Posts Tagged ‘Interleukin 6’

Author and Curator: Ritu Saxena, Ph.D

Although cancer stem cells constitute only a small percentage of the tumor burden, their self-renewal capacity and possible link with recurrence of cancer post treatment makes them a sought after therapeutic target in cancer. The post on cancer stem cells published on the 22nd of March, 2013, describes the identity of CSCs, their functional characteristics, possible cell of origin and biomarkers. This post focuses on the therapeutic potential of CSCs, their resistance to conventional anti-tumor therapies and current therapeutic targets including biomarkers, signaling pathways and niches.

CSCs Are Resistant to conventional anticancer therapies including chemotherapy, radiotherapy and surgery that are used either alone or in combination. However, these strategies have failed several times to eradicate CSCs resulting in metastasis and relapse, hence, a fatal disease outcome.

The properties of CSCs that contribute to or lead to chemoresistance include:

Quiescent Phenotype

Chemotherapeutic agents target fast-growing cells; however, some CSCs that remain in the dormant or quiescent stage are spared from lethal damage. Later, when the dormant CSCs enter cell cycle, tumor proliferation is stimulated.


Antiapoptotic proteins such as BCL-2 and some self-renewal pathways such as transforming growth factor β, Wnt/ β -catenin or BMI-1 are activated in CSCs. Consequently, DNA damage repair capability of CSCs is enhanced after genotoxic stress or activation of autocrine loops through the production of growth factors like epidermal growth factor (Moserle L, Cancer Lett, 1 Feb 2010;288(1):1-9).

Expression of Drug Efflux Pumps

CSCs express some proteins that have typically been known to contribute to multidrug resistance. The proteins are drug efflux pumps ABCC1, ABCG2 or MDR1. Multidrug resistance-associated proteins (ABCC subfamily) are members of the ATP-binding cassette (ABC) superfamily of transport proteins and act as cellular efflux transporters for a wide variety of substrates, in particular glutathione, glucuronide and sulfate conjugates of diverse compounds.

Radiotherapy is mainly used in breast cancer and glioblastoma multiforme. In glioblastoma multiforme, the properties of CSCs that contribute to radiotherapy resistance is the presence of CD133 marker. CD133+ CSCs preferentially activate DNA damage repair pathway and significantly induced checkpoint kinases that leads to reduced apoptosis in CSCs compared to the CD133- tumor cells (Bao S, Nature, 7 Dec 2006;444(7120):756-60).

Radiotherapy resistance in breast cancer is due to reduced levels of reactive oxygen species in CSCs. In addition, radiation resistance of progenitor cells in an immortalized breast cancer cell line was mediated by the Wnt/β catenin pathway proteins (Diehn M, et al, Nature, 9 Apr 2009;458(7239):780-3; Chen MS, et al, J Cell Sci, 1 Feb 2007;120(Pt 3):468-77).

As mentioned in the previous post on CSCs, CSC targeting therapy could either eliminate CSCs by either killing them after differentiating them from other tumor population, and/or by disrupting their niche. Efficient eradication of CSCs may require the combined ablation of CSCs themselves and their niches. Thus, identification of appropriate and specific markers of CSCs is crucial for targeting them and preventing tumor relapse. Table 1 (adapted from a review article on CSCs by Zhao et al) describes the currently used biomarkers for CSC-targeted therapy (Zhao L, et al, Eur Surg Res, 2012;49(1):8-15).

Table 1

Specific Target Cancer type Marker properties and therapy
Targeting cell markers
CD24+CD44+ESA+ Pancreatic cancer Pancreatic CSCs, elevated during tumorigenesis
CD44+CD24–ESA+ Breast cancer Breast CSCs
EpCAM high CD44+CD166+ Colorectal cancer
CD34+CD38– AML broad use as a target for chemotherapy
CD133+ Prostate cancer and breast cancer 5-transmembrane domain cell surface glycoprotein,also a marker for neuron epithelial, hematopoietic and endothelialprogenitor cells
Stro1+CD105+CD44+ Bone sarcoma
Nodal/activin Knockdown or pharmacological inhibition of its receptorAlk4/7 abrogated self-renewal capacity and in vivo tumorigenicity of CSCs.
Targeting signaling pathways
Hedgehog signaling Upregulated in several cancer types inhibitors: GDC-0449,PF04449913, BMS-833923, IPI-926 and TAK-441
Wnt/β-catenin signaling CML, squamous cell carcinoma Be required for CSC self-renewal and tumor growthinhibitors: PRI-724, WIF-1 and telomerase
Notch signaling Several cancer types An important regulator in normal development, adult stem cell maintenance,and tumorigenesis in multiple organs,inhibitors: RO4929097, BMS-906024, IPI-926 and MK0752
PI3K/Akt/PTEN/mTOR, Several cancer types The pathway is deregulated in many tumors and used to preferentially target CSCsinhibitors: temsirolimus, everolimus FDA-approved therapy for renal cell carcinoma
Targeting CSC Niche
Angiogenesis Niche Colon cancer, breast cancer, NSCLC Inhibitor: bevacizumab results in a disruption of the CSC niche, depleted vasculature and a dramatic reduction in the number of CSCs.
Hypoxia (HIF pathway) Ovarian cancer, lung cancer, cervical cancer Inhibitors: topotecan and digoxin have been approved for ovarian, lung and cervical cancer
Targeting Micro RNA
miR-200 family Inhibits EMT and cancer cell migration by direct targeting of E-cadherin transcriptional repressors ZEB1 and ZEB2
Let-7 family Regulates BT-IC stem cell-like properties by silencing more than one target
miR-124 Related to neuronal differentiation, targets laminin γ1 and integrin β1.
miR-21 Suppresses the self-renewal of embryonic stem cells

The challenge is to develop an effective treatment regimen that prevents survival, self-renewal and differentiation of CSCs and also disturbs their niche without damaging normal stem cells. In order to evaluate the efficiency of CSC-targeting therapies, in vitro models and mouse xenotransplantation models have been used for preclinical studies. Some potential CSC targeting agents in preclinical stages include notch inhibitors for glioblastoma stem cells and telomerase peptide vaccination after chemoradiotherapy of non-small cell lung cancer stem cells Stem Cells (Hovinga KE, et al, Jun 2010;28(6):1019-29; Serrano D, Mol Cancer, 9 Aug 2011;10:96). In addition, several phase II and phase III trials are currently underway to test CSC-targeting drugs focusing on efficacy and safety of treatment.


Bao S, Nature, 7 Dec 2006;444(7120):756-60).

Diehn M, et al, Nature, 9 Apr 2009;458(7239):780-3

Chen MS, et al, J Cell Sci, 1 Feb 2007;120(Pt 3):468-77

Zhao L, et al, Eur Surg Res, 2012;49(1):8-15

Hovinga KE, et al, Jun 2010;28(6):1019-29

Serrano D, Mol Cancer, 9 Aug 2011;10:96

Pharmaceutical Intelligence posts: Author and curator: Ritu Saxena, PhD Authors: Anamika Sarkar, PhD and Ritu Saxena, PhD Author: Ziv Raviv, PhD Reporter: Larry H Bernstein, MD Larry H Bernstein, MD Curator: Aviva Lev-Ari, PhD, RN Curator: Ritu Saxena, PhD Curator: Aviva Lev-Ari, PhD, RN Author and reporter: Tilda Barliya PhD Reporter and Curator: Stephen J. Williams, PhD Reporter: Ritu Saxena, PhD Reporter: Aviva Lev-Ari, PhD, RN Reporter: Ritu Saxena, PhD Aviva Lev-Ari, PhD, RN



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Author and Curator: Ritu Saxena, PhD


What are cancer stem cells?

Cancer is a debilitating disease estimated to be responsible for about 7.6 million deaths in 2008 (Jemal A, et al, CA Cancer J Clin, Mar-Apr 2011;61(2):69-90). Thus, extensive research is underway to deal with the various types of cancer. The concept of cancer stem cells (CSC) has surfaced in in the past decade after identification and characterization of CSC-enriched populations in several different types of cancer (Lapidot T, et al, Nature, 17 Feb 1994;367(6464):645-8; Reya T, et al, Nature, 1 Nov 2001;414(6859):105-11;  Trumpp A and Wiestler OD, et al, Nat Clin Pract Oncol, Jun 2008;5(6):337-47). Although there has been lot of debate on the cell of origin of CSC, according to the classical concept CSC are defined by their functional properties.

Functional properties of CSC

  • CSCs are at the top of tumor hierarchy. Regenerative tissues follow a hierarchical organization with adult stem cells at the top maintaining tissues and normal adult cells during homeostasis and regeneration during cell loss from injury. Similarly, several tumors follow the hierarchy with CSC at the top. Hierarchical organization has been reported in several cancer types including but not limited to breast cancer, brain cancer, colon cancer, leukemia and pancreatic cancer (Lapidot T, et al, Nature, 17 Feb 1994;367(6464):645-8; Al-Hajj M, et al, PNAS USA, 1 Apr 200;100(7):3983-8; Singh SK, et al, Nature, 18 Nov 2004;432(7015):396-401; Dalerba P, et al, PNAS USA, 12 Jun 2007;104(24):10158-63; Hermann PC, et al, Cell Stem Cell, 13 Sep 2007;1(3):313-23).
  • CSCs possess unlimited self-renewal capacity similar to that of physiological stem cells and unlike other differentiated cell types within the tumor. Cancer stem cells can also generate non-CSC progeny that is comprised of differentiated cells and forms tumor bulk.
  • Some CSs exhibit quiescent or dormant stage. Although not observed in all CSC types, some CSCs have been found to shuttle between quiescent, slow-cycling, and active states. The CSCs in their dormant and slow-cycling stage are less likely to be affected by conventional anti-tumor therapies which generally target rapidly dividing cells. Dormant stage is exhibited even in adult stem cells and the dormant normal stem cells can regain cell division potential during tissue injury (Wilson A, et al, Cell,  12 Dec 2008;135(6):1118-29). Thus, it has been speculated that dormant CSC might be a reason for tumor relapse even after pathologic complete response is observed post therapy.
  • Some CSCs are resistant to conventional anti-cancer therapies. This leads to accumulation of CSC that might result in relapse after anti-cancer therapy. For instance, Li et al (2008) reported that CSC accumulated in the breast of women with locally advanced tumors after cytotoxic chemotherapy had eliminated the bulk of the tumor cells (Li X,et al, J Natl Cancer Inst, 7 May 2008;100(9):672-9). A similar observation was made by Oravecz-Wilson et al (2009) stating that despite remarkable responses to the tyrosine kinase inhibitor imatinib, CML patients show imatinib refractoriness because leukemia stem cells in CML are resistant tyrosine kinase (Oravecz-Wilson KI, et al, Cancer Cell, 4 Aug 2009;16(2):137-48).
  • The CSC niche. CSC functional traits might be sustained by this microenvironment, termed “niche”. The niche is the environment in which stem cells reside and is responsible for the maintenance of unique stem cell properties such as self-renewal and an undifferentiated state. The heterogeneous populations which constitute a niche include both stem cells and surrounding differentiated cells. The necessary intrinsic pathways that are utilized by this cancer stem cell population to maintain both self-renewal and the ability to differentiate are believed to be a result of the environment where cancer stem cells reside. (Cabarcas SM, et al, Int J Cancer, 15 Nov 2011;129(10):2315-27). For instance, properties of CSC in glioma in a mouse xenograft model were maintained by vascular endothelial cells (Calabrese C, et al, Cancer Cell, Jan 2007;11(1):69-82). Several molecules including interleukin 6 have been observed to play a role in tumor proliferation and hence, participate in maintaining tumorigenic and self-renewal potential of CSC. Moreover, the CSC niche might not only regulate CSCs traits but might also directly provide CSC features to non-CSC population.

What is the origin of CSC?

According to current thinking, CSC result from epithelial-mesenchymal transition (EMT) when cells switch from a polarized epithelial to a non-polarized mesenchymal cell type with stem cell properties, including migratory behavior, self-renewal and generation of differentiated progeny, and reduced responsiveness to conventional cancer therapies (Scheel C and Weinberg RA, Semin Cancer Biol, Oct 2012;22(5-6):396-403; Crews LA and Jamieson CH, Cancer Lett, 17 Aug 2012). Evidence is accumulating that cancers of distinct subtypes within an organ may derive from different ‘cells of origin’. The tumor cell of origin is the cell type from which the disease is derived after it undergoes oncogenic mutation. It might take a series of mutations to achieve the CSC phenotype (Visvader JE, Nature, 20 Jan 2011;469(7330):314-22). Also, CSCs have been reported to originate from stem cells in some cases.

Biomarkers for CSC

CSC targeting therapy could either eliminate CSCs by either killing them after differentiating them from other tumor population, and/or by disrupting their niche. Efficient eradication of CSCs may require the combined ablation of CSCs themselves and their niches. Identifying appropriate biomarkers of CSC is a very important aim for CSCs to be useful as targets of anti-cancer therapies in order to possibly prevent relapse. Using cell surface markers, CSCs have been isolated and purified from cancers of breast, brain, thyroid, cervix, lung, blood (leukemia), skin (melanoma), organs of the gastrointestinal and reproductive tracts, and the retina. The challenge, however, is that CSCs share similar markers with normal cells which makes CSCs targeting difficult as it would harm normal cells in the process. More recently, advanced techniques such as signal sequence trap (SST) PCR screening methods have been developed to identify a leukemia-specific stem cell marker (CD96). After a small subset of human AML cells displayed tumorigenic properties, Leukemia Stem Cells (LSCs) were identified as leukemia cells with CD23+/CD38+ markers. These cells closely resemble hematopeotic stem cells (HSCs) (Bonnet D and Dick JR, Nat Med, Jul 1997;3(7):730-7). In solid tumors, a significant discovery was made when CSCs in breast cancer were identified within the ESA+/CD44+/CD24low-neg population of mammary pleural effusion and tumor samples (Al-Hajj M, et al, PNAS USA, 1 Apr 200;100(7):3983-8).

After these two landmark publications, CSCs were identified in many more solid and hematopoietic human tumors as well. In addition, within a tumor type, CSC-enriched populations display heterogeneity in markers. For example, only 1% of breast cancer cells simultaneously express both reported CSC phenotypes ESA+/CD44+/

CD24low-neg and ALDH-1+ (Ginestier C, et al, Cell Stem Cell, 1 Nov 2007;1(5):555-67). The discrepancy might be due to different techniques used to identify the markers and also a reflection of the molecular heterogeneity within the tumors. Recent advances in genome wide expression profiling studies have led to the identification of different subtypes in a particular type of cancer. Breast cancer was recently classified into different subtypes and this genetic heterogeneity is likely paralleled by a heterogeneous CSC complexity.


A lot of research is currently underway on various aspects of CSCs including biomarker identification, cell of origin, and clinical trials targeting CSC population in cancer. The concept of CSCs has evolved quite a bit since their discovery. Recently, identification of high genetic heterogeneity within a tumor has been in focus and subsequently it has been observed that several CSC clones can coexist and compete with each other within a tumor. Adding complexity to their identity is the fact that CSCs may have unstable phenotypes and genotypes. Taken together, the dynamics associated with CSCs makes it difficult to identify reliable and robust biomarkers and develop efficient targeted therapies. Thus, a major thrust of research should be to focus on the unfolding of the dynamic identity of CSCs in tumor types and at different that might lead to the identification and targeting of highly specific CSCs biomarkers.


Jemal A, et al, CA Cancer J Clin, Mar-Apr 2011;61(2):69-90

Reya T, et al, Nature, 1 Nov 2001;414(6859):105-11

Trumpp A and Wiestler OD, et al, Nat Clin Pract Oncol, Jun 2008;5(6):337-47

Lapidot T, et al, Nature, 17 Feb 1994;367(6464):645-8

Singh SK, et al, Nature, 18 Nov 2004;432(7015):396-401

Dalerba P, et al, PNAS USA, 12 Jun 2007;104(24):10158-63

Hermann PC, et al, Cell Stem Cell, 13 Sep 2007;1(3):313-23

Wilson A, et al, Cell,  12 Dec 2008;135(6):1118-29

Li X,et al, J Natl Cancer Inst, 7 May 2008;100(9):672-9

Oravecz-Wilson KI, et al, Cancer Cell, 4 Aug 2009;16(2):137-48

Cabarcas SM, et al, Int J Cancer, 15 Nov 2011;129(10):2315-27

Calabrese C, et al, Cancer Cell, Jan 2007;11(1):69-82

Scheel C and Weinberg RA, Semin Cancer Biol, Oct 2012;22(5-6):396-403

Crews LA and Jamieson CH, Cancer Lett, 17 Aug 2012

Visvader JE, Nature, 20 Jan 2011;469(7330):314-22

Bonnet D and Dick JR, Nat Med, Jul 1997;3(7):730-7

Al-Hajj M, et al, PNAS USA, 1 Apr 200;100(7):3983-8

Ginestier C, et al, Cell Stem Cell, 1 Nov 2007;1(5):555-67

Baccelli I and Trumpp AJ, Cell Biol, 6 Aug 2012;198(3):281-93

Zhao L, et al, Eur Surg Res, 2012;49(1):8-15

Pharmaceutical Intelligence posts:

Authors: Anamika Sarkar, PhD and Ritu Saxena, PhD Author: Ziv Raviv, PhD Reporter: Larry H Bernstein, MD

Larry H Bernstein, MD Curator: Aviva Lev-Ari, PhD, RN Curator: Ritu Saxena, PhD Curator: Aviva Lev-Ari, PhD, RN Author and reporter: Tilda Barliya PhD Reporter and Curator: Stephen J. Williams, PhD Reporter: Ritu Saxena, PhD Reporter: Aviva Lev-Ari, PhD, RN Reporter: Ritu Saxena, PhD Aviva Lev-Ari, PhD, RN

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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.

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.
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.
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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