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2 signalling pathway results in alloantigen-specific anergy in human t cells
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Targeting the ERK-1/2 Signalling Pathway Results in AlloantigenSpecific Anergy in Human T Cells P-M. Krieger, M.D. Sa¨emann, G.A. Bo¨hmig, F. Mu¨hlbacher, and G.J. Zlabinger
E
NGAGEMENT of the T-cell receptor induces activation of mitogen-activated protein kinases (MAPK) such as Jun-NH2 terminal kinase, p38, and extracellularrelated kinase (ERK), leading to transactivation of various gene products critical for proper T cell function.1 We have assessed the functional consequences of blocking the ERK pathway with a highly selective inhibitor with regard to T-cell alloreactivity. MATERIALS AND METHODS Human peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood. For assessment of T-cell proliferation, PBMC were cocultured with an equal number of irradiated allogeneic PBMC in the presence or absence of the selective ERK inhibitor UO126 (Sigma, Deisenhofen, Germany) or cyclosporine A (CsA). DNA synthesis was measured on day 7 by [3HT] uptake. Purified T cells were stimulated by immobilized CD3 and CD28 monoclonal antibodies (mAb) For secondary MLC, 1⫻106 PBMC were restimulated with allogeneic irradiated PBMC from the original or third-party donors. For cytokine measurement, supernatants were harvested after 24 hours and cytokines were determined by enzyme-linked immunosorbent assay. For evaluation of marker expression, cells were incubated with PE- and FITClabeled mAb and analyzed on a FACS flow cytometer (Becton Dickinson, San Jose, Calif). PHA blasts were generated with 2.5 g/mL PHA for 2 days then washed, and culture was started by addition of rIL-2.
INHIBITION OF THE ERK-PATHWAY PREVENTS ALLOANTIGEN-STIMULATED AND IL-2-DEPENDENT T-CELL PROLIFERATION
To assess the influence of ERK disruption on T-cell proliferation we used the highly selective inhibitor UO126. As shown in Fig 1, UO126 caused dose-dependent inhibition of proliferation, which could partially be reversed by addition
Fig 1. ERK-inhibition dose-dependently down-regulates T-cell proliferation as assessed in primary culture on day 7. The addition of exogenous rIL-2. (15U/mL) partially reverses downregulation of proliferation by ERK-blockade. Results are expressed as percentage of the control response. Each column reresents the mean ⫹ SD of eight independent experiments.
of rIL-2 (15 U/mL). We further revealed that blocking ERK impairs the upregulation of CD25, contributing to the diminished proliferation (data not shown). Interestingly, From the Institute of Immunology of Vienna, Department of Transplantsurgery and Internal Medicine III, University of Vienna, Vienna, Austria. Supported by grant P 14874 PAT. Address reprint requests to P.M. Krieger AKH Wien TXZentrum, Waehringer Guertel 18-20 10PO Wien, Australia.
© 2002 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/02/$–see front matter PII S0041-1345(02)02905-6
Transplantation Proceedings, 34, 1403–1404 (2002)
1403
KRIEGER, SA¨EMANN, BO¨HMIG ET AL
1404
Fig 2. ERK-blockade inhibits Il-2-dependent proliferation of T-cell blasts similar to rapamycine (1mol/L). CsA has no impact on IL-2-dependent proliferation.
inhibition of ERK down-regulates IL-2-dependent proliferation of T-cell blasts, indicating a major role for ERK in the transduction of the IL-2 receptor signal (Fig 2).
Fig 3. Pharmacologic disruption of ERK induces alloantigenspecific hyporesponsiveness upon restimulation with PBMC from the original donor. Response toward unrelated donors or PHA (2.5 g/mL) was unimpaired. (One representative result of six independent experiments.)
BLOCKING THE ERK-PATHWAY INDUCES ALLOANTIGEN-SPECIFIC HYPORESPONSIVENESS
DISCUSSION
We stimulated human PBMC for 7 days with alloantigenic PBMC in the presence or absence of UO126 and restimulated them with original or third-party alloantigens. Blockade of ERK during alloantigen priming led to alloantigenspecific tolerance (Fig 3), which was reversed by addition of rIL-2, indicating a classic state of anergy (data not shown). Proliferative response toward unrelated third party donors remained unimpaired (Fig 3). Interestingly, the calcineurininhibitor CsA did not prevent the induction of anergy and even promoted a deeper state of tolerance (data not shown).
We demonstrated that targeting the ERK-pathway inhibits T-cell proliferation in MLC, an in vitro correlate of acute allograft rejection by preventing IL-2 production, CD25 expression, and interference of IL-2 signalling. Importantly, disruption of the ERK-pathway induces a state of alloantigen-specific anergy, which is even promoted by the addition of CsA. These data suggest that distinct MAPK in T cells represent promising targets for future immunosuppressive therapy. REFERENCE 1. Dumont FJ, Staruch MJ, Fischer P, et al
Targeting the ERK-1/2 Signalling Pathway Results in AlloantigenSpecific Anergy in Human T Cells P-M. Krieger, M.D. Sa¨emann, G.A. Bo¨hmig, F. Mu¨hlbacher, and G.J. Zlabinger
E
NGAGEMENT of the T-cell receptor induces activation of mitogen-activated protein kinases (MAPK) such as Jun-NH2 terminal kinase, p38, and extracellularrelated kinase (ERK), leading to transactivation of various gene products critical for proper T cell function.1 We have assessed the functional consequences of blocking the ERK pathway with a highly selective inhibitor with regard to T-cell alloreactivity. MATERIALS AND METHODS Human peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood. For assessment of T-cell proliferation, PBMC were cocultured with an equal number of irradiated allogeneic PBMC in the presence or absence of the selective ERK inhibitor UO126 (Sigma, Deisenhofen, Germany) or cyclosporine A (CsA). DNA synthesis was measured on day 7 by [3HT] uptake. Purified T cells were stimulated by immobilized CD3 and CD28 monoclonal antibodies (mAb) For secondary MLC, 1⫻106 PBMC were restimulated with allogeneic irradiated PBMC from the original or third-party donors. For cytokine measurement, supernatants were harvested after 24 hours and cytokines were determined by enzyme-linked immunosorbent assay. For evaluation of marker expression, cells were incubated with PE- and FITClabeled mAb and analyzed on a FACS flow cytometer (Becton Dickinson, San Jose, Calif). PHA blasts were generated with 2.5 g/mL PHA for 2 days then washed, and culture was started by addition of rIL-2.
INHIBITION OF THE ERK-PATHWAY PREVENTS ALLOANTIGEN-STIMULATED AND IL-2-DEPENDENT T-CELL PROLIFERATION
To assess the influence of ERK disruption on T-cell proliferation we used the highly selective inhibitor UO126. As shown in Fig 1, UO126 caused dose-dependent inhibition of proliferation, which could partially be reversed by addition
Fig 1. ERK-inhibition dose-dependently down-regulates T-cell proliferation as assessed in primary culture on day 7. The addition of exogenous rIL-2. (15U/mL) partially reverses downregulation of proliferation by ERK-blockade. Results are expressed as percentage of the control response. Each column reresents the mean ⫹ SD of eight independent experiments.
of rIL-2 (15 U/mL). We further revealed that blocking ERK impairs the upregulation of CD25, contributing to the diminished proliferation (data not shown). Interestingly, From the Institute of Immunology of Vienna, Department of Transplantsurgery and Internal Medicine III, University of Vienna, Vienna, Austria. Supported by grant P 14874 PAT. Address reprint requests to P.M. Krieger AKH Wien TXZentrum, Waehringer Guertel 18-20 10PO Wien, Australia.
© 2002 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/02/$–see front matter PII S0041-1345(02)02905-6
Transplantation Proceedings, 34, 1403–1404 (2002)
1403
KRIEGER, SA¨EMANN, BO¨HMIG ET AL
1404
Fig 2. ERK-blockade inhibits Il-2-dependent proliferation of T-cell blasts similar to rapamycine (1mol/L). CsA has no impact on IL-2-dependent proliferation.
inhibition of ERK down-regulates IL-2-dependent proliferation of T-cell blasts, indicating a major role for ERK in the transduction of the IL-2 receptor signal (Fig 2).
Fig 3. Pharmacologic disruption of ERK induces alloantigenspecific hyporesponsiveness upon restimulation with PBMC from the original donor. Response toward unrelated donors or PHA (2.5 g/mL) was unimpaired. (One representative result of six independent experiments.)
BLOCKING THE ERK-PATHWAY INDUCES ALLOANTIGEN-SPECIFIC HYPORESPONSIVENESS
DISCUSSION
We stimulated human PBMC for 7 days with alloantigenic PBMC in the presence or absence of UO126 and restimulated them with original or third-party alloantigens. Blockade of ERK during alloantigen priming led to alloantigenspecific tolerance (Fig 3), which was reversed by addition of rIL-2, indicating a classic state of anergy (data not shown). Proliferative response toward unrelated third party donors remained unimpaired (Fig 3). Interestingly, the calcineurininhibitor CsA did not prevent the induction of anergy and even promoted a deeper state of tolerance (data not shown).
We demonstrated that targeting the ERK-pathway inhibits T-cell proliferation in MLC, an in vitro correlate of acute allograft rejection by preventing IL-2 production, CD25 expression, and interference of IL-2 signalling. Importantly, disruption of the ERK-pathway induces a state of alloantigen-specific anergy, which is even promoted by the addition of CsA. These data suggest that distinct MAPK in T cells represent promising targets for future immunosuppressive therapy. REFERENCE 1. Dumont FJ, Staruch MJ, Fischer P, et al