bluuebunny/crossref_metadata_2025 huggingface dataset:
The murine homolog of human Ep‐CAM, a homotypic adhesion molecule, is expressed by thymocytes and thymic epithelial cells
Authors: Nelson Andrew J. , Dunn Robert J. , Peach Robert , Aruffo Alejandro , Farr Andrew G.
Published: March 2007

In this report, we demonstrate that gp40, a molecule previously shown to be expressed by thymic epithelial cell lines in vitro and by thymic epithelial cells in vivo, is the murine homolog of human Ep‐CAM, a calcium‐independent homotypic adhesion molecule. gp40 is also expressed at low levels by thymocytes and peripheral T cells. In the adult thymus, gp40 expression was inversely related to the state of thymocyte maturation, with the highest levels associated with CD4− CD8− and CD4+CD8+ thymocyte populations. Ultrastructural immunohistochemistry revealed gp40 localization to areas of thymocyte/epithelial contact and demonstrated that gp40 is also expressed by thymic dendritic cells. During fetal development, thymocytes at days 14–16 of gestation expressed high levels of gp40. At later stages, the observed decline in the frequency of gp40+ cells and levels of expression correlated with the emergence of αβ+ thymocytes by day 18 of gestation. In short‐term cultures, stimulation of unfractionated adult thymocytes with concanavalin A increased gp40 expression, particularly among CD3hi and CD3int thymocyte populations. This demonstration that Ep‐CAM, initially considered to be expressed primarily by epithelial cells, is also expressed by thymocytes, T cells and antigen‐presenting cells, raises the possibility that Ep‐CAM may contribute to adhesive interactions between thymocytes and epithelial cells or dendritic cells, either in the context of thymocyte development or peripheral T cell trafficking and function.
Identification of overlapping epitopes in mutant ras oncogene peptides that activate CD4+ and CD8+ T cell responses
Authors: Abrams Scott I. , Stanziale Stephen F. , Lunin Scott D. , Zaremba Sam , Schlom Jeffrey
Published: March 2007

Mutant ras p21 proteins contain sequences which distinguish them from normal endogenous ras and, thus, may represent unique epitopes for T cell recognition of antigen bearing tumor cells. Here, we examined the capacity of a mutant K‐ras 9‐mer peptide to induce in vivo CD8+ cytotoxic T lymphocytes (CTL). The peptide chosen reflected positions 4–12 of the point‐mutated sequence of the K‐ras oncogene encoding the Gly to Val substitution at codon 12. The overall rationale for selecting this particular 9‐mer sequence was threefold: the mutant peptide contained a putative major histocompatibility complex (MHC) class I consensus anchor motif for murine H‐2Kd; specific binding to MHC class I may then create an immunogenic complex for the induction of anti‐ras CD8+ CTL; and finally, the mutant sequence overlapped with a newly characterized anti‐ras CD4+ T helper type 1 epitope, which may have implications for the coordination and activation of both anti‐ras immune mechanisms against the same target cell antigenic determinant. A functional interaction with H‐2Kd was demonstrated with the mutant ras4–12(V12) peptide, but not the normal ras4–12(G12) peptide, which specifically inhibited an H‐2Kd‐restricted, anti‐nucleoprotein NP147–155 CTL response in a dose‐dependent fashion. An anti‐ras CD8+ T cell line was then established from immune splenocytes of BALB/c (H‐2d) mice injected with ras4–12(V12) in adjuvant, which mediated peptide‐specific lysis of syngeneic P815 tumor targets. Cytotoxicity was restricted by H‐2Kd and strongly specific for the mutant ras peptide. Importantly, these anti‐ras CTL specifically lysed a syngeneic tumor line (i.e. A20 lymphoma) transduced with the corresponding point‐mutated ras oncogene, suggesting T cell receptor recognition of endogenously derived antigen. Overall, these data demonstrated that mutant ras p21 at codon 12 (Gly → Val) contained a peptide sequence which exhibited specific functional binding to a murine MHC class I molecule; the ability of the mutant, but not the normal sequence to bind selectively to murine MHC class I likely reflected the generation of a C‐terminal anchor residue; and the ras 4–12(V12) peptide was immunogenic for the production of antigen‐specific CD8+ CTL, which lysed in vitro a syngeneic tumor cell line harboring the mutant K‐ras oncogene.
Expression levels of stress protein gp96 are not limiting for major histocompatibility complex class I‐restricted antigen presentation
Authors: Lammert Eckhard , Arnold Danièle , Rammensee Hans‐Georg , Schild Hansjörg
Published: March 2007

Immunization of mice with gp96 induces CTL with specificity for proteins that are expressed in the cells from which gp96 was isolated (Arnold et al., J. Exp. Med. 1995. 182: 885, Udono et al., Proc. Natl. Acad. Sci. USA 1994. 91: 3077). Recently, it has been shown that gp96 from cells transfected with vesicular stomatitis virus (VSV) nucleocapsid protein as well as gp96 loaded in vitro with peptides containing an epitope of this protein are taken up by phagocytic cells which obtain thereby the capacity for stimulating VSV‐specific cytotoxic T lymphocytes (Suto and Srivastava, Science 1995. 269: 1585). The immunization experiments together with the peptide transfer from gp96/peptide complexes to major histocompatibility complex (MHC) class I molecules of phagocytic cells are consistent with the hypothesis that the endoplasmic reticulum‐resident protein gp96 plays a crucial role in the antigen presentation of a cell (Srivastava et al., Immunogenetics 1994. 39: 93). To examine the involvement of gp96 in class I‐restricted antigen presentation, we reduced gp96 RNA and protein levels by transfecting P13.1 cells with a vector containing part of gp96 cDNA in antisense orientation to the promoter. We found that antisense clones expressing strongly reduced levels of gp96 mRNA and gp96 protein show normal levels of MHC class I molecules on the cell surface and are recognized by T cells to the same extent as wild‐type cells. Thus, our results show that normal levels of gp96 expression in a cell are not limiting for class I‐restricted antigen presentation.
Regions of RAG1 protein critical for V(D)J recombination
Authors: Kirch Susan A. , Sudarsanam Priya , Oettinger Marjorie A.
Published: March 2007

The products of the recombination activating genes RAG1 and RAG2 are essential for activating V(D)J recombination, and thus are indispensable for the production of functional and diverse antigen receptors. To investigate the function of RAG1, we have tested a series of insertion and substitution mutations for their ability to induce V(D)J rearrangement on both deletional and inversional plasmid substrates. With these substrates we were also able to assess the effects of these mutations on both coding and signal joint formation, and to show that any one mutant affected all these reactions similarly. As defined previously, the core active regions of RAG1 and RAG2 permit the deletion of 40% and 25%, respectively, of well‐conserved sequence. We show here that this “dispensable” region of RAG1 is not necessary for coding joint formation or for recombination of an integrated substrate, and that this portion is not functionally redundant with the “dispensable” region of RAG2. Recombination with these core regions is also still subject to the 12/23 joining rule. Further, the minimal essential core region of RAG1 can be located within an even smaller portion of the gene.
The murine VpreB1 and VpreB2 genes both encode a protein of the surrogate light chain and are co‐expressed during B cell development
Authors: Dul Jeanne L. , Argon Yair , Winkler Thomas , ten Boekel Edwin , Melchers Fritz , Mårtensson Inga‐Lill
Published: March 2007

The surrogate light chain is composed of two polypeptides, VpreB and λ5. In the mouse there are two VpreB genes which are 99% identical within the coding regions. Extensive restriction enzyme mapping and sequencing of these two genes showed that only the coding region and immediate 5′ and 3′ flanking sequences exhibited such high homology. More distal sequences have diverged considerably. The region 5′ of the respective gene directed transcription of a reporter gene in a pre‐B cell line, indicating that it contained promoter, and perhaps enhancer function. The VpreB2 gene is functional, as it directed the production in COS cells of a 16‐kDa protein that assembled with λ5 and was recognized by a VpreB‐specific monoclonal antibody. Using transfected COS cells expressing either VpreB1 or VpreB2, a PCR assay was developed to examine the steady state level of transcripts from each gene. When this assay was applied to a number of cell lines representing early stages of B cell differentiation, co‐expression of the two genes was observed in every case. VpreB1 and VpreB2 were co‐expressed in the fetal liver of CB17 mice, where peak expression of each gene occurred at days 16–17 of gestation. Similarly, adult bone marrow from several strains of mice expressed both genes. In sorted bone marrow cells expression of both VpreB genes was detected in pro‐B/pre‐BI and large pre‐BII cells, while the RNA steady state levels were at least 100‐fold lower in small pre‐BII and immature/mature B cells. Finally, single‐cell reverse transcriptase‐polymerase chain reaction on such sorted bone marrow cells detected VpreB1 and VpreB2 expression in at least 30% of all pro‐B/pre‐BI cells and large Ig heavy chain, surrogate light chain (pre‐B receptor) expressing pre‐BII cells. These results demonstrate that the control of expression of the two VpreB genes overlaps during development. They suggest that both VpreB1 and VpreB2 polypeptides can assemble with λ5 and μ to form pre‐B cell receptor complexes.
Tolerance towards resident intestinal flora in mice is abrogated in experimental colitis and restored by treatment with interleukin‐10 or antibodies to interleukin‐12
Authors: Duchmann Rainer , Schmitt Edgar , Knolle Percy , Zum Büschenfelde Karl‐Hermann Meyer , Neurath Markus
Published: March 2007

There is now increasing evidence that hyperresponsiveness towards intestinal flora is a crucial event in the pathogenesis of inflammatory bowel disease (IBD). In support of this hypothesis, we recently described in humans that tolerance exists towards indigenous intestinal flora but is broken in active IBD lesions. In the present study, we have attempted to transfer this model into mice from different genetic backgrounds (BALB/c, SJL/J, C3H/HeJ). We found that mononuclear cells from spleen, small bowel and large bowel of mice do not proliferate, i.e. are tolerant when exposed to bacterial sonicates derived from autologous intestine (BsA) but do proliferate, i.e. are immune when exposed to bacterial sonicates derived from the heterologous intestine of syngenic littermates (BsH). Furthermore, we demonstrate that both local and systemic tolerance to BsA is broken in a murine model of chronic intestinal inflammation induced by the hapten reagent 2,4,6‐trinitrobenzene sulfonic acid (TNBS), which mimics several important characteristics of Crohn's disease. Tolerance to BsA was restored and TNBS‐induced colitis was abrogated in mice systemically treated with interleukin (IL)‐10 or antibodies to IL‐12. Treatment specifically restored tolerance to BsA, but did not suppress proliferation to BsH. In summary, we here report a new mouse model for the study of immunity and tolerance towards bacterial products. Our data suggest that tolerance to BsA is an important protective mechanism and that restoration of tolerance to resident intestinal flora by IL‐10 and antibodies to IL‐12 may be of potential therapeutic utility in patients with inflammatory bowel disease.
Human CD4 and human major histocompatibility complex class II (DQ6) transgenic mice: supersensitivity to superantigen‐induced septic shock
Authors: Yeung Rae S. M. , Penninger Josef M. , Kündig Thomas , Khoo Wilson , Ohashi Pamela S. , Kroemer Guido , Mak Tak W.
Published: March 2007

Rodents are significantly less sensitive to enterotoxin‐induced shock, and are thus not valid human disease models. Here, we describe a mouse strain carrying the human CD4 and human major histocompatibility complex (MHC) class II (DQ6) transgenes in an endogenous CD4‐ and CD8‐deficient background. T lymphocytes from these animals react to minute amounts (10–100 times less than control mice) of staphylococcal enterotoxin B (SEB) in vitro, similar to concentrations to which human cells react. In vivo, these double‐transgenic, double‐knockout mice succumb to normally sublethal amounts of SEB. This sensitivity is not due to a biased T cell receptor Vβ repertoire, increased T cell reactivity, or increased sensitivity to macrophage‐derived cytokines. Rather, tumor necrosis factor (TNF)‐α production by T cells and serum levels of TNF‐α correlate precisely with the clinical syndrome, showing a biphasic T cell‐dependent response. These data show that both human CD4 and MHC class II molecules can render mice supersensitive to superantigen‐induced septic shock syndrome. This animal model mimics the progression of septic shock in man by transforming normally resistant mice into hypersensitive SEB responders, a trait that is characteristic of humans. Mice that have been humanized by exchanging autochthonous superantigen ligands by their human equivalents may be useful to decipher superantigen responses in vivo and to assess the pathogenesis of superantigen‐associated diseases.
The macrophage cell surface glycoprotein F4/80 is a highly glycosylated proteoglycan
Authors: Haidl Ian D. , Jefferies Wilfred A.
Published: March 2007

Molecules whose expression is limited to particular leukocyte populations are of interest since they may perform unique functions for these cells. We therefore examined the biochemical nature of the F4/80 molecule, which is expressed solely on macrophage and dendritic cell subpopulations. Our study clearly indicates that post‐translational modifications, which can influence both a protein's structural and functional features, constitute a major component of the 160‐kDa cell‐surface F4/80 molecule. The F4/80 molecule is synthesized as a single polypeptide chain which acquires numerous intramolecular disulfide bonds and requires an extended time period (T1/2 = 60 min) for transport to an endoglycosidase H‐resistant form. The F4/80 molecule contains extensive N‐linked glycosylation which contributes approximately 40 kDa to the mature molecule. The N‐linked carbohydrates are of the branched, complex type, containing repeating N‐acetylglycosamine or N‐acetyllactosamine units which mediate the reactivity of the F4/80 molecule with Datura stramonium lectin. O‐linked glycosylation is also present and contributes approximately 10 kDa to the F4/80 molecule. Furthermore, the sialic acid modifications of the F4/80 molecule are primarily through α2‐6 linkages to galactose. Finally, we demonstrate that the F4/80 molecule is a proteoglycan modified by chondroitin sulfate glycosaminoglycans. In addition to clarifying the nature of the F4/80 molecule biochemically, these posttranslational modifications have specific implications for molecular recognition processes. We conclude that the modifications of the F4/80 molecule may mediate cell‐cell recognition, cell adhesion, or ligand binding independently of the F4/80 molecule protein core.
Divergent effects of FcγRIIIA ligands on the functional activities of human natural killer cells in vitro
Authors: Sulica Andrei , Metes Diana , Gherman Maria , Whiteside Theresa L. , Herberman Ronald B.
Published: March 2007

The Fcγ receptor (R)IIIA (CD16) plays an important role in regulating the cytotoxic and non‐cytotoxic functions of human natural killer (NK) cells. Some anti‐CD 16 monoclonal antibodies (mAb) have been shown to stimulate NK activity, while human monomeric (m) IgG induces dose‐dependent inhibition of NK activity. To explore further these interactions mediated via FcγRIIIA, purified NK cells were cultured for 2–3 days in the presence of mIgG, 3G8 mAb, interleukin‐2 (IL‐2) or a combination of mIgG or 3G8 with IL‐2. Binding of mIgG or 3G8 to FcγRIIIA induced divergent effects of functions of cultured NK cells: 3G8 mAb + IL‐2 induced dose‐dependent inhibition of proliferation attributable to apoptosis; in contrast, mIgG + IL‐2 significantly increased NK cell proliferation. Incubation of NK cells in the presence of mIgG up‐regulated expression of surface activation markers (CD69, IL‐2Rα, ICAM‐1), cytotoxicity, cytokine production (IL‐1β, IFN‐γ and TNF‐α) and release of soluble IL‐2R. Thus, mIgG binding to FcγRIIIA induced stimulatory signals in human NK cells, leading to up‐regulation of IL‐2Rα expression, cell proliferation and cytokine release.
Early message expression of interleukin‐4 and interferon‐γ, but not of interleukin‐2 and interleukin‐10, reflects later polarization of primary CD4+ T cell cultures
Authors: Gollob Kenneth J. , Dutra Walderez O. , Coffman Robert L.
Published: March 2007

The CD4+ T cell subpopulation identified by the Mel‐14high phenotype represents naive T cells that develop into T helper 1 (Th1) cells upon stimulation with anti‐CD3 and interleukin (IL)‐2; however, the addition of IL‐4 induces the development of high IL‐4, IL‐5 and IL‐10‐secreting cells (IL‐4 SC). Here, we investigate the timing of cytokine gene expression in purified naive, Mel‐14high CD4+ T cells after stimulation under conditions that induce one of three dramatically different populations following secondary stimulation: anti‐CD3 + IL‐2 (Th1), anti‐CD3 + IL‐2 and IL‐4 (IL‐4 SC) and anti‐CD3 + anti‐interferon (IFN)‐γ/anti‐IL‐4 (null population). The skewing toward IL‐4 SC development induced by IL‐4 in the primary stimulation was clearly visible by 40 h. The message for IL‐4 was absent at 20 h after stimulation, but was induced 30–100‐fold by 40 h and was accompanied by a decrease in IFN‐γ message. The message for IL‐5 was undetectable. The development of Mel‐14high T cells into a Th1 population follows kinetics similar to those of the development of IL‐4 SC in that IFN‐γ gene transcription was undetectable at 20 h and induced 100‐fold by 40 h post‐stimulation. This induction was inhibited by the inclusion of anti‐IFN‐γ/anti‐IL‐4 during the primary stimulation. Finally, IL‐10 message is present in both cultures that developed into IL‐4 SC and those that developed into Th1 populations, in contrast to the differentiated cultures where there is a segregation between IL‐10 and IFN‐γ expression. Undetectable levels of message for both IL‐4 and IFN‐γ were seen in the null cultures, which expressed both IL‐2 and IL‐10 message. T cells identified by the Mel‐14low phenotype develop into a population that secretes high levels of all cytokines tested, and this phenotype was revealed within 20 h after stimulation as determined by message analysis. These results demonstrate an early correlation of gene transcription for IL‐4 and IFN‐γ, with the development of IL‐4 SC and Th1 cells, and a lack of a correlation at these early time points for expression of IL‐2, IL‐5 and IL‐10.
Antigen‐specific B cells are required for the secondary response of T cells but not for their priming
Authors: Chowdhury Mohammed Golam Mowla , Maeda Ken‐Ichi , Yasutomo Koji , Maekawa Yoichi , Furukawa Atsuko , Azuma Miyuki , Nagasawa Hideyuki , Himeno Kunisuke
Published: March 2007

We studied the potential role of B cells in T cell responses using severe‐combined immunodeficient (SCID) mice grafted with the thymus from fetal C.B‐17 mice (TG mice). These mice developed both CD4+ and CD8+ T cells, but not B cells within 2 months after transplantation. TG mice showed normal delayed‐type hypersensitivity responses against the immunizing antigen ovalbumin (OVA). Lymph node (LN) cells of TG mice proliferated well in response to concanavalin A (Con A). Further, Con A stimulation induced the production of interleukin (IL)‐2, IL‐6 and interferon (IFN)‐γ and the expression of IL‐4 mRNA. Thus, TG mice were reconstituted without remarkable immunodeficiency. However, these T cells failed to proliferate to OVA stimulation. Response to OVA was also inhibited in SCID mice grafted with fetal C.B‐17 liver cells when B cells were depleted in the proliferation assay. Unresponsiveness against immunizing antigen was restored by the addition of antigen‐primed B cells, but not by naive B cells, lipopolysaccharide‐activated B cells or B cells primed with sheep red blood cells. Next, we examined whether antigen‐primed B cells could induce T cell responses without professional antigen‐presenting cells (APC). T and B cells were purified from OVA‐immunized mice by cell sorter. These T cells proliferated in response to OVA and produced IFN‐γ in the absence of non‐B APC. When anti‐CD80 or anti‐CD86 was added in the assay, proliferation and IFN‐γ production was inhibited. These results indicate that B cells activated specifically with antigen are required for the secondary response of T cells, but not for their priming.
A functionally significant allelic polymorphism in a T cell receptor Vβ gene segment
Authors: Vessey S. J. Rupert , Bell John I. , Jakobsen Bent K.
Published: March 2007

The effect of an allelic polymorphism in the BV1S1 gene segment on recognition of major histocompatibility complex (MHC)‐peptide complexes by a specific T cell receptor (TCR) was studied using RBL 2H3 cells transfected with TCR‐CD3ζ chimeric receptors. An HLA‐A2‐restricted human immunodeficiency virus (HIV) pol‐specific cytotoxic T lymphocyte (CTL) clone utilizing the BV1S1A2 gene in combination with AV2S1A2 was identified and the extracellular domains of the TCR were fused to CD3ζ. In degranulation assays RBL 2H3 transfectants expressing this receptor maintained the specificity of the parental CTL clone. The allelic variant BV1S1A1N1 containing a glutamine for histidine substitution at position 48 in the loop of the second complementarity‐determining region was generated by site‐directed mutagenesis. Transfection of this molecule as a CD3ζ chimera together with the original AV2S1A2 CD3ζ molecule resulted in cell surface expression of both chains but a loss of recognition of HLA‐A2 HIV pol peptide‐pulsed targets. The effect of this polymorphism on MHC‐peptide recognition supports current models of TCR MHC‐peptide interaction and provides evidence for a functional role for polymorphism in the TCRV genes.
Lipid vesicle‐entrapped influenza A antigen modulates the influenza A‐specific human antibody response in immune reconstituted SCID‐human mice
Authors: Walker William , Brewer James M. , Alexander James
Published: March 2007

This study was designed to investigate the capacity of purified influenza antigen in the presence and absence of adjuvant to induce human antibody responses in human‐PBL‐SCID mice. Non‐ionic surfactant vesicles (NISV) were used as adjuvant as they have been shown to promote the development of Th1 responses in mouse studies. Human peripheral blood lymphocyte‐SCID mice were inoculated with either purified influenza antigen (A/Texas, H3N2) or influenza antigen entrapped in NISV. Both vaccinated groups produced significantly higher plasma levels of influenza‐specific human IgG when individually compared with non‐vaccinated controls. However, similar comparisons revealed that specific IgM levels were significantly higher only in the group challenged with purified antigen. Further analysis of IgG subclasses also demonstrated an adjuvant‐dependent dichotomy in the responses of the vaccine groups when compared with non‐vaccinated controls. Thus, only influenza‐specific IgG1 antibodies (associated with Th1 responses in humans) were significantly increased above control levels using antigen with adjuvant, while both this subclass and antigen‐specific IgG4 (Th2 associated) were significantly increased with antigen alone. These results illustrate the suitability of this model for use in human vaccination studies and demonstrates that influenza antigen applied with NISV selectively promotes only Th1 responses, unlike free antigen which also promotes Th2 responses in vivo.
Co‐localization of the neonatal Fcγ receptor and IgG in human placental term syncytiotrophoblasts
Authors: Kristoffersen Einar K. , Matre Roald
Published: March 2007

Transfer of maternal IgG through the human placenta furnishes the newborn with passive immunity to a number of infectious agents. The exact mechanism of this transfer is still unknown, but it is agreed that it involves active receptor‐mediated transport. The neonatal Fc receptor is a major histocompatibility complex class I‐like receptor originally identified in the intestines of newborn rodents. A similar receptor has recently been detected in human placental syncytiotrophoblasts. Using multilabeling fluorescence immunohistochemistry and confocal laser scanning microscopy, we found that the neonatal Fc receptor co‐localizes with IgG and β2‐microglobulin in granules of human placental syncytiotrophoblast. The Fc receptor is not detected on syncytiotrophoblast apical plasma membrane. Localization to the outermost cellular barrier between the fetal and maternal blood further strengthens the role of the Fc receptor in transplacental transport of IgG.
Role of tumor necrosis factor‐α and platelet‐activating factor in neoangiogenesis induced by synovial fluids of patients with rheumatoid arthritis
Authors: Lupia Enrico , Montrucchio Giuseppe , Battaglia Edda , Modena Vittorio , Camussi Giovanni
Published: March 2007

The aim of the present study was to investigate in vivo in a mouse model the stimulation of neoangiogenesis by synovial fluids of patients with rheumatoid arthritis (RA) and to determine the role of tumor necrosis factor (TNF)‐α and platelet‐activating factor (PAF) in the formation of new vessels. Angiogenesis was studied in a mouse model in which Matrigel, injected subcutaneously, was used as a vehicle for the delivery of potential angiogenic stimuli. Synovial fluids of patients with RA but not with osteoarthritis (OA) were shown to induce neoangiogenesis. Since synovial fluid of patients with RA contained significantly higher levels of TNF‐α‐like bioactivity and of PAF than that of patients with OA, the role of these mediators was evaluated by using an anti‐TNF‐α neutralizing monoclonal antibody (mAb) and a PAF receptor antagonist, WEB 2170. When added to Matrigel, anti‐TNF‐α mAb and particularly WEB 2170 significantly reduced neoangiogenesis induced by synovial fluids of RA patients. Moreover, PAF extracted and purified from synovial fluid induced angiogenesis. These results suggest that the neoangiogenesis observed in rheumatoid synovitis may be due, at least in part, to the angiogenic effect of locally produced TNF‐α and PAF.
Transporter (TAP)‐independent processing of a multiple membrane‐spanning protein, the Epstein‐Barr virus latent membrane protein 2
Authors: Lee Steven P. , Thomas Wendy A. , Blake Neil W. , Rickinson Alan B.
Published: March 2007

Antigen presentation to CD8+ cytotoxic T lymphocytes (CTL) usually involves proteolytic cleavage of antigen in the cytosol and the delivery of epitope peptides onto major histocompatibility complex class I molecules in the endoplasmic reticulum (ER) via the heterodimeric peptide transporter TAP1/TAP2. In the few exceptional cases where TAP‐independent presentation of an endogenously expressed protein has been observed, the epitope‐containing domain of the protein either has naturally accessed or has been directed into the ER lumen where it is thought to become susceptible to ER proteases. Here, we describe a novel example of TAP‐independent processing involving the Epstein‐Barr virus (EBV) latent membrane protein LMP2, a multiple membrane‐spanning protein with minimal projection into the ER. Expression of LMP2 in the TAP− T2 cell line, whether from the resident EBV genome or from a recombinant vaccinia virus vector vacc‐LMP2, rendered the cells sensitive to recognition by CTL clones specific for two HLA‐A2.1‐restricted peptide epitopes, LMP2 329–337 or 426–434. Vacc‐LMP2‐mediated sensitization to lysis required expression of the antigen de novo in T2 cells and was blocked by brefeldin A. In the same experiments, two other EBV‐specific CTL epitopes, one derived from LMP2 but restricted through a different HLA allele (A11), the other restricted through A2.1 but derived from a different viral protein (BMLF1), did not display TAP‐independent processing. The results are discussed in relation to the unusual topology of LMP2 in the membrane and the position of the epitope peptides within that structure.
Peptide binding specificity of major histocompatibility complex class I resolved into an array of apparently independent subspecificities: quantitation by peptide libraries and improved prediction of binding
Authors: Stryhn Anette , Pedersen Lars Østergaard , Romme Tina , Holm Charlotte Bisgaard , Holm Arne , Buus Søren
Published: March 2007

Considerable interest has focused on understanding how major histocompatibility complex (MHC) specificity is generated and characterizing the specificity of MHC molecules with the ultimate goal being to predict peptide binding. We have used a strategy where all possible peptides of a particular size are distributed into positional scanning combinatorial peptide libraries (PSCPL) to develop a highly efficient, universal and unbiased approach to address MHC specificity. The PSCPL approach appeared qualitatively and quantitatively superior to other currently used strategies. The average effect of any amino acid in each position was quantitated, allowing a detailed description of extended peptide binding motifs including primary and secondary anchor residues. It also identified disfavored residues which were found to be surprisingly important in shaping MHC class I specificity. Assuming that MHC class I specificity is the result of largely independently acting subsites, the binding of unknown peptides could be predicted. Conversely, this argues that MHC class I specificities consist of an array of subspecificities acting in a combinatorial mode.
Somatic hypermutation occurs in B cells of terminal deoxynucleotidyl transferase‐, CD23‐, interleukin‐4‐, IgD‐ and CD30‐deficient mouse mutants
Authors: Texidó Gemma , Jacobs Heinz , Meiering Myriam , Kühn Ralf , Roes Jürgen , Müller Werner , Gilfillan Susan , Fujiwara Hiroshi , Kikutani Hitoshi , Yoshida Nobuaki , Amakawa Ryuichi , Benoist Christophe , Mathis Diane , Kishimoto Tadamitsu , Mak Tak W. , Rajewsky Klaus
Published: March 2007

We show through sequence analysis of rearranged immunoglobulin variable (Ig‐V) region genes from memory B cells of mouse mutants generated by gene targeting that terminal deoxynucleotidyl transferase, CD23, interleukin‐4, IgD and CD30 are dispensable for somatic hypermutation.
Subepithelial B cells in the human palatine tonsil. I. Morphologic, cytochemical and phenotypic characterization
Authors: Dono Mariella , Burgio Vito L. , Tacchetti Carlo , Favre Anna , Augliera Adriano , Zupo Simona , Taborelli Giuseppe , Chiorazzi Nicholas , Grossi Carlo E. , Ferrarini Manlio
Published: March 2007

This study describes the purification of a subset of tonsillar B cells which share phenotypic, morphologic and cytochemical features with subepithelial (SE) B cells. These cells, which represented the 5–10% of the total tonsillar B cells, were found in the Percoll gradient fraction of highest density, together with resting follicular mantle (FM) B cells. The latter B cells, however, expressed surface CD5 and could be removed by an immune rosetting procedure. The remaining small CD5− B cells had a surface phenotype (IgM+, IgD+, CD23−, CD38±, CD10−, CD44+) that was different from that of FM (IgM+, IgD+, CD23+, CD39+, CD38−, CD10−, CD44±) and of germinal center (GC) (CD23−, CD39−, CD38+, CD10+, CD44±, IgG+) B cells isolated from the same cell suspensions. Furthermore, the absence of surface activation markers (CD71 and CD69) and of surface IgG allowed us to distinguish small CD5− B cells from activated and memory cells migrating within Percoll fractions of lower density. In situ immunohistochemical studies revealed that B cells with an identical phenotype as that of small CD5− B cells could be detected predominantly in the SE region (lamina propria) of the tonsil, and also within the epithelium lining the cryptae. This area was also comprised of a relatively minor proportion of activated B cells, not found in the small CD5− B cell fraction owing to the separation procedure used. Consistent with the notion that the SE area could be a site of B cell activation was also the presence of activated macrophages and of plasma cells. Thirty to forty percent of small CD5− B cells isolated in suspension were positive for the endogeneous alkaline phosphatase (ALP) activity. In contrast, only a few FM B cells were ALP+, while GC cells were consistently ALP−. In situ studies also demonstrated a prevalent expression of ALP activity by the B cells in the SE area. At the ultrastructural level, small CD5− B cells were clearly different from both FM and GC B cells. They displayed a cytoplasm more extended than that of FM B cells with abundant endosomes and plasma membrane projections, and a speckled pattern of nuclear heterochromatin distribution. When fixed tissue sections were examined, cells with identical ultrastructural features could be demonstrated in the tonsillar lamina propria. Collectively, the above data demonstrate an identity of features between the small CD5− B cells isolated in suspension and SE B cells analyzed in situ. Since tonsillar SE B cells are generally thought to represent the homolog of the extrafollicular B cells (including those of the splenic marginal zone), these studies may provide new opportunities for functional studies on this so far incompletely characterized B cell subset.
Epitopes recognized by neutralizing therapy‐induced human anti‐interferon‐α antibodies are localized within the N‐terminal functional domain of recombinant interferon‐α2
Authors: Nolte Klaus‐Ulrich , Günther Gero , von Wussow Peter
Published: March 2007

During prolonged recombinant interferon (rIFN)‐α2 therapy, a minority of patients develop high‐titer neutralizing IFN‐α antibodies. Sera from nine IFN‐α antibody‐positive patients were studied to characterize the specificity of anti‐IFN‐α neutralizing antibodies by their ability to inhibit the antiviral and antiproliferative activity of different rIFN‐α subtypes and rIFN‐α1/α2 hybrids. These therapy‐induced antibodies (Tab) were compared with IFN‐α‐specific autoantibodies (Aab) from two patients with systemic lupus erythematosus who had never received any exogenous IFN‐α. Although IFN‐α subtypes are closely related in structure, Tab inhibited the antiviral activity of only recombinant (r)IFN‐α2 and rIFN‐α6, but not or slightly that of rIFN‐α1, ‐α7, ‐α8 and ‐α14. Furthermore, of four different rIFN‐α1/α2 hybrids tested, Tab inhibited only those which contained the N‐terminal residues 17–64 of rIFN‐α2. Comparison of the primary sequences of neutralized and not neutralized subtypes suggests an epitope involving the residues 22–31 of IFN‐α2 is recognized. Thus, Tab block rIFN‐α2 by reacting with only one of two functional domains. In contrast, Aab possessed a broad specificity and neutralized both the antiviral and antiproliferative activity of rIFN‐α2, ‐α6, ‐α7, ‐α8 and ‐α14. They also neutralized all four rIFN‐α1/α2 hybrids tested. These data demonstrate that Tab are highly specific for the therapeutic IFN‐α subtype and specifically neutralize rIFN‐α2 by binding to its N‐terminal functional domain.
Cyclosporin A sensitivity of the HIV‐1 long terminal repeat identifies distinct p56lck‐dependent pathways activated by CD4 triggering
Authors: di Somma M. Maddalena , Majolini M. Bernardetta , Burastero Samuele E. , Telford John L. , Baldari Cosima T.
Published: March 2007

The CD4 co‐receptor interacts with nonpolymorphic regions of major histocompatibility complex class II molecules on antigen‐presenting cells. This interaction results in the mobilization of a number of signaling mediators shared by the T cell receptor (TcR) signaling pathway and thus amplifies TcR‐generated signals. We have investigated the outcome of CD4 engagement on the activation of both cellular transcription factors and the HIV‐1 long terminal repeat (LTR). We show that CD4 triggering activates different pathways of HIV LTR activation which can be identified by their sensitivity to the immunosuppressant cyclosporin A. The response of the inducible cellular transcription factors involved in HIV LTR activation shows that both nuclear factor (NF)‐ϰB and NF‐AT mediate a cyclosporin A‐sensitive response to CD4, while AP‐1 is at least in part responsible for the cyclosporin A‐insensitive response. Both pathways can, however, be blocked by a kinase‐defective dominant negative p56lck mutant, supporting an essential role for p56lck kinase activity in CD4‐dependent signal transduction. A functional analysis of different CD4 epitopes using either anti‐CD4 mAb or HIV‐1 gp120 reveals a common epitope‐specific activation of both the LTR and of the transcription factors NF‐ϰB and NF‐AT.
Distinct mechanisms for rescue from apoptosis in Ramos human B cells by signaling through CD40 and interleukin‐4 receptor: role for inhibition of an early response gene, Berg36
Authors: Ning Zhi‐Qiang , Norton John D. , Li Jin , Murphy John J.
Published: March 2007

The role of interleukin‐4 (IL‐4) and CD40 signaling in negative regulation of apoptosis in human Ramos B cells induced in response to different agents was investigated. CD40 ligation protected cells from apoptosis induced by calcium ionophore through an initial, rapid and apparently Bcl‐2‐independent mechanism, associated with up‐regulation of Bcl‐xL. However, rescue from apoptosis induced by inhibition of macromolecular synthesis required several hours of prior stimulation with CD40 ligand/antibody and was accompanied by up‐regulation of Bcl‐2. In contrast, IL‐4 did not up‐regulate Bcl‐2 or Bcl‐xL and did not inhibit apoptosis induced by inhibitors of macromolecular synthesis. However, IL‐4 did protect Ramos cells from apoptosis induced by calcium ionophore and this effect was accompanied by inhibition of ionophore‐induced expression of an immediate early gene encoding a 36‐kDa zinc‐finger protein, Berg36. Anti‐sense blockade of Berg36 expression partially inhibited ionophore‐induced apoptosis to an extent commensurate with the level of IL‐4 protection, implicating Berg36 function as a requirement for apoptosis induced through calcium signaling and as a target for IL‐4 through which this cytokine inhibits apoptosis in Ramos B cells. These distinct mechanisms for rescue from apoptosis by CD40 and IL‐4 may help explain the co‐operative roles of these T cell‐derived signals for B cell survival.
Human follicular dendritic cells and vascular cells produce interleukin‐7: a potential role for interleukin‐7 in the germinal center reaction
Authors: Kröncke Rolf , Loppnow Harald , Flad Hans‐Dieter , Gerdes Johannes
Published: March 2007

Interleukin‐7 (IL‐7) has long been known as a potent growth factor in lymphocyte development. However, recent data obtained in vitro revealed additional functions for this cytokine, e.g. IL‐7 influences the generation of cytotoxic T cells and NK cells, and in higher concentrations, activates monocytes in a manner similar to bacterial lipopolysaccharide. Furthermore, human tonsillar B cells are able to proliferate upon stimulation by IL‐7 and cross‐linking of the B cell receptor. Considering the latter role of IL‐7 for B cell proliferation, we investigated which cells of the human immune system express IL‐7 in vivo. mRNA expression of tonsillar cells was analyzed using the reverse transcription polymerase chain reaction, and protein expression was assessed by immunohistochemical staining of frozen sections. Among a variety of different immune cell types isolated from human tonsils, only follicular dendritic cells (FDC) expressed specific IL‐7 products, whereas B and T cells were consistently negative. Immunohistochemical staining of tonsillar sections revealed the expression of immunoreactive IL‐7 protein on FDC, and a pronounced expression could also be detected in oral mucosa and vascular endothelial cells. For the latter, we could also demonstrate mRNA expression in primary cultured cells. In light of the previous finding that IL‐7 can act as a co‐stimulus for the induction of proliferation in tonsillar B cells, these data suggest a role of IL‐7 in the germinal center reaction. It is tempting to speculate that FDC may function as regulatory cells in B cell development in the tonsil, as epithelial nurse cells are thought to govern T cell development in the thymus.
Differential activation of human autoreactive T cell clones by altered peptide ligands derived from myelin basic protein peptide (87–99)
Authors: Vergelli Marco , Hemmer Bernhard , Utz Ursula , Vogt Anne , Kalbus Matthias , Tranquill Laura , Conlon Paul , Ling Nicholas , Steinman Lawrence , McFarland Henry F. , Martin Roland
Published: March 2007

We have examined the functional consequences induced by interaction of DR2a‐restricted myelin basic protein (MBP) (87–99)‐specific T cell clones (TCC) with altered peptide ligands (APL) derived from MBP peptide (87–99). The immunodominant MBP peptide (87–99) has been implicated as a candidate antigen in multiple sclerosis (MS) by several lines of evidence. In the present study, we have defined the T cell receptor (TCR) contact residues for DR2a‐restricted, (87–99)‐specific T helper type 1 T cells to design APL suitable to modify the functions of such T cells potentially relevant for the pathogenesis of MS. We show that neutral (L‐alanine substitutions) or conservative exchanges of the primary and secondary TCR contact residues lead to various alterations of T cell function, ranging from differences in interleukin‐2 receptor up‐regulation to anergy induction and TCR antagonism. The potential usefulness of APL as an immunomodulating therapy for DR2+ MS patients is discussed.
Cellular competition modulates survival and selection of CD8+ T cells
Authors: Freitas António A. , Agenes Fabien , Coutinho Graça C.
Published: March 2007

In this investigation we compare the repopulation of the CD8+ T cell compartments of bone marrow (BM) chimeras by either normal nontransgenic or T cell receptor (TcR) αβ‐transgenic (TG) CD8+ T cells, the fate of TG and non‐TG CD8+ T cells in different parabionts and the survival of TG and non‐TG peripheral CD8+ T cells after transfer into athymic hosts. We found that cellular competition among CD8 T cells occurs at several steps of T cell differentiation including (a) during the DN to DP transition, (b) positive selection in the thymus, (c) export from the thymus and (d) in the periphery. Comparison of the results obtained in the BM chimeras and in the parabionts shows that an important step of T cell selection occurs during seeding of peripheral lymphoid tissues. Once established, peripheral T cells resist replacement by recent thymus migrants, i.e. in the periphery, selection of T cell repertoires follows the rule “first come, first served”. Peripheral dominance correlates with T cell activation and division. Cell cycling and CD44 expression are more frequent among non‐TG CD8 T cells than TG CD8 T cells and within the latter, more frequent among P14 TG CD8 T cells than anti‐HYTG CD8 T cells. Thus, in the absence of intentional immunization, the frequencies of CD8+ T cells follow a hierarchy of selection in which non‐TG ⩾ P14 TG > anti‐HY TG. We also show that the equilibrium size and the fate of one CD8 T cell population differs according to the presence or absence of other CD8 T cell populations. Under these circumstances, selection of T cell repertoires and T cell survival and memory rely not only on the interactions of each T cell with their respective ligands, but also on the nature and number of other competing cells.
Major histocompatibility complex class I presentation of ovalbumin peptide 257–264 from exogenous sources: protein context influences the degree of TAP‐independent presentation
Authors: Wick Mary Jo , Pfeifer John D.
Published: March 2007

Peritoneal macrophages from C57BL/6 mice process antigens from bacteria or coated on polystyrene beads for presentation by major histocompatibility complex (MHC) class I molecules. To investigate this antigen processing pathway, peritoneal macrophages from homozygous TAP1−/− mice, which lack the transporter associated with antigen processing (TAP) and are defective in presenting endogenous antigens on MHC class I, were used. TAP1−/− or C57BL/6 macrophages were co‐incubated with either bacteria or polystyrene beads containing the 257–264 epitope from ovalbumin [OVA(257–264)], which binds the mouse class I molecule Kb. The source of the OVA(257–264) epitope was either the Crl‐OVA(257–264) (Crl‐OVA) fusion protein, the maltose binding protein (MBP)‐Crl‐OVA fusion protein, native OVA or bacterial recombinant OVA (rOVA); Crl‐OVA, MBP‐Crl‐OVA and rOVA were each expressed in bacteria, and Crl‐OVA and MBP‐Crl‐OVA purified from bacterial lysates and native egg OVA were coated onto polystyrene beads. The data reveal that peritoneal macrophages from C57BL/6 and TAP1−/− mice can process bacteria expressing Crl‐OVA, MBP‐Crl‐OVA and rOVA as well as beads coated with native OVA, purified Crl‐OVA, and purified MBP‐Crl‐OVA and present OVA(257–264) for recognition by OVA(257–264)/Kb‐specific T hybridoma cells, albeit with different relative processing efficiencies. The processing efficiency of TAP1−/− macrophages co‐incubated with bacteria or beads containing Crl‐OVA or MBP‐Crl‐OVA was reduced approximately three to five times compared to C57BL/6 macrophages, but OVA(257–264) was presented 100 times less efficiently when the source of OVA(257–264) was full‐length OVA. Chloroquine inhibition studies showed a differential requirement for acidic compartments in C57BL/6 versus TAP1−/− macrophages, which also depended upon the source of the OVA (257–264) epitope (Crl‐OVA versus full‐length OVA). These data suggest that TAP1−/− and C57BL/6 macrophages may process Crl‐OVA and full‐length OVA in different cellular compartments and that the protein context of the OVA(257–264) epitope influences the extent of TAP‐independent processing for MHC class I presentation.
Protective immunization with plasmid DNA containing the outer surface lipoprotein A gene of Borrelia burgdorferi is independent of an eukaryotic promoter
Authors: Simon Markus M. , Gern Lise , Hauser Pierre , Zhong Weimin , Nielsen Peter J. , Kramer Michael D. , Brenner Christiane , Wallich Reinhard
Published: March 2007

Plasmid DNA encoding the outer surface lipoprotein A (OspA) of Borrelia burgdorferi under the control of either strong eukaryotic/viral or its own bacterial promoter was injected intramuscularly (m. tibialis anterior) or intradermally into BALB/c and AKR/N mice. OspA‐specific antibodies and OspA‐reactive T helper 1 cells (Th1) were induced only with those plasmids containing the ospA structural gene including its own regulatory control region immediately upstream. In the absence of the ospA promoter, no or only marginal immune responses to OspA were obtained, even when strong eukaryotic promoter/enhancer elements were present. Together with the finding that the ospA promoter is active in a mouse B‐lymphoma line, the data suggest that spirochetes are able to express at least part of their genes in the mammalian environment. Mice previously vaccinated with the relevant ospA plasmid DNA were protected against subsequent experimental challenge with a virulent strain of B. burgdorferi, as measured by the appearance of antibodies to a prominent protective epitope (LA‐2) and the failure to re‐isolate spirochetes from ear biopsies. In addition, C.B‐17 severe‐combined immunodeficient mice could be protected against infection by passive transfer of immune sera from ospA plasmid DNA‐inoculated normal mice. Protective LA‐2‐related antibody titers obtained after repeated immunization persisted for 200 days and longer. This simple procedure of immunization using plasmid DNA consisting of a prokaryotic gene under the control of its own promoter holds great promise for the development of alternative subunit vaccines against bacterial infections, including Lyme disease. In addition, the availability of this novel prokaryotic promoter element now allows the study of the basis for the differential expression of bacterial genes in prokaryotic and eukaryotic environments.