bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross‐linking CD28 leads to activation of 70‐kDa S6 kinase
Authors: Pai Sung‐Yun , Calvo Victor , Wood Michael , Bierer Barbara E.
Published: March 2007

Proliferation of T lymphocytes in response to antigen/MHC complexes is dependent upon the presence of a co‐stimulatory signal; in its absence, T cells are rendered unresponsive to specific antigen. CD28 is a T cell surface glycoprotein that acts as a co‐stimulatory molecule when combined with signals initiated by the T cell receptor‐CD3 complex. While the biochemical signaling events following CD28 stimulation are still poorly defined, monoclonal antibodies (mAb) directed against CD28 have been shown to transduce a variety of early signals that are different in the presence of cross‐linking antibody or the presence of phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C (PKC). Stimulation of human T cells with cross‐linked anti‐CD28 mAb alone resulted in the activation of 70‐kDa (p70) S6 kinase, a rapamycin‐sensitive serine/threonine kinase that is believed to be important for cell cycle progression. Activation of p70 S6 kinase through CD28 was inhibited by rapamycin. Activation of p70 S6 kinase also increased in response to cross‐linked CD3, but followed a more rapid time course than activation via CD28. Cyclosporin A and FK506 had no effect on p70 S6 kinase activity initiated via either pathway. The combination of cross‐linked CD28 and cross‐linked CD3 had no more than an additive effect on the induction of p70 S6 kinase activity. Thus, recruitment of p70 S6 kinase activity appears to represent a common signal transduction event shared by both the CD28 and CD3 pathways of T cell activation.
Induction of HIV‐1 envelope (gp120)‐specific cytotoxic T lymphocyte responses in mice by recombinant CHO cell‐derived gp120 is enhanced by enzymatic removal of N‐linked glycans
Authors: Doe Barbara , Steimer Kathelyn S. , Walker Christopher M.
Published: March 2007

Priming of CD8+ cytotoxic T lymphocyte (CTL) responses with recombinant proteins has been facilitated by the development of novel adjuvants that deliver antigens into the class I major histocompatibility complex (MHC) pathway. However, the extent to which secondary structure or glycosylation of these proteins prevents priming of class I MHC‐restricted CTL responses is not clear. To address this issue, recombinant HIV‐1 gp120 envelope proteins produced in yeast, insect, or mammalian cells were compared for the ability to elicit CD8+ CTL activity in mice. Envelope‐specific CD8+ T lymphocytes were detected in BALB/c mice immunized with env 2‐3, a 55‐kDa yeast‐derived envelope protein that is not glycosylated and lacks a native conformation. This response was directed against a previously described epitope in the V3 region of gp120, as well as a newly identified epitope located near the carboxy‐terminus of the molecule. Similar levels of V3‐directed CTL activity were observed in mice immunized with recombinant gp120 produced in insect (Spodoptera fugiperda) cells using a baculovirus expression system (gp120BAC). In contrast, induction of CTL responses was considerably less efficient when mice were immunized with gp120CHO, a native, fully glycosylated envelope protein produced in mammalian CHO cells. Denaturation of gp120CHO prior to immunization was not sufficient to prime CTL responses. However, envelope‐specific CD8+ CTL activity was elicited when N‐linked glycans were removed by treatment with an endoglycosidase. Possible mechanisms by which N‐linked glycans influence delivery or processing of recombinant proteins for class I MHC presentation, and the implications of these findings for the design of subunit vaccines, are discussed.
Mechanisms in CD4 antibody‐mediated transplantation tolerance: kinetics of induction, antigen dependency and role of regulatory T cells
Authors: Scully Ralph , Qin Shixin , Cobbold Stephen , Waldmann Herman
Published: March 2007

CBA/Ca mice may be made tolerant to minor histoincompatible B10.BR skin grafts by treatment with a short course of non‐depleting anti‐mouse CD4 and CD8 monoclonal antibodies (mAb), during the transplantation period. We wished to determine when, in relation to antibody therapy, the T cells became tolerant. This was investigated by a series of adoptive transfer experiments in which mAb‐treated cells were removed from therapeutic antibody at defined times after skin grafting, and exposed to fresh antigen in the absence of further mAb treatment. We show here that T cells do not become fully tolerant until 5 weeks after skin grafting. If antibody therapy is continued for the full 5 weeks, T cell tolerance can still be established, suggesting that antibody therapy does not prevent lymphocytes from registering the presence of antigen. Once the tolerant state is established, it is difficult to break that tolerance by lymphocyte infusions from normal donors. This “resistance” is mediated by T cells of the tolerant host. We show that the maintenance of both tolerance and “resistance” requires a continuous supply of antigen. When tolerant cells were “parked” in T celldepleted mice, tolerance and “resistance” were eventually lost by 6 months. In contrast, “parked” cells exposed to fresh antigen at any time up to 4 months remained tolerant and “resistant” indefinitely. Finally, we wished to establish whether “resistance” was peculiar to this form of peripheral tolerance, or whether it might also be present in tolerance considered to be classically central. We observed resistance to be greater in the mAb‐treated peripherally tolerant group, but noted that some of the centrally tolerant animals also exhibited a level of resistance above that of T cell‐ablated controls. This suggests that a tolerance mechanism whose role is only minor in central tolerance may have a major role in antibody‐mediated peripheral tolerance.
The balance between deletion and activation of CD4+8+ thymocytes is controlled by T cell receptor‐antigen interactions and is affected by cyclosporin A
Authors: Curnow S. John , Schmitt‐Verhulst Anne‐Marie
Published: March 2007

The sensitivity of immature thymocytes to antigen‐induced deletion has been shown to correlate with their differentiation status. By using an in vitro approach we have investigated whether parameters of antigenic stimulation may also affect the response of thymocytes. Two T cell receptor (TcR)‐transgenic (Tg) mouse models have been compared, both of which recognize the allo‐antigen H‐2Kb but are functionally CD8“‐dependent” (KB5.C20‐Tg) and “‐independent” (BM3.3‐Tg). Presentation of the antigen H‐2Kb on the surface of fibroblasts, to thymocytes in vitro, resulted in the apoptosis of CD4+8+ thymocytes. In contrast to in vivo deletion, in vitro deletion was much greater for KB5.C20‐Tg than for BM3.3‐Tg. In the absence of engagement of CD8 (using an altered H‐2Kb‐α3 domain or CD8‐specific antibodies), the H‐2Kb‐induced deletion of CD4+8+ thymocytes was decreased for KB5.C20‐Tg, but no change in the pattern of deletion for BM3.3‐Tg occurred. CD4+8+ thymocytes which remained viable after in vitro exposure to antigen, were shown to have been activated. Cyclosporin A (CsA), which has been reported to inhibit activation‐induced cell death, did not affect antigen‐induced deletion of CD4+8+ thymocytes from KB5.C20‐Tg. More strikingly, deletion of CD4+8+ thymocytes from BM3.3‐Tg increased, whilst activation was partially inhibited by CsA. These results provide direct evidence that presentation of antigen to thymocytes can result in deletion or activation, depending on not only the differentiation status of the cell, but also parameters of TcR‐antigen interaction. Additionally, the effects of CsA suggest that activation can prevent the induction of deletion.
Comparison of the Jβ gene usage among different T cell receptor Vβ families in spleens of C57BL/6 mice
Authors: Kato Tomohiro , Suzuki Satoshi , Sasakawa Hiroko , Masuko Kayo , Ikeda Yoko , Nishioka Kusuki , Yamamoto Kazuhiko
Published: March 2007

To clarify whether there is a bias in the V‐D‐J combination of T cell receptor (TcR) genes, Jβ gene usage has been investigated in a total of 743 TcR β genes of Vβ2, Vβ8.2, and Vβ14 families expressed in C57BL/6 mouse spleens. Genes of TcR β chains, amplified by a reverse transcriptase‐polymerase chain reaction, were individually cloned into plasmids. Cloned genes (61 to 106), randomly selected in each respective Vβ family from three different mice, were tested by means of hybridization with 12 oligo DNA probes which were designed to differentiate 12 murine functional Jβ gene segments. The results are enumerated below. (1) The Jβ2.6 gene segment was found to be most frequently used (Vβ2, 19.8%; Vβ8.2, 21.2%; and Vβ14, 19.2%). In contrast, usage of the Jβ1.6 gene segment was most infrequent (Vβ2.1.9%; Vβ8.2, 2.9%; and Vβ14, 0.5%); (2) High frequency of the Jβ2.1 gene segment and low frequency of the Jβ1.3 and Jβ1.5 gene segments were also observed; (3) The Jβ2 cluster was used in preference to the Jβ1 cluster (usages of the Jβ2 cluster: Vβ2, 67.8%; Vβ8.2, 65.9%; and Vβ14, 70.4%); and (4) These biases were generally common to all three Vβ families examined and differences among individual mice were mostly small. Considering these findings, we conclude that the TcR Jβ gene segments in C57BL/6 mice splenocytes are selected with a bias, but are selected independently of the Vβ families.
Differential expression of mRNA encoding interleukin‐12 p35 and p40 subunits in situ
Authors: Bette Michael , Jin Shen‐Chu , Germann Tieno , Schäfer Martin K.‐H. , Weihe Eberhard , Rüde Erwin , Fleischer Bernhard
Published: March 2007

Interleukin‐12 (IL‐12) is a heterodimeric cytokine that plays an important role in the regulation of the immune response. For biological activity the expression of both subunits of IL‐12, p35 and p40, is required. Moreover, in the mouse the p40 chain of IL‐12 specifically inhibits the effects of the IL‐12 heterodimer. In the present study we have analyzed by in situ hybridization the expression of the p35 and p40 mRNA in the spleens of BALB/c and mutant (SCID, nude, beige) mice, unstimulated and after in vivo stimulation with lipopolysaccharide (LPS) and with staphylococcal enterotoxin B (SEB). In unstimulated spleens of BALB/c mice, p35 and p40 mRNA were only detectable in a few strongly stained single cells, p35 mRNA was expressed in addition weakly in the B cell areas. After injection of LPS or SEB, p40 mRNA was strongly induced in the T cell areas all over the spleen, whereas expression of p35 mRNA and its distribution pattern did not change. Surprisingly, most of the mRNA for p35 and p40 was localized in different areas of the spleen and was apparently produced by different cells. In macrophage‐depleted spleens the increased expression of p40 mRNA in response to LPS was reduced but still detectable, demonstrating that other cells besides macrophages can up‐regulate IL‐12 p40 mRNA. Nude mice showed a stronger expression of p35 mRNA, SCID mice lacked the weak p35 staining of the B cell areas but showed a strong basal expression of both p35 and p40 mRNA and a focal response to LPS. The pattern of IL‐12 mRNA expression in beige mice was the same as in normal mice. These data demonstrate a spatial dissociation of expression of the two chains of IL‐12 and are compatible with a regulatory role of the isolated IL‐12 p40 chain in vivo. In addition, they indicate that the demonstration of mRNA for both chains of IL‐12 in whole tissues or cell mixtures is not necessarily indicative of functional IL‐12.
Co‐stimulation with LFA‐1 triggers apoptosis in γδ T cells on T cell receptor engagement
Authors: Matsumoto Yoshihiro , Hiromatsu Kenji , Sakai Tetsu , Kobayashi Yasushi , Kimura Yuki , Usami Jun , Shinzato Toru , Maeda Kenji , Yoshikai Yasunobu
Published: March 2007

Stimulation of T cells through the T cell receptor (TcR) initiate activation pathways, and paradoxically can also result in activation‐induced cell death. Many factors influence a stimulated cell's decision to manifest one or the other. Here we show that co‐stimulation with LFA‐1 plays a key role in the choice between the two fates, differentiating between αβ and γδ T cells. Peripheral γδ T cells but not αβ T cells undergo apoptosis upon co‐cross‐linking of TcR and LFA‐1 in MRL lpr/lpr mice as well as +/+ mice. Our results suggest that apoptosis of γδ T cells is inducible by combined stimuli independent of the Fas‐mediated pathway.
Peptide antagonists that promote positive selection are inefficient at T cell activation and thymocyte deletion
Authors: Barnden Megan J. , Heath William R. , Rodda Stuart , Carbone Francis R.
Published: March 2007

We set out to determine whether thymocytes from T cell receptor (TCR) transgenic animals specific for a class I‐restricted determinant from ovalbumin (OVA) showed the same fine specificity for antigen‐driven deletion in single‐cell suspension culture as required for mature T cell activation. The transgenic TCR is specific for the Kb‐restricted peptide OVA257‐264 (SIINFEKL) which is known to have four TCR contact residues at position 1, 4, 6, and 7 from the crystal structure of this fragment in complex with Kb. OVA257‐264 analogs systematically substituted at each of these positions were assayed for their ability to promote immature double‐positive thymocyte deletion or mature T cell activation of a cytotoxic T lymphocyte line derived from this transgenic mouse. In the absence of additional antigen‐presenting cells, single‐cell thymocyte suspensions showed that the specificity for double‐positive thymocyte deletion and mature T cell activation was virtually identical, demonstrating a limited cross‐reactivity with a number of variants having conservative substitutions at these exposed residues. These peptides were considerably more efficient at both thymic deletion and mature T cell activation than a number of non‐conservative substitution analogs known to act as antagonists of OVA257‐264 and capable of selecting transgenic T cells in thymic organ culture. Therefore, both peripheral T cell activation and thymic deletion have an overall similar pattern of peptide specificity which differs from that required for positive selection. This suggests that a subset of major histocompatibility complex‐presented peptides could promote positive selection without causing either thymic deletion or peripheral activation of those selected T cells.
Induction of tolerance in freshly isolated alloreactive CD4+ T cells by activated T cell stimulators
Authors: Satyaraj Ebenezer , Rath Satyajit , Bal Vineeta
Published: March 2007

Activated human T cells express major histocompatibility complex class II proteins, and their potential to present antigens to T cell clones has been documented extensively. The effect of such T‐T presentation on responder T cell clones has been shown to be the induction of tolerance, sometimes accompanied by activation. To investigate whether freshly isolated responder T cells are also susceptible to such induction of tolerance by activated T cells functioning as antigen‐presenting cells (APC), we have used the capability of unprimed ex vivo T cells to respond in a proliferation assay in vitro to alloligands on professional APC. We show that purified human T cells ex vivo, when exposed to alloligand on activated T cells for primary allorecognition In vitro, fail to mount a proliferative response. Priming of responder CD4+ T cells with alloligand expressed on activated T cells results in the induction of nonresponsiveness to a subsequent challenge by competent allo‐APC. This ability of activated, HLA‐DR+ T cells to induce nonresponsiveness to subsequent challenge in bulk CD4+ T cell populations ex vivo has interesting implications for infections involving T cells such as human immunodeficiency virus.
The role of protein kinase C in the regulation of extracellular signal‐regulated kinase by the T cell antigen receptor
Authors: Izquierdo Manolo , Leevers Sally J. , Williams David H. , Marshall Chris J. , Weiss Arthur , Cantrell Doreen
Published: March 2007

The aim of this study was to explore the role of protein kinase C (PKC) in the activation of mitogen‐activated protein kinases (MAPK) in T lymphocytes. The MAPK extracellular signal‐regulated kinase‐2 (ERK2) is activated in response to phorbol esters which stimulate PKC, by transient expression of a constitutively active ras mutant, by cell activation via the G protein‐coupled type 1 muscarinic acetylcholine receptor (HM1R) or in response to triggering of the T cell antigen receptor (TCR). The relative contribution of PKC to TCR and HM1R regulation of ERK2 was explored by examining the effects of a PKC inhibitor (Ro 31‐8425) on ERK2 activation. The data demonstrate that phorbol ester and HM1R regulation of ERK2 was prevented by the PKC inhibitor, but that the inhibitor had no effect on ERK2 activation induced by expression of a constitutively active ras mutant p21v‐Ha‐ras. Furthermore, the TCR stimulates both PKC and p21ras but TCR regulation of ERK2 was only weakly suppressed by the PKC inhibitor. These data indicate that PKC has a potential but not a predominant role in TCR regulation of ERK2.
T cell receptor‐induced Fas ligand expression in cytotoxic T lymphocyte clones is blocked by protein tyrosine kinase inhibitors and cyclosporin A
Authors: Anel Alberto , Buferne Michel , Boyer Claude , Schmitt‐Verhulst Anne‐Marie , Golstein Pierre
Published: March 2007

Fas/APO‐1 is a member of the tumor necrosis factor receptor family of proteins, that induces apoptosis when cross‐linked with monoclonal antibody (mAb) or with its physiological ligand. Recently, both a perforin‐based and a Fas‐based mechanism have been proposed to account for T cell‐mediated cytotoxicity. In the present study we used a murine CD8+ cytotoxic T lymphocyte (CTL) clone (KB5.C20) specific for H‐2Kb and a T cell receptor (TcR)‐negative variant of the same clone (2005−D4) to test (i) whether the same cell can exert both cytotoxic effector mechanisms and (ii) the role of TcR engagement in the induction of Fas‐based cytotoxicity. We demonstrate that both the TcR+ and TcR− clones were able to express the Fas ligand after stimulation with phorbol 12‐myristate 13‐acetate (PMA)/ionomycin, and that TcR engagement of the KB5.C20 clone by means of antigen‐bearing cells or of its anticlonotypic mAb (Désiré‐1), which leads to Ca2+‐dependent, presumably perforin‐based, cytotoxicity, was also able to induce Fas‐based cytotoxicity. In addition, using inhibitors we investigated the signal transduction pathway(s) involved in the induction of Fas‐based cytotoxicity and expression of the Fas ligand mRNA in the CTL clones. The involvement of src‐like protein tyrosine kinases (PTK) in Fas ligand induction through TcR engagement, was strongly suggested by inhibition with the src‐like PTK inhibitor herbimycin A. Inhibition of Fas ligand induction by genistein, a more general TPK inhibitor, even upon stimulation by PMA plus ionomycin, suggested the possible involvement of PTK activities downstream of protein kinase C (PKC) in Fas ligand induction in CTL. Finally, the implication of the Ca2+/calmodulin‐dependent protein phosphatase calcineurin in Fas ligand induction was demonstrated by the partial inhibition of Fas ligand induction with cyclosporin A. Thus, in CTL clones, Fas ligand expression is inducible by TcR engagement through a pathway similar to that involved in expression of some lymphokine genes.
Triggering of human natural killer cells through CD16 induces tyrosine phosphorylation of the p72syk kinase
Authors: Ståhls Anders , Liwszyc Guillermo Eli , Couture Clément , Mustelin Tomas , Andersson Leif C.
Published: March 2007

Activation of protein tyrosine kinases (PTK) is an initial and obligatory event for the triggering of human natural killer (NK) cells to cytotoxicity. Of the different PTK detected in NK cells, only p56lck has previously been shown to participate in NK cell activation. Here we present evidence that another PTK, p72syk, is involved in activation of NK cells. Stimulation with a monoclonal antibody to the FcγRIII receptor (CD16) induced an increased tyrosine phosphorylation of p72syk. This phosphorylation correlated with an increased tyrosine kinase activity of p72syk towards a synthetic peptide substrate. A severalfold increase in the catalytic activity of p72syk was also seen after treatment of NK cells with an inhibitor of phosphotyrosine phosphatases, pervanadate. We conclude that triggering of the cytotoxic response in NK cells is associated with activation of p72syk.
Dichotomy between the T and the B cell epitopes of the synthetic polypeptide (T,G)‐A–L
Authors: Zisman Einat , Sela Michael , Ben‐Nun Avraham , Mozes Edna
Published: March 2007

Studies with the well‐characterized, synthetic, random‐multichain polypeptide poly(LTyr, LGlu)‐poly(DLAla)–poly(LLys) (T,G)‐A–L), led to the discovery of determinant‐specific genetic control of the immune response, as well as to other immunological phenomena. Moreover, the tetrapeptide TyrTyrGluGlu built on the same backbone (“T‐T‐G‐G)‐A–L”) was found to represent its major B cell epitope. We have recently shown that for interaction with major histocompatibility complex class II molecules and stimulation of T cells, (T, G)‐A–L requires proteolytic processing and the resulting T cell epitopes are close to the N termini of the branched polymer's side chains. Thus, we were interested to elucidate the major T cell epitope of (T, G)‐A–L, by using the ordered polypeptides (T‐T‐G‐G)‐A–L and (T‐G‐T‐G)‐A–L, in which only the two internal amino acids of the tetrapeptide attached to the side chains are switched. We established T cell lines to these antigens, and found that the ordered analog (T‐T‐G‐G)‐A–L, which was defined as the B cell epitope of (T,G)‐A–L, did not represent its T cell epitope, whereas (T‐G‐T‐G)‐A–L, to which only a minor anti‐(T,G)‐A–L Ab response was directed, was found to be its major T cell epitope. In addition, there was no cross‐reaction between (T‐G‐T‐G)‐A–L and (T‐T‐G‐G)‐A–L at the T cell level, similar to the lack of cross‐reaction of their antibodies. Analysis of the repertoire of the T cell receptors used by these lines revealed that the (T,G)‐A–L and the (T‐T‐G‐G)‐A–I specific T cell lines were not restricted in their Vα and Vβ TCR usage, whereas the (T‐G‐T‐G)‐A–L‐specific line was restricted by both Vα and Vβ T cell receptor gene products. This difference might be due to the thymus‐independent characteristics previously described for the latter antigen.
CD2‐CD48 interaction prevents apoptosis in murine B lymphocytes by up‐regulating bcl‐2 expression
Authors: Genaro Ana Maria , Gonzalo José Angel , Bosca Lisardo , Martinez‐A. Carlos
Published: March 2007

Antigen receptor engagement initiates clonal expansion and antibody secretion in B lymphocytes in response to foreign antigens. However, binding of self antigen to antigen receptors targets self‐reactive B cell clones for elimination or inactivation. The antigen‐triggered biochemical events and the eventual response of the cells are dependent on the simultaneous occupancy of co‐stimulatory receptors. CD2 is an intercellular adhesion molecule implicated in cell activation and expressed in human T and natural killer cells as well as in mouse B lymphocytes. Mouse B cells specific for allogeneic major histocompatibility complex (MHC) class I initiate a suicide program that leads to DNA fragmentation and cell death when confronted with soluble MHC class I while undergoing clonal expansion when the antigen is present on mitomycin C‐treated cells. Here we show that occupancy of CD2 in mouse B cells by the presence of either monoclonal antibody (mAb) specific for CD2, or soluble recombinant mouse CD48, its natural ligand in mouse, prevents the induction of apoptosis. Furthermore, the in vitro activation by mitomycin C‐treated allogeneic cells, is abrogated in the presence of anti‐CD48 mAb (OX78). These results indicate that a CD2‐CD48 interaction is involved in the control of B cell activation.
Modulation of cytokine expression in mouse dendritic cell clones
Authors: Granucci Francesca , Girolomoni Giampiero , Lutz Manfred B. , Foti Maria , Marconi Giulia , Gnocchi Paola , Nolli Luisa , Ricciardi‐Castagnoli Paola
Published: March 2007

Dendritic cells (DC) play an essential role in the induction of primary immune responses; however, very little information is available on cytokine production by DC. Here we determined the cytokine gene expression profile of two immortalized DC clones, CB1 and D2SC/1, both generated from mouse spleen but differing in their activation requirements. Among the cytokines tested, only transforming growth factor‐β1 was transcribed constitutively, but its production was detected only in D2SC/1 cells after treatment with granulocyte/macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF also promoted transcription and synthesis of interleukin (IL)‐1β in CB1 cells that need pretreatment with GM‐CSF to present major histocompatibility complex class II‐restricted antigens efficiently in vitro. Lipopolysaccharide (LPS) up‐regulated gene expression and induced release of tumor necrosis factor‐α in both DC clones. In addition, LPS induced transcription of IL‐1α and both gene expression and synthesis of IL‐1β in D2SC/1 cells. Interferon‐γ was ineffective in inducing cytokine gene expression, although it augmented the antigen‐presentation capacity of DC. IL‐4, IL‐10 and IL‐12 mRNA were not induced by any of the tested stimuli. The results suggest that DC have a limited cytokine gene expression pattern compared to macrophages and are heterogenous in some functional properties.
The human natural killer cell receptor for major histocompatibility complex class I molecules. Surface modulation of p58 molecules and their linkage to CD3 ζ chain, FcϵRI γ chain and the p56lck kinase
Authors: Bottino Cristina , Vitale Massimo , Olcese Lucia , Sivori Simona , Morelli Luigia , Augugliaro Raffaella , Ciccone Ermanno , Moretta Lorenzo , Moretta Alessandro
Published: March 2007

The natural killer cell (NK)‐specific p58 surface molecules, recognized by the GL183 and EB6 monoclonal antibodies (mAb), have been shown to represent the putative NK receptor for HLA‐C molecules. The interaction between p58 receptors and HLA‐C results in inhibition of the NK‐mediated target cell lysis. In this study, GL183−EB6+ clones (Cw4‐specific), after mAb‐induced surface modulation of EB6 molecules, acquired the ability to lyse the Cw4− C1R cells. In NK clones co‐expressing both GL183 and EB6 molecules and unable to kill Cw3‐protected target cells, the mAb‐induced modulation of EB6 molecules resulted both in selective co‐modulation of GL183 molecules and in the lysis of Cw3‐transfected P815 murine cells. In line with the co‐modulation experiments we also show that the GL183 and EB6 molecules can be co‐immunoprecipitated from GL183+/EB6+ clones after cell lysis in the presence of digitonin. The p58 receptor also revealed an association with molecules belonging to the ζ family (i.e. CD3 ζ and FcϵRI γ chains). Two‐dimensional diagonal gel analysis of the p58 complex immunoprecipitated from polyclonally activated p58+ NK cells indicated a preferential association with CD3 ζ chains either in the form of covalently linked ζ‐γ homodimers or in the form of ζ‐γ heterodimers, while γ‐γ homodimers were detectable in low amounts. However, p58+ clones displaying a unique association with γ‐γ homodimers could also be isolated. Probing the immunoprecipitated p58 complex with anti‐p56lck antibody also revealed an association with this member of the src family. In addition, mAb‐mediated signaling of NK clones via p58 molecules induced increments of p58/p56lck association. However, under the same experimental conditions that induced optimal in vivo tyrosine phosphorylation of the CD16‐associated CD3 ζ chains, no tyrosine phosphorylation was detected in the p58‐associated CD3 ζ, chains. In these in vivo experiments neither anti‐CD16 nor anti‐p58 mAb could induce tyrosine phosphorylation of the γ chains. Finally, the anti‐p58‐mediated inhibition of the NK cell triggering via CD16 molecules was not accompained by a down‐regulation of the tyrosine phosphorylation of the CD16‐associated CD3 ζ chains.
Structure of HLA‐B27‐specific T cell epitopes. Antigen presentation in B2703 is limited mostly to a subset of the antigenic determinants on B2705
Authors: Villadangos José A. , Galocha Begoña , García‐Hoyo Rosa , López Daniel , García Fernando , de Castro José A. Łópez
Published: March 2007

The structure of HLA‐B27‐specific epitopes recognized by anti‐B*2705 and anti‐B*2703 cytotoxic T lymphocytes (CTL) from three unrelated donors was examined with site‐specific mutants at various side‐chain pockets in the antigen‐binding site. The effect of any given mutation on allorecognition correlated strongly with its predictable effect on peptide binding. Acidic charges in the C/F pocket of HLA‐B27, which binds C‐terminal peptide residues, strongly modulated allorecognition. Anti‐B*2705 CTL from different donors were differently affected by some mutations, indicating individual differences in the structure of epitopes recognized by alloreactive CTL from each donor. Most anti‐B*2703 CTL recognized a subset of epitopes that were also present on B*2705, but differed from the bulk of allospecific epitopes on this subtype in their smaller dependence on pocket A structure, where the difference between these subtypes is located, and in their greater dependence on Glu45, in the B pocket. The structure of the very few epitopes on B*2703 not shared by B*2705 was quite different from that of the much more predominant cross‐reactive epitopes. The results strongly suggest that B*2703 is antigenically defective as compared with B*2705 and that this is due to the fact that the repertoire of peptides presented by B*2703 consists mainly of a subset of the B*2705‐bound peptides which do not critically require the canonic binding of the peptidic N‐terminus to a B*2705‐like A pocket, because they are sufficiently stabilized by other contacts through the peptide binding site.
Segregation of APO‐1/Fas antigen‐ and tumor necrosis factor receptor‐mediated apoptosis
Authors: Grell Matthias , Krammer Peter H. , Scheurich Peter
Published: March 2007

The cytokine tumor necrosis factor (TNF) and the APO‐1/Fas ligand represent typical inducers of apoptosis, i.e. programmed cell death. A limited sequence homology between the TNF receptor TR60 (p55) and the APO‐1/Fas (CD95) antigen in their intracellular domains suggests overlapping signaling pathways. The TNF‐sensitive cell line KYM‐1, which expresses high numbers of both TNF receptors and the APO‐1/Fas antigen, is highly sensitive towards triggering of apoptosis by each of the TNF receptors, but resistant to APO‐1/Fas stimulation. The opposite response pattern was obtained using the B lymphoid line SKW 6.4, which also co‐expresses all three cell surface molecules. Furthermore, no co‐modulation of APO‐1/Fas by down‐regulation of cell surface expressed TR60 and/or TR80 receptors, and vice versa, was found. These data argue against a physical interaction of TNF receptors with APO‐1/Fas and, in addition, demonstrate cell specific control of induction of apoptosis and usage of distinct signaling pathways by these receptor molecules.
Apoptotic elimination of Vβ8.2+ cells from the central nervous system during recovery from experimental autoimmune encephalomyelitis induced by the passive transfer of Vβ8.2+ encephalitogenic T cells
Authors: Tabi Zsuzsanna , McCombe Pamela A. , Pender Michael P.
Published: March 2007

A CD4+Vβ8.2+ T cell clone specific for the peptide 72–89 of guinea pig myelin basic protein (GMBP) was used to induce acute experimental autoimmune encephalomyelitis (EAE) in Lewis rats. To assess apoptosis in inflammatory cells infiltrating the central nervous system (CNS), we extracted cells from the spinal cord, enriched them for T cells and performed flow‐cytometric analysis of their DNA stained with propidium iodide. The presence of apoptosis was confirmed by the demonstration of DNA fragmentation on gel electrophoresis. A gradual increase in the proportion of apoptotic cells was observed between 4 and 7 days after the transfer of the encephalitogenic T cells. The highest frequency of apoptotic cells (9.2 ± 1.2%) was observed 7 days after cell transfer, when clinical recovery commenced. Passive transfer of ovalbumin‐specific cells resulted in only a background level (0.8%) of apoptosis in the CNS. We conclude that the apoptotic process selectively eliminates autoreactive T cells from the CNS as: (a) there was a selective disappearance of disease‐relevant CD5+Vβ8.2+ cells from the CNS during the course of EAE; (b) there was a decrease in the frequency of CNS‐infiltrating T cells reactive to the GMBP 72–89 peptide during the course of EAE, and in a standard proliferation assay there was a loss of in vitro reactivity of CNS‐infiltrating cells to this peptide, but not to a non‐CNS antigen (ovalbumin); (c) simultaneous surface labeling and DNA analysis of CNS‐infiltrating cells revealed that the frequency of Vβ8.2+ cells was about sevenfold higher in the apoptotic T cell population than in the normal (non‐apoptotic) T cell population; and (d) we were unable to detect recirculation of the Vβ8.2+ cells to lymphoid organs after their frequency decreased in the CNS. The selective apoptotic elimination of autoreactive T cells from the target organ of this spontaneously resolving autoimmune disease may have implications for the understanding of the mechanism by which an autoimmune attack is terminated and for the design of therapeutic strategies to facilitate this process.
Recognition of apoptotic cells by human macrophages: inhibition by a monocyte/macrophage‐specific monoclonal antibody
Authors: Flora Pauline K. , Gregory Christopher D.
Published: March 2007

Cells undergoing death by apoptosis are rapidly engulfed by phagocytes in vivo, a highly efficient process which prevents leakage of potentially dangerous intracellular contents from dying cells to neighboring tissue. We have tested a panel of monoclonal antibodies (mAb) specifying a range of human monocyte/macrophage surface antigens for their capacity to inhibit the in vitro recognition of apoptotic cells by human peripheral blood monocyte‐derived macrophages. The results identify the antigen defined by the 61D3 mAb, a widely‐used marker of monocyte/macrophage lineage cells, as an important mediator of apoptotic cell recognition. In our system, apoptotic, but not viable, cells were recognized by the cultured macrophages and 61D3 was found to inhibit the recognition of all apoptotic cell types tested, including Ca2+ ionophore‐treated or growth factor‐depleted B and T lymphocyte lines, tonsillar germinal center B cells, irradiated peripheral blood lymphocytes and senescing neutrophils. Furthermore, the apoptotic cell recognition pathway specified by 61D3 could be distinguished from that involving the macrophage αvβ3 vitronectin receptor which has been shown previously to play an important role in the recognition of apoptotic cells. These results provide further evidence that the mechanisms underlying rapid clearance of apoptotic cells involve multiple phagocyte receptors.
Fas ligation triggers apoptosis in macrophages but not endothelial cells
Authors: Richardson Bruce C. , Lalwani Narendra D. , Johnson Kent J. , Marks Rory M.
Published: March 2007

The reticuloendothelial system includes macrophages and endothelial cells. These cells are produced and destroyed in vivo with a precision that implies the existence of homeostatic mechanisms. The stimuli for endothelial cell proliferation and monocyte production are becoming well characterized. However, the mechanisms involved in eliminating these cells are poorly understood. One mechanism involved in cellular elimination is apoptosis, which can be triggered in some cells by ligation of the Fas molecule. In this report we have investigated whether macrophages and endothelial cells express the Fas molecule, and whether Fas transmits an apoptosis‐inducing signal in these cells. We demonstrate that macrophages express Fas and readily undergo apoptosis when cultured with anti‐Fas. In contrast, while endothelial cells can express the Fas molecule, Fas ligation is insufficient to induce apoptosis. These results suggest differential regulation of Fas function among cells of the reticuloendothelial system, and imply different mechanisms of homeostasis.
Characterization of cytokine‐producing cells in mucosal effector sites: CD3+ T cells of Th1 and Th2 type in salivary gland‐associated tissues
Authors: Hiroi Takachika , Fujihashi Kohtaro , McGhee Jerry R. , Kiyono Hiroshi
Published: March 2007

The major purpose of this study was to elucidate Th1 [interferon (IFN)‐γ and interleukin (IL)‐2] and Th2 (IL‐4, IL‐5 and IL‐6) cytokine‐producing CD3+ T cells in salivary glands, which are the major mucosal effector tissues in the oral region. Thus, CD3+ T cells were isolated from salivary gland‐associated tissues (SGAT) which consist of the submandibular gland (SMG: ∼46%), the periglandular lymph node (PGLN: ∼ 72 %), and the cervical lymph node (CLN: ∼ 90%). When SMG CD3+ T cells were examined by Th1 and Th2 cytokine‐specific ELISPOT and reverse transcriptase‐polymerase chain reaction assay, high levels of both cytokine‐specific spot‐forming cells (SFC) and mRNA for IFN‐γ, and for IL‐5 and IL‐6 were noted as representative Th1 or Th2 cytokines, respectively. Following stimulation with concanavalin A (Con A), SMG CD3+ T cells expressed mRNA and produced lymphokines for an array of Th1 (IFN‐γ and IL‐2) and Th2 (IL‐4, IL‐5 and IL‐6) cytokines. In comparison to the SMG CD3+ T cells, PGLN and CLN contain lower numbers of IFN‐γ‐, IL‐5‐ and IL‐6‐producing T cells. When these two tissues were compared, PGLN CD3+ T cells contained higher numbers of cytokine‐secreting cells than CLN. Further, IL‐2 and IL‐4 SFC and mRNA were also noted in addition to IFN‐γ, IL‐5 and IL‐6 after Con A activation. These findings showed that CD3+ T cells in SGAT, especially the SMG, are programmed to produce IFN‐γ, and IL‐5 and IL‐6 as Th1 and Th2 cytokines, respectively in vivo, although these cells are capable of producing other Th1 and Th2 cytokines after receiving appropriate T cell activation signals.
Restricted use of T cell receptor V genes in endomysial infiltrates of patients with inflammatory myopathies
Authors: Lindberg Christopher , Oldfors Anders , Tarkowski Andrzej
Published: March 2007

Inclusion body myositis (IBM), polymyositis (PM) and dermatomyositis (DM) are diseases characterized clinically by progressive muscle weakness and histologically by T lymphocyte infiltrates in striated muscle. The pathogenetic role of these cells is proposed to be cell‐mediated cytotoxicity in PM and IBM, but the exact mechanisms of their action are poorly understood. Characterization of the variable regions of T cell receptors (TcR) on the infiltrating lymphocytes may be expected to provide insights into the mechanisms of local activation of the immune system in inflammatory myopathies. Immunohistochemical analysis using a panel of monoclonal antibodies specific for 11 Vα/β TcR was performed on cryosectioned muscle biopsy specimens from eight patients with IBM, eight with PM and three with DM. In addition, TcR expression was studied in inflammatory infiltrates in skin biopsies obtained from some of the IBM patients challenged locally with tuberculin. Flow cytometry was used to assess expression of TcR on peripheral blood lymphocytes. All the patients displayed a clear restriction of TcR usage, preferentially limited to Vα2 and Vβ3 TcR families in the endomysial, but not in perivascular infiltrates, even within the same muscle specimen. Such a restriction was not found in skin punch biopsies or PBL from the same subjects. Our results suggest that T cells extravasate non‐selectively to the skeletal muscle, but once there, only certain TcR families proliferate, presumably after encounter with a locally exposed superantigen.
The diversity of antigen‐specific monoclonal antibodies from transgenic mice bearing human immunoglobulin gene miniloci
Authors: Wagner Simon D. , Popov Andrei V. , Davies Sarah L. , Xian Jian , Neuberger Michael S. , Brüggemanno Marianne
Published: March 2007

An approach to the preparation of antigen‐specific human monoclonal antibodies focuses on mice transgenic for human immunoglobulin gene miniloci; the V gene segments in these miniloci undergo productive rearrangement to yield mouse B cells expressing human immunoglobulin (Ig) chains. The general usefulness of this strategy hinges on whether it is feasible to obtain specific, high‐affinity antibodies following immunization of such animals with a variety of antigens. To test this, we have investigated the antigen‐specific responses in mice which carry human IgH miniloci (constaining just one or two VH segments) instead of a functional mouse IgH locus. Although serum responses were relatively weak, monoclonal antibodies were readily obtained to all immunogens tested (a hapten, foreign proteins and human lymphoma cells). The affinities of two of the hapten‐specific (anti‐2‐phenyl‐oxazol‐5‐one) antibodies were 60 and 160 nM, values intermediate between what is typically obtained in the primary and secondary response of normal mice. Sequence analysis of the rearranged V genes revealed that junctional events made a major contribution to diversity with a considerable amount of apparently non‐templated sequence at the V‐D and D‐J borders. Somatic hypermutation was also evident within the expressed V gene segments of many of the antigen‐specific hybridomas. These findings augur well for the general usefulness of the transgenic approach for the isolation of high‐affinity human antibodies to a wide range of antigens and suggests that the miniloci need not be particularly large.
An immortalized cell line with features of human follicular dendritic cells. Antigen and cytokine expression analysis
Authors: Orscheschek Karin , Merz Hartmut , Schlegelberger Brigitte , Feller Alfred C.
Published: March 2007

Follicular dendritic cells (FDC) are specialized cells residing primarily within lymphoid follicles. They bind immunocomplexes and play an important role in the presentation of antigen to follicular B cells. Isolation of FDC for in vitro studies, however, is difficult because they constitute only about 1% of the cells in lymphoid tissue and form tight clusters entrapping lymphocytes within their dendritic processes. The monoclonal antibody (mAb) Ki‐M4, which is highly restricted in its binding to FDC, is used to identify these cells.

In order to establish a new immortalized cell line with features of FDC, we applied a modified procedure to isolate and enrich FDC from human tonsils and fused them with the myeloma cell line SP2/0‐Ag14. The new hybrid cell line, designated FDC‐H1, is of both mouse lymphoid and human FDC origin. FDC‐H1 was found to have unlimited growth potential and to consistently express the Ki‐M4 antigen and other surface antigens of human FDC.

Semiquantitative reverse‐transcribed polymerase chain reaction (RT‐PCR) of enriched FDC and FDC‐H1 revealed the same highly restricted cytokine/mRNA profile for both, with detectable levels of interleukin (IL)‐1α and surface CD23 and a lack of mRNA for IL‐1β, IL‐2, IL‐3, IL‐4, IL‐7, IL‐9, IL‐10, interferon‐γ, tumor necrosis factor‐α, transforming growth factor‐β and granulocyte/macrophage‐colony‐stimulating factor. Additionally a weak but constant IL‐6 mRNA expression was found in the cell line FDC‐H1 by RT‐PCR. In situ hybridization experiments in tonsils revealed IL‐6 transcripts in cells with a staining pattern characteristic of a dendritic cell only in a few germinal centers.

To our knowledge, FDC‐H1 is the first cell line that constantly expresses surface antigens and a cytokine profile characteristic of FDC. It is, therefore, well suited for studying the biology of FDC and the functional relationship between FDC and normal or neoplastic lymphatic cells.
Two inhibitors of pro‐inflammatory cytokine release, interleukin‐10 and interleukin‐4, have contrasting effects on release of soluble p75 tumor necrosis factor receptor by cultured monocytes
Authors: Joyce David A. , Gibbons Deena P. , Green Patricia , Steer James H. , Feldmann Marc , Brennan Fionula M.
Published: March 2007

The biological activity of the pro‐inflammatory cytokine, tumor necrosis factor (TNF)‐α depends on the level of TNF‐α itself, the expression of the p55 and p75 cell surface receptors for TNF on target cells and the concentrations of the natural inhibitors of TNF‐α, the soluble p55 and p75 TNF receptors (TNF‐R). Interleukin (IL)‐10 and IL‐4 are known to inhibit TNF‐α production by monocytes. We, therefore, investigated the effects of IL‐10 and IL‐4 on the cell surface expression and release of TNF‐R by human monocytes to determine whether these cytokines also indirectly modulated the biological activity of TNF‐α. Exposure to IL‐10 (1‐10 U/ml) for 24 or 48 h increased soluble p75 TNF‐R expression and concomitantly reduced surface expression of p75 TNF‐R. Further, IL‐l α‐stimulated production of TNF‐α was diminished by IL‐10 and only a small proportion of this TNF‐α was bioactive, consistent with increased production of inhibitory soluble TNF‐R. IL‐10 also induced down‐regulation of surface p55 TNF‐R on monocytes, and increased release of soluble p55 TNF‐R. However, the expression of soluble p55 TNF‐R was much lower than soluble p75 TNF‐R, indicating that it contributed less importantly to neutralization of TNF‐α under these conditions. Like IL‐10, IL‐4 supressed the release of TNF‐α by monocytes. In contrast to IL‐10, however, IL‐4 (0.1‐10 ng/ml) supressed the release of soluble p75 TNF‐R from monocytes in a dose‐dependent manner. Release of soluble p55 TNF‐R was also supressed by IL‐4. IL‐10, therefore, reduces the pro‐inflammatory potential of TNF in three ways: by down‐regulating surface TNF‐R expression whilst increasing production of soluble TNF‐R and inhibiting the release of TNF‐α itself. This suggests that IL‐10 may be useful in the treatment of diseases where overexpression of TNF‐α occurs.
Evidence for the continuous recruitment and activation of T cells into the joints of patients with rheumatoid arthritis
Authors: Iannone Florenzo , Corrigall Valerie M. , Kingsley Gabrielle H. , Panayi Gabriel S.
Published: March 2007

Rheumatoid arthritis (RA) synovial fluid (SF) T cells express the activation markers CD69, HLA‐DR and very late antigen (VLA)‐1, but surprisingly few bear interleukin‐2 receptors (CD25). This unusual activation state is commonly assumed to be due to stimulation by local antigen, yet T cells activated in vitro express activation antigens in the clearly defined sequence: CD69, CD25, HLA‐DR and finally VLA‐1. Two possible explanations for the activation state of SF cells are: first, they comprise several subpopulations each expressing different activation antigens or, second, activation markers are up‐regulated by mechanisms other than antigen stimulation. To examine these hypotheses, double‐ and triple‐color immunofluorescence techniques were applied to four T cell populations: normal peripheral blood T cells activated in vitro, RA SF T cells, T cells from an in vivo model of migration [tuberculin purified protein derivative (PPD)‐induced skin blisters] and T cells co‐cultured with endothelial cells (EC). The results confirmed that in vitro activated T cells expressed activation markers in the sequence described above, with significant CD25 expression and few cells co‐expressing CD69 with HLA‐DR or VLA‐1. In contrast, almost half the SF T cells were CD69+HLA‐DR+ but CD25−; a significant minority were CD69+VLA−l+, T cells from PPD‐induced skin blisters were already HLA‐DR+ and VLA‐1+ at 24 h, although, in vitro, PPD‐activated T cells up‐regulated HLA‐DR and VLA‐1 only after 1 week, suggesting that pre‐activated T cells were preferentially recruited into the blisters. Finally, T cells were found to up‐regulate CD69 and, to a lesser extent, HLA‐DR after adhering to EC in vitro. In summary, the paradoxical activation state of SF T cells cannot be explained solely by single or multiple rounds of activation in situ. At least two other mechanisms, the preferential recruitment of pre‐activated T cells and the induction of HLA‐DR and especially CD69 by endothelial contact during migration, may also play a role.