bluuebunny/crossref_metadata_2025 huggingface dataset:
Genomic structure of the human Ig lambda 1 gene suggests that it may be expressed as an Ig lambda 14.1-like protein or as a canonical B cell Ig lambda light chain: implications for Ig lambda gene evolution.
Authors: Evans R J , Hollis G F
Published: June 2004

In pre-B cells, immunoglobulin mu (Ig mu) is associated with pre-B cell-specific proteins to form a multimeric complex that is found on the cell surface. One of these proteins is encoded by the three exon Ig lambda-like gene 14.1, whose expression is restricted to pre-B cells and occurs from an unrearranged gene. A comparison of the 14.1 gene structure to the seven-gene human Ig lambda locus revealed that the most 5' gene, Ig lambda 1, is organized in a three-exon structure very similar to the 14.1 gene. Transcription and splicing of these three-exon sequences would lead to an mRNA with an open reading frame which could encode a light (L) chain-like protein with a molecular weight of 23,045. Our analysis suggests that two transcripts may be produced from the Ig lambda 1 gene that share the same Ig lambda 1 constant region-containing third exon. One transcript would include all three 14.1-related exons and be expressed from the germline gene, and the second transcript would be produced after variable-joining (V-J) recombination has occurred to Ig lambda J1 and would encode a classic Ig lambda L chain protein. The conservation of the genomic organization of the human 14.1 and Ig lambda 1 genes and the mouse homolog, lambda 5, relative to the classic Ig lambda L chain genes provides insight into the evolution of Ig genes.
The kinetics of T cell antigen receptor expression by subgroups of CD4+8+ thymocytes: delineation of CD4+8+3(2+) thymocytes as post-selection intermediates leading to mature T cells.
Authors: Shortman K , Vremec D , Egerton M
Published: June 2004

Cortical thymocytes from adult mice, separated on the basis of coexpression of CD4 and CD8 or of binding of high levels of peanut agglutinin (PNA), were subdivided according to the level of expression of the T cell receptor (TCR)-CD3 complex. The incidence of dividing cells in the resultant subpopulations was determined by DNA staining. Precursor-product relationships and the timing of TCR-CD3 acquisition were studied using continuous in vivo [3H]TdR labeling and radioautography. The extent of intrathymic selection for TCR specificity in the subpopulations was determined from the incidence of cells bearing V beta 6 or V beta 17a in different mouse strains. The majority of dividing CD4+8+ blast cells expressed extremely low levels of TCR-CD3, indicating that TCR expression and specificity selection generally occurred after division ceased. The [3H]TdR-labeling studies indicated that postdivision TCR expression was rapid, and that those nondividing cortical thymocytes which had not expressed significant levels of TCR by day 1, remained extremely low or negative for their entire 3.6-d lifespan. Small cortical thymocytes which expressed moderate levels of TCR-CD3, were predominantly an unselected population with a lifespan of 3.8 d. A small subgroup of CD4+8+ PNA+ cortical thymocytes expressing high levels of TCR-CD3 was identified as a nondividing intermediate between the small cortical thymocytes expressing moderate levels of TCR and mature medullary thymocytes. These intermediates showed a 1-d lag in [3H]TdR labeling, then a 3.4-d transit time. The cell flux through this intermediate subpopulation was approximately 10(6) cells/d, similar to the rate of turnover of mature thymocytes; thus, although only 3-4% of thymocytes progressed to this intermediate state, once reaching it most then progressed to full maturity. In accordance with this, the incidence of the V beta selection markers within the intermediate subpopulation indicated that both positive and negative selection had already occurred. Selection for TCR specificity in the systems studied appeared to take place among CD4+8+ thymocytes expressing intermediate levels of TCR.
The mouse vitronectin receptor is a T cell activation antigen.
Authors: Moulder K , Roberts K , Shevach E M , Coligan J E
Published: June 2004

In this report, we demonstrate that the T cell activation antigen, recognized by monoclonal antibody H9.2B8, is the murine homologue of the vitronectin receptor (VNR) and, thereby, we provide initial evidence that VNR is expressed on lymphoid cells. VNR is expressed on a variety of T cell lines, tumors, and Con A-activated splenocytes, but not resting T cells, and is capable of binding to the extracellular matrix proteins fibronectin, fibrinogen, and vitronectin, via the tripeptide sequence RGD. There was no evidence of novel beta chains pairing with the VNR alpha chain, as has been demonstrated in some human cells. In view of recent studies demonstrating that this molecule functions as an accessory molecule in T cell activation, the VNR may play an important role in mouse T cell functions.
Identification of a 107-kD glycoprotein that mediates adhesion between stromal cells and hematolymphoid cells.
Authors: Kina T , Majumdar A S , Heimfeld S , Kaneshima H , Holzmann B , Katsura Y , Weissman I L
Published: June 2004

The mechanism of cell complex formation between lymphocytes and stromal cells was investigated. We found that lymphoid lines of both T and B lineages could form cell complexes with stromal cells from the thymus as well as bone marrow but not with macrophages or typical fibroblast lines. Formation of these cell complexes is temperature dependent and requires the presence of Mg2+, active cellular metabolism, and microfilament assembly of cytoskeleton. We raised an antiserum against a thymic stromal cell clone (BATE-2) in rats and found that, after absorption, this serum could effectively block cell complex formation between lymphocytes and stromal cells from both thymus and bone marrow. An efficient blocking was obtained only when the antiserum was added at the initial stage of cell interaction. From the blocking experiments and the SDS-PAGE analysis of immunoprecipitated materials from the stromal cell surface, we identified a unique 107-kD glycoprotein on the stromal cells as a molecule for mediating stromal cell-lymphocyte interaction. This is further supported by the findings that an antiserum raised in hamsters against the excised gel band corresponding to 107 kD, which specifically immunoprecipitated the 107-kD molecule, effectively blocked the lymphocyte-stromal cell interaction. The possible function of this molecule in hematolymphoid development is discussed.
Preferential utilization of specific immunoglobulin heavy chain diversity and joining segments in adult human peripheral blood B lymphocytes.
Authors: Yamada M , Wasserman R , Reichard B A , Shane S , Caton A J , Rovera G
Published: June 2004

We have examined at the molecular level the CDR3 and adjacent regions in peripheral blood B lymphocytes of normal individuals. A total of 111 sequences (12-28 sequences from six individuals) were obtained after cloning of the polymerase chain reaction-amplified segments into plasmids or phage. The average length of the VDJ joining was 109 nucleotides, with a range from 79 to 151. Approximately 75% of the sequences were in frame when translated into amino acids. Among the JH segments, JH4 was found most frequently (in 52.5% of the sequences), and JH1 and JH2 segments the least frequently (approximately 1% of the clones). A polymorphic JH6 gene with a one-codon deletion accompanied by a base change was present in two of six patients. Preferential breakpoints were found for JH2, JH3, JH4, and JH5, although the breakpoints of JH6 were distributed more heterogenously. In approximately 90% of the cases, significant homology of the D regions with published D sequences was found. Preferential usage of a particular coding frame was observed in in-frame sequences utilizing DA, D21/9, and DM1 segments. However, in general, all coding frames of germline D genes were used to generate CDR3s. Eight sequences that have a DN1-like D sequence with two base changes at the same positions were identified, suggesting the likely existence of a new germ line D gene belonging to the DN families. Using probes specific for a particular CDR3, the frequency of a specific B cell clone in the peripheral blood of normal individuals was estimated to be at most as high as 1/20,000.
5' regulatory region of a novel cytokine gene mediates selective activation by interferon gamma.
Authors: Wright T M , Farber J M
Published: June 2004

A newly described member of the platelet factor 4 family of cytokine genes, mig, is selectively induced by interferon gamma (IFN-gamma), and not IFN-alpha, in the mouse macrophage-like cell line RAW 264.7. Treatment of RAW 264.7 cells with IFN-gamma activated mig gene transcription as determined by nuclear run-on assays. mig genomic clones were isolated, and constructs containing genomic fragments that included the mig promoter region and the CAT reporter gene were prepared. In RAW 264.7 cells transfected with these constructs, CAT activity was found to be selectively induced by IFN-gamma. A 278-bp genomic fragment containing 235 nucleotides 5' of the transcription start site was sufficient for IFN-gamma-selective induction of CAT activity. Analysis of 5' deletion mutants localized a region essential for activation by IFN-gamma to within 64 nucleotides extending from -235 to -172. A genomic fragment containing this sequence was capable of conferring IFN-gamma inducibility to constructs with a heterologous promoter.
Somatic mutations in the variable regions of a human IgG anti-double-stranded DNA autoantibody suggest a role for antigen in the induction of systemic lupus erythematosus.
Authors: van Es J H , Gmelig Meyling F H , van de Akker W R , Aanstoot H , Derksen R H , Logtenberg T
Published: June 2004

The processes that govern the generation of pathogenic anti-DNA autoantibodies in human systemic lupus erythematosus (SLE) are largely unknown. Autoantibodies may arise as a consequence of polyclonal B cell activation and/or antigen-driven B cell activation and selection. The role of these processes in humoral autoimmunity may be studied by molecular genetic analysis of immunoglobulin (Ig) variable (V) regions of antibodies that are characteristic of SLE. We have analyzed the gene elements that encode a high affinity, IgG anti-double-stranded DNA autoantibody secreted by a monoclonal Epstein-Barr virus (EBV)-transformed cell line derived from a patient with active SLE. In addition, we have identified, cloned, and sequenced the germline counterparts of the VH and VL genes expressed in this autoantibody. The comparison of both sets of gene elements shows that the autoantibody VH and VL regions harbor numerous somatic mutations characteristic of an antigen-driven immune response. The light chain expressed in this autoantibody is a somatically mutated variant of the kv325 germline gene that is frequently associated with paraproteins having autoantibody activity and with Ig molecules produced by malignant B cells that express the CD5 antigen. Furthermore, the utilized DH segment has been repeatedly found in multireactive, low affinity IgM anti-DNA autoantibodies from SLE patients and healthy individuals. These results suggest that pathogenic IgG anti-DNA autoantibodies in human SLE may arise through antigen-driven selection of somatic mutations in the gene elements that frequently encode multireactive IgM autoantibodies.
Efficient and selective presentation of antigen-antibody complexes by rheumatoid factor B cells.
Authors: Roosnek E , Lanzavecchia A
Published: June 2004

Using Epstein-Barr virus B cell clones and antigen-specific T cell clones, we asked how antigen-antibody complexes are handled by B cells. We found that the only B cells capable of efficient presentation of antigen-antibody complexes are those that bind the complexes via membrane immunoglobulin, i.e., rheumatoid factor-producing B cells and, to a lower extent, antigen-specific B cells. On the contrary, nonspecific B cells, although capable of binding antigen-antibody complexes, fail to present them to T cells. Thus, rheumatoid factor B cells can present any antigen in the context of an immune complex and be triggered by T cells specific for a variety of foreign antigens. These results demonstrate a mechanism of intermolecular help that may be responsible for the production of rheumatoid factor and possibly of other types of autoantibodies.
Anti-CD4 mediates clonal anergy during transplantation tolerance induction.
Authors: Alters S E , Shizuru J A , Ackerman J , Grossman D , Seydel K B , Fathman C G
Published: June 2004

Depletion of CD4+ cells using anti-CD4 monoclonal antibodies leads to allograft tolerance. Here we show that anti-CD4-mediated tolerance to pancreatic islets of Langerhans transplanted from an A/J (IEk) donor to a diabetic C57B1/6 (B6) (IE-) recipient occurs in the absence of clonal deletion of the potentially IE-reactive V beta 11+ T cells. Instead, a state of clonal anergy is induced in both the CD4+V beta 11+ and CD8+V beta 11+ T cell subsets. This clonal anergy can be partially overcome in vitro by the addition of recombinant interleukin 2.
Extrahepatic transcription of human C-reactive protein.
Authors: Murphy T M , Baum L L , Beaman K D
Published: June 2004

We synthesized and cloned cDNA from human peripheral blood mononuclear cell (PBMC) transcripts that were hybrid selected by pCRP5, a liver C-reactive protein (CRP)-specific cDNA (Woo, P.,J.R. Korenberg, and A.S. Whitehead. 1985. J.Biol. Chem. 260:13384). Three hybrid-selected cDNA clones, HScDNA1, HScDNA3, and HScDNA8, were isolated and characterized. Nucleotide sequence analysis of the 5' end of the smaller clones, HScDNA1 and HScDNA8, demonstrated that these two PBMC clones are homologous to the 3' and 5' ends, respectively, of pCRP5. Our largest clone, HScDNA3, is larger than pCRP5, extending beyond both the 5' and 3' limits of pCRP5. Therefore, HScDNA3 was coded by human PBMC and not by the hybrid selection vehicle, pCRP5. HScDNA3 lacks the intervening sequence verifying that this clone is DNA made from a PBMC mRNA and not genomic DNA. The complete nucleotide sequence revealed that HScDNA3 is greater than 99% homologous to the CRP gene. These results demonstrate that PBMC express the CRP gene. Based on our previous report, which shows that peripheral blood cells synthesize a peptide recognized by anti-CRP (Kuta, A.E., and L.L. Baum. 1986. J. Exp. Med. 164:321), in conjunction with the data presented here, we conclude that human PBMC can synthesize CRP.
Expression of perforin and serine esterases by human gamma/delta T cells.
Authors: Koizumi H , Liu C C , Zheng L M , Joag S V , Bayne N K , Holoshitz J , Young J D
Published: June 2004

gamma/delta T cells have recently been described in association with a number of disorders, including autoimmune diseases. gamma/delta T cells are thought to play a cytotoxic role, but their mechanism of action is not known. Several granule mediators of cytotoxicity, including a pore-forming protein (perforin), and a family of serine esterases, have been isolated from cytotoxic T lymphocytes (CTL), lymphokine-activated killer (LAK) cells, and natural killer (NK) cells. We demonstrate here that gamma/delta T cells also express these mediators. Northern blots show that gamma/delta T cells express perforin, serine esterase 1 (SE 1), and SE 2. Three polyclonal antisera - raised against murine perforin, a peptide composed of amino acids 1-34 of human perforin, and human peforin expressed in bacteria - all reacted with a 70-kD protein in gamma/delta T cells on Western blots. Immunostaining with antiperforin antisera shows that primary gamma/delta T cells also contain perforin. Electron microscopy reveals that the granules of gamma/delta T cells resemble those of CTL, LAK, and NK cells. Gamma/delta T cells also resemble LAK cells in possessing inclusion bodies in their nuclei. These results imply that gamma/delta T cells resemble other cytolytic lymphocytes in their mechanism of action.
Junctional sequences influence the specificity of gamma/delta T cell receptors.
Authors: Rellahan B L , Bluestone J A , Houlden B A , Cotterman M M , Matis L A
Published: June 2004

T lymphocytes bearing the gamma/delta T cell receptor (TCR-gamma/delta) express a limited number of germline variable gene segments, generating receptor sequence diversity primarily through junctional mechanisms. To examine the role of V(D)J junctional sequences in antigen recognition by TCR-gamma/delta, we derived an alloreactive murine TCR-gamma/delta+ T cell line, LKD1, specific for the I-Ad class II major histocompatibility complex (MHC) molecule, and compared its receptor with that expressed by a previously characterized class II MHC alloreactive T cell line, LBK5, specific for I-Ek,b,s Ia molecules. Both LKD1 and LBK5 express receptors encoded by rearranged V gamma 1.2J gamma 2 and V delta 5D delta 2J delta 1 gene elements, differing in sequence only in the V(D)J junctional regions of the gamma and delta genes. These results demonstrate that junctionally encoded sequences corresponding to the putative third complementarity determining region can influence the antigen specificity of TCR-gamma/delta.
Interleukin 10: a novel stimulatory factor for mast cells and their progenitors.
Authors: Thompson-Snipes L , Dhar V , Bond M W , Mosmann T R , Moore K W , Rennick D M
Published: June 2004

We have characterized the mast cell stimulating activity of murine cytokine synthesis inhibitory factor, referred to as interleukin 10 (IL-10). It was found that IL-10 alone failed to support the growth of mast cell lines and mast cell progenitors. Nevertheless, it dramatically enhanced their growth when combined with IL-3 or IL-4. Moreover, IL-4 plus IL-10 supported the proliferation of mast cells as well as IL-3, suggesting that these two factors may provide a pathway for their development independent of IL-3. However, optimal mast cell growth was stimulated by the combination of IL-10, IL-4, and IL-3. This particular set of cytokines are coordinately produced by activated T cells and may constitute an effective network regulating early and late stages of mast cell development during certain immune responses.
Thy-1 supports adhesion of mouse thymocytes to thymic epithelial cells through a Ca2(+)-independent mechanism.
Authors: He H T , Naquet P , Caillol D , Pierres M
Published: June 2004

The aim of this study was to explore whether Thy-1, like other members of the Ig-like superfamily (e.g., CD2 and neural cell adhesion molecule), participates in cell-cell adhesion. This was investigated by measuring the binding of Thy-1+ probe cells (thymocytes or AKR1 T lymphoma cells) to Thy-1- cloned mouse thymic epithelial (MTE) cells using a quantitative cell adhesion assay. The results were as follows: (a) the thymo-epithelial cell interaction was found to be inhibitable (by 25-40%) by soluble Thy-1 molecules purified from phosphatidylinositol-specific phospholipase C-treated mouse thymocytes as well as by Fab' fragments of a Thy-1-specific mAb; (b) the binding of the Thy-1- AKR1 (Thy-1-d) mutant to MTE cells was found to be reduced (by 50%) as compared with that of the wild type T lymphoma; (c) the Thy-1-mediated adhesion pathway did not require Ca2+ and promoted the initial thymo-epithelial binding measured at 4 degrees C. These data provide the first direct evidence of an adhesive function of Thy-1 and suggest that this molecule, in addition to its T cell triggering properties, might play a role during the early T cell maturation by promoting thymocyte adhesion to thymic stroma.
Autonomous growth and tumorigenicity induced by P40/interleukin 9 cDNA transfection of a mouse P40-dependent T cell line.
Authors: Uyttenhove C , Druez C , Renauld J C , Hérin M , Noël H , Van Snick J
Published: June 2004

To test the transforming potential of deregulated P40/Interleukin 9 expression, we transfected a mouse P40-dependent T cell line with P40 cDNA, and examined the tumorigenicity of the resulting transfectants. When the cells, which grew autonomously in vitro, were injected intraperitoneally or subcutaneously into syngeneic mice, a very high tumor incidence was observed with as few as 10(4) cells per inoculum. Animals died as a result of widespread dissemination of lymphomatous tissue to abdominal and thoracic organs. The same P40-dependent cell line transfected with a control construct did not form tumors even after injection of 10(7) cells. These results indicate that uncontrolled expression of P40 can support T cell proliferation in vivo, and may be a transforming event involved in the development of certain T cell tumors.
Cytokines kill malaria parasites during infection crisis: extracellular complementary factors are essential.
Authors: Naotunne T S , Karunaweera N D , Del Giudice G , Kularatne M U , Grau G E , Carter R , Mendis K N
Published: June 2004

Malaria infection crisis, at which the parasitemia drops precipitously and the parasite loses infectivity to the mosquito vector, occurs in many natural malaria systems, and has not been explained. We demonstrate that in a simian malaria parasite (Plasmodium cynomolgi in its natural host, the toque monkey), the loss of infectivity during crisis is due to the death of circulating intraerythrocytic gametocytes mediated by crisis serum. These parasite-killing effects in crisis serum are due to the presence in the serum of cytokines tumor necrosis factor and interferon gamma, which are produced by the host as a result of the malaria infection. The killing activity of each cytokine is absolutely dependent upon the presence of additional, as yet unidentified factor(s) in the crisis serum.
Requirement of dendritic cells and B cells in the clonal deletion of Mls-reactive T cells in the thymus.
Authors: Mazda O , Watanabe Y , Gyotoku J , Katsura Y
Published: June 2004

The present study was performed to identify cells responsible for the elimination of T cells reactive with minor lymphocyte-stimulating (Mls) antigens during T cell development. Experiments were carried out in a fetal thymus organ culture (FTOC) system. To examine the tolerance-inducing activity, various populations of cells from adult CBA/J (Mls-1a) mice were injected into deoxyguanosine (dGuo)-treated FTOC of C3H/He (Mls-1b) mice with a microinjector, and 2 d later, the thymus lobes were injected with fetal thymus cells from C3H/He mice as T cell precursors. After 14 d of cultivation, cells were harvested and assayed for the expression of the T cell receptor V beta 6 element. The absence or marked reduction of T cells expressing V beta 6 at high levels (V beta 6high) was regarded as indicating the deletion of Mls-1a-reactive T cells. T cell-depleted populations of thymic as well as splenic cells from CBA/J mice were able to induce clonal deletion. Further characterization of the effector cells was carried out by fractionating the spleen cells before injecting them into dGuo-FTOC. None of the dish-adherent population, dish-nonadherent population, or purified B cells alone were able to induce clonal deletion, whereas the addition of purified B cells to adherent cells restored tolerance inducibility. It was further shown that a combination of CBA/J B cells and C3H/He dendritic cells was effective in eliminating Mls-reactive clones. These results indicate that for the deletion of clones reactive with Mls antigens during T cell development in the thymus, both DC and B cells are required.
Constitutive production of inflammatory and mitogenic cytokines by rheumatoid synovial fibroblasts.
Authors: Bucala R , Ritchlin C , Winchester R , Cerami A
Published: June 2004

Conditioned media obtained from fibroblasts cultured from rheumatoid and certain other inflammatory synovia were observed to stimulate [3H]thymidine incorporation in an indicator murine fibroblast line. Synovial fibroblasts derived from the joints of patients with osteoarthritis did not display this property. This effect persisted in culture for many weeks and occurred in the absence of co-stimulatory immune cells. Antibody neutralization studies implicated a role for basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-beta), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin 1 beta (IL-1 beta) in the increased proliferative activity of synovial fibroblast-conditioned media. Synovial cell synthesis of bFGF, TGF beta 1, GM-CSF, IL-1 beta, and IL-6 was confirmed by 35S-methionine labeling and immunoprecipitation. The constitutive production of inflammatory and mitogenic cytokines by synovial fibroblasts may represent the result of long-term, phenotypic changes that occurred in vivo. Persistent cytokine production by synovial fibroblasts may play an important role in the continued recruitment and activation of inflammatory cells in chronic arthritis and in the formation of rheumatoid pannus.
Differential endocytosis of CD4 in lymphocytic and nonlymphocytic cells.
Authors: Pelchen-Matthews A , Armes J E , Griffiths G , Marsh M
Published: June 2004

The endocytosis of the T cell differentiation antigen CD4 has been investigated in CD4-transfected HeLa cells, the promyelocytic HL-60 cell line, and in a number of leukemia- or lymphoma-derived T cell lines. CD4 internalization was followed using radioiodinated antibodies in an acid-elution endocytosis assay, or by covalently modifying cell surface proteins with biotin and analyzing CD4 distributions by immunoprecipitation; both approaches gave equivalent results. The assays demonstrated that in transfected HeLa cells and in HL-60 cells CD4 was constitutively internalized and recycled in the absence of ligand. Immunogold labeling and electron microscopy demonstrated that CD4 enters cells through coated pits. In contrast to the nonlymphocytic cells, T cell lines showed very little endocytosis of CD4. Measurements of fluid phase endocytosis and morphometric analysis of the endosome compartment indicated that the endocytic capacities of HeLa and lymphoid cells are equivalent and suggested that the low level of CD4 uptake in lymphocytic cells is due to exclusion of CD4 from coated pits. This conclusion was supported by experiments using truncated CD4 molecules, lacking the bulk of the cytoplasmic domain, which were internalized equally efficiently in both transfected lymphocytes and HeLa cells. Together, these results indicate that the cytoplasmic domain of CD4 mediates the different interactions with the endocytic apparatus in lymphoid and nonlymphoid cells. We suggest that the CD4-associated lymphocyte-specific protein tyrosine kinase p56lck may be involved in preventing CD4 endocytosis in T cells.
Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis.
Authors: Miyake K , Weissman I L , Greenberger J S , Kincade P W
Published: June 2004

Adhesion molecules are probably required for retention of maturing lymphocyte precursors in bone marrow, where they closely interact with and are dependent on stromal cells. Lymphomyeloid cell lines avidly adhere to cloned stromal cell lines in culture and screening pairs of these resulted in a selection strategy for a new monoclonal antibody to a leukocyte adhesion molecule. Immunoprecipitation analyses and comparison to a previously described antibody showed that it recognizes the alpha 4 chain of the integrin, VLA-4. This antibody totally inhibited lymphopoiesis and retarded myelopoiesis in long-term bone marrow cultures. A similar selection strategy resulted in two additional antibodies which define a single 100-kD species on stromal cells. This stromal cell adhesion molecule is a potential counter-receptor/ligand for VLA-4 on murine lympho-myeloid cells. Our findings suggest a new role for VLA-4 in lymphoid progenitor-microenvironment interactions. Recognition molecules that function in cell migration and inflammation in peripheral tissues may be important for steady-state lymphopoiesis within bone marrow.
T cell determinant structure: cores and determinant envelopes in three mouse major histocompatibility complex haplotypes.
Authors: Gammon G , Geysen H M , Apple R J , Pickett E , Palmer M , Ametani A , Sercarz E E
Published: June 2004

T lymphocytes recognize discrete regions on an antigen. The specificity of the T cell responses in three mouse strains of differing major histocompatibility complex (MHC) haplotype to a protein antigen, lysozyme, was analyzed using a series of peptides that walk the antigen in single amino acid steps. These peptide series were synthesized using the pin synthesis system, which was modified to allow the peptides to be cleaved from the pins into a physiological buffer free of toxic compounds. This methodology overcomes many of the problems associated with the production of peptides for screening proteins for antigenic determinants. The T cell determinants for the three strains were markedly different. This result points out the limitations of algorithms predicting determinants without reference to the MHC, and the importance of the empirical methodology. This analysis of the T cell response to lysozyme constitutes the most complete study of reactivity to a foreign protein to date and illustrates many important features of antigen recognition by T cells, e.g., presence of major and minor determinant regions. The outer boundaries of each immunogenic region, the determinant envelope, are difficult to define from recently immunized lymph nodes because of the heterogeneity in T cell recognition. However, core sequences common to all the immunogenic peptides in a continuous sequence can be easily defined.
Is cyclophilin involved in the immunosuppressive and nephrotoxic mechanism of action of cyclosporin A?
Authors: Sigal N H , Dumont F , Durette P , Siekierka J J , Peterson L , Rich D H , Dunlap B E , Staruch M J , Melino M R , Koprak S L
Published: June 2004

In this report we have approached two questions relating to the mechanism of action of cyclosporin A (CsA). First, we address whether the major cytosolic protein for CsA, cyclophilin, is directly involved in mediating the immunosuppressive activity of this drug, and, in particular, whether inhibition of this protein's peptidyl-prolyl cis-trans isomerase (PPIase) activity results in inhibition of murine T cell activation. Second, we ask whether the nephrotoxicity observed with CsA is related to inhibition of PPIase-dependent pathways in cells other than lymphocytes. Using a series of 61 cyclosporin analogues, we generally found a good correlation between cyclophilin binding and immunosuppressive activity for the majority of analogues analyzed. However, a number of compounds of distinct structural classes were found that could interact with cyclophilin but were much less immunosuppressive than expected. The inability of these analogues to inhibit lymphocyte activation could not be explained by their failure to enter the cell and bind to cyclophilin under the conditions used in the cellular assays. Surprisingly, a nonimmunosuppressive analogue, MeAla-6, which bound well to cyclophilin and was active as a PPIase inhibitor, did not induce renal pathology in vivo. Furthermore, another analogue, MeBm2t, which was immunosuppressive in vitro, possessed little or no activity as a PPIase inhibitor. These findings pose serious questions concerning a direct role of cyclosporin in mediating CsA's immunosuppressive and nephrotoxic activities. In addition, they raise doubts about whether PPIase has a direct function in lymphocyte signal transduction.
An isotype-specific trans-acting factor is defective in a mutant B cell line that expresses HLA-DQ, but not -DR or -DP.
Authors: Ono S J , Bazil V , Sugawara M , Strominger J L
Published: June 2004

The B lymphoblastoid cell line clone 13 (a subclone of the mutant cell line P3JHR-1) has been found to express high levels of HLA-DQ; by contrast, HLA-DR and -DP antigens are not expressed and cannot be induced by interferon gamma. Northern blot analysis using gene-specific probes indicated that the lack of surface expression of the DR and DP antigens is due to a marked decrease in the levels of steady-state RNA for both the alpha and beta chains. Southern blots demonstrated that none of the transcriptionally repressed genes are grossly deleted. Preparations of interspecific transient heterokaryons between clone 13 and the class II antigen-positive murine B cell lymphoma, A20, resulted in reactivation of the DRA gene and surface expression of both the DR and DP molecules. The efficiency of the DRA promoter relative to the DQB promoter is markedly and specifically diminished in clone 13 (P3JHR-1) as compared with the parental cell line, Jijoye, as assayed both by transient expression of appropriate chloramphenicol acetyltransferase gene (CAT) constructs and by in vitro transcription analysis. These data clearly demonstrate the existence of an isotype-specific trans-acting factor, and provide direct evidence that the highly homologous class II genes have distinct regulatory mechanisms.
Interferon gamma and tumor necrosis factor have a role in tumor regressions mediated by murine CD8+ tumor-infiltrating lymphocytes.
Authors: Barth R J , Mulé J J , Spiess P J , Rosenberg S A
Published: June 2004

We have investigated the mechanisms whereby adoptively transferred murine CD8+ lymphocytes mediate tumor regressions. Noncytolytic, CD8+ tumor-infiltrating lymphocytes (TIL) eradicated established lung tumors in irradiated mice. Many cytolytic and noncytolytic CD8+ TIL cultures specifically secreted interferon gamma (IFN-gamma) and tumor necrosis factor when stimulated with tumor cells in vitro. The effectiveness of TIL when adoptively transferred to mice bearing micrometastases correlated better with their ability to specifically secrete lymphokines than with their cytotoxicity in vitro. In 14 of 15 tests, therapeutically effective TIL specifically secreted IFN-gamma in vitro, whereas only 1 of 11 ineffective TIL specifically secreted IFN-gamma. In contrast, only 8 of 15 therapeutically effective TIL were cytolytic. Antibodies to TNF inhibited the effectiveness of two adoptively transferred TIL cultures. In five experiments, antibodies to IFN-gamma abrogated the ability of four different CD8+ TIL cultures to mediate tumor regressions, indicating that secretion of IFN-gamma is an essential part of the mechanism of action of TIL.
Tumor necrosis factor is a critical mediator in hapten induced irritant and contact hypersensitivity reactions.
Authors: Piguet P F , Grau G E , Hauser C , Vassalli P
Published: June 2004

We examined the role of cytokines in the cutaneous response to the application of trinitrochlorobenzene (TNCB) in both nonsensitized and sensitized mice, i.e., in the irritant reaction (IR) and contact hypersensitivity reactions (CH). When administered immediately before challenge, anti-tumor necrosis factor (TNF) antibody abrogated the ear swelling response in CH; antibody directed against interferon gamma or antibodies to both granulocyte/macrophage colony-stimulating factor and interleukin 3 (IL-3) had a partial inhibitory effect; anti-IL-2 receptor antibody had no effect. Anti-TNF prevented the various features of the CH, as seen on histological sections, e.g., leukocyte infiltration and hemorrhages within the dermis and keratinocytes necrosis. Anti-TNF antibody also prevented the IR. The presence of TNF mRNA was evaluated on Northern blots; TNF-alpha mRNA was detectable in an untreated ear, increased after the application of TNCB in nonsensitized mice, and was highest in sensitized mice. TNF mRNA accumulation, which was evident 0.5 h after hapten application and lasted greater than 72 h, was abolished by treatment with anti-TNF antibody, thus suggesting an auto-amplification of TNF production. The cellular origin of TNF mRNA was explored by in situ hybridization; basal keratinocytes showed the highest labeling, but TNF mRNA was also detectable in cells of the dermal infiltrate. After hapten (TNCB) application at sites susceptible (the ear) or resistant (the foot pad) to CH or IR, a close correlation was observed between TNF mRNA accumulation and the intensity of the inflammatory reaction. The major role played by TNF in both the CH and the IR explains the histologically similar aspects of these reactions and the extreme variability of these reactions at various anatomical sites.
T cell-T cell killing is induced by specific epitopes: evidence for an apoptotic mechanism.
Authors: Moss D J , Burrows S R , Baxter G D , Lavin M F
Published: June 2004

Epstein-Barr virus-specific cytotoxic T lymphocyte clones were shown to be an effective target for their own lysis when incubated in the presence of their specific epitopes but not in the presence of irrelevant epitopes. The mode of cell killing appeared to be by apoptosis and was prevented by previously described inhibitors of the process. Degranulation, as measured by serine esterase activity, was involved in this form of T cell-T cell killing. This is the first report of T cell-T cell killing by apoptosis and is only observed in the presence of a specific epitope. This result may be of significance in the use of peptide-based vaccines.
Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription.
Authors: Schlissel M S , Corcoran L M , Baltimore D
Published: June 2004

Virus-transformed pre-B cells undergo ordered immunoglobulin (Ig) gene rearrangements during culture. We devised a series of highly sensitive polymerase chain reaction assays for Ig gene rearrangement and unrearranged Ig gene segment transcription to study both the possible relationship between these processes in cultured pre-B cells and the role played by heavy (H) chain (mu) protein in regulating gene rearrangement. Our analysis of pre-B cell cultures representing various stages of maturity revealed that transcription of each germline Ig locus precedes or is coincident with its rearrangement. Cell lines containing one functional rearranged H chain allele, however, continue to transcribe and to rearrange the allelic, unrearranged H chain locus. These cell lines appear to initiate but not terminate rearrangement events and therefore provide information about the requirements for activating rearrangement but not about allelic exclusion mechanisms.
Characterization of somatically mutated S107 VH11-encoded anti-DNA autoantibodies derived from autoimmune (NZB x NZW)F1 mice.
Authors: Behar S M , Lustgarten D L , Corbet S , Scharff M D
Published: June 2004

We have studied 19 S107 heavy chain variable region gene (VH11)-encoded monoclonal antibodies from NZBWF1 mice. These studies show that a single VH gene can encode both antibodies to foreign antigens (anti-phosphorylcholine) and to self antigens (anti-double-stranded DNA) in the same animal. All of the anti-DNA antibodies contain many somatic mutations compared with the relevant germline genes. Since the anti-DNA antibodies were extensively somatically mutated and had undergone isotype switching, the response seems to be T cell dependent. While some of the antibodies appear to be the products of an antigen-driven and antigen-selected response, a number of characteristics of the antibodies suggest that forces other than antigen are contributing to the stimulation and selection of this response.
Circular DNA resulting from recombination between V-(D)-J joining signals and switch repetitive sequences in mouse thymocytes.
Authors: Davis D D , Yoshida K , Kingsbury L , Sakano H
Published: June 2004

During the course of analyzing circular DNA in mouse thymocytes, novel recombinants were identified with immunoglobulin heavy chain joining gene and switch region probes. These circles represent excision products of recombination between the heptamer-nonamer motif for V-(D)-J joining and a repetitive sequence for class switching. The molecular mechanisms that generate "hybrid circles" are discussed.
Listeria monocytogenes mutants lacking phosphatidylinositol-specific phospholipase C are avirulent.
Authors: Camilli A , Goldfine H , Portnoy D A
Published: June 2004

A number of bacterial species secrete phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we show that the facultative intracellular bacterial pathogen, Listeria monocytogenes, contains a gene, plcA, predicting a polypeptide with 31% amino acid identity to a Bacillus thuringiensis PI-PLC. Accordingly, L. monocytogenes secretes PI-PLC activity, while a mutant with a transposon insertion in plcA lacks detectable PI-PLC activity. In addition, expression of plcA in B. subtilis resulted in secretion of PI-PLC activity. The L. monocytogenes PI-PLC-defective mutant was three logs less virulent for mice and failed to grow in host tissues. The mutant was also defective for in vitro growth in mouse peritoneal macrophages. These results strongly suggest that PI-PLC is an essential determinant of L. monocytogenes pathogenesis. Whether the PI-PLC acts on a bacterial or host substrate remains to be determined.
The CD28 ligand B7/BB1 provides costimulatory signal for alloactivation of CD4+ T cells.
Authors: Koulova L , Clark E A , Shu G , Dupont B
Published: June 2004

Activation via the T lymphocyte cell surface molecule CD28 provides a potent amplification signal for interleukin 2 (IL-2) production in several in vitro systems. The B lymphocyte activation antigen, B7/BB1, is a natural ligand for CD28. Here we investigate the role of CD28 and B7/BB1 in primary activation of CD4+ T lymphocytes stimulated with allogeneic B lymphoblastoid cell lines. A subset of peripheral CD4+ T cells that is unresponsive to crosslinking of CD3/T cell receptor (TCR) with CD3 monoclonal antibody (mAb) does proliferate in response to allogeneic B lymphoblasts. TCR binding to allogeneic major histocompatibility complex antigens was an absolute requirement for activation of these cells because mAbs to either CD3 or human histocompatibility leukocyte antigen (HLA) class II completely inhibited activation. CD28 and B7/BB1 antibodies inhibited T cell proliferation 90% and 84%, respectively. Similar results were obtained with the total CD4+ T lymphocyte population. Crosslinking of HLA-DR antigens on small, resting B cells induced rapid expression of B7/BB1, which peaked at 6 h and returned to baseline levels within 18 h. These data demonstrate that CD28-B7/BB1 binding provides an important early second signal for alloactivation of CD4+ T lymphocyte by B lymphoblasts. The results also suggest that T cells interacting with allogeneic resting B cells may induce B7/BB1 expression in the alloantigen-presenting cell as a consequence of interaction between the TCR and class II molecules.
High levels of CD44 expression distinguish virgin from antigen-primed B cells.
Authors: Camp R L , Kraus T A , Birkeland M L , Puré E
Published: June 2004

The in vitro polyclonal stimulation of B cells through their surface immunoglobulin (Ig) induces substantial increases in CD44 protein levels within 24 hours, whereas other stimuli (e.g., lipopolysaccharide, phorbol 12,13 dibutyrate, and interleukin 4) fail to significantly upregulate CD44. The marked increase in CD44 protein expression on anti-Ig-treated B lymphocytes correlates with an increase in CD44-specific mRNA. Cell sorting experiments with B cells isolated from trinitrophenyl-keyhole limpet hemocyanin-immunized mice demonstrate that both short-term antigen-specific, IgG-secreting cells and long-term antigen-primed B cells are exclusively CD44high. We speculate that the rapid and sustained increase in CD44 expression mediated by surface Ig stimulation may alter the homing properties of antigen-primed B cells.
Phagocytosing neutrophils produce and release high amounts of the neutrophil-activating peptide 1/interleukin 8.
Authors: Bazzoni F , Cassatella M A , Rossi F , Ceska M , Dewald B , Baggiolini M
Published: June 2004

After phagocytosis of yeast opsonized with IgG, neutrophil leukocytes (polymorphonuclear leukocytes [PMN]) expressed high levels of neutrophil-activating peptide 1/interleukin 8 (NAP-1/IL-8) mRNA, which peaked after 3-5 h and were still elevated after 18 h. A similar but quantitatively less prominent effect was obtained with lipopolysaccharide (LPS). After phagocytosis, but not after exposure to LPS, the PMN progressively released considerable amounts of NAP-1/IL-8 into the culture medium (18.6-50 ng/ml in 18 h). The peptide released was biologically active, as indicated by the transient elevation of cytosolic-free calcium in PMN exposed to aliquots of the culture supernatants, and desensitization by prestimulation of the cells with recombinant NAP-1/IL-8. By producing NAP-1/IL-8 at sites where they phagocytose invading microorganisms, PMN could enhance the recruitment of new defense cells.
Messenger RNA expression of the cytokine gene cluster, interleukin 3 (IL-3), IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor, in allergen-induced late-phase cutaneous reactions in atopic subjects.
Authors: Kay A B , Ying S , Varney V , Gaga M , Durham S R , Moqbel R , Wardlaw A J , Hamid Q
Published: June 2004

Cryostat sections from skin biopsies from 24-h allergen-induced late-phase cutaneous reactions (LPR) in 14 human atopic subjects were hybridized with 35S-labeled RNA probes for a number of cytokines. mRNA was detected for interleukin 3 (IL-3) (8/14), IL-4 (10/14), IL-5 (11/14), and granulocyte/macrophage colony-stimulating factor (GM-CSF) (13/14). Only 5 of 14 gave hybridization signals for IL-2, and 0 of 14 for interferon gamma. Biopsies from diluent controls gave only occasional weak signals. These results suggest that cells infiltrating the site of the 24-h LPR transcribe mRNA for the IL-3, IL-4, IL-5, and GM-CSF gene cluster and support the hypothesis that atopy is associated with preferential activation of cells having a similar cytokine profile to the murine T helper type 2 subset.
DNA sequence analysis of NKG2, a family of related cDNA clones encoding type II integral membrane proteins on human natural killer cells.
Authors: Houchins J P , Yabe T , McSherry C , Bach F H
Published: June 2004

We have previously described the isolation of a cDNA clone, designated NKG2, that was expressed in all natural killer (NK) cells tested but not in T or B cells. In the present communication, the original isolate, when used to probe a cDNA library prepared from a CD3- NK cell clone, was found to crosshybridize with a family of transcripts that fell into four distinct groups designated NKG2-A, -B, -C, and -D. Full-length cDNA sequences were determined for each group, and the DNA and inferred peptide sequences were analyzed. All four transcripts encode type II membrane proteins of 215-233 amino acids. NKG2-A and -B peptides appear to be alternative splicing products of a single gene. NKG2-C is highly homologous with group A, having 94% homology in the external (COOH-terminal) domain and 56% homology throughout the internal and transmembrane regions. NKG2-D is distantly but significantly related (21% amino acid homology) to the first three groups. Therefore, NKG2-A, -C, and -D appear to be encoded by distinct genes within a family of NK cell-specific genes. Peptide sequence homology searches demonstrate that the NKG2 peptides are members of a supergene family that includes several other type II membrane proteins. This family is characterized by the presence of a C-type animal lectin domain, and several of its members have demonstrated transmembrane signaling capability.
A recombinant human receptor antagonist to interleukin 1 improves survival after lethal endotoxemia in mice.
Authors: Alexander H R , Doherty G M , Buresh C M , Venzon D J , Norton J A
Published: June 2004

Interleukin 1 (IL-1) is an endogenously produced cytokine that mediates a variety of physiological effects that may be beneficial or deleterious to the host. C57Bl/6 mice treated intravenously with a recently characterized human recombinant receptor antagonist protein to IL-1 (IL-1ra) had improved survival when treated after a lethal Escherichia coli endotoxin (lipopolysaccharide [LPS]) challenge. IL-1ra was effective when treatment was initiated after LPS, and intravenous administration every 4 h for 24 h was required. Serum levels of tumor necrosis factor (TNF) activity after LPS and in vitro TNF cytotoxicity were not altered by treatment with IL-1ra. These experiments provide direct evidence that the lethal effects of LPS may be mediated through the action of IL-1 and that the IL-1ra can provide a new treatment strategy for disease processes mediated via this cytokine.