bluuebunny/crossref_metadata_2025 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

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 > 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

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

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

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

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

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

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

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

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

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 > 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

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

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.
Autoantigen inhibits apoptosis of a human B cell leukemia that produces pathogenic rheumatoid factor.
Authors: Kobayashi R , Rassenti L Z , Meisenholder G , Carson D A , Kipps T J
Published: December 2022

We studied a variant CD5- B cell chronic lymphocytic leukemia (CLL) cell population that produces pathologic IgM kappa rheumatoid factor autoantibodies. In contrast to common CD5+ B cell CLL, this variant leukemia cell population displays intraclonal diversity in its expressed Ig V genes, similar to that noted for follicular B cell non-Hodgkin's lymphomas. Also, in contrast to common B cell CLL, these leukemia cells rapidly undergo cell death hours after being placed in tissue culture. We find that addition of Ag (aggregated human IgG) enhances significantly the survival of these cells in vitro. Leukemia cell survival also could be enhanced by exogenous IFN-gamma or anti-CD40 presented on Fc gamma RII (CDw32)-expressing L cells, but not by exogenous IL-4, IL-6, or monomeric human IgG. We find that Ag acts directly on the leukemia B cells to inhibit apoptosis. This effect could be mimicked by cross-linking the leukemia cells' surface IgM receptors with immobilized murine mAb specific for human Ig mu-chains, but not by immobilized mAb of irrelevant specificity. In contrast to most follicular NHL, this leukemia B cell population does not have evidence of bcl-2 gene rearrangement. Also, in contrast to non-Hodgkin's lymphomas and most B cell CLL, these cells do not express detectable amounts of bcl-2. Finally, although capable of inhibiting apoptosis, surface Ig receptor cross-linking does not induce expression of bcl-2 in these variant leukemia cells. We hypothesize that the lack of bcl-2 expression may render these leukemia cells particularly dependent upon the survival signal(s) derived from surface Ig receptor cross-linking. This state may represent an early stage in leukemia/lymphomagenesis, possibly accounting for the intraclonal diversity observed in the Ig V genes expressed by certain CD5- B cell leukemias and lymphomas.
A novel, antigen-presenting function of melanocytes and its possible relationship to hypopigmentary disorders.
Authors: Le Poole I C , Mutis T , van den Wijngaard R M , Westerhof W , Ottenhoff T , de Vries R R , Das P K
Published: December 2022

It is now well established that cultured human melanocytes are capable of expressing immunologically important cell surface molecules and that they can produce cytokines. Not all cells with the ability to express MHC class II molecules are capable of effective Ag presentation. However, the dendritic nature of melanocytes, their strategic position within the skin, and their phagocytic capacity seem to suggest a role for these cells in processing and presenting Ag. This study demonstrates that cultured normal human skin melanocytes can present peptide Ag, and process and present the mycobacterial HSP65 kDa protein and whole Mycobacterium leprae sonicate to CD4+ cytotoxic proliferative T cell clones in an Ag-specific and HLA-class II-restricted manner. T cell stimulation was dependent on costimulatory signals, i.e., LFA-3/CD2 and LFA-1/ICAM-1. Besides eliciting a T cell proliferative response, our studies further demonstrate that melanocytes can function as target cells for T cell-mediated cytotoxicity. The described Ag-processing and -presenting functions of melanocytes, taken together with in vivo behavior of melanocytes in hypopigmentation, provide new clues for the etiopathogenesis of melanin pigmentary disorders.
Conventional B cells, not B1 cells, are the source of autoantibodies in chronic graft-versus-host disease.
Authors: Reap E A , Sobel E S , Jennette J C , Cohen P L , Eisenberg R A
Published: December 2022

B1 (CD5+) B cells have been implicated as a source of certain autoantibodies in several murine and human studies. We have previously shown in the lpr model of autoimmunity, however, that conventional B cells, not B1 cells, were the source of autoantibodies directed at chromatin, ssDNA, and IgG. In the current study, we have investigated the origin of autoantibodies in chronic graft-versus-host (GVH) disease, induced in nonautoimmune mice by transferring la-incompatible spleen cells. GVH mice develop multiple autoantibodies and significant kidney damage. Therefore, this model allowed us to examine the B cell subset involved in both autoantibody production and tissue injury. We used two protocols to establish B cell chimeras that possessed immunoglobulin heavy chain (lgh) allotype-marked peritoneal (B1-cell source) cells and bone marrow-derived (conventional B cell source) cells from nonautoimmune C57BL/6kh (B6) congenic mice. In both types of chimera, chronic GVH was induced by giving mice alloreactive T cells i.p. All of the subsequent anti-chromatin, RF, and anti-ssDNA autoantibodies were produced by the conventional B cells and not by B1 cells. In addition, glomerular immune complex deposits of both IgM and IgG originated from the conventional B cells and not from B1 cells. These findings thus parallel those from our previous work on autoantibodies in lpr, and extend those findings by demonstrating that antibodies within pathogenic immune complexes in the kidneys are also exclusively of conventional B cell origin.
Normal T cell receptor-mediated signaling in T cell lines stably expressing HIV-1 envelope glycoproteins.
Authors: Tani Y , Tian H , Lane H C , Cohen D I
Published: December 2022

A series of T cell lines transfected to stably express HIV-1 envelope (env) glycoproteins were analyzed for viability and for T cell signaling. One transfectant was distinguished by its stable expression of gp120 and gp41, whereas the remainder of the T cell lines were similar to previously reported env-expressing T cells in synthesizing predominantly unprocessed env glycoprotein gp160. All of the transfectants were additionally constructed to express tat and rev proteins. None of these cell lines displayed growth abnormalities or spontaneous cell fusion, although the cell line synthesizing env gp120/gp41 could be induced to fuse and die when cocultured with a second cell expressing surface CD4. A cell line expressing only gp160 and the transfectant expressing gp160, gp120, and gp41 could be triggered normally via CD3-cross-linking as measured by protein tyrosine phosphorylation and by the induction of the CD69 activation marker. At levels of env protein expression sufficient to mediate syncytium formation and to kill cells, these HIV-1 env transfectants displayed no intrinsic T cell signaling abnormalities, suggesting that mechanisms other than a direct intracellular action of the tat or env proteins may be contributing to the deficit in Ag-specific T cell activation described subsequent to HIV infection in vivo and in vitro.
Anti-cytokine antibodies as carrier proteins. Prolongation of in vivo effects of exogenous cytokines by injection of cytokine-anti-cytokine antibody complexes.
Authors: Finkelman F D , Madden K B , Morris S C , Holmes J M , Boiani N , Katona I M , Maliszewski C R
Published: December 2022

Anti-cytokine antibodies that block interactions between cytokines and cytokine receptors have been used to inhibit endogenous cytokine function. However, injection of mice with mixtures of IL-4 and either of two neutralizing anti-IL-4 mAb, at a cytokine/anti-cytokine mAb molar ratio of approximately 2:1, enhances and prolongs in vivo IL-4 activity, as measured by induction of increased spleen cell Ia expression. Although splenocyte Ia expression returns to baseline two days after mice are injected with free IL-4, soluble IL-4-anti-IL-4 mAb complexes still induce several-fold increases in Ia expression 3 days after injection. Complexes that contain as little as 400 ng of IL-4 have considerable in vivo stimulatory activity, and a maximal effect on splenocyte Ia expression is induced by injection of 2 micrograms of complexed IL-4. The stimulatory effect of IL-4-containing complexes on splenocyte Ia expression can be blocked by increasing the ratio of anti-IL-4 mAb to IL-4, by injection of anti-IL-4R mAb, and by in vivo aggregation of the complexes. Complexes of IL-4 with a non-neutralizing anti-IL-4 mAb do not have increased IL-4 agonist activity in vivo. These observations are most consistent with the possibility that neutralizing anti-IL-4 mAb act as carrier proteins that increase the in vivo half-life of IL-4 by preventing its excretion, and possibly, by preventing modification of its active site. The enhanced agonist effect of IL-4-anti-IL-4 mAb complexes is not unique; complexes of IL-3 with a neutralizing anti-IL-3 mAb have a greatly increased ability, compared with free IL-3, to stimulate mucosal mastocytosis, and complexes of IL-7 with a neutralizing anti-IL-7 mAb have a greatly increased ability, compared with free IL-7 or IL-7 complexed with a non-neutralizing anti-IL-7 mAb, to stimulate an increase in pre-B cell number. These observations suggest that complexes of cytokines and neutralizing anti-cytokine mAb may provide a generally useful way to increase the magnitude and duration of cytokine effects in vivo.
CD4+/CD8- thymocytes dominate the fetal thymus treated with a combination of anti-T cell receptor-beta and anti-CD4 antibodies.
Authors: Punt J A , Hosono M , Hashimoto Y
Published: December 2022

Two major phenotypic changes characterize the development of a mature thymocyte from its CD4+/CD8+ (double positive or DP) precursor: the loss of expression of either CD4 or CD8 and the increase in the level of surface TCR. The specific surface interactions responsible for these changes are unknown, but studies using the fetal thymus as an experimental system have provided clues by identifying conditions that alter these maturational events. Development to the CD4+/CD8-/TCR-alpha beta high (single positive) phenotype is inhibited when thymocytes in fetal organ culture are exposed to antibodies directed against the CD4 molecule, the CD3 complex or the TCR-alpha/beta heterodimer. We show in this study, however, that treatment of fetal thymic lobes with a combination of anti-CD4 and anti-TCR-beta antibodies results in a marked increase in the proportion of mature CD4+/CD8-/TCR-alpha beta+ thymocytes and a decrease in the proportion of DP thymocytes. Although treatment of lobes with a combination of anti-CD4 and anti-CD3 epsilon antibodies also depletes cultures of DP thymocytes, the CD4+/CD8-/TCR+ population does not develop. Our results are consistent with the hypothesis that coengagement of CD4 and TCR biases development to the CD4 single positive phenotype and with observations that TCR engagement and CD3 engagement have different developmental consequences.
TCR-CD4 and TCR-TCR interactions as distinctive mechanisms for the induction of increased intracellular calcium in T-cell signalling.
Authors: Wolff C H , Hong S C , von Grafenstein H , Janeway C A
Published: December 2022

Specific activation of CD4 T cells involves recognition of a peptide: MHC class II complex by the heterodimeric alpha beta TCR and its CD4 co-receptor. The activation of T cells initiates a signal transduction cascade that includes a substantial and rapid increase in cytosolic calcium. In this work we study the role of the interactions between the TCR and the CD4 molecule in inducing this [Ca2+]i increase in the cloned CD4 T-cell line D10. Ligating CD3 or the TCR with mAb leads to proliferation of this cloned line and an increase in intracellular free calcium. An exceptional antibody, 16A, was able to stimulate proliferation but did not induce an increase in intracellular free calcium, even when extensively cross-linked with anti-Ig. However, when 16A was cross-linked to CD4 a rise in [Ca2+]i was observed. This demonstrated the ability of TCR-CD4 interactions to trigger a [Ca2+]i response in a situation where no signal was obtained from TCR-TCR interactions. Because CD4 molecules can associate with the TCR, we also studied whether CD4 is involved in the observed increases in intracellular free calcium obtained with other anti-TCR antibodies. This was examined in CD4-negative T cells transfected with cDNA encoding the D10 TCR. These cells could be induced to flux calcium by the same anti-TCR antibodies that gave this response in D10 cells, and again 16A failed to induce such a response. In the transfectants, anti-CD3 antibodies, in contrast to TCR antibodies, could induce a calcium signal in the absence of cross-linking by anti-Ig indicating that CD3 antibodies may signal distinctly from alpha beta TCR ligation. These studies document that antibodies binding different TCR/CD3 epitopes signal distinctively, and that CD4 can participate in, but is not absolutely required for, the induction of increased intracellular calcium.
Molecular characterization of anti-DNA antibodies induced in normal mice by immunization with bacterial DNA. Differences from spontaneous anti-DNA in the content and location of VH CDR3 arginines.
Authors: Gilkeson G S , Bloom D D , Pisetsky D S , Clarke S H
Published: December 2022

Immunization of normal mice with bacterial DNA induces a significant anti-DNA response that includes antibodies resembling some lupus anti-DNA in their binding properties, although lacking specificity for mammalian dsDNA. To determine the structure of these induced antibodies and their relationship to anti-DNA from lupus mice, we have characterized the clonality and selected V-region sequences of a panel of 20 anti-DNA antibodies from 3 BALB/c mice immunized with ssDNA from Escherichia coli. Southern blot analysis of H and L chain rearrangements indicated that two of the animals expressed pairs of clonally related antibodies. Amino acid sequences of 10 of the induced antibodies demonstrated predominant utilization of J558 family VH genes and JH4 in association with various DH, J kappa and V kappa genes. Among the VH CDR3 of these 10 antibodies, 4 displayed arginine residues as a result of N region additions. None of these antibodies, however, had more than one arginine residue in VH CDR3 nor arginines at positions 100 or 100a, characteristic features of lupus antibodies to dsDNA. These results suggest that normal mice immunized with bacterial DNA display certain facets of DNA Ag drive, although lacking the mechanisms for the production of antibodies to mammalian dsDNA.
Frequency of B cells expressing germ-line gamma 1 transcripts upon IL-4 induction.
Authors: Turaga P S , Berton M T , Teale J M
Published: December 2022

Murine B cells when activated with LPS and IL-4 have been shown to secrete IgG1 and IgE with corresponding H chain gene rearrangements. Using this system it has been previously demonstrated that transcription of gamma 1 and epsilon C region genes in their germ-line configuration occurs before switch recombination. Here, for the first time, the frequencies of B cells expressing germ-line gamma 1 transcripts are analyzed using in situ hybridization methodology. The results indicate that the combination of LPS and IL-4 induces a relatively low frequency of cells (5-12%) to express germ-line gamma 1 transcripts. Germ-line gamma 1 transcript-expressing cells were first detected on day 1 of culture with LPS and IL-4 and reached a maximum by days 3 to 5. The B cells that expressed germ-line gamma 1 transcripts appeared to be activated based on size and morphology. The frequency of B cells expressing germ-line gamma 1 transcripts approximated the frequency of C gamma 1+ plasma cells that appeared later in the cultures. These data may suggest a correlation between germ-line Ig transcript expression and the frequency of B cell precursors committed to the expression of a particular isotype.
Localization of an immunologically functional region of the streptococcal superantigen pepsin-extracted fragment of type 5 M protein.
Authors: Wang B , Schlievert P M , Gaber A O , Kotb M
Published: December 2022

Ten overlapping synthetic peptides, spanning the entire amino acid sequence of pepsin-extracted type 5 M protein, (pep M5), were used to delineate regions within this molecule that are important for the mitogenic activity of this streptococcal superantigen. Two of the overlapping peptides, SM5-8 and SM5-10, blocked the response of T cells to pep M5 in a dose-dependent manner. Neither peptide was toxic to T cells, but SM5-8 exhibited nonspecific inhibition of the T-cell response to either superantigen or polyclonal mitogens. In contrast, the inhibition by SM5-10 was pep M5-specific. Analysis of TCR V beta gene usage of the pep M5 response in the presence and absence of SM5-10 revealed that this peptide preferentially blocks the expansion of pep M5-specific V beta elements. The degree of inhibition of V beta expansion varied from one individual to the other, reflecting their TCR repertoire. SM5-10 did not significantly inhibit the response of T cells to other streptococcal superantigens. The data suggest that the region represented by SM5-10 of the pep M5 is important for superantigenic activity. In addition, the ability of this synthetic peptide to inhibit the response to pep M5 provides evidence that the mitogenic response is indeed mediated by this superantigen and is not a result of contamination with other streptococcal superantigens.
T cells involved in human autoimmune disease are resistant to tolerance induction.
Authors: Dayan C M , Chu N R , Londei M , Rapoport B , Feldmann M
Published: December 2022

Deletion of potentially self-reactive T-cell clones during intrathymic development provides an important mechanism of preventing autoreactivity. However, some potentially damaging cells may escape this process. Recent evidence suggests that these cells may be rendered 'anergic', that is, nonresponsive to Ag, in the absence of cell death. Such a mechanism may be particularly important in maintaining tolerance to organ-specific self Ag that are not expressed in the thymus. If so, the emergence of T cells resistant to anergy induction might be expected to result in autoimmune disease. It has previously been shown that anergy can be induced in human T cells in vitro by exposure to specific target peptide or bacterial enterotoxins in the absence of Ag-presenting cells. We have recently defined the antigenic specificity of multiple T-cell clones present at the site of a human organ-specific autoimmune disease, Graves' thyroiditis (Graves disease). In the current work, thyroid-derived T cells recognizing residues 535-551 of the thyroid tissue-specific enzyme, TPO3 have been used to examine whether cells actively involved in the autoimmune process are resistant to anergy induction, as defined by anergy induction with in vitro systems. Two systems were used. First, supraimmunogenic concentrations of peptide 535-551 (up to 1 mg/ml) failed to significantly anergize these T cells in the absence of APC. In addition, the bacterial enterotoxin SED that could stimulate these T cells in the presence of APC, failed to induce anergy when APC were not present. T cells from the peripheral blood of the same individual, in contrast, were anergizable with bacterial enterotoxins, using the same protocol. These results suggest that thyroid-infiltrating autoantigen-reactive T cells are refractory to induction of anergy, and the possibility is raised that this deficiency may be of importance in the development of autoimmunity.
Intravenous endotoxin suppresses the cytokine response of peripheral blood mononuclear cells of healthy humans.
Authors: Granowitz E V , Porat R , Mier J W , Orencole S F , Kaplanski G , Lynch E A , Ye K , Vannier E , Wolff S M , Dinarello C A
Published: December 2022

When administered parenterally, endotoxin stimulates the synthesis of IL-1, TNF-alpha, and IL-6. However, this initial injection induces tolerance; a second injection of endotoxin results in lower levels of circulating cytokines. In our study, five healthy male volunteers between the ages of 18 and 30 were injected with Escherichia coli endotoxin. Four subjects received only saline. Immediately before the injection and 3, 6, and 24 h afterward, PBMC were isolated and stimulated in vitro with endotoxin, IL-1, or toxic shock syndrome toxin-1. Inasmuch as CD14+ monocytes are the primary source of the cytokines induced by these stimuli, results are expressed as cytokine production per 10(6) CD14+ cells. Six h after endotoxin injection, endotoxin-stimulated CD14+ cells synthesized 66% less IL-1 beta (p < 0.01), 47% less TNF-alpha (p < 0.001), 56% less IL-6 (p < 0.01), and 49% less IL-8 (p < 0.01) than cells obtained before the injection. This suppression was not specific for endotoxin; IL-1 beta-induced IL-1 alpha and TNF-alpha were reduced by 84% (p = 0.01) and 68% (p < 0.001), respectively. A decrease in cytokine synthesis was also observed using toxic shock syndrome toxin-1 as a stimulus: 57% for IL-1 beta (p = 0.06), 70% for TNF-alpha (p < 0.01), 56% for IL-6 (p < 0.05), and 71% for IL-8 (p = 0.001). When data were expressed as cytokine production per 10(6) PBMC, cells isolated 3 h after endotoxin injection synthesized significantly less stimulus-induced IL-1, TNF-alpha, IL-6, and IL-8 than did PBMC from saline-injected controls. We conclude that endotoxin tolerance is due, in part, to changes in the stimulus-induced cytokine response of circulating CD14+ cells.
Induction of HIVMN neutralizing antibodies in primates using a prime-boost regimen of hybrid synthetic gp120 envelope peptides.
Authors: Haynes B F , Torres J V , Langlois A J , Bolognesi D P , Gardner M B , Palker T J , Scearce R M , Jones D M , Moody M A , McDanal C
Published: December 2022

We have tested synthetic peptides composed of Th (T1) and V3 loop B cell neutralizing determinants [SP10 MN(A)] of HIVMN gp120 and the fusogenic (F) domain of gp41 as immunogens in rhesus monkeys. After two immunizations with either HIV env peptide T1-SP10 MN(A) or F-T1-SP10 MN(A), rhesus monkey serum neutralization titers against the HIVMN isolate ranged from 1:160 to 1:1400, and in cell-cell syncytium inhibition assay ranged from 1:20 to 1:80. However, in contrast to animals immunized with T1-SP10 MN(A), animals immunized twice with F-T1-SP10 MN(A) had no rise in anti-gp120 and neutralizing antibodies with an additional immunization with F-T1-SP10 MN(A) peptide. One of 4 rhesus monkeys (18987) had anti-HIVMN antibodies that cross-neutralized divergent HIV isolates HIVIIIB and HIVRF. Serum from animal 18987 neutralized 5 of 10 HIV isolates tested, and neutralizing activity against HIVIIIB of 18987 serum was absorbed with the conserved gp120 loop V3 sequence IGPGRAF. Anti-HIV neutralizing antibodies were boosted after a 6-mo rest by 500 micrograms of T1-SP10 MN(A) in 4 of 4 animals previously immunized with T1-SP10 MN(A) and in 2 of 2 animals previously immunized with F-T1-SP10 MN(A). However, immunization after 6-mo rest of animal 18987 with 500 micrograms of T1-SP10 MN(A) peptide, although boosting anti-HIVMN neutralizing antibodies, selectively did not boost cross-neutralizing anti-HIVIIIB antibodies. Thus, synthetic peptides containing T and B cell epitopes of HIV gp120 can induce high levels of anti-HIVMN neutralizing antibodies in primates.
Cellular localization of CD4 in the human placenta. Implications for maternal-to-fetal transmission of HIV.
Authors: Lairmore M D , Cuthbert P S , Utley L L , Morgan C J , Dezzutti C S , Anderson C L , Sedmak D D
Published: December 2022

CD4 is a 55-kDa glycoprotein that serves as an important cellular differentiation Ag and cell signaling protein on T lymphocytes, as well as a principal receptor for HIV-1 on a variety of cell types including lymphocytes. CD4 receptor expression in syncytiotrophoblasts, the principal cellular barrier in the human placenta, has not been clearly defined. Knowledge concerning the expression of the CD4 receptor on placental trophoblasts is important to define potential mechanisms of transmission of the virus between maternal blood and fetal tissues. Both mature and immature placenta (n = 10) were examined using an avidin D-based immunohistochemical procedure that permits clear morphologic distinction of cell types in placental sections. Syncytiotrophoblasts were defined using anti-cytokeratin mAb (AE1/3), whereas endothelial cells in placental villi were distinctly identified using a mAb directed to CD31. Placental Hofbauer cells (macrophages) and other leukocytes were identified by mAb staining of leukocyte common Ag (CD45). CD4 expression (identified by staining with three separate anti-CD4 mAb) was exclusively localized using this immunohistochemical method to leukocytes in placental villi (e.g., Hofbauer cells); however, no CD4 staining was evident in syncytiotrophoblasts, cytotrophoblasts, or villus endothelial cells. Furthermore, immunoaffinity-purified trophoblasts were negative for CD4 receptor expression. CD4 RNA was not identified in purified trophoblasts using both Northern blot assay and a sensitive polymerase chain reaction method to identify CD4 RNA. In addition, time course studies of purified trophoblasts immediately after purification and at 24, 48, and 72 h in culture indicated that CD4 RNA was not present as a transient, but labile transcript in trophoblasts. These data indicate that the transmission of HIV-1 across syncytiotrophoblasts may occur by mechanisms other than by binding the CD4 receptor and that tissue leukocytes (in particular Hofbauer cells) are likely the principal CD4+ cellular target of HIV-1 in the placenta.