bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of human immunodeficiency virus type-1 to follicular dendritic cells in vitro is complement dependent.
Authors: Joling P , Bakker L J , Van Strijp J A , Meerloo T , de Graaf L , Dekker M E , Goudsmit J , Verhoef J , Schuurman H J
Published: December 2022

The authors studied the binding in vitro of HIV-1 virus particles, conjugated to fluorescein isothiocyanate, to follicular dendritic cells (FDC) isolated from human tonsils. Analysis was done using flow cytometry, fluorescence microscopy, and immunogold electron microscopy. The focus of study was on the effect of serum from various origins, including pooled fresh serum and heated serum from control donors and pooled heated serum from HIV-1-infected patients (containing anti-HIV-1 antibodies). In the presence of heated serum, either from controls or from HIV-1-infected patients, the fluorescence signal in flow cytometry was similar to the background value. In the presence of fresh serum, the signal was substantially increased, and an even higher signal was observed in the presence of fresh serum and serum from HIV-1-infected patients. This high fluorescence signal was also found in the presence of serum depleted of complement factor C5, but not with serum deficient in complement factor C3. The binding of HIV-1 virions to FDC in the presence of fresh serum was confirmed by fluorescence microscopy on cytospot preparations. After quenching of the extracellular fluorescence with trypan blue, the fluorescence was reduced to about 30% of the initial value, indicating that most of bound fluorescent virions were present extracellularly. Similar experiments using blood mononuclear cells showed that fluorescent HIV-1 particles after binding to these cells were present intracellularly. This flow cytometry data was confirmed in immunogold electron microscopy demonstrating that most HIV-1 gag p24 or FITC label was present at the outside of FDC and on adherent virus particles. We conclude that HIV-1 virions adhere to FDC in vitro in a complement component C3-dependent way. Anti-HIV-1 antibodies in serum from HIV-1 infected patients enhance binding but, by itself, are unable to mediate binding.
Proliferation of human T lymphocytes induced with superantigens is not dependent on costimulation by the CD28 counter-receptor B7.
Authors: Damle N K , Klussman K , Leytze G , Linsley P S
Published: December 2022

Staphylococcal enterotoxins, also known as superantigens (SAg), bind class II MHC molecules on APC and upon direct cell-to-cell contact stimulate proliferation of T cells expressing appropriate V beta gene products. The T cell surface molecule CD28 binds its costimulatory counter-receptor, B7 expressed on APC, and augments IL-2 production and T cell growth. Although the role of B7 costimulation during Ag-specific responses of T cells is established, its involvement during the activation of T cells with SAg has not been examined. Using a soluble Ig C gamma 1 chimera of CTLA-4, a second receptor for B7 and a homologue of CD28, this study examines the role of B7 expressed on APC during the induction of proliferation of CD4+ T cells upon stimulation with SAg (SAg/staphylococcal enterotoxins). CTLA-4lg, which has a higher avidity for B7 than CD28, had no effect on the synthesis of IL-2 as well as proliferative responses of CD4+ T cells induced by SAg presented on allogeneic EBV-transformed B cells, and IFN-gamma-activated endothelial cells. In contrast, T cell proliferation induced by alloAg presentation by the same APC was significantly inhibited by CTLA-4lg. mAb directed at the CD11a/CD18 molecule inhibited both SAg-induced and alloAg-induced proliferation of T cells. AlloAg-primed CD4+ T cells, which expressed both class II MHC and intercellular adhesion molecule-1 but not B7, presented SAg to and induced proliferation of both resting and SAg-primed T cells. These responses were inhibited by mAb directed at CD11a/CD18 but not by CTLA-4 Rg. These results suggest that SAg-induced responses differ from those induced by alloAg in that they are not obligatorily dependent on the costimulation by B7. In contrast, adhesive interaction between CD11a/CD18 on T cells and its counter-receptor on SAg-presenting cells is necessary and probably sufficient to support SAg-induced proliferation of T cells.
Isolation and characterization of human peripheral blood dendritic cells.
Authors: Thomas R , Davis L S , Lipsky P E
Published: December 2022

Dendritic cells (DC) are potent APC that can be purified from cultured peripheral blood non-T cells. Because no specific cell surface marker has been found, the lineage of DC remains obscure. The purpose of these studies was to determine the circulating blood cells that could give rise to functional human DC. DC were enriched when purified by standard techniques from non-T cells that were treated with L-leucyl-L-leucine methyl ester, known to be toxic to monocytes and cytolytic cells. To determine whether monocytes or B cells could give rise to DC, fresh non-T cells were sorted into CD14+ monocytes, CD19+ B cells, and CD14- and CD19- cells. Although there was some enrichment for APC function by cultured nonadherent CD14- or CD19- cells, a marked enrichment for cells with dendritic morphology and potent APC function was found in the population that was sorted by the absence of expression of CD14, CD19, CD3, and CD16. More than 90% of the CD14-CD19-CD16-CD3- sorted cells, and of control DC, expressed the myeloid markers CD13 and CD33. Therefore, fresh non-T cells were sorted based on the expression of these myeloid markers. In comparison with CD33-CD14- B cells, some of the CD33+ cells expressed CD14 dimly. However, they were easily distinguished from monocytes, which intensely expressed CD14. CD33+CD14dim cells developed dendritic processes and were more potent APC than control DC, CD33+CD14+, or CD33-CD14- cells. Although freshly isolated CD33+CD14dim DC expressed a number of cell surface molecules also expressed by CD14+ monocytes, they demonstrated lower levels of lysosomal enzymes and a lack of FcR-mediated phagocytosis in comparison with monocytes. Differentiation of morphology and phenotype of CD33+CD14dim cells occurred within 6 to 36 h in culture. However, the CD33+CD14dim cells could effectively function as APC without prolonged preincubation to develop dendritic morphology. These data indicate that human blood DC arise from precursors that express the myeloid lineage markers CD13 and CD33, but are functionally distinct from classic CD14+ monocytes.
Release of nitric oxide during the T cell-independent pathway of macrophage activation. Its role in resistance to Listeria monocytogenes.
Authors: Beckerman K P , Rogers H W , Corbett J A , Schreiber R D , McDaniel M L , Unanue E R
Published: December 2022

Immunodeficient mice are remarkably resistant to Listeria monocytogenes (LM) infection. We examined the role that nitric oxide (NO.) plays in the CB-17/lcr SCID (SCID) response to LM. SCID spleen cells produced large quantities of NO. (as measured by nitrite formation) when incubated in the presence of heat-killed LM. NO. production was dependent on the release of IFN-gamma by the SCID NK cells. When tested directly, macrophages produced large quantities of nitrite in response to LM, but only in the presence of IFN-gamma. The production of NO. induced by LM was not affected by neutralizing antibodies to TNF or IL-1. The production of NO. was inhibited by addition of either of two inhibitors of NO.synthase, NG-monomethyl arginine, or aminoguanidine. In a different situation, NK cells that were stimulated by TNF and Listeria products to release IFN-gamma did not produce NO.. Macrophages cultured with IFN-gamma killed live LM. This increased killing of LM was significantly inhibited by amino-guanidine. In vivo, administration of aminoguanidine resulted in a marked increase in the mortality and spleen bacterial loads of LM-infected SCID or immunocompetent control mice. We conclude that NO. is a critical effector molecule of T cell-independent natural resistance to LM as studied in the SCID mouse, and that the NO.-mediated response is essential for both SCID and immunocompetent host to survive after LM infection.
The fate of Borrelia burgdorferi, the agent for Lyme disease, in mouse macrophages. Destruction, survival, recovery.
Authors: Montgomery R R , Nathanson M H , Malawista S E
Published: December 2022

The macrophage is a known reservoir for a number of infectious agents, and is therefore a likely candidate site for persistence of Borrelia burgdorferi, the Lyme spirochete. We report that unopsonized B. burgdorferi enter macrophages rapidly, resulting mainly in degradation but occasionally in apparent intracellular persistence. We studied uptake of spirochetes by macrophages by simultaneously labeling infected cells with antibodies to B. burgdorferi and with sequential components of the endocytic pathway, and we examined optical sections (0.5-1.0 micron in thickness) of these cells by confocal fluorescence microscopy at multiple time points after infection. We found that only 5 min of incubation at 37 degrees C were required for nearly 100% of B. burgdorferi to enter a lysosomal glycoprotein-positive compartment, whereas 60 min were required for 90% of the spirochetes to appear in a cathepsin L-positive compartment under the same conditions. We also labeled infected living cells with acridine orange to distinguish live from killed intracellular organisms. Although the large majority of spirochetes within a given cell were dead, we saw occasional live ones up to 24 h (the longest interval examined) after all extracellular organisms had been lysed in distilled water. Moreover, we can reculture spirochetes from macrophages after infection. Persistence of spirochetes within macrophages provides a possible pathogenetic mechanism for chronic or recurrent Lyme disease in man.
Overexpression of CD3 eta during thymic development does not alter the negative selection process.
Authors: Hussey R E , Clayton L K , Diener A , McConkey D J , Howard F D , Rodewald H R , D'Adamio L , Dallenbach F , Stein H , Schmidt E V
Published: December 2022

To elucidate the role of CD3 eta in thymic development and to determine whether CD3 eta is involved in the negative selection process, CD3 eta was overexpressed > 100 fold in transgenic (tg) mice using a Thy-1 promoter and regulatory elements. CD3 eta was readily observed in the majority of cortical thymocytes and in a fraction of medullary thymocytes in tg mice by immunohistochemical staining with an anti-CD3 eta-specific mAb. In contrast, endogenous CD3 eta levels were too low to detect in normal littermates. Flow cytometric analysis demonstrated an increased level of TCR on thymocytes with intermediate TCR density in tg animals and parallel biochemical studies showed a marked increased in TCR-associated CD3 zeta-eta heterodimers and CD3 eta-eta homodimers relative to controls. Despite this change in surface TCR phenotype, there was no significant alteration in the total numbers or proportion of CD4+CD8+ double-positive or CD4+CD8- or CD4-CD8+ single-positive thymocytes or peripheral T cells. Percentages of SP V beta 5, V beta 6, and V beta 8 thymocytes in tg animals were unaltered compared to normal littermates when backcrossed either to C57BL/6 (H-2b) or DBA/2 (H-2d) backgrounds. Furthermore, induction of DNA fragmentation with anti-CD3 epsilon mAb treatment in vivo was not significantly different for tg and normal littermates. Collectively, these data imply that CD3 eta is not a limiting component of the negative selection process.
Structurally distinct T cell receptor complexes on developmentally distinct T cell populations in severe combined immunodeficiency mice expressing a TCR beta transgene.
Authors: Shores E W , Nakayama T , Wiest D L , Takahama Y , Sharrow S , Singer A
Published: December 2022

T cell differentiation and TCR expression were assessed in severe combined immunodeficiency (SCID) mice possessing an already rearranged TCRV beta 8 transgene. Unlike nontransgenic SCID mice, TCRV beta 8-transgenic SCID mice contained a broad spectrum of T cell populations, including both immature thymocytes and mature T cells. In TCRV beta 8-transgenic SCID mice, immature CD4-CD8- and CD4+CD8+ thymocytes expressed surface TCR complexes structurally distinct from those expressed by mature single-positive T cells. Immature CD4+CD8+ thymocytes expressed surface TCR beta chains without a clonotypic TCR partner chain and largely without associated CD3 components. In contrast, mature single-positive T cells expressed fully assembled surface TCR complexes containing disulfide-linked heterodimers consisting of transgenic TCR beta chains and endogenous TCR alpha chains. The surface TCR complexes on mature T cells were associated with CD3 components and were competent to transduce TCR-mediated proliferative signals. Thus, T cells at different stages of development in TCRV beta 8-transgenic SCID mice express structurally distinct surface TCR complexes, demonstrating that the developmental stage achieved by T cells in these mice is related to the structure of the surface TCR complexes they express. Indeed, the present results indicate that successful differentiation into single-positive T cells requires surface expression of fully assembled TCR complexes.
The cell-mediated response to schistosomal antigens at the clonal level. III. Identification of soluble egg antigens recognized by cloned specific granulomagenic murine CD4+ Th1-type lymphocytes.
Authors: Chikunguwo S M , Quinn J J , Harn D A , Stadecker M J
Published: December 2022

Granulomatous inflammation in schistosomiasis is a consequence of T cell-mediated hypersensitivity to parasite egg Ag. In the present study we used three consecutive independent chromatographic procedures to fractionate and identify the soluble egg Ag recognized by schistosome-specific, cloned, murine, CD4+ Th1-type lymphocytes, which had been shown previously to be capable of mediating granuloma formation in vivo when adoptively transferred to normal syngeneic hosts challenged with an i.v. injection of eggs. The stimulatory activity resided in two acidic egg molecules, with apparent molecular masses of 64 to 68 kDa and 38 to 42 kDa, each of which ran as a single band on SDS-PAGE after purification. Fast performance liquid chromatography and SDS-PAGE performed under reducing conditions suggested that the two molecules are related and that the 38- to 42-kDa molecule is a subunit of the 64- to 68-kDa molecule. Polyclonal lymphoid cells from schistosome-infected mice were similarly stimulated by the purified 64- to 68-kDa and 38- to 42-kDa molecules, implying that these are sensitizing Ag in the natural disease.
Activation of human peripheral blood mononuclear cells by nitric oxide-generating compounds.
Authors: Lander H M , Sehajpal P , Levine D M , Novogrodsky A
Published: December 2022

Recent work in this laboratory has identified immune-stimulatory properties of the oxidant hemin. In this study, we examined whether the nitrogen-based oxidant nitric oxide (NO) had inductive effects on human lymphocytes. We found that the NO-generating compounds sodium nitroprusside and S-nitroso-N-acetylpenicillamine rapidly enhanced the rate of glucose transport in resting human PBMC. In addition, NF-kappa B binding activity was induced by these agents as was the secretion of TNF-alpha. The data suggest that a cGMP-independent mechanism is involved as the cell permeant cGMP analogue, 8-Br-cGMP, had no effect in eliciting these inductive events. Activation of lymphocytes by these NO-generating compounds may be mediated through the protein tyrosine phosphorylation signal transduction pathway. We found that membrane-associated protein tyrosine phosphatase activity was enhanced in PBMC treated with sodium nitroprusside or S-nitroso-N-acetylpenicillamine and that the src family protein tyrosine kinase p56lck was activated in these cells. Inasmuch as p56lck activity is negatively controlled by tyrosine phosphorylation, its activation may be related to the enhancement of protein tyrosine phosphatase activity. 8-Br-cGMP had no effect on these enzymes. Taken together, these data suggest that NO may have immune-stimulatory properties and may signal through a hitherto undescribed cGMP-independent pathway.
Control of exocytosis in early neutrophil activation.
Authors: Sengeløv H , Kjeldsen L , Borregaard N
Published: December 2022

Exocytosis of human neutrophil secretory vesicles, gelatinase granules, specific granules, and azurophil granules was measured during modulation of the intracellular free calcium concentration. A strict rank order of exocytosis of the four compartments was observed when cytosolic calcium was elevated by an ionophore in Fura-2-loaded cells. The rank order was: secretory vesicles, gelatinase granules, specific granules, and azurophil granules. Secretory vesicles were exceptionally sensitive to cytosolic free calcium, being completely mobilized after small cytosolic calcium transients. Kinetic studies with FMLP or ionomycin as stimulus showed that the same rank order was valid concerning the rate of exocytosis. In contrast to the other granules, a major part of the secretory vesicles were exocytosed after FMLP stimulation when intracellular calcium was chelated with bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester. These data indicate that, in contrast to other neutrophil granules, secretory vesicles are mobilized completely after small isolated elevations of cytosolic calcium. On the other hand, complete elimination of the FMLP-derived calcium transient does not inhibit exocytosis of secretory vesicles markedly. Thus, the effect of FMLP on secretory vesicles is signaled via cytosolic free calcium and an alternative pathway activated in parallel. This may be the basis for the unique position of this compartment as a readily mobilizable store of components important in early neutrophil activation.
Presentation of endogenous peptides to MHC class I-restricted cytotoxic T lymphocytes in transport deletion mutant T2 cells.
Authors: Zweerink H J , Gammon M C , Utz U , Sauma S Y , Harrer T , Hawkins J C , Johnson R P , Sirotina A , Hermes J D , Walker B D
Published: December 2022

The ability of minigene-encoded viral peptide epitopes to be presented by class I molecules in the absence of MHC-encoded transporters has been evaluated in mutant T2 cells. These cells have a large deletion in the class II MHC region that includes the known transporter protein for antigenic peptides and proteasome genes and they are defective in presenting viral epitopes to CTL. T2 cells that express minigenes encoding the influenza virus matrix peptide 58-66 (GILGFVFTL) and two HTLV 1 Tax peptides 11-19 (LLFGYPVYV) and 12-19 were lysed by HLA-A2-restricted peptide-specific CTL. Minigene expression of a HLA-A2-restricted HIV reverse transcriptase peptide 476-484 (ILKEPVHGV) with three charged residues sensitized T2 cells poorly for lysis by HIV-specific CTL unless the peptide was preceded by an endoplasmic reticulum translocation signal sequence. Expression of an influenza virus nucleoprotein peptide 383-391 (SRYWAIRTR) with three charged arginine residues did sensitize HLA-B27+ T2 cells for lysis by peptide-specific CTL. These and other results with endogenously expressed peptide analogs in which hydrophobic and charged amino acids were interchanged demonstrate that antigenic peptides can be translocated from the cytoplasm into the class I Ag presentation pathway independent of MHC-encoded transporters; and that peptide hydrophobicity appears not to be a major determinant in selecting peptides for this alternate pathway.
Protease treatment of nuclear extracts distinguishes between class II MHC X1 box DNA-binding proteins in wild-type and class II-deficient B cells.
Authors: Hasegawa S L , Riley J L , Sloan J H , Boss J M
Published: December 2022

The X box region is critical for directing the expression of class II major histocompatibility complex genes in B lymphocytes. Although several class II promoter-specific DNA binding factors have been described, only the X box region factor, RFX, shows a genetic correlation with class II expression, being deficient in some B cell lines derived from patients with class II-deficient congenital immunodeficiency. To further evaluate the role of X box DNA-binding proteins in class II gene expression, the role of the X box region was examined in both class II-positive and -negative lymphoid cells. In addition to the wild-type B cell line Raji, two class II transcriptional mutant cell lines, SJO and RJ2.2.5, and Jurkat, a class II negative T cell line, were examined. In contrast to wild-type B cells, neither of the class II mutant cell lines could use the X box region to direct the expression of a transiently transfected reporter gene, indicating that the X box-dependent transcriptional pathway is defective in these cells. The binding activity of the X1 box DNA-binding protein RFX was examined and found to be present in wild-type B cells and the mutant RJ2.2.5 but was absent in SJO and Jurkat. However, other X1 box-specific activities were detected in all these cell lines. To determine whether these different X1 box activities represented distinct DNA binding proteins or multimeric forms of the same factor(s), protease treatment of the crude nuclear extracts followed by DNA-binding assays were carried out and demonstrated that B cell extracts contain at least two X1-specific factors. One of these cleaved products (band 1 pk) correlates with RFX activity. A similar comparison with protease-treated extracts prepared from Jurkat cells demonstrated the presence of the band 1pk activity despite an absence of the native RFX activity. In contrast, protease treatment and analysis of SJO extracts showed no detectable levels of the band 1pk activity. These results demonstrate that multiple X1 box-specific DNA-binding activities exist in all lymphoid cells, but the presence of an actively binding RFX species correlates with class II transcription.
IL-4 attenuates the transcriptional activation of both IFN-alpha and IFN-gamma-induced cellular gene expression in monocytes and monocytic cell lines.
Authors: Larner A C , Petricoin E F , Nakagawa Y , Finbloom D S
Published: December 2022

The interaction of IFN-alpha and IFN-gamma with monocytes results in several actions that significantly influence the course of an immune response. Many of these effects are proinflammatory and can contribute to the degree of tissue injury at a site of inflammation. Whereas recent investigations target IL-4 as a T cell product that can antagonize some of the responses induced by IFN, little is known regarding the mechanisms involved. We have taken advantage of two well defined systems: the transcriptional activation of the cellular genes ISG-54 by IFN-alpha and IP-10 by IFN-gamma. IL-4 treatment of both the monocytic leukemia cell line, THP-1, and normal peripheral blood monocytes resulted in inhibition of IFN-induced RNA levels for both genes. Nuclear run-on assays in THP-1 cells indicated that the effects of IL-4 were due to the inhibition of the transcriptional activation of these genes by both IFN-alpha and IFN-gamma. This inhibition was not due to alteration in the binding characteristics of IFN-alpha or IFN-gamma to the cell. In the IFN-alpha system, we were able to show that IL-4 treatment resulted in reduced formation of the transcriptional activator, IFN-stimulated gene factor 3. This reduction appears to be the result of a defect in the ability of IFN alpha to activate the IFN-stimulated gene factor 3 alpha component of IFN-stimulated gene factor 3.
Lactate transport in macrophages.
Authors: Loike J D , Kaback E , Silverstein S C , Steinberg T H
Published: December 2022

Macrophages perform phagocytic and effector activities in a number of different tissues. The environment of the inflammatory foci in which they function is often acidic and contains an abundance of lactate. We characterized the ability of thioglycollate-elicited mouse peritoneal macrophages to accumulate lactate from the medium and to use this lactate to maintain intracellular energy stores. Lactate uptake was stereospecific for L-lactate and was inhibited by the organic anion transport blocker probenecid but not by concentrations of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid that block anion exchangers. L-[14C]Lactate uptake was not affected by variation of the extracellular Na+ concentration but was enhanced by acidification of the extracellular medium, suggesting that lactate uptake was mediated by a proton cotransport system. The enhanced accumulation of [14C]-lactate seen in medium at pH 6.0 to 6.5 was inhibited by probenecid or by an excess of unlabeled L-lactate. When macrophages were incubated in PBS without glucose for 6 h, intracellular stores of phosphocreatine were 13 nmol/mg of protein, compared with 44 nmol/mg of protein in cells incubated in medium containing glucose. When lactate was substituted for glucose, phosphocreatine stores were 32 nmol/mg of protein. These studies reveal that macrophages take up L-lactate in a pH-dependent manner and that lactate uptake occurs via a probenecid-inhibitable monocarboxylate transporter; they suggest that macrophages can utilize this lactate as an energy source.
Influence of nephrectomy on tumor necrosis factor clearance in a murine model.
Authors: Bemelmans M H , Gouma D J , Buurman W A
Published: December 2022

TNF, a cytokine with multiple properties, is known to be rapidly inactivated in vivo. In patients with septic shock and kidney malfunction, conflicting data have been reported regarding the presence of TNF in the circulation. Recently, we have shown that these differences can be the result of the detection of free TNF vs TNF complexed with soluble TNF-R. Enhanced levels of soluble TNF-R have been reported in patients with kidney malfunction. Because the kidney is thought to play an important role in TNF and TNF-R metabolism, we investigated the influence of bilateral nephrectomy on TNF clearance and TNF-R regulation in a murine model. The data obtained show that bilateral nephrectomy results in circulating levels of immunologically detectable (ELISA), but not biologically active (bioassay), TNF. Injection of endotoxin results in significantly higher levels of immunologically detectable TNF in bilaterally nephrectomized mice, compared with sham-operated mice, whereas biologically active levels were similar. To investigate the roles of TNF-R1 (P55) and TNF-R2 (P75) in this process, clearance of TNF was studied by injection of murine and human TNF. Murine TNF injection leads to comparable clearance of bioactive TNF in nephrectomized and sham-operated mice (t1/2 = approximately 12 min). However, clearance of immunologically detectable murine TNF is significantly slower in nephrectomized mice, compared with sham-operated mice (t1/2 = 96 min vs 26 min, respectively; p < 0.05). Administration of human TNF results in a significantly lower clearance in nephrectomized mice, compared with sham-operated mice (t1/2 = 108 min vs 25 min, respectively; p < 0.05). This is observed for both bioactive human TNF and immunologically detectable human TNF. Based on the fact that murine TNF-R1 (55 kDa) has a similar affinity for murine and human TNF, whereas murine TNF-R2 (75 kDa) shows affinity only for murine TNF, the data obtained suggest an important role for TNF-R2 in inactivation and clearance of TNF by the kidney. Moreover, the data suggest that kidney malfunction affects TNF clearance, leading to increased amounts of circulating TNF-TNF-R complexes, which could function as a slow release reservoir for TNF.
Differential usage of T cell receptor V gene segments in CD4+ and CD8+ subsets of T lymphocytes in monozygotic twins.
Authors: Hawes G E , Struyk L , van den Elsen P J
Published: December 2022

The TCR confers immunity by the specific recognition of foreign Ag peptides in the context of self-MHC molecules. The mechanisms controlling TCR selection and repertoire generation are not clearly understood and seem to occur in an apparently random, (self) Ag-driven manner. To address the question to what extent the TCR repertoire is randomly shaped or genetically predetermined, we have analyzed the alpha beta TCR repertoire of the CD4+ and CD8+ subsets of peripheral blood lymphocyte cultures of monozygotic twins by using the polymerase chain reaction technique with TCR V region gene family-specific oligonucleotide primers. Our studies demonstrate that there is high concordance in the overall patterns of V gene usage within a pair of twins, particularly in V beta usage (mean V beta CD4+ R2 = 0.869 and CD8+ R2 = 0.833) and to a lesser extent V alpha usage (mean V alpha CD4+ R2 = 0.621 and CD8+ R2 = 0.627); whereas the patterns between unrelated individuals show more variability. This study has also demonstrated that the V alpha and V beta genes are not randomly used within the CD4+ and CD8+ subsets. We observed significant preferential skewing of several V alpha or V beta gene families to either the CD4+ or CD8+ subset in the majority of individuals analyzed (p-value range = 0.0476 to < 0.001). In particular, V alpha 11, 17, 22, and V beta 3, 9, 12, 18 were skewed to the CD4+ subset; whereas V alpha 2, 6, 12, 15, 20 and V beta 7, 14, 17 were skewed to the CD8+ subset. Furthermore, a number of the V genes showed patterns of skewing consistent only within a pair of twins. In three pairs of twins, V beta 2 was skewed to the CD4+ subset, whereas the fourth pair used almost equal frequencies of V beta 2 in both subsets. This observation was made for the V beta 2, 4, 5, 6, 8, 19 and V alpha 7, 16, 18, 21 families. Finally, the ratio of the relative V gene usage frequency that could be observed within an individual was conserved within the sets of twins; for instance, the relative amount of V beta 2 to that of V beta 3 was higher in both individuals of one set of twins, whereas it was lower in all of the other three sets. Together these observations suggest that the predominant influence shaping the TCR repertoire is genetically predetermined, of which, HLA-predicted selection mechanisms exerted during thymic maturation might be contributing factors.
Inhibition of cell adhesion by microspheres coated with recombinant soluble intercellular adhesion molecule-1.
Authors: Welder C A , Lee D H , Takei F
Published: December 2022

Murine recombinant soluble intercellular adhesion molecule-1 (sICAM-1) was produced and characterized. When immobilized on plastic microtiter wells, sICAM-1 efficiently mediated LFA-1-dependent cell adhesion, indicating that the purified protein retained the ability to bind to LFA-1. However, sICAM-1 in solution, at concentrations up to 100 micrograms/ml, was incapable of inhibiting the phorbol ester-induced homotypic aggregation of lymphocytes, the adhesion of T cells to plastic immobilized sICAM-1, and CTL effector function, all of which are mediated by intercellular adhesion molecule-1:LFA-1 interaction. In contrast, uniform polystyrene microspheres coated with sICAM-1 bound specifically to LFA-1+ cells and efficiently inhibited the adhesion of T cells to immobilized sICAM-1. The sICAM-1-coated microspheres also inhibited CTL function, but the inhibition was only partial. These results suggest that although monomeric sICAM-1 cannot competitively inhibit cell adhesion mediated by intercellular adhesion molecule-1 and LFA-1, microspheres coated with sICAM-1 can inhibit such cell adhesion.
HLA-DQ allelic polymorphisms constrain patterns of class II heterodimer formation.
Authors: Kwok W W , Kovats S , Thurtle P , Nepom G T
Published: December 2022

HLA-DQ class II molecules are complex heterodimers in which polymorphic alpha- and beta-polypeptides form noncovalent associations leading to cell surface expression. Using gene transfer with retroviral vectors, we demonstrate that surface expression of heterodimers among various DQ alpha and -beta polypeptides is not universal. Two types of allele-specific constraints were found: DQ beta chains that are associated with the DQw1 specificities do not form stable cell surface heterodimers with DQ alpha chains encoded on haplotypes that are associated with DQw2, DQw3, or DQw4. Similarly, surface expression of heterodimers composed of DQw2- or DQw3-associated DQ beta chains and DQw1 DQ alpha chains were not detected. The formation of mixed isotype heterodimers also showed allelic preference: DR alpha/DQw1 beta dimers were detected on the cell surface, but DR alpha/DQw3 beta dimers were not. Failure to express class II heterodimers on the cell surface was apparently due to either inefficient assembly of the alpha- and beta-chains or retention of the mismatched dimers in an intracellular compartment. DQ beta cDNA encoding DQw1- and DQw3-associated beta-polypeptides were modified to create chimeric expression vectors in which portions of the DQ beta polypeptides were exchanged between different alleles. The resulting chimeric DQ beta polypeptides were tested for their ability to form stable heterodimers with different DQ alpha polypeptides. A cluster of polymorphic amino acid residues encoded by the 3' end of the DQB1 second exon, corresponding to residues 60 to 91 in the DQ beta polypeptide, were found to be critical determinants constraining stable cell surface heterodimer formation among preferential alpha- and beta-combinations.
Murine C4b-binding protein. Mapping of the ligand binding site and the N-terminus of the pre-protein.
Authors: Ogata R T , Mathias P , Bradt B M , Cooper N R
Published: December 2022

We have constructed cell surface-bound forms of murine C4b-binding protein (mC4BP) that allowed us to monitor the binding of mC4BP to C4b with relatively simple erythrocyte rosette assays. We used two types of surface-bound mC4BP: one in which segments of mC4BP were fused directly to a peptide containing the transmembrane and cytoplasmic domains of human complement receptor CR2 (BPR1-type); and a second in which the same segments were fused to a longer peptide containing the five membrane-proximal short consensus repeats (SCR) of CR2 as well as the transmembrane and cytoplasmic domains (BPR2-type). COS cells transfected with either construct carrying all six mC4BP SCR rosetted with C4b-bearing EAC14 cells but not with C4b-lacking EAC1 cells; and rosetting was inhibited by excess inactivated C4 but not inactivated C3. COS cells transfected with BPR2 constructs carrying only SCR 1-3 or 1-4 gave similar rosetting behavior. However, rosetting was not observed with BPR2 constructs carrying only SCR 1-2 or 2-6, or with BPR1 constructs carrying only SCR 1-2, 1-3, 1-4, or 2-6. Finally, we found that alteration of the AUG sequence 56 triplet codons upstream of the putative N-terminus of mature mC4-BP eliminates rosetting whereas alteration of a second AUG sequence 13 codons upstream has no effect on rosetting. These results indicate that 1) SCR 1-3 of mC4BP are necessary and adequate for binding to C4b, 2) steric effects close to the cell surface may interfere with binding, and 3) mC4BP has an extraordinarily long 56 amino acid residue signal peptide.
Pure protein from Leishmania donovani protects mice against both cutaneous and visceral leishmaniasis.
Authors: Rachamim N , Jaffe C L
Published: December 2022

A protein purified from Leishmania donovani promastigotes, dp72, was shown to partially protect BALB/c mice against a challenge by this parasite. Immunized mice infected i.v. with 10(7) L. donovani promastigotes showed a 0, 60, and 78% reduction in liver parasite burden compared with the control mice at each time point examined after challenge, 1, 20, and 108 days, respectively. Western blotting demonstrated that the sera from the immune mice, which reacted specifically with dp72 in lysates of L. donovani, cross-reacted with one major band in total homogenates of Leishmania major and Leptomonas collosoma. Lymphocyte proliferation to crude and pure parasite Ag was also examined in mice immunized with dp72. Strong proliferation was found at low concentrations of crude L. donovani Ag (0.5 micrograms/ml) and with pure dp72. Proliferation at higher concentrations to crude L. major and L. collosoma Ag was observed. Little or no reaction (stimulation index < 1.0) was seen with other pure leishmanial Ag, including gp70-2, the promastigote surface protease, and lipophosphoglycan. Depletion in vitro of the CD4+ T cell subset from immune spleen cells abolished proliferation to dp72, whereas depletion of CD8+ T cells enhanced proliferation to the pure Ag. Experiments in vivo showed that immunized mice treated with antibodies to either CD4+, CD8+, both T cell subsets, or to IFN-gamma had larger LPB (178, 173, 176, and 130%, respectively) after challenge with L. donovani than nonimmunized controls. Mice immunized with dp72 but treated with either PBS or mouse Ig showed reduced LPB, 81 and 61%, respectively, compared with the nonimmunized animals. BALB/c mice immunized with dp72 were also protected against L. major which causes cutaneous leishmaniasis. Immunized mice infected with either 10(4) or 10(6) promastigotes did not develop lesions. Limiting dilution assays confirmed the protection.
Granulocyte-macrophage-colony-stimulating factor differentially regulates neutrophil migration across IL-1-activated and nonactivated human endothelium.
Authors: Yong K L , Linch D C
Published: December 2022

The directed migration of neutrophils across vascular endothelium to localize in inflammatory tissues is controlled by soluble mediators, including cytokines and growth factors. Granulocyte-macrophage CSF (GM-CSF) enhances and primes neutrophil functions, but its specific role in the movement and localization of neutrophils to infective sites has not been clarified. We demonstrate, using an in vitro model of the vascular endothelial barrier, that GM-CSF enhances neutrophil migration across unstimulated endothelium, increasing the percentage of migrating cells from 7.7 +/- 0.9% (mean +/- SE) in controls to 12.5 +/- 1.5% in the presence of GM-CSF (100 ng/ml) (n = 14, p < 0.0005). This effect is dose dependent, with maximal effects achieved at concentrations of 10 ng/ml or greater, and is independent of concentration gradients of GM-CSF. Transendothelial migration of neutrophils can also be increased by cytokine treatment of the endothelial cells. Preincubation of endothelial monolayers with IL-1 (10 U/ml) for 4 h increases the percentage of migrating cells to 16.8 +/- 1.4% (238 +/- 25% of base line, n = 7, p < 0.005). In the presence of GM-CSF, however, neutrophil migration across IL-1 treated endothelium (12.5 +/- 1.6%, n = 7) is no different from that across resting endothelium (11.6 +/- 1.6% in the same seven experiments). Hence GM-CSF acts to inhibit neutrophil migration across IL-1-activated endothelium, and almost completely abolishes IL-1-induced migration (n = 7, p < 0.0005). This differential effect of GM-CSF on neutrophil migration, depending upon the conditions of endothelial activation, does not relate to an effect on adhesion, because GM-CSF has no effect on the increased adhesion of neutrophils to IL-1-treated endothelium. The effect of GM-CSF on neutrophil migration across activated endothelium may be relevant to the clinical administration of human rGM-CSF.
Defects in the differentiation and function of antigen presenting cells in NOD/Lt mice.
Authors: Serreze D V , Gaskins H R , Leiter E H
Published: December 2022

Although T lymphocytes are the ultimate effectors of pancreatic beta cell destruction in autoimmune insulin-dependent diabetes, previous work has established that beta cell autoreactive T cells are generated in nonobese diabetic (NOD) mice as a result of APC dysfunctions. To determine if APC dysfunctions could result from developmental defects, we analyzed if macrophages (M phi) develop normally from NOD bone marrow stimulated with CSF-1 in the presence and absence of IFN-gamma. Due to interactions between the diabetogenic H-2g7 haplotype and background modifiers, NOD bone marrow cells were found to proliferate poorly to CSF-1 stimulation. IFN-gamma aberrantly increased CSF-1-stimulated proliferation of H-2g7 expressing bone marrow cells, although decreasing proliferation of bone marrow cells expressing diabetes resistant MHC haplotypes. FACS analysis indicated the diminished sensitivity of NOD hematopoietic precursors to CSF-1 was associated with a quantitative inability to generate phenotypically mature M phi. In addition to developmental defects, NOD M phi were also found to be functionally defective. Total MHC class I expression was aberrantly down-regulated in a tissue specific fashion in IFN-gamma-treated M phi from NOD mice, whereas MHC class I expression increased as expected in M phi from C57BL/KsJ (BKs) control mice. Total MHC class I expression also increased in IFN-gamma-treated M phi from NOR mice, a diabetes-resistant control strain that shares the H-2g7 haplotype of NOD, but contains BKs-derived genomic elements on chromosomes 2, 4, 11, and 12. This demonstrates differential trans-regulation of class I loci within the diabetogenic H-2g7 haplotype in NOD vs diabetes-resistant NOR mice. Aberrant down-regulation of MHC class I content in IFN-gamma-treated M phi from NOD mice was associated with decreased ability to activate CTL function. We propose these defects in M phi differentiation and function may interact with H-2g7 to generate APC in NOD mice that are unable to activate tolerogenic mechanisms, but remain capable of activating low level effector responses.
Manifestation of the MHC-unrestricted killing potential of a cytotoxic T cell clone requires activation in response to MHC-restricted self-presentation of antigen.
Authors: Dick T , Staege M S , Reichmann G , Reske-Kunz A B
Published: December 2022

10BK.1 T clone cells of (B10 x B10.BR)F1 genotype specifically react to peptides of OVA, frequently encountered in OVA preparations, and to the synthetic peptide OVA257-264 in an H-2Kb-restricted manner; the T clone cells produce lymphokines and proliferate in the absence of added APC, suggesting self-presentation of the Ag by the T cells. In accordance with their CD8+ CD4- phenotype 10BK.1 cells exhibit cytotoxic capacity. When the target cells were pretreated with OVA257-264 only cells bearing H-2Kb molecules were lysed. However, when the relevant OVA peptide was present during the 4-h 51Cr release assay 10BK.1 cells lysed target cells expressing H-2Kb molecules as well as target cells lacking H-2Kb elements. Likewise, 10BK.1 cells pretreated with OVA257-264 for 1 h and washed extensively to remove residual Ag were able to kill syngeneic and allogeneic target cells. Killing of allogeneic targets in the presence of Ag was inhibited by antibodies directed at H-2Kb. Allogeneic target cells were not killed when 10BK.1 cells were stimulated by peptide-pulsed syngeneic cells. Triggering of the TCR/CD3 complex of 10BK.1 cells was involved during the activation phase but not during the lytic phase. IL-2 did not participate in the MHC-unrestricted killing event. To explain the observed MHC-unrestricted cytotoxicity of 10BK.1 cells, the following model is proposed. In an initial step 10BK.1 cells present the relevant OVA peptide to one another in a H-2Kb-restricted fashion. The cells are thereby triggered to mobilize the lytic machinery. In a second step sensitized 10BK.1 cells lyse allogeneic target cells. Thus, the MHC-unrestricted cytotoxicity can be dissected into an MHC-restricted phase of 10BK.1 cell activation and an MHC-unrestricted lytic phase. Cytotoxic T cells with Ag self-presentation functions may account for tissue damage during bacterial infections.
The B cell immune response to an idiotype-inducing peptide epitope can be inhibited by immunodominance of a neighboring epitope.
Authors: Ju S T , Nonogaki T , Bernatowicz M S , Matsueda G R
Published: December 2022

Three synthetic peptides, EEYEYE (peptide 1), EEYEYEEEYEYE (peptide 2), and YEEEEY (peptide 3), were tested for their ability to induce common Id that had been previously characterized for murine antibodies specific to random synthetic polymers of glutamic acid, alanine, and tyrosine ((Glu,Ala,Tyr)n) (cGAT Id). Protein conjugates of either peptide 2 or peptide 3, but not peptide 1, induced cGAT Id. This unique approach directly identified two peptides capable of inducing cGAT Id antibodies. Previous immunization with peptide 1-protein conjugate inhibited the cGAT Id response to peptide 2 conjugated to a different protein carrier. Thus, a neighboring or overlapping epitope can be used to inhibit a cGAT Id-inducing epitope without the participation of carrier-specific Ts and without using anti-Id antibodies. In contrast, previous immunization with peptide 1-protein conjugate did not inhibit cGAT Id induction by peptide 3-protein conjugate or by (Glu,Ala,Tyr)n. This ruled out the participation of Id-specific Ts cells. The effectiveness of inhibition coincided with the avid binding of anti-peptide 1 antibodies to peptide 2, which was > 10 and 100 times stronger than the binding to peptide 3 and (Glu,Ala,Tyr)n, respectively. We hypothesize that the primed peptide 1-specific B cells capture and process peptide 2-protein efficiently and act as APC to Th cells specific to the protein of the challenging Ag, resulting in the selective and dominant activation of peptide 1-specific B cells. Thus, our data suggest that epitope priming can inhibit an Id response to a neighboring epitope by a mechanism of clonal dominance.
Induction of functional follicular dendritic cell development in severe combined immunodeficiency mice. Influence of B and T cells.
Authors: Kapasi Z F , Burton G F , Shultz L D , Tew J G , Szakal A K
Published: December 2022

Ag injected into immune mice immediately complexes with specific antibody. Immune complexes not phagocytosed by macrophages are transported by Ag transport cells to lymph node follicles for trapping by follicular dendritic cells (FDC). These FDC serve as a long term repository of unprocessed Ag that is believed to maintain both B cell memory and the secondary antibody response. Severe combined immunodeficiency mice lack functional B cells and T cells. Consequently, this mutation also appears to affect the ability to produce Ag-retaining FDC. to assess B and T cell requirements for FDC development and function, severe combined immunodeficiency mice were reconstituted with BM, or mature B and T cells. The development of a FDC reticulum, a three-dimensional network produced by the intertwining of FDC dendrites was assessed by Ag trapping on FDC using the histochemically detectable Ag horseradish peroxidase and quantitated by morphometry. The results showed that bone marrow transplants or B and T cells transferred together supply the required elements for the development of severe combined immunodeficiency FDC reticula that function in Ag trapping and the induction of the germinal center. In contrast, B cells or T cells injected separately induced a minimal development of FDC reticulum. The B and T cell requirements demonstrated here strongly indicate that B-T cell collaboration and the factors these cells produce are essential for FDC development.