bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin 4 down‐regulates the expression of CD14 in normal human monocytes
Authors: Lauener Roger P. , Goyert Sanna M. , Geha Raif S. , Vercelli Donata
Published: March 2007

Recombinant interleukin 4 (IL 4) down‐regulates the expression of CD 14 on normal human monocytes, as assessed by flow cytometry, binding assays with radiolabeled anti‐CD 14 monoclonal antibody (mAb), and immunoprecipitation of 125I‐labeled monocytes with anti‐CD14 mAb. In parallel, CD23 expression on monocytes was strongly increased by IL4 stimulation, as assessed by both flow cytometry and immunoprecipitation. Down‐regulation of surface CD14 was first detectable after 24–36 h of incubation with rIL 4, and was almost complete after 4 days of culture. None of the other recombinant lymphokines tested (IL 1, IL 2, IL 3, IL 5, IL 6, interferon‐γ, tumor necrosis factor α and β, granulocytemacrophage colony‐stimulating factor) decreased CD14 expression. Metabolic labeling studies with [35S]methionine showed that both the membrane‐associated and the soluble form of CD 14 are decreased by IL 4 stimulation. Northern blot analysis showed that incubation of monocytes with IL 4 induced a marked decrease in CD 14 mRNA. Nuclear run‐off assays revealed that the IL 4‐dependent down‐regulation of CD 14 resulted from decreased transcription. Thus, IL 4 exerts specific and opposite effects on the expression of monocytic antigens.
Differential effects of low and high concentrations of interleukin 6 on human B cells
Authors: Levy Yves , Fermand Jean‐Paul , Brouet Jean‐Claude
Published: March 2007

In this report we have studied the functional effects of a wide range of interleukin 6 concentrations on human B cells. B cells purified from tonsils and from five of six spleens proliferated directly in a dose‐dependent manner in the presence of low concentrations (1–100 ng/ml) of IL 6 whereas B cells from the sixth spleen proliferated only after in vitro anti‐μ antibodies activation. Only large B cells (presumably in vivo activated B cells) were responsive to IL 6. High concentrations of IL 6 (1–10 ng/ml) did not trigger B cell proliferation but were able to up‐regulate the expression of the B5 activation antigen on B cells, whereas the expression of two other B cell antigens (CD20 and CD23) was unchanged. At all concentrations of IL 6 tested (0.01–10 ng/ml) no B cell differentiation occurred after 7 days culture. Depletion before culture of B5+ cells from the B cell‐enriched spleen cell preparation abolished both the proliferative effect and up‐regulation of B5 antigen induced by IL 6, indicating that this lymphokine acted primarily on activated B cells. This differential effect of “low” and “high” concentrations of IL 6 on activated B cells may solve controversy on the role of IL 6 as an early or late agent in B cell maturation.
Spontaneous production of anti‐mouse red blood cell autoantibodies is independent of the polyclonal activation in NZB mice
Authors: Reininger Luc , Shibata Takanori , Schurmans StéPhane , Merino Ramon , Fossati Liliane , Lacour Marc , Izui Shozo
Published: March 2007

New Zealand Black (NZB) mice spontaneously develop an autoimmune hemolytic anemia together with a markedly increased production of polyclonal antibodies. The spontaneous generation of anti‐mouse red blood cells (MRBC), anti‐bromelain‐treated MRBC (BrMRBC) and anti‐DNA autoantibodies was compared to the polyclonal antibody formation in irradiated (800 rad) 2‐month‐old NZB mice reconstituted with bone marrow cells (BMC) from 2‐ or 10‐month‐old NZB mice. The injection of 10‐month‐old NZB BMC markedly accelerated the mortality rate in parallel with the progressive increase of anti‐MRBC and anti‐BrMRBC autoantibody production, but the spontaneous production of polyclonal IgM antibodies and anti‐DNA autoantibodies was completely abolished down to the levels of non‐autoimmune mice. In contrast, mice reconstituted with 2‐month‐old NZB BMC exhibited neither the acceleration of anemia nor the lack of polyclonal antibody production. These results strongly suggest that the spontaneous production of anti‐MRBC autoantibodies, including anti‐BrMRBC autoantibodies, in the NZB mouse occurs independently of the polyclonal B cell activation, and that they result from a specific immune stimulation, while the anti‐DNA autoantibody production is a consequence of polyclonal antibody formation.
The effect of mutations in the MHC class I peptide binding groove on the cytotoxic T lymphocyte recognition of the Kb‐restricted ovalbumin determinant
Authors: Nikolić‐Žugić Janko , Carbone Francis R.
Published: March 2007

The H‐2Kb‐restricted cytotoxic T lymphocyte (CTL) response directed against ovalbumin (OVA) is specific for a region contained within the sequence OVA253‐276. In this study we have characterized this response by examining the class I‐restricted presentation of OVA peptides by the naturally occurring Kb mutant (Kbm) glycoproteins Kbm1, Kbm3, Kbm5, Kbm8, Kbm10, Kbm11 and Kbm23. To facilitate this study we derived a series of somatic cell hybrid targets expressing the various Kbm class I molecules. Experiments using bulk OVA‐specific CTL from C57BL/6 mice demonstrated that all the Kbm molecules except for Kbm1 and Kbm8 could present OVA peptides for effective T cell recognition. Clonal analysis revealed a more complex and relatively diverse pattern of CTL recognition of the Kbm/peptide combinations. This diversity is unlikely to result from the existence of multiple, independent Kb‐restricted T cell determinants within OVA, since all CTL tested were specific for a single region between residues 259 and 273. Examination of the fine specificity of Kbm presentation identified individual changes at residues 77, 80 and 116 which affected T cell recognition. The results imply that these changes do not inhibit peptide binding since some clones could recognize peptide presented by a particular Kbm molecule, while other clones could not. All three residues reside within the peptide‐binding cleft of the class I protein and are not expected to directly contact the T cell receptor. Although we did not formally demonstrate that OVA binding by Kbm vs. Kb is quantitatively identical, our results are best explained by postulating that the changes at residues 77, 80 and 116 indirectly affect T cell recognition by altering peptide conformation. Taken together our results suggest that changes within the class I binding site can profoundly modify peptide presentation without significantly inhibiting peptide‐class I association.
Natural killer susceptibility is independent of HLA class I antigen expression on cell lines obtained from human solid tumors
Authors: Peña Jose , Alonso Corona , Solana Rafael , Serrano Rafael , Carracedo Julia , Ramirez Rafael
Published: March 2007

The susceptibility to natural killer (NK) cell‐mediated cytotoxicity of 20 cell lines obtained from human solid tumors and their class I histocompatibility antigen (HLA) levels were studied in an attempt to determine whether major histocompatibility complex (MHC) products expressed on cells derived from human solid tumors influence NK susceptibility. The effect of interferon‐γ (IFN‐γ) treatment on these elements was also analyzed. The MHC class I (HLA‐ABC, HLA‐A and HLA‐B) antigen levels and degree of NK lysis were very heterogeneous and no correlation was found on comparison. After treatment with IFN‐γ a marked decrease in NK susceptibility was observed in all the cell lines, including the control line K‐562. However, the level of HLA class I expression was not modified in any of the lines with the exception of the K‐562, which increased. In some cell lines the expression of HLA class I‐like antigens, CD1a, b and c, was also measured before and after IFN treatment; however, no correlation was found between CD1 levels and NK susceptibility. Consequently, from our results it is possible to conclude that HLA class I antigens do not play a decisive role in NK susceptibility of cell lines derived from human solid tumors and to suggest that molecules which are not HLA class I antigens but IFN‐γ inducible may confer NK resistance to these lines.
Functional implication for the topographical relationship between MHC class II and the low‐affinity IgE receptor: occupancy of CD23 prevents B lymphocytes from stimulating allogeneic mixed lymphocyte responses
Authors: Flores‐Romo Leopoldo , Johnson Gerald D. , Ghaderi Abbas A. , Stanworth Dennis R. , Veronesi Arianna , Gordon John
Published: March 2007

Following the observation of Bonnefoy et al. (J. Exp. Med. 1988. 167: 57), that the low‐affinity IgE receptor (CD23) on B lymphocytes can be coupled (with the use of chemical cross‐linking reagents) to major histocompatibility complex (MHC) class II DR molecules, we now report that ligands binding within the lectin‐homology region of CD23 prevent B cells from stimulating allogeneic mixed lymphocyte responses. Ligands capable of blocking mixed lymphocyte responses include the anti‐CD23 antibodies MHM6 and EBVCS 4 but not EBVCS 1 and 5, IgE itself, and small peptides representing sequences within the CH3 domain of IgE. The detailed topographical relationship between CD23 and MHC class II on the B lymphocyte surface was examined using dual immuno‐fluorescence labeling of cells and direct visualization of the staining by confocal laser scanning microscopy. On transformed B lymphoblasts, the two antigens were seen to co‐localize in discrete patches; on normal B cells which had been cultured for 2 days with interleukin 4, CD23 and MHC class II converged at a single pole which exhibited a tendency to pseudopod formation and provided a focus for homotypic cell‐cell interactions. The possibility that CD23 could serve as a co‐stimulatory‐adhesion molecule in antigen presentation by B lymphocytes is discussed with special reference to a potential role in the regulation of IgE synthesis.
J chain deficiency in human IgM monoclonal antibodies produced by Epstein‐Barr virus‐transformed B lymphocytes
Authors: Gloria Meng Y. , Criss Anne B. , Georgiadis Katy E.
Published: March 2007

Six human IgM monoclonal antibodies against Pseudomonas aeruginosa were purified and characterized. On agarose‐acrylamide sodium dodecyl sulfate (SDS) gels run under nonreducing conditions, IgM monoclonal antibodies showed variable amounts of a slower migrating form of IgM in addition to the one co‐migrating with plasma IgM. Protein blotting with anti‐J chain antibody showed that the slower migrating form did not contain J chain. Analysis of one of the monoclonal antibodies by sucrose gradient centrifugation showed that the J chain‐deficient form sedimented faster than the complete IgM. It is known that IgM preparations lacking J chain sediment faster by sucrose gradient centrifugation analysis and tend to form hexamers. The slower migrating form of IgM we observed on SDS gels under nonreducing conditions could be hexameric IgM. Further evaluation of this monoclonal antibody demonstrated that both forms of IgM had the same antigen‐binding activity. Glycosylation of the light chain was demonstrated in two of the monoclonal antibodies.
In vitro analysis of age‐related changes in the developmental potential of bone marrow thymocyte progenitors
Authors: Sharp Ayala , Kukulansky Tova , Globerson Amiela
Published: March 2007

Mechanisms underlying the age‐related decrease in the developmental capacity of thymocyte progenitors from the bone marrow (BM) were analyzed, focussing on interactions of these cells with the thymic microenvironment. We employed the experimental model in which mixtures of young and old mouse BM cells, congenic for the Thy‐1 marker, were seeded onto fetal thymus (FT) explants depleted of self lymphocytes and the levels of Thy‐1+ cells developing from each of the two donor types were measured. When cells from young and old BM donors were seeded simultaneously, in saturating quantities, a higher level of T cells developed from the young donors.

To find out whether there were originally more thymocyte progenitors in the young BM, we carried out the competitive colonization under limiting dilution conditions and found that the advantage of the young had diminished under these conditions, thus suggesting that the age‐related changes could not be related solely to quantitative differences. We then incubated the FT sequentially with old donor cells for 24 h, followed by young for an additional 48 h and found that the advantage of the young progenitors was eliminated. We thus established that the initial stage of colonization of the FT was important in determining the outcome of the subsequent development. The kinetics of simultaneous competition within the FT, however, revealed that the advantage of the young BM‐derived cells became significant only from day 7 in organ culture, thus suggesting that sequential divisions of these cells were at a higher level than those of the old. Recolonization of FT explants by young or old BM‐derived thymocytes obtained from the first colonization of the FT stroma showed a reduced, but still significant advantage for the young BM‐derived cells over the old. Thus, we concluded that the old BM thymocyte progenitors manifested a qualitative disadvantage which became apparent during competitive colonization of the FT.
Genetic recombination in the α2 domain of the Eα chain yields an Ed molecule with altered T cell activation
Authors: Chang Ming‐Der Y. , Kowal Czeslawa , Marzullo Laurie R. , Briner Thomas J. , Gefter Malcolm L. , Diamond Betty
Published: March 2007

We have used a novel T cell selection strategy to isolate a mutant of an H‐2d/f murine macrophage line defective in its ability to present antigen to some Ed‐restricted helper T cells. This mutant has an amino acid substitution in the α2 domain of the Ed molecule. The mutation changes the sequence at codon 177 from ACC to CAC, which results in a threonine to histidine substitution and appears to be the first in vitro mutation to have arisen by genetic recombination. Even though the mutation is distal to the proposed antigen‐binding groove, it affects antigen presentation, presumably by altering the scaffolding for the antigen‐binding groove. This type of mutant might not be readily isolated using other selection techniques.
Thyroid‐derived epithelial cells acquire alloantigen‐presenting capabilities following X‐irradiation and class II antigen induction
Authors: Czirjak Laszlo , Danko Katalin , Gaulton Glen N. , Stadecker Miguel J.
Published: March 2007

This work was undertaken to further define the antigen‐presenting capabilities of thyroid epithelial cells, as this is of paramount importance with regard to their potential to trigger autoimmune thyroiditis. For this purpose we developed the murine cloned thyroid‐derived epithelial cell line M.5 which, as previously reported, could be induced to express class II antigens with interferon (IFN)‐γ, but failed to stimulate a primary mixed leukocyte reaction.

We now show that M.5 cells acquired alloantigen‐presenting function, under conditions in which their replication was arrested by X‐irradiation, during a 4‐day period of exposure to UV‐inactivated reovirus, or to IFN‐γ, for induction of class II antigens.

The allostimulatory function by M.5 cells could not be explained on the basis of enhanced class II antigen expression, as equivalent numbers of M.5 cells, irradiated after the 4‐day exposure to reovirus, or to IFN‐γ, expressed higher amounts of class II antigens, but did not stimulate a primary mixed leukocyte reaction. Early X‐irradiation appeared to induce in the class II‐expressing M.5 cells a co‐stimulatory signal needed for T cell proliferation, similar to that otherwise provided by phorbol esters in this system. Preservation of alloantigenpresenting function by M.5 cells following fixation indicated that this costimulatory activity did not reside in a soluble molecule.

We surmise that M.5 epithelial cells must provide a least two signals in order to be able to stimulate lymphocyte populations. Consequently, the mode and conditions of epithelial cell activation may determine whether these cells acquire the capacity to serve as antigen‐presenting cells, and ultimately whether or not they are able to induce autoimmune disease.
Evidence that in X‐linked immunodeficiency with hyperimmunoglobulinemia M the intrinsic immunoglobulin heavy chain class switch mechanism is intact
Authors: Hendriks Rudolf W. , Kraakman Margriet E. M. , Craig Ian W. , Espanol Teresa , Schuurman Ruud K. B.
Published: March 2007

X‐linked immunodeficiency with hyperimmunoglobulinemia M (XHM) reflects an impairment of the immunoglobulin (Ig) heavy (H) chain class switch of B lymphocytes from IgM to IgG and IgA.

XHM is recessive; female carriers manifest normal IgG and IgA production. Due to random X chromosome inactivation in all somatic cells of females, about half of the lymphocytes of XHM carriers are not able to express an intact XHM gene. An intrinsic defect of the Ig H chain class switch mechanism in XHM B lymphocytes would thus lead to a skewed X chromosome inactivation pattern in the IgG‐ and IgA‐expressing B lymphocytes of female carriers.

IgM‐, IgG‐ and IgA‐expressing B lymphoblastoid cells (BLC) were established by Epstein‐Barr virus transformation of peripheral blood mononuclear cells of two female XHM carriers. In an analysis of differential methylation of the polymorphic DXS255 loci, random X chromosome inactivation patterns were found in populations of T lymphocytes, in IgM‐expressing B lymphocytes and in IgG‐ or IgA‐expressing B lymphocytes. The heterogeneity of Ig H chain rearrangements and the Ig light chain usage in the IgA‐ or IgG‐expressing BLC clones that had inactivated the X chromosome which carries the intact XHM gene and in BLC clones with the homologous X chromosome inactivated were similar.

The results indicated that the intrinsic Ig H chain class switch mechanism in XHM B lymphocytes is fully intact. We conclude that the XHM gene encodes a class switch inducer that is transferred to B lymphocytes.
Processing without proteolytic cleavage is required for recognition of insulin by T cells
Authors: Gradehandt Gernot , Hampl Johannes , Milbradt Silke , Rüde Erwin
Published: March 2007

Beef insulin as well as a chymotryptic A‐chain fragment [BI‐A1‐14(SSO3−)3] need uptake by antigen‐presenting cells (APC) for efficient presentation in combination with major histocompatibility complex class II molecules to insulinspecific T cells. This could be shown by the inability of aldehyde‐fixed APC to present these antigens to T cells. Furthermore, presentation of the insulin fragment as well as presentation of ovalbumin (OVA) was inhibited by treatment of APC with chloroquine, cerulenin or tunicamycin. This was not the case for a processing‐independent OVA peptide. Treatment of APC during antigen pulsing with various protease inhibitors, active on all classes of proteases, did not block presentation of insulin although some of these reagents did interfere with the presentation of OVA. Several inhibitors especially of serine or thiol proteases rather enhanced the presentation of insulin. This indicates that intracellular proteolytic cleavage of insulin does not seem to be required for generation of the antigenic determinant but, if it occurs, rather destroys the antigenic peptide. Insulin and its A‐chain fragment may, therefore, represent a model for a processing‐dependent antigen not requiring proteolytic cleavage but other modifications.
Cloning and sequencing of human λ immunoglobulin genes by the polymerase chain reaction
Authors: Songsivilai Sirirurg , Bye Jacqueline M. , Marks James D. , Hughes‐Jones Nevin C.
Published: March 2007

Universal oligonucleotide primers, designed for amplifying and sequencing genes encoding the rearranged human λ immunoglobulin variable region, were validated by amplification of the λ light chain genes from four human heterohybridoma cell lines and in the generation of a cDNA library of human Vλ sequences from Epstein‐Barr virus‐transformed human peripheral blood lymphocytes. This technique allows rapid cloning and sequencing of human immunoglobulin genes, and has potential applications in the rescue of unstable human antibody‐producing cell lines and in the production of human monoclonal antibodies.
VH and VL region structure of antibodies that recognize the (NANP)3 dodecapeptide sequence in the circumsporozoite protein of Plasmodium falciparum
Authors: Anker Roberto , Zavala Fidel , Pollok Brian A.
Published: March 2007

The sporozoite form of Plasmodium falciparum displays on its surface the circumsporozoite (CS) protein. The central domain of this protein possesses a reiterated tetrapeptide sequence Asn‐Ala‐Asn‐Pro (NANP), and > 90% of the sporozoite‐specific antibodies obtained from individuals living in malaria endemic areas recognize epitopes within this repeat sequence. Considering the highly repetitive structure of this naturally occurring antigen and its immunodominance, we were interested in analyzing the structural diversity of antibodies that bind to the (NANP)3 sequence. Molecular characterization of immunoglobulin heavy and light chain mRNA was performed for five hybridomas that produce antibodies with binding specificity for the dodecapeptide (NANP)3. These hybridomas were produced in BALB/c mice by inoculation with whole P. falciparum sporozoites. Sequence analysis and Northern blotting showed that for heavy chain, three hybridomas used VH elements that belong to the VHIX family and two to the VHJ558 family. Four different Vχ subgroups were represented among the light chains. Different D and Jχ segments are also utilized, while four heavy chain gene rearrangements involved the JH4 segment. These results indicated that multiple VH‐VL gene combinations can code for reactivity to the (NANP)3 sequence, demonstrating that the murine antibody response to this immunodominant region is structurally heterogeneous.
Efficient recognition by rat T cell clones of an epitope of mycobacterial hsp 65 inserted in Escherichia coli outer membrane protein PhoE
Authors: Hogervorst Els J. M. , Agterberg Marja , P. A. Wagenaar Josée , Adriaanse HenriëTte , Boog Claire J. P. , Zee Ruurd Van Der , Van Embden Jan D. A. , Eden Willem Van , Tommassen Jan
Published: March 2007

PhoE is a pore‐forming protein, abundantly expressed in the Escherichia coli outer membrane. Previous investigations have shown the possibility of inserting antigenic determinants in cell surface‐exposed regions of PhoE by recombinant DNA techniques without disturbing the biogenesis and the functioning of the protein. This method proved to be successful for foot‐and‐mouth disease virus B cell determinants.

We have now shown for the first time that PhoE can also be used as a carrier molecule for Tcell epitopes. A well‐characterized Tcell epitope (180–188) of the 65‐kDa heat‐shock protein (hsp 65) of Mycobacterium tuberculosis was expressed in PhoE and tested for recognition by specific Tcell clones. Specific and efficient T cell proliferation was found after stimulation with this protein construct in vitro. Interestingly, paraformaldehyde fixation of antigen‐presenting cells did not abrogate Tcell recogition. Thus, in contrast to hsp 65 itself, recognition of epitope 180–188 in the context of PhoE appeared to be independent of antigen‐processing events. At the level of polyclonal Tcell responses the epitope in the context of PhoE is recognized more efficiently than 180–188 as synthetic peptide or in the context of the hsp 65 molecule itself. These findings indicate that PhoE may serve as attractive vaccine carrier not only for B, but also for Tcell epitopes. Furthermore, the possibility for expression of PhoE constructs in attenuated Salmonella typhimurium strains offers the exciting prospect of new types of live oral vaccines expressing selected combinations of B and Tcell epitopes.
HLA class II molecules on monocytes regulate T cell proliferation through physical interaction in the CD3 activation pathway
Authors: Ruggiero Giuseppina , Racioppi Luigi , Manzo Ciro , Pirozzi Giuseppe , D'Oro Ugo , Ferrone Soldano , Zappacosta Serafino , Fontana Silvia
Published: March 2007

HLA class II molecules are involved in the OKT3‐induced T cell activation, since monoclonal antibodies (mAb) to monomorphic determinants of class II antigens are able to inhibit T cell proliferation. This effect involves several of the events leading to T cell activation and proliferation, i.e. interleukin (IL) 1, IL 6 and IL 2 secretion and IL 2 receptor expression. The main target of the inhibition is represented by monocytes, and the interference of anti‐class II mAb in the direct interaction of monocytes with T cells is likely to play a relevant role in the inhibition mechanism. To test this hypothesis, we investigated in the present study the effect of anti‐class II mAb on the proliferation of T cells stimulated with mAb OKT3 in the presence of paraformaldehyde‐treated monocytes. We show that the inhibition of proliferation is specific and dose dependent, and seems to involve particular class II epitopes. Addition of fixed monocytes to inhibited T cell cultures restores proliferation to a moderate extent, only if monocytes are added within the first 12 h of culture, suggesting that class II antigens or spatially related molecules deliver signals concurrently with the mitogenic stimulus. The blocking capability of anti‐class II mAb was not restricted to the CD4+ or the CD8+ T cell subsets, suggesting that, under inhibitory conditions, these mAb affect other structures on the T cell surface, relevant to the monocyte‐T cell interaction.
The effect of peripheral immunization with Mls‐12 on the emigration of antigen‐specific cells from the thymus
Authors: Kelly Katherine A. , Scollay Roland
Published: March 2007

Mature T cells found in the lymph nodes and spleen have the capacity to become activated and to proliferate in response to foreign antigens. The response of the thymus to such immunization is less well understood. We have examined one aspect of the thymic response by determining the effect of peripheral immunization upon cell emigration from the thymus. BALB/c (Mls‐1b) mice were injected with spleen cells from DBA/2 (Mls‐13) mice, and Vβ6+ (Mls‐13‐reactive) thymic emigrants were identified 3–30 days after immunization. Neither the rate of total cell migration from the thymus nor the proportion of Vβ6+ cells was altered, even though the immunizing spleen cells elicited an immune response in the draining (parathymic) lymph nodes. The same immunogen caused deletion of Vβ6+ cells in both the thymus and lymph nodes after intraperitoneal injection into the neonate. The inability of DBA/2 splenocytes to modify the development of adult thymocytes after intrathymic injection of the cells precluded the lack of entry into the thymus as the reason for the lack of any observed effect in the adult. Our results, therefore, indicate that the development of adult thymocytes is not modified by immunization, and suggest that the differing thymic response of mice injected as adults or neonates is related to changes in the intrathymic antigen presentation capacity associated with age.
Identification of a major antigenic epitope on CNBr‐fragment 11 of type II collagen recognized by murine autoreactive B cells
Authors: Burkhardt Harald , Holmdahl Rikard , Deutzmann Rainer , Wiedemann Hanna , Von Der Mark Helga , Goodman Simon , Von Der Mark Klaus
Published: March 2007

Immunization of certain strains of mice with native type II collagen (CII) induces both development of arthritis and an antibody response to autologous CII. The autoantibody response in a high‐responder strain, the DBA/1 mouse, has been described earlier, and a number of monoclonal antibodies have been characterized for arthritogenicity and autoreactive binding to cartilage in vivo and in vitro. Here we map the antigenic epitope of one of these arthritogenic monoclonal antibodies (CII‐C1). It belongs to a group of antibodies recognizing the CNBr fragment α1(II)‐CB11 of CII. Using the enzyme‐linked immunosorbent assay technique, we show that the antibody reacts only with native, triplehelical CII, but not with other collagens. The antibody is able to stain specifically the CB11 fragment by immunoblotting, suggesting some partial renaturation of the CNBr fragment into triple‐helical structures after blotting. The binding site of CII‐C1 on CB11 was further focused by rotary shadowing of antibody‐labeled CII to a site 89 ± 8 nm from the amino end of CII, corresponding to the middle of CB11. This location was confirmed by cleavage of CB11 with trypsin, separation of the tryptic peptides by high‐performance liquid chromatography and dot‐blot analysis of the antigenic peptides with the CII‐C1 antibody. Sequencing of the single positive peptide located the antigenic epitope within the sequence GFAGQAGPAGAT‐GAPGRP (residues 316–333). Assuming 0.29 nm per residue, this corresponds to a position within 92–96.5 nm from NH2 terminal end of CII. Apart from glycine residues, which are not exposed on the triple‐helical structure, only two amino acid residues (F‐x‐y‐Q) are conserved in CII from different species but are not found in the triple‐helix of other collagens except type IV collagen. Therefore, this structure is likely to be of critical importance for the binding of the CII‐C1 antibody. Of potential importance is that this structure is also found in certain other arthritogenic proteins such as 65‐kDa mycobacterial protein, in CMV and EBV.
The identification of the β2‐microglobulin binding antigen encoded by the human CDID gene
Authors: Bilsland Caroline A. G. , Milstein César
Published: March 2007

Human cluster of differentiation (CD1) is a family of cell surface glycoproteins composed of a 43–49‐kDa heavy chain non‐covalently associated with β2‐microglobulin. Five human CD1 genes have been detected and cloned. Three genes (CD1A, ‐B and ‐C) encode the serologically defined CD1a, ‐b and ‐c antigens. Thus two genes remain, CD1D and CD1E, whose protein products have not been characterized so far. This report describes how a β‐galactosidase‐CD1D fusion protein was used to raise specific antisera and a monoclonal antibody against the CD1D gene product. The monoclonal antibody defines a cell surface molecule expressed on a cortical thymocyte cell line and is composed of a 49‐kDa heavy chain associated with β2‐microglobulin, which is serologically distinct from CD1a.
Allorecognition of HLA‐DR and ‐DQ transfectants by human CD45RA and CD45R0 CD4 T cells: Repertoire analysis and activation requirements
Authors: Merkenschlager Matthias , Ikeda Hitoshi , Wilkinson David , Beverley Peter C. L. , Trowsdale John , Fisher Amanda G. , Altmann Daniel M.
Published: March 2007

We have investigated the requirements for allogeneic stimulation of human CD4 T cells using HLA class II products expressed on various cellular backgrounds. Human (class II‐negative RJ2.2.5 mutant) B cell lines transfected with HLA‐DR or ‐DQ cDNA clones were efficient stimulators for highly purified CD4 T cells. HLA‐DR‐transfected mouse L cells or IFN‐γ‐induced human fibroblasts, although able to function as accessory cells for T cell responses to the mitogen PHA, failed to stimulate strong T cell alloresponses. On the basis of these observations, we have employed class II transfectants to address the following questions: (a) do CD45RA and CD45R0 subpopulations differ in their allogeneic activation requirements, (b) are these subpopulations skewed in their recognition of HLA‐DQ vs. HLA‐DR in a manner which might support the concept that CD45RA T cells are involved in HLA‐DQ‐restricted suppressor inducer functions and (c) by using transfectants expressing individual HLA‐DR or ‐DQ heterodimers in combination with limiting dilution analysis, can one for the first time obtain estimates of precursor frequencies for allogeneic cells recognizing each of these class II isotypes? Our results show that CD45RA and CD45R0 T cells respond comparably to optimal numbers of stimulator cells. However, when CD45RA and CD45R0 T cell populations depleted of endogenous accessory cells were cultured with limiting numbers of stimulator cells, CD45R0 cells generally responded more strongly, consistent with the elevated levels of various adhesion molecules known to be expressed by this population. Further, we found a similar representation of responses to HLA‐DR and ‐DQ antigens among populations expressing CD45RA and CD45R0 isoforms. Finally, the precursor frequencies of allogeneic CD4 T cells responding to particular HLA‐DQ alleles were higher than to ‐DQ, but only by a factor of about 1.6, indicating that HLA‐DQ recognition may occur more frequently than implied from previous antibody blocking studies.
Lymphocytes infected with Theileria parva require both cell‐cell contact and growth factor to proliferate
Authors: Dobbelaere Dirk A. E. , Roditi Isabel J. , Coquerelle Thérr̀se M. , Kelke Christina , Eichhorn Margarete , Williams Richard O.
Published: March 2007

Lymphocytes infected with the intracellular parasite Theileria parva proliferate continuously as lymphoblastoid cell lines. We have previously shown that the continuous proliferation of the T. parva‐infected (Tpi) cell line TpM(803) is mediated in part by an autocrine mechanism (Dobbelaere, D. A. E. et al., Proc. Natl. Acad. Sci. USA 1988. 85: 4730). We now report that continuous proliferation also requires surface stimulation through cell‐cell contact. Under standard culture conditions this surface stimulus is provided by the infected cells themselves, but it can also be provided by uninfected lymphocytes or macrophages. The ability to respond to surface stimulation is critically dependent on the presence of the parasite in the host cell and is lost within 48 h after the elimination of the parasite from the host cell cytoplasm by treatment with the theilericidal drug BW720c. Tpi cells also secrete a growth factor which is able to support the proliferation of diluted Tpi cells. Growth factor secretion is rapidly lost upon elimination of the parasite. Moreover, inhibition experiments using antiinterleukin 2 (IL 2) antibodies show that IL 2 is involved in the proliferation of the Tpi cell lines TpM(803) and IN10. T cell proliferation is dependent on a number of costimulatory signals which are normally provided by accessory cells. The finding that Tpi cells can mutually stimulate each other to grow in the absence of conventional accessory cells helps to explain how they can escape the normal constraints on T cell growth, allowing them to invade and multiply in non‐lymphoid as well as lymphoid tissues.
Interleukin 4 induces interleukin 6 production by endothelial cells: Synergy with interferon‐γ
Authors: Howells Gareth , Pham Phuong , Taylor David , Foxwell Brian , Feldmann Marc
Published: March 2007

Interleukin (IL) 4 induces IL 6 production by human umbilical vein endothelial cells (HUVEC) in a dose‐dependent manner, as shown by bioassay and immunoprecipitation. Interferon (IFN)‐γ, which antagonizes IL 4 effects on leukocytes, synergized with IL 4 in the induction of IL 6 production by HUVEC. Contamination with endotoxin was excluded by heat‐inactivated IL 4, preincubating with anti‐IL 4 polyclonal antibody and the use of polymyxin B. The presence of IL 4 receptors on HUVEC was shown by affinity cross‐linking with 125I‐IL 4, revealing a 110‐kDa binding protein. However, compared with the amount seen on T cells the 60–70‐kDa cross‐linked doublet was present at much lower levels. Additional lower molecular weight cross‐linked proteins were isolated only with HUVEC, but the origin of these is unclear. IL 6 is a pluripotent cytokine produced by many cells which promotes the differentiation and growth of lymphocytes and the production of acute phase protein by hepatocytes, and is important in the regulation of immunity at the systemic and local levels. Since IL 4 and IFN‐γ are produced by T cells, which are frequently associated with vascular endothelium during chronic inflammation, IL 4 is likely to be an important cytokine in the regulation of IL 6 and perhaps other cytokine production by endothelium in vivo.
Proliferative responses induced by the activation of protein kinase C during the development of human T lymphocytes
Authors: González‐Fernández Africa , Diaz‐Espada Fernando , Kreisler Miguel , Deza Francisco Gambón
Published: March 2007

We have studied the effects of phorbol‐dibutyrate (PBu2), a protein kinase C (PKC) activator, on the proliferation of peripheral human T cells and thymocyte subpopulations selected by treatment with monoclonal antibodies and complement: pre‐thymocytes (CD1a−CD3−CD4−CD8−), cortical thymocytes (CD3−, class I− antigens) and medullary thymocytes (enriched as CD1a− cells). PBu2 induces a dose‐dependent proliferative response in human peripheral blood T cells at concentrations > 6 ng/ml, this proliferation being mediated by the autocrine interleukin 2 (IL2)/IL2 receptor (IL2R) pathway. Pre‐thymocytes respond to PBu2 in a way similar to T cells, being able to secrete IL2 in significant amounts and express the p55 chain of IL2R. On the other hand, cortical thymocytes are not induced to proliferate after PKC activation and neither expression of the p55 chain of IL2R nor IL2 secretion is observed. Human medullary thymoctes, phenotypically identical to peripheral blood T cells, show no proliferation in response to PBu2 at any concentration tested unless IL2 is supplied to the cultures. The activation of PKC induces the expression of IL2R in these cells, but not IL2 secretion. The implications of PKC activation in thymic maturation, the role of IL2 and the relevance of the differences between medullary thymocytes and peripheral blood T cells are discussed.
Role of the adhesion molecule ICAM‐1 (CD54) in staphylococcal enterotoxin‐mediated cytotoxicity
Authors: Dohlsten Mikael , Hedlund Gunnar , Lando Peter A. , Trowsdale John , Altmann Daniel , Patarroyo Manuel , Fischer Hans , Kalland Terje
Published: March 2007

Staphylococcal enterotoxin A (SEA) binds to major histocompatibility complex (MHC) class II molecules on target cells and directs human cytotoxic T lymphocytes (CTL) of irrelevant nominal specificity to mediate strong cytotoxicity against target cells. In this report we describe the importance of ICAM‐1(CD54) expression on the target cell in SEA‐dependent cell‐mediated cytotoxicity (SDCC), utilizing murine L cells co‐transfected with HLA‐DR and ICAM‐1. Human CTL mediated a low but significant cytotoxicity against HLA‐DR2‐and HLA‐DR7‐transfected cells after preincubation with SEA, but no reactivity towards uncoated HLA‐DR2 and HLA‐DR7 cells or SEA‐coated ICAM‐1‐transfected and untransfected L cells. In contrast, a strong cytotoxic response was mediated by CTL against L cells co‐transfected with HLA‐DR/ICAM‐1 and HLA‐DR7/ICAM‐1. Similar cytotoxic activity of the CTL was seen at a 30‐fold lower effector‐to‐target cell ratio when comparing the HLA‐DR2/ICAM‐1‐expressing cells with the HLA‐DR2‐expressing cells. SEA dose‐response analysis demonstrated that the HLA‐DR2/ICAM‐1‐expressing target cells enabled the CTL to respond to a 1000‐fold lower concentration of SEA in comparison to the HLA‐DR2‐expressing cells. CD3+CD4+ and CD3+CD8+ cytotoxic T cell lines were equally dependent on the expression of ICAM‐1 on the target cell. The strong CTL activity against HLA‐DR2/ICAM‐1‐transfected cells could be blocked by anti‐CD11a or anti‐CD18 monoclonal antibodies (mAb), but not by anti‐CD11b, anti‐CD11c, anti‐CD2 or unrelated control mAb. The great sensitivity of HLA‐DR2/ICAM‐1 expressing target cells to SDCC was strongly reduced by preincubation with various anti‐ICAM‐1 mAb but not by mAb against monomorphic HLA‐DR or murine MHC class I determinants. The result in this study clearly demonstrates that efficient re‐targeting of human CTL by SE is dependent on a proper interaction with the heterodimer CD11a/CD18 (Leu‐CAMa, LFA‐1) on the CTL and its target cell ligand ICAM‐1.
Identification and functional analysis of the transcriptional enhancer of the human T cell receptor β gene
Authors: Prosser Haydn M. , Lake Richard A. , Wotton David , Owen Michael J.
Published: March 2007

The productive rearrangement and transcription of T cell receptor (TcR) β genes is confined to T lymphocytes and is subject to both tissue‐specific and developmental regulation. In addition to their function in transcriptional control. cis‐acting elements are likely to play a role in the regulation of the rearrangement process. In this report we describe the location of a strong and inducible transcriptional enhancer 3′ to the human TcR Cβ2 gene segment. The core enhancer, defined by deletion analysis using a transient transfection assay, resided within 362 bp of DNA. This enhancer core was able to activate transcription from a heterologous promoter and functioned well in T and B lymphocytes, but only minimally in HeLa cells. In contrast, a longer fragment containing the enhancer core showed marked T cell specificity. The enhancer was highly inducible by phorbol esters, the molecular basis for the inducibility residing within a 118‐bp region of the enhancer core. This inducibility may be important in modulation of TcR β gene expression during T cell differentiation and/or activation.
Heterodimeric, disulfide‐linked α/β T cell receptors in solution
Authors: Slanetz Alfred E. , Bothwell Alfred L. M.
Published: March 2007

Structural and functional analysis of T cell receptor (TcR)‐ligand binding would be greatly advanced by the availability of an intact, assembled TcR in soluble form. We have produced such a molecule, by splicing the extracellular domains of a TcR to the glycosyl phosphatidylinositol membrane anchor sequences of Thy‐1. The molecule is expressed in the absence of CD3 on the cell surface, and can be cleaved from the membrane by treatment with phosphatidylinositol‐specific phospholipase C. The α and β chains of the soluble molecule are paired in the native conformation as judged by reactivity with the anti‐Vβ8 monoclonal antibody F23.1, and with the anti‐clonotypic monoclonal antibody 1B2; it is a disulfide‐linked dimer with a mol. mass of 95 kDa on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis under nonreducing conditions, and 47 kDa after reduction. We conclude that we have generated an α/β TcR in soluble form.
Inhibition of tumor necrosis factor‐α transcription by Epstein‐Barr virus
Authors: Gosselin Jean , Menezes José , D'Addario Mario , Hiscott John , Flamand Louis , Lamoureux Gilles , Oth Daniel
Published: March 2007

Tumor necrosis factor‐α (TNF‐α), which is produced mainly by monocyte/macrophage cells, has diverse physiological functions on lymphoid cells. Moreover, it has been shown that TNF‐α exhibits antiviral activities. Here we report that Epstein‐Barr virus (EBV), a B lymphotropic human herpes virus that interacts intimately with the immune system, exerts a strong inhibitory effect on TNF‐α production by lipopolysaccharide‐treated peripheral blood leukocytes as well as by monocytic cell lines, HL‐60 and U‐937. Flow cytometric analysis following staining with OKB7 monoclonal antibody showed that about 20% of cells from these monocytic lines express the CR2 antigen. Direct binding of fluorescein isothiocyanate‐labeled EBV indicated that the virus binds to approximately 22% of cells of both monocytic lines. However, no virus‐specific antigens were detected in the infected cells by immunofluorescence, suggesting that the infection was of the abortive type. The use of UV‐ or heat‐inactivated EBV and EBV treated with neutralizing antibodies resulted in the abrogation of the EBV inhibitory effect on TNF‐α synthesis. These results suggest that infectious virus is necessary to obtain such an inhibitory effect. Analysis of TNF‐α mRNA by polymerase chain reaction amplification indicated that the EBV suppressive effect is manifested at the transcriptional level. In contrast, EBV did not inhibit interleukin 1 mRNA production by these cells. These results indicate that EBV interacts directly with monocytes/macrophages to exert its immunomodulatory effect.
Isotype commitment of human B cells that are transformed by Epstein‐Barr virus
Authors: Miyawaki Toshio , Butler Joseph L. , Radbruch Andreas , Gartland G. Larry , Cooper Max D.
Published: March 2007

Epstein‐Barr virus (EBV) can transform a subpopulation of preactivated B cells thus promoting their growth and differentiation into plasma cells. In EBV‐transformed clones of IgM‐producing cells, the heavy chain constant region (CH) genes on the productive allele are fixed in germ‐line configuration, whereas in isotype‐switched clones the CH genes proximal to the expressed CH gene are deleted. In order to define more precisely the EBV‐susceptible B cells, we sorted subpopulations of B cells on the basis of their cell surface Ig (sIg) isotypes, infected them with EBV, and determined which isotypes they could produce following transformation. Most precursors of IgM‐producing plasma cells expressed both IgM and IgD on their surface, while a minority expressed IgM alone. Some B cell precursors of IgG‐ and IgA‐producing cells also expressed sIgM, but surprisingly none expressed IgD. Those precursors of IgG and IgA producers, which bore sIgM, expressed it in relatively low levels, whereas B cells expressing high levels of sIgM were incapable of generating IgG and IgA producers. All of the precursors of IgG and IgA plasma cells expressed these isotypes on their cell surface. Interestingly, precursor B cells capable of producing the IgG3 and IgA2 subclasses could be respectively enriched on the basis of the presence or absence of cell sIgM. These results demonstrate the isotype precommitment of EBV‐transformable B cells. They further suggest that residual IgM is transiently expressed on the surface of the IgG‐ and IgA‐committed B cell precursors, whereas sIgD expression is extinguished earlier in the process of isotype switching via CH gene deletion.
Dividing cells in bone marrow and spleen incorporate bromodeoxyuridine with high efficiency
Authors: Schittek Birgit , Rajewsky Klaus , Förster Irmgard
Published: March 2007

Labeling of dividing cells with the thymidine analogue 5‐bromo‐2′‐deoxyuridine (BrdUrd) has been frequently used to determine the life‐span of cells in various lymphocyte subsets. In this study we address the question of whether administration of BrdUrd in vivo allows labeling of proliferating lymphocytes at early as well as later stages of maturation with high efficiency. For this purpose, the proportion of BrdUrd‐labeled cells among all cells synthesizing DNA was determined in the bone marrow and spleen of mice that had received a single injection of BrdUrd or, alternatively, had been fed with BrdUrd for 1 week. On the average, 87% of bone marrow and 83% of spleen cells in the S phase of the cell cycle, as determined by DNA content, were labeled with BrdUrd 1.5 h following injection of the drug. We conclude from these data that administration of BrdUrd in vivo allows labeling of the vast majority of the dividing cells in both primary and secondary lymphoid organs and thus represents an efficient method to determine the life‐span of lymphoid cells in vivo.