bluuebunny/crossref_metadata_2025_split huggingface dataset:
T cell receptor V beta 2 and V beta 6 mediate tumor-specific cytotoxicity by tumor-infiltrating lymphocytes in ovarian cancer.
Authors: Peoples G E , Davey M P , Goedegebuure P S , Schoof D D , Eberlein T J 
Published: December 2022

Abstract
The interaction between T lymphocytes and the Ag-HLA complex on tumor cells is mediated by the TCR. The diversity and the specificity of the TCR are in part secondary to the gene rearrangement of the V region on the beta-chain (V beta). To determine whether a restricted number of TCR V beta genes are utilized in the recognition of ovarian cancer, tumor-infiltrating lymphocytes (TIL) were isolated from six consecutive untreated ovarian cancer patients. TIL were also cultured using repeated autologous tumor stimulation, and by 7 wk, five of six patients produced bulk cultures consisting of &gt; 50% CD8+ T cells and demonstrating an autologous tumor-specific pattern of cytotoxicity. TCR V beta gene usage was analyzed in the five patients yielding fresh TIL and corresponding 7-wk cultured, tumor-specific TIL; 22 primers specific for 20 TCR V beta gene families were employed and amplified by polymerase chain reaction and then quantitated by HPLC. A heterogeneous pattern of V beta usage was seen in the fresh TIL; however, V beta 2, V beta 3, V beta 6, V beta 7, V beta 8, and V beta 13.1 were found in increased proportions in at least three of five patients. In the 7-wk tumor-specific TIL, V beta analysis showed an increased usage of V beta 2, V beta 3, V beta 6, and V beta 7 in more than three of five patients. No significant change in V beta representation was seen in control populations that were not stimulated with tumor. Looking at the percent change in V beta usage between fresh and 7-wk tumor-specific cultures, V beta 2 and V beta 6 were augmented significantly in at least three of five patients (108% and 61%, respectively). To verify that the increase in representation of these V beta families was responsible for the increased cytotoxicity observed, mAb specific for V beta 2 and V beta 6 were used to block tumor lysis. Anti-V beta 6 and anti-V beta 2 significantly blocked cytotoxicity against autologous tumor cells in those TIL populations expressing increased levels of these V beta families. These data suggest that a selective repertoire of TCR V beta genes is used to recognize the Ag-HLA class 1 complexes on the surface of ovarian tumor cells, and specifically V beta 2 and V beta 6 appear to mediate antitumor activity. These findings may aid in the development of a more specific immunotherapy in ovarian cancer.
Signal transducing properties of the N-formyl peptide receptor expressed in undifferentiated HL60 cells.
Authors: Prossnitz E R , Quehenberger O , Cochrane C G , Ye R D 
Published: December 2022

Abstract
Differentiated HL60 cells respond to challenge with ligand by mobilizing intracellular second messengers, resulting in superoxide production, degranulation, and actin polymerization with subsequent chemotaxis and phagocytosis. The functional capabilities of undifferentiated HL60 cells have not been similarly characterized due to the absence of the cell surface receptors required to initiate these processes. To investigate these properties, undifferentiated HL60 cells were transfected with one of the better characterized neutrophil chemotactic receptors, the N-formyl peptide receptor (FPR). Expression of the recombinant FPR gene product in FPR-transfected HL60 cells and the absence of the endogenous FPR in vector-transfected HL60 cells was demonstrated by Northern blot and flow cytometric analyses. FPR-transfected HL60 cells retained their ability to undergo granulocytic differentiation with dibutyryl cAMP, as determined by FMLP- and PMA-stimulated superoxide production. Furthermore, incubation of FPR-transfected HL60 cells for 5 days in the presence of FMLP resulted in limited differentiation as evidenced by the expression of functional C5a receptors. Binding studies of FPR-transfected HL60 cells demonstrated the presence of two binding affinities with dissociation constants of 0.6 and 33 nM, similar to dibutyryl cAMP differentiated HL60 cells and human neutrophils but contrasting the single high affinity state of the FPR expressed in mouse L cell fibroblasts. FPR-transfected HL60 cells displayed FMLP-dependent calcium mobilization with an EC50 of 3 nM and actin polymerization with an EC50 of approximately 10 nM. Actin polymerization was not observed in FPR-transfected L cell fibroblasts or undifferentiated vector-transfected HL60 cells. Both calcium mobilization and actin polymerization were sensitive to treatment with pertussis toxin, indicating the requirement for a Gi-like protein. Stimulation of either undifferentiated or differentiated HL60 cells with ATP resulted in pertussis toxin-insensitive calcium mobilization but was ineffective in producing actin polymerization. The results described herein show for the first time that undifferentiated HL60 cells can respond to chemoattractant receptor stimulation with many of the properties of the mature neutrophil. Transfected HL60 cells will provide an excellent system to study the characteristics of chemotactic receptors as well as the functional properties of myeloid cells.
The majority of immature fetal thymocytes can be induced to proliferate to IL-2 and differentiate into cells indistinguishable from mature natural killer cells.
Authors: Brooks C G , Georgiou A , Jordan R K 
Published: December 2022

Abstract
Although immature thymocytes from day 14 embryonic mice (FTC) express IL-2R, they have generally been considered to be unresponsive to IL-2. We show here that they can in fact undergo substantial and prolonged growth in vitro provided that high doses of IL-2 are present. The ability of FTC to grow in IL-2 could be enhanced slightly by PMA and also by IL-4, but dramatically by the combination of IL-4 + PMA with IL-2. Pretreatment of FTC with IL-4 + PMA for as little as 24 h primed FTC for rapid and prolonged responsiveness to IL-2, permitting the establishment of long term lines. Kinetic and clonal analysis revealed that most individual FTC could grow under these conditions. Although proliferating cells expressed functional IL-2R of only very low affinity, and IL-2R alpha chains were undetectable by immunofluorescence, blocking experiments showed unambiguously that both IL-2R alpha and IL-2R beta were involved in signal transmission. FTC lines and clones developed in this manner lacked lineage-specific markers of mature T cells, B cells, and myeloid cells, but expressed the NK cell markers NK1.1 and asialo-GM1. They displayed potent cytolytic activity against NK-sensitive targets, and, when stimulated with PMA+ionomycin, secreted IL-3 and IFN-gamma, but not IL-2 or IL-4. After intrathymic injection they showed no evidence of growth or differentiation. These results demonstrate that most, if not all, immature thymocytes have the capacity to differentiate into cells which appear to be indistinguishable from mature NK cells. They suggest that T cells and NK cells derive from a common precursor which in the thymic environment differentiates into T cells and in the extrathymic environment into NK cells.
Nedocromil sodium selectively inhibits IgE and IgG4 production in human B cells stimulated with IL-4.
Authors: Kimata H , Mikawa H 
Published: December 2022

Abstract
We studied the effect of nedocromil sodium (NES) on human IgE and IgG subclass production. NES inhibited IgE and IgG4 production, without affecting IgM, IgA, IgG1, IgG2, or IgG3 production, by IL-4-stimulated mononuclear cells (MNC) obtained from non-atopic donors. Inhibition of IgE and IgG4 production by NES required the compound to be added on day 0 and to be present throughout the entire period of culture. Inhibition of IgE and IgG4 production was not mediated by known inhibitors, including IFN-alpha, IFN-gamma, transforming growth factor-beta, PGE2, or platelet-activating factor. Inhibition by NES required both T cells and monocytes because NES inhibited IgE production by B cells stimulated with IL-4 and anti-CD40 mAb in the presence, but not the absence, of T cells and monocytes. NES also inhibited IgE and IgG4 production in cultures stimulated with anti-CD3-treated T cells. However, in this case other isotypes were also inhibited by NES. In contrast to non-atopic donors' MNC, NES failed to inhibit spontaneous IgE and IgG4 production by MNC from atopic patients. However, NES was effective in inhibiting IL-4-induced IgE and IgG4 production in atopic patients' MNC without affecting IgM, IgA, IgG1, IgG2, or IgG3 production. NES inhibited IgE and IgG4 production by sIgE- and sIgG4-B cells, but not by sIgE+ and sIgG4+ B cells, respectively, stimulated with IL-4 or IL-4 plus anti-CD40 mAb in the presence of T cells and monocytes. These results suggest that NES may inhibit IL-4-induced switching of IgE and IgG4 production by sIgE- B cells and sIgG4- B cells, respectively, by affecting the interaction of B cells, T cells, and monocytes.
Role of dendritic cells in the priming of CD4+ T lymphocytes to peptide antigen in vivo.
Authors: Levin D , Constant S , Pasqualini T , Flavell R , Bottomly K 
Published: December 2022

Abstract
The contribution of dendritic cells (DC) in the initial priming of CD4+ T lymphocytes in vivo was examined. To assess the capacity of different APC to prime CD4+ T cells, a series of MHC class II I-E transgenic mice that differentially express I-E on APC were utilized. Transgenic mice that express I-E primarily on DC, on macrophages, and on B cells were primed with an I-E restricted peptide in vivo, and the extent of priming was determined by restimulation of CD4+ T cells in vitro with the same Ag. The results indicate that DC are required for priming of CD4+ T cells, and that the extent of priming correlates with the frequency of I-E+ DC. Moreover, DC alone can serve as APC for the peptide Ag, in that priming can be restored in an I-E negative mouse by the transfer of peptide-pulsed I-E+ DC. The potency of DC, compared with B cells and macrophages, to prime naive CD4+ T cells was confirmed with in vitro studies.
Differences among various lineages of antigen-presenting cells in processing exogenous antigen internalized through transferrin receptors.
Authors: McCoy K L , Page M S , Merkel B J , Inman J K , Stutzman R 
Published: December 2022

Abstract
The Ag, pigeon cytochrome c, was coupled to human ferric transferrin by a heteroligation technique to target Ag into the endosomal transport pathway via transferrin receptors. The ability of various types of APC that do or do not express transferrin receptors to process exogenous Ag in their endosomes was investigated by the stimulation of Ag-specific CD4+ T cells with the transferrin-Ag conjugate in a serum-free assay. When two B lymphoma cells were the source of APC, the conjugate was significantly more potent than native Ag in activating the T cells, agreeing with our previous finding using a third B lymphoma cell. The conjugate and Ag were similarly presented by splenic B cells that lack transferrin receptors to the T cells. However, both a macrophage hybridoma and a MHC class II-L cell transfectant hardly elicited a T cell response to the conjugate, although a response to native Ag was readily observed. These findings could not be attributed to an absence of transferrin receptors or receptor-mediated internalization of the conjugate, nor to differential expression of MHC class II molecules or li chain by the APC. The poor presentation of the conjugate by the L cell transfectants was associated with diminished catabolism of the conjugate, however, the macrophage hybridoma rapidly degraded the conjugate, similar to the B lymphoma cell. Peritoneal macrophages, which lack transferrin receptors, and the macrophage hybridoma induced a response to the conjugate only at concentrations that allowed internalization by fluid phase pinocytosis. The lower potency of the conjugate compared with native Ag with non-B-presenting cells suggest that these cell types process the conjugate by a different mechanism than used by B cells. Differences in the mechanism of Ag processing used by APC of distinct cell lineages may possibly influence immune responsiveness.
Triggering of the cytotoxic activity of murine natural killer and lymphokine-activated killer cells through the NK2.1 antigen.
Authors: Morelli L , Lemieux S 
Published: December 2022

Abstract
The NK2.1 alloantigen, one of the few NK-specific markers in the mouse, identifies a subset rather than all splenic NK cells. On the basis of the cell surface expression of a variety of Ag of the lymphocytic and monocytic lineages, we establish that BALB/c NK2.1+ cells are heterogeneous and that their phenotype is quite similar to the one reported for NK1.1+ cells. A NK2.1- cell population of the same size and related phenotype was also identified in fresh NK-enriched cell suspensions. After stimulation with IL-2, the only phenotypic trait that distinguishes NK2.1+ and NK2.1- cell subsets is precisely the expression of the NK2.1 Ag. To investigate whether NK2.1+ and NK2.1- lymphokine-activated killer cells would also be identical in their cytotoxic activity, NK-enriched spleen cells were cultured for 5 days with IL-2, sorted afterward on the basis of NK2.1 expression, and compared for their capacity to lyse YAC-1 targets. NK2.1+ cells are significantly more lytic than NK2.1- cells, suggesting that the anti-NK2.1 mAb, used for cell sorting, could have triggered NK2.1+ cells and enhanced their lytic activity. In support to this hypothesis we show that 1) immobilized anti-NK2.1 mAb induces granule exocytosis by LAK cells, 2) soluble anti-NK2.1 mAb specifically inhibits NK and LAK cell-mediated lysis of 4LO3311 hybridoma cells secreting anti-NK2.1 mAb, and 3) binding of anti-NK2.1 mAb selectively enhances the lysis of NK-sensitive targets. Furthermore, the successful activation of NK2.1+ cells induced by anti-NK2.1 F(ab')2 or F(ab) mAb fragments indicates that the triggering mechanism is different from reverse antibody-dependent cellular cytotoxicity and does not require cross-linking of the NK2.1 molecule. Our results strongly suggest that the NK2.1 molecule is implicated in a post-binding NK cell signaling event, and point out the possible functional relevance of this NK-specific Ag in non-MHC-restricted cytotoxicity.
Fc gamma RIIIA-mediated signaling involves src-family lck in human natural killer cells.
Authors: Pignata C , Prasad K V , Robertson M J , Levine H , Rudd C E , Ritz J 
Published: December 2022

Abstract
The cell surface receptor for IgG Fc domain (Fc gamma RIIIA, CD16) is present as a multimeric complex on human NK cells and is essential for antibody-dependent cellular cytotoxicity. Although CD16 engagement induces tyrosine phosphorylation of several substrates, the intracytoplasmic domains of the CD16 molecule lack intrinsic protein tyrosine kinase consensus sequences. We report here that, in human NK cells, engagement of the CD16 receptor induces tyrosine phosphorylation of p56lck tyrosine kinase. CD16 triggering through anti-receptor antibody enhances in vitro kinase activity of phosphotyrosyl proteins and autophosphorylation activity of anti-lck immunoprecipitates. Similar results were obtained after stimulation with IL-12. In vivo labeling experiments showed that CD16 cross-linking primarily induces in vivo phosphorylation of p56lck at the tyr-394 autophosphorylation site. Using in vitro kinase assay of CD16 immunoprecipitates from lysates of human NK cells, we demonstrated that a phosphorylated protein of 62 to 65 kDa was co-precipitated with CD16. Reprecipitation experiments also suggested that a small amount of p56lck may interact with this complex. These results indicate that CD16 triggering involves p56lck and that this kinase is therefore involved in the signaling pathways regulating NK cell cytolytic functions. However, additional kinases not yet identified are also likely to play a role in the early signaling events through CD16.
Expression and function of mixed isotype MHC class II molecules in normal mice.
Authors: Spencer J S , Freed J H , Kubo R T 
Published: December 2022

Abstract
Mixed isotype MHC class II molecules (E alpha dA beta d) occur at extremely low levels on the surface of normal mouse B cells and macrophages, as determined by surface staining with an E alpha dA beta d-specific hamster mAb, H71-258.41. The surface levels of mixed isotype on the B cell lymphoma line A20 are approximately 1 to 2% that of surface I-A, whereas the levels of these molecules on normal mouse B cells were estimated to be at least two to four times less than those on A20. Nevertheless, other investigators have recently reported that immunization of normal H-2d mice with the sperm whale myoglobin peptide 110-121 (SWM(110-121)) elicits T cells, predominantly, V beta 8.2+, that recognize the peptide only in context of E alpha A beta. We have characterized a large number of SWM(110-121)-specific T cell hybridomas from several strains of H-2d haplotype mice. All of the V beta 8.2+ 110-121-specific hybridomas were found to be restricted by E alpha dA beta d, whereas, of the V beta 8.2- 110-121-specific group, approximately half recognized the peptide through E alpha dA beta d whereas the remainder were restricted by either I-Ad or I-Ed. mAb inhibition experiments revealed that 14-4-4S (E alpha-specific) could block presentation by mixed isotype completely, while MK-D6 (A beta d-specific) and H71-258.41 (E alpha dA beta d-specific) only inhibited presentation when the concentration of peptide was limiting. Although A20 expresses very low levels of mixed isotype, 10 to 100 nmol of the peptide produced a detectable response, illustrating the remarkable efficiency in presenting this peptide through E alpha dA beta d. The ability of normal mouse APC to use this restriction element despite its extremely low expression has important implications for the activation of T cells by low levels of peptide-MHC complexes.
Specific induction of 80-kDa tumor necrosis factor receptor shedding in T lymphocytes involves the cytoplasmic domain and phosphorylation.
Authors: Crowe P D , VanArsdale T L , Goodwin R G , Ware C F 
Published: December 2022

Abstract
The 80-kDa TNFR (TNFR80) expressed by activated human T cells is constitutively phosphorylated and undergoes limited proteolytic cleavage (shedding) at the cell surface releasing a 40-kDa soluble TNF-binding protein. Triggering of activated T cells through the TCR rapidly increased the rate of TNFR80 shedding &gt; 20-fold more than nonstimulated cells, demonstrating that shedding is a specific, inducible process. The protein kinase inhibitor staurosporine inhibited constitutive phosphorylation and blocked inducible shedding of TNFR80, suggesting that phosphorylation may be important for cleavage of the extracellular domain. However, a deletion mutation of the entire cytoplasmic domain of human TNFR80 was shed when expressed in murine L929 cells, albeit relatively poorly compared with full length receptor. This demonstrates that the cytoplasmic domain is important but not essential for cleavage of the extracellular domain of TNFR80. Moreover, a requirement for phosphorylation of proteins other than the receptor was revealed by the finding that staurosporine completely blocked cleavage of the cytoplasmic deletion mutant. Collectively, these results demonstrate that protein phosphorylation is essential and the cytoplasmic domain is important for regulating the inducible production of soluble TNF-binding proteins by activated effector T cells.
Engineering Fab' fragments for efficient F(ab)2 formation in Escherichia coli and for improved in vivo stability.
Authors: Rodrigues M L , Snedecor B , Chen C , Wong W L , Garg S , Blank G S , Maneval D , Carter P 
Published: December 2022

Abstract
We previously developed an efficient route to humanized F(ab')2 fragments by high level secretion of the Fab' arms from Escherichia coli followed by directed chemical coupling in vitro. Here the number and type of interchain linkages in F(ab')2 molecules has been modified to simplify their production and improve their serum stability. All F(ab')2 variants had comparable binding affinity for the p185HER2 Ag and antiproliferative activity against p185HER2-overexpressing tumor cells. This was anticipated since the modifications are distant from the Ag-binding loops. Replacement of a single disulfide bridge between Fab' arms with a more stable thioether bridge increased the serum permanence time in normal mice by threefold to 2.1 h. Removal of the disulfide bond between L and H chains in the thioether-bridged F(ab')2 did not affect the pharmacokinetics, suggesting that the L chain remains associated with the H chain. An additional Fab' variant containing three repeats of the motif, CysProPro, was constructed with the aim of promoting efficient formation of F(ab')2 in E. coli. This Fab' (CPP)3 variant was recovered predominantly (up to 70%) as F(ab')2 directly from fermentation cell pastes, thus circumventing the need for in vitro coupling. The F(ab')2 (CPP)3 variant has a similar serum pharmacokinetics to the thioether-bridged molecules. The improvements described here for deriving F(ab')2 fragments from E. coli should enhance the clinical potential of these molecules.
Soluble intercellular adhesion molecule-1 inhibits MHC-restricted specific T cell/tumor interaction.
Authors: Becker J C , Termeer C , Schmidt R E , Bröcker E B 
Published: December 2022

Abstract
Intercellular adhesion molecule-1 (ICAM-1)/LFA-1-mediated cell-cell adhesion is essential for various immunologic functions. It was previously shown that some of these, including non-MHC-restricted cytotoxicity, could be inhibited by soluble forms of ICAM-1. Here, we characterize the effects of soluble ICAM-1 on MHC-restricted specific T cell/melanoma interactions. Tumor-infiltrating lymphocyte (TIL) clones, as well as melanoma lines from the same tumor specimens, were established. The TIL clones used were able to form conjugates with the autologous tumor and to kill it in an MHC-restricted way. TIL/melanoma interaction also induced increased cytokine production in TIL. Using this system, we could demonstrate that purified soluble ICAM-1 (950 ng/ml) or 12-fold concentrated cell-free melanoma supernatants, containing shed ICAM-1 (1000 ng/ml), were able to inhibit conjugate formation between T cell clones and the autologous melanoma cells as efficiently as mAb against CD11a. In addition, free ICAM-1 abrogated the MHC-restricted killing of the melanoma exerted by T cell clones and blocked the induction of cytokines in T cells due to the contact with autologous tumor cells.