Chronic activation of the kinase IKKβ impairs T cell function and survival

Abstract

Activation of the transcription factor NF-κB is critical for cytokine production and T cell survival after TCR engagement. The effects of persistent NF-κB activity on T cell function and survival are poorly understood. In this study, using a murine model that expresses a constitutively active form of inhibitor of NF-κB kinase β (caIKKβ) in a T cell-specific manner, we demonstrate that chronic inhibitor of NF-κB kinase β signaling promotes T cell apoptosis, attenuates responsiveness to TCR-mediated stimulation in vitro, and impairs T cell responses to bacterial infection in vivo. caIKKβ T cells showed increased Fas ligand expression and caspase-8 activation, and blocking Fas/Fas ligand interactions enhanced cell survival. T cell unresponsiveness was associated with defects in TCR proximal signaling and elevated levels of B lymphocyte-induced maturation protein 1, a transcriptional repressor that promotes T cell exhaustion. caIKKβ T cells also showed a defect in IL-2 production, and addition of exogenous IL-2 enhanced their survival and proliferation. Conditional deletion of B lymphocyte-induced maturation protein 1 partially rescued the sensitivity of caIKKβ T cells to TCR triggering. Furthermore, adoptively transferred caIKKβ T cells showed diminished expansion and increased contraction in response to infection with Listeria monocytogenes expressing a cognate Ag. Despite their functional defects, caIKKβ T cells readily produced proinflammatory cytokines, and mice developed autoimmunity. In contrast to NF-κB's critical role in T cell activation and survival, our study demonstrates that persistent IKK-NF-κB signaling is sufficient to impair both T cell function and survival.

Figure 1. Constitutive IKKβ signaling impairs mature T cell survival

a) Representative FACS plots of CD4 and CD8 expression on total thymocytes from WT and IKK mice. b) Absolute numbers of total thymocytes, and DN, DP, CD4SP and CD8SP thymocytes from WT and IKK mice. Mean ± SEM are calculated from four independent experiments. c) Representative FACS plots of CD4 and CD8 expression on total splenocytes from WT and IKK mice. d) Percentages and absolute numbers of CD4 and CD8 T cells in WT and IKK spleens. Mean ± SEM are calculated from four independent experiments. e) Representative photograph of spleens from 9 week old WT and IKK mice; spleen weights and absolute splenocyte numbers of mice 7 weeks or older. Each circle represents data from an individual mouse and horizontal bars indicate the mean ± SEM. f) Representative FACS plots of splenocytes stained with Invitrogen® Live/Dead marker; Percentages of Live/Dead⁺ cells from WT and IKK spleens. Mean ± SEM are calculated from six independent experiments.

Figure 2. Increased FasL expression contributes to enhanced IKK T cell apoptosis via the extrinsic pathway

a) Representative FACS plots of Live/Dead and AnnexinV staining of CD4 and CD8 T cells from WT and IKK spleens. b) Representative immunoblots of lysates prepared from purified WT and IKK T cells and probed for the indicated proteins. c) qPCR of Fas and FasL mRNA in CD4 and CD8 cells from WT and IKK spleen. Data shown are representative of two independent experiments. d) Representative histograms of Fas and FasL expression on CD4 and CD8 T cells from WT and IKK spleen. e and f) WT and IKK splenocytes were left unstimulated or stimulated with anti-CD3 (2C11) antibody for 65 hours in the presence of absence of 10 μg/ml anti-FasL blocking antibody, followed by CD4, CD8, and Live/Dead staining and FACS analysis. e) Representative FACS plots; f) Bar graph presentation of mean ± SEM from a single experiment in triplicate. Data shown are representative of three independent experiments.

Figure 3. Defects in TCR proximal signaling impair IKK T cell activation

a) Representative histograms of CD25 and CD69 expression on WT and IKK splenocytes stimulated overnight with anti-CD3 antibody. b) Representative histograms showing CFSE dilution in WT and IKK splenocytes stimulated for 72 hours with anti-CD3 antibody. c) Representative immunoblots of lysates prepared from purified WT and IKK T cells stimulated for indicated time with anti-CD3 (500A2) antibody, and probed for the indicated TCR signaling proteins. d) Representative FACS plots of calcium influx (monitored as a ratio of fluorescence at 405 and 510 nm) into Indo-1 loaded WT and IKK LN cells after TCR and CD4/CD8 cross-linking at the indicated time point. e) Representative immunoblots of nuclear and cytoplasmic fractions from purified WT and IKK T cells left unstimulated or stimulated for 6 hours with anti-CD3 (500A2) and anti-CD28 antibodies. Data shown are representative of at least three independent experiments.

Figure 4. Defective IL-2 production contributes to impairment in IKK T cell survival and proliferation

a) Representative FACS plots of IL-2 production by WT and IKK splenic T cells left unstimulated or stimulated as indicated in the presence of Golgi Plug for 4-6 hours. Percentage of viable CD4 and CD8 cells among WT and IKK splenocytes stimulated with anti-CD3 (2C11) antibody for 72 hours in the presence or absence of 100 U/ml exogenous IL-2 (b) or 0.5 μg/ml anti-CD28 antibody (c). Data shown are mean ± SEM from a single experiment in triplicate, and are representative of three independent experiments. d) Representative histograms showing CFSE dilution in WT and IKK splenocytes stimulated for 65 hours with anti-CD3 antibody, in the presence or absence of 100 U/ml exogenous IL-2 (b) or 0.5 μg/ml anti-CD28 antibody. All FACS plots shown are representative of three independent experiments.

Figure 5. Impaired antigen-specific IKK T cell responses in vivo

a) Schematic representation of experimental design showing individual (solid lines) and competitive (dotted lines) adoptive transfer models. (b-d) Individual adoptive transfers of sorted WT (Thy1.1⁺ Vα2⁺ CD8⁺) OT1 and IKK (Thy1.2⁺Vα2⁺CD8⁺) OT1 cells into WT Thy1.1⁺Th1.2⁺ recipients - Representative FACS analysis of peripheral blood samples to detect Vα2⁺ CD8 T cells (b) and congenic markers within the gated Vα2⁺ CD8 T cell population (c) at indicated time points after infection. d) Percentage of WT (Thy1.1⁺) and IKK (Thy1.2⁺) cells among total CD8⁺ cells in the peripheral blood. Mean ± SEM are calculated for 3 mice per group. Data shown are representative of two independent experiments. e-f) Competitive adoptive transfers. Sorted WT and IKK OT1 T cells were mixed in a 1:1 ratio, transferred into recipients and analyzed as in (b-d). Mean ± SEM are calculated for 3 mice per group. Data shown are representative of two independent experiments.

Figure 6. Blimp1 deficiency partially restores IKK T cell responsiveness to TCR stimulation

a) qPCR and representative FACS analysis of PD1 mRNA and protein expression in WT and IKK CD4 and CD8 splenic T cells. b) qPCR analysis of Blimp1/Prdm1 mRNA expression in WT and IKK CD4 and CD8 splenic T cells. Mean ± SEM for qPCR analyses are calculated from two independent experiments. c) Representative FACS plots of CD25 and CD69 expression on WT, IKK and IKK BlimpKO CD4 and CD8 splenic T cells left unstimulated or stimulated overnight with anti-CD3 antibody. d) Representative histograms showing CFSE dilution in WT, IKK and IKK BlimpKO CD4 and CD8 splenic T cells with or without anti-CD3 stimulation for 72 hours. Histograms shown are representative of at least two independent experiments.

Figure 7. Enhanced cytokine production and multi-organ infiltration in IKK mice

a) FACS plots of CD44 and CD62L expression on WT and IKK CD4 and CD8 splenic T cells. b) FACS plots showing intracellular staining for IFNγ and IL-17A in WT and IKK CD4 and CD8 T cells left unstimulated or stimulated for 6 hours in the presence of PMA, ionomycin and Golgi plug. c) Hematoxylin and eosin staining of lung, liver and pancreas sections from 6-month old WT and IKK mice. Photographs were taken using a Fisher Scientific Micromaster® microscope and Westover Scientific Micron software at 400x magnification. d) Immunofluorescence staining of liver sections from 6-month old WT and IKK mice. Photographs were taken using a Zeiss ApoTome Microscope and Axiovision software at 200x magnification. e) qPCR analysis of mRNA levels of indicated chemokines in purified WT and IKK CD4 and CD8 splenic T cells. Data shown are representative of at least four (a) or three (b, c, d, e) independent experiments.