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The induction of oral tolerance in mice
246
Mechanisms of tolerance and selection events
Results: We have previously shown that pedpheml T cells from 2C mice, injected intravenously with semi-allogeneic (BALB/c x C57BU6) Fl spleen cells (H-2d x b), are rendered hyporesponsive to a rechallenge with H-2d APCs (BALB/c). We now show that the hyporesponsive state can be reversed with PMA and lonomycin, suggesting T cell anergy rather than clonal deletion is a major factor contributing to the hypoproliferative MLR response. Enhanced tyrosine phosphorylation of distinct proteins are detected upon exposure of responsive 2C T cells to Ld molecules. In contrast, peripheral T cells from the hyporesponsive 2C mice demonstrate tyrosine phosphorylation patterns upon stimulation through the TCR and CD6 molecules which are distinct from those in T cells from naive 2C mice. Further, atterations in tyrosine phosphorylation in the tolerized cells are correlated with alterations in TCR-mediated signal transduction. Conclusions: Thus, we have found that 2C TCR transgenic mice can be rendered hyporesponsive to a dass I MHC alloantigen (H-2”) by prior intravenous administration of the allo-MHC/peptfde antigen on live lymphocytes. The hyporesponsive state is reversed with PMA/I suggesting anergy rather than deletion has been induced. Furthermore. a distinct pattern of protein tyrosine phosphorylation is correlated with the hyporesponsive or tolerized state in 2C mice.
m P 2 04 02
The induction of oral tolerance in mice
M. Saunders I, A.K. Lang2, N.A. Williams*. 16iophysics Research Unit, Bristol Oncology Centre, University of Bristol, UK, “Dept. of Pathology and Microbiotcg~ Univemity of Bristol, UK Introduction: Tolerance is a state of immunological unresponsiveness to antigens otherwise capable of inducing an active immune response and appears to prevent the induction of hypersensitivity to beneficial food antigens. The most reliable means of inducing immunological tolerance is via the oral route. The precise mechanisms involved in the induction of oral tolerance are still unclear but elucidation of these could lead to the development of suitable therapies for the development of oral vaccines and the prevention of autoimmune disease and allergies. Materials and Methods: NIH mice were fed one dose of 20 mg hen egg lysozyme (HEL) by gavage or PBS alone (controls). Peyer’s patch cells (PP), mesenteric lymph node cells (MLN), cervical lymph node cells (CLN) and inguinal lymph node cells (ILN) were removed 3, 6, 14 or 21 days after feeding. In vitro cell proliferation and cytokine (CK) production were measured in the presence or absence of HEL or keyhole limpet haemocyanin (KLH) over a timecourse of 3-6 days. Some groups of fed and control mice were given a footpad (FP) challenge with HEL or PBS in CFA 7 days after the initial feed. 14 days later, anti-HEL serum antibody levels, cell proliferation and CK production were examined or mice received a further challenge in the ear with HEL or PBS in order to assess the delayed type hypersensitivity response 24 hours later. Results: Cell proliferation data clearly demonstrates that an in vitro secondary response to HEL by PP. MLN, CLN and ILN cells occurs where the cells have been removed from mice between 3-6 days after feeding with HEL and is maintained by cells from mice up to 14 days after feeding. Primary kinetics in response to HEL only become evident 21 days after feeding, whereas the response to KLH demonstrates primary kinetic5 at each time-point. Cells from mice fed with PBS generated only primary responses to both HEL and KLH. Analysis of the cytokines produced within these cultures demonstrated that stimulation with HEL of cells from HEL-fed animals resulted in the production of significant amounts of IL4 and IL5. Measurements of serum antibodies in mice demonstrated that peak production of anti-HEL antibody occurred in groups of mice fed with HEL 14 days previously and that the principle component was IgGl. The antibody titres were then compared from groups of mice which were (1) fed HEL and FP challenged with PBS, (2) fed PBS and FP challenged with HEL (immunisation protocol) or (3) fed HEL and challenged with HEL (tolerisation protocol). A strong antibody response was detected in serum from group 2 with the major component being IgGl. The effect of feeding with HEL prior to immunisation (group 3) was to significantly diminish the antibody response to HEL. Conclusion: This data demonstrates that feeding a high dose of antigen appears to elicit transient priming in a variety of tissues. Cytokine and serum antibody data suggest that these may be Th2 type responses and could influence manipulation of the immune response in order to develop oral vaccines and prevent autoimmune disease and allergy.
P.2.04.03
Aiioantibody reactivity to endotheiiai MHC and non-MHC antigens in rat heart allograft rejection
J. Derhaag, A. Duijvesttjn, A. Gijsen, H. van der Heijden, J. Damoiseaux, P. van Breda Vriesman. Department of lmmunotcg~ University of Maastricht, f? 0. Bcx 616, 6200 MD, Maastricht, The Netherlands Introduction: During allograft rejection, vascular endothelium forms a major target for alloantibodies and allospecific cytotoxicT-cells, generated by the immune
24 June 1997 - Poster presentations
response of the recipient. We are studying the effect5 of alloantibody reactivity against endothelial cells (EC) in fully mismatched transplantations (Lewis (RTl’) hearts into BN (RTl”) recipients) and in MHC-matched but non-MHCmismatched transplantations (congenic LEW.l N (RTl”) hearts into BN (RTI “) recipients). Matsrlalsand Msthods: Experimental allosera, used as alloantibody source, were collected at the day of rejection, and studied in in v&o experiments with non-stimulated and cytokine (TNFa + IFNy) stimulated EC cell lines, derived from Lewis heart origin, so called RHEC lines. The alloantibody reactivity with EC was studied by FACS analysis. This technique was also used for isotyping alloantibodies reactive with EC, and for studying complement activation potentials of alloantibodies (C3 and membrane attack complex (MAC) detection) and induction of ICAM- and VCAM-1. Complement-mediatedcytotoxicity of alloantibodies generated during atlograft rejection was tested in a 51Cr release assay wlth complement of guinea pig (xenogeneic) or rat (syngeneic) origin, and either non-stimulated or cytokine stimulated EC. Results:In fullv mismatched heart transplantation, acute reiection (Tx-acute) occurred at day 7. In solely non MHC mismatched heart transplantation, d& layed rejection (TX-delayed)occurred day 24-35. Reactivity of allosera from Txdelayed with non-stimulated and cytokine stimulated EC was similar, whereas for TX-acute sera an increase intensity was seen with cytokine stimulated EC. In cytotoxtcity tests executed with xenogsneic complement, TX-acute sera lysed 27.2% of the unstimulated EC and 73% of cytokine-stimulated EC. TX-delayed sera showed lower cytotoxicity, 11% for non-stimulated EC and 16% for cytokine-stimulated EC. When syngeneic complement was used cytotoxicity was lower (10.1% for cytokine stimulated EC) with TX-acute sera, and nearly absent (2.5% for cytokine stimulated EC) with TX-delayed sera There were no major differences for IgG subclasses between TX-acute and TX-delayed alloantisera. But IgM alloantibodies were present in TX-acute and not, or only minor in Txdelayed sera. Experiments executed with isotype subfractions, IgG versus IgM, showed that IgM was responsible for complement-mediated cytotoxicity. TX-acute sera fixed complement as shown by C3 expression, however MAC was observed on a lower % of the cells than C3. TX-delayed sera did not, or only minody, induce complement activation. No indications were found that alloantibody reactivii or MAC formation induced ICAM- or VCAM-1 on EC. Conclusions: We conclude that in heart allograft rejection: 1) alloantibodies are formed that react with endothelial MHC and non-MHC antigens of graft origin, 2) the alloantibody IgM fraction in alloantisera is responsible for complement fixation and EC cvtotoxicitv. and 3) alloantibcdv reactivitv does not lead to adhesion molecule induction on EC.’Currently we investigate the potential role of TX-acute and TX-delayed alloantibodies in cytotoxicity against EC by ADCC mechanisms.
P.2.04.04
Central tolerance due to ectopic self antigen expression in the thymua preempts variable and leaky peripheral tolerance
L. Klein, U. Rirther ‘, B.A. Kyewski. Tumor lmmunolcgy Prcgmmme, German Cancer Research Centre, INF 280, D-69120 Heidelberg, FRG, ’ Institute ibr Molecular Biology, Hannover Medical S&co/, Hannove~ FRO
Introduction: MHC class II-restricted T cells specific for inducible, blood-borne self-antigens might escape central deletion due to lack of continuous presentation in the thymus. In order to study the fate of such T cells, we are investigating mice transgenic (tg) for human C-reactive protein (hCRP), an inducible protein secreted by hepatocytes. Results: Basal serum levels range from 51 nM in tg females to 500 nM in tg males, and are experimentally inducible 500-fold and 2C-fold, respectively. All tg males are tolerant of a dominant T cell epitooe of hCRP, whereas 30% percent of females are responsive. To characterlie the mechanism(s) of tolerfzation for this epitope, we generated T cell receptor (TCR) transgenic mice. In hCRP/TCR double-tg mice, TCR-tg thymocytes are already deleted at the early CD4/CD6 double positive stage. Thymus transplantation experiments revealed that ectopic hCRP-expression in thymic epithelium mediates this negative selection. Circulating, basal hCRP-levels by themselves induce an identical phenotype of deletion in male mice, whereas basal serum levels in female mice leave thymic maturation unaffected. Additional peripheral mechanisms of tolenzation were analyzed by transfer of mature tg-T cells into hCRP-transgenics. In all male mice, transferred T cells expand massively and disappear subsequently within one month, without leaving any memory population. In contrast, transferred cells are energized in 60% of female mice within as few as six days without preceding expansion, whereas the other female recipients remain responsive to hCRP. Conclusions: This model illustrates the complexity of tolerance induction depending on the expression-level and -pattern of an MHC class II-restricted epitope.
Mechanisms of tolerance and selection events
Results: We have previously shown that pedpheml T cells from 2C mice, injected intravenously with semi-allogeneic (BALB/c x C57BU6) Fl spleen cells (H-2d x b), are rendered hyporesponsive to a rechallenge with H-2d APCs (BALB/c). We now show that the hyporesponsive state can be reversed with PMA and lonomycin, suggesting T cell anergy rather than clonal deletion is a major factor contributing to the hypoproliferative MLR response. Enhanced tyrosine phosphorylation of distinct proteins are detected upon exposure of responsive 2C T cells to Ld molecules. In contrast, peripheral T cells from the hyporesponsive 2C mice demonstrate tyrosine phosphorylation patterns upon stimulation through the TCR and CD6 molecules which are distinct from those in T cells from naive 2C mice. Further, atterations in tyrosine phosphorylation in the tolerized cells are correlated with alterations in TCR-mediated signal transduction. Conclusions: Thus, we have found that 2C TCR transgenic mice can be rendered hyporesponsive to a dass I MHC alloantigen (H-2”) by prior intravenous administration of the allo-MHC/peptfde antigen on live lymphocytes. The hyporesponsive state is reversed with PMA/I suggesting anergy rather than deletion has been induced. Furthermore. a distinct pattern of protein tyrosine phosphorylation is correlated with the hyporesponsive or tolerized state in 2C mice.
m P 2 04 02
The induction of oral tolerance in mice
M. Saunders I, A.K. Lang2, N.A. Williams*. 16iophysics Research Unit, Bristol Oncology Centre, University of Bristol, UK, “Dept. of Pathology and Microbiotcg~ Univemity of Bristol, UK Introduction: Tolerance is a state of immunological unresponsiveness to antigens otherwise capable of inducing an active immune response and appears to prevent the induction of hypersensitivity to beneficial food antigens. The most reliable means of inducing immunological tolerance is via the oral route. The precise mechanisms involved in the induction of oral tolerance are still unclear but elucidation of these could lead to the development of suitable therapies for the development of oral vaccines and the prevention of autoimmune disease and allergies. Materials and Methods: NIH mice were fed one dose of 20 mg hen egg lysozyme (HEL) by gavage or PBS alone (controls). Peyer’s patch cells (PP), mesenteric lymph node cells (MLN), cervical lymph node cells (CLN) and inguinal lymph node cells (ILN) were removed 3, 6, 14 or 21 days after feeding. In vitro cell proliferation and cytokine (CK) production were measured in the presence or absence of HEL or keyhole limpet haemocyanin (KLH) over a timecourse of 3-6 days. Some groups of fed and control mice were given a footpad (FP) challenge with HEL or PBS in CFA 7 days after the initial feed. 14 days later, anti-HEL serum antibody levels, cell proliferation and CK production were examined or mice received a further challenge in the ear with HEL or PBS in order to assess the delayed type hypersensitivity response 24 hours later. Results: Cell proliferation data clearly demonstrates that an in vitro secondary response to HEL by PP. MLN, CLN and ILN cells occurs where the cells have been removed from mice between 3-6 days after feeding with HEL and is maintained by cells from mice up to 14 days after feeding. Primary kinetics in response to HEL only become evident 21 days after feeding, whereas the response to KLH demonstrates primary kinetic5 at each time-point. Cells from mice fed with PBS generated only primary responses to both HEL and KLH. Analysis of the cytokines produced within these cultures demonstrated that stimulation with HEL of cells from HEL-fed animals resulted in the production of significant amounts of IL4 and IL5. Measurements of serum antibodies in mice demonstrated that peak production of anti-HEL antibody occurred in groups of mice fed with HEL 14 days previously and that the principle component was IgGl. The antibody titres were then compared from groups of mice which were (1) fed HEL and FP challenged with PBS, (2) fed PBS and FP challenged with HEL (immunisation protocol) or (3) fed HEL and challenged with HEL (tolerisation protocol). A strong antibody response was detected in serum from group 2 with the major component being IgGl. The effect of feeding with HEL prior to immunisation (group 3) was to significantly diminish the antibody response to HEL. Conclusion: This data demonstrates that feeding a high dose of antigen appears to elicit transient priming in a variety of tissues. Cytokine and serum antibody data suggest that these may be Th2 type responses and could influence manipulation of the immune response in order to develop oral vaccines and prevent autoimmune disease and allergy.
P.2.04.03
Aiioantibody reactivity to endotheiiai MHC and non-MHC antigens in rat heart allograft rejection
J. Derhaag, A. Duijvesttjn, A. Gijsen, H. van der Heijden, J. Damoiseaux, P. van Breda Vriesman. Department of lmmunotcg~ University of Maastricht, f? 0. Bcx 616, 6200 MD, Maastricht, The Netherlands Introduction: During allograft rejection, vascular endothelium forms a major target for alloantibodies and allospecific cytotoxicT-cells, generated by the immune
24 June 1997 - Poster presentations
response of the recipient. We are studying the effect5 of alloantibody reactivity against endothelial cells (EC) in fully mismatched transplantations (Lewis (RTl’) hearts into BN (RTl”) recipients) and in MHC-matched but non-MHCmismatched transplantations (congenic LEW.l N (RTl”) hearts into BN (RTI “) recipients). Matsrlalsand Msthods: Experimental allosera, used as alloantibody source, were collected at the day of rejection, and studied in in v&o experiments with non-stimulated and cytokine (TNFa + IFNy) stimulated EC cell lines, derived from Lewis heart origin, so called RHEC lines. The alloantibody reactivity with EC was studied by FACS analysis. This technique was also used for isotyping alloantibodies reactive with EC, and for studying complement activation potentials of alloantibodies (C3 and membrane attack complex (MAC) detection) and induction of ICAM- and VCAM-1. Complement-mediatedcytotoxicity of alloantibodies generated during atlograft rejection was tested in a 51Cr release assay wlth complement of guinea pig (xenogeneic) or rat (syngeneic) origin, and either non-stimulated or cytokine stimulated EC. Results:In fullv mismatched heart transplantation, acute reiection (Tx-acute) occurred at day 7. In solely non MHC mismatched heart transplantation, d& layed rejection (TX-delayed)occurred day 24-35. Reactivity of allosera from Txdelayed with non-stimulated and cytokine stimulated EC was similar, whereas for TX-acute sera an increase intensity was seen with cytokine stimulated EC. In cytotoxtcity tests executed with xenogsneic complement, TX-acute sera lysed 27.2% of the unstimulated EC and 73% of cytokine-stimulated EC. TX-delayed sera showed lower cytotoxicity, 11% for non-stimulated EC and 16% for cytokine-stimulated EC. When syngeneic complement was used cytotoxicity was lower (10.1% for cytokine stimulated EC) with TX-acute sera, and nearly absent (2.5% for cytokine stimulated EC) with TX-delayed sera There were no major differences for IgG subclasses between TX-acute and TX-delayed alloantisera. But IgM alloantibodies were present in TX-acute and not, or only minor in Txdelayed sera. Experiments executed with isotype subfractions, IgG versus IgM, showed that IgM was responsible for complement-mediated cytotoxicity. TX-acute sera fixed complement as shown by C3 expression, however MAC was observed on a lower % of the cells than C3. TX-delayed sera did not, or only minody, induce complement activation. No indications were found that alloantibody reactivii or MAC formation induced ICAM- or VCAM-1 on EC. Conclusions: We conclude that in heart allograft rejection: 1) alloantibodies are formed that react with endothelial MHC and non-MHC antigens of graft origin, 2) the alloantibody IgM fraction in alloantisera is responsible for complement fixation and EC cvtotoxicitv. and 3) alloantibcdv reactivitv does not lead to adhesion molecule induction on EC.’Currently we investigate the potential role of TX-acute and TX-delayed alloantibodies in cytotoxicity against EC by ADCC mechanisms.
P.2.04.04
Central tolerance due to ectopic self antigen expression in the thymua preempts variable and leaky peripheral tolerance
L. Klein, U. Rirther ‘, B.A. Kyewski. Tumor lmmunolcgy Prcgmmme, German Cancer Research Centre, INF 280, D-69120 Heidelberg, FRG, ’ Institute ibr Molecular Biology, Hannover Medical S&co/, Hannove~ FRO
Introduction: MHC class II-restricted T cells specific for inducible, blood-borne self-antigens might escape central deletion due to lack of continuous presentation in the thymus. In order to study the fate of such T cells, we are investigating mice transgenic (tg) for human C-reactive protein (hCRP), an inducible protein secreted by hepatocytes. Results: Basal serum levels range from 51 nM in tg females to 500 nM in tg males, and are experimentally inducible 500-fold and 2C-fold, respectively. All tg males are tolerant of a dominant T cell epitooe of hCRP, whereas 30% percent of females are responsive. To characterlie the mechanism(s) of tolerfzation for this epitope, we generated T cell receptor (TCR) transgenic mice. In hCRP/TCR double-tg mice, TCR-tg thymocytes are already deleted at the early CD4/CD6 double positive stage. Thymus transplantation experiments revealed that ectopic hCRP-expression in thymic epithelium mediates this negative selection. Circulating, basal hCRP-levels by themselves induce an identical phenotype of deletion in male mice, whereas basal serum levels in female mice leave thymic maturation unaffected. Additional peripheral mechanisms of tolenzation were analyzed by transfer of mature tg-T cells into hCRP-transgenics. In all male mice, transferred T cells expand massively and disappear subsequently within one month, without leaving any memory population. In contrast, transferred cells are energized in 60% of female mice within as few as six days without preceding expansion, whereas the other female recipients remain responsive to hCRP. Conclusions: This model illustrates the complexity of tolerance induction depending on the expression-level and -pattern of an MHC class II-restricted epitope.