bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of HLA antigens on germ cells in human semen
Authors: Jassim Ali , Ollier William , Payne Annette , Biro Andrew , Oliver R. T. D. , Festenstein Hilliard
Published: March 2007

Nucleated cells other than sperm (NCOS) were isolated from human semen by centrifugation on a Ficoll density gradient. Using tissue‐specific monoclonal antibodies (mAb) > 99% of the NCOS were found to be sperm cell precursors (SpP). These cells were tested for the expression of class I and II (DR, DP and DQ) HLA antigens by using specific mAb. The anti‐HLA class I and II and anti‐β2‐microglobulin mAb reacted with < 1% of the NCOS. This was demonstrated by indirect immunofluorescence microscopy and fluorescence‐activated cell sorter analysis. These results were similar to those obtained from testing germ cells in frozen sections of normal adult testis using the same panel of mAb. In mixed lymphocyte‐NCOS cultures, the SpP failed to stimulate allogeneic lymphocytes even when different concentrations of cells were used. These results indicate little or no expression of HLA class I and II including the HLA‐D (T cell‐defined) determinant on the SpP, a phenomenon which could be of biological importance.
Intracellular events associated with inhibition of B cell activation by monoclonal antibodies to HLA class II antigens
Authors: Holte Harald , Blomhoff Heidi Kiil , Beiske Klaus , Funderud Steinar , Torjesen Peter , Gaudernack Gustav , Stokke Trond , Smeland Erlend B.
Published: March 2007

We have investigated several aspects of the inhibitory effects of monoclonal antibodies (mAb) directed against MHC class II antigens in B cell activation/proliferation, using a panel of mAb specifically reactive with antigens encoded by HLA class II loci (DP, DQ, DR). All mAb except the anti‐DP mAb inhibited significantly anti‐μ plus B cell growth factor‐induced DNA synthesis. Only one mAb, however, which was reactive with gene products of all three class II loci (DP, DQ, DR) inhibited anti‐μ‐induced DNA synthesis as well as c‐myc mRNA expression. In addition, the same mAb inhibited the early events induced by anti‐μ stimulation alone, including phosphatidylinositol turnover and elevation of [Ca2+]i. In contrast to previous findings in the murine system, none of the anti‐MHC class II mAb used in this study increased the cAMP levels.
Macrophage activation by interferon‐γ from host‐protective T cells is inhibited by interleukin (IL) 3 and IL 4 produced by disease‐promoting T cells in leishmaniasis
Authors: Liew Foo Y. , Millott Stephanie , Li Yun , Lelchuk Rosalia , Chan W. Ling , Ziltener Herman
Published: March 2007

BALB/c mice are highly susceptible to Leishmania major infection. They develop a progressive fatal disseminating disease even with a minimum infecting dose. However, these mice are able to contain the disease if they are exposed to sublethal γ‐irradiation shortly before infection. Earlier studies demonstrated that CD4+ T cells from mice which had recovered from infection (Tr) can adoptively transfer resistance. In contrast, CD4+ cells from mice with progressive disease (Ts) not only failed to protect, but can reverse the protective effect of the Tr cells.

Spleen cells from BALB/c mice which had recovered from L. major infection or which had progressive disease were cultured with leishmanial antigens in vitro. The culture supernatant from spleen cells of recovered mice (TrSN) contains high levels of macrophage‐activating factor (MAF) activity which can activate peritoneal macrophages to kill 51Cr‐labeled P815 cells and to eliminate intracellular parasites as measured by the reduction in [3H]thymidine uptake by residual parasites released from macrophages following sodium dodecyl sulfate treatment. The MAF activity of TrSN parallels that of recombinant interferon‐γ (IFN‐γ). In contrast, culture supernatant of spleen cells from mice with progressive disease (TsSN) contains no detectable MAF but it is able to neutralize the MAF activity of TrSN. The MAF‐inhibiting function of TsSN appears to be mediated by interleukin (IL) 3 and IL 4, since the MAF activity of TrSN and rIFN‐γ also can be inhibited by the addition of rIL 3 and rIL 4 but not by rIL 1 or rIL 2. Furthrmore, the MAF‐inhibiting activity of TsSN can be partially reversed by the addition of specific anti‐IL 3 or anti‐IL 4, but completely reversed by the combination of the two antibodies in vitro. These findings provide a mechanism for the immune regulation in leishmaniasis and a means by which the two subsets of CD4+ T cells influence each other through their modulation of macrophage function.
The VH gene sequences of anti‐DNA antibodies in two different strains of lupus‐prone mice are highly related
Authors: Eilat Dan , Hochberg Malka , Tron François , Jacob Laurent , Bach Jean‐François
Published: March 2007

The heavy and light chain V region sequences of an IgG anti‐DNA autoantibody (PME77), derived from a lupus‐prone (NZB x NZW)F1 mouse have been determined by mRNA sequencing. The Vx gene segment belongs to the Vx1A gene subgroup and is found in several (NZB x NZW)F1 and MRL lpr/lpr anti‐DNA antibodies, as well as in other antibodies of unrelated specificities. The VH gene segment appears to represent a unique gene or a subfamily of the large J558 VH gene family of the mouse, and is highly related to a germ‐line sequence of a major anti‐DNA idiotype (H130, IgM) of MRL mice. This anti‐DNA‐related VH segment has not been found, so far, to be expressed in antibodies with specificities for external or synthetic antigens; therefore, expression of such specificities may be regulated by powerful mechanisms of self tolerance in the healthy animal. In addition, both the heavy and light chain of the PME77 IgG antibody were found to contain somatic point mutations with a high ratio of replacement to silent mutations in complementarity determining regions. This IgM to IgG sequence relationship suggests an affinity maturation process, which is driven by the autoantigen.
Detection of perforin and granzyme A mRNA in infiltrating cells during infection of mice with lymphocytic choriomeningitis virus
Authors: Müller Christoph , Kägi David , Aebischer Toni , Odermatt Bernhard , Held Werner , Podack Eckhard R. , Zinkernagel Rolf M. , Hengartner Hans
Published: March 2007

The analysis of gene expression in cytotoxic T cells by in situ hybridization of serial liver and brain sections from mice infected with lymphocytic choriomeningitis virus (LCMV) and immunostaining with T cell marker‐ and virus‐specific antibodies revealed a close histological association of infiltrating lymphocytes expressing the perforin and granzyme A genes with virally infected cells. Maximal frequency of perforin and granzyme A mRNA‐containing cells on liver sections preceded by about 2 days maximal LCMV‐specific cytotoxicity of the lymphoid liver infiltrating cells. These results are most consistent with an involvement of perforin and granzyme A in cell‐mediated cytotoxicity in vivo.
Expression and ontogeny of murine CD2
Authors: Sen Jyoti , Arceci Robert J. , Jones William , Burakoff Steven J.
Published: March 2007

CD2 was first defined as the erythrocyte rosetting protein on the surface of human T cells. Recently, the rat and murine homologues have been identified by cDNA cloning. In this report we demonstrate that CD2 is expressed on the surface of most adult murine peripheral lymphocytes and thymocytes by indirect immunofluorescence using an anti‐murine CD2 antiserum. The expression of CD2 on murine B cells was unexpected since in rat and human species it has been defined as a T cell‐specific marker. Furthermore, CD2 appears very early on fetal thymocytes during development. The level of surface expression increases from day 13 of gestation to day 17, after which the surface density appears to reach a steady state. Thus, CD2 is expressed on day‐13 thymocytes at the same stage that Thy‐1, Pgp1 and the TcRγ/δ/CD3 complex have been shown to be expressed.
Thymocyte development: an analysis of T cell receptor gene expression in 519 newborn thymocyte hybridomas
Authors: Happ Mary Pat , Palmer Ed
Published: March 2007

We have examined the frequency of expression of individual α and β chain V gene families in a population of immature T cells that has not been selected or tolerized. To accomplish this, we generated 519 T cell hybridomas from freshly isolated thymocytes of newborn C57BL/10 mice and subjected RNA from these hybrids to hybridization analysis with 11 Vα, 16 Vβ, Cγ and Cδ probes. Comparison of the expressed repertoire of Vβ gene segments in this newborn thymocte population with similar data previously generated from adult peripheral T cells (Bill et al., Proc. Natl. Acad. Sci. USA 1988. 85: 9184.) revealed two Vβ genes, Vβ12 and Vβ15, whose expression is decreased in the periphery possibly due to the effects of tolerance. An additional Vβ gene segment (Vβ10) and a Vβ gene family (Vβ5.1, Vβ5.2) were expressed more frequently in the mature, peripheral population than in the newborn thymus. These findings may represent two instances of positive selection of T cells. Furthermore, unlike VH gene segments, Dβ‐proximal Vβ genes are not overrepresented on this collection of immature thymocyte hybridomas. A similar analysis of Vα gene family expression was hampered by the fact that Vα gene segments were expressed in only 15% of newborn thymocyte hybridomas (compared to 58% of adult hybridomas). An unexpectedly large fraction (57%) of those newborn hybrids expressing a Vα gene segment was also found to express Cδ mRNA and further examination revealed that several Vα gene probes were actually detecting δ chain mRNA. The most notable of these was the Vα7 gene family, which accounted for approximately one‐third of the expressed Vα genes but was expressed exclusively as part of a γ chain mRNA. We found no examples of hybridomas co‐expressing both full‐length β and δ chain mRNA, despite significant numbers of hybridomas co‐expressing full‐length β and γ chain transcripts. This observation suggests that a large number of mature α/β cells may have proceeded through development without having gone through a stage where a δ chain message is expressed, thereby precluding their development into γ/δ cells.
Immunoglobulin diversification in bursal duct‐ligated chickens
Authors: Lassila Olli , Lefkovits Ivan , Alanen Anna
Published: March 2007

The role of external antigen contact on immunoglobulin (Ig) diversification occurring in chicken bursal cells was evaluated. The entry of environmental antigens into the lumen of the bursa of Fabricius was prevented by ligating the bursal duct prior to hatching (BDL: bursal duct ligation). We used two‐dimensional gel electrophoresis to compare the heterogeneity of Ig molecules from bursa cells of normal and BDL chickens. We have found that Ig diversity obtained from BDL chickens' bursae in two‐dimensional gel analysis was similar to that of control birds. Furthermore, by using two monoclonal anti‐idiotype antibodies to study intrabursal Ig diversification we have shown that frequencies of the Cld‐1 and Cld‐2 idiotypes were also unaltered following bursal ligation. We conclude that primary B cell diversification in the bursa is independent of the external antigen flow from the bursal lumen.
Characterization of a novel myeloid antigen regulated during differentiation of monocytic cells
Authors: Cabañas Carlos , Sanchez‐Madrid Francisco , Bellon Teresa , Figdor Carl G. , Velde Anje A. Te , Fernandez Jesus M. , Acevedo Augustin , Bernabeu Carmelo
Published: March 2007

The monoclonal antibody HC1/6 generated against phorbol 12‐myristate 13‐acetatetreated U‐937 cells recognizes a new cell surface antigen with a broad relative molecular mass ranging from 100 to 150 kDa. This antigen is also present on monocytes, platelets and endothelial cells and is weakly expressed by granulocytes. In contrast, it is absent from T, B and erythroblastoid cells. The antigen HC1/6 is also expressed by normal tissue macrophages in tonsil, lung and kidney, as well as in skin biopsies from pathologies such as sarcoidosis and lepromatous leprosy. The expression of the HC1/6 antigen is increased up to 5‐fold when U‐937 (promonocytic) and HL‐60 (myelomonocytic) cell lines are stimulated with phorbol 12‐myristate 13‐acetate. Conversely, the expression of the HC1/6 antigen is down‐regulated in monocytes upon treatment with interferon‐γ. These findings are discussed in relation with other myeloid cell surface markers.
FcγRII expression in resting and activated B lymphocytes
Authors: Amigorena Sebastian , Bonnerot Christian , Choquet Daniel , Fridman Wolf Herman , Teillaud Jean‐Luc
Published: March 2007

In this report, we analyze the expression of the type II receptor for the Fc region of IgG (FcγRII) in resting and lipopolysaccharide (LPS)‐activated murine B lymphocytes. FcγRII is encoded by two genes, α and β. The β gene encodes two mRNA, β1 and β2, which are generated by alternative splicing. Using an S1 nuclease protection assay, we found that resting and activated B lymphocytes express predominantly the β1 transcript. Very low levels of the β2 mRNA were detected in this assay, while no expression of the α transcript could be detected. Quantitative Northern blot analysis showed that the amount of FcγRII β mRNA was increased 9‐fold in LPS‐activated B lymphocytes. The expression of FcγRII during the various phases of B cell activation was then studied by immunofluorescence using the monoclonal antibody 2.4G2. LPS stimulation induced an increase of the FcγRII cellular pool as well as of its expression at the surface of B lymphocytes. The rise in FcγRII surface expression occurred after the induction of class II antigens (la) and before transferrin receptor induction. FcγRII expression was found to be enhanced during the G1 phase of the cell cycle since (a) only large cells (i.e. those that had entered the G1 phase) expressed an increased amount of FcγRII and (b) blocking the entry of activated cells into the S phase (with the ion channel blocker quinine) did not affect the FcγRII induction by LPS. Furthermore, only B cell activators that induced cells to enter into G1 [LPS and F(ab')2 anti‐IgM antibodies, but not interleukin 4] caused an increase in the expression of FcγRII. These results show that the increase in the membrane expression of FcγRII occurs during the early G1 phase, establishing it as a marker for the entry of B lymphocytes into the cell cycle.
Interception of the development of self tolerance in fetal lambs
Authors: Mccullagh Peter
Published: March 2007

Investigation of the nature of immunological self tolerance has usually relied upon experimental protocols in which the tolerant state is interrupted in mature animals with the production of autoimmune disease. While such research has improved the understanding of those processes operative in overt autoimmunity, it has not been informative in relation to events associated with the establishment of self tolerance. Any description of this state which is to be based on observation will necessitate the use of experimental systems that permit observation of animals during the development of self tolerance.

The present experiment entailed intervention approximately one third of the way through the gestation period of fetal lambs. An earlier experiment had established that 54‐day fetal lambs would accept allografts of adult skin. This indicated that the capacity to discriminate between self and non‐self had not been acquired at that age. Fetuses at this stage of gestation were submitted to either partial or total removal of the thyroid gland. The excised tissue was then implanted in nude mice for periods of 5 to 9 weeks. It was subsequently replaced subcutaneously, either in the original donor or in another fetus at a comparable stage of gestation. At postmortem examination, several weeks later, self implants in lambs from which the thyroid gland had been completely removed displayed autoimmune thyroiditis of varying degrees of severity. However, self implants in partially thyroidectomized animals were uniformly free from autoimmune manifestations. This implied that these reactions had not been directed against contaminating murine tissues in the implants replaced in completely thyroidectomized lambs. All allogeneic implants were subject to vey heavy lymphocytic infiltration, usually with accompanying necrosis consistent with allograft rejection. This was taken as an indication that hypothyroid fetal lambs had become immunocompetent by the time of thyroid reimplantation. Spontaneous immunological reactivity against reimplanted self thyroid tissue by thyroidectomized lambs was interpreted as a failure to acquire the capacity for self recognition as a result of antigen deprivation.
Complex Ca2+ flux inhibition as primary mechanism of staurosporine‐induced impairment of T cell activation
Authors: Kubbies Manfred , Goller Bernhard , Russmann Eberhard , Stockinger Hubertus , Scheuer Werner
Published: March 2007

The inhibitory effect of the highly effective drug staurosporine on the early activation signal Ca2+ flux was investigated via multiparameter flow cytometry in human peripheral blood T lymphocytes. Staurosporine has been reported to be a specific inhibitor of protein kinase C. However, we show that it inhibits the Ca2+ influx in anti‐CD3 and phytohemagglutinin‐stimulated human CD4+ and CD8+ lymphocytes at concentrations between 1.0 and 10.0 ng/ml. Staurosporine decreases the number of Ca2+‐positive CD4+ and CD8+ lymphocytes as well as the Ca2+ influx per cell; the drug also delays the time of the maximum response to polyclonal stimulation. In addition, we demonstrate that staurosporine affects the primary Ca2+ response via inhibition of the release of the membrane‐bound Ca2+ from the endoplasmic reticulum in CD4+ and CD8+ lymphocytes. Binding studies of the anti‐CD3 antibody to T lymphocytes indicate normal binding capacities in the presence of staurosporine. With respect to the classical scheme of T cell activation via phospholipase C, our data suggest that staurosporine may inhibit T cell activation primarily by its effect on the early Ca2+ flux signal.
Regulation of hepatic synthesis of C3 and C4 during the acute‐phase response in the rat
Authors: Anthony Robert , El‐Omar Emad , Lappin David F. , Macsween Roderick N. M. , Whaley Keith
Published: March 2007

Following the i.p. injection of casein, rats showed increased serum levels of C4 and C3. C4 levels peaked on day 2 and returned to normal by day 4, while C3 levels peaked on day 3 before returning to normal on day 4. These changes were paralleled by changes in the hepatocyte synthesis rates of these two proteins.

Macrophages, isolated from the peritoneal cavities of rats on days 1 to 7 (day‐1 to day‐7 macrophages) following i.p. injection of casein, were cultured in vitro, and the peritoneal macrophage‐conditioned media (PMCM) assayed for their abilities to stimulate synthesis of C4 and C3 by hepatocytes from control rats. Day‐2 PMCM selectively stimulated synthesis of C4, while day‐3 and day‐4 PMCM selectively stimulated C3 synthesis. These activities were called C4‐hepatocyte stimulating factor (C4‐HSF) and C3‐HSF, respectively.

The addition of anti‐interleukin (IL) 1, tumor necrosis factor (TNF)‐α, TNF‐β, IL 6 or interferon (IFN)‐γ antibodies to day‐2 PMCM did not affect C4‐HSF activity, and none had any effect on C3‐HSF activity in day‐4 PMCM. However, the addition of anti‐IL 1 to day‐4 PMCM resulted in the re‐expression of C4‐HSF activity as well as loss of thymocyte proliferative activity. C4‐HSF activity could also be detected in day‐4 PMCM by separating it from IL1 activity using gel filtration chromatography. Furthermore the addition of recombinant IL1β to day‐2 PMCM prevented the expression of C4‐HSF activity. Thus IL 1 appears to play a regulatory role in the acutephase response in the rat, by preventing the expression of C4‐HSF activity.

The identities of C4‐HSF activity and C3‐HSF are still unknown but we believe that C3‐HSF activity could be IL 6 as it has a similar molecular weight (30 kDa) and purified human IL 6 was more effective than IL 1, TNF‐α or TNF‐β in stimulating C3 synthesis by rat hepatocytes. C4‐HSF activity appears to be a property of a previously undescribed cytokine. It is not IL 1α or β, TNF‐α or ‐β, IL 6 or IFN‐γ.
Endogenous interleukin 6 plays an obligatory role in interleukin 4‐dependent human IgE synthesis
Authors: Vercelli Donata , Jabara Haifa H. , Arai Ken‐Ichi , Yokota Takashi , Geha Raif S.
Published: March 2007

The lymphokine interleukin (IL) 4 plays a crucial role in the regulation of IgE synthesis. In the present study, the cellular and cytokine requirements for the IL 4‐dependent induction of IgE synthesis in humans were analyzed. Recombinant IL4 could induce IgE synthesis by peripheral blood mononuclear cells and autologous T/B cell mixtures, but not by highly purified B cells. IgE induction by IL4 was strongly decreased in monocyte‐depleted peripheral blood mononuclear cells. These results show that the induction of IgE synthesis by recombinant IL 4 is T cell dependent and optimal in the presence of monocytes. IL5 and IL6, but not IL2, IL1 and tumor necrosis factor‐α, strongly up‐regulated the IL4‐dependent synthesis of IgE, with modest effects on cell proliferation. An anti‐IL6 polyclonal antibody strongly inhibited IL4‐driven IgE production. Endogenous IL6 plays, therefore, an obligatory role in the IL4‐dependent induction of IgE. However, a combination of IL 4, IL 5 and IL 6 (with or without IL 1) at optimal concentrations could not induce IgE synthesis by purified normal B cells, indicating that cytokine‐mediated signals, although essential, are not sufficient for the IL 4‐dependent induction of IgE synthesis.
Activation of human B lymphocytes through CD40 and interleukin 4
Authors: Vallé Alain , Zuber Caroline E. , Defrance Thierry , Djossou Odile , Riem Menno De , Banchereau Jacques
Published: March 2007

We have produced and characterized a new CD40 monoclonal antibody, mAb 89, which in the presence of anti‐IgM antibodies co‐stimulates to induce B cell proliferation. mAb 89 activates resting B cells as shown by an increase in cell volume and an enhanced subsequent proliferation of B cells in response to anti‐IgM antibody. However, mAb 89 does not prepare B cells to respond to the growth‐promoting activity of interleukin (IL) 2 or IL4. Unlike IL 2 and IL 4, mAb 89 only weakly stimulates the proliferation of anti‐IgM pre‐activated B cells. Thus, the activating properties of anti‐CD40 are likely to explain its co‐stimulatory effect on B cells.

Interestingly, the anti‐CD40 mAb 89 was found to act in synergy with IL 4, but not with IL 2, in co‐stimulation and restimulation assays. In this respect, anti‐CD40 does not induce a significant increase of B cell surface IL 4 receptors while IL 4, but not IL 2, induces a twofold increase of the CD40 antigen expression. Thus the synergistic interaction between IL 4 and anti‐CD40 may be related to the IL 4‐dependent increase of CD40 antigen expression.
Molecules relevant for T cell‐target cell interaction are present in cytolytic granules of human T lymphocytes
Authors: Peters Peter J. , Geuze Hans J. , Van Donk Hans A. Der , Slot Jan W. , Griffith Janice M. , Stam Nico J. , Clevers Hans C. , Borst Jannie
Published: March 2007

An ultrastructural analysis of human cytotoxic T lymphocyte‐target cell (CTL‐TC) interaction has been undertaken to enable a better understanding of the killing mechanism. Attention was focused on granules in the CTL, which are known to contain lethal compounds. Within the membrane‐delimited cytotoxic granule an electron‐dense core as well as numerous membrane vesicles were identified. In CTL‐TC conjugates, specific membrane interactions take place, allowing the formation of intercellular clefts into which the granule cores and internal vesicles are released. T cell surface membrane molecules known to be involved in CTL‐TC interaction (T cell receptor, CD3 and CD8) are present on the membranes of the granule cores and internal vesicles, facing outward. An explanation for this localization of the membrane may be found in the fact that the granule is connected with an endocytotic pathway. Moreover, the lumen of the granule is rich in the enzyme cathepsin D, which indicates an association with a lysosomal compartment. Exocytosed vesicles and cores are seen to adhere to the plasma membrane of the TC. Although the exact contents of the granule vesicles and core remain to be identified, we suggest that specific interaction of CTL membrane molecules on the cytolytic granule components with molecules on the plasma membrane of the TC may ensure the unidirectional delivery of the lethal hit.
γ/δ T cells express a unique surface molecule appearing late during thymic development
Authors: Mackay Charles R. , Beya Marie‐Françoise , Matzinger Polly
Published: March 2007

The vast majority of T cells in man and mouse use the α/β form of T cell receptor (TcR), and express either CD4 or CD8, whereas the small subset of γ/δ T cells are usually CD4‐CD8‐. In contrast to man and mouse, the γ/δ subset in sheep, defined here using an anti‐γ/δ monoclonal antibody (mAb), comprises 30%–60% of T cells. We show that γ/δ T cells in sheep express a unique surface molecule termed T19 which is 215 kDa in size and unrelated to either CD45 or the TcR. The T19 molecule was expressed at a distinct stage during γ/δ T cell ontogeny within the thymus, since γ/δ thymocytes which appeared early in fetal ontogeny were T19‐ and also major histocompatibility complex (MHC) class I‐ and localized almost exclusively to the outer cortex and cortex of the thymus. “Mature‐type” γ/δ thymocytes which emerged late in thymic development were T19+ and MHC class I+ and localized predominantly to the thymic medulla. The sequence of events indicated that these cells were most likely derived from the early γ/δ thymocytes. These medullary γ/δ thymocytes showed a very distinctive association with Hassall's corpuscles, suggesting a role for these structures in γ/δ thymocyte maturation.

In the periphery, T19 was expressed exclusively within the γ/δ T cell subset, however some γ/δ T cells were T19‐. In particular, a large proportion of γ/δ T cells within intestinal epithelium lacked T19, indicating a correlation between T19 expression and either function or homing patterns of γ/δ T cells. Both T19+ and T19‐ γ/δ T cells were CD2‐, and expressed low levels of LFA‐1 and CD5. In addition, γ/δ T cells recirculated differently from other T cells, and appeared not to enter mesenteric lymph nodes at all from the blood. We propose that T19 is a maturation marker for γ/δ T cells. In addition, the exclusive expression of T19 by γ/δ T cells indicates that this molecule most likely serves a fundamental role in the interactions and function of γ/δ T cells.
Alleviation of autoimmunity in BXSB mice by monoclonal alloantibody to Ly‐5 (CD45)
Authors: Yakura Hidetaka , Ashida Tomofumi , Kawabata Isao , Katagiri Makoto
Published: March 2007

One of the hallmarks of immunological abnormalities in mice that are prone to syndromes resembling systemic lupus erythematosus is polyclonal activation of B cells and a switch of serum immunoglobulins or autoantibodies from IgM to IgG that coincides with onset of autoimmune disease. Therefore, we investigated whether the development of autoimmunity can be alleviated in such mice by administration of monoclonal alloantibody directed against Ly‐5, because this was known to reduce levels of polyclonal IgG antibody without affecting other immunoglobulin isotypes. Our results indicate that at least some forms of autoimmune disease may be effectively treated by thus reducing the concentration of IgG autoantibody in this manner.
Identical forms of the CD2 antigen expressed by mouse T and B lymphocytes
Authors: Altevogt Peter , Michaelis Marion , Kyewski Bruno
Published: March 2007

A monoclonal antibody (12‐15) reactive with the mouse CD2 was used to study the expression of the antigen in different lymphoid cell subsets. By two‐color immunofluorescence using B or T cell‐specific reagents and cell sorting in combination with biochemical analysis we provide evidence that the CD2 antigen is present on mouse B and T cells. The antigen is expressed by both subsets at similar density and appears to be biochemically indistinguishable.
Murine enterocytes can present soluble antigen to specific class II‐restricted CD4+ T cells
Authors: Kaiserlian Dominique , Vidal Karine , Revillard Jean‐Pierre
Published: March 2007

Highly purified mature epithelial cells (EC) from murine small intestinal villi (excluding Peyer's patch epithelium) were examined for their capacity to present foreign antigen to T cells. Cell suspensions composed of 99% Ia+T200‐ EC were able to present keyhole limpet hemocyanin to an antigen‐specific class II‐restricted L3T4+ T cell hybridoma, and stimulate interleukin 2 production. Antigen presentation by EC was inhibited by major histocompatibility complex class II‐specific monoclonal antibodies. It is concluded that EC express functional class II molecules and may activate adjacent CD4+ T cells to induce lymphokine synthesis.
Lymphokine gene expression related to CD4 T cell subset (CD45R/CDw29) phenotype conversion
Authors: Bettens Florence , Walker Christoph , Gauchat Jean‐François , Gauchat Dominique , Wyss Toni , Pichler Werner J.
Published: March 2007

In this study, we investigated whether the phenotype‐related differentiation of human 2H4+ (CD45R) naive cells to 2H4‐ (CDw29) memory CD4 cells corresponded to modulation of interleukin (IL) 2, IL 4, interferon (IFN)‐γ and granulocyte‐monocyte colony‐stimulating factor (GM‐CSF) gene expression, a phenomenon which might correlate to the distinct functional activities of naive cells or memory cells. To mimic in vitro CD4 T cell subset differentiation, freshly isolated 2H4+ and 2H4‐ CD4 cells were stimulated with the anti‐CD3 antibody (Leu‐4), expanded in IL 2‐containing medium and restimulated with Leu‐4 after 7 and 13 days. Absence of monocyte‐T cell interaction was compensated by adding monocyte supernatant to the culture medium and by cross‐linking the anti‐CD3 antibodies with goat anti‐mouse antibody coated on culture dishes.

It has been previously shown that in vitro stimulated 2H4+ cells acquire CDw29 surface antigens. Measurement of lymphokine gene expression by dot‐blot hybridization revealed that although stimulated 2H4+ cells proliferated less than stimulated 2H4‐ cells, and expressed less actin mRNA, they expressed more IL 2 but less IL 4 and GM‐CSF than 2H4‐ cells. No significant difference was observed between the two subsets for the expression of IFN‐γ. If subsets were restimulated with Leu‐4 antibodies, expression of IL 2 was decreased and expression of IL 4 was increased in both subsets; however, the differences among the subsets persisted. They were even more enhanced for IL 2 but less pronounced for GM‐CSF.

Thus, in spite of phenotype conversion, CD4 T cell subsets maintained a distinct capacity to express IL 2 and IL 4 genes.
Enumeration of cytokine‐secreting cells at the single‐cell level
Authors: Skidmore Barry J. , Stamnes Susan A. , Townsend Kay , Glasebrook Andrew L. , Sheehan Kathleen C. F. , Schreiber Robert D. , Chiller Jacques M.
Published: March 2007

A sensitive assay utilizing enzyme‐linked immunosorbent assay methodology has been developed for the quantitation of single cells secreting interferon (IFN)‐γ or tumor necrosis factor (TNF). Cloned T cells or cells from lymphoid organs were stimulated with antigen, concanavalin A, or phorbol myristate acetate and ionomycin in microwells coated with antibodies specific for IFN‐γ. Discrete “spots” overlying areas where cells secrete IFN‐γ were then developed by incubation with a second antibody to IFN‐γ, followed by an enzyme‐labeled antibody conjugate and substrate. Similarly, using TNF‐specific antibody reagents, TNF‐secreting cells were detected and quantitated in cell populations obtained from normal lymphoid tissues, bone marrow and peripheral blood, following activation with phorbol myristate acetate and ionomycin. Provided specific antibodies are available, this method has the potential to measure the frequency of cells secreting any cytokine.
C1q binding to chimeric monoclonal IgG3 antibodies consisting of mouse variable regions and human constant regions with shortened hinge containing 15 to 47 amino acids
Authors: Sandlie Inger , Aase Audun , Westby Christina , Michaelsen Terje E.
Published: March 2007

We have constructed four different deletion mutants of a chimeric mouse‐human IgG3 anti‐(4‐hydroxy‐3‐nitrophenyl)acetyl/(5‐iodo‐4 hydroxy‐3 nitrophenyl) acetyl (NP/NIP) antibody lacking one or more of the four exons coding for the hinge region. The mutant variants all retained intact hinge region epitopes since they all reacted with IgG3 hinge‐specific antibodies. Surprisingly, all the deletion mutants bound C1q equally well or even better than the wild type. Thus the high C1q binding activity of IgG3 compared to IgG1 is apparently not due to the total length of the IgG3 hinge, which is 62 amino acids, nor is it due to the length of the upper hinge which is the stretch from the end of CH1 to the first inter‐heavy chain disulfide bond.
Only dull CD3+ thymocytes bind to thymic epithelial cells. the binding is elicited by both CD2/LFA‐3 and LFA‐1/ICAM‐1 interactions
Authors: Nonoyama Shigeaki , Nakayama Masayuki , Shiohara Tetsuo , Yata Jun‐Ichi
Published: March 2007

In view of the necessity for thymocytes to interact with thymic epithelial cells to differentiate into mature T cells, this study analyzed the binding between human thymocytes, cultured thymic epithelial cells (CTEC) and the required adhesion molecules.

Immediately after separation, thymic epithelial cells (TEC) readily expressed ICAM‐1, which is one of the ligands of LFA‐1 cell adhesion molecules. However, the ICAM‐1 expression was gradually lost upon culture of TEC. IFN‐γ re‐induced ICAM‐1 on the CTEC, and the ability of CTEC to bind to thymocytes was also increased by IFN‐γ treatment. The increase in binding seemed to be caused by the LFA‐1/ICAM‐1 interaction, since it was inhibited by anti‐ICAM‐1 monoclonal antibody (mAb) and anti‐LFA‐1 mAb. This suggests that the LFA‐1/ICAM‐1 interaction is also involved in vivo with the binding of thymocytes to TEC, which have been shown to express ICAM‐1.

To better understand the nature of the cells involved in binding, thymocytes were sorted into CD3‐, CD3dull+, and CD3bright+ subsets (which are supposed to represent the immature, intermediate and mature stages of differentiation, respectively), and were examined for their binding to IFN‐γ‐treated CTEC. The result showed that only the CD3dull+ subset bound to CTEC. CD3‐, CD3bright+ cells and peripheral blood T lymphocytes did not bind, but they were induced to bind by neuramidase treatment. All these bindings were inhibited by anti‐LFA‐1 mAb and anti‐CD2 mAb. These findings indicate that CD3dull+ cells can bind to TEC via CD2/LFA‐3 and LFA‐1/ICAM‐1 interactions. Other cells seemed not to bind to TEC because of sialylation.
T cell activation by monoclonal antibodies directed to different epitopes on the human T cell receptor/CD3 complex: Evidence for two different modes of activation
Authors: Schlitt Hans JüRgen , Kurrle Roland , Wonigeit Kurt
Published: March 2007

The mouse monoclonal antibody (mAb) BMA031 (IgG2b) has recently been described to be directed against a monomorphic part of the human T cell receptor (TcR) α/β. In vitro analysis of its stimulatory potential for mononuclear cells revealed two patterns of responsiveness. Out of 35 tested individuals only 2 generated a proliferative response to low antibody concentrations (15 ng/ml; “high responders”), the others (“low responders”) responded only to high antibody concentrations (1.5 μg/ml); the anti‐CD3 mAb UCHT1 (IgG2b) stimulated only the two high responders. This response pattern to BMA031 was determined by the accessory cell compartment in the culture. Stimulation by BMA031 in low responders demonstrated some unusual features: (a) high antibody concentrations were required, (b) addition of autologous serum had no inhibitory effect and (c) vigorous depletion of macrophages reduced but did not abolish the proliferative response. These characteristics were shared by two other mAb, BMA032 and BW239/347, presumably directed against the TcR α/β but not by several other antibodies to the TcR/CD3 complex. Thus, the results demonstrate unusual stimulatory properties of three anti‐TcR α/β mAb, inducing a proliferative response without antibody cross‐linking. This suggests that the stimulatory effect of anti‐TcR/CD3 complex mAb is not only determined by their isotype, but also strongly depends on their epitope specificity.
Immunoglobulin‐specific T‐B cell interaction
Authors: Yurin Vitalij L. , Rudensky Alexander Y. , Mazel Svetlana M. , Blechman Janna M.
Published: March 2007

In the preceding report (Eur. J. Immunol. 1989. 19: 1677) we have demonstrated that normal B cells, including small B cells, are capable of presenting Igχ;‐1b allotypic determinants of their endogeneously synthesized Ig+ to Igχ;‐1b‐immune major histocompatibility complex (MHC) class II‐restricted T cells. A panel of Igχ;‐1b allotype‐specific T cell clones from August rats has been developed to study further the presentation of self surface Ig by B cells from Igχ;‐1‐congeneic August.1b rats. All the clones studied were of the T helper/inducer phenotype (W3/25+, OX8‐) and restricted by the RT‐1Bc molecule. These clones responded both to the serum IgG(Igχ;‐1b) in the presence of irradiated spleen cells (SC) from August rats and to the Igχ;‐1b‐bearing irradiated B cells from August. 1b rats. SC presentation of secreted IgG was much less effective than B cell presentation of membrane Ig. Using CNBr cleavage of isolated Cχ;(Igχ;‐1b) domain followed by high‐performance liquid chromatography fractionation of the derived antigenic peptides, the χ; chain sequence between amino acids 176 and 214 has been identified as the T cell epitope recognized by all T cell clones in association with RT‐1Bc. The fragment 176‐214 of the Igχ;‐1b allotype differs from that of Igχ;‐1a allotype by three amino acid substitutions at positions 184, 185, 188. T cell recognition of pLx‐1b(176‐214) required no additional processing by the antigen‐presenting cell: the efficient presentation of the peptide but not of intact IgG(Igχ;‐1b) by the paraformaldehyde‐fixed SC was observed. These data provide clear‐cut evidence for an absolute requirement of the processing of Ig molecules for T cell recognition to occur in our experimental system. Although the fixation of B cells from August. 1b rats diminished their Igχ;‐1b‐presenting ability, fixed Igχ;‐1b+ B cells were still able to induce Igχ;‐1b‐specific T cell clone responses. Our results suggest that B cells can express the processed form of self‐synthesized surface Ig in addition to intact surface Ig molecules. The former can be recognized by MHC‐restricted T cell.
Thrombocytopenia associated with the induction of neonatal tolerance to alloantigens: Immunopathogenic mechanisms
Authors: Merino JesúS , Qin Hui‐Yu , Schurmans Stephane , Gretener Denise , Grau Georges E. , Lambert Paul H.
Published: March 2007

BALB/c mice rendered tolerant to alloantigens by neonatal injection of semiallogeneic (C57BL/6 x BALB/c)F1 spleen cells develop a thrombocytopenia in association with an autoimmune lupus‐like syndrome. The possible mechanisms involved in the thrombocytopenia were investigated. The development of thrombocytopenia was first detected at 3 weeks of age coinciding with the start of the other autoimmune manifestations and was always related to a state of tolerance and B cell chimerism. There was a significant increase of megakaryocytes in bone marrow and spleens from thrombocytopenic tolerant mice and radiolabeled platelets from these mice were more rapidly eliminated from the bloodstream than normal platelets when injected into normal recipients. A significant correlation between the spleen weight and the decrease of the circulating platelets was observed, although some mice with severe thrombocytopenia had only a moderate spleen enlargement.

Thrombocytopenia significantly correlates with the levels of platelet‐associated IgG (PAIgG) but not with anti‐single‐stranded DNA antibodies or circulating immune complexes. Platelets from mice with high levels of PAIgG had a shorter life‐span when injected into normal mice than those from mice with low or normal PAIgG. The possibility that PAIgG are partially due to antibodies reacting specifically with platelet membrane components was analyzed. First, F(ab')2 Ig fragments from tolerant mice were shown to bind to normal platelets, in contrast to F(ab')2 Ig fragments from normal mice. Second, some monoclonal antibodies produced by hybridomas derived from tolerant mice reacted in vitro with platelets and induced a transient thrombocytopenia after i.v. injection into normal mice.

These data suggest that the thrombocytopenia observed in tolerant mice is the result of a peripheral hyperdestruction of platelets associated with (a) hypersplenism, (b) nonspecific fixation of immunoglobulins, probably as immune complexes and (c) with autoantibodies reacting specifically with platelets. It may represent an interesting model for human chronic idiopathic thrombocytopenia.
The involvement of Thy‐1 antigen in the activation of rat mast cells
Authors: Dráberová ĽUbica
Published: March 2007

The Thy‐1 antigen expressed on the surface of mouse T lymphocytes has been previously found to be involved in T cell activation. In the present study we have employed the anti‐Thy‐1.1 monoclonal antibody MRC OX7 and analyzed the expression and properties of Thy‐1 antigen on the surface of peritoneal and pleural rat mast cells. Direct radioantibody binding assays, indirect immunofluorescence studies and flow cytometry analysis revealed that isolated rat mast cells express on their surface large amounts of the Thy‐1 antigen. Scatchard analysis of the binding data indicated that at least one million Thy‐1 molecules are expressed per cell. Immunoprecipitation studies carried out on mast cells demonstrated that MRC OX7 antibody recognizes a surface molecule of approximately 25 kDa that appears to correspond to Thy‐1 antigen, and an additional molecule of 50 kDa. Incubation of the isolated mast cells with various concentrations of anti‐Thy‐1.1 antibody induced a rapid and concentration‐dependent increase in the intracellular free calcium concentration ([Ca2+]i). The early increase in [Ca2+]i was observed in both Ca2+‐supplemented and Ca2+‐free media. This indicated that the initial increase in [Ca2+]i is due to a release of Ca2+ from internal stores. The [Ca2+]i increase was followed by an increase in the histamine release which was also dependent on antibody concentration. These data suggest that Thy‐1 may act as an activation receptor on mast cells, analogously to its receptor function on T cells.
Studies on the thymus in chagas' disease
Authors: Savino Wilson , Leite‐de‐Moraes Maria Do Carmo , Hontebeyrie‐Joskowicz Mireille , Dardenne Mireille
Published: March 2007

Previous observations demonstrated severe thymocyte depletion in mice undergoing acute Chagas' disease. These data led us to investigate the status of the thymic microenvironment in these animals. Young adult C57BL/6 and C3H/HeJ mice were infected i.p. with 105 blood‐derived trypomastigote forms of Trypanosoma cruzi (CL strain) and killed 7–14 days after infection. Sera were then analyzed for thymic hormone (thymulin) levels, and frozen thymus sections were studied by immunohis‐tochemistry for the expression of functional antigens (thymulin and Ia), the distribution of distinct thymic epithelial cell subsets and extracellular matrix components. Infected mice exhibited a transient decrease in thymulin production and those with severe thymic atrophy showed a denser Ia‐bearing cellular network. In addition, an abnormal localization of the TR5 and CK18 antigens restricted to the medullary and cortical TEC subsets, respectively, was observed. Furthermore, an increase in the basement membrane proteins was detected within thymic lobules. We suggest that the thymic microenvironment is also affected during T. cruzi infection, extending the concept that the thymus should be regarded as a target in Chagas' disease.
Restriction fragment length polymorphism analysis of the VX locus in human lupus
Authors: Zouali Moncef , Madaio Michael P. , Canoso Rosa T. , Stollar B. David
Published: March 2007

To evaluate the degree of genetic polymorphism of the Vχ; repertoire in systemic lupus erythematosus (SLE), we performed Southern blot hybridizations with human gene probes corresponding to the four human Vχ; gene families. In a comparative analysis, non‐lymphoid cell DNA samples from three patients with idiopathic SLE, eight subjects with susceptibility to drug‐induced lupus and seven control individuals were digested with the restriction endonucleases Bam HI, Bg1 II, Eco RI and Hind III, and hybridized sequentially to the four Vχ; family‐specific probes. The restriction patterns on Southern blots revealed a low degree of polymorphism of the human Vχ; gene repertoires of SLE patients and control individuals. This analysis, together with previous parallel studies of the Vχ; locus in lupus‐prone mice, implies that autoantibody hyperproduction in lupus is not associated with major modifications in the structure or genomic organization of immunoglobulin light chain genes.
The gene for the t lymphocyte alloantigen, rt6, is not linked to either diabetes or lymphopenia and is not defective in the bb rat
Authors: Lang FrançOis , Kastern William
Published: March 2007

In an outcross between a diabetic BB/H rat and a healthy Long Evans Hooded rat, the segregation of the RT6 gene was studied in the 207 F2 animals to look for linkage with diabetes or lymphopenia. The recessive gene, albino (c), was used as a marker for the RT6 gene because of the close proximity of these two genes on chromosome 1. Though most of the albino F2 rats should have been homozygous for the BB RT6 gene, we found no increase in the incidence of diabetes or lymphopenia among them when compared to their hooded littermates. Therefore, the RT6 gene was not linked to diabetes or lymphopenia in the BB rat. Moreover, the non‐lymphopenic albino rats displayed normal RT6 expression when compared to the normal hooded rats showing that the RT6 gene from the BB/H grandfather was not defective. Any alteration in lymphocyte composition which could be specifically related to diabetes was studied by measuring all F2 rats for the major lymphocyte subsets including the RT6+ subset. We found that the typical pattern of lymphopenia described in diabetic BB rats was displayed by both diabetic and non‐diabetic lymphopenic rats in the F2 generation. Thus, all these lymphocyte abnormalities including the depletion in RT6+ T lymphocytes appeared as a consequence of lymphopenia alone and could not be specifically related to diabetes.