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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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NCD4lo cells resemble naïve CD4 T cells from aged mice in their phenotypic and functional attributes. A. CD4 levels on ANCD4 and YNCD4 cells (plotted on log on left and linear scale on right) stained as a mixture in a single well. B. Representative forward scatter histograms for ANCD4 and YNCD4 cells. C. Comparison of CD4 MFI values of ANCD4 and YNCD4 cells (Mean ± SE; data representative of three experiments). D. Data for forward scatter from individual mice (Mean ± SE; data representative of three experiments). E. Representative data showing levels of cytokines in supernatants of in vitro primed and recalled YNCD4 and ANCD4 cells. F. IFNγ/IL-13 ratios and mean ± SE calculated based on absorbance values. G. Histogram overlays of baseline ex vivo p-Erk (top) and total Erk (bottom) in ANCD4 and YNCD4 cells (plotted on log on left and linear scale on right). H. Pooled data, of paired samples showing baseline p-Erk MFI values (Mean ± SE) in ANCD4 and YNCD4 cells. I. ΔΔCt values of Real time RT-PCR for miR-181a normalized to miR-142 on ANCD4 and YNCD4 cells (Mean ± SE from five sets of independently sorted cells). J. 3H-Thymidine incorporation of ANCD4 and YNCD4 cells treated or not with BCI prior to activation (Mean ± SE of triplicate cultures). Data representative of six mice, one of three experiments. K. IFNγ/IL-13 and IFNγ/IL-4 ratio for YNCD4, BCI treated ANCD4 or BCI untreated ANCD4 cells during recall response. Data representing one of four independent experiments. ANCD4, naïve CD4 T cells from aged mice; BCI, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one; MFI, mean fluorescence intensity; SE, standard error; YNCD4, naïve CD4 T cells from young mice.
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Fig9: NCD4lo cells resemble naïve CD4 T cells from aged mice in their phenotypic and functional attributes. A. CD4 levels on ANCD4 and YNCD4 cells (plotted on log on left and linear scale on right) stained as a mixture in a single well. B. Representative forward scatter histograms for ANCD4 and YNCD4 cells. C. Comparison of CD4 MFI values of ANCD4 and YNCD4 cells (Mean ± SE; data representative of three experiments). D. Data for forward scatter from individual mice (Mean ± SE; data representative of three experiments). E. Representative data showing levels of cytokines in supernatants of in vitro primed and recalled YNCD4 and ANCD4 cells. F. IFNγ/IL-13 ratios and mean ± SE calculated based on absorbance values. G. Histogram overlays of baseline ex vivo p-Erk (top) and total Erk (bottom) in ANCD4 and YNCD4 cells (plotted on log on left and linear scale on right). H. Pooled data, of paired samples showing baseline p-Erk MFI values (Mean ± SE) in ANCD4 and YNCD4 cells. I. ΔΔCt values of Real time RT-PCR for miR-181a normalized to miR-142 on ANCD4 and YNCD4 cells (Mean ± SE from five sets of independently sorted cells). J. 3H-Thymidine incorporation of ANCD4 and YNCD4 cells treated or not with BCI prior to activation (Mean ± SE of triplicate cultures). Data representative of six mice, one of three experiments. K. IFNγ/IL-13 and IFNγ/IL-4 ratio for YNCD4, BCI treated ANCD4 or BCI untreated ANCD4 cells during recall response. Data representing one of four independent experiments. ANCD4, naïve CD4 T cells from aged mice; BCI, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one; MFI, mean fluorescence intensity; SE, standard error; YNCD4, naïve CD4 T cells from young mice.

Mentions: Average peripheral residence time of naive T cells in aged mice and older people is reported to be longer than their younger counterparts due to decreasing thymic output with age [16,17]. Since our data above suggest that NCD4lo cells from young mice are likely to be those with longer peripheral residence, we compared NCD4 cells from young (YNCD4) and aged (ANCD4) mice for functional and phenotypic features. We have already shown that ANCD4 cells respond poorly and die more easily post-activation [34]. We observed that ANCD4 cells had modestly but consistently lower CD4 levels than YNCD4 cells (Figure 9A log and linear plot, and 9C). ANCD4 cells were also consistently smaller than YNCD4 cells (Figure 9B and D). We also looked at the ability of sorted ANCD4 and YNCD4 cells to differentiate in vitro in response to non-polarizing activating conditions. Supernatants from cultures of ANCD4 and YNCD4 cells primed in vitro with anti-CD3 + anti-CD28 for three days and then restimulated with titrating doses of anti-CD3 were analyzed for IFNγ, IL-4 and IL-13. ANCD4 cells thus differentiated to effector cells produced more IL-4 and IL-13 compared to YNCD4 cells (Figure 9E). The relative Th1 dominance, indicated by the IFNγ/IL-13 ratio, was significantly lower in differentiated ANCD4 cells (Figure 9F). We also examined total and p-Erk levels. While total Erk levels (Figure 9G log and linear scale, bottom plot) were not different between ANCD4 and YNCD4 cells ex vivo, p-Erk levels in the resting state were lower in ANCD4 cells (Figure 9G log and linear scale, top plot). Differences in the pErk levels were significant (Figure 9H). In multiple independent experiments, relative levels of miR-181a were lower in ANCD4 cells compared to YNCD4 cells (Figure 9I) extending observations reported on human cells [8]. Consistent with the role of miRNA-181a in suppressing DUSP6 expression [8], ANCD4 cells responded better when treated with a pharmacological inhibitor of DUSP, BCI, prior to activation (Figure 9J). It is noteworthy that there was no apparent effect of BCI on YNCD4 cells, despite the fact that some of the YNCD4 cells would be expected to be CD4lo and would, therefore, have shown an effect of BCI. It is likely that the contribution of NCD4lo cells in the bulk YNCD4 cells may not be statistically apparent. Further, analysis of cytokines secreted by ANCD4 and YNCD4 cells treated with BCI and differentiated in vitro showed that the relative Th2 tendency of ANCD4 cells was partially reversed by BCI treatment (Figure 9K).Figure 9


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)

NCD4lo cells resemble naïve CD4 T cells from aged mice in their phenotypic and functional attributes. A. CD4 levels on ANCD4 and YNCD4 cells (plotted on log on left and linear scale on right) stained as a mixture in a single well. B. Representative forward scatter histograms for ANCD4 and YNCD4 cells. C. Comparison of CD4 MFI values of ANCD4 and YNCD4 cells (Mean ± SE; data representative of three experiments). D. Data for forward scatter from individual mice (Mean ± SE; data representative of three experiments). E. Representative data showing levels of cytokines in supernatants of in vitro primed and recalled YNCD4 and ANCD4 cells. F. IFNγ/IL-13 ratios and mean ± SE calculated based on absorbance values. G. Histogram overlays of baseline ex vivo p-Erk (top) and total Erk (bottom) in ANCD4 and YNCD4 cells (plotted on log on left and linear scale on right). H. Pooled data, of paired samples showing baseline p-Erk MFI values (Mean ± SE) in ANCD4 and YNCD4 cells. I. ΔΔCt values of Real time RT-PCR for miR-181a normalized to miR-142 on ANCD4 and YNCD4 cells (Mean ± SE from five sets of independently sorted cells). J. 3H-Thymidine incorporation of ANCD4 and YNCD4 cells treated or not with BCI prior to activation (Mean ± SE of triplicate cultures). Data representative of six mice, one of three experiments. K. IFNγ/IL-13 and IFNγ/IL-4 ratio for YNCD4, BCI treated ANCD4 or BCI untreated ANCD4 cells during recall response. Data representing one of four independent experiments. ANCD4, naïve CD4 T cells from aged mice; BCI, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one; MFI, mean fluorescence intensity; SE, standard error; YNCD4, naïve CD4 T cells from young mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4306244&req=5

Fig9: NCD4lo cells resemble naïve CD4 T cells from aged mice in their phenotypic and functional attributes. A. CD4 levels on ANCD4 and YNCD4 cells (plotted on log on left and linear scale on right) stained as a mixture in a single well. B. Representative forward scatter histograms for ANCD4 and YNCD4 cells. C. Comparison of CD4 MFI values of ANCD4 and YNCD4 cells (Mean ± SE; data representative of three experiments). D. Data for forward scatter from individual mice (Mean ± SE; data representative of three experiments). E. Representative data showing levels of cytokines in supernatants of in vitro primed and recalled YNCD4 and ANCD4 cells. F. IFNγ/IL-13 ratios and mean ± SE calculated based on absorbance values. G. Histogram overlays of baseline ex vivo p-Erk (top) and total Erk (bottom) in ANCD4 and YNCD4 cells (plotted on log on left and linear scale on right). H. Pooled data, of paired samples showing baseline p-Erk MFI values (Mean ± SE) in ANCD4 and YNCD4 cells. I. ΔΔCt values of Real time RT-PCR for miR-181a normalized to miR-142 on ANCD4 and YNCD4 cells (Mean ± SE from five sets of independently sorted cells). J. 3H-Thymidine incorporation of ANCD4 and YNCD4 cells treated or not with BCI prior to activation (Mean ± SE of triplicate cultures). Data representative of six mice, one of three experiments. K. IFNγ/IL-13 and IFNγ/IL-4 ratio for YNCD4, BCI treated ANCD4 or BCI untreated ANCD4 cells during recall response. Data representing one of four independent experiments. ANCD4, naïve CD4 T cells from aged mice; BCI, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one; MFI, mean fluorescence intensity; SE, standard error; YNCD4, naïve CD4 T cells from young mice.
Mentions: Average peripheral residence time of naive T cells in aged mice and older people is reported to be longer than their younger counterparts due to decreasing thymic output with age [16,17]. Since our data above suggest that NCD4lo cells from young mice are likely to be those with longer peripheral residence, we compared NCD4 cells from young (YNCD4) and aged (ANCD4) mice for functional and phenotypic features. We have already shown that ANCD4 cells respond poorly and die more easily post-activation [34]. We observed that ANCD4 cells had modestly but consistently lower CD4 levels than YNCD4 cells (Figure 9A log and linear plot, and 9C). ANCD4 cells were also consistently smaller than YNCD4 cells (Figure 9B and D). We also looked at the ability of sorted ANCD4 and YNCD4 cells to differentiate in vitro in response to non-polarizing activating conditions. Supernatants from cultures of ANCD4 and YNCD4 cells primed in vitro with anti-CD3 + anti-CD28 for three days and then restimulated with titrating doses of anti-CD3 were analyzed for IFNγ, IL-4 and IL-13. ANCD4 cells thus differentiated to effector cells produced more IL-4 and IL-13 compared to YNCD4 cells (Figure 9E). The relative Th1 dominance, indicated by the IFNγ/IL-13 ratio, was significantly lower in differentiated ANCD4 cells (Figure 9F). We also examined total and p-Erk levels. While total Erk levels (Figure 9G log and linear scale, bottom plot) were not different between ANCD4 and YNCD4 cells ex vivo, p-Erk levels in the resting state were lower in ANCD4 cells (Figure 9G log and linear scale, top plot). Differences in the pErk levels were significant (Figure 9H). In multiple independent experiments, relative levels of miR-181a were lower in ANCD4 cells compared to YNCD4 cells (Figure 9I) extending observations reported on human cells [8]. Consistent with the role of miRNA-181a in suppressing DUSP6 expression [8], ANCD4 cells responded better when treated with a pharmacological inhibitor of DUSP, BCI, prior to activation (Figure 9J). It is noteworthy that there was no apparent effect of BCI on YNCD4 cells, despite the fact that some of the YNCD4 cells would be expected to be CD4lo and would, therefore, have shown an effect of BCI. It is likely that the contribution of NCD4lo cells in the bulk YNCD4 cells may not be statistically apparent. Further, analysis of cytokines secreted by ANCD4 and YNCD4 cells treated with BCI and differentiated in vitro showed that the relative Th2 tendency of ANCD4 cells was partially reversed by BCI treatment (Figure 9K).Figure 9

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