Characterization of In Vivo Dlg1 Deletion on T Cell Development and Function
Figure 8
The acute or conditional loss of dlg1 results in differential Th1/Th2-type cytokine production.
(A) Expanded splenocytes from dlg1wt;BG or dlg1ko;BG mice were restimulated with plate-bound anti-CD3 and anti-CD28 antibodies or PMA/Ionomycin for 4 hours. Cells were stained with anti-CD8, permeabilized, and subsequently stained with anti-IFN-γ to determine intracellular cytokine levels (n≥3 each for dlg1wt;BG and dlg1ko;BG mice). Data represent mean +/− StDev. n.s. = no significance where p>0.05. (B) Expanded CD4+ T cells from dlg1flox/flox or dlg1flox/flox:CD4cre mice were restimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 24 hours. Cell supernatants were collected and cytokine production analyzed by ELISA. Standard deviations were calculated from triplicate stimulations and statistical significance determined using a paired Student’s t test. *p<0.05. (C) Th1 or Th2 polarized T cells from C57Bl/6 mice were transduced with either control- or Dlg1-miRNA retrovirus (right panel) and subsequently stimulated with plate-bound anti-CD3/CD28 for 6 hours. Cells were then surface stained with anti-CD4 followed by intracellular staining with antibodies against either IFNγ or TNFα (Th1 cells) or IL-4 (Th2 cells). Cells were analyzed by FACS and gated on CD4+ and GFP+ (miRNA vector) cells to determine the relative level of cytokine production (MFI) in control and knockdown populations (left panel) (IFNγ, TNFα n = 4; IL-4 n = 3). Data represent means from ≥3 experiments ± SEM. *p<0.05.