The present report describes developmental, phenotypic and functional features of unconventional CD4+ TCR alpha beta lymphocytes. In C57BL/6 mice, the majority of liver lymphocytes expressing intermediate intensity of TCR alpha beta (TCR alpha beta int) are CD4+ NK1.1+ and express a highly restricted TCR V beta repertoire, dominated by V beta 8 with some contribution by V beta 7 and V beta 2. Although these cells express the CD4 co-receptor, they are present in H2-1 A beta (A beta)-/- gene disruption mutants but are markedly reduced in beta 2-microglobulin (beta 2m)-/- mutant mice and hence are beta 2m dependent. Thymocytes expressing the CD4+ NK1.1+ TCR alpha beta phenotype are also beta 2m contingent, suggesting that these two T lymphocyte populations are related. The CD4+ NK1.1+ TCR alpha beta lymphocytes in liver and thymus share several markers such as LFA-1+, CD44+, CD5+, LECAM-1- and IL-2R alpha-. The CD4+ NK1.1+ TCR alpha beta int liver lymphocytes were not detected in athymic nu/nu mice. We conclude that beta 2m expression is crucial for development of the CD4+ NK1.1+ TCR alpha beta int liver lymphocytes and that thymus plays a major role. CD4+ TCR alpha beta int liver lymphocytes were also identified in NK1.1- mouse strains, there lacking the NK1.1 marker. We assume that the NK1.1 molecule is a characteristic marker of the CD4+ TCR alpha beta int liver lymphocytes in NK1.1+ mouse strains, although its expression is not obligatory for their development. The liver lymphocytes from beta 2m+/-, but not from beta 2m-/-, mice are potent IL-4 producers in response to CD3 or TCR alpha beta engagement and the IL-4 production by liver lymphocytes was markedly reduced by treatment with anti-NK1.1 mAb. We conclude that the CD4+ NK1.1+ TCR alpha beta int liver lymphocytes are capable of producing IL-4 in response to TCR stimulation.

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.

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-gamma. The number of CD4+ NK1+ TCR alpha/beta(intermediate) 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-gamma-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-gamma 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-gamma through IL-12 induction. Thus, CD4+ NKT cells may contribute to host resistance against intracellular bacteria prior to conventional IFN-gamma-producing Th1 cells.

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.