bluuebunny/crossref_metadata_2025 huggingface dataset:
A truncated T cell receptor repertoire reveals underlying immunogenicity of an antigenic determinant.
Authors: Nanda N K , Sercarz E
Published: June 2004

Induction of T cell responses to an antigenic peptide that is known to bind a major histocompatibility complex molecule is a function of either the T cell receptor (TCR) repertoire or regulatory influences by CD8 or CD4 regulatory T cells. We have tested the hypothesis that a lack of 10 TCR V beta gene segments in V beta a mice may result in an incomplete repertoire of regulatory T cells involved in maintaining peripheral tolerance. Such a hole in the repertoire of regulatory cells could result in expression of T cell responses to antigenic determinants that normally remain undetected in mice with a wild-type repertoire of TCR V beta gene segments. We show here that H-2d mice respond to the peptide 74-96 of hen egg-white lysozyme (HEL) when they are of V beta a haplotype at their TCR locus. The wild-type (V beta b) H-2d mice with their complete set of 20 TCR V beta gene segments fail to respond to HEL 74-96. The 74-96-specific T cell responsiveness was revealed in the wild-type (V beta b) mice when they were treated in vivo with anti-CD8 antibody, implicating the existence of regulatory cells that prevent expression of T cell responses specific for peptide 74-96. This is a demonstration that holes in the regulatory T cell repertoire can, in certain circumstances, become beneficial to the host, for example, in susceptibility against pathogens.
The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand.
Authors: O'Connell J , O'Sullivan G C , Collins J K , Shanahan F
Published: June 2004

Tumors escape immunological rejection by a diversity of mechanisms. In this report, we demonstrate that the colon cancer cell SW620 expresses functional Fas ligand (FasL), the triggering agent of Fas receptor (FasR)-mediated apoptosis within the immune system. FasL mRNA and cell surface FasL were detected in SW620 cells using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining, respectively. We show that SW620 kills Jurkat T cells in a Fas-mediated manner. FasR-specific antisense oligonucleotide treatment, which transiently inhibited FasR expression, completely protected Jurkat cells from killing by SW620. FasL-specific antisense oligonucleotide treatment of SW620 inhibited its Jurkat-killing activity. FasL has recently been established as a mediator of immune privilege in mouse retina and testis. Our finding that colon cancer cells express functional FasL suggests it may play an analogous role in bestowing immune privilege on human tumors. HT29 and SW620 colon cancer cells were found to express FasR mRNA and cell surface FasR using RT-PCR and immunofluorescence flow cytometry, respectively. However, neither of these cells underwent apoptosis after treatment by the anti-FasR agonistic monoclonal antibody CH11. Our results therefore suggest a Fas counterattack model for immune escape in colon cancer, whereby the cancer cells resist Fas-mediated T cell cytotoxicity but express functional FasL, an apoptotic death signal to which activated T cells are inherently sensitive.
T helper cells in murine germinal centers are antigen-specific emigrants that downregulate Thy-1.
Authors: Zheng B , Han S , Kelsoe G
Published: June 2004

After immunization, activated splenic T cells proliferate in periarteriolar lymphoid sheaths (PALS) and subsequently migrate to the lymphoid follicle where they enter nascent germinal centers. Analysis of TCR V(D)J gene rearrangements indicates extensive emigration, frequently involving more than a single white pulp region. These migrants constitute a unique set of T helper cells that express antigen-specific alpha beta TCR, CD3, and CD4, but little or no Thy-1, a differentiation antigen present on the great majority of peripheral murine T lymphocytes. The origin of CD4+ Thy-1 follicular T cells appears to be the Thy+ population in the PALS, as both sets commonly share identical V(D)J rearrangements.
A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1)
Authors: Bleul C C , Fuhlbrigge R C , Casasnovas J M , Aiuti A , Springer T A
Published: June 2004

Chemotactic factors are postulated to direct emigration of lymphocytes from the blood stream into sites of inflammation. Members of a family of chemotactic cytokines, termed chemokines, have been shown to attract lymphocytes but efficacy, i.e., the maximal percentage of attracted cells, has been low. We have identified a highly efficacious lymphocyte chemotactic activity in the supernatants of the murine bone marrow stroma cell line MS-5 which attracts 10-fold more lymphocytes in vitro than currently described lymphocyte chemoattractants. Purification of this chemotactic activity revealed identity to stromal cell-derived factor 1 (SDF-1). SDF-1 acts on lymphocytes and monocytes but not neutrophils in vitro and is both a highly efficacious and highly potent mononuclear cell attractant in vivo. In addition, SDF-1 induces intracellular actin polymerization in lymphocytes, a process that is thought to be a prerequisite for cell motility. Since SDF-1 is expressed constitutively in a broad range of tissues it may have a role in immune surveillance and in basal extravasation of lymphocytes and monocytes rather than in inflammation.
p95vav associates with tyrosine-phosphorylated SLP-76 in antigen-stimulated T cells.
Authors: Tuosto L , Michel F , Acuto O
Published: June 2004

p95vav, the product of the vav protooncogene, has been implicated in the T cell receptor (TCR)-mediated signaling cascade p95vav is phosphorylated on tyrosine residues after TCR stimulation by anti-TCR/CD3 antibodies and possesses a number of landmark features of signaling molecules such as a putative guanine nucleotide exchange factor domain, a pleckstrin homology domain, and an Sre homology (SH) 2 and two SH3 domains, which provide the capacity to form multimeric signaling complexes. However, the precise role of p95vav in TCR signaling remains unclear. In this work we show that physiological stimulation of T cell hybridomas with antigen presented by major histocompatibility complex class II molecules leads to a strong tyrosine phosphorylation of p95vav and its association with tyrosine-phosphorylated SLP-76. SLP-76 is a newly described SH2-containing protein that has been previously found to bind to the adapter molecule Grb2. Moreover, we provide evidence that p95vav-SI P-76 association is SH2-mediated by demonstrating that this interaction can be inhibited by a phosphopeptide containing a putative p95vav-SH2-binding motif (pYESP) present in SLP-76. Furthermore, in vitro experiments show that after antigen stimulation, phosphorylated p95vav-SLP-76 can bind to Grb2 in a complex that contains pp36/38 and pp116 proteins. Our data provide a clue to explain recent independent observations that overexpression of p95vav or SLP-76 enhances TCR-mediated gene activation.
The seven-span transmembrane receptor CD97 has a cellular ligand (CD55, DAF).
Authors: Hamann J , Vogel B , van Schijndel G M , van Lier R A
Published: June 2004

CD97 is an activation-induced antigen on leukocytes with a seven-span transmembrane (7-TM) region homologous to the secretin receptor superfamily. However, in contrast to this group of peptide hormone receptors, CD97 has an extended extracellular region with three EGF domains at the NH2 terminus, two of them with a calcium binding site. By demonstrating that lymphocytes and erythrocytes specifically adhere to CD97-transfected COS cells we here show that CD97 in parallel with its molecular evolution has acquired the ability to bind cellular ligands. A mAb selected on its capacity to block the adhesion between CD97 transfectants and red cells was found to be directed to the NH2-terminal short consensus repeat (SCR) of decay accelerating factor (DAF, CD55), a regulatory protein of the complement cascade. The specificity of the interaction of CD97 with CD55 was established by the observation that erythrocytes that lack CD55, obtained from patients with paroxysmal nocturnal hemoglobinuria (PNH) or the CD55, phenotype Inab, failed to adhere to CD97 transfectants. This is the first demonstration of a cellular ligand for a 7-TM receptor.
An Epstein-Barr virus-associated superantigen.
Authors: Sutkowski N , Palkama T , Ciurli C , Sekaly R P , Thorley-Lawson D A , Huber B T
Published: June 2004

More than 90% of adults are latently infected with Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, a self-limiting lymphoproliferative disease characterized by extensive T cell activation. Reactivation of this herpesvirus during immunosuppression is often associated with oncogenesis. These considerations led us to analyze the early events that occur after exposure of the immune system to EBV. Strong major histocompatibility complex (MHC) class II-dependent but not MHC-restricted, T cell proliferation was observed in vitro in response to autologous, lytically infected EBV-transformed B cells. By measuring the appearance of the early activation marker CD69 on individual T cell V beta subsets, we could demonstrate selective activation of human V beta 13- T cells. This was confirmed with murine T cell hybridomas expressing various human BV genes. While EBV- Burkitt's lymphoma cells were nonstimulatory, they induced V beta-restricted T cell activation after EBV infection. EBV specific activation was also demonstrated in cord blood cells, excluding a recall-antigen response. Thus, all of the characteristics of a superantigen-stimulated response are seen, indicating that induction of the EBV lytic cycle is associated with the expression of a superantigen in B cells. A model is presented proposing a role for the superantigen in infection, latency, and oncogenesis.
Human leukocyte elastase is an endogenous ligand for the integrin CR3 (CD11b/CD18, Mac-1, alpha M beta 2) and modulates polymorphonuclear leukocyte adhesion.
Authors: Cai T Q , Wright S D
Published: June 2004

Integrin CR3 (CD11b/CD18, Mac-1, alpha M beta 2) mediates the transient adhesion of polymorphonuclear leukocytes (PMN) to surfaces coated with fibrinogen, C3bi, ICAM-1, and other ligands. Recent studies (Cai, T.-Q., and S.D. Wright 1995. J. Biol. Chem. 270:14358) suggest that adhesion may be favored by stimulus-dependent changes in the kinetics of ligand binding by CR3. Cell detachment, on the other hand, must occur by a different mechanism because binding kinetics cannot affect cell adhesion after binding of ligand has occurred. We have sought a mechanism that would reverse binding of ligand to CR3 and report here that lysates of PMN contain an endogenous ligand that binds CR3 and competes the binding of C3bi. Purification and sequence analysis identified the structurally homologous azurophilic granule proteins, elastase, protease 3, and azurocidin as candidates. Studies with purified elastase and azurocidin showed that each bound specifically to purified, immobilized CR3. Elastase may play a role in modulating integrin-mediated cell adhesion because it is expressed at the cell surface, and the expression level is inversely proportional to cell adhesivity. Furthermore, a monoclonal antibody against elastase prevented detachment of PMN from fibrinogen-coated surfaces and blocked chemotaxis, confirming a role for this protein in regulating integrin-mediated adhesion. These studies suggest a model for release of integrin-mediated cell adhesion in which endogenous ligands such as elastase may release adhesion by "'eluting" substrate-bound ligand. A role for the proteolytic activity of elastase appears likely but is not demonstrated in this study.
Disturbed interaction of p21-rac with mutated p67-phox causes chronic granulomatous disease.
Authors: Leusen J H , de Klein A , Hilarius P M , Ahlin A , Palmblad J , Smith C I , Diekmann D , Hall A , Verhoeven A J , Roos D
Published: June 2004

Chronic granulomatous disease (CGD) is characterized by the failure of phagocytic leukocytes to generate superoxide, needed for the intracellular killing of microorganisms. This is caused by mutations in any one of the four subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In a rare, autosomal recessive form of CGD, a 67-kD cytosolic component of this enzyme (p67-phox) is missing. We here report on a patient with a mutation in the p67-phox gene that leads to expression of a nonfunctional p67-phox protein. The purified granulocytes of this patient failed to produce superoxide and contained about half of the normal amount of p67-phox. Analysis of the cDNA and genomic DNA of this patient showed that the patient is a compound heterozygote for a triplet nucleotide deletion in the p67-phox gene, predicting an in-frame deletion of lysine 58 in the p67-phox protein and a larger deletion of 11-13 kb in the other allele. Interestingly, the 58Lys deletion in p67-phox disrupts the interaction with p21-rac1, a ras-related protein involved in the activation of the NADPH oxidase. In contrast to normal neutrophils, in which p47-phox and p67-phox translocate to the plasma membrane upon cell activation, the cells of the patient did not show this translocation, indicating that an interaction between p67-phox and p21-rac1 is essential for translocation of these cytosolic proteins and activation of the NADPH oxidase. Moreover, this CGD patient represents the first case of disease caused by a disturbed binding of a ras-related protein to its target protein.
Colony growth of human hematopoietic progenitor cells in the absence of serum is supported by a proteinase inhibitor identified as antileukoproteinase.
Authors: Goselink H M , van Damme J , Hiemstra P S , Wuyts A , Stolk J , Fibbe W E , Willemze R , Falkenburg J H
Published: June 2004

Serum contains many growth factors and nutrients that stimulate colony formation of hematopoietic progenitor cells (HPC) in semisolid cultures. In the absence of serum, no proliferation of HPCs could be obtained in semisolid medium cultures of partially purified bone marrow cells in the presence of multiple hematopoietic growth factors, insulin, cholesterol, and purified clinical-grade human albumin. This appeared to be due to a suppressive activity induced by monocyte- and T lymphocyte-depleted accessory cells on CD34+ HPCs. Serum-free conditioned medium from the bladder carcinoma cellline 5637 could replace serum to support the growth of HPCs in these cultures. After gel filtration and reverse-phase high-performance liquid chromatography of 5637 supernatants, this activity could be attributed to a 15-kD protein that was further identified by NH2-terminal sequence analysis as the serine proteinase inhibitor antileukoproteinase (ALP). The growth-supportive activity from the 5637 conditioned medium and the (partially) purified fractions could be completely neutralized by a polyclonal rabbit IgG antibody against human ALP (huALP). Similar supportive effects on the growth of HPC could be obtained in the presence of recombinant huALP. We demonstrated that the COOH-terminal domain of ALP containing the proteinase inhibitory activity was responsible for this effect. alpha-1 proteinase inhibitor was capable of similar support of in vitro HPC growth. These results illustrate that proteinase inhibitors play an important role in the in vitro growth of hematopoietic cells by the neutralization of proteinases produced by bone marrow accessory cells. This may be of particular relevance for in vitro expansion of human hematopoietic stem cells in serum-free media.
Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF alpha in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response.
Authors: Pasparakis M , Alexopoulou L , Episkopou V , Kollias G
Published: June 2004

To investigate the role of TNF alpha in the development of in vivo immune response we have generated TNF alpha-deficient mice by gene targeting. Homozygous mutant mice are viable and fertile, develop lymph nodes and Peyer's patches and show no apparent phenotypic abnormalities, indicating that TNF alpha is not required for normal mouse development. In the absence of TNF alpha mice readily succumb to L. monocytogenes infections and show reduced contact hypersensitivity responses. Furthermore, TNF alpha knockout mice are resistant to the systemic toxicity of LPS upon D-galactosamine sensitization, yet they remain sensitive to high doses of LPS alone. Most interestingly, TNF alpha knockout mice completely lack splenic primary B cell follicles and cannot form organized follicular dendritic cell (FDC) networks and germinal centers. However, despite the absence of B cell follicles, Ig class-switching can still occur, yet deregulated humoral immune responses against either thymus-dependent (TD) or thymus-independent (TI) antigens are observed. Complementation of TNF alpha functioning by the expression of either human or murine TNF alpha transgenes is sufficient to reconstitute these defects, establishing a physiological role for TNF alpha in regulating the development and organization of splenic follicular architecture and in the maturation of the humoral immune response.
Interferon gamma regulates platelet endothelial cell adhesion molecule 1 expression and neutrophil infiltration into herpes simplex virus-infected mouse corneas.
Authors: Tang Q , Hendricks R L
Published: June 2004

In a mouse model of herpes simplex virus (HSV) 1 corneal infection, tissue destruction results from a CD4+ T cell-mediated chronic inflammation, in which interleukin 2 and interferon (IFN) gamma are requisite inflammatory mediators and polymorphonuclear leukocytes (PMN) are the predominant infiltrating cells. In vivo neutralization of IFN-gamma relieved inflammation at least in part through a specific block of PMN extravasation into HSV-1-infected corneas. Intercellular adhesion molecule (ICAM) 1 and platelet endothelial cell adhesion molecule (PECAM) 1 were upregulated on the vascular endothelium of inflamed corneas. Reduced PMN extravasation in anti-IFN-gamma-treated mice was associated with a dramatic reduction of PECAM-1 but not ICAM-1 expression on vascular endothelium. PMN accumulated in the lumen of corneal vessels after in vivo IFN-gamma neutralization. PECAM-1 was readily detectable on PMN inside the vessels but was not detectable on PMN that extravasated into the infected cornea. Moreover, flow cytometric analysis revealed reduced PECAM-1 expression but elevated major histocompatibility complex class I expression on PMN that recently extravasated into the peritoneal cavity when compared with PMN in the peripheral blood. We conclude that IFN-gamma contributes to HSV-1-induced corneal inflammation by facilitating PMN infiltration; this appears to be accomplished through upregulation of PECAM-1 expression on the vascular endothelium; and PMN downregulate PECAM-1 expression during the process of extravasation.
The importance of leukotrienes in airway inflammation in a mouse model of asthma.
Authors: Henderson W R , Lewis D B , Albert R K , Zhang Y , Lamm W J , Chiang G K , Jones F , Eriksen P , Tien Y T , Jonas M , Chi E Y
Published: June 2004

Inhalation of antigen in immunized mice induces an infiltration of eosinophils into the airways and increased bronchial hyperreactivity as are observed in human asthma. We employed a model of late-phase allergic pulmonary inflammation in mice to address the role of leukotrienes (LT) in mediating airway eosinophilia and hyperreactivity to methacholine. Allergen intranasal challenge in OVA-sensitized mice induced LTB4 and LTC4 release into the airspace, widespread mucus occlusion of the airways, leukocytic infiltration of the airway tissue and broncho-alveolar lavage fluid that was predominantly eosinophils, and bronchial hyperreactivity to methacholine. Specific inhibitors of 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) blocked airway mucus release and infiltration by eosinophils indicating a key role for leukotrienes in these features of allergic pulmonary inflammation. The role of leukotrienes or eosinophils in mediating airway hyperresponsiveness to aeroallergen could not be established, however, in this murine model.
The domain on the Duffy blood group antigen for binding Plasmodium vivax and P. knowlesi malarial parasites to erythrocytes.
Authors: Chitnis C E , Chaudhuri A , Horuk R , Pogo A O , Miller L H
Published: June 2004

Plasmodium vivax and the related simian malarial parasite P. knowlesi use the Duffy blood group antigen as a receptor to invade human erythrocytes and region II of the parasite ligands for binding to this erythrocyte receptor. Here, we identify the peptide within the Duffy blood group antigen of human and rhesus erythrocytes to which the P. vivax and P. knowlesi ligands bind. Peptides from the NH2-terminal extracellular region of the Duffy antigen were tested for their ability to block the binding of erythrocytes to transfected Cos cells expressing on their surface region II of the Duffy-binding ligands. The binding site on the human Duffy antigen used by both the P. vivax and P. knowlesi ligands maps to a 35-amino acid region. A 34-amino acid peptide from the equivalent region of the rhesus Duffy antigen blocked the binding of P. vivax to human erythrocytes, although the P. vivax ligand expressed on Cos cells does not bind rhesus erythrocytes. The binding of the rhesus peptide, but not the rhesus erythrocyte, to the P. vivax ligand was explained by interference of carbohydrate with the binding process. Rhesus erythrocytes, treated with N-glycanase, bound specifically to P. vivax region II. Thus, the interaction of P. vivax ligand with human and rhesus erythrocytes appears to be mediated by a peptide-peptide interaction. Glycosylation of the rhesus Duffy antigen appears to block binding of the P. vivax ligand to rhesus erythrocytes.
Inactivation of the IL-6 gene prevents development of multicentric Castleman's disease in C/EBP beta-deficient mice.
Authors: Screpanti I , Musiani P , Bellavia D , Cappelletti M , Aiello F B , Maroder M , Frati L , Modesti A , Gulino A , Poli V
Published: June 2004

Castleman's disease is a lymphoproliferative disorder thought to be related to deregulated production of IL-6. We have previously shown that mice lacking the trans-acting factor C/EBP beta, a transcriptional regulator of IL-6 and a mediator of IL-6 intracellular signaling, develop a pathology nearly identical to multicentric Castleman's disease, together with increasingly high levels of circulating IL-6. We describe here how the simultaneous inactivation of both IL-6 and C/EBP beta genes prevents the development of pathological traits of Castleman's disease observed in C/EBP beta-deficient mice. Histological and phenotypic analysis of lymph nodes and spleen of double mutant mice did not show either the lymphoadenopathy and splenomegaly or the abnormal expansion of myeloid, B and plasma cell compartments observed in C/EBP beta-/- mice, while B cell development, although delayed, was normal. Our data demonstrate that IL-6 is essential for the development of multicentric Castleman's disease in C/EBP beta-/- mice.
Expansion of natural (NK1+) T cells that express alpha beta T cell receptors in transporters associated with antigen presentation-1 null and thymus leukemia antigen positive mice.
Authors: Joyce S , Negishi I , Boesteanu A , DeSilva A D , Sharma P , Chorney M J , Loh D Y , Van Kaer L
Published: June 2004

Thymic selection of natural killer-1+ natural T cells that express alpha beta T cell receptors requires a conserved beta 2-microglobulin-associated molecule, presumably CD1d, displayed by CD4+8+ thymocytes. Here we demonstrate that positive selection of natural T cells occurs independent of transporters associated with antigen presentation-1 (TAP-1) function. Moreover, natural T cells in TAP-1o/o mice are numerically expanded. Several H-2 class Ib molecules function in a TAP-independent manner, suggesting that if expressed in TAP-1o/o thymocytes, they could play a role in natural T cell development. Of these class Ib molecules, H-2TL is expressed by TAP-1o/o thymocytes. Moreover, we find that thymi of TL+ mice congenic or transgenic for H-2T18 also have a numerically expanded natural T cell repertoire compared with TL- mice. This expansion, as in TAP-1o/o thymi, is evident in each of the limited T cell receptor V beta chains expressed by natural T cells, suggesting that TL and CD1d impact similar repertoires. Thus TL, in addition to CD1d, plays a role in natural T cell development.
Thymocytes can become mature T cells without passing through the CD4+ CD8+, double-positive stage.
Authors: Liu C P , Kappler J W , Marrack P
Published: June 2004

T cells bearing the class II-restricted, DO-T cell receptor (TCR) are CD4+ if their thymocyte precursors are positively selected on the class II protein, IAd, but they are almost all CD4- after positive selection on a class II for which they have higher avidity, IAb. DO-TCR+ T cells mature in H-2b mice lacking CD4. CD4- DO-TCR+ T cells appear in H-2b mice at the same rate as their CD4+ counterparts appear in H-2d animals, suggesting that the CD4- cells are not the product of some minor pathway of thymocyte development and selection. In H-2b CD4 knock out mice expressing human CD2 under the control of the mouse CD4 promoter, mature DO-TCR+ cells did not express human CD2. These results suggest that the CD4-CD8-, DO-TCR+ mature T cells have developed without ever passing through the equivalent of a CD4+,CD8+ stage. The early expression of alpha/beta receptors (TCRs) on thymocytes in TCR transgenic mice may allow maturation of this type. Passage through the equivalent of the CD4+ CD8+, double-positive stage is not essential for differentiation of thymocytes into mature T cells.
Interleukin (IL)-4-independent immunoglobulin class switch to immunoglobulin (Ig)E in the mouse.
Authors: Morawetz R A , Gabriele L , Rizzo L V , Noben-Trauth N , Kühn R , Rajewsky K , Müller W , Doherty T M , Finkelman F , Coffman R L , Morse H C
Published: June 2004

Immunoglobulin (Ig) class switching in B cells is regulated by stimuli transduced by cytokines and cell-cell contact. Among these stimuli, interleukin (IL)-4 has been considered an absolute prerequisite for class switching to IgE in the mouse. Here we report that IL-4-deficient (IL-4-/-) and wildtype mice had comparably elevated serum IgE levels during the course of a murine retrovirus-induced immunodeficiency syndrome, MAIDS. IgE switching in IL-4-/- mice was also induced by injection of anti-IgD antibody. Treatment with anti-IgD induced germline epsilon (g epsilon) transcripts with comparable efficiency in IL-4-/- mice and controls, but the levels of productive epsilon transcripts (p epsilon) were lower by a factor of 200 and serum IgE levels were lower by a factor of 300 in IL-4-/- mice as compared with controls. Induction of g epsilon after anti-IgD treatment of IL-4-/- mice was unaffected by simultaneous treatment with monoclonal antibodies to IL-4 and IL-4 receptor alpha chain. Infection of IL-4-/- mice with Nippostrongylus brasiliensis, a potent stimulus for IgE production, resulted in induction of g epsilon transcripts; however, p epsilon transcripts were barely detectable and serum IgE was not detected. These findings establish a novel IL-4-independent pathway for IgE switching in the mouse that is strongly activated in retroviral infection but weakly in nematode infection. This pathway appears to be dependent on distinct factors that separately control induction of g epsilon transcription and switch recombination to p epsilon.
Glu227-->Lys substitution in the acidic loop of major histocompatibility complex class I alpha 3 domain distinguishes low avidity CD8 coreceptor and avidity-enhanced CD8 accessory functions.
Authors: Shen L , Potter T A , Kane K P
Published: June 2004

Cytotoxic T lymphocyte (CTL) activation requires specific T cell receptor (TCR)-class I major histocompatibility complex (MHC) antigen complex interactions as well as the participation of coreceptor or accessory molecules on the surface of CTL. CD8 can serve as a coreceptor in that it binds to the same MHC class I molecules as the TCR to facilitate efficient TCR signaling. In addition, CD8 can be "activated" by TCR stimulation to bind to class I molecules with high avidity, including class I not recognized by the TCR as antigenic complexes (non-antigen [Ag] class I), to augment CTL responses and thus serve an accessory molecule function. A Glu/Asp227-->Lys substitution in the class I alpha 3 domain acidic loop abrogates lysis of target cells expressing these mutant molecules by alloreactive CD8-dependent CTL. Lack of response is attributed to the destruction of the CD8 binding site in the alpha 3 domain which is likely to disrupt CD8 coreceptor function. The relative importance of the class I alpha 3 domain acidic loop Glu227 in coreceptor as opposed to accessory functions of CD8 is unclear. To address this issue, we examined CTL adhesion and degranulation in response to immobilized class I-peptide complexes formed in vitro from antigenic peptides and purified class I molecules containing wild-type or Glu227-->Lys substituted alpha 3 domains. The alpha 3 domain mutant class I-peptide complexes were bound by CTL and triggered degranulation, however to much lower levels than wild-type class I-peptide complexes. In further experiments, it is directly demonstrated that the alpha 3 domain mutant class I molecules, which lack the Glu227 CD8 binding site, still serve as TCR-activated, avidity-enhanced CD8 accessory ligands. However, mutant class I peptide Ag complexes failed to effectively serve as CD8 coreceptor ligands to initiate TCR-dependent signals required to induce avidity-enhanced CD8 binding to coimmobilized non-Ag class I molecules. Thus the Glu227-->Lys mutation effectively distinguishes CD8 coreceptor and avidity-enhanced CD8 accessory functions.
Dual interaction of the malaria circumsporozoite protein with the low density lipoprotein receptor-related protein (LRP) and heparan sulfate proteoglycans.
Authors: Shakibaei M , Frevert U
Published: June 2004

Speed and selectivity of hepatocyte invasion by malaria sporozoites have suggested a receptor-mediated mechanism and the specific interaction of the circumsporozoite (CS) protein with liver-specific heparan sulfate proteoglycans (HSPGs) has been implicated in the targeting to the liver. Here we show that the CS protein interacts not only with cell surface heparan sulfate, but also with the low density lipoprotein receptor-related protein (LRP). Binding of 125I-CS protein to purified LRP occurs with a Kd of 4.9 nM and can be inhibited by the receptor-associated protein (RAP). Blockage of LRP by RAP or anti-LRP antibodies on heparan sulfate-deficient CHO cells results in more than 90% inhibition of binding and endocytosis of recombinant CS protein. Conversely, blockage or enzymatic removal of the cell surface heparan sulfate from LRP-deficient embryonic mouse fibroblasts yields the same degree of inhibition. Heparinase-pretreatment of LRP-deficient fibroblasts or blockage of LRP on heparan sulfate-deficient CHO cells by RAP, lactoferrin, or anti-LRP antibodies reduces Plasmodium berghei invasion by 60-70%. Parasite development in heparinase-pretreated HepG2 cells is inhibited by 65% when RAP is present during sporozoite invasion. These findings suggest that malaria sporozoites utilize the interaction of the CS protein with HSPGs and LRP as the major mechanism for host cell invasion.
Characterization of Cbl tyrosine phosphorylation and a Cbl-Syk complex in RBL-2H3 cells.
Authors: Ota Y , Beitz L O , Scharenberg A M , Donovan J A , Kinet J P , Samelson L E
Published: June 2004

Tyrosine phosphorylation of the Cbl protooncogene has been shown to occur after engagement of a number of different receptors on hematopoietic cells. However, the mechanisms by which these receptors induce Cbl tyrosine phosphorylation are poorly understood. Here we demonstrate that engagement of the high affinity IgE receptor (Fc epsilon R1) leads to the tyrosine phosphorylation of Cbl and analyze how this occurs. We show that at least part of Fc epsilon R1-induced Cbl tyrosine phosphorylation is mediated by the Syk tyrosine kinase, and that the Syk-dependent tyrosine phosphorylation of Cbl occurs mainly distal to the Cbl proline-rich region within the COOH-terminal 250 amino acids. Furthermore, we show by coprecipitation that Cbl is present in a complex with Syk before receptor engagement, that the proline-rich region of Cbl and a region of Syk comprised of the two SH2 domains and intradomain linker are required for formation of the complex, and that little or no tyrosine-phosphorylated Cbl is detected in complex with Syk. Overexpression of truncation mutants of Cbl capable of binding Syk has the effect of blocking tyrosine phosphorylation of endogenous Cbl. These results define a potentially important intramolecular interaction in mast cells and suggest a complex function for Cbl in intracellular signaling pathways.
Junctional diversity in signal joints from T cell receptor beta and delta loci via terminal deoxynucleotidyl transferase and exonucleolytic activity.
Authors: Candéias S , Muegge K , Durum S K
Published: June 2004

The site-specific V(D)J recombination reaction necessary to assemble the genes coding for immunoglobulin (Ig) and T cell receptor (TCR) variable regions is initiated by a precise double strand cut at the border of the recombination signals flanking the genes. Extensive processing of the coding ends before their ligation accounts for most of the Ig and TCR repertoire diversity. This processing includes both base additions to and loss from the coding ends. On the other hand, it has generally been thought that signal ends are not modified before they are fused, and that signal joints consist of a perfect head-to-head ligation of the recombination signals. In this study, we analyzed signal joints created during the rearrangement of different TCR-beta and TCR-delta genes in thymocytes. We show that a significant fraction (up to 24%) of these signal joints exhibits junctional diversity. This diversity results from N nucleotide additions for TCR-beta signal joints, and from N additions and exonucleolytic digestion for TCR-delta joints. Altogether, our findings suggest that: (a) signal ends can undergo some of the same modifications as coding ends, (b) inversional rearrangement generates more diversity than deletional events, and (c) fine differences exist in the recombinase/DNA complexes formed at each rearranging locus.
Local expression of transgene encoded TNF alpha in islets prevents autoimmune diabetes in nonobese diabetic (NOD) mice by preventing the development of auto-reactive islet-specific T cells.
Authors: Grewal I S , Grewal K D , Wong F S , Picarella D E , Janeway C A , Flavell R A
Published: June 2004

Lately, TNF alpha has been the focus of studies of autoimmunity; its role in the progression of autoimmune diabetes is, however, still unclear. To analyze the effects of TNF alpha in insulin-dependent diabetes mellitus (IDDM), we have generated nonobese diabetic (NOD) transgenic mice expressing TNF alpha under the control of the rat insulin II promoter (RIP). In transgenic mice, TNF alpha expression on the islets resulted in massive insulitis, composed of CD4+ T cells, CD8+ T cells, and B cells. Despite infiltration of considerable number of lymphoid cells in islets, expression of TNF alpha protected NOD mice from IDDM. To determine the mechanism of TNF alpha action, splenic cells from control NOD and RIP-TNF alpha mice were adoptively transferred to NOD-SCID recipients. In contrast to the induction of diabetes by splenic cells from control NOD mice, splenic cells from RIP-TNF alpha transgenic mice did not induce diabetes in NOD-SCID recipients. Diabetes was induced however, in the RIP-TNF alpha transgenic mice when CD8+ diabetogenic cloned T cells or splenic cells from diabetic NOD mice were adoptively transferred to these mice. Furthermore, expression of TNF alpha in islets also downregulated splenic cell responses to autoantigens. These data establish a mechanism of TNF alpha action and provide evidence that local expression of TNF alpha protects NOD mice from autoimmune diabetes by preventing the development of autoreactive islet-specific T cells.
CD81 expressed on human thymocytes mediates integrin activation and interleukin 2-dependent proliferation.
Authors: Todd S C , Lipps S G , Crisa L , Salomon D R , Tsoukas C D
Published: June 2004

Lymphocyte recognition of antigen by the antigen-specific T cell receptor (TCR) and coreceptor complexes rapidly alters the cell's adhesive properties facilitating high avidity cell-ligand interactions necessary for lymphocyte development and function. Here, we report the expression of CD81 (target of antiproliferative antigen [TAPA]-1) on human thymocytes and the physical association of CD81 with CD4 and CD8 T cell coreceptors. Antibody ligation of CD81 on thymocytes promotes the rapid induction of integrin-mediated cell-cell adhesion via lymphocyte function-associated molecule-1 (LFA-1). Cross-linking CD81 is also shown to be costimulatory with signaling through the TCR/CD3 complex inducing interleukin 2-dependent thymocyte proliferation. These data suggest that a CD81-mediated pathway in thymocytes is involved in the regulation of both cell adhesion and activation.
Dendritic Cell Development in Culture from Thymic Precursor Cells in the Absence of Granulocyte/Macrophage Colony-stimulating Factor
Authors: Saunders Dolores , Lucas Karen , Ismaili Jamila , Wu Li , Maraskovsky Eugene , Dunn Ashley , Shortman Ken
Published: June 2004

The earliest lymphoid precursor population in the adult mouse thymus had previously been shown to produce not only T cells, but also dendritic cell (DC) progeny on transfer to irradiated recipients. In this study, culture of these isolated thymic precursors with a mixture of cytokines induced them to proliferate and to differentiate to DC, but not to T lineage cells. At least 70% of the individual precursors had the capacity to form DC. The resultant DC were as effective as normal thymic DC in the functional test of T cell stimulation in mixed leukocyte cultures. The cultured DC also expressed high levels of class I and class II major histocompatibility complex, together with CD11c, DEC-205, CD80, and CD86, markers characteristic of mature DC in general. However, they did not express CD8α or BP-1, markers characteristic of normal thymic DC. The optimized mixture of five to seven cytokines required for DC development from these thymic precursors did not include granulocyte/macrophage colony stimulating factor (GM-CSF), usually required for DC development in culture. The addition of anti–GM-CSF antibody or the use of precursors from GM-CSF–deficient mice did not prevent DC development. Addition of GM-CSF was without effect on DC yield when interleukin (IL) 3 and IL-7 were present, although some stimulation by GM-CSF was noted in their absence. In contrast, DC development was enhanced by addition of the Flt3/Flk2 ligand, in line with the effects of the administration of this cytokine in vivo. The results indicate that the development of a particular lineage of DC, probably those of lymphoid precursor origin, may be independent of the myeloid hormone GM-CSF.
Systemic T Cell–independent Tumor Immunity after Transplantation of Universal Receptor–modified Bone Marrow into SCID Mice
Authors: Hege Kristen M. , Cooke Keegan S. , Finer Mitchell H. , Zsebo Krisztina M. , Roberts Margo R.
Published: June 2004

Gene modification of hematopoietic stem cells (HSC) with antigen-specific, chimeric, or “universal” immune receptors (URs) is a novel but untested form of targeted immunotherapy. A human immunodeficiency virus (HIV) envelope–specific UR consisting of the extracellular domain of human CD4 linked to the ζ chain of the T cell receptor (CD4ζ) was introduced ex vivo into murine HSC by retroviral transduction. After transplantation into immunodeficient SCID mice, sustained high level expression of CD4ζ was observed in circulating myeloid and natural killer cells. CD4ζ-transplanted mice were protected from challenge with a lethal dose of a disseminated human leukemia expressing HIV envelope. These results demonstrate the ability of chimeric receptors bearing ζ-signaling domains to activate non–T cell effector populations in vivo and thereby mediate systemic immunity.
Preferential Proliferation of Murine Colony-forming Units in Culture in a Chemically Defined Condition with a Macrophage Colony-stimulating Factor–negative Stromal Cell Clone
Authors: Takakura Nobuyuki , Kodama Hiroaki , Nishikawa Satomi , Nishikawa Shin-Ichi
Published: June 2004

The establishment of culture conditions that selectively support hematopoietic stem cells is an important goal of hematology. In this study, we investigated the possibility of using for this purpose a defined medium, mSFO2, which was developed for stromal cell–dependent bone marrow cultures. We found that a combination of epidermal growth factor (EGF), the OP9 stromal cell line, which lacks macrophage colony-stimulating factor, recombinant stem cell factor, and the chemically defined medium mSFO2 provides a microenvironment where c-Kit+ Thy-1+/lo Mac-1+/lo B220− TER119− commonβ+ IL-2Rγ+ gp130+ cells are selectively propagated from normal, unfractionated bone marrow cells. This cell population produced an in vitro colony at a very high efficiency (50%), whereas it has only limited proliferative ability in the irradiated recipient. Thus, the cells selected in this culture condition might represent colony-forming units in culture (CFU-c) with short-term reconstituting ability. Transferring this cell population into medium containing differentiation signals resulted in the rapid production of mature myelomonocytic and B cell lineages in vitro and in vivo. The fact that a similar culture condition was created by erb-B2–transduced OP9 in the absence of EGF indicated that EGF exerts its effect by acting on OP9 rather than directly on CFU-c. These results suggested that the balance between self-renewal and differentiation of CFU-c can be regulated by extracellular signals.
The Route of Antigen Entry Determines the Requirement for L-selectin during Immune Responses
Authors: Catalina Michelle D. , Carroll Michael C. , Arizpe Helen , Takashima Akira , Estess Pila , Siegelman Mark H.
Published: June 2004

L-selectin, an adhesion molecule constitutively expressed on leukocytes, is important for primary adhesion and extravasation of lymphocytes at specialized high endothelial venules within lymph nodes and other leukocytes at sites of inflammation. We have generated L-selectin–deficient mice by targeted disruption, and have confirmed a previously reported phenotype which includes strikingly impaired contact hypersensitivity (CHS) responses to reactive haptens (Tedder, T.F., D.A. Steeber, and P. Pizcueta. 1995. J. Exp. Med. 181:2259–2264; Xu, J.C., I.S. Grewal, G.P. Geba, and R.A. Flavell. 1996. 183:589–598.). Since the mechanism of this impairment has not been clarified, we sought to define the stage(s) at which the CHS response is affected in L-selectin–deficient mice. We show that epidermal Langerhans cells in L-selectin– deficient mice are normal in number, migrate to peripheral lymph nodes appropriately, and are functional in presenting allogeneic and haptenic antigens. Moreover, T cells, as well as neutrophil and monocyte effector populations, are fully capable of entry into the inflamed skin sites in the absence of L-selectin. Thus, antigen presentation and effector mechanisms are intact in L-selectin deficient mice. In contrast, virtually no antigen-specific T cells can be found within draining peripheral nodes after a contact challenge, suggesting that the defect resides primarily in the inability of antigen-specific T cells to home to and be activated in these nodes. Indeed, L-selectin–deficient mice mount completely normal CHS responses when alternate routes of immunization are used. These studies pinpoint the lesion in CHS to a discrete stage of the afferent limb of the response, clarify the role of L-selectin on effector populations, and illustrate the critical importance of the route of antigen entry to the successful execution of an immune response.
ON IMMUNITY TO TRANSPLANTABLE CHICKEN TUMORS
Authors: Rous Peyton , Murphy James B.
Published: June 2004

The phenomena of natural and acquired resistance to transplanted chicken tumors strikingly resemble those observed in the case of transplanted mammalian growths; and no more than those do they suggest that the tumors have an extrinsic cause.

That there may exist in fowls implanted with a chicken tumor a resistance directed against the tumor-causing agent distinct from the resistance manifested against the alien tumor cells has been shown in a previous article. Both sorts of resistance are present in a fowl in which a tumor has retrogressed, the resistance in such an instance being acquired. That directed against the agent is largely specific, giving little if any protection against the agents causing other tumors. There is some evidence that the conditions upon which a fowl's natural resistance depends are the same for the agents causing different chicken tumors.

It has proved impossible to protect chickens against the agent causing the simple sarcoma by injecting them with dried tumor material in which this agent has been attenuated by heat. The transfer of blood from resistant fowls to fowls with growing tumors is in our experience void of effect on the tumors.
THE INFLUENCE OF VARIOUS SUBSTANCES ON THE GROWTH OF MOUSE CARCINOMA
Authors: Fleisher Moyer S. , Loeb Leo
Published: June 2004

We have described the methods used in determining the influence of certain substances on tumor growth, and we measured approximately the degree of reliability of the quantitative method used. We examined with these methods various classes of substances,— distilled water, a number of inorganic salts, inorganic colloidal substances, various organic colloidal and non-colloidal substances, especially various proteids, tuberculin and hirudin alone as well as in combination with other substances. Distilled water, various inorganic sulphur preparations, and various inorganic salts did not show an inhibiting effect on tumor growth sufficient to be detected by means of our first method. Only in the case of gold potassium cyanide was there possibly a slightly retarding influence present. On the other hand, certain colloidal solutions of heavy metals (copper, platinum, gold) retard the growth of a number of tumors of injected animals. Certain combinations of copper salts and casein act in a similar manner. Of the organic substances used, casein, nucleoproteid, and hirudin were active, while the other proteids tested, as well as various other organic substances and tuberculin and lecithin, seemed to be either without effect or weaker than the other substances mentioned as retarding the tumor growth. Hirudin was active and caused in addition to its inhibiting influence the retrogression of a certain number of tumors. Especially active was a combination of hirudin with colloidal copper and of hirudin with nucleoproteid.

One single injection of casein or nucleoproteid, or of the Heyden preparation of colloidal copper, leads to a more or less marked edematous condition of a certain number of tumors, while hirudin caused in addition, in many cases, marked hemorrhages in or around the tumors. Other substances which we tested did not show this effect, although their inhibiting action on tumor growth may have been equally strong. Very young tumors (two to six days old) are not retarded in their growth through injection of colloidal copper or hirudin, while nine to thirteen days old tumors are, independently of their size on the ninth day, inhibited in approximately the same relative degree; absolutely, however, the more rapidly growing smaller tumors are more markedly inhibited than the normally more slowly growing larger tumors.