Masashi Emoto

Differential chemokine responses and homing patterns of murine TCR alpha beta NKT cell subsets.

NKT cells play important roles in the regulation of diverse immune responses. Therefore, chemokine receptor expression and chemotactic responses of murine TCRαβ NKT cells were examined to define their homing potential. Most NKT cells stained for the chemokine receptor CXCR3, while >90% of Vα14i-positive and ∼50% of Vα14i-negative NKT cells expressed CXCR6 via an enhanced green fluorescent protein reporter construct. CXCR4 expression was higher on Vα14i-negative than Vα14i-positive NKT cells. In spleen only, subsets of Vα14i-positive and -negative NKT cells also expressed CXCR5. NKT cell subsets migrated in response to ligands for the inflammatory chemokine receptors CXCR3 (monokine induced by IFN-γ/CXC ligand (CXCL)9) and CXCR6 (CXCL16), and regulatory chemokine receptors CCR7 (secondary lymphoid-tissue chemokine (SLC)/CC ligand (CCL)21), CXCR4 (stromal cell-derived factor-1/CXCL12), and CXCR5 (B cell-attracting chemokine-1/CXCL13); but not to ligands for other chemokine receptors. Two NKT cell subsets migrated in response to the lymphoid homing chemokine SLC/CCL21: CD4− Vα14i-negative NKT cells that were L-selectinhigh and enriched for expression of Ly49G2 (consistent with the phenotype of most NKT cells found in peripheral lymph nodes); and immature Vα14i-positive cells lacking NK1.1 and L-selectin. Mature NK1.1+ Vα14i-positive NKT cells did not migrate to SLC/CCL21. BCA-1/CXCL13, which mediates homing to B cell zones, elicited migration of Vα14i-positive and -negative NKT cells in the spleen. These cells were primarily CD4+ or CD4−CD8− and were enriched for Ly49C/I, but not Ly49G2. Low levels of chemotaxis to CXCL16 were only detected in Vα14i-positive NKT cell subsets. Our results identify subsets of NKT cells with distinct homing and localization patterns, suggesting that these populations play specialized roles in immunological processes in vivo.

Induction of IFN-gamma-producing CD4+ natural killer T cells by Mycobacterium bovis bacillus Calmette Guérin.

The CD4+ natural killer (NK)T cells in the liver are potent IL‐4 producers and hence may promote Th2 cell development. Following Mycobacterium bovis bacillus Calmette Guérin (BCG) infection, IL‐4‐producing CD4+ NKT cells become undetectable in liver mononuclear cells of normal density (interface between 40 and 70 % Percoll) by flow cytometry. The present study shows that M. bovis BCG infection changes the density of liver CD4+ NKT cells and shifts cytokine production from IL‐4 to IFN‐γ. The number of CD4+ NK1+ TCRα / βintermediate cells increased in the low‐density fraction (< 40 % Percoll density gradient) in parallel to the reduction of this cell population in the fraction of normal density. The number of IL‐4‐producing cells, however, was small and high frequencies of IFN‐γ‐secreting cells were identified in the low‐density fraction after TCR/CD3 ligation. Accordingly, selected low‐density CD4+ NKT cells encompassed high numbers of IFN‐γ producers and minute numbers of IL‐4‐secreting cells. Induction of low‐density CD4+ NKT cells by M. bovis BCG was abrogated by endogenous IL‐12 neutralization which also caused increased bacterial growth in the liver. We assume that M. bovis BCG infection changes cytokine secretion by the CD4+ NKT cell population from IL‐4 to IFN‐γ through IL‐12 induction. Thus, CD4+ NKT cells may contribute to host resistance against intracellular bacteria prior to conventional IFN‐γ‐producing Th1 cells.

Critical role of NK cells rather than V alpha 14(+)NKT cells in lipopolysaccharide-induced lethal shock in mice.

Although macrophages play a central role in the pathogenesis of septic shock, NK1+ cells have also been implicated. NK1+ cells comprise two major populations, namely NK cells and Vα14+NKT cells. To assess the relative contributions of these NK1+ cells to LPS-induced shock, we compared the susceptibility to LPS-induced shock of β2-microglobulin (β2m)−/− mice that are devoid of Vα14+NKT cells, but not NK cells, with that of wild-type (WT) mice. The results show that β2m−/− mice were more susceptible to LPS-induced shock than WT mice. Serum levels of IFN-γ following LPS challenge were significantly higher in β2m−/− mice, and endogenous IFN-γ neutralization or in vivo depletion of NK1+ cells rescued β2m−/− mice from lethal effects of LPS. Intracellular cytokine staining revealed that NK cells were major IFN-γ producers. The Jα281−/− mice that are exclusively devoid of Vα14+NKT cells were slightly more susceptible to LPS-induced shock than heterozygous littermates. Hence, LPS-induced shock can be induced in the absence of Vα14+NKT cells and IFN-γ from NK cells is involved in this mechanism. In WT mice, hierarchic contribution of different cell populations appears likely.

Phenotypic characterization of CD8(+)NKT cells.

We describe a novel CD8+NKT cell population expressing TCRα /β or TCRγ /δ . These CD8+NKT cells were prominent in the liver, and except for the thymus, virtually absent in other lymphoid organs. CD8+NKT cells expressed activation markers and comprised a high proportion of Ly49+ cells. The development of the majority of CD8+NKT cells expressing TCRα /β, but not TCRγ /δ, depended on classical MHC class I. No CD8+NKT cells were detectable in young athymic mice, whereas the cells expressing TCRγ /δ, but not TCRα /β, appeared randomly in aged athymic mice. CD8+NK1+ TCRα /β cells showed polyclonal TCRVβ usage and were virtually devoid of TCRVα14. CD8+NK1+ TCRγ /δ cells predominantly expressed TCRVγ1, 2 and 4, and Vδ4, 5, 6 and 7. CD8+NKT cells, in particular those expressing TCRγ /δ, were a major population in early life. IFN‐γ, but not IL‐4, was induced in CD8+NKT cells by in vitro stimulation, independent of the TCRα /β or TCRγ /δ lineage. Hence, these cells represent a unique, though heterogeneous T cell population that shares markers with, but is distinct from, both conventional NKT cells and conventional CD8+ T cells, and that may play a role in immune regulation.

Neutrophilia in LFA-1-Deficient Mice Confers Resistance to Listeriosis: Possible Contribution of Granulocyte-Colony-Stimulating Factor and IL-17

LFA-1 (CD11a/CD18) plays a crucial role in various inflammatory responses. In this study, we show that LFA-1−/− mice are far more resistant to Listeria monocytogenes infection than LFA-1+/− mice. Consistent with this, we found the following: 1) the numbers of granulocytes infiltrating the liver were markedly higher in LFA-1−/− mice than in LFA-1+/− mice, 2) increased antilisterial resistance in LFA-1−/− mice was abrogated by depletion of granulocytes, and 3) the numbers of granulocytes in peripheral blood, and the serum levels of both G-CSF and IL-17 were higher in LFA-1−/− mice than in LFA-1+/− mice. Neither spontaneous apoptosis nor survival of granulocytes from LFA-1−/− mice were affected by physiological concentrations of G-CSF. Our data suggest regulatory effects of LFA-1 on G-CSF and IL-17 secretion, and as a corollary on neutrophilia. Consequently, we conclude that increased resistance of LFA-1−/− mice to listeriosis is due to neutrophilia facilitating liver infiltration by granulocytes promptly after L. monocytogenes infection, although it is LFA-1 independent.

Interleukin‐4 and listeriosis

Summary: Experimental infection of mice with Listeria monocytogenes (L. monocytogenes) has served as an appropriate model for analyzing Thl nil driven immune responses. Generally, Th2 responses are absent and IL‐4 is not detectable. Here, we describe experimental settings under which IL‐4 is detectable in listeriosis. Our data suggest that IL‐4 is rapidly produced after infection. This prompt IL‐4 burst seems to stimulate chemokine responses and, therefore, may participate in the regulation of the early antilisterial host response. Soon thereafter, lL‐4 production wanes. At least partially this seems to be caused by downregulation of IL‐4–producing CD4+ NK1+ TCRαβint lymphocytes by IL‐12. In the absence of IFN‐γ responsiveness, IL‐4 production is demonstrable during acquired immunity against L. monocytogenes, and this elevated IL‐4 production apparently contributes to disease exacerbation. In conclusion, the data are consistent with a detrimental role of IL‐4 in listeriosis and active control of IL‐4 synthesis by the antilisterial immune response. The rapid, but transient, IL‐4 burst in listeriosis probably contributes to host defense without impairing development of the acquired T‐cell response because of its shortness.

Highly biased type 1 immune responses in mice deficient in LFA-1 in Listeria monocytogenes infection are caused by elevated IL-12 production by granulocytes.

LFA-1 (CD11a/CD18) plays a key role in various inflammatory responses. Here we show that the acquired immune response to Listeria monocytogenes is highly biased toward type 1 in the absence of LFA-1. At the early stage of listeriosis, numbers of IFN-γ producers in the liver and spleen of LFA-1−/− mice were markedly increased compared with heterozygous littermates and Vα14+NKT cell-deficient mice, and NK cells were major IFN-γ producers. Numbers of IL-12 producers were also markedly elevated in LFA-1−/− mice compared with heterozygous littermates, and endogenous IL-12 neutralization impaired IFN-γ production by NK cells. Granulocyte depletion diminished numbers of IL-12 producers and IFN-γ-secreting NK cells in the liver of LFA-1−/− mice. Granulocytes from the liver of L. monocytogenes-infected LFA-1−/− mice were potent IL-12 producers. Thus, in the absence of LFA-1, granulocytes are a major source of IL-12 at the early stage of listeriosis. We assume that highly biased type 1 immune responses in LFA-1−/− mice are caused by increased levels of IL-12 from granulocytes and that granulocytes play a major role in IFN-γ secretion by NK cells. In conclusion, LFA-1 regulates type 1 immune responses by controlling prompt infiltration of IL-12-producing granulocytes into sites of inflammation.

Participation of leukocyte function-associated antigen-1 and NK cells in the homing of thymic CD8+NKT cells to the liver.

A distinct CD8+NKT cell population expressing TCRα/β or TCRγ/δ has been identified in liver and thymus. We wondered whether cell adhesion molecules play a role in the homing of CD8+NKT cells to the liver. The number of liver CD8+NKT cells was markedly reduced in leukocyte function‐associated antigen (LFA)‐1–/– mice compared with wild‐type (WT) mice. The reduction was restricted to the liver only and no measurable alterations were found in other organs. In the liver of SCID mice reconstituted with thymocytes from LFA‐1–/– or WT mice, the number of donor‐derived CD8+NKT cells was comparable and the vast majority of these cells expressed TCRα/β. In a reciprocal radiation thymocyte reconstitution system with LFA‐1–/– and WT mice, LFA‐1 expressed on liver cells other than CD8+NKT cells appeared to be required for the homing of thymic CD8+NKT cells to the liver. The accumulation of donor thymocyte‐derived CD8+NKT cells in the liver of SCID mice was severely impaired by in vivo depletion of NK cells, but not of Kupffer cells. These results not only indicate that thymus provides a source for CD8+NKT cells expressing TCRα/β in the liver, but also suggest that LFA‐1 expressed on NK cells is involved in the homing of thymic CD8+NKT cells to the liver.

Cutting Edge: Contribution of NK Cells to the Homing of Thymic CD4+NKT Cells to the Liver

In contrast to peripheral lymphoid organs, in the liver a high proportion of T cells are CD4+NKT cells. We have previously reported that LFA-1 plays a pivotal role in the homing of thymic CD4+NKT cells to the liver. In the present study, we further assessed which cell type participates in the homing of thymic CD4+NKT cells to the liver. The accumulation of donor thymocyte-derived CD4+NKT cells in the liver of SCID mice that had been reconstituted with thymocytes from C57BL/6 mice was severely impaired by in vivo depletion of NK cells, but not Kupffer cells in recipients. These results suggest that NK cells participate in the homing of thymic CD4+NKT cells to the liver. We assume that LFA-1 expressed on NK cells is involved in this mechanism.

Rapid Development of a Gamma Interferon-Secreting Glycolipid/CD1d-Specific Vα14+ NK1.1− T-Cell Subset after Bacterial Infection

ABSTRACT The phenotypic and functional changes of glycolipid presented by CD1d(glycolipid/CD1d) specific Vα14+ T cells in the liver of mice at early stages of bacterial infection were investigated. After Listeria monocytogenes infection or interleukin-12 (IL-12) treatment, α-galactosylceramide/CD1d tetramer-reactive (α-GalCer/CD1d+) T cells coexpressing natural killer (NK) 1.1 marker became undetectable and, concomitantly, cells lacking NK1.1 emerged in both euthymic and thymectomized animals. Depletion of the NK1.1+ subpopulation prevented the emergence of α-GalCer/CD1d+ NK1.1− T cells. Before infection, NK1.1+, rather than NK1.1−, α-GalCer/CD1d+ T cells coexpressing CD4 were responsible for IL-4 production, whereas gamma interferon (IFN-γ) was produced by cells regardless of NK1.1 or CD4 expression. After infection, IL-4-secreting cells became undetectable among α-GalCer/CD1d+ T cells, but considerable numbers of IFN-γ-secreting cells were found among NK1.1−, but not NK1.1+, cells lacking CD4. Thus, NK1.1 surface expression and functional activities of Vα14+ T cells underwent dramatic changes at early stages of listeriosis, and these alterations progressed in a thymus-independent manner. In mutant mice lacking all α-GalCer/CD1d+ T cells listeriosis was ameliorated, suggesting that the subtle contribution of the NK1.1− T-cell subset to antibacterial protection is covered by more profound detrimental effects of the NK1.1+ T-cell subset.