bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of the allele‐specific antigen‐binding site on I‐Ak and I‐Ab molecules
Authors: Itoh Yasushi , Kajino Kiichi , Ogasawara Kazumasa , Katoh Masahito , Namba Kenichi , Takami Kimitaka , Iwabuchi Kazuya , Braunstein Ned S. , Onoé Kazunori
Published: March 2007

Residues 46 and 54 on a pigeon cytochrome c 43–58 analog, 50E, function as major histocompatibility complex class II contact sites. A peptide, 46F50E54A, with phenylalanine (F) at position 46 and alanine (A) at 54 on 50E bound to Ab and a peptide, 46D50E54A, with aspartic acid (D) at 46 and alanine at 54, bound to Ak. To determine the allele‐specific peptide contact sites on I‐A molecules corresponding to the I‐A contact sites of the peptides, we analyzed responses of Ak‐ and/or Ab‐restricted T cell hybridomas to 46F50E54A or 46D50E54A using L cell transfectants expressing recombinant I‐A molecules between Ak and Ab or point mutants of Ak as antigen presenting cells. It was shown that the N‐terminal half of the α helix of the Aα chain determined the allele‐specific T cell responses. Furthermore, with arginine (k type amino acid) or alanine (b type amino acid) at position 56 of the Ak α chain, these T cell hybridomas were stimulated predominantly by 46D50E54A (Ak binding peptide) or 46F50E54A (Ab binding peptide), respectively. Thus, the amino acid at position 56 of the Aα chain determines allele‐specific antigen presentation. This postulate was confirmed by direct binding analysis of 50E analogs of various I‐A molecules. A single amino acid change (arginine to alanine) at position 56 of the Ak α chain altered the peptide binding specificity (46D50E54A to 46F50E54A).
Human and rodent interferon‐γ as a growth factor for Trypanosoma brucei
Authors: Bakhiet Moiz , Olsson Tomas , Mhlanga Jama , Büscher Philippe , Lycke Nils , van der Meide Peter H. , Kristensson Krister
Published: March 2007

An example for the bidirectional exchange of activating signals between a pathogen and immunocompetent cells in the host is presented. Trypanosoma brucei, which include subspecies that cause African sleeping sickness, secrete a molecule that triggers lymphocytes to produce interferon (IFN)‐γ. We now report that proliferation of T. brucei is stimulated in axenic cultures by IFN‐γ. The growth‐enhancing effect on the pathogen is inhibited by anti‐IFN‐γ receptor (R) antibodies and does not occur after exposure to other cytokines, i.e. IFN‐α, IFN‐β and tumor necrosis factor (TNF)‐α. While rodent‐pathogenic T. brucei strains are stimulated by rat IFN‐γ, human pathogenic strains are more potently stimulated by human IFN‐γ. Rat and human IFN‐γ can partially block each others effects. Mice with disrupted IFN‐γ genes have reduced parasitemia and prolonged survival, while the outcome is reversed in mice that lack the IFN‐γR gene.
Elevated proliferation and interleukin‐4 release from CD4+ cells after chemotherapy in human Schistosoma haematobium infection
Authors: Grogan Jane L. , Kremsner Peter G. , Deelder Andre M. , Yazdanbakhsh Maria
Published: March 2007

Cellular immune responses to schistosomal antigens were examined in 110 Schistosoma haematobium‐infected individuals before and 5 weeks after treatment with praziquantel. Chemotherapy resulted in significant decrease in worm load as measured by egg output and circulating antigens. The proliferative responses to adult worm antigen (AWA) increased significantly after treatment p < (0.001) whereas purified protein derivative of tuberculin or phytohemagglutinin responsiveness was unaffected. Interleukin (IL)‐4 production in response to both AWA and soluble egg antigen (SEA) increased markedly after treatment (p < 0.001), but IL‐5 remained unchanged. None of the studied subjects released any measurable IL‐2 and only 21% produced interferon (IFN)‐γ in response to parasite antigens. The deficiency in either IFN‐γ or IL‐2 release was not restored by chemotherapy. Thus chronic infection with S. haematobium is associated with the reversible down‐regulation of T cell proliferative responses and IL‐4 release. Results from cell depletion experiments indicated that CD4+ T cells were the target of this down‐modulation.
Triggering of CD23b antigen by anti‐CD23 monoclonal antibodies induces interleukin‐10 production by human macrophages
Authors: Dugas Nathalie , Vouldoukis Ioannis , Bécherel Pierre , Arock Michel , Debré Patrice , Tardieu Marc , Mossalayi Djavad M. , Delfraissy Jean François , Kolb Jean Pierre , Dugas Bernard
Published: March 2007

The aim of this study was to evaluate the capacity of human macrophages to produce interleukin (IL)‐10 upon stimulation of membrane CD23. An anti‐CD23 monoclonal antibody (mAb) was found to elicit the expression of the specific mRNA for IL‐10 in CD23‐bearing macrophages, and to induce a time‐dependent production of this cytokine with a maximal effect reached after 12 h. Inasmuch as we previously reported that CD23 ligation evoked the generation of nitric oxide and of cAMP, the effect of the Rp diastereoisomer of adenosine 3′, 5′‐cyclic phosphorothioate (Rp‐cAMP, an inhibitor of the cAMP pathway) and of NG‐monomethyl‐L‐arginine (L‐NMMA, an inhibitor of the nitric oxide pathway) were evaluated on CD23‐induced IL‐10 production. In the presence of Rp‐cAMP, the CD23‐induced production of IL‐10 and of the pro‐inflammatory cytokine tumor necrosis factor‐γ (TNF‐γ) was totally abrogated, whereas, in the presence of L‐NMMA, IL‐10 production was enhanced and TNF‐γ production was suppressed. In addition, neutralization of IL‐10 with an anti‐IL‐10 mAb increased both the magnitude and duration of CD23‐driven TNF‐α production. Such an inducing effect was observed with different anti‐CD23 mAb (clone 135, MHM6 and 25), indicating that the triggering of the CD23 molecule at the surface of human macrophages induced the generation of IL‐10 through a cAMP‐dependent mechanism. Concomitantly this generation of IL‐10 was down‐regulated by nitric oxide, which was also produced after triggering of the CD23 antigen. Taken together these data indicated that human macrophages produced IL‐10 after triggering of the CD23 molecule and that this production could regulate the inflammatory state of these cells.
Epitope‐specific engagement of the protein tyrosine phosphatase CD45 induces tumor necrosis factor‐α gene expression via transcriptional mechanisms
Authors: Estoppey Olivier , Sauty Alain , Espel Enric , Menoud Véronique , Frei Philippe C. , Spertini François
Published: March 2007

The common leukocyte antigen CD45 plays a central role in T cell activation in coupling the T cell receptor (TCR) to the phosphatidylinositol pathway via interactions with TCR‐associated protein tyrosine kinases lck and fyn. We here demonstrate that engagement of CD45 by monoclonal antibodies (mAb) on activated T cells induces tumor necrosis factor (TNF)‐α as well as TNF‐β, interleukin (IL)‐2 and IL‐3 gene expression. When human alloreactive T cells are stimulated with mAb 4B2, which recognizes a determinant common to all CD45 isoforms, a vigorous production of TNF‐α mRNA was detected, which peaked 2 h later. Anti‐CD45 mAb cross‐linking was required. In contrast, neither mAb 10G10, which recognizes an epitope distinct from the one recognized by mAb 4B2, nor mAb UCHL‐1, a CD45RO‐specific antibody, induced any significant increase in TNF‐α transcription. Nuclear run‐on transcription assays demonstrated that CD45 cross‐linking caused transcriptional activation of the TNF‐α gene. De novo protein synthesis was not required, since incubation with cycloheximide (CHX) did not block transcriptional activation. CHX in contrast up‐regulated TNF‐α gene expression and increased transcript half‐life, an effect that was under the control of post‐transcriptional mechanisms. Engagement of CD45 by itself did not affect transcript stability. CD45 ligation resulted in TNF‐α secretion. These results indicate that in addition to its role in TCR/CD3‐mediated T cell activation, CD45, in an epitope‐specific manner, may act as a primary signaling molecule, leading to the transcriptional regulation and secretion of a major pro‐inflammatory cytokine.
Flt3 ligand supports the differentiation of early B cell progenitors in the presence of interleukin‐11 and interleukin‐7
Authors: Ray Robert J. , Paige Christopher J. , Furlonger Caren , Lyman Stewart D. , Rottapel Robert
Published: March 2007

B cell development is influenced by interactions between B cell progenitors and stromal cells. The precise mechanisms by which these interactions regulate B cell differentiation are currently unknown. Flt3 ligand (FL) is a growth factor which stimulates the proliferation of stem cells and early progenitors. Mice deficient for the FLT3 receptor exhibit severe reductions in early B lymphoid progenitors. We have previously described a clonal assay in vitro which allows us to follow the entire B cell differentiation pathway from uncommitted progenitors to mature, immunoglobulin‐secreting plasma cells. The growth factor combination of interleukin (IL)‐11, mast cell growth factor (MGF) and IL‐7 was shown to maintain the differentiation of these hematopoietic precursors into B cell progenitors capable of giving rise to functionally mature B cells in secondary cultures. Here, we show that FL in combination with IL‐11 and IL‐7 is sufficient to support the differentiation of uncommitted progenitors from day 10 yolk sac (AA4.1+) or day 12 fetal liver (AA4.1+ B220− Mac‐1− Sca‐1+) into the B lineage. The frequency of B cell progenitors obtained in these conditions was similar, if not better, than the frequency of B cell precursors that arose when cultured in IL‐11+MGF+IL‐7. Furthermore, the growth factor combination of IL‐11+FL+IL‐7 was able to maintain the potential of bipotent precursors giving rise to both the B and myeloid lineages in secondary cultures. We also show that FL synergizes with IL‐7 in the proliferation of committed B220+ pro‐B cells and may contribute to the maintenance of an earlier pro‐B cell population. Together, these results show that FL is important in supporting the differentiation and proliferation of early B cell progenitors in vitro.
Genetically resistant mice lacking interleukin‐12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response
Authors: Mattner Frank , Magram Jeanne , Ferrante Jessica , Launois Pascal , Padova Karin Di , Behin Reza , Gately Maurice K. , Louis Jacques A. , Alber Gottfried
Published: March 2007

Mice with homologous disruption of the gene coding for either the p35 subunit or the p40 subunit of interleukin‐12 (IL‐12) and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in resistance to infection and the differentiation of functional CD4+ T cell subsets in vivo. Wild‐type 129/Sv/Ev mice are resistant to infection with L. major showing only small lesions which resolve spontaneously within a few weeks and develop a type 1 CD4+ T cell response. In contrast, mice lacking bioactive IL‐12 (IL‐12p35−/− and IL‐12p40−/−) developed large, progressing lesions. Whereas resistant mice were able to mount a delayed‐type hypersensitivity (DTH) response to Leishmania antigen, susceptible BALB/c mice as well as IL‐12‐deficient 129/Sv/Ev mice did not show any DTH reaction. To characterize the functional phenotype of CD4+ T cells triggered in infected wild‐type mice and IL‐12‐deficient mice, the expression of mRNA for interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4) in purified CD4+ lymph node cells was analyzed. Wild‐type 129/Sv/Ev mice showed high levels of mRNA for IFN‐γ and low levels of mRNA for IL‐4 which is indicative of a Th1 response. In contrast, IL‐12‐ deficient mice and susceptible BALB/c mice developed a strong Th2 response with high levels of IL‐4 mRNA and low levels of IFN‐γ mRNA in CD4+ T cells. Similarly, lymph node cells from infected wild‐type 129 mice produced predominantly IFN‐γ in response to stimulation with Leishmania antigen in vitro whereas lymph node cells from IL‐12‐deficient mice and susceptible BALB/c mice produced preferentially IL‐4. Taken together, these results confirm in vivo the importance of IL‐12 in induction of Th1 responses and protective immunity against L. major.
Activated T cells induce expression of E‐selectin in vitro and in an antigen‐dependent manner in vivo
Authors: Sunderkötter Cord , Steinbrink Kerstin , Henseleit Ute , Bosse Roland , Schwarz Agatha , Vestweber Dietmar , Sorg Clemens
Published: March 2007

E‐selectin is an endothelial adhesion molecule for polymorphonuclear cells, monocytes and skin‐homing T cells. We have analyzed whether murine T cells are able to induce expression of E‐selectin in vitro and in vivo. Using models of inflammation in which T cells play either a significant or only a minor role, we compared induction of E‐selectin between normal mice and mice lacking functional T cells (athymic nude mice). In irritant contact dermatitis, a model without a major role for T cells, E‐selectin was transiently expressed within the first 24 h in both normal and nude mice. In experimental leishmaniasis (where specific T cells play an important role), a high expression of E‐selectin was maintained for 48 h in normal mice, whereas in nude mice expression was only transient. However, reconstitution of nude mice with 108 T cells from draining lymph nodes (LN) of Leishmania‐infected normal mice could restore sustained expression of E‐selectin. Transfer of T lymphocytes from normal LN or from LN of mice sensitized to the contact allergen trinitrochlorbenzene (TNCB) did not have this effect. T cells from TNCB‐sensitized mice, however, did induce sustained expression of E‐selectin in nude mice when TNCB was applied locally; here, reconstitution with Leishmania‐specific T cells had no effect. In vitro, T cells from infected or TNCB‐sensitized normal mice increased expression of E‐selectin on microvascular endothelial cells after 4 h of co‐culture. T cells from untreated mice were less effective. Induction was dependent on direct cell‐cell contact, but not on the action of interleukin‐1α, interleukin‐1β, tumor necrosis factor‐α or interferon‐γ. We conclude that sensitized T cells induce sustained expression of E‐selectin in vivo in an antigen‐dependent manner. This novel way of regulation could be relevant for cell‐mediated immunity and chronic disease. The mechanisms are unknown, but, as in vitro, might require direct cell‐cell contact.
Administration of anti‐interleukin‐2 receptor α antibody in vivo induces localized autoimmune disease
Authors: Taguchi Osamu , Takahashi Toshitada
Published: March 2007

Neonatal thymectomy (Tx) of mice at day 3 after birth (Tx‐3), but not day 7 (Tx‐7), induces organ‐localized autoimmune diseases such as oophoritis and gastritis. Lesions in Tx‐3 mice can be prevented by injection of splenic CD4+ cells from syngeneic normal mice, and this CD4+ population with suppressor activity is activated extrathymically by self antigens. Since it is speculated that these CD4+ T suppressor cells (Ts) express the interleukin‐2 receptor (IL‐2R) as an activated T cell population, an attempt was made to eliminate these Ts from the developing immune system of Tx‐7 mice and normal mice by i.p. injection of anti‐IL‐2Rα monoclonal antibodies. Interestingly, organ‐localized autoimmune disease with quite similar characteristics to those observed after neonatal Tx developed in not only Tx‐7 mice, but also normal mice. The results thus indicate that CD4+ cells expressing IL‐2Rα play an important role, as Ts in the periphery, in maintaining immune tolerance.
Permissive residues within the minimal epitopes of neutralizing monoclonal antibodies to the V3 loop of HIV‐1
Authors: Laisney Isabelle L. , Benjamin Howard , Gefter Malcom , Strosberg A. Donny
Published: March 2007

Neutralizing monoclonal antibodies (mAb) specific for the third variable (V3) domain of gp120, the HIV‐1 surface envelope protein, are mainly isolate specific. We have studied the composition and the permissivity of the minimal epitopes interacting with two of these, the 110‐A and 19.26.4 mAb, which are strictly LAI isolate specific. Screening a hexapeptide phage library displayed on the surface of filamentous phage with the 110‐A mAb has allowed selection of 49 phage sequences, permitting the definition of a consensus sequence. Based on this sequence, substituted synthetic peptides were prepared and used in binding assays. Our results show that both mAb interact with the same narrow region (316–320) of the V3 domain. The minimal epitope of the 110‐A mAb was identified as a five amino acid sequence, Hy x R G p, where Hy represents any non‐aromatic hydrophobic amino acid. By contrast, the minimal epitope of the 19.26.4 mAb was identified as x Q Pos G P, where Pos is any positively charged amino acid. Core residues of the epitope, critical for the binding to the mAb (written in uppercase letters), were set apart from permissive amino acid positions that tolerate substitutions (written in lowercase letters). Interestingly, the identified core residues Q2/317 (19.26.4 mAb) and R3/318 (110‐A mAb) do not tolerate substitution and correspond to the QR insertion in the V3 domain, characteristic of the LAI isolate as compared to other isolates. This result may explain the strict isolate specificity of most anti‐V3 LAI mAb. The two epitopes have totally different patterns of permissivity; thus, the effect of substitutions will differ depending on the mAb involved in the interaction. This suggests that the diversity of the antibody response is high enough to delay the emergence of HIV‐1 variants resistant to neutralization by V3‐specific antibodies.
OX40 is differentially expressed on activated rat and mouse T cells and is the sole receptor for the OX40 ligand
Authors: Al‐Shamkhani Aymen , Birkeland Marian L. , Puklavec Michael , Brown Marion H. , James William , Barclay A. Neil
Published: March 2007

OX40, a member of the tumor necrosis factor (TNF) receptor/nerve growth factor (NGF) receptor superfamily was first identified as a marker of activated rat CD4+ cells with the MRC OX40 monoclonal antibody (mAb). A ligand for OX40 (called OX40 ligand or OX40L) has recently been identified and has sequence similarity to TNF. Mouse OX40L‐immunoglobulin fusion protein (OX40L‐Ig) binds to activated mouse CD4+ and CD8+ cells (Baum, P. R. et al., EMBO J. 1994. 13: 3992) suggesting that OX40 could have a differential pattern of expression on mouse and rat T cells. This, however, did not rule out the presence of an alternative receptor on CD8+ cells that also binds the OX40L. We have compared the binding of the MRC OX40 mAb with that of OX40L‐Ig to activated rat lymph node cells and show that both recognize the same protein, namely OX40 which is expressed on CD4+ and CD4+ CD8α+ cells, but not on CD4−CD8+ cells. We have raised a new mAb (MRC OX86) using recombinant mouse OX40 protein and show by two‐color flow cytometry that mouse OX40 is expressed on CD4 and CD8 single‐positive cells. In addition, the new MRC OX86 mAb, unlike the MRC OX40 mAb, did not block binding of the OX40L. We conclude that OX40 is differentially expressed on activated mouse and rat T cells and is the sole receptor for the OX40L.
Translocation of long peptides by transporters associated with antigen processing (TAP)
Authors: Koopmann Jens‐Oliver , Post Markus , Neefjes Jacques J. , Hämmerling Günter J. , Momburg Frank
Published: March 2007

The major histocompatibility complex (MHC)‐encoded transporters associated with antigen processing (TAP) translocate peptides from the cytosol into the lumen of the endoplasmic reticulum (ER) where they associate with MHC class I molecules. The length of class I‐binding peptides is usually 8–11 amino acids, but examples of significantly longer peptides have been described. The preferred lengths and upper and lower size limits for peptides translocated by TAP have not been determined in detail because in the currently used test systems, peptides are subject to proteolytic degradation. In the present study, three sets of individual peptides or partially randomized peptide libraries ranging between 6 and 40 residues were used that contained a radiolabeled tyrosine and a consensus sequence for ER‐specific N‐glycosylation at opposite ends, thus ensuring that only nondegraded peptides were monitored in the transport/glycosylation assay. For three different transporters, rat TAP1/2a, rat TAP1/2u and hTAP, the most efficient ATP‐dependent transport was observed for peptides with 8–12 amino acids. Hexamers and longer peptides of up to 40 amino acids were also translocated, albeit less efficiently. For two of the three sets of peptides analyzed, rat TAP1/2a showed a less stringent length selection than rat TAP1/2u and human TAP. The superior transport of the decamer of the TNKT. Y series was not due to faster degradation or less efficient glycosylation of shorter or longer length variants. A binding assay with TAP‐containing microsomes revealed a high affinity for the radiolabeled decamer (KD = 580 nM), while other length variants were clearly inferior in their binding affinities. Thus, TAP binds and preferentially translocates peptides with a length suitable for binding to MHC class I molecules, but peptides that are considerably longer may also be substrates. About 105 peptide binding sites per cell equivalent of microsomes were determined, providing an estimate for the number of TAP complexes in the ER membrane.
Expression and function of the Fas receptor on human blood and tissue eosinophils
Authors: Hebestreit Holger , Yousefi Shida , Balatti Ivo , Weber Martina , Crameri Reto , Simon Dagmar , Hartung Konrad , Schapowal Andreas , Blaser Kurt , Simon Hans‐Uwe
Published: March 2007

The interaction between activated T cells and eosinophils has been proposed to play an important role in the pathogenesis of allergic diseases. T cell‐derived cytokines such as interleukin‐5 and granulocyte/macrophage colony‐stimulating factor inhibit eosinophil apoptosis and may therefore contribute to the development of tissue and blood eosinophilia in these disorders. Withdrawal of these cytokines leads to eosinophil apoptosis in vitro. In contrast, the mechanisms which actively induce apoptosis in eosinophils are at present not completely understood. In this study, we demonstrate that freshly isolated human eosinophils express mRNA and protein for the Fas receptor. Using anti‐Fas monoclonal antibody (mAb), we show that Fas activation accelerates apoptotic eosinophil death in vitro. Moreover, treatment of nasal polyps ex vivo with anti‐Fas mAb decreased eosinophilic tissue inflammation. However, we observed that blood as well as tissue eosinophils derived from some eosinophilic donors do not express functional Fas receptors, although Fas protein is normally expressed in these cells. This implies that the susceptibility of the Fas receptor is a matter of regulation in eosinophils as previously observed in other systems. These data suggest that Fas ligand/Fas interactions are involved in the regulation of eosinophil apoptosis and that defects in this system could contribute to the accumulation of these cells in allergic and asthmatic diseases.
Phenotypic and functional characterization of CD11c+ dendritic cell population in mouse Peyer's patches
Authors: Ruedl Christiane , Rieser Claudia , Böck Günther , Wick Georg , Wolf Hugo
Published: March 2007

The antigen‐presenting cell system in the gastrointestinal tract, one of three main sites (skin and lung being the others) of primary antigen contact, is poorly understood. Our study focused on dendritic cells (DC) as possible candidates for antigen uptake, processing and presentation in mucosal inductive sites, such as Peyer's patches (PP). To investigate the morphology, immunophenotype and stimulatory activity of intestinal DC, a procedure was developed to obtain a cell population by using collagenase digestion of PP, density centrifugation and cell sorting on the basis of CD11c expression. The resultant low‐density cell fraction consisted of a nonadherent cell population expressing different intensities of CD11c that could at least be characterized by typical DC morphology (e.g. abundant cytoplasma with veil‐like cytoplasmatic dendrites, irregularly shaped nuclei, multivesicular and multilamellar bodies), constitutive levels of surface MHC class II, the presence of macrophage‐specific markers, such as F4/80, Mac‐I and Fc receptors, respectively, on subpopulations of CD11c+ sorted cells and expression of adhesion and co‐stimulatory receptors like ICAM‐1 and CD44. The capability of this low‐density CD11c+ fraction to stimulate T cell responses was demonstrated in primary allogeneic mixed‐lymphocyte reactions (MLR). Herein, we show that the freshly isolated CD11c+ cells showed weak accessory function, but develop this capacity following short‐term culture in vitro in the presence of granulocyte/macrophage colony‐stimulating factor. Although the nature and functional capacity of the isolated CD11c+ needs further clarification, these preliminary results describing phenotype and accessory function provide some evidence that these cells isolated from the PP may be immature forms of DC and play a crucial role as antigen‐presenting cells with important implications for understanding the complex network regulating intestinal antigen uptake, processing and presentation.
Both activated and nonactivated leukocytes from the periphery continuously enter the thymic medulla of adult rats: phenotypes, sources and magnitude of traffic
Authors: Westermann Jürgen , Smith Tomas , Peters Ulrike , Tschernig Thomas , Pabst Reinhard , Steinhoff Gustav , Sparshott Sheila M. , Bell Eric B.
Published: March 2007

Although the thymus is primarily noted for the export of T cells to the periphery, a small influx of cells has also been observed. It is still a matter of debate whether entry into the thymus depends on prior activation. The phenotypes, sources and degree of immigration are largely unknown. We monitored by quantitative immunohistochemistry the entry of cells from the periphery into the rat thymus in three experimental models. We injected i.v. recirculating, small, nonactivated CD4+ T cell subsets, often referred to as naive (CD45RC+) and memory or antigen‐experienced (CD45RC−) cells, purified from thoracic duct lymph of allotype‐marked donors, allotype‐marked leukocytes released from spleen or lung transplants, or leukocytes labeled in the periphery for 12 weeks during the S‐phase of the cell cycle by oral application of 5‐bromo‐2‐deoxyuridine (BrdUrd). Early after i.v. injection (0.5 h), significantly more antigen‐experienced (CD45RC−) CD4+ T cells entered the thymus, and by 24 h four times as many cells from the CD45RC− subset as from the CD45RC+ subset had entered the thymus and localized to the medulla. None of the thymic entrants expressed the interleukin (IL)‐2 receptor. Following spleen transplantation ∼ 40% of donor cells entering the thymic medulla were T cells and ∼ 55% were B cells. In contrast, from a lung transplant, ∼ 85% of peripheral immigrants were T cells and ∼ 10% were B cells. After both procedures, a small number of NK cells and monocytes/macrophages were found among the immigrants (< 5%). Rats were fed BrdUrd continuously for 12 weeks, a procedure which labeled ∼ 30% of peripheral lymphocytes but not cortical thymocytes. BrdUrd‐labeled cells were localized almost exclusively to the thymic medulla and represented ∼ 10% of medullary cells. Of the thymic immigrants ∼ 50% were T cells, ∼ 30% were B cells (including ∼ 15% IgD+ cells), ∼ 15% were NK cells and the remainder (∼ 5%) were monocytes/macrophages. Only a quarter of BrdUrd‐labeled cells expressed the IL‐2 receptor. The thymus is continuously infiltrated by both activated and nonactivated leukocytes from the periphery, including T cells, B cells, NK cells and monocytes. These immigrants are supplied by lymphoid and nonlymphoid organs in a characteristic subset composition. Their entry is facilitated by prior antigen experience or activation. Thus, the participation of the thymic medulla in general leukocyte traffic suggests a mechanism by which the T cell repertoire could potentially be modulated by the peripheral tissues.
Human eotaxin represents a potent activator of the respiratory burst of human eosinophils
Authors: Elsner Jörn , Höchstetter Renate , Kimmig Daniela , Kapp Alexander
Published: March 2007

Increased numbers of eosinophils are found in parasitic infections, autoimmune diseases and allergic diseases such as allergic asthma. They are activated by distinct cytokines and chemokines leading to the immigration in the inflamed tissue and mediate tissue damage by releasing reactive oxygen species. Here, the effect of the recently cloned CC chemokine human eotaxin was investigated for its ability to affect different eosinophil effector functions and compared to the CC chemokines MCP‐3 and RANTES. Human eotaxin induced chemotaxis of human eosinophils in a dose‐dependent manner. The range of efficacy of the CC chemokines compared to the well‐known chemotaxin C5a was eotaxin = RANTES > MCP‐3 = C5a. In addition, eotaxin induced rapid and transient actin polymerization, a prerequisite for cell migration, in eosinophils in the same range of efficacy as observed for chemotaxis. To investigate whether eotaxin was able to activate the respiratory burst of eosinophils, release of reactive oxygen species was measured by lucigenin‐dependent chemiluminescence. Eotaxin induced production of significantly high amounts of reactive oxygen species at a concentration between 10 ng/ml and 500 ng/ml. Surprisingly, the effect of eotaxin was comparable to the well‐known eosinophil activator C5a. The range of efficacy of the CC chemokines compared to C5a in the activation of the respiratory burst was eotaxin = C5a > MCP‐3 > RANTES. Production of reactive oxygen species was inhibited by pertussis toxin, staurosporin, genestein and wortmannin. Furthermore, eotaxin induced transient increases in intracellular calcium concentration ([Ca2+]i) in human eosinophils. Therefore, pertussis toxinsensitive Gi‐proteins, protein kinase C, tyrosine kinase, phosphatidylinositol‐3‐kinase and transient increases in [Ca2+]i are involved in the signal transduction of eosinophils following stimulation with eotaxin. In summary, this study reveals the importance of the CC chemokine eotaxin as a potent activator of the respiratory burst, actin polymerization and chemotaxis. Eotaxin, therefore, plays an important role not only by attracting eosinophils to the site of inflammation but also by damaging tissue by its capacity to induce the release of reactive oxygen species.
Differential activation requirements of isotype‐switched B cells
Authors: Ehrhardt Rolf O. , Harriman Gregory R. , Inman John K. , Lycke Nils , Gray Belinda , Strober Warren
Published: March 2007

In the present studies, we compared the activation requirements of sIgM+/sIgD+ B cells with those of isotype‐switched sIgM−/sIgA+ B cells. We found that whereas sIgM+ B cells respond to T cell‐independent (TI) and T cell‐dependent (TD) Ag with no significant bias toward one stimulus, sIgA+ B cells were deficient in their ability to respond to antigen receptor cross‐linking but responded remarkably well to TD stimuli. Thus, dextran‐conjugated anti‐IgA antibody (anti‐IgA‐dextran), anti‐kappa‐dextran, or various immobilized anti‐IgA antibodies (Ab) induced only low‐level IgA B cell proliferation and no IgA secretion in the presence of various lymphokines; in marked contrast, sIgA+ B cells responded to cognate and noncognate T cell stimulation as well as to stimulation by CD40 ligand‐bearing fibroblasts by secreting large amounts of IgA (up to 240 000 ng/ml per 105 cells). This pattern of sIgA+ B cell responsiveness was noted with both germinal center peanut agglutininhi (PNAhi) and non‐germinal center PNAlo B cells. In confirmation of these results, whole Peyer's patch or lamina propria cell populations containing less than 15% sIgA+ B cells stimulated with a noncognate T cell stimulus or T cell membranes secreted mainly IgA (68%–94% of the total Ig secreted) and relatively little IgM. The strict T cell dependence of IgA B cell activation and differentiation provides important insights into immune responses of mucosal tissues and must be considered in the development of vaccines, particularly those designed to stimulate mucosal tissues containing large numbers of isotype‐switched B cells.
Expression cloning of the human C3a anaphylatoxin receptor (C3aR) from differentiated U‐937 cells
Authors: Crass Torsten , Raffetseder Ute , Martin Ulrich , Grove Melanie , Klos Andreas , Köhl Jörg , Bautsch Wilfried
Published: March 2007

A cDNA clone encoding the human C3a anaphylatoxin receptor (C3aR) was isolated from a pcDNAI/Amp expression library prepared from U‐937 cells which had been differentiated with dibutyryl cAMP to a macrophage‐like phenotype. The cDNA clone contained an insert of 4.3 kbp and was able to confer to transfected human HEK‐293 cells the capacity to bind specifically iodinated human C3a. Chinese hamster ovary cells co‐transfected with this cDNA clone and a G‐protein alpha subunit (Gα‐16) became functionally responsive to C3a and a C3a analog synthetic peptide, as measured by increased phosphoinositide hydrolysis. As inferred from the cDNA sequence, the clone encodes a 482‐residue polypeptide with seven hydrophobic membrane‐spanning helices and a high homology to the human C5a and formyl‐Met‐Leu‐Phe receptors. Uniquely among the family of G‐protein coupled receptors, the C3aR contains an exceptionally large second extracellular loop of approximately 175 residues. Northern hybridizations revealed an approximately 2.3‐kb transcript as the major and an additional ∼ 3.9 kb‐transcript as a minor transcription product of the C3aR. The C3aR appears to be widely expressed in different lymphoid tissues, as shown by Northern hybridizations, providing evidence for a central role of the C3a anaphylatoxin in inflammatory processes.
The functional half‐life of H‐2Kd‐restricted T cell epitopes on living cells
Authors: Eberl Gérard , Widmann Christian , Corradin Giampietro
Published: March 2007

The functional half‐life (t1/2) of different complexes formed by major histocompatibility complex (MHC) class I molecules and antigen on the surface of target cells was measured using specific cytotoxic T lymphocyte (CTL) clones in cytolysis, and interferon‐γ‐production and Ca2+‐mobilization assays. Functional t1/2 values of 5–10 h were obtained, which are in accordance with some previous estimations obtained from biochemical and immunochemical measurements. Moreover, these values were independent of the type of target cell, fixation of the target cells, or proteases able to degrade the peptides, suggesting that the unfolding of the peptide/MHC complexes at the cell surface alone determines the functional t1/2 of the CTL epitopes.
Abnormality of Oct‐1 DNA binding in T cells from Sjögren's syndrome patients
Authors: Flescher Eliezer , Vela‐Roch Norma , Ogawa Noriyoshi , Nakabayashi Toru , Escalante Agustin , Anaya Juan‐Manuel , Dang Howard , Talal Norman
Published: March 2007

Primary Sjögren's syndrome (SS) is an autoimmune rheumatic disease characterized by T cell hypoactivity. To understand the diminished T cell response to activation signals, we measured nucleoprotein DNA‐binding activities regulating gene expression during T cell activation using the electrophoretic mobility shift assay. Peripheral blood lymphocytes from 9/19 SS patients were found to be defective in their ability to bind an octomer sequence (Oct‐1). This Oct‐1‐binding phenotype remained stable in culture for up to 3 days prior to activation. This abnormality was not seen in resting T cells nor T cells from patients with systemic lupus erythematosus, rheumatoid arthritis (RA), or SS accompanied by RA. The SS Oct‐1 DNA‐binding abnormality correlated significantly with an inability of cells to exit the G0/G1 cell cycle phase when stimulated in vitro. Importantly, nucleoprotein extracts showing decreased DNA‐binding activity had normal amounts of Oct‐1 proteins as determined by immunoprecipitation, implying a functional defect in the Oct‐1 protein. Moreover, defective DNA binding was corrected by treatment with acid phosphatase.
T cell responses are governed by avidity and co‐stimulatory thresholds
Authors: Bachmann Martin F. , Sebzda Eric , Kündig Thomas M. , Shahinian Arda , Speiser Daniel E. , Mak Tak W. , Ohashi Pamela S.
Published: March 2007

We analyzed the avidity and CD28‐mediated co‐stimulatory requirements for the activation of T cells in vivo and in vitro. The strength of the T cell/antigen‐presenting cell interaction was varied by using altered peptide ligands for stimulation. Co‐stimulatory requirements were studied using T cells from CD28‐deficient mice. The results indicate that T cell activation is not an all‐or‐nothing event, but occurs in distinct steps. For each step, a certain avidity, co‐stimulatory threshold or both, must be met. Depending upon the strength of the interaction between the T cell receptor and the major histocompatibility complex/peptide and the presence of CD28 co‐stimulatory signals, T cells may undergo blast formation alone or proliferate or eventually both proliferate and differentiate to effector cells. Thus, T cell activation is governed by both avidity and co‐stimulatory thresholds.
Functional analysis of α1β1 integrin in human natural killer cells
Authors: Pérez‐Villar Juan J. , Melero Ignacio , Gismondi Angela , Santoni Angela , López‐Botet Miguel
Published: March 2007

Upon activation with interleukin (IL)‐2 human natural killer (NK) cells acquire on their surface the α1β1 and α2β1 integrins and down‐regulate the expression of α6β1. By employing α1β1‐specific monoclonal antibody (mAb) HP‐2B6, characterized in our laboratory, we examined the functional role of the α1β1 integrin in NK cells. Treatment with HP‐2B6 mAb partially interfered with attachment of cultured NK cells to type I collagen, and combined with an anti‐α2β1 (TEA 1/41) mAb, it completely abrogated cell adhesion to this extracelular matrix protein. In contrast, NK cell attachment to laminin was completely blocked by the anti‐β1 LIA 1/2 mAb, but was unaffected by α1 and α2‐specific mAb; as α3β1 and α6β1 were undetectable, the data indicate that the α1β1 integrin binding sites for type I collagen and laminin are different. Incubation with anti‐α1 HP‐2B6 or its F(ab')2 fragments specifically induced a rapid homotypic aggregation of NK cells that was dependent on active metabolism, an intact cytoskeleton and the presence of divalent cations (Ca2+ and Mg2+); homotypic cell adhesion was selectively blocked by anti‐CD18, CD11a or CD54 mAb. In addition, stimulation of cultured NK cells with the anti‐α1 HP‐2B6 enhanced TNF‐α production and induced tyrosine phosphorylation of a 110‐kDa protein. Pretreatment with specific inhibitors of protein tyrosine kinase (PTK) activity (tyrphostin 25 and herbimycin A) completely abrogated the functional effects induced by the anti‐α1 HP‐2B6 mAb. Our data show that ligation of the α1β1 integrin positively modulates IL‐2‐activated NK cell function via a PTK‐dependent pathway.
A shift to Th0 cytokine production by CD4+ cells in human longevity: Studies on two healthy centenarians
Authors: Paganelli Roberto , Scala Enrico , Rosso Rosamaria , Cossarizza Andrea , Bertollo Lorella , Barbieri Daniela , Fabrizi Assunta , Lusi Elena A. , Fagiolo Umberto , Franceschi Claudio
Published: March 2007

Centenarians, particularly healthy centenarians, constitute the example of successful aging and the study of their immune status can help to define the endpoint of the changes occurring throughout life. We characterized T cell clones (TCC) of two healthy centenarians, studying their phenotypes and production of representative Th1 and Th2 cytokines (IFN‐γ and IL‐4) and compared them with TCC obtained by three young normal subjects; in all 180 TCC were analyzed. In young donors, 35TCC were CD4+, 56 CD8+ and 2 were αβ4CD4−CD8− (double negative). In centenarians, we obtained 46 CD4+ TCC, 38 CD8+, 2 CD4+CD8+ (double positive) and 1 γδ+ double negative. Of the young subjects' TCC, 71% produced IFN‐γ but no IL‐4 (Th1 pattern) and this prevalence decreased to 39% in TCC from the centenarians. The number of clones showing the opposite Th2 pattern was similar in young and aged donors (3 out of 93 TCC and 2 out of 87 TCC, respectively). The intermediate profile of TCC producing both IL‐4 and IFN‐γ (Th0) was found in 25.8% of clones from young people, but it almost doubled to 58.6% in centenarians. The analysis shows that the Th profiles of CD8+ TCC is nearly superimposable in the two groups, whereas a major shift from a Th1 to a Th0 pattern is presented by CD4+ TCC. The balance provided by a majority of CD4+ TCC showing a Th0 pattern may ensure both humoral and cell‐mediated defences. In CD8+ TCC, however, a Th1 pattern still is present, possibly for efficient generation of cytotoxic responses. These findings should be extended by studying other centenarians and elderly subjects.
In interleukin‐4‐deficient mice, alum not only generates T helper 1 responses equivalent to Freund's complete adjuvant, but continues to induce T helper 2 cytokine production
Authors: Brewer James M. , Conacher Margaret , Satoskar Abhay , Bluethmann Horst , Alexander James
Published: March 2007

The role of interleukin (IL)‐4 in the activity of two frequently used vaccine adjuvants, Freund's complete adjuvant (FCA) and the aluminum hydroxide gels (alum), was studied using the standard antigen ovalbumin (OVA) in IL‐4 genedisrupted mice (IL‐4 ‐/‐). In the absence of adjuvant, there was an overall reduction in antibody production to OVA in IL‐4 −/− mice and significantly greater amounts of interferon (IFN)‐γ were produced following restimulation of splenocytes with antigen in vitro compared with immunocompetent controls (IL‐4 +/+). FCA and alum boosted the immune response to OVA in both IL‐4 −/− and IL‐4 +/+ mice. In IL‐4 +/+ mice, while FCA stimulated a wide‐spectrum immunoglobulin response, including both Th1‐associated IgG2a and Th2‐associated IgG1, alum enhanced only Th2 antibody production and no OVA‐specific IgG2a could be detected. In IL‐4‐deficient mice, however, not only was IgG2a production increased in all adjuvant‐treated groups, but alum was as potent at stimulating this antibody subclass as FCA. Similarly, increased production in vitro by splenocytes of the Th1 cytokine IFN‐γ, equivalent to that produced after inoculation with FCA/OVA, was only detected in IL‐4 −/− mice inoculated with alum/OVA. There was no IgE production in IL‐4 −/− mice and OVA‐specific IgG1 production, although still at significant levels, was reduced compared with wild‐type mice irrespective of the adjuvant used. However, although production of the Th2 cytokine IL‐5 was totally inhibited in IL‐4‐deficient mice inoculated with FCA/OVA, there was no significant difference in IL‐5 production between the two strains when alum was used as adjuvant.
Organ‐specific contribution to circulating C7 levels by the bone marrow and liver in humans
Authors: Naughton Michael A. , Walport Mark J. , Würzner Reinhard , Carter Martyn J. , Alexander Graeme J. M. , Goldman John M. , Botto Marina
Published: March 2007

Many cells types can produce complement component C7, although the major site of C7 synthesis is unknown. Conversion from recipient to donor allotype following organ transplantation has demonstrated the synthetic sites of several complement proteins, but in the case of C7 this was not possible until recently. A novel C7 polymorphism (C7 M/N) has been described based on the reactivity with the monoclonal antibody WU 4–15 which identifies in allotype of C7 (C7 M). Bone marrow and hepatic C7 production was quantified in bone marrow transplant and liver transplant recipients, respectively, where a mismatch for the C7 allotypes distinguished by the monoclonal antibody had occurred. In the bone marrow transplant group, one informative transplant was identified and donor‐derived C7 was detected by enzyme‐linked immunosorbent assay. It contributed to 18–27% of the total circulating C7 during the post‐transplant phase and was increased during episodes of inflammation. In the liver transplant group, the hepatic contribution to the C7 levels were 30% and 52%, respectively, in two patients identified prospectively. A further three informative liver transplant patients were identified retrospectively and in these individuals, 56–62% of the circulating C7 was liver‐derived. This study demonstrates that the majority of the circulating C7 is derived from the liver and bone marrow with a lesser contribution from other sources. These findings provide further support for the concept that locally secreted complement proteins have an important role in inflammation.
Influence of glycosylphosphatidylinositol‐linked H‐2Dd molecules on target cell protection and natural killer cell specificity in transgenic mice
Authors: Sentman Charles L. , Olsson‐Alheim Mats Y. , Lendahl Urban , Kärre Klas
Published: March 2007

The expression of certain major histocompatibility complex (MHC) class I ligands on target cells is one important determinate of their susceptibility to lysis by natural killer (NK) cells. NK cells express receptor molecules that bind to MHC class I. Upon binding to their MHC class I ligand, the NK cell is presumed to receive a signal through its receptor that inhibits lysis. It is unclear what role the MHC class I molecules of the effector and target cells play in signaling to the NK cell. We have investigated the role of the cytoplasmic and transmembrane domains of MHC class I molecules by producing a glycosylphosphatidylinositol (GPI)‐linked H‐2Dd molecule. The GPI‐linked H‐2Dd molecule is recognized by H‐2Dd‐specific antibodies and cytotoxic T lymphocytes. Expression of the GPI‐linked H‐2Dd molecule on H‐2b tumor cells resulted in protection of the tumor cells after transplantation into D8 mice (H‐2b, H‐2Dd) from rejection by NK cells. In addition, NK cells from mice expressing the GPI‐linked H‐2Dd molecule as a transgene were able to kill nontransgenic H‐2b lymphoblast target cells. The GPI‐linked MHC class I molecule was able to alter NK cell specificity at the target and effector cell levels. Thus, the expression of the cytoplasmic and transmembrane domains of MHC class I molecules are not necessary for protection and alteration of NK cell specificity.
R24 anti‐GD3 ganglioside antibody can induce co‐stimulation and prevent the induction of alloantigen‐specific T cell clonal anergy
Authors: Boussiotis Vassiliki A. , Pardo Nichole A. , Collins Heather , Houghton Alan , Ritz Jerome , Nadler Lee M. , Soiffer Robert J.
Published: March 2007

R24 is a monoclonal antibody directed against the cell surface ganglioside GD3. It can detect GD3 on the surface of a subset of T lymphocytes and can stimulate proliferation and secretion of cytokines in vitro. In the present report, we examined the effects of the R24 antibody upon antigen‐specific T cell response, employing an HLA‐DR7‐specific T cell clonal model. As previously shown, primary stimulation of HLA‐DR7‐specific alloreactive T cell clones by transfectants expressing HLA‐DR7 alone (t‐DR7) in the absence of B7 co‐stimulation resulted in anergy. Binding of cell surface GD3 on HLA‐DR7‐specific alloreactive T cell clones with R24 under these anergizing conditions resulted in interleukin‐2 (IL‐2) accumulation and prevented the induction of alloantigen‐specific T cell clonal anergy. Binding of GD3 by R24 also prevented anergy under conditions where B7:CD28 interactions were blocked by CTLA4‐Ig. The effect of R24 was abrogated in the presence of a combination of monoclonal antibodies for the α and β chains of the IL‐2 receptor (IL‐2R) or a neutralizing anti‐IL‐2 antibody. R24 does not appear to interact directly with the IL‐2R since incubation of T cell clones with R24 did not induce early activation of IL‐2R associated Jak kinases, Jak1 and Jak3, as was induced following incubation with IL‐2. In contrast, incubation of HLA‐DR7‐specific clones with t‐DR7 in the presence of R24 did result in phosphorylation of IL‐2R related Jak kinases after 24 h. Our data indicate that the membrane ganglioside GD3 structure recognized by R24 may play an important role in antigen‐specific T cell clonal response.