bluuebunny/crossref_metadata_2025 huggingface dataset:
A pathway of costimulation that prevents anergy in CD28- T cells: B7-independent costimulation of CD1-restricted T cells.
Authors: Behar S M , Porcelli S A , Beckman E M , Brenner M B
Published: June 2004

A class of molecules that is expressed on antigen presenting cells, exemplified by CD80 (B7), has been found to provide a necessary costimulatory signal for T cell activation and proliferation. CD28 and CTLA4 are the B7 counterreceptors and are expressed on the majority of human CD4+ T cells and many CD8+ T cells. The signal these molecules mediate is distinguished from other costimulatory signals by the finding that T cell recognition of antigen results in a prolonged state of T cell unresponsiveness or anergy, unless these costimulatory molecules are engaged. However, nearly half of the CD8+ and CD4-CD8- T cells lack CD28, and the costimulatory signals required for the activation of such cells are unknown. To understand the pathways of activation used by CD28- T cells, we have examined the costimulatory requirements of antigen-specific CD4-CD8- TCR(+)-alpha/beta circulating T cells that lack the expression of CD28. We have characterized two T cell lines, DN1 and DN6, that recognize a mycobacterial antigen, and are restricted not by major histocompatibility complex class I or II, but by CD1b or CD1c, two members of a family of major histocompatibility complex-related molecules that have been recently implicated in a distinct pathway for antigen presentation. Comparison of antigen-specific cytolytic responses of the DN1 and DN6 T cell lines against antigen-pulsed CD1+ monocytes or CD1+ B lymphoblastoid cell lines (B-LCL) demonstrated that these T cells recognized antigen presented by both types of cells. However, T cell proliferation occurred only when antigen was presented by CD1+ monocytes, indicating that the CD1+ monocytes expressed a costimulatory molecule that the B-LCL transfectants lacked. This hypothesis was confirmed by demonstrating that the T cells became anergic when incubated with the CD1(+)-transfected B-LCL in the presence of antigen, but not in the absence of antigen. The required costimulatory signal occurred by a CD28-independent mechanism since both the CD1+ monocytes and CD1+ B-LCL transfectants expressed B7-1 and B7-2, and DN1 and DN6 lacked surface expression of CD28. We propose that these data define a previously unrecognized pathway of costimulation for T cells distinct from that involving CD28 and its counterreceptors. We suggest that this B7-independent pathway plays a crucial role in the activation and maintenance of tolerance of at least a subset of CD28- T cells.
Low levels of HIV-1 infection in cutaneous dendritic cells promote extensive viral replication upon binding to memory CD4+ T cells.
Authors: Pope M , Gezelter S , Gallo N , Hoffman L , Steinman R M
Published: June 2004

Earlier work has identified a cell population that replicates HIV-1 in the absence of standard T cell stimuli. The system consists of dendritic cells and memory T lymphocytes that emigrate from organ cultures of human skin and together support a productive infection with HIV-1. These emigrants resemble cells that can be found in mucous membranes and that normally traffic in afferent lymph. Here, we report that a low level of infection in the dendritic cell can initiate extensive HIV-1 replication in cocultures with T cells. First we extended our earlier work to larger skin specimens from cadavers. As long as the organ cultures were set up within 36 h of death, the emigrant leukocytes were comparable to cells from fresh surgical specimens in number, phenotype, and function. These mixtures of dendritic cells and T cells provided the milieu for a productive infection with several virus isolates. When purified dendritic cells were separately pulsed with virus and then mixed with T cells that had not been pulsed with HIV-1, active infection ensued. The infectivity of HIV-pulsed dendritic cells persisted for at least 1.5 d in culture, but was blocked if AZT was added during that time to block reverse transcription in the dendritic cells. The number of copies of proviral DNA in the dendritic cells corresponded to < 100 copies per 5 X 10(4) cells, but upon mixing with T cells, > 10(4) copies were found 5-7 d later. By contacting syngeneic T cells, extralymphoid depots of dendritic cells--even with a low viral burden as has been reported in vivo--may contribute to chronic HIV-1 replication in infected individuals.
Evidence for a conformational change in a class II major histocompatibility complex molecule occurring in the same pH range where antigen binding is enhanced.
Authors: Boniface J J , Lyons D S , Wettstein D A , Allbritton N L , Davis M M
Published: June 2004

Many class II histocompatibility complex molecules bind antigenic peptides optimally at low pH, consistent with their exposure to antigen in acidic endosomal compartments. While it has been suggested that a partially unfolded state serves as an intermediate involved in peptide binding, very little evidence for such a state has been obtained. In this report, we show that the murine class II molecule IE becomes increasingly less stable to sodium dodecyl sulfate-induced dissociation since the pH is decreased in the same range that enhances antigenic peptide binding. Furthermore, at mildly acidic pH levels, IEk binds the fluorescent dye 1-anilino-naphthalene-8-sulfonic acid (ANS), a probe for exposed nonpolar sites in proteins, suggesting that protonation produces a molten globule-like state. The association of IEk with a single high-affinity peptide had only a small effect in these two assays, indicating that the changes that occur are distal to the peptide-binding groove. Circular dichroism analysis shows that a pH shift from neutral to mildly acidic pH causes subtle changes in the environment of aromatic residues but does not grossly disrupt the secondary structure of IEk. We propose a model in which perturbations in interdomain contacts outside the peptide-binding domain of IEk occur at acidic pH, producing a partially unfolded state that facilitates optimal antigen binding.
A structural transition in class II major histocompatibility complex proteins at mildly acidic pH.
Authors: Runnels H A , Moore J C , Jensen P E
Published: June 2004

Peptide binding by class II major histocompatibility complex proteins is generally enhanced at low pH in the range of hydrogen ion concentrations found in the endosomal compartments of antigen-presenting cells. We and others have proposed that class II molecules undergo a reversible conformational change at low pH that is associated with enhanced peptide loading. However, no one has previously provided direct evidence for a structural change in class II proteins in the mildly acidic pH conditions in which enhanced peptide binding is observed. In this study, susceptibility to denaturation induced by sodium dodecyl sulfate (SDS) detergent or heat was used to probe the conformation of class II at different hydrogen ion concentrations. Class II molecules became sensitive to denaturation at pH 5.5-6.5 depending on the allele and experimental conditions. The observed structural transition was fully reversible if acidic pH was neutralized before exposure to SDS or heat. Experiments with the environment-sensitive fluorescent probe ANS (8-anilino-1-naphthalene-sulfonic acid) provided further evidence for a reversible structural transition at mildly acidic pH associated with an increase in exposed hydrophobicity in class II molecules. IAd conformation was found to change at a higher pH than IEd, IEk, or IAk, which correlates with the different pH optimal for peptide binding by these molecules. We conclude that pH regulates peptide binding by influencing the structure of class II molecules.
The SH3 domain of p56lck binds to proline-rich sequences in the cytoplasmic domain of CD2.
Authors: Bell G M , Fargnoli J , Bolen J B , Kish L , Imboden J B
Published: June 2004

CD2, a cell surface glycoprotein expressed on T cells and natural killer cells, can couple to signaling pathways that result in T cell proliferation. An Src-like protein tyrosine kinase, p56lck, coprecipitates with CD2, and perturbation of CD2 by monoclonal antibodies results in an increase in the activity of p56lck, suggesting that an interaction with p56lck contributes to CD2-mediated signaling. Herein, we investigate the mechanism by which CD2 associates with p56lck. We demonstrate that CD2 and p56lck associate when coexpressed in nonlymphoid cells, that this association requires the cytoplasmic domain of CD2, and that the SH3 domain of p56lck mediates its interactions with CD2. Using truncation mutants of CD2, we identify two regions in the cytoplasmic domain of CD2 involved in binding p56lck. Each region contains a proline-rich sequence that, in the form of a synthetic peptide, directly binds p56lck. Thus, proline-rich sequences in the cytoplasmic domain of CD2 allow this transmembrane receptor to bind to the SH3 domain of p56lck.
CD16-mediated p21ras activation is associated with Shc and p36 tyrosine phosphorylation and their binding with Grb2 in human natural killer cells.
Authors: Galandrini R , Palmieri G , Piccoli M , Frati L , Santoni A
Published: June 2004

The Src homology (SH) 2/SH3 domain-containing protein Grb2 and the oncoprotein Shc have been implicated in a highly conserved mechanism that regulates p21ras activation. We investigated the involvement of these adaptor proteins in the signaling pathway induced by CD16 or interleukin (IL) 2R triggering in human natural killer (NK) cells. Both p46 and p52 forms of Shc were rapidly and transiently tyrosine phosphorylated upon CD16 or IL-2 stimulation with different kinetics. Shc immunoprecipitates from lysates of CD16- or IL-2-stimulated NK cells contained Grb2 and an unidentified 145-kD tyrosine phosphoprotein. Grb2 immunoprecipitates from anti-CD16-stimulated NK cells contained not only Shc, but also a 36-kD tyrosine phosphoprotein (p36). The interaction between Grb2 and Shc or p36 occurred via the Grb2SH2 domain as indicated by in vitro binding assays using a bacteriologically synthesized glutathione S-transferase-Grb2SH2 fusion protein. We also present evidence that p21ras is activated by CD16 and IL-2R cross-linking. Accumulation of guanosine triphosphate-bound Ras was detected within 1 minute and occurred with kinetics similar to inductive protein tyrosine phosphorylation and Grb2 association of Shc and p36 adaptor proteins.
Bcl-2+ tonsillar plasma cells are rescued from apoptosis by bone marrow fibroblasts.
Authors: Merville P , Déchanet J , Desmoulière A , Durand I , de Bouteiller O , Garrone P , Banchereau J , Liu Y J
Published: June 2004

Plasma cells represent the final stage of B lymphocyte differentiation. Most plasma cells in secondary lymphoid tissues live for a few days, whereas those in the lamina propria of mucosa and in bone marrow live for several weeks. To investigate the regulation of human plasma cell survival, plasma cells were isolated from tonsils according to high CD38 and low CD20 expression. Tonsillar plasma cells express CD9, CD19, CD24, CD37, CD40, CD74, and HLA-DR, but not CD10, HLA-DQ, CD28, CD56, and Fas/CD95. Although plasma cells express intracytoplasmic Bcl-2, they undergo swift apoptosis in vitro and do not respond to CD40 triggering. Bone marrow fibroblasts and rheumatoid synoviocytes, however, prevented plasma cells from undergoing apoptosis in a contact-dependent fashion. These data indicate that fibroblasts may form a microenvironment favorable for plasma cell survival under normal and pathological conditions.
An essential role for macrophage migration inhibitory factor in the tuberculin delayed-type hypersensitivity reaction.
Authors: Bernhagen J , Bacher M , Calandra T , Metz C N , Doty S B , Donnelly T , Bucala R
Published: June 2004

30 years ago, investigations into the molecular basis of the delayed-type hypersensitivity reaction (DTH) provided evidence for the first lymphokine activity: a lymphocyte-derived mediator called macrophage migration inhibitory factor (MIF), which inhibited the random migration of peritoneal macrophages. Despite the long-standing association of MIF with the DTH reaction and the cloning of a human protein with macrophage migration inhibitory activity, the precise role of MIF in this classic cell-mediated immune response has remained undefined. This situation has been further complicated by the fact that two other cytokines, interferon gamma and IL-4, similarly inhibit macrophage migration and by the identification of mitogenic contaminants in some preparations of cloned human MIF. Using recently developed molecular probes for mouse MIF, we have examined the role of this protein in a classical model of DTH, the tuberculin reaction in mice. Both MIF messenger RNA and protein were expressed prominently in DTH lesions, as assessed by reverse transcription polymerase chain reaction, in situ hybridization, and immunostaining with anti-MIF antibody. The predominant cellular origin of MIF appeared to be the monocyte/macrophage, a cell type identified recently to be a major source of MIF release in vivo. The administration of neutralizing anti-MIF antibodies to mice inhibited significantly the development of DTH, thus affirming the central role of MIF in this classic immunological response.
Peptide-pulsed dendritic cells induce antigen-specific CTL-mediated protective tumor immunity.
Authors: Celluzzi C M , Mayordomo J I , Storkus W J , Lotze M T , Falo L D
Published: June 2004

Cytotoxic T lymphocytes (CTLs) are a critical component of the immune response to tumors. Tumor-derived peptide antigens targeted by CTLs are being defined for several human tumors and are potential immunogens for the induction of specific antitumor immunity. Dendritic cells (DC) are potent antigen-presenting cells (APCs) capable of priming CTL responses in vivo. Here we show that major histocompatibility complex class I-presented peptide antigen pulsed onto dendritic APCs induces protective immunity to lethal challenge by a tumor transfected with the antigen gene. The immunity is antigen specific, requiring expression of the antigen gene by the tumor target, and is eliminated by in vivo depletion of CD8+ T cells. Furthermore, mice that have rejected the transfected tumor are protected from subsequent challenge with the untransfected parent tumor. These results suggest that immunization strategies using antigen-pulsed DC may be useful for inducing tumor-specific immune responses.
Association of tyrosine protein kinase Zap-70 with the protooncogene product p120c-cbl in T lymphocytes.
Authors: Fournel M , Davidson D , Weil R , Veillette A
Published: June 2004

Accumulating data show that the tyrosine protein kinase Zap-70 plays an essential role in T cell receptor-mediated signal transduction. However, the model of action, as well as the physiologically relevant substrates of Zap-70, have not been determined. We have attempted to identify a 120-kD tyrosine-phosphorylated protein (p120) that associates with Zap-70 in activated T lymphocytes. The results of our analyses showed that p120 is largely encoded by the c-cbl protooncogene. Furthermore, the association of Zap-70 with c-Cbl was shown to be induced by T cell receptor stimulation, implying that it required posttranslational modification of one or both of these products. FynT, but not Lck, also associated with c-Cbl in activated T cells. Finally, using a heterologous system, it was demonstrated that the ability of Zap-70 to cause tyrosine phosphorylation of p120c-cbl was dependent on Lck- or FynT-mediated signals. As c-Cbl can associate with several other signaling molecules, it may couple Zap-70 to downstream effectors during T cell activation.
Efficient destruction of human immunodeficiency virus in human serum by inhibiting the protective action of complement factor H and decay accelerating factor (DAF, CD55).
Authors: Stoiber H , Pintér C , Siccardi A G , Clivio A , Dierich M P
Published: June 2004

Activation of the human complement system leads to complement deposition on human immunodeficiency virus (HIV) and HIV-infected cells without causing efficient complement-mediated lysis. Even in the presence of HIV-specific antibodies, only a few particles are destroyed, demonstrating that HIV is intrinsically resistant to human complement. Here we report that, in addition to decay accelerating factor (DAF) being partially responsible, human complement factor H (CFH), a humoral negative regulator of complement activation, is most critical for this resistance. In the presence of HIV-specific antibodies, sera devoid of CFH (total genetic deficiency or normal human serum depleted of CFH by affinity chromatography) lysed free virus and HIV-infected but not uninfected cells. In the presence of CFH, lysis of HIV was only obtained when binding of CFH to gp41 was inhibited by a monoclonal antibody against a main CFH-binding site in gp41. Since CFH is an abundant protein in serum, and high local concentration of CFH can be obtained at the surface of HIV as the result of specific interactions of CFH with the HIV envelope, it is proposed that the resistance of HIV and HIV-infected cells against complement-mediated lysis in vivo is dependent on DAF and CFH and can be overcome by suppressing this protection. Neutralization of HIV may be achieved by antibodies against DAF and, more importantly, antibodies against CFH-binding sites on HIV envelope proteins.
Variation of gonococcal lipooligosaccharide structure is due to alterations in poly-G tracts in lgt genes encoding glycosyl transferases.
Authors: Yang Q L , Gotschlich E C
Published: June 2004

The lipooligosaccharide (LOS) expressed by gonococci spontaneously varies its structure at high frequency, but the underlying genetic mechanism has not been described. We have previously reported that the genes encoding the glycosyl transferases responsible for the biosynthesis of the variable alpha chain of the LOS of Neisseria gonorrhoeae are located in a locus containing five genes, lgtA, lgtB, lgtC, lgtD, and lgtE. Sequence analysis showed that lgtA, lgtC, and lgtD contained poly-G tracts within the coding frames, leading to the hypothesis that shifts in the number of guanosine residues in the poly-G tracts might be responsible for the high frequency variation in structure of gonococcal LOS. We now provide experimental evidence confirming this hypothesis.
CD8 T cell clones from young nonobese diabetic (NOD) islets can transfer rapid onset of diabetes in NOD mice in the absence of CD4 cells.
Authors: Wong F S , Visintin I , Wen L , Flavell R A , Janeway C A
Published: June 2004

T cells play an important role in the pathogenesis of diabetes in the nonobese diabetic (NOD) mouse. CD8 cytotoxic T cell lines and clones were generated from the lymphocytic infiltrate in the islets of Langerhans of young (7-wk-old). NOD mice by growing them on (NOD x B6-RIP-B7-1)F1 islets. These cells proliferate specifically to NOD islets and kill NOD islets in vitro. The cells are restricted by H-2Kd, and all bear T cell antigen receptor encoded by V beta 6. When these CD8 T cell lines and clones are adoptively transferred to irradiated female NOD, young NOD-SCID, and CB17-SCID mice, diabetes occurs very rapidly, within 10 d of transfer and without CD4 T cells.
HLA-derived peptides which inhibit T cell function bind to members of the heat-shock protein 70 family.
Authors: Nossner E , Goldberg J E , Naftzger C , Lyu S C , Clayberger C , Krensky A M
Published: June 2004

Synthetic peptides corresponding to sequences of HLA class I molecules have inhibitory effects on T cell function. The peptides investigated in this study have sequences corresponding to the relatively conserved region of the alpha 1 helix of HLA class I molecules that overlaps the "public epitope" Bw4/Bw6. These HLA-derived peptides exhibit inhibitory effects on T lymphocytes and have beneficial effects on the survival of allogenic organ transplants in mice and rats. Peptides corresponding to the Bw4a epitope appear most potent as they inhibit the differentiation of T cell precursors into mature cytotoxic T lymphocytes (CTL) and target cell lysis by established CTL lines and clones. To elucidate the mechanism through which these peptides mediate their inhibitory effect on T lymphocytes, peptide binding proteins were isolated from T cell lysates. We show that the inhibitory Bw4a peptide binds two members of the heat-shock protein (HSP) 70 family, constitutively expressed HSC70 and heat-inducible HSP70. Peptide binding to HSC/HSP70 is sequence specific and follows the rules defined by the HSC70 binding motif. Most intriguing, however, is the strict correlation of peptide binding to HSC/HSP70 and the functional effects such that only inhibitory peptides bind to HSC70 and HSP70 whereas noninhibitory peptides do not bind. This correlation suggests that small molecular weight HLA-derived peptides may modulate T cell responses by directly interacting with HSPs. In contrast to numerous reports of HSP70 expression at the surface of antigen-presenting cells and some tumor cells, we find no evidence that HSC/HSP70 are expressed at the surface of the affected T cells. Therefore, we believe that the peptides' immunodulatory effects are not mediated through a signaling event initiated by interaction of peptide with surface HSP, but favor a model similar to the action of other immunomodulatory compounds, FK506 and cyclosporin A, with a role for HSC/HSP70 similar to that for immunophilins, FKBPs and CyP40.
T cell responses in calcineurin A alpha-deficient mice.
Authors: Zhang B W , Zimmer G , Chen J , Ladd D , Li E , Alt F W , Wiederrecht G , Cryan J , O'Neill E A , Seidman C E , Abbas A K , Seidman J G
Published: June 2004

We have created embryonic stem (ES) cells and mice lacking the predominant isoform (alpha) of the calcineurin A subunit (CNA alpha) to study the role of this serine/threonine phosphatase in the immune system. T and B cell maturation appeared to be normal in CNA alpha -/- mice. CNA alpha -/- T cells responded normally to mitogenic stimulation (i.e., PMA plus ionomycin, concanavalin A, and anti-CD3 epsilon antibody). However, CNA alpha -/- mice generated defective antigen-specific T cell responses in vivo. Mice produced from CNA alpha -/- ES cells injected into RAG-2-deficient blastocysts had a similar defective T cell response, indicating that CNA alpha is required for T cell function per se, rather than for an activity of other cell types involved in the immune response. CNA alpha -/- T cells remained sensitive to both cyclosporin A and FK506, suggesting that CNA beta or another CNA-like molecule can mediate the action of these immunosuppressive drugs. CNA alpha -/- mice provide an animal model for dissecting the physiologic functions of calcineurin as well as the effects of FK506 and CsA.
Mononuclear phagocytes egress from an in vitro model of the vascular wall by migrating across endothelium in the basal to apical direction: role of intercellular adhesion molecule 1 and the CD11/CD18 integrins.
Authors: Randolph G J , Furie M B
Published: June 2004

Little is known about how mononuclear phagocytes (MP) are cleared from sites of inflammation as inflammatory lesions resolve. In this study, the possibility that MP could be cleared from tissues by migrating across endothelium in the basal to apical direction was investigated. In an in vitro model of a blood vessel wall consisting of human umbilical vein endothelial cells (HUVEC) grown on amniotic tissue, a majority of MP that initially transmigrated into the amnion later exited by migrating back across the endothelium in the basal to apical direction. MP that egressed from these cultures adhered to the apical surface of the endothelium or were found nonadherent in the medium above the endothelium. Egression of MP continued throughout the 4-d period examined, displaying higher than first order kinetics and a t(1/2) of approximately 24 h. These kinetics were decreased by increasing the volume of medium bathing the cultures, suggesting that a soluble factor(s) regulates the rate of egression. In contrast, the kinetics were accelerated by pretreating the endothelium with IL-1. The initial phase of this increased rate of egression was inhibited by antibodies to inter- cellular adhesion molecule 1 (ICAM-1) or CD18 by 100 and 71%, respectively. Immunostaining revealed that ICAM-1 was present on the apical and basal surfaces of umbilical vein endothelium in vitro and in situ. These data demonstrate that MP can traverse endothelium in the basal to apical direction, and lend insight into the mechanisms by which this process occurs.
Unresponsiveness to a self-peptide of mouse lysozyme owing to hindrance of T cell receptor-major histocompatibility complex/peptide interaction caused by flanking epitopic residues.
Authors: Moudgil K D , Grewal I S , Jensen P E , Sercarz E E
Published: June 2004

A self-peptide containing amino acid residues 46-61 (NRGDQSTDYGIFQINSR) of mouse lysozyme (ML) (p46-61, which binds strongly to the A(k) molecule but does not bind to the E(k) molecule), can induce a strong proliferative T cell response in CBA/J mice (A[k], E[k]) but no response at all in B10.A(4R) and CBA/J mice. The critical residues within p46-59 are immunogenic in both B10.A(4R) and CBA/J mice. The critical residues within p46-61 reside between amino acid positions 51 and 59. T cells of B10.A(4R) mice primed with the truncated peptides in vivo cannot be restimulated by p46-61 in vitro. This suggests that T cell receptor (TCR) contact (epitopic) residue(s) flanking the minimal 51-59 determinant within p46-61 hinder the interaction of the p46-61/A(k) complex with the appropriate TCR(S), thereby causing a lack of proliferative T cell response in this mouse strain. Unlike B10.A(4R) mice, [B10.A(4R) x CBA/J]F1 mice responded vigorously to p46-61, suggesting that thymic APC of B10.A(4R) mice do not present a self ligand to T cells resulting in a p46-61-specific hole in the T cell repertoire in B10.A(4R) or the F1 mice. Moreover, APC from B10.A(4R) mice are capable of efficiently presenting p46-61 to peptide-specific T cell lines from CBA/J mice. The proliferative unresponsiveness of B10.A(4R) mice to p46-61 is not due to non-major histocompatibility complex genes because B10.A mice (A[k], E[k]) respond well to p46-61. Interestingly, B10.A(4R) mice can raise a good proliferative response to p46-61 (R61A) (in which the arginine residue at position 61 (R61L/F/N/K), indicating that R61 was indeed responsible for hindering the interaction of p46-61 with the appropriate TCR. Finally, chimeric mice [B10.A(4R)-->B10.A] responded vigorously to p46-61, suggesting that thymic antigen presentation environment of the B10.A mouse was critical for development of a p46-61-reactive T cell repertoire. Thus, we provide experimental demonstration of a novel mechanism for unresponsiveness to a self peptide, p46-61, in the B10.A(4R) mouse owing to hindrance: in this system it is the interaction between the available TCR and the A(k)/p46-61 complex, which is hindered by epitopic residue(s) within p46-61. We argue that besides possessing T cells that are hindered by R61 of p46-61, CBA/J and B10.A mice have developed an additional subset of T cells bearing TCRs which are not hinderable by R61, presumably through positive selection with peptides derived from class II E(k), or class I D(k)/D(d) molecules. These results have important implications in self tolerance, shaping of the T cell repertoire, and in defining susceptibility to autoimmunity.
Human vascular adhesion protein 1 (VAP-1) is a unique sialoglycoprotein that mediates carbohydrate-dependent binding of lymphocytes to endothelial cells.
Authors: Salmi M , Jalkanen S
Published: June 2004

The regulated interactions of leukocytes with vascular endothelial cells are crucial in controlling leukocyte traffic between blood and tissues. Vascular adhesion protein-1 (VAP-1) is a novel, human endothelial cell molecule that mediates tissue-selective lymphocyte binding. Two species (90 and 170 kD) of VAP-1 exist in lymphoid tissues. Glycosidase digestions revealed that the mature 170-kD form of VAP-1 expressed on the lumenal surfaces of vessels is a heavily sialylated glycoprotein. The sialic acids are indispensable for the function of VAP-1, since the desialylated form of VAP-1 no longer mediates lymphocyte binding. We also show that L-selectin is not required for binding of activated lymphocytes to VAP-1 under conditions of shear stress. The 90-kD form of VAP-1 was only seen in an organ culture model, and may represent a monomeric or proteolytic form of the larger species. These data indicate that L-selectin negative lymphocytes can bind to tonsillar venules via the VAP- 1-mediated pathway. Moreover, our findings extend the role of carbohydrate-mediated binding in lymphocyte-endothelial cell interactions beyond the known selectins. In conclusion, VAP-1 naturally exists as a 170-kD sialoglycoprotein that uses sialic acid residues to interact with its counter-receptors on lymphocytes under nonstatic conditions.
Dissection of immunoglobulin E and T lymphocyte reactivity of isoforms of the major birch pollen allergen Bet v 1: potential use of hypoallergenic isoforms for immunotherapy.
Authors: Ferreira F , Hirtenlehner K , Jilek A , Godnik-Cvar J , Breiteneder H , Grimm R , Hoffmann-Sommergruber K , Scheiner O , Kraft D , Breitenbach M , Rheinberger H J , Ebner C
Published: June 2004

We dissected the T cell activation potency and the immunoglobulin (Ig) E-binding properties (allergenicity) of nine isoforms of Bet v 1 (Bet v 1a-Bet v 1l), the major birch pollen allergen. Immunoblot experiments showed that Bet v 1 isoforms differ in their ability to bind IgE from birch pollen-allergic patients. All patients tested displayed similar IgE-binding patterns toward each particular isoform. Based on these experiments, we grouped Bet v 1 isoforms in three classes: molecules with high IgE-binding activity (isoforms a, e, and j), intermediate IgE-binding (isoforms b, c, and f), and low/no IgE-binding activity (isoforms d, g, and 1). Bet v 1a, a recombinant isoform selected from a cDNA expression library using IgE immunoscreening exhibited the highest IgE-binding activity. Isoforms a, b, d, e, and 1 were chosen as representatives from the three classes for experimentation. The potency of each isoallergen to activate T lymphocytes from birch pollen-allergic patients was assayed using peripheral blood mononuclear cells, allergen-specific T cell lines, and peptide-mapped allergen-specific T cell clones. Among the patients, some displayed a broad range of T cell-recognition patterns for Bet v 1 isoforms whereas others seemed to be restricted to particular isoforms. In spite of this variability, the highest scores for T cell proliferative responses were observed with isoform d (low IgE binder), followed by b, 1, e, and a. In vivo (skin prick) tests showed that the potency of isoforms d and 1 to induce typical urticarial type 1 reactions in Bet v 1-allergic individuals was significantly lower than for isoforms a, b, and e. Taken together, our results indicate that hypoallergenic Bet v 1 isoforms are potent activators of allergen-specific T lymphocytes, and Bet v 1 isoforms with high in vitro IgE-binding activity and in vivo allergenicity can display low T cell antigenicity. Based on these findings, we propose a novel approach for immunotherapy of type I allergies: a treatment with high doses of hypoallergenic isoforms or recombinant variants of atopic allergens. We proceed on the assumption that this measure would modulate the quality of the T helper cell response to allergens in vivo. The therapy form would additionally implicate a reduced risk of anaphylactic side effects.
Lipoprotein e(P4) is essential for hemin uptake by Haemophilus influenzae.
Authors: Reidl J , Mekalanos J J
Published: June 2004

Heme uptake is a common means of iron and porphyrin acquisition by many pathogenic bacteria. The genus Haemophilus includes several important pathogenic bacterial species that characteristically require hemin-, protoporphyrin-, or heme-substituted proteins as essential growth factors under aerobic conditions. However, the mechanism of heme transport is not understood for Haemophilus. We have cloned a DNA fragment from H. influenzae that allows an Escherichia coli hemA mutant to employ exogenous hemin or protoporphyrin IX as sole sources of porphyrin. DNA sequencing of the cloned DNA fragment suggested that a previously characterized gene (hel) encoding an antigenic, outer membrane lipoprotein e(P4) was responsible for the complementation activity. Construction of hel insertion mutations in strain H. influenzae Rd demonstrated that hel is essential for growth under aerobic conditions but not under anaerobic conditions. The aerobic growth defect of hel mutants could be reversed by providing exogenous hemin in the presence of outer membrane. The analysis of hybrids between e(P4) and beta-lactamase demonstrated that a domain of e(P4) near its NH2' terminus was required for its function in hemin use. Within this domain is a short amino acid sequence that displays similarity to H. influenzae hemin binding protein HbpA, hemin-binding motifs present in eukaryotic transcription activator heme-activated protein, and the heme containing proteins hemoglobin (alpha-chain) and cytochrome C3, suggesting that this region may be involved in hemin binding and/or transport.
Costimulation by CD48 and B7-1 induces immunity against poorly immunogenic tumors.
Authors: Li Y , Hellstrom K E , Newby S A , Chen L
Published: June 2004

Genetic modification of many types of mouse tumors to express the B7-1 or B7-2 molecules, natural ligands for the T cell-costimulatory molecule CD28, increases their immunogenicity. However, even after transfection with the B7-1 and/or B7-2 genes, poorly immunogenic tumors fail to elicit and efficient immune response. We report here that two such tumors, the Ag104A sarcoma and the K1735-M2 melanoma, become immunogenic after transfection of the genes encoding murine B7-1 together with CD48, which is the natural ligand for CD2. Tumor-specific CD8+ cytotoxic T lymphocytes were readily generated and were effective for adoptive immunotherapy of metastasis induced by wild-type Ag104A sarcoma cells. A similar approach may be useful for developing therapy for other poorly immunogenic tumors, including those in humans.
The presentation of a hepatitis C viral peptide by distinct major histocompatibility complex class I allotypes from two chimpanzee species.
Authors: Cooper S , Kowalski H , Erickson A L , Arnett K , Little A M , Walker C M , Parham P
Published: June 2004

A cytotoxic T lymphocyte (CTL) line, derived from the liver of a common chimpanzee (Pan troglodytes) with hepatitis C, specifically recognized a hepatitis C viral 9-mer peptide (KHP-DATYSR in single-letter amino acid code) bound by the major histocompatibility complex (MHC) class I molecule, Patr-A04. This same CTL line also recognized the identical peptide bound by a structurally different class I molecule, Papa-A06, derived from the separate chimpanzee species, Pan paniscus or pygmy chimpanzee. These class I allotypes differ by six amino acids but, in spite of the structural differences, share the same antigen-presenting function. This is the first observation of antigen presentation to a given T cell receptor by different MHC class I allotypes from separate species.
Two levels of help for B cell alloantibody production.
Authors: Steele D J , Laufer T M , Smiley S T , Ando Y , Grusby M J , Glimcher L H , Auchincloss H
Published: June 2004

We have examined whether T cell stimulation by direct or indirect pathways contributes to alloantibody production by B cells after major histocompatibility complex (MHC)-disparate skin graft rejection in mice. Experiments were performed using normal mice, MHC class II-deficient mice, MHC class II-deficient mice with an intact peripheral CD4+ cell population (due to expression of class II antigens only on thymic epithelium), mice lacking the cytoplasmic tail of their MHC class II antigens, and mice depleted of CD4+ cells by anti-CD4 monoclonal antibody treatment. Depletion of recipient CD4+ cells reduced alloantibody production to barely detectable levels. Absence of donor MHC class II antigens did not affect the production of either immunoglobulin (Ig)M or IgG antibodies directed at class I alloantigens. Absence of recipient MHC class II antigens, however, led to production of only IgM but not IgG antibodies, even if the recipients had an intact CD4+ cell population. Absence of the cytoplasmic tail of the recipient's MHC class II antigens led to the production of slightly reduced amounts of IgG antibody. These findings indicate that (a) CD4+ cells are essential helper cells for B cell alloantibody production; (b) production of IgM alloantibody can occur with help from CD4+ cells, which recognize either donor class II antigens or modified recipient class II antigens; (c) isotype switching from IgM to IgG alloantibody requires help from CD4+ cells activated by antigens presented by recipient MHC class II molecules; and (d) the cytoplasmic domain of the recipient MHC class II molecules may be involved in the mechanism that leads to isotype switching by B cells. Thus, there are two levels of CD4-mediated help available for B cells responding to alloantigens: one (involving a noncognate interaction) can produce B cell activation, and a second (involving a cognate interaction) is required for differentiation and IgG alloantibody production.
Tumor necrosis factor alpha is a potent synergistic factor for the proliferation of primitive human hematopoietic progenitor cells and induces resistance to transforming growth factor beta but not to interferon gamma.
Authors: Snoeck H W , Weekx S , Moulijn A , Lardon F , Lenjou M , Nys G , Van Ranst P C , Van Bockstaele D R , Berneman Z N
Published: June 2004

Since tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and transforming growth factor (TGF)-beta have all been shown to be specific inhibitors of early human hematopoiesis, we wanted to investigate the interactions of these three cytokines on very primitive human adult bone marrow CD34++CD38- hematopoietic progenitor cells, using a pre-colony-forming cell (pre-CFC) assay, which detects the effects of these cytokines on the initial phases of the differentiation of these primitive progenitors, which are unresponsive to interleukin (IL) 3 alone. Surprisingly, TNF-alpha was a very potent stimulator of the proliferation of CD34++CD38- cells and was the most potent synergistic factor for the IL-3-induced proliferation of these cells of all cytokines tested (IL-1, IL-6, granulocyte colony-stimulating factor, kit ligand). TNF-alpha was the only cytokine that, as a single added factor, induced substantial proliferation in CD34++CD38- cells in the presence of IL-3, except for kit ligand, which induced very limited proliferation. TNF-alpha, moreover, induced a high degree of resistance to the inhibitory effects of TGF-beta in a dose-dependent way. The inhibitory effects of IFN-gamma, however, were not affected by the presence of TNF-alpha. We hypothesize that in situations of the hematopoietic stress, TNF-alpha may abrogate the inhibitory effect of ambient TGF-beta in the bone marrow microenvironment to allow primitive stem cells to proliferate and differentiate in response to an increased demand for mature blood cells.
A peptide recognized by human cytolytic T lymphocytes on HLA-A2 melanomas is encoded by an intron sequence of the N-acetylglucosaminyltransferase V gene.
Authors: Guilloux Y , Lucas S , Brichard V G , Van Pel A , Viret C , De Plaen E , Brasseur F , Lethé B , Jotereau F , Boon T
Published: June 2004

A cytolytic T lymphocyte (CTL) clone that lyses many HLA-A2 melanomas was derived from a population of tumor-infiltrating lymphocytes of an HLA-A2 melanoma patient. The gene coding for the antigen recognized by this CTL was identified by transfection of a cDNA library. It is the gene which has been reported to code for N-acetylglucosaminyltransferase V (GnT-V). Remarkably, the antigenic peptide recognized by the CTL is encoded by a sequence located in an intron. In contrast to the fully spliced GnT-V mRNA, which was found in a wide range of normal and tumoral tissues, the mRNA containing the intron region coding for the antigen was not found at a significant level in normal tissues. This mRNA was observed to be present in about 50% of melanomas. Our results suggest that a promoter located near the end of the relevant intron is activated in melanoma cells, resulting in the production of an mRNA coding for the antigen.
Transcription of the murine iNOS gene is inhibited by docosahexaenoic acid, a major constituent of fetal and neonatal sera as well as fish oils.
Authors: Khair-El-Din T , Sicher S C , Vazquez M A , Chung G W , Stallworth K A , Kitamura K , Miller R T , Lu C Y
Published: June 2004

Macrophage activation is deficient in the fetus and neonate when the serum concentrations of docosahexaenoic acid (DHA) are 150 microM, or 10-50-fold higher than in the adult. We now show that DHA inhibits production of nitric oxide (NO) by macrophages stimulated in vitro by IFNgamma plus LPS, or by IFNgamma plus TNFalpha. The half-maximal inhibitory activity of DHA was approximately 25 microM. There were strict biochemical requirements of the fatty acid for inhibition. Polyenoic fatty acids with 22 carbons were more inhibitory than those with 20 carbons. Among 22-carbon fatty acids, those with a greater number of double bonds and a double bond in the n-3 position were more inhibitory. DHA was the most inhibitory of the polyenoic acids we tested. Inducible nitric oxide synthase (iNOS) is the enzyme responsible for the production of NO by macrophages. NO production is initiated after new iNOS enzyme is synthesized following transcription of the iNOS gene. In macrophages stimulated by IFNgamma plus LPS, DHA inhibited accumulation of iNOS mRNA, as measured by Northern blotting, and iNOS transcription, as measured by nuclear run-on assays. We transfected RAW 264.7 macrophages with a construct containing the iNOS promoter fused to the chloramphenicol acetyl transferase gene. DHA inhibited activation of this promoter by IFN gamma plus LPS. By inhibiting iNOS transcription in the fetus and neonate, DHA may contribute to their increased susceptibility to infection.
Critical amino acids in the lymphocyte function-associated antigen-1 I domain mediate intercellular adhesion molecule 3 binding and immune function.
Authors: van Kooyk Y , Binnerts M E , Edwards C P , Champe M , Berman P W , Figdor C G , Bodary S C
Published: June 2004

We have identified amino acid residues within the evolutionarily conserved I domain of the alpha-chain (CD11a) of the leukocyte integrin leukocyte function-associated antigen (LFA) 1 that are critical for intercellular adhesion molecule (ICAM) 3 (CD50) binding. ICAM-3, a ligand of LFA-1, is thought to mediate intercellular adhesion essential for the initiation of immune responses. Using a panel of human/murine I domain chimeras and point mutants, we observed that the Ile-Lys-Gly-Asn motif, located in the NH2-terminal part of the CD11a I domain, is required for ICAM-3 but not ICAM-1 binding. These findings demonstrate that the I domain of CD11a contains distinct functional subdomains for ligand specific binding. An aspartic acid located at position 137, which is essential to ICAM-1/LFA-1 interactions (Edwards, C.P., M. Champe, T. Gonzalez, M.E. Wessinger, S.A. Spencer, L.G. Presta, P.W. Berman, and S.C. Bodary. 1995. J. Biol. Chem. 270:12635-12640), was also critical for ICAM-3 binding, whereas Ser at position 139 did not effect ICAM-1 or ICAM-3 binding. A synthetic peptide containing the Ile-Lys-Gly-Asn motif inhibited ICAM-3-dependent adhesion and proliferation of T cells at micromolar concentrations, suggesting that this peptide interferes with immune recognition. These observations underscore the importance of ICAM-3 in leukocyte function, and may lead to development of a new category of immunosuppressive agents.
An immunoglobulin E-dependent recombinant histamine-releasing factor induces interleukin-4 secretion from human basophils.
Authors: Schroeder J T , Lichtenstein L M , MacDonald S M
Published: June 2004

A novel recombinant histamine-releasing factor (rHFR), which stimulates secretion from a subpopulation of human basophils that express a particular type of immunoglobulin E (IgE) or IgE+, was found to induce interleukin-4 (IL-4) production from cells isolated from these same donors. The secretion of IL-4 protein induced by rHRF significantly correlated with histamine release and the amount of protein generated, and the kinetics were identical to those caused by anti-IgE activation. Furthermore, the ability of rHRF to induce IL-4 protein production from cells not normally responsive to this protein was transferred by passive sensitization with plasma containing IgE+ antibody. That this novel protein stimulates both mediator release and the secretion of IL-4 protein from human basophils suggests a prominent role for this molecule in allergic disease.
Low zone tolerance to contact allergens in mice: a functional role for CD8+ T helper type 2 cells.
Authors: Steinbrink K , Sorg C , Macher E
Published: June 2004

Normal skin is permeable to low molecular hydrophobic substances, including allergenic chemicals. Whereas such foreign matter appears to enter the skin naturally, it rarely induces contact hypersensitivity. This suggests that immunological tolerance would be the normal state of affairs. In search of a suitable model, we painted picryl chloride or oxazolone once or repeatedly on normal skin of BALB/c or C57B1/6 mice and found subsensitizing doses to be tolerogenic. The most effective doses in inducing tolerance were doses between those at the point of inflection from no responses to threshold sensitivity. But even doses three orders of magnitude lower than these suppressed subsequent sensitization if applied repeatedly. C57B1/6 mice (low responders) were consistently easier to make tolerant than BALB/c mice (high responders). The tolerant state established by a single painting was found to be fully developed at 48 h after initiation and long-lasting (>14 d). It could be adoptively transferred by intravenous injection of total spleen cells (SC), lymph node cells (LNC), or purified T cells and shown to be hapten specific. Pretreatment with cyclophosphamide (Cy) prevented tolerization. The T cells capable of transferring suppressive activity were found to be generated irrespective of the dose applied. On day 2 after painting, tolerance could be transferred with LNC from both tolerant and sensitized animals. On day 5, however, only cells from tolerant donors transferred tolerance. But by action of Cy, suppression was shown to be part of every sensitization, although masked. Production of hapten-specific antibodies was suppressed as well. Through depletion by monoclonal antibody in vitro the T suppressor cells were shown to belong to the murine CD8+ subset (Lyt2+). Upon restimulation in vitro by haptenized and irradiated normal SC, LNC from tolerant donors produced predominantly interleukin (IL)-4, IL-5, and IL-10. In contrast, LNC from sensitized donors produced preferentially IL-2 and interferon-gamma. Thus we demonstrate that painting subsensitizing doses of contact sensitizers on normal murine skin generates CD8+ Th2-like cells that give rise to hapten-specific tolerance. The model may have broader significance and apply to other species, including humans.