bluuebunny/crossref_metadata_2025 huggingface dataset:
CD44 regulates phagocytosis of apoptotic neutrophil granulocytes, but not apoptotic lymphocytes, by human macrophages.
Authors: Hart S P , Dougherty G J , Haslett C , Dransfield I
Published: January 2023

Phagocytosis of apoptotic neutrophil granulocytes by macrophages at inflammatory sites is an important determinant of the process by which inflammation resolves. We demonstrate that phagocytosis of apoptotic neutrophils, but not apoptotic lymphocytes, by human monocyte-derived macrophages is augmented rapidly following ligation of CD44 by bivalent Abs in vitro. Previously defined inhibitors of apoptotic cell recognition did not affect CD44-augmented phagocytosis of apoptotic neutrophils, suggesting that unique molecular recognition pathways are involved. These observations, together with the lack of effect of CD44 Abs upon macrophage phagocytosis of zymosan or Ig-opsonized erythrocytes, imply that CD44 may regulate the differential clearance of apoptotic leukocytes during evolution of inflammatory responses. This represents a novel role for CD44 in inflammation and tissue repair.
Role of the P2 residue of complement 1 inhibitor (Ala443) in determination of target protease specificity: inhibition of complement and contact system proteases.
Authors: Zahedi R , Wisnieski J , Davis A E
Published: January 2023

A dysfunctional C1 inhibitor (C1 INH) from a family in whom the propositus presented with systemic lupus erythematosus but without angioedema previously was shown to have diminished inhibitory activity toward isolated C1r and C1s, and intact C1. The mutation was identified as replacement of Ala443 (P2) with Val. This study further analyzed the reactivity of this mutant and characterized two mutants with Ser or Asp at this position. Ser at P2 does not interfere with binding of target proteases. However, the mutant with Asp at this position is unable to bind C1r and beta factor XIIa, and also has a decreased rate of reaction with C1s and kallikrein. Therefore, alteration of polarity alone had no effect on binding, while a bulky and/or charged side chain was not tolerated. Although defective in inhibition of C1r and C1s, the P2 A-->V mutant had acquired the ability to complex with trypsin. It also completely retained the ability to complex with kallikrein and factor XIIa. None of the 10 individuals expressing this mutant protein has ever had angioedema. This observation, combined with normal inhibition of contact system proteases and defective inhibition of complement proteases, suggests that angioedema is caused by bradykinin generated from contact system activation.
Involvement of laminin and its receptor in abrogation of heart graft rejection by autoreactive T cells from Trypanosoma cruzi-infected mice.
Authors: Silva-Barbosa S D , Cotta-de-Almeida V , Riederer I , De Meis J , Dardenne M , Bonomo A , Savino W
Published: January 2023

Extracellular matrix ligands and receptors have been identified as determining in vivo lymphocyte positioning and activation, including effector functions in alloreactive responses. Herein we evaluated the involvement of laminin and its receptor, the very late antigen 6 (VLA-6) integrin, in CD4+ T cell-dependent autoreactivity, using a transplantation model for the autoimmune myocarditis occurring in mice chronically infected with Trypanosoma cruzi. Previous work showed that syngeneic mouse hearts grafted in the ears of chronic chagasic recipients were rejected through a CD4+ T cell-dependent mechanism. Rejection also occurred when cells from chagasic animals were transferred adjacent to hearts transplanted into naive recipients. Here, we observed the formation of a thick laminin network during rejection, with donor-derived CD4+ T cells concentrated in the laminin-rich areas. Most importantly, anti-laminin as well as anti-laminin receptor Ab inhibited the rejection of syngeneic hearts by T cells from chagasic animals. Our results suggest that interaction of the VLA-6 molecule with laminin is involved in triggering the antimyocardial autoreactive process by driving the influx of CD4+ T cells to the heart. They also support the concept that an Ag-specific T cell response, even an autoreactive one, can be modulated by in vivo interactions involving extracellular matrix ligands and receptors. In this regard, our study represents, to our knowledge, the first in vivo evidence for laminin-mediated T cell echotaxis, with simultaneous experimental demonstration of ligand and receptor involvement. Lastly, our findings indicate that treatment with anti-VLA-6 Abs can be effective in suppressing autoimmune disease activity.
Regulation of C-C chemokine production by murine T cells by CD28/B7 costimulation.
Authors: Herold K C , Lu J , Rulifson I , Vezys V , Taub D , Grusby M J , Bluestone J A
Published: January 2023

C-C chemokines play an important role in recruitment of T lymphocytes to inflammatory sites. T lymphocytes secrete chemokines, but the activation requirements for chemokine production by T cells are uncertain. We studied the regulation of C-C chemokine production by CD28 costimulatory signals by murine T lymphocytes. Splenocytes from BALB/c mice cultured with anti-CD3 mAb expressed macrophage-inflammatory protein (MIP)-1alpha mRNA and secreted MIP-1alpha, which was inhibited by anti-B7-1 plus anti-B7-2 mAbs. MIP-1alpha production by Ag-stimulated T cells from DO.11.10 TCR transgenic mice was augmented by anti-CD28 mAb and increased compared with DO.11.10/CD28(-/-) cells. When T cell costimulation was provided by IL-2, MIP-1alpha was not enhanced. Studies with IL-2, IL-4, STAT4, and STAT6 knock-out mice suggested that chemokine production is controlled by pathways different from those regulating T cell differentiation. Thus, CD28 costimulation may amplify an immune response by stimulating T cell survival, proliferation, and production of chemokines that recruit T cells to inflammatory sites.
Distinct roles for B7-1 and B7-2 determinants during priming of effector CD8+ Tc1 and regulatory CD4+ Th2 cells for contact hypersensitivity.
Authors: Xu H , Heeger P S , Fairchild R L
Published: January 2023

Contact hypersensitivity (CHS) is a CD8+ T cell-mediated response to hapten sensitization and challenge of the epidermis. Both the IFN-gamma-producing effector CD8+ T cells and the IL-4/IL-10-producing CD4+ T (Th2) cells that restrict the magnitude and duration of the response are primed by hapten-presenting Langerhans cells (hpLC). hpLC isolated from hapten-sensitized animals expressed high levels of B7-2 and lower levels of B7-1, suggesting the availability of each molecule to provide costimulation during CD8+ and CD4+ T cell priming for CHS. Anti-B7-2 Ab given during hapten sensitization inhibited effector CD8+ and regulatory CD4+ T cell development and the magnitude of the CHS response. Cytokine production by CD8+ and CD4+ T cells was reduced in anti-B7-2 Ab-treated animals, indicating that costimulation for T cell development was not compensated by hpLC-expressed B7-1. Anti-B7-1 Ab inhibited CHS and increased the number of Th2 cells primed during hapten sensitization without inhibiting development of the IFN-gamma-producing CD8+ T cells. Depletion of CD4+ T cells, however, abrogated the inhibitory effect of anti-B7-1 Ab on the CHS response, indicating the amplified Th2 response mediated this inhibition. These results indicate a requirement for B7-2 costimulation by effector CD8+ and regulatory CD4+ T cells during priming for CHS. The ability of anti-B7-1 Abs to amplify the regulatory CD4+ Th2 component and inhibit CHS suggests a strategy to inhibit unwanted CD8+ T cell-mediated immune responses to Ags in the skin.
Human T cell leukemias with continuous V(D)J recombinase activity for TCR-delta gene deletion.
Authors: Breit T M , Verschuren M C , Wolvers-Tettero I L , Van Gastel-Mol E J , Hählen K , van Dongen J J
Published: January 2023

The so-called TCR-delta-deleting elements, deltaRec and psiJ alpha, flank the major part of the TCR-delta gene complex. By rearranging to each other, the deltaRec and psiJ alpha gene segments delete the TCR-delta gene complex and prepare the allele for subsequent TCR-alpha rearrangement. This intermediate rearrangement is thought to be a specific rearrangement event. In our studies on TCR-delta deletion mechanisms, we identified several T cell acute lymphoblastic leukemias (T-ALL) with continuous activity of the deltaRec-psiJ alpha rearrangement process. Extensive Southern blot, PCR, and sequencing analyses on the coding joints as well as the signal joints of the deltaRec-psiJ alpha rearrangements in these patients allowed us to prove that this continuous rearrangement activity occurred in the leukemic cells and that these cells, therefore, represent a polyclonal subpopulation within the otherwise monoclonal T-ALL. In additional studies, we also identified a T cell line (DND41) with continuous activity of the deltaRec-psiJ alpha rearrangement process. Our data suggest that the ongoing deltaRec-psiJ alpha gene rearrangements predominantly occur in T cells that cannot express a functional TCR-gammadelta, due to biallelic out-of-frame TCR-delta and/or TCR-gamma gene rearrangements. The described T-ALL and the T cell line can serve as an experimental model in further studies on the regulatory elements involved in the specific deletion of the TCR-delta gene complex.
Induction of STAT protein signaling through the CD40 receptor in B lymphocytes: distinct STAT activation following surface Ig and CD40 receptor engagement.
Authors: Karras J G , Wang Z , Huo L , Frank D A , Rothstein T L
Published: January 2023

Cross-linking CD40 mediates B lymphocyte growth and differentiation and regulates cell death pathways by an unknown mechanism. Previous reports have suggested that protein tyrosine kinase activity is critical for CD40-mediated biologic responses. We show here that CD40 engagement on murine B cells results in the rapid tyrosine phosphorylation of the STAT6 transcription factor and the transactivation of a reporter gene containing an IFN-regulatory factor-1 STAT-binding site. In earlier studies, surface Ig engagement was found to produce rapid activation of STAT6 accompanied by later induction of STAT1, which is not observed after CD40 ligation. Thus, these results define mitogenic receptor-specific induction of STAT proteins in B cells and identify a novel and direct signal transduction pathway from the cell surface to the nucleus activated in B cells stimulated through CD40 that regulates a gene previously shown to be involved in oncogenesis and programmed cell death.
Recombining sequence recombination in normal kappa-chain-expressing B cells.
Authors: Dunda O , Corcos D
Published: January 2023

Recombining sequence (RS) recombination is a DNA rearrangement that deletes one or two C kappa alleles in a large proportion of lambda-expressing B cells. Since its discovery, this recombination has been suggested to play a role in activating lambda gene rearrangements. A model involving a positive signal generated by RS recombination seems to be excluded, but another model that is still under consideration proposes that RS recombination removes DNA sequences within the kappa locus that would interfere with lambda gene assembly. Using PCR assays, we have found that kappa-expressing cells account for the majority of RS rearrangements in mouse spleen. RS rearrangements were also detected by Southern blot in kappa-secreting hybridomas. Quantification of rearrangements indicates that approximately 12% of kappa cells bear an RS recombination. From this finding, we infer that once a cell has performed an RS recombination on one kappa allele, it has a 3 times higher probability of rearranging functionally its other kappa allele rather than one of the lambda genes. These data call into question the role of RS recombination in the switch from kappa to lambda and suggest another function for this nonproductive rearrangement.
Cloning and characterization of the human recombination activating gene 1 (RAG1) and RAG2 promoter regions.
Authors: Zarrin A A , Fong I , Malkin L , Marsden P A , Berinstein N L
Published: January 2023

Recombination activating gene 1 (RAG1) and RAG2 are the essential and tissue-specific components of V(D)J recombination. We have characterized the genomic organization of the human RAG locus, mapped the transcriptional initiation sites, and partially sequenced and performed functional reporter assays on the 5' flanking regions of human RAG1 and RAG2. Transcription initiation sites were mapped by rapid amplification of 5' cDNA ends, primer extension, and/or RNase protection in normal thymocytes, three pre-B cell lines, and a mature B cell line. A single promoter region was used for RAG1 transcription. In contrast, transcription of RAG2 initiates at two distinct regions of the genome. The 5'-flanking region of the human RAG2 gene is TATA-less; however, there is a GATAA consensus at position -34 with respect to the major transcriptional initiation site of RAG1. Promoter regions of human RAG1 and RAG2 are active in both lymphoid and nonlymphoid cell lines, suggesting that an outside regulatory element is probably involved in the tissue-specific transcriptional regulation of the RAG genes.
Intracellular fate of vacuoles containing Toxoplasma gondii is determined at the time of formation and depends on the mechanism of entry.
Authors: Mordue D G , Sibley L D
Published: January 2023

We have characterized the intracellular fate of Toxoplasma in bone marrow-derived macrophages following two disparate modes of uptake: phagocytosis vs active invasion. The fate of parasite-containing vacuoles was followed by immunofluorescence localization of endogenous endocytic markers to track phagocytic processing in pulse-infected cells. During uptake of both opsonized and dead parasites, host cell plasma membrane MHC class II molecules and FcR were internalized into the phagosome and then gradually lost. Maturation of phagosomes containing Toxoplasma was a rapid, dynamic process of sequential fusion with early endosomes, late endosomes, and lysosomes that was complete within 15 min. Toxoplasma-containing phagosomes were transiently positive for transferrin receptor between 0 and 2.5 min, then contained the cation-independent mannose 6-phosphate receptor between 2.5 and 7.5 min, and finally matured to lysosome-like compartments containing lysosomal membrane glycoprotein 1 and the proton pump, but lacking cation-independent mannose 6-phosphate receptor. Toxoplasma-containing phagosomes also sequentially acquired host proteins that regulate endocytic fusion including rab5, N-ethylmaleimide-sensitive factor, and rab7. In marked contrast, MHC class II molecules and FcR were excluded from the parasitophorous vacuole formed by active parasite invasion. The parasitophorous vacuole also failed to acquire any host compartmental markers or fusion proteins analyzed. Our results indicate that Toxoplasma evades endocytic processing due to an absence of host regulatory proteins necessary to drive endocytic fusion, and that this divergence from normal maturation occurs during the formation of the primary vacuole.
Parasite-driven in vitro human lymphocyte cytotoxicity against autologous infected macrophages from mucosal leishmaniasis.
Authors: Brodskyn C I , Barral A , Boaventura V , Carvalho E , Barral-Netto M
Published: January 2023

Parasite-specific cytotoxicity in human leishmaniasis was evaluated in an autologous system. PBL from cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML) patients were exposed to Leishmania amazonensis-infected autologous macrophages for 7 days and then used as effector cells in a cytotoxic assay using 51Cr-labeled autologous infected macrophages as targets. Results are reported as LU per 10(7) PBMC. Cytotoxic activity is present in ML (9.7 +/- 2.1 LU/10(7) PBMC) but not in CL (1.5 +/- 2.4 LU/10(7) PBMC) patients' lymphocytes, and the differences were highly significant (p < 0.0001). Both CD8+ T cells and NK cells exhibited cytotoxic activity. Addition of rIL-12, but not of IFN-gamma, during the generation of effector cells increased cytotoxic responses against infected macrophages. On the other hand, addition of mAb against human IL-12 or IFN-gamma during the stimulation of PBL significantly decreased the cytotoxic responses. Addition of IL-10 led to diminished cytotoxic responses, whereas the addition of anti-IL-10 did not significantly increase the cytotoxic responses. The observation of parasite-driven autologous cytotoxic responses in patients with ML, the destructive form of leishmaniasis, but not in CL, suggests that this phenomenon is involved in tissue pathology rather than in protection. Understanding the regulation of cytotoxic responses in leishmaniasis may be relevant to strategies aimed at limiting pathologic tissue destruction.
Activation of p38mapk, MKK3, and MKK4 by TNF-alpha in mouse bone marrow-derived macrophages.
Authors: Winston B W , Chan E D , Johnson G L , Riches D W
Published: January 2023

TNF-alpha regulates the expression of many proinflammatory and profibrogenic gene products in macrophages, and hence plays a vital role in controlling the inflammatory response. We have shown previously that exposure of macrophages to TNF-alpha stimulates the activation of members of the mitogen-activated protein kinase (MAPK) family. In this study, we have investigated the mechanism of activation of the p38mapk by TNF-alpha in mouse bone marrow-derived macrophages. Exposure to TNF-alpha resulted in the activation of p38mapk, as measured by 1) the trans-phosphorylation of recombinant activating transcription factor-2 substrate by immunoprecipitated p38mapk and 2) specific tyrosine phosphorylation of immunoprecipitated p38mapk. In addition, selective ligation of the TNF-alpha receptor CD120a (p55) with human TNF-alpha was sufficient to induce p38mapk activation. Using an in vitro kinase assay with recombinant kinase-inactive p38mapk as substrate in the presence of [gamma-32P]ATP, the upstream kinases MKK3 (mitogen-activated protein kinase kinase 3) and MKK4 were found to be activated in response to TNF-alpha. These findings suggest that TNF-alpha transiently phosphorylates and activates the three members of the MAPK family, namely p42(mapk/erk2), p46 c-Jun amino-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38mapk following cross-linking of CD120a (p55), and that MKK3 and MKK4 are capable of phosphorylating p38mapk.
Sensitive responses of leukocytes to lipopolysaccharide require a protein distinct from CD14 at the cell surface.
Authors: Vasselon T , Pironkova R , Detmers P A
Published: January 2023

Responses of leukocytes to low concentrations of LPS require the expression of membrane-associated CD14 (mCD14) on the cell surface; mCD14 is, however, a glycosyl-phosphatidylinositol-anchored protein and, therefore, a poor candidate for transducing signals across the plasma membrane. The role of other cell surface proteins in responses of leukocytes to LPS was tested by measuring IL-6 secretion of cultured human monocytes and adhesion of PMN in response to LPS after treatment of the cells with trypsin. Trypsin abolished leukocyte integrin-mediated adhesion of PMN in response to LPS, but the trypsinized cells remained responsive to the alternate agonists TNF, formyl peptide, and PMA. Similarly, trypsin treatment of monocytes inhibited IL-6 production in response to LPS, but not to formyl peptide or PMA. No change in cell surface expression of mCD14 was observed by cytofluorometry, and no proteolysis of mCD14 was detected by immunoblot analysis. These results suggest that digestion of a cell surface protein distinct from mCD14 must account for the loss of responsiveness to LPS. The uptake by monocytes of [3H]LPS presented in LPS-soluble CD14 complexes was also inhibited by trypsin treatment. Monomeric LPS was taken up superstoichiometrically to mCD14 with a ratio of up to 15:1, and trypsin treatment decreased this uptake by more than half. This suggests that a cell surface protein may function in accepting LPS from mCD14. Together the results suggest that one or more trypsin-sensitive cell surface proteins distinct from CD14 participate in both the uptake of LPS by leukocytes and the initiation of the signaling process.
Synergy between L-selectin signaling and chemotactic activation during neutrophil adhesion and transmigration.
Authors: Tsang Y T , Neelamegham S , Hu Y , Berg E L , Burns A R , Smith C W , Simon S I
Published: January 2023

L-selectin enables capture and rolling of neutrophils on inflamed endothelium. This may facilitate the binding of agonists such as IL-8 and platelet-activating factor (PAF), which signal CD18-mediated firm adhesion and transmigration. Recent studies demonstrate that L-selectin can mediate transmembrane signaling. However, the functional effects of costimulation through agonist and L-selectin require further study. Here, we quantify cell adhesion, motility, and transmigration in response to co-activation through L-selectin and agonist. The surface expression of CD11b/CD18 increased and L-selectin decreased in proportion to the extent of L-selectin cross-linking. A flow cytometric assay was used to measure CD11b/CD18-dependent adhesion to fluorescent beads adsorbed with albumin. Neutrophil adhesion was detected within seconds of adding PAF (20 pM), IL-8 (50 pM), or cross-linking L-selectin. Costimulation through agonist and L-selectin potentiated by up to threefold the rate and extent of bead capture. Stimulation through L-selectin induced membrane ruffling, whereas PAF or IL-8 induced bipolar shape change. L-selectin cross-linking sustained the transient shape change induced by low concentrations (10-50 pM) of agonist. Chemokinesis stimulated by IL-8 was inhibited in the presence of cross-linking L-selectin. This was attributed to enhanced cell spreading following costimulation. Migration across HUVEC monolayers stimulated with IL-1 was also potentiated in the presence of L-selectin cross-linking. We propose that cross-linking of L-selectin and binding of agonist receptors may act synergistically to amplify neutrophil activation and emigration in the inflamed vasculature.
Regulation of anti-HIV-1 activity of RANTES by heparan sulfate proteoglycans.
Authors: Oravecz T , Pall M , Wang J , Roderiquez G , Ditto M , Norcross M A
Published: January 2023

The role of cell surface proteoglycans in CC chemokine-mediated anti-HIV-1 activity in T cells and macrophages was investigated. Enzyme digestion of heparan sulfate (HS), but not chondroitin sulfate, from the surface of PM1(CD26H) cells (a human T cell line selected for high CD26 expression) rendered them resistant to the antiviral effects of RANTES and macrophage-inflammatory protein-1beta at otherwise inhibitory chemokine concentrations. HIV-1 infection of macrophages, however, was inhibited only partially, even at high concentrations of RANTES, and this inhibition was not prevented by HS removal. Flow cytometry revealed that digestion of cell surface proteoglycans, including HS, prevented the binding of RANTES at 10 to 100 nM concentrations to PM1(CD26H) cells. However, the binding of RANTES to activated macrophages occurred only at higher concentrations (100-300 nM) and was mostly chondroitin sulfate, and not HS, dependent. These results support a role for HS in facilitating the interaction of CC chemokines with the cell surface and the consequent inhibition of HIV-1 infection. The absence of HS-dependent binding of RANTES at lower concentrations to macrophages is consistent with the resistance of these cells to the antiviral effects of chemokines.
Cutting Edge: Critical Role of NK1+ T Cells in IL-12-Induced Immune Responses In Vivo
Authors: Kawamura Toshihiko , Takeda Kazuyoshi , Mendiratta Sanjeev K. , Kawamura Hiroki , Van Kaer Luc , Yagita Hideo , Abo Toru , Okumura Ko
Published: January 2023

CD1-dependent NK1+ T cells rapidly produce IL-4 upon stimulation through the TCR. These cells may therefore play an important role in the initiation of Th2 responses. Here, we show that NK1+ T cells constitutively express receptors for IL-12 and IFN-γ, and that IL-12 induces production of perforin in these cells. Moreover, while IL-12 induces high levels of IFN-γ and cytotoxic activity of hepatic or splenic mononuclear cells against tumor cells, this effect of IL-12 is significantly reduced in CD1-deficient mice with impaired NK1+ T cells development. These results indicate that NK1+ T cells play a critical role in IL-12-induced production of IFN-γ to initiate Th1 immune responses and as IL-12-induced cytotoxic effector cells to initiate antitumor immunity.
Nuclear Factor of Activated T Cells (NFAT) as a Molecular Target for 1α,25-Dihydroxyvitamin D3-Mediated Effects
Authors: Takeuchi Atsuko , Reddy G. Satyanarayana , Kobayashi Tadashi , Okano Toshio , Park Jungchan , Sharma Surendra
Published: January 2023

The molecular basis of the immunomodulatory properties of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) remains elusive. We demonstrate here that 1α,25(OH)2D3-mediated suppressive effects on the inducible expression of cytokine genes in human T cells may, in part, be due to diminished activity of the transcription factor NFAT. The vitamin D3 receptor (VDR) and its heterodimeric partner retinoid X receptor α (RXRα) specifically bound to the distal NFAT site in the human IL-2 promoter, and this binding was abolished by mutating unique regions in the NFAT oligonucleotide. In vitro inhibition of NFAT complex formation was noted when VDR-RXRα heterodimers were added to DNA binding reactions containing nuclear extracts from activated B or T cells, whereas in vitro NFκB complex formation was not significantly influenced. Furthermore, 1α,25(OH)2D3 treatment of activated T cells resulted in decreased formation of NFAT complexes detected upon incubation of nuclear extracts from these cells with 32P-labeled probe. Transient expression of both VDR and RXRα, but not of a single component, was capable of inhibiting expression of a NFAT-driven reporter gene in stimulated Jurkat cells in a ligand-dependent manner. These results suggest that NFAT plays a crucial role in 1α,25(OH)2D3-mediated immunosuppressive activity.
Identification of Distinct Regions of 5′ Flanking DNA That Mediate Constitutive, IFN-γ, STAT1, and TGF-β-Regulated Expression of the Class II Transactivator Gene
Authors: Piskurich Janet F. , Wang Ying , Linhoff Michael W. , White Leigh C. , Ting Jenny P.-Y.
Published: January 2023

Class II transactivator (CIITA) is a master regulator required for constitutive and IFN-γ-inducible expression of class II MHC genes. Although the role of CIITA is greatly appreciated, the mechanisms underlying constitutive and IFN-γ-induced expression of CIITA are not understood. The study of CIITA induction is extremely important, but has been fraught with difficulty. This study describes for the first time a large (7-kb) fragment of 5′ flanking sequences that mediates the B cell-specific, IFN-γ-induced, and TGF-β-suppressed expression of CIITA. This pattern of expression matches the authentic expression of the endogenous gene. Within the 7-kb fragment, sequences that lie between nucleotides −545 and −113 relative to the transcriptional start site are critical for constitutive promoter expression in B cells. In contrast, inducible activation of CIITA by IFN-γ requires sequences contained in an additional 4 kb of upstream DNA. This region mediates an IFN-γ response when linked to either the endogenous CIITA promoter or a heterologous promoter. A role for STAT1 in regulation of the CIITA promoter is shown by the rescue of IFN-γ induction by expression of STAT1 in STAT1-defective U3A cells. TGF-β significantly inhibits IFN-γ-mediated induction of the CIITA promoter in 2fTGH fibroblasts, which indicates that the promoter is a target for TGF-β. This inhibition is achieved by suppression of the basal promoter. This study provides a focal point for understanding the mechanism of B cell-specific, IFN-γ-induced, and TGF-β-suppressed expression of CIITA.
Peptides That Mimic the Group B Streptococcal Type III Capsular Polysaccharide Antigen
Authors: Pincus Seth H. , Smith Michael J. , Jennings Harold J. , Burritt James B. , Glee Pati M.
Published: January 2023

Microbial polysaccharides are notably poor immunogens. We have developed an alternate route for the production of Abs to important carbohydrate epitopes. mAb S9, a protective mAb against the type III capsular polysaccharide of group B streptococci (GBS), was used to select epitope analogues from a peptide display phage library. Depending upon desorption conditions, two populations of phage were identified with displayed sequences of WENWMMGNA and FDTGAFDPDWPA. ELISA results demonstrated that these phage bound to S9 and no other Abs. Phage blocked the binding of S9 to type III GBS, but did not block binding by another anti-GBS mAb. Phage displaying the latter peptide sequence showed greater inhibition. Ab S9 and other monoclonal and polyclonal anti-GBS type III antisera bound the synthetic peptide FDTGAFDPDWPAC. The binding of S9 to GBS was inhibited by the free peptide with an IC50 of 30 μg/ml. The binding of polyclonal anti-GBS antibodies to peptide could be blocked by intact GBS as well as purified capsular polysaccharide. The peptide was conjugated to three different carriers and was used to immunize mice. All mice produced a significant antibody response to GBS and to the purified capsular polysaccharide following a single immunization. These data demonstrate that a peptide mimetic of the GBS capsular polysaccharide is both antigenic and immunogenic. The incorporation of such peptides into vaccine preparations may enhance the efficacy of vaccines in inducing Ab responses to important carbohydrate epitopes.
IL-18 (IFN-γ-Inducing Factor) Regulates Early Cytokine Production in, and Promotes Resolution of, Bacterial Infection in Mice
Authors: Bohn Erwin , Sing Andreas , Zumbihl Robert , Bielfeldt Claudia , Okamura Haruki , Kurimoto Masashi , Heesemann Jürgen , Autenrieth Ingo B.
Published: January 2023

IL-12-induced IFN-γ production is essential for clearance of Yersinia enterocolitica infection. Similar to IL-12, the recently described cytokine IL-18 (IFN-γ-inducing factor) is produced by macrophages and induces IFN-γ production in spleen cells. Therefore, we have investigated the role of IL-18 in Yersinia infection of mice. Heat-killed yersinia-triggered IL-18-promoted IFN-γ production of splenocytes was predominantly dependent on endogenous IL-12 production, whereas IL-12-promoted IFN-γ production was not IL-18 dependent. IL-18-induced IFN-γ production was to a higher degree dependent on IFN-γR-mediated mechanisms and in synergism with IL-2 resulted in at least fivefold higher IFN-γ levels as compared with the combination of IL-12 plus IL-2. Analysis of the effect of IL-18 on IL-12 production of LPS-stimulated peritoneal macrophages revealed that IL-18 decreased LPS-induced IL-12 production, indicating that IL-18 might be involved in negative regulation of IL-12 production. In vivo studies revealed that Yersinia-resistant C57BL/6 mice expressed fourfold higher IL-18 mRNA levels than did susceptible BALB/c mice. Administration of anti-IL-18 Abs caused a 100- to 1000-fold increase in bacterial counts in the spleen of infected mice but did not change IFN-γ production levels. Taken together, our data demonstrate that IL-18 is involved in regulation of cytokine production during the early phase of bacterial infections as well as in clearance of Yersinia infection.
Neutrophils but Not Eosinophils Are Involved in Growth Suppression of IL-4-Secreting Tumors
Authors: Noffz Gabriele , Qin Zhihai , Kopf Manfred , Blankenstein Thomas
Published: January 2023

Local expression of IL-4 by gene-modified tumor cells increases their immunogenicity by inducing an inflammatory response that is dominated by eosinophils. Eosinophils have been implicated as antitumor effector cells because the application of a granulocyte-depleting Ab inhibited rejection of IL-4 transfected tumors. This Ab did not discriminate between eosinophils and neutrophils and, therefore, this experiment could not exclude neutrophils as primary effector cells, whereas eosinophils were innocent bystander cells in IL-4 transfected tumors. We analyzed tumor growth suppression and granulocyte infiltration in IL-5-deficient (IL-5−/−) mice that had a deficiency of eosinophils, using two tumor lines (B16-F10 and MCA205) transfected to secrete IL-4. IL-4-expressing tumors were at least as efficiently rejected in IL-5−/− mice as in wild-type mice, despite an almost complete absence of tumor-infiltrating eosinophils. However, neutrophils were present in undiminished amounts and their depletion partially restored tumor growth. Furthermore, the growth of IL-5-secreting tumors was not impaired in either wild-type or IL-5−/− mice, even though it induced eosinophilia in both mouse strains. These findings demonstrate that eosinophils can be induced in IL-5−/− mice by exogenous IL-5 and argue against a compensatory effect of neutrophils in the absence of eosinophils. We conclude that 1) infiltration of IL-4 transfected tumors by eosinophils is completely IL-5 dependent, 2) eosinophils have no tumoricidal activity, and 3) neutrophils are responsible, at least in part, for tumor suppression.
Helminth Antigens Selectively Differentiate Unsensitized CD45RA+CD4+ Human T Cells in Vitro
Authors: Steel Cathy , Nutman Thomas B.
Published: January 2023

Human filarial helminth infections are characterized by type 2 immune responses to parasite Ag that can persist for the life of the individual; one possible cause for this may be prenatal exposure to the blood-borne microfilarial (Mf) stage of the parasite. To examine the relationship between early exposure to filarial Ag and subsequent immune responsiveness, CD45RA+CD4+ cells from normal unsensitized donors were stimulated in vitro with soluble microfilarial Ag (MfAg) from the filarial parasite Brugia malayi in the presence of APCs. MfAg alone induced proliferation and IFN-γ and IL-5 production in unsensitized CD45RA+CD4+ cells, demonstrating the ability of filarial Ags to prime naive T cells in the absence of exogenous cytokines and dendritic cells. Adding exogenous cytokine(s) (particularly IL-12 and IL-4) during priming was able to alter the MfAg-specific responses of CD45RA+CD4+ cells as well as subsequent responses to Ag. Interestingly, priming solely with MfAg led to enhanced IL-5 production following Ag restimulation, suggesting that MfAg preferentially primes for type 2 responses. These data demonstrate that filarial Ags by themselves can specifically prime CD45RA+CD4+ cells in vitro and do so in such a way as to deviate the immune response.
Effect of C1 Inhibitor on Inflammatory and Physiologic Response Patterns in Primates Suffering from Lethal Septic Shock
Authors: Jansen Patty M. , Eisele Bernd , de Jong Irma W. , Chang Alvin , Delvos Ulrich , Taylor Fletcher B. , Hack C. Erik
Published: January 2023

We evaluated the effect of C1 inhibitor (C1-inh), an inhibitor of the classical pathway of complement and the contact system, on the physiologic and inflammatory response in baboons suffering from lethal Escherichia coli sepsis. Five animals pretreated with 500 U/kg C1-inh (treatment group; n = 5), followed by a 9-h continuous infusion of 200 U/kg C1-inh subsequent to bacterial challenge, were compared with five controls receiving E. coli alone. Of the treatment group, one animal survived and another lived beyond 48 h, whereas all control animals died within 27 h. In four of five treated animals, less severe pathology was observed in various target organs. C1-inh administration did not prevent the hemodynamic or hematologic changes observed upon E. coli infusion. The activation of fibrinolysis and the development of disseminated intravascular coagulation were essentially unaffected by C1-inh. However, C1-inh supplementation significantly reduced decreases in plasma levels of factor XII and prekallikrein and abrogated the systemic appearance of C4b/c, indicating substantial inhibition of activation of the contact system and the classical complement pathway, respectively. Furthermore, treated animals displayed a reduced elaboration of various cytokines including TNF, IL-10, IL-6, and IL-8. Thus, the administration of C1-inh may have a beneficial but modest effect on the clinical course and outcome of severe sepsis in nonhuman primates. We suggest that activated complement and/or contact system proteases may, at least in part, contribute to the attendant manifestations of septic shock through an augmentation of the cytokine response.
Differential Activities of Secreted Lymphotoxin-α3 and Membrane Lymphotoxin-α1β2 in Lymphotoxin-Induced Inflammation: Critical Role of TNF Receptor 1 Signaling
Authors: Sacca Rosalba , Cuff Carolyn A. , Lesslauer Werner , Ruddle Nancy H.
Published: January 2023

Lymphotoxin (LT, LTα, TNFβ) is a member of the immediate TNF family that also includes TNF-α and lymphotoxin-β (LTβ). LT is produced by activated lymphocytes and functions as either a secreted homotrimer or a membrane-associated heterotrimer that includes the transmembrane protein LTβ. Secreted LTα3 can bind to two cell surface receptors, TNFR1 and TNFR2, while the membrane-bound heterotrimer LTα1β2 has been shown to interact with a distinct receptor, LTβR. LTα induces inflammation at the sites of expression of a rat insulin promoter-driven lymphotoxin (RIPLT) transgene in the pancreas and kidney. To determine the role of the various ligands and their receptors in LT-induced inflammation, mice deficient in either TNFR1, TNFR2, or LTβ were crossed to RIPLT-transgenic mice. Our results indicate that LTα-induced inflammation is dependent on the interaction of LTα3 with TNFR1, and there is no obvious role for TNFR2, since in its absence, LTα-induced inflammation is quantitatively and qualitatively similar to that seen in the wild type. However, the absence of LTβ results in accentuated infiltration of the kidney with an increase in the proportion of memory cells in the infiltrate. These data show a crucial role for the secreted LTα3 signaling via TNFR1 in LTα-induced inflammation, and a separate and distinct role for the membrane LTα1β2 form in this inflammatory process.
A New Role for B Cells in Systemic Autoimmunity: B Cells Promote Spontaneous T Cell Activation in MRL-lpr/lprMice
Authors: Chan Owen , J. Shlomchik Mark
Published: January 2023

A conventional view of the pathogenesis of systemic lupus erythematosus has emerged. The role of B cells is to secrete pathogenic autoantibodies, while the role of T cells is to provide help for autoantibody-producing B cells. A problem with this view is that spontaneous T cell activation as well as T cell infiltration of organs such as kidney and skin are prominent features in systemic lupus erythematosus patients and murine models of lupus. The identification of T cell infiltrates, in particular, suggests that autoantibody-mediated damage may be only part of the story and that T cells could also play a primary role in immune-mediated pathology. To test the role of B cells directly, we previously generated autoimmune-prone MRL-lpr/lpr mice that lack B cells. The complete absence of T cell infiltrates in these mice was surprising, and it prompted us to examine whether a key role of B cells in disease evolution is to prime autoreactive T cells. Here we demonstrate, by comparing B cell-deficient and control mice, that the expansion of activated and memory T cells in the MRL-lpr/lpr mouse is indeed highly dependent on B cells. These results suggest a novel role for B cells in autoimmune disregulation.
Cutting Edge: p21-Activated Kinase (PAK) Is Required for Fas-Induced JNK Activation in Jurkat Cells
Authors: Rudel Thomas , Zenke Frank T. , Chuang Tsung-Hsien , Bokoch Gary M.
Published: January 2023

The process of apoptosis is a critical component of normal immune system development and homeostasis, and in many cells this involves signaling through the c-Jun amino terminal kinase (JNK) pathway. In Jurkat T cells, Fas-induced JNK activity is dependent upon activation of the caspase cascades known to be central components of the apoptotic program. We show in Jurkat cell lines expressing a dominant negative PAK construct that PAK signaling is necessary for JNK activation in response to Fas receptor cross-linking. Inhibition of JNK activation induced by Fas does not impair cell death as assessed by DNA fragmentation. However, expression of the catalytically active C terminus of PAK2, which is generated through caspase action during Fas-mediated apoptosis, induces Jurkat cell apoptosis. We conclude that PAK activity resulting from caspase-mediated cleavage is a necessary component of JNK activation induced by Fas receptor signaling and that PAK2 can contribute to the induction of cell death.
Cutting Edge: TCR Stimulation by Antibody and Bacterial Superantigen Induces Stat3 Activation in Human T Cells
Authors: Gerwien Jens , Nielsen Mette , Labuda Tord , Nissen Mogens H. , Svejgaard Arne , Geisler Carsten , Röpke Carsten , Ødum Niels
Published: January 2023

Recent data show that TCR/CD3 stimulation induces activation of Stat5 in murine T cells. Here, we show that CD3 ligation by mAb and Staphylococcal enterotoxin (SE) induce a rapid, gradually accumulating, long-lasting tyrosine, and serine phosphorylation of Stat3 (but not Stat5) in allogen-specific human CD4+ T cell lines. In contrast, IL-2 induces a rapid and transient tyrosine and serine phosphorylation of Stat3. Compared with IL-2, CD3 ligation induces a delayed Stat3 binding to oligonucleotide probes from the ICAM-1 and IL-2Rα promoter. CD3-mediated activation of Stat3 is almost completely inhibited by a Src kinase inhibitor (PP1), whereas IL-2-induced Stat3 activation is unaffected. In conclusion, we show that CD3 ligation by mAb and SE triggers a rapid, PP1-sensitive tyrosine and serine phosphorylation of Stat3 in human CD4+ T cells. Moreover, we provide evidence that TCR/CD3 and IL-2 induce Stat3 activation via distinct signaling pathways.
Bruton’s Tyrosine Kinase Deficiency in Macrophages Inhibits Nitric Oxide Generation Leading to Enhancement of IL-12 Induction
Authors: Mukhopadhyay Sangita , George Anna , Bal Vineeta , Ravindran Balachandran , Rath Satyajit
Published: January 2023

We show that macrophages of X-linked immunodeficient mice with a mutant nonfunctional Bruton’s tyrosine kinase produce less NO than wild-type macrophages in response to a variety of stimuli. Induction of the inducible NO synthase (iNOS) protein, the transcription factor IFN regulatory factor-1 involved in iNOS expression, and the transcription factor STAT-1 involved in regulating IFN regulatory factor-1 induction are all poorer in X-linked immunodeficient than in wild-type macrophages. On the other hand, induction of IL-12 is higher in X-linked immunodeficient than in wild-type macrophages. Macrophage IL-12 induction is enhanced by iNOS inhibitors such as aminoguanidine and thiocitrulline and is inhibited by NO generation via sodium nitroprusside. There is relative enhancement of IFN-γ production by immune T cells from mice immunized under aminoguanidine cover. Our data thus suggest that Bruton’s tyrosine kinase participates in signaling for iNOS induction via IFN regulatory factor-1 in macrophages and that NO is an inhibitor of IL-12 induction.
Models for Antigen Receptor Gene Rearrangement. II. Multiple Rearrangement in the TCR: Allelic Exclusion or Inclusion?
Authors: Piper Hannah , Litwin Samuel , Mehr Ramit
Published: January 2023

This series of papers addresses the effects of continuous Ag receptor gene rearrangement in lymphocytes on allelic exclusion. The previous paper discussed light chain gene rearrangement and receptor editing in B cells, and showed that these processes are ordered on three different levels. This order, combined with the constraints imposed by a strong negative selection, was shown to lead to effective allelic exclusion. In the present paper, we discuss rearrangement of TCR genes. In the TCR α-chain, allelic inclusion may be the rule rather than the exception. Several previous models, which attempted to explain experimental observations, such as the fractions of cells containing two productive TCRα rearrangements, did not sufficiently account for TCR gene organization, which limits secondary rearrangement, and for the effects of subsequent thymic selection. We present here a detailed, comprehensive computer simulation of TCR gene rearrangement, incorporating the interaction of this process with other aspects of lymphocyte development, including cell division, selection, cell death, and maturation. Our model shows how the observed fraction of T cells containing productive TCRα rearrangements on both alleles can be explained by the parameters of thymic selection imposed over a random rearrangement process.
Strong TCR Ligation Without Costimulation Causes Rapid Onset of Fas-Dependent Apoptosis of Naive Murine CD4+ T Cells
Authors: Kishimoto Hidehiro , Sprent Jonathan
Published: January 2023

Activation-induced cell death of T cells typically occurs late in the primary response after a prior proliferative response. Here, we describe a novel form of cell death in which purified naive murine CD4+ cells undergo apoptosis within 18 h in vitro after strong TCR ligation. Such rapid-onset TCR-mediated death of T cells does not involve cell division and is Fas-dependent, inhibited by CD28 (and IL-6) costimulation and enhanced by IL-4 and IL-7; by contrast, spontaneous death of CD4+ cells cultured alone is Fas-independent and inhibited by IL-4 and IL-7. TCR-mediated Fas-dependent death of CD4+ cells is prevented by combined TCR/Fas ligation and by drugs that inhibit calcineurin-dependent signaling and mitogen-activated protein kinase MEK1 activation.
Priming MHC-I-Restricted Cytotoxic T Lymphocyte Responses to Exogenous Hepatitis B Surface Antigen Is CD4+ T Cell Dependent
Authors: Wild Jens , Grusby Michael J. , Schirmbeck Reinhold , Reimann Jörg
Published: January 2023

MHC-I (Ld)-restricted, S28–39-specific CTL responses are efficiently primed in H-2d BALB/c mice injected with low doses of native hepatitis B surface Ag (HBsAg) lipoprotein particles without adjuvants. Priming of this CTL response by exogenous HBsAg required CD4+ T cell “help” and IL-12: this CTL response could be neither induced in mice depleted of CD4+ T cells by in vivo Ab treatment, nor in (CD4+ T cell-competent or CD4+ T cell-depleted) IL-12-unresponsive STAT4−/− knockout BALB/c mice. Codelivery of oligonucleotides (ODN) with immunostimulating CpG sequences (ISS) with exogenous HBsAg reconstituted the CTL response to exogenous HBsAg in CD4+ T cell-depleted normal mice and in CD4+ T cell-competent and CD4+ T cell-depleted STAT4−/− BALB/c mice. Injection (by different routes) of “naked” pCI/S plasmid DNA encoding HBsAg into IL-12-responsive or –unresponsive BALB/c mice efficiently primed the MHC-I-restricted, HBsAg-specific CTL response. CTL priming was not detectable when CD4+ T cell-depleted animals were subjected to genetic immunization. In vivo priming of the well-characterized CD8+ CTL response to HBsAg in “high responder” BALB/c mice either by exogenous surface lipoprotein particles or by DNA vaccination is thus CD4+ T cell dependent. CTL priming by exogenous HBsAg, but not by genetic immunization, is IL-12 dependent. The dependence of CTL priming by exogenous HBsAg on CD4+ T cells can be overcome by codelivering ODN with ISS motifs, and this “adjuvants effect” operates efficiently in IL-12-unresponsive mice. The data characterize a feature of the adjuvant effect of ISS-containing ODN on CTL priming that may be of major interest for the design of CTL-stimulating vaccines with efficacy in immunodeficiency conditions.