bluuebunny/crossref_metadata_2025 huggingface dataset:
DR αβ dimers released from complexes with invariant chain fail to stimulate alloreactive T cell clones
Authors: Demotz Stéphane
Published: March 2007

To demonstrate that DR αβ dimers still complexed to invariant chain (Ii) have not yet acquired peptides recognized by alloreactive T cells, complexes between DR molecules and Ii isolated from Epstein‐Barr‐virus (EBV)‐transformed B cells were analyzed by affinity chromatography and gel filtration. First, it was shown that DR/Ii complexes inserted into artificial planar membranes (PM) failed to stimulate proliferative response of five alloreactive T cell clones and a polyclonal alloreactive Tcell line, while PM bearing mature DR αβ dimers from the same EBV‐B cells were stimulatory for the Tcell clones and the Tcell line. These findings indicate that either Ii inhibits binding of peptides to DR molecules or Ii hinders T cells recognition of peptide/DR complexes. Second, to discriminate between these two possibilities, DR αβ dimers, which were artificially released from complexes between DR molecules and Ii, were inserted into PM. These DR αβ dimers were devoid of alloreactive stimulatory capacity while fully capable of binding and presenting a tetanus toxin synthetic peptide to a specific Tcell clone, indicating that DR molecules released from complexes with Ii are empty. This study, by showing that DR molecules bound to Ii do not bear peptides recognized by alloreactive T cells, supports the notion that association of Ii with class II major histocompatibility complex (MHC) molecules prevents premature peptide loading and hence favors encounter with peptides derived from proteins of the extracellular compartment.

Since allogeneic class II MHC molecules released from complexes with Ii were not stimulatory for five out of five alloreactive Tcell clones and a polyclonal alloreactive Tcell line, these data also indicate that, in most cases, alloreactive T cells recognize ligands constituted by complexes between allogeneic class II MHC molecules and specific peptides which derive from the antigen‐presenting cells themselves or serum proteins.
Activator protein‐1 (AP‐1) is stimulated by microbial superantigens in human monocytic cells
Authors: Trede Nikolaus S. , Chatila Talal , Geha Raif S.
Published: March 2007

Microbial superantigens bind to major histocompatibility complex (MHC) class II molecules and activate gene transcription in monocytes. In search of transcription factors that potentially mediate the effects of superantigens at the nuclear level, we examined the capacity of staphylococcal superantigens to stimulate the activity of the transcriptional promoter factor activator protein‐1 (AP‐1). Electrophoretic mobility shift assays showed an increase in nuclear proteins that bound to the consensus AP‐1 motif within 5 min following the stimulation of the monocytic cell line THP‐1 with toxic shock syndrome toxin‐1 (TSST‐1) or staphylococcal entotoxin A. We show that mRNA levels for the subunits that compose AP‐1, the protooncogenes c‐fos and c‐jun, are up‐regulated by stimulation of THP‐1 cells with TSST‐1. The activated AP‐1 complexes were functional, as evidenced by the capacity of TSST‐1 to stimulate the expression of an AP‐1‐driven reporter gene construct transfected into THP‐1 cells. These results establish that the engagement of MHC class II molecules by superantigens increases the activity of functional AP‐1 complexes and that this may proceed in part by transcriptional activation of c‐fos and c‐jun protooncogenes.
The interleukin‐12 subunit p40 specifically inhibits effects of the interleukin‐12 heterodimer
Authors: Mattner Frank , Fischer Susanne , Guckes Stefanie , Jin Shenchu , Kaulen Hildegard , Schmitt Edgar , Rüde Erwin , Germann Tieno
Published: March 2007

The recently discovered cytokine interleukin (IL)‐12 is a heterodimeric protein of two disulfide‐bonded subunits of 35 and 40 kDa. IL‐12 has multiple effects on T cells and natural killer (NK) cells. In particular it appears to be a major factor for the development of cellular immunity. So far activity of the single subunits alone has not been described, however their expression is regulated independently. In this report we demonstrate for the first time that the mouse IL‐12 subunit p40 (IL‐12p40) specifically antagonizes the effects of the IL‐12 heterodimer in different assay systems. The proliferation of mouse splenocytes activated by phorbol ester and IL‐12 was inhibited by IL‐12p40, whereas the proliferation induced by phorbol ester and IL‐2 was not affected. Furthermore, the synthesis of interferon (IFN)‐γ by mouse splenocytes activated with IL‐2 and IL‐12 was suppressed by IL‐12p40. Purified mouse splenic CD4+ T cells produced IFN‐γ upon activation with plate‐bound anti‐CD3 monoclonal antibody which was enhanced more than tenfold in the presence of IL‐12. In this system IL‐12p40 inhibited only the enhancement caused by IL‐12 but not IFN‐γ synthesis of CD4+ T cells stimulated with anti‐CD3 alone. Moreover, IL‐12p40 inhibited the effects of IL‐12 on differentiated T helper type 1 (Th1) cells. IFN‐γ production by Th1 cells induced in a T cell receptor‐independent way by macrophages and IL‐2 or macrophages and IL‐12 was greatly reduced by IL‐12p40 providing evidence for the endogenous synthesis of IL‐12 in the Th1 cell, macrophage and IL‐2 co‐cultures. The specificity of inhibition was clearly demonstrated in the homotypic aggregation assay of Th1 cells. Incubation of Th1 cells with either IL‐2 and IL‐12 or IL‐2 and tumor necrosis factor induces LFA‐1/ICAM‐1‐dependent aggregation. Only IL‐2 + IL‐12 but not IL‐2 + tumor necrosis factor‐induced aggregation was inhibited in a dose‐dependent manner by IL‐12p40. Thus, the IL‐12 subunit p40 appears to be a specific inhibitor for the IL‐12 heterodimer.
Modification of the anti‐CD3‐induced cytokine release syndrome by anti‐interferon‐γ or anti‐interleukin‐6 antibody treatment: Protective effects and biphasic changes in blood cytokine levels
Authors: Matthys Patrick , Dillen Chris , Proost Paul , Heremans Hubertine , And Jo Van Damme , Billiau Alfons
Published: March 2007

Anti‐interferon‐γ (IFN‐γ) antibodies were found to protect mice against pathological changes induced by injection of anti‐CD3 antibody: incidence of diarrhea, severity of hypothermia and mortality rates were dramatically reduced. In anti‐IFN‐γ antibody‐treated mice, IFN‐γ blood levels were significantly reduced at 1.5 h post anti‐CD3 challenge, but more elevated levels were found from 4 to 24 h. This rebound‐like IFN‐γ response coincided with more profound hypoglycemia. Tumor necrosis factor and interleukin (IL)‐6 levels were not affected by anti‐IFN‐γ treatment. Exogenous IFN‐γ, administered within 3 h (but not later) of the anti‐CD3 challenge made the syndrome worse. Furthermore, inter‐mouse strain differences in sensitivity to the anti‐CD3 syndrome correlated with the ability of the strain to produce IFN‐γ. Anti‐IL‐6 antibodies provided only marginal protection against hypothermia and mortality, but did markedly reduce hypoglycemia. Levels of biologically active IL‐6 in serum were not influenced by anti‐IL‐6 antibody treatment during the first few hours after anti‐CD3 challenge, but were significantly increased at later times.

The data provide evidence that endogenous IFN‐γ is a critical element in the early phase of the anti‐CD3 syndrome; endogenous IL‐6, while possibly being involved in hypoglycemia, seems of lesser importance for the outcome of the syndrome.
Growth requirements for avian γδ T cells include exogenous cytokines, receptor ligation and in vivo priming
Authors: Kasahara Yoshihito , Chen Chen‐Lo H. , Cooper Max D.
Published: March 2007

These studies analyze growth requirements for the normal γδ T cell population in peripheral lymphoid tissues. Avian γδ T cells can respond well to T cell mitogens in the presence of αβ T cells, but our studies indicate that they do not grow well alone. Exogenous growth factors were required in order for γδ T cells to proliferate in response to receptor ligation by anti‐T cell receptor antibodies or other T cell mitogens. Interleukin‐2 was implicated as one of the necessary growth factors that the γδ cells cannot produce adequately on their own. The response to dual stimulation (receptor ligation plus exogenous T cell factors) was attributable to a discrete subpopulation of γδ T cells that could be identified by their cell surface CD8, major histocompatibility complex class II expression and relative increase in cell size. Conversely, non‐responsive γδ T cells did not exhibit these activation markers. These observations suggest a physiological basis for the relatively late appearance of γδ T cells in inflammatory responses and their failure as a population to match the growth potential of αβ T cells. More importantly, the results imply that the biological role of γδ T cells must be understood within the context of their interaction with αβ T cells.
Role of transforming growth factor‐βthe preferential induction of T helper cells of type 1 by staphylococcal enterotoxin B
Authors: Nageikerken Lex , Gollob Kenneth J. , Tielemans Margret , Coffman Robert L.
Published: March 2007

Stimulation of murine CD4+ T cells with staphylococcal enterotoxin B (SEB) results in the preferential development of T helper (Th) 1 cells [i.e. high interferon (IFN)‐γ and low interleukin (IL)‐4, IL‐5 and IL‐10]; whereas in response to plate‐bound anti‐CD3 or anti‐T cell receptor‐αβ, Th1 as well as Th2 cells develop. In the present study, we examined the mechanism which is responsible for the selective Th1 development in the SEB system. The addition of IL‐4 resulted in a strong development of Th2 cells showing that SEB stimulation can result inTh2 differentiation. Co‐stimulation with anti‐CD28 was insufficient in this regard. Lack of Th2 development in the SEB system was in part due to the inhibitory effect of endogenously produced transforming growth factor‐β (TGF‐β), because anti‐TGF‐β allowed the development of Th2 cells. Similarly, TGF‐β inhibited Th2 development and stimulated Th1 development in the anti‐CD3 system. This shift was only partially prevented by also including IL‐4 in the cultures. The effects of TGF‐β could only partially be explained by stimulation of IFN‐γ or inhibition of IL‐4 as intermediatory cytokines: (1) TGF‐β stimulated Th1 development even in the presence of anti‐IL‐4 and anti‐IFN‐γ, and (2) a strong inhibitory effect of anti‐TGF‐β on Th1 development was still observed when anti‐IL‐4 and IFN‐γ were simultaneously added to the cultures. It is concluded that SEB favors Th1 development by stimulation of TGF‐β production. Inhibition of Th2 development by TGF‐β is due, in part, to inhibition of IL‐4 and stimulation of IFN‐γ, and, in part, to a direct effect of TGF‐β on the responding T cells.
CD40 ligand is a T cell growth factor
Authors: Armitage Richard J. , Tough Teresa W. , Macduff Brian M. , Fanslow William C. , Spriggs Melanie K. , Ramsdell Fred , Alderson Mark R.
Published: March 2007

CD40 ligand (CD40L) is a 33‐kDa type II membrane glycoprotein induced on T cells upon activation. CD40L has previously been shown to induce proliferation of resting B cells, immunoglobulin (Ig) secretion from B cells cultured with cytokines and cytokine secretion and tumoricidal activity from monocytes. In this report CD40L is shown to be stimulatory for human T cells, inducing CD25 (p55 IL‐2R) and CD40L expression on resting peripheral blood T cells, enhanced expression of these molecules and CD69 on CD3‐activated cells and secretion of interferon‐y, tumor necrosis factor‐a and interleukin (IL)‐2 from T cells cultured in the presence of a sub‐mitogenic concentration of phytohemagglutinin A (PHA). Furthermore, stimulation with CD40L induces proliferation of CD3‐ or PHA‐activated T cells of blood, tonsillar or thymic origin. A similar proliferative response is observed with CD4− and CD8+ T cells and this effect is largely IL‐2 independent. A soluble construct of the extracellular domain of the CD40L has similar activity to that of membrane‐expressed ligand in the induction of T cell surface antigens and proliferation. The results presented here taken together with the various activities ascribed for CD40L on B cells and monocytes demonstrate that CD40L has pleiotropic biological activity for cells of the hemopoietic lineage.
Autoimmune syndrome after induction of neonatal tolerance to I‐E antigens
Authors: Gonzalez Antonio L. , Conde Carmen , Revilla Conchi , Ramos Angeles , Renedo Blanca , Merino Jesús
Published: March 2007

Neonatal injection of semiallogeneic spleen cells induces a state of specific tolerance to the parental alloantigens, but also the development of an autoimmune syndrome known as host‐versus‐graft disease (HVGD). The autoimmune features are a consequence of the allogeneic cooperation between persisting alloreactive host T helper type 2 (TH2) cells and donor semiallogeneic B cells. It has been established that I‐A alloantigens play a central role in the triggering of this HVGD. Here it was investigated if I‐E antigens, which have shown functional differences, regarding autoimmunity and alloreactivity, with respect to I‐A antigens, are also able to trigger this autoimmune syndrome. The injection of spleen cells from [B10.A(4R) X B10.A(2R)]F1 (I‐E+) hybrid mice into newborn B10.A(4R) (I‐E−) mice was accompanied by the establishment of chimerism and also by the development of a characteristic, but moderated, HVGD. The weak intensity of this HVGD is likely due to the moderation of the alloreactive responses induced against I‐E molecules. Moreover, the marked increase in the levels of IgE and in the titers of anti‐DNA IgGl antibodies strongly suggest that alloreactive TH2 cells play also a main role in the autoimmune syndrome following tolerization to I‐E antigens. Therefore, it is concluded that the I‐E and I‐A isotypes are functionally similar with respect to the allogeneic cellular interactions that account for the HVGD.
A mouse aminopeptidase N is a marker for antigen‐presenting cells and appears to be co‐expressed with major histocompatibility complex class II molecules
Authors: Hansen Anette Stryhn , Norén Ove , Sjöström Hans , Werdelin Ole
Published: March 2007

To analyze the expression of mouse aminopeptidase N (APN) on the cells of the immune system a panel of rat monoclonal antibodies against mouse intestinal APN was generated. These antibodies were used to affinity purify functional mouse APN from both intestine and kidney, and by flow cytometry to examine the APN expression of the cells of the mouse immune system. An APN closely related, perhaps identical, to the intestinal APN was expressed on a subpopulation of spleen cells and stimulated peritoneal exudate cells, primarily representing antigen‐presenting cells, such as B cells, macrophages, dendritic cells, and veiled cells. In contrast this APN expression could not be detected on thymocytes or spleen T cells. As a corollary, APN was expressed on monocyte, macrophage, and B lymphoma cell lines, but not on T hybridoma or thymoma cell lines. The expression of APN showed a striking correlation with the MHC class II expression in all the cell populations studied. This apparent co‐expression suggests a role for APN in antigen processing.
Autoimmune gld mutation uncouples suicide and cytokine/proliferation pathways in activated, mature T cells
Authors: Russell John H. , Wang Ruduan
Published: March 2007

Antigen receptor‐directed suicide plays an important role in the elimination of potentially autoaggressive immature T cells during thymic differentiation. Here we demonstrate evidence for a second pathway of receptor‐directed suicide in mature T cells that is missing in a mutant strain (gld) of mice with an “autoimmune” lymphoproliferative syndrome. The defect is evident within the gld activated T cell and does not require the presence of an antigen‐presenting cell for its expression. Receptor‐driven suicide is intact in immature T cells of animals with this mutation. These results support the significance of receptor‐directed suicide in the mature T cell compartment and suggest that the immune system may use three independent pathways for regulating programmed cell death in shaping and controlling the immune response.
Human CD38 is associated to distinct molecules which mediate transmembrane signaling in different lineages
Authors: Funaro Ada , De Monte Lucia B. , Dianzani Umberto , Forni Marco , Malavasi Fabio
Published: March 2007

The CD38 antigen displays restricted functional associations with surface molecules involved in immune system and complement. Capping of the CD38 molecule in normal or neoplastic T cells is followed by rapid and specific co‐modulation of the CD3‐Tcell receptor (TcR) complex. In normal and tumor cells of B lineage, CD38 was found to be also associated with surface Ig (sIg) and with the complement receptor 2 (CR2)/CD19 complex. The CD38 molecule expressed by purified natural killer cells displayed an association with the low affinity IgG Fc receptor (FcγRIII) CD16. These observations suggest that specialized areas in the plasma membrane contain co‐modulating structures, including different receptors involved in the transduction of extracellular signals. We propose a model whereby TcR, CR2 and CD16 are ligand binding structures in their respective lineages, while CD38 is a molecule involved in the intracellular transduction of the signals.
Long‐term thymic reconstitution by peripheral CD4 and CD8 single‐positive lymphocytes
Authors: Hilbert David M. , Holmes Kevin L. , Anderson Arthur O. , Rudikoff Stuart
Published: March 2007

Significant immigration of peripheral T cells into SCID thymus was observed following reconstitution with normal Peyer's patch, mesenteric lymph node or peripheral lymph node cells. Immunohistologic and flow cytometric analyses reveal that T cells from these tissues are found in the thymus for as long as 177 days and can account for up to 67% of intrathymic cells. The returning cells express the CD3/Tcell receptor α/β complex, indicative of mature cells, and are equally divided among helper (CD4+ CD8 ) and cytotoxic (CD4−/CD8+) phenotypes. The immigration of peripheral T cells is not accompanied by the appearance of immature, double‐positive (CD4+CD8+) thymocytes as seen in similar reconstitutions using bone marrow. Taken together, these results suggest that peripheral T cells from a variety of lymphoid organs may regularly re‐enter the thymus and, thus, possibly play a role in normal thymic development.
The monoclonal antibody CZ‐1 identifies a mouse CD45‐associated epitope expressed on interleukin‐2‐responsive cells
Authors: Brutkiewicz Randy R. , O'Donnell Carey L. , Maciaszek Joseph W. , Welsh Raymond M. , Vargas‐Cortes Mauricio
Published: March 2007

We have previously described a monoclonal antibody (mAb), CZ‐1, which reacts with an epitope expressed on most peripheral basophils, natural killer cells, B cells, and CD8+ T cells, but not with most thymocytes or peripheral CD4+ T cells. Here we show that mAb CZ‐1 defines a sialic acid‐dependent epitope associated with a subpopulation of CD45 molecules. This conclusion is based on the ability to block binding of mAb CZ‐1 by sialic acid, neuramin‐lactose, neuraminidase, and mAb to CD45RB, and by expression of the epitope on transfected W2 cells expressing exon B of CD45. The results suggest that the CZ‐1 epitope is a post‐translational modification expressed on a subpopulation of the CD45 molecules also expressing the B exon. Expression of the CZ‐1 epitope was required for freshly isolated lymphocytes to respond to interleukin‐2 (IL‐2). Depletion of CZ‐1+ cells by C or by cell sorting of thymocytes or splenocytes eliminated the IL‐2 responsive cells. The subpopulations of thymocytes and CD4+ splenocytes responding to IL‐2 were exclusively within the small CZ‐1+ subpopulation. mAb CZ‐1 was also used to subdivide CD45+ and CD45RB+ splenocytes into IL‐2‐responsive and ‐nonresponsive subpopulations. The CZ‐1 epitope was also expressed on virtually all lymphokine‐activated killer cell precursors. These data, thus, indicate that cells responsive to IL‐2 express this sialated modification of CD45.
Interferon‐γ regulation of C4 gene expression in cultured human glomerular epithelial cells
Authors: Zhou Wuding , Duncan Campbell R. , Martin John , Sacks Steven H.
Published: March 2007

The fourth component of human complement is mainly produced in liver, but extrahepatic gene expression has been reported in human renal tissue and other tissues, and is thought to contribute to the inflammatory reaction in local tissue. To identify the cellular origin of C4 synthesis in human kidney, we studied C4 gene expression and regulation by interferon‐γ (IFN‐γ) and C4 protein biosynthesis in isolated glomerular epithelial cells. cDNA/polymerase chain reaction analysis showed that C4 transcripts are present in glomerular epithelial cells (GEC), and that gene expression is up‐regulated by IFN‐γ. Metabolic labeling studies showed that GEC synthesize and secrete C4 as three polypeptide chains of ∼ 90, 70, and 30 kDa, which correspond to the bands produced by hepatoma cells. These results suggest that the fourth component of complement is produced by GEC. The GEC has an important role in the maintenance of glomerular barrier function, which is lost in a number of complement‐dependent conditions; glomerular epithelial synthesis of C4 could have a bearing on the possible physiological or pathological roles of complement under different circumstances.
Association of the p56lck protein tyrosine kinase with the FCγRIIIA/CD16 complex in human natural killer cells
Authors: Catherine Cone J. , Lu Yiling , Trevillyan James M. , Bjorndahl Jay M. , Phillips Catherine A.
Published: March 2007

The multimeric FcγRIIIA (CD16) complex is expressed on the surface of natural killer (NK) cells and is composed of a 50–70‐kDa transmembrane glycoprotein Fcγ receptor (CD16), the T cell receptor (TCR)‐ζ chain, and the FcεRIγ chain. Cross‐linking FcγRIIIA initiates the rapid tyrosine phosphorylation of multiple substrates including the ζ, subunit and causes subsequent cell activation and antibody‐dependent cellular cytotoxicity (ADCC). The subunits of the FcγRIIIA complex lack intrinsic protein tyrosine kinase (PTK) activity, suggesting that receptor‐induced tyrosine phosphorylation events are mediated by a nonreceptor PTK. We report here that the human FcγRIIIA is complexed with p56lck, a src‐family PTK previously found associated with the CD4 and CD8 receptors on T cells. Upon engagement of the CD16 receptor, p56lck is rapidly (within 30 s) and transiently phosphorylated on tyrosine residues. Several FcγRIIIA‐associated proteins are identified in immune complex kinase assays including the TCR‐ζ, subunit, a p70–90 ζ‐associated protein (ZAP), p50a (acidic) and p50b (basic), and p56lck. We demonstrate that the src‐family protein tyrosine kinase inhibitor, herbimycin A, blocks increased intracellular calcium levels and ADCC caused by CD16 cross‐linking on NK3.3 cells. Likewise cross‐linking CD16 with the protein tyrosine phosphatase CD45, abrogates CD16‐induced calcium mobilization. These data suggest that p56lck is physically associated with FcγRIIIA(CD16) and functions to mediate signaling events related to the control of NK cellular cytotoxicity.
Variable region gene selection of immunoglobulin G‐expressing B cells with specificity for a defined epitope on type II collagen
Authors: Mo John A. , Bona Constantin A. , Holmdahl Rikard
Published: March 2007

Immunization with type II collagen (CII) induces collagen‐induced arthritis (CIA) in animals, and B cells reactive with CII are involved in the induction and manifestation of the disease. In this study, B cell hybridomas producing IgG antibodies specific for a major epitope on mouse CII (the “C1” epitope, amino acid 316–333), were isolated 11 days after immunization from draining lymph nodes in DBA/1 mice. Injection into neonatal mice of purified and biotinylated monoclonal antibodies binding the C1 epitope led to a specific binding to joint cartilage, demonstrating that the antibodies interact with native antigen in vivo. cDNA sequencing of the B cell clones revealed that they all expressed the same combination of a variable heavy chain (VH J558 family) and light chain (Vx21 family) germ‐line gene, apparently lacking somatic mutations. The presence of isotype‐switched B cells expressing a certain combination of V genes encoding antibodies that bind epitopes in vivo, indicates that this B cell population has been peripherally selected.
Migration patterns of thymus‐derived γδ T cells during chicken development
Authors: Dunon Dominique , Cooper Max D. , Imhof Beat A.
Published: March 2007

Cell transfer experiments in congenic chick strains, one of which expresses the ov antigen marker, indicate that intestinal γ δ T cells are derived from γ δ + thymocytes in embryos and newly hatched birds, and this early intestinal colonization occurs in two discrete waves. Here, we extend these studies to show that splenic colonization by γ δ T cells occurs in essentially the same way. Following the engraftment of ov+ thymic lobes in thymectomized ov− recipients, γ δ T cells migrate both to the spleen and intestine. By 1 week after hatching, a third generation of thymus‐derived γ δ T cells begins to migrate to both peripheral lymphoid organs, and this thymus‐dependent seeding process is sustained over the first weeks of life. The survival time for splenic γ δ migrants is significantly less than for the intestinal migrants. Tissue section analysis indicates that γ δ T cells enter the intestinal epithelium at all villus levels. A shift in the γ δ intraepithelial lymphocyte distribution toward the villus tip in thymectomized birds suggests the comigration of enterocytes and γ δ intraepithelial lymphocytes. However, survival kinetics of the donor γ δ population and a relatively high division rate of intestinal γ δ T cells indicate that founder thymic migrants produce relatively long‐lived clones of intestinal γ δ T cells.
Immunization against tumor and minor histocompatibility antigens by eluted cellular peptides loaded on antigen processing defective cells
Authors: Franksson Lars , Petersson Max , Kiessling Rolf , Kärre Klas
Published: March 2007

Material eluted from RMA lymphoma or B6 spleen cells under acid conditions was fractionated by reverse phase high‐performance liquid chromatography, and tested for ability to restore the sensitivity to cytotoxic T lymphocytes of the processing/presentation defective mutant line RMA‐S. This allowed identification of three fractions (termed M1, M2 and M3) carrying B6 antigens recognized by cytotoxic T lymphocytes (CTL) elicited across the minor histocompatibility barrier A. BYanti‐B6 (both H‐2b) and one fraction (termed TI) carrying a tumor antigen recognized by B6 anti‐RMA CTL. By parallel runs of material from cell lysates over major histocompatibility complex class I affinity columns, the M2 and M3 antigens were defined as Kb restricted, and M1 and T1 as Db restricted. Isolated fractions loaded onto RMA‐S cells could be used to prime anti‐minor histocompatibility antigen and tumor CTL in vivo. They could also be used for in vitro restimulation of spleen cells from mice that had been primed either by antigen‐loaded RMA‐S, or by wild‐type RMA tumor cells and B6 splenocytes. The CTL generated by these methods were specific for the loading antigen, and they also recognized the antigen on the “physiological” target, i.e. RMA or B6 lymphoblasts. This system based on RMA‐S as an immunization and target antigen reporter cell may be used for dissection of complex CTL responses, e.g. in studies of clonal composition and epitope dominance, or for studies of tumors that are poor stimulators of immunity.
Post‐receptor occupancy events in leukocytes during β1 integrin‐ligand interactions
Authors: Sánchez‐Mateos Paloma , Arroyo Alicia G. , Balboa María A. , Sánchez‐Madrid Francisco
Published: March 2007

Leukocyte adhesion to and subsequent spreading on the endothelium are the initial steps in the migration of these cells to the surrounding tissues. We have investigated the participation of different VLA heterodimers in cell spreading by using the anti‐β1 TS2/16 monoclonal antibody (mAb) which induces a conformational change of different VLA integrin receptors, enabling a high‐affinity interaction with their ligands. Both VLA‐4 and VLA‐5 fibronectin (FN), as well as VLA‐2 collagen (COL) receptors mediated cell spreading and morphological changes. The spreading of U‐937 and α4‐transfected K‐562 cells was induced in both FN and VCAM‐1, suggesting that the morphological changes may be induced by cell‐cell as well as cell‐extracellular matrix (ECM) interactions. Furthermore, the β‐regulated cell spreading on VCAM‐1 and COL took place independently of the VLA‐5 FN receptor function. The enhancing effect on cell attachment induced by anti‐β1 TS2/16 mAb was observed in the presence of different doses of cytochalasin D, whereas cell spreading was abolished. Signal transduction during β1‐stimulated integrin‐ligand interaction was also investigated. We have found the co‐localization of β1 integrins and tyrosine‐phosphorylated proteins during the spreading of U‐937 and α2‐ and α4‐transfected K‐562 cells on both ECM (FN and COL) and cellular (VCAM‐1) ligands. Kinetic studies showed that tyrosine phosphorylation was almost coincident with cellular spreading. The tyrosine phosphorylation of polypeptides of 130 kDa and 77 kDa was triggered in U‐937 cells by the interaction of FN with the VLA‐5 receptor in a high‐affinity conformation. However, no signaling was observed by inducing the high‐affinity state of receptor in the absence of appropriate ligand. These data suggest that tyrosine kinase activation is a post‐receptor occupancy event that might be critical in regulating the adhesive properties of integrins.
Divergent effects of interleukin‐10 on cytokine production by mononuclear phagocytes and endothelial cells
Authors: Sironi Marina , Muñoz Cecilia , Pollicino Teresa , Siboni Andrea , Sciacca Francesca Luisa , Bernasconi Sergio , Yecchi Annunciata , Colotta Francesco , Mantovani Alberto
Published: March 2007

Interleukin‐10 (IL‐10), a product of T helper type 2 (TH2) cells and monocytes, inhibits cytokine production in mononuclear phagocytes. Given the similarities and interrelationship between cells of the monocyte‐macrophage lineage and endothelial cells, we examined the effects of IL‐10 on vascular endothelium. Murine IL‐10 induced low levels of IL‐6 production and amplified induction of IL‐6 by lipopolysaccharide (LPS) or IL‐1 in the murine tEND.1 endothelioma line, used for these studies because it retains properties of normal endothelium. The effect was more evident after prolonged (48–72 h) exposure to IL‐10. IL‐10 had similar activity on other endothelioma lines, whereas it inhibited IL‐6 production by peritoneal macrophages. Induction and amplification of cytokine production by IL‐10 was associated with higher levels of mRNA, which were maintained longer (up to 48 h) than in controls. In addition to IL‐6, murine IL‐10 induced or amplified expression of the chemoattractant cytokines monocyte chemotactic protein‐1 (MCP‐1) and KG Human IL‐10 inhibited IL‐6 release by LPS‐stimulated human peripheral blood mononuclear cells, whereas it did not interfere with cytokine production by LPS‐ or IL‐1‐stimulated human umbilical vein endothelial cells. The selective inhibitory action of IL‐10 on mononuclear phagocytes versus endothelial cells may play a role in the pathophysiology of TH2‐directed responses.
Staphylococcal enterotoxin B up‐regulates interleukin‐2 receptor β chain expression on tonsillar B cells
Authors: Franz Annette , Bryant Annette , Farrant John
Published: March 2007

The superantigen staphylococcal enterotoxin B (SEB) selectively up‐regulates the interleukin‐2 receptor (IL‐2R) β chain (p70) without up‐regulating the IL‐2R a chain (CD25) on a human tonsillar B cell population depleted of T cells. This action of SEB, probably mediated by binding to major histocompatibility complex class II, renders B cells sensitive to T cell‐derived IL‐2 and is sufficient for induction of vigorous DNA synthesis with low concentrations of IL‐2. This explains one of the mechanisms by which bacterial superantigens activate large numbers of B cells and may reflect a similar mechanism operative in cognate helper T cell/B cell interactions.
Interleukin‐10 inhibits the induction of monocyte procoagulant activity by bacterial lipopolysaccharide
Authors: Pradier Oliver , Gérard Catherine , Delvaux Anne , Lybin Myriam , Abramowicz Daniel , Capel Paul , Velu Thierry , Goldman Michel
Published: March 2007

Monocytes stimulated with bacterial lipopolysaccharide (LPS) generate a procoagulant activity (PCA) related to the induction of tissue factor (TF) expression at their surface. Since interleukin‐10 (IL‐10) was recently shown to inhibit LPS‐induced cytokine production and is currently considered as a potential therapeutic agent in septic shock, we were interested to determine its effects on LPS‐induced monocyte PCA. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with 1 μg/ml LPS in the presence of serial dilutions of recombinant human IL‐10 and PCA was determined after 6 h in a one‐stage clotting assay. IL‐10 inhibited in a dose‐dependent manner LPS‐induced TF‐dependent PCA: a significant effect was already observed with 30 pg/ml IL‐10 while 64–97% inhibition was achieved with 120 pg/ml IL‐10. In parallel flow cytometry experiments, IL‐10 was shown to block LPS‐induced TF expression at the surface of monocytes. In order to inhibit LPS‐induced PCA, IL‐10 had to be added to PBMC at least 6 h before LPS challenge. This inhibitory effect of IL‐10 was already apparent at the TF mRNA level and was prevented by co‐incubation with cycloheximide (20 μg/ml). These data suggest that IL‐10 acts via the induction of protein(s) which might interfer with TF gene transcription or mRNA stability. We conclude that the protective effects of IL‐10 in endotoxinemia might be related not only to cytokine synthesis blockade but also to inhibition of LPS‐induced PCA.
Age‐related increase of CD45RO+ lymphocytes in physically active adults
Authors: Gabriel Holger , Schmitt Barbara , Kindermann Wilfried
Published: March 2007

The CD45RO phenotype of peripheral blood CD4+ and CD8+ lymphocytes were determined in physically active males (n = 171) and females (n = 70) ranging in age from 15–68 years. Direct immunofluorescence and dual color flow cytometry was performed for analysis. Absolute cell numbers (r = 0.473) and percentages of CD4+ CD45RO+ (r = 0.648) within the CD4+ population, percentage of CD8+ CD45RO+ cells within the CD8+ lymphocytes (r = 0.498) and total lymphocyte number (r = −0.242) correlated significantly (p < 0.001) with age. We conclude that the age‐related increase of CD45RO+ T cells indicates a gradual increase of activated T cells.
Triggering via the alternative CD2 pathway induces apoptosis in activated human T lymphocytes
Authors: Wesselborg Sebastian , Prüfer Ulrike , Wild Martin , Schraven Burkhard , Meuer Stefan C. , Kabelitz Dieter
Published: March 2007

Activation of immature thymocytes or transformed (i.e. leukemic) T lymphocytes via CD3/T cell receptor (TcR) signaling can induce programmed cell death (apoptosis). Recent data indicate that anti‐CD3/TcR monoclonal antibodies (mAb) also trigger apoptosis in activated (but not resting) mature peripheral LT cells. We now report that interleukin‐2 (IL‐2) dependent human polyclonal T cell lines as well as T cell clones undergo programmed cell death when triggered via the alternative CD2‐dependent activation pathway. In the presence of exogenous IL‐2, a pair of mitogenic anti‐CD2 mAb suppressed the IL‐2‐driven proliferative response. Growth inhibition was associated with cell death and DNA fragmentation as revealed by propidium iodide staining and gel electrophoresis, respectively. Induction of apoptosis by anti‐CD2 mAb was prevented by cyclosporine A and FK 506. We conclude that programmed cell death can be initiated in activated human T cells by signaling via the CD2 pathway.
Rapid transformant selection by human complement using HRF20 (CD59) cDNA as a selection marker
Authors: Takizawa Hisao , Takahashi Kazuhiro , Murakami Tomoko , Okada Noriko , Okada Hidechika
Published: March 2007

The 20 kDa homologous restriction factor (HRF20, CD59) prevents formation of membrane attack complexes of human complement. Transfection of the cDNA of HRF20 to heterologous cells, which are sensitive to human complement, thereby renders these cells resistant to human complement attack. This property can then be used as a selection marker of transfection. In addition, the procedure is short and can be performed within 8 h. Since only 1 h is required for incubation with complement, the damage to the transfected cells is limited compared with damage resulting from selection for drug resistance which requires 10 days or more of incubation in the presence of harmful agents.
Coordinate expression of β1 and β2 integrin “activation” epitopes during T cell responses in secondary lymphoid tissue
Authors: Picker Louis J. , Treer John R. , Nguyen Mai , Terstappen Leon W. M. M. , Hogg Nancy , Yednock Ted
Published: March 2007

The monoclonal antibodies (mAb) 15/7 and 24 recognize unique activation‐dependent, conformational epitopes on β1 and β 2‐integrins, respectively. The expression of both of these epitopes closely correlates with the ligand binding ability of their respective integrins, and thus serves as indicators of functional integrin “activation”. Here, we have used six‐parameter flow cytometry to examine the expression of these epitopes and conventional β1‐ and β2‐integrin epitopes during human T cell activation in secondary lymphoid tissues in vivo, focusing particularly on the virgin to memory/effector cell transition. Fresh tonsil lymphocytes were stained with mAb against conventional or activation‐dependent integrin epitopes, followed by staining with mAb against CD3, CD45RA, and CD45RO, thus allowing the determination of integrin epitope expression on virgin (CD3+) T cells (CD45RA+/RO−to±), memory/effector (CD45RA−/RO++) T cells, and T cells undergoing the virgin to memory/effector transition: transition region‐1 (T1; CD45RA+to++/RO+); ‐2 (T2; CD45RA++/RO++); and ‐3 (T3; CD45RA+/RO++). Conventional β1‐ and β2‐integrin epitopes progressively increase during the virgin to T3 stages of the transition in tonsil, in keeping with the generally higher levels of these adhesion molecules on memory/effector vs. virgin T cells. Expression of both the β1 (15/7)‐and β2 (24)‐integrin activation epitopes first appears on transitional T cells, and is maintained on a relatively constant number of cells (averaging 25‐30%) throughout the T1‐T3 stages. These epitopes are also noted on a subset of activated memory/effector T cells. Importantly, on both transitional and activated memory/effector T cell subsets, the expression patterns of the 15/7 and 24 epitopes vs. a variety of T cell activation antigens are identical, and the expression of these epitopes relative to each other is linearly correlated, findings strongly supporting the coordinate activation of β1 and β2 integrins duringT cell activation in vivo. These results provide the first evidence of integrin activation during an in vivo immunologic response, and demonstrate the usefulness of mAb recognizing conformational epitopes and multiparameter flow cytometry in delineating the dynamic interplay of adhesion molecules during complex physiologic processes.
Global analysis of antibody repertoires. II. Evidence for specificity, self‐selection and the immunological “homunculus” of antibodies in normal serum
Authors: Nobrega Alberto , Haury Matthias , Grandien Alf , Malanchère Evelyne , Sundblad Anne , Coutinho Antonio
Published: March 2007

The serum IgM repertoires of C57BL/6, DBA/2 and BALB/c mouse strains were analyzed using a recently developed global and quantitative assay that measures antibody reactivities to a very large number of antigens. A characteristic repertoire could be assigned to each strain. The different repertoires could be successfully classified with multivariate statistics. Many common reactivities were also observed among the different strains, which allows the definition of a mouse‐specific repertoire. Analysis of human sera support this notion. To investigate the impact of minor genetic differences on the serum IgM repertoire, the congenic strains B10.D2/oSn and B10.D2/nSn, which differ in the expression of the C5 component of complement, were analyzed. The two strains could be separated based on the reactivity profiles obtained. The analysis of the results reveals that many antigenic proteins are not recognized at all by natural antibodies, while others are disproportionately reactive, the resulting patterns giving rise to what could be the definition of an “immunological homunculus”. The relevance of this type of analysis for clinical applications is discussed.
The human immunoglobulin χ locus. Characterization of the partially duplicated L regions
Authors: Huber Christian , Huber Erwin , Lautner‐Rieske Andrea , Schäble Karlheinz F. , Zachau Hans G.
Published: March 2007

The L regions are parts of the Cχ proximal (p) and distal (d) copies of the human immunoglobulin x locus and are therefore called the Lp and Ld regions. The two regions with their 25 Vχ genes and pseudogenes have now been cloned, thus completing the cloning of the x locus. Lp has been linked to the neighboring Ap and B regions, while Ld was linked to Ad. There is good evidence that at the other side of Ld, i.e. towards the centromere, the end of the locus has been reached. Most of the cloning and linking was achieved by chromosomal walking, employing cosmid and phage λ clones. No such clones could be found for three small gaps. Two of them were closed by a polymerase chain reaction strategy; the third one was characterized by genomic blot hybridization experiments and eventually bridged by a yeast artificial chromosome clone.

Early in evolution, a stretch of about 25 kb which comprised three VK genes near the 5′ end of the L region precursor must have been duplicated, such that the later duplication of large parts of the x locus resulted in the appeareance of two very similar three‐gene regions in each, Lp and Ld. Two deletions in the central parts of the L regions, on the other hand, must have occurred after the duplication of the locus, since they are found in Lp and Ld in different positions.
Multiple signaling pathways mediate anti‐Ig and IL‐4‐induced early response gene expression in human tonsillar B cells
Authors: Murphy John J. , Norton John D.
Published: March 2007

We have analyzed the relationship between the signaling pathways coupled to surface immunoglobulin and interleukin (IL)‐4 receptors in human B cells from the patterns of expression of a panel of phorbol ester‐inducible early response genes (ERG) activated by anti‐IgM and IL‐4 stimulation in vitro. Anti‐IgM stimulation led to the induction of all eleven ERG tested. Two of these, the proto‐oncogene, c‐fos and an anonymous ERG 1R20 were insensitive to protein kinase C (PKC) inhibition with the drug, staurosporine and retained inducibility after down‐regulation of PKC activity by purging with phorbol ester. These observations are consistent with previous data showing anti‐IgM signaling through both PKC‐dependent and PKC‐independent pathways, c‐fos and 1R20 were also the only ERG inducible in response to IL‐4 stimulation and whilst ionomycin induced only c‐fos, dibutyryl cyclic adenosine monophosphate stimulation led to induction of both c‐fos and 1R20. These observations lend support to a role for the adenylate cyclase pathway being important for coupling of IL‐4‐generated signals to B cells responses. None of the anti‐IgM‐responsive ERG was further induced when B cells were co‐stimulated with a combination of anti‐IgM and IL‐4, suggesting that the signaling cascades from these two agents are integrated downstream of third messenger pathways to synergistically promote B cell proliferation.
Patterns of major histocompatibility complex class II expression by T cell subsets in different immunological compartments. 2. Altered expression and cell function following activation in vivo
Authors: Hopkins John , McConnell Ian , Dalziel Robert G. , Dutia Bernadette M.
Published: March 2007

This study characterizes antigen‐induced phenotypic and functional aspects of major histocompatibility complex (MHC) class II expression on recirculating T cells in efferent lymph. In vivo secondary, but not primary challenge is associated with both kinetic and phenotypic alterations in class II expression by T cells. All three major T cell subsets, CD4+, CD8+ and T19+ (γδ T cell receptor), show an approximate four fold increase in the level of MHC class II expression during secondary responses. No changes in B cell expression of class II were seen. Resting efferent lymph T cells are predominantly either class II− or DR+DQ− but this changes to DR+DQ+ after antigenic challenge.

The antigen‐presenting function of these class II+ T cells was investigated at daily intervals after in vivo antigenic challenge. T cells from non‐activated lymph nodes could not induce proliferation of antigen‐specific T cells with soluble antigen but were weakly stimulatory in allo‐mixed lymphocyte reaction (MLR) at high (> 2:1) stimulator cell ratios. Activated T cells isolated during secondary in vivo responses, and expressing increased quantities of MHC class II, were positive stimulator cells in the MLR. In contrast these cells could not present soluble antigen or trypsin‐digested antigen to the T cell lines. In the MLR assays, the relative stimulation by class II+ T cells correlates with the levels of class II expression. We conclude from these experiments that both quantitative and qualitative changes in MHC class II, induced on T cells under physiological conditions, play a role in the regulation of the immune response in vivo but that that role is not simply one of presentation of soluble antigen.
Cyclosporin A enhances T cell‐mediated induction of E‐selectin
Authors: Doǧan Ahmet , MacDonald Thomas T. , Spencer Jo
Published: March 2007

In a fetal intestinal organ culture model of intestinal inflammation, activation of T cells by a lectin, superantigen or anti‐CD3 antibodies induces E‐selectin expression which peaks 6‐10 h after activation and disappears by 24 h. We now show that addition of cyclosporin A (Cys A) increases the number of E‐selectin‐expressing endothelial cells and the intensity of expression, and it prolongs expression up to at least 60 h after stimulation. The dynamics of E‐selectin expression on tumor necrosis factor activated human umbilical‐vein endothelial cell cultures is not affected by Cys A. Furthermore, other immunosuppressive agents FK 506 and dexamethasone inhibit E‐selectin expression, indicating that the enhancement observed in the presence of Cys A is not a consequence of general immunosuppression.