bluuebunny/crossref_metadata_2025 huggingface dataset:
Major histocompatibility complex class II‐restricted presentation of secreted and endoplasmic reticulum resident antigens requires the invariant chains and is sensitive to lysosomotropic agents
Authors: Humbert Martine , Bertolino Patrick , Forquet Frédérique , Rabourdin‐Combe Chantal , Gerlier Denis , Davoust Jean , Salamero Jean
Published: March 2007

We have tested the involvement of the invariant chains (Ii) p31 and p41 in the presentation of peptides derived from hen egg lysozyme (HEL) constructs targeted to different intracellular compartments within transfected fibroblasts. The endogenous HEL constructs were either present in the cytosol (HELc), secreted (HELs), or linked to the mammalian (KDEL C‐terminal sequence that causes retention of HEL in the endoplasmic reticulum (ER)/pre‐Golgi recycling compartment (HELr). Using Ii‐negative antigen‐presenting cells, the presentation of HELr to a HEL 46‐61 specific T cell hybridoma was far less efficient than the presentation of the HELs. High levels of Ii expression enhanced drastically the presentation of the HEL 46‐61 determinant derived from both HELr and HELs. HELr and HELs presentation was fully sensitive to lysosomotropic agents such as chloroquine, indicating that the formation of complexes between major histocompatibility complex (MHC) class II molecules and determinants derived from endogenous antigens entering the secretory pathway is taking place in an acidic compartment. The degradation and dissociation of Ii might be a prerequisite for the efficient presentation of endogenously derived determinants by MHC class II molecules, as for the presentation of most exogenous antigens. All our results are compatible with the notion that endogenous molecules being translocated into the lumen of the ER could be presented by class II molecules through a processing pathway involving an acidic compartment in which Ii chains dissociate from class II molecules.
Selection of internalization‐deficient cells by interleukin‐2‐Pseudomonas exotoxin chimeric protein: the cytoplasmic domain of the interleukin‐2 receptor β chain does not contribute to internalization of interleukin‐2
Authors: Furse Robert K. , Malek Thomas R.
Published: March 2007

To study the structural basis of ligand‐induced receptor‐mediated internalization of interleukin‐2 (IL‐2), a strategy has been developed to generate variant T cells that are deficient in internalization of this cytokine. IL‐2 receptor (IL‐2R) α‐ and β‐bearing EL4 cells, that express high‐affinity IL‐2R and internalize IL‐2, were treated with low doses of IL‐2‐Pseudomonas exotoxin chimeric protein (IL‐2‐PE40). This treatment resulted in isolation of a variant (CX1) that was unable to express high‐affinity IL‐2R or internalize IL‐2. Transfection of CX1 with the IL‐2Rβ cDNA led to surface expression of IL‐2Rβ and high‐affinity IL‐2R as well as the ability to internalize IL‐2. This finding indicates that the absence of the p subunit was the sole defect in CX1 responsible for its failure to internalize IL‐2. By transfecting CX1 with mutated β cDNA, several CX1 transfectants were produced that expressed a β‐subunit that lacked all amino acids of the intracytoplasmic region. These transfectants expressed high‐affinity IL‐2R and internalized IL‐2 at a rate comparable to cells expressing wild‐type β‐chain. These results demonstrate that internalization of IL‐2 is independent of any signals contained in the intracytoplasmic tail of the β subunit and raise the possibility that such signals may be entirely contained within the γ subunit.
HLA class II molecule‐mediated signal transduction mechanism responsible for the expression of interleukin‐1β and tumor necrosis factor‐α genes induced by a staphylococcal superantigen
Authors: Matsuyama Satoru , Koide Yukio , Yoshida Takato O.
Published: March 2007

Superantigens including staphylococcal enterotoxins (SE) bind to major histocompatibility complex class II molecules and interact with T cells bearing particular vβ chains. SEB was shown to induce the expression of interleukin (IL)‐1β and tumor necrosis factor (TNF)‐α genes in human peripheral blood monocytes bearing HLA class II molecules. Monoclonal antibodies directed against HLA‐DR and ‐DQ abolished the SEB‐induced expression of both the EL‐1β and TNF‐α genes, suggesting that the HLA class II molecules mediated the gene expression. Therefore, we investigated the signal transduction mechanism responsible for the expression of IL‐1β and TNF‐α genes induced by binding of SEB to the HLA class II molecules. Three protein tyrosine kinase (PTK) inhibitors, genistein, herbimycin A., and tyrphostin, each of which has a different mechanism of action, strongly inhibited the expression of the monokine mRNA induced by SEB. Analyses of PTK activity revealed that SEB induced a rapid increase of membrane‐associated PTK activity and this was blocked by tyrphostin. Furthermore, H‐7 inhibited the expression of the monokine mRNA induced by SEB, suggesting the involvement of protein kinase C (PKC) in the signaling pathway. The involvement of PKC was confirmed by the observations that phorbol 12‐myristate 13‐acetate (PMA), a direct activator of PKC, induced the expression of the monokine mRNA and that SEB evoked the activation of membrane‐associated PKC. Both activation of PKC and expression of the monokine mRNA induced by SEB appeared to be inhibited by tyrphostin, but those induced by PMA were not. Taken together, these findings indicate that both PTK and PKC play essential roles in HLA class II molecule‐mediated signal transduction elicited by SEB and that PTK activation may precede PKC activation in the signaling pathway.
Half‐life of antigen/major histocompatibility complex class II complexes in vivo: intra‐ and interorgan variations
Authors: Müller Klaus‐Peter , Schumacher Jochen , Kyewski Bruno A.
Published: March 2007

We have determined the half‐life in vivo of antigen/MHC class II complexes in different organ microenvironments. Mice were “pulsed” with myoglobin intravenously and MHC class II‐positive antigen‐presenting cell (APC) populations from different organs were isolated after various time intervals. Specific antigen/MHC complexes were quantitated by co‐cultivation of the APC subsets with myoglobin‐specific T‐T hybridoma cells in vitro. Half‐lives of antigen/MHC complexes differed both between organs and between compartments of the same organ. Half‐lives in peripheral organs (spleen and bone marrow) ranged between 3 and 8 h, whereas in the thymus half‐lives between 13 h (cortical epithelial cells) and 22 h (medullary dendritic cells) were observed. Half lives in vivo were independent of antigen processing, since intact protein or antigenic peptides yielded similar values. The considerably longer half‐life of peptide/MHC complexes in the thymus as compared to peripheral organs may reflect the distinct role which antigen presentation plays in both organs, i.e. induction of tolerance versus induction of immunity.
Switching capacity of FcϵRII‐positive and ‐negative murine B cells
Authors: Foy Teresa M. , Waldschmidt Thomas J.
Published: March 2007

In previous studies, our laboratory demonstrated the utility of the low affinity IgE Fc receptor (FcϵRII) in delineating a number of murine B cell subsets. In the spleen, the FcϵRII is expressed on mature conventional B cells but is absent on marginal zone B cells. In the peritoneal cavity, the receptor is present on all conventional B cells, but is not expressed on fresh peritoneal Ly1/sister B cells. The studies in this report compared the ability of these B cell poputations to isotype switch. Using a lipopolysaccharide (LPS)‐ and interleukin (IL)‐4‐driven system, sort‐purified FcϵRII‐ positive and ‐negative B cells from peritoneum and spleen were tested for switching to IgG1, IgE, and IgA. The results demonstrated that regardless of their source, FcϵRII+ B cells produced significant levels of IgG1 and IgE. Similar results were obtained with FcϵRII− (marginal zone) B cells obtained from spleen. In contrast, FcϵRII− (Ly1/sister) peritoneal B cells were found to produce IgG1 and IgA, but were incapable of secreting significant levels of IgE. Further studies tested for LPS and IL‐4‐induced expression of FcϵRII and Thy1 on the various B cell populations. These experiments demonstrated the induction of the FcϵRII on all B cells, regardless of their initial resting levels. Additionally, Thy1 was found to be induced only on those B cell subsets capable of producing IgE. Taken together, the results demonstrate a correlation between IgE secretion and Thy1 expression, and no apparent correlation between the presence of the FcϵRII and isotype commitment.
Peripheral clonal deletion of antiviral memory CD8+ T cells
Authors: Moskophidis Demetrius , Laine Elisabeth , Zinkernagel Rolf M.
Published: March 2007

Antiviral cytotoxic memory CD8+ T cells adoptively transferred to mice which are persistently infected with lymphocytic choriomeningitis virus WE or DOCILE initially proliferated extensively; they either caused the death of the recipient or, alternatively, disappeared within a few days. Apparently, the complete and coordinated induction and stimulation by widely distributed viral antigen caused these memory T cells to die before virus had been eliminated from the host. Thus memory T cells are as susceptible to peripheral exhaustion/deletion as unprimed T cells. These results indicate possible limitations of exclusively CD8+ T cell‐mediated adoptive immunotherapy against viral infections or tumors.
Elevated levels of T cell activation antigen CD27 and increased interleukin‐4 production in human lymphatic filariasis
Authors: Yazdanbakhsh Maria , Sartono Erliyani , Kruize Yvonne C. M. , Kumiawan Agnes , van der Pouw‐Kraan Tineke , van der Meide Peter H. , Selkirk Murray E. , Partono Felix , Hintzen Rogier Q. , van Lier René A. W. , Maizels Rick M.
Published: March 2007

To assess the immunological changes occurring during filarial infection with or without elephantiasis, 145 patients in different clinical groups from an endemic area in Indonesia were compared with respect to plasma levels of both soluble CD25 (sCD25) and sCD27; interleukin‐4 (IL‐4) and interferon‐γ release by peripheral blood mononuclear cells was also measured in a smaller subset of individuals. Levels of sCD27 were significantly elevated in elephantiasis and microfilaremic patients compared with endemic normals (p < 0.002), whereas sCD25 levels remained low in microfilaremics and was only slightly elevated in elephantiasis patients compared with endemic normals (p <0.02). As activated T cell populations release both sCD27 and sCD25, these findings imply that there is filarial‐driven activation of a Tcell subset that releases sCD27 rather than sCD25. The expansion of a particular Tcell population by filarial parasites is further suggested by the enhancement in both IL‐4‐producing and CD4+CD27−T cells in PBMC from elephantiasis and microfilaremic patients compared with endemic normals. More detailed characterization and comparison of CD27− lymphocytes from these individuals may identify mechanisms involved in the pathogenesis of lymphatic filariasis.
Identification of conserved T cell receptor CDR3 residues contacting known exposed peptide side chains from a major histocompatibility complex class I‐bound determinant
Authors: Kelly Janice M. , Sterry Sandra J. , Cose Stephen , Turner Stephen J. , Fecondo John , Rodda Stuart , Fink Pamela J. , Carbone Francis R.
Published: March 2007

We have analyzed the T cell receptor (TCR) repertoire found in the major histocompatibility complex class I‐restricted cytotoxic T lymphocyte (CTL) response to the protein ovalbumin (OVA). Despite skewing towards the expression of Vβ5.2+ TCR by OVA‐specific CTL from C57BL/6 mice, we found a relatively high degree of diversity in V(D)J usage in both TCR α‐ and β‐chains. Closer examination showed that the majority of these sequences encoded negatively and positively charged residues at their respective TCR α‐ and β‐chain VJ or VDJ junctions. These junctions form the third complementarity‐determining regions (CDR3) of the TCR polypeptides involved in the direct interaction with the class I‐bound peptide. Crystallographic analyses of Kb‐peptide complexes predict that the major determinant from OVA, peptide OVA257–264 (SIINFEKL), contains two exposed charged side chains which can contact the TCR. These are the negatively charged glutamic acid at determinant position 6 (P6) and the positively charged lysine at P7. To examine whether the TCR α‐chain makes contact with P7 lysine, we established a single chain TCR transgenic C57BL/6 mouse line where all T cells express a TCR β‐chain derived from the Vβ5.2+ clone B3. OVA‐specific T cells derived from in vivo primed transgenic mice preferentially expressed TCR α‐chains that also contained negatively charged junctional residues despite some further variation in Vα and Jα sequences. Stimulation of naive TCR β‐chain transgenic T cells with a P7 substitution peptide analogue induced a T cell response that was no longer cross‐reactive with the wild‐type OVA257–264 determinant, sugesting that the TCR α‐chain from the T cell clone B3 can determine the specificity for this residue. Consequently, these results reveal the existence of conserved residues in the CDR3 of TCR α‐ and β‐chains specific for OVA257–264 and identify their possible orientation over the peptide‐class I complex.
VH CDR3‐dependent positive selection of murine VH12‐expressing B cells in the neonate
Authors: Clarke Stephen H. , McCray Suzanne K.
Published: March 2007

Five to fifteen percent of peritoneal B1 (CD5+) cells from unmanipulated mice produce antibodies that bind bromelain‐treated mouse red blood cells and the hapten phosphatidylcholine (PtC). The majority of these B cells express either of two VH/Vϰ gene combinations, VH12/Vϰ4 or VHH/Vϰ9. Both the VH11 and VH12 genes are rearranged to JH1 and encode third complementarity determining regions (CDR3) of restricted length and sequence. These and other observations argue strongly that PtC‐specific Bl cells are antigen selected. To determine when selection of PtC‐specific Bl cells begins in mice we have used the polymerase chain reaction to amplify VH12‐D‐Jh1 rearrangements from livers of fetal and neonatal mice, and determined the CDR3 encoding sequences of individual clones. We find an unusually low ratio of productive (P) to non‐productive (NP) rearrangements (0.4–1.0) at both developmental stages. P rearrangements in day 1 neonates are biased in D gene use and in the sequence and length of their deduced VH CDR3. These biases are similar to those of PtC‐specific B1 cells in the adult peritoneum. D gene use and CDR3 length and sequence are significantly less biased among VH12 P rearrangements 2 to 3 days earlier in the day 18 fetal liver. We suggest that this rapid change in repertoire is due to positive ligand selection that is dependent on the sequence of VH CDR3. We suggest further that the majority of VH12‐expressing cells are not ligand selected and consequently undergo programmed cell death. The evidence of restriction in day 1 neonatal livers and the low P/NP ratio in the fetus suggests that selection of VH12‐expressing cells begins before birth.
Expression of the mucosal T cell integrin αM290β7 by a major subpopulation of dendritic cells in mice
Authors: Kilshaw Peter J.
Published: March 2007

Dendritic interdigitating cells isolated from lymph nodes, Langerhans' cells from skin and dendritic cells from the spleen have been tested for expression of the mucosal T cell integrin αM290β7. Almost all interdigitating cells isolated from mesenteric lymph nodes expressed the integrin at high levels; 50‐60% of those from lymph nodes draining non‐mucosal sites also expressed this adhesion molecule. In contrast, αM290β7 was not detected on either Langerhans' cells or splenic dendritic cells. Because this integrin has recently been shown to recognize a ligand that seems to be expressed mainly or exclusively by epithelial cells, it is proposed that this adhesion pathway may be important for antigen sampling by dendritic cells in mucosal tissues.
Costimulation by interleukin‐1 of multiple activation responses in a developmentally restricted subset of immature thymocytes
Authors: Rothenberg Ellen V. , Diamond Rochelle A.
Published: March 2007

An intriguing feature of thymocyte differentiation is that the competence to express both interleukin‐(IL)2 and CD25 is acquired even prior to T cell recept or (TcR) expression. When T cell receptor‐independent stimuli are used, immature cells can express IL‐2 at levels comparable to mature cells, but unlike the mature cells, immature cells require IL‐1 as a costimulus. Here we present evidence that IL‐1 affects a variety of responses by members of the CD25+ subset of immature thymocytes. Cells in this population are IL‐1 dependent not only for induction of IL‐2 expression, but also for high‐level maintenance of CD25 expression. CD25+ expression is amplified by IL‐1 through a mechanism highly sensitive to changes in Ca2+ ionophore concentration. The effects of IL‐1 on CD25 maintenance are not mediated by IL‐2, because of the divergent effects of cAMP on IL‐2 and CD25+ expression. IL‐1 costimulation also increases RNA accumulation in the cell cycle, and this effect too seems to be separable from the effects on IL‐2 and CD25+ expression. All these effects of IL‐1 are developmentally stage‐specific, manifest in the CD25+ subset of immature thymocytes but not in later‐stage thymocytes or splenic T cells. Multiparameter cell sorting experiments that dissect the transitional stages between immature and TcR+ thymocytes imply that all immature cells pass through an IL‐1 responsive state. Responsiveness to IL‐1 costimulation is then lost by these cells, apparently irreversibly, at a stage just prior to detectable cell‐surface TcR expression. These results indicate that IL‐1 responsiveness is a defining characteristic of the activation physiology of cells in a particularly important developmental stage.
Different proliferative response of human and chimpanzee lymphocytes after contact with human immunodeficiency virus type 1 gp120
Authors: Di Rienzo Anna Maria , Furlini Giuliano , Olivier René , Ferris Stephane , Heeney Jonathan , Montagnier Luc
Published: March 2007

T cell functional defects are a common aspect of human immunodeficiency virus (HIV) infection. Moreover, it has been suggested that indirect mechanisms are involved in CD4+ cell depletion. Unresponsiveness to proliferative stimuli of lymphocytes incubated with HIV particles or with viral proteins is well documented. Nevertheless, drawing a clear picture of the anergy phenomenon is difficult because of several unresolved and controversial questions. Here we report that recombinant gp120 induces anergy in T helper lymphocytes cultured with different stimuli. The proliferative responses to interleukin (IL)‐2, IL‐4, IL‐6, anti‐CD2, anti‐CD3 and phorbol 12‐myristate 13‐acetate are inhibited. Moreover, anergic cells show a different distribution in cell cycle phases as compared to control cells, leading us to suggest that the progresion in the cell cycle is hampered and that a pre‐mitotic block takes place. Furthermore, since chimpanzees are susceptible to HIV‐1 infection without showing immunodeficiency signs, we analyzed the proliferation of chimpanzee lymphocytes without observing anergy in cells preincubated with gp120. Taken together, these results support the hypothesis that anergy plays an important role in HIV infection in vivo.
Cross‐reactivity of IgG anti‐DNA‐secreting B cells in patients with systemic lupus erythematosus
Authors: Klinman Dennis M. , Shirai Akira , Conover Jacqueline , Steinberg Alfred D.
Published: March 2007

This study is the first to analyze the cross‐reactivity of in vivo activated B cells from patients with systemic lupus erythematosus. A chamber ELIspot assay was used to determine whether lymphocytes secreting antibodies that bound to DNA or 2,4,6‐trinitrophenol (TNP)‐keyhole limpet‐hemocyanin (KLH) could simultaneously bind to the unrelated antigens actin or ovalbumin.

IgM anti‐DNA‐, IgM anti‐TNP‐KLH‐ and IgG anti‐TNP‐KLH‐secreting B cells from patients and controls showed similar levels of cross‐reactivity (ranging from 6% to 23%, depending upon the antibody isotype and antigen pair examined). In general, IgG‐producing cells were less cross‐reactive than IgM producers from the same individual (on the average threefold, p < 0.001).

In contrast, IgG anti‐DNA‐secreting B cells from lupus patients (i) showed no decrease in cross‐reactivity when compared to IgM anti‐DNA‐secreting cells and (ii) were significantly more cross‐reactive than control IgG anti‐DNA‐secreting cells and IgG anti‐TNP‐KLH secreting cells from patients (p < 0.001). The degree of IgG anti‐DNA cross‐reactivity correlated with disease activity (r = 0.52, p < 0.02). The implications of these findings with respect to repertoire expression and disease pathogenesis are discussed.
The T cell antigen receptor α and β chains interact via distinct regions with CD3 chains
Authors: Manolios Nicholas , Kemp Owen , Li Zhan Guo
Published: March 2007

Selective pairwise interactions between CD3 chains and the clonotypic T cell antigen receptor (TCR)‐α, ‐β chains has recently been established. In this study, the region of interaction between clonotypic and CD3 chains involved with assembly was examined. To determine the site of protein interaction a variety of genetically altered TCR chains were constructed. These included: truncated proteins, lacking transmembrane and or cytosolic domains; chimeric proteins, in which extracellular, transmembrane or cytosolic domains were replaced with similar domains derived from either the Tac antigen or CD4; and point mutagenized TCR chains. COS‐1 cells were transfected with cDNA, metabolically labeled, and immunoprecipitates analyzed using non‐equilibrium pH gel electrophoresis (NEPHGE)‐SDS/PAGE. The results demonstrated that assembly between TCR‐α and TCR‐β chains occurred at the extracellular level. Assembly of the TCR‐α chain with CD3‐δ, and CD3‐ε was localized to an eight‐amino acid motif within the transmembrane domain of TCR‐α. Site‐specific mutations of the TCR‐α charged residues within this motif ( arginine, lysine) to leucine and similar point mutations of the transmembrane CD3‐ε and CD3‐δ charge groups resulted in the abrogation of assembly. In contast, TCR‐β and CD3‐ε binary complexes interacted via their extracellular domain. Analogous to TCR‐α, the site of TCR‐β and CD3‐δ assembly was at the transmembrane region. Despite multiple genetic manipulations on CD3‐γ and ζ; these proteins failed to assemble with TCR‐α. Similarly, there was no interaction between TCR‐β and ζ. These findings when coupled with the information on pairwise interactions and formation of higher order subcomplexes extend our model for the structure of the TCR complex.
Joint‐derived T cells in rheumatoid arthritis react with self‐immunoglobulin heavy chains or immunoglobulin‐binding proteins that copurify with immunoglobulin
Authors: van Schooten Wim C. A. , Devereux Deirdre , Ho Charles H. , Quan Jeanette , Aguilar Barbara A. , Rust Chantal J. J.
Published: March 2007

Rheumatoid arthritis patients were found to have CD4+ T cells that proliferate in response to autologous synovial fluid and plasma. T cell clones and polyclonal T cell lines were found to respond to antigen(s) eluted from protein A Sepharose and anti‐human immunoglobulin (Ig) antibody Sepharose. The antigen(s) was further resolved to fractions that contained intact Ig or Ig heavy chain since the T cells responded to > 100 kDa and 40‐‐60 kDa polypeptides derived from purified Ig under nonreducing and reducing conditions, respectively. These results indicated that the antigen(s) is either Ig heavy chain or Ig‐binding proteins that copurify with Ig and Ig subunits. Pepsin and papain digestion of the antigenic fractions eluted from protein A destroyed the T cell reactivity. Since most Fab regions are resistant to these enzymes, further analyses are required to localize the antigenic epitope(s). The presence of Ig‐ or Ig‐antigen complex‐reactive T cells in arthritic joints implies that B cells expressing anti‐Ig antibody (i.e. rheumatoid factor) may play an important role in antigen presentation to autoreactive T cells.
Assembly of monoclonal antibodies with IgG1 and IgA heavy chain domains in transgenic tobacco plants
Authors: Ma Julian K‐C. , Lehner Thomas , Stabila Paul , Fux Charles I. , Hiatt Andrew
Published: March 2007

The genes encoding the heavy and light chains of a murine monoclonal antibody (mAb Guy's 13) have been cloned and expressed in Nicotiana tabacum. Transgenic plants have been regenerated that secrete full‐length Guy's 13 antibody. By manipulation of the heavy chain gene sequence, constant region domains from an immunoglobulin alpha heavy chain have been introduced, and plants secreting Guy's 13 mAb with chimeric gamma/alpha heavy chains have also been produced. For each plant antibody, light and heavy chains have been detected by Western blot analysis and the fidelity of assembly confirmed by demonstrating that the antibody is fully functional, by antigen binding studies. Furthermore, the plant antibodies retained the ability to aggregate streptococci, which confirms that the bivalent antigen‐binding capacity of the full length antibodies is intact. The results demonstrate that IgA as well as IgG class antibodies can be assembled correctly in tobacco plants and suggest that transgenic plants may be suitable for high‐level expression of more complex genetically engineered immunoglobulin molecules. Since mAb Guy's 13 prevents streptococcal colonization in humans, transgenic plant technology may have therapeutic applications.
Infiltrating T cells from patients with primary Sjögren's syndrome express restricted or unrestricted T cell receptor Vβ regions depending on the stage of the disease
Authors: Legras Fabienne , Martin Thierry , Knapp Anne‐Marie , Pasquali Jean‐Louis
Published: March 2007

Organ‐specific autoimmune diseases are usually considered to be mediated by autoreactive T cells which infiltrate the target tissue. Conceivably, these T cells could represent pathogenic autoreactive cells which recognize their specific antigen (peptide or superantigen) within the pathological tissue. Extensive studies dealing with the clonality of the infiltrating autoreactive cells gave conflicting results both in humans and animals. One possibility for explaining these contradictory data could rely on the stage of the disease when the T cell population is studied. Here, we report on this parameter by analyzing T cell receptor β‐chain variable regions of infiltrating T cells involved during one of the most frequent human organ‐specific autoimmune disease, the primary Sjögren's syndrome. Six patients were selected on the basis of the duration of the disease before the biopsy procedure (two early and four late stages) to analyze initial and late T cell waves within the abnormal tissue. Using short‐term interleukin‐2‐stimulated T cells, polymerase chain reactions, Southern and sequence analysis, we conclude that: (a) there is a clear restriction in the Vβ usage by the infiltrating T cells only during the early stage of the disease, (b) this Vβ restriction is related to a monoclonal T cell expansion, (c) the expanded Vβ families are different from one patient to the other, and (d) there is no clear homology in length or amino acid composition in the CDR3 of the analyzed Vβ regions. These results could provide an explanation to conflicting results on the Vβ restriction usage during autoimmune diseases and could indicate time limitations in anti‐Vβ treatment. Furthermore, the monoclonal expansion of particular Vβ‐bearing T cells argues against a role for a superantigen during this disease.
CD45RA expression on γδ and αβ T cells emigrating from the fetal and postnatal thymus
Authors: Witherden Deborah A. , Abernethy Nevin J. , Kimpton Wayne G. , Cahill Ross N. P.
Published: March 2007

We have compared the expression of CD45RA on αβ and γδ T cells emigrating from the fetal and postnatal thymus. The fetal and postnatal thymus export both CD45RA+ and CD45RA‐ T cells. The number of γδ+CD45RA+ T cells was remarkably constant regardless of stage of ontogeny or T cell maturity. Around 5‐‐8% of γδ thymic emigrants, thymocytes and peripheral blood lymphocytes expressed CD45RA in both fetal and postnatal animals. In contrast to γδ T cells, up to one quarter of both fetal and postnatal αβ emigrants expressed CD45RA. Post‐thymic maturation of CD45RA expression on αβ emigrants, which occurred both before and after birth, appeared to be antigen independent.
Control of Mac‐2 surface expression on murine macrophage cell lines
Authors: Sato Sachiko , Hughes R. Colin
Published: March 2007

Mac‐2 antigen, a 32‐kDa murine macrophage cell‐surface protein expressed on thioglycollate‐elicited peritoneal exudate cells at higher levels than other macrophages, is a member of the S‐(soluble) galactoside‐binding lectin family with homologies to carbohydrate‐binding proteins of other cell types. Murine macrophage cell lines can be ordered in a linear differentiation sequence according to their expression of Mac‐2 and other surface markers (Leenen et al., Differentiation 1986. 32: 157.) We show here that antigen expression in macrophage cell lines can be regulated at the level of protein secretion. WEHI‐3 cells, classified as immature macrophages by virtue of their low level of surface Mac‐2 expression synthesize similar amounts of the antigen as more mature J774.2 and P388.D1 cells that express high amounts of surface Mac‐2, but unlike these latter cell lines WEHI‐3 cells fail to secrete the protein. Exogenously added Mac‐2 binds efficiently to WEHI‐3 cells and putative Mac‐2‐binding carbohydrates are expressed equally on WEHI‐3, J774.2 and P388.D1 cells as judged by binding of plant lectins of known carbohydrate‐binding specificities. Mac‐2 secretion and surface expression in WEHI‐3 cells is not significantly enhanced by calcium ionophore A23187, a powerful stimulator of Mac‐2 secretion in other cells and a moderate stimulator in J774.2 and P388. D1 cells. WEHI‐3 cells provide a valuable system for studying the mechanism of intracellular transport and secretion of Mac‐2, a protein that lacks a signal sequence and does not enter the classical secretory pathway.
Activation of phosphatidylinositol‐3‐kinase in Jurkat T cells depends on the presence of the p56lck tyrosine kinase
Authors: von Willebrand Maria , Baier Gottfried , Couture Clément , Burn Paul , Mustelinm Tomas
Published: March 2007

Activation of resting T lymphocytes by ligands to the T cell receptor (TcR)/CD3 complex is initiated by phosphorylation of a number of key regulatory proteins on specific tyrosine residues. One such protein is the heterodimeric enzyme phosphatidylinositol‐3‐kinase (PI3K). We recently found that this enzyme is also rapidly activated following TcR/CD3 triggering and that immunoprecipitated PI3K was activated in vitro by direct tyrosine phosphorylation. Here we show that TcR/CD3‐induced tyrosine phosphorylation and activation of PI3K in Jurkat T leukemia cells depend on the presence of the p56lck tyrosine kinase: in a variant of the Jurkat T cell line lacking p56lck, JCaM1, these responses were absent. We also show that p56lck directly activates PI3K purified from transfected COS‐1 cells, indicating that other T cell‐specific proteins are not required for the process. Finally, tryptic peptide maps show that p56lck phosphorylates three tyrosine residues in the p85α subunit of PI3K and two in p110 of PI3K. Our results suggest that p56lck is required for activation of PI3K in Jurkat T cells and can itself directly activate it by phosphorylating one or several stimulatory sites.
B cell differentiation induced by helper T cell membranes: evidence for sequential isotype switching and a requirement for lymphokines during proliferation
Authors: Hodgkin Philip D. , Castle Brian E. , Kehry Marilyn R.
Published: March 2007

Small dense B cells are stimulated to proliferate by membranes prepared from activated helper T (Th) cell clones. In combination with Th2 lymphokines, Th membranes stimulate B cells to differentiate to secrete predominantly immunoglobulin (Ig)M, IgG1 and IgE. The activity in Th membrane requires the expression of CD40 ligand by the T cells, and initiation of the B cell response occurs through the ligation of CD40 on the B cell surface. We have further characterized the properties of the B cell response and found that Th membranes stimulated B cell proliferation and Ig secretion in a cell density independent manner and the majority of the stimulated B cells underwent a limited number of division rounds between day 2 and 5 of culture. IgM‐secreting cells appeared in culture by day 3 and increased to reach a maximum, comprised of 30% of the viable cells, on day 5. IgG1‐secreting cells appeared 12‐‐24h after IgM‐secreting cells, and IgE‐secreting cells did not appear until day 5. These data are consistent with a sequential model of isotype switching related to cell division. As lymphokines were absolutely required for antibody production we were able to determine when during culture they were essential. Lymphokines needed to be present prior to and during B cell proliferation for differentiation to Ig‐secreting cells to proceed. This period corresponded closely to the time of maximum DNA synthesis. Consistent with sequential switching of Ig isotypes, differentiation to IgM secretion required the shortest exposure to lymphokines and IgE the longest. These experiments strongly suggest that the lymphokine‐induced commitment to differentiate is made before DNA synthesis begins, although the lymphokine‐delivered signals are necessary during DNA synthesis to support Ig class switching.
T cell receptor stimulation, but not CD28 costimulation, is dependent on LFA‐1‐mediated events
Authors: Green Jonathan M. , Zheng Xiang‐Guang , Shimizu Yoji , Thompson Craig B. , Turka Laurence A.
Published: March 2007

Accessory molecules play a crucial role in the development of the T cell response to antigenic challenge. In this manuscript we specifically examine the role of two accessory molecules, CD28 and LFA‐1, in modulating the T cell proliferative response to a variety of stimuli. We demonstrate that the proliferation induced by staphylococcal enterotoxins A and B in combination with CD28 costimulation is dependent on LFA‐1‐mediated events. This requirement for LFA‐1 is independent of T cell‐accessory cell adhesion. Similarly, an allogeneic mixed lymphocyte reaction, which has previously been shown to be a CD28‐dependent response, can be inhibited by blockade of LFA‐1. This suggests LFA‐1 plays an essential role in these responses, either by enhancing intercellular adhesion or by an independent signal transduction event. In contrast, when the primary activating stimulus is delivered by immobilized anti‐CD3 antibody or by PMA, and the secondary stimulus by either α‐CD28 or cell‐bound CD28 ligand B7, there is no requirement for LFA‐1. In addition, we demonstrate that cross‐linking of LFA‐1 with immobilized monoclonal antibody, or engagement of LFA‐1 with ICAM‐1 expressed on the surface of a CHO cell, provide an insufficient costimulus for T cell proliferation initiated by enterotoxin, immobilized α‐CD3 or phorbol ester. Our data suggests that LFA‐1, in contrast to CD28, functions not as a costimulatory molecule, but serves primarily to modulate the signal delivered through the T cell receptor.
Prominent T lymphocyte response to Borrelia burgdorferi from peripheral blood of unexposed donors
Authors: Roessner Karen , Russell Jennifer Q. , Cooper Sheldon M. , Budd Ralph C. , Fikrig Erol , Flavell Richard A.
Published: March 2007

The proliferative response of peripheral blood T cells to the spirochete, Borrelia burgdorferi, can be as pronounced in unexposed normal individuals as it is in Lyme disease patients. This finding was observed using three geographically distinct isolates of B. burgdorferi. The response is not due to a lipopolysaccharide effect of the spirochete, is sensitive to Proteinase K, and requires antigen processing. It does not result from cross‐reactivity of memory T cells that may be reactive to another antigen; the proliferative response to B. burgdorferi is equally distributed between naive (CD29−, CD45RO−) and memory (CD29+, CD45RO+) T cells, whereas the tetanus response is confined to the memory subset. In support of this notion, cord blood specimens that contain almost entirely naive T cells, respond as vigorously to B. burgdorferi as T cells from normal adult peripheral blood. A large panel of CD4+ T cell clones has been derived that are specific for B. burgdorferi. The majority of these clones are reactive to B. burgdorferi in the presence only of autologous HLA‐DR molecules. Collectively, these data suggest that the T cell response from normal individuals is more likely due to multiple antigenic epitopes within Borrelial proteins than a superantigen response.
Role of CD40 antigen and interleukin‐2 in T cell‐dependent human B lymphocyte growth
Authors: Blanchard Dominique , Gaillard Claude , Hermann Patrice , Banchereau Jacques
Published: March 2007

In the present study, we examined the participation of CD40 ligand (L)‐CD40 interaction in T cell‐dependent B cell responses. To this end, purified B lymphocytes were cultured over irradiated CD4+ cloned T cells activated with immobilized anti‐CD3 antibody. The anti‐CD40 mAb 89 strongly blocked, in a specific fashion, both proliferation and Ig secretion of tonsil B cells. Interestingly, proliferation of surface (s)IgD+ B cell was significantly less inhibited by anti‐CD40 than that of sIgD− cells. Preactivated T cells induced B cells to grow and secrete immunoglobulins preferentially in response to IL‐2. This contrasts with the CD40 system where B cells are essentially responsive to IL‐4 and IL‐10 but not to IL‐2 alone. Collectively, these data indicate that CD40L‐CD40 interaction plays an important role in IL‐2‐mediated T cell‐dependent B cell responses. However, the activation of a subset of sIgD+ cells may be independent of this interaction.
Differential effects of interleukin‐12 on the development of naive mouse CD4+ T cells
Authors: Schmitt Edgar , Hoehn Petra , Germann Tieno , Rüde Erwin
Published: March 2007

The influence of interleukin (IL)‐12 and IL‐4 on the differentiation of naive CD4+ T cells was studied in an accessory cell‐free in vitro system. Dense CD4+ T cells were purified from unimmunized mice and activated using immobilized anti‐CD3 monoclonal antibodies (mAb) in the presence of IL‐4, IL‐12, or a combination of both cytokines, and restimulated after 6 days by re‐exposure to anti‐CD3‐coated culture wells. T cells initially activated in the presence of IL‐4 produced substantial amounts of IL‐4 and trace amounts of interferon (IFN)‐γ after restimulation at day 6 with plate‐bound anti‐CD3 mAb. By contrast, T cells primed in the presence of IL‐12 produced high levels of IFN‐γ and only minimal amounts of IL‐4, thus indicating that IL‐12 and IL‐4 by acting directly on stimulated naive CD4+ T cells support the development of TH1 and TH2 cells, respectively. When naive CD4+ T cells were stimulated in the presence of IL‐12 together with IL‐4 in comparable concentrations, the effect of IL‐12 on TH1 differentiation was largely inhibited by IL‐4. On the other hand, IL‐12 exerted no inhibitory effect on IL‐4‐induced TH2 differentiation but rather enhanced the production of IL‐4 after restimulation of the respective T cells. Decreasing amounts of IL‐4 in combination with a high level of IL‐12 led to an increasing production of IFN‐γ by the emerging T cells and, simultaneously, to a relatively high production of IL‐4. These data were confirmed by time‐course experiments which revealed that the delayed addition of IL‐4 to IL‐12‐primed T cell cultures resulted in a gradual restoration of IFN‐γ production whereas in parallel the secretion of IL‐4 was not reduced over a wide period of delay (6–72 h). These results, therefore, demonstrate that (a) IL‐4 dominates the effect of IL‐12, (b) IL‐12 promotes the development of TH1 cells; however, in the presence of IL‐12 and relatively high levels of IL‐4 also the development of TH2‐like cells is slightly but significantly enhanced by IL‐12, and (c) high amounts of IL‐12 in combination with relatively low levels of IL‐4 give rise to a T cell population that upon rechallenge exhibited a cytokine profile resembling that of TH0 cells.
Genetic analysis of immune dysfunction in non‐obese diabetic (NOD) mice: Mapping of a susceptibility locus close to the Bcl‐2 gene correlates with increased resistance of NOD T cells to apoptosis induction
Authors: Garchon Henri‐Jean , Luan Jian‐Jian , Eloy Laure , Bédossa Pierre , Bach Jean‐François
Published: March 2007

The non‐obese diábetic (NOD) mouse strain provides a remarkable model for investigating the mechanisms of autoimmunity. Independent genetic analyses of this model have previously shown that chromosome 1‐linked loci were involved in the control of periinsulitis and sialitis on the one hand and of insulitis and diabetes on the other hand. In the present work, analysis of a [NOD × (NOD × C57BL/6)F1] backcross progeny allowed us to clearly dissociate two genetic regions: one was associated with periinsulitis and mapped to the middle region of chromosome 1, in the vicinity of the Bcl‐2 gene; the other was associated with insulitis and mapped to the proximal part of the chromosome. Three intermediate markers D1Mit18, D1Mit5 and D1Mit19 covering at least 25 centiMorgans between these two regions, were associated with neither periinsulitis nor insulitis. The role of the Bcl‐2‐linked region in the immune anomalies of NOD mice was further investigated in a (NOD × C57BL/6)F2 cross where the Bcl‐2nod haplotype was linked to elevated serum levels of IgG (p 0.0005). The middle region of chromosome 1 is, therefore, involved in the control of three phenotypes, including periinsulitis, sialitis and hyperIgG, pointing to Bcl‐2 as a good candidate for a cause of the NOD mouse disease. Consistent with the anti‐apoptotic function of the Bcl‐2 gene product, activated T lymphocytes from NOD mice showed a markedly increased resistance to induction of apoptosis following deprivation of interleukin‐2 when compared to those from non‐autoimmune strains. After the recent observation of the Fas gene alterations in the lpr and lprcg mutations, these findings indicate that deregulation of lymphoid cell apoptosis may be a general pathogenetic mechanism in autoimmune diseases.
Evidence for a stromal cell‐dependent, self‐renewing B cell population in lymphoid follicles of the ileal Peyer's patch of sheep
Authors: Griebel Philip J. , Ferrari Giorgio
Published: March 2007

Lymphoid follicles of the ileal Peyer's patch (PP) of young sheep function as the major source of B cells and a site of immunoglobulin (Ig) receptor diversification. However, extensive cell death in culture has restricted investigations of ileal PP follicular (iPf)B cell biology. We investigated the possibility that sustained iPfB cell proliferation may require an interaction with mesenchymal stromal cells (SC). Four SC lines, cloned from lymphoid follicles of the ileal PP, and various sheep and xenogeneic mesenchymal cells were used to characterize the nature of iPfB cell‐SC interactions. A sustained proliferative response was unique to iPfB cells, required iPfB cell‐SC contact, and SC membranes functioned as intact SC to either enhance or inhibit iPfB cell proliferative responses. The iPfB cell proliferation in SC co‐cultures was accompanied by extensive cell death and a slow decline in viable cell number. Flow cytometric analysis confirmed that viable lymphocytes, present in SC co‐cultures, were immature B cells that expressed surface IgM, with either λ or χ Ig light chain, and that SC co‐culture inhibited iPfB cell differentiation. Finally, addition of soluble anti‐sheep Ig to iPfB cell‐SC co‐cultures did not inhibit SC‐dependent iPfB cell proliferation or iPfB cell binding to SC. These data indicate that an interaction between specific SC membrane molecules and non‐Ig molecules of iPfB cells either supported or inhibited a self‐renewing proliferative response by immature (sIgMLo, BAQ44A−) iPfB cells. Finally, SC‐dependent iPfB cell proliferation was independent of T cells and extrinsic antigen which further suggests that a functionally distinct B cell population resides in lymphoid follicles of the ileal PP.
Interleukin‐2 prevention of apoptosis in human neutrophils
Authors: Pericle Federica , Liu Jin Hong , Blanchard D. Kay , Wei Sheng , Djeu Julie Y. , Diaz Jose I. , Forni Guido
Published: March 2007

Evidence is presented that interleukin (IL)‐2 maintains viability of human polymorphonuclear cells (PMN) in culture by preventing these cells from undergoing programmed cell death (PCD) and induces the synthesis of new RNA and protein. Our laboratory has recently discovered that human PMN constitutively express IL‐2β receptor and more importantly, PMN are able to respond functionally to IL‐2 by enhanced growth inhibitory activity against an opportunistic fungal pathogen, Candida albicans. We now report that IL‐2 was able to interfere with the PCD process and reduce the number of apoptotic PMN to < 40% in 72‐h culture. Freshly isolated PMN usually underwent a timedependent aging process and > 80% of PMN cultured in medium alone for 72 h showed morphologic features of PCD as depicted by hematoxylin and eosin staining as well as by electron microscopy. During the PCD process, untreated PMN not only exhibited condensed nuclear structure and decrease in cell size, but also displayed DNA fragmentation. DNA fragmentation in PMN was prevented by IL‐2. Prevention of PCD by IL‐2 was associated with an increase in new RNA and protein synthesis in PMN, which may reflect cytokine induction, such as tumor necrosis factor, as we have recently shown. Thus, our data expands our current understanding of PMN in that they may be an active component of the immune system, with a longer life‐span when activated than expected.
Evidence for cell surface association of CD2 and LFA‐1 (CD11a/CD18) on T lymphocytes
Authors: Altin Joseph G. , Pagler Eloisa B. , Parish Christopher R.
Published: March 2007

Previous studies have reported an association of the cell surface adhesion molecule CD2 with the T cell receptor and with CD45 on mouse and human T lymphocytes. In this study the association of CD2 with cell surface molecules was investigated using cell surface biotinylation of T lymphocytes, coupled with immunoprecipitation using two CD2‐specific monoclonal antibodies (mAb) (RM2–5 and 12–15) and analysis by SDS‐PAGE. Although both CD2 mAb immunoprecipitated CD2 from lysates of murine lymphocytes, it was found that mAb 12–15, but not RM2–5, co‐precipitated two other molecules of 95 and 180 kDa. Subsequent studies revealed that the 95‐ and 180‐kDa molecules were associated with a subspecies of CD2 (˜ 5%) on thymocytes, the antigen‐specific T cell line D10, and splenic T cells but not B cells. Two lines of evidence were obtained consistent with the 95‐ and 180‐kDa molecules being the β and α chains of LFA‐1. Firstly, an analysis of 12–15 mAb immunoprecipitates on 4–12% gels under reducing and nonreducing conditions shows that the 95‐ and 180‐kDa molecules have a molecular weight and migration pattern identical to LFA‐1. Secondly, depletion of LFA‐1 from lysates with LFA‐1 mAb abolished the ability of CD2 mAb 12–15 to co‐precipitate the 95‐ and 180‐kDa molecules, thereby identifying these as the β and α chains of mouse LFA‐1, respectively. These results provide evidence for the first time for an association of LFA‐1 and CD2 on mouse T lymphocytes, and suggest that the association occurs with an immunologically distinct subspecies of CD2 molecules.