human natural treg ( ntreg ) cells were purified from normal donor pbmc using the commercially available miltenyi treg kit with automacs ( available from miltenyi ). the resulting sample was enriched in ntreg cells relative to the original sample . similar results were obtained by depleting cd19 + cells and thereafter performing positive selection of cd25 + by automacs using miltenyi cd19 and cd25 beads . typically , 50 - 70 % of the enriched cells are foxp3 + as assessed by inteacellular foxp3 staining and flow cytometry analysis . in some studied , ntreg was enriched through facs sorting based on cd4 +, cd25high and cd127lo population . in this case , the foxp3 + cells consisted of greater than 90 % of the cd4 + cd25 + population . after expansion for three weeks time , ntreg cells experience over a one hundred fold population growth . about 50 % of the expanded cells were foxp3 + and the cultured cells exhibited potent inhibitory activities during in vitro functional assays . it has been reported ( bensinger et al . “ distinct il - 2 receptor signaling pattern in cd4 + cd25 + regulator t cells ” j . immunol 172 ( 9 ): 5287 - 96 ( 2004 )) that treg cells exhibit reduced akt activities compared with the activities of the convention effector cells under certain conditions . it was believed that the proposed signaling pathway was pi - 3k → akt → mtor . without wishing to be bound to any particular theory , applicants believe that t effector cells are sensitive to upstream / downstream inhibitors of the pi - 3k pathway . accordingly , inhibition of this pathway during the expansion of the population of ntreg cells may allow for the preferential growth of ntreg cells . several inhibitors of the pi - 3k - akt pathway were tested for activity during ntreg cell expansion , including ly294002 . ly294002 ( 2 -( 4 - morpholinyl )- 8 - phenyl - 4h - 1 - benzopyran - 4 - one ) is a cell - permeable , potent , and specific phosphatidylinositol 3 - kinase inhibitor ( ic 50 = 1 . 4 μm ) that acts on the atp - binding site of the enzyme . it also inhibits nonhomologous dna end - joining ( nhej ) activity of the 460 kda phosphatidylinositol 3 - like kinase dna - pkc s , which is the catalytic subunit of dna - activated protein kinase . it does not affect the activities of egf receptor kinase , map kinase , pkc , p1 4 - kinase , s6 kinase , and c - src even at 50 μm . it also blocks the proliferation of cultured rabbit aortic smooth muscle cells without inducing apoptosis ( cas 154447 - 36 - 6 ). several concentrations of ly294002 ( 2 -( 4 - morpholinyl )- 8 - phenyl - 4h - 1 - benzopyran - 4 - one , 10 mm in dmso , calbiochem product # 440204 ) ranging from 50 μm to roughly 100 nm final concentration ) was added to select cultures . concentrations greater than 50 μm were generally overly toxic to the cells . control cultures included no additives and 100 nm rapamycin . cultures were stimulated with anti - cd3 - cd28 coated beads ( dyna1 ) at 3 : 1 bead to cell ratio in the presence of 100 iu / ml of human r - il - 2 and placed in a 37 ° c . incubator . fresh il - 2 was supplied at the end of the first week and subsequently changed every three days or as needed . the purity of the ntreg cells was checked on days 6 - 7 of the culture and weekly thereafter . other pi - 3k inhibitors were also tested include pi - 103 . pi - 103 , ( p3 -( 4 -( 4 - morpholinyl ) pyrido [ 3 , 2 : 4 , 5 ] furo [ 3 , 2 - d ] pyrimidin - 2 - yl ) phenol ) is a cell - permeable pyridinylfuranopyrimidine compound that acts as a potent and atp - competitive inhibitor of dna - pk , pi3 - k , and mtor ( ic 50 = 2 , 8 , 88 , 48 , 150 , 26 , 20 , and 83 nm for dna - pk , p110α , p110β , p110δ , p110γ , pi3 - kc2b , mtorc1 , and mtorc2 , respectively ). it inhibits atr and atm only at much higher concentrations ( ic 50 = 850 and 920 nm , respectively ) and exhibits little activity towards a panel of more than 40 other kinases even at concentrations as high as 10 μm . it has also been shown to effectively block pi3 - k / akt signaling and cell proliferation in glioma cell lines both in vitro and in vivo ( cas 371935 - 74 - 9 ). as shown , addition of pi103 at 720 nm , 360 nm or 180 nm were able to enhance foxp3 expression as compared to their absence over a 3 week period of expansion culture of ntreg demonstrating the potential usefulness of this compound in the in vitro expansion of regulatory t cells as defined by a preculture presence of foxp3 expression . see fig1 . wortmannin as a broad and irreversible pi3k inhibitor was also tested and found to promote a dose dependent increase in foxp3 expression between 12 . 5 and 50 μm . as 605240 is a potent , cell permeable and atp - competitive inhibitor of phosphoinositide 3 - kinase □( pi ( 3 ) k □). it exhibits selectivity over other pi ( 3 ) k isoforms . as 252424 is a potent and selective phosphoinositide 3 - kinase ( pi ( 3 ) k ) p110 □ inhibitor shown to be selective for class ib pi ( 3 ) k - mediated cellular effects . are also pi3k inhibitors and were able to increase the percentage of cells in culture expressing foxp3 relative to the respective compounds absence . interestingly , ly303511 ( a derivative of ly294002 ) has been described to have an inhibitory effect on mtor similar to that described for ly294002 but does not inhibit pi - 3k . ly303551 did not permit for the selective growth of ntreg cells like its counterpart ly294002 . akt has been widely described as linking pi3k signal transduction to mtor complexes . in this respect it is interesting that the akt inhibitors that were tested were largely ineffective at promoting the expansion of ntreg cells . tested described akt inhibitors include but are not limited to akt inhibitor vii , tat - akt - in ( calbiochem product # 124014 ); akt inhibitor viii , isozyme - selective , akti - ½ calbiochem product # 124018 ; and akt inhibitor x ( 10 ( 4 -( n - diethylamino ) butyl )- 2 - chlorophenoxazine , hcl ; calbiochem product # 124020 ). additionally , the use of steroids ( e . g . dexamethasone or prednisolone ) had a synergistic or additive effect with respect to the % of foxp3 expressing cells obtained in expansion cultures when used with either rapamycin or ly294002 . this effect was optimal around 200 ng / ml ( prednisolone ) or 100 μm ( dexamethasone ). one of the characteristics of cd25 + and / or foxp3 + cd4 t cells is their ability to suppress cd4 t cell responses to antigen - specific as well as anti - cd3 stimulation in the presence of antigen presenting cells . similarly , cd25 + cells expanded in vitro have also been described as having similar suppressive capabilities . inclusion of rapamycin is such expanded cultures , in addition to enhancing foxp3 expression , also has been shown to enhance the suppressive capability of the expanded cells . applicants have discovered that ly294002 has a similar effect when combined with the expanded cd25 + cells of the invention . treg cultures were established as described elsewhere in this specification and expanded for two weeks . cells were harvested , washed two times and serial diluted onto a 96 - well round - bottom plate . cfse labeled purified cd4t cells were added to the wells at 1 × 10 5 cells per well to result in the treg : cd4 responder cell ratio &# 39 ; s shown in table 2 . cultured dendritic cells were added at 1 × 10 4 cells per well and okt3 mab was added at 1 μg per ml . percent response indicates the percentage of cfse labeled cd4t responder cells that failed to divide in the assay . while the invention has been described with reference to preferred embodiments , it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof to adapt to particular situations without departing from the scope of the invention . therefore , it is intended that the invention not be limited to the particular embodiments disclosed as the best mode contemplated for carrying out this invention , but that the invention will include all embodiments failing within the scope and spirit of the appended claims .
disclosed in this specification is a method to promote the growth of cd4 + cd25foxp3 + ntreg cells in a culture while treating with a pi - 3k antagonist . the resulting cells are useful in the treatment of immune - related diseases .