T-lymphocyte apoptosis is increased by non-interleukin-2-dependent induction in human mixed lymphocyte cultures

K. J. Woodside, M. Hu, Kristene Gugliuzza, G. C. Hunter, J. A. Daller

Research output: Contribution to journalArticle

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Abstract

Introduction. Transplant tolerance is dependent on the apoptotic deletion of allospecific T lymphocytes following interleukin-2 (IL-2)-dependent T-lymphocyte activation. Current immunosuppressive strategies block IL-2 and may prevent T-cell activation. We examined apoptotic alterations in mixed lymphocyte culture (MLC), a model of allospecific lymphocyte activation, by polyclonal rabbit antithymocyte antibody thymoglobulin (rATG) and monoclonal anti-IL-2 receptor antibody basiliximab. Methods. Human lymphocytes were isolated using Ficoll-Paque gradient. Cesium-irradiated (2500 rad) stimulator cells (106 cells/mL) were cocultured with equal numbers of responder cells. Results. Apoptosis was measured using annexin-V staining and propidium iodide exclusion using flow cytometry. Isolated protein was analyzed using Western blotting with densitometry. Apoptosis increased at days 3 and 7 in rATG MLC compared with control and basiliximab MLC. Fas was up-regulated in rATG MLC in a dose-dependent manner, whereas basiliximab did not alter fas. FasL was increased initially and at late time points in rATG MLC. Conclusions. Polyclonal rATG increased apoptosis and production of the proapoptotic proteins fas and fasL. In contrast, monoclonal basiliximab did not change lymphocyte apoptosis or apoptotic protein production. These results suggest that a specific IL-2 pathway blockade may prevent allospecific tolerance and that a non-IL-2 pathway blockade may encourage apoptosis of allospecifically activated T cells.

Original languageEnglish (US)
Pages (from-to)1949-1952
Number of pages4
JournalTransplantation Proceedings
Volume37
Issue number4
DOIs
StatePublished - May 2005

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Lymphocytes
Apoptosis
T-Lymphocytes
Rabbits
Antibodies
Interleukin-2
Lymphocyte Activation
Ficoll
Cesium
Fas Ligand Protein
Propidium
Densitometry
Interleukin-2 Receptors
Annexin A5
Immunosuppressive Agents
Flow Cytometry
Proteins
Cell Count
Western Blotting
Monoclonal Antibodies

ASJC Scopus subject areas

  • Surgery
  • Transplantation

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T-lymphocyte apoptosis is increased by non-interleukin-2-dependent induction in human mixed lymphocyte cultures. / Woodside, K. J.; Hu, M.; Gugliuzza, Kristene; Hunter, G. C.; Daller, J. A.

In: Transplantation Proceedings, Vol. 37, No. 4, 05.2005, p. 1949-1952.

Research output: Contribution to journalArticle

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AU - Daller, J. A.

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N2 - Introduction. Transplant tolerance is dependent on the apoptotic deletion of allospecific T lymphocytes following interleukin-2 (IL-2)-dependent T-lymphocyte activation. Current immunosuppressive strategies block IL-2 and may prevent T-cell activation. We examined apoptotic alterations in mixed lymphocyte culture (MLC), a model of allospecific lymphocyte activation, by polyclonal rabbit antithymocyte antibody thymoglobulin (rATG) and monoclonal anti-IL-2 receptor antibody basiliximab. Methods. Human lymphocytes were isolated using Ficoll-Paque gradient. Cesium-irradiated (2500 rad) stimulator cells (106 cells/mL) were cocultured with equal numbers of responder cells. Results. Apoptosis was measured using annexin-V staining and propidium iodide exclusion using flow cytometry. Isolated protein was analyzed using Western blotting with densitometry. Apoptosis increased at days 3 and 7 in rATG MLC compared with control and basiliximab MLC. Fas was up-regulated in rATG MLC in a dose-dependent manner, whereas basiliximab did not alter fas. FasL was increased initially and at late time points in rATG MLC. Conclusions. Polyclonal rATG increased apoptosis and production of the proapoptotic proteins fas and fasL. In contrast, monoclonal basiliximab did not change lymphocyte apoptosis or apoptotic protein production. These results suggest that a specific IL-2 pathway blockade may prevent allospecific tolerance and that a non-IL-2 pathway blockade may encourage apoptosis of allospecifically activated T cells.

AB - Introduction. Transplant tolerance is dependent on the apoptotic deletion of allospecific T lymphocytes following interleukin-2 (IL-2)-dependent T-lymphocyte activation. Current immunosuppressive strategies block IL-2 and may prevent T-cell activation. We examined apoptotic alterations in mixed lymphocyte culture (MLC), a model of allospecific lymphocyte activation, by polyclonal rabbit antithymocyte antibody thymoglobulin (rATG) and monoclonal anti-IL-2 receptor antibody basiliximab. Methods. Human lymphocytes were isolated using Ficoll-Paque gradient. Cesium-irradiated (2500 rad) stimulator cells (106 cells/mL) were cocultured with equal numbers of responder cells. Results. Apoptosis was measured using annexin-V staining and propidium iodide exclusion using flow cytometry. Isolated protein was analyzed using Western blotting with densitometry. Apoptosis increased at days 3 and 7 in rATG MLC compared with control and basiliximab MLC. Fas was up-regulated in rATG MLC in a dose-dependent manner, whereas basiliximab did not alter fas. FasL was increased initially and at late time points in rATG MLC. Conclusions. Polyclonal rATG increased apoptosis and production of the proapoptotic proteins fas and fasL. In contrast, monoclonal basiliximab did not change lymphocyte apoptosis or apoptotic protein production. These results suggest that a specific IL-2 pathway blockade may prevent allospecific tolerance and that a non-IL-2 pathway blockade may encourage apoptosis of allospecifically activated T cells.

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