Figure 7.

Reconstitution of responses to α-GalCer in IFN-γ-KO bone marrow cultures by CD1-dependent splenic T cells is mediated by IFN-γ. Bone marrow cells from IFN-γ-KO donor mice were collected after fractionation on Percoll gradients from the 60–75% Percoll interface (P2 layer), counted and adjusted to 10⁶·cells mL⁻¹. Spleens were collected from C57BL/6 (WT) or CD1KO (CD1) donor mice, and mononuclear cell suspensions were prepared by Ficoll-Hypaque centrifugation. T lymphocytes (T-cell) were further purified on nylon wool columns. Cultures were established as in Figure 6, panel (F), with 1 ng·mL⁻¹ IL-5, and in the absence or presence of 10⁻⁸M α-GalCer (+GalCer) Cultures contained 10⁶ P2 layer bone marrow cells alone (−), or co-cultured with 10⁵ T lymphocytes (+T-cell WT, +T-cell CD1). Where indicated, the co-cultures received anti-IFN-γ neutralizing antibody (100 ng·mL⁻¹; +anti-IFN-γ), or an equivalent amount of isotype-matched irrelevant antibody as a control (+isotype ctrl). Data are mean + SEM of EPO+ cell numbers recovered at day 7. *P ≤ 0.05, **P < 0.01, significant effect of α-GalCer; n = 5 for all means.