bluuebunny/crossref_metadata_2025 huggingface dataset:
Superantigen activation of immune cells evokes epithelial (T84) transport and barrier abnormalities via IFN-gamma and TNF alpha: inhibition of increased permeability, but not diminished secretory responses by TGF-beta2.
Authors: McKay D M , Singh P K
Published: December 2022

Superantigens (SAgs) are extremely potent stimulants of T cell activity that have been implicated in the etiopathophysiology of inflammatory disease. Here, we tested the hypothesis that Staphylococcus aureus enterotoxin B (SEB), a model SAg, can alter epithelial transport and/or barrier functions via immune stimulation. Confluent monolayers of the human colonic T84 epithelial cell line, grown on filter supports, were cocultured with SEB +/- PBMC. Subsequently, T84 transport (consisting of baseline short-circuit current (Isc, indicates net ion transport) and secretory responses to carbachol and forskolin) and barrier functions (consisting of transepithelial resistance and fluxes of 3H-labeled mannitol and 51Cr-EDTA) were examined in Ussing chambers. T84 monolayers cocultured with SEB-activated PBMC displayed a time- and dose-dependent decrease in secretory responses to carbachol and forskolin and a significant increase in permeability. These dramatic changes in epithelial function were not due to reduced epithelial viability. Neutralizing Abs to IFN-gamma partially prevented the transport abnormalities, and Abs to TNF-alpha inhibited the increase in epithelial permeability. Abs to IL-1beta and IL-6 did not modulate the SEB-activated PBMC-induced T84 pathophysiology. Addition of TGF-beta2 to conditioned medium from SEB-activated PBMC partially inhibited the increase in T84 permeability but did not affect the transport abnormalities. We conclude that SAgs can elicit epithelial irregularities characteristic of enteric inflammation and that IFN-gamma and TNF-alpha are key mediators in this coculture model of epithelial dysfunction. Additionally, we would highlight the role that TGF-beta2 may play in preventing prolonged increases in epithelial permeability.
Identification of residues in the class II-associated Ii peptide (CLIP) region of invariant chain that affect efficiency of MHC class II-mediated antigen presentation in an allele-dependent manner.
Authors: Gautam A M , Yang M , Milburn P J , Baker R , Bhatnagar A , McCluskey J , Boston T
Published: December 2022

Invariant chain (Ii) associates with class II MHC molecules and is crucial for Ag presentation by class II molecules. A general explanation for how invariant chain (Ii) associates with polymorphic MHC class II molecules has been suggested by the crystallographic structure of CLIP (class II-associated Ii peptide) complexed with an HLA class II molecule, HLA-DR3. We show here that methionine residues at positions 93 and 99 in Ii are important in MHC class II-mediated Ag presentation, but function in an allele-dependent manner. Introduction of a Met-->Ala mutation at position 99 in Ii (M99AIi) impaired presentation of peptides derived from exogenous proteins by I-Ad and I-Au class II molecules. Mutating Met-->Ala in Ii at position 93 (M93AIi) abrogated presentation by I-Au molecules, but not by I-Ad. Impaired Ag presentation was associated with conformationally altered expression of I-A molecules on the surface of cells expressing mutated Ii. Cell surface CLIP staining and immunoprecipitation studies showed that both I-Ad and I-Au molecules were associated with an increased abundance of Ii peptides, CLIP, in cells expressing mutated Ii. These results show that methionine 93 and methionine 99 play an important physiologic role in Ii association with class II molecules by regulating release of CLIP from class II in the endocytic compartments to allow binding of cognate peptides.
DNA-binding antibodies from viable motheaten mutant mice: implications for B cell tolerance.
Authors: Westhoff C M , Whittier A , Kathol S , McHugh J , Zajicek C , Shultz L D , Wylie D E
Published: December 2022

C57BL/6 me(v)/me(v) (viable motheaten) mice have defective hemopoietic cell protein-tyrosine phosphatase (HCP, SHP-1) and are a model in which to study the role of this phosphatase in immune regulation and hemopoietic development. Immune defects in these mice include hypergammaglobulinemia, production of multiple autoantibodies, and glomerular nephritis. The presence of self-reactive Abs in these mice is an enigma, since it has been demonstrated that they have increased negative selection against self-reactive transgenic Abs. It was of interest to examine autoantibodies from these mice to identify reactivity or structural features that might account for their persistence and expansion in the repertoire. Abs to DNA were selected for investigation, since they have been characterized extensively from normal and autoimmune strains of mice. Viable motheaten mice had high titers of IgM, anti-dsDNA Abs, and all 12 anti-DNA hybridomas generated were also IgM. They had high avidity for dsDNA, also bound to ssDNA, but were not polyreactive. They used V(H) and V(L) gene families found in anti-DNA Abs from other strains, which reinforces evidence that there is preferential use of a restricted number of germline elements in anti-DNA Abs. The results indicate that Hcph-mutant mice do not delete DNA-reactive B cells, nor are they able to maintain these B cells in a state of anergy. Evidence for positive selection and clonal expansion after V-D-J rearrangement was also seen, in that several Abs expressed the same heavy chain in combination with different light chains, which is also a hallmark of the autoimmune anti-DNA response.
Characterization of lymphotoxin-alpha beta complexes on the surface of mouse lymphocytes.
Authors: Browning J L , Sizing I D , Lawton P , Bourdon P R , Rennert P D , Majeau G R , Ambrose C M , Hession C , Miatkowski K , Griffiths D A , Ngam-ek A , Meier W , Benjamin C D , Hochman P S
Published: December 2022

The lymphotoxin-alpha beta complex (LT alpha beta) is found on the surface of activated lymphocytes and binds to a specific receptor called the LT beta receptor (LT beta R). In the mouse, signaling through this pathway is important for lymph node development and splenic organization, yet the biochemical properties of murine LT alpha and LT beta are essentially unknown. Here we have used soluble receptor-Ig forms of LT beta R and TNF-R55 and mAbs specific for murine LT alpha, LT beta, and LT beta R to characterize the appearance of surface LT alpha beta complexes and LT beta R on several common murine cell lines. Cells that bound LT beta R also bound anti-LT alpha and anti-LT beta mAbs in a FACS analysis. The ability of these reagents to discriminate between surface TNF and LT was verified by analysis of surface TNF-positive, LPS-activated murine RAW 264.7 monocytic cells. Primary mouse leukocytes from spleen, thymus, lymph node, and peritoneum were activated in vitro, and CD4+ and CD8+ T cells as well as B cells expressed surface LT ligand but not the LT beta R. Conversely, elicited peritoneal monocytes/macrophages were surface LT negative yet LT beta R positive. This study shows that on mononuclear cells, surface LT complexes and receptor are expressed similarly in mice and man, and the tools described herein form the foundation for study of the functional roles of the LT system in the mouse.
Antagonism of Immunostimulatory CpG-Oligodeoxynucleotides by Quinacrine, Chloroquine, and Structurally Related Compounds
Authors: Macfarlane Donald E. , Manzel Lori
Published: December 2022

Phosphorothioate oligodeoxynucleotides containing CpG (CpG-ODN) activate immune responses. We report that quinacrine, chloroquine, and structurally related compounds completely inhibit the antiapoptotic effect of CpG-ODN on WEHI 231 murine B lymphoma cells and inhibit CpG-ODN-induced secretion of IL-6 by WEHI 231. They also inhibit IL-6 synthesis and thymidine uptake by human unfractionated PBMC induced by CpG-ODN. The compounds did not inhibit LPS-induced responses. Half-maximal inhibition required 10 nM quinacrine or 100 nM chloroquine. Inhibition was noncompetitive with respect to CpG-ODN. Quinine, quinidine, and primaquine were much less powerful. Quinacrine was effective even when added after the CpG-ODN. Near-toxic concentrations of ammonia plus bafilomycin A1 (used to inhibit vesicular acidification) did not reduce the efficacy of the quinacrine, but the effects of both quinacrine and chloroquine were enhanced by inhibition of the multidrug resistance efflux pump by verapamil. Agents that bind to DNA, including propidium iodide, Hoechst dye 33258, and coralyne chloride did not inhibit CpG-ODN effect, nor did 4-bromophenacyl bromide, an inhibitor of phospholipase A2. Examination of the structure-activity relationship of seventy 4-aminoquinoline and 9-aminoacridine analogues reveals that increased activity was conferred by bulky hydrophobic substituents on positions 2 and 6 of the quinoline nucleus. No correlation was found between published antimalarial activity and ability to block CpG-ODN-induced effects. These results are discussed in the light of the ability of quinacrine and chloroquine to induce remission of rheumatoid arthritis and lupus erythematosus.
Requirement for In Vivo Production of IL-4, But Not IL-10, in the Induction of Proliferative Suppression by Filarial Parasites
Authors: MacDonald Andrew S. , Maizels Rick M. , Lawrence Rachel A. , Dransfield Ian , Allen Judith E.
Published: December 2022

Loss of T lymphocyte proliferation and the emergence of a host response that is dominated by a Th2-type profile are well-established features of human filariasis. We have previously reported that adherent peritoneal exudate cells (PEC) from mice transplanted with adult Brugia malayi parasites suppress the proliferation of lymphocytes without blocking Ag-cytokine production in vitro. We now show that infection of mice with the infective larval (L3) stage of B. malayi generates a similar population of PEC. Suppressive cells are generated within 7 days of infection and mediate their effects through a nitric oxide-independent pathway. Both L3 and adult infection elicit high levels of host IL-4 whereas the microfilarial stage of the parasite induces IFN-γ production and does not generate a similar form of suppression. Production of host IL-4 was necessary to allow the generation of suppressive PEC, given that IL-4-deficient mice implanted with adult parasites failed to induce proliferative block. However, IL-10-deficient mice implanted with adult parasites resulted in T cell suppression, indicating that IL-10 is not essential for the induction of hyporesponsiveness. Neither IL-4 nor IL-10 were directly responsible for ablating cellular proliferation in vitro, as the addition of neutralizing Ab to either cytokine did not reverse the proliferative block. Thus, IL-4 produced in vivo in response to filarial L3 and adult parasites is essential for the induction of proliferative suppression but is not itself the suppressive factor.
Development of Th1 and Th2 Populations and the Nature of Immune Responses to Hepatitis B Virus DNA Vaccines Can Be Modulated by Codelivery of Various Cytokine Genes
Authors: Chow Yen-Hung , Chiang Bor-Luen , Lee Yueh-Lun , Chi Wei-Kuang , Lin Wen-Chang , Chen Yen-Teen , Tao Mi-Hua
Published: December 2022

In this study, we provide direct evidence that the magnitude and nature of the immune response to a DNA vaccine can be differentially regulated by codelivery of various mouse cytokine genes. Mice immunized with a hepatitis B virus (HBV) DNA vaccine and the IL-12 or IFN-γ gene exhibited a significant enhancement of Th1 cells and increased production of anti-HBV surface IgG2a Ab, as well as a marked inhibition of Th2 cells and decreased production of IgG1 Ab. In contrast, coinjection of the IL-4 gene significantly enhanced the development of specific Th2 cells and increased production of IgG1 Ab, whereas Th1 differentiation and IgG2a production were suppressed. Coinjection of the IL-2 or the granulocyte-macrophage-CSF gene enhanced the development of Th1 cells, while the development of Th2 cells was not affected, and the production of IgG1 and IgG2a Ab were both increased. The CTL activity induced by HBV DNA vaccination was most significantly enhanced by codelivery of the IL-12 or IFN-γ gene, followed by the IL-2 or granulocyte-macrophage-CSF gene, whereas codelivery of the IL-4 gene suppressed the activity. When challenged with HBV surface Ag (HBsAg)-expressing syngeneic tumors, significant reduction of tumor growth was observed in mice that were coadministered the IL-12 gene but not the IL-4 gene. Taken together, these results demonstrate that application of a cytokine gene in a DNA vaccine formulation can influence the differentiation of Th cells as well as the nature of an immune response and may thus provide a strategy to improve its prophylactic and therapeutic efficacy.
Cutting Edge: Infection of Mice Lacking the Common Cytokine Receptor γ-Chain (γc) Reveals an Unexpected Role for CD4+ T Lymphocytes in Early IFN-γ-Dependent Resistance toToxoplasma gondii
Authors: Scharton-Kersten Tanya , Nakajima Hiroshi , Yap George , Sher Alan , Leonard Warren J.
Published: December 2022

Mice lacking the common cytokine receptor γ-chain (γc) gene exhibit defective development of NK cells and CD8+ T cells and greatly diminished production of IFN-γ. Because resistance of SCID mice to Toxoplasma gondii requires IL-12-dependent IFN-γ production by NK cells, we expected that γc-deficient mice would succumb rapidly to the parasite. Surprisingly, however, most γc-deficient mice survived the acute phase of T. gondii infection. As in wild-type mice, this resistance required IL-12 and IFN-γ; nevertheless, whereas wild-type mice depleted of CD4+ T cells survived, anti-CD4+ treated γc-deficient mice displayed diminished production of IFN-γ and all succumbed to acute infection. These data not only reveal a role for CD4+ T lymphocytes in IFN-γ-dependent host defense but also establish SCID and γc-deficient mice as powerful complementary tools for assessing the function of NK vs CD4+ T cells in immunopathophysiologic responses.
Local Synthesis of C3 Within the Splenic Lymphoid Compartment Can Reconstitute the Impaired Immune Response in C3-Deficient Mice
Authors: Fischer Michael B. , Ma Minghe , Hsu Nelson C. , Carroll Michael C.
Published: December 2022

Mice bearing a disrupted C3 locus (C3−/−) have an impaired Ab response to T-dependent Ags (bacteriophage φX 174 and nuclear protein-keyhole limpet hemocyanin) characterized by a reduction in number and size of germinal centers and impaired retention of Ag by follicular dendritic cells. To test the importance of C3 synthesized locally within the lymphoid compartment during an immune response to T-dependent Ag, we reconstituted C3−/− mice with wild-type bone marrow of MHC-identical littermates. Engraftment not only restored local C3 synthesis in the spleen, but also rescued the impaired humoral response. The major source of C3 mRNA was MOMA-2+ macrophages localized within the white pulp areas of the spleen. Interestingly, C3 expression is apparently regulated as C3 mRNA was not detected in splenic sections of nonimmune mice. Furthermore, local C3 synthesis by donor macrophages reversed the impaired Ag trapping by splenic follicular dendritic cells in C3-deficient mice.
Bone Marrow Transplantation Induces Either Clonal Deletion or Infectious Tolerance Depending on the Dose
Authors: Bemelman Frederike , Honey Karen , Adams Elizabeth , Cobbold Stephen , Waldmann Herman
Published: December 2022

The concept of immunologic tolerance arose from bone marrow transplantation in neonatal or irradiated mice, in which the predominant mechanism is clonal deletion of donor-specific T cells by donor hemopoietic cells in the recipient thymus. A short term treatment with nonlytic CD4 and CD8 mAbs can induce tolerance to tissue allografts or reversal of spontaneous autoimmunity. Such tolerance to skin or heart allografts is dependent on “infectious” tolerance mediated by regulatory CD4+ T cells. We show here, for multiple minor Ag differences, that while a large inoculum of donor marrow produces significant deletion of Ag-reactive cells as expected, a low marrow dose generates tolerance with little evidence of clonal deletion. Only this low dose tolerance can be transferred to unmanipulated recipients via CD4+ T cells, can be passed onto naive T cells as if infectious, and can act to suppress rejection of third party Ags when “linked” on F1 grafts.
A Common Immunoregulatory Locus Controls Susceptibility to Actively Induced Experimental Allergic Encephalomyelitis and Experimental Allergic Orchitis in BALB/c Mice
Authors: Teuscher Cory , Hickey William F. , Grafer Constance M. , Tung Kenneth S. K.
Published: December 2022

Previous studies have shown that differential susceptibility to actively induced experimental allergic encephalomyelitis (EAE) and experimental allergic orchitis (EAO) exists among various BALB/c substrains. Of eight substrains studied for EAE and 13 for EAO, BALB/cJ mice are phenotypically the most resistant to disease induction. Resistance to both diseases is controlled by single recessive mutations unlinked to any of the known alleles distinguishing BALB/cJ mice. In this study, segregation analysis employing a second generation backcross population shows that resistance to both EAE and EAO is due to a mutation in a common immunoregulatory gene. The role of immunoregulatory cells in controlling EAE resistance was examined using adoptive transfer protocols. BALB/cJ mice immunized with spinal cord homogenate plus adjuvants generate immunoregulatory spleen cells (SpC) that, when transferred to naive BALB/cByJ recipients, reduce the incidence and severity of EAE. Treatment of such cells with either cytotoxic monoclonal anti-Thy1.2 or anti-CD4 plus C′ before transfer abrogates the ability of BALB/cJ SpC to inhibit disease. In contrast, neither SpC from adjuvant-immunized BALB/cJ nor spinal cord homogenate- plus adjuvant-primed BALB/cByJ donors influences the incidence or severity of disease observed in recipients. In addition, the role of environment in influencing susceptibility to EAE and EAO in BALB/c mice is documented. Taken together, these results support the existence of a common disease susceptibility locus in the pathways leading to two autoantigenically distinct CD4+ T cell-mediated, organ-specific, autoimmune diseases.
Triggering of Peritoneal Macrophages with IFN-α/β Attenuates the Expression of Inducible Nitric Oxide Synthase Through a Decrease in NF-κB Activation
Authors: López-Collazo Eduardo , Hortelano Sonsoles , Rojas Armando , Boscá Lisardo
Published: December 2022

Triggering peritoneal macrophages with IFN-γ and a low concentration of LPS induced the expression of the inducible form of nitric oxide synthase (iNOS). This process was significantly inhibited when IFN-α/β was added during the initial 2 h after the start of IFN-γ/LPS activation. Evaluation of the transcriptional activity using run-on assays indicated that IFN-α/β inhibited the transcription of iNOS. Transfection experiments using a 1.7-kb promoter sequence corresponding to the 5′ flanking region of the murine iNOS gene showed decreased promoter activity in the presence of type I IFNs. Analysis of the transcription factors that participate in iNOS expression revealed a marked decrease of NF-κB activation, a nuclear factor required for the transcription of this gene. The degradation of IκBα and IκBβ, which is required for the translocation of NF-κB to the nucleus, was inhibited in the presence of IFN-α/β. However, the activity of other transcription factors such as IFN regulatory factor 1, which is involved in the expression of iNOS in response to IFN-γ, was not affected by IFN-α/β stimulation. These results suggest that in the presence of IFN-α/β, the activity of the iNOS promoter is impaired, and this attenuated nitric oxide synthase expression could be important in pathophysiologic situations in which secretion of type I IFNs occurs.
Distinct Homing Pathways Direct T Lymphocytes to the Genital and Intestinal Mucosae in Chlamydia-Infected Mice
Authors: Perry Linda L. , Feilzer Karen , Portis John L. , Caldwell Harlan D.
Published: December 2022

Immunity to genital tract infection with Chlamydia trachomatis is mediated by type 1 CD4+ T lymphocytes. To define the signals that govern lymphocyte trafficking to the genital mucosa, integrins expressed by infiltrating T cells and endothelial addressins displayed on local vasculature were characterized during the course of infection. All T cells expressed the αLβ2 heterodimer that binds vascular ICAM-1, and most displayed enhanced levels of the α4β1 integrin that interacts with VCAM-1. αE and β7low integrin chains were detected on approximately 15 and 30% of infiltrating T cells, respectively. Lymphocytes derived from the spleen or draining lymph nodes expressed this same integrin profile, suggesting that cells are recruited to the genital mucosa from the systemic circulation without significant selection pressure for these markers. Immunofluorescent staining for the corresponding vascular addressins revealed intense expression of VCAM-1 on small vessels within Chlamydia-infected genital tracts and up-regulation of ICAM-1 on endothelial, stromal, and epithelial cells. Mucosal addressin cell adhesion molecule-1 was not detected within genital tissues. These results indicate that T lymphocyte homing to the genital mucosa requires the interaction of αLβ2 and α4β1 with endothelial ICAM-1 and VCAM-1, respectively, which is the same pathway that directs lymphocytes to systemic sites of inflammation. Homing pathways defined for the intestinal mucosa and assumed to be relevant to all mucosal sites are not well represented in the genital tract. The identification of T lymphocyte trafficking pathways shared between systemic and mucosal tissues should facilitate vaccine strategies aimed at maximizing immune responses against Chlamydia and other pathogens of the urogenital tract.
Identification of a Sequence That Mediates Promiscuous Binding of Invariant Chain to MHC Class II Allotypes
Authors: Siebenkotten Ina M. , Carstens Cornelia , Koch Norbert
Published: December 2022

The invariant chain (Ii) shows promiscuous binding to a great variety of MHC class II allotypes. In contrast, the affinities of the Ii-derived fragments, class II-associated Ii peptides, show large differences in binding to class II allotypes. The promiscuous association of Ii to all class II polypeptides therefore requires an additional contact site to stabilize the interaction to the polymorphic class II cleft. We constructed recombinant molecules containing the class II binding site of Ii (CBS) and tested their association with HLA-DR dimers. The CBS fused to the transferrin receptor mediates binding of transferrin receptor-CBS to class II dimers. Within the CBS, deletion of a sequence N-terminal to the groove-binding motif abolished binding of Ii to DR. A promiscuous class II binding site was identified by reinsertion of the N-terminal residues, amino acids 81–87, of Ii into an Ii mutant that lacks the groove-binding segment. DR allotype-dependent association of Ii was achieved by insertion of antigenic sequences. The promiscuous association, in contrast to the class II allotype-dependent binding of Ii, is important to prevent interaction of class II dimers to nascent polypeptides in the endoplasmic reticulum.
A Bispecific Monoclonal Antibody Directed Against Both the Membrane-Bound Complement Regulator CD55 and the Renal Tumor-Associated Antigen G250 Enhances C3 Deposition and Tumor Cell Lysis by Complement
Authors: Blok Vanessa T. , Daha Mohamed R. , Tijsma Odette , Harris Claire L. , Morgan B. Paul , Fleuren Gert Jan , Gorter Arko
Published: December 2022

Tumor cells may inhibit the induction of a complement-mediated inflammatory response through overexpression of membrane-bound regulators of complement activation. Therefore, it is of interest to determine the most efficient approach to block these membrane-bound complement regulators on tumor cells. In the present study, we first generated a bispecific mAb directed against both CD55, using the functional blocking mAb MBC1, and the highly expressed HLA class I molecule as a model for a tumor-associated Ag, using the mAb W6/32. Tumor cells opsonized with bispecific mAb W6/32*MBC1, then exposed to complement and subsequently stained for C3 deposition, were assessed by flow cytometric analysis. We found that opsonization with W6/32*MBC1 resulted in a 92% enhancement of C3 deposition on renal tumor cells as compared with opsonization with W6/32 alone and a 17% enhancement of the C3 deposition as compared with incubation with a mixture of both parental mAb. Based on these results, we developed a bispecific mAb recognizing both CD55 and the relatively low expressed renal tumor-associated Ag G250. Increasing concentrations of the bispecific mAb G250*MBC1 resulted in a 25 to 400% increase in C3 deposition on renal tumor cells as compared with C3 deposition in the presence of the parental mAb G250 alone. G250*MBC1 enhanced C3 deposition by 21% in comparison with a mixture of both parentals. Furthermore, opsonization of tumor cells with G250*MBC1 rendered these cells more sensitive to complement-mediated lysis. In conclusion, the bispecific mAb G250*MBC1 induces deposition of C3 and tumor cell lysis more efficiently than G250 alone.
B Cell Sensitization to Helminthic Infection Develops In Utero in Humans
Authors: King Christopher L. , Malhotra Indu , Mungai Peter , Wamachi Alex , Kioko John , Ouma John H. , Kazura James W.
Published: December 2022

Human neonates are generally deficient in their ability to generate humoral immunity. This deficiency is thought to reflect physiologic immaturity of T and B cell function and lack of previous exposure to exogenous Ags. To determine whether neonatal humoral immunity can be modified by maternal helminth infection during pregnancy, we assessed Ig production by cord blood lymphocytes from healthy newborns of mothers living in an area of Kenya where schistosomiasis, bancroftian filariasis, and geohelminth infections are endemic. Twelve of 40 and 17 of 39 cord blood lymphocyte preparations from healthy newborns in Coast Province, Kenya, spontaneously made polyclonal IgE (range, 0.15–21 ng/ml) and IgG (1.6–10.1 ng/ml) in vitro. In vitro IgE synthesis by cord blood lymphocytes (CBL) was, on the average, 10-fold less than that of PBMC of Kenyan mothers (1.1–98 ng/ml) and was undetectable for CBL from newborns delivered in the United States. Schistosome and filarial Ags stimulated a 3- to >100-fold increase in the production of polyclonal IgE and parasite-specific IgG Abs by lymphocytes from 10 of 40 and 6 of 39 Kenyan newborns, respectively. CBL observed to have helminth Ag-driven B cell responses were more likely to be from newborns of schistosome- or filaria-infected mothers than from uninfected mothers (p < 0.05). These data indicate that the human fetus can be sensitized in utero to produce helminth-specific B cells and that neonatal B cells are intrinsically capable of IgE and IgG production.
Cutting Edge: Pig Islet Xenografts Are Susceptible to “Anti-Pig” But Not Galα(1,3)Gal Antibody Plus Complement in Gal o/o Mice
Authors: McKenzie Ian F. C. , Koulmanda Maria , Mandel Tom E. , Sandrin Mauro S.
Published: December 2022

Hyperacute rejection due to Galα(1,3)Gal (Gal) Ab plus complement (C′) is a major problem in xenografting vascularized organs from pigs to primates, but the fate of neovascularized xeno islets is unclear. Nonendocrine islet cells are Gal+, and there is a large rise in Gal Abs after transplantation, but graft remnants persist for some days in monkeys and humans. To define the role of αGal Ab plus C′ in porcine islet graft rejection, cultured porcine fetal islets were grafted to mice lacking the α(1,3)galactosyltransferase gene. Anti-Gal Ab plus C′ did not cause islet damage or rejection in mice lacking the α(1,3)galactosyltransferase gene, even when additional Ab plus C′ was given; in addition, hyperimmune mice (titer >1/20,000) did not reject pig islets, showing that islets are resistant to Gal Ab plus C′. However, islets can be destroyed by polyclonal mouse anti-pig Abs. Thus, the focus of islet xenografting should not be on Gal Ab plus C′.
The Alloreactive T Cell Response Against the Class Ib Molecule H2-M3 Is Specific for High Affinity Peptides
Authors: Dabhi Vikram M. , Hovik Rolf , Van Kaer Luc , Fischer Lindahl Kirsten
Published: December 2022

MHC class Ib molecule H2-M3 presents N-formylated peptides to CD8+ CTLs. Endogenous formylated peptides can come from the N-terminus of each of the 13 proteins encoded by the mitochondrial genome. In peptide competition assays, two of these peptides bind with high affinity, six bind with intermediate affinity, three bind with low affinity, and two do not bind measurably. Alloreactive CTLs from M3-specific, mixed lymphocyte cultures responded strongly against the two peptides with high affinity for M3, occasionally to peptides with intermediate affinity, and not at all to the rest. Long term lines and CTL clones reacted with only the high affinity peptides, demonstrating that alloreactive CTLs depend on specific peptides and that peptide affinity for class I correlates with alloantigenicity.
Multiple Sites of Vλ Diversification in Cattle
Authors: Lucier Mark R. , Thompson Rachel E. , Waire James , Lin Athena W. , Osborne Barbara A. , Goldsby Richard A.
Published: December 2022

Ig repertoire diversification in cattle was studied in the ileal Peyer’s patch (IPP) follicles of young calves and in the spleens of late first-trimester bovine fetuses. To investigate follicular diversification, individual IPP follicles were isolated by microdissection; Vλ diversity was examined by RT-PCR and subsequent cloning and sequencing. When 52 intrafollicular sequences from a 4-wk-old calf were determined and compared, two major groups, one of 23 members and the other of 25, could be delineated. An examination of these groups revealed clear genealogic relationships that implicated in situ diversification of Vλ sequences within the confines of an IPP follicle. Vλ expression was also examined in early (95 and 110 gestational day) fetal bovine spleens. Although earlier studies in cattle and sheep implicated the IPP as a likely site of Ab diversification, a close investigation of Vλ sequences in late first-trimester fetal calves revealed that diversity appears in the early fetal spleen before the establishment of a diverse repertoire in the ileum. When the sequences for the fetal spleen were compared with an existing pool of germline sequences, we found evidence of possible gene conversion events and possible untemplated point mutations occurring in sequences recovered from fetal spleens. We conclude that IPP is not the sole site of Vλ diversification in cattle. Also, as suggested for rabbits, cattle may use both gene conversion and untemplated somatic point mutation to diversify their primary Vλ repertoire.
pp56LckMediates TCR ζ-Chain Binding to the Microfilament Cytoskeleton
Authors: Rozdzial Moshe M. , Pleiman Chris M. , Cambier John C. , Finkel3 Terri H.
Published: December 2022

The TCR ζ-chain (ζ) on mature murine T lymphocytes binds to the microfilament cytoskeleton in response to Ag receptor ligation. Here, we report the role of Src family kinases in ζ-cytoskeletal binding, using mutant mice and a cell-free model system. Binding of ζ to actin in the cell-free system has a specific requirement for ATP and divalent cations, with an apparent Michaelis-Menton constant for ATP in the millimolar range, and can be disrupted by either EDTA or the microfilament poison, cytochalasin D, suggesting that microfilaments provide the structural framework for an active process involving cellular kinases. Indeed, tyrosine-phosphorylated ζ is a predominant form of the ζ-chain bound to polymerized actin, while challenge with alkaline phosphatase prevents ζ-chain association in solution and releases ζ-chain from the bound state. Phosphorylated Src-family kinase pp56Lck also associates with membrane skeleton upon TCR engagement and is a component of the reconstituted cytoskeletal pellet. ζ-Chain phosphorylation and ζ-cytoskeletal binding are abrogated in cell lysates with reduced levels of pp56Lck and in activated mutant murine T cells lacking pp56Lck, implicating pp56Lck as the kinase involved in ζ-chain tyrosine phosphorylation and ζ-cytoskeletal binding. Finally, recombinant Lck Src homology 2 domain preferentially inhibits reconstituted ζ-cytoskeleton association, suggesting that ζ-microfilament binding is dependent on interactions between phosphorylated tyrosine residues in ζ-chain activation motifs and the Src homology 2 domain of the Lck protein tyrosine kinase.
Pasteurella multocida Toxin Increases Endothelial Permeability via Rho Kinase and Myosin Light Chain Phosphatase
Authors: Essler Markus , Hermann Karin , Amano Mutsuki , Kaibuchi Kozo , Heesemann Jürgen , Weber Peter C. , Aepfelbacher Martin
Published: December 2022

Pasteurella multocida toxin (PMT) has been shown to induce actin reorganization through activation of the GTPase Rho. Here we investigated the involvement of the Rho target proteins Rho kinase and myosin light chain (MLC) phosphatase in the PMT-induced increase in endothelial permeability and the underlying actin reorganization of endothelial cells. Stimulation of endothelial layers with PMT enhanced transendothelial permeability >10-fold, and this was abolished by pretreatment with the specific Rho inactivator C3 transferase from Clostridium botulinum. The PMT-induced increase in endothelial permeability was associated with 1) inactivation of MLC phosphatase, 2) an increase in MLC phosphorylation, and 3) endothelial cell retraction and actin stress fiber formation. PMT-stimulated actin reorganization could be prevented by 1) pretreatment of cells with C3 transferase, 2) microinjection of the Rho binding domain and the pleckstrin homology domain of Rho kinase, and 3) microinjection of constitutively active MLC phosphatase. Together, these results suggest that PMT activates Rho/Rho kinase, which inactivates MLC phosphatase. The resulting increase in MLC phosphorylation causes endothelial cell retraction and a rise in endothelial permeability.
High Titer, Prostate Specific Antigen-Specific Human IgG Production byhu-PBL-SCID Mice Immunized with Antigen-Mouse IgG2a Complex-Pulsed Autologous Dendritic Cells
Authors: Coccia Marco A. , Brams Peter
Published: December 2022

We report here that immunization of human PBMC reconstituted SCID mice (hu-PBL-SCID mice) with in vitro cultured autologous dendritic cells (DC) pulsed with prostate specific antigen (PSA) complexed to a PSA-specific mouse IgG2a (PSA-IgG2a) consistently and reproducibly stimulates PSA-specific human IgG production. On day 0, female PBMC were used to reconstitute SCID mice and to generate DC in vitro. DC cultures were pulsed with PSA or PSA-IgG2a on day 6. The previously reconstituted hu-PBL-SCID mice were immunized with either PSA-pulsed DC and PSA, PSA-IgG2a-pulsed DC and PSA-IgG2a, or additional PBMC and PSA-IgG2a on day 7. Mice immunized with PSA-IgG2a-pulsed DC had, on the average, up to 31.5 times greater PSA-specific IgG serum concentrations than control mice. Competition ELISA confirmed the PSA specificity of serum IgG. Immunoblot analysis suggested that sera IgG preferentially recognized conformational epitopes on PSA. Therefore, our results represent a major step toward cloning human tumor-associated Ag-specific human mAbs from hu-PBL-SCID mice. In addition, flow cytometry showed that PSA-pulsed DC express significantly more B7.1, B7.2, CD40, and MHC class II surface molecules than mock-treated DC, but PSA-IgG2a-pulsed DC only had significantly enhanced B7.2 surface expression. Interestingly, PSA-specific IgG responses were reproducibly stimulated by DC expressing more B7.2, a molecule associated with Th2-type immune deviation, but not by those expressing more B7.1 and CD40, molecules associated with Th1-type immune deviation. Thus, our results show that stimulation with either Ag or Ag complexed to mAb yields DC with different phenotypes and APC effector functions.
TCR Transgenic Mice in Which Usage of Transgenic α- and β-Chains Is Highly Dependent on the Level of Selecting Ligand
Authors: Kersh Gilbert J. , Donermeyer David L. , Frederick Katherine E. , White J. Michael , Hsu Benjamin L. , Allen Paul M.
Published: December 2022

We have produced a TCR transgenic mouse that uses a TCR derived from a Th1 clone that is specific for residues 64 to 76 of the d allele of murine hemoglobin presented by I-Ek. Examination of these TCR transgenic mice on an H-2k/k background that expressed the nonstimulatory s allele of murine hemoglobin revealed that these mice express many endogenous TCR chains from both α and β loci. We found that this transgenic TCR is also very inefficient at mediating β selection, thereby showing a direct linkage between β selection and allelic exclusion of TCR β. We have also examined these mice on MHC backgrounds that have reduced levels of I-Ek and found that positive selection of cells with high levels of the transgenic TCR depends greatly on the ligand density. Decreasing the selecting ligand density is a means of reducing the number of available selecting niches, and the data reveal that the 3.L2 TCR is used sparingly for positive selection under conditions where the number of niches becomes limiting. The results, therefore, show a way that T cells may get to the periphery with two self-restricted TCRs: one that efficiently mediates positive selection, and another that is inefficient at positive selection with the available niches.
Isolation and Analysis of a T Cell Clone Variant Exhibiting Constitutively Phosphorylated Ser133 cAMP Response Element-Binding Protein
Authors: Belkowski Stanley M. , Rubin Charles S. , Prystowsky Michael B.
Published: December 2022

In driving T cell proliferation, IL-2 stimulates a new program of gene expression that includes proliferating cell nuclear antigen (PCNA), a requisite processivity factor for DNA polymerase δ. PCNA transcription is regulated in part through tandem CRE sequences in the promoter and CRE binding proteins; IL-2 stimulates CREB phosphorylation in the resting cloned T lymphocyte, L2. After culturing L2 cells for greater than 91 days, we consistently isolate a stable variant that exhibits constitutive CREB phosphorylation. L2 and L2 variant cells were tested for IL-2 responsiveness and rapamycin sensitivity with respect to specific kinase activity, PCNA expression and proliferation. In L2 cells, IL-2 stimulated and rapamycin inhibited the following: cAMP-independent CREB kinase activity, PCNA expression and proliferation. In L2 variant cells, CREB kinase activity was constitutively high; IL-2 stimulated and rapamycin blocked PCNA expression and proliferation. These results indicate that IL-2 induces a rapamycin-sensitive, cAMP-independent CREB kinase activity in L2 cells. However, phosphorylation of CREB alone is not sufficient to drive PCNA expression and L2 cell proliferation in the absence of IL-2.
The IL-1β-Converting Enzyme (Caspase-1) Inhibits Apoptosis of Inflammatory Neutrophils Through Activation of IL-1β
Authors: William R. , Watson G. , Rotstein Ori D. , Parodo Jean , Bitar Richard , Marshall John C.
Published: December 2022

IL-1β-converting enzyme (ICE), also known as caspase-1, subserves two dichotomous biologic roles. It processes newly synthesized pro-IL-1β to yield the active cytokine and, as the human homologue of the Caenorhabditis elegans gene product, ced-3, it also induces cellular apoptosis through the cleavage of key intracellular structural and regulatory proteins and through the catalytic activation of other caspase family members. We show here that two different proinflammatory stimuli, LPS and granulocyte-macrophage-CSF, up-regulate the expression of both ICE and IL-1β in human polymorphonuclear neutrophils, and that the ICE-dependent cleavage of pro-IL-1β results in delayed expression of the constitutive cell death program. The apoptotic delay can be blocked by inhibiting tyrosine kinases or NF-κB activation and by inhibiting protein synthesis. Since an antisense oligonucleotide for IL-1β, a blocking Ab to IL-1β, and preincubation with the IL-1R antagonist all prevent the delay in apoptosis, we conclude that IL-1β acts in an autocrine manner to inhibit granulocyte programmed cell death. We conclude that caspase-1 (ICE) subserves both pro- and antiapoptotic roles; the latter role is evident during inflammation as an inhibition of spontaneous neutrophil apoptosis through the processing of IL-1β. The ICE-dependent activation of IL-1β may represent a common autocrine pathway for the divergent stimuli that inhibit the constitutive expression of neutrophil programmed cell death during inflammation.
Cutting Edge: A Newly Identified Response Element in the CD95 Ligand Promoter Contributes to Optimal Inducibility in Activated T Lymphocytes
Authors: Norian Lyse A. , Latinis Kevin M. , Koretzky Gary A.
Published: December 2022

Inducible expression of CD95 ligand on activated T lymphocytes contributes to both cytotoxic effector mechanisms and peripheral T cell homeostasis. To understand better the transcriptional events that regulate this expression, we have examined the CD95 ligand promoter to determine which regions are required for its induced activity following T cell stimulation. We report here the identification of a new response element within the promoter that is required for its optimal function in activated Jurkat T cells. This region is bound by proteins contained in nuclear extracts of activated, but not resting, T cells. Multimerization of this sequence independently drives transcription in response to T cell activation, while mutation of it substantially decreases inducible promoter activity. Finally, we provide evidence that T cell activation-induced transcription of the CD95 ligand gene is regulated coordinately by this response element together with two previously defined sites for nuclear factor of activated T cells (NFAT).
Termination of Peripheral Tolerance to a T Cell Epitope by Heteroclitic Antigen Analogues
Authors: Zügel Ulrich , Wang Rongfang , Shih Grace , Sette Alessandro , Alexander Jeff , Grey Howard M.
Published: December 2022

Treating mice with an immunodominant T cell epitope from moth cytochrome c (MCC88–103) can induce T cell unresponsiveness under certain conditions of administration. In this report, we determined whether T cell tolerance to MCC88–103 in adult animals can be overcome by immunization with cross-reactive analogues of the tolerizing Ag. A panel of analogues of the tolerogen were tested for their capacity to terminate the tolerant state following in vivo immunization. As analyzed by their stimulatory capacity for a representative MCC88–103-specific T cell clone, this panel covered a wide range of cross-reactivity, including nonantigenic, antagonistic, weakly, and strongly antigenic peptides. Interestingly, only heteroclitic analogues, as measured in vitro by their enhanced antigenicity for the T cell clone that was specific for MCC88–103, were capable of breaking tolerance. Thus, an immune response to the cross-reactive, heteroclitic analogues of tolerized self Ags may represent a mechanism by which Ag molecular mimicry operates.
Multiple Signaling Pathways for the Activation of JNK in Mast Cells: Involvement of Bruton’s Tyrosine Kinase, Protein Kinase C, and JNK Kinases, SEK1 and MKK7
Authors: Kawakami Yuko , Hartman Stephen E. , Holland Pamela M. , Cooper Jonathan A. , Kawakami Toshiaki
Published: December 2022

Stimulation of the high affinity IgE receptor (FcεRI) as well as a variety of stresses induce activation of c-Jun N-terminal protein kinases (JNKs) stress-activated protein kinases in mast cells. At least three distinct signaling pathways leading to JNK activation have been delineated based on the involvements of Bruton’s tyrosine kinase (Btk), protein kinase C (PKC), and the JNK-activating cascades composed of multiple protein kinases. The PKC-dependent pathway, which is inhibited by a PKC inhibitor Ro31-8425 and can be activated by PMA, functions as a major route in FcεRI-stimulated mast cells derived from btk gene knockout mice. On the other hand, wild-type mouse-derived mast cells use both PKC-dependent and PKC-independent pathways for JNK activation. A PKC-independent pathway is regulated by Btk and SEK1 via the PAK→MEKK1→SEK1→JNK cascade, and is sensitive to phosphatidylinositol 3-kinase inhibitors, wortmannin and LY-294002, while the PKC-dependent pathway is affected to a lesser extent by both wortmannin treatment and overexpression of wild-type and dominant negative mutant SEK1 proteins. Another PKC-independent pathway involves Btk and MKK7, a recently cloned direct activator of JNK. Among the stresses tested, UV irradiation seems to activate Btk and JNK via the PKC-independent pathways.
Characterization of a Low Molecular Weight Isoform of IL-1 Receptor Antagonist
Authors: Malyak Mark , Guthridge Joel M. , Hance Kenneth R. , Dower Steven K. , Freed John H. , Arend William P.
Published: December 2022

IL-1R antagonist (IL-1Ra) exists in two well-characterized forms, 17-kDa secretory IL-1Ra (sIL-1Ra) and 18-kDa intracellular IL-1Ra (icIL-1Ra), that arise by alternative transcription of the same IL-1Ra gene. A third, lower molecular mass form (∼16 kDa) was detected by immunoblot within lysates of a variety of cells, including human monocytes and myelomonocytic cell lines. The 16-kDa isoform was designated icIL-1RaII, and the previously established 18-kDa form was designated icIL-1RaI. Intracellular IL-1RaII bound type I IL-1R up to fivefold less avidly than did sIL-1Ra and icIL-1RaI. Microsequencing of cyanogen bromide fragments of purified icIL-1RaII provided evidence consistent with initiation of protein translation at the second start site in either IL-1Ra mRNA. The results of site-directed mutation experiments established that icIL-1RaII could be derived by alternative translation initiation. In vitro transcription and translation of intact sIL-1Ra cDNA in rabbit reticulocyte lysates led to both pro-sIL-1Ra and icIL-1RaII proteins, whereas transcription and translation of icIL-1RaI cDNA produced both icIL-1RaI and icIL-1RaII proteins. Mutation of the first 5′ ATG in sIL-1Ra cDNA led to translation of only icIL-1RaII, while only sIL-1Ra was observed after mutation of the second ATG. These results indicate that icIL-1RaII is a third member of the IL-1Ra family and is a 16-kDa, 143-amino acid intracellular protein derived by alternative translation initiation from either sIL-1Ra mRNA or icIL-1Ra mRNA. The role in biology of either intracellular form of IL-1Ra remains unknown.
Fcε Receptor I on Dendritic Cells Delivers IgE-Bound Multivalent Antigens into a Cathepsin S-Dependent Pathway of MHC Class II Presentation
Authors: Maurer Dieter , Fiebiger Edda , Reininger Bärbel , Ebner Christof , Petzelbauer Peter , Shi Guo-Ping , Chapman Harold A. , Stingl Georg
Published: December 2022

In this study, we elucidate the FcεRI-mediated Ag uptake and presentation mechanisms of dendritic cells (DC). We found that FcεRI-bound IgE, after polyvalent but not after monovalent ligation, is efficiently internalized into acidic, proteolytic compartments, degraded, and delivered into organelles containing MHC class II, HLA-DM, and lysosomal proteins. To follow the fate of the fragmented ligand, we sought to interfere with invariant chain (Ii) degradation, a process critical for peptide loading of nascent MHC class II molecules. We found DC to express cathepsin (Cat) S, a cysteine protease involved in li processing by B cells. Exposure of DC to a specific, active-site inhibitor of Cat S resulted in the loss of anti-Cat S immunoreactivity, led to the appearance of an N-terminal Ii remnant, and decreased the export of newly synthesized MHC class II to the DC surface. Furthermore, inactivation of Cat S as well as blockade of protein neosynthesis by cycloheximide strongly reduced IgE/FcεRI-mediated Ag presentation by DC. Thus, multimeric ligands of FcεRI, instead of being delivered into a recycling MHC class II pathway, are channeled efficiently into MIIC (MHC class II compartment)-like organelles of DC, in which Cat S-dependent li processing and peptide loading of newly synthesized MHC class II molecules occur. This IgE/FcεRI-dependent signaling pathway in DC may be a particularly effective route for immunization and a promising target for interfering with the early steps of allergen presentation.