bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-5 induces a Pgp-1 (CD44) bright B cell subpopulation that is highly enriched in proliferative and Ig secretory activity and binds to hyaluronate.
Authors: Murakami S , Miyake K , June C H , Kincade P W , Hodes R J
Published: December 2022

Pgp-1 expression was examined in unstimulated B cell populations and in B cells activated with several polyclonal stimuli. Flow cytometry analysis demonstrated that Pgp-1 expression increased when B cells were activated with supernatant of cloned Th2 cells, with LPS, or with IL-5, stimuli that induced polyclonal proliferation and differentiation. IL-5-primed B cells were phenotypically unique and could be divided into two distinct subpopulations based on the brightness of Pgp-1 expression. Furthermore, sterile sorting experiments showed that proliferating and differentiating B cells were highly enriched in a Pgp-1-bright, Ia-dull, B220-dull subpopulation. The possibility that Pgp-1 expressed on activated B cells functions as an adhesion molecule was evaluated by assessing adhesion of activated B cells to defined substrates. It was found that IL-5-activated B cells bound strongly to hyaluronate-coated surface, and this binding was specifically inhibited by anti-Pgp-1 Ab. These findings suggest that Pgp-1 expression is a useful marker which, under defined conditions, identifies the proliferating and differentiating subset of activated B cells. Moreover, the Pgp-1 bright subset of IL-5-primed B cells binds to hyaluronate in a Pgp-1-dependent manner that suggests a potential role of Pgp-1 in the in vivo adherence and trafficking of activated B cells.
Rapid loss of IgM expression by normal murine B cells undergoing IgG1 and IgE class switching after in vivo immunization.
Authors: Snapper C M , Finkelman F D
Published: December 2022

Injection of mice with polyclonal goat anti-mouse IgD antibody (G alpha M delta) stimulates a potent T cell-dependent immune response characterized by large increases in serum IgG1 and IgE concentrations and by the generation of substantial numbers of membrane (m)IgG1+ B cells. The onset of this response occurs 6 days after G alpha M delta injection and peaks by day 7 to 8. Utilizing two color fluorescence analysis and cell sorting we demonstrate that most mIgG1-expressing B cells lack mIgM during the period of onset of Ig isotype switching (day 6). Both IgG1 and IgE are produced predominantly by mIgM- cells. On day 6, IgG1 and IgE are secreted predominantly by cells expressing mIgG1 and mIgE, respectively. By day 8, a majority of the IgG1 secretion occurs among the mIgG1- cells but virtually all IgE secretion continues to come from the mIgE+ population. B cells that strongly express mIgG1 secrete little IgM or IgE. Freshly harvested B cells expressing mIgG1, 6 days after G alpha M delta injection, have undergone substantial deletion of CH mu-specific DNA in contrast to their mIgG1- counterparts. Hence, the great majority of B cells that switch to the IgG1 or IgE isotypes in vivo rapidly lose their expression of IgM.
Mapping the site of interaction between murine IgE and its high affinity receptor with chimeric Ig.
Authors: Weetall M , Shopes B , Holowka D , Baird B
Published: December 2022

We have investigated the interaction of mouse (m) IgE with its Fc epsilon RI on rat basophilic leukemia cells using a set of chimeric Ig that were constructed by exchanging homologous H chain C domains between human (hu) IgG1 and mIgE. Binding affinities were examined with equilibrium and kinetic measurements, and we found that epsilon/C gamma 3 (mIgE with C epsilon 4 replaced by C gamma 3) was indistinguishable from mIgE. The huIgG1 and the other chimeric Ig, which did not contain both C epsilon 2 and C epsilon 3, did not bind detectably to rat basophilic leukemia cells (Ka less than 10(6) M-1). The ability of these chimeric Ig to stimulate a cellular response (degranulation) in the presence of multivalent Ag was also tested. The epsilon/C gamma 3 was indistinguishable from mIgE in eliciting a high level of degranulation, whereas the other chimeric Ig stimulated no response even when they were preaggregated to enhance their binding avidity. These results demonstrate that C epsilon 4 may be replaced by C gamma 3 without affecting the binding and cell activating properties of mIgE. The lack of binding by the other chimeric Ig indicates that both C epsilon 2 and C epsilon 3 are necessary for the binding interaction.
Recognition of a B cell lymphoma by anti-idiotypic T cells.
Authors: Wilson A , George A J , King C A , Stevenson F K
Published: December 2022

Idiotypic IgM derived from a B cell lymphoma can act as a tumor-associated Ag, in that immunization with this purified protein generates an anti-idiotypic immune response that specifically suppresses tumor development. Spleens of immune mice contain T cells that proliferate in response to idiotypic IgM. However, idiotypic Ag is presented to the T cells most efficiently in its natural form at the surface of the lymphoma cells, than as soluble IgM plus presenting cells. Variant tumors that display either little or no idiotypic IgM at the cell surface, but which are otherwise indistinguishable from parental tumor, induce a weak response or fail to stimulate the T cells, respectively. Anti-idiotypic lines and clones have been derived from the splenic T cells by growth in the presence of irradiated tumor cells. Phenotypic analysis revealed that cells from both lines and clones express CD3 and CD4 Ag, but not CD8. Recognition of tumor Id, which required no added presenting cells, was inhibited by antibody against MHC class II Ag, and variably by anti-CD4. Proliferative responses were inhibited by anti-idiotypic antibodies, but also by antibodies against the constant region of the mu H chain, indicating that perturbation of the surface IgM abrogates availability of idiotypic determinants to the T cells.
Cell surface modulation of CD26 by anti-1F7 monoclonal antibody. Analysis of surface expression and human T cell activation.
Authors: Dang N H , Torimoto Y , Sugita K , Daley J F , Schow P , Prado C , Schlossman S F , Morimoto C
Published: December 2022

In this paper, we examined in detail the ability of anti-1F7 to modulate 1F7 (CD26) surface expression as well as analyzed the functional relationship between the surface expression of CD3, CD2, and CD26 and human T cell activation. We showed that anti-1F7-induced modulation is an energy-dependent process that occurs via capping and internalization of the Ag-antibody complex. Although the recovery rate for Ag reexpression of 1F7 following optimal modulation is relatively delayed, reexpression of 1F7 is greatly accelerated following phorbol ester treatment. Most importantly, we demonstrated that modulation of the CD26 Ag leads to an enhancement in the proliferative activity of modulated human T cells treated with anti-CD3 or anti-CD2, which is preceded by an enhancement in Ca2+ mobilization. CD26 modulation also led to an increase in anti-CD3- or anti-CD2-mediated T cell clone proliferation. Finally, whereas modulation of the CD26 Ag has an effect on CD3- or CD2-induced T cell activation, modulation of the CD3/TCR complex inhibits the proliferative response of T cells incubated with anti-CD3 plus anti-1F7 or anti-CD2 plus anti-1F7. However, modulation of the CD2 structure does not affect anti-CD3- plus anti-1F7-induced human T cell activation. The above results thus provide additional evidence that the CD26 Ag plays an integral role in the regulation of human T cell activation.
Preferential proliferation of T cell receptor V gamma 3-positive cells in IL-2-stimulated fetal thymocytes.
Authors: Leclercq G , De Smedt M , Tison B , Plum J
Published: December 2022

Thymocyte cell suspensions, prepared from mice at different ages, were cultured in vitro with human rIL-2. This stimulation resulted in a cell population that contained almost 50% TCR-gamma delta-positive cells if thymocytes were taken from fetal day 17 until just after birth. Analysis of the variable (V gamma) region used by the TCR-gamma delta cells revealed that 90% of them expressed TCR-V gamma 3, and less than 5% expressed TCR-V gamma 2. Cells positive for TCR-alpha beta were barely detectable. If fetal day 18 organ cultured thymus lobes, instead of a cell suspension, were stimulated with IL-2, no rise in the number of TCR-V gamma 3+ or TCR-delta+ cells was observed, whereas a partial outgrowth of TCR-alpha beta+ cells occurred. From day 1 after birth, the number of TCR-gamma delta cells recovered from an IL-2-stimulated thymocyte cell suspension dropped to reach a plateau of 15% of the total cell number, whereas TCR-V gamma 3+ cells became undetectable in older animals. TCR-alpha beta+ cells, on the other hand, quickly rose in cell number after birth. Kinetic analysis showed that the preferential outgrowth of TCR-V gamma 3+ cells in IL-2-stimulated fetal day 18 thymocyte cell suspensions was present from the onset of the culture; a significant proliferation of CD4 or CD8 single positive TCR-alpha beta cells was never observed. This lack of proliferation of TCR-alpha beta cells was not due to inhibition by the activated TCR-V gamma 3+ cells. Throughout the IL-2 culture, one-fourth of the TCR-V gamma 3+ thymocytes was positive for CD8. Analysis of the DNA content and the IL-2 receptor (IL-2R) p55 expression showed that during the first days of culture the TCR-V gamma 3+ cells had a much higher proliferation rate than the TCR-V gamma 3- cells, although TCR-V gamma 3+ IL2R p55+ cells could not be detected. From day 3 to 4 of culture, the proliferation rate of TCR-V gamma 3+ cells equaled that of the rest of the cells and less than 20% of the TCR-V gamma 3+ cells expressed the IL-2R p55. The biologic significance of our findings is discussed.
Chemoprotective effects of recombinant human IL-1 alpha in cyclophosphamide-treated normal and tumor-bearing mice. Protection from acute toxicity, hematologic effects, development of late mortality, and enhanced therapeutic efficacy.
Authors: Futami H , Jansen R , MacPhee M J , Keller J , McCormick K , Longo D L , Oppenheim J J , Ruscetti F W , Wiltrout R H
Published: December 2022

In this study, recombinant human IL-1 alpha (rhIL-1 alpha) was used to protect normal and tumor-bearing BALB/c mice from the acute toxicity caused by lethal doses of cyclophosphamide (Cy) and 5-fluorouracil. Pretreatment of mice for 7 days with 10,000 U/day of rhIL-1 alpha protected 70 to 100% of mice from the acute death induced by lethal doses of both Cy (380 mg/kg) and 5-fluorouracil (250 mg/kg). In contrast, post-treatment of mice with single or multiple doses of rhIL-1 alpha was not chemoprotective. Pretreatment of mice with rhIL-1 alpha increased the acute LD90 of Cy from 380 mg/kg to greater than 500 mg/kg in normal mice, an LD90 dose-modifying effect of at least 1.25, was accompanied by a more rapid recovery from neutropenia and a less severe reduction in the number of bone marrow single lineage monocyte, myeloid, or myelomonocytic colonies. Some of the mice (10 to 50%) that were successfully protected by pretreatment with rhIL-1 alpha died after day 50. These mice consistently presented with extensive pulmonary inflammation and fibrosis at death. Mice bearing murine renal cancer (Renca) were also protected from the acute toxic effects of Cy (450 mg/kg) by pretreatment with rhIL-1 alpha. Renca-bearing mice pretreated with rhIL-1 alpha and either sublethal (300 mg/kg) or lethal (450 mg/kg) doses of Cy exhibited enhanced survival times over those of untreated Renca-bearing mice. Interestingly, the cause of death in Renca-bearing mice that ultimately failed treatment with rhIL-1 alpha plus 300 mg/kg Cy was recurrent tumor, whereas most mice treated with rhIL-1 alpha plus 450 mg/kg Cy had no detectable tumor, although several died from late pulmonary inflammation and fibrosis. Thus, the dose escalation of Cy in rhIL-1 alpha-pretreated mice results in greater antitumor effects of Cy. However, the dose escalation of some cytotoxic agents allowed by the use of myelostimulatory agents can result in late fatal complications not detected in acute toxicity testing.
IL-10, a novel growth cofactor for mature and immature T cells.
Authors: MacNeil I A , Suda T , Moore K W , Mosmann T R , Zlotnik A
Published: December 2022

We identified a new cytokine, B cell-derived T cell growth factor (B-TCGF), that is produced by a murine B cell lymphoma and induces proliferation of mature and immature thymocytes in the presence of IL-2 and IL-4. Both adult and day 15 fetal thymocytes (CD4-8-, CD4+8-, CD4-8+) proliferate strongly in the presence of IL-2, IL-4, and B-TCGF. B-TCGF alone does not stimulate thymocyte proliferation. B-TCGF appears to be identical to a novel cytokine whose cDNA was recently isolated at our institution, cytokine synthesis-inhibitory factor (CSIF; IL-10). rIL-10 has B-TCGF activity, and mAb specific for IL-10 inhibit the B-TCGF activity present in CH12 supernatants. Further studies have shown that day 15 fetal thymocytes cultured in the presence of IL-10, IL-2, and IL-4 remain CD4- and CD8- but exhibit increased CD3 expression. Adult CD4- CD8- thymocytes cultured under the same conditions proliferate whether they are CD3+ or CD3-. The CD3- population becomes enriched in CD3+ cells after 4 days of culture. IL-10 is secreted by day 15 fetal thymocytes, adult thymocytes, and adult splenocytes when stimulated via their TCR. IL-10 is strongly homologous to the EBV gene BCRFI, and BCRFI has CSIF activity. In contrast to IL-10, BCRFI does not exhibit detectable thymocyte-stimulating activity, suggesting the existence of at least two functional epitopes on the IL-10 molecule.
Evaluation of the mechanisms responsible for the reduction in erythrocyte complement receptors when immune complexes form in vivo in primates.
Authors: Cosio F G , Shen X P , Birmingham D J , Van Aman M , Hebert L A
Published: December 2022

Patients with immune complex-(IC) mediated diseases frequently have low levels of CR1 on E. The present study was undertaken to determine the role of circulating IC in causing low E-CR1 levels. E-CR1 were enumerated by measuring the binding of anti-CR1 mAb (E11) and rabbit anti-CR1 antibodies (RbaCR1) to E. In addition, the distribution of CR1 among E was assessed by flow cytometry of E stained with E11 and RbaCR1 and by evaluating the binding of E11-coated fluorescent beads (E11-beads) to E. E11-beads bind to clusters of CR1 on E. Five cynomolgus monkeys (CYN) were preimmunized to bovine gamma-globulin (BGG). E-CR1 changes in these animals were assessed: 1) acutely, during the first 60 min after an infusion of BGG and 2) chronically, during daily administration of BGG infusions over 2 wk. Acutely, there was a decrease in the number of E-CR1 as measured by E11 binding to E (E11/CR1). This decrease was not attributable to occupancy of CR1 by IC because the decrease in E11/CR1 number persisted after the IC had been cleared from E. By comparing the E11/CR1 levels in arterial blood to hepatic vein blood (n = 5), or in pulmonary artery blood (n = 1), we determined that the acute decrease in E11/CR1 number did not occur whereas E circulated through liver, spleen, or lung. The decrease in E11/CR1 number required the binding of IC to E because it did not occur after BGG was infused into nonimmunized CYN (n = 2) or into a preimmunized complement-depleted CYN. The decrease in E11/CR1 number was not due to loss of CR1 from E because E11/CR1 number recovered 24 h after infusion of BGG and in addition, enumeration of E-CR1 with RbaCR1 and E11-beads did not reflect a decrease in E-CR1 number. After several daily BGG infusions there was a persistent decrease in E-CR1 levels and that decrease appeared to be mainly the result of loss of CR1 from E because the decrease was confirmed with all methods of E-CR1 measurement and because E-CR1 levels recovered only slowly after BGG infusions were discontinued. Both in vitro and in vivo IC bound preferentially to subpopulations of E, identified by their ability to bind multiple E11-beads and by their high intensity staining with the anti-CR1 antibodies E11 and RbaCR1.(ABSTRACT TRUNCATED AT 400 WORDS)
Human and murine anti-DNA antibodies induce the production of anti-idiotypic antibodies with autoantigen-binding properties (epibodies) through immune-network interactions.
Authors: Puccetti A , Migliorini P , Sabbaga J , Madaio M P
Published: December 2022

To examine the potential role of immune-network interactions in the production of lupus autoantibodies, normal NZW rabbit antibody responses were analyzed after immunization with one of the following Ig preparations: human lupus serum anti-dsDNA antibodies, human lupus serum anti-ssDNA antibodies, a mixture of human lupus serum anti-dsDNA and anti-ssDNA antibodies, the MRL-lpr/lpr anti-dsDNA mAb H241, and the MRL-lpr/lpr anti-ssDNA mAb H130. Four of five rabbits produced Ig typical of lupus autoantibodies: individual rabbit Ig cross-reacted with multiple autoantigens including nucleic acids, cardiolipin, SmRNP, glomerular extract, laminin, and exogenous Ag. Rabbit anti-Id against human anti-dsDNA antibodies were highly specific for dsDNA. Notably, in each serum the autoantibody activity was confined to the anti-Id Ig fraction. A similar spontaneously occurring Id-anti-Id interaction was also found between anti-ssDNA and anti-dsDNA antibodies isolated from an individual lupus patient. These results indicate that lupus autoantibodies which share Ag binding properties with pathogenic Ig, including both cross-reactive and anti-dsDNA antibodies, can induce the production of Ig with similar autoantigen binding properties through immune-network interactions. This phenomenon, if unregulated, could lead to the amplification of pathogenic autoantibody production in individuals with systemic lupus.
The heavy metal lead exhibits B cell-stimulatory factor activity by enhancing B cell Ia expression and differentiation.
Authors: McCabe M J , Lawrence D A
Published: December 2022

Lead (Pb) has been shown to enhance B cell differentiation and to induce T cell proliferation in vitro. Our work demonstrates a direct enhancing effect of Pb on IgM production by T cell depleted-, LPS-activated B cells. Pb also caused a significant, dose-dependent increase in the B cell surface density of both products from the I-region of the murine MHC, I-A and I-E, that was comparable to and within the same time frame as other conventional B cell activators. Pb did not alter B cell surface density of MHC class-I molecules, but it did modulate the levels of other relevant activation Ag such as sIgD and Fc epsilon R. The modulation of the B cell phenotype caused by Pb was not due to release of IL-4 from residual T cells or endotoxin contamination of any culture reagents. Hg also increased cell surface MHC class II molecule density; however, neither Ni, Zn, nor Cd modulated Ia, indicating that immunomodulatory metals have different mechanisms of action. These results suggest that the mechanism of Pb immunopotentiation might be due to increased B-Th cell collaboration and that Pb can act as a B cell stimulatory factor. In addition to the obvious environmental implications of these results, determining Pbs mechanism of action on B cells may yield important information relevant to B cell activation in general.
Murine cytomegalovirus infection can enhance hybrid resistance through modulation of host natural killer activity.
Authors: Muluk S C , Hakim F T , Shearer G M
Published: December 2022

Studies were undertaken to assess the effect of murine cytomegalovirus (MCMV) in two different models involving injection of parental cells into F1 hosts. In both of these systems, MCMV-induced enhancement of hybrid resistance was found. In the first model, parent-into-F1 graft-vs-host reaction, MCMV infection of (C57BL/6 x C3H)F1 (B6C3F1) hosts was found to prevent the GVHR normally induced by injection of B6 parental splenocytes into the F1 hosts. The second model involved injection of parental bone marrow into lethally irradiated B6C3F1 and (C57BL/6 x DBA/2)F1 (B6D2F1) hosts. These irradiated hosts are known to exhibit resistance to engraftment by parental C57BL/6 (B6) bone marrow. This resistance was found to be markedly enhanced by injection of the hosts with MCMV 3 days before irradiation and bone marrow injection. In contrast, engraftment into B6C3F1 hosts of syngeneic marrow, or bone marrow from the C3H parent, was not affected by MCMV infection. Engraftment of DBA/2 marrow into B6D2F1 hosts was reduced at lower doses of injected marrow, suggesting enhanced resistance against the minor Hh Ag Hh-DBA. To test whether the MCMV-induced enhancement of resistance was mediated by NK cells, splenic NK activity (YAC-1 killing) and frequency (NK1.1 staining) were assessed. Both parameters were found to be elevated at 3 days after MCMV infection but to return to normal levels by 9 days. B6 bone marrow engraftment was in fact found to be normal when the marrow was administered to F1 mice 9 days after MCMV infection. Furthermore, anti-asialoGM1 administration prevented MCMV-induced enhancement of resistance to marrow engraftment. Thus, the NK enhancement resulting from MCMV infection appears to play a major role in the enhanced HR observed in the marrow engraftment model. This effect may be of importance in clinical bone marrow transplantation, a situation in which patients are susceptible to viral infection.
Combined effects of tumor necrosis factor-alpha, prostaglandin E2, and corticosterone on induced Ia expression on murine macrophages.
Authors: Zimmer T , Jones P P
Published: December 2022

Ia expression is an important marker of macrophage functional capacity. IFN-gamma induces Ia expression on perhaps all murine macrophages, whereas IL-4, granulocyte-macrophage CSF, and CSF-1 induce Ia on restricted sets of macrophages. Inhibitors of expression include PGE2, glucocorticoids, and IFN-beta. TNF has been found to augment Ia expression on several macrophage lineage cell lines but to inhibit expression on murine peritoneal macrophages. Our study shows that TNF can have opposite effects on Ia expression (induced by IFN-gamma) on thioglycollate-elicited peritoneal macrophages, depending on the length of time cells are treated and on the presence of other modulators. In particular, TNF augmented early expression induced by IFN-gamma but inhibited later expression. And although TNF synergized with PGE2 to markedly inhibit Ia induction on these cells, it partially antagonized the inhibition by corticosterone and IFN-beta. TNF and PGE2 also synergized to inhibit Ia expression induced on bone marrow-derived and splenic macrophages by either IFN-gamma or IL-4. In contrast to their effect on Ia expression, TNF and PGE2 had opposite effects on expression of gamma 2a FcR in macrophages. TNF blocked the increase in FcR expression due to any combination of PGE2, IFN-gamma, and IFN-beta. However, TNF and PGE2 both increased expression of gamma 2a FcR on WEHI-3 cells. If the different effects of TNF reflect the differentiation states of macrophages, its effects on Ia and FcR expression may vary with the progression of an immune response.
The in vivo elimination of CD4+ T cells prevents the induction but not the expression of carrier-induced epitopic suppression.
Authors: Leclerc C , Schutze M P , Deriaud E , Przewlocki G
Published: December 2022

Injection of mice with an immunogenic dose of carrier (keyhole limpet hemocyanin (KLH)) followed by immunization with hapten-carrier conjugate (TNP-KLH) selectively suppresses anti-hapten antibody response. In this study, the cellular basis of this epitopic suppression and also of the suppression induced by a high dose of carrier were analyzed by in vivo depletion of CD4+ or CD8+ T cell subsets by using mAb. The mAb treatments were performed either at the time of carrier priming or at the time of hapten-carrier immunization. The elimination of CD8+ T cells has not modified the anti-carrier antibody response, whether this treatment was performed at the time of KLH-priming or during TNP-KLH immunization. Moreover, the in vivo treatment with the anti-CD8 mAb did not modify the carrier-induced epitopic suppression induced either by a low immunogenic dose of KLH or by a high dose of this Ag. The elimination of CD4+ T cells at the time of KLH immunization has prevented the induction of a memory response to KLH, clearly establishing that CD4+ T cells are essential in memory B cell development to T-dependent Ag. Moreover, this treatment has totally abrogated the epitopic suppression induced either by low or high dosages of KLH. In contrast, the in vivo elimination of CD4+ T cells after carrier immunization did not abolish the secondary anti-carrier antibody response and did not prevent the expression of epitopic suppression. These data indicate that primed CD4+ T cells are required neither for memory B cell expression nor for the expression of suppression. Finally, once induced, the suppression can be evidenced after in vivo depletion of both primed CD4+ and CD8+ T cells. These data support the view that epitopic suppression is induced through the expansion of carrier-specific B cells and resulted from intramolecular antigenic competition between hapten and carrier epitopes.
Protein tyrosine phosphorylation and p56lck modification in IL-2 or phorbol ester-activated human natural killer cells.
Authors: Einspahr K J , Abraham R T , Dick C J , Leibson P J
Published: December 2022

Protein tyrosine kinases play fundamental roles in the transduction of signals that regulate cell growth, differentiation, and functional responses to a diversity of external stimuli. It is therefore likely that understanding protein tyrosine kinase activity in NK cells will be crucial in further defining the intracellular regulation of their unique and specialized functions. We investigated the role of protein tyrosine phosphorylation in receptor-mediated signal transduction using stimuli known to play major roles in regulating NK cell activation. Immunoblot analyses with antiphosphotyrosine antibodies demonstrated that IL-2, a potent stimulus for NK cell proliferation and an agent that enhances NK cytotoxic function, induced the tyrosine phosphorylation of at least eight proteins in clonal CD16+/CD3-human NK cells. In contrast, IL-4, which modulates NK cell function without inducing proliferation, had no apparent effect on protein tyrosine phosphorylation. Because protein kinase C (PKC) activation plays a prominent, yet distinct role in NK cell-mediated cytolytic reactions, we next investigated whether PKC activation affects NK cell protein tyrosine phosphorylation. Surprisingly, PKC-activating agents, including the phorbol esters 12-O-tetradecanoylphorbol-13-acetate and 4 beta-phorbol 12, 13-didecanoate, as well as the synthetic diacylglycerol,1-oleoyl-2-acetylglycerol, also induced the tyrosine phosphorylation of a distinct set of proteins. The 4 beta-phorbol 12,13-didecanoate homolog, 4 alpha-phorbol 12,13-didecanoate, which does not activate PKC, also failed to induce protein tyrosine phosphorylation. Further, the PKC inhibitor, 1-O-hexadecyl-2-O-methylglycerol blocked tyrosine phosphorylation induced by 1-oleoyl-2-acetylglycerol. In subsequent studies, both CD8+ and CD8- NK clones were found to express the src-family tyrosine kinase, p56lck, which was detected by immunoblot analysis with anti-p56lck antiserum. In both types of clonal NK cell lines, IL-2 and 12-O-tetradecanoyl-phorbol appeared to stimulate the differential phosphorylation of p56lck as evidenced by the appearance of higher molecular mass isoforms on SDS-polyacrylamide gels. Thus, our results identify and characterize a potential role for tyrosine phosphorylation and for the lymphocyte-specific tyrosine kinase p56lck in the signaling events that regulate NK cell activation.
Identification of a new V kappa gene family that is highly expressed in hybridomas from an autoimmune mouse strain.
Authors: Shefner R , Mayer R , Kaushik A , D'Eustachio P , Bona C , Diamond B
Published: December 2022

We have identified a new murine V kappa family that contains five to seven members, one member of which encodes the L chain V region of an anti-dsDNA antibody produced by a BALB/c hybridoma, C8.5. The cloned C8.5 V kappa gene exhibits highest homology with a human V kappa gene that was cloned from a nonproductive rearrangement but has never been seen in an expressed repertoire. Because this family was first identified in an autoantibody, we studied its expression in an autoimmune mouse strain. This V kappa family is expressed in 20% of hybridomas from NZB mice.
The importance of mast cells for the neutrophil influx in immune complex-induced peritonitis in mice.
Authors: Ramos B F , Qureshi R , Olsen K M , Jakschik B A
Published: December 2022

The role of mast cells in polymorphonuclear leukocyte (PMN) influx in Ag-antibody complex-induced peritonitis was evaluated in mast cell-deficient WBB6F1-W/Wv (W/Wv) mice and their normal littermates, WBB6F1-+/+ (+/+). Peritoneal cell influx was evaluated after i.p. injection of preformed immune complexes. The first significant elevation in the PMN count over PBS-treated controls in +/+ mice was observed 2 h after stimulation. During the period of maximum leukocyte concentrations (6 to 10 h), the increase in total cell count was 5-fold and in PMN 25-fold. In W/Wv mice the PMN influx started 2 h later than in the +/+ mice, and the maximum response (8 to 10 h) was only 50% of that in controls. Reconstitution of mast cells in W/Wv mice for 2 wk or more restored the PMN response to immune complexes. Mast cell release due to AG-antibody complexes was evaluated by measuring fluorescence intensity after berberine sulfate staining for heparin in mast cells from unstimulated as well as stimulated +/+ mice. There was a significant decrease in fluorescence intensity as early as 15 min after stimulation. By 30 min the fluorescence intensity had declined by 65%. This indicates extensive mast cell release that started before PMN mobilization. These experiments demonstrate that mast cells make a significant contribution to immune complex-induced inflammation.
The allorecognition of H-2Kb-specific CD4-CD8- T cell hybridomas is influenced by the substitution at residue 256 of MHC class I molecules.
Authors: Tanabe M , Minami M , Kano K , Takiguchi M
Published: December 2022

Our previous studies demonstrated that allorecognition of HTB176.10 and HTB177.2, H-2Kb-reactive CD4-CD8- T cell hybridomas is markedly influenced by the exchange of the alpha 3 domain between H-2Kb and H-2Dp. The recombinant genes of the exon 4 between H-2Kb and H-2Dp were constructed to determine the residues of the alpha 3 domain that influence the allorecognition of these T cell hybridomas. Seven recombinant genes of the exon 4 were generated by in vivo recombination in Escherichia coli. Chimeric genes containing these recombinants were transfected into L cells and the transfectants expressing equivalent amounts of chimeric molecules were selected by flow cytometry. Studies on responses of these T cell hybridomas to the chimeric molecules confirmed our previous observation that the primary structure of the alpha 3 domain influences the allorecognition by the hybridomas. Moreover, it was indicated that residue 256 on the alpha 3 domain markedly affects the allorecognition by the T cell hybridomas, although substitutions at residues 184, 193, 195, 197, 262, and 264 exerted some effects on the T cell recognition. Further studies with the use of a single amino acid mutant of H-2Kb at residue 256 confirmed the effect of substitution at residue 256 on allorecognition of the T cell hybridomas. Taken together, results of this study demonstrated that polymorphism of the alpha 3 domain is indeed involved in the formation of allodeterminants recognized by TCR.
Target cell lysis and IL-2 secretion by gamma/delta T lymphocytes after activation with bacteria.
Authors: Munk M E , Gatrill A J , Kaufmann S H
Published: December 2022

Peripheral blood T lymphocytes from healthy donors were stimulated with Mycobacterium tuberculosis in vitro and afterward analyzed phenotypically. Marked expansion of the gamma/delta T cell population (3- to 21-fold) was observed in 15/21 donors 7 to 10 days after stimulation. In addition to M. tuberculosis, Mycobacterium leprae (six of eight) as well as the gram-positive bacteria, Staphylococcus aureus (two of six), group A streptococci (seven of nine), and Listeria monocytogenes (four of eight) augmented gamma/delta TCR expression in peripheral blood T cells of many donors. gamma/delta T lymphocytes expressed IL-2R and secreted IL-2 upon restimulation with M. tuberculosis. Stimulation with M. tuberculosis evoked specific cytolytic activities in gamma/delta T lymphocytes because: gamma/delta T cells lysed M. tuberculosis pulsed but not unpulsed targets; high concentrations of TCR delta 1 mAb facilitated killing of unpulsed target cells; and low doses of anti-TCR delta 1 mAb blocked killing of pulsed targets. Furthermore, gamma/delta T cells from four donors, after activation with M. tuberculosis or with group A streptococci, respectively, only lysed targets pulsed with the homologous agents, whereas in other donors some cross-reactivity was observed. We conclude that, upon contact with mycobacteria and perhaps other microorganisms, gamma/delta T cells are activated which contribute to immunity against infection via IL-2 secretion and specific target cell lysis.
The adenovirus E3-14.7K protein is a general inhibitor of tumor necrosis factor-mediated cytolysis.
Authors: Gooding L R , Sofola I O , Tollefson A E , Duerksen-Hughes P , Wold W S
Published: December 2022

We have previously described a 14,700 m.w. protein (14.7K) encoded by the E3 region of adenovirus that prevents TNF-mediated cytolysis of adenovirus-infected C3HA mouse fibroblasts. In the studies described here we have extended our analysis of TNF cytolysis of C3HA cells and the circumstances under which 14.7K protects these cells from cytolysis. C3HA cells were killed by TNF in the presence of inhibitors of protein synthesis, in the presence of cytochalasin E (which disrupts the microfilaments), and when adenovirus E1A was expressed. As described for other cell types, pretreatment of C3HA cells with TNF prevented cytolysis by TNF plus cycloheximide or TNF plus cytochalasin E, indicating that TNF induces a response that protects against these treatments. Remarkably, when 14.7K was expressed in virus-infected cells, it also prevented TNF-induced lysis whether sensitivity to TNF was induced by inhibition of protein synthesis, disruption of the cytoskeleton by cytochalasin E, or expression of adenovirus E1A. The 14.7K protein also prevented TNF lysis of cells that are spontaneously sensitive to TNF lysis. Thus, 14.7K appears to be a general inhibitor of TNF cytolysis, and as such should be an important tool in unraveling the mechanism of TNF cytolysis. There was one exception; NCTC-929 cells were spontaneously sensitive to TNF lysis and that lysis was not affected by 14.7K even though the protein was made in large quantities and was metabolically stable in these cells. This suggests that there is heterogeneity among TNF-sensitive cell lines. The 14.7K protein was found in both the nuclear and cytosol fractions of TNF resistant as well as all spontaneously sensitive cells suggesting that 14.7K may have more than one site of action within the cell.
Cell specificity of granzyme gene expression.
Authors: Garcia-Sanz J A , MacDonald H R , Jenne D E , Tschopp J , Nabholz M
Published: December 2022

Granzymes are serine proteases present in secretory granules of cytolytic T lymphocyte lines. We have studied the expression of the granzyme family (granzyme A, B, C, D, E, F, and G) in different lymphoid cell populations and cell lines as well as in nonlymphoid cells and tissues. Our data show that with few exceptions expression of granzyme genes is restricted to T cells and their thymic precursors. In mature T cells granzymes are expressed only upon activation. The same is true for thymocytes, with the exception of grazyme A that is expressed also in non-stimulated cells. In T cells and thymocytes the distribution of mRNAs coding for different granzymes depends on the subpopulation tested and the activation protocol. Highly cytolytic PEL express granzymes A and B but none of the other granzymes.
Distinct subsets of accessory cells activate Thy-1+ triple negative (CD3-, CD4-, CD8-) cells and Th-1 delayed-type hypersensitivity effector T cells.
Authors: Matsushima G K , Stohlman S A
Published: December 2022

The SJL strain of mice possess a unique developmental delay in the ability to exhibit delayed-type hypersensitivity (DTH) responses after immunization with a wide variety of Ag. Similar to other models of DTH, the adoptive transfer of syngeneic Ag-pulsed macrophages from DTH-responsive mice into these DTH-unresponsive mice results in the activation of Ag-specific, CD4+ DTH effector Th1 T cells. The absence of other defects in APC-dependent immune responses indicate that the macrophages is the sole APC required for the induction of DTH effector T cells in SJL mice. The defect occurs during the sensitization phase of the DTH response; however, it has not been determined whether a Th cell, which is required for the induction of CD4+ DTH effector T cells, was present in the DTH unresponsive SJL mice. In this study, we have determined that the Thy-1+ helper cell is induced upon Ag stimulation of nonresponder mice and present evidence for the existence of an accessory cell distinct from the macrophage that induces CD4+ DTH effector T cells. Our data indicate that CD4+ DTH effector T cells are induced in an Ag-specific and MHC-restricted manner by an adherent macrophage that expresses the Mac-1+, Mac-2-, Mac-3+, I-A+ phenotype. Adoptive transfer of as few as 100 of the Mac-1+, Mac-2-, or Mac-3+ subsets from DTH responsive donors to DTH unresponsive recipients is able to overcome the DTH deficit. The activation of CD4+ DTH effector T cells in the SJL mouse cells also requires a Thy-1+, Lyt-1+, CD3-, CD4-, CD8-, helper cell. In contrast to the Mac-1+, Mac-3+, I-A+ accessory cell, this helper cell requires an adherent, irradiation resistant, accessory cell that expresses the Mac-1+, Mac-2-, Mac-3-, I-A- surface phenotype for activation. Further, the interaction between this accessory cell and the Thy-1+ helper cell is neither Ag-specific nor MHC restricted. This is the first demonstration of an accessory cell requirement for the Thy-1+, Lyt-1+, B220-, CD4-, CD8-, CD3- DTH Th cell. These data indicate that the activation of the triple negative helper cells and subsequent activation of the CD4+ effector T cells are regulated by two distinct macrophage subpopulations.
Diminished lectin-, epidermal growth factor-, complement binding domain-cell adhesion molecule-1 on neonatal neutrophils underlies their impaired CD18-independent adhesion to endothelial cells in vitro.
Authors: Anderson D C , Abbassi O , Kishimoto T K , Koenig J M , McIntire L V , Smith C W
Published: December 2022

To define further the molecular basis for abnormal interactions of cord blood or neonatal neutrophils with endothelial cells in vitro, we studied neutrophil adhesion and migration under experimental conditions specifically designed to evaluate CD18-independent mechanisms. Unstimulated cord blood neutrophils of healthy term neonates demonstrated significantly diminished adhesion to IL-1-stimulated endothelial cell monolayers under conditions of shear stress (congruent to 1.85 dynes/cm2); overall levels of migration by neonatal cells were also significantly diminished, although the adherent subpopulation of these cells migrated relatively normally. A mAb (DREG-56) against the human homologue of the murine MEL-14 antigen (termed lectin-, epidermal growth factor-, complement binding domain-cell adhesion molecule-1 (LECAM-1), a member of the LEC-CAM family of adhesion molecules) markedly inhibited adhesion of healthy adult but not cord blood neutrophils. In additional assessments of endothelial cell adhesion or migration in the absence of shear forces, cord blood neutrophils demonstrated significantly diminished values compared to adult controls. Moreover, mAb DREG-56 significantly diminished adhesion of healthy adult but not cord blood suspensions in the presence or absence of the anti-CD18 mAb R15.7. Immunofluorescence assessments of unstimulated cord blood neutrophils or neutrophils of neonates 12 to 48 h of age showed dramatically diminished levels of surface LECAM-1 compared to adult neutrophils. Chemotactic stimuli (FMLP, 10 nM, 15 min) consistently "down-regulated" surface LECAM-1 on adult neutrophils to levels approximately 10% of unstimulated suspensions and comparable to those of most unstimulated neonatal suspensions. Moreover, FMLP stimuli elicited little or no down-regulation of LECAM-1 on neonatal cells. In comparative studies, endothelial cell adhesion of unstimulated cord blood or adult control neutrophils (assessed under conditions of flow) was directly related to levels of neutrophil surface LECAM-1. Although FMLP stimulation significantly diminished both adhesion and LECAM-1 surface levels of adult control cells, the adhesion and LECAM-1 expression observed with cord blood cells were not significantly influenced by this stimulus. The mechanisms underlying diminished LECAM-1 expression and LECAM-1-dependent adhesion of neonatal neutrophils and the physiologic significance of these abnormalities deserve investigation.
IL-8 in septic shock, endotoxemia, and after IL-1 administration.
Authors: Van Zee K J , DeForge L E , Fischer E , Marano M A , Kenney J S , Remick D G , Lowry S F , Moldawer L L
Published: December 2022

Much effort has been directed toward elucidating the host response to sepsis and inflammation, resulting in the definition of a cascade of endogenous mediators that direct metabolic and immunological responses. Here we report that IL-8, a novel cytokine produced by a variety of cells in vitro in response to stimulation with bacterial LPS and the proinflammatory cytokines, appears in the circulation of primates in vivo during septic shock, sublethal endotoxemia, and after the administration of IL-1 alpha. The magnitude of the IL-8 response correlates with the severity of the insult, and levels of IL-8 peak relatively late, after those of TNF-alpha and IL-1 beta, and simultaneously with those of IL-6. IL-8 has been primarily defined as a selective activator and chemoattractant of neutrophils, and we demonstrate that after LPS or IL-1 alpha infusion, circulating neutrophil numbers rapidly recover from an initial neutropenia while IL-8 concentrations are maximal, supporting the hypothesis that IL-8 influences circulating leukocyte populations in vivo. We conclude that IL-8 is another participant in the cytokine cascade elicited by sepsis and inflammation and, as such, may play a significant role in host defense and disease.
Isolation, expression, and sequence of the TAP/Ly-6A.2 chromosomal gene.
Authors: McGrew J T , Rock K L
Published: December 2022

The murine Ly-6 locus controls the expression of a number of genes. One of the products of the Ly-6 locus, Ly-6A.2, has been implicated in the process of T cell activation. We have identified the chromosomal sequences encoding the Ly-6A.2 molecule using very stringent hybridization and washing conditions. We confirmed that this gene encoded the Ly-6A.2 molecule by transfection studies using a cell line genetically negative for the Ly-6A.2 gene as a DNA recipient. Sequence analysis showed that the Ly-6A.2 gene is made up of four exons. The start site of transcription was determined by primer extension analysis. The first exon does not contain protein coding sequences. The structure of the Ly-6A.2 gene supports previous speculation that various Ly-6 RNA can be generated by alternate splicing events. The Ly-6A.2 chromosomal gene is closely related to the previously characterized Ly-6C.1 chromosomal gene in the intron, exon, and 5' flanking regions. This analysis indicates that these genes have arisen as a consequence of gene duplication. Although endogenous Ly-6A.2 and Ly-6C genes are IFN responsive, only the latter contains a clearly identifiable IFN responsive element. A transfected Ly-6A.2 chromosomal gene that contains 3.9 kb of 5'-untranslated sequence is IFN responsive. However, a shorter chromosomal clone containing only 940 bp of 5' sequence is constitutively expressed in transfectants but does not respond to IFN.
Stimulation of murine natural killer (NK) cells by a monoclonal antibody specific for the NK1.1 antigen. IL-2-activated NK cells possess additional specific stimulation pathways.
Authors: Karlhofer F M , Yokoyama W M
Published: December 2022

NK cells lyse certain tumor cell targets but the effector cell surface molecules responsible for this reactivity remain uncertain. The allotypic NK1.1 Ag is the most specific serologic marker on murine cells that display non-MHC-restricted cytolysis of tumor cell targets, but no function has been previously ascribed to this Ag. In this report, we demonstrate that, in the presence of a mAb specific for the NK1.1 Ag (mAb PK136), freshly isolated and IL-2-activated NK cells from C57BL/6 mice can be induced to lyse an otherwise resistant target cell, Daudi. This phenomenon is effector and mAb specific because NK cells derived from BALB/c mice do not express the NK1.1 Ag and cannot be triggered by mAb PK136. We demonstrate that IL-2 activated but not freshly isolated NK cells express the Ly-6 and VEA Ag, originally described as T cell activation Ag. Moreover, mAb specific for Ly-6 and VEA induce target cell lysis by IL-2 activated but not freshly isolated NK cells. These mAb effects are specific, concentration dependent, and display kinetics that are similar to spontaneous cytolysis of NK-sensitive targets. The Fc portion of the activating antibodies and only FcR bearing target cells participate in mAb-induced activation, consistent with the mechanism of redirected lysis. Finally, analysis of Daudi cells transfected with beta 2-microglobulin gene demonstrate that the expression of MHC class I Ag by the target cell does not affect its sensitivity to mAb-induced lysis by NK cells. These data demonstrate that the NK1.1 Ag is functionally active on both freshly isolated and IL-2-activated NK cells and that IL-2-activated NK cells possess additional pathways of specific stimulation.
Human T cells in the SCID-hu mouse are phenotypically normal and functionally competent.
Authors: Krowka J F , Sarin S , Namikawa R , McCune J M , Kaneshima H
Published: December 2022

SCID-hu mice are heterochimeric animals that are constructed by transplanting human fetal thymus (Thy), liver (Liv), and/or lymph nodes into congenitally immunodeficient C.B-17 scid/scid (SCID) mice. Sensitive and specific two-color flow cytometric assays were used to evaluate human lymphocytes from peripheral blood of SCID-hu mice. Kinetic studies presented in this report show long term T lymphopoiesis in SCID-hu mice. Approximately one-half of SCID-hu mice constructed with Thy and Liv tissue develop detectable levels of circulating human T cells by 4 mo after transplantation. The average level of circulating human cells in SCID-hu mice is generally less than 2% of the total lymphoid cells in the peripheral blood of these mice. Some SCID-hu mice with as high as 13% human lymphocytes, however, have been detected. Nearly all human cells in the peripheral blood of SCID-hu mice are CD3+ cells that express TCR-alpha beta. The percentages of gamma delta+, CD4+, CD8+, CD25+, CD69+, and Leu-8+ cells among CD45+ cells in SCID-hu blood are similar to the levels found in adult peripheral blood. On average, 74% of SCID-hu T cells express CD45RA and 18% express CD29. Functional studies demonstrate that cells from SCID-hu Thy/Liv grafts or human T cells from SCID-hu peripheral blood are functionally competent to respond to mitogens or allogeneic human cells in vitro. They are similar to fetal thymocytes or adult T cells, respectively, in these responses. These studies demonstrate that the SCID-hu mouse is a useful model for the analysis of human immune differentiation and function in vivo.