Limits...
A regulatory role for TRAF1 in antigen-induced apoptosis of T cells.

Speiser DE, Lee SY, Wong B, Arron J, Santana A, Kong YY, Ohashi PS, Choi Y - J. Exp. Med. (1997)

Bottom Line: In the TNFR2-mediated signaling pathways, TRAF2 works as a mediator for activation signals such as NF-kappaB, but the role of TRAF1 has not been previously determined.Here we show in transgenic mice that TRAF1 overexpression inhibits antigen-induced apoptosis of CD8(+) T lymphocytes.Our results demonstrate a biological role for TRAF1 as a regulator of apoptotic signals and also support the hypothesis that the combination of TRAF proteins in a given cell type determines distinct biological effects triggered by members of the TNF receptor superfamily.

View Article: PubMed Central - PubMed

Affiliation: Ontario Cancer Institute, Departments of Medical Biophysics and Immunology, Toronto, Ontario M5G 2M9, Canada.

ABSTRACT
Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) and TRAF1 were found as components of the TNFR2 signaling complex, which exerts multiple biological effects on cells such as cell proliferation, cytokine production, and cell death. In the TNFR2-mediated signaling pathways, TRAF2 works as a mediator for activation signals such as NF-kappaB, but the role of TRAF1 has not been previously determined. Here we show in transgenic mice that TRAF1 overexpression inhibits antigen-induced apoptosis of CD8(+) T lymphocytes. Our results demonstrate a biological role for TRAF1 as a regulator of apoptotic signals and also support the hypothesis that the combination of TRAF proteins in a given cell type determines distinct biological effects triggered by members of the TNF receptor superfamily.

Show MeSH

Related in: MedlinePlus

In vitro proliferation and apoptosis of mature T cells. (A)  TRAF1 overexpression does not affect TCR-mediated proliferation of  mature T cells in vitro. T cells were isolated from lymph nodes of control  (open circle) and TRAF1.TG-10 transgenic (closed circle) littermates using  T cell enrichment columns (Biotex Laboratories, Inc.) and cultured in triplicate into anti-TCR Ab (H57-597) coated-tissue culture wells containing  mitomycin C–treated splenocytes (5 × 105/well). [3H]thymidine was  added 3 d later for 8–12 h. The culture was harvested and analyzed as described in Materials and Methods. The data show the mean cpm ± SEM  from the triplicate samples. The data are from one of several similar experiments using T cells from nontransgenic and TRAF1.TG-10 transgenic mice littermates (H-2d/b). Similar results were obtained when T  cells from TRAF1.TG-11 mice were used (data not shown). (B and C)  TRAF1 overexpression inhibits TCR-mediated apoptosis of CD8+ T  cells in vitro. A representative experiment showing the flow cytometric  plots displaying propidium iodide (PI) fluorescence versus forward light  scatter for TRAF1 transgenic and negative littermates after 48 h with IL-2  in either uncoated wells (medium) or wells coated with anti-CD3ε (10  μg/ml) is shown in B. Viable cells were quantified by PI exclusion using  a FACSCaliber® with CELLQUEST software and show low PI fluorescence. Percent of total cells in each region is indicated. In C, the percentages of cell survival in CD8− (CD4+) or CD8+ T cells were shown  as the mean ± SEM from the triplicate samples of three mice from each  group. Cells, cultured in either uncoated (medium) or anti-CD3ε–coated  plates as described in B, were recovered from the plates and stained with  anti-CD8 Ab (53-6.7; FITC conjugated). The CD8 staining was used to  gate the cells into CD8+ and CD8− pools; control staining showed that  virtually all CD8− cells were CD4+. Specific cell survival (%) was determined from the percentages of viable (PI-negative) cells and it equals 100 ×  (% survival after CD3 cross-linking/% survival without CD3 cross-linking).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196328&req=5

Figure 2: In vitro proliferation and apoptosis of mature T cells. (A) TRAF1 overexpression does not affect TCR-mediated proliferation of mature T cells in vitro. T cells were isolated from lymph nodes of control (open circle) and TRAF1.TG-10 transgenic (closed circle) littermates using T cell enrichment columns (Biotex Laboratories, Inc.) and cultured in triplicate into anti-TCR Ab (H57-597) coated-tissue culture wells containing mitomycin C–treated splenocytes (5 × 105/well). [3H]thymidine was added 3 d later for 8–12 h. The culture was harvested and analyzed as described in Materials and Methods. The data show the mean cpm ± SEM from the triplicate samples. The data are from one of several similar experiments using T cells from nontransgenic and TRAF1.TG-10 transgenic mice littermates (H-2d/b). Similar results were obtained when T cells from TRAF1.TG-11 mice were used (data not shown). (B and C) TRAF1 overexpression inhibits TCR-mediated apoptosis of CD8+ T cells in vitro. A representative experiment showing the flow cytometric plots displaying propidium iodide (PI) fluorescence versus forward light scatter for TRAF1 transgenic and negative littermates after 48 h with IL-2 in either uncoated wells (medium) or wells coated with anti-CD3ε (10 μg/ml) is shown in B. Viable cells were quantified by PI exclusion using a FACSCaliber® with CELLQUEST software and show low PI fluorescence. Percent of total cells in each region is indicated. In C, the percentages of cell survival in CD8− (CD4+) or CD8+ T cells were shown as the mean ± SEM from the triplicate samples of three mice from each group. Cells, cultured in either uncoated (medium) or anti-CD3ε–coated plates as described in B, were recovered from the plates and stained with anti-CD8 Ab (53-6.7; FITC conjugated). The CD8 staining was used to gate the cells into CD8+ and CD8− pools; control staining showed that virtually all CD8− cells were CD4+. Specific cell survival (%) was determined from the percentages of viable (PI-negative) cells and it equals 100 × (% survival after CD3 cross-linking/% survival without CD3 cross-linking).

Mentions: The TRAF1 transgenic mice revealed no gross anatomical abnormalities. Flow cytometry analysis of thymocytes from 6- to 8-wk-old TRAF1 transgenic or negative littermates did not indicate any significant difference in the total number of thymocytes (data not shown). Moreover, the percentage of double-negative (CD4−CD8−), double-positive (CD4+CD8+), and single-positive (CD4+ or CD8+) thymocytes in TRAF1 transgenic mice was comparable to that in negative littermates (data not shown). The numbers and ratios of T and B cells or CD4+ and CD8+ T cells from spleen or lymph nodes of TRAF1 transgenic mice were also comparable to those in negative littermates (data not shown), suggesting that TRAF1 overexpression did not dramatically affect lymphocyte development. When purified T cells from TRAF1 transgenic and negative littermates were tested for their ability to divide in response to anti-TCR antibody (H57-597) in vitro, T cells from TRAF1 transgenic and negative littermates responded similarly to antiTCR Ab (Fig. 2 A). These results demonstrate that TRAF1 overexpression did not affect TCR-mediated proliferation of mature T cells in vitro.


A regulatory role for TRAF1 in antigen-induced apoptosis of T cells.

Speiser DE, Lee SY, Wong B, Arron J, Santana A, Kong YY, Ohashi PS, Choi Y - J. Exp. Med. (1997)

In vitro proliferation and apoptosis of mature T cells. (A)  TRAF1 overexpression does not affect TCR-mediated proliferation of  mature T cells in vitro. T cells were isolated from lymph nodes of control  (open circle) and TRAF1.TG-10 transgenic (closed circle) littermates using  T cell enrichment columns (Biotex Laboratories, Inc.) and cultured in triplicate into anti-TCR Ab (H57-597) coated-tissue culture wells containing  mitomycin C–treated splenocytes (5 × 105/well). [3H]thymidine was  added 3 d later for 8–12 h. The culture was harvested and analyzed as described in Materials and Methods. The data show the mean cpm ± SEM  from the triplicate samples. The data are from one of several similar experiments using T cells from nontransgenic and TRAF1.TG-10 transgenic mice littermates (H-2d/b). Similar results were obtained when T  cells from TRAF1.TG-11 mice were used (data not shown). (B and C)  TRAF1 overexpression inhibits TCR-mediated apoptosis of CD8+ T  cells in vitro. A representative experiment showing the flow cytometric  plots displaying propidium iodide (PI) fluorescence versus forward light  scatter for TRAF1 transgenic and negative littermates after 48 h with IL-2  in either uncoated wells (medium) or wells coated with anti-CD3ε (10  μg/ml) is shown in B. Viable cells were quantified by PI exclusion using  a FACSCaliber® with CELLQUEST software and show low PI fluorescence. Percent of total cells in each region is indicated. In C, the percentages of cell survival in CD8− (CD4+) or CD8+ T cells were shown  as the mean ± SEM from the triplicate samples of three mice from each  group. Cells, cultured in either uncoated (medium) or anti-CD3ε–coated  plates as described in B, were recovered from the plates and stained with  anti-CD8 Ab (53-6.7; FITC conjugated). The CD8 staining was used to  gate the cells into CD8+ and CD8− pools; control staining showed that  virtually all CD8− cells were CD4+. Specific cell survival (%) was determined from the percentages of viable (PI-negative) cells and it equals 100 ×  (% survival after CD3 cross-linking/% survival without CD3 cross-linking).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196328&req=5

Figure 2: In vitro proliferation and apoptosis of mature T cells. (A) TRAF1 overexpression does not affect TCR-mediated proliferation of mature T cells in vitro. T cells were isolated from lymph nodes of control (open circle) and TRAF1.TG-10 transgenic (closed circle) littermates using T cell enrichment columns (Biotex Laboratories, Inc.) and cultured in triplicate into anti-TCR Ab (H57-597) coated-tissue culture wells containing mitomycin C–treated splenocytes (5 × 105/well). [3H]thymidine was added 3 d later for 8–12 h. The culture was harvested and analyzed as described in Materials and Methods. The data show the mean cpm ± SEM from the triplicate samples. The data are from one of several similar experiments using T cells from nontransgenic and TRAF1.TG-10 transgenic mice littermates (H-2d/b). Similar results were obtained when T cells from TRAF1.TG-11 mice were used (data not shown). (B and C) TRAF1 overexpression inhibits TCR-mediated apoptosis of CD8+ T cells in vitro. A representative experiment showing the flow cytometric plots displaying propidium iodide (PI) fluorescence versus forward light scatter for TRAF1 transgenic and negative littermates after 48 h with IL-2 in either uncoated wells (medium) or wells coated with anti-CD3ε (10 μg/ml) is shown in B. Viable cells were quantified by PI exclusion using a FACSCaliber® with CELLQUEST software and show low PI fluorescence. Percent of total cells in each region is indicated. In C, the percentages of cell survival in CD8− (CD4+) or CD8+ T cells were shown as the mean ± SEM from the triplicate samples of three mice from each group. Cells, cultured in either uncoated (medium) or anti-CD3ε–coated plates as described in B, were recovered from the plates and stained with anti-CD8 Ab (53-6.7; FITC conjugated). The CD8 staining was used to gate the cells into CD8+ and CD8− pools; control staining showed that virtually all CD8− cells were CD4+. Specific cell survival (%) was determined from the percentages of viable (PI-negative) cells and it equals 100 × (% survival after CD3 cross-linking/% survival without CD3 cross-linking).
Mentions: The TRAF1 transgenic mice revealed no gross anatomical abnormalities. Flow cytometry analysis of thymocytes from 6- to 8-wk-old TRAF1 transgenic or negative littermates did not indicate any significant difference in the total number of thymocytes (data not shown). Moreover, the percentage of double-negative (CD4−CD8−), double-positive (CD4+CD8+), and single-positive (CD4+ or CD8+) thymocytes in TRAF1 transgenic mice was comparable to that in negative littermates (data not shown). The numbers and ratios of T and B cells or CD4+ and CD8+ T cells from spleen or lymph nodes of TRAF1 transgenic mice were also comparable to those in negative littermates (data not shown), suggesting that TRAF1 overexpression did not dramatically affect lymphocyte development. When purified T cells from TRAF1 transgenic and negative littermates were tested for their ability to divide in response to anti-TCR antibody (H57-597) in vitro, T cells from TRAF1 transgenic and negative littermates responded similarly to antiTCR Ab (Fig. 2 A). These results demonstrate that TRAF1 overexpression did not affect TCR-mediated proliferation of mature T cells in vitro.

Bottom Line: In the TNFR2-mediated signaling pathways, TRAF2 works as a mediator for activation signals such as NF-kappaB, but the role of TRAF1 has not been previously determined.Here we show in transgenic mice that TRAF1 overexpression inhibits antigen-induced apoptosis of CD8(+) T lymphocytes.Our results demonstrate a biological role for TRAF1 as a regulator of apoptotic signals and also support the hypothesis that the combination of TRAF proteins in a given cell type determines distinct biological effects triggered by members of the TNF receptor superfamily.

View Article: PubMed Central - PubMed

Affiliation: Ontario Cancer Institute, Departments of Medical Biophysics and Immunology, Toronto, Ontario M5G 2M9, Canada.

ABSTRACT
Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) and TRAF1 were found as components of the TNFR2 signaling complex, which exerts multiple biological effects on cells such as cell proliferation, cytokine production, and cell death. In the TNFR2-mediated signaling pathways, TRAF2 works as a mediator for activation signals such as NF-kappaB, but the role of TRAF1 has not been previously determined. Here we show in transgenic mice that TRAF1 overexpression inhibits antigen-induced apoptosis of CD8(+) T lymphocytes. Our results demonstrate a biological role for TRAF1 as a regulator of apoptotic signals and also support the hypothesis that the combination of TRAF proteins in a given cell type determines distinct biological effects triggered by members of the TNF receptor superfamily.

Show MeSH
Related in: MedlinePlus