ovarian cancer is one of the most common and deadliest gynecologic cancers . in 2010 , 21880 new cases were diagnosed , and such cancer caused nearly 13850 deaths in the united states alone .
ovarian cancer usually has poor prognosis , and most patients were diagnosed at advanced stages .
the five - year survival rate for all stages of ovarian cancer is 46% in 2010 .
it has been well documented that patients ' clinical outcome and five - year survival rate are positively associated with the number of tumor - infiltrating lymphocytes ( tils ) , and the ratio of intraepithelial cd8 tils to treg cells , or negatively associated with tumor- infiltrating treg cells .
treg cells are also known as suppressor t cells which consist of a specific subpopulation of cells that functionally suppress the activation of immune system and maintain immune tolerance to self - antigens .
treg cells contain two major subsets known as natural treg cells ( ntreg ) and adaptive or induced treg cells ( itreg ) .
ntreg cells derived from thymus are considered as classic treg cells , by contrast , itreg cells develop in the periphery in response to self- or tumor antigens by converting naive cd4 t cells into treg cells . because most tumors express self - antigens , treg cells - mediated immunosuppression is believed to be one of the major contributors to immune evasion by tumors and becomes the main obstacle toward successful tumor immunotherapy . in this paper
, we will focus on human ovarian cancer and discuss the nature of treg cells including their subsets , trafficking , differentiation , and proliferation and the clinical application of manipulation of treg cells .
in early 1970s , gershon and kondo first described the existence of thymus - derived suppressive t cells ( later termed as treg cell ) in vivo [ 7 , 8 ] .
after more than a decade , sakaguchi et al . demonstrated that cd4 t cells expressing interleukin-2 ( il-2 ) receptor alpha - chain ( cd25 ) can be defined as the population of treg cells with immune - suppressive activities and maintaining immune tolerance to self - antigen . later in 2003 , hori et al .
found that the transcription factor forkhead box p3 ( foxp3 ) controls the development of treg cells and is crucial for maintaining the immune - suppressive function of treg cells .
natural treg cells differentiate in the thymus and migrate to periphery , which constitute 510% of cd4 t cells [ 1113 ] . in addition
identified another subset of treg cells , cd4  tr 1 cells , that suppress antigen - specific immune responses by producing high levels of il-10 .
in addition to cd4  treg , cd8 suppressive t cells have been found playing an important role in the regulation of autoimmune disease [ 7 , 15 ] .
cd8 suppressive t cells now refered to as cd8  treg cells are characterized as cd8cd25 , cd8cd122 , or cd8cd45rc  treg cells , which comprise less than 1% of peripheral cd8 t cells .
th3 treg cells have similar immune - suppressive function ; however , in contrast to natural treg cells , th3 exerts its suppressive capacity independent of cell membrane contact but mainly bases on the action of self - produced cytokine tgf .
treg cells consist of ~10% of peripheral cd4 t cells characterized as cd4cd25foxp3 t cells , which is important for the control of autoimmune reaction [ 9 , 11 ] .
dysregulation of treg can cause autoimmune diseases   and may contribute to tumor - initiated immune evasion . as demonstrated by in vivo mouse model , the deletion of treg cells results in tumor rejection .
however , the suppressive capacity of treg cells is also determined by the ratio of treg cells to effector t cells .
a high cd8/treg ratio is associated with favorable prognosis and improved survival [ 3 , 20 ] .
it has been reported that many human cancers are associated with high frequency of treg cells in the circulation or in the tumor tissues , including ovarian cancer , lung cancer , breast cancer , liver cancer , head and neck cancer , and lymphoma .
these increased levels of treg cells are linked to high death hazard and poor survival , while the depletion of tumor - infiltrated treg cells and the blockade of treg trafficking to tumors enhance anti - tumor immune response [ 4 , 26 ] .
ccr4 and its binding partners ccl22 and ccl17 are believed to be the most predominant axis in chemokine - mediated selective treg trafficking to the tumors .
have profiled chemotactic responses and chemokine receptors expression of human treg cells and found that treg cells specifically express chemokine receptors ccr4 and ccr8 .
chemokine ccl22 , the ligand for ccr4 , preferentially attracts activated - antigen - specific t cells to dendritic cells [ 28 , 29 ] .
it has also been shown that human ovarian cancer cells and tumor - associated microphages produce chemokine ccl22 , which mediates treg cells trafficking to tumor .
blockade of ccl22 in vivo significantly reduces human treg cells trafficking to tumors in ovarian carcinoma .
this chemokine - mediated treg trafficking has been also observed in other types of cancer , such as gastric cancer , hodgkin 's lymphoma , and breast cancer .
interestingly , in gastric cancer , ccl22 and ccl17 seem both important to recruit treg cells to the tumors as demonstrated by in vivo study as well as in vitro migration assay , and the levels of ccl22 and ccl17 within tumors are correlated to the increased levels of treg cells in early gastric cancer . besides ccr4 chemokine axis , ccr5/ccl5 axis may also selectively recruit treg cells to the tumors . using human pancreatic adenocarcinoma and murine pancreatic tumor model , it has been found that ccr5 is highly expressed in treg cells , while tumor cells produce elevated amount of ccl5 , and disruption of ccr5/ccl5 chemokine axis blocks treg cells migration and reduces tumor growth .
in addition , ccl20 chemokine shows high affinity to ccr6 and can also mediate selective ccr6 treg cells trafficking .
found that peripheral treg migrates from the thymus and appears in the periphery as early as 10th day of life .
they also found that cd4cd25  treg cells differentiation is totally dependent on il-2 , because il-2 knockout mice do not develop cd4cd25  treg  in vivo .
further evidences have been provided from the studies on irradiated rat model . in this study ,
autoimmune diseases were induced in rats by thymectomy and irradiation ; however the xenograft transfer of cd4 t cells from normal rats can abrogate the autoimmune responses .
these observations suggest that normal thymus - derived t cells have immune suppressive functions and thus prevent autoimmunity . in another model system ,
adoptive transfer of thymocytes or peripheral t cells depleted of cd4cd25  treg cells causes autoimmune diseases in mice , which provides further evidences of thymic origin of treg cells and their peripheral existence .
although there are several arguments about how and what stromal components are involved in thymic treg cell differentiation , thymic stromal cells , including cortical and medullary thymic epithelial cells and dendritic cells ( dcs ) , contribute to treg cells differentiation and selection .
jordan et al . used tcr - transgenic mice which express the receptor recognizing specific self - antigen and found that thymocytes bearing a tcr with high affinity to a specific self - antigen undergo selection and become cd4cd25  treg cells when interacting with a single self - antigen , but thymocytes bearing tcr with low affinity do not undergo selection .
for instance , tumor microenvironment favors the induction and differentiation of treg cells , and that has been extensively studied for several years . in the tumor microenvironment ,
dc differentiation and function were suppressed by tumor - associated factors il-10 , vegf , and tgf , resulting in immature / dysfunctional dc .
dysfunctional dc directly contributed to the induction of il-10-producing treg cells in vivo in human and in vitro [ 41 , 42 ] .
tumor - associated plasmacytoid dc also induced il-10  treg generation [ 43 , 44 ] .
tumor can convert dc into tgf-producing immature dc , which selectively promotes treg proliferation in tgf-dependent manner .
treg cells can also be converted from peripheral nave cd4cd25 t cells by the action of tgf. tumor microenvironment contains high levels of tgf which might mediate tumor - associated treg cells conversion .
in the mouse model , depletion of cd4cd25  treg cells using anti - cd25 antibody causes tumor regression , which correlated to the reduced number of treg cells [ 18 , 47 ] . using the recombinant il-2 diphtheria toxin conjugate dab(389)il-2 ( also known as denileukin diftitox and ontak ) , dannull et al
. demonstrated that dab(389)il-2 was capable of selectively eliminating cd25  treg cells from the pbmcs of cancer patients without inducing toxicity on other cellular subsets , and dab(389)il-2-mediated treg depletion enhanced anti - tumor immune responses and significantly reduced the number of treg cells present in the blood of cancer patients .
daclizumab ( also known as zenapex ) and basiliximab ( also called simulect ) are monoclonal antibodies against cd25 [ 49 , 50 ] , and the administration of daclizumab in patient with metastatic breast cancer enhanced anti - tumor immunity .
cytotoxic t - lymphocyte - associated antigen 4 ( ctla4 ) is constitutively expressed and restricted to cd4cd25  treg cells among all cd4 cells , and the immune - suppressive function of treg is mediated by ctla4 signaling [ 52 , 53 ] .
ctla4 binds to inhibitory b7 members on apc and transmits an inhibitory signal to t cells . in vivo administration of anti - ctla4 antibody
periodic infusions of anti - ctla4 antibody in previously vaccinated patients with cancer created clinically effective antitumor immune response .
patients with metastatic melanoma showed improved antitumor immunity and tumor regression by blockade of ctla-4 together with peptide vaccination .
glucocorticoid - induced tumor necrosis factor ( tnf ) receptor family - related protein ( gitr or dta-1 ) is predominantly expressed on the surface of treg cells .
an agonistic anti - gitr antibody administration in mice can abrogate treg - mediated immune suppression and enhance effective anti - tumor immunity in vivo [ 57 , 58 ] .
in addition , treatment with anti - gitr antibody in b16 mice elicited immune response and rejected tumor .
however gitr is not exclusively expressed on treg cell ; it is also expressed by various cd4 t cells and others .
therefore , the clinical therapeutic relevance of gitr blockade and its side effects on potential deficits of other effective immune cells remain to be determined .
ox40 ( cd134 ) also belongs to tnf receptor family and expressed on activated t cells .
similar to gitr , triggering ox40 by an agonistic antibody against ox40 reduces treg - mediated immune suppression and restores effector t - cell function both in vivo and in vitro .
it has been also shown that ox40 is necessary for treg development , homeostasis , and immune - suppressive activity .
however , stimulation of ox40 signal in nave t cells can abrogate treg - mediated suppression . clinical relevance of the depletion of treg cells has been further confirmed by the treatment of cyclophosphamide ( cy ) in the patients bearing tumor .
low dose of cy administration improved patients ' immune responses by reducing the number of tregcells and by decreasing the suppressive activity of treg cells .
effects of treg depletion on anti - tumor immune responses were further investigated by the study on b16 melanomas mouse model .
other immunosuppressants like cyclosporine a ( csa ) and azathioprine might also inhibit treg cells generation [ 64 , 65 ] .
for instance , high dose of csa abrogates treg cell generation ; by contrast , low dose of csa can promote treg cell development .
it is therefore important to determine whether lowdose of those agents can improve antitumor immunity in patients .
our group has demonstrated that human ovarian cancer cells and tumor - associated macrophage ( tam ) produced chemokine ccl22 , the ligand for ccr4 which functionally expressed on tumor treg cells , mediating treg cells trafficking to the tumor and ascites , and the blockade of ccl22 abrogated treg cells migration .
it has been demonstrated that chemokine receptor ccr4 is selectively expressed by treg cells , and the ccr4 and ccr4-associate chemokines axis is one of the most described tumor treg recruitment axes .
the administration of anti - ccr4 antibody effectively depletes ccr4 t cells and inhibits treg cells migration in hodgkin lymphoma .
furthermore , the significant correlation between ccl17 or ccl22 chemokines and the number of tumor - infiltrating treg cells was found in patients with neoplastic meningitis and gastric cancer [ 30 , 33 ] .
ccl5 and ccl20 chemokines are also involved in treg trafficking , and that blockade of those chemokines reduces treg cells trafficking and inhibits tumor growth [ 34 , 35 ] .
recently , a study has demonstrated that blockade of cxcr4 by a selective antagonist resulted in the significant reduction of intratumoral treg cells , which was associated with greatly increased antitumor immunity and an improved survival in an immunocompetent mouse model of ovarian cancer .
it is thought that blockade of tgf signaling pathway may alter treg phenotype and function and in turn enhances antitumor immunity .
notably , tgf is not only important for treg cell functional integrity , but also inhibits the proliferation and functional differentiation of t lymphocytes , nk cells , and macrophages [ 46 , 70 ] .
this may induce t - cell unresponsiveness to tcr stimulation , failure to produce th1 cytokines , and production of additional tgf .
tgf inhibitor ap 12009 was tested in a phase i / ii clinical trial for advanced pancreatic cancer and other malignancies .
ly2109761 , an inhibitor of tgf i / ii receptors , can suppress pancreatic cancer metastases . in a preclinical model
, we have shown that anti - tgf can reduce treg cells in tumors and tumor - draining lymph nodes .
however , as tgf is implicated in multiple layers of biological activities , the ultimate clinical therapeutic efficiency and side effects of tgf signaling blockade remain to be investigated .
the expression , regulation , functional , and clinical relevance of inhibitory b7 family members have been reviewed elsewhere .
human ovarian cancer and cancer - associated myeloid antigen - presenting cells express high levels of b7-h1 ( pd - l1 ) , which are negatively associated with patient survival [ 74 , 75 ] .
patients with high expression of b7-h1 had a significantly poor prognosis compared to the patients with low expression of b7h1 .
b7-h1 expression was also found inversely correlated to the intraepithelial cd8 t lymphocyte count , indicating that b7-h1 on tumor cells may suppress antitumor cd8 t cells .
the receptor , programmed death 1 ( pd-1 ) , is expressed on activated t - cell subsets , antigen - specific cd8 t cells , and treg .
interestingly , b7-h1/pd-1 has been reported to be involved in the development of induced treg cells . therefore , targeting b7-h1/pd-1 signaling pathway may reduce treg development and function . as anti - pd-1 is in clinical application to treat patients with melanoma , renal cell carcinoma , and other cancers , further mechanistic studies on these patients
will determine if the effects of anti - pd-1 on treg cells are mechanistically and clinically relevant .
in addition to b7-h1 , human ovarian cancer and cancer - associated myeloid antigen - presenting cells also express high levels of b7-h4 ( b7x , b7s1 ) , which are negatively associated with patient survival [ 74 , 80 , 81 ] .
interestingly , treg cells can induce il-10 expression by apcs and indirectly stimulate b7-h4 expression on apcs and convey suppressive activity to apcs [ 74 , 80 , 81 ] .
thus , it is tempting to speculate that blocking b7-h4 signaling pathway may disable the suppressive effects of treg cells on apcs .
notably , as the receptor for b7-h4 has not been identified , b7-h4 signaling is much less understood in both mouse and human system .
nonetheless , studies on ovarian cancer patients and preclinical cancer models suggest that interruption of b7-h4 signaling may lead to improved antitumor t - cell response and decreased treg suppressive function .
their phenotype , trafficking mechanism , suppressive activity , and clinical relevance have been defined in human cancer .
however , recent evidence indicates that treg cells may not be stable and are subject to environmental regulation . in this regard ,
although their action mode of mechanisms has been investigated in many different physiological and pathological scenarios , the key suppressive mechanisms may be differed in different tumors or / and in different stages .
nonetheless , targeting treg cells or / and reprogramming treg cells is an important strategy to treat patients with cancer .
it is suggested that combinatorial therapy by incorporating treg manipulation may be ideal direction to develop novel therapeutic regimen to efficiently treat patients with cancer .
 multiple layers of suppressive components including regulatory t ( treg ) cells , suppressive antigen - presenting cells , and inhibitory cytokines form suppressive networks in the ovarian cancer microenvironment . 
 it has been demonstrated that as a major suppressive element , treg cells infiltrate tumor , interact with several types of immune cells , and mediate immune suppression through different molecular and cellular mechanisms . in this paper 
 , we focus on human ovarian cancer and will discuss the nature of treg cells including their subsets , trafficking , expansion , and function 
 . we will briefly review the development of manipulation of treg cells in preclinical and clinical settings . 
1. Introduction
2. Regulatory T-Cell Subsets
3. Regulatory T-Cell Trafficking
4. Regulatory T-Cell Differentiation and Proliferation
5. Targeting Regulatory T Cells
6. Conclusions