NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy.

Spatiotemporal specificity of cAMP action is best explained by targeting protein kinase A (PKA) to its substrates by A-kinase-anchoring proteins (AKAPs). At synapses in the brain, AKAP79/150 incorporates PKA and other regulatory enzymes into signal transduction networks that include ␤-adrenergic receptors, ␣-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA), and N-methyl-D-aspartic acid receptors. We previously showed that AKAP79/150 clusters PKA with type 5 adenylyl cyclase (AC5) to assemble a negative feedback loop in which the anchored kinase phosphorylates AC5 to dynamically suppress cAMP synthesis. We now show that AKAP79 can associate with multiple AC isoforms. The N-terminal regions of AC5, -6, and -9 mediate this protein-protein interaction. Localized activation of PKA 2 triggers a plethora of intracellular signaling processes (1). Precise control of these phosphorylation events is often achieved by restricted activation of PKA in discrete microenvironments. AKAPs participate in this process by tethering the kinase close to preferred substrates. AKAPs now represent a family of 43 diverse but functionally related proteins that bind the regulatory subunit dimer of the PKA holoenzyme (2).AKAPs have been identified in a range of species, tissues, and cellular compartments. The AKAP79/150 group of anchoring proteins is perhaps the best understood member of this class of signal-organizing proteins. AKAP79/150 consists of three orthologs: bovine AKAP75, human AKAP79, and murine AKAP150. Although originally identified in the postsynaptic densities of neurons, this group of anchoring proteins is also expressed in a variety of other tissues. In addition to binding PKA, AKAP79 has the ability to bind protein phosphatase 2B (3) and protein kinase C (PKC) (4, 5). By organizing these signal transduction and signal termination enzymes in the same location, AKAP79 provides a platform to facilitate the bidirectional control of cAMP-and calcium-mediated signaling events.Although anchoring of PKA with its substrates provides an efficient mechanism for the spatial regulation necessary for selectivity of cAMP signaling, it was not clear how local pools of cAMP are managed. We have shown that AC isoforms can specifically interact with three different AKAP complexes, AKAP79, Yotiao, and mAKAP, to regulate events downstream of cAMP production (6 -8). We have also demonstrated that anchoring of AC5 to an AKAP79/150 complex provides negative feedback on AC5 via PKA phosphorylation of AC5 within the complex (6).Although characterization of the AKAP79-AC5 interaction has shed some light on the advantages gained by localizing different components of cAMP signaling pathways, several key issues remain unresolved. First of all, do other AC isoforms interact with AKAP79 or other anchoring proteins? Secondly, are AC isoforms recruited into larger signaling networks via their protein-protein interactions with AKAP79? AKAP79/150 has been shown to form a multiprotein signaling complex with AMPA and NMDA receptors (9 -11), adhesion mol...

Priapism is a condition of persistent penile erection in the absence of sexual excitation. Of men with sickle cell disease (SCD), 40% display priapism. The disorder is a dangerous and urgent condition, given its association with penile fibrosis and eventual erectile dysfunction. Current strategies to prevent its progression are poor because of a lack of fundamental understanding of the molecular mechanisms for penile fibrosis in priapism. Here we demonstrate that increased adenosine is a novel causative factor contributing to penile fibrosis in two independent animal models of priapism, adenosine deaminase (ADA)-deficient mice and SCD transgenic mice. An important finding is that chronic reduction of adenosine by ADA enzyme therapy successfully attenuated penile fibrosis in both mouse models, indicating an essential role of increased adenosine in penile fibrosis and a novel therapeutic possibility for this serious complication. Subsequently, we identified that both mice models share a similar fibrotic gene expression profile in penile tissue (including procollagen I, TGF-beta(1), and plasminogen activator inhibitor-1 mRNA), suggesting that they share similar signaling pathways for progression to penile fibrosis. Thus, in an effort to decipher specific cell types and underlying mechanism responsible for adenosine-mediated penile fibrosis, we purified corpus cavernosal fibroblast cells (CCFCs), the major cell type involved in this process, from wild-type mice. Quantitative RT-PCR showed that the major receptor expressed in these cells is the adenosine receptor A(2B)R. Based on this fact, we further purified CCFCs from A(2B)R-deficient mice and demonstrated that A(2B)R is essential for excess adenosine-mediated penile fibrosis. Finally, we revealed that TGF-beta functions downstream of the A(2B)R to increase CCFC collagen secretion and proliferation. Overall, our studies identify an essential role of increased adenosine in the pathogenesis of penile fibrosis via A(2B)R signaling and offer a potential target for prevention and treatment of penile fibrosis, a dangerous complication seen in priapism.-Wen, J., Jiang, X., Dai, Y., Zhang, Y., Tang, Y., Sun, H., Mi, T., Phatarpekar, P. V., Kellems, R. E., Blackburn, M. R., Xia, Y. Increased adenosine contributes to penile fibrosis, a dangerous feature of priapism, via A(2B) adenosine receptor signaling.

Signal transducer and activator of transcription 3 (STAT3) is considered a negative regulator of inflammation, as inhibition of STAT3 signaling enhances antitumor immunity. However, STAT3 activation is a key oncogenic pathway in natural killer (NK)-lineage large granular lymphomas, and we recently reported enhanced proliferation and function of human NK cells activated with IL-21, which signals primarily through STAT3. These IL-21-expanded NK cells also have increased NKG2D expression, which led us to focus our investigation on whether STAT3 regulates NKG2D. In this study, we show that modulation of STAT3 phosphorylation with cytokines and small-molecule inhibitors correlates with NKG2D expression on human NK cells, leading to altered NK-cell degranulation. Moreover, NKG2D expression on murine NK cells having conditional STAT3 ablation is lower than on NK cells from wild-type mice, and human NK cells carrying dominant-negative STAT3 mutations have decreased baseline NKG2D expression and blunted responses to IL-10 and IL-21. Lastly, we show binding of STAT3 to a predicted STAT3 binding site upstream of the NKG2D gene, which is enhanced by IL-10 and IL-21 and decreased by STAT3 inhibition. Taken together, these data show that NKG2D expression in NK cells is regulated at the transcriptional level by STAT3, resulting in a functional NK cell defect in patients with STAT3 mutations.