bluuebunny/crossref_metadata_2025 huggingface dataset:
The phosphoprotein phosphatase calcineurin controls calcium‐dependent apoptosis in B cell lines
Authors: Bonnefoy‐Berard Nathalie , Genestier Laurent , Flacher Monique , Revillard Jean Pierre
Published: March 2007

Group I Burkitt's lymphoma cell lines and the B104 lymphoma cell line which expresses a phenotype of immature B cells undergo apoptosis after cross‐linking of their surface immunoglobulin (Ig) receptors or after exposure to a calcium ionophore, while protein kinase C (PKC)‐activating phorbol esters prevent such apoptosis. We show here that blockade of the phosphoprotein phosphatase calcineurin or phosphatase 2B by cyclosporin A (CsA) also protects these B cell lines against Ca2+‐dependent apoptosis but not against apoptosis triggered by the PKC inhibitor chelerythrine or by serum deprivation. Okadaic acid, an inhibitor of phosphatases 1, 2A and 2C was ineffective. Among a series of human cytokines tested, only interferon‐α and tumor necrosis factor‐α were shown to protect against Ca2+‐dependent apoptosis when used alone or in combination with CsA. In contrast to phorbol esters which block the progression into the S/G2 phases of the cell cycle, CsA partially restored the proliferation of cells exposed to the calcium ionophore. Altogether these data provide indirect evidence for the control of B cell apoptosis by the serine/threonine phosphorylation status of yet undefined key cellular substrates.
The ability of H‐2Dd molecule to affect natural resistance to hemopoietic allografts is an intrinsic property shared by Ddm1 but not Ld
Authors: Milisauskas Vita K. , Nakamura Ichiro
Published: March 2007

F1 hybrid resistance (HR) to parental bone marrow grafts is mediated by natural killer (NK) cells, and thought to be controlled by the non‐class I hemopoietic histocompatibility (Hh) genes linked to the major histocompatibility complex (MHC). However, as in the in vitro NK cytotoxicity against hemopoietic targets, expression of certain class I MHC molecules does affect HR, although mechanisms underlying such an effect are not understood. In this study, we examine the relevance of the “self/non‐self” property of class I molecules and the molecular domains responsible for this function. H‐2b/Hh‐1b lymphoma cells were transfected with class 1 H‐2Dd or Ld gene, and its effect on the Hh‐1 phenotype was examined by testing the transfectant's ability to competitively inhibit the in vivo rejection of parental H‐2b/Hh‐1b bone marrow grafts by irradiated F1 hybrid hosts. Multiple independent clones of transfectants show that the genomic or cDNA of the Dd gene, but not of Ld, renders the Hh‐1b‐positive cells incapable of inhibiting HR in F1 mice, although both genes belong to the same region of the same haplotype. The same effect could be observed not only in H‐2b/d F1 mice for which Dd and Ld are self, but also in H‐2b/k F1 mice for which both Dd and Ld are non‐self. Thus, this function of the Dd molecule is an intrinsic property, not necessarily related to its self/non‐self characteristic relative to the effector cells. Furthermore, given the nature of the assay used in this study, the results favor a “target interference” model as the underlying mechanism of the Dd effect. To locate the relevant domain(s) of the Dd molecule, mutant Ddm1 gene was tested and found to have the same effect as the non‐mutant Dd. Ddm1 is a hybrid molecule between Dd and Ld, sharing with Dd only the α1 domain and a portion of the α2 domain. The two N‐terminal domains of Ddm1 differ from those of Dd by three amino acid substitutions, two of which affect the molecules' peptide‐binding properties.
4‐1BB T‐cell antigen binds to mature B cells and macrophages, and costimulates anti‐μ‐primed splenic B cells
Authors: Pollok Karen E. , Kim Young‐June , Hurtado José , Zhou Zhen , Kim Kack K. , Kwon Byoung S.
Published: March 2007

4‐1BB is expressed on activated murine T cells and may function as an accessory signaling molecule during T‐cell activation. To identify putative 4‐1BB ligands, a fusion protein consisting of the extracellular domain of 4‐1BB fused to human placental alkaline phosphatase (4‐1BB‐AP) was constructed. Alkaline phosphatase activity could then be used as an indicator of the relative amount of bound 4‐1BB. These studies indicated that 4‐1BB‐AP specifically bound to the surface of various mature B and macrophage cell lines. 4‐1BB‐AP bound at low levels to T cell lines (non‐activated and anti‐CD3‐activated), pre‐B‐cell lines, and an immature macrophage cell line. 4‐1BB‐AP did not bind to a glial tumor cell line, HeLa cells, or COS cells. In addition, 4‐1BB‐AP bound at higher levels to F(ab′)2 anti‐μ‐activated primary B cells compared to anti‐CD3‐activated primary T cells. Scatchard analysis indicated that the A20 B cell lymphoma expressed 3680 binding sites per cell with a Kd of 1.86 nM. Affinity cross‐linking studies demonstrated that a major cell surface species of 120 kDa bound to 4‐1BB‐AP 4‐1BB‐AP also bound to a minor species of approximately 60 kDa. The addition of paraformaldehyde‐fixed SF21 cells expressing recombinant 4‐1BB synergized with F(ab′)2 anti‐μ in inducing splenic B cell proliferation suggesting that 4‐1BB may function as a regulator of B cell growth.
T cell epitopes encompassing the mutational hot spot position 61 of p21 ras. Promiscuity in ras peptide binding to HLA
Authors: Iii Tobias Gedde‐Dahl , Spurkland Anne , Fossum Beate , Thorsby Erik , Gaudernack Gustav , Wittinghofer Alfred
Published: March 2007

Activated ras carry a point mutation either in codon 12, 13 or 61 which is tumor specific. Peptides derived from this oncoprotein are therefore potential tumor antigens. Essential for the feasibility of using ras‐derived peptides in therapy of cancer is whether p21 ras‐derived peptides can be processed, bind to human histocompatibility leukocyte antigen (HLA) and be recognized by T cells. Here we report the fine specificity and HLA restriction of several T lymphocyte clones (TLC) specific for a peptide which is derived from the second mutational hot spot in ras encoding residue 61. These TLC were generated from memory T cells present in the blood of a cancer patient and recognized a ras‐derived peptide carrying Leu instead of Gln at residue 61. By sequencing of the T cell receptor (TcR) genes three sets of “sister” TLC carrying highly different TcR were identified. Two of the TLC recognized a peptide carrying the 61 Leu mutation presented by HLA‐DQ8 and one recognized the same peptide presented by HLA‐DQ4. By using truncated peptides derived from residues 51 to 69 of p21 ras, partially overlapping minimal epitopes could be defined. All three TLC recognized the corresponding recombinant mutant p21 ras oncoprotein carrying Leu at residue 61 presented by autologous B‐lymphoblastoid cell lines (B‐LCL). This demonstrates that naturally derived ras peptides from this region of p21 ras encompass the three epitopes recognized by the TLC. These results indicate that immunogenic ras‐derived peptides may be used in immunotherapy of cancer where transforming ras oncoproteins are involved.
Murine dendritic cells pulsed in vitro with tumor antigen induce tumor resistance in vivo
Authors: Flamand VéRonique , Sornasse Thierry , Thielemans Kris , Demanet Christian , Bakkus Marleen , Bazin Hervé , Tielemans Françoise , Leo Oberdan , Urbain Jacques , Moser Muriel
Published: March 2007

The aim of this work is to induce tumor resistance to a B cell lymphoma in BALB/c mice using elements of the immune system. It has indeed been shown by us and by others that antigen‐presenting cells (APC) like dendritic cells can induce efficient immune responses and can even substitute for Freund's adjuvant. Here we show that mice immunized with syngeneic dendritic cells pulsed in vitro with tumor antigen (BCL1 idiotype expressed by lymphoma cells) are protected against a subsequent tumor inoculation. The in vivo resistance can be correlated with the induction of a humoral response specific for the idiotype expressed by the tumor. No such protection can be achieved when B cells are used as APC. These data show that effector cells in tumor‐bearing animals can be recruited and activated using dendritic cells, providing long‐lasting immune surveillance.
Developmental expression of the αIELβ7 integrin on T cell receptor γδ and T cell receptor αβ T cells
Authors: Lefrançois Leo , Barrett Terrence A. , Havran Wendy L. , Puddington Lynn
Published: March 2007

A novel monoclonal antibody, 2E7, was shown by immunoprecipitation to be reactive with the αIELβ7 integrin and was employed to analyze the expression of this integrin in lymphocyte subsets and during T cell ontogeny. In adult lymph nodes, αIEL was expressed at low levels by 40–70% of CD8+ T cells and < 5% of CD4+ T cells. However, virtually all intestinal intraepithelial lymphocytes and ˜20% of lamina propria CD4+ T cells were 2E7+, indicating a preferential expression of this integrin on mucosal T cells. Examination of αIEL integrin expression during thymus ontogeny revealed that ˜3–5% of fetal or adult thymocytes were 2E7+. Interestingly, early in fetal thymus ontogeny, ˜40% of 2E7+ cells expressed T cell receptor (TcR)‐γδ and this subset persisted through birth. A developmental switch occurred such that 2E7+ TcR− CD4−8+ cells detected on fetal day 19 were followed by 2E7+ TcR‐αβ CD4−8+ cells in the neonatal thymus. The latter population persisted throughout thymus ontogeny into adulthood. Interestingly, a subset of TcR‐γδ Vγ3+ day 16 fetal thymocyte dendritic epidermal cell (DEC) precursors were 2E7+, but all mature DEC expressed high levels of αIEL integrin, suggesting that the αIEL integrin was acquired late in DEC maturation. This possibility was strenghthened by immunohistochemical localization of the majority of 2E7+ γδ and αβ T cells to the medullary regions of the thymus. Overall, the results demonstrate a developmentally ordered expression pattern of the αIELβ7 integrin that suggests a common function for this integrin during TcR‐γδ and ‐αβ CD4−8+ T cell thymocyte development or perhaps in effector functions for these subsets.
Targeting major histocompatibility complex class II molecules to the cell surface by invariant chain allows antigen presentation upon recycling
Authors: Nijenhuis Marga , Calafat Jero , Kuijpers Karel C. , Janssen Hans , De Haas Marcel , Nordeng Tommy W. , Bakke Oddmund , Neefjes Jacques J.
Published: March 2007

We studied the functional consequences of targeting class II molecules to either the cell surface or to endocytic structures by expressing HLA‐DR1 in human kidney cells in the presence or absence of different forms of the invariant chain (Ii). Transfectants expressing class II molecules in the absence of Ii present influenza virus efficiently and co‐expression of full length Ii does not further increase antigen presentation. Chimeric Ii containing the cytoplasmic domain of the transferrin receptor (Tfr‐Ii) delivers class II molecules associated with Tfr‐Ii to endosomal compartments, but this does not result in efficient antigen presentation. When class II molecules are targeted to the cell surface by Ii lacking either 15 (Δ15Ii) or 23 (Δ23Ii) amino acids from the cytoplasmic domain, a fraction of free class II molecules is also observed. Whereas Δ15Ii did not affect antigen presentation by class II molecules, Δ23Ii inhibited, but did not abrogate, the response. We show that class II molecules expressed in the presence of Δ23Ii can be internalized, followed by degradation of Δ23Ii and return of free class II αβ heterodimers to the cell surface. A fraction of the resulting free class II molecules is sodium dodecyl sulfate stable, indicating that internalization and reappearance of class II molecules at the cell surface can be an alternative route for antigen presentation. In all transfectants, class II molecules were found in endocytic compartments that labeled for CD63 and resembled the multilaminar MIIC compartments found in B cell lines. Ii is not required for endosomal targeting of class II molecules. The number of class II molecules observed in the multilaminar compartments correlates with the efficiency of antigen presentation.
Self class I molecules protect normal cells from lysis mediated by autologous natural killer cells
Authors: Ciccone Ermanno , Pende Daniela , Vitale Massimo , Nanni Luca , Di Donato Carolina , Bottino Cristina , Morelli Luigia , Viale Oriane , Amoroso Antonio , Moretta Alessandro , Moretta Lorenzo
Published: March 2007

The surface expression of given HLA class I alleles protects target cells from lysis mediated by natural killer (NK) clones specific for these (or related) alleles. We could define two groups of NK clones specifically recognizing either Cw4 and related C alleles (“group 1”) or Cw3 and related C alleles (“group 2”), respectively. Monoclonal antibodies (mAb) to class I molecules should interfere with the interaction between NK receptors and class I molecules, thus resulting in lysis of protected target cells. However, none of the numerous available mAb to class I molecules had this effect. Therefore, we attempted to select new mAb on the basis of their ability to induce lysis of Cw4‐ or Cw3‐protected lymphoblastoid cell lines by “group 1” or “group 2” NK clones, respectively. From mice immunized with phytohemagglutinin (PHA)‐activated lymphocytes expressing either Cw3 or Cw4 alleles, two mAb were selected, the 6A4 (IgG1) and the A6‐136 (IgM), on the basis of their ability to induce lysis of protected target cell. Both mAb immunoprecipitated molecules which, in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, gave two bands of 45 and 12 kDa, typical of the class I heavy chain and β2 microglobulin, respectively. It has been proposed (but not proven), that self major histocompatibility complex class I molecules protect normal cells from autologous NK cell lysis. Thus, we used the 6A4 and A6‐136 mAb to assess this possibility directly. Cw4‐specific (“group 1”) and Cw3‐specific (“group 2”) NK clones were isolated from donors expressing the corresponding (or related) protective C alleles. None of these clones lysed autologous PHA‐induced blasts, used as target cells. However, addition of the F(ab′)2 of 6A4 mAb or the A6‐136 mAb resulted in lysis of autologous target cells by “group 1” or “group 2” NK clones, respectively. These data provide direct evidence that the expression of class I molecules protects normal cells from lysis by autologous NK cells.
Are primed CD4+ T lymphocytes different from unprimed cells?
Authors: Constant Stephanie , Zain Mona , West Jeff , Pasqualini Theresa , Ranney Patricia , Bottomly Kim
Published: March 2007

Primed and unprimed lymphocytes are usually classified as separate subsets of cells, based on phenotypic and functional distinctions. In the case of CD4+ T lymphocytes, primed cells are thought to proliferate more vigorously, quickly and easily, and to release a different profile of cytokines, than their naive equivalent. However, most of these data were obtained from studies in which populations of lymphocytes were compared before and after antigenic stimulation, and therefore did not distinguish between the effects resulting from the clonal expansion of specific precursor cells within such populations and those due to cell differentiation per se. We have investigated the contribution of precursor cell frequency to some of the functional changes observed in populations of CD4+ T cells following antigenic stimulation, using approaches in which antigen‐specific precursor frequencies are high in both primary and secondary stimulations: mixed leukocyte reaction responses and cells from αβ T cell receptor transgenic mice. Our data suggest that when equivalent numbers of antigen‐specific naive and previously primed CD4+ responder T cells are compared, there is no difference in their potency to proliferate but only the previously activated subset can generate cytokines such as interferon‐γ.
Clonal expansion of T lymphocytes causes arthritis and mortality in mice infected with toxic shock syndrome toxin‐1‐producing staphylococci
Authors: Abdelnour Arturo , Bremell Tomas , Holmdahl Rikard , Tarkowski Andrzej
Published: March 2007

Erosive arthritis is a common and feared complication of staphylococcal infection. The reason(s) for the progressive course of the arthritis is unknown. It has been recently established that enterotoxins produced by Staphylococcus aureus display superantigen properties leading to stimulation of T cells carrying distinct T cell receptor Vβ elements. This finding provides a potential connection between staphylococcal exoproteins and endogenous immune mechanisms participating in the infectious process. We have recently described successful induction of infectious arthritis in mice after intravenous inoculation of a toxic shock syndrome toxin‐1 (TSST‐1)‐producing S. aureus LS‐1 strain. Using this model we have now found a clonal expansion of T cells expressing Vβ11+ T cell receptor in the synovial tissue of arthritic mice. The role of TSST‐1 as a superantigen inducing oligoclonal expansion was confirmed in an in vitro culture system. The expansion of Vβ11+ T cells proved to be of arthritogenic significance since mice genomically deleted of the Vβ11+ T cells did not develop arthritis and since pretreatment of healthy mice with anti‐CD4 or anti‐Vβ11 monoclonal antibodies inhibited arthritis. In addition, CD4+ and Vβ11+ T cells showed themselves to be of pathogenic significance in staphylococcal‐induced mortality, since mice depleted of such populations showed increased survival. We propose that in hematogenously spread S. aureus‐induced arthritis the TSST‐1‐dependent clonal expansion of CD4+ Vβ11+ T cells is a driving pathogenic force.
Structure of the T cell receptor in a TiαVβ2,αVβ8‐positive T cell line
Authors: Hou Xiaohong , Dietrich Jes , Kuhlmann Jan , Wegener Anne‐Marie K. , Geisler Carsten
Published: March 2007

The T cell receptor (TcR) is composed of at least six different polypeptide chains consisting of the clonotypic Ti heterodimer (Tiαβ or Tiγδ) and the noncovalently associated CD3 chains (CD3γδϵζ). The exact number of subunits constituting the TcR is still not known; however, it has been suggested that each TcR contains two Ti dimers. To gain insight into the structure of the TcR we constructed a TiαVβ2,αVβ8‐positive T cell line which expressed the endogenous human TiVβ8 and the transfected mouse TiVβ2 both in association with the endogenous Tiα and CD3 chains at the cell surface. Preclearing experiments with radioiodinated cell lysate prepared with digitonin lysis buffer demonstrated that depleting the lysate of TiαVβ8 by immunoprecipitation with anti‐Vβ8 monoclonal antibody (mAb) did not reduce the amount of TiαVβ2 in the lysate, and likewise, depleting the lysate of TiαVβ2 with anti‐Vβ2 mAb did not reduce the amount of TiαVβ8. Comodulation experiments showed that Vβ8 and Vβ2 did not comodulate with each other. Furthermore, functional tests demonstrated that TcR containing Vβ8 and TcR containing Vβ2 mediated transmembrane activation signals independently of each other. These data demonstrate that mouse Vβ2 and human Vβ8 were not expressed in the same TcR in agreement with a TcR model where each TcR contains only one Ti dimer.
Transforming growth factor‐β2 induces apoptosis of murine T cell clones without down‐regulating bcl‐2 mRNA expression
Authors: Weller Michael , Constam Daniel B. , Malipiero Ursula , Fontana Adriano
Published: March 2007

Transforming growth factor‐β (TGFβ) is a potent immunosuppressive cytokine which inhibits the antigen (Ag)‐dependent expansion of T cells both in vitro and in vivo by mechanisms not well defined yet. Here we report that exposure of interleukin (IL)‐2‐dependent T cell lines to TGFβ2 results in apoptosis defined by morphology, nucleosomal size DNA fragmentation and in situ DNA end labeling. TGFβ2‐induced T cell apoptosis showed the following characteristics: (1) in contrast to the rapid evolution of apoptosis following IL‐2 deprivation, apoptosis of T cells triggered by TGFβ2 was delayed; (2) cycloheximide prevented TGFβ2‐induced apoptosis of CTLL‐2 but not of OVA‐7 T helper cells; (3) in contrast to apoptosis following IL‐2 deprivation, TGFβ2‐mediated T cell apoptosis was not associated with decreased expression of the proto‐oncogenes, bcl‐2 or c‐myc; (4) TGFβ2‐induced apoptosis was not restricted to IL‐2‐dependent T cell lines since the IL‐4‐dependent T cell line, CT.4S, as well as EL4 lymphoma cells, which grow independently of exogenous IL‐2, were also susceptible to TGFβ2‐mediated apoptosis. Taken together, these data may present a novel mechanism of TGFβ2‐mediated suppression of T cell expansion in response to Ag and IL‐2, the activation of the endogenous death program of apoptosis, which appears to operate independently of direct interactions of TGFβ2 with the IL‐2/IL‐2 receptor system.
The anti‐La response of a single MRL/Mp‐lpr/lpr mouse: Specificity for DNA and VH gene usage
Authors: Bloom Debra D. , Clair E. William St. , Pisetsky David S. , Clarke Stephen H.
Published: March 2007

Autoantibodies to ribonucleoproteins (RNP) occur prominently in human systemic lupus erythematosus and murine lupus models. In previous studies we demonstrated a relationship in MRL/Mp‐lpr/lpr (MRL/lpr) mice between antibodies to Sm, an RNP autoantigen, and antibodies to DNA. Thus, many anti‐Sm monoclonals bound DNA and expressed the same V region genes as anti‐DNA. In addition, many had multiple VHCDR3 Arg residues suggestive of selection by DNA, and some had somatic mutations suggesting selection for mutant B cells by DNA. To determine whether autoantibodies to other RNP antigens are also associated with the anti‐DNA response, we have analyzed the response to the La RNP. Six anti‐La B cell hybridomas were generated from a single MRL/lpr mouse. Southern blot analysis of Ig V gene rearrangements and V gene sequences indicated two clonally related pairs, suggesting an oligoclonal response. Antibodies from all six hybridomas bound single‐stranded DNA, while antibodies from five hybridomas bound double‐stranded DNA. Two hybridomas expressed a VH7183 gene which is used by members of two previously reported anti‐DNA clones and two anti‐Sm/DNA clones of MRL/lpr origin. These data demonstrate an association between the anti‐La and anti‐DNA responses in MRL/lpr mice, suggesting that cross‐reactive anti‐RNP and anti‐DNA responses are a general feature of autoimmunity in this lupus model.
Decreased anti‐donor major histocompatibility complex class I and increased class II alloantibody response in allograft tolerance in adult rats
Authors: Cuturi Maria‐Cristina , Josien Régis , Cantarovich Diego , Bugeon Laurence , Anegon Ignacio , Menoret Séverine , Smit Helga , Douillard Patrice , Soulillou Jean‐Paul
Published: March 2007

Permanent tolerance to allografts can be induced in adult rats by donor‐specific transfusions (DST) prior to transplantation. We have previously reported, in a model of heart allograft, the presence of a heavy leukocyte infiltrate, in the allograft which displayed a strong allospecific cytotoxicity when tested in vitro against donor cells, and a strong accumulation of mRNA for granzyme A and perforin in vivo. In contrast, there was a major decrease in the accumulation of mRNA for interleukin‐2 and interferon‐γ. These results suggested that the DST‐induced tolerance was associated with a decrease in type‐1 T helper (Th1) cell function. The major role of preformed antibodies in xeno and allorejection is clearly established. Nevertheless, the consequences of alloantibody production in acute rejection and tolerance induction remains to be elucidated. We here analyze the alloantibody response in rejecting and DST‐treated recipients. We show that, after transplantation, tolerant recipients, in contrast to rejecting ones, mount a low IgM alloresponse that switches to low IgG production. Detailed analysis of IgG alloantibodies in DST‐treated recipients revealed that their production decrease was not equally distributed. Whereas rejecting animals mounted a strong anti‐class I and II IgG alloantibody response, DST‐treated recipients produced anti‐class II and low titers of anti‐class I IgG alloantibodies. Furthermore, among IgG subclasses, tolerant recipients predominantly produced IgG2a, a profile which, in the rat, is compatible with a Th2‐controlled response. Finally, the passive transfer of immune serum from rejecting animals to DST‐treated recipients could abrogate the tolerance. We suggest that the absence of anti‐class I alloantibodies combined with preserved and/or increased anti‐class II production plays a major role in graft tolerance in this model. These results reinforced the role of alloantibodies in rejection and in induction of tolerance.
Properties of mouse CD40: Cellular distribution of CD40 and B cell activation by monoclonal anti‐mouse CD40 antibodies
Authors: Hasboldu Jhagvaral , Johnson‐Légeru Caroline , Atkinsu Chris J. , Clark Edward A. , Klaus Gerry G. B.
Published: March 2007

We describe here the derivation of a rat monoclonal antibody (mAb) against mouse CD40 (designated 3/23), which stains 45–50% of spleen cells of adult mice, approximately 90% of which are B cells. Interestingly, some 5–10% of both CD4+ and CD8+ T cells in the spleens of (some, but not all) adult, unimmunized mice are also CD40+, whereas CD40+ cells were not detectable in the thymus, even following collagenase digestion. Some 35–40% of lymphoid cells in the bone marrow of adult mice are CD40+ and virtually all of these are B220+, and hence of the B cell lineage: triple‐color flow cytometry showed that CD40 is expressed at low levels on some 30% of pre‐B cells, at intermediate levels on 80% of immature B cells and on essentially all mature B cells in the bone marrow. These results, therefore, suggest that in the mouse CD40 is expressed relatively late during the process of B cell differentiation. The mAb induced marked up‐regulation of major histocompatibility complex class II molecules, CD23 and B7.2 antigens on mature B cells. It also stimulated modest levels of DNA synthesis in mature B cells by itself: this was markedly enhanced by suboptimal concentrations of mitogenic (but not non‐mitogenic) anti‐μ and anti‐δ mAb, and moderately enhanced by co‐stimulation with interleukin‐4. Hyper‐cross‐linking of CD40 (using biotinylated mAb and avidin) also enhanced the proliferative response to anti‐CD40.
Insulin‐specific T cells are a predominant component of islet infiltrates in pre‐diabetic NOD mice
Authors: Wegmann Dale R. , Norbury‐Glaseru Mary , Danielf Dylan
Published: March 2007

Numerous investigations have demonstrated that T cells are involved in destruction of β cells in the NOD mouse, a widely studied model of type I diabetes. In this report we describe a series of islet‐specific T cell lines established from islet‐infiltrating lymphocytes obtained from individual pre‐diabetic NOD mice as well as a large panel of clones derived from these lines. Proliferation assays indicated that these nominally islet‐specific lines responded vigorously to porcine insulin. Furthermore, of 40 islet‐specific clones derived from lines established from 12‐week‐old mice, 22 (55%) responded to insulin. A similar analysis of islet‐specific clones established from 7‐week‐old mice indicated that 2 of 14 (14%) were insulin specific. These findings demonstrate that insulin‐specific T cells can comprise a major portion of the spontaneously arising T cell response to islets in NOD mice.
Phosphate is essential for stimulation of Vγ9Vδ2 T lymphocytes by mycobacterial low molecular weight ligand
Authors: Schoel Bernd , Sprengeru Silvia , Kaufmannf Stefan H. E.
Published: March 2007

T lymphocytes are divided into two subsets which express different T cell receptor heterodimers. In the peripheral blood of healthy individuals, the majority of T cells express the α/β T cell receptor (> 90%) while a minority have the γ/δ T cell receptor (< 10%). The γ/δ T cells of adults use preferentially the Vγ9Vδ2 chain combination. Although the stimulation requirements for γ/δ T lymphocytes are still undetermined, it has been reported that γ/δ T cells are not only stimulated, like α/β T cells, by conventional protein antigens and superantigens, but also by unusual ligands. Mycobacteria selectively stimulate Vγ9Vδ2 T cells, and a nonproteinacious low molecular weight fraction of 1–3 kDa has been identified as the tentative active component. Here, we confirm the nonproteinacious nature of this ligand, and show that it is comprised of unusual carbohydrate and phosphate. Importantly, cleavage of the terminal phosphate by alkaline phosphatase completely abrogates the stimulatory activity of the low molecular weight ligand for Vγ9Vδ2 T cells. Even mycobacterial whole lysate loses its stimulatory activity, for this T cell subset, after dephosphorylation with alkaline phosphatase. These findings identify phosphocarbohydrates as a novel molecular entity with selective stimlatory activity for a defined T cell subset.
Interleukin‐7 induces the association of phosphatidylinositol 3‐kinase with the α chain of the interleukin‐7 receptor
Authors: Venkitaraman Ashok R. , Cowling Rachel J.
Published: March 2007

The recently characterized receptor for interleukin (IL)‐7 (IL‐7R) includes a unique α chain as well as a common γ chain shared with the receptors for IL‐2 and IL‐4. Engagement of the IL‐7R activates the intracellular enzyme phosphatidylinositol (PtdIns) 3‐kinase but the mechanism of PtdIns 3‐kinase activation and the molecular basis of its interaction with IL‐7R are not known. Here we show that IL‐7 causes the 85‐kDa regulatory subunit of PtdIns 3‐kinase (p85), and PtdIns 3‐kinase activity, to associate with the IL‐7R. This interaction can be ascribed to ligand‐induced phosphorylation of a single Tyr residue in the receptor's unique α chain. Herbimycin A, a specific protein tyrosine kinase inhibitor, suppresses not only tyrosine phosphorylation of the IL‐7R but also its association with p85. A phosphopeptide corresponding to the sequence surrounding Tyr449 in the cytoplasmic tail of the IL‐7R α chain, but not its non‐phosphorylated analogue or phosphopeptides coincident with the sequences surrounding other α chain Tyr residues, efficiently competes out p85 binding. Replacement of Tyr449 with Phe results in a loss of p85 binding. Finally, soluble forms of the src homology 2 domains of p85, which bind directly to phosphotyrosyl peptides, specifically inhibit the association of p85 with the IL‐7R. Thus, PtdIns 3‐kinase recruitment occurs through a single, phosphotyrosine dependent recognition motif surrounding Tyr449 in the IL‐7R α chain. This motif corresponds to a canonical sequence for p85 binding, Tyr(P)‐X‐X‐Met. Since the closely related IL‐2R and IL‐4R also activate PtdIns 3‐kinase but are devoid of such canonical motifs, our results suggest that the mechanism by which IL‐7R recruits and activates PtdIns 3‐kinase differs fundamentally from that used by the other receptors. PtdIns 3‐kinase may, therefore, play a unique and important role in the biological response to IL‐7.
Moslecular and biological characterization of human 4‐1BB and its ligands
Authors: Alderson Mark R. , Smith Craig A. , Tough Teresa W. , Davis‐Smith Terri , Armitage Richard J. , Falk Ben , Roux Eileen , Baker Elizabeth , Sutherland Grant R. , Din Wenie S. , Goodwin Raymond G.
Published: March 2007

4‐1BB was originally described as a cDNA expressed by activated murine T cells and subsequently demonstrated to encode a member of the tumor necrosis factor receptor family of integral membrane proteins. Recently, we identified and cloned a murine ligand for 4‐1BB (mu4‐1BB‐L) and demonstrated it to be a member of an emerging family of ligands with structural homology to tumor necrosis factor. To characterize further the role of 4‐1BB in the immune response we undertook to clone the human homologue of 4‐1BB‐L. However, attempts to isolate a cDNA encoding the human 4‐1BB‐L by cross‐hybridization with the murine cDNA were unsuccessful. Therefore we first utilized cross‐species hybridization to isolate a cDNA encoding human 4‐1BB (hu4‐1BB). A fusion protein consisting of the extracellular portion of hu4‐1BB coupled to the Fc region of human immunoglobulin G1 (hu4‐1BB.Fc) was then used to identify and clone a gene for human 4‐1BB‐L from an activated CD4+ T cell clone using a direct expression cloning strategy. Human 4‐1BB‐L shows 36% amino acid identity with its murine counterpart and maps to chromosome 19p13.3. Scatchard analysis demonstrated high‐affinity binding of hu4‐1BB.Fc to either native or recombinant human 4‐1BB‐L. Both monoclonal antibody to hu4‐1BB and cells transfected with hu4‐1BB‐L induced a strong proliferative response in mitogen co‐stimulated primary T cells. In contrast, ligation of 4‐1BB on T cell clones enhanced activation‐induced cell death when triggered by engagement of the TCR/xsCD3 complex.
Induction of protective cytotoxic T cells with viral proteins
Authors: Bachmann Martin F. , Kündig Thomas M. , Freer Giulia , Li Yan , Kang Chil Yong , Bishop David H. , Hengartner Hans , Zinkernagel Rolf M.
Published: March 2007

Induction of CD8+, class I‐restricted T cells by non‐infectious, exogenous antigens has been documented for model protein antigens such as ovalbumin and for major histocompatibility complex restricted short peptides in viral and tumor systems. However, the protective capacity of cytotoxic T cells induced by conventional proteins has not been tested in vivo so far. We, therefore, evaluated the induction of protective cytotoxic T cells against three different full‐length recombinant viral proteins derived from a baculovirus expression system, i.e. the glycoprotein and nucleoprotein of lymphocytic choriomeningitis virus (LCMV) and the nucleoprotein of vesicular stomatitis virus (VSV). These viral proteins induced cytotoxic T cells in a T helper cell‐independent fashion which lysed infected target cells in vitro and protected mice from viral replication, immunopathological disease and growth of a tumor expressing the same antigen as a tumor antigen. These results are surprising, since it had been shown earlier for completely inactivated nonreplicating viral vaccines and again here for β‐propiolactone‐inactivated VSV or UV‐light inactivated LCMV that nonreplicating viral vaccines were incapable of inducing protective cytotoxic T cells. Our data show that immunization of mice with as little as 10 μg of non‐infectious viral proteins triggered long‐lasting CD8+ T cell‐mediated antiviral immunity. It was found that the protein alone was only weakly able to induce cytotoxic T cells, and that association with cellular debris functioned as an adjuvant. These findings may be relevant for our understanding of the phenomenon of cross‐priming and have obvious implications for vaccine strategies.
Inhibition of nitric oxide synthesis by interleukin‐4 may involve inhibiting the activation of protein kinase C epsilon
Authors: Sands William A. , Bulut Vedat , Severn Alison , Xu Damo , Liew Foo Y.
Published: March 2007

The murine macrophage cell line, J774, when activated with interferon‐γ (IFN‐γ), expressed high level of inducible nitric oxide synthase (iNOS) and bound significantly more [3H]‐phorbol‐dibutyrate (PBu2) compared to non‐activated cells. The increased PBu2 binding to the particulate fraction of the cells is a measure of activation and translocation of protein kinase C (PKC). Both the expression of iNOS and the enhanced PBu2 binding in the activated J774 cells were significantly inhibited by the pretreatment of the cells with murine recombinant interleukin‐4 (IL‐4). Stimulation of J774 cells by IFN‐γ and lipopolysaccharide results in the translocation predominantly of the epsilon isoform of PKC (PKC‐ϵ), and this is inhibited by IL‐4. The inhibition of PKC activation was also evident by measuring the PKC activity in the cytosolic fraction of the IL‐4‐treated cells. Activated J774 cells pretreated with IL‐4 or a PKC‐specific inhibitor (RO31‐8220) failed to express mRNA of iNOS analyzed by PCR. These results, therefore, suggest that the inhibition of nitric oxide synthesis in activated murine macrophages by IL‐4 is at the transcriptional level and may involve the inhibition of the activation of PKC‐ϵ.
T lymphocytes derived from skin lesions of patients with psoriasis vulgaris express a novel cytokine pattern that is distinct from that of T helper type 1 and T helper type 2 cells
Authors: Vollmer Sigrid , Menssen Antje , Trommler Paul , Schendel Dolores , Prinz Jörg Christoph
Published: March 2007

In various immunological disorders the pathomechanisms of tissue damage are causally associated with specific patterns of locally produced cytokines. To study the molecular and cellular mechanisms involved in the manifestation of psoriasis vulgaris we have assessed the cytokine mRNA profile expressed in lesional psoriatic skin and in T cell clones (TCC) that were established from skin lesions of patients with psoriasis. As demonstrated by use of the polymerase chain reaction (PCR), psoriasis lesions consistently exhibit transcription of a complex pattern of cytokines. It includes mediators selectively produced by T lymphocytes [interferon (IFN)‐γ, tumor necrosis factor (TNF)‐β, interleukin (IL)‐2, IL‐3 and IL‐5] as well as cytokines secreted by various cell types [transforming growth factor (TGF)‐α/‐β, TNF‐α, IL‐6/‐8 and granulocyte‐macrophage‐colony stimulating factor], while IL‐4 is missing. With the exception of TGF‐α, this cytokine profile was also observed in lesional psoriatic T cell clones yielding supernatants mitogenic for keratinocytes in vitro (MTCC), but not in T cell clones yielding supernatants that inhibited keratinocyte proliferation (STCC).

The congruent cytokine expression of psoriatic skin lesions and MTCC emphasizes that inflammation in psoriasis is driven by a sofar unrecognized regulatory T cell subset that may serve to control epidermal regeneration and convey immunosurveillance over epithelial surfaces. It is characterized by the combined expression of IFN‐γ, TGF‐β, IL‐2 and IL‐5 in the absence of IL‐4 and by its selective capacity to enhance keratinocyte proliferation. This newly defined combination of regulatory properties of a distinct T cell population cannot be reconciled with an immune response of the T helper cells (TH)0, TH1 or TH2 type.
Regulation of IgG antibody titers by the amount persisting of immune‐complexed antigen
Authors: Bachmann Martin F. , Kündig Thomas M. , Hengartner Hans , Zinkernagel Rolf M.
Published: March 2007

Antigens normally induce an immunoglobulin (Ig)G response which stays at an elevated level for several weeks or months, constituting an important part of the immunological memory. This study investigated factors influencing the level of neutralizing IgG titers against a virus and shows that within the range tested it was independent of the number of initially available and potentially responding T helper and B cells, but was regulated by the amount of specific IgG‐immune complexes forming depots of persisting antigen. These findings support the notion that the efficiency of vaccines in inducing long‐lasting protective IgG is regulated predominantly by the amount of persisting (and presumably follicular dendritic cell‐associated) antigen‐antibody complexes.
Cross‐reactivity between an HLA‐B27‐derived peptide and a retinal autoantigen peptide: a clue to major histocompatibility complex association with autoimmune disease
Authors: Wildner Gerhild , Thurau Stephan R.
Published: March 2007

Statistical correlations between the expression of various HLA antigens and certain autoimmune diseases have been observed for both HLA class I and II antigens. Autoimmune diseases like spondyloarthropathies and anterior uveitis are associated with HLA‐B27, but uveitis in Behçet's disease with HLA‐B51. We describe a peptide from disease‐associated HLA class I antigens sharing sequence homologies with a highly uveitogenic epitope from the retinal autoantigen S‐antigen. S‐antigen induces autoimmune uveitis in the animal model and is a major autoantigen in human disease. The HLA peptide induced uveitis in the Lewis rat and, moreover, suppressed S‐antigen‐induced disease when administered orally. Patients' PBL cross‐reacted with the HLA‐ and corresponding retinal peptide, explaining the organ specificity of the disease.
Regulation of ICAM‐3 (CD50) membrane expression on human neutrophils through a proteolytic shedding mechanism
Authors: del Pozo Miguel A. , Pulido Rafael , Muñoz Cecilia , Alvarez Vicente , Humbría Alicia , Campanero Miguel R. , Sánchez‐Madrid Francisco
Published: March 2007

The regulation of the cell surface expression of ICAM‐3 (CD50) was investigated in human neutrophils. Immunofluorescence flow cytometry analysis revealed a remarkable and very rapid down‐regulation of the ICAM‐3 cell surface expression upon neutrophil activation with stimulating agents such as phorbol myristate acetate (PMA) or calcium ionophore. A similar low expression of ICAM‐3 was observed on neutrophils from patients undergoing hemodialysis with cell‐activating cellulosic membranes. Internalization assays with 125I‐labeled anti‐ICAM‐3 monoclonal antibody (mAb) suggested that ICAM‐3‐down‐regulation was due to antigen release from the cell surface towards the outer milieu, rather than to antigen internalization. Immunoprecipitation studies confirmed this down‐regulatory effect, and revealed the presence of ICAM‐3 in cell‐free supernatants from activated neutrophils. Furthermore, the presence of a soluble form of ICAM‐3 with a range of concentrations of 0–296 ng/ml in the plasma from healthy human volunteers was detected by using a two‐site mAb radioimmunoassay. A proteolytic mechanism likely accounts for this process since protease inhibitors virtually abrogated the PMA‐induced down‐regulation of ICAM‐3. Functional studies showed that anti‐ICAM‐3 mAb were able to trigger homotypic neutrophil aggregation both before and after ICAM‐3 down‐regulation, indicating that the fraction of ICAM‐3 molecules remaining on the neutrophil surface upon activation are still capable of sustaining cell adhesion. In contrast, the loss of L‐selectin (CD62L) on activated neutrophils was almost complete, thus leading to an impairment of L‐selectin‐mediated neutrophil‐endothelial cell adhesion. These results indicate that ICAM‐3 is released to the medium upon neutrophil stimulation and that both ICAM‐3 and L‐selectin have a role in the neutrophil adhesive phenomena.
Macrophage – T cell interaction in experimental mycobacterial infection. Selective regulation of co‐stimulatory molecules on Mycobacterium‐ infected macrophages and its implication in the suppression of cell‐mediated immune response
Authors: Saha Bhaskar , Das Gobardhan , Vohra Harpreet , Ganguly Nirmal K. , Mishra Gyan C.
Published: March 2007

The most important immunopathological consequence of experimental mycobacterial infection is the suppression of T cell‐mediated immune response to both mitogens and mycobacterial antigens. We registered that there was decreased concanavalin A‐induced spleen cell proliferation in infected susceptible BALB/c mice as compared to normal mice. In resistant (C3H/HeJ) mice, infection with the bacteria did not induce any suppression in the mitogen‐induced lymphoproliferation. Likewise, delayed‐type hypersensitivity (DTH) responses, to keyhole limpet hemocyanin and mycobacterial crude soluble antigen were suppressed in infected BALB/c mice but not in C3H/HeJ mice. This depressed T helper cell function may either be due to defective T cell‐receptor occupancy by antigen‐Ia complex or altered co‐stimulatory signals provided by antigen‐presenting cells. In the present study, we have investigated the status of certain co‐stimulatory molecules on the infected macrophages from both susceptible and resistant mice. Our results demonstrate that upon mycobacterial infection, the macrophages are rendered incapable of delivering the co‐stimulatory signals to T helper cells, possibly due to the involvement of prostaglandin, as inhibition of its biosynthesis by indomethacin reversed the defect. Furthermore, the selective regulation was bacteria‐induced as killing of the bacteria by rifampicin abrogated the derangements in the expression of co‐stimulatory molecules on the Mycobacterium‐infected macrophages. Our observations revealed that upon infection with Mycobacterium tuberculosis, B7 was down‐regulated while ICAM‐1 was increased only in BALB/c but not in C3H/HeJ mice. Expression of VCAM‐1 did not change during the infection in either strain of mice. We found that these changes in ICAM‐1 and B7 expression on the surface of infected macrophages resulted in inhibition of DTH‐mediating functions of T helper cells from BALB/c mice. The results obtained in this study describe not only a novel immune evasion strategy adopted by Mycobacterium, but also open up the possibility of immunotherapy of mycobacterial infection by selective manipulation of co‐stimulatory molecules.
Soluble complement receptor type 1 (CD35) is released from leukocytes by surface cleavage
Authors: Danielsson Carina , Pascual Manuel , French Lars , Steiger Gertraud , Schifferli Jürg‐Alfred
Published: March 2007

The soluble form of complement receptor type 1 in human plasma (sCR1) might correspond to the shedding of the receptor by proteolytic cleavage at the cell surface. A new enzyme‐linked immunosorbent assay (ELISA) was established to specifically measure membrane‐bound CR1 using a rabbit polyclonal antibody against a 19‐amino acid peptide corresponding to the C‐terminal sequence of the intracellular domain of CR1 (mCR1‐ELISA). This ELISA measured CR1 from solubilized erythrocyte membranes, polymorphonucelar leukocytes (PMN), a B lymphocyte cell line and renal podocyte‐derived urinary vesicles in a dose‐dependent manner. In contrast, and similarly to recombinant soluble CR1 which lacks the intracellular domain of CR1, plasmatic sCR1 was not recognized, suggesting that sCR1 corresponds to an extracellular fragment of whole CR1. In vitro, PMN were shown to release a soluble form of CR1 which was also not recognized in the mCR1‐ELISA, and whose size was smaller (5 kDa) than the CR1 of PMN cell membranes. The release of soluble CR1 was highest for PMN and HL60 cells, followed by U937 cells and three different B lymphocyte cell lines, whereas T lymphocyte cell lines did not release soluble CR1. The levels of CR1 gene expression were also higher in PMN compared to remaining blood leukocytes and the different cell lines tested above. Incubation of PMN with formyl‐methionyl‐leucyl‐phenylalanine, tumor necrosis factor‐α or lipopolysaccharide accelerated the release of soluble CR1, and incubation with granulocyte/macrophage colony‐stimulating factor resulted in sustained CR1 gene expression and higher total soluble CR1 release. Our results suggest that soluble CR1 is produced by cleavage of cell surface CR1, and that a large fraction of human plasma sCR1 is cleaved from PMN. The release of sCR1 by leukocytes may play a role in the control of complement activation at sites of inflammation.
Gut‐homing CD4+ T cell receptor αβ+ T cells in the pathogenesis of murine inflammatory bowel disease
Authors: Rudolphi Angelika , Boll Guido , Poulsen Steen S. , Claesson Mogens H. , Reimann Jörg
Published: March 2007

We studied which T cell subsets from the gut‐associated lymphoid tissue (GALT) can migrate out of the gut mucosa and repopulate GALT compartments of an immunodeficient (semi)syngeneic host. Many distinct lymphocyte subsets were found in GALT of immunocompetent H‐2d (BALB/c, BALB/cdm2, C. B‐17+/+) mice. No antigen receptor‐expressing lymphoid cells were found in GALT of congenic C. B‐17 scid/scid (scid) mice. The heterotopic transplantation of a full‐thickness gut wall graft from the ileum or colon of immunocompetent (C. B‐17+/+, BALB/cdm2) donor mice onto immunodeficient scid mice selectively reconstituted a CD3+ T cell receptor αβ+ CD4+ T cell subset. CD4+ cells of this subset expressed the surface phenotype of mucosa‐seeking, memory T cells. In the immunodeficient scid host, this gut‐derived CD4+ T cell subset was found in spleen, peritoneal cavity, mesenteric lymph nodes (LN), epithelial layer and lamina propria of the small and large intestine, but not in peripheral LN. Scid mice heterotopically transplanted with gut from a congenic, immunocompetent donor developed clinical and histological signs of inflammatory bowel disease (IBD). Hence, the selective repopulation of GALT compartments with CD4+ T cells from normal GALT plays an essential role in the pathogenesis of IBD in an immunodeficient host.