bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of CD8+ T cells in the initiation of insulin‐dependent diabetes mellitus
Authors: Wang Bo , Gonzalez Antonio , Benoist Christophe , Mathis Diane
Published: March 2007

While it is generally accepted that T cells are critical for the development of diabetes in the non‐obese diabetic (NOD) mouse, the precise functions of the CD4+ and CD8+ subsets remain ill‐defined. Transfer experiments have provided evidence that CD4+ cells are the disease initiators, provoking massive mononuclear leukocyte infiltration into the pancreatic islets, while CD8+ cells play an effector role, responsible for the final destruction of islet beta cells. It was surprising, then, to find that NOD mice carrying a null mutation at the β2‐microglobulin (β2‐μ) locus, and thereby lacking major histocompatibility complex class I molecules and CD8+ T cells, developed neither insulitis nor diabetes. Here, we argue that the absence of insulitis in these animals results from their lack of CD8+ cells because islet infiltration is also absent when NOD mice are treated with an anti‐CD8 monoclonal antibody (mAb) at a young age. Interestingly, the anti‐CD8 effect is only observed when the mAb is injected during a discrete age window – 2 to 5 weeks after birth. Transfer experiments indicate that the lack of CD8+ cells during this period somehow alters the phenotype of CD4+ cells, preventing them from expressing their insulitic potential. This is not because they are generally immuno‐incompetent nor because they are generally more prone to differentiating into cells with Th2 characteristics. Given that neither the β2‐μ mutation nor anti‐CD8 treatment affect insulitis in a T cell receptor transgenic (tg) mouse strain with a CD4+ T cell repertoire highly skewed for an anti‐islet cell reactivity, the most straight‐forward interpretation of these observations is that CD8+ cells are required for effective priming and expansion of autoreactive CD4+ cells.
Differential induction of helper and killer T cells from isolated CD4+CD8+ thymocytes in suspension culture
Authors: Iwata Makoto , Kuwata Takeshi , Mukai Mutsumi , Tozawa Yuzuru , Yokoyama Minesuke
Published: March 2007

Thymocytes of T cell receptor transgenic mice with nonselecting and RAG‐2−/− backgrounds were developmentally arrested at the CD4+CD8+ stage before positive selection. These thymocytes underwent lineage commitment upon transient stimulation with a combination of ionomycin, a calcium ionophore, and phorbol 12‐myristate 13‐acetate (PMA), a protein kinase C activator, in suspension culture. The effective drug doses were limited within narrow ranges and much lower than those which induce proliferation of mature T cells. The doses corresponded to those which inhibit glucocorticoid‐induced apoptosis in these thymocytes. CD4 lineage commitment required longer duration, higher intensity of the stimulation, or both, than CD8 lineage commitment. Functional helper T cells (Th1 and Th2) were induced from the CD4 lineage‐committed cells upon secondary stimulation with a combination of ionomycin and PMA followed by lymphokine treatment. Cytotoxic T cells were induced from the CD8 lineage‐committed cells upon incubation with concanavalin A and irradiated splenic dendritic cells, but not with the combination of ionomycin and PMA. These results indicate that positive selection is mimicked by the pharmacological stimulation in the absence of other cell types, but that final maturation of CD8 T cells may require a different signal.
Severe combined immunodeficiency due to defective binding of the nuclear factor of activated T cells in T lymphocytes of two male siblings
Authors: Feske Stefan , Müller Judith M. , Graf Daniel , Kroczek Richard A. , Dräger Ruth , Niemeyer Charlotte , Baeuerle Patrick A. , Peter Hans‐Hartmut , Schlesier Michael
Published: March 2007

Peripheral blood lymphocytes (PBL) and alloreactive T cell lines of two male infants born to consanguinous parents and presenting with severe combined immunodeficiency (SCID) showed a pronounced deficiency in T cell activation. Although phenotypically normal, the proliferative response of the childrens' T cells was strongly reduced but could be improved by the addition of interleukin‐2 (IL‐2). Furthermore both childrens'T cells were unable to produce the cytokines IL‐2, interferon‐γ (IFN‐γ), IL‐4 and tumor necrosis factor‐α (TNF‐α). This multiple cytokine production deficiency could not be restored by IL‐2 or co‐stimulatory signals provided by antigen‐presenting cells (APC). Moreover, mRNA for IL‐2 and IFN‐γ could not be detected. In contrast, expression of the activation‐dependent cell surface markers CD25 and CD69 was within normal limits. To determine whether the functional defect of the patients' T cells was due to the absence or abnormal binding of transcription factors involved in cytokine gene expression, electrophoretic mobility shift assays were used to examine the DNA binding of AP‐1, Oct, CREB, SP1, NF‐ϰB and the nuclear factor of activated T cells (NF‐AT) to their respective response elements in the promoter of the IL‐2 gene. Whereas AP‐1, NF‐ϰB, Oct, CREB and SP1 displayed normal binding activities in nuclear extracts, the binding of NF‐AT to its IL‐2 promoter response element was barely detectable both before and after T cell stimulation. Our results strongly suggest that this NF‐AT/DNA binding defect is responsible for the multiple cytokine deficiency and the SCID phenotype observed in the two infant brothers.
Regulation of B cell growth and differentiation via CD21 and CD40
Authors: Axcrona Karol , Gray David , Leanderson Tomas
Published: March 2007

Stimulation in vitro of murine splenic B cells by lipopolysaccharide, anti‐ϰ Sepharose, anti‐CD40 or allo‐reactive T helper cells all up‐regulated CD21 and CD23 surface expression. Neither anti‐CD21 nor anti‐CD23 antibodies induced B cell growth or differentiation when added in soluble form or coupled to Sepharose. However, anti‐CD40‐stimulated B cells showed increased proliferation in the presence of anti‐CD21 antibodies coupled to Sepharose; co‐stimulation via CD21 also induced differentiation to immunoglobulin secretion in a fraction of anti‐CD40‐stimulated B cells. Furthermore, anti‐CD40 antibodies inhibited differentiation to immunoglobulin secretion induced by lipopolysaccharide and, hence, appears to be a dominant negative signal for B cell differentiation.
Correlation between immune maturation and idiotypic network recognition
Authors: Lange Hans , Solterbeck Meika , Berek Claudia , Lemke Hilmar
Published: March 2007

The maturation of T‐dependent humoral immune responses is mediated by somatic mutations. Antigen selection is one mechanism for the activation of B cell clones which express antibodies with progressively increased affinity and which are derived as somatic variants from germ‐line‐encoded genes. However, the emergence of B cell clones secreting rather low‐affinity antibodies and the shift to alternative germ‐line V region gene combinations during secondary and tertiary responses cannot be explained by antigen selection. It has been considered that idiotypic suppression may favor this clonal shift. Such an involvement would require that idiotypic recognition in the syngeneic host must be highly restricted to private idiotopes of each clone sequentially activated during immune maturation. To test this possibility, we produced 19 syngeneic anti‐idiotypic antibodies to the germ‐line‐encoded major Ox1 idiotype (IgM‐IdOx1 H11.5) of the anti‐2‐phenyl‐oxazolone (phOx) immune response in BALB/c mice. The fine specificity of these anti‐IdOx1 was tested with a set of anti‐phOx monoclonal antibodies, representing the first steps of maturation. About half of the anti‐IdOx1 showed almost no reactivity with the IdOx1 after the switch to IgG and none of the anti‐IdOx1 reacted with anti‐phOx antibodies which carried a glycine or histidine instead of arginine as the middle amino acid of the D region. These observations suggest a strong correlation between immune maturation and the idiotypic network. A model is presented in which idiotypic suppression may function as a driving force for diversification and maturation of the antigen‐induced immune response.
Complexity of the mouse gut T cell immune system: Identification of two distinct natural killer T cell intraepithelial lineages
Authors: Guy‐Grand Delphine , Cuénod‐Jabri Bana , Malassis‐Seris Michèle , Selz Françoise , Vassalli Pierre
Published: March 2007

Gut thymo‐dependent (CD8α+β+ or CD4+) or ‐independent (CD8α+β−) intraepithelial lymphocytes (IEL) mediate cytotoxicity following T cell receptor (TCR)‐CD3 signaling, but only TCRγδ+ and αβ+ thymo‐independent IEL show cytotoxicity of natural killer (NK) and antibody‐dependent cell‐mediated cytotoxicity types. Moreover, TCRαβ+ and γδ+ thymo‐independent IEL express NK receptors, and may therefore be referred to as NK‐TIEL. NK‐TIEL cytotoxicity is mediated through perforin, Fas, or both pathways. In contrast to that of other NK cells, this cytotoxicity is not negatively regulated by signals delivered through the recognition of major histocompatibility complex class I molecules. Thus, gut IEL include T cell subsets with unique specificities and functions, ontogenically distinct from other T cell lineages, which may increase the antigenic repertoire diversity of the immune system participating in the defense of the epithelial barrier.
Abnormal production of T helper 2 cytokines interleukin‐4 and interleukin‐5 by T cells from newborns with atopic parents
Authors: Piccinni Marie‐Pierre , Beloni Lucio , Giannarini Loretta , Livi Claudia , Scarselli Gianfranco , Romagnani Sergio , Maggi Enrico
Published: March 2007

T cell clones were generated from umbelical cord blood lymphocytes (UCBL) of nine newborns with atopic or nonatopic parents and their cytokine secretion profile was assessed. Both phytohemagglutinin‐induced and Dermatophagoides pteronyssinus‐spetific T cell clones from newborns with atopic parents exhibited an enhanced ability to produce the Th2 cytokines interleukin (IL)‐4 and IL‐5, compared to T cell clones from newborns with nonatopic parents. In contrast, the ability to produce interferon‐γ by UCBL from the two groups of newborns was not different. Of the five children who could be followed up to 3 years after birth, four with atopic parents developed clinical and/or biological atopic manifestations, whereas one without atopic parents did not. Thus, the pronounced production of IL‐4 and IL‐5 by UCBL not only appears to be related to the atopic status of parents, but also associates with the subsequent development of atopy in childhood.
L1 adhesion molecule on human lymphocytes and monocytes: expression and involvement in binding to αvβ3 integrin
Authors: Ebeling Olaf , Duczmal Andreas , Aigner Silke , Geiger Claudia , Schöllhammer Sandra , Kemshead John T. , Möller Peter , Schwartz‐Albiez Reinhard , Altevogt Peter
Published: March 2007

The L1 adhesion molecule is a member of the immunoglobulin (Ig) superfamily initially identified in the nervous system which contains six Ig‐like domains. Besides the known L1‐L1 homotypic interaction, L1 was recently shown to bind to very late antigen (VLA)‐5 in the mouse and αvβ3 in the human. The sixth Ig domain is critical for this function. We now demonstrate that human CD4+ peripheral blood T lymphocytes, monocytes and B lymphocytes, but not CD8+ T lymphocytes, express L1. When compared to the expression of CD31, another ligand for αvβ3 on T lymphocytes, only a small proportion of cells were CD31+L1+ double positive. L1 was also detected on the surface of human monocytic and lymphoid tumor lines and was shown to have a molecular mass of ∼220 kDa, similar to the molecule present on neuroblastoma cells. The function of the sixth Ig domain of human L1 as an integrin ligand was also investigated. Using an RGD‐containing peptide derived from the sixth Ig domain as well as a fusion protein of the sixth Ig domain of L1 and the Fc portion of human IgG1 (6.L1‐Fc), we demonstrated the binding of human MED‐B1 (αvβ3hi, α5β1lo) tumor cells and this binding was blocked by αv‐specific mAb. In contrast, human Nalm‐6 cells (αvβ3lo, α5β1hi) did not bind to the 6.L1‐Fc fusion protein. MED‐B1 cells could also be stained with the 6.L1‐Fc fusion protein. Our results suggest that human L1 binds predominantly to αvβ3 and that its presence on leukocytes could be important for adhesion and migration.
Tumor cells expressing a recall antigen are powerful cancer vaccines
Authors: Schweighoffer Tamás
Published: March 2007

Tumor cells were engineered to express a specific recall antigen molecule to serve as a target for the host's existing memory response, and then used as immunogens as a novel form of cancer vaccine. As recall antigen, the efficiently expressible hybrid protein Heat1 was employed, that contains an antigenic fragment of the Mycobacterium bovis 65‐kDa heat shock protein (hsp65), and thus can be recognized in mice immunized with live Bacillus Calmette‐Guérin (BCG) or its derivatives. Mouse M‐3 melanoma cells were transfected to express Heat1, irradiated, and used as anti‐melanoma vaccines. Vaccination elicited anti‐tumor immunity in mice, i.e. a subsequent challenge with the parental wild‐type M‐3 melanoma cells was rejected. Successful vaccination was dependent on the correct recognition of the recall antigen, since vaccination failed to prevent outgrowth of the challenge in mice possessing no memory response against the recall antigen. The results indicate that expression of a recall antigen can turn tumor cells into powerful vaccines. By using the Heat1 protein or other appropriate antigens, this general concept may be applied for human therapy.
Amino acid substitutions at position 97 in HLA‐A2 segregate cytolysis from cytokine release in MART‐1/Melan‐A peptide AAGIGILTV‐specific cytotoxic T lymphocytes
Authors: Maeurer Markus J. , Chan Huei‐Wie , Karbach Julia , Salter Russell D. , Knuth Alex , Lotze Michael T. , Storkus Walter J.
Published: March 2007

CD8+ T lymphocytes recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. Individual peptide termini appear to be fixed at the C‐ and N‐terminal ends. In contrast, central peptide side chains residues may point in different directions and exhibit limited flexibility, dependent on the MHC class I structural variation. For instance, position 97 in HLA‐A201 has been shown to shift individual peptide species into different coordinations, one oriented towards the peptide N terminus, or more towards the C‐terminal end. The conformational shape of such non‐anchor peptide residues may affect the affinity of MHC/peptide / TCR interaction, resulting in quantitative, or qualitative different T cell effector functions. To characterize the impact of different amino acid residues occupying position 97 in HLA‐A2 on peptide binding and presentation to CTL, we generated a panel of mutated HLA‐A2 molecules containing either M, K, T, V, G, Q, W, P or H at position 97. The HLA‐A0201 presented melanoma‐associated MART‐1/Melan‐A derived peptide AAGIGILTV was employed to assess the impact of such position‐97 mutations on HLA‐A2 in peptide binding measured in an HLA‐A2 reconstitution assay and presentation to AAGIGILTV‐specific polyclonal or clonal T lymphocytes as measured by cytotoxicity, or interferon (IFN)‐γ and granulocyte/macrophage colony‐stimulating factor (GM‐CSF) secretion. The high‐affinity AAGIGILTV peptide bound to all position‐97 mutants, albeit with differential efficiencies, and elicited specific release of IFN‐γ and GM‐CSF by CTL. CTL responses were triggered only by the HLA‐A2 wild type, by HLA‐A2‐H97 (histidine position 97 mutant), and HLA‐A2‐W97. The HLA‐A2‐M97 presenting molecule elicited enhanced cytokine release and CTL effector functions by polyclonal and by clonal effector T cells. These results indicate that MHC class I‐bound peptides can trigger specific cytokine release by effector T cells independently of their ability to induce cytolysis. We conclude that relatively minor changes in the MHC class I peptide binding groove, including substitutions at position 97, can affect recognition by antigen‐specific T cells. Mutant MHC class I molecules, such as those described here, may act as partial peptide antagonists and could be usefol for inducing T lymphocytes with qualitatively different effector functions.
Suppression of adjuvant arthritis in rats by induction of oral tolerance to mycobacterial 65‐kDa heat shock protein
Authors: Haque M. Azizul , Yoshino Shin , Inada Shigeyasu , Nomaguchi Hiroko , Tokunaga Osamu , Kohashi Osamu
Published: March 2007

Oral administration of mycobacterial 65‐kDa heat shock protein (HSP) given daily for 5 days prior to immunization with Mycobacterium tuberculosis (Mt) suppressed the development of adjuvant arthritis (AA) in rats. AA was significantly suppressed by 30 and 300 μg HSP, and variably by 0.3, 3 μg or 1 mg. Histological analysis of joint samples obtained from control and test rats confirmed the suppression of AA in the fed group. Feeding Mt or hen egg lysozyme (HEL) failed to affect AA, indicating that the suppression was HSP specific. The oral administration of 30 μg HSP decreased both delayed‐type hypersensitivity (DTH) reactions and proliferative responses to HSP and Mt. In addition, the proliferation of lymph node cells (LNC) from Mt‐sensitized rats was inhibited by the addition of spleen cells (SPC) from HSP‐fed animals, possibly by the secretion of transforming growth factor (TGF)‐β. Spleen cells obtained from tolerized donors were capable of transferring the tolerance to naive recipients. These results demonstrate that feeding HSP is an effective way to suppress AA and that the suppression of AA may be mediated by regulatory T cells generated following oral administration of mycobacterial 65‐kDa HSP.
CD9, CD63, CD81, and CD82 are components of a surface tetraspan network connected to HLA‐DR and VLA integrins
Authors: Rubinstein Eric , Le Naour François , Lagaudrière‐Gesbert Cécile , Billard Martine , Conjeaud Hélène , Boucheix Claude
Published: March 2007

CD9, CD63, CD81, and CD82 are glycoproteins of unknown function which belong to the tetraspan superfamily. These molecules have short cytoplasmic sequences, four transmembrane domains and two unequal extracellular regions. Here, we show that these molecules are associated with each other on cell surface and with other glycoproteins such as very late antigen (VLA) integrins and HLA‐DR antigens. Moreover, the VLA integrins and HLA‐DR antigens were also found to be associated. The interactions of these molecules were analyzed by transfection experiments. It is demonstrated that overexpression of CD9 antigen in Raji cells leads to a lower efficiency of precipitation of CD81 and CD82, suggesting a direct interaction between these molecules. In these cells, the coprecipitation of CD81 and CD82 was not modified, suggesting that these tetraspans did not compete for association. However, in COS‐7 cells, transfection of both CD81 and CD82 led to a marked reduction of the number of CD9/CD81 or CD9/CD82 complexes compared to single‐transfected cells, and this was associated with the appearance of CD81/CD82 complexes. Therefore, in this cellular system, CD9 competes with CD81 and CD82 for association with the other tetraspan proteins. Finally, the tetraspans do not compete for the association with integrins or HLA‐DR. Indeed, when CD9 was expressed in Raji cells, it was incorporated into the pre‐existing complexes of these molecules with CD81 and CD82. These data suggest the existence of a tetraspan network which, by connecting several molecules, may organize the positioning of cell surface proteins and play a role in signal transduction, cell adhesion, and motility.
CD4+CD8− thymocytes that express L‐selectin protect rats from diabetes upon adoptive transfer
Authors: Seddon Ben , Saoudi Abdelhadi , Nicholson Martin , Mason Don
Published: March 2007

Previous studies have shown that insulin‐dependent diabetes can be induced in normal PVG.RT1u rats by a protocol of adult thymectomy and irradiation. The injection of CD4+ T cells from non‐irradiated syngeneic donors prevents the onset of disease in approximately 50 % of pre‐diabetic recipients but all rats are protected if a particular subset of CD4+ cells is transferred. These protective cells express TCRαβ and have a memory phenotype, being CD45RClow RT6+. Further studies have demonstrated that the transfer of CD4+CD8− thymocytes, like that of unfractionated CD4+ peripheral T cells, also protects approximately half of recipients from diabetes suggesting that, as with the peripheral T cells, a functional heterogeneity may exist amongst CD4+CD8− thymocytes. In this study, we show that L‐selectin is expressed by 50–60 % of all CD4+CD8− thymocytes from 6‐week‐old rats. Adoptive transfer of these populations into thymectomized and irradiated rats revealed that the protection from diabetes observed by CD4+CD8− thymocytes was mediated almost entirely by the L‐selectin+ subset. Cells with this phenotype were also able to mediate both humoral and cell mediated responses, providing primed B cells with help for secondary antibody responses and mediating local graft‐versus‐host reactions. L‐selectin− CD4+CD8− thymocytes failed to mediate these responses. These data indicate that CD4+CD8− thymocytes must mature to the stage of L‐selectin expression, before they can mediate normal T cell function. The implications of these results are discussed with respect to the possible role of murine NK1.1+ thymocytes in the control of autoimmunity.
Vascular cell adhesion molecule‐1 expression by hematopoiesis‐supporting stromal cells is not essential for lymphoid or myeloid differentiation in vivo or in vitro
Authors: Friedrich Christof , Cybulsky Myron I. , Gutierrez‐Ramos Jose‐Carlos
Published: March 2007

Binding of myeloid and lymphoid precursors to stromal cells in bone marrow has been suggested to be mediated through α4 integrins (α4) and vascular cell adhesion molecule‐1 (VCAM‐1) expressed on hematopoietic progenitors and stromal cells, respectively. It has not been shown, however, how essential the VCAM‐1/α4 interaction is for hematopoiesis in vivo and whether or not other adhesion pathways can provide similar functional binding between stromal cells and hematopoietic progenitors. We addressed this issue by analyzing myeloid and lymphoid differentiation in vivo in mice with VCAM‐1‐null or ‐hypomorphic mutations and in vitro in long‐term hematopoietic cultures with stromal cell clones from wild‐type mice, which express or do not express VCAM‐1. Mice bearing VCAM‐1 mutations had no gross hematopoietic insufficiencies in the myeloid or lymphoid compartments and the distribution of myeloid progenitors between bone marrow and periphery was normal. In Dexter type long‐term bone marrow cultures from mutant mice, the formation of supportive stromal cell layers and myeloid proliferation and differentiation were not affected by the absence of VCAM‐1. Long‐term maintenance and proliferation of clonable pre‐B cells, cobblestone formation and differentiation to IgM‐secreting, mature B cells was equally possible on VCAM‐1+ and VCAM‐1− stromal cell clones. We conclude from our data that VCAM‐1 is not essential for the functional interaction between hematopoietic progenitors and stromal cells required for myeloid and B‐lymphoid development in vivo or in vitro.
‘Empty’ Ld molecules capture peptides from endocytosed hepatitis B surface antigen particles for major histocompatibility complex class I‐restricted presentation
Authors: Schirmbeck Reinhold , Reimann Jörg
Published: March 2007

Peptides recognized by CD8+ cytotoxic T lymphocytes in the context of major histocompatibility complex (MHC) class I molecules are usually derived from endogenous proteins synthesized within the cell. Exogenous 22‐nm hepatitis B surface antigen (HBsAg) particles are taken up by many cells, and are processed in a novel peptide‐transporter‐independent, endosomal or lysosomal pathway for class I (Ld)‐restricted epitope presentation. Here, we present evidence that ‘empty’ Ld molecules derived from the cell surface are involved in presenting antigenic peptides from endocytosed HBsAg particles. Intracellular assembly of presentation‐competent, trimeric Ld molecules required endocytosis of the exogenous antigen and ‘empty’m Ld molecules. These data assign a functional role to surface‐associated, ‘empty’ MHC class I molecules.
Differential interaction of the CD2 extracellular and intracellular domains with the tyrosine phosphatase CD45 and the ζ chain of the TCR/CD3/ζ complex
Authors: Verhagen Anne M. , Schraven Burkhart , Wild Martin , Wallich Reinhard , Meuer Stefan C.
Published: March 2007

T cell activation via CD2 requires interaction of CD2 with several signaling molecules. To investigate the structural requirements for an association of CD2 with the tyrosine phosphatase CD45 and the ζ chain of the T cell receptor (TCR)/CD3/ζ complex, we have expressed in mouse EL4 T cells a series of human CD2 chimeric and mutant proteins. Chimeric proteins in which the CD2 transmembrane and/or cytoplasmic domains were deleted or exchanged with analogous regions of CD4, CD28 or CD58 retained association with high levels of murine CD45 phosphatase activity, suggesting that the CD2 extracellular domain largely controls interaction with CD45. To a lesser extent, the cytoplasmic domain of CD2 was also shown to interact with CD45, as demonstrated by an increase in co‐immunoprecipitated phosphatase activity observed following replacement of the CD58 cytoplasmic domain with that of CD2. In contrast, the cytoplasmic domain of CD2 was found to be responsible for the majority of CD2 interaction with the ζ chain of the TCR/CD3/ζ complex. Deletion of the CD2 cytoplasmic domain, excluding the first three amino acids, removed virtually all CD2 associated ζ chain and approximately sevenfold higher levels of ζ chain were found in association with a CD58/58/2 chimera than with control human CD58 wild type. This study suggests that the CD2 extracellular and intracellular domains are differentially involved in regulating T cell activation through interaction with the tyrosine phosphatase CD45 and the ζ chain of the TCR/CD3/ζ complex.
Inhibition of experimental autoimmune myasthenia gravis by major histocompatibility complex class II competitor peptides results not only in a suppressed but also in an altered immune response
Authors: Wauben Marca H. M. , Hoedemaekers Astrid C. W. E. , Graus Yvo M. F. , Wagenaar Josee P. A. , van Eden Willem , De Baets Marc H.
Published: March 2007

To assess the capacity of major histocompatibility complex (MHC) class II‐binding competitor peptides in inhibiting antibody‐mediated disease processes, we studied experimental autoimmune myasthenia gravis in Lewis rats. Experimental autoimmune myasthenia gravis, a disease model mediated by T cell‐dependent autoantibodies against acetylcholine receptors, was induced by immunization with Torpedo californica acetylcholine receptor emulsified in complete Freund's adjuvant. The immunodominant acetylcholine receptor T cell epitope was recognized by T cells in the context of MHC class II RT1.BL. The disease inhibitory capacity of RT1.BL‐binding peptides not related to the acetylcholine receptor was determined upon co‐immunization with Torpedo acetylcholine receptor. Co‐immunization of peptide OVA323–339, a strong RT1.BL‐binding competitor peptide, resulted in complete disease inhibition. Although, the priming of the anti‐acetylcholine receptor T cell response was not fully inhibited, the kinetics of the response was changed. Moreover, besides a drastic reduction of the anti‐Torpedo acetylcholine receptor antibody titers, a shift in isotype distribution was found. These findings indicate that antibody‐mediated autoimmune processes can be suppressed by MHC class II competitor peptides. Furthermore, the administration of such peptides in vivo not only passively inhibits T cell activation, but also functionally alters the immune response.
Interleukin‐12 activates human γδ T cells: synergistic effect of tumor necrosis factor‐α
Authors: Ueta Chisato , Kawasumi Hiromi , Fujiwara Hiroshi , Miyagawa Toshi , Kida Hiroshi , Ohmoto Yasukazu , Kishimoto Susumu , Tsuyuguchi Izuo
Published: March 2007

γδ T cell populations are known to expand in response to intracellular bacterial infectious agents regardless of previous priming. We have shown previously that soluble factor(s) produced by Mycobacterium‐stimulated monocytes activate cord blood γδ T cells to proliferate. In this study, we investigated whether cytokines produced by monocytes are responsible for γδ T cell activation in vitro: interleukin (IL)‐1β, IL‐6, IL‐8, IL‐12, tumor necrosis factor (TNF)‐α and granulocyte/macrophage colony‐stimulating factor were examined. Recombinant human IL‐12 stimulated γδ T cells, but not αβ T cells in peripheral blood mononuclear cells, to express CD25 on their surfaces, and to expand in number in vitro. IL‐12‐primed γδ T cell numbers increased to a greater extent in the culture to which exogenous IL‐2 (5 U/ml) was added. Anti‐TNF‐α monoclonal antibody inhibited IL‐12‐induced up‐regulation of CD25 on γδ T cells, suggesting that endogenous TNF‐α may play a role in IL‐12‐induced activation of γδ T cells. Recombinant TNF‐α synergistically augmented IL‐12‐induced activation of γδ T cells. Furthermore, IL‐12 up‐regulated TNF receptors on γδ T cells in vitro: TNF‐α binding to its receptor induced CD25 expression on the γδ T cells in an autocrine or paracrine fashion, or perhaps both. It also became evident that both IL‐12 and TNF‐α were produced by mycobacterial lysate‐stimulated monocytes. Taken together, these results suggest that upon confrontation with mycobacterial organisms, γδ T cells can be quickly and antigen‐nonspecifically activated by soluble factors including IL‐12 and TNF‐α, both of which are produced by mononuclear phagocytes in response to mycobacterial organisms.
Characterization of the expression and gene promoter of CD22 in murine B cells
Authors: Andersson Kristin Brevik , Draves Kevin E. , Magaletti Dario M. , Fujioka Sean , Holmes Kevin L. , Law Che‐Leung , Clark Edward A.
Published: March 2007

CD22 is a B cell‐restricted surface molecule which may play an important role in interactions between B cells and other cells and in regulating signals through the B cell receptor (BCR) complex. Here we have examined whether the mouse is a suitable in vivo model for studying CD22 functions. In primary and secondary lymphoid organs of adult mice CD22 is on all mature B cells, including resting IgM+IgD+ B cells, IgG+ HSAlo memory B cells, syndecan+ plasma cells and CD5+ B cells, but it is not on immature IgM+IgD− B cells. Biochemical analysis revealed that murine CD22 is associated with the IgM receptor in some, but not all, CD22+ B leukemic and lymphoma cell lines; as with human CD22, murine CD22 is rapidly phosphorylated on tyrosine after ligation of the BCR. In the CD22− murine pro‐B cell line, FEMCL, CD22 expression was inducible by treatment with phorbol 12‐myristate 13‐acetate. A genomic fragment of the cd22b allele containing 1.3 kb 5′ of exon 1 was sequenced in order to identify potential DNA regulatory elements in the CD22 promoter region. Consensus sequences for transcription factor binding sites including PU.1, AP‐1, AP‐2, C/EBP and SP‐1 were present, but no classical TATA elements or initiator motifs were evident at relevant positions. The 1.3‐kb promoter fragment 5′ of exon 1 was sufficient for directing basal promoter activity in B and T cells. There was no significant sequence similarity between the murine and human cd22 gene promoters, although both contain repetitive elements and Sp‐1 and AP1 binding sites. Thus, murine CD22 shares a number of features with human CD22 and the mouse provides a suitable model system for elucidating the function of CD22 in vivo.
Split activity of interleukin‐10 on antigen capture and antigen presentation by human dendritic cells: Definition of a maturative step
Authors: Morel Anne‐Sophie , Quaratino Sonia , Douek Daniel Cesar , Londei Marco
Published: March 2007

Human CD1+ CD14‐ dendritic cells (DC) can be derived from CD14+ monocytes using granulocyte/monocyte colony‐stimulating factor and interleukin (IL)‐4. We have previously shown that IL‐10 pre‐treatment of such DC significantly inhibited their antigen‐presenting capacity to CD4+ T cell clones. In this study, we further analyze how IL‐10 influences antigen presentation. We first investigated whether IL‐10 could alter the early stage of antigen presentation, the capture of antigen. This can be mediated by mannose receptor (MR)‐mediated endocytosis and by fluid‐phase uptake through macropinocytosis. IL‐10‐treated DC showed an enhancement of both mechanisms of antigen capture, as indicated by the increase of fluorescein isothiocyanate‐dextran uptake through MR and lucifer yellow uptake. However, IL‐10‐treated DC, irradiated or glutaraldehyde‐fixed, were less efficient than untreated DC in stimulating mixed leukocyte reaction as well as in inducing the activation of peptide‐specific T cell clones, indicating that IL‐10 achieves its effects mainly by modifying the cell surface phenotype of DC. HLA class I and II, as well as intercellular adhesion molecule (ICAM)‐1, lymphocyte function‐associated antigen‐3, B7‐1, B7‐2 and ICAM‐3 expression were either significantly increased or essentially unchanged, and the ability to bind the epitope recognized by the T cell clones was also unaffected regardless of IL‐10 treatment. Our study also indicates that as‐yet unidentified accessory molecules may play an essential role in T cell activation. Thus, the IL‐10‐treated DC possess an increased capacity to capture antigen, with a concomitant decreased stimulatory activity. Our study suggests that IL‐10‐treated DC have the characteristics of highly immature DC (high capture ability, low stimulatory potency) and may represent an early maturative step of human DC of monocytic origin.
CD21 augments antigen presentation in immune individuals
Authors: Boackle Susan A. , Holers V. Michael , Karp David R.
Published: March 2007

CD21 (complement receptor 2, CR2) binds the C3 degradation products, iC3b and C3d, which are covalently linked to antigen or immune complexes in the process of complement activation. The ability of antigen‐nonspecific B cells to present immune complexes containing high titers of acquired antibodies was tested. Influenza virus was incubated with serum from immune donors to create complement‐containing complexes. These bound specifically to CD21 on transfected fibroblasts and B cell lines, as measured by microcytofluorimetry. Binding of immune complexes was ablated by inactivation of serum complement. In addition, the immunoglobulin in immune human serum blocked influenza binding to cells in the absence of complement, implying a minimal role for immunoglobulin‐Fc receptor interactions in this system. Significant immune complex binding required a threshold level of CD21 expression, suggesting that only those cells with the highest levels of CD21 are likely to participate in the processing of macromolecular antigens. B cells pulsed with complement‐influenza complexes elicited an augmented response from a panel of influenza‐specific, class II‐restricted T cell clones, as compared with those which had bound immunoglobulin‐influenza complexes lacking complement. This enhanced response did not require CD35. In addition, B cell lines expressing higher levels of CD21 were more efficient in processing antigen than those with lower levels. These findings suggest that presentation of antigen by B cells in immune individuals is dependent on the binding of complement‐antigen immune complexes to CD21.
Identification and analysis of the chicken CD3ε gene
Authors: Göbel Thomas W. F. , Fluri Monika
Published: March 2007

The chicken T cell receptor CD3ε gene was isolated using a degenerate polymerase chain reaction. The 1883 bp long cDNA encoded a transmembrane protein of 16.9 kDa lacking N‐linked glycosylation sites. Comparison of the chicken and mammalian CD3ε proteins revealed low homology in the extracellular domain with clusters of similarities located around the N‐terminal cysteine residue and proximal to the transmembrane region. The high conservation of the cytoplasmic domain included motifs important for signal transduction. The alignment of all CD3γ, CD3δ and CD3ε proteins allowed the identification of highly conserved residues and motifs. Southern blot analysis indicated the presence of a single copy CD3ε gene. The expression of the CD3ε transcript was limited to T cells and natural killer cells. A recessive mutation of the CD3ε gene in the CB chicken strain enabled the mapping of the epitope recognized by the CT3 monoclonal antibody. This analysis of the first non‐mammalian CD3ε gene provides novel information about evolutionary conserved structural features and its expression in natural killer cells.
Genetic defect in T lymphocyte‐specific homing into peripheral lymph nodes
Authors: Nakano Hideki , Tamura Toshiki , Yoshimoto Takayuki , Yagita Hideo , Miyasaka Masayuki , Butcher Eugene C. , Nariuchi Hideo , Kakiuchi Terutaka , Matsuzawa Akio
Published: March 2007

Lymphocytes circulating in the bloodstream home into lymph nodes (LN). T cells predominate in peripheral LN (PLN) and B cells in spleen or mucosal tissue, e.g. Peyer's patches (PP). DDD/1 mice are unique in marked paucity of LN cells, especially T cells. T cell frequency in PLN was 20–40% in this strain, compared to 60–80% in others. Immunohistochemistry confirmed the low density of T cells in the subcortical area but normal colonization of B cells in cortical area in PLN of DDD/1. In contrast, the T cell content of peripheral blood and spleen was higher in DDD/1 but that in PP was not significantly different compared to other strains. It was thus concluded that this abnormality in DDD/1 results from a homing defect of T cells into PLN but not from lymphopenia. Genetical analysis showed that the defect in T cell‐specific homing was regulated by a single autosomal recessive gene, tentatively designated plt (paucity of lymph node T cells). Reciprocal bone marrow transplantation indicated that the plt phenotype may arise from some defect in PLN stroma but not in lymphocytes. An in vivo homing assay using fluorescence‐labeled lymphocytes demonstrated that the homing defect was specific for T cells but not for B cells. A Stamper‐Woodruff assay revealed that the binding between lymphocytes and PLN high endothelial venules was normal and that L‐selectin and its ligand, peripheral node vascular addressin (PNAd), were expressed and functioned normally in DDD/1. These results taken together indicate that the T cell‐specific homing into PLN is disturbed at a post‐adhesion stage in DDD/1. The product of the plt locus may play a pivotal role at this stage.
Synergy between an antibody and CD8+ cells in eliminating an established tumor
Authors: Vasović Ljiljana V. , Dyall Ruben , Clynes Raphael A. , Ravetch Jeffrey V. , Nikolić‐Žugić Janko
Published: March 2007

We investigated the effector mechanisms operating during the rejection of a transplantable solid lymphoma E.G7 (H‐2b) which expresses the gene encoding chicken ovalbumin (OVA). Anti‐OVA cytotoxic T lymphocytes (CTL) completely and specifically protected animals from the onset of, but could not eradicate established, E.G7 tumors. The growth of the same lymphoma was also effectively prevented by the antibody GK1.5, whose target molecule, CD4, was expressed on E.G7 cells in vivo. Furthermore, GK1.5 was able to eradicate established solid E.G7 tumors. GK1.5‐mediated tumor elimination was due to its antitumor activity, and not to the elimination of regulatory CD4+ cells, based on unimpaired tumor growth in the absence of GK1.5 in animals that genetically lack CD4 T cells. In vitro, GK1.5 did not kill tumor cells: complement activation or apoptosis induction were not evident. In vivo, GK1.5‐mediated tumor regression did not depend on natural killer cells, but it absolutely required CD8+ cells and intact Fcγ receptor. We conclude that, in the E.G7 model, the collaboration of antibody and CTL immunity was crucial for the successful immunotherapy of established tumors. The mechanism of this collaboration is discussed.
T cell sensitivity to HLA class I‐mediated apoptosis is dependent on interleukin‐2 and interleukin‐4
Authors: Genestier Laurent , Paillot Romain , Bonnefoy‐Berard Nathalie , Waldmann Herman , Revillard Jean‐Pierre
Published: March 2007

Antibody interaction with a specific epitope of the HLA class I α1 domain triggers apoptosis of activated but not resting T and B cells by a pathway which involves neither Fas ligand nor tumor necrosis factor‐α. We have investigated at which stage of activation and proliferation T cells become sensitive to HLA class I‐mediated apoptosis, using two monoclonal antibodies (mAb) which recognize the same monomorphic epitope of the HLA class I α1 domain (mAb90, mouse IgG1, and YTH862, rat IgG2b) and can induce apoptosis of phytohemagglutinin (PHA)‐activated peripheral blood lymphocytes. Sensitivity to apoptosis develops after the expression of G1 markers (CD69 expression) but it is accelerated by addition of recombinant interleukin‐2 (rIL‐2). Blocking the IL‐2 pathway by cyclosporin A, FK506, rapamycin, anti‐IL‐2 or CD25 antibodies, prevented the development of sensitivity to apoptosis. Addition of IL‐2 and, to a lesser extent, IL‐4, reversed the inhibitory effect of cyclosporin A. Conversely, rIL‐7 and recombinant interferon‐γ restored proliferation of peripheral blood lymphocytes stimulated by PHA in the presence of cyclosporin A but did not restore sensitivity to class I‐mediated apoptosis. Finally cells stimulated in the presence of the DNA polymerase inhibitor aphidicolin did not enter into S phase of the cell cycle but secreted IL‐2 and underwent apoptosis when exposed to mAb90 or YTH862. Together, the data indicate that sensitivity of peripheral T cells to HLA class I‐mediated apoptosis depends on both activation signals and IL‐2 or IL‐4, but does not require cell proliferation. These data suggest that YTH862 and mAb90 might be used for achieving clonal deletion of antigen‐activated peripheral T cells in vivo, provided that the IL‐2 pathway is not blocked by other immunosuppressive agents.
A novel subset of adult γδ thymocytes that secretes a distinct pattern of cytokines and expresses a very restricted T cell receptor repertoire
Authors: Azuara Véronique , Levraud Jean‐Pierre , Lembezat Marie‐Pierre , Pereira Pablo
Published: March 2007

We have characterized the function, phenotype, ontogenic development, and T cell receptor (TCR) repertoire of a subpopulation of γδ thymocytes, initially defined by expressing low levels of Thy‐1, that represents around 5 % and 30 % of total γδ thymocytes in adult C57BL/6 and DBA/2 mice, respectively. Activation of FACS‐sorted Thy‐1dull γδ thymocytes from DBA/2 mice with anti‐γδ monoclonal antibodies in the presence of interleukin‐2 (IL‐2) results in the secretion of high levels of several cytokines, including interferon‐γ (IFN‐γ), IL‐4, IL‐10, and IL‐3. In contrast, only IFN‐γ was detected in parallel cultures of Thy‐1bright γδ thymocytes. Virtually all Thy‐1dull γδ thymocytes express high levels of CD44 and low levels of the heat‐stable antigen and CD62 ligand, while around half of them express the NK1.1 marker. Thy‐1dull γδ thymocytes are barely detectable in newborn animals, and their representation increases considerably during the first 2 weeks of postnatal life. The majority of Thy‐1dull γδ thymocytes from DBA/2 mice express TCR encoded by the Vγ1 gene and a novel Vδ6 gene named Vδ6.4. Sequence analysis of these functionally rearranged γ and δ genes revealed highly restricted Vδ‐Dδ‐Jδ junctions, and somewhat more diverse Vγ‐Jγ junctions. We conclude that Thy‐1dull γδ thymocytes exhibit properties that are equivalent to those of natural killer TCRαβ T cells. Both cell populations produce the same distinct pattern of cytokines upon activation, share a number of phenotypic markers originally defined for activated or memory T cells, display similar postnatal kinetics of appearance in the thymus and express a very restricted TCR repertoire.
Regulatory role of CD95 ligation on human B cells induced in vivo capable of spontaneous and highrate Ig secretion
Authors: Medina Francisco , Segundo Carmen , Rodríguez Carmen , Brieva José A.
Published: March 2007

CD95 ligation elicits apoptotic signals in many cell systems. This study analyzes the effect of anti‐CD95 mAb on human cells capable of spontaneous and highrate Ig secretion. Such cells have been induced in vivo and represent a highly mature B cell stage. Addition of the anti‐CD95 monoclonal antibody (mAb) CH11 to tonsil B cells inhibited 50–60% of their spontaneous Ig secretion. The effect was exerted early in the culture and could be reversed by a pre‐treatment with a neutralizing mAb. N‐acetyl‐D‐sphingosine (C2‐ceramide), although not a close analog, also reduced Ig secretion to a similar extent. The inclusion of a tetrapeptide inhibitor for certain interleukin‐1β‐converting enzyme proteases prevented the inhibitory effect of CH11 mAb on tonsil B cells. B cells capable of spontaneous Ab secretion obtained from blood of recently‐immunized volunteers were also inhibited by CH11 mAb and C2‐ceramide. In contrast, bone marrow (BM) B cells capable of spontaneous Ig secretion were unaffected by these agents. This CD95 ligation‐mediated inhibition of tonsil and blood Ig‐secreting B cells could not be reversed by cytokines with demonstrated activity on these B cells. Human mature B cells induced in vivo are identifiable as CD38hi cells. Flow cytometric analysis revealed that a fraction of tonsil CD38hi cells expressed low levels of CD95. Moreover, about 20% of these cells exhibited basal apoptosis, as defined by annexin V binding. This phenomenon was markedly increased by CD95 ligation. On the other hand, BM CD38hi cells showed neither CD95 expression nor CD95‐induced annexin V binding. These data suggest that CD95 ligation might play a role in the control of human humoral responses by inducing apoptosis in susceptible mature B cells.
Maternal IgG stimulates B lineage cell development in the progeny
Authors: Malanchère Evelyne , Huetz François , Coutinho Antonio
Published: March 2007

To examine the physiological role of maternal natural IgG antibodies on the development of B lineage cells of the progeny, we have bred homozygous μMT/μMT or heterozygous μMT/+ females to μMT/μMT or μMT/+ males, respectively. We could thus compare normal or B cell‐deficient mice born from Ig‐deprived (Ig−) or phenotypically normal mothers (Ig+). B cell‐deficient progeny of heterozygous mothers contain no detectable serum IgA or IgM, but IgG concentrations that peak at 2 mg/ml by 7–21 days of age, decay after weaning with a half‐life of 7 days, and remain detectable for 2 months after birth. At 7 days after birth, μMT/+ progeny born of Ig+ mothers contain two‐ to threefold higher numbers of bone marrow (BM) pre‐B and B cells, and of splenic B cells, compared to mice of the same age born from Ig− mothers. In contrast, the former progeny exhibit two to four times lower numbers of Ig‐secreting plasma cells in spleen and thymus, and contain sixfold lower serum IgM concentrations. A similar maternal IgG‐dependent stimulation of BM B cell precursors is also observed in μMT/μMT progeny. No significant differences were detected between the groups on day 3 after birth, suggesting the requirement for a minimal IgG concentration in the serum.