bluuebunny/crossref_metadata_2025 huggingface dataset:
Failure to synthesize the CD3-gamma chain. Consequences for T cell antigen receptor assembly, processing, and expression.
Authors: Geisler C
Published: December 2022

The TCR consists of the Ti alpha beta heterodimer and the associated CD3 chains, CD3 gamma delta epsilon zeta 2 or zeta eta. The structural relationships between the subunits of the Ti/CD3 complex are still not fully understood. To explore the roles of the individual CD3 chains for the assembly, intracellular processing, and expression of the TCR, mutants of the T cell line Jurkat were isolated. One variant, JGN, was found to produce all the Ti/CD3 components with the exception of CD3-gamma. The results indicate that: 1) the tetrameric form (Ti alpha beta-CD3 delta epsilon) of the Ti/CD3 complex is produced in the endoplasmic reticulum in the absence of CD3-gamma; 2) CD3-zeta does not associate with the Ti alpha beta-CD3 delta epsilon complex; 3) the Ti alpha beta-CD3 delta epsilon complex is not exported from the endoplasmic reticulum to the Golgi apparatus; and 4) CD3-gamma is required for cell surface expression of the Ti/CD3 complex. Transfection of the wild-type CD3-gamma gene into JGN reconstituted expression of functional Ti/CD3 complexes, and analysis of T cell lines producing different amounts of CD3-gamma indicated that CD3-gamma and CD3-delta competed for the binding to CD3-epsilon.
Regulation of C gamma 3 expression. Role of switch in the allotype-associated variation of human serum IgG3 levels.
Authors: Hassan M S , Islam K B , Hammarström L , Smith C I
Published: December 2022

Serum IgG3 levels are associated with G3m allotypes in humans. However, the molecular basis of this association has not been understood. In this study we have analyzed the biologic half-life, the secretion, the cell surface expression, the cytoplasmic content, and the mRNA expression of different allotypes. The biologic properties of the allotypes did not differ. However, the frequency of cells with membrane IgG3, cytoplasmic IgG3, and C gamma 3 mRNA was decreased in donors with a low serum IgG3, whereas the level of C gamma 3 mRNA expression in individual cells did not differ among cells of the different allotypes. As these findings indicated a pretranscriptional control of C gamma 3 expression, genomic DNA from donors with different allotypes were also studied. Despite the absence of gross, allotype-related differences in the I gamma 3 regions, we favor an upstream cis-element influencing C gamma 3 switching as the most likely explanation for variations in IgG3 serum levels.
Regulated expression of the mouse gamma 2b Ig H chain gene is influenced by polyA site order and strength.
Authors: Lassman C R , Milcarek C
Published: December 2022

Constructs with alterations in the normal order and spacing of polyadenylation sites of the mouse Ig-gamma 2b heavy chain gene were transfected into J558L cell tumor lines (myelomas) and A20 B cell tumor lines (lymphomas) representative of plasma and memory cells, respectively. When the membrane-specific (mb) polyA site was moved from its 3'-location to a position upstream (5') of the secretory (sec) polyA site, the mb site was used preferentially, even though the sec site was still efficiently transcribed. The relative strength of the mb polyA site seems to preclude efficient use of downstream elements. When two sec polyA sites are put in competition with each other in the same transcript, use of the first site predominates in both cell types, implying that the relative strength and the distance between the sites are important for normal regulation. When the sec polyA site is put upstream of the mb polyA site, in the absence of a competing splicing event, the sec polyA site is used preferentially in the myeloma cell but not the lymphoma cell, implying that its use is a regulated event. We therefore conclude that the B cell-regulated strength of the sec polyA site, as well as its 5'-location, relative to the unregulated, but very strong mb polyA site, are important parameters in the regulated expression of mb and sec mRNA in this system.
Single base pair substitutions within the HLA-DRA gene promoter separate the functions of the X1 and X2 boxes.
Authors: Sloan J H , Hasegawa S L , Boss J M
Published: December 2022

The class II MHC genes are expressed on the surfaces of B cells, activated T cells, and macrophages and may be induced in other cell types by IFN-gamma. The control of class II gene expression has been shown to be mediated by a series of conserved cis-acting sequences (W, X1, X2, and Y boxes) located immediately 5' to the genes. Although these sequences are conserved, the bp that are important for transcriptional regulation have yet to be identified. To address this issue with regard to the MHC gene HLA-DRA, a series of single bp substitutions spanning the conserved upstream sequences was created and analyzed for their effects on transcription in both B cells and IFN-gamma-treated fibroblasts. In addition, the effects of X1 and X2 box mutations on DNA/protein interactions were examined and compared to the transcriptional data. The results of these studies show that each of the conserved elements participate in maximal expression in B cells and that W, X1, and X2 boxes are important for IFN-gamma induction and expression in fibroblasts. Interestingly, some of the bp changes that altered B cell expression did not alter expression and IFN-gamma induction in fibroblasts, suggesting that different or altered factors control the expression of these genes in the different cell types. Mutant templates designed to eliminate the binding of X1- and X2-specific DNA binding proteins in vivo suggest that these elements and their factors may interact to promote transcription.
Enhancement of in vitro tumor-infiltrating lymphocyte cytotoxicity by heteroconjugated antibodies.
Authors: Reid I , Lundy J , Donohue J H
Published: December 2022

Tumor-infiltrating lymphocytes (TIL) were obtained from a mouse melanoma cell line (CL 62) transfected with the gene for the human melanoma Ag p97. TIL were cultured with anti-CD3 antibody and IL-2 for up to 38 days. Flow cytometry identified these TIL as Thy-1.2 + ve/CD4-ve/CD8 + ve cells. A heteroconjugated antibody 500A2 x 96.5, specific for both the CD3 Ag on TIL and the p97 Ag on CL 62 melanoma cells, was prepared using N-succinimidyl-3-(2-pyridyldithio)-propionate as a linking agent. TIL alone demonstrated low levels of cytotoxicity against autologous CL 62 tumor and also against the parental K1735 tumor and an allogeneic murine melanoma (B16). The addition of 500A2 x 96.5 heteroconjugated antibody enhanced TIL-mediated lysis of CL 62 tumor, but not of the K1735 or B16 tumors. This enhanced cytotoxicity was elicited at E:T ratios as low as 0.4:1, and in TIL cultured for 7 to 38 days. These results suggest that hetero-conjugated antibody may enhance the anti-tumor effect of TIL in vivo.
In vitro processing of insulin for recognition by murine T cells results in the generation of A chains with free CysSH.
Authors: Hampl J , Gradehandt G , Kalbacher H , Rüde E
Published: December 2022

Studies on the processing of insulin as an Ag for the presentation to MHC class II-restricted T cells revealed that the amino acid residues 1-14 of the insulin A chain are recognized by insulin-specific T cells. An A1-14 peptide containing three cys-residues that were protected by S-sulfonate groups still needed processing by APC for efficient presentation similar to native insulin. We suspected that reductive deblocking or opening of disulfide bonds that generates CysSH-residues may be an essential processing step for these Ag. Due to the instability of SH-groups it was not possible to test A chain peptides with free SH-groups in the usual way for processing-independent presentation by fixed APC. However, under acidic conditions (pH 5) during APC pulsing with the Ag we could demonstrate that the freshly reduced A1-14 fragment as well as reduced insulin are able to bind to Ia Ag and to stimulate appropriate T cells without further processing. Various substitutions of cys-residues by Ser within this peptide revealed that only CysA7 is critical for Ia binding and/or T cell recognition. In intact insulin, this residue links the A chain containing the T cell epitope to the B chain. Therefore, we propose that insulin processing is not dependent on proteolysis or on the generation of a conformational determinant but on the separation of A and B chains resulting in A chains whose cys-residues are converted into CysSH.
Tolerance induction in resting memory B cells specific for a protein antigen.
Authors: Johnson J G , Jemmerson R
Published: December 2022

Resting memory B lymphocytes specific for the model protein Ag cytochrome c have been shown to be susceptible to tolerance induction in in vitro splenic fragment cultures. This induction of nonresponsiveness is dependent upon the strength of the interaction between surface Ig and specific Ag, where concentration, valency, affinity, and time of exposure all appear to be important factors, as is the case for tolerance induction in immature or primary B cells. The induction of nonresponsivenes in greater than 80% of Ag-specific memory B cells was achieved by incubation with 1 microM cytochrome polymer for 24 h in the absence of T cell help. Not only were memory B cells unresponsive to specific Ag, they were also unable to become activated through nonspecific uptake and presentation of an Ag to which T cells have been primed, demonstrating that the induction of nonresponsiveness involves more than a modulation or blockade of surface Ig receptors. Although soluble factors collected from activated T cells failed to prevent memory B cells from becoming nonresponsive after surface Ig cross-linking, the direct activation of T cells within splenic fragment cultures did partially inhibit tolerance induction in splenic fragment memory B cells. In addition, the induction of tolerance was partially blocked by protein tyrosine kinase inhibitors, suggesting a physiologic change within the B cells associated with the state of nonresponsiveness and resulting from tyrosine-specific phosphorylation.
Humanized OKT3 antibodies: successful transfer of immune modulating properties and idiotype expression.
Authors: Woodle E S , Thistlethwaite J R , Jolliffe L K , Zivin R A , Collins A , Adair J R , Bodmer M , Athwal D , Alegre M L , Bluestone J A
Published: December 2022

Antibodies that possess the Ag-binding regions of OKT3 within the context of a human framework (Hu-OKT3 Ab) offer distinct advantages for optimizing anti-CD3 mAb therapy. First, manipulation of Ab genes to produce humanized Ab that retain Ag-binding activity may circumvent antigenicity problems. Second, Ab gene engineering provides a means for modifying functional properties, including T cell activation and immune suppression. The purpose of this study was to determine the functional properties of Hu-OKT3 Ab and to compare the functional properties and idiotypes of Hu-OKT3 Ab to those of murine OKT3. Three Hu-OKT3 IgG4 Ab, a chimeric OKT3 antibody (cOKT3-1) (grafted sequences comprising all OKT3 VH and VL regions) and two complementarity determining region (CDR)-grafted antibodies, gOKT3-5 and gOKT3-6 (grafted sequences comprising only OKT3 VH and VL CDR and some framework amino acids, were analyzed. Initial studies demonstrated that the cOKT3 and gOKT3-5 Ab bound selectively to T cells and competitively inhibited OKT3-FITC binding with avidities similar to that of murine OKT3. Binding avidity of the gOKT3-6 Ab was markedly less than that of the other two Hu-OKT3 Ab. Serologic analysis suggested that cOKT3 and gOKT3-5 Ab possess idiotypes (combining sites) similar to murine OKT3. T cell activation potency of all three Hu-OKT3 Ab was assessed by proliferation, induction of activation marker expression (IL-2R and Leu 23), and lymphokine production (TNF-alpha and IFN-gamma). The cOKT3 and gOKT3-5 Ab demonstrated T cell activation potencies similar to murine OKT3 as assessed by each parameter. CD3 coating and modulation by these two Ab was effective but somewhat less potent than that observed with OKT3. Finally, cOKT3 and gOKT3-5 Ab both inhibited CTL activity comparably to murine OKT3. In conclusion, these studies indicate that gOKT3-5 and cOKT3 Ab possess immune modulating properties similar to murine OKT3 and thus offer attractive alternatives to murine OKT3 for in vivo therapy.
A nuclear DNA-binding protein expressed during early stages of B cell differentiation interacts with diverse segments within and 3' of the Ig H chain gene cluster.
Authors: Liao F , Giannini S L , Birshtein B K
Published: December 2022

We have used electrophoretic mobility shift assays (EMSA) to detect B cell lineage-specific nuclear proteins that bind to diverse segments within and 3' of the Ig H chain gene cluster. DNA binding sites include sequences 5' of each of the following C region genes: mu, gamma 1, gamma 2a, epsilon, and alpha. For the most part, these binding sites lie 5' of CH-associated tandem repeats. Binding sites for the same B cell lineage-specific proteins have also been defined in the region 3' of C alpha, close to a recently described B cell-specific enhancer element. Cross-competition of EMSA indicates that the B cell lineage-specific nucleoprotein is indistinguishable from those described previously by others: S alpha-BP and BSAP. Because of the diverse sequences recognized by this protein, we term it NF-HB, B-lineage-specific nuclear factor that binds to Ig H gene segments. EMSA using segments 5' of S gamma 2a (5'S gamma 2a-176) and 3' of C alpha (3' alpha-88) shows multiple binding complexes, two of which are B cell lineage specific. The B cell-specific complex with fastest mobility contains only NF-HB, and the one with slowest mobility contains NF-HB together with a ubiquitous DNA-binding protein(s). The ubiquitous binding protein is different for 5' S gamma 2a-176 and for 3' alpha-88, representing the formation of protein-NF-HB complexes specific for these particular Ig DNA regions. Spleen cells show a single band upon EMSA with either 5'S gamma 2a-176 or 3' alpha-88. Upon LPS stimulation, additional binding complexes of slower mobility were formed resulting in a pattern comparable to those detected in pro-B, pre-B, and B cell lines. We hypothesize that NF-HB may promote physical interactions between the 3' alpha-enhancer and segments of the Ig H gene cluster.
Mapping of human H chain V region genes (VH4) using deletional analysis and pulsed field gel electrophoresis.
Authors: Van Dijk K W , Milner L A , Milner E C
Published: December 2022

We have used molecular genetic mapping techniques to establish the order and approximate chromosomal locations of VH4 elements on both chromosomes 14 from a single patient. A total of 10 BglII restriction fragments carrying VH4 elements was characterized. The genomic order of VH4-carrying restriction fragments was determined by analysis of the pattern of loss of hybridizing bands from the genomes of a panel of monoclonal lymphoblastoid cell lines which had well characterized rearrangements of the Ig locus on each chromosome. Some individual elements were identified using sequence-specific oligonucleotide probes. Physical dimensions were estimated by the assignment of these ordered elements to large (50-350-kb) restriction fragments using two-dimensional pulse field gel electrophoresis. Six such fragments spanning approximately 890 kb were physically linked and ordered. The chromosomes differed with respect to the complement of VH4 elements, although no evidence was found of major differences in organization. The establishment of a panel of well characterized deletion lines facilitates the rapid mapping of defined restriction fragments carrying VH elements.
Regulation of rat pulmonary artery endothelial cell transforming growth factor-beta production by IL-1 beta and tumor necrosis factor-alpha.
Authors: Phan S H , Gharaee-Kermani M , McGarry B , Kunkel S L , Wolber F W
Published: December 2022

Recent studies suggest that transforming growth factor-beta (TGF-beta) production is up-regulated at sites of tissue injury, inflammation and repair, or fibrosis. Endothelial cells represent a potentially important in vivo source of TGF-beta; however, the identity of endogenous modulators of TGF-beta production by these cells remains unclear. To address this issue, the effects of the cytokines, IL-1 beta, and TNF-alpha on TGF-beta production by rat pulmonary artery endothelial cells were examined. Conditioned media from cells treated with 0 to 20 ng/ml IL-1 beta and/or TNF-alpha were assayed for TGF-beta activity using a mink lung epithelial cell line. The results show that rat pulmonary artery endothelial cells secreted undetectable amounts of active TGF-beta in the absence of cytokines. However, upon acidification of the conditioned media before assay, a time-dependent increase in TGF-beta activity was noted in media from both untreated and cytokine-treated cells. However, both IL-1 beta and TNF-alpha treatment caused the secretion of significantly greater amounts of TGF-beta activity than control cells, in a dose-dependent manner, with maximal response obtained at cytokine doses of greater than 10 ng/ml. At equivalent doses of cytokine tested, the magnitude of the response was significantly greater with IL-1 beta. These responses were paralleled by increases in steady state mRNA levels for TGF-beta 1. Addition of both cytokines resulted in a synergistic response. Synergism with IL-1 beta was also noted with the fibrogenic agent bleomycin. Kinetic studies indicated that a minimum of 4 h of treatment with either IL-1 beta or TNF-alpha was required for detection of significant increases in either secreted TGF-beta activity or steady state TGF-beta 1 mRNA levels. Thus, endothelial cells could play a role in various TGF-beta-dependent processes in vivo, in situations wherein IL-1 beta and/or TNF-alpha may be present at comparable concentrations.
Species specificity in the interaction of CD8 with the alpha 3 domain of MHC class I molecules.
Authors: Newberg M H , Ridge J P , Vining D R , Salter R D , Engelhard V H
Published: December 2022

The alpha 1 and alpha 2 domains of the class I MHC molecule constitute the putative binding site for processed peptides and the TCR, although the alpha 3 domain has been implicated as a binding site for the CD8 molecule. Species specificity in the binding of CD8 to the alpha 3 domain has been suggested as an explanation for the low xenogeneic T cell response to class I molecules, but results on this point have been conflicting and controversial. We have addressed this issue using CTL lines from HLA-A2.1 transgenic mice that specifically recognize and lyse A2.1-expressing cells infected with influenza A/PR/8 or pulsed with influenza matrix peptide M1(57-68). Species specificity was examined using transfectants that expressed hybrid molecules containing the alpha 1 and alpha 2 domains from HLA-A2.1 and the alpha 3 domain from a murine class I molecule. Lower levels of M1(57-68) peptide were required to sensitize L cell transfectants expressing a chimera that contained an H-2Dd alpha 3 domain than targets expressing the intact A2.1 molecule. However, at high doses of peptide, lysis of these two targets was similar. However, no reproducible difference in sensitization was observed using EL4 or Jurkat transfectants expressing A2.1 or A2.1 chimeric molecules that contained an H-2Kb alpha 3 domain. In all cases, however, lysis of peptide-pulsed A2.1 expressing targets was more sensitive to inhibition with anti-CD8 mAb than lysis of cells expressing these chimeric molecules. Thus, under suboptimal conditions such as low Ag density or in the presence of anti-CD8 mAb, these CTL preferentially recognize class I molecules with a murine alpha 3 domain. This suggests that there is some species specificity in the interaction of CD8 with the alpha 3 domain of the class I molecule. However, CTL recognition was inhibited by point mutations in the alpha 3 domain of HLA-A2.1 that have been shown to inhibit binding of human CD8 and recognition by human CTL, suggesting that murine CD8 interacts to some degree with human alpha 3 domains, and that similar alpha 3 domain residues may be important for murine and human CD8 binding. The relevance of these results to an understanding of low xenogeneic responses is discussed.
Mechanisms of lipopolysaccharide-induced neutrophil retention. Relative contributions of adhesive and cellular mechanical properties.
Authors: Erzurum S C , Downey G P , Doherty D E , Schwab B , Elson E L , Worthen G S
Published: December 2022

Intravascular LPS rapidly induces neutrophil sequestration in pulmonary capillaries by mechanisms that, although currently unknown, must take into account the size difference between the neutrophil and capillary diameter. To determine whether LPS alters neutrophil stiffness, and hence the ability of neutrophils to traverse capillaries, neutrophil passage through pulmonary capillaries was modeled by passage through filters with 6.5-microns pores. LPS increased retention in the pores in a concentration-dependent fashion that required the presence of heat-inactivated platelet-poor plasma, and was evident as early as 10 min after stimulation. The effect of LPS on the structural properties of the neutrophil was then studied. LPS induced f-actin reorganization in neutrophils in the presence of plasma. Disruption of actin organization and assembly with cytochalasin D completely inhibited early LPS-induced retention and attenuated retention at later timepoints, indicating that LPS-stimulated retention depends on filament organization. LPS-induced actin assembly and retention were abrogated by an antibody directed against CD14, a putative LPS receptor. CD18-dependent adherence of neutrophils contributed significantly to retention only at later timepoints with no significant contribution to retention at 20 min as determined by inhibition of adherence with the mAb 60.3. Morphometric assessment of neutrophil accumulation in the lungs of rabbits given 1 microgram LPS showed a marked increase in apparent neutrophil number, which was unaltered by antibodies to CD18, suggesting that mechanisms other than adhesion may account for accumulation in vivo. Direct measurements showed that neutrophil stiffness increased with exposure to LPS in a fashion similar to LPS-induced retention and actin organization. Pretreatment of neutrophils with cytochalasin D attenuated the increased stiffness. These data suggest that reorganization of filamentous-actin induced by LPS leads to cell stiffening and retention in capillary-sized pores. Although the organization of f-actin continues to be important in retention at later time points, adherence of cells also contributes significantly to cell retention. The changes in mechanical properties of the neutrophil may be important in the sequestration of neutrophils in pulmonary capillaries noted in endotoxemia.
Expression of the murine homologue of the cell cycle control protein p34cdc2 in T lymphocytes.
Authors: Kim Y H , Proust J J , Buchholz M J , Chrest F J , Nordin A A
Published: December 2022

The mammalian homologue of the cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a p34cdc2 cyclin-dependent kinase that regulates the cell cycle of a wide variety of cell types. Resting murine T lymphocytes contained no detectable p34cdc2 protein, histone kinase activity, or specific mRNA for the cdc2 gene. Activation of the T cells by immobilized anti-CD3 resulted in the expression of specific mRNA late in the G1 phase of the cell cycle, and p34cdc2 protein was detectable at or near G1/S. At this point in the cell cycle, the protein was phosphorylated at tyrosine and displayed no H1 histone kinase activity. As the cells progressed through the cycle, the amount of specific mRNA and p34cdc2 increased, and H1 histone kinase activity was detectable when the cells were blocked at G2/M by nocodazole. The activation of T cells by phorbol dibutyrate induced the expression of IL-2R but failed to induce the synthesis of IL-2 or the expression of cdc2-specific mRNA. Under these conditions, the activated cells failed to enter the S phase of the cell cycle. Because the presence of IL-2 added exogenously during activation by phorbol dibutyrate resulted in the expression of cdc2-specific mRNA and progression through the cell cycle, either IL-2 or the interaction with IL-2R may be involved in the expression of cdc2 and regulation of the G1/S transition.
Modulatory effects of Epstein-Barr, herpes simplex, and human herpes-6 viral infections and coinfections on cytokine synthesis. A comparative study.
Authors: Gosselin J , Flamand L , D'Addario M , Hiscott J , Stefanescu I , Ablashi D V , Gallo R C , Menezes J
Published: December 2022

Herpesviruses such as EBV, HSV, and human herpes virus-6 (HHV-6) have a marked tropism for cells of the immune system and therefore infection by these viruses may result in alterations of immune functions, leading at times to a state of immunosuppression. We report the results of a comparative study in which we found that EBV, HSV-1, and HHV-6 act differentially on the immune system with regard to their effect on the synthesis of IL-1 beta, IL-6, and TNF-alpha, i.e., three immunoregulatory cytokines mainly secreted by activated monocytes/macrophages. Using the polymerase chain reaction technique, analyses of the mRNA levels for each of the three monokines after viral infection indicated that the effect exerted by each of these herpesviruses on cytokine synthesis by human PBMC was detectable at the transcriptional level. Different amounts of IL-1 beta protein were detected in infected PBMC cultures, HHV-6 being the strongest IL-1 beta up-regulatory among these three herpesviruses. Spontaneous releases of IL-6 and TNF-alpha were found reduced after infection by HHV-6 and EBV, respectively. In comparison to EBV and HHV-6, HSV-1 proved to be a weak monokine enhancer. Results of coinfection studies indicated that virus-induced suppressive effects on cytokine synthesis are dominant. In fact, EBV inhibited TNF-alpha synthesis even in the presence of HHV-6, a strong up-regulator of TNF-alpha synthesis. Similarly, EBV was unable to stimulate IL-6 production in the presence of HHV-6. Viral structural component(s) appeared to be responsible for the up-regulation of IL-6 by both EBV and HSV-1, and of TNF-alpha by HSV-1. Taken together, our observations illustrate that herpesviruses can selectively regulate cytokine synthesis thereby disturbing immune homeostasis; this effect may favor pathogenic events, including the reactivation and/or spread of other infectious agents within the host.
IL-4 inhibits macrophage-mediated killing of Plasmodium falciparum in vitro. A possible parasite-immune evasion mechanism.
Authors: Kumaratilake L M , Ferrante A
Published: December 2022

Although a number of mechanisms have been put forward for immunity to malaria, their importance remains to be clarified. One of the important findings is that nonactivated monocytes and macrophages showed marked antiplasmodial activity in vitro. Recently we postulated that parasites may induce host factors that may depress the natural antiplasmodial activity of monocytes. In this investigation we identify IL-4 as a lymphokine that could function in this capacity. Human monocytes and macrophages in the absence of antiplasmodial antibody showed substantial killing of the asexual erythrocytic forms of Plasmodium falciparum as determined by a radiometric assay. Suppression of this killing was seen if the mononuclear phagocytes were pretreated with human rIL-4 at concentrations of 10 to 250 U with optimum activity between 100 and 250 U/2 x 10(5) cells. Cells from some individuals were rendered completely inactive by the IL-4 treatment. In contrast, IL-4 did not affect the neutrophil-mediated anti-P. falciparum activity. Our work identifies a potentially important parasite immune evasion mechanism involving IL-4 suppression of macrophage antiparasite activity.
Predominance of VH-D-JH junctions occurring at sites of short sequence homology results in limited junctional diversity in neonatal antibodies.
Authors: Feeney A J
Published: December 2022

Sequence diversity at the junctions of Ig genes differs between newborn and adult mice in two respects: 1) fetal/newborn Ig lack N regions; and 2) these N- junctional sequences very often contain 1 to 6 nucleotides that could have been encoded by either of the two joined gene segments. We address the hypothesis that such short homologies preferentially direct recombination to that site, and we analyze the effect of such homology-directed recombination upon the neonatal Ig repertoire. We examined 546 CDR3 sequences that were generated from polymerase chain reaction-amplified DNA from fetal and newborn liver using primers from three different VH families: S107, 7183, and J558. All junctional sequences using 14 frequently occurring IgH V-D and D-J gene combinations were analyzed. In 12 of the 14 combinations analyzed, there were 1 to 3 short sequence homologies, and the junctional sequences that would be created by those homologies were observed with high frequency. The D-J junctions often had two to three predominant junctional sequences, whereas the V-D junctions had one dominant junctional sequence. The only exceptions were the VHJ558-D junctions, where homology-directed recombination using the sequence homology between VHJ558 genes and most D genes would result in an out-of-frame join, and most of our sequences were productive. This latter result further suggests that homology-directed recombination may play a role in the nonrandom VH gene usage observed in fetal and newborn mice. Thus, most neonatal IgH junctions show limited diversity, not only due to the lack of N regions, but also because of nonrandom junctional sequences. Inasmuch as the few adult N- junctions also show a high frequency of homology-directed junctional sequences, V-D-J recombination throughout life may involve pairing via short homologies, with addition of N regions obscuring its role in the formation of adult IgH junctions.
CD28-induced T cell activation. Evidence for a protein-tyrosine kinase signal transduction pathway.
Authors: Lu Y , Granelli-Piperno A , Bjorndahl J M , Phillips C A , Trevillyan J M
Published: December 2022

CD28 is a 44-kDa homodimeric receptor expressed on the majority of T cells. Engagement of the CD28 receptor by soluble anti-CD28 mAb in conjunction with PMA causes the induction of lymphokine/cytokine production and proliferation in resting T cells via signal transduction pathways independent of the TCR. The precise nature of the biochemical events that occur after perturbation of the CD28 receptor remain unclear. We report evidence for the coupling of CD28 to a protein-tyrosine kinase pathway. Multivalent cross-linking of the CD28 receptor or stimulation by soluble CD28 mAb plus PMA, but not PMA or soluble CD28 mAb alone, reproducibly caused the rapid (within 2 min) tyrosine phosphorylation of a 100-kDa cellular substrate. In some experiments, additional cellular substrates of 110, 85, 74, 68, 56, 43, and 29 kDa were also observed. The tyrosine phosphorylation of these substrates was completely inhibited by 12 h pretreatment of T cells with herbimycin A, a selective inhibitor of src-family protein-tyrosine kinases. Pretreatment of T cells with herbimycin was without effect on CD28 surface expression but did inhibit CD28 mAb plus PMA-induced IL-2 mRNA levels, IL-2R(CD25) up-regulation, and cell proliferation. The inhibition of IL-2 mRNA levels was likely at the level of transcription, because herbimycin inhibited NF-AT, AP-1, and CD28RC but not NF-kappa B or OCT-1 binding activities to their respective IL-2 enhancer region sequences. Herbimycin did not inhibit PMA-dependent events including CD69 surface expression, NF-kappa B nuclear binding activity or the level of CD25 induced by PMA alone, supporting the notion that herbimycin is acting to inhibit a CD28 initiated or regulated protein-tyrosine kinase pathway(s).
Independent regulation of c-myc, B-myb, and c-myb gene expression by inducers and inhibitors of proliferation in human B lymphocytes.
Authors: Golay J , Cusmano G , Introna M
Published: December 2022

Although a detailed picture is emerging about the nature of the second messengers involved in B cell activation and proliferation, little is yet known about the intracellular events taking place further downstream. The c-myb proto-oncogene, the structurally related B-myb gene, and c-myc probably code for transcription factors, have been demonstrated to be necessary for the proliferation of hemopoietic cells, and their expression is indeed induced after mitogenic stimulation of T and B lymphocytes. They are therefore likely to be key elements in the regulation of gene expression during proliferation. We have set out to study the regulation of the expression of these two myb genes and of that of c-myc in relation to entry into the different phases of the cell cycle during mitogenic stimulation of resting human B lymphocytes. Resting tonsillar B cells stimulated with the anti-CD20 antibody 1F5 alone are induced to enter the G1 but not the S phase of the cell cycle, whereas co-stimulation with the anti-CD40 antibody G28.5 further drives them to enter the S phase and proliferate. The G28.5 antibody alone has been reported to partially activate and increase the alertness of resting B cells without inducing them to enter G1. In this report we show that increasing the strength of the activating signal leads to progressive induction of the proliferation-related genes studied. Thus the G28.5 antibody alone induces c-myc mRNA only in resting B cells, 1F5 induces both c-myc and B-myb, and the full mitogenic signal given by both antibodies together is accompanied by increased expression of all three--c-myc, B-myb, and c-myb genes. In addition, using a semi-quantitative polymerase chain reaction method, we show that different inhibitors of B cell proliferation, namely, cyclosporin A, an anti-CD19 antibody (HD37), and transforming growth factor beta 1 (TGF-beta 1), inhibit differentially the induction of these same genes after mitogenic stimulation of B cells. Whereas cyclosporin A inhibits induction of all three genes, TGF-beta 1 specifically blocks B-myb induction and CD19 has little effect on either of the genes tested. We conclude that c-myb, B-myb, and c-myc are regulated independently from one another, that induction of c-myc and B-myb together is not sufficient to trigger B cell proliferation, and we suggest that expression of all three is a prerequisite for proliferation to occur.
Role of cytokines in the adoptive immunotherapy of an experimental model of human head and neck cancer by human IL-2-activated natural killer cells.
Authors: Rabinowich H , Vitolo D , Altarac S , Herberman R B , Whiteside T L
Published: December 2022

Peritumoral injection of human IL-2-activated natural killer cells into nude mice consistently induced regression of xenografts of human squamous cell carcinoma of the head and neck (SCCHN). To determine the mechanisms responsible for the tumor regression, the lymphoid cells infiltrating the tumor stroma at 24 to 48 h after adoptive immunotherapy were examined by in situ hybridization for the presence of mRNA for cytokines or IL-2R. Numerous lymphoid cells expressing cytokine or IL-2R genes were observed in these tumors, whereas the cultured IL-2-activated NK cells used for therapy were negative. Thus, it appeared that the transferred NK cells became activated in situ after coming into proximity with the tumor cells. To analyze this phenomenon, fresh or cultured human NK cells were coincubated in vitro with irradiated human SCCHN cell line, PCI-1, with or without the presence of IL-2. Expression of mRNA for IL-2R, perforin, and various cytokines was observed within 5 h. Contact with the tumor cells stimulated NK cells to proliferate, secrete IFN-gamma, TNF-alpha, and soluble IL-2R, up-regulate cell surface expression of IL2R p55 and p75 as well as CD16 Ag, and mediate higher levels of antitumor activity in 51Cr-release assays. In addition, supernatants of in vitro-activated NK cells significantly inhibited proliferation of SCCHN cell lines. By examining the effects of neutralizing mAb to various cytokines, this inhibitory activity was shown to be partially attributable to IFN-gamma. To determine the possible in vivo role of soluble factors produced by activated human NK cells, the supernatants (0.2 ml) or rIFN-gamma (10(5) U) were injected perilesionally each day for 2 wk into 3-day SCCHN established in immunosuppressed nude mice. These treatments caused significant (p less than 0.02) inhibition of tumor growth. The results of our studies indicate that human NK cells are strongly activated by SCCHN cells and that the consequent release of cytokines contribute to the regression of SCCHN growing in nude mice.
Identification and induction of keratinocyte-derived IL-10.
Authors: Enk A H , Katz S I
Published: December 2022

It has previously been demonstrated that the epidermis is a rich source of proinflammatory cytokines and growth factors and that complex interactions between these factors may affect inflammatory responses in skin. To investigate whether IL-10 (cytokine synthesis inhibitory factor) is part of this complex process, RNA was extracted from normal epidermis at various times after application of various chemicals to murine skin and mRNA signals for IL-10 were sought using a quantitative reverse-transcriptase-polymerase chain reaction technique. IL-10 signal strength was normalized to that of beta-actin in each sample. IL-10 mRNA signals were occasionally identified in normal epidermis but were uniformly enhanced 4 h after hapten application, and were maximal after 12 h. Contact allergens induced IL-10 mRNA signals whereas vehicles and irritants did not. Depletion of Langerhans cells, Thy-1+ dendritic epidermal cells, and T lymphocytes demonstrated that keratinocytes were the main source of IL-10 mRNA. IL-10 signals were also detected in mRNA derived from PAM 212 (spontaneously transformed keratinocyte) cells. IL-10 protein could be detected by immunoprecipitation, with an IL-10 mAB, of supernatants obtained 16 h after cultured epidermal cells were coupled with hapten. This study demonstrates that murine keratinocytes are capable of producing IL-10 mRNA and protein, and that signal strength of IL-10 mRNA is enhanced by hapten application.
The C/EBP family of transcriptional activators is functionally important for Ig VH promoter activity in vivo and in vitro.
Authors: Cooper C , Johnson D , Roman C , Avitahl N , Tucker P , Calame K
Published: December 2022

We have examined the functional importance of binding sites for C/EBP family members (E sites), in two Ig VH promoters: VH1, a member of the S107 family, and BCL1, a member of the J558 family. Mutation of the E site in the VH1 promoter diminishes transcription in vivo to 59% of wild-type and transcription from the BCL1 promoter in vitro is inhibited to an average of 39% of wild-type by competition with E site oligonucleotides. Purified E site binding proteins from plasmacytoma cells stimulated BCL1 transcription in vitro 2.3-fold. Although five C/EBP family proteins are known which bind the E site, antibody ablation of DNA:protein complexes resolved by electrophoretic mobility shift assays showed that Ig/EBP-1 is the only E site binding protein detectable in early B cell lines; more mature B cells contain Ig/EBP-1 and NF-IL6. We also show by antibody-depletion that Ig/EBP-1 activates the BCL1 promoter in vitro.
Patterns of antibody specificity during the BALB/c immune response to hen eggwhite lysozyme.
Authors: Newman M A , Mainhart C R , Mallett C P , Lavoie T B , Smith-Gill S J
Published: December 2022

We tested 49 BALB/c antilysozyme mAb from seven intervals during the immune response to lysozyme for patterns of specificity and avidity. We found that the antibody epitopes in composite covered at least 80% of the lysozyme surface, and their patterns of overlap suggest a continuum of potential antibody epitopes. Previously observed regional specificities, which emerged at different times in the immune response, were more discretely defined in late response antibodies, when the majority of mAb could be assigned to one of three functionally nonoverlapping complementation groups. The area covered by each antigenic region may be greater than an individual epitope, and may include multiple epitopes that overlap structurally and functionally to varying degrees. Connectivity between antigenic regions was seen in interactions among early and late stage antibodies, and among secondary stage mAb, but not among tertiary stage mAb from hyperimmunized mice. Patterns of overlap of early and late response antibodies suggest that the organization of antibody specificities change during the progression from primary to secondary to tertiary response. Over the same period in the response, the average relative avidity of IgG1 kappa mAb did not increase, suggesting that "affinity maturation" of serum antibodies reflects an increase in the number and diversity of antibodies, rather than an overall increase in the avidity of individual antibodies.
Neuropeptides modulate human eosinophil chemotaxis.
Authors: Numao T , Agrawal D K
Published: December 2022

To investigate the role of neuropeptides in allergic inflammation, we examined the effect of peptides on eosinophil chemotaxis. Eosinophils were purified from the blood of allergic and normal subjects using a discontinuous Percoll density gradients. Chemotaxis was induced by platelet-activating factor (PAF) and leukotriene B4, and was assayed by a modified Boyden's chamber technique. Four neuropeptides were examined in this study: substance P (SP), neurokinin A, calcitonin gene-related peptide (CGRP), and cholecystokinin octapeptide. Peptides alone (10 nM to 10 microM) were not chemotactic for eosinophils. However, when eosinophils were pre-treated with peptides (100 nM) at 37 degrees C for 30 min, chemotactic response to PAF (10 nM) was significantly enhanced (p < 0.01) in allergic subjects; % control by SP, neurokinin A, CGRP and cholecystokinin octapeptide was 269 +/- 42, 243 +/- 32, 227 +/- 21, and 251 +/- 42, respectively (n = 8). Similar results were obtained in leukotriene B4-induced eosinophil chemotaxis. In contrast, no enhancement was observed in normal subjects. Potentiating effect of SP and CGRP on PAF-induced eosinophil chemotaxis in allergic subjects was significantly attenuated by SP antagonist [D-Pro2,D-Trp7,9]-SP and human CGRP (8-37) receptor antagonist, respectively. Neutral endopeptidase inhibitors (phosphoramidon, leupeptin, and bestatin) failed to significantly augment the PAF-induced eosinophil chemotaxis when the cells were pretreated with various peptides and neutral endopeptidase inhibitors. The C-terminal fragment of SP (SP6-11) had an effect similar to that of the intact SP molecule, whereas no potentiating effect by the N-terminal of SP (SP1-9) was observed. These results suggest that neuropeptides may play a significant role in eosinophil infiltration by priming cells in allergic inflammation.
Antibodies and reactive T cells against the malaria heat-shock protein Pf72/Hsp70-1 and derived peptides in individuals continuously exposed to Plasmodium falciparum.
Authors: Behr C , Sarthou J L , Rogier C , Trape J F , Dat M H , Michel J C , Aribot G , Dieye A , Claverie J M , Druihle P
Published: December 2022

Pf72/Hsp70-1, a heat-shock protein of m.w. 72 kDa from Plasmodium falciparum is one of the Ag of interest to be included in a polyvalent vaccine against malaria. It is one of the major immunogens present in a fraction of purified blood stage parasites that elicited protection against experimental infection of Saimiri monkeys with blood stages of P. falciparum. It is present at all blood stages and one of its B cell epitopes is also detected on the surface of the infected hepatocyte. Moreover, Pf72 appears to be well conserved among different isolates of P. falciparum. We have examined the immune response against Pf72/Hsp70-1 in individuals from different age groups living in a holoendemic area (West Africa). The immune response against the native Ag (purified from schizonts and called Pf/Hsp70) was analyzed both at the humoral level by ELISA and at the cellular level by assessing in vitro proliferation and IFN-gamma production of PBMC. Of the individuals studied 52% had a statistically significant level of anti-Pf/Hsp70 antibodies as compared with unexposed individuals. These positive individuals showed a heterogeneous distribution because significant levels of antibodies were found in 70% of the adults but in only 26% of the children. The presence of Pf/Hsp70-specific reactive T cells in the blood was detected in 32% of the individuals. The total anti-Pf/Hsp70 antibody level (IgG+IgM) appeared strongly age related and correlated positively with parasite exposure, whereas the T cell response failed to correlate either with the antibody level or with age. Moreover, PBMC of donors responded to the Pf/Hsp70 in a dissociated way, namely, by either T cell proliferation or IFN-gamma production. Ten synthetic peptides based on sequences found in the C-terminal part of Pf72/Hsp70-1 were further tested as potential T cell epitopes. The proliferative response of PBMC from individuals continuously exposed to the parasite showed that three peptides more frequently trigger significant T cell proliferation (in 21% to 27% of the individuals) and three others less frequently (10%). None of these peptides allowed detection of reactive T cells in PBMC of Europeans with no previous exposure to malaria. Some of the stimulating peptides are highly similar to human heat-shock Hsc and Hsp70 with large stretches of identical amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)
Intercellular adhesion molecule-1 (ICAM-1)-dependent and ICAM-1-independent adhesive interactions between polymorphonuclear leukocytes and human airway epithelial cells infected with parainfluenza virus type 2.
Authors: Tosi M F , Stark J M , Hamedani A , Smith C W , Gruenert D C , Huang Y T
Published: December 2022

Acute respiratory virus infections are often associated with an early influx of neutrophils (PMN) into the airways. Maximal cytoxic injury by PMN depends on tight cell-cell adhesion. Infection of some cell types by respiratory and other viruses has been shown to increase PMN adhesion to these cells by undefined mechanisms. We studied adhesion by human PMN to monolayers of primary (1 degree) human tracheal epithelial cells (TEC) or an immortalized cell line derived from human TEC, 9HTEo-, that had been infected with parainfluenza virus type 2 (PiV2). PMN adhesion to uninfected 1 degree TEC was very low (< 5%), but PMN adhesion to PiV2-infected 1 degree TEC was greatly increased (89 +/- 7%). PMN adhesion to 9HTEo- cells was 47 +/- 6%, but increased, 87 +/- 8%, for PiV2-infected 9HTEo- cells. Surface intercellular adhesion molecule-1 (ICAM-1) expression on 1 degree TEC, as determined by immunofluorescence flow cytometry, was relatively low (23 fluorescence units) but doubled by 24 h after PiV2 infection and tripled by 48 h. The 9HTEo- cells constitutively expressed higher levels of surface ICAM-1 (120 units) which did not increase with PiV2 infection. Treatment of non-PiV2-infected 9HTEo- cells with mAb (R6.5) to ICAM-1 reduced PMN adhesion to these cells from 47 +/- 8 to 23 +/- 5%. Identical mAb treatment of either 1 degree TEC or 9HTEo- cells infected with PiV2 had no significant effect on PMN adhesion. Treatment of the PMN with mAb against CD11a, CD11b, or CD18 markedly reduced PMN adhesion to PiV2-infected 1 degree TEC and 9HTEo- cells. We conclude that PiV2 infection of human TEC causes a marked increase in their adhesive interactions with PMN by inducing increased surface expression of both ICAM-1 and one or more, as yet uncharacterized, non-ICAM-1 adhesion molecules that function as counter-receptors for CD11/CD18 on PMN. These mechanisms of adhesion may play a role in epithelial damage during acute respiratory virus infections.