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Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation.

Li J, Hardy K, Phetsouphanh C, Tu WJ, Sutcliffe EL, McCuaig R, Sutton CR, Zafar A, Munier CM, Zaunders JJ, Xu Y, Theodoratos A, Tan A, Lim PS, Knaute T, Masch A, Zerweck J, Brezar V, Milburn PJ, Dunn J, Casarotto MG, Turner SJ, Seddiki N, Kelleher AD, Rao S - J. Cell. Sci. (2016)

Bottom Line: Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation.This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood.Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4(+) T cells.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Education, Science, Technology & Mathematics, University of Canberra, Canberra, Australian Capital Territory 2617, Australia Department of Microbiology & Immunology, The Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria 3010, Australia.

No MeSH data available.


Related in: MedlinePlus

Colocalization of PKC-θ and NF-κB in T cells. (A) Representative immunoblots of PKC-θ, p50 and p65 protein levels in the nuclear extracts of activated Jurkat T cells (0, 2, 4 h) during primary (1°), and secondary (2°) stimulation with Sp1 as the loading control (representative graph of three independent repeats). The corresponding p65 and PKC-θ (normalized to Sp-1) densitometry is shown below (mean±s.e.m., n=3). *P≤0.05; ns, not significant (two-way ANOVA). (B) NF-κB (p65) activity detected in nuclear extracts from Jurkat T cells during day 0, primary, and day 9, secondary, activation (0, 2 and 4 h) with PMA and Ca2+ ionophore (+P/I). Representative graph of three independent replicates, error bars show s.e.m. of technical replicates. (C) The percentage of CD45RO− (naïve) and CD45RO+ (memory) ex-vivo-derived human CD4+ T cells with p50 and p65 staining as detected by using flow cytometry with (+P/I) or without (NS) PMA and Ca2+ ionophore (median±quartiles, n=6). The whiskers represent the first and fourth quartiles. **P≤0.01 (Wilcoxon matched-pairs signed rank test was used to compare groups). (D) p50 and p65 staining levels were calculated as fold-changes between human CD45RO− (naïve) and CD45RO+ (memory) T cells with (+P/I) or without (NS) PMA and Ca2+ ionophore (mean±s.e.m., n=6).
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JCS181248F4: Colocalization of PKC-θ and NF-κB in T cells. (A) Representative immunoblots of PKC-θ, p50 and p65 protein levels in the nuclear extracts of activated Jurkat T cells (0, 2, 4 h) during primary (1°), and secondary (2°) stimulation with Sp1 as the loading control (representative graph of three independent repeats). The corresponding p65 and PKC-θ (normalized to Sp-1) densitometry is shown below (mean±s.e.m., n=3). *P≤0.05; ns, not significant (two-way ANOVA). (B) NF-κB (p65) activity detected in nuclear extracts from Jurkat T cells during day 0, primary, and day 9, secondary, activation (0, 2 and 4 h) with PMA and Ca2+ ionophore (+P/I). Representative graph of three independent replicates, error bars show s.e.m. of technical replicates. (C) The percentage of CD45RO− (naïve) and CD45RO+ (memory) ex-vivo-derived human CD4+ T cells with p50 and p65 staining as detected by using flow cytometry with (+P/I) or without (NS) PMA and Ca2+ ionophore (median±quartiles, n=6). The whiskers represent the first and fourth quartiles. **P≤0.01 (Wilcoxon matched-pairs signed rank test was used to compare groups). (D) p50 and p65 staining levels were calculated as fold-changes between human CD45RO− (naïve) and CD45RO+ (memory) T cells with (+P/I) or without (NS) PMA and Ca2+ ionophore (mean±s.e.m., n=6).

Mentions: To quantify the relationship between nuclear translocation and gene-specific recruitment of PKC-θ and p65, nuclear p50 and p65 protein expression was examined in primary and secondary activated Jurkat T cells. Despite the basal expression of nuclear PKC-θ in non-stimulated cells, PKC-θ expression increased after primary and secondary activation. This was particularly associated with p65 nuclear translocation (Fig. 4A) and activity in secondary activated cells (Fig. 4B). In contrast, the levels of the p50 NF-κB subunit remained constant (Fig. 4A). ChIP-qPCR also demonstrated that the increase of PKC-θ enrichment in the secondary activated Jurkat T cells was colocalized with p65 and Pol II at the two-well characterized p65-binding promoters (Himes et al., 1993) IL2 and TNF, as well as at TNFSF9 and SATB1 (Fig. S4A), but not at a PKC-θ non-binding region in SMAD3 (Fig. S4B). We then sorted bulk naïve and memory human CD4+ T cells to show that higher levels of p65 and p50 were detected in resting human memory (CD45RO+) CD4+ T cells than naïve cells (CD45RO−), with a further increase demonstrable upon activation with PMA and Ca2+ ionophore (Fig. 4C,D). ChIP-PCR analysis showed a similar, but stimulus-dependent, increase in p65 enrichment at the TNF promoter in activated memory CD4+ T cells (Fig. S4C). Therefore, we found that PKC-θ and p65 recruitment was more effective in both transcriptional-memory-responsive Jurkat T cells and primary human CD4+ memory T cells.Fig. 4.


Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation.

Li J, Hardy K, Phetsouphanh C, Tu WJ, Sutcliffe EL, McCuaig R, Sutton CR, Zafar A, Munier CM, Zaunders JJ, Xu Y, Theodoratos A, Tan A, Lim PS, Knaute T, Masch A, Zerweck J, Brezar V, Milburn PJ, Dunn J, Casarotto MG, Turner SJ, Seddiki N, Kelleher AD, Rao S - J. Cell. Sci. (2016)

Colocalization of PKC-θ and NF-κB in T cells. (A) Representative immunoblots of PKC-θ, p50 and p65 protein levels in the nuclear extracts of activated Jurkat T cells (0, 2, 4 h) during primary (1°), and secondary (2°) stimulation with Sp1 as the loading control (representative graph of three independent repeats). The corresponding p65 and PKC-θ (normalized to Sp-1) densitometry is shown below (mean±s.e.m., n=3). *P≤0.05; ns, not significant (two-way ANOVA). (B) NF-κB (p65) activity detected in nuclear extracts from Jurkat T cells during day 0, primary, and day 9, secondary, activation (0, 2 and 4 h) with PMA and Ca2+ ionophore (+P/I). Representative graph of three independent replicates, error bars show s.e.m. of technical replicates. (C) The percentage of CD45RO− (naïve) and CD45RO+ (memory) ex-vivo-derived human CD4+ T cells with p50 and p65 staining as detected by using flow cytometry with (+P/I) or without (NS) PMA and Ca2+ ionophore (median±quartiles, n=6). The whiskers represent the first and fourth quartiles. **P≤0.01 (Wilcoxon matched-pairs signed rank test was used to compare groups). (D) p50 and p65 staining levels were calculated as fold-changes between human CD45RO− (naïve) and CD45RO+ (memory) T cells with (+P/I) or without (NS) PMA and Ca2+ ionophore (mean±s.e.m., n=6).
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JCS181248F4: Colocalization of PKC-θ and NF-κB in T cells. (A) Representative immunoblots of PKC-θ, p50 and p65 protein levels in the nuclear extracts of activated Jurkat T cells (0, 2, 4 h) during primary (1°), and secondary (2°) stimulation with Sp1 as the loading control (representative graph of three independent repeats). The corresponding p65 and PKC-θ (normalized to Sp-1) densitometry is shown below (mean±s.e.m., n=3). *P≤0.05; ns, not significant (two-way ANOVA). (B) NF-κB (p65) activity detected in nuclear extracts from Jurkat T cells during day 0, primary, and day 9, secondary, activation (0, 2 and 4 h) with PMA and Ca2+ ionophore (+P/I). Representative graph of three independent replicates, error bars show s.e.m. of technical replicates. (C) The percentage of CD45RO− (naïve) and CD45RO+ (memory) ex-vivo-derived human CD4+ T cells with p50 and p65 staining as detected by using flow cytometry with (+P/I) or without (NS) PMA and Ca2+ ionophore (median±quartiles, n=6). The whiskers represent the first and fourth quartiles. **P≤0.01 (Wilcoxon matched-pairs signed rank test was used to compare groups). (D) p50 and p65 staining levels were calculated as fold-changes between human CD45RO− (naïve) and CD45RO+ (memory) T cells with (+P/I) or without (NS) PMA and Ca2+ ionophore (mean±s.e.m., n=6).
Mentions: To quantify the relationship between nuclear translocation and gene-specific recruitment of PKC-θ and p65, nuclear p50 and p65 protein expression was examined in primary and secondary activated Jurkat T cells. Despite the basal expression of nuclear PKC-θ in non-stimulated cells, PKC-θ expression increased after primary and secondary activation. This was particularly associated with p65 nuclear translocation (Fig. 4A) and activity in secondary activated cells (Fig. 4B). In contrast, the levels of the p50 NF-κB subunit remained constant (Fig. 4A). ChIP-qPCR also demonstrated that the increase of PKC-θ enrichment in the secondary activated Jurkat T cells was colocalized with p65 and Pol II at the two-well characterized p65-binding promoters (Himes et al., 1993) IL2 and TNF, as well as at TNFSF9 and SATB1 (Fig. S4A), but not at a PKC-θ non-binding region in SMAD3 (Fig. S4B). We then sorted bulk naïve and memory human CD4+ T cells to show that higher levels of p65 and p50 were detected in resting human memory (CD45RO+) CD4+ T cells than naïve cells (CD45RO−), with a further increase demonstrable upon activation with PMA and Ca2+ ionophore (Fig. 4C,D). ChIP-PCR analysis showed a similar, but stimulus-dependent, increase in p65 enrichment at the TNF promoter in activated memory CD4+ T cells (Fig. S4C). Therefore, we found that PKC-θ and p65 recruitment was more effective in both transcriptional-memory-responsive Jurkat T cells and primary human CD4+ memory T cells.Fig. 4.

Bottom Line: Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation.This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood.Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4(+) T cells.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Education, Science, Technology & Mathematics, University of Canberra, Canberra, Australian Capital Territory 2617, Australia Department of Microbiology & Immunology, The Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria 3010, Australia.

No MeSH data available.


Related in: MedlinePlus