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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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Related in: MedlinePlus

NCD4lo cells show higher CD5 levels but no difference in proximal signaling. A. Ex vivo analysis of stained splenic cells to show gated NCD4lo and NCD4hi cells. B. Comparison of CD5 levels as log (left) and linear (right) plots between NCD4hi and NCD4lo cells. C. CD5 MFI values for NCD4hi and NCD4lo cells (Mean ± SE, one of three experiments). D. Comparison of forward scatter between NCD4hi and NCD4lo cells. E. Forward scatter values on NCD4hi and NCD4lo cells (Mean ± SE, one of five experiments). F. Comparison of TCRβ levels as log (left) and linear (right) plots between NCD4hi and NCD4lo cells. G. TCRβ MFI values for NCD4hi and NCD4lo cells (Mean ± SE, one of three experiments). H. Representative profile of Fluo-3/Fura-Red ratio in NCD4hi and NCD4lo cells at rest and post-activation. I. Pooled data from independent mouse spleens showing increase in Fluo-3/Fura-Red ratio with anti-CD3 (10 μg/ml) + anti-CD28 (3 μg/ml) treatment J. Pooled data for fold increase over background in pZap-70 staining MFI in NCD4hi and NCD4lo cells activated with anti-CD3 + anti-CD28, mean ± SE. MFI, mean fluorescence intensity; NCD4, naïve CD4 T cells; SE, standard error; TCR, T cell receptor.
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Fig2: NCD4lo cells show higher CD5 levels but no difference in proximal signaling. A. Ex vivo analysis of stained splenic cells to show gated NCD4lo and NCD4hi cells. B. Comparison of CD5 levels as log (left) and linear (right) plots between NCD4hi and NCD4lo cells. C. CD5 MFI values for NCD4hi and NCD4lo cells (Mean ± SE, one of three experiments). D. Comparison of forward scatter between NCD4hi and NCD4lo cells. E. Forward scatter values on NCD4hi and NCD4lo cells (Mean ± SE, one of five experiments). F. Comparison of TCRβ levels as log (left) and linear (right) plots between NCD4hi and NCD4lo cells. G. TCRβ MFI values for NCD4hi and NCD4lo cells (Mean ± SE, one of three experiments). H. Representative profile of Fluo-3/Fura-Red ratio in NCD4hi and NCD4lo cells at rest and post-activation. I. Pooled data from independent mouse spleens showing increase in Fluo-3/Fura-Red ratio with anti-CD3 (10 μg/ml) + anti-CD28 (3 μg/ml) treatment J. Pooled data for fold increase over background in pZap-70 staining MFI in NCD4hi and NCD4lo cells activated with anti-CD3 + anti-CD28, mean ± SE. MFI, mean fluorescence intensity; NCD4, naïve CD4 T cells; SE, standard error; TCR, T cell receptor.

Mentions: CD5 is a negative regulator of TCR signaling [10] and CD5 expression has been inversely correlated with T cell responsiveness [37]. We, therefore, tested the possibility that lower CD4 levels might correlate with higher CD5 expression. NCD4hi and NCD4lo cells from young B6 mice (Figure 2A) were tested for the levels of CD5. When gated NCD4hi and NCD4lo cells were analyzed for CD5 expression, it was apparent that NCD4lo cells had higher CD5 levels (Figure 2B, log plot on left, linear on right) in multiple mice analyzed (Figure 2C). This was despite the finding that NCD4hi cells were somewhat larger than NCD4lo cells (Figures 2D and E). We also examined the levels of a number of other molecular markers in these populations. NCD4lo cells showed lower TCRβ (Figure 2F, log and linear plots; and 2G) levels that NCD4hi cells, although both NCD4lo and NCD4hi cells were confirmed to be conventional CD4 T cells expressing TCRα/β. NCD4lo cells also expressed somewhat lower levels of MHC class I and CD2, but higher levels of CD54 (see Additional file 2: Figure S2A and S2B) in multiple mice examined. Another feature tested based on differences in cell size between NCD4lo and NCD4hi cells was mitochondrial mass and potential using indicator dyes, Mitotracker Green (MG) and Mitotracker Red (MR) [38,39]. However, NCD4lo cells did not show any striking differences from NCD4hi cells in mitochondrial content or potential (see Additional file 2: Figure S2C to S2E). These data indicated that the phenotypic differences between NCD4lo and NCD4hi cells were likely to be complexly regulated.Figure 2


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 show higher CD5 levels but no difference in proximal signaling. A. Ex vivo analysis of stained splenic cells to show gated NCD4lo and NCD4hi cells. B. Comparison of CD5 levels as log (left) and linear (right) plots between NCD4hi and NCD4lo cells. C. CD5 MFI values for NCD4hi and NCD4lo cells (Mean ± SE, one of three experiments). D. Comparison of forward scatter between NCD4hi and NCD4lo cells. E. Forward scatter values on NCD4hi and NCD4lo cells (Mean ± SE, one of five experiments). F. Comparison of TCRβ levels as log (left) and linear (right) plots between NCD4hi and NCD4lo cells. G. TCRβ MFI values for NCD4hi and NCD4lo cells (Mean ± SE, one of three experiments). H. Representative profile of Fluo-3/Fura-Red ratio in NCD4hi and NCD4lo cells at rest and post-activation. I. Pooled data from independent mouse spleens showing increase in Fluo-3/Fura-Red ratio with anti-CD3 (10 μg/ml) + anti-CD28 (3 μg/ml) treatment J. Pooled data for fold increase over background in pZap-70 staining MFI in NCD4hi and NCD4lo cells activated with anti-CD3 + anti-CD28, mean ± SE. MFI, mean fluorescence intensity; NCD4, naïve CD4 T cells; SE, standard error; TCR, T cell receptor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: NCD4lo cells show higher CD5 levels but no difference in proximal signaling. A. Ex vivo analysis of stained splenic cells to show gated NCD4lo and NCD4hi cells. B. Comparison of CD5 levels as log (left) and linear (right) plots between NCD4hi and NCD4lo cells. C. CD5 MFI values for NCD4hi and NCD4lo cells (Mean ± SE, one of three experiments). D. Comparison of forward scatter between NCD4hi and NCD4lo cells. E. Forward scatter values on NCD4hi and NCD4lo cells (Mean ± SE, one of five experiments). F. Comparison of TCRβ levels as log (left) and linear (right) plots between NCD4hi and NCD4lo cells. G. TCRβ MFI values for NCD4hi and NCD4lo cells (Mean ± SE, one of three experiments). H. Representative profile of Fluo-3/Fura-Red ratio in NCD4hi and NCD4lo cells at rest and post-activation. I. Pooled data from independent mouse spleens showing increase in Fluo-3/Fura-Red ratio with anti-CD3 (10 μg/ml) + anti-CD28 (3 μg/ml) treatment J. Pooled data for fold increase over background in pZap-70 staining MFI in NCD4hi and NCD4lo cells activated with anti-CD3 + anti-CD28, mean ± SE. MFI, mean fluorescence intensity; NCD4, naïve CD4 T cells; SE, standard error; TCR, T cell receptor.
Mentions: CD5 is a negative regulator of TCR signaling [10] and CD5 expression has been inversely correlated with T cell responsiveness [37]. We, therefore, tested the possibility that lower CD4 levels might correlate with higher CD5 expression. NCD4hi and NCD4lo cells from young B6 mice (Figure 2A) were tested for the levels of CD5. When gated NCD4hi and NCD4lo cells were analyzed for CD5 expression, it was apparent that NCD4lo cells had higher CD5 levels (Figure 2B, log plot on left, linear on right) in multiple mice analyzed (Figure 2C). This was despite the finding that NCD4hi cells were somewhat larger than NCD4lo cells (Figures 2D and E). We also examined the levels of a number of other molecular markers in these populations. NCD4lo cells showed lower TCRβ (Figure 2F, log and linear plots; and 2G) levels that NCD4hi cells, although both NCD4lo and NCD4hi cells were confirmed to be conventional CD4 T cells expressing TCRα/β. NCD4lo cells also expressed somewhat lower levels of MHC class I and CD2, but higher levels of CD54 (see Additional file 2: Figure S2A and S2B) in multiple mice examined. Another feature tested based on differences in cell size between NCD4lo and NCD4hi cells was mitochondrial mass and potential using indicator dyes, Mitotracker Green (MG) and Mitotracker Red (MR) [38,39]. However, NCD4lo cells did not show any striking differences from NCD4hi cells in mitochondrial content or potential (see Additional file 2: Figure S2C to S2E). These data indicated that the phenotypic differences between NCD4lo and NCD4hi cells were likely to be complexly regulated.Figure 2

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