bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytotoxic T lymphocytes (CTL) against a transforming gene product select for transformed cells with point mutations within sequences encoding CTL recognition epitopes.
Authors: Lill N L , Tevethia M J , Hendrickson W G , Tevethia S S
Published: June 2004

The 94-kD large tumor (T) antigen specified by simian virus 40 (SV40) is sufficient to induce cell transformation. T antigen contains four H-2Db-restricted cytotoxic T lymphocyte (CTL) recognition epitopes that are targets for CTL clones Y-1, Y-2, Y-3, and Y-5. These epitopes have been mapped to T antigen amino acids 207-215 (site I), 223-231 (sites II and III), and 489-497 (site V), respectively. Antigenic site loss variant cells that had lost one or more CTL recognition epitopes were previously selected by coculturing SV40-transformed H-2Db cells with the site-specific Db-restricted CTL clones. The genetic bases for T antigen CTL recognition epitope loss from the variant cells were identified by DNA amplification and direct sequencing of epitope-coding regions from variant cell DNAs. Cells selected for resistance to CTL clone Y-1 (K-1; K-1,4,5; K-3,1) carry deleted SV40 genomes lacking site I, II, and III coding sequences. Point mutations present within the site II/III coding region of Y-2-/Y-3-resistant cell lines specify the substitution of asparagine for lysine as T antigen amino acid 228 (K-2) or phenylalanine for tyrosine at position 230 (K-3). Point mutations identified within independently selected Y-5 resistant populations (K-5 and K-1,4,5) direct the substitution of isoleucine for asparagine at position 496 (K-5) or the substitution of phenylalanine for isoleucine at position 491 (K-1,4,5) of T antigen. Each substitution causes loss of the relevant CTL recognition epitope, apparently by compromising CTL T cell receptor recognition. These experiments identify specific amino acid changes within a transforming protein that facilitate transformed cell escape from site-specific CTL clones while allowing maintenance of cellular transformation. This experimental model system provides unique opportunities for studying mechanisms of transformed cell escape from active immunosurveillance in vivo, and for analysis of differential host immune responses to wild-type and mutant cell-transforming proteins.
T cell receptor-mediated recognition of self-ligand induces signaling in immature thymocytes before negative selection.
Authors: Abe R , Ishida Y , Yui K , Katsumata M , Chused T M
Published: June 2004

Shaping of the T cell repertoire by selection during intrathymic maturation involves T cell receptor (TCR) recognition of major histocompatibility complex/self-antigen complexes. In this communication, we studied the ability of minor lymphocyte stimulating (Mls) determinants to act as self-tolerogens in the selection of the T cell repertoire. We demonstrate that unprimed T cells from normal as well as TCR transgenic mice form Mls-specific conjugates with antigen-presenting cells, and that this TCR-ligand interaction leads to elevation of intercellular Ca2+ ([Ca2+]i). Peripheral T cells from TCR transgenic mice expressing receptors specific for self-Mls antigen show no reactivities to Mlsa. However, a proportion of immature thymocytes from these mice show specific binding and strong [Ca2+]i elevation in response to self-antigen-presenting cells, although these thymocytes do not proliferate. This self-reactivity of thymocytes is inhibited by antibodies specific for TCR, CD4, CD8, class II molecules, lymphocyte function-associated antigen 1, and intercellular adhesion molecule 1. These results demonstrate for the first time that before thymic negative selection, immature T cells can specifically interact with cells bearing self-antigen, and suggest that the resulting TCR-dependent signal transduction events provide a basis for negative selection of self-reactive T cells.
The alpha chain gene of H-2O has an unexpected location in the major histocompatibility complex.
Authors: Karlsson L , Peterson P A
Published: June 2004

A previously unknown major histocompatibility complex class II molecule consisting of the beta chain encoded by the H-2Ob gene and an unknown alpha chain was recently described. We now report that the alpha chain occurs in two allelic forms distinguished by charge difference. Using inbred recombinant mouse strains we were able to map the H-2Oa gene to a location between the A.TL and B10.MBR recombination points. Cosmids covering this region were used to isolate the gene. Sequence analysis revealed that the H-2Oa gene is the murine equivalent of the human HLA-DNA gene. These results indicate that the human HLA-DNA gene, the existence of which has long been known, is indeed coding for DO alpha, the alpha chain pairing with DO beta.
Lipopolysaccharide (LPS) partial structures inhibit responses to LPS in a human macrophage cell line without inhibiting LPS uptake by a CD14-mediated pathway.
Authors: Kitchens R L , Ulevitch R J , Munford R S
Published: June 2004

Lipopolysaccharides (LPS) that lack acyloxyacyl groups can antagonize responses to LPS in human cells. Although the site and mechanism of inhibition are not known, it has been proposed that these inhibitory molecules compete with LPS for a common cellular target such as a cell-surface binding receptor. In the present study, we used an in vitro model system to test this hypothesis and to evaluate the role of CD14 in cellular responses to LPS. Cells of the THP-1 human monocyte-macrophage cell line were exposed to 1,25 dihydroxyvitamin D3 to induce adherence to plastic and expression of CD14, a binding receptor for LPS complexed with LPS-binding protein (LBP). The uptake of picograms of [3H]LPS (agonist) and enzymatically deacylated LPS [3H]dLPS (antagonist) was measured by exposing the cells to the radiolabeled ligands for short incubation periods. The amounts of cell-associated LPS and dLPS were then correlated with cellular responses by measuring the induction of nuclear NF-kappa B binding activity and the production of cell-associated interleukin (IL)-1 beta. We found that similar amounts of [3H]LPS or [3H]dLPS were taken up by the cells. The rate of cellular accumulation of the ligands was greatly enhanced by LBP and blocked by a monoclonal antibody to CD14 (mAb 60b), yet no cellular responses were induced by dLPS or dLPS-LBP complexes. In contrast, LPS stimulated marked increases of NF-kappa B binding activity and IL-1 beta. These responses were enhanced by LBP and inhibited by mAb 60b. dLPS and its synthetic lipid A counterpart, LA-14-PP (also known as lipid Ia, lipid IVa, or compound 406) strongly inhibited LPS-induced NF-kappa B and IL-1 beta, yet neither antagonist inhibited the uptake of LPS via CD14. dLPS did not inhibit NF-kappa B responses to tumor necrosis factor (TNF) alpha or phorbol ester. Our results indicate that (a) both stimulatory and nonstimulatory ligands can bind to CD14 in the presence of LBP; (b) the mechanism of inhibition by dLPS is LPS-specific, yet does not involve blockade of LPS binding to CD14; and (c) in keeping with previous results of others, large concentrations of LPS can stimulate the cells in the absence of detectable binding to CD14. The findings indicate that the site of dLPS inhibition is distal to CD14 binding in the LPS signal pathway in THP-1 cells, and suggest that molecules other than CD14 are important in LPS signaling.
Mechanisms of mouse spleen dendritic cell function in the generation of influenza-specific, cytolytic T lymphocytes.
Authors: Nonacs R , Humborg C , Tam J P , Steinman R M
Published: June 2004

We have evaluated the capacity of dendritic cells to function as antigen-presenting cells (APCs) for influenza and have examined their mechanism of action. Virus-pulsed dendritic cells were 100 times more efficient than bulk spleen cells in stimulating cytotoxic T lymphocyte (CTL) formation. The induction of CTLs required neither exogenous lymphokines nor APCs in the responding T cell population. Infectious virus entered dendritic cells through intracellular acidic vacuoles and directed the synthesis of several viral proteins. If ultraviolet (UV)-inactivated or bromelain-treated viruses were used, viral protein synthesis could not be detected, and there was poor induction of CTLs. This indicated that dendritic cells were not capable of processing noninfectious virus onto major histocompatibility complex (MHC) class I molecules. However, UV-inactivated and bromelain-treated viruses were presented efficiently to class II-restricted CD4+ T cells. The CD4+ T cells crossreacted with different strains of influenza and markedly amplified CTL formation. Cell lines that lacked MHC class II, and consequently the capacity to stimulate CD4+ T cells, failed to induce CTLs unless helper lymphokines were added. Similarly, dendritic cells pulsed with the MHC class I-restricted nucleoprotein 147-155 peptide were poor stimulators in the absence of exogenous helper factors. We conclude that the function of dendritic cells as APCs for the generation of virus-specific CTLs in vitro depends measurably upon: (a) charging class I molecules with peptides derived from endogenously synthesized viral antigens, and (b) stimulating a strong CD4+ helper T cell response.
Differentiation of CD3-4-8- thymocytes in short-term thymic stromal cell culture.
Authors: Sen-Majumdar A , Lieberman M , Alpert S , Wiessman I L , Small M
Published: June 2004

We have investigated the ability of a heterogeneous thymic stromal cell (HTSC) culture system to promote in vitro differentiation of CD3-4-8- thymocytes. Culture of purified murine CD3-4-8- thymocytes on HTSC for 1 d resulted in the appearance of CD4+8+ cells, which did not occur when the sorted cells were maintained in medium alone. It is remarkable that when the culture period was extended to 2 d, CD3-4-8- progenitors differentiated further to CD4+8- and CD4-8+ cells, which also expressed high levels of TCR-CD3. This rapid differentiation on stroma in vitro appears to outpace parallel development in vivo. The differentiation potential of a subset of CD3-4-8- thymocytes that express high levels of a marker of normal and neoplastic thymic progenitors, the 1C11 antigen, was examined next. 1C11hiCD3-4-8- cells also gave rise to CD4-8+ and CD4+8+ populations after 1 d of culture on HTSC. Extending the culture period to 2 d resulted in a significant percentage of CD3-expressing cells that were CD4+8+, CD4+8- and CD4-8+ cells. These results suggest that in the in vitro HTSC culture system, various subsets of immature thymocytes can differentiate into all the mature phenotypes of cells normally found in the adult mouse thymus. This may provide a novel and rapid assay for thymic progenitors.
A fail-safe mechanism for maintaining self-tolerance.
Authors: Guerder S , Matzinger P
Published: June 2004

Using cytotoxic T lymphocyte (CTL) responses to the class I histocompatibility antigen Qa1 and to the minor histocompatibility antigen H-Y, we show that the immune system maintains a peripheral screening process that is able to tolerize a wide variety of potentially autoimmune CTL. The critical factor is the presence or absence of specific T helper cells. If T help is available, CTL precursors that recognize antigen are activated. In the absence of help, they are tolerized. Thus, T helper cells are guardians of peripheral tolerance in CTL.
Memory T cells are anergic to the superantigen staphylococcal enterotoxin B.
Authors: Lee W T , Vitetta E S
Published: June 2004

We have used staphylococcal enterotoxin B (SEB) to study the role of naive and memory T cells in the induction of peripheral tolerance. After administration of SEB to mice, the numbers of naive and memory T cells increase, as does the proportion of memory T cells, which are unresponsive to further stimulation with SEB in vitro. In addition, memory T cells generated in response to conventional antigen, which proliferate and provide help to B cells in the presence of the conventional antigen, fail to respond to superantigen. Hence, memory T cells, in general, are anergized by SEB. These results suggest that SEB-induced activation and anergy reflect the combined responses of naive and memory T cells. The differential activation vs. anergy of naive and memory T cells by superantigen may be related to cytokine production and may play an important role in the etiology of autoimmune diseases or immunodeficiency diseases such as acquired immune deficiency syndrome.
Evidence for an alpha/beta T cell-independent mechanism of resistance to mycobacteria. Bacillus-Calmette-Guerin causes progressive infection in severe combined immunodeficient mice, but not in nude mice or in mice depleted of CD4+ and CD8+ T cells.
Authors: Izzo A A , North R J
Published: June 2004

Depleting thymectomized mice of CD4+ T cells, or CD4+ plus CD8+ T cells, rendered them incapable of resolving Bacillus-Calmette-Guerin (BCG) infection in their lives, spleens, kidneys, and lungs. However, it did not render them incapable of stabilizing infection in the latter three organs after an initial period of BCG growth. Athymic nude mice showed a similar capacity to control BCG growth in these organs after a certain stage of infection. In contrast, congenitally severe combined immunodeficient (SCID) mice appeared to offer no resistance to BCG infection, in that the organism grew progressively in all organs of these mice and was lethal for them beginning on day 55 of infection. The results suggest that, although CD4+ T cells are important for resolving BCG infection, an alpha/beta T cell-independent mechanism of resistance can be acquired at 2-3 wk of infection that is capable of inhibiting further BCG growth in all organs except the lungs. Because this mechanism is absent from SCID mice, it is likely that it depends on the functions of gamma/delta T cells, B cells, or both types of cells. In keeping with this possibility is the additional finding that SCID mice engrafted with lymph node cells depleted of CD4+ or CD8+ T cells were capable of expressing an appreciable level of resistance against BCG infection.
Cytokine RANTES released by thrombin-stimulated platelets is a potent attractant for human eosinophils.
Authors: Kameyoshi Y , Dörschner A , Mallet A I , Christophers E , Schröder J M
Published: June 2004

Thrombin stimulation of human platelets results in the release of a preformed proteinaceous human eosinophil (Eo)-chemotactic activity. By the use of different high-performance liquid chromatography techniques, two Eo-chemotactic polypeptides (EoCPs), tentatively termed EoCP-1 and EoCP-2, were purified to homogeneity. Upon SDS-PAGE analysis, these chemotaxins showed molecular masses near 8 kD. NH2-terminal amino acid sequence analysis revealed identical sequences for both EoCP-1 and EoCP-2, which are also identical to that of RANTES, a cytokine that structurally belongs to the interleukin 8 superfamily of leukocyte selective attractants, and that is known to be a "memory-type" T lymphocyte-selective attractant. In the major Eo chemotaxin, EoCP-1, the residues 4 and 5, which in EoCP-2 were found to be serine residues, could not be identified. Electrospray mass spectrometry (ESP-MS) of EoCPs revealed for EoCP-2 a molecular mass of 7,862.8 +/- 1.1 daltons, which is 15.8 mass units higher than the calculated value of RANTES, indicating that EoCP-2 is identical to the full-length cytokine, and oxygenation, probably at methionine residue number 64, has taken place. Upon ESP-MS, EoCP-1 showed an average molecular mass of 8,355 +/- 10 daltons, suggesting O-glycosylation at these serine residues. Both natural forms of RANTES showed strong Eo-chemotactic activity (ED50 = 2 nM) with optimal chemotactic migration at concentrations near 10 nM, however, there were no significant migratory responses with human neutrophils. Chemotactic activity of RANTES for human Eos could be confirmed using recombinant material, which has been found to be as active as the natural forms. Since RANTES gene expression has been detected in activated T lymphocytes, and recombinant RANTES was shown to be a "memory" T lymphocyte-selective attractant, it is now tempting to speculate about an important role of RANTES in clinical situations such as allergene-induced late-phase skin reactions in atopic subjects or asthma, where in affected tissues both memory T cells and Eos are characteristic.
Mechanism of the rejection of major histocompatibility complex class I-disparate murine skin grafts: rejection can be mediated by CD4+ cells activated by allo-class I + II antigen in CD8+ cell-depleted hosts.
Authors: Kobayashi E , Kawai K , Ikarashi Y , Fujiwara M
Published: June 2004

In the preceding article, we analyzed the immunohistochemical rejection mechanism of major histocompatibility complex (MHC) class I (H-2K)-disparate murine skin grafts, and showed that only CD8+ cells infiltrated at the site of the epithelial tissue of MHC class I-disparate graft. We also showed that perfect survival of MHC class I-disparate grafts were attained in thymectomized recipients treated with anti-Lyt-2 monoclonal antibody. In this report, we showed that these long-surviving allo-class I grafts were rejected in the absence of CD8+ cells by stimulation with allo-MHC class I + II-disparate graft as the second stimulation. Furthermore, it was immunohistochemically revealed that under that condition, a large number of CD4+ cells infiltrated into the epithelial tissue of these long-surviving class I grafts, which were going to be rejected 2-5 d after the transplantation of a second graft with MHC class I + II difference. This result directly shows that CD4+ cells are able to became effectors for the rejection of allo-MHC class I (H-2K) skin graft.
Evaluation of the functional equivalence of major histocompatibility complex class II A and E complexes.
Authors: Cosgrove D , Bodmer H , Bogue M , Benoist C , Mathis D
Published: June 2004

Most mice display two conventional major histocompatibility complex class II isotypes, A and E. Several A+E- strains have been observed, but never any that are A-E+. Because of this and because of hints from several lines of functional analysis, it has been proposed that the two isotypes might not operate equivalently. This proposition has not been directly testable until now because of the lack of an E-only strain. We report the production of such mice, exploiting previously created class II-transgenic and class II-"knock-out" lines. A+E-, A-E-, and A-E+ littermates have been compared by a number of parameters. We find that E and A molecules are, for the most part, functionally equivalent. However, subtle differences are seen in their ability to engage CD4 molecules on immature thymocytes, and in the profile of receptors on T cells selected into the periphery.
Transfer of zymosan (yeast cell walls) to the parasitophorous vacuoles of macrophages infected with Leishmania amazonensis.
Authors: Veras P S , de Chastellier C , Rabinovitch M
Published: June 2004

Leishmania are flagellated protozoan parasites which, in their amastigote stages, survive and multiply within phagolysosome-like parasitophorous vacuoles (PV) of mammalian macrophages (MO). This study develops an earlier ultrastructural, incidental observation that zymosan particles (Z) were transferred to the PV of macrophages infected with Leishmania amazonensis. In the present report, a pulse-chase light microscopic assay was used to delineate several features of the Z transfer. The assay reflects both the movement of internalized particles to a position adjoining a PV, and their delivery to the vacuoles. Transfer was selective, in the sense that Z, beta-glucan or heat-killed yeast particles were transferred, whereas latex beads, aldehyde-fixed, or immunoglobulin G-coated erythrocytes were not. This selectivity may be related to the high density of carbohydrate ligands displayed on the surface of yeast-derived particles, to ligand resistance to lysosomal degradation or to signals encoded in the cytosolic tails of the receptors involved in particle recognition. A few Z particles could be found within PV after 1 h of incubation with infected MO, but chase periods of several hours at 34 degrees C were required for particle transfer to the PV in a substantial proportion of the MO. Ammonium chloride, chloroquine, or monensin, compounds which increase the pH in acidified compartments, substantially enhanced the transfer of Z particles. Finally, transfer was inhibited by cytochalasin D, but was unaffected by the antitubulin nocodazole. Although it is not yet known if particle transfer occurs by fusion of donor vesicles with PV or by interiorization of the former into the latter, the model described should be useful in the study of the interactions between large phagocytic vesicles and the modulation of those interactions by cellular, parasitic, and environmental signals.
Sialoadhesin on macrophages: its identification as a lymphocyte adhesion molecule.
Authors: van den Berg T K , Brevé J J , Damoiseaux J G , Döpp E A , Kelm S , Crocker P R , Dijkstra C D , Kraal G
Published: June 2004

In this study we present evidence that the mouse and rat sialoadhesin (originally named sheep erythrocyte receptor) on macrophages can function as a lymphocyte adhesion molecule. Lymphocytes were shown to bind to the splenic marginal zone, and lymph node subcapsular sinus and medulla in a frozen section assay. Selective depletion experiments showed that binding was mediated by macrophages. Adhesion was blocked by preincubation of the sections with monoclonal antibodies against mouse or rat sialoadhesin. Binding was temperature dependent, divalent cation independent, and involved sialic acid residues on the lymphocyte, as it could be inhibited by prior neuraminidase treatment or addition of the ganglioside GD1a. Binding to sialoadhesin was confirmed using the purified receptor and was observed among T cells, T blasts, B cells, and B blasts. Isolated macrophages or dendritic cells showed little binding. Sialoadhesin provides the first example of a macrophage-restricted lymphocyte adhesion molecule.
HLA-DR associates with specific stress proteins and is retained in the endoplasmic reticulum in invariant chain negative cells.
Authors: Schaiff W T , Hruska K A , McCourt D W , Green M , Schwartz B D
Published: June 2004

The major histocompatibility complex class II molecules are composed of two polymorphic chains which, in cells normally expressing them, transiently associate with a third, nonpolymorphic molecule, the invariant chain (Ii). To determine differences in the biology of class II molecules synthesized in the presence or absence of Ii, a comparative study was performed of BALB/c 3T3 cells that had been transfected with human class II HLA-DR molecules with or without cotransfection with human Ii. It was observed that in the absence of Ii, at least three high molecular weight proteins coimmunoprecipitate with HLA-DR molecules. These proteins did not coimmunoprecipitate with HLA-DR from cells cotransfected with Ii, nor did they coimmunoprecipitate with class I molecules from any of the transfectants. NH2-terminal sequence and/or Western blot analysis revealed the identity of two of the proteins as the endoplasmic reticulum (ER) resident stress proteins GRP94 and ERp72. Neither of these proteins was found to have an increased level of synthesis in the Ii- versus the Ii+ transfectants, indicating that their synthesis was not induced over constitutive levels. Fluorescence microscopy revealed that in the Ii- transfectants, the majority of the HLA-DR molecules were present in the ER, whereas in the Ii+ transfectants, the HLA-DR molecules were found in vesicular structures. We hypothesize that in the absence of Ii, ER resident stress proteins bind to class II molecules and retain them in the ER. This process, in turn, could prevent class II molecules from exiting the ER with endogenous peptides bound in their peptide binding cleft, and therefore could minimize autoimmune responses to endogenously processed self-peptides.
Disease inhibition by major histocompatibility complex binding peptide analogues of disease-associated epitopes: more than blocking alone.
Authors: Wauben M H , Boog C J , van der Zee R , Joosten I , Schlief A , van Eden W
Published: June 2004

Peptide analogues of disease-associated epitopes were studied for inhibition of experimental allergic encephalomyelitis (EAE) and adjuvant arthritis (AA) in Lewis rats. EAE- and AA-associated analogues were selected as competitors because of their in vitro inhibitory activity on proliferation of encephalitogenic and arthritogenic T cells. Although the EAE-associated competitor had a superior major histocompatibility complex (MHC) binding affinity, the AA-associated competitor was a better inhibitor of the in vitro proliferation of arthritogenic T cells. Furthermore, although in vivo EAE was inhibited by both competitors, AA was only inhibited by the AA-associated competitor. Remarkably, in contrast to what was expected of a regular MHC competitor peptide, the AA-associated peptide analogue also prevented AA upon immunization before disease induction and appeared to induce T cell responses that crossreacted with the original disease-associated epitope. Therefore, it is concluded that antigen-specific regulatory mechanisms were involved in synergy with MHC competition. The integration of both qualities into a single "competitor-modulator" analogue peptide may lead to the development of novel, more effective, disease-specific immunomodulatory peptides.
In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid sheath-associated foci and germinal centers.
Authors: Jacob J , Kelsoe G
Published: June 2004

In the genetically restricted response that follows immunization with (4-hydroxy-3-nitrophenyl)acetyl coupled to protein carriers, two distinct populations of B cells are observed in the spleens of C57BL/6 mice. By 48 h postimmunization, foci of antigen-binding B cells appear along the periphery of the periarteriolar lymphoid sheaths. These foci expand to contain large numbers of antibody-forming cells that neither bind the lectin, peanut agglutinin, nor mutate the rearranged immunoglobulin variable region loci. Germinal centers containing peanut agglutinin-positive B cells can be observed by 96-120 h after immunization. Although specific for the immunizing hapten, these B cells do not produce substantial amounts of antibody, but are the population that undergoes somatic hypermutation and affinity-driven selection. Both focus and germinal center populations are pauciclonal, founded, on average, by three or fewer B lymphocytes. Despite the highly specialized roles of the focus (early antibody production) and germinal center (higher affinity memory cells) B cell populations, analysis of VH to D to JH joins in neighboring foci and germinal centers demonstrate that these B cell populations have a common clonal origin.
Distinctive selection mechanisms govern the T cell receptor repertoire of peripheral CD4-CD8- alpha/beta T cells.
Authors: Huang L , Crispe I N
Published: June 2004

The T cell receptor (TCR) repertoire of CD4+ and CD8+ alpha/beta T cells is heavily influenced by positive and negative selection events that occur during T cell development in the thymus. The coreceptors CD4 and CD8 appear to be essential for this selection to occur. To gain insight into whether T cells that express TCR alpha/beta but lack either coreceptor (CD4- CD8- TCR alpha/beta or alpha/beta double-negative [DN] cells) are also subject to positive and negative selection, and whether selection can occur in the absence of coreceptors, we have performed an extensive immunogenetic analysis of the TCR V beta repertoire of alpha/beta DN cells in lymph nodes of normal mice. Our results show that alpha/beta DN cells appear to be unaffected by clonal deletion of V beta 5 and V beta 11 in I-E-expressing mice, and do not undergo deletion of V beta 6- and V beta 8.1-expressing T cells in Mls-1a-positive mice. They are also unaffected by positive selection of V beta 17a+ T cells in the context of I-Aq. The results suggest that most selection events require the participation of CD4 and CD8, while alpha/beta DN cells are unselected. This argues that most alpha/beta DN cells probably have never expressed CD4 or CD8. However, a unique form of repertoire selection occurs: enrichment of V beta 17a+ alpha/beta DN cells in I-E+ mice. This could be an instance of coreceptor-independent selection.
Cytomegalovirus prevents antigen presentation by blocking the transport of peptide-loaded major histocompatibility complex class I molecules into the medial-Golgi compartment.
Authors: del Val M , Hengel H , Häcker H , Hartlaub U , Ruppert T , Lucin P , Koszinowski U H
Published: June 2004

Selective expression of murine cytomegalovirus (MCMV) immediate-early (IE) genes leads to the presentation by the major histocompatibility complex (MHC) class I molecule Ld of a peptide derived from MCMV IE protein pp89 (Reddehase, M.J., J. B. Rothbard, and U.H. Koszinowski. 1989. Nature (Lond.). 337:651). Characterization of endogenous antigenic peptides identified the pp89 peptide as the nonapeptide 168YPHFMPTNL176 (del Val, M., H.-J. Schlicht, T. Ruppert, M.J. Reddehase, and U.H. Koszinowski. 1991. Cell. 66:1145). Subsequent expression of MCMV early genes prevents presentation of pp89 (del Val, M., K. Münch, M.J. Reddehase, and U.H. Koszinowski. 1989. Cell. 58:305). We report on the mechanism by which MCMV early genes interfere with antigen presentation. Expression of the IE promoter-driven bacterial gene lacZ by recombinant MCMV subjected antigen presentation of beta-galactosidase to the same control and excluded antigen specificity. The Ld-dependent presence of naturally processed antigenic peptides also in nonpresenting cells located the inhibitory function subsequent to the step of antigen processing. The finding that during the E phase of MCMV gene expression the MHC class I heavy chain glycosylation remained in an Endo H-sensitive form suggested a block within the endoplasmic reticulum/cis-Golgi compartment. The failure to present antigenic peptides was explained by a general retention of nascent assembled trimolecular MHC class I complexes. Accordingly, at later stages of infection a significant decrease of surface MHC class I expression was seen, whereas other membrane glycoproteins remained unaffected. Thus, MCMV E genes endow this virus with an effective immune evasion potential. These results also indicate that the formation of the trimolecular complex of MHC class I heavy chain, beta 2-microglobulin, and the finally trimmed peptide is completed before entering the medial-Golgi compartment.
The immunosuppressive and toxic effects of FK-506 are mechanistically related: pharmacology of a novel antagonist of FK-506 and rapamycin.
Authors: Dumont F J , Staruch M J , Koprak S L , Siekierka J J , Lin C S , Harrison R , Sewell T , Kindt V M , Beattie T R , Wyvratt M
Published: June 2004

FK-506 inhibits Ca(2+)-dependent transcription of lymphokine genes in T cells, and thereby acts as a powerful immunosuppressant. However, its potential therapeutic applications may be seriously limited by several side effects, including nephrotoxicity and neurotoxicity. At present, it is unclear whether these immunosuppressive and toxic effects result from interference with related biochemical processes. FK-506 is known to interact with FK-binding protein-12 (FKBP-12), an abundant cytosolic protein with cis-trans peptidyl-prolyl isomerase activity (PPIase) activity. Because rapamycin (RAP) similarly binds to FKBP-12, although it acts in a manner different from FK-506, by inhibiting T cell responses to lymphokines, such an interaction with FKBP-12 is not sufficient to mediate immunosuppression. Recently, it was found that the complex of FKBP-12 with FK-506, but not with RAP, inhibits the phosphatase activity of calcineurin. Here, we used L-685,818, the C18-hydroxy, C21-ethyl derivative of FK-506, to explore further the role of FKBP-12 in the immunosuppressive and toxic actions of FK-506. Although L-685,818 bound with high affinity to FKBP-12 and inhibited its PPIase activity, it did not suppress T cell activation, and, when complexed with FKBP-12, did not affect calcineurin phosphatase activity. However, L-685,818 was a potent antagonist of the immunosuppressive activity of both FK-506 and RAP. Moreover, L-685,818 did not induce any toxicity in dogs and rats or in a mouse model of acute FK-506 nephrotoxicity, but it blocked the effect of FK-506 in this model. Therefore, FK-506 toxicity involves the disruption of biochemical mechanisms related to those implicated in T cell activation. Like immunosuppression, this toxicity is not due to the inhibition of the PPIase activity of FKBP-12, but may be linked to the inhibition of the phosphatase activity of calcineurin by the drug FKBP-12 complex.
Both IgM and IgG anti-DNA antibodies are the products of clonally selective B cell stimulation in (NZB x NZW)F1 mice.
Authors: Tillman D M , Jou N T , Hill R J , Marion T N
Published: June 2004

Disease activity in systemic lupus erythematosus is closely associated with the appearance of immunoglobulin (Ig)G antibody to native DNA in both humans and mice. Like normal antibody responses, the anti-DNA autoantibody first appears as IgM and then switches to IgG. Structural studies of IgG anti-DNA suggest that these antibodies are the products of clonally selected, specifically stimulated B cells. The origins of the IgM anti-DNA have been less clear. To determine whether the earlier appearing IgM anti-DNA antibody in autoimmune mice also derives from clonally selected, specifically stimulated B cells or B cells activated by nonselective, polyclonal stimuli, we have analyzed the molecular and serological characteristics of a large number of monoclonal IgM anti-DNA antibodies from autoimmune (NZB x NZW)F1 mice. We have also analyzed IgM and IgG anti-DNA hybridomas obtained from the same individual mice to determine how the later-appearing IgG autoantibody may be related to the earlier-appearing IgM autoantibody within an individual mouse. The results demonstrate that: (a) IgM anti-DNA, like IgG, has the characteristics of a specifically stimulated antibody; (b) IgM and IgG anti-DNA antibodies have similar variable region structures and within individual mice may be produced by B cells derived from the same clonal precursors; (c) recurrent germline and somatically derived VH and VL structures may influence the specificity of anti-DNA monoclonal antibody for denatured vs. native DNA; and (d) the results provide a structural explanation for the selective development of IgG antibody to native DNA as autoimmunity to DNA progresses in (NZB x NZW)F1 mice.
NF-kappa B-like factors mediate interleukin 1 induction of c-myc gene transcription in fibroblasts.
Authors: Kessler D J , Duyao M P , Spicer D B , Sonenshein G E
Published: June 2004

Interleukin 1 (IL-1) is a pluripotent cytokine involved in mediating a variety of physiological processes, including induction of cell proliferation upon wound healing. Treatment of quiescent FS-4 human dermal fibroblast cells with IL-1 activates c-myc gene transcription, and nuclear localization of NF-kappa B. Previously, we have noted that the murine c-myc gene contains two functional NF-kappa B sites located at -1101 to -1081 bp (upstream regulatory element [URE]) and +440 to +459 bp (internal regulatory element [IRE]) relative to the P1 promoter. Here we have demonstrated that IL-1 treatment induced binding of NF-kappa B-like proteins (p50/p65) to these c-myc elements. Heterologous promoter-CAT constructs driven by multiple copies of either the URE or IRE were IL-1 inducible when transfected into FS-4 cells. In contrast, constructs harboring elements with two G to C residue conversions, such that they were no longer able to bind NF-kappa B, were not responsive to IL-1. Mutation of these two base pairs at both NF-kappa B sites within a c-myc promoter/exon I-CAT construct, resulted in loss of inducibility with IL-1 upon transfection into quiescent FS-4 cells. Thus, IL-1 significantly induces c-myc expression through positive regulation by NF-kappa B, suggesting a role for this family of factors in activation of proliferation associated with wound healing.
Long-lived complexes between peptide and class II major histocompatibility complex are formed at low pH with no requirement for pH neutralization.
Authors: Jensen P E
Published: June 2004

The binding of peptide antigens to class II histocompatibility glycoproteins can be markedly enhanced at pH values approximating those found in acidic endosomal compartments in antigen-presenting cells (APC). It has been proposed by others that low pH may increase the conformational flexibility of class II, facilitating both the association and dissociation of peptides. Neutralization of pH, as class II is expressed on the plasma membrane of APC, could then serve to trap peptide in a stable complex. If this were the only mechanism accounting for enhanced peptide binding at low pH, one would predict that there should be a concordance between the pH conditions required for enhanced binding and those associated with increased peptide dissociation. Furthermore, long-lived complexes of class II and peptide should not be observed at low pH without neutralization. In the present communization, I provide the data that support the generality of my previous conclusion that both affinity and maximal binding are regulated by pH in experiments using purified class II and biotin-labeled peptides. The pH profile for binding and dissociation using three different class II glycoproteins was analyzed, and the results demonstrated that enhanced binding is not coupled to enhanced dissociation. Peptide complexes were observed to be quite stable at pH 4.5 and above. This result was further substantiated in experiments where biotin-peptide/class II complexes were extensively dialyzed at low pH followed by analysis on Western blots probed with avidin. Finally, a low pH assay system was devised to analyze the formation of stable peptide/class II complexes without pH neutralization. Our results indicate that stable complexes can be formed at low pH without the requirement for a shift to neutral pH.
Antibody-resistant mutants of Borrelia burgdorferi: in vitro selection and characterization.
Authors: Sădziene A , Rosa P A , Thompson P A , Hogan D M , Barbour A G
Published: June 2004

We used polyclonal antisera and monoclonal antibodies (mAbs) to inhibit the growth of clonal populations of two strains of Borrelia burgdorferi, the Lyme disease agent, and thereby select for antibody-resistant mutants. mAbs were directed at the outer membrane proteins, OspA or OspB. Mutants resistant to the growth-inhibiting properties of the antibodies were present in the populations at frequencies ranging from 10(-5) to 10(-2). The several escape variants that were examined were of four classes. Class I mutants were resistant to all mAbs; they lacked OspA and OspB and the linear plasmid that encodes them. Two other proteins were expressed in larger amounts in class I mutants; mAbs to these proteins inhibited the mutant but not the wild-type cells. Class II mutants were resistant to some but not all mAbs; they had truncated OspA and/or OspB proteins. Class III mutants were resistant only to the selecting mAb; they had full-length Osp proteins that were not bound by the selecting antibody in Western blots. In two class III mutants resistant to different anti-OspA mAbs, missense mutations were demonstrated in the ospA genes. Class IV mutants were likewise resistant only to selecting antibody, but in this case the selecting antibody still bound in Western blots.
Monocyte-selective transendothelial migration: dissection of the binding and transmigration phases by an in vitro assay.
Authors: Muller W A , Weigl S A
Published: June 2004

We describe a quantitative assay of transendothelial migration (TEM) that allows us to selectively study the interaction of monocytes with confluent human endothelial cell (HEC) monolayers. The HEC are grown on hydrated collagen gels; the monocytes need not be purified. 100% of monocytes transmigrated the monolayer within 1 h at 37 degrees C and accumulated in the subendothelial collagen; TEM of lymphocytes was not detected within this time. Migration of neutrophils from the same donor was much slower and incomplete, with only 14% of PMN transmigrating in 2 h. This rapid TEM occurs in the absence of exogenous chemoattractants, and HEC in this system do not express cytokine-inducible leukocyte adhesion molecules. A slight modification of the TEM assay allowed us to separate binding to the apical HEC surface from TEM. We found that tight apical surface binding was the rate-limiting step for TEM. Two-thirds of this binding and TEM could be blocked by a monoclonal antibody against the leukocyte beta 2 integrin chain CD18. This assay will allow us to dissect the mechanisms of both the binding and transmigration stages of diapedesis.
The CD45 tyrosine phosphatase regulates phosphotyrosine homeostasis and its loss reveals a novel pattern of late T cell receptor-induced Ca2+ oscillations.
Authors: Volarević S , Niklinska B B , Burns C M , Yamada H , June C H , Dumont F J , Ashwell J D
Published: June 2004

CD45 is a transmembrane tyrosine phosphatase implicated in T cell antigen receptor (TCR)-mediated activation. In T cell variants expressing progressively lower levels of CD45 (from normal to undetectable), CD45 expression was inversely related to spontaneous tyrosine phosphorylation of multiple proteins, including the TCR zeta chain, and was directly correlated with TCR-driven phosphoinositide hydrolysis. The Ca2+ response in these cells was altered in an unexpected fashion. Unlike wild-type cells, stimulated CD45- cell populations did not manifest an early increase in intracellular Ca2+, but did exhibit a delayed and gradual increase in mean intracellular Ca2+. Computer-aided fluorescence imaging of individual cells revealed that CD45- cells experienced late Ca2+ oscillations that were not blocked by removal of extracellular Ca2+. CD45 revertants had the signaling properties of wild-type cells. Thus, CD45 has a profound influence on both TCR-mediated signaling and phosphotyrosine homeostasis, and its loss reveals a novel role for this tyrosine phosphatase in Ca2+ regulation.
Studies on T cell maturation on defined thymic stromal cell populations in vitro.
Authors: Jenkinson E J , Anderson G , Owen J J
Published: June 2004

We describe an in vitro system in which positive selection of developing T cells takes place on defined stromal cell preparations, which include major histocompatibility complex class II+ epithelial cells but exclude cells of bone marrow origin. In this system, maturation of double-positive T cell receptor negative (TCR-), CD4+8+ thymocytes into single-positive TCR+, CD4+ and CD8+ cells takes place together with the development of functional competence. As in vivo, this maturation is associated with the upregulation of TCR levels as cells progress from double-positive to single-positive status. We also show that class II+ epithelial cells in these cultures are less efficient than dendritic cells in mediating the deletion (negative selection) of V beta 8+ cells by the superantigen staphylococcal enterotoxin B. For the first time, this approach provides a model in which the cellular interactions involved in both positive and negative selection can be studied under controlled in vitro conditions.
Human T cells cannot act as autonomous antigen-presenting cells, but induce tolerance in antigen-specific and alloreactive responder cells.
Authors: Sidhu S , Deacock S , Bal V , Batchelor J R , Lombardi G , Lechler R I
Published: June 2004

The ability of two HLA-DR-expressing human T cell clones to function as antigen-presenting cells (APC) was investigated using highly purified T cells. The results demonstrated that these T cell clones are unable to act as autonomous APC, and that recognition of nominal or alloantigens on the surface of T cells leads to a state of nonresponsiveness. The first observation was that a T cell clone with specificity for the 306-324 peptide of influenza hemagglutinin (HA), and raised from a DR1 responder, exhibited apparent degeneracy of major histocompatibility complex restriction when cultured with peptide in the presence of peripheral blood mononuclear cells (PBMC) expressing a wide variety of structurally unrelated DR types. However, when the PBMC were pulsed with peptide and washed before coculture with the clone, peptide was exclusively recognized with DR1Dw1. This implied that in the presence of soluble peptide the T cells were displaying ligand to each other, and that the third-party APC were providing costimulatory signals. To test the ability of T cells to act as autonomous APC, accessory cell-free preparations of two DR1-restricted clones were cultured with peptide in the presence or the absence of added B cell APC. T cell purity was established by the absence of proliferation in response to the mitogen phytohemagglutinin (PHA). PHA-nonresponsive T cells were completely unable to proliferate in response to peptide alone; furthermore, preculture of the HA-specific clone, in the complete absence of accessory cells, with the same concentration of peptide (1 microgram/ml) that induced optimal proliferation when presented by conventional APC, led to profound nonresponsiveness. The same phenomenon was also observed when two of three anti-DR1 alloreactive T cell clones were precultured with a DR1-expressing T cell clone. The ability of the DR1-expressing clone to induce nonresponsiveness in anti-DR1 clones correlated with recognition of the DR1 alloantigen on the DR1-expressing clone.
A 68-kD GTP-binding protein associated with the T cell receptor complex.
Authors: Ohmura T , Sakata A , Onoue K
Published: June 2004

The identity of the guanine nucleotide-binding protein (G protein) involved in T cell activation pathways remains unclear. We identified a 68-kD GTP-binding protein associated with the T cell receptor (TCR)/CD3 complex using immunoprecipitation and GTP-affinity labeling techniques. Proteins coimmunoprecipitated with the TCR/CD3 complex in digitonin lysate of a human leukemic T cell line, MOLT 16, were incubated with alpha-[32P]GTP and irradiated with ultraviolet rays to covalently link the labeled GTP to GTP-binding proteins. They were then analyzed by electrophoresis. The 68-kD protein exhibited nucleotide specificity for GTP-binding and was insensitive to cholera and pertussis toxins. The 68-kD GTP-binding protein could be coimmunoprecipitated with the TCR/CD3 complex but not with other surface molecules such as major histocompatibility complex class I and lymphocyte function associated-1, which do not cause rapid Ca2+ mobilization. These suggest that the 68-kD GTP-binding protein is specifically associated with the TCR/CD3 complex.
Clostridium perfringens enterotoxin is a superantigen reactive with human T cell receptors V beta 6.9 and V beta 22.
Authors: Bowness P , Moss P A , Tranter H , Bell J I , McMichael A J
Published: June 2004

Candidate superantigens were screened for their ability to induce lysis of human histocompatibility leukocyte antigen class II-positive targets by human CD8+ influenza-specific cytotoxic T cell (CTL) lines. Clostridium perfringens enterotoxin (CPET) induced major histocompatibility complex unrestricted killing by some but not all CTL lines. Using "anchored" polymerase chain reactions, CPET was shown to selectively stimulate peripheral blood lymphocytes bearing T cell receptor V beta 6.9 and V beta 22 in five healthy donors. V beta 24, V beta 21, V beta 18, V beta 5, and V beta 6.1-5 appeared to be weakly stimulated. Antigen processing was not required for CPET to induce proliferation. Like the staphylococcal enterotoxins, CPET is a major cause of food poisoning. These data suggest that superantigenic and enterotoxigenic properties may be closely linked.