bluuebunny/crossref_metadata_2025 huggingface dataset:
Unraveling how Rfv3/Apobec3 promotes the retrovirus-specific neutralizing antibody response (105.12)
Authors: Santiago Mario , Smith Diana , Barrett Bradley , Guo Kejun , Heilman Karl , Benitez Robert , Montano Mauricio , Pelanda Roberta , Hasenkrug Kim , Greene Warner
Published: January 2023

Recovery from Friend retrovirus gene 3 (Rfv3) is a classical autosomal dominant gene that facilitates recovery from viremia and disease by promoting a more potent neutralizing antibody (NAb) response. We previously reported that Rfv3 is encoded by Apobec3, a deoxycytidine deaminase that can restrict a broad range of retroviruses. However, the mechanism for how Apobec3 promotes NAb responses and the nature of protective NAbs remain unclear. Apobec3 is related to Activation Induced Deaminase, but hapten-specific antibody affinity maturation is unaffected in Apobec3 deficient mice. In contrast, Apobec3 restriction of acute FV infection in immune cells is associated with more vigorous induction of germinal center B cells, increased B cell maturation and decreased hypergammaglobulinemia. Surprisingly, while Apobec3 reduced acute plasma infectious virus titers, plasma viral RNA loads were maintained. These plasma virions encounter an early reverse transcription block with minimum G-to-A mutations. Thus, Apobec3 appears to promote NAb responses by (1) reducing virus-induced immune dysfunction, while (2) facilitating substantial release of noninfectious viral particles, presumably to prime the B cell response. Finally, enhanced NAb responses in Rfv3/Apobec3 resistant mice correlated with virus-specific IgG1 and IgG3 titers. These findings highlight a fascinating interplay between innate Apobec3 restriction and humoral immunity, with potential implications for HIV vaccine development.
Migration of cytotoxic lymphocytes in cell cycle permits local MHC-I dependent control of division at sites of viral infection (105.21)
Authors: Kang Silvia , Herz Jasmin , Kim Jiyun , Nayak Debasis , Stewart-Hutchinson Phillip , Dustin Michael , McGavern Dorian
Published: January 2023

Following virus infection, cytotoxic T lymphocytes (CTL) divide rapidly to eradicate the pathogen and prevent the establishment of persistence. The magnitude of an anti-viral CTL response is thought to be controlled by the initiation of a cell cycle program within lymphoid tissues. However, it is presently not known whether this division program proceeds during migration or is influenced locally at sites of viral infection. Here we demonstrate that anti-viral CTL remain in cell cycle while transiting to infected tissues. One third of virus-specific CTL within blood were found to be in cell cycle following infection with lymphocytic choriomeningitis virus (LCMV). Using two-photon microscopy, we revealed that effector CTL divided rapidly upon arrest in the virus-infected central nervous system (CNS) as well as meningeal blood vessels. We also observed that MHC I-dependent interactions (but not costimulation) influenced the division program by advancing effector CTL through stages of cell cycle. These results demonstrate that CTL are poised to divide in transit and that their numbers can be influenced locally at the site of infection through interactions with cells displaying cognate antigen.
Human herpes virus 8 infection induces a Th2 skewed cytokine and chemokine response in primary B lymphocytes in the absence of virus replication (105.30)
Authors: Knowlton Emilee , Rappocciolo Giovanna , Piazza Paolo , Jenkins Frank , Jais Mariel , Rinaldo Charles
Published: January 2023

Objectives: Cytokines-chemokines play an important role in Kaposi’s sarcoma (KS), including tumor-cell proliferation, angiogenesis and vascular permeability. As DC-SIGN expressing B cells are natural targets for HHV-8, we examined the production of soluble factors by HHV-8 infected B cells and their relationship to HHV-8 replication. Methods: B cells were loaded with purified HHV-8, UV-light inactivated HHV-8 (UV-HHV-8) or soluble HHV-8 glycoprotein B (gB). Intracellular staining and flow cytometry were used to determine cytokine-chemokine production by B cells. Supernatants were examined for protein secretion by Cytometric Bead Array. Results: Peak levels of TNF-α, IL-6, IL-8, MIP-1α and MIP-1β of 570 (TNF-α) to 2.5 x 104 (MIP-1β) pg/ml were detected in HHV-8 infected cultures by 24-48h, which were 16 to 100 fold higher than in uninfected B cell cultures. Treatment with gB or UV-HHV-8 induced a similar pattern of cytokine-chemokine production by B cells. Conclusions: Elevated levels of several inflammatory cytokines and MIP chemokines were detected in HHV-8 infected B cells. Similar production of these factors by HHV-8 gB and UV-HHV-8 implies that full cycle virus replication is not necessary to elicit a strong immune response to HHV-8 infection. Determining the cytokine-chemokine response to HHV-8 infection of B cells could be significant to our understanding of HHV-8 pathogenesis, and could provide novel targets for vaccine development.
IFNγ regulates CXCL12 expression and cell trafficking during WNV infection (105.39)
Authors: Durrant Douglas , Klein Robyn
Published: January 2023

West Nile Virus (WNV), a mosquito-transmitted neurotropic flavivirus, causes severe neuroinvasive disease in the elderly and immuno-compromised. For viral clearance and survival, it is essential that virus-specific T cells enter into the central nervous system (CNS) parenchyma in both human and murine subjects. The chemokine CXCL12 is a potent immune factor that dictates homing and motility of lymphocytes to specific microenvironments. In the context of WNV encephalitis, we have shown that CXCL12-mediated localization of lymphocytes to the perivascular space modulates their ability to efficiently control infection. This localization regulates the parenchymal entry of infiltrating leukocytes during neuroinflammatory diseases. However, the means by which CXCL12 is regulated and how this regulation influences cell trafficking during WNV remains unclear. We show that CXCL12 is up-regulated within the spleen and down-regulated in the CNS on day 8 post-WNV infection. However, in the absence of IFN-γ, there is an increase in CXCL12 expression in the CNS of WNV-infected mice. We propose that during WNV infection, IFN-γ regulates CXCL12 expression both in the periphery and in the CNS, which has an effect on the localization of leukocytes and therefore influences the function of these cells to efficiently control infection.
HIV and SIV mutational analysis reveals functional decoupling of HIV-1 Nef-mediated T-cell activation and chemotaxis (105.46)
Authors: Park In-Woo , He Johnny
Published: January 2023

HIV-1 Nef has been shown to induce activation of CD4+ T cells but inhibit their migration toward SDF-1α. However, it is unclear how this protein orchestrates these two distinct activities. We found that HIV-1 Nef activated T cells only in the presence of CD3/ CD28 stimulation, while SIVpbj1.9 Nef activated T cells with or without CD3/CD28, indicating differential activation patterns between these two Nefs. We also observed that HIV-1 Nef inhibited T cell chemotaxis toward SDF-1α, while SIVpbj1.9 Nef did not. Moreover, we determined that CD3 stimulation diminished surface expression of CXCR4 in CD4+ T cells, leading to reduced SDF-1α-oriented migration. To further investigate the activation/chemotaxis interplay, we replaced the N-terminal half of HIV-1 Nef with the corresponding half of SIVpbj1.9 and determined its effects on chemotaxis and T cell activation. The resulting construct, hybrid HSNef, completely abrogated the chemotaxis blockade by HIV-1 Nef, while failing to activate T cells unless they were CD3/CD28 co-stimulated. Further analysis demonstrated that mutations at the myristoylation and SH3 binding sites in the N-terminal half of Nef were unresponsive to CD3/CD28 stimulation, but reversed the migration inhibition. However, a mutant for the basic rich domain of Nef was deficient for both activation and migration. Taken together, our findings indicate that the signals for T cell activation by Nef do not necessarily parallel those for T cell migration.
Viral persistence drives CD4 T cell differentiation toward T follicular helper cells (105.7)
Authors: Fahey Laura , Wilson Elizabeth , Elsaesser Heidi , Brooks David
Published: January 2023

CD4 T cells are central mediators of the adaptive immune response and are crucial to prevent and control viral infection. Yet, virus-specific CD4 T cell activity is considered to be lost during many persistent viral infections because they do not produce the classic Th 1 cytokines. Considering CD4 T cell help is critical for both CD8 T cell and B cell functions, it is unclear how CD4 T cells can lose responsiveness but continue to sustain long-term control of persistent viral replication. Recently, IL-21 was identified as a critical component of CD4 T cell help during viral persistence, suggesting that virus-specific CD4 T cells do not lose functionality but rather differentiate into a subset other than Th1. Thus, we sought to define how viral persistence impacts CD4 T cell differentiation such that it is capable of maintaining antiviral immunity during prolonged viral replication. We demonstrate that CD4 T cell function is not lost during viral persistence. Instead, viral persistence and prolonged T cell receptor stimulation progressively drives CD4 T cell development toward T follicular helper cells. The developmental program of CD4 T cells instituted during viral persistence is both molecularly and functionally distinct from that observed during an acute infection. Importantly, the progressive CD4 T cell differentiation and function is necessary to maintain B cell immunity and ultimately control persistent viral replication.
Treatment with Anti-CD137 (41BB) mAb enhances peptide vaccination against respiratory syncytial virus in aged mice (106.2)
Authors: Lee Sujin , Mittler Robert , Moore Martin
Published: January 2023

Respiratory syncytial virus (RSV) is the most important pathogen for lower respiratory tract illness (LRI) in infants and also a major cause of morbidity and mortality in the elderly. Using an aged mouse model of RSV pathogenesis, we found that aged mice had impaired antigen-specific CD8 T cell responses and delayed RSV clearance compared to young mice. We generated a peptide vaccine approach, TriVax, which is a co-mixture of a peptide representing an immunodominant RSV CD8 T cell epitope, a Toll-like receptor agonist, and a costimulatory anti-CD40 antibody. TriVax vaccination generated robust CD8 T cell responses and had a protective effect against RSV challenge in young BALB/c mice but not in aged BALB/c mice. We hypothesized that treatment of aged mice with agonistic CD137 (41BB) monoclonal antibody would stimulate T cells and enhance TriVax efficacy to RSV challenge in aged mice. We immunized 18-month old BALB/c mice twice with TriVax + anti-CD137 mAb. We found that co-administration of anti-CD137 mAb with TriVax enhanced antigen-specific CD8 T cell responses as well as showed a protective effect after RSV challenge. Thus, anti-CD137 mAb treatment rescued the deficient response to RSV TriVax vaccination in aged mice. The 41BB co-stimulatory pathway may be a target for enhancing T cell responses in the elderly.
The OmpA vaccine protects mice against lethal, extreme drug resistant-Acinetobacter baumannii infection (106.20)
Authors: Luo Guanpingsheng , Lin Lin , Ibrahim Ashraf , Spellberg Brad
Published: January 2023

Extreme drug resistant (XDR) Acinetobacter baumannii is a rapidly emerging pathogen causing infections with unacceptably high mortality rates due to inadequate available treatment. Prevention of such infections is a critical unmet medical need. Because diabetes is a clinical risk factor for A. baumannii infections, a diabetes ketoacidosis (DKA) mouse model of such infections was established to support a rational vaccine discovery program. Specific humoral immune responses to A. baumannii cell membrane proteins occurred during tail-vein infection with multiple clinical isolates in DKA mice. Western blots with paired pre-immune and immune sera identified outer membrane protein A (OmpA) as the immunodominant antigen. Vaccination of DKA mice with recombinant OmpA (rOmpA) with aluminum hydroxide adjuvant markedly improved survival, reduced tissue bacterial burden, and promoted inflammatory cytokine production in infected tissues. rOmpA is a promising vaccine candidate to prevent A. baumannii infections.
Schistosoma mansoni antigens induce IL-10 production and down-modulate the proinflammatory response in cutaneous leishmaniasis (106.21)
Authors: Araujo Maria Ilma , Bafica Aline , Varella Giuseppe , Oliveira Ricardo , Cardoso Luciana , Góes Alfredo , Loukas Alex , Carvalho Edgar , Oliveira Sérgio
Published: January 2023

S. mansoni infection or parasite products by induce regulatory cells and cytokines are able to down-modulate the Th1 inflammatory response involved in some autoimmune diseases. The objective of this study was to evaluate whether S. mansoni antigens are able to down-regulate the inflammatory immune response induced in vitro by the soluble Leishmania antigen (SLA) in cells of cutaneous leishmaniasis (CL) patients. Cytokines were measured in the supernatants of peripheral blood mononuclear cell (PBMC) culture stimulated with SLA in the presence and absence of S. mansoni recombinant antigens Sm29 and TSP2 and PIII, using a sandwich ELISA technique. Additionally, PBMC were evaluated for the expression of surface molecules CD14 and CD16, and also for intracellular cytokines (IL-12, TNF-alpha and IL-10). The addition of rSm29 and PIII to the cell cultures reduced the levels of IFN-γ in 54 and 58% of patients, respectively (mean reduction 49 and 56%). The three antigens used in the study were able to down modulate the production of TNF-alpha in 30% of CL patients and increased IL-10 production in 70 to 100% of them. The use of S. mansoni antigens also resulted in a lower frequency of classical and proinflammatory monocytes expressing IL-12 and TNF-alpha, while increased the frequency of monocytes expressing IL-10. The S. mansoni antigens used in this study down-modulated the inflammatory response in vitro induced by SLA in cells of CL patients.
Lactoferrin enhances BCG efficacy to protect against tuberculosis disease pathology (106.28)
Authors: Hwang Shen-An , Boyd Sydney , Kruzel Marian , Actor Jeffrey
Published: January 2023

Lactoferrin (LF), an iron binding protein found primarily in mucosal secretions and granules of neutrophils, possesses immune modulatory properties that include enhancement of the delayed type hypersensitivity against BCG antigens. Lactoferrin admixed to BCG enhanced host protection upon subsequent challenge with Erdman MTB, demonstrated by decreased organ CFU and reduced granuloma lesions. Splenic recall responses showed immunization with BCG/LF increased IFN-γ production. Additionally, dendritic cells cultured with BCG/LF increased expression of MHC II and CD86 compared to BCG alone. This study examines optimization of LF adjuvant to enhance efficacy of BCG by examining multiple vaccination parameters. C57BL/6 mice were immunized with or without booster at 8 weeks with BCG, BCG/bovine LF (1, 10 100μg), BCG/human recombinant lactoferrin (rhLF) sialylated (1, 10μg), or BCG/rhLF non-sialylated (1, 10μg). 12 weeks post-vaccination, mice were aerosol challenged with Erdman MTB and monitored through 24 weeks. At 6 weeks, splenic recall responses against BCG were examined. Peak LF adjuvant activity was observed at doses greater than 10μg/mouse, assessed by decreased pulmonary pathology post-MTB challenge. Splenic recall responses from BCG/bLF immunized mice showed increased IFN-γ and IL-17 with a concurrent decrease in IL-10 compared to BCG vaccinated and non-immunized controls. No significant differences observed between sialylated and non-sialylated human recombinant LF.
Intranasal vaccination with a defined live attenuated vaccine strain (ΔFTN0109) induces protective immunity against heterotypic pulmonary bacterial challenge. (106.29)
Authors: Signarovitz Aimee , Yu Jieh-Juen , Guentzel M. Neal , Klose Karl , Arulanandam Bernard
Published: January 2023

The need for an efficacious vaccine against the potential bioterrorism agent Francisella tularensis is a consequence of its low infectious dose, high mortality rate, and ability to be spread via aerosol, leading to pneumonic disease. FTN0109 is a novel protein that may be associated with the outer membrane of the bacterium and which is expressed across all subspecies of Francisella. In this study, we sought to determine the efficacy of ΔFTN0109 as a putative vaccine candidate. Using intramacrophage replication assays, ΔFTN0109 has been shown to be highly attenuated for growth. Moreover, the LD50 for this defined vaccine strain is >105 CFU in BALB/c and C57BL/6 mice following intranasal challenge in comparison to its parental strain U112 (LD50<10 CFU). Intranasal vaccination with ΔFTN0109 induces the expression of splenic antigen-specific IFN-γ and IL-2, with concurrent induction of preferential Th1 humoral responses (increased serum IgG2a over IgG1). Intranasal immunization with ΔFTN0109 confers complete protection in BALB/c mice against a subsequent pulmonary challenge with the heterotypic subspecies holarctica Live Vaccine Strain (LVS). Lungs, livers, and spleens of vaccinated and LVS-challenged mice exhibited significant reductions in bacterial burdens compared to mock-vaccinated and challenged animals. Current studies are ongoing to further clarify the mechanisms by which protection is conferred following vaccination with ΔFTN0109.
Effect of CCL1 antisense ODN on bacterial translocation in mice irradiated with whole body 137Cs γ-rays: III. Control of Enterococcus faecalis translocation (106.8)
Authors: Patel Niket , Asai Akira , Kogiso Mari , Cornforth Michael , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

In our previous studies, bacterial translocation and subsequent sepsis are demonstrated in whole body γ-irradiated mice (WBI-mice) orally infected with E. faecalis. M1Mϕ, which are major antibacterial effector cells against E. faecalis translocation, were induced by enterococcal antigen in mesenteric lymph nodes (MLN) of WBI-mice treated with CCL1 antisense ODN. Therefore, in this study, we tried to control E. faecalis translocation and subsequent sepsis in WBI-mice by CCL1 antisense ODN. WBI-mice were treated s.c. with CCL1 antisense ODN (10 μg/mouse) twice a day for 2 days starting 14 days after 5 Gy whole body γ-irradiation. From Day 1 to Day 10 after γ-irradiation, WBI-mice were decontaminated by antibiotics. One day after the final antisense ODN treatment, these mice were infected orally with 106 to 107 CFU/mouse of E. faecalis. The severities of infection were evaluated by the growth of the pathogen in MLN, kidneys and blood, as compared with those of WBI-mice treated with scrambled ODN. In the results, the great reduction of bacterial growth in MLN and kidneys of WBI-mice treated with CCL1 antisense ODN was demonstrated 1 to 3 days after infection, as compared with that of controls. Also, the pathogen was not detected in blood of WBI-mice treated with CCL1 antisense ODN. These results indicate that sepsis stemming from E. faecalis translocation in WBI-mice is controllable by CCL1 antisense ODN gene therapy.
Prevention of murine Sjögren's syndrome by local delivery of adeno-associated virus mediated Cytotoxic T-Lymphocyte Antigen 4 — immunoglobulin G fusion protein (107.1)
Authors: Yin Hongen , Nguyen Cuong , Samani Yuval , Uede Toshimitsu , Peck Ammon , Chiorini John
Published: January 2023

Dysfunction and lymphocytic infiltrations of the salivary and lachrymal glands are hallmarks of primary sjögren's syndrome (pSS), which is a chronic autoimmune epithelitis. Increasing evidence suggests that immunosuppressive agents can interrupt the progression of the disease. Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) is a key anti-inflammatory protein for shutting down the costimulatory pathway of B7:CD28, being required in immune suppression of T regulatory cells (Treg), and subsequent immune tolerance. To better understand the modulation of CTLA4-IgG on sjögren's syndrome, we generated a recombinant type 2 adeno-associated virus vector (AAV2) to express a chimera of mouse CTLA-4 fused with a human IgG (AAV2-CTLA4IgG). The recombinant CTLA4IgG was shown to be effective at blocking B7 expression on macrophages in vitro. Expression of the CTLA4IgG in the salivary glands of C57BL/6.NOD-Aec1Aec2 mice, a model of pSS, prevented the loss of salivary gland function and decreased both T-cell and macrophage infiltrates in the glands compared with control mice. Protection of the salivary glands was also accompanied by a decrease in Th1- and Th17- cell cytokines and an increase of TGF-β1 in CTLA4IgG treated mice compared with controls. This data suggests a potential role for CTLA-4IgG in preventing sjögren's syndrome by deactivating proinflammatory T cells and promoting suppressive Treg cells.
Murine bone marrow-derived dendritic cells generated in serum free conditions display an immature phenotype and protect from T1D. (107.11)
Authors: Looney Ben , Xia ChangQing
Published: January 2023

Dendritic cells possess the ability to modulate immune responses and therefore have been investigated as a cell based therapy to prevent or reverse Type 1 Diabetes. In the NOD mouse model, bone marrow-derived DCs (BM-DC) are generated from bone marrow precursors cultured with GM-CSF and IL-4 and also typically containing fetal calf serum (FCS). The effect of FCS on murine BM-DC has not been extensively investigated. In the present study we compare BM-DC generated in FCS free culture media (X-VIVO20) with BM-DC generated in culture media (RPMI1640) containing 10% FCS. Data show that CD11c+ BM-DC generated in a FCS free culture system express the same level of MHC Class II and CD80 and decreased CD86 expression compared to BM-DC derived from culture with 10% FCS. CD11c+ BM-DC from each culture system induce a similar level of Ag-specific CD4+ T cell proliferation and IFNγ production. CD11c+ BM-DC derived from FCS free culture media induce greater expression of IL-10 and IL-17. Additionally, we examine the ability of CD11c+ BM-DC, pulsed with the β cell-specific peptide GAD217-236, to prevent T1D onset. Data show 5 weekly IV administrations of both BM-DC delay T1D but only BM-DC generated in the FCS free culture system are able to prevent the onset of T1D in NOD mice when treatment begins at 9 weeks. Our results demonstrate that using BM-DC developed in FCS free medium not only avoids xenogeneic antigens but also provides better T1D protection.
Immunotherapy with Bet v 1-derived contiguous overlapping peptides leads to long term immunoregulatory responses (107.14)
Authors: Spertini Francois , Barbier Nathalie , Thierry Anne-Christine , Régine Audran , Charlon Vincent , Reymond Christophe
Published: January 2023

Allergen specific immunotherapy with whole pollen extract may induce anaphylaxis, is poorly standardized and of long duration. We thus designed a randomized, placebo-controlled phase I/II trial in volunteers with birch pollen allergic rhinitis and asthma to evaluate the safety and immunogenicity of a novel immunotherapy based on contiguous overlapping peptides (COPs) derived from Bet v 1, the major birch pollen allergen. A mixture of three COPs (AllerT™, Anergis SA, Switzerland) spanning the whole Bet v 1 molecule was selected for its inability to bind IgE. Prior to the pollen season, AllerT (in Alum) was injected subcutaneously to 15 adult volunteers at D0 (57 μg), D7, D14, D21 and D51 (95 μg each). Control volunteers (n=5) received only adjuvant. Overall AllerT was safe. No serious adverse events and no immediate allergic reactions were reported. AllerT induced a vigorous early Bet v 1 specific immune response marked by vaccine associated INFγ and IL-10 secretion. This contributed to a strong anti-Bet v 1-specific IgG4 enhancement. Moreover, two months after the second season post treatment (July 2010), serum Bet v 1 specific IgG4 response was still markedly increased as compared to pre-treatment values and to placebo whereas post seasonal Bet v 1 specific IgE titers were similar to baseline values. Taken together this indicates that immunotherapy with a mixture of three COPs derived from Bet v 1 (AllerT) was safe and immunogenic, and led to long term immunological memory.
Retinoic acid ameliorates experimental autoimmune hepatitis and suppresses the production of cytokine in NKT cells in mice (107.4)
Authors: Lee Kyoo-A , Song You Chan , Lee Yoon-Sook , Lee Jung-Mi , Kang Chang-Yuil
Published: January 2023

Retinoic acid(RA) has been demonstrated as a critical regulator of various immune cells. Especially RA regulates the response of helper T cells skewing to Th2 response during the priming. In spite of abundant studies regarding T cells, the role of RA on natural killer T(NKT) cell mediated response has not been assessed. In the present study, we demonstrated the effect of RA on the experimental autoimmune hepatitis and the response of NKT cells. We first examined whether pretreatment of RA could regulate concanavlin A(Con A)-induced hepatitis, which is initiated by NKT cells. The survival rate was increased dramatically and liver damage was ameliorated when RA was pretreated. And also the levels of IFN-gamma and IL-4 in serum were both reduced significantly in RA treated group and the cytokine expressing cells were reduced in various lymphocytes in liver, especially in NKT cells. Cytokine suppressive effects of RA were observed when NKT cell specific ligand was treated. These regulations were also detected when mononuclear cells of liver or splenocytes were treated with the stimulants in the presence of RA in vitro. These regulations are not related with the early activation of NKT cells. Rather, the apoptosis of lymphocytes in liver after the administration of con A were increased in RA-treated group. These findings implicates that RA ameliorates NKT cell mediated hepatitis and it could be used clinically to liver injury related with NKT cells.
Programming of induced pluripotent stem cells into regulatory T cell for arthritis prevention (107.6)
Authors: Haque Rizwanul , Lei Fengyang , Song Jianxun
Published: January 2023

Forkhead box p3 (FoxP3)-expressing regulatory T cells (Tregs) are involved in the regulation of central immune response and important for maintenance of peripheral immune response. Ectopic expression of FoxP3 conveys Treg phenotype to CD4+ T cells, lending the cells to therapeutic use in the prevention of autoimmune diseases. Previously we reported that induced pluripotent stem (iPS) cells have the capacity to develop into T cells. However, the potential of iPS cells differentiate into functional Tregs are not clear. In this study, we used retrovirus-mediated transduction to introduce mouse iPS cells with the FoxP3 gene and co-cultured the cells with the OP9 cells expressing the Notch ligand Delta like 1 (DL1) protein to induce Treg differentiation. In addition, we used a murine disease model to determine the role of iPS cell-derived Tregs in the treatment of arthritis. We found iPS cells-derived T cells expressed CTLA-4, CD25, and FoxP3 after three-week differentiation both in vitro and in vivo, and were able to secrete cytokines (e.g., IL-10). In addition, adoptive transfer of iPS cell-derived Tregs strongly suppressed arthritis development in the murine model of arthritis induced by collagen. These results indicate that iPS cells can differentiate into functional Tregs. The data suggest a potential novel approach to generate therapeutic Tregs from iPS cells for the treatment of autoimmune diseases Keywords: stem cells, regulatory T cells, FoxP3, T cell differentiation
iNKT cell-dependent activation of Kupffer cells in a mouse model of biliary obstruction (108.5)
Authors: Duwaerts Caroline , Gregory Stephen
Published: January 2023

Background: Previously we reported both Kupffer cells (KC), resident hepatic macrophages, and invariant natural killer T (iNKT) cells suppress liver injury in a mouse model of biliary obstruction. This study aims to clarify the interactions between these two cell populations during the course of cholestatic liver injury. Methods: Wild type (WT) and Ja18-/- (iNKT-deficient) female C57Bl/6 mice between 8 and 12 weeks of age were used for all experiments. Mice were injected with magnetic beads 3 days prior to sham- or bile duct ligation- (BDL) surgery to facilitate separation of KC from infiltrating mononuclear phagocytes. On day 3 post-surgery, animals were euthanized, and bead-containing KC were isolated and analyzed by flow cytometry. KC mRNA levels were analyzed using real-time quantitative PCR. Results: A shift in KC phenotype from CD11blo/Ly-6C- to CD11bhi/Ly-6C+ was more prominent in WT than Jα18-/- BDL mice. This shift was not seen in either sham groups. KC from WT BDL mice express higher mRNA levels of IL-1β, IL-6, IL-12, MCP-1, iNOS, and IL-10 compared to KC from Jα18-/- BDL mice. Conclusion: iNKT cell-activated KC express CD11bhi/Ly-6C+ and produce more pro- and anti-inflammatory cytokines in a mouse model of biliary obstruction.
A novel analog-sensitive allele of Csk reveals its kinase-independent role in the regulation of T cell receptor signaling (109.35)
Authors: Tan Ying Xim , Schoenborn Jamie , Zhang Chao , Shokat Kevan , Weiss Arthur
Published: January 2023

The earliest event in T cell receptor(TCR) signaling is the activation of the Src family kinase(SFK) Lck. Phosphorylation of a conserved tyrosine in the C-terminal tail of Lck inhibits its kinase activity and is reciprocally regulated by the protein tyrosine kinase, C-terminal Src kinase(Csk), and the protein tyrosine phosphatase, CD45. Using a chemical-genetic approach, our lab has generated a novel analog-sensitive dominant inhibitory allele of Csk(CskAS) that can be specifically inhibited by a small molecule. Surprisingly, inhibition of the CskAS allele leads to rapid T cell activation in the absence of any TCR stimulation, indicating that Lck activity is dynamically regulated by an equilibrium between Csk and CD45 activities and that there is an adaptive response to the reduced basal signaling imposed by the dominant inhibitory CskAS. Moreover, mutating the SH2 and SH3 domains of CskAS results in reduced T cell activation upon inhibition of CskAS, suggesting that protein-protein interactions are important for this ligand-independent activation. We have identified Dok-1(downstream of tyrosine kinases-1), an adaptor protein that inhibits tyrosine kinase signaling, as a candidate in controlling the adaptive response responsible for ligand-independent T cell activation. Dok-1 is hyperphosphorylated and interacts with Csk via its SH2 domain upon CskAS inhibition. We suggest that Dok-1 is part of a negative feedback loop that acts to dampen TCR signals.
Lipoteichoic acid from Lactobacillus plantarum induces nitric oxide release in interferon-γ-primed macrophages (110.12)
Authors: Kang Seok-Seong , Ryu Young Hee , Baik Jung Eun , Yun Cheol-Heui , Han Seung Hyun
Published: January 2023

Lactic acid bacteria are beneficial for human health by preventing microbial infections and reinforcing innate immunity in the gastrointestinal tract. Lipoteichoic acid (LTA) is known as a major immuno-stimulating agent of Gram-positive bacteria because of its potential to induce various inflammatory effector molecules. In the present study, we investigated whether highly-pure LTA from Lactobacillus plantarum induces nitric oxide (NO) in a murine macrophage-like cell line, RAW 264.7 cells. The LTA alone could not induce NO release even at 30 μg/ml but the combination of LTA and interferon (IFN)-γ enhanced NO release. The NO release was mainly mediated by inducible NO synthase (iNOS) since the NO release was inhibited by iNOS inhibitors, L-NIL or L-NAME, but not its stereoisomer, D-NAME. Upregulated mRNA and protein expression of iNOS was also observed in the presence of LTA and IFN-γ. In addition, LTA in the presence of IFN-γ induced IFN-γ expression and STAT-1 phosphorylation, both of which are the key pathways for inducing iNOS expression. Electrophoretic-mobility shift assay demonstrated that stimulation with the combination of LTA and IFN-γ remarkably increased the DNA-binding activities of NF-κB and AP-1 transcription factors which regulate the induction of iNOS gene expression. Collectively, these results indicate that LTA from L. plantarum alone has no inflammatory potential but it induces NO release under the inflamed conditions such as the presence of IFN-γ.
Protein kinase A RII directs prostaglandin E2 inhibitory effects on alveolar macrophage phagocytosis of Candida albicans via modulation of actin polymerization (110.16)
Authors: Serezani C. Henrique , Medeiros Alexandra , Kane Steve , Kim Sang-hoon , Lee Sang Pyo , Lewis Casey , Bourdonnay Emilie , Ballinger Megan , Peters-Golden Marc
Published: January 2023

Prostaglandin E2 (PGE2) is an eicosanoid that suppresses alveolar macrophage (AM) functions by activating two distinct stimulatory G protein (Gs)-coupled E prostanoid (EP) receptors (EP2 and EP4), increase in cyclic AMP (cAMP), and the actions of guanine nucleotide exchange protein activated by cAMP (Epac) and/or protein kinase A (PKA). Here, we investigated the role and cAMP effectors for PGE2 modulation of ingestion of C. albicans (Ca) and its effects on F-actin polymerization. PGE2 suppressed phagocytosis of Ca and agonists for both EP2 and EP4 receptors as well as PKA, but not Epac, mimicked this effect. Phagocytosis was augmented by the cyclooxygenase inhibitor indomethacin, antagonists of EP2 and EP4, inhibitors of adenylyl cyclase (AC), and PKA RII but not PKA RI. Confocal microscopy showed that Ca ingestion was associated with an increase in F-actin and a decrease in G-actin. COX and AC inhibitors further increased F-actin content, while PGE2 and PKA, but not Epac agonist decreased it. PGE2 decreased phosphorylation (associated with increased activity) of the actin depolymerizing factor cofilin-1 upon Ca challenge. PGE2-induced cofilin-1 dephosphorylation was mediated by the phosphatase PTEN. In summary, PGE2 inhibits Ca phagocytosis via an EP2/EP4-cAMP/PKARII axis which inhibits actin polymerization by increasing cofilin-1 activity. As PGE2 is overproduced in many immunosupressive states, these effects may contribute to impaired antifungal defense of the lung.
Novel quantitative proteomic analysis of influenza infected dendritic cells reveals a previously unappreciated immune modulatory protein and infection induced differential expression of DC proteins, with unexpectedly high membrane protein dynamics. (110.24)
Authors: Smallwood Heather , Lopez-Ferrer Daniel , Zink Erika , Pasa-Tolic Ljiljana , Thomas Paul
Published: January 2023

Airway dendritic cells (DC) initiate the immune response (IR) to respiratory pathogens (e.g.influenza). DC respond by releasing cytokines and presenting antigen to T cells, which activate on encountering the MCHI/peptide complex. This DC-T cell interaction dictates the IR to a given pathogen. Given that antigen presentation couples the innate and adaptive immune responses, a better understanding of these processes is vital to basic immune research and to therapeutic and vaccine development. To elucidate the underlying mechanisms of these processes we elected to identify DC proteins and quantify their changes in abundance using 18O labeling and LTQ-Orbit-trap-MS. Presuming the import of membrane protein dynamics on DCs, we developed a high pressure trypsinization technique to denature and digest insoluble proteins. We identified >1500 DC proteins, 20% of which change in abundance (i.e. ≥ 2 fold) early in infection. Unexpectedly this method detected infection induced changes in membrane proteins exceeding soluble protein dynamics. Analysis of these preliminary data indicates major shifts in DC metabolic and antigen processing and presentation pathways occur as a result of infection. Network analysis resulted in the identification of two critical modulator proteins; one of these was NFκB, a well known modulator of the immune response and another protein previously unidentified as an IR modulator. Together these data revealed new and important aspects of the immune response.
Overexpression of EAT-2, a SLAM family receptor adaptor molecule, suppresses CRACC receptor expression on Antigen Presenting Cells in an ERK MAP kinase dependent mechanism (110.26)
Authors: Aldhamen Yasser , Seregin Sergey , Liu Chyong-jy , Aylsworth Charles , Godbehere Sarah , Amalfitano Andrea
Published: January 2023

SLAM family receptors play a critical role in immune regulation. Their function is controlled primarily by SAP, EAT-2, and ERT adaptors. We have previously described a novel immunostimulatory function for EAT-2 in a vaccine model system. In the current work we sought to examine the mechanism of action of EAT-2 in APCs. Our results demonstrated that Ad vector infection itself of macrophages significantly (p<0.001) induced the transcription of the SLAM family member CRACC, but not other SLAM family members. In dramatic contrast, infection of macrophages with Ad-EAT2 significantly prevented Ad induced CRACC expression (p<0.001). FACS analysis confirmed that Ad mediated expression of EAT-2 also resulted in decreased protein levels of CRACC on APCs. By utilization of a variety of inhibitors, we confirmed that EAT-2 mediated suppression of CRACC required a functional ERK, but not Src-kinase, PI3K, or p38 pathways. In addition, blockade of ERK signaling abolished the ability of Ad-EAT2 to induce other innate immune response genes in APCs. Finally, utilization of immunoblot confirmed that Ad-EAT-2 significantly induced ERK activation in macrophages. Thus, EAT-2 over-expression by Adenovirus activates ERK pathway that inhibits Ad mediated induction of CRACC in APCs, an activity that correlates with increased induction of several innate immune response genes, as well the improved induction of antigen specific adaptive immune responses after Ad mediated transfer of immunogenic antigens.
Phosphorylation regulates RPS3-conferred specific NF-κB signaling during foodborne pathogen infection (110.7)
Authors: Wan Fengyi , Lenardo Michael
Published: January 2023

Rapid transcription of NF-kappaB target genes is vital for an effective immune response. We identified ribosomal protein S3 (RPS3) as an intrinsic NF-kappaB subunit that synergistically interacts with Rel subunit p65 to direct specific target gene transcription. Upon stimulation, RPS3 translocates to the nucleus, where it facilitates high affinity binding of NF-kappaB to certain kappaB sites. However, it still remains largely unknown how specific activating signals dissociate RPS3 from its cytoplasmic berth to execute the NF-kappaB “specifier” function in the nucleus. Here we report that the Inhibitor of kappaB (IkappaB) kinase 2 (IKK2), when activated, specifically phosphorylates RPS3. Serine phosphorylation of RPS3 augments its association with importin-alpha and subsequent nuclear translocation. Moreover, the foodborne pathogen Escherichia coli O157:H7 type III secretion system effector NleH1 specifically inhibits RPS3 phosphorylation to block subsequent RPS3 nuclear translocation. By selectively attenuating transcription of certain RPS3-dependent NF-kappaB target genes, E. coli O157:H7 alters the host innate NF-kappaB response in the gut to promote colonization and diarrhea but decrease mortality. In summary, our results indicate that the IKK2-dependent modification of a single amino acid in RPS3 promotes the specific recognition of certain kappaB sites by NF-kappaB, and provides new molecular insights into the complex pathogenetic mechanisms of foodborne pathogens.
Super resolution microscopy reveals that Caveolin-1 is required for antiviral immune response (110.8)
Authors: Gabor Kristin , Stevens Chad , Pietraszewski Matthew , Gould Travis , Lam Siew , Gong Zhiyuan , Hess Samuel , Kim Carol
Published: January 2023

Caveolae are integral to numerous signaling pathways but their function in antiviral signaling is largely unexplored. Understanding spatial distribution and dynamics of receptors within unperturbed membranes is essential for elucidating their role in antiviral signaling, but conventional studies of detergent-resistant membrane fractions cannot provide this information. While it is known that viruses exploit caveolae for entry into host cells, this study instead demonstrates an entry-independent mechanism for virus evasion of host cell defenses through disruption of clusters of signaling molecules organized within caveolae. Super-resolution microscopy has enabled the first single-molecule imaging of interactions between type I interferon receptors (IFN-R) and caveolae. In particular, cav-1b knockdown caused IFN-R clusters to disperse, suppressing antiviral immune response through abrogation of downstream signaling, a response strongly suggesting that IFN-R organization within caveolae is critical for IFN-mediated antiviral defense.
E. coli infection and endotoxic shock induce the expansion of regulatory dendritic cells (111.2)
Authors: Zhang Jiyan , Li Yajing , Guo Xiangrui
Published: January 2023

Regulatory dendritic cells (DCs) have been reported recently. However, whether regulatory DCs are induced in a natural immune response to an infectious disease or endotoxic shock remains to be explored. Here we report that regulatory CD11clowCD45RBhigh DCs become the predominant CD11c-expressing cells in the spleen during E. coli K12 infection, whereas i.p. injection of 2 ml of thioglycolate, which elicits aseptic peritonitis, does not induce regulatory CD11clowCD45RBhigh DCs. On the other hand, injection of isoproterenol for 5 consecutive days before E. coli K12 infection shows little effect on the induction of regulatory CD11clowCD45RBhigh DCs. A similar increase of regulatory CD11clowCD45RBhigh DCs is seen in mice injected with sublethal doses of LPS. The regulatory DCs secret little IL-10 and show marginal effect on the proliferation of T cells, but induce IL-10-expressing CD4 T cells. Taken together our data suggest that a natural immune response to an infectious disease or endotoxic shock induces regulatory CD11clowCD45RBhigh DCs.
Effect of CCL1 antisense ODN on bacterial translocation in mice irradiated with whole body 137Cs γ-rays: induction of M1 macrophages in translocation sites (111.21)
Authors: Kobayashi Makiko , Travis Lauren , Kogiso Mari , Cornforth Michael , Suzuki Fujio
Published: January 2023

M1Mϕ (IL-10-IL-12+ Mϕ) in mesenteric lymph nodes (MLN) of mice have been identified as major effector cells in the host defense against enterococcal translocation and subsequent sepsis. However, M1Mϕ are not easily generated in 5 Gy whole body γ-irradiated mice (WBI-mice) whose M2bMϕ (IL-10+CCL1+LIGHT+IL-12- Mϕ) predominate. M2bMϕ are inhibitory on resident Mϕ conversion to M1Mϕ. In our previous studies, M2bMϕ were not demonstrated in MLN of WBI-mice treated with CCL1 antisense ODN. In this study, therefore, we tried to induce M1Mϕ by enterococcal antigen in MLN of WBI-mice treated with CCL1 antisense ODN. WBI-mice were treated s.c. with CCL1 antisense ODN (10 μg/mouse) twice a day for 2 days starting 2 weeks after γ-irradiation. One day after the final treatment, these mice were stimulated orally with 107 heat-killed E. faecalis. Two days after stimulation, F4/80+ MLN-Mϕ were isolated from these mice and tested for their M1Mϕ properties based on their abilities to express intracellular IL-12, CXCL9 and iNOS mRNA. In the results, Mϕ from WBI-mice treated with scrambled ODN did not express iNOS mRNA, intracellular IL-12 and CXCL9, even when these mice were stimulated with enterococcal antigen. However, iNOS+IL-12+CXCL9+ Mϕ were isolated from the same antigen-stimulated WBI-mice treated with CCL1 antisense ODN. These results indicate that M1Mϕ are inducible by the enterococcal antigen in WBI-mice treated with CCL1 antisense ODN.
Systemic isotretinoin treatment modulates patients’ immune response to P. acnes (111.23)
Authors: Dispenza Melanie , Nelson Amanda , Gilliland Kathryn , Cong Zhaoyuan , Thiboutot Diane
Published: January 2023

Isotretinoin, a pro-drug for retinoic acid, is the only agent that induces a permanent remission of acne, but the mechanism underlying its long-term efficacy is unknown. We hypothesized that modulation of the immune response to the bacterium involved in acne, P.acnes, is key to isotretinoin’s ability to induce long-term or permanent remissions of acne. This study is the first to investigate changes in peripheral immune cells associated with isotretinoin treatment in acne patients in vivo. Peripheral blood samples were obtained from acne patients before starting isotretinoin therapy (baseline) and at 1, 4, 8, and 20 weeks after starting isotretinoin therapy. Monocytes and lymphocytes were isolated, activated in vitro, and analyzed by flow cytometry. Acne patients’ monocytes expressed higher levels of TLR-2 and secreted more IL-1β, IL-6, IL-8, and IL-10 in response to P. acnes than monocytes from normal volunteers. Isotretinoin therapy significantly decreased TLR-2 expression on acne patients’ monocytes compared to their baselines, and this decrease was maintained at six months after the cessation of therapy. Additionally, isotretinoin therapy significantly decreased the secretion of IL-1β, IL-6, IL-8, and IL-10 by untreated and P.acnes-treated monocytes from acne patients compared to baseline. These results suggest that modulation of the immune response to P.acnes may represent one mechanism of isotretinoin’s long-term efficacy.
Lactoferrin inhibits DNA-induced IFN-alpha production by human plasmacytoid dendritic cells (111.4)
Authors: Tewary Poonam , dela Rosa Gonzalo , Rodriguez Luis , Riboldi Elena , Shirota Hidekazu , Patrek Karel , Klinman Dennis , Yang De , Oppenheim Joost
Published: January 2023

Bacterial as well as self DNA is released at infection sites due to cell lysis. This DNA induces the production of IFN-α by TLR9+ Plasmacytoid dendritic cells (pDCs), which in turn can lead to exacerbated tissue damage as seen in psoriasis and lupus. Antimicrobial peptides like cathelicidins enhance the production of IFN-α, but little is known about the effect of other antimicrobial proteins stored in neutrophils. Here we present the inhibitory capacity of lactoferrin on DNA-induced IFN-α production by human pDCs. Confocal microscopy images showed lactoferrin binding to extracellular DNA molecules or CpG oligonucleotides, preventing their uptake by pDCs, and the subsequent activation of TLR-9. Flow cytometry experiments with labeled CpGs to analyze internalization dynamics indicate that lactoferrin prevented cathelicidin-DNA complexes from being internalized, circumventing over-activation of TLR-9. Moreover, when lactoferrin was supplemented in the diet, mice orally treated with the irritant DSS presented lower IFN-α levels in the mesenteric lymph nodes than controls. Therefore, we propose that lactoferrin acts as a scavenger molecule for DNA motifs, potentially reducing the undesirable effects of high levels of IFN-α.