bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for limited B-lymphopoiesis in adult rabbits.
Authors: Crane M A , Kingzette M , Knight K L
Published: June 2004

Rabbits are born with a limited VDJ gene repertoire formed primarily by rearrangement of one VH gene, VH1. The VDJ genes are undiversified at birth but become diversified by approximately 2 mo of age. To investigate more closely the time during which this diversity occurs, we determined the nucleotide sequences of VDJ genes from peripheral blood leukocytes taken from young rabbits at various time points, and we examined the extent of the diversification of the VDJ genes. At 4 wk of age there were, on average, 3 nucleotide changes per VH region, with approximately 75% of the genes showing some diversification. The number of nucleotide changes per VH region increased to 12 by 6-8 wk of age, and all but 1 of the 35 sequences analyzed were diversified. Because only a limited number of genes can be examined by nucleotide sequence analysis, we used an RNase protection assay to examine a large number of genes and we determined the level of undiversified VH1 mRNA in lymphoid organs of both young and adult rabbits. In young rabbits, we found a high level of undiversified VDJ genes, but the level was greatly reduced by 2 mo of age. By adulthood, essentially all VDJ genes of cells from appendix, peripheral blood, and bone marrow were diversified. Because we had expected B lymphopoiesis to be ongoing in the bone marrow of adult rabbits, we were surprised not to find undiversified VDJ genes from the newly generated B cells. Therefore, we searched for evidence of ongoing B lymphopoiesis in bone marrow by isolating and examining circular DNA for the presence of VD and DJ recombination signal joints. We found highly reduced levels of recombination signal joints in bone marrow of adult rabbits relative to the levels found in bone marrow of newborn rabbits. These data indicate that limited VD and DJ gene rearrangements occur in bone marrow of adult rabbits, and we therefore suggest that B lymphopoiesis is limited in adults.
CD40L-deficient mice show deficits in antiviral immunity and have an impaired memory CD8+ CTL response.
Authors: Borrow P , Tishon A , Lee S , Xu J , Grewal I S , Oldstone M B , Flavell R A
Published: June 2004

The ligand for CD40 (CD40L) is expressed on the surface of activated CD4+ T cells and its role in T-B cell collaborations and thymus-dependent humoral immunity is well established. Recently, by generating CD40L-knockout mice, we have confirmed its previously described role in humoral immunity and defined another important function of this molecule in the in vivo clonal expansion of antigen-specific CD4+ T cells. Here, we investigated the potential in vivo role of CD40L in antiviral immunity by examining the immune response mounted by CD40L-deficient mice following infection with lymphocytic choriomeningitis virus (LCMV), Pichinde virus, or vesicular stomatitis virus. Humoral immune responses of CD40L-deficient mice to these viruses were severely compromised, although moderate titres of antiviral IgM and some IgG2a were produced by virus-infected CD40L-deficient mice by a CD4+ T cell-independent mechanism. By contrast, CD40L-deficient mice made strong primary CTL responses to all three viruses. Interestingly however, although memory CTL activity was detectable in CD40L-deficient mice two months after infection with LCMV, the memory CTL response was much less efficient than in wild-type mice. Together, the results show that CD40-CD40L interactions are required for strong antiviral humoral immune responses, and reveal a novel role for CD40L in the establishment and/or maintenance of CD8+ CTL memory.
Development of insulitis without diabetes in transgenic mice lacking perforin-dependent cytotoxicity.
Authors: Kägi D , Odermatt B , Ohashi P S , Zinkernagel R M , Hengartner H
Published: June 2004

It is widely accepted that T cells play an important role in the destruction of beta cells leading to autoimmune type I diabetes, but the involved effector mechanisms have remained unclear. We addressed this issue by testing the role of perforin-dependent cytotoxicity in a disease model involving transgenic mice expressing glycoprotein of lymphocytic choriomeningitis virus (LCMV-GP) in the beta cells of the endocrine pancreas. In such mice, LCMV infection leads to a potent LCMV-GP-specific T cell response resulting in rapid development of diabetes. We report here that in perforin-deficient LCMV-GP transgenic mice, LCMV infection failed to induce diabetes despite the activation of LCMV-GP-specific T cells. Deletion of nu beta 6+ T cells in Mls-1a perforin-deficient mice and the activation of LCMV-GP-specific T cells in perforin-deficient LCMV-GP transgenic mice, however, indicated that thymic tolerance induction by negative selection was not affected by the disruption of the perforin gene and that there is no fundamental difference between the T cell repertoires of normal control and perforin-deficient mice. In addition, adoptive transfer of LCMV-GP-specific TCR transgenic perforin-deficient T cells activated by LCMV-GP recombinant vaccinia virus led to marked insulitis with infiltration of CD4+ and CD8+ T cells without the development of diabetes. These findings indicate that perforin-dependent cytotoxicity is not required for the initiation of insulitis but is crucial for the destruction of beta cells in the later phase of the disease process. Other mechanisms or soluble factors present in the inflammatory islet infiltrate apparently lack the ability to efficiently induce diabetogenic beta cell damage.
Rat blood neutrophils express very late antigen 4 and it mediates migration to arthritic joint and dermal inflammation.
Authors: Issekutz T B , Miyasaka M , Issekutz A C
Published: June 2004

Blood neutrophils contribute to joint injury in human and experimental models of arthritis. Neutrophil migration out of the blood in joint inflammation involves both the CD18 (beta2) integrins and a CD18 integrin-independent pathway. To investigate this migration, radiolabeled rat blood neutrophils were used to measure neutrophil accumulation in the inflamed joints of rats with adjuvant arthritis and the role of leukocyte integrins in migration to these joints and to dermal inflammation was determined. Neutrophils migrated rapidly (<2 h) to the inflamed joints 14-18 d after immunization with adjuvant. Blocking monoclonal antibodies (mAbs) to both LFA-1 and Mac-1 together, as well as a mAb to CD18, inhibited neutrophil accumulation in the inflamed joints by 50-75%. However, migration to dermal inflammation induced by C5a(des Arg)' tumor necrosis factor alpha, lipopolysaccharide, and poly-inosine:cytosine was inhibited by approximately 90%. Flow cytometry revealed the expression of low levels of very late antigen 4 (VLA-4) on nearly all rat blood neutrophils. Treatment with anti-VLA-4 plus anti-LFA-1 but neither mAb alone, strongly (60-75%) inhibited neutrophil accumulation in arthritic joints. This mAb combination also inhibited neutrophil migration to dermal inflammatory reactions by 30-70%. Blocking VLA-4 together with the CD18 integrins inhibited neutrophil accumulation by 95-99%, virtually abolishing neutrophil accumulation in cutaneous inflammation. A similar blockade of VLA-4 and CD18 decreased neutrophil accumulation in the inflamed joints by 70-83%, but a significant portion of the neutrophil accumulation to these joints still remained. In conclusion, rat blood neutrophils express functional VLA-4 that can mediate neutrophil migration to both inflamed joints and dermal inflammatory sites. VLA-4 appears to be able to substitute for LFA-1 in this migration and is particularly important for accumulation in inflamed joints. However, there exists an additional CD18- and VLA-4-independent pathway of neutrophil migration to arthritic joints that is not involved in acute dermal inflammation.
The human OX40/gp34 system directly mediates adhesion of activated T cells to vascular endothelial cells.
Authors: Imura A , Hori T , Imada K , Ishikawa T , Tanaka Y , Maeda M , Imamura S , Uchiyama T
Published: June 2004

Fresh leukemic cells from patients with adult T cell leukemia (ATL) and some ATL-derived T cell lines show adhesion to human umbilical vein endothelial cells (HUVECs) mainly through E-selectin, but a proportion of this binding remains unaffected by the addition of combinations of antibodies against known adhesion molecules. By immunizing mice with one of such cell lines, we established monoclonal antibodies (mAbs), termed 131 and 315, that recognize a single cell surface antigen (Ag) and inhibit the remaining pathway of the adhesion. These mAbs did not react with normal resting peripheral blood mononuclear cells (PBMC) or most of the cell lines tested except for two other human T cell leukemia virus type I (HTLV-I)-infected T cell lines. After stimulation with phytohemagglutinin (PHA), PBMC expressed Ag 131/315 transiently, indicating that these mAbs define a T cell activation Ag. Western blotting and immunoprecipitation revealed that Ag 131/315 has an apparent molecular mass of 50 kD. Expression cloning was done by transient expression in COS-7 cells and immunological selection to isolate a cDNA clone encoding Ag 131/315. Sequence analysis of the cDNA indicated that it is identical to human OX40, a member of the tumor necrosis factor/nerve growth factor receptor family. We then found that gp34, the ligand of OX40, was expressed on HUVECs and other types of vascular endothelial cells. Furthermore, it was shown that the adhesion of CD4+ cells of PHA-stimulated PBMC to unstimulated HUVECs was considerably inhibited by either 131 or 315. Finally, OX40 transfectants of Kit 225, a human interleukin 2-dependent T cell line, were bound specifically to gp34 transfectants of MMCE, a mouse epithelial cell line, and this binding was blocked by either 315 or 5A8, an anti-gp34 mAb. These results indicate that the OX40/gp34 system directly mediates adhesion of activated T cells or OX40+-transformed T cells to vascular endothelial cells.
An autosomal dominant locus, Nka, mapping to the Ly-49 region of a rat natural killer (NK) gene complex, controls NK cell lysis of allogeneic lymphocytes.
Authors: Dissen E , Ryan J C , Seaman W E , Fossum S
Published: June 2004

Natural Killer (NK) cells can recognize and kill MHC-incompatible normal bone marrow-derived cells. Presently characterized MHC-binding receptors on NK cells, including the Ly-49 family in the mouse, transmit inhibitory signals upon binding to cognate class I MHC ligands. Here we study in vivo NK-mediated lysis of normal allogeneic lymphocytes in crosses between alloreactivity-competent PVG rats and alloreactivity-deficient DA rats. NK cells from both strains are able to lyse standard tumor targets. We identify an autosomal dominant locus, Nka, that controls NK-mediated alloreactivity. Individuals carrying the dominant PVG allele in single dose were fully competent in eliminating allogeneic target cells, suggesting that Nka encodes or regulates a gene product inducing or activating alloreactivity. By linkage analysis and pulsed field gel electrophoresis, a natural killer gene complex (NKC) on rat chromosome 4 is described that contains the rat NKR-P1 and Ly-49 multigene families plus a rat NKG2D homologue. Nka maps within the NKC, together with the most telomeric Ly-49 family members, but separate from NKG2D and the NKR-P1 family. The Nka-encoded response, moreover, correlates with the expression of transcripts for Ly-49 receptors in NK cell populations, as Northern blot analysis demonstrated low expression of Ly-49 genes in DA NK cells, in contrast to high expression in alloreactivity-competent PVG, (DA X PVG)F1, and PVG.1AVI NK cells. The low Ly-49 expression in DA is not induced by MHC haplotype, as demonstrated by high expression of Ly-49 in the DA MHC-congenic PVG.1AVI strain. Finally, we have cloned and characterized the first four members of the rat Ly-49 gene family. Their cytoplasmic domains demonstrate substantial heterogeneity, consistent with the hypothesis that different Ly-49 family members may subserve different signaling functions.
Regulation of cell division cycle progression by bcl-2 expression: a potential mechanism for inhibition of programmed cell death.
Authors: Mazel S , Burtrum D , Petrie H T
Published: June 2004

Expression of the bcl-2 gene has been shown to effectively confer resistance to programmed cell death under a variety of circumstances. However, despite a wealth of literature describing this phenomenon, very little is known about the mechanism of resistance. In the experiments described here, we show that bcl-2 gene expression can result in an inhibition of cell division cycle progression. These findings are based upon the analysis of cell cycle distribution, cell cycle kinetics, and relative phosphorylation of the retinoblastoma tumor suppressor protein, using primary tissues in vivo, ex vivo, and in vitro, as well as continuous cell lines. The effects of bcl-2 expression on cell cycle progression appear to be focused at the G1 to S phase transition, which is a critical control point in the decision between continued cell cycle progression or the induction programmed cell death. In all systems tested, bcl-2 expression resulted in a substantial 30-60% increase in the length of G1 phase; such an increase is very substantial in the context of other regulators of cell cycle progression. Based upon our findings, and the related findings of others, we propose a mechanism by which bcl-2 expression might exert its well known inhibition of programmed cell death by regulating the kinetics of cell cycle progression at a critical control point.
Germinal center formation, immunoglobulin class switching, and autoantibody production driven by "non alpha/beta" T cells.
Authors: Wen L , Pao W , Wong F S , Peng Q , Craft J , Zheng B , Kelsoe G , Dianda L , Owen M J , Hayday A C
Published: June 2004

The production of class-switched antibodies, particularly immunoglobulin (Ig) G1 and IgE, occurs efficiently in T cell receptor (TCR) alpha-/- mice that are congenitally devoid of alpha/beta T cells. This finding runs counter to a wealth of data indicating that IgG1 and IgE synthesis are largely dependent on the collaboration between B and alpha/beta T cells. Furthermore, many of the antibodies synthesized in TCR alpha-/- mice are reactive to a similar spectrum of self-antigens as that targeted by autoantibodies characterizing human systemic lupus erythematosus (SLE). SLE, too, is most commonly regarded as an alpha/beta T cell-mediated condition. To distinguish whether the development of autoantibodies in TCR alpha-/- mice is due to an intrinsic de-regulation of B cells, or to a heretofore poorly characterized collaboration between B and "non-alpha/beta T" cells, the phenotype has been reconstituted by transfer of various populations of B and non-alpha/beta T cells including cloned gamma/delta T cells derived from TCR alpha-/- mice, to severe combined immunodeficient (SCID) mice. The results establish that the reproducible production of IgG1 (including autoantibodies) is a product of non-alpha/beta T cell help that can be provided by gamma/delta T cells. This type of B-T collaboration sustains the production of germinal centers, lymphoid follicles that ordinarily are anatomical signatures of alpha/beta T-B cell collaboration. Thus, non-alpha/beta T cell help may drive Ig synthesis and autoreactivity under various circumstances, especially in cases of alpha/beta T cell immunodeficiency.
Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles.
Authors: Pear W S , Aster J C , Scott M L , Hasserjian R P , Soffer B , Sklar J , Baltimore D
Published: June 2004

Notch is a highly conserved transmembrane protein that is involved in cell fate decisions and is found in organisms ranging from Drosophila to humans. A human homologue of Notch, TAN1, was initially identified at the chromosomal breakpoint of a subset of T-cell lymphoblastic leukemias/lymphomas containing a t(7;9) chromosomal translocation; however, its role in oncogenesis has been unclear. Using a bone marrow reconstitution assay with cells containing retrovirally transduced TAN1 alleles, we analyzed the oncogenic potential of both nuclear and extranuclear forms of truncated TAN1 in hematopoietic cells. Although the Moloney leukemia virus long terminal repeat drives expression in most hematopoietic cell types, retroviruses encoding either form of the TAN1 protein induced clonal leukemias of exclusively immature T cell phenotypes in approximately 50% of transplanted animals. All tumors overexpressed truncated TAN1 of the size and subcellular localization predicted from the structure of the gene. These results show that TAN1 is an oncoprotein and suggest that truncation and overexpression are important determinants of transforming activity. Moreover, the murine tumors caused by TAN1 in the bone marrow transplant model are very similar to the TAN1-associated human tumors and suggest that TAN1 may be specifically oncotropic for T cells.
T cell-mediated eradication of murine metastatic melanoma induced by targeted interleukin 2 therapy.
Authors: Becker J C , Pancook J D , Gillies S D , Furukawa K , Reisfeld R A
Published: June 2004

Induction of a T-cell mediated antitumor response is the ultimate goal for tumor immunotherapy. We demonstrate here that antibody-targeted IL2 therapy is effective against established pulmonary and hepatic melanoma metastases in a syngeneic murine tumor model. The effector mechanisms involved in this tumor eradication are not dependent on NK cells, since the therapeutic effect of antibody-IL2 fusion protein was not altered in NK cell-deficient mice. In contrast, T cells are essential for the observed antitumor effect, since therapy with antibody IL2 fusion proteins is unable to induce tumor eradication in T cell-deficient SCID mice. In vivo depletion studies characterized the essential effector cell population further as CD8 + T cells. Such CD8 + T cells, isolated from tumor bearing mice after antibody-directed IL2 therapy, exerted a MHC class I-restricted cytotoxicity against the same tumor in vitro. These data demonstrate the ability of antibody-targeted IL2 delivery to induce a T cell-dependent host immune response that is capable of eradicating established melanoma metastases in clinically relevant organs.
Hyaluronan fragments activate an NF-kappa B/I-kappa B alpha autoregulatory loop in murine macrophages.
Authors: Noble P W , McKee C M , Cowman M , Shin H S
Published: June 2004

Macrophages play an important role in the acute tissue inflammatory response through the release of cytokines and growth factors in response to stimuli such as lipopolysaccharide (LPS). Macrophage inflammatory effector functions are also influenced by interactions with the extracellular matrix (ECM). Such macrophage-ECM interactions may be important in regulating chronic inflammatory responses. Recent evidence has suggested that hyaluronan (HA), a glycosaminoglycan (GAG) component of ECM can induce inflammatory gene expression in murine macrophages. HA exists in its native form as a large polymer, but is found as smaller fragments under inflammatory conditions. The NF-kappa B/I-kappa B transcriptional regulatory system has been shown to be a critical component of the host inflammatory response. We examined the effects of high molecular weight HA and lower molecular weight HA fragments on NF-kappa B activation in mouse macrophages. Only the smaller HA fragments were found to activate NF-kappa B DNA binding activity. After HA stimulation, I-kappa B alpha mRNA was induced and I-kappa B alpha protein levels, which initially decreased, were restored. The induction of I-kappa Balpha expression was not observed for other GAGs. The time course of I-kappa B alpha protein regeneration in response to HA fragments was consistent with an autoregulatory mechanism. In support of this mechanism, in vitro translated murine I-kappa B alpha inhibited HA fragment-induced NF-kappa B DNA binding activity. The NF-kappa B DNA binding complex in HA-stimulated extracts was found to contain p50 and p65 subunits. Activation of the NF-kappa B/I-kappa B system in macrophages by ECM fragments may be an important mechanism for propagating the tissue inflammatory response.
RANTES and macrophage inflammatory protein 1 alpha selectively enhance immunoglobulin (IgE) and IgG4 production by human B cells.
Authors: Kimata H , Yoshida A , Ishioka C , Fujimoto M , Lindley I , Furusho K
Published: June 2004

We studied the effects of various chemokines including neutrophil-activating peptide 2 (NAP-2), beta-thromboglobulin (beta-TG), platelet factor 4 (PF-4), melanoma growth stimulating activity (GRO), gamma interferon-induced protein (IP-10), regulated on activation, normal T expressed and secreted (RANTES), macrophage inflammatory protein 1 alpha (MIP-1 alpha), MIP-1 beta, and monocyte chemotactic protein 1 (MCP-1) on Immunoglobulin (IgE) and IgG4 production by human B cells. None of these chemokines with or without interleukin (IL-4), anti-CD40 or -CD58 monoclonal antibody (mAb), induced IgE and IgG4 production by B cells from nonatopic donors. However, RANTES and MIP-1 alpha selectively enhanced IgE and IgG4 production induced by IL-4 plus anti-CD40 or -CD58 mAb without affecting production of IgM, IgG1, IgG2, IgG3, IgA1, or IgA2, whereas other chemokines failed to do so. Enhancement of IgE and IgG4 production by RANTES and MIP-1 alpha was specifically blocked by anti-RANTES mAb and anti-MIP-1 alpha antibody (Ab), respectively, whereas anti-IL-5 mAb, anti-IL-6 mAb, anti-IL-10 Ab, anti-IL-13 Ab, and anti-tumor necrosis factor-alpha mAb failed to do so. Purified surface IgE positive (slgE4) and slgG4+ B cells generated either in vitro or in vivo spontaneously produced IgE and IgG4, respectively, whereas sIgE- and sIgG4- B cells failed to do so. RANTES and MIP-1 alpha enhanced spontaneous IgE and IgG4 production in slgE+ and slgG4- B cells, respectively, whereas neither RANTES nor MIP-1 alpha did so in sIgE- or sIgG4- B cells. Purified sIgE4+ and sIgG4+, but not sIgE- or sIgG4- B cells, generated in vitro and in vivo expressed receptors for RANTES and MIP-1 alpha, whereas they failed to express receptors for other chemokines. These findings indicate that RANTES and MIP-1 alpha enhance IgE and IgG4 production by directly stimulating sIgE+ and sIgG4+ B cells.
The immunosuppressant 15-deoxyspergualin [correction of 1,5-deoxyspergualin] reveals commonality between preT and preB cell differentiation.
Authors: Wang B , Benoist C , Mathis D
Published: June 2004

15 [correction of 1,5] deoxyspergualin (DSG) is a potent immunosuppressant whose mechanism of action is still somewhat of a mystery. We have studied the generation of lymphocytes in mice treated with this drug. The differentiation of T cells in the thymus was blocked at an important early control point: the CD4-8- --> CD4+8+ transition, known to depend on the expression of a preTCR complex that includes the variable TCR-beta, but not TCR-alpha, chain. In clear contrast, a later control point, the CD4+8+ --> CD4+8- or CD4-8+ transition, dependent on the display of a conventional alpha:beta TCR complex, appeared unaffected, as did activation of mature T cells both in vitro and in vivo. Interestingly, preB cell differentiation in the bone marrow was blocked at a precisely equivalent point: the A-C --> C' transition, controlled by expression of a pre-receptor complex containing the Ig heavy, but not light, chain. Mature B cells seemed unperturbed. These findings have theoretical implications, suggesting common signaling pathways in early lymphocytes that are distinct from those employed by more mature cells, and are also of practical interest, to be considered in the design of DSG treatment protocols.
Regulated expression of telomerase activity in human T lymphocyte development and activation.
Authors: Weng N P , Levine B L , June C H , Hodes R J
Published: June 2004

Telomerase, a ribonucleoprotein that is capable of synthesizing telomeric repeats, is expressed in germline and malignant cells, and is absent in most normal human somatic cells. The selective expression of telomerase has thus been proposed to be a basis for the immortality of the germline and of malignant cells. In the present study, telomerase activity was analyzed in normal human T lymphocytes. It was found that telomerase is expressed at a high level in thymocyte subpopulations, at an intermediate level in tonsil T lymphocytes, and at a low to undetectable level in peripheral blood T lymphocytes. Moreover, telomerase activity is highly inducible in peripheral T lymphocytes by activation through CD3 with or without CD28 costimulation, or by stimulation with phorbol myristate acetate (PMA)/ionomycin. The induction of telomerase by anti-CD3 plus anti-CD28 (anti-CD3/CD28) stimulation required RNA and protein synthesis, and was blocked by herbimycin A, an inhibitor of S pi protein tyrosine kinases. The immunosuppressive drug cyclosporin A selectively inhibited telomerase induction by PMA/ionomycin and by anti-CD3, but not by anti-CD3/CD28. Although telomerase activity in peripheral T lymphocytes was activation dependent and correlated with cell proliferation, it was not cell cycle phase restricted. These results indicate that the expression of telomerase in normal human T lymphocytes is both developmentally regulated and activation induced. Telomerase may thus play a permissive role in T cell development and in determining the capacity of lymphoid cells for cell division and clonal expansion.
A mutated HLA-A2 molecule recognized by autologous cytotoxic T lymphocytes on a human renal cell carcinoma.
Authors: Brändle D , Brasseur F , Weynants P , Boon T , Van den Eynde B
Published: June 2004

Many human tumor cells have been shown to express antigens that are recognized by autologous cytotoxic T lymphocytes (CTL) and the molecular nature of a number of melanoma antigens has been defined recently. Here we describe the characterization of an antigen recognized on a renal cell carcinoma by autologous CTL clones. This antigen is encoded by the HLA-A2 gene present in the tumor cells. The sequence of this gene differs from the HLA-A2 sequence found in autologous peripheral blood lymphocytes by a point mutation that results in an arginine to isoleucine exchange at residue 170, which is located on the alpha-helix of the alpha 2 domain. Transfection experiments with the normal and mutated HLA-A2 cDNA demonstrated that this amino acid replacement was responsible for the recognition of the HLA-A2 molecule expressed on the tumor cells. The mutant HLA-A2 gene was also detected in the original tumor tissue from the patient, excluding the possibility that the mutation had appeared in vitro. Thus, HLA class I molecules carrying a tumor-specific mutation can be involved in the recognition of tumor cells by autologous CTL.
Autoreactive cytotoxic T lymphocytes in human immunodeficiency virus type 1-infected subjects.
Authors: di Marzo Veronese F , Arnott D , Barnaba V , Loftus D J , Sakaguchi K , Thompson C B , Salemi S , Mastroianni C , Sette A , Shabanowitz J , Hunt D F , Appella E
Published: June 2004

A subtractive analysis of peptides eluted from major histocompatibility complex (MHC) class I human histocompatibility leukocyte antigen (HLA)-A2.1 molecules purified from either human immunodeficiency virus type-1 (HIV-1)-infected or uninfected cells was performed using micro high-performance liquid chromatography and mass spectrometry. Three peptides unique to infected cells were identified and found to derive from a single protein, human vinculin, a structural protein not known to be involved in viral pathogenesis. Molecular and cytofluorometric analyses revealed vinculin mRNA and vinculin protein overexpression in B and T lymphocytes from HIV-1-infected individuals. Vinculin peptide-specific CTL activity was readily elicited from peripheral blood lymphocytes of the majority of HLA-A2.1+, HIV+ patients tested. Our observations suggest that atypical vinculin expression and MHC class I-mediated presentation of vinculin-derived peptides accompany HIV infection of lymphoid cells in vivo, with a resultant induction of antivinculin CTL in a significant portion of HIV+ (HLA-A2.1+) individuals.
Binding and cooperative interactions between two B cell-specific transcriptional coactivators.
Authors: Fontes J D , Jabrane-Ferrat N , Toth C R , Peterlin B M
Published: June 2004

The class II transactivator (CIITA) and B cell octamer-binding protein 1/octamer-binding factor 1/Oct coactivator from B cells (Bob1/OBF-1/OCA-B) represent two B cell-specific transcriptional coactivators. CIITA and Bob1 interact with proteins that bind to conserved upstream sequences in promoters of class II major histocompatibility genes and octamer-binding transcription factors Oct-1 and Oct-2, respectively. Both CIITA and Bob1 increase the expression from the DRA promoter, which is a prototypic class II promoter. Moreover, in the presence of CIITA, interactions between class II promoters and Bob1 are independent of the octamer-binding site. Using in vivo and in vitro binding assays, we confirm that Bob1 binds to CIITA. Thus, CIITA not only activates the expression of class II genes but recruits another B cell-specific coactivator to increase transcriptional activity of class II promoters in B cells.
Antigen compartmentation and T helper cell tolerance induction.
Authors: Oehen S , Feng L , Xia Y , Surh C D , Hedrick S M
Published: June 2004

The process of antigen recognition depends in part on the amount of peptide antigen available and the affinity of the T cell receptor for a particular peptide-major histocompatibility complex (MHC) molecule complex. The availability of self antigen is limited by antigen processing, which is compartmentalized such that peptide antigens presented by MHC class I molecules originate in the cytoplasm, whereas peptide antigens presented by MHC class II molecules are acquired from the endocytic pathway. This segregation of the antigen-processing pathways may limit the diversity of antigens that influence the development and selection of, e.g., CD4-positive, MHC class II-specific T cells. Selection in this case might involve only a subset of self-encoded proteins, specifically those that are plasma membrane bound or secreted. To study these aspects of immune development, we engineered pigeon cytochrome for expression in transgenic mice in two forms: one in which it was expressed as a type II plasma membrane protein, and a second in which it was targeted to the mitochondria after cytoplasmic synthesis. Experiments with these mice clearly show that tolerance is induced in the thymus, irrespective of antigen compartmentation. Using radiation bone marrow chimeras, we further show that cytoplasmic/mitochondrial antigen gains access to the MHC class II pathway by direct presentation. As a result of studying the anatomy of the thymus, we show that the amount of antigen and the affinity of the TCR affect the location and time point of thymocytes under-going apoptosis.
The pulmonary alveolar proteinosis in granulocyte macrophage colony-stimulating factor/interleukins 3/5 beta c receptor-deficient mice is reversed by bone marrow transplantation.
Authors: Nishinakamura R , Wiler R , Dirksen U , Morikawa Y , Arai K , Miyajima A , Burdach S , Murray R
Published: June 2004

Mice mutant for granulocyte macrophage colony-stimulating factor (GM-CSF) or the common receptor component (beta c) for GM-CSF, interleukin (IL)-3, and IL-5 exhibit a lung disorder similar to human pulmonary alveolar proteinosis, a rare disease with congenital, infantile, and adult forms. Bone marrow transplantation and hematopoietic reconstitution of beta c mutant mice with wild-type bone marrow reversed the established disease state in the lungs, defining this disease as hematopoietic in nature. It is likely that the disease involves alveolar macrophages, as donor myeloid cell engraftment into the lungs of mutant recipient mice correlated with reverting both the disease and an abnormal macrophage morphology seen in the lungs of affected animals. Recombination Activating Gene-2 mutant donor bone marrow, which lacks the potential to develop lymphocytes, reversed the pathology in the lungs to the same extent as whole bone marrow. These data establish that certain lung disorders, if of cell-autonomous hematopoietic origin, can be manipulated by bone marrow transplantation.
Activation protein 1-dependent transcriptional activation of interleukin 2 gene by Ca2+/calmodulin kinase type IV/Gr.
Authors: Ho N , Gullberg M , Chatila T
Published: June 2004

The Ca2+/calmodulin-dependent protein kinase (CaMK) type IV/Gr is selectively expressed in T lymphocytes and is activated after signaling via the T cell antigen receptor (TCR), indicating that it mediates some of the Ca(2+)-dependent transcriptional events that follow TCR engagement. Here we show that CaMKIV/Gr induces the transcription factor activation protein 1 (AP-1) alone or in synergy with T cell mitogens and with the p21ras oncoprotein. CaMKIV/ Gr signaling is associated with transcriptional activation of c-fos but is independent of p21ras or calcineurin. AP-1 is an integral component of the nuclear factor of activated T cells (NFAT) transcriptional complex, which is required for interleukin 2 gene expression in T cells. We demonstrate that CaMKIV/Gr reconstitutes the capacity of the cytosolic component of NFAT to direct transcription from NFAT sites in non-T cells. These results reveal a central role for CaMKIV/Gr as a Ca(2+)-regulated activator of gene transcription in T lymphocytes.
Partial signaling by CD8+ T cells in response to antagonist ligands.
Authors: Reis e Sousa C , Levine E H , Germain R N
Published: June 2004

Structural variants of an agonist peptide-major histocompatibility complex (MHC) molecule ligand can show partial agonist and/or antagonist properties. A number of such altered ligands appear to act as pure antagonists. They lack any detectable ability to induce T cell effector function and have been described as unable to induce calcium transients and turnover of inositol phosphates. This has been interpreted as an inability of these ligands to initiate any T cell receptor (TCR)-dependent signal transduction, with their antagonist properties ascribed to competition with offered agonist for TCR occupancy. Yet antagonists for mature CD8+ T cells can induce positive selection of thymocytes, implying active induction of T cell differentiation events, and partial agonists or agonist/antagonist combinations elicit a distinctive pattern of early TCR-associated tyrosine phosphorylation events in CD4+ T cells. We have therefore directly examined proximal TCR signaling in a CD8+ T cell line in response to various related ligands. TCR engagement with natural peptide-MHC class I agonist resulted in the same pattern of early TCR-associated tyrosine phosphorylation events as seen with CD4+ cells, including accumulation of both the p21 and p23 forms of phosphorylated zeta, phosphorylation of CD3 epsilon, and association of phosphorylated ZAP-70 with the TCR. Two antagonists that lacked the ability to induce any detectable CTL effector response (cytolysis, esterase release, gamma interferon secretion, interleukin-2 receptor alpha upregulation) were nevertheless found to also induce TCR-dependent phosphorylation events. In these cases, there was preferential accumulation of the p21 form of phospho-zeta without net phosphorylation of CD3 epsilon, as well as the association of nonphosphorylated ZAP-70 kinase with the receptor. These data show that variant ligands induce similar TCR-dependent phosphorylation events in CD8+ T cells as first observed in CD4+ cells. More importantly, they demonstrate that some putatively pure antagonists are actually a subset of partial agonists able to induce intracellular biochemical changes through the TCR. This delivery of a partial signal by antagonists raises the possibility that antagonism in some cases may result from active interference with stimulation of effector activity by agonist in mature T cells, while the same variant signal could selectively trigger intracellular events that allow positive without negative selection in thymocytes.
A human tumor necrosis factor p75 receptor agonist stimulates in vitro T cell proliferation but does not produce inflammation or shock in the baboon.
Authors: Welborn M B , Van Zee K , Edwards P D , Pruitt J H , Kaibara A , Vauthey J N , Rogy M , Castleman W L , Lowry S F , Kenney J S , Stüber D , Ettlin U , Wipf B , Loetscher H , Copeland E M , Lesslauer W , Moldawer L L
Published: June 2004

Tumor necrosis factor (TNF) is a potentially useful adjunct to anticancer therapies. However, the clinical utility of TNF has been limited by generalized toxicity and hypotension. Recently, studies have begun to dissect the individual proinflammatory and immunologic responses that result from TNF binding to its two cellular receptors, p55 and p75, in an attempt to develop TNF receptor agonists with reduced systemic toxicity. To evaluate a p75 receptor selective TNF mutant (p75TNF), TNF and p75TNF were administered to healthy anesthetized baboons. Intravenous infusion of the p75TNF produced none of the hemodynamic changes seen after the infusion of TNF. Infusion of p75TNF also failed to induce the plasma appearance of interleukins 6 and 8. However, p75TNF enhanced in vitro baboon thymocyte proliferation to concanavalin A, and infusion of p75TNF resulted in increased soluble p55 and p75 receptor plasma concentrations. Local skin necrosis and tissue neutrophil infiltration were seen after subcutaneous injections of TNF and p55TNF. Subcutaneous injection of p75TNF did not result in skin necrosis but did result in a modest dermal infiltration of lymphocytes and macrophages. The findings suggest that p75TNF may stimulate T cell proliferation without the systemic and local toxicity seen with TNF.
Interleukin-10 induces a long-term antigen-specific anergic state in human CD4+ T cells.
Authors: Groux H , Bigler M , de Vries J E , Roncarolo M G
Published: June 2004

Human CD4+ T cells, activated by allogeneic monocytes in a primary mixed lymphocyte reaction in the presence of exogenous interleukin (IL) 10, specifically failed to proliferate after restimulation with the same alloantigens. A comparable state of T cell unresponsiveness could be induced by activation of CD4+ T cells by cross-linked anti-CD3 monoclonal antibodies (mAbs) in the presence of exogenous IL-10. The anergic T cells failed to produce IL-2, IL-5, IL-10, interferon gamma, tumor necrosis factor alpha, and granulocyte/macrophage colony-stimulating factor. The IL-10-induced anergic state was long-lasting. T cell anergy could not be reversed after restimulation of the cells with anti-CD3 and anti-CD28 mAbs, although CD3 and CD28 expression was normal. In addition, restimulation of anergized T cells with anti-CD3 mAbs induced normal Ca2+ fluxes and resulted in increased CD3, CD28, and class II major histocompatibility complex expression, indicating that calcineurin-mediated signaling occurs in these anergic cells. However, the expression of the IL-2 receptor alpha chain was not upregulated, which may account for the failure of exogenous IL-2 to reverse the anergic state. Interestingly, anergic T cells and their nonanergic counterparts showed comparable levels of proliferation and cytokine production after activation with phorbol myristate acetate and Ca2+ ionophore, indicating that a direct activation of a protein kinase C-dependent pathway can overcome the tolerizing effect of IL-10. Taken together, these data demonstrate that IL-10 induces T cell anergy and therefore may play an important role in the induction and maintenance of antigen-specific T cell tolerance.
Platelet-activating factor exerts mitogenic activity and stimulates expression of interleukin 6 and interleukin 8 in human lung fibroblasts via binding to its functional receptor.
Authors: Roth M , Nauck M , Yousefi S , Tamm M , Blaser K , Perruchoud A P , Simon H U
Published: June 2004

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator of the lung. In this study, we demonstrate that PAF receptor mRNA and protein is expressed by human lung fibroblasts. Interaction of PAF with its specific receptor resulted in increases of tyrosine phosphorylation of several intracellular proteins, indicating that the PAF-receptor might be functionally active. PAF-induced transcription of protooncogenes c-fos and c-jun as well as of interleukin (IL)-6 and IL-8 genes in human fibroblasts. Transcription of the interleukins was followed by secretion of the respective proteins. Moreover, PAF enhanced proliferation of fibroblasts in a concentration-dependent manner. Using signaling inhibitors, we demonstrate that PAF-induced transcription of the c-fos, IL-6, and IL-8 genes, as well as proliferation, require activation of pertussis toxin-sensitive G proteins, tyrosine kinases, and protein kinase C (PKC). In contrast, transcription of c-jun was blocked by pertussis toxin, but not by inhibitors for tyrosine kinases or PKC. These data suggest that PAF stimulates distinct signaling pathways in human lung fibroblasts. In addition, the activation of human fibroblasts by PAF leads to enhanced proliferation and to the expression of proinflammatory cytokines, which may contribute to the pathophysiological changes in pulmonary inflammation.
An anti-platelet-endothelial cell adhesion molecule-1 antibody inhibits leukocyte extravasation from mesenteric microvessels in vivo by blocking the passage through the basement membrane.
Authors: Wakelin M W , Sanz M J , Dewar A , Albelda S M , Larkin S W , Boughton-Smith N , Williams T J , Nourshargh S
Published: June 2004

Platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) plays an active role in the process of leukocyte migration through cultured endothelial cells in vitro and anti-PECAM-1 antibodies (Abs) inhibit accumulation of leukocytes into sites of inflammation in vivo. Despite the latter, it is still not clear at which stage of leukocyte emigration in vivo PECAM-1 is involved. To address this point directly, we studied the effect of an anti-PECAM-1 Ab, recognizing rat PECAM-1, on leukocyte responses within rat mesenteric microvessels using intravital microscopy. In mesenteric preparations activated by interleukin (IL)-1 beta, the anti-PECAM-1 Ab had no significant effect on the rolling or adhesion of leukocytes, but inhibited their migration into the surrounding extravascular tissue in a dose-dependent manner. Although in some vessel segments these leukocytes had come to a halt within the vascular lumen, often the leukocytes appeared to be trapped within the vessel wall. Analysis of these sections by electron microscopy revealed that the leukocytes had passed through endothelial cell junctions but not the basement membrane. In contrast to the effect of the Ab in mesenteric preparations treated with IL-1 beta, leukocyte extravasation induced by topical or intraperitoneal administration of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine was not inhibited by the anti-PECAM-1 Ab. These results directly demonstrate a role for PECAM-1 in leukocyte extravasation in vivo and indicate that this involvement is selective for leukocyte extravasation elicited by certain inflammatory mediators. Further, our findings provide the first in vivo indication that PECAM-1 may have an important role in triggering the passage of leukocytes through the perivascular basement membrane.
A role for stem cell factor (SCF): c-kit interaction(s) in the intestinal tract response to Salmonella typhimurium infection.
Authors: Klimpel G R , Langley K E , Wypych J , Abrams J S , Chopra A K , Niesel D W
Published: June 2004

Cholera toxin (CT) has been shown to induce stem cell factor (SCF) production in mouse ligated intestinal loops. Further, SCF interaction(s) with its receptor (c-kit) was shown to be important for the intestinal tract secretory response after CT exposure. In this study, we have investigated whether SCF production is induced in the intestinal tract after exposure to Salmonella typhimurium and whether this production could be an important intestinal tract response to Salmonella infection. Using a mouse ligated intestinal loop model, increased levels of SCF mRNA were detected at 2-4 h post-Salmonella challenge. Intestinal fluid obtained from Salmonella-challenged loops contained high levels of SCF by ELISA. Human and murine intestinal epithelial cell lines were also shown to have increased levels of SCF mRNA after exposure to Salmonella. Inhibition of Salmonella invasion of epithelial cells was shown to be one potentially important role for SCF:c-kit interactions in host defense to Salmonella infection. Pretreatment of human or murine intestinal cell lines with SCF resulted in a cellular state that was resistant to Salmonella invasion. Finally, mice having mutations in the white spotting (W) locus, which encodes the SCF-receptor (c-kit), were significantly more susceptible to oral Salmonella challenge than their control littermates. Taken together, the above results suggest that an important intestinal tract response to Salmonella infection is an enhanced production of SCF and its subsequent interactions with c-kit.
B cell differentiation and isotype switching is related to division cycle number.
Authors: Hodgkin P D , Lee J H , Lyons A B
Published: June 2004

The mature, resting immunoglobulin (Ig) M, IgD+ B lymphocyte can be induced by T cells to proliferate, switch isotype, and differentiate into Ig-secreting or memory cells. Furthermore, B cell activation results in the de novo expression or loss of a number of cell surface molecules that function in cell recirculation or further interaction with T cells. Here, a novel fluorescent technique reveals that T-dependent B cell activation induces cell surface changes that correlate with division cycle number. Furthermore, striking stepwise changes are often centered on a single round of cell division. Particularly marked was the consistent increase in IgG1+ B cells after the second division cycle, from an initial level of < 3% IgG1+ to a plateau of approximately 40% after six cell divisions. The relationship between the percentage of IgG1+ B cells and division number was independent of time after stimulation, indicating a requirement for cell division in isotype switching. IgD expression became negative after four divisions, and a number of changes centered on the sixth division, including the loss of IgM, CD23, and B220. The techniques used here should prove useful for tracking other differentiation pathways and for future analysis of the molecular events associated with stepwise differentiation at the single cell level.
Interleukin-7 receptor alpha is essential for the development of gamma delta + T cells, but not natural killer cells.
Authors: He Y W , Malek T R
Published: June 2004

Mice that lack a functional gamma c subunit of the receptors for interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 display profound defects in lymphoid development. The IL-7/IL-7R system represents a critical interaction for conventional T and B cell development. In this report, the role of IL-7R alpha in the development of lymphoid lineages other than conventional T and B cells was examined. We demonstrate that gamma delta + T cells were absent in IL-7R alpha-deficient mice, whereas the development and function of natural killer cells were normal. Thus, IL-7R alpha function is required for the development of gamma delta + T cells but not natural killer cells.