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Peripheral residence of naïve CD4 T cells induces MHC class II-dependent alterations in phenotype and function.

Rane S, Das R, Ranganathan V, Prabhu S, Das A, Mattoo H, Durdik JM, George A, Rath S, Bal V - BMC Biol. (2014)

Bottom Line: It is not clear if these interactions result in alterations in their activation, survival and effector programming.Naïve CD4lo and CD4hi subsets of thymic single-positive CD4 T cells did not show differences whereas peripheral naïve CD4lo and CD4hi subsets of T cell receptor (TCR)-transgenic T cells did.Our findings also suggest the feasibility of potential pharmacological interventions for improved CD4 T cell responses during vaccination of older people via either anti-oxidant or DUSP inhibitor small molecules.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Immunology, New Delhi, 110067, India. sanketrn@nii.ac.in.

ABSTRACT

Background: As individual naïve CD4 T lymphocytes circulate in the body after emerging from the thymus, they are likely to have individually varying microenvironmental interactions even in the absence of stimulation via specific target recognition. It is not clear if these interactions result in alterations in their activation, survival and effector programming. Naïve CD4 T cells show unimodal distribution for many phenotypic properties, suggesting that the variation is caused by intrinsic stochasticity, although underlying variation due to subsets created by different histories of microenvironmental interactions remains possible. To explore this possibility, we began examining the phenotype and functionality of naïve CD4 T cells differing in a basic unimodally distributed property, the CD4 levels, as well as the causal origin of these differences.

Results: We examined separated CD4hi and CD4lo subsets of mouse naïve CD4 cells. CD4lo cells were smaller with higher CD5 levels and lower levels of the dual-specific phosphatase (DUSP)6-suppressing micro-RNA miR181a, and responded poorly with more Th2-skewed outcomes. Human naïve CD4lo and CD4hi cells showed similar differences. Naïve CD4lo and CD4hi subsets of thymic single-positive CD4 T cells did not show differences whereas peripheral naïve CD4lo and CD4hi subsets of T cell receptor (TCR)-transgenic T cells did. Adoptive transfer-mediated parking of naïve CD4 cells in vivo lowered CD4 levels, increased CD5 and reactive oxygen species (ROS) levels and induced hyporesponsiveness in them, dependent, at least in part, on availability of major histocompatibility complex class II (MHCII) molecules. ROS scavenging or DUSP inhibition ameliorated hyporesponsiveness. Naïve CD4 cells from aged mice showed lower CD4 levels and cell sizes, higher CD5 levels, and hyporesponsiveness and Th2-skewing reversed by DUSP inhibition.

Conclusions: Our data show that, underlying a unimodally distributed property, the CD4 level, there are subsets of naïve CD4 cells that vary in the time spent in the periphery receiving MHCII-mediated signals and show resultant alteration of phenotype and functionality via ROS and DUSP activity. Our findings also suggest the feasibility of potential pharmacological interventions for improved CD4 T cell responses during vaccination of older people via either anti-oxidant or DUSP inhibitor small molecules.

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The Erk-DUSP6-miR181a axis contributes to hyporesponsiveness of NCD4lo T cells. A. Histogram overlays of baseline ex vivo p-Erk (plotted on log on left and linear scale on right) in NCD4lo and NCD4hi cells. B. Pooled data of paired samples showing baseline p-Erk MFI values (Mean ± SE) in NCD4lo and NCD4hi cells. C. ΔΔCt values of Real time RT-PCR for miR-181a normalized to miR-142 on NCD4hi and NCD4lo cells (Mean ± SE from four sets of independently sorted cells). D. 3H-Thymidine incorporation of NCD4lo and NCD4hi cells treated or not with BCI prior to activation (Mean ± SE of triplicate cultures). Data representative of five mice, one of three experiments. E. Relative IFNγ/IL-4 and IFNγ/IL-13 ratios for BCI treated or untreated NCD4lo cells during recall response. Data from three independent experiments shown normalized to respective ratios for NCD4hi cells as 100%, shown as a dotted line. BCI, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one; DUSP6, dual-specific phosphatase 6; MFI, mean fluorescence intensity; NCD4, naïve CD4 T cells; SE, standard error.
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Fig8: The Erk-DUSP6-miR181a axis contributes to hyporesponsiveness of NCD4lo T cells. A. Histogram overlays of baseline ex vivo p-Erk (plotted on log on left and linear scale on right) in NCD4lo and NCD4hi cells. B. Pooled data of paired samples showing baseline p-Erk MFI values (Mean ± SE) in NCD4lo and NCD4hi cells. C. ΔΔCt values of Real time RT-PCR for miR-181a normalized to miR-142 on NCD4hi and NCD4lo cells (Mean ± SE from four sets of independently sorted cells). D. 3H-Thymidine incorporation of NCD4lo and NCD4hi cells treated or not with BCI prior to activation (Mean ± SE of triplicate cultures). Data representative of five mice, one of three experiments. E. Relative IFNγ/IL-4 and IFNγ/IL-13 ratios for BCI treated or untreated NCD4lo cells during recall response. Data from three independent experiments shown normalized to respective ratios for NCD4hi cells as 100%, shown as a dotted line. BCI, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one; DUSP6, dual-specific phosphatase 6; MFI, mean fluorescence intensity; NCD4, naïve CD4 T cells; SE, standard error.

Mentions: Phosphorylation of mitogen-activated protein (MAP) family tyrosine kinase Erk has been shown to be poor in many aging cells, including T cells [6,8,42,43] suggesting that T cell age may also be correlated with Erk phosphorylation. We examined baseline p-Erk levels ex vivo in NCD4hi and NCD4lo cells. NCD4lo cells show significantly lower levels compared to NCD4hi cells (Figure 8A log and linear plot, and 8B). The defective phosphorylation of Erk in human T cells is regulated by the activity of DUSP6, which in turn is controlled by miR-181a [8]. NCD4 cells from older people are reported to express lower levels of miR-181a (relative to a control miRNA, miR-142) than NCD4 cells from young people [8]. We therefore estimated the levels of miR-181a and miR-142 transcripts in mouse T cells using real-time RT-PCR assays. Levels of miR-181a transcripts (normalized to miR-142 transcript levels) were lower in NCD4lo cells compared to NCD4hi cells (Figure 8C), further suggesting that because of longer peripheral residence NCD4lo cells show a pattern similar to that reported in NCD4 T cells from older individuals [8]. Consistent with the role of miRNA-181a in suppressing DUSP6 expression [8], when NCD4lo and NCD4hi cells were treated with a pharmacological inhibitor of DUSP, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI) prior to activation, NCD4lo cells responded better following treatment with BCI (Figure 8D), while NCD4hi cells showed no effect of BCI. Further, analysis of cytokines secreted by NCD4lo and NCD4hi cells treated with BCI and differentiated in vitro showed that the relative Th2 tendency of NCD4lo cells was partially reversed by BCI treatment (Figure 8E).Figure 8


Peripheral residence of naïve CD4 T cells induces MHC class II-dependent alterations in phenotype and function.

Rane S, Das R, Ranganathan V, Prabhu S, Das A, Mattoo H, Durdik JM, George A, Rath S, Bal V - BMC Biol. (2014)

The Erk-DUSP6-miR181a axis contributes to hyporesponsiveness of NCD4lo T cells. A. Histogram overlays of baseline ex vivo p-Erk (plotted on log on left and linear scale on right) in NCD4lo and NCD4hi cells. B. Pooled data of paired samples showing baseline p-Erk MFI values (Mean ± SE) in NCD4lo and NCD4hi cells. C. ΔΔCt values of Real time RT-PCR for miR-181a normalized to miR-142 on NCD4hi and NCD4lo cells (Mean ± SE from four sets of independently sorted cells). D. 3H-Thymidine incorporation of NCD4lo and NCD4hi cells treated or not with BCI prior to activation (Mean ± SE of triplicate cultures). Data representative of five mice, one of three experiments. E. Relative IFNγ/IL-4 and IFNγ/IL-13 ratios for BCI treated or untreated NCD4lo cells during recall response. Data from three independent experiments shown normalized to respective ratios for NCD4hi cells as 100%, shown as a dotted line. BCI, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one; DUSP6, dual-specific phosphatase 6; MFI, mean fluorescence intensity; NCD4, naïve CD4 T cells; SE, standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4306244&req=5

Fig8: The Erk-DUSP6-miR181a axis contributes to hyporesponsiveness of NCD4lo T cells. A. Histogram overlays of baseline ex vivo p-Erk (plotted on log on left and linear scale on right) in NCD4lo and NCD4hi cells. B. Pooled data of paired samples showing baseline p-Erk MFI values (Mean ± SE) in NCD4lo and NCD4hi cells. C. ΔΔCt values of Real time RT-PCR for miR-181a normalized to miR-142 on NCD4hi and NCD4lo cells (Mean ± SE from four sets of independently sorted cells). D. 3H-Thymidine incorporation of NCD4lo and NCD4hi cells treated or not with BCI prior to activation (Mean ± SE of triplicate cultures). Data representative of five mice, one of three experiments. E. Relative IFNγ/IL-4 and IFNγ/IL-13 ratios for BCI treated or untreated NCD4lo cells during recall response. Data from three independent experiments shown normalized to respective ratios for NCD4hi cells as 100%, shown as a dotted line. BCI, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one; DUSP6, dual-specific phosphatase 6; MFI, mean fluorescence intensity; NCD4, naïve CD4 T cells; SE, standard error.
Mentions: Phosphorylation of mitogen-activated protein (MAP) family tyrosine kinase Erk has been shown to be poor in many aging cells, including T cells [6,8,42,43] suggesting that T cell age may also be correlated with Erk phosphorylation. We examined baseline p-Erk levels ex vivo in NCD4hi and NCD4lo cells. NCD4lo cells show significantly lower levels compared to NCD4hi cells (Figure 8A log and linear plot, and 8B). The defective phosphorylation of Erk in human T cells is regulated by the activity of DUSP6, which in turn is controlled by miR-181a [8]. NCD4 cells from older people are reported to express lower levels of miR-181a (relative to a control miRNA, miR-142) than NCD4 cells from young people [8]. We therefore estimated the levels of miR-181a and miR-142 transcripts in mouse T cells using real-time RT-PCR assays. Levels of miR-181a transcripts (normalized to miR-142 transcript levels) were lower in NCD4lo cells compared to NCD4hi cells (Figure 8C), further suggesting that because of longer peripheral residence NCD4lo cells show a pattern similar to that reported in NCD4 T cells from older individuals [8]. Consistent with the role of miRNA-181a in suppressing DUSP6 expression [8], when NCD4lo and NCD4hi cells were treated with a pharmacological inhibitor of DUSP, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI) prior to activation, NCD4lo cells responded better following treatment with BCI (Figure 8D), while NCD4hi cells showed no effect of BCI. Further, analysis of cytokines secreted by NCD4lo and NCD4hi cells treated with BCI and differentiated in vitro showed that the relative Th2 tendency of NCD4lo cells was partially reversed by BCI treatment (Figure 8E).Figure 8

Bottom Line: It is not clear if these interactions result in alterations in their activation, survival and effector programming.Naïve CD4lo and CD4hi subsets of thymic single-positive CD4 T cells did not show differences whereas peripheral naïve CD4lo and CD4hi subsets of T cell receptor (TCR)-transgenic T cells did.Our findings also suggest the feasibility of potential pharmacological interventions for improved CD4 T cell responses during vaccination of older people via either anti-oxidant or DUSP inhibitor small molecules.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Immunology, New Delhi, 110067, India. sanketrn@nii.ac.in.

ABSTRACT

Background: As individual naïve CD4 T lymphocytes circulate in the body after emerging from the thymus, they are likely to have individually varying microenvironmental interactions even in the absence of stimulation via specific target recognition. It is not clear if these interactions result in alterations in their activation, survival and effector programming. Naïve CD4 T cells show unimodal distribution for many phenotypic properties, suggesting that the variation is caused by intrinsic stochasticity, although underlying variation due to subsets created by different histories of microenvironmental interactions remains possible. To explore this possibility, we began examining the phenotype and functionality of naïve CD4 T cells differing in a basic unimodally distributed property, the CD4 levels, as well as the causal origin of these differences.

Results: We examined separated CD4hi and CD4lo subsets of mouse naïve CD4 cells. CD4lo cells were smaller with higher CD5 levels and lower levels of the dual-specific phosphatase (DUSP)6-suppressing micro-RNA miR181a, and responded poorly with more Th2-skewed outcomes. Human naïve CD4lo and CD4hi cells showed similar differences. Naïve CD4lo and CD4hi subsets of thymic single-positive CD4 T cells did not show differences whereas peripheral naïve CD4lo and CD4hi subsets of T cell receptor (TCR)-transgenic T cells did. Adoptive transfer-mediated parking of naïve CD4 cells in vivo lowered CD4 levels, increased CD5 and reactive oxygen species (ROS) levels and induced hyporesponsiveness in them, dependent, at least in part, on availability of major histocompatibility complex class II (MHCII) molecules. ROS scavenging or DUSP inhibition ameliorated hyporesponsiveness. Naïve CD4 cells from aged mice showed lower CD4 levels and cell sizes, higher CD5 levels, and hyporesponsiveness and Th2-skewing reversed by DUSP inhibition.

Conclusions: Our data show that, underlying a unimodally distributed property, the CD4 level, there are subsets of naïve CD4 cells that vary in the time spent in the periphery receiving MHCII-mediated signals and show resultant alteration of phenotype and functionality via ROS and DUSP activity. Our findings also suggest the feasibility of potential pharmacological interventions for improved CD4 T cell responses during vaccination of older people via either anti-oxidant or DUSP inhibitor small molecules.

Show MeSH
Related in: MedlinePlus