A Novel Role for Histone Deacetylase 6 (HDAC6) in the Regulation of IL-10 and Immune Tolerance Mediated by Antigen-Presenting Cells (APCs).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1360-1360
Author(s):  
Fengdong Cheng ◽  
Hongwei Wang ◽  
Noreen Luetteke ◽  
Javier Pinilla ◽  
Alan Kozikowski ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Gene Transcription ◽  
Immune Tolerance ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Inflammatory Status ◽  
C Terminus ◽  
Antigen Presenting

Abstract Abstract 1360 Poster Board I-382 There is now an undisputed recognition that bone marrow (BM) derived APCs can induce T-cell activation as well as T-cell tolerance to tumor antigens. The inflammatory status of the APC at the time of tumor antigen presentation, rather than its phenotype, has been proposed as explanation for the induction of such divergent T-cell outcomes. The molecular basis by which the APC regulate this critical decision remain however to be fully elucidated. Recently, we have focused our efforts to mechanistically understand the regulation of inflammatory/anti-inflammatory genes in their natural setting, the chromatin substrate, and how changes at this level could influence the overall inflammatory status of the APC. In particular, we have studied the consequences of chromatin modification by deacetylation of histone tails (mediated by histone deacetylases) upon expression of IL-10, an immunosuppressive cytokine that plays a central role in tolerance induction. By utilizing a reporter gene carrying the IL10 promoter fused to a luciferase gene, and plasmids encoding Flag-tagged versions of specific HDACs, we found that among all the HDACs evaluated, overexpression of HDAC6 in the APC resulted in transcriptional activation of IL-10 gene expression. Conversely, knockdown of HDAC6 in APCs using shRNA specific for murine HDAC6 resulted in abrogation of IL-10 gene transcription in response to LPS, as compared to APCs transduced with nontarget control (NT). Similar results were found when APCs were treated with the hydroxamate-based selective HDAC6 inhibitors, compound 3 (ST-3-06) and compound 7 (ST-292). Treatment of APCs with either compound resulted in a dose-dependent inhibition of IL-10 production in response to LPS. This effect was specific for IL-10, since no inhibition of other cytokines was observed in HDAC6 inhibitor-treated cells. Next, we evaluated the antigen-presenting capabilities of APCs in which HDAC6 was either knocked down or pharmacologically inhibited. In vitro culture of these APCs with naïve CD4+ T cells specific for a MHC class II restricted epitope of influenza hemagglutinin (HA) in the presence of cognate HA peptide resulted in enhanced activation of antigen-specific T cells since they produce higher levels of IL-2 and IFN-g ƒnrelative to clonotypic T cells encountering antigen on control APCs. Importantly, APCs lacking HDAC6 were capable of restoring the responsiveness of tolerized CD4+ T cells isolated from tumor-bearing mice. Our results suggest a role for HDAC6 in positively regulating IL-10 gene transcription in APCs, an effect that it is opposite to the recently described role of HDAC11 as a negative regulator IL-10 gene transcription1. To address whether a potential crosstalk between these two HDACs could represent a novel mechanism to tightly regulate IL-10 gene expression, we first performed confocal studies that revealed that HDAC6 and HDAC11 indeed colocalize in the cytoplasm. Coimmunoprecipitation confirmed that HDAC6 and HDAC11 interact. Furthermore, by using Flag-tagged HDAC6 wild type (1-1215) or Flag-tagged HDAC6 mutants lacking the C-terminus domain we demonstrated that the C-terminus portion of HDAC6 is required for its interaction with HDAC11. Taken together, we have demonstrated for the first time that HDAC6 regulates IL-10 gene expression, an effect that influences the overall inflammatory status of APC and determines antigen-specific T-cell responses. Importantly, inhibition of HDAC6 in APC with specific HDAC inhibitors represents a novel therapeutic approach to tip the balance towards immune activation rather than immune tolerance, a critical decision with significant implications for cancer immunotherapy. 1Villagra et al. Nature Immunology, 10:92-100, 2009 Disclosures Pinilla: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; exelixis: Research Funding.

A Novel Role of Histone Deacetylase 11 (HDAC11) in the Regulation of IL-10 Production and Immune Tolerance Mediated by Antigen-Presenting Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1330-1330
Author(s):  
Hongwei Wang ◽  
Fengdong Cheng ◽  
D. Nguyen ◽  
I. Suarez ◽  
K. Wright ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Histone Deacetylase ◽  
Immune Tolerance ◽  
Antigen Presenting Cells ◽  
Enhanced Production ◽  
Inflammatory Status ◽  
Antigen Presenting

Abstract Antigen-presenting cells (APC) can induce T-cell activation as well as T-cell tolerance. The induction of such a divergent outcomes is determined by the inflammatory status of the APC at the time of encounter with antigen specific T-cells. The molecular basis by which the APC regulate this critical decision of the immune system remain not well understood. Chromatin modification induced by acetylation/deacetylation of histones plays an important role in regulation of gene transcription, including genes involved in the inflammatory response. Histone deacetylases, a set of enzymes involved in histone modification are molecular targets for histone deacetylase inhibitors (HDI), novel compounds being evaluated as anticancer drugs. Interestingly, in addition to their antitumor properties, HDI have been also shown to modulate inflammatory responses. We evaluated therefore whether treatment with the hydroxamic acid analogue pan-HDAC inhibitor LAQ824 could influence the inflammatory status of the APC and their ability to determine CD4+ T-cell priming versus tolerance. In vitro treatment of APCs with LAQ824 resulted in enhanced acetylation of histones H-2A, H-2B, H3 and H4, increased expression of the co-stimulatory molecule B7.2 and enhanced production of pro-inflammatory mediators such as IL-1a, IL-1-b, IL-6, IL-12, TNF-a and RANTES in response to LPS stimulation. To our surprise, a dose-dependent inhibition of IL-10 mRNA and protein was observed in APCs treated with LPS and LAQ824. Chromatin immune precipitation (CHIP) assays indicate that this particular effect of LAQ824 involves histone modifications at the IL-10-promoter level. Given this inhibitory effect of LAQ824 and the central role of IL-10 in immune tolerance, we asked next whether a specific histone deacetylase(s) could predominantly influence IL-10 gene expression. By utilizing a reporter gene carrying the IL10 promoter fused to a luciferase gene, plasmids coding for Flag-tagged versions of all HDACs and plasmids carrying siRNA for specific silencing of HDACs, we found that among all the HDACs evaluated, HDAC11 negatively regulates the production of IL-10 in APCs. Importantly, treatment of APCs with LAQ824 resulted in increased expression of HDAC 11, diminished IL-10 production and the generation of APCs that effectively prime naive CD4+ T-cells and restore the responsiveness of tolerized antigen-specific T-cells from lymphoma bearing hosts. Taken together, we have demonstrated for the first time that HDAC11, a member of the HDAC family with no prior defined physiological role, is involved in regulation of IL-10 gene expression. Furthermore, our findings that HDAC11 expression in APCs can be manipulated by treatment of these cells with LAQ824, points to HDAC11 as a novel therapeutic target to influence immune activation versus immune tolerance, a critical decision with significant implications in autoimmunity, transplantation and cancer immunotherapy.

Tubastatin A, a Selective HDAC6 Inhibitor, Enhances Antigen-Presenting Cell (APC) Function and Restores the Responsiveness of Anergic CD4+ T Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 520-520
Author(s):  
Hongwei Wang ◽  
Zi Wang ◽  
Fengdong Cheng ◽  
Karrune V. Woan ◽  
Jennifer Rock-Klotz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Critical Role ◽  
T Cell Tolerance ◽  
Cell Tolerance ◽  
Inflammatory Status ◽  
Cognate Antigen ◽  
Hdac6 Inhibitor ◽  
Antigen Presenting

Abstract Abstract 520 Histones play a critical role in transcriptional regulation, cell cycle progression and developmental events. Histone acetylation/deacetylation alters chromatin structure and affects transcription factor access to DNA. Histone deacetylase inhibitors (HDI) induce growth arrest, cellular differentiation, and apoptosis and are being pursued as anticancer drugs. Interestingly, in addition to their antitumor properties, HDI have also been shown to modulate inflammatory responses. Recently, we have found that HDAC6 is required for the production of the immunosuppressive cytokine IL-10 by APCs. Given the role of this cytokine in T-cell tolerance induction we asked whether inhibition of HDAC6 with Tubastatin A, a potent and selective HDAC6 inhibitor would influence the inflammatory status of APCs and their ability to determine activation versus tolerance of antigen-specific CD4+ T cells in vitro. First, treatment of peritoneal elicited macrophages (PEM) with increasing concentration of Tubastatin A resulted in enhanced tubulin acetylation which was accompanied by enhanced expression of co-stimulatory molecules in treated cells. In addition, Tubastatin-A treated PEM were unable to produce IL-10 and TNF-a in response to LPS stimulation. In sharp contrast, Tubastatin-A treated PEM produce higher level of the pro-inflammatory cytokines IL-12 and IL-6. Next, we evaluated the ability of Tubastatin A-treated APCs to present cognate antigen to naïve and tolerant CD4+ T-cells specific for a MHC class II restricted epitope of influenza hemagglutinin (HA). We found that treatment of PEM with Tubastatin A significantly enhanced their antigen-presenting capabilities leading to effective priming of naïve CD4+ T-cells confirmed by their increased production of IL-2 and IFN-g in response to cognate antigen. More importantly, Tubastatin-A treated APCs were able to restore the responsiveness of tolerant CD4+ T cells isolated from lymphoma bearing mice. Taken together, selective HDAC6 inhibition with Tubastatin A provides a novel therapeutic approach to induce inflammatory APCs and overcome the significant barrier that T-cell tolerance has imposed to effective lymphoma immunotherapy. Disclosures: No relevant conflicts of interest to declare.

JQ 1, a Selective Bromodomain Inhibitor, Decreased the Expression of the Tolerogenic Molecule PDL1 in Antigen-Presenting Cells (APCs) and Restores the Responsiveness of Anergic CD4+ T Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2749-2749 ◽  
Author(s):  
Hongwei Wang ◽  
Fengdong Cheng ◽  
Limin Xing ◽  
Xiaohong Zhao ◽  
Alejandro Villagra ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Inflammatory Cytokine ◽  
Conflicts Of Interest ◽  
Specific Inhibitor ◽  
Inflammatory Status ◽  
Bet Inhibitors ◽  
Cognate Antigen ◽  
Antigen Presenting

Abstract Bromodomain and extraterminal (BET) is a protein domain that recognizes acetylated lysine residues such as those on the N-terminal tails of histones. This recognition is often a prerequisite for protein-histone association, chromatin remodeling and gene transcription. The role of BET proteins in regulating the response of inflammatory cytokine genes through translation of histone marks is poorly understood. Given that the inflammatory status of the APC is critical in determining T-cell activation versus T-cell tolerance and that epigenetic modifications of specific genes in the APC play a key role in this process, we recently determined the functional consequences of inhibiting BET in APCs. First, we evaluated the effects of JQ 1, a selective small-molecule BET bromodomain inhibitor on APC’s function and its regulation of antigen-specific CD4+ T-cells response. In vitro treatment of peritoneal elicited macrophages (PEM) or bone marrow derived dendritic cells (DCs) with increasing concentrations of JQ 1 resulted in decreased expression and protein production of the anti-inflammatory cytokine IL-10 and IL-6 in response to LPS stimulation. At the concentration used, JQ 1 did not affect the viability of treated APCs. Second, analysis of the expression of MHC class molecules and co-stimulatory molecules revealed a decreased expression of the tolerogenic PDL1 molecule in JQ 1- treated APCs as compared to untreated APCs. Third, we evaluated the ability of JQ 1 treated APCs to present cognate antigen to naïve or tolerant antigen-specific CD4+ T-cells. We found that treatment of either PEM or DC with JQ 1 enhanced their antigen-presenting capabilities leading to effective priming of naïve CD4+ T-cells confirmed by their increased production of IL-2 and IFN-gamma in response to cognate antigen. More importantly, JQ 1- treated APCs were able to restore the responsiveness of tolerant CD4+ T-cells isolated from lymphoma bearing hosts. Taken together, we have found that APCs treated with the Bromodomain specific inhibitor JQ 1 are more inflammatory, display lower expression of the immunosuppressive molecule PDL1 and more importantly, are capable of restoring the responsiveness of tolerant T-cells. Our studies therefore have unveiled a previously unknown immunological effect of BET inhibitors and have broadened their clinical scope as promising adjuvants in cancer immunotherapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Response of naive antigen-specific CD4+ T cells in vitro: characteristics and antigen-presenting cell requirements.

1992 ◽  
Vol 176 (5) ◽  
pp. 1431-1437 ◽  
Author(s):  
M Croft ◽  
D D Duncan ◽  
S L Swain
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cd4 Cells ◽  
Peptide Antigen ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Antigen Presenting

Because of the low frequency of T cells for any particular soluble protein antigen in unprimed animals, the requirements for naive T cell responses in specific antigens have not been clearly delineated and they have been difficult to study in vitro. We have taken advantage of mice transgenic for the V beta 3/V alpha 11 T cell receptor (TCR), which can recognize a peptide of cytochrome c presented by IEk. 85-90% of CD4+ T cells in these mice express the transgenic TCR, and we show that almost all such V beta 3/V alpha 11 receptor-positive cells have a phenotype characteristic of naive T cells, including expression of high levels of CD45RB, high levels of L-selectin (Mel-14), low levels of CD44 (Pgp-1), and secretion of interleukin 2 (IL-2) as the major cytokine. Naive T cells, separated on the basis of CD45RB high expression, gave vigorous responses (proliferation and IL-2 secretion) to peptide antigen presented in vitro by a mixed antigen-presenting cell population. At least 50% of the T cell population appeared to respond, as assessed by blast transformation, entry into G1, and expression of increased levels of CD44 by 24 h. Significant contributions to the response by contaminating memory CD4+ cells were ruled out by demonstrating that the majority of the CD45RB low, L-selectin low, CD44 high cells did not express the V beta 3/V alpha 11 TCR and responded poorly to antigen. We find that proliferation and IL-2 secretion of the naive CD4 cells is minimal when resting B cells present peptide antigen, and that both splenic and bone marrow-derived macrophages are weak stimulators. Naive T cells did respond well to high numbers of activated B cells. However, dendritic cells were the most potent stimulators of proliferation and IL-2 secretion at low cell numbers, and were far superior inducers of IL-2 at higher numbers. These studies establish that naive CD4 T cells can respond vigorously to soluble antigen and indicate that maximal stimulation can be achieved by presentation of antigen on dendritic cells. This model should prove very useful in further investigations of activation requirements and functional characteristics of naive helper T cells.

scholarly journals Distinct Roles of α7 nAChRs in Antigen-Presenting Cells and CD4+ T Cells in the Regulation of T Cell Differentiation

2019 ◽  
Vol 10 ◽  
Author(s):  
Masato Mashimo ◽  
Masayo Komori ◽  
Yuriko Y. Matsui ◽  
Mami X. Murase ◽  
Takeshi Fujii ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cd4 T Cells ◽  
Antigen Presenting Cells ◽  
T Cell Differentiation ◽  
Antigen Presenting ◽  
Α7 Nachrs

scholarly journals CD4+ T-cell killing of multiple myeloma cells is mediated by resident bone marrow macrophages

2020 ◽  
Vol 4 (12) ◽  
pp. 2595-2605 ◽  
Author(s):  
Ole Audun W. Haabeth ◽  
Kjartan Hennig ◽  
Marte Fauskanger ◽  
Geir Åge Løset ◽  
Bjarne Bogen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Antigen Presenting Cells ◽  
Bone Marrow Microenvironment ◽  
Cd4 T Cell ◽  
Myeloma Cells ◽  
Antigen Presenting

Abstract CD4+ T cells may induce potent antitumor immune responses through interaction with antigen-presenting cells within the tumor microenvironment. Using a murine model of multiple myeloma, we demonstrated that adoptive transfer of idiotype-specific CD4+ T cells may elicit curative responses against established multifocal myeloma in bone marrow. This finding indicates that the myeloma bone marrow niche contains antigen-presenting cells that may be rendered tumoricidal. Given the complexity of the bone marrow microenvironment, the mechanistic basis of such immunotherapeutic responses is not known. Through a functional characterization of antitumor CD4+ T-cell responses within the bone marrow microenvironment, we found that killing of myeloma cells is orchestrated by a population of bone marrow–resident CD11b+F4/80+MHC-IIHigh macrophages that have taken up and present secreted myeloma protein. The present results demonstrate the potential of resident macrophages as powerful mediators of tumor killing within the bone marrow and provide a basis for novel therapeutic strategies against multiple myeloma and other malignancies that affect the bone marrow.

scholarly journals Second Class Minors

2003 ◽  
Vol 197 (3) ◽  
pp. 375-385 ◽  
Author(s):  
Hiroeki Sahara ◽  
Nilabh Shastri
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
T Cell Responses ◽  
Cd4 T Cell ◽  
Interleukin 4 ◽  
General Strategy ◽  
Mhc Ii ◽  
Cell Responses ◽  
Antigen Presenting

CD4 T cells regulate immune responses that cause chronic graft rejection and graft versus host disease but their target antigens remain virtually unknown. We developed a new method to identify CD4 T cell–stimulating antigens. LacZ-inducible CD4 T cells were used as a probe to detect their cognate peptide/MHC II ligand generated in dendritic cells fed with Escherichia coli expressing a library of target cell genes. The murine H46 locus on chromosome 7 was thus found to encode the interleukin 4–induced IL4i1 gene. The IL4i1 precursor contains the HAFVEAIPELQGHV peptide which is presented by Ab major histocompatibility complex class II molecule via an endogenous pathway in professional antigen presenting cells. Both allelic peptides bind Ab and a single alanine to methionine substitution at p2 defines nonself. These results reveal novel features of H loci that regulate CD4 T cell responses as well as provide a general strategy for identifying elusive antigens that elicit CD4 T cell responses to tumors or self-tissues in autoimmunity.

IL-21 Gene Modified T Antigen Presenting Cells Enhance the Generation of Tumor-Specific Cytotoxic T Lymphocytes with a Memory Phenotype.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3709-3709
Author(s):  
Anjum S. Kaka ◽  
Ryan Hartmeier ◽  
Ann M. Leen ◽  
An Lu ◽  
Cliona M. Rooney ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Cultures ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Antigen Presenting Cells ◽  
Memory Phenotype ◽  
Cytotoxicity Assays ◽  
Antigen Presenting

Abstract IL-21 is a potent cytokine that augments the proliferation and effector function of NK cells and acts in synergy with other γ-chain cytokines to enhance the cytotoxicity of T lymphocytes. IL-21 is transiently produced by activated CD4+ T cells and may facilitate the generation of effector and memory T cells. Recently, T cells have been shown to be effective antigen presenting cells (TAPC) and we hypothesized that this characteristic may be enhanced through overexpression of IL-21 following genetic modification of TAPC. We demonstrate here that transduction of TAPC with IL-21 significantly enhances the generation of MART-1-specific CD8+ T cells suggesting a potential use for IL-21 in tumor immunotherapy protocols. IL-21 was cloned from CD3/CD28-activated CD4+ T cells and inserted into the SFG retroviral vector. To generate IL-21-producing T-APC, CD8-selected T cells from healthy, HLA-A2 donors were stimulated on αCD3/αCD28-coated plates in the presence of IL-2. After 2 days, activated cells were harvested and transduced on Retronectin-coated plates with IL-21 retroviral supernatant. On day 5, TAPC were washed and expanded in growth media supplemented by IL-2. Prior to use as APCs, TAPCs were CD4-depleted by MACS to eliminate residual IL-21 production by CD4+ T cells. IL-21-transduced and non-transduced (NT) CD8+ TAPC pulsed with MART-1 HLA-A2-restricted peptide (ELAGIGILTV) were irradiated and cocultured with autologous CD8+ peripheral blood T cells in media supplemented with IL-7 and IL-12. On day 7, responder T cell cultures were restimulated with peptide-loaded IL-21 or NT CD8+ TAPCs in the presence of IL-2 to induce expansion. Responder T cell cultures were then analyzed for MART-1 specificity by pentamer, ELISPOT and cytotoxicity assays and for their memory phenotype using monoclonal antibodies to CD27, CD28, CD62L, CD45RA, CD45RO, CD127 and CCR7. TAPC were efficiently expanded (>100-fold expansion) and transduced by retrovirus encoding IL-21 (>50% as measured by GFP). Gene modification of TAPC with IL-21 had minimal effect on MHC class I, II, CD80, CD83 and CD86 levels when compared to NT TAPC. However, there was increased expression of CD27, CD28 and CD62L, suggesting that IL-21 was biologically active. Seven days after stimulation with MART-1/ELA peptide-pulsed IL-21-TAPC and NT-TAPC, we observed a substantial increase (10±5-fold) in ELA-specific T cells in cultures stimulated with IL-21-TAPC compared to NT-TAPC when analyzed by FACS using ELA pentamers. Subsequent stimulation with IL-21-TAPCs amplified this effect, resulting in >50-fold increase in absolute ELA-specific T cell numbers when compared to NT-TAPC. ELA-specific CTL generated from IL-21-TAPC stimulation were functional as determined by IFN-γ ELISPOT and cytotoxicity assays. ELA-specific CTL generated from IL-21-TAPC exhibited a unique phenotype (CD45RA−, CD27high, CD28high, CD62Lhigh) as compared to CTL generated form NT-TAPC (CD45RA−, CD27low, CD28low, CD62Llow) suggesting that IL-21 may play a role in the development of T cell memory. In summary, IL-21 enhances the generation of tumor-specific CD8+ T cells which exhibit a central/effector memory phenotype. Our results indicate that IL-21 improves proliferation of antigen-specific T cells, possibly by maintaining CD28 expression allowing costimulation upon secondary antigen encounter.

Targeting STAT3 Signaling to Augment the Immunogenicity of B-Cell Lymphomas

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 708-708
Author(s):  
Hongwei Wang ◽  
F. Cheng ◽  
K. Wright ◽  
J. Tao ◽  
M. Smith ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Dna Binding ◽  
B Cell ◽  
Cd4 T Cells ◽  
Mutant Form ◽  
B Cell Lymphomas ◽  
Stat3 Signaling ◽  
Antigen Presenting

Abstract STAT3 signaling has emerged as a negative regulator of inflammatory responses in immune cells. In bone-marrow derived antigen-presenting cells (APCs), genetic or pharmacologic disruption of STAT3 led to inflammatory cells that effectively prime antigen-specific T-cell responses and restore the responsiveness of tolerized T-cells. In contrast, enhanced Stat3 activity in APCs resulted in increased production of the immunosuppressive cytokine IL-10 and induction of T-cell tolerance1. B-cell lymphomas being tumors derived from B-lymphocytes display intrinsic antigen-presenting capabilities. Augmentation of this APC function has been shown to result in effective anti-lymphoma immunity2. In this study we determined whether targeting Stat3 signaling might influence the intrinsic APC function of malignant B-cells and the responsiveness –or not- of antigen-specific CD4+ T-cells. First, we specifically block STAT3 signaling in A20 lymphoma B-cells by using a dominant negative variant of STAT3, Stat3b. Inhibition of STAT3 resulted in tumor cells capable not only of fully priming naïve antigen-specific CD4+T-cells but also able of restoring the responsiveness of tolerant T-cells from lymphoma bearing mice. Conversely, transfection of A20 B-cells with Stat3c, a constitutively activated mutant form of STAT3, led to T-cell unresponsiveness. Of note, manipulation of STAT3 in B cell tumors was associated with changes in the mRNA expression and protein levels of IL-10. Second, we evaluated the effects of two novel Stat3 inhibitors, CPA-7 (a platinum-containing compound that disrupts STAT3 DNA binding activity) and S3I-201 (inhibitor of Stat3:Stat3 complex formation and Stat3 DNA binding and transcriptional activities) in a murine model of Mantle Cell Lymphoma (MCL). In vitro treatment of FC-muMCL1 cells - derived from a tumor elicited in Em-Cyclin D1 transgenic mice- with increasing concentrations of either CPA-7 or S3I-201 resulted in an enhanced presentation of OVA-peptide to naïve CD4+ T-cells specific for a MHC class II restricted epitope of ovalbumin (OT-II cells). Indeed, these T-cells produce higher levels of IL-2 and IFN-gamma compared to anti-OVA T cells that encountered cognate antigen in untreated FC-muMCL1 cells. More importantly, MCL cells treated with CPA-7 restored the responsiveness of tolerized anti-OVA CD4+ T-cells. Finally, in vivo treatment of MCL-bearing mice with CPA-7 (5 mg/kg/iv given on days +21, +24 and +27 after tumor challenge) resulted in significant inhibition of p-Stat3 in malignant B-cells and augmentation of their APC function. Taken together, STAT3 signaling is involved in the regulation of the antigen-presenting capabilities of B-cell lymphomas and as such represents a novel molecular target to augment the immunogenicity of these tumors.

Induction and Break of Immune Tolerance to Human FVIII in a HLA-DRB1*1501 Humanized Hemophilic Mouse Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2280-2280
Author(s):  
Katharina Nora Steinitz ◽  
Brigitte Binder ◽  
Christian Lubich ◽  
Rafi Uddin Ahmad ◽  
Markus Weiller ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Mouse Model ◽  
Mhc Class Ii ◽  
Immune Tolerance ◽  
Cd4 T Cells ◽  
Hemophilia A ◽  
Class Ii ◽  
T Cell Epitopes

Abstract Abstract 2280 Development of neutralizing antibodies against FVIII is the major complication in the treatment of patients with hemophilia A. Although several genetic and environmental risk factors have been identified, it remains unclear why some patients develop antibodies while others do not. Understanding the underlying mechanisms that drive the decision of the immune system whether or not to make antibodies against FVIII would help to design novel therapeutics. We used a new humanized hemophilic mouse model that expresses the human MHC-class II molecule HLA-DRB1*1501 on the background of a complete knock out of all murine MHC-class II genes. Initial studies had indicated that only a fraction of these mice developed antibodies when intravenously (i.v.) treated with human FVIII. These findings which resemble the situation in patients with severe hemophilia A, evoked the question if the lack of antibody development in non-responder mice reflects the induction of specific immune tolerance after i.v. application of FVIII or represent non-responsiveness for other reasons. We addressed this question by choosing another application route (subcutaneous, s.c.) and by combining i.v. application with a concomitant activation of the innate immune system applying LPS, a well characterized ligand for toll-like receptor 4, together with FVIII. Both strategies resulted in the development of antibodies in all mice included in the study what suggested that non-responsiveness against i.v. FVIII does not reflect an inability to develop antibodies against FVIII. Next, we asked if i.v. FVIII does induce immune tolerance in non-responder mice. We pretreated mice with i.v. FVIII, selected non-responder mice and challenged them with s.c. FVIII. None of the mice developed antibodies what indicated that i.v. pretreatment had induced immune tolerance in non-responder mice. Currently, we test the hypothesis that immune tolerance after i.v. application is induced and maintained by FVIII-specific regulatory T cells. The differences in responder rates after i.v. and s.c. application of FVIII raised the question if there are differences in FVIII T-cell epitopes involved in the initial activation of FVIII-specific CD4+ T cells. We obtained spleen cells from mice treated with either i.v. or s.c. FVIII and generated CD4+ T-cell hybridoma libraries that were tested for peptide specificities. For this purpose we used a FVIII peptide library containing 15 mers with an offset of 3 amino acids. Our results indicate that the pattern of FVIII-specific T-cell epitopes involved in the activation of FVIII-specific CD4+ T cells after i.v. and s.c. application of FVIII is almost identical and represents a small set of FVIII peptides distributed over the A1, A2, B, A3 and C1 domains. Based on our results we conclude that the new HLA-DRB1*1501 hemophilic mouse model represents an interesting opportunity to uncover the mechanisms that drive the decision of the immune system whether or not to develop antibodies against FVIII. Disclosures: Steinitz: Baxter BioScience: Employment. Binder:Baxter BioScience: Employment. Lubich:Baxter BioScience: Employment. Ahmad:Baxter BioScience: Employment. Weiller:Baxter BioScience: Employment. de la Rosa:Baxter BioScience: Employment. Schwarz:Baxter BioScience: Employment. Scheiflinger:Baxter BioScience: Employment. Reipert:Baxter Innovations GmbH: Employment.

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