bluuebunny/crossref_metadata_2025 huggingface dataset:
Crossrecognition by CD8 T cell receptor αβ cytotoxic T lymphocytes of peptides in the self and the mycobacterial hsp60 which share intermediate sequence homology
Authors: Zügel Ulrich , Schoel Bernd , Yamamoto Shigeki , Hengel Hartmut , Morein Bror , Kaufmann Stefan H. E.
Published: March 2007

Immunization of C57BL/6 mice with the mycobacterial heat shock protein (hsp) 60 in immunostimulating complexes caused the in vivo activation of autoreactive major histocompatibility complex class I (H‐2Db)‐restricted CD8 T cell receptor (TcR) α/β cells. A CD8 TcR α/β clone with specificity for the mycobacterial hsp60 peptide499–508 was derived from this immunization, which, in addition, recognized syngeneic macrophages which had been stressed by interferon‐γ (IFN‐γ) stimulation. The stress‐induced, self peptide could be extracted from IFN‐γ‐stressed macrophages by acid elution, suggesting that the IFN‐γ‐induced self peptide is derived from an endogenous protein. Based on our observation that lysis of stressed target cells by this cytotoxic T lymphocyte (CTL) clone was specifically inhibited by hsp60‐specific antisense oligonucleotides, we used synthetic peptides representing amino acid (aa) sequences of the murine hsp60 for target cell sensitization and identification of the relevant self peptide. Synthetic peptides representing 9‐mer to 11‐mer aa sequences of the murine hsp60 with asparagine in anchor position 4 or 5 as the minimal requirement for H‐2Db binding were tested in CTL assays. The overlapping murine hsp60 peptides162–170/171 were stimulatory at a concentration as low as 10–100 pM. Seven other peptides of the murine hsp60 required intermediate peptide concentrations of 10–100 nM for recognition by the CTL clone. Although the murine and mycobacterial hsp60 peptides recognized by this CTL clone showed only intermediate homology (3 identical and 3 similar aa), our data suggest that endogenous hsp60 itself is the source of self peptide(s) presented by IFN‐γ‐stressed macrophages to the cross‐reactive CTL clone with promiscuous specificity. This notion is consistent with the idea of hsp as a link between infection and autoimmunity.
Phosphatidylinositol 3‐kinase activity is not essential for CD28 costimulatory activity in Jurkat T cells: studies with a selective inhibitor, wortmannin
Authors: Lu Yiling , Phillips Catherine A. , Trevillyan James M.
Published: March 2007

The interaction of CD28 with its counter‐receptor, B7‐1 (CD 80), on antigenpresenting cells induces a co‐signal in T cells required to promote antigendependent interleukin‐2 (IL‐2) production and to prevent clonal anergy. CD28 stimulation causes both protein‐tyrosine kinase and phosphatidylinositol3‐kinase (PI3‐K) activation, suggesting a possible role for these enzyme activities in CD28 co‐signal transduction. Here, we investigate the effect of wortmannin, a selective and irreversible PI3‐K inhibitor on CD28 co‐signaling events in the Jurkat T cell line. Wortmannin added to cell cultures partially inhibits CD28‐induced tyrosine phosphorylation of the putative p110 catalytic subunit of PI3‐K, but does not block CD28‐induced association of the p85 PI3‐K regulatory subunit with the CD28 receptor. Wortmannin inhibits in a dose‐dependent manner both total cellular PI3‐K activity and CD28‐induced receptor‐associated PI3‐K activity. Wortmannin (1 μM) inhibits cellular PI3‐K activity by 90% with complete inhibition achieved at 10 μM. The inhibitory effect of wortmannin on cellular PI3‐K activity is prolonged (> 18 h), suggesting that the drug is not readily metabolized by Jurkat T cells. Wortmannin, at concentrations that blocked PI3‐K activity, fails to inhibit the synergistic effect of CD28 on IL‐2 secretion in the presence of phorbol 12‐myristate 13‐acetate and ionomycin. These data demonstrate that CD28‐induced signaling events other than the activation of PI3‐K catalytic activity contribute to the control of IL‐2 secretion.
FK506 binding protein 12 mediates sensitivity to both FK506 and rapamycin in murine mast cells
Authors: Fruman David A. , Wood Michael A. , Gjertson Carl K. , Katz Howard R. , Burakoff Steven J. , Bierer Barbara E.
Published: March 2007

The immunosuppressive drugs FK506 and rapamycin bind to a family of intracellular proteins termed FK506‐binding proteins (FKBP). FK506 and rapamycin inhibit lymphocyte‐activation pathways by forming complexes with an FKBP; subsequently, the drug/FKBP complexes interact with target molecules involved in signal transduction. A key target of FK506/FKBP12 complexes is calcineurin, a calcium‐ and calmodulin‐dependent serine/threonine phosphatase. In mammalian cells, rapamycin treatment is associated with inhibition of the activity of several cellular serine/threonine kinases, including p70 S6 kinase. These kinases may function in signaling pathways involving TOR gene producs, which have been shown to interact with rapamycin/FKBP12 complexess in vitro. To determine if FKBP12 mediates the effects of both FK506 and rapamycin in mammalian cells, we overexpressed FKBP12 in a murine mast cell line. Increased expression of FKBP12 resulted in increased sensitivity to FK506 and rapamycin, as measured by inhibition of calcineurin activity and p70 S6 kinase activity, respectively. In contrast, overexpression of FKBP25 had no effect on sensitivity to either drug. Two distinct point mutations in FKBP12, one altering a hydrophobic residue within the drug‐binding pocket and the other changing a charged surface residue of FKBP12, abrogated its ability to mediate sensitivity to FK506 and rapamycin. These results establish that FKBP12 can mediate sensitivity to both FK506 and rapamycin in mammalian cells.
The third component of Xenopus complement: cDNA cloning, structural and functional analysis, and evidence for an alternate C3 transcript
Authors: Lambris John D. , Pappas John , Mavroidis Manolis , Wang Yang , Manzone Holly , Swager Joseph , Pasquier Louis Du , Silibovsky Randi
Published: March 2007

Although the third component of complement has been purified from two amphibian species, Xenopus laevis and the axolotl, only limited information is available about its primary structure in these species. We now present (a) 95% of the cDNA sequence encoding C3 from a Xenopus laevis/Xenopus gilli (Xenopus LG) hybrid (b) an analysis of the C3 convertase and factor I cleavage sites in Xenopus C3, and (c) evidence for an alternative form of C3. The Xenopus LG sequence has a 57% nucleotide and 52% amino acid sequence identity to human C3 and contains one potential N‐glycosylation site in the β‐chain. The deduced amino acid sequence showed that the C3 convertase and factor I cleavage sites (Arg‐Ser) are conserved in Xenopus C3 and protein sequencing of Xenopus C3 fragments fixed on zymosan during complement activation demonstrated that Xenopus C3 is indeed cleaved by C3 convertase and factor I at these sites. Our screening of a liver cDNA library identified an unusual C3 clone with a deletion of 2502 bp, suggesting the presence of a novel C3 transcript in Xenopus LG liver. The presence of this C3 transcript was confirmed by reverse transcription polymerase chain reaction using Xenopus LG liver mRNA and specific oligonucleotide probes. This transcript encoded a putative 102‐kDa protein comprising the β‐chain of C3, together with the first 59 residues and the last 103 residues of the α‐chain; it would therefore lack many of the ligand binding sites found in the intact <α‐chain. However, the molecule may be an analog of a truncated C3 molecule that is found in the serum of allergic dermatitis patients and acts as an inhibitor of eosinophil cytotoxicity and neutrophil adherence.
Induction in vitro of a primary human antiviral cytotoxic T cell response
Authors: Cerny Andreas , Fowler Patricia , Brothers Mary A. , Houghton Michael , Schlicht Hans J. , Chisari Francis V.
Published: March 2007

We report herein the successful priming of human anti‐viral cytotoxic T cells (CTL) in vitro using two induction strategies based on the stimulation of peripheral blood mononuclear cells isolated from uninfected donors with synthetic viral peptides. The peptides used contain HLA‐A2 binding motifs and have been identified as HLA‐A2‐restricted CTL epitopes in patients infected by the hepatitis B and C viruses. One approach uses repetitive long‐term stimulation and the other uses bulk cultures containing large numbers of naive peripheral blood mononuclear cells. Both approaches successfully induce HLA‐A2‐restricted CTL specific for several viral epitopes. Some CTL recognize endogenously synthesized antigen on target cells infected with recombinant vaccinia virus expressing the corresponding viral proteins. This simple technique permits easy analysis of the primary human CTL repertoire, and may be exploitable for production of specific CTL effector cells for adoptive immunotherapy and dissection of the cellular and molecular requirements for priming of naive human CTL.
Secondary rearrangements and post‐rearrangement selection contribute to restricted immunoglobulin DJH expression in young rabbit bone marrow
Authors: Fitts Matthew G. , Mage Rose G.
Published: March 2007

Extrachromosomal circular DNA purified from bone marrow cells of 2‐weeks‐old rabbits was assayed by polymerase chain reaction to determine the relative rearrangement frequencies of immunglobulin DH to JH genes in vivo. DH genes rearranged to individual JH genes with different frequencies. This bias did not correlate with potential sequence overlaps in the DH or JH coding sequences. The JH2 and JH4 genes were the preferred targets of recombination in primary rearrangements. Although primary rearrangements to JH6 were relatively infrequent, secondary rearrangements were detected. This assay also revealed previously undescribed JH pseudogenes with functional recombination signal sequences. Analyses of genomic VDJH indicated that B cells expressing VDJH4 heavy chains survived and dominated in the bone marrow environment due to secondary rearrangements and/or post‐rearrangement selection.
The role of the B cells in the adoptive transfer of collagen‐induced arthritis from DBA/1 (H‐2q) to SCID (H−2d) mice
Authors: Taylor Peter Charles , Plater‐Zyberk Christine , Maini Ravinder Nath
Published: March 2007

Collagen‐induced arthritis (CIA) can be transferred from DBA/1 to SCID mice when native type II collagen (CII) is administered together with spleen cells, arthritis appearing some 14 days after cell transfer. In the present study, we demonstrate that both donor T‐ and B‐lymphocyte populations play a role in this model, and that arthritis arises in SCID recipients of either murine or bovine native CII. Furthermore, the requirement for administration of soluble native CII can be replaced by subarthritogenic doses of serum from Wistar rats with CIA. In this case a fully developed arthritis appears as early as 2 days after cell transfer. However, protein G‐purified IgG from CIA rat serum together with splenocytes from arthritic DBA/1 mice does not transfer arthritis.

A key role of B cells in this model appears to be the production of a humoral arthritogenic factor since arthritis can be successfully transferred to SCID mice by CIA rat serum administered together with a B cell‐depleted splenocyte population consisting of T cells and donor‐histocompatible antigen‐presenting cells. By contrast, transfer of disease cannot be achieved by co‐administration of CIA rat serum and purified donor T cells, indicating that the presence of donor antigen‐presenting cells is a requirement for adoptive transfer of arthritis.

We propose that joint damage initiated by arthritogenic product(s) of the B cell lineage releases soluble antigens that are presented to T cells which perpetuate the disease. The finding that arthritis can be generated in SCID recipients of CIA rat serum together with splenocytes from non‐arthritic DBA/1 mice immunized with denatured CII supports the hypothesis that T cells with specificity for denatured joint components perpetuate disease initiated by humoral factors.
B lymphocyte recognition of cytochrome c: higher frequency of cells specific for self versus foreign antigen early in the immune response and V gene usage in the response to self antigen
Authors: Minnerath Jeanne M. , Mueller Carolyn M. , Buron Susan , Jemmerson Ronald
Published: March 2007

To study immunoglobulin gene usage in the antibody response of mice to the self antigen (Ag) mouse cytochrome c (cyt), B cell hybridomas were prepared from splenic B cells of immunized BALB/c mice prior to the onset of somatic mutation, i.e. 3 days after injecting ovalbumin (OVA)‐primed mice with mouse cyt coupled to OVA. Monoclonal antibodies (mAb) from all of the seven primary hybridomas we obtained were sensitive to a single amino acid substitution from aspartic acid to glutamic acid at position 62 in mouse cyt. This is the specificity of the vast majority of B cells responding to mouse cyt as determined from assays of B cells activated in splenic fragment cultures. Six of the mAb derive from the 19.1.2 J558 VH gene which is also used in the response to α (1 → 6) dextran and three of these mAb derive from the R9 Vx gene, a member of the VxOx‐1 family. The other mAb derive from distinct, although similar, Vx genes. Attempts to obtain hybridomas secreting primary (unmutated) mAb specific for cyt foreign to mice have been hampered by the much lower frequency of B cells responding early to foreign cyt in comparison to the self Ag. This suggests that, contrary to expectation of tolerance mechanisms, in naive BALB/c mice B lymphocytes specific for a single epitope on self cyt are present in higher frequency than B lymphocytes specific for similar epitopes on foreign cyt. Possible explanations for this result include biased expression in the B cell repertoire of the particular combination of V genes encoding mouse cyt‐specific mAb or to positive selection of developing B lymphocytes by endogenous Ag.
T cell receptor Vβ repertoire in mice lacking endogenous mouse mammary tumor provirus
Authors: Braun Michel Y. , Jouvin‐Marche Evelyne , Marche Patrice N. , MacDonald H. Robson , Acha‐Orbea Hans
Published: March 2007

When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg‐reactive T cell receptor (TcR) Vβ segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv‐6, −7, −8 and −9) and 38CH (Mtv−) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv‐6, −7, −8 or −9. The TcR Vβ chain (TcR Vβ) usage in these mice was analyzed using monoclonal antibodies specific for TcR Vβ2,Vβ3, Vβ4,β5,Vβ6,Vβ7,Vβ8,Vβ11,Vβ12 and Vβ14 segments. Both Mtv‐8+ mice and Mtv‐9+ mice deleted TcR Vβ5+ and Vβ11+ T cells. Moreover, we also observed the deletion of TcR Vβ12+ cells by Mtv‐8 and Mtv‐9 products. Mtv‐6+ and Mtv‐7+ animals deleted TcR Vβ3+ and Vβ35+ cells, and TcR Vβ6+,Vβ7+ and Vβ8.1+ cells, respectively. Unexpectedly, TcR Vβ8.2+ cells were also deleted in some backcross mice expressing Mtv‐7. TcR Vβ8.2 reactivity to Mtv‐7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn → Asp) in position 19 on the TcR Vβ8.2 fragment. Reactivities of BALB.D2 TcR Vβ8.2 and 38CH TcR Vβ8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared.

Finally, the observation of increased frequencies of TcR Vβ2+, Vβ4+ and Vβ8+ CD4+ T cell subsets in Mtv‐8+ and Mtv‐9+ mice, and TcR Vβ4+ CD4+ T cells in Mtv‐6+ and Mtv‐7+ mice, when compared with the T cell repertoire of Mtv− mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells.
Effects of interleukin (IL)‐13 on immediate‐early response gene expression, phenotype and differentiation of human mast cells. Comparison with IL‐4
Authors: Nilsson Gunnar , Nilsson Kenneth
Published: March 2007

The recently cloned interleukin 13 (IL‐13) shares most investigated biological activities on B lymphocytes and monocytes with IL‐4. In this study we investigated the potential role of IL‐13 in regulating human mast cell activities. The effects of IL‐13 on the expression of an immediate‐early response gene (c‐fos), proliferation, expression of mast cell‐associated cell surface antigen (CD54 and Kit), and in vitro differentiation of human mast cells, were investigated. We compared the effect of IL‐13 with that of IL‐4. Both IL‐13 and IL‐4 induced expression of c‐fos in cells from the human mast cell line HMC‐1. This indicates that mast cells express functional receptors for IL‐13. IL‐13 and IL‐4 decreased the proliferation rate of HMC‐1 cells. However, IL‐13 was less potent than IL‐4. Human mast cells constitutively express the adhesion molecule ICAM‐1 (CD54) and the receptor for stem cell factor (Kit) (CD117). The expression of CD54 was increased after treatment with IL‐13 or IL‐4, whereas the expression of Kit was decreased. Also in this action IL‐4 was more potent than IL‐13. By culturing mononuclear cells from cord blood in the presence of stem cell factor there is a differentiation of tryptase‐positive mast cells in the cultures. This process was inhibited when IL‐4 was present. In contrast, IL‐13 did not affect the expression of tryptase during differentiation of stem cell factor dependent cord blood‐derived mast cells. Taken together, these findings indicate that IL‐13 has regulatory effects on human mast cells. The effect overlaps with but is also different from that of IL‐4.
Simple strategy to induce antibodies of distinct specificity: Application to the mapping of gp120 and inhibition of HIV‐1 infectivity
Authors: Prieto Isidro , Hervás‐Stubbs Sandra , García‐Granero Marta , Berasain Carmen , Riezu‐Boj José‐Ignacio , Lasarte Juan‐José , Sarobe Pablo , Prieto Jesus , Borrás‐Cuesta Francisco
Published: March 2007

In this study 96 15‐mer peptides encompassing the entire sequence of HIV‐1 gp120 were synthesized and used to immunize BALB/c mice (i) alone or (ii) in conjunction with the T helper cell determinant FISEAIIHVLHSR (FIS) from sperm whale myoglobin, which is well recognized by major histocompatibility complex (MHC) class II molecules of BALB/c. Of these peptides 39 were immunogenic per se and 57 were not. Out of the 57 non‐immunogenic peptides 53 could be rendered immunogenic with the second immunization protocol. With the exception of 4 cases, the anti‐peptide antibody titers induced in (ii) were equal (14 cases) or higher (78 cases) than those induced in (i). From the 96 anti‐peptide antibodies tested, 12 were able to recognize recombinant gp120 with good antibody titers, a result in agreement with previously identified B cell epitopes from gp120 by anti‐peptide antibodies induced with longer peptides conjugated to a carrier protein. Moreover, 4 of the 12 anti‐peptide antisera that recognized gp 120 were able to neutralize HIV‐1 infectivity in vitro, showing that the strategy of co‐immunization with FIS may afford functional antibodies.
T cells expressing the γδ T cell receptor are not required for egg granuloma formation in schistosomiasis
Authors: Iacomini John , Ricklan David E. , Stadecker Miguel J.
Published: March 2007

Immunopathology in schistosomiasis consists of a granulomatous response around parasite eggs. It has been established that granuloma formation is mediated by CD4+ T helper cells. However, the role of T cells bearing the γδ T cell receptor (TCR) has not been determined. In this study we utilized mutant mice that lack either αβ or γδ T cells as a result of gene targeting to investigate the relative roles of αβ and γδ T cells in the induction of immunopathology related to schistosomiasis. Mutant and control mice were infected with Schistosoma mansoni and granuloma formation as well as lymph node cell proliferative responses to egg antigens were analyzed after 8 weeks. TCR δ mutant mice (lacking γδ T cells) displayed vigorous formation of egg granulomas that were not significantly different from those observed in normal controls, both in terms of granuloma size and cellular composition. In contrast, TCR α and TCR β mutant mice (lacking αβ T cells) were unable to form granulomas. Moreover, mesenteric lymph node cells from TCR δ mutant and control mice responded strongly to egg antigens in vitro, while TCR α and β mutant mice did not. Our studies show that in schistosomiasis granuloma formation and proliferative responses to egg antigens are strictly dependent on αβ T cells. They also suggest that γδ T cells by themselves can neither mediate a granulomatous inflammation, nor significantly modify one mediated by αβ T cells.
Regulation of interferon‐γ mRNA in a cytolytic T cell clone: Ca2+‐induced transcription followed by mRNA stabilization through activation of protein kinase C or increase in cAMP
Authors: Kaldy Pierre , Schmitt‐Verhulst Anne‐Marie
Published: March 2007

Activation pathways inducing the expression of the interferon (IFN)‐γ gene in a cytotoxic T lymphocyte (CTL) clone were studied for their effects on transcription and on mRNA stability. IFN‐γ was secreted by the CTL clone in response to the Ca2+ ionophore ionomycin when used in conjunction with either protein kinase C (PKC)‐activating phorbol 12‐myristate 13‐acetate (PMA) or with agents increasing cAMP, including prostaglandin E2. We describe that ionomycin induced IFN‐γ gene transcription, which was totally inhibited in the presence of cyclosporin A (CSA), an immunosuppressant forming a calcineurin‐inhibiting complex with cyclophilin. Ionomycin did not, however, permit accumulation of IFN‐γ mRNA. Activation of PKC by PMA or of cAMP‐dependent protein kinase through increase in cAMP had no transcription‐inducing effect, either alone or in conjunction with ionomycin, as measured in run on assays of the IFN‐γ gene. When transcription of the IFN‐γ gene, initiated in the presence of ionomycin and an agent increasing intracellular cAMP, was inhibited by CSA in the absence of PKC or cAMP‐dependent protein kinase activation, the IFN‐γ mRNA was rapidly degraded (half‐life = 30 min). When either PKC was activated or intracellular cAMP was increased at the time of inhibition with CSA, a stabilizing effect was observed on IFN‐γ mRNA, which led to an increase in secreted IFN‐γ. These effects were selective, they did not affect the rate of transcription of the actin gene, nor the accumulation of actin mRNA.

These results show that (i) post‐transcriptional events can be critical for IFN‐γ expression in activated lymphocytes, and (ii) specific stabilization of IFN‐γ mRNA can be mediated by activation of two different protein kinases involved in T cell activation.
Regulation of the p70zap tyrosine protein kinase in T cells by the CD45 phosphotyrosine phosphatase
Authors: Mustelin Tomas , Williams Scott , Tailor Pankaj , Couture Clément , Zenner Georg , Burn Paul , Ashwell Jonathan D. , Altman Amnon
Published: March 2007

Two classes of protein tyrosine kinases (PTK) are utilized by the T cell antigen receptor (TcR)/CD3 complex for initiation of the signaling cascade, the Src‐family PTK p56lck and p59fyn, and the Syk‐family PTK p70zap and p72syk. In addition, the CD45 phosphotyrosine phosphatase (PTPase) is required for the induction of tyrosine phosphorylation by the TcR/CD3, presumably by positively regulating Src‐family PTK. Here we report that CD45 also regulates the Syk‐family PTK p70zap (or ZAP‐70). In CD45‐negative T cells, p70zap was constitutively phosphorylated on tyrosine and co‐immunoprecipitated with the TcR‐ζ chain. In resting wild‐type CD45‐positive cells, p70zap was mainly unphosphorylated, but it was rapidly phosphorylated on tyrosine upon treatment of the cells with anti‐CD3 or PTPase inhibitors. Finally, p70zap co‐distributed with CD45 in intact T cells, and tyrosine phosphorylated p70zap was dephosphorylated by CD45 in vitro. These findings suggest that CD45 plays an important role, direct or indirect, in the regulation of p70zap and its function in TcR/CD3 signaling.
Immunoglobulins from rats that are resistant to adjuvant arthritis suppress the disease in arthritis‐susceptible rats
Authors: Ulmansky Rina , Naparstek Yaakov
Published: March 2007

The therapeutic effect of high doses of polyclonal immunoglobulins has been well established in various B cell‐associated autoimmune diseases. In the present work we have examined the effect of low doses of immunoglobulins in adjuvant arthritis, a T cell‐associated disease in the Lewis rat. Lewis rats were treated with purified rat immunoglobulins as well as their Fc and F(ab′)2 fragments and their protective effect on adjuvant arthritis was evaluated. We found that early as well as late treatment with low doses of rat immunoglobulins induced refractoriness to disease induction. The effect was found to be carried out by the F(ab′)2 part of the immunoglobulins and could be adsorbed by affinity purification on Mycobacterium tuberculosis. The protective antibodies were present in Fisher and BN rats that are resistant to adjuvant arthritis, but not in the arthritis susceptible Lewis and Wistar strains. We suggest that resistance to autoimmune arthritis is associated with the presence of protective immunoglobulins and that their effect is carried out through the antigen recognition part of the molecule.
Transforming growth factor‐β inhibits interleukin‐12‐induced production of interferon‐γ by natural killer cells: A role for transforming growth factor‐β in the regulation of T cell‐independent resistance to Toxoplasma gondii
Authors: Hunter Christopher A. , Bermudez Luiz , Beernink Hans , Waegell Wendy , Remington Jack S.
Published: March 2007

Severe‐combined immune deficient (SCID) mice have been found to resist infection with the intracellular protozoan parasite Toxoplasma gondii via interleukin (IL)‐12 stimulation of interferon (IFN)‐γ production by natural killer (NK) cells. Previously, we demonstrated the presence of increased levels of transcripts for transforming growth factor‐beta (TGF‐β) in the brains and lungs of SCID mice infected with T. gondii, leading us to investigate the role of TGF‐β in the mechanism of resistance to T. gondii in these mice. Stimulation of splenocytes from SCID mice with heat‐killed T. gondii resulted in production of low levels of IFN‐γ and a two to threefold increase in levels of TGF‐β in the culture supernatants. Production of IFN‐γ in these cultures was increased three to fourfold by addition of anti‐TGF‐β antibody. Stimulation of splenocytes from SCID mice with IL‐12 in combination with either TNF‐α or IL‐1β resulted in production of high levels of IFN‐γ. Addition of TGF‐β to these cultures inhibited production of IFN‐γ in a dose‐dependent manner. Immunohistochemical studies revealed increased levels of TGF‐β protein in the spleens of SCID mice 5 days after oral infection with the ME49 strain of T gondii, and brains of SCID mice at 18 days post‐infection. However, no difference was detected in the levels of TGF‐β transcripts in the spleens of uninfected mice or mice infected for 5 days. To test whether TGF‐β could antagonize IL‐12 mediated resistance to T. gondii in vivo, we administered TGF‐β to SCID mice infected with T. gondii. This treatment resulted in earlier mortality of infected mice and significantly reduced the ability of exogenous IL‐12 to delay time‐to‐death. Administration of anti‐TGF‐β to SCID mice, beginning 24 h prior to infection and every 2 days thereafter, delayed significantly time‐to‐death. Together, our data demonstrate that TGF‐β antagonizes the ability of IL‐12 to stimulate production of IFN‐γ by splenocytes from SCID mice, and suggest a role for TGF‐β in regulation of T cell‐independent resistance to T. gondii.
Heterogenous glycosylation of ICAM‐3 and lack of interaction with Mac‐1 and p150,95
Authors: de Fougerolles Antonin R. , Diamond Michael S. , Springer Timothy A.
Published: March 2007

Intercellular adhesion molecule (ICAM)‐1, ICAM‐2, and ICAM‐3 have been identified as counter‐receptors for the leukocyte integrin lymphocyte function‐associated antigen 1 (LFA‐1). The other leukocyte integrins, Mac‐1 and p150,95, also interact with ICAM‐1. ICAM‐1 and ICAM‐3 are highly homologous, and an undefined ligand for Mac‐1 is presnt on neutrophils where ICAM‐3 is well expressed. In addition, glycosylation has been shown to affect the interaction of ICAM‐1 with Mac‐1. We therefore sought to characterize ICAM‐3 heterogeneity and determine whether ICAM‐3 was a ligand for either Mac‐1 or p150,95. Despite extensive differences in N‐linked glycosylation, ICAM‐3 purified from lymphoid cells and from neutrophils supports adhesion of LFA‐1‐bearing cells equally well; however, neither supports adhesion of Mac‐1 or p150,95‐expressing chinese hamster ovary cell transfectants. Similarly, purified Mac‐1 does not support adhesion of ICAM‐2 or ICAM‐3‐expressing L cell transfectants. ICAM‐3 on neutrophils does not participate in Mac‐1‐dependent homotypic aggregation. Thus, ICAM‐3 is not a counter‐receptor for either Mac‐1 or p150,95.
Cytokine‐dependent regulation of growth and maturation in murine epidermal dendritic cell lines
Authors: Xu Shan , Ariizumi Kiyoshi , Edelbaum Dale , Bergstresser Paul R. , Takashima Akira
Published: March 2007

We have recently established dendritic cell (DC) lines (XS series) from the epidermis of newborn mice by repeated feeding with granulocyte/macrophage‐colony‐stimulating factor (GM‐CSF) and culture supernatants from skin‐derived stromal cell lines (NS series). XS lines resemble resident Langerhans cell (LC), which are immature DC that reside in epidermis, by their surface phenotype and antigen‐presenting profile. XS lines further resemble resident LC in that they express mRNA for interleukin‐1β and macrophage inflammatory protein (MIP)‐1α, and by the absence of mRNA for IL‐6. Their growth is promoted by GM‐CSF, colony‐stimulating factor‐1 (CSF‐1), or NS culture supernatant, and inhibited by interferon‐γ or tumor necrosis factor‐α. The expression by the XS lines of la molecules is up‐regulated by GM‐CSF, and down‐regulated by NS supernatant. These results suggest the existence of negative regulatory mechanisms in which the growth and/or maturation of DC is suppressed by selected cytokines.
Rolling of lymphocytes and neutrophils on peripheral node addressin and subsequent arrest on ICAM‐1 in shear flow
Authors: Lawrence Michael B. , Berg Ellen L. , Butcher Eugene C. , Springer Timothy A.
Published: March 2007

We studied leukocyte interactions in shear flow with peripheral lymph node addressin (PNAd), a mixture of glycoproteins expressed on high endothelial venules (HEV) that is required for lymphocyte homing and has been shown to contain a ligand for L‐selectin. T lymphocytes and neutrophils tether and roll on plastic‐immobilized PNAd and E‐selectin at 1.8 dyn/cm2 wall shear stress, but fail to interact with immobilized ICAM‐1, a ligand for LFA‐1 and Mac‐1, at the same flow rate. Cells roll faster on PNAd than on P‐selectin or E‐selectin. L‐selectin mAb inhibit T lymphocyte and neutrophil tethering to PNAd, but do not inhibit T lymphocyte tethering to purified E‐selectin. If allowed to interact with ICAM‐1 under static conditions, phorbol ester‐treated T lymphocytes, but not resting T lymphocytes, are able to form stationary adhesions that withstand the detachment force generated by 36 dyn/cm2 wall shear stress. In contrast, a wall shear stress of 7.3 dyn/cm2 detaches 50% of resting T lymphocytes bound to PNAd. Incubating T lymphocytes on PNAd and ICAM‐1 does not result in adhesion strengthening, suggesting that adhesion through PNAd by L‐selectin does not stimulate lymphocyte LFA‐1 avidity for ICAM‐1. Chemoattractant stimulation of neutrophils or phorbol ester stimulation of lymphoblasts rolling on co‐immobilized PNAd and ICAM‐1 results in rapid arrest and firm sticking, extending the model of sequential selectin‐mediated rolling and subsequent integrin‐mediated firm arrest to lymphocytes and ligands expressed on HEV.
Diacylglycerol lipase activation and 5‐lipoxygenase activation and translocation following TCR/CD3 triggering in T cells
Authors: Cifone Maria Grazia , Cironi Luisa , Santoni Angela , Testi Roberto
Published: March 2007

Arachidonic acid (AA) release was observed following T cell receptor (TCR)/CD3 complex cross‐linking in different tumor T cell lines as well as on purified peripheral T cells in vivo. Direct measurement of enzymatic activity in vitro of TCR/CD3‐stimulated Jurkat cell extracts on labeled vesicle substrates showed that TCR/CD3 cross‐linking resulted in AA release from sn‐1,2‐diacylglycerol (DAG) vesicles, as detected by TLC analysis, suggesting that DAG lipase was activated following TCR/CD3 stimulation and DAG generation. On the contrary, no phospholipase A2 activation was observed in response to TCR/CD3 stimulation, since no lyso‐phospholipids were generated in vitro from either phosphatidylcholine or phosphatidylinositol‐3,4‐bisphosphate, or from phosphatidic acid vesicles. Moreover, the 1‐DAG lipase inhibitor RHC80267 completely blocked TCR/CD3‐dependent AA release in vitro and in vivo, without effect upon TCR/CD3‐dependent inositol‐1,4,5‐trisphosphate (IP3) generation. Importantly, evidence for further metabolism of released AA was obtained, since synthesis and release of cysteinyl leukotrienes (CLT), but not of leukotriene B4 or cyclooxygenase products, could be detected by radioimmunoassay in different T cell lines and peripheral blood T cells following TCR/CD3 cross‐linking. Moreover, HPLC analysis revealed an accumulation of leukotriene E4 in TCR/CD3 stimulated Jurkat cells. This was associated with translocation of 5‐lipoxygenase from the cytosol to the cell membranes. Finally, TCR/CD3‐mediated CLT production was blocked by MK886, a specific inhibitor of 5‐LO translocation and activation.

Our data help define a further level in the fate of second messengers generated after TCR/CD3 triggering and suggest that additional mediators can play a role in the context of T cell activation.
Follicular dendritic cells and B cell chemotaxis
Authors: Burton Gregory F. , Kupp Leo I. , McNalley Erin C. , Tew John G.
Published: March 2007

B cells isolated from germinal centers (GC) of immune mice 2–5 days after antigen (Ag) challenge migrate in response to chemotactic signals, whereas GC B cells isolated at other times and resting B cells do not. Since B cells are in direct contact with follicular dendritic cells (FDC) in GC we reasoned that FDC might play a role in enabling B cells to become chemotactically active. Resting B cells were co‐cultured with FDC either with or without anti‐μ‐dextran (anti‐μ‐dex) as an Ag surrogate and/or recombinant interleukin (rIL)‐4 as a T cell surrogate. After 3 days, the B cells were isolated and their migration to chemotactic factors contained in zymosan‐activated serum assessed in microchemotaxis chambers. B cells incubated alone or with anti‐μ‐dex or rIL‐4 showed minimal migration, which could be increased if both anti‐μ‐dex and rIL‐4 were present. However, maximal migration was obtained when B cells were cultured with FDC, and this was not increased by addition of anti‐μ‐dex and/or rIL‐4, indicating that the FDC signal was a primary signal and did not require pre‐activation of the B cells. Checkerboard analysis using variation in concentration and location of the chemoattractant in chemotaxis chambers indicated that both chemotaxis and chemokinesis occurred. B cell migration began within 6 h of culture, peaked by 48 h and decreased thereafter. Removal of FDC or interference with FDC‐B cell contact ablated or significantly decreased induction of B cell migration. Furthermore, induction did not require functional T cells. These data indicate that FDC can induce resting B cells to become responsive to chemotactic signals.
Activation of virus‐specific major histocompatibility complex class II‐restricted CD8+ cytotoxic T cells in CD4‐deficient mice
Authors: Heemskerk Mirjam H. M. , Schilham Marco W. , Schoemaker Henriette M. , Spierenburg Gerrit , Spaan Willy J. M. , Boog Claire J. P.
Published: March 2007

Acute enteritic or respiratory disease is a consequence of coronavirus infection in man and rodents. Mouse hepatitis virus, stain A59 (MHV‐A59) causes acute hepatitis in mice and rats and induces a response of major histocompatibility complex (MHC) class II‐restricted CD4+ cytotoxic T cells, protecting mice against acute infection. In the present study we show that MHV‐A59 infection of mice that lack a functional CD4 gene activates effector cells of the CD8+ phenotype. These cytotoxic T cells lyse virus‐infected target cells in a MHC class II‐restricted fashion. The results indicate that CD8+ T cells have the potential to utilize MHC class II as restriction element, illustrating that the immune system can effectively deal with evading microorganisms, such as viruses which down‐regulate MHC class I.
Glycosylphosphatidylinositol‐linked Db does not induce an influenza‐specific cytotoxic T lymphocyte response or recycle membrane‐bound peptides
Authors: Motal Ussama M. Abdel , Sentman Charles L. , Zhou Xianzheng , Robinson Peter J. , Dahmén Jan , Jondal Mikael
Published: March 2007

Major histocompatibility complex (MHC) class I molecules, as well as MHC class I‐bound peptides, are known to recycle between the cell surface and an undefined, endosomal‐like compartment. Little is known about the functional significance of this process. We have explored this using two different forms of the H‐2Db molecule expressed in transgenic mice, either transmembranous (Db‐tm) or with a glycophosphatidylinositol (GPI)‐lipid anchor (Db‐GPI). The recycling capacity of peptides bound to Db‐tm and Db‐GPI was investigated using glycosylated Db‐binding glycopeptides, which were detected by flow cytometry. Only the tm form of Db was found to readily internalize and recycle glycopeptides to the cell surface. When transgenic mice were immunized with influenza A virus (PR8) strain and tested for cytotoxic T lymphocyte (CTL) responses against an immunedominant nucleoprotein epitope (366–374, ASNENMETM), onyl Db‐tm mice were found to generate specific CTL responses. The results support the idea that membrane recycling of MHC class I‐bound peptides on antigen‐presenting cells may be important for the generation of certain CTL responses.
Expression of heat‐stable antigen on tumor cells provides co‐stimulation for tumor‐specific T cell proliferation and cytotoxicity in mice
Authors: Wang Yi‐Chong , Zhu Lihua , McHugh Rebecca , Sell Kenneth W. , Selvaraj Periasamy
Published: March 2007

Heat‐stable antigen (HSA/J11d/possibly homologous to CD24), a cell adhesion molecule capable of providing a co‐stimulatory signal for T cell proliferation, is expressed on B cells, activated T cells, monocytes, granulocytes, Langerhans cells and thymocytes. Recent studies have demonstrated that co‐stimulatory signals provided by cell adhesion molecules such as B7‐1 play an essential role in generation of an anti‐tumor immune response. To examine whether the co‐stimulatory signal provided by HSA can induce an anti‐tumor immune response, we have transfected HSA cDNA into the murine melanoma cell line K1735M2, and examined the ability of this transfected cell line to induce tumor‐specific T cell responses. The results demonstrate that spleen cells from mice immunized with HSA‐transfected K1735M2 cells showed enhanced T cell proliferation in a mixed lymphocyte tumor reaction (MLTR) assay and also demonstrated a significant anti‐tumor cytotoxicity to the parent tumor cell (K1735M2). This anti‐tumor cytolytic activity could be abrogated by pretreatment of effector cells with anti‐mouse CD8 monoclonal antibody and complement. Under similar conditions, spleen cells from C3H mice immunized with vector‐transfected K1735M2 cells neither actively proliferate in an MLTR assay, nor did they exert significant cytolytic activity against the respective tumor cells. In summary, our study demonstrated that HSA can provide a co‐stimulatory signal for the T cell immune response against tumor cells in a murine model.
Anergy induction in encephalitogenic T cells by brain microvessel endothelial cells is inhibited by interleukin‐1
Authors: Bourdoulous Sandrine , Béraud Evelyne , Page Christine Le , Zamora Alfredo J. , Ferry Arnaud , Bernard Dominique , Strosberg A. Donny , Couraud Pierre‐Olivier
Published: March 2007

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) which can be induced, in susceptible strains like Lewis rats, by transfer of activated myelin basic protein (MBP)‐specific CD4+ T lymphocytes. The role of cerebral endothelium in the onset of EAE, with regard to adhesion, activation and infiltration in the CNS of encephalitogenic T lymphocytes, is not fully understood. When pretreated by interferon‐γ, the immortalized Lewis rat brain microvessel endothelial (RBE4) cells expressed major histocompatibility complex class II molecules and stimulated MBP‐specific proliferation and cytolytic activity of the syngeneic encephalitogenic T cell line, designated PAS. However, RBE4‐stimulated PAS lymphocytes subsequently entered an unresponsive state, known as anergy. When inoculated in syngeneic animals, anergic PAS cells, although still cytotoxic, failed to induce EAE, and no cell infiltration was detectable within CNS. The addition of interleukin‐1β (IL‐1β) during MBP presentation by RBE4 cells prevented T cell anergy induction, and maintained T cell encephalitogenicity, although PAS cells stimulated in these conditions caused delayed and attenuated clinical signs of EAE, with only discrete inflammatory lesions in the CNS, compared with EAE induced by PAS cells fully activated by thymic cells. Altogether, our results indicate that MBP presentation by brain microvessel endothelial cells to encephalitogenic T cells induces T cell anergy and loss of pathogenicity. In addition, IL‐1β co‐stimulation of T cells prevents anergy induction in vitro and at least partially maintains encephalitogenicity in vivo.
Interaction of B and T lymphocyte subsets with high endothelial venules in the rat: binding in vitro does not reflect homing in vivo
Authors: Walter Sebastian , Micheel Burkhard , Pabst Reinhard , Westermann Jürgen
Published: March 2007

Lymphocytes continuously migrate through the body, and their efficient extravasation from the blood via high endothelial venules (HEV) is essential for initiating an appropriate immune response. Most investigations have focused on the lymphocyte/HEV interaction in vitro. However, to what extent such systems reflect the situation in vivo is not known. It is also unclear whether lymphocyte subsets immigrate into the HEV in proportion to their presence in the blood, and whether import capacity is limited by the HEV. When rat mesenteric lymph node lymphocytes were incubated in vitro on cryostat sections, the well‐known preferential binding of B lymphocytes to HEV of Peyer's patches (PP) and T cells to HEV of axillary lymph nodes (axLN) was observed (axLN vs. PP: B lymphocytes 21.2 ± 5.0% vs. 40.6 ± 11.0%, T lymphocytes 84.6 ± 6.3% vs. 56.5 ± 12.9%). However, when labeled mesenteric lymph node lymphocytes were injected and their location within the HEV was analyzed 15 min later, no preferential interaction was seen. After injection of labeled thoracic duct lymphocytes, the percentage of labeled cells among B and T lymphocytes in the blood was significantly different (4.4 ± 0.9% vs. 8.9 ± 3.6%), whereas that in HEV of axLN (19.0 ± 6.4% vs. 16.6 ± 6.0%) and PP (30.6 ± 6.1% vs. 33.9 ± 4.4%) was comparable. Although the number of injected lymphocytes was similar in magnitude to the total blood lymphocyte pool, after injection there was no increase in lymphocyte numbers in the HEV. Thus, the adhesion assay in vitro does not completely reflect immigration into HEV in vivo. In addition, our data suggest that both the availability of lymphocyte subsets in small venules and the immigration rate into HEV are actively regulated in vivo.
Regulation of T cell receptor (TCR)‐β locus allelic exclusion and initiation of TCR‐α locus rearrangement in immature thymocytes by signaling through the CD3 complex
Authors: Levelt Christiann N. , Wang Baoping , Ehrfeld Angelika , Terhorst Cox , Eichmann Klaus
Published: March 2007

During thymocyte differentiation, the T cell receptor (TCR)‐β genes are rearranged before the TCR‐α genes. Immature CD4−8− double‐negative thymocytes with a productive rearrangement of the TCR‐β locus are selected to continue maturation to the CD4+8+ double‐positive stage, driven by signals through the pre‐TCR. The signals through the pre‐TCR can be synchronized by injection of mice with anti‐CD3ϵ monoclonal antibody. Using this approach, we demonstrated coordinated induction of a triad of responses in immature thymocytes: arrest of V to DJ rearrangement in the TCR‐β locus, transient down‐regulation of rearrangement‐activating gene (RAG)‐1 and RAG‐2 transcripts, and initiation of germ‐line transcription of the TCR‐α locus. These results suggest that the transition from TCR‐β to TCR‐α locus rearrangement is controlled by signal transduction through the pre‐TCR.
Soluble mouse major histocompatibility complex class II molecules produced in Drosophila cells
Authors: Wallny Hans‐Joachim , Sollami Giuseppina , Karjalainen Klaus
Published: March 2007

We have exploited Drosophila melanogaster. Schneider cells and compatible inducible expression vectors to produce large amounts of secreted major histocompatibility complex (MHC) class II molecules (I‐Ed). A simple two‐step purification protocol was developed. In the first step, recombinant molecules were enriched using a monoclonal anti‐class II antibody column followed by a nickel chelate column which further purified and concentrated the recombinant protein to several mg/ml. Characterization of the purified material indicates that the molecules are correctly assembled into αβ heterodimers. Further analysis shows that the recombinant MHC class II molecules are devoid of endogenous peptides and, therefore, homogenous peptide/MHC complexes could be prepared by adding exogenous I‐Ed‐specific peptides at slightly acidic pH. Upon peptide addition, molecules underwent a conformational change into a more compact form revealed by gel filtration analysis. In addition, the peptide/MHC complexes were biologically active. As little as 10 ng of these complexes coated on plastic from a 100 ng/ml solution were sufficient to trigger antigen‐specific T cell hybridomas. These MHC class II molecules, together with various forms of soluble T cell receptor (TcR) proteins, provide valuable tools to analyze the molecular details of TcR/antigen recognition.
Abnormalities of B cells and dendritic cells in SAMP1 mice
Authors: Haruna Hiroki , Inaba Muneo , Inaba Kayo , Taketani Shigeru , Sugiura Kikuya , Fukuba Yoh , Doi Hiroshi , Toki Junko , Tokunaga Rikio , Ikehara Susumu
Published: March 2007

The age‐related changes in the function of antigen‐presenting cells (APC) were examined using a substrain of senescence‐accelerated mouse (SAMP1). In the primary mixed lymphocyte reaction (MLR), dendritic cells (DC) from aged SAMP1 mice showed less stimulatory activity than those of age‐matched BALB/c or young SAMP1 mice. In the secondary MLR, the stimulatory activity of B cells was found to be lower in aged SAMP1 mice but not in age‐matched BALB/c or young SAMP1 mice. In addition, these age‐related decreases in the stimulatory activity of APC were found to be related to changes in the surface density of major histocompatibility complex class II and intercellular adhesion molecule‐1 (ICAM‐1) (but not B7‐1 or B7‐2 molecule) on APC (DC and B cells).