bluuebunny/crossref_metadata_2025 huggingface dataset:
The transcription factor Pit‐l/GHF‐1 is expressed in hemopoietic and lymphoid tissues
Authors: Delhase Mireille , Vergani Patricia , Malur Anagha , Hooghe‐Peters Elisabeth L. , Hooghe Robert J.
Published: March 2007

The expression of the Pit‐l/GHF‐1 transcription factor (hereafter Pit‐1), which controls the expression of growth hormone (GH) and prolactin (PRL) in the pituitary gland, has been documented in human and rat hemopoietic and lymphoid tissues and cell lines. Pit‐1 mRNA was detected by in situ hybridization in about 1 % of rat bone marrow cells and in the spleen red pulp and marginal zone. Pit‐1 was also expressed in human tonsils (mantle zone), in the thymus (rat and human, non‐lymphoid cells), in lipopolysaccharide‐stimulated rat peritoneal cells and in non‐hepatocyte cells in the liver (rat and human). A detailed investigation of the rat spleen showed a very similar distribution for Pit‐1, GH and PRL mRNA and Pit‐1, GH and PRL proteins (detected by immunocytochemistry). Using polymerase chain reaction followed by Southern hybridization, the expression of Pit‐1 could be confirmed in human and rat spleen, bone marrow and thymus. HL60 and RAJI leukemic cells were also positive. The sequence of fragments amplified from rat spleen and from human bone marrow completely matched published sequences of rat and human pituitary Pit‐1, respectively. Expression of GH and PRL in lymphoid tissues has been documented. The straightforward hypothesis would therefore be that Pit‐l's main function in lymphoid tissues is controlling GH and PRL expression, as in the pituitary gland. GH and PRL may be hemopoietic and lymphoid growth and differentiation factors.
Serum soluble interleukin‐6 receptor in MRL/lpr mice is elevated with age and mediates the interleukin‐6 signal
Authors: Suzuki Hiroshi , Yasukawa Kiyoshi , Saito Takashi , Narazak Masashi , Hasegawa Atsuhiko , Taga Tetsuya , Kishimoto Tadamitsu
Published: March 2007

The characteristics of soluble interleukin‐6 receptor (sIL‐6R) in murine sera were examined. To investigate a relationship between serum sIL‐6R level and autoimmune diseases, quantitative analysis of serum sIL‐6R in MRL/lpr mice was performed by an enzyme‐linked immunosorbent assay. The serum sIL‐6R level in MRL/lpr mice of both sexes was below the detection limit (< 1.0 ng/ml) at 8 weeks of age, but it increased in accordance with age and reached 42 ± 9.3 ng/ml in female and 31 ± 13 ng/ml in male mice at 30 weeks of age. In MRL/+ mice, although an age‐associated increase in serum sIL‐6R level was observed, it was much less extensive than that in MRL/lpr mice. Elevated serum sIL‐6R level at the age of 30 weeks was observed in female and male (NZB × NZW)F1 mice (32 ± 10 ng/ml and 17 ± 5.0 ng/ml, respectively), and male BXSB/Mpj Yaa mice (42 ± 18 ng/ml), suggesting that elevated serum sIL‐6R in aged mice is one of the characteristics of autoimmune‐prone mice. Quantitative analysis of serum IL‐6 in MRL/lpr revealed that the serum sIL‐6R level correlated well with the serum IL‐6 level. We also showed that sIL‐6R in the sera from MRL/lpr mice could mediate the IL‐6 functions through the IL‐6 signal‐transducing receptor component gpl30, suggesting that elevated production of sIL‐6R may partly contribute to development of autoimmune disease in MRL/lpr mice.
The level of N‐region diversity in T cell receptors is not pre‐ordained in the stem cell
Authors: Bogue Molly , Mossmann Horst , Stauffer Uta , Benoist Christophe , Mathis Diane
Published: March 2007

The αβ Tcell repertoires of adults and neonates are distinctly different. For example. Tcell receptors (TcR) from adult animals have substantial N‐nucleotide addition at their V‐D‐J junctions while those from neonatal animals do not. This dichotomy reflects a rather abrupt change in expression of the terminal deoxynucleotidyl transferase (TdT) gene in thymocytes on day 4 after birth. We have asked whether this change is due to the differentiation of successive waves of stem cells harboring different potentials for TdT expression, a scenario like the one proposed to explain developmental regulation of γδ Tcell repertoires. Reconstitution of adult severe combined immunodeficiency mice with either fetal liver or adult bone marrow precursors gave rise to T cells with substantial N‐region diversity in their TcR, even at the earliest points of reconstitution. It is most likely, then, that the abrupt change in TdT gene expression in day 4 thymocytes is due to an environmentally induced switch‐on.
Synovial IgG rheumatoid factors show evidence of an antigen‐driven immune response and a shift in the V gene repertoire compared to IgM rheumatoid factors
Authors: Randen Ingrid , Pascual Virginia , Victor Kimberly , Thompson Keith M. , Førre Øystein , Capra J. Donald , Natvig Jacob B.
Published: March 2007

We have established IgG rheumatoid factor (RF)‐secreting hybridoma cell lines from the synovial tissues of three patients from whom we have previously characterized several IgM RF. The IgG RF bind human and rabbit IgG and form intracellular complement‐fixing complexes indicative of a self association process in vivo. Nucleotide sequence analysis revealed that two IgG RF used VHIII gene segments, while one used a VHI gene segment. The VL gene usage consisted of a Vχ1, a Vλ2 and a Vχ4/Vχ6 hybrid, confirming our previous findings that many different VL genes can contribute to RF specificity. Although the IgG RF used VH genes from the same families that dominated the IgM RF response, two of the actual gene segments employed were not found among the IgM RF. In contrast to IgM RF, which in general were not very mutated, the IgG RF showed somatic mutations characteristic of an antigen‐driven immune response.
Membrane cofactor protein (MCP, CD46) in seminal plasma and on spermatozoa in normal and “sterile” subjects
Authors: Seya Tsukasa , Hara Tomoko , Matsumoto Misako , Kiyohara Hisakazu , Nakanishi Isao , Kinouchi Toshiaki , Okabe Masaru , Shimizu Akira , Akedo Hitoshi
Published: March 2007

A sperm protein of molecular mass 43 kDa (the spermatozoa membrane cofactor protein, smMCP) and a seminal plasma protein of 60 kDa (ssMCP) were identified by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) followed by immunoblotting with four monoclonal antibodies (mAb) against membrane cofactor protein (MCP, CD46).These proteins served as factor I cofactors for the cleavage of methylamine‐treated C3 (C3ma), the activity of which was blocked by M75, an MCP cofactor‐activity‐blocking mAb. Thus, these semen proteins are antigenic and functional homologoues of MCP. On SDS‐PAGE analysis these MCP migrated as single‐band proteins which differed from the two‐band forms of MCP expressed on other cells. smMCP was N‐glycosylated but not O‐glycosylated, while ssMCP was O‐glycosylated: after deglycosylation of these proteins bands were detected at 38‐40 kDa and 43 kDa on SDS‐PAGE, respectively. These semen MCP are therefore, structurally different from the conventional MCP. ssMCP in both normal and “sterile” subject groups was determined by sandwich enzyme‐linked immunosorbent assay. Seminal plasma in the two groups contained 250‐700 ng/ml ssMCP. The difference between the two groups was marginal, although samples from normal subjects tended to show higher concentrations of ssMCP than samples from “sterile” subjects. No molecular difference was observed with ssMCP and smMCP in the two groups by SDS‐PAGE/immunobloblotting analysis. Immunohistochemical analysis suggested that MCP was positive in glandular epithelial cells and the lumen of the prostate, and in most intra‐lumen cells of the testis. Using antibody M177, solubilized prostate and testis were analyzed by immunoblotting and compared with other cell MCP The major band of MCP in the testis, but not in the prostate, was of 60 kDa, which aligned with ssMCP. No band of testis or prostate MCP, however, aligned with smMCP. ssMCP may be produced in the testis, while the origin of smMCP remains unknown. We hypothesize that ssMCP is important in the survival of spermatozoa, protecting them against local secretion of immunoglobulin and complement in the female genital tract, and that smMCP, which is expressed on acrosome‐reacted spermatozoa, plays an essential role in the interaction of spermatozoa with oocytes.
Mouse Crry/p65 is a regulator of the alternative pathway of complement activation
Authors: Foley Sharon , Li Bin , Dehoff Marlin , Molina Hector , Holers V. Michael
Published: March 2007

Like man, mouse has evolved a unique set of regulatory proteins which provide protection from complement‐mediated damage to self membranes. The recently described mouse protein Crry/p65 has been shown to inhibit classical complement pathway C3 deposition on cell membranes in which it is expressed. In two distinct experimental systems, we now further delineate the regulatory activity of Crry/p65 and demonstrate its inhibitory effect on alternative complement pathway C3 activation. First, significant inhibition of mouse alternative pathway C3 deposition was demonstrated on neuraminidase‐treated human K562 cells expressing recombinant Crry/p65. Second, using a baculovirus technique, recombinant Crry/p65 was synthesized as a soluble molecule and then purified. This molecule was found to inhibit mouse C3 deposition on the surface of zymosan, a potent alternative complement pathway activator. These studies, combined with our earlier findings, demonstrate that Crry/p65 can regulate both the classical and alternative complement pathways. Crry/p65 must, therefore, exert its effects prior to, or at the level of, the C3 convertases, in a fashion similar to that of human membrane cofactor protein and/or decay‐accelerating factor. These studies provide further proof of the hypothesis that Crry/p65 is an evolutionarily unique, complement regulatory protein which has developed in mouse.
Cloning and sequencing of human immunoglobulin λ. gene segments
Authors: Williams Samuel C. , Winter Greg
Published: March 2007

To provide the building blocks for making synthetic antibody fragments we have used the polymerase chain reaction (PCR) to clone human variable (V) gene segments of λ light chains. The PCR primers were based on the sequences of known human Vλ segments, and were used to isolate 14 new Vλ segments (including 4 pseudogenes) from a single individual. We have compiled a sequence directory from this data and other sources to include all known human Vλ segments with open reading frames and we have identified a new Vx family (Vλ IX). Almost all of the segments (22/24) have different sequences in the complementarity‐determining regions, setting a lower limit to the structural diversity of the antigen binding sites encoded by human Vλ genes in the human population.
Variable region sequences of autoantibodies from mice with experimental systemic lupus erythematosus
Authors: Waisman Ari , Mozes Edna
Published: March 2007

We have sequenced nine monoclonal antibodies (mAb) derived from C3H.SW mice in which experimental systemic lupus erythematosus (SLE) was induced. The hybridomas were selected for binding to DNA or to HeLa nuclear extract (NE). Three mAb were found to bind DNA, and are shown to exhibit sequence characteristics of pathogenic anti‐DNA antibodies. One, mAb 2C4C2, is shown to use a heavy chain V region gene (VH) identical to the VH of anti‐DNA mAb isolated from other lupus‐prone mice, namely (NZB × NZW)F1. The light chain V region gene (VL) of mAb 2C4C2 is 98 % homologous to the VL of another anti‐DNA mAb, also isolated from (NZB × NZW)F1 mice. The other two anti‐DNA mAb, 5G12‐4 and 5G12‐6, share 93 % of their VH sequences with that of mAb 2C4C2. Six mAb bound proteins of HeLa NE. Four of these six antibodies were found to use the VH124 VH and V‐L7 VL. The nine mAb use a total of five VH and four VL germ‐line genes, demonstrating that the autoantibodies induced in mice with experimental SLE do not originate from one B cell clone. Three of these nine VH and VL were identical in sequence to germ‐line genes, while at least three others had somatic mutations. The latter suggests that the above autoantibodies arise in mice by both usage of existing (pre‐immune) B cells, and through an antigen‐driven process. Furthermore, it appears that autoantibodies found in mice with experimental SLE use genetic elements similar to those used by mAb that were isolated from mouse strains which develop lupus spontaneously.
Minute quantities of a single immunodominant foreign epitope are presented as large nested sets by major histocompatibility complex class II molecules
Authors: Vignali Dario A. A. , Urban Robert G. , Chicz Roman M. , Strominger Jack L.
Published: March 2007

The processing and presentation of immunogenetic peptides is an obligate event in the generation of an immune response. However, the degree of complexity with which an immunogenic foreign epitope is presented is still unclear. This question was addressed by analyzing the naturally processed peptides generated from exogenously‐derived hen egg white lysozyme (HEL) bound to the murine major histocompatibility complex (MHC) class II molecule, H‐2Ak. Using reversed‐phase chromatography (RPC), T cell hybridomas and mass spectrometry, 16 peptides were identified that contain the minimal MHC binding epitope 52–61. These peptides exhibited substantial N‐ and C‐terminal extensions and ranged from 13–28 amino acids in length. In contrast, MHC class I molecules present peptides of 8–11 residues and each foreign epitope appears to be represented by only a single peptide. The data here also show that only ∼ 0.8% of the total bound peptide was derived from this single HEL epitope. These findings provide direct evidence that relatively small amounts of processed peptide are required to stimulate an effective T cell response.
CD3‐mediated apoptosis of human medullary thymocytes and activated peripheral T cells: Respective roles of interleukin‐1, interleukin‐2, interferon‐γ and accessory cells
Authors: Groux Hervé , Monte Didier , Plouvier Benedicte , Capron Andre , Ameisen Jean‐Claude
Published: March 2007

Clonal deletion represents an important mechanism for the establishment of tolerance, by the elimination of autoreactive T cells. Deletion is accomplished by programmed cell death, termed apoptosis, induced by mobilization of the T cell receptor (TCR) on both thymocytes and mature T cells. The mechanism which drives T cells towards cell death or cell proliferation after TCR mobilization remains unclear. We show here that the mobilization of the CD3/TCR complex of both CD4+ and CD8+ single‐positive medullary human thymocytes and human mature activated T cells, in the absence of accessory cells, leads to an activation‐induced cell death process by apoptosis. In both cases, apoptosis was associated with interferon (IFN)‐γ gene expression and secretion in the absence of interleukin (IL)‐2 gene expression; and the addition of anti‐IFN‐γ antibody prevented cell death. Apoptosis could also be prevented by cyclosporin A (CsA) treatment and could be re‐induced by the addition of IFN‐γ to CsA‐treated cells. Addition of IL‐2 had two different effects, it prevented apoptosis and also allowed proliferation in response to CD3 monoclonal antibody. Addition of IL‐1, which induces IL‐2 gene expression and secretion or addition of accessory cells, had the same preventive effect. These results suggest that the uncoupling of IFN‐γ and IL‐2 gene expression following CD3/TCR mobilization initiates apoptosis of human T cells at several different stages during development and activation. We propose that co‐signals provided by accessory cells allow a coupling of IL‐2 gene and IFN‐γ gene expression, and that an essential role for IL‐2 secretion in T cell activation involves the inhibition of a death program induced by IFN‐γ secretion.
CD23 and CD21 function as adhesion molecules in homotypic aggregation of human B lymphocytes
Authors: Björck Pia , Elenström‐Magnusson Carina , Rosén Anders , Severinson Eva , Paulie Staffan
Published: March 2007

We have previously found that interleukin‐4 and CD40 monoclonal antibodies (mAb) are strong potentiatiors of homotypic B cell aggregation which is dependent on LFA‐1. We show here that CD23 mAb were also able to inhibit aggregation to a similar extent as LFA‐1 antibodies. This inhibition was restricted to the MHM6 epitope of CD23 and antibodies to other epitopes [Epstein‐Barr virus (EBV) CS‐1, EBV CS‐2, EBV CS‐5 and mAb 25] or occupation of the Fc‐binding site by IgE had no or a slightly enhancing effect on aggregation. When testing two antibodies to CD21, the recently defined ligand for CD23, one of these (BU32) was found to be inhibitory whereas the other (THB5) had no effect. By combining antibodies to LFA‐1 and CD23, aggregation was often completely inhibited. These data suggest that LFA‐1/ICAM‐1 and CD23/CD21 are the major molecules involved in homotypic aggregation of human B cells.
Cholera toxin adjuvant greatly promotes antigen priming of T cells
Authors: Hörnquist Elisabeth , Lycke Nils
Published: March 2007

Cholera toxin (CT) given perorally is a powerful mucosal immunogen and adjuvant. Information that explains the adjuvant effect of CT may be used for the development of more effective oral vaccines and might also contribute to our understanding of the mechanisms involved in regulating mucosal immunity. The present study was undertaken to investigate if CT administered together with keyhole limpet hemocyanin (KLH) would act to promote or inhibit priming of KLH‐specific T cells and whether the adjuvant effect of CT is restricted to mucosal immune responses or is a generalized phenomenon due to direct immunomodulating effects of CT. We found that CT adjuvant greatly augmented the effectiveness of a single oral priming immunization with KLH: re‐challenge with KLH in vitro 1 week following immunization gave several‐fold stronger proliferation in KLH‐specific spleen, mesenteric lymph node, Peyer's patch and gut lamina propria T cells from KLH + CT adjuvant as opposed to KLH only‐treated mice. Moreover, several‐fold stronger cytokine production, i.e. interleukin (IL)‐2, IL‐4, IL‐5, IL‐6, IL‐10 and interferon‐Y accompanied the enhanced proliferative response of T cells from CT adjuvant‐treated mice. The adjuvant effect of CT was not restricted to mucosal immune responses but was evident also following a single parenteral immunization with KLH + CT. Limiting dilution analysis revealed that CT adjuvant promoted a 20‐ to 40‐fold increase in the frequency of primed KLH‐specific T cells. Phenotypic and functional analyses clearly demonstrated that CT adjuvant primarily enhanced priming of CD4+ rather than CD8+ T cells and the pattern of lymphokine secretion disclosed that CT most probably promoted antigen priming of both Thl and Th2 type of CD4+ T precursor cells.
Interleukin‐4 and interleukin‐10 synergize to inhibit cell‐mediated immunity in vivo
Authors: Powrie Fiona , Menon Satish , Coffman Robert L.
Published: March 2007

The lack of cell‐mediated (Thl‐like) immunity that is often associated with strong humoral immune responses is thought to be due in part to the inhibition of Th1 effector function by the Th2‐derived cytokine interleukin‐10 (IL‐10). This hypothesis, however, is based entirely on results from in vitro studies, wherein IL‐10 has been shown to inhibit Thl cytokine synthesis. In this study we have compared the regulatory effects of both IL‐4 and IL‐10 on the development of a more complex Thl effector function in vivo, the development of delayed‐type hypersensitivity (DTH) to Leishmania major in mice immune to Leishmania. The results revealed two findings unexpected from in vitro studies with Thl clones. First, optimal inhibition of the DTH response (up to 70 %), assessed by footpad swelling and leukocytic infiltration, required the combination of IL‐4 and IL‐10, indicating that these two activities synergized to inhibit DTH reactivity. Second, IL‐4 inhibited interferon‐γ (IFN‐γ) production by lymph node cells draining the site of antigen challenge as well as did IL‐10. The combination of both cytokines was no more effective than either alone. The mechanism by which IL‐4 and IL‐10 acted to inhibit DTH responses did not appear to be through inhibition of IFN‐y or tumor necrosis factor production as treatment with antibodies which neutralized these activities failed to inhibit DTH responses. Inhibition of the DTH with IL‐4 and IL‐10 is the most effective specific regulator of DTH responses reported and the only one capable of modulating tuberculin DTH. These data establish IL‐4 and IL‐10 as potent inhibitors of Thl effector function in vivo and suggest their utility in controlling deleterious Thl‐mediated inflammatory responses such as occur in some infectious and autoimmune diseases.
Class I‐deficient resistant mice intracerebrally inoculated with Theiler's virus show an increased T cell response to viral antigens and susceptibility to demyelination
Authors: Pullen Lara C. , Miller Stephen D. , Dal Canto Mauro C. , Kim Byung S.
Published: March 2007

Intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV) results in immune‐mediated demyelination in susceptible mouse strains. The histology of TMEV‐induced demyelination is similar to that seen in patients suffering from multiple sclerosis. It was previously shown that the susceptibility of mice to TMEV‐induced demyelination in certain strain combinations is closely associated with the major histocompatibility complex (MHC) class I locus. Here we examine disease susceptibility of β2‐microglobulin (β2M)‐deficient transgenic mice lacking class I expression and functional CD8+ T cells. In contrast to TMEV‐infected parental C57BL/6 mice, the transgenics develop high levels of virus‐specific DTH and T cell proliferation accompanied by an increased frequency of central nervous system (CNS) demyelinating lesions. However, clinical signs of demyelination were not noted. Neither antibody titer nor viral persistance were significantly affected in the β2M‐deficient mice. These results suggest that in the absence of functional class I/CD8+ cells, the class II‐restricted T cell response to TMEV is enhanced and CNS pathogenesis is heightened, although the level is not severe enough to result in clinical disease. When the TMEV‐infected mice were subcutaneously immunized with virus, however, the β2M‐deficient mice displayed clinical symptoms. Therefore, our results strongly suggest that CD8+ T cells do not directly contribute to CNS demyelination. In contrast, such T cells appear to be primarily involved in down‐regulation of a potentially damaging CD4+ T cell response in resistant animals, although some of the T cells may play a role in clearing viral persistence in the CNS, resulting in the protection of the host from viral demyelination.
Identification and expression of two forms of the human transforming growth factor‐β‐binding protein endoglin with distinct cytoplasmic regions
Authors: Bellón Teresa , Corbi Angel , Lastres Pedro , Calés Carmela , Cebrián Marta , Vera Sonia , Cheifetz Sela , Massague Joan , Letarte Michelle , Bernabéu Carmelo
Published: March 2007

Endoglin is an homodimeric membrane antigen with capacity to bind transforming growth factor‐β (TGF‐β) and whose expression is up‐regulated on myeloid cells upon differentiation to macrophages. We have isolated full‐length cDNA clones from a λgtl0 library, prepared from phorbol 12‐myristate 13‐acetate‐differentiated HL60 cells by screening with an endoglin‐specific cDNA probe from endothelial cells. Sequencing of the largest clone (3073 bp), revealed that the leader sequence contains 25 residues and that the 586 amino acids of the extracellular and transmembrane domains were identical to those described for endothelial endoglin. However, the cytoplasmic tail encoded by this cDNA clone contains only 14 amino acids as opposed to the 47 residues previously reported, suggesting the existence of two alternative endoglin variants. The expression of these isoforms was demonstrated by polymerase chain reaction analyses on endothelial cells, myelomonocytic cell lines HL‐60 and U‐937, and placenta. Independent cDN A constructs corresponding to both forms were transfected into mouse fibroblasts leading to the expression of two distinct endoglin molecules. Both forms were shown to bind TGF‐β1 and, when overexpressed in transfected mouse fibroblasts, to form disulfide‐linked homodimers, indicating that the cysteine residues present in the extracellular domain are responsible for the dimerization.
Kinetic study of interleukin‐2 binding on the reconstituted interleukin‐2 receptor complexes including the human γ chain
Authors: Matsuoka Mitsuo , Takeshita Toshikazu , Ishii Naoto , Nakamura Masataka , Ohkubo Takao , Sugamura Kazuo
Published: March 2007

To gain an insight into roles of the interleukin‐2 (IL‐2) receptor γ chain in IL‐2 binding mechanisms, we examined association and dissociation rate constants for IL‐2 binding with a series of fibroblastoid L929 cell lines expressing IL‐2 receptor complexes reconstituted by transfection with the α, β and γ genes. The association rate constant (k) with the αβγ complex on L cells was fourfold larger than that with the αβ complex on L cells, and the dissociation rate constant (k') was one fifth of that with the αβ complex. These results indicate that the γ chain is involved in both mechanisms by which IL‐2 associates with and dissociates from receptors, resulting in the generation of the high‐affinity IL‐2 receptor along with the α and β chains. During the course of this study, we found that the IL‐2 dissociation from αβγ complex on lymphoid cells was a lot slower than that from the αβγ complex on fibroblast cells. A similar difference was observed with the βγ complex. These observations may indicate functional and constitutional differences of the high‐ and intermediate‐affinity IL‐2 receptor complexes between lymphoid and fibroblastoid cells.
Signaling via CD28 costimulates lymphokine production, but does not reverse unresponsiveness to interleukin‐2 in anti‐CD3 triggered Th1 cells
Authors: Sommer Frank , Röllinghoff Martin , Lohoff Michael
Published: March 2007

Previously, it has been described that the ability of murine Th1 cells to proliferate in response to exogenous interleukin (IL)‐2 is blocked when these cells are exposed to immobilized anti‐CD3 antibodies. In the present study we examined whether simultaneous triggering of the T cell antigen CD28 can prevent the induction of unresponsiveness to IL‐2 in Th1 cells. We report that costimulation of Th1 cells with anti‐CD28 monoclonal antibodies (mAb) did not overcome unresponsiveness to IL‐2 induced by various amounts of immobilized anti‐CD3 antibodies. However, stimulation with anti‐CD28 mAb strongly augmented IL‐2 and interferon‐γ production in anti‐CD3‐exposed Th1 cells. Thus, despite the fact that anti‐CD28 mAb is a potent costimulus for lymphokine production, signaling through CD28 does not seem to be sufficient to trigger proliferation in Th1 cells activated via the T cell receptor. These data suggest the existence of at least three signals to trigger Th1 cell activation. The first is mediated by ligation of the T cell receptor. One cosignal, delivered by the CD28 molecule, leads to IL‐2 production. A third, still undefined, signal is required for proliferation in response to IL‐2.
Surface phenotype and functions of tumor‐infiltrating dendritic cells: CD8 expression by a cell subpopulation
Authors: Chaux Pascal , Hammann Arlette , Martin François , Martin Monique
Published: March 2007

Although the function and significance of tumor‐infiltrating dendritic cells (TIDC) in the immune response to tumor have never been clearly demonstrated, their location suggests that they play a critical role in the presentation of tumor antigen to specific T cells. We studied the morphological and functional characteristics of interstitial dendritic cells (DC) located inside tumors obtained by injection of cancer cells into syngeneic rats. Single and double immunostaining of tumor sections revealed a dense network of cells which expressed class II major histocompatibility complex (MHC II) molecules. Cell morphology and surface markers were characteristic of DC populations in other tissues. These DC were in close contact with tumor cells and increased in number as the tumor grew larger. Unexpectedly, a subpopulation of morphologically characteristic TIDC expressed both CD8 and MHC II molecules. TIDC were purified from tumors by gradient centrifugation and immunobeads and characterized by morphology, ultrastructural study and surface markers studied by flow cytometry. TIDC were negative for the CD5 molecule (a pan T cell marker), and were not labeled with 3.2.3 monoclonal antibody (mAb) (an NK cell marker) or with Ki‐M2R mAb (a macrophage marker). A subpopulation of TIDC expressed the CD8 molecule, confirming the in situ results. TIDC expressed high levels of class I and class II MHC molecules and the adhesion molecule ICAM‐1. This expression is compatible with effective antigen presenting function. Purified TIDC triggered rapid and high levels of proliferation of tumor‐immune T cells in vitro, demonstrating the potential of these cells to constitutively process and present tumor‐associated antigens.
In vitro negative selection of αβ T cell receptor transgenic thymocytes by conditionally immortalized thymic cortical epithelial cell lines and dendritic cells
Authors: Tanaka Yujiro , Mamalaki Clio , Stockinger Brigitta , Kioussis Dimitris
Published: March 2007

We have established conditionally immortalized thymic cortical epithelial cell lines from transgenic mice carrying a temperature‐sensitive SV40 large Tantigen. One of these cell lines expresses cortical markers and produces IL‐1α, IL‐6, IL‐7, and TGF‐β1. These cells express class I major histocompatibility complex (MHC) constitutively and class II MHC upon induction with IFN‐μ. The cells appear to have a normal class I antigen presenting pathway since messages for both peptide transporter genes (TAP1, TAP2) were detected.

The ability of these cortical epithelial cells to present peptide antigen was compared to that of thymic dendritic cells. In suspension culture with αβ Tcell receptor (TcR) transgenic thymocytes, these epithelial cells and dendritic cells (pre‐pulsed with peptide cognate for the transgenic TcR) caused down‐regulation of CD4, CD8, and TcR in an antigen dose‐dependent and MHC‐restricted manner. CD4dullCD8dull cells were taken as evidence for negative selection because these cells contained apoptotic DNA. Concentration of peptide required for negative selection of thymocytes was similar between dendritic cells and cortical epithelial cells. In contrast, αβ transgenic spleen cells were activated only by dendritic cells but not by cortical epithelial cells.
Early B cell development requires μ signaling
Authors: Iglesias Antonio , Nichogiannopoulou Aliki , Stuart Williams G. , Flaswinkel Heinrich , Köhler Georges
Published: March 2007

In vitro studies with Abelson murine leukemia virus (AMuLV)‐transformed murine pre‐B cell lines demonstrated that wild‐type μ but not mutant μ chains lacking the first constant domain (μΔl) can efficiently induce Ig light (L) chain gene rearrangement. Using antibodies against the cytoplasmic tail of the immunoglobulin co‐receptor β (Igβ) chain we find μ., but not μA1 chains associated with Igμ. Since a heterodimer of surface‐labeled proteins was co‐precipitated with n we conclude that only wild‐type μ is associated with the Igα/Igβ co‐receptor on the surface of pre‐B cell lines. Mutant μΔ1 chains achieve their surface expression by utilizing a glycophospholipid anchor. In vivo analysis of transgenic mcie expressing either μ or μΔ1 transgenes revealed the expected “normal” B cell development in the case of wild‐type μ, transgenic lymphocytes, but a block in differentiation of μΔ1 transgenic lymphocytes. The maturation block occurs at the developmental transition of pre‐B lymphocytes from the CD43/S7+, CD45R/B220low stage to the CD43/S7−, B220low/hi8h stage in which the majority of L chain gene rearrangements occur. These results, together with the observed inability of the μΔ1 chains to signal activation of L chain gene joining and to associate Igα/Igβ in pre‐B cell lines suggest that signals mediated by the protein complex composed of μ/Igα/Igβ are crucial during differentiation of pre‐B lymphocytes.
The effect of bone marrow and thymus chimerism between non‐obese diabetic (NOD) and NOD‐E transgenic mice, on the expression and prevention of diabetes
Authors: Parish Nicole M. , Chandler Phil , Quartey‐Papafio Ruby , Simpson Elizabeth , Cooke Anne
Published: March 2007

The non‐obese diabetic (NOD) mouse is an established animal model of the autoimmune disease, insulin‐dependent diabetes mellitus (IDDM). The NOD‐E mouse is a transgenic mouse which expresses the I‐E molecule (absent in NOD mice). Expression of I‐E protects these mice from both insulitis and IDDM.We have investigated the possible mechanisms of this protection by constructing bone marrow, and combined bone marrow and thymus chimeras between NOD and NOD‐E mice.

Our data suggest that thymic epithelium may play no direct role in either protection against, or promotion of, IDDM. Protection from diabetes is provided either by NOD‐E donor bone marrow or NOD‐E recipient non‐thymic radioresistant cells. The means by which protection may be achieved in this system are discussed.
Frequent occurrence of in vivo clonal expansion of CD4− CD8− T cells bearing T cell receptor αβ chains in adult humans
Authors: Kusunoki Yoichiro , Hirai Yuko , Hayashi Tomonori , Kyoizumi Seishi , Takahashi Keiko , Morishita Yukari , Kodama Yoshiaki , Akiyama Mitoshi
Published: March 2007

We have previously reported 2 cases of healthy men showing in vivo monoclonal expansion of mature CD4− CD8− αβ T cells. In the present study, an additional 3 adults were found to exhibit such an expansion, among a total 464 adult donors studied. These 5 individuals were otherwise physiologically normal, with no history of severe illness and autoimmune disease at the time of examination. To investigate the mechanisms of the clonal expansion, further characterization of the clonal cells was attempted. No apparent preference for usage of the Tcell receptor β chain variable region was observed in the clonal T cells. These clonal T cells showed lectin‐dependent or redirected antibody‐dependent cell‐mediated cytotoxicities, whereas they could not lyse autologous lymphoblastoid cell lines. Failure of Fas antigen expression was not observed for any of these clones. These results suggest that clonal expansion of CD4− CD8− αβ T cells frequently occurs in the periphery without any T cell abnormalities.
Stimulation of the intracellular killing of Staphylococcus aureus by human monocytes mediated by Fcγ receptors I and II
Authors: Zheng Limin , Nibbering Peter H. , Van Furth Ralph
Published: March 2007

Previous studies have shown that intracellular killing of bacteria by monocytes is stimulated by interaction between IgG and Fey receptors (FcγR) in the membrane of these cells. In the present study anti‐FcγR monoclonal antibodies (mAb) were used to investigate the relative contributions of the various classes of FcγR to the intracellular killing of Staphylococcus aureus by human monocytes and the biochemical pathways involved. Anti‐FcγRI or anti‐FcγRII mAb, but not anti‐FcγRIII mAb, efficiently stimulated the intracellular killing of bacteria by monocytes. Cross‐linking FcγRI or FcγRII, but not FcγRIII, on monocytes with mouse anti‐FcγR mAb followed by bridging with F(ab′)2 fragments of goat anti‐mouse IgG enhanced this process. Since the NADPH oxidase inhibitor diphenyleneiodonium blocked the FcγR‐mediated intracellular killing of S. aureus, oxygen‐dependent bactericidal mechanisms are most probably involved. Cross‐linking FcγRI or FcγRII but not binding of the mAb to the FcγR on monocytes activated phospholipase C, as demonstrated by the increase in the intracellular concentration of inositol‐ (1,4,5)‐triphosphate. The enhanced intracellular killing stimulated by cross‐linking FcγR on monocytes was completely blocked by U‐73122, an inhibitor of phospholipase C‐dependent processes. Protein kinase C activity, but not the rise in the cytosolic free Ca++ concentration or pertussis toxin‐sensitive G proteins, is essential for the FcγR‐mediated intracellular killing of bacteria by monocytes.

Together, these results demonstrate that cross‐linking FcγRI or FcγRII is equally effective in stimulating the intracellular killing of bacteria by monocytes and that this stimulation is a phospholipase C‐dependent process.
T cell‐mediated cognate signaling of nitric oxide production by macrophages. Requirements for macrophage activation by plasma membranes isolated from T cells
Authors: Tao Xiang , Stout Robert D.
Published: March 2007

Macrophage generation of reactive nitrogen intermediates (RNI) represents a major effector mechanism in anti‐microbial immunity and non‐septic inflammatory reactions. The induction of macrophage RNI production has been demonstrated to require at least two signals which in microbial infections can be provided by interferon (IFN)‐γ and lipopolysaccharide (LPS). The current study demonstrates that, in the absence of LPS, T lymphocytes can provide cognate signal(s) which synergize with IFN‐γ in stimulating macrophage RNI production, as evidenced by the ability of plasma membranes from T cell clones to activate IFN‐γ‐primed macrophages. Although viable resting T cells can activate IFN‐γ‐primed macrophages by an interaction that is antigen specific, plasma membranes from resting T cells do not activate macrophages. Plasma membranes from T cells activated by immobilized anti‐CD3 were able to effectively induce RNI production in IFN‐γ‐primed macrophages. However, in contrast to the antigen‐specific interaction of macrophages with viable resting T cells, the activation of IFN‐γ‐primed macrophages by membranes from activated T cells does not display antigen specificity. Plasma membranes from activated T helper TH2 and from activated TH1 cells were equally effective in activating IFN‐γ‐primed macrophages, suggesting that the dominance of TH1 over TH2 cells in cell‐mediated responses involving macrophage effectors is not a reflection of differences in their ability to interact with macrophages but rather is a reflection of their different pattern of cytokine production. These results suggest that the T cell‐macrophage interaction involves reciprocal activation of both cells ‐ an antigen‐specific activation of the T cells which results in the acquisition of T cell membrane components involved in antigen‐nonspecific stimulation of the macrophages.
Engagement of major histocompatibility complex class II molecules leads to nitrite production in bone marrow‐derived macrophages
Authors: Hauschildt Sunna , Bessler Wolfgang G. , Scheipers Peter
Published: March 2007

The present study was designed to examine whether engagement of major histocompatibility complex (MHC) class II molecules can lead to induction of NO synthase in bone marrow‐derived macrophages. We treated the macrophages with toxic shock syndrome toxin 1 (TSST‐1), a superantigen which activates T cells in an MHC class II‐dependent manner. Upon addition of syngeneic spleen cells as a source of mature T cells to the TSST‐1‐treated macrophage culture, NO2− production was greatly increased. To test whether monoclonal antibodies (mAb) to MHC class II antigens also serve as an effective trigger signal for induction of NO synthase we incubated the cells with the anti‐I‐Ad/b mAb D3.137 and measured NO2− production in culture supernatants. The addition of the mAb D3.137 resulted in NO2− production which was completely suppressed by NG‐monomethyl‐L‐arginine, a homologue of L‐arginine, indicating that antibody‐induced NO2− production was due to activation of NO synthase. The ability of anti‐I‐A antibodies, which may imitate the effects of T cells, to induce NO2− production suggests that MHC class II molecules act as transmembrane signal transducers finally leading to induction of NO synthase.
Induction of T cell responses by chimeric bacterial proteins expressing several copies of a viral T cell epitope
Authors: Lo‐Man Richard , Martineau Pierre , Hofnung Maurice , Leclerc Claude
Published: March 2007

A viral T cell epitope was genetically inserted within the periplasmic MalE protein of Escherichia coli in two different permissive insertion sites and resulting hybrid proteins were used to study the in vitro and in vivo immunogenicity of the foreign T cell epitope. Purified hybrid MalE proteins containing the T cell epitope 120‐132 (PreS:T) from PreS2 region of hepatitis B virus HBsAg inserted alone or with its adjacent B cell epitope (132‐145) were able to induce strong peptide‐specific T cell responses in mice. In vitro stimulation of primed lymph node cells or specific T cell hybridomas by the hybrid proteins required processing of the inserted T cell epitope and was inhibited by antigen‐presenting cells fixation. The inserted T cell epitope was presented in vitro, in association with appropriate major histocompatibility complex molecules, as efficiently as free synthetic peptide. The in vitro immunogenicity of MalE hybrid proteins was increased by inserting four tandemly repeated copies of PreS:T, either at site 133 or 303. These results were confirmed in vivo by comparing the proliferative responses of lymph node cells from DBA/1 mice primed with MalE hybrid proteins containing one or four copies of PreS:T. Thus, the use of MalE hybrid proteins expressing multiple copies of a given foreign T cell epitope allows the induction of peptide‐specific T cell response with a lower dose of priming antigen.
Murine macrophages lack expression of the Cr2‐145 (CR2) and Cr2‐190 (CR1) gene products
Authors: Martin Brian K. , Weis John H.
Published: March 2007

Murine macrophages have been described as possessing complement receptors for C3b and iC3b. These binding activities have been assumed to be due to the presence of the CR1 and CR3 proteins, respectively. The mouse Cr2 gene produces two distinct gene products of approximately 190000 relative molecular mass (Mr) (Cr2‐190) and 145 000 Mr (Cr2‐145). Because of the similarity in size to human complement receptors, the Cr2‐190 protein has been dubbed murine CR1 while the murine Cr2‐145 product has been termed murine CR2. In order to define the complement receptor genes expressed by murine macrophages, we investigated the expression patterns of Cr2‐190, Cr2‐145 and another mouse complement receptor, Crry, in three different mouse macrophage populations: bone marrow‐derived macrophages, thioglycollate‐elicited peritoneal macrophages and the macrophage cell line, J774. Neither of the Cr2 gene transcripts encoding the Cr2‐145 and Cr2‐190 proteins could be detected in these populations by RT‐RPCR analysis although Crry transcripts were evident. 02‐145 and Cr2s‐190 proteins could not be detected on the surface of thioglycollate‐elicited macrophages using a monoclonal antibody that recognizes both proteins. Thus, contrary to previously published data, murine macrophages do not possess the Cr2 gene products.
Interleukin‐4 and interleukin‐10 synergize to inhibit cell‐mediated immunity in vivo
Authors: Powrie Fiona , Menon Satish , Coffman Robert L.
Published: March 2007

The lack of cell‐mediated (Thl‐like) immunity that is often associated with strong humoral immune responses is thought to be due in part to the inhibition of Thl effector function by the Th2‐derived cytokine interleukin‐10 (IL‐10). This hypothesis, however, is based entirely on results from in vitro studies, wherein IL‐10 has been shown to inhibit Thl cytokine synthesis. In this study we have compared the regulatory effects of both IL‐4 and IL‐10 on the development of a more complex Thl effector function in vivo, the development of delayed‐type hypersensitivity (DTH) to Leishmania major in mice immune to Leishmania. The results revealed two findings unexpected from in vitro studies with Thl clones. First, optimal inhibition of the DTH response (up to 70 %), assessed by footpad swelling and leukocytic infiltration, required the combination of IL‐4 and IL‐10, indicating that these two activities synergized to inhibit DTH reactivity. Second, IL‐4 inhibited interferon‐γ (IFN‐γ) production by lymph node cells draining the site of antigen challenge as well as did IL‐10. The combination of both cytokines was no more effective than either alone. The mechanism by which IL‐4 and IL‐10 acted to inhibit DTH responses did not appear to be through inhibition of IFN‐γ or tumor necrosis factor production as treatment with antibodies which neutralized these activities failed to inhibit DTH responses. Inhibition of the DTH with IL‐4 and IL‐10 is the most effective specific regulator of DTH responses reported and the only one capable of modulating tuberculin DTH. These data establish IL‐4 and IL‐10 as potent inhibitors of Thl effector function in vivo and suggest their utility in controlling deleterious Thl‐mediated inflammatory responses such as occur in some infectious and autoimmune diseases.
Ligation of B7 with CD28/CTLA‐4 on T cells results in CD40 ligand expression, interleukin‐4 secretion and efficient help for antibody production by B cells
Authors: de Boer Mark , Kasran Ahmad , Kwekkeboom Jaap , Walter Hugo , Vandenberghe Peter , Ceuppens Jan L.
Published: March 2007

It has been extensively shown that when T cells are co‐stimulated with B7‐CD28 interaction, a strong proliferative as well as cytolytic T cell response can be induced. In contrast, there exists only indirect evidence that the B7‐CD28 interaction is of importance for the induction of T cell helper functions in B cell responses. Here we have used mouse fibroblasts transfected with the human Fcγ receptor type II and human B7 to address this issue. We found that T cells, when activated through the T cell receptor (TcR)/CD3 complex with monoclonal antibodies and co‐stimulated by B7‐CD28 interaction, can provide efficient help for the induction of both IgM and IgG production by resting B cells. This helper activity is, at least in part, mediated by the interaction between the CD40 ligand on the T cells and CD40 on the B cells. We also demonstrate that more than one signal to the T cell is required for the induction of the CD40 ligand, one being delivered through the TcR/CD3 complex and the second by ligation of CD28 with the B7 molecule. In addition to the induction of cognate T helper function, we provide evidence that co‐stimulation of T cells with B7‐CD28 interaction can result in the secretion of both Th1‐ and Th2‐type lymphokines.
Lineage relationships of the fetal thymocyte subset that expresses the β chain of the interleukin‐2 receptor
Authors: Falk Ingrid , Levelt Christiaan N. , Eichmann Klaus
Published: March 2007

The β chain (p75) of the interleukin‐2 (IL‐2) receptor (IL‐2R) is expressed on up to 5‐7% of fetal thymocytes on day 16 of gestation, declining thereafter to a minute proportion of less than 1% around birth, and of 1‐2% of adult thymocytes. A significant part of fetal IL‐2R β+ thymocytes are γδ cells. The precursor‐progeny relationships of fetal DL‐2Rβ+ thymocytes to the αδ T cell lineage have not been previously studied, nor has their position within the developmental sequence been determined. Here we show that IL‐2Rβ is expressed on a subset of very immature cells, along with high amounts of Pgp1 and FCγRII/III, partially preceding the expression of intracellular CD3ϵ. IL‐2‐Rβ disappears before expression of IL‐2Rα. IL‐2Rβ+ cells, purified by sorting on day 15 of gestation, efficiently reconstituted fetal thymic lobes depleted of lymphoid cells by treatment with desoxyguanosine. They developed into T cell receptor (TCR)αβ+, TCRγδ+, and CD4/CD8 double‐ and single‐positive cells in similar proportions as did sorted IL‐2Rα+ day 15 fetal thymocytes. These data suggest that IL‐2Rβ expression marks a short period of very early thymocyte development, perhaps immediately after entry into the thymus.