Cell Surface Expression Of CD4 Research Articles

Potency and Selectivity of BTK Inhibitors in Clinical Development for B-Cell Malignancies

Background: Bruton tyrosine kinase (BTK) is a validated target for B-cell malignancies. The BTK inhibitor ibrutinib was approved in chronic lymphocytic leukemia, mantle cell lymphoma (MCL), and Waldenstrom macroglobulinemia. Acalabrutinib is a potent, highly selective, covalent BTK inhibitor with minimal off-target activity; it received accelerated FDA approval in October 2017 for the treatment of patients with MCL having ≥1 prior therapy. In addition to the approved covalent BTK inhibitors ibrutinib and acalabrutinib, clinical data in B-cell malignancies are available for spebrutinib (CC-292), tirabrutinib (ONO/GS-4059) and zanubrutinib (BGB-3111). We performed biochemical and cellular profiling of these 5 BTK inhibitors, investigating potency and selectivity. Methods: Two biochemical kinase assays assessed BTK inhibitor potency, with IC50 determination at a fixed time point (IMAP; Molecular Devices) or over time (LanthaScreen; Invitrogen); the latter was used to calculate binding kinetics. Kinome profiling was performed at a single dose (1 µM) using KINOMEscan (Eurofins DiscoverX). IC50 determinations were made using kinases with a Cys in the same position as the Cys481 residue in BTK, using assays developed in house (using IMAP and LanthaScreen) or at Thermo Fisher Scientific (Z9-LYTE) with IC50 determination at a fixed time point. On-target inhibition of BTK in cellular assays was evaluated using B-cell receptor-mediated activation of CD69 expression on peripheral B cells using human peripheral blood mononuclear cells (hPBMCs) or human whole blood (hWB). Off-target inhibition of epidermal growth factor receptor (EGFR) was evaluated in a cellular assay examining the effect of EGF-induced EGFR phosphorylation in A431 cells. Off-target inhibition of ITK and/or TXK was evaluated using T-cell receptor (TCR)-mediated activation of interleukin-2 expression in Jurkat T cells and CD25 cell surface expression using primary human peripheral T cells. Results: Based on biochemical binding kinetics, ibrutinib and zanubrutinib were the most potent BTK inhibitors (Table 1), followed by spebrutinib; acalabrutinib and tirabrutinib had comparable potency. Differences in potency were largely driven by differences in inactivation rates. However, differences in biochemical potency were lost (in part) in cellular assays using hPBMCs or hWB. EC50 values in hWB were Differences in overall kinase selectivity were observed among the BTK inhibitors (Figure 1; KINOMEscan). Acalabrutinib had the lowest hit rate; 1.5% of human wild-type kinases were inhibited >65% at 1 µM (excluding BTK). Kinase hit rate was also low for tirabrutinb (2.3%), whereas ibrutinib (9.4%), zanubrutinib (4.3%), and spebrutinib (8.3%) had higher hit rates. Acalabrutinib had a high selectivity for BTK over kinases with a Cys in the same position as the Cys481 residue in BTK (Table 2). Similar results were observed for tirabrutinib, whereas ibrutinib, spebrutinib and zanubrutinib were less selective in this panel of kinases with potential for off-target covalent binding by BTK inhibitors (Table 2). Acalabrutinib also had a higher selectivity for BTK over Src-family kinases than the other BTK inhibitors tested. Acalabrutinib and tirabrutinib had EC50 values >10 µM in the cellular assay of off-target inhibition of EGFR; ibrutinib, zanubrutinib, and spebrutinib had EC50 values of 0.07, 0.39, and 4.7 µM, respectively. No off-target inhibition was observed for acalabrutinib or tirabrutinib on TCR-mediated activation of T cells up to 10 µM. Ibrutinib, zanubrutinib, and spebrutinib had EC50 values Conclusion: BTK inhibitors in clinical development for B-cell malignancies had differing potency in biochemical assays, but these differences were lost (in part) in cellular assays, particularly in hWB. Among the BTK inhibitors tested, the greatest differentiation was observed in kinase selectivity profiles; acalabrutinib and tirabrutinib had the highest kinase selectivity. Disclosures Kaptein:Covaluation Pharma BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership; Apo-T BV: Consultancy. de Bruin:Acerta Pharma: Employment. Emmelot-van Hoek:Acerta Pharma: Employment. van de Kar:Acerta Pharma: Employment. de Jong:Acerta Pharma: Employment. Gulrajani:Acerta Pharma: Employment, Equity Ownership. Demont:Acerta Pharma: Employment. Covey:AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Mittag:Acerta Pharma: Employment, Equity Ownership. Barf:Covaluation Holding BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees.

Abstract 4750: The role of drug transporters in the acquired resistance to PBD dimer-containing antibody drug conjugates

Abstract Antibody drug conjugates (ADCs) delivering interstrand cross-linking pyrrolobenzodiazepine (PBD) dimers are being evaluated clinically in both hematological and solid tumors. These include ADCT-301, ADCT-402, MEDI3726 and rovalpituzumab tesirine (Rova-T) that contain the payload tesirine which releases the PBD dimer warhead SG3199. An important consideration in the future development of PBD ADCs is the development of clinical acquired resistance. The aim was to generate and characterize in vitro acquired resistant cell lines in both a hematological and solid tumor setting. To generate acquired resistant cells to ADCT-301 (targeting CD25) or warhead SG3199, CD25-positive Karpas-299 cells were incubated with an approximate GI50 dose of the drug for 96 h. Cells were washed and returned to fresh medium until normal cell growth was restored. This was repeated until a stable acquired resistance was established. The level of resistance achieved was >700-fold for the ADC, and 3-fold for the naked PBD. Cross-resistance between the agents was observed and also with an ADC delivering a different PBD dimer. Similarly, resistant cells to ADCT-502 (targeting HER2) or SG3199 were established using HER2-positive NCI-N87 cells incubated with GI50 doses for 144 h. The level of resistance achieved was 8 and 4-fold, respectively, and again, cross-resistance was observed. DNA interstrand cross-linking was measured in the parental and resistant lines treated with equi-molar doses of ADC or SG3199. In every case, the resistant lines produced fewer cross-links indicating an up-stream mechanism of resistance preventing SG3199 from reaching its DNA target. In the Karpas ADCT-301-resistant line, a small (15%) decrease in CD25 cell surface expression could not account for the level of resistance. RT2 Profiler PCR Array for human drug-transporters was used to probe the cell lines. In the Karpas ADCT-301-resistant line three gene products were significantly upregulated: ABCG2 (168-fold), SLCO2A1 (4.6) and ABCC2 (3.5). Significantly upregulated in the Karpas SG3199-resistant cells were SLCO2B1 (15.4), ABCC12 (6.4), ATP7A (4.6), SLC16A2 (2.8), ABCG2 (2.8), SLC7A9 (2.5), ABCB4 (2.3) and ABCC11 (2.1). Upregulation of ABCG2, ABCC2 and SLCO2B1 was confirmed by RT-PCR. In NCI-N87 ADCT-502-resistant cells, ABCG2 (91), SLC22A3 (65), ABCC2 (43), SLCO2B1 (30) and SLC7A7 (6.8) and in NCI-N87 SG3199-resistant cells ABCG2 (115), SLC22A3 (61), SLCO2B1 (54), ABCC2 (36), SLC7A7 (8), AQP7 (6.4) and SLC238A3 (2.9) were upregulated. Upregulation of ABCG2, ABCC2, SLCO2B1, SLC7A7 and SLC22A3 was confirmed by RT-PCR and for ABCG2 and ABCC2 also by western blot. ABCB1(MDR1) was not upregulated in any of the lines. These data show that acquired resistance to both PBD-ADCs and SG3199 can involve specific drug transporters. This has clinical implications as potential biomarkers of resistance and for the rational design of drug combinations. Citation Format: Simon Corbett, Francesca Zammarchi, Philip W Howard, Patrick H van Berkel, John A Hartley. The role of drug transporters in the acquired resistance to PBD dimer-containing antibody drug conjugates [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 4750.

Abstract 2194: Preclinical pharmacological profiling of ACP-5862, the major metabolite of the covalent BTK inhibitor acalabrutinib, displays intrinsic BTK inhibitory activity

Abstract Acalabrutinib is a potent and highly selective, oral covalent inhibitor of Bruton tyrosine kinase (BTK) that received accelerated approval for relapsed/refractory mantle cell lymphoma by the United States Food and Drug Administration in October 2017. ADME studies in humans, rat and dog, revealed extensive metabolism of acalabrutinib. The major circulating metabolite (M27, ACP-5862) was produced by CYP3A oxidation, resulting in a pyrrolidine ring-opened metabolite, with the butynamide electrophile still present. We investigated the on-target BTK inhibition and kinase selectivity profile of ACP-5862 using the same biochemical and cellular assays previously employed to profile acalabrutinib.1 The apparent BTK IC50 was determined over time using the LanthaScreen assay. Results indicate that ACP-5862, like acalabrutinib, is a covalent inhibitor of BTK. Binding kinetics derived from IC50 over time data, indicated that acalabrutinib and ACP-5862 have similar affinity (KI), however the BTK inactivation rate (kinact) for ACP-5862 was half that relative to acalabrutinib. This indicated a covalent BTK inhibition potency for ACP-5862 that was 2-fold lower than acalabrutinib. The overall kinome inhibition profile at 1 µM using KINOMEscan (Eurofins DiscoverX), as well as IC50 determinations on closely related kinases with a Cys in the same position as Cys481 in BTK (ITK, TXK, TEC, BMX, EGFR, ERBB2, ERBB4, BLK, JAK3), both revealed that the kinase selectivity profiles of acalabrutinib and ACP-5862 were similar. On-target inhibition in B cells was investigated measuring inhibition of B-cell antigen receptor-mediated activation of CD69 cell surface expression on human peripheral B cells using human peripheral blood mononuclear cells and human whole blood (hWB). The EC50 for anti-IgD-induced CD69 expression in hWB was 64 ± 6 nM for ACP-5862, compared to 9.2 ± 4.4 nM for acalabrutinib. The hWB EC90, representing near complete inhibition of BTK by acalabrutinib and ACP-5862 was observed at 72 ± 20 nM and 544 ± 376 nM, respectively. In conclusion, the results indicate that ACP-5862, the major metabolite of acalabrutinib, has intrinsic BTK inhibitory activity and a similar kinase selectivity profile as acalabrutinib. The contribution of ACP-5862 to on-target covalent inhibition of BTK in humans is unclear at present, but is most likely limited, since the EC90 in hWB for ACP-5862 approximated observed plasma Cmax in humans dosed with 100 mg acalabrutinib. The relative contribution of acalabrutinib and ACP-5862 to BTK pharmacodynamics is under further investigation. Terry Podoll and J. Greg Slatter are former Acerta Pharma employees. Reference 1. Barf T, Covey T, Izumi R, et al. Acalabrutinib (ACP-196): A covalent Bruton tyrosine kinase inhibitor with a differentiated selectivity and in vivo potency profile. J Pharmacol Exp Ther. 2017;363(2):240-252. Citation Format: Allard Kaptein, Terry Podoll, Gerjan de Bruin, Maaike Emmelot-van Hoek, Anouk de Jong, Bart van Lith, Niels Hoogenboom, Tjeerd Barf, Joseph Ware, J. Greg Slatter. Preclinical pharmacological profiling of ACP-5862, the major metabolite of the covalent BTK inhibitor acalabrutinib, displays intrinsic BTK inhibitory activity [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 2194.

Themis integrates T cell receptor and cytokine signals in CD8+ T cells

Abstract T cell receptor (TCR) signaling controls T cell development in the thymus and T cell functions in the periphery. Themis is a T-cell lineage-specific protein with a critical role in T cell development, where it regulates TCR signal strength though modulation of Shp1 phosphatase activity. The role of Themis in mature peripheral T cells is unknown. To address this, we generated Themis conditional knockout (cKO) mice, with post-selection Themis deletion mediated by distal Lck-Cre. The cKO mice have reduced CD8+ T cell numbers. Themis-deficient CD8+ T cells on the OT-I TCR transgenic background show normal functional responses to agonistic pMHC ligands of different affinities. However, Themis-deficient OT-I CD8+ T cells have reduced cell surface expression of CD5 and increased cell surface expression of CD8, suggesting decreased signal from self pMHC. Moreover, cKO OTI CD8+ T cells display severe reduction in lymphopenia-induced (homeostatic) proliferation in vivo, as well as reduction in proliferative responses to low affinity pMHC and pro-inflammatory cytokines in vitro. Signals from low affinity pMHC and cytokines synergistically induce phosphorylation of Akt, metabolic changes, and induction of c-Myc in CD8 T cells. This does not occur in the absence of Themis. Themis performs these functions through the phosphatase Shp1, as double deletion of Themis and Shp1 in peripheral T cells rescues the CD8+ T cell maintenance. This work shows a novel role for cytokines and very low affinity pMHC in altering cellular metabolism to drive CD8+ T cell proliferation. It also demonstrates that Themis and Shp1 act in concert to integrate T cell receptor and cytokine signals.

CD25 deficiency: A new conformational mutation prevents the receptor expression on cell surface

CD25 deficiency is a very rare autosomal recessive disorder that shows a clinical phenotype highly overlapping IPEX syndrome with an increased susceptibility to viral, bacterial, and fungal infections. It is due to mutations in the IL2Rα gene that codes for the α subunit of the IL2 receptor complex.Here we report the characterization of a novel IL2Rα gene mutation leading to a severe protein conformational alteration that abrogates its cell surface expression in a child presenting with early-onset IPEX-like disorder. Cytofluorimetric analysis revealed the total absence of CD25 cell surface expression and addressed IL2Rα molecular investigation.The early clinical and molecular diagnosis of CD25 deficiency in this patient promptly led to hematopoietic stem cell transplantation (HSCT), allowing complete resolution of the symptoms and definitive cure of the disease.

Physical fitness modulates the expression of CD39 and CD73 on CD4+ CD25- and CD4+ CD25+ T cells following high intensity interval exercise.

To investigate the impact of physical fitness on the mobilization of CD4+ CD25 - CD39 + and CD4 + CD25 + CD39 + T cells in response to acute exercise. Fifteen high physical fitness (25.3 ± 1.4 years) and 15 low physical fitness (26.1 ± 1.9 years) men performed a single bout of high-intensity interval exercise (HIIE, 10 bouts of 60 seconds at 85% HRmax intercepted by 75 seconds of recovery at 50% HRmax). Blood lymphocytes were isolated before, immediately after and 1 hour after exercise for assessment of cell surface expression of CD25, CD39, and CD73 on CD4+ T cells. Effector memory T cells (mTeff) were identified by CD4 + CD25 - CD39 + coexpression, and memory regulatory T cells (mTReg) were defined as CD4 + CD25 + CD39 + T cells. Exercise increased CD4+ and CD4 + CD25 + T cell frequencies immediately after followed by a decrease bellow to baseline values at 1 hour after the bout in both low and high physical fitness groups. At baseline, the proportions of mTeff were higher, while mTreg were lower in low physical fitness individuals. The frequency of mTreg increased immediately after HIIE in both groups, and remained higher 1 hour after the bout. However, high physical fitness individuals presented higher mTreg frequency in all periods evaluated. A significantly mobilization of mTeff cells was identified in both groups immediately after HIIE. High physical fitness individuals displayed a decrease in mTeff cells bellow to baseline, while the frequency of mTeff remained higher in low physical fitness group 1 hour after the bout. The peripheral frequency of CD4 + CD25 + CD73 + T cells increased in a similar way immediately after the bout in both groups, returning to the baseline values 1 hour after exercise. No differences in CD4 + CD25 - CD73 + T cells were observed after HIIE in both groups. Our results highlight the impact of physical activity status in the redistribution of CD4+ T cells expressing ectonucleotidases in response to HIIE.

The HIV gp41 Fusion Protein Inhibits T-Cell Activation through the Lentiviral Lytic Peptide 2 Motif.

The human immunodeficiency virus enters its host cells by membrane fusion, initiated by the gp41 subunit of its envelope protein. gp41 has also been shown to bind T-cell receptor (TCR) complex components, interfering with TCR signaling leading to reduced T-cell activation. This immunoinhibitory activity is suggested to occur during the membrane fusion process and is attributed to various membranotropic regions of the gp41 ectodomain and to the transmembrane domain. Although extensively studied, the cytosolic region of gp41, termed the cytoplasmic tail (CT), has not been examined in the context of immune suppression. Here we investigated whether the CT inhibits T-cell activation in different T-cell models by utilizing gp41-derived peptides and expressed full gp41 proteins. We found that a conserved region of the CT, termed lentiviral lytic peptide 2 (LLP2), specifically inhibits the activation of mouse, Jurkat, and human primary T-cells. This inhibition resulted in reduced T-cell proliferation, gene expression, cytokine secretion, and cell surface expression of CD69. Differential activation of the TCR signaling cascade revealed that CT-based immune suppression occurs downstream of the TCR complex. Moreover, LLP2 peptide treatment of Jurkat and primary human T-cells impaired Akt but not NFκB and ERK1/2 activation, suggesting that immune suppression occurs through the Akt pathway. These findings identify a novel gp41 T-cell suppressive element with a unique inhibitory mechanism that can take place post-membrane fusion.

IL-12 Enhances Immune Response by Modulation of Myeloid Derived Suppressor Cells in Tumor Microenvironment.

Myeloid derived suppressor cells (MDSCs) are a heterogenous population of immature cells that play a critical role in tumor associated immune suppression. In tumor conditions, the population of MDSCs increases. The main feature of these cells is their ability to suppress the T cell response in antigen specific or nonspecific manners depending on the condition of T cell activation. IL-12 can modulate MDSC in preliminary reports, so we investigated how IL-12 can affect MDSC in a tumor microenvironment. After implanting tumor based cells on syngeneic host, 4T-1/BALB/c or EL4/C57BL6 mice, MDSCs (Gr1+CD11b+) were isolated from splenocytes. Isolated MDSCs were treated with GM-CSF with or without IL-12 and analyzed based on their phenotypes and functions. Treatment of MDSC with IL-12 increased co-stimulatory molecules of CD80, CD86, OX-40L, enhancing the DC phenotype (CD11c) and maturation markers such as p-NF-κB and p-GSK3β. In addition to a change of surface markers, T-cell suppressive function of MDSC after IL-12 treatment was significantly improved compared with the control MDSC. In addition, PD-L1+F4/80+ macrophages, which show aninhibitory effect in phagocytosis, were decreased after IL-12 treatment. The changes of cell surface expression of CD80, CD86, MHC class II were also shown in vivo. Our results showed that the IL-12 can modulate MDSC into APC and recover the macrophage function. These results suggested that IL-12 plays a role in improving the tumor immune microenvironment through MDSC modulation.

Potency and Selectivity of BTK Inhibitors in Clinical Development for B-Cell Malignancies

Background: Bruton tyrosine kinase (BTK) is a validated target for B-cell malignancies. The BTK inhibitor ibrutinib was approved in chronic lymphocytic leukemia, mantle cell lymphoma (MCL), and Waldenstrom macroglobulinemia. Acalabrutinib is a potent, highly selective, covalent BTK inhibitor with minimal off-target activity; it received accelerated FDA approval in October 2017 for the treatment of patients with MCL having ≥1 prior therapy. In addition to the approved covalent BTK inhibitors ibrutinib and acalabrutinib, clinical data in B-cell malignancies are available for spebrutinib (CC-292), tirabrutinib (ONO/GS-4059) and zanubrutinib (BGB-3111). We performed biochemical and cellular profiling of these 5 BTK inhibitors, investigating potency and selectivity.Methods: Two biochemical kinase assays assessed BTK inhibitor potency, with IC50 determination at a fixed time point (IMAP; Molecular Devices) or over time (LanthaScreen; Invitrogen); the latter was used to calculate binding kinetics. Kinome profiling was performed at a single dose (1 µM) using KINOMEscan (Eurofins DiscoverX). IC50 determinations were made using kinases with a Cys in the same position as the Cys481 residue in BTK, using assays developed in house (using IMAP and LanthaScreen) or at Thermo Fisher Scientific (Z'-LYTE) with IC50 determination at a fixed time point.On-target inhibition of BTK in cellular assays was evaluated using B-cell receptor-mediated activation of CD69 expression on peripheral B cells using human peripheral blood mononuclear cells (hPBMCs) or human whole blood (hWB).Off-target inhibition of epidermal growth factor receptor (EGFR) was evaluated in a cellular assay examining the effect of EGF-induced EGFR phosphorylation in A431 cells.Off-target inhibition of ITK and/or TXK was evaluated using T-cell receptor (TCR)-mediated activation of interleukin-2 expression in Jurkat T cells and CD25 cell surface expression using primary human peripheral T cells.Results: Based on biochemical binding kinetics, ibrutinib and zanubrutinib were the most potent BTK inhibitors (Table 1), followed by spebrutinib; acalabrutinib and tirabrutinib had comparable potency. Differences in potency were largely driven by differences in inactivation rates. However, differences in biochemical potency were lost (in part) in cellular assays using hPBMCs or hWB. EC50 values in hWB were <10 nM for ibrutinib, acalabrutinib and zanubrutinib. Spebrutinib had an EC50 of 140 nM in hWB, showing the greatest loss of potency versus biochemical assays and hPBMCs, going to the physiologically most relevant cellular experimental conditions (Table 2).Differences in overall kinase selectivity were observed among the BTK inhibitors (Figure 1; KINOMEscan). Acalabrutinib had the lowest hit rate; 1.5% of human wild-type kinases were inhibited >65% at 1 µM (excluding BTK). Kinase hit rate was also low for tirabrutinb (2.3%), whereas ibrutinib (9.4%), zanubrutinib (4.3%), and spebrutinib (8.3%) had higher hit rates.Acalabrutinib had a high selectivity for BTK over kinases with a Cys in the same position as the Cys481 residue in BTK (Table 2). Similar results were observed for tirabrutinib, whereas ibrutinib, spebrutinib and zanubrutinib were less selective in this panel of kinases with potential for off-target covalent binding by BTK inhibitors (Table 2). Acalabrutinib also had a higher selectivity for BTK over Src-family kinases than the other BTK inhibitors tested.Acalabrutinib and tirabrutinib had EC50 values >10 µM in the cellular assay of off-target inhibition of EGFR; ibrutinib, zanubrutinib, and spebrutinib had EC50 values of 0.07, 0.39, and 4.7 µM, respectively.No off-target inhibition was observed for acalabrutinib or tirabrutinib on TCR-mediated activation of T cells up to 10 µM. Ibrutinib, zanubrutinib, and spebrutinib had EC50 values <1 µM in Jurkat T cells. Similar results were observed in primary human peripheral T cells.Conclusion: BTK inhibitors in clinical development for B-cell malignancies had differing potency in biochemical assays, but these differences were lost (in part) in cellular assays, particularly in hWB. Among the BTK inhibitors tested, the greatest differentiation was observed in kinase selectivity profiles; acalabrutinib and tirabrutinib had the highest kinase selectivity. [Display omitted] DisclosuresKaptein:Covaluation Pharma BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership; Apo-T BV: Consultancy. de Bruin:Acerta Pharma: Employment. Emmelot-van Hoek:Acerta Pharma: Employment. van de Kar:Acerta Pharma: Employment. de Jong:Acerta Pharma: Employment. Gulrajani:Acerta Pharma: Employment, Equity Ownership. Demont:Acerta Pharma: Employment. Covey:AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Mittag:Acerta Pharma: Employment, Equity Ownership. Barf:Covaluation Holding BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Monopoiesis in humans and mice.

Monocytes are a widely conserved cell population in vertebrates with important roles in both inflammation and homeostasis. Under both settings, monocytes continuously arise from hematopoietic progenitors in the bone marrow and, on demand, migrate into tissues through the bloodstream. Monocytes are classified into three subsets-classical, intermediate and non-classical-based on their cell surface expression of CD14 and CD16 in humans and Ly6C, CX3CR1 and CCR2 in mice. In tissues, monocytes differentiate further into monocyte-derived macrophages and dendritic cells to mediate innate and adaptive immune responses and maintain tissue homeostasis. Recently, the progenitors that strictly give rise to monocytes were identified in both humans and mice, thereby revealing the monocyte differentiation pathways.

Intrathymic Notch3 and CXCR4 combinatorial interplay facilitates T-cell leukemia propagation

Notch hyperactivation dominates T-cell acute lymphoblastic leukemia development, but the mechanisms underlying “pre-leukemic” cell dissemination are still unclear. Here we describe how deregulated Notch3 signaling enhances CXCR4 cell-surface expression and migratory ability of CD4+CD8+ thymocytes, possibly contributing to “pre-leukemic” cell propagation, early in disease progression. In transgenic mice overexpressing the constitutively active Notch3 intracellular domain, we detect the progressive increase in circulating blood and bone marrow of CD4+CD8+ cells, characterized by high and combined surface expression of Notch3 and CXCR4. We report for the first time that transplantation of such CD4+CD8+ cells reveals their competence in infiltrating spleen and bone marrow of immunocompromised recipient mice. We also show that CXCR4 surface expression is central to the migratory ability of CD4+CD8+ cells and such an expression is regulated by Notch3 through β-arrestin in human leukemia cells. De novo, we propose that hyperactive Notch3 signaling by boosting CXCR4-dependent migration promotes anomalous egression of CD4+CD8+ cells from the thymus in early leukemia stages. In fact, in vivo CXCR4 antagonism prevents bone marrow colonization by such CD4+CD8+ cells in young Notch3 transgenic mice. Therefore, our data suggest that combined therapies precociously counteracting intrathymic Notch3/CXCR4 crosstalk may prevent dissemination of “pre-leukemic” CD4+CD8+ cells, by a “thymus-autonomous” mechanism.

Abstract 1006: Adipsin enhanced clonogenicity and proliferation of human breast cancer patient-derived xenograft cells

Abstract Introduction: Tumor microenvironment plays a key role in the development and progression of tumors. Although adipose tissue is a predominant component of tumor microenvironment in mammary tissue, and secretes various cytokines, chemokines and growth factors, roles of adipocytes in the breast cancers remain to be elucidated. Methods: In this study, we identified adipsin, an adipokine predominantly expressed in the adipose-derived stem cells (ADSCs) of breast cancer patients, enhanced the clonogenicity, proliferation, and cancer stem cell-like properties of human breast cancer patient-derived xenograft (PDX) cells. Human breast cancer PDXs were established by the engraftment of surgical specimens of the breast cancer patients into immunodeficient mice. Human mammary adipose-derived stem cells (ADSCs) were isolated from the enzymatically dissociated adipose tissues of the surgical specimens of breast cancer patients. Results: When ADSCs with diverse patient backgrounds were co-cultured with human breast cancer PDX cells, ADSCs significantly enhanced the clonogenicity of breast cancer PDX cells (n = 9; p &amp;lt; 0.05). We found that adipsin, an adipokine that is involved in the generation of C3a and C3b in the complement alternative pathway, was highly expressed in the cultured supernatants of ADSCs when compared to those of breast cancer PDX cells (n = 9; p &amp;lt; 0.05). Accordingly, the amount of C3a was significantly higher in the cultured supernatants of ADSCs when compared to those of breast cancer PDX cells (n = 9; p &amp;lt; 0.05). Suppression of adipsin by a specific inhibitor or knockdown in mammary ADSCs significantly suppressed the sphere-forming abilities of breast cancer PDX cells; reduced the mRNA expression levels of the cancer stem cell marker CD44 and the chemokine receptor CXCR4 (p &amp;lt; 0.01); and reduced the cell surface expression of CD44 in breast cancer PDX cells, suggesting that cancer stem cell-like properties were regulated by adipsin. Although the suppression of CXCR4 by a specific inhibitor showed a tendency to suppress the sphere-forming abilities of breast cancer PDX cells, the dual suppression of adipsin/C3a and CXCR4 by their specific inhibitors showed no additive effects on the suppression of the clonogenicity of breast cancer PDX cells. Conclusion: These observations suggest that ADSCs are important components of tumor microenvironment in human breast cancers, and secrete adipsin that functions as regulators of the clonogenicity, proliferation, and cancer stem cell-like properties of human breast cancer cells. Citation Format: Hideaki Goto, Yohei Shimono, Toru Mukohara, Yohei Funakoshi, Yoshinori Imamura, Seishi Kono, Shintaro Takao, Akira Suzuki, Hironobu Minami. Adipsin enhanced clonogenicity and proliferation of human breast cancer patient-derived xenograft cells [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 1006.

Targeting the CD40-Costimulation Pathway by CFZ533 Prevents the Cross–Talk Between Follicular T Helper Cells and B Cells

Introduction Blockade of the costimulatory molecule CD40 by the novel fully human, Fc-silent, IgG1 antibody CFZ533 (Novartis Pharma) has shown to inhibit acute alloreactivity in non-human primates and is currently being investigated for prevention of rejection in a calcineurin-inhibitor-free regimen in kidney transplant patients. CD40 on B cells interacts with its ligand on T cells and serves as a necessary co-stimulatory factor for B cell activation, which promotes B cell proliferation, immunoglobulin class switching and differentiation. Materials and Methods In this study, we examined the effect of CFZ533 on humoral immunity by studying the cross-talk of follicular T helper cells (Tfh) with B cells and subsequent B cell activation parameters. By flow cytometry we determined the blockade of CD40 by CFZ533 on peripheral B cells, and its ability to inhibit their proliferation and differentiation after allogeneic stimulation. Results and Discussion Cell surface expression of CD40 on B cells was blocked by CFZ533 in a dose dependent manner. At a concentration of 1 ug/ml a maximal saturation of >95% was measured. While completely blocked by CFZ533, the presence of CD40 on B cells was confirmed by another antibody (clone HB14) directed against another epitope. Our T and B cell function assays revealed that blockade of CD40 by CFZ533 (1 ug/ml) inhibited alloantigen driven B cell proliferation (up to 70% and 90% inhibition, respectively). Next, we determined the impact of blockade of CD40 by CFZ533 on the T-B cell cross-talk. In the absence of Tfh cells, plasmablast (CD19+/-, CD27+ CD38+) differentiation was inhibited when stimulated with polyclonal antigen SEB or alloantigen, while in the presence of these Tfh cells, these stimuli led to B cells differentiation into Ig producing plasma blasts and exhibition of memory B cell class switching (loss of IgD-expression), confirming the T cell dependency of plasma blast formation. CFZ533 (1 ug/ml) inhibited plasma blast differentiation by 80% (mean) and partly inhibited class switching of B cells, SEB vs SEB+CFZ: mean 17% vs 9%, p= 0.02, respectively). Additionally, CFZ533 (1 ug/ml) diminished the expression of other B cell costimulatory markers such as OX40-L (member of the TNFR/TNF superfamily) and CD86 (CD28 superfamily) which both are necessary for T cell activation and survival, was diminished/lower by 42% and 59%, p = 0.05 and p<0.01, respectively, suggesting an inhibition of T-B cell interaction. Conclusion Our data show that blockade of CD40-blockade by CFZ533 effectively inhibits the functional Tfh-B cell interaction required for T cell dependent B cell activation and differentiation.

Monocyte-Derived Dendritic Cells Differentiated in the Presence of Lenalidomide Display a Semi-Mature Phenotype, Enhanced Phagocytic Capacity, and Th1 Polarization Capability.

Lenalidomide is an analog of thalidomide, with potent anticancer activity demonstrated in several hematological malignancies. It has immunomodulatory properties, being able to enhance the activation of different types of immune cells, which results in antitumor activities. Dendritic cells (DCs) are pivotal in the immune response, and different immunotherapeutic approaches targeting these cells are being developed. Since little is known about the effect of lenalidomide on DCs, the goal of the present work was to investigate the phenotype and function of human monocyte-derived DCs differentiated in the presence of lenalidomide (L-DCs). Our results showed that L-DCs display a unique phenotype, with increased cell surface expression of some maturation markers such as CD1d, CD83, CD86, and HLA-DR. This phenotype correlates with a lower expression of the E3 ubiquitin-ligase MARCH-I in L-DCs, upregulating the cell surface expression of CD86 and HLA-DR. In addition, immature L-DCs express higher amounts of DC-SIGN on the cell surface than control immature DCs. After LPS stimulation, production of IL-6 and TNF-α was severely decreased, whereas IL-12 and IL-10 secretion was dramatically upregulated in L-DCs, compared to that in the controls. Functionally, L-DCs are more effectively recognized by NKT cells in cytotoxicity experiments. Furthermore, L-DCs display higher opsonin-independent antigen uptake capability than control DCs. Mixed lymphocyte reaction experiments showed that L-DCs could stimulate naïve CD4 T-cells, polarizing them toward a predominant Th1 phenotype. In summary, DCs derived from monocytes in the presence of lenalidomide present a semi-mature phenotype, increased phagocytic capacity, reduced production of proinflammatory cytokines, and the ability to polarize T-cells toward predominant Th1-type responses; these are qualities that might be useful in the development of new immunotherapeutic treatments.

FGFR signaling regulates resistance of head and neck cancer stem cells to cisplatin.

Patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) have poor prognosis with less than 1-year median survival. Platinum-based chemotherapy remains the first-line treatment for HNSCC. The cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self renewing cell populations that constitute the bulk of the tumor. A small population of CSC exists within HNSCC that are relatively resistant to chemotherapy and clinically predicted to contribute to tumor recurrence. These head and neck CSCs (HNCSC) are identified by high cell-surface expression of CD44 and high intracellular activity of aldehyde dehydrogenase (ALDH) and termed ALDHhighCD44high. Here, we performed microarray analysis in two HNSCC cell lines (UM-SCC-1, UM-SCC-22B) to investigate molecular pathways active in untreated and cisplatin-resistant ALDHhighCD44high cells. Gene set enrichment analysis and iPathway analysis identified signaling pathways with major implications to the pathobiology of cancer (e.g. TNFα, IFN, IL6/STAT, NF-κB) that are enriched in cisplatin-resistant ALDHhighCD44high cells, when compared to control cells. FGF2 was also enriched in cisplatin-resistant ALDHhighCD44high, which was confirmed by ELISA analysis. Inhibition of FGF signaling using BGJ398, a pan-FGF receptor (FGFR) small-molecule inhibitor, decreased ALDHhighCD44high alone in UM-SCC-1 and preferentially targeted cisplatin-resistant ALDHhighCD44high cells in UM-SCC-22B. These findings suggest that FGFR signaling might play an important role in the resistance of head and neck CSC to cisplatin. Collectively, this work suggests that some head and neck cancer patients might benefit from the combination of cisplatin and a FGFR inhibitor.

Tissue-Resident Memory CD8+ T Cells: From Phenotype to Function.

Tissue-resident memory CD8+ T cells are an important first line of defense from infection in peripheral non-lymphoid tissues, such as the mucosal tissues of the respiratory, digestive, and urogenital tracts. This memory T cell subset is established late during resolution of primary infection of those tissues, has a distinct genetic signature, and is often defined by the cell surface expression of CD69, CD103, CD49a, and CD44 in both mouse and human studies. The stimuli that program or imprint the unique gene expression and cell surface phenotypes on TRM are beginning to be defined, but much work remains to be done. It is not clear, for example, when and where the TRM precursors receive these signals, and there is evidence that supports imprinting in both the lymph node and the peripheral tissue sites. In most studies, expression of CD49a, CD103, and CD69 on T cells in the tissues appears relatively late in the response, suggesting there are precise environmental cues that are not present at the height of the acute response. CD49a and CD103 are not merely biomarkers of TRM, they confer substrate specificities for cell adhesion to collagen and E-cadherin, respectively. Yet, little attention has been paid to how expression affects the positioning of TRM in the peripheral tissues. CD103 and CD49a are not mutually exclusive, and not always co-expressed, although whether they can compensate for one another is unknown. In fact, they may define different subsets of TRM in certain tissues. For instance, while CD49a+CD8+ memory T cells can be found in almost all peripheral tissues, CD103 appears to be more restricted. In this review, we discuss the evidence for how these hallmarks of TRM affect positioning of T cells in peripheral sites, how CD49a and CD103 differ in expression and function, and why they are important for immune protection conferred by TRM in mucosal tissues such as the respiratory tract.

A surface antigen of Orientia tsutsugamushi activates human monocyte-derived dendritic cells via nuclear factor-kB & p38 mitogen-activated protein kinase pathways.

Background & objectives:Scrub typhus is a chigger-borne disease caused by Orientia tsutsugamushi. The immunological reactions to O. tsutsugamushi infection are not completely understood. In this study, we investigated the response of dendritic cells (DCs) to a major 56-kDa scrub typhus antigen Sta56.Methods:Monocyte-derived human DCs were incubated with different concentrations of recombinant Sta56 and analyzed for maturation based on phagocytic capacity, the ability to induce T-cell proliferation, expression of surface markers, cytokine secretion and activation of toll-like receptor (TLR)-dependent signalling pathways.Results:Treatment of DCs with Sta56 induced cell surface expression of CD80, CD83, CD86 and MHC Class II increased the production of interleukin-12 (IL-12) p40, IL-12 p70 and IL-10 and decreased DC phagocytic capacity. Furthermore, Sta56 increased the ability of DCs to activate T-cell proliferation and interferon-γ secretion. TLR4-specific antibodies neutralized Sta56-elicited effects on DC maturation, suggesting direct interaction between Sta56 and TLR4. Moreover, Sta56 activated nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK) signalling as evidenced by decrease in Sta56-induced cytokine production and surface marker expression by specific inhibitors helenalin and SB203580, respectively, and increase in IκBα and p38 phosphorylation and NF-κB-DNA binding.Interpretation & conclusions:Our results showed that the surface antigen of O. tsutsugamushi activated DCs through interaction with TLR4 and activation of MAPK and NF-κB signalling, suggesting Sta56 as a potential candidate molecule for the development of vaccine against scrub typhus.

Vitamin D3 supplementation and the IL-2/IL-2R pathway in multiple sclerosis: Attenuation of progressive disturbances?

Vitamin D3 upregulates IL-2 receptor alpha (IL2RA, CD25)-expression on CD4+ T cells in vitro. We investigated effects of 48-weeks vitamin D3 supplements on CD25-expression by CD4+ T cells of patients with multiple sclerosis (MS). There was no significant difference between the vitamin D3 (n=30) and placebo group (n=23) in IL2RA mRNA-expression by PBMC. Likewise, CD25 cell surface-expression by conventional or regulatory T cells (Treg) did not differ between groups, although Treg CD25-expression and circulating soluble-CD25 levels decreased significantly in the placebo but not vitamin D3-group. We speculate that vitamin D3 may promote the maintenance of CD25-related immune homeostasis in MS.

Metalloprotease-dependent activation of EGFR modulates CD44+/CD24- populations in triple negative breast cancer cells through the MEK/ERK pathway.

The CD44+/CD24- cell phenotype is enriched in triple negative breast cancers, is associated with tumor invasive properties, and serves as a cell surface marker profile of breast cancer stem-like cells. Activation of Epidermal Growth Factor Receptor (EGFR) promotes the CD44+/CD24- phenotype, but the specific signaling pathway downstream of EGFR responsible for this effect is not clear. The purpose of this study was to determine the role of the MEK/ERK pathway in the expansion of CD44+/CD24- populations in TNBC cells in response to EGFR activation. Representative TNBC cell lines SUM159PT (claudin-low) and SUM149PT (basal) were used to evaluate cell surface expression of CD44 and CD24 by flow cytometry in response to EGFR and MEK inhibition or activation. EGFR and ERK phosphorylation levels were analyzed by Western blotting. The relationship between EGFR phosphorylation and MEK activation score in basal and claudin-low tumors from the TCGA database was examined. Inhibition of ERK activation with selumetinib, a MEK1/2 inhibitor, blocked EGF-induced expansion of CD44+/CD24- populations. Sustained activation of ERK by overexpression of constitutively active MEK1 was sufficient to expand CD44+/CD24- populations in cells in which EGFR activity was blocked by either erlotinib, an EGFR kinase inhibitor, or BB-94, a metalloprotease inhibitor that prevents generation of soluble EGFR ligands. In basal and claudin-low tumors from the TCGA database, there was a positive correlation between EGFR_pY1068 and MEK activation score in tumors without genomic loss of DUSP4, a negative regulator of ERK, but not in tumors harboring DUSP4 deletion. Our results demonstrate that ERK activation is a key event in EGFR-dependent regulation of CD44+/CD24- populations. Furthermore, our findings highlight the role of ligand-mediated EGFR signaling in the control of MEK/ERK pathway output in TNBC tumors without DUSP4 loss.

Bitter Melon Enhances Natural Killer-Mediated Toxicity against Head and Neck Cancer Cells.

Natural killer (NK) cells are one of the major components of innate immunity, with the ability to mediate antitumor activity. Understanding the role of NK-cell-mediated tumor killing in controlling of solid tumor growth is still in the developmental stage. We have shown recently that bitter melon extract (BME) modulates the regulatory T cell (Treg) population in head and neck squamous cell carcinoma (HNSCC). However, the role of BME in NK-cell modulation against HNSCC remains unknown. In this study, we investigated whether BME can enhance the NK-cell killing activity against HNSCC cells. Our results indicated that treatment of human NK-cell line (NK3.3) with BME enhances ability to kill HNSCC cells. BME increases granzyme B accumulation and translocation/accumulation of CD107a/LAMP1 in NK3.3 cells exposed to BME. Furthermore, an increase in cell surface expression of CD16 and NKp30 in BME-treated NK3.3 cells was observed when cocultured with HNSCC cells. Collectively, our results demonstrated for the first time that BME augments NK-cell-mediated HNSCC killing activity, implicating an immunomodulatory role of BME. Cancer Prev Res; 10(6); 337-44. ©2017 AACR.