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. 2012 Jun 29;287(27):23010-9.
doi: 10.1074/jbc.M112.350538. Epub 2012 May 8.

Opposing roles for TRAF1 in the alternative versus classical NF-κB pathway in T cells

Ann J McPherson  1 , Laura M Snell, Tak W Mak, Tania H Watts
Affiliations

Opposing roles for TRAF1 in the alternative versus classical NF-κB pathway in T cells

Ann J McPherson et al. J Biol Chem. .

Abstract

T cells lacking TRAF1 hyperproliferate in response to T cell receptor signaling but have impaired signaling downstream of specific TNFR family members such as 4-1BB. Here we resolve this paradox by showing that while TRAF1 is required for maximal activation of the classical NF-κB pathway downstream of 4-1BB in primary T cells, TRAF1 also restricts the constitutive activation of NIK in anti-CD3-activated T cells. Activation of the alternative NF-κB pathway is restricted in unstimulated cells by a cIAP1/2:TRAF2:TRAF3:NIK complex. Using knockdown of NIK by siRNA we show that in activated CD8 T cells TRAF1 is also involved in this process and that constitutive activation of the alternative NF-κB pathway is responsible for costimulation independent hyperproliferation and excess cytokine production in TRAF1-deficient CD8 T cells compared with WT CD8 T cells. The T cell costimulatory molecule 4-1BB critically regulates the survival of activated and memory CD8 T cells. We demonstrate that stimulation through 4-1BB induces cIAP1-dependent TRAF3 degradation and activation of the alternative NF-κB pathway. We also show that while both TRAF1 and cIAP1 have non-redundant roles in suppressing the alternative NF-κB pathway in T cells activated in the absence of costimulation, activation of the classical NF-κB pathway downstream of 4-1BB requires TRAF1, whereas cIAP1 plays a redundant role with cIAP2. Collectively these results demonstrate that TRAF1 plays a critical role in regulating T cell activation both through restricting the costimulation independent activation of NIK in activated T cells and by promoting the 4-1BB-induced classical NF-κB pathway.

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Figures

FIGURE 1.
FIGURE 1.
4-1BB induced activation of the canonical NF-κB pathway requires TRAF1 and either cIAP1 or cIAP2. WT, TRAF1−/−, or cIAP1−/− OT-I splenocytes were stimulated with SIINFEKL peptide for 48 h and rested for 24 h. A and B, cells were stimulated in the presence of agonistic anti-4-1BB antibody or a rat isotype control for the indicated time and actin and IκB levels were analyzed in WT or TRAF1−/− T cells (A) or WT and cIAP−/− T cells (B) at the times indicated. C, cIAP1−/− OT-I T cells were further transfected with one of three variants of siRNA targeting cIAP2 or with scrambled RNA, and 20 h later cIAP2 protein levels were assessed by Western blot. D, C variant siRNA was used to knockdown cIAP2 expression and 20 h later IκB expression was assessed. E, activated OT-I CD8 T cells were isolated and 4-1BB expression determined. Data in A–E are representative of at least three independent experiments.
FIGURE 2.
FIGURE 2.
Activated TRAF1−/− CD8 T cells display costimulation independent hyperproliferation and cytokine production. Purified primary naïve CD8 T cells (106/ml) from WT and TRAF1−/− mice were CFSE labeled and stimulated for 2 days with the indicated agonistic antibodies. A, proliferation was measured by CFSE dilution, (B) IFNγ production and (C) TNFα production were measured by intracellular staining. The numbers in the upper-boxed quadrant indicate the percent of cells staining for the cytokine. Data are representative of at least three experiments.
FIGURE 3.
FIGURE 3.
4-1BB signaling leads to cIAP1-dependent TRAF3 degradation and activation of the non-canononical NF-κB pathway. A, purified primary CD8 T cells (106/ml) from WT C57BL/6 mice were stimulated for 18 h with immobilized anti-CD3 (1 μg/ml) alone or with anti-CD3 plus immobilized anti- 4-1BB (10 μg/ml) or with anti-CD3 plus anti-CD28 (10 μg/ml) and lysates examined for p100 and p52 as well as actin. B, OT-I T cells were activated as in Fig. 1 and stimulated with agonistic anti-4-1BB antibody for the indicated time. Activation of alternative NF-κB was determined by Western blotting for p100/p52 as well as for levels of the upstream NF-κB regulators, TRAF2 (B) and TRAF3 (C). D, activated WT and cIAP1−/− OT-I T cells were stimulated for 3 h and TRAF3 expression analyzed. Data are representative of at least three independent experiments.
FIGURE 4.
FIGURE 4.
Costimulation- independent activation of the alternative NF-κB pathway in CD8 T cells is restrained by TRAF1. Activation of the alternative NF-κB pathway was determined by Western blotting for p100/p52. A, purified naïve CD8 T cells from WT and TRAF1−/− mice were stimulated for 2 days with anti-CD3 and the indicated costimulatory antibodies. B, antigen-activated WT or TRAF1−/− OT-I T cells, prepared as in Fig. 1, were further stimulated with agonistic anti-4-1BB antibody for 6 h and lysates analyzed for p100 processing to p52. Data are representative of three experiments.
FIGURE 5.
FIGURE 5.
Role of NIK in the proliferation and function of activated WT and TRAF1−/− CD8 T cells. Purified naïve CD8 T cells from WT or TRAF1−/− mice were CFSE labeled and transfected with siRNA targeting NIK or a scrambled control. Cells were rested for 20 h and then stimulated for 2 days with the indicated agonistic antibodies. A, analysis of p100 processing in stimulated cells with or without NIK knockdown was analyzed by Western blot as a surrogate for measuring NIK levels. B, WT CD8 T cells and (C) TRAF1−/− CD8 T cells were transfected with scrambled siRNA or siRNA targeting NIK, proliferation of T cells was measured by CFSE dilution and IFNγ and TNFα production was measured by intracellular staining. The frequency of IFNγ and TNFα producing cells is shown as an average of three replicate wells. Data are representative of at least two experiments.
FIGURE 6.
FIGURE 6.
cIAP1−/− CD8 T cells show increased effector function but still respond to costimulation. A, purified primary CD8 T cells (106/ml) from WT and cIAP1−/− mice were stimulated for 18 h with immobilized anti-CD3 (1 μg/ml) alone or with anti-CD3 plus immobilized anti-4-1BB (10 μg/ml) or with anti-CD3 plus anti-CD28 (10 μg/ml) and lysates examined for p100 and p52 as well as actin expression. B, purified primary CD8 T cells from WT or cIAP1−/− mice were labeled with CFSE and stimulated as in A and cell division monitored by CFSE dilution. C, cIAP1−/− CD8 T cells were transfected with scrambled siRNA or siRNA targeting NIK, proliferation of T cells was measured by CFSE dilution and IFNγ and TNFα production was measured by intracellular staining. The frequency of IFNγ and TNFα-producing cells is shown as an average of three replicate wells. Data are representative of at least two experiments.
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