bluuebunny/crossref_metadata_2025 huggingface dataset:
Spontaneous T cell antigen receptor variants of a human T leukemia cell line.
Authors: Maecker H T , Levy R
Published: December 2022

We have sought to address the question of clonal variation of TCR within a human T leukemia cell line, HPB-ALL. To do so, a panel of anti-idiotypic antibodies was produced and the cell line examined for variants. We isolated both spontaneous idiotype and receptor-negative variants without applying mutagens or any selective pressure other than sorting the cells. These sorted and cloned populations are all clonally related to each other as shown by their beta-TCR locus gene rearrangements. The idiotype variants have alpha-chains which are differentially glycosylated, but they have the same size core protein after treatment with peptide N-glycosidase F to remove their carbohydrate side chains. This probably accounts for their idiotypic difference, since the antibody that distinguishes them appears dependent upon glycosylation for its binding, as shown by immunoprecipitation in the presence versus the absence of tunicamycin, which inhibits glycosylation from occurring. The idiotype variants differed from one another in variable region sequences by only a single amino acid substitution in the beta-chain, which is likely not important for the idiotypic difference. The receptor-negative variant produces both alpha- and beta-mRNA and cytoplasmic protein for TCR, but fails to transport this protein to the cell surface. We conclude that idiotype and receptor-negative variants of a T cell clone can occur in the absence of appreciable somatic mutation.
Structural and functional studies of HLA-DR restricted antigen recognition by human helper T lymphocyte clones by using transfected murine cell lines.
Authors: Lechler R I , Bal V , Rothbard J B , Germain R N , Sekaly R , Long E O , Lamb J
Published: December 2022

Murine L cells expressing the products of transfected HLA-DR1 genes functioned as APC for two influenza-specific, human Th cell clones with comparable efficiency to a DR1-expressing human lymphoblastoid cell line. In order to investigate the restriction specificity of the two Th clones, a transfectant expressing the species-mismatched MHC class II dimer DR1:I-E was tested as an APC. Both T cells showed no loss of Ag sensitivity due to substitution of the murine chain. One of the Th clones, TLC 72, showed even greater degeneracy by responding to Ag in the context of I-Ek. Taking into account the lower level of MHC class II expression on the I-Ek transfectant, there is remarkably little loss of efficiency of Ag-induced T cell activation due to the substitution of I-E for DR as restriction element. The Ag-specific responses of both clones were inhibited by anti-CD4 antibody when DR-transfected L cells or human lymphoblastoid cells were used as APC. This inhibition was also seen when Ag was presented to TLC72 by the I-Ek-expressing transfectant. Whether this inhibition is the result of negative signaling or of blocking an interaction between human CD4 and I-Ek is discussed. Similarly the inhibitory effects of mAb against the T cell accessory molecule LFA/1 were the same for both clones when either the transfectants or the lymphoblastoid cell line were used as APC, suggesting that L cells may express a molecule that is capable of acting as a ligand for human LFA/1. The results presented here further illustrate the value of transfectants in analyzing T cell recognition and accessory cell requirements. The patterns of degeneracy of MHC restriction exhibited by these clones provides a platform for a more detailed analysis of key residues involved in MHC class II-restricted T cell Ag recognition.
Antitubulin antibodies. II. Natural autoantibodies and induced antibodies recognize different epitopes on the tubulin molecule.
Authors: Matthes T , Wolff A , Soubiran P , Gros F , Dighiero G
Published: December 2022

Natural and induced antitubulin antibodies were compared for their epitope recognition on alpha- and beta-tubulin subunits by immunoenzymatic assays and Western blot techniques on partially digested tubulin molecules. Our results indicated that natural autoantibodies recognized different epitopes from those recognized by induced antibodies, because: 1) all polyspecific natural autoantibodies tested so far recognized the same or very overlapping epitopes in the central part of both alpha- and beta-subunits (between positions 100 and 300 on the tubulin amino acid sequence) and that this epitope differed from the various epitopes recognized by induced antitubulin antibodies on the amino-terminal or carboxy-terminal parts of the tubulin subunits; 2) one human myeloma protein (monoclonal (m)IgA, kappa) with a monospecific antitubulin activity bound to an epitope around position 310 on both alpha- and beta-subunits and a second human mIg (mIgM, kappa) with a monospecific anti-beta activity bound to an epitope on the carboxy-terminal part of the subunit around amino acid position 350. Both epitopes differed from epitopes recognized by induced antitubulin antibodies. These results thus confirmed our previous findings indicating that natural and induced antitubulin antibodies do not share cross-reactive idiotopes.
Antipeptide antibody specific for the N-terminal of soluble immune response suppressor neutralizes concanavalin A and IFN-induced suppressor cell activity in an in vitro cytotoxic T lymphocyte response.
Authors: Devens B H , Semenuk G , Webb D R
Published: December 2022

Soluble immune response suppressor (SIRS) is a nonspecific immunosuppressive lymphokine produced by Lyt-2+ lymphocytes after exposure to Con A, IFN-alpha, or IFN-gamma. The N-terminal 21 residues of SIRS have recently been elucidated and antisera specific for this sequence have been raised in rabbits by using a synthetic peptide coupled to an immunogenic carrier protein. In a series of experiments, we have established that this antiserum blocks the suppressive activity of Con A- or IFN-activated suppressor cells. These data establish that Con A- or IFN-activated suppressor cells exert some or all of their suppressive effects via SIRS. Further studies show that SIRS acts primarily during the induction of CTL and not at the effector phase. Last, we show that SIRS is not involved in the radiation-resistant Ag-specific suppressor cell circuit that can be induced during the in vitro MLR.
Monoclonal antipeptide antibodies as tools to dissect closely related gene products. A model using peptides encoded by the calcitonin gene.
Authors: Ghillani P , Motté P , Bohuon C , Bellet D
Published: December 2022

We have explored the possibility of using mAb as tools for distinguishing between closely related gene products. We utilized calcitonin (CT) gene products as a model, because this 32-amino-acid-amidated hormone is biosynthesized by post-translational processing of a larger precursor. By using CT as a hapten, we had previously identified a mAb (CT07) with restricted specificity to mature CT, and had shown that another mAb (CT08) directed to a different epitope bound to both CT and the CT precursor. In this study, we used synthetic peptides analogous to various regions of biosynthetic intermediates of CT as haptens, and generated a library of mAb which define distinct epitopes. First, we identified two separate epitopes located in either the 1-11 or the 12-21 region of the C-terminal flanking peptide of CT (katacalcin, KC), and which were recognized by mAb KC01 and KC04, respectively. Second, we identified a conformational epitope in the C-terminal region of the putative glycine-extended form of CT (CT-Gly). This epitope was recognized by mAb CT19 and was shared by mature CT but not by CT precursors. Third, we identified an epitope restricted to CT-Gly and recognized by mAb CT20. For dissecting between related products of the CT gene, we designed different monoclonal immunoradiometric assays (m-IRMA) based on CT08 as the radiolabeled indicator antibody. A first m-IRMA based on CT07 as the capture antibody specifically recognized mature CT and did not cross-react with CT precursors. Conversely, another m-IRMA with KC01 as the capture antibody was specific for CT precursors and did not cross-react with either mature CT or CT-Gly. A third assay based on CT20 as the capture mAb was specific for CT-Gly and was not affected by the presence of either CT precursors or mature CT. We also used these antibodies to demonstrate that neoplastic C cells incompletely released processed CT precursors in serum, in addition to mature CT. This study demonstrates that mAb can be used as tools to selectively recognize closely related gene products. These findings might be applied to the study of other molecules biosynthesized by enzymatic modifications of a larger precursor.
C1q induces chemotaxis and K+ conductance activation coupled to increased cytosolic Ca2+ in mouse fibroblasts.
Authors: Oiki S , Okada Y
Published: December 2022

Cultured mouse fibroblasts (L cells) respond to whole C with a slow hyperpolarization. Among the C components tested, C1q was found to be most effective. In contrast, the cell did not respond to C1, in which the collagen-like region of the C1q molecule is masked. The C1q-induced hyperpolarizing response was inhibited by collagen or C1q-specific antisera. Human diploid skin fibroblasts (Flow 1,000 cells) also exhibited similar membrane potential changes in response to whole C or C1q. After repeated applications of C1q, the cell membrane became unresponsive (desensitized). The treatment of L cells with pronase E inhibited the C1q-induced response, whereas the response to ATP, which is known to interact to its own receptor, was still preserved. The reversal potential of C responses was close to the K+ equilibrium potential. The hyperpolarizing response was inhibited by a blocker of Ca2+-activated K+ channels in fibroblasts (quinine), by deprivation of extracellular Ca2+ or by a Ca2+ channel blocker (nifedipine). By means of Ca2+-selective microelectrodes, the cytosolic free Ca2+ concentration was found to increase from 126 to 206 nM upon stimulation of L cells with C1q. Using an agarose-well method, L cells were observed to migrate predominantly toward C1q or whole C. It is concluded that the fibroblasts have the C1q receptor sensitive to pronase E and that activation of C1q receptors gives rise to Ca2+ influx, triggering an increase in the cytosolic free Ca2+ ions, which in turn induces a hyperpolarizing response as a result of the stimulation of Ca2+-activated K+ channels and initiates chemotaxis to C1q.
Identification of T epitopes within a potential Plasmodium falciparum vaccine antigen. A study of human lymphocyte responses to repeat and nonrepeat regions of Pf155/RESA.
Authors: Rzepczyk C M , Ramasamy R , Ho P C , Mutch D A , Anderson K L , Duggleby R G , Doran T J , Murray B J , Irving D O , Woodrow G C
Published: December 2022

PBMC from Melanesians who had high antibody reactivities to fusion proteins encompassing the 3' and the 5' repeat regions of the ring infected E surface antigen (Pf155/RESA), were tested for their ability to respond to synthetic and recombinant peptides representing regions of Pf155/RESA. The aim was to identify T cell epitopes within the Ag. Most of the synthetic peptides from the nonrepeat regions of Pf155/RESA were selected for study on the basis of their tendency to form amphipathic alpha-helices. Peptides representing immunodominant B cell epitopes were also tested. Three-quarters of the Melanesian donors responded to the recombinant peptides (Ag 1505 and Ag 632-100) and to the 8 x 4 mer, a synthetic peptide representative of the 3' repeat region. Whereas all the remaining eight peptides tested elicited a response in at least one donor, three peptides (M40, M42, and BTA3) representing sequences in the nonrepeat regions showed greatest promise as potentially useful T epitopes. Responses in control donors were also observed to most of the peptides but the percentage of responders was lower. T cell bulk lines specific to Ag 1505 and Ag 632-100 were established. All donors were HLA tissue typed, but no obvious correlations between responsiveness and HLA type were observed. Our results suggest that there are T cell epitopes within and outside the repeat regions of Pf155/RESA.
Escherichia coli-specific T lymphocytes in experimental pyelonephritis.
Authors: Kurnick J T , McCluskey R T , Bhan A K , Wright K A , Wilkinson R , Rubin R H
Published: December 2022

We have assessed the phenotype and specificity of infiltrating mononuclear cells in a model of unilateral ascending acute pyelonephritis induced in rats with nephritogenic Escherichia coli or Pseudomonas aeruginosa. Histologic examination showed a predominance of mononuclear cells in the interstitium at all periods examined (4, 8, 15, 21, and 25 days), although at 4 and 8 days neutrophils were also abundant. Most of the mononuclear cells had the morphologic appearance of large lymphocytes. Immunoperoxidase studies with mAb showed that most of the mononuclear cells were W3/25+; many were W3/13+ and a small proportion were OX8+. Many of the mononuclear cells were Ia+. T cells were propagated in IL-2-containing media from small fragments of renal tissue with pyelonephritic lesions. Most of the propagated cells were W3/25+; fewer than (10%) were OX8+ or Ia+. T cells propagated from kidneys infected with E. coli responded, in proliferation assays, to the infecting strain or other E. coli strains, but not to P. aeruginosa or enterococci. The response to non-p-pilus-bearing E. coli was as great or greater than to E. coli with adhesins. T cells derived from lesions induced by P. aeruginosa responded to the infecting organisms, but not to E. coli. The response to the infecting organism (E. coli or P. aeruginosa) was MHC restricted, as indicated by the requirement for syngeneic APC. The results show that large numbers of T lymphocytes, especially with the "helper/inducer" phenotype, accumulate in the lesions of acute pyelonephritis in rats. Among the infiltrating T lymphocytes are activated cells and cells with specific reactivity to the infecting bacteria (or related strains). The findings indicate that T lymphocytes play a role within the kidney in response to the invading bacteria.
Changes in gene expression associated with IFN-beta and IL-2-induced augmentation of human natural killer cell function.
Authors: Kornbluth J , Hoover R G
Published: December 2022

NK function can be augmented by a variety of agents, including the cytokines IL-2 and IFN. The mechanisms associated with IL-2- and IFN-mediated augmentation of NK function are largely unknown. In order to learn more about the regulation of NK activity, we have studied changes in gene expression that occur upon treatment of a cloned line of NK cells (NK 3.3) with rIL-2 and rIFN-beta. Both IL-2 and IFN-beta induced rapid augmentation of lysis mediated by NK 3.3, which was significant within 1 h, peaked at 6 h of treatment, and declined by 12 h. This enhancement of lytic function was independent of proliferation and associated with a corresponding increase in steady state levels of RNA coding for both the nuclear proto-oncogene c-myb and for the IL-2R. These changes were specific in that RNA levels of another nuclear proto-oncogene, c-myc, were increased by IL-2 but not by IFN-beta, whereas HLA class I RNA levels were relatively unchanged by either IL-2 or IFN-beta treatment. Treatment of NK 3.3 with the combination of IL-2 and IFN enhanced both lysis and c-myb expression in an additive fashion. These findings suggest that c-myb may play a regulatory role in the cytolytic activity of NK cells.
Effect of human recombinant IL-2 on murine macrophage precursors. Involvement of a receptor distinct from the p55 (Tac) protein.
Authors: Baccarini M , Schwinzer R , Lohmann-Matthes M L
Published: December 2022

Homogeneous populations of bone marrow-derived murine macrophage precursors are able to respond to human rIL-2. A prolonged exposure to the lymphokine results in proliferation and in the final differentiation of macrophage precursors to mature macrophages, as shown by follow-up of the antigenic profile by means of cytofluorograph analysis. The frequency of the IL-2-responding cells was 1/48 as determined by limiting dilution analysis, and no cell death was evidenced in soft agar cultures. An analysis of the IL-2-binding sites conducted on bone marrow-derived macrophage precursors revealed the presence of 4000 binding sites with intermediate affinity (Kd approximately 3 x 10(-9)). Bone marrow-derived macrophage precursors did not react, even after prolonged culture in IL-2-conditioned media, with mAb recognizing the binding site (AMT-13, 3C7) or a binding site-associate molecule (7D4) of a Mr 50,000 to 60,000 component of the IL-2R expressed by the IL-2-dependent T cell line CTLL-2. Cross-linking studies showed the presence of a single binding protein, with an apparent Mr of 70,000. Macrophage precursors also proved able to internalize human rIL-2 within the first 20 min of incubation at 37 degrees C. The implications of these findings are discussed.
Identification of alpha 2-macroglobulin as a carrier protein for IL-6.
Authors: Matsuda T , Hirano T , Nagasawa S , Kishimoto T
Published: December 2022

In this report we demonstrate that alpha 2-macroglobulin (alpha 2M) is a carrier protein for IL-6. IL-6 was found to bind plasma proteins and an immunoblot analysis revealed that the complex between IL-6 and plasma proteins contains alpha 2M. Furthermore, purified alpha 2M bound IL-6. alpha 2M did not inhibit IL-6 activity or its binding to homologous receptor. IL-6 bound to alpha 2M retained its biologic activity and became resistant to treatment with proteases, although free IL-6 was easily degraded. These findings indicate that alpha 2M plays an important role as a carrier protein for IL-6 in serum and makes IL-6 produced at the local inflammatory site available to lymphocytes, hepatocytes, and hematopoietic stem cells, resulting in the induction of the coordinate systemic host defense reactions, such as immune response, acute phase reaction, and hematopoiesis.
IL-3 and stromal cell-derived factor synergistically stimulate the growth of pre-B cell lines cloned from long-term lymphoid bone marrow cultures.
Authors: Rennick D , Jackson J , Moulds C , Lee F , Yang G
Published: December 2022

The addition of IL-3 to modified Whitlock-Witte long-term lymphocyte cultures was found to enhance the growth of a small but significant number of B cell precursors supported by an adherent stromal cell monolayer. Several pre-B cell lines were cloned from IL-3-treated long-term lymphocyte cultures. The growth requirements and physical properties of one representative clone, BL/3, are described. BL/3 cells were shown to be unresponsive to IL-3 except when it is used at very high concentrations. In contrast, significant growth was stimulated by stromal cell conditioned medium previously shown to contain a pre-B cell growth factor. Optimal growth of the pre-B cell clone was stimulated by stromal cell conditioned medium plus IL-3. Synergy between the stromal cell-derived factor and IL-3 occurred when IL-3 was used over a wide range of concentrations including a relatively low amount that was ineffective as a growth stimulus by itself. The finding that more than one factor is required to sustain optimal growth of some pre-B cells parallels the complex growth requirements reported for some primitive myeloid/erythroid progenitors.
Absence of induction of IL-1 production in human monocytes by complement fragments.
Authors: Arend W P , Massoni R J , Niemann M A , Giclas P C
Published: December 2022

The ability of C fragments to induce IL-1 production in human monocytes was examined by using various approaches to carefully exclude the role of contaminating endotoxin. The presence of IL-1 activity in monocyte supernatants and lysates was assayed by the augmentation of PHA-induced proliferation of murine thymocytes. SRBC were opsonized with IgM rabbit antibodies and various human C components to prepare EAC reagents that contained less than 25 pg LPS/ml of EAC at 5 x 10(8) cells/ml. EAC1q, EAC4b, EAC4b2aoxy, EAC4b2aoxy C3b, EAC4b2aoxyC3bi, and EAC4b2aoxyC3d all failed to induce IL-1 production when incubated at 10- to 100-fold excess with adherent human monocytes. Similarly, LPS-free purified C3a, C5a, and C5a des Arg all showed no IL-1-inducing activities at concentrations up to 25 micrograms/ml. However, the same C5a preparations were active on human monocytes in the induction of chemotaxis, and C3a and C5a both induced skin-blueing in guinea pigs. Fragment Ba and Bb preparations purified by gel filtration chromatography contained approximately 100 pg LPS/micrograms Ba or Bb. These Ba and Bb preparations at 10 and 50 micrograms/ml, respectively, induced IL-1 production in the presence of 5 micrograms/ml polymyxin B (PMB). However, Ba and Bb preparations purified by affinity chromatography and HPLC contained lower levels of endotoxin contamination and displayed IL-1-inducing activities at Ba and Bb concentrations of 50 and 100 micrograms/ml, respectively, that were almost completely inhibited by PMB. To explore further the role of contaminating endotoxin, a Bb preparation was adsorbed with PMB-4B in the presence of a dialyzable detergent to remove LPS bound to the Bb. This LPS-free Bb preparation failed to induce IL-1 production while maintaining intact enzymatic activities. These results indicate that various solid phase or soluble C fragments, including C3b, iC3b, C3d, C3a, C5a, Ba or Bb do not induce IL-1 production in human monocytes in the absence of contaminating endotoxin.
Identification and localization of allergenic determinants on grass group I antigens using monoclonal antibodies.
Authors: Esch R E , Klapper D G
Published: December 2022

Epitopes recognized by five mAb which block the binding of human IgE antibodies to grass group I (GpI) Ag were characterized and partially mapped. Site specificity studies defined four apparently non-overlapping blocking antibody binding sites on the meadow fescue GpI molecule, Fes e I. One of these sites (site A) was localized to a 14,000 m.w. fragment designated P3 generated by CNBr cleavage of purified Fes e I. The P3 peptide possessed human IgE binding sites as well as other epitopes (non-site A) defined by 19 other anti-GpI mAb. All of the P3 reactive antibodies recognized cross-reactive determinants found on GpI Ag isolated from five different grasses suggesting that P3 is a conserved portion of grass GpI molecules. The P3 fragment from Fes e I was used to immunize mice and induced antibodies which reacted with intact GpI Ag from all 5 different grasses currently being studied in this laboratory.
Characterization of the enhanced susceptibility of paroxysmal nocturnal hemoglobinuria erythrocytes to complement-mediated hemolysis initiated by cobra venom factor.
Authors: Parker C J , Stone O L , Bernshaw N J
Published: December 2022

When whole serum C is activated by cobra venom factor complexes (CoFBb), paroxysmal nocturnal hemoglobinuria (PNH) III E (the most C-sensitive type) are hemolyzed, but normal and PNH II E (the intermediately sensitive type) are not. Previous studies have shown that after exposure to CoFBb and serum, PNH III E bind relatively large amounts of the trimolecular C complex, C5b67, whereas normal and PNH II E bind virtually none. In the studies reported herein, we have observed that when normal and PNH III E are incubated with isolated C5, C6, and 125I-C7 in the presence CoFBb, the normal E bind more C5b-7 than the PNH cells. When C7-deficient serum is included in the reaction mixture, however, the PNH E are once again observed to bind much greater amounts of C5b-7. These observations suggest that plasma and membrane factors act in concert to restrict the assembly of the trimolecular C5b-7 complex on human E. PNH III E appear to be deficient in the membrane component of this inhibitory system.
Fc gamma R-mediated killing by eosinophils.
Authors: Graziano R F , Looney R J , Shen L , Fanger M W
Published: December 2022

In this report we present data on the ability of the different Fc gamma R present on eosinophils to mediate killing of erythroid and tumor targets, and on a comparison of eosinophil and neutrophil Fc gamma R-mediated killing. Erythroid target killing was assessed using chicken erythrocytes (CE) and heteroantibodies composed of Fab fragments of anti-CE antibodies covalently coupled to Fab fragments of anti-Fc gamma R antibodies. Such anti-CE x anti-Fc gamma R reagents permit linkage of CE target cells with the FcR molecules on the eosinophil or neutrophil effector cells. Tumor target killing was assessed using hybridoma cell lines (HC) bearing anti-Fc gamma R antibodies on their cell surface. Freshly isolated eosinophils and neutrophils constitutively express similar amounts of the low affinity Fc gamma R, Fc gamma RII on their cell surface, but neither cell type expresses the high affinity Fc gamma R, Fc gamma RI. In contrast, eosinophils have only about 5% as much of the low affinity Fc gamma R found on human granulocytes and large granular lymphocytes (Fc gamma RIII) as neutrophils. Untreated, freshly prepared eosinophils or neutrophils did not lyse any of the anti-Fc gamma R bearing HC nor did they lyse CE in the presence of anti-Fc gamma R containing heteroantibodies. Upon treatment with granulocyte monocyte-CSF (GM-CSF), both cell types lysed HC-bearing antibody to Fc gamma RII (HC IV.3A) and CE in the presence of anti-CE x anti-Fc gamma RII heteroantibodies. However, neither cell type lysed HC-bearing antibody to Fc gamma RI or Fc gamma RIII, or CE in the presence of anti-CE x anti-Fc gamma RI HA. Treatment with GM-CSF did not significantly alter the number of Fc gamma R on either cell type. Treatment of neutrophils with IFN-gamma for 18 h induced the expression of Fc gamma RI on these cells and their ability to lyse anti-Fc gamma RI- or Fc gamma RII-bearing HC and CE through Fc gamma RI, Fc gamma RII, and Fc gamma RIII. In contrast, 6-h treatment of eosinophils or neutrophils with IFN-gamma induced neither Fc gamma RI expression on either cell type nor killing of HC or CE through Fc gamma R. In summary, incubation with GM-CSF, induced eosinophils and neutrophils to kill anti-Fc gamma RII-bearing HC and to lyse CE through Fc gamma RII. This augmented killing was not associated with enhanced expression of Fc gamma RII.(ABSTRACT TRUNCATED AT 400 WORDS)
Secretion of novel and homologous neutrophil-activating peptides by LPS-stimulated human endothelial cells.
Authors: Schröder J M , Christophers E
Published: December 2022

Human umbilical vein endothelial cells in culture produce two chemotactic polypeptides when stimulated with LPS. The chemotactic factors could be purified to apparent homogeneity by HPLC techniques and were identified as 7.5-kDa and 15-kDa polypeptides by SDS-PAGE under nonreducing conditions. Both factors are potent chemotaxins for human neutrophils demonstrating half-maximal chemotaxis at 2 ng/ml and g ng/ml, respectively. In addition both peptides elicited release of azurophilic granule constituents when neutrophils were pretreated with cytochalasin B. Cross-desensitization experiments by using human neutrophils revealed cross-reactivities between both chemotaxins, not, however, with C5a or FMLP, indicating that both endothelial cell-derived neutrophil activating peptides (ENAP) are homologous. In addition, the 7.5-kDa factor (beta-ENAP) proved to be the quantitatively dominating and more potent chemotaxin as compared to the 15 kDa factor (alpha-ENAP). beta-ENAP shows biochemical and biologic similarities to monocyte- and lymphocyte-derived neutrophil-activating peptides MONAP and LYNAP, which recently were purified and sequenced.
IFN-mediated differential regulation of the expression of HLA-B7 and HLA-A3 class I genes.
Authors: Hakem R , Le Bouteiller P , Barad M , Trujillo M , Mercier P , Wietzerbin J , Lemonnier F A
Published: December 2022

The regulation by IFN of the expression of HLA-B7 and HLA-A3 class I molecules was studied in Jurkat human T lymphoma cells, HHK EBV-transformed human B lymphocytes, and murine HLA-B7 HLA-A3 co-transfected L fibroblasts. Jurkat cells express constitutively low level of HLA class I molecules and treatment with human IFN resulted in preferential increase of the expression of HLA-B7 molecules, the expression of the HLA-A3 molecules being relatively unchanged. Similar treatment of HHK cells, which express constitutively large amount of HLA class I molecules, resulted in a marginal increase of the expression of both HLA-B7 and HLA-A3 molecules. HLA-B7 HLA-A3 co-transfected L cells express relatively low level of HLA class I molecules, expression of both however was significantly increased after treatment with murine INF-alpha, the augmentation being more accentuated for HLA-B7 molecules. In all cases, variations of cell surface expression were related to parallel modifications of the level of HLA-B7 and HLA-A3 RNA transcripts. Important nucleotide differences exist between the IFN consensus sequences associated with the HLA-B7 and HLA-A3 class I genes. Using oligonucleotides corresponding to these sequences two patterns of retarded bands were observed by the gel mobility shift assay, suggesting that the IFN-mediated differential regulation of the expression of the HLA-B7 and HLA-A3 genes could be due to different nuclear regulatory factors.
HLA-B51 and HLA-Bw52 differ by only two amino acids which are in the helical region of the alpha 1 domain.
Authors: Hayashi H , Ennis P D , Ariga H , Salter R D , Parham P , Kano K , Takiguchi M
Published: December 2022

Genes encoding the serologically cross-reactive HLA-B51 and HLA-Bw52 molecules were isolated and the exons sequenced. HLA-B51 genes obtained from Caucasian and Oriental individuals were identical. HLA-Bw52 differs from HLA-B51 by four nucleotide substitutions in exon 2 encoding the alpha 1 domain. These comprise one isolated silent substitution in codon 23 and a cluster of three coding substitutions in codons 63 and 67. Amino acid substitutions of N----E at position 63 and F----S at position 67 are the only differences between HLA-B51 and HLA-Bw52 and these residues are postulated to form HLA-B51 specific epitopes. HLA-B51 could have been formed from HLA-Bw52 by the combination of a genetic exchange with HLA-B8 and a point mutation. Similarity of HLA-B51 and HLA-Bw52 with HLA-Bw58 suggest they also share a common ancestor.
An H-2Ld hybrid molecule with a Qa-2 alpha-3 domain and phosphatidyl-inositol anchor is not recognized by H-2Ld-specific cytotoxic T lymphocytes.
Authors: Mann D W , Stroynowski I , Hood L , Forman J
Published: December 2022

Ld/Q7d, a hybrid molecule consisting of alpha-1 and alpha-2 domains from H-2Ld and alpha-3 and carboxy-end components from Q7d, was expressed on the surface of CRL-3A rat liver cells. This molecule retained serologic H-2Ld epitopes. The Ag is attached to the cell membrane through a phosphatidyl-inositol linkage, characteristic of Qa-2 molecules. Both bulk cultured and cloned H-2Ld alloreactive CTL as well as H-2Ld restricted vesicular stomatitis virus-specific CTL lyse CRL-3A cells which express H-2Ld but show little or no lytic activity on cells which express the Ld/Q7d hybrid. These cells also fail to act as cold target competitors for alloreactive anti-H-2Ld CTL. However, cells expressing Ld/Q7d are not resistant to CTL mediated lysis because they can be killed in the presence of lectin. These data indicate that recognition of polymorphic class I CTL epitopes in the alpha-1 and alpha-2 domains are influenced by the structure of the carboxy-end of the molecule.
Precursor immigration and thymocyte succession during larval development and metamorphosis in Xenopus.
Authors: Turpen J B , Smith P B
Published: December 2022

The developing thymus in Xenopus was examined at four different levels: 1) precursor immigration of cytogenetically distinct embryonic stem cells; 2) waves of colonization during tadpole life and metamorphosis; 3) inter-thymic exchange of cells between separate lobes; and 4) development of cortical and medullary thymocytes. Based on the flow cytometric analysis of cytogenetically distinct thymocytes, there were at least two periods of stem cell immigration into the thymus, one during early larval life and the second before or during metamorphosis. Within the thymus, cohorts of cells derived from the first wave of immigration expanded at different times. The initial expansion occurred before 35 days of development. Cells involved in the second period of expansion were also derived from the initial immigrants, expanded after 35 days, and resulted in a turnover of thymocytes during the larval period. Precursor cells entering the thymus during metamorphosis expanded and resulted in an additional replacement of thymocytes. Cortical and medullary thymocytes were isolated from animals that received embryonic stem cell grafts. No differences in the presence or absence, or in the percentages, of donor thymocytes in these different fractions were observed. When limiting numbers of stem cells were transplanted, several cases of asymmetrical thymic lobe colonization were observed. These data suggested that an inter-thymic exchange of cells did not occur during larval life.
Non-specifically activated human peripheral blood mononuclear cells are cytotoxic for human keratinocytes in vitro.
Authors: Kalish R S
Published: December 2022

Cultured human keratinocytes were lysed by activated PBMC in a 4-h 51Cr release assay. PBMC were activated by incubation with 50 U/ml of rIL-2 for 4 days. The cytotoxic precursors were found to be NKH1+ and included both CD2+ and CD2- phenotypes. This cytotoxicity was not genetically restricted, as cells killed both allogeneic and autologous keratinocytes without priming. Cytotoxicity was blocked by pre-incubation of effector cells with mAb against LFA-1 alpha-(TS1/22) and beta-chains (TS1/18), but not by antibodies directed against CD4, CD8, or leukocyte common Ag (T200) suggesting that LFA-1 is an important interactive molecule in this cytotoxicity. IFN-gamma is reported to upregulate ICAM-1, the ligand for LFA-1. Pre-treatment of target keratinocytes with IFN-gamma was also found to greatly increase the sensitivity of keratinocytes to lysis. This increased sensitivity to lysis was blocked by anti-LFA-1 and anti-ICAM-1, but not by anti-DR (L243), and thus was not the result of increased DR expression. Such treated targets were lysed at low levels (15 to 18%) by an Ag-specific CD8+ cytotoxic clone as well as a T cell line derived from a skin lesion of allergic contact dermatitis. In contrast, control keratinocytes were only sensitive to IL-2-activated PBMC as described above. The above findings may be relevant to a variety of conditions in which epidermal damage is associated with lymphocytic infiltrate. These conditions include graft-vs-host disease, erythema multiforme, and lupus erythematosus. DR+ keratinocytes, which may be a marker for IFN-gamma are also found in the above conditions. It is suggested that epidermal pathology may be mediated by non-specific cytotoxicity induced in the course of an immune response.
T cell receptor beta-chain selection in human allograft rejection.
Authors: Miceli M C , Finn O J
Published: December 2022

We have analyzed a series of T cell lines established from renal needle biopsies taken from renal allograft recipients with clinical signs of rejection. These T cells show strong cytotoxicity directed against donor HLA and their proliferative capacity in vitro is highly correlated with irreversible graft rejection. A total of 10 of 12 lines examined by Southern blot analysis using a J beta C beta DNA probe show predominant beta-chain rearrangements. In one instance DNA was isolated from cell lines generated from sequential biopsies taken from the same patient at different times of rejection. These lines show the same predominant beta-chain rearrangements. To determine whether these predominant rearrangements are due to expansion of a single clone or different T cell clones rearranging similar beta-chains, the same blots were analyzed with a J gamma probe. Cell line MH3 shows two predominant beta-chain rearrangements and at least seven of eight possible rearranged gamma-chain bands, implying that multiple clones share similar beta-chains. In contrast, the cell line King shows a single beta-chain and a single gamma-chain rearrangement. Many of the other cell lines fall between these two extremes, indicating that both beta-chain selection and clonal dominance are operating during graft rejection, resulting in the appearance of predominant beta-chain rearrangements.
Steroid sensitivity of thymocyte subpopulations during intrathymic differentiation. Effects of 17 beta-estradiol and dexamethasone on subsets expressing T cell antigen receptor or IL-2 receptor.
Authors: Screpanti I , Morrone S , Meco D , Santoni A , Gulino A , Paolini R , Crisanti A , Mathieson B J , Frati L
Published: December 2022

We have studied the effects of the steroid hormones, 17 beta-estradiol and dexamethasone, on the relative proportion of thymocyte expression of CD4 (L3T4), CD8 (Ly-2), TCR and IL-2R, used to identify different stages of thymocyte differentiation. After short-term in vivo steroid treatment, a significant decrease in the number and proportion of the CD4+/CD8+, double positive subpopulation was observed in parallel with a proportional increase in the percentage of the CD4+/CD8- single positive, of the CD4-/CD8-, double negative and, to a lesser extent, of the CD8+/CD4- subsets. Either steroid treatment increased the proportion of cells expressing either the epsilon-chain of the CD3 complex and/or the beta-chain of the TCR (beta-TCR) (TCR+/CD3+) and the 55 kDa protein of the IL-2R (IL-2R+), related to the increase of CD4+ SP thymocytes and of DN cells, respectively. Furthermore, the increased proportion of CD3+ cells could also be partially related to the increase of both the CD4+ and DN subsets. A decrease of the effect on either DN/IL-2R+ cells or CD4+ SP cells was selectively observed after long-term treatments with 17 beta-estradiol or DEX, respectively. It is concluded that after short-term administration, different steroid hormones mediate a similar selective depletion of DP, TCR-/CD3-, IL2R- cells presumably in an intermediate stage of differentiation. However, either steroid effects evolve differently in long-term treatment schedules, resulting in different effects on early (DN/IL2R+) and late (SP/IL2R-) steps of thymocyte differentiation.
A role for class I MHC antigens in responses of activated B lymphocytes.
Authors: Southern S O , Dutton R W
Published: December 2022

Expression of the H-2 D protein was increased over 10-fold in B cells activated by IL-4 IL-5, IFN-gamma or LPS. Differences in the regulation of H-2 D expression and in growth rates were detected during lymphokine-triggered responses of subpopulations of splenic B cells. The enhancement of H-2 D expression on activated B cells was regulated at the transcriptional level and required synthesis of an intermediate cellular protein(s). B cell growth and differentiation were inhibited by antibodies to the H-2 D Ag suggesting a signaling function for the class I molecules on activated B lymphocytes. In T cells, a similar elevation of H-2 D expression occurred after stimulation with IL-5 or Con A.
Induction of allergen-specific IgE and IgG responses by anti-idiotypic antibodies.
Authors: Nagpal S , Shanthi K N , Kori R , Schroder H , Metcalfe D D , Subba Rao P V
Published: December 2022

In order to explore idiotypic, anti-idiotypic, and anti-anti-idiotypic responses to allergens, BALB/c mice were immunized with affinity-purified human idiotypic antibodies directed against a highly purified shrimp allergen. This resulted in the production of anti-idiotypic antibodies which were quantitated by using rabbit idiotypic antibodies raised against the same purified allergen. The mouse anti-idiotypic antibodies recognized shrimp-specific human idiotypic antibodies of the IgE isotype from 18 of 20 individuals, and IgG antibodies from 14 of 20 shrimp-sensitive patients. Immunization of BALB/c mice with affinity-purified, allergen-specific anti-idiotypic antibodies induced anti-allergen IgE and IgG responses in the absence of the allergen. This paper thus presents evidence that anti-idiotypic antibodies raised against allergen-specific idiotypic antibodies may substitute for the original allergen in the induction of allergen-specific idiotypic antibodies. The demonstration of shared idiotopes on IgG and IgE antibodies in the sera of shrimp-sensitive patients supports the use of allergen-specific anti-idiotypic antibodies as surrogate allergens.
Binding and internalization of gold-labeled IFN-gamma by human Raji cells.
Authors: Filgueira L , Groscurth P , Aguet M
Published: December 2022

Binding and internalization of gold-labeled IFN-gamma (IFN-gamma/Au) by human Raji cells was examined by scanning and transmission electron microscopy. For SEM, visualization of gold particles was enhanced by the silver enhancement technique and by backscattered electron imaging. Binding studies revealed distinct labeling of microvilli-bearing cells after incubation with at least 10 U/ml IFN-gamma/Au, whereas cells with a smooth surface showed substantially lower labeling. After application of higher IFN-gamma (greater than 200 U/ml) concentrations, labeling intensity remained constant, which is consistent with the concentration of radiolabeled IFN-gamma required for saturating receptors on Raji cells. The specificity of IFN-gamma/Au binding was demonstrated by complete displacement with unlabeled IFN-gamma and by partial inhibition of labeling with a monoclonal anti-IFN-gamma R antibody. Thus, colloidal gold represents a valuable tag for visualizing the interaction of IFN-gamma with its receptor. Internalization of IFN-gamma/Au was initiated by accumulation of gold particles in coated pits which occurred within 10 min after warming of Raji cells. Additional incubation at 37 degrees C (up to 2 h) led to the appearance of gold particles in endocytic vesicles and lysosomes. Thus, our studies indicate that IFN-gamma/Au enters the Raji cells via the typical endocytotic pathway.
Comparison of transforming growth factor-beta and a macrophage- deactivating polypeptide from tumor cells. Differences in antigenicity and mechanism of action.
Authors: Tsunawaki S , Sporn M , Nathan C
Published: December 2022

A factor in medium conditioned by mouse tumor cells was shown previously to suppress the capacity of mouse peritoneal macrophages to undergo a respiratory burst and to kill protozoal pathogens (macrophage deactivation factor, MDF). Recently, pure transforming growth factor-beta (TGF-beta) proved to be a potent macrophage deactivator as well. Two lines of evidence suggest that MDF is not identical with TGF-beta. First, rabbit anti-TGF-beta IgG neutralized the respiratory burst-suppressing activity of TGF-beta without affecting the bioactivity of MDF, even when the latter was treated with acid to activate potentially latent TGF-beta. Second, in contrast to MDF, which decreases the affinity of the NADPH oxidase for NADPH, permeabilized macrophages that had been deactivated with TGF-beta displayed the same Km and Vmax of the oxidase as activated macrophages. As with MDF, TGF-beta had no effect on two other potential control points over the secretion of respiratory burst products, namely, hydrogen peroxide catabolism or glucose uptake. Finally, neither MDF nor TGF-beta affected the extent or affinity of binding of phorbol diesters to macrophages, the activity or cofactor requirements for protein kinase C, or the ability of protein kinase C to translocate quantitatively from cytosol to membrane fractions in response to phorbol diesters. Thus, 1) MDF is not identical with TGF-beta, and 2) in contrast to the activation or deactivation of macrophages by numerous other agents, TGF-beta regulates macrophage respiratory burst capacity at a level other than the apparent affinity of the oxidase for its substrate.
Epitope clusters of Qa-2 antigens defined by a panel of new monoclonal antibodies.
Authors: Sharrow S O , Arn J S , Stroynowski I , Hood L , Sachs D H
Published: December 2022

A recently derived intra-MHC recombinant mouse strain, the C3H.KBR was found to produce a surprisingly high titer of anti-Qa antibodies when immunized with C3H.SW lymphocytes. By using this immunization combination, a panel of 10 mAb with specificity for determinants encoded by the Q region was produced. These reagents were analyzed for strain distribution by microcytotoxicity, immunofluorescence, and flow cytometry assays. Competitive inhibition analyses, performed by using fluorescein-labeled antibodies and normal spleen cells, defined at least three epitope clusters, or groups of spatially related determinants, detected by this panel. One epitope cluster was unique to this new series of antibodies in that it was not detected with seven previously described anti-Qa mAb. These antibodies also have been analyzed for reactivity with products of isolated Q-region genes by using transfected cell lines. The data indicate that the Q6d, Q7d, and Q10d genes encode determinants reactive with one or more mAb and that two of the three epitope clusters defined with normal cells map to the N and/or C2 domains of these molecules. The third epitope cluster is presumed to map to the C2 domain. These reagents should be useful in determining the number of Q-region genes expressed and in analyses of Q gene expression in subpopulations of normal cells, in transfected cell lines, and during differentiation and ontogeny.
Recombinant IL-3 induces histamine release from human basophils.
Authors: MacDonald S M , Schleimer R P , Kagey-Sobotka A , Gillis S , Lichtenstein L M
Published: December 2022

Human rIL-3 induces histamine release from some human basophils, with cells from atopics responding to a greater extent than non-atopic donors. The dose response curves were highly variable. IL-3 was active on purified basophils and the release process was slower and required more calcium than anti-IgE. Removal of surface IgE from basophils rendered them unresponsive to IL-3. The response could be restored by passive sensitization of basophils with IgE+, IgE known to bind histamine-releasing factors, and not IgE-, IgE unreactive with histamine-releasing factors. Thus, IL-3 uncovers IgE heterogeneity. IL-3 does not, however, directly interact with IgE+. Rather, passive sensitization with IgE+ or stimulation of basophils with low concentrations of several secretagogues renders the cells sensitive to IL-3. IL-3 may well play a pro-inflammatory role by potentiating the effects of IgE+ or various secretagogues.