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Posts Tagged ‘CTLA-4’


Cyclic Dinucleotides and Histone deacetylase inhibitors

Curators: Larry H. Bernsten, MD, FCAP and Aviva Lev-Ari, PhD, RN

LPBI

 

New Class of Immune System Stimulants: Cyclic Di-Nucleotides (CDN): Shrink Tumors and bolster Vaccines, re-arm the Immune System’s Natural Killer Cells, which attack Cancer Cells and Virus-infected Cells

Reporter: Aviva Lev-Ari, PhD, RN

The Immunotherapeutics and Vaccine Research Initiative (IVRI), a UC Berkeley effort funded by Aduro Biotech, Inc.

https://pharmaceuticalintelligence.com/2016/04/24/new-class-of-immune-system-stimulants-cyclic-di-nucleotides-cdn-shrink-tumors-and-bolster-vaccines-re-arm-the-immune-systems-natural-killer-cells-which-attack-cancer-cells-and-virus-inf/

A new class of immune system stimulants called cyclic di-nucleotides have shown promise in shrinking tumors and bolstering vaccines against tuberculosis, and research that could help re-arm the immune system’s natural killer cells, which normally attack cancer cells and virus-infected cells, to better fight tumors.

Much of the excitement around combining these two areas — the immunology of cancer and the immunology of infectious disease — comes from the amazing success of immunotherapy against cancer, which started with the work of James Allison when he was a professor of immunology at UC Berkeley and director of the Cancer Research Laboratory from 1985 to 2004. Allison, now at the University of Texas MD Anderson Cancer Center, discovered a way to release a brake on the body’s immune response to cancer that has proved highly successful at unleashing the immune system to attack melanoma and is being tried against other types of cancer. Allison’s technique uses an antibody that blocks an immune suppressor called CTLA4, antibodies that block another immune suppressor, PD1. This has been successful in treating melanoma, renal cancer and a type of lung cancer. Both CTLA4 and PD1 antibodies are now FDA-approved as cancer therapies.

Another promising avenue involves a protein in cells that responds to foreign DNA to launch an innate immune response — the first response of the body’s immune system. The protein, dubbed STING, is triggered by small molecules called cyclic di-nucleotides (CDN), and makes immune cells release interferon and other cytokines that activate disease-fighting T cells and stimulate the production of antibodies that together kill invading pathogens and destroy cancer cells. Listeria bacteria, for example, secrete a CDN directly into infected cells that activates STING.

Russell Vance, a UC Berkeley professor of molecular and cell biology and current head of the Cancer Research Laboratory, discovered several years ago that the chemical structure of these di-nucleotides is critical to their ability to work in humans. Aduro has since developed a CDN designed to work in humans and found that injecting it directly into a tumor in mice caused the tumor to shrink.

Sarah Stanley, an assistant professor of public health, has found evidence that CDNs can help improve the imperfect vaccines we have today against tuberculosis.

 

Researchers at UC Berkeley will have access to Aduro’s novel technology platforms for research use, including its STING pathway activators, proprietary monoclonal antibodies and the engineered listeria bacteria, referred to as LADD (listeria attenuated double-deleted). David Raulet, professor of molecular and cell biology and director of IVRI has contributed to making these cells a new focus of cancer research. As tumors advance, NK cells inside the tumors appear to become desensitized, he said. Recent research shows that some cytokines and other immune mediators Raulet discovered are able to “wake them up” and get them to resume their elimination of cancer cells.

 

Histone deacetylase inhibitors: molecular mechanisms of action

W S Xu1,2, R B Parmigiani1,2 and P A Marks1

Oncogene (2007) 26, 5541–5552; http://dx.doi.org:/10.1038/sj.onc.1210620

This review focuses on the mechanisms of action of histone deacetylase (HDAC) inhibitors (HDACi), a group of recently discovered ‘targeted’ anticancer agents. There are 18 HDACs, which are generally divided into four classes, based on sequence homology to yeast counterparts. Classical HDACi such as the hydroxamic acid-based vorinostat (also known as SAHA and Zolinza) inhibits classes I, II and IV, but not the NAD+-dependent class III enzymes. In clinical trials, vorinostat has activity against hematologic and solid cancers at doses well tolerated by patients. In addition to histones, HDACs have many other protein substrates involved in regulation of gene expression, cell proliferation and cell death. Inhibition of HDACs causes accumulation of acetylated forms of these proteins, altering their function. Thus, HDACs are more properly called ‘lysine deacetylases.’ HDACi induces different phenotypes in various transformed cells, including growth arrest, activation of the extrinsic and/or intrinsic apoptotic pathways, autophagic cell death, reactive oxygen species (ROS)-induced cell death, mitotic cell death and senescence. In comparison, normal cells are relatively more resistant to HDACi-induced cell death. The plurality of mechanisms of HDACi-induced cell death reflects both the multiple substrates of HDACs and the heterogeneous patterns of molecular alterations present in different cancer cells.

histone deacetylase, histone deacetylase inhibitor, apoptosis, mitotic cell death, senescence, angiogenesis

Acetylation and deacetylation of histones play an important role in transcription regulation of eukaryotic cells (Lehrmann et al., 2002;Mai et al., 2005). The acetylation status of histones and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyl-transferases (HATs). HATs add acetyl groups to lysine residues, while HDACs remove the acetyl groups. In general, acetylation of histone promotes a more relaxed chromatin structure, allowing transcriptional activation. HDACs can act as transcription repressors, due to histone deacetylation, and consequently promote chromatin condensation. HDAC inhibitors (HDACi) selectively alter gene transcription, in part, by chromatin remodeling and by changes in the structure of proteins in transcription factor complexes (Gui et al., 2004). Further, the HDACs have many non-histone proteins substrates such as hormone receptors, chaperone proteins and cytoskeleton proteins, which regulate cell proliferation and cell death (Table 1). Thus, HDACi-induced transformed cell death involves transcription-dependent and transcription-independent mechanisms (Marks and Dokmanovic, 2005Rosato and Grant, 2005Bolden et al., 2006;Minucci and Pelicci, 2006).

Table 1 – Nonhistone protein substrates of HDACs (partial list).   Full table

http://www.nature.com/common/images/table_thumb.gif

In humans, 18 HDAC enzymes have been identified and classified, based on homology to yeast HDACs (Blander and Guarente, 2004;Bhalla, 2005Marks and Dokmanovic, 2005). Class I HDACs include HDAC1, 2, 3 and 8, which are related to yeast RPD3 deacetylase and have high homology in their catalytic sites. Recent phylogenetic analyses suggest that this class can be divided into classes Ia (HDAC1 and -2), Ib (HDAC3) and Ic (HDAC8) (Gregoretti et al., 2004). Class II HDACs are related to yeast Hda1 (histone deacetylase 1) and include HDAC4, -5, -6, -7, -9 and -10 (Bhalla, 2005Marks and Dokmanovic, 2005). This class is divided into class IIa, consisting of HDAC4, -5, -7 and -9, and class IIb, consisting of HDAC6 and -10, which contain two catalytic sites. All class I and II HDACs are zinc-dependent enzymes. Members of a third class, sirtuins, require NAD+ for their enzymatic activity (Blander and Guarente, 2004) (see review by E Verdin, in this issue). Among them, SIRT1 is orthologous to yeast silent information regulator 2. The enzymatic activity of class III HDACs is not inhibited by compounds such as vorinostat or trichostatin A (TSA), that inhibit class I and II HDACs. Class IV HDAC is represented by HDAC11, which, like yeast Hda 1 similar 3, has conserved residues in the catalytic core region shared by both class I and II enzymes (Gao et al., 2002).

HDACs are not redundant in function (Marks and Dokmanovic, 2005Rosato and Grant, 2005Bolden et al., 2006). Class I HDACs are primarily nuclear in localization and ubiquitously expressed, while class II HDACs can be primarily cytoplasmic and/or migrate between the cytoplasm and nucleus and are tissue-restricted in expression.

The structural details of the HDAC–HDACi interaction has been elucidated in studies of a histone deacetylase-like protein from an anerobic bacterium with TSA and vorinostat (Finnin et al., 1999). More recently, the crystal structure of HDAC8–hydroxamate interaction has been solved (Somoza et al., 2004Vannini et al., 2004). These studies provide an insight into the mechanism of deacetylation of acetylated substrates. The hydroxamic acid moiety of the inhibitor directly interacts with the zinc ion at the base of the catalytic pocket.

This review focuses on the molecular mechanisms triggered by inhibitors of zinc-dependent HDACs in tumor cells that explain in part: (I) the effects of these compounds in inducing transformed cell death and (II) the relative resistance of normal and certain cancer cells to HDACi induced cell death. HDACi, for example, the hydroxamic acid-based vorinostat (SAHA, Zolinza), are promising drugs for cancer treatment (Richon et al., 1998Marks and Breslow, 2007). Several HDACi are in phase I and II clinical trials, being tested in different tumor types, such as cutaneous T-cell lymphoma, acute myeloid leukemia, cervical cancer, etc (Bug et al., 2005Chavez-Blanco et al., 2005Kelly and Marks, 2005;Duvic and Zhang, 2006) (Table 2). Although considerable progress has been made in elucidating the role of HDACs and the effects of HDACi, these areas are still in early stages of discovery.

Table 2 – HDACi in clinical trials.  Full table

http://www.nature.com/common/images/table_thumb.gif

Recent phylogenetic analyses of bacterial HDACs suggest that all four HDAC classes preceded the evolution of histone proteins (Gregoretti et al., 2004). This suggests that the primary activity of HDACs may be directed against non-histone substrates. At least 50 non-histone proteins of known biological function have been identified, which may be acetylated and substrates of HDACs (Table 1) (Glozak et al., 2005Marks and Dokmanovic, 2005;Rosato and Grant, 2005Bolden et al., 2006Minucci and Pelicci, 2006Zhao et al., 2006). In addition, two recent proteomic studies identified many lysine-acetylated substrates (Iwabata et al., 2005Kim et al., 2006). In view of all these findings, HDACs may be better called ‘N-epsilon-lysine deacetylase’. This designation would also distinguish them from the enzymes that catalyse other types of deacetylation in biological reactions, such as acylases that catalyse the deacetylation of a range of N-acetyl amino acids (Anders and Dekant, 1994).

Non-histone protein targets of HDACs include transcription factors, transcription regulators, signal transduction mediators, DNA repair enzymes, nuclear import regulators, chaperone proteins, structural proteins, inflammation mediators and viral proteins (Table 1). Acetylation can either increase or decrease the function or stability of the proteins, or protein–protein interaction (Glozak et al., 2005). These HDAC substrates are directly or indirectly involved in many biological processes, such as gene expression and regulation of pathways of proliferation, differentiation and cell death. These data suggest that HDACi could have multiple mechanisms of inducing cell growth arrest and cell death (Figure 1).

Figure 1.  Full figure and legend (90K)

Multiple HDACi-activated antitumor pathways. See text for detailed explanation of each pathway. HDAC6, histone deacetylase 6; HIF-1, hypoxia-induced factor-1; HSP90, heat-shock protein 90; PP1, protein phosphatase 1; ROS, reactive oxygen species; TBP2, thioredoxin binding protein 2; Trx, thioredoxin; VEGF, vascular endothelial growth factor.

http://www.nature.com/onc/journal/v26/n37/images/1210620f1.jpg

HDACi have been discovered with different structural characteristics, including hydroximates, cyclic peptides, aliphatic acids and benzamides (Table 2) (Miller et al., 2003Yoshida et al., 2003Marks and Breslow, 2007). Certain HDACi may selectively inhibit different HDACs. For example, MS-275 preferentially inhibits HDAC1 with IC50, at 0.3 m, compared to HDAC3 with an IC50 of about 8 m, and has little or no inhibitory effect against HDAC6 and HDAC8 (Hu et al., 2003). Two novel synthetic compounds, SK7041 and SK7068, preferentially target HDAC1 and 2 and exhibit growth inhibitory effects in human cancer cell lines and tumor xenograft models (Kim et al., 2003a). A small molecule, tubacin, selectively inhibits HDAC6 activity and causes an accumulation of acetylated -tubulin, but does not affect acetylation of histones, and does not inhibit cell cycle progression (Haggarty et al., 2003). No other HDACi for a specific HDAC has been reported.

HDACi selectively alters gene expression

HDACi-induced antitumor pathways

  • HDACi induces cell cycle arrest
  • HDACi activates the extrinsic apoptotic pathways
  • HDACi activates the intrinsic apoptotic pathways
  • HDACi induces mitotic cell death
  • HDACi induces autophagic cell death and senescence
  • ROS, thioredoxin and Trx binding protein 2 in HDACi-induced cell death
  • Antitumor effects of HDAC6 inhibition
  • Activation of protein phosphatase 1
  • Disruption of the function of chaperonin HSP90
  • Disruption of the aggresome pathway
  • HDACi inhibits angiogenesis

HDACi can block tumor angiogenesis by inhibition of hypoxia inducible factors (HIF) (Liang et al., 2006). HIF-1 and HIF-2 are transcription factors for angiogenic genes (Brown and Wilson, 2004). The oxygen level can control HIF activity through two mechanisms. First, under normoxic conditions, HIF-1 binds to von Hippel–Lindau protein (pVHL) and is degraded by the ubiquitination–proteasome system. Second, HIF activity depends on its transactivation potential (TAP), which is affected by the interaction with the coactivator p300/CBP among others. This complex can be disrupted by Factor Inhibiting HIF (FIH). Hypoxic conditions activate HIF through repression of the hydroxylases responsible for HIF degradation and loss of function.

 

Combination of HDACi with other antitumor agents

The HDACi have shown synergistic or additive antitumor effects with a wide range of antitumor reagents, including chemotherapeutic drugs, new targeted therapeutic reagents and radiation, by various mechanisms, some unique for particular combinations (Rosato and Grant, 2004Bhalla, 2005Marks and Dokmanovic, 2005Bolden et al., 2006).

Clinical development of HDACi

At least 14 different HDACi are in some phase of clinical trials as monotherapy or in combination with retinoids, taxols, gemcitabine, radiation, etc, in patients with hematologic and solid tumors, including cancer of lung, breast, pancreas, renal and bladder, melanoma, glioblastoma, leukemias, lymphomas, multiple myeloma (see National Cancer Institute website for CTEP clinical trials, ctep.cancer.gov or clinicaltrials.gov, and website of companies developing HDACi; Table 2).

The resistance to HDACi

Conclusions and perspectives

HDACs have multiple substrates involved in many biological processes, including proliferation, differentiation, apoptosis and other forms of cell death. Indeed, the fact that HDACs have histone and multiple nonhistone protein substrates suggests these enzymes should be referred to as ‘lysine deacetylases’. HDACi can cause transformed cells to undergo growth arrest, differentiation and/or cell death. Normal cells are relatively resistant to HDACi. HDACi are selective in altering gene expression, which may reflect, in part, the proteins composing the transcription factor complex to which HDACs are recruited. Both altered gene expression and changes in non-histone proteins caused by HDACi-induced acetylation play a role in the antitumor activity of HDACi. This is reflected in the different inducer-activated antitumor pathways in transformed cells (Figure 1). The functions of HDACs are not redundant. Thus, a pan-HDAC inhibitor such as vorinostat may activate more antitumor pathways and have therapeutic advantages compared to HDAC isotype-specific inhibitors.

Almost all cancers have multiple defects in the expression and/or structure of proteins that regulate cell proliferation and death. Compared to other antitumor reagents, the plurality of action of HDACi potentially confers efficacy in a wide spectrum of cancers, which have heterogeneity and multiple defects, both among different types of cancer and within different individual tumors of the same type. The multiple defects in a cancer cell may be the reason for transformed cells being more sensitive than normal cells to HDACi. Thus, given the relatively rapid reversibility of vorinostat inhibition of HDACs, normal cells may be able to compensate for HDACi-induced changes more effectively than cancer cells.

HDACi have synergistic or additive antitumor effects with many other antitumor reagents – suggesting that combination of HDACi and other anticancer agents may be very attractive therapeutic strategies for using these agents. Complete understanding of the mechanisms underlying the resistance and sensitivity to HDACi has obvious therapeutic importance. Targeting resistant factors will enhance the antitumor efficacy of HDACi. Identifying markers that can predict response to HDACi is a high priority for expanding the efficacy of these novel anticancer agents.

References  ….

NEWS AND VIEWS   Blocking HDACs boosts regulatory T cells

Nature Medicine News and Views (01 Nov 2007)

RESEARCH   

Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug

Nature Biotechnology Research (01 Jan 2007)

Comments of reviewer:

 

The complexity of cancer has been known for almost a century, in large part from the seminal work of Otto Warburg in the 1920s using manometry, and following the work of Louis Pasteur 60 years earlier with fungi.

 

The volume of work and our unlocking of mitotic activity, apoptosis, mitochondria, and the cytoskeleton has taken us further into the cell interior, cell function, metabolic regulation, and pathophysiology.  Despite the enormous contributions to our knowledge of genomics, there is a large body of work in pathways of cell function that resides in no small part in activity of protein catalysts and enzymes.

 

The work that has been described covers only cyclic dinucleotides and HDACi’s.  Some of the activities described have relevance to microorganisms as well as cancer.  As we have seen, blocking HDACs boosts the activity of regulatory T-cells. There are many specific functional alterations elucidated above.

 

The first presentation is of an antibody that blocks an immune suppressor called CTLA4, antibodies that block another immune suppressor, PD1. This also involves a protein in cells that responds to foreign DNA to launch an innate immune response — the first response of the body’s immune system. The protein, dubbed STING, is triggered by small molecules called cyclic di-nucleotides (CDN), and makes immune cells release interferon and other cytokines that activate disease-fighting T cells and stimulate the production of antibodies that together kill invading pathogens and destroy cancer cells. Listeria bacteria, for example, secrete a CDN directly into infected cells that activates STING.

 

The second is resident in acetylation status of histones and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyl-transferases (HATs). HATs add acetyl groups to lysine residues, while HDACs remove the acetyl groups. In general, acetylation of histone promotes a more relaxed chromatin structure, allowing transcriptional activation. HDACs can act as transcription repressors, due to histone deacetylation, and consequently promote chromatin condensation. HDAC inhibitors (HDACi) selectively alter gene transcription, in part, by chromatin remodeling and by changes in the structure of proteins in transcription factor complexes (Gui et al., 2004).  The description focuses on the molecular mechanisms triggered by inhibitors of zinc-dependent HDACs in tumor cells that explain in part: (I) the effects of these compounds in inducing transformed cell death and (II) the relative resistance of normal and certain cancer cells to HDACi induced cell death.

 

HDACs have multiple substrates involved in many biological processes, including proliferation, differentiation, apoptosis and other forms of cell death. Indeed, the fact that HDACs have histone and multiple nonhistone protein substrates suggests these enzymes should be referred to as ‘lysine deacetylases’. HDACi can cause transformed cells to undergo growth arrest, differentiation and/or cell death. Normal cells are relatively resistant to HDACi. HDACi are selective in altering gene expression, which may reflect, in part, the proteins composing the transcription factor complex to which HDACs are recruited. Both altered gene expression and changes in non-histone proteins caused by HDACi-induced acetylation play a role in the antitumor activity of HDACi.

 

 

 

 

 

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IRF-1 Deficiency Skews the Differentiation of Dendritic Cells

Reporter: Larry H Bernstein, MD, FCAP

 

 

IFN Regulatory Factor-1 Negatively Regulates CD4+CD25+ Regulatory T Cell Differentiation by Repressing Foxp3 Expression1

 

Alessandra Fragale*, Lucia Gabriele†, Emilia Stellacci*, Paola Borghi†,…. and Angela Battistini2,*
The Journal of Immunology   Aug 1, 2008; 181(3): 1673-1682

Regulatory T (Treg) cells are critical in inducing and maintaining tolerance. Despite progress in understanding the basis of immune tolerance,

  • mechanisms and molecules involved in the generation of Treg cells remain poorly understood.

IFN regulatory factor (IRF)-1 is a pleiotropic transcription factor implicated in the regulation of various immune processes. In this study, we report that IRF-1 negatively regulates CD4+CD25+ Treg cell

  • development and function by specifically repressing Foxp3 expression.

IRF-1-deficient (IRF-1−/−) mice showed a selective and marked increase of highly activated and differentiated CD4+CD25+Foxp3+ Treg cells in thymus and in all peripheral lymphoid organs. Furthermore,

  • IRF-1−/− CD4+CD25− T cells showed extremely high bent to differentiate into CD4+CD25+Foxp3+ Treg cells, whereas
  • restoring IRF-1 expression in IRF-1−/− CD4+CD25− T cells
    • impaired their differentiation into CD25+Foxp3+ cells.

Functionally, both isolated and TGF-β-induced CD4+CD25+ Treg cells from IRF-1−/− mice

  • exhibited more increased suppressive activity than wild-type Treg cells.

Such phenotype and functional characteristics were explained at a mechanistic level by the finding that

  • IRF-1 binds a highly conserved IRF consensus element sequence (IRF-E) in the foxp3 gene promoter in vivo and
  • negatively regulates its transcriptional activity.

We conclude that IRF-1 is a key negative regulator of CD4+CD25+ Treg cells

  • through direct repression of Foxp3 expression.
Introduction

Tolerance is critical for prevention of autoimmunity and maintenance of immune homeostasis by active suppression of inappropriate immune responses. Suppression has a dedicated population of  T cells that

  • control the responses of other T cells.

This cell population, referred to as regulatory T (Treg)3 cells, actually comprises several subsets, including naturally occurring CD4+CD25+ Treg cells that arise in thymus. Once generated,

  • thymic Treg cells are exported to peripheral tissues, and
  • comprise 5–10% of peripheral CD4+ T cells (1, 2, 3).

CD4+CD25+ Treg cells are characterized by

  • constitutive expression of IL-2Rα (CD25), CTLA-4, and glucocorticoid-induced TNFR family-related gene; moreover,
  • they express CD62 ligand (CD62L) and are mainly CD45RBlow (4).

In contrast to cell surface markers, which can be shared with other T cells populations,

  • the forkhead/winged-helix family transcriptional repressor Foxp3 is
  • specifically expressed in CD4+CD25+ Treg cells and
  • rigorously controls their development and function (5, 6, 7).

Functionally after TCR stimulation, CD4+CD25+ Treg cells can

  • mediate strong suppression of proliferation and
  • IL-2 production by CD4+ T cells both in vivo and in vitro (8).

Although mechanisms of suppression are not fully understood,

  • they appear to be cell contact-mediated, whereas
  • the relative contribution of soluble cytokines remains controversial
    • with differences between in vitro and in vivo results (1, 8, 9).

Indeed, the involvement of cytokines in the suppressor function of CD4+CD25+ Treg cells has been proposed in vivo,

  • where they are able to produce IL-10 and TGF-β (10, 11, 12), and
  • importantly, IL-10 activity has been recently associated with the function of TGF-β-induced CD4+CD25−CD45RBlow cells (13).

Beside naturally occurring CD4+CD25+ Treg cells, CD4+CD25+ Treg cells can also be

  • induced (inTreg) in vivo or in vitro after TCR stimulation and TGF-β treatment,
  • acquiring expression of CD25 and Foxp3 both in mice (14, 15, 16) and humans (17, 18, 19, 20),
    • although with characteristic functional differences (20).

Despite extensive studies on the role of Foxp3 in inducing and maintaining tolerance, little information on regulation of its expression is available. Transcription factors of the IFN regulatory factor (IRF) family participate in

  • the early host response to pathogens,
  • in immunomodulation and
  • hematopoietic differentiation (21).

Nine members of this family have been identified based on a unique helix-turn-helix DNA binding domain, located at

  • the N terminus that is responsible for binding to the IRF consensus element (IRF-E) (21).
The first member of the family, IRF-1, was originally identified as a protein that binds
  • the cis-acting DNA elements in the ifnβ gene promoter and the IRF-E (also referred to as the IFN-stimulated response element; ISRE),
  • in the promoters of IFN-αβ-stimulated genes (22).

IRF-1 is expressed at low basal levels in all cell types examined, but

  • accumulates in response to several stimuli and cytokines including IFN-γ, the strongest IRF-1 inducer (22).
Intensive functional analyses conducted on this transcription factor have revealed a remarkable functional diversity in the
  • regulation of cellular responses through the
  • modulation of different sets of genes,
  • depending on
    1. cell type,
    2. state of the cell, and/or
    3. nature of the stimuli (21).
We and others have shown that IRF-1 affects the differentiation of both lymphoid and myeloid lineages (22, 23, 24, 25, 26, 27, 28). In particular, studies in knockout (KO) mice have implicated IRF-1
in the regulation of various immune processes:
  1. impairment of CD8+ T cell and NK cell maturation,
  2. impaired IL-12 macrophage production,
  3. exclusive Th2 differentiation, and
  4. defective Th1 responses…………. have all been observed (22, 23, 24, 25, 26).
As a result, IRF-1−/− mice are highly susceptible to infections, for which effective host control
    • is associated with a Th1 immune response (24).
In contrast, these mice are characterized by
  • increased resistance to several autoimmune diseases such as
  1. collagen-induced arthritis,
  2. experimental autoimmune encephalomyelitis,
  3. Helicobacter pylori-induced gastritis,
  4. induced lymphocytic thyroiditis,
  5. insulitis, or
  6. diabetes (29, 30, 31, 32).
Recently, we reported that IRF-1−/− mice display a prevalence of
  • dendritic cell (DC) subsets with immature and tolerogenic features that were
    • unable to undergo full maturation after stimulation.
Moreover, IRF-1−/− DC conferred
    • increased suppressive activity to CD4+CD25+ Treg cells (33).
Because there is growing evidence that immature or partially matured DC can induce tolerance (34, 35), we hypothesized that IRF-1 could play a role in
  • Treg development and function.
In this study, we analyzed the CD4+CD25+ compartment in IRF-1−/− mice and
  • we found that in vivo IRF-1 deficiency resulted in a
  • selective and marked increase in highly differentiated and activated CD4+CD25+Foxp3+ Treg cells, whereas
reintroduction of IRF-1 by retrovirus transduction
    • impaired TGF-β-mediated differentiation of IRF-1−/− CD4+CD25− T cells into CD4+CD25+Foxp3+ Treg cells.
At molecular level, we show that IRF-1 plays a direct role in the generation and expansion of CD4+CD25+ Treg cells
    • specifically repressing Foxp3 transcriptional activity.
Our results, therefore, highlight a unique role for IRF-1 as regulator of Foxp3, thus pointing to IRF-1 as a specific tool to control altered tolerance.
Results
CD4+CD25+ Treg from IRF-1−/− mice are increased and functionally more suppressive than WT Treg cells
The distribution and the phenotype of CD4+CD25+Foxp3+ Treg in lymphoid organs of IRF-1−/− mice were determined by flow cytometry.
the number of ex vivo double positive CD4+CD25+ cells was significantly increased in spleens and skin draining and mesenteric lymph nodes (2.8-, 2.3-, and 2.1-fold increase, respectively), and to a lesser extent, in thymus (1.6-fold increase) of IRF-1−/− mice as compared with WT mice. Consistently with previous reports (23, 41), no differences in CD4+ T cell and total cell numbers in all lymphoid organs from WT or IRF-1−/− mice were found (data not shown). Strikingly, intracellular analysis of Foxp3 expression showed that this factor was increasingly expressed in CD4+CD25+ Treg cells from spleens as well as from other lymphoid organs of IRF-1−/− mice
FACS analysis of splenic magnetically sorted CD4+CD25+ Treg cells was performed to evaluate the expression of activation markers.  IRF-1−/− Treg cells were to a large extent characteristic of a marked activated and differentiated phenotype.
Because there is accumulating evidence that activity of CD4+CD25+ Treg cells in vivo involves some immunosuppressive cytokines (9, 10, 11, 12), we also compared the cytokine profile of IRF-1−/− CD4+CD25+ Treg cells with the profile of WT counterparts . Lower levels of proinflammatory cytokines, such as TNF-α and IFN-γ, whereas higher levels of IL-4 were expressed in CD4+CD25+ Treg cells as well as in CD4+CD25− T lymphocytes from KO as compared with WT cells. Notably, only IRF-1−/− Treg cells showed a clear-cut increase in the expression of IL-10. By contrast, TGF-β was expressed at similar levels in CD4+CD25+ Treg cells from both IRF-1−/− and WT mice. Accordingly with mRNA data, IL-10 secretion in supernatants of TCR-stimulated CD4+CD25+ cocultures from IRF-1−/− mice was significantly increased (3-fold), whereas
    • IFN-γ secretion was decreased (2.5-fold) compared with cocultures from WT mice (Fig. 2⇑C).
As the functional hallmark of Treg cells is their ability to suppress the expansion of effector T cells, we next evaluated this activity performing suppression assays (1, 2, 3, 8). Importantly, CD4+CD25+ Treg cells from IRF-1−/− mice were found significantly more efficient than WT Treg cells in suppressing the proliferation of syngeneic CD4+CD25− responder T cells in a dose-dependent fashion. Next, to verify whether IRF-1−/− Treg cells suppression ability was retained vs WT responder T cells, we performed suppression assays using IRF-1−/− Treg and WT responders and vice versa. The suppressive activity of IRF-1−/− Treg cells toward WT responders was dose-dependently increased, as well.
IRF-1−/− CD4+CD25− T cells show high bent to convert into CD4+CD25+ Treg cells
It has been reported in mice and human that TGF-β promotes the induction of peripheral CD4+CD25− T cells into CD4+CD25+ Treg cells (inTreg), that acquire Foxp3 expression and regulatory functions.
In presence of TGF-β, 44.2% of CD4+CD25+ inTreg cells were generated in the coculture of CD4+CD25− T cells from IRF-1−/− mice, whereas
  • only 24% of double positive cells were detected in the corresponding coculture from WT mice.
Notably, even in absence of TGF-β, 25.4% CD4+CD25+ inTreg were generated in the coculture of CD4+CD25− T cells from IRF-1−/− mice, as
  • compared with 16.5% of Treg cells generated in WT cocultures.
Importantly, an increased number of CD4+CD25+-gated Foxp3+ cells were observed in IRF-1−/− inTreg cells in the presence (4.5-fold increase) or in the absence (8-fold increase) of TGF-β compared with WT inTreg cells. Next, to evaluate quantitatively Foxp3 expression levels in TGF-β-induced Treg vs ex vivo freshly purified Treg cells, quantitative real-time PCR was performed. A clear-cut
induction of Foxp3 mRNA (4.5-fold increase) was detected in TGF-β-treated IRF-1−/− cells compared with WT cells. Of note, these levels were comparable with those present in freshly isolated IRF-1−/− CD4+CD25+ cells. Strikingly, also untreated IRF-1−/− T cells showed higher levels of Foxp3 mRNA than WT untreated cells (6-fold increase) and similar to levels present in freshly purified WT CD4+CD25+ Treg cells.
The functionality of CD4+CD25+Foxp3+ inTreg cells was then assessed by suppression assays. TGF-β-treated IRF-1−/− inTreg cells were significantly more effective than the WT counterpart cells
  • in suppressing proliferation of effector T cells in a dose-dependent way.
Interestingly, a saturating amount of anti-IL-10 m Abs neutralized the suppression ability of  inTreg cells from both IRF-1−/− and WT mice even though the effect was much more marked in IRF-1−/− inTreg cells. Control Abs did not exhibit any effect.
Restoring IRF-1 expression in IRF-1−/− CD4+CD25− T cells impairs their differentiation into CD4+CD25+Foxp3+ cells
To address the specificity of IRF-1 role in differentiation of CD4+CD25+ Treg cells from CD25− cells, we investigate whether
  • forced expression of IRF-1 in CD4+CD25− IRF-1−/− T cells could rescue the WT phenotype.
  • bicistronic retroviral vectors expressing murine IRF-1 and human CD8 protein as surface marker (MigR1 IRF-1-CD8) or CD8 alone (MigR1 EV-CD8) were generated.
Splenic CD4+CD25− cells from IRF-1−/− mice were stimulated with plate-bound anti-CD3 and anti-CD28 Abs and infected with either retrovirus.
  • 31.6% of MigR1 EV-CD8 CD4+ retrovirus-infected cells were CD25+, by contrast
  • only 17.7% of MigR1 IRF-1-CD8 retrovirus-infected cells were double positive.
Consistently, Foxp3 expression in CD8+-gated cells was significantly decreased in MigR1 IRF-1-CD8-infected cells as compared with
  • those infected with MigR1 EV-CD8 vectors,
  • strongly supporting the evidence that IRF-1 specifically impairs CD4+CD25+ cell differentiation.
IRF-1 binds an IRF-E on the Foxp3 core promoter and inhibits its transcriptional activity
To shed light on the molecular mechanisms responsible for the striking effect exerted by IRF-1 on the development and function of CD4+CD25+ Treg cells, we investigated whether IRF-1, which is a regulator of key immunomodulatory genes (21), could directly regulate the foxp3 gene promoter activity. The proximal promoter of human foxp3 gene has been recently characterized and localized at −511/+176 bp upstream of the 5′ untranslated region (38). By the Genomatix software, we analyzed this region and found an IRF-E spanning from −234 to −203 bp . This region has been found highly homologous to mouse and rat foxp3 promoter, and of note, the IRF-E is perfectly conserved between humans and these species (38). To determine whether IRF-1 could bind this sequence, DNA affinity purification assays were performed with cell extracts from Jurkat T cells, which display discrete basal levels of IRF-1, and from the same cells treated with IFN-γ to maximally stimulate IRF-1 expression. A total of 200 μg of nuclear extracts was incubated with oligonucleotides containing the WT or the a mutated version of IRF-E. The isolated complexes were then examined by immunoblotting against IRF-1. A specific binding of IRF-1 to Foxp3 oligonucleotide was evident. The binding was strongly stimulated by IFN-γ treatment and, interestingly, it was comparable to that obtained when the same extracts were incubated with a synthetic oligonucleotide corresponding to C13, the canonical IRF-1 consensus sequence (21). IRF-1 binding was highly specific because a mutated version of the Foxp3/IRF-E, or an unrelated oligonucleotide corresponding to the STAT binding site present on the β-casein gene promoter, did not retain any protein from the same extracts. To functionally characterize the specific binding of IRF-1 to the foxp3 gene promoter, we cloned the encompassing part of the proximal promoter containing the IRF-E from −296 to +7 bp of foxp3 gene promoter upstream the luciferase reporter gene. The effect of IRF-1 was evaluated in Jurkat T cells transiently cotransfected with the luciferase reporter gene and increasing doses of an IRF-1-expressing vector.
The results indicated that the basal transcriptional activity of the foxp3 gene promoter
    • was substantially reduced in the presence of IRF-1 and the effect was dose-dependent.
Conversely, the basal activity of the foxp3 gene promoter construct mutated in the IRF-E
    • was not affected by IRF-1 overexpression.
Interestingly, IRF-2, a repressor of IRF-1 transcriptional activity on most promoters (21), neither affected the promoter activity nor counteracted the inhibitory effect exerted by IRF-1.  IRF-1, IRF-2, as well as the IFN-γ treatment drastically reduced the transcriptional activity of the il4 gene promoter, whereas
  • the low molecular mass polypeptide lmp2 construct was stimulated by IRF-1 and by IFN-γ treatment, but it was not affected by IRF-2.

All together these results demonstrate the specificity and functional relevance of IRF-1 binding to the foxp3 proximal promoter.

Foxp3 is a direct target of IRF-1 in human and mouse primary CD4+CD25− T cells and CD4+CD25+ Treg cells
To assess the biological relevance of the the reported effects of IRF-1 on Treg development and on the regulation of Foxp3 expression, we performed experiments with primary cells. We first assessed by Western blot IRF-1 expression levels in CD4+CD25+ Treg cells vs CD4+CD25− T cells magnetically sorted from PBMC of healthy donors or from mice spleens. Strikingly, we found that IRF-1 was down-regulated in double positive cells as compared with CD4+CD25− T cells both in mouse and human primary cells. To determine whether IRF-1 binds the Foxp3 oligonucleotides in primary Treg cells, pull-down assays with the same extracts were then performed. IRF-1 binding to Foxp3 oligonucleotide was significantly decreased in primary CD4+CD25+ Treg cells compared with CD4+CD25− T cells from both species. Foxp3 staining of CD4+CD25− T cells and CD4+CD25+ human Treg cells confirmed that these cells expressed low and high levels of Foxp3, respectively, and
  • Foxp3 expression was further increased by IL-2 treatment.
To test whether IRF-1 expression was also down-modulated during the acquisition of Treg cell phenotype upon TGF-β treatment, freshly purified TCR-activated CD4+CD25− T cells from both species were cultured with TGF-β, or left untreated, for 3 days and Western blot analysis was performed. When cells were cultured in presence of TGF-β, IRF-1 expression was substantially decreased, as compared with untreated cells. Pull-down assays revealed that IRF-1 binding to Foxp3 oligonucleotide was decreased in TGF-β-treated primary cells compared with untreated cells, as well. Consistently, FACS analysis of these cultures indicated that ∼35% of TGF-β-treated CD4+ cells were Foxp3+ in human and ∼10% in mouse TGF-β treated cultures, respectively. By contrast, even though 46.3% of human untreated cells were CD25+ only 5% were Foxp3+.
Next, we assessed the in vivo IRF-1 binding to foxp3 gene in human and mouse primary magnetically sorted CD4+CD25− T cells and CD4+CD25+ Treg cells, using ChIP assay with anti-IRF-1 Abs. After DNA immunoprecipitation, subsequent real-time PCR amplification of the foxp3 gene surrounding the IRF-E site showed significant IRF-1 binding to Foxp3 promoter in CD4+CD25−Foxp3− T cells, and by contrast, a 5-fold decrease of IRF-1 binding in CD4+CD25+Foxp3high human Treg cells (Fig. 6⇑C). Similarly, the binding of IRF-1 to the Foxp3 promoter in the mouse Treg cells was decreased by ∼50%.
Finally, to assess the functionality of the in vivo IRF-1 binding, negatively selected primary human and mouse CD4+ T lymphocytes were nucleofected with the Foxp3 luciferase reporter gene along with expression vector for IRF-1. Fig. 6⇑E shows the results obtained with T cells from three different healthy donors and Fig. 6⇑F shows a representative experiment with mouse T cells from three independent experiments. In all samples, a discrete basal activity of foxp3 gene promoter was present and this activity was significantly repressed by IRF-1.
Discussion
The identification of molecules controlling Treg differentiation and function is important not only in understanding host immune responses in malignancy and autoimmunity but also in shaping immune response.
In this study, we have shown that IRF-1, a transcription factor involved in the IFN signaling, selectively affects CD4+CD25+ Treg cell development and function, unraveling a novel immunoregulatory function of IRF-1 in addition to its well-established role in balancing Th1 vs Th2 type immune responses. Several lines of evidence support this conclusion:
1) IRF-1−/− mice show a selective and marked increase in all lymphoid organs of CD4+CD25+Foxp3+ Treg cells; 2) CD4+CD25+ from IRF-1−/− mice are characterized by a highly activated and differentiated  phenotype and higher levels of Foxp3 that make them to be functionally more suppressive than WT Treg cells;
3) after TGF-β treatment, and importantly also in its absence, CD4+CD25− T cells from KO mice promptly converted into CD4+CD25+Foxp3+ Treg with a higher suppressive activity than WT cells;
4) forced retrovirus-mediated expression of IRF-1 in IRF-1−/− CD4+CD25− T cells impairs their differentiation into CD25+Foxp3+ cells; and 5) IRF-1 directly regulates transcriptional activity of the foxp3 gene promoter.
The phenotypical and functional characteristics of IRF-1−/− Treg cells strongly support the conclusion that IRF-1 can be considered a key negative regulator of CD4+CD25+ Treg cells.
The increased frequency of differentiated and activated CD4+CD25+ Treg cells characterized by an immunosuppressive cytokine profile described in this study
    • may provide a mechanistic base for the reduced incidence and severity of several autoimmune diseases characterizing IRF-1−/− mice .
In this regard, it has been recently shown that CD4+CD25+ Treg cells were increased in IRF-1−/− mice backcrossed with the MRL/lpr mice, which showed reduced glomerulonephritis.
The increased production of the immunosuppressive cytokine IL-10 by isolated Treg cells from IRF-1−/− mice and the reverted suppression ability of inTreg by anti-IL-10 Abs suggest that this cytokine could play a key role in their suppressor function. Consistently, IL-10 activity has been recently associated with the function of TGF-β-induced CD4+CD25−CD45RBlow cells because their suppressive activity was abrogated with anti-IL-10R Ab treatment (13). Moreover, several reports focused on the in vivo IL-10 role in peripheral CD4+CD25+ Treg cell function in various autoimmunity models (10, 11, 12), although IL-10 seems not required for the functions of thymically derived Treg cells (1). In contrast with the increased IL-10 production, T cells from IRF-1−/− mice failed to produce significant amounts of proinflammatory cytokines such as IFN-γ or TNF-α. Accordingly, an inverse relationship between in vivo IFN-γ administration and generation or activation of CD4+CD25+ Treg cells has been recently shown (45). Moreover, in humans, it has been reported that TNF-α inhibits the suppressive function of both naturally occurring CD4+CD25+ Treg and TGF-β-induced Treg cells, and an anti-TNF Ab therapy reversed their suppressive activity by down-modulating the expression of Foxp3 (46). These latter and our results are apparently in contrast with what was recently reported on the stimulating role of IFN-γ on Foxp3 induction and conversion of CD4+CD25− T cells to CD4+ Treg cells in the IFN-γ KO model (47). In this regard, it is noteworthy to underline that, as it has been also suggested, although knocking down genes involved in up-regulation of IFN-γ expression do not significantly influence autoimmunity, by contrast the absence of genes expressed in response to IFN-γ, including IRF-1, lead to greatly reduced autoimmunity (48). Thus, although the exact mechanism underlying IFN-γ and TNF-α interference with the elicitation of Treg cells remains to be defined, we can speculate that induction of IRF-1 expression, which is up-regulated by IFN-γ and TNF-α, may represent a mechanism through which proinflammatory cytokines negatively affect Foxp3 expression, thereby influencing generation or activation of CD4+CD25+ Treg cells.
It is well known that Foxp3 plays a pivotal role in the regulatory functions of CD4+CD25+ T cells both in humans and in animal models. Thus, the key question in the field of Treg biology is which are molecules and signals that govern Foxp3 transcription.
We identify Foxp3 as specific target of IRF-1 and we show
    • that it binds to foxp3 gene promoter in vitro and in vivo and represses its expression.
Structure of the human foxp3 gene promoter and elements necessary for its induction in T cells have been reported. We have identified an IRF-E sequence at 203 bp upstream of the transcriptional start site that is highly conserved. This element is bound by IRF-1 as proven by pull-down experiments and by ChIP analysis in intact cells, and IRF-1 binding resulted in a specific,
  • dose-dependent repression of the foxp3 proximal promoter.
Notably, treatments with IFN-γ, a major IRF-1 inducer, significantly inhibited foxp3 gene promoter transcriptional activity, whereas IRF-2 did not have any effects. It is noteworthy that the foxp3 gene is highly conserved between mouse and man species, and in particular, the core promoter and the IRF-E identified in this study are perfectly conserved between mouse and human. Such conservation underscores the importance of this motif as regulatory element and provides additional evidence for the role of IRF-1 in regulating foxp3 gene expression.  IRF-1 binds this sequence and negatively regulates its expression in both human and mouse cells. The molecular interactions enabling IRF-1 to inhibit Foxp3 are not yet identified, although our preliminary results show that IRF-1 may compete with c-Myb for the binding to the same overlapping consensus sequence on the foxp3 gene promoter.
In summary, the current study provides evidence that IRF-1 affects CD4+CD25+ development and function by Foxp3 repression. Thus, our data demonstrate a new important contribution by which IRF-1 affects T cell differentiation and provide new important insights into molecular mechanisms controlling immune homeostasis.


Th1-Th2-Th17-Treg origin

Th1-Th2-Th17-Treg origin (Photo credit: Wikipedia)

 

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