Briefly, cleared T cell lysates were incubated for 20?min at 37?C with or without 0

Briefly, cleared T cell lysates were incubated for 20?min at 37?C with or without 0.5?mM GTP in the presence of 20?M Taxol and layered on a sucrose cushion. activation, primarily by non-classical antigen-presenting cells. Thus, AKAP9-dependent TCR trafficking drives efficient T cell re-activation and extends their retention at sites of inflammation with implications for disease pathogenesis. Maturation, differentiation and trafficking of T lymphocytes are critical for generating an effective immune response1,2. Dendritic cells (DCs) take up and process antigen at the site of inflammation and emigrate into secondary lymphoid organs, including lymph nodes. Circulating na?ve T cells enter lymph nodes and differentiate and expand upon encountering their specific antigen loaded on major histocompatibility complex (MHC) class II molecules on DCs3. Mature effector T cells then leave lymphoid organs, enter the bloodstream, and migrate to sites of inflammation. There is mounting evidence that T cell recruitment to inflamed tissue occurs through a process that is largely antigen-independent4,5,6, whereas antigen recognition by tissue-resident antigen-presenting cells (APCs) results in T cell re-activation that elicits effector functions7,8. Effector T cells that fail to be activated exit the NVP-231 inflamed tissue via afferent lymphatics and accumulate in the draining lymph node (dLN)9,10,11,12,13, guided by CCR7-CCL19/21 NVP-231 chemokine receptor/ligand cues10,12. However, intracellular molecular mechanisms that coordinate effector T cell retention versus egress remain largely unknown. Several T cell functions including T cell homing and motility, conjugate formation with APCs, T cell antigen receptor (TCR) recycling and migration into inflamed tissues are coordinated by the actin and microtubule (MT) network14. MTs are dynamic structures that undergo growth and catastrophe, which are important for cell division, vesicular trafficking and migration15. The scaffold protein A kinase anchoring protein 9 (AKAP9, AKAP450), present in the Golgi and centrosome of most cells, is emerging as a regulator of MTs emanating from these MT organizing centres15,16,17, particularly the cis-Golgi15. AKAP9 has been implicated in processes that may rely on MTs such as the polarization and migration of T cells18 as well as the formation of the immune synapse with APCs via effects on a T cell integrin, LFA-1 (ref. 19) in human T cell lines. MTs from the Golgi represent a distinct MT subpopulation that does not rely on centrosomal nucleation and regulates specific cellular tasks, which are beginning to be elucidated20. Thus, AKAP9 may regulate a subset of MTs that impact defined cellular functions in T cells and other cell types. Indeed, the normal viability of AKAP9 global-deficient mice21 infer circumscribed rather than global roles for AKAP9 in MT functions. To explore the physiological role of AKAP9 in T cell functions, we generated mice with a conditional deletion of AKAP9 specifically in CD4 and CD8 T cells using Cre-driven by the CD4 promoter22, which we refer to as AKAP9cko/CD4. We show that AKAP9 deficiency did not impair T cell priming, expansion or migration into tissues. Rather, it prevented re-activation and retention of T cells in inflamed tissue in two clinically relevant disease models, anti-glomerular basement membrane (GBM) nephritis and experimental autoimmune encephalitis (EAE), a model of multiple sclerosis. The impaired retention in AKAP9cko/CD4 mice correlated with protection from developing organ damage. (Supplementary Fig. 3cCf). Consistent with these findings, T cell priming was intact in AKAP9cko/CD4 mice following immunization with keyhole limpet hemocyanin or myelin oligodendrocyte glycoprotein (MOG) peptide (Fig. 1aCc). Open in a separate window Figure 1 Priming of CD4+ T cells is unaffected in AKAP9cko/CD4 mice.(a) Proliferation of T cells in lymph node suspensions recovered 4 days after foot pad immunization with MOG, keyhole limpet hemocyanin (KLH) or PBS from draining inguinal lymph nodes and co-incubated with increasing concentrations of the immunizing peptide (cells from PBS immunized mice were incubated with MOG peptide). Data are presented as mean uptake of 3H-Thymidine s.e.m., differentiated AKAP9wt and AKAP9cko/CD4 TH1 cells were adoptively co-transferred via Rab12 tail vein injection at day 10 after induction of glomerulonephritis. We observed equal accumulation of AKAP9cko/CD4 and AKAP9wt cells (Fig. 2c), confirming intact recruitment of cells in the absence of AKAP9. No T cell accumulation of either genotype was observed in the dLNs at this NVP-231 early time point. Differences in apoptosis did not account for the reduced number of effector cells in the kidney as the amount of Annexin V+.