NK Cell Infiltration Research Articles

Intravesical Mycobacterium brumae triggers both local and systemic immunotherapeutic responses against bladder cancer in mice

The standard treatment for high-risk non-muscle invasive bladder cancer (BC) is the intravesical administration of live Mycobacterium bovis BCG. Previous studies suggest improving this therapy by implementing non-pathogenic mycobacteria, such as Mycobacterium brumae, and/or different vehicles for mycobacteria delivery, such as an olive oil (OO)-in-water emulsion. While it has been established that BCG treatment activates the immune system, the immune effects of altering the mycobacterium and/or the preparation remain unknown. In an orthotopic murine BC model, local immune responses were assessed by measuring immune cells into the bladder and macromolecules in the urine by flow cytometry and multiplexing, respectively. Systemic immune responses were analyzed by quantifying sera anti-mycobacteria antibody levels and recall responses of ex vivo splenocytes cultured with mycobacteria antigens. In both BCG- and M. brumae-treated mice, T, NK, and NKT cell infiltration in the bladder was significantly increased. Notably, T cell infiltration was enhanced in OO-in-water emulsified mycobacteria-treated mice, and urine IL-6 and KC concentrations were elevated. Furthermore, mycobacteria treatment augmented IgG antibody production and splenocyte proliferation, especially in mice receiving OO-in-water emulsified mycobacteria. Our data demonstrate that intravesical mycobacterial treatment triggers local and systemic immune responses, which are most significant when OO-in-water emulsified mycobacteria are used.

Immunomodulatory role of histamine H4 receptor in breast cancer

BackgroundAlthough the role of histamine H4 receptor (H4R) in immune cells is being extensively investigated, its immunomodulatory function in cancer is completely unknown. This study aimed to investigate the role of H4R in antitumour immunity in a model of triple-negative breast cancer.MethodsWe evaluated growth parameters, histological characteristics and the composition of tumour, splenic and tumour draining lymph node (TDLN) immune subsets, in a syngeneic model, developed orthotopically with 4T1 cells in H4R knockout (H4R-KO) and wild-type mice.ResultsMice lacking H4R show reduced tumour size and weight, decreased number of lung metastases and percentage of CD4+ tumour-infiltrating T cells, while exhibiting increased infiltration of NK cells and CD19+ lymphocytes. Likewise, TDLN of H4R-KO mice show decreased CD4+ T cells and T regulatory cells (CD4+CD25+FoxP3+), and increased percentages of NK cells. Finally, H4R-deficient mice show decreased Tregs in spleens and non-draining lymph nodes, and a negative correlation between tumour weight and the percentages of CD4+, CD19+ and NK splenic cells, suggesting that H4R also regulates antitumour immunity at a systemic level.ConclusionsThis is the first report that demonstrates the participation of H4R in antitumour immunity, suggesting that H4R could be a target for cancer treatment.

Abstract 4546: The tumor inflammation signature is predictive of anti-PD1 treatment benefit in the CERTIM pan-cancer cohort

Abstract Purpose of the study : The 18-gene Tumor Inflammation Signature (TIS) (Ayers et al, J Clin Invest 2017; 127:2930) is a clinical research assay that enriches for clinical response to immune checkpoint blockade. In this work, the performance of the TIS was evaluated on standard tumor specimens for its ability to predict clinical benefit of immunotherapy in a cohort of cancer patients treated with PD1 checkpoint inhibitors in routine clinical care. Experimental procedures : Consecutive metastatic cancer patients treated with anti-PD1 in the outpatient monocentric CERTIM cohort with available formalin fixed paraffin embedded tumor specimen were selected for analysis. RNA were extracted from tissue samples with High Pure FFPET RNA Isolation Kit (Roche) and 25-100 ng RNA was hybridized to a pilot version of the NanoString® PanCancer Immuno-oncology 360 Panel. The association between individual gene expression (GE), TIS and other GE signatures with outcome (response to therapy and overall survival) was assessed with logistic resgression model, LogRank tests and Cox model. Results : A total of 69 patients with non-small cell lung carcinoma (NSCLC, n=42), small cell lung carcinoma (n=2), melanoma (n=9), renal cell carcinoma (n=10), urothelial carcinoma (n=5) or colorectal carcinoma (n=1), who had received nivolumab (n=59) or pembrolizumab (n=10) were analyzed. All samples but one SCLC could be successfully analyzed, demonstrating the feasibility of the testing even in small biopsy samples with little available RNA (>30 ng). In the whole cohort, clinical response (PR or CR versus SD or progression) was significantly associated with TIS score [OR=2.002, 95%CI=(1.123,3.977), p =0.029] as well as with the individual expression of PDL1, CTLA4, IDO1 or GE scores characterizing Th1, cytotoxic, NK cells infiltration, immuno-proteasome or antigen processing machinery. Receiver operating characteristics for response status demonstrated the high discriminatory ability of the TIS signature in this unselected series of patients (area under the ROC curve 0.71). Patients with a high TIS score (> 9.39) showed a longer OS (median OS 20.2 months) compared to those with lower scores (median OS 5.6 months, p value = 0.0017). A high TIS score was also significantly associated with clinical response when the analysis was limited to the NSCLC (n=42, [OR=3.156, 95%CI=(1.168,11.455), p-value=0.042] - or showed a strong trend in lung adenocarcinoma (n=29 [OR=11.251, 95%CI=(0.962,131.534), p-value=0.054] patients. Conclusion : Due to costs and toxicity, it is mandatory to identify the patients who benefit of immune checkpoint inhibitors. Here we demonstrate that the Tumor Inflammation Signature, that reflects key processes involved in anti-tumor immune response, may be used in routine samples to predict clinical benefit and prolonged survival of cancer patients. Citation Format: diane damotte, jennifer arrondeau, pascaline boudou-rouquette, audrey lupo, jerome biton, hanane ouakrim, Marco Alifano, Gervais Claire, Audrey Bellesoeur, Nora Kramkimel, Angeline Duche, Franck Letourneur, Xing Ren, Pascale Morel, Alessandra Cesano, Sarah Warren, François Goldwasser, Karen Leroy. The tumor inflammation signature is predictive of anti-PD1 treatment benefit in the CERTIM pan-cancer cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4546.

Abstract 5549: Antitumor efficacy of the oncolytic peptide LTX-315 in genetic mouse models that resist conventional chemo- and immunotherapeutic treatments

Abstract LTX-315 is an oncolytic peptide that induces immunogenic cancer cell death and is currently evaluated in phase 1 clinical trials for its ability to trigger recruitment of tumor-specific T cells to tumors. Previous experimental studies identified LTX-315's ability to control cancer in mice grafted with various tumor cell lines. Here we extend our analyses to conditional genetic mouse models of both melanoma (driven by Braf and Pten alterations) and soft tissue sarcoma (driven by Kras and P53 alterations). In these genetic models, cancer cells are derived from somatic cells that are transformed in their normal tissue microenvironment and progress to high-grade tumors that are poorly infiltrated by T cells and typically resist prescribed chemo- and immunotherapeutic treatments. We report that LTX-315 not only delays tumor progression substantially in both models, but also profoundly alters the tumor microenvironment, most notably characterized with CD8+ T cell, NK cell and dendritic cell infiltration. Transition from a ‘cold' to a ‘hot' tumor microenvironment was also observed in patients with melanoma, sarcoma and breast cancer. Furthermore, CD8+ T cell depletion in mice abrogated long-term antitumor efficacy of LTX-315, indicating that these cells are required for drug-induced tumor control. Importantly, the ability to convert non-T-cell-infiltrated tumors into ones that display antitumor T cell immunity opens the possibility to prime and make unresponsive tumors sensitive for systemic immune treatments. Citation Format: Mikael J. Pittet, Hsin-Wei Liao, Baldur Sveinbjørnsson, Øystein Rekdal. Antitumor efficacy of the oncolytic peptide LTX-315 in genetic mouse models that resist conventional chemo- and immunotherapeutic treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5549.

Abstract 121: Angiogenin and the mmp9-timp2 axis are strongly upregulated in pro-angigoenic dnk-like cells isolated from colorectal cancer patients

Abstract Natural Killer (NK) cells are effector lymphocytes involved in tumor immunosurveillance. In solid malignancies, tumor associated (TANK; peripheral blood) and tumor infiltrating (TINK) NK cells have compromised functions. We functionally and molecularly characterize TINK and TANKs from blood and tissue samples of colorectal cancer (CRC) patients, a tumor type where inflammation and angiogenesis have clinical relevance, compared to NK isolated from control and non-oncologic inflammatory bowel disease patients. NK subset distribution and cytokine profiling were performed by multicolor flow cytometry, using peripheral blood and tissue samples from CRC patients, for surface antigen and cytokine profiling characterization. Conditioned media (CM) from FACS-sorted NKs were used either for secretomic profiling using antibody membrane arrays or in functional in vitro angiogenesis assays. CRC TINKs/TANKs showed decreased activation marker NKG2D, impaired degranulation activity, a decidual-like NK polarization toward the CD56brightCD16dim/- CD9+CD49+ subset. TINKs and TANKs supernatants induced endothelial cell proliferation, migration, adhesion and formation of capillary-like structures in vitro. It has been reported that dNK release proangiogenic factors and metastasis-associated (MMP-9, TIMP1-2) proteins. We describe for the first time the expression of angiogenin and MMP2, MMP9, TIMP1 and TIMP2 by CRC derived NK cells. This could be a phenotype relevant to their invasive capabilities and pro-angiogenic function. STAT-3/STAT-5 activation was observed in TANKs, and inhibition of the STAT5 pathway by pimozide, an antipsychotic drug, reduced proangiogenic factor VEGF and angiogenin production and capillary-like structure formation, but did not affect the levels of TIMP-1, TIMP-2 and MMP-9. Citation Format: Antonino Bruno, Giuseppe Pelosi, Luigi Boni, Lorenzo Dominioni, Lorenzo Mortara, Douglas Noonan, Adriana Albini. Angiogenin and the mmp9-timp2 axis are strongly upregulated in pro-angigoenic dnk-like cells isolated from colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 121.

Abstract 1698: Loss of MHC-I expression is associated with altered immune microenvironment and decreased response to PD-1 checkpoint inhibition

Abstract Cancer immunotherapies, such as PD-1 inhibitors, achieve durable responses in a subset of cancer patients, however, a significant proportion of patients fail to respond to treatment. Defects in antigen presentation, such as the loss of MHC-I expression, are a major mechanism of resistance to PD-1 inhibitors. Developing new therapeutic strategies that overcome the lack of antigen presentation will improve cancer treatment and patient survival. To develop a pre-clinical model to evaluate therapies that could overcome resistance, we have generated a unique PD-1 blockade sensitive, syngeneic immunocompetent melanoma mouse model. We generated defects in antigen presentation using the CRISPR/Cas9 system, creating B2m-deficient melanoma cell lines. We demonstrated that B2m is necessary for IFN-gamma induced MHC-I surface expression. Unlike control tumors, we found that B2m-deficient tumors were resistant to PD-1 blockade, leading to decreased mouse overall survival. Using multi-parameter flow cytometry, we showed an increase in NK cell, regulatory T cell and dendritic cell infiltration in B2m-deficient tumors compared to controls, while the other immune cell population were unchanged. Since NK cell activity is negatively regulated by MHC-I, our findings suggest that additional factors are required to further invigorate infiltrating NK cells and to trigger a NK cell-mediated anti-tumor response. We are currently evaluating new approaches targeting NK cells and regulatory T cells on B2m-deficient tumors. This pre-clinical model could enable the development of a therapeutic strategy serving to improve the treatment of tumors lacking antigen presentation. Citation Format: Cecile Gstalder, Priya Pancholi, Dorota Sadowicz, Rizwan Haq. Loss of MHC-I expression is associated with altered immune microenvironment and decreased response to PD-1 checkpoint inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1698.

Immunoprotective responses against murine sarcocystosis by β - Irradiated sporocysts

This study aimed to induce protective immunity against infection with Sarcocystis muris in experimental mice using β-irradiated sporocysts. Mice were vaccinated with 50 sporocysts of S. muris which were exposed to 1.84 μSv β-irradiation for 2, 4 and 8 h. After challenge infection, different samples were taken for evaluation. Serum and intestinal wash were assayed for IFN-γ and IgA, respectively. Mesenteric lymph nodes (MLNs) and spleen were investigated for CD4+ and CD8+ T cells using immunohistochemistry. For liver, the morphological changes in parasitic stages and the count of infiltrated CD8+ T, NK1.1+ and FasL+ cells were also investigated. Real time (RT) - PCR was used for detection of liver MHC I, CD1d, IFN-γ, perforin and FasL as well as the parasite 18S ribosomal(r) RNA in liver and muscle tissues. Alterations of liver parasitic stages as well as a decrease in the infection with the parasite in both of liver and muscle tissues were dependent on radiation exposure time. An investigation for the mechanism of immunoprotection showed an increase in liver NK1.1+ & FasL+ cells, serum IFN-γ and intestinal IgA, while CD4+ and CD8+ T showed a remarkable increase in MLNs and spleen. FasL expression increased in the liver dependently on radiation exposure time, while perforin, MHC I and CD1d were not. β-irradiated sporocysts with 1.84 μSv for 8 h s could induce the highest protection against infection with Sarcocystis. This could be largely relied on the increased infiltration of NK cells and associated higher expression of FasL in the liver.

α-Galactosylceramide and peptide-based nano-vaccine synergistically induced a strong tumor suppressive effect in melanoma

α-Galactosylceramide (GalCer) is a glycolipid widely known as an activator of Natural killer T (NKT) cells, constituting a promising adjuvant against cancer, including melanoma. However, limited clinical outcomes have been obtained so far. This study evaluated the synergy between GalCer and major histocompatibility complex (MHC) class I and MHC class II melanoma-associated peptide antigens and the Toll-Like Receptor (TLR) ligands CpG and monophosphoryl lipid A (MPLA), which we intended to maximize following their co-delivery by a nanoparticle (NP). This is expected to improve GalCer capture by dendritic cells (DCs) and subsequent presentation to NKT cells, simultaneously inducing an anti-tumor specific T-cell mediated immunity.The combination of GalCer with melanoma peptides and TLR ligands successfully restrained tumor growth. The tumor volume in these animals was 5-fold lower than the ones presented by mice immunized with NPs not containing GalCer. However, tumor growth was controlled at similar levels by GalCer entrapped or in its soluble form, when mixed with antigens and TLR ligands. Those two groups showed an improved infiltration of T lymphocytes into the tumor, but only GalCer-loaded nano-vaccine induced a prominent and enhanced infiltration of NKT and NK cells. In addition, splenocytes of these animals secreted levels of IFN-γ and IL-4 at least 1.5-fold and 2-fold higher, respectively, than those treated with the mixture of antigens and adjuvants in solution. Overall, the combined delivery of the NKT agonist with TLR ligands and melanoma antigens via this multivalent nano-vaccine displayed a synergistic anti-tumor immune-mediated efficacy in B16F10 melanoma mouse model. Statement of SignificanceCombination of α-galactosylceramide (GalCer), a Natural Killer T (NKT) cell agonist, with melanoma-associated antigens presented by MHC class I (Melan-A:26) and MHC class II (gp100:44) molecules, and Toll-like Receptor (TLR) ligands (MPLA and CpG), within nanoparticle matrix induced a prominent anti-tumor immune response able to restrict melanoma growth. An enhanced infiltration of NKT and NK cells into tumor site was only achieved when the combination GalCer, antigens and TLR ligands were co-delivered by the nanovaccine.

Anti-PD-1 therapy redirects macrophages from an M2 to an M1 phenotype inducing regression of OS lung metastases.

Osteosarcoma (OS) pulmonary metastasis translates into poor patient survival. The implication of PD‐1‐PD‐L1 pathway in the context of NK cells and/or macrophages in OS is unknown. We investigated the effect of anti‐PD‐1 in OS lung metastasis and the role of NK cells and/or macrophages in anti‐PD‐1 responses. A human LM7 OS mouse model was used. Immunohistochemistry for tissues (PD‐L1, caspase‐3, Ki‐67, NK cells, macrophages), and Western blotting for OS lung tumors (p‐Stat3, p‐Erk1/2) was performed. NK and macrophages were assessed using flow cytometry. NK cell and macrophage depletion were conducted using anti‐asialo GM1 and clodrosome, respectively. PD‐L1 expression was observed in human OS cells and OS patient lung metastases. Anti‐PD1 antibody led to a significant decrease in the number of OS lung metastases, enhanced tumor apoptosis, decreased tumor cell proliferation, and p‐STAT‐3/p‐Erk1/2 signaling blockade in OS lung tumors. NK cells and macrophages in OS lung tumors expressed PD‐1 and anti‐PD1 increased NK cell and macrophage tumor infiltration. Increased numbers of antitumor M1 macrophages and decreased pro‐inflammatory M2 macrophages were seen. NK depletion did not affect therapeutic effect of anti‐PD‐1, suggesting that NK cells were not directly involved. However, macrophage depletion significantly compromised anti‐PD1 efficacy, confirming their role in efficacy of anti‐PD‐1 against OS lung metastasis. Our findings suggest that OS lung metastases regression by anti‐PD1 can be attributed to activated tumor M1 macrophages and reduced M2 macrophages. Owing to the co‐relation of M1 macrophages with OS patient outcome, we provide a novel mechanism of PD‐1 blockade and a basis for future clinical trials for anti‐PD‐1 antibodies in OS.

Gene expression profiling and immune cell-type deconvolution highlight robust disease progression and survival markers in multiple cohorts of CTCL patients

ABSTRACTCTCL follows different courses depending on the clinical stage at the time of diagnosis. Patients with early stage Mycosis Fungoides (MF) variant of CTCL may experience an indolent course over decades, whereas patients with advanced MF and Sézary Syndrome (SS) disease at diagnosis, often succumb within 5 years. Even within early stage CTCL/MF, a minority of patients will progress to more advanced stages. We recently generated RNA sequencing data on 284 CTCL-relevant genes for 157 patients and identified differentially expressed genes across stages I-IV. In this study, we aim to validate robust molecular markers linked to disease progression and survival. We performed multiple hypothesis testing-corrected analysis of variance (ANOVA) on the expression of individual genes across all CTCL samples and early stage (≤IIA) CTCL/MF patients. We used in silico immune cell-type deconvolution from gene expression data to estimate immune cell populations. Based on the analysis of all CTCL samples, we identified TOX, FYB, and CD52 as predictors of disease progression and poor survival. Among early stage (≤IIA) CTCL/MF patients, these 3 genes, along with CCR4, were valuable to predict disease progression. We validated these 4 genes in 3 independent, external Sézary Syndrome patient cohorts with RNA-Sequencing data. In silico immune cell-type deconvolution revealed that neutrophil infiltration in early stage MF conveyed a higher risk for disease progression. Also, NK cell infiltration in late stage MF/SS correlated with improved survival. TOX, FYB, CCR4 and CD52 are robust disease progression and decreased survival biomarkers in CTCL.

Donor-specific antibody mediates chronic, but not acute, kidney allograft rejection in the absence of natural killer cells

Abstract Dysregulated donor-specific antibody (DSA) responses are induced in B6.CCR5−/− mice transplanted with complete MHC mismatched kidney allografts and NK cells play a critical role in acute graft injury and failure. The current study investigated the consequence of high DSA titers on graft outcomes in the presence vs. absence of NK cell activation within the kidney graft. B6.CCR5−/− recipients of complete MHC mismatched A/J allografts or semi allogeneic (A/J x B6)F1 kidney grafts responded with equivalent serum DSA titers that reached peak by day 14 post-transplant. B6.CCR5−/− recipients rejected A/J allografts between days 15–30, whereas B6 isografts and (A/J x B6)F1 semi allogeneic grafts survived past day 65. On day 7 post-transplant NK cell infiltration into A/J allografts was composed of distinct Ly49b+ cell populations expressing low and high levels of NK1.1 and both NK1.1high and NK1.1low cells increased in A/J allografts with time to rejection. In contrast, NK cell infiltration into (A/J x B6)F1 grafts on day 7 post-transplant was composed entirely of NK1.1high cells that decreased to background/isograft numbers thereafter. (A/J x B6)F1 grafts at day 65 post-transplant had severe chronic injury with prominent interstitial fibrosis, glomerulopathy and arteriopathy that was accompanied by gene expression of profibrogenic factors. These results indicate that NK cells synergize with DSA to cause acute kidney allograft failure whereas high titers of DSA in the absence of NK cell activation cannot cause acute antibody-mediated rejection but induce the indolent development of interstitial fibrosis and glomerular injury that leads to graft dysfunction and failure at later times after transplantation.

Dual TLR2/9 Recognition of Herpes Simplex Virus Infection Is Required for Recruitment and Activation of Monocytes and NK Cells and Restriction of Viral Dissemination to the Central Nervous System.

The importance of TLR2 and TLR9 in the recognition of infection with herpes simplex virus (HSV) and HSV-caused diseases has been described, but some discrepancies remain concerning the benefits of these responses. Moreover, the impact of TLR2/9 on innate and adaptive immune responses within relevant mucosal tissues has not been elucidated using natural mucosal infection model of HSV. Here, we demonstrate that dual TLR2/9 recognition is essential to provide resistance against mucosal infection with HSV via an intravaginal route. Dual TLR2/9 ablation resulted in the highly enhanced mortality with exacerbated symptoms of encephalitis compared with TLR2 or TLR9 deficiency alone, coinciding with highly increased viral load in central nervous system tissues. TLR2 appeared to play a minor role in providing resistance against mucosal infection with HSV, since TLR2-ablated mice showed higher survival rate compared with TLR9-ablated mice. Also, the high mortality in dual TLR2/9-ablated mice was closely associated with the reduction in early monocyte and NK cell infiltration in the vaginal tract (VT), which was likely to correlate with low expression of cytokines and CCR2 ligands (CCL2 and CCL7). More interestingly, our data revealed that dual TLR2/9 recognition of HSV infection plays an important role in the functional maturation of TNF-α and iNOS-producing dendritic cells (Tip-DCs) from monocytes as well as NK cell activation in VT. TLR2/9-dependent maturation of Tip-DCs from monocytes appeared to specifically present cognate Ag, which effectively provided functional effector CD4+ and CD8+ T cells specific for HSV Ag in VT and its draining lymph nodes. TLR2/9 expressed in monocytes was likely to directly facilitate Tip-DC-like features after HSV infection. Also, dual TLR2/9 recognition of HSV infection directly activated NK cells without the aid of dendritic cells through activation of p38 MAPK pathway. Taken together, these results indicate that dual TLR2/9 recognition plays a critical role in providing resistance against mucosal infection with HSV, which may involve a direct regulation of Tip-DCs and NK cells in VT. Therefore, our data provide a more detailed understanding of TLR2/9 role in conferring antiviral immunity within relevant mucosal tissues after mucosal infection with HSV.

Dose intensification of TRAIL-inducing ONC201 inhibits metastasis and promotes intratumoral NK cell recruitment.

ONC201 is a first-in-class, orally active antitumor agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. ONC201 has demonstrated safety and preliminary efficacy in a first-in-human trial in which patients were dosed every 3 weeks. We hypothesized that dose intensification of ONC201 may impact antitumor efficacy. We discovered that ONC201 exerts dose- and schedule-dependent effects on tumor progression and cell death signaling in vivo. With dose intensification, we note a potent anti-metastasis effect and inhibition of cancer cell migration and invasion. Our preclinical results prompted a change in ONC201 dosing in all open clinical trials. We observed accumulation of activated NK+ and CD3+ cells within ONC201-treated tumors and that NK cell depletion inhibits ONC201 efficacy in vivo, including against TRAIL/ONC201-resistant Bax-/- tumors. Immunocompetent NCR1-GFP mice, in which NK cells express GFP, demonstrated GFP+ NK cell infiltration of syngeneic MC38 colorectal tumors. Activation of primary human NK cells and increased degranulation occurred in response to ONC201. Coculture experiments identified a role for TRAIL in human NK-mediated antitumor cytotoxicity. Preclinical results indicate the potential utility for ONC201 plus anti-PD-1 therapy. We observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients after ONC201 treatment. The results offer what we believe to be a unique pathway of immune stimulation for cancer therapy.

Driving Natural Killer cells toward the melanoma tumor battlefield: Autophagy as a valuable therapeutic target

ABSTRACTNatural Killer (NK) cell-based cancer immunotherapies were often disappointing in the clinic mostly due to insufficient NK cell infiltration into tumors. We found that targeting autophagy induced a massive infiltration of NK cells into melanoma tumors. These findings highlight autophagy inhibition as a cutting-edge approach to fully exploit the anti-tumor properties of NK cells in a wide variety of cancer patients.

Multiple genetically modified GTKO/hCD46/HLA-E/hβ2-mg porcine hearts are protected from complement activation and natural killer cell infiltration during ex vivo perfusion with human blood.

In pig-to-human xenotransplantation, early cellular rejection reactions are mediated by natural killer cells (NK cells). Human NK cells are inhibited by HLA-E via CD94/NKG2A receptors. To protect porcine grafts against human NK cell responses, transgenic GTKO pigs expressing hCD46 and HLA-E have been generated. The aim of this study was to test the effect of this genetic modification on xenogeneic, and in particular human NK cell response, using an ex vivo perfusion model of pig hearts with human blood. Cardiopleged and explanted genetically modified (gm) pig hearts (GTKO/hCD46/HLA-E/hβ2-microglobulin) and wild-type (wt) controls (n=6 each) were reperfused and tested in an 8hours ex vivo perfusion system using freshly drawn human blood. Cardiac function was evaluated during a 165-minute period in working heart mode. Myocardial damage, antibody deposition, complement activation, and coagulation parameters were evaluated histologically at the end of perfusion. The number of NK cells in the perfusate was determined by flow cytometry at baseline and at 8hours; tissue infiltration by NK cells was quantified by immunofluorescence microscopy using NKp46 staining of frozen sections. Deposition of IgG (1.2±1×107 vs 8.8±2.9×106 ; P<.01), IgM (4.4±3.7×106 vs 1.7±1.2×106 ; P<.01), and the complement activation product C4b/c (3.5±1.3×106 vs 2.3×106 ±9.4×105 ; P>.01) was lower in gm than wt hearts. NK cell percentages of leukocytes in the perfusate decreased from 0.94±0.77% to 0.21±0.25% (P=.04) during xenoperfusion of wt hearts. In contrast, the ratio of NK cells did not decrease significantly in the gm hearts. In this group, NK cell myocardial infiltration after 480minutes of perfusion was lower than in wt organs (2.5±3.7×104 /mm3 vs 1.3±1.4×105 /mm3 ; P=.0001). The function of gm hearts was better preserved compared to wt organs, as demonstrated by higher cardiac index during the first 2hours of ex vivo perfusion. GTKO, hCD46, and HLA-E expression in porcine hearts reduced complement deposition, complement dependent injury, and myocardial NK cell infiltration during perfusion with human blood. This tested combination of genetic modifications may minimize damage from acute human-anti-pig rejection reactions and improve myocardial function after xenotransplantation.

The immunomodulatory activity of bemcentinib (BGB324): A first-in-class selective oral AXL inhibitor in patients with relapsed/refractory acute myeloid leukemia or myelodysplastic syndrome.

70 Background: The tyrosine kinase AXL acts as suppressive immune checkpoint of the innate immune system. AXL signalling leads to immunosuppression and tumour immune escape by downregulating dendritic cell activity, modulating efferocytosis as well as favouring an immunosuppressive chemokine profile and M-MDSC expansion. Therefore it represents an interesting novel immune oncology target. Bemcentinib (BGB324) is a first-in-class, highly selective and orally bioavailable small molecule AXL inhibitor in phase II clinical development. In pre-clinical models, inhibition of AXL signalling with bemcentinib reversed multiple established immune suppressive mechanisms leading to increased infiltration of CTLs, NK and NKT cells and decreased infiltration of M-MDSCs (Wnuk-Lipinska, 2017). We explored safety, PK, efficacy and the effects of treatment with bemcentinib on the T- and B cell repertoire in patients with relapsed AML or MDS. Methods: BGBC003 (NCT02488408) is an open-label, dose finding study of bemcentinib in patients with R/R AML or MDS. Three dose levels have been explored and a loading dose of 400 mg on days one to three followed by 200 mg daily thereafter has been established as safe and recommended phase 2 dose. The TCRß repertoire was quantified by NGS of DNA isolated from PBMNCs using an Illumina MiSeq sequencer. TCRß genes and the IGH repertoire were analysed with BIOMED2-TCRß-A and –B and BIOMED2-FR1/-FR3 primer pools, respectively. Using genomic DNA as template, the amplicons were tagged with Illumina adapters and indices in two consecutive PCR reactions. Demultiplexing and FastQ formated data output was generated by the MiSeq reporter. Analysis of TCRß and IGH data was performed on a Microsoft Cloud using our in-house analysis pipeline Pippa. Results: Early evidence of antileukemic activity was seen. Diversification of the TCR and IGH repertoire could be detected in peripheral blood at day 21 of therapy compared to pre-treatment in three and two out of five matched samples, respectively. Conclusions: Bemcentinib shows preliminary evidence of immune modulation in AML. Clinical trial information: NCT02488408.

Lymphoid environment in molecular subtypes ofbreast cancer.

Recently, alarge body ofevidence has shown that themicroenvironment ofinvasive breast carcinoma affects its development and thepatient's outcome, and vice versa - cancer cells express factors that modulate tumour milieu in terms ofits composition and function. We performed animmunohistochemical (IHC) staining of108 formalin-fixed, paraffin-embedded (FFPE) tissue samples to investigate therelationships between T-cell, B-cell, and NK-cell infiltrate, invasive breast carcinomas molecular subtypes, and other prognostic indicators. Themain findings ofour study were as follows: thesignificantly higher infiltrate oftheanalysed immune cell subsets in triple-negative (TNBC), HER2-positive, non-luminal and luminal B/HER2+ breast carcinomas than in luminal Acancers; their higher densities in poorly differentiated lesions; correlations between lymphoid cells and theexpression ofhormonal receptors, HER2 receptor status, and marker ofcancer proliferation. Furthermore, we observed T-cell numbers to be associated with greater tumour diameter. In summary, theresults ofour study indicate associations between tumoural lymphoid infiltration and theunfavourable intrinsic subtypes as well as other detrimental prognostic factors in invasive breast carcinomas.

Intrahepatic recruitment of cytotoxic NK cells contributes to autoimmune hepatitis progression

Autoimmune hepatitis (AIH) is a chronic autoimmune disease with the primary focus on autoreactive Th1-mediated response and cytotoxic T cells reaction. The contribution of natural killer cells (NK cells) to AIH remains poorly understood. In this project, we revealed that the frequency of peripheral NK cells, more accurately CD56dimNK subset, was reduced in AIH patients. The reduction of CD56dimNK was negatively correlated with disease progression. In experimental autoimmune hepatitis (EAH), hepatic accumulation of NK cells is observed along with a decrease of NK cells in the periphery. In addition, infiltrated NK cells are almost conventional CXCR3− NK cells, the counterpart of CD56dimNK cells in the human. Furthermore, conventional CXCR3-NK cells of infiltration and liver resident NK cells are activated progressively at active phase. Therefore, recruitment of cytotoxic NK cells into the liver, but not liver resident NK cells expansion, may partly account for AIH progression.

Experimental study on the effect of Yiqi Wenyang Decoction on nasal mucosa infiltration of NK cells in mice with allergic rhinitis

Objective: To observe the effect of Yiqi Wenyang Decoction on the infiltration and activation of NK cells in nasal mucosa of mouse model with allergic rhinitis (AR), and to explore the potential mechanism for effective intervention of AR with Yiqi Wenyang Decoction. Methods: Fourty-eight mice were randomly divided into blank group, model group, low, medium and high dose of Yiqi Wenyang Decoction group and Cetirizine group, with 8 rats in each group. After modeling of AR, the model group was filled with 0.9% sodium chloride solution. Yiqi Wenyang Decoction groups of each dose were given different concentrations of Yiqi Wenyang Decoction water extract, while the Cetirizine group was given aqueous solution of Cetirizine. The behavior, morphological changes of nasal mucosa and infiltration of NK cells in nasal mucosa were observed. The levels of IL-4 and INF-γ in nasal lavage fluid were measured. Besides, the drug safety was observed by acute toxicity test. Results: In the respect of behavioral scoring, middle and high dose of Yiqi Wenyang Decoction group were superior to the model group (number of sneezing: q value was 7.189, 8.748, respectively; number of scratching nose: q value was 12.074, 14.560, respectively; all P<0.05). In middle and high dose of Yiqi Wenyang Decoction group, the infiltration of NK cells and nasal lavage fluid IL-4 levels were lower than those in model group (IOD: q value was 10.073, 12.322, respectively; IOD/Area: q value was 10.954, 14.073, respectively; IL-4: q value was 4.705, 6.801, respectively; all P<0.05). There was no significant difference in nasal lavage fluid of INF-γ among each group (Fv=1.166, P>0.05). In acute toxicity test, no obvious poisoning symptoms and death occurred in mice. Conclusion: Yiqi Wenyang Decoction can control the nasal symptom, reduce the local NK cell infiltration of nasal mucosa and inhibit the expression of the 2-type cytokines released by NK cells, which may be related with the potential mechanism of effective intervention of AR with Yiqi Wenyang Decoction.

Two immune-enhanced molecular subtypes differ in inflammation, checkpoint signaling and outcome of advanced head and neck squamous cell carcinoma

ABSTRACT The immune environment of primary tumor is associated with the clinical response and benefit of immunotherapy. This study aims to investigate the intratumoral immune profile and its clinical relevance in advanced head and neck squamous cell carcinoma (HNSCC). Gene expression profiles of 401 HNSCCs at stage III-IVB from two cohorts (The Cancer Genome Atlas, TCGA, n = 203; the Leipzig Head and Neck Group, LHNG, n = 198) were involved in this analysis. Based on the global immune-related genes, four gene expression subtypes (C1-4) were identified in HNSCCs. Overall, subtypes C2 and C3 showed upregulation of immune profiles and increased tumor lymphocyte infiltration, exhibiting an enhanced immune microenvironment (EIME). However, the two EIME subtypes revealed differences in immune markers and clinical features. Subtype C2 showed higher expression of macrophage signature, whereas subtype C3 was more associated with B cell infiltration. T cell and NK cell infiltration was not different between C2 and C3 subtypes. The subtype C2 tumors were characterized by inflammation compared with subtype C3. Although the checkpoint receptors PD1 and CTLA4 expressed equally between the EIME subtypes, their ligands (PD-L1/PD-L2, CD86/CD80) were significantly upregulated in subtype C2 compared with C3. HPV-positive tumors were predominantly enriched in subtype C3 but not in C2. Furthermore, patients in subtype C2 had a worse outcome than those in C3. In summary, two immune-enhanced subtypes with different immune characteristics and clinical features were identified in advanced HNSCC. The different immune microenvironments among HNSCC subgroups may provide new insights into the strategy of immunotherapy.