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. 2021 May 5;22(9):4907.
doi: 10.3390/ijms22094907.

Cord Blood T Cells Expressing High and Low PKCζ Levels Develop into Cells with a Propensity to Display Th1 and Th9 Cytokine Profiles, Respectively

Affiliations

Cord Blood T Cells Expressing High and Low PKCζ Levels Develop into Cells with a Propensity to Display Th1 and Th9 Cytokine Profiles, Respectively

Khalida Perveen et al. Int J Mol Sci. .

Abstract

Low Protein Kinase C zeta (PKCζ) levels in cord blood T cells (CBTC) have been shown to correlate with the development of allergic sensitization in childhood. However, little is known about the mechanisms responsible. We have examined the relationship between the expression of different levels of PKCζ in CBTC and their development into mature T cell cytokine producers that relate to allergy or anti-allergy promoting cells. Maturation of naïve CBTC was initiated with anti-CD3/-CD28 antibodies and recombinant human interleukin-2 (rhIL-2). To stimulate lymphocyte proliferation and cytokine production the cells were treated with Phytohaemagglutinin (PHA) and Phorbol myristate acetate (PMA). Irrespective of the PKCζ levels expressed, immature CBTC showed no difference in lymphocyte proliferation and the production of T helper 2 (Th2) cytokine interleukin-4 (IL-4) and Th1 cytokine, interferon-gamma (IFN-γ), and influenced neither their maturation from CD45RA+ to CD45RO+ cells nor cell viability/apoptosis. However, upon maturation the low PKCζ expressing cells produced low levels of the Th1 cytokines, IFN-γ, IL-2 and tumour necrosis factor-alpha (TNF), no changes to levels of the Th2 cytokines, IL-4, IL-5 and IL-13, and an increase in the Th9 cytokine, IL-9. Other cytokines, lymphotoxin-α (LT-α), IL-10, IL-17, IL-21, IL-22 and Transforming growth factor-beta (TGF-β) were not significantly different. The findings support the view that low CBTC PKCζ levels relate to the increased risk of developing allergic diseases.

Keywords: PKCζ; T cell maturation; Th1 and Th2 subsets; allergy; cord blood T cells; cytokines.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Comparison of proliferation and cytokine production in naïve cord blood T cells (CBTC) in Protein Kinase C zeta (PKCζ) low or high group. (a) Comparison of PKCζ between cord blood (CB) and adult blood (AB) T cells, n = 24 for each AB or CB. (b) Shows lymphoproliferation as a Stimulation index (SI) and disintegrations per minute (DPM) in 3H-thymidine pulsed cultures stimulated with Phytohaemagglutinin (PHA) and Phorbol myristate acetate (PMA) (c) Purified CBCTs were stained with Carboxyfluorescein succinimidyl ester (CFSE) dye and stimulated with immobilized anti-CD3/-CD28 antibodies for 3 days. Gating and representative histogram for CFSE dilution after exclusion of doublets and dead cells. Overlaid histograms for stained unstimulated and unstained stimulated samples were used as control and for gating the non-proliferating cells and for auto-fluorescence, respectively. (d) Naïve CB CD3+ T cells were stimulated with PHA/PMA (18 h) and percentage of CD3+ T cells producing interleukin-4 (IL-4) and Interferon-gamma (IFN-γ) and median fluorescent intensity (MFI) were examined by flow cytometry assays. (e) On day 5 of CFSE stained culture (anti-CD3/-CD28), cells were re-stimulated with PHA/PMA (18 h) for detection of intracellular cytokine. Representative flow dot plots and data for CFSE dye dilution and IFN-γ producing cells in high and low PKCζ group. Data mean ± SD of n = 3 for of low and n = 4 high PKCζ group. ** p < 0.01. ns: not significant (Student’s t-test).
Figure 2
Figure 2
Comparison of viability, apoptosis, and maturation in CBTC expressing low or high levels of PKCζ during maturation in culture. Purified CB CD3+ T cells were matured with anti-CD3/-CD28 and recombinant human interleukin-2 (rhIL-2) for 7 days. (a) Representative flow dot plots and percentage CB CD3+ T cells was analyzed at indicated times and expressed as percentage of dead cells (7-aminoactinomycin D (7-AAD+) and apoptotic cells (Annexin V+). n = 3 for low PKCζ and n = 5 for high PKCζ (b) Cell counts during maturation assay. (c) Representative dot plot for naïve and maturation expression in CB CD3+ T cells on day seven. Data mean ± SD of n = 4 for low and n = 4 high PKCζ group. ns: not significant (a,b): Student’s t-test, (c): one-way ANOVA with post hoc Tukey’s multiple comparisons test.
Figure 3
Figure 3
Detection of Th1 and Th2 cytokine in matured PKCζ low and high CBTC. Purified CB CD3+ T cells were matured with anti-CD3/-CD28 antibodies with recombinant human interleukin-2 (rhIL-2). On day seven, cells were left unstimulated or stimulated with PHA/PMA overnight. Dead cells were excluded by gating on Fixable Viability Stain 510 (FVS510) negative population and cytokines were analysed by flow cytometry. Representative histograms and graphs for (a) Th1 and (b) Th2 cytokines in PKCζ low and high samples showing the gating for the analysis of % positive cells for respective cytokines percentage and their MFI. Shaded and empty histograms represent unstimulated and stimulated samples stained with a cytokine-specific antibody. Graphs represent the means ± SD of n = 6 for low and 5 for high. * p < 0.05. ns: not significant. (Student’s t-test).
Figure 4
Figure 4
Other T cell cytokines expressed by matured cord blood T cells, which express either low or high levels of PKCζ. Cells were matured by treating with anti-CD3/-CD28 and rhIL-2. After maturation, they were stimulated as per Figure 3. (a) Representative histograms for Th or Treg cytokines in PKCζ low and high samples showing the gating for the analysis of graphs for respective cytokine positive cells percentage and their MFI. Shaded and empty histograms represent unstimulated and stimulated samples stained for the respective cytokines. Graphs represent the means ± SD of n = 6 for low and 5 for high. ns: not significant. (Student’s t-test).
Figure 5
Figure 5
Correlation analyses of levels of PKCζ expression with Th1 cytokines production. Data from Figure 3 were subjected to correlation analysis. The first column represents the Pearson correlation of PKCζ (MFI) with representative cytokines (percentage of CD3+ T cells for each cytokine) and the second column represents the MFI of the positive gated cells. * p < 0.05, ** p < 0.01. ns: not significant. Correlations were performed using the two-tailed Pearson correlation coefficient.
Figure 6
Figure 6
Correlation of PKCζ expression with Th2/Th9 cytokines. Data from Figure 4 were subjected to correlation analysis. The first column represents the Pearson correlation of PKCζ (MFI) with representative cytokines (percentage of CD3+ T cells for each cytokine) and the second column represents the MFI of the positive gated cells. ** p < 0.01, *** p < 0.001. ns: not significant. Correlations were performed using the two-tailed Pearson correlation coefficient.
Figure 7
Figure 7
The ratio of Th2 vs. Th1 in low vs. high PKCζ CBTC. Purified CB CD3+ T cells were overnight stimulated as in Figure 1 (naive T cells) and Figure 4 and Figure 5 (mature T cells). The cells were gated for total CD3+ T cells producing IL-4 and IFN-γ as percentage of T cells or MFI as examined by flow cytometry assays. Comparison of percentage decrease in IL-4: IFN-γ in mature T cells (normalized against naïve T cells) and compared between CB samples expressing low vs. high PKCζ. n = 3 for low and n = 4 for high PKCζ group. * p < 0.05. ns: not significant. (Student’s t-test).

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