bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostaglandin E2 differentially modulates cytokine secretion profiles of human T helper lymphocytes.
Authors: Snijdewint F G , Kaliński P , Wierenga E A , Bos J D , Kapsenberg M L
Published: December 2022

PGE2 is a well known immunomodulator that has multiple effects on the immune system. We demonstrate that PGE2 selectively and dose dependently inhibits IL-2 and IFN-gamma production by mitogenically stimulated human PBL and CD4+ TLC, although at low concentrations IL-4 production is not affected and IL-5 production is even up-regulated. In the tested TLC, PGE2 induced a dramatic elevation (up to 85-fold) of the intracellular cAMP levels. The action of PGE2 may, therefore, be associated with elevation of intracellular cAMP levels, affecting IL-4 and IL-5 differentially from IL-2 and IFN-gamma production. To test this hypothesis we investigated cytokine production by TLC in the absence or presence of agents that affect cAMP levels, either directly (2'-O-dibutyrylcAMP) or through activation of adenylate cyclase (forskolin) or by blocking of phosphodiesterase (3-isobutyl-1-methyl-xanthine). Similar to PGE2, forskolin, 2'-O-dibutyrylcAMP, and 3-isobutyl-1-methyl-xanthine induced inhibition of IL-2 production by TLC and up-regulation of IL-5 production. However, in contrast to PGE2, these agents suppressed IL-4 production although IFN-gamma production was only moderately affected. No significant differences were found between intracellular cAMP levels of mitogenically stimulated Th1 cell clones, which predominantly secrete IL-2 and IFN-gamma, and those of Th2 cell clones, which mainly secrete IL-4 and IL-5. Our results indicate that PGE2 selectively modulates cytokine secretion profiles of human T cells and that elevation of cAMP levels has an important, but possibly not exclusive, regulatory role in this phenomenon.
Structural comparison and chromosomal localization of the human and mouse IL-13 genes.
Authors: McKenzie A N , Li X , Largaespada D A , Sato A , Kaneda A , Zurawski S M , Doyle E L , Milatovich A , Francke U , Copeland N G
Published: December 2022

The genomic structure of the recently described cytokine IL-13 has been determined for both human and mouse genes. The nucleotide sequence of a 4.6-kb DNA segment of the human gene is described. The human IL-13 gene (IL13) occurs as a single copy in the haploid genome and maps to human chromosome 5. A 4.3-kb DNA fragment of the mouse IL-13 gene (IL13) has been sequenced and found to occur as a single copy, mapping to mouse chromosome 11. Intrachromosomal mapping studies revealed that both genes contain four exons and three introns and show a high degree of sequence identity throughout their length. Potential recognition sequences for transcription factors that are present in the 5'-flanking region and are conserved between both genes include IFN-responsive elements, binding sites for AP-1, AP-2 and AP-3, and NF-IL 6 site, and a TATA-like sequence. Both genes map to chromosomal locations adjacent to genes encoding other cytokines, including IL-3, GM-CSF, IL-5, and IL-4, suggesting that IL-13 is another member of this cytokine gene family that may have arisen by gene duplication.
CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus saimiri gene.
Authors: Rouvier E , Luciani M F , Mattéi M G , Denizot F , Golstein P
Published: December 2022

To detect novel molecules involved in immune functions, a subtracted cDNA library between closely related murine lymphoid cells was prepared using improved technology. Differential screening of this library yielded several clones with a very restricted tissue specificity, including one that we named CTLA-8. CTLA-8 transcripts could be detected only in T cell hybridoma clones related to the one used to prepare the library. Southern blots showed that the CTLA-8 gene was single copy in mice, rats, and humans. By radioactive in situ hybridization, the CTLA-8 gene was mapped at a single site on mouse chromosome 1A and human chromosome 2q31, in a known interspecific syntenic region. The CTLA-8 cDNA sequence indicated the presence, in the 3'-untranslated region of the mRNA, of AU-rich repeats previously found in the mRNA of various cytokines, growth factors, and oncogenes. The CTLA-8 cDNA contained an open reading frame encoding a putative protein of 150 amino acids. This protein was 57% homologous to the putative protein encoded by the ORF13 gene of herpesvirus Saimiri, a T lymphotropic virus. These findings are discussed in the context of other genes of this herpesvirus homologous to known immunologically active molecules. More generally, CTLA-8 may belong to the growing set of virus-captured functionally important cellular genes related to the immune system or to cell death and cell survival.
Syngeneic response to SJL follicular center B cell lymphoma (reticular cell sarcoma) cells is primarily in V beta 16+ CD4+ T cells.
Authors: Tsiagbe V K , Asakawa J , Miranda A , Sutherland R M , Paterson Y , Thorbecke G J
Published: December 2022

The growth of SJL B cell lymphomas (RCS, reticulum cell sarcoma) in vivo and in vitro is known to depend on cytokine production by RCS-responsive host CD4+ T cells. The high frequency of RCS responsive cells in normal SJL lymph nodes prompted us to prepare a set of 21 RCS-specific T cell hybridomas. Like normal SJL T cells, these hybridoma cells respond to RCS, but not to normal syngeneic B cells; produce IL-2, IL-4, and IL-5; and promote growth of RCS in gamma-irradiated syngeneic hosts. A superantigen-like stimulation by RCS cells was borne out by the fact that all the RCS-specific hybridomas used V beta 16 in their TCR. RCS cells did not stimulate I-As-restricted, V beta 17a+ KLH-specific, or V beta 1+ heme-specific hybridomas, but were excellent Ag presents for these cells. Preincubation of RCS cells with high concentrations of the KM core peptide (high affinity for I-As) did not interfere with the ability of RCS to stimulate RCS-specific hybridomas. The relative representation of mRNA for V beta 1, 4, 10, 15, 16, and 17a was evaluated in RNA extracted from normal SJL lymph node cells responding to Con A or to RCS cells. Only V beta 16 was specifically enriched in the response to RCS. Moreover, the degree of responsiveness to RCS cells in lymph node cells from F1 hybrids of SJL and I-E transgenic SJL mice, corresponds to the relative abundance of V beta 16 in mRNA, but not of V beta 17a mRNA.
IFN-gamma-mediated antimicrobial response. Indoleamine 2,3-dioxygenase-deficient mutant host cells no longer inhibit intracellular Chlamydia spp. or Toxoplasma growth.
Authors: Thomas S M , Garrity L F , Brandt C R , Schobert C S , Feng G S , Taylor M W , Carlin J M , Byrne G I
Published: December 2022

The role of indoleamine 2,3-dioxygenase (IDO) in IFN-gamma-mediated inhibition of intracellular parasite growth has been examined previously, although earlier work has been largely correlative. In this study, we defined more completely the role of IDO in the IFN-antimicrobial response. Two mutant cell lines, derived from ME180 cells and exhibiting reduced IDO activity (IR3B6A, IR3B6B) were characterized to determine if they retained the capacity to inhibit intracellular Chlamydia and Toxoplasma growth. Mutant cells treated with IFN-gamma exhibited reduced capacity to suppress pathogen growth. The expression of several IFN-regulated genes also was measured to confirm that the inability to inhibit pathogen growth was because of the lack of IDO. The expression of class II MHC, intracellular adhesion molecule-1, MxA, and P68 kinase genes was induced in the IFN-gamma-treated wild type ME180 cells, but was variable in the mutant cell lines, supporting the hypothesis that IFN-gamma-induced production of IDO is a key IFN-gamma-mediated antimicrobial mechanism.
Four different classes of inhibitors of receptor-mediated endocytosis decrease tumor necrosis factor-induced gene expression in human endothelial cells.
Authors: Bradley J R , Johnson D R , Pober J S
Published: December 2022

We have investigated the relationship between receptor-mediated endocytosis of TNF and TNF-induced gene expression in cultured human endothelial cells. Exposure of cells to hypertonicity, to cytoplasmic acidification, to treatment with phenylarsine oxide, or to treatment with primary amines such as putrescine or dansylcadaverine each inhibited receptor-mediated endocytosis by 30 to 75%, as measured by uptake of acetylated Dil-low density lipoprotein or of 125I-TNF. All four treatments also inhibited TNF-induced surface expression of ELAM-1 by 50 to 100%. Among these four treatments, only hypertonicity inhibited pinocytosis, as measured by uptake of fluorescein-BSA, and only phenylarsine oxide irreversibly inhibited protein synthesis, as measured by [35S]methionine incorporation. Notably, acidification or treatment with primary amines selectively inhibited the response to TNF, compared with the response to PMA, a drug that induces ELAM-1 through a pathway that bypasses surface receptors. Primary amines, which can be used for sustained periods under physiologic culture conditions without causing toxicity, were investigated further. Pretreatment of endothelial cells with 10 mM putrescine or 100 microM dansylcadaverine also inhibited TNF induction of ICAM-1 expression and VCAM-1 expression. Primary amines also inhibited IL-1-induced increases in ELAM-1, ICAM-1, and VCAM-1 measured 4 to 6 h after treatment and inhibited IFN-beta- and IFN-gamma-mediated induction of class I MHC molecules and IFN-gamma-mediated induction of class II MHC molecules measured 72 h after treatment with cytokine. Levels of mRNA encoding cytokine-inducible molecules were also selectively reduced by primary amines. A constitutively expressed surface molecule, gp96, was not affected in the same cells. These data are consistent with a role for receptor-mediated endocytosis in TNF-mediated gene induction and suggest a new potential target for anti-inflammatory therapy.
Expression of prolactin receptors in murine lymphoid cells in normal and autoimmune situations.
Authors: Gagnerault M C , Touraine P , Savino W , Kelly P A , Dardenne M
Published: December 2022

We have analyzed the expression of prolactin receptors (PRL-R) on murine lymphoid cells by using flow cytofluorometry analysis with biotinylated anti-PRL-R mAb raised against several epitopes of the extracellular domain of the PRL-R and by using polymerase chain reaction amplification. We demonstrated that PRL-R were universally expressed in normal rat and mouse hematopoietic tissues. In both primary lymphoid organs, namely, thymus and bone marrow, > 90% of cells were labeled by the anti-PRL-R mAb, but the density of PRL-R (assessed by fluorescence intensity) was lower on thymocytes than on bone marrow cells. In peripheral lymphoid organs there were smaller proportions of cells bearing PRL-R and we could clearly distinguish cell subsets of various fluorescence intensities. By using classical markers for lymphoid cell populations, we noted that all B cells and macrophages from spleen, lymph nodes, and blood strongly expressed the PRL-R. Regarding T cell populations, large proportions (> 85%) of PRL-R+ cells were detected in the four thymocyte subsets, thus contrasting with the smaller proportions (50 to 65%) of T cells labeled in the periphery. Similar percentages of PRL-R+ cells were observed in CD4+ and CD8+ peripheral lymphocyte subsets. Importantly, the stimulation of thymocytes and spleen cells with the T cell mitogen Con A promoted an enhancement of the density of PRL-R molecules on the cell membranes. Because hyperprolactinemia is associated with some autoimmune diseases, we also investigated PRL-R expression in the NZB autoimmune mouse. In contrast to the pattern observed in normal animals, the frequencies of PRL-R-bearing T cells as well as the density of PRL-R per cell increased with age in NZB mice, suggesting that some imbalances of PRL/PRL-R interaction might occur in autoimmune situations. In conclusion, our data provide a molecular basis for a better understanding on the mode of action of PRL within the immune system in physiologic and pathologic situations.
Characterization of T cells infiltrating the heart in rats with experimental autoimmune myocarditis. Their similarity to extrathymic T cells in mice and the site of proliferation.
Authors: Hanawa H , Tsuchida M , Matsumoto Y , Watanabe H , Abo T , Sekikawa H , Kodama M , Zhang S , Izumi T , Shibata A
Published: December 2022

A model of experimental autoimmune myocarditis, which resembles fatal giant cell myocarditis in humans, was previously established in rats immunized by s.c. injection of human cardiac myosin. We characterized herein the surface phenotype of lymphocytes infiltrating the heart and pericardial cavity as well as of mononuclear cells in various organs by using mAb in conjunction with immunofluorescence tests. Since profound thymic atrophy always accompanied the diseased states, attention was focused on characterization of T cells with properties similar to those of extrathymic T cells. In mice, extrathymic T cells were activated in association with thymic atrophy, expressed high levels of LFA-1 and IL-2R beta-chains, and contained a significant proportion of double negative CD4-CD8- T cells. In diseased rats, a large proportion of activated T cells that expressed high levels of LFA-1 and IL-2R was demonstrated in the pericardial effusion and heart tissue. Such T cells were rare in the other organs. Light scatter and microscopic observation revealed that activated lymphoblasts were most abundant in the pericardial effusion. Moreover, one-fourth of such T cells in the pericardial effusion displayed double negative phenotype. These cells in rats might correspond to the extrathymic T cells in mice. However, only a limited population of such activated T cells infiltrated the heart tissue. Concerning the location of such T cells mainly in the outer layer of the heart, it raised the possibility that extrathymic T cell differentiation in these autoimmune rats might occur in the pericardial cavity, and the differentiated cells then migrated to the sites of the cardiac lesion.
Religation of the T cell receptor after primary activation of mature T cells inhibits proliferation and induces apoptotic cell death.
Authors: Radvanyi L G , Mills G B , Miller R G
Published: December 2022

The proliferative response of murine splenic T cells, initially activated by cross-linking the TCR complex with either antibodies, mitogenic lectin, or alloantigen was severely inhibited when the activated cells were recovered and given an additional activation signal by recross-linking the TCR complex, or by adding Ca2+ ionophore and phorbol ester. Under the same conditions, cross-linking other T cell surface determinants such as CD4, CD8, or class I MHC on preactivated T cells had no effect. Assessment of cell viability using vital dye exclusion together with the detection of DNA fragmentation revealed that the reduction of the proliferative responses was associated with an induction of apoptotic-like cell death in the activated T cell population and not due to a blockade of cell division. Accumulation of eosin stained (dead) cells did not occur immediately upon replating the activated cells, but began after a lag period during which at least two cell divisions occurred. In addition, perturbation of T cell proliferation after activation depended on how the cells were initially activated. Only T cells activated in the presence of additional cells found in spleen and lymph node were susceptible to inhibition; T cells activated after nylon wool purification were not susceptible. These results have potential implications for understanding self-tolerance and immunoregulation.
Usage of T cell receptor beta-chain variable gene is highly restricted at the site of inflammation in murine autoimmune uveitis.
Authors: Rao N A , Naidu Y M , Bell R , Lindsey J W , Pararajasegaram G , Sun Y , Steinman L
Published: December 2022

Improved molecular methods allow identification of the specific autoaggressive T cells involved in autoimmune inflammations. In this study of interphotoreceptor retinoid-binding protein-induced uveitis, TCR V beta usage was studied using RNA-polymerase chain reaction amplification of transcripts derived directly from ocular tissues and from T cell lines obtained from the spleen. Specific V beta 5' primers from the major murine TCR V beta families were coupled with a common 3' primer from the V beta C region. Amplification of rearranged TCR V beta-D beta-J beta-C beta sequences was confirmed by Southern blot analysis. In ocular tissue from sensitized mice, TCR V beta expression was limited mainly to one to three V beta families, with predominant expression of V beta 2, V beta 12, and V beta 15. In most animals there was similar, albeit limited, TCR gene usage in both the recognition of autoantigen in uveitogenic T cell lines and at the site of inflammation in the eye. Identification of a limited TCR V beta repertoire in Ag-reactive T cell lines correlated with TCR usage at the target site of autoimmune expression. The gene products of the restricted TCR V beta rearrangements found in lesions and in the cell lines may serve as the target for selective immunotherapy.
Preferential activation of Th2 cells in chronic graft-versus-host reaction.
Authors: De Wit D , Van Mechelen M , Zanin C , Doutrelepont J M , Velu T , Gérard C , Abramowicz D , Scheerlinck J P , De Baetselier P , Urbain J
Published: December 2022

The injection of DBA/2 parental lymphocytes into adult, immunologically intact (C57BL/6 x DBA/2) F1 hybrid mice results in a chronic graft-vs-host reaction (GVHR) characterized by a deficiency in CD4+ T cell functions and a B cell activation leading to autoantibody production. The discovery that distinct subpopulations of Th cells may regulate the effector immune functions led us to investigate whether the chronic GVHR differentially affects Th subsets. Data are presented indicating that mice undergoing a GVHR spontaneously produced lymphokines of Th2 origin. IL-4 and IL-10 mRNA were detected in the spleens of GVH mice, and IL-4 was shown to be responsible for the increased expression of class II Ag on B cells. Moreover, upon polyclonal activation in vitro, GVH T cells exhibited defective IL-2 and IFN-gamma production but elevated IL-4 production. We conclude that the chronic GVHR is characterized by a selective deficiency in cells secreting IL-2 and IFN-gamma and a hyperactivation of Th2 cells. The simultaneous production of IL-4 and IL-10 might explain the association between B cell hyperactivity and impairment of Th1-like activities in various models that associate autoimmunity and immunosuppression, such as GVHR and HIV infection.
IgA antibodies to a protective antigen in human Schistosomiasis mansoni.
Authors: Grzych J M , Grezel D , Xu C B , Neyrinck J L , Capron M , Ouma J H , Butterworth A E , Capron A
Published: December 2022

The specific IgA antibody responses to the protective recombinant Schistosoma mansoni 28-kDa glutathione-S-transferase (Sm28GST) Ag and to derived synthetic peptides have been evaluated before and 6 mo after chemotherapy in S. mansoni-infected patients from Kenya. These studies revealed a parallelism between the age-dependent evolution of IgA antibody levels to Sm28GST and to one synthetic peptide (115-131) and the acquisition of resistance to reinfection. Functional analysis revealed that IgA antibodies to Sm28GST displayed a potent neutralizing effect on the enzymatic properties of the molecule, and also markedly impaired schistosome fecundity, by limiting both the egg laying of mature worms and the hatching capacity of schistosome eggs into viable miracidia. These results suggest that, in addition to IgE, IgA antibodies might participate in the protective immune response against schistosomiasis.
Macrophage colony-stimulating factor in human fetal astrocytes and microglia. Differential regulation by cytokines and lipopolysaccharide, and modulation of class II MHC on microglia.
Authors: Lee S C , Liu W , Roth P , Dickson D W , Berman J W , Brosnan C F
Published: December 2022

CSF-1 is a growth factor that selectively promotes the proliferation, survival, and differentiation of cells of the mononuclear phagocyte series. As part of a study on the role of cytokine and hematopoietic growth factors in central nervous system (CNS) development and inflammation, we examined the expression of CSF-1 in dissociated glial cells cultured from human fetal CNS tissue. CSF-1 mRNA and protein were constitutively expressed by astrocytes. The steady state level of CSF-1 mRNA was markedly up-regulated by both IL-1 beta and TNF-alpha in a time- and dose-dependent manner, whereas only a minimal increase was detected after stimulation with LPS. In unstimulated astrocyte cultures, CSF-1 protein levels gradually increased to 3.5-fold base-line values by 96 h and were significantly increased by all three stimulants in the order of IL-1 > or = TNF > LPS. Low levels of CSF-1 mRNA and protein were also detected in unstimulated microglia cultures. In contrast to astrocyte cultures, CSF-1 mRNA and protein increased significantly after stimulation with LPS, but changed only minimally after exposure to TNF-alpha or IL-1 beta. The effect of CSF-1 on cell proliferation, morphology, and class II MHC Ag expression was determined in highly enriched cultures of microglia and astrocytes. Microglia treated with CSF-1 showed a modest level of proliferation and differentiation into rod-shaped cells, whereas neither cell number nor shape was changed in astrocyte cultures. Interestingly, marked inhibition of both basal and IFN gamma-induced class II MHC Ag expression was observed in microglial cells cultured in the presence of CSF-1, whereas no effect was detected in astrocytes. These results suggest the possibility that in situ production of CSF-1 in the CNS may regulate normal glial cell development and contribute to the immunologic status of the CNS through the down-regulation of class II MHC expression.
Soluble E-selectin is found in supernatants of activated endothelial cells and is elevated in the serum of patients with septic shock.
Authors: Newman W , Beall L D , Carson C W , Hunder G G , Graben N , Randhawa Z I , Gopal T V , Wiener-Kronish J , Matthay M A
Published: December 2022

A quantitative sandwich ELISA for E-selectin in the fluid phase (soluble E-selectin, sEs) has been developed that is sensitive to 100 pg/ml. The assay shows no reactivity with either L- or P-selectins. We have used this to determine the fate of E-selectin after cell-surface expression and to test whether levels measured in vivo may represent the state of endothelial activation. E-selectin was first detectable in supernatants of IL-1-stimulated endothelial cells at 24 h, and increased slowly up until 72 h. However, over this time period the total E-selectin detectable in the system (cells plus supernatants) declined dramatically. 125I-surface-labeled endothelial cells cultured for 24 h show an E-selectin of reduced m.w. in the supernatant, indicating that the molecule is shed from the surface. The shed form also appears to be slightly smaller than the intact membrane form as determined from immunoprecipitation and molecular sieving studies. In addition, the cytoplasmic domain of the molecule found in supernatants of activated endothelial cells and in serum is not intact as determined by loss of reactivity with an antipeptide antibody specific for the cytoplasmic domain. We have examined the sera of 71 normal individuals. Without exception, sEs was found in serum in the range of 0.13 to 2.8 ng/ml, suggesting that even in the absence of overt inflammatory processes E-selectin is being synthesized and released into the bloodstream. In addition, bacteremic patients with hypotension, but not those without, showed markedly elevated sEs values. As determined by cell-binding studies, the blood-derived form of E-selectin is biologically active.
Detection and quantitation of cells secreting IL-6 under physiologic conditions in BALB/c mice.
Authors: Shirai A , Holmes K , Klinman D
Published: December 2022

IL-6 is a pleiotropic cytokine involved in the regulation of hematopoiesis and lymphocyte activation. An extremely sensitive ELIspot assay was developed to detect and enumerate individual cells secreting IL-6 in normal BALB/c mice. Under physiologic conditions, the frequency of cells producing IL-6 in the bone marrow, spleen, and mesenteric lymph nodes of normal adult animals was approximately 0.5, 0.1, and 0.01%, respectively. FACS analysis revealed that macrophages and Mac1- B220- cells accounted for the vast majority of IL-6 production in the bone marrow, whereas T and B lymphocytes accounted for 4% and 38% of IL-6 production in the spleen. Simultaneous studies comparing cell number with IL-6 levels indicated that the average activated cell produced approximately 85 molecules of IL-6/s.
Cytokine secretion by genetically modified nonimmunogenic murine fibrosarcoma. Tumor inhibition by IL-2 but not tumor necrosis factor.
Authors: Karp S E , Farber A , Salo J C , Hwu P , Jaffe G , Asher A L , Shiloni E , Restifo N P , Mulé J J , Rosenberg S A
Published: December 2022

Recent investigations have demonstrated that the in vivo growth of weakly immunogenic murine tumors can be inhibited by genetic manipulations that enable them to secrete a variety of cytokines. Inasmuch as most human tumors fail to elicit a detectable host immune response we questioned whether the growth of a nonimmunogenic murine tumor could be inhibited by the secretion of cytokines. We have thus inserted the cDNA encoding for human IL-2 or TNF into the nonimmunogenic murine fibrosarcoma MCA 102. Tumor cells secreting IL-2 failed to grow in vivo despite normal in vitro growth. This growth inhibition required an intact immune system as tumors grew progressively in mice sublethally irradiated before tumor injection. Tumor inhibition was abrogated by the in vivo depletion, by specific mAb before tumor injection, of either CD8+ T cells or NK cells, but not CD4+ T cells. IL-2 secretion by tumor afforded a significant survival benefit to the animal, and IL-2-secreting tumor limited the growth of admixed nonsecreting parental tumor. Histologic evidence and FACS analyses revealed a dense lymphocytic infiltration of IL-2-secreting tumors composed of both CD4+ and CD8+ T cells. In contrast, secretion of TNF failed to inhibit the growth of MCA 102, and similar lymphocyte subset depletions, or administration of specific anti-TNF mAb had no effect on the growth of TNF secreting MCA 102. In summary, these investigations demonstrated that the host response to this nonimmunogenic tumor can be markedly enhanced by the genetic manipulation of the tumor cells to secrete IL-2, but not TNF. This strategy has potential application for the development of immunotherapies for nonimmunogenic tumors.
Biphasic Ca2+ responses in human basophils. Evidence that the initial transient elevation associated with the mobilization of intracellular calcium is an insufficient signal for degranulation.
Authors: MacGlashan D , Botana L M
Published: December 2022

Human basophils exhibited biphasic cytosolic Ca2+ responses after stimulation with both the bacterial peptide, FMLP and IgE cross-linking agents such as goat polyclonal anti-IgE PS myeloma (anti-IgE) antibody and Ag. The biphasic response to anti-IgE antibody was not apparent unless supraoptimal concentrations of anti-IgE antibody were used to stimulate the cells. Biphasic response characteristics were also observed in single cells. As in many cell types, the first phase of the Ca2+ response appeared independent of extracellular Ca2+. Challenging basophils with anti-IgE in the presence of EGTA, added simultaneously or 3 to 5 min before the stimulus, had little effect on the first transient Ca2+ elevation while completely eliminating the second phase of the response. We noted that while simultaneous addition of EGTA had little effect on the histamine release after challenge with FMLP, there was no histamine release after the simultaneous addition of anti-IgE and EGTA. An examination of the kinetics of histamine release after anti-IgE also demonstrated that no histamine release occurs during the initial transient response. Since the initial Ca2+ transient was intact after both stimuli in the presence of EGTA, we concluded that the initial transient was not sufficient to induce histamine release. The hypothesis that distinct sources of calcium were responsible for the initial Ca2+ transient that followed challenge with FMLP peptide or anti-IgE antibody was also tested. We found that prior treatment of basophils with thapsigargin inhibited the initial Ca2+ response that followed both FMLP and anti-IgE antibody. Furthermore, stimulation with FMLP, in the presence of EGTA, depleted the calcium source responsible for the anti-IgE response. The experiment conducted in reverse order showed that anti-IgE antibody could discharge the calcium source responsible for the initial FMLP Ca2+ response. These experiments indicated that FMLP and anti-IgE antibody use the same internal source of calcium to produce the initial Ca2+ response. Taken together with previous studies on FMLP, anaphylatoxin split product of the C component, C5; and platelet activating factor-induced release in human basophils, these results suggest that the initial transient elevation, which is relatively independent of extracellular Ca2+, is not sufficient to induce degranulation.
Peptide-priming of cytolytic T cell immunity in vivo using beta 2-microglobulin as an adjuvant.
Authors: Rock K L , Fleischacker C , Gamble S
Published: December 2022

CTL recognize oligopeptides bound to MHC class I molecules. Immunization of animals with antigenic peptides has often failed to stimulate CTL responses. We confirm that immunizations with several peptides, including natural and optimally active antigenic sequences, do not prime cytotoxic immunity in mice. However, immunization with peptides together with human beta 2-microglobulin primes Ag-specific CTL. Priming is observed when animals receive injections either i.v. with ex vivo peptide/beta 2-microglobulin-pulsed cells or s.c. with an admixture of peptide and beta 2-microglobulin. beta 2-Microglobulin promotes the priming of CTL immunity if it is added with peptide, but not if it is added after cells are exposed to peptide. Synthetic peptides and mixtures of peptides from enzymatically cleaved Ag are immunogenic. When a tryptic digest of OVA or the synthetic peptide (OVA258-276) are used as immunogens, the CTL that respond recognize the endogenously processed epitope presented by an OVA-transfected target cell. The peptide + beta 2-microglobulin-primed CTL are CD4-CD8+ and are class I MHC restricted. Using the immunization protocol with beta 2-microglobulin, we have primed CTL responses with peptides from OVA, Sendai virus, and vesicular stomatitis virus. These results may explain previous failures to prime CTL with peptides in vivo and provide a novel approach for developing peptide-based vaccines for viral diseases.
Fine specificity of the IgE interaction with the low and high affinity Fc receptor.
Authors: Nissim A , Schwarzbaum S , Siraganian R , Eshhar Z
Published: December 2022

The characterization of the site(s) on the IgE molecule that accommodate the high (Fc epsilon RI) and low (Fc epsilon RII) affinity receptors for IgE should allow the design of IgE analogues that can be used to block the onset of the allergic response or to regulate IgE production. To identify the IgE domain responsible for receptor binding, we generated a series of chimeric IgE antibodies in which constant region domains were interchanged between the human and mouse molecules. Binding studies with these chimeras revealed that both the high and low affinity receptor binding-sites reside primarily in the third constant domain of IgE (C epsilon 3). Additional chimeric IgE molecules were generated in which different parts of the human C epsilon 3 domain were exchanged with their murine homologues. Binding experiments with these chimeras suggest that not only the sequence of a particular C epsilon 3 fragment, but the entire C epsilon 3 domain in its native configuration is essential for binding to the Fc epsilon RI. The amino acid residues determining the species specificity of the Fc epsilon RII are not contained in the first 16 amino acids of the C epsilon 3 domain.
Isolation from Trypanosoma cruzi-infected mice of CD8+, MHC-restricted cytotoxic T cells that lyse parasite-infected target cells.
Authors: Nickell S P , Stryker G A , Arevalo C
Published: December 2022

Recent in vivo depletion studies in mice demonstrated that CD8+ T cells play a critical role in acute resistance to experimental Trypanosoma cruzi infections. As part of efforts to characterize these protective CD8+ T cell effector populations, we report here that splenic lymphocytes from mice chronically infected with T. cruzi can be induced to express high levels of cytolytic activity after stimulation in vitro with irradiated T. cruzi-infected macrophages. Cytolytic activity can either be detected using a nonspecific lectin-dependent 51Cr-release assay or using 51Cr-labeled T. cruzi-infected target cells. Fresh splenocytes from chronically infected mice stimulated with T. cruzi-infected macrophages exhibit relatively "promiscuous" killing activity inasmuch as significant lysis of both T. cruzi-infected and uninfected syngeneic and allogeneic cells is detected. However, subsequent rounds of in vitro stimulation lead to the expression of lytic activity that is T. cruzi Ag-specific and MHC-restricted. Several short term in vitro maintained cytolytic T cell lines were shown to have mixed phenotypes by FACS analysis; approximately 50% to 75% of the cells in these populations were CD4-, CD8+, whereas 20% to 40% were CD4-, CD8-. Experiments in which effector cells were positively selected by adherence to anti-CD8 mAb-treated plates confirmed that CD8+ T cell could exhibit Ag-specific cytolytic activity against T. cruzi-infected target cells. Efforts are under way to clone these CTL to test their in vivo function and to determine their Ag specificity.
Antimetastatic vaccination of tumor-bearing mice with two types of IFN-gamma gene-inserted tumor cells.
Authors: Porgador A , Bannerji R , Watanabe Y , Feldman M , Gilboa E , Eisenbach L
Published: December 2022

IFN-gamma genes were introduced through retroviral vectors into the high metastatic, low H-2Kb class I expressor, and poorly immunogenic 3LL-D122 clone. Two types of IFN-gamma infectants were isolated: IFN-gamma high secretors (128 to 256 IU/ml) and IFN-gamma non- or very low secretors (< 2 IU/ml). Both manifested high expression of MHC class I Ag. IFN-gamma secretors showed significant decrease in tumorigenicity and metastatic growth, whereas IFN-gamma nonsecretors retain tumorigenicity and were more metastatic than parental D122 cells. However, both groups, when inoculated in an irradiated form, induced similar high levels of CTL and protected mice to the same degree against a subsequent graft of parental cells. This indicates that enhanced expression of MHC class I and related genes caused by IFN-gamma is the major participant in CTL induction. Immunization of mice carrying an established tumor of parental D122 cells with IFN-gamma infectants is capable of almost completely preventing lung metastasis. Immunotherapy of tumor-bearing hosts is more effective when IFN-gamma secreting cells are used as immunogens, indicating that mechanisms, in addition to CTL, are stimulated by secreted IFN-gamma. Moreover, immunization with IFN-gamma high secretors of postoperative mice, carrying established micrometastases, almost completely cured these mice. Support for the participation of non-T cell effectors in the response to IFN-gamma secretors derives from the reduced tumorigenicity of these cells in CD1 nude mice. These data provide a rationale for the use of IFN-gamma gene-transferred tumor cells as a modality for cancer therapy.
Clonal heterogeneity in LFA-3 and ICAM-1 requirement for lysis by alloreactive T lymphocytes.
Authors: Galocha B , López D , López de Castro J A
Published: December 2022

HLA-B27-specific CTL are heterogeneous in their capacity to lyse murine P815 cells transfected with HLA-B27. Failure to kill murine transfectants could be caused by insufficient avidity of the human effector cells towards murine targets. Alternatively, it may imply alteration of allospecific T cell epitopes upon expression of HLA-B27 on mouse cells. To discern between these alternatives, P815 cells were co-transfected with HLA-B27, human ICAM-1, and LFA-3, and the transfectants were used as target cells with a series of HLA-B27-specific alloreactive CTL. Thirty-seven percent of the CTL tested significantly lysed HLA-B27(+)-P815 cells, without requiring simultaneous expression of human adhesion molecules. Twenty-one percent of the CTL showed significant lysis of only the murine transfectants expressing ICAM-1 or ICAM-1 + LFA-3. These CTL were shown by mAb-blocking analysis to have lower avidity than CTL from the previous group. In addition, they recognized HLA-B27 on murine cells not expressing human adhesion molecules, as assessed by cold target competition assays. As many as 42% of the CTL were unable to kill, or did so very inefficiently, P815 transfectants regardless of the presence of human ICAM-1 and LFA-3. With one detected exception, these CTL did not recognize HLA-B27 on murine cells in cold target competition assays. In contrast, they were able to recognize HLA-B27(+)-M1 fibroblast transfectant cells in direct cytotoxicity or cold target competition assays. Failure to kill murine transfectants by CTL from this group did not correlate with lower avidity, relative to CTL from the other groups, as shown by blocking experiments with mAb against human T cell adhesion molecules and their counter-receptors. These results indicate that lack of lysis of murine transfectants expressing class I HLA molecules by alloreactive CTL can be accounted for by low avidity of interspecies cell interactions in some cases but, more often, it is caused by alteration of allospecific T cell epitopes. Most likely, the basis for such alteration is allorecognition of HLA-B27-bound peptides that are expressed on human but not on mouse cells, mainly as a consequence of phylogenetic protein divergence between both species.
Recent thymic emigrants in the rat express a unique antigenic phenotype and undergo post-thymic maturation in peripheral lymphoid tissues.
Authors: Hosseinzadeh H , Goldschneider I
Published: December 2022

Studies of the functional properties and developmental potentials of immediate post-thymic cells have been hampered by the lack of reliable markers with which to distinguish recent thymic emigrants (RTE) from the bulk of peripheral T cells. In the present study, the intrathymic FITC-labeling technique was used in concert with three-color flow-cytometric analysis to identify, phenotypically characterize, and study the short term fate of RTE in normal rats. The results indicated that between 3 and 4% of total T cells in lymph node and spleen of 5- to 8-wk-old rats had been released from the thymus within the preceding 24 h. Unlike the bulk of the peripheral T cells, which had a Thy1-, CD45RC+, and/or RT6+ phenotype, these RTE were Thy1+, CD45RC-, and RT6-. Furthermore, two discrete subsets of RTE were defined: a major subset (approximately 95%) of CD4+ or 8+ (single positive), TCR-alpha beta hi T cells that resembled medullary thymocytes; and a minor subset (approximately 5%) of CD4+ and 8+ (double positive), TCR-alpha beta low T cells that resembled cortical thymocytes. The following evidence suggested that most if not all Thy1+ T cells in young adult rats are RTE and their immediate descendants: 1) thymectomy (but not sham thymectomy) selectively depleted Thy1+ T cells from lymph node within 3 to 7 days, even in adrenalectomized rats; 2) most FITC-labeled RTE differentiated into Thy1-, CD45RC+, RT6+ T cells within 7 days of release from the thymus; 3) transitional phenotypes of Thy1+ T cells, including Thy1low, CD45RC+, and RT6+, were observed in normal, as well as in intrathymic, FITC-injected rats; 4) most T cells in neonatal rats were Thy1+ and RT6-, whereas their descendants were Thy1- and RT6+. These experiments demonstrate that most RTE in normal rats are phenotypically (and presumably developmentally) immature at the time of release from the thymus, and progressively acquire the phenotypic attributes of more mature T cells post-thymically. These unique phenotypic attributes should expedite the isolation of RTE and their immediate descendants for definitive studies of their developmental and functional properties.
Control of IFN-inducible MxA gene expression in human cells.
Authors: Ronni T , Melén K , Malygin A , Julkunen I
Published: December 2022

MxA is an IFN-induced human protein which is located in the cytoplasm of induced cells. MxA makes the cells resistant to infection by influenza and vesicular stomatitis viruses. In the present work we used baculovirus expression system to produce MxA protein. The protein was purified to homogeneity and highly specific polyclonal anti-MxA antibodies were prepared. In human mononuclear cells, and A549 lung carcinoma cells expression of MxA protein is induced by very low (< 1 IU/ml) doses of leukocyte IFN-alpha (nIFN-alpha), whereas IFN-gamma does not seem to induce it or potentiate the induction by IFN-alpha. In mononuclear cells stimulated with high doses of leukocyte IFN-alpha concentrations, the amount of MxA mRNA was induced 10-fold at 4 h after IFN induction and up to 10-fold higher MxA protein levels were observed at 24-48 h postinduction. The gene can be reinduced by IFN-alpha 24 h after the initial induction suggesting for a lack of negative feedback after this time point. The protein is very stable, the half-life being approximately 2.3 days. Flow cytometric analysis revealed that monocytes have higher basal and induced MxA protein levels than lymphocytes but the dose-dependency of MxA expression is very similar in both cell types. Granulocytes are producing very low amounts of MxA protein.
Impaired cell-mediated immunity in mice bearing healthy orthotopic corneal allografts.
Authors: Sonoda Y , Streilein J W
Published: December 2022

A very high proportion of orthotopically grafted, histoincompatible corneas are accepted in healthy condition for prolonged intervals (> 8 weeks) by naive untreated BALB/c mice. Because these grafts form the anterior surface of the anterior chamber of the eye and because alloantigens placed in the anterior chamber elicit a deviant, stereotypic, systemic immune response known as anterior chamber-associated immune deviation, we have examined the state of alloantigen-specific cell-mediated immunity in BALB/c mice with accepted corneal allografts, as well as mice that had rejected their corneal grafts. Attempts to induce alloantigen-specific delayed hypersensitivity (DH) by s.c. injections of lymphoid cells, genetically identical to the grafts, resulted in positive responses only in "rejector" mice; "acceptor" mice failed to develop or display DH. Spleens cells from acceptors and rejectors were then assayed for the ability to suppress local adoptive transfer of alloantigen-specific DH. It was found that transfer reactions failed to develop if spleen cells from acceptor mice were added to the inoculum, whereas spleen cells from rejected mice had no similar inhibitory effects. The suppression mediated by acceptor spleen cells in local adoptive transfer assays was determined to be specific for the alloantigens expressed on the long-standing corneal allografts. We conclude that, when orthotopic corneal allografts are accepted indefinitely by adult mice, graft success correlates with the induction of alloantigenically specific anterior chamber-associated immune deviation. It is proposed that suppression of alloantigen-specific DH, which is characteristic of mice with accepted grafts, plays a critical role in the success of grafted corneas.
Defective T cell receptor-mediated signaling and differential induction of T cell functions by murine AIDS virus superantigen.
Authors: Kanagawa O , Wiebenga M E , Vaupel B A
Published: December 2022

A B cell line, B6-1710, expressing murine AIDS virus superantigen stimulates T cell hybridomas derived from a Ld-specific CTL clone to produce IL-2 regardless of their CD4/CD8 phenotype. However, B6-1710 cell did not stimulate the original CTL clone, L3, in either proliferation or cytolytic assays. Both B6-1710 and Ld+ P815 cells stimulated a significant Ca2+ influx in the T cell hybridomas. In contrast, only P815 stimulated tyrosine phosphorylation of a 19-kDa protein in the T cell hybridoma. The addition of the nonspecific protein kinase C activator PMA restored the proliferative response of L3 cells to B6-1710. PMA did not induce the lysis of B6-1710 cells by L3. These results suggest that murine AIDS virus encoded superantigen elicits a TCR-mediated signal different from that caused by nominal Ag. This different signaling by viral superantigen may be important for the development of viral pathogenesis in vivo.
IgE production in human helminth infection. Reciprocal interrelationship between IL-4 and IFN-gamma.
Authors: King C L , Low C C , Nutman T B
Published: December 2022

To determine whether there is a reciprocal relationship between IL-4 and IFN-gamma production in persons with parasite-induced elevations in serum IgE, PBMC were obtained from helminth-infected individuals with a broad range of serum IgE levels that fell into two distinct groups--extremely elevated (HI; n = 9; range, 4,129 to 18,400 ng/ml) or elevated (EL; n = 6; range, 403 to 1,018 ng/ml), relative to control subjects (NL; n = 7; range, 4 to 159 ng/ml). PBMC were stimulated in vitro, with Ag or mitogens, and IL-4 and IFN-gamma were measured by ELISA in culture supernatants. Helminth Ag- (but not tetanus toxoid) stimulated IL-4 production in eight of nine HI (range, 49 to 150 pg/ml) but was undetectable in either EL or NL (p < 0.01). In contrast, PBMC from EL produced significant levels of IFN-gamma to helminth Ag (GM = 358 pg/ml) compared with HI (GM = 89 pg/ml; p = 0.02) and NL (GM = 9 pg/ml; p < 0.001). Tetanus toxoid induced comparable levels of IFN-gamma among the three groups. Mitogen-driven IL-4 production was ninefold greater in HI [geometric mean (GM) = 913 pg/ml] versus EL (GM = 111 pg/ml; p < 0.01) and NL (GM = 193 pg/ml; p < 0.001) and correlated with serum IgE levels (r = 0.8; p < 0.01). Mitogen-driven IFN-gamma synthesis was equivalent among the groups. Although parasite Ag-driven IL-4 secreting CD4+ cells were detected (by ELISPOT) among infected subjects with both high and low serum IgE levels, the number of IL-4 exceeded that of IFN-gamma-secreting cells among individuals with elevated serum IgE levels, whereas the opposite relationship existed among subjects with normal serum IgE. In a subpopulation of infected individuals (n = 4), parasite-Ag added to PBMC cultures induced polyclonal IgE that was directly associated with the parasite-Ag-driven IL-4 production and inversely related to IFN-gamma synthesis in PBMC supernatants from parallel cultures. Furthermore, neutralizing anti-IFN-gamma antibody augmented both parasite-driven IL-4 synthesis and IgE production in vitro (n = 4). The data indicate that helminth-induced serum IgE levels are directly related to an increased capacity by PBMC to produce IL-4 and inversely associated to IFN-gamma production. It further supports the concept that IL-4 and IFN-gamma reciprocally regulate IgE in vivo.