The gene for hSK4, a novel human small conductance calcium-activated potassium channel, or SK channel, has been identified and expressed in Chinese hamster ovary cells. In physiological saline hSK4 generates a conductance of approximately 12 pS, a value in close agreement with that of other cloned SK channels. Like other members of this family, the polypeptide encoded by hSK4 contains a previously unnoted leucine zipper-like domain in its C terminus of unknown function. hSK4 appears unique, however, in its very high affinity for Ca 2؉ (EC 50 of 95 nM) and its predominant expression in nonexcitable tissues of adult animals. Together with the relatively low homology of hSK4 to other SK channel polypeptides (approximately 40% identical), these data suggest that hSK4 belongs to a novel subfamily of SK channels.In mammals, small conductance calcium-activated potassium channels, or SK channels, are thought to underlie currents that have been described in a wide range of tissues, including brain (1-13), peripheral nervous system (14-16), skeletal muscle (17-19), adrenal chromaffin cells (20)(21)(22), leukocytes (23-28), erythrocytes (29-32), colon (33, 34), and airway epithelia (35,36). Pharmacologically, certain types of SK channels have been distinguished by their sensitivities to the bee venom apamin (5, 7-23, 37), whereas other functionally related conductances appear insensitive (7,24,27,34). Features that distinguish members of this family from their closest phenotypic neighbors, the maxi-K calcium-activated, or BK, potassium channels, are the SK channels' low conductance (less than 50 pS), the weak or negligible dependence of their activity on membrane voltage, and their high affinity for Ca 2ϩ (EC 50 Ͻ 1 M) (3, 19-23, 25, 26, 33-40).Fragments of SK genes first were identified in computerbased searches of GenBank's database of expressed sequence tags (ESTs) for cDNAs encoding sequences resembling the pore domains of known families of K ϩ channels (41). We have extended this work by identifying ESTs including the gene encoding human SK4 (hSK4), a member of a novel subfamily of SK channels, and expressing one of these cDNAs in Chinese hamster ovary cells. In addition, we cloned the full-length gene of rSK1 (41).Members of the first subfamily to be described are predominantly expressed in excitable tissues and are half-maximally activated at cytosolic free Ca 2ϩ concentrations in the range of 600-700 nM (41). The hSK4 channel differs from these in that its transcript is found in nonexcitable tissues and is halfactivated at 95 nM free Ca 2ϩ , indicating it is likely to be open at resting levels of Ca 2ϩ in certain types of cells. The hyperpolarization resulting from the activity of hSK4 suggests that this channel could regulate electrogenic transport. METHODSCloning of SK Genes. The two P regions of the yeast TOK channel were used to screen the EST database of GenBank using the BLAST algorithm (42). One of the ESTs that was identified as a novel potential mammalian K ϩ channel cDNA was labeled by random prim...

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Large-conductance calcium-activated potassium channels (maxi-K channels) have an essential role in the control of excitability and secretion. Only one gene Slo is known to encode maxi-K channels, which are sensitive to both membrane potential and intracellular calcium. We have isolated a potassium channel gene called Slack that is abundantly expressed in the nervous system. Slack channels rectify outwardly with a unitary conductance of about 25-65 pS and are inhibited by intracellular calcium. However, when Slack is co-expressed with Slo, channels with pharmacological properties and single-channel conductances that do not match either Slack or Slo are formed. The Slack/Slo channels have intermediate conductances of about 60-180 pS and are activated by cytoplasmic calcium. Our findings indicate that some intermediate-conductance channels in the nervous system may result from an interaction between Slack and Slo channel subunits.

Although dendritic cell (DC) dysfunction in cancer is a well-recognized consequence of cancer-associated inflammation that contributes to immune evasion, the mechanisms that drive this process remain elusive. Here, we show the critical importance of tumor-derived TLR2 ligands in the generation of immunosuppressive IL-10-producing human and mouse DCs. TLR2 ligation induced two parallel synergistic processes that converged to activate STAT3: stimulation of autocrine IL-6 and IL-10 and upregulation of their respective cell surface receptors, which lowered the STAT3 activation threshold. We identified versican as a soluble tumor-derived factor that activates TLR2 in DCs. TLR2 blockade in vivo improved intra-tumor DC immunogenicity and enhanced the efficacy of immunotherapy. Our findings provide a basis for understanding the molecular mechanisms of DC dysfunction in cancer and identify TLR2 as a relevant therapeutic target to improve cancer immunotherapy.

Kv1.3 is a voltage-gated potassium channel with roles in human T cell activation/proliferation, cell-mediated cytotoxicity, and volume regulation and is thus a target for therapeutic control of T cell responses. Kv1.3 is also present in some mouse thymocyte subsets and splenocytes, but its role in the mouse is less well understood. We report the generation and characterization of Kv1.3-deficient (Kv1.3 ؊/؊ ) mice. In contrast to wild-type cells, the majority of Kv1.3 ؊/؊ thymocytes had no detectable voltage-dependent potassium current, although RNA and protein for several potassium channel subunits were found in the thymocyte population. Surprisingly, the level of chloride current in the Kv1.3 ؊/؊ thymocytes was increased approximately 50-fold over that in wildtype cells. There were no abnormalities in lymphocyte types or absolute numbers in thymus, spleen, and lymph nodes and no obvious defect in thymocyte apoptosis or T cell proliferation in the Kv1.3 ؊/؊ animals. The compensatory effects of the enhanced chloride current may account for the apparent lack of immune system defects in Kv1.3 ؊/؊ mice.The voltage-gated K ϩ channel, Kv1.3, is expressed in B and T cells, natural killer cells, macrophages (for review, see Ref.

Dendritic cells (DC) play a pivotal role in the tumor microenvironment (TME). As the primary antigen-presenting cells in the tumor, DCs modulate anti-tumor responses by regulating the magnitude and duration of infiltrating cytotoxic T lymphocyte responses. Unfortunately, due to the immunosuppressive nature of the TME, as well as the inherent plasticity of DCs, tumor DCs are often dysfunctional, a phenomenon that contributes to immune evasion. Recent progresses in our understanding of tumor DC biology have revealed potential molecular targets that allow us to improve tumor DC immunogenicity and cancer immunotherapy. Here, we review the molecular mechanisms that drive tumor DC dysfunction. We discuss recent advances in our understanding of tumor DC ontogeny, tumor DC subset heterogeneity, and factors in the tumor microenvironment that affect DC recruitment, differentiation, and function. Finally, we describe potential strategies to optimize tumor DC function in the context of cancer therapy.

Objective Ankylosing spondylitis (AS) is an inflammatory arthritis in which men have a higher risk of developing progressive axial disease than women. Transcriptomic studies have shown reduced expression of cytotoxic cell genes in the blood of AS patients. HLA–B27 contributes the greatest risk for AS, suggesting a role for CD8+ T cells. This study was undertaken to profile AS patient cytotoxic cells with the hypothesis that an alteration in CD8+ T cells might explain the aberrant cytotoxic profile observed in patients. Methods Whole blood was examined for GZM and PRF1 gene expression by quantitative polymerase chain reaction. Serum and synovial fluid (SF) were examined for granzyme and perforin 1 expression by bead array, and blood and SF mononuclear cells were examined for granzyme and perforin 1 expression by fluorescence‐activated cell sorting (FACS). Results GZM and PRF1 gene expression were both reduced in AS patients compared to healthy controls, especially in men. Perforin 1, but not granzyme, protein levels were reduced in AS patient serum. Granzymes were elevated in AS SF, but not in rheumatoid arthritis or osteoarthritis SF. FACS revealed a reduction in granzyme‐positive and perforin 1–positive lymphocytes, but not an intrinsic defect in CD8+ T cell granzyme or perforin 1 production. CD8+ T cell frequency was reduced in the blood and increased in the SF of AS patients. Conclusion Our findings indicate that AS patients have an altered cytotoxic T cell profile. These data suggest that CD8+ T cells with a cytotoxic phenotype are recruited to the joints, where they exhibit an activated phenotype. Thus, a central role for CD8+ T cells in AS may have been overlooked and deserves further study.

IntroductionDendritic cells (DCs) play a central role in initiating and regulating immune responses to foreign and self-antigens. 1 Under steady-state conditions, lymphoid and nonlymphoid tissues contain stable numbers of DCs, which is achieved through a dynamic interplay between the influx of new precursors, DC emigration, and death. Bone marrow (BM)-derived pre-cDCs, the immediate precursor of conventional DCs (cDCs), migrate via blood and enter peripheral lymphoid and nonlymphoid tissues, where they differentiate into cDCs that proliferate for several generations. [2][3][4] Monocytes also participate in the generation of cDCs in some tissues. [5][6][7] Nonlymphoid tissue cDCs emigrate continuously via lymphatics into draining lymph nodes 8 ; similar to the resident populations of lymphoid tissue DCs, most migrant cDCs die in lymphoid tissue and do not reenter the blood circulation. 1 Whether all peripheral tissue cDCs home to lymph nodes or some are destined to die in their local milieu is unclear. Dysregulation or loss-of-function of various cell types and molecules can alter DC homeostasis. 9,10 Chief among them is the cytokine Flt3 ligand, which regulates the number of early progenitors in BM and the rate of DC proliferation in peripheral tissues. 11,12 During acute inflammatory processes, DC numbers fluctuate markedly. Strong inflammatory stimuli can cause an abrupt decrease in the number of lymphoid cDCs, a consequence of maturation-induced apoptosis and migration. 13,14 DC numbers usually rebound within 2 to 3 days, then expand through recruitment of new precursors, augmentation of cDCs proliferation in situ, and differentiation of monocytes into "inflammatory" DCs. 3,[15][16][17][18] Increased expression of granulocyte/macrophage colony stimulating factor (GM-CSF) is considered key to this response. 19 Inflammation also causes egress of tissue DCs into lymph nodes by modulating chemokine receptor expression and altering the structure of regional lymphatics and lymph nodes. [20][21][22] With resolution of inflammation, DC numbers normalize through apoptosis and cytotoxic T-cell mediated killing. 23,24 In chronic inflammatory processes, such as in cancer, DC fate remains poorly understood.The current model of DC ontogeny indicates that the monocyte/ macrophage and DC lineages diverge from a common monocyte-DC progenitor (MDP) in BM. 25 The subsequent defined progenitors in the DC lineage, common DC precursors (CDP or pro-DC) followed by pre-cDCs, are considered irreversibly committed to become terminal DCs. The general assumption of this model is that death completes the life cycle of all DCs. In this study, we report a new termination pathway for cDCs. We found that immunostimulatory CD11c ϩ MHC class II ϩ cDCs retain the capacity to evolve into CD11c Ϫ MHC class II Ϫ macrophage-like cells with potent immunosuppressive activity. We show the critical importance of GM-CSF in maintaining DC identity. By tracking genetically tagged CD11c ϩ cells in vivo, we found that tumors induce a high proportion of cDCs...