bluuebunny/crossref_metadata_2025 huggingface dataset:
Inflammatory cerebrospinal fluid t cells have activation requirements characteristic of cd4+cd45ra‐ t cells
Authors: Chofflon Michel , González Victor , Weiner Howard L. , Hafler David A.
Published: March 2007

It has long been recognized that T cells in the cerebrospinal fluid (CSF) and other inflammatory compartments cannot be stimulated by mitogen and the reason for this has remained unknown. This question was investigated using mononuclear cells (MNC) isolated from the CSF of subjects with multiple sclerosis and other inflammatory brain diseases which predominantly express the CD4 and CDw29 but not CD45RA determinants. CSF and blood cells were stimulated by either the CD3/T cell receptor complex, the CD2 activation pathway, calcium ionophore, or an activator of protein kinase C, phorbol myristate acetate (PMA). CSF MNC proliferated less than blood MNC following stimulation by phytohemagglutinin in subjects with inflammation in the CSF, but not in subjects with non‐inflammatory CNS diseases. Moreover, CSF MNC were not induced to proliferate through stimulation of the CD2 pathway by anti‐T112 + anti‐T113 monoclonal antibodies (mAb). This was not due to defects in either interleukin 2 receptors, interleukin 2 secretion, or to T cell pre‐activation in vivo. Instead, the refractory activation state of inflammatory CSF T cells was corrected by PMA. That CSF contains predominantly CD4+CDw29+CD45RA‐ cells suggests that PMA may be co‐stimulatory with anti‐CD2 mAb to activate this population of T cells. This was confirmed in experiments with sorted T cells from normal subjects. These data suggest that the inability of mitogens or anti‐CD2 mAb to stimulate inflammatory CSF T cells, which can be corrected by an inducer of protein kinase C, is related to the relative absence of CD4+CD45RA+ cells in the CSF. Alterations of protein kinase C and protein phosphorylation may exist in inflammatory T cell populations that regulate the immune response.
Analysis of human t cell response to two plasmodium falciparum merozoite surface antigens
Authors: Rzepczyk Christine M. , Ramasamy Ranjan , Mutch David A. , Ho Paul C‐L. , Battistutta Diana , Anderson Karen L. , Parkinson David , Doran Trevor J. , Honeyman Margo
Published: March 2007

Eight novel human T cell epitopes were identified within the two major merozoite surface antigens (MSA1 and MSA2) of Plasmodium falciparum using synthetic peptides. All except one of the peptides conformed structurally to an amphipathic α helix and three out of the four MSA1 peptides also contained sequences containing the Rothbard motif. Peptide MSA2/2, which fitted none of these criteria, was recognized by our donors to a similar degree as the other peptides. This peptide also contains a B cell epitope. Proliferative responses were obtained in both immune and nonimmune donors, however, the number of responses in the immune donor group was significantly higher. There was no correlation between the level of proliferation and antibody titers to these antigens. No peptides were preferentially recognized in association with specific HLA class II antigens.
Activation of human t cells by toxic shock syndrome toxin‐1: the toxin‐binding structures expressed on human lymphoid cells acting as accessory cells are hla class ii molecules
Authors: Uchiyama Takehiko , Imanishi Ken'Ichi , Saito Shinji , Araake Minako , Yan Xiao‐Jie , Fujikawa Hiroshi , Igarashi Hideo , Kato Hidehito , Obata Fumiya , Kashiwagi Noboru , Inoko Hidetoshi
Published: March 2007

Toxic shock syndrome toxin‐1 (TSST‐1)‐binding assay using 125I‐labeled TSST‐1 showed the presence of specific TSST‐1 binding in a B cell fraction of human peripheral blood mononuclear cells and L cells transfected with DR2 genes or DR4 genes but not in a T cell fraction and control L cells. Fixation with paraformaldehyde, an inhibitor of antigen processing, did not remove TSST‐1‐binding activity of the transfectants. Binding of 125I‐labeled TSST‐1 to the transfectants was reduced by an anti‐DR monoclonal antibody. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed the presence of a single band with TSST‐1‐binding activity and the same migration pattern as DR heterodimers. TSST‐1‐induced T cell responses, proliferation and interleukin 2 (IL2) production were observed in the presence of the transfectants but not in the presence of control L cells, while concanavalin A‐induced IL2 production was observed in the presence of either the transfectants or control L cells. Presence of an anti‐DR monoclonal antibody inhibited the TSST‐1‐induced responses. Paraformaldehyde‐fixed Daudi cells were effective in supporting TSST‐1‐induced IL2 production by T cells. These results indicate that HLA class II molecules directly bind intact TSST‐1 and perform an essential role as the TSST‐1‐binding structures on accessory cells in T cell activation by the toxin.
A heat shock‐like protein from the human malaria parasite plasmodium falciparum induces autoantibodies
Authors: Mattei Denise , Scherf Artur , Bensaude Oliver , Silva Luiz Pereira Da
Published: March 2007

The humoral immune response to a 72‐kDa heat shock‐like protein of Plasmodium falciparum has been analyzed using mouse monoclonal antibodies (mAb) and human immune sera. Three regions of the molecule containing B cell epitopes were identified by screening a sublibrary encoding the COOH‐terminal half of the antigen with the mAb. One B cell epitope mapped to a region poorly conserved between the parasite 72‐kDa polypeptide and mammalian heat‐shock proteins (Hsp 70). Another mAb, G10C9, reacted with an amino acid region that has a high degree of homology with mouse (87.5%) and human (81.2%) Hsp 70. Both mouse and human cells were recognized by this mAb when analyzed by indirect immunofluorescence and by two‐dimensional immunoblots. Sera from humans infected with malaria also recognized the human Hsp 70. Thus, our results indicate that autoantibodies directed against host Hsp 70 can be induced by the homologous parasite protein.
T cell differentiation in athymic nude rats (rnu/rnu): demonstration of a distorted t cell subset structure by flow cytometry analysis
Authors: Schwinzer Reinhard , Hedrich Hans‐JüRgen , Wonigeit Kurt
Published: March 2007

The expression of T cell‐associated antigens was analyzed on extrathymically differentiated T cells from athymic nude (rnu/rnu) rats. Two‐color flow cytometry and monoclonal antibodies were used to compare the subset structure of the rnu/rnu T cell population with that of normal T cells. In adult nude rats CD4+ and CD8+ cells were found which co‐expressed the OX19 antigen (CD5) clearly defining them as T cells. Double‐positive (CD4+CD8+) cells were not detected in nylon wool‐enriched rnu/rnu T cells. The ratio of CD4+ to CD8+ cells in nude rat lymph node cells did not significantly differ from normal T cells. However, the composition of the nude rat CD4+ and CD8+ subsets [as identified by the subset‐dividing monoclonal antibodies OX22 and P4/16 (anti‐RT6)] differed from that found in control rats. The most striking observation among the rnu/rnu CD4+ subset was an inversion in the ratio of OX22+ to OX22‐ cells (0.5) compared to normal rats (2.3). Only 61% of the nude rat CD4+ cells were found to be RT6+ compared to 85% of rnu/+ CD4+ cells. In the rnu/rnu CD8 subset the proportion of RT6 co‐expressing cells was also markedly reduced (38% vs. 77% of the CD8+ subset). Furthermore, the nude rat CD8+ subset contained a substantial number of OX22‐ cells which were nearly absent in normal rats. The demonstration of cells bearing T cell markers in adult athymic rats further confirms the existence of an extrathymic pathway of T cell differentiation. The unusual T cell subset composition found in athymic rats, however, indicates that T cell subset generation and/or maintenance along this pathway differs from normal T cell development.
Human thymic dendritic cell‐thymocyte association: ultrastructural cell phenotype analysis
Authors: Landry Diane , Lafontaine Marielle , Barthélémy Hughes , Paquette Nicole , Chartrand Claude , Pelletier Micheline , Montplaisir Serge
Published: March 2007

In rodent thymus, associations between dendritic cells (DC) and thymocytes have been suggested to be implicated in differentiation and/or maturation processes. In this study, we report intimate associations formed between human thymic DC and thymocytes in culture and we analyze their ultrastructural cell phenotype. Observations by phase contrast microscopy showed that DC present long and thin dendrites and bind many thymocytes. Transmission (TEM) and scanning electron microscopy (SEM) revealed that both cellular populations were in close connection and tight membrane contact could be observed. The phenotype of DC and attached thymocytes was characterized with a series of monoclonal antibodies by protein A‐gold TEM and SEM immunolabelings. Quantitative evaluation of immunolabeling (number of gold granules/μm of cellular membrane) suggests the presence of two subpopulations of CD1+ thymic DC (strong and weak), whereas this discrepancy is not observed in DR+ and CD4+ DC populations. On the other hand, the majority of thymocytes bound to DC strongly express the CD1, CD4, CD8 and CD2 antigens and weakly the CD3 antigen, indicating that they represent double‐positive immature thymocytes. Uniform distribution of DC and thymocytes membrane antigens was confirmed with a backscattered SEM study. This morphological and immunolabeling TEM and SEM analysis demonstrates that human thymic DC may form associations with CD4+CD8+CD3weak thymocytes and raises questions about their physiological relationship.
Isolation of early immunoglobulin λ‐like gene transcripts in human fetal liver
Authors: Schiff Claudine , Milili Michèle , Fougereau Michel
Published: March 2007

In an attempt to identify early events of human Ig gene expression, we have screened a human fetal liver cDNA library (< 90 days of gestation) with Cμ‐, Cγ‐, Cχ‐, Cλ‐specific probes and we report the characterization of two clones, Fλ1 and Fλ8, that hybridized with a human Cλ gene. These two clones, which are only 85% homologous to the functional Cλ genes, were shown to be additional nonallelic members of the 14.1/16.1 Cλ‐like family. Using pulsed field gel electrophoresis these three Cλ‐like genes were shown to be present on a 200‐kb DNA fragment, defining a cluster distinct from that of the Cλ one. Fλ1 and Fλ8 contained an identical Cλ‐like region, and differed from each other by a splicing event which joins a Jλ‐like to the Cλ‐like exon in the Fλ1 clone in the absence of any rearrangement. Homologies observed between Fλ1 and the mouse λ5 gene suggest that this human clone may contain the exon 2 and 3 equivalents of λ5. Since λ5 is selectively expressed in pre‐B cells, our proposal is also supported by the early expression of this clone, together with the presence of full‐length μ and γ transcripts and the absence of functional Ig light chain transcripts. The presence of one nucleotide deletion in the C region of Fλ1 conferring it a pseudogene status, the actual λ5 equivalent might be either one of the 14.1 or 16.1 human Cλ‐like genes, the function of which is so far unknown.
Regulation of surface igm‐ and igd‐mediated inositol phosphate formation and ca2+ mobilization in murine b lymphocytes
Authors: Harnett Margaret M. , Holman Mary J. , Klaus Gerry G. B.
Published: March 2007

Cross‐linking of surface IgM or IgD receptors on B cells initiates a signaling cascade involving the activation of an (uncharacterized) G‐protein: this in turn activates a polyphosphoinositide‐specific phosphodiesterase (PPI‐PDE), thereby leading to the release of inositol phosphates. In order to investigate if the two isotypes of sIg share a common G‐protein, we stimulated B cells sequentially with anti‐μ and anti‐δ antibodies. Ligation of either class of receptor for 1 h led to the activation of the PPI‐PDE, which persisted for several hours. However, this was accompanied by inhibition of further stimulation of the enzyme via the heterologous receptors. This desensitization was shown to operate at the level of the coupling between G‐protein and the PPI‐PDE. These effects waned after 4–8 h of stimulation, when signaling via the heterologous receptors had essentially returned to normal. In addition, stimulation of B cells by anti‐μ and anti‐δ together did not elicit additive responses, either in terms of increases in inositol phosphate production, or in terms of increases in intracellular Ca2+ levels. Taken together, these results indicate that sIgM and IgD receptors share a common G‐protein and that signaling via these receptors is under both positive and negative feedback control. The mechanisms involved are unknown, but these effects may well be due to modulation of the activities of components of the signaling cascade by protein kinase C.
Major histocompatibility complex‐linked and t cell‐dependent selection of antibody repertoires. quantitation of i‐e‐related specificities in normal mice*
Authors: Rossi Claudia Peña , Pereira Pablo , Portnoï Denis , Coutinho António
Published: March 2007

Autoreactive B cell repertoires with major histocompatibility complex (MHC) class II (I‐E)‐related specificities were investigated by quantitating frequencies of specific B lymphocyte clonal precursors in unmanipulated normal and athymic BALB/c mice and in I‐E‐negative, MHC‐congenic BALB.B10 mice. Clonal culture supernatants containing anti‐I‐E antibodies were identified by their selective binding to I‐Ekα Edβ‐transfected fibroblasts, and those containing anti‐anti‐I‐E antibodies were detected by their selective binding to anti‐I‐E monoclonal antibodies. Analysis of splenic B lymphocytes from BALB/c mice revealed high frequencies of both specificities in the compartment of large, naturally activated cells, but not among small, resting lymphocytes. The selection of such clones was found to be MHC linked because of their absence in BALB.B10 mice, and T cell dependent because of their reduced frequency in athymic BALB/c mice. The positive selection of V regions representing complementarities and mimicries of self‐class II antigens may suggest a set of mechanisms participating in the maintenance of natural tolerance.
Syngeneic t cell transfer of diabetes into nod newborn mice: in situ studies of the autoimmune steps leading to insulin‐producing cell destruction
Authors: Bedossa Pierre , Bendelac Albert , Bach Jean‐François , Carnaud Claude
Published: March 2007

To overcome the limitations of in situ studies during the chronic spontaneous autoimmune process leading to insulin cell destruction and diabetes in non‐obese diabetic (NOD) mice, we have developed a model of acute transfer of diabetes into healthy syngeneic newborns. The injection of 20 × 106 T cells from adult diabetic mice produced synchronous insulitis within 3 weeks and diabetes within 4–5 weeks in young recipients, at a time when non‐injected control mice do not even exhibit histological changes in their pancreases. Sequential studies of pancreases from T cell‐transferred mice showed that lymphoid infiltration was preceded by a strong tissue expression of Ia antigen which was restricted to the vessel‐associated cells limiting the islet of Langerhans, and which might play a role in the recruitment of circulating T cells inside the islets. Acute destruction of most of the insulin‐producing cells, leading to diabetes, could take place within a few days after insulitis had begun. A majority of the inflammatory cells were T lymphocytes, ∼30% of which expressed interleukin 2 receptors. L3T4+ T cells largely predominated at the early phase of islet invasion whereas the proportion of Ly‐2+ T cells substantially increased later when β cell destruction occurred. In contrast, only a minority of B cells and macrophages participated to the inflammatory process. These data are in keeping with previous demonstrations that both T cell subsets contribute to the autoimmune disease. Furthermore, they suggest that β cell injury is mediated through a cytotoxic process, which requires the sequential involvement of L3T4+ (helper) and Ly‐2+ (cytotoxic) T cells.
Conformational analysis of t immunogenic peptides by circular dichroism spectroscopy
Authors: Abergel Chantal , Loret Erwann , Claverie Jean‐Michel
Published: March 2007

The structure of two T‐immunogenic peptides, one from the gag p24 protein of the human immunodeficiency virus, the other from the 11.1 gene product of Plasmodium falciparum, was studied by circular dichroism spectroscopy in various pH and solvent conditions. Although both sequences are predicted to adopt an alpha‐helical conformation and one of them is a repeat of a perfect alpha‐amphipathic sequence pattern, these two peptides exhibit a strong propensity to adopt an extended, turn or aperiodical conformation in solution.
Transcription of unrearranged t cell receptor ∂ genes in cd3‐ major histocompatibility complex‐unrestricted cytotoxic cells
Authors: Biassoni Roberto , Ferrini Silvano , Sekaly Rafick P. , Long Eric O.
Published: March 2007

Transcription of the T cell receptor (TcR) δ gene was detected in clones and in enriched populations of CD3‐ major histocompatibility complex‐unrestricted cytotoxic cells. The transcripts were derived, at least in part, from unrearranged TcR δ genes, as shown by hybridization to a synthetic oligonucleotide probe corresponding to germ‐line sequences just upstream of Jδ1. Transcripts from unrearranged TcR δ genes are similar in size to those from productively rearranged genes. The fact that the bulk of δ transcripts was derived from unrearranged genes was supported by the observation that TcR δ gene rearrangements were not detected in DNA isolated from an enriched CD3‐ cytotoxic cell population. Transcription of the TcR δ gene in CD3‐ cytotoxic cells is consistent with the notion that these cells belong to a T cell lineage.
Recruitment of alloreactive cytotoxic T lymphocytes by an antigenic peptide
Authors: Staerz Uwe D. , Zepp Fred , Schmid Rita , Hill Mark , Rothbard Jonathan
Published: March 2007

To investigate the requirements for induction of cytotoxic T lymphocytes (CTL) by peptides we chose the 16‐residue nucleoprotein peptide (NPP; 365–380) from the influenza virus A/NT/60/68 as model substrate that is recognized in conjunction with major histocompatibility complex H‐2d. Here we present that CTL can be raised from naive animals by repeated in vitro stimulation with high concentrations of peptide. The frequency of this response can be boosted by immunization of the animals with NPP‐conjugated to ovalbumin as a carrier. However, in contrast to NPP‐specific CTL lines raised from virus‐primed animals none of the peptide‐induced CTL lines were able to lyse virus‐infected targets. Although they did not show an apparent difference in fine specificity of the peptide recognized, their affinity to the target cells was 100‐fold lower than that of CTL from virus‐primed animals as estimated from the peptide concentration needed to achieve significant lysis. In addition, the activity of peptide‐induced CTL was very sensitive to blocking by anti‐CD8 antibodies as compared to virus‐specific CTL. Furthermore, all peptide‐induced CTL showed a high second reactivity for allogeneic H‐2k targets. Therefore, it is argued that high epitope density achieved by high peptide concentrations can in vitro recruit lymphocytes of another specificity. For the tested peptide the reactive T lymphocytes showed high alloreactivity.
Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells
Authors: Panina‐Bordignon Paola , Tan Agnes , Termijtelen Annemarie , Demotz Stefan , Corradin Giampietro , Lanzavecchia Antonio
Published: March 2007

To understand the effect of human MHC class II polymorphism on antigen recognition, we analyzed the memory T cell response to three tetanus toxin epitopes defined by three short synthetic peptides (p2, p4 and p30). We found that p2 and p30 are universally immunogenic, since they are recognized by all primed donors, irrespective of their MHC haplotypes. The analysis of specific clones indicates that both peptides are very promiscuous in their capacity to bind to class II. p30 can be recognized in association with DRw11(5), 7, 9 and with DPw2 and DPw4, while p2 can be recognized in association with DR1, DRw15(2), DRw18 (3), DR4Dw4, DRw11(5), DRw13(w6), DR7, DRw8, DR9, DRw52a and DRw52b. On the contrary, the third peptide, p4, can be recognized by only half of the donors in association with only DRw52a and DRw52c.

Analysis of truncated peptides shows that p30 contains three distinct epitopes, each recognized in association with different class II molecules. Therefore, the restriction specificity is already set at the level of the peptide‐MHC complex and, in all cases, T cells discriminate p30 bound to different class II molecules. On the contrary, p2 contains only one epitope, which is recognized in association with all DR molecules. In this case we found two different restriction patterns. Some clones are monogamous, since they recognize the peptide in association with one DR allele, while others are promiscuous, since they recognize the peptide in association with several different DR molecules. Thus, in this case, the restriction specificity is also set at the level of the T cell receptor.

We suggest that both the promiscuous binding of peptides and the promiscuous recognition by T cells are dependent on the particular structure of the DR molecules, having a monomorphic α chain associated with a polymorphic β chain.
Mast cell stimulation by monoclonal antibodies specific for the Fcϵ receptor yields distinct responses of arachidonic acid and leukotriene C4 secretion
Authors: Ortega Enrique , Hazan Bella , Zor Uriel , Pecht Israel
Published: March 2007

The release of arachidonic acid ([3H]AA) and leukotriene C4 (LTC4) from the rat mucosal mast cells of the RBL‐2H3 line stimulated by Fcϵ receptor‐specific monoclonal antibodies (mAb), by IgE and multivalent antigen, or by Ca2+ ionophores, was investigated. In parallel, secretion of the granular enzyme β‐hexosaminidase was also assayed. The release of [3H]AA and LTC4 in response to stimulation by three FcϵRI‐specific mAb shows similar quantitative differences to those observed for the secretion of granule‐stored mediators. The mAb F4 induced a substantial release of both [3H]AA and LTC4, which is as high as that induced by IgE and multivalent antigen. mAb J17 and H10 were found to induce an insignificant release of [3H]AA, but while J17 did induce release of LTC4, H10 failed to induce it, even though both J17 and H10 caused substantial release of β‐hexosaminidase. Ca2+ ionophores were found to be relatively more effective in inducing release of [3H]AA and LTC4 than in causing the secretion of granular mediators, as compared to cell stimulation by FcϵRI aggregation. These results illustrate that the cell responses of degranulation and de novo synthesis and release of mediators have different sensitivities to stimulation by (a) configurationally distinct FcϵRI dimers, (b) FcϵRI aggregates induced by IgE and multivalent antigen, and (c) Ca2+ ionophores.
Recombinant soluble receptors for the Fcγ portion inhibit antibody production in vitro
Authors: Varin Nadine , Sautès Catherine , Galinha Annie , Even Jos , Hogarth P. Mark , Fridman Wolf H.
Published: March 2007

The problem of the structural relationship between suppressive IgG‐binding factor and low‐affinity receptors for the Fc portion of IgG (FcγRII) has not yet been solved. In the present work we have isolated a recombinant soluble FcγRII containing only the two external domains of FcγRII, and analyzed its biochemical characteristics and biological activity. A cDNA encoding FcγRII was mutated by the creation of a stop codon at the Lys175 codon. L cells have been transfected with this cDNA inserted into an expression vector.

A cell line was obtained that secretes recombinant soluble FcγRII which reacts with a monoclonal anti‐FcγRII antibody and binds to IgG1, IgG2a and IgG2b murine isotypes but not to IgG3 or F(ab')2 fragments of IgG2a. The secreted molecule contains two molecular species of relative molecular mass (Mr) 44 000 and 34 000–38 000 and of pI 4.5 and 6.3. They correspond to different glycosylations of a single polypeptide of Mr 19 000. After purification to homogeneity, soluble FcγRII has found to suppress secondary and primary in vitro antibody responses in a dose‐dependent way. The present work shows that recombinant soluble FcγRII has biochemical characteristics, immunoreactivity and biological activity similar to those of suppressive IgG‐binding factor.
Heterogeneity in the ability of IgG1 monoclonal anti‐D to promote lymphocyte‐mediated red cell lysis
Authors: Kumpel Belinda M. , Leader Katherine A. , Merry Anthony H. , Hadley Andrew G. , Poole Geoffrey D. , Blancher Antoine , Goossens Dominique , Hughes‐Jones Nevin C. , Bradley Benjamin A.
Published: March 2007

Thirty‐four IgG anti‐D human monoclonal antibodies (mAb) derived from 18 donor were assessed for their ability to mediate lysis of D+ red cells by lymphocytes in antibody‐dependent cell‐mediated cytotoxicity assays. Cell‐bound antibody was quantified and the mAb were compared at similar levels of sensitization. The majority (23/31) of IgG1 and all (3/3) IgG3 mAb were ineffective; two donors produced both lytic and non‐lytic anti‐D mAb. Greater sensitivity was achieved using fluid‐phase antibody (as culture supernatants) in the assay than was obtained with pre‐sensitized red cells. Minimum levels of 2000 anti‐D molecules per cell were required for lysis using pre‐sensitized cells. Partial D red cells (DIVa, DVa and DVI) were lysed by three mAb that were lytic with normal D+ cells. There was no relationship between lytic ability and Gm allotype or D epitope specificity of the antibodies. Four mAb to other blood group specificities were tested: two (anti‐E and anti‐G) were lytic and two (anti‐c and anti‐Kell) were not lytic. Possible reasons for the heterogeneity of the lytic activity by the mAb are discussed.
Subsets of thymopoietic rat thymocytes defined by expression of the CD2 antigen and the MRC OX‐22 determinant of the leukocyte‐common an
Authors: Law Debbie A. , Spruyt Louise L. , Paterson David J. , Williams Alan F.
Published: March 2007

The MRC OX‐22 monoclonal antibody recognizes a restricted determinant of the rat leukocyte‐common antigen (L‐CA, CD45), which is expressed on most peripheral T cells and all B cells. In contrast only 2%–3% of thymocytes are OX‐22+ and these are now shown to be mostly of the immature CD4−CD8− (double‐negative, DN), phenotype with very few of the mature phenotype cells being OX‐22+. Analysis of immunoperoxidase sections suggests that the DN OX‐22+ cells are located in the cortex. Among the DN cells about 60% are OX‐22+ and a similar percentage are positive for CD2 antigen. Double staining showed that OX‐22+CD2−, OX‐22+CD2+, and OX‐22−CD2+ populations can be defined and that these three sets account for ∼95% of the DN cells. Measurement of the thymopoietic activity of DN subsets showed that OX‐22+CD2− and OX‐22+CD2+ cells have regenerative capacity whilst OX‐22−CD2+cells do not.
T cell receptor gene expression in sheep: differential usage of TcR 1 in the periphery
Authors: Hein Wayne R. , Dudler Lisbeth , Beya Marie‐FranÇOise , Marcuz Anne , Grossberger Dario
Published: March 2007

We have isolated cDNA clones for each chain of the sheep T cell receptor (TcR) and used these to analyze TcR gene expression in thymocyte and peripheral T cell subsets. Outer cortical thymocytes expressed a low level of mature message for all TcR chains suggesting that intrathymic precursors for the α/β and γ/δ lineages occur in this population. Inner cortical and medullary thymocytes expressed high levels of mature α/β transcripts, low levels of mature δ transcripts but no detectable γ message. Mature α/β transcripts were detected in peripheral CD4+ lymphocytes and these as well as CD8+ cells expressed a surface heterodimer of 85 kDa which resolved into 40‐ and 50‐kDa subunits after reduction. Peripheral CD4−CD8− lymphocytes, which in sheep are marked by the T19 antigen and may account for up to 60% of T cells in blood, expressed a surface heterodimer of 75 kDa. The T19+ cells had high levels of mature δ and truncated β transcripts in their cytoplasm but did not express the Cγ gene detected in DN thymocytes, although they seem to share Vγ and/or Jγ elements. Both forms of the sheep TcR are associated with CD3 molecules on the cell surface. These results show that (a) in contrast to the situation in rodents and humans, a large proportion of peripheral sheep lymphocytes use TcR γ/δ; (b) the proportion of T cells in the periphery which use TcR γ/δ is greater than in thymus; and (c) CD4−CD8− cells in thymus and periphery (T19+) use the same Cδ gene but appear to use different Cγ genes, suggesting that in sheep there may be more than one lineage of lymphocytes expressing TcR γ/γ.
Structure and expression of the T cell receptor gamma locus in pre‐B and early hemopoietic cells
Authors: Mccubrey James A. , Steelman Linda S. , Risser Rex G. , Mckearn John P.
Published: March 2007

The genetic structure and expression of the T cell receptor (TcR) loci were examined in pre‐B and early hemopoietic cells. Thirty‐eight percent of Abelson murine leukemia virus‐transformed pre‐B cell lines were rearranged at TcRγ. Moreover, many pre‐B cell lines were rearranged at two distinct γ loci, Vγ1.2 and Vγ2. The γ rearrangements in the pre‐B cell lines were similar to those observed previously in T cell lines. V + C‐containing γ mRNA was detected in two pre‐B cell lines. In all other pre‐B and interleukin (IL) 3‐dependent lymphoid and myeloid lines examined, smaller Cγ‐containing mRNA were detected. These Cγ transcripts were independent of the genetic configuration of the γ locus. In contrast, the TcR α and β loci were in the germ‐line configuration in all non‐T cell lines examined and mRNA encoding these loci were not detected. When IL 3‐dependent lymphoid and myeloid cell lines were transformed to growth factor independence by a non‐autocrine mechanism, no mRNA transcripts encoding TcR Cγ were detected. However, TcR Cγ mRNA transcripts were detected in factor‐independent cell lines that arose by an autocrine mechanism. The cell cycle expression of Cγ was compared with protooncogenes and other marker genes previously shown to be cell cycle specific. mRNA transcripts encoding Cγ were detected in the highest amounts 4–8 h after IL 3, but not phorbol myristate acetate, addition. A similar time period of expression was observed with ornithine decarboxylase which has been shown to be expressed in G1 phase. These observations indicate that TcRγ is often rearranged in pre‐B cell lines and may be directly regulated by IL 3 in IL 3‐dependent cells.
In vitro production of IL 1β, IL 1α, TNF and IL 2 in healthy subjects: distribution, effect of cyclooxygenase inhibition and evidence of independent gene regulation
Authors: Endres Stefan , Cannon Joseph G. , Ghorbani Reza , Dempsey Roy A. , Sisson Stephen D. , Lonnemann Gerhard , Meer Jos W. M. Van Der , Wolff Sheldon M. , Dinarello Charles A.
Published: March 2007

Numerous studies have reported altered in vitro cytokine production in various diseases. In the present study we used specific immunoassays to quantitate production of interleukin 1β (IL 1β), IL 1α, tumor necrosis factor (TNF) and IL 2 from human peripheral blood mononuclear cells (PBMC). The distribution of cell‐associated and secreted cytokines was studied in PBMC of 21 individuals; in response to lipopolysaccharide (LPS) the proportion of cell‐associated IL 1β ranged from 13% to 56%, for IL 1α 29% to 98%, and for TNF 2% to 17%. In a larger cohort of 32 subjects, the total amount of immunoreactive cytokines produced in response to LPS or phytohemagglutinin was normally distributed within the study group. Mean production of IL 1α in response to LPS was 10.1 ng/ml and exceeded production of IL 1β (5.6 ng/ml) and TNF (2.2 ng/ml). The distribution pattern was characterized by high intersubject variability extending over two orders of magnitude and the presence of high and low “producers”. Production of IL 1α and IL 1β correlated (R = 0.69). In contrast, production of IL 1β did not correlate with production of TNF or IL 2. Indomethacin present during stimulation of PBMC increased the amount of IL 1β produced and showed a high correlation (R = 0.83) compared to cultures without indomethacin. Thus, low production of IL 1β in certain subjects appears not to be due to inhibitable levels of cyclooxygenase products. In a retrospective study, PBMC from 12 subjects who had taken oral cyclooxygenase inhibitors during the preceding 7 days produced 43% more IL 1β than subjects who did not take these drugs (p < 0.05). These studies demonstrate that the amount of cytokine synthesized by PBMC (a) is regulated independently for IL 1, TNF and IL 2; (b) correlates for IL 1β and IL 1α; (c) is intrinsic for low and high “producers”, and (d) production of IL 1β increases with the use of oral cyclooxygenase inhibitors.
The α3 domain of major histocompatibility complex class I molecules plays a critical role in cytotoxic T lymphocyte stimulation
Authors: Samberg Nancy L. , Scarlett Elizabeth C. , Stauss Hans J.
Published: March 2007

Allogeneic major histocompatibility complex class I molecules induce strong cytotoxic T lymphocyte (CTL) responses whereas xenogeneic molecules do not. We have tested a series of mouse/human hybrid molecules for their ability to stimulate mouse CTL. The molecules with murine α3 domains consistently stimulated stronger CTL responses than those with human α3 domains, independent of the species origin of the N‐terminal α1 or α2 domains. We have found that the ability of class I molecules to induce strong cytotoxic responses correlates positively with their ability to stimulate expansion of the CD8+CD4− T cell subset. The results indicate that mouse T cells can recognize class I molecules with human α1 and/or α2 domains, but for efficient stimulation of these T cells it is important that the immunizing molecule contains a murine α3 domain. We suggest that T cell priming requires an efficient interaction of CD8 with the class I α3 domain, and this shows some species restriction.
An antigenic peptide of the HIV‐1 NEF protein recognized by cytotoxic T lymphocytes of seropositive individuals in association with different HLA‐B
Authors: Culmann BÉAtrice , Gomard Elisabeth , Kiény Marie‐Paule , Guy Bruno , Dreyfus FrançOis , Saimot Adrien‐GÉRard , Sereni Daniel , Lévy Jean‐Paul
Published: March 2007

By using target cells that expressed only the regulatory NEF protein of the human immunodeficiency virus (HIV), we were able to identify specific cytolytic T cells (CTL) from most of the seropositive patients tested. This NEF‐specific cytolytic activity was mediated by CD8+ lymphocytes. In two different donors, we identified a unique peptide in the NEF protein that could be recognized in association with two different HLA class I molecules. The definition of such a peptide should prove useful in evaluating vaccine strategies.
Murine macrophage precursor characterization. I. Production, phenotype and differentiation of macrophage precursor hybrids
Authors: Leenen Pieter J. M. , Slieker Walentina A. T. , Melis Marleen , van Ewijk Willem
Published: March 2007

This study reports on the earliest stages of mononuclear phagocyte differentiation. A crucial question in this developmental process is whether mature macrophage (Mϕ) heterogeneity is already appointed at the precursor cell level. For this purpose, we produced clonal populations of mononuclear phagocytes from bone marrow culture by somatic cell hybridization with two hypoxanthine, aminopterin, thymidine‐sensitive myeloid cell lines. A panel of 22 stable hybrids was obtained from these fusions. Differentiation stage analysis of the hybrids indicated that all cell lines had immature mononuclear phagocyte characteristics. The hybrids exhibited typical myeloid morphology and mainly nonadherent growth. Mature Mϕ features, such as expression of the cell surface antigens Mac‐1, Mac‐2 and F4/80, phagocytosis of latex beads, and expression of nonspecific esterase and acid phosphatase activity, were virtually absent. The immature Mϕ markers Thy‐1, MIV 25 and MIV 52, on the other hand, were readily expressed, although heterogeneity was observed among different hybrid cell lines.

We then analyzed the differentiation potential of seven hybrids by culture of the cells in the presence of post‐lipopolysaccharide serum supplemented with interferon‐γ and found that the expression of mature Mϕ characteristics was induced. However, the various hybrids showed divergent patterns of mature Mϕ marker induction. ROC2 cells, for instance, showed extensive morphological and phenotypical differentiation without concomitant induction of phagocytosis. In contrast, W1C4 cells showed significant induction of phagocytosis without simultaneous increase of phosphatase and esterase activity. R1C1 cells were unique in the strong induction of Ia antigen expression.

Together, our data indicate that (a) early Mϕ differentiation stages can be rescued by somatic cell hybridization, and that (b) the obtained cell lines are able to mature according to divergent differentiation programs.
Murine macrophage precursor characterization. II. Monoclonal antibodies against macrophage precursor antigens
Authors: Leenen Pieter J. M. , Melis Marleen , Slieker Walentina A. T. , van Ewijk Willem
Published: March 2007

The aim of the present study was the phenotypical analysis of early stages in macrophage (Mϕ) differentiation. For this purpose, we produced a panel of syngeneic rat hybridomas, which secreted antibodies (mAb) against Mϕ precursor antigens. As immunogens we used immortalized Mϕ precursors (P. J. M. Leenen et al., Eur. J. Immunol. 1990. 20: 15). We screened the obtained mAb in the following in vitro models of Mϕ differentiation: (a) a panel of Mϕ cell lines ordered in a linear differentiated sequence; (b) immature and mature mononuclear phagocytes obtained from bone marrow (BM) culture; (c) a panel of Mϕ precursor hybrids, and (d) differentiated and control Mϕ precursor hybrid cells.

Four mAb, ER‐MP12, ER‐MP20, ER‐MP54 and ER‐MP58, were selected. These mAb recognize antigens which disappear in the course of Mϕ differentiation. Next, we investigated whether these mAb also recognized Mϕ precursors in normal BM. For this purpose, ER‐MP‐positive and ‐negative BM fractions were isolated using a fluorescence‐activated cell sorter. Fractions were cultured in Mϕ‐colony‐stimulating factor‐containing conditioned medium, and the resulting mature Mϕ progeny was quantified using the MTT assay. The present experiments indicate that ER‐MP12 and ER‐MP20 detect a subpopulation of BM Mϕ precursors, whereas ER‐MP58 stains virtually all Mϕ precursors.

Biochemical analysis of radioiodinated antigens revealed that these mAb recognize different molecules. ER‐MP12 and ER‐MP20 bound to single‐chain (glyco)proteins of 140 kDa and 14 kDa, respectively. ER‐MP54 precipitated multiple polypeptides, of which the major chains have an apparent molecular mass of 90, 80‐85 and 70‐75 kDa. Based on the molecular mass of the recognized antigens and the mAb specificities we conclude that ER‐MP12, ER‐MP54 and ER‐MP58 recognize hitherto unknown antigens of murine Mϕ precursor cells. The antigen detected by ER‐MP20 is most likely identical to Ly‐6C.
Selective elimination of double‐positive immature thymocytes by a thymic epithelial cell line
Authors: Nakashima Manabu , Mori Keiko , Maeda Kenichi , Kishi Hiroyuki , Hirata Kazuho , Kawabuchi Masaru , Watanabe Takeshi
Published: March 2007

A cloned epithelial cell line, TEL‐2, has been established from the stroma tissues of normal mouse thymus. Incubation of mouse thymocytes on TEL‐2 cells resulted in the selective elimination of double‐positive (CD4+CD8+ cells from the culture, whereas single‐positive (CD4+ CD8− or CD4−CD8) thymocytes remaining in the culture were concentrated in non‐integrated cell population. The CD3− or CD3 low‐positive thymocytes were also eliminated by the TEL‐2 cells from the culture, followed by the concentration of CD3 high‐positive cells in the culture. Only intact viable thymocytes were integrated into TEL‐2 cells. Electron microscopic examination showed that the integrated cells into TEL‐2 cytoplasm were gradually degenerated. Mature single‐positive T cells, mature B cells or double‐negative thymocytes were not integrated into TEL‐2 cells. The TEL‐2 cell may provide information on the mechanism of selective disappearance of double‐positive immature cells from the thymus.
Activation of bone marrow‐derived mouse macrophages by bacterial lipopeptide: cytokine production, phagocytosis and Ia expression
Authors: Hauschildt Sunna , Hoffmann Petra , Beuscher H. Ulrich , Dufhues Gabriele , Heinrich Peter , Wiesmüller Karl‐Heinz , Jung Günther , Bessler Wolfgang G.
Published: March 2007

The lipopeptide N‐palmitoyl‐S‐(2,3‐bis(palmitoyloxy)‐(2RS)‐propyl)‐(R)‐cysteinyl‐alanyl‐glycine (Pam3Cys‐Ala‐Gly), a synthetic analogue of the N‐terminal part of bacterial lipoprotein, induces the secretion of interleukin (IL) 1, IL 6 and tumor necrosis factor (TNF)‐α in bone marrow‐derived macrophages that have been cultured in vitro for up to 20 days. IL 6 and TNF‐α secretion increased from day 6 to day 20 whereas IL 1 secretion increased until day 13 and decreased on day 20. In contrast to the enhancement of cytokine production, phagocytosis of IgG‐coated sheep erythrocytes and Ia expression were found to be diminished after treatment with lipopeptide for 24 h. Morphological studies revealed lipopeptide‐induced changes of macrophage cultures. The data presented here show the potential of the lipopeptide as a strong activator of bone marrow‐derived macrophages.
Specificity and cross‐reactive idiotypes of anti‐glomerular basement membrane autoantibodies in HgCl2‐induced autoimmune glomerulonephritis
Authors: Guéry Jean‐Charles , Druet Elvira , Glotz Denis , Hirsch François , Mandet Chantal , De Heer Emile , Druet Phillippe
Published: March 2007

Murcury‐induced autoimmune glomerulonephritis in the Brown‐Norway (BN) rat is characterized by the successive appearance of linear and granular glomerular IgG deposits. Anti‐laminin autoantibodies represent the major part of the anti‐glomerular basement membrane (GBM) antibodies produced in this model. Fusions were performed in this model and four anti‐GBM monoclonal antibodies (mAb) were obtained. Three of them were laminin specific. Using rabbit anti‐idiotype antibodies, cross‐reactive idiotypes (CRId) were characterized on anti‐laminin antibodies. They were expressed on the three anti‐laminin mAb, on kidney‐eluted and circulating anti‐laminin antibodies. CRId‐bearing immunoglobulins were detected transiently in the circulation and paralleled the anti‐laminin antibody activity. By immunofluorescence studies on kidney cryostat sections two different CRId were defined. One was localized close to the antigen‐combining site since it was not revealed on kidney‐bound antibodies, in contrast with the second CRId. This latter CRId was also found deposited in a typical linear pattern in the early phase of the disease and in a granular pattern in the late phase, demonstrating that these CRId are components of immune deposits. Taken together, these results suggest that in this model of T‐dependent polyclonal B cell activation, restricted sets of V genes encode for at least a part of the anti‐GBM autoantibodies.
Rat anti‐glomerular basement membrane antibodies in toxin‐induced autoimmunity and in chronic graft‐vs.‐host reaction share recurrent idiotypes
Authors: Guéry Jean‐Charles , Tournade Hervé , Pelletier Lucette , Druet Elvira , Druet Philippe
Published: March 2007

Cross‐reactive idiotypes (CRId) borne on autoanti‐glomerular basement membrane antibodies of Brown‐Norway (BN) rats with mercury‐induced glomerulonephritis have been described in the preceding study (Guéry, J.‐C. et al., Eur. J. Immunol. 1990. 20: 93). BN rats treated with sodium aurothiopropanol sulfonate or D‐penicillamine, as well as (LEW×BN)F1 hybrids transferred with BN rat spleen cells, developed quite similar autoimmune abnormalities. In the present study, it is shown that immunoglobulins bearing such “public” idiotypes are also produced and deposited in the kidney in these three models. The CRId here described may, therefore, be considered as a marker of sets of recurrently expressed V region genes during the course of these autoimmune disorders. Anti‐self class II T cells are present in the three models of toxin‐induced autoimmunity and anti‐allo class II T cells are responsible for the chronic graft‐vs.‐host reaction. The same B cell clones are probably triggered during these processes as a consequence of a polyclonal B cell activation mediated by anti‐class II T cells.
Specific high‐affinity receptors for interferon‐γ on mouse bone marrow‐derived mast cells: inhibitory effect of interferon‐γ on mast cell precursors
Authors: Nafziger Joëlle , Arock Michel , Guillosson Jean‐Jacques , Wietzerbin Juana
Published: March 2007

Cultured mast cells derived from murine bone marrow were investigated for the presence of specific interferon‐γ (IFN‐γ) receptors, and for the effects of IFN‐γ on mast cell proliferation. 125I‐labeled recombinant IFN‐γ (125I‐Mu‐rIFN‐γ) was shown to bind to high‐affinity receptors on these cells. Scatchard analysis of binding data indicated the presence of about 500 homogeneous binding sites per cell, with an apparent equilibrium dissociation constant of 3×10−10 M. The binding of 125I‐Mu‐rIFN‐γ to mast cells was inhibited by unlabeled Mu‐rIFN‐γ but not by unlabeled Mu‐IFN‐α/β. Cross‐linking of 125I‐Mu‐rIFN‐γ to mast cell membrane proteins using a cross‐linking agent yielded a predominant complex of 100 ± 10 kDa on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and autoradiography which IFN‐γ‐receptor complex. To assess the biological significance of these receptors, we studied the effects of Mu‐rIFN‐γ on mast cell proliferation, which was markedly inhibited in mast cell precursors but not in mature mast cells. These in vitro results are in agreement with the antiproliferative effect of IFN‐γ previously reported for other hematopoietic progenitors, and suggest that IFN‐γ could find its application in the treatment of human systemic mastocytosis.
Characterization of rat tissue cultured mast cells
Authors: Chan Bosco M. C. , Ra Chisei , Hu Weining , McNeill Karol , Miller Hugh R. P. , Befus Dean , Kinet Jean Pierre , Froese Arnold
Published: March 2007

Twelve continuous rat tissue cultured mast cell (MC) lines were established by prolonged culture of rat peritoneal MC in the absence of added factors or feeder layers. Two of these lines, RCMC1 and RCMC2, have been briefly described previously, seven others are now also described. Both RCMC1 and RCMC2 lack a marker chromosomes present on RBL‐CA10.7 cells. All lines were found to express the phenotype of mucosal MC as defined by alcian blue‐positive and safranin 0‐negative staining, the presence of rat MC protease II and a low histamine content. When analyzed for high‐(FcϵRI) and low‐affinity (FcϵRL) receptors for IgE, the various lines yielded a variety of receptor patterns. Northern blot analysis of the RNA of RCMC1, RCMC2 and RBL‐CA10.7 revealed that all three cell lines contained the same mRNA species for the α, β and γ subunits for FcϵRI previously found in another rat basophilic leukemia cell line. Quantitation of the relative amounts of α, β and γ mRNA did not correlate with the expression of the relative amounts of FcϵRI(α) in these cells. The relative amounts of mRNA for all these subunits of RCMC2 were equal or higher than those of RCMC1, suggesting that the low expression of FcϵRI(α) on the former was a consequence of post‐transcriptional events. Analysis of a RCMC1 clone over a 6‐month period revealed changes in the expression of both FcϵRI(α) and FcϵRL.