iNKT cells have an early requirement for vitamin D and 1,25D3. Based on the inability of early 1,25D3 treatment to recover iNKT cell numbers in the D− mice, we conclude that vitamin D is required before the appearance of the first tetramer positive cells in the thymus (d5). Vitamin D deficiency results in a cell intrinsic defect in iNKT cells. Our data suggests that a vitamin D regulated committed precursor for iNKT cells may be present in the bone marrow. Timed pregnancies show that vitamin D is required before embryonic day 13 for normal numbers of iNKT cells to develop. Factors that affect very early iNKT cell development are beginning to be identified. iNKT cell development requires rearrangement of the Vα14Jα18 TCRα chain. The Jα18 rearrangement is a secondary rearrangement that occurs only if the primary rearrangements fail to generate a productive TCRα chain. Because the Jα18 rearrangement takes longer than primary TCRα rearrangements, factors that prolong DP cell survival have been shown to be required for iNKT cell development (30
). Several transcription factors have been shown to affect DP thymocyte survival and in particular HEB has been shown to be important in the development of iNKT cells (31
). Interestingly, HEB deficient thymocytes had reduced DP thymocyte survival, defective iNKT cells and normal conventional T cells although the T cells showed limited TCR diversity (31
). It would be interesting to determine whether vitamin D regulates HEB.
Vitamin D deficient CD24−
iNKT cell precursors undergo increased apoptosis compared to the vitamin D sufficient CD24−
iNKT cells. The increase in apoptosis leads to a reduced pool of more mature iNKT cells that can go on to expand and mature by upregulating CD44 and NK1.1. Signals that down-regulate CD24 expression on iNKT cells have not been well studied. It has been shown that in the absence of early growth response 2 (Egr2), iNKT cell precursors failed to down-regulate CD24 expression (34
). However, unlike D− iNKT cells, Egr2KO iNKT cells are functionally defective (34
). Therefore, it seems unlikely that vitamin D regulates iNKT cell development through the Egr2 pathway. More recently c-Myc has been identified as a factor that controls expansion of CD24−
iNKT cells that leads to the further maturation of the iNKT cells (35
). It is possible that the VDR and cMyc interact to regulate proliferation and survival of the iNKT cell precursors.
There were fewer iNKT cells in the D− mice than in the 1,25D3 deficient mice. Increased levels of 25(OH)D3
in the D+ CypKO mice led to more iNKT cells. 25(OH)D3
can bind to the VDR but with a much lower affinity than 1,25D3. Binding of 25(OH)D3
has been shown to occur readily in vitro when there is no 1,25D3 present in the culture media. In vivo evidence for an effect of 25(OH)D3
on vitamin D targets has recently been shown (29
). CypKO mice fed diets that contained pharmacological levels of vitamin D were able to normalize calcium homeostasis and prevent the development of osteomalacia (29
). In the present experiments excess vitamin D was not added to the diets of CypKO mice. None-the-less our work provides an additional example of the ability of 25(OH)D3
to bind to the VDR in vivo in the absence of conversion to 1,25D3.
The VDR KO mice have a block in iNKT cell development. The failure of VDR KO iNKT cells to develop normally is reflected in the poor activation of the iNKT cell hybridoma by VDR KO DP thymocytes but not by 1,25D3 deficient or D− derived DP thymocytes (27
). There are other examples in the literature where VDR and vitamin D deficiency result in disparate results. Vitamin D deficiency has been shown to increase susceptibility to EAE while VDR deficiency makes the animals more resistant (7
). In experimental allergic asthma, vitamin D deficiency has no effect while VDR deficient mice fail to develop allergic asthma (37
). The VDR is a nuclear receptor that regulates transcription and there are at least two possibilities of how the unliganded VDR regulates iNKT development/function. Unliganded VDR might bind to other protein(s) (nuclear receptor co-repressor (N-CoR) is a possibility) that normally inhibit the induction of iNKT cells by DP thymocytes. The other possibility is that VDR functions as a heterodimer with the retinoid X receptor (RXR). In the absence of the VDR excess RXR would be available to dimerize with one of the many other nuclear receptors that require RXR for activity. This other nuclear receptor (estrogen receptor, retinoic acid receptor, glucocorticoid receptor) would then be an inhibitor of the induction of iNKT cells by the DP thymocytes.
Early exposure of neonatal mice to vitamin D is required for mice to develop optimal numbers of iNKT cells. Vitamin D deficiency results in epigenetic changes in iNKT cells that cannot be rescued by later exposure to vitamin D or 1,25D3. The reduced numbers of iNKT cells is a result of increased apoptosis of early iNKT cell precursors in the thymus of the D− host. Expression of the VDR is required for normal antigen presentation by DP thymocytes while ligand deficiency has no effect on DP thymocyte presentation. The data presented here suggest that the amount of vitamin D available in the environment early during development of iNKT cells dictates the number of iNKT cells. The implications for humans are that vitamin D might be a factor that affects NKT cell numbers, and along with the correlations in humans between reduced numbers of NKT cells and increased autoimmunity, might explain in part the relationship between vitamin D, the environment, NKT cells and autoimmune disease prevalence.