bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of complement receptor 2 (CR2) on HTLY‐1‐infected lymphocytes and transformed cell lines
Authors: McNearney Terry A. , Ebenbichler Christoph F. , Tötsch Martin , Dierich Manfred P.
Published: March 2007

The expression of complement receptor 2 (CR2) has been demonstrated in established HTLV‐1‐transformed cell lines and in 12 studied de novo infected peripheral blood lymphocytes cultures, using 2 HTLV‐1 sources. The simultaneous detection of CR2 and HTLV‐1 antigens in both co‐cultivated and supernatant‐infected peripheral blood lymphocytes suggest that the increased CR2 expression is in tandem with the increasing HTLV‐1 antigen expression. CR2 up‐regulation seen during polyclonal activation is presumably in response to a viral protein, although a cellular factor has not been ruled out. Increasing CR2 expression during early infection suggests its possible involvement in selection or development of subsequent transformation events. Variable levels of CR2 in immortalized cell lines argue against its obligate expression of function in the maintenance of the transformed state. The expression of CR2 in cellular activation of T cells may be stage restricted. This study also expands the cellular distribution for CR2.
Alloreactive cytotoxic T cells can develop and function in mice lacking both CD4 and CD8
Authors: Schilham Marco W. , Fung‐Leung Wai‐Ping , Rahemtulla Amin , Kuendig Thomas , Zhang Li , Potter Julia , Miller Richard G. , Hengartner Hans , Mak Tak W.
Published: March 2007

Using the technique of homologous recombination in embryonic stem cells, a mouse strain without functional CD4 and CD8 genes has been generated. Surprisingly, these mice contain significant numbers of αβ T cells. Although mice deficient for CD8 only do not show any cytotoxic response when their T cells are stimulated with either alloantigen or viral antigen, the CD4−8− mice do generate alloreactive cytotoxic T cells. These cytotoxic T cells bear the αβ T cell receptor and recognize major histocompatibility complex class I antigens. In addition fully allogeneic skin transplants were rejected but skin transplants expressing only minor transplantation antigens were not. Virus‐specific cytotoxic T lymphocytes were also not detected. It seems that alloreactive cytotoxic T cells can be induced and exert their effector function in vitro and in vivo in the absence of CD8, and that they can develop and mature in vivo without the CD8 molecule or the signals it might provide.
Developmental relationship between cytotoxic α/β T cell receptor‐positive intraepithelial lymphocytes and Peyer's patch lymphocytes
Authors: Cuff Christopher F. , Cebra Christopher K. , Rubin Donald H. , Cebra John J.
Published: March 2007

Following intraduodenal priming of mice with reovirus, precursor cytotoxic T lymphocytes (pCTL) rapidly appear in intraepithelial lymphocytes (IEL) and Peyer's patches. These cells express CTL activity after secondary in vitro stimulation with reovirus‐infected cells. Adoptive transfer of Peyer's patch lymphocytes from normal BALB/c mice into reovirus‐infected CB.17 severe combined immunodeficiency mice results in the infection‐dependent appearance of large numbers of both CD8+Thy‐1+ and CD8−Thy‐1+, IEL that express the α/β T cell receptor (TcR). Phenotypic and functional characterization of IEL derived from conventionally reared, reovirus‐infected mice also points to extensive similarities in the pCTL derived from Peyer's patches and IEL. As in the Peyer's patches, pCTL are persistent in the IEL compartment for up to 4 weeks after infection. A large percentage of IEL that are recovered from reovirus‐primed mice after in vitro culture are CD8+Thy‐1+ cells that express α/β TcR. Furthermore, depletion experiments demonstrate that the CD8+Thy‐1+ population mediates the virus‐specific CTL activity. Using limiting dilution analyses, it was estimated that 7 days after intraduodenal infection the average frequency of virus‐specific pCTL was 197/106 CD8+Thy‐1+ IEL and 190/106 CD8+Thy‐1+ Peyer's patch lymphocytes. Taken together, these observations provide evidence that specific cellular immunity to reovirus in IEL is mediated, at least in part, by conventional cytotoxic T lymphocytes and that these cells are functionally and phenotypically similar to the pCTL derived from the Peyer's patches.
Antigen processing and presentation of human rhinovirus to CD4 T cells is facilitated by binding to cellular receptors for virus
Authors: Hastings Gillian Z. , Francis Michael J. , Rowlands D. J. , Chain Benjamin M.
Published: March 2007

Human rhinovirus serotypes (HRV) fall into two distinct groups, major and minor, by virtue of their cell receptor‐binding ability. In this study minor receptor‐binding group viruses are demonstrated to bind directly to cells of the murine immune system, including lymphoid dendritic cells which act as antigen‐presenting cells, although they do not produce a productive infection in murine cells. This binding is specific and can be blocked by other serotypes of minor‐group HRV Pre‐treatment of HRV 1A, a minor‐group virus, with HRV 1A‐specific antibodies inhibited the cellular proliferation of murine virus primed T helper cells, whereas antibody treatment of HRV 15, a non‐binding major serotype, gave no inhibition. The cell binding ability of minor‐group HRV played a role in the overall immunogenicity of this virus group, which was shown to be enhanced compared to the immunogenicity of major‐group viruses in mice.
Association of CD22 with the B cell antigen receptor
Authors: Peaker Christopher J. G. , Neuberger Michael S.
Published: March 2007

The antigen receptor on B lymphocytes is composed of membrane immunoglobulin sheathed by an α/β heterodimer. This structure is in several respects analogous to the antigen receptor on T cells except that, in the case of the T cell but not the B cell receptor, several receptor‐associated proteins have been described which may modulate the effects of antigen interaction (e.g. CD4, CD8, CD2 and CD5). To screen for specific associations with the B cell antigen receptor that might be of only low stoichiometry, we have exploited the sensitivity of in vitro kinase assays. We show that the B cell antigen receptor associates with CD22.The association is specific and stable, but Western blotting reveals it to be of low stoichiometry (0.2 to 2% of membrane immunoglobulin is CD22 associated).The CD22/antigen receptor association was demonstrated with multiple isotypes (IgM, IgD and IgG) and was evident both in Burkitt lymphoma lines and in tonsil cells. Whilst the significance of the association is unknown, it is notable that CD22 is a B cell‐specific adhesion molecule which we find contains within its cytoplasmic domain a sequence bearing high homology to the “Reth motif implicated in signal transduction. Indeed, CD22 becomes tyrosine phosphorylated less than one minute after antigen‐receptor cross‐linking. Thus, it is tempting to speculate that interactions involving CD22 assist in the antigen‐mediated triggering of B cell activation.
T cell receptor β chain dimers on immature thymocytes from normal mice
Authors: Groettrup Marcus , Boehmer Harald Von
Published: March 2007

During T cell development the T cell receptor (TCR) β chain is expressed before the TCRα chain. Experiments in TCRβ transgenic severe combined immune deficiency (SCID) mice have shown that the TCRβ protein can be expressed on the cell surface of immature thymocytes in the absence of the TCRα chain and that the TCRβ protein controls T cell development with regard to cell number, CD4/CD8 expression and allelic exclusion of the TCRβ chain. Subsequent experiments have shown that on the surface of thymocytes fromTCRβ transgenic SCID mice the TCRβ protein can be expressed in a monomelic and dimeric form whereas only the dimeric form was found on the surface of a TCRβ‐transfected, immature T cell line. The results presented here show that normal thymocytes from 16‐day‐old fetuses likewise express only the dimeric form and that the monomelic form on the surface of thymocytes from transgenic mice results from glycosyl phosphatidylinositol linkage. Our results show for the first time that under physiological conditions a TCRβ dimer can be expressed on the cell surface without the TCRα chain.
Identification of the T cell surface signal‐transducing glycoprotein sgp‐60 as CD48, a counter‐receptor for mouse CD2
Authors: Kato Kazunori , Tamura Naoto , Okumura Ko , Yagita Hideo
Published: March 2007

We recently showed that CD48 is the major counter‐receptor for CD2 in the murine system. To examine whether sgp‐60, which has been proposed as the murine homologue of human LFA‐3, is another ligand for mouse CD2, we performed the characterization of sgp‐60 by using mouse CD2‐human IgG chimeric protein (mCD2Rg) and anti‐mouse monoclonal antibodies (mAb). Anti‐sgp‐60 mAb 5‐8A10, as well as anti‐mouse CD48 mAb HM48‐1 completely inhibited the binding of mCD2Rg at the ligand site. 5‐8A10 immunoprecipitated the same molecule as that recognized by HM48‐1 and reacted with mouse CD48 cDNA‐transfected Chinese hamster ovary cells, indicating that sgp‐60, recognized by 5‐8A10, is identical to mouse CD48. The epitope recognized by 5‐8A10 was different from that recognized by HM48‐1 and OX78, suggesting that the different T cell activating property of these anti‐mouse CD48 mAb depends on the epitopes they recognize. The identification of sgp‐60 as CD48 further suggests a role of CD48 in regulating T cell activation.
Allergen exposure induces the activation of allergen‐specific Th2 cells in the airway mucosa of patients with allergic respiratory disorders
Authors: Del Prete Gianfranco F. , De Carli Marco , D'Elios Mario M. , Maestrelli Piero , Ricci Mario , Fabbri Leonardo , Romagnani Sergio
Published: March 2007

Biopsy specimens were obtained from the bronchial or the nasal mucosa of three patients with grass pollen‐induced bronchial asthma or rhinitis 48 h after positive bronchial or nasal provocation test with grass pollen extract. T cell clones (TCC), derived from these and control specimens, were then assessed for their phenotype, allergen‐specificity, profile of cytokine secretion and ability to provide B cell help for IgE synthesis. Out of 50 and 61 CD4+ TCC derived from the bronchial mucosa of the two patient with atopic asthma 11 (22%) and 19 (31%), respectively, showed both proliferation and cytokine production in response to grass pollen allergens under major histocompatibility complex‐restricted conditions. Of these 21 (70%) exhibited a clear‐cut type 2 T helper (Th2) profile and induced IgE synthesis in autologous peripheral blood B cells in the presence of grass allergens. All the other 9 grass‐specific clones showed a Th0 pattern of cytokine secretion, but only 1 provided moderate help for IgE synthesis. In contrast, the majority of TCC, derived under the same experimental conditions from the bronchial mucosa of two nonatopic patients with toluene diisocyanate‐induced asthma, were CD8+ and most of them produced interferon‐γ or interferon‐γ and interleukin‐5, but not interleukin‐4, in response to nonspecific stimulation. Of 22 CD4+ TCC 3 (14%) derived from the grass‐stimulated mucosa of the patient with allergic rhinitis, but none of those derived from the unstimulated nostril of the same patient, exhibited proliferation and cytokine production in response to grass allergens. All had a clear‐cut Th2 profile and provided help for IgE synthesis by autologous B cells.

These data indicate that inhalation of the relevant allergen results in the activation of allergen‐specific Th2 lymphocytes in the airway mucosa of patients with allergic respiratory disorders. These cells may play a central role in determining the nature of the inflammatory response in the airways of atopic patients.
Enhanced triggering of mucosal immune responses by reducing splenic phagocytic functions
Authors: Van den Dobbelsteen Germie P. J. M. , Brunekreef Karin , Kroes Hilde , van Rooijen Nico , Van Rees Emmelien P.
Published: March 2007

The role of the spleen in the rat mucosal immune response was investigated to three structural different pneumococcal polysaccharides, type 3, 4, and 14. Following immunization with pneumococcal polysaccharides, a larger amount of free antigen was found in several lymphoid tissues and an increased trapping of immune complexes was seen in follicles of splenectomized animals, as compared to control animals. Thus, clearance of the polysaccharides seems to be less effective after splenectomy. An increase in specific IgA antibody‐containing cells (ACC) was found in mesenteric lymph nodes, villi and Peyer's patches in splenectomized rats. Apparently, splenectomy and subsequent decreased clearance of the antigen causes a prolonged stay of the antigen in the system and therefore specific ACC can be induced in different lymphoid tissues. After splenectomy the specific IgM and IgG antibody titers in serum decreased significantly for pneumococcal polysaccharides types 4 and 14, but not for type 3. Furthermore, the serum IgA antibody titers against the three types of polysaccharides under study were not affected. After elimination of macrophages in the spleen by treatment with dichloromethylene diphosphonate liposomes no ACC against type 14 were evoked in the marginal zone of the spleen, and again, an increase was observed in specific IgA ACC in mucosa‐associated lymphoid tissues. The IgA antibody titers were also enhanced.

In conclusion, IgA responses against pneumococcal polysaccharides can be elicited in absence of the spleen, i.e. at mucosal sites or in the draining lymph nodes. Furthermore, polysaccharide‐specific IgA responses are enhanced after reduction of splenic phagocytic functions.
Up‐regulated recombination‐activating gene expression in slg− variants of a human mature B cell line undergoing secondary Igλ, rearrangements in cell culture
Authors: Stiernholm Niclas B. J. , Berinstein Neil L.
Published: March 2007

To study the role of surface immunoglobulin (sIg) expression in the control of rearrangement activity at the Ig light chain loci, we established rare sIg− clones (λ−) from a human sIg+ B cell line (μl+). Upon expansion of these sIg− clones, surface immunofluorescence analysis revealed a gradual emergence of sIg+ subpopulations, differing from the original tumor cell line both in their idiotypes and Cλ isotypes. DNA analysis revealed that this sIg heterogeneity resulted from a process of ongoing Igλ, rearrangements. That is, one of the Igλ rearrangements in the parental cell line was replaced by novel Igλ rearrangements in the sIg− clones, which in turn were replaced by yet additional Igλ rearrangements in the sIg+ variants. Northern analysis demonstrated that while the expression of the recombination‐activating genes RAG1 and RAG2 was relatively low in the parental cell line, their expression was significantly increased in both the sλ g+ variants and their sIg+ progenies. We thus describe a human mature B cell line, in which differential RAG expression allows sIg heterogeneity to be generated through secondary Igλ gene rearrangements. Our results indicate that the induction of RAG expression may be inversely associated with sIg expression, but that sIg expression, alone, is not sufficient to down‐regulate this expression.
Immune response in mouse and malaria‐exposed humans to peptides derived from Pfl1‐1, a highly repetitive megadalton protein of Plasmodium falciparum
Authors: Scherf Artur , Behr Charlotte , Sarthou Jean‐Louis , Pla Marika , Rogier Christophe , Trape Jean‐François , Silva Luiz Pereira Da , Dubois Philippe
Published: March 2007

We have investigated the immune response against the Plasmodium falciparum gametocyte‐specific antigen Pf11‐1. This megadalton parasite molecule has been implicated in the process of erythrocyte rupture during gametogenesis. The molecule is composed in great part of degenerated nonapeptide motifs which are tandemly repeated several hundred times. A computer algorithm searching for T sites predicted that the entire repeat region of the Pf11‐1 represents potential T cell antigenic major histocompatibility complex class II‐binding sites. To test this hypothesis, synthetic peptides corresponding to two nonamer subtype repeats, differing only at two amino acid positions, were used to immunize congenic mouse strains. Both peptides were shown to contain both B and T cell epitopes. The immune response is restricted to the H‐2d andH‐2khaplotypes. The T cell response against the peptides appeared to be highly specific, clearly discriminating between the two similar nonamer repeat sequences, whereas the humoral response produced cross‐reacting antibodies. We also investigated the humoral and T cell reactivities of P. falciparum‐primed individuals in West Africa against the synthetic Pf11‐1 peptides. Among 51 individuals 35 had antibodies to at least one of the two peptides and a majority of them (28) had antibodies reacting with both peptides. The cellular response was analyzed by [3H] thymidine incorporation or interferon‐y release. There was considerable variation in the response to the two peptides. Among the human samples 36% responded to one repeat subtype, while only 13% responded to the second subtype. Interestingly, in individual donors the T cell response to both peptides are associated, suggesting that, as shown for mice, the response is restricted by a genetic element. The data obtained on the two subtypes of the nonamer repeat region suggest that the entire Pfll‐1 molecule might induce an unusually heterogenous B and T cell response during natural infection in man.
The invasin protein of Yersinia spp. provides co‐stimulatory activity to human T cells through interaction with β integrins
Authors: Brett Sara J. , Mazurov Alexey V. , Charles Ian G. , Tite John P.
Published: March 2007

The invasin proteins of Yersinia spp. are outer membrane proteins which are involved in the penetration of these bacteria into mammalian cells (Cell 1990. 60: 861). Invasin binds to several different β integrins with extremely high affinity, the integrin‐binding domain of invasin has been mapped to the C‐terminal 192 amino‐acids of the molecule (J. Biol. Chem. 1991.266:24367). Expression of this fragment alone on the cell surface of non‐invasive bacteria is enough to confer the invasive phenotype on these strains (EMBOJ. 1990. 9:1979). Here we show that the carboxy‐terminal 192 amino acids of invasin expressed as a fusion protein with the maltose binding protein of E. coli is capable of delivering co‐stimulatory signals to human T cells through the β integrins. Co‐stimulation was assayed by the ability of invasin to augment the response of highly purified CD4+ and CD8+ T cells to co‐immobilized anti‐CD3 antibody. Antibody blocking studies indicated that the co‐stimulation was mediated through β integrins. The proliferation induced by co‐stimulation of CD4+ T cells was accompanied by the synthesis of the cytokines tumor necrosis factor‐a and interferon‐y, whereas the activation of CD8+ T cells led to the generation of cytotoxic effectors. The region of the invasin molecule involved in T cell activation was further mapped using synthetic peptides. A region of the invasin molecule containing the residues TAKSKKFP‐SY could substitute for invasin in T cell activation. The co‐stimulation by peptide could also be inhibited by anti‐integrin antibodies. The observation that an outer membrane protein of a bacterium which is associated with reactive arthritis and other autoimmune spondyloarthropathies can act as a T cell co‐stimulus may have implications for the etiology of these diseases.
T cell receptor targeting to thymic cortical epithelial cells in vivo induces survival, activation and differentiation of immature thymocytes
Authors: Müller Klaus‐Peter , Kyewski Bruno A.
Published: March 2007

We report that targeting of T cell receptors (TcR) to non‐major histocompatibility complex (MHC) molecules on thymic cortical epithelial cells by hybrid antibodies in vivo and in fetal thymic organ cultures results in phenotypic and functional differentiation of thymocytes. A single pulse with hybrid antibodies rescues immature, CD4/8 double‐positive thymocytes from their programmed death in vivo, induces expression of the early activation antigen CD69 followed by TcR up‐regulation, concomitant down‐regulation of CD8 or CD4 and their conversion to functional mature T cells by day 3. This temporal sequence of maturation only affects small thymocytes without co‐induction of blastogenesis. TcR targeting to MHC class II‐positive epithelial cells predominantly induces CD4‐positive T cells. This generation of CD4 single‐positive T cells occurs also in MHC class II‐deficient mice and thus is independent of CD4‐MHC class II interactions. Moreover, in the presence of a specific deleting antigen (Mls 1a),TcR targeting results in transient activation of immature thymocytes, however, not in subsequent TcR (Vβ6) up‐regulation and development of single‐positive T cells. Our findings imply that TcR cross‐linking to cortical epithelial cells is sufficient to confer a differentiation signal to immature thymocytes. Futhermore, this approach distinguishes two independent TcR‐mediated intrathymic events: activation and subsequent deletion of the same thymocyte subset.
Antigen‐presenting cells in adoptively transferred and spontaneous autoimmune diabetes
Authors: Lo David , Reilly Christina R. , Scott Bernadette , Liblau Roland , McDevitt Hugh O. , Burkly Linda C.
Published: March 2007

Histological techniques were used to identify antigen‐presenting cells (APC) in adoptively transferred diabetes in NOD mice and Ins‐HA transgenic mice, and in spontaneously diabetic NOD mice. In adoptively transferred disease, CD4+ Tcells and F4/80+ macrophages dominated early infiltrates. By contrast, in spontaneously developing diabetes in NOD mice, lymphocytic infiltrates appeared to be well organized around a network of VCAM‐1+ NLDC‐145+ ICAM‐1+ dendritic cells. Thus, the primary APC spontaneous autoimmune disease appears to be the strongly stimulatory dendritic cell rather than the normally resident macrophage. Next, we used chimeric animals to demonstrate that insulitis and diabetes could occur even when responding T cells were unable to recognize islet‐specific antigen directly on β cells. Altogether, the results demonstrate that immune‐mediated damage does not require direct contact between CD4+ T cells and β cells. Moreover, despite the induction of ICAM‐1, VCAM‐1, and class II on vascular endothelium near islet infiltrates, these experiments show that recruitment of lymphocytes occurs even when antigen presentation is not possible on vascular endothelium.
TAP2‐defective RMA‐S cells present Sendai virus antigen to cytotoxic T lymphocytes
Authors: Zhou Xianzheng , Glas Rickard , Momburg Frank , Hämmerling Günter J. , Jondal Mikael , Ljunggren Hans‐Gustaf
Published: March 2007

The murine antigen‐processing‐defective mutant cell line RMA‐S is leaky in the presentation of certain endogenously synthesized minor histocompatibility and viral antigens to major histocompatibility complex (MHC) class I‐restricted cytotoxic T lymphocytes (CTL). The viral antigens include influenza virus nucleoprotein, vesicular stomatitis virus (VSV) nucleocapsid and Rauscher murine leukemia virus (MuLV) antigen. Here we demonstrate Sendai virus antigen presentation by the HAM2 (murine TAP2, transporter associated with antigen presentation type 2)‐defective RMA‐S cell line and compare antigen presentation after restoration of the defect by murine TAP1/2 gene transfection. Kinetic studies revealed that RMA‐S cells required 2‐3 h longer incubation and approximately 10 times higher doses of Sendai virus to reach the same level of killing as the RMA parental line. After transfection of RMA‐S cells with the murine TAP1/2 gene, Sendai virus antigen presentation was restored to levels of the RMA wild‐type line with regard to time of virus infection and dose of virus needed for sensitizing target cells. The presentation of Sendai virus antigen in RMA‐S cells was sensitive to brefeldin A (BFA), suggesting that the presentation was mediated via the endogenous pathway. Our findings comfirmed leakiness of antigen presentation in RMA‐S cells and extended it to Sendai virus. The results underscored the role for intact expression of the TAP 1/2 molecules for efficient MHC class I‐mediated antigen presentation.
Antigen processing mutant T2 cells present viral antigen restricted through H‐2K
Authors: Zhou Xianzheng , Glas Rickard , Liu Tianmin , Ljunggren Hans‐Gustaf , Jondal Mikael
Published: March 2007

Cytotoxic T lymphocytes (CTL) recognize foreign antigens as short peptides presented by class I molecules of the major histocompatibility complex (MHC). T2 cells are profoundly defective in the presentation of endogenously synthesized antigens to CTL due to a deletion of MHC class II‐encoded genes for transporters associated with antigen presentation (TAP1/TAP2). Surprisingly, we here demonstrate that T2 cells, after infection with Sendai virus, are readily killed by H‐2Kb restricted CD8+ T cells. In contrast to classical class I‐mediated antigen presentation, the presentation of Sendai virus antigen inT2Kb cells is brefeldin A (BFA) insensitive. The present findings may suggest the presence of an alternative pathway for MHC class I‐mediated antigen presentation in T2 cells.
Cytokine production and heterogeneity of primary stromal cells that support B lymphopoiesis
Authors: Witte Pamela L. , Frantsve Lisa M. , Hergott Muriel , Rahbe Sandra M.
Published: March 2007

Many reports document that bone marrow stromal cells or their cytokine products can influence the formation of B cells in vitro. Most of this data comes from studies using lines or clones of stromal cells after multiple passage in culture, which could alter gene expression. Our aim in the present study was to determine which cytokines are produced by normal stromal cells under conditions that promote B lymphopoiesis. Primary cultured stromal cells were isolated on FACS from active Whitlock cultures. These cells proved to be relatively homogeneous in expression of cell surface antigens (CD44, VCAM‐1, MECA10, and a molecule marked by hamster anti‐mouse 8.28 monoclonal antibody). RNA from unselected Whitlock cultured adherent cells and sorted stromal cells from the same cultures were subjected to reverse transcriptase polymerase chain reaction to assess constitutive expression of several cytokine genes. Transcripts for interleukin‐1β (IL‐1β), IL‐7, macrophage (M)‐colony‐stimulating factor (CSF), stem cell growth factor (SCGF), insulin‐like growth factor 1 (IGF‐1) and occasionally leukemia inhibitory factor were detected in RNA from intact cultures. Messages for IL‐7, M‐CSF, and SCGF were selectively contained within the isolated stromal cell fraction; whereas, IL‐1β was found solely within the non‐stromal cell fraction. IGF‐1 was transcribed by both stromal cells and macrophages in Whitlock cultures. No evidence was found for constitutive expression of IL‐1 α, IL‐4, IL‐6, or granulocyte‐macrophage‐CSF. This is in contrast to some reported stromal cell lines and clones. To determine if all primary stromal cells from active lymphopoietic cultures produced IL‐7, the isolated cells were stained to reveal cytoplasmic IL‐7 protein. A majority of the cells produced IL‐7, but about 20 % had no detectable IL‐7 protein. Taken together, our results suggest that the primary stromal cells are a distinguishable cell type but functional subsets may exist. In regard to the differences in IL‐7 production, the primary cell phenotype appears to mirror at least one division noted among the stromal cell lines.
Association of a 70‐kDa tyrosine phosphoprotein with the CD16:ζ:γ complex expressed in human natural killer cells
Authors: Vivier Eric , da Silva Antonio J. , Ackerly Melissa , Levine Herbert , Rudd Christopher E. , Anderson Paul
Published: March 2007

The CD16: ζ: γ receptor complex allows natural killer (NK) cells to recognize and eliminate antibody‐coated target cells. Whereas the ectodomain of CD16 is the receptor for Fcγ domains of immunoglobulins, disulfide‐linked homo‐ and heterodimers composed of ζ and γ are required for the cell surface expression, and signal transduction properties of the complex. Engagement of CD16 activates the tyrosine kinase pathway, which induces the tyrosine phosphorylation of several substrates, including the ζ subunit and the phospholipase C γ‐1 and γ‐2 isoforms. Here we show that CD 16 stimulation of either peripheral blood NK cells, leukemic NK cells, or Jurkat transformants expressing a CD16:ζ:γ receptor complex, results in the tyrosine phosphorylation of a 70 kDa ζ‐associated protein (pp70). Similarly, a 70‐kDa ζ‐associated phosphoprotein in T cells has been shown to be a tyrosine kinase (ZAP‐70). Peptide mapping analysis indicates that the 70‐kDa ζ‐associated phosphoproteins from T cells and NK cells are structurally indistinguishable. We conclude that the CD16:ζ:γ complex may use a ZAP‐70‐related non‐receptor tyrosine kinase, in the CD16 signaling cascade leading to NK cell activation.
Regulation of perforin gene expression in a T cell hybrid with inducible cytolytic activity
Authors: García‐Sanz José A. , Podack Eckhard R.
Published: March 2007

A mouse × rat T cell hybrid (PC60) that does not require interleukin (IL)‐2 for proliferation, was used as a model to study regulation of perforin gene expression. Perforin mRNA is barely detectable in non‐induced PC60 cells; however, a 30‐fold induction is observed after stimulation with IL‐1 alone. Peak perforin mRNA levels were reached after 10 h of induction with IL‐1, and these levels were maintained for as long as the stimulus was present. IL‐2 by itself has no detectable effect. However, in combination with IL‐1 it shows the same induction kinetics as IL‐1 alone for the first 10 h, subsequently there is synergism (100‐fold induction) between IL‐1 and IL‐2. The induction response was mainly due to increased transcriptional rates of the perforin gene, and require newly synthesized proteins. The half‐life of perforin mRNA in this system is about 5 h. In addition, we confirm the existence of two types of mouse perforin mRNA that differ in their 5′ untranslated regions, and show evidence that both mRNA are translated in vivo with similar efficiencies.
Cachexia and the acute‐phase protein response in inflammation are regulated by interleukin‐6
Authors: Oldenburg Hester S. A. , Rogy Michael A. , Lazarus Douglas D. , van Zee Kimberly J. , Keeler Bonnie P. , Chizzonite Richard A. , Lowry Stephen F. , Moldawer Lyle L.
Published: March 2007

Cachexia and the acute‐phase response are common manifestations of inflammation and are presumed to be the product of increased synthesis and release of cytokines, including tumor necrosis factor (TNF), interleukin‐1 (IL‐1) and interleukin‐6 (IL‐6). IL‐1 receptor blockade has been previously shown to attenuate the weight loss, anorexia and acute‐phase protein responses associated with a turpentine abscess. However, IL‐1 receptor blockade was also associated with a reduced plasma IL‐6 response, suggesting that the benefit achieved by IL‐1 receptor blockade may be mediated by reduced systemic IL‐6 production. To gain a better understanding of the role of IL‐6 in this model of inflammation, C57BL/6 mice were passively immunized with either a monoclonal anti‐IL‐6 antibody (20F3), an anti‐IL‐1 type I receptor monoclonal antibody (35F5), a non‐immune rat IgG, or a combined therapy of 35F5 and 20F3, before receiving a sterile turpentine abscess. IL‐6 or IL‐1 receptor blockade equally spared body weight and food intake. Compared to IL‐1 receptor blockade, passive immunization against IL‐6 further reduced the hepatic acute‐phase protein response, as represented by serum amyloid P and complement 3. Combined blockade of IL‐6 and IL‐1 receptor did not result in a further sparing of body weights or improvement of food intake. These results confirm that IL‐1 contributes to host cachexia and the acute‐phase response following a turpentine abscess, but also show that these actions are dependent upon an IL‐6 response. We onclude that the influence of IL‐1 on cachexia and the acute‐phase response is mediated, at least in part, through IL‐6 and, thus, IL‐6 may play a pivotal role in the cachexia and acute‐phase response to inflammation.
Regulation of T cell production in T cell receptor transgenic mice
Authors: Kelly Katherine A. , Pircher Hanspeter , von Boehmer Harald , Davisand Mark M. , Scollay Roland
Published: March 2007

The thymus produces many more cells than it releases into the periphery. According to generally accepted models of T cell development most of this loss occurs in the thymic cortex, among CD4+8+ thymocytes. An interesting situation arises in the case of T cell receptor (TcR) transgenic mice in which all cells can potentially be positively selected, leading to a theoretical increase of about 30‐fold in the survival rate of CD4+8+ cells and in their transition to mature CD4+8−or CD4−8+ thymocytes. This in turn should lead to a 30‐fold increase in the size of the thymic medulla, in the emigration rate and in the size of the peripheral Tcell pool. Increases in medullary or peripheral pool sizes of this magnitude are not seen in TcR transgenic mice. The question was therefore asked whether some form of homeostatic process regulated the size of the mature T cell pool and at what level it might operate. In this report we demonstrate that the increased rate of double‐positive to single‐positive transition in the TcR transgenic mice is directly reflected in an increased emigration rate, and that the medulla seems to be relatively efficient regardless of the number of cells passing through it. However, the potential increases in emigrant numbers in TcR transgenic mice are offset by the reduced size of the CD4+8+ thymocyte pool. It would appear then that regulation of T cell production, if it occurs, probably does so through regulation of the size of the CD4+8+ thymocyte pool. Mechanisms for regulation of this kind are not yet known.
Diversity of expressed V and J regions of immunoglobulin light chains in Xenopus laevis
Authors: Stewart Sue E. , Du Pasquier Louis , Steiner Lisa A.
Published: March 2007

In Xenopus laevis, two immunoglobulin light chain isotypes, designated L1 or ϱ and L2 or σ, have been identified. The genomic organization of the L1 locus has been described previously: a constant (C) gene segment is preceded by a joining (J) gene segment; in addition, there are many cross‐hybridizing variable (V) gene segments. To evaluate the extent of sequence diversity of L1 V regions, we screened three cDNA libraries, constructed from mitogen‐stimulated Xenopus splenocytes, with probes for the C or the J gene segment. Eighteen cDNA clones that contain complete or truncated V regions were chosen for sequence analysis. The C regions of all clones are identical or nearly identical to the genomic C gene segment; the V regions are greater than 80 % identical in nucleotide sequence and are presumably derived from a single family of V gene segments. Although framework regions are nearly identical, complementarity‐determining regions are quite diverse. The expressed J segments fall into distinct groups, suggesting the presence of more than one germ‐line J segment. Therefore, a genomic library was screened with a J region probe. A clone overlapping with the previously identified J‐ C clone, and containing four additional J gene segments, was isolated. All five J gene segments are very similar and three are identical in nucleotide sequence. Each of the three distinct germ‐line J sequences is represented in the set of cDNA clones, suggesting that combinatorial diversification occurs; imprecision of V‐J joining also appears to contribute to variability. Overall, these results suggest that the immunoglobulin repertoire in this species is not significantly restricted by a limitation in the diversity of light chain V regions.
Interleukin‐6 production in “normal” and HTLV‐1 tax‐expressing brain‐specific endothelial cells
Authors: Rott Ortwin , Tontsch Ulrike , Fleischer Bernhard , Cash Evelyne
Published: March 2007

Abnormal cytokine production can contribute in many instances to the development of pathology. Our study focuses on the regulation of interleukin (IL)‐6 production in vitro in brain‐specific endothelial cells (BEC) under physiological conditions and in a model of human T leukemia virus‐1 (HTLV‐1) infection. IL‐6 production was strongly up‐regulated in a dose‐dependent mode upon exposure to recombinant IL‐1β although nearly not detectable in unstimulated BEC. This induction of IL‐6 production could be achieved by reagents known to increase intracellular levels of cAMP, such as for skolin, prostaglandin E or pentoxifylline. Furthermore, transcription and production of IL‐6 was inducible by addition of dibutyryl cAMP, but not by addition of calcium ionophores or diacylglycerol. To assess a potential role of HTLV‐1‐infected BEC in the pathogenesis of tropical spastic paraparesis (TSP), the HTLV‐1 tax gene was expressed in BEC. Tax gene‐expressing BEC produced constitutively very high amounts of IL‐6, which were not longer hyperinducible by IL‐ip or cAMP derivates. Our results indicate that HTLV‐1 tax induces hyperproduction of IL‐6 in brain‐specific endothelial cells directly by an intracellular mechanism which subsequently renders IL‐6 production independent of exogenous stimuli or activators of (cAMP‐dependent) second messenger levels. On the basis of these findings we suggest that tax‐mediated hyperactivation of IL‐6 production in BEC contributes to elevated IL‐6 levels found in serum and cerebrospinal fluid of patients with TSP and might have a significance in the immune pathogenesis of the disease.
The murine immune response to the male‐specific antigen mouse testicular cytochrome c
Authors: Kelner Gregory S. , Minnerath Jeanne M. , Jenkins Marc K. , Jemmerson Ronald
Published: March 2007

Male and female A/J mice were examined for their ability to elicit T lymphocyte and antibody (Ab) responses to the male‐specific Ag, mouse testicular cytochrome c (Mt cyt).T lymphocytes from both male and female mice primed in vivo responded to the Ag in in vitro proliferation assays, and the dose‐response curves were statistically indistinguishable. In addition, similar levels of Ab to Mt cyt were observed in immunized male and female mice. The B cells producing the Ab had switched isotypes to IgG1 and IgG2a, indicating that the self‐reactive T helper (Th) cells in male mice were functional. Thus, male mice do not appear to be immunologically tolerant to Mt cyt, at least at the Th and B lymphocyte levels. No evidence for disease was found in male mice primed with Mt cyt. Major histocompatibility complex (MHC) class II‐positive antigen‐presenting cells are present in the testes and these were shown in vitro to process and present Mt cyt to a Tcell hybridoma specific for the synthetic peptide Mt cyt 93‐104. However, the hybridoma was not activated in the absence of exogenous Mt cyt 93‐104 or Mt cyt, indicating that endogenous Mt cyt is not normally processed in sufficient quantity to effectively load MHC class II molecules with this particular Mt cyt‐derived peptide. Notwithstanding any immunologic privilege of the testes, the lack of tolerance to Mt cyt and its failure to elicit an autoimmune disease could extend from the low levels of processed Mt cyt Ag available for T cell recognition. The T cell response elicited by Mt cyt contrasts the lack of response to mouse somatic cytochrome c which differs from Mt cyt at 13 amino acid residues and is expressed in most tissues and at higher levels.
Induction of macrophage nitric oxide production by interferon‐γ and tumor necrosis factor‐α is enhanced by interleukin‐10
Authors: Corradin Sally Betz , Fasel Nicolas , Buchmüller‐Rouiller Yolande , Ransijn Adriana , Smith Josiane , Mauël Jacques
Published: March 2007

Interleukin‐10 (IL‐10) has been reported to inhibit nitric oxide (NO) synthesis and microbicidal activity of interferon‐γ (IFN‐γ)‐stimulated macrophages (MΦ) by preventing the secretion of tumor necrosis factor‐α (TNF‐α) which serves as an autocrine activating signal. We have examined the effects of recombinant IL‐10 on the capacity of IFN‐γ together with exogenous TNF‐α to induce NO synthesis by bone marrow‐derived MΦ. Under these conditions and in contrast to its reported deactivating potential, IL‐10 strongly enhanced NO synthesis measured as nitrite (NO) release (half maximal stimulation at approximately 10 U/ml). IL‐10 further increased NO production by MΦ stimulated in the presence of optimal concentrations of prostaglandin E2, a positive modulator of MΦ activation by IFN‐γ/TNF‐α. Increased steady state levels of NO synthase mRNA were observed in 4‐h IFN‐γ/TNF‐α cultures and enhanced NO release was evident 24 h but not 48 h after stimulation. These results suggest that the effects of IL‐10 on MΦ function are more complex than previously recognized.
The Mycobacterium tuberculosis 71‐kDa heat‐shock protein induces proliferation and cytokine secretion by murine gut intraepithelial lymphocytes
Authors: Beagley Kenneth W. , Fujihashi Kohtaro , Black C. Allen , Lagoo Anand S. , Yamamoto Masafumi , McGhee Jerry R. , Kiyono Hiroshi
Published: March 2007

Murine intraepithelial lymphocytes (IEL) respond poorly to T cell mitogens and to monoclonal antibody stimulation of T cell receptor (TCR)‐ and CD3‐associated molecules. In contrast, we found that a soluble extract of Mycobacterium tuberculosis (Mtb), but not purified protein derivative of tuberculin, induced significant proliferative responses in IEL cultures. The active component was apparently a heat shock protein (HSP), since recombinant 71‐kDa HSP from Mtb induced IEL to proliferate, while 65‐kDa HSP from M. bovis and M. leprae did not. Both α/β and α/δ TCR‐enriched IEL gave proliferative responses to 71‐kDa HSP. Further, culture supernatants from IEL stimulated with 71‐kDa HSP contained elevated levels of interleukin‐(IL)‐3/granulocyte‐macrophage colony‐stimulating factor, interferon‐y and IL‐6, but not IL‐2, IL‐4, IL‐5 or transforming growth factor‐β Finally, several IEL T cell clones have been maintained for up to 6 weeks, when stimulated with 71‐kDa HSP, IL‐2 and feeder cells. Our results show that the 71‐kDa HSP of Mtb induces IELT cells to divide and to secrete cytokines and this may offer a model for cloning and study of IEL T cells in vitro.
A possible role of two hydrophobic amino acids in antigen recognition by synovial T cells in rheumatoid arthritis
Authors: Maruyama Toshiaki , Saito Ichiro , Miyake Sachiko , Hashimoto Hiroshi , Sato Kazuto , Yagita Hideo , Okumura Ko , Miyasaka Nobuyuki
Published: March 2007

Synovial T cells play a crucial role in the pathogenesis of rheumatoid arthritis (RA) synovitis. We have quantitatively analyzed the T cell receptor (TcR) variable (V) region gene repertoire of freshly isolated synovial fluid (SF) T cells, comparing it with that of peripheral blood (PB) T cells in RA. The TcR V gene repertoire of PB and SF T cells in RA and osteoarthritis was heterogeneous. In contrast, Vail in SF was expressed to a greater degree in three of five RA patients, and increased levels of Vp6, 1‐3 were found in the SF of four of six RA, compared with paired PB. Of note, Vβ6, 1–3 was universally used in four RA patients with a disease duration of less than 10 years, irrespective of their HLA‐DR types. This was in contrast to two other RA patients, suffering for more than 20 years, who showed different Vα and Vβ usages. β‐chain sequence analysis in RA patients with a preference for Vβ6, 1–3 has shown that a few clones dominated in SF, whereas polyclonality was observed in PB. These findings suggest oligoclonal expansion of T cells in response to specific antigen(s) in the SF of these patients with RA of relatively short duration. Concomitant use of two hydrophobic amino acids, leucine and valine, in the Dβ region was noticeable among the predominant SF clones. These two amino acids might directly contact a peptide specific for the induction of synovitis in RA patients. TcR‐directed therapy may, therefore, be useful for the treatment of early RA synovitis.
Distribution of perforin‐containing cells in normal and pregnant mice*
Authors: Zheng Li Mou , Ojcius David M. , Young John Ding‐E.
Published: March 2007

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells elaborate a cytolytic protein named perforin or cytolysin. It was widely held that, in vivo, high quantities of perforin are not present in resting lymphocytes and are usually produced only by activated lymphocytes found under pathological conditions. Until now, only one tissue was known to synthesize abundant quantities of perforin under nonpathological conditions, the uterus during pregnancy. To investigate the possibility that perforin might also be synthesized by other tissues, several tissues besides the uterus from pregnant and normal mice were tested by immunofluorescence and immunoperoxidase for the presence of perforin. The tissues studied were the ears, brain, nasal epithelium, tongue, salivary gland, larynx, thymus, stomach, liver, spleen, small intestine, and lymph nodes; two cell populations with different sizes and levels of perforin expression were found. Large cells, displaying the NK cell phenotype and expressing high levels of perforin, were detected not only in the uterus but also in the salivary gland and lungs of pregnant mice. Small cells, expressing low levels of perforin, were detected mainly in the stomach and small intestine, and they were expressed in both pregnant and normal mice. Taken together, these results imply that perforin‐containing cells exist in vivo under nonpathological conditions, and that the immune system is endowed with heretofore unknown mechanisms for stimulating the activation of NK cells in a limited number of tissues during pregnancy.
Selective activation of resting human γδ T lymphocytes by interleukin‐2
Authors: Kjeldsen‐Kragh Jens , Quayle Alison J. , Skålhegg BjøRn S. , Sioud Mouldy , Førre Øystein
Published: March 2007

In rheumatoid arthritis and other inflammatory diseases we and others have found that γδ T cells express activation antigens, suggesting that they are involved in the pathogenesis of these disorders. In this study we have stimulated peripheral blood mononuclear cells from normal donors with recombinant interleukin‐2 (rIL‐2) to see whether such a stimulus alone could activate γδ T cells. Short‐term exposure (24‐96 h) to rIL‐2 selectively stimulated the γδ but not the αβ T cells to express activation antigens (CD69, CD25 and HLA‐DR). Long‐term culture (2 weeks) in rIL‐2‐containing medium caused a selective increase in the proportion of the γδ T cells and a corresponding reduction of the fraction of αβ T cells. Limiting dilution analysis revealed that approximately 1/60 of the γδ T cells responded to IL‐2 in contrast to only 1/250 of the αβ T cells. Comparison of the expression of the IL‐2 receptor (IL‐2R) a and P chains showed that there was a similar expression of the α chain on γδ and αβ T cells whereas the relative density of the β chain was more than twice as high on γδ T cells. Both the IL‐2‐induced proliferation of γδ T cells and the expression of activation antigens on these cells could be inhibited by an anti‐IL‐2Rβ monoclonal antibody (mAb) but not by an anti‐IL‐2Rα mAb. Expression of CD69 on γδ T cells was dependent neither on the presence of B cells, monocytes, nor αβ T cells. Finally, we found that the IL‐2‐induced expression of CD69 was inhibited by activation of cAMP‐dependent protein kinase and by inhibition of the Src‐family of the tyrosine protein kinase, but not by inhibition of protein kinase C or by activation of the CD45 associated tyrosine phosphatase. The ability of γδ T cells to be activated by IL‐2 is a feature which they have in common with natural killer cells. Moreover, it may be possible that the expression of activation antigens on γδ T cells in inflammatory diseases is an epiphenomenon secondary to IL‐2 produced by activated αβ T cells.