Invariant NKT (iNKT) cells are tissue-resident innate-like T cells critical to the host immune response. We previously identified a 6.6 Mbp region on chromosome 1 as a major regulator of iNKT cell number and function in C57BL/6 and 129X1/SvJ mice. Here, we fine-mapped this locus by assessing the iNKT cell response to alpha-galactosylceramide (αGalCer) in a series of B6.129 congenic lines. This analysis revealed the presence of at least two genetic elements that regulate iNKT cell cytokine production in response to αGalCer. While one of these genetic elements mapped to the B6.129c6 interval containing Slam genes, the dominant regulator in this region mapped to the 0.14 Mbp B6.129c3 interval. In addition, we found that numbers of thymic iNKT cells and DP thymocytes were significantly lower in B6.129c3 mice, indicating that this interval also regulates iNKT cell development. Candidate gene analysis revealed a fivefold increase in Fcgr3 expression in B6.129c3 iNKT cells, and we observed increased expression of FcγR3 protein on B6.129c3 iNKT cells, NK cells, and neutrophils. These data identify the B6.129c3 interval as a novel locus regulating the response of iNKT cells to glycosphingolipid, revealing a link between this phenotype and a polymorphism that regulates Fcgr3 expression.

NKT cells play a critical role in tissue-specific immune responses by rapidly producing cytokines in response to glycolipid stimulation. We previously reported that host genetic background played a substantial role in the NKT cell response to the agonist glycolipid α-galactosylceramide (αGalCer). A 6.6 Mbp region on chromosome 1 was identified as a major regulator of the NKT cell cytokine response between C57BL/6 (B6) and 129X1/SvJ mice. To fine-map this locus, we generated several B6.129 congenic lines with overlapping 129X1/SvJ intervals from 0.1 to 1.1 Mbp, and assessed the response of NKT cells to αGalCer. An analysis of serum and intracellular cytokine production after αGalCer challenge revealed significantly reduced levels of all cytokines tested in the B6.129c3 (c3) congenic strain that possessed the minimal 129 interval containing 5 genes. In vitro analysis of sorted splenic NKT cells indicated that the reduced cytokine production was NKT cell-intrinsic. In addition, we observed significantly fewer thymic NKT cells in the B6.129c3 congenic mice suggesting that the c3 interval may also regulate NKT cell development. Analysis of candidate genes within the c3 interval revealed a 5-fold increase in Fcgr3 gene expression on c3 NKT cells, which was associated with increased expression of FcγR3 protein on B6.129c3 NKT, NK and neutrophils. Taken together, these data identify the c3 interval as a novel locus regulating the response of NKT cells to glycosphingolipid, and reveal a link between this phenotype and an expression polymorphism that regulates Fcgr3 expression.

Signaling lymphocyte activation marker family member 6 (Slamf6) is a cell surface signaling receptor that plays an important role in NKT cell development. Upon activation with the CD1d ligand α-galactosylceramide (αGalCer), NKT cell Slamf6 expression increases dramatically (~10-fold) and it maintains this high level of expression for at least 5 days after activation. The mechanisms through which Slamf6 regulates NKT cell function are largely unknown. To investigate the effect of Slamf6 on peripheral NKT cell populations, we challenged C57BL/6 (B6) or B6.Slamf6−/− mice with the NKT-specific agonist αGalCer. Examination of liver NKT cell numbers 3 days after challenge revealed a 50-fold increase in B6 mice over vehicle-treated controls. In contrast, we observed a 270-fold increase in NKT cells in B6.Slamf6−/− mice versus controls. An analysis of spleen NKT cells yielded similar results. A comparison of in vivo BrdU uptake by NKT cells between B6 and B6.Slamf6−/− mice revealed no significant differences in proliferation. In addition, NKT cell intracellular IFN-γ, IL-4, and TNF production between B6 and B6.Slamf6−/− mice after administration of αGalCer revealed no difference in the production of these cytokines. We next evaluated the Slamf6-specific effect on NKT cell expansion using an in vitro co-culture system. Cross-linking of Slamf6 on purified B6 NKT cells resulted in diminished NKT cell expansion and increased numbers of TUNEL+ NKT cells. Taken together, these data support a model where Slamf6 acts as a negative regulator of NKT cell expansion in the presence of a strong agonist by regulating the death threshold of NKT cells. These data suggest that Slamf6 blockade could be a useful tool to manipulate the expansion of NKT cells in vivo.

ABSTRACT Pseudomonas aeruginosa is an important human opportunistic pathogen, accounting for a significant fraction of hospital-acquired lung infections. CD1d-restricted NKT cells comprise an unusual innate-like T cell subset that plays important roles in both bacterial and viral infections. Previous reports have differed in their conclusions regarding the role of NKT cells in clearance of P. aeruginosa from the lung. Since there is significant strain-dependent variation in NKT cell number and function among different inbred strains of mice, we investigated whether the role of NKT cells was dependent on the host genetic background. We found that NKT cells did indeed play a critical role in the clearance of P. aeruginosa from the lungs of BALB/c mice but that they played no discernible role in clearance from the lungs of C57BL/6 mice. We found that the strain-dependent role of NKT cells was associated with significant strain-dependent differences in cytokine production by lung NKT cells and that impaired clearance of P. aeruginosa in BALB/c CD1d−/− mice was associated with an increase in neutrophil influx to the lung and increased levels of proinflammatory cytokines and chemokines after infection. Finally, we found that the role of alveolar macrophages was also dependent on the genetic background. These data provide further support for a model in which the unusually high level of variability in NKT cell number and function among different genetic backgrounds may be an important contributor to infectious-disease susceptibility and pathology.

CD1d-restricted NKT cells comprise an innate-like T cell population that exerts significant influence over early events in the developing immune response. The frequency of NKT cells is highly variable in humans and in mice, but the basis for this variability remains unclear. In this study, we report a striking deficiency of type I NKT cells in the wild-derived inbred strains PWD/PhJ, SPRET/EiJ, and CAST/EiJ. Investigation of the underlying basis for the lack of type I NKT cells revealed that one strain, PWD/PhJ, exhibited a significant impairment in thymocyte and splenocyte CD1d gene and protein expression. Accordingly, both thymocytes and bone marrow–derived dendritic cells from PWD mice exhibited a significant impairment in the ability to present α-galactosylceramide to NKT cells. The impaired PWD CD1d gene expression was due to impaired CD1d promoter activity. Fine-mapping of the promoter activity revealed that two single nucleotide substitutions at positions −331 and −164 in the proximal promoter were each sufficient to account for the diminished PWD CD1d promoter activity. Examination of the strain distribution pattern of these polymorphisms revealed that, of 19 strains analyzed, only PWD and PWK mice possessed both CD1d promoter polymorphisms. A subsequent examination of the PWK strain revealed that it also exhibited impaired thymocyte CD1d expression and very low numbers of NKT cells. Taken together, these results provide new insight into the control of CD1d gene expression, and they have implications for the evolution of CD1d and type I NKT cells.

NKT cells are known to rapidly produce a large amount of cytokines upon activation. Although a number of signaling pathways that regulate the development of NKT cells have been identified, the signaling pathways involved in the regulation of NKT cell cytokine production remain unclear. In this study, we show that the p38 MAPK pathway is dispensable for the development of NKT cells. However, NKT cell cytokine production and NKT-mediated liver damage are highly dependent on activation of this pathway. p38 MAPK does not substantially affect cytokine gene expression in NKT cells, but it regulates the synthesis of cytokines through the Mnk–eIF4E pathway. Thus, in addition to gene expression, translational regulation by p38 MAPK could be a novel mechanism that contributes to the overall production of cytokine by NKT cells.