Figure 5: AKAP9 is required for restimulation by non-classical APCs.

(a) Proliferation under T_H1-differentiating conditions of naïve AKAP9^wt and AKAP9^cko/CD4 CD4⁺ T cells after 3 days of TCR/CD3ɛ-crosslinking and an additional 2 days of incubation, presented as mean fold expansion (cell number/cell number at day 0) ±s.e.m., n=5. (b) Left panel: Proliferation following 7 days of co-incubation with splenocytes in conditions as in a in the absence or presence of SEB, n=5. Right panel: Representative micrographs of T cell clusters on day 5 of culture. Scale bar, 100 μm. (c) T cell proliferation with SEB over time, n=3. (d) Mixed lymphocyte reaction: Proliferation after 6 days of incubation with allogeneic Balb/c splenocytes, n=4. (e) Re-stimulation reaction: Naïve AKAP9^wt and AKAP9^cko/CD4 CD4⁺ T cells were incubated with SEB-loaded bmDCs for 5 days, rested for 3 days (Priming) and then re-stimulated with SEB-loaded splenocytes (Re-stimulation). ±s.e.m., n=5. (f) Conjugate formation with SEB-pulsed DCs. Left panel: Representative FACS plots of CD11c versus CD4 (pre-gated for TCR Vβ8.1/8.2). Right panel: Mean percentage of CD11c-positive CD4⁺TCR Vβ8.1/8.2⁺ events ±s.e.m., n=4. (g) Conjugate formation with SEB-pulsed splenocytes at indicated T cell:splenocyte ratios, presented as mean percentage of CD19⁺ events of CD4⁺ T cells ±s.e.m., n=3. (h) Phosphorylation of ZAP70 and ERK in AKAP9^wt and AKAP9^cko/CD4 T cells after co-incubation with SEB-pulsed splenocytes for the indicated times in minutes. Quantification is the relative density of phospho-specific signals to corresponding total protein signals ±s.e.m., n=3. #P<0.05. NS, not significant.