bluuebunny/crossref_metadata_2025 huggingface dataset:
p40, a novel surface molecule involved in the regulation of the non‐major histocompatibility complex‐restricted cytolytic activity in humans
Authors: Poggi Alessandro , Pella Nicoletta , Morelli Luigia , Spada Franca , Revello Valentino , Sivori Simona , Augugliaro Raffaella , Moretta Lorenzo , Moretta Alessandro
Published: March 2007

Four monoclonal antibodies (mAb) termed NKTA255, NKTA72, 1F1 and 1B1 were selected on the basis of their ability to inhibit the cytolytic activity of natural killer (NK) cell clones against P815 target cells. These mAb selectively reacted with normal or tumor cells of hematopoietic origin and displayed a cellular distribution similar to that of CD45 or CD11a/CD18 antigens. Immunoprecipitation experiments showed that they reacted with molecules with an apparent molecular mass of 40 kDa under both reducing and nonreducing conditions (“p40” molecules), thus differing from CD45 or CD11a/CD18 antigens as well as from the “inhibitory” receptors for HLA class I molecules (i.e. p58, CD94 and NKB1 molecules). Double‐immunofluorescence analysis of peripheral blood mononuclear cells allowed the identification of three distinct populations on the basis of the fluorescence intensity of cells stained with anti‐p40 mAb. p40bright cells were homogeneously HLA‐DR‐positive, p40medium cells were HLA‐DR‐negative but co‐expressed CD56 antigens, while p40dull cells were all CD3+. Anti‐p40 mAb strongly inhibited the lysis of K562 target cells, mediated by fresh NK cells, as well as the lysis of P815 target cells by NK or T cell clones. In addition, in redirected killing assays, anti‐p40 mAb strongly reduced the anti‐CD16 mAb‐induced cytolytic activity of NK cell clones. On the contrary, they did not inhibit either the anti‐CD3 or anti‐T cell receptor mAb‐mediated cytolytic activity of T cell clones or the lysis of allogeneic phytohemagglutinin blasts mediated by specific cytolytic T cell clones. The p40‐induced inhibition of the NK cytotoxicity required optimal cross‐linking, as anti‐p40 mAb could inhibit the lysis of Fcγ receptor (FcγR)‐positive but not of FcγR‐negative target cells. In addition, (Fab')2 fragments of anti‐p40 mAb failed to inhibit the lysis of FcγR‐positive target cells. In conclusion, p40 molecules represent a new type of inhibitory surface molecule that appears to play a general regulatory role in the NK‐mediated cytolysis.
B7‐1 expression of Langerhans cells is up‐regulated by proinflammatory cytokines, and is down‐regulated by interferon‐γ or by interleukin‐10
Authors: Chang Chung‐Hsing , Furue Masutaka , Tamaki Kunihiko
Published: March 2007

Langerhans cells (LC) act as potent antigen‐presenting cells (APC) for primary and secondary T cell‐dependent immune responses. LC express several costimulatory and/or adhesion molecules such as B7/BB1, which has been implicated as one of the important determinants for professional APC. Recent studies have shown that B7/BB1 antigens comprise three distinct molecules termed B7‐1, B7‐2, and B7‐3. We have examined the regulatory properties of B7‐1 expression in LC using various cytokines including interleukin (IL)‐1α, IL‐1β, IL‐2, IL‐3, IL‐4, IL‐5, IL‐6, IL‐7, IL‐10, interferon (IFN)‐γ, granulocyte/macrophage colony‐stimulating factor (GM‐CSF), and tumor necrosis factor (TNF)‐α. We have demonstrated: 1) that the B7‐1 expression of LC is reproducibly up‐regulated by either GM‐CSF, TNF‐α, IL‐1α, IL‐1β, or IL‐4 in a dose‐ and time‐dependent manner, 2) that GM‐CSF exhibits the most active effect on B7‐1 up‐regulation in each experiment, 3) that IFN‐γ or IL‐10 profoundly inhibits the B7‐1 expression of LC in a dose‐ and time‐dependent manner, and 4) that the down‐regulatory ability of IFN‐γ or IL‐10 neutralizes the activity of up‐regulatory cytokines. The enhancing or inhibitory action of these cytokines on B7‐1 expression occurs selectively because none of the cytokines consistently affects I‐A expression of LC. These data suggest that the B7‐1 expression of LC may be dynamically regulated by these up‐ and down‐regulatory cytokines in normal and inflammatory epidermal microenvironment.
αEβ7 and α4β7 integrins associated with intraepithelial and mucosal homing, are expressed on macrophages
Authors: Tiisala Sinikka , Paavonen Timo , Renkonen Risto
Published: March 2007

The two β7 integrins αEβ7 and α4β7 are the most recently described members of the integrins participating in intercellular binding. Their expression has been shown to be restricted to leukocytes and they have been suggested to be predominantly found in lymphocytes associating with the epithelium. Expression of β7 has mainly been studied on lymphocytes whereas macrophages have been reported not to express the β7 integrins. In this paper we have studied the expression of β7 integrins in monocytoid cells. The myelomonocytic cell lines HL‐60 and THP‐1 did not express β7 mRNA or protein, but differentiation of these cell lines to macrophages with phorbol 12‐myristate 13‐acetate (PMA) led to a strong induction of the β7 mRNA expression. A clear but less pronounced up‐regulation of β7 mRNA‐expression was also seen after treatment of HL‐60 and THP‐1 cells with interferon‐γ (IFN‐γ). However, its up‐regulating effect on the surface expression of α4β7 and αE7 complexes (detected by the monoclonal antibodies Act I and HML‐1, respectively) exceeded that observed with PMA. To verify the in vitro cell line observations with normal cells, we also studied peripheral blood monocytes and tissue macrophages. Peripheral blood monocytes were Act I− and HML‐1− in flow cytometry, but their expression was increased after a 72‐h culture in the presence of PMA or IFN‐γ. Also, several Act I+ and HML‐1+ macrophages were found in immunohistochemical stainings of both liver and edemic lung biopsies as well as in lymph node sinuses. We therefore conclude that while monocytes do not express β7 integrins the more differentiated cells of the monocyte‐macrophage lineage do express both the α4β7 and αEβ7 integrins, which might play a role in their intraepithelial homing.
Hypomethylation of the interferon‐γ gene correlates with its expression by primary T‐lineage cells
Authors: Melvin Ann J. , McGurn Mary E. , Bort Susan J. , Gibson Christine , Lewis David B.
Published: March 2007

To determine the potential role of methylation in the regulation of interferon‐γ (IFN‐γ) gene transcription by T cells, primary T‐lineage cell populations were analyzed for the extent of methylation of three CpG sites within or near transcriptional activator elements in the 5′ flank and first intron of the human IFN‐γ gene. A striking correlation was observed between the capacity of the IFN‐γ gene to be expressed and the degree of hypomethylation. The IFN‐γ gene was virtually completely methylated at all sites in thymocytes, neonatal T cells, and adult CD45RAhiCD45R0lo (antigenically naive) CD4 T cells, cell types that all have a low or undetectable capacity to express the IFN‐γ gene. In contrast, there was substantial hypomethylation in T‐lineage cell types with relatively high capacities to express the IFN‐γ gene, including adult CD8 T cells and adult CD45RAloCD45R0hi (memory/effector) CD4 T cells. These results suggest that hypomethylation of the IFN‐γ genetic locus may be an important determinant of IFN‐γ gene expression in vivo by T‐lineage cells.
Thymic epithelium induces full tolerance to skin and heart but not to B lymphocyte grafts
Authors: Thomas‐Vaslin Véronique , Salaün Josselyne , Gajdos Benoǐt , le Douarin Nicole , Coutinho Antonio , Bandeira Antonio
Published: March 2007

Athymic nude mice reconstituted at birth with allogeneic thymic epithelia (TE) from day 10 embryos (E10), show life‐long specific tolerance to skin and heart grafts, but eliminate B lymphocytes of the TE donor haplotype, nearly as well as those from a third strain. Previous immunizations with B cells do not alter the state of tolerance to skin grafts, but specifically accelerate elimination of lymphocytes. In contrast, transplantation of E15 allogeneic thymuses already seeded by hematopoietic cells resulted in chimeras tolerant to both skin and B lymphocytes. In vitro reactivities towards stimulator spleen cells of the haplotype of the thymus were observed in both E10 TE and E15 thymus chimeras.

We conclude that induction of full in vivo tolerance to B cells requires hematopoietic cells, while this is not the case for induction of tolerance to skin and heart tissues; furthermore, in vitro reactivity to stimulator spleen cells of the tolerized haplotype is independent of in vivo tolerance.
Role of CD44 cytoplasmic domain in hyaluronan binding
Authors: Perschl Astrid , Lesley Jayne , English Nicole , Trowbridge Ian , Hyman Robert
Published: March 2007

The hyaluronan (HA) binding activity of mutant CD44 constructs expressed in AKR1 T‐lymphoma cells was evaluated by flow cytometry using fluoresceinconjugated HA (Fl‐HA). Previous studies showed that wild‐type hematopoietic CD44 bound Fl‐HA when expressed in AKR1, but that truncated “tailless” CD44, lacking all but six amino acids of the cytoplasmic domain, did not bind. Here, we show that a disulfide‐bonded dimer of CD44, formed by substituting the transmembrane region of CD3ζ chain for that of CD44, binds Fl‐HA, even when the cytoplasmic domain of the CD44 dimer is absent. We conclude that dimerization of CD44 abrogates the requirement for the cytoplasmic domain, suggesting that the cytoplasmic domain of CD44 may contribute to HA binding by promoting CD44 clustering. These results suggest that changes in the distribution of CD44 on the cell surface, induced by molecular interactions either from within the cell or from outside, may regulate its role as a receptor. Further studies sought to localize the region of the CD44 cytoplasmic domain contributing to HA binding by the construction of a series of cytoplasmic domain truncation mutants and internal deletion mutants. All of the mutant CD44 molecules bound Fl‐HA similarly to wild‐type CD44. Thus, it was not possible to assign the function mediating HA binding to a specific region of the cytoplasmic domain, suggesting either that multiple regions of the cytoplasmic domain can promote enhancement of HA binding, or that the role of the cytoplasmic domain in mediating this function does not require a specific amino acid sequence.
Expression of TAP1 by human trophoblast
Authors: Clover Linda M. , Sargent Ian L. , Townsend Alain , Tampé Robert , Redman Chris W. G.
Published: March 2007

Successful placentation in the human is dependent on the trophoblast evading recognition and destruction by the maternal immune system. However, invasive cytotrophoblast express HLA‐G which may be able to present peptide to T cells. Transporter proteins are essential for peptide presentation and major histocompatibility complex (MHC) class I assembly. We have determined their expression by trophoblast in relation to HLA‐G, using immunohistochemistry. Antitransporter protein antibody (TAP1) labeling closely paralleled that of MHC class I, but the intensity of its expression was much greater on the HLA‐G+ extravillous cytotrophoblast than any other fetal or maternal tissue in the first trimester and at term. This suggests that the extravillous cytotrophoblast are very actively assembling MHC class I antigens with peptides. However, expression of MHC class I by the cytotrophoblast was not correspondingly elevated. This pattern could result from HLA‐G being shed from the surface of the trophoblast, a process which may play a central role in protecting the fetus from maternal immune attack.
Ovalbumin injected with complete Freund's adjuvant stimulates cytolytic responses
Authors: Ke Yong , Li Ying , Kapp Judith A.
Published: March 2007

CD8+ cytotoxic T lymphocytes (CTL) recognize antigens (Ag) associated with class I major histocompatibility complex (MHC) molecules. Endogenously synthesized protein Ag are processed into peptides in the cytoplasm and transported to the endoplasmic reticulum where they are bound by class I proteins. Exogenous Ag do not enter the class I processing pathway of most cells and thus do not activate CD8+ CTL. Nevertheless, several investigators have reported that immunization with exogenous Ag can activate CD8+ T cells that have immunoregulatory activity. To determine how exogenous Ag entered the class I pathway in vivo and whether immunosuppressive CD8+ T cells were cytolytic, we have shown in this report that injection with OVA emulsified in the complete Freund's adjuvant (CFA) primed CD8+, class I MHC‐restricted, OVA‐specific CTL in mice. These CTL recognize the OVA257–264 epitope, produce tumor necrosis factor‐α and interferon‐γ upon activation. Both oil and mycobacteria components in CFA were required for inducing CTL responses. Priming was not attributed to direct sensitization of class I‐bearing cells by contaminating peptides. Rather, phagocytic cells, but not CD4+ helper T cells, were required for priming CD8+ CTL by OVA‐CFA. Thus, OVA in CFA is taken up by antigen‐presenting cells and processed into the class I pathway by phagocytic cells in vivo. In addition, CTL induced by OVA‐CFA suppressed the antibody response to OVA in adoptive recipients. These results suggest that CD8+ CTL specific for exogenous proteins might be routinely stimulated by injecting proteins in conventional adjuvants and that such cells have the potential to regulate immune responses in vivo.
Mimicking the humoral immune response in vitro results in antigen‐specific isotype switching supported by specific autologous T helper cells: generation of human HIV‐1‐neutralizing IgG monoclonal antibodies from naive donors
Authors: Chin Lie‐Te , Malmborg Ann‐Christin , Kristensson Karin , Hinkula Jorma , Wahren Britta , Borrebaeck Carl A. K.
Published: March 2007

Molecular and cellular requirements for antigen‐specific isotype switch of human B cells have been investigated by mimicking signaling occurring in germinal centers. Peripheral blood mononuclear cells from healthy seronegative blood donors were first primary immunized in vitro, using a synthetic immunogen containing both a T and B cell epitope, which generated specific IgM‐secreting B cells. We used the apex of the V3 loop of gp120 as B cell epitope linked to a promiscuous T helper epitope from tetanus toxin. In parallel, CD4+ T helper cell clones specific for the T epitope of the immunogen were established. In a secondary in vitro stimulation period, we co‐cultured the antigen‐specific T and B cells on CD32‐transfected fibroblasts, together with an anti‐CD40 monoclonal antibody. This resulted in isotype switching and human antigen‐specific, IgG‐secreting B cells were detected. This response was strictly dependent upon the presence of autologous T helper cells and the immunogen. Antigen‐specific human B cells derived from this primary and secondary in vitro immunization were subsequently subjected to electrofield‐induced somatic cell hybridization and hybridomas secreting human anti‐V3 IgG monoclonal antibodies were isolated. One human antibody was further characterized and shown to be specific for the immunizing antigen with an affinity constant of 24 nM. This antibody also effectively neutralized different isolates of HIV‐1, achieving a 50% neutralization at 0.46 μg/ml.
Regulation of the interaction between Th1 and Th2 T cell clones to provide help for antibody production in vivo
Authors: Rizzo Luiz V. , Dekruyff Rosemarie H. , Umetsu Dale T. , Caspi Rachel R.
Published: March 2007

On the premise that an individual with an intact immune system has the capability to develop both cellular and antibody immune responses supported by the balance between the lymphokines secreted by T helper (Th) cells, we studied the interaction between different types of Th cell clones in vivo and the parameters that may affect this interaction. We used an adoptive transfer system in which nude or lethally irradiated mice were reconstituted with histocompatible CD4+ keyhole limpet hemocyanin (KLH)‐specific T cell clones with defined lymphokine profiles. This approach allowed us to study the effects of the cognate interaction between T and B cells in the presence of a defined set of lymphokines. We demonstrated that the co‐transfer of both subsets of Th cells resulted in increased production of IgA, and decreased production of IgE and IgG2a. The concomitant presence of both cell types also increases their functional survival in vivo. We have shown that in the presence of a Th2 clone, higher immunization doses (above 100 μg trinitrophenol (TNP)‐KLH/mouse) result in increased production of IgE and IgG1. In contrast, when a Th1 clone is present, low immunization doses (less than 50 μg TNP‐KLH/mouse) resulted in increased production of IgG2a. We were also able to show that the neutralization of interleukin‐4 (IL‐4) and or interferon‐γ (IFN‐γ) was sufficient to abrogate most of the regulatory effects caused by the Th2 or the Th1 clone respectively. Our results indicate that the subset of T cell(s) transferred determines the type of response obtained. In addition, the data presented indicate that the antigen dose used for immunization can modulate the quantitative parameters of the response. Furthermore, we have shown that the interaction between the two subsets of T cells in vivo is characterized by both antagonistic and agonistic effects and that most of the regulatory effects exerted by one subset over the other are mediated by IL‐4 or IFN‐γ.
Molecular characterization of leukocyte adhesion deficiency in six patients
Authors: Wright Ann H. , Douglass Wendy A. , Taylor G. Malcolm , Lau Yu‐Lung , Higgins David , Davies Kevin A. , Law S. K. Alex
Published: March 2007

Leukocyte adhesion deficiency (LAD) is caused by defects in the CD18 gene, which codes for the common β2 subunit of the leukocyte integrins LFA‐1, Mac‐1 and p150,95. Failure to produce a functional β2 subunit results in the defective expression of all three leukocyte integrins, and the leukocytes of LAD patients have subnormal adhesion properties. Six patients with LAD were studied. Patient B was homozygous and carried a G284S mutation. A two‐bp (GA) deletion at position 1256 (1256ΔGA) was found in the cDNA of patient C, who also had an abnormally large mRNA of 4.3 kb. Patients E and K were siblings and were heterozygous at the genomic level. One defective allele contained a mutation in intron 6/7 which created a preemptive 3′ splice site. The resulting mRNA has 12 extra bases at the junction of exons 6 and 7, coding for four extra residues PSSQ in the protein. The same allele also carried a R586W mutation. The other allele was transcribed at a low level and was not characterized. Patient G carried a L149P mutation in one allele; again, the other allele was not characterized due to low transcription levels. Patient R carried two mutant alleles with G284S and R593C mutations respectively. The G284S mutation and the 1256ΔGA deletion have not been reported previously. CD18 cDNA carrying the abnormalities were cotransfected with normal CD11a or CD11b cDNA into COS cells. Expression of the LFA‐1 (CD11a/CD18) and Mac‐1 (CD11b/CD18) antigens on COS cells was not detected, suggesting that these two mutations are sufficient to account for LAD.
Anti‐CD40 plus interleukin‐4‐activated human naive B cell lines express unmutated immunoglobulin genes with intraclonal heavy chain isotype variability
Authors: Galibert Laurent , van Dooren Joelle , Durand Isabelle , Rousset Françoise , Jefferis Royston , Banchereau Jacques , Lebecque Serge
Published: March 2007

Combination of anti‐CD40 antibody and interleukin‐4 (IL‐4) induces B cell clonal expansion reminiscent of the T‐dependent proliferation following antigenic challenge in vivo. We have analyzed the usage of CH genes and the presence or absence of somatic mutations within the progeny of a single human naive B cell activated with anti‐CD40+IL‐4. To address this issue, single‐cell cultures of naive (sIgD+) tonsillar B lymphocytes expressing the VH1‐restricted G8 idiotype were set up. After culture and RNA extraction, VH1+ Ig mRNA were reverse‐transcribed, amplified by polymerase chain reaction and sequenced. A single sIgD+ B cell could generate clones expressing μ, γ1, γ3 or ε, illustrating that the progeny of a single cell can express different isotypes in response to the same stimulus in vitro. The rate of somatic mutations affecting the immunoglobulin variable heavy chain gene was indistinguishable from the background of errors introduced by Taq polymerase.
Integration vectors for antibody chimerization by homologous recombination in hybridoma cells
Authors: Kardinal Christian , Hooijberg Erik , Lang Peter , Zeidler Reinhard , Mocikat Ralph
Published: March 2007

Gene targeting in hybridoma cells provides a tool for generating chimeric antibodies with great ease and at high yield. We present an evaluation of integration vectors for the chimerization of the immunoglobulin heavy chain locus which are universally applicable to hybridomas of different isotypes and mouse strains. There are three problems arising with vector integration: (i) the frequent persistence of the parental isotype; (ii) an isotype‐dependent aberrant replacement‐like recombination giving rise to antibodies devoid of the CH1 domain; and (iii) secondary recombinations leading to excision of the integrated sequence. To overcome these problems, we have systematically evaluated the consequences of extending the vector flank. Although the homology length clearly determines the recombination frequency, this effect is counteracted by the secondary recombination, which also correlates to the homology length. In contrast, the truncating recombination events are not dependent on the homology length and never lead to re‐excision of the construct. To take advantage of the increased genetic stability obtained with short flanks, we constructed an enrichment vector which yields high recombination efficiencies despite using a short flanking sequence. In addition, irradiation of the cells enhanced homologous recombination. The problem of the co‐production of two isotypes was overcome by a two‐step targeting reaction.
Clonal expansion of myelin basic protein‐reactive T cells in patients with multiple sclerosis: Restricted T cell receptor V gene rearrangements and CDR3 sequence
Authors: Vandevyver Caroline , Mertens Nadja , van den Elsen Peter , Medaer Robert , Raus Jef , Zhang Jingwu
Published: March 2007

Myelin basic protein (MBP)‐reactive T cells are thought to play an important role in the pathogenesis of multiple sclerosis (MS). In some patients with MS, these autoreactive T cells display a limited heterogeneity in their epitope recognition and T cell receptor (TCR) variable (V) gene usage. These individual‐dependent properties of MBP‐reactive T cells have led to the speculation that they may represent clonal expansion in vivo in some MS patients. In the present study, 51 MBP‐reactive T cell clones derived from patients with MS and healthy individuals were examined for their epitope recognition and the TCR Vα and Vβ gene rearrangements. The V gene junctional region sequences of identified α and β genes were further analyzed to probe their clonal origins, as the sequences are unique for individual clones. Our data showed that 26 clones derived from nine patients with MS shared a predominant reactivity to the immunodominant regions of MBP, 84–102, 110–129 and 143–168, and used various TCR Vα and Vβ rearrangements. The V gene usage of the clones was restricted to certain Vα Vβ combination(s) in a given MS patient, but varied among different patients. The sequence analysis revealed that the clones generated from a given patient shared a limited or a single junctional region sequence pattern(s), indicating their oligoclonal or monoclonal origin(s). In contrast, 25 MBP‐reactive T cell clones derived from normal individuals exhibited unfocused epitope recognition and V gene usage. Thus, the limited heterogeneity of MBP‐reactive T cells in their structural and functional charactertistics reflects their clonal expansion in vivo in some patients with MS.
Intranasal immunization with liposomes induces strong mucosal immune responses in mice
Authors: Vadolas Jim , Davies John K. , Wright Peter J. , Strugnell Richard A.
Published: March 2007

BALB/c mice were immunized intranasally with either soluble ovalbumin (OVA) or OVA entrapped in liposomes. The effect of adding Sigma cholera toxin B subunit (sCT‐B), which contained low amounts of cholera holotoxin (CT), or recombinant CT‐B (rCT‐B) which was free from CT, as mucosal adjuvants was also investigated. The mucosal [lung enzyme‐linked immunospot assay (ELISPOT), lung washing] and systemic (serum antibody and spleen ELISPOT) responses of immunized mice to OVA and CT‐B were determined. Results showed that soluble OVA and liposome‐entrapped OVA were poor inducers of mucosal or systemic responses unless CT‐B was added as adjuvant. The types of responses augmented by sCT‐B and rCT‐B were different. CT‐B containing low levels of CT (i.e. sCT‐B) boosted both mucosal and systemic IgA and IgG responses, whereas rCT‐B only increased IgG responses, unless antigen was entrapped in liposomes. Although rCT‐B was unable to adjuvant IgA responses against soluble OVA, it was able to induce IgA responses against itself. These data show that mucosal responses can be increased by addition of CT‐B containing low levels of CT to antigen preparations given intranasally, suggesting a direct role for CT‐A in isotype switching. Furthermore, the ability of CT‐B to adjuvant IgA responses against added antigens and its ability to induce responses against itself appear to be separate phenomena. The results from this study should assist the rational formulation of mucosal vaccines which induce potent mucosal and systemic immune responses.
Chronic relapsing experimental autoimmune encephalomyelitis with a delayed onset and an atypical clinical course, induced in PL/J mice by myelin oligodendrocyte glycoprotein (MOG)‐derived peptide: Preliminary analysis of MOG T cell epitopes
Authors: de Rosbo Nicole Kerlero , Mendel Itzhack , Ben‐Nun Avraham
Published: March 2007

Myelin basic protein (MBP) and proteolipid protein (PLP), the most abundant proteins of central nervous system (CNS) myelin, have been extensively studied as possible primary target antigens in multiple sclerosis (MS), a primary demyelinating autoimmune disease of the CNS. However, there is increasing evidence to suggest that autoimmune reactivity against the quantitatively minor myelin component, myelin oligodendrocyte glycoprotein (MOG), can also play a role in the pathogenicity of MS. We recently demonstrated a predominant response to MOG by peripheral blood lymphocytes from patients with MS tested for their reactivity against various myelin antigens, including MBP and PLP. To ascertain whether or not T cell reactivity to MOG in MS is a potentially pathogenic response, we have tested the ability of synthetic MOG peptides (pMOG) representing potential T cell epitopes, to induce neurological disease in mice. Both strains of mice tested (SJL/J and PL/J mice) were able to mount a primary T cell response to some of the five MOG peptides synthesized, pMOG 1–21, 35–55, 67–87, 104–117 and 202–218. T cell lines could be raised in both strains to pMOG 35–55 and 67–87, but epitope definition revealed that each strain recognized a different minimal epitope within these two peptides. T cell lines to pMOG 1–21 and 202–218 could also be raised in SJL/J and PL/J mice, respectively. T cell reactivity to pMOG 104–117 was not observed in either mouse strain. None of the peptides tested induced detectable clinical signs in SJL/J mice. In contrast, an MS‐like chronic relasping‐remitting disease could be induced in PL/J mice with pMOG 35–55. The disease presented with a delayed onset and with clinical signs which differed significantly in their progression and expression from the typical ascending paralysis of experimental autoimmune encephalomyelitis induced with other myelin components, such as MBP and PLP. Histological examination of CNS tissue from mice injected with pMOG 35–55 revealed only mild neuropathological signs with few inflammatory foci in brain and spinal cord. Some myelin splitting and edema were detected upon electron microscopic examination in the spinal cord and cerebellum. Transfer of pMOG 35–55 reactive T cells into naive PL/J mice resulted in pathological changes characterized by inflammatory foci in the brain and spinal cord. This passively induced disease was clinically silent, as was also reported for Lewis rats injected with T cells specific for the same MOG peptide. These data, which demonstrate unequivocally the encephalitogenic activity of MOG, support our contention that MOG may be as important as MBP or PLP in disease pathogenesis and could be a primary target antigen in autoimmune diseases of the CNS.
Selective induction of interleukin‐1 receptor antagonist and interleukin‐8 in human monocytes by normal polyspecific IgG (intravenous immunoglobulin)
Authors: de Souza Valéria Ruiz , Carreno Marie‐Paule , Kaveri Srinivas V. , Ledur Annick , Sadeghi Hoss , Cavaillon Jean‐Marc , Kazatchkine Michel D. , Haeffner‐Cavaillon Nicole
Published: March 2007

We have investigated the effects of intravenous immunoglobulin (IVIg), a therapeutic preparation of normal human polyspecific IgG, on the synthesis and release of cytokines by peripheral blood monocytes. IVIg was found to selectively induce gene transcription and secretion of interleukin‐1 receptor antagonist (IL‐1ra) and IL‐8 in cultures of normal human monocytes. The addition of IVIg to cultures of purified monocytes induced a dose‐dependent secretion of IL‐1ra and IL‐8 without stimulating the production of IL‐1α, IL‐1β, tumor necrosis factor‐α or IL‐6. The effects of IVIg required both the Fc and F(ab′)2 portions of IgG. IVIg‐induced production of IL‐8 by monocytes was enhanced by lipopolysaccharide (LPS), although LPS inhibited the secretion of IL‐1ra, suggesting that IVIg and LPS stimulate distinct intracellular pathways in monocytes. Induction of IL‐1ra and IL‐8 by IVIg was enhanced in the presence of autologous T lymphocytes. Our observations document the selectivity of the effects of IVIg on the synthesis of cytokines and cytokine antagonists by human monocytes. Induction of IL‐1ra and IL‐8 by IVIg may contribute to the anti‐inflammatory effects of immunoglobulin therapy in patients with autoimmune and systemic inflammatory disorders.
Inhibition of natural killer cell‐mediated bone marrow graft rejection by allogeneic major histocompatibility complex class I, but not class II molecules
Authors: Öhlén Claes , Höglund Petter , Sentman Charles L. , Carbone Ennio , Ljunggren Hans‐Gustaf , Koller Beverly , Kärre Klas
Published: March 2007

The role of major histocompatibility complex (MHC) class I and class II molecules in natural killer (NK) cell‐mediated rejection of allogeneic, semi‐syngeneic and MHC‐matched bone marrow grafts was investigated. The use of β2‐microglobulin (β2m) ‐/‐ and β2m +/‐ mice as bone marrow donors to MHC‐mismatched recipients allowed an analysis of whether the presence of semi‐syngeneic and allogeneic MHC class I gene products would be triggering, protective or neutral, in relation to NK cell‐mediated rejection. Loss of β2m did not allow H‐2b bone marrow cells to escape from NK cell‐mediated rejection in allogeneic (BALB/c) or semi‐allogeneic (H‐2Dd transgenic C57BL/6) mice. On the contrary, it led to stronger rejection, as reflected by the inability of a larger bone marrow cell inoculum to overcome rejection by the H‐2‐mismatched recipients. In H‐2‐matched recipients, loss of β2m in the graft led to a switch from engraftment to rejection. At the recipient level, loss of β2m led to loss of the capability to reject H‐2‐matched β2m‐deficient as well as allogeneic grafts. When MHC class II‐deficient mice were used as donors, the response was the same as that against donors of normal MHC phenotype: allogeneic and semi‐syngeneic grafts were rejected by NK cells, while syngeneic grafts were accepted. These data suggest a model in which allogeneic class I molecules on the target cell offer partial protection, while certain syngeneic class I molecules give full protection from NK cell‐mediated rejection of bone marrow cells. There was no evidence for a role of MHC class II molecules in this system.
The role of bcl‐xL in CD40‐mediated rescue from anti‐μ‐induced apoptosis in WEHI‐231 B lymphoma cells
Authors: Choi Michael S. K. , Boise Lawrence H. , Gottschalk Alexander R. , Quintans José , Thompson Craig B. , Klaus Gerry G. B.
Published: March 2007

The phenotypically immature B cell lymphoma WEHI‐231 undergoes apoptotic cell death when cultured with anti‐immunoglobulin (Ig) antibodies, via a bcl‐2‐independent mechanism. We have therefore studied the role of the bcl‐2‐related protein bcl‐x in controlling cell death in WEHI‐231. We find that overexpression of the long form of bcl‐x (bcl‐xL) renders these cells refractory to anti‐Ig‐induced cell death. Stimulation of WEHI‐231 via CD40 has similar protective effects. We show here that ligation of CD40 rapidly induces the appearance of the bcl‐xL protein in WEHI‐231, while stimulation via sIgM, sIgD, CD5 or CD45 receptors, or with phorbol esters plus ionomycin does not. WEHI‐231 cells also rapidly undergo massive apoptosis following culture with thapsigargin, a specific inhibitor of the Ca2+‐ATPase of the endoplasmic reticulum: this is also reversed by anti‐CD40, or by overexpression of bcl‐xL. We, therefore, conclude that bcl‐xL plays a key role in the regulation of antigen receptor‐mediated apoptosis via CD40 in WEHI‐231. In addition, the fact that this protein is not induced in WEHI‐231 in response to phorbol dibutyrate plus ionomycin points to a fundamental signaling defect in these cells, which could conceivably be a reflection of their immature, apoptosis‐susceptible phenotype.
Suppression of experimental autoimmune encephalomyelitis in Lewis rats by antibodies against CD2
Authors: Jung Stefan , Toyka Klaus , Hartung Hans‐Peter
Published: March 2007

The immunotherapeutic potential of three anti‐rat CD2 monoclonal antibodies (mAb) (OX34, OX54, OX55) and the combination of OX54 with OX55 was tested in Lewis rat experimental autoimmune encephalomyelitis (EAE). In actively induced EAE, a single injection of OX34 2 days before immunization with myelin basic protein (MBP) in complete Freund's adjuvant (CFA) completely prevented or greatly attenuated EAE in all animals. Injection of OX54 acted moderately suppressive while OX55 or OX54/55 did not affect disease severity. Abrogation of EAE by OX34 was not restricted to its application before immunization. Therapeutic administration of all three mAb and the Ab combination from onset of first clinical signs efficiently blocked progression of disease and prevented all animals from developing hind limb paresis. In adoptive transfer EAE induced with in vitro activated cells of an encephalitogenic T helper line, clinical and histological signs were completely prevented by injection of OX34 on the day of cell transfer and 4 days later, underlining the strong impact of anti‐CD2 mAb on the effector phase of disease.

Immunocytofluorometric analysis of peripheral blood lymphocytes after a single Ab injection demonstrated that all mAb induced a variable degree of transient reduction in T cell numbers and modulation of CD2 antigens. In contrast to the other mAb, OX34 persisted on lymphocytes for at least 11 days, which may explain its unique suppressive effect on EAE after a single injection before immunization. The assumption that prophylactic administration of OX34 also inhibits MBP‐induced EAE, due to persistence into the effector phase, was substantiated by the finding that none of the mAb prevented generation of an antigen‐specific cellular response in MBP/CFA‐immunized animals. Since none of the Ab induced T cell unresponsiveness or inhibited T cell activation by antigen‐ or Ab‐mediated stimulation of the T cell receptor, we suggest that their marked action on the effector phase of EAE may rely on inhibition of T cell infiltration into the central nervous system. The demonstrated efficacy of these anti‐CD2 mAb in EAE suggests a potential therapeutic role that may be equal to that of anti‐CD4 or anti‐T cell receptor Ab.
HIV‐1 glycoprotein gp120 disrupts CD4‐p56lck/CD3‐T cell receptor interactions and inhibits CD3 signaling
Authors: Hubert Pascale , Bismuth Georges , Körner Marie , Debré Patrice
Published: March 2007

Using the CD4+ human T cell clone P28, we demonstrated that the HIV‐1 glycoprotein gp120 inhibited CD3‐induced inositol trisphosphate production, calcium influx and T cell proliferation. Additionally, gp120 was shown to dissociate the tyrosine kinase p56lck from CD4 in CEM cells, with a concommittant inhibition of CD4‐linked kinase activity. We have addressed the question whether disruption of CD4/p56lck or CD4/CD3‐T cell receptor interactions, or both, could account for the inhibitory effect of gp120 in P28 cells. By comparing the effects of various anti‐CD4 monoclonal antibodies (mAb) with those of gp120, we show that gp120 and IOT4a modulate CD4 expression, and decrease CD4‐associated p56lck and CD4‐linked kinase activity at the plasma membrane. In contrast, OKT4A and OKT4 anti‐CD4 mAb have no inhibitory effect. Interestingly, gp120 also inhibits CD3‐induced Lck activation and cellular tyrosine phosphorylation, particularly of phosphoinositide‐specific phospholipase C‐γ‐1. Kinetic experiments reveal that the inhibitory effect of gp120 on CD3‐induced tyrosine phosphorylation appears as early as 30 min, but culminate when CD4‐p56lck complexes disappear from the cell surface after 4 h. These results suggest that a negative signal is triggered by gp120 that results, after a few hours, in down‐modulation of CD4‐p56lck complexes and the impairment of CD3 signaling. Supporting this hypothesis, gp120 inhibits CD3‐linked kinase activity as shown by the inhibition of the phosphorylation of CD3 chains, leading to the inhibition of subsequent signal transduction.
Characterization of the human interleukin‐5 gene promoter: involvement of octamer binding sites in the gene promoter activity
Authors: Gruart‐Gouilleux Valérie , Engels Peter , Sullivan Michael
Published: March 2007

To investigate the human interleukin(IL)‐5 gene promoter, we have constructed a plasmid with the firefly luciferase reporter gene linked to human IL‐5 5′ flanking sequence (nucleotides −507 to + 44). We have used this plasmid to transfect the mouse EL4 T cell line, which can, under certain conditions, produce IL‐5 transcripts. Phorbol 12‐myristate 13‐acetate, A23187 and N6,2′‐O‐dibutyryl‐adenosine 3′:5′‐cyclic monophosphate co‐stimulation of EL4 cells transfected with the human IL‐5/luciferase reporter gene construct resulted in maximal induction of the luciferase gene. Deletion analysis of the IL‐5 promoter revealed the presence of negative regulatory elements between nucleotides −404 and −312 and two regions, located between nucleotides −312 and −227 and between nucleotides −80 and −35, that are involved in the positive regulation of the IL‐5 promoter. Using electrophoretic mobility shift assays, we show that the positive element located between nucleotides −312 and −227 involves the binding of factors antigenically related to Oct1, Oct2A and Oct2B, to a perfect octamer motif located at position −244/−237. Introduction of three point mutations in the octamer motif of the IL‐5/luciferase reporter gene plasmid, which results in the loss of competition for the factors binding to the IL‐5 promoter sequence, reduced the production of luciferase from stimulated, transfected EL4 cells, by 90%. Octamer factors can also bind within the second positive regulatory region.
A 17‐kDa CD4‐binding glycoprotein present in human seminal plasma and in breast tumor cells
Authors: Autiero Monica , Cammarota Giancarlo , Friedlein Arno , Zulauf Martin , Chiappetta Gennaro , Dragone Vincenzo , Guardiola John
Published: March 2007

We previously isolated gp17, a human seminal plasma glycoprotein, which specifically interacts with the D1‐D2 region of CD4, a T cell surface molecule involved in antigen recognition mediated by helper T cells also acting as a receptor for the human immunodeficiency virus. In this study we report that monoclonal antibodies (mAb) reacting with gp17 are able to inhibit the binding of gp17 to immobilized soluble CD4. An immunohistochemical analysis shows that gp17 is also expressed in mammary tumor cells upon hormone treatment and in biopsies from breast cancer patients. A structural characterization of gp17, including amino acid sequencing, indicates that the protein has an extensive structural similarity with a glycoprotein designated as seminal actin‐binding protein (SABP), also secreted by male sexual glands. SABP is in turn identical to gross cystic disease fluid protein‐15 (GCDFP‐15) or prolactin‐inducible protein (PIP), a factor known as a highly specific and sensitive marker of primary and metastatic apocrine breast cancer. To establish further the correspondence of gp17 and GCDFP‐15/PIP/SABP, the latter was expressed in bacteria from a cloned cDNA and purified by affinity chromatography to either anti‐gp17 mAb‐Sepharose or CD4‐Sepharose. The purified recombinant protein is shown to inhibit the binding of labeled, pure g17 to immobilized soluble CD4. The finding that breast cancer cells express a protein able to interact with the CD4 domains involved in the recognition of class II major histocompatibility antigens suggests a possible mechanism by which a tumor may affect the activity of tumor‐infiltrated CD4+ T cells.
No role of interleukin‐4 in CD23/IgE‐mediated enhancement of the murine antibody response in vivo
Authors: Hjulström Susanna , Landin Andreas , Jansson Liselotte , Holmdahl Rikard , Heyman Birgitta
Published: March 2007

Antigen‐specific IgE up‐regulates the specific IgM, IgG1, IgG2a and IgE response in vivo when given to mice together with antigen. The enhancement is mediated by the low‐affinity receptor for IgE, FcϵRII or CD23, as demonstrated both in CD23‐deficient mice and by blocking CD23 with anti‐CD23 monoclonal antibodies. A possible mechanism behind the regulatory effects of CD23 is that the IgE/CD23/antigen complex is endocytosed by B cells, leading to increased antigen processing and presentation on major histocompatibility complex (MHC) class II molecules toT helper cells. In the present study we have found that the expression of CD23 is reduced fivefold on splenic B cells in mice genetically deficient for IL‐4. When IL‐4‐deficient mice and normal littermates were immunized with 2,4,6‐trinitrophenyl (TNP)‐specific IgE followed by bovine serum albumin (BSA)‐TNP or with BSA‐TNP alone, the BSA‐specific IgG1 and IgG2a responses were equally well augmented by IgE in all mice. In addition, a low but significant IgE response was seen even in the IL‐4‐deficient mice. Thus, enhancement of the antibody response through IgE and CD23 occur in the absence of IL‐4 and is not dependent on CD23 up‐regulation.
Purification and characterization of endogenous peptides extracted from HLA‐DR isolated from the spleen of a patient with rheumatoid arthritis
Authors: Gordon Robert D. , Young Janice A. , Rayner Stephen , Luke Richard W. A. , Crowther Mandy L. , Wordsworth Paul , Bell John , Hassall Giles , Evans Jonathan , Hinchliffe Sue A. , Culbert Eric J. , Davison Matthew D.
Published: March 2007

We have purified HLA‐DR from the spleen of a patient with rheumatoid arthritis. The patient had Felty's syndrome and was heterozygous for the DR4Dw4 antigen. We have isolated endogenous peptides from purified HLA‐DR molecules. The peptides were purified by reverse phase HPLC and the major peaks were subjected to N‐terminal sequencing. The peptides were derived from a variety of proteins: human serum albumin, human erythroid protein 4.1, 60S ribosomal proteins L31 and L35, VCAM‐1, human immunoglobulin λ chain and cathepsin‐S. A peptide corresponding to the sequence of human serum albumin (HSA) residues 106–120 was synthesized and shown to bind to HLA‐DR4Dw4 (IC50 = 1.41 μM). We have confirmed and refined current ideas about the structural motif for the binding of peptides to HLA‐DR and HLA‐DR4Dw4.
Transforming growth factor‐β1‐induced expression of the mucosa‐related integrin αE on lymphocytes is not associated with mucosa‐specific homing
Authors: Austrup Frank , Rebstock Sabine , Kilshaw Peter J. , Hamann Alf
Published: March 2007

The integrin αE (HML‐1, αIEL, αM290) is largely expressed on lymphocytes in epithelial sites, especially the gut mucosa. We investigated whether αE has any role in homing or delineates a phenotype with distinct migratory behavior. Lymph node T cells were stimulated for 5 days with anti‐CD3 in the presence or absence of transforming growth factor (TGF)‐β1 to generate αE+ or αE− cells, respectively. The two populations were then tested for their homing properties in mice. Both αE+ (TGF‐β‐treated) and αE− (control) cells of either CD4+ or CD8+ subset had a low capacity to enter the gut and showed the same homing behavior with respect to a variety of other organs. The same was true for αE+ and αE− cells that had been briefly stimulated with anti‐CD3 (24 h) and then allowed to return to a resting state before injection, though in this case both populations showed a greater capacity to recirculate through lymphoid tissue than was seen with fully activated cells. The results indicate that αEβ7 does not act as a homing receptor, and that the expression of the site‐specific marker αE does not correlate with a distinct homing behavior.
Lack of L‐selectin expression by cells transferring diabetes in NOD mice: insights into the mechanisms involved in diabetes prevention by Mel‐14 antibody treatment
Authors: Lepault Françoise , Gagnerault Marie‐Claude , Faveeuw Christelle , Bazin Hervé , Boitard Christian
Published: March 2007

The process of mononuclear cell extravasation from the blood into the islets of Langerhans in nonobese diabetic (NOD) mice is dependent on the expression of a set of molecules, most of which remain to be defined. The observation that vascular addressins are expressed in inflamed islets raises the issue of the involvement of one of their ligands, L‐selectin, in the pathogenesis of autoimmune diabetes. Treatment of NOD females with Mel‐14, an antibody specific for L‐selectin, reduced the spontaneous development of both insulitis and diabetes. Pretreatment of diabetic donors with Mel‐14 decreased the capacity of their splenocytes to transfer the disease. However, the treatment of recipients had no effect on the transfer of diabetes by untreated diabetogenic splenocytes. To reconcile these apparently conflicting results, we fractionated spleen T cells from diabetic mice according to L‐selectin expression. Diabetogenic cells were found only in the L‐selectin subpopulation. Thus, diabetogenic cells in adult mice share phenotypic characteristics with activated/memory cells, and enter the pancreas using L‐selectin‐independent migratory pathways.