bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of High Density Lipoprotein-Bound and Soluble RT6 Released Following Administration of Anti-RT6.1 Monoclonal Antibody
Authors: Lesma Elena , Moss Joel , Brewer H. Bryan , Bortell Rita , Greiner Dale , Mordes John , Rossini Aldo A.
Published: December 2022

RT6 is a rat lymphocyte glycosylphosphatidylinositol (GPI)-anchored alloantigen with nicotinamide adenine dinucleotide (NAD) glycohydrolase (NADase) and auto-ADP-ribosyltransferase activities. RT6 may have immunoregulatory properties based in part on the observation that injection of diabetes-resistant (DR)-BB rats with depleting doses of anti-RT6.1 mAb induced autoimmune diabetes and thyroiditis. We now report that injection of DR-BB rats with anti-RT6.1 mAb increased plasma NADase activity, which localized, by fluid phase liquid chromatography fractionation, to the high density lipoprotein (HDL) fraction. Following ultracentrifugation in high salt, however, RT6 was found in the nonlipoprotein fraction, where it existed, under nondenaturing conditions, as a 200-kDa complex and, by SDS-PAGE, as a 30- to 36-kDa species. Thy-1, another GPI-linked protein, and proteins that reacted with anti-GPI-oligosaccharide Abs also translocated from HDL to the nonlipoprotein fraction under similar conditions. Injection of anti-RT6.1 mAb into thymectomized DR and diabetes-prone-BB rats increased soluble RT6 to levels comparable to those observed in euthymic DR-BB rats, suggesting that HDL-bound RT6 is not derived from peripheral lymphocytes. In agreement, NADase activity in the plasma of eviscerated DR-BB rats did not increase following injection of anti-RT6 mAb. These data suggest that HDL is a carrier of plasma RT6 and other GPI-linked proteins, with equilibrium between the lipoprotein and nonlipoprotein fractions being salt dependent. Since GPI-linked proteins in HDL can transfer to cells in a functionally active form, the presence of RT6 in HDL is consistent with it having a role in signaling in nonlymphoid cells.
Human Melanoma-Reactive CD4+ and CD8+ CTL Clones Resist Fas Ligand-Induced Apoptosis and Use Fas/Fas Ligand-Independent Mechanisms for Tumor Killing
Authors: Rivoltini Licia , Radrizzani Marina , Accornero Paola , Squarcina Paola , Chiodoni Claudia , Mazzocchi Arabella , Castelli Chiara , Tarsini Paolo , Viggiano Vincenzo , Belli Filiberto , Colombo Mario P. , Parmiani Giorgio
Published: December 2022

Tumor cells have been shown recently to escape immune recognition by developing resistance to Fas-mediated apoptosis and acquiring expression of Fas ligand (FasL) molecule that they may use for eliminating activated Fas+ lymphocytes. In this study, we report that tumor-specific T lymphocytes isolated from tumor lesions by repeated in vitro TCR stimulation with relevant Ags (mostly represented by normal self proteins, such as MART-1/Melan A and gp100) can develop strategies for overcoming these escape mechanisms. Melanoma cells (and normal melanocytes) express heterogeneous levels of Fas molecule, but they result homogeneously resistant to Fas-induced apoptosis. However, CD4+ and CD8+ CTL clones kill melanoma cells through Fas/FasL-independent, granule-dependent lytic pathway. In these lymphocytes, Ag/MHC complex interaction with TCR does not lead to functional involvement of FasL, triggered, on the contrary, by T cell activation with nonspecific stimuli such as PMA/ionomycin. Additionally, melanoma cells express significant levels of FasL (detectable on the cell surface only after treatment with metalloprotease inhibitors), although to a lesser extent than professional immune cells such as Th1 clones. Nevertheless, antimelanoma CTL clones resist apoptosis mediated by FasL either in soluble form or expressed by Th1 lymphocytes or FasL+ melanoma cells. These results demonstrate that CD4+ and CD8+ antimelanoma T cell clones can be protected against Fas-dependent apoptosis, and thus be useful reagents of immunotherapeutic strategies aimed to potentiate tumor-specific T cell responses.
The Leukocyte Function-Associated Antigen-1 (LFA-1)-Binding Site on ICAM-3 Comprises Residues on Both Faces of the First Immunoglobulin Domain
Authors: Bell Elaine D. , May Andrew P. , Simmons David L.
Published: December 2022

ICAM-3 (CD50), a member of the Ig superfamily, is a major ligand for the leukocyte integrin LFA-1 (CD11a/CD18). This interaction represents one of several Ig superfamily/integrin ligand-receptor pairs that have been described to date. ICAM-3 is highly expressed on resting leukocytes and on APCs. In addition to an adhesive function, ICAM-3 can act as a signal-transducing molecule on T cells, providing a costimulatory signal for cell proliferation. Eighteen point mutations in ICAM-3 were generated, and residues important for binding of functional blocking Abs were identified. Mutation of seven of the residues reduced or abrogated adhesion to LFA-1, including three residues that are located on strand A of the ABED face of domain 1. In contrast, extensive mutagenesis analysis of ICAM-1 has shown that only residues on the GFC face interact with LFA-1. Our results provide evidence for a more extensive binding interface between ICAM-3 and LFA-1 than has previously been described. ICAM-3 appears to be unique among the ICAMs in utilizing residues on both faces of domain 1 for interaction with its ligand LFA-1.
Bcl-2-Mediated Inhibition of Apoptosis Prevents Immunogenicity and Restores Tumorigenicity of Spontaneously Regressive Tumors
Authors: Bonnotte Bernard , Favre Nathalie , Moutet Monique , Fromentin Annie , Solary Eric , Martin Monique , Martin François
Published: December 2022

Tumor cell clones from a rat colon carcinoma differ in their tumorigenicity and immunogenicity. The PRO clones give rise to progressive tumors, whereas the REG clones yield tumors that regress in a few weeks through a specific immune response. REG cells were more sensitive than PRO cells to apoptosis triggered by serum withdrawal in vitro. Furthermore, a fraction of REG cells, but no PRO cells, underwent apoptosis in the hours following injection into syngeneic rats. To further analyze the role of apoptosis, we overexpressed the antiapoptotic protein Bcl-2 in REG cells. Unlike parental or fake-transfected REG cells, Bcl-2-overexpressing REG cells resisted serum withdrawal-induced apoptosis, did not undergo apoptosis at 48 h postinjection into naive syngeneic rats, and gave rise to progressive, metastatic, and lethal tumors. Interestingly, REG-bcl2 cells were rejected by syngeneic hosts that had been preimmunized by an injection of parental REG cells, indicating that Bcl-2 overexpression did not alter tumor cell sensitivity to the effector cells of the immune response. Taken together, these observations indicate that tumor cell apoptosis may contribute to immunogenicity.
Gene Knockout B Cell-Deficient Mice Demonstrate That B Cells Play an Important Role in the Initiation of T Cell Responses toChlamydia trachomatis(Mouse Pneumonitis) Lung Infection
Authors: Yang Xi , Brunham Robert C.
Published: December 2022

T cell-mediated immunity as measured by delayed-type hypersensitivity, and IFN-γ production has been shown to be critical for host defense against Chlamydia trachomatis infection in both human and animal studies. Using gene-targeted B cell-deficient mice, we examined the role of B cells in protective immunity to C. trachomatis (mouse pneumonitis) (MoPn) lung infection. B cell-deficient mice were observed to have a significantly higher mortality rate and in vivo chlamydial growth than did wild-type mice following MoPn lung infection. Interestingly, B cell-deficient mice not only lacked Ab responses but also failed to mount an efficient delayed-type hypersensitivity response following chlamydial lung infection. In contrast to results obtained from MoPn-infected wild-type C57BL/6 mice, spleen cells from infected B cell-deficient mice failed to produce Th1-related (IFN-γ) or Th2-related (IL-6 and IL-10) cytokines after Chlamydia-specific in vitro restimulation. Moreover, unlike wild-type mice, B cell-deficient mice were not immune to rechallenge infection following recovery from primary chlamydial infection. The data indicate that B cells play an important role in host defense to primary and secondary chlamydial infection and suggest that B cells are crucial for the initiation of early T cell responses to chlamydial infection. This study provides evidence for the role of B cells in the in vivo priming of T cells during infection with the intracellular bacterial pathogen, C. trachomatis.
Absence of T Lymphocyte-Derived Cytokines Fails to Diminish Macrophage 12/15-Lipoxygenase Expression In Vivo
Authors: Sendobry Sandra M. , Cornicelli Joseph A. , Welch Kathryn , Grusby Michael J. , Daugherty Alan
Published: December 2022

IL-4 and IL-13 are the only known activators of 15-lipoxygenase (LO) expression in cultured macrophages. To determine whether these lymphocyte-derived cytokines regulate 15-LO expression in vivo, the abundance of the murine homologue (12/15-LO) was assessed in peritoneal macrophages from immune-deficient strains of mice. Macrophages were harvested from recombinase activator gene (RAG)-2−/− mice that do not develop mature lymphocytes and cannot secrete activation-dependent cytokines. Unexpectedly, 12/15-LO protein and activity were significantly increased in peritoneal macrophages from RAG-2−/− mice compared with strain-matched controls. This increase was related to phenotypic differences between cells from RAG-2+/+ and RAG-2−/− mice. After 3 h in culture, RAG-2+/+ macrophages were of two distinct sizes, with only the larger cells immunostaining for 12/15-LO. However, all RAG-2−/− cells were distributed in the large size range, and all were immunoreactive for the enzyme. The activation of 12/15-LO expression appears to be related to prolonged residence within the peritoneum, since there were fewer resident peritoneal macrophages in RAG-2−/− than in RAG-2+/+ mice, and newly recruited macrophages elicited by the administration of Sephacryl to RAG-2−/− mice did not immunostain for 12/15-LO. To determine whether 12/15-LO expression was due to IL-4 or IL-13 from nonlymphoid cells, the abundance of the enzyme was quantified in peritoneal macrophages from STAT6−/− mice that have attenuated responses to both cytokines. STAT6 deficiency did not influence the abundance of the protein in macrophages. Therefore, neither IL-4 nor IL-13 secretion is a requirement for macrophage 15-LO expression in vivo.
A Novel T Cell-Regulated Mechanism Modulating Allergen-Induced Airways Hyperreactivity in BALB/c Mice Independently of IL-4 and IL-5
Authors: Hogan Simon P. , Matthaei Klaus I. , Young Janine M. , Koskinen Aulikki , Young Ian G. , Foster Paul S.
Published: December 2022

The immunoregulatory functions of IL-4 and IL-5 have identified these cytokines as primary targets for the resolution of airways inflammation and bronchial hyperreactivity in asthma. However, the individual contribution of each of these cytokines and of IL-5-regulated eosinophilia to the induction of airways hyperreactivity in mouse models of asthma remains highly controversial. In this investigation, we have used IL-4- and IL-5-deficient mice of the same genetic background in combination with inhibitory mAbs to these cytokines to identify unequivocally the contribution of these factors to the induction of airways hyperreactivity. Sensitization and aeroallergen challenge of wild-type mice with OVA induced pathological changes to the respiratory epithelium, airways eosinophilia, and hyperreactivity to β-methacholine. Inhibition of the actions of IL-4 and/or IL-5 did not abolish airways hyperreactivity, and in the case of IL-4-deficient mice pretreated with anti-IL-5 mAb, airways hyperreactivity persisted in the absence of pronounced airways inflammation. Airways hyperreactivity was abolished only by anti-CD4+ mAb treatment. However, aeroallergen challenge of IL-5−/− mice showed that morphologic changes to the airways were critically linked to IL-5 and eosinophilia. This investigation demonstrates the existence in BALB/c mice of a novel CD4+ T cell pathway for modulating airways hyperreactivity. These findings may provide an explanation for the dissociation of airways eosinophilia from the development of airways hyperreactivity observed in some cases of asthma and in animal models of this disease.
Antigen Processing of Two H2-IEd-Restricted Epitopes Is Differentially Influenced by the Structural Changes in a Viral Glycoprotein
Authors: Chianese-Bullock Kimberly A. , Russell Helena I. , Moller Christopher , Gerhard Walter , Monaco John J. , Eisenlohr Laurence C.
Published: December 2022

The factors that influence the intracellular location(s) of MHC class II-restricted epitope loading remain poorly understood. We present evidence that two I-Ed-restricted epitopes of the influenza hemagglutinin (HA) molecule, termed site 1 (S1; encompassing amino acid residues 107–119) and site 3 (S3; encompassing amino acid residues 302–313), are generated in distinct endocytic compartments. By means of an epitope-specific mAb, we show that S1 becomes detectable in late endocytic/lysosomal vesicles; using a mutant cell line, we also show that the presentation of S1 is dependent upon H2-DM expression. In contrast, S3; presentation is H2-DM-independent and appears in early endosomes as a result of acid-induced structural changes in HA. Presentation of both epitopes can be made H2-DM-independent by denaturing HA and made H2-DM-dependent by preventing the acid-induced conformational changes from occurring. These findings indicate that the structural context of a given epitope can determine where it is processed.
The Role of Apoptosis in the Resolution of T Cell-Mediated Cutaneous Inflammation
Authors: Orteu Catherine H. , Poulter Len W. , Rustin Malcolm H. A. , Sabin Caroline A. , Salmon Mike , Akbar Arne N.
Published: December 2022

We have investigated cutaneous purified protein derivative-induced delayed-type hypersensitivity (DTH) responses in healthy volunteers to determine features associated with both the generation and resolution of the reaction. The clinical peak of the response occurred at day 3; however, T cell numbers were maximal on day 7. There was a preferential increase of CD4+CD45RO+ T cells on day 7, which was largely due to proliferation, since a mean of 19% was in cycle. The proliferation of this subset was associated with the presence of IL-15, which was expressed as early as 12 h, and IL-2, which showed peak expression at 7 days. By day 14, there was a significant decrease in both the mean T cell number/unit area and IL-2 and IL-15 expression in perivascular infiltrates. Maximal CD95 (Fas/Apo-1) ligand and TNF-α expression were observed at 7 days and were associated with the presence of 1.83% (range 0.81–2.48%) apoptotic T cells. At 14 days, CD95 ligand and TNF-α expression were reduced significantly, and the presence of 2.5% (range 1.5–3.75%) of apoptotic T cells at this time was probably due to cytokine deprivation, associated with decreased Bcl-2 relative to Bax expression. The induction and resolution of the Mantoux reaction may depend on the expression of cytokines, such as IL-2 and IL-15, which regulate both proliferation and apoptosis in T cells. Failure to control either of these phases of the Mantoux reaction may contribute to the chronicity of inflammatory responses in certain cutaneous diseases.
Resistance to HgCl2-Induced Autoimmunity in Haplotype-Heterozygous Mice Is an Intrinsic Property of B Cells
Authors: Hanley Gregory A. , Schiffenbauer Joel , Sobel Eric S.
Published: December 2022

Exposure to low doses of mercury chloride induces autoantibodies to the nucleolar protein fibrillarin in H-2s, but not in H-2b, mice. Surprisingly, F1 crosses between resistant and sensitive haplotypes are resistant. Previously, we have shown that the resistance in these F1 mice was due to coexpression of the resistant class II allele. Using adoptive transfer techniques we have examined several mechanisms by which the resistant haplotype could be down-regulating the antifibrillarin response in F1 (s/b) mice. Similar to other autoimmune models, mercury-induced autoimmunity requires cognate MHC-restricted T cell help. The absence of autoantibody production in F1 mice was not due to a difference in thymic education or to the absence of antifibrillarin-specific T cell help. These results suggest that the resistance is due to an intrinsic property of the haplotype-heterozygous B cells.
Antibodies to TR2 (Herpesvirus Entry Mediator), a New Member of the TNF Receptor Superfamily, Block T Cell Proliferation, Expression of Activation Markers, and Production of Cytokines
Authors: Harrop Jeremy A. , Reddy Manjula , Dede Kimberly , Brigham-Burke Michael , Lyn Sally , Tan Kong B. , Silverman Carol , Eichman Christopher , DiPrinzio Rocco , Spampanato Jay , Porter Terence , Holmes Stephen , Young Peter R. , Truneh Alemseged
Published: December 2022

TR2 (TNFR-related 2) is a recently identified member of the TNFR family with homology to TNFRII. We have demonstrated previously that TR2 mRNA is expressed in resting and activated human T cells and that TR2-Ig partially inhibits an allogeneic mixed leukocyte proliferation response. We now characterize TR2 further by the use of specific mAbs. Flow-cytometry analysis using TR2 mAbs confirmed that resting PBL express high levels of cell surface TR2, and that TR2 is widely expressed on all freshly isolated lymphocyte subpopulations. However, stimulation of purified T cells with either PHA or PHA plus PMA resulted in decreased surface expression within 48 h of activation before returning to resting levels at 72 h. TR2 mAbs inhibited CD4+ T cell proliferation in response to stimulation by immobilized CD3 or CD3 plus CD28 mAbs. Assay of culture supernatants by ELISA showed inhibition of TNF-α, IFN-γ, IL-2, and IL-4 production, which, for IL-2 and TNF-α was also confirmed by intracellular cytokine staining. Furthermore, expression of activation markers on CD4+ T cells, including CD25, CD30, CD69, CD71, and OX40 (CD134), was inhibited. TR2 mAbs inhibited proliferation in a three-way MLR, and a response to soluble recall Ag, tetanus toxoid. In conclusion, these results suggest that TR2 is involved in the activation cascade of T cell responses and TR2 mAbs prevent optimal T cell proliferation, cytokine production, and expression of activation markers.
Rejection of H-Y Disparate Skin Grafts by Monospecific CD4+ Th1 and Th2 Cells: No Requirement for CD8+ T Cells or B Cells
Authors: Zelenika Diana , Adams Elizabeth , Mellor Andrew , Simpson Elizabeth , Chandler Phillip , Stockinger Brigitte , Waldmann Herman , Cobbold Stephen P.
Published: December 2022

We wished to determine whether CD4+ T cells could reject a skin graft that was discordant for a single minor transplantation Ag in the absence of CD8+ T cells or Ab. Transgenic A1(M) mice were constructed that express the rearranged Vβ8.2 and Vα10 TCR genes from a T cell clone that is specific for the male Ag (H-Y) in the context of H2-Ek. In addition, the RAG-1−/− background was bred onto these mice to eliminate any endogenous TCR rearrangements. As expected, clonal deletion was found to be complete in the thymus of male A1(M)×RAG-1−/− mice, while only CD4+ T cells were positively selected and found in the periphery of females. Female A1(M)×RAG-1−/− mice were able to rapidly reject (in <14 days) male (but not female) skin grafts in a CD4-dependent fashion. After multiple grafts, it was confirmed that no CD8+ T cells or surface Ig+ B cells were present. An immunofluorescent analysis of spleen cells after grafting showed that the majority of T cells expressed activation markers (CD44, CD25, and intracytoplasmic IL-2) and a significant proportion were making IFN-γ and IL-4. Surprisingly, the transfer of either Th1 or Th2 CD4+ T cell lines from these mice into T cell-depleted recipients was sufficient to cause a specific rejection of male skin.
Augmentation and Suppression of Immune Responses to an HIV-1 DNA Vaccine by Plasmid Cytokine/Ig Administration
Authors: Barouch Dan H. , Santra Sampa , Steenbeke Tavis D. , Zheng Xin X. , Perry Helen C. , Davies Mary-Ellen , Freed Daniel C. , Craiu Abie , Strom Terry B. , Shiver John W. , Letvin Norman L.
Published: December 2022

The use of cytokines has shown promise as an approach for amplifying vaccine-elicited immune responses, but the application of these immunomodulatory molecules in this setting has not been systematically explored. In this report we investigate the use of protein- and plasmid-based cytokines to augment immune responses elicited by an HIV-1 gp120 plasmid DNA vaccine (pV1J-gp120) in mice. We demonstrate that immune responses elicited by pV1J-gp120 can be either augmented or suppressed by administration of plasmid cytokines. A dicistronic plasmid expressing both gp120 and IL-2 induced a surprisingly weaker gp120-specific immune response than did the monocistronic pV1J-gp120 plasmid. In contrast, systemic delivery of soluble IL-2/Ig fusion protein following pV1J-gp120 vaccination significantly amplified the gp120-specific immune response as measured by Ab, proliferative, and CTL levels. Administration of plasmid IL-2/Ig had different effects on the DNA vaccine-elicited immune response that depended on the temporal relationship between Ag and cytokine delivery. Injection of plasmid IL-2/Ig either before or coincident with pV1J-gp120 suppressed the gp120-specific immune response, whereas injection of plasmid IL-2/Ig after pV1J-gp120 amplified this immune response. To maximize immune responses elicited by a DNA vaccine, therefore, it appears that the immune system should first be primed with a specific Ag and then amplified with cytokines. The data also show that IL-2/Ig is more effective than native IL-2 as a DNA vaccine adjuvant.
Identification of the Integral Membrane Protein RM3/1 on Human Monocytes as a Glucocorticoid-Inducible Member of the Scavenger Receptor Cysteine-Rich Family (CD163)
Authors: Högger Petra , Dreier Jens , Droste Anne , Buck Friedrich , Sorg Clemens
Published: December 2022

The RM3/1 Ag is a membrane glycoprotein restricted to human monocytes and macrophages that evolve in the late phase of inflammation. Peptide sequence analysis of the RM3/1 protein revealed similarity to CD163, a member of the scavenger receptor cysteine-rich family. Using specific Abs (RM3/1, Ki-M8), we demonstrate an identical cellular regulation for the RM3/1 and the CD163 protein. Most notably, we show for the first time that CD163 is significantly up-regulated by glucocorticoids. In contrast, the protein is down-regulated by the immunosuppressant cyclosporin A and by phorbol esters, while the inflammatory mediator LPS has no significant influence on the expression. We describe the first isolation of a full-length cDNA of CD163 and expression of the corresponding protein. Several splice variants of CD163 exist, and we elucidated the kinetics of induction of three major mRNA splice variants by fluticasone propionate; another splice variant was proved to be unresponsive to this glucocorticoid. Taken together with a previous result showing an involvement of RM3/1 in adhesion of monocytes to the activated endothelium, we discuss that CD163 might play an important role in inflammatory processes.
IL-4 Protects Adult C57BL/6 Mice from ProlongedCryptosporidium parvumInfection: Analysis of CD4+αβ+IFN-γ+ and CD4+αβ+IL-4+ Lymphocytes in Gut-Associated Lymphoid Tissue During Resolution of Infection
Authors: Aguirre Shirley A. , Perryman Lance E. , Davis William C. , McGuire Travis C.
Published: December 2022

Resistance of adult C57BL/6 mice to severe Cryptosporidium parvum infection is dependent on CD4+αβ+ TCR lymphocytes. In this study, we demonstrated that treatment with anti-IFN-γ mAb extended oocyst excretion 18 days longer, and anti-IL-4 mAb extended oocyst excretion at least 11 days longer than isotype control mAb treatment. Analysis of the specific activity of anti-IFN-γ mAb present in treated mouse sera suggested that IFN-γ may have a limited role in the resolution phase of infection. Changes were also documented in numbers of CD4+αβ+IFN-γ+ and CD4+αβ+IL-4+ lymphocytes in Peyer’s patches and intraepithelium of adult C57BL/6 mice during resolution of C. parvum infection. Resistance to initial severe infection was associated with CD4+αβ+IFN-γ+ lymphocytes, and eventual resolution of infection was associated with CD4+αβ+IL-4+ lymphocytes. Analysis of cytokine expression following in vitro stimulation with C. parvum Ags during resolution of infection demonstrated consistent increases in CD4+αβ+IL-4+ lymphocytes, but not CD4+αβ+IFN-γ+ lymphocytes. The relevance of CD4+αβ+IL-4+ lymphocytes in protection against C. parvum was then evaluated in C57BL/6 IL-4 gene knockout mice (IL-4−/−). Adult IL-4−/− mice excreted oocysts in feces approximately 23 days longer than IL-4+/+ mice. Further, anti-IFN-γ mAb treatment increased the severity and the duration of infection in IL-4−/− mice compared with those in IL-4+/+ mice. Together, the data demonstrated that IFN-γ was important in the control of severity of infection, and either IFN-γ or IL-4 accelerated termination of infection. However, neither IL-4 nor IFN-γ was required for the final clearance of infection from the intestinal tract of adult mice.
Role of Stress-Activated Mitogen-Activated Protein Kinase (p38) in β2-Integrin-Dependent Neutrophil Adhesion and the Adhesion-Dependent Oxidative Burst
Authors: Detmers Patricia A. , Zhou Dahua , Polizzi Elizabeth , Thieringer Rolf , Hanlon William A. , Vaidya Sanskruti , Bansal Vinay
Published: December 2022

Bacterial LPS elicits both rapid activation of the stress-activated MAP kinase p38 in polymorphonuclear leukocytes (PMN) and rapid adhesion of the PMN to ligands for the leukocyte integrin CD11b/CD18. The functional correlation between these two events was examined. The time course for tyrosine phosphorylation of p38 in PMN in response to 10 ng/ml LPS in 1% normal human serum was consistent with participation in signaling for leukocyte integrin-dependent adhesion, with transient phosphorylation peaking at 10 to 20 min. The concentration dependence of p38 phosphorylation also resembled that for PMN adhesion, with <1 ng/ml LPS eliciting a response. Phosphorylation was inhibited by mAb 60b against CD14, but not by mAb 26ic, a nonblocking anti-CD14. The function of p38 in integrin-dependent adhesion and the adhesion-dependent oxidative burst was tested using a specific inhibitor of p38, SB203580. SB203580 inhibited adhesion by diminishing the initial rate of adherence in response to both LPS and TNF, with a half-maximal concentration in the range of 0.1 to 0.6 μM. It did not, however, block adhesion in response to formyl peptide or PMA. The p38 inhibitor also blocked the adhesion-dependent oxidative burst with a half-maximal concentration similar to that for adhesion. Timed delivery of the compound during the lag phase preceding H2O2 production suggested that p38 kinase activity was required throughout the lag but not after the oxidase was assembled. These results suggest that p38 functions in PMN to signal leukocyte integrin-dependent adhesion and the subsequent massive production of reactive oxygen intermediates.
Inducible Nitric Oxide: An Autoregulatory Feedback Inhibitor of Vascular Inflammation
Authors: Peng Hai-Bing , Spiecker Martin , Liao James K.
Published: December 2022

Inducible nitric oxide (iNO) is produced at sites of vascular inflammation by resident and nonresident vascular wall cells, but its role in the inflammatory process is not known. In this study, we show that a novel function of iNO is to terminate inflammatory processes. We find that iNO produced by murine macrophage-like cells, RAW264.7, can inhibit cytokine-induced endothelial cell activation in a separated and mixed endothelial-RAW264.7 coculture system. Both iNO production and endothelial VCAM-1 expression were induced simultaneously with bacterial LPS and murine-specific IFN-γ. Inhibition of iNO synthase (iNOS) activity with Nω-monomethyl-l-arginine in endothelial-RAW264.7 cocultures, stimulated with murine-specific IFN-γ and LPS, decreased iNO production by 86%, augmented VCAM-1 and iNOS expression in endothelial and RAW264.7 cells, respectively, and increased monocyte adhesion to the endothelial cell surface. Transient transfection studies using various VCAM-1 promoter constructs demonstrated that inhibitory effects of iNO on VCAM-1 gene transcription were mediated, in part, by inhibitory effects of iNO on κB cis-acting elements. Immunofluorescence studies using an Ab to the RelA (p65) subunit of nuclear factor-κB revealed that iNO inhibited the activation of nuclear factor-κB. These studies indicate that iNO attenuates iNOS expression in macrophages and inhibits monocyte adhesion to endothelial cells, and suggest that endogenously derived iNO may be an important autoregulatory inhibitor of vascular inflammation.
Vaccination with BV8S2 Protein Amplifies TCR-Specific Regulation and Protection Against Experimental Autoimmune Encephalomyelitis in TCR BV8S2 Transgenic Mice
Authors: Offner Halina , Adlard Kirsten , Bebo Bruce F. , Schuster Jeanette , Burrows Gregory G. , Buenafe Abigail C. , Vandenbark Arthur A.
Published: December 2022

TCR determinants overexpressed by autopathogenic Th1 cells can naturally induce a second set of TCR-specific regulatory T cells. We addressed the question of whether immune regulation could be induced naturally in a genetically restricted model in which a major portion of TCR-specific regulatory T cells expressed the same target TCR BV8S2 chain as the pathogenic T cells specific for myelin basic protein (MBP). We found vigorous T cell responses to BV8S2 determinants in naive mice that could be further potentiated by vaccination with heterologous BV8S2 proteins, resulting in the selective inhibition of MBP-specific Th1 cells and protection against experimental encephalomyelitis. Moreover, coculture with BV8S2-specific T cells or their supernatants reduced proliferation, IFN-γ secretion, and encephalitogenic activity of MBP-specific T cells. These results suggest that immune regulation occurs through a nondeletional cytokine-driven suppressive mechanism.
Differential Requirements of CD45 Protein Tyrosine Phosphatase for Cytolytic Activities and Intrathymic and Extrathymic Development of Intestinal Intraepithelial Lymphocytes
Authors: Yada Shinichiro , Kishihara Kenji , Kong Young-Yun , Nomoto Kikuo
Published: December 2022

CD45 is a transmembrane protein tyrosine phosphatase essential for Ag receptor-mediated signaling in both T and B cells. In this study we investigated roles of CD45 in development and cytolytic activities of murine intestinal intraepithelial lymphocytes (i-IEL) using CD45 exon 6 knockout (CD45−/−) mice. Interestingly, the total cell number of i-IEL was significantly reduced in CD45−/− mice during aging (10–20 wk of age), whereas the i-IEL number was normally increased in the wild-type littermates. Especially, the number of γδTCR+ i-IEL decreased markedly in CD45−/− mice during aging. The i-IEL in CD45−/− mice were more susceptible to in vitro spontaneous apoptosis than the normal i-IEL, implying that CD45 is required for maintenance of the cellularity of i-IEL. Results from in vivo analyses of the extrathymic and intrathymic development of i-IEL suggested that CD45-mediated signaling is required for the intrathymic, but not the extrathymic, development of i-IEL. Moreover, the whole i-IEL from CD45−/− mice showed a significantly reduced cytolytic activity, and the residual cytolytic activity was completely diminished by depleting CD45+ i-IEL, suggesting that CD45 is indispensable for the TCR-mediated cytolytic activity of i-IEL. Furthermore, we found differential contributions of CD45 and p56lck to development and induction of cytolytic activities of i-IEL.
On Histocompatibility Barriers, Th1 to Th2 Immune Deviation, and the Nature of the Allograft Responses
Authors: Li Xian Chang , Zand Martin S. , Li Yongsheng , Zheng Xin Xiao , Strom Terry B.
Published: December 2022

In the present study, we have sought to determine the basis for the frequent failure of Th1 to Th2 immune deviation to blunt the severity of allograft rejection, as such immune deviation has proven highly effective in the treatment of several T cell-dependent autoimmune states. Our study demonstrates that treating islet allograft recipient mice with anti-IL-12 mAb is highly effective in producing Th1 to Th2 immune deviation in several model systems (i.e., fully MHC, partially MHC, or multiple minor Ag barriers). Nevertheless, anti-IL-12 failed to prolong the engraftment of fully MHC-mismatched islet allografts. However, anti-IL-12-treated recipients carrying MHC-matched but multiple minor Ag-mismatched allografts experienced prolonged engraftment; allograft tolerance was frequently achieved in the DBA/2J (H-2d) to BALB/c (H-2d) strain combination. In another model, in which the host response was dominated by CD4+ T cells responding to donor allopeptides presented upon host APCs in the context of self MHC class II molecules, anti-IL-12 treatment proved to be extremely potent. Thus, Th1 to Th2 immune deviation produces prolonged engraftment as compared with recipients of MHC-mismatched allografts when rejection is dependent upon indirectly presented allogeneic peptides and a reduced mass of responding alloreactive T cells.
The Role of Enhancer A in the Locus-Specific Transactivation of Classical and Nonclassical HLA Class I Genes by Nuclear Factor κB
Authors: Gobin Sam J. P. , Keijsers Vivian , van Zutphen Marlijn , van den Elsen Peter J.
Published: December 2022

HLA class I expression is tightly controlled at the transcriptional level by several conserved regulatory elements in the proximal promoter region. In this study, the two putative κB motifs of enhancer A (κB1 and κB2) of the classical and nonclassical HLA class I genes were investigated for their binding properties of transcription factors and tested for their contribution to the NF-κB-induced route of transactivation. It was shown that NF-κB-induced transactivation through enhancer A is most important for the HLA-A locus, which contains two NF-κB binding sites. Although the enhancer A of HLA-B contains only one NF-κB binding site (κB1), there was still a moderate transactivation by NF-κB. Since HLA-F, which also possesses one NF-κB binding site but lacks protein binding to its κB2 site, was not transactivated by NF-κB, the NF-κB-mediated transactivation through the κB1 motif in HLA-B is most probably facilitated by binding of the transcription factor Sp1 to the upstream κB2 site. Thus, transcriptional regulation of HLA class I genes by NF-κB is restricted to the HLA-A and HLA-B loci.
Specific Antiviral Activity Demonstrated by TGTP, A Member of a New Family of Interferon-Induced GTPases
Authors: Carlow Douglas Alan , Teh Soo-Jeet , Teh Hung-Sia
Published: December 2022

The GTPase superfamily includes a diversity of molecules whose functions are regulated through the binding and hydrolysis of GTP. This superfamily can be segregated into families of functionally related molecules that typically share amino acid sequence similarity within and around the nucleotide-binding domains. A new family of putative GTPases, including IRG-47, LRG-47, IGTP, and TGTP/Mg21, has recently emerged that share significant sequence identity (25–40%). Expression of these molecules has been shown to be selectively induced by IFN-γ and in some cases by IFN-αβ or bacterial LPS. This induction pattern implicates these putative GTPases as part of the innate defense of cells to infection, but their role in such defense has not yet been defined. We have previously described the cloning of TGTP and now confirm its intrinsic activity as a GTPase. We found that TGTP is strongly induced by endogenous IFN-αβ produced in response to standard lipofection of plasmid DNA or polyinosinic polycytidilic acid. The ability of endogenously produced IFN-αβ to efficiently induce expression of TGTP under these conditions suggested that TGTP might participate in defense against viral infection. This proposal was borne out when TGTP-transfected L cells displayed relative resistance to plaque formation by vesicular stomatitis virus but not herpes simplex virus. This observation places TGTP among a small family of innate antiviral agents and has implications for the functions of other members of this family of GTPases.
The Mannose Receptor Mediates Induction of IFN-α in Peripheral Blood Dendritic Cells by Enveloped RNA and DNA Viruses
Authors: Milone Michael C. , Fitzgerald-Bocarsly Patricia
Published: December 2022

Peripheral blood dendritic cells (DC) produce IFN-α in response to challenge by many enveloped viruses including herpes simplex virus (HSV) and HIV, whereas Sendai virus predominantly stimulates IFN-α production by monocytes. Glycosylated viral envelope proteins are known to be important for the induction of IFN-α. In this study we demonstrate that stimulation of IFN-α synthesis by HSV is inhibited by a number of monosaccharides, including fucose, N-acetylglucosamine, and N-acetylgalactosamine as well as the yeast polysaccharide mannan, supporting a role for lectin(s) in the IFN-α stimulation pathway. Furthermore, antiserum to the mannose receptor (MR) also inhibited HSV, vesicular stomatitis virus, and HIV-induced IFN-α production, but failed to inhibit the IFN-α induced by Sendai virus. We further demonstrated that freshly isolated blood DC and IFN-α-producing cells responding to HSV stimulation express the MR. This study therefore implicates the MR as an important receptor for the nonspecific recognition of enveloped viruses by DC and the subsequent stimulation of IFN-α production by these viruses. Thus, the MR probably serves as a critical link between innate and adaptive immunity to viruses, especially given the role of the MR in Ag capture by DC and the importance of IFN-α in shaping immunity.
CTL Response toMycobacterium tuberculosis: Identification of an Immunogenic Epitope in the 19-kDa Lipoprotein
Authors: Mohagheghpour Nahid , Gammon Dawn , Kawamura Loyce Masae , van Vollenhoven Annika , Benike Claudia J. , Engleman Edgar G.
Published: December 2022

The successful resolution of infection with Mycobacterium tuberculosis (M.tb) is believed to involve the induction of CTLs that are capable of killing cells harboring this pathogen, although little information is known about the MHC restriction or fine specificity of such CTLs. In this study, we used knowledge of the HLA-A*0201-binding motif and an immunofluorescence-based peptide-binding assay to screen for potential HLA-A*0201-binding epitopes contained in the 19-kDa lipoprotein of M.tb (M.tb19). CD8+ T cells derived from HLA-A*0201+ patients with active tuberculosis (TB) as well as tuberculin skin test-positive individuals who had no history of TB were used as effector cells to determine whether these epitopes are recognized by in vivo-primed CTLs. An in vitro vaccination system using HLA-A*0201+ dendritic cells (DCs) as APCs was used to determine whether these epitopes can sensitize naive CD8+ T cells in vitro, leading to the generation of Ag-specific CTLs. The results show that an HLA-A*0201-binding peptide comprised of residues 88 to 97 of M.tb19 (P88–97) is recognized by circulating CD8+ CTLs from both healthy tuberculin skin test-positive individuals and patients with active TB but not by tuberculin skin test-negative subjects. Moreover, dendritic cells pulsed with this peptide induced class I MHC-restricted CTLs from the T cells of healthy unsensitized persons. Finally, CTL lines that were specific for P88–97 were shown to lyse autologous monocytes that had been infected acutely with the H37Ra strain of M.tb. These results demonstrate that M.tb19 elicits HLA class I-restricted CTLs in vitro and in vivo that recognize endogenously processed Ag. Epitopes of the type identified here may prove useful in the design of an M.tb vaccine.
B7-1, But Not CD28, Is Crucial for the Maintenance of the CD4+ T Cell Responses in Human Leprosy
Authors: Schlienger Katia , Uyemura Koichi , Jullien Denis , Sieling Peter A. , Rea Thomas H. , Linsley3 Peter S. , Modlin Robert L.
Published: December 2022

We used human leprosy as a model to compare patterns of costimulatory molecule expression in respect to the clinical/immunologic spectrum of disease. We found that B7-1, B7-2, and CD28 transcripts dominated in tuberculoid leprosy patients, who have potent T cell responses to Mycobacterium leprae. In contrast, CTLA-4 was more strongly expressed in lesions from lepromatous patients, who manifest specific T cell anergy to the leprosy bacterium. T cell clones from tuberculoid lesions were CD4+CD28+ or CD4+CD28−, and T cell clones from lepromatous lesions were predominantly CD8+CD28−. The M. leprae-specific recall response of CD4+ T cell clones from tuberculoid lesions was blocked by anti-B7-1 mAb, but not by anti-B7-2 mAb or CTLA-Ig. However, anti-CD28 and anti-CTLA-4 mAbs did not block activation of clones from tuberculoid lesions, suggesting that B7-1 may utilize another costimulatory pathway. Peripheral blood T cell responses in the lepromatous form were strongly regulated by CD28 during T cell activation, in contrast to the tuberculoid form. Thus, B7-1 costimulation could play a role in maintaining a strong immune response to the pathogen.
Pathogenic Mycobacterium tuberculosis Evades Apoptosis of Host Macrophages by Release of TNF-R2, Resulting in Inactivation of TNF-α
Authors: Balcewicz-Sablinska M. Katarzyna , Keane Joseph , Kornfeld Hardy , Remold Heinz G.
Published: December 2022

Infection by Mycobacterium tuberculosis (MTB) induces human alveolar macrophage (AMφ) apoptosis by a TNF-α-dependent mechanism. The apoptotic response is postulated to be a defense mechanism, limiting the growth of this intracellular pathogen. Consistent with that model, recent studies showed that the virulent MTB strain H37Rv induces substantially less AMφ apoptosis than the attenuated strain H37Ra. We now report that AMφ infection with either H37Rv or H37Ra induces comparable levels of TNF-α measured by ELISA but that TNF-α bioactivity is reduced in supernatants of H37Rv-infected AMφ. Differential release of soluble TNFR2 (sTNFR2), with formation of inactive TNF-α-TNFR2 complexes accounted for the difference in TNF-α bioactivity in these cultures. Release of sTNFR2 by H37Rv-infected AMφ was IL-10 dependent since it was inhibited by neutralizing anti-IL-10 Ab. Thus, the effect of TNF-α produced by AMφ following infection can be modulated by virulent MTB, using IL-10 as an upstream mediator.
cDNA Encoding a Single-Chain Antibody to HIV p17 with Cytoplasmic or Nuclear Retention Signals Inhibits HIV-1 Replication
Authors: Tewari Deepanker , Goldstein Simoy L. , Notkins Abner L. , Zhou Paul
Published: December 2022

HIV-1 gag p17 protein is an attractive target for molecular intervention, because it is involved in the viral replication cycle at both the pre- and postintegration levels. In the present experiments, we targeted p17 by intracellularly expressing a cDNA encoding an Ab to p17. cDNA from a hybridoma-secreting Ab to p17 was cloned, sequenced, reconstructed as a single-chain Ab fragment (scFv), and expressed in the cytoplasm or nucleus with appropriate retention signals. The expressed scFvs had no effect on T cell growth or CD4 expression and bound specifically to HIV-1 p17. Human CD4+ Jurkat T cells that expressed scFvs and were infected with HIV-1 showed a marked reduction in virus replication compared with cells expressing vector alone. The inhibition of virus replication was more pronounced when scFvs were expressed in the cytoplasm rather than the nucleus. From these studies, we conclude that the intracellular expression of a single-chain Ab to p17 inhibits HIV replication; in addition, the degree of inhibition is related to the intracellular targeting site.
Cloning of BY55, a Novel Ig Superfamily Member Expressed on NK Cells, CTL, and Intestinal Intraepithelial Lymphocytes
Authors: Anumanthan Anukanth , Bensussan Armand , Boumsell Laurence , Christ Andreas D. , Blumberg Richard S. , Voss Stephan D. , Patel Amish T. , Robertson Michael J. , Nadler Lee M. , Freeman Gordon J.
Published: December 2022

Expression of the BY55 protein has been shown to be tightly associated with NK and CD8+ T lymphocytes with cytolytic effector activity. To determine the function of this protein, we molecularly cloned BY55 cDNA. The cDNA sequence predicts a cysteine-rich, glycosylphosphatidylinositol-anchored protein of 181 amino acids with a single Ig-like domain weakly homologous to killer inhibitory receptors. Reduction and carboxyamidomethylation of immunoprecipitated BY55 gave a band of 27 kDa, whereas reduction alone led to an 80-kDa species, suggesting that BY55 is a tightly disulfide-linked multimer. RNA blot analysis revealed BY55 mRNAs of 1.5 and 1.6 kb whose expression was highly restricted to NK and T cells. BY55 was expressed on the CD56dim, CD16+ subset of NK cells, which have high cytolytic activity, but was not expressed and was not induced on the CD56bright, CD16− subset of NK cells, a subset with high proliferative, but low cytolytic, capacity. In human tissues, BY55 mRNA was expressed only in spleen, PBL, and small intestine (in gut lymphocytes). BY55 was expressed on all intestinal intraepithelial lymphocytes, which were predominantly CD3+TCRα/β+CD4−CD8+CD11b+CD28−CD45RO+CD56−CD101+CD103+ (αEβ7 integrin). In addition, BY55 was expressed on most CD8+CD28− peripheral blood T cells. These phenotypic relationships suggest that CD8+CD28+ precursor CTL may terminally differentiate into CD8+CD28−BY55+ effector CTL and that some of the peripheral blood CD8+CD28− subset may represent recirculation from mucosal epithelial immune sites.
An Improved Retroviral Gene Transfer Technique Demonstrates Inhibition of CD4−CD8− Thymocyte Development by Kinase-Inactive ZAP-70
Authors: Sugawara Takehiko , Di Bartolo Vincenzo , Miyazaki Tadaaki , Nakauchi Hiromitsu , Acuto Oreste , Takahama Yousuke
Published: December 2022

ZAP-70 is a Syk family tyrosine kinase that plays an essential role in initiating TCR signals. Deficiency in ZAP-70 causes a defect in the development at CD4+CD8+ thymocytes due to defective TCR-mediated positive and negative selection. Using a newly devised retrovirus gene transfer and an efficient green fluorescence protein detection technique in fetal thymus organ cultures, the present study shows that forced expression in developing thymocytes of a catalytically inactive mutant of ZAP-70, but not wild-type ZAP-70, inhibits T cell development at the earlier CD4−CD8− stage. The ZAP-70 mutant blocked the generation of CD4+CD8+ thymocytes even in the absence of endogenous ZAP-70. Thus, the present results demonstrate a novel technique for gene transfer into developing T cells and suggest that ZAP-70/Syk family tyrosine kinases are involved in the signals inducing the generation of CD4+CD8+ thymocytes.
The Inability of the Nonobese Diabetic Class II Molecule to Form Stable Peptide Complexes Does Not Reflect a Failure to Interact Productively with DM
Authors: Peterson Mary , Sant Andrea J.
Published: December 2022

Sequence variability in MHC class II molecules plays a major role in genetically determined susceptibility to insulin-dependent diabetes mellitus (IDDM). It is not yet clear whether MHC class II polymorphism allows selective binding of diabetogenic peptides or regulates some key intracellular events associated with class II-restricted Ag presentation. In this study, we have employed gene transfer techniques to analyze the intracellular events that control peptide acquisition by the unique class II molecule expressed by nonobese diabetic mice (I-Ag7). This structurally unique class II molecule fails to demonstrate stable binding to antigenic peptides and fails to undergo the conformational change associated with stable peptide binding to class II molecules. The experiments reported here demonstrate that I-Ag7 can productively associate with two protein cofactors important in class II-restricted Ag presentation, invariant chain (Ii) and DM. DM participates in the removal of the Ii-derived class II-associated Ii chain peptide and the p12 degradation product from the I-Ag7 molecule. In addition, I-Ag7 undergoes a conformational change when DM is expressed within the APC. Finally, DM can mediate accumulation of peptide/class II complexes on the surface of APCs. Collectively, our experiments indicate that the failure of the I-Ag7 molecule to stably bind peptide cannot be attributed to a failure to interact with the DM or Ii glycoproteins.