NF-κB is crucial in proximal T-cell signaling for calcium influx and NFAT activation

Abstract

In the accepted model of T-cell activation, parallel signal-transduction pathways activate the transcription factors NF-κB, NFAT, and AP-1 to drive clonal expansion of T cells in response to Ag. Genome-wide transcriptional profiling following Ag-induced CD8(+) T-cell activation in C57BL/6 mouse T cells revealed that genes regulated by NFAT were also reduced in the absence of NF-κB p50 and cRel subunits. Importantly, p50(-/-) cRel(-/-) CD8(+) T cells had significantly diminished NFAT and AP-1 activation compared with WT or PKCθ(-/-) CD8(+) T cells. Attenuated NFAT activation after TCR engagement was associated with reduced calcium influx, PLCγ and Zap70 activation. Interestingly, pharmacological bypass of PLCγ-regulated pathways largely rescued p50(-/-) cRel(-/-) T-cell proliferative defects. These results indicate a crucial and unexpected requirement for NF-κB p50 and cRel subunits in proximal TCR signaling and calcium responses. They further suggest that key defects in T cells in the absence of NF-κB pathway components may be due to impaired proximal T-cell signaling.

Keywords: Gene expression; NF-κB; Signal transduction; T-cell activation; Transcription factors.

Figure 1. NF-κB p50^−/−cRel^−/− CD8⁺ T cells exhibit pronounced clonal expansion defects in vitro and in vivo

(A) In vitro proliferation of OT-1 WT, PKCθ^−/− and p50^−/−cRel^−/− CD8⁺ T cells cultured alone or in the presence of DC + OVA. CPM values represent ³H-thymidine incorporation. (B) Relative amount of IL-2 mRNA present in WT OT-1, PKCθ^−/− and p50^−/−cRel^−/− CD8⁺ T cells after 18 h culture without or with DC + OVA, determined by microarray and normalized to 18S ribosomal RNA. (C) Clonal expansion of transplanted OT-1 WT, PKCθ^−/− and p50^−/−cRel^−/− CD8⁺ T cells was determined in peripheral blood 7 days post TriVax administration by flow cytometry; values represent % of endogenous (45.1) and transplanted (45.2) OVA-Tetramer⁺ CD8⁺ T cells/viable (DAPI-) PBMCs. (D) Percentage of transplanted (CD45.2) OVA-Tetramer⁺ CD8⁺ T cells/PBMC over 5 weeks, determined by flow cytometry. ((A–D) Data are shown as mean + SEM (n=4 mice per genotype) and are representative of two independent experiments. Statistical significance is indicated as *p<0.05, **p<0.01, ***p<0.001, Student’s t-test.

Figure 2. p50^−/−cRel^−/− T cells have defects in NFAT and AP-1 activation and calcium influx

(A) Unstimulated and stimulated OT-1 WT, PKCθ^−/− and p50^−/−cRel^−/− CD8⁺ T cells after 18 h culture with DC and OVA were used to prepare nuclear lysates and EMSA was performed to detect NF-κB, NFAT and AP-1. (B) Same as (A) except polyclonal WT, PKCθ^−/−, PKCα^−/−PKCθ^−/− and p50^−/−cRel^−/− CD8⁺ T cells were used, and stimulated with 1 μg/mL anti-CD3/CD28 for 18 h. (C) Western blotting to determine NFATc1 in nuclear fraction of WT and p50^−/−cRel^−/− CD8⁺ T cells unstimulated or stimulated with 1 μg/mL CD3/CD28 for 18 h. (D) WT, PKCθ^−/−, and p50^−/−cRel^−/− polyclonal CD8⁺ T cells were analyzed for the ability to influx calcium: the first 60 s represent a baseline after which the following components were added to the culture: (*)10 μg/mL CD3/CD28 and (#) 9 mM EGTA. Points are Arbitrary Fluorescent Units (AFU) and lines represent one of three repeats within each assay. (E) Same as (D) except polyclonal CD8⁺ cells were cultured for 48 h with 1 μg/mL CD3/CD28, rested for 2 h, and then assayed for calcium after anti-CD3/CD28 treatment. (F–H) Same as (D) except OT-1 T cells were used and graphs compare calcium influx based on T-cell:DC interaction ± OVA. (A–H) Data are from one experiment (n=4 mice/genotype/pooled and spread across treatments), representative of three independent experiments.

Figure 3. Reduced PLCγ and Zap70 activation in p50^−/−cRel^−/− CD8+⁺ T cells

(A) WT and p50^−/−cRel^−/− CD8⁺ T cells untreated or stimulated with 10 μg/ml anti-CD3/CD28 for 2, 5 or 10 min after which lysates were made and western blotting was performed to detect pPLCγ, total PLCγ, pZap70 and total Zap70. (B) Same as (A), but only the 5 min time-point was used for detection of pZap70, total Zap70, pLAT (Y132), pLAT (Y171), and total LAT. (C) WT and p50^−/−cRel^−/− CD8⁺ T cells were assayed for the ability to influx calcium as in Figure 2C, in unstimulated or either 10 μg/mL CD3/CD28 or 50 ng PMA and 500 ng ionomycin stimulated cells. (D) WT and p50^−/−cRel^−/− CD8+ ⁺ T cells were cultured alone or with 50 ng PMA and 500 ng ionomycin for 6 h; nuclear lysates were made and western blotting was performed to detect NFAT, RelA and c-Jun. (E) In vitro proliferation of WT and p50^−/−cRel^−/− CD8⁺ T cells cultured alone or with 1 μg/mL anti-CD3/CD28 and/or 50 ng PMA and 500 ng ionomycin for 48 h; values represent CPM of ³H-thymidine incorporation and are given as mean + SD. (A–E) Data are from one experiment (n=2–4 mice/genotype), representative of three independent experiments. Statistical significance is indicated as *p<0.05, **p<0.01, ***p<0.001, Student’s t-test.