bluuebunny/crossref_metadata_2025 huggingface dataset:
Mouse NKR-P1B, a Novel NK1.1 Antigen with Inhibitory Function
Authors: Carlyle James R. , Martin Alberto , Mehra Arun , Attisano Liliana , Tsui Florence W. , Zúñiga-Pflücker Juan Carlos
Published: January 2023

The mouse NK1.1 Ag originally defined as NK cell receptor (NKR)-P1C (CD161) mediates NK cell activation. Here, we show that another member of the mouse CD161 family, NKR-P1B, represents a novel NK1.1 Ag. In contrast to NKR-P1C, which functions as an activating receptor, NKR-P1B inhibits NK cell activation. Association of NKR-P1B with Src homology 2-containing protein tyrosine phosphatase-1 provides a molecular mechanism for this inhibition. The existence of these two NK1.1 Ags with opposite functions suggests a potential role for NKR-P1 molecules, such as those of the Ly-49 gene family, in regulating NK cell function.
Cleavage of the Glycosylphosphatidylinositol Anchor Affects the Reactivity of Thy-1 with Antibodies
Authors: Kukulansky Tova , Abramovitch Shirley , Hollander Nurit
Published: January 2023

Thy-1 protein, a member of the Ig superfamily, is bound to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. We demonstrate that following anchor cleavage by phospholipase C, the reactivity of the solubilized Thy-1 with several mAbs is lost, and its reactivity with polyclonal anti-Thy-1 Abs is markedly decreased. Hence, solubilized Thy-1 cannot be detected by a range of mAbs. In contrast, enzymatic cleavage of biotinylated Thy-1 yields an intact solubilized protein that can be detected by streptavidin. These results exclude a possible proteolytic degradation of solubilized Thy-1 and suggest that the marked decrease in Thy-1 immunoreactivity following delipidation is due to conformational changes in the Thy-1 protein. We further demonstrate that addition of phospholipase C to preformed Ab-Ag complexes causes dissociation and removal of Thy-1 from the complex, indicating that delipidation of Thy-1 induces a conformational change in Thy-1 that is sufficient to dissociate bound Ab. The possibility should therefore be considered that the GPI anchor affects the conformation of a protein to which it is linked.
Two Stages of Increased IgA Transfer During Lactation in the Marsupial,Trichosurus vulpecula(Brushtail Possum)
Authors: Adamski Frances M. , Demmer Jerome
Published: January 2023

The polymeric Ig receptor (pIgR) and J chain molecules are involved in the transfer of IgA across the mammary gland epithelia into milk. The J chain binds two IgA molecules to form dimeric IgA, and the pIgR transports this complex through epithelial cells. We report here the cloning of the first marsupial homologues for the pIgR and J chain from the brushtail possum. Marsupial young are born after a short gestation and are less developed than eutherian newborn. The pouch young is completely dependent on milk as its sole source of nutrition during early lactation and this phase can be considered to be equivalent to an external gestation. Two periods of increased expression of pIgR, J chain, and IgA heavy chain mRNAs were observed in the mammary gland during lactation. The first occurs for a brief period after birth of the pouch young and is likely to reflect IgA transfer via the colostrum. The second period of increased expression, which is unique to marsupials, occurs after the early lactation period and just before young exit the pouch. We propose that this represents a second colostral-like phase at the end of the external gestation.
Immunomodulatory Role of C10 Chemokine in a Murine Model of Allergic Bronchopulmonary Aspergillosis
Authors: Hogaboam Cory M. , Gallinat Chad S. , Taub Dennis D. , Strieter Robert M. , Kunkel Steven L. , Lukacs Nicholas W.
Published: January 2023

The immunomodulatory role of the chemokine C10 was explored in allergic airway responses during experimental allergic bronchopulmonary aspergillosis (ABPA). The intratracheal delivery of Asperigillus fumigatus Ag into A. fumigatus-sensitized mice resulted in significantly increased levels of C10 within the bronchoalveolar lavage, and these levels peaked at 48 h after A. fumigatus challenge. In addition, C10 levels in BAL samples were greater than 5-fold higher than levels of other chemokines such as monocyte-chemoattractant protein-1, eotaxin, and macrophage-inflammatory protein-1α. From in vitro studies, it was evident that major pulmonary sources of C10 may have included alveolar macrophages, lung fibroblasts, and vascular smooth muscle cells. Experimental ABPA was associated with severe peribronchial eosinophilia, bronchial hyperresponsiveness, and augmented IL-13 and IgE levels. The immunoneutralization of C10 with polyclonal anti-C10 antiserum 2 h before the intratracheal A. fumigatus challenge significantly reduced the airway inflammation and hyperresponsiveness in this model of ABPA, but had no effect on IL-10 nor IgE levels. Taken together, these data suggest that C10 has a unique role in the progression of experimental ABPA.
Human Cytomegalovirus Inhibits IFN-α-Stimulated Antiviral and Immunoregulatory Responses by Blocking Multiple Levels of IFN-α Signal Transduction
Authors: Miller Daniel M. , Zhang Yingxue , Rahill Brian M. , Waldman W. James , Sedmak Daniel D.
Published: January 2023

The type I IFNs represent a primordial, tightly regulated defense system against acute viral infection. IFN-α confers resistance to viral infection by activating a conserved signal transduction pathway that up-regulates direct antiviral effectors and induces immunomodulatory activities. Given the critical role of IFN-α in anti-human cytomegalovirus (HCMV) immunity and the profound ability of HCMV to escape the host immune response, we hypothesized that HCMV blocks IFN-α-stimulated responses by disrupting multiple levels of the IFN-α signal transduction pathway. We demonstrate that HCMV inhibits IFN-α-stimulated MHC class I, IFN regulatory factor-1, MxA and 2′,5-oligoadenylate synthetase gene expression, transcription factor activation, and signaling in infected fibroblasts and endothelial cells by decreasing the expression of Janus kinase 1 and p48, two essential components of the IFN-α signal transduction pathway. This investigation is the first to report inhibition of type I IFN signaling by a herpesvirus. We propose that this novel immune escape mechanism is a major means by which HCMV is capable of escaping host immunity and establishing persistence.
IL-1 Is an Effective Adjuvant for Mucosal and Systemic Immune Responses When Coadministered with Protein Immunogens
Authors: Staats Herman F. , Ennis Francis A.
Published: January 2023

Mucosal immunization with soluble protein Ag alone may induce Ag-specific tolerance, whereas mucosal immunization with Ag in the presence of a mucosal adjuvant may induce Ag-specific systemic and mucosal humoral and cell-mediated immune responses. The most widely used and studied mucosal adjuvant is cholera toxin (CT). Although the mechanism of adjuvanticity of CT is not completely understood, it is known that CT induces mucosal epithelial cells to produce the proinflammatory cytokines IL-1, IL-6, and IL-8 and up-regulates macrophage production of IL-1 and the costimulatory molecule B7.2. Because IL-1 may duplicate many of the activities of CT, we evaluated IL-1α and IL-1β for their ability to serve as mucosal adjuvants when intranasally administered with soluble protein Ags. IL-1α and IL-1β were as effective as CT for the induction of Ag-specific serum IgG, vaginal IgG and IgA, systemic delayed-type hypersensitivity, and lymphocyte proliferative responses when intranasally administered with soluble protein Ag. Our results indicate that IL-1α and IL-1β may be useful as mucosal vaccine adjuvants. Such an adjuvant may be useful, and possibly required, for vaccine-mediated protection against pathogens that infect via the mucosal surfaces of the host such as HIV.
Selection of HIV-Specific Immunogenic Epitopes by Screening Random Peptide Libraries with HIV-1-Positive Sera
Authors: Scala Giuseppe , Chen Xueni , Liu Weimin , Telles Jean Noel , Cohen Oren J. , Vaccarezza Mauro , Igarashi Tatsu , Fauci Anthony S.
Published: January 2023

Efforts to develop a protective HIV-1 vaccine have been hindered by difficulties in identifying epitopes capable of inducing broad neutralizing Ab responses. In fact, the high mutation rate occurring in HIV-1 envelope proteins and the complex structure of gp120 as an oligomer associated with gp41 result in a high degree of antigenic polymorphism. To overcome these obstacles, we screened random peptide libraries using sera from HIV-infected subjects to identify antigenic and immunogenic mimics of HIV-1 epitopes. After extensive counterscreening with HIV-negative sera, we isolated peptides specifically recognized by Abs from HIV-1-infected individuals. These peptides behaved as antigenic mimics of linear or conformational HIV-1 epitopes generated in vivo in infected subjects. Consistent with these findings, sera of simian HIV-infected monkeys also recognized the HIV-specific epitopes. The selected peptides were immunogenic in mice, where they elicited HIV-specific Abs that effectively neutralized HIV-1 isolates. These results demonstrate that pools of HIV-1 mimotopes can be selected from combinatorial peptide libraries by taking advantage of the HIV-specific Ab repertoire induced by the natural infection.
Th2-Induced Airway Mucus Production Is Dependent on IL-4Rα, But Not on Eosinophils
Authors: Cohn Lauren , Homer Robert J. , MacLeod Heather , Mohrs Markus , Brombacher Frank , Bottomly Kim
Published: January 2023

Mucus hyperproduction in asthma results from airway inflammation and contributes to clinical symptoms, airway obstruction, and mortality. In human asthmatics and in animal models, excess mucus production correlates with airway eosinophilia. We previously described a system in which TCR transgenic CD4 Th2 cells generated in vitro were transferred into recipient mice and activated in the respiratory tract with inhaled Ag. Th2 cells stimulated airway eosinophilia and a marked increase in mucus production, while mice that received Th1 cells exhibited airway inflammation without eosinophilia or mucus. Mucus could be induced by IL-4−/− Th2 cells at comparable levels to mucus induced by IL-4+/+ Th2 cells. In the current studies we dissect further the mechanisms of Th2-induced mucus production. When IL-4−/− Th2 cells are transferred into IL-4Rα−/− mice, mucus is not induced, and BAL eosinophilia is absent. These data suggest that in the absence of IL-4, IL-13 may be critical for Th2-induced mucus production and eosinophilia. To determine whether eosinophils are important in mucus production, IL-5−/− Th2 cells were transferred into IL-5−/− recipients. Eosinophilia was abolished, yet mucus staining in the epithelium persisted. These studies show definitively that IL-5, eosinophils, or mast cells are not essential, but signaling through IL-4Rα is critically important in Th2 cell stimulation of mucus production.
T Cell-Tropic HIV gp120 Mediates CD4 and CD8 Cell Chemotaxis through CXCR4 Independent of CD4: Implications for HIV Pathogenesis
Authors: Iyengar Sujatha , Schwartz David H. , Hildreth James E. K.
Published: January 2023

HIV entry is determined by one or more chemokine receptors. T cell-tropic viruses bind CXCR4, whereas macrophage-tropic viruses use CCR5 and other CCRs. Infection with CXCR4 and CCR5-tropic HIV requires initial binding to CD4, and chemotaxis induced by the CCR5-tropic envelope has been reported to be strictly dependent on CD4 binding. We demonstrate that, in contrast to CD4-dependent gp120 signaling via CCR5, envelope signaling through CXCR4 is CD4 independent, inducing chemotaxis of both CD4 and CD8 T cells. Signaling by virus or soluble envelope through CXCR4 may affect pathogenesis by attracting and activating target and effector cells.
Long Term Prevention of Allergic Lung Inflammation in a Mouse Model of Asthma by CpG Oligodeoxynucleotides
Authors: Sur Sanjiv , Wild James S. , Choudhury Barun K. , Sur Nilanjana , Alam Rafeul , Klinman Dennis M.
Published: January 2023

Asthma is an inflammatory disease of the airways that is induced by Th2 cytokines and inhibited by Th1 cytokines. Despite a steady increase in the incidence, morbidity, and mortality from asthma, no current treatment can reduce or prevent asthma for a prolonged period. We examined the ability of unmethylated CpG oligodeoxynucleotides (ODN), which are potent inducers of Th1 cytokines, to prevent the inflammatory and physiological manifestations of asthma in mice sensitized to ragweed allergen. Administration of CpG ODN 48 h before allergen challenge increased the ratio of IFN-γ to IL-4 secreting cells, diminished allergen-induced eosinophil recruitment, and decreased the number of ragweed allergen-specific IgE-producing cells. These effects of CpG ODN were sustained for at least 6 wk after its administration. Furthermore, there was a vigorous Th1 memory response to the recall Ag, inhibition of peribronchial and perivascular lung inflammation, and inhibition of bronchial hyperresponsiveness 6 wk after administration of CpG ODN. Administration of CpG ODN in IFN-γ −/− mice failed to inhibit eosinophil recruitment, indicating a critical role of IFN-γ in mediating these effects. This is the first report of a treatment that inhibits allergic lung inflammation in presensitized animals for a prolonged period and thus has relevance to the development of an effective long term treatment for asthma.
Phosphatidylinositol 3-Kinase-Dependent and -Independent Cytolytic Effector Functions
Authors: Fuller Claudette L. , Ravichandran Kodimangalam S. , Braciale Vivian L.
Published: January 2023

Two distinct forms of short-term cytolysis have been described for CD8+ CTLs, the perforin/granzyme- and Fas ligand/Fas (CD95 ligand (CD95L)/CD95)-mediated pathways. However, the difference in signal transduction events leading to these cytolytic mechanisms remains unclear. We used wortmannin, an irreversible antagonist of phosphatidylinositol 3-kinase (PI3-K) activity, to investigate the role of PI3-K in influenza-specific CD8+ CTL cytolytic effector function. We found that the addition of wortmannin at concentrations as low as 1 nM significantly inhibited both the Ag/MHC-induced cytolysis of CD95− target cells and serine esterase release. In strong contrast, W did not inhibit the Ag/MHC-induced CD95L expression or the CD95L/CD95-mediated cytolysis of CD95+ targets. A combination of wortmannin and blocking mAb against CD95L inhibited the cytolysis of CD95+ targets, indicating that the wortmannin-independent cytolysis was due to CD95L/CD95 mediated cytolysis. These findings suggest a differential role for PI3-K in mediating cytolysis and, thus far, the earliest difference between perforin/granzyme- and CD95L/CD95-dependent cytolysis. Our data reinforce the idea of a TCR with modular signal transduction pathways that can be triggered or inhibited selectively, resulting in differential effector function.
Effective Stimulation for IL-12 p35 mRNA Accumulation and Bioactive IL-12 Production of Antigen-Presenting Cells Interacted with Th Cells
Authors: Yamane Hidehiro , Kato Takuma , Nariuchi Hideo
Published: January 2023

Bioactive IL-12 is composed of two subunits, p35 and p40. In the APC-Th cell interaction, p40 mRNA accumulation in APC was shown to be up-regulated by stimulation with CD40 ligand (CD40L) on Th cells. However, the CD40-CD40L interaction scarcely induced p35 mRNA accumulation in APC. In the present experiments, p35 mRNA accumulation was induced in splenic macrophages/dendritic cells by the interaction with paraformaldehyde-fixed Th1 cells in the presence of Ag, and the p35 mRNA accumulation was abrogated by the inclusion of anti-I-A in cultures to block TCR/MHC class II interaction. The accumulation was also induced by the stimulation with agonistic anti-I-A. These results indicate that the interaction of the MHC class II molecule with TCR evokes an activation signal for p35 mRNA accumulation in APC. Furthermore, the production of bioactive IL-12 in macrophages/dendritic cells stimulated with CD40L was enhanced by the inclusion of agonistic anti-I-A. The p35 mRNA accumulation and IL-12 production of macrophages/dendritic cells induced by stimulation with OVA-specific fixed Th1 clone expressing CD40L were also enhanced by adding OVA in cultures. These results indicate that the p35 mRNA accumulation induced by MHC class II stimulation plays a role in bioactive IL-12 production.
In Vivo Inhibition of NF-κB in T-Lineage Cells Leads to a Dramatic Decrease in Cell Proliferation and Cytokine Production and to Increased Cell Apoptosis in Response to Mitogenic Stimuli, But Not to Abnormal Thymopoiesis
Authors: Ferreira Valérie , Sidénius Nicolai , Tarantino Nadine , Hubert Pascale , Chatenoud Lucienne , Blasi Francesco , Körner Marie
Published: January 2023

To understand the role of NF-κB complexes in T cell development and activation, we have generated transgenic mice in which RelA and c-Rel complexes were selectively inhibited in the T-lineage cells by specific expression of a trans-dominant form of IκBα. Transgene expression did not affect the thymic development, but led to lowered numbers of splenic T cells and to a dramatic decrease in the ex vivo proliferative response of splenic T lymphocytes. Analysis of IL-2 and IL-2Rα expression demonstrated that the perturbation of the proliferation response was not attributable to an abnormal expression of these genes. In contrast, expression of IL-4, IL-10, and IFN-γ was strongly inhibited in the transgenic T cells. The proliferative deficiency of the transgenic T cells was associated with an increased apoptosis. These results point out the involvement of NF-κB/Rel family proteins in growth signaling pathways by either regulating proteins involved in the IL-2 signaling or by functionally interfering with the cell cycle progression.
Igα and Igβ Are Required for Efficient Trafficking to Late Endosomes and to Enhance Antigen Presentation
Authors: Siemasko Karyn , Eisfelder Bartholomew J. , Stebbins Christopher , Kabak Shara , Sant Andrea J. , Song Wenxia , Clark Marcus R.
Published: January 2023

The B cell Ag receptor (BCR) is a multimeric complex, containing Igα and Igβ, capable of internalizing and delivering specific Ags to specialized late endosomes, where they are processed into peptides for loading onto MHC class II molecules. By this mechanism, the presentation of receptor-selected epitopes to T cells is enhanced by several orders of magnitude. Previously, it has been reported that, under some circumstances, either Igα or Igβ can facilitate the presentation of Ags. However, we now demonstrate that if these Ags are at low concentrations and temporally restricted, both Igα and Igβ are required. When compared with the BCR, chimeric complexes containing either chain alone were internalized but failed to access the MHC class II-enriched compartment (MIIC) or induce the aggregation and fusion of its constituent vesicles. Furthermore, Igα/Igβ complexes in which the immunoreceptor tyrosine-based activation motif tyrosines of Igα were mutated were also incapable of accessing the MIIC or of facilitating the presentation of Ag. These data indicate that both Igα and Igβ contribute signaling, and possibly other functions, to the BCR that are necessary and sufficient to reconstitute the trafficking and Ag-processing enhancing capacities of the intact receptor complex.
Modulation of CD28 Expression: Distinct Regulatory Pathways During Activation and Replicative Senescence
Authors: Vallejo Abbe N. , Brandes Johann C. , Weyand Cornelia M. , Goronzy Jörg J.
Published: January 2023

The costimulatory molecule CD28 has a restricted tissue distribution and is expressed on T cells and some plasmacytoma cells. Although CD28 is constitutively expressed, its expression is transiently down-regulated following T cell activation and declines progressively with in vitro senescence. In vivo, CD8+ T cells and, less frequently, CD4+ T cells may completely lose CD28 surface expression during chronic infections and with aging. This correlates with changes of nuclear protein-binding activities to two motifs, site α and β, within the CD28 minimal promoter. Both α- and β-bound complexes are found only in lymphoid tissues, in CD28+ T cells, and in some transformed B cells. These complexes are coordinately expressed except during replicative senescence, which is characterized by the down-modulation of site β- but not site α-binding activities. In contrast, T cell activation induces a parallel decline in both site α- and β-binding activities. CD4+ and CD8+ T cells differ in their β-binding profiles, which may explain the more pronounced down-regulation of CD28 in senescent CD8+ T cells. In vivo expanded CD4+CD28null and CD8+CD28null T cells uniformly lack α- and β-bound complexes, resembling the pattern seen in chronically activated cells and not of senescent cells.
Combination Gene Therapy with CD86 and the MHC Class II Transactivator in the Control of Lung Tumor Growth
Authors: Martin Brian K. , Frelinger John G. , Ting Jenny P.-Y.
Published: January 2023

Early reports suggest that the costimulatory molecule CD86 (B7-2) has sporadic efficacy in tumor immunity, whereas changes in cancer immunity mediated by the MHC class II transactivator (CIITA) have not been extensively investigated. CIITA activates MHC class II expression in most cells; however, in the Line 1 lung carcinoma model system, CIITA activates MHC class I and well as class II. Here we show that CD86 is very effective in inducing a primary immune response against Line 1. Tumor cells expressing CD86 grew in only 50% of the mice injected with live cells, and those mice that developed tumors did so with significantly delayed kinetics. Furthermore, irradiated CD86-expressing Line 1 cells served as an effective tumor vaccine, demonstrating that CD86 is effective in inducing tumor immunity in the Line 1 system. These data suggest that if CIITA and CD86 cooperate, enhanced tumor immunity could be achieved. CIITA alone was mildly beneficial in slowing primary tumor growth but only when expressed at low levels. Clones expressing high levels of class II MHC grew as fast as or faster than parental tumor, and CIITA expression in a tumor vaccine assay lacked efficacy. When CIITA and CD86 were coexpressed, there was no cooperative immune protection from tumor growth. Cells that coexpress both genes also failed as a cancer vaccine, suggesting a negative role for CIITA in this lung carcinoma. These data suggest that human cancer vaccine trials utilizing CIITA gene therapy alone or in combination with CD86 should be approached with caution.
Mycobacterium tuberculosis CDC1551 Induces a More Vigorous Host Response In Vivo and In Vitro, But Is Not More Virulent Than Other Clinical Isolates
Authors: Manca Claudia , Tsenova Liana , Barry Clifton E. , Bergtold Amy , Freeman Sherry , Haslett Patrick A. J. , Musser James M. , Freedman Victoria H. , Kaplan Gilla
Published: January 2023

Mycobacterium tuberculosis CDC1551, a clinical isolate reported to be hypervirulent and to grow faster than other isolates, was compared with two other clinical isolates (HN60 and HN878) and two laboratory strains (H37Rv and Erdman). The initial (1–14 days) growth of CDC1551, HN60, HN878, and H37Rv was similar in the lungs of aerosol-infected mice, but growth of Erdman was slower. Thereafter, the growth rate of CDC1551 decreased relative to the other strains which continued to grow at comparable rates up to day 21. In the lungs of CDC1551-infected mice, small well-organized granulomas with high levels of TNF-α, IL-6, IL-10, IL-12, and IFN-γ mRNA were apparent sooner than in lungs of mice infected with the other strains. CDC1551-infected mice survived significantly longer. These findings were confirmed in vitro. The growth rates of H37Rv and CDC1551 in human monocytes were the same, but higher levels of TNF-α, IL-10, IL-6, and IL-12 were induced in monocytes after infection with CDC1551 or by exposure of monocytes to lipid fractions from CDC1551. CD14 expression on the surface of the monocytes was up-regulated to a greater extent by exposure to the lipids of CDC1551. Thus, CDC1551 is not more virulent than other M. tuberculosis isolates in terms of growth in vivo and in vitro, but it induces a more rapid and robust host response.
Mice Lacking NK Cells Develop an Efficient Th1 Response and Control CutaneousLeishmania majorInfection
Authors: Satoskar Abhay R. , Stamm Luisa M. , Zhang Xingmin , Satoskar Anjali A. , Okano Mitsuhiro , Terhorst Cox , David John R. , Wang Baoping
Published: January 2023

NK cells are believed to play a critical role in the development of immunity against Leishmania major. We recently found that transplantation of wild-type bone marrow cells into neonatal tgε 26 mice, which are deficient in T and NK cells, resulted in normal T cell development, but no or poor NK cell development. Using this novel model we analyzed the role of NK cells in the development of Th1 response and control of cutaneous L. major infection. Mice selectively lacking NK cells (NK−T+) developed an efficient Th1-like response, produced significant amounts of IL-12 and IFN-γ, and controlled cutaneous L. major infection. Administration of neutralizing IL-12 Abs to NK−T+ mice during L. major infection resulted in exacerbation of the disease. These results demonstrate that NK cells are not critical for development of protective immunity against L. major. Furthermore, they indicate that IL-12 can induce development of Th1 response independent of NK cells in NK−T+ mice following L.major infection.
C5a Suppresses the Production of IL-12 by IFN-γ-Primed and Lipopolysaccharide-Challenged Human Monocytes
Authors: Wittmann Miriam , Zwirner Jörg , Larsson Vivi-Ann , Kirchhoff Konstanze , Begemann Gabriele , Kapp Alexander , Götze Otto , Werfel Thomas
Published: January 2023

IL-12 is a key mediator of the immune response, skewing T lymphocytes toward a type 1 cytokine pattern. Priming with IFN-γ or GM-CSF is required for expression of IL-12p70 by cells in which IL-12 is inducible by bacterial products such as LPS. We here show for the first time that the production of bioactive IL-12 by human monocytes can be significantly suppressed by C5a if applied to IFN-γ-primed monocytes before LPS stimulation. There was a dose-dependent suppression by IL-12 (p70) on the levels of intracellular cytokine production and cytokine secretion. mRNA studies consistently showed a reduction of IL-12p40 and IL-12p35 expression by stimulation in the presence of C5a. The results of several different experimental approaches suggest that IL-12 down-regulation was not due to endogenous IL-10, IL-4, or PGE2 production induced by C5a. Moreover, stimulation of IFN-γ-primed monocytes with C5a did not lead to a down-regulation of the CD14 Ag, which is an LPS receptor. These findings show that the anaphylatoxin C5a has the capacity to directly interact with the complex regulation of IL-12.
Mechanisms of IL-10 Production in Human Microglia-T Cell Interaction
Authors: Chabot Sophie , Williams Gary , Hamilton Mark , Sutherland Garnette , Yong V. Wee
Published: January 2023

IL-10, a cytokine with important anti-inflammatory properties, is generated within the CNS during neuroinflammation. The mechanism for its production is poorly understood. Since infiltrating lymphocytes come into close proximity with the macrophage-like cells of the CNS, the microglia, we have used an in vitro human microglia-T cell coculture system to address the mechanisms of IL-10 production. We demonstrate that microglia or activated T cells alone secrete negligible amounts of IL-10, but that their coculture results in significant IL-10 production, which was effected by both cell types. IL-10 generation was cell contact dependent, and treatment with anti-CD40, CTLA-4-Fc, or anti-CD23 decreased the IL-10 content in microglia-T cell cocultures. The combination of anti-CD40 and CTLA-4-Fc reduced IL-10 levels to the negligible amounts seen with T cells or microglia in isolation. By also measuring TNF-α levels, specificity of cytokine regulation was observed; while anti-CD40 and CTLA-4-Fc reduced IL-10 and TNF-α levels, anti-CD23 did not affect TNF-α while attenuating IL-10 generation. Anti-very late Ag-4, which decreased TNF-α levels, did not affect IL-10. These results implicate the CD40, B7, and CD23 pathways in IL-10 production following microglia-T cell encounter and have relevance to the regulation of an anti-inflammatory response within the CNS.
Cyclosporin A-Induced Autologous Graft-Versus-Host Disease: A Prototypical Model of Autoimmunity and Active (Dominant) Tolerance Coordinately Induced by Recent Thymic Emigrants
Authors: Wu Dianna Y. , Goldschneider Irving
Published: January 2023

Cyclosporin A (CSA)-induced autologous graft-vs-host disease (autoGVHD) is an autoimmune syndrome initiated by autoeffector T cells presumed to be exported from the thymus during CSA treatment. The appearance of noncytotoxic immunoregulatory T cell activity after cessation of CSA treatment is also thymus dependent. In the present study, we have tested the hypothesis that both autoeffector and immunoregulatory T cells in CSA-treated rats are recent thymic emigrants (RTEs). Local syngeneic graft-vs-host reaction (synGVHR) and timed thymectomy (Tx) assays revealed that autoeffector T cells appear initially in the thymus and are promptly exported to lymph nodes (LN) during the first week of CSA treatment. In contrast, immunoregulatory thymocytes are first detectable by local synGVHR inhibition assays during the second week of CSA treatment but are not exported to LN until ∼4 days post-CSA. Both the autoeffector and immunoregulatory T cells in LN express Thy-1, a selective marker for RTEs in the rat. However, the autoeffector RTEs have a CD4+8+ phenotype, whereas the immunoregulatory RTEs have a CD4+8− phenotype. Thus, the coordinate formation in and release from the thymus cortex and medulla of autoeffector and immunoregulatory T cells in CSA-treated rats directly demonstrates that centrally induced, nondeletional tolerance can serve as a fail-safe mechanism by which clones of autoeffector T cells that have escaped intrathymic negative selection for self-MHC class II Ag can be suppressed postthymically.
Treatment of Breast Cancer with Fibroblasts Transfected with DNA from Breast Cancer Cells
Authors: de Zoeten Edwin , Carr-Brendel Victoria , Markovic Dubravka , Taylor-Papadimitriou Joyce , Cohen Edward P.
Published: January 2023

This investigation was based on the hypothesis that weakly immunogenic, breast cancer-associated Ags, the products of mutant or dysregulated genes in the malignant cells, will be expressed in a highly immunogenic form by semiallogeneic IL-2-secreting fibroblasts transfected with DNA from breast cancer cells. (Classic studies indicate that transfection of genomic DNA can stably alter both the genotype and the phenotype of the cells that take up the exogenous DNA.) To investigate this question, we transfected LM mouse fibroblasts (H-2k) modified to secrete IL-2 with genomic DNA from a breast adenocarcinoma that arose spontaneously in a C3H/He mouse (H-2k). To increase their nonspecific immunogenic properties, the fibroblasts were also modified before transfection to express allogeneic MHC determinants (H-2Kb). Afterward, the IL-2-secreting semiallogeneic cells were cotransfected with DNA from the spontaneous breast neoplasm, along with a plasmid (pHyg) conferring resistance to hygromycin. Pooled colonies of hygromycin-resistant cells were then tested in C3H/He mice for their immunotherapeutic properties against the growth of the breast neoplasm. The results indicated that tumor-bearing mice immunized with the transfected cells survived significantly longer than mice in various control groups. Similar beneficial effects were seen in C57BL/6 mice injected with a syngeneic breast carcinoma cell line (EO771) and semiallogeneic, IL-2-secreting fibroblasts transfected with DNA from EO771 cells. The immunity was mediated by CD8+ T cells since immunized mice depleted of CD8+ cells failed to resist tumor growth.
The p65 Subunit of NF-κB Is Redundant with p50 During B Cell Proliferative Responses, and Is Required for Germline CH Transcription and Class Switching to IgG3
Authors: Horwitz Bruce H. , Zelazowski Piotr , Shen Yi , Wolcott Karen M. , Scott Martin L. , Baltimore David , Snapper Clifford M.
Published: January 2023

B cells lacking individual NF-κB/Rel family members exhibit defects in activation programs. We generated small resting B cells lacking p65 or p50 alone, or lacking both p50 and p65, then evaluated the ability of these cells to proliferate, secrete Ig, and undergo Ig class switching. B cells lacking p65 proliferated well in response to all stimuli tested. However, these cells demonstrated an isolated defect in switching to IgG3, which was associated with a decrease in γ3 germline CH gene expression. Whereas, previously reported, B cells lacking p50 alone had a severe proliferative defect in response to LPS, a moderate defect in response to CD40 ligand (CD40L), and normal proliferation to Ag receptor cross-linking using dextran-conjugated anti-IgD Abs (αδ-dex), B cells lacking both p50 and p65 exhibited severely impaired proliferation in response to LPS, αδ-dex, and CD40L. This defect could be overcome by simultaneous administration of αδ-dex and CD40L. In response to this latter combination of stimuli, B cells lacking both p50 and p65 secreted Ig and underwent isotype switching to IgG1 as efficiently as B cells lacking p50 alone. These data demonstrate a role for the p65 subunit of NF-κB in germline CH gene expression as well as functional redundancy between p50 and p65 during proliferative responses.
Fas Ligand Induction in Human NK Cells Is Regulated by Redox Through a Calcineurin-Nuclear Factors of Activated T Cell-Dependent Pathway
Authors: Furuke Keizo , Shiraishi Mitsuhiro , Mostowski Howard S. , Bloom Eda T.
Published: January 2023

Fas ligand (FasL) on cytotoxic lymphocytes is important for mediating apoptosis of activated lymphocytes and other target cells. We have reported that NK cell functions, such as proliferation, cell death, and killing activity, are subject to regulation by cellular redox status. Here, we report that expression of FasL protein and mRNA in activated NK cells is also regulated by redox. Ligation of CD16 on IL-2-preactivated NK cells resulted in reduction of intracellular peroxide level as well as induction of FasL expression. This CD16-induced FasL expression was suppressed by oxidative stress, including thiol deprivation or treatment with hydrogen peroxide (H2O2). Addition of thiol-reducing compounds, such as l-cystine, 2-ME, or N-acetyl cysteine, restored FasL expression. These data suggest that CD16 stimulation requires cellular reducing status for FasL induction in NK cells. Because FasL gene activation following CD16 cross-linking is regulated by the NF of activated T cells (NFAT), we examined the effect of oxidative stresses on NFAT activation. Electrophoretic mobility shift assays revealed that both thiol insufficiency and H2O2 treatment suppressed DNA-binding activity of NFAT and that addition of thiol-reducing compounds reversed or even enhanced it. Furthermore, these oxidative stresses inhibited activity of calcineurin, a serine/threonine phosphatase that regulates NFAT activation. These results suggest that suppression of calcineurin and NFAT activation is a mechanism by which oxidative stress inhibits FasL induction in activated NK cells and further support the hypothesis that thiol-reducing compounds might be required for maintenance of optimal NK functions under physiologic oxidative conditions.
Human Effector Memory T Cells Express CD86: A Functional Role in Naive T Cell Priming
Authors: Jeannin Pascale , Herbault Nathalie , Delneste Yves , Magistrelli Giovanni , Lecoanet-Henchoz Sybille , Caron Gersende , Aubry Jean-Pierre , Bonnefoy Jean-Yves
Published: January 2023

The glycoprotein CD86 expressed on APCs provides a costimulatory signal necessary for an efficient activation of naive T cells. In contrast, there is controversy about the condition of expression and the function of CD86 on T cells. In this study, we have analyzed the phenotype and the biological activity of CD86+ T cells generated from human PBMC. Results show that CD86 expression on T cells is induced by long term stimulation via CD3 and IL-2R and is down-regulated as the cells become quiescent. The CD86-expressing cells are memory effector T cells: 1) they express CD45RO and high levels of the activation markers CD25, CD54, and HLA-Dr; 2) they selectively express CD30, CD40-ligand, and CD70; and 3) in response to stimulation, most of them produce IFN-γ before dying by apoptosis. We then analyzed whether CD86 expressed on T cells is functional. Results show that paraformaldehyde-fixed CD86+ T cells enhance the proliferation and production of IFN-γ by anti-CD3 mAb-stimulated naive T cells and induce proliferation of resting allogenic T cells. All these effects are prevented by neutralizing anti-CD86 mAbs. In contrast, we report no autocrine effect of CD86 in CD86+ T cell activation. In conclusion, these data show that human memory effector T cells express a functional form of CD86 that can costimulate naive T cell responses.
β1 Integrin Cross-Linking Inhibits CD16-Induced Phospholipase D and Secretory Phospholipase A2 Activity and Granule Exocytosis in Human NK cells: Role of Phospholipase D in CD16-Triggered Degranulation
Authors: Milella Michele , Gismondi Angela , Roncaioli Paola , Palmieri Gabriella , Morrone Stefania , Piccoli Mario , Frati Luigi , Cifone Maria Grazia , Santoni Angela
Published: January 2023

Recent data indicate that integrin-generated signals can modulate different receptor-stimulated cell functions in both a positive (costimulation) and a negative (inhibition) fashion. Here we investigated the ability of β1 integrins, namely α4β1 and α5β1 fibronectin receptors, to modulate CD16-triggered phospholipase activation in human NK cells. β1 integrin simultaneous cross-linking selectively inhibited CD16-induced phospholipase D (PLD) activation, without affecting either phosphatidylinositol-phospholipase C or cytosolic phospholipase A2 (PLA2) enzymatic activity. CD16-induced secretory PLA2 (sPLA2) protein release as well as its enzymatic activity in both cell-associated and soluble forms were also found to be inhibited upon β1 integrin coengagement. The similar effects exerted by specific PLD pharmacological inhibitors (2,3-diphosphoglycerate, ethanol) suggest that in our experimental system, sPLA2 secretion and activation are under the control of a PLD-dependent pathway. By using pharmacological inhibitors (2,3-diphosphoglycerate, wortmannin, ethanol) we also demonstrated that PLD activation is an important step in the CD16-triggered signaling cascade that leads to NK cytotoxic granule exocytosis. Consistent with these findings, fibronectin receptor engagement, by either mAbs or natural ligands, resulted in a selective inhibition of CD16-triggered, but not of PMA/ionomycin-induced, degranulation that was reversed by the exogenous addition of purified PLD from Streptomyces chromofuscus.
Serial TCR Engagement and Down-Modulation by Peptide:MHC Molecule Ligands: Relationship to the Quality of Individual TCR Signaling Events
Authors: Itoh Yasushi , Hemmer Bernhard , Martin Roland , Germain Ronald N.
Published: January 2023

In the present study, we examined the relationships among quantitative aspects of TCR engagement as measured by receptor down-modulation, functional responses, and biochemical signaling events using both mouse and human T cell clones. For T cells from both species, ligands that are more potent in inducing functional responses promote TCR down-modulation more efficiently than weaker ligands. At low ligand density, the number of down-modulated TCR exceeds the number of available ligands by as much as 80–100:1 in the optimal human case, confirming the previous description of serial ligand engagement of TCR (Valitutti, et al. 1995. Nature 375:148–151). A previously unappreciated relationship involving TCR down-modulation, the pattern of proximal TCR signaling, and the extent of serial engagement was revealed by analyzing different ligands for the same TCR. Functionally, more potent ligands induce a higher proportion of fully tyrosine phosphorylated ζ-chains and a greater amount of phosphorylated ZAP-70 than less potent ligands, and the number of TCR down-modulated per available ligand is higher with ligands showing this full agonist-like pattern. The large number of receptors showing partial ζ phosphorylation following exposure to weak ligands indicates that the true extent of TCR engagement and signaling, and thus the amount of sequential engagement, is underestimated by measurement of TCR down-modulation alone, which depends on full receptor activation. These data provide new insight into T cell activation by revealing a clear relationship among intrinsic ligand quality, signal amplification by serial engagement, functional T cell responses, and observable TCR clearance from the cell surface.
Identification of Residues in the First Domain of Human Fcα Receptor Essential for Interaction with IgA
Authors: Wines Bruce D. , Hulett Mark D. , Jamieson Gary P. , Trist Halina M. , Spratt Joanne M. , Hogarth P. Mark
Published: January 2023

The FcR family contains multiple receptors for Igs, of which the most distantly related (∼20%) is the IgA receptor (human FcαR), being more homologous (∼35%) to another family of killer-inhibitory receptor-related immunoreceptors with a 19q13.4 chromosomal location in humans. This study of the FcαR demonstrated that, like several IgG receptors, FcαR is a low affinity receptor for Ab (Ka ∼ 106 M−1). Rapid dissociation of the rsFcαR:IgA complex (t1/2 ∼ 25 s) suggests that monomer IgA would bind transiently to cellular FcαRs, while IgA immune complexes could bind avidly. Mutagenesis of histidyl 85 and arginyl 82, in the FG loop of domain 1, demonstrated that these residues were essential for the IgA-binding activity of FcαR, while arginyl 87 makes a minor contribution to the binding activity of the receptor. This site is unusual among the Fc receptors (FcγRII, FcγRIII, and FcεRI), in which the ligand binding site is in domain 2 rather than domain 1, but like FcαR, the FG loop comprises part of the ligand binding site. The putative F and G strands flanking the FcαR ligand binding site are highly homologous in the other killer-inhibitory receptor-related immunoreceptors, suggesting they comprise a conserved structural element on which divergent FG loops are presented and participate in the specific ligand interactions of each of these receptors.
Position Effects of Variable Region Carbohydrate on the Affinity and In Vivo Behavior of an Anti-(1→6) Dextran Antibody
Authors: Coloma M. Josefina , Trinh Ryan K. , Martinez Alexander R. , Morrison Sherie L.
Published: January 2023

IgG is a glycoprotein with an N-linked carbohydrate structure attached to the CH2 domain of each of its heavy chains. In addition, the variable regions of IgG often contain potential N-linked carbohydrate addition sequences that frequently result in the attachment of V region carbohydrate. Nonetheless, the precise role of this V region glycan remains unclear. Studies from our laboratory have shown that a naturally occurring somatic mutant of an anti-dextran Ab that results in a carbohydrate addition site at Asn58 of the VH has carbohydrate in the complementarity-determining region 2 (CDR2) of the VH, and the presence of carbohydrate leads to an increase in affinity. However, carbohydrate attached to nearby positions within CDR2 had variable affects on affinity. In the present work we have extended these studies by adding carbohydrate addition sites close to or within all the CDRs of the same anti-dextran Ab. We find that carbohydrate is attached to all the novel addition sites, but the extent of glycosylation varies with the position of the site. In addition, we find that the position of the variable region carbohydrate influences some functional properties of the Ab, including those usually associated with the V region such as affinity for Ag as well as other characteristics typically attributed to the Fc such as half-life and organ targeting. These studies suggest that modification of variable region glycosylation provides an alternate strategy for manipulating the functional attributes of the Ab molecule and may shed light on how changes in carbohydrate structure affect protein conformation.
Mycobacterium bovisBacille Calmette-Guérin Enhances Pathogenicity of Simian Immunodeficiency Virus Infection and Accelerates Progression to AIDS in Macaques: A Role of Persistent T cell Activation in AIDS Pathogenesis
Authors: Zhou Dejiang , Shen Yun , Chalifoux Laura , Lee-Parritz David , Simon Meredith , Sehgal Prabhat K. , Zheng Leiya , Halloran Matilda , Chen Zheng W.
Published: January 2023

It has recently been proposed that Mycobacterium tuberculosis may enhance the pathogenicity of HIV infections and accelerate the course of HIV disease. This hypothesis has been tested in the present study using a simian immunodeficiency virus of macaques (SIVmac)/Mycobacterium bovis bacille Calmette-Guérin (BCG)-coinfected macaque model. Naive and chronically SIVmac-infected monkeys were evaluated. Following BCG inoculation, the SIVmac-infected monkeys exhibited the dominant responses of TCR-β complementarity-determining region 3-restricted T cell subpopulations. This BCG-driven T cell activation correlated with a marked increase in viral loads in SIVmac-infected monkeys. Moreover, the prolonged T cell activation coincided with the enhanced decline of CD4+ PBL counts and the accelerated progression to clinical AIDS in the coinfected monkeys, suggesting that Mycobacterium-driven T cell activation may be the mechanism underlying the enhanced pathogenicity of AIDS virus infection in the coinfected individuals. Within 2 to 7 mo after BCG coinfection, all chronically SIVmac-infected monkeys died from SIV-induced AIDS including tuberculosis-like disease. Surprisingly, the naive monkeys manifested a T cell activation-related toxic shock syndrome and a profound depletion of CD4+ lymphocytes 2 wk after simultaneous SIVmac/BCG inoculation. These naive animals died 2 mo after SIVmac/BCG inoculation, with the evidence of the persistent SIV p27 antigenemia and SIVmac-induced disease. In contrast, the normal monkeys not infected with SIVmac survived BCG infection; the control SIVmac-infected animals showed a natural course of chronic SIV infection. Thus, results from this SIV/BCG coinfection model strongly support the hypothesis that active coinfection with HIV and Mycobacterium can impact remarkably on the AIDS virus-induced disease.
Mitogenic Activity of Purified Capsular Polysaccharide A from Bacteroides fragilis: Differential Stimulatory Effect on Mouse and Rat Lymphocytes In Vitro
Authors: Brubaker Jeffery O. , Li Qiao , Tzianabos Arthur O. , Kasper Dennis L. , Finberg Robert W.
Published: January 2023

Bacteroides fragilis, a Gram-negative colonic bacterium, induces the formation of abscesses associated with intra-abdominal sepsis in humans. The singular ability of this organism to modulate abscess formation in experimental rodent models resides in the structurally distinct and ionically charged capsular polysaccharides A (PS A) and B (PS B). The regulation of abscess formation in animals is dependent on T lymphocytes. However, the manner in which PS A interacts with T cells remains unknown. We therefore tested the T cell stimulatory capacity of purified PS A on mouse and rat lymphocytes in cellular proliferation assays and found that the PS A molecule possesses mitogenic characteristics distinguishable from those of the polyclonal B cell activator LPS, the T cell mitogen Con A, and staphylococcal enterotoxin A superantigen. Further, PS A stimulated proliferation of normal mouse and rat lymphocytes differentially. Mouse B cells responded to PS A in a fashion that did not require exogenous APC function, while rat T lymphocyte responses to PS A required APC function derived from autologous or xenogenic feeder cells. Cellular depletion experiments showed that the CD4+ subset of rat spleen cells was the primary responder cell type to PS A in vitro. The differential stimulatory effects of PS A on mouse and rat lymphocytes may reflect its ability to stimulate different lymphocyte subsets in vivo through the activities of receptor/counter-receptor pairs present on responder lymphocytes and cognate APC.