- Email: [email protected]
HIV-1 gp120 induces CD4 association with several molecules on the T cell surface and modulates T cell interaction with endothelium and homing
Monday, 23 June 1997 - Oral presentations
AIDS
follows a decline in effective parasite specific immunity. This immunosupression can be explained, in part, by the polydonal activation of B lymphocytes. lmmunogiobulin produced by such activated B cells is directed against parasite antigens and a variety of unrelated (including setf) antigens. Such autoantibody activity could have potential long term pathological consequences for the host and thus any immune recognition events leading to the production of such autoantibody would have to be rigidly controlled and quickly suppressed. Evidence shows that in most circumstances, apoptosis serves a biologically meaningful, homeostatic function. Thus, the role of apoptosis during a malaria infection was investigated. Materialsand Methods: Plasmodiumchabaudi chabaudi (AS strain) was maintained and used as a cloned line in CBA/Ca mice. Mice were infected with 5 x l@ Rc. chabaudi AS infected cells or sham infected to be used as controls. Parasitaemia was monitored by light microscopy of air dried, methanol fixed, thin tail-blood smears stained with 10% Giemsa’s stain. When infected with this number of parasites naive mice produce an initial acute primary patasitaemia which reaches its peak levels on day 8-10, drops rapidly over the next few days and is partially resolved by day 18-l 8. After a short period of low grade or subpatent infection, the parasitaemia recrudesces producing a second major peak of parasitaemia. Plasma taken during the course of such an infection was used to analyse the binding capacity of antibody to the surface of infected and non-infected red blood cells by FACS. On day 15 of infection groups of 5 infected and control mice were killed by cervical dislocation and the spleens removed, formalin-fixed and paraffin embedded for tissue sectioning. Detection of apoptosis was performed by the TUNEL assay. Reeultr: Our FACS assay demonstrated an increase in antibody activity from day 8 to day 12 which was then maintained until day 15 but which had been reduced to low levels by day 18 post-infection. The number of apoptotic ceils in the spleens from infected mice was 30 times more than in the spleen from non-infected mice and corresponds to T cells and macrophages. Cor~~luslon: In our model we observe an increase of apoptosis in infected mice at the same time that antibody responses start to decrease. Thus, our results may suggest that apoptosis could be one possible determinant of immunosupression in malaria infections. We are using the PC. chabaudiAS model to investigate the cellular and molecular bases of this phenomena such as main T cell subset undergoing apoptosis as well the pathway used in the system (~53 dependent or independent pathway).
Room E/F
14:00-16:OO
0.4.06
0.4.06.1
AIDS
HIV-1 gpl20 induces CD4 association with several mole&es on the T ceil surface and modulates T ceil interaction with endotheiium and homing
M. Bragardo, D. Buonfiglio, M.J. Feito, V. Redoglia, G. Garbatino, S. Bonissoni, U. Dianzani. University of Torino at Novam, /ta/y
Introduction:Migration of lymphocytes is crucial for the immune response and is determined by a complex network of adhesive interactions in which several receptors operate simultaneously as lymphocyte homing traffic signals. This study analyzes the effects of the gpl2O/CD4 binding on the aggregation of several adhesion molecules on the T cell surface and evaluates the effect on T cell interaction with vascular endothelium (VE) both in vitroand in viw. Materials and Methoda:Intermolecular associations were evaluated by co-capping and co-immunoprecipitation. In vitro binding assays were perfoned on h;mbilical vein endothelial cells using 51Cr-labelled CD4+ T cells. A sfafic binding assay evaluated stable interactions mediated by integrtns, and a dynamic binding assay, evaluated weak interactions mediated bv constitutivelv active molecules. such-as selectins and CD38. In viw homing assays weri performed usins 51Cr-labelled2D4 cells, a CD4-CD8- mouse T cell clone transfected with the human CD4. Reeutt% We found that CD4+ Tcell treatment with gp120 specifically induces CD4 association with several surface molecules. GplPOs derived from different HIV-l strains induce different association patterns, as shown by both co-capping and immunoprecipitation. Since several of these molecules were involved in lymphocyte interaction with VE. we examined the possibility that gp12O/CD4 binding alters CD4+ T ceil interaction witi VE in vitro and in viw. Gpl20 increased dynamic binding and inhibited static binding to VE of peripheral blood CD4+ T cells and SUPT-1 cells. Binding inhibition with mAbs showed that the gp120 effect on @mamicbinding involved CD38, CD31, and CD48d, whereas the effect on static binding involved CD31, CDlla, and CD49d. In viw experiments showed that treatment of 2D4 cells with gpl20 increased their
29
homing into the spleen, gut, and mesenteric lymph nodes, whereas it decreased homing into peripheral lymph nodes. Conclusions: Alteration of lymphocyte homing may contribute to immune deficiency in HIV+ patients by decreasing the probability of encounter between antigens and lymphocytes and inhibiting the spread of effector lymphocytes into tissues.
0.4.06.2
Both perforin- and Fas-dependent cytotoxicities are mediated by a human anti-HIV specific CD8+ T ceil line
S. Garcia’, M. F6vrier2, G. Dadaglio’, H. Lecoeur I, Y. Rivi&e2, M.-L. Gougeon ’ ’ Unit6d’Onco/ogie Vi&e, lnstitut Pasteur Pads, France, 2Unit6 de Vitvkqie ef d’lmmunologie Cellulaire, InsMut Pasteur Paris, France Introduction: Two mechanisms of CTL-mediated killina have been identified: the first one involves pore-forming perforin and gran&es molecules both released towards targets cells, and the second one the transduction of a death signal upon the ligation of the Fas receptor on target cells by the Fas ligand on effector ceils. Although both mechanisms are initiated upon TCR ligation and lead ultimately to the apoptosis of target cells, they display different characteristics. The perforin-based cell death may rather contribute to specific host defense, while Fas-based cell death would rather participate to the regulation of immune response by eliminating Fas+ activated cells. In spite of this functional duality, both mechanisms were shown to coexist within cytotoxic murine CD8+ T cells. Nevertheless, until now, there is no evidence that both perforin and Fasdependent cytotoxicities can be mediated simultaneously by human CTL. In the present study we tested the ability of a specific anti-Nef CD8+ CTL line, derived from an HIV-infected donor, to kill Fas+ compliant target cells. Materialand Method: An anti-Nef peptide CD& T cell line. named Pl, was obtained after specific stimulations oi PBMC from an HIV-infected don& by autologous EBV blasts coated with a Nef peptide. The specific cvtotoxicitv of this T ceil line was tested using a 5’Cr relesase assay. The cytotox& dir&ed to Fas+ compliant ceils was assessed using the human Fas+ T cell line Jurkat as target cells. EGTAfMg2+was used as Ca2+chelator during cytotoxicity assay. The Fas and perfotin expressions were analysed by flowcytometry, and Fas-L expression was analysed by Western Blot. Reautts: We first showed that Jurkat Fas+ hi cells were killed by dlls Fas-L+ murtne effector cells, while Molt-4 Fas+‘Ocells were not killed, which confirms the previously reported sensitivity of Jurkat cells to Fas-mediated lysis. Then, we showed that Pl cells killed not only Nef peptide coated EBV target cells, but also J&at cells, while mdt-4 cells were resistant to Pl induced lysis. Moreover, the lysis directed to the Nef peptide was inhibited by EGTA/Mg”‘, while the lysis directed to Jurkat cells was resistant to this treatment. Finally, this dual cytotoxicity was correlated with the dual expression of perfotin and Fas-L by Pi cells. Altogether, these results indicate the coexistence of perforin and Fas-mediated mechanisms within a specific CD8+ CTL line. Conclusions: We showed for the first time that human specific CD8+ CTL, generated during an in vivo infection, were able to induce both perforin and Fas-mediated ceil death. This finding suggests that both mechanims could be potentially efficient and concomitent in vivo. Since these mechanisms are thought to play a different role, involved at different phases during the course of immune response, each mechanim should be highly regulated to occur adequately. A dysruption of this control in favor of Fas-mediated pathway could lead to oatholooical situations. In oarticular. the constant activation of saecific CTL byantigens during chronic HI’V infection could induce a continuous Fas-L expression on these cells which could subsequently be responsible for the bystander lysis of uninfected Fas+ compliant cells. -
10.4.06.3 1 Adaptations of CTL responses to drug-induced variants of HIV-1 reverse transcriotase J. Duntze’, G. Haas’, A. Hosmalin ‘, R.Grimm5, M. Magierowska ‘, G. Jung4, K.H. Wiesmiiller4, C. Katlama3, H. Agut’, P. Debti l, B. Autran 1 ’ lmmunologie Cell., URA CNRS625, 2Dept of Virol~ 3L&pt Mal. Infect, Hop. PiM-Salp&i&e, Paris, France, 4Dept. Organ. Chem., TUbingen, sHewlelt-Packard GmbH, Waldbronn, Germany
Introduction:Mutations related to the antiretroviral drugs AZT, ddC, ddl or 3TC mostly occur at the site of enzymatic activity in the N-terminal half of HIV-l reverse transcriptase (RT). The aim of the study was to determine the immunogenicity and therapeutic potential of CTL epitopes in this part of the molecule containing anti-retroviral drug-induced viral mutations. Mods: The evolution of HIV-specific CTL responses was followed over 4 years in the trench lmmunoco cohort of 47 patients (CD4+ counts: 1050-15O/mm3). The recognition of RT vaccinia virus encoded minigenes and specific RT peptides was tested after stimulation with autologous irradiated PHA blasts or specific peptides. Synthetic peptides containing viral mutations known to induce resistance to antiretroviral drugs as AZr, ddC and 3TC were designed according to MHC motifs for HLA A2 and A3. MHC-peptlde interactions were
AIDS
follows a decline in effective parasite specific immunity. This immunosupression can be explained, in part, by the polydonal activation of B lymphocytes. lmmunogiobulin produced by such activated B cells is directed against parasite antigens and a variety of unrelated (including setf) antigens. Such autoantibody activity could have potential long term pathological consequences for the host and thus any immune recognition events leading to the production of such autoantibody would have to be rigidly controlled and quickly suppressed. Evidence shows that in most circumstances, apoptosis serves a biologically meaningful, homeostatic function. Thus, the role of apoptosis during a malaria infection was investigated. Materialsand Methods: Plasmodiumchabaudi chabaudi (AS strain) was maintained and used as a cloned line in CBA/Ca mice. Mice were infected with 5 x l@ Rc. chabaudi AS infected cells or sham infected to be used as controls. Parasitaemia was monitored by light microscopy of air dried, methanol fixed, thin tail-blood smears stained with 10% Giemsa’s stain. When infected with this number of parasites naive mice produce an initial acute primary patasitaemia which reaches its peak levels on day 8-10, drops rapidly over the next few days and is partially resolved by day 18-l 8. After a short period of low grade or subpatent infection, the parasitaemia recrudesces producing a second major peak of parasitaemia. Plasma taken during the course of such an infection was used to analyse the binding capacity of antibody to the surface of infected and non-infected red blood cells by FACS. On day 15 of infection groups of 5 infected and control mice were killed by cervical dislocation and the spleens removed, formalin-fixed and paraffin embedded for tissue sectioning. Detection of apoptosis was performed by the TUNEL assay. Reeultr: Our FACS assay demonstrated an increase in antibody activity from day 8 to day 12 which was then maintained until day 15 but which had been reduced to low levels by day 18 post-infection. The number of apoptotic ceils in the spleens from infected mice was 30 times more than in the spleen from non-infected mice and corresponds to T cells and macrophages. Cor~~luslon: In our model we observe an increase of apoptosis in infected mice at the same time that antibody responses start to decrease. Thus, our results may suggest that apoptosis could be one possible determinant of immunosupression in malaria infections. We are using the PC. chabaudiAS model to investigate the cellular and molecular bases of this phenomena such as main T cell subset undergoing apoptosis as well the pathway used in the system (~53 dependent or independent pathway).
Room E/F
14:00-16:OO
0.4.06
0.4.06.1
AIDS
HIV-1 gpl20 induces CD4 association with several mole&es on the T ceil surface and modulates T ceil interaction with endotheiium and homing
M. Bragardo, D. Buonfiglio, M.J. Feito, V. Redoglia, G. Garbatino, S. Bonissoni, U. Dianzani. University of Torino at Novam, /ta/y
Introduction:Migration of lymphocytes is crucial for the immune response and is determined by a complex network of adhesive interactions in which several receptors operate simultaneously as lymphocyte homing traffic signals. This study analyzes the effects of the gpl2O/CD4 binding on the aggregation of several adhesion molecules on the T cell surface and evaluates the effect on T cell interaction with vascular endothelium (VE) both in vitroand in viw. Materials and Methoda:Intermolecular associations were evaluated by co-capping and co-immunoprecipitation. In vitro binding assays were perfoned on h;mbilical vein endothelial cells using 51Cr-labelled CD4+ T cells. A sfafic binding assay evaluated stable interactions mediated by integrtns, and a dynamic binding assay, evaluated weak interactions mediated bv constitutivelv active molecules. such-as selectins and CD38. In viw homing assays weri performed usins 51Cr-labelled2D4 cells, a CD4-CD8- mouse T cell clone transfected with the human CD4. Reeutt% We found that CD4+ Tcell treatment with gp120 specifically induces CD4 association with several surface molecules. GplPOs derived from different HIV-l strains induce different association patterns, as shown by both co-capping and immunoprecipitation. Since several of these molecules were involved in lymphocyte interaction with VE. we examined the possibility that gp12O/CD4 binding alters CD4+ T ceil interaction witi VE in vitro and in viw. Gpl20 increased dynamic binding and inhibited static binding to VE of peripheral blood CD4+ T cells and SUPT-1 cells. Binding inhibition with mAbs showed that the gp120 effect on @mamicbinding involved CD38, CD31, and CD48d, whereas the effect on static binding involved CD31, CDlla, and CD49d. In viw experiments showed that treatment of 2D4 cells with gpl20 increased their
29
homing into the spleen, gut, and mesenteric lymph nodes, whereas it decreased homing into peripheral lymph nodes. Conclusions: Alteration of lymphocyte homing may contribute to immune deficiency in HIV+ patients by decreasing the probability of encounter between antigens and lymphocytes and inhibiting the spread of effector lymphocytes into tissues.
0.4.06.2
Both perforin- and Fas-dependent cytotoxicities are mediated by a human anti-HIV specific CD8+ T ceil line
S. Garcia’, M. F6vrier2, G. Dadaglio’, H. Lecoeur I, Y. Rivi&e2, M.-L. Gougeon ’ ’ Unit6d’Onco/ogie Vi&e, lnstitut Pasteur Pads, France, 2Unit6 de Vitvkqie ef d’lmmunologie Cellulaire, InsMut Pasteur Paris, France Introduction: Two mechanisms of CTL-mediated killina have been identified: the first one involves pore-forming perforin and gran&es molecules both released towards targets cells, and the second one the transduction of a death signal upon the ligation of the Fas receptor on target cells by the Fas ligand on effector ceils. Although both mechanisms are initiated upon TCR ligation and lead ultimately to the apoptosis of target cells, they display different characteristics. The perforin-based cell death may rather contribute to specific host defense, while Fas-based cell death would rather participate to the regulation of immune response by eliminating Fas+ activated cells. In spite of this functional duality, both mechanisms were shown to coexist within cytotoxic murine CD8+ T cells. Nevertheless, until now, there is no evidence that both perforin and Fasdependent cytotoxicities can be mediated simultaneously by human CTL. In the present study we tested the ability of a specific anti-Nef CD8+ CTL line, derived from an HIV-infected donor, to kill Fas+ compliant target cells. Materialand Method: An anti-Nef peptide CD& T cell line. named Pl, was obtained after specific stimulations oi PBMC from an HIV-infected don& by autologous EBV blasts coated with a Nef peptide. The specific cvtotoxicitv of this T ceil line was tested using a 5’Cr relesase assay. The cytotox& dir&ed to Fas+ compliant ceils was assessed using the human Fas+ T cell line Jurkat as target cells. EGTAfMg2+was used as Ca2+chelator during cytotoxicity assay. The Fas and perfotin expressions were analysed by flowcytometry, and Fas-L expression was analysed by Western Blot. Reautts: We first showed that Jurkat Fas+ hi cells were killed by dlls Fas-L+ murtne effector cells, while Molt-4 Fas+‘Ocells were not killed, which confirms the previously reported sensitivity of Jurkat cells to Fas-mediated lysis. Then, we showed that Pl cells killed not only Nef peptide coated EBV target cells, but also J&at cells, while mdt-4 cells were resistant to Pl induced lysis. Moreover, the lysis directed to the Nef peptide was inhibited by EGTA/Mg”‘, while the lysis directed to Jurkat cells was resistant to this treatment. Finally, this dual cytotoxicity was correlated with the dual expression of perfotin and Fas-L by Pi cells. Altogether, these results indicate the coexistence of perforin and Fas-mediated mechanisms within a specific CD8+ CTL line. Conclusions: We showed for the first time that human specific CD8+ CTL, generated during an in vivo infection, were able to induce both perforin and Fas-mediated ceil death. This finding suggests that both mechanims could be potentially efficient and concomitent in vivo. Since these mechanisms are thought to play a different role, involved at different phases during the course of immune response, each mechanim should be highly regulated to occur adequately. A dysruption of this control in favor of Fas-mediated pathway could lead to oatholooical situations. In oarticular. the constant activation of saecific CTL byantigens during chronic HI’V infection could induce a continuous Fas-L expression on these cells which could subsequently be responsible for the bystander lysis of uninfected Fas+ compliant cells. -
10.4.06.3 1 Adaptations of CTL responses to drug-induced variants of HIV-1 reverse transcriotase J. Duntze’, G. Haas’, A. Hosmalin ‘, R.Grimm5, M. Magierowska ‘, G. Jung4, K.H. Wiesmiiller4, C. Katlama3, H. Agut’, P. Debti l, B. Autran 1 ’ lmmunologie Cell., URA CNRS625, 2Dept of Virol~ 3L&pt Mal. Infect, Hop. PiM-Salp&i&e, Paris, France, 4Dept. Organ. Chem., TUbingen, sHewlelt-Packard GmbH, Waldbronn, Germany
Introduction:Mutations related to the antiretroviral drugs AZT, ddC, ddl or 3TC mostly occur at the site of enzymatic activity in the N-terminal half of HIV-l reverse transcriptase (RT). The aim of the study was to determine the immunogenicity and therapeutic potential of CTL epitopes in this part of the molecule containing anti-retroviral drug-induced viral mutations. Mods: The evolution of HIV-specific CTL responses was followed over 4 years in the trench lmmunoco cohort of 47 patients (CD4+ counts: 1050-15O/mm3). The recognition of RT vaccinia virus encoded minigenes and specific RT peptides was tested after stimulation with autologous irradiated PHA blasts or specific peptides. Synthetic peptides containing viral mutations known to induce resistance to antiretroviral drugs as AZr, ddC and 3TC were designed according to MHC motifs for HLA A2 and A3. MHC-peptlde interactions were