scholarly journals Sunitinib Exerts In Vitro Immunomodulatory Activity on Sarcomas via Dendritic Cells and Synergizes With PD-1 Blockade

2021 ◽  
Vol 12 ◽  
Author(s):  
Darina Ocadlikova ◽  
Mariangela Lecciso ◽  
Javier Martin Broto ◽  
Katia Scotlandi ◽  
Michele Cavo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cell Lines ◽  
Effector T Cells ◽  
Sarcoma Cell ◽  
Ifn Γ ◽  
Sarcoma Cells

BackgroundHigh-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD-1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in in vitro cocultures of sarcoma.MethodsThe human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-γ production, before and after nivolumab exposure, were analyzed.ResultsAlong with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib-treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-γ. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-γ-producing effector T cells.ConclusionsTaken together, our in vitro data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-γ-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.

PD-1/PD-1-Ligand Interaction Contributes to Immunosuppressive Microenvironment of Hodgkin Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 379-379
Author(s):  
Ryo Yamamoto ◽  
Momoko Nishikori ◽  
Toshio Kitawaki ◽  
Tomomi Sakai ◽  
Masakatsu Hishizawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Signaling System ◽  
Peripheral T Cells ◽  
Ifn Γ ◽  
Immunosuppressive Microenvironment

Abstract Programmed death-1 (PD-1), a member of the CD28 costimulatory receptor superfamily, inhibits T cell activity by providing a second signal to T cells in conjunction with signaling through the T-cell receptor. PD-1/PD-1 ligand (PD-L) signaling system is indicated to be involved in the functional impairment of T cells such as in chronic viral infection or tumor immune evasion. We hypothesized that this signaling system is also involved in the pathogenesis of Hodgkin lymphoma (HL). We examined expression of B7-H1 and B7-DC, two known PD-Ls, in lymphoid cell lines using RT-PCR and flow cytometry. They were expressed in HL and several T-cell lines, whereas most B-NHL lines lacked their expression. Immunohistochemical staining of HL tissues demonstrated that PD-Ls were also expressed in primary H/RS cells. As gene expression of B7-H1 and B7-DC was increased in Epstein-Barr virus (EBV)-transformed lymphoblastoid B-cell lines, we examined the effect of EBV latent membrane proteins on their gene regulation. By luciferase reporter assay, both LMP1 and LMP2A were shown to enhance promoter activity of B7-H1 and B7-DC genes. This finding implies that in cases of EBV-positive HL, latent membrane proteins may help H/RS cells escape from host immune surveillance by upregulating PD-L gene expression. We next analyzed PD-1 expression of tumor-infiltrating T cells of HL tissue samples by flow cytometry, and found that PD-1+ cells were elevated markedly in these cells. As HL patients are well recognized as having defective cellular immunity, we compared PD-1 expression level in peripheral blood T cells of HL patients with those of healthy volunteers and B-NHL patients. PD-1 was significantly elevated in peripheral T cells of HL patients compared to the other two groups. PD-1+ T cells were highest in patients with active disease, and tended to decline along with treatment. Although regulatory T cells are reported to play a part in the pathogenesis of HL, FOXP3+ T cells were not significantly elevated in peripheral T cells of HL patients, and PD-1+ T cells did not overlap with these regulatory population. To elucidate whether the PD-1/PD-L signaling pathway is functional in the immunosuppressive microenvironment of HL, we finally examined the effect of blockade of this pathway. After culturing bulk HL tumor cells with anti-PD-L blocking antibodies, IFN-γ production was measured by ELISA. Blockade of PD-Ls augmented IFN-γ production of HL-infiltrating T cells. We concluded that anti-tumor activity of HL-infiltrating T cells was inhibited via the PD-1/PD-L pathway, and this inhibition could be successfully relieved by PD-L blockade. Taken together, our observations indicate that “T-cell exhaustion” is essential to the pathogenesis of HL, and tumor-infiltrating T cells around H/RS cells seem to be kept in balance by this inhibitory signaling. Our findings provide a potentially effective and clinically applicable strategy for the immunotherapy of HL.

scholarly journals Apoptotic Cells for the Prevention of Cytokine Release Syndrome (CRS) in CAR T-Cell Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1626-1626
Author(s):  
Dror Mevorach ◽  
Veronique Amor ◽  
Yehudith Shabat
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Inflammatory Cytokines ◽  
Apoptotic Cells ◽  
Car T ◽  
Ifn Γ ◽  
Pro Inflammatory Cytokines

Abstract Background: Chimeric antigen receptor (CAR)-modified T cells with specificity against CD19 have demonstrated dramatic promise against highly refractory hematologic malignancies. Clinical responses with complete remission rates as high as 90% have been reported in children and adults with relapsed/refractory acute lymphoblastic leukemia (ALL). However, very significant toxicity has been observed and as many as 30% in average developing severe forms of CRS and possibly related neurotoxicity. CRS is occurring due to large secretion of pro-inflammatory cytokines, mainly from macrophages/monocytes, and resembles macrophage-activating syndrome and hemophagocytosis in response to CAR T-secreting IFN-g and possibly additional cytokines. To better understand the mechanisms leading to CRS and to treat or prevent it, we have developed in vitro and in vivo models of CRS with and without CAR-modified T cells. Early apoptotic cells that have been successfully tested for the prevention of acute GVHD, including in 7 ALL patients, were tested in these models for their effect on cytokines and CAR T cell cytotoxicity. Methods: CD19-expressing HeLa cells were used alone or with co-incubation with human macrophages for in vitro experiments and intraperitoneal experiments. Raji was used in vivo for leukemia induction. LPS and IFN-γ were used to trigger additional cytokine release. CD19-specific CAR-modified cells were used (ProMab) for anti-tumor effect against CD19-bearing cells. Cytotoxicity assay was examined in vivo using 7-AAD with flow cytometry and in vitro by survival curves and analysis of tumor load in bone marrow and liver. CRS occurred spontaneously or in response to LPS and IFN-γ. Mouse IL-10, IL-1β, IL-2, IP-10, IL-4, IL-5, IL-6, IFNα, IL-9, IL-13, IFN-γ, IL-12p70, GM-CSF, TNF-α, MIP-1α, MIP-1β, IL-17A, IL-15/IL-15R, and IL-7, as well as 32 human cytokines were evaluated by Luminex technology using the MAPIX system analyzer (Mereck Millipore) and MILLIPLEX Analyst software (Merek Millipore). Mouse IL-6Rα, MIG (CXCL9), and TGF-β1 were evaluated by Quantikine ELISA (R&D systems). Bone marrow and liver were evaluated using flow cytometry and immunohistochemistry. The IFN-γ effect was evaluated by STAT1 phosphorylation and biological products. Human macrophages and dendritic cells were generated from monocytes. Early apoptotic cells were produced as shown in GVHD clinical trial; at least 50% of cells were annexin V-positive and less than 5% were PI-positive. Results: Apoptotic cells had no negative effect in vitro or in vivo on CAR-modified T cells with specificity against CD19. There were comparable E/T ratios for CAR T in the presence or absence of apoptotic cells in vitro, and comparable survival curves in vivo. On the other hand, significant downregulation (p<0.01) of pro-inflammatory cytokines, including IL-6, IP-10, TNF-a, MIP-1α, MIP-1β, was documented. IFN-γ was not downregulated, but its effect on macrophages and dendritic cells was inhibited at the level of phosphorylated STAT1 and IFN-γ-induced expression of CXCL10 and CXCL9 was reduced. Conclusion: CRS evolves from several factors, including tumor biology, interaction with monocytes/macrophages/dendritic cells, and as a response to the CAR T cell effect and expansion. Apoptotic cells decrease pro-inflammatory cytokines that originate from innate immunity and inhibit the IFN-γ effect on monocyte/macrophages/ dendritic cells without harming IFN-γ levels or CAR-T cytotoxicity. Disclosures Mevorach: Enlivex: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Amor:Enlivex: Employment. Shabat:Enlivex: Employment.

Decreased Expression of Indoleamine 2,3-Dioxygenase 1 (IDO 1) in Dendritic Cells From Patients with Immune Thrombocytopenia (ITP) Correlates with Impaired Regulatory T Cells Development

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2770-2770
Author(s):  
Lucia Catani ◽  
Daria Sollazzo ◽  
Antonio Curti ◽  
Sara Trabanelli ◽  
Cecilia Evangelisti ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Dendritic Cells ◽  
Regulatory T Cells ◽  
Healthy Subjects ◽  
Effector T Cells ◽  
Pathogenetic Role ◽  
Chronic Itp ◽  
Ido1 Expression

Abstract Abstract 2770 Recent studies suggest a bi-directional interaction between regulatory T cells (Tregs) and dendritic cells (DCs). Despite their role as inducers of immunity, DCs may also be critical in maintaining tolerance to self-antigens by inducing Tregs. Specifically, Indoleamine 2,3-dioxygenase 1 (IDO 1) expressing DCs expand Tregs. In turn, Tregs, which express CTLA4, engage its receptors B7-1(CD80)/B7-2(CD86), thus inducing the expression of IDO1 enzyme in DCs. IDO1 expression leads to tryptophan depletion and immunosuppressive kynurenine enrichment in the tissue. Both factors drive the de novo generation of Tregs from CD4+ T cells. In addition, Tregs may modulate the maturation and/or function of DCs, which are required for the activation of effector T cells. Tregs out compete with naïve T cells in aggregating around DCs. After forming aggregates, Tregs specifically downregulate the expression of CD80 and CD86 on DCs. The result of the interaction between Tregs and DCs is the impaired capacity of the antigen presenting cells to establish long lasting interactions with effector T cells. Therefore, given the crucial role of DCs and Tregs in initiating, regulating, maintaining or repressing immune responses, investigations on their interaction is of great importance for better elucidating the pathogenesis of ITP. Of note, in ITP this interaction and its pathogenetic role have never been investigated in depth. In this paper, we enumerated and functionally characterized Treg subsets, exploring whether in ITP abnormal Tregs interactions with DCs and/or effector T cells might play a pathogenetic role. Specifically, we studied whether, in ITP patients: 1) DCs maturation is differentially modulated by Tregs from ITP patients as compared with healthy donors; 2) the mechanism of Tregs generation is altered. Forty adult ITP patients, newly diagnosed (14 cases) or with persistent (20 cases) or chronic ITP (9 cases) were studied after informed consent. At the time of the study, patients with persistent or chronic ITP were out off therapy by at least two months. None of the patients were splenectomized. The median platelet count at the time of the study was 53×109/L (range 8–99). Allogeneic Mixed Leukocyte Reaction (MLR) was performed to test the suppressive activity of Tregs. To analyze the in vitro ability of highly purified CD4+CD25+ T cells to inhibit DCs maturation, normal immature CD14-derived DCs were cultured alone and with allogeneic CD4+CD25+ T cells from healthy subjects or ITP patients in the presence of Lipopolysaccaryde. CD80 and CD86 expression of CD14-derived DCs was then tested at flow cytometry. To evaluate the in vitro conversion of non Tregs into Tregs, CD4+CD25- T cells were cultured alone and with autologous mature CD14-derived DCs from ITP patients and healthy subjects. The percentages of CD4+CD25highFoxP3+ T cells were quantitated at flow cytometry. mRNA IDO1 expression of immature and mature CD14-derived DCs was then evaluated by Real-time RT-PCR. To determine IDO1 enzyme activity kynurenine levels were measured in the supernantants of mature CD14-derived DCs. We found that in ITP Tregs show lower ability to suppress T cell proliferation and to inhibit CD14-derived DCs maturation because they do not affect the expression of the costimulatory molecules CD80 and CD86. We show that the absolute number of Tregs was significantly decreased in ITP patients in comparison to healthy subjects (CD4+CD25highFoxp3+ T cells (5.5±4.3 vs 11.6±6.9 cells/microL; p below 0.01) and CD4+CD25highCD127low/negative T cells (50.9±27.3 vs 80.5±37.7 cells/microL; p below 0.02)). In addition, in ITP patients we document that the low number of circulating Tregs may be due to the reduced ability of mature CD14-derived DCs to convert non-Treg cells (CD4+CD25-) into Tregs (CD4+CD25highFoxP3+ Tregs). This is related to reduced expression and activity of the IDO1 enzyme in mature CD14-derived DCs from ITP patients, since DCs expressing IDO1 favour Treg generation. In conclusion, taken together our data demonstrate that in ITP the cross-talk between Tregs and DCs is impaired and plays a pathogenetic role. As a consequence, we found the generation of more immunogenic DCs and defective Tregs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals AB0045 ULTRASOUND GUIDED BIOPSIES OF RA JOINTS AT TIME OF CLINICAL DIAGNOSIS CONTAIN PROFOUND T CELL CLONALITIES

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1325.1-1325
Author(s):  
S. Turcinov ◽  
E. Af Klint ◽  
A. De Bondt ◽  
M. S. Mia ◽  
A. Catrina ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd4 T Cells ◽  
Clonal Expansion ◽  
Ultrasound Guided ◽  
Alpha Beta

Background:Rheumatoid arthritis (RA) is a disease characterized by synovial joint inflammation, mainly affecting small joints. Histological findings in synovial biopsies ranges from inflammatory infiltration including ectopic lymphoid structures, to a cell sparse fibroid phenotype. T cells in affected joints are non-naïve and have by flow cytometry approaches been shown to have a wide TCR-beta chain gene usage. New technologies allow for analyses of paired TCR sequences and their antigen-specificities.Objectives:To study the alpha/beta-T cell receptor repertoire in single sorted T cells from synovial biopsies at time of RA-diagnosis.Meth ods:Synovial biopsies were taken, primarily using an ultrasound guided technique, from seventeen patients (12 ACPA+, 5 ACPA-) with rheumatoid arthritis. Fresh biopsies were enzymatically digested, followed by mild mechanical treatment, prior to flow cytometry cell sorting. Single cell index sorting of T cells was made into 384-well plates with PCR-buffer followed by a nested PCR and deep sequencing of the TCR amplicons. TCR-receptor sequences showing clonal expansion from four ACPA+ HLA-DRB1*0401 patients were further cloned into SKW3 cells for studies of their reactivity byin vitrostimulation with peptides of viral and citrullinated origin from the literature. A positive response, as measured by CD69-up regulation or IL-2 production, was used to define specificity.Results:Fourteen of the assessed joints were small (1 MTP, 4 MCP and 8 wrists), whereas the remaining three were large joints (2 knees and 1 ankle), table 1. Individual T cells could be isolated from all of these biopsies, with a variating CD4:CD8 ratio. Based on the flow cytometry phenotyping we could identify CD4 T cells of both Treg and T peripheral helper phenotype already at this early time point. Productive alpha/beta-TCR sequences could be retrieved from 16 out of 17 patients and clonal expansion (>1 copy/TCR) was seen in all but one of these patients, with clone sizes ranging between 2 – 34 copies of each TCR.Table 1.Patient characteristics.PatientsGender(F/M)HLA-SE allelesJointsJoint swelling prior to biopsy (months)Stiffness specific joint (median VAS)Pain specific joint (median VAS)ACPA+ (n = 12)9/3*0401, *0404, *0408, *01 and *101 MTP, 4 MCP, 6 wrists, 1 knee4 (1-12)a46 (0-84)45 (22-99)ACPA- (n = 5)3/2*04011 MCP, 2 wrists, 1 ankle, 1 knee5 (0.25-7)59 (15-73)47 (33-81)SKW3 cell lines(patients n = 4)4/0*0401/0404 n=2*0401 n=21 MTP, 3 wrists2 (1-6)50.5 (42-84)50 (40-99)aData not available for one patient. One patient with prior RA-diagnosis, but after 9 months of treatment remission lasting for 20 years.Artificial T cell lines were generated from the expanded clones of HLA-DRB1*04:01 RA subjects. Ourin vitrostimulation protocol identified virus specific CD4 T cells in all samples. So far, no citrulline reactivity has been found. HCMV, followed by HHV were the most commonly found viral reactivities, whereas others were found only in one donor (e.g. JCV, EBV). The majority of clones are thus “orphans”, to which we are still seeking the driving antigen.Conclusion:Clonally expanded T cells are found in the synovium of early RA patients and include virus-specific CD4+ T cells. Our data show that the local T cell repertoire is broad already at the time of RA diagnosisDisclosure of Interests:Sara Turcinov: None declared, Erik af Klint Paid instructor for: Abbvie (courses and lectures), An De Bondt Employee of: Janssen., Muhammad Sohel Mia: None declared, Anca Catrina: None declared, Frederik Stevenaert Employee of: Janssen, Vivianne Malmström Grant/research support from: VM has had research grants from Janssen Pharmaceutica

scholarly journals Functional specialization of gut CD103+ dendritic cells in the regulation of tissue-selective T cell homing

2005 ◽  
Vol 202 (8) ◽  
pp. 1063-1073 ◽  
Author(s):  
Bengt Johansson-Lindbom ◽  
Marcus Svensson ◽  
Oliver Pabst ◽  
Caroline Palmqvist ◽  
Gabriel Marquez ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Oral Route ◽  
Effector T Cells ◽  
Mesenteric Lymph Nodes ◽  
T Cell Homing ◽  
Almost All

Gut-associated lymphoid tissue (GALT) dendritic cells (DCs) display a unique ability to generate CCR9+α4β7+ gut-tropic CD8+ effector T cells. We demonstrate efficient induction of CCR9 and α4β7 on CD8+ T cells in mesenteric lymph nodes (MLNs) after oral but not intraperitoneal (i.p.) antigen administration indicating differential targeting of DCs via the oral route. In vitro, lamina propria (LP)–derived DCs were more potent than MLN or Peyer's patch DCs in their ability to generate CCR9+α4β7+ CD8+ T cells. The integrin α chain CD103 (αE) was expressed on almost all LP DCs, a subset of MLN DCs, but on few splenic DCs. CD103+ MLN DCs were reduced in number in CCR7−/− mice and, although CD8+ T cells proliferated in the MLNs of CCR7−/− mice after i.p. but not oral antigen administration, they failed to express CCR9 and had reduced levels of α4β7. Strikingly, although CD103+ and CD103− MLN DCs were equally potent at inducing CD8+ T cell proliferation and IFN-γ production, only CD103+ DCs were capable of generating gut-tropic CD8+ effector T cells in vitro. Collectively, these results demonstrate a unique function for LP-derived CD103+ MLN DCs in the generation of gut-tropic effector T cells.

The Antioxidant n-Acetylcysteine Blocks Direct Regulatory T Cell Mediated Suppression of CD4+ Effector T Cells In Vitro

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2765-2765
Author(s):  
Todd W. Kelley ◽  
Olga Efimova
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Effector T Cells ◽  
T Cell Subsets ◽  
Potential Contribution ◽  
Tgf Beta ◽  
Treg Suppression ◽  
Dose Dependent

Abstract Abstract 2765 Background: CD4+CD25+FOXP3+ regulatory T cells (Tregs) employ a range of suppressive strategies including factors that are cytotoxic to target CD4+CD25−FOXP3− effector T cells (Teff) such as perforin and granzyme B and those that suppress proliferation, differentiation and/or cytokine production including TGF-beta1, IL-10 and CTLA-4. The relative contribution of each of these mechanisms to Treg function is unclear but from the available data their importance appears context specific. Because T cells are very sensitive to the redox state of the microenvironment, and display a pattern of impaired activation under conditions of oxidative stress, we investigated the potential contribution of an oxidant dependent suppressive pathway on direct Treg mediated suppression of Teff in vitro using the antioxidant n-acetylcysteine (NAC) to block reactive oxidants. Methods: Tregs and Teff were derived from the spleens of 2–4 month old C57BL/6 mice maintained in pathogen free conditions. T cell subsets were isolated using magnetic bead based techniques. Purity was assessed by flow cytometry. For suppression assays, Tregs were co-cultured with CFSE-labeled Teff at the indicated ratios and stimulated with anti-CD3/CD28 coated beads for 3 days. Proliferation was measured by flow cytometric evaluation of CFSE dilutional staining. Suppression was calculated by comparing proliferation of Teff cultured alone to those co-cultured with Tregs (% suppression= 1 − Tregs:Teff/Teff alone). In other experiments, CFSE-labeled Teff were cultured alone in the indicated conditions with TGF-beta +/− NAC and proliferation was assessed by CFSE staining. Intracellular reactive oxygen species (ROS) were measured by flow cytometry using the redox sensitive cell permeable indicator dye 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (DCFDA). Results: The presence of NAC prevented Treg suppression of Teff in a dose dependent fashion at Treg:Teff ratios of 1:2, 1:1 and 2:1 (see figure). Suppression was significantly decreased at 0.1mM NAC (p<0.05 at 1:1; p<0.01 at 2:1) and essentially absent at 1mM NAC. Proliferation of Teff was markedly higher in the setting of 1mM NAC compared to conditions without NAC, even during co-culture with Tregs at a 2:1 ratio of Tregs:Teff. Treg suppression of Teff proliferation was dependent on TGF-beta as neutralizing antibodies reversed the effect (p<0.001). The presence of NAC was sufficient to overcome the suppressive effects of exogenous TGF-beta on CD3/CD28 stimulated Teff proliferation. Treatment of CD3/CD28 stimulated Teff with TGFbeta resulted in a significant dose dependent increase in the levels of intracellular ROS (p<0.0001 at 10ng/mL TGF-beta) that inversely correlated with the degree of proliferation. Conclusion: NAC blocks Treg mediated TGF-beta dependent suppression in vitro. This suggests that TGF-beta may function to suppress proliferation of Teff via a ROS dependent mechanism and raises the possibility that targeted delivery of antioxidants may have clinical utility for modulating the effects of Tregs in vivo. Disclosures: No relevant conflicts of interest to declare.

Ex vivo isolation of RCC-reactive CD8+ CTL clones from HLA-identical allogeneic donor T cell lines stimulated by CD80-transfected tumor cells

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 15625-15625 ◽  
Author(s):  
S. S. Tykodi ◽  
J. A. Thompson ◽  
B. M. Sandmaier ◽  
M. B. Maris ◽  
R. Storb ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Ex Vivo ◽  
Unrelated Donor ◽  
T Cell Antigens ◽  
Ifn Γ ◽  
T Cell Lines ◽  
Anti Tumor Activity

15625 Background: Regression of metastatic renal cell carcinoma (mRCC) is observed in a minority of patients treated by immunotherapies such as interleukin-2 (IL-2), interferon-a (IFN-a), or reduced-intensity allogeneic hematopoietic cell transplantation. However, the development of specific cellular immunotherapies for mRCC has been hindered by the lack of molecularly characterized T cell antigens with preferential expression on RCC cells. We have developed an ex vivo strategy for the isolation of RCC-reactive CD8+ CTL clones that may facilitate the identification of novel RCC-associated T cell antigens. Methods: RCC tumor lines were established from two patients with mRCC presenting to our institution for allogeneic HCT received from either an HLA-matched sibling or volunteer unrelated donor. Irradiated RCC tumor lines that were unmodified or transfected with a cDNA for human CD80 were used to stimulate responder CD8+ T cells isolated from pretransplant patient (autologous) or donor-derived (allogeneic) blood samples in mixed lymphocyte/tumor cell (MLTC) cultures supplemented with recombinant human IL-7 and IL-12 (stimulation #1) or IL-2 (2nd and subsequent stimulations). T cell lines with anti-tumor activity measured by IFN-γ ELISA were then cloned by limiting dilution. Results: After two or more in vitro stimulations, allogeneic CD8+ T cell lines stimulated by CD80- transfected RCC tumor cells, but not the other MLTC culture combinations tested demonstrated tumor-specific IFN-γ release. CD3+/CD8+/TCRaβ+ CTL clones with potent in vitro anti-tumor activity for unmodified RCC tumor were isolated from both sibling- and unrelated- donor derived T cell lines. Three such clones with unique specificities for allogeneic targets recognized the unmodified RCC tumor but not LCL or fibroblast target cells isolated from the same patient suggesting tumor-restricted expression of the target antigens. Conclusions: Ex vivo MLTC culture utilizing CD80-transfected RCC tumor and HLA- matched allogeneic responder CD8+ T cells warrants further study as a strategy to isolate CTL clones that may be used to identify novel RCC-associated T cell antigens. No significant financial relationships to disclose.

T Helper Type 1-Bias in a Regulatory T Cell-Deficient Mouse Model for Immune Thrombocytopenia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 524-524
Author(s):  
Tetsuya Nishimoto ◽  
Fumiaki Kumagai ◽  
Masayoshi Monno ◽  
Tsutomu Takeuchi ◽  
Masataka Kuwana
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Autoantibody Response ◽  
Ifn Γ ◽  
Culture Supernatants ◽  
Deficient Mice ◽  
Platelet Autoantibodies

Abstract Abstract 524 Background: Immune thrombocytopenia (ITP) is a T cell-mediated autoimmune disorder, in which IgG autoantibodies to platelet surface glycoproteins promote platelet clearance in the reticuloendthelial system. CD4+CD25+Foxp3+ regulatory T cells (Tregs) are known to play a crucial role in the maintenance of immune homeostasis to self-antigens. Several lines of recent evidence have shown that Tregs are decreased in number and are functionally impaired in patients with ITP. Recently, we have found that approximately one third of Treg-deficient mice spontaneously develop thrombocytopenia with increased platelet-associated IgG and proportion of reticulated platelets. Platelets eluates and culture supernatants of splenocytes prepared from thrombocytopenic mice contain IgG antibodies capable of binding to intact platelets, which are not detected in non-thrombocytopenic mice. The main target of anti-platelet autoantibodies is GPIb, and some mice also produce anti-GPIIIa antibodies. However, detailed mechanisms that elicit ITP during immune reconstitution through homeostatic proliferation in the absence of Tregs remain uncertain. Purpose: To evaluate T-helper (Th) cell balance that promotes anti-platelet autoantibody response in a Treg-deficient mouse model for ITP. Methods: Treg-deficient mice were prepared by inoculation of Treg-depleted CD4+ T cells obtained from BALB/c mice into syngeneic T cell-deficient nude mice. Platelet count was determined using flow cytometry 4 weeks after inoculation, and Treg-deficient mice with platelet count < 0.33 × 106/ul were regarded as ITP mice. Treg-deficient mice without thrombocytopenia were also used as a control. To evaluate cytokine profiles of Th cells, proportions of Th subsets in the freshly prepared splenic CD4+ T cells were evaluated by intracellular staining for IFN-γ, IL-4, and IL-17 followed by flow cytometry. Th1, Th2, and Th0 cells were defined as IFN-γ+IL-4−, IFN-γ−IL-4+, IFN-γ+IL-4+ cells, respectively, and Th17 and Th1/17 cells were defined as IFN-γ−IL-17+ and IFN-γ+IL-17+, respectively. In addition, CD4+ T cells were isolated from splenocytes using magnetic activated cell sorting, and were stimulated with phorbol 1,2-myristate 1,3-acetate and ionomycin for 4 days. The culture supernatants were subjected to a cytokine bead array to measure levels of interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17, interferon (IFN)-γ, and tumor necrosis factor (TNF). Finally, to determine IgG subclasses of anti-platelet autoantibodies, splenocyte culture supernatants were incubated with platelets derived from BALB/c mice, followed by incubation with fluorescence-conjugated antibodies to IgG1, IgG2a, IgG2b, or IgG3. Then, the antibodies bound to platelets was detected by flow cytometry. Results: Fourteen ITP mice and 8 control mice were used at 6–8 weeks after inoculation. The proportions of Th1, Th2, and Th0 cells did not differ significantly between ITP and control mice, while the Th1/Th2 ratio was significantly increased in ITP mice than in control mice (8.3 versus 3.2, p < 0.01). The proportions of Th17 and Th1/17 cells were comparable between ITP and control mice. There was no difference in the in vitro production levels of cytokines except IL-4, which was lower in ITP mice compared to control mice (140 versus 600 pg/ml, p = 0.02). Increase in the IFN-γ/IL-4 ratio was noted in the culture supernatants from ITP mice, compared to those from control mice (15.6 versus 9.2, p = 0.04). The Th1/Th2 ratio detected by flow cytometric measurement and the IFN-γ/IL-4 ratio in in vitro cultures were correlated with each other (r = 0.85, p < 0.01). IgG subclasses of anti-platelet autoantibodies were heterogeneous among individual ITP mice, but IgG2a was the predominant subclass in the majority of ITP mice. Interestingly, a high Th1/Th2 ratio was associated with production of IgG2b anti-platelet antibodies, while the mice with a low Th1/Th2 ratio produced IgG1 anti-platelet antibodies. Conclusions: These findings suggest that induction of IgG anti-platelet autoantibody response in Treg-deficient mice is associated with Th1 bias, which is analogous to the Th balance in patients with primary ITP. The Th1/Th2 balance may modulate the autoimmune responses during expansion of CD4+ T cells in the absence of Tregs. Disclosures: No relevant conflicts of interest to declare.

Programming of Donor T Cells Using Allogeneic Delta-like Ligand 4-Positive Dendritic Cells to Reduce Gvhd but Retain GVL Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 233-233
Author(s):  
Kazuhiro Mochizuki ◽  
Lijun Meng ◽  
Izumi Mochizuki ◽  
Qing Tong ◽  
Shan He ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Effector T Cells ◽  
Cd4 T Cell ◽  
Leukemic Cells ◽  
Alloreactive T Cells ◽  
Donor T Cells ◽  
Ifn Γ

Abstract Host antigen-presenting cells (APCs) are critical for inducing a potent graft-versus-leukemia (GVL) response after allogeneic hematopoietic stem-cell transplantation (allo-HSCT). In this setting, host APCs activate donor T cells to become effector T cells that recognize and react to antigens in malignant cells. However, alloreactive T cells also mediate graft-versus-host disease (GVHD), which causes significant morbidity and mortality after allo-HSCT. Many studies suggest that if alloreactive T cells have reduced capacity to expand in local tissues, they will be unable to trigger severe GVHD. Thus, it is possible that host APC induction of qualitative changes in donor T cells can potentially modify their anti-host toxicities while retaining the GVL effect. Here we report the establishment of a cellular programming approach that reduces the GVHD toxicity of donor T cells using host dendritic cells (DCs) that express high levels of Dll4 (named Dll4hi DCs). We have previously identified inflammatory Dll4hi DCs. They occurred in HSCT mice early during GVHD induction and had a greater ability than Dll4-negative DCs to induce IFN-γ and IL-17 in alloantigen-activated T cells. However, only approximately 0.03 X 105 Dll4hi DCs were recovered from one HSCT mouse. To provide adequate numbers of Dll4hi DCs for therapeutic translation, we developed a novel culture system capable of producing large number of Dll4hi DCs (about 100.0 X 105) from the bone marrow (BM) of one mouse using Flt3L and the TLR agonists lipopolysaccharide (LPS) and R848, which activate TLR4 and TLR7/8, respectively. Dll4hi DCs showed significantly different phenotype as compared to conventional DCs derived from GM-CSF-stimulated BM cells (named GM-DCs), as evidenced by expressing higher levels of Dll4, Ifnb, Il4, Il6 and Ido, and producing lower levels of iNOS and arginase I. When cultured with C57BL/6 (B6) mouse CD4+ T cells (H2b) at a T cell: DC ratio of 4:1 for 5 days, BALB/c mouse Dll4hi DCs (H2d) induced 3- to 5-fold more in frequency of alloreactive effector T cells producing high levels of IFN-γ and IL-17 compared to GM-DCs. Following transfer, allogeneic Dll4hi DC-induced CD4+ T cells were unable to mediate severe GVHD in BALB/c recipients, with all of them surviving 60 days after allo-HSCT. In contrast, both unstimulated B6 CD4+ T cells and allogeneic GM-DC-induced B6 CD4+ T cells caused lethal GVHD in all BALB/c recipients, indicating that GM-DCs could not be used for reducing the GVHD toxicity of donor CD4+ T cells. Mechanistic analysis showed that Dll4hi DC-induced CD4+ T cell recipients showed 2- to 6-fold less donor CD4+ T cells in the spleen, liver, and intestine 12 days after transplantation compared to unstimulated CD4+ T cell recipients. This reduction of Dll4hi DC-induced CD4+ T cells was associated with markedly increased apoptosis in recipient mice. IFN-γ production by Dll4hi DC-induced CD4+ T cells was essential for their anti-GVHD effects. Absence of T cell IFN-γ led to improved survival and expansion of Dll4hi DC-induced CD4+ T cells in transplant recipients and caused lethal GVHD. Finally, we demonstrated that Dll4hi DC-induced alloreactive T cells had acquired the ability to kill A20 leukemic cells in BALB/c recipients and control growth of P815 mastocytoma cells in the second model of BDF1 recipients, leading to significantly improved survival of mice receiving allo-HSCT. Furthermore, in the third mouse model of GVHD directed against minor histocompatibility antigens, B6 Dll4hi DC-induced C3H.SW CD8+ T cells produced high levels of IFN-γ, had reduced capacity to mediate GVHD in B6 recipients, but preserved GVL activity against C1498 myeloid leukemic cells. In summary, our findings demonstrate that in vitro Dll4hi DC programming represents a novel and effective platform to reduce toxicities of donor T cells. This strategy has several potential advantages compared to current and developing methods for the modification of donor T cells to reduce GVHD, including a relatively short period of culture, no requirement for T cell subset selection and no need of viral transduction. Importantly, this method may lead to new strategies that can produce large amount of leukemic cell-reactive donor T cells with decrease capability of causing severe GVHD. Disclosures No relevant conflicts of interest to declare.

scholarly journals The interaction of macrophage and non-macrophage tropic isolates of HIV-1 with thymic and tonsillar dendritic cells in vitro.

1996 ◽  
Vol 183 (4) ◽  
pp. 1851-1856 ◽  
Author(s):  
P U Cameron ◽  
M G Lowe ◽  
F Sotzik ◽  
A F Coughlan ◽  
S M Crowe ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cell Lines ◽  
Viral Entry ◽  
Response Selection ◽  
T Cell Lines ◽  
Hiv 1

Dendritic cells isolated from thymus and tonsil were tested for susceptibility to HIV-1 strains that are tropic for macrophages or for T cell lines. DCs were purified by cell sorting and before infection expressed high levels of CD4 and HLA-DR and lacked markers for T, B, NK cells, or macrophages. Viral entry and reverse transcription was found after pulsing with strains of HIV-1 that could infect macrophages. During the first 36 h the PCR signals for gag sequences increased in DCs and macrophages. In contrast little if any viral DNA was found after pulsing macrophages or DCs with HIV-1 that was able to infect T cell lines. DCs pulsed with HIV-1 were able to transmit infection to responding T cells during an allogeneic or superantigen response. Selection for virus able to infect lymphoid DCs and other DCs expressing CD4 and its transfer to T cells during subsequent immune responses may provide a mechanism for the observed predominance of macrophage-tropic HIV-1 after in vivo transmission.

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