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Perturbation of the T lymphocyte lineage in transgenic mice expressing a constitutive repressor of nuclear factor (NF)-kappaB.

Boothby MR, Mora AL, Scherer DC, Brockman JA, Ballard DW - J. Exp. Med. (1997)

Bottom Line: In addition to deregulated T cell growth and survival, transgene expression impairs the development of normal T cell populations as evidenced by diminished numbers of TCRhi CD8 single-positive thymocytes.This defect was significantly amplified in the periphery and was accompanied by a decrease in CD4(+) T cells.Taken together, these in vivo findings indicate that the NF-kappaB/Rel signaling pathway contains compensatory components that are essential for the establishment of normal T cell subsets.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.

ABSTRACT
Members of the nuclear factor (NF)-kappaB/Rel family transcription factors are induced during thymic selection and in mature T lymphocytes after ligation of the T cell antigen receptor (TCR). Despite these findings, disruption of individual NF-kappaB/Rel genes has revealed no intrinsic defect in the development of mature T cells, perhaps reflecting functional redundancy. To circumvent this possibility, the T cell lineage was targeted to express a trans-dominant form of IkappaBalpha that constitutively represses the activity of multiple NF-kappaB/Rel proteins. Transgenic cells expressing this inhibitor exhibit a significant proliferative defect, which is not reversed by the addition of exogenous interleukin-2. Moreover, mitogenic stimulation of splenocytes leads to increased apoptosis of transgenic T cells as compared with controls. In addition to deregulated T cell growth and survival, transgene expression impairs the development of normal T cell populations as evidenced by diminished numbers of TCRhi CD8 single-positive thymocytes. This defect was significantly amplified in the periphery and was accompanied by a decrease in CD4(+) T cells. Taken together, these in vivo findings indicate that the NF-kappaB/Rel signaling pathway contains compensatory components that are essential for the establishment of normal T cell subsets.

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Increased apoptosis among activated T cells from IκBα(ΔN)  transgenic mice. (A) Splenocytes from either NTg (open bars) or Tghi  (closed bars) mice were cultured in the presence of Con A (2.5 μg/ml).  After 40 h in culture, cells were divided equally, stained with PE-labeled  antibodies against CD4 or CD8, and subjected to TUNEL analysis. The  data represent the mean percentage (± SEM) of TUNEL-positive cells in  each T cell subset. The mean was calculated using data from 19 individual  Tghi mice or an equal number of NTg controls (10 independent experiments with 6–8-wk-old mice). The increased frequencies of apoptotic  cells among transgenic CD4+ and CD8+ cells were statistically significant  when compared with nontransgenic CD4+ or CD8+ cells (P <0.001).  (B) Splenocytes were activated using plate-bound anti-CD3, then processed for TUNEL assays as in A. Results represent cumulative data from  12 individual Tghi mice or an equal number of NTg controls (six independent experiments with 6-8-wk-old mice). The increased frequencies  of apoptotic cells among transgenic CD4+ and CD8+ cells were statistically significant when compared with nontransgenic CD4+ or CD8+ cells  (P ⩽0.001).
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Figure 4: Increased apoptosis among activated T cells from IκBα(ΔN) transgenic mice. (A) Splenocytes from either NTg (open bars) or Tghi (closed bars) mice were cultured in the presence of Con A (2.5 μg/ml). After 40 h in culture, cells were divided equally, stained with PE-labeled antibodies against CD4 or CD8, and subjected to TUNEL analysis. The data represent the mean percentage (± SEM) of TUNEL-positive cells in each T cell subset. The mean was calculated using data from 19 individual Tghi mice or an equal number of NTg controls (10 independent experiments with 6–8-wk-old mice). The increased frequencies of apoptotic cells among transgenic CD4+ and CD8+ cells were statistically significant when compared with nontransgenic CD4+ or CD8+ cells (P <0.001). (B) Splenocytes were activated using plate-bound anti-CD3, then processed for TUNEL assays as in A. Results represent cumulative data from 12 individual Tghi mice or an equal number of NTg controls (six independent experiments with 6-8-wk-old mice). The increased frequencies of apoptotic cells among transgenic CD4+ and CD8+ cells were statistically significant when compared with nontransgenic CD4+ or CD8+ cells (P ⩽0.001).

Mentions: It is now well recognized that programmed cell death can occur in response to primary stimulation through the TCR (34–36) or after activated T cells are exposed to certain proapoptotic agents (37). Moreover, evidence from studies with established cell lines suggests that NF-κB may either inhibit (38, 39) or enhance their susceptibility to apoptosis (40–42). Accordingly, we next investigated the influence of IκBα(ΔN) transgene expression on entry into the apoptotic pathway after primary activation of resting T cells with mitogens. When the prevalence of apoptotic T cells was determined by TUNEL assays after mitogenic stimulation with Con A, we observed that expression of the IκBα(ΔN) transgene led to a two-fold increase in the frequency of T cell apoptosis (Fig. 4 A). This enhancement in programmed cell death applied to both CD4+ and CD8+ T cells. Similar results were obtained using immobilized antiTCR antibodies as the stimulus, albeit with a more pronounced effect on the CD8+ subset (Fig. 4 B). Selective gating on lymphoblasts revealed a doubling in the frequency of apoptotic CD8+ T cells derived from the transgenic mice as compared with controls. Thus, the increased sensitivity to apoptosis observed under these experimental conditions cannot be attributed solely to the failure of these cells to undergo blast transformation. We conclude that the expression of IκBα(ΔN) in transgenic T cells is associated with enhanced apoptosis of T cells in response to primary TCR stimulation (35, 36).


Perturbation of the T lymphocyte lineage in transgenic mice expressing a constitutive repressor of nuclear factor (NF)-kappaB.

Boothby MR, Mora AL, Scherer DC, Brockman JA, Ballard DW - J. Exp. Med. (1997)

Increased apoptosis among activated T cells from IκBα(ΔN)  transgenic mice. (A) Splenocytes from either NTg (open bars) or Tghi  (closed bars) mice were cultured in the presence of Con A (2.5 μg/ml).  After 40 h in culture, cells were divided equally, stained with PE-labeled  antibodies against CD4 or CD8, and subjected to TUNEL analysis. The  data represent the mean percentage (± SEM) of TUNEL-positive cells in  each T cell subset. The mean was calculated using data from 19 individual  Tghi mice or an equal number of NTg controls (10 independent experiments with 6–8-wk-old mice). The increased frequencies of apoptotic  cells among transgenic CD4+ and CD8+ cells were statistically significant  when compared with nontransgenic CD4+ or CD8+ cells (P <0.001).  (B) Splenocytes were activated using plate-bound anti-CD3, then processed for TUNEL assays as in A. Results represent cumulative data from  12 individual Tghi mice or an equal number of NTg controls (six independent experiments with 6-8-wk-old mice). The increased frequencies  of apoptotic cells among transgenic CD4+ and CD8+ cells were statistically significant when compared with nontransgenic CD4+ or CD8+ cells  (P ⩽0.001).
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Figure 4: Increased apoptosis among activated T cells from IκBα(ΔN) transgenic mice. (A) Splenocytes from either NTg (open bars) or Tghi (closed bars) mice were cultured in the presence of Con A (2.5 μg/ml). After 40 h in culture, cells were divided equally, stained with PE-labeled antibodies against CD4 or CD8, and subjected to TUNEL analysis. The data represent the mean percentage (± SEM) of TUNEL-positive cells in each T cell subset. The mean was calculated using data from 19 individual Tghi mice or an equal number of NTg controls (10 independent experiments with 6–8-wk-old mice). The increased frequencies of apoptotic cells among transgenic CD4+ and CD8+ cells were statistically significant when compared with nontransgenic CD4+ or CD8+ cells (P <0.001). (B) Splenocytes were activated using plate-bound anti-CD3, then processed for TUNEL assays as in A. Results represent cumulative data from 12 individual Tghi mice or an equal number of NTg controls (six independent experiments with 6-8-wk-old mice). The increased frequencies of apoptotic cells among transgenic CD4+ and CD8+ cells were statistically significant when compared with nontransgenic CD4+ or CD8+ cells (P ⩽0.001).
Mentions: It is now well recognized that programmed cell death can occur in response to primary stimulation through the TCR (34–36) or after activated T cells are exposed to certain proapoptotic agents (37). Moreover, evidence from studies with established cell lines suggests that NF-κB may either inhibit (38, 39) or enhance their susceptibility to apoptosis (40–42). Accordingly, we next investigated the influence of IκBα(ΔN) transgene expression on entry into the apoptotic pathway after primary activation of resting T cells with mitogens. When the prevalence of apoptotic T cells was determined by TUNEL assays after mitogenic stimulation with Con A, we observed that expression of the IκBα(ΔN) transgene led to a two-fold increase in the frequency of T cell apoptosis (Fig. 4 A). This enhancement in programmed cell death applied to both CD4+ and CD8+ T cells. Similar results were obtained using immobilized antiTCR antibodies as the stimulus, albeit with a more pronounced effect on the CD8+ subset (Fig. 4 B). Selective gating on lymphoblasts revealed a doubling in the frequency of apoptotic CD8+ T cells derived from the transgenic mice as compared with controls. Thus, the increased sensitivity to apoptosis observed under these experimental conditions cannot be attributed solely to the failure of these cells to undergo blast transformation. We conclude that the expression of IκBα(ΔN) in transgenic T cells is associated with enhanced apoptosis of T cells in response to primary TCR stimulation (35, 36).

Bottom Line: In addition to deregulated T cell growth and survival, transgene expression impairs the development of normal T cell populations as evidenced by diminished numbers of TCRhi CD8 single-positive thymocytes.This defect was significantly amplified in the periphery and was accompanied by a decrease in CD4(+) T cells.Taken together, these in vivo findings indicate that the NF-kappaB/Rel signaling pathway contains compensatory components that are essential for the establishment of normal T cell subsets.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.

ABSTRACT
Members of the nuclear factor (NF)-kappaB/Rel family transcription factors are induced during thymic selection and in mature T lymphocytes after ligation of the T cell antigen receptor (TCR). Despite these findings, disruption of individual NF-kappaB/Rel genes has revealed no intrinsic defect in the development of mature T cells, perhaps reflecting functional redundancy. To circumvent this possibility, the T cell lineage was targeted to express a trans-dominant form of IkappaBalpha that constitutively represses the activity of multiple NF-kappaB/Rel proteins. Transgenic cells expressing this inhibitor exhibit a significant proliferative defect, which is not reversed by the addition of exogenous interleukin-2. Moreover, mitogenic stimulation of splenocytes leads to increased apoptosis of transgenic T cells as compared with controls. In addition to deregulated T cell growth and survival, transgene expression impairs the development of normal T cell populations as evidenced by diminished numbers of TCRhi CD8 single-positive thymocytes. This defect was significantly amplified in the periphery and was accompanied by a decrease in CD4(+) T cells. Taken together, these in vivo findings indicate that the NF-kappaB/Rel signaling pathway contains compensatory components that are essential for the establishment of normal T cell subsets.

Show MeSH
Related in: MedlinePlus