bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibody binding to a collagen type‐II epitope gives rise to an inhibitory peptide for autoreactive T cells
Authors: Burkhardt Harald , Yan Tan , Bröker Barbara , Beck‐Sickinger Annette , Holmdahl Rikard , von der Mark Klaus , Emmrich Frank
Published: March 2007

It is well documented that antigen recognition by T cells requires small peptides which are generated by protein cleavage in antigen‐presenting cells. These peptides have to associate with major histocompatibility complex (MHC) molecules in order to be recognized. An inhibitory peptide may bind to the same site of the MHC‐encoded protein but is not recognized by the T cell. Here we describe a stimulatory and an inhibitory peptide sequence within human collagen type II (CII) as defined by means of the same autoreactive human T cell clone. Most interestingly, the inhibitory peptide is not generated by regular processing in peripheral blood mononuclear cells but only in the presence of an antibody that binds to the same domain and thereby seems to protect the inhibitory sequence. This finding may indicate that certain autoantibodies have the potential to block autoreactive T cells with specificity for a distinct epitope on the same antigen.
Expression of the adhesion molecules ICAM‐1 and ICAM‐2 on tumor cell lines does not correlate with their susceptibility to natural killer cell‐mediated cytolysis: evidence for additional ligands for effector cell β2 integrins
Authors: Akella Rama , Hall Robert E.
Published: March 2007

The LFA‐1 molecule (CD11a/CD18), a member of the leukocyte (β2) integrin subfamily of the integrin supergene family, has been shown to subserve important function(s) in natural killer (NK) and lymphokin‐activated killer (LAK) effector cells based on monoclonal antibody inhibition and other studies. Presently, two cellular ligands for LFA‐1 have been identified, termed ICAM‐1 and ICAM‐2. In this study, we have examined the role of target cell ICAM‐1 (CD54) and ICAM‐2 in NK‐mediated target lysis. Using a panel of tumor target cell lines, ICAM‐1 surface protein and transcript expression did not correlate with sensitivity to NK lysis. Compared to ICAM‐1, ICAM‐2 transcript expression was very low or undetectable in tumor cell targets, and also did not correlate with sensitivity to NK lysis. ICAM‐1+ K562 cells and K562 cells which were rendered surface ICAM‐1− with an antisense oligonucleotide were equally sensitive to NK lysis. Finally, human ICAM‐1− P815 cells were stably transfected with the human ICAM‐1 gene, and both ICAM‐1− P815 (wild type) and ICAM‐1+ stable transfectants were equally insensitive to NK lysis. These studies provide evidence that ICAM‐1 and ICAM‐2 are not important target cell ligands for NK effector cell LFA‐1 and that other target cell ICAM may exist.
Ipr T cells vaccinate against lupus in MRL/lpr mice
Authors: de Alborán Ignacio M. , Gutierrez José C. , Gonzalo José A. , Andreu José L. , Marcos Miguel A. R. , Kroemer Guido , Martínez‐A Carlos
Published: March 2007

MRL/MP‐lpr/lpr mice are homozygous for the lpr mutation that results in the accumulation of phenotypically abnormal cells (CD3+CD4+CD8−) in all lymphoid issues. Although no major abnormalities in the T cell receptor repertoire expressed by such lpr cells have been reported, the lpr mutation is a major disease‐accelerating factor. Finally, intravenous administration of irradiated lpr cells recovered from the hyperplastic lymph nodes of adult diseased animals to young MRL/Mp‐lpr/lpr mice resulted in a highly significant amelioration of disease parameters. This “T cell vaccination” approach resulted in a selective depletion of cells expressing products of the Vβ8.2 subfamily among lymph node T cells, in addition to eliciting a surge in peripheral T cells capable of conferring disease protection in adoptive transfer experiments. Thus, a strategy aimed at specifically reducing the frequency of lpr cells proved successful in mitigating the autoimmune process. These findings add to the involvement of lpr cells in the autoimmune process and constitute the first report that T cell vaccination may be beneficial to a spontaneously occurring autoimmune disease.
Phenotypical and biochemical characterization of a variant CD45R expression pattern in human leukocytes
Authors: Schwinzer Reinhard , Schraven Burkhart , Kyas Urte , Meuer Stefan C. , Wonigeit Kurt
Published: March 2007

As a result of a regulatory polymorphism a variant pattern of CD45R expression can be found in 8% of healthy individuals. We have previously reported that all resting T cells of these individuals react with CD45RA monoclonal antibodies (mAb) that are directed to the high‐molecular mass isoforms (220 and 205 kDa) of the leukocyte common antigen (CD45). In addition, their T cells remain CD45RA+ after in vitro activation. In this report we studied the regulatory CD45R polymorphism in more detail. Flow cytometry studies revealed that CD45RA antigens are weakly expressed on normal monocytes but are absent from granulocytes. In individuals displaying the variant CD45R pattern both monocytes and granulocytes were CD45RA+. In controls and variant T cells CD45RA mAb precipitated two chains with molecular masses of about 220 and 205 kDa. In activated T cells from controls (CD45RA−) neither p220 nor the p205 chain of CD45RA could be detected. However, activated T cells from variant individuals (CD45RA+) did not express p220 but expressed p205 of CD45RA. These data indicate that there is a failure to down‐regulate p205 CD45RA in individuals displaying the variant CD45R expression pattern. Since their T cells lost p220 CD45RA during activation the data also suggest that the two isoforms detected by CD45RA mAb can be regulated independently.
Molecular cloning of a novel member of the immunoglobulin gene superfamily homologous to the polymeric immunoglobulin receptor
Authors: Jackson David G. , Hart Derek N. J. , Starling Gary , Bell John I.
Published: March 2007

The CMRF35 monoclonal antibody recognizes a cell membrane antigen present on the surface of monocytes, neutrophils, a proportion of peripheral blood T and B lymphocytes and lymphocytic cell lines. Initial studies with CMRF35 suggested an unusual pattern of serological reactivity which did not correspond to any of the known leukocyte differentiation antigen clusters. We describe here the cloning and sequencing of a cDNA encoding the CMRF35 antigen by means of expression in COS cells and immunoselection with the CMRF35 monoclonal antibody. The cDNA encodes a novel integral membrane glycoprotein of 224 amino acids that represents a new member of the immunoglobulin (Ig) gene superfamily. The molecule comprises (a) a single extracellular Ig variable domain remarkably similar to the Fc receptor for polymeric IgA and IgM, (b) a membrane‐proximal domain containing a high proportion of proline, serine and threonine residues that was predicted to be heavily O‐glycosylated, (c) an unusual transmembrane anchor that contained a glutamic acid and a proline residue and (d) a short cytoplasmic tail. Transcripts encoding the CMRF35 protein were detected in early monocytic cell lines, in peripheral blood T cells and in some B lymphoblastoid cell lines, confirming the results of immunocytological staining. However, the level of CMRF35 expression on peripheral blood T cells was shown to decrease in response to mitogenic stimulation. The likelihood that the CMRF35 antigen shares a common evolutionary ancestor with the poly Ig Fc receptor is discussed.
An alternative pathway of B cell activation: stilbene disulfonates interact with a Cl− binding motif on AEn‐related proteins to stimulate mitogenesis
Authors: Deane Katherine H. O. , Mannie Mark D.
Published: March 2007

Stilbene disulfonates are known to competitively inhibit Cl−/HCO3− flux through Band 3‐related anion exchange (AE) proteins. To study the role of AE in lymphocyte activation, stilbene disulfonates were added to cultures of rat splenocytes (SPL). Four different stilbene derivatives were tested and each directly stimulated mitogenic proliferative responses of SPL. The mitogenic activity of these analogs paralleled their known patterns of interaction with Band 3‐related AE proteins, as measured by; (a) their effective mitogenic concentrations, (b) their rank order of mitogenic potency [DIDS > SITS > DNDS ⋍ DAzDS], (c) their patterns of nonreversible binding to the mitogenic receptor [DIDS ≫ SITS, DNDS], and (d) the specific, noncompetitive inhibition of their activity by the antagonist niflumic acid. Stilbene disulfonates directly activated purified B cell populations but not isolated T cells and furthermore, acted in synergy with anti‐IgM to stimulate proliferation of SPL. These findings show that stilbene disulfonates represent a novel class of mitogens that interact with AEn‐related proteins to stimulate an alternative activation pathway in B cells. These studies also indicate that immunomodulating activities of nonsteroidal anti‐inflammatory drugs such as niflumic acid may by mediated, in part, by their interactions with AEn‐related proteins.
High‐level expression of a human immunoglobulin γ1 transgene depends on switch region sequences
Authors: Gram Hermann , Zenke Gerhard , Geisse Sabine , Kleuser Beate , Bürki Kurt
Published: March 2007

We describe that chimeric mouse‐human immunoglobulin heavy chain (IgH) genes lacking a switch region and controlled by an IgH promoter and the intronic enhancer are only weakly expressed in transgenic mice. Insertion of part of the human Cγ1 or murine Cμ switch region into the major intron of the chimeric IgH gene results in a 102‐to 103‐fold increase in transgene expression. Analysis of B cell hybridoma clones from transgenic mice suggests that switch sequences influence IgH transgene expression at the cellular level. However, the effect of switch region sequences on IgH gene expression observed in vivo is not apparent in transfected B cell lines. These results indicate that switch region sequences which are located proximal to the constant part of the gene and which are normally present in a rearranged IgH gene after class switching represent a novel type of regulatory element that plays a critical role in IgH gene expression in vivo.
Immune regulation in self tolerance: functional elimination of a self‐reactive, counterregulatory CD8+ T lymphocyte circuit by neonatal transfer of encephalitogenic CD4+ T cells lines
Authors: Qin Yufen , Sun Deming , Wekerle Hartmut
Published: March 2007

Transfer of encephalitogenic, CD4+ T lymphocyte lines into syngeneic adult Lewis rats not only leads to the development of experimental autoimmune encephalomyelitis (EAE), but, in addition, to the expansion of counterregulatory, CD8+ T lymphocyte clones which are able to lyse specifically the encephalitogenic T cells in vitro and to neutralize their encephalitogenic capacity in vivo. In striking contrast, in neonatal rats, which still lack myelin (autoantigens), injection of the same encephalitogenic lines neither mediates EAE, nor confers protection in later life against the myelin‐specific T cells. In fact, this treatment results in the life‐long functional elimination of counterregulatory, clonotypic CD8+ T lympholcytes, which cannot even be reinduced by repeated injections of the relevant CD4+ T line. These data seem to point to a self‐protective T cell control mechanism which is developed within the immune system prior to, and thus independent of the appearance of the appropriate self antigen.
Expression and function of the neural cell adhesion molecule L1 in mouse leukocytes
Authors: Kowitz Aiga , Kadmon Guni , Eckert Marion , Schirrmacher Volker , Schachner Melitta , Altevogt Peter
Published: March 2007

The neural cell adhesion molecule L1 is a cell surface glycoprotein of the immunoglobulin superfamily which mediates adhesion between neural cells. The possibility that similar cell‐cell recognition mechanisms may be shared by the nervous and immune systems prompted us to study the expression and function of L1 in cells of the hematopoietic system. Immunofluorescence analysis using monoclonal L1 antibody revealed that the molecule is expressed in the bone marrow, spleen, and thymus of the mouse. This observation was confirmed by amplifying cDNA derived from these organs by the polymerase chain reaction with L1‐specific oligonucleotide primers. Two‐color fluorescence analysis indicated that bone marrow lymphoid and granulocyte precursor cells express low and high levels of L1, respectively. In the thymus L1 is primarily expressed by mature cells that have a strong expression of CD3 and in the spleen both B cells and T cells express L1.

The possible function of L1 in lymphoid cells was studied using subcloned ESb‐MP lymphoma cells having high or low densities of L1 on the cell surface as well as activated splenic B lymphoblasts. Parental and subcloned ESb‐MP cells that strongly expressed L1 could form homotypic aggregates in the presence of low Ca2+ levels, whereas subcloned ESb‐MP cells with a weak expression of L1 did not aggregate, suggesting that L1 mediates the Ca2+‐independent aggregation of the parental ESb‐MP cells. Furthermore, the aggregation of activated B lymphoblasts under physiological concentrations of Ca2+ and Mg2+ was inhibited by 30% in the presence of Fab fragments of polyclonal L1 antibodies, implying that L1 also mediates adhesion among normal lymphoid cells. A possible role of L1 on lymphocytes in stimulating the innervation of lymphoid organs is discussed.
Inhibitory and stimulatory signaling via immunoglobulin receptors: dichotomous responses elicited in clonal B cell populations
Authors: Van Endert Peter M. , Moldenhauer Gerhard
Published: March 2007

The paradox that cross‐linking of IgM antigen receptors on B cells by native or surrogate antigen can inhibit, as well as stimulate, B cell functions has previously been attributed to changes during maturation and activation, programming either a negative or a positive response at defined developmental stages. In contrast to this concept, we show here that some B cells possess the potential for both types of response at the same stage. In three clonal malignant human B cell populations, bivalent soluble monoclonal antibodies to IgM or idiotype, but not IgD completely inhibited spontaneous DNA synthesis, but significantly induced [3H]thymidine uptake when coupled to insoluble compounds. In co‐incubation experiments mitogenic stimuli were dominant over inhibitory ones and were still effective after prolonged pretreatment of the B cells with inhibitory reagents. Ionomycin, known to increase intracellular calcium levels, and low doses of phorbol ester, described to activate protein kinase C, also suppressed DNA synthesis. High doses of phorbol ester alone or in combination with ionomycin, however, induced DNA synthesis in two of the lymphomas. We conclude that some B cells may respond to cross‐linking of surface IgM in a dose‐dependent manner so that all signals increase DNA synthesis once a threshold has been reached. These dose‐dependent effects may in part involve signaling via breakdown of membrane inositol phosphates.
Tumor necrosis factor‐α production induced in T lymphocytes through the AIM/CD69 activation pathway
Authors: Santis Ana G. , Campanero Miguel R. , Alonso Jose L. , Tugores Antonio , Alonso Miguel A. , Yagüe Enriqueta , Pivel Juan P. , Sánchez‐Madrid Francisco
Published: March 2007

Human activation inducer molecule (AIM/CD69), a dimeric glycoprotein of 33 and 27 kDa, is the earliest inducible cell surface antigen expressed during lymphocyte activation, which has been also involved in lymphocyte proliferation. Although AIM is absent from peripheral blood resting lymphocytes, it is expressed by in vivo activated lymphocytes infiltrating sites of chronic inflammation in several pathologies, as well as by lymphocytes after in vitro activation with different stimuli. We have investigated the possibility that tumor necrosis factor‐α (TNF‐α) gene expression and protein secretion could be induced in peripheral blood T cells through the AIM/CD69 molecule. Anti‐AIM monoclonal antibodies (mAb) were able to induce TNF‐α secretion in T cells when protein kinase C (PKC) was simultaneously activated by treatment with phorbol esters. TNF‐α secretion was detected at 24 h and peaked at day 3 upon T lymphocyte activation with anti‐AIM mAb. Immunoprecipitation studies with an anti‐TNF‐α mAb from surface iodinated T cells activated through AIM, demonstrated that TNF‐α first appeared as a cell surface molecular form of 26 kDa, which is subsequently released to the extracellular medium as the 17‐kDa molecular form of TNF‐α. AIM stimulation dramatically increased TNF‐α mRNA levels, and this mRNA induction and subsequent TNF‐α secretion were virtually abrogated by the immunosuppressive drug cyclosporin A. Taken together these results indicate that AIM constitutes a novel molecular pathway in T lymphocytes for induction of TNF‐α, and suggest a relevant pathologic role for AIM+ lymphocytes located at sites of tissue injury in the pathogenesis of different chronic inflammatory diseases.
Capacity of antigen uptake by B cells, fibroblasts or macrophages determines efficiency of presentation of a soluble self antigen (C5) to T lymphocytes
Authors: Stockinger Brigitta
Published: March 2007

Self antigens in the body fluids must be taken up, processed and presented by antigen‐presenting cells (APC) in order to induce T cell tolerance. For self antigens like the fifth component of complement (C5) which is not picked up by APC via antigen‐specific receptors, presentation has to rely on uptake by nonspecific means. C5 was used as a model soluble self antigen to study the capacity of different APC (B lymphoma cells, fibroblasts and macrophages) of taking up, processing and presenting low concentrations of soluble C5 to C5 specific T cell hybrids. Under conditions of limiting antigen amounts macrophages and fibroblasts exhibited similar presentation capacity for soluble C5 while B cells did not. C5 presentation by macrophages was enhanced in the presence of C5‐specific antibody and augmented further if antigen was added in the form of particulate latex‐antigen‐antibody complexes indicating enhanced uptake via Fc receptor‐mediated endocytosis or phagocytosis. B cells presented soluble C5 only in the presence of C5‐specific antibody. Uptake of C5 under these conditions occurred via Fc receptors type II. This pathway of antigen uptake did not operate with other antigens which were presented efficiently after nonspecific endocytosis.

In light of these findings it seems reasonable to propose that nonspecific endocytosis of serum proteins like C5 by B cells is normally limited in order to avoid interference with their critical role in antigen receptor‐mediated uptake and presentation for the initiation of an antibody response. It seems likely that presentation of soluble self antigens present in the circulation may normally be the task of dendritic cells and macrophages depending on the physical shape of the antigen.
Diversity of novel recombining elements suggests developmentally programmed expression of the T cell receptor α/δ locus
Authors: Arden Bernhard
Published: March 2007

During fetal ontogeny, the first wave of γδ T lymphocytes appears in the thymus at day 14 of gestation assembling predominantly T cell receptors (TcR) with Vγ3 and Vδ1. To identify Vδ gene segments that are transcribed at day 16, subsequent to the first wave of Vδ1 expression, δ chain cDNA was amplified by the anchored polymerase chain reaction with single‐sided specificity for Cδ. Unexpectedly, most of the cDNA clones do not contain V gene segments. In some cDNA clones an alternative splice from the leader exon to the Cδ exon has deleted the whole variable region exon. In other cDNA clones, multiple non‐V‐like elements are juxtaposed to the Dδ2 and Jδ1 gene segments. A large number of these diverse elements appear to be rearranged in fetal thymocytes, bringing Vα gene segments located upstream of the recombining element into proximity to the Jα locus. It is proposed that these rearrangements make irreversiblethe commitment to the TcR αβ lineage and determine a programmed read out of different clusters of Vα gene segments.
Among naive precursor cell subpopulations only progenitors of memory B cells originate germinal centers
Authors: Linton Phyllis‐Jean , Lo David , Lai Lily , Thorbecke G. Jeanette , Klinman Norman R.
Published: March 2007

Immunization leads to the generation of both antibody‐forming cells (AFC) and memory B cells which are thought to arise in germinal centers within lymphoid follicles. The findings that the precursors to memory B cells reside in the J11Dlo subpopulation of the spleens in non‐immune mice and that this subpopulation is distinct from conventional AFC precursors, including CD5+ B cells, suggest that the precursors of germinal centers might also reside in the J11Dlo subpopulation. To test this hypothesis, SCID mice were repopulated with CD4+ carrier‐primed T cells and T‐depleted J11Dlo, J11Dhi or CD5+ B cells and immunized with a hapten‐carrier conjugate. Only the J11Dlo population was enriched for cells that produced germinal centers. Thus, the subpopulation of precursors that generates memory B cells also originates germinal centers.
Enrichment of primary lymphocyte‐supporting stromal cells and characterization of associated B lymphocyte progenitors
Authors: Funk Phillip E. , Witte Pamela L.
Published: March 2007

Studies of Whitlock/Witte long‐term bone marrow cultures have revealed the necessity of two cell types for B lymphopoiesis, a stem cell and the stromal cell. While a number of stromal cell lines exist they have been found to be heterogeneous with respect to cell surface marker expression and growth factor production. Separation and analysis of fresh bone marrow stromal cells is, therefore, necessary to understand the regulation of lymphopoiesis in vivo. Here we report the early stages of such studies. We demonstrate that stromal cells, as assessed by morphology and alkaline phosphatase reactivity after short‐term culture, are enriched in cellular aggregates that can be separated from bone marrow suspensions. Stromal cells are present in aggregates at a frequency of one per thousand cells, whereas marrow from which the aggregates have been removed contains only one stromal cell per fifty‐thousand cells. These aggregates are able to form Whitlock cultures from greatly reduced numbers of initiating cells, indicating that they contain culturable B lineage precursors as well as stromal cells capable of supporting B lymphopoiesis. The aggregates appear to be naturally formed and provide a means to examine native B cell precursor‐stromal cell contacts. We find little evidence for sequestering of late‐stage B cell precursors within the aggregates. Terminal deoxynucleotidyl transferase‐positive cells, on the other hand, are approximately three times more frequent in bone marrow aggregates, suggesting close contact between very early B cell progenitors and stromal cells within the aggregates. The finding that stromal cells are enriched in cellular aggregates is an important first step in the ultimate isolation of these cells from marrow suspensions, which is vital to understanding stromal cell function in vivo.
Peptide recognition, T cell receptor usage and HLA restriction elements of human heat‐shock protein (hsp) 60 and mycobacterial 65‐kDa hsp‐reactive T cell clones from rheumatoid synovial fluid
Authors: Quayle Alison J. , Wilson Kay Black , Li Shu Guang , Kjeldsen‐Kragh Jens , Oftung Fredrik , Shinnick Thomas , Sioud Mouldy , Førre Øsystein , Capra J. Donald , Natvig Jacob B.
Published: March 2007

A commonly held postulate regarding the etiology of rheumatoid arthritis (RA) is that of antigenic mimicry. Recent interest has focused on the mycobacterial 65‐kDa heat‐shock protein (hsp) as a putative causal agent. The 65‐kDa hsp has over 40% sequence homology with the human hsp 60, and elevated synovial T cell responses to both antigens have been demonstrated in RA and juvenile rheumatoid arthritis patients. Such T cells should, therefore, be specific for shared epitopes on the two antigens. To investigate this, we screened synovial fluid mononuclear cells from two early RA patients with peptides of the 65‐kDa hsp which have the greatest homology with the human hsp 60. We also raised a panel of T cell clones from one of the patients with the 65‐kDa hsp. The synovial T cell population from both patients and one of the T cell clones recognized a peptide representing the amino‐acid sequence 241–255. This clone also responded to the peptide of the equivalent human sequence, and was restricted by HLA‐DQ. A second T cell clone recognized an adjacent epitope (amino acid sequence 251–265) which is also highly homologous with the human sequence, but this clone was restricted by HLA‐DR. The clones utilized different Vβ gene segments but the same Dβ and Jβ gene elements, and both exhibited specific cytotoxicity against autologous antigen‐pulsed macrophages. Our findings, therefore, do not disagree with the postulate that autoimmune disease could possibly be triggered by bacterial epitopes with homology to self protein. However, it is also noted that there are alternative interpretations of this data.
Induction of high‐affinity paf receptor expression during T cell activation
Authors: Calabresse Cynthia , Nguer Momar Cheik , Pellegrini Olivier , Benveniste Jacques , Richard Yolande , Thomas YolèNe
Published: March 2007

Activated human T cells via the CD2 or the CD3 pathways exhibited a higher capacitythan resting T lymphocytes to incorporate and metabolize [3H] pafacether (paf) at 37°C. Resting T lymphocytes lacked specific binding capacity for paf, yet high‐affinity paf receptors (paf‐R) were inducedon CD3‐ or CD2‐dependent activation. This up‐regulation in the number of paf‐R became apparent by day 1 of culture, reached a maximum of about 25000 sites cell by days 4 to 6 and subsequently declined. Interestingly, human recombinant interleukin‐2 in a dose‐dependent manner prevented the decrease of high‐affinity paf‐R expression on T cells. By contrast, the receptor affinitywas constant throughout the culture period. Thus, paf‐R at different stages of Tcell activation were indistinguishable with respect to receptor‐ligand interaction, and differed only in their number. Together, these data demonstrate that after activation human T cells develop membrane high‐affinity paf‐binding sites. They also suggest for the first time that expression of the paf‐R are coupled to T cell activation and/or differentiation.
Non‐additive effects of multiple amino acid substitutions on antigen‐antibody recognition
Authors: Mateu Mauricio G. , Andreu David , Carreno Cristina , Roig Xavier , Cairó Jordi‐Joan , Camarero Julio A. , Giralt Ernest , Domingo Esteban
Published: March 2007

Synthetic peptides have been used to mimic the main antigenic site of foot‐and‐mouth disease virus (FMDV) of serotype C and of several variant isolates. This region includes multiple continuous B cell epitopes. The effect of single amino acid replacements, individually or in combination, on antigen specificity has been evaluatedusing monoclonal antibodies. Quantitative enzyme immunodot assays have shown that both additive and non‐additive effects of multiple replacements occur in continuous Bcell epitopes, with regard to antibody recognition. Antigenically critical single replacements may be compensated by other, non‐critical replacements. Thus, the role of a single amino acid on antibody recognition depends on the sequence context in the antigenic domain. The non‐additive effects of multiple replacements may modulate the extent of antigenic diversification of highly variable RNA viruses, and keep virusesconfined within antigenic groups by precluding linear antigenic divergence.
Interleukin‐1 induces protein tyrosine phosphorylation in T cells
Authors: Mũnoz Eduardo , Zubiaga Ana , Huang Chi‐Kuang , Huber Brigitte T.
Published: March 2007

Interleukin (IL)‐1α activates multiple signal transmission pathways inthe T helper type 2 cell line, D10A, and these pathways are linked to two separate IL‐1 receptors (IL‐1R). In the present report we show that IL‐1 induces the activation of tyrosine kinase in these cells, leading to tyrosine phosphorylationof a subset of proteins of 38, 75, 97 and 115 kDa. This type of phosphorylation is prevented by a monoclonal antibody directed against the 80‐kDa IL‐1R and by tyrphostins which are specific inhibitors of tyrosine kinases. In addition, this inhibitor blocks IL‐1‐ and IL‐2‐induced proliferation in D10A cells as well as the c‐myc and c‐myb proto‐oncogene mRNA expression inresponse to IL‐1. Interestingly, the inhibitor of cAMP‐dependent kinase, H‐8, only blocks IL‐1‐induced c‐myb, but not c‐myc mRNA expression. Altogether, our results demonstrate that the activation of a tyrosine kinase(s) is an early and major event that happens after IL‐1/IL‐1R interaction, leading to an increase in intracellular cAMP which results in c‐myb and IL‐5 mRNA expression. Independent of cAMP, by tyrosine phosphorylation of specific substrates IL‐1 also induces c‐myc andIL‐6 mRNA expression and cellular proliferation.
Cytokine enhancement of complement‐dependent phagocytosis by macrophages: synergy of tumor necrosis factor‐α and granulocyte‐macrophage colony‐stimulating factor for phagocytosis of Cryptococcus neoformans
Authors: Collins Helen L. , Bancroft Gregory J.
Published: March 2007

We have examined the regulation of complement dependent phagocytosis by macrophage‐activating cytokines. Tumor necrosis factor (TNF)‐α and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), but not interferon‐γ, interleukin‐4 or macrophage‐CSF, stimulated ingestion of the encapsulated fungal pathogen Cryptococcus neoformans by resident peritoneal macrophages in vitro. This was dependent upon opsonization of the yeasts with complement, 72 h of incubation with the cytokines for maximum effect, and the obligate involvement of the macrophage CR3 receptor. TNF‐α and GM‐CSF synergized at low concentrations, resulting in dramatic up‐regulation of phagocytosis when compared to either cytokine alone. Supernatants from C. neoformans‐specific T cells also increased macrophage phagocytic efficiency. Finally, the administration ofneutralizing mAb specific for TNF‐α and GM‐CSF increased mortality in C. neoformans‐infected mice, and induced the rapid progression of disease with involvement of the brain and meninges. We conclude that TNF‐α andGM‐CSF are potent regulators of complement‐dependent phagocytosis by murine macrophages. Macrophage activation with these two cytokines can completely overcome the anti‐phagocytic properties of the virulent yeasts. Our results, therefore, implicate TNF‐α and GM‐CSF as important mediators of resistance to encapsulated pathogens such as C. neoformans where ingestion of the organism is acritical process in host resistance.
Membrane molecules which trigger the production of interleukin‐1 and tumor necrosis factor‐α by lipopolysaccharide‐stimulated human monocytes
Authors: Couturier Christine , Jahns Gerlinde , Kazatchkine Michel D. , Haeffner‐Cavaillon Nicole
Published: March 2007

We have investigated the role of the membrane molecules CD11/CD18 and CD14 whichmay mediate the binding of lipopolysaccharide (LPS) to human monocytes, in the induction of the production and release of interleukin (IL)‐1 and tumor necrosis factor‐α (TNF‐α) by LPS‐stimulated cells. Blockade of CD11a, CD11b and CD18 with saturating concentrations of specific mAb did not inhibit the releaseof cytokines from LPS‐stimulated monocytes. In contrast, inhibition of the release of IL‐1β and TNF‐α occurred in monocytes cultures that had been pretreated with either of two monoclonal antibodies (mAb) recognizing different epitopes on the CD14 molecule. The binding of LPS to CD14 has been previously shown to require serum factors. In the present study, we found that serum had an enhancing effect on the release of IL‐1 and TNF‐α from LPS‐stimulated cultures of normal human monocytes. The inhibitory effect of anti‐CD14 mAb was, however, observed in cultures performed in the presence or in the absence of serum, suggesting that triggering of IL‐1/TNF‐α release by CD14 is independent of LPS‐binding proteins or other serum proteins. IL‐1β and TNF‐α were also released from LPS‐stimulated cultures of monocytes from patients with paroxysmal nocturnal hemoglobinuria lacking expression of CD14. Thus, CD14 but notCD11/CD18 can trigger serum‐dependent and independent cytokine release from endotoxin‐stimulated normal human monocytes; CD14 is not, however, the only LPS receptor that is involved in the secretory response of endotoxin‐stimulated cells.
Both LFA‐1‐positive and ‐deficient T cell clones require the CD2/LFA‐3 interaction for specific cytolytic activation
Authors: Kemenade Elly Van De Wiel‐van , Te Velde Anje A. , De Boer Annemiek J. , Weening Ron S. , Fischer Alain , Borst Jannie , Melief Cornelis J. M. , Figdor Carl G.
Published: March 2007

We investigated the capacity of T lymphocytes from a leukocyte adhesion‐deficient (LAD) patient to respond to alloantigen. Leukocytes of this patient completely lacked LFA‐1 surface expression due to the absence of mRNA coding for theLFA‐1 β chain. Despite the absence of LFA‐1, T lymphocytes obtained from this patient, cultured with allogeneic stimulator cells (lymphoblastoid B cells JY), were capable of lysing JY cells. Furthermore, two T cell clones (one CD4+ and one CD8+), generated from this lymphocyte culture, specifically lysed theallogeneic lymphoblastoid JY cells. The cytolytic capacity of LFA‐1‐negative T lymphocytes and T cell clones was comparable to that of control LFA‐1‐positive T cells with allospecificity against JY. Detailed analysis of the CD4 positive and LFA‐1‐negative T cell clone demonstrated that it specifically recognized HLA‐DQ. Antibody inhibition studies showed that the CTL/target cell interaction was mediated through the CD2/LFA‐3 adhesion pathway. LFA‐1 expressed by the target cells did not participate in the CTL/target cell conjugate formation and contributed only minimally to the cytotoxic activity. Moreover, when allogeneic LFA‐1‐deficient B cells, bearingthe appropriate HLA‐DQ alloantigen, were used as target cells, significant levels of specific cytotoxicity were measured, further excluding a role for LFA‐1 in this interaction. The adhesion molecules, VLA‐4, CD44 and L‐selectin (LECAM1) were not involved.

These results demonstrate that LFA‐1‐negative T lymphocytes can exert allospecific cytotoxicity and that CTL/target cell contact is mediated through the CD2/LFA‐3 route. This observation may explain in part why in LAD patients viralinfections, cleared largely by T cells, are less frequently observed than bacterial infections, in which phagocytic cells play a major role.
HLA‐DR peptide‐induced alloreactive T cell lines reveal an HLA‐DR sequence that can be both “dominant” and “cryptic”: evidence for allele‐specific processing
Authors: de Koster H. Saskia , Rood Jon J. Van , Termijtelen Annemarie
Published: March 2007

Previously, we reported on a T cell line, ThoU6, which we obtained through stimulation of DPw3+ cells with a synthetic” DR3 peptide “ with a sequence identical to the third hypervariable region of the DRB1*0301 chain. This T cell line recognizes both the synthetic peptide presented by DPw3 as well as DR3+ DPw3+ stimulator cells. This implies that the synthetic DR3 peptide has a natural counterpart in DR3‐positive cells. Here we describe the recognition pattern of another T cell line that was sensitized with the same synthetic DR3 peptide. This T cell line, BieU6, shows both HLA‐DRw13/Dw18 (self)‐restricted recognition of the synthetic DR3 peptide and allorecognition towards DR13/Dw19, a molecule which is highly homologous to Dw18, in the absence of synthetic peptide. These results suggest that the epitope formed by the Dw18 molecule plus the synthetic DR3 peptide and recognized by T cell line BieU6 mimics the Dw19 molecule. The potential role for a Dw19‐specific peptide is discussed. The inability of T cell line BieU6 to recognize Dw18+ DR3+ cellsindicates that, in this case, the synthetic DR3 peptide is “cryptic”, i.e. does not have a natural counterpart that is effectively presented to T cells. Mapping of the shortest peptides recognized by T cell lines ThoU6 and BieU6 indicate thatthese sequences are fully overlapping. We, therefore, suggest that the antigen‐presenting molecules, HLA‐DPw3 and HLA‐Dw18, differ in their accessibilityfor self peptides derived from the third hypervariable region of DR molecules. These observations may be explained by allele‐specific processing.
T cell responses to human recombinant acetylcholine receptor‐α subunitin myasthenia gravis and controls
Authors: Sun Jia‐Bin , Harcourt Gillian , Wang Zeng‐Yu , Hawke Simon , Olsson Tomas , Fredrikson Sten , Link Hans
Published: March 2007

Antibodies against the nicotinic acetylcholine receptor (AChR) of the neuro‐muscular junction are detectable in most patients with myasthenia gravis (MG) and assumed to participate in the destruction of the AChR, thereby, causing the characteristic signs and symptoms of the disease. The extent and importance of T cell responses to AChRand its subunits in MG are still unsettled. We have now examined T cell reactivities using human recombinant AChR‐α subunit as antigen. Upon recognition of appropriate antigen in an MHC‐class II‐restricted fashion, memory T cells secrete interferon‐γ (IFN‐γ). Adopting this principle in an immunospot assay we found that 73% of MG patients had recombinant human AChR‐α subunit‐reactive T cells at a median value of 1 per 56000 blood mononuclear cells, while only 27% of the MG patients responded to the α subunit in a conventionallymphocyte proliferation assay. This compares with even lower numbers of AChR‐reactive T cells and 14% positivity in the proliferation assay among control subjects. The T cell responses to the control antigens purified protein derivative and myelin basicprotein did not differ between MG and controls, underlining the specificity of an augmented T cell reactivity to AChR‐α subunit in MG. α Subunit‐specific T cell lines and clones propagated from patients with MG and healthy controls yielded a high proportion of α subunit‐reactive T cells in the IFN‐γ immunospot aassay. Their appearance was inhibited by the addition of monoclonal anti‐MHC class II antibodies, demonstrating that an MHC‐restricted T cell response was measured. Our data underline that the AChR‐α subunit is a major T cell autoantigen in MG.
Non‐random features of the repertoire expressed by the members of one Vx gene family and of the V‐J recombination
Authors: Milstein César , Even Jos , Jarvis John M. , Gonzalez‐Fernandez Africa , Gherardi Ermanno
Published: March 2007

The 5' and 3' flanking sequences of 14 members of the VxOx (VK 4/5) gene family of BALB/c mice have been established. The family was unusual in the number of bases between the codon for Pro 95 and the heptamer sequence; most members contained four but there were also examples of none. A conserved leader sequence was used toamplify the genomic DNA of rearranged genes in order to analyze the spleen B cell repertoire of non‐immunized animals. The library contained many members with virtually identical sequences to one or other of the already known members of the family. In addition, there were repeats of other sequences, allowing the definition of 12 hitherto undefined members of the family. Only 3 out of 96 could have originated by gene conversion, or as artefacts of the amplification procedure, and only 2 were putative somatic mutants.

The frequency of expression of different members of the VxOx gene family was not random, and some germ‐line genes were unrepresented in the library. The high frequency of VxOx1‐Jx5 is in line with the dominance of this combination in the oxazolone response.

An analysis of the junctional segment showed that although in most cases the diversity was due to trimming, there were exceptions indicating de novo additions (N or P bases). The average number of bases trimmed from the Vx and the Jx segments was not the same. There was no correlation in the number of bases trimmed from Vx or Jx in each recombination. The implications of asymmetric trimming in terms of the mechanism of recombination are discussed.
Cytotoxic T lymphocytes against the antigen‐processing‐defective RMA‐S tumor cell line
Authors: Sijts Alice J. A. M. , De Bruijn Marloes L. H. , Nieland John D. , Kast W. Martin , Melief Cornelis J. M.
Published: March 2007

RMA‐S is an antigen processing‐defective cell line, obtained from a Rauscher virus‐induced tumor. The cells express only a low level of cell surface major histocompatibility complex (MHC) class I molecules, which are supposed to be devoid of internally derived antigenic peptides. We investigated Rauscher virus expression and Rauscher peptide presentation to virus‐specific cytotoxic T lymphocytes (CTL) by this cell line. Viral proteins are expressed properly, both intracellularly and atthe cell surface of RMA‐S. Rauscher peptides are presented tovirus‐specific CTL in the groove of both the class I H2Kb and Db molecules, but at a low level. Culture of RMA‐S cells at room temperature increases theirsusceptibility to CTL. The RMA‐S defect thus affects, but not totally abrogates, Rauscher peptide presentation by MHC class I molecules via the endogenous pathway. Thisindicates that the RMA‐S antigen processing deficit is not absolute.
A cross‐species functional interaction between the murine major histocompatibility complex class I α3 domain and human CD8 revealed by peptide‐specific cytotoxic T lymphocytes
Authors: Moots Robert J. , Samberg Nancy L. , Pazmany Laszlo , Frelinger Jeffrey A. , McMichael Andrew J. , Stauss Hans J.
Published: March 2007

The monomorphic cell surface glycoprotein CD8 acts as co‐receptor in the recognition of peptide‐major histocompatibility complex (MHC) class I complexes by cytotoxic lymphocytes (CTL) by binding to the monomorphic α3 domain of the class I molecule. Positions 227 and 245 in the class I α3 domain appear to be especially important for this interaction. Recent reports suggest there is no interspecies recognition between CD8 and MHC class I.

In this study, hybrid genes from human class I HLA‐A0201 and murine class I H2Kb were transfected into human and mouse cells and tested in Cr‐release assays using HLA‐A0201‐restricted influenza A matrix peptide‐specific CTL as effectors. Transfected cells expressing chimeric genes comprising the α1 and α2 domains from HLA‐A0201 together with the H2Kb α3 domain were lysed as effectively as wild‐type HLA‐A0201 and in both cases, killing was blocked by anti‐CD8 antibodyequally well. These results indicate that human CD8 can interact with the α3 domain of murine class I to the same level as human class I.
The variable occurrence of CD45RA on CD8+ thymocytes correlates with the presence of Mtv sequences; its expression on other thymocytes is rare
Authors: Huby Russell D. J. , Goff Lindsey K.
Published: March 2007

While all thymocytes express CD45, only a small fraction (<3% in mice) bear the high molecular weight isoform, CD45RA. It has been suggested that these cells alone constitute the generative thymocyte lineage and should, therefore, occur within every ontogenic subset. To test this, we determined CD45RA expression among thymocyte subsets defined by CD4 and CD8. In some strains, exemplified by C57BL/Icrf, very few (< 0.2%) Tthymocytes expressed CD45RA and were mostly CD4−CD8− or CD4+CD8+, inconsistent with them constituting the generativelineage. In others, exemplified by BALB/c, CD45RA was expressed on up to 3% of T thymocytes, which were mostly CD4−CD8+. The limited occurrence of CD4−CD8+CD45RA+ thymocytes suggests that they are a nonconstitutive subset playing a role in the thymus of only some strains. Their occurrence correlates with that of Mtv proviruses within the mouse genome; however, their T cell receptor Vβ repertoire is diverse, suggesting they are not uniquely selected by Mtv superantigens. We propose that they may be mature CD8 T cells, possibly responsible for introducing viral superantigens into the thymus.
High frequency of cord blood lymphocytes against mycobacterial 65‐kDa heat‐shock protein
Authors: Fischer Heinrich Peter , Sharrock Claire E. M. , Panayi Gabriel S.
Published: March 2007

A high frequency of nonadherent mononuclear cells in human cord blood proliferates in response to mycobacterial 65‐kDa heat‐shock protein. The frequency range in cord blood is not different from that in peripheral blood of Bacillus Calmette‐Guérin vaccinated adults. In comparison we found 10 to 100 times lower frequencies to purified protein derivative in nonadherent cord blood mononuclear cells thanin adult peripheral blood mononuclear cells. These findings may provide experimental support for Cohen's theory of the immunological homonculus.
Anti‐arthritic effects demonstrated by an interleukin‐2 receptor‐targeted cytotoxin (DAB486IL‐2) in rat adjuvant arthritis
Authors: Bacha Patricia , Forte Serene E. , Perper Stuart J. , Trentham David E. , Nichols Jean C.
Published: March 2007

DAB486IL‐2 is an interleukin‐2 receptor‐specific cytotoxin which selectively targets and kills cells which bear the high‐affinity form of the IL‐2 receptor. Since elimination of activated T lymphocytes may be useful in the treatment of rheumatoid arthritis, the effect of DAB486IL‐2 treatment in an animal model of arthritis was investigated. We demonstrated that rats treated with DAB486IL‐2 during the induction phase of disease have delayed onset of symptoms and significantly reduced severity of inflammation as well as a depressed proliferative response to mycobacterial stimulation in vitro. In addition, the presence of preexisting antibodies to the molecule had no impact on the anti‐arthritic effects observed in this model. These data suggest that DAB486IL‐2 may have therapeutic potential in the treatment of rheumatoid arthritis.
Minimal growth requirements of mature T lymphocytes: interleukin (IL)‐1 and IL‐6 increase growth rate but not plating efficiency of CD4 cells stimulated with anti‐CD3 and IL‐2
Authors: Soldaini Elisabetta , Robson MacDonald H. , Nabholz Markus
Published: March 2007

We show that interleukin (IL)‐2 is necessary and sufficient for the proliferation of both CD4 and CD8 subsets of peripheral murine T cells activated by plastic‐bound anti‐CD3 monoclonal antibodies (mAb). The frequency of proliferating cells (f) was 0.32 for CD4 cells and 0.63 for CD8 cells. These frequencies were not increased by the addition of IL‐1 or IL‐6, alone or in combination. These cytokines were unable to induce responsiveness to IL‐2 in T cells confirming that they cannot substitute for the signal delivered via the TcR/CD3 complex. On the other hand, IL‐1 and IL‐6 increase the growth rate of CD4 cells. The addition of IL‐6 significantly lowered the mean doubling time (dt) of CD4 cells (dt: 26 h vs. 38 h in the presence of IL‐2 alone, p < 0.01), while the addition of IL‐1, ineffective by itself, combined with IL‐6 further increased the growth rate of CD4 cells (dt: 23 h, p < 0.001). The growth rate of CD8 cells stimulated with anti‐CD3 and IL‐2, was markedly faster than that of CD4 cells (dt: 18 h vs. 38 h, p < 0.001) and was not significantly influenced by addition of IL‐1 and/or IL‐6.