bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin‐2 secretion by human B lymphocytes occurs as a late event and requires additional stimulation after CD40 cross‐linking
Authors: Kindler Vincent , Matthes Thomas , Jeannin Pascale , Zubler Rudolf H.
Published: March 2007

While Epstein‐Barr virus (EBV)‐immortalized B cell lines have been shown to secrete interleukin (IL)‐2 after stimulation with either teleocidin or phorbol myristate acetate (PMA) and ionomycin, experimental conditions leading to IL‐2 production by normal human B cells have not been reported. In the present study we investigated various Bcell activating conditions, including – by analogy to EBV‐immortalized B lymphocytes – stimulation of B cells that are already proliferating (in cultures with IL‐4 and immobilized anti‐CD40 monoclonal antibody; the anti‐CD40 system). This approach showed that B lymphocytes secreted IL‐2 in the culture medium, but only if they were first activated for more than 24 h in the anti‐CD40 system before exposure to PMA plus ionomycin. The production rate of IL‐2 by B lymphocytes reached a maximum after 6 days of priming in such cultures followed by 48 h of stimulation with PMA plus ionomycin, corresponding to 7% or 15% of that of fresh CD4+ T cells activated, respectively, with phytohemagglutinin plus PMA, or with PMA plus ionomycin for 48 h. This IL‐2 production could not be attributed to T cell contamination nor to EBV‐infected B cells according to flow cytometric and reverse transcriptase‐polymerase chain reaction analysis of cultured B cells. Lower IL‐2 expression (detected only as mRNA synthesis) was also induced in the cultured B lymphocytes after incubation with cross‐linking anti‐IgM antibodies instead of PMA plus ionomycin. The appearance of IL‐13 mRNA, but not IL‐4 mRNA, was detected under the same stimulation conditions as for IL‐2 mRNA. These results show that the production of IL‐2 by normal B lymphocytes occurs as a late event relative to their activation and proliferation, and is in this respect subject to regulation different to that found in T lymphocytes.
Phagocyte‐induced antigen‐specific activation of unprimed CD8+ T cells in vitro
Authors: De Bruijn Marloes L. H. , Jackson Michael R. , Peterson Per A.
Published: March 2007

The strict segregation of the major histocompatibility complex (MHC) class I and class II loading pathways has been challenged by recent reports indicating that MHC class I molecules can acquire antigen in the phagocytic pathway. We now show that this alternative peptide loading pathway can be used efficiently to generate macrophages able to activate unprimed antigen‐specific cytotoxic T cells in vitro. Short peptides (8–11 residues), administered in the phagocytic pathway at nanomolar concentrations, were found to be effective in specifically activating naïve cytotoxic T lymphocytes (CTL) in vitro, but longer peptides or whole protein antigen were not. Whole protein antigen coated on beads did, however, render macrophages susceptible to lysis by an antigen‐specific CTL clone. This indicates that proteolysis in the phagocytic pathway has limited capability for class I‐restricted presentation. We propose a model for class I loading in the phagocytic pathway consisting of direct trafficking of nascent MHC class I from the trans‐Golgi network to the phagosome, prior to appearance at the cell surface, and the use of the narrow cavity between bead and phagosomal membrane as a peptide exchange/loading compartment. Targeting immunogenic class I‐binding peptide to the phagocytic pathway of macrophages facilitates presentation in association with class I. This is a useful tool for CTL response induction in vitro.
T lymphocyte development in p56lck deficient mice: allelic exclusion of the TcR β locus is incomplete but thymocyte development is not restored by TcR β or TcR αβ transgenes
Authors: Wallace Valerie A. , Kawai Kazuhiro , Levelt Christiaan N. , Kishihara Kenji , Molina Thierry , Timms Emma , Pircher Hanspeter , Penninger Josef , Ohashi Pamela S. , Eichmann Klaus , Mak Tak W.
Published: March 2007

The protein tyrosine kinase, p56lck, is involved in signal transduction in mature T cells and in the molecular events controlling early thymocyte differentiation. Thymuses of mice deficient for p56lck expression (p56lck‐/‐) consist of immature CD4‐CD8‐ double‐negative (DN) and CD4+CD8+ double‐positive (DP) thymocytes and are severely reduced in total cell number. In this report we have studied DN thymocytes from p56lck‐/‐ mice and found an increase in the proportion of the CD44−CD25+ subset, suggesting that transit through this stage, which is known to require T cell receptor (TcR) β expression, may be delayed in the absence of p56lck expression. In addition, the expression of a transgenic TcR β chain or TcR αβ pair did not restore thymic development in p56lck‐/‐ mice. However, in contrast to mice expressing a dominant negative isoform of p56lck in which DP thymocytes do not develop, DP thymocytes still develop in nontransgenic and TcR transgenic p56lck‐/‐ mice. These results demonstrate that expansion of the DP subset is impaired in p56lck‐/‐ mice. In contrast, allelic exclusion is not severely compromised. Although there was an increase in the number of peripheral T cells expressing more than one Vβ chain in TcR transgenic p56lck‐/‐ mice, we found that inhibition of endogenous TcR β gene rearrangement was almost complete in thymocytes of Vβ transgenic p56lck‐/‐ mice and we could not detect any peripheral T cells that expressed more than one Vβ chain in non‐transgenic p56lck‐/‐ mice.
Restoration of endogenous antigen processing in Burkitt's lymphoma cells by Epstein‐Barr virus latent membrane protein‐1: coordinate up‐regulation of peptide transporters and HLA‐class I antigen expression
Authors: Rowe Martin , Khanna Rajiv , Jacob Catherine A. , Argaet Victor , Kelly Adrian , Powis Stephen , Belich Monica , Groom‐Carter Deborah , Lee Steve , Burrows Scott R. , Trowsdale John , Moss Denis J. , Rickinson Alan B.
Published: March 2007

Group I Burkitt lymphoma (BL) lines retaining the original BL tumor cell phenotype are unable to present endogenously expressed antigens to HLA class I‐restricted cytotoxic T cells (CTL) but can be recognized if the relevant HLA class I/peptide epitope complex is reconstituted at the cell surface by exogenous addition of synthetic target peptide. Endogenous antigen‐processing function is restored in BL lines that have undergone Epstein‐Barr virus (EBV)‐induced drift in culture to the group III phenotype typically displayed by EBV‐transformed lymphoblastoid cell lines (LCL) of normal B cell origin. We compared group I versus group III cells for their expression of proteasome components, transporter proteins and HLA‐class I antigens, all of which are thought to be involved in the endogenous antigen processing pathway. By Western blot analysis, there were not consistent differences in the low molecular mass protein subunits of proteasomes (lmp)‐2, lmp‐7 and δ, although the mb‐1 proteasome subunit was regularly present at higher levels in group I BL lines relative to group III lines or LCL. By contrast there were marked differences in the expression of peptide transporter‐associated proteins (Tap), with down‐regulation of Tap‐1 and Tap‐2 in 8/8 and 7/8 group I BL lines, respectively. Surface levels of HLA class I antigens were also consistently lower in group I cells; this was not associated with an intracellular accumulation of free HLA heavy chains, such as is seen in the Tap‐deficient T2 processing‐mutant line, but instead reflected a reduced rate of HLA class I synthesis in group I cells. Analysis of EBV gene transfectants of the B lymphoma lines BJAB and BL41 showed that the virus‐encoded latent membrane protein‐1 (LMP1), which is one of several EBV antigens expressed in group III but not in group I cells, was uniquely able to up‐regulate expression both of the Tap proteins and HLA class I. Furthermore, this was accompanied by a restoration of antigen‐processing function as measured by the ability of these cells to present an endogenously expressed viral antigen to CTL. These effects of LMP1 were similar to those induced in the same cell lines by interferon‐γ treatment. The results implicate both Tap and HLA class I expression as factors limiting the antigen‐processing function of BL cells, and suggest that the accessibility of other EBV‐associated malignancies to CTL surveillance may be critically dependent upon their LMP1 status.
Oral tolerance to haptens: intestinal epithelial cells from 2,4‐dinitrochlorobenzene‐fed mice inhibit hapten‐specific T cell activation in vitro
Authors: Galliaerde Veronique , Desvignes Cyril , Peyron Eric , Kaiserlian Dominique
Published: March 2007

The mechanisms underlying the induction of immunological tolerance after feeding soluble exogenous antigens, including proteins and haptens, are still unclear. Using a model of oral tolerance to the contact‐sensitizing hapten 2,4‐dinitrochlorobenzene (DNCB), we have compared the ability of intestinal epithelial cells and of Peyer's patch APC to present DNCB in vitro or ex vivo after oral feeding, to specific peripheral lymph node T cells from DNCB‐sensitized mice. In contrast to Peyer's patch APC, which induce efficient hapten‐specific T cell activation upon exposure to the hapten either in vitro or in vivo, mature MHC class‐II‐positive intestinal epithelial cells were unable to induce T cell activation in either case. Interestingly, enterocytes from DNCB‐fed mice exerted a dramatic inhibitory effect on the proliferative response of hapten‐primed T cells in response to dinitrobenzene sulfonate presented by syngeneic spleen cells. This inhibitory effect, which was also observed with supernatant of intestinal epithelial cells from DNCB‐fed mice, could be reversed by neutralizing anti‐transforming growth factor (TGF)‐β antibodies. In addition, pre‐incubation of hapten‐sensitized T cells with enterocytes from DNCB‐fed mice induced T cell anergy, which could be reversed by exogenous interleukin‐2 or interleukin‐4. These data demonstrate that intestinal epithelial cells activated in vivo by oral administration of DNCB are able to block proliferation of activated T cells through secretion of immunosuppressive cytokines such as TGF‐β. It is proposed that intestinal epithelial cells may play a significant role in oral tolerance by limiting T cell‐mediated hypersensitivity responses.
CD28/CTLA‐4 ligands: the gene encoding CD86 (B70/B7.2) maps to the same region as CD80 (B7/B7.1) gene in human chromosome 3q13‐q23
Authors: Fernández‐Ruiz Elena , Somoza Chamorro , Sánchez‐Madrid Francisco , Lanier Lewis L.
Published: March 2007

CD86 (B70/B7.2) is an antigen of the immunoglobulin superfamily expressed on monocytes, dendritic cells and activated B,T, and natural killer cells. CD86 was recently identified as a second ligand for the T cell antigens CD28 and CTLA‐4, and plays an important role in the co‐stimulation of T cells in a primary immune response. We report here the assignment of the CD86 gene to human chromosome 3 using Southern blot analysis on a panel of hamster × human somatic cell hybrid genomic DNA. Fluorescence hybridization in situ on metaphase chromosomes coupled with GTG banding (G‐bands by trypsin using Giemsa staining) confirmed this assignment and localized the CD86 gene to 3q13‐q23 region. The CD86 gene is, therefore, located in the proximity of the CD80 (B7/B7.1) gene, the first identified ligand for CD28 and CTLA‐4, previously mapped to chromosome 3q13.3‐q21. Deletions, inversions and insertions of chromosome 3q21‐q26, as well as translocations of 3q21 with other chromosomes have been described in many cases of acute myeloid leukemia (AML), acute nonlymphocytic leukemia (ANLL), chronic myeloid leukemia (CML) and myelodisplastic syndromes (MDS), suggesting that this region contains several genes involved in the leukemic process.
Selection of T cell receptor variable gene‐encoded amino acids on the third binding site loop: a factor influencing variable chain selection in a T cell response
Authors: Callan Margaret F. C. , Reyburn Hugh T. , Bowness Paul , Rowland‐Jones Sarah , Bell John I. , McMichael Andrew J.
Published: March 2007

The 3′ end of the T cell receptor Vβ7.1 gene contains the five nucleotides CAAGA between the broadly conserved consensus sequence of nucleotides TGC/T GCC AGC AGC (which encode cysteine, alanine, serine and serine at positions 92–95 of the β chain) and the heptamer that signals rearrangement. These nucleotides are frequently preserved during gene rearrangement, resulting in the common presence of glutamine at position 96 and of aspartate or glutamate at position 97 of the Vβ7.1 chain CDR3 loop in peripheral blood lymphocytes. There is selection of Vβ7.1 and of the Vβ7.1 gene‐encoded glutamate at position 97 of the β chain CDR3 loop in the cytotoxic T lymphocyte response to the HLA B2705‐restricted influenza A nucleoprotein epitope SRYWAIRTR. Our results indicate that selection of Vβ7.1 gene‐encoded amino acid residues on CDR3 loops may be one factor driving selection of Vβ7.1 in this response.
Phosphotyrosine‐dependent association between CD22 and protein tyrosine phosphatase 1C
Authors: Campbell Mary‐Ann , Klinman Norman R.
Published: March 2007

CD22 is a B lymphocyte‐specific cell surface glycoprotein that becomes tyrosine phosphorylated upon B cell activation. To determine if tyrosine phosphorylated CD22 couples signaling through membrane immunoglobulin (mIg) to downstream elements, we looked for molecules coprecipitating with CD22 after anti‐Ig stimulation. We found that a 60‐kDa molecule was stably associated with CD22 following cross‐linking of mIg and have identified this molecule as protein tyrosine phosphatase 1C (PTP1C). The association between PTP1C and CD22 is dependent upon tyrosine phosphorylation of CD22, but does not appear to require tyrosine phosphorylation of PTP1C.
The interaction of beta 2‐microglobulin (β2m) with mouse class I major histocompatibility antigens and its ability to support peptide binding. A comparison of human and mouse β2m
Authors: Pedersen Lars Ø. , Stryhn Anette , Holtet Thor L. , Etzerodt Mikael , Gerwien Jens , Nissen Mogens H. , Thøgersen Hans C. , Buus Seren
Published: March 2007

The function of major histocompatibility complex (MHC) class I molecules is to sample peptides derived from intracellular proteins and to present these peptides to CD8+ cytotoxic T lymphocytes. In this paper, biochemical assays addressing MHC class I binding of both peptide and β2‐microglobulin (β2m) have been used to examine the assembly of the trimolecular MHC class I/β2m/peptide complex. Recombinant human β2m and mouse β2m2 have been generated to compare the binding of the two β2m to mouse class I. It is frequently assumed that human β2m binds to mouse class I heavy chain with a much higher affinity than mouse β2m itself. We find that human β2m only binds to mouse class I heavy chain with slightly (about 3‐fold) higher affinity than mouse β2m. In addition, we compared the effect of the two β2m upon peptide binding to mouse class I. The ability of human β2m to support peptide binding correlated well with its ability to saturate mouse class I heavy chains. Surprisingly, mouse β2m only facilitated peptide binding when mouse β2m was used in excess (about 20‐fold) of what was needed to saturate the class I heavy chains. The inefficiency of mouse β2m to support peptide binding could not be attributed to a reduced affinity of mouse β2m/MHC class I complexes for peptides or to a reduction in the fraction of mouse β2m/MHC class I molecules participating in peptide binding. We have previously shown that only a minor fraction of class I molecules are involved in peptide binding, whereas most of class I molecules are involved in β2m binding. We propose that mouse β2m interacts with the minor peptide binding (i.e. the “empty”) fraction with a lower affinity than human β2m does, whereas mouse and human β2m interact with the major peptide‐occupied fraction with almost similar affinities. This would explain why mouse β2m is less efficient than human β2m in generating the peptide binding moiety, and identifies the empty MHC class I heavy chain as the molecule that binds human β2m preferentially.
Soluble form of the human high‐affinity receptor for IgE inhibits recurrent allergic reaction in a novel mouse model of type I allergy
Authors: Naito Koji , Hirama Minoru , Okumura Ko , Ra Chisei
Published: March 2007

The recombinant soluble form of the human high‐affinity receptor for IgE α subunit (sFcϵRIα) is able to block the binding of IgE to the cell surface FcϵRI and prevent the activation of mast cells and basophils. To evaluate its anti‐allergic effects in vivo, we established a novel model for type I allergy by transplanting antigen‐specific IgE‐secreting hybridomas into syngeneic mice. The hybridomas continuously produced anti‐2,4,6‐trinitrophenyl IgE in vivo, and ear swelling responses could be elicited upon applying picryl chloride to the ears of these mice, which reached their maximum 1–2 h after the antigen challenge. The second swelling response, in extent comparable to the first response, was induced by the second antigen challenge several days later. When the sFcϵRIα was intravenously administered before the first challenge and periodically betweeen the first and the second challenges, the ear swelling response to the second challenge, but not to the first challenge, was suppressed almost completely. Immunohistochemical examination revealed that treatment with the sFcϵRIα blocked the binding of IgE to the cell surface IgE receptors after the first challenge. Thus, our results indicate that the sFcϵRIα suppresses recurrent allergic reactions by preventing IgE binding to the cell surface FcϵRI.
Invriant chain peptides enhancing or inhibiting the presentation of antigenic peptides by major histocompatibility complex class II molecules
Authors: Adams Sharlene , Humphreys Robert E.
Published: March 2007

Two soluble invariant chain (Ii) peptides with overlapping sequences had contrasting effects on the presentation of antigenic peptides by murine Ad, Ak, Ed, and Ek major histocompatibility complex (MHC) class II molecules. Naturally produced class II‐associated invariant chain peptides human (h)Ii81–104/murine (m)Ii80–103 inhibited antigen presentation on these MHC class II alleles in a manner consistent with competitive inhibition. The Ii‐4 peptides hIi77–92/mIi76–91 enhanced presentation of antigenic peptides on I‐E class II alleles by promoting the exchange of peptides at the cell surface. Treatment of antigenpresenting cells (APC) with Ii‐4 before the addition of antigenic peptide greatly enhanced subsequent T cell responses, while treatment of APC with Ii–4 after antigenic peptide binding decreased subsequent T cell responses. The hIi81–104 and mIi80–103 peptides inhibited T cell responses in both types of assays. The binding of biotinylated antigenic peptide to MHC class II‐transfected L cells, as measured by flow cytometry, was inhibited by mIi80‐103 and enhanced by mIi‐4. Segments of Ii fragments remaining associated with MHC class II, or released Ii peptides, appear to regulate the formation of stable antigenic peptide/MHC class II complexes either positively or negatively through interactions at or near the antigenic peptide binding site. These findings open a pathway for the design of novel therapeutics based on the structure and function of natural and rationally designed fragments of Ii.
Expression and functional significance of an activation‐dependent epitope of the β1 integrins in chronic inflammatory diseases
Authors: Arroyo Alicia G. , García‐Vicuña Rosario , Marazuela Mónica , Yednock Ted A. , González‐Amaro Roberto , Sánchez‐Madrid Francisco
Published: March 2007

The avidity of VLA integrins for their ligands can be increased by their transition to an active conformational state. This conformational change can be detected with a novel monoclonal antibody (mAb), termed 15/7, that recognizes an activation‐dependent conformational epitope on the common β1 polypeptide of different VLA αβ1 integrins. In an attempt to understand the possible role of the active conformational state of β1 integrins in vivo, we first investigated the expression of 15/7 epitope on T lymphocytes from patients with chronic inflammatory joint diseases. An enhanced expression of the 15/7 epitope was found in the synovial fluid (SF) T lymphocytes from these patients as compared to their peripheral blood (PB) T cells. The effect of different cytokines on the appearance of the 15/7 activation epitope in PB T lymphocytes was subsequently analyzed; interferon‐γ, interleukin‐2 and, to a lower extent, tumor necrosis factor‐α were able to induce an increased expression of the 15/7 epitope. This enhanced 15/7 expression correlated with a higher binding ability to fibronectin of cytokine‐activated T cells. The presence of this activation epitope was detected in a small proportion of T lymphocytes scattered within inflammatory foci of synovial membrane from rheumatoid arthritis and thyroid glands from Hashimoto's chronic thyroiditis. We then analyzed the possible role of 15/7 epitope expression on cell adhesion in vitro. Immunofluorescence studies showed that the 15/7 epitope displayed a spot‐like distribution, selectively decorating adhesive contacts of U‐937 myelomonocytic cells attached to the 80 kDa proteolytic fragment of fibronectin (FN80). Furthermore, the anti‐β1 15/7 mAb was able to induce both T lymphocyte, Jurkat and U‐937 cellular binding and spreading on FN80. Altogether these results indicate that an activated conformation of β1 integrins is detected in vivo in lymphocyte infiltrates from chronic inflammatory conditions. The active conformations of β1 integrins are regulated by physiologic mediators such as cytokines, play an important role in cellular attachment and spreading, and appear to be involved in the development of inflammatory processes.
TAP1‐independent loading of class I molecules by exogenous viral proteins
Authors: Bachmann Martin F. , Oxenius Annette , Pircher Hanspeter , Hengartner Hans , Ashton‐Richardt Philip A. , Tonegawa Susumu , Zinkernagel Rolf M.
Published: March 2007

Presentation of peptides derived from endogenous proteins on class I molecules needs functional TAP peptide transporters. To reveal whether class I‐associated presentation of exogenous proteins also required the presence of TAP transporters, we assessed in vitro the ability of spleen cells and macrophages from TAP1‐deficient mice (TAP1−/−) to present peptides derived from exogenous recombinant viral proteins on their class I molecules. We found that recombinant glycoand nucleoprotein from lymphocytic choriomeningitis virus and nucleoprotein of vesicular stomatitis virus were presented as efficiently by TAP1 −/− cells as by control cells. Peptide regurgitation was not involved. Since particulate, nonreplicating antigens can efficiently prime anti‐viral cytotoxic T cells in vivo, this new, TAP‐independent pathway of class I‐associated antigen presentation may be applicable for vaccine strategies.
Cloning of ATAC, an activation‐induced, chemokine‐related molecule exclusively expressed in CD8+ T lymphocytes
Authors: Müller Sabine , Dorner Brigitte , Korthäuer Ulf , Mages Hans W. , D'Apuzzo Massimo , Senger Gabriele , Kroczek Richard A.
Published: March 2007

A cDNA clone, designated ATAC, was isolated from a collection of human T cell activation genes. Analysis of tissue distribution determined that ATAC mRNA of approxiamtely 0.9 kb is exclusively expressed in activated CD8+ T cells. Induction of the ATAC gene requires stimulation by both phorbol 12‐myristate 13‐acetate and Ca2+ ionophore A23187 (“two‐signal gene”) and is fully abrogated by the immunosuppressive agent cyclosporin A. Upon stimulation, ATAC mRNA is detectable within 30 min, maximal expression is seen after 4 h. The polypeptide encoded by the open reading frame of ATAC mRNA is 114 amino acids long with a calculated Mr of 12.52 kDa. The structural features predict the cleavage and secretion of a mature ATAC protein of approximately 10 kDa from the 12.52‐kDa precursor. ATAC is highly similar to a very recently identified murine molecule designated lymphotactin both at the cDNA (73.8% identity) and the protein (61.4% identity) levels, and related to members of the C‐C and C‐X‐C chemokine families. Two variants of the ATAC protein were expressed and tested for chemotaxis and Ca2+ release on a variety of target cells. The ATAC gene was located to chromosome 1q23.
A soluble form of TRAP (CD40 ligand) is rapidly released after T cell activation
Authors: Graf Daniel , Müller Sabine , Korthäuer Ulf , van Kooten Cees , Weise Christoph , Kroczek Richard A.
Published: March 2007

TRAP is a tumor necrosis factor (TNF)‐related, 33‐kDa type II transmembrane protein almost exclusively expressed on the surface of activated CD4+ T lymphocytes. Interaction of TRAP with CD40 on B cells is of paramount importance for immunoglobulin class switching and subsequent synthesis of IgG, IgA or IgE in vivo. We now provide evidence that activated T cells not only express cell membrane‐associated TRAP but also a soluble form of TRAP (sTRAP). After generating monoclonal antibodies against TRAP and establishing a TRAP‐specific enzyme‐linked immunosorbent assay we were able to detect substantial amounts of sTRAP in the supernatants of activated T cells. The onset and rate of sTRAP release was found to parallel the expression of TRAP on the cell surface. sTRAP, an 18‐kDa protein, is generated by proteolytic proccessing of full‐length TRAP in an intracellular compartment. Starting with methionine 113 of fulllength TRAP, sTRAP lacks the transmembrane region and a part of the extracellular domain but contains the entire TNF‐α homology region and can, therefore, bind to CD40. Like other members of the TNF superfamily (e.g. TNF‐α, Fas/APO‐1 ligand), TRAP thus has the potential to be biologically active not only in a transmembrane form but also as a soluble molecule.
Immunodominance with progenitor B cell diversity in the neutralizing antibody repertoire to influenza infection
Authors: Patera Andriani C. , Graham Christine M. , Thomas D. Brian , Smith Claire A.
Published: March 2007

We report striking immunodominance in the neutralizing antibody responses of major histocompatibility complex congenic mice to natural infection with influenza virus (H3N2 subtype), as deduced by sequencing the hemagglutinin (HA) genes of monoclonal antibody (mAb)‐selected mutant viruses. A majority of mAb, established from individual BALB/c (H‐2d) mice, select mutant viruses containing the same single amino acid substitution in the membrane distal ectodomain, HA1 198 A→E, whereas changes at either HA1 158 G→E or HA1 198 A→E are selected for by mAb from BALB.K (H‐2k) donors. The structural basis for immunodominance, and potential diversity of progenitor B cells, was investigated by sequence analysis of H and L chain gene rearrangements in mAb specific for HA1 158 or HA1 198. No correlation was found between antibody specificity and VH or VL gene usage, and a minimum of three to six progenitor cells contributed to the individual's repertoire for a single antigenic site. However, in a further analysis of the HA1 158‐specific antibody response of CBA/Ca (H‐2k) donors, there was highly restricted light chain gene usage. Focusing of the immune repertoire to limited regions of the HA molecule during a primary viral infection may be a significant factor in immune pressure for antigenic variation, particularly since there is no evident restriction in the antibody response to immunization.
Distinct signaling properties identify functionally different CD4 epitopes
Authors: Baldari Cosima T. , Milia Elisabetta , di Somma M. Maddalena , Baldoni Fabiana , Valitutti Salvatore , Telford John L.
Published: March 2007

The CD4 coreceptor interacts with non‐polymorphic regions of major histocompatibility complex class II molecules on antigen‐presenting cells and contributes to T cell activation. We have investigated the effect of CD4 triggering on T cell activating signals in a lymphoma model using monoclonal antibodies (mAb) which recognize different CD4 epitopes. We demonstrate that CD4 triggering delivers signals capable of activating the NF‐AT transcription factor which is required for interleukin‐2 gene expression. Whereas different anti‐CD4 mAb or HIV‐1 gp120 could all trigger activation of the protein tyrosine kinases p561ck and p59fyn and phosphorylation of the Shc adaptor protein, which mediates signals to Ras, they differed significantly in their ability to activate NF‐AT. Lack of full activation of NF‐AT could be correlated to a dramatically reduced capacity to induce calcium flux and could be complemented with a calcium ionophore. The results identify functionally distinct epitopes on the CD4 coreceptor involved in activation of the Ras/protein kinase C and calcium pathways.
Characterization of critical residues in the cytoplasmic domain of the human interleukin‐5 receptor α chain required for growth signal transduction
Authors: Cornelis Sigrid , Fache Ina , van der Heyden José , Guisez Yves , Tavernier Jan , Devos René , Fiers Walter , Plaetinck Geert
Published: March 2007

Interleukin (IL)‐5 binds to a cell surface receptor composed of two polypeptide chains, α and β, both belonging to the hemopoietic cytokine receptor family. Mouse cells expressing common mouse β chain (AIC2B) that were transfected with human IL‐5 receptor (R)α cDNA proliferated in response to picomolar concentrations of human IL‐5, indicating that a functional receptor was reconstituted. We show that in these cells, human (h)IL‐5 as well as mouse (m)IL‐3 induce tyrosine phosphorylation of β chain and JAK 2 kinase. Phosphorylated β receptor was co‐precipitated with anti‐JAK 2 antibodies, suggesting that both molecules were physically associated. IL‐5 and IL‐3 also induce cytosolic DNA binding activity as measured by an electrophoretic mobility shift assay using the interferon‐γ responsive region of human Fc γ1 gene DNA element. A deletion mutant of hIL‐5Rα lacking the cytoplasmic part could bind hIL‐5 normally in association with the β chain, but was unable to transmit a biological signal. The cytoplasmic domain was also indispensable for tyrosine phosphorylation and activation of DNA binding proteins. A membrane‐proximal proline‐rich element of the hIL‐5Rα cytoplasmic domain that is conserved among different members of the hemopoietic cytokine receptor family was essential for biological activity. Point mutations in this motif also knocked out IL‐5‐inducible JAK 2 phosphorylation.
CD20 monoclonal antibodies decrease interleukin‐4‐stimulated expression of the low‐affinity receptor for IgE (FcϵRII/CD23) in human B cells by increasing the extent of its cleavage
Authors: Bourget Isabelle , di Berardino Wilma , Breittmayer Jean‐Philippe , Grenier‐Brossette Nicole , Plana‐Prades Montserrat , Bonnefoy Jean‐Yves , Cousin Jean‐Louis
Published: March 2007

CD20 monoclonal antibody (mAb) B1 is known to inhibit B cell proliferation. We show that B1 reduced both anti‐μ + interleukin‐4 (IL‐4)‐induced DNA synthesis and the concomitant expression of CD23 at the surface of human tonsillar B cells. B1 mAb had no effect on CD23 mRNA levels. The disappearance of CD23 molecule from the surface correlates with an increase of soluble CD23 fragments in the culture medium, indicating that CD20 mAb B1 stimulated the cleavage of the molecule. B1 also inhibits IgE production by peripheral blood mononuclear cells cultured in the presence of IL‐4. Suppression of IgE synthesis and enhancement of CD23 cleavage are concomitant but appear not to be functionally related.
Intra‐ and extra‐thymic expression of the pre‐T cell receptor α gene
Authors: Bruno Ludovica , Rocha Benedita , Rolink Antonius , von Boehmer Harald , Rodewald Hans‐Reimer
Published: March 2007

We have analyzed pre‐T cell receptor α (pTα) gene expression in cells from various anatomical sites to investigate the lineage specificity of pTα RNA as well as its presence in pro‐T cells and in sites of extrathymic T cell development. pTα RNA is found in precursors of αβ T cells but is absent from mature αβ T cells as well as T cells that express the γδ T cell receptor on the cell surface. pTα expression is exquisitely T lineage specific in that mature and immature B cells, myeloid cells, NK cells and pluripotent stem cells are pTα negative. On the other hand, pTα expression is found in pro‐T cells outside the thymus as well as in intra‐ and extra‐thymic sites of T cell development. The latter finding is consistent with the notion that early steps of T cell development within and outside the thymus may be similar.
Preferential recognition of specific DNA motifs by anti‐double‐stranded DNA autoantibodies
Authors: Herrmann Martin , Winkler Thomas H. , Fehr Holger , Kalden Joachim Robert
Published: March 2007

Although antibodies (Ab) specific for double‐stranded (ds) DNA are thought to be involved in the etiopathogenesis of systemic lupus erythematosus (SLE), the fine structure of their DNA targets remains elusive. We have adapted a polymerase chain reaction (PCR)‐assisted immunoprecipitation method to define the binding sites in DNA sequences recognized by high affinity anti‐dsDNA Ab of SLE patients. SLE sera were used to bind templates from a pool of double‐stranded oligonucleotides (ON). A central part of 20 base‐pair random sequence was flanked by restriction endonuclease recognition sites and sequences complementary to predefined PCR primers. Immunoselected ON were precipitated, isolated from the immune complexes and then subjected to a further immunoprecipitation step after amplification by PCR. After five cycles of immunoprecipitation and PCR, the resulting ON were cloned. Sequence analysis revealed that sera from SLE patients and two human monoclonal anti‐dsDNA Ab obtained from SLE patients preferentially select sequences expected to form non‐B‐DNA structures. Inhibition studies of the Farr assay confirmed the increased affinity of the selected epitopes for anti‐DNA Ab as compared to random B‐DNA.
Prevention and treatment of Lewis rat experimental allergic encephalomyelitis with a monoclonal antibody to the T cell receptor Vβ8.2 segment
Authors: Imrich Horst , Kugler Christine , Torres‐Nagel Nora , Dörries Rüdiger , Hünig Thomas
Published: March 2007

The predominance of T cell receptor (TCR) Vβ8.2 utilization by encephalitogenic T cells induced in Lewis rats by immunization with myelin basic protein (MBP) is controversial. Thus, both an almost exclusive usage of Vβ8.2 [Burns, F. R., Li, X., Shen, N., Offner, H., Chou, Y. K., Vandenbark, A. A. and Heber‐Katz, E., J. Exp. Med. 1989. 169: 27; Chluba, J., Steeg, C., Becker, A., Wekerle, H. and Epplen, J. T., Eur. J. Immunol. 1989. 19: 279] and a quite diverse Vβ composition of CD4 T cells causing experimental autoimmune encephalomyelitis (EAE) [Sun, D., Gold, P. D., Smith, L., Brostoff, S. and Coleclough, C., Eur. J. Immunol. 1992. 22: 591; Sun, D., Le, J. and Coleclough, C., Eur. J. Immunol. 1993. 23: 494] have been reported. Using a recently developed monoclonal antibody (mAb) specific for TCR Vβ8.2, we show that postnatal treatment effectively eliminates Vβ8.2‐bearing cells and prevents MBP‐induced EAE in the majority of Lewis rats. Moreover, treatment of adult Lewis rats with Vβ8.2‐specific mAb as late as on day 12 after MBP immunization suppressed the development of neurological symptoms. Thus, Vβ8.2‐bearing cells do play a decisive role in Lewis rat EAE, and suppression of the small (5%) Vβ8.2‐expressing T cell subset provides an effective therapeutic strategy.
Self‐reactive anti‐class II T helper type 2 cell lines derived from gold salt‐injected rats trigger B cell polyclonal activation and transfer autoimmunity in CD8‐depleted normal syngeneic recipients
Authors: Saoudi Abdelhadi , Castedo Maria , Nochy Dominique , Mandet Chantal , Pasquier Régine , Druet Philippe , Pelletier Lucette
Published: March 2007

Brown Norway (BN) rats given gold salts develop an autoimmune syndrome with an immune complex‐type glomerulonephritis in the context of a polyclonal B cell activation that was suspected to be due to the emergence of anti‐self major histocompatibility complex (MHC) class II T cells. In the present study, six anti‐self MHC class II T cell lines have been derived from six gold salttreated rats by repeated stimulations with normal syngeneic MHC class II‐bearing cells. The T cell lines proliferated in the presence of self MHC class II‐positive B cell‐enriched or B cell‐depleted cells and the proliferation was inhibited by preincubating stimulator cells with an anti‐IA monoclonal antibody. The T cell lines produced interleukin (IL)‐4 only or IL‐4 and some interferon (IFN)‐γ and could, therefore, be considered as T helper type 2 (Th2) and Th0 cells, respectively. They triggered normal syngeneic B cells to produce in vitro IgE, anti‐DNA, anti‐laminin and anti‐2,4‐6‐trinitrophenol antibodies through, at least in part, cognate interactions. More interestingly, these lines when transferred into normal BN rats induced an autoimmune syndrome similar to or even more severe than the one observed in the active gold model, provided the recipients were CD8 depleted. These manifestations included a dramatic increase in serum IgE concentration and the production of anti‐DNA and anti‐laminin antibodies. In addition, all recipients displayed an autoimmune glomerulonephritis due to anti‐laminin antibodies, granular IgG deposits in the interstitium, in the vessel walls and along the tubular basement membranes and a severe tubulointerstitial nephritis with marked mononuclear cell infiltration. An anti‐ovalbumin T cell line that produced IL‐4 and low amounts of IFN‐γ was used as a control and did not induce autoimmunity. These results demonstrate for the first time the ability of autoreactive Th2 as well as Th0 cell lines to induce antibody‐mediated autoimmunity. They also show that CD8+ cells play a crucial role in the control of such autoreactive cells. Finally, this work suggests that Th2 cells could initiate cell‐mediated reactions either directly or indirectly.
The role of lymphocyte subsets and adhesion molecules in T cell‐dependent cytotoxicity mediated by CD3 and CD28 bispecific monoclonal antibodies
Authors: Renner Christoph , Jung Wolfram , Sahin Ugur , van Lier Rene , Pfreundschuh Michael
Published: March 2007

The cure of human Hodgkin's tumors heterotransplanted into SCID mice can be achieved by two bispecific monoclonal antibodies (Bi‐mAb) directed against the tumor‐associated CD30 antigen and CD3 and CD28, respectively, and normal peripheral human blood T cells. We investigated the role of lymphocyte subsets and adhesion molecules in this Bi‐mAb‐mediated cytolysis. CD4+ lymphocytes were the most rapidly expanding subpopulation, but Bi‐mAb‐directed cytotoxicity was mediated preferentially by CD8+ lymphocytes and effector cells belonging to the CD45RO+ “memory” pool. Blocking of the LFA‐1/ICAM‐1 or CD2/LFA‐3 adhesion pathways by mAb decreased Bi‐mAb‐mediated cytotoxicity. This was not due to inhibition of aggregate formation between Bi‐mAb‐coated T lymphocytes and target cells. Cross‐linking of LFA‐1 or CD2 molecules on lymphocytes prestimulated with Bi‐mAb bound to CD3 and CD28 antigen lead to a more pronounced and prolonged rise in the intracellular concentration of free Ca2+. Additional CD2 cross‐linking resulted in the tyrosine phosphorylation of distinct proteins. These findings indicate that adhesion molecules play a critical role and function as co‐stimulatory signals rather than as cellular contact mediators in CD3 and CD28 Bi‐mAb‐stimulated T lymphocytes.
Spreading of the immune response to different myelin basic protein peptides in chronic experimental autoimmune encephalomyelitis in B10.RIII mice
Authors: Jansson Liselotte , Diener Per , Engström Åke , Olsson Tomas , Holmdahl Rikard
Published: March 2007

B10.RIII mice develop chronic and relapsing experimental autoimmune encephalomyelitis (EAE) after immunization with the myelin basic protein (MBP) peptide 89‐101 (VHFFKNIVTPRTP). To investigate the basis for the chronicity of the disease, the subsequent development of an immune responses to other parts of the MBP protein were investigated. Onset of disease occurs 9‐25 days after immunization with MBP89‐101. T cell responses towards a series of MBP peptides were assessed in an enzyme‐linked immunospot assay detecting single cells secreting IFN‐γ. There were responses not only to MBP89‐101, but also towards peptides derived from sequences outside of MBP89‐101. These peptides were of two kinds: those with sequences completely outside the 89‐101 stretch of MBP; and those sharing a short sequence with MBP89‐101 depending on alternative splicing of MBP mRNA. Immunization with these peptides also produced chronic EAE and a spreading of the immune response to other MBP peptides. Immunization with stepped peptides around the relevant region (MBP87‐110) showed that peptides sharing a 6‐amino‐acid motif induced EAE after immunization. After MBP 89‐101 peptide immunization, T cells isolated from lymph nodes did not cross‐react in vitro to the other peptides sharing this motif. We suggest that one mechanism for the development of relapses during the disease course is the recruitment of new T cells with specificity for MBP peptides not derived from the peptide used for immunization. This is the first time such a mechanism has been demonstrated in a chronic autoimmune disease model.
Processing of a viral glycoprotein in the endoplasmic reticulum for class II presentation
Authors: Bartido Shirley M. , Diment Stephanie , Reiss Carol S.
Published: March 2007

Endogenous processing of viral glycoproteins for presentation to CD4+ T cells is a poorly investigated aspect of antigen processing and presentation. This pathway may involve not only pathogens, but also self proteins, and may thus be involved in self‐tolerance. We have characterized the processing of the endoplasmic reticulum‐restricted glycoprotein (G) of vesicular stomatitis virus, termed poison tail (Gpt), biochemically and enzymatically, and by T cell recognition assays. Expressed with a vaccinia vector, Gpt remains endoglycosidase H‐sensitive and does not mature to endoglycosidase D sensitivity. The protein is degraded in the ER with a T1/2 of 4 h. Gpt peptides are not secreted since Gpt‐infected cells are unable to sensitize uninfected antigen‐presenting cells in an innocent bystander assay. Using flow cytometry, Gpt is undetectable on the plasma membrane; in contrast, wild‐type G is readily found on the surface or secreted into the milieu as soluble G following infection of A20 cells with a vaccinia recombinant expressing G. The degradation of Gpt is sensitive to the thiol reagent diamide and occurs optimally at physiological pH. A series of proteolytic inhibitors were tested: 3,4‐dichloroisocoumarin and 1‐chloro‐3‐tosylamido‐7‐amino‐2‐heptanone inhibited degradation, which suggests the involvement of a serine protease. The degradation does not require transport to the Golgi complex, and is not sensitive to a variety of lysosomotropic agents. We show that the degradation products include the immunogenic epitopes recognized by a panel of T cell clones and hybridomas.
Self‐reactive T cell clones in a restricted population of interleukin‐2 receptor β+ cells expressing intermediate levels of the T cell receptor in the liver and other immune organs
Authors: Kawachi Yasuyuki , Watanabe Hisami , Moroda Tetsuya , Haga Manabu , Liai Tsuneo , Hatakeyama Katsuyoshi , Abo Toru
Published: March 2007

T cells expressing high levels of the T cell receptor (TCRhigh) differentiate in the major intrathymic pathway and then distribute to the peripheral immune organs, whereas T cells expressing intermediate levels of the TCR (TCRint) differentiate in both extrathymic pathways and an alternative intrathymic pathway and localize in unique sites, including the liver and thymic medulla. Since TCRint cells constitutively express interleukin‐2 receptor β‐chain (IL‐2R), two‐color staining for CD3 (or TCR) and IL‐2Rβ clearly distinguished IL‐2RßT+ CD3int (or TCRint) cells from IL‐2Rβ−, CD3high cells. CD3int cells may be considered to be primordial T cells based on their phenotype, morphology and other functional properties. In this study, using anti‐Vβ mAb in conjunction with the endogenous superantigen Mls, the distribution of self‐reactive clones among T cells generated in all of the above pathways was investigated in mice. Self‐reactive T cell clones were confined to IL‐2Rβ+, CDint cells, in all of the organs tested. A significant proportion of self‐reactive clones was never identified among CD3high cells in the thymus and peripheral immune organs in either young (8 week old) or old (50 week old) mice. Possibly reflecting their self‐reactivity, CD3int cells, but neither NK cells nor CD3high cells had a potent cytotoxic effect against a syngeneic hepatoma in the presence of anti‐CD3 mAb. These results raise the possibility that CD3int cells seen in the liver and thymus might belong to a similar primordial lineage of T cells, and that self‐reactive clones are not generated through the major intrathymic pathway, but only through extrathymic pathways and an alternative intrathymic pathway.
Tumor cells cotransfected with interleukin‐7 and B7.1 genes induce CD25 and CD28 on tumor‐infiltrating T lymphocytes and are strong vaccines
Authors: Cayeux Sophie , Beck Christian , Aicher Alexandra , Dörken Bernd , Blankenstein Thomas
Published: March 2007

Interleukin‐7 (IL‐7) and the membrane molecule B7 are both able to provide proliferation and activation signals for T cells. However, tumor cells transfected to express either molecule alone are not reliably rejected in syngeneic hosts or are not sufficiently immunogenic to serve as potent tumor vaccines. Since IL‐7 and B7 have shown synergistically to induce activation and proliferation of T cells in vitro, we have expressed B7.1 by means of a retrovirus in the mammary adenocarcinoma TS/A which arose spontaneously in a BALB/c mouse and in the plasmacytoma J558L and their IL‐7‐transfected sublines to improve vaccine efficacy. Expression of IL‐7 or B7.1 alone in tumor cells decreased tumorigenicity, but nevertheless tumors grew in a substantial number of mice. In contrast, IL‐7/B7.1 cotransfected cells did not grow as tumor in a single case. This inhibition of tumor growth was completely T cell dependent, because TS/A‐IL‐7/B7.1 cells retained their full tumorigenic potential in T cell‐deficient mice. Analysis of tumor‐infiltrating T lymphocytes revealed increased numbers of T cells in B7, IL‐7 and IL‐7/B7 transfected compared to parental tumors. In IL‐7/B7 transfected tumors, T cell numbers were not further increased compared to that in singlegene‐transfected tumors. However, T cells in B7 and IL‐7 transfected tumors differed phenotypically with respect to activation markers. In B7 transfected tumors, T cells were predominantly CD28+ and CD25+, while in IL‐7‐transfected tumors, T cells were mainly CD28− and CD25+. In IL‐7/B7 cotransfected tumors, the majority of T cells was CD28+ and CD25+. Thus, IL‐7 and B7 induced an anti‐tumor immune response by complementary T cell directed pathways in a cooperative fashion. Importantly, immunization of mice with the transfected cells and subsequent contralateral challenge with parental tumor cells showed that IL‐7/B7 co‐expressing cells induced the most strongly protective immunity, which is superior to that induced by single‐gene transfectants and to the adjuvant Corynebacterium parvum. Vaccine efficacy was abrogated when irradiated cells were used for vaccination. Together, our results show that IL‐7 and B7.1 transfected tumor cells induce strong T cell activation and tumor immunity.