bluuebunny/crossref_metadata_2025 huggingface dataset:
The CD27 membrane receptor, a lymphocyte‐specific member of the nerve growth factor receptor family, gives rise to a soluble form by protein processing that does not involve receptor endocytosis
Authors: Loenen Wil A. M. , de Vries Evert , Gravestein Loes A. , Hintzen Rogier Q. , Van Lier René A. W. , Borst Jannie
Published: March 2007

CD27 is a transmembrane glycoprotein found exclusively on human T and B lymphocytes. It belongs to a recently identified receptor family, whose members are involved in cell differentiation and survival. This family includes the nerve growth factor receptor, two different types of tumor necrosis factor, receptors the Fas antigen, and the B cell‐specific protein CD40. T cell activation via the antigen receptor strongly enhances CD27 membrane expression, suggesting a role for CD27 during T cell differentiation. A soluble form of CD27 (sCD27) is released into the supernatant of activated T cells, and detected in serum and urine of healthy individuals and patients.

We have investigated the mechanism underlying the generation of sCD27. One mRNA encodes both the transmembrane receptor and sCD27, as shown by cDNA transfection. In line with this, only one CD27 precursor protein is found, that is processed to the mature receptor by extensive O‐linked glycosylation. All newly synthesized protein is rapidly transported to the plasma membrane; no internal pool of mature protein is detectable. The transmembrane form gives rise to sCD27 after arrival at the cell surface, most likely via a proteolytic event, that does not involve receptor internalization.
Glycosylation of CD45: carbohydrate processing through Golgi apparatus is required for cell surface expression and protein stability
Authors: Pulido Rafael , Sánchez‐Madrid Francisco
Published: March 2007

The importance of glycosylation on cell surface expression, protein stability and function of the CD45 phosphotyrosine phosphatase leukocyte common antigen, has been studied in the K562 erythroleukemic cell line. Cell treatment with the N‐glycosylation inhibitor tunicamycin generated unglycosylated CD45 polypeptides of 130 and 140 kDa. Immunofluorescence flow cytometry and Scatchard techniques revealed that CD45 cell surface expression was decreased in a time‐and dose‐dependent manner upon tunicamycin incubation. Moreover, a remarkable decrease in CD45 phosphatase activity was detected in tunicamycin‐treated cells, which correlated with the diminished CD45 cell surface expression. Pulse‐chase biosynthetic assays demonstrated that the fate of the unglycosylated CD45 proteins underwent a late non‐lysosomal degradation, since it was not prevented either by monensin, an inhibitor of Golgi transport, or by the lysosomal function inhibitor chloroquine. These results demonstrate that intact glycosylation of CD45 molecules through Golgi compartment is required for stability and proper transport towards the plasma membrane of these phosphotyrosine phosphatase proteins.
Characterization of the bovine peripheral lymph node homing receptor: a lectin cell adhesion molecule (LECAM)
Authors: Walcheck Bruce , White Michael , Kurk Sandy , Kishimoto Takashi K. , Jutila Mark A.
Published: March 2007

The phenomenon of tissue‐specific homing of lymphocyte populations has been most clearly shown in larger domestic animals, such as the sheep and cow, yet the molecular interactions which control these processes in these animals have not been defined. Here we tested the cross‐reactivity of four anti‐human peripheral lymph node homing receptor (LECAM‐1) (also known as LAM‐1, LEC‐CAM‐1, Leu‐8, TQ‐1, or human equivalent of gp90 MEL‐14) antibodies on bovine lymphocytes. These antibodies stained all bovine neutrophils and monocytes, and variable numbers of peripheral blood lymphocytes, as determined by flow cytometry. In young calves (< 1 month old) virtually all circulating lymphocytes expressed LECAM‐1, whereas the percentage of positive lymphocytes in older animals (> 1 year) varied from 17%–67%. Bovine LECAM‐1 was rapidly lost from the cell surface of PMA‐activated and chymotrypsin‐treated cells. Anti‐LECAM‐1 monoclonal antibody blocked > 80% of bovine lymphocyte binding to peripheral lymph node high endothelial venules (HEV). Since the lectin domain of LECAM‐1 is thought to mediate lymphocyte‐HEV adhesion, we sought to establish further the similarity of the bovine, mouse, and human molecules by comparing nucleotide sequences in this region of the molecule. The polymerase chain reaction (PCR) was used to specifically clone the bovine lectin domain from single‐strand cDNA. Subsequent sequencing showed an identity of >80% at the nucleotide level with the human and mouse molecules. The predicted amino acid sequences were also highly conserved. Though striking similarities were seen between the bovine, mouse and human molecule, indicating evolutionary conservation of this family of proteins, notable differences were detected. The nucleotide sequence of the bovine lectin domain predicts one additional N‐linked glycosylation site compared to mouse and human. Preliminary analysis suggested a more tissue‐restricted expression of LECAM‐1 in the compared to the human and mouse, which correlates with a better separation of lymphocyte homing phenotypes seen in these larger animals. Virtually all peripheral lymph node lymphocytes in 6‐month‐old calves expressed LECAM‐1, whereas, ileal Peyer's patch lymphocytes were predominantly negative. Finally, by testing anti‐human LECAM‐1 antibodies in a different species we have established the co‐expression of antigenic epitopes on leucocyte LECAM‐1 and a molecule(s) expressed by endothelial cells.
Superior cross‐protective effect of nasal vaccination to subcutaneous inoculation with influenza hemagglutinin vaccine
Authors: Tamura Shin‐Ichi , Asanuma Hideki , Ito Yuji , Hirabayashi Yoshihiro , Suzuki Yujiro , Nagamine Takashi , Aizawa Chikara , Kurata Takeshi , Oya Akira
Published: March 2007

Intranasal (i.n.) vs. subcutaneous (s.c.) administration of influenza hemagglutinin (HA) vaccine was systematically compared in BALB/c mice. Mice were immunized with different vaccines, together with cholera toxin B subunit as an adjuvant, and 4 weeks later were challenged with either a small (2 μl) or a large (20 μl) volume of mouse‐adapted A/Guizhou‐X (H3N2) virus, each of which gave virgin mice either a nasal or a lung predominant infection. Both i.n. and s.c. inoculations of A/Guizhou‐X vaccine conferred almost complete protection against both challenges. i.n. inoculation of A/Fukuoka (H3N2) or A/Sichuan (H3N2) vaccine conferred almost complete cross‐protection against 2‐μl challenge and a partial cross‐protection against 20‐μl challenge, whereas the s.c. inoculation conferred no cross‐protection against 2‐μl challenge with a partial cross‐protection against 20‐μl challenge. Moreover, i.n. immunization of PR8 (H1N1) vaccine gave a slight cross‐protection against 2‐μl challenge, while the s.c. inoculation did not. The degree of protection was easily improved by i.n. inoculation of higher doses of vaccine, but not by the s.c. inoculation. In parallel with the protection, the i.n. vaccination produced a high level of cross‐reacting IgA and IgG antibody to A/Guizhou‐X HA in nasal and broncho‐alveolar washes, while the s.c. vaccination produced the cross‐reacting IgG antibody alone. Thus, i.n. inoculation with inactivated vaccines, which induces crossreacting anti‐HA IgA antibody as well as IgG antibody, is more effective than s.c. vaccination for providing cross‐protection against drift viruses.
Mouse autoreactive γ/δ T cells II. Molecular characterization of the T cell receptor
Authors: Ezquerra Angel , Wilde David B. , McConnell Thomas J. , Sturmhöfel Knut , Valas Robert B. , Shevach Ethan M. , Coligan John E.
Published: March 2007

A panel of dendritic epidermal T cell (DETC) lines, and hybridomas derived from them, has been shown to spontaneously secrete lymphokines in the absence of added stimuli, which suggests that these cells are autoreactive. These cell lines are characterized by the expression of a Vγ1.1Cγ4/Vδ6 type T cell receptor (TcR), but several of the DETC lines also express a second TcR. Sequence analyses of these γ/δ TcR revealed that the γ chains were identical and that the δ chains, while not identical, were quite restricted in diversity, indicating that these receptors may recognize a common or closely related group of antigens. Analysis of hybridomas derived from newborn thymocytes identified six hybridomas that spontaneously secrete lymphokines. Five hybrids expressed a Vγ1.1Cγ4/Vδ6 receptor and one hybrid a Vγ1.1Cγ4/Vδ4 receptor that had a close structural relationship to the DETC γ/δ TcR associated with spontaneous lymphokine secretion. γ/δ TcR of the Cγ4 type expressed by splenic hybridomas that did not spontaneously secrete lymphokines revealed no such relationship. Curiously, like the DETC, several of the thymocyte hybridomas that spontaneously secreted lymphokines expressed a second TcR, Vγ2Cγ1 or Vγ3Cγ1, apparently in association with the same δ chain that paired with the Cγ4 chain. The presence of spontaneous lymphokinesecreting γ/δ T cells with such highly homologous TcR in both the thymus and skin suggests a thymic origin for the autoreactive DETC and that these cells recognize a common or closely related group of self‐antigens.
Regulation of immunoglobulin gene expression in normal lymphocytes II. Mechanisms of down‐regulation of immunoglobulin secretion after engagement of the B cell antigen receptor
Authors: Berberich Ingolf , Schimpl Anneliese
Published: March 2007

When B cells are stimulated with lipopolysaccharide (LPS) they start to proliferate and mature into immunoglobulin (Ig)‐secreting cells. Co‐stimulation with F(ab')2 fragments of antibodies directed against the B cell antigen receptor leads to an inhibition of Ig secretion but not of proliferation. This effect can be mimicked by phorbol esters alone or by a combination of phorbol esters and the Ca2+ ionophore ionomycin, which activate protein kinase C. Here we report that co‐stimulation with phorbol esters and ionomycin differentially affects a group of genes normally up‐regulated during the course of LPS‐dependent B cell activation. Thus, the mRNA coding for the membrane‐bound form of IgM and the interleukin 2 receptor (55‐kDa protein) continue to be expressed at the levels typical of LPS‐stimulated cells, while the mRNA coding for the secreted form of IgM (μs) and for the J chain are reduced. The loss of μS mRNA is attributable to an altered processing behavior with respect to the μ precursor and/or a decreased stability of the mRNA itself.
Variation in human T cell receptor Vβ and Jβ repertoire: analysis using anchor polymerase chain reaction
Authors: Rosenberg William M. C. , Moss Paul A. H. , Bell John I.
Published: March 2007

Anchor polymerase chain reaction has been applied to the study of human T cell receptor β chain repertoire in peripheral blood. The use of this technique has demonstrated that considerable variation in Vβ and Jβ usage exists, both within and between individuals. Particular Vβ families, including Vβ6, Vβ4 and Vβ12 are commonly utilized, while families such as Vβ10, Vβ11 and Vβ15 are rare in all individuals studied. Marked interindividual variation in Vβ usage was detected for Vβ12 and Vβ4. Biased usage of Jβ elements is a prominent feature of peripheral repertoire, while there is no evidence for preferential Vβ‐Jβ recombination events. Biased Jβ usage in expressed Vβ‐Dβ‐Jβ‐Cβ transcripts, subject to selection, was the same as that in aberrant, unselected Dβ‐Jβ‐Cβ transcripts, implying that bias resulted from events relating to rearrangement itself, in the absence of selection. N‐region diversity showed some evidence for preferential insertion of deoxyguanosine, consistent with the action of terminal deoxytransferase. No P‐nucleotide incorporation was seen in association with intact Jβ elements. These data provide evidence of some of the variation in human T cell receptor β chain repertoire and provide a basis for comparisons with sequences which may be obtained in autoimmune and superantigen‐mediated diseases.
Selective reactivity of CD8‐independent T lymphocytes to a cytotoxic T lymphocyte‐selected H‐2Kb mutant altered at position 222 in the α3 domain
Authors: Shepherd Sara E. , Sun Rui , Nathenson Stanley G. , Sheil James M.
Published: March 2007

To study the structural basis for specificity and affinity of cytotoxic T lymphocytes for major histocompatibility complex/peptide complexes, we have employed a cytotoxic T lymphocyte (CTL)‐mediated immunoselection approach to obtain H‐2Kb structural mutants which are resistant to lysis by a Kb‐specific alloreactive CTL clone. In this study we describe the Kb structural mutant, designated R8.60.14, recovered following immunoselection using the CD8‐dependent CTL clone 60 as a selective agent. Although serologically unaltered with respect to Kb expression, R8.60.14 is not recognized by CD8‐dependent, Kb‐specific CTL. DNA sequence analysis revealed a single Glu → Lys amino acid substitution at position 222 in the Kb α3 domain of this variant. To determine if a direct correlation exists between CD8 dependence of a Kb specific CTL and its failure to respond to R8.60.14, we examined the lytic response against R8.60.14 by CD8‐independent, Kb‐specific CTL obtained from long‐term culture in the presence of anti‐CD8 monoclonal antibody, 3.155. CD8‐independent CTL exhibit no difference in their response against the R8 parent and R8.60.14 variant. This study demonstrates unequivocally that Kb‐specific recognition of R8.60.14 by CD8‐independent CTL is unaltered, while the response by CD8‐dependent CTL is completely abrogated. Thus, the sole basis for emergence of this variant in the CTL‐mediated immunoselection approach used in this study resides in the alteration of a single CD8‐binding site residue at position 222 in the Kb α3 domain. The functional importance of this Glu222 residue for the interaction between the CD8 molecule on CD8‐dependent CTL and the Kb α3 domain is further reinforced by virtue of the recovery of the R8.60.14 variant on the basis of its resistance to lysis by a CD8‐dependent CTL clone in this CTL‐mediated immunoselection approach.
Interaction of CD4:lck with the T cell receptor/CD3 complex induces early signaling events in the absence of CD45 tyrosine phosphatase
Authors: Deans Julie P. , Kanner Steven B. , Torres Raul M. , Ledbetter Jeffrey A.
Published: March 2007

Antibody‐mediated ligation of the CD3/T cell antigen receptor (TcR) activates phospholipase C (PLC) via a tyrosine kinase signaling pathway that requires expression of the transmembrane tyrosine phosphatase CD45. In normal T cells, CD3‐mediated PLC activation is significantly augmented by co‐ligation of CD3 with the CD4 co‐receptor; however, unlike CD3‐associated tyrosine kinases, antibody‐induced activation of the CD4‐associated tyrosine kinase p56lck does not require CD45 expression. To explore the role of CD45 in the CD3 and CD4 activation pathways further, we examined the effect of CD3/CD4 cross‐linking on tyrosine phosphorylation and activation of phospholipase C in CD45− mutant cells of the T cell leukemia line HPB.ALL. In accord with previous observations, anti‐CD3 stimulation of the CD45‐deficient cells failed to activate tyrosine kinases, or PLC as measured by mobilization of intracellular calcium. However, we show here that ligation of CD3 with CD4 leads to tyrosine phosphorylation of PLCγ1 and elevation in the intracellular free Ca2+ concentration in CD45− cells that is in excess of that seen in CD45+ cells. Since CD4 stimulation alone did not activate PLC, a component of the CD3 signaling pathway must be independent of CD45. Anti‐CD4‐induced tyrosine phosphorylation and activation of CD4‐associated lck was also enhanced in CD45− cells, suggesting that increased lck activation compensates for the defect in CD3/TcR signaling, such that interaction of the CD3 signaling pathway with the CD4‐associated pathway activates PLC even in the absence of CD45. The data demonstrate that the requirement for CD45 in coupling CD3/TcR to the PI‐PLC activation cascade is not absolute, but rather substantiates a role for CD45 in modifying molecular interactions that control T cell activation.
Weak positive selection of transgenic T cell receptor‐bearing thymocytes: importance of major histocompatibility complex class II, T cell receptor and CD4 surface molecule densities
Authors: Bogen Bjarne , Gleditsch Lars , Weiss Siegfried , Dembič Zlatko
Published: March 2007

We have produced αβ T cell receptor (TcR)‐transgenic mice and studied MHC‐dependent positive selection of T cells bearing this receptor. The α and β transgenes were isolated from an I‐Ed‐restricted, CD4+ BALB/c (H‐2d/d) T cell clone specific for a peptide consisting of the 91–101 residues of the λ2 immunoglobulin light chain of MOPC315. Mice which carry the transgenes on a BALB/c background, but with H‐2d/d, H‐2b/d or H‐2b/b major histocompatibility complex (MHC) haplotypes, were investigated for TcR expression in thymocytes and peripheral T cells. The thymocytes expressing the transgene‐encoded αβ receptor are weakly positively selected when compared with previous findings in other TcR‐transgenic mice models. Thus, αβ thymocytes vary in their efficacy of being positively selected by their restriction element. Furthermore, the density of TcR and CD4 on thymocytes, as well as the density of I‐Ed molecules on thymic epithelial cells, appear critical for the extent of positive selection. A possible explanation is that the transgenic TcR has a marginal affinity for self‐MHC molecules on thymic epithelium, and that this may be compensated for by an increase in the number of CD4/TcR/MHC ternary complexes forming between the maturing thymocyte and the cortical epithelial cells.
Suppression and transient induction of lymphokines in cancer patients after administration of polyethylene glycolated interleukin‐2
Authors: Shih Yihshing , Konrad Michael W. , Kim Warren M. , Childs Anne , Paradise Carolyn , Meyers Frederick J. , Groves Eric S.
Published: March 2007

Polyethylene glycolated (pegylated) interleukin‐2 (PEG IL‐2) was administered as a weekly i.v. bolus to patients with metastatic cancer in a phase‐I trial. Efficacy, toxicity and pharmacokinetics have been described previously. To explore mechanism of IL‐2 action and discover predictors of efficacy, the levels of several lymphokines were measured in pharmacokinetic serum samples. IL‐1β and IL‐6 were elevated in many patients before PEG IL‐2 administration, forming a continuous, log‐normal distribution among patients. The levels of the two lymphokines were strongly correlated. However, no significant correlation could be found between these levels, clinical chemistry, or tumor regression seen after PEG IL‐2 administration. Three hours after PEG IL‐2 administration, IL‐1β and IL‐6 levels, if elevated, fell to normal. In all patients, independent of initial levels, IL‐6 and IFN‐γ, but not IL‐1β, increased 4 to 6 h after the injection and then fell rapidly, even though PEG IL‐2 levels were high and often changed only slightly during this period. This suggests an active shut down of lymphokine synthesis, or an increase in elimination rate. After the fourth administration of PEG IL‐2, the peak level of IFN‐γ was 2 to 20 times higher than after the first, while the peak level of IL‐6 did not change in a consistent direction. Responding patients had typical peak levels of IL‐6 and IFN‐γ. Low levels of TNF and IL‐4 were occasionally seen before and after PEG IL‐2 administration, but no consistent pattern was evident.
Disruption of CD8‐dependent negative and positive selection of thymocytes is correlated with a decreased association between CD8 and the protein tyrosine kinase, p56lck
Authors: van Oers Nicolai S. C. , Garvin Alex M. , Davis Craig B. , Forbush Katherine A. , Carlow Douglas A. , Littman Dan R. , Perlmutter Roger M. , Teh Hung‐Sia
Published: March 2007

The CD4 and CD8 coreceptor molecules on immature thymocytes participate in T cell repertoire selection. To examine more definitively the role of CD4 and CD8 in the negative and positive selection of immature thymocytes, we generated transgenic mice with elevated surface CD4 expression and mated them with mice expressing a transgenic T cell receptor. Augmented CD4 expression was found to markedly alter CD8‐dependent negative and positive selection of T cells specific for the male (H‐Y) antigen presented by H‐2Db major histocompatibility complex class I molecules. Moreover, the cytoplasmic tail of CD4 was essential for effecting these alterations, since the overexpression of tailless CD4 molecules failed to influence the outcome of CD8‐dependent selection. The inhibition of positive and negative selection in double‐transgenic mice expressing the full‐length CD4 molecule was associated with a decreased interaction between the protein tyrosine kinase p56lck and CD8. These results strongly implicate p56lck in T cell repertoire selection.
Immunization with soluble simian virus 40 large T antigen induces a specific response of CD3+ CD4− CD8+ cytotoxic T lymphocytes in mice
Authors: Schirmbeck Reinhold , Zerrahn Jens , Kuhröber Andreas , Kury Evelyn , Deppert Wolfgang , Reimann Jörg
Published: March 2007

C57BL/6 (B6) mice (H‐2b) were immunized with the large tumor antigen (T Ag) of simian virus 40 (SV40). Intraperitoneal or subcutaneous sensitization with soluble T Ag specifically primed cytotoxic lymphocyte precursors (CTLp). T Ag‐specific cytotoxic T lymphocytes (CTL) were detected in a cytotoxicity assay after specific in vitro restimulation of effector cell populations from mice immunized with 2–10 μg purified, soluble T Ag and boosted with an injection of 2 μg T Ag 2–4 weeks after priming. Cells used for in vitro restimulation and as targets in cytotoxicity assays were syngeneic (B6‐derived) RBL5 lymphoma cells expressing SV40 T Ag after transfection with a T Ag‐encoding expression vector. Effector cells of this response were H‐2 class I‐restricted CD3+ CD4− CD8+ CTL. The magnitude of the anti‐T Ag CTL response of B6 mice stimulated by soluble virus protein was comparable to the anti‐T Ag CTL response of SV40‐infected B6 mice. Injections of denatured or native T Ag protein primed CTLp equally well, but immunization with an equal dose of antigen emulsified in incomplete Freund's adjuvants inefficiently stimulated CTLp.
CD59 expressed by human endothelial cells functions as a protective molecule against complement‐mediated lysis
Authors: Brooimans Ricardo A. , van der Ark Arnoldus A. J. , Tomita Motowo , van Es Leendert A. , Daha Mohamed R.
Published: March 2007

CD59 is a 18–20‐kDa membrane glycoprotein that inhibits formation of the membrane attack complex of complement (C) on homologous cells. In the present study we analyzed the expression and function of CD59 on human endothelial cells. Immunohistochemical analysis of renal cortex demonstrated a predominant expression of CD59 on peritubular capillary endothelial cells and glomerular endothelial cells. Flow cytometry analysis showed that human umbilical vein endothelial cells (HUVEC) expressed CD59 and the fluorescence intensity was approximately four times that of peripheral blood lymphocytes. CD59 is detected on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis as a single 20‐kDa molecule in 2 % deoxycholate extracts of HUVEC. CD59 was released from the surface of HUVEC by phosphatidylinositol‐specific phospholipase C, demonstrating that it is attached to the cell membrane by means of a glycolipid anchor. The functional activity of CD59 expressed on HUVEC was studied. Blocking of CD59 antigen with F(ab')2 fragments of polyclonal anti‐CD59 enhanced markedly the susceptibility of HUVEC to C‐mediated lysis. This effect was dependent on the amount of blocking antibodies added. Northern blot analysis revealed the presence of three species of mRNA expressed in HUVEC, which hybridized to a cDNA probe specific for CD59, with sizes of about 800, 1400 and 2000 bp. These findings suggest that CD59 may be important in protection of endothelial cells against C‐mediated damage at local sites of inflammation, thereby maintaining the vascular integrity in vivo.
Regulation of L‐selectin expression on cultured bone marrow leukocytes and their precursors
Authors: Salmi Marko , Jalkanen Sirpa
Published: March 2007

L‐selectin (LECAM‐1, LAM‐1, MEL‐14 antigen, Dreg antigen) is one of the molecules controlling lymphocyte homing from the blood to peripheral lymph nodes and granulocyte adhesion to inflamed endothelium. In this work, regulation of L‐selectin expression on mouse bone marrow cells was studied. L‐selectin‐negative cells were isolated by panning technique, cultured for 1–7 days with cytokines and mitogens, and L‐selectin expression was analyzed by immunofluorescence staining. When cultured for 3 days with interleukin (IL) 1, IL 2, IL 5, IL 6, phytohemagglutinin, pokeweed mitogen or in the medium alone, 75%–85% of L‐selectin‐negative large cells (including granulocytes, macrophages/monocytes, blasts and their precursors) became L‐selectin positive. In contrast, IL 3, IL 4, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and lipopolysaccharide (LPS) prevented the induction of L‐selectin in a time‐ and dose‐dependent manner. GM‐CSF was the most potent inhibitor and only 10%–15% of cells became L‐selectin positive after 3 days of culture. Furthermore, L‐selectin was down‐regulated on cultured unselected bone marrow cells by IL 3, IL 4, GM‐CSF and LPS stimulation. After culture, the relative molecular mass of L‐selectin was 100 kDa, similar to the size of the granulocyte form of this antigen. Cultured cells adhered to high endothelial venules (HEV) only 10%–32% as effectively as freshly isolated bone marrow cells despite high levels of L‐selectin expression. The phenotypic analysis and the HEV binding data indicate that after culturing L‐selectin was almost exclusively expressed on bone marrow leukocytes of myeloid series, and on these cells it was not functional in mediating peripheral lymph node HEV binding. Overall, these results show that the expression of L‐selectin can be modulated by regulating the maturation and differentiaton of the cells in vitro. This supports the idea that different cytokines and mitogens may also be important in controlling migrational status of leukocytes in vivo.
Cross‐linking of surface IgM or IgD causes differential biological effects in spite of overlap in tyrosine (de)phosphorylation profile
Authors: Alés‐Martinez José E. , Scott David W. , Phipps Richard P. , Casnellie John E. , Kroemer Guido , Martinez‐A Carlos , Pezzi Luis
Published: March 2007

Although displaying similar amounts of surface IgM and IgD, ECH 408‐1 cells only succumb to apoptosis after cross‐linking of IgM (not IgD), suggesting that different signaling pathways couple to both receptors. Immunoprecipitation studies revealed the presence of several proteins selectively associated with IgM and IgD, thus ruling out that the lack of inhibitory signaling mediated by IgD might be due to membrane expression in the absence of associated proteins belonging to the B cell receptor complex. 32P metabolic labeling and immuno‐precipitation studies demonstrated that IgM and IgD are associated with phosphoproteins of 32–33 kDa in an isotype‐specific fashion. Kinetic analyses of tyrosine kinase activity showed that cross‐linking of surface IgM or IgD resulted in the rapid (1–3 min) phosphorylation of several protein substrates on tyrosine residues, followed by a dephosphorylation step. Isotype‐specific changes of the phosphorylation status specifically affected molecules in the 32–33 kDa range, i.e. IgM (not IgD) cross‐linking affected a ∼ 32‐kDa protein, whereas IgD (not IgM) cross‐linking induced phosphorylation of a protein exhibiting a slightly lower mobility (33 kDa). These results suggest that isotype‐specific immuno‐globulin‐associated molecules could be involved in the second messenger cascade leading to different biological effects upon IgM and IgD cross‐linking.
Role of the transmembrane and cytoplasmic domains of surface IgM in endocytosis and signal transduction
Authors: Dubois Patrice M. , Stepinski Jan , Urbain Jacques , Sibley Carol Hopkins
Published: March 2007

The cross‐linking of membrane IgM (mIgM) triggers the activation and differentiation of B lymphocytes. One very rapid result of the cross‐linking is the activation of phospholipase C, the subsequent mobilization of free calcium from internal stores and the activation of protein kinase C. This is followed by a redistribution of the receptor‐ligand complexes to a small cap on the B cell surface, the first step in endocytosis and antigen processing. Cross‐linking of major histocompatibility complex (MHC) class I neither stimulates the release of intracellular calcium nor does it induce capping and endocytosis of the cell surface receptors. In this study, we sought to determine the role of the two carboxyterminal domains of the μ heavy chain in signal transduction, capping and endocytosis of mIgM. We took advantage of the clear differences between MHC class I molecules and mIgM, replacing the transmembrane and cytoplasmic domains of μ by their MHC class I equivalents. Our results show that the hybrid heavy chain could still associate with light chains and assemble into a tetramer on the cell surface. However, cross‐linking of the hybrid cell receptor produced neither release of calcium from internal stores, nor capping and endocytosis. These observations demonstrate that the two carboxy‐terminal domains of μ are critical to both signal transduction and modulation of the mIgM‐ligand complexes from the surface of B lymphocytes.
Analysis of the T cell receptor (TcR) regions in the NOD, NON and CTS mouse strains define new TcR Vα haplotypes and new deletions in the TcR Vβ region
Authors: Lund Torben , Shaikh Sanober , Hattori Masakazu , Makino Susumu
Published: March 2007

We have analyzed the T cell receptor (TcR) Vα and TcR Vβ regions in the spontaneous mouse model for insulin‐dependent diabetes mellitus, the NOD mouse, and compared it to the regions in the two sister strains, the NON and CTS strains. Based on restriction fragment length polymorphism analysis the TcR Vα region in the NOD mouse is essentially identical to that of the SJL/J strain. In contrast both the NON and CTS strains have a unique TcR Vα haplotype. Whereas the NOD and NON strains apparently contains all the TcR Vβ genes, the CTS mouse has three deletions in the Vβ region. Our analysis does not give any indications for the diabetic phenotype of the NOD mouse.
Differential modulation by glucocorticoids of alternative complement protein secretion in cells of the monocyte/macrophage lineage
Authors: Lemercier Claudie , Julen Nathalie , Coulpier Muriel , Dauchel Hélène , Ozanne Denyse , Fontaine Marc , Ripoche Jean
Published: March 2007

The effect of the synthetic glucocorticoid dexamethasone (DXM) on the secretion by human monocytes of alternative complement proteins C3, factor B and factor H was investigated. Results indicated that DXM modulates this secretion in a direction which would be consistent with its anti‐inflammatory properties. DXM, at therapeutic concentrations, had a suppressive effect on C3 and factor B secretion and a stimulatory effect on factor H secretion by monocytes. This differential modulation on C3, factor B and factor H secretion was similar in mature macrophages. Together with previous studies showing that DXM had a suppressive effect on C3 and factor B secretion and a stimulatory effect on factor H secretion by human endothelial cells, our results indicate that DXM appears to have the general property of regulating local production of complement components so as to control complement activation.
The CD27− subset of peripheral blood memory CD4+ lymphocytes contains functionally differentiated T lymphocytes that develop by persistent antigenic stimulation in vivo
Authors: de Jong Rolien , Brouwer Miranda , Hooibrink Berend , van der Pouw‐Kraan Tineke , Miedema Frank , van Lier René A. W.
Published: March 2007

On the basis of expression of the T cell differentiation antigen CD27, human peripheral blood CD4+ memory cells can be divided into two subsets, a large CD45RA−CD27+ (82%) and a small CD45RA−CD27− (18%) population. Analysis of the functional properties of these memory T cell subsets showed that proliferative responses to the recall antigen tetanus toxoid (TT), shortly after booster immunization, were mainly confined to the CD27− population. Also, in atopic individuals, proliferative responses to allergens for which these individuals are sensitized, were limited to the CD45RA−CD27− population. After stimulation with CD3 monoclonal antibody and phorbol ester, CD27+ cells produced vast amounts of interleukin (IL)‐2 but minimal amounts of IL‐4, whereas in marked contrast, CD27− T cells secreted low levels of IL‐2 and high levels of IL‐4. The capacity of the vast majority of these latter cells to produce IL‐4 was found to be a stable feature since high IL‐4‐secreting T cell clones were generated from the CD27− subset. These findings suggest that upon renewed as well as chronic antigenic stimulation in vivo, memory T cells acquire the CD45RA−CD27− phenotype and that, as a consequence, in this subset functionally differentiated CD4+ T cells are compartmentalized. Our results predict that analysis of the small CD27− subset of memory cells, that makes up approximately 10% of the peripheral blood T cell population, will provide information on the specificity and function of responding CD4+ T cells at a given point in time in healthy and diseased individuals.
Expansion and clonal deletion of peripheral T cells induced by bacterial superantigen is independent of the interleukin‐2 pathway
Authors: Gonzalo José Angel , de Alborán Ignacio Moreno , Alés‐Martínez José E. , Martínez‐A. Carlos , Kroemer Guido
Published: March 2007

Injection of the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) into mice provokes a rapid expansion and subsequent contraction of the pool of SEB‐reactive T cells bearing T cell receptor (TcR) Vβ8 gene products. Given that interleukin 2 (IL‐2) stimulates proliferation, abolishes anergy, and counteracts apoptotic cell death in T cells in vitro, we tested whether the IL‐2 synthesis inhibitor cyclosporin A (CsA) or a vaccinia virus recombinant releasing high amounts of human IL‐2 modulate SEB responses in vivo. Surprisingly, neither IL‐2 nor CsA were able to change the in vivo kinetics and magnitude of SEB‐induced expansion, unresponsiveness to SEB, and peripheral clonal deletion of T cells expressing products of the SEB‐reactive TcR Vβ8 gene family. In accord with these in vivo observations, IL‐2 is incapable of reversing “anergy” and apoptotic cell death of Vβ8+ SEB‐reactive T cells isolated from SEB‐primed mice in vitro. Accordingly, upon SEB injection Vβ8+ T cells expand rapidly, without expressing IL‐2 receptor (IL‐2R)α chains in vivo, although SEB induces IL‐2R α in vitro. Altogether, these results indicate that the IL‐2/IL‐2R‐mediated pathway is not involved in T cell repertoire modulation by bacterial superantigens. Moreover, the data suggest that unresponsiveness of Vβ8+ T cells from SEB‐primed mice is not a reversible process, but involves an unreversible commitment to programmed cell death. Absence or presence of IL‐2 responsiveness could be a hallmark to distinguish truly reversible anergy and peripheral clonal deletion.
The human immunoglobulin x locus. Characterization of the duplicated A regions
Authors: Lautner‐Rieske Andrea , Huber Christian , Meindl Alfons , Pargent Walter , Schäble Karlheinz F. , Thiebe Rainer , Zocher Ines , Zachau Hans G.
Published: March 2007

The central regions of the x locus, the so‐called A regions, have been fully characterized on cosmid and phage λ clones. The regions, which are parts of the Cx‐proximal and‐distal copies of the locus and are, therefore, called Ap and Ad regions, comprise about 140 kb each and contain together 30 Vx genes and pseudogenes. The A regions have been linked on their 5′ sides to the O regions and on their 3′ sides to the L regions. Chromosomal walking has eliminated a previous gap in the Ap region. Detailed restriction maps of the Ap and Ad regions and the sequences of 9 Vx genes are reported. Four events, which have occurred in evolution probably after the duplication of the A region, were identified: the insertion of an Alu element in Ad; the insertion of part of a LINE element in Ap; the deletion of a 17.5‐kb fragment including one Vx gene from Ap; the sequence divergence of duplicated Vx gene regions which ranges among the five pairs studied here from 0 to 14 bp per kb and converted two genes to pseudogenes while their duplicates stayed functional. An analysis of the A regions of the lymphoid cell lines RPMI 6140 and GM607 confirmed the previous finding that the Vx‐Jx rearrangement in these cell lines had occurred by deletion and inversion mechanisms, respectively. Thus, the structural data contribute to the understanding of the evolution and the functioning of the A regions of the x locus.
Repertoire of Vα and Vβ regions of T cell antigen receptors on CD4+ and CD8+ peripheral blood T cells in a novel X‐linked combined immunodeficiency disease
Authors: Goldman Armond S. , Palkowetz Kimberly H. , Rudloff Helen E. , Brooks Edward G. , Schmalstieg Frank C.
Published: March 2007

The repertoire of V regions of α/β+ T cell receptor (TcR) on CD4+ and CD8+ T cells from the peripheral blood of six patients with a novel X‐linked combined immunodeficiency was investigated by flow cytometry. The relative frequencies of V region subfamilies Vα2a on CD4+ T lymphocytes and Vβ5b and Vβ12a on CD8+ T lymphocytes from the peripheral blood from the affected males were decreased significantly. Also, the relative frequencies of certain other V region subfamilies on CD4+ or CD8+ T cells from the peripheral blood of some patients were either considerably below or above the ranges found in normal subjects.

Although there may be other explanations including an extrathymic event, we suggest that the abnormalities in the TcR repertoire of peripheral blood T cells are consistent with a dysregulation of the intrathymic maturation/selection of T cells that leads to deficiencies in T cell populations in the peripheral blood of males with this disease.
VLA‐2 is the integrin used as a collagen receptor by leukocytes
Authors: Goldman Rachel , Harvey Joanna , Hogg Nancy
Published: March 2007

Cultured T cells and freshly isolated mononuclear leukocytes are able to bind collagen specifically. These leukocytes express equivalent levels of the integrins VLA‐1, VLA‐2 and VLA‐3 which are collagen‐binding receptors on other cells. However, only solubilized VLA‐2 is able to bind collagen and only monoclonal antibodies specific for α2 or β1 subunits are able to block the binding of intact cells to collagen. This restriction provides another example of the dependence of integrin specificity on the cell type on which it is expressed.

It was also speculated that the inserted or I domain on the α subunits of VLA‐1, VLA‐2 and the β2 integrin family might have a role in collagen binding on the basis of its sequence homology to other types of collagen binding proteins. However, LFA‐1, CR3 and p150,95 showed no collagen binding activity, suggesting that the I domain has another function.
Inflammation but not autoimmunity occurs in transgenic mice expressing constitutive levels of interleukin‐2 in islet β cells
Authors: Allison Janette , Malcolm Leonie , Chosich Nikola , Miller Jacques F. A. P.
Published: March 2007

Transgenic mice expressing murine interleukin (IL)‐2 constitutively in islet β cells were generated (RIP‐IL‐2 mice). They died at an early age, when higher levels of IL‐2 were produced, because of a predominant macrophage inflammatory response that destroyed the exocrine pancreas. Animals with lower levels of IL‐2 survived and had islets that became increasingly infiltrated with lymphocytes over time. However, in spite of the presence of impressive peri‐ and intra‐islet infiltrates, autoimmunity to islet antigens was not seen. Autoimmunity was also not induced to extrathymic H‐2Kb molecules known to induce tolerance by a peripheral mechanism when the RIP‐IL‐2 mice were mated to other mice expressing H‐2Kb in islet β cells (RIP‐Kb mice). Apparently, IL‐2 can act only on activated T cells and is unable to reverse tolerance in T cells that have been made unresponsive through inappropriate presentation of antigen.
Interaction between Epstein‐Barr virus and a T cell line (HSB‐2) via a receptor phenotypically distinct from complement receptor type 2
Authors: Hedrick Joseph A. , Watry Debbie , Speiser Catherine , O'Donnell Patrick , Lambris John D. , Tsoukas Constantine D.
Published: March 2007

Epstein‐Barr virus (EBV), the causative agent of mononucleosis and several human cancers, infects cells via complement receptor type 2 (CR2, CD21) which also serves as the receptor for the third complement component, C3. Expression of this receptor is restricted to B lymphocytes, immature thymocytes, and certain epithelial cells. In the present investigation, we describe the presence of a seemingly novel EBV receptor which is phenotypically distinct from CR2. Among various leukemic T cells studied, one, HSB‐2, demonstrates no reactivity to several anti‐CR2 antibodies, yet it reacts strongly with EBV as detected by incubation with biotin‐conjugated virus and streptavidin‐phycoerythrin. The virus binding is specific as demonstrated by blocking with anti‐EBV antibodies and with non‐conjugated virus. Aggregated C3 also binds HSB‐2 and is capable of partially inhibiting EBV binding. The absence of CR2 on HSB‐2 is further supported by the lack of expression of specific mRNA, assessed by Northern blotting analysis and polymerase chain reaction. Viral internalization and infection is demonstrated with electron microscopy, with detection of EBV‐DNA by Southern blotting, and with detection of EBNA‐1 transcripts by the polymerase chain reaction. Even though HSB‐2 does not express CR2, it nevertheless displays transcripts which have some homology to a CR2 cDNA probe under low stringency hybridization conditions. This probe encompasses approximately the N‐terminal half of CR2 which includes the EBV‐binding epitope(s). The HSB‐2 message is 5.2 kb, a size distinct from the 4.7‐kb message of B cell CR2s. In contrast, the 5.2‐kb message in not seen, under similar hybridization conditions, with a probe comprising the C‐terminal half of CR2. Collectively, the data indicate that a receptor molecule having distinct phenotypic characteristics from the known CR2 protein on B cells is utilized by EBV to target human T lymphocytes.
Cell cycle‐dependent regulation of CDw75 (β‐galactoside α‐2,6‐sialyltransferase) on human B lymphocytes
Authors: Erikstein Bjørn K. , Funderud Steinar , Beiske Klaus , Aas‐Eng Anne , De Lange Davies Catharina , Blomhoff Heidi Kiil , Smeland Erlend B.
Published: March 2007

Within the hematopoietic system, CDw75 is primarily expressed on cells of the B cell lineage. Cloning and sequencing of the gene has shown CDw75 to be a β‐galactoside α‐2,6‐sialyltransferase. This enzyme plays an important role in the intracellular terminal glycosylation pathways in various cell types. In this article, we demonstrate that COS cells transfected with the CDw75 cDNA clone displayed sialyltransferase activity, in contrast to mock‐transfected cells. We also found that activated B cells displayed an increased enzyme activity compared to resting cells, in accordance with the staining data. Moreover, CDw75 expression was found to be up‐regulated approximately 7‐9‐fold from early G1 to the G2/M phases of the cell cycle in peripheral blood leukocyte B cells. This was shown by staining of in vitro activated B cells with the anti‐CDw75 monoclonal antibody HH2, using cell fractions corresponding to different stages of the cell cycle. Using a combination of Hoechst 33258 and propidium iodide after bromodeoxyuridine incorporation, it is possible to distinguish between different phases of the first and second cell cycle. By combining this with HH2 immunofluorescence staining, using a multistation multiparameter flow cytometry program, we confirmed the cell cycle‐dependent expression of CDw75. Immunocytochemical stainings of cytospin specimens of elutriated B cells showed that the antigen was up‐regulated in late G1 before the appearance of the nuclear activation antigen Ki67. Finally, we showed that activated B cells secreted soluble CDw75 into the medium, as demonstrated by a specific blocking of HH2 staining of B cells using suboptimal concentrations of HH2. In accordance with this, we observed small, but detectable levels of soluble sialyltransferase activity in supernatants of activated B cells.
Regulation of the p59fyn protein tyrosine kinase by the CD45 phosphotyrosine phosphatase
Authors: Mustelin Tomas , Pessa‐Morikawa Tiina , Autero Matti , Gassmann Martin , Andersson Leif C. , Gahmberg Carl G. , Burn Paul
Published: March 2007

Triggering of the T cell antigen receptor/CD3 (TcR/CD3) complex leads to rapid tyrosine phosphorylation of regulatory proteins that participate in initiating T cell activation and proliferation. This signal transduction event requires the presence of the TcR/CD3‐associated protein tyrosine kinase p59fyn. There is also evidence that the CD45 phosphotyrosine phosphatase is involved in TcR/CD3 signalling. We show here by capping experiments using double indirect immunofluorescence techniques that the receptor phosphotyrosine phosphatase CD45 and the intracellular protein tyrosine kinase p59fyn specifically co‐distribute in functional T lymphocytes. Furthermore, we provide evidence that isolated p59fyn is a substrate for CD45 as indicated by the rapid dephosphorylation of the regulatory Tyr531 of p59fyn by CD45. This dephosphorylation is accompanied by a severalfold increase in the catalytic activity of p59fyn as measured by its autophosphorylation and phosphorylation of an exogenous substrate. We also demonstrate that CD45‐mediated dephosphorylation and activation of p59fyn apparently occurs at a slow basal rate in resting T cells. This represents the first identification of a physiologic regulator of p59fyn and implies a mechanism for the role of CD45 in TcR/CD3 signal transduction.
Analysis of cytokine mRNA levels in interleukin‐4‐transgenic mice by quantitative polymerase chain reaction
Authors: Platzer Cornelia , Richter Günther , Überla Klaus , Müller Werner , Blöcker Helmut , Diamantstein Tibor , Blankenstein Thomas
Published: March 2007

Interleukin (IL)‐4‐transgenic mice were used as a model system to study the consequences of low levels of IL‐4 expression for the expression of other cytokines examined by quantitative polymerase chain reaction (PCR). For this purpose, a plasmid was constructed which contains, in tandem array, 5′ and 3′ primer sequences specific for the cytokine genes IL‐1 to IL‐6, tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN)‐γ and β‐actin. During co‐amplification, target and control DNA complete for the primers and the amount of PCR product is proportional to the amount of input DNA. Competitive PCR was performed first to adjust the cDNA to be compared to identical concentrations of β‐actin cDNA and subsequently to determine cytokine mRNA levels from spleen cells of normal and IL‐4‐transgenic animals. The sensitivity of this approach was demonstrated by the capability to detect a twofold difference in IL‐4 mRNA levels between IL‐4‐transgenic heterozygous and homozygous animals. Upon lipopolysaccharide activation, the IL‐4 transgene which is expressed essentially in B lymphocytes was induced approximately 50‐fold. Several cytokine mRNA such as those coding for IL‐5, IL‐6, IFN‐γ and also the IL‐4 receptor were found to be up‐regulated in IL‐4‐transgenic mice, whereas IL‐1, IL‐2, IL‐3, TNF and LT mRNA levels did not seemed to be influenced by IL‐4. A possible functional significance of the elevated IFN‐γ mRNA was demonstrated by showing that (a) CD23 expression was not increased, and (b) Mac‐1+ cells were markedly increased in the spleen of transgenic mice.