Cytotoxic T lymphocytes (CTL) respond to antigenic peptides presented on MHC class I molecules. On most cells, these peptides are exclusively of endogenous, cytosolic origin. Bone marrow-derived antigen-presenting cells, however, harbor a unique pathway for MHC I presentation of exogenous antigens. This mechanism permits cross-presentation of pathogen-infected cells and the priming of CTL responses against intracellular microbial infections. Here, we report a novel diphtheria toxin-based system that allows the inducible, short-term ablation of dendritic cells (DC) in vivo. We show that in vivo DC are required to cross-prime CTL precursors. Our results thus define a unique in vivo role of DC, i.e., the sensitization of the immune system for cell-associated antigens. DC-depleted mice fail to mount CTL responses to infection with the intracellular bacterium Listeria monocytogenes and the rodent malaria parasite Plasmodium yoelii.

The role played by resident macrophages (Mφ) in the initiation of peritoneal inflammation is currently unclear. We have used a conditional Mφ ablation strategy to determine the role of resident peritoneal Mφ in the regulation of neutrophil (PMN) recruitment in experimental peritonitis. We developed a novel conditional Mφ ablation transgenic mouse (designated CD11bDTR) based upon CD11b promoter-mediated expression of the human diphtheria toxin (DT) receptor. The murine DT receptor binds DT poorly such that expression of the human receptor confers toxin sensitivity. Intraperitoneal injection of minute (nanogram) doses of DT results in rapid and marked ablation of F4/80-positive Mφ populations in the peritoneum as well as the kidney, and ovary. In experimental peritonitis, resident Mφ ablation resulted in a dramatic attenuation of PMN infiltration that was rescued by the adoptive transfer of resident nontransgenic Mφ. Attenuation of PMN infiltration was associated with diminished CXC chemokine production at 1 h. These studies indicate a key role for resident peritoneal Mφ in sensing perturbation to the peritoneal microenvironment and regulating PMN infiltration.

The Ca2+ -activated K+ channel KCa3.1 is required for Ca2+ influx and the subsequent activation of B and T cells. Inhibitors of KCa3.1 are in development to treat autoimmune diseases and transplant rejection, underscoring the importance in understanding how these channels are regulated. We show that nucleoside diphosphate kinase B (NDPK-B), a mammalian histidine kinase, functions downstream of PI(3)P to activate KCa3.1. NDPK-B directly binds and activates KCa3.1 by phosphorylating histidine 358 in the carboxyl terminus of KCa3.1. Endogenous NDPK-B is also critical for KCa3.1 channel activity and the subsequent activation of CD4 T cells. These findings provide one of the best examples whereby histidine phosphorylation regulates a biological process in mammals, and provide an example whereby a channel is regulated by histidine phosphorylation. The critical role for NDPK-B in the reactivation of CD4 T cells indicates that understanding NDPK-B regulation should uncover novel pathways required for T cell activation.

Myotubularins (MTMs) belong to a large subfamily of phosphatases that dephosphorylate the 3 position of phosphatidylinositol 3-phosphate [PI(3)P] and PI(3,5)P 2 . MTM1 is mutated in X-linked myotubular myopathy, and MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth syndrome. However, little is known about the general mechanism(s) whereby MTMs are regulated or the specific biological processes regulated by the different MTMs. We identified a Ca 2؉ -activated K channel, K Ca 3.1 (also known as KCa4, IKCa1, hIK1, or SK4), that specifically interacts with the MTMR6 subfamily of MTMs via coiled coil (CC) domains on both proteins. Overexpression of MTMR6 inhibited K Ca 3.1 channel activity, and this inhibition required MTMR6's CC and phosphatase domains. This inhibition is specific; MTM1, a closely related MTM, did not inhibit K Ca 3.1. However, a chimeric MTM1 in which the MTM1 CC domain was swapped for the MTMR6 CC domain inhibited K Ca 3.1, indicating that MTM CC domains are sufficient to confer target specificity. K Ca 3.1 was also inhibited by the PI(3) kinase inhibitors LY294002 and wortmannin, and this inhibition was rescued by the addition of PI(3)P, but not other phosphoinositides, to the patch pipette solution. PI(3)P also rescued the inhibition of K Ca 3.1 by MTMR6 overexpression. These data, when taken together, indicate that K Ca 3.1 is regulated by PI(3)P and that MTMR6 inhibits K Ca 3.1 by dephosphorylating the 3 position of PI(3)P, possibly leading to decreased PI(3)P in lipid microdomains adjacent to K Ca 3.1. K Ca 3.1 plays important roles in controlling proliferation by T cells, vascular smooth muscle cells, and some cancer cell lines. Thus, our findings not only provide unique insights into the regulation of K Ca 3.1 channel activity but also raise the possibility that MTMs play important roles in the negative regulation of T cells and in conditions associated with pathological cell proliferation, such as cancer and atherosclerosis.Myotubularins (MTM) are a large family of evolutionarily conserved lipid phosphatases (PT) that specifically dephosphorylate the 3Ј position of phosphatidylinositol 3-phosphate [PI(3)P] and PI(3,5)P 2 (28, 39). Fourteen MTMs in mammalian cells have been identified, and they can be divided into six subgroups based on sequence alignment and phylogenetic comparison. Members of one of these subgroups lack phosphatase activity due to a mutation in a critical residue within the phosphatase domain. MTM1, the founding member of this gene family, is mutated in X-linked myotubular myopathy, and MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth (CMT) syndrome type 4B (3, 30, 37). In addition to containing a phosphatase domain, most MTMs are composed of a GRAM domain which may mediate association of MTMs with membranes, a Rac-induced localization domain which mediates the association with Rac-induced membrane ruffles, and a C-terminal coiled coil (CC) domain (10,28,29,39). Recent data have indicated that the MTM CC domains mediate specific heterodimerization between a MTM (PT ac...

Th cells are the major effector cells in transplant rejection and can be divided into Th1, Th2, Th17, and Treg subsets. Th differentiation is controlled by transcription factor expression, which is driven by positive and negative cytokine and chemokine stimuli at the time of T cell activation. Here we discovered that chemokine platelet factor 4 (PF4) is a negative regulator of Th17 differentiation. PF4-deficient and platelet-deficient mice had exaggerated immune responses to cardiac transplantation, including increased numbers of infiltrating Th17 cells and increased plasma IL-17. Although PF4 has been described as a platelet-specific molecule, we found that activated T cells also express PF4. Furthermore, bone marrow transplantation experiments revealed that T cell-derived PF4 contributes to a restriction in Th17 differentiation. Taken together, the results of this study demonstrate that PF4 is a key regulator of Th cell development that is necessary to limit Th17 differentiation. These data likely will impact our understanding of platelet-dependent regulation of T cell development, which is important in many diseases, in addition to transplantation.