ROLE OF STAT3 IN HUMAN NK CELL FUNCTIONS

Natural Killer (NK) cells are cytotoxic lymphocytes, which play a critical role in the immune response against malignant cells and microbial infections. NK cells are equipped with activating receptors, which upon detecting ligands expressed on stressed cells induce cytolytic activity of NK cells. Stimulation of NK cell proliferation and priming of NK cytolytic capability are accomplished by cytokines, which mediate their signals mainly through JAK-STAT signaling pathway. Previously, we found that K562 cells genetically modified to express membrane bound IL-21 (mbIL-21), which predominantly activates STAT3, induce robust expansion and activation of human NK cells. Further investigations revealed role of STAT3 in the transcriptional regulation of NKG2D, a primary activating NK receptor. Based on these findings, I hypothesized that STAT3 signaling plays a critical role in human cytolytic function and proliferation. I analyzed NK cells from Job syndrome patients to test my hypothesis. Job syndrome caused by dominant negative STAT3 mutations is a naturally occurring STAT3 deficient genetic model. Assessment of cytolytic activity revealed impaired cytolytic function in Job Syndrome patients’ NK cells. Investigations into the probable underlying causes of impaired cytotoxicity showed deficient NKG2D receptor expression and impaired polarization of cytolytic granules to the immune synapse formed between Job syndrome patients’ NK cell and target cell. I validated these findings in STAT3 knock-down primary human NK cells, which also displayed impaired cytolytic function and cytolytic granule polarization. Expansion of Job syndrome patients’ NK cells with mbIL21 stimulation restored NKG2D expression and cytolytic granule polarization to normal levels and enhanced cytolytic activity. As constitutively active STAT3 is oncogenic, STAT3 is major drug target in cancer therapeutics. To assess a probable side effect of pharmacological inhibition of STAT3, I assessed its effect on human NK cell cytolytic function. Treatment of primary human NK cells with small molecule STAT3 inhibitor S3I-201 suppressed NK cytolytic function. I employed pharmacological and genetic models of STAT3 deficiency to study the role of STAT3 in human NK cell proliferation. Treatment with STAT3 inhibitor S3I-201 reduced expansion of human NK cells stimulated with mbIL21 and membrane bound IL-15 (mbIL15). mbIL21 and mbIL15 induced expansion was also deficient in Job syndrome patients’ NK cells. Thus, both pharmacological and genetic models complemented each other in underlying role of STAT3 signaling in human NK cell proliferation. Employing pharmacological and genetic approaches, I showed that STAT3 deficiency in primary human NK cells causes impairment of cytolytic function and cytokine induced expansion. This is the first report to demonstrate the role of STAT3 in the transport of cytolytic granules in NK cell and NK cell functional deficiency in Job syndrome patients, which may provide an immunological basis for their proclivity to cancer. Restoration of NK cell function upon mbIL21 stimulation, suggests adoptive NK cell therapy as a treatment option for Job syndrome patients. By assessing the effect of pharmacological STAT3 inhibition on NK cytotoxicity and proliferation, this study provides potential biomarkers for monitoring side effects of STAT3 inhibition, which is fast emerging as a therapeutic approach in cancer treatment

Role of adenosine signaling in penile erection and erectile disorders.

INTRODUCTION: Penile erection is a hemodynamic process, which results from increased flow and retention of blood in the penile organ due to the relaxation of smooth muscle cells. Adenosine, a physiological vasorelaxant, has been shown to be a modulator of penile erection. AIM: To summarize the research on the role of adenosine signaling in normal penile erection and erectile disorders. MAIN OUTCOME MEASURES: Evidence in the literature on the association between adenosine signaling and normal and abnormal penile erection, i.e., erectile dysfunction (ED) and priapism. METHODS: The article reviews the literature on the role of endogenous and exogenous adenosine in normal penile erection, as well as in erectile disorders namely, ED and priapism. RESULTS: Adenosine has been shown to relax corpus cavernosum from various species including human in both in vivo and in vitro studies. Neuromodulatory role of adenosine in corpus cavernosum has also been demonstrated. Impaired adenosine signaling through A(2B) receptor causes partial resistance of corpus cavernosum, from men with organic ED, to adenosine-mediated relaxation. Increased level of adenosine has been shown to be a causative factor for priapism. CONCLUSION: Overall, the research reviewed here suggests a general role of exogenous and endogenous adenosine signaling in normal penile erection. From this perspective, it is not surprising that impaired adenosine signaling is associated with ED, and excessive adenosine signaling is associated with priapism. Adenosine signaling represents a potentially important diagnostic and therapeutic target for the treatment of ED and priapism

Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells

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

Increased adenosine contributes to penile fibrosis, a dangerous feature of priapism, via A2B adenosine receptor signaling

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-β1, 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 A2BR. Based on this fact, we further purified CCFCs from A2BR-deficient mice and demonstrated that A2BR is essential for excess adenosine-mediated penile fibrosis. Finally, we revealed that TGF-β functions downstream of the A2BR to increase CCFC collagen secretion and proliferation. Overall, our studies identify an essential role of increased adenosine in the pathogenesis of penile fibrosis via A2BR 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 A2B adenosine receptor signaling

Schema for NK cell manufacturing with aAPCs.

<p>Artificial antigen-presenting cells (aAPCs) were produced by genetic modification of K562 to express costimulatory molecules and membrane-bound cytokines. To expand NK cells <i>ex</i> vivo, unfractionated PBMC are stimulated weekly with irradiated PBMC, inducing rapid proliferation of NK cells and in some cases non-specific expansion of T cells. Contaminating T cells may be depleted, and the remaining purified NK cells may be stimulated weekly by the aAPCs as needed to obtain sufficient numbers. Expanded NK cells may be used directly or cryopreserved for future use.</p

Gene expression in NK cells stimulated with mbIL15 or mbIL21 as assessed using the nCounter platform.

<p>Paired aliquots of purified NK cells from 4 donors were stimulated for one week with either Clone 4 (mbIL15) or Clone 9.mbIL21. Total RNA was purified and equal quantities hybridized for detection of expression of 96 genes. Gene expression was normalized to LDH (mean 6,076 copies detected), and the remaining data for each gene plotted as mean +/− SEM for each expansion condition. Genes with detection below background (set at ≤10 detected copies) were excluded as not biologically significant (grey box). The ten highest-expressed genes in addition to LDH are labeled, as are genes that are differentially expressed by >2 fold (red) or <0.5 fold (blue) in mbIL21-expanded cells. Differentially expressed genes were replotted (inset) for comparison, and two-tailed <i>t-</i>test applied.</p

NK cell expansion and purity after repeated weekly stimulation with aAPCs expressing membrane-bound cytokines.

<p><i>A</i>, Mean expansion of CD3^neg/CD16-or-56^pos NK cells from 22 donors expanded for 21 days using aAPCs bearing mbIL15 or mbIL21. NK cells from five donors were also expanded with aAPCs expressing both mbIL15 and mbIL21. <i>B</i>, Mean expansion of NK cells from 4 donors expanded for 42 days using aAPCs bearing mbIL15 or mbIL21. <i>C</i>, Percent of CD3^pos T/NKT cells and CD3^neg/CD16-or-56^pos NK cells in the starting PBMC product (day 0) and at the end of 21 days of expansion on Clone 9.mbIL21 from 20 donors. <i>D</i>, Mean expansion of CD3^neg/CD16-or-56^pos NK cells on Clone 9.mbIL21 from unfractionated PBMC (n = 19), unfractionated PBMC followed by NK purification at day 14 of expansion (n = 3), or from NK cells purified from PBMC at day 0 prior to expansion (n = 13). Mean +/− SD is shown for each plot. All p values indicated are for <i>t-</i>test of fold expansion at the end of the expansion period.</p

Direct cytotoxicity and cytokine secretion of fresh NK cells, mbIL15-expanded, and mbIL21-expanded NK cells.

<p>NK cells were purified from four normal donor buffy coats and assessed directly, and then retested after being expanded for 21 days with Clone 9.mbIL21 and Clone 4 (mbIL15) (with CD3 depletion on day 14). NK cells were assessed for cytotoxicity against 721.221 (A) or cytokine secretion in response to K562 (B). NK cells from another four donors were purified and cryopreserved, and expanded with Clone 9.mbIL21 and then cryopreserved. Fresh (circles) and expanded (squares) NK cells were then thawed and activated in parallel for 24 h in 50 IU/mL IL-2, and then tested for cytotoxicity against HLA^null 721.221 targets (C), C*0702-transduced (Group C1) 721.221 targets (D), or B*5801-transduced (Group Bw4) 721.221 targets (E). All data points shown are mean +/− SEM of the means of four donors tested in triplicate.</p