INFERTILITY TREATMENT BASED ON REGULATORY TCELLS AND IMMUNE TOLERANCE IN PREGNANCY

By : Sarah Kamilah 030 0! "#$ FACULTY OF MEDICINE TRISAKTI UNI%ERSITY &AKARTA "0##

ABSTRACT

Infertility became one of the issues in reproductive health that have not found a solution to overcome them. Several studies have been done to solve these problems including by researching the Treg cells and immune tolerance of the maternal immune response. Review of the literature on Treg cells in the ovary, testis, uterus and the pregnancy tissue and its association with infertility has been done as well as studies in mice which showed that Treg cells is critical to maternal immune tolerance in pregnancy. Inadequate numbers of Treg cells or their functional deficiency are linked with

infertility, miscarriage and pre-eclampsia. ecause of important role of Treg cells and it function in maternal immune response, this potential to be one of interest for future treatment in the world of
reproductive health.

INTRODUCTION

!regnancy is a unique immunological challenge in which an antigenically distinct fetus and placenta develop in the uterus of the mother. "nderstanding the mechanisms responsible for tolerance of the male and female gametes poses a similar challenge, since these also e#press antigens perceived as foreign by the immune system, but are not re$ected. %ne emerging focus is the role of speciali&ed populations of T-lymphocytes termed regulatory T-cells. These cells are potent suppressors of inflammatory immune responses, and are essential in preventing destructive immunity in all the tissues of the body. This homeostatic regulation is pivotal in tissues containing endogenous physiological antigens that escape detection as 'self( due to their tissue-restricted e#pression, and in epithelial surfaces where tolerance of non-dangerous foreign antigens is essential to normal function. )*+

DISCUSSION

R'()la*+ry T-,'ll- a./ imm).' -)00r'--i+. The term 'regulatory T-cells( refers to a family of T-lymphocyte populations with suppressive,regulatory properties that are devoted to maintaining antigen-specific T-cell tolerance. -t least three subsets of ./01 regulatory T-cells with distinct suppressive mechanisms are distinguished by their phenotype, cytokine secretion and tissue origin. These are type * regulatory T-cells )Tr*+ cells, T-helper 2 )Th2+ cells and ./01 ./341 regulatory T-cells )Treg cells+. 5ach of these populations has the characteristic capacity to actively inhibit the proliferation and effector function of other T-cells.)*+ %f the three cell populations, Treg cells have fast become established as perhaps the most potent and widespread suppressive cell lineage in the immune system. "nlike Th2 and Tr* cells which appear to represent altered differentiation states of conventional ./0 1 ./341 T-cells, Treg cells are believed to comprise a distinct and unique T-cell lineage. They have now been implicated in critical protective functions in auto-immune disease, transplantation tolerance, gastro-intestinal homeostasis and inflammatory disease, and conversely, are a pivotal component of the pathophysiological immune tolerance induced by tumours. %f the three cell subsets, Treg cells are distinguished by their apparently essential role in reproductive events. )*+ Treg cells are defined as a separate cellular subset on the basis of their surface phenotype as well as functional characteristics. Treg cells are one of the four ma$or classes of ./0 1 T-cells, together with Th* cells, Th3 cells and Th*6 cells, distinguished from the other classes by their role in tolerance as opposed to immunity.
)3+

/espite the unique suppressive properties of Treg

cells compared with other lymphocytes, their cellular features are less distinct. Treg cells are generally identified on the basis of their constitutive e#pression of surface markers.

-t least two distinct pathways of Treg generation appear to e#ist. 7irst, 'natural( Treg cells can originate via a selective process in the thymus based on the structure of their individualT-cell receptors )[Link]+. 8owever, the thymus is not the sole source of Treg cells and there is now compelling evidence that the ma$ority are 'inducible( Treg cells generated within peripheral tissues, particularly in later life when the thymus undergoes e#tensive involution. -s a result, the gross pool of Treg cells at any one time is the sum of the cells selected for survival in the thymus and those cells generated in the periphery. )*+ T-cell survival in the thymus is the result of asequential selection process based on the interaction between na9ve T-cells and the thymic stromal epithelium. /epending on the avidity of the interaction between the T.R of an individual T-cell and the :8.,self-antigen peptide comple# e#pressed by stromal cells, neglect, positive selection or negative selection can ensue. 7ailure to interact with :8. molecules presenting self-antigen on the thymic epithelial cells leads to apoptosis of the T-cell through the lack of T.R signalling )neglect+. !ositive selection is the result of a T.R signal of sufficient avidity to signify a T-cell(s ability to recogni&e self-:8. comple#es, whereas a high avidity T.R,antigen interaction is indicative of e#cessive reactivity to self and hence a potentially pathological self-reactive T-cell, leading again to apoptosis of the T-cell )negative selection+. In summary, 'the thymus selects the useful, neglects the useless and destroys the harmful(. )*+ The role of peripheral tissues in sustaining Treg cell populations was elegantly demonstrated by the observation that ablation of a tissue depletes the ability of donor Treg cells to prevent organ-specific autoimmune disease after adoptive transfer into intact recipients, compared with Treg cells from unmodified donors. This shows that the presence of peripheral tissue antigens is essential for the de novo generation of antigen-specific regulatory T-cells from

na9ve T-cell precursors. The importance of tissue-specifc antigens for generation of Treg cells to protect against autoimmunity is also shown in other tissues, including the ovary. - similar conclusion is drawn from T.R transgenic mouse models, which show that with persistent low levels of antigenic stimulation, naive cells can be induced to e#press the hallmark Treg cell marker 7o#p2 and take on a suppressive phenotype. )*+ M',ha.i-m +1 Imm).' S)00r'--i+. 2y Tr'( ,'lloth innate and adaptive immune cells are targets of Treg-cell-mediated suppression, and Treg cells are known to employ a variety of mechanisms to mediate these effects. Treg cells directly suppress many functions of ./01 and ./;1 T cells, ranging from their proliferation to their differentiation into T-helper )T8+-*, T83, and T8*6 subsets. In some cases, Treg cells induce apoptosis of responding T cells. Treg cells also suppress the activation of cells, thereby

inhibiting humoral immune responses. %ther prominent targets of Treg cells include dendritic cells, macrophages, natural killer cells, mast cells, and osteoblasts. Treg cells are also involved in tissue repair and in the resolution of tissue inflammation, suggesting a potential role for Treg cells in the regulation of nonimmune cells. In fact, Treg cells can inhibit the development of transplant vasculopathy, a comple# process that involves many immune and nonimmune cells, supporting a broad role for Treg cells in tissue remodeling. )2+ Treg cells probably employ several different mechanisms to suppress pathogenic Teffector cells. In vitro assays have demonstrated that activation of Treg cells via T.R stimulation is required to mediate suppression of T-effector cells and that this suppression requires strict cell<cell contact. "nder some conditions, Treg cells have been shown to deprive T-effector cells of survival and growth factors or to directly kill activated T cells via gran&yme-dependent mechanisms. 7urthermore, Treg cells e#press ./2= and ./62, the ectoen&ymes that break down

the e#tracellular -T! into adenosine. This process has been shown to turn an -T!-rich inflammatory milieu to one that is immunosuppressive, as adenosine inhibits the activation of dendritic cells and macrophages, which in turn prevents T-cell priming. Treg cells also e#press .T>--0 on their surface, which can directly engage the peripheral membrane protein antigen-presenting cells )-!.s+ to inhibit -!. activation via different mechanisms. )2+ In addition, Treg cells can produce copious amounts of immune suppressive cytokines, including T?7-@, I>-*A, and I>-24, which are known to inhibit a wide spectrum of cellular activities. T?7-@ e#erts broad antiproliferative and anti-inflammatory effects. I>-*A strongly inhibits activation of macrophages and dendritic cells, and I>-24 is a key mediator of Treg-cellinduced immunosuppression. /espite advances in our understanding of Treg-cell function, the processes by which this catalog of in vitro mechanisms contributes to in vivo immunosuppression by Treg cells is still not known. )*+ R'()la*+ry T,'ll- i. R'0r+/),*i3' Pr+,'--'The maternal system shows evidence of activation against paternal :8. and minor antigens during pregnancy in mice and women. !eripheral tolerance mechanisms are clearly involved in preventing maternal immune re$ection of the fetus in pregnancy. The qualities and functions of regulatory T-cells make these cells perfectly suited to controlling the immune response to reproductive antigens, and there is now substantial evidence confirming their important roles in regulating tolerance to male and female gametes and to the conceptus. )*+ Tr'( ,'ll- i. *h' *'-*'- a./ +3ary The ovary has been informative in understanding the roles of Treg cells in protecting tissues e#pressing reproductive antigens from autoimmune destruction. In mice, removal of the thymus shortly after birth causes spontaneous ovarian autoimmune disease, but this can be prevented by 6 on

passive transfer of T-lymphocytes from spleens of normal adult mice. -utoimmune ovarian disease is believed to result from operational failure of the normal immune regulatory mechanisms required for normal tissue homeostasis. )*+ The converse situation occurs with male reproductive antigens, where spleen cells from normal male donors are more effective in suppressing epididymitis and orchitis than the Treg cells from female. Sertoli cells may be instrumental in activating testicular Treg cell responses due to their e#pression of the immune-regulatory co-stimulatory molecule programmed death ligand * )!/>*, also known as ./360 or 6-8*+ which binds programmed death * )!/*+

e#pressed by Treg cells and inhibits proliferation of ./;1 T-cells. )*+ Tr'( ,'ll- i. h)ma. 0r'(.a.,y. %bservations on ./01 ./341 cells in human pregnancy described an increase in this subset in early pregnancy decidual tissue. %ther reports demonstrated an increase in circulating ./01 ./341 cells during early pregnancy with a peak phase at the second trimester and a decline post-partum to levels slightly higher than pre-pregnancy levels. The elevation during the first and second trimesters has been confirmed in studies more precisely identifying Treg cells as ./34high cells, and a clear decline in ./01 ./34high Treg cells occurs during the weeks $ust prior to delivery. The ./01 ./341 T-cells comprising the e#panded pool in pregnancy are highly enriched for 7%B!2 and e#ert suppressive function in vitro. )*+ The percentage of ./21 lymphocytes that e#press ./0 and ./34 are significantly decreased in deciduas in spontaneous vaginal delivery compared with elective .aesarean section. These findings were recently confirmed by a study showing a sharp decrease in ./0 1 ./34 high Treg cells towards the end of pregnancy and an increase in ./0 1 ./34low T-cells. This implies a potential role for Treg cells in the immunological changes preceding labour, and prompts

speculation that their decline might be a causal factor in fetal e#pulsion from the maternal tissues. )*+ Tr'( ,'ll- i. i.1'r*ili*y4 mi-,arria(' a./ 0r'(.a.,y ,+m0li,a*i+.Several studies have reported that ./01 ./34high T-cells are significantly reduced in both the peripheral blood and decidual tissue of pre-eclamptic patients compared with normal pregnant women. %bservations that the tryptophan cataboli&ing en&yme I/% is decreased in pregnancies complicated by pre-eclampsia is consistent with diminished Treg cell activity in pregnancy with decreased I/% potentially caused by, and contributing to, insufficient Treg cell activity. - preliminary report suggests a possible bias towards Th*6 cells over Treg cells in women suffering third trimester pre-eclampsia+. )*+ Recurrent spontaneous miscarriage has also been associated with a mal-adaptation in the maternal immune system. Several studies report increased numbers of ./21 ./341 cells in the decidua at the time of spontaneous abortion compared with decidua recovered at therapeutic abortion. 8owever, the proportion of Treg cells among these cells is evidently reduced, since both decidual and peripheral blood ./01 ./34high T-cells are lower in tissues recovered after spontaneous abortion compared with induced abortions and non pregnant women. In a comprehensive study, women e#periencing repeated miscarriage were shown to have a reduced frequency of Treg cells within the peripheral blood ./0 1 pool, and reduced suppressive capacity, compared with normal fertile women. )*+ Ori(i. a./ a.*i('. -0',i1i,i*y +1 Tr'( ,'ll- i. 0r'(.a.,y /efining the pathways of Treg generation and the consequences for specific functional roles is critical for understanding how Treg cells operate in reproductive processes, and for designing possible interventions to e#ploit these cells to improve fertility and pregnancy

outcomes. Ce have only incomplete knowledge of the tissue origins of decidual Treg cells and the roles of conceptus antigens and other factors including cytokines and hormones in driving their e#pansion in early pregnancy, but there is sufficient emerging information to build a working model. )*+ Treg cells accumulate in the uterus during the estrus phase of the reproductive cycle as evidenced by elevated 7o#p2 mRD- e#pression, potentially in response to estrogen-induced e#pression of several chemokines that target the ..R4 chemokine receptor e#pressed by Treg cells. In women, a comparable e#pansion in ./0
1

./341 - 7%B!21 Treg cells occurs in the

peripheral blood during the late follicular phase of the menstrual cycle, when cell abundance tightly correlates with serum 53 levels, and then is followed by a dramatic decline at the luteal phase. It is important to note that since Treg cells require e#posure to antigen to e#ert their full suppressive function, estrogen alone would be insufficient to activate Treg cells for pregnancy, and instead can be viewed as potentiating Treg cells in preparation for stimulation by pregnancyassociated antigens. )*+ The thymus appears not to be the origin of the elevated numbers of Treg cells in pregnancy, so by default a peripheral tissue pathway of generation must occur, in line with studies implicating the uterus draining lymph nodes as the predominant site of Treg pool e#pansion. The relatively elevated numbers of Treg cells in tissues of mice bearing allogeneic pregnancies suggest fetal alloantigens act to drive Treg cell proliferation. Don-:8. antigens and non-classical :8. molecules e#pressed by placental trophoblast cells are implicated, and indeed a population of ./;1 T cells with regulatory properties that is activated in response to costimulation by the carcinoembryonic antigen family present on trophoblast cells has been

described in women. 7urthermore, !/>* is strongly e#pressed by human placental trophoblast cells and a role in influencing cytokine e#pression by decidual regulatory T-cells is indicated. )*+ In the decidual tissue, there are some mature myeloid /.s population which e#press markers indicative of a tolerogenic phenotype which is likely to be maintained by the ?:-.S7, I>-*A and I>-0-dominated cytokine environment of the decidual tissue. - notable role for an immunoregulatory glycan binding protein, galectin-* in inducing tolerogenic /.s and supporting their capacity to generate decidual Tr* cells is shown by loss of allogeneic pregnancies in mice deficient in galectin-*. )*+ The mechanisms for recruitment of Treg cells in the decidual tissue after embryo implantation are likely to be modified compared with the non-pregnant state. 5#pression of chemokine ).<. motif+ receptor 4 )..R4+ may facilitate accumulation and retention of antigenactivated effector Treg cells in the implantation site since Treg cells in the gravid uterus are predominantly ..R41. This marker is associated with a highly suppressive phenotype and may be a marker for those cells that have been activated by paternal alloantigen. The Treg cells that accumulate in the decidual tissue in pregnancy show specificity in their suppressive function for fetus alloantigens, indicating there is preferential recruitment from maternal peripheral blood, or possibly local e#pansion of Treg cells reactive with conceptus tissue. )*+ .onsistent with a possible role for normal embryonic development in regulating Treg numbers, ./341 ./21 cells are decreased in the decidual tissues of women with ectopic pregnancies, compared to tissues from women with normal pregnancies, while numbers in the peripheral blood were unchanged. 8owever, interpretation of this study is limited by lack of utili&ation of a specific marker for Treg cells. .onversely, Treg cells were more abundant in

implantation sites in partial and complete molar pregnancies, where Treg cell numbers positively correlated with ./;1 cytoto#ic T-cells. )*+ R+l' +1 -'[Link] 1l)i/ i. a,*i3a*i.( Tr'( ,'ll- 1+r 0r'(.a.,y Seminal fluid elicits an inflammation-like response in the female reproductive tract associated with recruitment of /.s into the endometrial and cervical tissues. These /.s are capable of processing male antigens in seminal fluid and activating T-cells in draining lymph nodes. Recent observations in mice e#posed to seminal fluid in the absence of conception support a role for seminal fluid in driving Treg cell activation and proliferation, resulting in which promotes tolerance of paternal alloantigens at the time of embryo implantation. The high levels of T?7b and prostaglandin 5 in seminal fluid are likely to be important in skewing the Tcell response towards the Treg cell phenotype. )*+ Chen seminal fluid applied in the conte#t of pregnancy, the notion of bystander tolerance conferred by Treg cells might e#plain why repeated e#posure of the female reproductive tract to a limited panel of paternal antigens in seminal fluid, or alternatively prolonged e#posure to a small number of trophoblast antigens early in pregnancy, may be sufficient to prime the maternal immune system to tolerate a wider repertoire of additional antigens e#pressed later in gestational tissues. )*+

Th'ra0')*i, 0+*'.*ial +1 r'()la*+ry T-,'ll- i. r'0r+/),*i3' m'/i,i.'

- range of strategies is under investigation to e#pand Treg cell numbers, in both an antigen-specific and a non-specific manner. .urrent approaches typically involve cell-based therapies using either adoptive transfer of e# vivo manipulated Treg cells or induction of 7%B!2 e#pression in naive lymphocytes. The attractive approach of increasing Treg cell numbers by recovery and e#pansion of an individual patient(s Treg cells e# vivo is currently limited by the ability to isolate Treg cells from human samples with high purity. )*+ Typical isolation protocols use magnetic beads that purify cells based on the e#pression of surface markers such as ./0, using positive selection for these cell types or elimination of non-./0E cells, followed by multiple rounds of selection for ./34 1 cells. 8owever, this approach fails to yield sufficient purity as ./34 also isolates activated T-cells and fails to discriminate between the ./34high cell population, which includes the ma$ority of human Treg cells, and ./34intermediate cells that largely comprise non-regulatory T-cells. .onsequently, when the isolated cells are e#panded in vitro, the anergic nature of Treg cells means that any contaminating T-cell subsets e#pand faster and result in a low percentage of the desired suppressive populations.)*+ Several strategies for increasing Treg cell activity are in development as novel tissue transplantation therapies or treatments for autoimmune disease hese include )*+ administration of I/%-inducing agents or cytokines )such as ?-.S7+ to e#pand the pool of tolerogenic /.sF )3+ delivery of tolerogenic /.s after pulsing with specific antigenF )2+ delivery of antigen in a form such as apoptotic cellular material known to stimulate Treg proliferation and )0+ delivery of antigen-specific Treg cells after recovery and e# vivo e#pansion. /., dendritic cell. )*+ -nother protocol for isolating Treg cells uses multiple surface markers in combination including ./0, ./34, .T>-0 and ./*36, followed by fluorescent-activated cell sorting.

-lthough this technique can produce a highly purified population, the quantity of cells generated is substantially lower than with magnetic bead protocols, and as a result, significant levels of in vitro e#pansion are required. )*+ Pr+m+*i.( /'3'l+0m'.* +1 Tr'( ,'ll- i. 3i3+ a./ i. 3i*r+ -lternative strategies for augmenting Treg cell activity involve the manipulation of naive ./01 T-cells to induce 7%B!2 e#pression. 5ctopic lenti-viral-induced e#pression of 7%B!2 is another proposed technique. There is a growing body of evidence that 7%B!2 1 cells can be generated from naive T-cells in the periphery through stimulation with low levels of antigen or by delivering antigens in association with /.s. The efficacy of this approach both in vitro and in vivo is improved when /.s are conditioned into a tolerogenic phenotype by addition of e#ogenous cytokines or by transfection with immunosuppressive cytokine genes. )*+ -nother alternative for driving endogenous regulatory T-cell production might be administration of granulocyte colony-stimulating factor )?-.S7+. ?-.S7 treatment is reported to induce development of tolerogenic /.s, which in turn elicit potently suppressive regulatory Tcells reminiscent of Tr* cells. 8owever, an important pitfall of this approach is the possibility of phenotype reversion in ?-.S7-induced /.s, leading to the opposite effect of inducing immunity. )*+ Pr+-0',* 1+r )*ili5i.( Tr'( ,'ll- i. *r'a*m'.* 1+r i.1'r*ili*y a./ -)21'r*ili*y The efforts to devise Treg therapies for tissue transplantation show e#citing promise and the lessons learnt will be valuable for developing analogous therapies for reproductive disorders. It seems reasonable that boosting the number and,or activity of Treg cells reactive with appropriate conceptus antigens should confer stronger immune tolerance in women prone to une#plained infertility, miscarriage or preeclampsia due to intrinsic tolerance deficiency. )*+

The first reported clinical study targeting the regulatory T-cell ne#us has utili&ed ?-.S7 treatments in women e#periencing recurrent miscarriage. -dministration of 7ilgrastim )?-.S7+ for the first 2A days of pregnancy showed promise in a small pilot study in reducing the incidence of subsequent miscarriage as e#pected, ?-.S7 treatment appears to be associated with an increase in circulating ./01 ./341 Treg cells and /.s. )*+ -nother strategy worthy of clinical evaluation would involve recovery and in vitro priming of /.s with male partner :8. antigen, in an approach analogous to treatments under development for allogeneic graft re$ection, )utili&ing cytokines to condition /.s into a tolerogenic phenotype prior to transfer.)*+ 8owever, it is important to caution that any attempts in women to alter Treg cell number or function during or prior to pregnancy must be based in a sound biological rationale, informed by knowledge of the antigens against which pregnancy-associated Tregs are normally targeted, and taking into account the natural ontogeny, timing of activation and regulators governing these cells. )*+ .omprehensive clinical trials will be essential to ensure that no adverse effects result from manipulating Treg cells. 7inally, since reproductive pathologies result from multiple aetiologies, robust and practical diagnostics for defining individual women or couples at risk of Treg cell-deficient pregnancy will need to be developed. )*+

CONCLUSION Treg cells have an important role in successful pregnancy. 8owever, the underlying mechanisms governing Treg cell numbers and function in the implantation site, and the reasons for their deficiency in some reproductive pathologies, are not clear. To understand the function and dynamics of Treg cells during pregnancy, more research is needed such as defining the nature and significance of Treg cell interactions with other uterine leukocyte populations, notably /.s, DG cells and mast cells. Treg is interesting treatment for e#isting problems during pregnancy and
reproduction. Their most important role in reproduction could be a new way to reduce fertility disorders associated with immune tolerance.

REFERENCES

*. ?uerin et al. Regulatory T-cells and immune tolerance in pregnancyH a new target for infertility treatmentI 8uman Reproductive "pdate, Jol. *4 Do. 4. !H 4*6-424 3. Khu L. !aul C5. ./0 T cellsH fates, functions, and faults. Blood 2008; **3H*446-*4M= 2. %(?arra -, Jieira !. Regulatory T cells and mechanisms of immune system controlF 3AA0. !. ;A*-4