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Apoptosis in the thymus of mice during the growth of syngeneic transplantable tumor
72
Regulation of apoptosis
Interestingly, the strongest apoptotic mAbs B-030, B-627, B-L25 and B-E28 recognize a similar or adjacent epitope on the CD95 antigen. The presence of goat anti-mouse antibodies (GAM) enhanced the apoptotic activll wlth all mAbs CD95 used at sub-optimal concentration except for mAb 8634, which remained silent. To further investigate the mAb valency required for apoptotic activity and the role of the Fc portion in the programmed cell death, the activity of F(ab’,‘h or Fab fragments of agonistic mAbs such as B-627. B-G30 and B-E28 were tested. The F(f(ab’)* fragments of mAb B-027, B-630 and B-E28 still induced cell death, whereas only Fab fragments of mAb B-G27 still triggered apoptosis in SKW6.4. The addition of GAh4antibodies restored the induction of cell death with Fab fragments of mAbs B-630 and B-E28. The regulation pathway(s) of CD95 mAb induced cell death was also investigated. Two regulation pathways of CD95 mAb induced cell death were observed. Firstly, the apoptosis induced by mAb B-G27 mAb was significantly reduced by mAbs B-D29 and B-G34, whereas the other CD95 mAbs had no effect. Secondly, the apoptosis induced by mAbs B-G30, B-l25 or B-E28 was significantly enhanced by mAbs ED29, B-G34 and B-K14.
P. 1.08.18
Possible mechanisms for induction of epoptosis after CDSmediated T-cell activation and its
reduction by CDS&costlmulation Y. Miiller’, E. Wierenga3, R. Mailhammer’, W. Wilmanns’~*. G. Jung’. ’ Medizinische Klinik III der Univetsibit, Miinchen, Germany, * GSF-Research Center for Environment and Health, M&when, German)! 3Laboratory for Ceil Biology; University of Amstenlam, The Netherlands Introduction: We have previously observed, that stimulation of resting human Tcells with high density anti-CD3 leads to proliferation (CD28minus-proliferation) and subsequent anergy and apoptosis. This negative signaling could be counteracted if cells were stimulated with low density submitogenic anti-CD3 together with CDP&antlbody (CD28plus-proliferation). Here we further explore these two modes of stimulation as a model for “productive” vs. “abortive” T- cell pmllleration. Materials and Methods: Apoptosis was determined by FACS analysis of omoidium iodide stained cells. lvmohokine oroduction bv auantitation of c-DNA iPdFl) and secreted proteins @LISA). Bcl-i, ~53 and bcl-&, were detected by western blot analysis. Reeults: The apoptosis preventing effect of CM&ostimulation is lost at low cell concentrations. Production of 11-2, 11-4, ylFN and TNFu varies considerablv between T-cells of diierent donors but is consistentlv hiaher durina CD26pl;s proliferation. No consistent difference in the expression if ~53 was observed. The expression of bcl-a, and bcl-2 seems to be elevated during CD26plus-proliferation. Concluolon: Apoptosis induced after (CDPBminus)-Tcell pmliieration (see definition above) is reduced in the presence of CD28-costlmulation, possibly due to increased lymphoklne production and bcl-XLexpression.
P.1.08.17
Regulation of thymocyte apoptosis by CD3/TCR complex, zinc and glucocorticoids in old mice: Implications for thymic lnvolutlon
M. Pmvinciali, G. Di Stefano, S. Stronati. lmmunologv Center INRCA Gerontology Research Department, Ancona, Italy The involution and atrophy of the thymic gland arising during aging has been associated with a reduction of the proportion and number of CD4+CD8+doublepositive (DP) thymccytes. Apoptosis represents the main mechanism involved in the regulation of the intrathymic deletion of DP thyrnocytes. Aim of this work was to evaluate whether the decline with aging in the fraction of DP thymocytes is associated with an alteration in the responsiveness of thymocytes to factors regulating the apoptotic cell death, such as antibodies to CD3JTcell receptor complex, zinc and dexamethasone (DEX). Balb/c mice were used at the age of 2 mo (young), 21-22 mo (old), and 24-26 mo (very-old). Thymocytes from these mice were incubated overnight with anti CD3 monoclonal antibody (8 &ml), Zn2+ (150 PM). or DEX (IO-’ M) and then analysed for the number of apoptotic nuclei, the cell cycle phase, and the percentage of CD4+CD8+ cells by flow cytometty. A significant decrease of either the total number and the proportion of CD4+Co8+ DP thymocytes was present in old and vety-old mice. Antibodies to CD3 antigen induced apoptosis of thymocytes from both young and old mice. The stimulation of CD3/TCR complex was more effective in giving apoptosis in old than young mice. An impairment of the inhibiting effect of zinc on either apoptosis induced by seturn deprivation, anti CD3 antibodies, or DEX, was found in old ages and in particular in vely-old mice. A reduced DEX-induced apoptosis was also present in old age: this effect was more evident in very-old than in old mice. The simultaneous administration of anti CD3 antibodies and DEX did not modify the apoptosis induced by each factor alone in both young and old mice. The boosting of apoptosis by DEX in aged mice required protein synthesis being blocked through cycloeximide. The effect of zinc and DEX on apoptosis was
23 June 1997 - Poster presentations
exerted on thymocytes in a specific cell cycle phase, i.e., on GO/G1phase cells.
Either anti CD3 antibodies. zinc and DEX regulated apoptosis by modulating the pmportlon of DP thymocyies. The results demonstrate an altered responsiveness of thymocytes from old and very-old mice to CD3/TCR complex, zinc and glucocorticoids suggesting that the age-related reduction in the number of DP thymocytes may be due, at least in part, to an increased apoptosis of this population occurring with increasing age.
1.. P 1 08 .18 ( Stimulation of CD40 in human bladder carcinoma cells inhibits anti-F&APO-l
induced apoptosis
G. JBnsson, E. Jakobson, E. Gelius, P. Bjarck, S. Paulie. Dept. ofirnrnuno/~ Stockholm Univarsi& S- 106 9 1 Stockholm, Sweden CD46 is a cell surface receptor expressed on B lymphocytes, certain eplthelial carcinomas, bone-marrow derived dendritic calls and on follicular dendritic cells. CD40 mediates a strong gmwth stimulatory signal in B cells and triggeling of the receptor prevents germinal centre B cells from undergoing apoptosis. We were interested to see whether CD40 could prevent apoptosis induced via the Fas/APO-1 antigen. Here we report that ligation of CD40 on human bladder carcinoma cells with antibodies or with soluble construct of the CD40 ligand inhibited cell death induced via the Fas/APO-I antigen. Protection appeared to occur independent of bcl-2 upregulation, since rescue could be obtained by adding CD40 agonists immediately before the anti-Fas/APO-I antibodies. Two of the cell lines were found to be inherently resistant to apoptosis in spite of a similar expression of the Fas/APO-1 antigen on these cells aa on the sensitive lines. Preliminary data show that these resistant cell lines also express the Fas/APO-1 ligand. The results show that CD40 stimulation may protect bladder carcinoma cells from apoptcsis actively induced via Fas/APO-1 and by expressing the Fas/APO-I ligand these cells could escape immune response.
P.1.08.19
Apoptosis of T cdl8 in the first trimester human troohoblast durina abortion ~roc888
M. Jerzak ‘, J. Kotarski *, K. Us-Lachowicz 3, M. Kasplzycka 3, A. Got&l 3. ’ LIept Clin Immunolo9y, Univ School of Med., Lublin, Poland, * Uepr Gyn Surg. Univ School of Med. L&in, pdsntf, 3Dept C/in Immunol~ Transplantation lnstibte, Univ. School of Med. Warsew,Poland Introduction: Apoptosis has been proposed as a mechanism for maintaining the tolerance in the immune system. Induction of the apoptotic cell death is also a possible outcome of the lymphocyte activation. Recent data suggest that human foetal membranes express Fas (CD95), a cell surface receptor that mediates apoptosis. Expression of Fas L by human trophoblast has been also proposed as a mechanism providing protection against the &tic action of decidual immune cells. In this sense, reduction of Fas or Fas L function can be associated with pregnancy 1089. MaterlaIr and Methods: We studied apoptosis of T cells isolated from the first trimester trophoblast in I2 women after spontaneous abortion. We used gel electmphomsis to detect DNA fragmentation, which is associated with apoptosis. Cells undergoing DNA fragmentation were also identified by DNA content analysis using flow cytometry. This method was based on the accumulation of ethanol-fixed apoptotlc cells in the sub-GdGl peak of the DNA content histogram as a msult of loss of DNA fragments out of the cells and because of a reduced DNA “stainability” by propidium iodide (PI). Results: We did not detect apoptosis by the “ladder” technique. However, apoptotic index (percentage positive/total cells) ranged from 2 to 18% using Row cytometry. Conclusion: Trophoblast cells usually fail to stimulate alloantigen-specific T cells, but they may express non-classical MHC alloantigens to which mothers produce IgG alloantibody and that requires T helper cell activation. Minimal apoptosis of T cells in the human tmphoblast might have been a defence mechanism against rejection of the foetal allograff by maternal immune system.
P.1.08.20
Apoptosls in the thymus of mice during the growth of syngeneic transplantable tumor
E.P. Kisseleva, R.P. Ogurtsov, A.N. Suvomv. lnstifute for Experimental Medicine, St. F@fe&urg, Russia Intmductfon: Apoptosis (A) of immature autoreactive thymocytes has been proposed as one of the mechanisms of negative selection associated with T cell differentiation. Studies of the thymic involution in tumor-beating mice have been mostly limited to the h&pathologic level and the intrathymlc development of A remained undefined. The aim of the study was to evaluate the rate of A in thymocytes during the growth of mtine syngeneic tumor. Materials and Msthodo:CBHA mice were S.C. inoculated with IO5 cells of weakly immunogenic solid hepatoma 22a. At weekly intervals thymuses
23 June 1997 - Poster presentations
were investigated histdogically in paraffin sections. DNA fragmentation was measured by gel electrophoresis and diphenylamine test (DFA). The number of apoptotic cells was calculated using microscopy in cells stained with a mixture of acddine orang&thidium bromide. Rwuftm Starting from the third week after tumor inoculation the progressive thymic involution occured. Histological studies revealed striking delymphatfxation of thymlc cortex, reduced number of mkozes in the cortex. The number of pyknoses was increased two-three times in the cortex but stfll remained under the same low rate in tumor-bearers as well as in controls. Therefore we could not reveal any difference in apoptotic cell numbers either by staining with fluorescent dyes or by DNA fragmentation in gel electrophoresis. In order to study the difference between thymocytes of tumor-bearing and control mice, cells were incubated in vftro during 2 h at 3X in medium containing 10% of fetal calf serum. The development of spontaneous A in thymccytes from tumor bearers was two times higher than in control animals as evaluated by fluorescent test and showed more intensive “ladder” development by electrophoresis. This difference was evident from the first week after tumor inoculation before the signes of involution appeared. After the 24 h incubation in vftro the spontaneous development of A was also higher in thymes from tumor-bearers as registered by DFA-test. Oppositely to the previous two, this test did not reveal the early changes and showed the enhancement of A only at the stage of developing thymic involution. Conduslon: This was the ffrst atempt to evaluate A in the thymus during the growth of syngeneic tumor. Being a chronic process the tumor development did not result in a massive increase of A and showed only slight rise of the number of pyknotic cells by histological studies. Three other tests were not enough sensitive to show any difference. Incubation of thymocytes in vitro allowed to register the enhanced ability of thymocytes from tumor-bearers for the development of spontaneous A. It happened at the early stage of tumor development and became more prominent later. Enhanced readiness to A may reflect the changes in thymocyte differentiation induced by intrathymic microenvironment in response to antigenic stimuli from increased tumor burden.
P.1.08.21
All forms of TGF-P are co-stimulatory to rat T-tymphocytes by increasing viability and preventing apoptosis
A. Schidtt I, H.-O. Sj6gmn ‘, M. Lfndvall 1.2.’ The Wallenberg Laboratory, Depatiment of Tumor lmmunolo~ University of Lund, Lund, &eden, 2Astra Draco AB, Lund, Sweden Introductton: TGF-p is a growth factor with known immuno-suppressive effect on lymphocytes. We and others have found the opposite effect of TGF-p. In our system TGF-p is increasing the prolfferatfve response of rat T-lymphocytes to Staphylococcal enterotoxfn A, but only if a certain amount of monocytes is added. In this study we compared the three isoforms TGF+l, TGF-fi2 and TGF-,93 in their ability to costimulate, affect apoptosis and viability of T-lymphocytes. Material and Methods: Wlstar/Furth rat spleen lymphocytes stimulated with Staphylccoccal enterotoxin A, TGF-@I (natural human), TGF-,92 and TGF-B3 (both recombinant human). Apoptotic cells am revealed by positive Annexin-V binding and negative ethidiumbromide binding. Resuh The addition of all three isofonns of TGF-,3 to SEA stimulated T-lymphocytes increase the proliferative response almost 10 times. An increase of SEA concentration is increasing the proliferation but also the amount of apoptotic cells. The addition of all isoforms of TGF-fi decrease the amount of apoptotic cells caused by SEA. TGF-fi2 and TGF;BB am reaching both these responses wfth one tenth of the TGF-j31 concentration. Antibodies neutrilizlng the cytokines IFN-x and TNFa am both decreasing the amount of apoptotic cells both with SEA alone and with SEA and TGF-pl in proliferation. Conclusion:All three isofomts of TGF-6 acts co-stimufatory on rat T-lymphocytes accompanied by a high viability and low amount of apoptotic cells. TGF-BP and TGF$3 am almost IO-fold mom potent than TGF-Bl. TGF-fi acts through an IFN-y and TNFa independant pathway when acting cc-stimulatory and anti-apoptotic.
IP.1.08.22
Intravenous prepamtions of normal IgG (IVlg) induces apoptosis in human leukemic cells: A role for Fas and a&ration of caboases
N. Prasad ‘, N. Bervas ’ , G. Papoff 2, G. Rubstti *, M.D. Kazatchkfne ‘, S.V. Kaved ’ 1INSERM lJ430, Hdpital Broussais, Paris, France, 2Mtuto di Biolqia Cellulare, CNR, Rome, Italy Introductfon: Therapeutic preparations of pooled normal immunoglobulin for intravenous use (IVlg) is beneficial in several autoimmune diseases and lymphoproliferatiie disorders. IVlg exhibits anti-proliferative effect on cultured T lymphocytes. This study was aimed at gaining further insight into the observed anti-proliferative effect of IVlg.
Regulation of apoptosis
13
Baterlalr and Mathode:Human leukemic cell lines CEM (T cells), Raji (B cells) and MM6 (monocytes) were used. Apoptosis was detected by dye exclusion assays, by analysis of DNA fragmentation, and by analysis of Annexin V binding to phosphotkfylserines on apoptotic cells. To test any role for Fas, supematants from fibroblasts transfected wkh soluble Fas clones which have been shown to block Fas-induced apoptosis and Fas sensitive cell line HUT78 and a Fas-resistant variant Bl were used. Specific inhibitors of macromolecular synthesis, actinomycfnD and cycloheximide and that of caspases (ICE-like proteases) wem employed. Results: The study cfeady indicated that IVlg actively induces apoptosis in these cells in vitro and is characterized by classical DNA ladder formation and by exposure of phosphotktyfserine on the cell surface. IVlg induces apoptosis in the presence of actinomycinD and cycloheximide, suggesting that no new gene transcription or protein synthesis is required for IVlgmediated apoptosis. In CEM T cells, IVlg-induced apoptosis was markedly inhibited by supematants containing soluble Fas molecules as compared to the negative contmf. The extent of IVlg-mediated apoptosis was significantfy lower in HUT 7651 Fasresistant cells compared to Fas sensitive ceils. Further, specific peptide inhibitors of caspases, ICE and YAMA, modestly inhibited IVlg-induced apoptosis in CEM T cells. In addition, specific cleavage of one of the substrates of YAMA, Poly ADP-Ribcse Polymerase (PARP) into a 86 kDa signature apoptotfc fragment was observed upon IVlg treatment. Conclusion: Our data indicate that therapeutic preparations of pooled normal immunoglobulins (IVlg) induce apoptosis in human leukemic cell lines and further suggests a role for Fas and for the activation of ICE-like proteases in IVlg-induced apoptosis. P.1.08.23
a-CD40 Ab treatment prevente in a concentration dependent manner the BCR-ligation induced apoptosis of a human folllcular fymphoma cell line of mature phenotype
M. Eray ‘.‘, A. Ripatti’, L.C. Andersson 1,4,M. Kaartinen*, J. Pelkonen3. ’ Department of Pathology, Haartman Institute, University of Helsinki, Finland, * Deparimenf of Bactedolog): Haartman Institute, University of Helsinki, Finland, ‘Department of Clinical Microbiology Univer&y of Kuopio, Finland, 4Institute for Oncokqy and Pathology, Karolinska Institute, Sweden An unique human follicular lymphoma line (HF-1.3.4) which is easily induced to apoptosis by cross-linking its smlg with relevant monoclonal or polyclonal antibodies (Eray et al. Int. lmmunofogy 6: 1617) was studied. a-CD40 antibody was able to prevent the (Y-Kinduced apoptosis of HF-1.3.4 cells completely at a concentration of 800 ndml. A partfal prevention of apoptosis was seen in ab concentrations as low as 100 @ml. The expression of several cytokines and cytokine receptors (112, 114, 115, 116, ILlO, IL13, INF-y, TNFa, ll2RB and 112&x) was measured during apop tosis induction and prevention through CD40 in HF-1.3.4 cells. The expression of the same cytokines and receptors, in similarly treated BCR-ligation induced apoptosis msistent control lymphoma cells was also studied. For all measumments a quantitative RT-PCR-assays utillsing plasmid based internal standards was used. The HF-1.3.4 cells express in excess Bcl-2 oncoprotein. Its expression was followed during apoptosis induction in untreated and a-CD40 treated HF-1.3.4 cells. Results am discussed in detail.
P.l.08.24
Expression of 8220 on activated T cell blast8 precedes apoptosis
T. Renno, A. Attinger, M. Hahne ‘, J. Tschopp ’ , H.R. MacDonald. From Luoivig institute for Cancer Research, Lausanne Branch, Epalinges, Switzerland, ’ lnstituta of Biochemistr)! University of Lausanne, Epalinges, Switzerland Superantigens am bacterial or viral products that polyclonally activate T cells beating certain TCR B chain variable elements. For instance, Vfi8+ T cells proliferate in response to staphylococcal entemtoxin B (SEB) in vivo then undergo Fas- or TNF-mediated apoptosis. We have recently shown that apoptotic SEB-reactive T cells express the B cell marker 8220 and other markers. Hem we report the identification of a novel subset of B220+ T cell blasts that am the precursors of these apoptotic cells in SEB immunized mice. We also show that the B220 molecule expressed by these T cell blasts’is biochemically similar to that exomssed on the surface of CD4-CD8- double neoative (DN) cells that accumulate in the lymphoid organs of the Fas-L-defective gld mlce,‘but Is distinct from that displayed on the surface of El cells. Collectively, our data support a model whereby upon activation, T cells upregulate 8220 before undergoing aoootosis.
’ ’
Regulation of apoptosis
Interestingly, the strongest apoptotic mAbs B-030, B-627, B-L25 and B-E28 recognize a similar or adjacent epitope on the CD95 antigen. The presence of goat anti-mouse antibodies (GAM) enhanced the apoptotic activll wlth all mAbs CD95 used at sub-optimal concentration except for mAb 8634, which remained silent. To further investigate the mAb valency required for apoptotic activity and the role of the Fc portion in the programmed cell death, the activity of F(ab’,‘h or Fab fragments of agonistic mAbs such as B-627. B-G30 and B-E28 were tested. The F(f(ab’)* fragments of mAb B-027, B-630 and B-E28 still induced cell death, whereas only Fab fragments of mAb B-G27 still triggered apoptosis in SKW6.4. The addition of GAh4antibodies restored the induction of cell death with Fab fragments of mAbs B-630 and B-E28. The regulation pathway(s) of CD95 mAb induced cell death was also investigated. Two regulation pathways of CD95 mAb induced cell death were observed. Firstly, the apoptosis induced by mAb B-G27 mAb was significantly reduced by mAbs B-D29 and B-G34, whereas the other CD95 mAbs had no effect. Secondly, the apoptosis induced by mAbs B-G30, B-l25 or B-E28 was significantly enhanced by mAbs ED29, B-G34 and B-K14.
P. 1.08.18
Possible mechanisms for induction of epoptosis after CDSmediated T-cell activation and its
reduction by CDS&costlmulation Y. Miiller’, E. Wierenga3, R. Mailhammer’, W. Wilmanns’~*. G. Jung’. ’ Medizinische Klinik III der Univetsibit, Miinchen, Germany, * GSF-Research Center for Environment and Health, M&when, German)! 3Laboratory for Ceil Biology; University of Amstenlam, The Netherlands Introduction: We have previously observed, that stimulation of resting human Tcells with high density anti-CD3 leads to proliferation (CD28minus-proliferation) and subsequent anergy and apoptosis. This negative signaling could be counteracted if cells were stimulated with low density submitogenic anti-CD3 together with CDP&antlbody (CD28plus-proliferation). Here we further explore these two modes of stimulation as a model for “productive” vs. “abortive” T- cell pmllleration. Materials and Methods: Apoptosis was determined by FACS analysis of omoidium iodide stained cells. lvmohokine oroduction bv auantitation of c-DNA iPdFl) and secreted proteins @LISA). Bcl-i, ~53 and bcl-&, were detected by western blot analysis. Reeults: The apoptosis preventing effect of CM&ostimulation is lost at low cell concentrations. Production of 11-2, 11-4, ylFN and TNFu varies considerablv between T-cells of diierent donors but is consistentlv hiaher durina CD26pl;s proliferation. No consistent difference in the expression if ~53 was observed. The expression of bcl-a, and bcl-2 seems to be elevated during CD26plus-proliferation. Concluolon: Apoptosis induced after (CDPBminus)-Tcell pmliieration (see definition above) is reduced in the presence of CD28-costlmulation, possibly due to increased lymphoklne production and bcl-XLexpression.
P.1.08.17
Regulation of thymocyte apoptosis by CD3/TCR complex, zinc and glucocorticoids in old mice: Implications for thymic lnvolutlon
M. Pmvinciali, G. Di Stefano, S. Stronati. lmmunologv Center INRCA Gerontology Research Department, Ancona, Italy The involution and atrophy of the thymic gland arising during aging has been associated with a reduction of the proportion and number of CD4+CD8+doublepositive (DP) thymccytes. Apoptosis represents the main mechanism involved in the regulation of the intrathymic deletion of DP thyrnocytes. Aim of this work was to evaluate whether the decline with aging in the fraction of DP thymocytes is associated with an alteration in the responsiveness of thymocytes to factors regulating the apoptotic cell death, such as antibodies to CD3JTcell receptor complex, zinc and dexamethasone (DEX). Balb/c mice were used at the age of 2 mo (young), 21-22 mo (old), and 24-26 mo (very-old). Thymocytes from these mice were incubated overnight with anti CD3 monoclonal antibody (8 &ml), Zn2+ (150 PM). or DEX (IO-’ M) and then analysed for the number of apoptotic nuclei, the cell cycle phase, and the percentage of CD4+CD8+ cells by flow cytometty. A significant decrease of either the total number and the proportion of CD4+Co8+ DP thymocytes was present in old and vety-old mice. Antibodies to CD3 antigen induced apoptosis of thymocytes from both young and old mice. The stimulation of CD3/TCR complex was more effective in giving apoptosis in old than young mice. An impairment of the inhibiting effect of zinc on either apoptosis induced by seturn deprivation, anti CD3 antibodies, or DEX, was found in old ages and in particular in vely-old mice. A reduced DEX-induced apoptosis was also present in old age: this effect was more evident in very-old than in old mice. The simultaneous administration of anti CD3 antibodies and DEX did not modify the apoptosis induced by each factor alone in both young and old mice. The boosting of apoptosis by DEX in aged mice required protein synthesis being blocked through cycloeximide. The effect of zinc and DEX on apoptosis was
23 June 1997 - Poster presentations
exerted on thymocytes in a specific cell cycle phase, i.e., on GO/G1phase cells.
Either anti CD3 antibodies. zinc and DEX regulated apoptosis by modulating the pmportlon of DP thymocyies. The results demonstrate an altered responsiveness of thymocytes from old and very-old mice to CD3/TCR complex, zinc and glucocorticoids suggesting that the age-related reduction in the number of DP thymocytes may be due, at least in part, to an increased apoptosis of this population occurring with increasing age.
1.. P 1 08 .18 ( Stimulation of CD40 in human bladder carcinoma cells inhibits anti-F&APO-l
induced apoptosis
G. JBnsson, E. Jakobson, E. Gelius, P. Bjarck, S. Paulie. Dept. ofirnrnuno/~ Stockholm Univarsi& S- 106 9 1 Stockholm, Sweden CD46 is a cell surface receptor expressed on B lymphocytes, certain eplthelial carcinomas, bone-marrow derived dendritic calls and on follicular dendritic cells. CD40 mediates a strong gmwth stimulatory signal in B cells and triggeling of the receptor prevents germinal centre B cells from undergoing apoptosis. We were interested to see whether CD40 could prevent apoptosis induced via the Fas/APO-1 antigen. Here we report that ligation of CD40 on human bladder carcinoma cells with antibodies or with soluble construct of the CD40 ligand inhibited cell death induced via the Fas/APO-I antigen. Protection appeared to occur independent of bcl-2 upregulation, since rescue could be obtained by adding CD40 agonists immediately before the anti-Fas/APO-I antibodies. Two of the cell lines were found to be inherently resistant to apoptosis in spite of a similar expression of the Fas/APO-1 antigen on these cells aa on the sensitive lines. Preliminary data show that these resistant cell lines also express the Fas/APO-1 ligand. The results show that CD40 stimulation may protect bladder carcinoma cells from apoptcsis actively induced via Fas/APO-1 and by expressing the Fas/APO-I ligand these cells could escape immune response.
P.1.08.19
Apoptosis of T cdl8 in the first trimester human troohoblast durina abortion ~roc888
M. Jerzak ‘, J. Kotarski *, K. Us-Lachowicz 3, M. Kasplzycka 3, A. Got&l 3. ’ LIept Clin Immunolo9y, Univ School of Med., Lublin, Poland, * Uepr Gyn Surg. Univ School of Med. L&in, pdsntf, 3Dept C/in Immunol~ Transplantation lnstibte, Univ. School of Med. Warsew,Poland Introduction: Apoptosis has been proposed as a mechanism for maintaining the tolerance in the immune system. Induction of the apoptotic cell death is also a possible outcome of the lymphocyte activation. Recent data suggest that human foetal membranes express Fas (CD95), a cell surface receptor that mediates apoptosis. Expression of Fas L by human trophoblast has been also proposed as a mechanism providing protection against the &tic action of decidual immune cells. In this sense, reduction of Fas or Fas L function can be associated with pregnancy 1089. MaterlaIr and Methods: We studied apoptosis of T cells isolated from the first trimester trophoblast in I2 women after spontaneous abortion. We used gel electmphomsis to detect DNA fragmentation, which is associated with apoptosis. Cells undergoing DNA fragmentation were also identified by DNA content analysis using flow cytometry. This method was based on the accumulation of ethanol-fixed apoptotlc cells in the sub-GdGl peak of the DNA content histogram as a msult of loss of DNA fragments out of the cells and because of a reduced DNA “stainability” by propidium iodide (PI). Results: We did not detect apoptosis by the “ladder” technique. However, apoptotic index (percentage positive/total cells) ranged from 2 to 18% using Row cytometry. Conclusion: Trophoblast cells usually fail to stimulate alloantigen-specific T cells, but they may express non-classical MHC alloantigens to which mothers produce IgG alloantibody and that requires T helper cell activation. Minimal apoptosis of T cells in the human tmphoblast might have been a defence mechanism against rejection of the foetal allograff by maternal immune system.
P.1.08.20
Apoptosls in the thymus of mice during the growth of syngeneic transplantable tumor
E.P. Kisseleva, R.P. Ogurtsov, A.N. Suvomv. lnstifute for Experimental Medicine, St. F@fe&urg, Russia Intmductfon: Apoptosis (A) of immature autoreactive thymocytes has been proposed as one of the mechanisms of negative selection associated with T cell differentiation. Studies of the thymic involution in tumor-beating mice have been mostly limited to the h&pathologic level and the intrathymlc development of A remained undefined. The aim of the study was to evaluate the rate of A in thymocytes during the growth of mtine syngeneic tumor. Materials and Msthodo:CBHA mice were S.C. inoculated with IO5 cells of weakly immunogenic solid hepatoma 22a. At weekly intervals thymuses
23 June 1997 - Poster presentations
were investigated histdogically in paraffin sections. DNA fragmentation was measured by gel electrophoresis and diphenylamine test (DFA). The number of apoptotic cells was calculated using microscopy in cells stained with a mixture of acddine orang&thidium bromide. Rwuftm Starting from the third week after tumor inoculation the progressive thymic involution occured. Histological studies revealed striking delymphatfxation of thymlc cortex, reduced number of mkozes in the cortex. The number of pyknoses was increased two-three times in the cortex but stfll remained under the same low rate in tumor-bearers as well as in controls. Therefore we could not reveal any difference in apoptotic cell numbers either by staining with fluorescent dyes or by DNA fragmentation in gel electrophoresis. In order to study the difference between thymocytes of tumor-bearing and control mice, cells were incubated in vftro during 2 h at 3X in medium containing 10% of fetal calf serum. The development of spontaneous A in thymccytes from tumor bearers was two times higher than in control animals as evaluated by fluorescent test and showed more intensive “ladder” development by electrophoresis. This difference was evident from the first week after tumor inoculation before the signes of involution appeared. After the 24 h incubation in vftro the spontaneous development of A was also higher in thymes from tumor-bearers as registered by DFA-test. Oppositely to the previous two, this test did not reveal the early changes and showed the enhancement of A only at the stage of developing thymic involution. Conduslon: This was the ffrst atempt to evaluate A in the thymus during the growth of syngeneic tumor. Being a chronic process the tumor development did not result in a massive increase of A and showed only slight rise of the number of pyknotic cells by histological studies. Three other tests were not enough sensitive to show any difference. Incubation of thymocytes in vitro allowed to register the enhanced ability of thymocytes from tumor-bearers for the development of spontaneous A. It happened at the early stage of tumor development and became more prominent later. Enhanced readiness to A may reflect the changes in thymocyte differentiation induced by intrathymic microenvironment in response to antigenic stimuli from increased tumor burden.
P.1.08.21
All forms of TGF-P are co-stimulatory to rat T-tymphocytes by increasing viability and preventing apoptosis
A. Schidtt I, H.-O. Sj6gmn ‘, M. Lfndvall 1.2.’ The Wallenberg Laboratory, Depatiment of Tumor lmmunolo~ University of Lund, Lund, &eden, 2Astra Draco AB, Lund, Sweden Introductton: TGF-p is a growth factor with known immuno-suppressive effect on lymphocytes. We and others have found the opposite effect of TGF-p. In our system TGF-p is increasing the prolfferatfve response of rat T-lymphocytes to Staphylococcal enterotoxfn A, but only if a certain amount of monocytes is added. In this study we compared the three isoforms TGF+l, TGF-fi2 and TGF-,93 in their ability to costimulate, affect apoptosis and viability of T-lymphocytes. Material and Methods: Wlstar/Furth rat spleen lymphocytes stimulated with Staphylccoccal enterotoxin A, TGF-@I (natural human), TGF-,92 and TGF-B3 (both recombinant human). Apoptotic cells am revealed by positive Annexin-V binding and negative ethidiumbromide binding. Resuh The addition of all three isofonns of TGF-,3 to SEA stimulated T-lymphocytes increase the proliferative response almost 10 times. An increase of SEA concentration is increasing the proliferation but also the amount of apoptotic cells. The addition of all isoforms of TGF-fi decrease the amount of apoptotic cells caused by SEA. TGF-fi2 and TGF;BB am reaching both these responses wfth one tenth of the TGF-j31 concentration. Antibodies neutrilizlng the cytokines IFN-x and TNFa am both decreasing the amount of apoptotic cells both with SEA alone and with SEA and TGF-pl in proliferation. Conclusion:All three isofomts of TGF-6 acts co-stimufatory on rat T-lymphocytes accompanied by a high viability and low amount of apoptotic cells. TGF-BP and TGF$3 am almost IO-fold mom potent than TGF-Bl. TGF-fi acts through an IFN-y and TNFa independant pathway when acting cc-stimulatory and anti-apoptotic.
IP.1.08.22
Intravenous prepamtions of normal IgG (IVlg) induces apoptosis in human leukemic cells: A role for Fas and a&ration of caboases
N. Prasad ‘, N. Bervas ’ , G. Papoff 2, G. Rubstti *, M.D. Kazatchkfne ‘, S.V. Kaved ’ 1INSERM lJ430, Hdpital Broussais, Paris, France, 2Mtuto di Biolqia Cellulare, CNR, Rome, Italy Introductfon: Therapeutic preparations of pooled normal immunoglobulin for intravenous use (IVlg) is beneficial in several autoimmune diseases and lymphoproliferatiie disorders. IVlg exhibits anti-proliferative effect on cultured T lymphocytes. This study was aimed at gaining further insight into the observed anti-proliferative effect of IVlg.
Regulation of apoptosis
13
Baterlalr and Mathode:Human leukemic cell lines CEM (T cells), Raji (B cells) and MM6 (monocytes) were used. Apoptosis was detected by dye exclusion assays, by analysis of DNA fragmentation, and by analysis of Annexin V binding to phosphotkfylserines on apoptotic cells. To test any role for Fas, supematants from fibroblasts transfected wkh soluble Fas clones which have been shown to block Fas-induced apoptosis and Fas sensitive cell line HUT78 and a Fas-resistant variant Bl were used. Specific inhibitors of macromolecular synthesis, actinomycfnD and cycloheximide and that of caspases (ICE-like proteases) wem employed. Results: The study cfeady indicated that IVlg actively induces apoptosis in these cells in vitro and is characterized by classical DNA ladder formation and by exposure of phosphotktyfserine on the cell surface. IVlg induces apoptosis in the presence of actinomycinD and cycloheximide, suggesting that no new gene transcription or protein synthesis is required for IVlgmediated apoptosis. In CEM T cells, IVlg-induced apoptosis was markedly inhibited by supematants containing soluble Fas molecules as compared to the negative contmf. The extent of IVlg-mediated apoptosis was significantfy lower in HUT 7651 Fasresistant cells compared to Fas sensitive ceils. Further, specific peptide inhibitors of caspases, ICE and YAMA, modestly inhibited IVlg-induced apoptosis in CEM T cells. In addition, specific cleavage of one of the substrates of YAMA, Poly ADP-Ribcse Polymerase (PARP) into a 86 kDa signature apoptotfc fragment was observed upon IVlg treatment. Conclusion: Our data indicate that therapeutic preparations of pooled normal immunoglobulins (IVlg) induce apoptosis in human leukemic cell lines and further suggests a role for Fas and for the activation of ICE-like proteases in IVlg-induced apoptosis. P.1.08.23
a-CD40 Ab treatment prevente in a concentration dependent manner the BCR-ligation induced apoptosis of a human folllcular fymphoma cell line of mature phenotype
M. Eray ‘.‘, A. Ripatti’, L.C. Andersson 1,4,M. Kaartinen*, J. Pelkonen3. ’ Department of Pathology, Haartman Institute, University of Helsinki, Finland, * Deparimenf of Bactedolog): Haartman Institute, University of Helsinki, Finland, ‘Department of Clinical Microbiology Univer&y of Kuopio, Finland, 4Institute for Oncokqy and Pathology, Karolinska Institute, Sweden An unique human follicular lymphoma line (HF-1.3.4) which is easily induced to apoptosis by cross-linking its smlg with relevant monoclonal or polyclonal antibodies (Eray et al. Int. lmmunofogy 6: 1617) was studied. a-CD40 antibody was able to prevent the (Y-Kinduced apoptosis of HF-1.3.4 cells completely at a concentration of 800 ndml. A partfal prevention of apoptosis was seen in ab concentrations as low as 100 @ml. The expression of several cytokines and cytokine receptors (112, 114, 115, 116, ILlO, IL13, INF-y, TNFa, ll2RB and 112&x) was measured during apop tosis induction and prevention through CD40 in HF-1.3.4 cells. The expression of the same cytokines and receptors, in similarly treated BCR-ligation induced apoptosis msistent control lymphoma cells was also studied. For all measumments a quantitative RT-PCR-assays utillsing plasmid based internal standards was used. The HF-1.3.4 cells express in excess Bcl-2 oncoprotein. Its expression was followed during apoptosis induction in untreated and a-CD40 treated HF-1.3.4 cells. Results am discussed in detail.
P.l.08.24
Expression of 8220 on activated T cell blast8 precedes apoptosis
T. Renno, A. Attinger, M. Hahne ‘, J. Tschopp ’ , H.R. MacDonald. From Luoivig institute for Cancer Research, Lausanne Branch, Epalinges, Switzerland, ’ lnstituta of Biochemistr)! University of Lausanne, Epalinges, Switzerland Superantigens am bacterial or viral products that polyclonally activate T cells beating certain TCR B chain variable elements. For instance, Vfi8+ T cells proliferate in response to staphylococcal entemtoxin B (SEB) in vivo then undergo Fas- or TNF-mediated apoptosis. We have recently shown that apoptotic SEB-reactive T cells express the B cell marker 8220 and other markers. Hem we report the identification of a novel subset of B220+ T cell blasts that am the precursors of these apoptotic cells in SEB immunized mice. We also show that the B220 molecule expressed by these T cell blasts’is biochemically similar to that exomssed on the surface of CD4-CD8- double neoative (DN) cells that accumulate in the lymphoid organs of the Fas-L-defective gld mlce,‘but Is distinct from that displayed on the surface of El cells. Collectively, our data support a model whereby upon activation, T cells upregulate 8220 before undergoing aoootosis.
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