown that TNFa, Fas/FasL, and the perforin/granzyme pathways are dispensable for viral control and susceptibility to MPyV-induced tumors. Here, we investigated whether IFN-γ may be an important effector mechanism for limiting viral burden and tumorigenesis. Using Western analyses for early region nonstructural viral T antigens, as well as FACS analyses of cells infected by a novel recombinant MPyV carrying an intra-T antigen HA epitope tag, we found that IFN-γ reduces expression of viral T antigens in a dose-dependent fashion. Compared to MPyV-infected wild type C57BL/6 mice, IFN-γ receptor KO mice had a 1-log higher viral load during acute infection in most organs, as well as during the persistent phase of MPyV infection; notably, 3-log higher viral genome copies were present in the kidneys, a major site of persistence by both murine and human polyomaviruses. This increase in viral burden was not due to a lack of responding cells, as fully functional virus-specific CD8 T cells were found during the entire course of infection in IFN-γR KO mice at levels similar to those seen in wild type mice. Studies are underway to determine if IFN-γ receptor KO mice are susceptible to PyV tumorigenesis and to investigate the importance of IFN-γ as a critical CD8 antiviral effector function in MPyV infection.
Cellular immunogenicity of an Ad35GGRIN+ENV and MVA-CMDR prime-boost vaccine regimen. (42.3)
Authors: Kim Silvia , Currier Jeffrey , Cox Josephine , Excler Jean-Louis , Sayeed Eddy , Robb Merlin , Kim Jerome , Michael Nelson , Marovich Mary
Published: January 2023

Newer HIV-1 vaccine strategies use heterologous prime-boost regimens to stimulate broader immune responses. We tested 2 heterologous vectors with heterologous HIV-1 gene inserts: Ad35GRIN+Ad35ENV and MVA-CMDR. The Ad35GRIN+Ad35ENV vaccine is comprised of two vectors containing sequences from HIV-1 subtype A (GRIN=gag, rt, int and nef and ENV=env). MVA-CMDR contains genes from HIV-1 subtype CRF01_AE (gag, env and pol). Balb/c mice (5/group) were immunized according to the following schema: Group 1:MVA-CMDR-MVA-CMDR; Group 2:MVA-CMDR-Ad35GRIN+ENV; Group 3:Ad35GRIN+ENV-MVA-CMDR; Group 4:Ad35GRIN+ENV-Ad35GRIN+ENV. Immunogenicity testing was performed at 2 weeks post boost: ELISPOT, ICS (CD107a, IFNγ, TNFα and IL-2) and pentamer (H-2Kd) Gag epitope staining were used. A variety of stimuli were used to define responses to inserts and vectors. Insert-specific immune responses were detected in all groups. The best combination was Group 3. Notably, there was a shift from a focused vector response in Group 1 to an insert response in Group 3 (0.78% CD8+INFγ gag pool response and 4.24% CD8+INFγ MVA response in Group 1 vs 5.74% CD8+INFγ gag pool response and 0.61% CD8+INFγ MVA response in Group 3). The relative contributions of vector versus insert are not clear. It appears that the insert response is dominant in the heterologous vector format and that subtle differences in cross-reactive populations can be revealed using the pentamer (H-2Kd) Gag epitope staining.
Investigation of TLR ligand-generated inflammatory signals for the induction of high magnitude memory CD8+ T lymphocyte responses (42.8)
Authors: Nelson Michelle , Johnson Alison , Chu Chin-Fun , Milligan Gregg
Published: January 2023

Most currently used vaccines elicit antibody and CD4+ T lymphocytes, but protection against some pathogens may also require a CD8+ T cell response. We are examining the ability of specific TLR agonists to stimulate a specific third signal, such as IL-12 and type 1 interferons, in bone marrow-derived dendritic cells (BMDC) necessary for appropriate development of antigen-specific memory CD8+ T cells against HSV-2. TLR4 (MPL) and TLR9 (CpG) ligands elicit high IL-12 and low IFN-α/β, while TLR3 (Poly(I:C)) ligands generate the opposite cytokine response. Initial in vitro studies demonstrated that gB peptide-specific CD8+ T cells co-cultured with gB-pulsed, TLR ligand-treated BMDC had a more activated phenotype, but there was little difference in CD8+ T cell proliferation when compared to media-treated BMDC cultures. Upon restimulation, the activated CD8+ T cells produced high quantities of TNF-α, IFN-γ, and granzyme B, but variation between groups was minor. These results suggest the choice of TLR ligand for stimulation had little impact on CD8+ T cells in vitro. Further experiments will focus on examining the lymphoid tissues to which our CD8+ T cells reside, their activation status, cytokine production profiles, and cell surface chemokine receptor expression directed by in vivo delivery of gB-pulsed, TLR ligand-treated BMDC. We believe that different TLR ligands will differentially shape the memory response, in turn, providing greater protection from subsequent infection.
Plant Polysaccharide Activates Macrophage to Clear Francisella tularensis Infections (42.9)
Authors: Skyberg Jerod , Holderness Jeff , Rollins MaryClare , Jutila Mark , Pascual David
Published: January 2023

Pulmonary F. tularensis infections are highly lethal in untreated patients, and current antibiotic regimens are not always effective. Thus, we hypothesize that innate immunity augmentation with natural compounds could improve protective immunity to Francisella and complement antibiotic therapies. Screening of a natural compound library revealed a plant polysaccharide with the ability to activate human γδ T cells. The addition of this plant polysaccharide to both mock- and Francisella-infected murine macrophages enhanced surface expression of CD11b, CD40, CD80 and CD86 along with augmenting production of IL-6, TNF-α, and IL-1β. Plant polysaccharide treatment also augmented clearance of F. tularensis from infected murine macrophages, an effect that was time-, dose-, and nitric oxide-dependent, but independent of MyD88. In addition, this plant polysaccharide could enhance clearance of Francisella from infected primary human macrophages when co-cultured with autologous NK cells, which were found to have elevated expression of IFN-γ mRNA. Therefore, this plant polysaccharide holds potential as a therapeutic agonist that can enhance resistance to F. tularensis infection. This work supported by NIH U54AI-65357, P01AT-04986 and NIH Contract HHSN2662004000009/N01-AI40009.
A novel model to study lethal vs. non-lethal viral encephalitis (43.1)
Authors: Taylor Katherine , Kolokoltsova Olga , Borisevich Vika , Peng Bi-Hung , Smith Jennifer , Estes Mark , Yun Nadya , Poussard Allison , Paessler Slobodan
Published: January 2023

Venezuelan equine encephalitis virus (VEEV) generally causes a mild febrile illness; however, a subset of patients,primarily children,develops lethal encephalitis. The virus requires high level biocontainment (BSL3) limiting the use of many research tools. A model of VEEV-like encephalitis that can be utilized to determine the protective host response to disease development is needed to study the mechanisms of immunoprotection. We hypothesized that intranasal inoculation of TC83, an attenuated strain of VEEV,in α/B TCR KO mice would mimic VEEV encephalitis. Infected α/B TCR KO developed signs of illness including significant weight loss,as compared to controls,but all mice fully recovered from infection. Viral titers at the peak of illness reached surprisingly high levels in the brain (log 9- log 10). These levels were reduced (log 3- log 5). Surprisingly, similar low levels of virus were maintained indicating the ability of TC83 to persist in the brain. Visceral organs had low level of virus present during acute infection only. The reduction of infection occurred in the absence of T cells and detectable neutralizing antibody levels. Infection-induced transcriptomes in the brain of the murine host as well as cytokine protein levels demonstrated elevated levels of IL-12(p40), INF-β, MCP-1, MIP-1α, MIP-1β, and RANTES. TGF-β was below the limit of detection. This model represents a unique situation to study acute viral response and chronic viral persistence in the CNS.
IgD-bearing B cell populations in the channel catfish, Ictalurus punctatus (43.17)
Authors: Edholm Eva-Stina , Sahoo Manoranjan , Bengten Eva , Wilson Melanie
Published: January 2023

Channel catfish, Ictalurus punctatus, express two different IgD+ B cell populations. Catfish IgM+/IgD+ B cells are small and agranular, while IgM-/IgD+ B cells are larger and exhibit plasmablast morphology. The use of cell sorting, flow cytometry and RT-PCR demonstrated that IgD+ B cell expression varies between individual catfish. For example, some catfish have little to no IgD+ B cells in their peripheral blood leukocytes (PBL), while in others the IgM-/IgD+ B cell population can represent as much as 72%. This contrasts to the situation in mammals where IgM-/IgD+ B cells are rare. Also, transfection studies show IgD functions as a typical BCR since Igδ chains associate with accessory molecules CD79a and CD79b. Furthermore, IgM-/IgD+ B cells preferentially express IgL σ chains, while IgM+/IgD+ B cells can express any of the four catfish IgL isotypes. As assessed by 5'-RACE, all membrane IgD transcripts from sorted IgM-/IgD+ B cells contained viable VH rearrangements with no bias in family member usage. However, all secreted IgD transcripts were V-less and began with a leader sequence spliced to the Igδ CH1 domain and it is likely that these transcripts encode for the secreted form of IgD previously found in catfish serum. Importantly, the Igδ leader mediated IgD protein secretion when transfected into catfish B cells. This characterization of distinct IgM+/IgD+ and IgM-/IgD+ B cells in teleosts provides an important first step in understanding the evolution of IgD function.
Characterization of intestinal distension in ELT-2kd Caenorhabditis elegans upon infection with Pseudomonas aeruginosa (43.27)
Authors: Elliott Samantha , Sturgeon Craig , Montgomery Madeline
Published: January 2023

Caenorhabditis elegans has become a popular model for studying innate immunity. Recently, the transcription factor ELT-2, which is homologous to human GATA-4, -5 and -6, has been shown to play a role in the nematode immune response, along with its established role in intestinal development. One characteristic of ELT-2’s role in innate immunity is the accumulation of bacteria and distension of the intestinal lumen when ELT-2 is downregulated in the nematode by RNA interference. However, the mechanism of this distension remains unclear. In this study, we characterize the timeline of intestinal distension in ELT-2kd and ELT-7kd nematodes during Pseudomonas aeruginosa infection. We show that intestinal distension is seen in ELT-2kd but not ELT-7kd nematodes during P. aeruginosa infection. Significant distension in ELT-2kd nematodes is seen within 30 hours of pathogen exposure. A detailed comparison of this distension with survival of the nematodes on the pathogen will be presented. These data provide new insight into the role of ELT-2 in protecting C. elegans against pathogenic bacteria.
Evidence for NK cells in equine endometrial cups (43.29)
Authors: Noronha Leela , Huggler Kate , de Mestre Amanda , Antczak Douglas
Published: January 2023

Placentation during equine pregnancy is distinguished by a unique subset of trophoblast cells that invade the endometrium and form discrete structures termed the endometrial cups. Shortly after the trophoblasts invade, there is a dramatic infiltration of maternal leukocytes which accumulate around the cups. Our lab has previously characterized the infiltrating leukocytes as primarily CD3+ cells with substantial CD4+ and CD8+ subpopulations. Here, we identify expression of the NK cell marker NKp46 in peripheral blood mononuclear cells (PBMC) and endometrial cup lymphocytes (ECL). PCR primers were developed to detect the equine homolog of NKp46 and expression was detected in resting and stimulated lymphocytes using standard PCR. Quantitative real-time PCR primers and standards were used to quantitate expression in CD3+ and CD3- sorted lymphocytes. NKp46 gene expression in CD3- lymphocytes was 10-fold higher than in CD3+ cells. Levels comparable to CD3-depleted lymphocytes were detected in frozen tissue sections of endometrial cups. The highest levels of expression were detected in leukocytes isolated from the endometrium surrounding the cups of five mares during early pregnancy (days 43-46). NKp46 expression levels were 7-43 fold higher in ECL compared to paired PBMC (p=0.04). Coincident CD3γ expression was lower in all ECL compared to paired PBMC (p=0.03). These data provide the first evidence for the presence of NK cells in the equine endometrium during pregnancy.
The puzzle of TIR domain-containing proteins in bacteria (43.3)
Authors: Zhang Qing , Zmasek Christian , Cai Xiaohui , Godzik Adam
Published: January 2023

Toll/interleukin-1 receptor (TIR) domain-containing proteins play important roles in defense against pathogens in both animals and plants, connecting the immunity signaling pathways via a chain of specific protein-protein interactions. Among them is SARM, the only TIR domain-containing adaptor that can negatively regulate TLR signaling. By extensive phylogenetic analysis, we show here that SARM clusters with bacterial TIR domains, suggesting a relatively recent lateral gene transfer from bacteria to animals. We also show evidence of several additional, independent transfer events, none of which, however, survived in vertebrates. TIR domain-containing proteins had been characterized in bacterial pathogens, where they were shown to represent a new class of virulence factors that can suppress animal Toll-like receptor (TLR) dependent host defenses. An evolutionary relationship between the animal SARM adaptor and bacterial proteins with TIR domains illustrates the possible role that bacterial TIR-containing proteins play in regulating eukaryotic immune responses and how this mechanism was possibly adapted by the eukaryotes themselves.
Origin of diversified vertebrate immune signaling and regulation in a basal chordate (43.30)
Authors: Xu Anlong , Yuan Shaochun , Yu Cuiling , Huang Shengfeng , Chen Shangwu
Published: January 2023

To well understand how the cytoplamic network for immune signaling and regulation originated from and evolved among species. Our group has dedicated extensive effort to address this question by studying amphioxus immunity at basal chordate. We first characterized a novel IgSF named as VCP, which could recognize microbes and induced phosphorylation of tyrosines in its intracellular region, as a possible precursor of vertebrate IgSF in lymphocytes. We then showed that amphioxus MyD88 can be utilized by most of the vertebrate-specific TLRs leading to the activation of the NF-κB pathway, which could be negatively regulated by SARM and the duplicated TRAFs. We further found the disparate subcellular localization and signaling activities of two amphioxus FADD homologs by showing that over-expression of amphioxus FADD1 resulted in the formation of death effecter filamentous structures in the cytoplasm and the activation of NF-κB, while translocation of the FADD2 from nuclear to cytoplasm could induce apoptosis. In all, our studies not only showed the evolutionary origin of lymphocyte signaling, the conservation of NF-κB activation and the apoptosis induction, but also revealed the regulation difference of a basic innate immune pathway and the dynamic change of apoptotic network at basal chordate depending on its huge gene expansion.
Phylogenetic and developmental study of CD4, CD8α and CD8β T cell co-receptor homologs in two amphibian species, Silurana (Xenopus) tropicalis and Xenopus laevis (43.5)
Authors: Chida Asiya , Goyos Ana , Robert Jacques
Published: January 2023

Although CD4 and CD8 gene homologs have been identified in many jawed vertebrates, phylogeny and functional conservation of these molecules is not fully understood. We have characterized CD4, CD8α and CD8β gene homologs in two species of the subfamily Xenopodinae, S. tropicalis, whose full genome is sequenced, and X. laevis. Multiple alignments of amino acid sequences, in these two species, as in other nonmammalian species, reveal a poor conservation of the residues of CD4 and CD8α that bind to MHC class II and class I molecules, respectively, suggesting species-specific coevolution. Phylogenetic analysis indicates that all three co-receptor genes are more related to other tetrapods than to bony fish. Furthermore, developmental and cell-specific expression patterns of these genes in X. laevis are very similar to that of mammals. X. laevis CD4 is mainly expressed by peripheral non-CD8 T cells and detected in the thymus at the onset of organogenesis, 5 days post-fertilization (dpf). The CD8β gene is specifically expressed by adult CD8 positive T cells and thymocytes, and is first detected in the thymus and intestine at 6 dpf. TCRα and TCRγ transcripts are also detected at 6 dpf, before TCRβ (9 dpf). TCRγ transcripts cloned from 7dpf are fully rearranged and in frame. This suggests that T cell subsets, possibly γ/δ T cells or even α/β T cells, reside in the intestine before the onset of thymocyte differentiation, and may contribute to mucosal immunity early in development.
The residue 96Gln of chicken CD132 is critical to bind to chicken IL-2 molecule (43.9)
Authors: Gu Jianyou , Huang Zhenyu , Ruan Xizhen , Zhou Jiyong
Published: January 2023

The common cytokine receptor γ chain (γc) is shared by at least six cytokine receptors and plays a critical role in the regulation of immune responses. In this study, the functional domains of chicken γc (chCD132) were mapped with monoclonal antibodies (mAb), a synthetic peptide, and a phage display peptide library. One functional mAb to chCD132 was produced using recombinant rchCD132 ectodomain protein expressed in prokaryotic cells as an immunogen and used to finely map the functional domain of the chCD132 protein. The mimotope of anti-chCD132 mAb, QEHQNL, was identified by phage display. The corresponding native epitope in the chCD132 molecule, Q84E94L95Q96N97L98, was identified by peptide-competitive ELISA. Lymphocyte proliferation and inhibition assays further confirmed that the binding of chIL-279-96 to chCD132 molecule was inhibited by chCD13282-101. Site-specific mutation indicates that chCD13282-101 changed the residue Q96 lacked the ability binding to chIL-2 molecule. These findings demonstrates that chIL-279-96 and chCD13282-101 are the functional domains of the interaction between chIL-2 and chCD132 molecules, and that the residue Q96 within chCD13282-101 is the critical site for its bioactivity and chIL-2-binding.
IL-4 stimulates production of TGF-β-induced (TGFBI) mRNA and protein in T cells but not in monocytes, fibroblasts, or pulmonary epithelial cells (44.16)
Authors: Bocharov Alexander , Luzina Irina , Lockatell Virginia , Highsmith Kendrick , Atamas Sergei
Published: January 2023

TGFBI is an extracellular matrix and an adhesion protein produced by a variety of cell types. It is not known whether cytokines other than TGF-β may regulate TGFBI production. RT-Q-PCR and Western blotting assays revealed production of TGFBI mRNA and protein, respectively, in cultured purified primary monocytes, primary pulmonary fibroblasts, and a pulmonary epithelial cell line A549. Stimulation of these cultures with 300 ng/ml of rhIL-4 did not cause a significant change in TGFBI mRNA after 3, 6, 12, or 24 h, or in TGFBI protein after 24 or 48h, (p > 0.05 in all cases, two separate cell donors tested each on two independent occasions). Primary T cell (> 95% purity) cultured from 4 separate donors and tested each on at least three occasions produced basal TGFBI mRNA and protein, although at the levels 10-15 fold below those observed in other tested cell types. Stimulation of T cells or PBMC with IL-4 revealed time dependent increases in the steady-state level of TGFBI mRNA and protein, but not in TGF-β mRNA. The maximal effect of IL-4 on TGFBI mRNA (2-6 fold increase, p < 0.05) was observed at 3 h and 6 h; and on TGFBI protein at 24 and 48 h of stimulation. The effect of IL-4 on TGFBI production was not attenuated by a TGF-β-blocking antibody 1D11. Thus, IL-4 induces TGFBI production in T cells in a TGF-β-independent fashion. T cells may use TGFBI as an adhesion molecule for binding to known TGFBI receptors integrins αVβ3, αVβ5, and α3β1, collagens, and fibronectin.
Matrix metalloproteases mediate migration of pulmonary T cells in response to influenza virus infection (44.7)
Authors: Baaten Bas , Deiro Mia , Bradley Linda
Published: January 2023

Influenza viruses cause significant annual illness and death with recurrent seasonal epidemics and sporadic devastating pandemics. The efficiency of highly pathogenic influenza viruses has been attributed to potent and aberrant inflammatory responses, which can result in severe immunopathological damage and death in humans. T cells can contribute to pulmonary damage by lysis of infected cells and the production of pro-inflammatory cytokines. Thus, migration of T cells and their effector responses in situ are crucial steps towards immunity, but could also contribute to the high morbidity and mortality associated with pathogenic strains. We show that migration of T cells to efficiently cross the lung endothelial barrier and move through the basement membrane, which is essential for their function, is regulated by matrix metalloproteases (MMPs). MMPs have the proteolytic capability to mediate cleavage of the collagen network that makes up the basement membrane. In vitro, polarized T cells express MMP2 and MMP9 activity. We demonstrate that MMP2 and MMP9 are expressed following infection and that inhibition of activity results in decreased T cell recruitment to the respiratory tract. The discovery of the roles of MMPs in T cell function during influenza virus pathogenesis could elucidate the balance between immunity and pathology and may therefore have considerable therapeutic relevance.
Impact of sickle-cell trait on humoral immunity against Plasmodium falciparum in children living in malaria endemic areas of Mali (45.10)
Authors: Miura Kazutoyo , Diakite Mahamadou , Moretz Samuel , Zhou Hong , Diouf Ababacar , Tullo Gregory , Lopera-Mesal Tatiana , Anderson Jennifer , Fairhurst Rick , Long Carole
Published: January 2023

Previous studies have shown that children with sickle-cell trait (heterozygosity for normal hemoglobin A and sickle hemoglobin S, genotype AS) are resistant to P. falciparum malaria when compared with AA children. Passive transfer studies have established the importance of antibodies against blood-stage parasites. However, the impact of sickle-cell trait on immunity against parasite antigens has not been extensively studied. In a longitudinal study in rural Mali, we followed 73 AS and 126 AA children for an entire 6-month transmission season. Before the transmission season, antibody titers were measured against 4 parasite blood-stage antigens (AMA1, MSP1, EBA175 and MSP2). AS children experienced significantly fewer malaria episodes than AA children. Because there is a significant effect of age on antibody titers, we stratified children in 3 age groups (3-5, 6-8 and 9-11 years). Compared to AA children aged 9-11 years, AS children showed significantly lower antibody titers to all 4 antigens. For the 6-8 years group, AS children showed significantly lower antibody titers to EBA175 and MSP2. No differences were observed between AA and AS children 3-5 years old for any antigen. These data suggest that the antibody titers against these major blood-stage antigens before transmission season are not involved in reducing the incidence of malaria in AS children. The functional activity of antibodies (i.e., in vitro parasite growth-inhibitory activity) will be also presented.
Long-term maintenance of protective memory CD8+ T cells in the absence of CD4+ T cells in mice vaccinated against Blastomyces dermatitidis (45.3)
Authors: Nanjappa Som , Heninger Erika , Wuthrich Marcel , Klein Bruce
Published: January 2023

CD8+- and CD4+- T cell mediated immunity is major contributing factor for defense against pathogenic fungi. Our previous studies have shown that CD8+ T cells in the absence of CD4+ T cells protected vaccinated mice from lethal challenge with Blastomyces dermatitidis. However, it has also been shown that CD4+ T-cell help is required for maintenance of memory CD8+ T cells after viral and intracellular bacterial infections. To explore the requirements for maintenance of CD8+ T cell memory against pathogenic fungi, we tested whether fungal-specific memory CD8+ T cell persist in the absence of CD4+ T cell help. Here, we show that protective CD8+ T cell memory endures without CD4+ T-cell help for at least six months after fungal vaccination in mice. Additionally, we show that type I cytokines (IFNγ and TNFα), which are requisite for mediating protection, remained intact in the memory CD8+ T cells after a recall response to a lethal pulmonary challenge in vaccinated mice. The phenotypic markers CD27, CD62L, CD122, and CD127 of memory CD8+ T cells were similar to those on CD8+ T-cells induced with helper CD4+ T cells. These findings imply that CD4+ T cell help is not required for the generation, maintenance, and recall responses of protective memory CD8+ T cells after fungal vaccination. Our studies have potential implications for the usage of fungal vaccines in immune-compromised hosts.
A Unique Population of Tissue-Memory CD4+ T cells in the Airways after Influenza Infection that is Dependent on the Integrin VLA-1 (45.8)
Authors: Topham David , Chapman Timothy
Published: January 2023

During the immune response to influenza infection, activated T cells are distributed to both lymphoid and extralymphoid tissues, including the infected airways where direct recognition of viral antigen-bearing cells takes place. The collagen binding α1β1 integrin VLA-1 is essential for the development of memory CD8+ T cells in the airways but while expressed by some CD4+ T cells, its significance has not been demonstrated. We investigated the role of VLA-1 on virus-specific CD4+ T cells during and after primary or secondary influenza infection of mice. The proportion of CD4+ cells expressing CD49a (α1 integrin) was low in all tissues sampled during primary infection, but increased in the airways after viral clearance. Furthermore, during the first 24hr of a secondary influenza challenge, the majority of IFN-γ secreting effector CD4+ T cells from the airways were in the CD49a+ population. Airway CD49a+ CD4+ cells also expressed reduced markers of apoptosis compared to CD49a- cells, and fewer memory or effector CD4+ cells could be recovered from airways of α1-/- mice, though lymphoid tissues appeared unaffected. These data suggest VLA-1 expression defines a population of tissue-memory CD4+ T cells that act as rapid effectors upon re-infection, and VLA-1 expression is integral to their accumulation in the airways.
Generation of intestinal effector and memory CD8 T cells following oral Listeria monocytogenes infection (46.1)
Authors: Sheridan Brian , Jellison Evan , Lefrançois Leo
Published: January 2023

The intestinal mucosa contains unique inductive and effector sites designed for antigen presentation and promotion of primary and memory CD8 T cell responses. Thus, generation of adaptive immunity within these tissues likely results in T cell responses with distinct cellular outcomes. Here, we utilized an oral Listeria monocytogenes (LM) infection model in which internalin A is modified to promote invasion of murine intestinal epithelial cells. Oral infection induced a robust CD8 T cell response. In peripheral nonmucosal tissues, the majority of LM-specific CD8 T cells resemble short-lived effector cells (SLEC; CD127- KLRG1+). Surprisingly, the majority of effector CD8 T cells in the intestinal mucosa resembled memory precursor effector cells (MPEC; CD127+ KLRG1-) following oral infection. Splenectomy failed to impact the mucosal or systemic T cell response suggesting an intestinal tissue as the major site of T cell priming. Of the inductive sites examined, only Peyer’s patch (PP) LM-specific CD8 T cells expressed CD69 and CD103. Moreover, expression of these receptors was maintained on memory CD8 T cells from the PP where they reside along the border of the T cell and B cell zones. Contrary to systemic recall via i.v. infection, oral recall resulted in preferential expansion of CD8 T cells expressing both CD127 and KLRG1. The maintenance of a stable memory population uniquely tailored to respond to intestinal pathogens may be a hallmark of oral challenge.
Co-delivery of genetic adjuvants enhances mucosal immune responses induced by PMED DNA vaccination (46.15)
Authors: Franchini Anthony , Reinhart Todd , Stagnar Cristy , Narendran Amithi , Fuller Deborah
Published: January 2023

Mucosal immune responses play a vital role in protecting against natural influenza infection. We have shown that particle mediated epidermal delivery (PMED) of a DNA vaccine induces systemic and mucosal immune responses. PMED results in the direct transfection of epidermal cells, including skin-resident Langerhans cells (LC) to induce an immune response. Inclusion of a DNA plasmid expressing the heat-labile enterotoxin (LT) of E. coli improves systemic immune responses. We hypothesize that LT and other genetic adjuvants would similarly enhance the mucosal immunogenicity of PMED DNA vaccines. In this study, we assessed the ability of LT and the chemokine CCL20, which is involved in attracting dendritic cells and influencing T cell trafficking to the mucosa, for the ability to enhance systemic and lung immune responses. Mice were vaccinated with a influenza A nucleoprotein (NP) plasmid with or without LT and/or CCL20 as a genetic adjuvants. LT, not CCL20, enhanced IFNγ T cell responses in the lung. Th1/Th2/Th17 responses measured by ELISA showed PMED DNA vaccination induced broad T cell responses differently modulated by the adjuvants. Only LT enhanced antibody responses measured by HAI and ELISA in the lung and periphery. Enhanced adjuvant activity was correlated to higher frequency of activated APC (MHCII+/CD80+) in the draining lymph node. These data show LT as an effective genetic adjuvant for enhancing mucosal responses in the lung by modulating APC activation.
Eimeria maxima recombinant Gam82 gametocyte antigen vaccine protects against coccidiosis and augments humoral and cell-mediated immunity (46.19)
Authors: Lillehoj H. , Jang S. , Lee S. , Lee K. , Park M. , Subramanian B. , Sriraman R. , Srinivasan V. , Lillehoj E.
Published: January 2023

Intestinal infection with Eimeria, the etiologic agent of avian coccidiosis, stimulates protective immunity to subsequent colonization by the homologous parasite, whilst cross-protection against heterologous species is poor. This study was designed to assess a purified recombinant protein from E. maxima gametocytes (Gam82) in stimulating immunity against experimental infection with live parasites. Following Gam82 intramuscular immunization and oral parasite challenge, body weight gain, fecal oocyst output, gut lesions, serum antibody response, cytokine production, and lymphoproliferation were assessed to evaluate vaccination efficacy. Animals vaccinated with Gam82 and challenged with E. maxima showed lower oocyst shedding and reduced intestinal pathology compared with non-vaccinated and parasite-challenged animals. Gam82 vaccination also stimulated the production of antigen-specific serum antibodies, induced greater levels of IL-2 and IL-15 mRNAs, and increased splenic lymphoproliferation compared with non-vaccinated controls. These results demonstrate that the Gam82 recombinant protein protects against coccidiosis and augments humoral and cell-mediated immunity.
A role for macrophage IκB kinase in the mucosal adjuvant activity of edema toxin for sublingual vaccines (46.2)
Authors: Jee Junbae , Steiner Haley , Bonnegarde Astrid , Fial Michael , Boyaka Prosper
Published: January 2023

It has been demonstrated that nuclear factor κB (NF-κB) activation by IκB kinase (Ikk)β deletion in macrophages increases pro-inflammatory cytokines responses and enhances pathogen clearance. To address whether this pathway also regulates the mucosal adjuvant activity of edema toxin (EdTx), LysMcre x Ikkβf/f (IkkβΔMø) mice with Ikkβ-deleted macrophages were sublingually immunized with 50µg of Yersinia pestis F1V fusion protein with or without 15µg of EdTx as an adjuvant. Both control and IkkβΔMø mice immunized with F1V+EdTx developed higher levels of serum Abs, i.e., IgG, IgG1, IgG2a and IgA, when compared to mice immunized with F1V alone. The IkkβΔMø mice exhibited higher serum IgG2a levels (and IgG2a:IgG1 ratio) than control counterpart immunized with F1V+EdTx, suggesting that immune responses to F1V are biased toward Th1-type. These mice also showed elevated levels of F1V-specific serum IgA Abs than control littermates. The effect of Ikkβ deletion was more pronounced in secretory Abs since EdTx as adjuvant promoted higher fecal IgA Abs, as well as IgG and IgA Ab titers in saliva and vaginal washes of IkkβΔMø mice. These studies reveal a new role for macrophages and NF-κB in shaping mucosal immune responses induced by EdTx derivative adjuvants via the sublingual mucosa.
Modulation of trafficking receptor imprinting on T cells during Bordetella pertussis infection (46.7)
Authors: Abramson Tzvia , Waheed Sana , Kwong Brian , Tarlton Nicole , Ravindra Dipti , Green Caroline
Published: January 2023

Despite widespread vaccination against Bordetella pertussis (Bp), whooping cough is reemerging. Trafficking receptors (TR) direct cell migration to infection sites, generating protective immunity. To test our hypothesis that pertussis toxin (PTX) modifies dendritic cell (DC) imprinting of TR on T cells, leading to compromised homing to respiratory mucosa, we established a murine model for Bp and B. parapertussis (Bpp)-a strain that lacks PTX and analyzed TR expression by FACS on memory CD4+ CD44+ cells in blood vs lungs during peak infection and recovery (days 5 and 25). While blood memory CD4 cells from Bpp infection revealed 40-100% increased expression of β7, β1, α4, α1, α6, CCR6, CCR5 and CXCR3 at 5 days post infection, while cells from Bp had reduced levels of most TR and expressed α4 and β1 only later at 25 days post infection. However, expression of β7, α4, β1 and CCR6 was elevated in lung memory T cells from Bp infected mice at 5 and 25 days post infection, suggesting a local up-regulation of these molecules. In-vitro co-cultures of lung DC from 25 day post infection with Bp but not Bpp resulted in a significant imprinting of α6, CD103, α4, β1, CCR6, CD106 and LFA-1. In conclusion, TR imprinting on blood memory T cells is compromised at early Bp infection, however imprinting of TR by DC is efficient at later phase of infection supporting the prolonged manifestation of the natural disease.
Helicobacter felis-associated gastric pathology in gnotobiotic mice (46.8)
Authors: Durham Carolyn , Schmitz Julia , Schoeb Trenton , Soltau Thomas , Wolf Kyle , McCracken Vance , Lorenz Robin
Published: January 2023

Human gastric infection with Helicobacter pylori is associated with pathologic consequences, such as gastritis, dysplasia, and adenocarcinoma. This led to the classification of H. pylori as a type-1 carcinogen, but the role of additional non-helicobacter factors in the disease is unclear. In this study we utilized three models in C57BL/6(B6) mice to evaluate the role of gastric microbiota in H. felis disease development: germfree/gnotobiotic(B6.GB), colonized with Altered Schaedler Flora®(B6.ASF), and specific pathogen free model(B6.SPF). Despite similar histological changes over 24 weeks of infection, bacterial colonization of the three groups was different. While B6.GB and B6.ASF mice did not eradicate the helicobacter, the B6.SPF mice cleared it and acquired Lactobacillus spp, indicating that H. felis is sufficient to cause a dysplastic phenotype but alone is not causing histological damage seen in the B6.SPF mice. Over 24 weeks, IL-17 transcription decreased significantly in the B6.SPF and B6.ASF, whereas in B6.GB mice continued to be upregulated. Additionally, B6.SPF mice had very high IgG2c, while in the other two groups, IgG1 was dominant. This data suggests that though H. felis is sufficient to cause gastric inflammation, bacterial clearance is mediated not only by a CD4+ response, but also by other bacteria taking advantage of the gastric environment that has been established by the helicobacter infection.
Toll-like receptor-2 and interleukin-1 signaling in keratinocytes during Staphylococcus aureus infections (46.9)
Authors: Jensen Liselotte
Published: January 2023

Staphylococcus aureus is a significant human pathogen. Subcutaneous infections in mice trigger neutrophil recruitment in an IL-1β, but not IL-1α, dependent manner. Since S. aureus can colonize the skin we explored the role keratinocytes play as first responders to bacterial challenges. In mice, topical application of IL-1, S. aureus or the bacterial cell wall components lipoteichoic acid (LTA) and peptidoglycan (PGN) triggered neutrophil recruitment to the skin surface. In human tissue culture models IL-1α and IL-1β increased mRNA and protein production of the neutrophil chemotactic CXCL1, CXCL2 and IL-8. S. aureus, LTA and PGN induced similar expression profiles in a TLR2 dependent manner. The S. aureus activated chemokine production was preceded by significant IL-1α and IL-1β secretion. Expression of IL-1α was significantly higher than that of IL-1β. IL-1RI neutralization experiments revealed that S. aureus derived LTA and PGN induced chemokine expression requires IL-1RI signaling. Surprisingly, we further found that LTA and PGN initiated chemokine secretion is dependent upon endocrine IL-1α, but not IL-1β, signaling. Our data demonstrate that the innate immune response of keratinocytes is regulated differently than those of subcutaneous tissues. A multifaceted IL-1 scheme involving IL-1α in the skin and IL-1β in underlying tissues may represent a fail-safe system, which protects the host against genetic variation and immune evasion mechanisms developed by pathogens.
Multivariate analysis identifies biomarkers of intestinal inflammation (47.10)
Authors: McBee Megan , Zeng Yu , Parry Nicola , Nagler Cathryn , Tannenbaum Steven , Schauer David
Published: January 2023

Few predictive indicators are available for diagnosis of the chronic intestinal inflammation that characterizes inflammatory bowel disease (IBD). Identification of predictive serum biomarkers may allow for therapeutic intervention prior to disease onset in individuals bearing IBD susceptibility genes. Discriminatory multivariate modeling of 23 cytokines (plus chlorotyrosine [CT] and nitrotyrosine [NT], protein adducts from reactive oxygen and nitrogen species) in serum and tissue from two murine models of colitis was performed to identify disease- associated biomarkers. Citrobacter rodentium infection of C57BL/6J mice and Helicobacter-dependent spontaneous colitis in TLR4 x IL10 deficient mice were used as the acute and chronic colitis models. A subset of serum factors identified in the acute colitis model (NT, G-CSF, KC, IL-12/23p40 and IL-17) was also influential in the serum from mice with chronic disease. Surprisingly, the most influential acute tissue factors (11 total) were also influential in chronic tissue (15 total), and were dominated by Th17- and neutrophil-associated factors. Additionally, multivariate models generated from the influential serum or tissue factors predicted the severity of histological colonic lesions. This study has therefore identified neutrophil and Th17 associated factors in serum as predictive biomarkers of intestinal inflammation, in agreement with the presence of neutrophils and Th17 cells in the inflamed colonic tissue of patients with IBD.
Inducible ablation of Langerhans cells enhances skin immune responses (48.15)
Authors: Kaplan Daniel , Bobr Aleh , Olvera-Gomez Irlanda , Hogquist Kristin , Igyarto Botond
Published: January 2023

Although initially thought to be required for the development of contact hypersensitivity (CHS) responses, recent data obtained from two Langerhans cell (LC)-deficient mouse models has cast this role in doubt. We have previously described huLangerin-DTA mice in which LC are constitutively ablated but other Langerin+ DC are unaffected. In these mice, the absence of LC leads to the development of increased CHS responses. In the second model, muLangerin-DTR mice, LC and Langerin+ dermal dendritic cells (dDC) are inducibly ablated by administration of DT. LC, unlike Langerin+ dDC, repopulate slowly after diphtheria toxin (DT) administration. By comparing early and late time-points after DT administration, it appears that Langerin+ dDC are required for optimal CHS responses and efficient epicutaneous immunization but that LC are redundant. One major difference between these two models that could account for the disparate results is the timing of LC ablation: inducible (muLangerin-DTR) vs. constitutive (huLangerin-DTA). We now report the development of a new line of mice (huLangerin-DTR) in which administration of DT leads to inducible ablation of LC without affecting Langerin+ dDC. The acute and selective ablation of LC in these mice results in increased CHS responses. Thus, LC-mediated suppression does not require their absence during ontogeny or during the steady state. Rather, LC actively limit cutaneous immune responses and may function to terminate ongoing cutaneous immune responses and/or prevent responses against epidermal antigens.
The PD-Ligand Pathway regulates the development, maintenance and function of induced regulatory T cells in vitro and in vivo (49.16)
Authors: Francisco Loise , Salinas Victor , Brown Keturah , Vanguri Vijay , Freeman Gordon , Kuchroo Vijay , Sharpe Arlene
Published: January 2023

Both the PD-1:PD-Ligand pathway and regulatory T cells (Tregs) are instrumental to the maintenance of peripheral tolerance. PD-1 has two ligands, PD-L1 and PD-L2. We demonstrate that PD-L1 plays a pivotal role in the de novo differentiation of regulatory T cells and the maintenance of Treg function. PD-L1-/- APCs minimally convert naïve CD4 T cells to become induced-regulatory T cells (iTregs). Moreover, PD-L1-coated beads profoundly increase the CD4+Foxp3+ Treg population, mediated by attenuation of the Akt-mTOR axis and concomitant with upregulation of PTEN. These PD-L1-iTregs suppress T effector cell proliferation more efficiently than control-iTregs and express high levels of CD25, PD-1, B7-1 and GITR. Furthermore, when control or PD-L1-induced Tregs are sorted and re-cultured with PD-L1-coated beads, PD-L1 enhances Foxp3 expression and suppressive function of iTregs. The obligatory role for PD-L1 in controlling iTreg development and function in vivo is illustrated by a marked reduction in iTreg conversion and rapid onset of a fatal inflammatory phenotype in PD-Ligand-/- Rag-/- recipients of naïve CD4 T cells. Parallel studies with PD-L2 reveal similar effects on the de novo differentiation of iTregs. Thus, the PD-1:PD-Ligand pathway can inhibit T cell responses by promoting both the induction and maintenance of induced Tregs. These studies define a novel mechanism for iTreg development and function, as well as a new strategy for controlling Treg plasticity.
Altered intestinal homing molecule expression prevents effective Treg trafficking in the FVB.mdr1a-/- model of colitis. (49.22)
Authors: Tanner Scott , Lorenz Robin
Published: January 2023

The FVB.mdr1a-/- mouse, lacking the small molecule pump P-glycoprotein, is a commonly used model for the study of spontaneous T cell mediated colitis. Our lab has shown that a defect in regulatory T cells (Treg) in this model could be a cause of disease. One possible mechanism for the defect in immune regulation is the inability of Tregs to traffic to the gut where they are needed to effectively maintain homeostasis. To investigate this, we examined the numbers of Tregs within various lymphoid organs in the FVB.mdr1a-/- mice. While splenic Treg percentages remained similar between knockout and wild type controls, a decrease in Treg percentages was observed within the MLN, Peyer’s patches and lamina propria. We then adoptively transferred FVB effector T cells (Teff) + FVB or FVB.mdr1a-/- Treg cells into FVB.Rag-/- recipients. After 4 weeks, tissue was harvested to monitor Treg trafficking. Significantly fewer Tregs were observed in the lamina propria of FVB.mdr1a-/- recipients (9.47±5.26% vs. 2.83±0.70%). In addition, FVB.mdr1a-/- Treg recipients showed significantly less expression of the gut homing receptors CCR9 (63.36±3.85% vs. 29.69±2.19%) on splenic Tregs and α4β7 (49.85±2.49% vs. 12.26±5.61%) within lamina propria Tregs. Interestingly, an increase in total splenic cell numbers was observed in the recipients of FVB.mdr1a-/- Tregs. This suggests mdr1a deficient Tregs remain in the spleen and are unable to utilize CCR9 and α4β7 to traffic to the intestine.
Defects of Regulatory T Cell function In The Wiskott-Aldrich Syndrome. (49.25)
Authors: Adriani Marsilio , Jones Krystin , Kirby Martha , Anderson Stacie , Silvin Christopher , Wincowitch Stephen , Candotti Fabio
Published: January 2023

Natural Tregs from both WAS patients and WASp-deficient (WKO) mice were previously reported as unable to suppress the proliferation of conventional T-cells in vitro. Because WAS T-cells have a known defect of IL-2 production and this cytokine is important for the generation of Treg cells induced from naïve T-cells, we assessed the efficiency with which WKO naïve T-cells can differentiate in Tregs and observed a significant defect compared to WT. Such defect was not corrected by increasing concentration of anti-CD3 and TGF-β. Preliminary experiments indicated a role for endogenous IL-2 production in the generation of iTregs and pointed again to the IL-2 pathway as a major determinant of the Treg cells defects in WAS. Furthermore, we previously observed that the impaired suppression of T-cell proliferation could be partially rescued by pre-activation of nTregs. Because activated nTregs are known to be able to induce B-cell apoptosis, we set out to evaluate the function of WKO nTregs on B-cells. We found that pre-activated WKO Treg cells fail to effectively suppress B-cell proliferation. The reduced granzyme-B secretion by WKO nTregs compared to WT, suggest that the defective suppressive activity on B-cells may be due to a degranulation defect of WKO cells. In summary, our data suggest that both natural and induced arms of T regulatory cells are defective in WAS and that defective peripheral Treg induction may be involved in the pathogenesis of autoimmunity in this disease.
Interleukin-35-mediated induction of a novel regulatory T cell population (49.7)
Authors: Collison Lauren , Henderson Abigail , Chaturvedi Vandana , Turk Mary , Vignali Dario
Published: January 2023

Regulatory T cells (Tregs) play a critical role in the maintenance of immunological self tolerance and immune homeostasis. Due to their potent immunosuppressive properties, the ex vivo generation of regulatory T cells is an important goal of immunotherapy. We show that treatment of conventional T cells (Tconv) with the inhibitory cytokine IL-35 induces IL-35 expression and confers suppressive capacity, in the absence of Foxp3, IL-10 and TGFβ expression, upon Tconv cells. IL-35-dependent induced Tregs, termed iTR35, are strongly suppressive in vitro and in in vivo models including IBD, EAE, and a B16 tumor model. Treg-mediated suppression induces the generation of iTR35 in an IL-35- and IL-10-dependent manner in vitro and within the tumor microenvironment. Human IL-35 can mediate the generation of human iTR35 that express IL-35 and are suppressive. iTR35 may constitute a key mediator of infectious tolerance and ex vivo generated iTR35 may possess therapeutic utility in various human diseases.