bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-12 synthesis by human Langerhans cells.
Authors: Kang K , Kubin M , Cooper K D , Lessin S R , Trinchieri G , Rook A H
Published: December 2022

IL-12 is a 70-kDa heterodimeric cytokine composed of a p35 chain and p40 chain. This cytokine exerts a powerful positive regulatory influence on the development of Th1 helper T-cell immune responses and is a potent inducer of IFN-gamma production and cytotoxic T cell differentiation and function. Because epidermal Langerhans cells (LC) are important members of the dendritic APC lineage family critical for initiating cell mediated immune responses, we examined LC for their ability to produce IL-12. Epidermal cell (EC) suspensions obtained from volunteers were enriched for, or depleted of, Langerhans cells (CD1a+ EC). Enriched populations contained > 90% CD1a+ cells, whereas depleted populations contained < 1% CD1a+ cells. As assessed by reverse transcription-PCR amplification, IL-12 p40 mRNA was constitutively expressed in LC RNA extracted immediately following keratome harvest, and increased spontaneously after overnight incubation. Radioimmunoassay (RIA) of IL-12 p40 protein on supernatants revealed IL-12 release by CD1a-enriched fractions of epidermal cells. Ab specific for p40 clearly demonstrated IL-12 in epidermal LC by flow cytometry. A bioassay for the functional IL-12 heterodimer (p70) indicated that LC could produce IL-12 biologic activity, which was neutralized by anti-IL-12 Ab. These results indicate that epidermal LC, in particular cultured LC maturing into dendritic cells, express IL-12 p40 mRNA, as well as p40 and functional p70 protein, and suggest that this is one mechanism behind the high potency of dendritic APCs, such as LC, to initiate Th1 type immune responses under appropriate conditions.
Class II MHC antigen (Ia)-bearing dendritic cells in the epithelium of the rat intestine.
Authors: Maric I , Holt P G , Perdue M H , Bienenstock J
Published: December 2022

Many tissues are found to contain populations of cells with an unusual dendritic shape, high levels of surface expression of MHC class II (Ia) gene products, and strong accessory function for the stimulation of specific clones of quiescent T lymphocytes. Dendritic cells (DC) represent major population of "professional" APC in various lymphoid and nonlymphoid tissues, distinct from cells of the monocyte/macrophage lineage. Among the best characterized nonlymphoid dendritic cells are epidermal Langerhans cells, but it has been shown that interstitium and epithelium of other organs also contain irregularly shaped, strongly MHC class II positive cells. In recent years, DC have been localized to alveolar septa in the lung, as well as within and just beneath airway epithelium, comprising a tightly meshed network that is reminiscent of epidermal Langerhans cells. In the gastrointestinal tract, conventional immunohistochemical analysis of mucosal class II MHC (Ia) staining reveals a morphologically heterogeneous pattern of staining in the lamina propria. DC that exhibit strong Ag-presenting activity in vitro have been extracted from enzymatic digests of colonic mucosa, but no previous reports of MHC class II-positive cells with pleiomorphic morphology have been recorded within the epithelium of the intestine. Employing a novel combination of nonconventional section planes, pre-embedding fixation, and immunohistochemical techniques, we now demonstrate Ia staining of cells with classical DC morphology within the epithelium of the intestine in normal specific pathogen-free rats. Our investigation suggests that cells with the morphologic and phenotypical characteristics of DC are present within the mucosal epithelium of the rat jejunum and colon, comprising a significant organized network. The number of DC within epithelium of the colon was 117 +/- 20 per 10-microns-thick cross-section. These findings have important theoretical implications for research on Ag processing and T cell activation in the context of allergic and infectious diseases in the gastrointestinal tract.
Postnatal development of T cells in dogs with X-linked severe combined immunodeficiency.
Authors: Somberg R L , Tipold A , Hartnett B J , Moore P F , Henthorn P S , Felsburg P J
Published: December 2022

X-linked severe combined immunodeficiency disease (XSCID) in both humans and dogs results from mutations in the common gamma-chain, gamma c, which is a common component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. Although human and canine XSCID share similar features, such as a failure to thrive, hypogammaglobulinemia, an absent T cell mitogenic response, and thymic dysplasia, near normal percentages of T cells are observed in some affected dogs, whereas XSCID boys have few, if any, circulating T cells. In this study, PBL were analyzed by flow cytometry beginning shortly after birth until 9 wk of age. XSCID dogs < 3 wk of age had an elevated number of B cells and were nearly devoid of T cells, phenotypically resembling most human XSCID patients. At 5 wk of age, however, T cells appeared in approximately one-half of the XSCID dogs, although the absolute number of T cells was one-third of normal in these dogs. While the percentage of CD45RA+ T cells in normal dogs gradually decreased with age from > 90% in neonates to < 40% by 3 to 5 yr of age, in XSCID dogs a rapid decline in the percentage of CD45RA+ T cells was observed, resulting in < 10% CD45RA+ T cells by 7 to 9 wk of age. Maternal engraftment was not detected in any of the XSCID dogs by using a sensitive PCR assay. The appearance of nonmaternally derived T cells in XSCID dogs that undergo a rapid switch from CD45RA+ to CD45RA- suggests that limited thymic emigration and peripheral expansion of T cells can occur in the absence of a functional gamma c.
In vivo blockade of TNF-alpha by intravenous infusion of a chimeric monoclonal TNF-alpha antibody in patients with rheumatoid arthritis. Short term cellular and molecular effects.
Authors: Lorenz H M , Antoni C , Valerius T , Repp R , Grünke M , Schwerdtner N , Nüsslein H , Woody J , Kalden J R , Manger B
Published: December 2022

Due to the unknown etiology of RA, specific treatment is not available. Recently, in a double-blinded, placebo-controlled clinical trial, in vivo blockade of TNF-alpha by a single infusion of a chimeric TNF-alpha-blocking mAb, cA2, has proven to be highly effective in the treatment of RA. In parallel to this trial, we tested the consequences of cA2 infusion in ex vivo and in vitro experiments. In this paper, we describe an increase in CD4+ and CD8+ T lymphocyte counts on day 1 and a marked decrease in monocyte counts preferentially on day 7 after cA2 treatment, without major changes in B lymphocyte or NK cell counts. In addition, we found an increased responsiveness of PBMC to CD28 mAb/PMA, but not to CD3 mAb, superantigen staphylococcus enterotoxin B, or PHA on day 1 after infusion. The increase in DNA synthesis of PBMC was paralleled by increased IL-2 mRNA and IL-4 mRNA expression and IL-2 protein secretion in culture supernatants after in vitro stimulation of PBMC with CD28 mAb/PMA. In PBMC, we did not find any significant changes in mRNA or protein expression of CD28 Ag or CD28 ligands, B7-1 and B7-2. Serum concentrations of IL-1 beta, IL-6, and soluble CD14 were significantly diminished after in vivo TNF-alpha blockade. We did not see relevant changes in granulocyte function in vitro after cA2 infusion. Finally, we observed a statistically significant decrease in slCAM-1 molecules in the serum of patients treated with verum compared with that in the serum of subjects given placebo. This change in slCAM-1 concentration was evident on days 1 and 7 after the infusion of 10 mg/kg cA2, whereas it occurred only on day 7 in the serum of patients treated with the low dose (1 mg/kg) of cA2.
Biochemical characterization of antigen-specific glycosylation-inhibiting factor from antigen-specific suppressor T cells. II. The 55-kDa glycosylation-inhibiting factor peptide is a derivative of TCR alpha-chain and a subunit of antigen-specific glycosylation-inhibiting factor.
Authors: Ishii Y , Nakano T , Ishizaka K
Published: December 2022

We isolated a unique TCR alpha-chain derived from the OVA-specific Ts hybridoma, 231F1. The TR alpha-chain consists of V alpha 11.3, a unique J alpha and a complete sequence of C alpha region. Transfection of the TCR-alpha cDNA into a TCR-alpha-, TCR-beta+ T cell line, 175.2, resulted in the expression of TCR-alpha beta, and the transfectant contained a 35-kDa peptide having the TCR- alpha-specific antigenic determinant. However, the stable transfectant failed to release a peptide with the TCR-alpha determinant upon stimulation with anti-CD3. In contrast, overexpression of the cDNA in the 231 F1 cells markedly increased the formation of the 55-kDa peptide, which reacted with both anti-glycosylation-inhibiting factor (GIF) and the mAb H28-710. Definitive evidence for the relationship between the 55-kDa peptide and the TCR alpha-chain was obtained by transfection of the cDNA of the TCR alpha-chain with histidine tag into the 231F1 cells. The 55 kDa GIF peptide formed by stable transfectants of the TCR-alpha-tag cDNA bound to Ni+-nitrilotriacetic acid-agarose. Upon stimulation with anti-CD3, a stable transfectant of the TCR-alpha cDNA formed OVA-specific GIF which contained the 55-kDa GIF peptide, and bound not only to anti-TCR-alpha column but also to anti-TCR-beta column. The results indicate that the OVA-specific GIF consists of the TCR-alpha+ 55-kDa GIF and another peptide with TCR-beta determinant. It was found that the association of the TCR-beta+ peptide with the 55-kDa GIF is required for binding of the factor to OVA, but not essential for the formation and release of the latter peptide.
CD4low TCRint thymocytes do not belong to the CD8 lineage maturation pathway.
Authors: Lucas B , Marodon G , Penit C
Published: December 2022

Thymocytes with a low expression of CD4 and an intermediate density of the TCR (CD4low TCRint) were analyzed for phenotype, MHC dependence, production kinetics, and TCR repertoire to investigate their position in the intrathymic T cell maturation process. Comparison of normal and MHC-deficient mice showed that the CD4low TCRint cell subset was MHC class II dependent, as this subpopulation could not be defined in MHC class II- or double (class I and II)-deficient mice. These thymocytes were heat-stable Aghigh and CD69+, thus immature and recently engaged in a TCR interaction, probably with MHC class II molecules. Their generation kinetics were studied in two systems: development of exogenous bone marrow cells transferred into RAG-2-/- mice, and pulse labeling with bromodeoxyuridine. In both systems, CD4low TCRint cells were produced well before CD4low TCRhigh cells, the direct precursors of CD8 single-positive cells. Their production paralleled that of CD4high TCRint cells, but they were different than these thymocytes in their smaller cell size. Moreover, they had the same V beta 6 frequency in Mls-1a and Mls-1b mice, suggesting that these cells could be undergoing a negative selection process. The data here clearly demonstrate that CD4low TCRint thymocytes do not belong to the CD8 lineage maturation pathway, and suggest that these cells could represent a MHC class II-restricted dead-end subset.
Haptoglobin phenotyping by newly developed monoclonal antibodies. Demonstration of haptoglobin uptake into peripheral blood neutrophils and monocytes.
Authors: Wagner L , Gessl A , Parzer S B , Base W , Waldhäusl W , Pasternack M S
Published: December 2022

We have generated two IgG murine mAbs that recognize native human haptoglobin (Hp). These mAbs, 3A8 and 4B2, efficiently bind to the Hp complex regardless of the serum donor's phenotype. The specificity of mAb 3A8 was confirmed by immunoaffinity purification of 3A8-binding material from human serum and subsequent N-terminal amino acid sequencing of the invariant 40-kDa chain. mAb 3A8 and 4B2 were also reactive with cell-associated Hp when studied by immunocytochemistry. When human peripheral blood leukocytes were tested, 90% of the granulocytes and a lesser (and variable) fraction of monocytes displayed an intense intracytoplasmatic granular staining. This was confirmed by flow cytometric analysis of permeabilized leukocytes and by demonstrating the presence of Hp (of the expected Hp serum phenotype) in extracts of washed granulocytes by immunoblotting. Leukocytes obtained from a Hp phenotype 2-2 donor, incubated in culture medium supplemented with 10% serum from a donor possessing the Hp 1-1 phenotype, contained both Hp phenotypes when analyzed by immunoblotting after a 6-h incubation period. In addition, Hp was actively exocytosed by granulocytes following their exposure to Candida albicans. These observations suggest that exogenous Hp is concentrated within granulocytes and not synthesized de novo and is, in turn, exocytosed following neutrophil activation. Northern blotting analysis is consistent with the lack of haptoglobin gene transcription in granulocytes. These findings together with the earlier observations that Hp modulates granulocyte activity suggest that Hp levels may be enhanced locally at sites of inflammation to modulate granulocyte activity.
Development of CD8 alpha alpha+ intestinal intraepithelial T cells in beta 2-microglobulin- and/or TAP1-deficient mice.
Authors: Fujiura Y , Kawaguchi M , Kondo Y , Obana S , Yamamoto H , Nanno M , Ishikawa H
Published: December 2022

The development of CD8+ intestinal intraepithelial T lymphocytes (IEL) was analyzed in mice that are deficient in the expression of MHC class I molecules, owing to either a mutated beta 2-microglobulin (beta 2m) gene or a mutated transporter associated with Ag processing 1 (TAP1) gene, and in mice doubly homozygous for beta 2m and TAPI mutations. In all mutant mice, the population size of major CD8 alpha alpha+ and CD8 alpha beta+ alpha beta-IEL subsets was reduced drastically, and this resulted in a conspicuous decrease in the total number of alpha beta-IEL. Concomitantly, a compensatory two- to threefold increase in the number of gamma delta-IEL consisting mostly of CD8 alpha alpha+ subset was noted. In radiation bone marrow chimeras, this wild-type/mutant phenotype was determined by the genotype of radioresistant host cells, but was not determined by the genotype of reconstituting bone marrow-derived cells. In beta 2m X TCR-delta double mutant mice, however, the CD8 alpha alpha+ but not CD8 alpha beta+ alpha beta-IEL subset expanded dramatically. Thus, in the absence of gamma delta-IEL, alpha beta-IEL in beta 2m-deficient mice outnumbered those in wild-type littermates. These results indicate that the generation of CD8 alpha alpha+ lymphocyte population of alpha beta- and gamma delta-IEL is not dependent, but that of CD8 alpha beta+ lymphocyte population of alpha beta-IEL is dependent on beta 2m- and/or TAP1-dependent MHC class I molecules, expressed by the controlling cells present in the anatomical site, where the development of IEL takes place.
Mutational analysis of TNF-alpha gene reveals a regulatory role for the 3'-untranslated region in the genetic predisposition to lupus-like autoimmune disease.
Authors: Jacob C O , Lee S K , Strassmann G
Published: December 2022

Lupus-prone mice show reduced production of TNF-alpha and, upon long-term treatment with recombinant TNF-alpha, significant protection from disease development. Mutational analysis of the 5'-untranslated region (UTR) and 3'-UTR of the mouse TNF-alpha gene reveals a marked degree of polymorphism. Transient expression experiments in the RAW 264.7 macrophage-like cell line using the luciferase reporter system suggest an important role for the mutations in the 3'-UTR in the biosynthesis of TNF-alpha, and provide a molecular explanation for the reduced TNF-alpha production in lupus-prone NZW mice.
A human killer inhibitory receptor specific for HLA-A1,2.
Authors: Döhring C , Scheidegger D , Samaridis J , Cella M , Colonna M
Published: December 2022

Killer inhibitory receptors (KIRs) are transmembrane glycoproteins, expressed on NK cells and a small subset of T cells, that inhibit cell-mediated cytotoxicity upon binding to polymorphic MHC class I determinants on target cells. Although human KIRs specific for HLA-C and HLA-B molecules have been characterized, none have been shown to interact with HLA-A. Here we demonstrate that a member of the KIR cDNA family, designated NKAT4, encodes a 70-kDa receptor specific for HLA-A3.
Peptide-based, but not whole protein, vaccines elicit immunity to HER-2/neu, oncogenic self-protein.
Authors: Disis M L , Gralow J R , Bernhard H , Hand S L , Rubin W D , Cheever M A
Published: December 2022

HER-2/neu, an overexpressed oncogenic protein, has been proposed as a human cancer vaccine target. HER-2/neu is a "self" protein, however, and methods of vaccine strategies that would be effective in immunizing patients to a "self" tumor Ag have not been established. Many of the tumor Ags defined in humans are nonmutated self proteins, e.g., MAGE, and overcoming tolerance may be key in the generation of effective anti-tumor immunity. One theory states that tolerance to self proteins is directed only to dominant epitopes of proteins and not to every portion of the protein. Accordingly, tolerance can be circumvented by immunization to peptide fragments, but not whole protein. The studies outlined here demonstrate rat neu-specific immunity could be elicited in rats by vaccination with immunogenic rat neu peptides, but not by immunization with the intact protein. A rat model was used since rat neu protein is 89% homologous to human HER-2/neu protein and has a similar tissue distribution and level of expression. Rats were immunized with groups of peptides derived from the amino acid sequence of the intracellular domain or extracellular domain of rat neu protein and both groups developed CD4+ T cell immunity and Ab immunity to rat neu peptides and protein. Animals immunized in a similar fashion with intact purified rat neu protein did not develop Ab or T cell immunity to rat neu. Furthermore, rats that developed neu-specific immunity showed no histopathologic evidence of autoimmunity directed against organs expressing basal levels of rat neu protein. These studies suggest an immunization strategy that might be effective in human cancer vaccines targeting self tumor Ag.
In vivo self-renewal of c-Kit+ Sca-1+ Lin(low/-) hemopoietic stem cells.
Authors: Osawa M , Nakamura K , Nishi N , Takahasi N , Tokuomoto Y , Inoue H , Nakauchi H
Published: December 2022

The long term marrow-repopulating ability of mouse bone marrow c-Kit+ Sca-1+ Lin(low/-) cells was studied. Injection of as few as 100 c-Kit+ Sca-1+ Lin(low/-) cells could rescue a lethally irradiated recipient mouse and reconstitute both myeloid and lymphoid cells in their peripheral blood for at least 10 mo. When limiting dilution analysis was performed in the presence of host-derived rescue cells, as low as five c-Kit+ Sca-1+ Lin(low/-) cells were shown to be capable of repopulating lymphohemopoietic cells. Subsequently, we examined whether c-Kit+ Sca-1+ Lin(low/-) cells had a capacity for self-renewal in vivo. After transplantation of 500 c-Kit+ Sca-1+ Lin(low/-) Ly-5.1+ cells into lethally irradiated Ly-5 congenic mice, the expansion of cells with the same phenotypes as the injected cells was monitored. By day 28 post-transplantation, more than 50,000 donor type c-Kit+ Sca-1+ Lin(low/-) Ly-5.1+ cells were found in the spleen and over 18,000 cells were found in bone marrow. The expansion in spleen, however, was transient in that by day 60 cells of the donor phenotype were found only in bone marrow. The c-Kit+ Sca-1+ Lin(low/-) LY-5.1+ cells expanded in spleen or bone marrow contained as many high proliferative potential colony-forming cells as the originally injected cells. They also contained cells with LTRA, but the frequency appeared to be less compared with naive c-Kit+ Sca-1+ Lin(low/-) cells. These data provide evidence that c-Kit+ Sca-1+ Lin(low/-) cells in bone marrow are capable of multilineage differentiation as well as self-renewal and that spleen is a primary site of stem cell expansion after transplantation.
Proteolytic cleavage of alpha-actinin by calpain in T cells stimulated with anti-CD3 monoclonal antibody.
Authors: Selliah N , Brooks W H , Roszman T L
Published: December 2022

Stimulation of the TCR/CD3 complex on T cells initiates rearrangement of the actin cytoskeleton. The results presented show that a temporal increase in the appearance of filamentous actin begins immediately after stimulation of T cells with immobilized anti-CD3 mAb. The formation of filamentous actin in these stimulated cells reaches a steady state within 30 min after anti-CD3 mAb stimulation. At this time, pseudopod formation is observed and becomes progressively more evident over the next several hours. Experiments were done to investigate the role of the actin cytoskeletal associated proteins, alpha-actinin, vinculin, and talin, in the assembly of the actin cytoskeleton in anti-CD3 mAb-stimulated T cells. Using immunofluorescence, these three proteins are detected throughout the cytosol in resting T cells. However, after anti-CD3 mAb stimulation of the T cells, these proteins move to one pole of the cell. Electrophoresis followed by immunoblotting of T cell lysates prepared from resting as well as anti-CD3 mAb-stimulated cells revealed that alpha-actinin, but not vinculin or talin, was modified as a consequence of cell activation. Results show that alpha-actinin exists as a 105-kDa subunit in resting T cells, but that anti-CD3 mAb stimulation of T cells leads to the appearance of an 80-kDa lower molecular form of alpha-actinin. Experiments show that this occurs as a consequence of the 105-kDa subunit being proteolytically cleaved by calpain.
Analysis of the costimulatory role of IL-2 and IL-15 in initiating proliferation of resting (CD56dim) human NK cells.
Authors: Warren H S , Kinnear B F , Kastelein R L , Lanier L L
Published: December 2022

IL-15 is a newly described cytokine produced by monocytes and other non-T cells that utilizes the IL-2R beta- and common gamma-chains, thereby stimulating many NK cell functions previously ascribed to IL-2. Thus, IL-15 may promote NK cell activity during innate immune responses, before the activation of T lymphocytes and subsequent production of IL-2. This study investigated the ability of rIL-15 to substitute for rIL-2 in initiating proliferation of resting human NK cells cocultured with various stimulator cells. NK cell proliferation could not be initiated with rIL-15 as the sole costimulatory cytokine. However, NK cell proliferation was initiated with rIL-15 and either rIL-10 or rIL-12, cytokines also produced by monocytes and other APC and implicated in innate immune responses. Individually, rIL-10, rIL-12, and rIL-15 are effective initiators of NK cell proliferation when combined with submitogenic concentrations of rIL-2, indicating their potential involvement in NK cell proliferation at early stages of an Ag-specific T cell immune response. NK cells proliferating in the different cytokine combinations or optimum concentrations of rIL-2 were indistinguishable in terms of phenotype and cytotoxic activity, but differed in whether they secreted IFN-gamma or IL-5. IFN-gamma was secreted in cultures containing rIL-12, whereas IL-5 secretion was dependent upon interaction of IL-2 with the high affinity IL-2R. These results support the notion that NK cell proliferation occurs at different phases of the immune response with the particular cytokine milieu influencing the repertoire of NK cell-secreted cytokines.
Unusual uniformity of the N-linked oligosaccharides of HLA-A, -B, and -C glycoproteins.
Authors: Barber L D , Patel T P , Percival L , Gumperz J E , Lanier L L , Phillips J H , Bigge J C , Wormwald M R , Parekh R B , Parham P
Published: December 2022

MHC class I glycoproteins possess an invariant site for N-linked oligosaccharide addition at position 86 of the heavy chain. For human HLA-A, -B, and -C class I glycoproteins, position 86 is the only site of N-linked glycosylation. Comparison of the size and relative abundance of oligosaccharides associated with nine HLA-A, -B, or -C allotypes isolated from EBV-transformed B cell lines and mixtures of HLA-A, -B, and -C allotypes isolated from pooled PBLs revealed a very restricted set of structures. Allotypes encoded by the HLA-A and -B loci have two predominant glycan structures that were almost exclusively di-sialylated. In contrast, HLA-C allotypes have four glycan structures, comprising those associated with HLA-A and -B and two additional glycans. Identical oligosaccharides were present on different allotypes of a class I HLA locus, and in particular, HLA-C allotypes defining two inhibitory specificities for NK cells were shown to possess the same set of oligosaccharides. The uniformity of oligosaccharide structure associated with different HLA-A, -B, and -C products and the relative lack of heterogeneity for any given allotype are unusual features for a mammalian glycoprotein. Particularly striking is that such conserved oligosaccharide structures juxtapose the major regions of amino acid sequence variation within the Ag recognition site, including the polymorphisms of the alpha 1 helix that determine the inhibitory ligands for human NK cells.
Decreased production of TGF-beta 1 by human alveolar macrophages compared with blood monocytes.
Authors: Toossi Z , Hirsch C S , Hamilton B D , Knuth C K , Friedlander M A , Rich E A
Published: December 2022

As the main immunocytes lining pulmonary alveoli, alveolar macrophages (AM) are critical to the maintenance of immune hemostasis of the lung. This study examined the capacity of AM obtained from healthy individuals in comparison with autologous blood monocytes (MN) to produce transforming growth factor-beta 1 (TGF-beta), a pivotal molecule in regulation of immune responses and in promotion of fibrosis. AM produced negligible TGF-beta in response to LPS at both 24 and 72 h of culture. In contrast, LPS induced significant levels of TGF-beta in MN cultures (79.5 +/- 35 pg/ml in AM vs 890 +/- 162 pg/ml in MN, p less than 0.001, at 24 h). AM also produced significantly less TGF-beta than MN in response to phorbol ester and Con A. By northern blot analysis, constitutive expression of TGF-beta mRNA was lower in AM than MN at the time of isolation and after 24 h of culture. Lower expression of steady state TGF-beta message was not due to a more rapid decay of its mRNA in AM. Furthermore, TGF-beta mRNA expression was up-regulated by rTGF-beta in MN but was not induced in AM. In contrast to TGF-beta, LPS-stimulated AM produced sixfold higher levels of TGF-alpha at 24 h than MN (p less than 0.01). Production of IL-10 by LPS-stimulated AM was sixfold lower than MN (p less than 0.005) at 24 h of culture, but was comparable with MN at 72 h. Both 10-day cultured monocytes and peritoneal macrophages also had reduced capacity to produce TGF-beta. Therefore, the inability to produce TGF-beta may be a feature of more differentiated mononuclear phagocytes. In health, the reduced expression of TGF-beta by AM and the intact ability to produce TGF-alpha and IL-10 may favor a timely and regulated host response to inhaled pathogens while limiting potentially deleterious inflammatory responses.
Four IgG anti-islet human monoclonal antibodies isolated from a type 1 diabetes patient recognize distinct epitopes of glutamic acid decarboxylase 65 and are somatically mutated.
Authors: Madec A M , Rousset F , Ho S , Robert F , Thivolet C , Orgiazzi J , Lebecque S
Published: December 2022

The selective destruction by an autoimmune process of the beta cells in the pancreas is the hallmark of the type 1 insulin-dependent diabetes mellitus. What triggers islet cell-specific autoreactive T and B cells, however, remains unclear. Identification of the targets of the anti-islet cell autoantibodies frequently found in insulin-dependent diabetes mellitus patients and analysis of their sequences should provide some insights into the nature of this disease. We have combined EBV transformation with CD40 activation of peripheral B cells from one patient with insulin-dependent diabetes mellitus to isolate four B cell clones that secrete islet cell-specific autoantibodies, These four human monoclonal autoantibodies are of the IgG1 isotype, and they each recognize a different epitope of the glutamic acid decarboxylase enzyme. Analysis of their variable gene sequences shows that, while clonally unrelated, three of the four human monoclonal autoantibodies use a member of the VH4 family, and two have rearranged the same delta light chain variable gene. The IgG1 isotype of the four autoantibodies as well as the presence of somatic mutations in both heavy and light chain genes provide concrete evidence for their derivation by a T cell-dependent immune response.
Suppression of antigen-induced arthritis in rabbits by ex vivo gene therapy.
Authors: Otani K , Nita I , Macaulay W , Georgescu H I , Robbins P D , Evans C H
Published: December 2022

Gene therapy offers a novel approach to treating human joint diseases such as rheumatoid arthritis. In the present study, we have used the retrovirus, MFG-IRAP, to transfer the human IL-1 receptor antagonist protein (IRAP) gene to rabbits' knees and have assessed its impact on inflammatory and chondrodestructive aspects of the acute phase of antigen-induced arthritis in these joints. Surprisingly, intra-articular expression of IRAP was three- to fivefold higher in arthritic knees than in nonarthritic knees, accumulating to levels of over 20 ng/knee in the highest expressing joints. This level of expression produced a marked chondroprotective effect but a milder anti-inflammatory one. Both the increased cartilage matrix catabolism and the inhibition of matrix synthesis that occur in antigen-induced arthritis were abrogated in the presence of the IRAP gene; the latter effect was particularly strong. Of the indices of inflammation that were examined, only leukocyte influx into the joint space was inhibited, and this effect declined with time. Concentrations of rabbit IL-1 were reduced by the IRAP gene, suggesting inhibition of an autocrine induction loop. These data demonstrate that the course of arthritic disease in the rabbit knee can be altered by genetic manipulation, thus encouraging the further development of gene treatments for human joint diseases.
The role of p56lck in the development of gamma delta T cells and their function during an infection by Listeria monocytogenes.
Authors: Fujise S , Matsuzaki G , Kishihara K , Kadena T , Molina T , Nomoto K
Published: December 2022

We investigated roles of p56lck tyrosine kinase (Lck) on the development and function of gamma delta T cells in adult mice using lck gene knockout (lck -/-) mice. The mature gamma delta T cells (heat-stable Ag negative) were generated significantly in the thymi of adult lck -/- mice. When Listeria monocytogenes was infected i.p., gamma delta T cells were induced in the peritoneal cavity of the lck -/- mice. Interestingly, the repertoire of gamma delta T cells was obviously different in the lck +/+ and lck -/- mice; i.e., the gamma delta T cells of the lck -/- mice induced by the listerial infection dominantly expressed V delta 1 while those of the lck +/+ mice dominantly expressed V delta 6. The V delta 1 + gamma delta T cells in the lck -/- mice were extrathymically generated, supported by their appearance in thymectomized irradiated mice reconstituted with bone marrow cells from the lck -/- mice. Furthermore, the gamma delta T cells of the lck +/+ mice were protective in the early stage of the listerial infection, while the gamma delta T cells in the lck -/- mice were not protective against the listerial infection because the depletion of the gamma delta T cells from the lck -/- mice did not influence the bacterial burden in the spleens. These observations thus suggest that 1) gamma delta T cells can develop in adult mice through the intrathymic and extrathymic pathways even in the absence of Lck, 2) Lck influences the expansion and the repertoire of gamma delta T cells, and 3) the gamma delta T cells raised in the absence of Lck are not protective against L. monocytogenes.
Analysis of the site of interaction of CD28 with its counter-receptors CD80 and CD86 and correlation with function.
Authors: Kariv I , Truneh A , Sweet R W
Published: December 2022

CD28 and CTLA-4 are homologue members of the Ig superfamily of molecules, containing a single V-like domain, transmembrane, and cytoplasmic regions. Both receptors associate with the counter-receptors CD80 and CD86, but the avidity of interaction for CD28 is about 20-fold lower than for CTLA-4. The interaction between CD28 and its cognate receptors provides a costimulatory signal for optimal T cell activation. Our previous mutational analysis of CD28 defined the highly conserved "MYPPPY" motif in the CDR3-region of the V-like domain as a key site of common and selective recognition. We have extended our analysis to cover all residues in the membrane distal loops of the V region, examining their effect on association with CD80/CD86 in cell adhesion and novel protein-based binding assays, and determining correlation between binding and functional response. Conservative F substitutions at either Y residue in the MYPPPY motif selectively reduced binding to CD86, but mutation of the three amino acids immediately C-terminal to Y 104 equivalently reduced binding to both co-receptors. The conservative F substitution of Y 26 in the CDR1-like region also reduced binding to CD80 and CD86. Other substitutions in the CDR1 loop and mutations spanning the CDR2 and DE loops had no effect. We conclude that the CDR1 and CDR3 regions contribute to a common binding site for CD80/CD86, and that the CDR3 region also carries determinants for selective recognition of these counter-receptors within the MYPPPY motif. Furthermore, for CD28, the strength of functional response, as measured by IL-2 production, directly correlates with binding avidity.
Nerve growth factor and cytokines mediate lymphoid tissue-induced neurite outgrowth from mouse superior cervical ganglia in vitro.
Authors: Kannan Y , Bienenstock J , Ohta M , Stanisz A M , Stead R H
Published: December 2022

Superior cervical ganglia (SCG) from neonatal mice were cultured with adult murine lymphoid tissue explants in Matrigel (Collaborative Biomedical, Bedford, MA). After 1 and 2 days in culture, many neurites grew toward thymus and spleen. Normal mesenteric lymph node (MLN) induced a smaller effect; however, activated MLN (isolated from mice 10 days after infection with Nippostrongylus brasiliensis; Nb-MLN-10d) caused significantly increased neurite outgrowth. To determine the roles of nerve growth factor (NGF) and cytokines in the promotion of neuritogenesis by lymphoid tissues, anti-NGF and various anti-cytokines were added to cocultures. Anti-NGF inhibited most of the neurite outgrowth toward thymus and spleen but only partially that toward Nb-MLN-10d. Anti-mouse IL-1 beta also significantly reduced the number of neurites growing toward thymus, spleen, and normal MLN. The number of neurites growing toward Nb-MLN-10d was significantly reduced by anti-IL-1 beta, anti-IL-3, anti-IL-6, or anti-GM-CSF. Exogenous IL-1 beta and IL-3 caused neurite outgrowth in single SCG cultures; and the IL-1 beta-, but not the IL-3-, mediated effect was completely blocked by anti-NGF. In one-day thymus/SCG cocultures, endogenous IL-1 was not detectable at concentrations sufficient to cause nerve growth; however, ample NGF was present in the thymic tissues and culture supernatants, but not in SCG. These data suggest that IL-1 mediates NGF production in lymphoid tissues, which in turn induces the growth of sympathetic nerves. Moreover, IL-3, IL-6, or GM-CSF produced during inflammation might also play important roles in the stimulation of nerve growth in vivo.
5-Oxo-eicosatetraenoate is a broadly active, eosinophil-selective stimulus for human granulocytes.
Authors: O'Flaherty J T , Kuroki M , Nixon A B , Wijkander J , Yee E , Lee S L , Smitherman P K , Wykle R L , Daniel L W
Published: December 2022

5-Oxo-eicosatetraenoate (5-oxoETE) is gaining recognition as a chemotactic factor for eosinophilic (Eo) as well as neutrophilic (Neu) polymorphonuclear leukocytes. We found that the eicosanoid was far stronger than C5a, platelet-activating factor (PAF), leukotriene B4 (LTB4), or FMLP in stimulating Eo chemotaxis. Moreover, it had weak intrinsic degranulating effects on otherwise unstimulated Eo, produced prominent degranulation responses in Eo primed by granulocyte-macrophage CSF, and enhanced the Eo-degranulating potencies of PAF, C5a, LTB4, and FMLP by up to 10,000-fold. Low picomolar levels of 5-oxoETE also induced Eo to activate mitogen-activated protein kinases (MAPKs), as defined by shifts in the electrophoretic mobility and tyrosine phosphorylation of two immunodetectable proteins, p44 and p42. 5-OxoETE was > or = 100-fold weaker or unable to stimulate any of these responses in Neu. Finally, 5-oxo-15-hydroxy-ETE and 5-hydroxy-ETE activated both cell types, but were weaker than 5-oxoETE and had Eo/Neu potency ratios approaching unity. 5-OxoETE, thus, is uniquely potent and selective in promoting Eo not only to migrate, but also to release granule enzymes and activate MAPKs. By triggering MAPK activation, the eicosanoid may also influence the production of anaphylactoid lipids (e.g., PAF), arachidonic acid metabolites, and cytokines. 5-OxoETE therefore possesses a biologic profile well suited for mediating Eo-dominated allergic reactions in vivo.
Unstimulated peripheral blood mononuclear cells from patients with the hyper-IgD syndrome produce cytokines capable of potent induction of C-reactive protein and serum amyloid A in Hep3B cells.
Authors: Drenth J P , van der Meer J W , Kushner I
Published: December 2022

The hyper-IgD and periodic fever syndrome (HIDS) and familial Mediterranean fever (FMF) are both characterized by attacks of periodic fever accompanied by acute phase responses that are substantially higher in HIDS than in FMF. To determine whether this difference could be due to differences in production of acute phase protein-inducing mediators, we studied PBMC from HIDS and FMF patients in the inactive phase of disease. Unstimulated PBMC from patients with inactive HIDS released significantly more IL-1 beta, IL-6, and TNF-alpha than did PBMC from patients with FMF, but unstimulated PBMC from the latter group released significantly more IL-1 beta and IL-6 compared with controls. Conditioned medium (CM) derived from PBMC of patients with inactive HIDS induced significantly greater CRP production and significantly higher mRNAs for CRP and SAA in Hep3B cells than did CM derived from the PBMC of patients with inactive FMF. Stimulation of PBMC with LPS led to further increases in cytokine production and in acute phase protein-inducing ability in both patient groups and in controls. These findings suggest that the greater acute phase response seen in HIDS compared with FMF reflects greater production of acute phase protein-inducing cytokines in the former patients and indicates that PBMC from inactive HIDS patients are already activated in vivo. Finally, the finding of both quantitative and qualitative differences in cytokine production by unstimulated PBMC from HIDS and FMF patients supports the likelihood of different pathogeneses of these diseases.
Impaired production of both normal and mutant C1 inhibitor proteins in type I hereditary angioedema with a duplication in exon 8.
Authors: Ernst S C , Circolo A , Davis A E , Gheesling-Mullis K , Fliesler M , Strunk R C
Published: December 2022

In the autosomal dominant disorder type I hereditary angioedema, reduced levels of C1 inhibitor may be due in part to increased turnover and decreased synthesis of normal C1 inhibitor protein. A type I hereditary angioedema patient was recently described in whom the C1 inhibitor mutation consisted of a 20-bp duplication of nucleotides 1414 to 1433 in exon 8 that introduced a frame shift predicting the loss of a normal stop codon and the translation of a protein 52 amino acids longer than normal. In this study, we analyzed the expression of C1 inhibitor in fibroblasts obtained from a skin biopsy of this patient. Two proteins of approximately 78 and 94 kDa were found intracellularly, corresponding to the products of normal and mutated alleles, respectively. Pulse-chase analysis showed a complete lack of secretion of the mutated form. In addition, there was decreased extracellular production of the normal C1 inhibitor, suggesting either decreased secretion or increased intracellular catabolism of the normal protein because of the presence of the mutant allele. The production of other complement proteins was normal. This study provides a model for further analysis of autosomal dominant genetic disorders in which production of the functional protein may be affected by the product of the mutated allele.
B4B, a novel growth-arrest gene, is expressed by a subset of progenitor/pre-B lymphocytes negative for cytoplasmic mu-chain.
Authors: Ruegg C L , Wu H Y , Fagnoni F F , Engleman E G , Laus R
Published: December 2022

We subjected PBMC of normal adults to density fractionation to enrich for an immunoblast fraction that would include early immune lineage precursors. Differential display PCR experiments identified one transcript that is expressed specifically in this immunoblast fraction. This cDNA, designated B4B, encodes a novel gene product containing four putative transmembrane-spanning domains. B4B+ cells, detected with anti-B4B Ig, were found at very low frequency in PBMC (0.01%) and were enriched significantly in intermediate density fractions (0.1-1.0%). B4B+ cells were shown to be CD19+CD45+HLA-DR+ and negative for CD20, cytoplasmic mu-chain, CD3, CD16, CD56, CD34, and CD68 (monocyte), consistent with a progenitor/pre-B lymphocyte subset that does not express cytoplasmic mu-chain and thus may lack productive Ig rearrangement. This phenotypic description of the B4B+ subset agrees with our finding that the frequency of B4B+ cells was greatly increased in bone marrow (3-10%) as compared with PBMC (0.01%). The B4B polypeptide sequence exhibits significant homology to only one known protein, PMP-22/gas-3, a Schwann cell-specific protein that induces cell growth arrest. Transient expression of B4B specifically inhibited cellular proliferation by more than 50%. Based on its antiproliferative effect and pattern of expression restricted to a subpopulation of immature B cells, the B4B gene product may be involved in the elimination of B cells before productive VDJC rearrangement of Ig loci or, alternatively, in the growth arrest of transformed progenitor B cells.
Human T cells require IL-2 but not G1/S transition to acquire susceptibility to Fas-mediated apoptosis.
Authors: Fournel S , Genestier L , Robinet E , Flacher M , Revillard J P
Published: December 2022

The interaction between Fas ligand and Fas, both expressed on activated T cells, is the major pathway in the regulation of activation-induced cell death. However, activated T cells that express membrane Fas are initially resistant to anti-Fas-induced apoptosis and become susceptible only after proliferation in vitro. Since IL-2 is known to regulate activation-induced cell death, we studied the effect of IL-2 on anti-Fas-mediated apoptosis. Interference with the IL-2 pathway was achieved by 1) inhibition of cytokine synthesis using cyclosporin A or FK506, 2) neutralization of IL-2 by anti-IL-2 Ab, 3) inhibition of binding to IL-2R by CD25 mAb, and 4) blocking of IL-2R signaling by rapamycin. We show that Fas expression is independent of the IL-2 pathway, whereas Fas-mediated apoptosis does not develop in the presence of inhibitors of IL-2 production or signaling. While the addition of rIL-2 reversed the inhibitory effect of cyclosporin A and FK506, the addition of rIL-4, rIL-7, or rIFN-gamma did not, although these cytokines induced progression into the S phase of the cell cycle. Aphidicolin-treated activated T cells that do not progress into the S phase were susceptible to Fas-mediated apoptosis. Therefore, Fas-mediated apoptosis is controlled by signals generated by IL-2 in agreement with the reported alteration of apoptosis in mice deficient in IL-2 or IL-2R.
Functional CD40 ligand expressed by human blood dendritic cells is up-regulated by CD40 ligation.
Authors: Pinchuk L M , Klaus S J , Magaletti D M , Pinchuk G V , Norsen J P , Clark E A
Published: December 2022

We examined whether human blood dendritic cells (DCs) express a functional ligand for CD40 (CD40L). Human blood DCs expressed significant amounts of cell surface CD40L identical to that expressed on activated T cells, as detected by mAb to CD40L or a chimeric CD40.Ig fusion protein (CD40.Ig). Stimulation through CD40 up-regulated protein and mRNA CD40L expression in DCs, B cells, and B cell lines. CD40-mediated CD40L expression was inhibited by a protein tyrosine kinase inhibitor, herbimycin, in a dose-dependent manner, suggesting that the induction of CD40L expression via CD40 requires protein tyrosine kinase activity. CD40L surface expression correlated with constitutive or inducible levels of CD40L-specific mRNA, as determined by reverse transcribed PCR analysis (RT-PCR) using CD40L-homologous primers. Furthermore, CD40L on DCs was functional, since CD40L+ DCs, unlike CD40L- DCs, induced B cell IgG and IgA production, and this induction could be inhibited by blocking CD40L-CD40 interactions with mAb to CD40L. Thus, CD40L on DCs and CD40L induced by crosslinking CD40 may regulate B cell activation and maturation. The importance of DC CD40L expression on B cell function is discussed.
Development of CD4-CD8- alpha beta TCR+NK1.1+ T lymphocytes: thymic selection by self antigen.
Authors: Schulz R J , Parkes A , Mizoguchi E , Bhan A K , Koyasu S
Published: December 2022

Development of CD4-CD8- double negative (DN) alpha beta TCR+ cells were examined by cell transfer experiments using an Ly-5 congenic mouse system. Purified DN alpha beta TCR+ thymocytes injected intrathymically emigrated from the thymus to the spleen. The same cells did not return to the thymus when injected i.v. Similarly, peripheral DN alpha beta TCR+ cells from spleen and liver did not go to the thymus when injected i.v. but migrated to the spleen. These results indicate that DN alpha beta TCR+ thymocytes develop within the thymus and emigrate to peripheral organs. It is thus likely that peripheral DN alpha beta TCR+ cells are at least partly of thymic origin. DN alpha beta TCR+ thymocytes are unique in that they express a natural killer cell marker, NK1.1, which is not found on conventional T cells. We further examined the thymic selection of DN alpha beta TCR+NK1.1+ thymocytes by using an anti-HY TCR-transgenic (tg)/Rag-2(-/-) mouse system with H-2 backgrounds that were negative, positive, or nonselecting for conventional T cells. The number of DNtg TCR alpha beta+NK1.1+ cells was m prominent in male H-2b animals in which conventional T cells are deleted by HY/H-2Db recognition. Fewer DNtg TCR alpha beta+NK1.1+ cells were found in H-2b females (positive selecting background), and almost no DNtg TCR alpha beta+NK1.1+ cells were detected in H-2d animals (nonselecting background). Unlike conventional T cells, DNtg TCR alpha beta+NK1.1+ cells from anti-HY/Rag-2(-/-) H-2b mice express Fc(epsilon)RI(gamma) and CD3zeta as DN alpha beta TCR+NK1.1+ cells from normal C57BL/6 mice. Our results indicate that DNtg TCR alpha beta+NK1.1+ cells are positively selected by self Ag/MHC and emigrate to the peripheral organs.