Prolonged MHC class II deprivation leads to progressive defects in T cell activation and proliferation in response to antigen-bearing DCs. OT-II cells were transferred into MHC class II-deficient, CD4-deficient, or wild-type B6 mice on indicated days before i.v. injection of 1 × 10⁶ Act-mOVA DCs on day 0. In the circumstance where DCs were transferred on the same day as T cells, the T cells were injected before the DCs. (A) Spleens were harvested 3 days after DC injection. Similar results were found with peripheral lymph nodes (data not shown). (Left) Histograms show CFSE content of OT-II cells (CD4+ Thy1.1+) transferred into MHC class II-deficient (unshaded) and CD4-deficient (shaded) recipients. The mean number of cell divisions (M) as experienced by an average OT-II cell recovered on day 3 was calculated according to the following formula: M = Σ_0→n(T_n × n)/Σ_0→nT_n, where T_n represents the number of OT-II cells within each cell division bin n; n = number of cell divisions. The graph shows data from spleens of CD4-deficient (filled squares) and MHC class II-deficient (open circles) recipients. Each data point shows the mean (±SD) of four animals. These data are representative of five independent experiments. (B) Spleens were harvested 18 h after injection of Act-mOVA DCs. Histograms show CD69 expression by OT-II (CD4+ Thy1.1+) cells transferred into MHC class II−/−, CD4−/−, and wild-type B6 recipients. Each histogram is representative of two animals per group and three independent experiments.

OT-II T cells fail to form clusters with antigen-bearing DCs after transfer into MHC class II-deficient recipients. CFSE-labeled OT-II cells were adoptively transferred into MHC class II-deficient or CD4−/− mice on the day of (day 0) (A, B, D, and E), 1 day before (day −1), or 4 days before (day −4) (C and F) injection of seminaphthorhodafluor (SNARF)-labeled Act-mOVA DCs. (A–F) Photomicrographs show CFSE-labeled OT-II cells (green) and SNARF-labeled mOVA DCs (red) in spleens 24 h after DC injection into CD4−/− (A–C) mice and MHC class II−/− (D–F). High-power view of an individual cluster (B and E) is representative of multiple clusters formed within the periarterial lymphoid sheaths of the spleen. (Scale bars: A, C, D, and F, 50 μm; B and E, 25 μm.) (G) The percent of DC (mean ± SD) with no OT-II cells making contact in CD4−/− (filled squares) or MHC class II−/− (open circles) recipients is indicated. This graph shows combined data from three individual experiments. ∗, P < 0.001.

MHC class II deprivation impairs CD4 T cell motility. (A and B) Time-lapse images of CFSE-labeled OT-II T cells in explanted lymph nodes of wild-type (A) and MHC class II-deficient (B) mice. The center of mass of individual cells (colored dots) was tracked in 3D and plotted on z projections of the imaging volume (200 × 225 × 75 μm). Seven representative T cell tracks (colored lines) are shown for both and a time stamp (hr:min:sec:msec) is placed in the upper right corner of each frame. (C and D) Three-dimensional plots of day 7 OT-II T cell trajectories of B6 (C) and MHC class II-deficient (D) mice. (E–G) Two-dimensional projections of OT-II T cell tracks (colored lines) are plotted normalized to their starting positions OT-II T cell trajectories in wild-type hosts (E) and MHC class II-deficient hosts on day 2 (F) and day 7 (G). Representative tracks are shown ranging from 20 to 45 min in length. The axes are in micrometers. Mean velocity (v) for each group is shown with SE and the total number of velocity measurements in the imaging record (n). These data are representative of three independent experiments.

MHC class II availability promotes basal Rap1 and Rac1 activity. OT-II CD4 T cells were purified from transgenic mice treated with Y3P or mouse IgG (control) for 7 days. Samples containing 2–7 × 10⁶ cells were lysed and basal active Rap1 (A), Rac1 (B), or Ras (C) was precipitated with a GST-RalGDS-RBD fusion protein, GST-Pak1 fusion protein, or GST Raf-RBD fusion protein respectively, as previously described (49). The resulting precipitates were analyzed by Western blotting with an anti-Rap1, anti-Rac1, or anti-Ras antibody to assess the content of active Rap1, Rac1, or Ras in samples. Aliquots of total lysates were analyzed in the same way to assess the total Rap1, Rac1, or Ras content. Bands were quantified and the total number of pixels in each image from two independent experiments was used to calculate the average active to total ratio. Error bars represent standard deviations.