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The Migratory Properties and Numbers of T Regulatory Cell Subsets in Circulation Are Differentially Influenced by Season and Are Associated With Vitamin D Status - PubMed

️Wed Jan 01 2020

The Migratory Properties and Numbers of T Regulatory Cell Subsets in Circulation Are Differentially Influenced by Season and Are Associated With Vitamin D Status

Abigail A Lamikanra et al. Front Immunol. 2020.

Abstract

The control of peripheral immune responses by FOXP3⁺ T regulatory (Treg) cells is essential for immune tolerance. However, at any given time, Treg frequencies in whole blood can vary more than fivefold between individuals. An understanding of factors that influence Treg numbers and migration within and between individuals would be a powerful tool for cellular therapies that utilize the immunomodulatory properties of Tregs to control pathology associated with inflammation. We sought to understand how season could influence Treg numbers and phenotype by monitoring the proportion of natural thymus-derived Tregs (nTregs) defined as (CD3⁺CD4⁺CD25⁺FOXP3⁺CD127^-/low ) cells as a proportion of CD4⁺ T cells and compared these to all FOXP3⁺ Tregs (allTregs, CD3⁺CD25⁺FOXP3⁺CD127^-/low ). We were able to determine changes within individuals during 1 year suggesting an influence of season on nTreg frequencies. We found that, between individuals at any given time, nTreg/CD4⁺ T cells ranged from 1.8% in February to 8.8% in the summer where median nTreg/CD4 in January and February was 2.4% (range 3.75-1.76) and in July and August was 4.5% (range 8.81-3.17) p = 0.025. Importantly we were able to monitor individual nTreg frequencies throughout the year in donors that started the year with high or low nTregs. Some nTreg variation could be attributed to vitamin D status where normal linear regression estimated that an absolute increase in nTreg/CD4⁺ by 0.11% could be expected with 10 nmol increase in serum 25 (OH) vitamin D3 (p = 0.005, 95% CI: 0.03-0.19). We assessed migration markers on Tregs for the skin and/or gut. Here cutaneous lymphocyte associated antigen (CLA⁺) expression on CD25⁺FOXP3⁺CD4⁺/CD4⁺ was compared with the same population expressing the gut associated integrin, β7. Gut tropic CD25⁺FOXP3⁺β7⁺Tregs/CD4⁺ had similar dynamics to nTreg/CD4⁺. Conversely, CD25⁺FOXP3⁺CLA⁺Tregs/CD4⁺ showed no association with vitamin D status. Important for cellular therapies requiring isolation of Tregs, the absolute number of β7⁺CD4⁺CD25⁺FOXP3⁺Tregs was positively associated with 25(OH)vitamin D3 (R² = 0.0208, r = 0.184, p = 0.021) whereas the absolute numbers of CLA⁺CD4⁺CD25⁺FOXP3⁺Tregs in the periphery were not influenced by vitamin D status. These baseline observations provide new opportunities to utilize seasonal variables that influence Treg numbers and their migratory potential in patients or donors.

Keywords: Tregs; migration; regulatory T cells; seasons; tolerance; vitamin D3.

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Figures

**FIGURE 1**
Schematic representing platelet donor recruitment and analysis of data. The median age range of all participants was 58 (range 21–72). The median number of samples taken during 1 year from each participant was 6 (range 2–9). Data obtained from these visits were pooled to provide an average value for parameters determined each month or every 2 months for 1 year. Treg and vitamin D3 metabolite variation of individual participants were determined in 32 of these donors where the median age was 59 years (range 23–72).

**FIGURE 2**
Monthly changes in whole blood parameters determined using an automated hematological analyzer. The variation in numbers of white blood cells (WBC), lymphocytes, monocytes and neutrophils are shown.

**FIGURE 3**
Variation in absolute numbers of T cells each month. The estimate of absolute numbers of T cell subsets are shown for each month and were determined after immune-phenotyping and analysis as described in Materials and Methods for all CD3⁺ T cells **(A)**, CD4⁺ T cells **(B),** CD8⁺ T cells **(C),** and nTregs **(D)**. In D the p-values are shown using one way ANOVA analysis of log transformed values where the p-values for changes in nTregs/μL between January and June, July and August are 0.010, 0.004, and 0.004, respectively; between July and December p = 0.032 and between August and December, p = 0.026.

**FIGURE 4**
Bimonthly changes in immune-regulatory T cells between January and December of 1 year. The variation of **(A)** all FOXP3⁺ T cells that includes thymic and peripherally induced CD4⁺ and CD8⁺Tregs is compared with **(B)** non-activated CD25^hi nTregs and **(C)** with CD4⁺ T cells that express the Vα24 chain found on i-NKT cells. The p-values for non-transformed values following use of the ordinary one way ANOVA is shown where n is between 34 and 74 at each time point. Box and whisker bar graphs are used to show the median values with outliers lower than the 10th percentile and greater than the 90th percentile. **(D)** The proportions of nTregs in circulation vary between individuals as well as between seasons. The pattern of Treg levels in 1 year is shown for 43 donors that attended clinic multiple times in 1 year. Q1 represents donors that had mean nTregs less than the 25th percentile of annual nTregs and are compared with donors that had levels between the 25th and 50th percentile (Q2), between the 50th and 75th percentile (Q3) or above the 75th percentile (Q4) of nTregs in 1 year. Error bars show the standard error of the means at each time point.

**FIGURE 5**
CD25⁺FOXP3⁺ Treg subsets show distinct changes through the year with differences in proportions of naïve and tropic marker expression. **(A)** CD45RA⁺ Tregs, **(B)** β7⁺ Tregs, **(C)** and CLA⁺ Tregs are shown as a proportion of CD4 + helper T cells. Absolute numbers of **(D)** CD45RA⁺ Tregs, **(E)** β7⁺ Tregs and **(F)** CLA⁺ Tregs are also shown. The ordinary one way ANOVA was used to determine p-values of non-transformed data and bar graphs with error bars to show outliers below and above the 10th and 90th percentiles, respectively.

**FIGURE 6**
Differences in the dynamics of Treg subsets within the CD3 + CD4 + CD25⁺FOXP3⁺ (Total Treg) population. CLA⁺ Tregs are more likely to change substantially compared with CD45RA + and β7⁺ Tregs, where CLA⁺ Tregs have declined and β7⁺ Tregs may peak during late summer/autumn. Left side: Bimonthly variation of Treg subsets/Total Tregs determined between individuals **(A)** CD45RA⁺ Tregs, **(B)** β7⁺ Tregs, **(C)** CLA⁺ Treg. ns, p = 0.601. Right side: Bi-monthly changes in Treg subsets/Total Treg within individuals that are in each quartile **(D)** CD45RA⁺Tregs, **(E)** β7⁺Tregs, and **(F)** CLA⁺Tregs is shown. The mean ± SEM of Treg subset/Total Tregs is shown for donors that had an annual mean frequency of subset⁺ Tregs/Total Tregs below the 25th percentile (Q1) between the 25th and 50th percentile (Q2), between the 50th and 75th percentile (Q3) and above the 75th percentile (Q4).

**FIGURE 7**
Seasonal variation of vitamin D status and Treg dynamics within the CD4 + helper population. **(A)** Daylight hours in Oxfordshire, **(B)** vitamin D status of donors attending clinic in the months shown. The association of 25 (OH) vitamin D metabolites with **(C)** all FOXP3⁺ T cells/CD3⁺ T cells R² = 0.165, r = 0.406, p < 0.0001 **(D)** CD127^–/low nTregs/CD4⁺ R² = 0.042, r = 0.178, p = 0.023. Also shown is the association of 25 (OH) vitamin D metabolites with **(E)** CD45RA⁺Tregs/CD4⁺ T cells R² = 0.008, r = 0.085, p = 0.284, **(F)** with β7⁺Tregs/CD4⁺ T cells R² = 0.067, r = 0.267, p = 0.001 and **(G)** with CLA⁺ Tregs/CD4⁺ T cells R² = 0.0011, r = 0.051, p = 0.519. Box and whisker plots with error bars to show outliers below the 10th percentile and outliers above the 90th percentile.

**FIGURE 8**
Vitamin D status is positively associated with the absolute number of CD45RA + and β7 + Tregs. The association of 25 (OH) vitamin D metabolites with **(A)** CD45RA⁺Tregs/Tregs, R² = 0.008 r = 0.069, p = 0.388, **(B)** β7⁺Tregs/Total Tregs, R² = 0.002, r = 0.057, p = 0.476 and **(C)** CLA⁺Tregs/Total Tregs, R² = 0.029, r = –0.167, p = 0.034 is compared with the association of 25 (OH) vitamin D metabolites with absolute numbers/μL of **(D)** CD45RA⁺Tregs/μL, R² = 0.021, r = 0.153, p = 0.052 **(E)** β7⁺Tregs/μL, R² = 0.021, r = 0.184, p = 0.021, and **(F)** CLA⁺Tregs/μL, R² = 0.010, r = 0.056, p = 0.488.

**FIGURE 9**
Seasonal variation of cortisol and Treg dynamics within the CD4+ helper population. **(A)** Bimonthly cortisol levels in sera collected between 08:30 am and 11:30 am. Also shown is the association of cortisol with **(B)** all FOXP3 + T cells/CD3 R² = 0.041, r = –0.222, p = 0.005, **(C)** CD127^–/low nTregs/CD4 + R² = 0.061, r = –0.257, p = 0.001, **(D)** CD45RA⁺Tregs/CD4⁺ T cells (R² = 0.055, r = –0.320, p < 0.0001), **(E)** with β7⁺Tregs/CD4⁺ T cells R² = 0.018, r = –0.221, p = 0.005 and **(F)** with CLA⁺ Tregs/CD4⁺ T cells R² = 0.009, r = –0.132, p = 0.094. Box and whisker plots are with error bars to show outliers below the 10th percentile and outliers above the 90th percentile.

**FIGURE 10**
Cortisol levels in sera are negatively associated with the absolute number of CD45RA + and β7 + Tregs. The association of cortisol with **(A)** CD45RA⁺Tregs/Tregs, R² = 0.016, r = −0.189, p = 0.016, **(B)** β7⁺Tregs/Tregs, R² = 0.004, r = −0.076, p = 0.341, and **(C)** CLA⁺Tregs/Tregs, R² = 0.001, r = 0.045, p = 0.573 is compared with the association of cortisol with absolute numbers/μL of **(D)** CD45RA⁺Tregs/μL, R² = 0.020, r = −0.171, p = 0.030, **(E)** β7⁺Tregs/μL, R² = 0.023, r = −0.165, p = 0.039, and **(F)** CLA⁺Tregs/μL, R² = 0.005, r = −0.023, p = 0.771.

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The Migratory Properties and Numbers of T Regulatory Cell Subsets in Circulation Are Differentially Influenced by Season and Are Associated With Vitamin D Status - PubMed