- Email: [email protected]
Orally induced, HLA-peptide specific gamma-delta T-cells suppress experimental autoimmune uveitis in the rat
Monday, 23 June 1997 - Oral presentations
Materials and Methods:In this study, the adjuvant arthritis model and the experimental autoimmune encephalomyelitis model in Lewis rats wem used as experimental models of tieumatoid arthritis and multiple sclerosis, respectively. Anergy was induced in the arthritogenic T cell clone A2b, or the encephalitogenic T cell clone Zla, by incubating the T cells in the absence of APCs with a supra-optimal dose of their respective stimulatoly peptides, the mycobacterial HSP85 peptide 178-l 90, or a stimulatory and encephalitogenic peptfde analog of MBP72-85 (MBP72-85s,&. Reaulta: Co-culture experiments of A2b cells with anergic A2b cells showed suppression of the peptide specific A2b response. Co-culturing A2b cells with non allergic A2b cells, or with allergic Zla cells, did not lead to suppression. Similarly, specifii suppression was observed when Zla responders were cocultured with anergic Zla cells. These findings were extended to a polyclonal immune response. Polyckmal lymphnode cells, delived from animals immunized with Mycobactetium tubetou/osis (Mt) in IFA, were co-cultured with anergic A2b cells. Such polyclonal lymphnode cells respond to peptide 178-l 90, but to a much lesser extend than to whole Mt. Strikingly, anergic A2b cells completely suppressed polyclonal responses to whole Mt. This indicated that the observed suppression was not rest&ted to T cells specific for epitope 178-190, but was spread to T cells recognizing other epitopes. Conclusion:We show that anergic T cells are able to actively modulate the response of autoreactive T cells. The suppression is not restricted to a certain mtide specific response, but can spread to T cells recognizing other epitopes. This feature might have important implications for therapy of autoimmune diseases.
0501.7
Orally Induced, HLA-peptlde specific gamma-delta T-cells stm~re88 ex~rlmental autolmmune uveitis In the rat - .
Effector phase of the allergic response
35
mononuclear cells were restimulated by culture in ovalbumin (ova) peptide, whole ova protein or anti-CD3. Results: Following 4-8 consecutive days of aerosol challenge the pmliferation of lung mononuclear cells in response to culture with ova protein, ova peptide 323-339 or immobilised anti-CD3 was lost. The magnitude of this reduction ranged from 90-97% and was dependent on the dose of aerosol administered. Moreover, this effect was specific to the lung since it was not observed in lymph node or spleen cells of me mice. Reduced levels of pmliferation were associated with a decrease in the level of IL-2 production by lung mononuclear cells, but not spleen or lymph node cells. However, the addition of exogenous IL-2 did not mtore T cell responses, implying that the reduced proliferative responses were not a consequence of a failure to produce IL-2. The decrease in pulmonary T cell responses to antigen did not result from T cells migrating away from the tissue since the number of ova specific T cells in me lung mucosa of challenged mice did not differ significantly from naive mice. The unresponsivness was transient since animals mat had been aerosol challenged and subsequently “rested” for a P-week period displayed normal responses. The failure of me T cells to respond to antigen was due to active inhibition since removal of adherant cells resulted in a total resoration of the antigen-driven responses. Using indomethacin or monomethyl arginine, inhibition of mediator production by the adherant cells, which comprised both rnacrophages and dendritic cells, did not restore responses. Conclusion: Aerosol challenge of TCR transgenic mice resulted in the induction of a lung T cell unresponsiveness. This effect was not a consequence of changes in the number of either antigen presenting cells or T cells in the lung. The reduction resulted from the action of adhemnt cells that inhibited T cell responses to antigen by a process mat did not involve secretion of immunosuppressive mediators. The mechanism of this down-regulation of pulmonaty T cell resoonses will be discussed.
G. Wildner l, T. Hiinig *, S.R. Thurau ‘. ’ Section of Immunobiolog)! University Eye Hospital, LudwigMawimiians-University, Munich, Germany 21nstitutefor Virology and Immunobiology; University of Wikzburg, Witzburg, Germany Introduction: Experimental autoimmune uveitis (EAU) is a CD4+-T cell mediated inflammatory autoimmune disease affecting the eye, where destruction of the retinal tissue decreases vision and sometimes ends LIDin blindness. The retinal autoantigens are well characterized, and we recently described an HLA-oeotide mimickino a uveitooenic retinal oeotide. EAU in Lewis rats can be supp&ed by oral application 2 retinal auto&&ens and their peptfdes as well the peptide derived from me sequence of uveitis-associated HLA-antigens. Our aim was to further characterize the cell population conferring suppression by oral application of antigen. Msterlalsand Methods: Lewis rats were fed with either a 14mer peptide detived from the sequence of retinal S-Antigen (PDSAg), a crossreactive, disease-associated HLA-peptide (B27PD) and a peptide from non-uveitis-associated HIA-Class l-antigens (B7PD) as a control. cz/Band y/S TCR+ cells were isolated from the spleens of tolerized rats by MACS-separation and transferred to naive recipients. which were subsequently immunized with peptide PDSAg for induction of uveitis. The surface phenotype of these cells was analyzed and their antigen specificity investigated by in vitro-proliferation. Results: Adoptive transfer of y/6-T cells from the spleens of rats orally tolerized with S-Antigen-peptide or with the crossmactive HLA-peptide B27PD resulted in reduced incidence and severity of disease, as compared to cells gained from rats tolertzed with control peptide B7PD. Q-T cells from neither group showed any ameliorating effect. The y/6 cells isolated from the spleens of orally tolerized rats proliferated in vitm only in coculture with their respective antigen and an n/fJ-T cell line with the same antigen specificity. Conduslon: In the rat model of a T cell mediated autoimmune disease, peptide specific y/&T lymphocytes am a major down-regulatory cell population in oral tolerance.
10:00-l
2:oo
0.502
Effector phase of the allergic response
as
) 0501.8
1 Aerosol challenge of T cell receptor transgenic mice results in down-regulation of pulmonary T cell resDonse8
S.-C. Lee, Z. Jaffar, S.T. Holgate, K. Roberts. University Medicine, Southampton General Hospital, Southampton, UK Intmduotlon: To study the T call response to aemsolised antigens entering the lung we have used an ovalbumin specific T cell receptor (TCR) transgenic mouse DO.11.10. The TCR transgenes am expressed in BALB/c mice (H-2d haplotype) which facilitates me aerosol challenge of transgenic mice directly. The principle advantages of adopting this approach am that the magnitude of T cell response is largely amplified and the ova specific T cells can be identified using an anti-cfonotypic antibody. Materfals and Methods: DO.11.lO mice were exposed to aerosols of ova solution for 20 min a day for up to 8 consequtive days. Lung tissue was then removed and mononuclear cells isolated by enzymic dissociation of the tissue and subsequent purification by density gradient cenbifugation. Isolated lung
71 0
5 02
0.5.02.2
Room E/F
1
No abstract available
Activated human mast cells (HMC1) inhibit prollferatlon but Increase IFNr production by CD8+ T cells
F.L. de Pater-Huijsen 1.2,M.J. de Riemer*, R.M.R. Reijneke 2,3, M. Pompen’,
R.Lutter’,‘, H.M. Jansen ‘, T.A. Out2,3. ’ Depar?mentof Pu/mono/~ Academic Medical Center, Amsterdam, the Netherlands, 2Clinical and Laboratory lmmunokqy Unit, Academic Medical Center, Amsterdam, the Nethedands, 3Labomtory for Experimental and Clinical immunology (CLB), Amsterdam, the Netherlands
Introduction:In patients with allergic asthma, both mast cells and T cells am believed to be involved in the immunological processes in me lungs. Materials and Methods: To obtain information on the interactions between mast cells and CD8+ T lymphocytes we investigated the modulatory capacity of irradiated mast cells (human mast cell line HMC-1; dr. J.H. Butterfield) on CD8+ T cell proliferation and cytokine production in vitro. We performed our study using highly purified polycional CD8+ T cell populations (over 99% CD8+ T cells) and CD8* T cell clones (all obtained from peripheral blood from healthy controls). Results: The proliferation of the polyclonal CDS+ T cell lines and the CD8+ T-cell clones was specifically decreased by stimulated mast cells. IFN-y production, however, was specifically increased (till 33-fold at 1 day) in both lines and clones. IL-4 production was decreased by not-stimulated mast cells and slightly increased by stimulated mast cells. We found mat me effects were due to soluble factors released by actiiated mast cells. IL-12 appeared not to be involved. Mast cell effects could not be mimicked by adding mediators like PGEP, PGM, TNFa. IL-lo, IL-i/I and IL-8 (all known to be produced by HMC-1 cells) separately to the T-cell cultures. Next we studied the kinetics of IFN-y mRNA appearance in cultures of polyclonal CD8+ T-cells with or without stimulated mast cells. RNA was isolated at different time-points and a semi-quantitative RT-PCR was performed. IFN-y mRNA was found to be increasing from 30 minutes up to 4 hours (the latest time-point studied so far) in the co-cultures of T-cells with stimulated mast cells. At all these time-points the quantity of IFN-y
Materials and Methods:In this study, the adjuvant arthritis model and the experimental autoimmune encephalomyelitis model in Lewis rats wem used as experimental models of tieumatoid arthritis and multiple sclerosis, respectively. Anergy was induced in the arthritogenic T cell clone A2b, or the encephalitogenic T cell clone Zla, by incubating the T cells in the absence of APCs with a supra-optimal dose of their respective stimulatoly peptides, the mycobacterial HSP85 peptide 178-l 90, or a stimulatory and encephalitogenic peptfde analog of MBP72-85 (MBP72-85s,&. Reaulta: Co-culture experiments of A2b cells with anergic A2b cells showed suppression of the peptide specific A2b response. Co-culturing A2b cells with non allergic A2b cells, or with allergic Zla cells, did not lead to suppression. Similarly, specifii suppression was observed when Zla responders were cocultured with anergic Zla cells. These findings were extended to a polyclonal immune response. Polyckmal lymphnode cells, delived from animals immunized with Mycobactetium tubetou/osis (Mt) in IFA, were co-cultured with anergic A2b cells. Such polyclonal lymphnode cells respond to peptide 178-l 90, but to a much lesser extend than to whole Mt. Strikingly, anergic A2b cells completely suppressed polyclonal responses to whole Mt. This indicated that the observed suppression was not rest&ted to T cells specific for epitope 178-190, but was spread to T cells recognizing other epitopes. Conclusion:We show that anergic T cells are able to actively modulate the response of autoreactive T cells. The suppression is not restricted to a certain mtide specific response, but can spread to T cells recognizing other epitopes. This feature might have important implications for therapy of autoimmune diseases.
0501.7
Orally Induced, HLA-peptlde specific gamma-delta T-cells stm~re88 ex~rlmental autolmmune uveitis In the rat - .
Effector phase of the allergic response
35
mononuclear cells were restimulated by culture in ovalbumin (ova) peptide, whole ova protein or anti-CD3. Results: Following 4-8 consecutive days of aerosol challenge the pmliferation of lung mononuclear cells in response to culture with ova protein, ova peptide 323-339 or immobilised anti-CD3 was lost. The magnitude of this reduction ranged from 90-97% and was dependent on the dose of aerosol administered. Moreover, this effect was specific to the lung since it was not observed in lymph node or spleen cells of me mice. Reduced levels of pmliferation were associated with a decrease in the level of IL-2 production by lung mononuclear cells, but not spleen or lymph node cells. However, the addition of exogenous IL-2 did not mtore T cell responses, implying that the reduced proliferative responses were not a consequence of a failure to produce IL-2. The decrease in pulmonary T cell responses to antigen did not result from T cells migrating away from the tissue since the number of ova specific T cells in me lung mucosa of challenged mice did not differ significantly from naive mice. The unresponsivness was transient since animals mat had been aerosol challenged and subsequently “rested” for a P-week period displayed normal responses. The failure of me T cells to respond to antigen was due to active inhibition since removal of adherant cells resulted in a total resoration of the antigen-driven responses. Using indomethacin or monomethyl arginine, inhibition of mediator production by the adherant cells, which comprised both rnacrophages and dendritic cells, did not restore responses. Conclusion: Aerosol challenge of TCR transgenic mice resulted in the induction of a lung T cell unresponsiveness. This effect was not a consequence of changes in the number of either antigen presenting cells or T cells in the lung. The reduction resulted from the action of adhemnt cells that inhibited T cell responses to antigen by a process mat did not involve secretion of immunosuppressive mediators. The mechanism of this down-regulation of pulmonaty T cell resoonses will be discussed.
G. Wildner l, T. Hiinig *, S.R. Thurau ‘. ’ Section of Immunobiolog)! University Eye Hospital, LudwigMawimiians-University, Munich, Germany 21nstitutefor Virology and Immunobiology; University of Wikzburg, Witzburg, Germany Introduction: Experimental autoimmune uveitis (EAU) is a CD4+-T cell mediated inflammatory autoimmune disease affecting the eye, where destruction of the retinal tissue decreases vision and sometimes ends LIDin blindness. The retinal autoantigens are well characterized, and we recently described an HLA-oeotide mimickino a uveitooenic retinal oeotide. EAU in Lewis rats can be supp&ed by oral application 2 retinal auto&&ens and their peptfdes as well the peptide derived from me sequence of uveitis-associated HLA-antigens. Our aim was to further characterize the cell population conferring suppression by oral application of antigen. Msterlalsand Methods: Lewis rats were fed with either a 14mer peptide detived from the sequence of retinal S-Antigen (PDSAg), a crossreactive, disease-associated HLA-peptide (B27PD) and a peptide from non-uveitis-associated HIA-Class l-antigens (B7PD) as a control. cz/Band y/S TCR+ cells were isolated from the spleens of tolerized rats by MACS-separation and transferred to naive recipients. which were subsequently immunized with peptide PDSAg for induction of uveitis. The surface phenotype of these cells was analyzed and their antigen specificity investigated by in vitro-proliferation. Results: Adoptive transfer of y/6-T cells from the spleens of rats orally tolerized with S-Antigen-peptide or with the crossmactive HLA-peptide B27PD resulted in reduced incidence and severity of disease, as compared to cells gained from rats tolertzed with control peptide B7PD. Q-T cells from neither group showed any ameliorating effect. The y/6 cells isolated from the spleens of orally tolerized rats proliferated in vitm only in coculture with their respective antigen and an n/fJ-T cell line with the same antigen specificity. Conduslon: In the rat model of a T cell mediated autoimmune disease, peptide specific y/&T lymphocytes am a major down-regulatory cell population in oral tolerance.
10:00-l
2:oo
0.502
Effector phase of the allergic response
as
) 0501.8
1 Aerosol challenge of T cell receptor transgenic mice results in down-regulation of pulmonary T cell resDonse8
S.-C. Lee, Z. Jaffar, S.T. Holgate, K. Roberts. University Medicine, Southampton General Hospital, Southampton, UK Intmduotlon: To study the T call response to aemsolised antigens entering the lung we have used an ovalbumin specific T cell receptor (TCR) transgenic mouse DO.11.10. The TCR transgenes am expressed in BALB/c mice (H-2d haplotype) which facilitates me aerosol challenge of transgenic mice directly. The principle advantages of adopting this approach am that the magnitude of T cell response is largely amplified and the ova specific T cells can be identified using an anti-cfonotypic antibody. Materfals and Methods: DO.11.lO mice were exposed to aerosols of ova solution for 20 min a day for up to 8 consequtive days. Lung tissue was then removed and mononuclear cells isolated by enzymic dissociation of the tissue and subsequent purification by density gradient cenbifugation. Isolated lung
71 0
5 02
0.5.02.2
Room E/F
1
No abstract available
Activated human mast cells (HMC1) inhibit prollferatlon but Increase IFNr production by CD8+ T cells
F.L. de Pater-Huijsen 1.2,M.J. de Riemer*, R.M.R. Reijneke 2,3, M. Pompen’,
R.Lutter’,‘, H.M. Jansen ‘, T.A. Out2,3. ’ Depar?mentof Pu/mono/~ Academic Medical Center, Amsterdam, the Netherlands, 2Clinical and Laboratory lmmunokqy Unit, Academic Medical Center, Amsterdam, the Nethedands, 3Labomtory for Experimental and Clinical immunology (CLB), Amsterdam, the Netherlands
Introduction:In patients with allergic asthma, both mast cells and T cells am believed to be involved in the immunological processes in me lungs. Materials and Methods: To obtain information on the interactions between mast cells and CD8+ T lymphocytes we investigated the modulatory capacity of irradiated mast cells (human mast cell line HMC-1; dr. J.H. Butterfield) on CD8+ T cell proliferation and cytokine production in vitro. We performed our study using highly purified polycional CD8+ T cell populations (over 99% CD8+ T cells) and CD8* T cell clones (all obtained from peripheral blood from healthy controls). Results: The proliferation of the polyclonal CDS+ T cell lines and the CD8+ T-cell clones was specifically decreased by stimulated mast cells. IFN-y production, however, was specifically increased (till 33-fold at 1 day) in both lines and clones. IL-4 production was decreased by not-stimulated mast cells and slightly increased by stimulated mast cells. We found mat me effects were due to soluble factors released by actiiated mast cells. IL-12 appeared not to be involved. Mast cell effects could not be mimicked by adding mediators like PGEP, PGM, TNFa. IL-lo, IL-i/I and IL-8 (all known to be produced by HMC-1 cells) separately to the T-cell cultures. Next we studied the kinetics of IFN-y mRNA appearance in cultures of polyclonal CD8+ T-cells with or without stimulated mast cells. RNA was isolated at different time-points and a semi-quantitative RT-PCR was performed. IFN-y mRNA was found to be increasing from 30 minutes up to 4 hours (the latest time-point studied so far) in the co-cultures of T-cells with stimulated mast cells. At all these time-points the quantity of IFN-y