Figure 7.

The induction of Th1 and Th2 type cells in vitro. Lymph node cells from an MBP-specific TCR transgenic mouse were stained with CD8, B220, Mac-1, CD69, and CD44, and the negative cells were collected by flow cytometry as naive, CD4⁺ T cells (reanalysis of cells by flow cytometry indicated a population >98% CD4⁺). These cells were cultured in vitro at a concentration of 10⁵ CD4⁺ cells and 10⁶ irradiated nontransgenic syngeneic splenocytes per well in 24-well plates with varying concentrations of Ac1-11, Ac1-11[4A], or Ac1-11[4Y]. 10 d later, cells were washed several times and restimulated at a concentration of 5 × 10⁴ T cells plus 3 × 10⁵ irradiated splenocytes per well in 96-well round-bottom plates with 10 μM Ac1-11, Ac111[4A], or Ac1-11[4Y]. Supernatants were collected at 48 h, and cytokines were detected by ELISA. Black bars, cytokine response to Ac1-11 in secondary challenge; shaded bars, cytokine response to Ac1-11[4A] in secondary challenge; cross-hatched bars, cytokine response to Ac1-11[4Y] in secondary challenge. In general, Ac1-11 induced Th1 type responses regardless of primary concentration, Ac1-11[4A] induced Th2 type responses at high concentrations and Th1 type responses at low concentrations, and Ac1-11[4Y] induced primarily Th2 type responses, except at the lowest two doses. nd, not determined. Previous experiments indicated that too few live cells were recovered at the doses for which cytokine levels were not determined to test a secondary response.