bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential response of dendritic cells to Enterococcus faecalis harboring a pathogenicity island encoded transcriptional regulator. (42.5)
Authors: Awasthi Shanjana , Baghdayan Arto , Coburn Phillip , Shankar Nathan
Published: January 2023

Enterococcus faecalis is responsible for the vast majority (65-80%) of nosocomial infections caused by antibiotic-resistant enterococci and treatment failure often leads to severe clinical consequences. Host immune factors can be important in preventing infection and dissemination, however, the immune mechanisms in play during E. faecalis infections are not well understood. In an earlier in vivo study, using a wild type clinical E. faecalis strain (E99) and its isogenic mutant (DBS01), deficient in a pathogenicity island encoded transcriptional regulator (PerA), we showed that the mutant strain was significantly less virulent and attenuated for survival within macrophages. In the present study we investigated how these strains interacted with dendritic cells (DCs), another key component of the innate immune system. Using flow cytometry and fluorescence microscopy we examined the phagocytic uptake of GFP-labeled E. faecalis by cultured JAWS II DCs as well as measured the expression of cell-surface DC-markers. Although, no significant differences were observed in the levels of expression of DC-markers and cytokines between the two bacterial strains, DCs phagocytosed more mutant bacteria as compared to the wild type strain. These results suggest that the virulence of strain E99 may in part be explained by its ability to circumvent the host immune system by preventing uptake and processing by DCs.
Severity of intestinal disease following Campylobacter jejuni infection correlates with level of TNF-α production and macrophage apoptosis (42.6)
Authors: Tabbara Khaled , Al Muraikhi Amal , Bazzi Ali , Botta Giuseppe
Published: January 2023

Campylobacter jejuni causes diarrhea ranging from mild to bloody.Penetration of epithelial barrier allows organisms to interact with macrophages inducing proinflammatory cytokines. Infection of macrophages induces apoptosis, potentially leading to immune evasion or suppression. We investigated invasiveness, cytokine production and apoptosis of the human monocytic cell line THP-1 using three human C. jejuni isolates. To determine invasiveness,THP-1 cells were infected in vitro,treated with Gentamicin, lysed and plated. Cytokine mRNA expression was determined by semi quantitative multiplex RT-PCR and protein by ELISA.Apoptosis was determined by flow cytometry following Annexin V and TUNEL staining of infected cells. In vitro invasiveness of isolates correlated with disease severity. The isolate from severest disease(3915) was most invasive and the least invasive isolate was from the patient with mildest disease(115) while the third isolate from a patient with intermediate severity(417) fell in between. Cytokine message and expression(IL-1β,TNF-α and IL-8) was highest with 3915 followed by 417 and the least being 115. Differences between the three isolates were most prominent in TNF-α where mRNA expression following 3915 infection was about 3x of 417 and about 7x of 115. ELISA showed similar differences. 60.7% of 3915 cells underwent apoptosis while 417 induced 42.3% and 115 only 30%. We suggest that severity of disease is related to levels of TNF-α production and apoptosis.
IMMUNOSUPPRESSIVE CYTOKINE PRODUCTION IN BOVINE VITAMIN A -DEFICIENT CHALLENGED WITH M. BOVIS (43.13)
Authors: Silva Ediane , Reis Michelle , Waters W Ray , Nonecke Brian , Junqueira-Kipnis Ana Paula , Estes Don Mark
Published: January 2023

It has been shown that vitamin A could induce T regulatory (Treg) cells, especially in the tuberculosis infection. One of action mechanism these cells are due the cytokines presence as IL-10, TGF-β and FOXP3. In this study was evaluated the production of these cytokines in vitamin A sufficient and deficient tuberculosis calves. Animals of 1 - 2 weeks old from a TB-free source were fed with a diet containing or not vitamin A. PBMC were cultivated with or without PPD, and then cells were stained for CD8, WC1γδ T and NK cells receptors for flow cytometry analysis. The cells culture supernatant was evaluated by ELISA for TGF-β. qRT-PCR assay was performed from lung and lynphonod tissues to detect the IL-10, TGF-β and FOX-P3 expression. According to our results Vitamin A supplied animals had higher number of IL10 secreting cells, especially CD8+ (2.27±1.36) and WC1γδ T (2.44±1.48) cells PPD stimulated. Vitamin A deficient animals showed higher numbers of NK+IL10+ (3.46±2.77) cells. The TGF-β in the supernatant from bovines vitamin A supplied was higher (125.15 pg/ml) than vitamin A deficient (16.5 pg/ml). The TGF-β and IL-10 mRNA expression was the same for Vitamin A supplied and deficient, in the lung as well as the lymph node. The IL10 secreting role seems to be more associated to WC1 γδ T and NK cells. Vitamin A diet influences the immunosuppressive cytokine production, especially the TGF-β, when the cells are activated.
Immunogenicity of Anaplasma marginale T4SS proteins (43.18)
Authors: Brown Wendy , Morse Kaitlyn , Sutten Eric , Norimine Junzo
Published: January 2023

For many bacterial pathogens, including Anaplasma marginale, outer membranes (OM) can induce protective immunity. Key questions are what outer membrane proteins are immunogenic and whether linked recognition of associated membrane proteins is important for protection. The type IV secretion system is a virulence factor comprised of approximately 12 proteins spanning the inner and outer membranes of many Gram-negative bacteria. A reverse genetic approach identified immunogenic type IV secretion system (T4SS) proteins VirB9-1, VirB9-2, and VirB10 recognized by sera and T cells of A. marginale OM vaccinated cattle. Recognition of these by sera from infected cattle suggests surface expression. Additional T4SS proteins were expressed, and recombinant VirB2, VirV7, VirB11, and VirD4 also stimulated significant T-cell responses in OM vaccinees. These proteins were not generally recognized by immune sera. Strong IgG2 responses to VirB9-1 or VirB9-2 in the absence of CD4 T cell responses in two OM vaccinees suggests that a T4SS protein partner associated with VirB9-1 provides T cell help for isotype switching. In Agrobacterium tumefaciens VirB9 associates with VirB10 and VirB7, and we have data indicating associations of both VirB9 proteins with each other and with VirB10. The use of T4SS proteins and protein complexes as immunogens to prevent or reduce infection is a novel strategy for developing vaccines for many pathogenic bacteria.
Innate and adaptive immune defenses against a fungus linked to global amphibian declines in the South African Clawed Frog, Xenopus laevis (43.24)
Authors: Ramsey Jeremy , Reinert Laura , Harper Laura , Woodhams Douglas , Rollins-Smith Louise
Published: January 2023

Many amphibian species have suffered population declines due to an emerging infectious disease, chytridiomycosis, caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd). Little is known about immune defenses against this skin pathogen. Here we show that antimicrobial peptides in the skin mucus of the South African clawed frog, Xenopus laevis, inhibited Bd growth in vitro, and skin peptides secreted under natural conditions were within the range of concentrations required to inhibit Bd growth. Peptide secretion was induced by norepinephrine stimulation in a dose-dependent fashion. Maximal stimulation resulted in peptide depletion and increased susceptibility to infection. Frogs required approximately 28-50 days for peptide renewal. Following experimental exposure, infection intensity in the skin was greatest from 20-30 days and declined significantly by 45 days. Thus, immune defenses effectively cleared the infection by seven weeks. Sublethal X-irradiation of frogs resulted in decreased leukocyte numbers in the spleen and greater susceptibility to infection. Immunization against Bd resulted in elevated IgM and IgY antibody responses in vivo that persisted for greater than four weeks. Mucus secretions from X. laevis previously exposed to Bd five months earlier contained IgM, IgY, and IgX antibodies. These data strongly suggest that both innate and adaptive immune defenses are critical in the resistance of X. laevis to lethal Bd infections.
Evidence for somatic hypermutation at shark T cell receptor α locus (43.6)
Authors: Criscitiello Michael , Eubanks Jeannine , Coots Andrea , Flajnik Martin
Published: January 2023

Somatic hypermutation (SHM) is a mechanism B cells use while responding to antigen for affinity maturation, and is also employed in the diversification of the primary B cell repertoire in the gut associated lymphoid tissues of ruminants. Recently it was discovered that SHM operates at the T cell receptor γ locus of the sandbar shark. We have looked for evidence of this AID-mediated mechanism at other T cell receptor loci in the nurse shark, Ginglymostoma cirratum. Our initial hypothesis invoked SHM at the γ and δ loci, but not at the (presumably) MHC-restricted α and β loci. Surprisingly, we find evidence for mutation at the α locus. We see evidence for canonical point mutations as well as the tandem mutations that have been a hallmark of SHM at traditional immunoglobulin, IgNAR and now T cell receptor loci of cartilaginous fish. Ongoing studies are assaying SHM at α in the shark thymus to determine if this phenomenon is being employed for affinity maturation of αβ T cell responses (a process we deem unlikely with MHC restriction). Alternatively, SHM may be used along with receptor editing at the α locus in the thymus to generate primary repertoire paratopes that can mutate to positive selection. The thymus was suggested as a “mutant breeding” organ for self tolerance by Neils Jerne forty years ago - future comparative studies will determine if SHM is an original tool of vertebrate T cells for thymic diversification or an adaptation singular to sharks.
Blood dendritic cells in cardiovascular disease risk (44.13)
Authors: Carrion Julio , Zeituni Amir , Bear Adam , Cutler Christopher
Published: January 2023

An estimated 80 million U.S. adults have one or more types of cardiovascular diseases (CVD) with the total cost to the U.S. for 2009 estimated to be $475.3 billion. Atherosclerosis (ATH) is the single most important contributor to CVD; however, only 50% of ATH patients have currently identified risk factors. Chronic periodontitis (CP), a common inflammatory disease, is linked to an increased CVD risk. Dendritic cells (DCs) which infiltrate arterial walls and destabilize atherosclerotic plaques, have been implicated in CVD. While the source of atherogenic DCs is presently unclear, we propose that dermal DCs from peripheral inflamed sites, such as CP tissues are a source. We therefore designed a clinical study to elicit, quantitate and isolate DCs in the peripheral blood of otherwise healthy CP patients. Ten patients with CP were consented according to IRB standards and enrolled in the study. Peripheral blood collected at baseline and 24 hrs after mechanical debridement (MD) was used to identify and sort blood DCs. After PBMC separation, DCs were labeled with DC-SIGN-APC and BDCA-1-PE and quantitated by FACS analysis. We found that after MD there was a 5-6% increase in the numbers of blood DCs in this patient population. This increase in the blood may represent mobilization of DCs from bone marrow stores, as well as from CP tissues. We are presently analyzing the DC transcriptome of sorted DCs to identify markers of atherogenesis.
Thioredoxin inhibits epidermal Langerhans’ cell (LC) migration in man and mouse (44.6)
Authors: Dearman Rebecca , Cumberbatch Marie , Del Val Greg , Almond Rachael , Griffiths Christopher , Kimber Ian
Published: January 2023

Epidermal LC play a critical role in the initiation of skin immune responses. LC migration is dependent upon cytokine signals, provided by tumour necrosis factor-α (TNF-α) and interleukin (IL)-1β. We have now examined the ability of human recombinant thioredoxin (hTrx), a redox-active protein, to influence LC migration. Wild type hTrx and various cysteine to alanine mutants were produced in E coli. LC migration in response to chemical contact allergen (oxazolone; Ox) or to intradermal (id) injection of recombinant murine TNF-α or IL-1β was assessed in BALB/c mice. LC migration was provoked in healthy human volunteers (n=10) by id injection of recombinant human TNF-α. Various forms of hTrx were administered by topical application 2h prior to the migratory stimuli. LC densities were assessed in epidermal sheets by immunofluorescent staining for MHC class II (mouse) or CD1a (human). Topical application of hTrx inhibited significantly Ox- and TNF-α-induced LC migration in BALB/c strain mice, but failed to impact on IL-1β-induced migration. Use of mutant forms of hTrx demonstrated that this ability was redox-independent. Application to the skin of wild type hTrx to human volunteers inhibited TNF-α-induced LC migration also. Inhibition of LC migration was selective for IL-1β-dependent stimuli (contact allergen and TNF-α). These data suggest that thioredoxin possesses novel immunomodulatory properties as a result of its ability to modulate IL-1β production and/or signalling.
L-selectin expression on CD4+ T cells is critical for antibody production, whereas L-selectin expression on both CD4+ and CD8+ T cells is important for dermatitis in a mouse model of atopic dermatitis (44.9)
Authors: Kadono Takafumi , Tomota Manabu , Ohmatsu Hanako , Kai Hiromichi , Asano Yoshihide , Sugaya Makoto , Sato Shinichi
Published: January 2023

L-selectin is a cell adhesion molecule that plays an important role in leukocyte migration especially to peripheral lymph nodes. L-selectin deficiency is known to decrease subcutaneous immune responses, however, the role of L-selectin in atopic dermatitis model is unknown. Moreover, it is not clear on which cell subset the expression of L-selectin is critical for cutaneous inflammation. Here, we showed that in mouse atopic dermatitis model elicited by epicutaneous sensitization with ovalbumin (OVA) OVA-specific antibody production and dermatitis is completely lost with L-selectin deficiency. We also found that L-selectin expression on CD4+ T cells is critical for antibody production, whereas L-selectin expression on both CD4+ and CD8+ T cells is important for dermatitis. Moreover, as few as 100 OVA-specific CD4+ T cells were able to elicit OVA-specific antibody production in L-selectin deficient mice. When wild-type CD4+ T cells were intravenously injected to L-selectin deficient mice, OVA-specific antibody-forming cells existed mostly in draining lymph nodes, suggesting that L-selectin-positive CD4+ T cells recruited to draining lymph nodes could muster antibody-forming cells independent of L-selectin. These results suggested that this mouse atopic dermatitis model is completely L-selectin dependent with L-selectin on CD4+ T cells and CD8+ T cells playing distinctive roles, and targeting L-selectin may be an option for treating atopic dermatitis.
IL-17 is critical for the generation of protective Th1 immunity against intracellular pathogens (45.1)
Authors: Lin Yinyao , Gopal Radha , Khader Shabaana
Published: January 2023

The importance of T helper type 1 (Th1) cell immunity in host resistance to the intracellular pathogens is well established. However, the relative roles of Interleukin (IL)-12-Th1 and IL-23-T helper type 17 (Th17) cell responses in immunity to intracellular pathogens is not well described. The IL-23-Th17 pathway is critical for protective immunity against extracellular bacterial infections. In contrast, the IL-23-Th17 pathway is dispensable for protection against intracellular pathogens such as Mycobacterium tuberculosis and Listeria. However, our data suggest that IL-17 regulates the Th1 pathway and is required for protective immunity against some intracellular pathogens such as Francisella tularensis Live Vaccine Strain and M.bovis BCG. IL-23-dependent IL-17 induces IL-12 in myeloid cells and is required for the generation of IFNγ producing adaptive immune responses. These findings illustrate a novel biological function for IL-17 in regulating IL-12-Th1 immunity and host responses against intracellular pathogens.
Regulation of proinflammatory cytokine Interleukin-6 (IL-6) induction by NF-kappaB signaling in pandemic H1N1 influenza A virus-infected human bronchial epithelial cells (45.29)
Authors: Xing Zheng , Gao Wei , Qu Bingqian , Li Xue , Jin Yu , Yang Ku , Cardona Carol
Published: January 2023

The influenza A (H1N1) pandemic of 2009 is caused by a novel swine influenza virus, which has resulted in thousands of mortalities worldwide. Pulmonary failure due to over-induction of proinflammatory factors appeared to be the main cause in severe cases leading to death. To elucidate the pathogenesis of the novel H1N1 virus in humans, we infected human bronchial epithelial cells with A/Nanjing/NJU108/2009 (H1N1), isolated from a patient in Nanjing, China, and analyzed expression profiles of various cytokines and chemokines. We found that among proinflammatory cytokines, IL-6 was induced up to hundred and thousand fold after infection, while the levels of IL-1beta and IL-8 remained unchanged. This study aims to understand how NF-kappa B (NFκB) signaling pathway regulates the induction of IL-6 upon infection. Surprisingly, when a specific IKKalpha/beta inhibitor was used, IL-6 induction was not affected. IL-6 induction was, however, greatly suppressed by an NFκB1/p100 inhibitor, which indicates that the NFκB signaling was involved in the regulation of IL-6 but the classical NFkB pathway seems to be only partially implicated. We further examined the phosphorylation and activation of IKKs, NFκB1 and NFκB2, and other regulators in NFκB signaling. Our data demonstrated that a novel mechanism was employed in NFκB signaling pathway which regulates IL-6 induction and may play a critical role in pathogenesis in humans infected with the novel pandemic H1N1 influenza A virus.
Recycling endosomal membranes contribute to Toxoplasma gondii parasitophorous vacuole formation in mast cells (45.4)
Authors: Smith Norah , Butcher Barbara , Denkers Eric , Baird Barbara , Holowka David
Published: January 2023

For the obligate intracellular parasite, Toxoplasma gondii, elucidating its initial interactions with host cells and their parasitophorous vacuole membrane (PVM) is important for better understanding T. gondii infection. In many cell types, recycling endosomal (RE) trafficking often contributes to plasma membrane remodeling during processes such as phagocytosis. Previously, we characterized RE trafficking in RBL-2H3 mast cells and demonstrated IgE/antigen-stimulated outward trafficking of FITC-cholera toxin B (CTxB) bound to GM1 on RE membranes. When these cells are infected with virulent Type I strain tachyzoites, we find that invading parasites have an accumulation of FITC-CTxB on the PVM at early times after infection. This response is substantially enhanced when FITC-CTxB labels the RE pool in addition to the plasma membrane, providing evidence that RE membranes contribute to the nascent PVM. Consistent with earlier studies, IgE bound to its receptor at the plasma membrane shows no detectable accumulation at the PVM during this early stage (first hour) of infection. Under these conditions, antigen-stimulated degranulation is substantially inhibited, suggesting that parasitic infection interferes with normal signaling responses in these cells. Our ongoing research aims to further investigate the possible link between the PVM and RE trafficking and the potential immunomodulatory effects of T. gondii on infected mast cell signaling.
Depot versus dispersal: biodegradable polymer vaccination effects of size and chemistry (46.17)
Authors: Huntimer Lucas , Phanse Yashdeep , Petersen Latrisha , Carrillo-Conde Brenda , Ramer-Tait Amanda , Narasimhan Balaji , Wannemuehler Michael
Published: January 2023

Development of safe and efficacious vaccine regimen including delivery vehicles, routes, and adjuvants is paramount to public health and preventive medicine. This study evaluated the whole body distribution of unique, surface eroding polymers prepared from aliphatic sebacic acid (SA) and aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH). The inherent surface erosion kinetics permit development of a single dose vaccine based on tailored release kinetics of immunogen, adjuvanticity and sparing of protein dose, and sustained antigen presentation. Fabrication methods were used to create a size range (400 nm to 10 µm) of particles encapsulating LSS 640 fluorophore. These particles were used to evaluate in vivo distribution following intranasal (IN) or subcutaneous (SC) administration to mice. Using live animal imaging at multiple time points, fluorescent analysis at the injection and distal anatomical sites establish that microparticles distributed in a pattern consistent with a depot effect that was dependent upon the injection route. In contrast, nanoparticles distributed systemically regardless of route. Ex vivo analysis of excised tissues verified nanoparticle distribution to distal sites. The ability to assess the depot and dispersal effect of this novel delivery platform in real time consequent to the induction of an antibody response will provide insight into how size and polymer chemistry can be used therapeutically to effect targeting of a vaccine to desired immune tissues.
Novel role of galectin-4: A potent inducer of monocytes differentiation and activation. (47.12)
Authors: Hong So-Hee , Do Ji-Hye , Shin Jun-Seop , Park Chung-Gyu
Published: January 2023

Galectin-4 (Gal-4) is one of β-galactoside-binding proteins (Galectin family) and is specifically expressed in the gastrointestinal tracts. Although intensive studies have been done on other isoforms of Galectin (Gal-1, -3, -9), the role of Gal-4 in immune system remains unknown. Here, we found that Gal-4 specifically affects the differentiation and activation of monocytes. In addition to prolonging monocytes cell survival, Gal-4 induced morphological changes of monocytes into macrophage-like cells. Notably, Gal-4 entered into the cytoplasm of monocytes after binding and increased the production of pro-inflammatory cytokine such as IL-6 and TNF-a. Gal-4 also increased the expression of co-stimulatory molecule such as CD40, 80 and CD86 in CD14+ monocytes. Furthermore, we provide the evidence that Gal-4 mediated its modulatory function through MAPK signaling cascade (p38 and c-Jun) via MyD88 by Western blot analyses. As such, Gal-4 did not increase macrophage differentiation markers in MyD88-deficient monocytes from MyD88 knockout mice. In conclusion, our study is the first to describe the specific effect of galectin-4 on monocytes and further suggests that Gal-4 expression might modulate mucosal immunity via monocytes activation and differentiation.
A functional impairment of Foxp3+CD4+ regulatory T cell may lead to the exacerbated inflammation in ARDS. (47.2)
Authors: Elhadad Sonia , London Lucille
Published: January 2023

Acute Respiratory Distress Syndrome (ARDS) is a clinical syndrome characterized by an intense host inflammatory response to an infectious, non-infectious, or extrapulmonary insult. It is associated with the development of lung damage, the rapid onset of severe respiratory and organ failure. Neutrophils and lymphocytes have been shown to accumulate in the airways. However, the mechanisms that lead to this aberrant inflammation and the nature of the lymphocyte subsets involved in the recruitment of neutrophils to the lung and in their activation are not yet clearly defined. Using a unique mouse model of virus-induced ARDS that recapitulates the histopathology observed in patients with ARDS, we hypothesized, that the inflammation observed in ARDS is the result of (i) a defective regulatory Foxp3+CD4+ T cell population (T-reg), and (ii) an imbalance in the modulation of T-regs and the proinflammatory TH17 population. Therefore, we evaluated the presence of Foxp3+CD4+ T-regs in the lungs and BAL fluid of infected mice. We observed a difference in the phenotype of the regulatory T cell population in the lungs at different stages of the disease from progression to recovery in our virus-induced ARDS model. We concluded that the inflammation observed in our model, does not result from the absence of regulatory T cells, but may result from an impairment of the function of the Foxp3+CD4+ regulatory T cells, which may contribute to the uncontrolled inflammation observed in ARDS.
Microbiota antigen specific Th17 cells induce colitis and promote Th1 cell response through IL-17 induction of innate cell IL-12 and IL-23 production (47.3)
Authors: Feng Ting , Qin Hongwei , Wang Lanfang , Benveniste Etty , Elson Charles , Cong Yingzi
Published: January 2023

Both Th1 and Th17 cells have been implicated in the pathogenesis of IBD and experimental colitis. However, the complex relationship between Th1 and Th17 cells has not been completely analyzed. Although it has been shown recently that Th17 cells can convert into Th1 cells, the underlying in vivo mechanisms and the role of Th1 cells converted from Th17 cells in colitis pathogenesis are still largely unknown. By using Thy1.1-IFNγ reporter mice and IL-17 capture technique, we generated Th1 and Th17 cells of high purity from immunodominant microbiota antigen CBir1 flagellin specific TCR transgenic mice. Highly purified Th1 and Th17 cells differentially induced colitis, in that Th1 cells induced mild, whereas Th17 cells induced severe colitis when transferred into TCRβxδ-/- mice. In the colitic Th17 recipients, substantial IFNγ+ Th1 cells emerged in intestinal lamina propria. Colonic tissues of Th17 recipients produced high levels of IL-12 and IL-23. IL-17 treatment induced BMDC IL-12 and IL-23 production in vitro. Administration of anti-IL-17 mAb to Th17 recipients abrogated colitis development, blocked colonic IL-12 and IL-23 production, and inhibited T cell IFNγ production. Furthermore, colon-conditioned medium of colitic Th17 cell recipients promoted IFNγ production by Th17 cells, which was inhibited by the addition of anti-IL-12 and anti-IL-23 mAb. These data indicate that Th17 cells promote Th1 cells through IL-17 induction of mucosal innate cell IL-12 and IL-23 production.
Role of MKP-1 in an IL-10 knockout murine inflammatory bowel disease model (47.7)
Authors: Wancket Lyn , Matta Ranyia , Barnard John , Grieves Jessica , Nelin Leif , Cato Andrew , Liu Yusen
Published: January 2023

IBD is a chronic intestinal inflammatory disease that often has extra-intestinal manifestations. We examined the role of the MAPK phosphatase Mkp-1 in a mouse model of IBD. Mkp-1+/+/Il-10+/+, Mkp-1-/-/Il-10+/+, Mkp-1+/+/Il-10-/-, and Mkp-1-/-/Il-10-/- (dKO) mice on a 129 background were housed in a specific pathogen-free environment. Colitis signs were evaluated using a clinical scoring system, histological examination, and cytokine analysis. Most dKO mice developed severe rectal prolapse, peri-ocular lesions, and high clinical scores, signs generally not seen in Il-10 KO mice. dKO colons had extensive chronic and neutrophilic colitis, mucosal hyperplasia, and higher histological IBD scores than Il-10 KO colons. dKO eyelids and conjunctival mucosa were thickened and had neutrophilic and lymphoplasmocytic conjunctivitis. After LPS treatment, splenocytes and lymph node cells isolated from dKO mice had more robust production of Th-1 cytokines than cells from Il-10 KO mice. dKO colons contained higher levels of Th-1 cytokines and IL-17, and exhibited greater p38 and ERK activation, than did Il-10 KO colons. These data indicate that loss of Mkp-1 skews host immunity towards an exaggerated Th-1/Th-17 response and provide novel insights on the interaction between Mkp-1 and IL-10 in colitis models. Our studies support a pivotal role of Mkp-1 as a brake in the mucosal immune response to limit IBD development.
Conversion of conventional T cells to Tregs within the eye and the dual role of the vision related molecule, retinoic acid, in ocular immune privilege (48.1)
Authors: Zhou Ru , Horai Reiko , Mattapallil Mary , Rigden Rachael , Villasmil Rafael , Caspi Rachel
Published: January 2023

Ocular immune privilege protects the eye from immune damage in part through TGFβ. Retinoic acid (RA) is abundant in the eye, as it participates in the chemistry of vision. However, its role, if any, in immune responses involving the eye is unknown. RA-synthesizing enzymes RALDH1, 2 and 3 were expressed in the cornea and retina. Aqueous humor (AH) massively induced Foxp3 expression in purified CD4+ T cells stimulated through the T cell receptor, while suppressing their proliferation and acquisition of effector function, as judged by reduction of lineage-specific transcription factors and effector cytokines for Th1, Th2 and Th17. AH-induced Foxp3+ T cells expressed CD25, GITR, CTLA4, CD103 and were functionally suppressive. The effect of AH was reversed by antagonizing either RA or TGFβ signaling and involved TGFβ-driven upregulation of RARα. Foxp3 expression was reduced upon removal of AH from culture, but was stabilized by the DNA demethylating agent 5-AZA. Importantly, naive retina-specific (but not polyclonal) T cells, injected into the eyes of live mice, were converted in vivo into Foxp3+ cells. In contrast, previously primed T cells exposed to AH on 2nd stimulation were resistant to suppressive effect of AH. Thus, RA in the eye plays a dual role: in vision and in immune privilege, by local induction of Tregs. However, primed effector cells are relatively insensitive to AH, helping to explain occurrence of uveitis in the face of an inhibitory ocular microenvironment.
Nuclear and cytoplasmic antibodies in severe periodontytis (48.16)
Authors: Aguilera galaviz Luis , Padilla Bernal María , Aceves Medina María , Sanchez Rodríguez Sergio , Lopez Luna María
Published: January 2023

Periodontitis is bacterial infection of tooth-supporting tissues, the immunopathologic mechanisms include inflammatory cells and chemical mediators; persistent inflammation develops a local autoimmune, the presence of autoantibodies against extracellular matrix components, ANCA, anti-DNA was detected. Objetives: To determine the presence of auto-antibodies in severe periodontyti. Methods: Ten patients with sever periodontytis were clinically evaluated and 20 healthy people, blood sample was collected to set up IFI for ANCA, antiepithelial antibodies, ANA in Hep2 cells and Western Blot to determine the molecular profile using leucocite and keratynocytes extracts. Results: 57% were positives for ANCA, the total of sera were positives for nuclear and/or cytoplasmic antibodies and 57% presented anti-epithelial antibodies, the sera recognized proteins in a range of molecular weight 10, 30, and 70 kD in leucocyte extract, and 30 and 70 kD in mouse skin extract, mostly in the range of the Hsp family. Conclusions: The nature of the immune response in periodontal disease is influenced by the microflora, however the production of antibodies against self estructures, could be involved in the pathogenic mechanism; these autoantibodies could be associated with necrotic or apoptotic phenomena.Struchture and metabolic activity of microorganism should be involved in mimetic or cross reactions, the antigen procesesing in situ could be eventually stimulate self reactive lymphocytes.
Deficient virus specific IgA production following murine gammaherpesvirus 68 infection of the respiratory tract (48.9)
Authors: Sundararajan Aarthi , Huan Lifang , Joo Hyemee , Speck Samuel , Sangster Mark
Published: January 2023

The B cell response to viruses that replicate in the respiratory tract typically includes an IgA component. Murine gammaherpesvirus 68 (MHV68)infection in the mouse provides a model for studying gammaherpesvirus immunobiology. Following intranasal (i.n.)infection, infectious MHV-68 is cleared from the lung, but the virus establishes latency in B cells, macrophages and dendritic cells. Reactivation of latent virus and shedding in secretions may be critical for virus transmission. The respiratory mucosa is central to MHV68 pathogenesis, but little is known about B cell-mediated mucosal immunity to the virus. We have analyzed this using ELISPOT assay and ELISA to measure antibody secreting cell(ASC)numbers and antibody(Ab)levels, respectively. Although the virus generated a vigorous IgG response in draining lymph nodes, there was little, if any IgA production. In addition, virus-specific IgA was absent from serum and mucosal surfaces of the upper and lower respiratory tract. IgA was prominent in the response to i.n. immunization with inactivated MHV68, indicating that factors associated with virus replication modulate IgA response. Analysis of an MHV68 mutant did not identify genes that may down-regulate IgA production. An IgA response in IFNγR-/- mice following i.n. MHV68 infection indicated a role for strong Th1 type environment in reducing IgA production. Down-regulation of IgA production by MHV68 may reflect a high level of viral adaptation that favors virus transmission.
Nuclear factor-κB drives the development of Foxp3+ regulatory T cells through the c-Rel enhanceosome (49.1)
Authors: Ruan Qingguo , Kameswaran Vasumathi , Tone Yukiko , Li Li , Liou Hsiou-Chi , Greene Mark , Tone Masahide , Chen Youhai
Published: January 2023

Regulatory T (Treg) cells are essential for maintaining immune homeostasis and preventing autoimmune diseases. Although Foxp3 expression marks the commitment of progenitors to Treg cell lineage, how Treg cells are generated during lymphocyte development remains enigmatic. We report here that two members of the nuclear factor (NF)-κB family, i.e., c-Rel and p65 (also known as RelA), controlled the development of Treg cells by promoting the formation of a Foxp3-specific enhanceosome. This enhanceosome contained c-Rel, p65, NFAT, Smad, and CREB. Although Smad and CREB first bound to Foxp3 enhancers, they later moved to the promoter to form the c-Rel enhanceosome. c-Rel-deficient mice had up to 90% reductions of Treg cells compared to wild-type mice, and c-Rel-deficient T cells were compromised in Treg cell differentiation. Thus, Treg cell development is controlled by a Foxp3-specific enhanceosome, and strategies targeting NF-κB can be effective for manipulating Treg cell function.
The Transcriptional Repressor BCL6 Controls the Suppressive Function of Regulatory T cells and Inhibits Th2 Cytokine Expression by Regulatory T cells (49.10)
Authors: Dent Alexander , Sawant Deepali
Published: January 2023

Regulatory T cells (Tregs) expressing the transcription factor FOXP3 represent a distinct lineage of T helper cells indispensable for the maintenance of immune homeostasis and the inhibition of autoimmunity. The transcriptional repressor protein BCL6 critically regulates the immune response. BCL6-deficient mice develop severe and spontaneous inflammation early in life that is frequently lethal. BCL6 is a potent inhibitor of Th2 cell differentiation and is required for differentiation into the follicular helper lineage. However, the role of BCL6 in other T cell subsets is less clear, and little is known about the role of BCL6 in Treg cells despite the fact that BCL6 is an inhibitor of inflammation. We have found that BCL6 is an important regulator of Treg gene expression and function. BCL6-deficient Tregs are hyper-proliferative, are prone towards Th2 cytokine expression and do not suppress inflammation in vivo. Our data indicate that BCL6 is a key inhibitor of Th2 cytokine expression in Treg cells, and that lack of BCL6 activity in Treg cells can lead to abnormal Th2-type inflammatory responses.
Control of Treg by TNFRSF25 (49.12)
Authors: Wolf Dietlinde , Schreiber Taylor , Deyev Vadim , Tsai Matthew , Podack Eckhard
Published: January 2023

TNF-receptor superfamily member 25 (TNFRSF25, DR3), as a member of the TNF receptor superfamily, is constitutively expressed on CD4+ T cells at low levels and upregulated after activation. Signaling through TNFRSF25 enhances Th2 and Th17 cytokine production and contributes to the pathology of several autoimmune diseases. Here we investigate the role of TNFRSF25 on CD4+FoxP3+ Treg cells, where it is constitutively expressed at high levels. We show that TNFRSF25 is unique among several TNFRSF members in its ability to expand Treg in vivo to 30-35% of CD4+ T cells. For TNFRSF25 signaling we use an agonistic α-TNFRSF25 antibody, clone 4C12. The expansion of CD4+FoxP3+ cells is due to increased proliferation of CD4+FoxP3+CD25int cells that express high levels of GITR and CD103. 4C12 expanded CD4+FoxP3+ cells retain TGFβ-dependent suppressive activity ex vivo, except in the continued presence of 4C12. Furthermore, 4C12 rescued TL1A-/- mice from susceptibility to dextran sodium sulfate induced colitis. These data highlight the plasticity of the CD4+FoxP3+ regulatory T cell pool and provide first evidence of a physiological pathway leading to Treg-specific expansion in vivo. Therefore, using TNFRSF25 agonists to modulate Treg function may have important applications for the treatment of autoimmune diseases, chronic infection, transplantation and cancer.
CD4+ T cells expressing VEGFR1 (Vascular Endothelial Growth Factor Receptor1) (49.23)
Authors: Shin Jin-Young , Lim Jong-Hyoung , Cho Hyoung-Soo , Park Chung-Gyu
Published: January 2023

Regulatory T cells are a specialized subpopulation of T cells that control immune response and thereby maintain immune system homeostasis and tolerance to self-antigens. The isolation and expansion of CD4+ Treg cells requires specific surface markers. Currently, CD25, CTLA-4 and GITR are used, but they are also expressed on the activated T cells. In this study, we identified vascular endothelial growth factor receptor 1 (VEGFR1) as a new surface marker for CD4+ regulatory T cells. We found that VEGFR1, a receptor for VEGF involved in angiogenesis, is constitutively expressed on the surface of T cells. CD4+VEGFR1high T cells (1.0 % ± 0.5% of CD4+ T cells) are hyporesponsive and can suppress CD4+ responder T cell (Tres) proliferation as efficiently as CD4+CD25+ regulatory T cells (Treg cells). VEGFR1highCD4+ T cells are also able to mediate potent suppression of Tres in a contact-independent manner. CD4+ VEGFR1high T cells are not overlapped with CD4+Foxp3+ or CD4+CD25+ T cells and secrete IL-10. We suggest that VEGFR1 can be a useful surface marker for CD4+ regulatory T cells and CD4+ VEGFR1high T cells can be a new subset of regulatory T cells.
T cell epitopes (Tregitopes) Suppress Immune Responses in vivo by Activating Natural Tregs (49.24)
Authors: Su Yan , Moise Leonard , Li Xin , Rossi Robert , Skupsky Jonathan , Martin William , DeGroot Annie , Scott David
Published: January 2023

A set of putative natural T regulatory epitopes (Tregitopes, derived from IgG) have been identified, which have suppressive effects both in vitro and in vivo. These Tregitopes are promiscuous, bind highly to MHC class II and are highly conserved in multiple species. In this study, we further investigated the mechanisms of suppression by Tregitopes. We first demonstrated that neither CFA nor IFA emulsified Tregitopes peptides per se are immunogenic in vivo. Administration of Tregitopes by IFA upregulated FoxP3+ T cells and reduced specific CD4+ T cell responses in OVA TCR transgenic and normal Balb/c mice. Interestingly, we found that Tregitopes activated natural, but not inducible Tregs. Furthermore, B cells that are pulsed with Tregitopes and a target antigen OVA in vitro acquired tolerogenic phenotypes. These B cells can suppress both T cell and antibody responses to OVA protein in vivo. In NOD mice, administration of Tregitopes resulted in delayed onset of type I diabetes and even reversed hyperglycemia in the majority of new onset diabetic mice. Together, our results suggest that Tregs induced by Tregitopes represents a new paradigm for treatment of autoimmune disease (Supported by NIH grants RO1 AI035622, RO1 DK068343 and R43 DK081261).
Antigen dose differentially controls the induction of specific Treg and Teff populations (49.5)
Authors: Turner Michael , Morel Penelope
Published: January 2023

The concept that Th1/Th2 differentiation may be affected by antigen dose was introduced a decade ago (O’Garra, Bottomly). Recently, we reported that antigen dose also controls Treg induction whereby low antigen doses elicit weak signals via the TCR/Akt/mTOR pathway, allowing Foxp3 expression and expansion of Treg. METHODS: We expanded our studies on antigen dose effects to include regulatory and effector populations that have been recently added to the CD4+ repertoire. Purified CD4+ T cells from BDC2.5 TCR transgenic mice were stimulated by DC pulsed with increasing doses of peptide antigen. After 7 days of priming, T cells were re-stimulated with anti-CD3 and anti-CD28 and analyzed for cytokine production and Foxp3 expression. RESULTS: We found that altering the antigen dose qualitatively affected the CD4+ T cell response in a hierarchical manner. Low dose stimulation led to Foxp3+ Treg expansion; intermediate doses induced IL-13+ Th2 and IL-10+ Tr1 cells; and higher antigen doses favored Th1/Th17 responses. In addition, when low-dose-stimulated T cells were subsequently re-stimulated with a high antigen dose, the Th1/Th17 response remained diminished. CONCLUSIONS: Our results indicate that Tr1, Th17 and Treg are included in the repertoire of CD4+ T cells that are affected by antigen dose. These findings have broad implications for vaccine design and we are in the process of determining the in vivo significance of these qualitative changes to the CD4 compartment.
In vivo administration of mitogenic anti-CD3 monoclonal antibody generates granzyme B expressing CD4+Foxp3+ regulatory T cells and depletes dendritic cells (49.6)
Authors: Suvas Susmit , Vietto Nicholas , Channappanavar Rudragouda , Twardy Brandon
Published: January 2023

CD3 specific monoclonal antibody therapy uses multiple mechanisms to induce immune tolerance. In this report, we determined that intra-peritoneal administration of 10 µg of mitogenic anti-CD3 monoclonal antibody (mAb) into C57BL/6 mice resulted in a significant reduction in the frequency of splenic dendritic cells (CD11chi) when compared to PBS treated mice at 24h post-injection. Interestingly, the proportion of CD11chi cells undergoing apoptosis (as determined by Apo-stat assay) was significantly higher in anti-CD3 treated mice in comparison to PBS treated control group. Recently, it was shown that in vitro activation of CD4+CD25+ regulatory T cells with anti-CD3 mAb induces granzyme B expression in regulatory T cells and transforms them into cytolytic T cells. To determine the underlying mechanism of increased apoptosis of dendritic cells in anti-CD3 mAb treated mice, we next ascertained the expression of granzyme B in CD4+Foxp3+ regulatory T cells (Treg) derived from the spleen tissue of anti-CD3 and PBS treated groups of mice. Our results showed that when restimulated in vitro with anti-CD3 mAb (1 µg/ml) for 6h, frequency of Treg expressing cell surface CD107a and intracellular granzyme B was significantly higher in anti-CD3 treated mice in comparison to PBS treated control group. Taken together, our results determined that in vivo administration of anti-CD3 mAb resulted in the induction of granzyme B expressing Treg pool and enhanced apoptosis of dendritic cells.
β-catenin inhibits T cell activation by selective interference with LAT-PLC-γ1 phosphorylation. (50.12)
Authors: Gajewski Thomas , Zheng Yan , Locke Frederick , Cannon Judy , Gounari Fotini , Driessens Gregory
Published: January 2023

The functional role of β-catenin in post-thymic T cells is poorly understood. We observed that β-catenin protein was constitutively degraded in peripheral T cells. Introduction of a stabilized mutant of β-catenin into primary T cells resulted in reduced proliferation and cytokine secretion following TCR/CD28 stimulation, and interfered with effector cell differentation. The biochemical mechanism of blunted T cell activation was investigated. Stable β-catenin was predominantly localized to the cytosol, and cytokine production was rescued with PMA+Ionomycin, arguing for an inhibitory effect on early TCR signaling. Analysis of specific signaling events revealed a selective inhibition of LAT phosphorylation on tyrosine 136, which was associated with defective PLC-γ1 phosphorylation and calcium signaling but normal ERK activation. Stable β-catenin was detected constitutively in regulatory T cells, and conditional deletion of β-catenin in these cells caused rapid cell death. Contrary to a recent report using GSK-3 inhibitors, we saw no effect on T cell memory differentiation with direct genetic manipulation of β-catenin. Our findings indicate that β-catenin negatively regulates effector T cell activation via a unique biochemical mechanism. Functionally, our observations suggest that conditions under which β-catenin might be inhibited in vivo would potentiate T cell-based immunity at the level of both effector and regulatory T cells.
Regulation of TCR signaling and CARD11 by the kinesin GAKIN (50.16)
Authors: Lamason Rebecca , Kupfer Abraham , Pomerantz Joel
Published: January 2023

Lymphocyte signaling from the T cell receptor (TCR) to NF-κB is required for mounting an appropriate immune response and is regulated at multiple steps. One such regulatory mechanism involves the activation and synapse recruitment of protein signaling complexes, including one that contains CARD11, a multi-domain scaffold protein required for TCR signaling to NF-κB. To identify novel regulators of CARD11, we conducted an expression-cloning screen that yielded the kinesin family member, guanylate kinase-associated kinesin (GAKIN). We show that GAKIN is a novel inhibitor of NF-κB activation that can interact with CARD11 in a signal dependent manner, can compete with Bcl10 for CARD11 association, and may regulate the dynamic localization of CARD11 to the immunological synapse. This study indentifies GAKIN as a novel component of the TCR to NF-κB pathway and reveals a previously unrecognized mechanism for controlling the strength or duration of T cell signaling.
Persistent Changes in T Cell Receptor Surface Organization Visualized by Quantum Dot Probes with k-Space Image Correlation Spectroscopy (50.2)
Authors: Perica Karlo , Bieler Joan , Li Yu , Selya Aaron , Edidin Michael , Schneck Jonathan
Published: January 2023

There is growing evidence that the surface pattern and spatial distribution of T cell receptors (TCRs) on cell surfaces plays a critical role in T Cell antigen detection and effector function, from micron-sized Supramolecular Adhesion Clusters to microclusters 100’s of nanometers in diameter. To date, TCR organization patterns have been primarily examined in the context of antigen stimulation or immediately thereafter, on a timescale of seconds to minutes, despite evidence from binding studies that shows changes persist many days post-activation. Here, we present a method for imaging long-term TCR lateral surface organization on live cells at two relevant length scales: 10’s and 100’s of nms. TCR are labeled with monomeric biotin-MHC/peptide or antibody fragments, followed by streptavidin-quantum dots (QD). We have found that QD “blinking” is a useful parameter for detecting TCR organization on a nm scale, since electronic coupling between blinking states of adjacent QDs reports single or small clusters of receptors. Simultaneously, we quantify organization at the scale of 100’s of nms by epifluorescense. As proof of principle, we have demonstrated that in vitro activated T cells have enhanced clustering at the 10’s and 100’s nm scales compared to naïve counterparts, supporting results from MHC dimer binding assays. Clustering can also be modulated by IL-2 signaling for active, but not naïve, T cells.