scholarly journals TOFACITINIB INHIBITS INFLAMMATORY CYTOKINES FROM ULCERATIVE COLITIS AND HEALTHY MUCOSAL EXPLANTS AND IS ASSOCIATED WITH P-STAT1/3 REDUCTION IN T-CELLS

2020 ◽  
Author(s):  
Rhonda M Brand ◽  
Beverly A Moore ◽  
Ashley Zyhowski ◽  
Aaron Siegel ◽  
Shikhar Uttam ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
T Cells ◽  
Logistic Regression ◽  
Mononuclear Cells ◽  
Dynamic Range ◽  
Ex Vivo ◽  
Target Engagement ◽  
Mixed Effect ◽  
Colorectal Tissue ◽  
Colorectal Biopsies

Introduction: Poor translatability of animal disease models has hampered development of new inflammatory bowel disorders (IBD) therapeutics. We describe a preclinical, ex vivo system using freshly obtained and well-characterized human colorectal tissue from ulcerative colitis (UC) and healthy control (HC) participants to test potential therapeutics for efficacy and target engagement, using the JAK/STAT inhibitor Tofacitinib (TOFA) as a model therapeutic. Methods: Colorectal biopsies from HC and UC were cultured and stimulated with multiple mitogens +/-TOFA. Soluble biomarkers were detected using a 29-analyte multiplex ELISA. Target engagement in CD3+CD4+ and CD3+CD8+ T cells was determined by flow cytometry in PBMC and isolated mucosal mononuclear cells (MMC) following activation of STAT1/3 phosphorylation. Data were analyzed using linear mixed effect modeling, t-test, and analysis of variance. Biomarker selection was performed using penalized and Bayesian logistic regression modeling, with results visualized using uniform manifold approximation and projection (UMAP). Results: Under baseline conditions, 27/29 biomarkers from UC were increased versus HC. Explant stimulation increased biomarker release magnitude, expanding the dynamic range for efficacy and target engagement studies. Logistic regression analyses identified the most representative UC baseline and stimulated biomarkers. TOFA inhibited biomarkers dependent on JAK/STAT signaling. STAT1/3 phosphorylation in T-cells revealed compartmental differences between PBMC and MMC. Conclusions: Immunogen stimulation increases biomarker release in similar patterns for HC and UC, while enhancing the dynamic range for therapeutic efficacy studies. This work demonstrates the power of ex vivo human colorectal tissue as preclinical tools for evaluating target engagement and downstream effects of new IBD therapeutic agents.

scholarly journals Targeting CD38 is lethal to Breg-like chronic lymphocytic leukemia cells and Tregs, but restores CD8+ T-cell responses

2020 ◽  
Vol 4 (10) ◽  
pp. 2143-2157 ◽  
Author(s):  
Alak Manna ◽  
Timothy Kellett ◽  
Sonikpreet Aulakh ◽  
Laura J. Lewis-Tuffin ◽  
Navnita Dutta ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Lymphocytic Leukemia ◽  
Dependent Manner

Abstract Patients with chronic lymphocytic leukemia (CLL) are characterized by monoclonal expansion of CD5+CD23+CD27+CD19+κ/λ+ B lymphocytes and are clinically noted to have profound immune suppression. In these patients, it has been recently shown that a subset of B cells possesses regulatory functions and secretes high levels of interleukin 10 (IL-10). Our investigation identified that CLL cells with a CD19+CD24+CD38hi immunophenotype (B regulatory cell [Breg]–like CLL cells) produce high amounts of IL-10 and transforming growth factor β (TGF-β) and are capable of transforming naive T helper cells into CD4+CD25+FoxP3+ T regulatory cells (Tregs) in an IL-10/TGF-β-dependent manner. A strong correlation between the percentage of CD38+ CLL cells and Tregs was observed. CD38hi Tregs comprised more than 50% of Tregs in peripheral blood mononuclear cells (PBMCs) in patients with CLL. Anti-CD38 targeting agents resulted in lethality of both Breg-like CLL and Treg cells via apoptosis. Ex vivo, use of anti-CD38 monoclonal antibody (mAb) therapy was associated with a reduction in IL-10 and CLL patient-derived Tregs, but an increase in interferon-γ and proliferation of cytotoxic CD8+ T cells with an activated phenotype, which showed an improved ability to lyse patient-autologous CLL cells. Finally, effects of anti-CD38 mAb therapy were validated in a CLL–patient-derived xenograft model in vivo, which showed decreased percentage of Bregs, Tregs, and PD1+CD38hiCD8+ T cells, but increased Th17 and CD8+ T cells (vs vehicle). Altogether, our results demonstrate that targeting CD38 in CLL can modulate the tumor microenvironment; skewing T-cell populations from an immunosuppressive to immune-reactive milieu, thus promoting immune reconstitution for enhanced anti-CLL response.

scholarly journals Oxidative stress response in regulatory and conventional T cells: a comparison between patients with chronic coronary syndrome and healthy subjects

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Anna K. Lundberg ◽  
Rosanna W. S. Chung ◽  
Louise Zeijlon ◽  
Gustav Fernström ◽  
Lena Jonasson
Keyword(s):  
Oxidative Stress ◽  
T Cells ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Thioredoxin Reductase ◽  
Healthy Controls ◽  
Antioxidant Genes ◽  
Consistent Finding ◽  
Coronary Syndrome ◽  
Thioredoxin Reductase 1

Abstract Background Inflammation and oxidative stress form a vicious circle in atherosclerosis. Oxidative stress can have detrimental effects on T cells. A unique subset of CD4+ T cells, known as regulatory T (Treg) cells, has been associated with atheroprotective effects. Reduced numbers of Treg cells is a consistent finding in patients with chronic coronary syndrome (CCS). However, it is unclear to what extent these cells are sensitive to oxidative stress. In this pilot study, we tested the hypothesis that oxidative stress might be a potential contributor to the Treg cell deficit in CCS patients. Methods Thirty patients with CCS and 24 healthy controls were included. Treg (CD4+CD25+CD127−) and conventional T (CD4+CD25−, Tconv) cells were isolated and treated with increasing doses of H2O2. Intracellular ROS levels and cell death were measured after 2 and 18 h, respectively. The expression of antioxidant genes was measured in freshly isolated Treg and Tconv cells. Also, total antioxidant capacity (TAC) was measured in fresh peripheral blood mononuclear cells, and oxidized (ox) LDL/LDL ratios were determined in plasma. Results At all doses of H2O2, Treg cells accumulated more ROS and exhibited higher rates of death than their Tconv counterparts, p < 0.0001. Treg cells also expressed higher levels of antioxidant genes, including thioredoxin and thioredoxin reductase-1 (p < 0.0001), though without any differences between CCS patients and controls. Tconv cells from CCS patients were, on the other hand, more sensitive to oxidative stress ex vivo and expressed more thioredoxin reductase-1 than Tconv cells from controls, p < 0.05. Also, TAC levels were lower in patients, 0.97 vs 1.53 UAE/100 µg, p = 0.001, while oxLDL/LDL ratios were higher, 29 vs 22, p = 0.006. Conclusion Treg cells isolated from either CCS patients or healthy controls were all highly sensitive to oxidative stress ex vivo. There were signs of oxidant-antioxidant imbalance in CCS patients and we thus assume that oxidative stress may play a role in the reduction of Treg cells in vivo.

scholarly journals The Anthrax Vaccine Adsorbed Vaccine Generates Protective Antigen (PA)-Specific CD4+ T Cells with a Phenotype Distinct from That of Naïve PA T Cells

2008 ◽  
Vol 76 (10) ◽  
pp. 4538-4545 ◽  
Author(s):  
William W. Kwok ◽  
Junbao Yang ◽  
Eddie James ◽  
John Bui ◽  
Laurie Huston ◽  
...  
Keyword(s):  
T Cells ◽  
Mononuclear Cells ◽  
Protective Antigen ◽  
Ex Vivo ◽  
Cytokine Profile ◽  
T Cell Epitopes ◽  
Peripheral Blood Mononuclear ◽  
Anthrax Vaccine ◽  
Cellular Immune

ABSTRACT Cellular immune responses against protective antigen (PA) of Bacillus anthracis in subjects that received the anthrax vaccine adsorbed (AVA) vaccine were examined. Multiple CD4+ T-cell epitopes within PA were identified by using tetramer-guided epitope mapping. PA-reactive CD4+ T cells with a CD45RA− phenotype were also detected by direct ex vivo staining of peripheral blood mononuclear cells (PBMC) with PA-specific tetramers. Surprisingly, PA-specific T cells were also detected in PBMC of nonvaccinees after a single cycle of in vitro PA stimulation. However, PA-reactive CD4+ T cells in nonvaccinees occurred at lower frequencies than those in vaccinees. The majority of PA-reactive T cells from nonvaccinees were CD45RA+ and exhibited a Th0/Th1 cytokine profile. In contrast, phenotyping and cytokine profile analyses of PA-reactive CD4+ T cells from vaccinees indicated that vaccination leads to commitment of PA-reactive T cells to a Th2 lineage, including generation of PA-specific, pre-Th2 central memory T cells. These results demonstrate that the current AVA vaccine is effective in skewing the development of PA CD4+ T cells to the Th2 lineage. The data also demonstrated the feasibility of using class II tetramers to analyze CD4+ cell responses and lineage development after vaccination.

scholarly journals Cytokine production in ex-vivo stimulated fresh and cryopreserved T-cells

2021 ◽  
Vol 67 (2) ◽  
pp. 95-101
Author(s):  
Monica Vuță ◽  
Ionela-Maria Cotoi ◽  
Ion Bogdan Mănescu ◽  
Doina Ramona Manu ◽  
Minodora Dobreanu
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Gradient Centrifugation ◽  
Intracellular Cytokine Staining ◽  
Interleukin 2 ◽  
Middle Aged

Abstract Objective: In vitro cytokine production by peripheral blood mononuclear cells (PBMCs) is an important and reliable measure of immunocompetence. PBMC can be stimulated directly after isolation or frozen for later use. However, cryopreservation may affect cell recovery, viability and functionality. This study aims to investigate cytokine synthesis in ex-vivo stimulated fresh and cryopreserved CD4+ and CD4- T cells. Methods: PBMCs were obtained by Ficoll gradient centrifugation from heparinized peripheral blood of 6 middle-aged clinically healthy subjects. Half of these cells (labeled “Fresh”) was further processed and the other half (labeled “Cryo”) was cryopreserved at -140°C for up to 3 months. Fresh-PBMCs were activated with Phorbol-Myristate-Acetate/Ionomycin/Monensin for 5 hours immediately after isolation while Cryo-PBMCs were identically activated after thawing and cell resting. Activated cells were fixed, permeabilized and intracellular cytokine staining was performed using Phycoerythrin (PE)-conjugated antibodies for Interleukin-2 (IL-2), Tumor Necrosis Factor-alpha (TNF-a), and Interferon-gamma (IFN-g). All samples were analyzed within 24 hours by flow cytometry. Results: Both Fresh and Cryo CD3+CD4+/CD3+CD4- sub-populations partially produced each of the three cytokines. A higher percentage of CD4+ T cells produced IL-2 and TNF-a and a greater percentage of CD4- T cells were found to produce IFN-g. A significantly higher percentage of Cryo-lymphocytes was shown to produce TNF-a in both CD3+CD4+ (31.4% vs 24.9%, p=0.031) and CD3+CD4- (22.7% vs 17.9%, p=0.031) subpopulations. No notable difference was found for IL-2 and IFN-g production between Fresh and Cryo T cells. Conclusion: Cryopreservation for up to 3 months significantly increases TNF-a production of T-cells in clinically healthy middle-aged subjects.

scholarly journals Oxidative Stress Response in Regulatory and Conventional T Cells: A Comparison Between Patients with Chronic Coronary Syndrome and Healthy Subjects

2020 ◽  
Author(s):  
Anna K Lundberg ◽  
Rosanna WS Chung ◽  
Louise Zeijlon ◽  
Gustav Fernström ◽  
Lena Jonasson
Keyword(s):  
Oxidative Stress ◽  
T Cells ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Thioredoxin Reductase ◽  
Healthy Controls ◽  
Antioxidant Genes ◽  
Consistent Finding ◽  
Coronary Syndrome ◽  
Thioredoxin Reductase 1

Abstract BackgroundInflammation and oxidative stress form a vicious circle in atherosclerosis. Oxidative stress can have detrimental effects on T cells. A unique subset of CD4+ T cells, known as regulatory T (Treg) cells, has been associated with atheroprotective effects. Reduced numbers of Treg cells is a consistent finding in patients with chronic coronary syndrome (CCS). However, it is unclear to what extent these cells are sensitive to oxidative stress. In the present study, we tested the hypothesis that oxidative stress might be a potential contributor to the Treg cell deficit in CCS patients. MethodsThirty patients with CCS and 24 healthy controls were included. Treg (CD4+CD25+CD127-) and conventional T (CD4+CD25-, Tconv) cells were isolated and treated with increasing doses of H2O2. Intracellular ROS levels and cell death were measured after 2 and 18 h, respectively. The expression of antioxidant genes was measured in freshly isolated Treg and Tconv cells. Alxo, total antioxidant capacity (TAC) was measured in fresh peripheral blood mononuclear cells. ResultsAt all doses of H2O2, Treg cells accumulated more ROS and exhibited higher rates of death than their Tconv counterparts, p < 0.0001. Treg cells also expressed higher levels of antioxidant genes, including thioredoxin and thioredoxin reductase-1 (p < 0.0001), though without any differences between CCS patients and controls. Tconv cells from CCS patients were, on the other hand, more sensitive to oxidative stress ex vivo and expressed more thioredoxin reductase-1 than Tconv cells from controls, p < 0.05. Also, TAC levels were lower in patients, 0.97 vs 1.53 UAE/100 µg, p = 0.001. ConclusionTreg cells isolated from either CCS patients or healthy controls were all highly sensitive to oxidative stress ex vivo. There were however signs of oxidant-antioxidant imbalance in CCS patients and we thus assume that oxidative stress plays a role in the reduction of Treg cells in vivo.

scholarly journals Ex Vivo Expanded Human Vγ9Vδ2 T-Cells Can Suppress Epithelial Ovarian Cancer Cell Growth

2019 ◽  
Vol 20 (5) ◽  
pp. 1139 ◽  
Author(s):  
Tsui Mao ◽  
Carol Miao ◽  
Yi Liao ◽  
Ying Chen ◽  
Chia Yeh ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Γδ T Cells ◽  
Ovarian Cancer Cells ◽  
Cytotoxic Activities ◽  
Antitumor Effects ◽  
Peripheral Blood Mononuclear

γδ-T-cells have attracted attention because of their potent cytotoxicity towards tumors. Most γδ-T-cells become activated via a major histocompatibility complex (MHC)-independent pathway by the interaction of their receptor, Natural Killer Group 2 Member D (NKG2D) with the tumor-specific NKG2D ligands, including MHC class I-related chain A/B (MICA/B) and UL16-binding proteins (ULBPs), to kill tumor cells. However, despite their potent antitumor effects, the treatment protocols specifically targeting ovarian tumors require further improvements. Ovarian cancer is one of the most lethal and challenging female malignancies worldwide because of delayed diagnoses and resistance to traditional chemotherapy. In this study, we successfully enriched and expanded γδ-T-cells up to ~78% from peripheral blood mononuclear cells (PBMCs) with mostly the Vγ9Vδ2-T-cell subtype in the circulation. We showed that expanded γδ-T-cells alone exerted significant cytotoxic activities towards specific epithelial-type OVCAR3 and HTB75 cells, whereas the combination of γδ-T cells and pamidronate (PAM), a kind of aminobisphosphonates (NBPs), showed significantly enhanced cytotoxic activities towards all types of ovarian cancer cells in vitro. Furthermore, in tumor xenografts of immunodeficient NSG mice, γδ-T-cells not only suppressed tumor growth but also completely eradicated preexisting tumors with an initial size of ~5 mm. Thus, we concluded that γδ-T-cells alone possess dramatic cytotoxic activities towards epithelial ovarian cancers both in vitro and in vivo. These results strongly support the potential of clinical immunotherapeutic application of γδ-T-cells to treat this serious female malignancy.

A Randomized Phase II Study of Xcellerated T Cells™ with or without Prior Fludarabine Therapy in Patients with Relapsed or Refractory Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2410-2410
Author(s):  
James R. Berenson ◽  
Ivan M. Borrello ◽  
Ravi Vij ◽  
Asad Bashey ◽  
Thomas Martin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Magnetic Beads ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
New Drugs ◽  
M Protein ◽  
High Dose ◽  
T Cell Therapy

Abstract Background: T cells from myeloma subjects can be activated and expanded ex vivo using the Xcellerate™ Process, in which peripheral blood mononuclear cells are incubated with anti-CD3 and anti-CD28 antibody-coated magnetic beads (Xcyte™-Dynabeads®). In a previous study (Borrello et al., ASCO 2004), Xcellerated T Cells administered to myeloma subjects following high dose chemotherapy and autologous stem cell transplantation led to accelerated lymphocyte recovery and restoration of the T cell receptor repertoire. In the current study, subjects with relapsed or refractory myeloma were randomized to Xcellerated T Cells with or without one cycle of fludarabine prior to Xcellerated T Cells. Fludarabine is being used to assess the influence of lymphoablation on the anti-tumor and immune reconstitution effects of T cell therapy; it has previously been reported to have no significant activty in myeloma (Kraut et al., Invest. New Drugs, 1990). Methods: Approximately 30 subjects are planned to receive treatment. Each receives a single dose of 60–100 x 109 Xcellerated T Cells. Subjects on the fludarabine arm receive a single cycle (5 days at 25 mg/m2), completed 4 days prior to the Xcellerated T Cell infusion. Results: 17 subjects have been enrolled and 13 treated to date, with median last f/u visit of 28 days (range 0–140). Xcellerated T Cells were successfully manufactured in all subjects, with T cell expansion 136 ± 61 fold (mean ± SD), with 79.2 ± 13.8 x 109 cells infused, and final product 98.0 ± 2.0% T cells (n=13). There have been no reported serious adverse events related to Xcellerated T Cells. In the fludarabine arm, lymphocytes decreased from 1,228 ± 290/mm3 (mean ± SEM) to 402 ± 164 following fludarabine, and then increased to 1,772 ± 278 on Day 14 following T cell infusion (n=7). In the non-fludarabine arm, lymphocyte counts increased from 1,186 ± 252 to 3,204 ± 545 on Day 14 (n=4). Lymphocytes were comprised of both CD4+ and CD8+ T cells. Increases were observed in NK cells from 77 ± 26 to 121 ± 25, monocytes from 166 ± 44 to 220 ± 30 and platelets from 218 ± 16 to 235 ± 24 by Day 14 (n=11). In the non-fludarabine arm, neutrophils increased from 3.6 ± 0.9 to 4.8 ± 0.6 on Day 1. On the fludarabine arm, 3 of 6 subjects developed Grade 4 neutropenia and one developed Grade 3 thrombocytopenia. Seven subjects were evaluable for serum M-protein measurements to Day 28. One of three fludarabine treated subjects had an M-protein decrease of 38%. Conclusions: Xcellerated T Cells were well-tolerated and led to increased lymphocytes, including T cells and NK cells. Increases in other hematologic parameters, including neutrophils and platelets were also observed. In this patient population, fludarabine is lymphoablative and also can cause neutropenia and thrombocytopenia. The fludarabine schedule has been decreased from 5 to 3 days. A decrease in M-protein has been observed in one of three fludarabine-treated subjects; data on additional subjects will be presented.

Impaired Alloantigen Presenting Activity of Cord Blood Nucleated Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2203-2203
Author(s):  
Sandeep Chunduri ◽  
Dolores Mahmud ◽  
Javaneh Abbasian ◽  
Damiano Rondelli
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Solid Organ Transplantation ◽  
T Cell Response ◽  
Solid Organ ◽  
Cd34 Cells

Abstract Transplantation of HLA-mismatched cord blood (CB) nucleated cells has limited risk of severe acute graft-versus-host disease and graft rejection. This may depend on naïve T cells not yet exposed to many antigens and on immature antigen-presenting cells (APC) not delivering appropriate signals to allogeneic T cells. In order to test the APC activity of human circulating CB cells in-vitro, we initially used irradiated CB mononuclear cells (MNC) or immunomagnetically selected CD34+ cells, CD133+ cells, or CD14+ monocytes to stimulate the proliferative response of incompatible blood T cells in mixed leukocyte culture (MLC). CB MNC failed to induce allogeneic T cell proliferation, while CD34+ and CD133+ progenitors or CD14+ monocytes induced potent T cell alloresponses. Nevertheless, since allogeneic T cell response was not restored after depletion of CD3+ cells in the CB, nor the add-back of irradiated CB MNC to CD34+ or CD14+ stimulators inhibited allo-T cells, a direct suppressive effect of CB MNC was excluded. Allogeneic peripheral blood cytotoxic T-lymphocyte (CTL) responses were not induced after 7 days of stimulation with irradiated CB MNC, although after 4 weekly rechallenges with CB MNC, on average a 23% lysis of antigen-specific CB PHA-blasts was observed at the highest effector:target ratio (50:1). To test the tolerogenic potential of CB MNC, T cells initially exposed to CB MNC were rechallenged in secondary MLC with CB MNC, or CD34+ cells, or monocyte-derived dendritic cells (Mo-DC) generated in liquid culture with GM-CSF and IL-4. Allogeneic T cells were still unresponsive upon rechallenge with CB MNC, but proliferated upon 3 days of restimulation with CD34+ cells or Mo-DC from the same CB. Surprisingly, the supernatant of these latter MLCs did inhibit completely a 3rd party MLC. Instead, the supernatant of blood T cells that had been activated by CB CD34+ cells or Mo-DC both in primary and secondary MLC did not. These results show an impaired allo-APC activity of CB MNC but not CB CD34+ cells, and suggest that T cells releasing immunosuppressive cytokines may be activated by CB MNC and then expanded by a second more potent stimulation with professional APC. This hypothesis could explain the sustained engraftment of HLA-mismatched CB stem cell transplants in humans. Based on these results, the in-vivo or ex-vivo downregulation of T cell alloreactivity induced by CB MNC will be tested in experimental models of stem cell, as well as solid organ transplantation.

Isopentenyl Pyrophosphate (IPP), a Metabolite produced in Myeloma Cells Induces the Chemotaxis of γδT Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2766-2766
Author(s):  
Eishi Ashihara ◽  
Tatsuya Munaka ◽  
Shinya Kimura ◽  
Masaki Kanai ◽  
Hirohisa Abe ◽  
...  
Keyword(s):  
T Cells ◽  
Mononuclear Cells ◽  
Mevalonate Pathway ◽  
Ex Vivo ◽  
Time Lapse ◽  
Dependent Manner ◽  
Peripheral Blood Mononuclear ◽  
Isopentenyl Pyrophosphate ◽  
Γδt Cells ◽  
Cellular Analysis

Abstract Abstract 2766 γδT cells, which control the innate immune system, are classified into three subtypes on the basis of Vγ chain. Of these subtypes, Vγ2Vδ9 T cells display anti-tumor immunity. We have demonstrated that nitrogen-containing bisphosphonate (N-BP) treatment expands Vγ2Vδ9 T cells ex vivo and that these expanded cells can kill tumor cells in a major histocompatibility complex-unrestricted manner (Sato, Int J Cancer, 2005; Uchida, Biochem Biophys Res Commun, 2007; Sato, Cancer Immunol Immunother, 2008.). N-BP inhibits farnesyl pyrophosphate synthase in the mevalonate pathway, resulting in the accumulation of isopentenyl pyrophosphate (IPP), which is a stimulatory antigen for Vγ2Vδ9T cells. In the present study, we investigated the chemotactic factors for Vγ2Vδ9T cells by using a micro total analysis system-based microfluidic cellular analysis device (Kanai, Sens Actuators A, 2004; Munaka, Analyst, 2007.). This microchip possesses a minute-volume (240 nL) chamber integrated with a micro-sample injector that permits the injection of a small amount (several nL) of a solute (Figure 1). Because of the minute size of this chamber, a concentration gradient can be maintained free from the influence of fluid convection and stirring, and the solute can consequently spread in a diffusion-dependent manner. Therefore, administration of a humoral factor via the sample injector mimics its release from the cell surface. We first investigated whether the supernatant of RPMI8226 multiple myeloma (MM) cells treated with zoledronic acid (ZOL) induced chemotaxis of γδT cells. We treated RPMI8226 MM cells with ZOL (1 mM) overnight and collected the supernatant. Human γδT cells were obtained by the culture of peripheral blood mononuclear cells as previously reported (Uchida, Biochem Biophys Res Commun, 2007.), and these cells were cultured in the microchip. After the injection of supernatant, γδT cells migration was observed under a microscope and continuous time-lapse recording was performed for 30 min. γδT cells migrated toward the injector, indicating that the supernatant of ZOL-treated RPMI8226 cells includes a chemoattractant factor for γδT cells. We next applied soluble MICA (sMICA), sICAM-1, sVCAM-1, and IPP and examined the migration of γδT cells. Among them, sMICA and IPP were chemoattractive for γδT cells, and the velocity of γδT cell migration was increased by the injection of IPP compared to the solvent control (Figure 2). These observations indicate that IPP, a metabolite of the mevalonate pathway in MM cells, or sMICA is a chemotactic factor for γδT cells when the target MM ells are treated with ZOL. Disclosures: Munaka: Shimadzu Corporation: Employment. Kanai:Shimadzu Corporation: Employment. Abe:Shimadzu Corporation: Employment.

Robust Induction Of Th1-Like γδ T Cells With Anti-Myeloma Activity By Lenalidomide In Combination With HMB-PP As Well As Zoledronic Acid

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 129-129
Author(s):  
Takeshi Harada ◽  
Qu Cui ◽  
Shingen Nakamura ◽  
Hirokazu Miki ◽  
Asuka Oda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Zoledronic Acid ◽  
Cytotoxic Activity ◽  
Immune Cells ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Γδ T Cells ◽  
Bone Marrow Microenvironment

Abstract Multiple myeloma (MM) still remains incurable even with the implementation of novel therapeutic modalities, leading to the idea to develop various forms of immunotherapies. In this regard, γδ T cells bearing Vγ9Vδ2 TCR expanded from peripheral blood mononuclear cells (PBMCs) have attracted attention as potent effectors available in a novel immunotherapy against MM. Human Vγ9Vδ2 γδ T cells can be expanded ex vivo by aminobisphosphonates in combination with IL-2, and effectively target and impair MM cells. However, MM cells appear to protect themselves from external insults by immune cells in a unique bone marrow microenvironment created by the accumulation of mesenchymal stem cells/bone marrow stromal cells (BMSCs) with defective osteoblastic differentiation and acid-producing osteoclasts. To improve the therapeutic efficacy of γδ T cells, therefore, we need to develop a maneuver to effectively enhance the expansion and activity of γδ T cells while disrupting the MM cell-bone marrow interaction. Lenalidomide (Len), a novel immunomodulatory anti-MM agent, shows pivotal anti-MM activity by targeting immune cells as well as the interaction of MM cells and their surrounding cells in the bone marrow. The present study was undertaken to explore the efficacy of Len in combination with zoledronic acid (Zol) or a precursor of isopentenyl pyrophosphate (IPP) (E)-4 hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), a microbial antigen for Vγ9Vδ2 TCR, on the induction and expansion of Th1-like γδ T cells with enhanced cytotoxic activity against MM cells in the skewed bone marrow microenvironment in MM. When combined with Zol (1μM), clinically relevant doses of Len (around 1 μM) substantially expanded γδ T cells from PBMCs to the levels similar to IL-2 (100 U/ml). Len was able to expand γδ T cells more robustly in combination with HMB-PP (1 μM) than Zol from PBMCs from the majority of normal donors. However, Len alone did not show any significant effects on γδ T cell expansion and activation, suggesting a costimulatory role of Len on Zol or HMB-PP-primed γδ T cells. The surface expression of LFA-1, and the cytotoxicity-associated molecules NKG2D, DNAX accessory molecule-1 (DNAM-1; CD226) and TRAIL were up-regulated in the expanded γδ T cells. Although functional diversity has been demonstrated in γδ T cells expanded by various stimuli, Len in combination with either Zol or HMB-PP enhanced intracellular IFN-γ along with the surface NKG2D but not Foxp3 in γδ T cells at higher levels than IL-2, suggesting robust induction of Th1-like γδ T cells by Len. Importantly, γδ T cells expanded with the combinatory treatments with Len and Zol or HMB-PP exerted potent cytotoxic activity against MM cells but not normal cells surrounding MM cells in bone marrow samples from patients with MM. Such treatments with Len was able to maintain the cytotoxic activity of the γδ T cells against MM cells in acidic conditions with lactic acid, and restored their anti-MM activity blunted in the presence of BMSCs. Interestingly, the expanded γδ T cells markedly suppressed the colony formation in semi-solid methylcellulose assays of RPMI8226 and KMS-11 cells [81±1 (mean ± SD) vs. 0±0 and 40±1 vs. 16±4 colonies/dish, respectively, p<0.01], and decreased in size their side populations, suggesting targeting a drug-resistant clonogenic MM cells. These results collectively demonstrate that Len and HMB-PP as well as Zol are an effective combination for ex vivo expansion of Th1-like γδ T cells with potent anti-MM activity, and suggest that Len in combination with Zol may maintain their in vivo anti-MM activity in the bone marrow where MM cells reside. The present results warrant further study on Len-based immunotherapy with γδ T cells. Disclosures: No relevant conflicts of interest to declare.

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