bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoglobulin production in severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood mononuclear cells
Authors: Abedi Mohammad R. , Christensson Birger , Islam Khalid B. , Hammarström Lennart , Edvard Smith C. I.
Published: March 2007

Immunodeficient C.B‐17 scid/scid (SCID) mice were reconstituted with human peripheral blood mononuclear cells and analyzed for humoral immunity. The majority of adoptively transferred animals had serum levels of 1–4 mg/ml of human IgG 8–12 weeks after i.p. reconstitution with 20 × 106 PBMC, whereas the IgM and especially IgA concentrations were considerably lower. The half‐lives of human IgG, IgM, and IgA in SCID mice were 12 days, 36, and 23 hours, respectively. Furthermore, IgA was rapidly secreted into bile indicating that the low IgA concentration was mainly caused by a high turnover rate. IgG subclass distribution in mouse serum was similar to that found in the donor serum. Irradiation with 3 Gy prior to adoptive transfer resulted in increased levels of human IgG early after reconstitution, whereas both IgM and IgA concentrations were impaired. A polyclonal serum Ig pattern was found 4 weeks after transfer of human cells later frequently followed by a predominance of oligoclonal bands. Unexpectedly, oligoclonal bands were also found using donors with a negative Epstein‐Barr virus serology. Human cells were found to reside in the peritoneal cavity for several months. Within 2 weeks of reconstitution, human cells were also identified in lymphoid structures in the vicinity of the liver hilus with a later spread to other lymphoid organs. Homing of human cells to skin and gut was not seen.
Cell surface marker analysis of mouse thymic dendritic cells
Authors: Ardavin Carlos , Shortman Ken
Published: March 2007

Cell surface markers of mouse thymic dendritic cells have been studied by flow cytometry after isolation by collagenase digestion, separation of the low‐density cell fraction and differential adherence. The dendritic cell preparation had a purity of > 90%, the contaminating population being essentially composed of thymocytes, macrophages constituting <1%. Dendritic cells displayed high forward and low‐intermediate side angle scatter, and expressed high levels of major histocompatibility complex (MHC) class I and class II molecules, the heat‐stable antigen (HSA), the adhesion molecules Pgp‐1 (CD44), LFA‐1, ICAM‐1 and low levels of Mac‐1 and the leukocyte common antigen CD45. Thymic dendritic cells are negative for the stem cell antigen‐2 (Sca‐2), the B cell‐specific form of CD45 (B220), the mouse macrophage markers Fc receptor and F4/80, and the granulocyte marker Gr‐1. However, although they do not express the T cell markers Thy‐1, CD2, CD3, CD4 and CD5, 20%–30% of dendritic cells are positive for the interleukin 2 receptor α chain (CD25), and about 30% express intermediate levels of CD8.

These results are discussed with regard to the functional significance of the expression of CD8 by thymic dendritic cells, and the existence of different dendritic cell subpopulations in the murine thymus.
Impaired post‐transcriptional expression of interleukin‐2 receptor in pokeweed mitogen‐activated T cells
Authors: de la Calle‐Martín Oscar , Alberola‐Ila José , Engel Pablo , Inglés Julia , Fabregat Virginia , Barceló Joan Josep , Lozano Francisco , Gallart Teresa
Published: March 2007

The expression and role of interleukin‐2/interleukin‐2 receptor (IL‐2/IL‐2R) system in the pokeweed mitogen(PWM)‐induced T cell mitogenesis was studied. In the absence of monocytes (Mo), both soluble and Sepharose‐bound PWM fail to induce T cell mitogenesis even when exogenous IL‐2 or IL‐1 or IL‐1+IL‐2 or IL‐4 are also present. In the presence of Mo, PWM stimulation of T lymphocytes (highly depleted of B lymphocytes) induces as much IL‐2 mRNA as phytohemagglutinin (PHA), but results in higher and persistent IL‐2 levels in culture supernatants despite the concomitant T cell mitogenesis, suggesting that PWM‐activated T cells do not utilize the IL‐2 they produce. Confirming this notion, Mo‐dependent PWM‐preactivated T cells, as compared to PHA‐preactivated ones: (a) failed to consume exogenous IL‐2 and their mitogenic response did not increase upon exposure to exogenous IL‐2; (b) exhibited very low numbers of high‐affinity IL‐2R; and (c) showed lower expression of IL‐2R p55 and undetectable expression of IL‐2R p75 on their surface. Moreover, the PWM‐induced T cell mitogenesis was not inhibited by anti‐IL‐2 or CD25 antibodies and only partially (50%–60%) inhibited by cyclosporin A, while these treatments abrogated the PHA‐induced one. PWM‐activated T cells, as compared to the PHA‐activated ones, exhibited as high (p55) or even higher (p75) mRNA expression of both IL‐2R p55 and p75 subunits. The possibility that PWM interferes with IL‐2R subunits once expressed on the T cell surface was excluded. Thus, intracellular PWM‐related events are likely to impair IL‐2R expression post‐transcriptionally. Possible explanations for this effect and its relation with the capacity of PWM to induce T cell‐dependent B cell differentiation are discussed.
The cell‐mediated response to schistosomal antigens at the clonal level: development and characterization of a panel of egg antigen‐specific murine T cell clones
Authors: Chikunguwo Silas M. , Harris Timothy S. , Brodeur Peter H. , Harn Donald A. , Stadecker Miguel J.
Published: March 2007

The cellular basis of the immune response underlying the granulomatous hypersensitivity in experimental murine schistosomiasis caused by Schistosoma mansoni was investigated by examining a panel of 16 egg antigen‐specific T cell clones. The clones were derived from a sensitized T cell line by limiting dilution, and were selected on the basis of their strong responses against schistosomal egg antigens. By cytofluorographic analysis, it was determined that all clones were T helper cells and expressed the CD3+CD4+CD8− phenotype. Lymphokine analysis revealed that some clones secreted either interleukin (IL)‐2 or IL‐4, but a surprisingly large number were double producers. Southern blot analysis verified the clonality of these T cells and indicated that the clones examined included at least five independent clones by the criterion of T cell receptor β gene rearrangements. Despite their diversity, the clones responded strongly, and virtually exclusively, to egg antigen components with isoelectric points in the limited range of 4.7 to 5.2. The relevant antigenic egg molecules were shown to require processing by accessory cells for presentation to, and stimulation of, the T cell clones.
Inhibition of superantigen recognition by peptides of the variable region of the T cell receptor β chain
Authors: Macneil Donna , Fraga Ester , Singh Bhagirath
Published: March 2007

T cells bearing certain variable (V) regions of the T cell receptor (TcR), including Vβ3, Vβ6, Vβ8.1 and Vβ9, are stimulated by one or more forms of the endogenous superantigen, mouse lymphocyte stimulatory (Mls) locus, encoded by the mouse mammary tumor virus, in the context of a non‐polymorphic region of the class II molecules of the major histocompatibility complex (MHC). To identify putative sites of interaction of TcR‐Vβ region and Mls‐1a, we examined the effect of peptides derived from the protein sequence of Vβ6 on recognition of Mls‐1a by T cell hybridomas and show that three peptides corresponding to amino acid positions 1 to 20, 48 to 75, and 58 to 75 of the Vβ6 peptide sequence interfere with the activation of several Vβ6+ hybridomas by Mls‐1a‐bearing spleen cells, but not with that of a Vβ8+ hybridoma. The Mls‐reactive hybridomas are specific for a synthetic peptide poly‐18, poly EYK(EYA)5 and its peptide (EYA)5, in the context of I‐Ad. This peptide does not require processing and the peptides 1–20, 48–75, and 58–75 do not inhibit recognition of (EYA)5 by the same Vβ6+ T cell hybridomas. The two sequences 1–20 and 58–75 are proposed to lie outside the putative binding domain of processed antigen, indicating that recognition by TcR of Mls‐1a is different from the classical MHC‐restricted recognition of processed antigen. These results suggest that the recognition of superantigen/class II MHC by T cells can be inhibited by peptides related to the site of interaction of the TcR, suggesting that such peptides could have possible regulatory effects on the induction and regulation of immune responses.
Opposing effects of tumor necrosis factor‐α and nerve growth factor upon leukotriene C4 production by human eosinophils triggered with N‐formyl‐methionyl‐leucyl‐phenylalanine
Authors: Takafuji Shigeru , Bischoff Stephan C. , de Weck Alain L. , Dahinden Clemens A.
Published: March 2007

Tumor necrosis factor (TNF) as well as the hematopoietic growth factors interleukin‐3, interleukin‐5, and granulocyte‐macrophage colony‐stimulating factor affect several eosinophil functions. We previously reported (J. Exp. Med. 1989. 170: 467; 1990. 172: 1577) that the hematopoietic growth factors also potentiate leukotriene C4 (LTC4) formation by eosinophils as well as basophils stimulated with soluble chemotactic peptides such as N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP), but whether TNF also modulates lipid mediator generation in normodense eosinophils triggered with FMLP is unkonwn. Here we show that a short preincubation (10 min) of human eosinophils purified from healthy donors with low concentrations of TNF (5‐150 pM) strongly enhances LTC4 formation in response to FMLP. However, basophil mediator release is not affected by TNF preincubation. Nerve growth factor (NGF), the receptor of which is structurally related to the TNF receptors, tended to suppress lipid mediator synthesis in eosinophils, in contrast to its profound potentiating capacity on basophil mediator release. Thus, the present study demonstrates a first difference in susceptibility of eosinophils and basophils towards cytokines, indicating that TNF and NGF may regulate the relative importance of effector functions of these otherwise closely related cell types.
Somatically mutated IgG anti‐DNA antibody clonally related to germ‐line encoded IgM anti‐DNA antibody
Authors: Taki Shinsuke , Hirose Sachiko , Kinoshita Kohji , Nishimura Hiroyuki , Shimamura Toshiro , Hamuro Junji , Shirai Toshikazu
Published: March 2007

Among 15 anti‐DNA antibody‐producing hybridomas derived from a single NZB × NZW F1 mouse, an IgM and an IgG were shown to use the same VH gene of the Q52 family. Using a combination of two primers both consisting of a mixture of oligonucleotides (one complementary to the 5′ end of VH segment and one to the 3′ end of VH segment of Q52 family) we determined the sequences of several members of germ‐line VH genes in the Q52 family derived from NZB and NZW strains. Comparison of the sequences with those of cloned VH cDNA obtained from the hybridomas revealed that the VH sequence of the IgM anti‐DNA antibody was identical to that of a cloned NZW germ‐line VH gene, except for the priming sites. In contrast, the VH sequence of the IgG counterpart contained somatically mutated nucleotides. Because the IgG anti‐DNA antibody showed a higher DNA binding activity than did the IgM antibody, we conclude that these changes in nucleotide sequences were induced and selected through an antigen‐driven mechanism as is the case in a normal immune response. It is tempting to speculate that the germ‐line encoded, low‐affinity IgM autoantibody undergoes somatic mutations and isotype switching, resulting in generation of pathogenic, high‐affinity autoantibodies in autoimmune diseases.
The stromal cell line S17 supports the growth of lipopolysaccharide‐stimulated CBA/N spleen cell colonies in vitro
Authors: Narendran Arumugavadivel , Cumano Ana , Dorshkind Kenneth , Paige Christopher J.
Published: March 2007

The expression of an X‐linked defect in the CBA/N strain of mice has been found to result in a number of immune abnormalities. These include low responsiveness to antigens and a greatly reduced ability to respond to many of the common B cell mitogens. An in vitro manifestation of this condition is the virtual inability of CBA/N B cells to form colonies in lipopolysaccharide (LPS)‐containing semisolid agar cultures. In this report we show evidence that the colony‐forming ability of CBA/N spleen cells can be effectively restored by the bone marrow stromalderived cell line S17. Spleen cells from 4–5‐week‐old homozygous CBA/N female mice were grown in double‐layer agar cultures containing S17 feeder layers. Control cultures contained the fibroblast‐like cell line 95.17 or were treated with medium alone. It was found that at an input cell concentration of 104 cells per plate, CBA/N colony formation was increased from a frequency of approximately 1 in 5000 to 1 in 50 total splenic cells. Studies with purified surface immunoglobulin‐positive cells indicate the direct involvement of S17 in this process. The CBA/N colonies formed were dependent on the presence of a mitogen (LPS) and secreted detectable amounts of IgM. Major implications of these findings and the application of this assay system to study the CBA/N defect have been discussed.
Interleukin‐7 expression during mouse thymus development
Authors: Wiles Michael V. , Ruiz Patricia , Imhof Beat A.
Published: March 2007

We have monitored the expression of interleukin‐7 (IL‐7) in the developing embryonic mouse thymus by a combination of quantitative polymerase chain reaction (PCR) and immunofluorescence microscopy. A strong specific signal for IL‐7 mRNA was detected by day 12 in the developing fetal thymus. IL‐7 mRNA was found to be maximally expressed on day 15, and then decreased over the next 5 days. Immunofluorescence staining of fetal thymus sections using an anti‐IL‐7 antibody confirmed these PCR data. IL‐7 protein expression was first detected at day 13 of development. At 14 days the intensity of the staining increased by a factor of three and stayed at this level over the next 4 days. The same anti‐IL‐7 antibody used for immunofluorescence, blocked the proliferation of fetal thymocytes in organotypic cultures. In addition, we detected mRNA coding for IL‐2 and SCF (also known as the steel factor or KL) in embryonic thymocytes. The implications of these findings for early thymocyte growth are discussed.
A lymphocyte differentiation and activation antigen, CZ‐1, that distinguishes between CD8+ and unstimulated CD4+ T lymphocytes
Authors: Vargas‐Cortes Mauricio , O'Donnell Carey L. , Appel Michael C. , Maciaszek Joseph W. , Yurkunas Kate S. , Welsh Raymond M.
Published: March 2007

We report the generation and cellular reactivity of a novel rat IgM monoclonal antibody (mAb), CZ‐1, made against mouse natural killer (NK) cells activated in vivo. mAb CZ‐1 recognizes a molecule whose properties are consistent with that of a trypsin‐sensitive, non‐phosphatidyl inositol‐linked sialoglycoprotein. The expression of the antigen recognized by mAb CZ‐1 is restricted mostly to cells of the lymphoid lineage. The antigen is expressed on 10%–25% of bone marrow cells and 3%–5% of thymocytes. Analysis of thymocyte subpopulations indicates expression of the CZ‐1 antigen on 100% of the NK1.1+, 27% of the CD4−CD8−, 1.1% of the CD4+CD8+, 1.1% of the CD4+CD8−, and 33% of the CD4−CD8+ cells. In the spleen, the CZ‐1 antigen is expressed on B lymphocytes, NK cells, and virtually all CD8+ T lymphocytes. Most unstimulated CD4+ splenic T lymphocytes, monocytes and polymorphonuclear cells, with the notable exception of basophils, do not react with mAb CZ‐1. CD4+ T cells activated in vivo by virus infection or in vitro by anti‐CD3 and interleukin‐2 express the CZ‐1 antigen. These results indicate that mAb CZ‐1 identifies a novel inducible lymphocyte activation/differentiation antigen that distinguishes between thymic and unstimulated splenic CD4+ and CD8+ T lymphocytes. This mAb will be a useful tool in the identification of lymphocyte subpopulations and in the study of the ontogeny and activation of these cells.
Differential expression of granzyme A and granzyme B proteases and their secretion by fresh rat natural killer cells (NK) and lymphokine‐activated killer cells with NK phenotype (LAK‐NK)
Authors: Velotti Francesca , Palmieri Gabriella , D'Ambrosio Daniele , Piccoli Mario , Frati Luigi , Santoni Angela
Published: March 2007

Granzymes A—G are a family of serine proteases localized in the cytoplasmic granules of cytotoxic T lymphocytes (Masson, D. et al. Cell 1987. 49: 679) and granzyme A is secreted by T lymphocytes in response to antigenic stimulation. Granzyme A is also expressed by natural killer (NK) and lymphokine‐activated killer (LAK) cells. Here we show that fresh rat NK cells constitutively express granzyme B and that granzyme A and granzyme B are differentially regulated in unstimulated NK cells vs. LAK cells with NK phenotype (LAK‐NK cells). We also show that both granzymes A and B are secreted in a calcium‐dependent manner, by NK and LAK‐NK cells in response to stimuli which trigger NK cell functions.
Increased numbers of T cells recognizing multiple myelin basic protein epitopes in multiple sclerosis
Authors: Olsson Tomas , Sun Jiabin , Hillert Jan , Höjeberg Bo , Ekre Hans‐Peter , Andersson Gudrun , Olerup Olle , Link Hans
Published: March 2007

Myelin basic protein (MBP)‐autoreactive T cells have a crucial pathogenetic role in experimental allergic encephalomyelitis (EAE) and certain MBP epitopes may be immunodominantly recognized. The heterogeneity and quantity of the T cell response to different epitopes of MBP in multiple sclerosis (MS) and non‐MS controls is not so clearly defined. We now study T cell reactivity to six different peptides of MBP in MS compared to controls in short‐term cultures of blood mononuclear cells by measuring numbers of T cells that secrete interferon‐γ in response to antigen. In comparison with controls, MS patients showed dramatically increased numbers of MBP peptide‐reactive T cells with mean values varying between 10.4 and 22.5 per 105 blood mononuclear cells. Among those MBP peptides examined (amino acid 1–20, 63–88, 89–101, 96–118, 110–128 and 148–165), no single peptide is preferentially recognized. Neither is any preferential response apparent after subdivision of the MS patients according to their HLA‐DR genotype. Our findings suggest that a quantitative increase of a broad repertoire of myelin‐autoreactive T cells with capacity to secrete IFN‐γ can be important for the pathogenesis of MS.
Membranes of activated CD4+ T cells expressing T cell receptor (TcR) αβ or TcR γδ induce IgE synthesis by human B cells in the presence of interleukin‐4
Authors: Gascan Hugues , Aversa Gregorio G. , Gauchat Jean‐Françlois , Van Vlasselaer Peter , Roncarolo Maria‐Grazia , Yssel Hans , Kehry Marilyn , Spits Hergen , De Vries Jan E.
Published: March 2007

In the present study it is demonstrated that human B cells can be induced to switch to IgE production following a contact‐mediated signal provided by activated T cell receptor (TcR) γδ+, CD4+ and TcR αβ+, CD4+ T cell clones and interleukin (IL)‐4. The signal provided by these T cell clones was antigen nonspecific, indicating that the TcR αβ/CD3 or TcR γδ/CD3 complexes were not involved in these T‐B cell interactions. Activated TcR αβ+, CD8+, and TcR γδ+, CD4−CD8−, or resting CD4+ T cell clones were ineffective. Intact TcR αβ+ or TcR γδ+, CD4+ T cell clones could be replaced by plasma membrane‐enriched fractions isolated from these activated CD4+ T cell clones. In contrast, membranes isolated from resting TcR αβ+, CD4+, TcR γδ+, CD4+ T cell clones or an Epstein‐Barr virus (EBV)‐transformed B cell line (EBV‐LCL) failed to provide the costimulatory signal that, in addition to IL‐4, is required for induction of IgE synthesis. As described for intact CD4+ T cells, CD4+ T cell membranes induced purified surface IgM+ B cells to switch to IgG4‐ and IgE‐ but not to IgA‐producing cells, excluding the possibility of a preferential outgrowth of IgG4‐ and IgE‐committed B cells. The membrane activity was inhibited by protease or heat treatment. Induction of IgE synthesis by B cells co‐cultured with both TcR αβ+, CD4+ and TcR γδ+, CD4+ T cell clones and membrane preparations of these cells was blocked by anti‐class II major histocompatibility complex (MHC) monoclonal antibodies (mAb), whereas various anti‐CD4 mAb had differential blocking effects. Murine L cells, or EBV‐LCL transfected with CD4 could not replace CD4+ T cell clones. These results indicate that, although CD4 and class II MHC antigens are required for productive CD4+ T cell clone‐B cell interactions, an additional signal, provided by a membrane associated (glyco)protein that is induced by activation of both TcR αβ and TcR γδ, CD4+ T cells, is needed for induction of IgE production in the presence of IL‐4.
Interleukin‐1 up‐regulates transcription of its own receptor in a human fibroblast cell line TIG‐1: role of endogenous PGE2 and cAMP
Authors: Takii Takemasa , Akahoshi Tohru , Kato Koichi , Hayashi Hidetoshi , Marunouchi Tohru , Onozaki Kikuo
Published: March 2007

The regulation of interleukin‐1 receptor (IL‐1R) mRNA expression by IL‐1 in a human lung fibroblast cell line (TIG‐1) was investigated. After 2 h of stimulation with human recombinant IL‐1α or IL‐1β, the levels of T cell/fibroblast‐type IL‐1R mRNA increased, and the elevation was sustained for at least 72 h. IL‐1 also stimulated synthesis of prostaglandin E2 (PGE2) and secondary cAMP accumulation. Exogenously added PGE2 increased the levels of both IL‐1R mRNA and intracellular cAMP. Forskolin, cholera toxin and 8‐Bromo adenosine (8‐Br‐cAMP) all increased IL‐1R mRNA levels. Indomethacin blocked IL‐1 stimulation of IL‐1R mRNA expression, PGE2 production and cAMP. 125I‐labeled IL‐1α‐binding studies showed that this cell line expresses 2.6 × 104 IL‐1R per cell with a Kd of 5.1 × 10−10 M. After treatment of the cells with IL‐1, the level of IL‐1R increased over that of control cells. PGE2 also increased IL‐1R without alteration in its affinity. Cross‐linking experiments indicate that this cell line expresses the 80‐kDa receptor molecule before and after treatment with PGE2; the molecular mass corresponds to the T cell/fibroblast type IIL‐1R. These results indicate that IL‐1 does not directly stimulate expression of IL‐1R mRNA or cell surface IL‐1R, but only indirectly by stimulation of endogenous PGE2.
Origin of CD5+ B cells and natural IgM‐secreting cells: reconstitution potential of adult bone marrow, spleen and peritoneal cells
Authors: Thomas‐Vaslin Véronique , Coutinho Antonio , Huetz François
Published: March 2007

The bulk of natural IgM secretion is currently attributed to peritoneal CD5+ B cells and their progeny, believed to be independent of adult bone marrow precursors. We have compared the capacity of peritoneal or splenic cells from normal adult mice to generate serum IgM after transfer into allotype‐congenic, irradiated and bone marrow‐protected mice. Recipients of either cell population produced donor‐allotype IgM‐secreting cells in the spleen, and had donor‐derived serum IgM. In both cases as well, recipient IgM secretion recovered to control levels. Since the spleen cell‐derived natural IgM production could result from expansion of CD5+ B cells present in the inoculum, we next investigated the ability of Ig− bone marrow (BM) cells (Ig− BM) to reconstitute natural IgM secretion in irradiated mice. This cell population was most efficient in reconstituting donor‐derived IgM secretion.

The origin and phenotype (IgM, CD5) of B cells present in spleen and peritoneum of recipient mice were also analyzed. In agreement with the high level of donor IgM‐secreting cells, transfers of splenic and Ig− BM cells fully reconstitute donor B cells in spleen and peritoneum and inhibit reconstitution from host origin. In contrast, donor peritoneal cells reconstitute B cells very poorly in spleen and allow for reconstitution by host cells. Furthermore, Ig− BM cells as well as splenic or peritoneal donor cells, all reconstitute CD5+ B cells in the peritoneum of recipient mice. Interestingly, the fraction of IgM+ cells of each allotype that differentiate to IgM secretion varies widely, but normal levels of IgM are established even when the number of donor B cells present in the animal is very limited.
An experimentally validated panel of subfamily‐specific oligonucleotide primers (Vα1‐w29/Vβ1‐w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction
Authors: Genevée Catherine , Diu Anita , Nierat Janine , Caignard Anne , Dietrich Pierre‐Yves , Ferradini Laurent , Roman‐Roman Sergio , Triebel Frédéaric , Hercend Thierry
Published: March 2007

We report here the characterization of a series of T cell receptor (TcR) Vα or Vβ subfamily‐specific oligonucleotide primers. Criteria that have guided the design of each oligonucleotide include appropriate thermodynamic parameters as well as differential base‐pairing scores with related and unrelated target sequences. The specificity of the oligonucleotides for each Vα or Vβ subfamily was tested by polymerase chain reaction (PCR) on both a series of TcR encoding plasmid DNA and clonal T cell populations. Unexpected cross‐reactivities were observed with plasmid cDNA sequences corresponding to unrelated subfamily gene segments. This led to the synthesis of additional series of oligonucleotides to obtain a relevant panel. A series of Vα1‐w29/Vβ1‐w24 TcR subfamily‐specific oligonucleotides was eventually selected which generates little, if any, cross‐reactivity. The use of Cα or Cβ primers for the amplification of internal positive control templates (i.e. Cβ for the Vα series and Cα for the Vβ series) has been tested in PCR performed with cDNA derived from peripheral blood lymphocytes; it was shown not to alter the amplification of the V subfamily‐specific DNA fragments. This panel of oligonucleotides will be helpful in the study of TcR V gene segment usage and, thus, may lead to a better characterization of T cell responses in physiological and pathological situations.
Rapid activation‐independent shedding of leukocyte L‐selectin induced by cross‐linking of the surface antigen
Authors: Palecanda Aiyappa , Walcheck Bruce , Bishop D. Keith , Jutila Mark A.
Published: March 2007

L‐selectin (also termed LAM‐1, Leu‐8, TQ‐1, gp90MEL‐14, peripheral lymph node homing receptor and LECAM‐1) is an adhesion protein thought to be important in leukocyte entry into lymphoid tissues and sites of inflammation. We, as well as others, have shown that leukocyte activation by chemotactic factors results in rapid shedding (release) of L‐selectin from the cell surface. Here we have used flow cytometry, enzyme‐linked immunosorbent assay, and SDS‐PAGE/Western blot analysis to determine whether cross‐linking of L‐selectin in the absence of activation causes shedding. We found that rapid loss of leukocyte L‐selectin expression (down‐regulation) could be induced by treating cells with a chemical cross‐linker [bis (sulfosuccinimidyl) suberate]. L‐selectin down‐regulation via cross‐linking could occur at 4°C and in the absence of detectable cell activation (increased expression of CD11b/18). The loss of L‐selectin expression was due to shedding of the molecule from the leukocyte cell surface. Cross‐linking of L‐selectin with specific monoclonal antibodies also caused loss of surface expression of L‐selectin at 37°C. Finally, shed L‐selectin was detected in the plasma of healthy adults whose peripheral blood leukocytes demonstrated no obvious signs of activation. Our results suggest that activation‐independent shedding of leukocyte L‐selectin may occur in vivo and a possible mechanism could involve cross‐linking of leukocyte L‐selectin. This provides a novel mechanism for rapid regulation of expression of a leukocyte‐endothelial cell adhesion receptor.
Origin of thymic and peritoneal Ly‐1 B cells
Authors: Than Soe , Inaba Muneo , Inaba Kayo , Fukuba Yoh , Adachi Yasushi , Ikehara Susamu
Published: March 2007

Murine Ly‐1 B cells were first found in the peritoneal cavity (Per C). It has been thought that Ly‐1 B cells are responsible for the development of autoimmune diseases, since they spontaneously produce autoantibodies. However, we have found that such Ly‐1 B cells are present in the thymus of normal mice, and that they play a crucial role in negative selection in the Mls system. In addition, thymic Ly‐1 B cells and Per C Ly‐1 B cells have been found to differ in function, ontogeny and location. In this communication, we report on the different origins of these cells. C57BL/6 mice are lethally irradiated and reconstituted with fetal (day 14) liver cells or bone marrow cells from 4‐, 6‐ or 12‐week‐old mice. Fetal liver cells are shown to have the capacity to reconstitute both thymic and Per C Ly‐1 B cells, whereas bone marrow cells from 6‐ or 12‐week‐old mice have the capacity to reconstitute thymic Ly‐1 B cells, but not Per C Ly‐1 B cells, indicating that fetal hemopoietic stem cells (HSC) differ from HSC in the bone marrow of adult mice, and that thymic and Per C Ly‐1 B cells have different origins.
A common ancestor of the mammalian transcription factors TCF‐1 and TCF‐1α/LEF‐1 expressed in chicken T cells
Authors: Gastrop Jan , Hoevenagel Ronald , Young John R. , Clevers Hans C.
Published: March 2007

Several mammalian T cell‐specific transcription factors have been cloned recently. Two of these, TCF‐1 and TCF‐1α/LEF‐1, display a moderate level of overall sequence similarity, and contain virtually identical versions of a novel type of DNA‐binding domain, the HMG box. To study evolutionary aspects of the TCF transcription factors in relation to lymphoid differentiation, we have isolated chicken TCF clones from a spleen cDNA library. Low‐stringency screening with human probes as well as a polymerase chain reaction‐aided strategy resulted in the cloning of a single chicken TCF (chTCF) gene. Sequence comparison revealed that chTCF contained a TCF‐1α‐like N terminus, and a TCF‐1‐like C terminus. Furthermore, TCF‐1 and TCF‐1α were more homologous to chTCF than to each other. We postulate that chTCF is the direct descendant of a single ancestral gene, which has been duplicated in mammals to yield TCF‐1 and TCF‐1α.
Functional characterization of non‐human primate erythrocyte immune adherence receptors: implications for the uptake of immune complexes by the cells of the mononuclear phagocytic system
Authors: Edberg Jeffrey C. , Kimberly Robert P. , Taylor Ronald P.
Published: March 2007

Erythrocytes from primates express an immune adherence (IA) receptor that binds complement‐opsonized immune complexes (IC) both in vivo and in vitro.We have analyzed the immunochemical and functional properties of theIA receptor from erythrocytes from species that have been used for in vivo IC clearance studies and have compared these properties to the human IA receptor (which is called complement receptor type 1, CR1). Erythrocytes from all species (chimpanzee, baboon, rhesus and cynomolgus monkey) bind antibody/double‐stranded DNA IC when opsonized with autologous complement. However, IC which are bound to chimpanzee erythrocytes are not released upon addition of chimpanzee serum (which contains factor I activity), while IC bound to baboon erythrocytes and human erythrocytes are released upon addition of autologous serum. Anti‐human CR1 monoclonal antibodies (mAb) E11 and HB8592 bind to erythrocytes from all species examined and the number of mAb epitopes per erythrocyte correlated with the number of IC that could bind to the erythrocyte under saturating conditions. However, a number of interesting differences between the species are observed withother mAb. The anti‐CR1 mAb 1B4 and 3D9, which block recognition of ligand by CR1, did not bind to chimpanzee erythrocytes and bound partially to rhesus and cynomolgusmonkey erythrocytes. In addition, the ability of autologous serum to induce release of erythrocyte‐bound IC correlates with the presence of these epitopes. These findings, taken in context with previous clearance studies, suggest that serum‐mediated release may not be required for the rapid transfer of the IC from the erythrocyte tothe mononuclear phagocytic system.
Murine epidermal Langerhans cells are potent stimulators of an antigen‐specific T cell response to Leishmania major, the cause of cutaneous leishmaniasis
Authors: Will Antje , Blank Christine , Röllinghoff Martin , Moll Heidrun
Published: March 2007

Cutaneous leishmaniasis is initiated by the bite of an infected sandfly and inoculation of Leishmania major parasites into the mammalian skin. Macrophages are knownto play a central role in the course of infection because they are the prime host cellsand function as antigen‐presenting cells (APC) for induction of the cell‐mediated immune response. However, in addition to macrophages in the dermis, the skin contains epidermal Langerhans cells (LC) which can present antigen (Ag) to T cells. Therefore, using a murine model of cutaneous leishmaniasis, we analyzed the ability of epidermal cells to induce a T cell response to L.major. The results demonstrated that freshly isolated LC, but not cultured LC, are highly active in presenting L.major Ag in vitro to T cells from primed mice and toa L.major‐specific T cell clone. Furthermore, freshly isolated LC had theability to retain L.major Ag in immunogenic form for at least 2 days. Their efficiency was much greater than that of irradiated spleen cells, a standard population of APC. LC stimulated both Tcell proliferation and production of the lymphokines interleukin (IL)‐2 and IL‐4. The response was Ag specific and could be induced by lysate of L.major parasites and by live organisms. The data suggest that epidermal LC are important APC in cutaneous leishmaniasis. They may perform a critical function by capturing L.major Ag in the skin and presenting it either to quiescent T cells circulating through the draining lymph node or locally to T effector cells infiltrating the cutaneous lesion.
Bee venom phospholipase A2‐specific T cell clones from human allergic and non‐allergic individuals: cytokine patterns change in response to the antigen concentration
Authors: Carballido José M. , Carballido‐Perrig Nicole , Terres GeróNimo , Heusser Christoph H. , Blaser Kurt
Published: March 2007

Protein antigens with both allergenic and immunoprotective properties represent appropriate molecules to study IgE and IgG regulation. We have established a panel of T cell clones specific to bee venom phospholipase A2 (PLA) from human individuals allergic, hyposensitized or immune (protected) to bee sting. All clones obtained were CD3+, CD4+ and expressed α,β T cell receptor. Depending on the T cell clone, maximal stimulation required 1 to 100 μg/ml of PLA, and the addition of interleukin (IL)‐2 and/or IL‐4 increased their antigen‐dependent proliferation. Following antigen stimulation, the clones produced IL‐4, interferon‐γ (IFN‐γ) and granulocyte‐macrophage colony‐stimulating factor. Most clones also produced tumor necrosis factor α (TNF‐α) and tumor necrosis factor β (TNF‐β), and some produced IL‐5 and/or IL‐2. Both absolute and relative amounts of secreted cytokines depended on the antigen concentration. At low antigen doses, IL‐4 was producedbut little or not IFN‐γ, whereas at higher PLA concentrations significant amounts of both IL‐4 and IFN‐γ were obtained. Thus, these PLA‐specific Tcell clones could be classified according to the changes in the ratio of IL‐4/IFN‐γ production in response to increasing antigen concentrations. Clones derived from allergic and hyposensitized individuals required higher critical amounts of antigen for IFN‐γ induction, and expressed increasing IL‐4/IFN‐γ ratios with increasing concentrations of PLA. Modulation of cytokine patterns by thedose of the antigen may be a driving force for IgE or IgG formation resulting in allergy or immunoprotection.
Mycobacterial heat‐shock proteins as carrier molecules. II: The use of the 70‐kDa mycobacterial heat‐shock protein as carrier for conjugated vaccinescan circumvent the need for adjuvants and Bacillus Calmette Guérin priming)
Authors: Barrios Christy , Lussow Alexander R. , Embden Jan Van , Zee Ruurd Van Der , Rappuoli Rino , Costantino Paolo , Louis Jacques A. , Lambert Paul‐Henri , Giudice Giuseppe Del
Published: March 2007

In a recent work, we have shown that mycobacterial heat‐shock proteins (hsp)of 65‐kDa (GroEL‐type) and 70‐kDa (DnaK‐type) acted as carrier molecules in mice, previously primed with Mycobacterium tuberculosis var. bovis (bacillus Calmette‐Guérin, BCG), for the induction of high andlong‐lasting titers of IgG against the repetitive malaria synthetic peptide (NANP)40. Anti‐peptide antibodies were induced when the malaria peptide, conjugated to the mycobacterial hsp, was given in the absence of any adjuvants (Lussow etal., Eur. J. Immunol. 1991. 87: 2960). In this report, we show that mice immunized with peptides or oligosaccharides conjugated to the 70‐kDa hsp produced high titers of IgG antibodies in the absence of any previous priming with BCG.The anti‐peptide antibody response persisted for at least 1 year. This adjuvant‐free carrier effect of the 70‐kDa hsp was T cell dependent, since no anti‐peptide nor anti‐70‐kDa IgG antibodies were induced in athymic nu/nu mice. Previous immunization of mice with the 65‐kDa or 70‐kDa hsp did not have any negative effect on the induction of anti‐peptide IgG antibodies after immunization with hsp‐peptide conjugates in the absence of adjuvants. Furthermore, preimmunization with the 65‐kDa hsp could substitute for BCG in providingan effective priming for the induction of anti‐(NANP) antibodies. Finally, both the 65‐kDa and 70‐kDa hsp acted as carrier molecules for the induction ofIgG antibodies to group C meningococcal oligosaccharides, in the absence of adjuvants. These findings strongly suggest that the use of hsp as carriers in conjugated constructs for the induction of anti‐peptide and anti‐oligosaccharide antibodies could be of value in the design of new vaccines for eventual use in humans.
Interaction of C1q receptor with lung surfactant protein A
Authors: Malhotra Rajneesh , Haurum John , Thiel Steffen , Sim Robert B.
Published: March 2007

Earlier we reported the purification of C1q receptor (C1qR) from U937 cells and human tonsil lymphocytes (Malhotra, R. and Sim, R. B., Biochem. J. 1989. 218: 625) and showed that C1qR interacts with the ligands C1q, mannose‐binding protein, conglutinin and lung surfactant protein A (SP‐A) (Malhotra, R., Thiel, S., Reid, K. B. M. and Sim, R. B., J. Exp. Med. 1990. 172: 955). C1qR was characterized as an acidic glycoprotein, which, when solubilized, exists as a dimer of Mr 115000 under non‐denaturing conditions. In this article we provide evidence for binding of radioiodinated SP‐A to U937 cells and show that binding of radioiodinated SP‐A to U937 cells is specific, saturable, salt dependent and is inhibited by purified C1qR and byC1q. The interaction of SP‐A withU937 cells was found to up‐regulate the surface expression of C1qR. Incubation of SP‐A with U937 cells at 37°C for 80 min was found to increase the receptornumber per cell. Increase in receptor number was inhibited in the presence of sodium azide and monensin. Incubation of cells with calcium ionophore A 23187 induced increased surface expression in the absence of SP‐A. The results indicate that interactionof SP‐A with U937 cells triggers the expression of an intracellular pool of C1qR.
Molecular cloning and expression of the murine RANTES cytokine: structural and functional conservation between mouse and man
Authors: Schall Thomas J. , Simpson Nancy J. , Mak John Y.
Published: March 2007

The infiltration and activation of monocytes is a hallmark of chronic inflammation, including that associated with a variety of disease states such as rheumatoid arthritis, atherosclerosis, and various autoimmune conditions. Recently, a family of small molecular mass proteins has been described which appear to have inflammatory properties, including chemoattractant effects on monocytes. We report here on the molecular cloning, characterization, and functional expression of muRANTES, a new murine member of this family. muRANTES expressed in a mammalian expression system is an approximately 8‐kDaprotein exhibiting immune cross‐reactivity with a rabbit polyclonal antiserumgenerated against human RANTES. Boyden chamber chemotaxis experiments reveal some lack of species specificity in monocyte chemoattractant potential, as recombinant muRANTES attracts human monocytes in a dose‐dependent fashion in vitro. muRANTES and its human homolog share approximately 85% amino acid identity, a higher level of conservation than that seen with any other species homologs in this cytokine family, and second only to transforming growth factor‐β among reported immune cytokines.
Suppression of lymphocyte proliferation by a retroviral p15E‐derived hexapeptide
Authors: Oostendorp Robert A. J. , Schaaper Wim M. M. , Post Jacob , Von Blomberg B. Mary E. , Meloen Rob H. , Scheper Rik J.
Published: March 2007

CKS‐17 (LQNRRGLDLLFLKEGGL), a synthetic peptide derived from a conserved region of retroviral transmembrane proteins, has previously been shown to suppress severaldifferent immune effector mechanisms. The present study was undertaken to further delineate immunosuppressive site(s) of CKS‐17. Overlapping hexapeptides covering the complete sequence of CKS‐17 were synthesized. One CKS‐17‐derived hexapeptide, LDLLFL, suppressed ligand [CD3, interleukin (IL)‐2]‐induced lymphocyte proliferation. Spontaneous proliferation of transformed lymphoid cell lines, as well as cell lines from myeloid or epitheloid origin, was not inhibited byLDLLFL. Full suppression required the continuous presence of LDLLFL during culturing, and did not involve interference with monocyte function. Radiolabeling studies showed that the hexapeptide did not compete with IL‐2 for IL‐2 receptor binding. Most likely the LDLLFL motif inteferes with steps shared bythe IL‐2 and CD3 receptor‐induced signaling pathways. Since LDLLFL displays multiple immunosuppressive activities, it may constitute a biologically relevant immunosuppressive site of retroviral transmembrane proteins.
Tissue‐specific and allelic expression of the complement regulator CD46 is controlled by alternative splicing
Authors: Russell Sarah M. , Sparrow Rosemary L. , McKenzie Ian F. C. , Purcell Damian F. J.
Published: March 2007

CD46 (membrane cofactor protein) is a human cell surface glycoprotein withcofactoractivity for factor I‐mediated cleavage of complement components C3b andC4b. The CD46 protein from normal lymphocytes resolves on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis as two major bands of 66 and 56 kDa. CD46 cDNA encodes four extracellular short consensus repeat domains, a Ser/Thr/Pro (STP) ‐ rich region, a transmembrane region and a cytoplasmic tail. We now show that exquisite control ofmRNA splicing is responsible for the heterogeneous expression of CD46 isoforms. Differential splicing of 5 exons generates at least 14 CD46 mRNA variants whose expression is stringently regulated by allelic, tissue‐specific and malignancy‐related factors, as: (a) leukemic cells and Epstein‐Barr virus‐transformed B cells preferentially incorporate the first of three STP exons (exon 7) into mRNA, and produce a larger CD46 isoform of 74 kDa, (b) an allelic difference in the proportion of 66‐ and 56‐kDa CD46 isoforms on lymphocytes corresponds to the preferential inclusion or exclusion of the second STP exon (exon 8), (c) the third STP exon (exon 9) isspecifically deleted in some placentae, (d) spermatozoa delete both exons 12 and 13, encoding a shorter transmembrane region and a unique cytoplasmic tail and (e) all tissues tested differentially splice exon 13, resulting in two alternative cytoplasmic tails. The distribution of the 14 alternatively spliced RNA transcripts correlated with the presence of protein isoforms of the predicted size, indicating that alternative splicing leads to heterogeneity of CD46 glycoproteins.
T cell recognition of donor major histocompatibility complex class Ipeptides during allograft rejection
Authors: Fangmann Josef , Dalchau Rosemarie , Sawyer Greta J. , Priestley Carol A. , Fabre John W.
Published: March 2007

LEW (RTI1) recipients of DA (RTIavl) skin and kidney allografts were tested for the capacity of their T lymphocytes to proliferate to three 22–24‐amino acid peptides from the hypervariable regions of the RTI‐Aavl classical MHC class I molecule. Ten days after rejecting second‐set DA kidneyallografts, spleen cells (but interestingly not lymph node cells) from LEW recipients showed strong, LEW antigen‐presenting cell‐dependent, CD4+ Tcell proliferation to peptide 1 (from the α helical region of the α1 domain). CD8+ T cells showed no response to peptide 1. There was no response by the spleen cells to peptide 2 (from the β sheet of the α2 domain) or peptide 3 (from the α helical region of the α2 domain). Immunization of LEW rats with pure RTI‐Aavl class I H chain in Freund's adjuvant gave responses identical to that seen after grafting, i.e. good CD4+ T cell proliferation to peptide 1, but none to peptides 2 and 3. However, immunization of LEW rats with peptides 1, 2 and 3 in Freund's adjuvant resulted in good CD4+ T cell proliferation responses to each of the peptides. These data demonstrate that indirect allorecognition can be stimulated by allograft rejection, and emphasize that the physiological processing of donor antigens will influence which peptides will be important in indirect allorecognition in transplantation.
Soluble anti‐μ monoclonal antibodies prime resting B cells to secrete immunoglobulins in response to interleukins‐4 and ‐5
Authors: Phillips Catherine , Klaus Gerry G. B.
Published: March 2007

Soluble anti‐immunoglobulin (Ig) antibodies have been generally found to inhibit Ig secretion in B cells, via largely unknown mechanisms. To investigate this phenomenon further a two‐step culture system was used in which B cells are primed for24–72 h with various soluble monoclonal or polyclonal anti‐Ig antibodies: after washing the cells were placed in readout cultures with a combination of interleukin (IL)‐5 and IL‐4. Using this protocol B cells primedwith (mitogenic ornonmitogenic) anti‐μ monoclonal antibodies differentiated into large numbers of IgM‐secreting cells, comparable to responses to lipopolysaccharide. In contrast, priming with polyclonal rabbit anti‐Ig or monoclonal anti‐x antibodies, markedly inhibited Ig secretion induced by IL‐4 + IL‐5. In addition, anti‐μ was markedly inhibitory if left in thereadout cultures with the two lymphokines. These results, therefore, indicate that appropriate cross‐linking of surface IgM receptors on B cells can prime the cells tosecrete Ig when they are restimulated by T cell‐derived lymphokines in the absence of anti‐μ. Incontrast co‐ligation of both surface IgM and surface IgD receptors apparently results in powerful inhibition of Ig secretion, which is not reversed by stimulation withIL‐4 plus IL‐5.
Ongoing Vx−Jx recombination after formation of a productiveVx−Jx coding joint
Authors: Huber Christian , Klobeck Hans‐Gustav , Zachau Hans G.
Published: March 2007

Vx genes of man can recombine with the Jx gene segments eitherbyan inversion or by a deletion mechanism. Back‐to‐back fusion products of the respective recombination signal sequences (signal joints) are retained on the chromosome after the formation of a Vx−Jx coding joint by an inversion. Our knowledge of the structure of the human x locus and the application of the polymerase chain reaction allowed us now to establish a direct relationshipbetween different x recombination products in the lymphoid cell line JI. Two consecutive inversions fully explain the existence of two coding joints and two signal joints on the same chromosome of this cell line. Although the initially formed coding joint is productively rearranged and expressed, a second Vx−Jx rearrangement took place which leads to an aberrant joint. In this process a Jx gene segment of the signal joint that had been created in the first Vx−Jx joining was used as the recombination target. The sequence of the two rearrangements is unequivocal since a product of the first (productive) reaction is a partner in the second (aberrant) one.