bluuebunny/crossref_metadata_2025 huggingface dataset:
Down‐modulation of CD4+ T helper type 2 and type 0 cells by T helper type 1 cells via Fas/Fasligand interaction
Authors: Hahn Sinuhe , Stalder Thomas , Wernli Marion , Bürgin Diana , Tschopp Jürg , Nagata Shigekazu , Erb Peter
Published: March 2007

Fas was recently demonstrated to be the major target molecule engaged by CD4+ cytolytic T lymphocytes (CTL). We examined Fas expression on various cloned T cell subpopulations and their susceptibility to lysis by CD4+ or CD8+ CTL. A reciprocal relationship in Fas and Fas‐ligand expression was observed in CD4+ T helper (Th)1‐ and Th2‐type clones, and Fas mRNA was predominantly detected in Th2 clones, whereas Fas‐ligand mRNA was principally found in Th1 clones. The two Th0 clones tested expressed both Fas and Fas‐ligand, but only one exhibited cytolytic activity, whereas both were sensitive to CD4‐mediated lysis. A functional consequence of the inverse Fas‐Fas‐ligand expression pattern was that Th2 and Th0 cells were sensitive to lysis by both Th1 CD4+ CTL and a CD8+ CTL clone in a Fas‐dependent manner. These results suggest that cytolytic CD4+ Th1 cells may play an immunomodulatory role, regulating a Th2/Th0 response by Fas‐mediated lysis.
Exogenous human immunodeficiency virus type‐1 Tat protein selectively stimulates a phosphatidylinositol‐specific phospholipase C nuclear pathway in the Jurkat T cell line
Authors: Zauli Giorgia , Previati Marizio , Caramelli Elisabetta , Bassini Alessandra , Falcieri Elisabetta , Gibellini Davide , Bertolaso Lucia , Bosco Domenico , Robuffo Iole , Capitani Silvano
Published: March 2007

We investigated the effect of extracellular Tat protein of human immunodeficiency virus‐type 1 (HIV‐1) on the phosphatidylinositol (PI) cycle, which represents a major signal transduction pathway in lymphoid cells. Recombinant Tat, recombinant HIV‐1 p24 and cross‐linked anti‐CD3 monoclonal antibody (mAb) were added in culture for 1–60 min to Jurkat lymphoblastoid CD4+ T cells. The stimulation of T cell receptor by cross‐linked anti‐CD3 mAb resulted in a rapid increase of the phosphatidylinositol‐specific phospholipase C (PI‐PLC) activity in whole cell lysates. On the other hand, Tat protein, either alone or in combination with anti‐CD3 mAb, showed little effect on the PI turnover of whole cell extracts. Tat, however, selectively stimulated a nuclear‐specific PI‐PLC with a peak of activity after 30 min from the addition in culture to Jurkat cells. Interestingly, this time corresponded to that required for the uptake and nuclear localization of recombinant Tat protein, as demonstrated by electron microscope immunocytochemistry experiments with anti‐Tat mAb. Moreover, exogenous Tat reached the nucleus of Jurkat cells in a bioactive form, as shown in a HIV‐1 long terminal repeat‐chloramphenicol acetyl transferase transactivation assay. The specific increase of a nuclear PI‐PLC activity was further demonstrated by the ability of Tat to stimulate PI turnover also when added directly to isolated nuclei. As a whole, these data demonstrate that Tat selectively stimulates a nuclear polyphosphoinositide hydrolysis, which appears to be independent of the cellular PI turnover. The relevance of these findings for a better understanding of the biological functions of extracellular Tat is discussed.
Polymorphonuclear granulocytes enhance lipopolysaccharide‐induced soluble p75 tumor necrosis factor receptor release from mononuclear cells
Authors: Lien Egil , Liabakk Nina‐Beate , Johnsen Ann‐Charlotte , Nonstad Unni , Sundan Anders , Espevik Terje
Published: March 2007

Lipopolysaccharide (LPS), a part of the Gram‐negative bacteria cell wall, is a potent inducer of tumor necrosis factor (TNF). TNF is an important mediator in Gram‐negative infections such as meningococcal septic shock, but its harmful action can be prevented by the natural occurring soluble (s) TNF receptors (sTNFR) sp55 and sp75. In this study, the effect of LPS on release of sTNFR was investigated. First, we found a selective increase in human whole‐blood sp75 TNFR levels following LPS stimulation, accompanied by no increase in sp55. Separating the different blood cell populations, mononuclear cells (PBMC) selectively released sp75 upon LPS stimulation, while LPS induced a minor increase in sp75 release from polymorphonuclear granulocytes. Interestingly, in co‐cultures of PBMC and granulocytes, the release of LPS‐induced sp75 TNFR was enhanced. Second, adherent monocytes were also found to selectively release sp75 TNFR upon LPS stimulation, where Neisseria meningitidis LPS was found to be 100‐1000 times more potent in inducing sp75 release than Escherichia coli LPS. Using flow cytometry, the monocyte membrane distribution of both TNFR were found to be increased after LPS stimulation. Third, human umbilical vein endothelial cells selectively released sp55 TNFR after stimulation with LPS. We conclude that mononuclear and endothelial cells might be the main sources of soluble p75 and p55 TNFR, respectively, observed in Gramnegative sepsis, although these receptors are released in vivo more rapidly than they are in vitro.
In vitro cultured stromal cells from human tonsils display a distinct phenotype and induce B cell adhesion and proliferation
Authors: Lisignoli Gina , Monaco Maria C. G. , Facchini Andrea , Toneguzzi Stefania , Cattini Luca , Hilbert David M. , Lavaroni Stefano , Belvedere Ornella , Degrassi Alberto
Published: March 2007

Peripheral lymphoid tissues contain a fibroblastic cell type referred to as stromal cells or reticulum cells which interact with lymphocytes as part of the lymphoid microenvironment. After isolation from human tonsils and expansion in vitro we analyzed the surface phenotype, extracellular matrix components, cytoskeletal products, cytokine production, binding and functional interaction with B lymphocytes of in vitro cultured stromal cells (HTSC) both in resting condition and after activation with tumor necrosis factor (TNF)‐α and interferon (IFN)‐γ. Our results show that HTSC do not express specific myeloid, lymphoid, endothelial or epithelial markers. HTSC express CD54 (ICAM‐1), CD49a (VLA‐1), CD49b (VLA‐2), CD49c (VLA‐3), CD49e (VLA‐5), CD49f (VLA‐6), CD29, CD51, CD44 and produce vinculin, β‐tubulin, α‐actin, vimentin, fibronectin, laminin and collagen types I, III and IV. Activation of HTSC up‐regulated CD54 (ICAM‐1) and induced HLA‐DR and CD106 (VCAM‐1).

HTSC constitutively produce interleukin (IL)‐6 which is enhanced upon activation with TNF‐α. IL‐8 and granulocyte/macrophage colony‐stimulating factor are detected only in the supernatants of activated HTSC. Reverse transcriptase polymerase chain reaction analysis revealed that HTSC display mRNA for IL‐1α, leukemia inhibitory factor and IL‐7. The adhesion of tonsillar B lymphocytes to activated HTSC is mediated by CD11a/CD18 and CD54. Furthermore, HTSC can induce maximal proliferation of IL‐2‐activated B lymphocytes cocultured in direct cell‐cell contact with HTSC. These results clearly distinguish in vitro cultured HTSC from common fibroblasts and other non‐lymphoid elements present in the lymphoid parenchyma, such as follicular dendritic cells, and show that HTSC actively participate in the lymphoid microenvironment. In vitro cultures of HTSC could therefore be a useful model system for detailed analysis of the interactions between stromal cells and lymphocytes under physiological and pathological conditions.
Calcium‐dependent lectin activity with novel distribution on thymocyte subsets
Authors: Mallett Susan , Rosen Steven D. , Hyun William
Published: March 2007

In this study we use carbohydrate probes to search for novel cell surface lectins in the immune system. Many carbohydrate binding proteins are members of the C‐type lectin superfamily, whose ligand binding is dependent on calcium. To identify potential new members of this superfamily, fluorescein‐conjugated carbohydrate polymers were used to probe for calcium‐dependent cell surface binding. This approach offers advantages over the use of monoclonal antibody probes since only carbohydrate binding proteins would be identified. We have identified a cell surface lectin, referred to as thy‐lec, detected by the probe fucoidan‐fuorescein isothiocyanate. This calcium‐dependent lectin has a novel distribution on thymocyte subsets. It is present on the surface of immature CD4+8+ and on large, cycling CD4−8− cells and CD8+4− cells, but not on small, mature phenotype CD8+4− or CD4+8− thymocytes. This lectin is not found on mature T cells or other leukocytes in lymph nodes, spleen or bone marrow. It is proposed that this novel cell surface has a function in the maturation of T cells in the thymus.
Autoreactive T cell specificity in autoimmune hemolytic anemia of the NZB mouse
Authors: Perry Frances E. , Barker Robert N. , Mazza Graziella , Day Michael J. , Wells Angela D. , Shen Chia‐Rui , Schofield Ann E. , Elson Christopher J.
Published: March 2007

Splenic T cells from Coombs'‐positive New Zealand Black (NZB) mice proliferated consistently in vitro in response to the integral red blood cell (RBC) membrane protein Band 3, the antigen previously shown to be the target for the pathogenic RBC autoantibodies. The responding cells predominantly express CD4 and the proliferative response is blocked by antibodies to the NZB major histocompatibility complex class II but not by antibodies to an irrelevant H‐2 haplotype. NZB splenic T cells also proliferated in response to the internal membrane skeleton protein spectrin. By contrast, T cells from BALB/c and DBA2 mice, which bear the same H‐2 haplotype as NZB mice, but which do not develop autoimmune hemolytic anemia (AIHA), fail to respond to Band 3. It is considered that these results support the hypothesis that Band 3‐reactive T cells provide help for the production of pathogenic anti‐Band 3 autoantibodies in NZB mice. T cells from Coombs'‐negative NZB mice as young as 3 weeks old proliferated in response to Band 3; moreover, the RBC from Coombs'‐negative mice bore elevated levels of autoantibody as judged by a sensitive direct enzyme‐linked anti‐globulin test. Thus, the pathology of AIHA develops at a much earlier age than was thought previously.
Expression of the common cytokine receptor γ chain by murine dendritic cells including epidermal Langerhans cells
Authors: Mohamadzadeh Mansour , Ariizumi Kiyoshi , Sugamura Kazuo , Bergstresser Paul R. , Takashima Akira
Published: March 2007

The common cytokine receptor γ chain (γc) is an indispensable component of interleukin (IL)‐2, IL‐4, IL‐7, IL‐9, and IL‐15 receptors, and its expression has been detected in several leukocyte populations, including T cells, B cells, monocytes, natural killer cells, and neutrophils. The purpose of this study was to determine whether γc receptors are expressed by dendritic cells (DC). Constitutive γc mRNA expression was observed by reverse transcription polymerase chain reaction and/or Northern blotting for: (a) Ia+ epidermal Langerhans cells (LC), (b) 4F7+ splenic DC, (c) granulocyte/macrophage colony‐stimulated factor‐propagated bone marrow‐derived DC, and (d) the epidermal‐derived DC line, XS52, which retains important functions of epidermal LC. Exposure of XS52 cells to recombinant IL‐4 induced a rapid up‐regulation of c‐myc mRNA expression, and this IL‐4‐dependent signaling was blocked almost completely by anti‐γc monoclonal antibody (mAb) TUGm2 in a soluble form. Moreover, c‐myc up‐regulation was inducible in XS52 cells by the same mAb in an immobilized form. These results imply that molecules recognized by this antibody (i.e. γc receptors) are expressed on XS52 cell surfaces. We thus conclude that DC express functional γc receptors, which then mediate cytokine‐dependent regulation of DC functions.
Non‐tolerant B cells cause autoimmunity in anti‐CD8 IgG2a‐transgenic mice
Authors: Battegay Manuel , Fiedler Petra , Kalinke Ulrich , Brombacher Frank , Zinkernagel Rolf M. , Peter Hans‐Hartmut , Köhler Georges , Eibel Hermann
Published: March 2007

Using a pair of γ2a/x immunoglobulin genes, transgenic mice were generated to study tolerance induction in B cells that express IgG2a autoantibodies. The transgenic IgG2a specifically binds CD8 α chains of the CD8.2 allotype expressed on the surface of CD8+ T cells, but not CD8 molecules expressed by the CD8.1 allele. Thus, IgG2a transgenic mice expressing the CD8.1 allele were used as controls to monitor B cell development and mice expressing CD8.2 were used to study B cell tolerance. Both types of mice showed transgenic γ2a expression on the surface of B cells. Expression of endogenous heavy chain alleles was strongly inhibited in immature B cell subsets, whereas mature B cells co‐expressed transgenic γ2a and endogenous IgM/D. The transgenic x chain expression leads only to partial allelic exclusion of endogenous light chains. B cells that express high levels of transgenic CD8.2‐specific IgG2a were identified using soluble CD8‐Ig. In CD8.1+ and in CD8.2+ mice, we found no differences in expression and maturation of transgenic anti‐CD8.2 IgG2a+ B cells. High levels of serum anti‐CD8.2 IgG2a antibodies led to the elimination of CD8+ T cells, causing a severe defect in cytotoxic immune responses. These results show that tolerance induction is incomplete in the CD8.2+ mice, either because IgG2a+ B cells are resistant to censoring mechanisms or because the secreted CD8‐specific IgG2a antibodies render the CD8 autoantigen inaccessible to the B cells. This contrasts strongly with the efficient induction of B cell tolerance in mice expressing anti‐CD8.2 IgM autoantibodies.
Biological function and distribution of human interleukin‐12 receptor β chain
Authors: Wu Chang‐You , Warrier Rajeev R. , Carvajal Daisy M. , Chua Anne O. , Minetti Lisa J. , Chizzonite Richard , Mongini Patricia K. A. , Stern Alvin S. , Gubler Ueli , Presky David H. , Gately Maurice K.
Published: March 2007

We previously described the cloning of a cDNA encoding an interleukin‐12 receptor (IL‐12R) subunit, designated β, that bound IL‐12 with low affinity when expressed in COS cells. We now report that a pair of monoclonal antibodies (mAb), 2B10 and 2.4E6, directed against different epitopes on the IL‐12Rβ chain, when used in combination, strongly inhibited IL‐12‐induced proliferation of activated T cells, IL‐12‐induced secretion of interferon‐γ by resting peripheral blood mononuclear cells (PBMC), and IL‐12‐mediated lymphokine‐activated killer cell activation. The mAb had no effect on lymphoblast proliferation induced by IL‐2, −4, or −7. Thus, the IL‐12Rβ chain appears to be an essential component of the functional IL‐12R on both T and natural killer (NK) cells. We previously observed that high affinity IL‐12R were expressed on activated T and NK cells, but not B cells. Studies using flow cytometry and reverse transcription‐polymerase chain reaction analysis showed that IL‐12Rβ chain was expressed on several human T, NK, and (surprisingly) B cell lines, but not on non‐lymphohematopoietic cell lines. The Kit225/K6 (T cell) and SKW6.4 (B cell) lines were found to express the greatest amounts of IL‐12Rβ chain (800–2500 sites/cell); however, Kit225/K6 but not SKW6.4 cells bound IL‐12. Similar to SKW6.4 B cells, activated tonsillar B lymphocytes expressed IL‐12Rβ chain but, consistent with previous results, did not display detectable IL‐12 binding. Likewise, up to 72% of resting PBMC from normal volunteer donors expressed IL‐12Rβ, but did not bind measurable amounts of IL‐12. These results indicate that expression of IL‐12Rβ is essential, but not sufficient, for expression of functional IL‐12R. We speculate that expression of functional, high‐affinity IL‐12R may require the presence of a second subunit that is more restricted in its expression than IL‐12Rβ.
Co‐localization of β2‐microglobulin and IgG in human placental syncytiotrophoblasts
Authors: Kristoffersen Einar K. , Matre Roald
Published: March 2007

The fetal syncytiotrophoblast cells in close contact with maternal blood circulation apparently lack surface expression of HLA molecules, including the HLA light chain β2‐microglobulin. This is thought to contribute significantly to a successful pregnancy. We find that syncytiotrophoblasts do express β2‐microglobulin. β2‐microglobulin is primarily localized intracellularly in apical granules, and co‐localize with human IgG. The origin and function of syncytiotrophoblast β2‐microglobulin is unknown, but its localization in the syncytiotrophoblasts may implicate β2‐microglobulin in the transplacental transport of IgG in conjunction with a recently identified class I HLA‐like receptor for IgG/Fc. Alternatively, β2‐microglobulin may associate with a hitherto unidentified class I HLA molecule.
A monoclonal antibody, 3/22, to rabbit CD11c which induces homotypic T cell aggregation: evidence that ICAM‐1 is a ligand for CD11c/CD18
Authors: Blackford Jeanette , Reid Hugh W. , Pappin Darryl J. C. , Bowers Florence S. , Wilkinson J. Michael
Published: March 2007

The rabbit CD11c molecule has been characterized by use of a new monoclonal antibody, mAb 3/22. Expression of the p150,95 integrin (CD11c/CD18) has been shown by flow cytometry and immunohistochemistry to be restricted to monocytes, macrophages, dendritic cells and a small population of lymphocytes in peripheral blood. No expression on neutrophils could be demonstrated. Incubation of the newly derived CD8+ T cell line, BJ/873, with mAb 3/22 causes homotypic aggregation, which has been shown to be a cell surface event that is not dependent on intracellular signaling or on receptor cross‐linking. Inhibition studies show that the ligands responsible for this aggregation are CD11c/CD 18 and ICAM‐1, both of which are expressed on BJ/873. One other rabbit T cell line, K34, that also expresses p150,95 and ICAM‐1, shows a similar aggregation response when stimulated with 3/22. Cell lines that express p150,95 but not ICAM‐1 do not aggregate. These observations suggest that ICAM‐1 is a ligand for activated p150,95.
Nitric oxide‐mediated suppression of T cell responses during Trypanosoma brucei infection: soluble trypanosome products and interferon‐γ are synergistic inducers of nitric oxide synthase
Authors: Sternberg Jeremy M. , Mabbott Neil A.
Published: March 2007

African trypanosome infections result in lymphocyte unresponsiveness and anemia in the mammalian host. In murine infections, these effects are mediated by suppressor macrophages releasing nitric oxide (NO). We investigated the mechanism of activation of macrophages to produce NO during trypanosomiasis in vitro. A soluble component of trypanosome lysates induced NO synthesis in peritoneal macrophage cultures only when the macrophages were co‐stimulated with interferon‐gamma (IFN‐γ). The macrophage‐activating factor was also released in a soluble form by live bloodstream‐form trypanosomes, but not procyclic trypanosomes. When splenocyte cultures were exposed to IFN‐γ and trypanosomes, an NO‐dependent suppression of T cell proliferation occurred. This is similar to the suppression observed in the spleens of trypanosome‐infected mice, suggesting that a combination of trypanosome‐released macrophage‐activating factors and IFN‐γ are a trigger of immune dysfunction in trypanosomiasis.
Different cytokines regulate antigen uptake and presentation of a precursor dendritic cell line
Authors: Lutz Manfred B. , Aßmann Caroline U. , Girolomoni Giampiero , Ricciardi‐Castagnoli Paola
Published: March 2007

Langerhans cells (LC) and dendritic cells (DC) need to be activated in order to perform their antigen‐presenting function. In this study, we explored the influence of cytokines on the uptake and presentation of protein antigens by the retrovirally immortalized myeloid cell line FSDC. This cell line was generated from mouse fetal skin and was previously shown to have the characteristics of early DC precursors. Both FSDC and bone marrow‐derived DC (BM‐DC) were more effective in the pinocytosis of FITC‐conjugated ovalbumin (FITC‐OVA) and dextran (FITC‐DX) than B cells or macrophages. Pretreatment of FSDC with granulocyte/macrophage colony‐stimulating factor (GM‐CSF) ± interleukin (IL)‐4 enhanced the pinocytic uptake of FITC‐OVA and FITC‐DX, but did not induce antigen‐presenting capacity. In contrast, untreated FSDC or FSDC pre‐incubated with GM‐CSF ± IL‐4 suppressed T cell responses. Treatment of FSDC with IFN‐γ reduced pinocytosis but increased the expression of MHC and co‐stimulatory/adhesion molecules and promoted efficient presentation of OVA protein or peptide to the specific DO11.10 T cell hybridoma or to naive CD4+ T cells from DO11.10 TCR‐transgenic mice. The results suggest that antigen uptake and antigen presentation in DC are regulated by different cytokine signals provided by the surrounding tissue.
Tolerance is overcome in beef insulin‐transgenic mice by activation of low‐affinity autoreactive T cells
Authors: Poplonski Lorraine , Vukusic Brian , Pawling Judy , Clapoff Susan , Roder John , Hozumi Nobu , Wither Joan
Published: March 2007

To gain insight into the factors controlling the maintenance or loss of T cell self tolerance we produced beef insulin (BI)‐transgenic BALB/c mice. Transgenic mice express BI under control of the human insulin promoter and secrete physiological amounts of beef insulin. Although these mice are tolerant to BI, as evidenced by the lack of insulin‐specific IgG antibody production following intraperitoneal immunization, tolerance is not complete. Footpad immunization results in a weak antigen‐specific T cell proliferative response, indicating the presence of self‐ reactive BI‐specific T cells in the periphery. These T cells are functional in vivo, providing support for IgG1, IgG2a, and IgG2b BI‐specific antibody production, but require higher concentrations of antigen than nontransgenic T cells (both in vivo and following recall responses in vitro) to become activated. In vitro, BI‐specific T cell proliferation in BI‐transgenic mice can be largely restored by addition of interleukin‐2, indicating that a significant component of T cell tolerance is mediated by anergy. To characterize the autoreactive T cells that become activated when tolerance is broken, BI‐specific T cell hybridomas were generated from transgenic mice and compared to a panel of hybridomas previously derived from nontransgenic BALB/c mice. The majority of BI‐transgenic hybridomas recognized the immunodominant A1–14 beef insulin peptide but with lower avidity than BALB/c hybridomas. Consistent with this, none of the dominant T cell receptor rearrangements found in the BALB/c BI‐specific T cell receptor repertoire were found in the transgenic hybridomas. These results indicate that, despite evidence for clonal inactivation of many BI‐specific T cells in BI‐transgenic mice, loss of tolerance results from activation of low‐affinity antigen‐specific T cells that appear to have escaped this process.
Negative feedback mechanism suppresses interleukin‐12 production by antigen‐presenting cells interacting with T helper 2 cells
Authors: Hino Ayako , Nariuchi Hideo
Published: March 2007

Interleukin‐12 (IL‐12) has been shown to be produced by monocytes by the ligation of CD40. In the present experiments, IL‐12 is shown to be produced by murine spleen antigen‐presenting cells (APC) by interaction with T helper 1 (Th1) 1 clones through CD40‐CD40 ligand (CD40L) interaction, but not with Th2 clones. The IL‐12 production induced by the Th1 clone interaction was inhibited by the addition of exogenous IL‐10. Th2 clones were shown to produce a sufficient amount of IL‐10 to inhibit the IL‐12 production induced by Th1 clones. In the presence of anti‐IL‐10 monoclonal antibodies splenic APC interacting with Th2 clones produced IL‐12. These results indicate that IL‐10 produced by Th2 cells stimulated with antigen suppress IL‐12 production of APC interacting with Th2 cells. IL‐12 is composed of two subunits, p35 and p40. In our experiments, p40 mRNA accumulation was shown to be affected by IL‐10 more severely than the accumulation of p35 mRNA, indicating that IL‐10 regulates IL‐12 production by APC mainly by affecting p40 mRNA accumulation.
Expression of bcl‐x during mouse B cell differentiation and following activation by various stimuli
Authors: Choi Michael S. K. , Holman Mary , Atkins Chris J. , Klaus Gerry G. B.
Published: March 2007

We have studied the expression of the novel anti‐apoptotic protein bcl‐x during mouse B cell differentiation and activation. We find that bcl‐x is expressed throughout all stages of B cell differentiation in the bone marrow, and is only down‐regulated in mature (sIgD+) B cells. Immature peripheral B cells express low levels of bcl‐x even in adult animals, whereas mature resting B cells do not. Mature B cells re‐express the protein following activation, achieving maximal levels after 36–48 h. The highest levels of bcl‐x are observed with potent comitogenic stimuli (such as anti‐CD40 + anti‐Ig): B cells first express bcl‐x in the G1 phase of the cell cycle and contain maximal levels in S phase. In addition, B cells from CBA/N mice, which do not proliferate when stimulated with anti‐Ig, anti‐CD40, or both, exhibited only low levels of the protein following culture with these stimuli. To investigate the functional significance of bcl‐x in activated B cells, we tested their sensitivity to apoptosis induced by the Ca2+ ATPase inhibitor thapsigargin: B cell blasts activated with anti‐CD40 and anti‐Ig were resistant to this agent. The available data therefore suggest that bcl‐x fulfils two roles in B cells: it promotes survival of immature B cells (which lack bcl‐2) and secondly, it apparently plays an additional role in protecting activated mature B cells (perhaps those in germinal centers) from apoptotic stimuli.
Expression of high‐ and low‐affinity receptors for C3a on the human mast cell line, HMC‐1
Authors: Legler Daniel F. , Loetscher Marcel , Jones Simon A. , Dahinden Clemens A. , Arock Michel , Moser Bernhard
Published: March 2007

The proteolytic cleavage product of complement component 3, (C3a), like C4a and C5a, is a potent anaphylatoxin and induces the production of inflammatory mediators in phagocytes. Notably, mast cells respond to C3a with the release of vasoactive substances, including histamine. We have examined the function and receptor binding of C3a in a human leukemic mast cell line, HMC‐1. Similar to chemoattractant agonists in leukocytes, C3a induced rapid cytosolic free calcium concentration increases in HMC‐1 cells. EGTA did not diminish this response, indicating that mobilizable Ca2+ was from intracellular stores. Receptors for C3a in HMC‐1 cells couple in part to Bordetella pertussis toxin‐sensitive G‐proteins and, therefore, appear to belong to the family of serpentine receptors that require G‐proteins for signal transduction. HMC‐1 cells express two types of C3a receptors, C3aR1 and C3aR2, that were shown to bind 125I‐C3a with high‐(Kd1 = 2.1–4.8 nM) or low‐affinity (Kd2 = 30–150 nM), and both receptors are expressed at high level: 3 × 105–6 × 105 C3aR1/cell and 5 × 105–2.3 × 106 C3aR2/cell. Results from cross‐linking experiments with 125I‐C3a fully agree with the presence of two different classes of C3a receptors in HMC‐1 cells. Two membrane proteins with apparent molecular masses of 54–61 kDa (p57) and 86–107 kDa (p97) could be covalently modified with 125I‐C3a, and this cross‐linking was inhibited with an excess of unlabeled C3a. Many of the known agonists for leukocytes including 13 chemokines (IL‐8, NAP‐2, GROα, ENA‐78, IP10, PF4, MCP‐1, 2 and 3, RANTES, MIP‐1α, MIP‐1β and 1309), three neuropeptides (neuropeptide Y, somatostatin and calcitonin), as well as C5a, did not activate HMC‐1 cells, indicating that C3a is one of a few protein ligands for which this cell line expresses specific receptors. The apparent selectivity for C3a and the abundant expression of C3a receptors make the HMC‐1 cell line an excellent choice for the cloning of the receptor genes.
Murine epidermal Langerhans cells do not express inducible nitric oxide synthase
Authors: Blank Christine , Bogdan Christian , Bauer Carmen , Erb Klaus , Moll Heidrun
Published: March 2007

In Leishmania‐infected macrophages (MΦ), the formation of reactive nitrogen intermediates by the inducible isoform of nitric oxide synthase (iNOS) is critical for the killing of the intracellular parasites. We have recently shown that, in addition to MΦ, epidermal Langerhans cells (LC) can phagocytose Leishmania major, but they do not allow parasite replication. Therefore, we analyzed whether LC and MΦ display the same leishmanicidal effector mechanism. Unlike MΦ, stimulation of unselected epidermal cells with interferon‐γ/lipopoly‐saccharide did not lead to the release of nitric oxide (NO), and inhibition of NO production had no effect on the rate of infection of LC. iNOS mRNA was clearly detectable in MΦ as well as unselected epidermal cells (the majority of which consists of keratinocytes) after stimulation with different cytokines. In contrast, pure LC obtained by single‐cell picking from cytokine‐activated or L. major‐infected epidermal cells did not express iNOS mRNA. Addition of the NO donor S‐nitroso‐N‐acetylpenicillamine to already‐infected LC did not alter their rate of infection, indicating that LC do not utilize exogenous NO for the control of intracellular Leishmania. These results suggest that in the L. major‐infected skin, activated MΦ and keratinocytes, but not LC have the ability to express iNOS activity. Therefore, an as yet unidentified, NO‐independent mechanism appears to be responsible for the control of parasite replication in LC.
Evidence that induction of tolerance in vivo involves active signaling via a B7 ligand‐dependent mechanism: CTLA4‐Ig protects Vβ8+ T cells from tolerance induction by the superantigen staphylococcal enterotoxin B
Authors: Lane Peter , Haller Corinne , McConnell Fiona
Published: March 2007

Co‐stimulation through CD28 is thought to be necessary for the activation of unprimed CD4+ T cells, which are otherwise rendered tolerant. However, we previously found that CD4+ T cell priming was normal or augmented in mice which overexpressed a soluble form of CTLA4 where co‐stimulation through CD28 was abrogated. To investigate this CD4+ T cell response, we exploited the capacity of the superantigen staphylococcal enterotoxin B to stimulate T lymphocytes bearing Vβ8+, which represent ϰ30% of all CD4+ T cells. In littermate controls of CTLA4‐Ig transgenic mice, immunization with staphylococcal enterotoxin B leads to expansion, followed by deletion of Vβ8+ T cells, and the remaining cells are tolerant when stimulated in vitro. Comparable expansion and deletion of Vβ8+ T cells occurs in CTLA4‐Ig transgenic mice. However, in contrast to normal mice, the remaining Vβ8+ T cells from CTLA4‐Ig transgenic mice are not anergic and remain responsive to superantigen in vitro.
Tracing interactions of thymocytes with individual stromal cell partners
Authors: Merkenschlager Matthias
Published: March 2007

Cellular interactions in T cell development can be analyzed using thymus chimeras prepared in vitro, in which stromal cells and T cell precursors are manipulated separately. In an earlier study, we showed that for optimal T cell maturation, most ‐ if not all ‐ stromal cells must display appropriate (selecting) major histocompatibility complex (MHC) molecules: the substituion of selecting by nonselecting stromal cells leads to a proportional decrease in mature T cell production. These data imply that the availability of selecting stromal micro‐environments is rate limiting for positive selection, and that in positive selection, each thymocyte engages only one (rather than multiple) stromal cell partners. To test this hypothesis, we developed a tracing sytem for thymocyte/stromal cell interactions, based on the acquisition by thymocytes of stroma‐derived MHC class II determinants. When MHC class II‐deficient precursors are placed in H‐2b × k F1 environments (where all stromal cells co‐express H‐2b and H‐2k), individual thymocytes acquire class II determinants of both haplotypes. In striking contrast, when placed in mosaic stromal environments (where stromal cells express either H‐2b or H‐2k evenly interspersed), individual thymocytes preferentially acquire MHC class II determinants of one or the other haplotypes, but rarely both. This provides strong evidence that thymocytes have intimate interactions with individual stromal cells: having engaged one stromal cell niche, thymocytes do not (or only rarely) have promiscuous liaisons with others.
IL‐12 inhibits endotoxin‐induced inflammation in the eye
Authors: Whitcup Scott M. , Rizzo Luiz V. , Lai James C. , Hayashi Seigi , Gazzinelli Ricardo , Chan Chi‐Chao
Published: March 2007

Interleukin‐12 (IL‐12) is a heterodimeric cytokine that induces interferon (IFN)‐γ production and an increased generation of Th1 cells. Both IL‐12 and IL‐12 antagonists are being studied for the treatment of allergic reactions, autoimmune disease and malignancy. The goal of the present experiments was to examine the importance of IL‐12 in endotoxin‐induced ocular inflammation. The number of inflammatory cells infiltrating eyes with endotoxin‐induced uveitis (EIU) was significantly increased in animals treated with intraperitoneal anti‐IL‐12 antibody when compared to control animals, but there was no difference in infiltrating inflammatory cells in the eyes of animals treated with IL‐12 when compared to controls. In contrast, intraocular injection of IL‐12 significantly inhibited the development of endotoxin‐induced intraocular inflammation. The infiltrating inflammatory cells were reduced in the eyes of animals receiving intraocular IL‐12 when compared to controls. Cytokine analysis of the aqueous humor obtained from eyes with EIU showed increased levels of IFN‐γ and decreased levels of IL‐6 in eyes receiving intraocular IL‐12. These data show that IL‐12 has an inhibitory effect on endotoxin‐induced inflammation in the eye and suggest that IL‐12 can have an immunoregulatory function in some forms of inflammatory disease.
Use of monoclonal antibodies to study interleukin ‐1β‐converting enzyme expression: only precursor forms are detected in interleukin‐1β‐secreting cells
Authors: Miossec Christine , Decoen Marie‐Claude , Durand Laurence , Fassy Florence , Diu‐Hercend Anita
Published: March 2007

We have generated a series of monoclonal antibodies (mAb) using recombinant interleukin (IL)‐1β‐converting enzyme (ICE) p20 and p10 subunits as immunogens. The mAb have been selected for further study based on their reactivity with ICE in transfected COS cells and their lack of cross‐reactivity with TX, the closest ICE homolog known to date. Two anti‐p20 and one anti‐p10 mAb have been used to study ICE expression by Western blotting and immunodetection. In ICE‐transfected COS cells, the mAb recognize the p45 ICE precursor and the maturation products (p20 or p10 subunits) for which they are specific. In monocytes and cell lines expressing ICE, only precursor forms are detected and intracellular immunostaining followed by confocal microscopy shows that they are located in the cytoplasm. Quantification experiments show that THP1 cells express approximately 67000 molecules of ICE precursor per cell, with an estimated precursor to mature ratio of at least 100. In these cells as well as in monocytes, lipopolysaccharide stimulation did not change the pattern of ICE expression, although efficient secretion of mature IL‐1β was measured. However, upon cell disruption, precursor maturation was observed. Our results, therefore, show that ICE is present in cells as a large pool of intracytoplasmic precursor, and that very limited amounts of mature ICE protein are present, but nevertheless sufficient to allow efficient IL‐1β cleavage. Altogether, these observations suggest that post‐translational maturation of the precursor protein could represent a specific step in the regulation of ICE enzymatic activity.
Analysis of hypermutation in immunoglobulin heavy chain passenger transgenes
Authors: Johnston J. Martin , Ihyer Sudhakar R. , Smith Robin S. , Tai Kuei‐Fang , Farmer Tara , Korsmeyer Stanley J. , Nadon Nancy L. , Carroll William L.
Published: March 2007

Somatic hypermutation of immunoglobulin (Ig) genes plays a critical role in the maturation of the human antibody response. The molecular basis of this important process is, however, unknown. To identify cis‐acting sequences that initiate and target hypermutation, we have made three minitransgenes containing different portions of an Ig heavy chain (IgH) locus. Each transgene is a passenger, bearing a nonsense mutation preventing its translation; thus, transgene mutations reflect the endogenous mutational process and are not subject to affinity selection. To study transgenes after their circulation through the compartment associated with hypermutation in vivo, we rescued B cells as hybridomas after hyperimmunizing mice with the hapten 4‐hydroxy‐3‐nitrophenyl acetyl (NP). Hybridoma transgene and endogenous variable regions were amplified by polymerase chain reaction, subcloned, and sequenced. Endogenous anti‐NP VDJ regions show the expected, at times extensive degree of base substitution. In mice bearing the smallest construct, which includes 2.4 kb of 5′ IgH sequences, a rearranged VDJ region, the 5′ matrix attachment region, and the intron enhancer, one of four evaluable hybridomas demonstrates two base substitutions in the V segment of one transgene copy. The two larger constructs include additional 3′ IgH sequences (an α constant region and the 3′ enhancer) and either the original VDJ segment or a substituted T cell receptor β segment. Ten hybridomas derived from mice bearing these larger constructs demonstrate no evidence of targeted mutation, despite demonstrable transgene transcription in all hybridomas. In our system, mutation of a rearranged VDJ segment and surrounding promoter/enhancer regions is not increased by the juxtaposition of a constant region segment and the IgH 3′ enhancer.
The protein interactions of the immunoglobulin receptor family tyrosine‐based activation motifs present in the T cell receptor ζ subunits and the CD3 γ,δ and ϵ chains
Authors: Osman Narin , Turner Helen , Lucas Susan , Reif Karin , Cantrell Doreen A.
Published: March 2007

Immunoglobulin family tyrosine‐based activation motifs (ITAM), which define the conserved signaling sequence EX2YX2L/IX7YX2L/I, couple the T cell antigen receptor (TCR) to cellular proteins including protein tyrosine kinases (PTK) and adapter molecules. The TCR is a multichain complex with four invariant chains CD3γ, δ and ϵ that each contain a single ITAM and the TCR ζ chain that contains three ITAM. The present study explores the protein interactions of the doubly phosphorylated CD3 γ, δ, ϵ ITAM to determine whether they have common or unique biochemical properties. The data show that the doubly phosphorylated ITAM all bind the PTK ZAP‐70, but the ITAM also variably bind the PTK p59fyn and the adapters Shc, Grb‐2 and the p85 regulatory subunit of phosphoinositol 3′ kinase. The CD3 and ζ ITAM display a hierarchy of ZAP‐70 binding: ζ1 = γ = δ > ζ3 > ζ2 = ϵ. Shc, Grb‐2 and p85 could bind the ζ ITAM and the CD3 γ and δ ITAM, but not the CD3 ϵ ITAM. There were also subtle differences in the hierarchy of reactivity of these adapters for the CD3 γ,δ and ζ ITAM that show that the ζ, CD3 γ, δ and ϵ ITAM have different binding properties. The present study thus shows that the different ITAM of the TCR/CD3 complex can interact with different cytosolic effectors, indicating that differential ITAM phosphorylation during T cell activation could be a mechanism to generate signaling diversity by the TCR complex.
Induction of T cell unresponsiveness by anti‐CD3 antibodies occurs independently of co‐stimulatory functions
Authors: Andris Fabienne , Van Mechelen Marcelle , De Mattia Fabrizio , Baus Erika , Urbain Jacques , Leo Oberdan
Published: March 2007

Antibodies to the T cell receptor (TcR)‐associated CD3 molecules represent potent immunosuppressive agents in vivo in both human and animals models, in spite of their well‐characterized mitogenic properties. We demonstrate in this report that antibodies to the B7.2 molecule inhibit IL‐2 production in vivo caused by anti‐CD3 administration, suggesting that anti‐CD3 monoclonal antibodies (mAb) stimulate naive T cells in vivo in a co‐stimulation‐dependent fashion. To characterize better the mechanisms by which antibodies to CD3 induce antigen unresponsiveness in naive T cells, we developed a model of activation‐induced T cell unresponsiveness in vitro. Our data indicate that following interaction with mitogenic anti‐CD3 mAb in vitro, naive purified CD4+ T cells become refractory to a further stimulus. This unresponsive state develops independently of co‐stimulatory functions, as neither B7‐expressing antigen‐presenting cells nor anti‐CD28 mAb are able to prevent anergy induction in this model. We therefore conclude that induction of unresponsiveness in naive T cells by anti‐CD3 mAb is not a consequence of co‐stimulus‐deficient stimulation, but may develop following a productive response both in vivo and in vitro. Unresponsive T cells display a defective calcium mobilization upon TcR triggering, suggesting that anergy is maintained in these cells through receptor desensitization. The potential role of co‐stimulation‐independent TcR desensitization in the down‐regulation of immune responses in vivo is briefly discussed.
Demonstration of local clonality of mucosal T cells in human colon using DNA obtained by microdissection of immunohistochemically stained tissue sections
Authors: Dogan Ahmet , Dunn‐Walters Deborah K. , MacDonald Thomas T. , Spencer Jo
Published: March 2007

Intraepithelial lymphocytes have been shown to be oligoclonal and to be disseminated widely along the human intestine. However, studies using monoclonal antibodies have suggested that superimposed on the widespread clones, there is local variability in the mucosal T cell population. We have investigated the possibility that local dominant clones of T cells are present in the colonic mucosa by polymerase chain reaction (PCR) amplification of T cell receptor β chain junctional regions using DNA extracted from microdissected fragments of tissue sections. Colon from two right hemicolectomy specimens was sampled at 7‐cm intervals. Adjacent areas of mucosa were microdissected from sections from each colon sample. When the PCR products were separated according to size on polyacrylamide gels, bands of identical size were often observed when DNA extracted from adjacent fragments of mucosa had been used. Different bands were present when the different samples of colon had been studied. Sequencing of the PCR products confirmed that clonally related T cells were present in adjacent areas of mucosa, whereas different clones dominated at distant sites. DNA extracted from cells microdissected from the T cell zone of Peyer's patch was treated identically. The sequences obtained from the Peyer's patch, as expected, were diverse. However, one of the sequences identified was identical to that of one of the clones in the colon, implying that this clone was either trafficking through the Peyer's patch or possibly originated from the Peyer's patch. In this study, we also identified the Peyer's patches as a site of proliferation of CD4+ T cells. No T cell division was observed in the lamina propria. The molecular and immunohistochemical observations together support the hypothesis that the Peyer's patches are a source of mucosal T cells.
Interleukin‐12 and B7.1 co‐stimulation cooperate in the induction of effective antitumor immunity and therapy of established tumors
Authors: Zitvogel Laurence , Robbins Paul D. , Storkus Walter J. , Clarke Martha R. , Maeurer Markus J. , Campbell Ronna L. , Davis C. Geoffrey , Tahara Hideaki , Schreiber Robert D. , Lotze Michael T.
Published: March 2007

Interleukin‐12 (IL‐12) promotes specific and long‐lasting anti‐tumor immunity mediated by T cells in a variety of murine tumor models. IL‐12 also synergizes with B7.1 (CD80) co‐stimulation to induce proliferation and cytokine production by both human and murine T cells in vitro. We evaluated the combined antitumor efficacy of IL‐12 and B7.1 gene delivery in two apparently poorly immunogenic tumor models (TS/A and MCA207). In both of these models, expression of B7.1 and production of IL‐12 in the inoculum led to improved anti‐tumor immunity, with up to 80% long‐term tumor‐free animals (vs 0– 20% of mice remaining tumor free when inoculated with either B7.1‐ or IL‐12‐transfected tumors alone). Tumor‐free mice were capable of rejecting a subsequent rechallenge with the wild‐type tumor in 66% of the cases. Cooperativity was dependent upon the level of IL‐12 secreted by engineered cells. IL‐12 delivery required B7 expression of therapeutic effects to be observed in these models. Vaccines provided at a site distal to a control, non‐transfected tumor slowed (TS/A) or abrogated (MCA207) the progression of wild‐type tumors. The synergistic anti‐tumor effects associated with combined application of B7.1‐ and IL‐12‐transfected tumors were partially negated by systemic administration of the CD28‐B7.1/B7.2 antagonist CTLA4‐Ig or by inoculation with neutralizing antibodies directed against murine interferon‐γ or tumor necrosis factor‐α, two cytokines elicited in response to IL‐12 stimulation. These data support the potential clinical utility of combined gene therapy using IL‐12‐ and B7.1‐engineered autologous cells (tumor or fibroblasts) as a vaccine to elicit specific anti‐tumor immunity.
T cell recognition of Tn‐glycosylated peptide antigens
Authors: Jensen Teis , Galli‐Stampino Luisa , Mouritsen Søren , Frische Klaus , Peters Stefan , Meldal Morten , Werdelin Ole
Published: March 2007

The mouse hemoglobin‐derived decapeptide Hb (67–76), VITAFNEGLK, which binds well to Ek and is non‐immunogenic in CBA/J mice, was O‐glycosylated with the tumor‐associated carbohydrate Tn (α‐D‐N‐acetylgalactosamine, or α‐D‐GalNAc). Each of the ten positions in the peptide was substituted with serine or threonine having the Tn antigen attached. The complete set of Tn‐glycosylated peptides were then studied for binding to Ek and for immunogenicity in CBA/J mice. All of those glycopeptides which had the Tn attached to serine or threonine at a position in the peptide where, according to the crystal structure determinations, the amino acid side chain was oriented downwards into the binding site of the major histocompatibility complex (MHC) molecule, completely lost their capacity for binding to Ek. This was the case for the glycopeptides with Tn attacted at position 68 and 76, which are the major anchor residues and for those with Tn attached at position 71 and 73, which function as secondary anchor residues. Those glycopeptides which had Tn attached to serine or threonine at positions where the side chain pointed away from the binding site maintained their capacity for binding to Ek, except for those with Tn attached at position 70 and 74. Furthermore, some of the MHC‐binding glycopeptides were immunogenic. In particular, this was the case for the glycopeptide with Tn attached to the central position 72 in the decapeptide. From previous studies, this is known to be the dominant T cell receptor contact residue of Hb (67–76). The results suggest that T cells may be capable of recognizing epitopes which are partially defined by a small glycan group.