bluuebunny/crossref_metadata_2025 huggingface dataset:
Murine thymocytes possess specific cell surface‐associated exoaminopeptidase activities: Preferential expression by immature CD4−CD8− subpopulation
Authors: Bauvois Brigitte
Published: March 2007

Murine thymocytes are shown to possess at least three well‐defined exo‐N‐aminopeptidase activities on their surface. One of them cleaves the prolyl bond in the synthetic dipeptide nitroanilide Gly‐Pro‐pNA (Km 0.95 mM and Vmax 8 nmol/h at pH 7.4 and 37°C) and is specifically inhibited by phenylmethane sulfonyl fluoride, diprotin A, Gly‐Pro‐Ala and Gly‐Pro‐Gly‐Gly. These data further support identification of this enzyme with a serine exopeptidase dipeptidyl peptidase IV (DPP IV), previously reported to be specific for collagen. The two other forms of N‐exopeptidase activities are detected when Ala‐pNA and Leu‐pNA are used as substrates. Leu‐aminopeptidase activity (Km 1.4 mM, Vmax 15 nmo/h) and Ala‐aminopeptidase activity (Km 4.0 mM, Vmax 20 nmo/h) are inhibited by inhibitors for thiol‐ and trypsin‐like proteinases, i.e. tosyl lysyl chloromethyl ketone, leupeptin and N‐ethylmaleimide. Addition inhibition of Leu‐aminopeptidase activity by pepstatin, a known inhibitor of carboxyl proteases, suggests that aminopeptidase activity detected with Leu‐pNA is different in part from Ala‐aminopeptidase activity. Among the various lymphoid cell populations tested, the three aminopeptidase activities are increased by three‐ to fourfold in the immature CD4−CD8− thymocyte subset as well as in the thymoma BW5147. In contrast, cortisone‐resistant thymocytes, lymph node and spleen cells exhibit levels of activities almost similar to that of unfractionated thymocytes. During ontogeny, the levels of these activities are increased four‐ to sevenfold on fetal thymocytes (from days 14 to 16). Finally, when thymocytes or spleen cells are cultured with a mitogenic concentration of concanavalin A, their proliferative responses are correlated with an enhancement of the aminopeptidase activities (1.3‐ to 5‐fold). From these results, a correlation between the presence of these peptidases on the cell surface of immature and mature lymphoid cells and biological responsivenesses is suggested.
Specific deletion of the J‐Cδ locus in murine α/β T cell clones and studies using transgenic mice
Authors: Ohashi Pamela S. , Wallace Valerie A. , Broughton Heather , Ohashi Cara T. , Ferrick David A. , Jost Veronica , Mak Tak W. , Hengartner Hans , Pircher Hanspeter
Published: March 2007

A deletion event in the T cell receptor (TcR) δ locus has been characterized in a panel of mouse α/β cytotoxic T lymphocyte (CTL) clones. Data presented here shows that Jδ1, Jδ2 and Cδ are absent from functional CTL clones while a germ‐line Dδ1 fragment is retained, thus suggesting a specific deletion of this region.

We have investigated the possible significance of the J‐Cδ deletion by generating T cell lines from TcR α/β transgenic mice. Unlike control T cell lines which included a T cell line derived from a β transgenic mouse, the lines expressing the transgenic α/β heterodimer have not deleted the Cδ region. This strongly suggests that the J‐Cδ deletion event is not responsible for directing T cells to the α/β lineage, but rather is involved in the rearrangement or transcriptional activity of the α locus. In addition, to ensure that the α/β transgene does not have any inhibitory affects on the rearrangement of the δ loci in general, the γ/δ expressing dendritic epithelial Tcell (DETC) population was examined in TcR α/β transgenic mice and alterations in this T cell subset were not found. This finding that normal γ/δ DETC cells are present in α/β transgenic mice, together with the data showing that the Dδ1 region remains in an unrearranged germ‐line configuration in functional α/β CTL, suggests that commitment to the α/β or γ/δ lineage is predetermined at a particular stage in early T cell ontogeny.
Intact, 45‐kDa (membrane) form of CD23 is consistently mitogenic for normal and transformed B lymphoblasts
Authors: Cairns Jennifer A. , Gordon John
Published: March 2007

When isolated from lysates of the RPMI 8866 B lymphoblastoid cell line, the CD23 antigen was found to be present be in two forms corresponding to the intact 45‐kDa membrane species and to a 25‐kDa product that is more usually found as a released fragment in the extracellular medium. By contrast with preparations of extracellular species of CD23 which were seen to be variable in their biological activity, cell‐associated CD23 was consistently mitogenic both for autogenous transformed B lymphoblasts and for pre‐activated normal B cells. Addition to the normal cocktail of protease inhibitors of Nα‐tosyl‐L‐lysine chloromethylketone (TLCK), which has selectivity for trypsin‐like serine proteases, resulted in preparations of CD23 from RPMI 8866 cell lysates that were exclusively in the 45‐kDa intact form; such material retained full and reliable activity in the biological assays. The implications of these observations for the autocrine control of B lymphocyte growth are discussed.
Interleukin 4 receptors on normal human B lymphocytes: Characterization and regulation
Authors: Zuber Caroline E. , Galizzi Jean‐Pierre , Vallé Alain , Howard Nobuyuki , Howard Maureen , Banchereau Jacques
Published: March 2007

Human interleukin 4 (IL 4) up‐regulates the expression of CD23 on both resting and “in vivo” activated B cells but induces proliferation and/or differentiation only on “in vitro” activated B lymphocytes. Resting B cells express 360 high‐affinity IL 4 receptors (R) per cell (Kd = 25–75 pM). Activation of resting B cells with anti‐IgM antibody or Staphylococcus aureus Cowan I (SAC) results in a two‐to‐threefold increase of IL 4R number without alteration of IL 4R affinity for IL 4. Flow cytometric analysis of biotinylated IL 4 binding shows that IL 4R expression is up‐regulated on virtually all anti‐IgM‐stimulated B cells, but only on a subpopulation of larger cells among SAC‐activated B lymphocytes. Culturing cells for 40 h with optimal concentrations of IL 4 does not significantly affect IL 4R levels on resting and anti‐IgM‐preactivated B lymphocytes but triples IL 4R levels on SAC‐preactivated B cells. Removal of IL 4 from cell cultures results in a two‐to‐fourfold increase of IL 4R levels 2 h later, suggesting an increase in IL 4R turnover. Resting and activated B cells degrade 1251‐labeled IL 4 at 37 °C. Sodium dodecyl sulfate‐polyacrylamide gel electrophoretic analysis of IL 4 binding molecules on resting, “in vivo” activated and anti‐IgM‐activated B cells reveals the same three species of 130, 80‐75, 70‐65 kDa. Thus, IL 4 displays its different biological activities on resting and activated B cells through IL 4R of the same affinity, gross biochemical structure and ability to mediate IL 4 degradation.
Fine manipulation of antibody affinity for synthetic epitopes by altering peptide structure: Antibody binding to looped peptides
Authors: Jemmerson Ronald , Hutchinson Robert M.
Published: March 2007

Linear peptides weakly imitate antibody binding sites on globular proteins when the peptides are shown to be effective at all. As a step toward enhancing the ability of peptides to mimic epitopes, we have examined the effects of various alterations in peptide structure on antibody binding. Synthetic peptides containing the core amino acid sequence of residues 41 to 48 from horse cytochrome c were examined for their ability to bind antibodies elicited against the 41–48 peptide coupled to bovine serum albumin (BSA). Since residues 41–48 in native cytochrome c are part of an omega loop, in some peptides cysteines were incorporated for intrachain disulfide bonding to stabilize loop structure. In additional cases, glycine was incorporated as a spacer between the natural sequence and the cysteine residues with the intent of relaxing loop structure slightly. Eleven analogues containing the 41–48 sequence were tested. These included native cytochrome c and the 1‐80 and 1‐65 cyanogen bromide‐cleaved fragments. The native protein did not bind the anti‐41–48 antibodies. The other analogues differed by over three orders of magnitude in their binding. The affinity of binding was inversely related to the extent of predicted loop structure indicating that the antibodies were elicited against the 41–48 sequence in a more unfolded conformation despite the Pro Gly sequence at positions 44 and 45 that generally favors a β turn. Surprisingly, the immunizing peptide, containing residues 41–48 only, was the poorest binding peptide. The relative impotence of 41–48 was shown to be largely due to differences at the amino terminus between the free and BSA‐coupled peptides as the antibodies were elicited against the latter. The distinctions among the synthetic peptides containing the 41–48 sequence show the exquisite sensitivity of antibody binding to amino acid changes that may occur outside of an epitope and suggest modifications in peptide structure at the periphery of an epitope that can lead to desired changes in antibody affinity.
Type 6 and 19 pneumococcal polysaccharides coupled to erythrocytes elicit pneumococcal cell wall‐specific primary IgM responses and capsular polysaccharide‐specific secondary IgG responses
Authors: Milligan Gregg N. , Fairchild Robert L. , Sterner Kay E. , Braley‐Mullen Helen
Published: March 2007

Previous results have shown that the primary murine antibody responses to vaccine preparations of type 6 (S6; Danish type 6A) or type 19 (S19; Danish type 19F) pneumococcal capsular polysaccharides consist entirely of IgM antipneumococcal cell wall carbohydrate (PnC)‐specific antibodies. No capsular polysaccharide‐specific IgM antibodies were detectable by plaque‐forming cell or enzyme‐linked immunosorbent assay techniques. In this report, antibodies specific for S6 and S19 capsular polysaccharides were induced in secondary responses to chicken erythrocyte (CRBC) conjugates of S6 and S19. Essentially all detectable IgG produced in the secondary response was capsular polysaccharide specific and included all subclasses of IgG. In contrast, all detectable IgM produced in the primary response to S6‐CRBC and S19‐CRBC, and the IgM produced in the secondary response to S6‐CRBC was not capsular polysaccharide specific since it reacted with PnC. Thus, there is a major change in the specificity of the primary IgM response compared to the secondary IgG response of mice immunized with S6‐CRBC or S19‐CRBC. Injection of PnC or any PnC‐containing polysaccharide prior to immunization with S6‐CRBC or S19‐CRBC resulted in suppression of the primary IgM response. In contrast, only the capsular polysaccharide used in the immunizing polysaccharide‐erythrocyte conjugate suppressed induction of the capsular polysaccharide‐specific secondary IgG response. These results suggest that S6 and S19 capsular polysaccharidespecific IgG‐producing memory B cells derive from capsular polysaccharidespecific precursors which do not produce detectable antibody after primary immunization.
Synergy of tumor necrosis factor with protein kinase C activators on T cell activation
Authors: Angeles Muñoz‐Fernández M. , Pimentel‐Muiños Felipe X. , Alonso Miguel A. , Campanero Miguel , Sánchez‐Madrid Francisco , Silva August , Alonso José Luis , Fresno Manuel
Published: March 2007

The ability of tumor necrosis factor (TNF)‐α to activate T lymphocytes in combination with other stimuli has been studied. TNF was strongly co‐mitogenic with low doses of anti‐CD3 antibodies or phorbol esters (those which are strong activators of protein kinase C, PKC) but poorly with phytohemagglutinin or concanavalin A. No synergism was seen with the calcium ionophore A23187. TNF was co‐mitogenic with several phorbol esters known to activate PKC but was uneffective with inactive phorbol esters such as methyl‐phorbol 12‐myristate 13‐acetate. Furthermore, H‐7 a known inhibitor of PKC, inhibited the proliferative response of T cells induced by esters plus TNF. This effect took place at low doses of TNF and was also observed with purified T lymphocytes indicating that the effect of TNF was not dependent on accessory cells. This proliferative effect of TNF was inhibited by an anti‐interleukin 2 receptor (IL2R) antibody, MAR 108, which blocks IL 2 binding to its receptor. Although PKC activation induced CD25 (IL 2R) expression but very little IL 2 synthesis, TNF did not synergize by augmenting the synthesis of this lymphokine in peripheral blood lymphocytes stimulated with phorbol esters. By contrast, TNF strongly increased the membrane level of CD25 and to a lesser extent that of the activation antigen, 4F2, over the levels already induced by phorbol esters on T cells. More interestingly, TNF significantly increased the number of high‐affinity IL 2R on purified T cells in the presence of phorbol 12,13‐dibutyrate.

Our results indicate that TNF is co‐mitogenic with those stimuli which strongly activate PKC and suggest that TNF may play a role on T cell activation increasing the number of effective IL 2/IL 2R interactions when these are limiting.
Influence of adenosine deaminase inhibition on the phosphoinositide turnover in the initial stages of human T cell activation
Authors: Buc Hélène A. , Moncion Arlette , Hamet Michèle , Houllier Anne‐Marie , Thuilier Laure , Perignon Jean‐Luis
Published: March 2007

An experimental model of adenosine deaminase deficiency was established on the human T cell line Jurkat by using 2'‐deoxycoformycin, a strong specific inhibitor of the enzyme. When deoxyadenosine was added to the inhibited cells, the nucleotide profile was modified reproducing that found in lymphocytes from adenosine deaminase‐deficient children. The metabolism of phosphoinositides, analyzed by either the release of [3H]inositol phosphates or the breakdown of 32P‐prelabeled phosphatidyl inositides, was compared in normal and modified cells where dATP was accumulated. No modification in 32P labeling of phosphoinositides was detectable within the 32P‐loading period. However, when the cells were stimulated by phytohemagglutinin or anti‐CD3 monoclonal antibody, the phosphoinositide hydrolysis was strongly reduced in the dATP‐containing lymphoblasts. This decrease was correlated with the intracellular dATP concentration.
Identification and immunogenic properties of an 80‐kDa surface antigen on a drug‐treated tumor variant: Relationship to MuLV gp70
Authors: Grohmann Ursula , Ullrich Stephen J. , Mage Michael G. , Appella Ettore , Fioretti Maria C. , Puccetti Paolo , Romani Luigina
Published: March 2007

Highly immunogenic tumor variants are generated by in vitro or in vivo treatment of L5178Y murine lymphoma cells with triazene derivatives. Most of these variants express new transplantation antigens which are not present on the original L5178Y tumor cells. In this study, a polyclonal syngeneic antiserum raised to one such variant (L5178Y/DTIC) was employed in immunoprecipitation studies of cell surface and metabolically labeled L5178Y/DTIC cells. One‐ and two‐dimensional electrophoretic analyses of the immunoprecipitates detected a surface antigen of approximately 80 kDa. Additionally, a 45‐kDa component was detected in the lysate of [35S]methionine‐labeled cells. Anti‐xenotropic MuLV gp70 serum precipitated material whose electrophoretic pattern was similar to that of the 80‐kDa surface antigen. Sequential immunoprecipitation analysis revealed that the molecules reactive with the variant‐specific antiserum were removed by the anti‐xenotropic gp70 antibodies, whereas immunodepletion was only partial when the cell extract was first treated with the variant‐specific antibodies. After Western blotting, the 80‐ and 45‐kDa antigens precipitated by the variant‐specific antibodies were injected intrasplenically into recipient mice. Only the animals sensitized with the 80‐kDa antigen developed specific immunity to L5178Y/DTIC cells in that they displayed an increased frequency in CTL precursors (CTLp) to the variant cells. Sera from mice sensitized to the 80‐kDa protein specifically inhibited the development of a primary CTL response to L5178Y/DTIC cells.
Expression of high molecular weight isoforms of CD45 by mouse thymic progenitor cells
Authors: Goff Lindsey K. , Larsson Lena , Fisher Amanda G.
Published: March 2007

We have studied the expression of isoforms of CD45 (leukocyte common antigen, LCA) among T cell precursors using the organ culture system of Jenkinson et al. (Eur. J. Immunol. 1982. 12: 583). These experiments show that cells capable of recolonizing alymphoid embryonic thymic lobes in vitro can be detected in the thymus of fetal and adult mice and are enriched when thymocytes are depleted of cells bearing CD4 or CD8. These data are consistent with results from in vivo experiments of Fowlkes et al. (J. Exp. Med. 1985. 162: 802) indicating that T cell precursors lie within the double‐negative (CD4−CD8−) compartment. No precursors were detected among the reciprocal populations of cells bearing CD4 and/or CD8 (single and double positives). Double‐negative cell fractions were then divided on the basis of reactivity with monoclonal antibodies RA3‐2C2 and RA3‐3A1. These antibodies recognize the high molecular weight species of the LCA or, more accurately, a product defined by exon A of the CD45 gene. Recolonizing cells were found predominantly in the CD45RA+ (RA3‐2C2 and RA3‐3A1 reactive) fraction of double‐negative thymocytes; CD45RA− enriched populations had increased efficiency of recolonization and CD45RA− depleted populations had decreased ability to recolonize as compared with the whole CD4−CD8− fraction. To clarify whether progenitors enriched in the CD45RA+ fraction were capable of giving rise to mature CD4+, CD8+ and CD4+CD8+ cells, we analyzed the progeny of lobes seeded with CD4−CD8−CD45RA+ fractions. After 7–9 days in organ culture the proportion of CD4+, CD8+ or CD4+CD8+ cells had increased to 35.2%, 18.6% and 23.7%, respectively (mean of five experiments), indicating that progenitors among the CD45RA+ population were indeed multipotent. These results suggest that the majority of T stem cells in the thymus are among thymocytes that express the CD45RA molecule, an hypothesis supported by our finding that removal of CD45RA‐expressing cells (using complement and antibody) eliminated recolonizing capacity of thymic cell fractions.
Hypothermia and hypoglycemia induced by anti‐CD3 monoclonal antibody in mice: Role of tumor necrosis factor
Authors: Alegre Marisa , Vandenabeele Peter , Flamand Véronique , Moser Muriel , Leo Oberdan , Abramowicz Daniel , Urbain Jacques , Fiers Walter , Goldman Michel
Published: March 2007

The possible involvement of tumor necrosis factor‐α (TNF) in the metabolic disturbances induced by anti‐CD3 monoclonal antibodies (mAb) was analyzed in DBA/2 mice injected with 50 μg of the anti‐murine CD3 mAb 145‐2C11. First, we found that 145‐2C11 induces a profound hypothermia maximal between 3 h and 6 h after the injection (at 3 h: − 3.0 ± 0.1 °C) as well as hypoglycemia (blood glucose levels at 6 h and 24 h: 76 ± 13 mg/100 ml and 92 ± 22 mg/100 ml, respectively, p < 0.001 as compared with control values). These metabolic changes are preceded by the release of TNF into the circulation (peak serum TNF levels at 2 h: 50 ± 23 pg/ml, p < 0.01 as compared with controls). The release of TNF induced by 145‐2C11 depends on the effect of the mAb on T cells as it is not observed in athymic nude mice while lipopolysaccharide‐resistant C3H/HeJ mice also display a significant rise in serum TNF (peak levels at 2 h: 59 ± 44 pg/ml). Pretreatment of DBA/2 mice with 12 mg of rabbit anti‐murine TNF antibodies completely prevents the hypothermia while the hypoglycemia is significantly attenuated. Finally, F(ab')2 fragments of 145‐2C11 induce only a transient hypoglycemia (blood glucose levels at 6 h: 109 ± 14, p < 0.001 as compared with controls) but neither hypothermia nor significant TNF release. We conclude that TNF is a major mediator of the acute metabolic changes induced by the intact form of 145‐2C11.
Organization of the chimpanzee C4‐CYP21 region: Implications for the evolution of human genes
Authors: Kawaguchi Hiroshi , Golubic Mladen , Figueroa Felipe , Klein Jan
Published: March 2007

We prepared a cosmid library from chimpanzee DNA and screened it with a mouse probe specific for the complement component 4 (C4)‐encoding gene. We isolated 29 clones and constructed restriction maps for 20 of these. The clones could be arranged into two overlapping clusters covering the entire C4 region of both chromosomes in this particular heterozygous chimpanzee. The region is about 100 kilobases (kb) long and contains two C4 and two CYP21 genes, the latter coding for the enzyme 21‐hydroxylase. Using oligonucleotide probes we identified the genes as corresponding to human C4A, C4B, CYP21 and CYP21P genes. The last gene apparently contains an 8‐base pair (bp) deletion (as does the corresponding human gene), which renders it a pseudogene. The genes are arranged in the order C4A…︁ CYP21P…︁ C4B…︁ CYP21. Each of the two C4 genes is 16 kb long and thus corresponds to the short version of the human C4 genes. We suggest that the duplication of the basic C4‐CYP21 unit that generated the standard arrangement of the human C4‐CYP21 region occurred before the separation of the evolutionary lineages leading to humans and chimpanzees (i.e., more than five million years ago). We suggest further that the original form of the C4 gene was of the long variety and was generated by the insertion of a 6.8‐kb element into one of the C4 introns. The element was subsequently excised in the ancestors of the chimpanzees and in at least one lineage of the human C4B gene. We speculate that the presence of the 6.8‐kb insert in the human C4A and some C4B genes might largely be responsible for the great instability of this chromosomal region which leads to frequent duplications and deletions, some of which cause 21‐hydroxylase deficiency.
Inhibition of T cell developmemt in thymic organ culture: Implications for the mechanism of action of cyclosporin A
Authors: Siegel Richard M. , Yui Katsuyuki , Tenenholz Drew E. , Kubo Ralph , Greene Mark I.
Published: March 2007

We have examined the effects of the immunosuppressive drug cyclosporin A (CsA) on the phenotypic maturation of T cells in thymic organ cultures begun at day 16 of gestation. CsA specifically inhibited the generation of cells expressing high levels of α/β TcR/CD3 complexes and a mature phenotype defined by CD4 and CD8 surface markers. Adding interleukin (IL) 1β, IL 2 or IL 4 failed to reverse the effects of CsA, and major histocompatibility complex class II expression in the thymic medulla was preserved. Possible mechanisms of CsA‐mediated inhibition of T cell development are discussed.
Interleukin 2 receptor‐targeted cytotoxicity. Receptor binding requirements for entry of a diphtheria toxin‐related interleukin 2 fusion protein into cells
Authors: Waters Cory A. , Schimke Patricia A. , Snider Catherine E. , Itoh Kyogo , Smith Kendall A. , Nichols Jean C. , Strom Terry B. , Murphy John R.
Published: March 2007

The receptor binding requirements for entry of the NAD+ ADP‐ribosyltransferase component of DAB486‐IL 2 into target cells were examined. Experiments utilizing cell lines bearing either high‐affinity or individual subunits of the interleukin 2 receptor (IL 2R) as well as human peripheral blood mononuclear cells with natural killer activity demonstrate that the high‐affinity receptor facilitates delivery of fragment A from DAB486‐IL 2 to the cytosol approximately 1000 times more efficiently than either the intermediate‐(p75) or low‐affinity (p55) forms of the IL 2R. We show that elongation factor 2 (EF‐2) in these cells is not quantitatively or qualitatively altered indicating that the relative resistance to intoxication displayed by IL 2R variant cell lines cannot be attributed to an altered intracellular target of the hybrid toxin. We also demonstrate that an alteration in the binding of DAB486‐IL 2 to the p75 subunit of the IL 2R may account for the selective cytotoxicity of DAB486‐IL 2 for cells bearing the heterodimeric high‐affinity IL 2R.
Regulation of murine lymphokine production in vivo II. Dehydroepiandrosterone is a natural enhancer of interleukin 2 synthesis by helper T cells
Authors: Daynes Raymond A. , Dudley Donald J. , Araneo Barbara A.
Published: March 2007

Dehydroepiandrosterone (DHEA) is an adrenal steroid hormone produced in abundance by humans and most other warm‐blooded animals, is uniquely sulfated (DHEAS) prior to export into the plasma, and exhibits an age‐related decline in production with progressive age. No major physiological functions have been ascribed to the activity of this steroid, although DHEA is known to serve as an intermediary in sex steroid synthesis. Studies on the effects of glucocorticoids (GCS) on the immune system led us to question whether DHEA had effects on the ability of activated lymphocytes to produce interleukin 2 (IL 2). We determined that (a) lymphocytes from DHEA‐ or DHEAS‐treated mice consistently produced much greater levels of IL 2 than normals in response to stimulation, (b) direct lymphocyte exposure to DHEA at low doses (10−10 – 10−7 M) caused an enhanced capacity to secrete IL 2 following activation, (c) IL 2 production by activated cloned T cell lines could be augmented by DHEA treatment, and (d) GCS‐induced depressions in IL 2 synthesis by T cells or T cell clones could be overcome in vitro and in vivo by exposure to the effects of DHEA. These findings suggest that DHEA, presumably through receptor‐mediated mechanisms similar to other types of seroid hormones, may represent a natural and important regulator of IL 2 production in both normal and pathologic conditions.
Class I (H‐2Kb) gene transfection reduces susceptibility of YAC‐1 lymphoma targets to natural killer cells
Authors: Carlow Douglas A. , Payne Urula , Hozumi Nobumichi , Roder John C. , Czitrom Andrei A.
Published: March 2007

A “hybrid gene” (MTKb) comprised of the human metallothionein IIA promoter ligated to the genomic sequence of the major histocompatibility complex class I (H‐2Kb) gene was subcloned into the expression vector pSV2neo and transfected into the natural killer (NK) cell‐sensitive YAC‐1 lymphoma. The Kb gene product was readily detectable on the cell surface of G418‐resistant transfectants using both Kb‐specific monoclonal antibodies and H‐2b‐specific cytolytic T cells. Unlike control pSV2neo transfectants, MTKb‐pSV2neo transfectants were relatively resistant to lysis by NK cells from H‐2a, H‐2b, H‐2k or H‐2 (a × b)F1 haplotype mice. These data strongly suggest that the effects of MHC expression on susceptibility to NK cells can be mediated by a single and well‐defined class I molecule, Kb.
Human monoclonal rheumatoid factors derived from the polyclonal repertoire of rheumatoid synovial tissue: Incidence of cross‐reactive idiotopes and expression of VH and Vϰ subgroups
Authors: Thompson Keith M. , Randen Ingrid , Natvig Jacob B. , Mageed Rizgar A. , Jefferis Roy , Carson Dennis A. , Tighe Helen , Forre Oystein
Published: March 2007

A panel of 14 human monoclonal IgM rheumatoid factors (RF) derived from the synovial tissue of rheumatoid arthritis (RA) patients was studied for the expression of cross‐reactive idiotopes (CRI) and variable heavy (VH) and variable kappa (Vx) subgroups. Four of the twelve kappa RF expressed light chains of the Vx subgroup. Three of these were characterized as belonging to the VxIIIb sub‐subgroup, and expressed the VxIIIb associated CRI, 17.109. None of the RF expressed the VxIIIa‐associated CRI, 6B6.6. One of the fourteen RF expressed the VH‐associated CRI, G6, and five expressed either or both the VHIII‐associated CRI, B6 and D12. Seven RF bound to protein A (SpA), which indicates the expression of VHIII subgroup V regions. Together the data indicated that 9/11 RF express VHIII regions and 2/11 express VHI regions. There was no obvious correlation between V region usage or CRI expression and fine specificity of the RF for human IgG subclasses. These data indicate a difference in V gene usage by RF derived from RA patients compared with RF paraproteins derived from non‐RA patients. There is not a bias towards variable chains of the VxIII subgroup, but a marked preference for variable heavy chains of the VHIII subgroup is seen. Further studies may elucidate the pathological significance of these findings in RA.
Interferon‐γ‐activated human granulocytes kill ingested Mycobacterium fortuitum more efficiently than normal granulocytes
Authors: Geertsma Minke F. , Nibbering Peter H. , Pos Odette , Van Furth Ralph
Published: March 2007

Although shortly after the onset of a mycobacterial infection granulocytes are present at the site of inflammation, the role of granulocytes in the elimination of mycobacteria is not well understood. In vitro studies with, for example Mycobacterium tuberculosis or M. bovis, are hampered by the slow proliferation and clumping of the bacteria. To avoid these disadvantages, we developed a model using the atypical mycobacterium M. fortuitum. The present study concerned two questions: whether human granulocytes are able to phagocytose and intracellularly kill opsonized M. fortuitum and whether intracellular killing of these bacteria can be enhanced by treatment of the granulocytes with recombinant human interferon‐γ (rIFN‐γ). The results showed that normal granulocytes phagocytosed opsonized M. fortuitum rapidly, but did not kill these bacteria effectively. The intracellular killing of M. fortuitum was significantly enhanced by incubation of the granulocytes with rIFN‐γ for 18 h before the start of the killing assay. Since these rIFN‐γ‐pretreated granulocytes did not release more O2− and H2O2 upon stimulation with phorbol 12‐myristate 13‐acetate or opsonized M. fortuitum than control granulocytes, non‐oxidative killing mechanisms are probably involved in the enhanced killing of M. fortuitum.
Identification of FcγRIIa, a product of the murine αFcγR gene
Authors: Daëron Marc , Bonnerot Christian , Latour Sylvain , Benhamou Marc , Fridman Wolf H.
Published: March 2007

Two genes, α and β, encode murine low‐affinity receptors for the Fc portion of IgG (FcγRII). The amino acid sequences deduced from the nucleotidic sequences of α and β cDNA are highly homologous in extracellular domains. As a consequence, the protein product of the αFcγR gene has not yet been distinguished from that of the βFcγR gene, α and β cDNA, however, show no homology in sequences coding for intracellular portions. We therefore raised antibodies against a synthetic peptide corresponding to the 26 intracytoplasmic amino acids of the expected product of the murine αFcγR gene. F(ab')2 fragments of α‐specific antibodies thus obtained stained specifically membrane proteins which were present in cells containing α transcripts but not in cells containing β transcripts only; they bound molecules carrying the 2.4G2 epitope, characteristic of the extracellular domains of murine FcγRII; they immunoprecipitated material which migrated as heterogeneous glycosylated proteins of 45–55 kDa when native and, when deglycosylated, as a single polypeptide with an apparent molecular mass of 29 kDa, which is compatible with the calculated molecular mass of the protein expected to be translated from αFcγR transcripts. These criteria identify a product of the murine αFcγR gene as a subtype of FcγRII which can be designated FcγRIIa.
Nurse cells of the bursa of Fabricius: Do they exist?
Authors: Wuorela Maarit , Jalkanen Sirpa , Pelliniemi Lauri J. , Toivanen Paavo
Published: March 2007

Cell‐cell interactions in B lymphocyte development have so far been incompletely characterized, mostly due to lack of a special organ for B cell maturation in the mammalian species. Certain well‐known lymphostromal interactions in the thymus have raised the question whether similar interactions with nurse cells would also operate in the development of B cells. We have tested this hypothesis in the chicken bursa of Fabricius, an organ specific for the B cell maturation. To identify possible nurse cells, with viable lymphocytes enclosed, the cells in the bursa of Fabricius were dispersed with collagenase and trypsin. Light and electron microscopic examination of bursa cell suspensions showed four types of aggregates, identified by low magnification light microscopy as potential nurse cell‐like complexes. Electron microscopy revealed that all aggregates consisted of epithelial cells, and complexes of epithelial cells with lymphocytes enclosed were not observed. These findings indicate that interactions similar to those seen in the avian and mammalian thymus between epithelial nurse cells and T lymphocytes are not a part of the avian B cell differentiation process.
Characterization of Vβ‐bearing cells in athymic (nu/nu) mice suggests an extrathymic pathway for T cell differentiation
Authors: Rocha Benedita
Published: March 2007

In the present article, the expression of the T cell receptor (TcR) β chain and other T cell molecules was evaluated in surface immunoglobulin‐negative spleen cell populations of young and old BALB/c and C57BL/6 nude mice, using a panel of monoclonal antibodies. The results obtained show that in young nude mice, most Thy‐1high cells do not express other T cell markers. These mice have, however, a sizable population of Thy‐1low cells with the same phenotype of α/β+, CD4−CD8− thymocytes or MRL/lpr peripheral T cells, expressing predominantly genes of the Vβ8 family. The evolution of α/β+ cells in aging nudes is strongly suggestive of an extrathymic pathway of differentiation of these cells since (a) the acquisition of high density TcR and CD3, as well as Thy‐1 or CD4CD8 antigens at the cell surface of nude Vβ+ T cells is not simultaneous; (b) α/β+ cells in nude mice co‐express other T cell markers at random and, even in old mice, they never completely resemble to the predominant high Thy‐1+ CD3+ TcR α/β+, CD4+CD8+ cells of euthymic controls; and (c) BALB/c nude T cells express Vβ11 genes, that are deleted in euthymic BALB/c mice. This latter finding may also indicate differences in the mechanisms of selection of T cell specificities in the thymus vs. the peripheral pools.
Retroviral interleukin 4 gene transfer into an interleukin 4‐dependent cell line results in autocrine growth but not in tumorigenicity
Authors: Blankenstein Thomas , Li Weiqun , Müller Werner , Diamantstein Tibor
Published: March 2007

A retroviral vector has been constructed containing the mouse interleukin 4 (IL 4) gene under the transcriptional control of the thymidine kinase promoter. Infection of the IL 4‐dependent T cell line CT4S by the thymidine kinase IL 4 construct resulted in factor‐independent growth. The infected cells grow as a consequence of continous consumption of the endogenously produced IL 4. Clones were established secreting different amounts of IL 4 but none of them was capable of growing in vivo. The lack of correlation between factor‐independent growth and tumorigenicity distinguishes IL 4 from other growth factors and could reflect the recently reported activity of IL 4 to suppress tumor growth in vivo.
The phosphorylation of the CD3 γ chain of T lymphocytes is modulated by β‐endorphin
Authors: Kavelaars Annemieke , Eggen Bart J. L. , De Graan Pierre N. E. , Gispen Willem H. , Heijnen Cobi J.
Published: March 2007

The neuropeptide β‐endorphin can modulate the response of T and B cells to mitogenic or antigenic stimulation. In the present report we describe a novel mechanism by which β‐endorphin can interfere with T cell activation. It is shown here that β‐endorphin can modulate the phorbol ester‐induced phosphorylation of the γ chain of the CD3 complex. The effect of β‐endorphin is dose dependent and appears to be mediated via interaction of β‐endorphin with an opiate receptor on lymphocytes. Evidence is presented that the modulatory effect of β‐endorphin is specific for the phosphorylation of the CD3 γ chain. β‐Endorphin does not affect the phosphorylation of total cell protein, nor does it have any effect on the phosphorylation of the CD4 determinant on T cells. The possible consequence of a change in CD3 γ chain phosphorylation is discussed.
Identity of PB76 differentiation antigen and lymphocyte alkaline phosphatase
Authors: Márquez Carlos , De La Hera Antonio , Leonardo Esther , Pezzi Luis , Strasser Andreas , Martínez‐a Carlos
Published: March 2007

Alkaline phosphatases (APases, EC 3.1.3.1) are ecto‐enzymes bound to cell membranes by a phosphatidyl‐inositol anchor. We have previously shown that APase is present on activated murine B cells and its expression correlates with the process of B cell differentiation into immunoglobulin secretion. Recently, a monoclonal antibody (mAb), G‐5‐2, that recognizes a 76‐kDa molecule preferentially expressed on the surface of pre‐B and plasma cells (PB76) was described. Some features shared by APase and PB76 differentiation antigen suggest that the G‐5‐2 mAb might be specific for lymphocyte APase.

Here, we have analyzed this possibility and found an absolute correlation between PB76 expression in cells and their APase activity. Although PB76 has been described as a B cell‐restricted marker, PB76 is also expressed on some T cells, such as the YAC‐1 T cell lymphoma, that are known to bear APase. Treatment of YAC‐1 cells with phosphatidylinositol‐specific phospholipase C resulted in a quantitatively correlated removal of both APase and PB76 antigens. Moreover, we demonstrate that PB76 antigen has APase activity using an enzyme‐antigen immunoassay with the G‐5‐2 mAb. We conclude that PB76 and lymphocyte APase are one and the same antigen.
Transcription, translation and secretion of both IgA subclasses in polyclonally activated human lymphocytes
Authors: Islam Khalid Bin , Hassan Mohammed Sawkat , Engström Per‐erik , Hammarström Lennart , Edvard Smith C. I.
Published: March 2007

To analyze human IgA subclass‐specific mRNA we have developed RNA probes for quantitation of α1 and α2 heavy chain constant region genes by solution hybridization. Under our assay conditions, we can detect as little as 3 pg of specific mRNA andthere is less than 2% cross‐reactivity between the two IgA subclasses. Using this method, the relative proportions of IgA1 and IgA2 mRNA in pokeweed mitogen(PWM)‐stimulated cells were found to be 66% and 34%, respectively, while in the Epstein‐Barr virus (EBV)‐stimulated cells they were 77% and 23%, respectively. Cytoplasmic staining of the plasma cells and determination of IgA subclass distribution by flow cytometry revealed an almost even distribution of the two IgA subclasses as induced by both activators. The culture supernatant contained 72% IgA1 and 28% IgA2 after PWM stimulation, while EBV stimulation induced 85% IgA1 and 15% IgA2. This report thus describes a method for the quantitative analysis of IgA subclass‐specific mRNA. Furthermore, we present evidence that in response to in vitro stimulation of peripheral blood lymphocytes by polyclonal activators the IgA‐producing B cells not only synthesize both isotypes but also have the potential to secrete them.
Extensive splenic B cell activation in IgMtransgenic mice
Authors: Forni Luciana
Published: March 2007

Two lines of IgMtransgenic mice were analyzed for the state of activation of their splenic compartment, with regard to the frequency of large cells in the different lymphoid subpopulations, and to the isotype distribution of background plasma cells. We observed an extensive B cell activation preferentially involving B lymphocytes co‐expressing transgenic and endogenous IgM (IgD), and resulting in massive immunoglobulin classswitch. Nearly all splenic plasma cells contain endogenous immunoglobulins, withfrequencies of IgG and IgA plasma cells significantly higher than in normal mice. There are virtually no plasma cells that produce only the transgenic IgM. Moreover, only a proportion ofplasma cells producing endogenous immunoglobulins co‐express the transgenic product. In addition to these observations that apply to both transgenic lines, differences were found between the two lines concerning the quantitative expression of the transgenic IgM, the frequency of cells expressing the transgene and the magnitude of switch. These data are indicative of the complexity of the IgMtransgenic mouse model, in which the phenomenology may depend on the transgene insertional position, on B cell physiology and on immunological mechanisms of recognition, induction and regulation.
A CD4+ TH2 cell line isolated from mice chronically infected with Trypanosoma cruzi induces IgG2 polyclonal response in vivo
Authors: Spinella Sylviane , Milon Geneviève , Hontebeyrie‐joskowicz Mireille
Published: March 2007

In our study we describe further characteristics of a CD4+ T cell line (G‐05) isolated from lymph nodes of C3H/HeJ mice chronically infected with Trypanosoma cruzi. This T cell line secreted lymphokines such as interleukin (IL)4 and IL 5 and could be defined as a TH2 type of helper T cells. By passive transferinto naive mice, the G‐05 line was able to induce a polyclonal B cell activation in the spleen. This splenic B cell activation was quite similar to that seen in chronically T. cruzi‐infected animals, where the isotypic pattern presents a large increase of IgG2a and IgG2b isotypes. Moreover, it was possible to reproduce this kind of polyclonal B cell activation in vivo, with the supernatant of G‐05 T cells cultured in the presence of T. cruzi extract, accessory cells andexogenous IL2. Analysis of this supernatant showed the presence of large amounts of IL 4,IL 5, IL 3 and IL 6 but not of interferon‐γ, and residual IL 2 activity was not significant. These results suggest that the G‐05 T cells considered as TH2 cells on the basis of their lymphokine production are involved in the development of the in vivo polyclonal B cell activation in T. cruzi infection.
Signaling through CD19, Fc receptors or transforming growth factor‐β: each inhibits the activation of resting human B cells differently
Authors: Barrett Thomas B. , Shu Geraldine L. , Draves Kevin E. , Pezzutto Antonio , Clark Edward A.
Published: March 2007

To understand further the roles that negative regulatory signals may play in B cell immune responses, we compared three inhibitors of B cell proliferation: cross‐linking CD19 with monoclonal antibody (mAb), signaling through Fc receptors by intact anti‐μ mAb, and transforming growth factor‐β (TGF‐β). Each agent was tested for its ability to block proliferation and specific activation events induced in human tonsilar B cells activated by either cross‐linking surface immunoglobulin, signaling through CD20, or direct activation of protein kinase C (PKC) by phorbol 12‐myristate 13‐acetate. We found that each inhibitor was functionally distinct. Bothanti‐CD19 mAb and anti‐μ mAb inhibited antiimmunoglobulin activated cells and anti‐CD20‐activated cells, but neither inhibited cells activated by phorbol 12‐myristate 13‐acetate. TGF‐β, on the other hand, inhibited equally profoundly cells activated by each of the three regimens. These results suggest that TGF‐β blocks B cell activation at a step following the activation ofPKC, whereas both signaling through CD19 and Fc receptor block early steps in the PKC activation pathway. Signaling through anti‐CD19 mAb was unique in that proliferation of anti‐immunoglobulin‐activated cells was reduced on day 3 and then augmented subsequently. With all other inhibitory combinations the block was permanent. We conclude that each of these three inhibitors has unique important functions and therefore suggest that the effectiveness of negative signaling in B cell immune regulation will dependon the combinations of specific inhibitors modulating a specific activation program.
Subsets of CD3+ (T cell receptor α/β or γ/δ) and CD3− lymphocytes isolated from normal human gut epithelium display phenotypical features different from their counterparts in peripheral blood
Authors: Jarry Anne , Cerf‐bensussan Nadine , Brousse Nicole , Selz Françoise , Guy‐grand Delphine
Published: March 2007

Intestinal intraepithelial lymphocytes (IEL) were studied, after isolation in humans for their surface antigens with a large variety of monoclonal antibodies. They show peculiar characteristics when compared with peripheral blood lymphocytes and intestinal laminapropria lymphocytes. Although a majority of human intraepithelial lymphocytes (IEL) express an α/β type of T cell receptor (TcR), 13% express a γ/δTcR, a percentage which was significantly higher than that found in blood and in lamina propria. In contrast to observations in mice, there was no evidence that normal human TcR γ/δ+ intestinal IEL might use preferential variable segments of γ genes. About 10% of human intestinal IEL expressed the α chain but not the β chain of CD8, thus resembling a subset of CD8 α+β− IEL, which was recently described in mice and found to be of thymoinde‐pendent origin. In addition, 10% of human IEL had a unique phenotype of immature T cells, as they bore only CD7, but no other T cell or natural killer cell markers. Finally, even the major population of IEL which expressed the usual markers of the T cell lineage (CD3,TcR α/β, CD2, CD4 or CD8 α/β) differed from peripheral blood T lymphocytes by their peculiar expression of surface antigens associated with activation. Indeed, 80% of IEL were CD45R0+, CD45A−, but co‐expression of CD11a, CD29 and LFA‐3 was inconstant. In addition, 90% of IEL expressed HML‐1.
Interleukin 2 production by a marmoset B cell Iine
Authors: Brent Lawrence H. , Miyawaki Toshio , Everson Michael P. , Butler Joseph L.
Published: March 2007

Several B cell lines constitutively secreting interleukin (IL) 2 were derived from the Epstein‐Barr virus‐positive marmoset B cell line, B95–8. A representative line, KRC‐18, was cloned by limiting dilution and found to be 40% surface IgM+, 60% cytoplasmic IgM+, > 95% DR+, weakly Tac+ and devoid of T cell, monocyte and NK cell surface antigens. Supernatant from KRC‐18 cells supported the long‐term growth of an IL 2‐dependent murine T cell linc, HT‐2, and contained 7—8 units/ml of IL 2 activity when compared to recombinant IL 2. The supernatant was fractionated by Sephadex G‐75 gel filtration, and maximal proliferation of HT‐2 cells was supported by the 20—22‐kDa column fraction. The proliferative response of HT‐2 cellsto KRC‐18 supernatant was inhibited by monoclonal antibodies to human IL 2 or the murine IL 2 receptor in a dose‐dependent manner, suggesting that the KRC‐18IL 2 has epitopes that are similar to human IL 2, and that its activity is mediated through binding to the IL 2 receptor of the target cell line. When KRC‐18 cells were analyzed for cytoplasmic IL 2, >90% of the cells contained intracellular IL 2 in amountsequivalent to, or greater than, mitogen‐activated T cells. These data indicate that certain B lineage cell lines are capable of IL 2 synthesis and secretion.
Elevated expression of Pgp‐1 (Ly‐24) by murine peritoneal B lymphocytes
Authors: Murphy Timothy P. , Kolber Donna L. , Rothstein Thomas L.
Published: March 2007

B cell expression of the surface glycoprotein, Pgp‐1 (Ly‐24), was evaluated using flow cytometric analysis. Pgp‐1 expression on naive, conventional (splenic) B cells was low but could be increased by mitogenic stimulation. Pgp‐1 expression on naive peritoneal B cells was higher than expression by unmanipulated conventionalB cells, suggesting the possibility that peritoneal B cells have been activated in vivo. However, the elevated expression of Pgp‐1 by peritoneal B cells was not accompanied by increased expression of surface Ia but was correlated with Ly‐1 status. Further, Pgp‐1 expression by B cells from germ‐free mice did not differ from that of normal animals. The elevated expression of Pgp‐1 by peritoneal B cellsis superficially similar to that provoked by mitogenic stimulation of conventional B cells; however, several characteristics suggest that the elevated expression of Pgp‐1 by peritoneal B cells does not reflect prior activation of these cells.