Three-color immunofluorescence analysis was performed using a FACScan ® flow cytometer (Becton Dickinson & Co., Mountain View, CA) and analyzed using CellQuest software. Roland (Institute Pasteur, Paris, France). FITC–anti-Ly49A (JR9-319) was the gift of J. Cells were stained using combinations of directly conjugated mAbs: FITC–anti-TCR-αβ (clone H57), biotin–anti-heat-stable antigen (HSA, clone J11d), and biotin– anti-Vβ8 (clone F23.1) FITC–anti-IL-7Rα chain ( 21) (purified and locally conjugated by standard methods), PE–anti-HSA, biotin–anti-TCR-αβ, FITC–anti-IL-2Rβ, FITC–anti-Ly49C, and PE–anti-NK1.1 (all from PharMingen, San Diego, CA) PE–antiCD4, FITC–anti-CD8, and Tricolor–streptavidin (Caltag Laboratories, San Francisco, CA). Liver lymphocytes were isolated using discontinuous Percoll gradients ( 20) with minor modifications. Thymocyte and splenocyte (red cell–depleted) suspensions were prepared aseptically in HBSS after pressing through sterile mesh filters. ![]() This prompt IL-4 production by NK T cells has suggested a model in which these cells are one of the major determining factors influencing the final TH1/TH2 profile of immune responses ( 13, 14). NK T cells are remarkable for their ability to produce large amounts of cytokines after TCR stimulation ( 12), notably IL-4 ( 12, 13). Studies using T cell hybridomas derived from NK T cells have clearly identified the nonpolymorphic MHC class Ib CD1 molecule as the ligand recognized by these peculiar TCR ( 11). ![]() The limited TCR-αβ diversity of NK T cells suggested that these cells interact with a similarly nonpolymorphic ligand ( 4, 10). The TCRαβ repertoire of NK T cells is markedly restricted: TCR-β chain usage includes Vβ8, Vβ7, and Vβ2, whereas the TCR-α chain is mostly an invariant α chain using the Vα14 and Jα281 segments with a conserved junctional sequence ( 9, 10). Moreover, although the level of TCR expression on conventional T cells is high, NK T cells express intermediate TCR levels (TCR-αβ int). NK T cells express high levels of the IL-2Rβ molecule, a shared cytokine receptor chain used by IL-2 and IL-15, which is also found on NK cells and TCR-γδ T cells, but at low levels on conventional TCR-αβ T cells ( 8). NK T cells comprise both CD4 −CD8 − (double negative, DN) and CD4 + TCR-αβ bearing T cells ( 4, 5), which coexpress a cluster of NK cell markers, including receptors of the C-lectin Ly49 and NKR-P1 (including the NK1.1 antigen) families ( 1– 3, 6, 7). NK T cells are a specialized subset of T cells that share surface markers with the NK cells and have unique properties with respect to their TCR diversity and specificity, as well as their ultimate biological functions ( 1– 3). These results suggest a stepwise pattern of differentiation for thymically derived NK T cells: primary selection via their invariant TCR to confer the IL-4–producing phenotype, followed by acquisition of NK-associated markers and maturation/export to the periphery. We also failed to detect peripheral NK T cells in these mice, demonstrating that γc-dependent interactions are required for export and/or survival of NK T cells from the thymus. Moreover, these data suggest a closer ontogenic relationship of NK T cells to TCR-αβ T cells than to NK cells with respect to cytokine dependency. These results define a maturational progression of NK thymocyte differentiation where intrathymic selection and IL-4–producing capacity can be clearly dissociated from the acquisition of the NK phenotype. Despite these phenotypic abnormalities, γc − NK thymocytes could produce normal amounts of IL-4. However, γc-deficient NK thymocytes fail to coexpress the NK-associated markers NKR-P1 or Ly49, yet retain characteristic expression of the cytokine receptors interleukin (IL)-7Rα and IL-2Rβ. ![]() NK thymocytes differentiate in γc − mice as shown by the normal percentage of TCR Vβ8 + CD4 −CD8 − cells and the normal quantity of thymic Vα14–Jα281 mRNA that characterize the NK T repertoire. For this purpose, we studied common γ chain (γc)-deficient mice, which demonstrate a selective defect in CD3 − NK cell development relative to conventional TCR-αβ T cells. In this report, we have assessed the lineage relationships and cytokine dependency of natural killer (NK) T cells compared with mainstream TCR-αβ T cells and NK cells.
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