bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct pathways of B cell differentiation. I. Residual T cells in athymic mice support the development of splenic germinal centers and B cell memory without an induction of antibody.
Authors: Stedra J , Cerny J
Published: December 2022

B cell memory to T cell-dependent Ags develops in the germinal centers (GC). Here we report that thymus-deficient, nu/nu mice immunized with phosphorylcholine coupled to keyhole limpet hemocyanin (EPC-KLH) develop GC in the spleen in the absence of Ab-forming cell (AFC) response. However, the formation of GC on EPC-KLH immunization requires T cells, because 1) CB.1.7-scid mice reconstituted with B lymphocytes failed to develop GC without a supplement of CD4+ cells and 2) in vivo administration of an anti-CD4 mAb abolished the GC response in euthymic mice. Thus, it appears that the formation of GC in nu/nu mice was due to a low number of T cells that were detectable in situ within the splenic lymphoid follicles. The numbers of GC in individual Ag-stimulated nu/nu mice appeared to correlate with the density of T cells in the splenic sections. The B cells in these GC expressed T15, the dominant Id of anti-PC Ab, and became primed for an anamnestic response. Secondary challenge with EPC-KLH resulted in an increased number of GC without detectable AFC. However, when the Ag-primed nu/nu mice received CD4+ lymphocytes 1 day before the challenge, they demonstrated a vigorous AFC response that was predominantly IgM and significantly higher than the secondary response of nu/nu mice that had been reconstituted with CD4+ cells during both primary and secondary immunizations. Therefore, it appears that immunization of nu/nu mice may lead to an early step of B cell activation and memory development even though the T lymphocytes in these mice are incompetent to provide help for Ab formation. The memory and Ab pathways of B cell differentiation may involve different mechanisms of T cell help.
Migration of so-called naive and memory T lymphocytes from blood to lymph in the rat. The influence of IFN-gamma on the circulation pattern.
Authors: Westermann J , Persin S , Matyas J , van der Meide P , Pabst R
Published: December 2022

It has been suggested that naive T lymphocytes migrate directly from the blood into the lymph nodes, whereas memory T lymphocytes arrive via the afferent lymphatics. However, these routes were defined indirectly by determining only the frequencies of naive and memory T lymphocytes in the afferent and efferent lymph of sheep. In this study naive (CD45RC+) and memory (CD45RC-) CD4+ lymphocytes obtained by thoracic duct cannulation of the rat were injected i.v. and their reappearance in the thoracic duct lymph (efferent lymph) was monitored in absolute numbers over 5 days. In addition, it was investigated whether continuous application of IFN-gamma influences the migration of the two subsets differently. After i.v. injection both naive and memory T lymphocytes returned to the thoracic duct in considerable numbers and with similar kinetic patterns. Interestingly, within 5 days at least 25% of memory phenotype lymphocytes converted to naive phenotype lymphocytes. When naive and memory T lymphocytes were injected in animals receiving a continuous IFN-gamma infusion, only the reappearance of the naive T lymphocytes but not that of memory T lymphocytes in the thoracic duct lymph was reduced. Thus, by investigating the migration of labeled lymphocytes, data were obtained that contradict conclusions drawn by analyzing preferential accumulation of lymphocyte subsets only. Our results suggest that in unstimulated animals naive and memory thoracic duct lymphocytes migrate at a similar tempo and to a comparable extent from the blood via the lymph node into the efferent lymph. IFN-gamma only influences the traffic of naive lymphocytes, indicating that the migration of the two populations is regulated by different mechanisms.
Fas antigen and p55 TNF receptor signal apoptosis through distinct pathways.
Authors: Wong G H , Goeddel D V
Published: December 2022

The Fas Ag and the p55 TNF receptor (TNF-R1) are related molecules that can signal apoptosis. Some tumor cell lines are selectively killed by Fas activation and others by TNF-R1 activation even though both receptors are often co-expressed. TNF-R1-mediated cytotoxicity can be selectively inhibited under conditions in which Fas-mediated cell death is not affected. Activation of both receptors results in synergistic signaling of apoptosis. These results indicate that different biochemical pathways are activated by Fas and TNF-R1. Combination treatment with agonists of Fas and TNF-R1 may have therapeutic potential.
The cloning of CD70 and its identification as the ligand for CD27.
Authors: Bowman M R , Crimmins M A , Yetz-Aldape J , Kriz R , Kelleher K , Herrmann S
Published: December 2022

CD70 is a surface Ag found on activated but not resting T and B lymphocytes. The biologic activity of this Ab-defined cell surface molecule on lymphocytes has not been established. Therefore, in an effort to understand the function of the CD70 protein, a mAb defining the CD70 Ag was used to isolate by expression cloning the cDNA responsible for the CD70 molecule. The predicted protein product is a type II transmembrane protein. Bioassays demonstrated that the CD70 cDNA clone expressed in African green monkey kidney cells would induce the proliferation of PHA-costimulated T cells. Comparison with known sequences indicates identity with the CD27 ligand. Therefore the molecule defining the CD70 Ag is identical to the recently defined ligand for CD27.
Antigen-specific CD8+ T cell clone protects against acute Toxoplasma gondii infection in mice.
Authors: Khan I A , Ely K H , Kasper L H
Published: December 2022

Encephalitis caused by the obligate intracellular protozoan, Toxoplasma gondii, is a major cause of morbidity and mortality in those afflicted with AIDS. The cell-mediated response is critical for host immunity against this parasite. A panel of T cell clones that proliferate in response to a major parasite protein, p30 (SAG-1) were isolated from mice. One of these clones (C3) bearing the CD8+ phenotype was able to induce nearly 100% protection against acute infection when adoptively transferred into naive mice. In contrast, an Ag-specific CD4+ T cell clone (C2) failed to show significant protection in spite of its ability to produce similar quantities of IFN-gamma. Depletion of host CD4+ T cells with mAb had no effect on CD8(+)-mediated adoptive protection, whereas treatment with anti-IFN-gamma completely abrogated this protection. These results indicate that Ag-specific CD8+ T cells in combination with IFN-gamma are able to induce significant protection against acute toxoplasmosis in the experimental murine model.
Acute inflammatory activity of the S100 protein CP-10. Activation of neutrophils in vivo and in vitro.
Authors: Devery J M , King N J , Geczy C L
Published: December 2022

The murine S100 chemotactic protein of m.w. 10,000 termed (CP-10), has potent chemotactic activity for murine and human myeloid cells. We examined the ability of a synthetic CP-10 hinge region peptide CP-10(42-55) and rCP-10 to act as chemotactic agents and induce expression of the adhesion molecule Mac-1 (CD 11b/CD 18) in vivo. Maximal neutrophil (PMN) accumulation occurred between 2 to 8 h after mouse footpad injection of rCP-10 (10(-7) M) or CP-10 peptide (10(-6) M). The infiltrating PMN expressed high levels of Mac-1, and low levels of the murine L-selectin Mel-14. Injection of CP-10 peptide i.p. also induced infiltration of PMNs that expressed high levels of Mac-1. Cell suspensions obtained after i.p. injection of CP-10 peptide could be significantly inhibited from adhering to fibrinogen-coated plates when incubated with anti-Mac-1 antibody. The chemotactic activity of CP-10 peptide toward murine inflammatory PMN in vitro was also inhibited by anti-Mac-1 antibody. Neither CP-10 analogue stimulated or primed murine inflammatory or human blood neutrophils for superoxide production or granular enzyme release. The localization of CP-10 in vivo was examined using murine footpads injected with LPS and was found to be concentrated around the endothelial cells of the small blood vessels. This distribution suggests that the accumulated CP-10 the may contribute to the generation of a chemotactic gradient.
Properties of protein kinase C isoforms (beta II, epsilon, and zeta) in a macrophage cell line (J774) and their roles in LPS-induced nitric oxide production.
Authors: Fujihara M , Connolly N , Ito N , Suzuki T
Published: December 2022

Northern analysis of poly(A)+ RNA extracted from J774 cells (a mouse macrophage cell line) showed that this cell line constitutively expresses mRNAs specific for protein kinase C (PKC)-beta I, -beta II, -epsilon and -zeta, but not those for PKC-alpha, -gamma or -delta. Western analysis of the total cell lysate showed that J774 cells express PKC-beta II, -epsilon and -zeta isoenzymes, but failed to show the expression of PKC-beta I. The exposure of J774 cells to > 10 nM PMA led to a loss of immunoreactive PKC-beta II in 4 h. The down-regulation of immunoreactive PKC-epsilon required more than 8 h of the exposure to > 100 nM PMA. Immunoreactive PKC-zeta was most resistant to PMA treatment and was not significantly reduced after the exposure to 300 to 600 nM PMA for 24 h. PMA-mediated, persistent down-regulation of PKC-beta II is probably a result of the inhibition of PKC-beta II biosynthesis at the posttranscriptional level, because PMA-exposed cells were found to gradually increase the expression of PKC-beta II specific mRNA. PMA-pretreated cells responded to a low dose (10 ng/ml), but not to a high dose (1 microgram/ml), of LPS by significantly lower expression of mRNA specific for the inducible nitric oxide synthase (i-NOS) gene and production of nitric oxide (NO) than the control cells did. Thus, PKC could be a part of the signal transduction apparatus involved in LPS-induced inducible nitric oxide synthase gene activation.
Deletion and editing of B cells that express antibodies to DNA.
Authors: Chen C , Radic M Z , Erikson J , Camper S A , Litwin S , Hardy R R , Weigert M
Published: December 2022

We previously demonstrated that in mice transgenic for genes coding for an anti-ssDNA autoantibody B cells were functionally inactivated but not physically deleted. We have now extended this model by introducing an arginine into the CDR2 of the heavy chain transgene. This change alters the specificity of the Ab from anti-ssDNA to anti-dsDNA and increases the affinity for ssDNA. Mice carrying this transgene displayed a significant reduction of peripheral B cells and anti-dsDNA B cells were not recovered from the spleens. The remaining B cells escape deletion by revising their Ag receptors in several ways: 1) elimination of the transgenic heavy chain gene via intrachromosomal recombination, followed by rearrangement and expression of endogenous VH genes; 2) ongoing rearrangement of endogenous kappa light chain genes to generate a non-dsDNA-binding Ab; and 3) expression of a rare V lambda gene, V lambda x, to generate a non-DNA-binding Ab.
Development and characterization of channel catfish long term B cell lines.
Authors: Miller N W , Rycyzyn M A , Wilson M R , Warr G W , Naftel J P , Clem L W
Published: December 2022

The establishment of channel catfish long term cloned B cell lines, the first such cell lines from ectothermic vertebrates, is described. These diploid cell lines were developed by in vitro LPS stimulation of B cells from normal channel catfish peripheral blood in the absence of overt attempts to transform or immortalize the cells. The resultant cell lines were cloned and maintained continuously in vitro for more than 12 mo without restimulation, feeder cells, or exogenous factors. Southern blot analyses of the parental cell lines revealed multiple mu-chain gene rearrangements, suggesting a polyclonal origin for the cell lines. Additional evidence for polyclonal development was provided by the demonstration that the parental cell lines transcribed mRNA for all of the six known channel catfish VH gene families. The characterization of several clonal cell lines revealed mRNA expression for both the secreted and membrane forms of the catfish mu-chain; however, the cloned cell lines each expressed only a single VH gene and analysis of the Ig H chain locus was consistent with allelic exclusion having occurred in these cells. Flow cytometry demonstrated that the cloned and uncloned cell lines produced both cytoplasmic and cell surface IgM. This IgM contained only one of the two L chain isotypes of the channel catfish, suggesting preferential L chain usage. Although these cells did not appear morphologically to be plasma cells, they secreted moderate levels of IgM in culture. These cell lines have considerable potential for addressing questions concerning the evolution of B cell function.
MTS-32 monoclonal antibody defines CD4+8- thymocyte subsets that differ in their maturation level, lymphokine secretion, and selection patterns.
Authors: Vicari A , Abehsira-Amar O , Papiernik M , Boyd R L , Tucek C L
Published: December 2022

We have previously described MTS-32 as identifying an Ag on both thymic stromal cells and thymocytes. In contrast with CD4+8+ and CD4-8+ thymocytes, of which the vast majority are MTS-32+, a notable subset of CD4+8- thymocytes is MTS-32-. Here we show that with regard to heat-stable Ags, Qa-2, and CD69 expression CD4+8- MTS-32- thymocytes are phenotypically enriched in mature cells when compared with their MTS-32+ counterparts. Moreover, sorted CD4+8- MTS-32+ thymocytes are unable to respond to anti-CD3 cross-linking, whereas MTS-32- CD4+8- thymocytes respond to the same stimulus by producing IL-4, IL-5, IL-10, IFN-gamma, and trace amounts of IL-2. In addition, MTS-32- CD4+8- and CD4-8- TCR-alpha beta+ thymocytes differ in their TCR V beta repertoire on a Mls-1a selecting background. We therefore suggest that the MTS-32 ligand is involved in signals consecutive with TCR recognition in the thymus, i.e., selection, activation, and lymphokine production.
Role of infused CD8+ cells in the induction of peripheral tolerance.
Authors: Zhang L , Shannon J , Sheldon J , Teh H S , Mak T W , Miller R G
Published: December 2022

The CD8 molecule when present on a cell recognized by a mature T cell has been shown to have an inhibitory effect on the development of an in vitro immune response by that T cell. To test whether CD8 has a role in the induction of peripheral tolerance in vivo, we have studied tolerance induction in B6 female mice carrying a transgenic TCR specific to male H-Y Ag. Previous studies have shown that male Ag reactive cells are inactivated in these mice after the i.v. injection of viable male lymphoid cells. Here, B6 female transgenic mice were injected with B6 male lymph node cells from CD4 or CD8 gene knockout mice, and the fate of male Ag-specific T cells was followed in vivo. Our results indicate that injection of male CD4 knockout (express CD8) lymphoid cells but not CD8 knockout (do not express CD8) lymphoid cells induced a significant reduction of male H-Y Ag reactive cells in the periphery, suggesting that CD8 plays an important role in the induction of peripheral tolerance of class I-restricted T cells. These results imply that peripheral tolerance can be induced not just by absence of signals required for activation but by the presence of additional signals.
Regulation of IFN-gamma-induced nuclear expression of IFN consensus sequence binding protein in murine peritoneal macrophages.
Authors: Politis A D , Ozato K , Coligan J E , Vogel S N
Published: December 2022

IFNs are well characterized macrophage-activating agents. Their varied effects are largely mediated via the induction of many genes, whose products act in concert to induce macrophage differentiation. Homologous DNA sequences have been found upstream of the promoter in many of these IFN-inducible genes and bind a family of trans-acting proteins. Interferon consensus sequence binding protein (ICSBP) is one member of this family of interferon regulatory factors (IRF) and is structurally related within the DNA-binding domain to the other members, IRF-1, IRF-2, and ISGF3 gamma. ISCBP mRNA levels become elevated in response to IFN-gamma; however, little is known about the regulation of ICSBP expression at the protein level. In this study, anti-ICSBP peptide Abs were used to quantify and localize ICSBP in murine peritoneal exudate macrophages. Western blot analysis of cytoplasmic and nuclear extracts from treated and control cells revealed ICSBP to be induced by IFN-gamma and not by IFN-alpha and to exist primarily in the nucleus. The regulation of ICSBP induction by IFN-gamma was consistent with the characteristics found at the mRNA level; inhibition by IFN-alpha or glucocorticoids and the requirement for protein kinase C (as determined pharmacologically). The time course of IFN-gamma-induced ICSBP showed an induction of protein that required approximately 12 h to reach maximal levels. Induced ICSBP was relatively stable, exhibiting a half-life of approximately 48 h. Indirect immunofluorescence also demonstrated ICSBP to be an IFN-gamma-inducible protein that is strongly localized to the nucleus.
Purification and cloning of a novel serine protease, RNK-Tryp-2, from the granules of a rat NK cell leukemia.
Authors: Sayers T J , Wiltrout T A , Smyth M J , Ottaway K S , Pilaro A M , Sowder R , Henderson L E , Sprenger H , Lloyd A R
Published: December 2022

We have biochemically purified a 27-kDa serine protease (designated RNK-Tryp-2) from the granules of the rat large granular lymphocyte leukemia cell line (RNK-16) which has tryptase activity. Utilizing molecular sieve chromatography and reverse-phase HPLC, we purified RNK-Tryp-2 to homogeneity and sequenced 33 NH2-terminal amino acids. Oligonucleotide primers were used in the PCR to generate a 528-bp cDNA clone encoding a novel serine protease from RNK-16 mRNA. This cDNA clone was used to isolate an 884-bp RNK-Tryp-2 cDNA from an RNK-16 lambda-gt11 library. The open reading frame predicts a mature protein of 233 amino acids which does not have potential sites for N-linked glycosylation. The cDNA encodes a leader peptide of at least 25 amino acids. The characteristic Ile-Ile-Gly-Gly amino acids of the N-terminus, and the His, Asp, and Ser amino acids that form the catalytic triad of serine proteases, are conserved. The amino acid sequence has less than 45% identity with any other member of the serine protease family, indicating that RNK-Tryp-2 is distinct protease. Southern blot analysis suggests the existence of one or more related genes. A single 1.3-kb mRNA transcript was detected by Northern blot analysis of total cellular RNA from the in vivo passaged RNK-16, rat splenocytes, lung and liver nonparenchymal cells, as well as in highly purified rat LGL and T cells. RNK-Tryp-2 is a novel serine protease that is expressed in the granules of large granular lymphocytes.
Unusual topology of an HLA-B27 allospecific T cell epitope lacking peptide specificity.
Authors: Villadangos J A , Galocha B , López de Castro J A
Published: December 2022

Recognition of MHC + peptide complexes by TCRs is thought to involve a large surface formed by exposed residues from the bound peptide and from the alpha-helices of the MHC protein. This interaction appears to be essentially symmetrical in the positioning of the TCR relative to the MHC molecule. In this study the topology of HLA-B27 recognition by an alloreactive TCR, 64.8P, has been analyzed with a panel of site-specific mutants that have changes at multiple positions along the peptide binding site of HLA-B27. Abrogation of transfectant target cell lysis by CTL 64.8P was obtained only with some mutations in the peptide side chain binding pockets A and B, whereas little or no effect was observed with mutations outside these pockets. CTL 64.8P efficiently lysed murine transfectant cells, including HLA-B27+ RMA-S cells. Recognition of this latter transfectant was more efficient upon increased HLA-B27 expression at 26 degrees C. The uneven distribution of mutations affecting HLA-B27 allorecognition by CTL 64.8P strongly suggests an asymmetrical topology in the interaction of this TCR with HLA-B27, in which most of the binding energy is provided by contacts with HLA-B27 and/or peptide residues located close to pockets A and B, with little contribution from other areas of the MHC or peptide molecules. Its conservation in RMA-S cells further suggests that the epitope recognized by CTL 64.8P is either peptide-independent or requires any of a set of peptides having the same amino-terminal residues.
The role of IL-2 secreted from genetically modified tumor cells in the establishment of antitumor immunity.
Authors: Bannerji R , Arroyo C D , Cordon-Cardo C , Gilboa E
Published: December 2022

Although the importance of CD4+ T cells for the induction of an effective CD8+ cytolytic T cell response is well documented, the mechanism by which MHC class II-negative tumor cells recruit CD4+ T help is not well understood. We have previously shown that IL-2 or IFN-gamma gene-modified CMS-5 tumor cells do not grow in syngeneic mice; however, mice which rejected the cytokine-secreting tumor cells develop a protective immune response against a challenge with parental tumor cells. Here we show that rejection of IL-2-secreting CMS-5 cells is not mediated by T cells. However, establishment of a protective immune response against CMS-5 tumor cells requires the presence of both CD4+ and CD8+ T cell subsets during the period of immunization with IL-2-secreting CMS-5 cells as well as during the effector phase. Extensive histologic analysis has failed to detect the presence of T cells at the site of immunization with either IL-2- or IFN-gamma-secreting CMS-5 cells. The main infiltrate at the site of inoculation with IL-2-secreting CMS-5 cells consisted of NK cells that appeared to play a role in their rejection. The predominant infiltrate at the site of inoculation with IFN-gamma-secreting CMS-5 cells consisted of macrophages. These observations argue against a direct role for the intact tumor cell in presenting either T helper or CTL epitopes to the immune system, and support the view that specialized APC are responsible for the in vivo priming of a T cell response against MHC class I-restricted Ag.
Differential cytokine expression in maternal blood and placenta during murine gestation.
Authors: Delassus S , Coutinho G C , Saucier C , Darche S , Kourilsky P
Published: December 2022

To evaluate the influence of gestation on peripheral blood T cells, we measured the mRNA production of several cytokines (IL-2, IL-4, IL-10, IFN-gamma) and the p55 subunit of IL-2R at different time points in the blood of pregnant mice and in the placenta. This was made possible by the use of a new PCR technique that is precise and quantitative. Our results show that pregnancy induces profound changes in the expression of these genes in peripheral blood cells. During the first week of gestation, there is an increase in the levels of all the cytokines, followed by a state of immunodepression characterized by levels of cytokines below normal (nonpregnant mice). In the placenta low levels of IL-2 and IL-10 are detected. IFN-gamma mRNA production is higher than the blood IFN-gamma mRNA in the last week of pregnancy. However, the main difference is found for IL-4 mRNA expression where the placenta levels are 5- to 10-fold higher than the blood mRNA expression. We discuss these results in the context of the placenta as a privileged immune site, where IL-4, being the main cytokine, may play a major regulatory role.
Cefdinir (CI-983), a new oral amino-2-thiazolyl cephalosporin, inhibits human neutrophil myeloperoxidase in the extracellular medium but not the phagolysosome.
Authors: Labro M T , el Benna J , Charlier N , Abdelghaffar H , Hakim J
Published: December 2022

Cefdinir, a new oral 2-amino-5-thiazolyl cephalosporin, inhibited the luminol-amplified chemiluminescence (LACL) response of human neutrophils stimulated by PMA but not opsonized zymosan, in a concentration-dependent but not time-dependent manner. The LACL response to opsonized zymosan in cytochalasin B-treated neutrophils was, however, inhibited by cefdinir. Various cephalosporins, regardless of the presence of a 2-amino-5-thiazolyl moiety, did not significantly alter the neutrophil LACL response triggered by PMA and zymosan. The LACL response induced by the calcium ionophore A23187 and FMLP was also impaired by cefdinir, and this impairment was increased in cytochalasin B-treated neutrophils. Superoxide anion generation by neutrophils, measured in terms of lucigenin-amplified chemiluminescence and cytochrome c reduction, was not altered. Spontaneous and FMLP-induced neutrophil degranulation, assessed by lysozyme and beta-glucuronidase release, were not modified by cefdinir. Furthermore, cefdinir inhibited LACL generation in cell-free systems consisting of H2O2, NaI, and either horseradish peroxidase or a myeloperoxidase-containing neutrophil extract. Orthodianisidine oxidation in these two acellular systems was inhibited by cefdinir. Cefdinir did not alter neutrophil bacterial killing at concentrations that inhibited myeloperoxidase-containing neutrophil extract-dependent reactions induced by soluble stimuli. Taken together, these data strongly suggest that cefdinir directly inhibits the activity of myeloperoxidase-containing neutrophil extract released into the extracellular medium during neutrophil stimulation by soluble mediators, but has no effect on that released into the phagolysosome during phagocytosis. This unusual property of a member of the beta-lactam family could be of interest in modulating the exaggerated inflammatory process often associated with infectious diseases.
Neutrophil-activating peptides NAP-2 and IL-8 bind to the same sites on neutrophils but interact in different ways. Discrepancies in binding affinities, receptor densities, and biologic effects.
Authors: Petersen F , Flad H D , Brandt E
Published: December 2022

IL-8 and the neutrophil-activating peptide 2 (NAP-2) are members of the chemokine family of host defense cytokines. Although IL-8 was shown to interact with two different high affinity receptors on polymorphonuclear neutrophil granulocytes (PMN), direct demonstration of specific binding sites for NAP-2 is difficult, because the NAP-2 molecule lacks iodinable side chains. Here we present a modified labeling procedure for the chemokine that does not affect its biologic activity. The 125I-labeled NAP-2 specifically bound to PMN with two different affinities (KD = 0.65 and 22.4 nM). We observed complete cross-competition of unlabeled IL-8 with 125I-labeled-NAP-2 and of unlabeled NAP-2 with 125I-labeled IL-8, indicating the absence of monospecific binding sites for either chemokine. However, in contrast to former work by others, the total number of accessible sites was considerably lower for NAP-2 (13,000/cell) than for IL-8 (59,000/cell). In addition, PMN prepared from heparinized blood expressed significantly more receptors for NAP-2 than cells prepared from citrated blood, whereas receptor numbers for IL-8 were unchanged. Desensitization experiments suggested a regulatory role for the NAP-2 high affinity site. Short-term priming of PMN with a nonstimulatory dose of NAP-2 (or MGSA) but not with IL-8 led to drastic down-regulation of the subsequent degranulation response, challenged by higher dosages of NAP-2, MGSA, or IL-8. Reduced functional responsiveness of cells correlated with the rapid down-regulation and internalization of NAP-2 and IL-8 high affinity binding sites. Thus, our data indicate that chemokines could mediate by individual modes of interaction with common receptor's different biologic functions.
Cross-linking of ICAM-1 induces co-signaling of an oxidative burst from mononuclear leukocytes.
Authors: Rothlein R , Kishimoto T K , Mainolfi E
Published: December 2022

Cell adhesion molecules were first described as accessory molecules simply to bridge one cell to another. More recently, it has been realized that these molecules also transmit signals from outside of the cell to inside. We show that cross-linking of the ICAM-1 on the cell membrane with anti-ICAM-1 mAb and F(ab')2 fragments of goat anti-MIgG in the presence of suboptimal levels of the bacterial peptide FMLP results in co-stimulation of an oxidative burst from CD14 expressing PBMCs. The amplitude of the oxidative response was less than the oxidative burst induced by CD18 cross-linking, whereas the response was more prolonged. On the other hand, cross-linking by anti-L-selectin mAb plus F(ab')2 fragments of goat anti-MIgG induced a minimal oxidative burst that was not significantly greater than the response generated by anti-L-selectin mAb alone. The addition of an excess of soluble ICAM-1 to compete for the anti-ICAM-1 mAb inhibits the oxidative burst in response to ICAM-1 cross-linking but not to CD18 cross-linking. These results suggest that ICAM-1 is capable of delivering a transmembrane signal into CD14-positive PBMC.
Characterization of rabbit complement component C8. Functional evidence for the species-selective recognition of C8 alpha by homologous restriction factor (CD59).
Authors: White R V , Kaufman K M , Letson C S , Platteborze P L , Sodetz J M
Published: December 2022

Protection of host cells from lysis by the C5b-9 cytolytic complex is provided by a membrane-associated protein (CD59) that interacts with homologous C8 and C9 during C5b-9 assembly. This interaction restricts normal formation and function of the complex, thereby protecting cells from attack by homologous C. In this study, rabbit C8 was purified and characterized and used to investigate the role of the individual C8 subunits in homologous restriction and the basis for species selectivity by human CD59. Exchanging the disulfide-linked C8 alpha-gamma dimer in human C8 with one from rabbit was found to be sufficient to overcome restriction by human Es. Activity of the hybrid C8 and rabbit C8 toward these target cells was equivalent, thus establishing that restriction is not dependent on the species of C8 beta. Because C8 gamma was previously shown to have no role in restriction, these results suggest that within C8, structural determinant(s) recognized by CD59 reside solely on C8 alpha. Sequences determined from full-length cDNA clones for rabbit C8 alpha, C8 beta, and C8 gamma support this conclusion. All three subunits are strikingly similar to human with regard to length, m.w., N- and C-terminal residues, conserved cysteines, and overall sequence. However, when sequences are compared under high stringency, a single segment of extended sequence dissimilarity is detected between the two species of C8 alpha. Within human C8 alpha, this 37-residue segment resides near the putative membrane-interacting region and may constitute a human CD59 recognition site.
IFN-gamma, TNF, and IL-6 production by vaccinia virus immune spleen cells. An in vitro study.
Authors: Carpenter E A , Ruby J , Ramshaw I A
Published: December 2022

The production of cytokines by virus immune spleen cells after in vitro restimulation was investigated. Vaccinia virus-primed spleen cells from CBA/H mice were stimulated in vitro with virus-infected UV-irradiated syngeneic cells. Both CD4+ and CD8+ T cell populations proliferated after restimulation. TNF, IL-6, and IFN-gamma were detected within 12 h of restimulation with maximal levels reached by 24 h. Adherent cells were major producers of IL-6 and TNF, whereas IFN-gamma production was dependent on CD4+ T cells and adherent cells. The IFN-gamma response was Ag specific, whereas the production of TNF and IL-6 was not. Stimulation with vaccinia virus constructs encoding IFN-gamma or TNF altered the levels of cytokines produced, in that IFN-gamma expression by stimulator cells led to increased IFN-gamma production, whereas TNF expression by stimulator cells augmented both TNF and IFN-gamma production by responder cells.
Changes in glucose transport and transporter isoforms during the activation of human peripheral blood lymphocytes by phytohemagglutinin.
Authors: Chakrabarti R , Jung C Y , Lee T P , Liu H , Mookerjee B K
Published: December 2022

We have explored the mechanism of stimulation of glucose transport during PHA stimulation of human peripheral blood lymphocytes (HPBT) enriched in T cells. Equilibrium exchange flux of 3-O-methyl glucose (3-O-MG) was stimulated two- and fourfold at 24 and 48 h after PHA stimulation, respectively. The increase was transient in that the flux rate returned to control (unstimulated) levels by 96 h. Immunoblotting and immunoprecipitation using specific Abs revealed that resting HPBT expresses glucose transporter isoforms GLUT-2 and GLUT-3 but not GLUT-1. After PHA stimulation, GLUT-1 expression was induced predominantly in the plasma membrane, whereas GLUT-3 expression was simultaneously down-regulated. GLUT-1 expression was detectable at 24 h, peaked at 48 h, and disappeared at 96 h. The total number of glucose transporters per cell measured as the total capacity of D-glucose displaceable cytochalasin B binding did not change significantly at any time after PHA stimulation. PHA stimulation also caused expression of high affinity IL-2R and secretion of IL-2. The IL-2 secretion was transient, which peaked at 24 h, slightly preceding the GLUT-1 expression peak and disappeared at 72 h. In PHA-activated HPBT cells synchronized at G0-G1, GLUT-1 was not expressed but was rapidly induced by exposure to IL-2. This induction did not occur in the presence of cyclosporin A, which inhibits IL-2 secretion. Based on these observations, we conclude that PHA stimulation increases glucose transport partly by inducing the expression of GLUT-1 instead of GLUT-3 and that GLUT-1 expression is induced by signals generated by IL-2 binding to its high affinity receptors.
IL-12 influences intrathymic T cell development.
Authors: Godfrey D I , Kennedy J , Gately M K , Hakimi J , Hubbard B R , Zlotnik A
Published: December 2022

IL-12 has been implicated in the maturation and activation of peripheral T lymphocytes and NK cells. In the present study we have investigated the potential role of IL-12 in intrathymic T cell development. Treatment of mouse fetal thymic organ culture with IL-12 caused a significant reduction in size and cell number compared with the untreated controls. Flow cytometric analysis of the thymocytes recovered from these lobes showed differential effects on individual thymocyte subsets, most but not all of which were significantly decreased. In contrast, however, we observed an increase in both the percentage and cell number of alpha beta TCR+CD4-CD8+ thymocytes. This effect could be neutralized with an anti-IL-12 antibody, demonstrating the specificity of the influence of IL-12 in the fetal thymic organ culture system. IL-12 caused proliferation of isolated thymocyte subsets, particularly CD3+CD4-CD8+ cells in the presence of IL-2 and IL-4. Additionally, IL-12 induced significant proliferation of early CD3-CD4-CD8- triple negative CD44+CD25+ pro-T cells in combination with stem cell factor. We show that both the p35 and p40 chains of IL-12 are produced in the fetal and adult mouse thymus and that thymic stromal cells are a potential source of this cytokine.
Differential activation of p21ras in CD45RA+ and CD45RO+ human T lymphocytes.
Authors: Patel H R , Renz H , Terada N , Gelfand E W
Published: December 2022

CD45RA+ and CD45RO+ human T cells differ in their functional responses to different forms of mitogenic stimulation. However, little is known about the intracellular signaling events related to the functional differences in these two subsets. In the present study, we examined the ability of different Abs to the TCR/CD3 complex to activate the p21ras proto-oncogene product in CD45RA+ and CD45RO+ T cells. Stimulation with soluble OKT3 anti-CD3 mAb induced activation of p21ras in CD45RA+ cells but not in CD45RO+ cells, although soluble anti-CD3 was able to induce both activation of CD3-associated kinases and IL-2 responsiveness in CD45RO+ cells. Similar results were obtained by using UCHT1, a different anti-CD3 Ab. In contrast, cross-linked anti-CD3 or the combination of phorbol ester and ionomycin were found to activate p21ras equally in both T cell subsets. Our results demonstrate a selective inability of CD45RO+ human T cells to activate p21ras in response to soluble anti-CD3 mAb.
Immunologic and molecular characterization of Amb p V allergens from Ambrosia psilostachya (western Ragweed) pollen.
Authors: Ghosh B , Rafnar T , Perry M P , Bassolino-Klimas D , Metzler W J , Klapper D G , Marsh D G
Published: December 2022

We have purified and characterized the Amb p V allergen (A1 variant) from western ragweed (Ambrosia psilostachya) pollen. This allergen was found to be highly cross-reactive with the Amb a VA1 allergen from short ragweed (A. artemisiifolia) pollen in a competitive double-Ab radioimmunoassay (DARIA) and the two allergens showed concordant allergenic potency in histamine-release experiments. We cloned and sequenced several Amb p V genes from western ragweed pollen and flowers by direct PCR of genomic DNA. The amino acid sequences deduced from the nucleotide sequences indicated the presence of multiple forms of Amb p V that could be broadly classified into two groups: Amb p VA and Amb p VB variants. The sequences of the Amb p VA variants are highly homologous to Amb a V (about 90% identity) and very similar to the protein sequence that we obtained. The Amb p VB variants share approximately 65% amino acid homology with Amb a V and have five to seven cysteine residues as compared with the eight found in Amb a V and Amb t V. Two cysteine residues that form disulfide bonds in other Amb Vs (positions 19 and 43 in Amb a V) are replaced by serine and alanine in the Amb p VB1 and Amb p VB2 variants. We have generated model structures of Amb p VA1, VA2, VA3, and VB1 variants from the nuclear magnetic resonance-derived structure of Amb a VA1 by homology modeling. Comparison of antigenic epitopes predicted for the structures of Amb p V variants and Amb a VA1 explains the observed cross-reactivity of the two ragweed proteins and suggests the epitopes likely to be involved in Ab recognition.
Exploration of requirements for peptide binding to HLA DRB1*0101 and DRB1*0401.
Authors: Hill C M , Liu A , Marshall K W , Mayer J , Jorgensen B , Yuan B , Cubbon R M , Nichols E A , Wicker L S , Rothbard J B
Published: December 2022

The individual amino acid contacts responsible for peptide binding to DRB1*0101 and/or DRB1*0401 were defined using a quantitative binding assay. The differential contribution of each amino acid in two well studied T cell determinants, HA307-319 and RMBP 90-102, was delineated by comparing the IC50 values of analogues of varying length. This analysis confirmed the importance of a hydrophobic amino acid located near the amino-terminus for binding to both alleles and revealed that the contribution of the carboxyl-terminal amino acids differed between DRB1*0101 and DRB1*0401. Taking advantage of previous experiments demonstrating that all of the residues could be replaced with alanine, with the exception of the key hydrophobic amino acid, simplified analogues composed of polyalanines were used to prove 1) optimal binding depended on the position of the hydrophobic side chain relative to the amino- and carboxyl-termini; 2) aromatic amino acids were superior to aliphatic side chains at this position; and 3) a significant amount of free energy of binding arises from hydrogen bonding between the class II binding site and the amide bonds of the ligand. The role of each carbonyl and amide nitrogen was measured by assaying analogues containing reduced peptide bonds or N-methyl amino acids. Serine, but not glycine, could be used as a framework amino acid for peptide ligands, indicating that the beneficial aspects of these simplified structures was the combination of retaining the correct orientation of the peptide bonds, the restriction of the conformational freedom by limiting the possible phi/psi angles of the peptide, and avoidance of deleterious side-chain contacts. Collectively, these data were consistent with the peptide binding in a nonrepeating conformation with the vast majority of the free energy of binding arising from hydrogen bonds with the peptide backbone and a single, key hydrophobic side chain interacting in a conserved pocket in both DRB1*0101 and DRB1*0401.
Biased utilization of DHQ52 and JH4 gene segments in a human Ig transgenic minilocus is independent of antigenic selection.
Authors: Tuaillon N , Miller A B , Longberg N , Tucker P W , Capra J D
Published: December 2022

We have assessed the effect of antigenic selection on human Ig heavy chain D and JH gene segment utilization in mice that contain transgenes composed of 2 VH (psi VH3-105 and VH5-251), 10 D, 6 JH, C mu, and C gamma 1 human gene segments. Human heavy chains using the functional VH5-251 gene segment are expressed in the serum and on the surface of murine B cells. The second VH gene segment (psi VH3-105) is not expressed as a protein but is rearranged and transcribed into mRNA. We previously reported that the functional VH5-251 mu transcripts preferentially used the DHQ52 and JH4 gene segments similar to their use in the human repertoire. Here, we demonstrate that the nonfunctional (psi VH3-105) gene segment shows the same bias in D and JH gene segment utilization. Because transcripts using the pseudo-VH gene segment cannot be subjected to antigenic selection, we conclude that the restricted repertoire observed is Ag independent. Analysis of pseudo VH gene segment recombination products reveals no bias in D gene segment reading frame utilization. Finally, we demonstrate that in the human transgene coding sequence complementarities can affect the recombination site and that D inversion is common.
Characterization of the T cell determinants in the induction of autoimmune arthritis by bovine alpha 1(II)-CB11 in H-2q mice.
Authors: Brand D D , Myers L K , Terato K , Whittington K B , Stuart J M , Kang A H , Rosloniec E F
Published: December 2022

Collagen-induced arthritis (CIA) is an experimental autoimmune disease elicited in genetically susceptible strains of mice by immunization with heterologous type II collagen. This experimental disease is mediated by the immune response of both T and B cells, and susceptibility is restricted by the class II molecules of the MHC. To study the T cell determinants of bovine type II collagen (CII) that mediate the autoimmune response in H-2q mice, we have identified a cyanogen bromide fragment of bovine CII, CII(124-402), that induces arthritis in DBA/1 mice. Using an overlapping set of peptides to map the T cell response to CII(124-402), we have determined that the I-Aq-restricted T cell response to this collagen fragment is mediated by a single immunodominant antigenic determinant. Consequently, this determinant plays a central role in promoting the production of the collagen-specific Abs and the induction of CIA in H-2q mice. Characterization of this immunodominant determinant revealed that the core residues required for T cell stimulation consists of only eight amino acids and is located at amino acids 260 through 267 of bovine CII. The systematic analysis of the contribution of each of these amino acids, in conjunction with sequences of other peptides known to bind to I-Aq, have allowed us to propose a peptide binding motif for the collagen arthritis susceptibility allele, I-Aq.
Class I-restricted cytotoxic T lymphocytes are directly cytopathic for their target cells in vivo.
Authors: Ando K , Guidotti L G , Wirth S , Ishikawa T , Missale G , Moriyama T , Schreiber R D , Schlicht H J , Huang S N , Chisari F V
Published: December 2022

Although CD8-positive, class I-restricted CTL play a critical role in viral clearance and immunopathology in many model systems, they have not been shown to directly kill their target cells in vivo at the single cell level. Using a hepatitis B surface Ag transgenic mouse model of class I-restricted, CTL-mediated liver disease, we now demonstrate that CD8-positive, Ld-restricted hepatitis B surface Ag-specific CTL bind and kill their target cells in vivo by triggering them to undergo degenerative cytologic changes compatible with apoptosis. Unexpectedly, the data also indicate that the pathologic consequences of this direct, Ag-specific CTL effect are much less severe than the cytodestructive, Ag-nonspecific inflammatory response that they induce when they are activated by Ag recognition.
Heat-stable antigen is an important costimulatory molecule on epidermal Langerhans' cells.
Authors: Enk A H , Katz S I
Published: December 2022

Heat-stable antigen (HSA), expressed by activated B cells, has been described as a costimulatory molecule for CD4+ T cells. Because epidermal Langerhans cells (LC) are known to express HSA, we determined whether LC HSA also served as a costimulator of Th cells. We have confirmed that HSA is expressed by freshly prepared (fresh) and, to a lesser extent, short-term cultured (cultured) LC and we demonstrate that costimulatory effects of HSA are prominent on fresh and 1-day cultured LC, whereas 2- to 4-day cultured LC exhibit less HSA-costimulatory activity. The anti-HSA mAb 20C9 almost completely blocked the proliferative response of Th1 or lymph node T cells induced by fresh or cultured LC. 20C9 also specifically inhibited LC-dependent IL-2 production by Th1 cells. The inhibitory effect of 20C9 was not observed when Th2 cells were substituted for Th1 cells or peripheral lymph node T cells. Furthermore, Th1 cells rescued from cocultures of T cells and 20C9-treated, Ag-pulsed LC, (but not from control-treated cocultures) were anergic to restimulation with untreated Ag-pulsed LC. These data suggest that LC HSA is an important costimulatory molecule in Th1 cell-dependent cutaneous immune reactions.
CD28-mediated costimulation is necessary for optimal proliferation of murine NK cells.
Authors: Nandi D , Gross J A , Allison J P
Published: December 2022

CD28, a cell surface molecule expressed on all murine T cells, plays a critical role in T cell activation. We show here that NK-1.1+ cells, a subpopulation of SCID splenocytes, and IL-2-activated NK cells express CD28, although at lower levels than alpha beta T cells. Optimal proliferation of highly purified asialo GM1+ NK cells from the SCID spleen was observed in response to stimulation with IL-2 and PMA, together with anti-CD28 or L-B7+ cells. Thus, in addition to IL-2, murine NK cells require CD28-mediated costimulatory signals for optimal proliferation. IL-2-activated NK cells produced enhanced levels of IFN-gamma and TNF in response to stimulation with anti-CD28 and PMA. On the other hand, we were unable to demonstrate that CD28 signaling had any effect on NK-mediated cytotoxicity. We conclude that the CD28 costimulatory pathway is functional in NK cells and plays an important role in their proliferation and cytokine production.
Assessment of CD4 expression by early T precursor cells and by dendritic cells in the human thymus.
Authors: Sotzik F , Rosenberg Y , Boyd A W , Honeyman M , Metcalf D , Scollay R , Wu L , Shortman K
Published: December 2022

The adult mouse thymus contains a minute population of early lymphoid precursor cells that express moderate levels of CD4. We searched for a corresponding population of early T precursors in the infant human thymus, by first depleting the majority of more mature thymocytes, then using immunofluorescence and flow cytometry to analyze cells bearing a range of early T lineage markers. No discrete population of early T precursors expressing CD4 was observed, in contrast to the murine thymus. Most putative very early human thymocytes were CD4-8-3-1-2lo44+34+7hi class I MHChi class II MHC-. However, a distinct population of human thymic dendritic cells expressing high levels of CD4 was isolated. These were CD4hi8-3-1-2-44+34-7- class I MHChi class II MHChi, and lacked markers of B cells, NK cells, or myeloid cells. They were large cells that exhibited dendritic morphology after brief periods of culture, and they were efficient stimulators of allogeneic T cells. The biologic implications of CD4 expression by thymic dendritic cells are discussed.