bluuebunny/crossref_metadata_2025 huggingface dataset:
The T cell receptor associated CD3‐ϵ protein is phosphorylated upon T cell activation in the two tyrosine residues of a conserved signal transduction motif
Authors: Sancho Jaime , Franco Rafael , Chatila Talal , Hall Craig , Terhorst Cox
Published: March 2007

Signal transduction through the Tcell receptor for antigen, the TcR/CD3 complex, involves phosphorylation of tyrosine residues in the CD3‐ϵ chain. Since both CD3‐ϵand the ζ, chain contain a tyrosine‐based signaling motif, we examine phosphorylation of CD3‐ϵ in human T cells. Engagement of the TcR/CD3 complex induced tyrosine phosphorylation of CD3‐ϵ in vivo. Induction of CD3‐ϵ phosphorylation followed similar kinetics to that of the ζ, chain phosphorylation. In contrast to ζ, CD3‐ϵ phosphorylation was strictly dependent upon cell surface expression of this member of the TcR/CD3 complex. Chemical and proteolytic cleavage combined with peptide‐specific Western blotting established that CD3‐ϵ phosphorylation occurred in the two tyrosine residues located in the signal transduction motif in the C‐terminal portion of the molecule. Taken together, these data indicated that phosphorylation of CD3‐ϵ by tyrosine protein kinases may serve to couple the TcR/CD3 complex to other effector molecules in the signaling cascade.
Molecular characterization of the early activation antigen CD69: A type II membrane glycoprotein related to a family of natural killer cell activation antigens
Authors: Ziegler Steven F. , Ramsdell Fred , Hjerrild Kathryn A. , Armitage Richard J. , Grabstein Kenneth H. , Hennen Kathryn B. , Farrah Terry , Fanslow William C. , Shevach Ethan M. , Alderson Mark R.
Published: March 2007

CD69 is a disulfide‐linked homo‐dimer expressed on the surface of activated T cells, B cells, natural killer cells, neutrophils and platelets. Antibody cross‐linking of CD69 in the presence of phorbol ester results in cellular activation events including proliferation and the induction of specific genes. Using an expression cloning strategy we have isolated cDNA encoding human CD69 from a CD4+ T cell clone. Transfection of the cDNA clone in CV‐1/EBNA cells results in the expression of a covalently linked homodimer. The cDNA insert hybridizes to a 1.7‐kb mRNA in phorbol 12‐myristate 13‐acetate‐ or phytohemoagglutinin‐stimulated human T cells. Using the human clone we have isolated cDNA encoding mouse CD69, which, when expressed in human T cells allowed those cells to respond to anti‐mouse CD69 antibodies by secreting interleukin‐2 and interferon‐γ. Sequence analysis showed that both mouse and human CD69 are type II membrane glycoproteins related to the NKR‐P1 and Ly‐49 families of natural killer cell activation molecules.
Development of γδ T cells in the adult murine thymus
Authors: Zorbas Michelle , Scollay Roland
Published: March 2007

The development of T cells belonging to the γδ lineage is not well understood. We have analyzed the cells in the adult murine thymus which express the γδ TcR on the surface in order to learn more about this process. Our data demonstrate a number of clear subpopulations of γδ expressing cells in the thymus based on the expression of Thy‐1 and HSA (heat‐stable antigen). Only one of these subpopulations, the one expressing both Thy‐1 and HSA, contains dividing cells or has a significant rate of turnover. Together with the fact that emigrant γδ cells are HSA+Thy‐1+, this suggests that this thymic subpopulation is the sole, or major, source of exported cells. However, the turnover of cells from this population is 5 × 104 ‐ 10 × 104 cells per day, while previous estimates of the rate of export of γδ cells are in the order of 104 cells per day. Furthermore the Vγ profile of recent γδ+ emigrants differs from that of the thymic HSA+Thy‐1+ cells. This raises the possibility that only a selected subpopulation of the thymic γδ+HSA+Thy‐l+ population is exported, and that some γδ cells may die in situ in the thymus. The function of the other γδ thymic subpopulations, which are turning over very slowly or not at all, (i.e. the HAS−Thy‐l− and HAS−Thy‐l+ subpopulations) remains unclear.
Restricted expression of transgenic HLA‐DRA gene in thymic epithelial cells and its role in acquisition of T cell tolerance to self‐superantigens and processed DRα‐derived peptide
Authors: Fukui Yoshinori , Yamamoto Ken , Yokoyama Nobuhiko , Iwanaga Tomohisa , Kurashima Chieri , Esaki Yukio , Kimura Akinori , Akashi Takumi , Hirokawa Katsuiku , Sasazuki Takehiko
Published: March 2007

We have established a set of transgenic mouse lines in which the HLA‐DRA gene was expressed in different cell types. In one line (DRα‐24), DRαEβb molecules were expressed on thymic medullary and cortical epithelial cells and all lineages of bone marrow‐derived antigen‐presenting cells (APC) except for thymic macrophages. By contrast, expression of the molecules in another line (DRα‐30) was found on thymic medullary and cortical epithelial cells but not on bone marrow‐derived APC in the thymus and periphery. To evaluate the role of thymic epithelial cells in acquisition of T cell tolerance, comparative analysis of DRα‐24 and DRα‐30 was performed. In DRα‐30, T cells expressing TcR Vβ5 and Vβ11 were eliminated to comparable levels to those in DRα‐24, suggesting that expression of the DRαEβb molecules on thymic epithelial cells are sufficient for clonal deletion of the self‐superantigen‐reactive T cells. In addition, CD4+ T cells from DRa‐30 as well as those from DRα‐24 were tolerant to DRα‐derived peptide/I‐Ab complex expressed on spleen cells from DRα‐24 even in the presence of exogenous interleukin‐2. These observations suggest that expression of the DRα chain in thymic epithelial cells could induce T cell tolerance directed toward naturally processed DRα‐derived peptide bound to I‐Ab molecules, probably via clonal deletion of the self‐reactive T cells.
Interleukin‐9 potentiates the interleukin‐4‐induced immunoglobulin (IgG, IgM and IgE) production by normal human B lymphocytes
Authors: Dugas Bernard , Renauld Jean Christophe , Pène Jerome , Bonnefoy Jean Yves , Peti‐Frère Corinne , Braquet Pierre , Bousquet Jean , Van Snick Jacques , Mencia‐Huerta Jean Michel
Published: March 2007

IgE production by normal peripheral blood lymphocytes (PBL) is known to be triggered upon stimulation by interleukin (IL)‐4. In the present study we showed that IL‐9, another T cell‐derived cytokine, markedly potentiated IgE production induced by suboptimal doses of IL‐4, whereas no effect of IL‐9 was observed in the absence of IL‐4. The potentiating effect of IL‐9 appeared to be associated with the increased frequency of IgE‐producing cells, as revealed by a specific ELISA‐spot assay. Under the same experimental conditions, IL‐9 also enhanced the IL‐4‐induced IgG production but did not elicit IgM production. However, IL‐9 did not amplify the IL‐4‐dependent expression of membrane‐bound and soluble low affinity receptor for IgE (CD23). IL‐4‐induced IgE production was also potentiated by IL‐6 but not by tumor necrosis factor‐α and IL‐β The possibility that the activity of IL‐9 was mediated by IL‐6 released from accessory cells was excluded by the observations that monocyte depletion did not abolish the effect of IL‐9 and that IL‐9 was still active on fluorescence assisted cell sorted CD20+ B lymphocytes co‐cultured with irradiated murine EL4 cells. In addition, IL‐9 was shown to potentiate the IL‐4‐induced IgG and IgM production by normal human B lymphocytes preactivated with Staphylococcus aureus Cowan strain. Taken together, these data suggest that IL‐9 plays a regulatory role in the IL‐4‐dependent immunoglobulin production.
The influence of rearranged T cell receptor αβ transgenes on early thymocyte development
Authors: Nikolić‐Žugić Janko , Andjeli Sofija , Teh Hung‐Sia , Jain Nada
Published: March 2007

Most murine thymocytes and mature T cells originate from a numerically minor population of CD8−4− (double‐negative, DN) thymocytes. In this study, we investigated the effects of rearranged T cell receptor (TcR) α and β transgenes on early T cell development. We analyzed the precursor potential, the expression of CD25 and TcR at mRNA and/or protein level in DN thymocyte subsets in TcR transgenic (Tg) mice. We report the following observations: (i) despite a large overrepresentation of total DN cells in TcR Tg mice, precursor‐containing CD25+ DN and CD810410 thymocytes are reduced to a third of the nontransgenic control numbers; (ii) like in the normal mice, CD25+ DN and CD810410 can, and TcR+ DN cells cannot generate other thymic subsets; (iii) TcR α mRNA and TcR α/β protein levels are quantitatively increased, but their developmental expression is similar to that in normal mice; and (iv) surface TcR αβ expression becomes detectable as the thymocytes down‐regulate CD25, paralleling the situation in normal mice. Our findings implicate stringent transcriptional control, rather than TcR gene rearrangement, as a decisive regulator of TcR αβ expression in early ontogeny.
Interaction of cutaneous stromal cells and γ/δ T cell receptor (TcR)‐positive cells. I. Vγ5−γ/γTcR+ T cells migrating from organ‐cultured murine skin proliferate by co‐culture with cutaneous stromal cells in the presence of interleukin‐2
Authors: Nakagawa Satoshi , Hara Masahiro , Seki Makiko , Yagita Hideo , Tagami Hachiro , Aiba Setsuya
Published: March 2007

It has been reported that Thy‐1+CD3+CD4−CD8− cells as well as Langerhans cells migrate from organ‐cultured murine skin into culture medium. We examined whether these Thy‐1+ populations of migrating cells were derived from Thy‐l+ dendritic epidermal T cells (Thy‐1+ DEC) and found that they were Thy‐1+CD3+ CD4−CD8−γ/ΔTcR+ (γΔ+T) cells but did not express Vγ/5TcR, which was used by a vast majority of Thy‐1+DEC Recently, a unique interaction between stromal cells and lymphohemopoietic progenitors has been reported in bone marrow and thymus. In this study, we established fibroblastoid cutaneous stromal cell (CSC) lines and clones from murine skin and examined the interaction between CSC and γδ+T cells. When these γδ+T cells were co‐cultured with CSC, a marked proliferation of small lymphoid cells was observed only in the presence of interleukin (IL)‐2. Neither CSC alone nor IL‐2 alone could induce a similar proliferation. Flow cytometry revealed that they were Thy‐1+CD3+ CD4−CD8−γ/5TcR− but Vγ5TcR−. Analysis of the major segments of their TcR by polymerase chain reaction demonstrated that Vγ1,Vγ2,Vγ4 and all of the Vδ chains from Vδ1 to Vδ7 were used without any predominant pattern. These data indicate the possible presence of yδ+T cells other thanVγ5TcR+Thy‐l+ DEC in the murine skin and the unique capacity of the CSC to support the growth of these migrating y/δ+ T cells.

The nomenclature of murine T cell receptor γ chain is according to Reilly et al. (Nature 1986. 527:878). The relationship between the different nomenclature systems is summarized in Takagi et al. (J. Immunol. 1989. 747:2112).
In vivo activation of mouse dendritic epidermal T cells in sites of contact dermatitis
Authors: Kaminski Marek J. , Bergstresser Paul R. , Takashima Akira
Published: March 2007

Adult mouse epidermis contains a population of dendritic,Thy‐l+,CD3+,CD4−, CD8− and T cell receptor (TcR) Vγ3/Vδ1+ leukocytes termed dendritic epidermal T cells (DETC). DETC isolated from skin and placed into culture will proliferative vigorously in response to T cell mitogens and T cell growth factors. In the present study, we examined whether DETC can be activated in situ by modulating their epidermal environment. Ear skin of CBA mice was painted with the chemical irritant, croton oil, and the epidermal cells (EC) isolated from such sites were then tested for proliferative responses to exogenous interleukin‐2 (IL‐2), in the absence of added mitogens. Cells from croton oil‐treated skin showed marked IL‐2 responsiveness, whereas cells from phosphate‐buffered saline‐treated skin failed to proliferate. IL‐2 responses were seen as early as 2 days after croton oil treatment and peaked between days 5 and 10. γδ TcR‐bearing cells, most likely resident DETC, were the major population to respond to IL‐2, since depletion of γδ TcR+ cells, but not γδ TcR+ cells, abolished that responsiveness, and since γδ TcR+ cell numbers increased markedly in the cultures that contained added IL‐2. These results indicate that DETC in normal skin, which are at a state of rest, may be activated when their residential epidermal environment is disrupted externally. This process of DETC activation may be a critical step in the maturation of DETC into effector leukocytes in vivo.
Pathogenic autoantibodies in the NZB mouse are specific for erythrocyte Band 3 protein
Authors: Barker Robert N. , De Oliveira Geraldo G. SÁ , Elson Christopher J. , Lydyard Peter M.
Published: March 2007

NZB mice spontaneously develop autoimmune hemolytic anemia (AIHA). The red blood cell (RBC) autoantigen bound by pathogenic IgG autoantibodies, previously designated “X”, was identified by immunoprecipitation. Autoantibody eluted from the RBC of AIHA‐positive NZB mice precipitated a 105‐kDa antigen that was identical in apparent molecular mass to Band 3, the RBC anion channel protein. Furthermore, the immunoblotted antigen also reacted specifically with BRIC 132, a monoclonal antibody against Band 3. The results, therefore, demonstrate that Band 3 bears autoantigenic epitopes that are important in the pathogenesis of AIHA in the NZB mouse.
Presentation of mouse tum− P91A antigen from chimeric proteins with different subcellular localizations by class I molecules of the major histocompatibility complex
Authors: Godelaine Danièle , Van Pel Aline , Van Malderen Marianne , Beaufay Henri
Published: March 2007

Like many antigens presented by class I molecules the mouse Ld‐binding turn−antigen P91A derives from a cytosolic protein. To decide how stringent this localization is for presentation to cytotoxic lymphocytes (CTL) the P91A template has been inserted in the cDNA of rat esterase ES‐10, a protein located in the endoplasmic reticulum (ER), and in the cDNA of mouse interleukin‐9, a secretory product of lymphocytes. The esterase construct was also engineered to replace the C‐terminal leucine by arginine, which causes secretion of the protein, or to delete the N‐terminal presequence, which prevents transfer of the nascent chain to the ER. After cell‐free transcription‐translation, or transfection in COS cells, the products of the chimeric cDNA had the expected size and localization; however, the truncated form of esterase remained undetected in COS cells. The various chimeric templates were transfected in Pl.HTR cells (H‐2d); upon challenge with Ld‐restricted anti‐P91A CTL the cells were lyzed almost as efficiently as cells transfected with the full‐length P91A cDNA. We conclude that peptide fragments that bind to class I molecules of the major histocompatibility complex can be generated in the ER.
Evidence for selective pressure in the appearance of monoclonal immunoglobulins during aging: Studies in M54 μ‐transgenic mice
Authors: Gueret Roland , Grandien Alf , Andersson Jan , Coutinho Antonio , Radl Jiri , Weksler Marc E.
Published: March 2007

Serum monoclonal immunoglobulins (M‐Ig) appear during aging but little is known about the immunological factors which lead to their development. We have investigated whether such M‐Ig occur as a clonally random process or result from V‐region‐directed selective pressures. We have analyzed a μ‐transgenic mouse strain in which over 95% of all splenic B cells express the transgenic μ chain. All endogenous repertoire and unchain diversity are generated from the 5% of the B cells which express endogenous μ chains. Not one of the M‐Ig detected in these mice were of transgene origin alone; 11 of the 14 M‐Ig did not express a μ‐ chain and none of the μ chain containing M‐Ig expressed the transgene allotype alone. This observation suggests that the B cells giving rise to M‐Ig are heavily selected from among the small number of B cells which express endogenous Ig. The selective factors that might act on the endogenous B cell pools are discussed.
In vivo enhancement of the specific antibody response via the low‐affinity receptor for IgE
Authors: Heyman Birgitta , Tianmin Liu , Gustavsson Susanne
Published: March 2007

The ability of antigen‐specific IgE antibodies to modulate the in vivo antibody response was studied by comparing the antibody response in mice immunized with 2,4,6‐trinitrophenyl (TNP)‐specific monoclonal IgE followed by bovine serum albumin (BSA)‐TNP or with BSA‐TNP alone. The serum IgG antibody response against BSA, measured in enzyme‐linked immunosorbent assay ELISA, was enhanced up to 100‐fold in groups receiving IgE. The enhancement required specific interaction between IgE and antigen, since no effect was seen when unconjugated BSA was used as antigen. Polyclonal activation by IgE/antigen complexes did not occur. IgE given 24 h after specific antigen had no stimulatory capacity. Pretreatment of the mice with Fee receptor type II (FcϵRII)‐specific monoclonal antibody completely inhibited the IgE‐mediated enhancement. Thus, the data demonstrate for the first time an in vivo role for FcϵRII in enhancement of specific antibody production.
Phosphodiesterase inhibitor pentoxifylline, a selective suppressor of T helper type 1‐ but not type 2‐associated lymphokine production, prevents induction of experimental autoimmune encephalomyelitis in Lewis rats
Authors: Rott Ortwin , Cash Evelyne , Fleischer Bernhard
Published: March 2007

The phosphodiesterase inhibitor pentoxifylline (POX), which is known to have pharmacological effects in animal models of multiorgan failure and endotoxin‐mediated shock, was tested for its immunosuppressive potential on T lymphocyte activation in vitro and in vivo. POX was found to have a profound inhibitory effect on both mitogen‐ and antigen‐induced proliferation of CD4+ T cells in vitro. This inhibitory activity of the drug could be reproduced by treating T lymphocytes with cAMP analogues during stimulation. Responses of repeatedly in vitro stimulated cells were much more strongly inhibited by the drug and by cAMP analogues than responses of fresh resting lymphocytes. Furthermore, POX could drastically down‐regulate tumor necrosis factor regulate production and to a lesser extent interleukin (IL)‐2 secretion in activated T cells, but an excess of exogenous IL‐2 did not override the antiproliferative effect of the drug. In contrast, the same doses of POX had no inhibitory effect on spontaneous or induced IL‐4 and IL‐6 production by short‐term cultured T lymphocytes, indicating a selective sparing of T helper type 2 (Th2)‐associated lymphokine functions by the drug. To test a potential use of POX as an antiinflammatory agent in T cell‐mediated autoimmune disease, the influence of POX on myelin basic protein (MBP)‐induced experimental autoimmune encephalomyelitis (EAE) was assessed. The onset of EAE in Lewis rats could almost completely be abrogated by oral administration of POX during the induction phase of disease. Lack of clinical symptoms in POX‐treated animals coincided with a marked suppression of MBP‐specific T cell reactivity in vitro, without any evidence for a generalized impairment of T cell activity. Collectively, our data suggest the potential use of xanthine derivatives of the POX type as a supporting antiinflammatory therapeutic agent in Th1 CD4+ T cell‐mediated autoimmune diseases in animal models and possibly in man.
Reduced expression of major histocompatibility complex class I free heavy chains and enhanced sensitivity to natural killer cells after incubation of human lymphoid lines with β2‐microglobulin
Authors: Carbone Ennio , Stuber György , Andrée Sofia , Franksson Lars , Klein Eva , Beretta Alberto , Siccardi Antonio G. , Kärre Klas
Published: March 2007

Enhancement of major histocompatibility complex (MHC) class I expression leads to protection from recognition by natural killer (NK) cells in several systems. MHC class I gene products can be expressed in different forms at the cell surface ‐ for example as “empty” β2‐microglobulin (β2m)‐associated heterodimers or free heavy chains. To study the role of different class I heavy chain forms in NK target interactions, we have used lymphoblastoid target cell lines preincubated with β2m. This was found to shift the equilibrium between β2m‐associated and nonassociated ‐ heavy chains in favor of the former. In parallel, there was a significant increase in NK sensitivity. The recognition of MHC class I‐deficient cell lines was not affected by β2m, arguing against a general nonspecific effect of fern on NK sensitivity. Our data indicate that protection against NK recognition correlates with target cell expression of free heavy chains (i.e. devoid of β2m) rather than with expression of complexes.
Induction of protective immunity against coronavirus‐induced encephalomyelitis: Evidence for an important role of CD8+ T cells in vivo
Authors: Flory Egbert , Pfleiderer Michael , And Albert Stühler , Wege Helmut
Published: March 2007

Coronavirus MHV‐JHM infections of rats provide useful models to study the pathogenesis of virus‐induced central nervous system disease. To analyze the role of the immune response against defined MHV‐JHM antigens, we tested the protective efficacy of vaccinia virus (VV) recombinants expressing either the nucleocapsid (N) or the spike (S) protein. A strong protection was mediated in animals by immunization with recombinant VV encoding a wild‐type S protein (VV‐SWildtype), whereas VV recombinant expressing a mutant S354CR protein (VV‐S354CR) had no protective effect. Recombinant VV encoding N protein (VV‐N) induces a humoral and a CD4+ T cell response, but did not prevent acute disease regardless of the immunization protocol. In these experiments, challenge with an otherwise lethal dose of MHV‐JHM was performed prior to the induction of virus‐neutralizing antibodies and studies with the anti‐CD8+ monoclonal antibody, MRC OX8 showed that elimination of the CD8+ subset of T cells abrogates the protective effect. This result indicates that CD8+ T cells primed by recombinant VV expressing wild‐type S protein are a primary mechanism of immunological defense against MHV‐JHM infection in rats.
Interleukin‐12/T cell stimulating factor, a cytokine with multiple effects on T helper type 1 (Th1) but not on Th2 cells
Authors: Germann Tieno , Gately Maurice K. , Schoenhaut David S. , Lohoff Michael , Mattner Frank , Fischer Susanne , Jin Shen‐Chu , Schmitt Edgar , Rüde Erwin
Published: March 2007

At least two subsets of CD4+ T helper cell lymphocytes termed Th1 and T h, 2 exist in the mouse and probably in humans. They are characterized by the secretion of different lymphokines and by their functional behavior. Dysregulated expansion of one or the other subset may be one reason for the development of certain diseases. Thus, it is of importance to define the signals involved in the differentiation and activation of the two Th cell subsets. It is known and has been confirmed in this report that the cytokine interleukin (IL)‐1 acts onTh2 cells but not on Th1 cells. We now report that a previously identified cytokine which was provisionally termed T cell stimulating factor is identical with IL‐12 and exhibits a reciprocal behaviour to IL‐1. IL‐12 has several effects on Th1 cells. It can induce the proliferation of certain Th1 cells in combination with IL‐2. Synthesis of interferon (IFN)‐γ by Th1 cells can be triggered by IL‐2 plus IL‐12. In contrast to the IFN‐γ production observed after T cell receptor (TcR) CD3 stimulation of Th1 cells with lectin Concanavalin A the IFN‐γ production induced by IL‐12+IL‐2 is insensitive to the immunosuppressive drug cyclosporin A. Furthermore, IL‐12 enhances the TcR/CD3‐induced synthesis of IFN‐γ of several Th1 clones. Finally, IL‐12 (+ IL‐2) induces homotypic cell aggregation of Th1 clones. This type of cell aggregation depends on the participation of LFA‐1 and ICAM‐1 molecules. In all activation systems with Th1 cells no effect of IL‐1 was demonstrable. In contrast, only IL‐1 but not IL‐12 served as a co‐stimulatory signal for several Th2 cell lines activated via the TcR/CD3 complex.
A soluble, single‐chain Kd molecule produced by yeast selects a peptide repertoire indistinguishable from that of cell‐surface‐associated Kd
Authors: Abastado Jean‐Pierre , Ojcius David M. , Casrouge Armanda , Yeh Patrice , Schumacher Ton N. M. , Ploegh Hidde L. , Kourilsky Philippe
Published: March 2007

Peptide binding to a soluble, single‐chain Kd protein produced by the yeast strain Kluyveromyces lactis, and to Kd molecules on Kd‐expressing cells (P815) was studied using radiolabeled Kd‐restricted peptides. The stability of the peptide‐Kd complexes formed was monitored in the absence and presence of unlabeled competitor peptides. Radioiodination of the Tyr anchor residue in position 2 of the peptide interferes with binding. A Kd‐biased peptide library and a modified antigenic peptide in which a second Tyr was added in positions 6 and 8, respectively, were therefore used to assay binding. Recombinant and cell‐associated Kd molecules are very similar in the following respects: the ease with which the proteins can be loaded with labeled peptide; the spectrum of peptides selected from a peptide library; the stability of the labeled peptide‐Kd complex formed; and the ability to partially dissociate the class 1‐peptide complex with exogenous, unlabeled peptides. These results imply that measurements of peptide binding to soluble Kd molecules are a reliable indicator of the peptide‐binding properties of Kd proteins on living cells. The large quantities of soluble recombinant Kd protein currently available represent an invaluable tool not only for dissecting the molecular mechanisms of antigen presentation but also for vaccinations and the design of T cell‐specific toxins.
Protection from insulin‐dependent diabetes mellitus is linked to a peptide transporter gene
Authors: Caillat‐Zucman Sophie , Bertin Eric , Timsit José , Boitard Christian , Assan Roger , Bach Jean‐François
Published: March 2007

HLA class II association with insulin‐dependent diabetes mellitus (IDDM) is well established but is still difficult to map to a particular locus. Polymorphism of the genes coding for transporter associated with antigen processing (TAP1 and TAP2), and located in the HLA class II region, was studied in 167 IDDM patients (116 adult‐onset and 51 childhood‐onset patients) and 98 normal controls using oligotyping after genomic amplification. A dominant protective effect was observed for theTAP2*0201 allele [relative risk (RR)=0.3, corrected probability (pc) < 0.001]. Conversely, susceptibility to IDDM was associated with apparent homozygosity for the TAP2*0101 allele (RR=3.4, pc < 0.001). Protection was independent from but additive to the protection conferred by the DRB1*02 DQB1*0602 haplotype (RR=0.06, pc<0.05), and antagonistic to the DRB1*03 DQB1*0201 and DRB1*04 DQB 1*0302 haplotypes predisposing effect (RR=1.1, not significant), arguing in favor of an absence of linkage disequilibrium between TAP2 and HLA class II genes. This was assessed by x2 analysis. TAP1 allelic distribution was not different among diabetics and controls. A significant association was observed between the presence of TAP2*0101 and that of islet cell antibodies (p < 0.05). These data suggest that the TAP2 gene, which encodes protein required for delivery of antigen peptides to class I molecules in the endoplasmic reticulum, could modulate the autoimmune response leading to β cell destruction. From a practical point of view, they make the combined screening of HLA class II and TAP2 loci a highly valuable tool in IDDM prediction.
Enhancing effect of natural killer cell stimulatory factor (NKSF/interleukin‐12) on cell‐mediated cytotoxicity against tumor‐derived and virus‐infected cells
Authors: Chehimi Jihed , Valiante Nicholas M. , D'Andrea Annalisa , Rengaraju Manthrasalam , Rosado Zenaida , Kobayashi Michiko , Perassia Bice , Wolf Stanley F. , Starr Stuart E. , Trinchieri Giorgio
Published: March 2007

Natural killer cell stimulatory factor (NKSF) or interleukin‐12 (IL‐12) is a heterodimeric cytokine with pleiomorphic effects on T and NK cells, including induction of lymphokine production, mitogenesis, and enhancement of spontaneous cytotoxic activity. Similarly to IL‐2, NKSF/IL‐12 enhances NK cell‐mediated cytotoxicity within a few hours and independently from induced proliferation. This effect is independent from other induced cytokines, because it is not prevented by antibodies neutralizing interferon (IFN)‐α, IFN‐β IFN‐γ, IL‐2 or tumor necrosis factor (TNF)‐α and, unlike the induction of IFN‐γ production by peripheral blood lymphocytes, it does not require HLA class II‐positive accessory cells. Enhanced cytotoxicity is accompanied by morphologic changes in NK cells, including a significant increase in the number of cytoplasmic granules. In addition to the previously described ability to enhance the cytotoxic activity of NK cells against tumor‐derived target cells, NKSF/IL‐12 is also a potent stimulator of cytotoxicity against virus‐infected cells, either fibroblasts acutely infected with herpes viruses or T cell lines chronically infected with human immunodeficiency virus‐1. NK cell‐mediated antibody‐dependent cytotoxicity or anti‐CD16 antibody‐redirected lysis is not significantly enhanced by NKSF/IL‐12. However, the ability of resting peripheral blood T cells to mediate anti‐CD3 antibody‐redirected lysis is enhanced by 18‐h incubation with NKSF/IL‐12, indicating that this lymphokine can modulate the cytotoxic capability of both NK and T cells.
Gene expression and secretion of cytokines and cytokine receptors from highly purified CD56+ natural killer cells stimulated with interleukin‐2, interleukin‐7 and interleukin‐12
Authors: Naume Bjørn , Johnsen Ann‐Charlotte , And Terje Espevik , Sundan Anders
Published: March 2007

Interleukin (IL)‐2 IL‐7 and IL‐12 stimulate the generation of lymphokine‐activated killer activity and proliferation in natural killer (NK) cells by different mechanisms. In this study, we have compared the ability of IL‐2, IL‐7 and IL‐12 to induce expression of cytokines and cytokine receptors both at the gene and protein level. IL‐2 and IL‐12 stimulated the CD56+ NK cells to release significant amounts of soluble p55 and p75 tumor necrosis factor receptor (TNFR), whereas less amounts of soluble TNFR were detected in IL‐7‐stimulated cultures. The p55 and p75 TNFR mRNA were expressed in resting NK cells, and no further induction was observed after cytokine‐stimulation. Compared to the effects of IL‐2, IL‐7 induced lower, but substantial levels of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) mRNA, and IL‐7 was a more potent GM‐CSF‐inducing stimulus than IL‐12. IL‐12 induced higher levels of interferon‐γ (IFN‐γ) mRNA than did IL‐2, and IL‐7 only weakly influenced the IFN‐γ expression. In accordance with the mRNA studies, IL‐7 induced the secretion of high amounts of GM‐CSF and no or low levels of IFN‐γ, whereas high amounts of IFN‐γ and low levels of GM‐CSF were detected in supernatants from IL‐12‐stimulated NK cells. In conclusion, IL‐2, IL‐7 and IL‐12 differentially regulate expression of cytokines and cytokine receptors both at the gene and protein level.
Bacterial expression of a single‐chain Fv fragment which efficiently protects the acetylcholine receptor against antigenic modulation caused by myasthenic antibodies
Authors: Mamalaki Avgi , And Nikos Trakas , Tzartos Socrates J.
Published: March 2007

Monoclonal antibodies (mAb) against the main immunogenic region (MIR) of the acetylcholine receptor (AChR) are very potent in inducing antigenic modulation of the AChR in animals and in muscle cell cultures. A recombinant antibody fragment of the rat anti‐MIR mAb198 was cloned by polymerase chain reaction and expressed as soluble single‐chain Fv fragment (scFv198) in E. coli and affinity purified. DNA sequencing was used to define the VH (IB) and VL (K2) chain gene usage. scFv198 was found immunologically and biologically active. Its binding affinity for the Torpedo AChR (KD=2 ± 0.6 nM) was very similar with that of the intact mAb198 (KD=1.8 ± 0.6 nM) while for the human AChR(KD=80.7 ± 16.6 nM) it was about four times lower than that of the intact mAb198 (KD=21.6 ± 6.6 nM). This fragment was capable of efficiently protecting the AChR in human cell cultures, against antigenic modulation caused by the intact mAb198 or by the antibodies from a myasthenic patient. The produced scFv198 fragment is, therefore, potentially useful in therapeutic applications for myasthenia gravis after appropriate genetic manipulations.
Expansion of the CD4−, CD8− γδ T cell subset in the spleens of mice during non‐lethal blood‐stage malaria
Authors: van der Heyde Henri C. , Elloso M. Merle , Roopenian Derry C. , Manning Dean D. , Weidanz William P.
Published: March 2007

Splenic γδ T cells (CD4−, CD8−) increased more that 10‐fold upon resolution of either Plasmodium chabaudi adami or P. c. chabaudi infections in C57BL/6 mice compared to controls. Similarly, a 10‐ to 20‐fold expansion of the γδ T cell population was observed in β2‐microglobulin deficient (β2‐m0.0) mice that had resolved P. c. adami, P. c. chabaudi or P. yoelii yoelii infections. In contrast, increases in the number of splenic αβ T cells in these infected mice were only two to three‐fold indicating a differential expansion of the γδ T cell subset during malaria. Because nucleated cells of β2‐m0/0 mice lack surface expression of major histocompatibility complex class I and class Ib glycoproteins, our findings suggest that antigen presentation by these glycoproteins is not necessary for the increasing number of γδ T cells. Our observation that after resolution of P. c. adami malaria, C57BL/6 mice depleted of CD8+ cells by monoclonal antibody treatment had lower numbers of γδ T. cells than untreated controls suggests that the demonstrated lack of CD8+ cells in β2‐m0/0 mice does not contribute to the expansion of the γδ T cell population during non‐lethal malaria.
Evaluation of functional heterogeneity in the CD8 subset with T cells from T cell receptor‐transgenic mice
Authors: Gaugler Béatrice , Schmitt‐Verhulst And Anne‐Marie , Guimezanes Annick
Published: March 2007

The question of functional differentiation within the CD8 subset has been addressed in a model of TcR‐transgenic (TcR‐tg) mice expressing a TcR specific for H‐2Kb (Ti). CD8+ Ti+ T cells present in the periphery of these mice have no cytotoxic T lymphocyte (CTL) activity unless they are stimulated with H‐2Kb‐expressing cells. In contrast to T cells from normal H‐2k littermates, alloantigen induction of CTL from TcR‐tg mice is independent of CD4+ T helper (Th) cells and is accompanied by high level secretion of interleukin‐(IL)‐2 by Ti+ CD8+ T cells. Precursor frequency analysis performed on CD8+ cells from TcR‐tg mice revealed a high frequency of Th as compared to CTL precursors. This raised the possibility of the existence of distinct subpopulations within CD8+ precursors with different requirements for differentiation to functional CTL. FACS analyses (performed on resting and on in vitro stimulated T cells from normal and TcR‐tg mice) demonstrated a heterogeneous expression of Ly‐6C on CD8+ cells with a large enrichment of Ly‐6C− cells among the Ti+ cells which persisted after stimulation with H‐2b cells in conditions that led to a homogeneous expression of the activation markers pgp‐1 and CD69. The possibility that Ly‐6C expression could mark functionally different subpopulations in CD8+ T cells was investigated. Stimulation of sorted populations of Ly‐6C− and Ly‐6C+ cells allowed detection of CTL precursors in both these subsets and the majority of limiting dilution wells containing one pCTL also scored positive for IL‐2 secretion. Thus, for CD8+ T cells expressing the same TcR, differentiation led to acquisition of both IL‐2 secretion and CTL function and there was no evidence for the existence of a distinct population of helper‐dependent CTL precursors.
B‐1a and conventional B cells from autoimmune NZB. H‐2bm12 mice exhibit similar functional characteristics in vivo
Authors: Klinman Dennis M. , Holmes Kevin L. , Conover Jacqueline , Chiang Bor‐Luen , Gershwin M. Eric
Published: March 2007

NZB. H‐2bm12 mice develop an autoimmune syndrome characterized by the overproduction of anti‐DNA antibodies and the expansion of B‐1 B cells. Thus, these animals provide a useful model to examine the antigenic specificity, cross‐reactivity and functional capability of B‐l versus conventional lymphocytes. Neither the repertoire expressed by in vivo activated Ly‐1+ splenic lymphocytes, nor their cross‐reactivity, differed significantly from that of conventional splenic B cells. When Ly‐1+ cells were cultured in vitro in the presence of lipopolysac‐charide plus interleukin‐4 or interferon γ, they underwent isotype switching at the same frequency as conventional B cells. Of interest, B‐l cells from the peritoneal cavity were significantly less likely to undergo isotype switching than those from the spleen. These findings indicate that in vivo activated B‐la and conventional B cells from mice with lupus manifest similar functional characteristics.
Antibody‐induced activation of the hyaluronan receptor function of CD44 requires multivalent binding by antibody
Authors: Lesley Jayne , Kincade Paul W. , Hyman Robert
Published: March 2007

CD44 can function as a receptor for hyaluronan (HA). However, many cell lines and normal hematopoietic cells that express CD44 do not constitutively bind HA. A monoclonal antibody (mAb) specific for CD44 (IRAWB14) has been described previously which induces CD44‐mediated binding of HA rapidly (seconds to minutes) in some cell lines and in normal murine T cells. Of 16 CD44‐specific mAb tested in the present study, only 3 exhibited this activity. Monovalent Fab fragments were prepared from two IgG2a antibodies that induce HA binding (IRAWB 14 and IRAWB 26) and used to determine whether multivalent binding was required for induction of HA receptor function. Fab from both antibodies had a tendency to form multivalent aggregates. After addition of iodoacetamide to prevent further aggregation, multimeric and monovalent forms were separated by gel filtration. This made it possible to compare the inducing activity of monovalent and multivalent antibody fragments of identical composition in the absence of Fc determinants. Multimeric forms were very active at inducing binding of fluorescein‐conjugated HA (Fl‐HA). Monovalent Fab fragments of both antibodies had 20‐ to 50‐fold lower binding activity than intact antibody or multimer. IRAWB 26 Fab monomers were completely inactive in the induction of HA‐binding. The observed weak inducing activity of IRAWB 14 Fab monomer could be attributed to very low levels of contaminating multimer. Induction of HA binding could also be achieved by using anti‐immunoglobulin to cross‐link Fab monomers of IRAWB 26. Thus, multivalent binding was required for the activation of HA binding by CD44‐specific antibody, suggesting that the distribution of CD44 molecules on the cell surface is important for HA receptor function.

In kinetic studies, induction of HA receptor function occurred simultaneously with antibody binding at 0°C (ice water bath). Furthermore, antibody could induce HA binding in paraformaldehyde‐fixed cells, which were permeable to propidium iodide and trypan blue, suggesting that intracellular signaling mechanisms were not involved in induction of receptor function. We conclude, therefore, that these CD44‐specific antibodies are inducing HA binding by directly influencing the distribution of CD44 on the cell surface. The possibility of a concurrent change in CD44 conformation is not ruled out. We discuss possible mechanisms by which CD44 might be activated to bind HA in vivo.
Expression of immunoglobulin kappa and lambda chains in mink
Authors: Bovkun Ludmila A. , Peremislov Valery V. , Nayakshin Alexander M. , Belousov Evgenij S. , Mechetina Ludmila V. , Aasted Bent , Taranin Alexander V.
Published: March 2007

The ratio of ϰ and λ chains of immunoglobulins varies significantly from one species to another. It has previously been thought that λ was only type expressed in mink. We tested mink immunoglobulin light chains using two monoclonal antibodies G80 and G88. It has been shown that G80 and G88 specifically recognize two antigenically different subpopulations of the light chains. Immunochemical analysis of these subpopulations separated by affinity chromatography suggested that they represent λ and ϰ types of light chains, respectively. Screening of a mink cDNA library with monoclonal antibody G88 resulted in the isolation of clone pIGK‐1 containing ϰ chain‐encoding sequence. The cDNA insert of pIGK‐1 included most of the V segment, as well as the J, C and 3′ untranslated sequences. Mink Vϰ sequence shown the highest homology with the human VϰII subgroup genes (76‐79%). Mink Cϰ sequence was 53‐63% homologous to Cϰ of other species. The striking feature of mink Cϰ chain is the presence of glutamine in the C‐terminal position. Southern blot analysis suggested that mink haploid genome has one Cϰ gene and multiple Vϰ genes. The ϰ: λ chain ratio in the 12 minks studied was, on the average, 46:54. The same ratio was observed for the ϰ‐ and X‐producing cells in the mesenteric lymph nodes. The five previously identified mink light chain allotypes were assigned to the λ chains, thereby confirming that λ chains in this species are additionally subdivided into several subtypes.
Characterization and purification of T lymphocyte aminopeptidase B : A putative marker of T cell activation
Authors: Belhacene Nathalie , Mari Bernard , Rossi Bernard , Auberger Patrick
Published: March 2007

We have previously described that human T lymphocytes express membrane‐associated peptidase activities (Mari et al., EMBO J., 1992,11:3875).We show in this report that intact Jurkat T cells readily cleaved H‐Arg‐paranitroanilide, an aminopeptidase B (AP‐B) substrate. The identification of the hydrolyzing activity as AP‐B was confirmed by its sensitivity to both arphamenine B and bestatin in the nanomolar range. Significant AP‐B activity was released in the supernatant upon incubation of intact T lymphocytes at 37°C. However, AP‐B activity was found mainly in the cytosolic fraction of Jurkat T cells. Cytosolic T cell AP‐B was purified to homogeneity and exhibited a molecular mass of 72 kDa. Purified AP‐B cleaved N‐terminal basic amino acid‐containing peptides such as thymopentin (H‐Arg‐Lys‐Asp‐Val‐Tyr‐OH), indicating that it might play a role in the regulation of the concentration of important soluble mediators of T cell activation. A rabbit polyclonal antibody was shown to recognize AP‐B as assessed by both immunoprecipitation and Western blot experiments. Finally, we found that AP‐B was up‐regulated during activation of normal and leukemic T lymphocytes.
T cell immunity after a viral infection versus T cell tolerance induced by soluble viral peptides
Authors: Kyburz Diego , Aichele Peter , Speiser Daniel E. , Hengartner Hans , Zinkemagel Rolf M. , Pircher Hanspeter
Published: March 2007

The fate of in vivo activated CD8+ cytotoxic T cells was studied in transgenic mice expressing a T cell receptor (TCR) specific for the lymphocytic choriomeningitis virus (LCMV) glycoprotein peptide 33‐41 presented by major histocompatibility complex (MHC) class I molecules. LCMV infection of TCR transgenic mice induced LCMV‐specific effector and memory T cells whereas injection of soluble LCMV glycoprotein peptide 33‐41 resulted in tolerance by peripheral deletion and anergy of LCMV‐specific T cells after an initial expansion phase. Similarly, LCMV peptide 33‐41‐specific tolerance could be achieved in normal C57BL/6 mice and was not abrogated by an LCMV infection. These results obtained with a classically MHC‐restricted peptide antigen parallel previous findings with retroviral or bacterial superantigens and indicate a possibility to modulate specifically mature peripheral cytotoxic T lymphocytes in vivo.
CD4+8− help prevents rapid deletion of CD8+ cells after a transient response to antigen
Authors: Kirberg Jörg , And Ludovica Bruno , von Boehmer Harald
Published: March 2007

We have followed the fate of mature CD8+ T cells with a male‐specific transgenic T cell receptor after antigenic stimulation with hemopoietic cells in the absence or presence of help. Our data show that mature CD8+ T cells can be deleted after a 3‐week period of transient activation and that help, e.g. in the form of interleukin‐2, can considerably delay the deletion. These experiments have implications for the design of protocols aiming at the establishment of specific immunological tolerance in T cells.
Thymus influences the development of extrathymically derived intestinal intraepithelial lymphocytes
Authors: Lin Tesu , Matsuzaki Goro , Kenai Hiroyuki , Nakamura Takao , Nomoto Kikuo
Published: March 2007

Overwhelming evidence suggests that the majority of murine small intestinal intraepithelial lymphocytes (IEL) are extrathymically derived. These IEL include those with T cell receptor (TCR) γδ and someTCR αβ (CD8αα and Thy‐1−). In contrast, congenitally athymic nude mice have low numbers of γδ TCR IEL as well as very few αβ TCR IEL, far less than that would be expected if one assumes that γδ TCR IEL and αβ TCR (CD8αα and Thy‐1−) IEL in euthymic mice are extrathymically derived. To examine this discrepancy, we followed extrathymic IEL differentiation in IEL of day 3‐thymectomized (NTX) mice as another athymic mouse model and found that γδ TCRIEL and extrathymically derived αβ TCR IEL in NTX mice are markedly reduced, almost to the level of nude mice. We further show that it is probably the absence of a thymic stroma that is responsible for the lower amounts of extrathymically derived IEL in nude mice, as the low amounts can be corrected to euthymic levels by syngeneic fetal thymus grafting but not by direct injection of F1 thymocytes. Lastly, unlike TCR/CD3+ extrathymically derived IEL, we noted a large proportion of extrathymic CD3−CD8− and CD3−CD8+ IEL; they were threefold more frequent in nude and NTX than in euthymic mice. This suggests that the thymus influences extrathymically derived IEL in its development from CD3− to CD3+ at the small intestinal epithelium.
Pairing of VH gene families with the λ light chain: Evidence for a non‐stochastic association
Authors: Yurovsky Vladimir V. , Kelsoe Garnett
Published: March 2007

The frequencies at which four VH gene families pair with the λ1 light (L) chain were determined by sequential hybridization of VH‐ and λ1‐specific DNA probes to mitogen‐induced colonies of B cells. Analysis of pair frequencies indicates that the repertoire of λ L chain antibodies is generated by the stochastic pairing of smaller 3′‐to‐mid‐locusVH gene families (X‐24, S107, Q52). However, the large 5′ VH J558 family appeared to associate with the λ1L chain non‐stochastically; the frequency of VhJ558/λ1+ colonies among all λ1+ colonies was significantly lower than the frequency of J558 expression among all (Cμ+) B cell colonies. This difference suggests that selection, either intrinsic at the level of rearrangement or heavy and L chain pairing, or extrinsic following surface immunoglobulin expression, may operate to shape the λ antibody repertoire prior to the introduction of exogenous antigen.