bluuebunny/crossref_metadata_2025 huggingface dataset:
Raf‐1 and ERK2 kinases are required for phorbol 12,13‐dibutyrate‐stimulated proliferation of rat lymphoblasts. ERK2 activation precedes Raf‐1 hyperphosphorylation
Authors: Lisbona Carlos , Alemany Susana , Calvo Victor , Fernandez‐Renart Margarita
Published: March 2007

Rat lymphoblasts are arrested in the G1 phase of the cell cycle and can be promoted to proceed up to the S phase, when they are stimulated by phorbol ester. In this work, we have studied some details of the phorbol 12,13‐dibutyrate (PBu2)‐stimulated proliferation. We show that in response to PBu2 at least four different protein kinase C (PKC) isoforms translocate to the membrane. A specific PKC ζ antibody recognizes two bands of 75 and 82 kDa. These two activities are separated using a Mono Q chromatography and we show that p75 is the classical PKC ζ isoform, while p82 might be a related isoform which is PBu2 sensitive. Our data show that there is a correlation between the ability of PBu2 to promote mitogenesis and to activate ERK2 kinase, suggesting that ERK2 kinase might be the limiting step of the process. We also show that ERK kinase activation precedes Raf‐1 kinase hyperphosphorylation, suggesting that Raf‐1 kinase activation is not required for ERK kinase activation. This idea was checked using a Raf‐1 kinase antisense (AS) oligonucleotide. The results obtained with the Raf‐1 AS oligonucleotide indicate that this serine/threonine kinase is dispensable for ERK kinase activation, but needed for the PBu2 mitogenic signaling even as late as 7 h after the delivery of the signal.
CR1(CD35) and CR2(CD21) complement C3 receptors are expressed on normal human thymocytes and mediate infection of thymocytes with opsonized human immunodeficiency virus
Authors: Delibrias Catherine‐Charlotte , Mouhoub Amal , Fischer Elizabeth , Kazatchkine Michel D.
Published: March 2007

The present study demonstrates that the C3b receptor CR1 (CD35) and the C3dg/Epstein‐Barr virus receptor CR2 (CD21) are expressed by 25% and 70% of normal human thymocytes, respectively. The expression of CR2 extends to both CD1+ and CD1− cells in the thymus. Two subsets of CR2+ thymocytes were defined expressing low and high density of the receptor. The CR2++ subset represented 20% of CR2+ thymocytes and co‐expressed the CR1 receptor. CR2++ thymocytes expressed an immature CD1dull, CD3−, CD4dull, CD8−, CD7++ phenotype and included a subpopulation of large cells expressing CD34. Twenty percent of thymocytes expressed the CD21 epitope defined by monoclonal antibody BU32, which is involved in the binding of CD23 to CD21. These observations provide a basis for a role for CD21 in the proliferation and differentiation of thymocytes at early stages of maturation. The functionality of CR1 and CR2 on thymocytes was evidenced by the ability of the receptors to mediate infection of cells with complement‐opsonized human immunodeficiency virus (HIV). The results may be relevant to the immunopathogenesis of HIV infection.
Expression of major histocompatibility complex class II molecules in rat T cells
Authors: Reizis Boris , Schramm Christoph , Cohen Irun R. , Mor Felix
Published: March 2007

The expression of major histocompatibility complex (MHC) class II molecules in murine T cells has been controversial. We therefore reexamined the transcription, synthesis and surface expression of MHC class II determinants in rat T cells both in vivo and in vitro. In naive rats, a large proportion of small CD4+8+ and mature CD4+8−/CD4−8+ thymocytes was found to be MHC class II positive. At least some of the MHC class II molecules found on thymocytes were actively synthesized. The synthesis of MHC class II proteins was detected in peripheral T cells activated in vivo during induction of experimental allergic encephalomyelitis (EAE). A proportion of T cells from the inflammatory lesion of EAE exhibited MHC class II on the surface. A panel of helper T cell lines and clones was shown to synthesize MHC class II proteins. In a prototypic clone, a weak constitutive expression of MHC class II was observed. During activation, the rate of endogenous MHC class II synthesis increased and passive absorption of surface MHC class II from other cells occurred. Our data demonstrate the expression of MHC class II molecules in rat T cells in both the thymus and periphery. Since the primary function of MHC class II molecules is the presentation of peptide epitopes to T cells, these results call attention to the possible role of MHC class II molecules in T‐T interactions during T cell maturation and activation.
Leishmania major infection in mice primes for specific major histocompatibility complex class I‐restricted CD8+ cytotoxic T cell responses
Authors: da Conceição‐Silva Fátima , Perlaza Blanca L. , Louis Jacques A. , Romero Pedro
Published: March 2007

This report shows that lymphoid tissues of mice which have resolved a primary infection with Leishmania major contain parasite‐specific major histocompatibility complex (MHC) class I‐restricted cytolytic CD8+ T cell precusors that can be expanded after specific restimulation in vitro with syngeneic antigen‐presenting cells pulsed with a cyanogen bromide digest of L. major. In H‐2b mice, two distinct populations of CD8+ T cells were identified which both lysed target cells pulsed with L. major‐derived peptides but were restricted by a different H‐2b class I gene product. Interestingly, these two populations appear to recognize different parasite‐derived peptides. It is noteworthy that one K°‐restricted CD8+ T cell line was able to specifically lyse syngeneic macrophages infected with viable L. major, indicating that some L. major‐derived peptides may reach the MHC class I pathway of presentation from the phagolysosomal compartment where the parasites are confined in infected macrophages. The importance of these parasite‐specific MHC class I restricted cytolytic CD8+ T cells for the elimination of L. major by the infected host remains to be determined.
B cell antigen receptor cross‐linking induces tyrosine phosphorylation and membrane translocation of a multimeric Shc complex that is augmented by CD19 co‐ligation
Authors: Lankester Arjan C. , Van Schijndel Gijs M. W. , Rood Pauline M. L. , Verhoeven Arthur J. , Van Lier René A. W.
Published: March 2007

The SH2 domain‐containing transforming Shc protein has been implicated in mitogenic signaling via several surface receptors through p21ras. Following tyrosine phosphorylation by either receptor or non‐receptor tyrosine kinases, Shc may interact with the adaptor protein Grb2, which is linked to Sos1, a guanine nucleotide exchange factor for human ras. Ligation of the antigen receptor complex on B cells (BCR) is known to activate various intracellular signaling pathways, which may accumulate in mitogenic responses. With respect to the initial steps, the activation of BCR‐associated non‐receptor tyrosine kinases appears to be indispensible. In this report we show that Shc proteins become tyrosine phosphorylated after BCR ligation on both transformed and normal human B cells. This is accompanied by the association of Shc with Grb2 proteins and a yet unidentified 145‐kDa tyrosine phosphorylated protein. Subcellular fractionation revealed that this activation‐induced multimeric Shc complex rapidly translocates towards the plasma membrane. Co‐ligation of the BCR with the CD19 molecule results in a marked increase of these events, whereas CD19 cross‐linking alone does not induce Shc tyrosine phosphorylation or translocation. Thus, in B cells the Shc complex may represent a molecular junction between the BCR and the mitogenic p21ras cascade.
Transfer of Sjögren's syndrome‐like autoimmune lesions into SCID mice and prevention of lesions by anti‐CD4 and anti‐T cell receptor antibody treatment
Authors: Hayashi Yoshio , Haneji Norio , Hamano Hironori , Yanagi Kumiko
Published: March 2007

We describe the successful transfer of murine Sjögren's syndrome‐like autoimmune lesions from MRL/lpr mice (H‐2k) to severe combined immunodeficiency (SCID) mice (H‐2d) and prevention of lesions by anti‐CD4 and ‐T cell receptor Vβ8 antibody treatment. Mononuclear cells (1 × 106) isolated from the inflammed submandibular salivary gland tissues of MRL/lpr mice were transferred intraperitoneally into SCID mice. Autoimmune lesions resembling those seen in Sjögren's syndrome developed in the salivary and lacrimal glands of SCID mice 8 weeks after the injection, whereas other organs did not show any lesion. This pathology resembles Sjögren's syndrome in humans involving both the salivary and lacrimal glands. Immunohistochemically, a major proportion of these infiltrating cells in transferred SCID mice were CD4+ and Vβ8+. When the spleen cells from MRL/lpr mice were injected, severe inflammatory lesions, probably resulting from a graft‐versus host reaction, were observed in multiple organs of SCID mice. The disease could not be induced by intraperitoneal administration of the sera from MRL/lpr mice, or of the spleen cells from C3H/He (H‐2k) and BALB/c (H‐2d) mice. We detected autoantibody production specific for the salivary gland tissue in sera from transferred SCID mice. Moreover, we found that the lesions were prevented by administration of the isolated cells treated in vitro with anti‐CD4 and anti‐Vβ8 monoclonal antibodies. These results suggest that CD4‐ and Vβ8‐bearing T cells are involved in recognizing an autopeptide and triggering autoimmunity in the salivary and lacrimal glands, and therapies designed with anti‐CD4 and anti‐Vβ8 antibodies may prove effective in treating the murine autoimmune disease.
Monoclonal IgA class‐switch variants against bacterial surface antigens: molecular forms and transport into murine respiratory secretions
Authors: Steinmetz Ivo , Albrecht Frank , Häußler Susanne , Brenneke Birgit
Published: March 2007

The present study describes a new model for passive immunization of the respiratory tract with IgA in comparison to other isotypes. Monoclonal IgA‐isotype‐switch variants were isolated from different IgG‐producing hybridoma clones specific for surface epitopes of bacterial respiratory tract pathogens. Analysis of the molecular form of the IgA variants revealed the simultaneous production of monomeric, dimeric and higher polymeric IgA by a single‐cell line with predominance of the polymeric forms. The specificities of the IgA variants were identical to the parent IgG antibodies as demonstrated by inhibition experiments. The IgA variant antibodies were separated into monomers and polymers by gel filtration. Intravenous injection of the different molecular forms of IgA and of IgG into mice were used to investigate the transport characteristics of IgA into murine upper and lower respiratory tract secretions by the physiological route in comparison to IgG. Polymeric IgA variant, monomeric IgA variant and IgG were detected in immunologically active form in both nasal secretion and bronchoalveolar fluid as evidenced by binding to their antigens in an enzyme‐linked immunosorbent assay. The relative contribution of the specific exogenous monoclonal IgA and monoclonal IgG to total IgA and IgG, respectively, was determined in secretions. Comparison of the secretion to serum transport ratios clearly indicates selective transport of polymeric IgA variant into nasal secretions relative to IgG parent antibody. Molecular and functional characteristics of the IgA variants make them ideal for passive mucosal immunization experiments and identification of protective epitopes in mucosal immunity.
Antigenic and immunogenic mimicry of the HER2/neu oncoprotein by phage‐displayed peptides
Authors: Orlandi Rosaria , Ménard Sylvie , Colnaghi Maria I. , Boyer Cinda M. , Felici Franco
Published: March 2007

To recover peptides that antigenically and immunogenically mimic the p185HER2 oncoprotein, we selected the phage‐peptide libraries pVIII‐9aa and pVIII‐9aa. Cys using murine monoclonal antibodies (mAb) MGr2 and MGr6, directed against two distinct epitopes of the p185HER2 extracellular domain. Phagedisplayed peptides containing consensus amino acid motifs were recovered and shown to compete specifically for mAb binding on tumor cells that overexpress p185HER2. The deduced amino acid sequence of the peptides suggests that both epitopes defined by the mAb on p185HER2 are discontinuous and that hydrophobic interactions are involved in binding with the mAb. A phage clone displaying the GPLDSLFAQ peptide elicited a specific immune response against the p185HER2 in BALB/c mice, demonstrating that this phage‐displayed peptide represents an immunological equivalent of the MGr2 epitope on p185HER2 and might be used as a substitute for this oncoprotein in in vitro and in vivo immunological studies.
Structural profile of idiotype, anti‐idiotype and anti‐anti‐idiotype monoclonal antibodies in the HLA‐DQ3 antigenic system
Authors: Iwasaki Yoshiaki , Takabatake Hiroyuki , Shinji Toshiyuki , Monestier Marc , Ferrone Soldano
Published: March 2007

Interest in the characterization of idiotype cascades in the HLA antigenic system has been stimulated by their potential role in the immune response to mismatched HLA allospecificities and in the survival of kidney allografts. Since no information is available about the structural organization of idiotypic cascades in the HLA system, we have sequenced the variable regions of the heavy (VH) and light (VL) chains of mouse anti‐HLA‐DQ3 monoclonal antibody (mAb) KS13 elicited by cell membrane‐bound antigens, of syngeneic anti‐HLA‐DQ3 mAb S2B154 elicited by anti‐idiotypic (anti‐id) mAb K03‐34 and of five syngeneic anti‐id mAb elicited by mAb KS13. mAb KS13 and S2B154, which have been previously shown to be very similar in their specificity and idiotypic profile, share several structural characteristics. Their VH and VL regions are encoded by the same VH. VK and JH genes, display relatively similar V(D)J rearrangements and differ only through a few amino acid substitutions. Among the five anti‐id mAb elicited by mAb KS13, mAb R1‐38 and R18‐9 utilize multiple genetic elements that are different from those used by anti‐id mAb KO3‐34, K03‐256 and K03‐335. These results indicate that diverse V region combinations can confer an anti‐id specificity in the antigenic system analyzed. mAb K03‐34, K03‐256 and K03‐335 originate from the same B cell clone, since they use the same V, D and J genes and possess identical V(D)J rearrangements. The latter three anti‐id mAb differ only by point mutations, which have dramatic effects on the HLA‐DQ3 antigen mimicry properties of the three anti‐id mAb. mAb K03‐34 is the only one to induce anti‐HLA‐DQ3 antibodies both in syngeneic and xenogeneic hosts. The antigen mimicry properties of anti‐id mAb K03‐34 depend upon its three‐dimensional conformation, since no significant amino acid sequence homology has been found between its VH and VL regions and α1 and β1 domains of HLA‐DQ3 antigens.
Inhibition of proliferation and differentiation during early T cell development by anti‐transferrin receptor antibody
Authors: Brekelmans Pieter , Van Soest Peter , Leenen Pieter J. M. , van Ewijk Willem
Published: March 2007

Proliferating cells require iron and, therefore, express the transferrin receptor (CD71) that mediates cellular iron uptake. Cycling thymocytes, which have the CD4−8−3−, CD4−8+3−, or CD4+8+3− phenotypes, also express CD71. The importance of CD71‐mediated iron uptake for proliferation and maturation of thymocytes was studied using fetal thymus organ cultures at day 14 of gestation and treating them for 7 days with a CD71 monoclonal antibody (mAb). The intracellular iron deficiency caused by this treatment, inhibits both proliferation and maturation of the thymocytes. Cell recovery was reduced by 60%, but cells still expanded tenfold during the culture. Remarkably, the final maturation of αβ T cells was completely blocked as no thymocytes with low or high CD3/αβTcR expression developed. Moreover, only few cells reached the CD4+8+3− stage of T cell development. CD4−8−3− thymocytes, however, as well as its CD44−25+ subset developed in normal numbers, suggesting that CD44−25+ CD4−8−3− cells, or their immediate progeny, were most vulnerable to CD71 mAb treatment. The development of γδ T cells, which also express CD71, was not affected in these cultures. This suggests that γδ T cells are either less iron‐dependent or possess alternative iron‐uptake mechanisms. Thus, our observations indicate that CD71 treatment, causing decreased intracellular iron levels, severely inhibits the major proliferation phase from the CD44−25+ CD4−8−3− to the CD4+8+3− cells, and completely abrogates the final maturation of CD4+8+3− cells into αβTcR‐expressing cells. In contrast, proliferation and differentiation of the earliest thymic precursors into CD44−25+ CD4−8−3− cells is not affected by CD71 treatment.
GM1, a ganglioside that specifically enhances immunoglobulin production and proliferation in human plasma cells
Authors: Kimata Hajime
Published: March 2007

The effects of gangliosides on human plasma cell responses were studied. Among the various gangliosides tested, only GM1 enhanced immunoglobulin (Ig) production and proliferation in the human plasma cell lines, IM‐9 and AF‐10, while other gangliosides (GM2, GM3, GD1a, GD1b, GD3, GT1b, and GQ1b) had no effect. Among the various cytokines tested, including interleukin (IL)‐1β, IL‐2, IL‐3, IL‐4, IL‐5, IL‐6, IL‐9, IL‐10, IL‐12, IL‐13, interferon (IFN)‐α and IFN‐γ, only IL‐6 enhanced Ig production and proliferation in IM‐9 and AF‐10 cells. However, the enhancement of plasma cell responses by GM1 was specific and was not mediated by IL‐6, since GM1 activity was blocked by anti‐GM1 monoclonal antibody (mAb), but not by control IgM, anti‐IL‐6 Ab or the anti‐IL‐6 receptor mAb, PM1. Conversely, the enhancement by IL‐6 was blocked by anti‐IL‐6 Ab and PM1, but not by anti‐GM1 mAb. GM1, but not other gangliosides, also enhanced Ig production and proliferation in freshly separated plasma cells from patients with plasma cell leukemia and in plasma cells generated in vitro. These actions of GM1 were specifically blocked by anti‐GM1 mAb, but not by anti‐IL‐6 Ab or PM1. These results indicate that GM1 may be an important regulator of plasma cell responses.
Ionizing radiation induces apoptotic cell death in human TcR‐γ/δ+ T and natural killer cells without detectable p53 protein
Authors: Seki Hidetoshi , Kanegane Hirokazu , Iwai Kazuyuki , Konno Akihiro , Ohta Kunio , Yachie Akihiro , Taniguchi Noboru , Miyawaki Toshio
Published: March 2007

The p53 tumor suppressor gene has been shown to be involved in programmed cell death, apoptosis, in murine immature thymocytes after treatment with ionizing radiation. Ionizing radiation also induces apoptosis in peripheral mature lymphocytes. In this work, we investigated the p53 participation in radiation‐induced apoptosis in human peripheral blood lymphocytes (PBL) subpopulations. Exposure to γ‐irradiation resulted in an appreciable induction of apoptotic cell death in TcR‐α/β+ (CD4+ and CD8+) T cells, TcR‐γ/δ+ T cells, B cells and natural killer (NK) cells, as assessed by DNA fragmentation as well as the morphological characteristics. Importantly, it was found that there was a marked difference among PBL subpopulations as regards the induction of p53 protein by γ‐irradiation. Similar to previous observations for murine thymocytes, p53 induction in TcR‐α/β+ T cells and B cells after γ‐irradiation was evident by Western blot analysis. Radiation‐induced apoptosis in TcR‐α/β+ T cells and B cells was efficiently inhibited by cycloheximide, indicating the requirement of de novo protein synthesis, including p53 protein, for radiation‐induced apoptosis in both subpopulations. In marked contrast, no identifiable levels of p53 protein were induced in either TcR‐γ/δ+ T or NK cells after γ‐irradiation. In addition, it was demonstrated that radiation‐induced cell death in TcR‐γ/δ+ T and NK cells could be prevented by interleukin‐2, but not by cycloheximide. These results imply that radiation‐induced lymphocytic apoptosis can be mediated by p53‐dependent or ‐independent mechanisms.
Deletion of CD4+ T cells and thymocytes by apoptosis in mouse mammary tumor virus (C4)‐infected Vβ2 transgenic mice
Authors: Wang Hong , Gill Randall F. , Lichlyter Darcy , Iglesias Antonio , Bluethmann Horst , Wei Wei‐Zen
Published: March 2007

Mouse mammary tumor virus MMTV(C4) encodes a Vβ2‐specific superantigen. In Vβ2 transgenic (TG2) mice more than 98 % of peripheral T cells express Vβ2. Infection of Tg2 mice with MMTV(C4) at birth through their mothers' milk or at 6–8 weeks of age by intravenous injection resulted in massive deletion of peripheral CD4+ T cells and suppressed thymopoiesis. The number of peripheral CD8+ T cells was not affected in neonatally infected mice. In older mice injected with MMTV(C4), splenic CD8+ T cells were significantly elevated. Suppressed thymopoiesis was observed in both neonatally infected and older mice injected with MMTV(C4). Thymocytes which expressed high level CD3 or Vβ2 were deleted. To determine if T cells or thymocytes were deleted through apoptosis, DNA fragmentation was examined by flow cytometry and diphenylamine (DPA) binding assay. Approximately 31 % of CD4+ T cells from MMTV(C4)‐infected Tg2 mice as compared to 6% from normal Tg2 mice contained fragmented nuclear DNA by flow‐cytometric analysis. The DPA binding assay showed significantly increased total soluble DNA in lymph node cells and thymocytes from MMTV(C4)‐infected mice. The kinetics of T cell and thymocyte apoptosis correspond to their deletion, supporting apoptosis as the mechanism of T cell and thymocyte deletion. CD4+ T cell and thymocyte deletion by MMTV(C4) in Tg2 mice provides a sensitive system for the analysis of retrovirus superantigen‐induced apoptosis.
Stimulated natural killer cells secrete factors with chemotactic activity, including NAP‐1/IL‐8, which supports VLA‐4‐ and VLA‐5‐mediated migration of T lymphocytes
Authors: Somersalo Kristina , Carpén Olli , Saksela Eero
Published: March 2007

In vivo, natural killer (NK) cells dominate among the early invading cells in allografts and virus‐infected tissues, and they are followed later by an influx of T cells. The same sequence of events was seen in our modified Boyden chamber assay. The migration of both CD3+/CD4+ and CD3+/CD8+ cells through fibronectin‐coated filters increased after co‐culture with NK cells. The migratory response to a soluble factor from NK cells supernatants was predominantly chemotactic rather than chemokinetic. Endogenous NK cells, purified in the presence of human serum albumin, did not induce T cell chemotaxis, but NK cells which were purified in the presence of 10% fetal calf serum (FCS), or which were activated in the absence of FCS with 10−4 M histamine, with 300 IU/ml interleukin (IL)‐2, or with a combination of 10 IU/ml IL‐2 and 10 μg/ml CD16 monoclonal antibody increased T cell migration by 30–70%. Both the random and chemotactic migration were dependent on fibronectin receptors VLA‐4 and VLA‐5 on T cells. About 60% of the chemotactic was neutralized by NAP‐1/IL‐8 polyclonal antibody. Northern blot analysis revealed IL‐8 mRNA expression in highly purified, stimulated NK cells; dimeric IL‐8 protein secreted by NK cells was detected by immunoblotting, and, in immunofluorescence staining IL‐8 was visualized in NK cells. These observations suggest that NK cells, early invaders in the foci of injury, participate in the initiation of a specific immune response by facilitating T cell recruitment.
T helper cell unresponsiveness: Rapid induction in antigen‐transgenic and reversion in non‐transgenic mice
Authors: Bachmann Martin F. , Rohrer Urs Hoffmann , Steinhoff Ulrich , Bürki Kurt , Skuntz Susan , Arnheiter Heinz , Hengartner Hans , Zinkernagel Rolf M.
Published: March 2007

T cell tolerance is usually established by clonal deletion of self‐specific T cells in the thymus, or some times, in the periphery. Alternatively, tolerance may also be achieved by induction of clonal T cell unresponsiveness by a poorly understood mechanism called “anergy”. We found that transgenic mice expressing a soluble form of vesicular stomatitis virus (VSV) glycoprotein (G) predominantly in liver and kidney exhibited normal B cell responses. VSV‐G‐specific T help‐independent neutralizing IgM responses were within normal ranges, but no T help‐dependent neutralizing IgG antibodies were generated upon immunization with recombinant VSV‐G protein and recombinant vaccinia virus expressing VSV‐G. This demonstrated absence of B cell tolerance but presence of T helper cell unresponsiveness. After adoptive transfer of transgenic spleen cells into thymectomized immuno‐incompetent hosts, the unresponsive T helper cells regained function and switched the neutralizing IgM response to IgG, comparably to control T helper cells, within 7 days. Conversely, when naive non‐transgenic spleen cells were transferred into transgenic mice, VSV‐G‐specific T helper cells became unresponsive within 3–4 days. These results suggest that VSV‐G‐specific T helper cells are rendered unresponsive within a few days in the VSV‐G transgenic host also outside of the thymus and that this unresponsiveness was reversed by transfer into antigen‐free recipients.
Shared amino acid sequences in the NDβN and Nα regions of the T cell receptors of tumor‐infiltrating lymphocytes within malignant glioma
Authors: Ebato Michimasa , Nitta Taizo , Yagita Hideo , Sato Kiyoshi , Okumura Ko
Published: March 2007

The purpose of this study was to assess the V‐(D)‐J junctional region of the T cell receptor (TCR), the CDR3 region, which is responsible for glioma‐specific antigen contact in αβ TCR‐mediated recognition. We sequenced the TCR α and β chians of Vα7, and Vβ13.1 cDNA derived from tumor‐infiltrating lymphocytes (TIL) of 12 glioma patients and also the corresponding clones from the patients' peripheral blood lymphocytes (PBL). A shared Vβ13.1 DJ sequence of the CDR3 region, NDβN, was demonstrated in 49 of 66 Vβ13.1+ clones (74.2 %) from the glioma TIL, whereas only 4 of 33 clones (12.1 %) were observed in the Vβ13.1+ clones from the PBL (p < 0.001). A common VDJ sequence, FCASS (Vβ13.1)‐YRLPWGTSDS (NDβN)‐GELFF(Jβ2.2), was observed not only in the gliomas from each patient, but also among all the patients with a preference for Vβ13.1. In contrast, the amino acid sequences of the Vβ13.1+ PBL clones were diverse and random. Next, we sequenced subclones from other Vβ subfamilies randomly selected to compare their VDJ region rearrangements (Vβ3 and Vβ5.1). In contrast to Vβ13.1, the amino acid sequences of these junctional regions were completely different in these subclones. The V‐J junctional region of the α chain is dominated by a few clones in some patients, and no shared amino acid sequences were detected in the TCR Vα junctional region. However, in the Nα region of the Vα7‐bearing TIL clones, arginine was used in 27 of 44 clones (61.4%) compared to only 3 of 12 clones from the PBL (p < 0.05). These results are consistent with the hypothesis that a clonal expansion/accumulation of glioma lineage‐specific T cells occurred in vivo at the tumor site and that these T cells may be recognizing glioma‐specific antigens.
Co‐stimulation by anti‐immunoglobulin is required for B cell activation by CD40Llow T cells
Authors: Poudrier Johanne , Owens Trevor
Published: March 2007

During cognate B : T interactions, B cells encounter antigen (Ag) through surface immuno‐globulin (sIg) and present antigenic peptides to T helper (Th) cells. However, most in vitro systems used to study contact events involved in the delivery of T help for B cells circumvent the requirement for T cell Ag specificity by using anti‐CD3/T cell receptor (TcR) monoclonal antibodies (mAb) to activate T cells. To study the role of sIg engagement in the responsiveness of B cells to T help, we pre‐treated small resting B cells with soluble anti‐χ mAb prior to contact with an activated Th1 clone. By reducing the concentration of anti‐TcR mAb we obtained low levels of CD40 ligand (CD40Llow) on Th cells, comparable to those expressed by lymph node T cells activated in vitro (ex vivo T cells). In contrast to untreated B cells, which did not respond to CD40Llow Th, anti‐Ig‐treated B cells responded strongly. Low buoyant density B cells also responded to CD40Llow Th cells. There was no B cell response to resting Th cells. mAb against CD54/intercellular adhesion molecule‐1 or major histocompatibility complex (MHC) class II completely inhibited B cell responses to CD40Llow Th1 cells, equivalent to the effects of blocking CD40 interactions. This contrasts with mAb blocking responses to CD40LhighTh, where CD40 effects predominate. Our data show that sIg engagement is necessary for the induction of B cell response to CD40Llow Th cells. Anti‐CD3‐activated ex vivo T cells that were also CD40Llow did not provide help to small resting B cells, but did induce responses from sIg‐stimulated B cells. Thus, our data support a requirement for sIg signaling in physiological B cell activation, and further confirm previous work showing CD40 ligation to be necessary but not sufficient for delivery of T help to B cells.
Activation‐induced proteolysis of the cytoplasmic domain of ζ in T cell receptors and Fc receptors
Authors: Taupin Jean‐Luc , Anderson Paul
Published: March 2007

The CD3‐T cell receptor (TCR) complex on T cells and the Fcγ receptor type III (FcγRIII)‐ζ‐γ complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide‐linked dimers composed of the related subunits ζ and γ. Immunochemical analysis of receptor complexes separated on two‐dimensional diagonal gels allowed the identification of a previously uncharacterized ζ‐p14 heterodimer. ζ‐p14 is a component of both CD3‐TCR and FcγRIII‐ζ‐γ. Peptide mapping analysis shows that p14 is structurally related to ζ, suggesting that it is either: (i) derived from ζ proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding ζ express ζ‐p14 supports the former possibility. The expression of CD3‐TCR complexes including ζ‐p14 increases following activation with phorbol 12‐myristate 13‐acetate or concanavalin A, suggesting that proteolysis of ζ may contribute to receptor modulation or desensitization.
Maternal immunization modulates the primary immune response to 2‐phenyl‐oxazolone in BALB/c mice
Authors: Lemke Hilmar , Lange Hans , Berek Claudia
Published: March 2007

The development of the antibody repertoire in newborn mice is greatly influenced by idiotypic network interactions. It has been demonstrated that anti‐idiotypic antibodies either directly injected or transferred from the mother may alter the repertoire for life. For an elucidation of the underlying mechanisms we have analyzed the primary immune response to 2‐phenyl‐5‐oxazolone (phOx) coupled to chicken serum albumin (CSA) in BALB/c mice after complete disappearance of maternal antibodies which originated from different stages of affinity maturation. Depending on the serum titers of the mothers after primary (1° mo), secondary (2° mo) or tertiary (3° mo) immunization, maternal anti‐phOx IgG persisted in F1 mice for up to 9 months. In addition, F1 mice born to 2° mo developed – even without immunization – an anti‐phOx IgM titer which reached levels similar to an antigen‐induced primary response. An enhancement of the early primary anti‐phOx as well as anti‐CSA response was seen in F1 mice born from 1° mo, whereas the response was delayed when born to 2° mo and 3° mo. The antibody titers in the latter group of mice remained at a lower level for 3 months. In contrast, mice of the F2 generation which received a smaller amount of the same collection of maternal antibodies as F1 mice from 3° mo exhibited a quite different primary response: (i) They showed an earlier onset in their anti‐CSA response. (ii) Whereas normally a plateau in antibody titer was reached by the 4th weak after immunization, in 55 % of the F2 mice a prolonged increase of the anti‐phOx and anti‐CSA antibody titers was observed. At 12 weeks after antigenic challenge, titers reached plateau levels of 6 × 105 which were never before seen in a primary phOx or CSA response. Thus, depending on its own immunological experience, the maternal immune system induces a state of memory in the offspring which results in a faster and/or enhanced immune response in the F1 and F3 generations.
CD8+ T cell‐mediated protection against an intracellular bacterium by perforin‐dependent cytotoxicity
Authors: Kägi David , Ledermann Birgit , Bürki Kurt , Hengartner Hans , Zinkernagel Rolf M.
Published: March 2007

Growth of Listeria monocytogenes is mainly controlled by macrophages, which are activated by specific T cells. A potential role of CD8+ T cells by direct lysis of infected cells was investigated in perforin‐deficient mice generated by homologous recombination. The absence of perforin‐mediated cytotoxicity resulted in delayed clearance of Listeria from the spleen but not the liver after primary infection, overall susceptibility to Listeria however was not increased. Protection against a secondary infection was drastically impaired in perforin‐deficient mice. Adoptive transfer of immune spleen cells to recipients revealed that anti‐Listeria protection by CD8+ T cells from perforin‐deficient versus normal mice was about 10‐fold reduced in livers and about 100‐fold reduced in the spleen of recipients. CD4+ T cells from immune control and perforin‐deficient mice conferred comparable protection. These results indicate that the protective effect of CD8+ T cells against an intracellular bacterium mainly evident in secondary infection is mediated by a perforin‐dependent pathway, presumably cytotoxicity, and less by other direct or indirect effector mechanisms.
Interleukin‐2 induces tyrosine phosphorylation and nuclear translocation of stat3 in human T lymphocytes
Authors: Nielsen Mette , Svejgaard Arne , Skov Søren , Ødum Niels
Published: March 2007

An early biochemical event associated with T cell activation through the interleukin‐2 receptor (IL‐2R) is tyrosine phosphorylation of several intracellular substrates. The exact mechanism by which IL‐2 regulates transcription of different genes is presently unknown. Here, we report that stimulation through the IL‐2R induced tyrosine phosphorylation and subsequent nuclear translocation of stat3, a newly identified member of the signal transducers and activators of transcription (STAT) family of proteins. In contrast, stat1 proteins were not tyrosine phosphorylated after IL‐2 ligation, whereas tyrosine‐phosphorylated stat1 proteins (91 and 84 kDa proteins) were translocated to the nucleus following interferon‐γ treatment of HeLa cells.

Apart from stat3, another cytoplasmic protein was tyrosine phosphorylated and subsequently translocated to the nucleus in response to IL‐2. This protein had an apparent molecular mass of 84 kDa and was not recognized by stat3 or stat1 mAb or antisera. Since IL‐2 induced nuclear translocation of the 84 kDa protein and stat3 followed identical kinetics, p84 is a candidate for a new, yet undefined, member of the STAT family. Taken together, we report that IL‐2 induces tyrosine phosphorylation and subsequent nuclear translocation of stat3 and an as yet undefined 84‐kDa protein in antigen‐specific human T cell lines.
T lymphocyte‐mediated antiviral immune responses in mice are diminished by treatment with monoclonal antibody directed against the interleukin‐2 receptor
Authors: Utermöhlen Olaf , Tárnok Attila , Bönig Lutz , Lehmann‐Grube Fritz
Published: March 2007

Blocking the interleukin‐2 receptor's α‐chain in lymphocytic choriomeningitis virus‐infected mice by treatment with monoclonal antibodies diminished the increase of numbers of CD8+ T lymphocytes in spleens and prevented CD8+ T lymphocyte‐mediated virus clearance from organs as well as generation of virus‐specific cytotoxic T lymphocytes. Also, the CD8+ T cell‐mediated early phase of the delayed‐type hypersensitivity footpad swelling reaction was decreased. The same treatment had no effect on the number of CD4+ spleen T lymphocytes, which, however, did not enlarge during infection, but these cells' heightened DNA synthesis and cytokine production were reduced by antibody treatment; yet the generation of antiviral antibodies remained unaffected, and the CD4+ T lymphocyte‐mediated second part of the footpad reaction was somewhat augmented. We conclude that blocking of the interleukin‐2 receptor by antibody in lymphocytic choriomeningitis virus‐infected mice diminishes both CD8+ and CD4+ T cell‐mediated antiviral immune responses, the former more than the latter.
Expression of Bruton's tyrosine kinase protein within the B cell lineage
Authors: Genevier Helen C. , Hinshelwood Steve , Gaspar Hubert B. , Rigley Kevin P. , Brown Derek , Saeland Sem , Rousset Francoise , Levinsky Roland J. , Callard Robin E. , Kinnon Christine , Lovering Ruth C.
Published: March 2007

Defects in the gene encoding Bruton's tyrosine kinase (Btk), normally expressed in B cells, cause X‐linked agammaglobulinemia (XLA). The phenotype of XLA is characterized by a lack of circulating B cells and immunoglobulin. It has been suggested that B cell maturation from the pre‐B cell stage to more mature stages is dependent on the appropriate expression of this gene. The Btk mRNA is expressed in B cells and myeloid cells, but protein expression in relation to B cell maturation has not been determined. Moreover, expression of the Btk protein has so far only been investigated in human Epstein‐Barr virus‐transformed B cell lines, and in murine splenocytes and B cell lines. We have developed an antiserum which recognizes the human Btk protein and shown that normal human tonsillar B cells, peripheral blood monocytes and myeloid cells express the protein, whereas tonsil‐derived T cells do not. We also show that the protein is present in early and mature human B cell lines, but is absent in terminally differentiated plasma cell lines. Furthermore, expression is reduced or absent in three B lineage cell lines derived from two patients with defined genetic mutations in Btk and suffering from XLA.
Low‐level infection with Trichuris muris significantly affects the polarization of the CD4 response
Authors: Bancroft Allison J. , Else Kathryn J. , Grencis Richard K.
Published: March 2007

Resistance and susceptibility to the intestinal nematode Trichuris muris has been shown to be dependent upon the induction of T helper type 2 (Th2) or Th1 cells, respectively. This study demonstrates that in a normally resistant strain of mouse, i.e. BALB/K which mounts a dominant Th2 response, sub‐threshold levels of infection (< 40 eggs) can survive and become sexually mature adult worms (10–20 adults). The immunological basis of this phenomenon was found to be a dramatically altered polarization of the CD4 response. The Th2‐response characteristic of this strain of mouse infected with T. muris was shown to be significantly down‐regulated as assessed by in vitro cytokine production [interleukin (IL)‐4, IL‐5 and IL‐9]. In contrast, Th1 parameters of infection such as in vitro interferon‐γ production and the presence of parasite‐specific IgG2a were greatly up‐regulated in these mice.
Involvement of a serine protease in the synthesis of platelet‐activating factor by endothelial cells stimulated by tumor necrosis factor‐α or interleukin‐1α
Authors: Bussolino Federico , Arese Marco , Silvestro Lugi , Soldi Raffaella , Benfenati Emilio , Sanavio Fiorella , Aglietta Massimo , Bosia Amalia , Camussi Giovanni
Published: March 2007

It has been shown that production of platelet‐activating factor PAF, 1‐O‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine) by endothelial cells (EC) stimulated with tumor necrosis factor (TNF)‐α and interleukin (IL)‐1α requires the synthesis of new proteins and is regulated by anti‐proteinases. Here, we demonstrate that TNF‐α and IL‐1α induce the expression by EC of a 34‐kDa diisopropyl fluorophosphate‐binding protein immunoprecipitated by an antihuman elastase antibody. This protein is released in the medium and cleaves the chromogenic substrate N‐methoxysuccinyl‐ Ala‐Ala‐Pro‐Val p‐anilide, which is specific for elastase. The generation of this elastase‐like protein seems to be important for the synthesis of PAF induced by TNF‐α and IL‐lα, as suggested by the following observations: (a) it precedes the synthesis of PAF; (b) the inhibitors of serine protease and anti‐human elastase antibody prevent the synthesis of PAF and the activation of 1‐O‐alkyl‐2‐lyso‐glycerophosphocholine acetyl‐CoA: acetyltransferase, which is a key enzyme of the PAF remodelling pathway; (c) elastase, at concentrations similar to that detectable in the medium of cytokine‐activated EC, elicits a rapid synthesis of PAF by EC. High‐performance liquid chromatography‐tandem mass spectrometric analysis of bioactive PAF demonstrates that the molecular species produced after stimulation of EC with TNF‐α, IL‐1α or elastase are similar, with a predominant synthesis of the alkyl species. These results indicate that TNF‐α and IL‐1α stimulate the production of a serine protease which is critical in the activation of enzymes involved in PAF synthesis, suggesting the potential involvement of this mechanism in the regulation of EC functions.
Freshly isolated mouse 4F7+ splenic dendritic cells process and present exogenous antigens to T cells
Authors: Mohamadzadeh Mansour , Pavlidou Anastassia , Enk Alexander , Knop Jürgen , Rüde Erwin , Gradehandt Gernot
Published: March 2007

The antibody 4F7 was reported to recognize an epitope expressed on dendritic cells (DC) from various tissues. To study the ability of splenic 4F7+ dendritic cells to process antigen for presentation to CD4+ T cells, DC were enriched using a separation procedure avoiding overnight culture which could lead to an altered phenotype. These DC were used as antigen‐presenting cells (APC) in stimulation cultures of major histocompatibility complex class II‐restricted T cells. It was found that they induce antigen‐dependent lymphokine production by T cells and therefore could present exogenous antigens. These processing takes place intracellularly, because fixation abrogates presentation to T cells. Moreover, antigen presentation needs intracellular processing within endo‐ or lysosomes as chloroquine‐treatment prevents T cell activation. Titration of APC numbers revealed that contaminating APC most likely did not account for antigen‐specific T cell activation by DC. No evidence was found for release of antigenic peptides or for partial antigen processing possibly done by cell surface located enzymes on DC. In conclusion, these results indicate that freshly enriched DC are able to process antigens similarly to other APC.
α2,3‐Sialyl and α1,3‐fucosyltransferase‐dependent synthesis of sialyl Lewis x, an essential oligosaccharide present on L‐selectin counterreceptors, in cultured endothelial cells
Authors: Majuri Marja‐Leena , Pinola Mari , Niemelä Ritva , Tiisala Sinikka , Natunen Jari , Renkonen Ossi , Renkonen Risto
Published: March 2007

Sialyl Lewis x (sLex) oligosaccharides have been shown to be present in counterreceptors for L‐selectin. We and others have previously shown that high endothelial cells in lymph nodes and at sites of inflammation express sLex. Here we show that also cultured human umbilical vein endothelial cells (HUVEC) express sLex on their cell surface. This oligosaccharide is formed by sequential action of α2,3‐sialyl‐ (α2,3‐ST) and α1,3‐fucosyltransferases (α1,3‐FT) on N‐acetyllactosamine. At least two of the several α2,3‐ST and four of the several α1,3‐FT are present in HUVEC. In functional assays both α2,3‐ST and α1,3‐FT activities were observed in HUVEC lysates with exogenous lactosamine and sialyllactosamine acceptors, leading to the generation of the sialyllactosamine and sLex sequences, respectively. TNF stimulation increased the level of mRNA expression of FT VI, and the α1,3‐FT activity in HUVEC. Taken together these data show that endothelial cells express sLex and that they possess mRNA as well as enzyme activities of several α2,3‐ST and α1,3‐FT necessary in the final steps of sLex synthesis. Furthermore, inflammatory cytokines such as TNF can enhance transferase activities relevant in generating putative L‐selectin counterreceptors.
Characterization of two Epstein‐Barr virus epitopes restricted by HLA‐B7
Authors: Hill Ann , Worth Austen , Elliott Tim , Rowland‐Jones Sarah , Brooks Jill , Rickinson Alan , McMichael Andrew
Published: March 2007

We have identified two epitopes for Epstein‐Barr virus specific cytotoxic T lymphocytes (CTL) restricted by the common allele HLA‐B7. They are EBNA 3C 881–9 (QPRAPIRPI) and EBNA 3A 379–387 (RPPIFIRRL). The epitopes conform well to the recently described motif for HLA‐B7‐binding peptides (Huckzo et al., J. Immunol. 1993. 151: 2572). Titration of the peptides in CTL assays and detergent lysate binding assays revealed that extending the peptides at either the N or C terminus did not reduce their affinity for HLA‐B7. This behavior contrasted with HLA‐B51, which binds peptides with a similar motif to B7, and has identical amino acid residues at sites expected to form the “F” pocket of the peptide‐binding groove. HLA‐B51 also bound the peptide EBNA 3C 881–9, but was unable to bind peptides extended at the C terminus.
Antigen‐processing organelles from DRB1*1101 and DRB1*1104 B cell lines display a differential degradation activity
Authors: Barbey Catherine , Watts Colin , Corradin Giampietro
Published: March 2007

We have developed an in vitro assay for tetanus toxin (tt) C fragment (C‐fr) degradation. Purified endosomes (abbreviated endosomes 1101 or 1104) and lysosomes (abbreviated lysosomes 1101 or 1104) from the DRB1*1101 (Gly 86) and DRB1*1104 (Val 86) B cell lines were used to degrade 125I‐labeled C‐fr in vitro. Using three distinct methods of analysis, we show that the capacity of endosomes and lysosomes to degrade the tt C‐fr or tt synthetic Y‐P30 peptide differed. Using sodium dodecylsulfate‐polyacrylamide gel electrophoresis, 125I‐labeled C‐fr degradation patterns observed either with endosomes 1101/1104 or lysosomes 1101/1104 are distinct both in terms of the number of fragments and the kinetics of generation of the fragments. These results were confirmed by high‐performance liquid chromatography analysis, where we observed that the elution profiles of the 125I‐labeled Y‐P30 peptide digested by endosomes 1101/1104 were different compared to those obtained with lysosomes 1101/1104. Furthermore, the kinetics of degradation of 125I‐labeled Y‐P30 were faster with lysosomes 1104 than with lysosomes 1101. This difference in activity of the 1101 and 1104 organelles was also found in a functional assay where we showed that the activation capacity of the P30 peptide was diminished when digested by lysosome 1104, regardless of the antigen‐presenting cell (APC) used, whereas endosomes 1101 or lysosomes 1101 modified P30 peptide in a form that discriminated between presentation by 1101 or 1104 APC. Taken together, these results suggest that the differential processing and presentation displayed by the DRB1*1101 and DRB1*1104 APC is due partly to a different enzymatic content and partly to the dimorphism at position DRβ86.
Protein kinase C is not a downstream effector of p21ras in activated T cells
Authors: Williams David H. , Woodrow Melissa , Cantrell Doreen A. , Murray Edward J.
Published: March 2007

The aim of this present study was to investigate the role of protein kinase C (PKC), downstream of p21ras, in activating interleukin‐2 (IL‐2) gene expression. It has been reported that PKC is an effector of p21ras in T cells. Data is presented, using the potent and selective PKC inhibitor Ro 31‐8425 and transient expression of a constitutively active ras mutant, which clearly shows that PKC is not downstream of p21ras in the induction of NF‐AT and AP‐1 transcriptional activity and in the expression of IL‐2 in human Jurkat T cells. Reporter gene experiments demonstrated that NF‐χB transcriptional activity is not affected by expression of activated p21ras. The signaling pathways involving PKC activation, calcium mobilization and ras activation combine to provide the necessary components for production of IL‐2 during T cell activation.