Autocrine IL-6 Production Research Articles

Importance of IL-6 trans-signaling and high autocrine IL-6 production in human osteoarthritic chondrocyte metabolism

ObjectiveNeutralization of Interleukin (IL)-6-signaling by antibodies is considered a promising tool for the treatment of osteoarthritis (OA). To gain further insight into this potential treatment, this study investigated the effects of IL-6-signaling and IL-6 neutralization on chondrocyte metabolism and the release of IL-6-signaling-releated mediators by human chondrocytes. DesignChondrocytes were collected from 49 patients with advanced knee/hip OA or femoral neck fracture. Isolated chondrocytes were stimulated with different mediators to analyze the release of IL-6, soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130). The effect of IL-6 and IL-6/sIL-6R complex as well as neutralization of IL-6-signaling on the metabolism was analyzed. ResultsOA chondrocytes showed high basal IL-6 production and release, which was strongly negatively correlated with the production of cartilage-matrix-proteins. Chondrocytes produced and released sIL-6R and sgp130. The IL-6/sIL-6R complex significantly increased nitric oxide, prostaglandin E2 and matrix metalloproteinase 1 production, decreased Pro-Collagen Type II and mitochondrial ATP production, and increased glycolysis in OA chondrocytes. Neutralization of IL-6-signaling by antibodies did not significantly affect the metabolism of OA chondrocytes, but blocking of gp130-signaling by SC144 significantly reduced the basal IL-6 release. ConclusionAlthough IL-6 trans-signaling induced by IL-6/sIL-6R complex negatively affects OA chondrocytes, antibodies against IL-6 or IL-6R did not affect chondrocyte metabolism. Since inhibition of gp130-signaling reduced the enhanced basal release of IL-6, interfering with gp130-signaling may ameliorate OA progression because high cellular release of IL-6 correlates with reduced production of cartilage-matrix-proteins.

Tumor-educated Gr1+CD11b+ cells drive breast cancer metastasis via OSM/IL-6/JAK-induced cancer cell plasticity.

Cancer cell plasticity contributes to therapy resistance and metastasis, which represent the main causes of cancer-related death, including in breast cancer. The tumor microenvironment drives cancer cell plasticity and metastasis, and unraveling the underlying cues may provide novel strategies for managing metastatic disease. Using breast cancer experimental models and transcriptomic analyses, we show that stem cell antigen-1 positive (SCA1+) murine breast cancer cells enriched during tumor progression and metastasis had higher in vitro cancer stem cell-like properties, enhanced in vivo metastatic ability, and generated tumors rich in Gr1hiLy6G+CD11b+ cells. In turn, tumor-educated Gr1+CD11b+ (Tu-Gr1+CD11b+) cells rapidly and transiently converted low metastatic SCA1- cells into highly metastatic SCA1+ cells via secreted oncostatin M (OSM) and IL-6. JAK inhibition prevented OSM/IL-6-induced SCA1+ population enrichment, while OSM/IL-6 depletion suppressed Tu-Gr1+CD11b+-induced SCA1+ population enrichment in vitro and metastasis in vivo. Moreover, chemotherapy-selected highly metastatic 4T1 cells maintained high SCA1+ positivity through autocrine IL-6 production, and in vitro JAK inhibition blunted SCA1 positivity and metastatic capacity. Importantly, Tu-Gr1+CD11b+ cells invoked a gene signature in tumor cells predicting shorter overall survival (OS), relapse-free survival (RFS), and lung metastasis in breast cancer patients. Collectively, our data identified OSM/IL-6/JAK as a clinically relevant paracrine/autocrine axis instigating breast cancer cell plasticity and triggering metastasis.

Autocrine IL-6 drives cell and extracellular matrix anisotropy in scar fibroblasts

Fibrosis is associated with dramatic changes in extracellular matrix (ECM) architecture of unknown etiology. Here we exploit keloid scars as a paradigm to understand fibrotic ECM organization. We reveal that keloid patient fibroblasts uniquely produce a globally aligned ECM network in 2-D culture as observed in scar tissue. ECM anisotropy develops after rapid initiation of a fibroblast supracellular actin network, suggesting that cell alignment initiates ECM patterning. Keloid fibroblasts produce elevated levels of IL-6, and autocrine IL-6 production is both necessary and sufficient to induce cell and ECM alignment, as evidenced by ligand stimulation of normal dermal fibroblasts and treatment of keloid fibroblasts with the function blocking IL-6 receptor monoclonal antibody, tocilizumab. Downstream of IL-6, supracellular organization of keloid fibroblasts is controlled by activation of cell-cell adhesion. Adhesion formation inhibits contact-induced cellular overlap leading to nematic organization of cells and an alignment of focal adhesions. Keloid fibroblasts placed on isotropic ECM align the pre-existing matrix, suggesting that focal adhesion alignment leads to active anisotropic remodeling. These results show that IL-6-induced fibroblast cooperativity can control the development of a nematic ECM, highlighting both IL-6 signaling and cell-cell adhesions as potential therapeutic targets to inhibit this common feature of fibrosis.

CD4 Effector TCR Avidity for Peptide on APC Determines the Level of Memory Generated.

Initial TCR affinity for peptide Ag is known to impact the generation of memory; however, its contributions later, when effectors must again recognize Ag at 5-8 d postinfection to become memory, is unclear. We examined whether the effector TCR affinity for peptide at this "effector checkpoint" dictates the extent of memory and degree of protection against rechallenge. We made an influenza A virus nucleoprotein (NP)-specific TCR transgenic mouse strain, FluNP, and generated NP-peptide variants that are presented by MHC class II to bind to the FluNP TCR over a broad range of avidity. To evaluate the impact of avidity invivo, we primed naive donor FluNP in influenza A virus-infected host mice, purified donor effectors at the checkpoint, and cotransferred them with the range of peptides pulsed on activated APCs into second uninfected hosts. Higher-avidity peptides yielded higher numbers of FluNP memory cells in spleen and most dramatically in lung and draining lymph nodes and induced better protection against lethal influenza infection. Avidity determined memory cell number, not cytokine profile, and already impacted donor cell number within several days of transfer. We previously found that autocrine IL-2 production at the checkpoint prevents default effector apoptosis and supports memory formation. Here, we find that peptide avidity determines the level of IL-2 produced by these effectors and that IL-2Rα expression by the APCs enhances memory formation, suggesting that transpresentation of IL-2 by APCs further amplifies IL-2 availability. Secondary memory generation was also avidity dependent. We propose that this regulatory pathway selects CD4 effectors of highest affinity to progress to memory.

Gene Regulation and Suppression of Type I Interferon Signaling By STAT3 in Diffuse Large B-Cell Lymphoma

The Janus kinas (JAK)/signal transducer and activator of transcription (STAT) pathway is central to signaling by receptors of diverse cytokines, growth factors and related molecules, which is critical for normal hematopoiesis and immune response. However, this signaling pathway is deregulated in several B cell lymphomas. In activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL), autocrine production of IL-6 and IL-10 can result from high NF-κB activity caused by a variety of genetic alterations, such as mutations in MYD88 and CARD11. Our recent work has demonstrated that JAK1 is an active kinase downstream of IL-6 and IL-10 signaling that activates STAT3. Interestingly, JAK1 can function in the nucleus to regulate gene expression through phosphorylating histone H3 on tyrosine 41. There are nearly 3,000 JAK1 epigenetic target genes including MYD88, IRF4 and MYC, of which expression is essential for cancer cell survival and proliferation.Here we have investigated the canonical gene regulatory mechanism by STAT3 in ABC DLBCL cells. We performed genome-wide analysis and identified 2,251 STAT3 direct target genes, all of which involve B cell activation, survival, proliferation, differentiation and migration. Whole transcriptome profiling revealed that STAT3 acts as both a transcriptional activator and suppressor, with a comparable number of up- and down-regulated genes. STAT3 regulates multiple oncogenic signaling pathways, including NF-κB, cell cycle checkpoint, PI3K/AKT/mTORC1 and STAT3 itself. These results suggest that STAT3 is involved in the positive feedback regulation of IL-6/IL-10 signaling and shows crosstalk with NF-κB signaling pathways in ABC DLBCL cells. In addition, STAT3 negatively regulates the lethal type I interferon signaling pathway by inhibiting expression of IRF7, IRF9, STAT1 and STAT2, all of which are critical transcription factors in the type I interferon pathway. Inhibition of STAT3 activity by ruxolitinib synergizes with the type I interferon inducer lenalidomide in growth inhibition of ABC DLBCL cells in vitro . In an ABC DLBCL xenograft mouse model, combination treatment using ruxolitinib and lenalidomide completely inhibits the tumor growth during the period of treatment.In summary, we demonstrate that STAT3 is a critical transcriptional regulator of ABC DLBCL. Gene regulation by STAT3 in ABC DLBCL accentuates survival signaling pathways while dampening the lethal type I interferon pathway. Knowledge of these STAT3 regulated genes has led to our demonstration that a small molecule inhibitor in the JAK1-STAT3 signaling pathway synergizes with lenalidomide, suggesting a new therapeutic strategy for ABC DLBCL, a subtype that is particularly difficult to treat and has poor prognosis. DisclosuresNo relevant conflicts of interest to declare.

Autocrine and paracrine IL-2 signals collaborate to regulate distinct phases of CD8 T cell memory

Abstract Differential IL-2 signaling is implicated in memory CD8 T cell fates. Autocrine IL-2 production is a hallmark of polyfunctional, long-lived lymphoid memory cells. Whether the IL-2 signals perceived by CD8 T cells come from autocrine or paracrine sources, the timing of these signals, and their differential impact on the qualitative outcomes of CD8 T cell responses are not clearly understood. We used two distinct models of germline and conditional autocrine IL-2 ablation in post-thymic CD8 T cells to specifically dissect the requirement and timing of autocrine IL-2. Our studies show that autocrine IL-2 is uniquely required during primary CTL responses to program the key memory property of robust recall responses. Ablation of autocrine IL-2 in fully differentiated wild type memory cells immediately prior to rechallenge did not impact their recall expansion potential. Furthermore, we found that paracrine IL-2 could effectively supplant autocrine IL-2 for primary expansion, effector differentiation and memory maintenance, but not recall expansion. Likewise, our data highlight the redundancy of autocrine and paracrine IL-2 in optimally driving equivalent metabolic programs associated with effector and memory states. While CD4 licensing of DCs is needed for optimal programming of autocrine IL-2 in CD8 T cells, we found that the requirement for CD4 help is less critical for autocrine IL-2 production in the context of inflammatory infections. These findings have implications in adoptive T cell immunotherapies where expansion of engineered cells is largely driven by paracrine IL-2, which can potentially impact their long-term sentinel function.

Tracking the in vivo history and fate of IL-2 producing CD8 T cells during memory differentiation

Abstract IL-2 is a key driver of T cell responses critical for the conversion of resting antigen specific T cells into glycolytic powerhouses that undergo massive expansion and elicit cytolytic function to clear infections. Recent studies have used ex vivo antigen challenge combined with somatic IL-2 deletion to show that both paracrine and autocrine IL-2 signals are indispensable for the formation of highly functional memory CD8 T cells. Despite this critical role of autocrine IL-2 for memory T cell fate, the in vivo history and fate of autocrine IL-2 producing CD8 T cells remains unknown. We generated a mouse model in which autocrine IL-2 production leads to transcription of TdTomato, leading to an indelibly marked T cell. Following acute infection, a proportion of antigen specific CD8 T cells made autocrine IL-2, however, more than half of the T cell response had no history of IL-2 production. Furthermore, contrary to their ex vivo functionality, memory precursor and terminal effector T cells had similar history of in vivo IL-2 production. Despite autocrine IL-2 production being a benchmark of highly functional memory T cells, we observed no increase in the ex vivo polyfunctionality of T cells that produced in vivo IL-2 during infection. Finally, while strong TCR engagement is critical for the recruitment of robust T cell responses, we saw that in vivo autocrine IL-2 production is more effectively elicited by lower affinity TCR signals. Thus, we see that while proportion of T cells rely on autocrine IL-2 during infection, the ability of a CD8 T cell to recruit autocrine IL-2 function heavily depends on the avidity of the TCR to the peptide-MHC complex. Together these data provide the first insights into the in vivo function of IL-2 in antigen specific CD8 T cells.

Autocrine IL-2 programs survival of exhausted CD8 T cells during chronic infection

Abstract Loss of T cell function is a hallmark of chronic infections (such as HIV, HCV) and cancers. Chronic antigenic stimulation leads to a progressive, hierarchical loss of function in T cells – properties such as autocrine IL-2 production and ability to lyse infected target cells are lost first, followed by the loss of TNF-α and then IFN-γ production. The sum total of these events, known as exhaustion, is followed by the eventual physical deletion of antigen-specific CTLs. Our current clinical checkpoint blockade therapies are heavily hinged upon the very presence of the exhausted T cells that can be re-invigorated to regain function. Hence, it is critical to define the survival signals that prevent the deletion of exhausted antigen-specific T cells. While IL-2 is historically known to be a key survival cytokine for T cells, autocrine expression of IL-2 is extinguished early during T cell exhaustion. Thus, it has been long believed that IL-2 is dispensable for the maintenance of exhausted CTLs during chronic infections. Surprisingly, deletion of il2 specifically in antigen-specific CD8 T cells (CD8IL2−/−) led to a precipitous decline in the numbers of these exhausted CD8 T cells compared to their wild-type counterparts (CD8IL2+/+) within the same environment. This paradoxical need for autocrine IL-2 in the long-term sustenance of an exhausted CD8 T cell pool is ascribed to its instructional role in programming key aspects of CD8 T cell survival and metabolism early during priming. These findings are of high consequence to checkpoint blockade immunotherapeutic strategies, where success is heavily dependent on the presence of an exhausted T cell population that can be re-functionalized to fight off the chronic infection or tumor.

Epigenetic gene regulation by Janus kinase 1 in diffuse large B-cell lymphoma

Janus kinases (JAKs) classically signal by activating STAT transcription factors but can also regulate gene expression by epigenetically phosphorylating histone H3 on tyrosine 41 (H3Y41-P). In diffuse large B-cell lymphomas (DLBCLs), JAK signaling is a feature of the activated B-cell (ABC) subtype and is triggered by autocrine production of IL-6 and IL-10. Whether this signaling involves STAT activation, epigenetic modification of chromatin, or both mechanisms is unknown. Here we use genetic and pharmacological inhibition to show that JAK1 signaling sustains the survival of ABC DLBCL cells. Whereas STAT3 contributed to the survival of ABC DLBCL cell lines, forced STAT3 activity could not protect these cells from death following JAK1 inhibition, suggesting epigenetic JAK1 action. JAK1 regulated the expression of nearly 3,000 genes in ABC DLBCL cells, and the chromatin surrounding many of these genes was modified by H3Y41-P marks that were diminished by JAK1 inhibition. These JAK1 epigenetic target genes encode important regulators of ABC DLBCL proliferation and survival, including IRF4, MYD88, and MYC. A small molecule JAK1 inhibitor cooperated with the BTK inhibitor ibrutinib in reducing IRF4 levels and acted synergistically to kill ABC DLBCL cells, suggesting that this combination should be evaluated in clinical trials.

STAT3 and NF-κB cooperatively control in vitro spontaneous apoptosis and poor chemo-responsiveness in patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an adult disease characterized by in vivo accumulation of mature CD5/CD19/CD23 triple positive B cells and is currently incurable. CLL cells undergo spontaneous apoptosis in response to in vitro cell culture condition but the underlying mechanism is unclear. We hypothesize that the sensitivity of CLL cells to spontaneous apoptosis may be associated with the constitutive activities of transcription factors STAT3 and/or NF-κB. We now show that the sensitivity of fresh CLL cells to spontaneous apoptosis is highly variable among different patients during 48 hours’ cell culture and inversely correlated with in vivo constitutively activated STAT3 and NF-κB (p < 0.001). Both activated STAT3 and NF-κB maintain the levels of anti-apoptotic protein Mcl-1/Bcl-xL and autocrine IL-6 production. CLL cells with higher susceptibility to in vitro spontaneous apoptosis show the greatest chemosensitivity (p < 0.001), which is reflected clinically as achieving a complete response (CR) (p < 0.001), longer lymphocyte doubling times (p < 0.01), time to first treatment (p < 0.01), and progression free survival (p < 0.05). Our data suggest that the sensitivity of CLL cells to in vitro spontaneous apoptosis is co-regulated by constitutively activated STAT3 and NF-κB and reflects the in vivo chemo-responsiveness and clinical outcomes.

Potential crosstalk of the interleukin-6-heme oxygenase-1-dependent mechanism involved in resistance to lenalidomide in multiple myeloma cells.

Interleukin (IL)-6 is one of the most important survival factors in multiple myeloma (MM), and determines the poor prognosis of MM. IL-6 mainly has a paracrine bone marrow stromal cell origin and an autocrine MM cell origin. As an enzyme having cytoprotective effects, heme oxygenase-1 (HO-1) promotes the growth and drug resistance of various malignant tumors. HO-1 expression levels in bone marrow CD138(+) cells of MM patients were significantly higher than those in healthy donors, and positively correlated with both serum IL-6 and intracellular IL-6 mRNA expression levels. Culture of U266, RPMI8226 and CD138(+) cells with exogenous IL-6 in vitro induced high HO-1 expression levels and allowed them to resist lenalidomide. It is hypothesized that this was probably attributable to IL-6-mediated activation of the Janus kinase 2-signal transducer and activator of transcription 3 pathway. In contrast, without IL-6 coculture, enhanced HO-1 expression in U266, RPMI8226 and bone marrow CD138(+) cells from MM patients significantly inreased IL-6 mRNA expression levels and facilitated autocrine IL-6 production. The findings were associated with high HO-1 expression-enhanced p38 mitogen-activated protein kinase phosphorylation. Reduced HO-1 expression sensitized MM cells to lenalidomide. Therefore, we postulated that IL-6 in the bone marrow microenvironment of MM patients stimulated high HO-1 expression in MM cells and their resistance to lenalidomide. High HO-1 expression also stimulated autocrine IL-6 production, and exacerbated drug resistance and disease. This study supports the use of HO-1 as a possible marker for both MM prognosis and drug resistance, and as a potential therapeutic target.

The Quantity of Autocrine IL-2 Governs the Expansion Potential of CD8+ T Cells.

Adequate responsiveness of CD8(+) T cell populations is of utmost importance for the efficacy of many vaccines and immunotherapeutic strategies against intracellular pathogens and cancer. In this study, we show in mouse models that the relative number of IL-2-producing cells within Ag-specific CD8(+) T cell populations predicts the population expansion capacity upon challenge. We further demonstrate that IL-2 producers constitute the best responding subset. Notably, we show that elevated production of IL-2 by CD8(+) T cells results in concomitant improved population expansion capacity and immunity. The amount of IL-2 produced on a per-cell basis essentially connects directly to the superior CD8(+) T cell population expansion. Together, our findings identified that autocrine IL-2 production operates in a dose-dependent fashion to facilitate the expansion potential of Ag-specific CD8(+) T cell populations, which may instigate ways to augment therapies depending on fit CD8(+) T cells.

A Paracrine Role for IL6 in Prostate Cancer Patients: Lack of Production by Primary or Metastatic Tumor Cells.

Correlative human studies suggest that the pleiotropic cytokine IL6 contributes to the development and/or progression of prostate cancer. However, the source of IL6 production in the prostate microenvironment in patients has yet to be determined. The cellular origin of IL6 in primary and metastatic prostate cancer was examined in formalin-fixed, paraffin-embedded tissues using a highly sensitive and specific chromogenic in situ hybridization (CISH) assay that underwent extensive analytical validation. Quantitative RT-PCR showed that benign prostate tissues often had higher expression of IL6 mRNA than matched tumor specimens. CISH analysis further indicated that both primary and metastatic prostate adenocarcinoma cells do not express IL6 mRNA. IL6 expression was highly heterogeneous across specimens and was nearly exclusively restricted to the prostate stromal compartment--including endothelial cells and macrophages, among other cell types. The number of IL6-expressing cells correlated positively with the presence of acute inflammation. In metastatic disease, tumor cells were negative in all lesions examined, and IL6 expression was restricted to endothelial cells within the vasculature of bone metastases. Finally, IL6 was not detected in any cells in soft tissue metastases. These data suggest that, in prostate cancer patients, paracrine rather than autocrine IL6 production is likely associated with any role for the cytokine in disease progression.

IL-2 production by activated naïve and effector CD8 T cells forecasts memory formation. (LYM5P.700)

Abstract The clearance and control of many intracellular pathogens and tumors rely on the development of efficacious CD8 T cell responses. These responses are functionally diverse, however, whether and how this heterogeneity forecasts effector formation, memory potential, and protective capacity has not yet been definitively determined. To address this, we utilized IL-2 and IFN-γ double cytokine reporter mice to separate viable IL-2 and IFN-γ producing CD8 T cell subsets, and performed a series of adoptive transfer experiments to track their memory potential and fate. We discovered that within hours of activation, naïve CD8 T cells undergo a burst of cytokine synthesis and distinct populations develop that produce only IL-2, only IFN-γ, co-produce IL-2 and IFN-γ, or do not produce either cytokine. This diversity is consequential as newly activated naïve CD8 T cells that fail to produce IL-2 preferentially attain effector features. Conversely, autocrine IL-2 production by recently activated naïve CD8 T cells is associated with the formation of memory traits. Similarly, the ability of CD8 T cells to produce IL-2 during the effector phase also signified memory potential and the ability to mount vigorous secondary proliferative responses. Together these data demonstrate that functional heterogeneity influences the differentiation of CD8 T cell responses and that effector and memory formation can be predicted by the pattern of autocrine IL-2 production.

390 Epigenetic and Post-Translational Modification of STAT3 Regulates Its Spatiotemporal Trafficking and Transcriptional Activity in Mesenchymal Cells of Crohn's Disease and Animal Models of Crohn's Disease

STAT3 activity is governed by phosphorylation and acetylation. Post-translational modifications determine localization to the nucleus for transcriptional activity, or to signaling endosomes for regulation of signaling pathways. We recently identified a pattern of high pSTAT3(S727) and low pSTAT3(Y705) levels due to autocrine IL-6 production in mesenchymal cells of strictures in fibrostenotic Crohn's disease. The opposite is seen in the normal intestine in the same patient. This pattern characterizes B2 from other Crohn's phenotypes. We recently showed that STAT3 regulates transcription of TGFβ1 and collagen I. TheAIM of this study is to determine the post-translational and epigenetic modifications that determine the subcellular trafficking and function of STAT3. METHODS: Subepithelial myofibroblasts (SEMF) and smooth muscle cells (SMC) were isolated from strictured intestine and normal intestine of patients with fibrostenotic Crohn's disease and from intestine of TNBS treated C57Bl/6J mice and RAG(-/-) mice following transfer of CD4+CD45RBhi T-cells both after 8 weeks. Isolated cells were used to prepare RNA and cell lysates. Histologic sections were also prepared for immunofluorescence. Cell lysates and immunofluorescent staining of primary cultured cells were used to determine STAT3 subcellular localization of phosphorylated Y705 and S727 and acetylated K685. RESULTS: In both animal models expression of TGF-β1 and Collagen I increased in mesenchymal cells after 8 weeks similar to Crohn's disease. Similar patterns of S727 and Y705 phosphorylation and K685 acetylation and subcellular localization were observed in SEMF and SMC in B2 Crohn's disease (Figure 1), TNBS colitis and T-cell transfer. Phospho S727 and Acetyl K685 were increased and phospho Y705 was decreased in strictures in mice. Phospho S727 and acetyl K685 were localized to the nucleus, phospho Y705 was localized to endosomes in humans (Figrue 2) and mice. The opposite pattern was seen in normal intestine and control mice. IL-6-induced STAT3 nuclear localization of phospho S727 and acetyl K685 was enhanced by inhibition of histone deacetylation with Trichostatin A (500nM) and blocked by the inhibition of acetylation with the p300/CBP acetylation inhibitor, C646 (2.5 μM). IL-6-induced Erk1/2 activation and mesenchymal cell proliferation was inhibited by transfection of a dominant negative STAT3(Y705F) mutant or by pharmacologic inhibition of STAT3 phosphorylation. CONCLUSIONS: Activity of STAT3 is regulated by post-transcriptional modification of phosphorylation state and by epigenetic modification of acetylation of lysine residues that determine its subcellular localization and activity. Distinct subcellular localization to nucleus or signaling endosome differentially regulate transcription of TGF-β1 and collagen IαI, and proliferation of mesenchymal cells, respectively.

Ikaros imposes a barrier to CD8+ T cell differentiation by restricting autocrine IL-2 production.

Naive CD4(+) T cells require signals from the TCR and CD28 to produce IL-2, expand, and differentiate. However, these same signals are not sufficient to induce autocrine IL-2 production by naive CD8(+) T cells, which require cytokines provided by other cell types to drive their differentiation. The basis for failed autocrine IL-2 production by activated CD8(+) cells is unclear. We find that Ikaros, a transcriptional repressor that silences IL-2 in anergic CD4(+) T cells, also restricts autocrine IL-2 production by CD8(+) T cells. We find that CD8(+) T cell activation in vitro in the absence of exogenous cytokines and CD4 help leads to marked induction of Ikaros, a known repressor of the Il2 gene. Naive murine CD8 T cells haplo-insufficient for Ikzf1 failed to upregulate Ikaros, produced autocrine IL-2, and differentiated in an IL-2-dependent manner into IFN-γ-producing CTLs in response to TCR/CD28 stimulation alone. Furthermore, Ikzf1 haplo-insufficient CD8(+) T cells were more effective at controlling Listeria infection and B16 melanoma growth in vivo, and they could provide help to neighboring, non-IL-2-producing cells to differentiate into IFN-γ-producing effectors. Therefore, by repressing autocrine IL-2 production, Ikaros ensures that naive CD8(+) T cells remain dependent on licensing by APCs and CD4(+) T cells, and it may therefore act as a cell-intrinsic safeguard against inappropriate CTL differentiation and immunopathology.

Pancreatic Cancer-Associated Stellate Cells Promote Differentiation of Myeloid-Derived Suppressor Cells in a STAT3-Dependent Manner

Pancreatic stellate cells (PSC) are a subset of pancreatic cancer-associated fibroblasts. These cells provide prosurvival signals to tumors; however, little is known regarding their interactions with immune cells within the tumor microenvironment. We hypothesized that factors produced by human PSC could enhance myeloid-derived suppressor cell (MDSC) differentiation and function, which promotes an immunosuppressive microenvironment. Primary PSC cell lines (n = 7) were generated from human specimens and phenotypically confirmed via expression of vimentin, α-smooth muscle actin (α-SMA), and glial fibrillary acidic protein (GFAP). Luminex analysis indicated that PSC but not human fetal primary pancreatic fibroblast cells (HPF; negative controls) produced MDSC-promoting cytokines [interleukin (IL-6), VEGF, macrophage colony-stimulating factor (M-CSF) ] and chemokines (SDF-1, MCP-1). Culture of peripheral blood mononuclear cells [peripheral blood mononuclear cell (PBMC), n = 3 donors] with PSC supernatants or IL-6/granulocyte macrophage colony-stimulating factor (GM-CSF; positive control) for 7 days promoted PBMC differentiation into an MDSC (CD11b+CD33+) phenotype and a subpopulation of polymorphonuclear CD11b+CD33+CD15+ cells. The resulting CD11b+CD33+ cells functionally suppressed autologous T-lymphocyte proliferation. In contrast, supernatants from HPF did not induce an MDSC phenotype in PBMCs. Culture of normal PBMCs with PSC supernatants led to STAT3 but not STAT1 or STAT5 phosphorylation. IL-6 was an important mediator as its neutralization inhibited PSC supernatant-mediated STAT3 phosphorylation and MDSC differentiation. Finally, the FLLL32 STAT3 inhibitor abrogated PSC supernatant-mediated MDSC differentiation, PSC viability, and reduced autocrine IL-6 production indicating these processes are STAT3 dependent. These results identify a novel role for PSC in driving immune escape in pancreatic cancer and extend the evidence that STAT3 acts as a driver of stromal immunosuppression to enhance its interest as a therapeutic target.

4-Chlorobenzoyl berbamine, a novel berbamine derivative, induces apoptosis in multiple myeloma cells through the IL-6 signal transduction pathway and increases FOXO3a-Bim expression

Multiple myeloma (MM) is an incurable hematopoietic malignancy, although many novel therapeutic agents have been explored. In the present study, we showed that 4-chlorobenzoyl berbamine (BBD9), a novel derivative of berbamine, inhibited the growth of 4 MM cell lines (U266, RPMI 8226, MM1.R and MM1.S). After a 24-h treatment with BBD9, the half maximal inhibitory concentration (IC(50)) values were 1.8, 2.3, 1.5 and 2.4 µg/ml, respectively, using MTT assays. In BBD9-treated U266 and RPMI 8226 cells, Annexin V (AV)-propidium iodide (PI) staining and FACS analysis demonstrated that apoptosis was involved in this inhibition. This was confirmed by western blot analysis indicating activation and cleavage of caspase-3, -8, -9 and PARP. BBD9 also induced G2/M phase cell cycle arrest in these cells. To investigate the mechanisms responsible for BBD9-induced apoptosis, U266 cells were incubated with 0, 1 or 2 µg/ml of BBD9 combined with 0 or 150 ng/ml of interleukin (IL)-6. MTT assays showed that IL-6 partially abrogated the BBD9-induced cell growth inhibition. Furthermore, BBD9 inhibited autocrine IL-6 production, and downregulated membrane IL-6 receptor (IL-6R) expression. Crucial proteins downstream of the IL-6 signaling pathway, including AKT and STAT3, were inactivated in BBD9-treated U266 cells, although exogenous IL-6 did not abrogate this effect. Forkhead transcription factor class 3a (FOXO3a), a nuclear transcription factor downstream from AKT, was upregulated in the nuclei of BBD9-treated U266 cells. Bim, the target gene of FOXO3a, was upregulated at both the protein and mRNA levels, as shown by western blot analysis and quantitative PCR. These results suggest that BBD9 induces apoptosis in MM cells through the inhibition of the IL-6 signaling pathway, leading to FOXO3a activation and upregulation of pro-apoptotic Bim.

Abstract 2874: Pancreatic cancer associated stellate cells promote differentiation of myeloid-derived suppressor cells in a STAT3-dependent manner.

Abstract Pancreatic cancer is the fourth most common cause of cancer-related death in the United States with many patients that are diagnosed dying within one year. Pancreatic ductal adenocarcinoma is an inflammatory disease and includes several stromal elements such as lymphocytes and other immune cells, fibroblasts, and stellate cells. Pancreatic stellate cells (PSC), also known as cancer associated fibroblasts, can provide pro-survival signals to tumor cells, however their interactions with immune cells within the tumor microenvironment have not been explored in detail. We hypothesized that factors produced by human PSC could enhance myeloid-derived suppressor cell (MDSC) differentiation and function, which promotes immunosuppression in the tumor microenvironment. Primary PSC were harvested from human specimens and morphology was confirmed via immunofluorescent microscopy and staining for vimentin, alpha-smooth muscle actin (α SMA), and glial fibrillary acidic protein (GFAP). We analyzed soluble factors from PSC and human fetal primary pancreatic fibroblast cell line (HPPFC; negative controls) using a Luminex assay. Normal donor peripheral blood mononuclear cells (PBMC, n=3 donors) were cultured with 5 and 10% PSC supernatants, 10% HPPFC supernatants (negative control), or IL-6/GM-CSF (positive control) for 7 days, and assessed for MDSC phenotype by flow cytometry. The FLLL32 STAT3 inhibitor was used to determine whether PSC-mediated MDSC differentiation or PSC viability was STAT3-dependent. Stellate cell lines (n=7) were generated from patients and validated. Luminex analysis indicated that PSC produced cytokines involved in MDSC differentiation (IL-6, VEGF, MCSF) and chemotaxis (SDF-1, MCP-1). Culture with PSC supernatants for 7 days promoted PBMC differentiation into an MDSC (CD11b+CD33+) phenotype (p&amp;lt;0.01) and a sub-population of polymorphonuclear CD11b+CD33+CD15+ cells (p&amp;lt;0.05). Supernatants from a HPPFC were used as a negative control, which when cultured with PBMC did not induce an MDSC phenotype. The resulting CD11b+CD33+ cells were functional as they suppressed autologous T cell proliferation (p&amp;lt;0.05). Culture of normal PBMCs with PSC supernatants led to STAT3 but not STAT1 or STAT5 phosphorylation. Finally, the FLLL32 STAT3 inhibitor abrogated PSC-mediated MDSC differentiation, PSC viability, and reduced autocrine IL-6 production indicating these processes are STAT3 dependent (p&amp;lt;0.01). These data demonstrate a novel role for PSC in supporting immunosuppression associated with cancer and suggest that STAT3 within both stromal and immunosuppressive cells represents a therapeutic target. Citation Format: Thomas A. Mace, Zeenath Ameen, Wendy Frankel, Amy Collins, Sylwia Wojcik, Markus Mair, Gregory S. Young, James R. Fuchs, Tim D. Eubank, Tanios Bekaii-Saab, Mark Bloomston, Gregory B. Lesinski. Pancreatic cancer associated stellate cells promote differentiation of myeloid-derived suppressor cells in a STAT3-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2874. doi:10.1158/1538-7445.AM2013-2874

B7–CD28 Costimulatory Signals Control the Survival and Proliferation of Murine and Human γδ T Cells via IL-2 Production

γδ T cells play key nonredundant roles in immunity to infections and tumors. Thus, it is critical to understand the molecular mechanisms responsible for γδ T cell activation and expansion in vivo. In striking contrast to their αβ counterparts, the costimulation requirements of γδ T cells remain poorly understood. Having previously described a role for the TNFR superfamily member CD27, we since screened for other nonredundant costimulatory receptors in γδ T cell activation. We report in this article that the Ig superfamily receptor CD28 (but not its related protein ICOS) is expressed on freshly isolated lymphoid γδ T cells and synergizes with the TCR to induce autocrine IL-2 production that promotes γδ cell survival and proliferation in both mice and humans. Specific gain-of-function and loss-of-function experiments demonstrated a nonredundant function for CD28 interactions with its B7 ligands, B7.1 (CD80) and B7.2 (CD86), both in vitro and in vivo. Thus, γδ cell proliferation was significantly enhanced by CD28 receptor agonists but abrogated by B7 Ab-mediated blockade. Furthermore, γδ cell expansion following Plasmodium infection was severely impaired in mice genetically deficient for CD28. This resulted in the failure to mount both IFN-γ-mediated and IL-17-mediated γδ cell responses, which contrasted with the selective effect of CD27 on IFN-γ-producing γδ cells. Our data collectively show that CD28 signals are required for IL-2-mediated survival and proliferation of both CD27(+) and CD27(-) γδ T cell subsets, thus providing new mechanistic insight for their modulation in disease models.