bluuebunny/crossref_metadata_2025 huggingface dataset:
Synergistic Activation of the Germline ε Promoter Mediated by Stat6 and C/EBPβ
Authors: Mikita Thomas , Kurama Masae , Schindler Ulrike
Published: December 2022

Transcription of the Ig H chain germline transcripts is a prerequisite for class switching. Expression of the ε germline transcript is induced by IL-4 and requires the integrity of a composite IL-4 response element. The element is bound by the IL-4-inducible transcription factor Stat6 and one or more members of the CAAT/enhancer-binding protein (C/EBP) family, a constitutively expressed class of transcription factors. Here, we show that Stat6 and C/EBPβ cooperate to synergistically activate transcription from the ε element. The effect was most pronounced in lymphoid cells, and the activation domains of both proteins were required to achieve this synergy. Although other members of the C/EBP family are able to bind the element, very little cooperativity was seen with C/EBPα and none with C/EBPγ. In fact, C/EBPγ was able to inhibit IL-4-induced reporter activity. Stat6 and C/EBPβ bind the IL-4 response element simultaneously. The fast dissociation rate apparent when Stat6 binds this DNA element alone is slowed when C/EBPβ binds at the neighboring site. These data suggest a mechanism whereby C/EBPβ stabilizes Stat6 binding at this element, thereby increasing the likelihood that both of their activation domains will interact, possibly with other factors, to activate transcription in an IL-4-dependent manner.
Fc Receptors Are Not Required for Antibody-Mediated Protection Against Lethal Malaria Challenge in a Mouse Model
Authors: Rotman Harris L. , Daly Thomas M. , Clynes Raphael , Long Carole A.
Published: December 2022

The mechanisms by which Abs mediate protection during blood-stage malaria infections is controversial, with some evidence pointing to the direct effect of Abs on parasite invasion and growth, while other studies suggest that Abs act in cooperation with monocytes to achieve parasite inhibition. To determine whether the effector phase of protection in vivo to the rodent parasite Plasmodium yoelii yoelii requires Fc receptor bearing cells, we passively transferred immune sera into FcR γ-chain knockout mice. Inflammatory macrophages from these knockout mice were unable to mediate phagocytosis or Ab-dependent cell-mediated cytotoxicity (ADCC) through FcγRI, FcγRII, or FcγRIII. Passive transfer of either P. y. yoelii hyperimmune sera or anti-GST-PYC2 sera directed to the major merozoite surface protein (MSP-1) of this parasite enabled both BALB/cByJ mice and FcR γ-chain-deficient mice to resist lethal P. y. yoelii 17XL (Py17XL) challenge. mAb302, a protective IgG3 Ab, also passively protected both strains of mice. Most of these samples contain Ab isotypes that would not be able to protect mice if their protective effects required Ab-dependent cell-mediated cytotoxicity. These results establish that, in this infection, protection is directly mediated by Abs and does not require the participation of Fc receptors.
IFNs Are Critical Regulators of IL-1 Receptor Antagonist and IL-1 Expression in Human Microglia
Authors: Liu Judy S. H. , Amaral Terry D. , Brosnan Celia F. , Lee Sunhee C.
Published: December 2022

Because IL-1 is implicated in the pathogenesis of multiple sclerosis, and IFNs are known to alter disease course, we sought to determine whether IFNs can regulate the expression of IL-1 and IL-1R antagonist (IL-1Ra) in primary cultures of human microglia and astrocytes. We found that IL-1 and IL-1Ra are products of microglia but not astrocytes, and IFN-β and IFN-γ differentially modulate LPS- and cytokine-induced IL-1 and IL-1Ra. IFN-β induces IL-1Ra and augments LPS- and IL-4-induced IL-1Ra, but suppresses LPS- and IL-1-induced IL-1, shifting the balance toward the expression of the IL-1Ra. Like IFN-β, IFN-γ suppresses the expression of both LPS and IL-1-induced IL-1β. However, IFN-γ also suppresses the expression of IFN-β- and IL-4-induced IL-1Ra so that IFN-γ may enhance or suppress IL-1 activity depending on the other cytokines present. IL-4 has similar effects to IFN-β; however, other anti-inflammatory cytokines, did not regulate IL-1 or IL-1Ra in human microglia. Our data demonstrate a novel suppressive effect of IFN-β and IL-4 on IL-1 activity in human microglia, suggesting that IFN-β, a therapeutic agent used for multiple sclerosis, could have wider applications in the treatment of other central nervous system disorders in which IL-1 activity has been implicated in the pathogenesis.
Molecular Analysis of Polyreactive Monoclonal Antibodies from Rheumatic Carditis: Human Anti-N-Acetylglucosamine/Anti-Myosin Antibody V Region Genes
Authors: Adderson Elisabeth E. , Shikhman Alexander R. , Ward Kent E. , Cunningham Madeleine W.
Published: December 2022

Anti-myosin Abs are associated with inflammatory heart diseases such as rheumatic carditis and myocarditis. In this study, human cross-reactive anti-streptococcal/anti-myosin mAbs 1.C8, 1.H9, 5.G3, and 3.B6, produced from peripheral blood lymphocytes of patients with rheumatic carditis, and mAb 10.2.5, produced from a tonsil, were characterized, and the nucleotide sequences of their VH and VL genes were analyzed. Human mAbs 1.C8, 1.H9, 10.2.5, and 3.B6 reacted with human cardiac myosin while mAb 5.G3 did not. The mAbs were strongly reactive with N-acetyl-β-d-glucosamine, the dominant epitope of the group A streptococcal carbohydrate. mAb 1.H9 was moderately cytotoxic to rat heart cells in vitro in the presence of complement. The anti-myosin mAbs from rheumatic carditis were found to react with specific peptides from the light meromyosin region of the human cardiac myosin molecule. Anti-streptococcal/anti-myosin mAbs from normal individuals reacted with distinctly different light meromyosin peptides. The mAbs were encoded by VH3 gene segments V3-8, V3-23, and V3-30 and by the VH4 gene segment V4-59. The variable region genes encoding the anti-streptococcal/anti-myosin repertoire were heterogeneous and exhibited little evidence of Ag-driven somatic mutation.
Induction of Species-Specific Host Accommodation in the Hamster-to-Rat Xenotransplantation Model
Authors: Yin Dengping , Ma Lian Li , Blinder Leonard , Shen JiKun , Sankary Howard , Williams James W. , Chong Anita S.-F.
Published: December 2022

The combination of two immunosuppressants, leflunomide and cyclosporin A (CsA), completely inhibits immune xenoreactions in the hamster-to-Lewis rat xenotransplantation model. In addition, the control of acute xenograft rejection with this combination of immunosuppressants subdues early T-independent xenoreactivity and uncovers a late immune response that can be controlled by CsA alone. We attribute this acquired responsiveness to CsA to a modification in the recipient’s humoral response to the xenograft, and refer to this change as host accommodation. Host accommodation can be induced in Lewis rats receiving hamster hearts by the combination of leflunomide and CsA. A 7-day treatment with leflunomide and CsA was able to convert xenoreactivity from one that was resistant to CsA treatment into one that was controlled by CsA. The presence of the hamster xenograft was critical for the induction of host accommodation since the immunosuppressive regimen, either alone or in combination with a transfusion with donor-specific spleen cells, was unable to modify the anti-hamster reactivity in Lewis rats. When accommodation was induced in the presence of hamster hearts, these accommodated rats were able to acutely reject third-party mouse hearts while under CsA therapy, thus indicating that the host accommodation is species specific. Finally, we demonstrate that host accommodation is associated with a loss in the ability to produce species-specific, T-independent xenoantibodies. These novel observations suggest that xenoreactive T-independent humoral responses can be deleted selectively without significant loss of other innate, Ag-specific T-independent humoral responses.
EAE TCR Motifs and Antigen Recognition in Myelin Basic Protein-Induced Anterior Uveitis in Lewis Rats
Authors: Buenafe Abigail C. , Offner Halina , Machnicki Michael , Elerding Heather , Adlard Kirsten , Jacobs Ray , Vandenbark Arthur A. , Adamus Grazyna
Published: December 2022

T cells infiltrating the iris/ciliary body of Lewis rats with anterior uveitis (AU) that had been induced by myelin basic protein (MBP) immunization were previously found to share surface markers common to the T cells that cause experimental autoimmune encephalomyelitis (EAE). To determine whether these AU-associated T cells are in fact the same as those that infiltrate the central nervous system to cause EAE, we examined TCR V gene expression in T cells infiltrating the anterior chamber in rats with AU. As with EAE, we found a biased expression of Vβ8.2 and Vα2 in the iris/ciliary body and, although one would expect an influx of nonspecific inflammatory T cells, these biases were still evident at the peak of AU. An analysis of the TCR Vβ8.2 and Vα2 sequences derived from the iris/ciliary body demonstrated the presence of the same complementarity determining region 3 motifs found in MBP-specific T cells that are pathogenic for EAE and found in T cells derived from the central nervous system of rats with EAE. Finally, T cells isolated from the iris/ciliary body of rats with AU were found to proliferate in a specific fashion to MBP Ags. Thus, it appears that MBP-specific T cells are pathogenic for AU as well as EAE in the Lewis rat. In addition, the long-term presence of this highly restricted MBP response in the iris/ciliary body indicates that distinct immunoregulatory mechanisms exist in the environment of the eye. This provides an interesting model with which to address questions pertaining to the nature of T cells infiltrating the eye and their regulation during EAE and other systemic diseases.
Constitutive IL-10 Production Accounts for the High NK Sensitivity, Low MHC Class I Expression, and Poor Transporter Associated with Antigen Processing (TAP)-1/2 Function in the Prototype NK Target YAC-1
Authors: Petersson Max , Charo Jehad , Salazar-Onfray Flavio , Noffz Gabriele , Mohaupt Mariette , Qin Zhihai , Klein George , Blankenstein Thomas , Kiessling Rolf
Published: December 2022

Tumor cells that are treated with rIL-10 or transfected with the IL-10 gene show phenotypic changes. These include low but peptide-inducible expression of MHC class I, low sensitivity to specific CTL-mediated lysis, and increased NK sensitivity. In vitro-established mouse tumor lines were screened for IL-10 expression and production, and a large proportion of plasmocytomas or T cell lymphomas were found to produce IL-10. Since one of these lines was the prototype NK target cell YAC-1, we investigated whether the high IL-10 production of this cell line was related to its high NK sensitivity and its defects in MHC class I expression. The decrease in H-2 expression following the in vitro culture of in vivo-passaged YAC-1 cells was accompanied by a gradual increase in IL-10 production, whereas the reverse was found when passing in vitro-grown YAC-1 in vivo as an ascites tumor in syngenic mice. In addition, differences in YAC-1 MHC class I expression correlated with alterations in the functional activity of TAP-1/2 proteins. YAC-1 cells that were transduced with a retroviral IL-10 antisense construct (Y-IL-10 AS) only produced about half of the IL-10 that was produced by YAC-1 transduced with the control construct (Y-IL-10 Mock). Relative to Y-IL-10 Mock cells, the expression of H-2 on Y-IL-10 AS cells was markedly increased, and NK sensitivity was decreased. These data argue for a mechanism wherein IL-10 production is causally related to the low H-2 expression, decreased TAP function, and high NK sensitivity of YAC-1 cells.
Switch of CD4+ T Cell Differentiation from Th2 to Th1 by Treatment with Cathepsin B Inhibitor in Experimental Leishmaniasis
Authors: Maekawa Yoichi , Himeno Kunisuke , Ishikawa Hiroyuki , Hisaeda Hajime , Sakai Tohru , Dainichi Teruki , Asao Tetsuji , Good Robert A , Katunuma Nobuhiko
Published: December 2022

When activated, CD4+ T helper cells differentiate functionally into one of two subsets, Th1 or Th2. Before the Th differentiation, Ags must be processed into peptide epitopes and presented to CD4+ T cells in association with MHC class II molecules. However, the proteases responsible for this Ag processing have not been well defined. When BALB/c mice susceptible to infection with Leishmania major were treated with a specific inhibitor (CA074) of cathepsin B, a lysosomal cysteine protease that digests exogenous antigenic proteins, those mice acquired resistance against infection with L. major and showed the shift of immune responses from Th2 to Th1; that is, they produced specific IgG2a Ab and generated IFN-γ in contrast to untreated and infected mice that produced IgG1 and IgE and generated IL-4. CA074 interfered with the digestion of L. major Ags with lysosomal enzymes in vivo as well as in vitro. However, this inhibitor did not show any direct influence on the growth of L. major and the functions of T cells stimulated with anti-CD3 Ab. These findings indicate that cathepsin B inhibitor could switch CD4+ T cell differentiation from Th2 to Th1, suggesting that the alteration in Ag processing modulates the polarity of Th differentiation.
Autoimmunity Without Diabetes in Transgenic Mice Expressing β Cell-Specific CD86, But Not CD80: Parameters that Trigger Progression to Diabetes
Authors: Guerder Sylvie , Eynon Elizabeth E. , Flavell Richard A.
Published: December 2022

To define more clearly the roles of CD80 (RIP-CD80) and CD86 (RIP-CD86) in the activation of autoreactive T cells in vivo, we generated transgenic mice expressing either or both costimulatory molecules on the β cells of the pancreas. While RIP-CD80 mice do not show any sign of autoimmunity, at the age of 7 mo RIP-CD86 transgenic mice develop a lymphoid infiltrate with both IFN-γ- and IL-4-positive cells in the vicinity of the islets; these mice, however, never progress to diabetes. This fundamental difference in the ability of CD80 and CD86 to activate self-reactive T cells in vivo is, however, obliterated when the level of TCR signaling is increased by either TNF-α or transgenic MHC class II expression. These results support the suggestion that CD80 and CD86 mainly differ at the level of the intensity of the signals they deliver.
Natural Killer Cell Lysis of Cytomegalovirus (CMV)-Infected Cells Correlates with Virally Induced Changes in Cell Surface Lymphocyte Function-Associated Antigen-3 (LFA-3) Expression and Not with the CMV-Induced Down-Regulation of Cell Surface Class I HLA
Authors: Fletcher Jean M. , Prentice H. Grant , Grundy Jane E.
Published: December 2022

CMV and other viruses down-regulate the cell surface expression of class I HLA, and while this allows them to evade CTL, it may make infected cells more susceptible to lysis by NK cells, due to the failure to engage class I inhibitory receptors on the NK cell. We studied CMV infection and found that fibroblasts infected with virus strains Towne, Toledo, Davis, and C1FE were refractory to NK lysis, while those infected with strains AD169, C1F, or R7 were susceptible. All viral strains down-regulated class I HLA to a similar extent, and we concluded that there was no evidence for any correlation between the latter and susceptibility to NK lysis. In contrast, there was a strong correlation between NK killing of CMV-infected cells and cell surface levels of lymphocyte function-associated antigen-3 (LFA-3). Fibroblasts infected with the Towne, Toledo, Davis, and C1FE strains of CMV down-regulated LFA-3 expression and were refractory to lysis, while strains AD169, C1F, and R7 up-regulated LFA-3 and were susceptible to NK killing. U373 MG (malignant glioma) cells expressed constitutively high levels of LFA-3 and were sensitive to NK lysis when infected with any of the above-listed CMV strains. We estimated that a minimum of between 29,000 and 71,000 LFA-3 molecules per target cell were needed for NK susceptibility. The effects on LFA-3 expression were due to immediate early/early viral gene products. We also demonstrated that fibroblasts infected with the strains Towne, Toledo, Davis, and C1FE expressed a ganciclovir-sensitive late CMV gene product, which delivered an inhibitory signal to NK cells.
The Roles of L-Selectin, β7 Integrins, and P-Selectin in Leukocyte Rolling and Adhesion in High Endothelial Venules of Peyer’s Patches
Authors: Kunkel Eric J. , Ramos Carroll L. , Steeber Douglas A. , Müller Werner , Wagner Norbert , Tedder Thomas F. , Ley Klaus
Published: December 2022

Lymphocyte trafficking into Peyer’s patches requires β7 integrins and L-selectin. Here, we use intravital microscopy to examine leukocyte rolling and adhesion in Peyer’s patch high endothelial venules (HEV) of wild-type, L-selectin-deficient (L−/−), β7 integrin-deficient (β7−/−), and β7/L−/− mice. Although the leukocyte rolling flux fraction was reduced by 70%, Peyer’s patches in L−/− mice were of normal size and cellularity. In β7−/− mice, the rolling flux fraction was normal, but the number of adherent leukocytes in HEV was greatly reduced. The median leukocyte rolling velocity was reduced in L−/− mice and increased in β7−/− mice, suggesting that β7 integrins and L-selectin mediate rolling in Peyer’s patch HEV at different velocities. β7/L−/− exhibited both a low rolling flux fraction and low adhesion and had severely reduced Peyer’s patch size and cellularity. The residual rolling in these mice was completely blocked by a P-selectin mAb. A significant P-selectin component was also detected in the other genotypes. Twenty-six percent of B and T lymphocytes isolated from Peyer’s patches of wild-type mice expressed functional ligands for P-selectin, and this fraction was increased to 57% in β7/L−/− mice. Peyer’s patch HEV were found to express P-selectin under the conditions of intravital microscopy, but not in situ. Our data suggest a novel P-selectin dependent mechanism of lymphocyte homing to Peyer’s patches. In situ, β7 integrins and L-selectin account for all lymphocyte homing to Peyer’s patches, but P-selectin-dependent rolling, as induced by minimal trauma, may support trafficking of effector T lymphocytes to Peyer’s patches.
IL-2 Induces T Cell Adherence to Extracellular Matrix: Inhibition of Adherence and Migration by IL-2 Peptides Generated by Leukocyte Elastase
Authors: Ariel Amiram , Yavin Eran J. , Hershkoviz Rami , Avron Ann , Franitza Suzanne , Hardan Izhar , Cahalon Liora , Fridkin Mati , Lider Ofer
Published: December 2022

Migration of inflammatory cells requires cell adhesion and their subsequent detachment from the extracellular matrix (ECM). Leukocyte activation and migration must be terminated to stop inflammation. Here, we report that IL-2 enhances human T cell adherence to laminin, collagen type IV, and fibronectin (FN). In contrast, neutrophil elastase, an enzyme activated during inflammation, degrades IL-2 to yield IL-2 fractions that inhibit IL-2-induced T cell adhesion to FN. The amino acid composition of two of these IL-2 fractions, which appear to block T cell adherence to FN, were analyzed, and three peptides were consequently synthesized. The three peptides IVL, RMLT, and EFLNRWIT, but not the corresponding inversely synthesized peptides, inhibited T cell adhesion to FN induced by a variety of activators: IL-2, IL-7, macrophage inflammatory protein (MIP)-1β, and PMA, as well as anti-CD3 and anti-β1 integrin-activating mAb. Moreover, these IL-2 peptides inhibited T cell chemotaxis via FN-coated membranes induced by IL-2 and MIP-1β. Inhibition of T cell adherence and migration apparently involves abrogation of the rearrangement of the T cell actin cytoskeleton. Thus, the migrating immune cells, the cytokines, and the ECM can create a functional relationship in which both inflammation-inducing signals and inhibitory molecules of immune responses can coexist; the enzymatic products of IL-2 may serve as natural feedback inhibitors of inflammation.
TNF-α Released by Comigrating Monocytes Promotes Transendothelial Migration of Activated Lymphocytes
Authors: Green David M. , Trial JoAnn , Birdsall Holly H.
Published: December 2022

We investigated mechanisms that increase motility and transendothelial trafficking of activated lymphocytes. Freshly isolated lymphocytes stimulated with immobilized anti-CD3 for 2 h migrate into polymerized collagen in 1.99 ± 0.25-fold greater numbers and across confluent endothelial monolayers in 4.8 ± 0.5-fold greater numbers compared with leukocytes incubated with nonspecific IgG. Activated lymphocytes form clusters with monocytes, and their increased motility was dependent on the presence of comigrating monocytes. Five lines of evidence support the idea that monocytes modulate lymphocyte motility through the release of TNF-α: 1) flow-cytometric analyses, using highly specific and avid mAbs to probe permeabilized whole blood leukocytes, showed that >80% of circulating monocytes contain intracellular TNF-α, whereas <5% contain IL-1 and none contain IL-6; 2) stimulation with immobilized anti-CD3 that was intended to activate lymphocytes also induced monocytes to release increased quantities of TNF-α; 3) rTNF-α, added in doses of 1 to 20 pg/ml to purified anti-CD3-stimulated lymphocytes, reproduced, in a dose-dependent manner, the motility-enhancing effect of adding monocytes; 4) the transient increase in the expression of TNF R-I on CD3-activated T lymphocytes parallels their transiently increased motility; and 5) addition of anti-TNF-α, anti-TNF R-I, anti-TNF R-II, or soluble TNF R-I decreased the motility of stimulated lymphocytes. These results suggest that T lymphocyte stimulation via the CD3-TCR complex signals nearby monocytes to release TNF-α, which feeds back on the lymphocytes to increase their locomotor activity.
Lipopolysaccharide and Ceramide Use Divergent Signaling Pathways to Induce Cell Death in Murine Macrophages
Authors: Lakics Viktor , Vogel Stefanie N.
Published: December 2022

Ceramide is a well-known apoptotic agent that has been implicated in LPS signaling. Therefore, we examined whether LPS-induced macrophage cytotoxicity is mediated by mimicking ceramide. Both LPS and the cell-permeable ceramide analogue, C2 ceramide, induced significant cell death in IFN-γ-activated, thioglycollate-elicited peritoneal macrophages after 48 and 24 h, respectively. Ceramide-induced cell death was neither accompanied by DNA fragmentation nor phosphatidyl serine externalization, characteristics of apoptosis. In contrast, LPS induced a significant fraction of cells to undergo apoptosis, as demonstrated by DNA fragmentation and quantified by DNA analysis on FACS, yet the majority of the cells died in a necrotic fashion. C3H/HeJ Lpsd macrophages were resistant to LPS-induced cell death and less sensitive to C2 ceramide-evoked cytotoxicity, when compared with Lpsn macrophages. C2 ceramide plus IFN-γ failed to activate release of nitric oxide (NO·), whereas LPS-induced cell death, but not C2-induced cytotoxicity, was blocked by an inhibitor of inducible NO· synthase (iNOS), NG-monomethyl-l-arginine. Macrophages from IFN regulatory factor-1 (−/−) mice shown previously to respond marginally to LPS plus IFN-γ to express iNOS mRNA and NO·, were refractory to LPS plus IFN-γ-induced cytotoxicity and apoptosis. These data suggest that although LPS may mimic certain ceramide effects, signal transduction events that lead to cytotoxicity, as well as the downstream mediators, diverge.
Short-Term and Long-Term Cytokine Release by Mouse Bone Marrow Mast Cells and the Differentiated KU-812 Cell Line Are Inhibited by Brefeldin A
Authors: Zhu Fu-Gang , Gomi Kaede , Marshall Jean S.
Published: December 2022

Mast cells and basophils produce a wide range of cytokines, including large amounts of both IL-6 and granulocyte-macrophage CSF (GM-CSF). However, the route by which cytokines are secreted is poorly understood. In the current study, we used two inhibitors of vesicular transport, brefeldin A and monensin, to examine the routes of secretion of IL-6 and GM-CSF in the differentiated KU812 human cell line and cultured mouse bone marrow mast cells (mBMMC). Studies of cytokine production over 6 to 24 h demonstrated that IL-6 and GM-CSF release from both cell types were inhibited by brefeldin A (BFA) following activation with calcium ionophore, A23187. Monensin had similar inhibitory effects to that of BFA on the initial and ongoing IL-6 release from KU812 cells. In contrast, the amount of each cytokine remaining within the cells was significantly enhanced. Similar results were obtained following IgE-mediated activation of mBMMC. BFA significantly inhibited both the constitutive secretion of IL-6 and the immediate ionophore-induced increase in IL-6 release from KU812 cells at 20 min postactivation. However, treatment with these agents did not alter the release of histamine and β-hexaminidase from either mBMMC or KU812 cells. These studies suggest that both the initial 20-min release of IL-6 and secretion of IL-6 and GM-CSF over up to 24 h by mBMMC and differentiated KU-812 cells occur predominately through a vesicular transport-dependent mechanism, and that little, if any, IL-6 and GM-CSF is released through degranulation.
Macrophage-Inflammatory Protein-3β/EBI1-Ligand Chemokine/CKβ-11, a CC Chemokine, Is a Chemoattractant with a Specificity for Macrophage Progenitors Among Myeloid Progenitor Cells
Authors: Kim Chang H. , Pelus Louis M. , White John R. , Broxmeyer Hal E.
Published: December 2022

Chemoattractants are potential factors influencing cell migration. Stromal cell-derived factor-1, a CXC chemokine, is the only chemokine reported to have chemotactic activity for hemopoietic progenitor cells (HPC). We report in this work another chemokine of the CC subfamily, which is chemotactic for HPC. Macrophage-inflammatory protein (MIP)-3β/EBI1-ligand chemokine/CKβ-11 attracted bone marrow and cord blood CD34+ cells. In contrast to stromal cell-derived factor-1, which attracts multiple types of HPC, MIP-3β attracted mainly CFU granulocyte macrophage, but not other HPC such as burst-forming unit erythrocyte or CFU granulocyte, erythrocyte, macrophage, and megakaryocyte. Chemoattracted CD34+ cells formed CFU granulocyte macrophage-like colonies, which were morphologically determined as large macrophages. These progenitors were selectively responsive to stimulation by macrophage CSF, demonstrating that MIP-3β attracts macrophage progenitors. Expression of CCR7, the receptor for MIP-3β, was detected at a mRNA level in the attracted CD34+ cells as well as input CD34+HPC. Expression of MIP-3β mRNA was not constitutive, but was inducible in bone marrow stromal cells by inflammatory agents such as bacterial LPS, IFN-γ, and TNF-α. Taken together, our findings suggest that MIP-3β is expressed in the bone marrow environment after induction with certain inflammatory cytokines and LPS, and may play a role in trafficking of macrophage progenitors in and out of the bone marrow in inflammatory conditions.
Ex Vivo Anti-CD3 Antibody-Activated Donor T Cells Have a Reduced Ability to Cause Lethal Murine Graft-Versus-Host Disease but Retain Their Ability to Facilitate Alloengraftment
Authors: Drobyski William R. , Majewski David , Ozker Kutlan , Hanson Gerald
Published: December 2022

The purpose of this study was to determine whether ex vivo anti-CD3 Ab-activated T cells behaved in a biologically similar manner as naive T cells with respect to causing graft-vs-host disease (GVHD) and facilitating engraftment after allogeneic marrow transplantation. This question was addressed using two well-defined MHC-incompatible murine models of GVHD (C57BL/6 (H-2b)→B10.BR (H-2k)) and engraftment (C57BL/6 (H-2b)→AKR/J (H-2k)). Transplantation with anti-CD3-activated T cells significantly reduced GVHD compared with that in animals transplanted with equivalent numbers of naive T cells. Protection from GVHD was not T cell subset dependent, as highly enriched populations of either activated CD4+ or CD8+ T cells caused less lethal GVHD than comparable numbers of purified naive CD4+ or CD8+ T cells. Transplantation with activated T cells also resulted in protection from LPS-mediated GVH lethality in unirradiated F1 recipients. Analysis of immune recovery indicated that animals transplanted with activated T cells had thymic and splenic B cell reconstitution that compared favorably to that in non-GVHD control mice. When engraftment was analyzed, equivalent degrees of donor cell engraftment were observed when animals were transplanted with limiting numbers (5 × 105) of naive vs activated B6 T cells. Further studies indicated that activated CD8+ T cells were exclusively responsible for enhancing engraftment and that facilitation of engraftment was dependent upon the direct recognition of host MHC alloantigens. Collectively, these data demonstrate that transplantation with anti-CD3 Ab-activated T cells results in a reduction in GVHD, but these cells retain their ability to facilitate alloengraftment. The use of this approach in allogeneic marrow transplantation may represent an alternative strategy to mitigate GVHD without compromising engraftment.
Maternal B Lymphocytes Specific for Paternal Histocompatibility Antigens Are Partially Deleted During Pregnancy
Authors: Aït-Azzouzene Djemel , Gendron Marie-Claude , Houdayer Monique , Langkopf Anja , Bürki Kurt , Nemazee David , Kanellopoulos-Langevin Colette
Published: December 2022

Although genetically different from its mother, a mammalian fetus bearing paternal alloantigens is normally not rejected. To investigate one of the many possible mechanisms involved in this important biologic phenomenon, we analyzed the consequences of fetal alloantigen recognition on maternal B lymphocytes. We used transgenic mice expressing a unique B cell receptor with a relatively high affinity for the MHC class I molecule H-2Kk on most B lymphocytes. We provide the first evidence for an alloantigen-specific B cell deletion in the spleens and bone marrow of transgenic mothers bearing H-2Kk-positive fetuses. This highly reproducible deletion affects ≤80% of Id-bearing B cells, starts at midpregnancy, and is only observed until term. Such a specific maternal B cell deletion could contribute to the success of the fetal allograft.
Sam68 Association with p120GAP in CD4+ T Cells Is Dependent on CD4 Molecule Expression
Authors: Jabado Nada , Jauliac Sébastien , Pallier Annaïck , Bernard Frédéric , Fischer Alain , Hivroz Claire
Published: December 2022

p120 GTPase-activating protein (p120GAP) is a major negative regulator of p21ras activity in several cell types including T cells. Catalytic activity of this enzyme is regulated in part by its interaction with several associated tyrosine-phosphorylated proteins. Sam68 was initially described as associated with p120GAP. It has been further established that Sam68 is a substrate of src kinases in mitosis and that it is not associated with p120GAP in transformed fibroblasts. We describe herein that Sam68 associates with p120GAP and PLCγ1 in human mature T cells and in a T cell line expressing the CD4 molecule HUT78 CD4+. This association is present in nonactivated cells and increases after anti-CD3 activation. It is dependent on CD4 expression and, in part, on the association of CD4 with p56lck, as shown by the strongly decreased association of Sam68 with p120GAP in the CD4− mutants, HUT78 CD4−, and by the reduced association of Sam68 with both p120GAP and p56lck in the HUT78 T cell line expressing a CD4 mutant unable to interact with p56lck, HUT78 C420/22. We propose that recruitment of Sam68, via CD4/p56lck, to the inner face of the plasma membrane may permit, via its docking properties, the correct association of key signaling molecules including PLCγ1 and p120GAP. This formation of transduction modules will enable the activation of different signaling cascades including the p21ras pathway and an array of downstream events, ultimately leading to T cell activation.
Differential Expression of Fas and Fas Ligand in Acute and Chronic Graft-Versus-Host Disease: Up-Regulation of Fas and Fas Ligand Requires CD8+ T Cell Activation and IFN-γ Production
Authors: Shustov Andrei , Nguyen Phuong , Finkelman Fred , Elkon Keith B. , Via Charles S.
Published: December 2022

The parent-into-F1 model of acute and chronic graft-vs-host disease (GVHD) was used as an example of in vivo cell-mediated or Ab-mediated responses, respectively, and the roles of Fas and Fas ligand (FasL) were investigated. Using both flow cytometry and PCR methodologies, we found that acute GVHD mice exhibited significant up-regulation of Fas and FasL, whereas Fas/FasL up-regulation in chronic GVHD mice was equal to or marginally greater than that in uninjected mice. Functional studies confirmed that Fas/FasL contributed to the anti-host CTL activity of splenocytes from acute GVHD mice, although a perforin-dependent pathway was also identified. Despite the presence of FasL on both donor CD4+ and CD8+ T cells in acute GVHD mice, depletion studies demonstrated that all the in vitro anti-host CTL activity resided in the CD8+ population. Furthermore, injection of CD8-depleted B6 spleen cells into F1 mice blocked Fas/FasL up-regulation and IFN-γ production, resulting in chronic GVHD. Lastly, up-regulation of Fas/FasL in acute GVHD mice could be blocked by anti-IFN-γ mAb in vivo. Thus, in this in vivo model of alloantigen immune responsiveness, Fas/FasL up-regulation is critically dependent on Ag-specific (donor) CD8+ T cell activation and IFN-γ production. Donor CD4+ T cell activation in the absence of CD8+ T cell activation results in an autoantibody-mediated response, no significant Fas/FasL up-regulation, impaired elimination of autoreactive B cells, and persistent humoral autoimmunity.
Clonal Analysis of a Human Antibody Response. III. Nucleotide Sequences of Monoclonal IgM, IgG, and IgA to Rabies Virus Reveal Restricted Vκ Gene Utilization, Junctional VκJκ and VλJλ Diversity, and Somatic Hypermutation
Authors: Ikematsu Wataru , Kobarg Jörg , Ikematsu Hideyuki , Ichiyoshi Yuji , Casali Paolo
Published: December 2022

In previous work, we generated four IgM, five IgG1, and one IgA1 mAbs to rabies virus using B cells from four subjects vaccinated with inactivated rabies virus, a thymus-dependent (TD) mosaic Ag, and sequenced the mAb VHDJH genes. Here, we have cloned the VκJκ and VλJλ genes to complete the primary structure of the Ag-binding site of these mAbs. While the anti-rabies virus mAb selection of Vλ genes (2e.2.2 twice, DPL11, and DPL23) reflected the representation of the Vλ genes in the human haploid genome (stochastic utilization), that of Vκ genes (O2/O12 twice, O8/O18, A3/A19, A27, and L2) did not (p = 0.0018) (nonstochastic utilization). Furthermore, the selection of both Vκ and Vλ genes by the anti-rabies virus mAbs vastly overlapped with that of 557 assorted VκJκ rearrangements, that of 253 VλJλ rearrangements in λ-type gammopathies, and that of other Abs to thymus-dependent Ags, including 23 anti-HIV mAbs and 51 rheumatoid factors, but differed from that of 43 Abs to Haemophilus influenzae type b polysaccharide, a prototypic thymus-independent (TI) Ag. The anti-rabies virus mAb VκJκ and VλJλ segments displayed variable numbers of somatic mutations, which, in mAb58 and the virus-neutralizing mAb57, entailed a significant concentration of amino acid replacements in the complementarity-determining regions (p = 0.0028 and p = 0.0023, respectively), suggesting a selection by Ag. This Ag-dependent somatic selection process was superimposed on a somatic diversification process that occurred at the stage of B cell receptor for Ag rearrangement, and that entailed V gene 3′ truncation and N nucleotide additions to yield heterogeneous CDR3s.
Sustained TCR Signaling Is Required for Mitogen-Activated Protein Kinase Activation and Degranulation by Cytotoxic T Lymphocytes
Authors: Berg Nancy N. , Puente Lawrence G. , Dawicki Wojciech , Ostergaard Hanne L.
Published: December 2022

Requirements for T cell activation are not fully established. One model is that receptor occupancy and down-regulation are essential for activation, and another, not necessarily mutually exclusive, model is that sustained signals are important. Here we examine the importance of signal duration in T cell activation. First, we demonstrate that immobilized, but not soluble cross-linked, Abs to CD3 stimulate degranulation by CTL. The cross-linked Abs are not deficient in their ability to signal since they stimulate the same tyrosine phosphorylation pattern as immobilized Ab, but it is very transient relative to that stimulated by immobilized Ab. Furthermore, novel decreased migratory forms of Lck occur to a significant extent only after stimulation with immobilized Abs. A dramatic difference in the duration of signals is very evident when mitogen-activated protein kinase (MAPK) activity is examined. Immobilized anti-CD3 stimulates very high levels of MAPK activation that is still detectable 1 h after stimulation. In contrast, cross-linked Ab stimulates only transient and incomplete activation of MAPK. Taken together, these results suggest that TCR engagement and induction of tyrosine phosphorylation alone are not sufficient for T cell activation and that the duration of TCR-stimulated signals is critical to attain a functional response.
HIV-1 Tat Inhibits Human Natural Killer Cell Function by Blocking L-Type Calcium Channels
Authors: Zocchi M. Raffaella , Rubartelli Anna , Morgavi Paola , Poggi Alessandro
Published: December 2022

Herein we show that functional phenylalkylamine-sensitive L-type calcium channels are expressed by human NK cells and are involved in the killing of tumor targets. Blocking of these channels by phenylalkylamine drugs does not affect effector/target cell binding but inhibits the release of serine esterases responsible for cytotoxicity. Interestingly, treatment of NK cells with HIV-1 Tat, which is known to affect several calcium-mediated events in immune cells, impairs their cytotoxic activity. In addition, Tat inhibits the rise in intracellular free calcium concentration upon cross-linking of the adhesion molecule CD11a, engaged during effector/target cell interaction, and the activation molecule CD16. Exogenous Tat does not influence NK-target cell binding but prevents NK cell degranulation. We propose that the molecular structure(s) on NK cells mediating the inhibitory effects HIV-1 Tat belong to L-type calcium channels, based on three lines of evidence: 1) binding of phenylalkylamine derivatives to these channels is cross-inhibited by Tat; 2) L-type calcium channels from NK cell lysates bind to Tat linked to Sepharose columns; 3) the inhibitory effect of HIV-1 Tat on NK cell function is prevented by the agonist of L-type calcium channels, Bay K 8644. Altogether, these results suggest that exogenous Tat is deeply involved in the impairment of NK cell function during HIV-1 infection.
Construction of a Lipopolysaccharide Reporter Cell Line and Its Use in Identifying Mutants Defective in Endotoxin, But Not TNF-α, Signal Transduction
Authors: Delude Russell L. , Yoshimura Atsutoshi , Ingalls Robin R. , Golenbock Douglas T.
Published: December 2022

Gram-negative bacterial LPS is a potent activator of inflammatory responses. The binding of LPS to CD14 initiates signal transduction; however, the molecular processes immediately following this event remain unclear. We engineered an LPS-inducible fibroblast reporter cell line to facilitate the use of molecular genetic techniques to study the LPS signaling pathway. A plasmid containing the human Tac Ag cDNA under transcriptional control of the human E selectin promoter was cotransfected into Chinese hamster ovary (CHO)-K1 cells together with a CD14 expression plasmid. A cell line was obtained, 3E10, which up-regulated expression of Tac following stimulation with LPS. Pools of mutagenized cells were exposed to LPS and then labeled with anti-Tac mAb. Cells that failed to up-regulate Tac expression were enriched by flow cytometry. Thirty clonal mutant cell lines were identified that continued to express CD14 and bind LPS, but failed to express Tac or translocate nuclear factor-κB (NF-κB) following LPS exposure. TNF-α-treated mutant cells continued to express Tac and translocate NF-κB. An analysis of LPS-induced NF-κB activity in heterokaryons derived from polyethylene glycol-fused cell lines indicated that recessive mutations in genes encoding components of the LPS signaling pathway accounted for the signaling defects. To date, two complementation groups have been identified from 11 cell lines analyzed. These data demonstrate that the TNF-α signaling pathway diverges from the LPS pathway early in the signal-transduction cascade despite similarities in LPS- and TNF-α-induced responses. Identification of the genes affected in these mutant reporter cells should identify heretofore-elusive components of the LPS signaling cascade.
Gene-Modified Tumor Vaccine with Therapeutic Potential Shifts Tumor-Specific T Cell Response from a Type 2 to a Type 1 Cytokine Profile
Authors: Hu Hong-Ming , Urba Walter J. , Fox Bernard A.
Published: December 2022

Vaccination with a poorly immunogenic/nonimmunogenic tumor fails to protect the host from a subsequent challenge with the same tumor. The mechanisms underlying the failure of these tumors to sensitize therapeutic T cells are not clearly understood, but the inability of host T cells to recognize tumor has been implicated. In this study, vaccination with the poorly immunogenic B16BL6-D5 (D5 H-2b) tumor fails to generate therapeutic T cells from the tumor vaccine-draining lymph nodes (TVDLN) in our adoptive immunotherapy model. However, if vaccination is performed with an allogeneic MHC class I gene (H-2 Kd)-modified tumor, the T cells obtained from the TVDLN are therapeutic after activation with anti-CD3 and IL-2. Lymph nodes (LN) draining both D5 and D5-Kd tumor vaccines contained increased numbers of cells with reduced expression of L-selectin (L-selectinlow/−) compared with naive LN. This implies that vaccination led to sensitization of T cells even in LN draining the unmodified D5 tumor. L-selectinlow/− cells from D5-Kd, but not D5, TVDLN were therapeutic in our animal model. No antitumor activity was seen in the high level L-selectin T cells. L-selectinlow/− T cells exhibited tumor-specific cytokine release that was type 2 (IL-4, IL-10) following vaccination with native D5 and type 1 (IFN-γ) following vaccination with gene-modified D5-Kd. Our data suggest that the failure of unmodified D5 to generate therapeutic T cells is not due to an inability to recognize tumor Ags, but, rather, to the induction of an immune response that is ineffective in mediating tumor regression, i.e., immune deviation.
Activation of Cutaneous Dendritic Cells by CpG-Containing Oligodeoxynucleotides: A Role for Dendritic Cells in the Augmentation of Th1 Responses by Immunostimulatory DNA
Authors: Jakob Thilo , Walker Patricia S. , Krieg Arthur M. , Udey Mark C. , Vogel Jonathan C.
Published: December 2022

Genetic vaccination depends at least in part on the adjuvant properties of plasmids, properties that have been ascribed to unmethylated CpG dinucleotides in bacterial DNA. Because dendritic cells (DC) participate in the T cell priming that occurs during genetic vaccination, we reasoned that CpG-containing DNA might activate DC. Thus, we assessed the effects of CpG oligodeoxynucleotides (CpG ODN) on Langerhans cell (LC)-like murine fetal skin-derived DC (FSDDC) in vitro and on LC in vivo. Treatment with CpG ODN as well as LPS induced FSDDC maturation, manifested by decreased E-cadherin-mediated adhesion, up-regulation of MHC class II and costimulator molecule expression, and acquisition of enhanced accessory cell activity. In contrast to LPS, CpG ODN stimulated FSDDC to produce large amounts of IL-12 but only small amounts of IL-6 and TNF-α. Injection of CpG ODN into murine dermis also led to enhanced expression of MHC class II and CD86 Ag by LC in overlying epidermis and intracytoplasmic IL-12 accumulation in a subpopulation of activated LC. We conclude that immunostimulatory CpG ODN stimulate DC in vitro and in vivo. Bacterial DNA-based vaccines may preferentially elicit Th1-predominant immune responses because they activate and mobilize DC and induce them to produce large amounts of IL-12.
Differential Regulation of Monocyte Matrix Metalloproteinase and TIMP-1 Production by TNF-α, Granulocyte-Macrophage CSF, and IL-1β Through Prostaglandin-Dependent and -Independent Mechanisms
Authors: Zhang Yahong , McCluskey Kevin , Fujii Karen , Wahl Larry M.
Published: December 2022

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) produced by monocytes are believed to be involved in the migration of these cells through the basement membrane and the ensuing destruction of connective tissue in chronic inflammatory lesions. Because monocytes encounter a variety of cytokines at these sites, we examined the effect of cytokines either alone or in combination on the production of monocyte MMPs and TIMP-1. TNF-α, granulocyte-macrophage-CSF (GM-CSF), or IL-1β when added individually enhanced the endogenous levels of 92-kDa gelatinase (MMP-9) and TIMP-1 but failed to induce interstitial collagenase (MMP-1). However, GM-CSF, when added with either TNF-α or IL-1β, induced MMP-1 and synergistically enhanced MMP-9 and TIMP-1. Th2 cytokines, such as IL-4, inhibited the induction of MMPs and TIMP-1 by TNF-α, GM-CSF, and IL-1. Cytokine stimulation of MMP-1 was due, at least in part, to an increase in the release of arachidonic acid and PG E2 (PGE2), because inhibition of MMP-1 by indomethacin could be reversed by exogenous PGE2. In contrast to MMP-1, cytokine stimulation of MMP-9 and TIMP-1 was unaffected by indomethacin. The PGE2-independent induction of monocyte MMP-9 and TIMP-1 by these cytokines differed from stimulation of MMP-9 and TIMP-1 by LPS, which is in large part PG-dependent. In addition, LPS stimulated higher levels of MMP-1 whereas cytokines induced higher levels of MMP-9 and TIMP-1. This is the first demonstration that monocyte MMP-1 can be induced by cytokines and that MMP-1, MMP-9, and TIMP-1 are differentially regulated by cytokines through PG-dependent and -independent mechanisms.
Different Molecular Events Result in Low Protein Levels of Mannan-Binding Lectin in Populations from Southeast Africa and South America
Authors: Madsen Hans O. , Satz M. Leonardo , Hogh Birthe , Svejgaard Arne , Garred Peter
Published: December 2022

Previous studies have shown that three point mutations in exon 1 and a particular promoter haplotype of the mannan-binding lectin (MBL) gene lead to a dramatic decrease in the serum concentration of MBL. In this study, MBL genotypes and serum concentrations were determined in unrelated individuals in a population from Mozambique (n = 154) and in two native Indian tribes from Argentina (i.e., the Chiriguanos (n = 43) and the Mapuches (n = 25)). In both populations, the MBL concentrations were low compared with those found in Eskimo, Asian, and European populations. In Africans, the low serum concentrations were due to a high allele frequency (0.24) of the codon 57 (C) variant, which resulted in a high frequency of individuals with MBL deficiency (0.06), and were also due to the effect of a relatively high frequency (0.13) of low-producing promoter haplotypes. The low concentrations in the South American populations were primarily due to an extremely high allele frequency of the codon 54 (B) variant in both the Chiriguanos (0.42) and the Mapuches (0.46), resulting in high frequencies of individuals with MBL deficiency (0.14 and 0.16, respectively). In the search for additional genetic variants, we found five new promoter mutations that might help to elucidate the evolution of the MBL gene. Taken together, the results of this study show that different molecular mechanisms are the basis for low MBL levels on the two continents.