Seasonal variation in vitamin D₃ levels is paralleled by changes in the peripheral blood human T cell compartment

Abstract

It is well-recognized that vitamin D₃ has immune-modulatory properties and that the variation in ultraviolet (UV) exposure affects vitamin D₃ status. Here, we investigated if and to what extent seasonality of vitamin D₃ levels are associated with changes in T cell numbers and phenotypes. Every three months during the course of the entire year, human PBMC and whole blood from 15 healthy subjects were sampled and analyzed using flow cytometry. We observed that elevated serum 25(OH)D₃ and 1,25(OH)(2)D₃ levels in summer were associated with a higher number of peripheral CD4+ and CD8+ T cells. In addition, an increase in naïve CD4+CD45RA+ T cells with a reciprocal drop in memory CD4+CD45RO+ T cells was observed. The increase in CD4+CD45RA+ T cell count was a result of heightened proliferative capacity rather than recent thymic emigration of T cells. The percentage of Treg dropped in summer, but not the absolute Treg numbers. Notably, in the Treg population, the levels of forkhead box protein 3 (Foxp3) expression were increased in summer. Skin, gut and lymphoid tissue homing potential was increased during summer as well, exemplified by increased CCR4, CCR6, CLA, CCR9 and CCR7 levels. Also, in summer, CD4+ and CD8+ T cells revealed a reduced capacity to produce pro-inflammatory cytokines. In conclusion, seasonal variation in vitamin D₃ status in vivo throughout the year is associated with changes in the human peripheral T cell compartment and may as such explain some of the seasonal variation in immune status which has been observed previously. Given that the current observations are limited to healthy adult males, larger population-based studies would be useful to validate these findings.

Figure 1. Seasonal variation in serum vitamin D₃ levels and the amount of daylight.

Median serum A) 25(OH)D₃ and B) 1,25(OH)₂D₃ concentrations of 15 healthy volunteers during each of the four seasons. C) Duration of daylight in the study region in a month prior to serum vitamin D₃ concentration assay (source: the Royal Netherlands Meteorological Institute). * p<0.05 as compared to winter.

Figure 2. Peripheral T cell (subset) numbers throughout the four seasons.

A) Percentage (of live gate) and absolute numbers of CD4⁺ T cells. B) Percentage (of live gate) and absolute numbers of CD8⁺ T cells, over time. C) Percentage (within CD4⁺ T cells) and absolute counts, of CD4⁺CD45RA⁺ T cells. D) Percentage (within CD4⁺ T cells) and absolute counts, of CD4⁺CD45RO⁺ T cells. E) Percentage and absolute counts of Ki-67-expressing CD4⁺CD45RA⁺ T cells. Whole blood samples obtained from 15 healthy volunteers during each season were analyzed for the respective markers using flow cytometry. Ki-67 analysis was performed on PBMC. Data show results of viable cells from 15 healthy donors. * p<0.05 as compared to winter.

Figure 3. Seasonal variation in numbers and Foxp3 expression of Treg during the four seasons.

A) Percentage (within CD4⁺ T cells) and absolute numbers of CD4⁺CD25^hiCD127⁻ Treg and B) level of Foxp3 expression (mean fluorescence intensity; MFI). Whole blood and PBMC isolated from 15 healthy volunteers during each season were analyzed for the respective markers using flowcytometry. Data show results from 15 healthy donors. * p<0.05 as compared to winter.

Figure 4. Skin, lymphoid tissue and gut homing receptor expression on CD4⁺ T cells.

Percentage and level of expression (MFI) of A) CCR4, B) CCR6, C) CLA, D) CCR7 and E) CCR9 by CD4⁺ T cells during the different seasons of the year. Whole blood from 15 healthy volunteers during each season was analyzed for the respective markers using flow cytometry. Data show results from 15 healthy donors. * p<0.05 as compared to winter.

Figure 5. Skin, lymphoid tissue and gut homing receptor expression on CD4⁺CD25^hiCD127⁻ regulatory T cells.

Percentage of Treg (within CD4⁺ T cells) and their level of expression (MFI) of A) CCR4, B) CCR6, C) CLA, D) CCR7 and E) CCR9 during the four seasons of the year. Whole blood from 15 healthy volunteers during each season was analyzed for the respective markers using flow cytometry. Data show results from 15 healthy donors. * p<0.05 as compared to winter.

Figure 6. Seasonal variation in cytokine profile of CD4⁺ and CD8⁺ T cells.

Percentage and the level of production on a per cell basis (MFI) of A) IFNγ, B) IL-2 and C) IL-17 by CD4⁺ T cells; and of D) IFNγ and E) IL-2 by CD8⁺ T cells analyzed by flow cytometry. PBMC isolated from 10 healthy volunteers and intracellular staining for cytokines was performed after the cells were stimulated with PMA plus ionomycin in the presence of brefeldin A. CD4⁺ T cells were defined as CD3⁺CD8⁻. Data show results from 10 healthy donors. * p<0.05 as compared to winter.