Increased CD69 Expression Research Articles

No Improvement in Survival for T-PLL Patients over the Last Two Decades

BACKGROUND: T-cell prolymphocytic leukemia (T-PLL) is a rare, aggressive, post thymic lymphoid malignancy, with an incidence of approximately 0.1/100,000 people. T-PLL accounts for ~2% of all mature lymphocytic leukaemias in adults >30 years. Median overall survival (OS) ~20 months and long term remissions are infrequent. Intravenous alemtuzumab, a monoclonal antibody directed against CD52 remains the most effective treatment in T-PLL. At the Royal Marsden Hospital (RMH) we have been using alemtuzumab for T-PLL since the early 1990s. We describe our experience over the last 3 decades. METHODS: We included 174 T-PLL patients that were diagnosed or treated at RMH between 1989-2019. Immunophenotyping data was available through our hematological malignancy diagnostic service (HMDS) from 1998 onwards. Kaplan-Meier analysis was used for OS and disease free interval (DFI) RESULTS: There were 174 patients in total. Mean age at diagnosis was 61 years old (range 32-88) and M: F ratio was ~2:1 (113:61). 90 patients had reliable information on complete blood count (CBC) at diagnosis with median white blood cell count of 74 x 109/L (range 10-918), median hemoglobin 126 g/L (range 59-175) and median platelet count 116 x 109/L (range 7-513). Sufficient immunophenotyping data was available for analysis in 135/174 patients. The results showed predictably high expression of CD2, CD3, CD5, CD7, CD52 and TCRαβ. CD25 was positive in 67/135 (50%) of cases which is higher than the 18-35% seen in the current literature. CD4+/CD8- cases comprised 83/135 (61%) with CD4-/CD8+ 19/135 (14%), CD4+/CD8+ 32/135 (23%) and CD4-/CD8- 2/135 (2%). These results are summarized in table 1. Karyotyping/FISH (fluorescent in-situ hybridization) records showed a clonal result in 84 patients and of these 65/84 (77%) had an aberration of chromosome 14. These comprised 48/65 with inv(14), 6/65 t(14;14), 2/65 t(X;14), 1/65 TCL1 gene rearrangement by FISH and 8/65 with TRA/TRD involvement by FISH. Other frequent abnormalities seen were i(8)(q10) in 31/84 (39%) and complex karyotypic abnormality in 40/84 (48%). Lower frequency abnormalities seen were monosomy 11 in 8/84 (9%), monosomy 12 in 8/84 (9%), trisomy 8 in 6/84 (7%), 17p loss in 5/84 (6%) and mononsomy 13 in 4/84 (5%). Alemtuzumab was used in 116 patients, 69/116 (59%) receiving it as frontline treatment and 47/116 (41%) as salvage therapy. In 50/116 (43%) alemtuzumab was used as single agent, the remaining 66/116 (57%) receiving combination therapy, mostly pentostatin. Mean time from diagnosis to receiving alemtuzumab was 6.8 months (range 0-53 months). Overall response rate (ORR) to alemtuzumab was 94/116 (81%) with complete remission (CR) 69/116 (59%), partial remission (PR) 25/116 (22%) and no response (NR) 20/116 (17%). Alemtuzumab produced better response rates when used as frontline therapy. Table 2 summarizes these results and OS and DFI are shown in figures B and C. Allogeneic stem cell transplant (allo-SCT) was performed in 34 patients. Median OS post allo-SCT was 22 months and median DFI 31 months. Relapse rate post allo-SCT was 47%, non-relapse mortality 38% and transplant related mortality 29%. Figures D and E show OS and DFI post allo-SCT. Although relapse rates are high post allo-SCT there is a small cohort of patients who are achieving long term remission. We analysed OS by decade of diagnosis covering 3 decades 1990-1999, 2000-2009 and 2010-2019. The median OS for the whole cohort was 20.6 months with median OS of 21 months, 23 months and 19 months for each decade respectively. Results are shown in figure A. There was no statistically significant difference between the curves by log-rank analysis. DISCUSSION: Immunophenotyping results were similar to previous published data on T-PLL except for an increase in CD25 expression at 50%. Our data shows that outcomes in T-PLL have remained unchanged since the first decade of alemtuzumab usage, with no improvement in OS in the last 20 years. Despite improvements in diagnostic techniques and supportive care median OS is static at approximately 20 months. These results highlight the need for novel therapies in T-PLL. Given the rarity of T-PLL, international, multi-center, randomised trials are needed to improve outcomes. Disclosures Cross: Royal Marsden Cancer Charity: Other: MD Residency, Research Funding. Iyengar:Abbvie: Honoraria; Janssen: Honoraria. Dearden:Janssen: Honoraria; Genentech: Honoraria; Abbvie: Honoraria; Sanofi: Honoraria.

EGFL7 Antagonizes NOTCH Signaling and Represents a Novel Therapeutic Target in Acute Myeloid Leukemia (AML)

Epidermal growth factor-like domain 7 (EGFL7) is a secreted protein. We previously found that EGFL7 is up-regulated in primary AML blasts and that a high mRNA expression correlates with shorter event-free and overall survival in AML patients (Papaioannou et al. PNAS 2017;114(23)). However, the underlying mechanism by which EGFL7 promotes leukemogenesis is largely unknown. To identify EGFL7 binding partners, we took an unbiased high-throughput approach by using an antibody interaction-array to measure the ability of EGFL7 to bind directly ~400 proteins expressed by primary AML blasts. Using this strategy, we found in cell lysates of 3 AML patients that EGFL7 binds several signaling proteins important for normal and malignant hematopoiesis including NOTCH (in all patients P<0.001 compared to control). The direct interaction between EGFL7 and NOTCH1 as well as NOTCH2 receptor was confirmed in co-immunoprecipitation assays in THP1 and primary patient cells. Next, we stimulated AML blasts from 3 patients with recombinant EGFL7 (rEGFL7) and found that it reduced NOTCH intracellular domain (NICD1/2, Figure 1A), indicating that EGFL7 inhibits NOTCH activation. In line with this, we found that stimulation of AML patients samples' with rEGFL7 resulted in a decreased expression of its well-known downstream target gene HES1 (Figure 1B; in all patients P<0.05 compared to unstimulated (unstim) cells). On the other hand, treatment with an anti-EGFL7 blocking antibody (AB) caused increased levels of NICD1/2 (Figure 1C) and expression of HES1 (Figure 1D, in all patients P<0.05 compared to cells treated with IgG) indicating a reactivation of NOTCH signaling. Moreover, the treatment of THP1 cells with the anti-EGFL7 blocking AB resulted in increased apoptosis (1.9 fold increase compared to IgG control, P<0.05) and differentiation (14.9 fold increase in CD11B expression compared to IgG control, P<0.05; 5.6 fold increase in CD14 expression compared to IgG control, P<0.05). To determine whether blocking EGFL7 could provide a new targeted therapy for patients with AML, we treated 3 independent AML models (i.e. an AML cell line based xenotransplant model (n=4 mice per group), a primary murine AML model (n=7 mice per group), and a patient derived xenotransplant model (n=4 mice per group)) with anti-EGFL7 or IgG1 control to determine whether anti-EGFL7 could prolong survival in vivo. In all models, we demonstrated that in vivo treatment with anti-EGFL7 results in prolonged overall survival (cell line model: P<0.01; primary murine AML model: P<0.01; for the patient derived xenotransplant model: P<0.05). In conclusion, our data demonstrate that EGFL7 contributes to NOTCH silencing in AML by antagonizing canonical NOTCH ligand binding. Reactivation of NOTCH signaling in vivo using anti-EGFL7 results in prolonged survival of leukemic mice, supporting EGFL7 might be a novel therapeutic target in AML. Figure 1 A AML patient blasts were cultured in the presence or absence (Unstim) of rEGFL7. Total proteins were extracted for immunoblotting with β-ACTIN as loading control. B Total RNA was extracted for quantitative real time (qRT-PCR) analysis of HES1 mRNA normalized to β-ACTIN control (*P<0.05, **P<0.01, ***P<0.001) stimulated with +rEGFL7 vs Unstim. C Patient blasts were cultured in presence of 100 μg/ml of normal IgG or 100 μg/ml anti-EGFL7 antibody (@EGFL7) for 4 hours. Total protein was extracted for immunoblotting of NICD with b-ACTIN as loading control. D Patient blasts were cultured with 100 μg/ml of normal IgG or anti-EGFL7 antibody for 2 hours and 10 hours. Total RNA was extracted for qRT-PCR analysis of HES1 mRNA with b-ACTIN as internal control. *P<0.05, ***P<0.001 vs IgG control. E Treatment with anti-EGFL7 blocking antibody prolonged survival compared to IgG1 controls in 3 independent mouse models. Disclosures No relevant conflicts of interest to declare.

Abstract 4960: Studying the impact of gut microbiome to hepatocellular cancer in an orthotopic murine model

Abstract Background and Objective: As one of the most important components of human body, gut microbiota play a vital role in human health by participating in metabolomics and maintaining immunological homeostasis. Microbiota dysbiosis is significantly associated with initiation and progression of different diseases. Due to close anatomical and functional connection between gut and liver (gut-liver axis), gut microbiota are found to impact the development of liver diseases including hepatocellular cancer (HCC) without fully understanding mechanisms. The overall objective of present study is to investigate how microbiota modulate HCC growth by regulating intrahepatic immune response. Methods: Administration of carbon tetrachloride in combination with transplant of oncogenic hepatocytes by intrasplenic injection was used to make an orthotopic mouse model of HCC. A specific antibiotics cocktail (ABX) was selected to inhibit some kinds of gut microbiota. 16s rRNA sequencing was used to characterize gut microbiota profile in mice with the different treatments. Magnetic resonance imaging (MRI) was used to monitor tumor growth. Adoptive transfer of tumor antigen-specific CD8+ T cells following the treatment was used to investigate how ABX administration impacted intrahepatic immune response. Flow cytometry and multiplex cytokine assay were used to detect the alteration of effector CD8+ T cell in phenotype, proliferation, and function. Immunohistochemistry (IHC) was used to investigate immune cell tumor-infiltration. Results: The established murine model reflects the typical features of human HCC. ABX oral administration significantly changed gut microbiota composition, particularly in abundance of P. aeruginosa and Muribaculaceae. ABX-mediated alteration of gut microbiota significantly retarded HCC progression. Interestingly, ABX treatment was found to prevent adoptively transferred tumor antigen-specific CD8+ T cell from tumor-induced immune tolerance, as higher frequency of effector CD8+ T cell was detected in ABX-treated tumor-bearing mice compared to that in control tumor-bearing mice. Correspondingly, ABX treatment resulted in the increase in expression of CD69 and enhanced production of IFN-γ in tumor antigen-specific CD8+ T cells relevant to that in control mice. Conclusion: Modulating microbiota with antibiotics is able to shape intrahepatic immune responses and therapeutically suppress tumor growth, providing a new option in HCC control. Citation Format: Xiaoqiang Qi, Ming Yang, Joseph Stenberg, Guangfu Li, Kevin Staveley-O’Carroll. Studying the impact of gut microbiome to hepatocellular cancer in an orthotopic murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4960.

Antigen Priming with Enantiospecific Cationic Lipid Nanoparticles Induces Potent Antitumor CTL Responses through Novel Induction of a Type I IFN Response.

Certain types of cationic lipids have shown promise in cancer immunotherapy, but their mechanism of action is poorly understood. In this study, we describe the properties of an immunotherapeutic consisting of the pure cationic lipid enantiomer R-1,2-dioleoyl-3-trimethyl-ammonium-propane (R-DOTAP) formulated with modified viral or self-peptide Ags. R-DOTAP formulations with peptide Ags stimulate strong cross-presentation and potent CD8 T cell responses associated with a high frequency of polyfunctional CD8 T cells. In a human papillomavirus tumor model system, a single s.c. injection of tumor-bearing mice with R-DOTAP plus human papillomavirus Ags induces complete regression of large tumors associated with an influx of Ag-specific CD8 T cells and a reduction of the ratio of regulatory/Ag-specific CD8 T cells. R-DOTAP also synergizes with an anti-PD1 checkpoint inhibitor, resulting in a significant inhibition of B16 melanoma tumor growth. We found that R-DOTAP stimulates type I IFN production by dendritic cells in vivo and in vitro. s.c. injection of R-DOTAP results in an IFN-dependent increase in draining lymph node size and a concomitant increase in CD69 expression. Using knockout mice, we show that type I IFN is required for the induction of CD8 T cell activity following administration of R-DOTAP plus Ag. This response requires Myd88 but not TRIF or STING. We also show that R-DOTAP stimulates both TLR7 and 9. Collectively, these studies reveal that R-DOTAP stimulates endosomal TLRs, resulting in a Myd88-dependent production of type I IFN. When administered with Ag, this results in potent Ag-specific CD8 T cell responses and antitumor activity.

Immune modulation caused by synthetic opioids during CD4+ T cell activation

Abstract Opioids are heavily prescribed medications used to treat chronic pain. Since their additional response includes euphoria, there is an exceedingly high addiction rate that has led to a country wide epidemic of opioid misuse. Opioid effects are not limited to the nervous system, since leukocytes express opioid receptors, which are G-protein coupled receptors known as μ, δ, and κ, suggesting that natural and synthetic opioids might regulate the immune system. Based on previously published reports, we hypothesized that opioids induce distinct regulatory pathways in CD4+ T cells that results in inhibition of the immune response. CD4+ T cells are isolated by negative selection from peripheral blood mononuclear cell and opioid receptor expression is assessed on resting and activated T cells using flow cytometry. Two models of opioid exposure in vitro were developed to study the effects of opioids on CD4+ T cell activation. Cells were treated with a δ opioid receptor specific agonist (DPDPE) for 24 h before or after activation and harvested 24 h after the second treatment and stained for expression of a well characterized early T cell activation marker – CD69. Cells treated with an opioid after activation showed no major shifts in CD69 intensity, suggesting opioids had no influence on a previously activated T cell. However, CD4+ T cells treated with an opioid before activation exhibited an increase in CD69 expression, suggesting an unexpected enhancement of T cell activation. In conclusion, opioids seem to play distinct biological activities depending on the activation state of the CD4+ T cell, and may function as both immunoinhibitory and immunostimulatory agents. Future studies will identify the mechanisms by which opioids regulate immunity.

Donor T and NK Cells are Derived from the Donor Lung Parenchyme and Represent Tissue-Resident Memory Cells - An Important Feature for Tolerance Development

Purpose In previous studies, we identified donor lymphocytes in peripheral blood of recipients directly after transplantation and persisting at least three weeks that were characterized by the tissue retention marker CD69. In order to determine their origin, we hypothesized that donor T and NK cells have a peculiar tissue-resident memory phenotype that is shared between cells derived from lung perfusates, trachea parenchyma and lymph nodes. Methods Donor lymphocytes in recipient blood were determined in 27 lung transplant patients at T0, T24, 3 weeks by staining of donor HLA class I molecules in combination with lineage- and tissue-specific markers using flow cytometry. The phenotype of T and NK cells in perfusates (n=30), donor trachea (n=5), and lymph node (n=10), recipient explanted parenchyma (n=15) was compared to circulating cells using the same markers. Results In peripheral blood of all lung transplant recipients, donor derived T and NK cells were detected at T0, T24 and 3 weeks and had higher CD69 expression compared to recipient cells (p=0.001 to 0.03) and were mostly CD25−. This phenotype was similar to T and NK cells in corresponding perfusates, with significantly increased CD69 expression compared to circulating PBMCs (all p Conclusion Our results suggest that donor T and NK cells found in the periphery of lung transplant recipients are derived from lung parenchyma and represent tissue-resident memory cells. This transient chimerism in recipient blood might be clinically relevant for tolerance induction after transplantation due to unique features of TRM T and NK cells.

Variable Bruton Tyrosine Kinase (BTK) Resynthesis across Patients with Chronic Lymphocytic Leukemia (CLL) on Acalabrutinib Therapy Affect Target Occupancy and Reactivation of B-Cell Receptor (BCR) Signaling

Inhibitors of B-cell receptor (BCR) signaling, in particular of Bruton tyrosine kinase (BTK), are effective in patients with chronic lymphocytic leukemia (CLL). Acalabrutinib is a highly selective, covalent BTK inhibitor with rapid absorption and fast elimination (Barf et al., J Pharmacol Exp Ther 2017; 363:240-252). In a previous clinical trial of patients with relapsed/refractory CLL, acalabrutinib therapy was well tolerated and efficacious and had high target occupancy (Byrd et al., N Engl J Med 2016; 374:323-21). Here, we report on BTK occupancy, BTK resynthesis rates, and the relationship between BTK occupancy and BCR signaling in patients with CLL enrolled in a single center, phase 2 study of acalabrutinib monotherapy (NCT02337829).Patients were randomized to receive either 200 mg of acalabrutinib once daily (QD) or 100 mg twice daily (BID). The percentage of BTK bound by acalabrutinib (target occupancy) was measured with the aid of a biotin-tagged analogue probe. After 3 days of dosing, the median BTK occupancy in peripheral blood at peak (4 hours postdose) was 99% in both dosing groups (n=21 per group). However, at the drug trough time points (12 hours for BID; 24 hours for QD), median occupancy was greater in the BID than QD group (95% vs 87%, respectively; P<0.0001), with less interpatient variance.As acalabrutinib binds covalently to BTK, reactivation of the pathway requires de novo synthesis of BTK. We estimated the rate of BTK synthesis in vivo by measuring the accumulation of free BTK (% unbound by acalabrutinib) in serial blood samples taken 4, 12, 24, 36, and 48 hours postdose during a preplanned dose interruption on days 4 and 5. BTK was synthesized at a median rate of 13.4% per day, which was highly variable (fast vs slow) across patients (Figure). Interestingly, we found the median BTK de novo synthesis rate to be roughly twice as fast as observed in B cells from healthy volunteers (P<0.0001). BTK occupancy levels between matched peripheral blood and lymph node samples in individual patients were highly correlated (R=0.83; P<0.0001), with less than a 3% difference overall, suggesting a similar BTK synthesis rate in the lymph nodes and peripheral blood of patients with CLL. Similar results were observed for matched bone marrow and peripheral blood samples.We next correlated BTK occupancy levels and activity of BCR signaling in 10 representative patients. On Day 3, four hours after receiving the drug, significant reductions were observed in phospho-BTK (P=0.05), pNF-κB (P=0.03), and CD69 expression (P=0.001) compared with pretreatment. Next, we evaluated the change in BCR signaling from trough (for each dose group) to 36 (BID) or 48 (QD) hours after dosing (i.e., during the short window of drug withholding). A trend for increased signaling was driven by individual patients with low occupancy (≲70%). To mimic microenvironmental activation, we stimulated overnight with anti-IgM, all the peripheral blood samples collected during drug withholding (4 to 48 hours postdose). We found that upon BCR activation, B-CLL cells showed the ability to reactivate BCR signaling as measured by an increase in CD69 expression. Moreover, the extent of reactivation in BCR signaling positively correlated with the percentage of free BTK (R=0.73; P=0.0001).In conclusion, higher target coverage was achieved when acalabrutinib was administered 100 mg BID compared with 200 mg QD. Given that BTK de novo synthesis rates do vary across patients with CLL, and that reactivation of BCR signaling correlates with lower occupancy (higher free BTK), BID dosing provides the highest target coverage for a greater number of patients.This work was supported by the Intramural Research Program of National Heart, Lung, and Blood Institute, the National Institutes of Health, and Acerta Pharma. We thank our patients for donating blood and tissue samples to make this research possible. [Display omitted] DisclosuresCheung:Acerta Pharma: Employment, Equity Ownership; AstraZeneca: Equity Ownership. Gulrajani:Acerta Pharma: Employment, Equity Ownership. Davies-Hill:NIH/NCI: Employment. Izumi:Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Covey:AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

Inecalcitol Enhances Daratumumab-Induced Antibody-Dependent Cell Cytotoxicity Towards Multiple Myeloma and Acute Myeloid Leukemia Cell Lines

Daratumumab (DARA), the first monoclonal antibody directed against CD38, received accelerated approval in 2015 and regular approval in 2016 by the Food and Drug Administration for the treatment of relapsed/refractory multiple myeloma (MM) (Bhatnagar et al., 2017); its therapeutic indications were recently extended to first-line newly diagnosed MM patients in combination with standards of care following a positive clinical trial in this setting (Mateos et al., 2018). Therefore, DARA has brought extensive evidence that targeting CD38 is a highly successful strategy against MM (Frerichs et al., 2018; Plesner & Krejcik, 2018; Touzeau & Moreau, 2017). Interestingly, the pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) as compared to patients who achieved less than PR (Nijhof et al., 2016); furthermore, low level of CD38 expression has recently been identified as an adverse prognostic factor with high significance (p < 0.001) among MM patients (Arana et al., 2018). Besides MM, there are growing preclinical hints that DARA can be efficacious against acute myeloid leukemia (AML) cell lines (Fatehchand et al., 2016; Buteyn et al., 2018) or even T-cell acute lymphoblastic leukemia cells obtained from children patients (Bride et al., 2018), provided that enough CD38 molecules are present at the surface of the leukemic cells.Inecalcitol (INE: 14epi-,19nor-,23yne-,1,25dihydroxy-cholecalciferol), a vitamin D receptor agonist characterized by potent anti-proliferative and pro-differentiating properties and by a low calcemic potential (Okamoto et al., 2012; Ma et al., 2013; Medioni et al. 2014), has been shown to stimulate CD38 expression levels in 37 out of 38 MM cell lines (ASH 2017) and in 10 out of 11 AML cell lines (ASH 2016, AACR 2018). The next question was to know if this increase in CD38 expression induced by INE resulted in an increase in DARA-induced antibody-dependent cell cytotoxicity (ADCC).We have tested two MM (MM1.S, L-363) and seven AML (HL-60, U-937, THP-1, Molm-13, UOC-M1, OCI-AML2, Kasumi-1) cell lines representative of various levels of spontaneous CD38 cell surface density, as measured in thousands of CD38 molecules per cell by fluorescent anti-CD38 labeling (HIT2 clone) followed by quantitative flow cytometry using Cellquant calibrator beads (BioCytex). They ranged from very high CD38-positive (MM.1S, 51.0 ± 1.6 and UOC-M1, 46.5 ± 4.9) to high (OCI-AML2, 16.6 ± 1.5), low (L-363, 7.1 ± 0.1 and THP-1, 7.0 ± 0.2), very low (Molm-13, 2.1 ± 1.0 and Kasumi-1, 1.1 ± 1.2) and CD38-negative (HL-60, U-937). After exposure to 10 nM INE or vehicle for 72h, DARA-induced ADCC was assessed using either the calcein staining of target cells exposed to healthy donors peripheral blood mononuclear cells (PBMC) as effector cells, or the ADCC Reporter Bioassays Kit from Promega (G7018).After control vehicle incubation, DARA-induced ADCC was concentration-related in the range of 0.01 to 1 µg/mL in all cell lines and achieved a maximum ranging from 7% in THP-1, although a CD38-negative cell line, to respectively 27% and 28% in MM1.S and UOC-M1, the two highest CD38-positive cell lines. However, the correlation between maximal DARA-induced ADCC and CD38 surface density did not reach statistical significance (Spearman's rank correlation r = 0.6134, p = 0.09, n = 9).After incubation with 10 nM INE, the initially CD38-negative U-937 and HL-60 cells were found to express low (7.9 ± 0.3) and high (25.6 ± 7.3) surface CD38 levels, respectively. All the CD38-positive cell lines have shown a marked increase in the number of surface CD38 molecules: MM1.S (228 ± 12), OCI-AML2 (76,7 ± 6.5), UOC-M1 (68.8 ± 3.1), L-363 (63.9 ± 1.6), Molm-13 (38.2 ± 8.9), THP-1 (17.9 ± 0.9) and Kasumi-1 (15.7 ± 3.3) (paired Student's t-test, p = 0.02, n = 7). DARA-induced ADCC was concentration-related in the same 0.01 to 1 µg/mL range. Maximal DARA-induced ADCC was increased by 10 nM INE in all cell lines (paired Student t-test, p = 0.0025, n = 9), ranged from 20% in the lowest CD38-expressing cell line U-937 to 42% in the highest CD38-expressing cell line MM1.S, and was significantly correlated with the number of CD38 molecules/cell (Spearman's rank correlation r = 0.8333, p < 0.01, n = 9).Altogether, these results suggest that inecalcitol could represent a therapeutic agent of choice to enhance the clinical response of MM and AML patients to anti-CD38 antibodies such as daratumumab. DisclosuresMouly:Hybrigenics: Employment. Planquette:Hybrigenics: Employment, Equity Ownership, Patents & Royalties: inventor, but no royalties. Rousseau:Hybrigenics: Employment. Delansorne:Hybrigenics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: inventor, but no royalties.

Antigen-loaded dendritic cells triggers a specific cytotoxic Tlymphocytes immune response against hepatocellular carcinoma: in vitro study.

Hepatocellular carcinoma (HCC) is one of the common malignancies, characterized by poor response to conventional therapeutic options. Immunotherapy with dendritic cells (DCs)-vaccines is one of the most successful strategies used for the treatment of HCC. However, the methods applied in the preparation of antigen-loaded DCs are important factors for optimization of DCs vaccines. The present study was conducted to investigate the effect of HCC-whole tumor cell lysate prepared using rapid repetitive freeze-thaw cycles on the immunogenicity of DCs and evaluate the ability of whole tumor cell lysate-pulsed DCs vaccine to induce a specific cytotoxic Tlymphocytes (CTLs) response against HepG2 cell line. Immature DCs generated from peripheral blood monocytes were randomized into two groups: control DCs and whole tumor cell lysate-pulsed DCs. Phenotypic analysis of the DCs' cell maturation marker CD83 and co-stimulatory molecule CD86 was performed. HCC-specific cytotoxic activity of CD8+ CTLs was measured in vitro. Loading of DCs with necrotic whole cell lysate resulted in non-significant changes in DCs' expression of CD83, but a significant increase in expression of CD86. In addition, CD8+ CTLs stimulated with whole tumor cell lysate-pulsed DCs showed a high cytotoxic activity that specifically attack HepG2 cells. Our findings indicated that pulsation of DCs with whole tumor cell lysate prepared by repetitive freeze-thaw cycles could efficiently enhance the ability of DCs to induce proliferation and clonal expansion of CD8+ CTLs. Data herein, also indicated that whole tumor cell lysate-pulsed DCs triggers a specific CD8+ CTLs against HCC tumor cells.

Bioinstructive microparticles for self-assembly of mesenchymal stem Cell-3D tumor spheroids

3D multicellular tumor spheroids (3D-MCTS) that closely mimic in vitro the complex lung tumor microenvironment (TME) are highly desirable for screening innovative anti-cancer therapeutics. Despite significant improvements in mimicking lung TME, few models have combined tumor-infiltrating mesenchymal stem cells from bone marrow (hBM-MSCs) with heterotypic 3D tumor spheroid models containing ECM mimetic components. Herein, we engineered hybrid 3D-MCTS that combine, for the first time, A549:fibroblasts:hBM-MSCs in heterotypic tri-culture, with bioinstructive hyaluronan microparticles that act as tumor-ECM mimetics and as cell-anchoring hotspots. The obtained results indicated that 3D microspheres provided proper support for cells to self-assemble into compact 3D microtissues and promoted an increase in CD44 expression, emulating the presence of native-ECM hyaluronan. 3D-MCTS size and sphere-like morphology was reproducible and tri-culture models presented the characteristic solid tumors necrotic core. Mesenchymal stem cells tracking demonstrated that hBM-MSCs migrate to different regions in 3D microtumors mass exhibiting dynamic interactions with cancer cells and stromal fibroblasts, alike in human tumors. Importantly, doxorubicin administration revealed hBM-MSCs effect on cytotoxic responses in 3D tri-culture models and in dual cultures of hBM-MSCs:A549 at 10:1 ratio. Such findings evidence the relevance of including hBM-MSCs in combination with cancer-stromal fibroblasts in 3D in vitro tumor models and the importance to test different cell-to-cell ratios to mimic tumor heterogeneity. In addition, bioinstructive hyaluronan-microparticles were also effective as cell-agglomerating scaffolds and showed potential to be used as an enabling technology for including different ECM components in 3D in vitro models in the future.

Focal and segmental glomerulosclerosis in murine models: a histological and ultrastructural characterization with immunohistochemistry correlation of glomerular CD44 and WT1 expression

ABSTRACT Aim : Focal segmental glomerulosclerosis (FSGS) is a common progressive chronic renal disease. Podocyte injury and loss are the postulated pivotal events that trigger FSGS. In this study, the authors aim to examine the evolution of FSGS in murine models histologically, ultrastructurally and immunohistochemically with special emphasis on podocytes and parietal epithelial cells (PECs). Material and methods : FSGS resembling primary FSGS in humans was initiated in Wistar rats using intravenous Adriamycin injections. Blood and urine analysis were performed at 0, 8, and 12 weeks. Both the control kidneys and the test kidneys were harvested at 8 and 12 weeks, examined histologically and ultrastructurally and the findings correlated with the glomerular expression of immunostains specific for podocytes (WT-1) and for activated PECs (CD44). Results : FSGS developed in both 8 and 12 weeks test groups showing progressive proteinuria, podocytopathy and segmental glomerular scarring. There was a decrease in the glomerular expression of WT-1 with a concurrent increase in the glomerular expression of CD44, indicating podocyte loss with synchronous increase in activated PECs. The evolving FSGS correlated negatively with podocytes and positively with activated PECs. Conclusion : Our study shows that with podocyte injury there is podocyte effacement and loss, proteinuria, glomerular segmental adhesion and scarring, all culminating in FSGS. In addition, there is activation, hyperplasia and hypertrophy of PECs. This demonstrates that both podocyte loss and PEC activation promote FSGS. Our findings are consistent with recent investigations. More studies are required to further understand the role of these cells in the evolution of FSGS and subsequently introduce new targeted treatment modalities.

Characterization of two potential immunotherapeutics in tumor infiltrating lymphocytes (TILs) using flow cytometry and Meso Scale Discovery.

e15052 Background: Inter-K Peptide Therapeutics has developed two synthetic compounds that modulate kinase activity. To investigate the potential of these compounds as cancer immunotherapeutics, a flow cytometric intracellular cytokine staining (ICS) assay was developed to investigate the effect of the compounds on immune cells isolated from tumors from subjects with non-small cell lung cancer (NSCLC). In addition, the concentration of various cytokines was measured in cell culture supernatant after treatment with the compounds. Methods: After optimizing the assays in PBMC from healthy donors, the assay was used for clinical specimens. Tumor infiltrating lymphocytes (TILs) were isolated from two resected tumors by mechanical disaggregation. TILs were stimulated with or without anti-CD3/CD28 antibodies with and without Inter-K Compound A or Compound B for 72 hours. Following stimulation, cell culture supernatants were analyzed for 10 proinflammatory-related cytokines using the Meso Scale Discovery (MSD) assay. TILs were analyzed by flow cytometry with a 10-color immunophenotyping panel to measure the expression of functional markers CD25, IL-2, IFNg and Ki67 in NK, NKT, CD4 T and CD8 T cells. Results: Immunomodulation induced by Compound A treatment included an over two-fold increase in CD25 expression in CD4 T cells, statistically significant increases in IL-6 and IL-8 production while a decrease in Ki67 expression in CD8 T cells and a reduction in IL-2, IFNg, TNFa, IL-4, IL-10 and IL-13 production was observed. Compound B did not appear to have immunomodulatory properties. Conclusions: This work provides an example of the utility of flow cytometry in compound evaluation. In this case, preliminary data suggests that Compound A but not B was able to modulate the response in TIL samples in vitro, and supports the continued investigation of Compound A as an immunotherapeutic. Although TIL specimens are challenging to work with due to tumor availability and low cellular recovery, as the target for the immunotherapy, it is important that compound characterization be conducted in TIL in addition to healthy donor PBMC and cell lines.

Expression of adhesion molecules on granulocytes and monocytes following myocardial infarction in rats drinking white wine.

Neutrophils and monocytes through their CD15s, CD11b and CD44 adhesion molecules are implicated in the initiation and resolution of cardiac inflammation as well as in healing processes after the myocardial infarction (MI). The aim of this study was to determine the effect of white wine consumption on granulocyte and monocyte CD15s, CD11b, and CD44 expression 24h after the surgically inflicted MI. Granulocytes and monocytes were analyzed by flow cytometry, using whole blood of male Sprague–Dawley rats that consumed white wine for 4 weeks. This group was compared with water only drinking controls, sham animals (subject to surgery without myocardial infarction) and baseline group (intact animals that received no intervention prior to being sacrificed). Sham animals did not differ from baseline animals in CD11b+CD44+ percentage and CD44+ median fluorescence intensity. Wine drinking was associated with striking increase in CD44 expression on monocyte subpopulations. Its expression was three and fourfold increased on monocytes and large monocytes, respectively, relative to the water only drinking controls. Because of known role of CD44 on suppression of post-infarction inflammation, its upregulation on granulocytes and monocytes may significantly contribute to the microenvironment favourable for the cardiac regeneration.

Hyaluronan impairs the barrier integrity of brain microvascular endothelial cells through a CD44-dependent pathway

Hyaluronan (HA) constitutes the most abundant extracellular matrix component during brain development, only to become a minor component rapidly after birth and in adulthood to remain in specified regions. HA signaling has been associated with several neurological disorders, yet the impact of HA signaling at the blood–brain barrier (BBB) function remains undocumented. In this study, we investigated the impact of HA on BBB properties using human-induced pluripotent stem cell (iPSC) -derived and primary human and rat BMECs. The impact of HA signaling on developmental and mature BMECs was assessed by measuring changes in TEER, permeability, BMECs markers (GLUT1, tight junction proteins, P-gp) expression and localization, CD44 expression and hyaluronan levels. In general, HA treatment decreased barrier function and reduced P-gp activity with effects being more prominent upon treatment with oligomeric forms of HA (oHA). Such effects were exacerbated when applied during BMEC differentiation phase (considered as developmental BBB). We noted a hyaluronidase activity as well as an increase in CD44 expression during prolonged oxygen–glucose deprivation stress. Inhibition of HA signaling by antibody blockade of CD44 abrogated the detrimental effects of HA treatment. These results suggest the importance of HA signaling through CD44 on BBB properties.

The adjuvant system AS01 up-regulates neutrophil CD14 expression and neutrophil-associated antigen transport in the local lymphatic network.

The liposome-based adjuvant system AS01 is under evaluation for use in several vaccines in clinical development. We have shown previously that AS01 injected with hepatitis B surface antigen (HBsAg) induces a distinct cellular signature within the draining lymphatics that enhances local lymphocyte recruitment and antigen-specific humoral immunity. Here, we show that AS01-induced neutrophil recruitment is associated with increased expression of CD14 and enhanced antigen uptake capacity in neutrophils from both afferent and efferent lymphatic compartments during the first 48h after vaccination. Significant and transient increases in CD14 expression on systemic neutrophils were also observed following primary and boost vaccination with HBsAg-AS01; however, they were not observed following additional encounter with HBsAg-alone or HBsAg-alum. These results show that following immunization with AS01, neutrophils expressing higher levels of CD14 are both more abundant and efficient at antigen uptake, warranting further investigation into the role of neutrophil-associated CD14 in the adjuvanticity of AS01.

Hyaluronan-binding by CD44 reduces the memory potential of activated murine CD8 T cells.

Expansion and death of effector CD8 T cells are regulated to limit immunopathology and cells that escape contraction go on to generate immunological memory. CD44, a receptor for the extracellular matrix component hyaluronan, is a marker of activated and memory T cells. Here, we show with a murine model that the increase in CD44 expression and hyaluronan binding induced upon CD8 T cell activation was proportional to the strength of TCR engagement, thereby identifying the most strongly activated T cells. When CD44-/- and CD44+/+ OT-I CD8 T cells were adoptively transferred into mice challenged with Listeria-OVA, there was a slight increase in the percentage of CD44+/+ cells at the effector site. However, CD44+/+ cells were out-competed by CD44-/- cells after the contraction phase in the lymphoid tissues, and the CD44-/- cells preferentially formed more memory cells. The hyaluronan-binding CD44+/+ CD8 effector T cells showed increased pAkt expression, higher glucose uptake, and were more susceptible to cell death during the contraction phase compared to non-binding CD44+/+ and CD44-/- OT-I CD8 T cells, suggesting that CD44 and its engagement with hyaluronan skews CD8 T cells toward a terminal effector differentiation state that reduces their ability to form memory cells.

Interleukin (IL)-23 Stimulates IFN-γ Secretion by CD56bright Natural Killer Cells and Enhances IL-18-Driven Dendritic Cells Activation.

Interleukin (IL)-23 is a member of the IL-12 family of cytokines that, as the other members of this family, is secreted by monocytes, macrophages, and dendritic cells (DC) upon recognition of bacterial, viral, and fungal components. IL-23 is critical during immunity against acute infections, and it is also involved in the development of autoimmune diseases. Although immunoregulatory effects of IL-23 on mouse natural killer (NK) cells have been described, the effect of IL-23 on human NK cells remains ill-defined. In this study, we observed that monocytes stimulated with LPS secreted IL-23 and that blockade of this cytokine during monocyte and NK cell coculture led to a diminished production of IFN-γ by NK cells. Accordingly, rIL-23-induced NK cell activation and stimulated IFN-γ production by CD56bright NK cells. This effect involved MEK1/MEK2, JNK, PI3K, mammalian target of rapamycin, and NF-κB, but not STAT-1, STAT-3, nor p38 MAPK pathways. Moreover, while NK cell-mediated cytotoxicity remained unaltered, antibody-dependent cellular cytotoxicity (ADCC) was enhanced after IL-23 stimulation. In addition, IL-23 displayed a synergistic effect with IL-18 for IFN-γ production by both CD56bright and CD56dim NK cells, and this effect was due to a priming effect of IL-23 for IL-18 responsiveness. Furthermore, NK cells pre-stimulated with IL-18 promoted an increase in CD86 expression and IL-12 secretion by DC treated with LPS, and IL-23 potentiated these effects. Moreover, IL-23-driven enhancement of NK cell “helper” function was dependent on NK cell-derived IFN-γ. Therefore, our results suggest that IL-23 may trigger NK cell-mediated “helper” effects on adaptive immunity, shaping T cell responses during different pathological situations through the regulation of DC maturation.

Capturing the systemic immune signature of a norovirus infection: an n-of-1 case study within a clinical trial.

The infection of a participant with norovirus during the adaptive study of interleukin-2 dose on regulatory T cells in type 1 diabetes (DILT1D) allowed a detailed insight into the cellular and cytokine immune responses to this prevalent gastrointestinal pathogen. Serial blood, serum and peripheral blood mononuclear cell(PBMC) samples were collected pre-, and post-development of the infection. To differentiate between the immune response to norovirus and to control for the administration of a single dose of aldesleukin (recombinant interleukin-2, rIL-2) alone, samples from five non-infected participants administered similar doses were analysed in parallel. Norovirus infection was self-limited and resolved within 24 hours, with the subsequent development of anti-norovirus antibodies. Serum pro- and anti-inflammatory cytokine levels, including IL-10, peaked during the symptomatic period of infection, coincident with increased frequencies of monocytes and neutrophils. At the same time, the frequency of regulatory CD4 + T cell (Treg), effector T cell (Teff) CD4 + and CD8 + subsets were dynamically reduced, rebounding to baseline levels or above at the next sampling point 24 hours later. NK cells and NKT cells transiently increased CD69 expression and classical monocytes expressed increased levels of CD40, HLA-DR and SIGLEC-1, biomarkers of an interferon response. We also observed activation and mobilisation of Teffs, where increased frequencies of CD69 + and Ki-67 + effector memory Teffs were followed by the emergence of memory CD8 + Teff expressing the mucosal tissue homing markers CD103 and β7 integrin. Treg responses were coincident with the innate cell, Teff and cytokine response. Key Treg molecules FOXP3, CTLA-4, and CD25 were upregulated following infection, alongside an increase in frequency of Tregs with the capacity to home to tissues. The results illustrate the innate, adaptive and counter-regulatory immune responses to norovirus infection. Low-dose IL-2 administration induces many of the Treg responses observed during infection.

Capturing the systemic immune signature of a norovirus infection: an n-of-1 case study within a clinical trial

Background: The infection of a participant with norovirus during the adaptive study of interleukin-2 dose on regulatory T cells in type 1 diabetes (DILT1D) allowed a detailed insight into the cellular and cytokine immune responses to this prevalent gastrointestinal pathogen.Methods: Serial blood, serum and peripheral blood mononuclear cell (PBMC) samples were collected pre-, and post-development of the infection. To differentiate between the immune response to norovirus and to control for the administration of a single dose of aldesleukin (recombinant interleukin-2, rIL-2) alone, samples from five non-infected participants administered similar doses were analysed in parallel.Results: Norovirus infection was self-limited and resolved within 24 hours, with the subsequent development of anti-norovirus antibodies. Serum pro- and anti-inflammatory cytokine levels, including IL-10, peaked during the symptomatic period of infection, coincident with increased frequencies of monocytes and neutrophils. At the same time, the frequency of regulatory CD4+T cell (Treg), effector T cell (Teff) CD4+and CD8+subsets were dynamically reduced, rebounding to baseline levels or above at the next sampling point 24 hours later. NK cells and NKT cells transiently increased CD69 expression and classical monocytes expressed increased levels of CD40, HLA-DR and SIGLEC-1, biomarkers of an interferon response. We also observed activation and mobilisation of Teffs, where increased frequencies of CD69+and Ki-67+effector memory Teffs were followed by the emergence of memory CD8+Teff expressing the mucosal tissue homing markers CD103 and β7 integrin. Treg responses were coincident with the innate cell, Teff and cytokine response. Key Treg molecules FOXP3, CTLA-4, and CD25 were upregulated following infection, alongside an increase in frequency of Tregs with the capacity to home to tissues.Conclusions: The results illustrate the innate, adaptive and counter-regulatory immune responses to norovirus infection. Low-dose IL-2 administration induces many of the Treg responses observed during infection.

Capturing the systemic immune signature of a norovirus infection: an n-of-1 case study within a clinical trial

Background: The infection of a participant with norovirus during the adaptive study of interleukin-2 dose on regulatory T cells in type 1 diabetes (DILT1D) allowed a detailed insight into the cellular and cytokine immune responses to this prevalent gastrointestinal pathogen. Methods: Serial blood, serum and peripheral blood mononuclear cell (PBMC) samples were collected pre-, and post-development of the infection. To differentiate between the immune response to norovirus and to control for the administration of a single dose of aldesleukin (recombinant interleukin-2, rIL-2) alone, samples from five non-infected participants administered similar doses were analysed in parallel. Results: Norovirus infection was self-limited and resolved within 24 hours, with the subsequent development of anti-norovirus antibodies. Serum pro- and anti-inflammatory cytokine levels, including IL-10, peaked during the symptomatic period of infection, coincident with increased frequencies of monocytes and neutrophils. At the same time, the frequency of regulatory CD4 + T cell (Treg), effector T cell (Teff) CD4 + and CD8 + subsets were dynamically reduced, rebounding to baseline levels or above at the next sampling point 24 hours later. NK cells and NKT cells transiently increased CD69 expression and classical monocytes expressed increased levels of CD40, HLA-DR and SIGLEC-1, biomarkers of an interferon response. We also observed activation and mobilisation of Teffs, where increased frequencies of CD69 + and Ki-67 + effector memory Teffs were followed by the emergence of memory CD8 + Teff expressing the mucosal tissue homing markers CD103 and β7 integrin. Treg responses were coincident with the innate cell, Teff and cytokine response. Key Treg molecules FOXP3, CTLA-4, and CD25 were upregulated following infection, alongside an increase in frequency of Tregs with the capacity to home to tissues. Conclusions: The results illustrate the innate, adaptive and counter-regulatory immune responses to norovirus infection. Low-dose IL-2 administration induces many of the Treg responses observed during infection.