scholarly journals Extracorporeal photopheresis as a non-specific immune therapy of autoimmune diseases and skin T-cell lymphoma (a review of the literature and own studies)

2019 ◽  
Vol 47 (5) ◽  
pp. 419-434
Author(s):  
A. V. Kil'dyushevskiy ◽  
V. A. Molochkov ◽  
T. A. Mitina ◽  
Ya. G. Moysyuk ◽  
A. V. Molochkov
Keyword(s):  
T Cells ◽  
T Cell ◽  
Autoimmune Diseases ◽  
Immune Tolerance ◽  
Immune Therapy ◽  
Effector Memory ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Extracorporeal Photopheresis ◽  
Antigen Presenting

Aim: To present well-known and disputable mechanisms of the effects of extracorporeal photopheresis (ECP) in heterogeneous clinical conditions, as well as to demonstrate its advantages over conventional hormonal, immunosuppressive and cytostatic treatments, with a recommendation to widely implement it into practical management of autoimmune disease and cutaneous T-cell lymphomas (CTCLs).Key points: Despite convincing evidence of the ECP efficacy in the treatment of T-cell mediated disorders, a unifying concept of its mechanism has not been established so far. In this review, we attempted to determine the value of multiple, sometimes contradictory and equivocal points of view to immunobiochemical processes underlying the restoration of mechanism of immune tolerance in some autoimmune diseases and CTCLs. We focused our attention on our own clinical and immunological data obtained during a 20-years' experience with the use of ECP in clinical departments of MONIKI (Russia). Based on this, we have shown that ECP is more effective in autoimmune diseases than conventional treatment approaches with hormones, immunosuppressants and cytostatics. Unlike them, ECP is selectively targeted to auto-aggressive T-cells without induction of systemic immunosuppression. The leading role is played by the transformation of activated (immunogenic) myeloid dendrite cells (DC) into tolerogenic cell associated with their synthesis of inhibitor cytokines. The interplay of the cytokines with an antigen results in polarization of CD4+ Т lymphocytes via the Th2 pathway with restoration of the Th1/Th2 balance and their cytokine production. ECP triggers regulatory anti-clonotypic effector memory cells at the end stage of CD3+/CD8+/CD27-/CD28-/CD62L+ differentiation, that provide and maintain the peripheral immune tolerance, by deletion of the clone of auto-reactive cytotoxic lymphocytes and inducing their apoptosis. In autoimmune disorders, ECP results in reduction of the expression of integrin adhesion molecules on auto-reactive cell membranes with subsequent loss of their ability to migrate through the endothelium to their target cells. In its turn, it leads to decreasing immunoinflammatory response in the lesion. Both clinical and experimental data indicate that the mechanism of ECP action against CTCLs is characterized by activation of tumor cell apoptosis, unblocking of co-activation receptors on the antigen-presenting DC providing the functioning of the second signaling pathway for T lymphocyte activation. This results in proliferation of anti-tumor effector cells pool, production of DC activating cytokines that participate in the CD4+ polarization via Th1 pathway. In addition, this review considers the mechanism of the immunomodulating effect of ECP in the context of its influence at the levels of transcription and translation of proteins contributing to the pathophysiology of the disorders, based on molecular immunogenetic studies. Thus, ECP is able to induce antigen-specific immunological tolerance through the transformation of antigen-presenting cells, modulation of cytokine profile, adhesion and activation molecules, as well as through formatting of the regulatory T cells (Tregs).Conclusion: Undoubtedly, the immunobiological ECP technique has significant advantages over well-known conventional hormonal, immunosuppressive, and cytostatic therapies of autoimmune diseases and CTCLs.

Generation of Cytomegalovirus (CMV)-Specific CD4 and CD8 T Cell Lines Using Protein-Spanning Pools of pp65 and IE1 Derived Peptides.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 477-477
Author(s):  
Erica Dander ◽  
Giuseppina Li Pira ◽  
Ettore Biagi ◽  
Fabrizio Manca ◽  
Andrea Biondi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cell ◽  
Effector Memory ◽  
Target Cells ◽  
Pulsed Dc ◽  
Ifn Γ ◽  
T Cell Lines

Abstract BACKGROUND: Reactivation of latent CMV in immunocompromised recipients of allogeneic stem cell transplantation remains a major cause of morbidity and mortality. Reconstitution of immunity by CMV specific immunotherapy is an attractive alternative to drugs currently used, which show high toxicity and are sometimes ineffective. It has been demonstrated that CD4 helper T-cell function is crucial for the persistence of in vivo transferred CD8 CMV-specific CTL. Based on this finding, we have explored the feasibility of generating both anti-CMV CD4 and anti-CMV CD8 T-cell lines. METHODS: Dendritic Cells (DC) were generated from donor peripheral blood (PB) monocytes after a 7-day culture in the presence of GM-CSF plus IL-4 and matured with TNF-α, IFN-α, IFN-γ, IL1-β, POLI I:C. Matured-DC were then pulsed with a pool of 50 peptides spanning pp65 and IE1 proteins which are recognised by both CD4 and CD8 T lymphocytes. Donor T cells were stimulated three times at a T cell/DC ratio of 1:6 on day 0, +7 and +14 with mature peptide pulsed-DC. At the end of the culture the specificity of generated T cells was determined as percentage of pentamer-positive cells and intracellular IFN-γ production after incubation with peptide pulsed-DC. Cultured T cells were also analysed for their ability to proliferate in response to peptide pulsed-target cells, to kill them in a standard citotoxicity assay and to migrate in response to inflammatory (CXCL9, CCL3 and CCL5) and constitutive (CXCL12) chemokines. RESULTS: CMV-specific T cell lines were generated from five CMV seropositive donors. In four cases CD4 and CD8 CMV-specific T cell lines were expanded successfully. Cultured T cells expressed CD8 (mean= 70%, range 60–81%) and CD4 (mean= 20%, range 15–28%) and showed a CD45RA- CCR7- Effector Memory phenothype (mean=26%, range 19–30%) or a CD45RA+ CCR7- T Effector Memory RA-Positive phenothype (mean=67%, range 59–77%). An enriched CMV-specific T cell population was observed after staining with pentamers (7–45% pentamer-positive T cells). Furthermore, 90% of CD8+ and 40% of CD4+ T cells expressed high levels of intracytoplasmatic perforin and granzyme. In 4/5 cases tested, cutured T cells showed a cytolitic activity against CD8-peptide pulsed target cells (average lysis=50%, range 40–55%) and to a lesser extent against CD4-peptide pulsed target cells (average lysis=35%, range 30–40%). In addition, cultured T lymphocytes were able to proliferate and to produce intracytoplasmic IFN-γ (average production=50%, range 35–60%) after exposure to peptide-pulsed DC. Finally, Cultured T cells strongly migrated in response to chemokines (CXCL9, CCL3 and CCL5) involved in the recruitment of effector cells during viral infection. DISCUSSION: In conclusion, a great advantage of this method is represented by the possibility to generate anti-CMV CD4+ T cells, which could support in vivo the persistence of re-infused CMV-specific CTL. Moreover, the possibility of generating peptides under GMP conditions would facilitate the translation of this approach into clinical intervention.

Constitutive JAK3 Activation as a Mouse Model for Human Peripheral T-Cell Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1612-1612
Author(s):  
Melanie G. Cornejo ◽  
Michael G. Kharas ◽  
Benjamin Lee ◽  
Sandra A. Moore ◽  
Gary Gilliland ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Therapeutic Potential ◽  
Cell Lymphoma ◽  
Skin Lesions ◽  
Lymphoproliferative Disease ◽  
T Cell Lymphoma ◽  
Effector Memory ◽  
Target Cells ◽  
Proliferative Potential

Abstract Molecular pathogenesis of human peripheral T-cell malignancies remains poorly understood. We aimed to establish a model for these disorders by using the recently identified JAK3A572V allele. This mutation, which lies in the pseudokinase (JH2) domain of JAK3, transforms Ba/F3 cells to factor-independent growth and causes a striking lymphoproliferative phenotype in a murine bone marrow transplantation assay. Further characterization of JAK3A572V animals revealed that the aberrantly expanded cell population consisted of a mature effector/memory subtype of CD8+T-cells that infiltrated all major lymphoid and several non-lympoid organs and could be transplanted into secondary and tertiary recipients. These JAK3A572V T-cells had increased proliferative capacities and displayed enhanced phosphorylation of common JAK3 targets, such as STAT5 and S6-kinase. Proliferation of primary T-cells transformed by JAK3A572V was effectively inhibited with a small molecule JAK inhibitor that had no effect on the proliferative potential of control cells transduced with a wildtype JAK3 allele. Furthermore, the mutant cells showed increased production of cytotoxic cytokines, such as IFN-γ and TNF-α, compared to wildtype counterparts, which correlated with an increased cellular cytotoxicity towards allogeneic target cells. Of particular interest, JAK3A572V animals presented with skin lesions and histopathologic analysis showed aberrant skin-homing T-cells tagging along the epidermal/dermal junctions. Mice receiving Rag1-deficient donor cells transduced with the JAK3A572V allele also developed a lethal lymphoproliferative disease characterized by the expansion of immature CD3−TCRβ−CD4+/−CD8+ cells, suggesting that the JAK3A572V-dependent lymphoproliferation does not require proper TCR rearrangement. Altogether these results indicate that in this murine model, constitutive activation of JAK3 results in peripheral/cutaneous T-cell lymphoma (PTCL) that closely resembles the human disease. These findings suggest the possibility that the molecular basis of human PTCL could include aberrant JAK3 signaling and might provide a useful platform for deciphering the molecular and cellular mechanisms and requirements for peripheral lymphoid disease development and progression. Furthermore, it provides an opportunity to investigate the therapeutic potential of selective JAK3 inhibitors for this subset of lymphoid disorders, whose treatment remains a challenge.

scholarly journals Peptides Derived From S and N Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 Induce T Cell Responses: A Proof of Concept for T Cell Vaccines

2021 ◽  
Vol 12 ◽  
Author(s):  
Yu-Sun Lee ◽  
So-Hee Hong ◽  
Hyo-Jung Park ◽  
Ho-Young Lee ◽  
Ji-Yeon Hwang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Severe Acute Respiratory Syndrome ◽  
T Cell Responses ◽  
Effector Memory ◽  
Target Cells ◽  
Dependent Manner ◽  
Peptide Vaccines ◽  
Cell Responses ◽  
Ifn Γ

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that escape vaccine-induced neutralizing antibodies has indicated the importance of T cell responses against this virus. In this study, we highlight the SARS-CoV-2 epitopes that induce potent T cell responses and discuss whether T cell responses alone are adequate to confer protection against SARS-CoV-2 and describe the administration of 20 peptides with an RNA adjuvant in mice. The peptides have been synthesized based on SARS-CoV-2 spike and nucleocapsid protein sequences. Our study demonstrates that immunization with these peptides significantly increases the proportion of effector memory T cell population and interferon-γ (IFN-γ)-, interleukin-4 (IL-4)-, tumor necrosis factor-α (TNF-α)-, and granzyme B-producing T cells. Of these 20 peptides, four induce the generation of IFN-γ-producing T cells, elicit CD8+ T cell (CTL) responses in a dose-dependent manner, and induce cytotoxic T lymphocytes that eliminate peptide-pulsed target cells in vivo. Although it is not statistically significant, these peptide vaccines reduce viral titers in infected hamsters and alleviate pulmonary pathology in SARS-CoV-2-infected human ACE2 transgenic mice. These findings may aid the design of effective SARS-CoV-2 peptide vaccines, while providing insights into the role of T cells in SARS-CoV-2 infection.

scholarly journals Targeting Antigen Presenting   Cells to Treat Autoimmune  Inflammation

2021 ◽  
Author(s):  
◽  
Aras Toker
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Mhc Class Ii ◽  
T Cell Proliferation ◽  
Target Cells ◽  
Antigen Presenting

<p>Glatiramer acetate (GA) is approved for the treatment of relapsing-remitting multiple sclerosis (MS), and can suppress experimental autoimmune encephalomyelitis (EAE), a murine model of human MS. GA treatment is associated with the induction of anti-inflammatory TH2 responses and with the antigen specific expansion of regulatory T cells that counteract or inhibit pathogenic events in MS and EAE. These T cell mediated mechanisms of protection are considered to be a result of modulation of antigen presenting cells (APCs) by GA, rather than direct effects on T cells. However, it is unknown if GA preferentially targets a specific APC subset or can act through multiple APCs in vivo. In addition, GA-modulated innate cells may also exhibit direct antigen non-specific suppression of autoreactive cells. One objective of this study was to identify the in vivo target cell population of GA and to assess the potential of the target cells to antigen non-specifically suppress immune responses. Fluorophor-labelled GA bound to monocytes after intravenous injections, suggesting that monocytes may be the primary target of GA in vivo. In addition, intravenous GA treatment enhanced the intrinsic ability of monocytes to suppress T cell proliferation, both in vitro and in vivo. The findings of this study therefore suggest that GA-induced monocytes may contribute to GA therapy through direct mechanisms of antigen non-specific T cell immunosuppression. A further objective of this work was to investigate the potential of an in vivo drug targeting approach. This approach was hypothesised to increase the uptake of GA by the target cells and substantially improve GA treatment through antigen specific mechanisms such as induction of TH2 or regulatory T cells. Targeting antigens to professional APCs with an anti-MHC class II antibody resulted in significantly enhanced T cell proliferation in vitro. However, no EAE suppression occurred when GA was targeted to MHC class II in vivo. In addition, targeting GA specifically to monocytes also failed to suppress EAE. These findings suggest that GA treatment may selectively modulate monocytes to enhance their ability to inhibit autoreactive T cells, which could be part of the mechanism by which GA ameliorates MS. Targeting GA to a specific cell type may not be a powerful approach to improve treatment, because increased proliferation of GA specific T cells is not sufficient for disease suppression, and conjugation to antibodies may functionally reduce GA to a mere antigen devoid of immunomodulatory capacity.</p>

The Immunomodulatory Drug Lenalidomide (CC5013; Revlimid), Enhances Antigen-Presenting Cell’s Function Leading to Effective Priming of CD4+ T-Lymphocytes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2391-2391
Author(s):  
Hongwei Wang ◽  
Aung Naing ◽  
Fengdong Cheng ◽  
Pedro Horna ◽  
Ildelfonso Suarez ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Immune Tolerance ◽  
T Cell Responses ◽  
T Cell Tolerance ◽  
Cell Tolerance ◽  
Cell Responses ◽  
Cognate Antigen ◽  
Antigen Presenting

Abstract Professional antigen-presenting cells (APCs) play an important role in the initiation of antigen-specific T-cell responses. The demonstration that these cells are also required for the induction of T-cell tolerance, placed APCs at the crossroads of immune activation versus immune tolerance. Recent studies have demonstrated that the inflammatory status of the APC at the time of antigen presentation is the central determinant of T-cell priming versus T-cell tolerance. As such, therapeutic induction of inflammatory APCs might override immune tolerance and enhance the efficacy of immunotherapeutic strategies targeting hematologic tumors. Lenalidomide (CC5013) is a thalidomide analogue with immunomodulatory properties. Phase I and Phase II clinical trials in patients with myelodysplastic syndrome (MDS) have shown high frequency of erythropoietic responses, particularly in patients with 5q31 deletion associated with emergence of polyclonal lymphoid infiltrate in responding patient bone marrows. This observation raised the question as to whether immunological mechanism(s) may mediate, at least in part, the beneficial effect of CC5013 in patients with MDS. To gain further insight into the effects of Lenalidomide on APC’s function and regulation of antigen-specific CD4+ T-cell responses, we treated peritoneal elicited macrophages (PEM) and bone marrow-derived dendritic cells (DCs) with escalating concentration of Lenalidomide in vitro. Enhanced expression of both B7.1 and B7.2 co-stimulatory molecules was observed in Lenalidomide-treated APCs relative to untreated APCs. No difference in the expression of MHC class II molecules or CD40 was detected. Assessment of cytokine production by ELISA showed that Lenalidomide-treated APCs produce higher levels of TNF-a, IL-6 and IL-10 in response to LPS stimulation as compared to untreated APCs. Next, we evaluated the ability of Lenalidomide-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 either PEM or DC with low doses of Lenalidomide (range: 1.5–12.5 uM) 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-gamma in response to cognate antigen. Taken together, our results shows that by inducing inflammatory APCs, Lenalidomide directs the outcome of antigen-specific T-cell responses. Furthermore, they have broadened the scope of this drug as a promising adjuvant in cancer immunotherapy.

scholarly journals D-mannose ameliorates autoimmune phenotypes in mouse models of lupus

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Haiting Wang ◽  
Xiangyu Teng ◽  
Georges Abboud ◽  
Wei Li ◽  
Shuang Ye ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Autoimmune Diseases ◽  
Mouse Models ◽  
Effector Memory ◽  
T Cell Proliferation ◽  
Autoantibody Production ◽  
Models Of Lupus

Abstract Background Systemic lupus erythematosus is an autoimmune disease characterized by an overproduction of autoantibodies resulting from dysregulation in multiple immune cell types. D-mannose is a C− 2 epimer of glucose that exhibits immunoregulatory effects in models of autoimmune diseases, such as type 1 diabetes, induced rheumatoid arthritis, and airway inflammation. This study was conducted to evaluate the efficacy of D-mannose treatment in mouse models of lupus. Results Firstly, the effect of D-Mannose was evaluated by flow cytometry on the in vitro activation of non-autoimmune C57BL/6 (B6) bone marrow-derived dendritic cells (BMDCs) and their ability to induce antigen-specific CD4+ T cell proliferation and activation. D-mannose inhibited the maturation of BMDCs and their induction of antigen-specific T cell proliferation and activation. In vivo, D-mannose increased the frequency of Foxp3+ regulatory T cells in unmanipulated B6 mice. To assess the effect of D-mannose in mouse models of lupus, we used the graft-versus-host disease (cGVHD) induced model and the B6.lpr spontaneous model. In the cGVHD model, D-mannose treatment decreased autoantibody production, with a concomitant reduction of the frequency of effector memory and follicular helper T cells as well as germinal center B cells and plasma cells. These results were partially validated in the B6.lpr model of spontaneous lupus. Conclusion Overall, our results suggest that D-mannose ameliorates autoimmune activation in models of lupus, at least partially due to its expansion of Treg cells, the induction of immature conventional dendritic cells and the downregulation of effector T cells activation. D-Mannose showed however a weaker immunomodulatory effect in lupus than in other autoimmune diseases.

scholarly journals Immunomodulatory Role and Therapeutic Potential of Non-Coding RNAs Mediated by Dendritic Cells in Autoimmune and Immune Tolerance-Related Diseases

2021 ◽  
Vol 12 ◽  
Author(s):  
Yifeng Liu ◽  
Xiaoze Wang ◽  
Fan Yang ◽  
Yanyi Zheng ◽  
Tinghong Ye ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Autoimmune Diseases ◽  
Immune Tolerance ◽  
Therapeutic Potential ◽  
The Body ◽  
Cytokines And Chemokines ◽  
Non Coding Rnas ◽  
And Function ◽  
Antigen Presenting

Dendritic cells (DCs) are professional antigen-presenting cells that act as a bridge between innate immunity and adaptive immunity. After activation, DCs differentiate into subtypes with different functions, at which point they upregulate co-stimulatory molecules and produce various cytokines and chemokines. Activated DCs also process antigens for presentation to T cells and regulate the differentiation and function of T cells to modulate the immune state of the body. Non-coding RNAs, RNA transcripts that are unable to encode proteins, not only participate in the pathological mechanisms of autoimmune-related diseases but also regulate the function of immune cells in these diseases. Accumulating evidence suggests that dysregulation of non-coding RNAs contributes to DC differentiation, functions, and so on, consequently producing effects in various autoimmune diseases. In this review, we summarize the main non-coding RNAs (miRNAs, lncRNAs, circRNAs) that regulate DCs in pathological mechanisms and have tremendous potential to give rise to novel therapeutic targets and strategies for multiple autoimmune diseases and immune tolerance-related diseases.

scholarly journals Emerging Therapeutics for Immune Tolerance: Tolerogenic Vaccines, T cell Therapy, and IL-2 Therapy

2021 ◽  
Vol 12 ◽  
Author(s):  
Cody D. Moorman ◽  
Sue J. Sohn ◽  
Hyewon Phee
Keyword(s):  
T Cells ◽  
T Cell ◽  
Autoimmune Diseases ◽  
Immune Tolerance ◽  
Immunosuppressive Agents ◽  
Antigen Receptors ◽  
Chimeric Antigen Receptors ◽  
Biologic Drugs ◽  
T Cell Therapy ◽  
Tnf Α

Autoimmune diseases affect roughly 5-10% of the total population, with women affected more than men. The standard treatment for autoimmune or autoinflammatory diseases had long been immunosuppressive agents until the advent of immunomodulatory biologic drugs, which aimed at blocking inflammatory mediators, including proinflammatory cytokines. At the frontier of these biologic drugs are TNF-α blockers. These therapies inhibit the proinflammatory action of TNF-α in common autoimmune diseases such as rheumatoid arthritis, psoriasis, ulcerative colitis, and Crohn’s disease. TNF-α blockade quickly became the “standard of care” for these autoimmune diseases due to their effectiveness in controlling disease and decreasing patient’s adverse risk profiles compared to broad-spectrum immunosuppressive agents. However, anti-TNF-α therapies have limitations, including known adverse safety risk, loss of therapeutic efficacy due to drug resistance, and lack of efficacy in numerous autoimmune diseases, including multiple sclerosis. The next wave of truly transformative therapeutics should aspire to provide a cure by selectively suppressing pathogenic autoantigen-specific immune responses while leaving the rest of the immune system intact to control infectious diseases and malignancies. In this review, we will focus on three main areas of active research in immune tolerance. First, tolerogenic vaccines aiming at robust, lasting autoantigen-specific immune tolerance. Second, T cell therapies using Tregs (either polyclonal, antigen-specific, or genetically engineered to express chimeric antigen receptors) to establish active dominant immune tolerance or T cells (engineered to express chimeric antigen receptors) to delete pathogenic immune cells. Third, IL-2 therapies aiming at expanding immunosuppressive regulatory T cells in vivo.

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.

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