Presence Of IL-2 Research Articles

Abstract 2753: Interleukin-10 and transforming growth factor-beta do not suppress tumor killing activity of PBMC-derived SUPLEXA cells

Abstract Introduction: SUPLEXA cells are peripheral blood mononuclear cells (PBMCs) being developed by Alloplex Biotherapeutics as an autologous adoptive therapy for cancer. The novel nature of SUPLEXA cells is that they are developed from peripheral blood mononuclear cells (PBMCs) that have been activated and expanded by engineered leukocyte stimulator cell lines (ENLIST cells), which are melanoma cell lines that express immunomodulatory proteins to program the tumor killing phenotype of SUPLEXA cells. We find that the SUPLEXA cell manufacturing process reproducibly results in a mixture of NK cells, CD8 T cells, and γδ T cells with broad tumor cell killing activity. In this study, we evaluated the effects of two major immunosuppressive cytokines found in the tumor microenvironment (IL-10 and TGF-β) on the tumor cytolytic function and cytokine release by SUPLEXA cells. Results: SUPLEXA cells were prepared by a 14-day process of PBMC induction by ENLIST cells for 5 days followed by 9 days expansion in cytokines. SUPLEXA cell mediated tumor cytolytic assays using red fluorescent protein (RFP) expressing M14 melanoma cells as targets were performed in the absence or presence of IL-10 or TGF-β (1.25 - 10 ng/ml). Cytokine levels in 48-hour culture supernatants from cytolytic reactions and SUPLEXA or M14 target cells alone were measured using a 36-plex Luminex assay. Without cytokine addition, SUPLEXA mediated cytolysis was 47.3% at 8:1 and 30.9% at 4:1 effector to target (E:T) ratios. Significant increases in SUPLEXA cell tumoricidal activity was observed with addition of IL-10 (74.3% at 8:1 and 51.7% at 4:1 E:T ratios at 2.5 ng/ml IL-10 added, P &It 0.001 by two-way ANOVA). Adding TGF-β caused a reduction in cytolytic activity that was not significant (36.5% at 8:1 and 20.8% at 4:1 E:T ratios with 2.5 ng/ml TGF-β added). Supernatants from cytolysis assays showed that SUPLEXA cells produced IFNγ, MIP-1α, RANTES, IL-8, Gro-α, MIG, and IP-10 in response to tumor cells. IL-10 significantly augmented IFNγ, RANTES, and MIP-1α production by SUPLEXA cells in a dose-dependent fashion. In contrast, TGF-β reduced IFNγ, MIP-1α, Gro-α, RANTES, and IP-10 production, but increased IL-8 production by SUPLEXA cells. Conclusions: An in vitro approach was used as a surrogate for what might occur in patients with solid tumors to test whether potent immune suppressive cytokines that are present in the tumor microenvironment might inhibit SUPLEXA cell function. Our findings indicate that IL-10 and TGF-β do not significantly reduce the tumor killing activity of SUPLEXA cells. Instead, we discovered that IL-10 significantly enhances SUPLEXA cell function and cytokine production, which suggests that SUPLEXA cells may acquire heightened tumoricidal activity and promote immune surveillance by augmenting cytokine and chemokine levels within the tumor microenvironment when given therapeutically to cancer patients. Citation Format: Frank Borriello, Joshua W. Keegan, James A. Lederer. Interleukin-10 and transforming growth factor-beta do not suppress tumor killing activity of PBMC-derived SUPLEXA cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2753.

Abstract 2527: Ovarian cancer ascites display altered immune environment featured by enhanced soluble factors and suppressive cellular context

Abstract Background: Tumor microenvironment (TME) of epithelial ovarian cancer is unique among solid tumors as tumor cells create their "malignant ascites" TME. These ascites, acting as a complex mixture of soluble factors and cell components, are known to be rich in macrophages which were suggested to skew to a M2-like phenotype involved in resistance and metastases. However, while macrophages can adopt different phenotypes, it's now postulated that tumor-associated macrophages (TAM) from ovarian cancer patient ascites may even acquire mixed M1/M2 properties, providing a particular pro-inflammatory and tumor-promoting microenvironment. Characterization of cell composition along with the growth factors present in the microenvironment is thus crucial to understand ovarian cancer biology and more particularly the immunosuppressive pathways that would underlie the altered immune cell activity. Methods & Results: Through flow cytometry-based marker expression analysis and quantitation of soluble mediators in ovarian cancer ascites, we investigated here the cell component nature as well as the presence of several growth factors. While these ascites were interestingly enriched in major "immunosuppressive" cell subsets including T and myeloid populations, TAM were intriguingly shown to display a mixed phenotype characterized by high expression of CD163, unrelated to M1/M2 categorization since also expressing Arg1, CD80, and iNOS markers. The immunosuppressive phenotype was also linked to IL6, LIF and IL10 among other factors present in the ascitic environment. In order to assess their role in the occurrence of such an immunosuppressive cell status, soluble mediators were then investigated on healthy monocytes undergoing M1 polarization. We demonstrated that, while LIF did not directly affect IL6 and IL10 levels released by the cells, interestingly IL6 alone or in co-presence with LIF decreased IL6 supernatant content, with respect to the control. These changes were concomitant with a decrease and increase in the expression of CD80 and CD163, respectively. Furthermore, CD14 and CD206 were found to increase in the presence of IL6+/-LIF. Conclusion: All these data highlight a switching activity of IL6 skewing cells from M1 polarization and resulting in a proper macrophage phenotype exhibiting a mixed M1/M2 status and would suggest these ascite-containing factors as keys conferring an immunosuppressive cell phenotype. Altogether, these translational findings highlight ovarian patient-derived ascites as a valuable tool to understand the mechanisms of suppression and to allow for the identification of novel markers to develop innovative targeted therapies. Citation Format: Imane Nafia, Assia Chaibi, Christophe Rey, Jean-Philippe Guegan, Alban Bessede, Coriolan Lebreton, Antoine Italiano. Ovarian cancer ascites display altered immune environment featured by enhanced soluble factors and suppressive cellular context [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2527.

Abstract 5510: Preclinical study of allogeneic CD19-CAR-DNT cells as an off-the-shelf immunotherapy drug for NHL

Abstract Introduction Allogeneic Double Negative T (DNT, CD3+CD4-CD8-) cells have shown great potential as a novel off-the-shelf immunotherapy drug candidate in clinic trials for r/r/AML patients. This is our first report to demonstrate that CD19-CAR-DNT cells with high percentage of Tscm+Tcm phenotypes have been successfully developed with strong immunotherapeutic effects on Raji-luciferase tumor cells from non-Hodgkin’s lymphoma both in vitro and in vivo. Methods DNT cells were expanded ex vivo from healthy donors and then were transduced with CD19-CAR lentivirus in the presence of IL-2. The CD19-CAR-DNT cell expansions were performed under GMP condition and then harvested and cryo-preserved by CryStor®CS5 in liquid nitrogen for future use. Summary The ex vivo expanded CD19-CAR-DNT cells has more than 50% of Tscm and Tcm phenotypes. The viability and CAR+ percentage of CD19-CAR-DNT cells were 84.40% and 34.98% respectively. The killing of CD19-CAR-DNT cells on Raji-luciferase cells was 77.92% (n=6) at the effector to target ratio of 4:1. Elevated IFN-γ release from CD19-CAR-DNT cells after overnight co-culture with Raji cells was observed. The antitumor efficacy was evaluated in the xenografted NHL mice model. After the first infusion of the cells, the tumor growth inhibitory rates (TGI %) of CD19-CAR-DNT cells at 1×106, 5×106 and 1×107 per mouse on day14 were 80.54%, 98.31% and 99.05% respectively, the killing effects were in a dose-dependent manner. After the second infusion of the CD19-CAR-DNT cells at day 14, median survival times (MST) of the treated three groups mice were elongated till Day60, that is twice longer than that of the xenografted vehicle group. In the subsequent PKPD study, infusion CD19-CAR-DNT cells into Raji-luciferase cell xenografted mice were primarily distributed in lungs, peripheral blood, spleen, and bone marrow within 72h, homing back to liver and spleen on Day36. All animal body weights in both fresh and frozen reconstituted CD19-CAR-DNT cells treated mice were increased with no observed toxicities. Conclusions ex vivo expansion of CD19-CAR-DNT cells were successfully developed to explore its potential as a novel off-the-shelf universal CART drug for malignant B cells leukemia. The cell subsets consist of more than 50% of Tscm and Tcm cells whereas most immunological check point inhibitors were not elevated after priming by target tumor cells except a slight increase of PD-1 molecule. Fresh or frozen CD19-CAR-DNT cells can kill Raji cells in vitro and in vivo studies in a dose-dependent manner although the frozen one showed a delayed killing in vivo. The tumor cells were almost depleted and the MST was significantly increased with NOAEL at 1x107 cells/mouse. The amounts of CD19-CAR-DNT cells are re-distributed from early lungs, peripheral blood to live, spleen later. Citation Format: Liming Yang, Dan Wang, Shangzhi Xu, Liuyang Wang, Jianjun Tong, Honglin Zhu, Man Xu, Xiancai Li, Letu Ji, Chuntao Zheng, Zhiqiang Xiang, Qinghua Sun, Hengcai Wang. Preclinical study of allogeneic CD19-CAR-DNT cells as an off-the-shelf immunotherapy drug for NHL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5510.

Differential Effects of Alarmins on Human and Mouse Basophils.

Epithelial-derived alarmins (IL-33, TSLP, and IL-25) play an upstream role in the pathogenesis of asthma. Basophil-derived cytokines are a pivotal component of allergic inflammation. We evaluated the in vitro effects of IL-33, TSLP, and IL-25, alone and in combination with IL-3 on purified peripheral blood human basophils (hBaso) and bone marrow-derived mouse basophils (mBaso) in modulating the production of IL-4, IL-13, CXCL8 or the mouse CXCL8 equivalents CXCL1 and CXCL2. IL-3 and IL-33, but not TSLP and IL-25, concentration-dependently induced IL-4, IL-13, and CXCL8 release from hBaso. IL-3 synergistically potentiated the release of cytokines induced by IL-33 from hBaso. In mBaso, IL-3 and IL-33 rapidly induced IL-4 and IL-13 mRNA expression and protein release. IL-33, but not IL-3, induced CXCL2 and CXCL1 from mBaso. Differently from hBaso, TSLP induced IL-4, IL-13, CXCL1 and CXCL2 mRNA expression and protein release from mBaso. IL-25 had no effect on IL-4, IL-13, and CXCL1/CXCL2 mRNA expression and protein release even in the presence of IL-3. No synergism was observed between IL-3 and either IL-25 or TSLP. IL-3 inhibited both TSLP- and IL-33-induced CXCL1 and CXCL2 release from mBaso. Our results highlight some similarities and marked differences between the effects of IL-3 and alarmins on the release of cytokines from human and mouse basophils.

Counteractive Effects of IL-33 and IL-37 on Inflammation in Osteoarthritis.

Osteoarthritis (OA) is a chronic inflammatory disease where pro-inflammatory cytokines, damage-associated molecular patterns (DAMPs), and macrophages play a crucial role. However, the interactive role of these mediators, the exact cause precipitating OA and definitive treatment for OA are not known yet. Moreover, the interactive role of interleukin (IL)-33 and IL-37 with other factors in the pathogenesis of OA has not been discussed elaborately. In this study, we analyzed the expression of IL-33 and IL-37 in human OA knee and hip joint cartilage tissues. The effect of increased DAMPs, IL-33, and IL-37 on IL-6, tumor necrosis factor (TNF)-α, toll-like receptors (TLRs), and matrix metalloproteinases (MMPs) expression was delineated using human normal and osteoarthritic chondrocytes. The effect of anti-inflammatory cytokine IL-37 on various mediators of inflammation in the presence of IL-33, rHMGB-1, and LPS was investigated to delineate the effects of IL-37. Further, the effects of blocking IL-33 downstream signaling and the effects of IL-33 and IL-37 on macrophage polarization were assessed along with examining the macrophage phenotypes in human OA cartilage tissues. The results of this study revealed increased expression of IL-33 in OA cartilage and that IL-33 increases IL-6, TNF-α, TLRs, and MMPs expression and favors phenotypic conversion towards the M1 phenotype, while IL-37 and blocking IL-33 receptor ST2 have opposite effects. Overall, the results suggest that blocking IL-33 and increasing IL-37 act synergistically to attenuate inflammation and might serve as potential therapeutics in OA.

Heterogeneity of Latency Establishment in the Different Human CD4+ T Cell Subsets Stimulated with IL-15.

ABSTRACTHIV integrates into the host genome, creating a viral reservoir of latently infected cells that persists despite effective antiretroviral treatment. CD4-positive (CD4+) T cells are the main contributors to the HIV reservoir. CD4+ T cells are a heterogeneous population, and the mechanisms of latency establishment in the different subsets, as well as their contribution to the reservoir, are still unclear. In this study, we analyzed HIV latency establishment in different CD4+ T cell subsets stimulated with interleukin 15 (IL-15), a cytokine that increases both susceptibility to infection and reactivation from latency. Using a dual-reporter virus that allows discrimination between latent and productive infection at the single-cell level, we found that IL-15-treated primary human CD4+ T naive and CD4+ T stem cell memory (TSCM) cells are less susceptible to HIV infection than CD4+ central memory (TCM), effector memory (TEM), and transitional memory (TTM) cells but are also more likely to harbor transcriptionally silent provirus. The propensity of these subsets to harbor latent provirus compared to the more differentiated memory subsets was independent of differential expression of pTEFb components. Microscopy analysis of NF-κB suggested that CD4+ T naive cells express smaller amounts of nuclear NF-κB than the other subsets, partially explaining the inefficient long terminal repeat (LTR)-driven transcription. On the other hand, CD4+ TSCM cells display similar levels of nuclear NF-κB to CD4+ TCM, CD4+ TEM, and CD4+ TTM cells, indicating the availability of transcription initiation and elongation factors is not solely responsible for the inefficient HIV gene expression in the CD4+ TSCM subset.IMPORTANCE The formation of a latent reservoir is the main barrier to HIV cure. Here, we investigated how HIV latency is established in different CD4+ T cell subsets in the presence of IL-15, a cytokine that has been shown to efficiently induce latency reversal. We observed that, even in the presence of IL-15, the less differentiated subsets display lower levels of productive HIV infection than the more differentiated subsets. These differences were not related to different expression of pTEFb, and modest differences in NF-κB were observed for CD4+ T naive cells only, implying the involvement of other mechanisms. Understanding the molecular basis of latency establishment in different CD4+ T cell subsets might be important for tailoring specific strategies to reactivate HIV transcription in all the CD4+ T subsets that compose the latent reservoir.

A critical role of energy sensor AMPKα1 in rapamycin dependent memory CD8+ T cell survival

Abstract Energy sensors mTORC1 and AMPKα1 regulate T-cell metabolism and differentiation, while rapamycin (Rapa), an inhibitor of mTORC1, supports T-cell memory. However, the underlying pathway, the exact mechanisms, and the role of AMPKα1 in Rapa-induced T-cell memory is not well understood. Using the Listeria monocytogenes (rLmOVA) infection model and genetic and pharmaceutical tools, we demonstrate that Rapa promotes T-cell memory in mice post-rLmOVA infection. When T-cells stimulated in vitro in the presence of IL-2 without rapamycin (IL-2/T) or with rapamycin (IL-2+Rapa /T) and transferred into mice, it differentiated into short-term effector T (TE) [IL-7R−CD62L−KLRG1+] and long-lived memory T (TM) [IL-7R+CD62L+KLRG1−] cells, respectively. We determined that rapamycin-treated T cells activated transcriptional factors, FOXO1, TCF1 and Eomes and metabolic pAMPKα1(T172), pULK1(S555), and ATG7 molecules and promoted mitochondrial biogenesis and oxidative phosphorylation (OXPHOS). Using Seahorse-real time metabolic analyzer, we found that rapamycin-treated AMPKα-deficient TM cells up-regulated transcription factor HIF-1α and induced a metabolic switch from OXPHOS to glycolysis. Interestingly, despite the rapamycin treatment, AMPKα-deficient TM cells lost their cell survival capacity. Altogether, our data provide a mechanistic explanation for enhanced T-cell survival after rapamycin treatment and suggest that rapamycin promotes T-cell memory via transcriptional FOXO1-TCF1-Eomes programs and AMPKα1-ULK1-ATG7 metabolic axis. And AMPKα1 plays a critical role in rapamycin-induced increased survival and metabolic switching of CTLs during the transition of effector to memory T cells. Supported by Canadian Institutes of Health Research grant (409228). Saskatchewan Health Research Foundation (SHRF) postdoctoral fellowship.

Identification of a subset of tumor-reactive CD8 TILs expressing NKG2A in HNSCC and CRC

Abstract Even though blocking the interaction of NKG2A with its ligand HLA-E is a promising strategy to restore the function of cytotoxic T cells and induce tumor cell killing, several aspects of the biology of NKG2A+ CD8 T cells remain to be understood to fully benefit from this therapeutic approach. Here, we characterize the expression of NKG2A on tumor-infiltrating CD8 T cells (CD8 TILs) in patients with head and neck squamous cell carcinoma (HNSCC) and microsatellite-stable colorectal cancer (CRC). Our results show that NKG2A is primarily expressed by a population of tumor-reactive CD8 TILs identified by coexpression of CD39 and CD103 (DP CD8). NKG2A expression was stable and maintained during in vitro expansion. Interestingly, while TCR stimulation in presence of IL-15 and TGF-β are thought to be responsible for NKG2A upregulation, our in vitro results suggest that other factors might be necessary to induce its expression on naive CD8 T cells. Ex vivo analysis of NKG2A+ DP CD8 TILs by scRNAseq and flow cytometry revealed that those cells were activated, more differentiated, and displayed a cytolytic profile suggesting a role in tumor recognition and killing. Finally, we found that, even though there was a high degree of clonal overlap between NKG2A+ and NKG2A− DP CD8, some CDR3 TCR sequences were enriched in one versus the other subset. Consequently, HPV-reactive cells were preferentially enriched in NKG2A− DP CD8 TILs. Altogether, our results provide evidence that NKG2A is expressed by tumor-reactive CD8 TILs in HNSCC and CRC. However, NKG2A-inducing factor(s) remain to be identified and might provide new potential drug targets to bolster anti-tumor immune responses in cancer patients. Supported by the Providence Portland Medical Foundation.

Select Amino Acid Formulation Influenced Anion Secretion Induced by Th2 Cytokines in Human Bronchial Epithelial Cells (HBECs)

IntroductionAirway surface liquid is tightly regulated by ion channels and/or transporters, and plays a critical role in inflammatory airway diseases, such as ARDS, and in edema fluid clearance during lung injury. Although beta‐adrenergic agonists have been shown to improve alveolar fluid clearance, they have limited effect in late stages of ARDS due to reduced epithelial beta‐adrenergic receptor function or agonist delivery to target regions. CFTR, CLCA2, TMEM16A, and alternative Cl‐/HCO3‐ exchangers (e.g., SLC4 family) are known to be expressed in lung epithelia. Their relative contribution during late stages of lung injury where TH2 cytokines such as IL13 are elevated is not fully understood. The expression pattern of these anion channels are dysregulated in ARDS, and restoration of channel activities could improve fluid accumulation within the lung. In the present study, various anion channel and/or exchanger activities were measured in HBECs exposed to IL13. We have shown that select amino acids influence anion channel activity. Therefore, the effect of a proprietary amino acid formulation on specific anion channels were studied in IL13‐treated HBECs.MethodsTransepithelial short‐circuit current (Isc) was sequentially blocked using inhibitors for ENaC (benzamil), CFTR CFTRInh172), TMEM16A (CaCCInhA01) and Na+/K+/2Cl‐ co‐transporter (bumetanide) in differentiated HBECs mounted in Ussing chambers. Patch‐clamp whole‐cell recordings were performed to determine anion channel activity after IL13 treatment, and channel and/or exchanger expression was analyzed by immunofluorescence.ResultsIL13 increased benzamil‐insensitive (anion) Isc (87.5 ± 4.7 µA.cm‐2 vs 21.9 ± 1.8 µA.cm‐2; P <0.001) and decreased CFTR Isc (1.4 ± 0.52 µA.cm‐2 vs 12.2 ± 0.83 µA.cm‐2; P <0.001) compared to controls. Amino acids reduced benzamil‐insensitive Isc (55.9 ± 4.8 µA.cm‐2; P <0.001) and increased CFTR Isc (6.7 ± 0.75 µA.cm‐2; P <0.001). There was no change in TMEM16A Isc with IL13 treatment. Bumetanide‐sensitive Isc was higher in IL13‐treated cells compared to controls (52.5 ± 3.6 µA.cm‐2 vs 5.5 ± 0.62 µA.cm‐2; P <0.001) suggesting anion secretion via alternate mechanisms, and amino acids decreased bumetanide‐sensitive anion secretion (20.5 ± 2.9 µA.cm‐2; P <0.001). Similarly, whole‐cell patch clamp recordings showed that amino acids restored the CFTR response in IL13‐treated cells. Immunofluorescence imaging demonstrated decreased CFTR expression with IL13, while amino acids recovered CFTR at the apical membrane.ConclusionTreatment with IL13 increased anion secretion while reducing CFTR channel activity in HBECs. Amino acid formulation restored CFTR activity and decreased total anion current in the presence of IL13. These studies suggest anion secretion via alternate anion secretory pathways. Future studies should be directed at understanding the link between CFTR and alternate anion channels in conditions associated with elevated IL13, and how amino acids regulate these channel and/or exchanger functions

Batf Stabilizes the Th17 Cell Developmental Program through Impairment of Stat5-dependent Recruitment of Ets1-Runx1 Complexes

Abstract The emergence of alternative CD4 T cell fates is controlled by transcriptional networks activated downstream of TCR and cytokine signaling inputs. Th17 cells develop from antigen-activated naive CD4 T cells in the presence of IL-6 and TGF-b. An important facet of the Th17 cell program is its intrinsic instability; early development of Th17 cells shares overlapping features with that of Treg cells and Th17 cells are prone to transdifferentiation into Th1-like cells. The basic leucine zipper transcription factor ATF-like (Batf) contributes to the transcriptional programming of multiple effector T cells but is indispensable for Th17 cell development. Although Batf is required for development of Th17 cells, its mechanisms of action to underpin the Th17 program are incompletely understood. Here, we find that in addition to activating core genes of the Th17 program, Batf also acts to restrict expression of genes of the Treg and Th1 programs through its actions to restrain IL-2-induced Stat5 activation, thereby limiting Stat5-dependent recruitment of Ets1-Runx1 factors to Th1 and Treg cell-specific gene loci. Accordingly, Batf acts both to pioneer regulatory elements in Th17-specific genes and limit assembly of a Stat5-Ets1-Runx1 enhanceosome that appears to be required for optimal expression of Th1 and Treg cell-specific genes. Our findings define a new role for Batf in T cell differentiation and unveil an important role for Stat5-Ets1-Runx1 interactions in transcriptional networks that define alternative T cell developmental fates. Supported by NIH R01 DK115172 and T32 AI007051-37

HIV-1 Vpr drives a tissue residency-like phenotype during selective infection of resting memory Tcells.

SummaryHIV-1 replicates in CD4+ T cells, leading to AIDS. Determining how HIV-1 shapes its niche to create a permissive environment is central to informing efforts to limit pathogenesis, disturb reservoirs, and achieve a cure. A key roadblock in understanding HIV-T cell interactions is the requirement to activate T cells in vitro to make them permissive to infection. This dramatically alters T cell biology and virus-host interactions. Here we show that HIV-1 cell-to-cell spread permits efficient, productive infection of resting memory T cells without prior activation. Strikingly, we find that HIV-1 infection primes resting T cells to gain characteristics of tissue-resident memory T cells (TRM), including upregulating key surface markers and the transcription factor Blimp-1 and inducing a transcriptional program overlapping the core TRM transcriptional signature. This reprogramming is driven by Vpr and requires Vpr packaging into virions and manipulation of STAT5. Thus, HIV-1 reprograms resting T cells, with implications for viral replication and persistence.

IL-33 regulates M1/M2 chemokine expression via mitochondrial redox-related mitophagy in human monocytes

Interleukin (IL)-33 is an epithelial-derived cytokine that enhances T helper (Th) 2 responses. Allergens and other agents induce IL-33 in asthma. Excessive production of reactive oxygen species (ROS) leads to airway inflammation. Mitophagy is the selective degradation of mitochondria by autophagy and often occurs in defective mitochondria, followed by ROS production. In the present study, we examined the effects of IL-33 on ROS production and mitophagy in human monocytes, and the detailed mechanisms were investigated. Human monocyte cell line THP-1 was pretreated with different concentrations of IL-33. ROS production was measured by flow cytometry. Mitochondrial involvement and the mitophagy and intercellular pathway activation were evaluated by quantitative real-time PCR, western blotting, and confocal microscopy, and cytokine/chemokine concentrations were detected by ELISA. The data showed that IL-33 alone could induce ROS expression in THP-1 cells. The expression of complex II and V mRNA was increased in the presence of IL-33. The mitophagy-related proteins PINK1, Parkin, and LC3 were regulated by IL-33 through the AMPK pathway. IL-33 significantly decreased M1-related cytokines CXCL-10 and TNF-α production and significantly increased M2-related cytokine CCL-22 production. In conclusion, IL-33 induces ROS production and subsequently influences mitophagy through AMPK activation, altering the macrophage-polarization phenotype of monocytes.

Elotuzumab, a potential therapeutic humanized anti-SLAMF7 monoclonal antibody, enhances natural killer cell-mediated killing of primary effusion lymphoma cells.

Primary effusion lymphoma (PEL) is a rare aggressive B-cell non-Hodgkin's lymphoma with no optimal treatment. Signaling lymphocytic activation molecule-F7 (SLAMF7, CD319), a type I transmembrane glycoprotein highly expressed in multiple myeloma (MM), represents a promising target for mAb-based immunotherapy. SLAMF7 also expresses on several hematopoietic lineages including NK cells. Elotuzumab (Elo), a humanized antibody targeting SLAMF7, is approved by FDA for MM treatment. In this study, we analyzed the expression of SLAMF7 on seven PEL cell lines. All PEL cells and NK cells showed high expression of SLAMF7. NK cells were enriched from PBMCs of healthy donors by MACS and expanded by co-culturing with MHC-class I negative K562 cells in the presence of IL-2 and IL-15. Expanded NK cells showed direct killing, and Elo demonstrated potent ADCC against PEL in an Effector:Target (E:T) dependent manner. Surface expression of CD107a on NK cells also increased in the process of ADCC. We also examined SLAMF7 expression of NK subpopulations and found that the CD56+CD16+ NK subpopulation demonstrated the highest SLAMF7 expression. Full-length-Elo but not F(ab')2-Elo exerts direct engagement to the expressing SLAMF7 on NK cells, promotes CD107a expression, and further augments NK cytotoxicity toward PEL. Elo enhanced survival of PEL-bearing immunodeficient mice with adoptive transfer of human NK cells. Taken together, our results show that NK cells play roles in PEL killing, and Elo causes ADCC/SLAMF7 ligation to boost NK cytotoxicity against PEL, offering promising preclinical evidence of Elo as a therapeutic monoclonal antibody treatment for PEL.

A Tale of Two Loads: Modulation of IL-1 Induced Inflammatory Responses of Meniscal Cells in Two Models of Dynamic Physiologic Loading.

Meniscus injuries are highly prevalent, and both meniscus injury and subsequent surgery are linked to the development of post-traumatic osteoarthritis (PTOA). Although the pathogenesis of PTOA remains poorly understood, the inflammatory cytokine IL-1 is elevated in synovial fluid following acute knee injuries and causes degradation of meniscus tissue and inhibits meniscus repair. Dynamic mechanical compression of meniscus tissue improves integrative meniscus repair in the presence of IL-1 and dynamic tensile strain modulates the response of meniscus cells to IL-1. Despite the promising observed effects of physiologic mechanical loading on suppressing inflammatory responses of meniscus cells, there is a lack of knowledge on the global effects of loading on meniscus transcriptomic profiles. In this study, we compared two established models of physiologic mechanical stimulation, dynamic compression of tissue explants and cyclic tensile stretch of isolated meniscus cells, to identify conserved responses to mechanical loading. RNA sequencing was performed on loaded and unloaded meniscus tissue or isolated cells from inner and outer zones, with and without IL-1. Overall, results from both models showed significant modulation of inflammation-related pathways with mechanical stimulation. Anti-inflammatory effects of loading were well-conserved between the tissue compression and cell stretch models for inner zone; however, the cell stretch model resulted in a larger number of differentially regulated genes. Our findings on the global transcriptomic profiles of two models of mechanical stimulation lay the groundwork for future mechanistic studies of meniscus mechanotransduction, which may lead to the discovery of novel therapeutic targets for the treatment of meniscus injuries.

Qi Ling decreases paclitaxel resistance in the human prostate cancer by reversing tumor-associated macrophages function.

Tumor-associated macrophages (TAMs) are critical immune cells infiltrated into tumor. In present study, we evaluated the effects of Qi Ling (QL), a traditional Chinese medicine on paclitaxel resistance in prostate cancer cells and explored the underlying mechanisms. We administrated QL to rats and collected the serum from QL-treated rats (QL-serum). We established the co-culture system of TAMs/paclitaxel resistant prostate cancer cells. We treated the TAMs with QL-serum and measured the viability of paclitaxel resistant prostate cancer cells after exposing to paclitaxel. We monitored the expression of M1 and M2 markers, the expression and activation of IL-6/STAT3 signaling pathways in TAMs after QL treatment. We treated TAMs with QL-serum together with interleukin (IL)-6, measured the expression of M1 and M2 markers, and the viability of paclitaxel resistant prostate cancer cells. In co-culture system, QL-serum-treated TAMs decreased the paclitaxel resistance in the human prostate cancer cells. QL-serum treatment significantly up-regulated the expression of M1 markers inducible nitric oxide synthase and tumor necrosis factor α while decreased the expression of M2 markers IL-10 and chemokine (C-C motif) ligand 22. QL-serum suppressed the activation of IL-6/ signal transducer and activator of transcription 3 signaling pathway. All these effects of QL-serum were abolished in the presence of IL-6. Qi Ling re-programmed TAMs and decreases paclitaxel resistance in prostate cancer cells.

Targeting an IL-6/JAK/STAT/PIM1 pathway in renal cell carcinoma.

376 Background: Renal cell carcinoma (RCC) is the most common cancer of the kidney and accounts for over 430,000 new cases worldwide and 179,000 deaths each year. PIM1 overexpression is associated with poor clinical outcomes in patients with RCC. PIM1 is a constitutively active effector serine/threonine kinase with roles in tumor progression including cell proliferation, apoptosis, invasion, and migration. Yet, the mechanisms that underlie PIM1 expression and its function in RCC are not fully understood. Previous studies have implicated the cytokine IL-6 as an activator of STAT3 and stimulator of PIM1 expression in pancreatic cancer. The aim of the present study is to explore expression of PIM1 in RCC and identify potential signaling pathways that influence PIM1 expression. Methods: To evaluate the contribution of PIM1 expression in RCC we first established baseline PIM1 levels across four RCC cell lines (786-O, 769-P, CAKI-1, and RCC-4). Next, we quantified the presence of IL-6 in cell culture supernatants for each cell line using an ELISA assay. IL-6 signals through the JAK/STAT pathway, consequently we then used immunoblotting to determine whether ruxolitinib, a JAK1/2 inhibitor, would affect PIM1 expression. Finally, we examined cell viability post-ruxolitinib therapy using an MTS assay. Results: Our results show that 769-P, 786-O, and CAKI-1 but not RCC-4 cells overexpress PIM1 relative to normal renal proximal tubule epithelial cells. Results from the ELISA assay show that 786-O, CAKI-1, and to a lesser degree RCC-4 cells secrete IL-6 into their media. Interestingly, 769-P cells had little to no IL-6 present in their media. Based on these differences we sought to understand what effect inhibiting JAK may have on PIM1 expression. After treating cells with ruxolitinib, we found that 786-O and CAKI-1 cells show a time and dose dependent decrease in PIM1 levels whereas 769-P cells did not. To understand if these differences correlate with cell viability, we performed an MTS assay after treating cells with ruxolitinib. Preliminary results show that at 8hr post-treatment, CAKI-1 but not 769-P cells display a time and concentration dependent loss of cell viability. Conclusions: Our initial studies suggest that differential expression of PIM1 among RCC cell lines may be linked to autocrine IL-6 secretion. In CAKI-1 cells JAK inhibition decreases PIM1 expression and cell viability. In the context of 769-P cells with high PIM1 expression but low IL-6 secretion levels, JAK inhibition did not affect cell viability. These data suggest that in 769-P cells there exists a non-IL-6 dependent mechanism stimulating PIM1 expression. Further investigation is required to elucidate the contexts of these PIM1 regulatory pathways in order to develop IL-6/JAK/PIM1 targeted anti-cancer strategies in RCC.

Identification of nafamostat mesylate as a selective stimulator of NK cell IFN-γ production via metabolism-related compound library screening

Natural killer (NK) cells play important roles in controlling virus-infected and malignant cells. The identification of new molecules that can activate NK cells may effectively improve the antiviral and antitumour activities of these cells. In this study, by using a commercially available metabolism-related compound library, we initially screened the capacity of compounds to activate NK cells by determining the ratio of interferon-gamma (IFN-γ)+ NK cells by flow cytometry after the incubation of peripheral blood mononuclear cells (PBMCs) with IL-12 or IL-15 for 18 h. Our data showed that eight compounds (nafamostat mesylate (NM), loganin, fluvastatin sodium, atorvastatin calcium, lovastatin, simvastatin, rosuvastatin calcium, and pitavastatin calcium) and three compounds (NM, elesclomol, and simvastatin) increased the proportions of NK cells and CD3+ T cells that expressed IFN-γ among PBMCs cultured with IL-12 and IL-15, respectively. When incubated with enriched NK cells (purity ≥ 80.0%), only NM enhanced NK cell IFN-γ production in the presence of IL-12 or IL-15. When incubated with purified NK cells (purity ≥ 99.0%), NM promoted NK cell IFN-γ secretion in the presence or absence of IL-18. However, NM showed no effect on NK cell cytotoxicity. Collectively, our study identifies NM as a selective stimulator of IFN-γ production by NK cells, providing a new strategy for the prevention and treatment of infection or cancer in select populations.

Human CD4 T Cells From Thymus and Cord Blood Are Convertible Into CD8 T Cells by IL-4.

Commitment to the CD4+ or CD8+ T cell lineages is linked to the acquisition of a functional program broadly defined by helper and cytotoxic properties, respectively. The mechanisms underlying these processes in the human thymus remain largely unclear. Moreover, recent thymic emigrants are thought to have some degree of plasticity, which may be important for the shaping of the immune system and adjustment to specific peripheral needs. We show here that IL-4 induces proliferation-independent de novo synthesis of CD8αβ in human CD4 single-positive (SP) thymocytes, generating a stable CD8SP population that features a diverse TCRαβ repertoire, CD4 expression shut-down and ThPOK downregulation. IL-4 also promotes an innate-like program in both CD4SP and CD8SP thymocytes, characterized by Eomes upregulation in the absence of T-bet, in line with its recognized role in the generation of thymic innate-like CD8+ T cells. The clinical relevance of these findings is further supported by the profile of IL-4 production and IL-4 receptor expression that we identified in the human thymus. Importantly, human cord blood CD4+ T cells preserve the ability to generate Eomes+ CD8+ T cells in the presence of IL-4, with implications in neonatal immunity. Our results support a role for IL-4 in the dynamic regulation of human thymocyte plasticity and identify novel strategies to modulate immune responses.

Interfering with alternatively activated macrophages by CSF-1R inhibition exerts therapeutic capacity on allergic airway inflammation

PurposeAllergic asthma is a chronic inflammatory disorder with airway hyperresponsiveness and tissue remodeling as the main pathological characteristics. The etiology of asthma is relatively complicated, involving genetic susceptibility, epigenetic regulation, environmental factors, and immune imbalance. Colony stimulating factor 1 receptor (CSF-1R), highly expressed in myeloid monocytes, plays an important role in regulating inflammation. However, the pathological role of CSF-1R and the therapeutic effects of CSF-1R inhibitor in allergic airway inflammation remain indistinct. MethodsThe house dust mite (HDM)-triggered allergic airway inflammation model was conducted to fully uncover the efficacies of CSF-1R inhibition, as illustrated by histopathological examinations, biochemical analysis, ELISA, RT-PCR, Western blotting assay, immunofluorescence, and flow cytometry. Furthermore, bone marrow-derived macrophages (BMDMs) were differentiated and polarized upon IL-4/IL-13 induction to clarify the underlying mechanisms of CSF-1R inhibition. ResultsHerein, we presented that the expression of CSF-1R was increased in HDM-induced experimental asthma and inhibition of CSF-1R displayed dramatic effects on the disease severity of asthma, referring to suppressing the secretion of allergic mediators, dysfunction of airway epithelium, and infiltration of inflammatory cells. Furthermore, CSF-1R inhibitor could markedly restrain the polarization and expression of transcriptional factors of alternatively activated macrophages (AAMs) in the presence of IL-4/IL-13 and reduce the recruitment of CSF-1R-dominant macrophages, both in acute and chronic allergic airway inflammation model. ConclusionCollectively, our findings demonstrated the molecular pathological mechanism of CSF-1R in allergic airway diseases and suggested that targeting CSF-1R might be an alternative intervention strategy on the homeostasis of airway immune microenvironment in asthma.

Interleukin-4 Responsive Dendritic Cells Are Dispensable to Host Resistance Against Leishmania mexicana Infection.

IL-4 and IL-13 cytokines have been associated with a non-healing phenotype in murine leishmaniasis in L. mexicana -infected BALB/c mice as demonstrated in IL-4−/−, IL-13−/− and IL-4Rα-/- global knockout mouse studies. However, it is unclear from the studies which cell-type-specific IL-4/IL-13 signaling mediates protection to L. mexicana. Previous studies have ruled out a role for IL-4-mediated protection on CD4+ T cells during L. mexicana infections. A candidate for this role may be non-lymphocyte cells, particularly DCs, as was previously shown in L. major infections, where IL-4 production drives dendritic cell-IL-12 production thereby mediating a type 1 immune response. However, it is unclear if this IL-4-instruction of type 1 immunity also occurs in CL caused by L. mexicana, since the outcome of cutaneous leishmaniasis often depends on the infecting Leishmania species. Thus, BALB/c mice with cell-specific deletion of the IL-4Rα on CD11c+ DCs (CD11ccreIL-4Rα-/lox) were infected with L. mexicana promastigotes in the footpad and the clinical phenotype, humoral and cellular immune responses were investigated, compared to the littermate control. Our results show that CL disease progression in BALB/c mice is independent of IL-4Rα signaling on DCs as CD11ccreIL-4Rα-/lox mice had similar footpad lesion progression, parasite loads, humoral responses (IgE, IgG1, IgG 2a/b), and IFN-γ cytokine secretion in comparison to littermate controls. Despite this comparable phenotype, surprisingly, IL-4 production in CD11ccreIL-4Rα-/lox mice was significantly increased with an increasing trend of IL-13 when compared to littermate controls. Moreover, the absence of IL-4Rα signaling did not significantly alter the frequency of CD4 and CD8 lymphocytes nor their activation, or memory phenotype compared to littermate controls. However, these populations were significantly increased in CD11ccreIL-4Rα-/lox mice due to greater total cell infiltration into the lymph node. A similar trend was observed for B cells whereas the recruitment of myeloid populations (macrophages, DCs, neutrophils, and Mo-DCs) into LN was comparable to littermate IL-4Rα-/lox mice. Interestingly, IL-4Rα-deficient bone marrow-derived dendritic cells (BMDCs), stimulated with LPS or L. mexicana promastigotes in presence of IL-4, showed similar levels of IL-12p70 and IL-10 to littermate controls highlighting that IL-4-mediated DC instruction was not impaired in response to L. mexicana. Similarly, IL-4 stimulation did not affect the maturation or activation of IL-4Rα-deficient BMDCs during L. mexicana infection nor their effector functions in production of nitrite and arginine-derived metabolite (urea). Together, this study suggests that IL-4 Rα signaling on DCs is not key in the regulation of immune-mediated protection in mice against L. mexicana infection.