Manjiri Sathe

IL-23 is essential for T cell–mediated colitis and promotes inflammation via IL-17 and IL-6

Uncontrolled mucosal immunity in the gastrointestinal tract of humans results in chronic inflammatory bowel disease (IBD), such as Crohn disease and ulcerative colitis. In early clinical trials as well as in animal models, IL-12 has been implicated as a major mediator of these diseases based on the ability of anti-p40 mAb treatment to reverse intestinal inflammation. The cytokine IL-23 shares the same p40 subunit with IL-12, and the anti-p40 mAbs used in human and mouse IBD studies neutralized the activities of both IL-12 and IL-23. IL-10-deficient mice spontaneously develop enterocolitis. To determine how IL-23 contributes to intestinal inflammation, we studied the disease susceptibility in the absence of either IL-23 or IL-12 in this model, as well as the ability of recombinant IL-23 to exacerbate IBD induced by T cell transfer. Our study shows that in these models, IL-23 is essential for manifestation of chronic intestinal inflammation, whereas IL-12 is not. A critical target of IL-23 is a unique subset of tissue-homing memory T cells, which are specifically activated by IL-23 to produce the proinflammatory mediators IL-17 and IL-6. This pathway may be responsible for chronic intestinal inflammation as well as other chronic autoimmune inflammatory diseases.

IL-23 induces spondyloarthropathy by acting on ROR-γt + CD3 + CD4 − CD8 − entheseal resident T cells

The spondyloarthropathies are a group of rheumatic diseases that are associated with inflammation at anatomically distal sites, particularly the tendon-bone attachments (entheses) and the aortic root. Serum concentrations of interleukin-23 (IL-23) are elevated and polymorphisms in the IL-23 receptor are associated with ankyosing spondylitis, however, it remains unclear whether IL-23 acts locally at the enthesis or distally on circulating cell populations. We show here that IL-23 is essential in enthesitis and acts on previously unidentified IL-23 receptor (IL-23R)+, RAR-related orphan receptor γt (ROR-γt)+CD3+CD4−CD8−, stem cell antigen 1 (Sca1)+ entheseal resident T cells. These cells allow entheses to respond to IL-23 in vitro—in the absence of further cellular recruitment—and to elaborate inflammatory mediators including IL-6, IL-17, IL-22 and chemokine (C-X-C motif) ligand 1 (CXCL1). Notably, the in vivo expression of IL-23 is sufficient to phenocopy the human disease, with the specific and characteristic development of enthesitis and entheseal new bone formation in the initial complete absence of synovitis. As in the human condition, inflammation also develops in vivo at the aortic root and valve, which are structurally similar to entheses. The presence of these entheseal resident cells and their production of IL-22, which activates signal transducer and activator of transcription 3 (STAT3)-dependent osteoblast-mediated bone remodeling, explains why dysregulation of IL-23 results in inflammation at this precise anatomical site.

IL-27 Blocks RORc Expression to Inhibit Lineage Commitment of Th17 Cells

IL-27 is secreted by APCs in response to inflammatory stimuli and exerts a proinflammatory Th1-enhancing activity but also has significant anti-inflammatory functions. We examined the molecular mechanism by which IL-27 regulates TGFβ plus IL-6- or IL-23-dependent Th17 development in the mouse and human systems. IL-27 inhibited the production of IL-17A and IL-17F in naive T cells by suppressing, in a STAT1-dependent manner, the expression of the Th17-specific transcription factor RORγt. The in vivo significance of the role of IL-27 was addressed in delayed-type hypersensitivity response and experimental autoimmune encephalomyelitis (EAE). By generating mice deficient for the p28 subunit of IL-27, we showed that IL-27 regulated the severity of delayed-type hypersensitivity response and EAE through its effects on Th17 cells. Furthermore, up-regulation of IL-10 in the CNS, which usually occurs late after EAE onset and plays a role in the resolution of the disease, was notably absent in IL-27p28−/− mice. These results show that IL-27 acts as a negative regulator of the developing IL-17A response in vivo, suggesting a potential therapeutic role for IL-27 in autoimmune diseases.

Anterior gradient-2 plays a critical role in breast cancer cell growth and survival by modulating cyclin D1, estrogen receptor-α and survivin

IntroductionAnterior-gradient 2 (AGR2) is an estrogen-responsive secreted protein. Its upregulation has been well documented in a number of cancers, particularly breast cancer, for which mixed data exist on the prognostic implications of AGR2 expression. Although emerging evidence indicates that AGR2 is associated with poor prognosis, its function and impact on cancer-relevant pathways have not been elucidated in breast cancer.MethodsTo investigate the biologic role of AGR2 in breast cancer, AGR2 was transiently knocked down, by using siRNA, in T47 D and ZR-75-1 (estrogen receptor-α (ER)-positive) and MDA-MB-231 and SK-BR-3 (ER-negative) human breast cancer cell lines. The impact of silencing AGR2 was evaluated in both anchorage-dependent and anchorage-independent growth (soft agar, spheroid) assays. Cell-cycle profiles in ER-positive cell lines were determined with BrdU incorporation, and cell death was measured with Annexin V, JC-1, and F7-26 staining. After transiently silencing AGR2 or stimulating with recombinant AGR2, modulation of key regulators of growth and survival pathways was assessed with Western blot. Combination studies of AGR2 knockdown with the antiestrogens tamoxifen and fulvestrant were carried out and assessed at the level of anchorage-dependent growth inhibition and target modulation (cyclin D1, ER).ResultsAGR2 knockdown inhibited growth in anchorage-dependent and anchorage-independent assays, with a more-pronounced effect in ER-positive cell lines. Cyclin D1 levels and BrdU incorporation were reduced with AGR2 knockdown. Conversely, cyclin D1 was induced with recombinant AGR2. AGR2 knockdown induced cell death in ZR-75-1 and T47 D cells, and also downregulated survivin and c-Myc. Evidence of AGR2-ER crosstalk was demonstrated by a reduction of ER at the protein level after transiently silencing AGR2. AGR2 knockdown in combination with fulvestrant or tamoxifen did not preclude the efficacy of the antiestrogens, but enhanced it. In addition, p-Src, implicated in tamoxifen resistance, was downregulated with AGR2 knockdown.ConclusionsTransiently silencing AGR2 in ER-positive breast cancer cell lines inhibited cell growth and cell-cycle progression and induced cell death. Breast cancer drivers (ER and cyclin D1) as well as cancer-signaling nodes (pSrc, c-Myc, and survivin) were demonstrated to be downstream of AGR2. Collectively, the data presented support the utility of anti-AGR2 therapy in ER-positive breast cancers because of its impact on cancer-relevant pathways.

Modulation of Paired Immunoglobulin-Like Type 2 Receptor Signaling Alters the Host Response to Staphylococcus aureus-Induced Pneumonia

ABSTRACT Paired immunoglobulin-like type 2 receptors (PILRs) inhibitory PILRα and activating PILRβ are predominantly expressed on myeloid cells. Their functions in host defense and inflammation are largely unknown, and in this study, we evaluated their roles in an acute Staphylococcus aureus pneumonia model. Compared to their respective controls, Pilrb−/− mice or mice in which PILRα was activated with an agonistic antibody showed improved clearance of pulmonary staphylococci and improved survival. These mice had reduced serum or bronchoalveolar lavage fluid levels of interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and IL-6 and elevated levels of gamma interferon (IFN-γ), IL-12, and IL-10. In contrast, mice in which PILRβ was activated had increased lung bacterial burdens and higher mortality coupled with an intense proinflammatory response with highly elevated levels of IL-1β, TNF-α, and IL-6. Treatment groups with reduced bacterial burdens had higher levels of Keratinocyte-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2), and MIP-1α in bronchoalveolar lavage fluid and an increased influx of neutrophils and macrophages to the lungs. Consistent with our in vivo findings, bone marrow-derived macrophages from Pilrb−/− mice released significantly less IL-1β and TNF-α and more IFN-γ and IL-12 than did the wild-type macrophages when directly stimulated with heat-killed S. aureus. To our knowledge, this is the first evidence that S. aureus directly interacts with PILRβ. It provides a mechanism by which manipulating the balance in favor of an inhibitory PILR signal, by activation of PILRα or deletion of PILRβ, helps to control acute S. aureus-mediated pneumonia and attenuate the inflammatory response. These results highlight the importance of PILRs in innate immunity and the control of inflammation.

Cancer-induced Expansion and Activation of CD11b(+)Gr-1(+) Cells Predispose Mice to Adenoviral-triggered Anaphylactoid-type Reactions

Intravascular delivery (1.5 x 10(9) particles and higher) of recombinant adenovirus (rAd) induces myeloid cell mediated, self-limiting hemodynamic responses in normal mice. However, we observed anaphylactoid-type reactions and exacerbated hemodynamic events following rAd injection in mice bearing malignant 4T1 mammary carcinoma. Because 4T1 tumors induce significant CD11b(+)Gr-1(+) myeloid cell expansion and activation, we set to determine whether this causes rAd-induced exaggerated responses. When treated with a single intravenous dose (1 x 10(10) particles) of rAd, mice implanted with 4T1 carcinoma succumbed due to the anaphylactoid-type reactions. In contrast, normal mice and mice implanted with a related mammary carcinoma (66cl4) that does not induce CD11b(+)Gr-1(+) cell expansion, showed minimal responses. Depletion of phagocytic CD11b(+)Gr-1(+) cells prior to rAd delivery protected 4T1 tumor-bearing animals, whereas passive transfer of CD11b(+)Gr-1(+) cells from 4T1 tumor-bearing animals was sufficient to convey susceptibility to anaphylactoid-type reactions in normal animals. We further show that there is upregulation of nitric oxide and leukotriene signaling pathways in the 4T1 tumor-induced CD11b(+)Gr-1(+) myeloid cells and that pretreating mice with inhibitors of nitric oxide synthetase and leukotrienes can attenuate the anaphylactoid-type reactions. These data show that malignant tumor growth can alter CD11b(+)Gr-1(+) myeloid cells, rendering hosts susceptible to anaphylactoid-type reactions upon intravascular treatment with rAd.Intravascular delivery (1.5 × 109 particles and higher) of recombinant adenovirus (rAd) induces myeloid cell mediated, self-limiting hemodynamic responses in normal mice. However, we observed anaphylactoid-type reactions and exacerbated hemodynamic events following rAd injection in mice bearing malignant 4T1 mammary carcinoma. Because 4T1 tumors induce significant CD11b+Gr-1+ myeloid cell expansion and activation, we set to determine whether this causes rAd-induced exaggerated responses. When treated with a single intravenous dose (1 × 1010 particles) of rAd, mice implanted with 4T1 carcinoma succumbed due to the anaphylactoid-type reactions. In contrast, normal mice and mice implanted with a related mammary carcinoma (66cl4) that does not induce CD11b+Gr-1+ cell expansion, showed minimal responses. Depletion of phagocytic CD11b+Gr-1+ cells prior to rAd delivery protected 4T1 tumor-bearing animals, whereas passive transfer of CD11b+Gr-1+ cells from 4T1 tumor-bearing animals was sufficient to convey susceptibility to anaphylactoid-type reactions in normal animals. We further show that there is upregulation of nitric oxide and leukotriene signaling pathways in the 4T1 tumor-induced CD11b+Gr-1+ myeloid cells and that pretreating mice with inhibitors of nitric oxide synthetase and leukotrienes can attenuate the anaphylactoid-type reactions. These data show that malignant tumor growth can alter CD11b+Gr-1+ myeloid cells, rendering hosts susceptible to anaphylactoid-type reactions upon intravascular treatment with rAd.

The Myeloid Receptor PILRβ Mediates the Balance of Inflammatory Responses through Regulation of IL-27 Production

Paired immunoglobulin-like receptors beta, PILRβ, and alpha, PILRα, are related to the Siglec family of receptors and are expressed primarily on cells of the myeloid lineage. PILRβ is a DAP12 binding partner expressed on both human and mouse myeloid cells. The potential ligand, CD99, is found on many cell types, such as epithelial cells where it plays a role in migration of immune cells to sites of inflammation. Pilrb deficient mice were challenged with the parasite Toxoplasma gondii in two different models of infection induced inflammation; one involving the establishment of chronic encephalitis and a second mimicking inflammatory bowel disease in order to understand the potential role of this receptor in persistent inflammatory responses. It was found that in the absence of activating signals from PILRβ, antigen-presenting cells (APCs) produced increased amounts of IL-27, p28 and promoted IL-10 production in effector T cells. The sustained production of IL-27 led ultimately to enhanced survival after challenge due to dampened immune pathology in the gut. Similar protection was also observed in the CNS during chronic T. gondii infection after i.p. challenge again providing evidence that PILRβ is important for regulating aberrant inflammatory responses.

PD-1/PD-L1 and immune-related gene expression pattern in pediatric malignant brain tumors: clinical correlation with survival data in Korean population

BackgroundPD-L1 expression has been evaluated as a predictive biomarker for immunotherapy in numerous tumor types. However, very limited data are available in pediatric brain tumors. The aim of this study was to characterize PD-1 and PD-L1 expressions of four pediatric malignant brain tumors and gene expression profile.MethodsThis study included 89 pediatric patients receiving standard treatment at Seoul National University Children’s Hospital and Seoul National University Bundang Hospital between 1990 and 2014: atypical teratoid/rhabdoid tumor (AT/RT) 20; ependymoma (EPN) 20; high grade glioma (HGG) 21; and medulloblastoma (MBL) 28. We performed immunohistochemistry assays for PD-1 and PD-L1. To characterize the gene expression, a custom immune-response focused gene panel was used.ResultsPD-1 expression was positive in 7 (35%) AT/RT, 7 (35%) EPN, 4 (19%) HGG, and 3 (11%) MBL patients. PD-L1 expression was positive in 8 (40%) AT/RT, 4 (20%) EPN, and 4 (19%) HGG; negative in all MBL patients. There was no statistically significant difference in the overall survival of PD-L1 positive patients. The gene expression analysis demonstrated differences in two clustering functional categories: cell–cell signaling and antigen presentation pathway.ConclusionsAT/RT, EPN, and HGG showed a relatively higher expression rate of PD-L1 (19–40%). This suggests these tumor types might be good candidates for PD-1 checkpoint blockade. We determined that gene expression may potentially serve as a molecular tool in predicting which patients will respond to immunotherapy. Further investigation is required to better understand the predictive and prognostic role of PD-L1 in pediatric brain tumors.

Establishment of engineered cell-based assays mediating LAG3 and PD1 immune suppression enables potency measurement of blocking antibodies and assessment of signal transduction

LAG3 is an important regulator of T cell homeostasis and studies in mouse tumor models have demonstrated that simultaneously antagonizing LAG3 and PD1 can augment tumor-specific T cell responses and induce tumor rejection. The combined use of LAG3 antagonist antibodies with established anti-PD1 therapies is currently being evaluated in human clinical trials. A functional assay for human LAG3 was developed by co-culture of a Jurkat T-cell lymphoma line overexpressing LAG3 with a Raji B-cell lymphoma line in the presence of staphylococcal enterotoxins. Reversal of LAG3 repression was measured as an increase in IL-2 production or NFAT activation in response to treatment with MK-4280, an anti-human LAG3 antagonist antibody. Changes in cytokines, chemokines, and other mRNA transcripts were in agreement with published in vitro and in vivo models for LAG3 biology which highlights the physiological relevance of the Jurkat functional assay. Additional engineering of PD1 and PDL1 components into the LAG3 assay resulted in a bi-functional assay that is capable of inducing a 10-fold response to individual antibodies blocking either PD1 or LAG3. Importantly, when MK-4280 and pembrolizumab were combined to block both pathways, a synergistic 50-fold increase in response was observed.

Preclinical to clinical translation of anti-PD-1 blockade

Meeting abstracts Pembrolizumab (MK-3475), a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1), is currently being studied in clinical trials across more than 30 types of cancers. Immunotherapy with anti–PD-1 monoclonal antibodies such as pembrolizumab shows robust,