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Perturbations in gamma-IFN and IL-2 synthesis in HIV-infected patients: Correlations with disease progression and T cell apoptosis
134
AIDS
late the expression of other known activation antigens, i.e. CD25 CD36 and HLA-DR. Although after 4h-incubation with PHA an average of 2-3% cells were in S-phase as evaluated by propidium iodide staining, it is not currently known which percentage of CD69+ lymphocytes will ultimately progress to cell cycle or, alternatively, undergo activation-induced apoptosis. Preliminary data suggest that the eariy expression of CD69 is not predictive of the commitment to cell proliferation.
1P-4.06.1 5 ( Comparative analysis of flow cytometric methods for quantltatlng and phenotyping apoptotlc peripheral lymphocytes from HIV-Infected persons Her& Lecoeur, Marie-Use Gougeon. Unif&‘Onco/ogie Vim/e, DBparfemenf SIDA et R&vvirus, lnstitut Pasteur 28 rue du Dr. Roux, 75724 Paris Cadex 15,
23 June 1997 - Poster presentations
of 112~secretingT cells increased and was correlated to the intensity of the immunodeficiency, whereas susceptibility of alpha-TNF or gamma-IFN T cells to apoptosis was not associated with the stage of the disease. Conclusion: Our data suggest that the progression of HIV disease is associatsd with concomitant and opposite modifications in the production of two “Thl” cytokines, IL2 and gamma-IFN. Increased 96 of “Th2” ILCsecreting cells seems to be a minor and late phenomenon, which may be related to associated infections or hypersensitivity reactions. Furthermore, the decrease in the % of I@-secrstina T cells aooears to be closelv related to the maioration of T cell apoptosis. The reversion ‘of these abnorm&ties in patients under protease inhibitors therapy is actually investigated.
P.4.06.17
FCWCl3
Introduction: HIV infection is associated with early functionnal defects of the immune system, including increased apoptosis in Peripherical Blood Lymphocytes. This programmed cell death occurs in both CD4+ and CD6+ T cells from HIV-infected persons, and was shown to be a marker of disease progression. The precise detection, quantification, and phenotyping of apoptotic lymphocytes can be accessed by several cytofluotimetric techniques now available. However, some of them are not adapted to studies in PBLa from HIV-infected subjects. Msterlals end Methods: We have evaluated five cytofluorimetric methods of apoptosis quantification in 24 hour cultured PBLs from controls and HIVinfected subjects. These techniques detected distinct cellular alterations of the apoptosis process. The reduced DNA stainability has been detected either with the PI assay on isolated nuclei, or with the AO/EB dual staining method on entire and non-fixed cells. DNA fragmentation was detected following in sifu nick translation. Membrane alterations such as the increased permeability was evaluated following 7-AAD incorporation, and the loss of phosphatidylserine asymetry after Annexin-V fixation. Resuits: These methods proved to be reliable and gave statistically similar apoptosis quantitation on PBLs from healthy donors. However, in HIV-infected persons, discordant results were observed with PI and 7-AAD staining assays. We found that after ficoll isolation, PBLs from AIDS patients were enriched in crythrocytes and granulocytes. On one hand, granulccytes were found responsible for a bad apoptosis estimation with the PI assay and erythrccytes accountable for an underestimation rate of apoptosis in the 7-AAD assay. To prevent these interferences, we propose modifications which render these methods more suitable for me application to PBLs from HIV-infected patients. Applications to immuno phenotyping of surface antigens are also discussed. Conclusion: Altogether these observations underline that it is essential to assess critically the apoptosis quantitation methods with respect to their applicability to complex lymphoid populations such as those from AIDS patients.
1P.4.06.16 ( perturbations In gamma-IFN and IL-2 synthesis In HIV-Infected patients: Correlations wlth disease progression and T cell apoptosls E. Ledru I, S. Boullier’, R.Roue’, T. Debord’, M.L. Gougeon ‘. ’ Viral Oncology Unit, Pasteur Institute, Paris, France, * lnfectous and Tropical Diseases Department, Begin Military Hospital, 3 Man&, France Introduction: Studies of cytokine production by T cell clones and by peripheral blood mononuclear cells have suggested that Thl to Th2 switch could be an important feature in HIV disease pathogenesis. To assess this hypothesis, we investigated the cytokine production at single cell level in HIV-infected patients at different stages of the disease. Since it has been suggested that Thl cytokines could restore functional defects via T cells protection from apoptosis, we also analyzed in single cell assay relationships between cytokine synthesis and apoptosis. Methods: Flow cvtometdc evolution of cvtokine svnthesis (lL2.lL4. oammaIFN, alpha-TNF) ani apoptosis in differeni T cell subsets &as ~~rf&&d by triple staining of peripheral blood mononuclear cells after stimulation by PMA + ionomycine + PHA. 43 HIV-infected patients (before therapy with protsase inhibitors) and 22 healthy donors were analysed in a cross-sectional study. Results: (i) cyto/dne synthesis: We observed a decrease in the % of T cells secreting IL2 in HIV+ patients compared to controls. This decrease was correlated to the progression of the disease, estimated by the ex vivo % of CD4 cells. Furthermore, we reported a positive correlation between the progression of the disease and the % of gamma-IFN-secreting T cells. Few T cells were able to produce simultaneously gamma-IFN and 112.CD6+ T cells, which represent the majority of T cells in patients with advanced disease, were still able to synthetize large amounts of gamma-IFN but were low I@producers. There was also a slight decrease in alpha-TNF synthesis in HIV+ patients, whereas no increase in ihe % of lL4-prodicing cells-was observed, excepted. in some patients with AIDS. (ii) relationships between cytokine synthesis and apoptosis: In controls, susceptibility to apaptosis was lower in IliZ-secreting than in gamma-IFN or in alpha-TNF-secreting T cells. In HIV-infected patients, susceptibility to apoptosis
patterns of lymphocyte cytoklne productlon at the single cell level In HIV1 Infection
A.E. Sousa, R.M.M. Victorino. Cellular Immunology Unit, CEElPAh?iversify Hospital Santa Maria, Medicine 2. Lisbon, Portugal
Introduction:The imbalance of cytokines produced during HIV infection is a major factor contributing to the HIV induced dysrsgulation of the immune system and a promising area for immunointervention. Flow cytometric analysis of the cytokine production at the single cell level offers a new approach to a better definition of cytokine production patterns dutfng HIV infection. MaterialandMethods:Cytokine secretion by peripheral blood mononuclear cells from HIV1 infected patients and from healthy controls were analysed at the single cell level by flow cytometry (FACScan, BD) after a short time (4 h) culture in the presence of an acessoty cell independent stimulus (PMA plus lonomycin) and Brefeldin A. Analysis was done within a lymphogate and also within CD69+ cells and results were expressed as the percentage of cells producing IFNy, IIZ!, 114, IL10 and IFNy/lL4 simultaneously within distinct cell subsets (CD3+, CD4+, CDB+, CD56+, CD45RO+). Resufts: In our experimental condiions, the percentage of cytokine producing calls in healthy subjects varies within a very narrow range. In HIVl+ patients this range is much wider particularly in the group with t500 CD4+ cella/mm3. The percentage of IFNy+ cells is enhanced both in CD4+ and CD6+ cells and increases with progression of disease. The percentage of lL2+ cells is reduced. particularly in patients with ~500 CD4+cells/mm3.The percentage of lL4+ and ILiO+ cells is markedly increased in a subset of patients irrespective of their CD4+ level. THO cells (IFNy+ lL4+) are increased particularly in patients with CD4+ cells <500/mm3. The relevance of analysing cytokines in CD3+/CD69+ gated cells is documented in view of the bias due to the observed disturbances in cell activation. Conclusion: The alteration in the patterns of cytokine production in HIVl+ patients is not characterized by a simple shift from a THlTTH2 pattern. THO cells are expanded with disease progression as well as the cells producing individually IFNy, IL4 and ILlO; this is consistent with a “terminal” differentiation of peripheral blood lymphocytes in HIV1 infection. Supported by a grant from Praxis/JNICT, Lisbon, Portugal
P.4.06.16
Reduced sensltlvlty of CD8+ but not CD4+ T cells from HIV-infected patients to 11-2, IL-7 and IL-12: Implications for actlvatlon and apoptosis
J. Vingerhcets, E. Bisalinkumi, G. Penne, E. Bosmans’, B. Colebunders, L. Kestens, G. Vanham. Laboratory of Immunology, Institute of Tropical Medicine, Antwerpen, Belgium, ’ Ewogenetics, Tessenderio,Belgium Introduction: The production of regulatory cytokines during HIV infection and their potential immunomodulatory effects have been studied extensively. However, few studies were focused on whether the T cells from HIV-infected persons displayed an altered sensitivity to these cytokines. Therefore, we wanted to compare me responsiveness of CD4+ and CD6+ T cells from HIV-infected patients and HIV-neaative controls to 11-2. IL-7 and IL-12. We measured the resoonse to these csokines in PBMC cuhures with regard to T cell proliferation (with or without additional TcWCD3 triggering), upregulation of the activation marker CD25 induction of blasts, effects on apoptosis and production of IFNy, IL-4 and IL-lo. Cytokine receptor-chain expression and co-expression with CD26 was determined on resting T cells. hlethods:Proliferation was determined by [3H]Thy-incorporation. CD4+ or CD6+ T cells were analyzed separately using flow cytometry. For each sample, bright CD25 expression and blast formation (as parameters of activation) were detemined simultaneously with the measurement of apoptosis (by TUNEL assay or light scatter analysis). Cytokine production was determined by ELISA. Cytokine receptor expression on fresh cells was determined by flow cytometry. Resufts: The co-stimulaton effect of IL-7 and IL-12 on suboptimal TcWCD3 triggering was lowered in PBMc from patients. IFNy-production after stimulation with 11-12, alone or in combination with IL-2 or IL-7, was significantly lower in patient PBMC. IL-IO production after stimulation with IL-2 plus IL-12 tended to be higher, in patients as compared to controls. Activation of CD& T cells from
AIDS
late the expression of other known activation antigens, i.e. CD25 CD36 and HLA-DR. Although after 4h-incubation with PHA an average of 2-3% cells were in S-phase as evaluated by propidium iodide staining, it is not currently known which percentage of CD69+ lymphocytes will ultimately progress to cell cycle or, alternatively, undergo activation-induced apoptosis. Preliminary data suggest that the eariy expression of CD69 is not predictive of the commitment to cell proliferation.
1P-4.06.1 5 ( Comparative analysis of flow cytometric methods for quantltatlng and phenotyping apoptotlc peripheral lymphocytes from HIV-Infected persons Her& Lecoeur, Marie-Use Gougeon. Unif&‘Onco/ogie Vim/e, DBparfemenf SIDA et R&vvirus, lnstitut Pasteur 28 rue du Dr. Roux, 75724 Paris Cadex 15,
23 June 1997 - Poster presentations
of 112~secretingT cells increased and was correlated to the intensity of the immunodeficiency, whereas susceptibility of alpha-TNF or gamma-IFN T cells to apoptosis was not associated with the stage of the disease. Conclusion: Our data suggest that the progression of HIV disease is associatsd with concomitant and opposite modifications in the production of two “Thl” cytokines, IL2 and gamma-IFN. Increased 96 of “Th2” ILCsecreting cells seems to be a minor and late phenomenon, which may be related to associated infections or hypersensitivity reactions. Furthermore, the decrease in the % of I@-secrstina T cells aooears to be closelv related to the maioration of T cell apoptosis. The reversion ‘of these abnorm&ties in patients under protease inhibitors therapy is actually investigated.
P.4.06.17
FCWCl3
Introduction: HIV infection is associated with early functionnal defects of the immune system, including increased apoptosis in Peripherical Blood Lymphocytes. This programmed cell death occurs in both CD4+ and CD6+ T cells from HIV-infected persons, and was shown to be a marker of disease progression. The precise detection, quantification, and phenotyping of apoptotic lymphocytes can be accessed by several cytofluotimetric techniques now available. However, some of them are not adapted to studies in PBLa from HIV-infected subjects. Msterlals end Methods: We have evaluated five cytofluorimetric methods of apoptosis quantification in 24 hour cultured PBLs from controls and HIVinfected subjects. These techniques detected distinct cellular alterations of the apoptosis process. The reduced DNA stainability has been detected either with the PI assay on isolated nuclei, or with the AO/EB dual staining method on entire and non-fixed cells. DNA fragmentation was detected following in sifu nick translation. Membrane alterations such as the increased permeability was evaluated following 7-AAD incorporation, and the loss of phosphatidylserine asymetry after Annexin-V fixation. Resuits: These methods proved to be reliable and gave statistically similar apoptosis quantitation on PBLs from healthy donors. However, in HIV-infected persons, discordant results were observed with PI and 7-AAD staining assays. We found that after ficoll isolation, PBLs from AIDS patients were enriched in crythrocytes and granulocytes. On one hand, granulccytes were found responsible for a bad apoptosis estimation with the PI assay and erythrccytes accountable for an underestimation rate of apoptosis in the 7-AAD assay. To prevent these interferences, we propose modifications which render these methods more suitable for me application to PBLs from HIV-infected patients. Applications to immuno phenotyping of surface antigens are also discussed. Conclusion: Altogether these observations underline that it is essential to assess critically the apoptosis quantitation methods with respect to their applicability to complex lymphoid populations such as those from AIDS patients.
1P.4.06.16 ( perturbations In gamma-IFN and IL-2 synthesis In HIV-Infected patients: Correlations wlth disease progression and T cell apoptosls E. Ledru I, S. Boullier’, R.Roue’, T. Debord’, M.L. Gougeon ‘. ’ Viral Oncology Unit, Pasteur Institute, Paris, France, * lnfectous and Tropical Diseases Department, Begin Military Hospital, 3 Man&, France Introduction: Studies of cytokine production by T cell clones and by peripheral blood mononuclear cells have suggested that Thl to Th2 switch could be an important feature in HIV disease pathogenesis. To assess this hypothesis, we investigated the cytokine production at single cell level in HIV-infected patients at different stages of the disease. Since it has been suggested that Thl cytokines could restore functional defects via T cells protection from apoptosis, we also analyzed in single cell assay relationships between cytokine synthesis and apoptosis. Methods: Flow cvtometdc evolution of cvtokine svnthesis (lL2.lL4. oammaIFN, alpha-TNF) ani apoptosis in differeni T cell subsets &as ~~rf&&d by triple staining of peripheral blood mononuclear cells after stimulation by PMA + ionomycine + PHA. 43 HIV-infected patients (before therapy with protsase inhibitors) and 22 healthy donors were analysed in a cross-sectional study. Results: (i) cyto/dne synthesis: We observed a decrease in the % of T cells secreting IL2 in HIV+ patients compared to controls. This decrease was correlated to the progression of the disease, estimated by the ex vivo % of CD4 cells. Furthermore, we reported a positive correlation between the progression of the disease and the % of gamma-IFN-secreting T cells. Few T cells were able to produce simultaneously gamma-IFN and 112.CD6+ T cells, which represent the majority of T cells in patients with advanced disease, were still able to synthetize large amounts of gamma-IFN but were low I@producers. There was also a slight decrease in alpha-TNF synthesis in HIV+ patients, whereas no increase in ihe % of lL4-prodicing cells-was observed, excepted. in some patients with AIDS. (ii) relationships between cytokine synthesis and apoptosis: In controls, susceptibility to apaptosis was lower in IliZ-secreting than in gamma-IFN or in alpha-TNF-secreting T cells. In HIV-infected patients, susceptibility to apoptosis
patterns of lymphocyte cytoklne productlon at the single cell level In HIV1 Infection
A.E. Sousa, R.M.M. Victorino. Cellular Immunology Unit, CEElPAh?iversify Hospital Santa Maria, Medicine 2. Lisbon, Portugal
Introduction:The imbalance of cytokines produced during HIV infection is a major factor contributing to the HIV induced dysrsgulation of the immune system and a promising area for immunointervention. Flow cytometric analysis of the cytokine production at the single cell level offers a new approach to a better definition of cytokine production patterns dutfng HIV infection. MaterialandMethods:Cytokine secretion by peripheral blood mononuclear cells from HIV1 infected patients and from healthy controls were analysed at the single cell level by flow cytometry (FACScan, BD) after a short time (4 h) culture in the presence of an acessoty cell independent stimulus (PMA plus lonomycin) and Brefeldin A. Analysis was done within a lymphogate and also within CD69+ cells and results were expressed as the percentage of cells producing IFNy, IIZ!, 114, IL10 and IFNy/lL4 simultaneously within distinct cell subsets (CD3+, CD4+, CDB+, CD56+, CD45RO+). Resufts: In our experimental condiions, the percentage of cytokine producing calls in healthy subjects varies within a very narrow range. In HIVl+ patients this range is much wider particularly in the group with t500 CD4+ cella/mm3. The percentage of IFNy+ cells is enhanced both in CD4+ and CD6+ cells and increases with progression of disease. The percentage of lL2+ cells is reduced. particularly in patients with ~500 CD4+cells/mm3.The percentage of lL4+ and ILiO+ cells is markedly increased in a subset of patients irrespective of their CD4+ level. THO cells (IFNy+ lL4+) are increased particularly in patients with CD4+ cells <500/mm3. The relevance of analysing cytokines in CD3+/CD69+ gated cells is documented in view of the bias due to the observed disturbances in cell activation. Conclusion: The alteration in the patterns of cytokine production in HIVl+ patients is not characterized by a simple shift from a THlTTH2 pattern. THO cells are expanded with disease progression as well as the cells producing individually IFNy, IL4 and ILlO; this is consistent with a “terminal” differentiation of peripheral blood lymphocytes in HIV1 infection. Supported by a grant from Praxis/JNICT, Lisbon, Portugal
P.4.06.16
Reduced sensltlvlty of CD8+ but not CD4+ T cells from HIV-infected patients to 11-2, IL-7 and IL-12: Implications for actlvatlon and apoptosis
J. Vingerhcets, E. Bisalinkumi, G. Penne, E. Bosmans’, B. Colebunders, L. Kestens, G. Vanham. Laboratory of Immunology, Institute of Tropical Medicine, Antwerpen, Belgium, ’ Ewogenetics, Tessenderio,Belgium Introduction: The production of regulatory cytokines during HIV infection and their potential immunomodulatory effects have been studied extensively. However, few studies were focused on whether the T cells from HIV-infected persons displayed an altered sensitivity to these cytokines. Therefore, we wanted to compare me responsiveness of CD4+ and CD6+ T cells from HIV-infected patients and HIV-neaative controls to 11-2. IL-7 and IL-12. We measured the resoonse to these csokines in PBMC cuhures with regard to T cell proliferation (with or without additional TcWCD3 triggering), upregulation of the activation marker CD25 induction of blasts, effects on apoptosis and production of IFNy, IL-4 and IL-lo. Cytokine receptor-chain expression and co-expression with CD26 was determined on resting T cells. hlethods:Proliferation was determined by [3H]Thy-incorporation. CD4+ or CD6+ T cells were analyzed separately using flow cytometry. For each sample, bright CD25 expression and blast formation (as parameters of activation) were detemined simultaneously with the measurement of apoptosis (by TUNEL assay or light scatter analysis). Cytokine production was determined by ELISA. Cytokine receptor expression on fresh cells was determined by flow cytometry. Resufts: The co-stimulaton effect of IL-7 and IL-12 on suboptimal TcWCD3 triggering was lowered in PBMc from patients. IFNy-production after stimulation with 11-12, alone or in combination with IL-2 or IL-7, was significantly lower in patient PBMC. IL-IO production after stimulation with IL-2 plus IL-12 tended to be higher, in patients as compared to controls. Activation of CD& T cells from