bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a bovine surface antigen uniquely expressed on CD4−CD8− T cell receptor γ/δ+ T lymphocytes
Authors: Clevers Hans , Machugh Niall D. , Bensaid Albert , Dunlap Sabrina , Baldwin Cynthia L. , Kaushal Anita , Iams Keith , Howard Christopher J. , Ivan Morrison W.
Published: March 2007

In this study, two monoclonal antibodies, IL‐A29 and CC15, are described that identify a novel bovine cell surface marker of 215/300 kDa. The antibodies reacted with a discrete population of resting lymphocytes in peripheral blood which, in young animals, constituted about 25% of the mononuclear cells. Thymus, lymph nodes and spleen contained < 5% positive cells. These cells were negative for surface Ig, a monocyte/granulocyte marker, and the T lymphocyte antigens CD2, CD6, CD4 and CD8. Immunohistological analyses revealed the presence of IL‐A29/CC15‐positive lymphocytes in the thymic medulla, in the outer cortex of lymph nodes, in the marginal zones of the spleen, in the dermal and epidermal layers of the skin and in the lamina propria of the gut. The IL‐A29/CC15+ cells in unfractionated blood mononuclear cells responded in autologous and allogeneic mixed lymphocyte cultures, and when purified they responded to concanavalin A in the presence of recombinant interleukin 2. These observations suggested this population of cells belonged to the T cell lineage. In order to unambiguously define their lineage, cDNA clones encoding bovine T cell receptor (TcR) and CD3 proteins were isolated. Northern blot analyses of IL‐A29/CC15+ cell populations and of established cell lines of various lineages demonstrated that they expressed TcR δ and CD3γ, δ and ϵ mRNA; TcR α was not expressed, whereas only a truncated form of TcR β mRNA was present. These results indicate that the IL‐A29 and CC15 antibodies define a unique population of CD4−CD8−, γ/δ T cells.
New B cell epitopes in the Plasmodium falciparum malaria circumsporozoite protein
Authors: Stüber Dietrich , Bannwarth Wilhelm , Pink J. Richard L. , Meloen Rob H. , Matile Hugues
Published: March 2007

Most antibodies directed against the Plasmodium falciparum circumsporozoite (CS) protein react with its central domain, which contains about 40 repeats of the tetrapeptide Asn‐Ala‐Asn‐Pro (NANP). To search for new epitopes in the non‐repetitive part of the CS protein, we expressed the non‐repetitive regions of the protein in E. coli as fusion proteins with mouse dihydrofolate reductase linked to six adjacent histidine residues. These fusion proteins were obtained at > 70% purity by a single Ni‐chelate affinity chromatography step. Of the new epitopes defined in the C‐terminal portion of the CS protein, three are located in a stretch of 65 amino acids immediately C‐terminal of the protein's central repetitive domain. Pooled sera from inhabitants of a malaria‐endemic area reacted with epitopes in this region of the molecule, and four mouse monoclonal antibodies to this region also reacted with the native CS protein on sporozoites. Two of the monoclonal antibodies reacted with a peptide PNDPNRNVD derived from a conserved region of the CS protein. The other two antibodies showed different reactivities to sporozoites of the NF54 and Ro59 parasite isolates. One, which reacted with a peptide ENANANNAV, recognized Ro59 but not NF54 sporozoites, while the other reacted with a small percentage of NF54 but not Ro59 sporozoites. Antibodies which react with non‐repetitive regions of the CS protein could contribute to maintaining its genetic variability.
Selective expansion of a specific anti‐tumor CD8+ cytotoxic T lymphocyte clone in the bulk culture of tumor‐infiltrating lymphocytes from a melanoma patient: Cytotoxic activity and T cell receptor gene rearrangements
Authors: Gervois Nadine , Heuze Françoise , Diez Elisabeth , Jotereau Francine
Published: March 2007

In vitro culture of metastatic melanoma fragments with 150 units of recombinant interleukin 2 resulted in the successive expansion of CD4+ and then CD8+ tumor‐infiltrating lymphocytes (TIL) throughout a 2‐month period. TIL cultured for 43 days and consisting of 95% CD8+ and 10% CD4+ T lymphocytes were cloned by limiting dilution (LD). Thirteen CD8+ and thirty‐one CD4+ clones were obtained, indicating that the frequency of clonogenic CD8+ proliferative T lymphocytes was much lower than that of their CD4+ homologues. When LD was performed in the presence of autologous melanoma cells the frequency of CD8+ clones was increased by factor 4. The DNA from TIL of day 43 bulk culture and of six CD8+ clones was hybridized with T cell receptor (TcR) β and γ probes. Identical configuration of the nonfunctional γ and functional β TcR genes was found in “bulk culture” and cloned TIL. The CD8+ clones therefore derived from a clonal population of CD8+ cells which had expanded in vitro before the LD.

All the CD8+ clones tested were strongly cytotoxic for autologous melanoma cells but did not kill autologous fibroblasts or concanavalin A blasts, or any of the 10 allogeneic tumor targets tested, including 5 melanomas, 2 breast cell lines, 1 neuroblastoma, K‐562 and the Epstein‐Barr virus‐transformed cell line used as a feeder. Furthermore, specific killing was inhibited by monoclonal antibodies against CD3, CD8, TcR α/β and against class I major histocompatibility complex antigens indicating that these cytotoxic T lymphocyte clones recognized autologous tumor cells through the TcR, in an HLA class I‐restricted manner. These data show that it is feasible to obtain tumor‐specific cytotoxic T lymphocytes from melanoma TIL with a simple culture technique and that a single clone could be expanded to more than 1010 cells which should allow testing of immunotherapeutic potential of these cells by adoptive transfer into melanoma patients.
Immunoglobulin‐specific T‐B cell interaction III. B cell activation by immunoglobulinrecognizing T cell clones
Authors: Mazel Svetlana M. , Rudensky Alexander Y. , Yurin Vitalij L.
Published: March 2007

Immunoglobulin (Ig)‐specific T‐B cell interactions were studied in the model of T cell recognition of Ig ϰ chain Igϰ‐1b allotype in Igϰ‐1‐congenic rats. Using Igϰ‐1b‐recognizing major histocompatibility complex (MHC)‐restricted T helper clones from August rats we have shown that Igϰ‐1b+ B cells from congenic August. 1b rats presented Igϰ‐1b epitope of the processed self‐synthesized Ig to T clones. This interaction was found to be a bidirectional regulatory event inducing specific MHC‐dependent proliferation of both interacting T cell and B cell as well as Ig(Igϰ‐1b) synthesis. Small Igϰ‐1b+ B cells were capable of inducing T clone proliferation and becoming activated in response to the same T clone. Limiting dilution analysis suggested that every tenth cell in Igϰ‐1b+ B cell population is involved in this interaction. The bystander activation of Igϰ‐1a+ B cells by T clones in the presence of irradiated Igϰ‐1b+ spleen cells, if observed, was less than the level of specific Igϰ‐1b+ B cell proliferation. In contrast to a 20‐fold increase of Ig(Igϰ‐1b) levels upon stimulation of Igϰ‐1b+/1a+ B cell population from heterozygous (August × August.1b)F1 rats by T clones, a “nonspecific” increase of Ig(Igϰ‐1a) was not observed. This result demonstrates the requirement for direct T‐B contact for B cell activation to occur. The data suggest a great functional potency of T‐B interactions mediated by T cell recognition of Ig‐derived peptide/MHC class II complexes on the B cell surface. The implication of the data for idiotypic regulation enables us to propose the existence of putative idiopeptidic network T‐B cell interactions.
Absence of demonstrable phospholipid turnover in B cells stimulated by low mitogenic concentrations of dextran‐anti‐immunoglobulin conjugates
Authors: Brunswick Mark , Bonvini Ezio , Francis Mark , Felder Christian C. , Hoffman Thomas , Mond James
Published: March 2007

Previously we have demonstrated that when anti‐immunoglobulin (Ig) is conjugated to high molecular weight dextran (Dex) it stimulates B cell activation at pg/ml concentrations in the absence of detectable phosphoinositide hydrolysis or increases in intracellular ionized calcium. To study carefully whether anti‐Ig‐Dex recruited a phosphoinositide‐dependent pathway of activation, we stimulated B cells that were labeled with 32P and [3H]glycerol with anti‐Ig‐Dex conjugates at concentrations ranging from 1—1 × 10−4 μg/ml. Thirty seconds to thirty minutes after stimulation lipids were extracted and analyzed by thin layer chromatography and spots correlating with known lipid standards were isolated and counted. There was a four‐ and tenfold increase in the ratio of 32P/3H incorporated into phosphatidic acid (a metabolite of diacylglycerol) and phosphatidylinositol, respectively, when cells were stimulated with 0.1–1.0 μg/ml of anti‐Ig‐Dex for 30 min. Below 1 ng/ml there was no detectable increase in the turnover of these metabolites despite the fact that in parallel cultures B cells were stimulated to proliferate by this concentration of anti‐Ig‐Dex. To determine whether a cAMP‐dependent pathway was recruited by low concentrations of conjugates, we evaluated cAMP levels from B cells that were stimulated with anti‐Ig‐Dex for 5–60 min using a radioimmunoassay. While cholera toxin stimulated a 50–100‐fold increase in the levels of cAMP, we observed no alteration in cAMP in anti‐Ig‐stimulated cells. These results support and extend our previous findings by demonstrating that B cell activation that is induced by cross‐linking of surface Ig may not stimulate phosphoinositide‐dependent or cAMP‐dependent pathways of activation. Possible alternative mechanisms of activation will be discussed.
Protein sequence and gene structure for mouse leukosialin (CD43), a T lymphocyte mucin without introns in the coding sequence
Authors: Cyster Jason , Somoza Chamorro , Killeen Nigel , Williams Alan F.
Published: March 2007

A partial cDNA clone for mouse leukosialin was isolated by use of a rat leukosialin cDNA probe. The mouse cDNA was then used to isolate genomic clones that corresponded to the two mouse genes detected in Southern blots. One gene encoded an open reading frame for the homologue of rat leukosialin and this gene was notable for the absence of introns within the coding sequence. A lack of introns has previously been observed for the human leukosialin gene (Shelley, C. S., Remold‐O'Donnell, E., Rosen, F. S. and Whitehead, A. S., Biochem. J., submitted). The other mouse gene was an intronless pseudogene for a leukosialinrelated sequence. The presence of only one functional gene that lacked coding‐region introns established that molecular heterogeneity in mouse leukosialin could not arise from multiple genes or alternative splicing of exons. The sequence of mouse leukosialin suggested an extracellular segment with a high content of O‐linked carbohydrate, as is the case in the rat and human. In addition the mouse molecule had one possible N‐linked glycosylation site. The cytoplasmic domain of 124 amino acids was highly conserved between rodent and human leukosialins for the functional genes but not for the pseudogene. This suggests an important functional role for the cytoplasmic domain.
Microheterogeneity of human interleukin 6 synthesized by transfected NIH/3T3 cells: Comparison with human monocytes, fibroblasts and endothelial cells
Authors: Schiel Xaver , Rose‐John Stefan , Dufhues Gabi , Schooltink Heidi , Gross Volker , Heinrich Peter C.
Published: March 2007

A cDNA containing the entire coding region of human interleukin 6 (IL 6) was stably expressed in murine NIH/3T3 fibroblasts using a bovine papilloma virus‐based expression vector with a metallothionein promoter. Expression of IL 6 in transfected cells was highly inducible by heavy metals like cadmium as measured at mRNA and protein levels. Cadmium‐stimulated transfected NIH/3T3 cells synthesized and exported biologically active IL 6 (1.7 × 104 U/106 cells/24 h). IL 6 from the culture medium of transfected NIH/3T3 cells exhibited at least eight bands on Western blots after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, indicating that human IL 6 expressed in NIH/3T3 cells shows a complex glycosylation pattern. Using glycosidases and the N‐glycosylation inhibitor tunicamycin it was possible to discriminate between five species carrying N‐linked carbohydrate side chains and two species carrying only O‐linked side chains. In addition, a substantial amount of unglycosylated IL 6 was observed. IL 6 from transfected NIH/3T3 cells differed markedly in its glycosylation pattern from those of stimulated human monocytes, fibroblasts and endothelial cells.
Absolute numbers of lymphocyte subsets migrating through the compartments of the normal and transplanted rat spleen
Authors: Willführ Karl Ulrich , Westermann Jürgen , Pabst Reinhard
Published: March 2007

Lymphocyte migration is one of the basic principles of the immune system. Up to now lymphocyte migration experiments have been performed either in a quantitative way, determining whole organ recoveries of radiolabeled lymphocytes without histologic localization, or based on autoradiography which does not provide absolute numbers of immigrant lymphocytes. In this study the traffic of lymphocyte subsets through the splenic compartments: red pulp (RP), marginal zone (MZ), periarteriolar lymphatic sheath (PALS) and follicle was evaluated in absolute numbers. In normal spleens and splenic transplants fluorescein isothiocyanate (FITC)‐labeled immigrant lymphocytes were localized and characterized immunohistochemically in cryostat sections by light microscopy. In addition morphometry of the splenic compartments was performed and the recovery of 51Cr‐labeled lymphocytes in the spleen was determined. The combination of these methods allowed total numbers of immigrant subset cells to be calculated in individual splenic compartments.

At 15 min about 17% of the injected B lymphocytes were found in the MZ. This is the largest fraction of an injected lymphocyte subset found in a single splenic compartment. At 24 h immigrant B cells were not only found in the follicle, but they had reached comparable numbers in the three compartments: follicle, RP and MZ. Most immigrant T lymphocytes were found in the PALS, which from 6 h after injection onwards contained more T cell immigrants than any single organ of the body. CD4+ and CD8+ lymphocytes showed a similar distribution throughout the splenic compartments at early time points. At 24 h CD4+ lymphocytes homed preferentially to the PALS, whereas CD8+ cells seemed to prefer the RP and MZ. Both CD4+ and CD8+ cells also migrated into the follicles. In regenerated splenic tissue after autotransplantation lymphocyte immigration was reduced in all compartments and to the MZ in particular. An impaired lymphocyte migration to the MZ in splenic transplants may be one reason for the lack of protection provided against bacterial infections. Thus examining lymphocyte migration in absolute numbers provides additional information which cannot be gained by determining labeling indices or percentages of lymphocyte subsets alone.
Selective expression of CD8α (Ly‐2) subunit on activated thymic γ/δ cells
Authors: Robson MacDonald H. , Schreyer Magali , Howe Rawleigh C. , Bron Claude
Published: March 2007

Although most cells of the T cell receptor (TcR) γ/δ lineage are CD4−CD8−, some γ/δ cells express CDS. We show here that mouse thymic γ/δ cells express CD8 following in vitro activation by concanavalin A. Staining with monoclonal and polyclonal antibodies to the CD8 α (Ly‐2) and CD8 β (Ly‐3) subunits indicates that only CD8 α is expressed by these activated TcRγ/δ cells. Furthermore, immunoprecipitation and reduced/nonreduced gel analysis reveals that thymic γ/δ cells express CD8 α as a homodimer. In contrast, similarly activated TcR δ/β cells express a conventional CD8 α/CD8 β heterodimer.
Type II collagen‐specific human T cell lines established from healthy donors
Authors: Lacour Michael , Rudolphi Ulrike , Schlesier Michael , Peter Hans‐Hartmut
Published: March 2007

Four type II collagen‐specific T cell lines established from the peripheral blood of a healthy donor were studied in detail. These CD4+ T cell lines with an α/β T cell receptor proliferated in response to native and denatured human and chicken type II collagen and human type I collagen, but not to human type IV collagen or other potentially arthritogenic antigens. The T cell response showed typical dose response characteristics, peaked between 30 and 48 h, was major histocompatibility complex class II restricted and not due to a mitogenic effect. Type II collagen‐reactive T cells could hardly be detected in freshly isolated peripheral blood mononuclear cells from healthy donors, as revealed by limiting dilution analysis (frequency < 1/100000). By prestimulation in bulk cultures for 10 days, type II collagen‐reactive T cells could be approximately 1000‐fold enriched. However, in these limiting dilution cultures, collagen‐reactive T cells could only be observed in a narrow window of cell concentrations, suggesting that type II collagen‐reactive T cells may be controlled by suppressive mechanisms in healthy persons.
Production and regulation of interleukin 6 in human B lymphoid cells
Authors: Hutchins David , Cohen Brian B. , Michael Steel C.
Published: March 2007

Human B cell lines were screened for production of interleukin 6 (IL 6) by the B9 hybridoma cell bioassay. Some long‐established lines such as RPMI 1788, CESS and recently established early‐passage Epstein‐Barr virus (EBV)‐transformed lymphoblastoid lines were constitutive IL 6 producers. Other long‐established lymphoblastoid lines such as RPMI 8866 and SKW6.4 and all Burkitt lymphomalines tested were nonproducers of IL 6.

Constitutive production of IL 6 in early passage lines could be enhanced by the phorbol ester phorbol 12‐myristate 13‐acetate (PMA) and recombinant (r)IL 4 but not by rIL 1α or rIL 1β. Nonproducing EBV‐transformed lymphoblastoid cell lines could be induced to IL 6 production by PMA, rIL 1α, IL 1β and IL 4. Amongthe nonproducing lines SKW6.4 was induced to IL 6 production by PMA, rIL 1α, rIL 1β and rIL 4, whereas RPMI 8866 was induced only by PMA, to a limited extent by rIL 1α and rIL 1β but not at all by rIL 4. There was no induction of IL 6 by any recombinant cytokine in the two Burkitt lymphoma lines but measurable production of IL 6 was induced by PMA in one of them (EB4). Cytokines which wereneither enhancers nor inducers of cell lines on their own included rIL 2, rIL 5, interferon (rIFN)‐γ, native purified IFN‐α, tumor necrosis factor (rTNF)‐α, rTNF‐β and purified platelet‐derived transforming growth factor‐β. rIL 4 synergized with either rIL 1α or rIL 1β in the induction of IL 6 in the nonproducing line SKW6.4. Similar effects were also seen in this line with combinations of (a) rIFN‐γ and rIL 4 and (b) IFN‐α and both rIL 1α and rIL 1β. rIL 4, with or without rIL 1, was more effective than rIL 6 in the induction of IgM synthesis in SKW6.4 and the effect was only partially inhibited by anti‐IL 6antiserum at a dose which totally inhibited IL 6‐induced IgM production.

Normal peripheral blood lymphocyte populations pre‐activated by anti‐immunoglobulin rosetting exhibited enhanced production of IL 6 in the presence of rIL 4 and PMA but not in the presence of rIL 1, in contrast to the behavior of adherent mononuclear blood cells which showed IL 4‐induced down‐regulation of IL 6 production. Human B lymphoblastoid cell‐derived IL 6 was shown by Western blotting (using a polyvalent goat antiserum) to be composed of at least three distinct bands between 21 and 30 kDa, andboth constitutively produced and induced activities were neutralized by the same antiserum.

The finding that human B cell lines differ in their constitutive and inducible productionof IL 6, a cytokine implicated in both the growth and differentiation of these cells, is discussed with respect to its possible role as an autocrine factor.
Effect of bacterial toxins on human B cell activation I. Mitogenic activity of pertussis toxin
Authors: Kolb Jean‐pierre , Genot Elisabeth , Petit‐koskas Elisabeth , Paul‐eugene Nathalie , Dugas Bernard
Published: March 2007

Pertussis toxin (PT) was found to elicit an increased thymidine uptake in resting B lymphocytes purified from human peripheral blood. A significant mitogenic effect was detected for toxin concentrations > 100 ng/ml (1 nM) and a plateau of stimulation was reached at 1000 ng/ml (10 nM). B cell blasts, activated by a first signal such as Staphylococcus aureus Cowan I or insolubilized anti‐μ chain antibody, were also stimulated to DNA synthesis by PT in the same range of concentrations. At lower sub‐mitogenic concentrations, the toxin potentiated the response to the low‐molecular weight B cell growth factor (LMW‐BCGF or 12‐kDa BCGF), a progression factor for activated B cells. The “A” or catalytic subunit was devoid of any activity on B cells, suggesting the stimulatory effect of the toxin might be associated with the binding or “B” subunit, as it has been shown for T cells. This hypothesis was strengthened by the observation that, as in T cell, the whole toxin but not the “A” promoter, was able to induce calcium influx in these cells. In addition, the purified “B” oligomer alone was found to promote DNA synthesis in B cells. Finally, a fragment of the soluble cleaved form of the CD23 molecule (FcϵRII) could be involved in the process of PT mitogenicity for B cells.
Selective peripheral expansion and activation of B cells expressing endogenous immunoglobulin in μ‐transgenic mice
Authors: Grandien Alf , Coutinho António , Andersson Jan
Published: March 2007

Two different lines of C57BL/6 mice (IgHb) carrying complete rearranged μ chain genes from BALB/c (IgMa) were analyzed for the expression and secretion of endogenous as well as transgenic immunoglobulins at the level of single cells. Quantitation of B cells expressing endogenous IgMb by cytofluorometry, limiting dilution analyses of clonal precursors and secretory cell assays revealed a marked selective expansion, activation and terminal differentiation of those cells producing endogenous immunoglobulins. Thus, the very infrequent IgMb−bearing B cells produced in bone marrow of transgenic mice accumulate in spleen, where they are activated and account for roughly half of all natural immunoglobulin‐secreting cells.

These observations indicate that μ‐transgenic mice are valuable in studies of the antibody repertoire selection operating in unprimed animals but their use could be misleading in the analyzing ''monoclonal'' immune system.
Induction of cell‐associated interleukin 1 through stimulation of the adhesion‐promoting proteins LFA‐1 (CD11a/CD18) and CR3 (CD11b/CD18) of human monocytes
Authors: Couturier Christine , Haeffner‐cavaillon Nicole , Weiss Laurence , Fischer Elisabeth , Kazatchkine Michel D.
Published: March 2007

Serum‐free culture of human monocytes in the presence of monoclonal antibodies to the LFA‐1 α chain (CD11a), CR3 α chain (CD11b) or β chain (CD18) bound to Sepharose induced the dose‐dependent production of cell‐associated interleukin (IL) 1 activity and of IL 1α and IL 1β antigens, but no release of extracellular IL 1 activity or antigen in the culture medium. Triggering of IL 1 production was also observed with insolubilized anti‐CD11/CD18 F(ab')2 antibodies. Two cross‐linked antibodies recognizing distinct epitopes on the CD11b molecule induced cell‐associated IL 1. Soluble antibodies did not induce IL 1 production. The kinetics of induction of IL 1 by stimulation of adhesion‐promoting proteins differed from those of IL 1 induction by adhesion to plastic. The lack of induction of IL 1 release by stimulation of the CD11/CD18 molecules resembled the intracellular accumulation of IL 1 induced by lipid A. Induction of IL 1 by adhesive processes may be a mechanism by which T cells trigger IL 1 production by monocytes during antigen presentation.
Antigen‐induced inhibition of autoimmune response to rat male accessory glands: distinct role of I‐A and I‐E‐positive peritoneal cells
Authors: Ferro Maria Elena , Romero‐piffiguer Marta , Riera Clelia M.
Published: March 2007

The present report describes the structural and functional characteristics of the population of peritoneal cells (PC) from rats injected intraperitoneally (i.p.) 2 h or 24 h previously with a suppressor dose of a purified fraction (FI) of rat male accessory glands (RAG; FI‐PC2h and FI‐PC24h, respectively). Both populations of PC showed the presence of the autoantigens of RAG on their membrane. The study of cell surface marker showed increase of OX17 (I‐E) in FI‐PC2h and OX6 (I‐A) in FI‐PC24h. I.p. injection of FI‐PC2h 10 and 3 days prior to immunization with chemically modified (diazotized arsanilic and sulfanilic acid) RAG (MRAG) in complete Freund's adjuvant induced suppression of the delayed‐type hypersensitivity (DTH) response to MRAG, whereas the i.p. injection of FI‐PC24h induced potentiation of DTH responseto MRAG when compared with controls (animals injected 10 and 3 days prior to immunizationwith PC from rats injected i.p. with an unrelated antigen). The treatment of FI‐PC2h with OX17 and FI‐PC24h with OX6 prior to the transfer blocked the suppression and potentiation described above. These findings show that the i.p. injection of an FI of RAG at a suppressor dose induces PC with immunologically opposite characteristics, depending on the time of obtention.
Antigenic phenotyping of human follicular dendritic cells isolated from nonmalignant and malignant lymphatic tissue
Authors: Petrasch Stephan , Perez‐alvarez Carmelo , Schmitz Jörn , Kosco Marie , Brittinger Günter
Published: March 2007

Follicular dendritic cells (FDC) are located within follicles of secondary lymphoid tissue and in lymph nodes of patients with germinal center cell‐derived non‐Hodgkin lymphomas. Reliable antigenic phenotyping of FDC within tissue sections has been difficult due to simultaneous labeling of the surrounding germinal center cells. Using anenzyme cocktail to digest human tonsils and cervical lymph nodes with subsequent fractionation by albumin gradient centrifugation, cell isolates containing up to 20% FDC were obtained. This preparation allowed the determination of antigenic phenotype on individual FDC. Molecules expressed by FDC were detected by an isotype‐specific immunocytochemical double‐labeling procedure, i.e. a monoclonal antibody (mAb) specific for FDC (KiM4 or DRC1) in conjunction with a mAb reactive against an additional antigenic determinant. Nonspecific binding of mAb to immunoglobulin Fc receptors located on FDC membranes was avoided by incubation of cells with human IgG aggregates prior to immunostaining. The results revealed that isolated FDC from these lymphoid tissues express transferrinreceptors, the intercellular adhesion molecule 1, class II antigens, the B cell antigens CD20 and CD21, and the myelomonocytic properties CD11b and CD14. Immunoglobulin μ or γ heavy chains and the B cell antigens CD23 and CD24 are detected on 50% of an isolated FDC population. These FDC are negative for the T helper cell antigen CD4, the B cell cell antigens CD19 and CD22, the immunolobulinα and δ chains and the S‐100 protein.

FDC isolated from lymph nodes of patients with low‐grade malignant non‐Hodgkin lymphoma, identified by DRC1 or KiM4 mAb, presented the same antigenic profile as seen on FDC from nonmalignant tissue. This suggests that FDC from lymphoma tissue isolated in this manner have the same properties as those found in normal tissue.
VH gene family repertoires of “viable motheaten” (mev) mice
Authors: Freitas Antonio A. , Sidman Charles L.
Published: March 2007

Mutant viable motheaten (mev) mice provide an useful experimental model to study the origin and molecular properties of autoantibodies. In the present investigation we have compared by in situ hybridization VH gene family usage in lipopolysaccharide‐activated B cells (available repertoire) and spontaneously immunoglobulin‐secreting (actual repertoire) B cells in the spleen of 6—8‐week‐old BALB/c and mutant BALB/c‐mev mice. We have found that while sharing identical available splenic repertoires and expressing a diversified set of VH families, mev mice differ from control BALB/c animals in VH family representation in the actual plasma cell repertoires where they showed a decreased utilization of VH7183 genes and an increased representation of the VHJ606 family when compared to control BALB/c animals. These results indicate that selection of actual repertoires may indeed differ between autoimmuneand control mice, but do not establish whether such changes are the primary cause of the disease or whether they are secondary to the initiating of the autoimmune process.
Analysis of type II collagen‐reactive T cells in the mouse I. Different regulation of autoreactive vs. non‐autoreactive anti‐type II collagen T cells in the DBA/1 mouse
Authors: Andersson Mikael , Holmdahl Rikard
Published: March 2007

The T cell reactivity against type II collagen (CII) was analyzed in the collagen‐induced arthritis‐susceptible mouse strain DBA/1. It was shown that the proliferative response in lymph node cells from rat CII‐immunized mice was mainly directed against a foreign determinant present on all heterologous CII tested but not on autologous CII. A T cell line with this reactivity reacted with high sensitivity with CII and the determinant was mapped to the CB11 fragment of CII. A weak autoreactive response could be detected in the primary cultures using high concentrations of mouse CII and this reactivity remained after several stimulations with high concentrations of rat CII but not with low concentrations of rat CII. A similar response against mouse CII but with only limited cross‐reactivity to rat CII was seen when culturing the cells with mouse CII as antigen. The optimal concentration for the autoreactive response was always more than 100‐fold higher than for the response of the T cells specific for heterologous CII. An anti‐CII T cell response could also be detected in spleen cells from unimmunized mice and the strongest response was obtained using autologous CII. These results suggest that T cells recognizing self CII are normally activated in the DBA/1 mouse and possibly as a consequence exhibit a clonal anergy pattern with a weak proliferative response only at high concentrations of CII.
Phytohemagglutinin induces major short‐term protease‐sensitive lymphocyte traffic involving high endothelium venule‐like blood vessels in acute delayed‐type hypersensitivity‐like reactions in skin and other tissues
Authors: Binns Richard M. , Licence Stephen T. , Peter Wooding F. B.
Published: March 2007

Injection of phytohemagglutinin induces dose‐dependent acute delayed‐type hypersensitivity‐like reactions in young pig skin which attract large numbers of labeled peripheral blood lymphocytes and lymphoblasts but not red cells. Similar reactions are induced in the gut wall, joints, and draining lymph nodes, and also in the skin of sheep and cattle, and by concanavalin A. Labeled peripheral blood lymphocyte entry starts within 2 h, is maximal at 6—18 h and is effectively over by 48 h and, both earlyand late, is markedly inhibited in a selective way by trypsinization, like high endothelium venule‐mediated entry to lymphoid tissues. Electron microscopy showed development of high endothelium venule‐like blood vessels with intramural lymphoid cells inmononuclear infiltrates. Inductive processes in this model short‐term peripheral defense mechanism are being investigated.
Cross‐linking of the surface immunoglobulin on lymphocytes from the bursa of Fabricius results in second messenger generation
Authors: Ratcliffe Michael J. H. , Tkalec Lydia
Published: March 2007

The bursa of Fabricius represents the major site of B lymphocyte development in avianspecies. Although the vast majority of bursal lymphocytes express cell surface immunoglobulin (sIg), it is generally considered that the bursa does not represent a significant site of antigen‐induced B cell maturation to Ig secretion. However, the question as to whether antigen, either exogenous or self, can induce positive or negative selection of bursal lymphocytes in such a way as to modify the peripheral B cell repertoire remains open. Clearly, such intrabursal selection would require that bursal lymphocyte sIg have the molecular machinery to transduce signals into the cell as a consequence of its interaction with antigen.

In this report we demonstrate that exposure of bursal lymphocytes to antibodies directed against sIg induced a rapid increase in cytosolic free calcium ion concentrations [Ca2+]i. Furthermore, such antibodies also induced a rapid increase in intracellular phosphatidic acid concentrations followed by a rise in intracellular phosphatidylinositol. Increased [Ca2+]i, phosphatidic acid and phosphatidylinositol levels required the cross‐linking of sIg and were not induced by antibodies to other bursal cell surface antigens. Thus, cross‐linkingof the sIg on bursal lymphocytes results in second messenger generation, demonstrating that bursal sIg is a functional signal transduction element.
Induction of germ‐line immunoglobulin heavy chain transcripts by mitogens and interleukins prior to switch recombination
Authors: Severinson Eva , Fernandez Carmen , Stavnezer Janet
Published: March 2007

It has recently been postulated that immunoglobulin class switching is preceded by transcription from unrearranged heavy chain genes. In this report, we have investigated the conditons under which RNA transcribed from unrearranged Cγ3, Cγ1, Cγ2b, Cγ2a, Cϵ and Cα genes are induced in normal spleen cells by mitogens and/or interleukin (IL)4, IL5 and interferon‐γ. Lipopolysaccharide (LPS) plus IL4 induced germ‐line γ1 and ϵ transcripts. LPS induced γ2b and γ3 transcripts and high doses of IL4 suppressed these LPS‐induced transcripts. Interferon‐γ induced low levels of germ‐line γ2a transcripts and profoundly suppressed the γ1 and ϵ transcripts induced by LPS and IL4. IL5 alone or in combination with IL4 and/or LPS did not induce germ‐line α transcripts. Spleen cells of the partially immunodeficient mice CBA/N and C3H/HeJ, which do not express IgG3 could be induced, however, by polyclonal activators to express germ‐line γ3 and γ2b transcripts. The data indicate that the capacity of a ligand to induce/suppress transcription of a particular unrearranged heavychain gene is a good indicator of its capacity to induce switching to the corresponding Ig isotype. However, it is also clear that control of switching can be carried out at other levels.
Correlations of autoimmunity with H2 and T cell receptor β chain genotypesin (NZB x NZW) F2 mice
Authors: Noonan Daniel J. , McConahey Patricia J. , Cardenas Gustavo J.
Published: March 2007

Accelerated autoimmunity as expressed by the classical autoimmune strain mouse (NZB XNZW)F1 is thought to be the result of major histocompatibility complex (MHC)‐associated NZW genes acting on a genetic predisposition for autoimmunity as expressedby the NZB mouse. To evaluate more accurately both H2 and T cell receptor (TcR) β chain involvement in F1 disease, we studied the segregation of NZB (H2d, TcRB) and NZW (H2w, TcRW) haplotypes of these genetic elements and the development of autoimmunity in (NZB X NZW)F2 generation mice. F2 mice with the H2d/z genotype lived shorter average life‐spans and expressed elevated levels of antibodies to gp70, ssDNA and dsDNA, while those with the TcRW/W genotype (homozygous for the NZW TcR deletion) expressed elevated levels of autoantibodies but had relatively long life‐spans. On the other hand, mice with the TcRB/B genotype (homozygous for the NZB TcR) produced consistently low levels of autoantibodies but died at an early age. The most severely affected F2 population were the mice carrying both the TcRB/B and H2d/z alleles. These mice died on an average within the first 5 months of life, but produced the lowest levels of antibodies to gp70, single‐stranded DNA and double‐stranded DNA. These data confirm the contribution of NZW H2linked genes to accelerated autoimmunity in the F1 hybrid and, furthermore, define NZB TcR‐linked components as primary developers of this phenomenon. They also suggest a limited, if any, contribution of both the NZW TcR deletion and traditional autoantibodies to F1 accelerated autoimmunity.
Eosinophil cytotoxicity enhancing factor: purification, characterization and immunocytochemical localization on the monocyte surface
Authors: Elsas P. Xavier , Capella Elsas M. I. Gaspar , Dessein Alain J.
Published: March 2007

The monokine eosinophil cytotoxicity enhancing factor (ECEF) increases antibody‐dependent cytotoxicity of eosinophils towards helminth larvae. A monokine biochemicallyindistinguishable from ECEF increases the release of leukotriene C4 and other arachidonicacid metabolites by eosinophils. We have developed monoclonal antibodies (mAb) to these monokines by immunizing mice with ECEF made by the U‐937 histiocytic lymphoma cell line. mAb 81.10.C9 (IgG2b) and 9A6G (IgG1) inhibit the effect of the monokine on release of A A products. Both mAb bind ECEF, which appears after affinity chromatography purification as a major 13—14‐kDa and a minor 62‐kDa component (13—14 kDa and 52 kDa after reduction) in silver‐stained gels. An additional component of 30 kDa is detectable after radioiodination of the immunopurified material. The specificity of both mAb was studied in several ways. In immunoprecipitation, bothrecognize the 13—14‐kDa and the 30‐kDa components, while the 62–(52)‐kDa protein is not significantly precipitated. Both mAb react in enzyme‐linked immunosorbent assay with products secreted by peripheral blood mononuclear cells and monocytes, as well as with those secreted by phorbol 12‐myristate 13‐acetateand lipopolysaccharide‐stimulated U‐937 cells and with the immunopurified proteins. These were separated in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, electroeluted and assayed for ECEF activity. Activity was associated with the 13—14‐kDa and the 30‐kDa fractions, as seen by increased eosinophil antibody‐dependent adherence to schistosomula and cytotoxicity. Granulocyte‐monocyte‐colony‐stimulating factor and interleukin 1, but not tumor necrosisfactor, could be detected in crude U‐937 supernatants. However, active immunopurified ECEF has no activity in assays for granulocyte‐monocyte‐colony‐stimulating factor, interleukin 1 or tumor necrosis factor. Immunocytochemical localization of ECEF employing the mAb shows strong surface staining of viable monocytes and U‐937 cells, suggesting that ECEF is associated to the cell surface. These properties distinguish ECEF from other monokines previously reported to activate eosinophils.
A novel pathway of human B cell activation initiated by CK226 surface antigen
Authors: Poggi Alessandro , Maggi Enrico , Biagiotti Roberta , Giudizi Maria Grazia , Almerigogna Fabio , Pella Nicoletta , Caligaris‐cappio Federico , Romagnani Sergio , Moretta Lorenzo
Published: March 2007

In this study we analyzed the effect of CK226 monoclonal antibody (mAb) on human B cell activation and proliferation. This mAb was shown to recognize a 75‐kDa surface molecule expressed on both T and B lymphocytes and to mediate T lymphocyte activation andproliferation. Flow cytometry analysis of B cell populations isolated from peripheral blood, tonsil and spleen showed that CK226 surface antigen is highly expressed on 40—80% of surface Ig+ cells. When purified B cells were cultured in the presence of CK226 mAb, up‐regulation of major histocompatibility complex class II and CD23 surface structures and the de novo expression of CD25 antigen could be detected within 48 h. In addition, B cells underwent proliferation ([3H] thymidine uptake) in the absence of either T cells or exogenous lymphokines. Proliferation was potentiated by the addition of suboptimal concentrations (0.5 ng/ml) of phorbol 12‐myristate 13‐acetate (PMA). Cells recovered at day 5 were surface Ig+ and no CD3+ cells could be detected. CK226‐induced proliferation (either in the presence or in the absence of PMA) was not inhibited by anti‐CD25 mAb. Addition of exogenous interleukin 2 to CK226‐stimulated B cells resulted in further increase of B cell proliferation. On the other hand, CK226 mAb did not display a co‐stimulatory effect with submitogenic concentrations of either anti‐Ig antibody or Staphylococcus aureus Cowan strain I bacteria. In addition proliferation induced by mitogenic concentrations of the above stimuli was inhibited in a dose‐dependent fashion by CK226 mAb.
T cell receptor‐bearing cells among rat intestinal intraepithelial lymphocytes are mainly α/β+ and are thymus dependent
Authors: Vaage John Torgils , Dissen Erik , Ager Ann , Roberts Ian , Fossum Sigbjørn , Rolstad Bent
Published: March 2007

Rearrangement of both the β and γ chain T cell receptor (TcR) genes was detected in intestinal intraepithelial lymphocytes (IEL) from normal euthymic rats. Flow cytometric analyses showed that about 73% of the IEL were CD3+ (1F4) and that67% were TcR α/β+ (R73). About 5% of the IEL were found to be CD3+, TcR α/β− in double‐labeling experiments suggesting that a small fraction of IEL in the rat express the alternative TcR γ/δ. More than 70% of the IEL were granular implying that many CD3+ IEL are granular. In IEL from athymic nude rats no rearrangement of either the TcR β or γ chain genes or surface expression of CD3 or TcR α/β was detected despite the fact that about 95% of the cells were granular and morphologically similar to those in normal rats. Taken together our data suggest that the majority of IEL in the rat express the conventional TcR α/β and that TcR‐bearingcells in the gut epithelium are thymus dependent.
Cytofluorometric and cytomorphologic analysis of human bone marrow cells derived fromstromal cultures stimulated by granulocyte‐macrophage colony‐stimulating factor, interferon‐γ and splenopentin pentapeptide
Authors: Maciejewski Jaroslaw , Wächter Marlies , Elstner Elena , Weber Harald , Diezel Wolfgang , Seiler Friedrich R. , Volk Hans‐dieter
Published: March 2007

We studied the influence of human recombinant granulocyte‐macrophage colony‐stimulating factor (hrGM‐CSF), human recombinant interferon‐γ (hrIFN‐γ) and splenopentin pentapeptide (Sp‐5), either alone or in combination, on the proliferation and differentiation of human bone marrow cells in modified Dexter's cultures. After 10, 14 and 21 days cells were analyzed by classical staining according to Pappenheim and by cytofluorometry with a set of different monoclonal antibodies. IFN‐γ inhibited the proliferation of progenitor cells and provided signals promoting monocytic differentiation, whereas GM‐CSF induced the proliferation of blastoid elements which expressed HLA‐DR and M2 (VIM‐2 monoclonal antibody), but progressively lost surface CD34. Furthermore, an increase of CD15+ cells was also observed. When GM‐CSF was tested in combination with IFN‐γ, it abolished the inhibitory effect of IFN‐γ and both cytokines synergized to promote the expression of CD11c, CD14 and M2 surface antigens. Sp‐5 alone had only a marginal activity, but it potentiated the effects of GM‐CSF. These findings suggest that GM‐CSF may induce the transition from stem cells to committed myeloid progenitors. In contrast to IFN‐γ, Sp‐5 can serve as an additional proliferative signal with negligible effects on cell maturation.
Study of the thymocyte cell cycle by bivariate analysis of incorporated bromodeoxyuridine and DNA content
Authors: Baron Christophe , Pénit Claude
Published: March 2007

The in vivo cell cycle of normal murine thymocytes was studied by bivariate analysis of bromodeoxyuridine and total DNA content in the 24 h following a single injection of the thymidine analogue. Bromodeoxyuridine incorporation was strictly limited to cells in S phase and 98% of S phase cells were labeled, demonstrating high efficiency and specificity. Cell‐cycle parameters were determined by measuring the DNA content of bromodeoxyuridine‐labeled cells, related to their distribution in the different phases. The changes of this distribution as a function of time reflected the progression of the cells along the cell cycle. The duration of total cycle, S phase, and both G2/M and G1 was 10 h, 6.5 h and 1.5 h, respectively. All thymocytes labeled inS phase entered G2/M, divided and returned to the G0/G1. Seventy percent of them remained in the resting state, and the other 30% reentered a second S phase. Cell‐cycle parameters of isolated CD4−CD8− cells were also determined. No evidence of cell loss during S or G2/M phase was found, suggesting that intrathymic cell death is not directly linked to the proliferative phases of differentiation.
Allergen‐directed expression of Fc receptors for IgE (CD23) on human T lymphocytes is modulated by interleukin 4 and interferon‐γ
Authors: Prinz Jörg C. , Baur Xaver , Mazur Gertraud , Peter Rieber E.
Published: March 2007

T lymphocytes bearing Fc receptors (FcR) for immunoglobulins are known to have immunoglobulin class‐specific regulatory functions. Here we report that expression on T cells of the low‐affinity FcR for IgE (FcϵRII/CD23) is preferentially induced by stimulation with antigens that cause an IgE response. T cells from eight patientsallergic to the hemoglobin of Chironomus thummi thummi mosquito larvae (CHIT I) were analyzed for reactivity with the anti‐FcERII/CD23 monoclonal antibody (mAb)M‐L25 under various conditions. No FcϵRII/CD23+ T cells were observed among freshly isolated, resting peripheral blood mononuclear cells (PBMC). Stimulation of PBMC with CHIT I, however, induced a marked although transient FcϵRII/CD23 expression on a large portion of the allergen‐activated T lymphocytes. It reached a maximum of 37.2 ± 4.6% FcϵRII/CD23+ T cell blasts on day 5 of culture. The selectivity of this expression became evident when compared to non‐allergenic control antigens: after stimulation of PBMC with tetanus toxoid or purified protein derivative from tuberculin a maximum of 4.6% ± 1.4% and 4.2% ± 1.1% T cell blasts was found to express FcϵRII/CD23, respectively. Activation by an anti‐CD3 mAb was insufficient to induce FcϵRII/CD23 on T cells. The allergen‐stimulated FcϵRII/CD23+ T cells exclusively belonged to the CD4+CD29+ helper inducer T cell subset.

Using a cDNA probe coding for the B cell FcϵRII/CD23, Northern blot analysis revealed a 1.7‐kb FcϵRII/CD23 mRNA in extracts of highly purified allergen‐stimulated T cells. It was of the same size as FcϵRII/CD23 mRNA of the lymphoblastoid B cell line WI‐L2.

Of several cytokines tested [interleukin (IL) 1 to IL 6, interferon‐γ (IFN‐γ), tumor necrosis factor‐α] only IL 4 and IFN‐γ significantly modified allergen‐induced FcϵRII/CD23 expression onT cells. The latter was enhanced nearly twofold in the presence of IL 4, and was almost completely abrogated by IFN‐γ. IL 4, however, could not increase the number of FcϵRII/CD23+ T lymphocytes either alone or in combination with an anti‐CD3 mAb. Taken together, the selective induction of FcϵRII/CD23 onT cells by allergen and its inclusion in the regulatory network of cytokines point to an important role of FcϵRII/CD23+ T lymphocytes in the human IgE response.