bluuebunny/crossref_metadata_2025 huggingface dataset:
Bispecific Molecules Directed to the Fc Receptor for IgA (FcαRI, CD89) and Tumor Antigens Efficiently Promote Cell-Mediated Cytotoxicity of Tumor Targets in Whole Blood
Authors: Deo Yashwant M. , Sundarapandiyan Karuna , Keler Tibor , Wallace Paul K. , Graziano Robert F.
Published: December 2022

The FcR for IgA (FcαRI, CD89) is primarily expressed on cytotoxic immune effector cells. By chemically cross-linking F(ab′) fragments of the FcR for IgA (FcαRI)-specific mAb (A77) with tumor Ag-specific mAb (anti-HER2/neu and anti-epidermal growth factor receptor), we have developed bispecific molecules (BSM) that simultaneously bind to respective tumor Ags and FcαRI-expressing effector cells in whole blood. These BSM mediated up to 55% of specific lysis of appropriate tumor Ag-expressing target cells (from a variety of tumors) with purified polymorphonuclear leukocytes, monocytes, or whole blood effector cells without preactivation with exogenous cytokines. To our knowledge, this is the first demonstration of Ab-dependent cell-mediated cytotoxic activity via FcαRI in whole blood. Also, monocyte-derived macrophages mediated phagocytosis of HER2/neu-expressing tumor cells (>95% tumor cell loss). These BSM-mediated cytotoxic activities were completely inhibited by F(ab′)2 of A77, demonstrating the specific role of FcαRI as a trigger molecule. Furthermore, the binding of these BSM to monocytes or polymorphonuclear leukocytes in whole blood did not induce modulation of FcαRI in the absence of the target Ag. Therefore, immune effector cells may be “armed” with FcαRI-directed BSM in whole blood. These FcαRI-directed BSM may offer new treatment options for various malignancies and other disease conditions.
Antigen-Primed T Cells from B Cell-Deficient JHD Mice Fail to Provide B Cell Help
Authors: Macaulay Anne E. , DeKruyff Rosemarie H. , Umetsu Dale T.
Published: December 2022

B cells are effective in presenting Ag to primed T cells, but a specific role for B cells in priming naive T cells has not been clearly established. In this report we demonstrate that Ag administration to B cell-deficient JHD mice primes T cells for Ag-specific proliferative responses, but such primed T cells fail to provide help for isotype switching and IgG production in B cells. Although T cells primed in the absence of B cells could proliferate in response to Ag presented by B cells and could induce Ag-specific IgM production, such T cells failed to produce high levels of IL-4 as are normally induced in T cells by Ag-presenting B cells. These findings suggest that while B cells are not absolutely required for T cell priming, they provide signals to T cells that are not replicated by other APCs and that influence the subsequent ability of T cells to interact with B cells.
CD40 Ligand/CD40 Stimulation Regulates the Production of IFN-γ from Human Peripheral Blood Mononuclear Cells in an IL-12- and/or CD28-Dependent Manner
Authors: McDyer John F. , Goletz Theresa J. , Thomas Elaine , June Carl H. , Seder Robert A.
Published: December 2022

CD40 ligand (CD40L)/CD40 costimulation is an important regulator of Th1 responses. Two mechanisms by which CD40L/CD40 stimulation may enhance IFN-γ are via direct induction of IL-12 and augmentation of the expression of costimulatory molecules such as B7 from APCs. We examined the ability of CD40L/CD40 stimulation to regulate the production of IFN-γ through IL-12 and/or CD28 costimulation from human PBMCs stimulated with T cell-specific stimuli. The roles of exogenous and endogenous CD40L/CD40 stimulation were evaluated using a trimeric soluble CD40L agonist (CD40T) and an anti-CD40L Ab, respectively. The presence of CD40T in cultures increased the production of IL-12 and IFN-γ from PBMCs stimulated with varying amounts of PHA. The mechanism, however, by which CD40T enhanced IFN-γ varied according to the level of T cell activation. Under maximal stimulatory conditions (PHA, 1/100), an IL-12-dependent pathway was dominant. At relatively low levels of T cell stimulation (PHA, 1/500 and 1/1000), however, an additional IL-12-independent CD28-dependent pathway was elucidated. We further studied the role of exogenous CD28 stimulation in regulating the production of IFN-γ. The enhancement of IFN-γ production induced by direct CD28 stimulation was primarily dependent on endogenous IL-12 or CD40L/CD40 stimulation. Together, these data suggest that the production of IFN-γ involves a complex interaction between two interdependent, yet distinct, costimulatory pathways and provide evidence that CD40T may be an effective adjuvant for the enhancement of responses.
Resistance to Polyoma Virus-Induced Tumors Correlates with CTL Recognition of an Immunodominant H-2Dk-Restricted Epitope in the Middle T Protein
Authors: Lukacher Aron E. , Wilson Christopher S.
Published: December 2022

The natural mouse pathogen polyoma virus is highly oncogenic in H-2k mice carrying the endogenous superantigen encoded by the mouse mammary tumor provirus Mtv-7. This superantigen results in deletion of Vβ6 TCR-expressing polyoma-specific CD8+ CTL, which appear to be critical effectors against polyoma tumorigenesis. Here we have isolated cloned lines of CD8+ T cells from resistant (i.e., Mtv-7−) H-2k mice that specifically lyse syngeneic polyoma virus-infected cells and polyoma tumor cells. Nearly all these CTL clones express Vβ6 and are restricted in their recognition of virus-infected cells by H-2Dk. Screening a panel of synthetic peptides predicted to bind to Dk, for which no consensus peptide binding motif is known, we identified a peptide corresponding to a nine-amino acid sequence in the carboxyl-terminus of the middle T (MT) protein (amino acids 389–397) that was recognized by all the Vβ6+CD8+ CTL clones. The inability of MT389–397-reactive CTL to recognize cells infected with a mutant polyoma virus encoding a MT truncated just proximal to this sequence indicates that MT389-397 is a naturally processed peptide. The frequencies of precursor CTL specific for polyoma virus and MT389–397 peptide were similar, indicating that MT389–397 is the immunodominant epitope in H-2k mice. In addition, polyoma-infected resistant mice possess a 10- to 20-fold higher MT389-397-specific precursor CTL frequency than susceptible mice. This highly focused CTL response to polyoma virus provides a valuable animal model to investigate the in vivo activity of CTL against virus-induced neoplasia.
Role of CD80 (B7.1) and CD86 (B7.2) in the Immune Response to an Intracellular Pathogen
Authors: Subauste Carlos S. , de Waal Malefyt Rene , Fuh Franklin
Published: December 2022

The costimulatory ligands CD80 and CD86 play a crucial role in the initiation and maintenance of an immune response. We demonstrate that whereas infection of human monocytes with viable tachyzoites of Toxoplasma gondii resulted in rapid induction of expression of CD80 and up-regulation of expression of CD86, incubation with killed organisms failed to alter the levels of expression of these costimulatory ligands. The T. gondii-mediated changes in levels of expression of these molecules are critical to the T cell response to the parasite. Proliferation of resting T cells in response to parasite-infected cells was dependent on both CD80 and CD86. More importantly, early production of IFN-γ in response to T. gondii by T cells from T. gondii-seronegative individuals occurred only after stimulation with monocytes that exhibited increased expression of CD80 and CD86 (monocytes infected with viable parasites) and was almost completely ablated by the combination of anti-CD80 plus anti-CD86 mAb. Moreover, proliferation and IFN-γ production by CD4+ CD45RA+ T cells from unexposed individuals were dependent on both CD80 and CD86. These data indicate that pathogen-monocyte interaction influences the ensuing T cell response.
Morphine Enhances Macrophage Apoptosis
Authors: Singhal Pravin C. , Sharma Puneet , Kapasi Aditi A. , Reddy Krishna , Franki Nicholas , Gibbons Nora
Published: December 2022

Laboratory data indicate that morphine decreases the number of peritoneal and alveolar macrophages (Mφ) and compromises their phagocytic capability for immune complexes and bacteria. We hypothesize that morphine decreases the number of, as well as compromises the phagocytic capability of, Mφ by programming their death. We studied the effect of morphine on Mφ apoptosis in vivo as well as in vitro. Peritoneal Mφ harvested from morphine-treated rats showed DNA fragmentation. Morphine enhanced murine Mφ (J 774.16) apoptosis in a dose-dependent manner. Human monocytes treated with morphine showed a classic ladder pattern in gel electrophoretic and end-labeling studies. Morphine promoted nitric oxide (NO) production both under basal and LPS-activated states. NG-nitro-l-arginine methyl ester (l-NAME) and NG-monomethyl-l-arginine monoacetate (l-NMMA), inhibitors of NO synthase, attenuated the morphine-induced generation of NO by Mφ. Morphine also enhanced Mφ mRNA expression of inducible NO synthase (iNOS). Since morphine-induced Mφ apoptosis was inhibited by L-NAME and L-NMMA, it appears that morphine-induced Mφ apoptosis may be mediated through the generation of NO. Morphine promoted the synthesis of Bax and p53 proteins by Mφ. Moreover, IL-converting enzyme (ICE)-1 inhibitor attenuated morphine-induced Mφ apoptosis. These studies suggest that morphine activates the induction phase of the apoptotic pathway through accumulation of p53. The effector phase of morphine-induced apoptosis appears to proceed through the accumulation of Bax and activation of ICE-1. The present study provides a basis for a hypothesis that morphine may be directly compromising immune function by promoting Mφ apoptosis in patients with opiate addiction.
CD14-Dependent Internalization of Bacterial Lipopolysaccharide (LPS) Is Strongly Influenced by LPS Aggregation But Not by Cellular Responses to LPS
Authors: Kitchens Richard L. , Munford Robert S.
Published: December 2022

We analyzed the impact of ligand aggregation and LPS-induced signaling on CD14-dependent LPS internalization kinetics in human monocytic THP-1 cells and murine macrophages. Using two independent methods, we found that the initial rate and extent of LPS internalization increased with LPS aggregate size. In the presence of LPS binding protein (LBP), large LPS aggregates were internalized extremely rapidly (70% of the cell-associated LPS was internalized in 1 min). Smaller LPS aggregates were internalized more slowly than the larger aggregates, and LPS monomers, complexed with soluble CD14 in the absence of LBP, were internalized very slowly after binding to membrane CD14 (5% of the cell-associated LPS was internalized in 1 min). In contrast, the initial aggregation state had little or no effect on the stimulatory potency of the LPS. Previous studies suggest that LPS-induced signal responses may influence the intracellular traffic and processing of LPS. We found that elicited peritoneal macrophages from LPS-responsive (C3H/HeN) and LPS-hyporesponsive (C3H/HeJ) mice internalized LPS with similar kinetics. In addition, pre-exposure of THP-1 cells to LPS had no effect on their ability to internalize subsequently added LPS, and pre-exposure of the cells to the LPS-specific inhibitor, LA-14-PP, inhibited stimulation of the cells without inhibiting LPS internalization. In these cells, LPS is thus internalized by a constitutive cellular mechanism(s) with kinetics that depend importantly upon the physical state in which the LPS is presented to the cell.
Ox-40 Ligand: A Potent Costimulatory Molecule for Sustaining Primary CD4 T Cell Responses
Authors: Gramaglia Irene , Weinberg Andrew D. , Lemon Michael , Croft Michael
Published: December 2022

Ox-40 and Ox-40 ligand (Ox-40L) are thought to be involved in T cell-APC interactions. However, their exact role in T cell responses is undefined. Using fibroblast transfectants expressing Ox-40L and/or B7-1, and CD4 cells from TCR transgenic mice, we investigated the effect of Ox-40 signaling on primary responses to the Ag pigeon cytochrome c. Ox-40 expression on naive CD4 cells peaked 2 to 3 days after activation, and was lost by 4 to 5 days. APCs with Ox-40L promoted partial activation of naive T cells with some IL-2 secretion, but were unable to enhance proliferation, unlike those with B7-1. APCs coexpressing Ox-40L with B7-1 induced large quantities of IL-2 and promoted proliferative responses that persisted for several days. Effector cells taken 5 days after naive T cell activation reexpressed Ox-40 within 4 h and responded strongly to APCs expressing Ox-40L, whereas B7-1 had little effect. Synergy was also seen between Ox-40L and B7-1, with primarily IL-2 being elevated, although IL-4 and IL-5 were also up-regulated. The most striking action was on effector T cell proliferation, which continued at high levels for up to 4 days, with little proliferation evident at this time in the absence of Ox-40 signals. These data suggest that Ox-40/Ox-40L interactions act after initial activation events to prolong clonal expansion and enhance effector cytokine secretion, and may be involved in promoting long-lived primary CD4 responses.
Antagonistic Roles for Phospholipase D Activities in B Cell Signaling: While the Antigen Receptors Transduce Mitogenic Signals Via a Novel Phospholipase D Activity, Phosphatidylcholine-Phospholipase D Mediates Antiproliferative Signals
Authors: Gilbert Jonathan J. , Pettitt Trevor R. , Seatter Sandra D. , Reid Steven D. , Wakelam Michael J. O. , Harnett Margaret M.
Published: December 2022

Cross-linking of the Ag receptors on B cells induces DNA synthesis and proliferation. Butanol trap experiments suggest that one or more phospholipase D activities play a key role in this process. Although phosphatidylcholine-phospholipase D has been shown to play a central role in the transduction of proliferative responses for a wide variety of calcium-mobilizing receptors, we show that the Ag receptors are not coupled to this phospholipase. In addition, phosphatidylcholine-phospholipase D is not stimulated under conditions that mimic T cell-dependent B cell activation. In contrast, ATP, which inhibits surface Ig (sIg)-mediated DNA synthesis in murine B cells via P2-purinoceptors, activates phosphatidylcholine-phospholipase D. Phosphatidylcholine-phospholipase D is therefore associated with antiproliferative signal transduction in mature B cells, but it does not transduce early signals associated with sIg-mediated growth arrest or apoptosis in immature B cells. Mitogenic stimulation of sIg is, however, coupled to a novel nonphosphatidylcholine-hydrolyzing phospholipase D activity. The resultant sIg-generated phosphatidic acid, unlike the phosphatidylcholine-derived phosphatidic acid generated via the purinoceptors, is converted to diacylglycerol. These data provide the first evidence that while the novel sIg-coupled phospholipase D and resultant diacylglycerol generation may play a role in B cell survival and proliferation, phosphatidylcholine-phospholipase D may transduce, via phosphatidic acid, negative immunomodulatory signals in mature B lymphocytes.
Induction or Protection from Experimental Autoimmune Encephalomyelitis Depends on the Cytokine Secretion Profile of TCR Peptide-Specific Regulatory CD4 T Cells
Authors: Kumar Vipin , Sercarz Eli
Published: December 2022

Autoimmune diseases can result from the breakdown of regulation and subsequent activation of self-antigenic determinant-reactive T cells. During the evolution of the autoimmune response to myelin basic protein (MBP) in B10.PL mice, several distinct T cell populations expand: the effectors mediating experimental autoimmune encephalomyelitis (EAE) are MBP-reactive, CD4+, and predominantly TCR Vβ8.2+; in addition, at least two regulatory populations can be detected—one comprised of Vβ14+ CD4 T cells, reactive to a framework region 3 determinant on the Vβ8.2 chain, and a second that is CD8+ and reactive to another Vβ8.2 determinant. The combined action of these two regulatory cell types controls disease-causing effectors, resulting in spontaneous recovery from disease. In this report, we reveal that the cytokine secretion pattern of TCR peptide-specific regulatory CD4 T cells can profoundly influence whether a type 1 or type 2 population predominates among MBP-specific CD4 effectors. The priming of type 1 regulatory T cells results in deviation of the Ag-specific effector T cell population in a type 2 direction and protection from disease. In contrast, induction of type 2 regulatory T cells results in exacerbation of EAE, poor recovery, and an increased frequency of type 1 effectors. Thus, the encephalitogenic potential of the MBP-reactive effector population is crucially and dominantly influenced by the cytokine secretion phenotype of regulatory CD4 T cells. These findings have important implications in understanding peripheral tolerance to self-Ags as well as in the design of TCR-based therapeutic approaches.
Clonal Expansion Within CD4+ and CD8+ T Cell Subsets in Human T Lymphotropic Virus Type I-Infected Individuals
Authors: Eiraku Nobutaka , Hingorani Ravi , Ijichi Shinji , Machigashira Koichi , Gregersen Peter K. , Monteiro Joanita , Usuku Koichiro , Yashiki Shinji , Sonoda Shunro , Osame Mitsuhiro , Hall William W.
Published: December 2022

To investigate the diversity of the T cell repertoire involved in human T lymphotropic virus type I (HTLV-I) infections, peripheral blood T cell subsets were analyzed by using a PCR-based assay that permits determination of complementarity-determining region 3 (CDR3) length variation in TCR Vβ transcripts. In two of four asymptomatic HTLV-I carriers and in four of five patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), mono- or oligoclonal expansions were detected in the CD4+ T cell subset. In one patient with adult T cell leukemia, a specific clone bearing Vβ7 was detected in the CD4+ T cell subset. In contrast, clonal expansion was not observed in the CD4 T cell subsets of three individuals with asymptomatic HTLV-II infection or in our previous studies of a large number of uninfected individuals. Oligoclonal expansions in the CD8+ T cell subset were detected in all subjects, including the patient with adult T cell leukemia. No differences in the number of expanded clones were noted between asymptomatic carriers and in patients with HAM/TSP and there was no obvious restriction in the TCR V region usage. Direct sequencing revealed no significant bias in the CDR3 motifs utilized by the predominant clones. This report is the first direct demonstration of clonal expansions within fractionated T cell subsets (CD4+ and CD8+) in HTLV-I infections and suggests that 1) clonal expansion of CD4+ T lymphocytes likely occurs as a direct result of infection and 2) polyclonal CD8+ T cell expansion occurs frequently and independently of disease association.
Intrinsic Defects in Macrophage IL-12 Production Associated with Immune Dysfunction in the MRL/++ and New Zealand Black/White F1 Lupus-Prone Mice and theLeishmania major-Susceptible BALB/c Strain
Authors: Alleva David G. , Kaser Steven B. , Beller David I.
Published: December 2022

We have demonstrated that macrophages (Mφ) from young, prediseased, lupus-prone MRL/++ and New Zealand Black/White F1 mice display defective production of TNF-α, IL-1, and IL-6, but normal production of IL-10. In an attempt to determine the potential functional implications of this phenotype for autoimmunity, we demonstrate here that endotoxin-activated Mφ from these lupus-prone mice showed dramatically reduced expression of IL-12, a cytokine essential for Th1 responses that may be defective during lupus. IL-12 production was also reduced by Mφ from the control BALB/c strain, compatible with the concept that a genetically programmed deficit in IL-12 levels may underlie the IL-4-dominated BALB/c response to infection by the parasite Leishmania major. Although both IL-12 and TNF-α expression defects by Mφ from lupus-prone strains are expressed rapidly after activation, treatment with each cytokine demonstrated that only TNF-α contributes to the subsequent dysregulation of Mφ IL-1 and IL-6 expression in these strains, and that the reduced autocrine activity of defective IL-12 or TNF-α levels was not causal to each other. Although the intrinsic defect in IL-12 expression by lupus-prone and BALB/c Mφ may lead to defective Th1 responses, these Mφ responded to the Th1-derived cytokine, IFN-γ, in a normal fashion suggesting a defective role in the induction, rather than the propagation, of Th1 responses in these mice. Our finding of a conserved intrinsic defect in IL-12 production by Mφ from the two principal mouse models of multigenic lupus provides insight into how excessive humoral responses may develop, and perhaps be prevented, in systemic autoimmune disease.
Isolation and Characterization of a Variant HL60 Cell Line Defective in the Activation of the NADPH Oxidase by Phorbol Myristate Acetate
Authors: Tardif Marianne , Rabiet Marie-Josèphe , Christophe Thierry , Milcent Marie-Danielle , Boulay François
Published: December 2022

Promyelocytic human leukemia HL60 cells can be differentiated into neutrophil-like cells that exhibit an NADPH oxidase activity through direct stimulation of protein kinase C (PKC) with PMA or through formyl peptide receptor activation. We have isolated a variant HL60 clone that exhibited a conditional PMA-induced oxidative response depending on the agent used for the differentiation. While cells differentiated with DMSO responded to either PMA or N-formyl peptide (N-formyl-Met-Leu-Phe-Lys or fMLFK), cells differentiated with dibutyryl-cAMP (Bt2cAMP) responded to fMLFK but very poorly to PMA. However, in Bt2cAMP-differentiated cells, the expression of the different PKC isoforms was similar to that observed in DMSO-differentiated cells. Moreover, PMA was able to induce a normal phosphorylation of the cytosolic factor p47phox and to fully activate extracellular signal-regulated kinases (Erk1/2). Interestingly, Bt2cAMP-differentiated cells exhibited a strong and sustained O2− production when costimulated with PMA and suboptimal concentrations of fMLFK which were, per se, ineffective. This sustained response was only slightly reduced by the conjunction of the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor PD98059 and wortmannin, a phosphatidylinositol-3 kinase (PI3K) inhibitor. Variant HL60 cells that were stably transfected with a constitutively active form of Rac1 were able, when differentiated with Bt2cAMP, to secrete oxidant following PMA stimulation. Altogether, the results suggest that, in addition to the phosphorylation of p47phox, the activation of NADPH oxidase requires the activation of a Rac protein through a pathway that diverges at a point upstream of MEK and that is independent of the activation of wortmannin sensitive PI3K.
Persistent Expression of Experimental Autoimmune Encephalomyelitis (EAE)-Specific Vβ8.2 TCR Spectratype in the Central Nervous System of Rats with Chronic Relapsing EAE
Authors: Kim Giok , Kohyama Kuniko , Tanuma Naoyuki , Arimito Hirohiko , Matsumoto Yoh
Published: December 2022

Monitoring the TCR repertoire is indispensable for the assessment of T cell-associated autoimmune diseases and subsequent TCR-based immunotherapy. In the present study, we examined the TCR repertoire of spinal cord T cells of Lewis rats by CDR3 spectratyping during chronic relapsing experimental autoimmune encephalomyelitis (EAE) induced by immunization with spinal cord homogenate. It was found that Vβ8.2 spectratype with the shortest CDR3 expanded oligoclonally throughout the course of the disease. In addition, Vβ12 spectratype expansion was observed at the first and second attacks of EAE. Sequence analysis revealed that clones with the DSSYEQYF sequence, which is a representative sequence of myelin basic protein (MBP)-reactive T cell clones, constituted the predominant population in the Vβ8.2 family. Surprisingly, Vβ12 also used the identical amino acid sequence in the CDR3 region. These findings indicate that although infiltrating T cells in the central nervous system are activated polyclonally, the TCR repertoire remains unchanged throughout the course. Moreover, the finding that the predominant CDR3 amino acid sequence of Vβ8.2 and Vβ12 spectratypes is identical with that of MBP-induced EAE suggests that a single Ag in spinal cord homogenate, possibly MBP, is involved in disease development.
Characterization of the Pathogenic Autoreactive T Cells in Cyclosporine-Induced Syngeneic Graft-Versus-Host Disease
Authors: Chen Weiran , Thoburn Christopher , Hess Allan D.
Published: December 2022

Administration of the immunosuppressive drug cyclosporine after syngeneic bone marrow transplantation paradoxically elicits a systemic autoimmune syndrome resembling graft-vs-host disease (GVHD). This syndrome, termed syngeneic GVHD, is associated with the development of CD8+ cytolytic T lymphocytes that promiscuously recognize MHC class II molecules in association with a peptide from the invariant chain (CLIP). Clonal analysis reveals a major subset of cells that are pathogenic and require the N-terminal flanking region of CLIP for activation, while there is a minor subset of nonpathogenic T cells that require the C-terminal flanking region. The present studies show that pathogenic T cells produce type 1 cytokines (IL-2; IFN-γ), while the nonpathogenic clones produce type 2 cytokines (IL-4; IL-10). Moreover, the repertoire of the pathogenic T cells is highly conserved with respect to Vβ and Vα TCR gene expression. The vast majority of clones express Vβ8.5 (12/12) and Vα11 (11/12). Although a limited number was evaluated, the nonpathogenic clones have only a Vα restriction. Sequence analysis of the pathogenic T cell clones reveals a marked heterogeneity in the complementarity-determining region 3 domain and differential J region gene expression for both TCR α- and β-chains. Evaluation of the specificity of these clones suggests that the functional interaction between the N-terminal flanking region of CLIP (defined by the amino acid sequence -KPVSP-) and the V region of the TCR is critical, allowing effective target cell recognition and tissue destruction in syngeneic GVHD.
CD16 Cross-Linking Blocks Rearrangement of the TCRβ Locus and Development of αβ T Cells and Induces Development of NK Cells from Thymic Progenitors
Authors: Durum Scott K. , Lee Chong-Kil , Geiman Theresa M. , Murphy William J. , Muegge Kathrin
Published: December 2022

Mouse thymocytes normally develop into T lymphocytes, but the embryonic thymus also contains precursor cells capable of developing into NK cells. Here, we describe conditions that induce pro-T cells to develop into NK cells. CD16 is expressed on thymic pro-T cells. We observed that CD16 cross-linking during culture of embryonic thymic organs suppressed rearrangement of the TCRβ locus (but did not inhibit TCRγ locus rearrangement). Rearrangement of the TCRβ locus is normally required for development to the CD4+CD8+, and this development was also suppressed by CD16 cross-linking. The ability of CD16 cross-linking to block αβT cell development was not attributable to toxic effects, but rather was accompanied by promotion of development into NK cells, identified based on molecular and functional criteria. These results suggest that common lymphoid precursors can respond to environmental signals to commit to the αβT vs NK developmental pathways.
Epidermal Growth Factor (EGF) Modulates Fetal Thymocyte Growth and Differentiation: Partial Reversal by Insulin, Mimicking by Specific Inhibitors of EGF Receptor Tyrosine Kinase Activity, and Differential Expression of CD45 Phosphatase Isotypes
Authors: Freitas Claudia S. , Dalmau Sergio R. , Savino Wilson
Published: December 2022

We have recently reported that epidermal growth factor (EGF) modulates thymocyte development in fetal thymus organ cultures. Exogenously added EGF arrested thymocyte growth and differentiation, acting at the transition from the CD4−CD8− (double-negative (DN)) to the CD4+CD8+ (double-positive (DP)) phenotype. In this study, we further investigate some molecular aspects of this blockade. This inhibitory effect could be mimicked by tyrphostins, which are selective inhibitors of EGF receptor kinase activity. An attempt to use insulin (INS) as a synergizing effector resulted in partial restoration of lobe cellularity, leading to expansion of the CD44−CD25+ DN subset. However, INS did not overcome the EGF-driven blockade of the thymocyte DN → DP transition. Analysis of CD45 phosphatase showed that this transition was preceded by a rise in CD45RB isotype expression. At the end of a 7-day culture, the remaining DN cells from both EGF- and EGF+INS-treated fetal thymus organ cultures showed a CD45RB− phenotype and were negative for the EGF-immunoreactive molecule described previously on the fetal thymocyte surface. This finding implies that neither molecule is related to the growth capability of cells at this early developmental stage; it is more likely that the molecules are related to subsequent events in the thymocyte pathway to the DP phenotype. Thus, our data support the concept that EGF receptor-related circuitry may be relevant in thymus ontogeny. Additionally, evidence is provided for the duality between growth and differentiation at this particular early stage of thymocyte development.
IL-12 Up-Regulates IL-18 Receptor Expression on T Cells, Th1 Cells, and B Cells: Synergism with IL-18 for IFN-γ Production
Authors: Yoshimoto Tomohiro , Takeda Kiyoshi , Tanaka Takashi , Ohkusu Kazunobu , Kashiwamura Shin-ichiro , Okamura Haruki , Akira Shizuo , Nakanishi Kenji
Published: December 2022

IL-18 is a product of macrophages and with IL-12 strikingly induces IFN-γ production from T, B, and NK cells. Furthermore, IL-18 and IL-12 synergize for IFN-γ production from Th1 cells, although this combination fails to affect Th2 cells. In this study, we show that IL-12 and IL-18 promptly and synergistically induce T and B cells to develop into IFN-γ-producing cells without engaging their Ag receptors. We also studied the mechanism underlying differences in IL-18 responsiveness between Th1 and Th2 cells. Pretreatment of T or B cells with IL-12 rendered them responsive to IL-18, which induces cell proliferation and IFN-γ production. These IL-12-stimulated cells had both high and low affinity IL-18R and an increased IL-18R mRNA expression. In particular, IL-12-stimulated T cells strongly and continuously expressed IL-18R mRNA. However, when T cells developed into Th1 cells after stimulation with anti-CD3 and IL-12, they lowered this IL-12-induced-IL-18R mRNA expression. Then, such T cells showed a dominant response to anti-CD3 by IFN-γ production when they were subsequently stimulated with anti-CD3 and IL-18. In contrast, Th2 cells did not express IL-18R mRNA and failed to produce IFN-γ in response to anti-CD3 and IL-18, although they produced a substantial amount of IFN-γ in response to anti-CD3 and IL-12. However, when Th1 and Th2 cells were stimulated with anti-CD3, IL-12, and IL-18, only the Th1 cells markedly augmented IFN-γ production in response to IL-18, suggesting that IL-18 responsiveness between Th1 and Th2 cells resulted from their differential expression of IL-18R.
Impaired TCR-Mediated Apoptosis and Bcl-XL Expression in T Cells Lacking the Stress Kinase Activator SEK1/MKK4
Authors: Nishina Hiroshi , Radvanyi Laszlo , Raju Kaliannan , Sasaki Takehiko , Kozieradzki Ivona , Penninger Josef M.
Published: December 2022

The dual specificity kinase SEK1 (MKK4) is a direct activator of stress-activated protein kinases (SAPK/JNK) in response to environmental stresses or mitogenic factors. We show in Sek1−/−Rag−/− chimeric mice that a Sek1 null mutation augments the susceptibility of peripheral T cells to TCR/CD3 religation-induced apoptosis. Sek1−/− T cells failed to induce expression of the death suppressor Bcl-XL in response to Ag receptor activation. The Sek1 mutation did not alter the induction of apoptosis in response to etoposide, cisplatinum, Adriamycin, and γ-irradiation. Moreover, we show that CD3ε activation alone leads to SEK1 activation in Sek1+/+ T cells. These results suggest that SEK1 transduces cellular survival signals during T cell stimulation.
Carboxyl-Terminal 15-Amino Acid Sequence of NFATx1 Is Possibly Created by Tissue-Specific Splicing and Is Essential for Transactivation Activity in T Cells
Authors: Imamura Ryu , Masuda Esteban S. , Naito Yoshiyuki , Imai Shin-ichiro , Fujino Tadahiro , Takano Toshiya , Arai Ken-ichi , Arai Naoko
Published: December 2022

NFAT regulates transcription of a number of cytokine and other immunoregulatory genes. We have isolated NFATx, which is one of four members of the NFAT family of transcription factors and is preferentially expressed in the thymus and peripheral blood leukocytes, and an isoform of NFATx, NFATx1. Here we provide evidence showing that 15 amino acids in the carboxyl-terminal end of NFATx1 are required for its maximum transactivation activity in Jurkat T cells. A fusion between these 15 amino acids and the GAL4 DNA binding domain was capable of transactivating reporters driven by the GAL4 DNA binding site. Interestingly, this 15-amino acid transactivation sequence is well conserved in NFAT family proteins, although the sequences contiguous to the carboxyl-terminal regions of the NFAT family are much less conserved. We also report three additional isoforms of NFATx, designated NFATx2, NFATx3, and NFATx4. This transactivation sequence is altered by tissue-specific alternative splicing in newly isolated NFATx isoforms, resulting in lower transactivation activity in Jurkat T cells. NFATx1 is expressed predominantly in the thymus and peripheral blood leukocyte, while the skeletal muscle expressed primarily NFATx2. In Jurkat cells, transcription from the NFAT site of the IL-2 promoter is activated strongly by NFATx1 but only weakly by NFATx2. These data demonstrate that the 15-amino acid sequence of NFATx1 is a major transactivation sequence required for induction of genes by NFATx1 in T cells and possibly regulates NFAT activity through tissue-specific alternative splicing.
Identification of Two NF-κB Sites in Mouse CD95 Ligand (Fas Ligand) Promoter: Functional Analysis in T Cell Hybridoma
Authors: Matsui Ken , Fine Alan , Zhu Bangmin , Marshak-Rothstein Ann , Ju Shyr-Te
Published: December 2022

Fas ligand (FasL) gene expression is critically involved in peripheral T cell tolerance and lymphocyte homeostasis. Previous studies have suggested that nuclear translocation of NF-κB during T cell activation is a critical event for FasL gene activation. In the present study we have identified two NF-κB sites (designated FasL-κB1 and FasL-κB2) on the promoter (∼700 bp) of FasL. The NF-κB sites were identified by electrophoretic mobility shift assay. Transient transfection reporter analyses showed that the FasL promoter activity was comparable between a construct that contains both sites and a shorter construct (433 bp) that contains only the FasL-κB1 site. Furthermore, elimination of FasL-κB1 by site-directed mutagenesis significantly inhibited FasL promoter activity. These observations provide strong evidence that NF-κB directly binds to the FasL-κB1 site and up-regulates FasL gene expression.
lck-Independent Inhibition of T Cell Antigen Response by the HIV gp120
Authors: Gratton Sophie , Julius Michael , Sékaly Rafick-Pierre
Published: December 2022

Binding of the HIV envelope glycoprotein gp120 to CD4 inhibits T cell activation. We have used a murine T cell clone transfected with either wild-type human CD4 or mutated forms of CD4 to characterize the pathways involved in this inhibitory effect of gp120. Ag-induced proliferation of T cell clones transfected with human CD4 was completely inhibited in the presence of gp120, even though stimulation of this clone is independent of a CD4/MHC class II interaction. In addition, our results demonstrate that the inhibition by gp120 is not due to the sequestration of lck from TCR and does not require activation of lck by gp120. This suggests that CD4 can regulate the initiation of T cell activation independently of its interaction with lck. Moreover, we demonstrate that the nonresponsiveness induced by gp120 can be reversed by soluble CD4 when added early after onset of stimulation and that gp120 exerts its inhibitory effect when cells are in the G0 ≥ 1 phase of the cell cycle.
In Vivo and In Vitro Activities of the gp130-Stimulating Designer Cytokine Hyper-IL-6
Authors: Peters Malte , Blinn Guido , Solem Fian , Fischer Martina , zum Büschenfelde Karl-Hermann Meyer , Rose-John Stefan
Published: December 2022

IL-6 is a multifactorial cytokine mediating acute inflammatory responses in the liver. When IL-6 binds to a specific receptor (IL-6R), the IL-6/IL-6R complex associates with the signal transducer gp130, initiating intracellular signaling. A soluble form of the IL-6R (sIL-6R) renders target cells sensitive to IL-6 that do not express the IL-6R on their surfaces. A designer cytokine, termed Hyper-IL-6, consisting of IL-6 covalently linked to the sIL-6R was fully active on gp130-expressing cells at 100- to 1000-fold lower concentrations than unlinked IL-6 and IL-6R. Mice were injected i.p. with Hyper-IL-6 or IL-6. Upon injection of Hyper-IL-6 into mice, the acute phase response, as measured by haptoglobin mRNA expression in the liver, was markedly increased and lasted significantly longer compared with that in mice injected with a 10-fold higher dose of IL-6 alone. On human hepatoma cells, Hyper-IL-6 caused similar effects, indicating that the longer lasting response to the fusion protein could not only be explained by the longer plasma half-life of the fusion protein. Experiments using iodinated IL-6 and Hyper-IL-6 revealed that Hyper-IL-6 bound with high affinity to gp130 and was less efficiently internalized. This effect might explain the longer lasting activity of this protein on cells. The highly active IL-6/sIL-6R designer protein might be of significant clinical importance for the stimulation of cells that are more responsive to the IL-6/sIL-6R complex than to IL-6 alone. Such cells include hemopoietic progenitor cells and hepatocytes.
Soluble ICAM-1 Activates Lung Macrophages and Enhances Lung Injury
Authors: Schmal Hagen , Czermak Boris J. , Lentsch Alex B. , Bless Nicolas M. , Beck-Schimmer Beatrice , Friedl Hans P. , Ward Peter A.
Published: December 2022

Because of the important role of rat ICAM-1 in the development of lung inflammatory injury, soluble recombinant rat ICAM-1 (sICAM-1) was expressed in bacteria, and its biologic activities were evaluated. Purified sICAM-1 did bind to rat alveolar macrophages in a dose-dependent manner and induced production of TNF-α and the CXC chemokine, macrophage inflammatory protein-2 (MIP-2). Alveolar macrophages exhibited cytokine responses to both sICAM-1 and immobilized sICAM-1, while rat PBMCs failed to demonstrate similar responses. Exposure of alveolar macrophages to sICAM-1 resulted in NFκB activation (which was blocked by the presence of the aldehyde peptide inhibitor of 28S proteosome and by genistein, a tyrosine kinase inhibitor). As expected, cross-linking of CD18 on macrophages with Ab resulted in generation of TNF-α and MIP-2. This response was also inhibited in the presence of the proteosome inhibitor and by genistein. Alveolar macrophages showed adherence to immobilized sICAM-1 in a CD18-dependent manner. Finally, airway instillation of sICAM-1 intensified lung injury produced by intrapulmonary deposition of IgG immune complexes in a manner associated with enhanced lung production of TNF-α and MIP-2 and increased neutrophil recruitment. Therefore, through engagement of β2 integrins, sICAM-1 enhances alveolar macrophage production of MIP-2 and TNF-α, the result of which is intensified lung injury after intrapulmonary disposition of immune complexes.
A RANTES-Antibody Fusion Protein Retains Antigen Specificity and Chemokine Function
Authors: Challita-Eid Pia M. , Abboud Camille N. , Morrison Sherie L. , Penichet Manuel L. , Rosell Karen E. , Poles Tina , Hilchey Shannon P. , Planelles Vicente , Rosenblatt Joseph D.
Published: December 2022

The successful eradication of cancer cells in the setting of minimal residual disease may require targeting of metastatic tumor deposits that evade the immune system. We combined the targeting flexibility and specificity of mAbs with the immune effector function of the chemokine RANTES to target established tumor deposits. We describe the construction of an Ab fusion molecule with variable domains directed against the tumor-associated Ag HER2/neu, linked to sequences encoding the chemokine RANTES (RANTES.her2.IgG3). RANTES is a potent chemoattractant of T cells, NK cells, monocytes, and dendritic cells, and expression of RANTES has been shown to enhance immune responses against tumors in murine models. RANTES.her2.IgG3 fusion protein bound specifically to HER2/neu Ag expressed on EL4 cells and on SKBR3 breast cancer cells as assayed by flow cytometry. RANTES.her2.IgG3 could elicit actin polymerization of THP-1 cells and transendothelial migration of primary T lymphocytes. RANTES.her2.IgG3 prebound to SKBR3 cells also facilitated migration of T cells. RANTES.her2.IgG3 bound specifically to the CCR5 chemokine receptor, as demonstrated by flow cytometry, and inhibited HIV-1 infection via the CCR5 coreceptor. RANTES.her2.IgG3, alone or in combination with other chemokine or cytokine fusion Abs, may be a suitable reagent for recruitment and activation of an expanded repertoire of effector cells to tumor deposits.
Cytokine-Induced Protein Kinase B Activation and Bad Phosphorylation Do Not Correlate with Cell Survival of Hemopoietic Cells
Authors: Hinton Heather J. , Welham Melanie J.
Published: December 2022

Activation of phosphoinositide-3 kinases (PI3Ks), their downstream target protein kinase B (PKB), and phosphorylation of Bad have all been implicated in survival signaling in many systems. However, it is not known whether these events are sufficient or necessary to universally prevent apoptosis. To address this issue, we have used three different factor-dependent hemopoietic cell lines, MC/9, BaF/3, and factor-dependent (FD)-6, which respond to a range of cytokines, to investigate the relationship between PI3K, PKB, and Bad activity with survival. The cytokines IL-3, IL-4, stem cell factor (SCF), GM-CSF, and insulin all induced the rapid and transient activation of PKB in responsive cell lines. In all cases, cytokine-induced PKB activation was sensitive to inhibition by the PI3K inhibitor, LY294002. However, dual phosphorylation of the proapoptotic protein Bad was found not to correlate with PKB activation. In addition, we observed cell-type-specific differences in the ability of the same cytokine to induce Bad phosphorylation. Whereas IL-4 induced low levels of dual phosphorylation of Bad in FD-6, it was unable to in MC/9 or BaF/3. Insulin, which was the most potent inducer of PKB in FD-6, induced barely detectable Bad phosphorylation. In addition, the ability of a particular cytokine to induce PKB activity did not correlate with its ability to promote cell survival and/or proliferation. These data demonstrate that, in hemopoietic cells, activation of PKB does not automatically confer a survival signal or result in phosphorylation of Bad, implying that other survival pathways must be involved.
Human Vascular Endothelial Cells Favor Clonal Expansion of Unusual Alloreactive CTL
Authors: Biedermann Barbara C. , Pober Jordan S.
Published: December 2022

We have shown previously that cultured HUVEC or mixtures of endothelial cells (EC) and B lymphoblastoid cells (BLC) induce the differentiation of purified CD8+ PBL into allospecific, class I MHC-restricted CTL that lyse EC, but not BLC autologous to EC. Furthermore, these EC-selective CTL lines secrete little IFN-γ after target cell contact. In the present study, we have analyzed these polyclonal populations at a single cell level by cloning at limiting dilution and propagating the resulting CTL clones in the absence of EC. Phenotypically stable, alloreactive EC-selective CTL preferentially emerge from cocultures in which EC or EC + BLC are the initial stimulating cell types compared with cocultures stimulated by BLC alone (p = 0.005). Compared with BLC-stimulated CTL, EC-stimulated CTL clones often fail to secrete IFN-γ after target cell contact (p = 0.0006) and constitutively express CD40 ligand (CD40L) at rest (p = 0.0006). The absence of IFN-γ secretion does not result from a switch to IL-4 secretion. The expression of CD40L inversely correlates with the secretion of IFN-γ after target cell contact (p = 0.0001), but correlations of CD40L expression and failure to secrete IFN-γ with EC-selective killing did not reach statistical significance. We conclude that in a microenvironment in which allogeneic EC are in close contact with infiltrating CD8+ T cells, such as within a graft arterial intima, CTL subsets may emerge that display EC selectivity or express CD40L and secrete little IFN-γ after Ag contact.
Reactive Oxygen Intermediates Enhance Fcγ Receptor Signaling and Amplify Phagocytic Capacity
Authors: Pricop Luminita , Gokhale Jayashree , Redecha Patricia , Ng Sonia C. , Salmon Jane E.
Published: December 2022

Receptors for the Fc region of IgG (FcγR) mediate internalization of opsonized particles by human neutrophils (PMN) and mononuclear phagocytes. Cross-linking of FcγR leads to activation of protein tyrosine kinases and phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) within FcγR subunits, both obligatory early signals for phagocytosis. Human PMN constitutively express two structurally distinct FcγR, FcγRIIa and FcγRIIIb, and can be induced to express FcγRI by IFN-γ. We have previously shown that stimulation of PMN through FcγRIIIb results in enhanced FcγRIIa-mediated phagocytic activity that is inhibited by catalase. In the present study, we have tested the hypothesis that reactive oxygen intermediates (ROI) have the capacity to regulate FcγR responses and defined a mechanism for this effect. We show that H2O2 augmented phagocytosis mediated by FcγRIIa and FcγRI in PMN and amplified receptor-triggered tyrosine phosphorylation of FcγR-associated ITAMs and signaling elements. Generation of endogenous oxidants in PMN by cross-linking FcγRIIIb similarly enhanced phosphorylation of FcγRIIa and Syk, a tyrosine kinase required for phagocytic function, in a catalase-sensitive manner. Our results provide a mechanism for priming phagocytes for enhanced responses to receptor-driven effects. ROI generated in an inflammatory milieu may stimulate quiescent cells to rapidly increase the magnitude of their effector function. Indeed, human monocytes incubated in the presence of stimulated PMN showed oxidant-induced increases in FcγRIIa-mediated phagocytosis. Definition of the role of oxidants as amplifiers of FcγR signaling identifies a target for therapeutic intervention in immune complex-mediated tissue injury.