class 1a pi3ks are directly linked to lymphocyte activation mainly through receptor tyrosine kinases , such as the antigen and cytokine receptors ( engelman et al . , 2006 ; huang and sauer , 2010 ; so and fruman , 2012 ) .
class 1a pi3ks , hereafter referred to as pi3k , are comprised of a catalytic subunit of 110 kda ( of which there are three isoforms ) that generates phosphatidylinositol 3,4,5 phosphate ( pip3 ) from its main substrate phosphatidylinositol 4,5 phosphate ( pip2 ) , and a regulatory subunit ( of which there are five isoforms ) .
pten encodes a protein with a lipid phosphatase function that directly opposes pi3k signaling by dephosphorylating pip3 at the 3 position to generate pip2 .
cells lacking pten have elevated levels of pip3 and constitutive activation of pi3k signaling pathways ( stambolic et al . , 1998 ; cantley and neel , 1999 ) . with increasing age , mice heterozygous for pten develop t cell lymphomas and cancers in multiple tissues , and develop a lethal polyclonal autoimmune disorder , similar to that seen in fas - deficient mice ( di cristofano et al .
, 1998 , 1999 ; podsypanina et al . , 1999 ; suzuki et al . ,
germline mutations in pten occur in a group of autosomal dominant syndromes known as the pten hamartoma tumor syndromes , which include cowden syndrome , proteus syndrome , proteus - like syndrome , and bannayan  riley  ruvalcaba syndrome , demonstrating the importance of pten as a tumor suppressor .
consistent with this , genomic amplification and mutation of either pi3k or akt has been reported in a large number of cancers as well ( samuels et al .
, 2004 ; lee et al . , 2005 ; carpten et al . , 2007
2009 ) , although the role of pten as a tumor suppressor is now believed to involve more than its ability to oppose pi3k signaling .
while disruption of regulation by pten has the overt phenotype of cancer progression , pten also plays an important role in maintaining t cell tolerance at multiple stages within the t cell compartment .
upon ligation of the t cell receptor ( tcr ) in the presence of co - stimulatory molecules , pi3k recruitment and activation leads to the production of lipid products which in turn recruit downstream ph - domain containing targets such as pdk1 and akt ( see figure 1 ) . ultimately , these events lead to activation of the mtor pathway and inactivation of members of the foxo family , inducing growth and proliferation of t cells and acquisition of effector function ( for review , see engelman et al . , 2006 ; finlay and cantrell , 2010 ; so and fruman , 2012 ) .
in the absence of pten , tcr stimulation alone results in hyperactivation of the pi3k pathway , resulting in effective cytokine production and proliferation independent of co - stimulation ( buckler et al . , 2006 ) . thus , put another way , negative regulation of pi3k signaling by pten enforces the requirement for co - stimulation in nave t cells .
however , the notion that pi3k signaling is required for co - stimulation to mediate its effects has been challenged by the generation of mice which lack all isoforms of the regulatory subunit of class 1a pi3k in t cells ( deane et al . , 2007 ) .
t cells from these mice are able to proliferate under co - stimulatory conditions in the absence of detectable akt signaling .
these mice also maintained a normal anti - viral response upon mhv infection , although in vivo t helper function to b cell antibody responses was impaired . whether the relative lack of defects is due to the fact that lack of pi3k was genetic , rather than acquired ,
however , these studies indicate that potential therapies targeting pi3k for inflammatory diseases and cancer may not compromise all aspects of cellular immunity , and further suggest that pi3k signaling has specialized functions in the context of t cell activation .
pten / pi3k signaling in mature t cells.class 1a pi3ks are activated by receptor tyrosine kinases that drive co - stimulatory , t cell receptor ( tcr ) , and cytokine signaling pathways .
pten directly opposes pi3k signaling by converting pi(3,4,5)p3 to pi(4,5)p2 . through their ph - domains , pdk1 and akt bind pip3 , enabling akt to be phosphorylated by pdk1 on ser308 .
akt can then target downstream substrates such as foxos in the nucleus , leading to their inactivation and export from the nucleus .
this results in attenuation of a foxo - dependent gene program normally acting to promote pathways involved in , but not limited to , t cell homeostasis ( in resting t cells ) , apoptosis ( upon , for example , cytokine withdrawal ) , treg induction , and homing to secondary lymphoid organs ( in circulating , nave t cells ) .
akt also targets tsc2 and pras40 ( not shown ) , negative regulators of mtorc1 , allowing for activation of mtorc1 .
activation of s6k and 4ebps by mtorc1 results in increased protein translation , allowing growth and proliferation , and upregulation of hif-1 , important for glycolytic metabolism .
inhibitory targeting of bad and gsk3 also contribute to akt s effect on cell cycle , survival and metabolism .
pi3k activation is critical for optimal responses of t cells to cytokines which utilize the common gamma chain .
for example , engagement of the il-2r on activated t cells results in activation of the jak / stat pathway , as well as the pi3k and mapk pathways , and the combination of these signals is required for the observed proliferation and cell survival in response to il-2 .
thus , appropriate responses to tcr ligation and cytokines require downregulation of pten , which is constitutively expressed in nave t cells .
this normally occurs as a consequence of tcr stimulation itself , which terminates detectable pten expression within 2448 h. the importance of this is demonstrated by two findings .
first , retroviral mediated  enforced  expression of pten renders activated il-2r t cells unable to fully respond to il-2 stimulation .
second , regulatory t cells , which normally do not divide in response to il-2 alone , have complete responses and signaling to il-2 restored solely by genetic ablation of pten ( bensinger et al . , 2004 ; walsh et al . , 2006
this demonstrates that control of pi3k through pten plays an important role not only in modulating the degree of activation signals within lymphocytes , but in maintaining traits of a specific lineage .
deletion of pten , or expression of constitutively active myristoylated - akt , within the cd8 t cell compartment inhibits the development and survival of memory cd8 t cells ( hand et al . , 2010 ) .
on the contrary , akt is required for a transcriptional program leading to upregulation of cytolytic effector , chemokine , and adhesion molecules ( macintyre et al . , 2011 ) .
conditional deletion of pten using ox40-cre has demonstrated that pten plays a critical role in limiting the expansion of tfh cells and in maintaining control over gc reactions , indicating that t cell intrinsic roles of pten are crucial for maintaining global levels of tolerance ( rolf et al . , 2010 ) .
lastly , the promotion of itreg generation and maintenance by pd - l1 is associated with downregulation of akt signaling and concomitant upregulation of pten expression ( francisco et al . , 2009 ) .
these examples all provide evidence that the balance of akt signaling within multiple stages of development and differentiation states helps determine cellular fate .
pten also has been shown to play an important role in central tolerance and in regulating proliferation of developing cells in the thymus ( suzuki et al . , 2001 ; hagenbeek et al . ,
signaling , loss of pten allows cells to bypass the -selection checkpoint , indicating that pi3k signaling is crucial for the ongoing development of early t cell precursors .
interestingly , thymic cellularity and subset percentages are relatively unperturbed prior to tumor development , which likely occurs at the dp stage during thymic maturation , suggesting that secondary events emerge in this context of pten loss to promote transformation ( discussed below ; hagenbeek et al .
, 2004 ; hagenbeek and spits , 2008 ; xue et al . , 2008 ; guo et al . ,
members of the foxo family of transcription factors play a critical role in dna damage and oxidative stress responses ( tran et al . , 2002 ; miyamoto et al . , 2007
2009 ) and in preventing entry into cell cycle ( medema et al . , 2000 ) , acting as bona fide tumor suppressors as demonstrated in hematopoietic and epithelial tissues ( coffer , 2003 ; accili and arden , 2004 ; paik et al . , 2007 ) .
it is becoming increasingly appreciated that foxos play important roles within the t cell compartment as well , regulating homing ( fabre et al .
, 2008 ; sinclair et al . , 2008 ; finlay et al . , 2009 ; kerdiles et al . , 2009 ; finlay and cantrell , 2010 ) , survival ( hedrick , 2009 ; kerdiles et al . , 2009 ; dejean et al . , 2011 ) , and the development and function of effector and memory subsets ( kerdiles et al . , 2010 ; ouyang et al . , 2010 ; rao et al . , 2012 ) .
in quiescent cells , foxos are active within the nucleus , where they maintain the pattern of chemokine and adhesion molecule expression , and expression of the il-7r , needed for the migration and survival of circulating t cells ( fabre et al . , 2008 ;
akt phosphorylation on ser473 , mediated by mtorc2 , is critical for foxo inactivation , implicating mtor in control over foxos , and placing pten upstream in this pathway .
importantly , mtorc2 has been shown to be critical for the phenotype in models of pten loss .
prostate cancer caused by conditional pten deletion in prostate epithelium requires mtorc2 , and deletion of one copy of rictor , required for mtorc2 complex assembly and activity , was sufficient to protect pten heterozygous mice from prostate cancer ( guertin et al . , 2009 ) .
the role of foxos in t cells was first described using mice in which retroviral gene - trap targeting of embryonic stem cells was used to generate a null foxo3 allele , although the authors could not rule out the possibility that undetectable levels of a truncated form of foxo3 was produced ( lin et al . , 2004 ) .
these mice exhibited spontaneous lymphoproliferation and multi - organ lymphocyte infiltration , indicating that foxo3 is important for the control of t cell tolerance and homeostasis .
this hyper - activated phenotype was correlated with decreased expression of ib and ib , leading to increased nf-b activation .
as ikk has been shown to phosphorylate and inactivate foxos ( hu et al . , 2004 ) , this implicates foxos in a critical negative feedback loop that may serve to limit inflammatory responses in certain settings , and is potentially important in the context of tumors which maintain low levels of akt activation .
other studies using foxo3-deficient strains did not find the same immunological defects ( hosaka et al .
, 2004 ; dejean et al . , 2009 ) , attesting first to the redundancy of members of this family and , second , to the possibility of dominant - negative effects of undetected foxo3 gene products in the aforementioned mice .
t cell - specific loss of foxo1 was shown to severely disrupt t cell homeostasis , resulting in multi - organ lymphocyte infiltration as well as exocrine pancreatitis and hind limb paralysis ( kerdiles et al . , 2010 ) .
this was attributed to a defect in both the development and function of foxp3 regulatory t cells , and this phenotype was exacerbated by combined deletion of foxo1 and foxo3 ( kerdiles et al . , 2010 ;
more recently , foxo3a has been mechanistically linked to anergy induction in t cells through upregulation of sirt1 ( gao et al . , 2012 ) .
il-2 was shown to reverse t cell anergy through the pi3k pathway by inactivation of foxos , which prevented transcriptional upregulation of sirt1 . in mouse
embryonic fibroblasts and lymphoid cells , growth factor and cytokine withdrawal upregulates bh3-only pro - apoptotic mediators puma and bim in a foxo3a - dependent manner that requires downregulation of pi3k / akt signaling ( you et al . , 2006 ) .
these findings strongly support the idea that impaired downregulation of pi3k activity , as seen in t cells lacking pten , results in survival effects and apoptotic resistance in response to cytokine deprivation through inactivation of foxo family members .
fasl has been shown to be another important target of foxo ( brunet et al . , 1999 ) .
given that pten heterozygous mice display decreased sensitivity to fas - mediated aicd , similar to lpr and gld mice ( van parijs and abbas , 1996 ) , constitutive inactivation of foxos due to loss of pten is likely a partial mechanism underlying the phenotype observed in these mice .
additionally , the finding that a deficiency in foxos is sufficient to drive the development of hemangiomas , similar to what is seen in patients with cowden disease and bannayan  zonana syndrome , and thymic lymphomas , similar to mice with a t cell - specific deletion of pten , as well suggests that inactivation of foxos may be critical for the phenotype brought on by loss of pten ( paik et al . , 2007 ) .
genetic studies in mice expressing conditional alleles of pten have been crucial for studying the tissue specific role of pten in tumorigenesis .
deletion of pten during hematopoiesis using mx-1-cre results in myeloproliferative disease and transplantable leukemia , and has shown that pten is required for maintaining the hematopoietic stem cell ( hsc ) compartment ( yilmaz et al . , 2006 ; lee et al . ,
similar to the mx-1-cre model of pten loss , vec - cre - mediated pten loss , in which nearly 40% of fetal liver hscs were subject to deletion of pten , led to impaired hsc self - renewal and the development of a myeloproliferative disorder followed by leukemia ( guo et al . , 2008 ) .
it is notable that this phenotype shares striking similarity to mice in which all six alleles of foxo1/3a/4 were deleted by mx-1-cre ( tothova et al . , 2007 ) , and to foxo3a mice ( miyamoto et al . , 2007 )
importantly , this study reported the same t(14;15 ) chromosomal translocation ( seen within a subset of human t - all ) , in all blast - crisis samples analyzed from these mice , suggesting a critical genomic destabilizing event potentially independent of pten s role in controlling pi3k / akt signaling .
similarly , this translocation , involving the c - myc and tcr/ loci and resulting in constitutively high levels of c - myc , was found to recur with 100% incidence in t cell lymphomas from mice in which pten was deleted specifically in t cells ( cd4-cre  pten mice , hereafter referred to as pten-t mice ; liu et al . , 2010 ) .
thus , well after t lineage commitment in mice subject to lymphoma and lymphoproliferative autoimmune disease , pten is critical to prevent the emergence of the same genetic abnormality present in hscs that correlates with the selective pressure of these cells for the development of leukemia .
a deficiency in rag1 or tcr prevented the t(14;15 ) translocation event in pten-t mice , but did not inhibit malignant transformation , although lymphomagenesis developed with delayed onset and was primarily restricted to the thymus ( liu et al .
interestingly , mature t cells from 3-week - old pten-t mice that had not undergone transformation , when transferred into either immunocompetent or immunoincompetent recipients , did not develop a malignant phenotype throughout the duration of the host s life , suggesting that malignancy arises within the thymus after a period of latency in these mice , consistent with an earlier report demonstrating that transplanted pten thymocytes gave rise to t cell lymphomas in immunodeficient recipients ( hagenbeek and spits , 2008 ) . supporting this , pten-t mice thymectomized at 3 weeks of age did not develop lymphoma , although later in life these mice exhibited signs of systemic autoimmunity .
thus , within distinct developmental stages , pten is required for the prevention of lymphoma and autoimmunity . in the context of dna damage , loss of pten
enables cells to bypass the normal g2/m checkpoint enforced by chk1 as a result of akt - mediated chk1 sequestration in the cytoplasm ( puc et al . , 2005 ) .
akt has also been shown to promote prosurvival responses following dna double - strand breaks ( dsbs ; bozulic et al . , 2008 ) .
it is possible that secondary mutations or other genetic alterations occurred in rag1 or tcr  pten-t mice as a result of genetic instability due to pten loss , and that hyper - active akt maintains the survival of these cells that would normally undergo apoptosis .
spectral karyotyping analyses , however , failed to detect any chromosomal translocations in these mice to support this idea .
induction of the notch pathway in malignant cells from these mice , however , was shown to increase cellular levels of c - myc , suggesting that pi3k and c - myc cooperate in multiple models of pten loss to promote lymphomagenesis and lymphoproliferative autoimmune disorder .
disruption of foxo function has been shown to accelerate c - myc - driven lymphomagenesis ( bouchard et al . , 2007 ) , and constitutively active mutants of foxo3a resistant to inactivation by akt directly repress multiple target genes of c - myc and block c - myc - dependent proliferation and transformation ( bouchard et al . , 2004 ; jensen et al . , 2011 ) .
together , this offers the possibility that c - myc and pi3k cooperate in tumorigenesis through inactivation of foxos .
whether or not restoration of foxo signaling in mice lacking pten in t cells has an effect on metabolism , survival , infiltrative capacity , lymphomagenesis , or the prevention of autoimmunity remains to be seen . a recent study in which pten  cd4-cre mice were crossed to pdk1 mice
has shown that while lymphoma did not develop in this model of t cell - specific pten deletion , pdk1/akt signaling was dispensable for the survival , proliferation and differentiation of t cell progenitors , and in vivo expansion of peripheral t cells ( finlay et al . , 2009 ) .
the mechanism for control of cell metabolism independent of pdk1 is unclear in these mice , although thymocyte proliferation in the absence of pten was shown to require rhoa - dependent pathways , and a dependence on c - myc , which was recently shown to play a pivotal role in t cell metabolism following activation ( wang et al . , 2011 ) ,
mice which express a single hypomorphic pdk1 allele similarly prevented a wide range of tumors when crossed to pten heterozygous mice , indicating that the requirement for pdk1 in the context of tumor formation brought on by loss of pten is not limited to its role in controlling migratory capacity of t cells ( bayascas et al . , 2005 ) .
additionally , deletion of s6k1 in the mx-1-cre model of pten loss resulted in delayed development of leukemia , indicating that an mtorc1-mediated pathway , in this context shown to involve induction of a hif-1-dependent glycolytic program , contributes to leukemogenesis in pten - deficient cells ( tandon et al . ,
  pten nuclear function , independent of its lipid phosphatase activity , has been shown to contribute to its tumor - suppressive effects and , in particular , in maintaining genomic stability .
control of dna stability and dsb repair has in part been attributed to interaction of pten with an integral kinetochore protein , cenp - c .
mutants originally identified in cowden disease patients demonstrated that the c - terminus of pten , but not the phosphatase domain , was required for interaction with centromeres and with cenp - c , and that dna instability and dsbs could be prevented by a phosphatase - dead pten mutant which retained its ability to bind to cenp - c ( shen et al . , 2007 ) . additionally , nuclear exclusion of pten , but not a phosphatase - inactive mutant , was shown to impair the tumor - suppressive apc - cdh1 nuclear complex , again demonstrating tumor - suppressive capability of catalytically inactive pten ( song et al . , 2011 ) .
additional protein  protein interactions within the nucleus , independent of pten catalytic activity , have been shown to increase the activity and stability of p53 ( li et al . , 2006 ; salmena et al . ,
2008 ) , and upregulate expression of rad51 , an essential component of the dsb repair machinery ( baker , 2007 ; shen et al . , 2007 ) .
a lysine to glutamate mutation in pten ( k289e ) identified in a cowden syndrome family , which lead to a dramatic decrease in nuclear import without disrupting phosphatase activity or
membrane localization of pten , as well strongly supports the idea that nuclear pten can be critical for tumor suppression independent of its role in opposing pi3k signaling in certain contexts ( trotman et al .
. molecular and phenotypic consequences of loss of pten phosphatase - dependent and -independent functions during hematopoiesis and within the t cell compartment.loss of pten phosphatase activity results in increased levels of pip3 , causing hyperactivation of the akt pathway and subsequent activation of the mtor pathway , and inactivation of the foxo family of transcription factors .
disruption of pten phosphatase - independent function accounts for loss of much of its nuclear activity , compromising genetic stability and dsb repair , leading to chromatid breaks and translocations .
that mature t cells do not undergo transformation , and that lymphoma in t cell - specific pten knockout mice is of thymic origin , suggests that pten phosphatase - independent function contributes less to the phenotype at the mature t cell stage , and that loss of t cell tolerance can likely be attributed to loss of pten phosphatase activity .
whether depletion of hematopoietic stem cells ( hscs ) and development of leukemia stem cells ( lscs ) , as well lymphomagenesis , depends on the absence of pten phosphatase activity ( which likely maintains survival of genetically aberrant cells ) , pten phosphatase - independent activity ( promoting genetic instability ) , or both , remains an open question .
inactivation of pten phosphatase activity is sufficient to abrogate its tumor - suppressive effects , emphasizing the importance of pten s role in downregulation of pi3k signaling .
this has been demonstrated by a subset of cowden disease patients that harbor a missense mutation at a cysteine residue ( c124 ) critical for phosphatase activity .
a c124s mutant has been shown to form a stable complex with pip3 , potentially protecting it from dephosphorylation by other lipid phosphatases to account for higher cellular levels of pip3 compared to conditions in which pten is completely absent ( myers et al . , 1998 ) .
as well , the importance of akt activity in tumor development induced by loss of pten has been reported in a number of studies ( stiles et al . , 2002 ; bayascas et al . , 2005
constitutive akt activation in t cells and thymocytes has been shown to be sufficient to drive autoimmunity and lymphoma ( rathmell et al . ,
2003 ) and thymomas ( malstrom et al . , 2001 ) , respectively , and bone marrow chimera experiments have demonstrated that enforced expression of constitutively active akt in hscs was sufficient for the development of myeloproliferative disease , t cell lymphoma , or aml ( kharas et al . , 2010 ) .
pten-t mice crossed onto the rag1 background , which do not harbor the t(14;15 ) translocation , potentially serve as an example in which akt signaling is not sufficient to drive cancer progression ( liu et al . , 2010 ) .
in vitro studies using malignant cells from these mice demonstrated a dependence on notch , suggesting that akt at the very least must cooperate with other oncogenic pathways to allow tumor growth in this model , similar to what has been suggested in an mmtv - myrakt breast cancer model with respect to tumorigenesis induced by loss of pten ( blanco - aparicio et al . , 2007 ) .
in contrast to this , in a model of notch - induced tumorigenesis , genetic loss of pten induced an oncogene addiction switch that rendered t - all cells resistant to notch inhibition through gsis and dependent on pi3k / akt signaling to maintain tumor growth ( palomero et al . , 2007 ) .
this idea is perhaps emphasized by the observation that pten-t mice , regardless of whether the t(14;15 ) translocation event occurs , maintain constitutively high levels of c - myc .
the reliance on c - myc in the context of pten loss remains an important question .
in tumors driven by inducible c - myc activation in a zebrafish model of t - all , constitutive activation of the akt pathway through genetic disruption of pten or transgenichexpression of akt2 rendered cells in this model independent of c - myc for tumor progression ( gutierrez et al .
conversely , conditional genetic disruption of both pten and c - myc in mice , while still subject to myeloproliferative and lymphoproliferative disorders , prevented the development of hematopoietic malignancies , highlighting an important dependence on c - myc in this context of pten loss ( zhang et al .
whether loss of pten allows the survival of cells that , through other mechanisms , have acquired genetic aberrations , such as translocations resulting in constitutive c - myc , or whether pten , independent of its role in pi3k downregulation , prevents genetic instability that collaborates with dysregulation of pi3k to allow cancer progression , remains an open question that needs to be addressed in a context - dependent manner , with consideration of what function of pten is lost , changes in levels of expression , and in which tissues abnormal growth originates .
pten is the one of the most frequently mutated or lost genes in human cancer . given the emerging roles of pi3k in immune system regulation , it is possible that pten is also playing a prominent role in the prevention of autoimmune disease and inflammatory / lymphoproliferative syndromes .
the finding that pten did not play a role in lymphomagenesis in mature t cells from mice which lack pten specifically in t cells suggested that pten has a specific function in preventing lymphoma within a distinct time frame in this compartment .
that these non - malignant t cells were able to provoke autoimmunity demonstrates that well beyond the stage at which pten is required for prevention of lymphomagenesis in this model , pten plays a critical role in the maintenance of t cell tolerance .
it will be important to determine which biochemical requirements pten fulfills in each stage of protection , and whether these requirements are distinct within different stages from cells of the same lineage , or whether lack of particular features leads to different outcomes depending on developmental stage . given the importance of pi3k signaling in both normal immunity and the development of cancer , it will be interesting to see how closely pten s role as a lipid phosphatase is tied to prevention of disease , and how integral pi3k signaling remains throughout disease progression . uncovering the specific biochemical functions of pten within these contexts will be key to the development of targeted therapies for the prevention and treatment of t cell malignancies and autoimmune disease .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 the generation of lipid products catalyzed by pi3k is critical for normal t cell homeostasis and a productive immune response . 
 pi3k can be activated in response to antigen receptor , co - stimulatory , cytokine , and chemokine signals . 
 moreover , dysregulation of this pathway frequently occurs in t cell lymphomas and is implicated in lymphoproliferative autoimmune disease . 
 akt acts as a central mediator of pi3k signals , downstream of which is the mtor pathway , controlling cell growth and metabolism . 
 members of the foxo family of transcription factors are also regulated by akt , thus linking control over homing and migration of t cells , as well cell cycle entry , apoptosis , and dna damage and oxidative stress responses , to pi3k signaling . 
 pten , first identified as a tumor suppressor gene , encodes a lipid phosphatase that , by catalyzing the reverse of the pi3k  reaction ,  
 directly opposes pi3k signaling . 
 however , pten may have other functions as well , and recent reports have suggested roles for pten as a tumor suppressor independent of its effects on pi3k signaling . through the use of models in which pten is deleted specifically in t cells , it is becoming increasingly clear that control over autoimmunity and lymphomagenesis by pten involves multi - faceted functions of this molecule at multiple stages within the t cell compartment . 