bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeted neutralization of the complement membrane attack complex inhibitor CD59 on the surface of human melanoma cells
Authors: Junnikkala Sami , Hakulinen Juha , Meri Seppo
Published: March 2007

Major problems in the immunotherapy of human tumors with complement‐activating monoclonal antibodies (mAb) are (i) inherent resistance of tumor cells to complement cytolysis and (ii) a possible undiscriminatory attack against normal cells. In the present study we have developed a procedure to simultaneously direct the complement membrane attack complex and neutralize its inhibitor CD59 (protectin) on human melanoma cells in vitro. G361 melanoma cells were selectively recognized in heterogenous cell mixtures by a complement‐fixing mAb (R24) against the tumor cell GD3‐ganglioside. Biotinylated anti‐CD59 mAb (YTH53.1) was directed to the tumor cells with a high‐affinity biotin‐avidin bridge using a proportion of R24 as a biotinylated targeting mAb and avidin as a linker. Biotinylated anti‐CD59 mAb lost its ability to activate complement, but retained its CD59‐neutralizing activity. Thus, it was possible to avoid nonspecific lysis of surrounding erythrocytes and endothelial cells and direct the CD59‐neutralizing effect to the tumor cells. As a result the tumor cells were efficiently killed by R24 plus complement while the bystander cells remained viable. These results suggest that it is possible to target an unrestricted complement membrane attack against GD3‐ and CD59‐positive melanoma cells.
Early degenerate selection of thymocytes by class I major histocompatibility complex
Authors: Grassi Fabio , Barbier Eliane , Cazenave Pierre‐André
Published: March 2007

Ontogeny of T cells is accomplished in the thymus by a process of positive selection, in which interaction of the T cell receptor (TcR) expressed on CD4+8+ thymocytes with self major histocompatibility complex (MHC), expressed on cortical epithelial cells, determines the progress along the maturation pathway and confers self restriction to T cells. Conversely, cells behaving as self reactive by interaction with bone marrow‐derived antigen‐presenting cells are negatively selected by apoptosis. We show here that the presence of a class I‐restricted soluble TcR (sTcR) in the fetal thymic microenvironment, early in T cell ontogeny, determines an enhanced negative selection of a sizeable number of CD4+8+ thymocytes, which have been previously subjected to a positive‐selection event. We hypothesize that the generation of the mature thymic T cell repertoire stems from an interaction of TcR, under a critical affinity threshold, with a self peptide‐MHC complex which is common to a great number of TcR specificities using the same restriction element. A shift in this affinity threshold, caused by sTcR, results in the generation of cells acting in a self‐reactive manner, which are then deleted. In extended fetal thymus organ culture in the presence of sTcR, we have also observed the appearance of mature CD8+ T cells, which once adoptively transferred to syngeneic nude mice are expanded in the periphery, consistent with an enhanced avidity of these cells for self MHC.
Engagement of MHC class II molecules by staphylococcal superantigens activates src‐type protein tyrosine kinases
Authors: Morio Tomohiro , Geha Raif S. , Chatila Talal A.
Published: March 2007

Staphylococcal exotoxins (SE) are superantigens that bind to monomorphic determinants on major histocompatibility complex (MHC) class II molecules and stimulate human peripheral blood T lymphocytes in a Vβ‐specific manner. SE also deliver activation signals via MHC class II molecules that initiate cell adhesion and cytokine gene transcription. These events are preceded by tyrosine phosphorylation and are antagonized by inhibitors of tyrosine kinases, indicating an essential role for these kinases in signaling via class II molecules. We report that stimulation of human peripheral blood monocytes with SE induced rapid and selective activation of the src‐related protein tyrosine kinases (PTK) fgr and hck. SE also induced the activation of fgr and lyn in B cells. PTK activation by SE required MHC class II expression, and was greatly potentiated in the presence of T cells bearing toxin‐specific Vβ chains. These results indicate that in addition to their antigen and superantigen‐presenting function, MHC class II molecules act as signal‐transducing receptors that are coupled to src‐type PTK.
Production of nitric oxide and superoxide by activated macrophages and killing of Leishmania major
Authors: Assreuy Jamil , Cunha Fernando Q. , Epperlein Monica , Noronha‐Dutra Alberto , O'Donnell Catherine A. , Liew Foo Y. , Moncada Salvador
Published: March 2007

Murine macrophages can be activated to produce nitric oxide (NO) and superoxide and these two radicals can react to form peroxynitrite, a powerful oxidant which may be involved in parasite killing. We now show that murine macrophages activated with zymosan and interferon‐γ (ZYM/IFN‐γ) produced both superoxide (peaking 1–2 h after stimulation, then rapidly declining) and NO (barely detectable at 6 h, peaking by 24 h). Macrophages activated with ZYM alone produced only superoxide, while stimulation with lipopolysaccharide (LPS) and IFN‐γ induced NO but not superoxide. Cells stimulated with ZYM/IFN‐γ or LPS/IFN‐γ killed Leishmania major to a similar degree, an effect that was completely blocked by the addition of N‐iminoethyl‐L‐ornithine. However, macrophages stimulated with ZYM alone were unable to kill L. major. S‐nitroso‐acetyl‐penicillamine, which release NO, was highly leishmanicidal when added directly to the parasites. 3‐morpholino‐sydnonimine hydrochloride which releases both NO and superoxide simultaneously, was also efficient at killing L. major and this cytotoxicity was greatly enhanced by the addition of superoxide dismutase. Finally, authentic peroxynitrite failed to induce any cytotoxic effect, even at a high concentration. Thus macrophages can produce either NO, superoxide or both, depending on the stimulus. However, the killing of L. major is dependent only on the production of NO.
The human OX40 homolog: cDNA structure, expression and chromosomal assignment of the ACT35 antigen
Authors: Latza Ute , Dürkop Horst , Schnittger Susanne , Ringeling John , Eitelbach Florian , Hummel Michael , Fonatsch Christa , Stein Harald
Published: March 2007

Tissue distribution and expression on mitogen and virally stimulated lymphocytes render the ACT35 molecule a human lymphocyte activation antigen which as yet could not be clustered. Expression cloning of the ACT35 antigen from a pCDM8 library of the HUT‐102 cell line revealed strong homology of the cDNA and its encoded protein sequence with the formerly described rat OX40 antigen. The 1.4‐kb nucleotide sequence and the deduced 277‐amino acid sequence of the single transmembrane protein were 65% and 63% identical, in human and in rat, respectively. Conservation included one N‐linked glycosylation site and one protein kinase C phosphorylation site. When expressed in COS‐1 cells, the cDNA presented properties comparable to the native ACT35 antigen and the rat OX40 molecule (relative molecular mass 48000). Thus, the ACT35 protein corresponds to the hitherto unknown human OX40 antigen and is, therefore, another member of the tumor necrosis factor/nerve growth factor receptor (TNFR/NGFR) family. After applying fluorescence in situ hybridization, the human ACT35/OX40 gene could be mapped to chromosome band 1p36 and is, thus, linked to the genes for TNFR II and CD30.
The effect of rapamycin on T cell development in mice
Authors: Luo Hongyu , Duguid William , Chen Huifang , Maheu Michel , Wu Jiangping
Published: March 2007

Rapamycin (RAPA) is a strong immunosuppressant with a chemical structure similar to that of FK506, although it acts by a mechanism different from both FK506 and cyclosporin A. The effect of RAPA on T cell development in mice was investigated in this study. RAPA caused significant thymic atrophy. The major histological change in the RAPA‐treated thymus was thinning of the cortex. No other apparent damage in the cortex or medulla was observed. Consistent with these histological findings, in vivo thymocyte cycling was blocked by RAPA before the S phase, and the ex vivo and in vitro proliferation of the thymocytes was also strongly repressed by the drug. According to electron microscopy and DNA fragmentation assays, RAPA did not induce apoptosis. These results indicate that the repressed thymocyte proliferation is a major mechanism causing RAPA‐induced thymic atrophy. Further, RAPA had no effect on thymocyte apoptosis induced by anti‐CD3 or ionomycin, and the drug did not interfere with deletion of CD4+8+ thymocytes or peripheral Vβ6+ T cells induced by anti‐CD3 or Mls‐1a, respectively. These data suggest that RAPA does not hamper the negative selection. There was a relative increase in the CD3− fraction of the de novo developing CD4 and CD8 double‐positive (DP) thymocytes in the RAPA‐treated mice. Moreover, there were relative increases of the CD3− fractions of the CD4 or CD8 single‐positive (SP) cells in both the thymi and periphery. The generation of the CD3− SP under the influence of RAPA could be used as a useful model for further study of the function and signal transduction of these CD3‐defective SP cells.
D‐penicillamine‐ and quinidine‐induced antinuclear antibodies in A.SW (H‐2s) mice: similarities with autoantibodies in spontaneous and heavy metal‐induced autoimmunity
Authors: Monestier Marc , Novick Kristine E. , Losman Michele J.
Published: March 2007

Ten percent of human lupus syndromes occur in patients as a result of treatment with certain medications. H‐2s mice can produce autoantibodies following treatment with various drugs or heavy metals and they are a potential animal model of drug‐induced lupus. We have examined nine anti‐chromatin monoclonal antibodies (mAb) from A.SW mice that had been treated with either D‐penicillamine or quinidine, two lupus‐inducing drugs in humans. These mAb are specific either for DNA or histone‐DNA complexes corresponding to nucleosomes or subnucleosome particles. Only one mAb reacts with an unknown chromatin antigen. The V region sequences of six of these mAb were studied and are notable by several features. As previously observed in spontaneous autoantibodies to DNA or histone‐DNA complexes, arginine or asparagine residues are found at critical locations throughout the V regions. Many of these residues, potentially important for binding to DNA or DNA‐histone complexes, result either from somatic mutations or atypical VH‐D‐JH rearrangements. Another significant characteristic is that the VH genes of several D‐penicillamine‐ or quinidine‐induced mAb are most similar to those of anti‐nucleolar mAb obtained from mercury‐injected A.SW mice. The implications of these findings for the pathogenesis of spontaneous or induced autoimmunity are discussed.
Virus or a hapten‐carrier complex can activate autoreactive B cells by providing linked T help
Authors: Steinhoff Ulrich , Burkhart Christoph , Arnheiter Heinz , Hengartner Hans , Zinkernagel Rolf
Published: March 2007

We investigated the mechanism leading to an IgG autoantibody response in two transgenic mouse lines expressing the glycoprotein of vesicular stomatitis virus (VSV‐G). Previous experiments have shown that these animals do not mount a transgene‐specific IgG response upon stimulation with purified VSV‐G or infection with recombinant vaccinia virus expressing VSV‐G. However, infection of VSV‐G transgenic animals with wild‐type vesicular stomatitis virus, serotype Indiana, readily induced VSV‐G‐specific, neutralizing IgG autoantibodies. We have tested whether this labile state of tolerance reflected differential availability of VSV‐G‐specific T help. For this, we immunized transgenic mice with the self‐antigen VSV‐G covalently coupled to sperm‐whale myoglobulin (VSV‐G‐SWM), to provide new T helper epitopes that are linked to the B cell epitope; co‐injected uncoupled VSV‐G and SWM served as control. High titers of VSV‐G specific IgG autoantibodies were detected in serum of mice immunized with VSV‐G‐SWM but not after co‐injection of uncoupled VSV‐G and SWM. Transgenic animals depleted of CD4+ T cells prior to injection of VSV‐G‐SWM failed to mount an IgG response. Priming of transgenic mice with the foreign carrier did not accelerate the IgG autoantibody response to VSV‐G‐SWM, suggesting that B cells were limiting the rate of the response. Thus, self‐reactive B cells could be triggered to produce IgG, if they received linked T help specific for a foreign carrier determinant provided either by a classical carrier determinant or a virus.
T helper type 1 development of naive CD4+ T cells requires the coordinate action of interleukin‐12 and interferon‐γ and is inhibited by transforming growth factor‐β
Authors: Schmitt Edgar , Hoehn Petra , Huels Christoph , Goedert Sigrid , Palm Norbert , Rüde Erwin , Germann Tieno
Published: March 2007

It was observed in vitro and in vivo that both interferon (IFN)‐γ and interleukin (IL)‐12 can promote the development of T helper type 1 (TH1) cells. Since IL‐12 was shown to be a costimulator for the production of IFN‐γ by T or natural killer (NK) cells, IL‐12 might play only an indirect role in TH1 differentiation by providing IFN‐γ which represents the essential differentiation factor. Using anti‐CD3 monoclonal antibody (mAb) for activation of naive CD4+ T cells in the absence of accessory cells we could demonstrate that costimulation by IFN‐γ alone results only in marginal TH1 development. Similarly, IL‐12 in the absence of IFN‐γ is only a poor costimulator for inducing differentiation towards the TH1 phenotype. Our data indicate that both cytokines are required to allow optimal TH1 development and that IL‐12 has a dual role, it promotes differentiation by direct costimulation of the T cells and also enhances the production of IFN‐γ which serves as a second costimulator by an autocrine mechanism. Another cytokine that was reported to favor TH1 differentiation in certain experimental systems is transforming growth factor (TGF)‐β. With naive CD4+ T cells employed in this study TGF‐β strongly inhibited the production of IFN‐γ triggered by IL‐12 as well as the IL‐12‐induced TH1 development. When TGF‐β was combined with anti‐IFN‐γ mAb for neutralization of endogenous IFN‐γ the TH1‐inducing capacity of IL‐12 was completetly suppressed.
A directory of human germ‐line Vχ segments reveals a strong bias in their usage
Authors: Cox Jonathan P. L. , Tomlinson Ian M. , Winter Greg
Published: March 2007

From the genomic DNA of a single individual, we have amplified, cloned and sequenced 37 human germ‐line Vχ segments. Four of these segments were new. We then compiled a comprehensive directory of all germ‐line Vχ segments and identified 50 different sequences with open reading frames. Comparison with 236 rearranged sequences revealed that no more than 24 of these germ‐line sequences could be assigned rearranged counterparts, that some of these were rarely used, and that only about 11 sequences are used frequently. This suggests that the expressed Vχ repertoire is mainly derived from a limited number of segments. Most surprisingly, the Jχ‐distal region of the locus appears to be rarely used: we could unambiguously assign 162 rearranged sequences to Vχ segments of the Jχ‐proximal region, but only 5 to segments of the Jχ‐distal region.
Engagement of the CD4 receptor inhibits the interleukin‐2‐dependent proliferation of human T cells transformed by Herpesvirus saimiri
Authors: Bröker Barbara M. , Tsygankov Alexander Y. , Fickenscher Helmut , Chitaev Nikolai A. , Müller‐Fleckenstein Ingrid , Fleckenstein Bernhard , Bolen Joseph B. , Emmrich Frank
Published: March 2007

Infection with Herpesvirus saimiri, a tumor virus of non‐human primates, transformed human CD4+ T cell clones to permanent interleukin (IL)‐2‐dependent growth without need for restimulation with antigen and accessory cells. The IL‐2‐dependent proliferation of these cells was dramatically inhibited by soluble anti‐CD4 whole antibodies, F(ab′)2 and Fab fragments, and also by gp 120 of human immunodeficiency virus. The inhibition was not due to cell death and could be overcome by high concentrations of exogenous IL‐2. Cell surface expression of CD4, and to a lesser degree the density of the IL‐2 receptor α chain, were reduced upon anti‐CD4 treatment. After long lasting (>12h) incubation with anti‐CD4, abundance and activity of CD4‐bound p56lck were diminished while the free fraction of p56lck remained unchanged. Since IL‐2 binding to its receptor activated only the CD4‐bound fraction of p56lck, the IL‐2‐induced p56lck activity was diminished after long‐term CD4 ligation. Taken together, our results suggest a cross talk between CD4‐ and IL‐2 receptor‐mediated signaling via p56lck.
Ca2+ mobilization in physiologically stimulated single T cells gradually increases with peptide concentration (analog signaling)
Authors: Røtnes Jan S. , Bogen Bjarne
Published: March 2007

We have investigated Ca2+ mobilization in single T cells stimulated with their physiological ligand, i.e. antigenic peptide bound to major histocompatibility complex (MHC) molecules on antigen‐presenting cells (APC). Fibroblasts expressing I‐Ed class II molecules were pulsed with a peptide derived from the λ2315 immunoglobulin light chain. Onto such antigen‐pulsed fibroblasts were sedimented cloned Th1 cells loaded with Fura‐2. Changes in cytosolic Ca2+ concentration in single T cells were continually monitored by use of an imaging system based on fluorometry. Ca2+ mobilization was both peptide‐specific and MHC‐restricted. Within seconds of the initial APC‐T cell contact, a Ca2+ spike could be observed. The Ca2+ response gradually declined over a 25‐min period, during which oscillations were noted. Various parameters characterizing the magnitude of the Ca2+ response (latency, increase rate, max and mean Ca2+ increase, frequency and period of oscillations) all correlated with the amount of peptide used for pulsing the fibroblasts. Thus, Ca2+ mobilization in single T cells appears not to be an all or none phenomenon. Rather, activation is incremental (analog signaling), the degree of Ca2+ mobilization probably being related to the number of stimulatory peptide‐MHC complexes on the surface of the APC. The extent of calcium mobilization and lymphokine production (interleukin (IL)‐2, IL‐3, interferon‐γ) correlated, at least at the population level.
The progressive differentiation of primed T cells is associated with an increasing susceptibility to apoptosis
Authors: Salmon Mike , Pilling Darrell , Borthwick Nicola J. , Viner Nick , Janossy George , Bacon Paul A. , Akbar Arne N.
Published: March 2007

Recent studies have suggested that T cell memory for recall antigens resides in clones of primed T cells with a short inter‐mitotic half‐life. In humans such cells express an isoform of the leukocyte common antigen termed CD45RO. Nevertheless, little is known of the fate of these primed T cells after initial activation, since no markers are available to distinguish recently primed cells from long‐established clones. This report is focused on a spectrum of primed CD4+ T cells characterized by an inverse relationship between the expression of two CD45 epitopes: CD45RB and CD45RO. We show that primed CD4+ T cells progress through many cycles of division from a CD45RBbrightOdull to a CD45RBdullObright state, resulting in a highly skewed distribution of the T cell receptor variable region usage within this particular population. The progressive differentiation defined by the shift from CD45RBbright to CD45RBdull is paralleled by the gradual loss of bcl‐2 and gain of Fas expression, two features associated with an increased propensity for apoptosis. At the same time, the highly differentiated CD45RBdull cells selectively lose the capacity to synthesize interleukin (IL)‐2, a cytokine which is particularly effective in preventing T cell apoptosis, although they produce high levels of IL‐4. The inability to produce adequate levels of IL‐2 leads to the apoptosis of primed CD45RBdull cells, when they are stimulated in the absence of exogenous IL‐2. These observations show the crucial dependence of highly differentiated T cells on the availability of exogenous IL‐2, and suggest both a major constraint for the persistence of T cell memory maintained by continually cycling primed cells, and an important mechanism contributing to the maintenance of T cell homeostasis in vivo.
Lymphokine production induced by streptococcal pyrogenic exotoxin‐A is selectively down‐regulated by pooled human IgG
Authors: Skansén‐Saphir Ulrika , Andersson Jan , Björk Lars , Anderson Ulf
Published: March 2007

The influence of pooled human IgG preparations for intravenous use (IVIg) on cytokine production induced by streptococcal pyrogenic exotoxin‐A (SPE‐A) was studied at the single‐cell level using cytokine‐specific monoclonal antibodies and indirect immunofluorescence or immunohistochemical staining. Mononuclear cells from healthy adult blood donors were stimulated with SPE‐A alone or in the presence of IVIg. IVIg was added either prior to stimulation or 24 h after initiation of cultures, in an attempt to evaluate whether IVIg treatment could influence an already established systemic streptococcal disease. Cells were harvested after 48 or 72 h of culture and stained for the following cytokines: interleukin(IL)‐1α, IL‐1β, IL‐1ra, IL‐6, IL‐8, IL‐2, tumor necrosis factor interferon(IFN)‐γ and TNF‐α and TNF‐β and granulocyte macrophage‐colony‐stimulating factor. Stimulation with SPE‐A lead to extensive lymphokine and monokine production. With the addition of IVIg prior to stimulation there was a strong reduction of blast transformation and an almost complete inhibition of lymphokine production, in particular in the synthesis of IFN‐γ and TNF‐β while the synthesis of IL‐1 and IL‐8 was either unaffected or increased. Adding IVIg 24 h after SPE‐A stimulation also resulted in reduced blast transformation and decreased synthesis of IFN‐γ and TNF‐β. These results indicate an immunomodulatory potential by IVIg on streptococcally induced T cell activation and lymphokine production.
gld/gld mice are unable to express a functional ligand for Fas
Authors: Ramsdell Fred , Seaman Michael S. , Miller Robert E. , Tough Teresa W. , Alderson Mark R. , Lynch David H.
Published: March 2007

Mice homozygous for either the lpr or gld genes develop phenotypically identical autoimmune disorders. The gene responsible for the pathology in lpr/lpr mice encodes the Fas antigen, a protein associated with the induction of programmed cell death. To determine if the defect associated with gld represents a mutation in the ligand for Fas, we have assessed the ability of lymphoid cells from homozygous gld/gld mice to lyse target cells in a Fas‐dependent manner. Using an antagonistic antibody to Fas, we demonstrate that activated T cells from normal and lpr mice are capable of inducing Fas‐mediated lysis of tumor target cells. In contrast, activated T cells from gld/gld mice fail to induce lysis of tumor targets, although cells from gld mice are able to lyse specific allogeneic targets following mixed lymphocyte culture. In addition, activated T cells from gld/gld homozygous animals are not capable of binding to a Fas.Fc fusion protein at high levels, whereas activated T cells from normal and lpr/lpr animals bind Fas.Fc efficiently. These data indicate that mice homozygous for gld are unable to express a functional ligand for Fas.
T cell receptor antagonist peptides are highly effective inhibitors of experimental allergic encephalomyelitis
Authors: Franco Alessandra , Southwood Scott , Arrhenius Thomas , Kuchroo Vijay K. , Grey Howard M. , Sette Alessandro , Ishioka Glenn Y.
Published: March 2007

The feasibility of using T cell receptor (TcR) antagonist peptides to inhibit autoimmune disease has been examined. First, the fine antigenic structure of the I‐As‐restricted encephalitogenic determinant proteolipid protein (PLP) 139–151 has been analyzed. It was found that residues 145 and 148 were I‐As anchor residues, and residue 144 appeared to be especially critical in T cell activation. Residues 142, 143, 146, and 147 were found to be crucial for activation of some, but not all, of the T cells studied. Next, good I‐As‐binding nonantigenic analogs were tested for TcR antagonism. Accordingly, several single substitution analogs were identified which could act as TcR antagonists. Moreover, when two such analogs were combined, the resulting TcR antagonist pool inhibited most of the PLP 139–151‐specific T cell clones in vitro. When the efficacy of this TcR antagonist pool in inhibiting EAE induction in vivo was examined, it was found that the analog pool was a remarkably potent inhibitor of disease induction. The TcR antagonist pool was approximately 10‐fold more potent than our best major histocompatibility complex blocker and was still capable of significant inhibition when injected in equimolar amounts with the encephalitogenic PLP 139–151 determinant.
Distinct roles for CD4 and CD8 as co‐receptors in T cell receptor signalling
Authors: Maroun Christiane R. , Julius Michael
Published: March 2007

We demonstrate that CD4 and CD8 modify signals induced through the T cell receptor for antigen (TCRαβ) in distinct fashions. Pretreatment of CD4+ lymph node T cells with CD4‐specific monoclonal antibody results in a tenfold inhibition of DNA synthesis induced by anti‐TCRαβ. In contrast, pretreatment of CD8+ T cells with CD8‐specific mAb has no effect on DNA synthesis subsequently induced through TCRαβ. While inhibiting late activation signals, pretreatment with anti‐CD4 does not detectably alter the pattern of anti‐TCRαβ‐induced tyrosine phosphorylation of cellular proteins, nor subsequent Ca2+ mobilization. The distinct biological consequences of anti‐CD4 and anti‐CD8 pretreatment correlate with the differential association of their respective ligands with the cellular protein tyrosine kinase, p56lck. While both T cell lineages contain similar levels of cellular p56lck, tenfold more is associated with CD4 than with CD8. This difference is associated with the differential effects of pretreatment with anti‐CD4 and anti‐CD8 on the distribution and activity of p56lck. Further, antibody‐mediated aggregation of TCRαβ on CD4+ T cells induces the appearance of a p56lck species with decreased mobility in sodium dodecylsulfate‐polyacrylamide gel electrophoresis. This effect is observed in CD4+ T cells exclusively and involves the fraction of p56lck which is not associated with CD4. The results presented here demonstrate that the signalling elements which couple the antigen receptor to second messenger‐generating systems are under distinct physical and/or functional constraints in the two T cell lineages.
Anti‐LFA‐1 (CD11a) monoclonal antibody interferes with neonatal induction of tolerance to alloantigens
Authors: Schurmans Stephane , Gonzalez Antonio , Revilla Conchi , Ramos Angeles , Lambert Paul Henri , Merino Jesus
Published: March 2007

The injection of (C57BL/6 × BALB/c)F1 spleen cells into newborn BALB/c mice results in the induction of a specific cytotoxic T lymphocyte (CTL) tolerance to the alloantigens. On the contrary, alloreactive CD4+ T cells persist in the host and are still able to activate autoreactive F1 B cells to produce autoantibodies. This state of “split tolerance” is closely associated with the development of a lupus‐like autoimmune syndrome. The LFA‐1 integrin plays a relevant role in homing, intercellular adhesion and tranduction of co‐stimulatory signals in leukocytes. Because of the beneficial effects of anti‐LFA‐1 monoclonal antibodies (mAb) treatment in various models of organ transplantation and autoimmune disease, we have investigated if such a treatment could interfere with the induction of neonatal tolerance or the development of the autoimmune syndrome in F1 cell‐injected newborn mice. For this purpose, BALB/c mice neonatally injected with F1 cells were treated from day 1 up to day 15 with a non‐cytotoxic anti‐LFA‐1 (CD11a) mAb. Anti‐LFA‐1 mAb treatment interfered with the persistence of a stable chimerism and with the establishment of CTL tolerance, as shown by rejection of allogeneic skin grafts and F1 B cells, and by a normal in vitro CTL activity against the corresponding alloantigens. As a consequence, these mice did not develop the characteristic autoimmune features seen in close association with an effective induction of CTL tolerance to alloantigens. These results stress the importance of the interactions between LFA‐1 and its ligands during the neonatal induction of tolerance to alloantigens.
Interleukin‐10 inhibits B7 and intercellular adhesion molecule‐1 expression on human monocytes
Authors: Willems Fabienne , Marchant Arnaud , Delville Jean‐Pierre , Gérard Catherine , Delvaux Anne , Velu Thierry , De Boer Mark , Goldman Michel
Published: March 2007

There is evidence that interleukin −10(IL‐10) interferes with the costimulatory properties of antigen‐presenting cells and, thereby, inhibits their ability to induce T cell activation. To determine whether this effect might involve modulation of the expression of accessory molecules, we analyzed by flow cytometry the influence of human IL‐10 on the basal expression of intercellular adhesion molecule 1 (ICAM‐1) as well as on the interferon γ(IFN‐γ)‐induced up‐regulation of ICAM‐1 and B7 at the surface of human monocytes. IL‐10 inhibited both the basal expression and the IFN‐γ‐induced ICAM‐1 up‐regulation. IL‐10 also reduced B7 up‐regulation on IFN‐γ‐stimulated monocytes. The inhibitory effect of IL‐10 both on ICAM‐1 and B7 expression was shown to be dose dependent. We conclude that the ability of IL‐10 to decrease both ICAM‐1 and B7 expression on monocytes might contribute to its immunosuppressive properties.
T cells of staphylococcal enterotoxin B‐tolerized autoimmune MRL‐lpr/lpr mice require co‐stimulation through the B7‐CD28/CTLA‐4 pathway for activation and can be reanergized in vivo by stimulation of the T cell receptor in the absence of this co‐stimulatory signal
Authors: Zhou Tong , Weaver Casey , Linsley Peter S. , Mountz John D.
Published: March 2007

The CD28/CTLA‐4 receptors on T cells interact with the B7 molecule on antigen‐presenting cells (APC) to produce a co‐stimulatory signal that determines the outcome of activation. The role of this co‐stimulatory signal in T cell activation and loss of tolerance in autoimmune MRL‐lpr/lpr mice has not been investigated previously. The present study examines the contribution of the CD28/CTLA‐4 co‐stimulatory pathway to the loss of T cell tolerance in Vβ8 transgenic MRL‐lpr/lpr and ‐+/+ mice in which neonatal tolerance has been induced by the superantigen staphylococcal enterotoxin B (SEB). An artificial APC transfected with the murine B7 gene, and a CTLA‐4‐Ig fusion protein were used to analyze the significance of the CD28/CTLA‐4 pathway in vitro. The CTLA‐4‐Ig fusion protein was also used to inhibit the pathway in vivo. Our results demonstrate that CD28 and CTLA‐4 mRNA was overexpressed in the lymph nodes of lpr/lpr mice (MRL, C57BL/6, C3H and AKR), but not in +/+ mice of the same background strain. Lymph node T cells and thymocytes from SEB neonatally tolerized MRL‐lpr/lpr mice that had undergone tolerance loss, proliferated when cultured with SEB and B7+ fibroblasts in vitro, but did not proliferate when the SEB was presented in the context of B7− fibroblasts. This in vitro tolerance loss could be prevented by blocking of B7 signaling by CTLA‐4‐Ig. This loss of tolerance did not occur in lymph node T cells from thymectomized MRL‐lpr/lpr mice. SEB challenge of tolerized MRL‐lpr/lpr mice in vivo led to weight loss, increased serum cytokine levels and depletion of Vβ8+ T cells. These effects were blocked by blocking of the co‐stimulatory pathway by treatment with the CTLA‐4‐Ig fusion protein prior to and during challenge with SEB. T cells from thymus and lymph nodes of these mice did not proliferate later in response to stimulation in vitro with SEB even in the presence of B7+ APC. Nonresponsiveness was not due to deletion of Vβ8+ CD28+ T cells, as the number of these cells was increased after treatment with SEB and the CTLA‐4‐Ig fusion protein. These results suggest that the response of autoreactive T cells in the thymus and lymph nodes depends on signaling by B7 in vivo and in vitro and that SEB‐reactive T cells can be reanergized in vivo by stimulation of the T cell receptor in the absence of signaling through the CD28/CTLA‐4 co‐stimulatory pathway.
Mutagenesis of the human interleukin‐6 fourth predicted α‐helix: Involvement of the Arg168 in the binding site
Authors: Fontaine Véronique , Ooms Josette , Content Jean
Published: March 2007

Random substitutions of amino acid 161–184 of human interleukin‐6 (hIL‐6) have been generated at the cDNA level using oligonucleotide‐directed mutagenesis. Among the majority of the mutant proteins showing a reduced biological activity on murine hybridoma cells, only those having a substitution of Met161, Arg168, Arg179 or Met184, retained a tertiary structure similar to the IL‐6 folding. These residues are thus probably involved in the interaction with the IL‐6 receptor. However, the contacts established by Arg168 and Arg179 seem far more important for the biological activity. According to Bazan's model of cytokine folding and the receptor binding site on the fourth α‐helix, based on growth hormone similarity, we propose that Arg168 and Arg179 are located on the exposed surface of this presumed helix.
Selective stimulation of murine cytotoxic T cell and antibody responses by particulate or monomeric hepatitis B virus surface (S) antigen
Authors: Schirmbeck Reinhold , Melber Karl , Mertens Thomas , Reimann Jörg
Published: March 2007

In the murine system, we tested in vivo the immunogenicity of different preparations of the yeast‐derived surface antigen (S‐antigen or S‐protein) of hepatitis B virus (HBV). Native S‐protein molecules self‐assemble into stable 22‐nm particles. BALB/c mice immunized with low doses of native S‐particles without adjuvants efficiently generated an H‐2 class I‐restricted CD8+ cytotoxic T lymphocyte (CTL) response, and developed easily detectable serum antibody titers against conformational determinants of the native S‐particle or linear epitopes of the denatured S‐protein. Disruption of S‐particles with sodium dodecyl sulfate and β‐2‐mercaptoethanol generated p24 S‐monomers. Injection of an equal dose of S‐monomers into mice efficiently primed CTL, but did not stimulate an antibody response against conformational or linear epitopes of the native or denatured S‐protein. In vivo priming of CTL by S‐particles or S‐monomers required “endogenous” processing of the antigen because the injection of an equimolar (or higher) dose of an antigenic, S‐derived 12‐mer peptide into mice did not prime CTL. Native (particulate) or denatured (monomeric) S‐antigen injected with mineral oil (incomplete Freund's adjuvant) or aluminum hydroxide failed to stimulate a CTL response. Hence, different preparations can be produced from a small protein antigen which specifically stimulate selected compartments of the immune system.
Allergen‐induced CD23 on CD4+ T lymphocytes and CD21 on B lymphocytes in Patients with allergic asthma: Evidence and regulation
Authors: Gagro Alenka , Rabatić Sabina
Published: March 2007

Interaction of CD4+ T cells and B cells is necessary for IgE production. It has been recently demonstrated that cell surface antigen CD21 is a ligand for CD23 (FcϵRII) and that the pairing of these molecules may participate in the control of IgE production. In this study we investigated the effect of the Dermatophagoides pteronyssinus (Dpt) allergen and recombinant interleukin(rIL)‐4 on the expression of CD21 and CD23 on T and B cells of asthmatic patients allergic to Dpt and of healthy controls. Peripheral blood mononuclear cells (PBMC) were incubated alone or with Dpt allergen (100 biological units/ml) and/or rIL‐4 (100 U/ml) for up to 7 days. The flow‐cytometric analysis of double‐fluorescence staining revealed that Dpt allergen and/or rIL‐4 induced CD23 on CD4+ T lymphocytes only in allergic patients. The allergen‐induced CD23 on T cells is de novo synthesized antigen since no induction of CD23 on T cells was observed in cultures with 0.4 μg/ml actinomycin D. Moreover, 100 U/ml of interferon‐γ inhibited the induction of CD23 on CD4+ T cells. T cells obtained from healthy donors did not express CD23 or CD21 antigen upon incubation with allergen and/or rIL‐4. Although rIL‐4 also induced CD23 in controls, the expression was only observed on CD20+ cells. The allergen alone induced a significant elevation of the mean fluorescence intensity of both CD21 and CD23 only in allergic in dividuals. When the cell proliferation was analyzed, a slightly increased stimulation index upon cultivation of PBMC was obtained from non‐allergic donors as well, but less than in allergic patients. The co‐expression of major histocompability complex class II molecules and CD23 on CD4+ T lymphocytes in allergic patients, as assessed by the three‐color immunofluorescence analysis, indicates that these cells were activated.

We conclude that CD4+ T lymphocytes possess a unique capability to express CD23 upon exposure to allergen. Moreover, the allergen‐mediated induction of CD23 on T cells observed only in allergic patients may be the reason for the increase of IgE production. This would not occur in non‐allergic individuals as there is no CD23 expression on T cells.
The biased Vγ gene usage in the synovial fluid of patients with rheumatoid arthritis
Authors: Kageyama Yasunori , Koide Yukio , Miyamoto Shigehito , Inoue Tetsuo , Yoshida Takato O.
Published: March 2007

Taking advantage of the reverse transcriptase‐polymerase chain reaction (RT‐PCR), we have analyzed T cell receptor γ‐chain mRNA of synovial fluid γ/δ T cells from patients with rheumatoid arthritis (RA) in comparison with those of peripheral blood mononuclear cells (PBMC) from RA patients and healthy individuals. The quantitative RT‐PCR method in conjunction with nucleotide sequencing revealed the frequent usage of the Vγ3 gene segment in RA synovial fluid mononuclear cells (SFMC) (p < 0.01) which in PBMC of healthy individuals occurred rarely. PBMC of most healthy individuals expressed the Vγ9 gene predominantly (p < 0.01) as expected. However, only half of RA patients showed elevated levels of the Vγ9 gene expression in their PBMC. The γ‐chain mRNA containing the Vγ3 gene in RA SFMC showed no conserved junctional sequence (complementarity‐determining region 3). To investigate the nature of ligands recognized by the Vγ3‐bearing T cells, we analyzed Vγ gene usage of RA SFMC, RA PBMC, and normal PBMC stimulated with Mycobacterium tuberculosis (MT) or MT plus interleukin‐2 since there is mounting evidence of high reactivity of RA SFMC to MT and mycobacterial heat‐shock protein 65. However, the Vγ usage appeared to be mostly Vγ9 in RA SFMC, RA PBMC and normal PBMC. Taken together these results suggest that an as yet unknown antigen(s) (other than MT) might select γ/δ T cells expressing the Vγ3 gene in RA SFMC.
Sialyl Lewisx‐ and L‐selectin‐dependent site‐specific lymphocyte extravasation into renal transplants during acute rejection
Authors: Turunen Juha Pekka , Paavonen Timo , Majuri Marja‐Leena , Tiisala Sinikka , Mattila Pirkko , Mennander Ari , Gahmberg Carl G. , Häyry Pekka , Tamatani Takuya , Miyasaka Masayuki , Renkonen Risto
Published: March 2007

Kidney allograft rejection is an inflammatory process dominated by lymphocytes. During rejection lymphocytes preferentially adhere to the peritubular capillary endothelium (PTCE), which acquires morphological features common to high endothelium. These observations indicate that PTCE is the site of lymphocyte entry into the rejecting renal allograft. Of the identified endothelial adhesion molecules, ICAM‐1 was already expressed on the endothelium of normal kidneys, and its expression was strongly enhanced during rejection without site‐specific restriction. VCAM‐1 was not expressed on the endothelium of normal or syngeneic kidneys, but its expression was induced during allograft rejection not only in PTCE, but occasionally also on the endothelium of larger vessels. Sialyl Lewisx (sLex) showed a very restricted pattern of expression; endothelium was sLex‐negative both in control and syngeneic kidneys. On the other hand, PTCE reacted strongly with anti‐sLex antibody in allografts. When kidney frozen sections were treated with sialidase the binding of lymphocytes decreased by 70%. Low‐dose chymotrypsin treatment of lymphocytes, known to remove L‐selectin from the lymphocyte surface, decreased their binding to PTCE by 60%. Likewise lymphocyte adhesion to PTCE was inhibited by 70% by anti‐sLex‐ and anti‐L‐selectin‐antibodies and by sLex tetrasaccharide. Finally PTCE in the allografts, but not in syngeneic grafts or normal kidneys, bound an L‐selectin‐IgG fusion protein, indicating that ligands for L‐selectin were induced during rejection.
Interferon‐γ‐ and interleukin‐4‐producing T cells can be primed on dendritic cells in vivo and do not require the presence of B cells
Authors: Ronchese Franca , Hausmann Barbara , Gros Graham Le
Published: March 2007

The antigen‐presenting cell (APC) requirements for the in vivo induction of Th1‐and Th2‐type responses were investigated using a severe combined immunodeficiency (SCID)mouse chimera model. SCID mice adoptively transferred with either T cells [SCID(T)] or T + B cells [SCID(T + B)] and immunized with antigen in adjuvant were able to generate antigen‐specific T cells which could produce both interferon (IFN)‐γ and interleukin (IL)‐4 upon in vitro restimulation. This suggests that B cell APC are not necessary for the priming of either IFN‐γ‐ or IL‐4‐producing T cells in vivo. The ability of different APC to activate Th2‐dependent effector mechanisms was also investigated. SCID(T) and SCID(T + B) mice were infected with the nematode parasite Nippostrongylus brasiliensis and analyzed for the development of IL‐5‐dependent peripheral blood eosinophilia. Following infection both SCID(T) and SCID(T + B) mice generated similar numbers of peripheral blood eosilnophils, suggesting that similar amounts of IL‐5 had been produced. Therefore, B cell APC are also not required for the in vivo activation of Th2 cells to lymphokine production. To establish more precisely which APC prime T cells to produce IFN‐γ and IL‐4, normal mice were immunized by injection of syngeneic splenic dendritic cells which had been pulsed with antigen in vitro. T cells from these immunized mice were able to produce good IFN‐γ and IL‐4 responses upon in vitro restimulation with specific antigen; therefore, dendritic cells appear to be sufficient APC for the in vivo priming of both IFN‐γ‐ and IL‐4‐producing T cells.
A specific intercellular pathway of apoptotic cell death is defective in the mature peripheral T cells of autoimmune lpr and gld mice
Authors: Gillette‐Ferguson Illona , Sidman Charles L.
Published: March 2007

Homozygosity for either of the unlinked murine autosomal recessive mutations lpr or gld leads to autoimmunity characterized by peripheral accumulation of CD4−/CD8− “double‐negative” T cells, autoantibodies and various forms of tissue pathology. Recently, the gene affected by lpr was identified as fas, whose product acts as a trigger for programmed cell death or apoptosis. Data reported here indicate that the Fas receptor and its ligand, the wild‐type form of the gld gene product, are essential for antigen‐stimulated peripheral T cell apoptosis. Furthermore, the wild‐type gld gene product is a non‐cell‐autonomous protein that is produced by activated T cells. Apoptotic elimination of antigen‐receptor‐triggered peripheral T cells appears to be abnormal in lpr and gld mice, and this deficiency causes peripheral T cells to accumulate resulting in lymphadenopathy. These findings support the importance of apoptotic regulation of lymphocyte persistence after antigen encounter in vivo.
Anergic B cells constitutively present self antigen: Enhanced immunoglobulin receptor‐mediated presentation of antigenic determinants by B cells is hierarchical
Authors: Kanost Dawn , McCluskey James
Published: March 2007

Presentation of hen egg lysozyme (HEL) by HEL‐specific B cells was studied in transgenic mice expressing anti‐HEL immunoglobulin (Ig‐transgenic). In T hybridoma assays, presentation of the HEL46–61 determinant by B cells from Ig‐transgenic mice required 103−104‐fold lower concentrations of HEL than were required for presentation by B cells from non‐transgenic mice. In contrast, presentation of the HEL determinants 112–129 and 25–43 by HEL‐specific B cells was either not significantly enhanced, or enhanced only 10‐fold compared with B cells from non‐transgenic mice. Enhanced presentation of HEL determinants by B cells from Ig‐transgenic donors was specific for HEL, since keyhole limpet hemocyanin or synthetic HEL46–61 peptide were presented comparably by B cells from Ig‐transgenic mice and non‐transgenic littermates. A minimum of 1–4% Ig‐transgenic B cells was required to detect enhanced presentation of HEL46–61 in vitro. Constitutive presentation of the HEL46–61 determinant, but not the HEL25–43 or HEL112–129 determinants, was detectable on anergic HEL‐specific B cells from double (HEL/Ig)‐transgenic mice. In the presence of exogenously added HEL, anergic B cells presented all three HEL determinants. Constitutively presented HEL46–61 was not due to endogenous synthesis of HEL antigen by anergic B cells from double‐transgenic mice, as comparable levels of the HEL46–61 determinant were constitutively presented by B cells from Ig‐Tg → HEL‐Tg irradiation bone marrow chimeric mice. Firstly, these results indicate that the enhanced antigen presentation mediated by Ig receptors on B cells is not equivalent for all antigenic determinants. Secondly, the data demonstrate that anergic, autoreactive B cells efficiently process and present nominal antigens in addition to constitutively presenting specific self antigen in vivo.
The Y chromosome‐linked “autoimmune accelerating” yaa gene suppresses collagen‐induced arthritis
Authors: Jansson Liselotte , Holmdahl Rikard
Published: March 2007

The Y‐linked autoimmune accelerating gene mutation (yaa), first discovered in the BXSB mouse strain, is known to accelerate spontaneous autoantibody production and subsequent development of lupus disease. We have investigated the role of the yaa gene in the development of the type II collagen (CII)‐induced arthritis (CIA), which is used as a model for rheumatoid arthritis. In contrast to the accelerating effects on development of lupus autoimmunity we can show that the presence of BXSB Y chromosome carrying the yaa gene block development of CIA in F1 crosses with three normally CIA‐susceptible strains, DBA/1, C3H.Q and B10.Q. Backcross experiments showed an additional modulatory effect from other BXSB genes or possibly from DBA/1 × chromosome. To evaluate the effect mediated by the yaa gene alone, the BXSB Y chromosome was bred into the DBA/1 gene background. The DBA/1 congenic DBA/1.yaa male mice were less susceptible to arthritis development than their DBA/1 counterparts. (B10.QxDBA/1.yaa)F1 acquired resistance to arthritis development similar to that of DBA/1.yaa, indicating a role for the yaa gene alone. The serum levels of autoantibodies to CII were significantly suppressed in all strains carrying yaa. In DBA/1.yaa mice a reduced number of T cells were found to produce interferon‐γ after in vitro stimulation with CII. Thus, although autoreactive B cells are important in both diseases they play different roles in murine lupus and in CIA.