bluuebunny/crossref_metadata_2025 huggingface dataset:
Early separation of B and T lymphocyte precursors in chick embryo.
Authors: Houssaint E , Mansikka A , Vainio O
Published: June 2004

Embryonic chimeras were used to demonstrate an early separation of chicken T and B cell precursors. Genetically polymorphic cell surface antigens, Bu-1 and Ov, which are expressed on cells of the B and T lineage, respectively, are useful markers in adoptive cell transfer studies. Allelic products Bu-1a and Bu-1b can be detected with monoclonal antibodies (mAbs) L22 and 11G2, respectively, and the Ov antigen with mAb 11A9. Chimeric chickens were constructed by reconstituting irradiated 14-d Ov- H.B19 embryos with the sorted Bu-1+ or Bu-1- fractions of spleen cells from age-matched H.B19 Ov+ embryos. Chimeras were analyzed, 3-4 wk after hatching, for the presence of Ov+ cells in the bursa, thymus, spleen, and peripheral blood lymphocytes. T cell precursors giving rise to thymocytes and peripheral T cells were present only in the Bu-1-, but not in the Bu-1+, fraction. We previously demonstrated that, in contrast, all B cell precursors in spleen from 14-d embryos are exclusively present in the Bu-1+ fraction. We also analyzed the immunoglobulin light chain gene rearrangement in these populations by polymerase chain reaction. We show here that VJ recombination occurs in the Bu-1+, but not in the Bu-1-, fraction of spleen. These data demonstrate an early commitment to the B cell lineage, which occurs before the colonization of the bursa of Fabricius. Segregation of B cell precursors from the other hemopoietic precursors, and consequently separation of T and B cell precursors, occurs before the colonization of the primary lymphoid organs.
T cell contact with Ia antigens on nonhemopoietic cells in vivo can lead to immunity rather than tolerance.
Authors: Gao E K , Kosaka H , Surh C D , Sprent J
Published: June 2004

Long-term H-2-heterozygous a----(a x b)F1 bone marrow (BM) chimeras prepared with supralethal irradiation (1,300 rad) are devoid of Ia+ host BM-derived antigen-presenting cells (APC), but show quite strong host Ia expression in germinal centers, probably on follicular dendritic cells (a class of nonhemopoietic stromal cells). To examine whether Ia expression on these non-BM-derived cells is capable of inducing post-thymic tolerance of T cells, thymectomized irradiated (a x b)F1 mice were reconstituted with parent alpha stem cells and then, 6 mo later, given parent alpha thymus grafts. As measured by primary mixed lymphocyte reactions and V beta expression, the CD4+ cells differentiating in the thymus-grafted mice showed no detectable tolerance to the H-2 (Ia) antigens of the host. To examine whether the thymus-grafted mice contained immunologically significant quantities of host Ia antigens, long-term alpha----(alpha x b)F1 chimeras were injected with normal strain alpha CD4+ cells; the donor cells were recovered from thoracic duct lymph of the chimeras and tested for host reactivity in vitro. The results showed that Ia expression in the chimeras was sufficient to cause selective trapping of a substantial proportion of host-Ia-reactive CD4+ cells soon after transfer and, at later stages, to induce strong priming. Tolerance was not seen. The data place constraints on the view that T cell recognition of antigen expressed on cells other than typical BM-derived APC leads to tolerance induction.
Accumulation of adoptively transferred adherent, lymphokine-activated killer cells in murine metastases.
Authors: Basse P , Herberman R B , Nannmark U , Johansson B R , Hokland M , Wasserman K , Goldfarb R H
Published: June 2004

While close contact between lymphokine-activated killer (LAK)/adherent, lymphokine-activated killer (A-LAK) cells and tumor cells is believed to be a prerequisite for initiating the events leading to tumor cell lysis, clear evidence for the ability of these effector cells to infiltrate tumors or tumor metastases in vivo still has to be obtained. In the present study, we report that a significant fraction of adoptively transferred A-LAK cells, labeled with fluorochromes for identification, accumulates in lung and liver metastases of the B16 melanoma, the MCA 102 sarcoma and the Lewis lung carcinoma lines. Thus, 5- to 10-fold higher numbers of A-LAK cells were found in the malignant lesions compared to the surrounding normal tissue. The infiltration seemed very heterogeneous after intravenous injection of moderate numbers of A-LAK cells (15 x 10(6)). However, after adoptive transfer of 45 million A-LAK cells, an A-LAK cell/tumor cell ratio higher than 1:1 in most metastases was observed. Surprisingly, approximately 5% of the lung metastases seemed totally resistant to infiltration even though neighboring metastases were highly infiltrated. While substantial infiltration of lung metastases was seen after i.v. injection, significant infiltration of liver metastases was seen only after intraportal injection of the A-LAK cells indicating impaired traffic of intravenous injected A-LAK cells through the lung capillaries. These results present direct evidence that A-LAK cells, upon a proper route of administration, have the potential to migrate to and heavily infiltrate metastases from murine tumors of different origin.
Endogenously synthesized peptide with an endoplasmic reticulum signal sequence sensitizes antigen processing mutant cells to class I-restricted cell-mediated lysis.
Authors: Anderson K , Cresswell P , Gammon M , Hermes J , Williamson A , Zweerink H
Published: June 2004

The HLA-A2-positive human mutant cell line T2 is not lysed by influenza virus-specific HLA-A2-restricted cytotoxic lymphocytes after virus infection. However, lysis does occur when cells are incubated with the antigenic influenza matrix protein-derived peptide M57-68. To examine the nature of this defect, T2 cells were transfected with two different plasmids. One plasmid encoded the peptide M57-68, and the other encoded the same peptide preceded by an endoplasmic reticulum translocation signal sequence. Mutant T2 cells expressing the M57-68 peptide without the signal sequence were not susceptible to lysis by M57-68-specific HLA-A2-restricted cytotoxic T lymphocytes, whereas T2 cells expressing the M57-68 peptide plus signal sequence were lysed effectively. Lysis of parental T1 cells with either plasmid was equally effective. These results suggest that the T2 mutant cells are defective in the transport of antigenic peptides from the cytosol into the secretory pathway.
Lupus antiribosomal P antisera contain antibodies to a small fragment of 28S rRNA located in the proposed ribosomal GTPase center.
Authors: Chu J L , Brot N , Weissbach H , Elkon K
Published: June 2004

The ribosomal P proteins are necessary for GTPase activity during protein synthesis. In addition to antibodies to the P proteins, sera from lupus patients contain anti-rRNA activity. To determine whether lupus antiribosomal sera recognize the region of 28S rRNA recently proposed to form part of the ribosomal GTPase center, an rRNA fragment corresponding to nucleotides (nt) 1922-2020 was transcribed in vitro and tested for antigenicity. 18 of 24 (75%) lupus sera containing anti-P antibodies, but only 2 of 24 (8%) lupus sera without anti-P, immunoprecipitated this rRNA fragment (p less than 0.001). The binding was specific, since no significant differences were observed between anti-P positive and negative lupus sera in binding to the RNA fragment transcribed in the antisense orientation or to a control region of rRNA. The majority of sera tested protected a rRNA fragment of approximately 68 nucleotides. To evaluate the fine specificity of the anti-28S antibodies, deletions and site-directed mutations were made in the RNA fragment. The anti-28S antisera required nt 1944-1955 for recognition and were remarkably sensitive to destabilizing as well as nondestabilizing mutations in the stems of the RNA fragments. Detection of antiprotein and anti-RNA antibodies directed against a functionally related domain in the ribosome, together with the remarkable specificity of anti-28S antibodies, strongly suggests a direct role for this region of the ribosome in initiating and/or maintaining antiribosomal autoantibody production.
Characterization of antigen-specific CD4+ effector T cells in vivo: immunization results in a transient population of MEL-14-, CD45RB- helper cells that secretes interleukin 2 (IL-2), IL-3, IL-4, and interferon gamma.
Authors: Bradley L M , Duncan D D , Tonkonogy S , Swain S L
Published: June 2004

In previous studies we demonstrated that, following activation by mitogens or alloantigens, helper T cell precursors proliferate and differentiate in vitro to produce a population of effector cells that secrete high titers of lymphokines upon restimulation. In this report, we demonstrate that a similar effector population develops in vivo following primary antigen stimulation. When restimulated with specific antigen in vitro, CD4+ T cells from mice primed 5 to 7 days previously by subcutaneous administration of keyhole limpet hemocyanin (KLH) in adjuvant, produced high levels of interleukin 2 (IL-2), IL-4, and IL-3, and little or no interferon gamma (IFN-gamma) or IL-5. The effector T cells provided excellent helper activity for in vitro antibody responses of 4-hydroxy-5-iodo-nitrophenyl acetic acid-primed B cells with the production principally of the immunoglobulin G1 (IgG1) and IgM isotypes, small quantities of IgG3, and no detectable IgG2a, or IgG2b. Antigen-specific secretion of IL-2, IL-3, and IL-4 by in vivo effectors was detectable by 12 hours following in vitro restimulation. IFN-gamma and IL-5 were not detected until 48 and 72 hours of culture, respectively, and low levels of these lymphokines were produced. Lymphokine production by primed CD4+ T cells could be induced as early as 3 days following immunization, peaked on day 5, and declined thereafter. The kinetics of in vivo appearance of effector CD4+ T cells that produce lymphokines upon restimulation in vitro were similar for each of the lymphokines examined. Mice depleted of precursor CD4+ T cells by adult thymectomy exhibited limited capacity to generate lymphokine secreting CD4+ T cells in response to primary immunization with KLH, suggesting that the majority of lymphokine producing T cells arise from short-lived and/or precursor cells. Separation of CD4+ T cells from KLH-primed mice on the basis of expression of the lymph node-specific homing receptor, MEL-14, revealed that antigen-specific production of IL-2, IL-3, IL-4, and IFN-gamma was exclusively associated with the MEL-14- subset of CD4+ T cells. Separation on the basis of CD45RB expression, demonstrated that antigen-specific lymphokine production was primarily associated with the minor CD45RB- population, which has been previously associated with memory activity. Our results indicate that primary in vivo immunization leads to the development of a transient population of helper-effectors with a unique phenotype that can produce large quantities of lymphokines and mediate excellent helper activity for B cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Selection of a human T helper type 1-like T cell subset by mycobacteria.
Authors: Haanen J B , de Waal Malefijt R , Res P C , Kraakman E M , Ottenhoff T H , de Vries R R , Spits H
Published: June 2004

Mycobacteria elicit a cellular immune response in their hosts. This response usually leads to protective immunity, but may sometimes be accompanied by immunopathology due to delayed type hypersensitivity (DTH). A striking example in man is tuberculoid leprosy, which is characterized by high cellular immunity to Mycobacterium leprae and immunopathology due to DTH. Skin lesions of patients suffering from this disease have the characteristics of DTH reactions in which macrophages and CD4+ T lymphocytes predominate. In animal models, it has been shown that DTH responses are associated with the presence of a particular subset of CD4+ T cells (T helper type 1 [Th1]) that secrete only certain cytokines, such as interleukin 2 (IL-2), interferon gamma (IFN-gamma), and lymphotoxin, but no IL-4 or IL-5. We studied the cytokine release of activated M. leprae-reactive CD4+ T cell clones derived from tuberculoid leprosy patients. These T cell clones, which were reactive with mycobacterial heat shock proteins, exhibited a Th1-like cytokine secretion pattern with very high levels of IFN-gamma. Half of these clones secreted low levels of IL-4 and IL-5, but the ratio of IFN-gamma to IL-4 and IL-5 was much higher than that of T cell clones reactive with nonmycobacterial antigens. A Th1-like cytokine secretion pattern was also observed for T cell clones and polyclonal T cell lines from control individuals that recognized both heat shock and other mycobacterial antigens. The levels of IFN-gamma secreted by these clones were, however, significantly less than those of patient-derived T cell clones. This Th1-like pattern was not found with T cell clones from the same patients and healthy individuals generated in the same manner, but reactive with nonmycobacterial antigens. Our data thus indicate that mycobacteria selectively induce human T cells with a Th1-like cytokine secretion profile.
Borrelia burgdorferi activates a T helper type 1-like T cell subset in Lyme arthritis.
Authors: Yssel H , Shanafelt M C , Soderberg C , Schneider P V , Anzola J , Peltz G
Published: June 2004

18 cloned T cell lines reactive with Borrelia burgdorferi proteins, all CD3+4+8-TCR-alpha/beta+ and restricted by HLA class II proteins, were isolated from four patients with chronic Lyme arthritis. Analysis of these T cell clones indicated that the T cell response to the Lyme disease spirochete is not oligoclonally restricted; yet all produced the same pattern of lymphokines, resembling that of murine type 1 T helper cells, after antigen-specific or nonspecific stimulation. Therefore, a subset of human CD4+ T cells, with a distinct profile of lymphokine secretion, is selectively activated by the pathogen inciting this chronic inflammatory disease.
H-2Kd-restricted antigenic peptides share a simple binding motif.
Authors: Romero P , Corradin G , Luescher I F , Maryanski J L
Published: June 2004

We have defined structural features that are apparently important for the binding of four different, unrelated antigenic epitopes to the same major histocompatibility complex (MHC) class I molecule, H-2Kd. The four epitopes are recognized in the form of synthetic peptides by cytotoxic T lymphocytes of the appropriate specificity. By analysis of the relative potency of truncated peptides, we demonstrated that for each of the four epitopes, optimal antigenic activity was present in a peptide of 9 or 10 amino acid residues. A comparison of the relative competitor activity of the different-length peptides in a functional competition assay, as well as in a direct binding assay based on photoaffinity labeling of the Kd molecule, indicated that the enhanced potency of the peptides upon reduction in length was most likely due to a higher affinity of the shorter peptides for the Kd molecule. A remarkably simple motif that appears to be important for the specific binding of Kd-restricted peptides was identified by the analysis of peptides containing amino acid substitutions or deletions. The motif consists of two elements, a Tyr in the second position relative to the NH2 terminus and a hydrophobic residue with a large aliphatic side chain (Leu, Ile, or Val) at the COOH-terminal end of the optimal 9- or 10-mer peptides. We demonstrated that a simple peptide analogue (AYP6L) that incorporates the motif can effectively and specifically interact with the Kd molecule. Moreover, all of the additional Kd-restricted epitopes defined thus far in the literature contain the motif, and it may thus be useful for the prediction of new epitopes recognized by T cells in the context of this MHC class I molecule.
Increased frequency of T cell receptor V alpha 12.1 expression on CD8+ T cells: evidence that V alpha participates in shaping the peripheral T cell repertoire.
Authors: DerSimonian H , Band H , Brenner M B
Published: June 2004

The T cell receptor repertoire has a potential for vast diversity. However, this diversity is limited by the fact that the majority of thymocytes die as the repertoire is shaped by positive and negative selection events during development. Such thymic selection affecting TCR V beta gene segment usage has been demonstrated in the mouse. However, similar data has not been forthcoming in man, and little is known about the role of the TCR alpha chain in antigen/major histocompatibility complex (MHC) recognition in any species. Here, we used a monoclonal antibody recognizing the TCR V alpha 12.1 gene product to assess the expression of this gene in the peripheral blood of man. In most individuals tested, the percentage of cells expressing V alpha 12.1 was significantly higher in CD8+ T cells than in CD4+ T cells. That the V alpha gene product itself was responsible for this increased expression in CD8+ T cells was underscored by the lack of substantial skewing of V beta usage in the V alpha 12.1-bearing T cells. Moreover, the skewed expression of V alpha 12.1 was already present at birth, indicating that it was likely to be due to a developmental process rather than the result of exposure to environmental antigens. Based on the established role for CD8 in binding to class I MHC molecules, we suggest that increased expression of V alpha 12.1 on CD8+ T cells points to a role for TCR's using V alpha 12.1 in class I MHC/Ag recognition. These results indicate that V alpha gene usage in the peripheral blood of man is not random, and they support a role for V alpha as a participant in the self-MHC recognition process that shapes the TCR repertoire.
Evidence that natural murine soluble interleukin 4 receptors may act as transport proteins.
Authors: Fernandez-Botran R , Vitetta E S
Published: June 2004

The present studies were undertaken to determine whether the interleukin 4 binding proteins (IL-4BPs) previously identified in the biological fluids of mice are soluble forms of IL-4Rs. We also studied the binding properties of IL-4BPs in order to gain insight into their physiological role in vivo. Affinity-purified IL-4BPs and recombinant soluble IL-4Rs generated similar one-dimensional (Cleveland) peptide maps after digestion with either Staphylococcus aureus V8 protease or trypsin, indicating structural similarities. Furthermore, a rat mAb directed against the murine IL-4Rs immunoprecipitated the IL-4BPs and completely inhibited binding of 125I-IL-4 to a purified preparation of IL-4BPs. Taken together these data indicate that the IL-4BPs are soluble IL-4Rs. At 4 degrees C the IL-4BPs competitively inhibited the binding of IL-4 to membrane IL-4Rs but their ability to prevent binding of IL-4 to cells at 37 degrees C, at the same concentrations, was significantly reduced. Kinetic binding studies of soluble IL-4BPs vs. membrane IL-4Rs disclosed important differences in their rates of dissociation from IL-4. Whereas dissociation at 4 degrees C was slow for both, dissociation of IL-4 from IL-BPs at 37 degrees C was considerably faster (t 1/2 of 2 min) than dissociation of IL-4 from membrane IL-4Rs (t 1/2 of approximately 69 min). Temperature-dependent changes in dissociation kinetics were reversible, and could not be accounted for by either inactivation of the IL-4BPs at 37 degrees C or receptor internalization. Additional experiments also demonstrated that when IL-4BPs bind to IL-4 at 37 degrees C, the IL-4/IL-4BPs complex can rapidly dissociate, allowing IL-4 to bind to membrane IL-4Rs. In addition, binding of IL-4 by the IL-4BPs protects IL-4 from proteolytic degradation. Taken together, these results suggest that the IL-4BPs are naturally occurring forms of soluble IL-4Rs and that some of their properties (fast dissociation kinetics and protection of IL-4 from proteolysis) are consistent with a potential role as carrier proteins for IL-4 in the circulation.
Bacterial entry and intracellular processing of Neisseria gonorrhoeae in epithelial cells: immunomorphological evidence for alterations in the major outer membrane protein P.IB.
Authors: Weel J F , Hopman C T , van Putten J P
Published: June 2004

The fate of the major outer membrane protein of the gonococcus, P.IB, during the adherence, entry, and intracellular processing of the bacteria in infected epithelial cells was investigated using post-embedding immunoelectron microscopy. Various domains of the P.IB molecule were probed at different stages in the infection. These studies revealed that P.IB epitope exposure remained unaltered during the initial attachment of the bacteria to the host cells. In contrast, upon secondary attachment of the bacteria to the eukaryotic cells, apparent zones of adhesion were formed between the gonococci and the host cell membrane, which were characterized by loss of a defined P.IB epitope. These zones of adhesion with the altered P.IB immunoreactivity continued to exist and increased in number during cellular penetration, suggesting that they were essential to bacterial invasion into the eukaryotic cells. After bacterial entry, two classes of gonococci could be recognized; morphologically intact, P.IB-positive bacteria and disintegrated organisms that showed a change in, and, in a later stage, a complete loss of P.IB immunoreactivity. The intracellular alterations in the P.IB antigen could be prevented by treatment of the host cells with the lysosomotropic agent chloroquine. These observations point to a mechanism by which a subpopulation of intracellular gonococci can escape the epithelial cell defense by preventing or resisting exposure to host cell proteolytic activity.
Presentation of viral antigen to class I major histocompatibility complex-restricted cytotoxic T lymphocyte. Recognition of an immunodominant influenza hemagglutinin site by cytotoxic T lymphocyte is independent of the position of the site in the hemagglutinin translation product.
Authors: Hahn Y S , Braciale V L , Braciale T J
Published: June 2004

Class I major histocompatibility complex (MHC) restricted T lymphocytes preferentially recognize fragments of polypeptides processed through a nonendosomal presentation pathway. At present the intracellular compartment(s) in which polypeptide fragmentation occurs and factors which influence the formation of an antigenic epitope are not well understood. To assess the role of residues flanking an antigenic site in the generation of the antigenic moiety recognized by class I MHC restricted T lymphocytes we have moved the coding sequence for an immunodominant H-2Kd restricted site on the influenza A/JAPAN/57 hemagglutinin (residues 202-221) by site-directed mutagenesis to six different positions along the coding sequence of the hemagglutinin gene. We have found that all six classes of mutants are recognized by MHC class I restricted T cells as efficiently as the wild type hemagglutinin gene product. Thus neither N-terminal to C-terminal position within the translation product nor sequences flanking the antigenic site influence processing.
Granulocyte/macrophage colony-stimulating factor and interleukin 3 release from human peripheral blood eosinophils and neutrophils.
Authors: Kita H , Ohnishi T , Okubo Y , Weiler D , Abrams J S , Gleich G J
Published: June 2004

Human peripheral blood eosinophils released eosinophil survival-enhancing activity when stimulated with the calcium ionophore, ionomycin. The release of activity was detected as early as 3 h after stimulation and was inhibited by an immunomodulating agent, cyclosporin A. The survival-enhancing activity was completely abolished by treatment with anti-interleukin 3 (IL-3) and anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) monoclonal antibodies. Moreover, IL-3 and GM-CSF were measurable in ionomycin-stimulated eosinophil supernatants by immunoassay. Eosinophils produced approximately one-half as much IL-3 and one-fifth as much GM-CSF as ionomycin-stimulated mononuclear cells. Neutrophils also produced IL-3 and GM-CSF, but the amounts were less than those produced by eosinophils. These observations suggest a novel role for eosinophils in pathophysiology of allergic inflammation and host defense mechanisms.
Surface expression of a T cell receptor beta (TCR-beta) chain in the absence of TCR-alpha, -delta, and -gamma proteins.
Authors: Punt J A , Kubo R T , Saito T , Finkel T H , Kathiresan S , Blank K J , Hashimoto Y
Published: June 2004

The antigen receptor expressed by mature T cells has been described as a disulfide-linked alpha/beta or gamma/delta heterodimer noncovalently associated with CD3, a complex of transmembrane proteins that communicates signals from the T cell receptor (TCR) to the cell interior. Studies suggest that all component chains must assemble intracellularly before surface expression can be achieved. We described, however, a CD4+/CD8+ transformed murine thymocyte, KKF, that expresses surface TCR-beta chains in the absence of gamma, delta, and alpha proteins; these beta chains are only weakly associated with CD3-epsilon and CD3-zeta. Furthermore, KKF responds differently to stimulation through TCR-beta and CD3-epsilon, a functional dissociation that has been ascribed to a CD4+/CD8+ subpopulation of normal thymocytes. KKF's unique TCR structure may offer an explanation for the functional anomalies observed.
Defective intracellular transport as a common mechanism limiting expression of inappropriately paired class II major histocompatibility complex alpha/beta chains.
Authors: Sant A J , Hendrix L R , Coligan J E , Maloy W L , Germain R N
Published: June 2004

Distinct combinations of class II major histocompatibility complex (MHC) alpha and beta chains show widely varying efficiencies of cell surface expression in transfected cells. Previous studies have analyzed the regions of the class II chains that are critically involved in this phenomenon of variable expression and have shown a predominant effect of the NH2-terminal domains comprising the peptide-binding site. The present experiments attempt to identify the post-translational defects responsible for this variation in surface class II molecule expression for both interisotypic alpha/beta combinations failing to give rise to any detectable cell membrane molecules (e.g., E alpha A beta k) and intraisotypic pairs with inefficient surface expression (e.g., A alpha d A beta k). The results of metabolic labeling and immunoprecipitation experiments using L cell transfectants demonstrate that in both of these cases, the alpha and beta chains form substantial amounts of stable intracellular dimers. However, the isotype- and allele-mismatched combinations do not show the typical post-translational increases in molecular weight that accompany maturation of the N-linked glycans of class II MHC molecules. Studies with endoglycosidase H reveal that no or little progression to endoglycosidase H resistance occurs for these mismatched dimers. These data are consistent with active or passive retention of relatively stable and long-lived mismatched dimers in a pre-medial-Golgi compartment, possibly in the endoplasmic reticulum itself. This retention accounts for the absent or poor surface expression of these alpha/beta combinations, and suggests that conformational effects of the mismatching in the NH2-terminal domain results in a failure of class II molecules to undergo efficient intracellular transport.
Allelic exclusion at DNA rearrangement level is required to prevent coexpression of two distinct T cell receptor beta genes.
Authors: van Meerwijk J P , Romagnoli P , Iglesias A , Bluethmann H , Steinmetz M
Published: June 2004

In mice double transgenic for functionally rearranged T cell receptor (TCR) V beta 2 and V beta 8.2 genes we found that most T lymphocytes express both TCR beta chains simultaneously. These T cells show no abnormality in thymic selection in vivo and their TCRs are capable of transducing activation signals in vitro. These results indicate that multispecific T cells may appear in the periphery if allelic exclusion of TCR beta genes is not established at the level of gene rearrangement.
Leukemia inhibitory factor improves survival of retroviral vector-infected hematopoietic stem cells in vitro, allowing efficient long-term expression of vector-encoded human adenosine deaminase in vivo.
Authors: Fletcher F A , Moore K A , Ashkenazi M , De Vries P , Overbeek P A , Williams D E , Belmont J W
Published: June 2004

Low recovery and poor retroviral vector infection efficiency of hematopoietic stem cells has hindered application of gene therapy for disease affecting blood-forming tissues. Developmental restriction (or death) of stem cells during ex vivo infection has contributed to these difficulties. In these studies we report that the cytokine leukemia inhibitory factor (LIF) directly or indirectly supported the survival of hematopoietic stem cells during culture of bone marrow with vector-producing fibroblasts, resulting in efficient recovery of stem cells able to compete for engraftment in irradiated recipient animals. The infection efficiency of hematopoietic stem cells recovered from these cultures was approximately 80%; and all recipients (20/20) of the LIF-treated marrow were stably engrafted with the progeny of provirus-bearing stem cells. Expression of vector-encoded human adenosine deaminase (hADA) was detected in all recipients at levels averaging 15-50% of endogenous murine ADA in all their hematolymphoid tissues. Survival of stem cells in untreated cultures was approximately 10% of that observed from LIF-treated cultures, resulting in poor engraftment of recipient animals with transplanted cells. The infection efficiency of the few stem cells recovered from untreated cultures, however, was high (approximately 80%), suggesting that LIF did not have an effect on infection efficiency per se, but acted at the level of stem cell survival. Consistent with the poor engraftment observed in the control animals, expression of vector-encoded ADA was only approximately 4-20% of the endogenous levels. These results support the postulated role of LIF as a regulator of hematopoiesis and suggest that cytokine stimulation can positively affect inefficient retroviral vector transduction in hematopoietic stem cells.
Distinct effects of two CD44 isoforms on tumor growth in vivo.
Authors: Sy M S , Guo Y J , Stamenkovic I
Published: June 2004

Tumor growth is dependent in part on interactions between tumor cells and the extracellular matrix of host tissues. Expression of the cell surface glycoprotein CD44/Pgp-1, which mediates cell-substrate interactions is increased in many types of malignancies, but the role of CD44 in tumor growth is largely undefined. Recently, two isoforms of CD44 have been identified: an 80-90 kD form, which has high affinity for cell bound hyaluronate and a 150 kD form which does not mediate attachment to hyaluronate-coated surfaces. In this work, human B cell lymphoma cells stably transfected with cDNA clones encoding either of the two CD44 isoforms were compared for tumorigenicity and metastatic potential in nude mice. Expression of the 80-90 kD form but not the 150 kD form of CD44 greatly enhanced both local tumor formation and metastatic proclivity of the lymphoma cells. Our results suggest that CD44 polypeptides may play an important role in regulating primary and metastatic tumor development in vivo.
Specialized functions of major histocompatibility complex class I molecules. II. Hmt binds N-formylated peptides of mitochondrial and prokaryotic origin.
Authors: Shawar S M , Vyas J M , Rodgers J R , Cook R G , Rich R R
Published: June 2004

The physiological functions of the mouse telomeric major histocompatibility complex (MHC) class I molecules, including Hmt, are unknown. Hmt presents a polymorphic, N-formylated peptide encoded by the mitochondrial gene ND1 forming the cell surface maternally transmitted antigen (Mta). Because the N-formyl moiety is required for Hmt binding, we proposed that Hmt may function generally in presentation of N-formylated antigens. This hypothesis was validated by a competitive binding assay, demonstrating that synthetic N-formyl peptides from other mitochondrial genes also bound Hmt. Bacteria similarly initiate protein synthesis with N-formylmethionine; indeed, we established that Hmt can also present prokaryotic peptides in an N-formyl-dependent manner. These results indicate biochemical specialization of this MHC-peptide interaction and suggest a unique role for Hmt in prokaryotic host defenses.
The lymphocyte glycoprotein CD6 contains a repeated domain structure characteristic of a new family of cell surface and secreted proteins.
Authors: Aruffo A , Melnick M B , Linsley P S , Seed B
Published: June 2004

The isolation, characterization, and expression of a full-length cDNA encoding the human T cell glycoprotein CD6 is described. COS cells transfected with the CD6 clone express a 90-kD protein that reacts with all available anti-CD6 monoclonal antibodies. RNA blot hybridization analysis indicates that CD6 transcripts are predominantly restricted to cells in the T lineage. The predicted CD6 sequence is 468 amino acids long, with the typical features of a type I integral membrane protein. The cytoplasmic domain of CD6 contains two serine residues, one or both of which are substrates for phosphorylation during T cell activation. The extracellular domain of CD6 is significantly related to the extracellular domain of the human and mouse T cell antigen CD5, the cysteine-rich domain of the bovine and mouse type I macrophage scavenger receptor, the extracellular domain of the sea urchin spermatozoa protein that crosslinks the egg peptide speract, the mammalian complement factor 1, and the human lung tumor antigen L3. These molecules, therefore, constitute a new gene superfamily that is well conserved across species boundaries.
An unusual lineage of alpha/beta T cells that contains autoreactive cells.
Authors: von Boehmer H , Kirberg J , Rocha B
Published: June 2004

In male mice that express a transgenic alpha/beta T cell receptor (TCR) specific for a male-specific peptide presented by class I Db major histocompatibility complex (MHC) molecules, we describe an unusual lineage of alpha/beta T cells that are thymus dependent but do not require selection by Db MHC molecules on thymic epithelium in the absence of the specific peptide (positive selection). These cells express the transgenic alpha/beta TCR and have the CD4-8- or CD4-8low phenotype. Cells with the latter phenotype are only detected when hemopoietic cells express both the male-specific peptide as well as Db MHC molecules. In fact, these cells are autoreactive, as they expand relatively slowly after transfer into male nude mice. Also in male but not female alpha/beta TCR transgenic mice, the CD8+ cells with the transgenic TCR bear the Pgp1 marker characteristic of mature T cells activated by antigen. CD4-8- as well as CD4-8low cells do not respond significantly when cultured with male stimulator cells but proliferate vigorously when stimulated by TCR antibodies. By this latter criterion, cells in the periphery of male alpha/beta TCR transgenic mice differ from mature male-specific T cells from female alpha/beta TCR transgenic, which become intrinsically anergic when transferred into male nude mice and cannot be stimulated significantly by TCR antibodies. Thus, intrathymic deletion does not eliminate all autoreactive T cells and it is possible that cells with an apparently "benign" autoreactivity may be involved in certain forms of autoimmunity.
Phenolic glycolipid-1 of Mycobacterium leprae binds complement component C3 in serum and mediates phagocytosis by human monocytes.
Authors: Schlesinger L S , Horwitz M A
Published: June 2004

Previous studies from this laboratory have demonstrated that Mycobacterium leprae, an obligate intracellular bacterial parasite, enters human mononuclear phagocytes via complement receptors on these host cells and bacterium-bound C3. The present study investigates the role of M. leprae surface molecules in C3 fixation and phagocytosis. By enzyme-linked immunosorbent assay, C3 binds selectively to phenolic glycolipid-1 (PGL-1), a major surface molecule of the leprosy bacillus. C3 fixation to PGL-1 is serum concentration dependent and is abolished in heat-inactivated serum or serum containing ethylenediaminetetraacetic acid. C3 fixation is also abolished in serum containing ethyleneglycol-bis (beta-aminoethyl ether)N,N,N'-tetraacetic acid and MgCl2 indicating that isolated PGL-1 fixes C3 via the classical complement pathway. The capacity of PGL-1 to fix C3 is dependent upon its terminal trisaccharide since sequential removal of monosaccharide units of the trisaccharide results in a stepwise reduction in C3 fixation. Deacylation of PGL-1 also abolishes C3 fixation. C3 fixes to the trisaccharide of PGL-1 that is chemically linked to bovine serum albumin via the chemical carrier, 8-methoxycarbonyloctanol. PGL-1 mediates C3 fixation to polystyrene microspheres, and PGL-1 and C3 together mediate ingestion of polystyrene microspheres by human monocytes, wherein these inert test particles reside in membrane-bound phagosomes. Thus, complement receptors on mononuclear phagocytes, complement component C3, and PGL-1 comprise a three-component receptor-ligand-acceptor molecule system for mediating phagocytosis of M. leprae.
Severe combined immunodeficiency (SCID) in man: B cell-negative (B-) SCID patients exhibit an irregular recombination pattern at the JH locus.
Authors: Schwarz K , Hansen-Hagge T E , Knobloch C , Friedrich W , Kleihauer E , Bartram C R
Published: June 2004

Human severe combined immunodeficiency (SCID) patients were analyzed by a polymerase chain reaction assay for their recombination capability at the DHQ52-JH region of the immunoglobulin heavy chain locus. Five patients with B cells (B+ SCID) exhibited a recombination pattern also observed in healthy persons. In contrast, six patients lacking B cells (B- SCID) showed a grossly altered rearrangement pattern characterized by the (partial) absence of regular DHQ52-JH recombinations and the presence of abnormal rearrangements. These events were caused by deletions surpassing the boundaries of immunoglobulin coding elements and thus resemble the pattern of deletional recombinations previously described in SCID mice.
Lysozyme is an inducible marker of macrophage activation in murine tissues as demonstrated by in situ hybridization.
Authors: Keshav S , Chung P , Milon G , Gordon S
Published: June 2004

This study demonstrates the induction of lysozyme mRNA expression in situ in tissue macrophages (M phi) of mice following in vivo stimulation. The resting resident tissue M phi of most tissues do not contain enough lysozyme mRNA to be detected by in situ hybridization using 35S-labeled RNA probes. Following Bacille Calmette Guerin or Plasmodium yoelli infection, however, M phi recruited to liver and spleen hybridize strongly to the lysozyme probe. Within 24 h of infection, cells found in the marginal zone of the spleen begin to produce lysozyme mRNA. This response is also evoked by a noninfectious agent (intravenously injected sheep erythrocytes), and is possibly the result of an early phagocytic interaction. Later in the infection, other cells in the red and white pulp of the spleen, and cells in granulomas in the liver, become lysozyme-positive. Kupffer cells are rarely lysozyme-positive. Lysozyme mRNA levels in liver granulomas remain relatively constant during the infection, and lysozyme is produced by most granuloma cells. This contrasts with tumor necrosis factor alpha (TNF alpha) mRNA, which is produced by fewer cells in the granuloma, and which can be massively induced by lipopolysaccharide administration. The production of lysozyme, previously considered a constitutive function of M phi, is therefore an indicator of M phi activation in vivo, where immunologically specific and nonspecific stimuli both stimulate lysozyme production at high levels in subpopulations of cells occupying discrete anatomical locations.
The Salmonella typhimurium locus mviA regulates virulence in Itys but not Ityr mice: functional mviA results in avirulence; mutant (nonfunctional) mviA results in virulence.
Authors: Benjamin W H , Yother J , Hall P , Briles D E
Published: June 2004

The virulent Salmonella typhimurium strain WB600 carries the mviA allele of the gene mouse virulence A. As shown here, the virulent phenotype of WB600 is the result of a nonfunctional mviA gene. As compared to the functional allele mviA+, mviA increases virulence in Itys mice, but not in Ityr mice. A specific BglII site, mviA4185, between osmZ and galU, located at approximately 35 min on the salmonella chromosome, was within mviA. Insertion of an antibiotic cassette in the mviA4185 site of mviA+ or the homologous mviA4093 site of mviA DNA resulted in virulence when either cassette was recombined into the chromosome. When mviA and mviA+ were both expressed in the same strain with one carried in the chromosome and the other on a plasmid, avirulence was dominant. Replacement of the mviA allele of strain WB600 using P22 transductions of linked antibiotic cassettes cloned into the chromosome of virulent S. typhimurium strains (SR-11, TML, SL1344, C5, ATCC14028, W118-2, and WB600) showed that all but WB600 contained the avirulent mviA+ allele. Southern hybridizations provided no evidence for a second mviA allele anywhere in the genome of the six non-WB600 strains.
Antisense oligonucleotides from the stage-specific myeloid zinc finger gene MZF-1 inhibit granulopoiesis in vitro.
Authors: Bavisotto L , Kaushansky K , Lin N , Hromas R
Published: June 2004

Zinc finger proteins are transcriptional regulators of other genes, often controlling developmental cascades of gene expression. A recently cloned zinc finger gene, MZF-1, was found to be preferentially expressed in myeloid cells. Using complementary radiolabeled MZF-1 RNA hybridized to human bone marrow smears in situ, it was discovered that the expression of MZF-1 is essentially limited to the myelocyte and metamyelocyte stages of granulopoiesis. Antisense but not sense oligonucleotides from MZF-1 significantly inhibited granulocyte colony-stimulating factor-driven granulocyte colony formation in vitro.
T cell receptor V-segment frequencies in peripheral blood T cells correlate with human leukocyte antigen type.
Authors: Gulwani-Akolkar B , Posnett D N , Janson C H , Grunewald J , Wigzell H , Akolkar P , Gregersen P K , Silver J
Published: June 2004

We compared T cell receptor (TCR) V-segment frequencies in human leukocyte antigen (HLA) identical siblings to sibling pairs who differ at one or both HLA haplotypes using four V beta-specific and one V alpha-specific monoclonal antibody. In every one of nine families HLA-identical sibs had the most similar patterns of V-segment frequencies in their peripheral blood, whereas totally mismatched sibs were, in general, the most dissimilar; HLA haploidentical sibs tended to be intermediate between the two groups. The degree of similarity among HLA-identical sibs was comparable to that observed among three pairs of identical twins suggesting that HLA is the major genetic component influencing TCR V-segment frequency. Consistent with this observation, it was found that the frequency of T cells expressing particular V beta segments was skewed towards either CD4+ or CD8+ cells indicating that T cells expressing some V beta genes may be positively selected primarily by class I or class II major histocompatibility complex proteins. Finally, it was observed that individuals who express the HLA class I specificity, B38, tend to express high levels of V alpha 2.3+ cells among their CD8+ T cells. These observations represent definitive proof that human V-segment frequencies are profoundly influenced by the HLA complex.
Adenovirus infection inhibits the phosphorylation of major histocompatibility complex class I proteins.
Authors: Lippé R , Luke E , Kuah Y T , Lomas C , Jefferies W A
Published: June 2004

Major histocompatibility complex (MHC) class I molecules act as peptide receptors to direct the recognition of foreign antigens by cytolytic T cells. The cell surface expression and trafficking of these peptide receptors is thought to be controlled by the conformation of the MHC molecule and possibly by the phosphorylation of the cytoplasmic portion of the heavy chain protein. It is of some interest that adenoviruses (Ads) have evolved proteins that interfere with the expression of MHC molecules. One of these proteins, called E3/19k, binds to newly synthesized MHC molecules in the rough endoplasmic reticulum (RER) and inhibits their trafficking to the cell surface. Here we show that during the infection of a human cell line with Ad2, the phosphorylation of the endogenous MHC molecules is inhibited. We also observe that the phosphorylation of the endogenous HLA molecules is grossly impaired in a human cell line transfected with the Ad2 EcoRI D fragment containing the E3/19k gene. We conclude that the E3/19k protein inhibits the phosphorylation of the MHC heavy chains and that this may be one of the important functions of this protein in infected cells. In addition, we show that a mutant of the E3/19k protein, which lacks an RER retention signal but which retains its ability to bind to HLA molecules, does not inhibit the phosphorylation of HLA molecules and that phosphorylated molecules are not Endo H sensitive. This suggests that HLA molecules are phosphorylated after leaving the medial-Golgi compartment, thus providing the most compelling evidence yet that HLA molecules are phosphorylated at or near the cell surface. Finally, to our knowledge, this is the first study under which the phosphorylation of MHC molecules is shown to be altered and may have some relevance for other pathogenic conditions.
Perforin-expressing granulated metrial gland cells in murine deciduoma.
Authors: Zheng L M , Joag S V , Parr M B , Parr E L , Young J D
Published: June 2004

It has previously been shown that granulated metrial gland (GMG) cells of the pregnant uterus express abundant quantities of the lymphocyte pore-forming protein, perforin. No perforin was present before implantation of the embryo, but large numbers of perforin-producing GMG cells were observed after implantation, which coincides with decidualization of the uterus. The possible source of the activation factors responsible for perforin gene induction in GMG cells was studied here with the pseudopregnancy model, in which cervical stimulation of mice during estrus leads to a series of hormonal changes resembling those seen in pregnancy, but in the absence of an embryo. Subsequent stimulation of the uterus of pseudopregnant mice with oil causes the stimulated portion of the endometrium to differentiate into decidual tissue. Perforin-containing GMG cells were in fact present in the deciduomata, but not in adjacent nondecidualized tissues of the same mice. These results suggest that maternal factors associated with decidual tissue are responsible for the local expression of perforin in GMG cells.
Human autoantibody to RNA polymerase I transcription factor hUBF. Molecular identity of nucleolus organizer region autoantigen NOR-90 and ribosomal RNA transcription upstream binding factor.
Authors: Chan E K , Imai H , Hamel J C , Tan E M
Published: June 2004

In dividing eukaryotic cells, nucleoli disperse before mitosis and reform in daughter cells at sites of ribosomal RNA (rRNA) gene clusters that are at the secondary constrictions of chromosomes, called nucleolus organizer regions (NORs). In this study, cDNA clones for a NOR autoantigen (NOR-90) were selected using a specific human autoantibody probe and were subsequently identified to encode an alternative form of the reported human upstream binding factor (hUBF). Results from immunoprecipitation showed that anti-NOR-90 antibodies recognized both forms of hUBF/NOR-90. Our data therefore showed that UBF, a critical factor in the regulation of rRNA transcription, was tightly bound to NOR during mitosis even when rRNA synthesis was thought to be minimal. Furthermore, we identified a nucleolar transcription factor as a novel target for human autoimmune response.