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Resistance of Foxp3+ regulatory T cells to Nur77-induced apoptosis promotes allograft survival.

Tao R, Hancock WW - PLoS ONE (2008)

Bottom Line: Overexpression of Nur77 in the T cell lineage decreased numbers of peripheral CD4 and CD8 T cells by approximately 80% compared to wild-type (WT) mice.Upon activation through the T cell receptor in vitro or in vivo, Nur77Tg T cells showed only marginally decreased proliferation but significantly increased apoptosis.Allografts in Nur77Tg recipients had significantly increased expression of multiple Treg-associated genes, including Foxp3, Foxp1, Tip60 and HDAC9.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Stokes Research Institute and Biesecker Pediatric Liver Center, Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
The NR4A nuclear receptor family member Nur77 (NR4A1) promotes thymocyte apoptosis during negative selection of autoreactive thymocytes, but may also function in mature extrathymic T cells. We studied the effects of over-expression of Nur77 on the apoptosis of murine peripheral T cells, including thymic-derived Foxp3+ regulatory (Treg) cells. Overexpression of Nur77 in the T cell lineage decreased numbers of peripheral CD4 and CD8 T cells by approximately 80% compared to wild-type (WT) mice. However, the proportions of Treg cells were markedly increased in the thymus (61% of CD4+Foxp3+ singly positive thymocytes vs. 8% in WT) and secondary lymphoid organs (40-50% of CD4+Foxp3+ T cells vs. 7-8% in WT) of Nur77 transgenic (Nur77Tg) mice, and immunoprecipitation studies showed Nur77 was associated with a recently identified HDAC7/Foxp3 transcriptional complex. Upon activation through the T cell receptor in vitro or in vivo, Nur77Tg T cells showed only marginally decreased proliferation but significantly increased apoptosis. Fully allogeneic cardiac grafts transplanted to Nur77Tg mice survived long-term with well-preserved structure, and recipient splenocytes showed markedly enhanced apoptosis and greatly reduced anti-donor recall responses. Allografts in Nur77Tg recipients had significantly increased expression of multiple Treg-associated genes, including Foxp3, Foxp1, Tip60 and HDAC9. Allograft rejection was restored by CD25 monoclonal antibody therapy, indicating that allograft acceptance was dependent upon Treg function in Nur77Tg recipients. These data show that compared to conventional CD4 and CD8 T cells, Foxp3+ Tregs are relatively resistant to Nur77-mediated apoptosis, and that tipping the balance between the numbers of Tregs and responder T cells in the early period post-transplantation can determine the fate of the allograft. Hence, induced expression of Nur77 might be a novel means to achieve long-term allograft survival.

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Increased apoptosis of Nur77Tg T cells upon activation in vitro and in vivo.Splenocytes from WT or Nur77Tg mice were stimulated with PMA/ionomycin for 72 h, followed by flow cytometry using T cell markers and Annexin-V and 7-AAD staining. (a) Live cells were gated based on forward vs. side scatter, and (b) T cell apoptosis was based on Annexin-V and 7-AAD staining. (c) CFSE-labeled B6 or Nur77Tg spleen and lymph node cells were adoptively transferred into B6/DBA F1 mice. After 72 h, donor-derived live CD4+ or CD8+ T cells were identified by gating on H-2Kd (-) and H-2Dd (-) cells; figure in each square indicates percentage of the gated population. Data are representative of 3 experiments with similar results.
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pone-0002321-g002: Increased apoptosis of Nur77Tg T cells upon activation in vitro and in vivo.Splenocytes from WT or Nur77Tg mice were stimulated with PMA/ionomycin for 72 h, followed by flow cytometry using T cell markers and Annexin-V and 7-AAD staining. (a) Live cells were gated based on forward vs. side scatter, and (b) T cell apoptosis was based on Annexin-V and 7-AAD staining. (c) CFSE-labeled B6 or Nur77Tg spleen and lymph node cells were adoptively transferred into B6/DBA F1 mice. After 72 h, donor-derived live CD4+ or CD8+ T cells were identified by gating on H-2Kd (-) and H-2Dd (-) cells; figure in each square indicates percentage of the gated population. Data are representative of 3 experiments with similar results.

Mentions: Although Nur77 over-expression causes increased thymocyte apoptosis during development, its effects on peripheral T cell apoptosis after activation are unclear. Whole splenocytes from WT or Nur77Tg mice were stimulated for 72 h with soluble CD3 mAb (1 µg/ml) alone or CD3 plus CD28 mAbs (0.5 µg/ml). Far fewer live Nur77Tg cells were recovered after in vitro stimulation as compared to WT splenocytes (Figure 2a). Similar differences were seen after stimulation for 72 h with PMA plus ionomycin; both CD4+ and CD8+ T cells from Nur77Tg mice showed significantly increased apoptosis after activation as compared to WT T cells (Figure 2b). We used a parent-to-F1 adoptive transfer model to assess the effects of Nur77 over-expression on T cell survival upon allogeneic stimulation in vivo. Use of Nur77Tg mice as a source of donor T cells led to a markedly reduced recovery of T cells after 72 h as compared to use of T cells from WT mice (Figure 2c), suggesting markedly increased death of Nur77Tg T cells as a result of alloantigen-induced T cell activation and proliferation in vivo.


Resistance of Foxp3+ regulatory T cells to Nur77-induced apoptosis promotes allograft survival.

Tao R, Hancock WW - PLoS ONE (2008)

Increased apoptosis of Nur77Tg T cells upon activation in vitro and in vivo.Splenocytes from WT or Nur77Tg mice were stimulated with PMA/ionomycin for 72 h, followed by flow cytometry using T cell markers and Annexin-V and 7-AAD staining. (a) Live cells were gated based on forward vs. side scatter, and (b) T cell apoptosis was based on Annexin-V and 7-AAD staining. (c) CFSE-labeled B6 or Nur77Tg spleen and lymph node cells were adoptively transferred into B6/DBA F1 mice. After 72 h, donor-derived live CD4+ or CD8+ T cells were identified by gating on H-2Kd (-) and H-2Dd (-) cells; figure in each square indicates percentage of the gated population. Data are representative of 3 experiments with similar results.
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Related In: Results  -  Collection

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pone-0002321-g002: Increased apoptosis of Nur77Tg T cells upon activation in vitro and in vivo.Splenocytes from WT or Nur77Tg mice were stimulated with PMA/ionomycin for 72 h, followed by flow cytometry using T cell markers and Annexin-V and 7-AAD staining. (a) Live cells were gated based on forward vs. side scatter, and (b) T cell apoptosis was based on Annexin-V and 7-AAD staining. (c) CFSE-labeled B6 or Nur77Tg spleen and lymph node cells were adoptively transferred into B6/DBA F1 mice. After 72 h, donor-derived live CD4+ or CD8+ T cells were identified by gating on H-2Kd (-) and H-2Dd (-) cells; figure in each square indicates percentage of the gated population. Data are representative of 3 experiments with similar results.
Mentions: Although Nur77 over-expression causes increased thymocyte apoptosis during development, its effects on peripheral T cell apoptosis after activation are unclear. Whole splenocytes from WT or Nur77Tg mice were stimulated for 72 h with soluble CD3 mAb (1 µg/ml) alone or CD3 plus CD28 mAbs (0.5 µg/ml). Far fewer live Nur77Tg cells were recovered after in vitro stimulation as compared to WT splenocytes (Figure 2a). Similar differences were seen after stimulation for 72 h with PMA plus ionomycin; both CD4+ and CD8+ T cells from Nur77Tg mice showed significantly increased apoptosis after activation as compared to WT T cells (Figure 2b). We used a parent-to-F1 adoptive transfer model to assess the effects of Nur77 over-expression on T cell survival upon allogeneic stimulation in vivo. Use of Nur77Tg mice as a source of donor T cells led to a markedly reduced recovery of T cells after 72 h as compared to use of T cells from WT mice (Figure 2c), suggesting markedly increased death of Nur77Tg T cells as a result of alloantigen-induced T cell activation and proliferation in vivo.

Bottom Line: Overexpression of Nur77 in the T cell lineage decreased numbers of peripheral CD4 and CD8 T cells by approximately 80% compared to wild-type (WT) mice.Upon activation through the T cell receptor in vitro or in vivo, Nur77Tg T cells showed only marginally decreased proliferation but significantly increased apoptosis.Allografts in Nur77Tg recipients had significantly increased expression of multiple Treg-associated genes, including Foxp3, Foxp1, Tip60 and HDAC9.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Stokes Research Institute and Biesecker Pediatric Liver Center, Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
The NR4A nuclear receptor family member Nur77 (NR4A1) promotes thymocyte apoptosis during negative selection of autoreactive thymocytes, but may also function in mature extrathymic T cells. We studied the effects of over-expression of Nur77 on the apoptosis of murine peripheral T cells, including thymic-derived Foxp3+ regulatory (Treg) cells. Overexpression of Nur77 in the T cell lineage decreased numbers of peripheral CD4 and CD8 T cells by approximately 80% compared to wild-type (WT) mice. However, the proportions of Treg cells were markedly increased in the thymus (61% of CD4+Foxp3+ singly positive thymocytes vs. 8% in WT) and secondary lymphoid organs (40-50% of CD4+Foxp3+ T cells vs. 7-8% in WT) of Nur77 transgenic (Nur77Tg) mice, and immunoprecipitation studies showed Nur77 was associated with a recently identified HDAC7/Foxp3 transcriptional complex. Upon activation through the T cell receptor in vitro or in vivo, Nur77Tg T cells showed only marginally decreased proliferation but significantly increased apoptosis. Fully allogeneic cardiac grafts transplanted to Nur77Tg mice survived long-term with well-preserved structure, and recipient splenocytes showed markedly enhanced apoptosis and greatly reduced anti-donor recall responses. Allografts in Nur77Tg recipients had significantly increased expression of multiple Treg-associated genes, including Foxp3, Foxp1, Tip60 and HDAC9. Allograft rejection was restored by CD25 monoclonal antibody therapy, indicating that allograft acceptance was dependent upon Treg function in Nur77Tg recipients. These data show that compared to conventional CD4 and CD8 T cells, Foxp3+ Tregs are relatively resistant to Nur77-mediated apoptosis, and that tipping the balance between the numbers of Tregs and responder T cells in the early period post-transplantation can determine the fate of the allograft. Hence, induced expression of Nur77 might be a novel means to achieve long-term allograft survival.

Show MeSH