bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of a memory B cell response does not require primed helper T cells
Authors: Leclerc Claude , Sedlik Christine , Lo‐Man Richard , Charlot Bernadette , Rojas Marie , Dériaud Edith
Published: March 2007

The use of universally immunogenic T cell epitopes, such as those identified in tetanus toxin or malaria circumsporozoite protein, could represent a major improvement in the development of synthetic vaccines. However, one limitation of this approach is the lack of T cell cross‐reactivity between the vaccine and the pathogen. To determine whether the memory B cell response elicited by immunization with a synthetic peptide containing a B cell epitope linked to a T cell epitope can be restimulated by the same B cell epitope linked to different T cell epitope(s), we used a synthetic peptide which contains non‐overlapping B and T cell determinants from hepatitis B surface antigen (HBsAg) of hepatitis B virus (HBV). The results of this study clearly show that primed T cells can increase the antibody response against a B cell epitope linked to the priming T cell determinant. However, the antibody response obtained was weaker than that obtained after two injections of the peptide containing both B and T cell epitopes, showing the important role played by memory B cells in secondary antibody responses. Moreover, a strong antibody response against the B cell epitope was elicited by boosting mice with the B cell epitope linked to a heterologous carrier, thus demonstrating that a strong B cell memory response can be revealed in the absence of primed T cells. These results therefore provide new important information for the design of synthetic or recombinant vaccines.
CD8+ Type 1 CD44hi CD45 RBlo T lymphocytes control intracellular Brucella abortus infection as demonstrated in major histocompatibility complex class I‐ and class II‐deficient mice
Authors: Oliveira Sergio C. , Splitter Gary A.
Published: March 2007

Genetically engineered mice with a targeted disruption in the β2‐microglobulin (β2‐m) gene or the H2‐I‐Aβ chain (αβ) which lack functional CD8+ or CD4+ T cells, respectively, were used to assess the role of T cell subsets in Brucella abortus infection. Murine brucellosis was markedly exacerbated in β2‐m‐deficient mice (β2‐m−/−) compared to Aβ mutant (Aβ−/−) or C57BL/6 mice, strongly indicating that optimal resistance to B. abortus requires CD8+ T cells. Splenocytes from Brucella‐primed β2‐m−/−, αβ−/− and C57BL/6 mice exhibited a type 1 cytokine profile marked by elevated IFN‐γ mRNA expression and protein production, and basal levels of IL‐2 and IL‐4 transcripts. B. abortus did not induce secretion of TGF‐β1, but substantial IL‐10 activity was detected in spleen cell supernatants from all mouse strains studied. CD8+ T cells from Aβ−/− and C57BL/6 mice displayed a CD44hi CD45RBlo phenotype and a type 1 cytokine transcription profile featuring high levels of IFN‐γ mRNA. Additionally, we have shown the ability of C57BL/6 CD8+ CTL to kill Brucella‐infected macrophages. This study illustrates the predominant role of MHC class I‐restricted T cells in controlling B. abortus infection.
Normal clonal expansion but impaired Fasmediated cell death and anergy induction in interleukin‐2‐deficient mice
Authors: Kneitz Burkhard , Herrmann Thomas , Yonehara Shin , Schimpl Anneliese
Published: March 2007

Despite a normal development of all major lymphoid subsets, with time, interleukin‐2 (IL‐2)‐deficient mice develop a fatal immunopathology. The disease phenotype is characterized by lymphoadenopathy, splenomegaly, T cell infiltration of various organs, overproduction of a number of cytokines and autoantibody formation. Phenotypically, CD4+ and CD8+ T cells exhibit features characteristic of antigenically experienced cells. The accumulation of cells with a memory phenotype together with the previous suggestion of an involvement of IL‐2 in the termination phase of immune responses prompted us to study the fate of superantigen‐reactive T cells in IL‐2‐deficient mice in comparison to their IL‐2‐producing littermates. We show that expansion in vivo of CD4+ and, to a lesser extent, CD8+ T cells reactive to the superantigens staphylococcal enterotoxin A and B (SEA and SEB) proceeds normally in the absence of IL‐2, but that fewer CD4+ cells are subsequently deleted. The residual superantigenreactive cells fail to become anergic as measured by proliferation in vitro in response to the same superantigen. T cell blasts generated in vitro from lymph node cells of IL‐2‐deficient mice by superantigen stimulation in the absence of exogenous IL‐2 also fail to become anergic. In contrast to cells from IL‐2‐producing littermates, they do not exhibit Fas‐induced apoptosis when cultured on anti‐Fas antibody‐coated plates, although Fas expression by IL‐2‐deficient cells is normal or even elevated compared to the IL‐2‐producing control cells. The data suggest that activation of T cells in the absence of IL‐2 fails to generate a signal which is necessary to activate the apoptotic pathway and thus leads to an accumulation of antigen‐experienced cells and the chronic inflammatory responses observed in IL‐2‐deficient mice.
T lymphocytes from normal human peritoneum are phenotypically different from their counterparts in peripheral blood and CD3− lymphocyte subsets contain mRNA for the recombination activating gene RAG‐1
Authors: Hartmann Johannes , Maaßen Volker , Rieber Peter , Fricke Harald
Published: March 2007

The surface antigens of peritoneal lymphocytes of healthy human individuals were studied. B lineage cells comprised 2.3% of the total peritoneal lymphocyte population. Although the majority of peritoneal cavity lymphocyte (PCL) T cells expressed αβ T cell receptor (TcR), up to 17% expressed γδ TcR. The majority of PCL exhibited markers of the thymus‐dependent lineage (CD2+ CD3+ Tcαβ+ CD4+ or CD8α+β+) and surface antigens associated with memory and activation (CD45RO+ CD11a+ CD18+ CD49d+ HLA‐DR). Up to 92% of both CD4+ and CD8+ T cells bore CDw60, thus characterizing the T cell subset containing helper activity for mitogen‐driven B cell differentiation. The majority of PCL T cells were CD8+ and, in addition, up to 60% of this population expressed the homodimeric CD8α+β−. Messenger RNA for the recombination activating gene RAG‐1 was examined in CD3− PCL depleted of CD19+ lineage cells. The PCL population which comprised cells containing RAG‐1 mRNA transcripts was CD19−, surface IgM−, cytoplasmic IgM− and CD2− CD3− CD4− CD8− CD56−. However, this population was CD7+ (approx. 75%), and contained both CD7−CD34+ (up to 3%) and CD7+ CD34+ (up to 3%) cells. These findings are compatible with the hypothesis that the adult human peritoneum provides a microenvironment capable of supporting a thymus‐indenpendent differentiation of T lymphocytes.
Epstein‐Barr virus/C3d receptor (CR2, CD21) activated by its extracellular ligands regulates pp105 phosphorylation through two distinct pathways
Authors: Bouillie Sylvie , Barel Monique , Drane Pascal , Cassinat Bruno , Balbo Michelle , Holers V. Michael , Frade Raymond
Published: March 2007

We previously demonstrated that human C3d or pep16, a 16‐amino acid synthetic peptide derived from human C3d, induced in vivo and in vitro tyrosine phosphorylation of pp105, an intracellular component found only in human cells that express CR2 at their surface. To determine the contribution of CR2 molecules to this enzymatic regulation, we first analyzed whether activation of CR2 by other extracellular CR2 ligands could trigger such regulation in cell extracts. Subsequently, we used cell extracts of either CR2‐positive cells depleted in CR2 molecules by absorption with anti‐CR2 antibodies or CR2‐negative cells transfected with CR2 cDNA. We demonstrate here that pp105 phosphorylation was induced when CR2 was activated by C3d and pep16 as well as by gp350, the Epstein‐Barr virus capsid protein or OKB7, an anti‐CR2 monoclonal antibody (mAb). HB5, another anti‐CR2 mAb, which did not activate B lymphocytes through CR2, did not induce pp105 phosphorylation. Thus, C3d, pep16, gp350, and OKB7 presented similar properties in activating CR2 to trigger pp105 phosphorylation and in regulating B lymphocyte proliferation, while HB‐5 had no effect on either assays. Furthermore, our data demonstrate that the presence of CR2 activated by its extracellular ligands regulates pp105 phosphorylation through two distinct pathways: one which also requires the presence of non‐activated CD19, and one which is independent of CD19. The involvement of CD19 in the first pathway was not due to the formation of putative CR2‐CD19 complexes. Both pathways were TAPA‐1 independent. This is the first demonstration that activated CR2 molecules can play a regulatory role in enzymatic function, such as phosphorylation, despite the absence of CD19 and TAPA‐1.
Monocyte activation: rapid induction of α1/β1 (VLA‐1) integrin expression by lipopolysaccharide and interferon‐γ
Authors: Rubio Miguel Angel , Sotillos Marisol , Jochems Gijs , Alvarez Vicente , Corbií Angel L.
Published: March 2007

Monocytes play a key role in inflammation, tissue injury and remodelling and wound healing, and most monocyte effector functions are dependent on adhesive interactions. We have analyzed the changes in the pattern of β1 integrin expression that take place during monocyte activation and demonstrated that lipopolysaccharide (LPS) and interferon (IFN)‐γ specifically induce the expression of the α1/β1 integrin, which was detectable on the monocyte membrane as early as 12 h after monocyte activation. The up‐regulated α1/β1 expression was not dependent on monocyte adherence to solid surfaces, and Northern blot analysis revealed that LPS and IFN‐γ induce the α1 mRNA de novo. Monocyte deactivating cytokines such as interleukin (IL)‐4 or IL‐10, could only minimally inhibit the LPS‐ or IFN‐γ mediated up‐regulation of α1/β1, suggesting that cytokine release subsequent to monocyte activation does not play a major role in the integrin induction. Interestingly, the LPS‐induced expression of α1/β1 was found to be dependent on the redox state of the cell, since it was inhibited by antioxidants which also altered the morphological changes that take place during monocyte culture in vitro. The rapid induction of α1 in LPS‐activated monocytes suggests that α1/β1 might be involved not only in monocyte/extracellular matrix interactions during inflammatory reactions, but also in contributing to further monocyte activation and cytokine production during septic shock syndrome.
Characterization of thymic stromal‐derived lymphopoietin (TSLP) in murine B cell development in vitro
Authors: Ray Robert J. , Furlonger Caren , Williams Douglas E. , Paige Christopher J.
Published: March 2007

B cell development is dependent on both direct interactions with stromal cells and their secreted cytokines. The precise mechanisms by which these interactions regulate B cell differentiation are currently unknown. We report here that a novel growth factor thymic stromal‐derived lymphopoietin (TSLP) can replace the activity of interleukin‐7 (IL‐7) in supporting B cell development in vitro. TSLP was found to promote the proliferation and differentiation of committed B220+ B cell progenitors from day 15 fetal liver. Phenotypic analysis of these cells revealed that they are at the pro‐B cell stage of differentiation and express cell surface markers characteristic of pro‐B cells cultured in IL‐7. TSLP can replace the activity of IL‐7 in supporting the progression of B lymphocytes from uncommitted bipotential precursors. In the absence of either TSLP or IL‐7, the progeny of cells that give rise to mature B lymphocytes fail to develop from these bipotential precursors. Moreover, TSLP can substitute for IL‐7 in supporting the sustained proliferative response exhibited by B cell progenitors from CBA/N mice. Together these results show that TSLP can replace the requirement for IL‐7 during in vitro B cell development.
Phenotypic and functional analysis of B lymphopoiesis in interleukin‐7‐transgenic mice: expansion of pro/pre‐B cell number and persistence of B lymphocyte development in lymph nodes and spleen
Authors: Mertsching Elisabeth , Grawunder Ulf , Meyer Valerie , Rolink Ton , Ceredig Rhodri
Published: March 2007

Transgenic mice in which mouse interleukin (IL)‐7 cDNA is expressed under the control of the mouse major histocompatibility complex (MHC) class II (Eα) promoter develop a lymphoproliferative disease characterized by the early polyclonal expansion of T cells followed in many cases by the development of lymphomas of immature B cells. Here, we have analyzed B cell development in these transgenic mice. Phenotypic analysis using monoclonal antibodies to B220, IgM, IgD, c‐kit, IL‐7 receptor, MHC class II, AA4.1, CD19, CD23, CD25, CD40 and CD43 shows that B lymphopoiesis in the bone marrow is dramatically altered and the number of pro/pre‐B and immature B cells is significantly increased. Interestingly, pro/pre‐B and immature B cells persist in the spleens of adult transgenic mice and are also present in lymph nodes and blood. Cell cycle analysis of lymph node cells shows that subpopulations of developing B cells retain the cell cycle profiles of their bone marrow counterparts. Limiting dilution analysis shows that the number of clonable pre‐B cells is significantly increased and that at limiting dilution, growth of transgenic pre‐B cells is still dependent on exogenous IL‐7. Using semiquantitative polymerase chain reaction (PCR) and in situ hybridization, the level of IL‐7 transcripts in the spleen was found to decrease between 2 and 4 weeks in control mice with levels in transgenics mice being approximately 50 times greater. These transgenic mice represent an interesting model with which to study the effects of IL‐7 overexpression in the bonemarrow and raise interesting questions regarding the regulation of B lymphopoiesis in normal mice.
Human eosinophils express messenger RNA encoding RANTES and store and release biologically active RANTES protein
Authors: Ying Sun , Meng Qiu , Taborda‐Barata Luis , Corrigan Christopher J. , Barkans Julia , Assoufi Basil , Moqbel Redwan , Durham Stephen R. , Kay A. Barry
Published: March 2007

Eosinophils synthesize and store various cytokines with potential autocrine activity. We hypothesized that eosinophils synthesize and store RANTES, a CC chemokine with potent eosinophil chemotactic activity. Expression of RANTES mRNA in highly purified eosinophil populations was detected by reverse transcription followed by polymerase chain reaction analysis. In situ hybridization (ISH) with 35S‐labeled RANTES‐specific riboprobes showed that 6.8–10% of peripheral blood eosinophils obtained from atopic subjects expressed RANTES mRNA, increasing to 25% after incubation (16 h) with interferon(IFN)‐γ, but not ionomycin in vitro. Peripheral blood eosinophils also showed specific immunoreactivity with an anti‐RANTES monoclonal antibody, consistent with translation of the mRNA. By enzyme‐linked immunosorbent assay, blood eosinophils were shown to contain a median of 7300 pg (range 5200–8800) RANTES per 106 cells, of which a mean of 24% was released into culture supernatants after stimulation of the cells with serum‐coated particles in vitro. These culture supernatants exhibited eosinophil chemotactic activity which was inhibited (mean 68%) by a specific anti‐RANTES antibody. Sequential immunocytochemistry and ISH on biopsies obtained from allergen‐induced late‐phase cutaneous reactions showed that 55–75% of the infiltrating RANTES mRNA+ cells were EG2+ eosinophils. Allergen, but not diluent challenge, was also associated with a time‐dependent increase in the number of cells showing RANTES immunoreactivity. Of these cells, 55% were identified as eosinophils by morphological criteria. Thus, human eosinophils have the capacity to synthesize, store and secrete physiologically relevant quantities of RANTES, and may therefore be an important source of this chemokine in allergic inflammation.
Differences and similarities in the A2.1‐restricted cytotoxic T cell repertoire in humans and human leukocyte antigen‐transgenic mice
Authors: Wentworth Peggy A. , Vitiello Antonella , Sidney John , Keogh Elissa , Chesnut Robert W. , Grey Howard , Sette Alessandro
Published: March 2007

HLA‐A2.1‐binding peptides (n = 38) were screened for immunogenicity with human peripheral blood mononuclear cells in cytotoxic T lymphocyte (CTL) induction experiments in vitro and with splenocytes from HLA‐A2.1/Kb transgenic mice following immunization in vivo. These data were compiled and analyzed to determine the level of overlap between the A2.1‐restricted CTL repertoire of A2.1/Kb‐transgenic mice and A2.1+ humans. In both humans and mice, a major histocompatibility complex affinity threshold of approximately 500 nM appears to determine the capacity of a peptide to elicit a CTL response. Good concordance between the human data in vitro and mouse data in vivo was observed with 85% of the high‐binding peptides, 58% of the intermediate binders, and 83% of the low/negative binders. Although some peptides immunogenic for mouse CTL but not for humans (and vice versa) could be identified, the data as a whole suggest an extensive overlap between T cell receptor repertoires of mouse and human CTL and support the use of HLA‐transgenic mice for the identification of potential human CTL epitopes.
Expression of gp40, the murine homologue of human epithelial cell adhesion molecule (Ep‐CAM), by murine dendritic cells
Authors: Borkowski Teresa A. , Nelson Andrew J. , Farr Andrew G. , Udey Mark C.
Published: March 2007

Dendritic cells (DC) can be distinguished from other antigen‐presenting cells (APC) by their morphology, motility and ability to initiate primary responses in naive T cells. Certain cell surface proteins (e.g. major histocompatibility complex antigens, co‐stimulatory/adhesion molecules and DEC205) are selectively expressed by DC, and may contribute to the potent APC activity of these leukocytes. As an outgrowth of studies of adhesion molecules expressed by epithelia and Langerhans cells (LC), we examined DC from murine epidermis and various lymphoid tissues for evidence of expression of gp40, a glycoprotein recently identified as the murine homologue of human epithelial cell adhesion molecule (Ep‐CAM). gp40 was detected on freshly‐obtained LC, cultured LC and LC that migrated from skin explants, as well as on keratinocytes. In skin‐associated lymph nodes, gp40 was selectively expressed by some DC in T cell‐dependent areas. DC‐enriched preparations from skin‐associated lymph nodes and spleen contained many cells that co‐expressed DC markers (CD11c and DEC205) and high levels of gp40. Lower levels of gp40 were present on DC from gut‐associated lymph nodes. These results demonstrate that the putative homophilic adhesion molecule gp40 is expressed by subpopulations of DC in selected tissues; we propose that gp40 expression may have functional consequences for DC.
Tepoxalin blocks neutrophil migration into cutaneous inflammatory sites by inhibiting Mac‐1 and E‐selectin expression
Authors: Zhou Lubing , Pope Barbara L. , Chourmouzis Erika , Fung‐Leung Wai‐Ping , Lau Catherine Y.
Published: March 2007

Inflammation is characterized by the migration of polymorphonuclear leukocytes from the vasculature into the tissue causing profound injury. Adhesion and migration of neutrophils across the vascular bed are governed by a series of complex events including cytokine/chemokine production which in turn orchestrates the temporal expression of a cohort of adhesion molecules mediating the migration. Many of these adhesion molecules and their inducers are under the control of inflammatory response transcriptional factors such as NFXB and AP‐1. Recently we showed tepoxalin, previously known as a dual cyclooxygenase/lipoxygenase (CO/LO) inhibitor, to be a potent inhibitor of NFXB‐induced transcription in vitro. In this study, we demonstrated that when administered in vivo, tepoxalin but not naproxen (a nonsteroidal anti‐inflammatory drug, NSAID) or zileuton (an LO inhibitior), effectively inhibits neutrophil migration into inflammatory sites in murine skin stimulated by either lipopolysaccharide (LPS) or tumor necrosis factor‐α. Immunohistochemical analysis indicates that 10–50 mg/kg of tepoxalin inhibits neutrophil migration. It also effectively blocks the up‐regulation of Mac‐1 (CD11b/CD18) on neutrophils. Quantitative polymerase chain reaction Mac‐1 analysis shows that LPS‐induced transcription of E‐selectin mRNA was dramatically suppressed by both 25 and 50 mg/kg of tepoxalin, whereas the level of ICAM‐1 was only affected by 50 mg/kg of tepoxalin. Since it has been documented that the expression of E‐selectin and Mac‐1 is regulated either directly or indirectly by the transcription factor NFXB, our studies provide in vivo evidence that tepoxalin is a potent inhibitor of NFXB‐mediated events in animal models and this novel molecular mechanism clearly defines it as a new class of anti‐inflammatory compounds.
Disease in the scurfy (sf) mouse is associated with overexpression of cytokine genes
Authors: Kanangat Sivadasan , Blair Patrick , Reddy Ramani , Deheshia Massoud , Godfrey Virginia , Rouse Barry T. , Wilkinson Erby
Published: March 2007

The murine X‐linked lymphoproliferative disease scurfy is similar to the Wiskott‐Aldrich syndrome in humans. Disease in scurfy (sf) mice is mediated by CD4+ T cells. Based on similarities in scurfy mice and transgenic mice that overexpress specific cytokine genes, we evaluated the expression of cytokines in the lesions of sf mice by Northern blotting, quantitative reverse‐transcription polymerase chain reaction (RT‐PCR) and by hybridization in situ. Overall, the phenotypic characteristics of scurfy disease correlated well with increased interleukin (IL)‐4 (lymphadenopathy), IL‐6 (B cell proliferation, hypergammaglobulinemia), IL‐7 (dermal inflammatory cell infiltration), and high levels of tumor necrosis factor‐α (wasting).
High doses of interleukin‐12 inhibit the development of joint disease in DBA/1 mice immunized with type II collagen in complete Freund's adjuvant
Authors: Hess Henry , Gately Maurice K. , Rüde Erwin , Schmitt Edgar , Szeliga Jacek , Germann Tieno
Published: March 2007

Collagen‐induced arthritis (CIA) is an (autoimmune) joint disease readily elicited in DBA/1 mice by immunization with type II collagen (CII) emulsified with complete Freund's adjuvant. It is a destructive arthritis involving about 50% of the limbs and occurs with an incidence of 70% to 100%. In this study we evaluated the effect of mouse recombinant interleukin‐12 (mrIL‐12) on CIA. Administration of mrIL‐12 at high doses (1 μg/mouse, daily) for 2 or 3 weeks delayed the onset and reduced the incidence of CIA. Furthermore, the severity of CIA was much milder and in most cases restricted to single digits of the paws. Short‐term administration of high doses of IL‐12 exerted some, but less pronounced, disease‐suppressing effect. In contrast, 10‐fold lower doses of IL‐12 given during the first 3 weeks, or high doses of IL‐12 administered therapeutically proved to be ineffective. Only those regimens of IL‐12 treatment that ameliorated CIA were associated with a down‐regulation of the CII‐specific antibody response. A strong inhibition of CII‐specific IgG1 antibodies (10‐ to 20‐fold) and a moderately (2‐ to 6‐fold) suppressed IgG2b response was observed, whereas the level of CII‐specific IgG2a antibodies remained high. Taken together, the results indicate that some initial events in the induction of CIA in DBA/1 mice injected with CII emulsified with CFA are suppressed by treatment with high doses of IL‐12.
Lack of intermediate‐affinity interleukin‐2 receptor in mice leads to dependence on interleukin‐2 receptor α, β and γ chain expression for T cell growth
Authors: Chastagner Patricia , Moreau Jean‐Louis , Jacques Yannick , Tanaka Toshiyuki , Miyasaka Masayuki , Kondo Motonari , Sugamura Kazuo , Thèze Jacques
Published: March 2007

An interleukin (IL)‐4 dependant mouse T cell clone 8.2 derived from an IL‐2‐dependent T cell line was characterized. As measured by flow cytometric analysis and Northern blotting, it expresses IL‐2 receptor β (IL‐2Rβ) and γ (IL‐2Rγ) chains, but has lost expression of IL‐2 receptor α chain (IL‐2Rα). To investigate the properties of the mouse IL‐2Rβγ complex and the role of IL‐2Rα gene expression, this clone was further studied. T cell clone 8.2 has lost the capacity to bind 125I‐labeled human IL‐2 under experimental conditions able to detect intermediate‐affinity IL‐2R in human cells. Mouse IL‐2 is unable to block the binding of mAb TMβ1 to 8.2 cells. Under the same experimental conditions, mouse IL‐2 blocks the binding of TMβ1 to C30‐1 cells expressing the IL‐2αβγ complex. Since TMβ1 recognizes an epitope related to the IL‐2 binding site of IL‐2Rβ, these results can be taken as a demonstration that mouse IL‐2Rβγ does not bind mouse IL‐2. Furthermore, T cell clone 8.2 does not proliferate in response to recombinant mouse or human IL‐2. On the other hand, T cell transfectant lines expressing heterospecific receptors made of the human IL‐2Rβ and mouse IL‐2Rγ chains bind 125I‐labeled human IL‐2 and proliferate in response to IL‐2. This establishes the difference between mouse and human IL‐2Rβ chains. Transfection of T cell clone 8.2 with human IL‐2Rα genes restores their capacity to proliferate in response to IL‐2. In addition, all transfectants grown in IL‐2 express the endogeneous mouse IL‐2Rα chain. When grown in IL‐4, the endogeneous mouse IL‐2Rα gene remains silent in all these transfectants. These results show that, contrary to the human, the mouse does not express an intermediate‐affinity IL‐2R. Expression of the IL‐2Rα gene is therefore required for the formation of the functional IL‐2R in mice.
Self‐renewal of B‐1 lymphocytes is dependent on CD19
Authors: Krop Ian , De Fougerolles Antonin R. , Hardy Richard R. , Allison Michael , Schlissel Mark S. , Fearon Douglas T.
Published: March 2007

The B‐1 subset of B lymphocytes is maintained by self‐renewal of mature cells, and this process may involve signaling through membrane immunoglobulin (mIg). We determined whether CD19, a membrane protein that co‐stimulates B cells by mIg, has a role in this process. Pre‐natal treatment of mice with 1D3, a rat anti‐mouse CD19 monoclonal antibody, down‐regulated CD19 expression and reduced by sixfold the number of B‐1a cells at birth; B‐2 cells were relatively unaffected. Prolonged treatment of adult mice with 1D3 caused the loss of approximately 2% per day of peritoneal B‐1a cells, without diminishing the recovery of splenic B‐2 cells. The loss of B‐1a cells was associated with inhibition of their replication rather than with accelerated turnover. Therefore, CD19 is involved in the development and self‐renewal of B‐1a cells, perhaps through its ability to amplify signaling through mIgM.
Selectively impaired development of intestinal T cell receptor γδ+ cells and liver CD4+ NK1+ T cell receptor αβ+ cells in T cell factor‐1‐deficient mice
Authors: Ohteki Toshiaki , Wilson Anne , Verbeek Sjef , MacDonald H. Robson , Clevers Hans
Published: March 2007

T cell factor‐1 (Tcf‐1) is a transcription factor that binds to a sequence motif present in several T cell‐specific enhancer elements. In Tcf‐1‐deficient (Tcf‐1−/‐) mice, thymocyte development is partially blocked at the transition from the CD4−8+ immature single‐positive stage to the CD4+8+ double‐positive stage, resulting in a marked decrease of mature peripheral T cells in lymph node and spleen. We report here that the development of most intestinal TCRγδ+ cells and liver CD4+ NK1.1+TCRαβ+ (NK1+T) cells, which are believed to be of extrathymic origin, is selectively impaired in Tcf‐1−/‐ mice. In contrast, thymic and thymus‐derived (splenic) TCRγδ+ cells are present in normal numbers in Tcf‐1−/‐ mice, as are other T cell subsets in intestine and liver. Collectively, our data suggest that Tcf‐1 is differentially required for the development of some extrathymic T cell subsets, including intestinal TCRγδ+ cells and liver CD4+ NK1+ T cells.
Administration of interleukin‐10 abolishes innate resistance to Listeria monocytogenes
Authors: Kelly John P. , Bancroft Gregory J.
Published: March 2007

We have used severe‐combined immunodeficient (SCID) mice to examine the immunoregulatory effects of interleukin (IL)‐10 on innate resistance to infection with Listeria monocytogenes. Addition of heat killed Listeria to spleen cells from naive SCID mice resulted in secretion of interferon (IFN)‐γ from natural killer cells in vitro. This response was enhanced up to 15‐fold in the presence of exogenous IL‐2, but was completely ablated by addition of IL‐10 with an IC50 of < 0.5 U/ml. Infection of SCID mice with viable Listeria in vivo resulted in a prolonged course of infection eventually causing death by 12–14 days, whereas daily administration of IL‐10 increased bacterial replication in the liver and spleen by up to 1000‐fold resulting in death by day 4 post‐infection. The immunosuppressive actions of IL‐10 in vivo were also observed in immunocompetent BALB/c mice, where doses as low as 100 U/day converted a sublethal infection to 100% mortality. To study the events controlling expression of endogenous IL‐10, peritoneal macrophage monolayers were challenged with Listeria after pre‐incubation with a panel of recombinant cytokines. IFN‐γ primed macrophages for enhanced tumor necrosis factor (TNF) secretion, but inhibited IL‐10 production, whereas granulocyte/macrophage colony‐stimulating factor (CSF), macrophage CSF and also IL‐4 enhanced macrophage IL‐10 responses after ingestion of Listeria in vitro. Finally, monoclonal antibody neutralization of IFN‐γ during infection of SCID mice with Listeria inhibited TNF‐α mRNA, but augmented expression of IL‐10 mRNA in infected tissues. These results demonstrate that exogenous IL‐10 is a potent immunosuppressive cytokine in the context of infection with an intracellular bacterium and that expression of endogenous IL‐10 versus TNF is differentially regulated by the cytokine environment of the macrophage.
CD40/CD40 ligand interactions are required for T cell‐dependent production of interleukin‐12 by mouse macrophages
Authors: Kennedy Mary K. , Picha Kathleen S. , Fanslow William C. , Grabstein Kenneth H. , Alderson Mark R. , Clifford Ky N. , Chin Wilson A. , Mohler Kendall M.
Published: March 2007

We have previously shown that T cell receptor‐activated mouse T helper (Th)1 clones induce the production of interleukin (IL)‐12 by splenic antigen‐presenting cells (APC). Here, we show that the expression of CD40L by activated T cells is critical for T cell‐dependent IL‐12 production by mouse macrophages. IL‐12 was produced in cultures containing alloreactive Th1 clones stimulated with allogeneic peritoneal macrophages, or in cultures of splenocytes stimulated with anti‐CD3. Anti‐CD40L monoclonal antibodies (mAb) inhibited the production of IL‐12, but not IL‐2, in these cultures by ⩾90% and had dramatic inhibitory effects on antigen‐dependent proliferation of Th1 clones. In addition, both activated T cells and a Th1 clone derived from CD40L knockout mice failed to induce IL‐12 production from splenic APC or peritoneal macrophages. Finally, macrophages cultured in the absence of T cells produced IL‐12 upon stimulation with soluble recombinant CD40L in combination with either supernatants from activated Th1 clones or with interferon‐γ and granulocyte/macrophage colony‐stimulating factor. Thus, both CD40L‐dependent and cytokine‐mediated signals from activated T cells are required to induce the production of IL‐12 by macrophages. A blockade at the level of IL‐12 production may explain, at least in part, the dramatic ability of anti‐CD40L mAb to inhibit disease in animal models that are dependent upon the generation of a cell‐mediated immune response. Moreover, a defect in T cell‐dependent induction of IL‐12 may contribute to the immune status of humans that lack functional CD40L.
A study of complexes of class II invariant chain peptide: Major histocompatibility complex class II molecules using a new complex‐specific monoclonal antibody
Authors: Eastman Susan , Deftos Michael , Deroos Paul C. , Hsu Di‐Hwei , Teyton Luc , Braunstein Ned S. , Hackett Charles J. , Rudensky Alexander
Published: March 2007

Complexes of major histocompatibility complex (MHC) class II molecules containing invariant chain (Ii)‐derived peptides, known as class II‐associated invariant chain peptides (CLIP), are expressed at high levels in presentation‐deficient mutant cells. Expression of these complexes in mutant and wild‐type antigen‐presenting cells suggests that they represent an essential intermediate in the MHC class II antigen‐presenting pathway. We have generated a monoclonal antibody, 30‐2, which is specific for these complexes. Using this antibody, we have found quantitative differences in CLIP: MHC class II surface expression in mutant and wild‐type cells. Our experiments also show that CLIP: MHC class II complexes are preferentially expressed on the cell surface similar to total mature MHC class II molecules. These complexes are found to accumulate in the endosomal compartment in the process of endosomal Ii degradation. Analysis of the fine specificity of the antibody indicates that these complexes have Ii peptide bound to the peptide‐binding groove.
Membrane exon sequences of the three Xenopus Ig classes explain the evolutionary origin of mammalian isotypes
Authors: Mußmann Rainer , Wilson Melanie , Marcuz Anne , Courtet Michèle , Du Pasquier Louis
Published: March 2007

We have cloned and sequenced the genes corresponding to the membrane exons of the three immunoglobulin (Ig) heavy chain isotypes (μ, ν and χ) of Xenopus. Among membrane Ig (mIg) polypeptides, the transmembrane domain are the most highly conserved. The transmembrane and cytoplasmic domains of Xenopus mIgM are similar to the corresponding domains of all known vertebrate mIgM molecules, supporting the idea that amphibian μ gene is homologous, not just analogous, to the μ gene of higher vertebrates. The membrane forms of the two other Ig isotypes mIgX and mIgY exhibit the specific structure found in all Ig membrane exons, but are not homologous with any specific mammalian non‐μ Ig isotype; they are most similar to Xenopus mIgM. Based on the conserved transmembrane domains of Xenopus mIgX, mIgY, we suggest that first the ν and later the χ genes arose by duplication from the original μ gene. The transmembrane and the 37‐amino‐acid‐long cytoplasmic domains of Xenopus mIgY have conserved residues found in avian mIgY and mammalian mIgG and mIgE, suggesting that the modern isotypes might share a common ancestor with amphibian mIgY. However, while the sequence similarity between the membrane exons of avian mIgY and mammalian mIgG and IgE is striking, the overall similarity with Xenopus mIgY is very low. Thus, the genes giving rise to Xenopus mIgY and those eventually leading to avian mIgY and mammalian mIgG and mIgE must have diverged early in evolution, probably at the level of the primitive amphibians or before.
Expression and function of B7‐1 (CD80) and B7‐2 (CD86) on human epidermal Langerhans cells
Authors: Rattis Frédérique‐Marie , Péguet‐Navarro Josette , Staquet Marie‐Jeanne , Dezutter‐Dambuyant Colette , Courtellemont Pascal , Redziniak Gérard , Schmitt Daniel
Published: March 2007

In addition to T cell receptor triggering, activation of T cells requires co‐stimulatory signals that have been shown to be mainly initiated through CD28. We analyzed the expression and function of the two ligands for CD28, B7‐1 (CD80) and B7‐2 (CD86), on human Langerhans cells (LC), the antigen‐presenting cells from epidermis. Human LC freshly isolated from epidermis (fLC) expressed significant level of B7‐2, which was increased upon a short culture in vitro. In contrast, B7‐1 was undetectable on fLC but appeared at the cell surface after a 3‐day culture in vitro. Pre‐incubation of 18‐h cultured LC with anti‐B7‐2 monoclonal antibodies (mAb) was sufficient to abrogate the binding of CTLA4‐Ig fusion protein, while a combination of both mAb against B7‐1 and B7‐2 was necessary to obtain a complete inhibition of CTLA4‐Ig binding on 3‐day cultured LC, showing the absence of a third CTLA4 ligand. The function of B7‐1 and B7‐2 on human LC has been analyzed by adding mAb at the beginning of mixed epidermal cell lymphocyte reactions. Anti‐B7‐2 mAb and CTLA4‐Ig, but not anti‐B7‐1 mAb, strongly inhibited allogeneic, as well as recall antigen‐induced T cell proliferation supported by fLC or 3‐day cultured LC. Collectively, these results demonstrate that B7‐2 is the major ligand for CD28/CTLA4 at the LC surface and that it plays a crucial role in human LC co‐stimulatory function with little, if any, dependence on B7‐1 expression.
P‐selectin directs T lymphocyte‐mediated injury in delayed‐type hypersensitivity responses: studies in glomerulonephritis and cutaneous delayed‐type hypersensitivity
Authors: Tipping Peter G. , Huang Xiao R. , Berndt Michael C. , Holdsworth Stephen R.
Published: March 2007

The role of P‐selectin in T lymphocyte accumulation and injury was studied in delayed‐type hypersensitivity (DTH) responses in the skin and glomeruli of rats. Sprague Dawley rats were sensitized to sheep globulin and challenged 5 days later in the skin by subcutaneous injection and simultaneously in glomeruli by intravenous injection of a subnephritogenic dose of sheep anti‐rat glomerular basement membrane globulin. This resulted in cutaneous and glomerular T lymphocyte‐dependent macrophage influx and injury characteristic of DTH. Up‐regulation of P‐selectin expression on endothelial cells was observed in both inflammatory lesions. Treatment of rats with anti‐CD5 antibody immediately prior to antigen challenge prevented the development of injury as assessed by measurement of proteinuria and skin swelling, as well as local T cell and macrophage accumulation in the glomerulus and in the skin, but did not block up‐regulation endothelial cell P‐selectin. Treatment with anti‐CD4 antibody produced similar results. Blocking P‐selectin in vivo with a functionally inhibitory antibody prevented development of proteinuria and skin swelling following antigen challenge. Local accumulation of T cells and macrophages was markedly attenuated in glomeruli and the skin and up‐regulation of endothelial cell P‐selectin was prevented. These data demonstrate that P‐selectin is locally up‐regulated on endothelial cells in T cell‐dependent glomerular and cutaneous inflammation and suggests a pivotal functional role for P‐selectin in local T cell recruitment and subsequent injury in DTH.
Anti‐parasitic effector mechanisms in human brain tumor cells: Role of interferon‐γ and tumor necrosis factor‐α
Authors: Däubener Walter , Remscheid Christine , Nockemann Susanne , Pilz Korinna , Seghrouchni Samira , Mackenzie Colin , Hadding Ulrich
Published: March 2007

Toxoplasma gondii, an obligate intracellular parasite, is able to replicate in human brain cells. We recently showed that interferon (IFN)‐γ‐activated cells from glioblastoma line 86HG39 were able to restrict Toxoplasma growth. The effector mechanism responsible for this toxoplasmostatic effect was shown by us to be the IFN‐γ‐mediated activation of indolamine 2,3‐dioxygenase (IDO), resulting in the degradation of the essential amino acid tryptophan. In contrast, glioblastoma 87HG31 was unable to restrict Toxoplasma growth after IFN‐γ activation, and IFN‐γ‐mediated IDO activation was weak. We observed that tumor necrosis factor (TNF)‐α alone is unable to activate IDO or to induce toxoplasmostasis in any glioblastoma cell line tested. Interestingly, we found that TNF‐α and IFN‐γ were synergistic in the activation of IDO in glioblastoma cells 87HG31, 86HG39 and U373MG and in native astrocytes. This was shown by the measurement of enzyme activity as well as by the detection of IDO mRNA in TNF‐α + IFN‐γ activated cells. This IDO activity results in a strong toxoplasmostatic effect mediated by glioblastoma cells activated simultaneously by both cytokines. Antibodies directed against TNF‐α or IFN‐γ were able to inhibit IDO activity as well as the induction of toxoplasmostasis in glioblastoma cells stimulated with both cytokines. Furthermore, it was found that the addition of L‐tryptophan to the culture medium completely blocks the antiparasitic effect. We therefore conclude that both TNF‐α and IFN‐γ may be involved in the defense against cerebral toxoplasmosis by inducing IDO activity as an antiparasitic effector mechanism in brain cells.
Immunization with glycosylated Kb‐binding peptides generates carbohydrate‐specific, unrestricted cytotoxic T cells
Authors: Abdel‐Motal Ussama M. , Berg Louise , Rosén Anders , Bengtsson Marita , Thorpe Cristopher J. , Kihlberg Jan , Dahmén Jan , Magnusson Göran , Karlsson Karl‐Anders , Jondal Mikael
Published: March 2007

Cytotoxic T cells (CTL) recognize target proteins as short peptides presented by major histocompatibility complex (MHC) class I restriction elements. However, there is also evidence for peptide‐independent T cell receptor (TCR) recognition of target proteins and non‐protein structures. How such T cell responses are generated is presently unclear. We generated carbohydrate (CHO)‐specific, MHC‐unrestricted CTL responses by coupling di‐ and trisaccharides to Kb‐ or Db‐binding peptides for direct immunization in mice. Four peptides and three CHO have been analyzed with the CHO either in terminal or central positions on the carrier peptide. With two of these glycopeptides, with galabiose (Galα1‐4Gal; Gal2) bound to a homocysteine (via an ethylene spacer arm) in position 4 or 6 in a vesicular stomatitis virus nucleoprotein‐derived peptide (RGYVYQGL binding to Kb), CTL were generated which preferentially killed target cells treated with glycopeptide compared to those treated with the core peptide. Polyclonal CTL were also found to kill target cells expressing the same Gal2 epitope in a glycolipid. By fractionation of CTL, preliminary data indicate that glycopeptide‐specific Kb‐restricted CTL and unrestricted CHO‐specific CTL belong to different T cell populations with regard to TCR expression. The results demonstrate that hapten‐specific unrestricted CTL responses can be generated with MHC class I‐binding carrier peptides. Different models that might explain the generation of such responses are discussed.
Interleukin‐5 mRNA expressed by eosinophils and γ/δ T cells in parasite‐immune sheep
Authors: Bao Shisan , McClure Susan J. , Emery David L. , Husband Alan J.
Published: March 2007

Interleukin (IL)‐5 is produced by a variety of cell types and contributes to both lymphocyte development and eosinophil terminal differentiation in vitro. The coincidence of worm expulsion and eosinophilia in sheep infected with the gastrointestinal nematode Trichostrongylus colubriformis suggests that eosinophils may be involved as effector cells in host immunity against parasite infection. The role of IL‐5 in this process was investigated by observing the distribution of IL‐5 mRNA+ cells in the small intestine, mesenteric lymph nodes (MLN) and Peyer's patches (PP) by an in situ hybridization technique using a murine IL‐5 riboprobe. IL‐5 mRNA+ cells were distributed throughout the lamina propria (LP) of the small intestine from the tips of the villi to the muscularis mucosae and in the parafollicular areas of MLN and PP in both naive and immune sheep. The phenotypes of IL‐5 mRNA+ cells was explored by simultaneous eosin and immunohistochemical staining using a monoclonal antibody recognizing the T19 marker, which identifies a major subset of γ/α TCR+ cells in sheep. In immune sheep, there was about a five‐fold increase in the number of eosinophils and IL‐5 mRNA+ cells in the LP, but there was no significant change in numbers of T19+ cells. Most of the IL‐5 mRNA+ cells in the LP were eosinophils, but many of the T19+ cells also expressed IL‐5 mRNA. In contrast, there were fewer eosinophils than T19+ cells in MLN of immune sheep and, compared to controls, a threefold increase in T19+ cells and a five‐fold increase in T19+/IL‐5 mRNA+ double‐positive cells was observed in immune sheep. In PP, there were very few eosinophils but substantial numbers of T19+ cells; however, no significant differences in numbers of eosinophils, T19+ or IL‐5 mRNA+ cells were observed between control and immune sheep. These results indicate that in sheep, both eosinophils and γ/δ T cells are capable of IL‐5 expression and suggest that IL‐5 is an important regulatory factor in autocrine and paracrine activation of effector cells involved in parasite immune expulsion.
Mycobacteria‐reactive γδ T cells in HIV‐infected individuals: lack of Vγ9 cell responsiveness is due to deficiency of antigen‐specific CD4 T helper type 1 cells
Authors: Wesch Daniela , Kabelitz Dieter , Friese Klaus , Pechhold Klaus
Published: March 2007

Human γδ T lymphocytes expressing the variable T cell receptor elements Vγδ paired with Vδ2 are activated by antigen derived from Mycobacterium tuberculosis (M. tb.) and presented by antigen‐presenting cells (APC). The subsequent proliferation is strictly dependent on the presence of CD4+TCRαβ+ T helper type 1 (Th1) cells producing interleukin‐2 (IL‐2). In this study, we report that the reactivity of Vγ9 cells to M. tb. stimulation in vitro was drastically decreased or absent in the majority of the analyzed HIV‐1‐infected individuals (CDC stages III and IV). We show that the failure of Vγ9 cells frim HIV− individuals to proliferate following M. tb. stimulation was not due to an intrinsic qualitative or quantitative defect of γδ T cells but rather to a deficiency of M. tb.‐reactive CD4 Th1 cells. Thus, Vγ9 responsiveness could be restored if cultures of M. tb.‐stimulated T cells from HIV+ donors were reconstituted with one of the following: (i) exogenous IL‐2; (ii) purified CD4T cells from allogeneic donors; or (iii) T cell‐depleted APC from allogeneic donors. In the majority of HIV+ patients, the defective Th1 activity of M. tb.‐stimulated CD4 T cells could be increased neither by cytokines known to favor Th1 development (IL‐12, interferon‐γ) nor by neutralization of the Th1‐suppressing Th2 cytokine IL‐10. We suggest that measurement of Vγ9 cell expansion within M. tb.‐stimulated peripheral blood mononuclear cells provides a sensitive assay for the functional capacity of antigen (M. tb.)‐specific CD4 Th1 cells in HIV‐infected individuals.
Macrophage‐T cell interaction in murine salmonellosis: selective down‐regulation of ICAM‐1 and B7 molecules in infected macrophages and its probable role in cell‐mediated immunity
Authors: Gupta Sudhir , Vohra Harpreet , Saha Bhaskar , Nain Chander Kanwal , Ganguly Nirmal Kumar
Published: March 2007

Vaccine development and understanding of cellular immune modulatory mechanisms in salmonella infections have been impeded due to the paucity of data on antigens capable of eliciting effective immune responses. The present study was done to evaluate the efficacy of five major purified salmonella antigens (porins, pili, flagella, outer membrane proteins and heat shock proteins) in modulating T cell‐macrophage interactions which play a central role in resistance to and recovery from infection with several intracellular pathogens, including salmonella. The results showed that the T cells recovered 10 days post‐immunization (D10 T cells) from mice immunized with porins and outer membrane proteins showed maximum proliferation in the presence of macrophages incubated with dead bacteria; however, this response was decreased when T cells were co‐cultured with live Salmonella typhimurium‐infected macrophages. Delayed‐type hypersensitivity responses, as measured by increased footpad thickness at 24 h, though induced effectively by porins, pili and flagella, were completely abrogated when D10 T cells were pre‐incubated with macrophages infected with live bacteria. The phagocytic and bactericidal ability of normal macrophages, when grown in presence of T cell supernatants, was not influenced by the immunizing agents, but T cell supernatants obtained from mice immunized with porins and heat‐shock protein triggered increased bactericidal activity. Further, the expression of the co‐stimulatory molecules ICAM‐1 and B7 increased with increasing bacteria (dead): macrophage ratio, but this expression was down‐regulated upon incubation with live bacteria.