bluuebunny/crossref_metadata_2025 huggingface dataset:
Identity of Toxoplasma gondii antigens that elicit CD4 T cells and their protective potential (130.13)
Authors: Satija Harshita , Blanchard Nicolas , Gonzalez Federico , Robey Ellen , Shastri Nilabh
Published: January 2023

Toxoplasma gondii (Toxo) can cause severe disease in immunocompromised individuals. Previous studies in various strains of mice have shown that genetic resistance to disease is linked to the MHC locus and protection is largely mediated by Toxo-specific CD8 T cells. In B10.D2 (H-2d) mice, CD8 T cells recognize an immunodominant peptide derived from the Toxo-protein, GRA6. However, a GRA6-specific response is not detected in C57BL/6 (B6, H-2b) mice. Instead, Toxo immunization of B6 mice elicits primarily a CD4 T cell response to unknown Toxo antigens. To identify the CD4 T cell stimulating antigens, we generated a Toxo-specific, lacZ inducible, CD4 T cell hybridoma and used it as a probe to screen a Toxo-cDNA library in bone marrow derived dendritic cells. The results reveal that CD4 T cells can be elicited by a hypothetical protein in the Toxo sequence database. We have defined the minimal peptide presented by MHC class II molecules and we will discuss the protective potential of this peptide-specific CD4 T cells in Toxo infected mice.
Retinoic acid and α-galactosylceramide differentially regulate CD19+ B cell activation in vitro and augment antibody production in vivo (130.20)
Authors: Chen Qiuyan , Ross A. Catharine
Published: January 2023

All-trans retinoic acid (RA) promotes the maturation and differentiation of B cells, which are known as a class of professional antigen-presenting cells. Here we have shown that CD1d, a MHC-class I-like molecule that presents lipid antigens, is expressed in the spleen mainly on B cells, and is increased by RA. Thus we hypothesized that RA and the CD1d ligand, α-galactosylceramide (αGalCer), could interact to promote the differentiation, maturation, and antibody response of antigen-activated B cells. αGalCer alone markedly stimulated and RA further increased B cell proliferation, synergizing with the B-cell antigen receptor. Combined treatment with RA and αGalCer also enriched a CD19hi B cell subpopulation, with higher CD1d expression, that exhibited a more differentiated phenotype in terms of sIgG1, CD138/syndecan-1 and PNA/Fas expression, as well as γ1 GLT, Pax-5, AID, and IRF-4, genes that control B cell proliferation and plasmacytic cell differentiation. In vivo, mice treated with RA and/or αGalCer during primary immunization with tetanus toxoid produced a higher anti-tetanus IgG response and, after reimmunization, had more bone marrow anti-tetanus antibody-secreting cells, indicative of heightened long-term memory. Thus, RA together with αGalCer can effectively regulate B cell proliferation and differentiation, ultimately promoting a more efficient antibody response to protein antigen.
Editing the pMHCI repertoire in the ER by tapasin and ERAAP (130.24)
Authors: Kanaseki Takayuki , Camfiled Lind Kristin , Shastri Nilabh
Published: January 2023

The peptide/MHC class I complexes (pMHCI) displayed on the cell surface are essential for eliciting CD8+ T cell responses for adaptive immunity. How appropriate pMHCI are assembled within the peptide loading complex (PLC) in the endoplasmic reticulum (ER) is unclear. Here we show that tapasin, the core component of the PLC, edits the pMHCI repertoire in vivo. Compared to cells from wild-type mice, cells from tapasin-deficient mice express an immunologically distinct pMHCI repertoire. Tapasin-deficient cells lack many normal pMHCI and also express a set of novel pMHCI on the surface. Thus, CD8+ T cells responses are elicited in reciprocal immunizations between wild-type and tapasin-deficient mice. Interestingly, this set of pMHCI ligands is distinct from that found earlier in mice lacking ERAAP, the ER aminopeptidase associated with antigen processing. Taken together these findings suggest that the pMHC I repertore is edited independently by tapasin and ERAAP in the ER.
The Contribution of Peptide-MHC Stability to Immunodominance and the Duration of Cross-Priming (130.31)
Authors: Watson Alan , Thompson Megan , Mylin Lawrence , Schell Todd
Published: January 2023

CD8+ T-cell responses are often multivalent, directed toward epitopes contained in one or several proteins. The response to Simian virus 40 large-tumor (T) antigen is directed toward four H-2b restricted epitopes (designated sites I, II/III, IV and V). Site V is immunorecessive since specific T cells are not detected following immunization unless the three dominant epitopes are inactivated in T antigen. Site V was previously shown to form unstable peptide-MHC (pMHC) complexes and to be inefficiently cross-presented. In this study, we identified point mutations within site V that increase the stability of pMHC while conserving recognition by site V-specific T cells. These mutations overcame the immunorecessive nature of site V within the context of full length T antigen. Increased immunogenicity of the site V variants was associated with an increase in both the proportion of naïve T cells that were primed and the duration of cross-priming in-vivo. We are currently investigating the contribution of the increased duration of epitope V cross-priming toward more effective naïve T cell recruitment. A model is proposed in which immunization approaches that extend the duration of cross-priming may promote effective T cell responses against otherwise weak epitopes.
Administration of a vaccine composed of dendritic cells pulsed with premalignant oral lesion lysate results in an increase in CD4+ and CD8+ T cells and a decrease in Foxp3+ cells in tongues of mice bearing carcinogen-induced premalignant oral lesions. (131.24)
Authors: Clark Anna-Maria , Young M. Rita
Published: January 2023

Most studies with dendritic cell vaccines have focused on eliminating or preventing progression of established malignancies. This is complicated by the immune suppression often exhibited in the presence of solid carcinomas. The goal of this study is to determine if an immune response can be elicited by administering a DC vaccine during the premalignant stages of oral squamous cell carcinoma, prior to development of systemic immune suppression. Mice treated with the carcinogen 4NQO in drinking water develop premalignant oral lesions which progress to OSCC. As previous studies in our lab demonstrated that premalignant lesions and OSCC overexpress common tumor antigens, BM-derived DCs were pulsed with premalignant lesion lysate and administered to 4NQO-treated C57/BL6 mice exhibiting premalignant lesions. Immunohistochemical analysis demonstrated an increase in CD8+ and CD4+ cells, as well as a decrease in Foxp3+ cells, in the tongues of 4NQO-treated mice immunized with premalignant lesion-pulsed dendritic cells (DCpm) compared to 4NQO-treated control mice. In addition, splenocytes from 4NQO-treated DCpm mice released greater amounts of IFN-γ compared to splenocytes from 4NQO-treated control mice in response to stimulation with antigen. These studies indicate that DCpm vaccination may be able to elicit a stimulatory immune response in 4NQO-treated mice bearing premalignant lesions.
Accumulation of a unique mature B cell population in aged mice. (132.2)
Authors: Hao Yi , O’Neill Patrick , Cancro Michael
Published: January 2023

Humoral immune responses and protective immunity are compromised in aged individuals, but the basis for this remains unclear. We have discovered a novel CD21/35-CD23-CD43-AA4.1-CD19+B220+ B cell population that gradually increases with age, expanding more than 5-fold in magnitude by 22 months of age. Despite sharing phenotypic features with mature follicular and marginal zone B cells, this novel subset is largely unresponsive to BCR, TLR or CD40 mediated stimulation. In addition, this population is markedly less reliant on the survival factor BLyS both in vitro and in vivo. The expansion of this unusual population does not reflect intrinsic features of cells derived from aged BM progenitors, but instead appears to be derived from a very small subset of FO B cells after exhaustive expansion. Together these findings reveal that primary B cell pools in aged individuals are increasingly populated by a unique population with the characteristics of “exhausted” B cells.
CD38 modulates CXCR4-mediated signals and homing of chronic lymphocytic leukemia (CLL) cells. (133.5)
Authors: Vaisitti Tiziana , Aydin Semra , Rossi Davide , Cottino Francesca , Audrito Valentina , Serra Sara , D'Arena Giovanni , Brennan Paul , Pepper Chris , Gaidano Gianluca , Malavasi Fabio , Deaglio Silvia
Published: January 2023

Human CD38 is a glycoprotein endowed with enzymatic and receptorial functions. Its engagement leads to activation/proliferation signals in T, B, NK and dendritic cells. In chronic lymphocytic leukemia (CLL) cells, CD38 drives proliferation and survival signals and its expression defines a subset of cells with higher migration and signaling ability in response to CXCL12, a critical chemokine in the re-circulation of leukemic cells from blood to lymphoid organs. The aim of this work is to define the role of CD38 in the regulation of CLL cell homing, an essential step in the maintenance and progression of the disease. Results indicate that i) CD38 signals stabilize early (ERK1/2 phosphorylation) and late (chemotaxis) functional responses to CXCL12. ii) The use of CLL clones with bimodal CD38 expression confirmed that the migrating population is CD38+. Furthermore, iii) de novo expression of CD38 using a lentiviral technique is followed by increased sensitivity to CXCL12 and ultimately chemotaxis. iv) In CLL cells, CD38 and CXCR4 are part of the same supra-molecular complex, that also includes the tetraspanin CD81 and the integrin CD49d. These results indicate that CD38 synergizes with the CXCR4 signaling pathway, through a specific molecular organization on CLL cells. The functional and physical association between CD38 and CXCR4 represents a novel mechanism in the regulation of neoplastic lymphocyte chemotaxis and homing and potentially a novel therapeutic target.
Role of CCR9-CCL25 axis in Prostate Cancer Metastasis (133.7)
Authors: Singh Shailesh , Sharma Praveen , Singh Rajesh , Grizzle William
Published: January 2023

Bone metastasis is major cause of prostate cancer (PCa) associated deaths and handful options are available to deal with this condition. Therefore, better understating of metastatic process and identification of molecular signature on metastatic cells are urgently needed to improve therapeutic. We have shown that chemokine receptor CCR9 is highly expressed by the PCa tissue and cells and play an important role in cell migration and invasion under the chemotactic gradient of CCL25, which is the only natural ligand for CCR9. CCL25 is primarily expressed by the thymus, which involutes with age. For the first time we show higher serum CCL25 in PCa patients in comparison to normal healthy donors and secretion of CCL25 within the primary prostate cancer in a paracrine manner. Interestingly, CCL25 was highly produced by the bone marrow cells of tumor bearing mice in comparison to non-tumor bearing mice. Furthermore, inhibition of CCR9-CCL25 interaction in vivo showed significant inhibition of bone metastasis and tumor establishment. These results provide clinical and biological significance of CCR9-CCL25 axis in prostate cancer metastasis. Higher production of CCL25 by the bone marrow cells of the tumor bearing mice confirms that CCL25 produced the bone marrow stromal cells are induced by the factors produced by the tumor cell to facilitate metastasis to the bone. Therefore, developing CCR9 directed therapies, may improve the therapeutic outcome of prostate cancer.
IL-16 Mediates CD4 T Lymphocyte Accumulation in the lymph nodes by desensitizing S1P1 (134.1)
Authors: Green Daniel , Richmond Jillian , Harstine Bo , Center David , Cruikshank William
Published: January 2023

IL-16 is a cytokine generated at sites of inflammation. Originally characterized as a CD4 ligand that induces migration, it is now known that IL-16 can alter TCR-induced cell activation and inhibit many chemokine receptors via cross-receptor desensitization. Previous work has demonstrated that stimulation by HIV-1 gp120 results in greater CCL21 induced migration with concomitant inhibition of sphingosine-1-phosphate (S1P) induced migration. Injection of gp120 into SCIDhu mice resulted in sequestration of CD4+ T cells within lymph nodes. We now investigated whether a natural ligand for CD4, IL-16, could similarly affect T cell homing. We found that IL-16 could not augment CCL21; however, was capable of inhibiting S1P induced migration. This effect was dependent on p56lck signaling and did not affect S1P receptor expression. IL-16 intratracheal instillation resulted in initial recruitment of CD4+ cells into the lung with subsequent recruitment into lung draining lymph nodes. Instillation of eotaxin-2 resulted in lung accumulation but not lymph node accumulation of T cells. The effect by IL-16 was CD4 dependent as no accumulation was seen using CD4-/- mice. Accumulation within the nodes occurred 2 h post instillation and resolved by 24 h. We propose that this is the first description of cytokine desensitization of S1P stimulation and further, suggests an additional mechanism by which IL-16 can regulate recruitment of cells and mediate the inflammatory process.
Differential contribution of IL-18 receptor beta to protective immunity and immunopathology during infection with intracellular bacteria (134.18)
Authors: Ismail Nahed , Trochtenberg Alyssa , Ghose Purnima , Jones Abrill
Published: January 2023

IL-18 has been shown to play a critical role in cytokine-induced organ failure during endotoxic shock. Human monocytotropic ehrlichiosis (HME) is a tick-borne infectious disease caused by E. chaffeenesis. HME is modeled in B6 mice using Ehrlichia muris (EM) that cause mild disease, and Ixodes ovatus Ehrlichia (IOE) that cause fatal shock-like syndrome. As Ehrlichia-induced septic shock and tissue injury is associated with increased IL-18 production, we examined the effect of blocking IL-18 signaling via IL-18Rβ on host defense & immune responses following sublethal (EM) and lethal (IOE) infection. Compared to IOE-infected isotype controls, in vivo IL-18Rβ neutralization in IOE-infected mice decreased bacterial burden, increased frequency of IFN-γ producing NK and NKT cells and enhanced production of TNF-α by T and non T cells on day 2 post-infection. On day 7 post-infection, blocking IL-18Rβ in lethally infected mice did not significantly influence the acquired pathogenic immune responses compared to lethally-infected isotype controls. Interestingly, blocking IL-18Rβ in the EM-infected mice increased bacterial burden, which was associated with suppressed early production of TNF-α and IFN-γ by different cell subsets compared to EM-infected isotype controls. These data suggest that IL-18Rβ may play dual protective and pathogenic roles in sublethal and lethal ehrlichial infection, respectively, through regulation of IFN-γ and TNF-α production by different innate cell subsets.
Distinct roles for IL-13 and IL-17 in chronic inflammation and fibrosis (134.8)
Authors: Wynn Thomas , Ramalingam Thirumalai , Madala Satish , Thompson Robert , Mentink Margaret , Barron Luke , Cheever Allen , Wilson Mark
Published: January 2023

TH2 cytokines regulate inflammation, mucus production, tissue remodeling, and fibrosis. Consequently, they have become important therapeutic targets for a number of debilitating illnesses, including asthma, idiopathic pulmonary fibrosis, ulcerative colitis, liver fibrosis, and other diseases in which TH2 cytokines are over-produced. In schistosomiasis, numerous studies have shown that TH2 cytokines play a critical role in the pathogenesis of the disease. Thus, understanding how the TH2 cytokine response is regulated during infection as well as the mechanism of action of each TH2 cytokine (IL-4, IL-5, IL-9, IL-10, IL-13, IL-21, IL-25) has been a major goal of our group over the past several years. Interestingly, in contrast to schistosomiasis-induced lung and liver fibrosis, bleomycin induced pulmonary fibrosis is associated with the development of an IL-17A-producing CD4+ and γδ+ T cell response. Studies conducted with C57BL/6 il17a-/- mice confirmed an essential role for IL-17A in bleomycin-induced fibrosis. Bleomycin-induced IL-17A production was also TGFβ dependent and recombinant IL- 17A-mediated fibrosis required TGFβ, suggesting cooperative roles for IL-17A and TGFβ in the development of fibrosis. We also found that fibrosis induced by IL-1β, which mimics bleomycin-induced fibrosis, is highly dependent on IL-17A. Together, these results suggest distinct non-overlapping immunological roles for IL-13 and IL-17A in the pathogenesis of fibrosis.
Eosinophils are required for the development of myosin-induced myocarditis and dilated cardiomyopathy (135.28)
Authors: Baldeviano Geral , Barin Jobert , Srinivasan Sachin , Chen Ping , Talor Monica , Zheng Donfeng , Bejda Djahida , Gabrielson Kathleen , Rose Noel , Cihakova Daniel
Published: January 2023

Eosinophilic infiltration has been associated with severe myocarditis and depressed cardiac function; however, demonstrating a causal relationship between eosinophils and myocarditis and dilated cardiomyopathy (DCM) has long remained elusive. We show here that Interleukin-5 (IL-5) is dispensable for the recruitment of eosinophils into the inflamed heart, the development of myosin-induced myocarditis (EAM) and the progression of EAM to DCM. However, IL-5-overexpressing transgenic mice developed peripheral eosinophilia, severe eosinophilic myocarditis, increased myocardial fibrosis and accelerated progression to DCM. In addition, eosinophil-deficient (dblGATA1) mice developed attenuated EAM, decreased myocardial fibrosis and greatly improved left ventricular function. Treatment with resorcinol, a specific inhibitor of eosinophilic peroxidase, significantly decreased myocarditis and myosin-induced heart hypertrophy. We conclude that eosinophils are required for the development of EAM, and the progression to DCM, at least in part through their secretion of eosinophilic peroxidase.
Abrogation of integrin α9β1-mediated signaling skews the generation of functional Th17 cells in regional lymph nodes of arthritic mice (135.3)
Authors: Kanayama Masashi , Morimoto Junko , Uede Toshimitsu
Published: January 2023

Th17 cells are essential regulator of autoimmune diseases. It is known that antigen presenting cells including conventional dendritic cells (cDCs) play a critical role in the differentiation of Th17 cells as the supplier of several Th17-polarizing cytokines such as IL-6 and IL-23. We have previously shown that the interaction between α9 integrin (α9) and its ligands such as tenascin-C (TN-C) enhance the production of a variety of cytokines including IL-6 by fibroblasts and macrophages. To examine the in vivo significance of α9 in Th17 development, we used murine collagen-induced arthritis (CIA). We demonstrated that α9 and TN-C were expressed by cDCs in the regional lymph nodes and the interaction between α9 and TN-C promoted the production of IL-6 from cDCs. The administration of anti-α9 antibody (55A2C) before and 2 days after first immunization with typeII collagen significantly suppressed the gene expression of IL-6 and IL-23 and the generation of Th17, but not Th1 cells in regional lymph nodes at day 7. Surprisingly at day 24, Th17, but not Th1 cell was significantly accumulated in regional lymph nodes of 55A2C-treated mice due to the down-regulation of CCR6 on Th17 cells. Nevertheless, arthritis was significantly attenuated in 55A2C-treated mice. These data suggest that α9-mediated signaling contributes to the generation of functional Th17 cells through the activation of cDCs in regional lymph node.
NODckitW/Wv mast cell deficient mice fail to develop type 1 diabetes (135.39)
Authors: Hatfield Julianne , Quirion Mary , Rao Kavitha , Brown Melissa
Published: January 2023

Mast cells (MCs) are innate immune cells typically known for their role in allergic responses and their ability to facilitate early immune responses to pathogens. Yet they also play a role in modifying many adaptive immune responses, including those that mediate autoimmune disorders such as EAE, the murine model of the human demyelinating disease, multiple sclerosis. We have transferred the c-kitW/Wv mutations, two independent mutations that affect MC development, onto the NOD mouse, a strain that exhibits a high incidence of spontaneous diabetes in females. NODc-kitW/Wv mice are mast cell deficient in all tissues examined, have similar mild hematopoietic deficits (macrocytic anemia) to those of the WBB6 parent strain and have normal thymic T cell compartments. In addition, they have inherited all of the 15 Idd loci tested. We demonstrate that NODc-kitW/Wv mice exhibit significantly reduced insulitis compared to wild type NOD mice and fail to develop hyperglycemia. These features correlate with the higher activation state and differential activation induced expression of inflammatory mediators in NOD mice compared to mast cells derived from the congenic diabetes resistant NOR strain of mice. Our data suggest that, as in EAE, mast cells are critical determinants of diabetes development.
Antiphospholipid (aPL) antibodies promote the release of plasma membrane-derived vesicles which inhibit the phagocytosis of apoptotic cells - role in Systemic Lupus Erythematosus (SLE) (135.9)
Authors: Antwi-Baffour Samuel , Kholia Sharad , Inal Jameel
Published: January 2023

Plasma Membrane-derived Vesicles (PMVs) are membrane-coated vesicles of diameter 0.1 to 1 µm, carrying various proteins inherent in their parental cells. PMVs are released when cells undergo activation/apoptosis via blebbing and shedding. Exposition of phosphotidylserine (PS) on the outer membrane leaflet could explain a role for PMVs in phagocytosis and thrombosis. Increased PMV release, although controlled, is a sign of cellular dysfunction and indicator of cell injury or stress. Patients with inflammatory disease such as Systemic Lupus Erythematosus (SLE) show increased PMV levels in plasma, accompanied by persistent apoptosis and defective clearance which correlates with disease activity. Although still unclear, impaired clearance of apoptotic cells (ACs) may represent a mechanism for the development or enhancement of SLE. Both PMVs and ACs express PS on the outer leaflet of the lipid bilayer as a marker for phagocytosis. We found that PMVs dose dependently inhibit the phagocytosis of ACs by competing for the PS receptor on macrophages. Phosphotidylserine is directly involved as annexin V-labelled PMVs reversed the PMV-mediated inhibition of phagocytosis. We further confirmed elevated PMV levels in SLE patient plasma and showed that the aPL antibodies from the plasma of such patients induced the release of PMVs. These findings show that PMVs can modulate phagocytosis of ACs and suggest an alternative mechanism for PMV enhancement of inflammatory responses.
Modulation of the RIG-I/MDA5-MAVS-dependent signaling pathway by distinct LCMV components shapes type I interferon responses (136.12)
Authors: Zhou Shenghua , Cerny Anna , Fitzgerald Katherine , Welsh Raymond , Kurt-Jones Evelyn , Finberg Robert
Published: January 2023

Early and efficient induction of type I IFN and other interferon-stimulated genes following virus infection is essential for the control of the viral infection, and this induction is mostly controlled by the innate immune system via pattern-recognition receptors (PRRs), including the endosomal Toll-like receptors and the retinoic acid inducible gene-I like RNA helicases RIG-I and MDA5. The prototypic arenavirus, Lymphocytic choriomeningitis virus (LCMV), is an excellent system to study the impact of virus-host interactions on viral pathogenesis. Although most aspects of LCMV model have been extensively studied and the findings obtained from this model have been used to interpret the pathogenesis of human viral diseases, the molecular mechanisms by which LCMV modulates type I IFN responses are not clear. Better understanding of these mechanisms is critical for the development of novel vaccines and effective antiviral therapeutic strategies against arenavirus infection. In the present study, we demonstrate that LCMV genomic RNA activates the RIG-I/MDA5-MAVS-dependent signaling pathway to induce type I IFN responses. We further demonstrate that LCMV nucleoprotein inhibits LCMV RNA induced type I IFN responses by physically interacting with both RIG-I and MDA5 signaling molecules. These results indicate that interactions between LCMV viral proteins and viral genomic RNA and the host signaling molecules are critical to regulate the virus induced innate immune response.
Expression patterns of Human Interferon-alpha and Interferon-lambda subtypes by monocytes, dendritic cells and B cells. (136.14)
Authors: Schramm Lynnsie , Mane Viraj , Bykadi Srikant , Navarro Maria , Jubin Ronald , Rabin Ronald
Published: January 2023

Human interferon-alpha comprises a family of thirteen highly homologous genes and twelve subtypes (coding sequences of IFN-alpha1 and -13 are identical). We employed two modifications of fluorescent-labeled probe technology--molecular beacons (MB) and locked-nucleic acids (LNA) to develop a highly efficient, sensitive, and specific quantitative RT-PCR assay for each human IFN-alpha subtype as well as the three subtypes of IFN-lambda, and for completion, IFN-beta and -gamma. The assay is formatted to a 384-well plate that measures 16 samples/plate and includes four-point standards for each IFN. Using this assay, we determined expression patterns of these IFN by monocytes, monocyte-derived macrophages and dendritic cells (MDM and MDDC), primary mDC, pDC and B cells in response to ligands for TLR 3, 4, and 9. All the myeloid cells, including mDC expressed high levels of IFN-beta and -lambda1, and moderate levels of IFN-alpha. pDC expressed high levels of almost all IFN, although with quantitative and in one case, a qualitative difference in between the response to CpG (TLR9) vs. imiquod (TLR7). B cells also expressed IFN—more subtypes than myeloid cells, but and fewer than pDC. Type I IFN expression was always accompanied by type III IFN expression. We conclude that IFN expression patterns are both ligand and cell-type specific. Subtypes alone or in combination may have unique roles in the innate immune response.
Identification of glycolipid ligands for human MINCLE. (136.22)
Authors: O'Callaghan Chris , Anita Mistry , Dunbar Rod , Feizi Ten , Liu Yan , de Sa Palma Maria , Fenton-May Angharad
Published: January 2023

We are studying the role of C-type lectin-like molecules in innate immunity. We have produced recombinant human MINCLE (CLEC4E) protein and generated a specific antibody against this. We find limited expression in a range of immune cells. Human MINCLE co-immunoprecipitates with the FcR gamma chain. Recombinant MINCLE was screened for binding to a variety of glycan arrays and found to bind to several glycolipids, including synthetic glycolipids. Using a quantitative cellular reporter assay, these glycolipids were shown to cause potent cellular activation through MINCLE. Further investigation are underway to assess the relevance of the interactions of MINCLE in human disease. These ligands have great potential as adjuvants.
NLRP3 inflammasome participates in lysosomal destabilization in macrophages exposed to the bacterial toxin tetanolysin O (136.30)
Authors: Heid Michelle , Chu Jessica , Sun Chengqun , Thomas L. , Nuñez Gabriel , Watkins Simon , Salter Russell
Published: January 2023

The NLRP3 inflammasome is a multiprotein complex that includes the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), which binds with capase-1 via CARD-CARD interactions and is involved with capase-1 activation. Our lab has previously shown that exposure of murine bone marrow derived macrophages (BMDM) with the cholesterol dependent cytolysin tetanolysin O (TLO), produced by Clostridium tetani, activates the NLRP3 inflammasome and induces IL-1beta secretion. Furthermore, we have shown that TLO treatment of BMDM results in the loss of acidity in lysosomes. In this study, our objective was to determine if the effects of TLO on the lysosome in BMDM are dependent upon NLRP3 and whether the inflammasome plays a role in lysosomal destabilization. Our data show that in NLRP3-deficient macrophages lysosomal permeabilization is diminished following toxin exposure relative to equivalently treated wild type cells. The results to be presented further our understanding of how NLRP3 functions in macrophages exposed to stressful stimuli such as bacterial toxins.
Size and shape matter: Role of surface curvature in inflammasome activation by synthetic microparticles (136.39)
Authors: St. Pierre Christine , Zhu Jinato , Hayward Ryan , Kurt-Jones Evelyn
Published: January 2023

The human immune system is poised to recognize and respond to foreign particulate substances, like crystals, pollen, bacteria or fungal spores. While some particles can induce significant inflammation others are relatively non-inflammatory. The physical and chemical characteristics that determine the response to different particles are unclear. In this study, we sought to systematically examine the roles that particle size, shape, and surface texturing play in uptake efficiency and subsequent immune cell activation. Using a novel micro-capillary flow focusing technique, we generated microparticles of similar composition but varying in shape from spherical to budding. We discovered that particles with high surface curvature (budding particles) were associated with and phagocytosed by macrophages at higher frequency than particles with low surface curvature, i.e., smooth particles. Remarkably, budding particles also induced stronger IL-1β secretion than smooth particles, through activation of the Nalp3 inflammasome-signaling complex. In vivo, budding particles induced more rapid neutrophil recruitment to the injection site, a hallmark of acute inflammation, than spherical particles. Although currently underappreciated, these findings demonstrate a remarkable and pronounced role for particle shape and surface curvature, independent of chemical composition and surface ligand recognition, in immune cell activation.
TLR3 Reduces Hepatitis C Viral Replication in Human Hepatocytes (136.7)
Authors: Eksioglu Erika , Bayouth Lily , Bess Jennifer , Zhu Haizhen , Liu Hongyan , Darwiche Houda , Nelson David , Liu Chen
Published: January 2023

Background: During an acute viral infection the intracellular innate immune defense is activated through the production of type I IFN and not well understood. We have developed an experimental model showing that HCV can induce IFN in LH86 cells, which have wild type TLR3 and RIG-I expression. The aim of this study is to examine how HCV induces IFN and the role of TLR in acute infection. Methods: LH86 and Huh7.5 cells and their transfected counter parts (LH86 siRIG-I, siTLR3 and siTLR7and Huh7.5 RIG-I, TLR3 and TLR7) were tested in an infectious JFH-1 HCV cell culture system. The IFN induction and antiviral efficacy of RIG-I in HCV infection was determined using real-time PCR. We also measured apoptosis by light microscopy and by flow cytometric analysis of Annexin-V expression. Results: Our data show that TLR3 is important for the induction of IFNβ in LH86 cells since silencing it prevents its production as well as the cytopathic effects normally observed in this cell line. Forced stable expression of wild type TLR3 and TLR7 in Huh7.5 cells partially restored the induction of IFN and significantly reduced the replication of the virus in an 80 day culture, with TLR3 having the greatest impact. The induction of IFN is strongly correlated with this antiviral effect. At the same time the virus decreases the expression of TLRs. Conclusion: Our study indicates that TLR3 and RIG-I in liver cells play a key role in anti-HCV defense.
Intravital 2P-imaging reveals anti-viral CTL mediate fatal pathology by recruiting myelomonocytic cells (137.17)
Authors: McGavern Dorian , Kang Silvia , Kim Jiyun , Dustin Michael
Published: January 2023

Many viruses can cause severe neurological complications in the central nervous system (CNS) through initiation of immunopathology. A case in point is the fatal meningitis induced by lymphocytic choriomeningitis virus (LCMV) - a disease associated with vascular breakdown and seizures. Although LCMV-specific CNS-infiltrating cytotoxic lymphocytes (CTL) are essential for disease, it is not known precisely how CTL mediate fatal immunopathology. Recently, it has become possible to directly observe immunity and immunopathology using two photon laser scanning microscopy (TPLSM). We initiated our real time studies of LCMV-induced meningitis by visualizing LCMV-specific CTL dynamics in the meninges during the onset of symptoms. Our findings yielded a novel and unexpected mode of pathogenesis defined by CTL trafficking into the meningeal space followed by a massive secondary recruitment of pathogenic myelomonocytic cells (i.e., monocytes and neutrophils). Importantly, myelomonocytic cells were responsible for severe vascular leakage and rapid onset disease during meningitis, and CTL directly participated in their recruitment by releasing myelomonocytic cell chemoattractants. Our results demonstrate that a CD8+ T cell dependent disorder can proceed in the absence of CTL effector mechanisms and rely instead on secondarily recruited myelomonocytic cells.
CD11c high dendritic cells regulate the reestablishment of vascular quiescence and stabilization after lymph node vascular expansion (138.10)
Authors: Tzeng Te-Chen , Chyou Susan , Tian Sha , Webster Brian , Carpenter April C. , Lu Theresa T.
Published: January 2023

Lymph node expansion during immune responses is accompanied by rapid vascular expansion. The maturation of the newly expanded vasculature and the regulatory mechanisms involved have not been well studied. We show that while initiation of vascular expansion in immune-stimulated nodes is associated with upregulated endothelial cell proliferation, increased high endothelial venule trafficking efficiency and VCAM-1 expression, and disrupted perivascular fibroblastic reticular cell organization, the reestablishment of vascular quiescence and stabilization after expansion is characterized by reversal of these phenomena. While CD11c medium cells are associated with the initiation of vascular expansion, CD11c hi MHCII med dendritic cells accumulate later and their short-term depletion in mice abrogates the reestablishment of vascular quiescence and stabilization. CD11c hi MHCII med cells promote endothelial cell quiescence in vitro and, in vivo, mediate quiescence at least in part by mediating reduced lymph node VEGF. Disrupted vascular quiescence and stabilization in expanded nodes is associated with attenuated T cell-dependent B cell responses. These results describe a novel mechanism whereby CD11c hi MHCII med dendritic cells regulate the reestablishment of vascular quiescence and stabilization after lymph node vascular expansion and suggest that these dendritic cells function in part to orchestrate the microenvironmental alterations required for successful immunity.
Neural control of acetylcholine release by T cells attenuates TNF production in endotoxemia (138.24)
Authors: Rosas-Ballina Mauricio , Ochani Mahendar , Olofsson Peder , Valdés-Ferrer Sergio , Chavan Sangeeta , Tracey Kevin
Published: January 2023

The cholinergic anti-inflammatory pathway regulates pro-inflammatory cytokine production through vagus nerve signaling via the α7 subunit of the nicotinic acetylcholine receptor expressed on macrophages. Vagus nerve stimulation requires an intact splenic nerve to attenuate TNF production by spleen macrophages. Despite exhaustive search, cholinergic fibers have not been observed in spleen. Here, we used transgenic mice that express enhanced green fluorescent protein (eGFP) under control of transcriptional regulatory elements of choline acetyltransferase, the enzyme that synthesizes acetylcholine. eGFP was detected in spleen B and T cells, some of which were located in close proximity to catecholaminergic nerve endings in the parenchyma of the white pulp. Vagus nerve stimulation significantly increased acetylcholine levels in spleen. In vitro, adrenergic stimulation induced the release of acetylcholine by spleen lymphocytes through a β2 adrenergic receptor dependent mechanism. Vagus nerve stimulation failed to suppress TNF in endotoxemic nude mice, and transfer of CD3+ T cells into nude mice restored vagus nerve control of cytokine production. Together, these data identify T cells as an essential component of the cholinergic anti-inflammatory pathway, and suggest that neural signals increase acetylcholine release from spleen T cells, which in turn attenuates TNF production in spleen via α7 expressed on innate immune cells.
The phagocyte NADPH oxidase (NOX2) regulates adaptive immune response at the level of both T cells and APCs (138.7)
Authors: Williams Mark , Shatynski Kristen , Chen Haiyan
Published: January 2023

Absence of phagocyte NADPH oxidase (NOX2) activity causes chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent bacterial infections. In addition to this innate immune dysfunction, CGD patients or animal models display an altered, perhaps augmented, adaptive immune response. It has been previously shown that T cells express the phagocyte oxidase and also been proposed that NOX2 expression alters APC function. The goal of this study was to determine if the differences in adaptive responses are inherent to T cells, or if other cells create an environment that promotes T helper polarization. Anti-CD3 activation of purified naive T cells from NOX2-deficient mice led to augmented IFNγ and IL-17 secretion. NOX2-deficient antigen presenting cells (APC) showed selective increases in GM-CSF, IL-6, TNF and MCP-1 induced by LPS challenge in vitro. APC function was measured using OVA-specific OT-II T cells, and oxidase deficient APC induced more IFNγ and IL-17 upon antigen stimulation. Adoptive transfer of OT-II T cells into wild type or NOX2(-/-) hosts followed by immunization with OVA in the presence of either CFA or Alum revealed altered cytokine profiles of T cells after restimulation in vitro. These results suggest roles for the phagocyte NADPH oxidase in shaping the adaptive and innate immune responses in both a T cell and APC dependent fashion.
Calcium Signaling Promotes Targeted Recycling of the Lateral Border Recycling Compartment (LBRC) During Diapedesis (140.1)
Authors: Han Fei , Florey Oliver , Feng Gong , Muller William
Published: January 2023

Leukocyte transendothelial migration (diapedesis, TEM) is a crucial step in both the innate and adaptive immune responses. We have shown that targeted recycling of membrane from a reticulum localized close to the endothelial cell (EC) lateral border (the lateral border recycling compartment, LBRC) surrounds the transmigrating leukocyte during TEM (Mamdouh, Z., et al 2003. Nature). Targeted recycling is required for TEM to proceed. However the mechanisms underlying recruitment of the LBRC are not entirely clear. An increase of cytosolic free calcium (Ca) in ECs and the resulting activation of Ca/Calmodulin-dependent myosin light chain kinase (MLCK) are crucial for diapedesis to occur. Here we address the role of Ca signaling in the process of targeted recycling. The cell permeant Ca chelator BAPTA-AM was loaded into ECs under conditions that blocked calcium flux in response to histamine, but did not impair cytoskeletal or junctional integrity. Under these conditions, blocking of cytosolic free Ca flux in ECs abolished targeted recycling of the LBRC, as well as leukocyte transendothelial migration to the same extent (> 80%). Furthermore, blockade of MLCK with ML7 (1uM) also blocked targeted recycling and TEM by > 80%. This study demonstrates that Ca signaling is upstream of LBRC targeted recycling, and MLCK activation (presumably by Ca/Calmodulin dependent mechanism) is required for targeted recruitment of the LBRC.
The S1P1 receptor antagonist W146 induces lymphopenia in mice. Demonstration that functional antagonism of S1P1 is the mechanism of lymphopenia evoked by fingolimod-like compounds. (140.15)
Authors: Godessart Nuria , Mustafa Sanam , Dolgachev Vlad , Aulí Mariona , Aguilar Núria , Cabedo Judit , Calbet Marta , Domènech Teresa , Domínguez María , López Rosa , Prats Neus , Milligan Graeme , Kunkel Steven , Tarrasón Gema
Published: January 2023

Agonists of sphingosine 1-phosphate receptor 1 (S1P1) are a novel class of immunosuppressants. S1P is the endogenous agonist of S1P1 and promotes lymphocyte egress from lymph nodes (LN) into blood. However, synthetic agonists like fingolimod prevent the egress, causing systemic lymphopenia. This apparent contradiction is explained by the concept of functional antagonism, according to which synthetic S1P1 agonists induce receptor internalization, making cells unresponsive to the endogenous S1P. If this mechanism is true, S1P1 antagonists should induce lymphopenia. We have fully characterized compound W146, a S1P1 antagonist, by binding and cellular assays. We then administered W146 and several S1P1 agonists to mice and measured blood lymphocytes and plasma levels of compounds in different experimental conditions. All compounds induced lymphopenia in blood and a concomitant increase of CD4+ and CD8+ T cells in LN. These results indicated that lymphopenia evoked by W146 was due to S1P1 blockade and not to another mechanism, like cytotoxicity. By Western blot, FACS and immunocytochemistry we showed that all compounds but W146 induce S1P1 internalization. W146 is able to prevent the internalization induced by an agonist. We report, for the first time, lymphopenia with a S1P1 antagonist. Plasma levels needed to surmount the effects of endogenous S1P are achieved in our experimental conditions. Our studies strongly support the validity of the concept of functional antagonism.
Endotoxin Tolerance represents a state of alternative polarization in mononuclear cells (142.4)
Authors: Pena Olga , Raj Disha , Pistolic Jelena , Fjell Christopher , Hancock Robert
Published: January 2023

Classical (M1) and alternative (M2) polarization of mononuclear cells such as monocyte and macrophages, mainly occurs as a response to micro-environmental challenges such as the incursion of a pathogen. Lipopolysaccharide, also known as endotoxin (LPS), is a potent inducer of inflammation and M1 polarization. LPS can also generate an effect in mononuclear cells known as endotoxin tolerance, defined as the reduced capacity of the cell to respond to LPS activation following a first exposure to this stimulus. Using systems biology approaches in peripheral blood mononuclear cells and monocyte derived macrophages, we identified that endotoxin tolerance is characterized by a similar state of alternative polarization, featuring expression of genes and proteins critical for the development of key M2 functions, including reduced production of pro-inflammatory mediators, expression of genes involved in phagocytosis as well as tissue remodeling. Moreover, expression of different methallothionein gene isoforms, known for their role in the control of oxidative stress and immunomodulation was also found consistently up-regulated during endotoxin tolerance. These results favor the hypothesis that, after an initial inflammatory stimulus, mononuclear cells undergo an alternative polarization most probably to control and balance prospective hyper-inflammation.
Increased severity of spontaneous autoimmune thyroiditis (SAT) and salivary gland infiltration in NOD.H-2h4 mice lacking regulatory T cells (T reg) (143.25)
Authors: Ellis Jason , Braley-Mullen Helen
Published: January 2023

NOD.H-2h4 mice given NaI in the drinking water develop SAT with chronic inflammation of the thyroid by T and B cells and production of anti-thyroglobulin (Tg) autoantibody. CD28-/- NOD.H2-4 mice, which have greatly reduced numbers of CD4+CD25+FoxP3+ Treg, were developed in order to examine the role of T reg in SAT development in WT and B cell-deficient (B-/-) mice. CD28-/- NOD.H2-h4 mice given NaI water develop more severe SAT than WT mice with collagen deposition (fibrosis) and low serum T4. Lymphocyte infiltration of salivary glands is also increased compared to WT mice. Most 4-6 mo old CD28-/- mice not given NaI water also develop SAT, whereas WT NOD.H-2h4 mice not given NaI water rarely develop SAT until much later. Although anti-Tg autoantibody levels generally correlate with SAT severity in WT mice, CD28-/- mice have reduced anti-Tg autoantibodies. Over time, the percentage of splenic follicular B cells decreases and marginal zone B cells increase. B -/- NOD.H-2h4 mice are resistant to SAT, but they develop SAT after transient depletion of T reg. CD28-/-B-/- mice, which lack most T reg, develop SAT comparable to WT NOD.H-2h4 mice. In B-/- mice, a lack of B cells as APC during the initiation phase of SAT may lead to preferential activation of Treg leading to reduced activation of effector T cells and reduced SAT severity. T reg also regulate SAT development if B cells are present since severe SAT develops in CD28-/- mice lacking T reg. (Supported by NIH RO1 AI076395)
CD137 expression distinguishes two functional CD4+CD25+ T regulatory subsets in Type one diabetes. (143.3)
Authors: Kachapati Kritika , Wu Yuehong , Mittler Robert , Wicker Linda , Ridgway William
Published: January 2023

The treatment of nonobese diabetic (NOD) mice with anti-CD137 monoclonal antibody prevented diabetes by increasing the number of T regulatory cells, which transferred protection from disease. We show here that two subsets of Tregs, CD137pos and CD137neg, differ phenotypically and functionally. CD137pos Tregs are distinctly CD69hi, CD62Llow and produce significantly higher levels of IL-10 and ICOS. There is significant decline in the number of CD137pos Tregs in NOD spleen but not thymus with age. This decline is not observed in an NOD.Idd9.3 congenic strain that is protected from diabetes and has a B10 Cd137 allele. The NOD.Idd9.3 strain has significantly higher numbers of CD137pos Tregs in both spleen and thymus compared to NOD mice. Functionally, NOD CD137pos Tregs are more suppressive than CD137neg cells. Here we dissect the mechanism of suppression and find that in a contact independent transwell system, the combination of anti-IL-10R and anti-TGF-β blocking antibodies decreases but does not abolish suppression of CD137pos Tregs. The loss of peripheral CD137pos Tregs with age in NOD mice, the increase in the frequency of CD137pos Tregs with anti-CD137 antibody treatment, and the persistently higher numbers of CD137pos Tregs in NOD.Idd9.3 congenic mice suggest that this functionally superior Treg subset could act to prevent diabetes when increased by genetic or environmental means. Therefore CD137pos Tregs are a therapeutic target in T1D and other autoimmune diseases.