No abstract

SummaryThe programmed cell death 1 (PD‐1) receptor plays a major role in regulating T cell activation. Our aim was to determine how inflammation influences PD‐1‐mediated T cell suppression. Flow cytometry analysis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA) synovial fluid (SF) mononuclear cells showed an increase in the percentage of PD‐1+ cells within the CD4+ and CD8+ T cell compartment compared to paired peripheral blood (PB). Upon in‐vitro T cell receptor (TCR) stimulation of healthy control (HC) CD4+ T cells in the presence of plate‐bound PD‐L1fc chimera, significantly decreased proliferation and interferon (IFN)‐γ secretion was observed. In contrast, CD4+ T cells from RA and PsA PB and SF appeared resistant to such PD‐1‐mediated inhibition. Addition of the proinflammatory cytokines tumour necrosis factor (TNF)α, interleukin (IL)‐6 and IL‐1β, which were increased in RA and PsA SF compared to osteoarthritis (OA) SF, consistently abrogated PD‐1‐mediated suppression in HC CD4+ T cell cultures. This effect was reversed by inhibitors of these cytokines. Soluble PD‐1 (sPD‐1) levels were increased in cell culture supernatants from TNFα and IL‐6‐stimulated cultures compared to untreated controls, and also in RA and PsA, but not in OA, serum and SF. Functionally, addition of sPD‐1fc counteracted PD‐1‐mediated suppression of HC CD4+ T cells, and increased T cell proliferation in HC CD4+ T cell/monocyte co‐cultures. These in‐vitro findings indicate that CD4+ T cells from patients with RA and PsA show increased resistance to PD‐1‐mediated suppression, which may be explained in part by the presence of soluble PD‐1 in the inflammatory environment.

Natural killer (NK) cells are the primary effectors of the innate immune response against virus infected cells or cells that have undergone malignant transformation. NK cells recognize their targets through a complex array of activating and inhibitory receptors, which regulate the intensity of the effector response against individual target cells. However, many studies have shown that tumor cells can escape immune cell recognition through a variety of mechanisms, developing resistance to NK cell killing. Using a lentiviral shRNA library, we previously demonstrated that several common signaling pathways modulate susceptibility of tumor cells to NK cell activity. In this study, we focused on one of the genes (PI3KCB), identified in this genetic screen. The PI3KCB gene encodes an isoform of the catalytic subunit of PI3K called P110β. The PI3K pathway has been linked to diverse cellular functions, but has never been associated with susceptibility to NK cell activity. Gene silencing of PI3KCB resulted in increased susceptibility of several tumor cell lines to NK cell lytic activity and induced increased IFN-γ secretion by NK cells. Treatment of primary tumor cells with two different PI3K inhibitors also increased target cell susceptibility to NK cell activity. These effects are due, at least in part, to modulation of several activating and inhibitory ligands and appear to be correlated with PI3K signaling pathway inhibition. These findings identify a new and important role of PI3KCB in modulating tumor cell susceptibility to NK cells and open the way to future combined target immunotherapies.

3960 Multiple myeloma (MM) is a B cell neoplasm characterized by clonal expansion of malignant plasma cells in the bone marrow. Despite the use of new drugs such as lenalidomide and bortezomib, MM remains an incurable disease. Successful treatment of MM with allogeneic stem cell transplantation suggests that MM is susceptible to immunologic approaches. NK cells are the primary effectors of the innate immune response against infectious pathogens and malignant transformation. Unlike T and B cells, NK cells do not recognize antigens in the context of classical major histocompatibility complex (MHC) but lyse target cells without specific antigen recognition. Nevertheless, MM cells have developed mechanisms to evade innate immune surveillance and the molecular basis for target resistance to NK cell-mediated lysis is not well understood. To identify novel pathways that modulate MM cell resistance to the immune system, we previously developed a genetic screen to detect cell-cell interactions using a large lentiviral shRNA library containing a total of 6,144 shRNAs targeting more than 1,000 human genes. Using this approach we found that silencing JAK1 and JAK2 results in significantly increased MM cell susceptibility to NK cell lysis. This effect was not noted when JAK3 and TYK2 were targeted. JAK1, JAK2 JAK3 and TYK2 are members of a family of tyrosine kinases that are constitutively associated with many membrane cytokine receptors. After activation, JAK proteins regulate phosphorylation/activation of STAT proteins, which subsequently initiate gene transcription. To understand JAK1 and JAK2 involvement in MM resistance to NK cells, we undertook a series of experiments to analyze the JAK signaling pathway in MM cells. We first analyzed the activation status of STAT proteins in a series of MM cell lines (IM-9, KM12BM, RPMI 8226, U266) in which JAK1 and JAK2 expression was reduced by specific shRNAs. Constitutive activation of STAT proteins was not affected by JAK1 or JAK2 gene silencing suggesting that these kinases were not activated in the absence of cytokine receptor-mediated signaling. Since JAK1 and JAK2 are associated with the IFN-γ receptor and we previously showed that JAK1 and JAK2 silencing induces increased secretion of IFN-γ from NK cells, we pre incubated MM cell lines with NK activated supernatant or recombinant IFN-γ and tested them for STAT activation. 15 min incubation was sufficient to initiate phosphorylation of STAT1 but no other STATs were activated. Silencing of JAK1 or JAK2 with specific shRNAs prevented STAT1 activation. To validate this finding, we tested primary MM cells treated with different concentrations of Jak inhibitor 1 (0 nM, 10 nM, 30 nM and 40 nM). These cells had a similar STAT profile at their basal level when compared with the previously tested MM cell lines. Pre-incubation with NK activated supernatant or IFN-γ also induced rapid activation of STAT1, which was completely inhibited when cells were pre-treated with Jak inhibitor 1. Treatment of MM cells with 10, 30 and 40 nM of Jak inhibitor enhanced killing by NK cells by 46.6%, 51% and 53%, compared to untreated cells (p=0.0036, p=0.0011 and p=0.0010 respectively). These findings demonstrate that IFN-γ signals rapidly enhance resistance of MM cells to NK cells but inhibition of this pathway at the level of JAK1 and JAK2 reverses this effect and induces susceptibility to NK cell mediated lysis. Disclosures: No relevant conflicts of interest to declare.

NK cells are the primary effectors of the innate immune response against infections pathogens and malignant transformation through their efficient cytolytic activity and cytokine secretion. Nevertheless, tumor cells have developed mechanisms to evade innate immune surveillance and the molecular basis for target resistance to NK cell-mediated lysis is not yet completely understood. To identify novel pathways that modulate tumor cell resistance to NK cells, we previously developed a cell-cell interaction based screening approach using a large sub-set of a lentiviral shRNA library containing multiple independent shRNAs targeting more than 1,000 human genes. Using this approach we found that silencing JAK1 and JAK2 significantly increased secretion of INF-γ from NK cells and increased tumor cell susceptibility to NK cell lysis. To examine the role of the JAK signaling pathway in the modulation of tumor cell susceptibility to NK lysis, we analyzed down-stream signaling pathways in several cell lines (IM9, U937, K562, RPMI, MM1S KM12BM) and primary tumor cells (AML, MM, ALL). In the absence of NK cells, silencing JAK1 or JAK2 did not affect the basal activation of STAT proteins (STAT1(pY701), STAT1(pS727), STAT3(pY705), STAT3(pS727), STAT4(pY693), STAT5(pY694), STAT6(pY641)) or AKT(pS473) and ERK1/2(pT202/pY204) or expression of activating or inhibitory ligands on tumor cells. Because JAK1 and JAK2 transduce signals downstream of the IFN-γ receptor, we hypothesized that JAKs may play a role in tumor cell evasion of NK cell activities such as cytolysis and IFN-γ secretion. To test this hypothesis we pre-incubated various tumor cell lines or primary tumor cells with activated NK supernatant or recombinant human IFN-γ. Tumor cell activation in this fashion resulted in activation of STAT1 (pSTAT1(pY701)) but none of the other STATs, ERK or AKT. As expected, STAT1 activation was blocked when JAK1 or JAK2 were silenced or inhibited by a JAK inhibitor. Silencing of STAT1 with 2 independent shRNAs also resulted in increased tumor susceptibility to NK cell cytolysis in 3 different tumor cell lines tested. To confirm that IFN-γ secreted by activated NK cells induced resistance in tumor cell targets we used a blocking IFN-γ antibody (D9D10). 10μg/ml D9D10 completely blocked STAT1 phosphorylation and in different experiments using U937, IM-9, KM12BM, MM1S and RPMI we found that D9D10 significantly increased specific NK target cell lysis by 51.8%, 78.5%, 25.1%, 20.6% and 28.5% compared to IgG1 isotype controls. Similar results were obtained whit different primary tumor cells. To determine whether IFN-γ stimulation affected expression of ligands involved in NK cell recognition of tumor cells, we analyzed the effect of activated NK supernatant or IFN-γ on the expression of MHC Class I, β2M, HLA-C, HLA-A2, NKG2D, NKP44, NKP46, NKP30 ligands using chimeric FC proteins, MICA/B, DNAM-1 ligands (CD112, CD155), 2B4 ligand (CD48), TRAIL ligands (TRAIL-R1, TRAIL-R2), Fas ligand (CD95) and PD1 ligands (PDL1, PDL2, B7H3, B7H4). The basal expression of these ligands varied among the various tumor cell lines or primary tumors tested but the only ligand that was significantly up-regulated in every tumor sample tested was PDL1. PDL1 expression by tumor cells is known to inhibit T cell immunity. To test whether increased levels of PDL1 could also inhibit NK cell killing, we co-cultured primary NK cells with U937, IM9, KM12BM, RPMI, K562, MM1S, primary MM, AML and ALL cells with or without 10μg/ml anti-PDL1 antibody (recombinant mab with Fc mutated to eliminate FcR-mediated effects). Blocking PDL1 significantly increased NK cell killing of U937, IM9, KM12BM, RPMI, MM, AML and ALL (p=0.03, p=0.02, p=0.03, p=0.005, p=0.009, p=0.03 and p=0.02 respectively). NK cell killing activity did not further increase when a JAK inhibitor was added to the co-culture. These results show that NK cell secretion of IFN-γ results in IFN receptor signaling and activation of JAK1, JAK2 and STAT1 in the tumor cell targets, followed by rapid up-regulation of PDL1 expression and increased resistance to NK cell lysis. Blockade of JAK pathway activation prevents subsequent PDL1 up-regulation resulting in increased susceptibility of tumor cells to NK cell activity suggesting that JAK pathway inhibitors may work synergistically with other immunotherapy regimens by eliminating IFN-induced PDL1 mediated immunoinhibition. Disclosures: Freeman: Bristol-Myers-Squibb/Medarex: Patents & Royalties; Roche/Genentech: Patents & Royalties; Merck: Patents & Royalties; EMD-Serrono: Patents & Royalties; Boehringer-Ingelheim: Patents & Royalties; Amplimmune: Patents & Royalties; CoStim Pharmaceuticals: Patents & Royalties; Costim Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.