CXCL16-positive dendritic cells enhance invariant natural killer T cell-dependent IFNγ production and tumor control.

Crosstalk interactions between dendritic cells (DCs) and invariant natural killer T (iNKT) cells are important in regulating antitumor responses elicited by glycolipid antigens. iNKT cells constitutively express the chemokine receptor CXCR6, while cytokine-activated DCs upregulate the transmembrane chemokine ligand, CXCL16. This study examined the co-stimulatory role of CXCR6/CXCL16 interactions in glycolipid-dependent iNKT cell activation and tumor control. Spleen and liver DCs in wild-type mice, but not iNKT cell deficient (Jα18(-/-)) mice, transiently upregulated surface CXCL16 following in vivo administration of the glycolipid antigen α-galactosylceramide. Recombinant CXCL16 did not directly induce iNKT cell activation in vitro but enhanced interferon (IFN)-γ production when mouse or human iNKT cells were stimulated with plate-bound anti-CD3. Compared with glycolipid-loaded CXCL16(neg) DCs, CXCL16(hi) DCs induced higher levels of IFNγ production in iNKT cell cultures and following adoptive transfer in vivo. The number of IFNγ(+) iNKT cells and expansion of T-bet(+) iNKT cells were reduced in vivo when CXCL16(-/-) DCs were used to activate iNKT cells. Enhanced IFNγ production in vivo was not dependent on CXCR6 expression on natural killer (NK) cells. Adoptive transfer of glycolipid-loaded CXCL16(hi) DCs provided superior protection against tumor metastasis compared to CXCL16(neg) DC transfers. Similarly, wild-type DCs provided superior protection against metastasis compared with CXCL16(-/-) DCs. These experiments implicate an important role for CXCR6/CXCL16 interactions in regulating iNKT cell IFNγ production and tumor control. The selective use of CXCL16(hi) DCs in adoptive transfer immunotherapies may prove useful for enhancing T helper (Th) type 1 responses and clinical outcomes in cancer patients.

Acquisition of MHC-specific receptors on murine natural killer cells.

NK cells in adult mice express two families of MHC class I-specific receptors, namely, Ly49 and CD94/NKG2. Co-expression of these receptors in various combinations generates diverse receptor repertoires. The expression of individual receptors is mostly stochastic and independent of each other. NK cells acquire the receptors as they develop from progenitors in the bone marrow in adult mice. In vivo as well as in vitro studies have shown that the acquisition of the receptors is ordered and regulated by the host MHC class I. Developing NK cells first acquire CD94/NKG2 and subsequently various Ly49 receptors in an ordered manner. Unlike adult NK cells, most fetal and neonatal NK cells express CD94/NKG2 but not Ly49. During the first several weeks after birth, NK cells expressing various Ly49 receptors slowly accumulate, while CD94/NKG2+ NK cells decrease to approximately 50% of the population. The acquisition of NK receptors following hematopoietic stem cell transplantation is also a slow and apparently preprogrammed process, mimicking the ontogeny, regardless of whether stem cells from fetal liver or adult bone marrow are used as donors. The regulation of the transcription of individual receptor genes is rather complex, since two promoters have been identified for the genes encoding Ly49 and CD94.

Unique subset of natural killer cells develops from progenitors in lymph node.

Natural killer (NK) cells have been thought to develop from committed progenitors in the bone marrow. However, a novel pathway of thymus-dependent NK-cell development that produces a unique subset of NK cells expressing CD127 has recently been reported. We now have identified 2 populations of NK progenitors, one in the thymus and the other in the lymph node (LN). Immature double-negative 2 (CD4(-)CD8(-)CD44(+)CD25(+)) thymocytes have potential to produce NK cells with rearranged T-cell receptor gamma genes (Tcrgamma(+)) in vitro. Tcrgamma(+) NK cells are rare in spleen but relatively abundant in the thymus and LN. Approximately 20% of LN NK cells are Tcrgamma(+), and they are found at similar levels in both CD127(+) and CD127(-) subsets. Moreover, a subpopulation of LN cells resembling immature thymocytes differentiates into Tcrgamma(+) NK cells in vitro and also repopulates the NK compartment in lymphopenic mice. Athymic mice lack the LN NK progenitors expressing CD127 as well as Tcrgamma(+) NK cells. These results suggest that Tcrgamma(+) NK cells may be generated from unique progenitors in the thymus as well as in the LN.

Critical role for the chemokine receptor CXCR6 in homeostasis and activation of CD1d-restricted NKT cells.

NK T (NKT) cells play important roles in the regulation of diverse immune responses. However, little is known about the mechanisms that regulate homeostasis and activation of these cells. Thymic NKT cells up-regulated the chemokine receptor CXCR6 following positive selection and migrated toward CXCL16 in vitro. However, CXCR6 was not essential for thymic development or maturation. In contrast, liver and lung NKT cells were depleted in CXCR6+/- and CXCR6-/- mice. The reduction in liver and lung NKT cells coincided with an increase in bone marrow NKT cells, suggesting a redistribution of NKT cells in CXCR6-/- animals. In wild-type mice, CXCL16 neutralization reduced accumulation of mature NK1.1+, but not immature NK1.1- NKT cell recent thymic emigrants in the liver. Given that thymic NKT cells are preferentially exported as NK1.1- cells, this suggests an additional role for CXCR6/CXCL16 in maturation or survival of immature liver NKT cells. CXCL16 blockade did not deplete resident NK1.1+ NKT cells, indicating that CXCR6/CXCL16 are not required to retain mature NKT cells in the liver. Cytokine production by liver and spleen NKT cells was impaired in CXCR6-/- mice following in vivo stimulation with alpha-galactosylceramide, implicating a novel role for CXCR6 in NKT cell activation. Reduced IFN-gamma production was not due to an intrinsic defect as production was normal following PMA and ionomycin stimulation. Preformed transcripts for IL-4, but not IFN-gamma, were reduced in CXCR6-/- liver NKT cells. These data identify critical roles for CXCR6/CXCL16 in NKT cell activation and the regulation of NKT cell homeostasis.

Expression of rearranged TCRgamma genes in natural killer cells suggests a minor thymus-dependent pathway of lineage commitment.

Natural killer (NK) cells are thought to develop from common lymphoid progenitors in the bone marrow. However, immature thymocytes also retain NK potential. Currently, the contribution of the thymus-dependent pathway in normal steady-state NK-cell development is unknown. Here, we show that TCRgamma genes are rearranged in approximately 5% of neonatal and 1% of adult mouse splenic NK cells, and similar levels are detected in NK cells from TCRbeta,delta double-knockout mice, excluding the possibility of T-cell contamination. NK-cell TCRgamma gene rearrangement is thymus dependent because this rearrangement is undetectable in nude mouse NK cells. These results change the current view of NK-cell development and show that a subset of NK cells develops from immature thymocytes that have rearranged TCRgamma genes.

Murine CD160, Ig-like receptor on NK cells and NKT cells, recognizes classical and nonclassical MHC class I and regulates NK cell activation.

Human and mouse NK cells use different families of receptors to recognize MHC class I (MHC I) on target cells. Although human NK cells express both Ig-like receptors and lectin-like receptors specific for MHC I, all the MHC I-specific receptors identified on mouse NK cells to date are lectin-like receptors, and no Ig-like receptors recognizing MHC I have been identified on mouse NK cells. In this study we report the first MHC I-specific Ig-like receptor on mouse NK cells, namely, murine CD160 (mCD160). The expression of mCD160 is restricted to a subset of NK cells, NK1.1+ T cells, and activated CD8+ T cells. The mCD160-Ig fusion protein binds to rat cell lines transfected with classical and nonclassical mouse MHC I, including CD1d. Furthermore, the level of mCD160 on NK1.1+ T cells is modulated by MHC I of the host. Overexpression of mCD160 in the mouse NK cell line KY-2 inhibits IFN-gamma production induced by phorbol ester plus ionomycin, whereas it enhances IFN-gamma production induced by NK1.1 cross-linking or incubation with dendritic cells. Cross-linking of mCD160 also inhibits anti-NK1.1-mediated stimulation of KY-2 cells. Anti-mCD160 mAb alone has no effect. Thus, mCD160, the first MHC I-specific Ig-like receptor on mouse NK cells, regulates NK cell activation both positively and negatively, depending on the stimulus.