bluuebunny/crossref_metadata_2025 huggingface dataset:
Major histocompatibility complex class II-associated p41 invariant chain fragment is a strong inhibitor of lysosomal cathepsin L.
Authors: Bevec T , Stoka V , Pungercic G , Dolenc I , Turk V
Published: June 2004

The invariant chain (Ii) is associated with major histocompatibility complex class II molecules during early stages of their intracellular transport. In an acidic endosomal/lysosomal compartment, it is proteolytically cleaved and removed from class II heterodimers. Participation of aspartic and cysteine proteases has been observed in in vitro degradation of Ii, but the specific enzymes responsible for its in vivo processing are as yet undefined. We have previously isolated a noncovalent complex of the lysosomal cysteine protease cathepsin L with a peptide fragment derived from the p41 form of Ii from human kidney. Here we show that this Ii fragment, which is identical to the alternatively spliced segment of p41, is a very potent competitive inhibitor of cathepsin L (equilibrium inhibition constant Ki = 1.7 X 10(-12) M). It inhibits two other cysteine proteases, cathepsin H and papain, but to much lesser extent. Cysteine proteases cathepsins B, C, and S, as well as representatives of serine, aspartic, and metalloproteases, are not inhibited at all. These findings suggest a novel role for p41 in the regulation of various proteolytic activities during antigen processing and presentation. The Ii inhibitory fragment shows no sequence homology with the known cysteine protease inhibitors, and may, therefore, represent a new class.
CD4+ T cell activation and tolerance induction in B cell knockout mice.
Authors: Phillips J A , Romball C G , Hobbs M V , Ernst D N , Shultz L , Weigle W O
Published: June 2004

B cell knockout mice microMT/microMT were used to examine the requirement for B cell antigen (Ag) presentation in the establishment of CD4+ T cell tolerance. CD4+T cells from microMT mice injected with exogenous protein Ag in adjuvant responded to in vitro challenge by transcription of cytokine mRNA, cytokine secretion, and proliferation. Peripheral tolerance could be established in microMT mice with a single dose of deaggragated protein. This tolerance was manifested by a loss of T cell proliferation and cytokine production (including both T helper cell type 1 [Th1]- and Th2-related cytokines), indicating that B cells are not required for the induction of peripheral T cell tolerance and suggesting that the dual zone tolerance theory is not applicable to all protein Ags and is not mediated through Ag presentation by B cells.
Impaired neutrophil response and CD4+ T helper cell 1 development in interleukin 6-deficient mice infected with Candida albicans.
Authors: Romani L , Mencacci A , Cenci E , Spaccapelo R , Toniatti C , Puccetti P , Bistoni F , Poli V
Published: June 2004

To define the role of interleukin (IL)6 in Candida albicans infection, IL-6 deficient mice were assessed for susceptibility to systemic or gastrointestinal infection, as well as for parameters of elicited T helper cell (Th) immunity. IL-6-deficient mice were more susceptible than wild-type mice to either type of infection caused by virulent C. albicans. In response to systemic challenge with a live vaccine strain of yeast, IL-6-deficient mice failed to mount Th1-associated protective immunity, but the resulting Th2-biased response could be redirected to the Th1 phenotype by IL-10 neutralization. Severe impairment of the macrophage and neutrophil response to infection was observed in IL-6-deficient mice, but administration of IL-6 would increase both neutrophil response and resistance to infection. IL-6 seems to oppose the Th2-promoting role of IL-10 in candidiasis, its early regulatory activity involving effects on neutrophil function.
Therapy of murine tumors with p53 wild-type and mutant sequence peptide-based vaccines.
Authors: Mayordomo J I , Loftus D J , Sakamoto H , De Cesare C M , Appasamy P M , Lotze M T , Storkus W J , Appella E , DeLeo A B
Published: June 2004

The BALB/c Meth A sarcoma carries a p53 missense mutation at codon 234, which occurs in a peptide, termed 234CM, capable of being presented to cytotoxic T lymphocytes (CTL) by H-2Kd molecules (Noguchi, Y., E.C. Richards, Y.-T. Chen, and L.J. Old. 1994. Proc. Natl. Acad. Sci. USA. 91:3171-3175). Immunization of BALB/c mice with bone marrow-derived dendritic cells (DC), generated in the presence of granulocyte macrophage colony-stimulating factor and interleukin 4, and prepulsed with the Meth A p53 mutant peptide, induced CTL that specifically recognized peptide-pulsed P815 cells, as well as Meth A cells naturally expressing this epitope. Immunization with this vaccine also protected naive mice from a subsequent tumor challenge, and it inhibited tumor growth in mice bearing day 7 subcutaneous Meth A tumors. We additionally determined that immunization of BALB/c mice with DC pulsed with the p53 peptide containing the wild-type residue at position 234, 234CW, induced peptide-specific CTL that reacted against several methylcholanthrene-induced BALB/c sarcomas, including CMS4 sarcoma, and rejection of CMS4 sarcoma in vaccination and therapy (day 7) protocols. These results support the efficacy of DC-based, p53-derived peptide vaccines for the immunotherapy of cancer. The translational potential of this strategy is enhanced by previous reports showing that DC can readily be generated from human peripheral blood lymphocytes.
Concurrent engagement of CD40 and the antigen receptor protects naive and memory human B cells from APO-1/Fas-mediated apoptosis.
Authors: Lagresle C , Mondière P , Bella C , Krammer P H , Defrance T
Published: June 2004

Naive and memory B cells were isolated from human tonsils and examined for expression of APO-1/Fas and for their sensitivity to the APO-1-dependent apoptosis. APO-1 was found to be constitutively expressed on memory but not on naive B cells. The susceptibility of both cell types to the APO-1 apoptotic pathway was acquired upon CD40 triggering and was correlated with increased expression of the APO-1 receptor. Both naive and memory B cells were protected from the APO-1-mediated death signal after dual ligation of the Ag receptor adn CD40. Our findings suggest that the APO-1 pathway controls the specificity of B cell responses to T-dependent Ags and that occupancy of the Ag receptor dictates the outcome of APO-1-ligation on B cell survival.
The function of the soluble interleukin 6 (IL-6) receptor in vivo: sensitization of human soluble IL-6 receptor transgenic mice towards IL-6 and prolongation of the plasma half-life of IL-6.
Authors: Peters M , Jacobs S , Ehlers M , Vollmer P , Müllberg J , Wolf E , Brem G , Meyer zum Büschenfelde K H , Rose-John S
Published: June 2004

Interleukin 6 (IL-6) is considered an important mediator of acute inflammatory responses. Moreover, IL-6 functions as a differentiation and growth factor of hematopoietic precursor cells, B cells, T cells, keratinocytes, neuronal cells, osteoclasts, and endothelial cells. IL-6 exhibits its action via a receptor complex consisting of a specific IL-6 receptor (IL-6R) and a signal transducing subunit (gp130). Soluble forms of both receptor components are generated by shedding and are found in patients with various diseases such as acquired immune deficiency syndrome, rheumatoid arthritis, and others. The function of the soluble (s)IL-6R in vivo is unknown. Since human (h)IL-6 acts on human and murine target cells, but murine IL-6 on murine cells only, we constructed transgenic mice expressing the hsIL-6R. We report here that in the presence of hsIL-6R, mice are hypersensitized towards hIL-6, mounting an acute phase protein gene induction at significantly lower IL-6 dosages compared to control animals. Furthermore, in hsIL-6R transgenic mice, the detected acute phase response persists for a longer period of time. The IL-6/IL-6R complex prolongs markedly the Il-6 plasma half-life. Our results reinforce the role of the hsIL-6R as an agonistic protein, help to understand the function of the hsIL-6R in vivo, and highlight the significance of the receptor in the induction of the acute phase response.
Requirement of Lyn and Syk tyrosine kinases for the prevention of apoptosis by cytokines in human eosinophils.
Authors: Yousefi S , Hoessli D C , Blaser K , Mills G B , Simon H U
Published: June 2004

In allergic diseases, the cytokines interleukin (IL)5 and granulocyte/macrophage colony-stimulating factor (GM-CSF) are upregulated and have been proposed to cause blood and tissue eosinophilia by inhibition of eosinophil apoptosis. We demonstrate herein, in freshly isolated human eosinophils, that the IL-3/IL-5/GM-CSF receptor beta subunit interacts with cytoplasmic tyrosine kinases to induce phosphorylation of several cellular substrates, including the beta subunit itself. The Lyn and Syk intracellular tyrosine kinases constitutively associate at a low level with the IL-3/IL-5/GM-CSF receptor beta subunit in human eosinophils. Stimulation with GM-CSF or IL-5 results in a rapid and transient increase in the amount of Lyn and Syk associated with the IL-3/IL-5/GM-CSF receptor beta subunit. Lyn is required for optimal tyrosine phosphorylation and activation of Syk. In contrast, Syk is not required for optimal tyrosine phosphorylation and activation of Lyn. These data suggest that Lyn is proximal to Syk in a tyrosine kinase cascade that transduces IL-3, IL-5, or GM-CSF signals. Compatible with this model, both Lyn and Syk are essential for the activation of the antiapoptotic pathway(s) induced through the IL-3/IL-5/GM-CSF receptor beta subunit in human eosinophils.
LFA-1-deficient mice show normal CTL responses to virus but fail to reject immunogenic tumor.
Authors: Schmits R , Kündig T M , Baker D M , Shumaker G , Simard J J , Duncan G , Wakeham A , Shahinian A , van der Heiden A , Bachmann M F , Ohashi P S , Mak T W , Hickstein D D
Published: June 2004

The leukocyte integrin LFA-1 (CD11a/CD18) plays an important role in lymphocyte recirculation and homotypic interactions. Leukocytes from mice lacking CD11a displayed defects in in vitro homotypic aggregation, in proliferation in mixed lymphocyte reactions, and in response to mitogen. Mutant mice mounted normal cytotoxic T cell (CTL) responses against systemic LCMV and VSV infections and showed normal ex vivo CTL function. However, LFA-1-deficient mice did not reject immunogenic tumors grafted into footpads and did not demonstrate priming response against tumor-specific antigen. Thus CD11a deficiency causes a selective defect in induction of peripheral immune responses whereas responses to systemic infection are normal.
Different superantigens interact with distinct sites in the Vbeta domain of a single T cell receptor.
Authors: Hong S C , Waterbury G , Janeway C A
Published: June 2004

CD4 T cell receptors (TCRs) recognize antigenic peptides presented by self major histocompatibility complex (MHC) class II molecules as well as non-self MHC class II molecules. The TCRs can also recognize endogenous retroviral gene products and bacterial toxins known collectively as superantigens (SAGs) that act mainly on the Vbeta gene segment-encoded portion of the Vbeta domain; most SAGs also require MHC II class for presentation. We have studied the interaction of the TCR from a well-characterized CD4 T cell line with SAGs by mutational analysis of its Vbeta domain. This appears to separate viral (v)SAG from bacterial (b)SAG recognition. T cells having a TCR with glycine to valine mutation in amino acid residue 51 (G51V) in complementarity determining region 2 of the TCR Vbeta domain fail to respond the bSAGs staphylococcal enterotoxin B (SEB), SEC1, SEC2, and SEC3, whereas they retain the ability to respond to non-self MHC class II molecules and to foreign peptides presented by self MHC class II molecules. It is interesting to note that T cells expressing mutations of both G51V and G53D of V beta regain the response to SEB and partially that to SEC1, but do not respond to SEC2, and SEC3, suggesting that different bacterial SAGs are viewed differently by the same TCR. These results are surprising, because it has been generally believed that SAG recognition by T cells is mediated exclusively by hypervariable region 4 on the exposed, lateral face of the TCR Vbeta domain. Response to the vSAG Mtv-7 was generated by mutation in Vbeta residue 24 (N24H), confirming previously published data. These data show that the vSAG Mtv-7 and bSAGs are recognized by different regions of the TCR Vbeta domain. In addition, various bSAGs are recognized differently by the same TCR. Thus, these mutational data, combined with the crystal structure of the TCR beta chain, provide evidence for distinct recognition sites for vSAG and bSAG.
The role of interleukin 12 and nitric oxide in the development of spontaneous autoimmune disease in MRL/MP-lpr/lpr mice.
Authors: Huang F P , Feng G J , Lindop G , Stott D I , Liew F Y
Published: June 2004

MRL/MP-lpr/lpr (MRL/lpr) mice develop a spontaneous autoimmune disease. Serum from these mice contained significantly higher concentrations of nitrite/nitrate than serum from age-matched control MRL/MP-+/+ (MRL/+), BALB/c or CBA/6J mice. Spleen and peritoneal cells from MRL/lpr mice also produced significantly more nitric oxide (NO) than those from the control mice when cultured with interferon (IFN) gamma and lipopolysaccharide (LPS) in vitro. It is interesting to note that peritoneal cells from MRL/lpr mice also produced markedly higher concentrations of interleukin (IL) 12 than those from MRL/+ or BALB/c mice when cultured with same stimuli. It is striking that cells from MRL/lpr mice produced high concentrations of NO when cultured cells from MRL/+ or BALB/c mice. The enhanced NO synthesis induced by IFN-gamma/LPS was substantially inhibited by anti-IL-12 antibody. In addition, IL-12-induced NO production can also be markedly inhibited by anti-IFN-gamma antibody, but only weakly inhibited by anti-tumor necrosis factor alpha antibody. The effect of IL-12 on NO production was dependent on the presence of natural killer and possibly T cells. Serum from MRL/lpr mice contained significantly higher concentrations of IL-12 compared with those of MRL/+ or BALB/c control mice. Daily injection of recombinant IL-12 led to increased serum levels of IFN-gamma and NO metabolites, and accelerated glomerulonephritis in the young MRL/lpr mice (but not in the MRL/+ mice) compared with controls injected with phosphate-buffered saline alone. These data, together with previous finding that NO synthase inhibitors can ameliorate autoimmune disease in MRL/lpr mice, suggest that high capacity of such mice to produce IL-12 and their greater responsiveness to IL-12, leading to the production of high concentrations of NO, are important factors in this spontaneous model of autoimmune disease.
Chronic inflammation caused by lymphotoxin is lymphoid neogenesis.
Authors: Kratz A , Campos-Neto A , Hanson M S , Ruddle N H
Published: June 2004

In presenting a unifying concept for chronic inflammation and lymphoid organogenesis, we suggest that lymphotoxin's (LT, LT-alpha, TNF-beta) crucial role in these processes is pivotal and similar. Chronic inflammatory lesions that developed in the kidney and pancreas at the sites of transgene expression in rat insulin promoter-LT (RIP-LT) mice resembled lymph nodes with regard to cellular composition (T cells, B cells, plasma cells, and antigen-presenting cells), delineated T and B cell areas, primary and secondary follicles, characteristic morphologic and antigenic (ICAM-1, VCAM-1, MAdCAM-1, and PNAd) features of high endothelial venules, and ability to respond to antigen and undergo Ig class switching when obtained from mice immunized with SRBC. The vascular changes, with the exception of PNAd, appear to be the direct consequence of transgene derived LT expression, as they were also observed in RIP-LT mice lacking mature T and B cells. These data show that LT-induced chronic inflammation has the characteristics of organized lymphoid tissue.
Resistance to cutaneous graft-vs.-host disease is not induced in T cell receptor delta gene-mutant mice.
Authors: Shiohara T , Moriya N , Hayakawa J , Itohara S , Ishikawa H
Published: June 2004

The function of murine dendritic epidermal cells (dEC) remains largely speculative, probably because of the lack of a suitable in vivo model, although previous studies suggest that gamma/delta+ dEC may have originally evolved to serve as a self-protection mechanism(s). Our previous study demonstrated that the epidermis of mice that had spontaneously recovered from cutaneous graft-vs-host disease (GVHD) induced by local injection of CD4+ autoreactive T cells contained unexpectedly large numbers of dEC and became resistant to subsequent attempts to induce GVHD in a site-restricted manner, suggesting that the resistance is mediated by dEC. However, because alpha/beta+ dEC as well as gamma/delta+ dEC were greatly increased in number in the epidermis, it was unclear whether gamma/delta+ dEC are indeed responsible for this protection. The availability of this murine model and mice selectively lacking gamma/delta T cells as a result of disruption of the T cell receptor C delta gene segment allowed us to investigate the role of gamma/delta+ dEC. In the epidermis of gamma/delta T cell-deficient mice (delta-/-), a congenital lack of gamma/delta+ dEC was substituted for by alpha/beta+ dEC of either a CD4-8+ or a CD4-8- phenotype. After intradermal injection of the autoreactive T cells, delta-/- mice developed significantly enhanced delayed-type hypersensitivity responses and cutaneous GVHD, which persisted longer than in heterozygous littermate controls (delta+/-). Surprisingly, resistance to the cutaneous GVHD was not induced in the epidermis of delta-/- mice after spontaneous recovery from the GVHD, whereas the "susceptible" epidermis of delta-/+ mice contained large numbers of alpha/beta dEC comparable to those in "resistant" epidermis of delta+/- mice. Injection of day 16 fetal thymocytes from wild-type mice into delta-/- mice resulted in the appearance of donor-type gamma/delta+ dEC in the epidermis, and reconstitution with gamma/delta+ dEC restored the protective immune response of the epidermis against the GVHD to nearly normal levels. These results indicate that gamma/delta+ dEC are responsible for the site-restricted protection against cutaneous GVHD.
Exochelins of Mycobacterium tuberculosis remove iron from human iron-binding proteins and donate iron to mycobactins in the M. tuberculosis cell wall.
Authors: Gobin J , Horwitz M A
Published: June 2004

To multiply and cause disease in the host, Mycobacterium tuberculosis must acquire iron from the extracellular environment at sites of replication. To do so, the bacterium releases high-affinity iron-binding siderophores called exochelins. In previous studies, we have described the purification and characterization of the exochelin family of molecules. These molecules share a common core structure with another type of high-affinity iron-binding molecule located in the cell wall of M. tuberculosis: the mycobactins. The water-soluble exochelins differ from each other and from water insoluble mycobactins in polarity, which is dependent primarily upon the length and modifications of an alkyl side chain. In this study, we have investigated the capacity of purified exochelins to remove iron from host high-affinity iron-binding molecules, and to transfer iron to mycobactins. Purified desferri-exochelins rapidly removed iron from human transferrin, whether it was 95 or 40% iron saturated, its approximate percent saturation in human serum, and from human lactoferrin. Desferri-exochelins also removed iron, but at a slower rate, from the iron storage protein ferritin. Purified ferri-exochelins, but not iron transferrin, transferred iron to desferri-mycobactins in the cell wall of live bacteria. To explore the possibility that the transfer iron from exochelins to mycobactins was influenced by their polarity, we investigated the influence of polarity on the iron affinity of exochelins. Exochelins of different polarity exchanged iron equally with each other. This study supports the concept that exochelins acquire iron for M. tuberculosis by removing this element from host iron-binding proteins and transferring it to desferri-mycobactins in the cell wall of the bacterium. The finding that ferri-exochelins but not iron transferrin transfer iron to mycobactins in the cell wall underscores the importance of exochelins in iron acquisition. This study also shows that the variable alkyl side chain on the core structure of exochelins and mycobactins, the principal determinant of their polarity, has little or no influence on their iron affinity.
Mitochondrial control of nuclear apoptosis.
Authors: Zamzami N , Susin S A , Marchetti P , Hirsch T , Gómez-Monterrey I , Castedo M , Kroemer G
Published: June 2004

Anucleate cells can be induced to undergo programmed cell death (PCD), indicating the existence of a cytoplasmic PCD pathway that functions independently from the nucleus. Cytoplasmic structures including mitochondria have been shown to participate in the control of apoptotic nuclear disintegration. Before cells exhibit common signs of nuclear apoptosis (chromatin condensation and endonuclease-mediated DNA fragmentation), they undergo a reduction of the mitochondrial transmembrane potential (delta psi m) that may be due to the opening of mitochondrial permeability transition (PT) pores. Here, we present direct evidence indicating that mitochondrial PT constitutes a critical early event of the apoptotic process. In a cell-free system combining purified mitochondria and nuclei, mitochondria undergoing PT suffice to induce chromatin condensation and DNA fragmentation. Induction of PT by pharmacological agents augments the apoptosis-inducing potential of mitochondria. In contrast, prevention of PT by pharmacological agents impedes nuclear apoptosis, both in vitro and in vivo. Mitochondria from hepatocytes or lymphoid cells undergoing apoptosis, but not those from normal cells, induce disintegration of isolated Hela nuclei. A specific ligand of the mitochondrial adenine nucleotide translocator (ANT), bongkreik acid, inhibits PT and reduces apoptosis induction by mitochondria in a cell-free system. Moreover, it inhibits the induction of apoptosis in intact cells. Several pieces of evidence suggest that the proto-oncogene product Bcl-2 inhibits apoptosis by preventing mitochondrial PT. First, to inhibit nuclear apoptosis, Bcl-2 must be localized in mitochondrial but not nuclear membranes. Second, transfection-enforced hyperexpression of Bcl-2 directly abolishes the induction of mitochondrial PT in response to a protonophore, a pro-oxidant, as well as to the ANT ligand atractyloside, correlating with its apoptosis-inhibitory effect. In conclusion, mitochondrial PT appears to be a critical step of the apoptotic cascade.
The requirement for proteasome activity class I major histocompatibility complex antigen presentation is dictated by the length of preprocessed antigen.
Authors: Yang B , Hahn Y S , Hahn C S , Braciale T J
Published: June 2004

Accumulating evidence has implicated the proteasome in the processing of protein along the major histocompatibility complex (MHC) class I presentation pathway. The availability of potent proteasome inhibitors provides an opportunity to examine the role of proteasome function in antigen presentation by MHC class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). We have investigated the processing and presenting of antigenic epitopes from influenza hemagglutinin in target cells treated with the inhibitor of proteasome activity MG132. In the absence of proteasome activity, the processing and presentation of the full-length hemagglutinin was abolished, suggesting the requirement for proteasome function in the processing and presentation of the hemagglutinin glycoprotein. Epitope-containing translation products as short as 21 amino acids when expressed in target cells required proteasome activity for processing and presentation of the hemagglutin epitope to CTLs. However, when endogenous peptides of 17 amino acids or shorter were expressed in target cells, the processing and presentation of epitopes contained in these peptides were insensitive to the proteasome inhibitor. Our results support the hypothesis that proteasome activity is required for the generation of peptides presented by MHC class I molecules and that the requirement for proteasome activity is dependent on the size of the translation product expressed in the target cell. The implications of these findings are discussed.
Nasal administration of glutamate decarboxylase (GAD65) peptides induces Th2 responses and prevents murine insulin-dependent diabetes.
Authors: Tian J , Atkinson M A , Clare-Salzler M , Herschenfeld A , Forsthuber T , Lehmann P V , Kaufman D L
Published: June 2004

We previously demonstrated that a spontaneous Th1 response against glutamate decarboxylase (GAD65) arises in NOD mice at four weeks in age and subsequently T cell autoimmunity spreads both intramolecularly and intermolecularly. Induction of passive tolerance to GAD65, through inactivation of reactive T cells before the onset of autoimmunity, prevented determinant spreading and the development of insulin-dependent diabetes mellitus (IDDM). Here, we examined whether an alternative strategy, designed to induce active tolerance via the engagement of Th2 immune responses to GAD65, before the spontaneous onset of autoimmunity, could inhibit the cascade of Th1 responses that lead to IDDM. We observed that a single intranasal administration of GAD65 peptides to 2-3-wk-old NOD mice induced high levels of IgG1 antibodies to GAD65. GAD65 peptide treated mice displayed greatly reduced IFN gamma responses and increased IL-5 responses to GAD65, confirming the diversion of the spontaneous GAD65 Th1 response toward a Th2 phenotype. Consistent with the induction of an active tolerance mechanism, splenic CD4+ (but not CD8+) T cells from GAD65 peptide-treated mice, inhibited the adoptive transfer of IDDM to NOD-scid/scid mice. This active mechanism not only inhibited the development of proliferative T cell responses to GAD65, it also limited the expansion of autoreactive T cell responses to other beta cell antigens (i.e., determinant spreading). Finally, GAD65 peptide treatment reduced insulitis and long-term IDDM incidence. Collectively, these data suggest that the nasal administration of GAD65 peptides induces a Th2 cell response that inhibits the spontaneous development of autoreactive Th1 responses and the progression of beta cell autoimmunity in NOD mice.
Localization of the binding site for the monocyte immunoglobulin (Ig) A-Fc receptor (CD89) to the domain boundary between Calpha2 and Calpha3 in human IgA1.
Authors: Carayannopoulos L , Hexham J M , Capra J D
Published: June 2004

Immunoglobulin (Ig) A serves as the first line of humoral defense at all mucosal surfaces and is present in large quantities of blood. In playing its role in humoral immunity, IgA interacts with a variety of effector molecules present both in serum and on the surfaces of immune and inflammatory cells. To study these interactions, we previously established expression of human IgA1 in insect cells using recombinant baculoviruses and showed that the expressed antibody is a structurally and functionally intact polypeptide useful for examining the molecular properties of IgA. Indeed, since the C alpha 2 N-linked glycosylation site lies near the Fab-distal pole of C alpha 2, the inability of a mutant IgA1 lacking C alpha 2 N-glycosylation to bind its cognate receptor suggested that the monocyte Fc alpha receptor (mFcalphaR) recognizes IgA at a hinge-distal site encompassing the boundary between the C alpha 2 and C alpha 3 domains. In this report, we utilize both domain-swapped IgA/IgG and point-mutated IgA chimeras to verify the above hypothesis. Using an antigen-specific rosetting assay and a mFc alpha R-expressing cell line, we show that (a) C alpha 2 and C alpha 3 together are necessary and sufficient for binding; (b) neither the IgA hinge nor the tailpiece is necessary for binding; (c) mutations away from the interdomain boundary do not affect binding; and (d) mutations located near the three-dimensional boundary between C alpha 2 and C alpha 3 completely disrupt binding. Taken together, these results localize the mFc alpha R recognition site on IgA to the boundary region between the second and third constant domains--a site analogous to that recognized by Staphylococcus aureus protein A on IgG. The use of this hinge-distal site is, to date, unique among Fc receptors of the Ig superfamily.
Major histocompatibility complex class II-expressing endothelial cells induce allospecific nonresponsiveness in naive T cells.
Authors: Marelli-Berg F M , Hargreaves R E , Carmichael P , Dorling A , Lombardi G , Lechler R I
Published: June 2004

The role of endothelial cells (EC) in initiating a primary T cell response is of importance in clinical transplantation and autoimmunity since EC are the first allogeneic target encountered by the recipient's immune system and may display tissue-specific autoantigens in the context of an inflammatory response. In this study, we have investigated the antigen-presenting cell function of human umbilical vein-derived EC (HUVEC), depleted of constitutively major histocompatibility complex class II+ cells and induced to express class II molecules by interferon-gamma. The results show that HUVEC do not express B7 but can support proliferation by antigen-specific T cell clones. In contrast, they were unable to initiate a primary alloresponse using three independent HUVEC cultures and MHC class II-mismatched CD4+ T cells from eight donors. The response to HUVEC was reconstituted by trans-costimulation provided by DAP.3 transfectants expressing human B7.1. Coculture of peripheral blood T cells with EC expressing allogeneic DR molecules had markedly different effects on CD45RO+ and RA+ subsets. Subsequent reactivity of the RO+ T cells was unaffected by exposure to EC, indicating a neutral encounter. In contrast, culture with DR+ EC induced allospecific nonresponsiveness in RA+ T cells.
Rabies superantigen as a Vbeta T-dependent adjuvant.
Authors: Astoul E , Lafage M , Lafon M
Published: June 2004

Recently we reported evidence that nucleocapsid (NC) of rabies virus is a Vbeta8-specific exogenous superantigen (SAg) in humans and a Vbeta6-specific SAg in BALB/c mice. NC was also found to stimulate rabies vaccination by enhancing the rabies neutralizing antibody response. In this study, we tested the hypothesis that the stimulating effect of NC and its SAg properties are linked. To do this, we studied the effect of rabies SAg on the immune response to an unrelated antigen, the influenza virus, and compared the response in two congenic strains of mice, BALB/c and BALB/D2. BALB/c mice are rabies SAg responsive, whereas BALB/D2 mice are not responsive to SAg activation by rabies NC because they lack the SAg recognition element, the Vbeta6 T cell receptor. In BALB/c mice, coinjection of rabies SAg with inactivated influenza virus resulted in a rapid and long-term increase in (a) the titres of influenza virus-specific antibodies (IgG and IgM), including protective hemagglutination-inhibiting antibodies, (b) antigen-specific proliferation and, (c) IL-2 and IL-4 secretion by lymph node lymphocytes, when compared to mice that received influenza virus only. In contrast, in BALB/D2 mice, neither antibody nor lymphocyte responses were stimulated. Moreover, during establishment of the primary response, the increase in influenza-primed T cells was mainly restricted to those bearing a Vbeta6 TCR. These data establish that rabies SAg can stimulate both T and B cell-specific responses to an unrelated antigen, depending on expression of the SAg target (Vbeta6 T lymphocytes). This is the first report linking NC adjuvant properties with its SAg mechanism.
Breaking self-tolerance in nonobese diabetic mice.
Authors: Ridgway W M , Fassò M , Lanctot A , Garvey C , Fathman C G
Published: June 2004

Unresponsiveness to self is maintained through two mechanisms of immune regulation: thymic-negative selection and peripheral tolerance. Although thymic-negative selection is a major mechanism to eliminate self-reactive T cells, normal mice have readily detectable populations of T cells reactive to self-proteins but do not exhibit autoimmune responses. It has been postulated that autoimmune disease results from breakdown or loss of peripheral tolerance. We present data that demonstrate that peripheral tolerance or unresponsiveness to self can be broken in nonobese diabetic (NOD) mice. Immunization of NOD mice (but not of conventional mice) with self-peptides caused an immune response to self-peptide with resultant autoproliferation of peripheral lymphocytes. Autoproliferation of self-reactive T cells in NOD mice resulted from the recognition and proliferation of the activated T cells to endogenously processed and presented self-antigens. This loss of self-tolerance demonstrated in vitro may well be the basis of NOD autoimmune disease in vivo.
Distinct T cell receptor signaling requirements for perforin- or FasL-mediated cytotoxicity.
Authors: Esser M T , Krishnamurthy B , Braciale V L
Published: June 2004

A diverse array of signals are generated in a cytotoxic T lymphocyte (CTL) after the T cell receptor (TCR) engages the class I major histocompatibility complex (MHC) peptide complex. These signals result in a multitude of CTL effector functions, including cellular cytotoxicity, cell surface receptor expression, and cytokine secretion. We have examined signaling through the TCR in a wild type CD8+, MHC-restricted, antigen-specific CTL clone, 14-7, and its interleukin 2-dependent variant clone 14-7FD. We report here that 14-7FD is unable to kill via the perforin mechanism of killing, yet is able to kill via the Fas ligand/Fas mechanism and secrete interferon-gamma in an antigen-specific manner. 14-7FD has cytolytic granules that contain perforin and serine esterases, which are secreted after phorbol ester and Ca2+ ionophore treatment. Lastly, to investigate which TCR signaling requirements were operational in 14-7FD, we examined TCR-triggered intracellular Ca2+ mobilization in the two clones. After TCR engagement, 14-7FD failed to mobilize intracellular Ca2+, which may be the cause for its inability to trigger the perforin/granule exocytosis mechanism of killing. These results indicate that the signal transduction events that trigger perforin killing and the signaling requirements to induce FasL expression are distinct. We hypothesize that these two distinct TCR signal transduction requirements allow for separate activation of these two mechanisms of killing relating to their role in eradication of infected cells or regulation of immune responses.
CD45-null transgenic mice reveal a positive regulatory role for CD45 in early thymocyte development, in the selection of CD4+CD8+ thymocytes, and B cell maturation.
Authors: Byth K F , Conroy L A , Howlett S , Smith A J , May J , Alexander D R , Holmes N
Published: June 2004

The CD45 transmembrane glycoprotein has been shown to be a protein phosphotyrosine phosphatase and to be important in signal transduction in T and B lymphocytes. We have employed gene targeting to create a strain of transgenic mice that completely lacks expression of all isoforms of CD45. The spleens from CD45-null mice contain approximately twice the number of B cells and one fifth the number of T cells found in normal controls. The increase in B cell numbers is due to the specific expansion of two B cell subpopulations that express high levels of immunoglobulin (IgM) staining. T cell development is significantly inhibited in CD45-null animals at two distinct stages. The efficiency of the development of CD4-CD8- thymocytes into CD4+ CD8+ thymocytes is reduced by twofold, subsequently the frequency of successful maturation of the double positive population into mature, single positive thymocytes is reduced by a further four- to fivefold. In addition, we demonstrate that CD45-null thymocytes are severely impaired in their apoptotic response to cross-linking signals via T cell receptor (TCR) in fetal thymic organ culture. In contrast, apoptosis can be induced normally in CD45-null thymocytes by non-TCR-mediated signals. Since both positive and negative selection require signals through the TCR complex, these findings suggest that CD45 is an important regulator of signal transduction via the TCR complex at multiple stages of T cell development. CD45 is absolutely required for the transmission of mitogenic signals via IgM and IgD. By contrast, CD45-null B cells proliferate as well as wild-type cells to CD40-mediated signals. The proliferation of B cells in response to CD38 cross-linking is significantly reduced but not abolished by the CD45-null mutation. We conclude that CD45 is not required at any stage during the generation of mature peripheral B cells, however its loss reveals a previously unrecognized role for CD45 in the regulation of certain subpopulations of B cells.
Requirement for CD8+ T cells in the development of airway hyperresponsiveness in a marine model of airway sensitization.
Authors: Hamelmann E , Oshiba A , Paluh J , Bradley K , Loader J , Potter T A , Larsen G L , Gelfand E W
Published: June 2004

To study the role of CD8+ T cells in allergic sensitization, we examined the effects of in vivo depletion of CD8+ T cells prior to sensitization on IgE production, immediate type cutaneous hypersensitivity and development of altered airway responsiveness. BALB/c mice were thymectomized and treated with anti-CD8 antibody resulting in depletion of CD8+ T cells (<1%) in spleen and lymphoid tissues. In these mice, sensitization to ovalbumin (OVA) via the airways still resulted in IgE anti-OVA responses and immediate cutaneous reactions to OVA, but the animals were unable to develop airway hyperresponsiveness, eosinophil infiltration of the lung parenchyma, or IL-5 production in the local lymph nodes of the airway. Transfer of CD8+ T cells from naive animals during sensitization (on day 8 of the 10-d protocol) fully restored the ability to develop airway hyperresponsiveness and this was accompanied by IL-5 production and eosinophil accumulation in the lung. These data indicate a critical role for CD8+ T cells in the production of IL-5 and the development of altered airway responsiveness after antigen sensitization through the airways.
The half-life of RAG-1 protein in precursor B cells is increased in the absence of RAG-2 expression.
Authors: Grawunder U , Schatz D G , Leu T M , Rolink A , Melchers F
Published: June 2004

Site-specific recombination of immunoglobulin and T cell receptor gene segments in B and T lymphocytes is dependent on the expression of two recombinant activation genes, Rag-1 and Rag-2. Here, we show that RAG-1 protein turnover in pre-B cells depends on the expression of RAG-2. The apparent half-life of RAG-1 protein is increased when RAG-2 is not expressed in differentiating pre-B cells.
Transcriptional regulation of the Icam-1 gene in antigen receptor- and phorbol ester-stimulated B lymphocytes: role for transcription factor EGR1.
Authors: Maltzman J S , Carmen J A , Monroe J G
Published: June 2004

Intercellular adhesion molecule (ICAM) 1/CD54 plays an important role in T cell dependent B cell activation and for function of B lymphocytes as antigen-presenting cells. ICAM-1 expression is upregulated as a consequence of B lymphocyte antigen receptor (BCR) signaling, thereby serving to render antigen-stimulated B cells more receptive to T cell-mediated costimulatory signals. We have investigated BCR-induced expression of the Icam-1 gene in primary B cells and B cell lines and have found it to be dependent on BCR-induced expression of the transcription factor EGR1. Icam-1 transcription, induced by BCR cross-linking or bypassing the BCR with phorbol ester, is absent in a B cell line in which the EGR1-encoding gene (egr-1) is methylated and not expressed. A potential EGR1-binding site was located at -701 bp upstream of the murine Icam-1 gene transcription start site and shown by electrophoretic mobility shift assay to bind to murine EGR1. Mutation of this site in the context of 1.1 kb of the Icam-1 promoter significantly abrogated transcriptional induction by phorbol ester and anti-mu stimulation in primary B cells. A direct effect of EGR1 on the Icam-1 promoter is suggested by the ability of EGR1 expressed from an SV40-driven expression vector transactivate the wild-type Icam-1 promoter, whereas mutation of the EGR1 mutation of the EGR1 binding motif at -701 bp markedly compromises this induction. These data identify EGR1 as a signaling intermediate in BCR-stimulated B cell functional responses, specifically linking BCR signal transduction to induction of the Icam-1 gene. Furthermore, similar findings for BCR-induced CD44 gene induction (Maltzman, J.S., J.A. Carman, and J.G. Monroe. 1996. Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes. Mol. Cell. Biol. In press) suggest that EGR1 may be an important signaling molecule for regulating levels of migration and adhesion molecules during humoral immune responses.
Patr-A and B, the orthologues of HLA-A and B, present hepatitis C virus epitopes to CD8+ cytotoxic T cells from two chronically infected chimpanzees.
Authors: Kowalski H , Erickson A L , Cooper S , Domena J D , Parham P , Walker C M
Published: June 2004

Common chimpanzees (Pan troglodytes) infected with hepatitis C virus (HCV) show a disease progression similar to that observed for human patients. Although most infected animals develop a chronic hepatitis, virus persistence is associated with an ongoing immune response, for which the beneficial or detrimental effects are uncertain. Lines of virus-specific cytotoxic CD8+ T lymphocytes (CTL) have been previously established from liver biopsies of two common chimpanzees chronically infected with HCV-1. The viral epitopes recognized by six lines of CTL have been defined using synthetic peptides and shown to consist of 8 to 9-residue peptides derived from various viral proteins. Five of the epitopes derive from sequences that vary among strains of HCV. The majority of the corresponding variant epitopes from different HCV strains were either recognized less efficiently or not at all by the CTL, suggesting their response may have limited potential for controlling replication of HCV variants. Complementary DNAs encoding class I alleles of the two common chimpanzees, Patr-A, -B, and -C were cloned, sequenced, and transfected individually into a class I-deficient human cell line. Analysis of peptide presentation by the class I transfectants to CTL identified the Patr class I allotypes that present the six epitopes defined here and an additional epitope defined previously. The assignment of epitopes to class I allotypes based upon analysis of the transfected cells correlates precisely with the segregation of antigen-presenting function within a panel of common chimpanzee cell lines and the expression of class I heavy chains as defined by isoelectric focusing. Five of the HCV-1 epitopes are presented by Patr-B allotypes, two epitopes are presented by a Patr-A allotype, and none is presented by Patr-C allotypes.
A predictable sequential determinant spreading cascade invariably accompanies progression of experimental autoimmune encephalomyelitis: a basis for peptide-specific therapy after onset of clinical disease.
Authors: Yu M , Johnson J M , Tuohy V K
Published: June 2004

The development of autoimmune disease is accompanied by the acquired recognition of new self-determinants, a process commonly referred to as determinant spreading. In this study, we addressed the question of whether determinant spreading is pathogenic for progression of chronic-relapsing experimental autoimmune encephalomyelitis (EAE), a disease with many similarities to multiple sclerosis (MS). Our approach involved a systematic epitope mapping of responses to myelin proteolipid protein (PLP) as well as assaying responses to known encephalitogenic determinants of myelin basic protein (MBP 87-89) and myelin oligodendrocyte glycoprotein (MOG 92-106) at various times after induction of EAE in (SWR X SJL)F1 mice immunized with PLP 139-151. We found that the order in which new determinants are recognized during the course of disease follows a predictable sequential pattern. At monthly intervals after immunization with p139-151, responses to PLP 249-273, MBP 87-99, and PLP 137-198 were sequentially accumulated in al mice examined. Three lines of evidence showed that determinant spreading is pathogenic for disease progression: (a) spreading determinants mediate passive transfer of acute EAE in naive (SWR X SJL)F1 recipients; (b) an invariant relationship exists between the development of relapse/progression and the spreading of recognition to new immunodominant encephalitogenic determinants; and (c) after EAE onset, the induction of peptide-specific tolerance to spreading but not to nonspreading encephalitogenic determinants prevents subsequent progression of EAE. Thus, the predictability of acquired self-determinant recognition provides a basis for sequential determinant-specific therapeutic intervention after onset of the autoimmune disease process.
A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis.
Authors: Süss G , Shortman K
Published: June 2004

Dendritic cells (DC), the most efficient antigen-presenting cells, are well equipped for activation of naive CD4+ T cells by their expression of high levels of major histocompatibility complex and costimulator molecules. We now demonstrate that some DC are equally well equipped for killing these same T cells. Murine splenic DC consist of both conventional CD8alpha- DC and a major population of CD8alpha+ DC. Whereas CD8- DC induce a vigorous proliferative response in CD4 T cells, CD8+ DC induce a lesser response that is associated with marked T cell apoptosis. By using various mixtures of T cells and DC from Fas-mutant lpr/lpr mice and Fas-ligand (FasL) mutant gld/gld mice, we show this death is due to interaction of Fas on activated T cells with FasL on CD8+ DC. Furthermore, we show by direct surface staining that CD8+ DC, but not CD8- DC, express FasL at high levels. These findings indicate that FasL+ CD8+ DC are a specialized subgroup of DC with a role in the regulation of the response of primary peripheral T cells.
Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.
Authors: Goodell M A , Brose K , Paradis G , Conner A S , Mulligan R C
Published: June 2004

Hematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow and replenish all adult hematopoietic lineages throughout the lifetime of the animal. While experimenting with staining of murine bone marrow cells with the vital dye, Hoechst 33342, we discovered that display of Hoechst fluorescence simultaneously at two emission wavelengths revealed a small and distinct subset of whole bone marrow cells that had phenotypic markers of multipotential HSC. These cells were shown in competitive repopulation experiments to contain the vast majority of HSC activity from murine bone marrow and to be enriched at least 1,000-fold for in vivo reconstitution activity. Further, these Hoechst-stained side population (SP) cells were shown to protect recipients from lethal irradiation at low cell doses, and to contribute to both lymphoid and myeloid lineages. The formation of the Hoechst SP profile was blocked when staining was performed in the presence of verapamil, indicating that the distinctly low staining pattern of the SP cells is due to a multidrug resistance protein (mdr) or mdr-like mediated efflux of the dye from HSC. The ability to block the Hoechst efflux activity also allowed us to use Hoechst to determine the DNA content of the SP cells. Between 1 and 3% of the HSC were shown to be in S-G2M. This also enabled the purification of the G0-G1 and S-G2M HSC had a reconstitution capacity equivalent to quiescent stem cells. These findings have implications for models of hematopoietic cell development and for the development of genetic therapies for diseases involving hematopoietic cells.