Extracellular purine metabolism and signaling of CD73-derived adenosine in murine Treg and Teff cells

CD73-derived adenosine acts as potent inhibitor of inflammation, and regulatory T cells (Treg) have been shown to express CD73 as a novel marker. This study explored the role of endogenously formed adenosine in modulating NF-κB activity and cytokine/chemokine release from murine Treg and effector T cells (Teff) including key enzymes/purinergic receptors of extracellular ATP catabolism. Stimulating murine splenocytes and CD4+ T cells with anti-CD3/anti-CD28 significantly upregulated activated NF-κB in CD73−/− T cells (wild type: 4.36 ± 0.21; CD73−/−: 6.58 ± 0.75; n = 4; P = 0.029). This was associated with an augmented release of proinflammatory cytokines IL-2, TNF-α, and IFN-γ. Similar changes were observed with the CD73 inhibitor APCP (50 μM) on NF-κB and IFN-γ in wild-type CD4+ T-cells. Treatment of stimulated CD4+ T-cells with adenosine (25 μM) potently reduced IFN-γ release which is mediated by adenosine A2a receptors (A2aR). AMP (50 μM) also reduced cytokine release which was not inhibited by APCP. In Teff, A2aR activation (CGS21680) potently inhibited the release of IL-1, IL-2, IL-3, IL-4, IL-12, IL-13, IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), CCL3, and CCL4. However, in Treg, CGS21680 did not alter cytokine/chemokine release. In summary, CD73-derived adenosine tonically inhibits active NF-κB in CD4+ T-cells, thereby modulating the release of a broad spectrum of proinflammatory cytokines and chemokines. Downregulation of P2X7 and upregulation of CD73 in Treg after antigenic stimulation may be an important mechanism to maintain the ability of Treg to generate immunosuppressive adenosine.

Download Full-text

Granulocyte-Macrophage Colony-Stimulating Factor-Deficient Mice Have Impaired Resistance to Blood-Stage Malaria

Infection and Immunity ◽

10.1128/iai.69.1.129-136.2001 ◽

2001 ◽

Vol 69 (1) ◽

pp. 129-136 ◽

Cited By ~ 49

Author(s):

Julie Riopel ◽

MiFong Tam ◽

Karkada Mohan ◽

Michael W. Marino ◽

Mary M. Stevenson

Keyword(s):

Blood Stage ◽

Colony Stimulating Factor ◽

Necrosis Factor Alpha ◽

Gm Csf ◽

Tnf Α ◽

Macrophage Colony Stimulating Factor ◽

Ifn Γ ◽

Macrophage Colony ◽

Stimulating Factor ◽

Blood Stage Malaria

ABSTRACT The contribution of granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic and immunoregulatory cytokine, to resistance to blood-stage malaria was investigated by infecting GM-CSF-deficient (knockout [KO]) mice with Plasmodium chabaudi AS. KO mice were more susceptible to infection than wild-type (WT) mice, as evidenced by higher peak parasitemia, recurrent recrudescent parasitemia, and high mortality. P. chabaudiAS-infected KO mice had impaired splenomegaly and lower leukocytosis but equivalent levels of anemia compared to infected WT mice. Both bone marrow and splenic erythropoiesis were normal in infected KO mice. However, granulocyte-macrophage colony formation was significantly decreased in these tissues of uninfected and infected KO mice, and the numbers of macrophages in the spleen and peritoneal cavity were significantly lower than in infected WT mice. Serum levels of gamma interferon (IFN-γ) were found to be significantly higher in uninfected KO mice, and the level of this cytokine was not increased during infection. In contrast, IFN-γ levels were significantly above normal levels in infected WT mice. During infection, tumor necrosis factor alpha (TNF-α) levels were significantly increased in KO mice and were significantly higher than TNF-α levels in infected WT mice. Our results indicate that GM-CSF contributes to resistance to P. chabaudi AS infection and that it is involved in the development of splenomegaly, leukocytosis, and granulocyte-macrophage hematopoiesis. GM-CSF may also regulate IFN-γ and TNF-α production and activity in response to infection. The abnormal responses seen in infected KO mice may be due to the lack of GM-CSF during development, to the lack of GM-CSF in the infected mature mice, or to both.

Download Full-text

Interferon γ–independent Rejection of Interleukin 12–transduced Carcinoma Cells Requires CD4+ T Cells and Granulocyte/Macrophage Colony–stimulating Factor

Journal of Experimental Medicine ◽

10.1084/jem.188.1.133 ◽

1998 ◽

Vol 188 (1) ◽

pp. 133-143 ◽

Cited By ~ 41

Author(s):

Chiara Zilocchi ◽

Antonella Stoppacciaro ◽

Claudia Chiodoni ◽

Mariella Parenza ◽

Nadia Terrazzini ◽

...

Keyword(s):

T Cells ◽

Tumor Regression ◽

Tumor Rejection ◽

Interleukin 12 ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Ifn Γ ◽

Macrophage Colony ◽

Stimulating Factor

We analyzed the ability of interferon (IFN)-γ knockout mice (GKO) to reject a colon carcinoma transduced with interleukin (IL)-12 genes (C26/IL-12). Although the absence of IFN-γ impaired the early response and reduced the time to tumor onset in GKO mice, the overall tumor take rate was similar to that of BALB/c mice. In GKO mice, C26/IL-12 tumors had a reduced number of infiltrating leukocytes, especially CD8 and natural killer cells. Analysis of the tumor site, draining nodes, and spleens of GKO mice revealed reduced expression of IFN- inducible protein 10 and monokine induced by γ-IFN. Despite these defects, GKO mice that rejected C26/IL-12 tumor, and mice that were primed in vivo with irradiated C26/IL-12 cells, showed the same cytotoxic T lymphocyte activity but higher production of granulocyte/macrophage colony–stimulating factor (GM-CSF) as compared with control BALB/c mice. Treatment with monoclonal antibodies against GM-CSF abrogated tumor regression in GKO but not in BALB/c mice. CD4 T lymphocytes, which proved unnecessary or suppressive during rejection of C26/IL-12 cells in BALB/c mice, were required for tumor rejection in GKO mice. CD4 T cell depletion was coupled with a decline in GM-CSF expression by lymphocytes infiltrating the tumors or in the draining nodes, and with the reduction and disappearance of granulocytes and CD8 T cells, respectively, in tumor nodules. These results suggest that GM-CSF can substitute for IFN-γ in maintaining the CD8–polymorphonuclear leukocyte cross-talk that is a hallmark of tumor rejection.

Download Full-text

Dimethyl fumarate suppresses granulocyte macrophage colony-stimulating factor–producing Th1 cells in CNS neuroinflammation

Neurology Neuroimmunology & Neuroinflammation ◽

10.1212/nxi.0000000000000729 ◽

2020 ◽

Vol 7 (4) ◽

pp. e729 ◽

Cited By ~ 3

Author(s):

Farinaz Safavi ◽

Rodolfo Thome ◽

Zichen Li ◽

Guang-Xian Zhang ◽

Abdolmohamad Rostami

Keyword(s):

T Cells ◽

Dimethyl Fumarate ◽

Th1 Cells ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Healthy Donors ◽

Macrophage Colony Stimulating Factor ◽

Ifn Γ ◽

Macrophage Colony ◽

Stimulating Factor

ObjectiveTo study the immunomodulatory effect of dimethyl fumarate (DF) on granulocyte macrophage colony-stimulating factor (GM-CSF) production in CD4+ T cells in experimental autoimmune encephalomyelitis (EAE) and human peripheral blood mononuclear cells (PBMCs).MethodsWe collected splenocytes and CD4+ T cells from C57BL/6 wild-type and interferon (IFN)-γ–deficient mice. For human PBMCs, venous blood was collected from healthy donors, and PBMCs were collected using the Percoll gradient method. Cells were cultured with anti-CD3/28 in the presence/absence of DF for 3 to 5 days. Cells were stained and analyzed by flow cytometry. Cytokines were measured by ELISA in cell supernatants. For in vivo experiments, EAE was induced by myelin oligodendrocyte glycoprotein35–55 and mice were treated with oral DF or vehicle daily.ResultsDF acts directly on CD4+ T cells and suppresses GM-CSF–producing Th1 not Th17 or single GM-CSF+ T cells in EAE. In addition, GM-CSF suppression depends on the IFN-γ pathway. We also show that DF specifically suppresses Th1 and GM-CSF–producing Th1 cells in PBMCs from healthy donors.ConclusionsWe suggest that DF exclusively suppresses GM-CSF–producing Th1 cells in both animal and human CD4+ T cells through an IFN-γ–dependent pathway. These findings indicate that DF has a better therapeutic effect on patients with Th1-dominant immunophenotype. However, future longitudinal study to validate this finding in MS is needed.

Download Full-text

Multiplex determination of murine seminal fluid cytokine profiles

Reproduction ◽

10.1530/rep.1.00959 ◽

2006 ◽

Vol 131 (3) ◽

pp. 613-621 ◽

Cited By ~ 29

Author(s):

Nadia Gopichandran ◽

Uma V Ekbote ◽

James J Walker ◽

David Brooke ◽

Nicolas M Orsi

Keyword(s):

Seminal Fluid ◽

Cell Mediated Immunity ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Inflammatory Protein ◽

Tnf Α ◽

Ifn Γ ◽

Macrophage Colony ◽

Antigen Presenting ◽

Stimulating Factor

Seminal fluid is known to be responsible for orchestrating mating-induced immunomodulation. Central to this process are numerous cytokines that modulate uterine leukocyte recruitment and trafficking. Despite this, a comprehensive analysis of the cytokine profile of murine seminal fluid is lacking. This study addressed this issue by using multiplex immunoassays to characterise the profile of interleukin (IL)-1α , IL-1β , IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, eotaxin, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon (IFN)-γ, keratinocyte-derived chemokine (KC), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α , MIP-1β , regulated upon activation normal T-cell expressed and secreted (RANTES), and tumour necrosis factor (TNF)-α in fluid drawn from the seminal vesicles of single mice (n = 18). Their levels and ratios were compared with those found in serum. IL-1α , IL-1β , IL-2, IL-5, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-17, GM-CSF, IFN-γ, MCP-1 and TNF-α levels were significantly higher in serum; IL-4, G-CSF, eotaxin, KC and RANTES exhibited the opposite trend. Based on these findings, we propose a model of mating-induced immunomodulation that implicates seminal eotaxin, RANTES and MIP-1α in the relocation and concentration of extravasated migrating endometrial eosinophils to the luminal epithelium. Furthermore, KC may participate in uterine neutrophil chemotaxis and activation. Eotaxin and MIP-α , together with IL-1β and IL-9, may also enhance further cytokine synthesis for endometrial antigen-presenting cell recruitment for processing paternal ejaculate antigens. IL-4 and G-CSF could also minimise deleterious cell-mediated immunity and modulate IFN-γ production, thereby supporting the establishment of pregnancy.

Download Full-text

Idiotype Immunization Combined With Granulocyte-Macrophage Colony-Stimulating Factor in Myeloma Patients Induced Type I, Major Histocompatibility Complex–Restricted, CD8- and CD4-Specific T-Cell Responses

Blood ◽

10.1182/blood.v91.7.2459.2459_2459_2466 ◽

1998 ◽

Vol 91 (7) ◽

pp. 2459-2466 ◽

Cited By ~ 6

Author(s):

Anders Österborg ◽

Qing Yi ◽

Lotta Henriksson ◽

Jan Fagerberg ◽

Susanne Bergenbrant ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Response ◽

Type I ◽

Gm Csf ◽

Histocompatibility Complex ◽

Macrophage Colony Stimulating Factor ◽

Ifn Γ ◽

Macrophage Colony ◽

Stimulating Factor

Idiotypic structures expressed on the myeloma Ig protein might be regarded as a tumor-specific antigen. Five patients with IgG myeloma were immunized with the purified serum M-component by repeated intradermal injections together with soluble granulocyte-macrophage colony-stimulating factor (GM-CSF). All patients developed an idiotype (Id)-specific T-cell immunity, defined as blood T cells predominantly secreting interferon-γ (IFN-γ) and interleukin-2 (IL-2) (type I cells). Id-specific DNA synthesis was induced in one patient. Delayed-type hypersensitivity against the Id was not evoked. The specific IFN-γ/IL-2 T-cell response was inhibited (46% to 100%) by a major histocompatibility complex (MHC) class I monoclonal antibody (MoAb) in all five patients. A 5% to 37% inhibition by an MHC class II MoAb was seen in four patients. CD4+ as well as CD8+ T cells enriched by magnetic microbeads contained Id-specific cells. The T cells recognized peptides corresponding to the complementarity-determining regions 1, 2, and 3 of the heavy chain of the Id. There was a transient rise of B cells producing IgM anti-idiotypic antibodies in all patients. The results indicate that immunization of myeloma patients using the autologous M-component and soluble GM-CSF may evoke an Id-specific predominantly MHC class I–restricted type I T-cell response.

Download Full-text

Cytokine and Chemokine Levels Correlate with Apoptosis Markers in Platelets of Pediatric Patients with Immune Thrombocytopenia

Blood ◽

10.1182/blood.v128.22.1371.1371 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1371-1371

Author(s):

Nadine Goelz ◽

Jeannine Winkler ◽

Julia J.M. Eekels ◽

Margaret L. Rand ◽

Oliver Speer ◽

...

Keyword(s):

T Cells ◽

Platelet Count ◽

Platelet Activation ◽

Plasma Levels ◽

Immune Thrombocytopenia ◽

Caspase 3 ◽

Gm Csf ◽

Cytokines And Chemokines ◽

Tnf Α ◽

Acute Itp

Abstract Immune thrombocytopenia (ITP) is defined as an autoimmune disease that leads to platelet clearance by macrophages. It is well known that auto-reactive B cells and CD4+ T-helper (Th) cells, and in particular their cytokines, have been associated with ITP. Cytokines and chemokines are key players in our immune response to recruit different cells, e.g. macrophages and monocytes, to respond to certain inflammatory signals. Specific cytokines (i.e. TNF-a, IFN-g) are known to induce apoptosis in nucleated blood cells. To understand the role of cytokines and chemokines in acute ITP, we studied their plasma levels in 10 pediatric ITP patients at diagnosis with a median platelet count of 3 x 109/L (range < 1 to 22 x 109/L) and compared them with controls: healthy children (n=9; platelet count > 150 x 109/L); and chemotherapy-induced thrombocytopenia patients (cTP; n=9; median platelet count of 12 x 109/L; range: 3-53 x 109/L). ITP patients fulfilled the criteria for acute ITP and presented with mild to moderate bleeding symptoms. The median age at diagnosis was 3.4 yrs (range: 1.6 - 6.5 yrs); blood samples were taken prior to treatment. Luminex technology was used to measure plasma levels of 42 cytokines and chemokines. Markers of platelet apoptosis - activated caspase-3, -8 and -9 - and of platelet activation - CD62P and CD63 expression and PAC-1 binding - were measured by flow cytometry. Distinct plasma cytokine/chemokine patterns were observed in ITP patients compared with controls. Significantly increased levels of the Th1 cell commitment cytokines TNF-α (p < 0.01) and IFN-g (p < 0.05), as well as of the Th2 cytokines IL-6 (p < 0.01), IL-10 (p < 0.01) and IL-13 (p < 0.05), were identified in ITP patients. We have previously shown that there is activation of platelet caspase-3, -8 and -9 at diagnosis in acute paediatric ITP patients compared with controls (Winkler et al, Br J Haematol 2012;156:508). In ITP patients, but not in controls, a negative correlation between eotaxin and caspase- 3 (r2 = 0.72), -8 (r2 = 0.76) and -9 (r2 = 0.53) activity were observed, as well as a negative correlations between GM-CSF and caspase-8 (r2 = 0.52) and -9 (r2 = 0.33). Furthermore, we found a correlation between IL-13 and platelet activation as measured by CD62P (r2 = 0.87) and CD63 (r2 = 0.67) expression in ITP patients but not in controls. In summary, increased plasma levels of the cytokines TNF-α, IFN-g, IL-6, IL-10, and IL-13 were observed in ITP patients at initial presentation, suggesting that these cytokines contribute to the pathogenesis of the disease. Since IL-6 and IFN-g are known to activate macrophages, while higher levels TNF-α, IL-10 and IL-13 in the plasma are signs for activated T cells, our findings are consistent with the current model of ITP, in which activated macrophages induce B and T cells to produce anti-platelet autoantibodies. Our goal is to study the interplay between the immune system and the reduction of platelet count in ITP and to further define apoptosis/activation-related pathways in ITP platelets. Furthermore, we showed a correlation in ITP patients between the chemokine eotaxin and GM-CSF with caspase-3, -8 and -9 activity, and a correlation between IL-13 and platelet activation. Our results imply, that apoptosis and platelet activation at diagnosis in ITP play a role in the development of ITP, but the underlying mechanisms are still unknown and needs to be further evaluated by increasing the patient cohort in our study. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Idiotype Immunization Combined With Granulocyte-Macrophage Colony-Stimulating Factor in Myeloma Patients Induced Type I, Major Histocompatibility Complex–Restricted, CD8- and CD4-Specific T-Cell Responses

Blood ◽

10.1182/blood.v91.7.2459 ◽

1998 ◽

Vol 91 (7) ◽

pp. 2459-2466 ◽

Cited By ~ 148

Author(s):

Anders Österborg ◽

Qing Yi ◽

Lotta Henriksson ◽

Jan Fagerberg ◽

Susanne Bergenbrant ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Response ◽

Type I ◽

Gm Csf ◽

Histocompatibility Complex ◽

Macrophage Colony Stimulating Factor ◽

Ifn Γ ◽

Macrophage Colony ◽

Stimulating Factor

Abstract Idiotypic structures expressed on the myeloma Ig protein might be regarded as a tumor-specific antigen. Five patients with IgG myeloma were immunized with the purified serum M-component by repeated intradermal injections together with soluble granulocyte-macrophage colony-stimulating factor (GM-CSF). All patients developed an idiotype (Id)-specific T-cell immunity, defined as blood T cells predominantly secreting interferon-γ (IFN-γ) and interleukin-2 (IL-2) (type I cells). Id-specific DNA synthesis was induced in one patient. Delayed-type hypersensitivity against the Id was not evoked. The specific IFN-γ/IL-2 T-cell response was inhibited (46% to 100%) by a major histocompatibility complex (MHC) class I monoclonal antibody (MoAb) in all five patients. A 5% to 37% inhibition by an MHC class II MoAb was seen in four patients. CD4+ as well as CD8+ T cells enriched by magnetic microbeads contained Id-specific cells. The T cells recognized peptides corresponding to the complementarity-determining regions 1, 2, and 3 of the heavy chain of the Id. There was a transient rise of B cells producing IgM anti-idiotypic antibodies in all patients. The results indicate that immunization of myeloma patients using the autologous M-component and soluble GM-CSF may evoke an Id-specific predominantly MHC class I–restricted type I T-cell response.

Download Full-text

Measurement of Phenotype and Absolute Number of Circulating Heparin-Binding Hemagglutinin, ESAT-6 and CFP-10, and Purified Protein Derivative Antigen-Specific CD4 T Cells Can Discriminate Active from Latent Tuberculosis Infection

Clinical and Vaccine Immunology ◽

10.1128/cvi.00607-14 ◽

2014 ◽

Vol 22 (2) ◽

pp. 200-212 ◽

Cited By ~ 15

Author(s):

Paul Hutchinson ◽

Timothy M. S. Barkham ◽

Wenying Tang ◽

David M. Kemeny ◽

Cynthia Bin-Eng Chee ◽

...

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Interleukin 2 ◽

Heparin Binding ◽

Content Type ◽

Purified Protein Derivative ◽

Gm Csf ◽

Tnf Α ◽

Latent Tb ◽

Ifn Γ

ABSTRACTThe tuberculin skin test (TST) and interferon gamma (IFN-γ) release assays (IGRAs) are used as adjunctive tests for the evaluation of suspected cases of active tuberculosis (TB). However, a positive test does not differentiate latent from active TB. We investigated whether flow cytometric measurement of novel combinations of intracellular cytokines and surface makers on CD4 T cells could differentiate between active and latent TB after stimulation withMycobacterium tuberculosis-specific proteins. Blood samples from 60 patients referred to the Singapore Tuberculosis Control Unit for evaluation for active TB or as TB contacts were stimulated with purified protein derivative (PPD), ESAT-6 and CFP-10, or heparin-binding hemagglutinin (HBHA). The CD4 T cell cytokine response (IFN-γ, interleukin-2 [IL-2], interleukin-17A [IL-17A], interleukin-22 [IL-22], granulocyte-macrophage colony-stimulating factor [GM-CSF], and tumor necrosis factor alpha [TNF-α]) and surface marker expression (CD27, CXCR3, and CD154) were then measured. We found that the proportion of PPD-specific CD4 T cells, defined as CD154+TNF-α+cells that were negative for CD27 and positive for GM-CSF, gave the strongest discrimination between subjects with latent and those with active TB (area under the receiver operator characteristic [ROC] curve of 0.9277;P< 0.0001). Also, the proportions and absolute numbers of HBHA-specific CD4 T cells were significantly higher in those with latent TB infection, particularly CD154+TNF-α+IFN-γ+IL-2+and CD154+TNF-α+CXCR3+. Finally, we found that the ratio of ESAT-6- and CFP-10-responding to HBHA-responding CD4 T cells was significantly different between the two study populations. In conclusion, we found novel markers ofM. tuberculosis-specific CD4 cells which differentiate between active and latent TB.

Download Full-text

Cytokines and Chemokines in Periodontitis

European Journal of Dentistry ◽

10.1055/s-0040-1712718 ◽

2020 ◽

Vol 14 (03) ◽

pp. 483-495 ◽

Cited By ~ 2

Author(s):

Doaa Elsayed Ramadan ◽

Ninuk Hariyani ◽

Retno Indrawati ◽

Rini Devijanti Ridwan ◽

Indeswati Diyatri

Keyword(s):

Immune System ◽

Proinflammatory Cytokines ◽

Alveolar Bone ◽

Interferon Γ ◽

Large Network ◽

Cytokines And Chemokines ◽

Tnf Α ◽

Wide Range ◽

Macrophage Colony ◽

Soft Tissue Inflammation

AbstractPeriodontitis is a common inflammatory periodontal disease affecting a wide range of population all over the world. The causing bacteria releases chemicals which activate the innate immune system to release proinﬂammatory cytokines contributing to more progression. This activates the acquired immune system leading to more progression of periodontitis. As the immune response goes on, released cytokines and chemokines can damage the periodontal ligaments, gingiva, and alveolar bone. There are many types of cytokines and chemokines in periodontitis. Cytokines are peptide mediators who are responsible for cell signaling and communication. Chemokines are a large subfamily of cytokines having the ability to coordinate leukocyte recruitment and activation. This paper is a narrative review of the literature.This review ensures that inflammatory mediators in the case of periodontitis can cause a noticeable damage in the whole apparatus of the periodontium. It causes soft tissue inflammation and bone damage affected by the mediators of both innate and acquired immune system.The inflammatory process is accompanied by large network of cytokines and chemokines. There is high expression of proinflammatory cytokines such as interleukin (IL)-1α, IL-1β, IL-6, IL-12, tumor necrosis factor (TNF)-α, and regulatory cytokines such as IL-4, IL-1(RA) receptor antagonist, IL-10, and induced protein (IP)-10. There is also increased production of cytokines IL-10, IL-12, interferon-γ, IP-10, IL-1RA, and IL-4. Cytokines IL-17, IL-6, IL-1β, TNF-α, macrophage colony-stimulating factor, and prostaglandin E2 trigger the osteoclast activity causing bone resorption.

Download Full-text

Genetically Modified DCs Engineered To Express PSA and/or PSMA Can Induce a Potent Immune Response Against Prostate Cancer Cells.

Blood ◽

10.1182/blood.v106.11.3075.3075 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3075-3075

Author(s):

Jagdeep S. Walia ◽

Jianhui Cai ◽

Daniel H. Fowler ◽

Jeffrey A. Medin4

Keyword(s):

Prostate Cancer ◽

Immune Response ◽

T Cells ◽

Immune System ◽

Tumor Cells ◽

Murine Model ◽

Cytolytic Activity ◽

Gm Csf ◽

Tnf Α ◽

Ifn Γ

Abstract Prostate cancer (Pca) is the most frequently diagnosed cancer in American men, with an estimated 230,110 cases expected in 2004. Despite various treatment strategies for patients including androgen ablation, radical prostatectomy, radiotherapy, and chemotherapy, the incidence of recurrence remains high and there is limited impact on survival, specially for metastatic disease. Our strategy involves the use of genetically-modified dendritic cells (DCs) to induce an immune response. We have previously demonstrated in a murine model that mature DCs engineered to express prostate tumor-associated antigens (TAAs) can stimulate immune system to specifically target TAA-expressing tumor cells. In view of the heterogenous nature of Pca, we hypothesized that stimulating the immune system against two antigens simultaneously may augment the anti-tumor activity. We generated murine DCs from whole bone marrow from mice by culturing them in granulocytemonocyte colony stimulating factor (GM-CSF) and IL-4 (20ng/ml each) and later with TNF-α. During the DC development, they were transduced with a concentrated oncoretrovirus that engineers the coexpression of prostate specific antigen (PSA) and CD25 (a cell surface marker for tranduced cells) (DC-PSA) or solely the expression of prostate specific membrane antigen (DC-PSMA). Transductions of DCs resulted in 30–60% expression of the either CD25 or PSMA as checked by flowcytometry. These DCs displayed high expression of DC markers like CD11c, CD80, CD86, CD40 and MHC class II molecules. There was no change in their allostimulatory capacity as checked by mixed lymphocyte reaction. Later, mice were injected either with DC non-transduced(NT), DC-PSA, with DC-PSMA. After two immunizations at different time points, the splenocytes were collected from all the groups one week after the last immunization. These splenocytes were stimulated to become effectors and were subsequently analysed to check for IFN-γ secretion, IL-10 secretion and cytolytic assays, using the targets as syngeneic murine prostate tumor cells, RM1 engineered to express PSA and PSMA. The effectors showed high IFN-γ and high cytolytic activity low IL-10 secretion as compared to controls. Our next step will be to test the increase of the levels of IFN-γ secretion and cytolytic activity in the mice immunized with DC-PSA and DC-PSMA both as compared to DC-PSA alone and DC-PSMA alone. To show clinically feasibility of our approach, we extended our work to human cells. HuDCs were generated using human CD34+ hematopoietic cells by culturing them in GM-CSF, SCF, Flt3L and TNF-α for 12 days. During DC production, they were transduced to express PSA or PSMA using a concentrated oncoretrovirus. They were checked for DC markers and the expression of the respective TAAs i.e PSA (CD25) or PSMA. Later, these cells were co-cultured with autologous T-cells. When these immunized T cells were used as effectors against the HLA-matched prostate cancer cell lines expressing PSA and PSMA, they showed high IFN-γ secretion and Low IL-10 secretion as compared controls. Thus, we have found that human DCs can be used to sensitize T cells to show antitumor responses and we are going to test in murine model the augmentation of such antitumour response by using multiple antigen immunotherapy approach.

Download Full-text