bluuebunny/crossref_metadata_2025 huggingface dataset:
Possible Involvement of Matrix Metalloproteinase-9 in Langerhans Cell Migration and Maturation
Authors: Kobayashi Yasunobu , Matsumoto Motonobu , Kotani Mayumi , Makino Taketoshi
Published: January 2023

Epidermal Langerhans cells (LC) are potent dendritic cells in the induction of primary T cell-mediated immune responses in the skin. They capture foreign Ags and migrate to regional lymph nodes to carry and present these Ags to naive T cells. We investigated the role of matrix metalloproteinase-9 (MMP-9) in LC migration using an anti-MMP-9 mAb. Intradermal injection of anti-MMP-9 mAb before rhodamine B or oxazolone painting markedly inhibited these hapten-induced decreases in LC number in the epidermis and the accumulation of dendritic cells in the regional lymph nodes, indicating that MMP-9 plays some important roles in LC migration in the induction phase of contact sensitization. Treatment with anti-MMP-9 mAb also blocked the increase in cell size, dendrite development, and the enhanced expression of MHC class II Ags in LC induced by hapten painting. In addition, intradermal injection of purified MMP-9 induced marked increases in cell size, dendrite extension, and enhanced expression of MHC class II Ags in LC. These results strongly suggested that MMP-9 is involved not only in LC migration, but also in their morphological and phenotypic maturation in the skin.
Differential Impact of Substitution of Amino Acids 9–13 and 91–101 of Human CD14 on Soluble CD14-Dependent Activation of Cells by Lipopolysaccharide
Authors: Stelter Felix , Loppnow Harald , Menzel René , Grunwald Uwe , Bernheiden Martin , Jack Robert S. , Ulmer Artur J. , Schütt Christine
Published: January 2023

The soluble form of the endotoxin receptor CD14 is required for the LPS-induced activation of cells lacking membrane-bound CD14. It has been shown that a deletion mutant of human CD14 consisting of the N-terminal 152 amino acids has the capacity to mediate the stimulation of different cell types by LPS. To identify the structural domains of the molecule related to this functional property, we screened a set of alanine substitution mutants using CD14-negative U373 astrocytoma cells. We show that 3 of 18 soluble mutants of human CD14 failed to mediate the LPS-induced IL-6 production in U373 cells. These mutants were located in two regions of the molecule (aa 9–13 and 91–101) that are not essential for LPS binding. In addition, the mutants had a reduced capacity to mediate LPS-stimulated IL-6 production in human vascular endothelial and SMC. In contrast, the potential of sCD14(91–94,96)A, and sCD14(97–101)A to signal LPS-induced activation of human PBMC was not significantly reduced. These results show that the regions 9–13 and 91–101 are involved in the sCD14-dependent stimulation of cells by LPS but that the mechanisms by which different cell types are activated may not be identical.
Reduction of the Nonspecific Animal Toxicity of Anti-Tac(Fv)-PE38 by Mutations in the Framework Regions of the Fv Which Lower the Isoelectric Point
Authors: Onda Masanori , Kreitman Robert J. , Vasmatzis George , Lee Byungkook , Pastan Ira
Published: January 2023

Anti-Tac(Fv)-PE38, also called LMB-2, is a very active recombinant immunotoxin that has produced eight responses, including a durable clinical complete remission in a recently completed phase I trial of leukemias and lymphomas. Dose escalation was limited by liver toxicity. We have noted that the Fv of anti-Tac has an isoelectric point (pI) of 10.2. We hypothesize that the overall positive charge on the Fv portion of anti-Tac(Fv)-PE38 contributes to nonspecific binding to liver cells and results in dose-limiting liver toxicity. We have used a mouse model to investigate the basis of this toxicity and found that lowering the pI of the Fv of anti-Tac from 10.2 to 6.82 by selective mutation of surface residues causes a 3-fold decrease in animal toxicity and hepatic necrosis. This change in pI did not significantly alter the CD25 binding affinity, the cytotoxic activity toward target cells, or antitumor activity, resulting in a 3-fold improvement in the therapeutic index. If this decreased toxicity occurs in humans, it should greatly increase the clinical utility of this immunotoxin.
Subversion of Monocyte Functions byCoxiella burnetii: Impairment of the Cross-Talk Between αvβ3 Integrin and CR3
Authors: Capo Christian , Lindberg Frederik P. , Meconi Sonia , Zaffran Yona , Tardei Gratiela , Brown Eric J. , Raoult Didier , Mege Jean-Louis
Published: January 2023

Several intracellular pathogens exploit macrophages as a niche for survival and replication. The success of this strategy requires the subversion or the avoidance of microbicidal functions of macrophages. Coxiella burnetii, the agent of Q fever, is a strictly intracellular bacterium that multiplies in myeloid cells. The survival of C. burnetii may depend on the selective use of macrophage receptors. Virulent C. burnetii organisms were poorly internalized but survived successfully in human monocytes, whereas avirulent variants were efficiently phagocytosed but were also rapidly eliminated. The uptake of avirulent organisms was mediated by leukocyte response integrin (αvβ3 integrin) and CR3 (αMβ2 integrin), as demonstrated by using specific Abs and RGD sequence-containing peptides. The phagocytic efficiency of CR3 depends on its activation via αvβ3 integrin and integrin-associated protein. Indeed, CR3-mediated phagocytosis of avirulent C. burnetii was abrogated in macrophages from integrin-associated protein−/− mice. In contrast, the internalization of virulent C. burnetii organisms involved the engagement of αvβ3 integrin but not that of CR3. The pretreatment of monocytes with virulent C. burnetii organisms prevented the CR3-mediated phagocytosis of zymosan particles and CR3 activation assessed by the expression of the 24 neo-epitope. We conclude that the virulence of C. burnetii is associated with the engagement of αvβ3 integrin and the impairment of CR3 activity, which probably results from uncoupling αvβ3 integrin from integrin-associated protein. This study describes a strategy not previously reported of phagocytosis modulation by intracellular pathogens.
Inhibition of Oligodendrocyte Apoptosis by Sublytic C5b-9 Is Associated with Enhanced Synthesis of Bcl-2 and Mediated by Inhibition of Caspase-3 Activation
Authors: Soane Lucian , Rus Horea , Niculescu Florin , Shin Moon L.
Published: January 2023

We have previously shown that generation of sublytic C5b-9, the membrane attack complex of complement, induces oligodendrocytes to enter cell cycle and reduces apoptotic cell death in vitro. In the present study, the cellular factors involved in apoptosis of oligodendrocyte progenitor cells and oligodendrocytes, and the inhibitory effect of C5b-9 on apoptotic process were investigated. Oligodendrocyte progenitor cells identified by mAb A2B5 that were isolated from neonatal rat brains were differentiated into oligodendrocytes in serum-free defined medium. The differentiation, which occurs simultaneously with apoptotic cell death, was associated with a rapid loss of bcl-2 mRNA and increased expression of caspase-3 mRNA. Activation of caspase-3 in differentiating cells was demonstrated by the generation of 17- and 12-kDa fragments of caspase-3 proenzyme and by cleavage of poly(ADP-ribose) polymerase, a specific caspase-3 substrate. Cell death associated with differentiation was inhibited by the caspase-3 inhibitor DEVD-CHO in a dose-dependent manner. Assembly of sublytic C5b-9 resulted in inhibition of caspase-3 activation. In addition, synthesis of BCL-2 protein in oligodendrocytes was significantly increased by C5b-9. The TNF-α-induced apoptosis of oligodendrocytes was also inhibited by C5b-9. These results indicate that up-regulation of BCL-2 protein and inhibition of caspase-3 activation are potential mechanisms by which C5b-9 increases survival of oligodendrocyte in vitro and possibly in vivo during inflammation and immune-mediated demyelination affecting the CNS.
Endogenous Monocyte Chemoattractant Protein-1 (MCP-1) Protects Mice in a Model of Acute Septic Peritonitis: Cross-Talk Between MCP-1 and Leukotriene B4
Authors: Matsukawa Akihiro , Hogaboam Cory M. , Lukacs Nickolas W. , Lincoln Pamela M. , Strieter Robert M. , Kunkel Steven L.
Published: January 2023

We investigated the involvement of monocyte chemoattractant protein (MCP)-1 in a murine model of septic peritonitis induced by cecal ligation and puncture (CLP). Initial studies demonstrated that CLP induced a dramatic increase in MCP-1 production in the peritoneum, followed by an increase in the recruitment of leukocytes. MCP-1 blockade with anti-MCP-1 antiserum significantly decreased the survival rate following CLP, which was accompanied by an enhanced recovery of viable bacteria from the peritoneum. This was likely due to the reduction in the recruitment and activation of both macrophages and neutrophils. To understand the mechanisms whereby MCP-1 may influence neutrophil infiltration, levels of chemokines known to attract neutrophils were monitored, which showed that peritoneal levels of macrophage-inflammatory protein (MIP)-2, KC, and MIP-1α were not altered with anti-MCP-1 Abs. However, anti-MCP-1 Abs reduced the peritoneal levels of leukotriene B4 (LTB4) by 59%. The i.p. injection of MCP-1 into normal mice resulted in elevated levels of LTB4 in the peritoneum. In vitro, MCP-1 stimulated the production of LTB4 from peritoneal macrophages, in a dose-dependent manner. A specific LTB4 receptor antagonist (CP-105,696) inhibited CLP-induced recruitment of both neutrophils and macrophages, which was accompanied by a reduced level of MCP-1 in the peritoneum. Finally, administration of CP-105,696 was extremely detrimental to the survival of mice following CLP. These experiments demonstrate that endogenous MCP-1 serves as an indirect mediator to attract neutrophils via the production of LTB4, and suggest the cross-talk can occur between MCP-1 and the lipid mediator LTB4 during septic peritonitis.
NH2- and COOH-Terminal Truncations of Murine Granulocyte Chemotactic Protein-2 Augment the In Vitro and In Vivo Neutrophil Chemotactic Potency
Authors: Wuyts Anja , D’Haese Anne , Cremers Valerie , Menten Patricia , Lenaerts Jean-Pierre , De Loof Arnold , Heremans Hubertine , Proost Paul , Van Damme Jo
Published: January 2023

Chemokines are important mediators of leukocyte migration during the inflammatory response. Post-translational modifications affect the biological potency of chemokines. In addition to previously identified NH2-terminally truncated forms, COOH-terminally truncated forms of the CXC chemokine murine granulocyte chemotactic protein-2 (GCP-2) were purified from conditioned medium of stimulated fibroblasts. The truncations generated 28 natural murine GCP-2 isoforms containing 69–92 residues, including most intermediate forms. Both NH2- and COOH-terminal truncations of GCP-2 resulted in enhanced chemotactic potency for human and murine neutrophils in vitro. The truncated isoform GCP-2(9–78) was 30-fold more potent than intact GCP-2(1–92)/LPS-induced CXC chemokine (LIX) at inducing an intracellular calcium increase in human neutrophils. After intradermal injection in mice, GCP-2(9–78) was also more effective than GCP-2(1–92)/LIX at inducing neutrophil infiltration. Similar to human IL-8 and GCP-2, murine GCP-2(9–78) and macrophage inflammatory protein-2 (MIP-2) induced calcium increases in both CXCR1 and CXCR2 transfectants. Murine GCP-2(9–78) could desensitize the calcium response induced by MIP-2 in human neutrophils and vice versa. Furthermore, MIP-2 and truncated GCP-2(9–78), but not intact GCP-2(1–92)/LIX, partially desensitized the calcium response to human IL-8 in human neutrophils. Taken together, these findings point to an important role of post-translationally modified GCP-2 to replace IL-8 in the mouse.
Transcriptional and Translational Regulation of Inflammatory Mediator Production by Endogenous TGF-β in Macrophages That Have Ingested Apoptotic Cells
Authors: McDonald Patrick P. , Fadok Valerie A. , Bratton Donna , Henson Peter M.
Published: January 2023

We recently reported that phagocytosis of apoptotic cells inhibits the release of inflammatory cytokines by human macrophages. In this paper we show that apoptotic cell uptake by mouse J774 macrophages also inhibits the synthesis and secretion of the chemokines, macrophage inflammatory protein-2 (Mip-2), KC, and Mip-1α (but not that of monocyte chemoattractant protein-1 (MCP-1)/JE), and increases TGF-β formation. Anti-TGF-β neutralizing Abs largely reversed the inhibitory effect of apoptotic cell uptake, and accordingly, exogenous TGF-β down-regulated the synthesis of the same mediators. Apoptotic cell ingestion or TGF-β also inhibited Mip-2 and Mip-1α gene expression in LPS-treated J774 cells, whereas TNF-α mRNA levels were unaffected. Importantly, TGF-β pretreatment of J774 cells did not significantly alter chemokine and TNF mRNA stability. Finally, we found that apoptotic cell uptake and TGF-β did not modulate NF-κB or AP-1 DNA binding in J774 cells. We conclude that the decreased production of chemokines and TNF resulting from apoptotic cell ingestion is largely mediated by a common event, i.e., feedback inhibition by endogenous TGF-β, but involves different mechanisms. Whereas TNF-α production appears to be translationally down-regulated, the suppression of most chemokines investigated appears to reflect transcriptional inhibition. In a broader context, the impairment of chemokine and TNF generation by apoptotic cell uptake might represent an important mechanism contributing to the resolution of inflammation. An additional consequence could be the selective recruitment of monocytes into inflammatory sites, as MCP-1/JE production by mouse macrophages was unaffected by apoptotic cell uptake, in contrast to other chemokines.
Urokinase Receptor (CD87) Aggregation Triggers Phosphoinositide Hydrolysis and Intracellular Calcium Mobilization in Mononuclear Phagocytes
Authors: Sitrin Robert G. , Pan Pauline M. , Harper Hollie A. , Blackwood R. Alexander , Todd Robert F.
Published: January 2023

Leukocytes utilize urokinase receptors (uPAR; CD87) in adhesion, migration, and matrix proteolysis. uPAR aggregate at cell-substratum interfaces and at leading edges of migrating cells, so this study was undertaken to determine whether uPAR aggregation is capable of initiating activation signaling. Monocyte-like U937 cells were labeled with fluo-3-acetoxymethyl ester to quantitate intracellular Ca2+ concentrations ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by mAb cross-linking. uPAR aggregation induced highly reproducible increases in [Ca2+]i of 103.0 ± 10.9 nM (p < 0.0001) and >3-fold increases in cellular d-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Similar increases in [Ca2+]i were also elicited by uPAR aggregation in human monocytes, but cross-linking a control IgG2a had no effect on [Ca2+]i. Selectively cross-linking uPA-occupied uPAR with an anti-uPA mAb produced smaller increases in [Ca2+]i, but fully saturating uPAR with exogenous uPA enhanced the [Ca2+]i response to equal the effect of aggregating uPAR directly. Increased [Ca2+]i was inhibited by thapsigargin, herbimycin A, and U73122, but only partially reduced by low extracellular [Ca2+], indicating that uPAR aggregation increases [Ca2+]i by activating phospholipase C through a tyrosine kinase-dependent mechanism, generating Ins(1,4,5)P3 and releasing Ca2+ from Ins(1,4,5)P3-sensitive intracellular stores. Cross-linking the β2 integrin CR3 could not duplicate the effect of uPAR cross-linking, and uPAR-triggered Ca2+ mobilization was not blocked by anti-CR3 mAbs. These results indicate that uPAR aggregation initiates phosphoinositide hydrolysis by mechanisms that are not strictly dependent on associated uPA or CR3.
H-Ras Signals to Cytoskeletal Machinery in Induction of Integrin-Mediated Adhesion of T Cells
Authors: Tanaka Yoshiya , Minami Yasuhiro , Mine Shinichiro , Hirano Hideyasu , Hu Chang-Deng , Fujimoto Hiroko , Fujii Koichi , Saito Kazuyoshi , Tsukada Junichi , van Kooyk Yvette , Figdor Carl G. , Kataoka Tohru , Eto Sumiya
Published: January 2023

The adhesive function of integrins is regulated through cytoplasmic signaling. The present study was performed to investigate the relevance of cytoplasmic signaling and cytoskeletal assembly to integrin-mediated adhesion induced by chemokines. Adhesion of T cells induced by chemokines macrophage inflammatory protein (MIP)-1α and MIP-1β was inhibited by pertussis toxin, wortmannin, and cytochalasin B, suggesting that both G protein-sensitive phosphatidylinositol (PI) 3-kinase activation and cytoskeletal assemblies are involved. The chemokine-induced T cell adhesion could be mimicked by expression of small G proteins, fully activated H-RasV12, or H-RasV12Y40C mutant, which selectively binds to PI 3-kinase, in T cells, inducing activated form of LFA-1α and LFA-1-dependent adhesion to ICAM-1. H-Ras expression also induced F-actin polymerization which colocalized with profilin in T cells. Adult T cell leukemia (ATL) cells spontaneously adhered to ICAM-1, which depended on endogenous MIP-1α and MIP-1β through activation of G protein-sensitive PI 3-kinase. H-Ras signal pathway, leading to PI 3-kinase activation, also induced active configuration of LFA-1 and LFA-1-mediated adhesion of ATL cells, whereas expression of a dominant-negative H-Ras mutant failed to do. Profilin-dependent spontaneous polymerization of F-actin in ATL cells was reduced by PI 3-kinase inhibitors. In this paper we propose that H-Ras-mediated activation of PI 3-kinase can be involved in induction of LFA-1-dependent adhesion of T cells, which is relevant to chemokine-mediated signaling, and that profilin may form an important link between chemokine- and/or H-Ras-mediated signals and F-actin polymerization, which results in triggering of LFA-1 on T cells or leukemic T cells.
Chemokine and Chemokine Receptor Interactions Provide a Mechanism for Selective T Cell Recruitment to Specific Liver Compartments Within Hepatitis C-Infected Liver
Authors: Shields Philip. L. , Morland Clare M. , Salmon Michael , Qin Shixin , Hubscher Stefan G. , Adams David H.
Published: January 2023

The role played by chemokines in regulating the selective recruitment of lymphocytes to different tissue compartments in disease is poorly characterized. In hepatitis C infection, inflammation confined to portal areas is associated with a less aggressive course, whereas T cell infiltration of the liver parenchyma is associated with progressive liver injury and cirrhosis. We propose a mechanism to explain how lymphocytes are recruited to hepatic lobules during bursts of necroinflammatory activity in chronic hepatitis C infection. We report here that lymphocytes infiltrating hepatitis C-infected liver express high levels of the chemokine receptors CCR5 and CXCR3. However, whereas the CCR5 ligands macrophage inflammatory protein-1α and -1β were largely confined to vessels within portal tracts, the CXCR3 ligands IFN-inducible protein-10 and monokine-induced by IFN-γ were selectively up-regulated on sinusoidal endothelium. In vitro, human hepatic sinusoidal endothelial cells secreted IFN-inducible protein-10 and monokine-induced by IFN-γ in response to stimulation with IFN-γ in combination with either IL-1 or TNF-α. This suggests that intrahepatic Th1 cytokines drive the increased expression of IFN-inducible protein-10 and monokine-induced by IFN-γ and thereby promote the continuing recruitment of CXCR3-expressing T cells into the hepatic lobule in chronic hepatitis C infection.
Increased Vaccine-Specific T Cell Frequency After Peptide-Based Vaccination Correlates with Increased Susceptibility to In Vitro Stimulation But Does Not Lead to Tumor Regression
Authors: Lee Kang-Hun , Wang Ena , Nielsen Mai-Britt , Wunderlich John , Migueles Steven , Connors Mark , Steinberg Seth M. , Rosenberg Steven A. , Marincola Francesco M.
Published: January 2023

Although in vitro sensitization assays have shown increased melanoma Ag (MA)-specific CTL reactivity after vaccination with MA peptides, clinical responses have been uncommon. This paradox questions whether data obtained from the in vitro stimulation and expansion of T cells lead to an overestimation of the immune response to vaccines. Using HLA/peptide tetramer (tHLA), we enumerated MA-specific T cell precursor frequency (TCPF) directly in PBMC from 23 melanoma patients vaccinated with gp100:209–217(210M) (g209–2M) peptide. Vaccine-specific TCPF was higher in postvaccination PBMC from seven of seven patients treated with peptide alone and four of five patients treated with peptide plus IL-12 (range of postvaccination TCPF, 0.2–2.4% and 0.2–2.5%, respectively). The increased TCPF correlated with enhanced susceptibility to in vitro stimulation with the relevant epitope. Paradoxically, no increase in postvaccination TCPF was observed in most patients who had been concomitantly treated with IL-2 (1 of 11 patients; range of postvaccination TCPF, 0.02–1.0%), a combination associated with enhanced rates of tumor regression. The lack of increase in TCPF seen in these patients corresponded to inability to elicit expansion of vaccine-specific T cells in culture. This study shows that a peptide-based vaccine can effectively generate a quantifiable T cell-specific immune response in the PBMC of cancer patients, though such a response does not associate with a clinically evident regression of metastatic melanoma.
HLA-E and HLA-G Expression on Porcine Endothelial Cells Inhibit Xenoreactive Human NK Cells Through CD94/NKG2-Dependent and -Independent Pathways
Authors: Sasaki Hitomi , Xu Xiao-Chun , Mohanakumar T.
Published: January 2023

Human NK cells contribute a significant role to host defense as well as xenogeneic cytotoxicity. Previous studies using human 721.221 cell line have shown that peptides derived from the leader sequence of the HLA-G binds and up-regulates the surface expression of HLA-E molecules, which was considered to consequently provide negative signals to human NK cells. However, the direct role of HLA-G in inhibiting human NK cells remains controversial. In this study, we showed that the expression of HLA-G or HLA-E in porcine endothelial cells directly protected sensitive porcine cells from human NK cell-mediated xenogeneic cytotoxicity. Ab blocking assays using F(ab′)2 of the HLA class I-specific mAb PA2.6 indicated that the protection was directly mediated by the expression of HLA-G and HLA-E on the porcine cells. The HLA-E-mediated protection was blocked by anti-human CD94 Ab. In addition, the engagement of HLA-E lead to the phosphorylation of the CD94/NKG2 complex and the recruitment of SH2 domain-containing protein phosphatase 1 (SHP-1) to the complex. Therefore, HLA-E protected porcine cells from xenoreactive human NK cells through a CD94/NKG2-dependent pathway. In contrast, HLA-G inhibited human NK cells in the absence of CD94/NKG2 phosphorylation or SHP-1 recruitment, and the inhibition was not blocked by anti-CD94 Ab. Therefore, HLA-G protected porcine cells from human NK cells through a CD94/NKG2-independent pathway. These results demonstrated that both HLA-E and HLA-G could directly inhibit human NK cells in the absence of other endogenous HLA class I molecules. These results also have practical implications in preventing xenograft rejection mediated by human NK cells.
Anti-Double-Stranded DNA Antibodies and Immunostimulatory Plasmid DNA in Combination Mimic the Endogenous IFN-α Inducer in Systemic Lupus Erythematosus
Authors: Vallin Helena , Perers Anders , Alm Gunnar V. , Rönnblom Lars
Published: January 2023

Patients with systemic lupus erythematosus (SLE) have increased blood levels of IFN-α, which correlate to disease activity. We previously identified an IFN-α-inducing factor (IIF) in the blood of SLE patients that activated the natural IFN-α-producing cells in cultures of normal PBMC. The SLE-IIF contained DNA and IgG, possibly as small immune complexes. In our study, we demonstrated that SLE-IIF correlated to the presence of anti-dsDNA Abs in patients and contained anti-dsDNA Abs as an essential component. Purified anti-DNA Abs or SLE-IgG caused only a weak IFN-α production in cultures of normal PBMC in the presence of costimulatory IFN-α2b. However, they converted the plasmid pcDNA3, which itself induced no IFN-α production in PBMC, into an efficient IFN-α inducer. A human monoclonal anti-ss/dsDNA Ab had the same effect. This IFN-α-inducing activity of the plasmid was abolished by methylation, suggesting that unmethylated CpG DNA motifs were important. Like IIF in SLE serum, the combination of SLE-IgG and pcDNA3 appeared to stimulate IFN-α production in natural IFN-α-producing cells, a unique cell population resembling immature dendritic cells. The IFN-α production was greatly enhanced by IFN-α2b and IFN-β, and for SLE-IIF it was also enhanced by GM-CSF but inhibited by IL-10. We have therefore identified a new function of DNA-anti-DNA Ab complexes, IFN-α induction, that might be important in the pathogenesis of SLE.
Virus-specific production of IFN-gamma and IL-17 by CD4+ T cells from patients infected with West Nile virus (128.18)
Authors: Latham Lesley  P , Bramson Jonathan
Published: January 2023

The mechanisms that underlie the pathology of neuroinvasive West Nile virus (WNV) remain unknown. The goal of this study was to characterize the CD4+ T cell response to WNV in individuals with neuroinvasive and non-neuroinvasive symptoms. WNV-infected subjects were screened for CD4+ T cell reactivity with peptide pools derived from a library spanning the WNV polyprotein. Virus-specific CD4+ T cells were directed primarily against E, NS1, and NS3. Most WNV-specific CD4+ T cells produced IFN-γ. Surprisingly, IL-17 production was primarily associated with NS3-reactive cells. Single cell analysis revealed three distinct populations: IFN-γ+/IL-17-, IFN-γ+/IL-17+, IFN-γ-/IL-17+. Preliminary analysis suggests that elevated frequencies of virus-specific CD4+ T cells producing both IFN-γ and IL-17 are associated with protection against WNV neuroinvasive disease. This work was funded by a contract from NIAID (N01-AI-40066).
Role of lipid structure in the humoral immune response in mice to covalent lipid-peptides derived from the membrane proximal region of HIV-1 gp41 (128.2)
Authors: Watson Douglas , Szoka Francis
Published: January 2023

The membrane proximal region (MPR) of HIV-1 gp41 is a desirable target for development of a vaccine that elicits neutralizing antibodies since the patient-derived monoclonal antibodies, 2F5 and 4E10, bind to MPR and neutralize primary isolates. The 2F5 and 4E10 antibodies cross-react with lipids and structural studies suggest that MPR immunogens may be presented in a membrane environment. We hypothesized that covalent attachment of lipid anchors would enhance the immunogenicity of MPR-derived peptides presented in liposomal bilayers. In a comparison of eight different lipids conjugated to an extended 2F5 epitope peptide, a sterol, cholesterol hemisuccinate (CHEMS), was found to promote the strongest anti-peptide immune response in BALB/C mice. Differences in immunogenicity amongst the conjugates could not be explained solely by any one factor, such as: the presence of a phosphate in the lipid, membrane partitioning of the lipid anchor or peptide secondary structure. Conjugation to CHEMS also rendered a 4E10 epitope peptide immunogenic. Finally, conjugation of CHEMS to a helically constrained immunogen spanning both epitopes elicited antibodies that bound to each of the individual 4E10 and 2F5 epitopes as well as to an oligomeric recombinant envelope protein. Further research into the mechanism of how structure influences the immune response to the MPR may lead to immunogens that could be useful in prime-boost regimens for focusing the immune response in an HIV vaccine.
Quantitative bioimaging of the WR and IHDJ vaccinia virus dissemination and the effects of prophylactic immunoglobulin treatments (128.23)
Authors: Zaitseva Marina , Meseda Clement , Kapnick Senta , Manischewitz Jody , Nelson Henriette , Merchlinsky Michael , Weir Jerry , Golding Hana
Published: January 2023

Protection from lethal challenge with recombinant WR-Luciferase (TK+) and IHDJ-Luciferase (TK+) vaccinia viruses (VV) was studied by detecting firefly luciferase activity in live Balb/c mice using IVIS technology. High correlation (R2=0.8-0.9) was observed between viral loads in the organs measured by plaque assay and bioluminescence. WR- and IHDJ VV that generate predominantly IMV and EEV forms, respectively, induced lethality within 8-10 days after intranasal challenge with 10^5 pfu yet their dissemination and susceptibility to therapies differed. WR persisted at significantly higher levels than IHDJ at the site of challenge, while IHDJ replicated better in the liver in agreement with the roles of EEV and IMV in host dissemination and inter host transmission. Vaccinia immunoglobulin (VIGIV) at Day -2 completely protected from the WR but not from IHDJ challenge. Sym002, a mixture of 26 human anti-vaccinia mAbs (Symphogen, Denmark), completely protected from WR challenge at 1000 lower dose than VIGIV, but only partially protected from IHDJ. Protection by Sym002 correlated with a significantly reduced flux at the nasopharyngeal area and in the liver. Importantly, our study detected significantly higher bioluminescence at the nasopharyngeal area and in the lungs in non-survivors than in survivors suggesting that bioimaging can substitute for lethality endpoint for evaluation of anti-smallpox treatments.
CD8+ T cell inflation and differentiation during persistent CMV infection is independent of viral replication (128.29)
Authors: Snyder Christopher M , Cho Kathy S. , Bonnett Elizabeth L , Hill Ann B.
Published: January 2023

Murine cytomegalovirus establishes systemic, chronic infection in mice and drives the dramatic accumulation of virus-specific CD8+ T cells over time. Though the virus is thought to achieve true latency, a small amount of persistent viral replication may occur as well. We have been investigating the development and maintenance of the inflationary CD8+ T cells that result from chronic infection. These cells remain functional, but appear to be differentiated into effector cells by repeated antigen stimulation. We have recently shown that most of these T cells do not divide during chronic infection and have a short life span in circulation. Dying cells can be replaced by newly primed T cells. However, further evidence suggests that the majority of the short-lived T cells in circulation are replaced by the progeny of clones primed early infection. Thus our data suggest that the inflationary T cells are maintained by continuous production of short-lived, effector T cells from a memory population. We had assumed that this equilibrium would be dependent on persistent viral replication to drive the differentiation of new T cells. However, blocking viral replication with anti-viral drugs or infecting with a replication deficient virus still resulted in CD8+ T cell inflation and differentiation. These data suggest that viral persistence and CD8+ T cell accumulation does not require viral replication.
Specificity-dependent cross reactive capacity of HIV-specific CTLs (128.9)
Authors: Motozono Chihiro , Takiguchi Masafumi , Ueno Takamasa
Published: January 2023

Although CTLs having a potent cross reactive capacity toward HIV variants is important for control of viral replication, what inherent characteristics of antigenic peptides influence it remains elusive. We addressed this issue based on immunodominant HLA-B35-restricted CTLs specific for 2 overlapping Nef epitopes, VY8 (VPLRPMTY) and RY11 (RPQVPLRPMTY). The TCR analysis of CD8 T cells from 6 HLA-B35+ HIV-infected subjects showed that the majority of VY8-specific cells had a same TCR-α variable segment whereas RY11-specific cells had diverse TCR clonotypes. We then transduced 6 representative pairs of TCR-αβ chains specific for VY8 and RY11 into TCR-deficient T cells and analyzed their individual fine specificity toward an array of variant peptides. Each RY11-specific TCR retained its reactivity toward lager numbers of variant peptides than any VY8-specific TCRs, indicating the highly cross-reactive nature of individual RY11-specific TCR clonotypes. Moreover, unlike VY8-specific TCRs, each individual RY11-specific TCR had the ability to recognize different peptide variants. Taken together, the overall cross reactive capacity of the oligoclonal CTL populations specific for RY11 is more potent than those specific for VY8, highlighting the specificity-dependent cross reactive capacity of antiviral CTL responses.
Select human anthrax protective antigen (PA) epitope-specific antibodies provide protection from lethal toxin challenge in vitro and in vivo (129.16)
Authors: Crowe Sherry R , Ash Linda L , Engler Renata J M , Ballard Jimmy D , Harley John B , Farris A Darise , James Judith A
Published: January 2023

The release of Bacillus anthracis spores can cause significant morbidity and mortality and an effective vaccine as well as directed immunotherapeutics are needed. The currently licensed vaccine requires six primary vaccinations, annual boosters, and has been associated with serious adverse events. Thus there is increasing interest in developing an improved vaccination strategy which can generate lasting protective immunity with reduced vaccinations. This study seeks to identify the major humoral targets of B. anthracis that provide protection. To this end, plasma was obtained from two hundred vaccinated individuals and thirty unvaccinated controls. Ninety-five percent of all vaccinated individuals had anti-protective antigen antibodies and these antibodies correlated with in vitro protection against lethal toxin. Utilizing overlapping decapeptides, we identified common antigenic regions within those individuals with high toxin neutralizing activity. Epitope-specific antibodies directed against three of these regions were isolated and were able to mediate protection in vitro and in a mouse model of in vivo lethal toxin challenge. Identification of common, protective, peptide-specific humoral immune responses provide key information for further immunotherapeutic and vaccine development. NIAID: 5U19AI062629
Human CD1-restricted T cells from leprosy lesions display distinct and complementary functions in comparison to MHC-restricted T cells (129.28)
Authors: Sieling Peter A , Hunger Robert , Modlin Robert L
Published: January 2023

CD1 proteins are a family of MHC-like polypeptides that present lipids rather than peptides to T cells. We have examined the role of CD1-restricted T cells in the context of mycobacterial infection since T cells mediate host defense against mycobacterial infection and the mycobacterial envelope contains a high concentration of lipids. To determine whether CD1 and MHC-restricted T cells represent distinct or complementary functional populations, T cells derived from lesions of leprosy patients were activated through their CD3 receptor, gene expression patterns were evaluated, and protein expression measured using ELISA. CD1- and MHC-restricted T cells exhibited a similar profile of cytokine production, particularly Th1 and Th2 cytokines. In contrast, the two populations of T cells were more distinct in their chemokine expression. CD1-restricted T cells produced CCL20, which shares a receptor with antimicrobial β-defensins; in contrast, MHC-restricted T cells produced CXCL10, a chemokine whose receptor is expressed on activated Th1 T cells. Our data reveal both complementary and distinct functions of CD1- and MHC-restricted T cells and suggest that CD1-restricted T cells function in the rapid response to microbial infection in contrast to MHC-restricted T cells, which regulate adaptive immunity.
Generation of non-specific and specific B cell responses to the Trypanosoma cruzi B cell Mitogen TcPRAC (129.4)
Authors: Bryan Marianne A , Norris Karen A
Published: January 2023

Acute Trypanosoma cruzi infection results in polyclonal B cell activation and a delay of specific humoral immunity. TcPRAC, a T. cruzi B cell mitogen may contribute to this dysfunction. Stimulation of splenocytes with TcPRAC induced B cell proliferation, secretion of IgM and IL-10, and up-regulation of CD69 and CD86. During experimental infection, TcPRAC specific IgG was undetectable when responses to other T. cruzi antigens developed. Genetic immunization generated TcPRAC specific IgG without apparent B cell dysfunction. TcPRAC immune sera inhibited TcPRAC-induced B cell proliferation in vitro. These results suggest that TcPRAC may contribute to delayed specific antibody development during experimental infection by non-specifically activating B cells and inducing IL-10 and non-specific IgM secretion. In addition, we demonstrated that while TcPRAC was not antigenic during experimental infection, delivery of TcPRAC via genetic immunization elicited specific antibodies. Further experiments will analyze the effect of TcPRAC immunization and neutralization on the generation of T. cruzi specific humoral immunity during early acute phase infection.
Distinct bacterial genes influence on Th1 and Th2 responses during Listeria monocytogenes infection (129.6)
Authors: Kanoh Makoto , Shen Hau , Maruyama Saho , Matsumoto Akira , Shinomiya Hiroto , Cossart Pascale , Goebel Werner , Asano Yoshihiro
Published: January 2023

CD4+T cells are divided into four types according to the set of lymphokines they produce, i.e., Th1 cells, Th2 cells, Th17 cells and Treg cells. The differentiation process of T cells into each subset is controlled by cytokines produced during the innate immune response in its early phase. We showed a two-step induction mechanism for differentiation of T cell subsets during pathogenic infection. The first step is induced by the basal function of dendritic cells and is antigen-independent and antigen-nonspecific. This step affects the precursor frequency of Th1 and Th2 T cell subsets. In the present study, we investigated bacterial genes, which involved in the host-pathogen interaction and is required for the initiation of T cell subset differentiation in the early phase of pathogenic infection. We demonstrate that TLR2 and MyD88 molecules are not essentially required for the induction of Th1 cell differentiation during Listeria infection, although those molecules are required for early inflammatory gene induction. It is also demonstrated that genes involved in the expression of the pathogenisity of Listeria are not required for the induction of Th1 cell subset. Finally, it is demonstrated that the induction of Th1 cell subset and the inhibition of Th2 cell subset induction are separate phenomenon and are mediated by distinct genes carried by Listeria genome.
Role of IL-17 receptor alpha (IL-17Rα) signaling in Helicobacter pylori-host interactions (129.7)
Authors: Algood Holly M Scott , Allen Shannon Sedberry , Washington Kay , Peek Richard , Cover Timothy L
Published: January 2023

H. pylori persistently colonizes the human stomach and induces a gastric mucosal inflammatory response. To investigate a potential role of Th17 responses in modulating H. pylori-host interactions, wild-type C57Bl/6 (WT) mice and IL-17Rα-/- mice were orogastrically infected with H. pylori. By 3 months post-infection, H. pylori colonization density was significantly higher in IL-17Rα-/- mice than in WT mice. Gastric inflammation, assessed by histologic staining, was significantly more severe in H. pylori-infected IL-17Rα-/- mice than in infected WT mice. FACS analysis indicated that infected IL-17Rα-/- mice had significantly higher numbers of gastric CD4+ cells and B cells and significantly lower numbers of gastric neutrophils, compared to infected WT mice. IFNγ, IL-4, and multiple chemokines (KC, LIX, MIP2) were expressed at significantly higher levels in stomachs of infected WT mice than in infected IL-17Rα-/- mice. In contrast, gastric IL-17 transcript levels were 9-fold higher in infected IL-17Rα-/- mice than in infected WT mice. Primary gastric epithelial cells (PGEC) harvested from WT mice expressed KC and GM-CSF in response to recombinant IL-17, whereas cells from IL-17Rα-/- mice did not. PGECs from both WT and IL-17Rα-/- mice expressed IL-6, IL-1β, and MIP2 in response to IL-17. These data indicate that IL-17Rα signaling has an important role in modulating the gastric mucosal response to H. pylori.
CD8+ T cells clear human intestinal Cryptosporidium infection through cytotoxic granule release (129.8)
Authors: Pantenburg Birte , Castellanos-Gonzalez Alejandro , Dann Sara M. , Connelly Rhykka L. , Lewis Dorothy E. , Ward Honorine D. , White, Jr Arthur Clinton
Published: January 2023

INTRODUCTION: A key factor in the human adaptive immune response towards Cryptosporidium, associated with resistance to infection, is IFNg. Its function and the cells involved in clearing the parasite from the human intestinal epithelium are unknown. As CD4+ and CD8+ cells produce IFNg after re-stimulation with parasite antigens in vitro we hypothesized that CD8+ cells contribute to control of the infection. METHODS: We studied CD8+ cell cytotoxicity by 51Chromium (51Cr) release assay and fluorescent microscopy. Targets were 51Cr labeled CaCo2 cells. Effectors were CD8+ cells isolated from peripheral blood mononuclear cells of sensitized or naïve volunteers after 6 day culture with IL-2 + IL-15 +/- C. hominis glycoprotein 15 (gp15). RESULTS: Gp15 stimulated CD8+ cells from a sensitized donor matched at HLA-A and HLA-B killed infected cells, with killing increasing with effector numbers, in contrast to CD8+ cells from a naïve donor. In microscopy experiments the same primed CD8+ cells released cytolytic granules into infected cells. CD8+ cells from sensitized donors matched at HLA-A, but not at HLA-B did not kill infected targets as efficiently. HLA-A and HLA-B antibodies inhibited cytotoxicity. CONCLUSION: The data suggest that re-stimulated CD8+ cells from sensitized individuals kill infected cells through release of cytotoxic granules. HLA-B seems to be at least partially required for peptide recognition.
Aging increases susceptibility to systemic infection with L. monocytogenes (129.9)
Authors: Smithey Megan J , Foster Sarah , Rudd Brain , Nikolich-Zugich Janko
Published: January 2023

Aging is accompanied by changes that affect components of both the innate and adaptive immune system, often resulting in a variable state of poorly-understood immunodeficiency. Using the murine model of systemic infection with the intracellular bacterium L. monocytogenes, we have investigated several parameters of the primary and secondary CD8 T-cell response to determine the impact of age on this arm of adaptive immunity. Comparison of these responses in young adult (8 week) and old (18 month) C57BL/6 mice revealed a dose-dependent decrease in survival, as well as a delayed ability to clear the bacterium in old animals. Kinetic analysis of the antigen-specific CD8 T-cell response revealed a markedly reduced magnitude of expansion in the primary effector T-cell population in old mice; however, antigen-specific CD8 T cells differentiating in old animals exhibited production of IFNγ and TNFα equivalent to those in adult counterparts. These results suggest that the quality of the CD8 T-cell response in old animals may be maintained with age. However, reduced primary expansion of Ag-specific CD8 T-cells and/or reduced precursor frequency of these cells may result in an effector CD8 T-cell population that is insufficient in size to protect against high-dose exposure to a new pathogen. Further experiments are in progress to test this hypothesis.
Expansion of influenza M1-specific memory cells cross-reactive with Epstein-Barr Virus (EBV) lytic epitopes correlates with severity of EBV-induced infectious mononucleosis (130.14)
Authors: Aslan Nuray , Watkin Levi , Pishtari Jonila , Xu Huiqing , Sullivan John L , Luzuriaga Katherine
Published: January 2023

Cross-reactive memory T cells specific to previously encountered influenza A contribute to the lymphoproliferation characteristics of EBV-associated infectious mononucleosis (IM). We showed that cross-reactive CD8 T cell responses commonly occur during EBV infection. Strong cross-reactive T cell responses specific to influenza M1 (Flu-M1) and EBV-specific lytic epitope EBV-BRLF-1 have been observed in 10/10 HLA-A2+ IM patients. We also detected cross-reactive T cell responses that recognized both Flu-M1 and lytic EBV-BMLF-1 epitopes in 14/26 HLA-A2+ IM patients. In addition, 8/19 IM patients demonstrated some intra-viral cross-reactivity between EBV BRLF-1 and BMLF-1 specific epitopes. Cross-reactive T cell responses between Flu-M1 and EBV-BRLF-1/EBV-BMLF-1 epitopes were observed in all IM patients but at different time points after infection. Our analyses showed that Flu-M1 and EBV-BMLF-1 used few Vbeta TCR families that were stable overtime. However, EBV-BRLF-1-specific Vbeta TCR repertoire used multiple Vbeta families that varied between individuals and varied during the time course of IM in the same patient. Disease severity of IM directly correlated with percentage of Flu-M1-specific CD8 T cells but did not correlate with EBV-BMLF-1 or BRLF-1-specific responses.
Human T cell immunity to influenza: magnitude, breadth, and antigenic variation (130.17)
Authors: Subbramanian Ramu A. , Brady Rebecca , Basha Saleem , Shata Tarek , Bernstein David I
Published: January 2023

Unraveling the nature of T cell immunity to influenza is currently an active area of research. Several lines of evidence now suggest that cell mediated immunity plays a significant role in controlling influenza disease in humans. While influenza hemagglutinin (HA)-specific humoral responses have been studied in considerable detail, our understanding of the coexistent cellular responses elicited by HA in humans is woefully limited. Utilizing quantitative approaches including ELISPOT and multiparametric flow cytometry we determine the magnitude of cell mediated responses to HA, the lineage of the responding T cells, and their frequency of prevalence in a tightly-defined cohort of healthy subjects. We demonstrate that the magnitude of HA-specific T cell memory populations are maintained at a low, finite frequency and vary significantly among individuals. HA-specific humoral- and T cell immunity do not correlate with each other. Importantly, the contribution of the T cell responses elicited by the variant and conserved regions of HA varies significantly among high- and low-responders. The ramifications of these observations to our understanding of influenza immunity in humans and development of effective vaccines against this pathogen will be discussed.
Effect of plant extracts on epithelial cell response to influenza infection (130.6)
Authors: Warren Kristi J , Naik Shibani , Kohut Marian
Published: January 2023

Plant extracts derived from Echinacea and Prunella have been used to treat and/or prevent various ailments including respiratory infection. In this study we examined whether plant extracts obtained from Echinacea purpurea (E.pur), E. angustifolia (E.ang), E. pallida (E.pal) or Prunella (Prun) altered cytokine and chemokine response to influenza infection of human bronchial epithelial cells (A549). A549 cells were treated with the plant extracts for 2 hours and subsequently exposed to virus (~ 40 HAU of Influenza A/PR/8/34) or media alone. Cell supernatants were collected 24 hrs later and assayed for cytokines/chemokines with a multiplex assay. The results showed that two extracts (E. ang & E. pal) increased IL-6, IL-8, G-CSF, IP-10 and RANTES, in cell cultures of media alone. In cultures with influenza virus, IL-1β, IL-15 and MIP1β were increased by E. ang, whereas IL-6, IL-8, IP-10 & RANTES were increased by all four plant extracts. These findings suggest that the plant extracts may have direct effects on epithelial tissue in addition to the known effects on monocytes and macrophages. It is possible that the early induction of these cytokines/chemokines promotes an earlier or more potent innate immune response.