A survey approach utilizing 38 classical and wild-derived inbred mouse strains was used to further understand the genetic control of iNKT-cell development. Our primary finding is that the degree of variation in iNKT-cell frequency across multiple inbred mouse strains is similar to what has been observed in humans 23-25, 29, 32
. Since our mice were housed in a well-controlled specific pathogen free environment, the genetic background is expected to be the primary contributor to the observed strain-dependent variation. Previous studies in identical twins also suggested that the genetic background contributes more significantly than environmental factors to the variation of iNKT-cell frequencies in humans 23
. Taken together, these results indicate that development of iNKT-cells in both humans and mice is a complex trait that requires further studies to more fully understand its genetic basis. An important finding of the current study stems from the comparison of iNKT-cell levels in PB and lymphoid organs. These analyses revealed while there is some correlation, levels of total iNKT cells in PB do not necessarily predict their frequency in lymphoid tissue. We also confirm that compared to most of the strains analyzed, autoimmune T1D prone NOD mice have severely reduced levels of iNKT-cells. However, NOD mice exhibited a higher PB:spleen ratio of total iNKT cells compared to many other strains (). This observation was in line with a previous report 30
. While weak for total iNKT-cells, there is a strong correlation between PB and spleen in the proportion of the CD4+
subset. Taken together, these results suggest that in PB-based human studies analyses of the proportion of CD4+
rather than total iNKT-cells may provide a more consistent and reliable readout of their levels in lymphoid tissues.
A somewhat surprising finding in our current studies is that most wild-derived inbred mouse strains had very low frequencies of iNKT-cells (). Some of them had barely detectable iNKT-cells. Polymorphisms in the CD1d1
gene found in wild-derived inbred strains of Mus spretus
and Mus musculus castaneus
origins affect its antigen presentation function 40
. When congenically expressed in C57BL/6 mice, Mus spretus
-derived CD1d1 molecules had a diminished ability to select iNKT-cells resulting a ~50% reduction of these cells in the thymus 40
. Therefore, the allele difference in CD1d1
could contribute to the overall low frequencies of iNKT-cells in the wild-derived inbred strains examined here. However, the extremely low levels of iNKT-cells in some of those strains, such as SPRET/EiJ (Mus spretus), can not be explained solely by the previously reported polymorphisms in the CD1d1
gene. Furthermore, the MSM/Ms strain that belongs to the Mus musculus molossinus
subspecies previously found to have the same CD1d1
allele as that in C57BL/6 mice virtually lacked iNKT-cells (). GWA studies did not reveal an association of any analyzed iNKT phenotype with a genetic control component on Chr 3 where the CD1d1
gene is located. While this could be explained by the exclusion of SPRET/EiJ from the mapping studies, it also suggests that the overall contribution of the CD1d1
polymorphisms from other wild-derived inbred strains, such as CAST/EiJ, is relatively weak to reach the threshold for the whole genome association study. Collectively, these results indicate that genetic components other than CD1d1
polymorphisms contribute more strongly to the largely reduced iNKT-cell levels in wild-derived inbred strains. Therefore, identification of the other genetic components that inhibit the development of iNKT-cells in the wild-derived inbred strains represents an area warranting further studies.
Genome-wide association mapping studies identified loci on on Chr. 4, 6, 7, 10, 12, and 19 were found to contain a gene(s) regulating the levels of both thymic and splenic iNKT-cells. In this regard the proximal end of Chr. 7 is of particular interest as it contains a cluster of loci linked to levels of both thymic and splenic iNKT-cells. This Chr. 7 cluster includes a region between 3.10-3.62Mb where several microRNAs (Mir290 to Mir295) are located. This may be of significance as microRNAs are reported to be important for normal iNKT-cell development 41, 42
. In addition, the most significant region detected in our entire analyses (peak score = 17.24) was for a locus on Chr 12 regulating levels of thymic iNKT-cells. Genes within these collective regions will be prioritized for future studies.
Previous studies using a backcross approach from C57BL/6 to NOD.Nkrp1b
mice identified two loci respectively on Chr 1 and 2 significantly linked to the frequency of thymic iNKT-cells 43
. Both loci (Nkt1
) were subsequently confirmed by congenic analysis, and Slamf1
within the Nkt1
locus have also been shown to regulate iNKT-cell development 17, 44-46
. To our surprise, neither locus was detected by our genome-wide association mapping across strains for thymic iNKT-cell frequencies. One possible explanation is that the statistical power in our study was not high enough to detect Nkt1
due to the increased genetic complexity and an insufficient number of inbred strains used in our study. In a genetically more complex but overall small population as in our case, contributions from other loci may exceed Nkt1
in terms of ability to regulate thymic iNKT-cells. A non-mutually exclusive possibility is that other genetic variants present in some of the strains analyzed here mask the effect of Nkt1
. As a result, the likelihood to detect these two loci in our analysis is reduced. In any case, it can be predicted that more loci than Nkt1
can be identified if different pairs of inbred mouse strains were chosen for an F2 or first-backcross analyses to map genes controlling the development of iNKT-cells. Our results also further indicate the development and anatomical distribution of iNKT-cells are complex traits that remain to be fully dissected. While genome-wide association studies (GWAS) have provided a tremendous amount of information to our understanding of the genetic control of several disease-related traits in humans, the same approach remains a challenge when a limited number of mouse strains are used. Nevertheless, our strain survey results and GWAS provide a starting point for the future identification and functional characterization of genes controlling various aspects of iNKT-cell development.
In conclusion, we showed a large variation in iNKT-cell frequency across multiple inbred mouse strains similar to the case in humans. Relatively poor correlation in the frequency of total iNKT-cells between PB and the spleen or thymus further emphasizes the importance of comprehensive analysis of lymphoid organs in humans when possible. Finally, our results will facilitate the selection of mouse strains for future studies mapping genes regulating iNKT-cell development.