bluuebunny/crossref_metadata_2025 huggingface dataset:
A Th0/Th2-like function of CD4+CD7- T helper cells from normal donors and HIV-infected patients.
Authors: Autran B , Legac E , Blanc C , Debré P
Published: December 2022

We previously reported the expansion during HIV infection of a subset of CD4+ T cells characterized by a lack of CD7 cell surface expression. This CD4+CD7- subset showed in normal donors a lower cell proliferation than CD4+CD7+ autologous cells after CD3 triggering or CD28 costimulation. Our aim was to further characterize the Th function of this CD4+CD7- T cell subset, both in normal donors and in HIV-infected patients. Their CD4+CD7- cell proliferation and cytokine secretion were analyzed after cell-sorting and co-stimulation of the CD3 and CD28 pathways. In normal donors, the IL-2 produced by CD4+CD7- cells in response to a CD3 plus CD28 costimulus represented 50 +/- 28% of the autologous CD4+CD7+ cell IL-2 secretion. In addition, the CD4+CD7+ T cells produced higher amounts of IL-4 and IL-10 than the CD4+CD7+ cells with mean CD4+CD7-: CD4+CD7+ ratios of 5.4 +/- 4.5 and 26 +/- 25, for IL-4 and IL-10, respectively, whereas the IFN-gamma production was similar in both subsets. After a triggering of the CD3 complex alone, significant amounts of IL-2 were detectable in CD4+CD7+ cell supernatants only; conversely, IL-4 and IL-10 could be detected only in the culture medium from CD4+CD7- T cells. This profile of cytokine production was maintained both over time and at the clonal cell level in the CD4+CD7- T cell subset. In HIV-infected patients, the CD4+CD7- T cell expansion observed in relationship with disease progression was associated to an in vivo activation of the CD4+CD7- cells, as assessed by cell surface expression of the HLA-DR, CD25, CD71, and CD57 markers. The CD4+CD7- T cells from HIV-seropositive patients showed the same imbalance of cell proliferation and IL-2, IL-4, and IL-10 production as observed in normal donors despite low levels of proliferation and cytokine production. Together, our data indicate that the lack of CD7 expression defines a CD4+ Th cell subset with a Th0/Th2-like profile of cytokine secretion in normal individuals. The CD4+CD7- subset is expanded during HIV infection and characterized by the same although impaired profile of cytokine production. These CD4+CD7- T cells might play a role in the Th cell dysfunctions observed in HIV infection.
Influence of IL-1 receptor blockade on the human response to endotoxemia.
Authors: Van Zee K J , Coyle S M , Calvano S E , Oldenburg H S , Stiles D M , Pribble J , Catalano M , Moldawer L L , Lowry S F
Published: December 2022

Although the experimental administration of IL-1 induces several aspects of the inflammatory response, such as fever, tachycardia, and acute phase proteinemia, the contribution of IL-1 to the human responses to injury or infection remains unclear. A specific IL-1R antagonist (IL-1ra), which effectively blocks the actions of IL-1, was utilized to evaluate the influence of endogenous IL-1 during experimental human endotoxemia. Eighteen healthy volunteers each underwent one control study day, followed 3 days later by one of three randomly chosen treatments: a 6-h infusion of IL-1ra alone (133 mg/h), 20 U/kg national reference endotoxin alone, or both endotoxin and IL-1ra infusion. IL-1ra administration alone was not associated with any observable response. Despite achieving high circulating levels of IL-1ra (34 +/- 3 micrograms/ml), there were no significant differences in hemodynamic parameters, core temperature, or resting energy expenditure in those endotoxemic volunteers receiving IL-1ra when compared with those who did not. Furthermore, leukocyte kinetic and circulating cytokine, acute phase protein, and endocrine responses were similar in both endotoxemic groups. However, IL-1 blockade did significantly reduce the subjective severity of symptoms experienced by the endotoxemic volunteers (p < 0.05). This study demonstrates that an endogenous IL-1 response does not play a significant role in the hemodynamic, immunologic, and metabolic responses to mild endotoxemia in humans.
Distinct signal transduction in mouse CD4+ and CD8+ splenic T cells after CD28 receptor ligation.
Authors: Abe R , Vandenberghe P , Craighead N , Smoot D S , Lee K P , June C H
Published: December 2022

Current evidence suggests that recognition of Ag/MHC by the TCR alone is insufficient to lead to T cell proliferation or effector function. For a Th cell to produce sufficient IL-2 to allow autocrine-driven clonal expansion, there is a requirement for so-called "costimulatory" or "accessory" signals in addition to TCR ligation by Ag/MHC. Although the first signal delivered by TCR ligation has been well characterized, information regarding the biochemical nature of second signals is limited. In the present report, using a newly generated mAb specific for CD28, signal transduction by the CD28 receptor has been studied in mouse splenic T cells. When freshly isolated splenic T cells were assessed, cross-linking of CD28 by mAb did not induce increases in intracellular calcium concentration were assessed, cross-linking of CD28 by mAb did not induce increases in intracellular calcium concentration whereas TCR cross-linking was able to induce calcium mobilization. In contrast, when T cells were activated by phorbol ester treatment or by in vitro culture, CD28 ligation was able to induce calcium mobilization in 60 to 70% of splenic T cells. Unexpectedly, the CD28-induced calcium response was mainly limited to T cells of the CD4+ subset, whereas both CD4+ and CD8+ T cell subsets showed increases in [Ca2+]i of similar magnitude after CD3 epsilon ligation. The temporal nature of the CD28-induced signal was also different from TCR-induced calcium mobilization. CD28-induced signals were delayed in onset and sustained in duration in contrast to TCR signals that had short latency and brief duration. Differential expression of CD28 on the surface of activated CD4+ or CD8+ T cells did not appear to account for the differences in signal transduction between the two T cell subsets. The preferential responsiveness of the CD4+ T cell population to CD28-induced signaling was also observed in downstream events such as the induction of IL-2R, CD69 expression, and in cellular proliferation. These results indicate that the costimulatory signal delivered by CD28 may have fundamentally different biochemical properties in CD4 and CD8 T cell subsets, and therefore the functional role of CD28 may differ in these T cell subsets.
Gamma delta T cells in normal spleen assist immunized alpha beta T cells in the adoptive cell transfer of contact sensitivity. Effect of Bordetella pertussis, cyclophosphamide, and antibodies to determinants on suppressor cells.
Authors: Askenase P W , Szczepanik M , Ptak M , Paliwal V , Ptak W
Published: December 2022

Our prior studies showed that gamma delta T cells were required to assist alpha beta T cells in the successful adoptive cell transfer of contact sensitivity (CS) responsiveness. These TCR-gamma delta+ regulatory T cells in immune spleen and lymph node were CD3+, CD4-, CD8+, nonantigen-specific, and non-MHC-restricted. In the current work, experiments were conducted to determine the mechanisms of how the gamma delta T cells were required to assist the alpha beta T cells in CS. We found that similar regulatory gamma delta T cells were in the spleen of normal mice, but not in the spleen of nude nor SCID mice, suggesting that the regulatory gamma delta T cells were present before immunization and required the thymus for differentiation, and also required rearrangements of gamma delta V gene segments. Treatment of cell transfer recipient mice with Bordetella pertussis (Bp), or with a low dose of cyclophosphamide (50 mg/kg), restored the ability of alpha beta+ gamma delta- T cells to transfer CS. This and other results suggested that Bp caused the CS-assisting gamma delta T cells to leave the lymphoid organs (such as the spleen) and enter the circulation, and only then to be able to assist the TCR-alpha beta+ CS-effector T cells. This effect needed the simultaneous i.v. injection of the CS-effector alpha beta T cells and the CS-assisting gamma delta T cells. The results also suggested that treatment with cyclophosphamide inactivated suppressor T cells in the recipients that acted to inhibit the alpha beta T cell transfer of CS, and thus that the CS-assisting gamma delta T cells acted by protecting the CS-effector alpha beta T cells from this endogenous suppression. This suppression of CS transfers also was eliminated by treatment of recipients with two different mAbs to determinants on suppressor T cells. In conclusion, we have described regulatory TCR-gamma delta+ CS-assisting/protecting T cells that are non-antigen-specific, non-MHC-restricted, CD3+, CD8+ gamma delta T cells that may assist adoptive transferring CS-effector alpha beta T cells by making these effector T cells resistant to suppressor T cells in the normal recipients.
CD27-CD27 ligand/CD70 interactions enhance alloantigen-induced proliferation and cytolytic activity in CD8+ T lymphocytes.
Authors: Brown G R , Meek K , Nishioka Y , Thiele D L
Published: December 2022

To study the role of CD27-CD27 ligand (CD27L)/CD70 interactions in the generation of murine allospecific T cell responses, SF9 cells or cell membranes expressing recombinant human CD70 were added to in vitro MLC containing C57BL/6 (H-2b) responder cells and class I and II MHC disparate H-2b/d stimulator cells. Alloantigen-specific CTL generation, CD8+ T cell proliferation, and levels of N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl esterase activity were enhanced in the presence of human CD27L/CD70 expressed on SF9 cell membranes. Enhancement of CD8+ T cell responses occurred in the absence of any discernible effects on CD4+ T cell proliferation or IL-2 responses. Additional studies demonstrated that CD27L/CD70-expressing membranes enhanced proliferative responses to class I MHC differences but had no effect on proliferative responses to class II MHC disparities. Enhancement of allospecific CTL generation was also observed when CD27L/CD70-expressing membranes were added only during the last 24 to 48 h of 5-day MLC. Thus, the present studies suggest that CD27-CD27L/CD70 interactions can selectively enhance differentiation of Ag-specific CD8+ T cell effector mechanisms under conditions in which Th cell responses are not altered.
Induction of TCR-gamma delta expression on triple-negative (CD3-4-8-) human thymocytes. Comparative analysis of the effects of IL-4 and IL-7.
Authors: He W , Zhang Y , Deng Y , Kabelitz D
Published: December 2022

It is well established that IL-4 and IL-7 control the differentiation of TCR-gamma delta-expressing cells from CD3/TCR-negative thymic precursors. In this study, we have compared the in situ expression of IL-4 and IL-7 mRNA in human postnatal thymus with the in vitro effect of IL-4 and IL-7 on the expansion of TCR-gamma delta + cells from highly purified CD3-4-8- triple-negative thymocytes. IL-4 mRNA expression was restricted to subcapsular regions of the human thymus, whereas cells expressing IL-7 mRNA were distributed throughout the thymic tissue, with some enrichment in subcapsular and cortical regions. Epithelial cells of the outer layer of Hassall's corpuscle strongly expressed IL-7 mRNA. IL-7 but not IL-4 or IL-2, stimulated strong proliferative activity and cellular expansion of triple-negative thymocytes. All three induced the appearance of TCR-gamma delta + cells within 4 days of culture. In the presence of IL-4 or IL-2, 30 to 55% of viable cells were TCR-gamma delta + after 5 to 9 days, whereas only 10 to 15% triple-negative thymocytes cultured with IL-7 expressed TCR-gamma delta. However, comparable absolute numbers of viable TCR-gamma delta + thymocytes were recovered when triple-negative thymocytes were cultured with IL-4 or IL-7, but not with IL-2. Taken together, our results suggest that IL-4 and IL-7 (both of which are produced in situ in thymic tissue) play equally important roles in the in vitro generation of TCR-gamma delta + thymocytes from triple-negative precursor cells.
Inhibition of stem cell factor-induced proliferation of primitive murine hematopoietic progenitor cells signaled through the 75-kilodalton tumor necrosis factor receptor. Regulation of c-kit and p53 expression.
Authors: Jacobsen F W , Dubois C M , Rusten L S , Veiby O P , Jacobsen S E
Published: December 2022

TNF-alpha is a pleiotropic cytokine with stimulatory as well as inhibitory effects on hematopoiesis. We have previously demonstrated that TNF-alpha directly inhibits CSF-induced proliferation of primitive murine lineage-negative bone marrow progenitors (Lin-) and stem cell antigen-1 hematopoietic progenitors through the 75-kDa TNF receptor (TNF-R2), whereas TNF-alpha-induced inhibition of more committed Lin- progenitors is mediated through the 55-kDa TNF-R (TNF-R1), indicating a differential role of the two TNF-Rs in hematopoiesis. Numerous studies have demonstrated the ability of stem cell factor (SCF), a key regulator of hematopoiesis signaling through c-kit, to synergize with other hematopoietic growth factors, but little is known about cytokines capable of inhibiting hematopoiesis induced by SCF. While TNF-alpha has been demonstrated to enhance SCF-induced proliferation of myeloid leukemia blasts, the present report demonstrates that TNF-alpha, by signaling through TNF-R2, inhibits SCF-induced proliferation of normal murine Lin- and stem cell antigen-1 hematopoietic progenitors. SCF-stimulated proliferation of the hematopoietic cell line FDC-P1 was also potently inhibited by TNF-alpha and was accompanied by down-regulation of c-kit cell surface expression as well as c-kit mRNA levels. Finally, treatment of the FDC-P1 cell line with TNF-alpha resulted in increased levels of the tumor suppressor p53 mRNA, suggesting another mechanism by which hematopoietic effects of TNF-alpha may be mediated.
Degradation of lamin B1 precedes oligonucleosomal DNA fragmentation in apoptotic thymocytes and isolated thymocyte nuclei.
Authors: Neamati N , Fernandez A , Wright S , Kiefer J , McConkey D J
Published: December 2022

Chromatin condensation and nuclear envelope breakdown are characteristic features of apoptotic cell death, but the mechanisms underlying these phenomena have not been identified. Solubilization of nuclear lamin is responsible for both events in mitosis. In this work, we report that glucocorticoids stimulate rapid degradation of lamin B1 that occurs before oligonucleosomal DNA fragmentation in apoptotic thymocytes. Protease inhibitors and the Ca2+ buffering agent BAPTA-AM block lamin degradation and DNA fragmentation, indicating that the processes are regulated by similar or identical mechanisms. Incubation of isolated thymocyte nuclei with Ca2+ stimulates lamin degradation before the detection of oligonucleosomal DNA fragments. However, in contrast to lamin dissolution during mitosis and some other forms of apoptosis, glucocorticoid-induced degradation of lamin B1 in thymocytes is not accompanied by dephosphorylation-mediated activation of cdc2. Our results demonstrate that lamin degradation is an early feature of apoptosis in thymocytes and suggest that chromatin condensation and breakdown of the nuclear envelope may occur as a result of disruption of nuclear lamina architecture.
Do HLA genes play a prominent role in determining T cell receptor V alpha segment usage in humans?
Authors: Gulwani-Akolkar B , Shi B , Akolkar P N , Ito K , Bias W B , Silver J
Published: December 2022

Previous studies in humans have demonstrated that HLA genes can profoundly influence the TCR V beta repertoire. To similarly assess the influence of HLA genes on the TCR V alpha segment repertoire, the V alpha repertoires of 12 individuals from three unrelated families were determined by quantitative PCR. Each family contained at least one pair of HLA-identical and -nonidentical siblings. Repertoire analysis was performed on purified CD4+ and CD8+ cells by using V alpha-specific primers. We were unable to demonstrate more similar V alpha repertoires between HLA-identical siblings than between HLA-nonidentical siblings. In contrast, when a similar analysis was performed on the same individuals for the V beta repertoire, HLA-identical siblings were found to have significantly more similar repertoires than HLA-nonidentical siblings. Furthermore, both the V alpha and V beta repertoires of monozygotic twins showed striking similarity. Despite our inability to show an influence of HLA genes on the V alpha repertoire, we did observe a very strong skewing in terms of preferential expression on CD4+ or CD8+ cells of several V alpha segments, notably TCRAV1, -2, -5, -6, -7, -11, -12, and -13. These studies suggest that HLA genes play less of a role in determining V alpha segment usage than V beta. Nevertheless, the pronounced skewing of V alpha segment expression in the CD4+ or CD8+ populations suggests some role for HLA genes in determining the V alpha TCR repertoire. Furthermore, the striking similarity of V alpha repertoires of identical twins suggests a major role for non-HLA genes in determining the V alpha repertoire.
Cell surface expression of class II MHC proteins bound by a single peptide.
Authors: Ignatowicz L , Winslow G , Bill J , Kappler J , Marrack P
Published: December 2022

On normal cells, the peptide-binding grooves of class II MHC proteins contain a wide spectrum of peptides. For some purposes, however, it would be helpful to have cells bearing class II proteins engaged by only one peptide species. In an attempt to make such cells we constructed a gene for a MHC class II beta-chain, IA beta b, covalently linked to a peptide, E alpha 52-68, which is known to bind to the peptide-binding groove of IAb. This gene, together with the gene for IA alpha b, was transfected into B lymphoma cells and fibroblasts. The IAb-E alpha complex was expressed on the surfaces of these cells where it could be recognized by a mAb and T cells specific for IAb plus E alpha 52-68. Most of the peptide on fibroblasts remained covalently attached to the IAb beta-chain, but the covalent linker and/or peptide were degraded to some extent on B lymphoma cells. Nearly all of the IAb expressed by transfected fibroblasts was occupied by the E alpha peptide. Of 16 IAb-reactive T cell hybridomas, only 3 could respond to the IAb-E alpha complex on fibroblasts, confirming the idea that recognition of MHC may often involve recognition of the peptides bound to the MHC as well.
Cloning and characterization of the gene for mouse macrophage migration inhibitory factor (MIF).
Authors: Mitchell R , Bacher M , Bernhagen J , Pushkarskaya T , Seldin M F , Bucala R
Published: December 2022

An emerging body of data indicates that the protein mediator described originally as macrophage migration inhibitory factor (MIF) exerts a central and wide ranging role in host inflammatory responses. MIF is a major constituent of corticotrophic cells within the anterior pituitary gland and is secreted into the circulation in a hormone-like fashion. MIF also exists performed in monocytes/macrophages and is a pivotal mediator in the host response to endotoxic shock. To gain further insight into the biologic expression of this protein that encompasses components of both the immune and the endocrine systems, we have cloned the mouse MIF gene and identified potential regulatory sequences present within the 5'-proximal promoter region. The gene for mouse MIF is located on chromosome 10, spans approximately 1 kb, and shares a high degree of structural homology with its human counterpart. Of note, the consensus enhancer/promoter motifs identified include both inflammatory/growth factor-related elements and sites associated with the genes for certain peptide hormones. We also report the structures of two MIF pseudogenes that account for early observations suggesting that mouse MIF is encoded by a highly homologous multigene family.
Structure of the MHC A and B locus promoters in hominoids. Insights on the evolution of the class I MHC multigene family.
Authors: Vallejo A N , Pease L R
Published: December 2022

The expansion and contraction of mammalian class I multigene families raises the issue as to what determines the loss or retention of family members. We propose that accumulating changes in regulatory regions result in the loss of expression of the gene products during times critical to selection, leading to the extinction of misregulated genes. The structures of promoter regions of MHC class I genes in nonhuman primates support this view. The B promoters are more homogeneous and contain regulatory elements also found in the promoters of the homologous class I genes of more distant mammals, whereas the A locus promoters were significantly more heterogeneous, have fewer sequence motifs related to known transcription factor-binding sites and have accumulated nucleotide substitutions within one of the widely conserved class I promoter elements. These findings are consistent with the view that the more polymorphic B locus is the principal MHC locus encoding functional class I Ag-presenting molecules whereas the less polymorphic A locus is assuming a secondary role as a consequence of promoter defects.
Importance of TNF production for the curative effectiveness of low dose melphalan therapy for mice bearing a large MOPC-315 tumor.
Authors: Gorelik L , Rubin M , Prokhorova A , Mokyr M B
Published: December 2022

We have previously shown the importance of the acquisition of CD8+ T cell-dependent tumor-eradicating immunity for the curative effectiveness of low dose melphalan (L-PAM, L-phenylalanine mustard) for mice bearing a large MOPC-315 tumor. Here we show the importance of TNF production for the curative effectiveness of low dose L-PAM for such tumor-bearing mice. Studies regarding the mechanism(s) through which TNF exerts its antitumor effects in L-PAM-treated MOPC-315 tumor-bearing mice (L-PAM TuB mice) revealed that MOPC-315 tumor cells are not sensitive to the cytotoxic effects of TNF either before or after the chemotherapy. However, TNF is essential for the in vitro generation of potent CTL activity by CD8+ T cells from L-PAM TuB mice. Moreover, addition of exogenous TNF to in vitro stimulation cultures of spleen cells from untreated mice bearing a large MOPC-315 tumor resulted in the generation of a greatly enhanced CTL activity against MOPC-315-associated Ags. Finally, we have previously shown that TCR V beta 8+/CD8+ T cells are involved in the curative effectiveness of low dose L-PAM for mice bearing a large MOPC-315 tumor. Here we show that TNF is important for the ability of MOPC-315-specific V beta 8+/CD8+ T cell lines from L-PAM TuB mice to bring about tumor eradication upon adoptive transfer into tumor-bearing mice. Taken together, these studies illustrate that low dose L-PAM mediates its therapeutic effectiveness for mice bearing a large MOPC-315 tumor, at least in part, via TNF production (e.g., by V beta 8+/CD8+ T cells), which in turn promotes the generation of anti-MOPC-315 CTL activity.
Neutrophil deactivation by influenza A virus. Role of hemagglutinin binding to specific sialic acid-bearing cellular proteins.
Authors: Hartshorn K L , Liou L S , White M R , Kazhdan M M , Tauber J L , Tauber A I
Published: December 2022

Bacterial superinfections are the most common cause of mortality during influenza epidemics. Depression of phagocyte functions by influenza A viruses (IAVs) is a likely contributory cause of such infections. We used an in vitro model of viral depression of neutrophil respiratory burst responses to FMLP and PMA to examine the mechanism of IAV-induced phagocyte deactivation. Respiratory burst responses or intracellular calcium mobilization were triggered by the virus itself, but these were not causally related to deactivation. By treating neutrophils with neuraminidase, and by use of purified IAV hemagglutinin (HA) preparations, cross-linking of sialic acid-bearing neutrophil surface components by the IAV HA was shown to be responsible for deactivation. IAV competed for binding to neutrophils with Abs directed against CD43, sialyl-Le(x), CD45, and gangliosides. Deactivation could be reproduced by treating neutrophils with anti-CD43 or -sialyl-Le(x) Abs in the absence of IAV. However, treatment of neutrophils with elastase markedly reduced CD43 expression, without affecting overall IAV binding or the ability of IAV to cause deactivation. Hence, although IAV binding to CD43 can account for deactivation, other IAV-binding proteins exist (e.g., those bearing sialyl-Le(x)) that can independently mediate functional depression.
Protection from lethal coronavirus infection by immunoglobulin fragments.
Authors: Lamarre A , Talbot P J
Published: December 2022

Molecular mechanisms of in vitro and in vivo virus neutralization by specific Ab remain largely undefined. Murine coronaviruses provide an excellent animal model for such studies. To determine the role of Ab bivalency and the contribution of its Fc portion in the neutralization of viral infectivity and passive protection of mice by an in vitro neutralizing and in vivo protective mAb (7-10A), F(ab')2 and Fab fragments were generated and their biologic properties were examined. The two fragments reacted in ELISA like the whole Ab against viral Ag or specific anti-idiotypic Abs. The affinity constants of the different Ab preparations were determined by surface plasmon resonance using immobilized anti-idiotypic Abs. The apparent affinity constant of the whole Ab molecule was 7.0 x 10(9) M-1 and was reduced 2-fold for F(ab')2 fragments and 14-fold for Fab molecules. Like whole Ab, both F(ab')2 and Fab fragments could neutralize virus in vitro and passively protect mice in vivo. However, the efficiency of in vivo neutralization by Fab fragments was reduced compared with the bivalent molecules, despite almost identical half-lives of both types of Ab fragments. These results demonstrate that in vitro and in vivo virus neutralization mechanisms by this Ab are independent of Fc-mediated functions and bivalency, but are probably influenced by Ab avidity. Also, this is the first report of in vivo protection against a viral infection by Fab fragments of antiviral Ab.
CTL specific for the tyrosinase autoantigen can be induced from healthy donor blood to lyse melanoma cells.
Authors: Visseren M J , van Elsas A , van der Voort E I , Ressing M E , Kast W M , Schrier P I , Melief C J
Published: December 2022

CTL that lyse melanoma cells were previously isolated from several melanoma patients. Such CTL recognize autologous proteins, indicating the occurrence of autoreactive T cells in melanoma patients. We have now raised CTL, using responding T lymphocytes from healthy donor blood, that lysed not only cells incubated with an HLA-A*0201-binding tyrosinase peptide but also melanoma cells endogenously processing and presenting the epitope. Our results suggest that autoreactive CTL precursors are present in healthy donor blood and can be activated in vitro with synthetic peptides presented on appropriate APCs. Therefore, tissue-specific, autoantigen-derived peptides might be useful in immunotherapy of both poorly and nonimmunogenic tumors.
IL-12 exacerbates rather than suppresses T helper 2-dependent pathology in the absence of endogenous IFN-gamma.
Authors: Wynn T A , Jankovic D , Hieny S , Zioncheck K , Jardieu P , Cheever A W , Sher A
Published: December 2022

To assess the role of IFN-gamma in the in vivo regulation of Th subset differentiation by IL-12, schistosome egg-induced Th2 responses and granuloma formation were studied in IFN-gamma knock-out (gamma KO) mice in which the absence of endogenous IFN-gamma is assured. Rather than suppressing pathology and eosinophilia as observed in wild-type animals, exogenous IL-12 in egg-injected gamma KO mice exacerbated Th2-dependent granuloma formation while failing to reduce peak tissue eosinophilia. Similarly, instead of inhibiting its production, IL-12 caused a dramatic increase in serum IgE levels in gamma KO animals after egg injection. Although the suppressive effects of IL-12 on Th2 responses were blocked in the absence of IFN-gamma, lymphocyte proliferation and IL-2 production were enhanced, a phenomenon which may underlie the observed exacerbation of egg-induced pathology. These findings formally establish that IL-12 inhibits Th2 development indirectly in vivo through the stimulation of IFN-gamma synthesis. In contrast, its promotion of Th1-associated responses seems to be at least partly a result of the direct action of the cytokine.
Protective CTL-dependent immunity and enhanced immunopathology in mice immunized by particle bombardment with DNA encoding an internal virion protein.
Authors: Zarozinski C C , Fynan E F , Selin L K , Robinson H L , Welsh R M
Published: December 2022

Anti-viral CTL were induced in vitro using a particle bombardment device or "gene-gun" to deliver plasmid DNA encoding the nucleoprotein of the lymphocytic choriomeningitis virus (LCMV). Using this plasmid we were able to study T cell-mediated immunity in the absence of a neutralizing Ab response. Upon a single DNA immunization, a nearly 2 log10 reduction in splenic viral titers was observed 3 days after LCMV infection. After two or three immunizations a greater than 3 log10 inhibition of viral titers in the spleen was observed, with most animals having no detectable virus. C57BL/6 mice immunized with DNA encoding the nucleoprotein gene were also challenged with LCMV intracranially. Upon intracranial challenge, vaccinated animals displayed either protection or enhanced immunopathology leading to accelerated kinetics of death. Using limiting dilution analysis it was possible to detect LCMV-specific CTL precursors in both the spleen and lymph nodes of vaccinated animals. C57BL/6 mice inoculated with DNA demonstrated an anamnestic CTL response detectable at day 4 after LCMV challenge. Thus DNA vaccines are capable of inducing an anti-viral T cell response that can inhibit viral replication and mediate either protective immunity or immunopathology. Vaccination with DNA may therefore provide a useful alternative to current viral or subunit vaccines once the efficacy of immunization with DNA is optimized.
Mobilization of granules and secretory vesicles during in vivo exudation of human neutrophils.
Authors: Sengeløv H , Follin P , Kjeldsen L , Lollike K , Dahlgren C , Borregaard N
Published: December 2022

The extent of mobilization of four different intracellular compartments was measured during in vivo exudation of neutrophils into skin chambers and compared with resting neutrophils obtained from blood. Exudation of neutrophils induced increased surface expression of alkaline phosphatase, complement receptor 1, and Mac-1, and a complete loss of L-selectin. The increase in the content of surface molecules in the plasma membrane is in accordance with complete mobilization of secretory vesicles. Granule matrix proteins were secreted into the chamber fluid by the exudated neutrophils and the exocytosed proteins were recovered in the skin chamber fluid. Release of gelatinase from gelatinase granules was 38.1%, lactoferrin release from specific granules was 21.9%, and myeloperoxidase release from azurophil granules was 7.0%, clearly illustrating a hierarchy in mobilization among granules. When exudate neutrophils were stimulated with FMLP, additional mobilization of granules was observed and the rank order regarding release was preserved. This is the first report to evaluate the mobilization of secretory vesicles during in vivo exudation of human neutrophils. It is shown that secretory vesicles are regulated exocytotic vesicles that are fully mobilized during in vivo exudation. Once exocytosed, secretory vesicles are not re-formed within a period of 6 h.
Human IL-17: a novel cytokine derived from T cells.
Authors: Yao Z , Painter S L , Fanslow W C , Ulrich D , Macduff B M , Spriggs M K , Armitage R J
Published: December 2022

A cDNA encoding human IL-17 (hIL-17) was cloned from a CD4+ T cell library. The predicted 155-amino acids sequence contains an N-terminal signal peptide and exhibits 72% amino acid identity with HVS13, an open reading frame from a T-lymphotropic Herpesvirus saimiri, and 63% with murine CTLA8. High levels of hIL-17 were induced from primary peripheral blood CD4+ T cells upon stimulation. When expressed in CV1/EBNA cells, recombinant hIL-17 was secreted in both glycosylated and nonglycosylated forms. A hIL-17.Fc fusion protein and supernatants from cells transfected with hIL-17 induced IL-6 and IL-8 production and enhanced the surface expression of the intracellular adhesion molecule-1 (ICAM-1) in human fibroblasts.
Natural killer cell ontogeny in the athymic rat. Relationship between functional maturation and acquired resistance to E1A oncogene-expressing sarcoma cells.
Authors: Cook J L , Iklé D N , Routes B A
Published: December 2022

Newborn nude rats are NK cell deficient and highly susceptible to tumor induction by NK-susceptible, E1A oncogene-expressing, BHK-21 sarcoma cells, whereas adults have normal NK activity and are tumor resistant. This model of NK cell-related tumor rejection was used to characterize age-related functional maturation of NK cells during nude rat ontogeny. Small numbers of cytolytic incompetent, NKR-P1+ cells were detected in the spleens of newborns. During the first month of life, there was an age-related increase in the numbers of splenic NK cells, followed by a stepwise maturation in their cytolytic competence. Three functional stages in NK cell cytolytic activity were apparent. A nonlytic newborn stage was followed by a transitional stage, during which absent or relatively weak spontaneous cytolytic activity was augmented by prolonged NK cell exposure to susceptible target cells. In the final stage, both spontaneous and augmented cytolytic activity increased toward the adult level that was attained by 4 wk of age. The data suggest the existence of a transient stage in NK cell ontogeny when triggering through the NKR-P1 molecule is functionally immature, and confirm the importance of a cytolytic competent, NK cell response for nude rat rejection of E1A oncogene-expressing tumor cells.
Tumor-associated E6 protein of human papillomavirus type 16 contains an unusual H-2Kb-restricted cytotoxic T cell epitope.
Authors: Gao L , Walter J , Travers P , Stauss H , Chain B M
Published: December 2022

We previously showed that CTL from H-2b mice immunized against the E6 protein of human papilloma virus (HPV) type 16 recognized a 10-mer peptide corresponding to amino acids 131 to 140. We show in this study that the minimal epitope, 130 to 137, is a 8-mer peptide presented by H-2Kb class I molecules. At position P8, the 130 to 137 peptide contains a hydrophobic methionine anchor residue, but P3 and P5 do not contain the typical anchor residues that are frequently found in Kb-bound peptides. Analysis with alanine-substituted peptides indicates that the tryptophan at P3 acts as an alternative anchor mediating Kb binding, while an arginine at P2 is a TCR contact residue. Synthetic 9-mer peptides corresponding to residues 130 to 138 are as efficiently recognized by CTL as 130 to 137 peptides. Analysis of extracts of E6-expressing cells suggests that Ag processing may produce multiple peptides containing the minimal 130 to 137 epitope. In vitro binding studies indicate that Kb binding of peptide 130 to 137 is approximately five orders of magnitude less efficient than Kb binding of previously identified CTL epitopes. In contrast, the E6 protein contains another potential CTL epitope in the region of amino acids 41 to 50. A synthetic peptide spanning this region binds very strongly to Kb and is capable of stimulating a strong peptide-specific CTL response. In the context of the whole protein, however, this epitope remains cryptic.
Surrogate light chain-dependent selection of Ig heavy chain V regions.
Authors: Keyna U , Beck-Engeser G B , Jongstra J , Applequist S E , Jäck H M
Published: December 2022

Bone marrow B cell precursors frequently rearrange the Ig heavy chain variable (VH) gene segment VH81X. It is puzzling, therefore, that mature B cells in adult mice rarely express mu-heavy chains bearing this VH gene segment. We show in this work in transformed pre-B cell lines that two VH81X/mu-chains that differ in their VH-D-JH joining sequences are not assembled covalently with the B cell precursor-specific surrogate light (SL) chain and are not expressed on the cell surface. From these findings, we propose that a B cell precursor clonally expands and proceeds to the next developmental stage only if it expresses a mu-chain with a VH domain that, together with the SL chain, directs the formation of a signal-transducing mu/SL chain membrane complex. Therefore, a checkpoint exists early in B cell development, at which SL chain not only screens B cell precursors for the presence of a full-length mu-chain, but also for a VH domain that promotes the assembly of a mu/SL chain complex.
Breaking immunologic ignorance to an antigenic peptide of simian virus 40 large T antigen.
Authors: Soldevila G , Geiger T , Flavell R A
Published: December 2022

Expression of antigenic proteins in the periphery may result in immunologic tolerance, immunologic ignorance, or autoimmunity. It is not clear why some Ags induce tolerance, whereas others activate Ag-reactive T cells. The physical nature of the Ag, the developmental timing of Ag expression, the priming of reactive lymphocytes, and the level of Ag expression are possible factors determining the immunologic response to extra-thymic Ags. Expression of the whole SV40 large T Ag (SV40-T) induces transformation of T antigen-expressing cells in vivo, and this phenomenon has been postulated to be the triggering event that leads to autoimmunity in some transgenic mouse models. Here we present a model in which a nononcogenic, yet antigenic, fragment of the SV40-T (SV40-Tfrag) is expressed specifically in pancreatic islet beta-cells. In contrast to whole SV40-T transgenic mice, SV40-Tfrag mice that are also transgenic for a TCR specific for the SV40-T are ignorant of Ag in vivo. They do not respond in vivo to the tissue-specific SV40-Tfrag Ag even after priming, but are fully responsive in vitro. This immunologic ignorance cannot be broken after activated SV40-T reactive T cells are transferred into sublethally irradiated mice expressing the islet-specific SV40-Tfrag. However, similar adoptive transfer experiments in mice co-expressing B7-1 and SV40-Tfrag on islet cells specifically lead to Ag recognition and diabetes. This demonstrates that in some circumstances the presence of (primed) reactive T cells is not sufficient to break tolerance; rather, costimulation is additionally required to elicit an autoimmune response. This also suggests that SV40-T-induced cellular transformation is important for the autoimmune response directed against SV40-T in other tissue-specific transgenic models.
In vivo association between p56lck and MAP kinase during IL-2-mediated lymphocyte proliferation.
Authors: Taieb J , Blanchard D A , Auffredou M T , Chaouchi N , Vazquez A
Published: December 2022

We previously reported that p56lck expression is upregulated in human B lymphocytes upon mitogenic stimulation. In this report, we characterized the molecules associated with p56lck in vivo in leukemic B cells costimulated with anti-mu Ab and IL-2 for 72 h. In vitro phosphorylation after p56lck immunoprecipitation indicated that p56lck is associated in vivo with the beta chain of the IL-2 receptor and p42 MAP kinase as well as a number of other proteins. Moreover, p56lck-associated MAP kinase is tyrosine and threonine phosphorylated, suggesting that it is activated. Prevention of DNA synthesis with aphidicolin abrogated this molecular association, and furthermore, cell cycle analysis with IL-2-dependent T cells showed that in cells in G1, MAP kinase was not associated to p56lck, whereas this p56lck-MAP kinase association was observed when cells are in S phase. Thus, p56lck and MAP kinase are only associated during S phase. These data suggest that MAP kinase in association with p56lck is directly involved in the control of IL-2-mediated DNA synthesis of both B and T lymphocytes.
IL-12 promotes enhanced recovery from vesicular stomatitis virus infection of the central nervous system.
Authors: Bi Z , Quandt P , Komatsu T , Barna M , Reiss C S
Published: December 2022

To investigate the role of a cytokine in host defense against the vesicular stomatitis virus (VSV) infection of the central nervous system (CNS), IL-12 was injected i.p. into groups of 10 BALB/c mice on days -1, 0, 1, 2, and 3 postinfection. Four days postinfection, mice were examined. IL-12 strongly enhanced immunity to VSV infection in the CNS as demonstrated by 1) decreased VSV titers in brain homogenate of IL-12-injected mice compared with those of controls; 2) increased expression of inducible nitric oxide synthase in the CNS; 3) enhanced expression of both MHC class I and class II Ags in the CNS; 4) increased T cell infiltration in the CNS, especially in the olfactory bulb; and 5) diminished VSV-induced apoptosis in olfactory bulb. No detrimental effect was observed even with the 200 ng/mouse dose of IL-12. Protective effects of IL-12 were dose dependent. Collectively, these results demonstrate that exogenously added IL-12, even when injected peripherally, significantly enhances recovery from VSV infection of the CNS.
Impact of interferon-gamma receptor deficiency on experimental Staphylococcus aureus septicemia and arthritis.
Authors: Zhao Y X , Tarkowski A
Published: December 2022

The role of IFN-gamma in the regulation of host resistance of Staphylococcus aureus was studied using IFN-gamma receptor-deficient (IFN-gamma R-/-) mice in a model of S. aureus-induced septicemia and arthritis. IFN-gamma R-/- mice and wild-type controls were inoculated intravenously with a toxic shock syndrome toxin-1-producing S. aureus LS-1 strain. IFN-gamma R-/- mice displayed significantly more frequent and more severe arthritis compared with wild-type littermates (p < 0.01) throughout the course of infection. Notably, IFN-gamma R-/- mice developed severe sepsis with high mortality early after the inoculation with staphylococci. However, the mortality of wild-type mice became significantly higher at later stages of the disease compared with IFN-gamma R-/- mice (p < 0.05). This differential outcome of sepsis-related mortality was associated with deficiencies of bacterial elimination from blood and parenchymatous organs and correlated well to serum levels of IL-6 and spleen IL-1 beta and TNF-beta mRNA expression. Thus, bacterial growth and proinflammatory cytokines IL-1 beta, TNF-beta, and IL-6 were higher at the early stage of infection in IFN-gamma-/- mice but increased at the later stage in wild-type littermates. Our data indicate that the absence of IFN-gamma R leads to harmful as well as beneficial effects in S. aureus infection, depending on the stage of the disease and the localization of the infection.
IL-10 is involved in the protective effect of dibutyryl cyclic adenosine monophosphate on endotoxin-induced inflammatory liver injury.
Authors: Arai T , Hiromatsu K , Kobayashi N , Takano M , Ishida H , Nimura Y , Yoshikai Y
Published: December 2022

The effects of exogenous cAMP, dibutyryl cAMP (DBcAMP) on LPS-induced liver injury were examined in mice made hypersensitive to LPS by treatment with i.v. injection of Propionibacterium acnes. In vivo administration of DBcAMP significantly protected P. acnes-treated mice from LPS-induced liver injury, including apoptosis of hepatocytes. DBcAMP significantly increased circulating IL-10 level in correlation with suppression of the TNF-alpha level after LPS challenge in P. acnes-treated mice. Treatment with anti-IL-10 mAb abrogated the protective effect of DBcAMP on LPS-induced liver injury. Similar to in vivo findings, addition to DBcAMP to in vitro culture of liver adherent cells from P. acnes-treated mice enhanced IL-10 synthesis after LPS stimulation. These results suggest that the increment in IL-10 production by liver adherent cells is involved in the protective effect of DBcAMP on LPS-induced inflammatory liver injury.
Subcellular distribution of docking/fusion proteins in neutrophils, secretory cells with multiple exocytic compartments.
Authors: Brumell J H , Volchuk A , Sengelov H , Borregaard N , Cieutat A M , Bainton D F , Grinstein S , Klip A
Published: December 2022

Neutrophils contain at least four distinct types of secretory organelles, which undergo exocytosis during infection and inflammation. The signaling pathways leading to secretion of individual granules and their kinetics of exocytosis vary greatly, causing temporal and regional differences in docking and fusion with the plasma membrane. As a step toward understanding the processes underlying differential granular secretion in neutrophils, we assessed the presence and distribution of a number of proteins reported to be involved in vesicular docking and/or fusion in other systems. Specific Abs were used for immunoblotting of cells fractionated by density gradients and free-flow electrophoresis, and for localization by confocal immunofluorescence and electron microscopy. Syntaxin 1, VAMP (vesicle-associated membrane protein)-1, synaptosome-associated protein-25 (SNAP-25), synaptophysin, and cellubrevin were not detectable in human neutrophils. In contrast, syntaxin 4, VAMP-2, and the 39-kDa isoform of secretory carrier membrane protein (SCAMP) were present. SCAMP was found mainly in secondary and tertiary granules and in a fraction containing secretory vesicles, but was virtually absent from the primary (lysosomal) granules. This profile is consistent with the proposed "post-Golgi" distribution of SCAMP. VAMP-2 was largely absent from primary and secondary granules, but concentrated in tertiary granules and secretory vesicles. This pattern of distribution parallels the increasing sensitivity of these exocytic compartments to intracellular free calcium. Accordingly, ionomycin induced translocation of VAMP-2 toward the plasma membrane. Syntaxin 4 was found almost exclusively in the plasma membrane, and it accumulated in lamellipodia of migrating cells. This regional accumulation may contribute to localized secretion into the phagosomal lumen.
Characterization of mutant forms of recombinant human properdin lacking single thrombospondin type I repeats. Identification of modules important for function.
Authors: Higgins J M , Wiedemann H , Timpl R , Reid K B
Published: December 2022

Properdin is a serum glycoprotein that up-regulates the alternative pathway of complement by stabilizing the C3b-Bb complex. It also binds sulfated glycoconjugates, such as sulfatide, in vitro. Properdin is composed of cyclic dimers, trimers, and tetramers of a 53-kDa monomeric subunit. The monomer contains an N-terminal region of no known homology and six thrombospondin type 1 repeats (TSRs) of approximately 60 amino acids. To identify the regions of properdin important for function, we have expressed human properdin, and mutant forms each lacking a single TSR, in Chinese hamster ovary cells. In addition, limited tryptic digestion yielded "nicked" properdin by the cleavage of one peptide bond in TSR5. The structural and functional properties of these altered forms of properdin were investigated. Properdin "nicked" in TSR5 is unable to bind C3b but retains its overall structure and its ability to bind sulfatide. The removal of TSR5 prevents C3b and sulfatide binding. Properdin lacking TSR4 is unable to stabilize the C3b-Bb complex but is able to bind C3b and sulfatide, and shows the presence of monomers and dimers in an electron microscope. Properdin without TSR3 is able to stabilize the C3b-Bb complex, to bind C3b and sulfatide, and forms dimers, trimers, and tetramers. Properdin lacking TSR6 is unable to form oligomers. The N-linked carbohydrate of properdin is not required for oligomerization or stabilization of the C3b-Bb complex. The results implicate TSR5 in both C3b and sulfatide binding, and suggest that TSR4 may also be involved in stabilization of the C3b-Bb complex.