Patent Application: US-80098504-A

Abstract:
the invention concerns the identification of a pathway where the co - receptor cd28 inactivates glycogen synthase 3 and where a reagent that inhibits gsk - 3 can enhance , or substitute for cd28 - dependent t - cell mediated immune responses . altered t - cell responses are applied to increased proliferation , cytokine production and in the generation of increased cytolytic t - cell responses in the context of infection by virus &# 39 ;, bacterial , fungi or prions . for example , the use of gsk - 3 inhibitor sb415286 preferentially cooperated with cd28 costimulation to increase interleukin 2 transcription , and to provide cd28 - replacement signals leading to increased il - 2 transcription . similarly , inhibitors sb415286 and lithium chloride increased the cd28 - dependent generation of cytolytic t - cell responses against a virally or baterially infected cells . overall , the invention relates to a cd28 induced signaling transduction pathway that inactivates glycogen synthase kinase and the application of this finding to the modulation of t - cell activation , proliferation and the generation of cytolytic t - cells .

Description:
the following reagents are widely available . nonidet p - 40 , phenyl methyl sulfonyl fluoride ( pmsf ) ( sigma , st . louis , mo . ), sodium dodecyl sulfate ( sds ), acrylamide and bisacrylamide ( national diagnostics , manville , n . j . ), protein a sepharose beads , ficoll - paque ( pharmacia , n . j . ), phosphatidyl inositol gamma - 32p - atp ( specific activity , 3000 ci / mmol ) ( nen , mass .). gsk - 3 inhibitor sb415286 was purchased commercially from glaxosmithkline ( london ). monoclonal antibodies were employed which are specific for cd28 : ( 9 . 3 ) ( becton dickinson , calif . ); and for rabbit anti - mouse antisera ( coulter immunology , hialeah , fla .). t lymphoblastoid cell lines , e . g ., jurkat ( atcc tib 152 ) were cultured in rpmi - 1640 containing 10 % ( v / v ) fetal bovine serum , l - glutamine ( 2 mm ), penicillin ( 50 u / ml ) and streptomycin ( 50 mg / ml ) at 37 . degree c and 5 % co . sub . 2 . peripheral blood t - cells were isolated from the buffy coat by lymphocyte separation medium ( ficoll - paque ) density gradient centrifugation . the non - adherent cells were cultured in rpmi 1640 supplemented with 10 % ( v / v ) fbs , 100 u / ml penicillin , 100 mg / ml streptomycin and 2 mm l - glutamine . alternatively , blood was taken from voluntary donors and separated by ( ficoll - paque ) density gradient centrifugation using standard techniques . for immunoprecipitations , cells were lysed in ice cold lysis buffer containing 1 % tritonx - 100 in 20 mm tris - hcl ph 8 . 3 , 150 mm nacl . the lysis buffer contained protease and phosphatase inhibitors as described in prasad et al ., 1993 , proc . natl . acad . sci . usa 90 : 7366 . postnuclear lysates were incubated for 1 hour at 4 ° c . with the indicated antibodies . protein a - sepharose beads ( 30 ul , pharmacia ), were added and incubated for 1 hour at 4 ° c . the beads were washed three times in cold lysis buffer and proteins were eluted by boiling for 5 min in sds sample buffer . cell lysates ( 106 cells / lane ) or immunoprecipitates were separated by sds - page and transferred to nitrocellulose for immunoblotting . the membranes were blocked with 5 % milk in tbs ( 10 mm tris - hcl ph 7 . 6 , 150 mm nacl ) and incubated with the indicated antibody . bound antibody was revealed with hrp - conjugated rabbit anti - mouse or donkey anti - rabbit antibodies using enhanced chemiluminescence ( ecl ; amersham , arlington heights , ill .) or another appropriate secondary antibody , and protein was visualized by enhanced chemiluminescence ( ecl , amersham ). for receptor - crosslinking experiments , peripheral t - cells or jurkat cells were suspended at a density of 20 . times . 10 . sup . 6 cells / ml in rpmi - 1640 media containing fetal calf serum ( fcs ) ( 2 % v / v ) and incubated with an anti - cd28 mab ( 2 - 5 ug / ml ) or anti - cd3 ( 2 - 8 ug / ml ) antibody in combination with rabbit anti - mouse ( 1 - 2 ug / ml ) for various times ( usually between 5 - 1 smin ) at 37 degree c . a sample of rabbit anti - mouse alone served as a negative control . purified naive t - cells were cultured in 96 - well plates at a density of 2 × 10 5 cells and activated with anti - cd3 / cd28 / igg2α and anti cd3 / cd28 / ctla - 4 coated beads for 48 hours . after this period of time , cells were washed , and beads were removed by using a magnet . cells were then restimulated with anti - cd3 / cd28 coated beads for further 48 hours . to assay proliferation , cells were pulsed with 1 uci of [ 3 h ] thymidine for the last 18 hours of the indicated periods of time . in parallel , cells were stained for surface gm1 expression as described above . jurkat and peripheral t cells were transfected as described ( raab et al . immunity 15 : 921 - 933 , 2001 ). briefly , gstwt and gsks9a purified cdnas ( 10 ug / 10 7 cells ) and / or 3 × nfat / ap - 1 luciferase reporter ( 2 . 5 g / 10 7 cells ) from promega were used . cells were electroporated using btx gene pulser at 250v , 800 f in 10 % fcs , cultured for 15 - 18 hr , stimulated with okt3 and / or cd28 and 2 g / ml rabbit anti - mouse at 37 ° c . for 6 hr , followed by a measurement of luciferase activity using a dual luciferase system kit ( promega , madison , wis .) and a luminometer from microlumat , ( eg & amp ; g berthold , wildbad , germany ). luciferase units of the experimental vector were normalized to the level of the control vector in each sample . [ heading - 0074 ] induction and measurement of cytolytic t - cell responses to tumor cells peripheral blood lymphocytes ( pbls ) were isolated by ficoll gradient centrifugation from buffy coats , or from blood samples ( 60 - 120 cc ) taken from healthy volunteers . leukemic cells k562 were first treated with 50 ug / ml mitomycin c ( sigma ) for 1 hour in 5 % co 2 incubator at 37 ° c . and then washed at least three times with rpmi 1640 to remove the presence of drug . washed k562 cells and pbls ( 1 : 5 ratio ) were then resuspended in rpmi 1640 with 5 % fetal calf serum , 1 % penicillin - streptomycin and 1 % l - glutamine . pbls and leukemic cells were transferred into 24 well plate . cultures were either incubated alone , or in the presence of different concentrations of the inhibitor sb415286 ( glaxosmithkline ) or the inhibitor lithium chloride ( 1 mm and 5 mm ) ( sigma ) were added to the cells . occassionally , pbls would be preincubated with inhibitor for periods of time ( i . e . 5 - 24 hours ) prior to incubation with k562 cells . in either case , cell cultures were then allowed to incubate for 8 days in 5 % co 2 incubator at 37 ° c . prior to an assessment of cytotoxic function . over this period , the k562 cells die due to the treatment with mitomycin c . for an assessment of cytotoxity , effector cells ( pbls ) and fresh target cell k562 were added into 96 well plates at various ratios ( 50 : 1 ; 25 : 1 etc .). culture medium , effector cells and target cells were added into separate wells as background controls . cytotoxicity was assessed using a cytotoxicity assay kit ( promega ) as outlined by the manufacturer . to assess whether cd28 ligation could induce gsk - 3 phosphorylation , the t - cell line jurkat was subjected to anti - cd28 crosslinking followed by immunoblotting with an anti - phospho - specific monoclonal antibody that recognizes phospho - serine 21 on gsk - 3α and phospho - serine 9 on gsk - 3β ( fig1 ). as shown in fig1 a ( left panel ), anti - cd28 ligation induced an increase in phosphorylation of phospho - serine 9 on gsk - 3β and gsk - 3α on ser - 21 in jurkat t - cells ( lanes 1 , 2 vs 3 ). further , increasing amounts of the antibody ( 4 and 8 ug / ml ) used in crosslinking increased the level of phosphorylation ( lane 1 and 2 , respectively ). rabbit anti - mouse served as a negative control ( lane 3 ). these observations indicate that the ligation of cd28 alone can induce the phosphorylation of gsk - 3α and β on sites that inhibit the activity of the kinase . a comparison of gskα phosphorylation was then made under conditions where anti - cd3 , anti - cd28 and a combination of anti - cd3 / cd28 antibodies were used to crosslink receptors on the surface of cells ( fig1 a , right panels ). anti - cd28 ligation induced an increase in phosphorylation of gsk - 3α on ser - 21 at levels similar to that induced by anti - cd3 ligation ( lane 3 vs 2 ). immunoblotting was also conducted using an antibody that recognizes gsk - 3 α / β independent of the phosphorylation of the protein ( lower panels ). this allowed for the identification of the gsk - 3β and gsk - 3α forms of the kinase . cd28 ligation also induced gsk - 3α phosphorylation on inhibitory sites in a mouse t - cell dc27 . 10 ( fig1 b ). in this case , anti - cd3 had little if any effect on the phosphorylation status of gsk - 3α ( lane 2 vs 1 ). anti - cd28 induced a marked change in the phosphorylation of gsk - 3α on ser - 21 ( lane 3 vs 1 ). the combination of anti - cd3 and cd28 ligation induced levels of phosphorylation similar to that observed with anti - cd28 alone ( lane 4 vs . 3 ). these observations indicate that anti - cd28 crosslinking alone is sufficient to induce marked changes in the phosphorylation of gsk - 3 on inhibitory sites . immunoblotting conducted using an antibody that recognizes gsk - 3 α / β independent of the phosphorylation allowed for the identification of the gsk - 3α form of the kinase . to confirm that cd28 could also induced site specific phosphorylation of gsk - 3 in normal primary cells , human peripheral t - cells were subjected to anti - cd28 ligation followed by immunoblotting with an anti - phospho - specific monoclonal antibody that recognizes phospho - serine 21 on gsk - 3α and phospho - serine 9 on gsk - 3β ( fig2 , upper panels ). in this case , cd28 induced primarily the phosphorylation of gsk - 3β on serine 9 ( fig2 , lane 3 vs 1 ). in this case , the level of phosphorylation in control cells was negligible ( lane 1 ), while anti - cd28 ligation alone induced greater phosphorylation than observed for anti - cd3 crosslinking ( lane 2 vs 3 ). overall , our data shown that these data clearly demonstrate that cd28 ligation can each induce the phosphorylation of the kinase gsk - 3β on ser 9 in human peripheral t - cells . two distinct cd28 signaling pathways have been described , one dependent on calcium and cyclosporin sensitive and another one cyclosporine a ( csa ) insensitive ( ghosh et al . blood 99 : 4517 - 4524 , 2002 ). in an attempt to assess whether pi 3 - kinase and / or the calcinuerin - cyclophilin pathway operates upstream of the cd28 - gsk - 3 pathway , cells were exposed to the by pi 3 - kinase inhibitor , wortmannin and the calcinuerin - cyclophilin inhibitor cyclosporin a . treatment of peripheral t - cells with wortmannin at 100 and 200 nm for 30 min partially decreased cd28 induced phosphorylation of gsk - 3 on ser - 9 ( fig2 , lanes 5 , 6 ). by contrast , cells were treated for 30 min with various concentrations of cyclosporin a and then stimulated with anti - cd28 mabs showed the same level of phosphorylation as found in the untreated samples . gsk - 3 β phosphorylation was insensitive to cyclosporin a , even at a concentration of 40 _m cyclosporine ( lanes 7 - 9 ). as a control for gsk - 3 expression , immunoblotting of lysates showed equal amounts of gsk - 3 in the different cells ( lower panel ). these data show that cd28 inactivation of gsk3β depends on pi 3k activity , but not on the tcr - driven calcinuerin - cyclophilin pathway . this further underlines the independence of the tcr and cd28 mediated pathways . [ heading - 0084 ] cd28 cooperation with sb415286 in the potentiation of gsk - 3 inactivation the ability of cd28 to regulate gsk - 3 might have an effect on the enhancing effect of cd28 on tcr driven interleukin 2 ( il - 2 ) transcription . to address this , dc27 . 10 cells that had been stably transfected with human cd28 were transiently transfected with either wild - type , or constitutively active gsk - 3 ( gsk - 3s9a ) in the presence of a nfat - ap - 1 - luciferase based il - 2 reporter plasmid . transcriptional activity assessed upon stimulation with anti - cd3 , anti - cd28 and anti - cd3 / cd28 mabs ( fig3 a ). under these conditions , anti - cd3 induced il - 2 transcription , which in turn was greatly potentiated by anti - cd28 co - ligation . by contrast , over - expression of gsk - 3wt and gsk - 3s9a significantly reduced both tcr / cd3 and tcr / cd3 - cd28 mediated il - 2 transcription when compared to mock transfected cells . importantly , the expression of wild - type , or constitutively active gsk - 3 ( gsk - 3s9a ) also inhibited the potentiating effect of anti - cd28 alone on activation . in the presence of gsks9a , anti - cd3 / cd28 co - crosslinking induced levels of il - 2 transcription that were even below the level observed with tcr / cd3 ligation in mock transfected cells and 7 - 8 times lower than that observed with combined ligation of tcr and cd28 on mock transfected cells . as a control for expression , anti - gst showed the presence of expressed gskwt and gsks9a ( upper right panel ). overall , these data confirm by a different approach that cd28 and tcr x cd28 co - stimulation employs the gsk pathway in its amplification of il - 2 transcription . to further assess whether the cd28 co - stimulation is connected to gsk - 3 , t - cells were activated by anti - cd3 , or anti - cd3 / cd28 , in the presence or absence of 100 um gsk - 3 inhibitor sb415286 . as above , an assessment of il - 2 gene activation was determined using cells that had been transfected with a nf - at / ap - 1 il - 2 promoter construct . under these conditions , anti - cd3 activated transcription was slightly increased by co - ligation with anti - cd28 . however , the addition of sb415286 preferentially increased the response in the context of cd28 co - ligation ( fig3 b ). a slight effect was observed on anti - cd3 induced gene activation ; however , this was some 10 - fold lower than that observed in the context of anti - cd28 co - ligation . overall , these finding demonstrate a preferential connection between cd28 co - ligation and the ability of inactivators of gsk - 3 to potentiate t - cell cytokine responses . [ heading - 0087 ] sb415286 enhances cytolytic t - cell responses against tumor cell line k562 in a cd28 dependent manner previous studies have documented a role of cd28 is enhancing proliferation and the generation of a cytotoxic t - cell response to tumor cells ( chen et al cell 1992 71 : 1093 - 102 ). these anti - tumor ctl responses against both melanoma and carcinoma tumours are crucially dependent on cd28 mediated co - signaling ( haynes j immunol 2002 169 : 5780 - 6 ; chen et al cell 1992 71 : 1093 - 102 ; hu et al j immunol 2002 169 : 4897 - 904 ). given the ability of cd28 to inactivate gsk - 3 , it was next assessed whether the inactivation of gsk - 3 could play the crucial role in potentiating these t - cell responses to tumor cells . peripheral t - cells were exposed to the bcr - abl induced tumor erythroleukemia cell line k562 ( that had been treated with mitomycin c ) and assessed for proliferation and the generation of a ctl response ( fig4 ). t - cells were found to undergo proliferation from 48 - 96 hours as measured by 3h - thymidine incorporation . under these conditions , the response of t - cells was greatly enhanced by exposure to sb415286 . treatment with sb415286 increased the level of response by two - three fold at 48 hours . these data clearly show that inhibition of gsk - 3 can potentiate the proliferative response to tumor cells . in addition to proliferation , sb415286 enhanced the development of a ctls response against the k562 cells ( fig5 ). peripheral t - cells were co - cultured with k562 cells in the absence or presence of the gsk - 3 inhibitor sb415286 for 6 days followed by a measurement of cytolytic t - cell function . cytotoxicity was measured by cytotox96 ® assay . under these conditions , the inhibitor had a major effect in potentiating cytolytic t - cell response ( fig5 a ). the response was potentiated with 10 um by some 5 - 6 - fold in response at a 50 : 1 effector target ratio and at 50 um at a 25 : 1 ratio . further , the cd28 - dependency of the response was shown by the fact that anti - cd28 blocked the cytotoxicity at all t - cell / apc ratios ( fig5 b ). this dependency on cd28 is consistent with the ability of anti - cd28 to induce gsk - 3 phosphorylation on ser - 9 ( fig1 ) and the ability of sb415286 to cooperate more effectively with anti - cd3 / cd28 co - ligation than anti - cd3 alone ( fig3 b ). it also demonstrates that the potentiation of cytolytic activity is not simply due to the clonal expansion of reactive t - cells ( which will also occur ) since the cytolytic assay was normalised on a per cell basis . lastly , consistent with an effect mediated via gsk - 3 , another inhibitor of gsk - 3 also potentiated ctl killing of targets ( fig6 ). the invention may be used to regenerate and enhance an immune response involving the production of cytokines ( interleukin 2 , 4 , interferony etc ), the development of cytotoxic t - cells , and the reconstitution of immune cells in an immunocompromised individual with immunodeficiency due to chemotherapy or as caused by an infectious agent scuh as the human immunodeficiency virus ( hiv - 1 ). peripheral blood lymphocytes and / or purified t - cells purified from human blood may be exposed in vitro to various concentrations of a drug that inhibits gsk - 3a / b , either in the absence or presence of reagents that ligate cd28 such as anti - cd28 , or cd80 / 86 . cells are left for various times at 37 ° c . or rt , and re - infused into the patient at various cell concentrations . the invention may be used to enhance the development of an immune response and the generation of a cytotoxic t - cell response ( ctl ) in the treatment of patients with various infections caused by virus &# 39 ;, bacteria , fungi or prions . peripheral blood lymphocytes and / or purified t - cells purified from human blood may be exposed in vitro to various concentrations of a drug that inhibits gsk - 3a / b , either in the absence or presence of reagents that ligate cd28 such as anti - cd28 , or cd80 / 86 . cells are left for various times at 37 ° c . or rt , and re - infused into the patient at various cell concentrations . alternatively , exposure to the inhibitor would be combined with the presentation of specific antigens associated with the virus or bacteria , alone or in conjunction with dendritic cell vaccines ( ie dendritic cells displaying specific antigens ). dendritic cells could also be infected with the appropriate virus and used as a vaccine . the invention may be used to boost the development of an immune response and the generation of a cytotoxic t - cell response ( ctl ) in the treatment of patients with a defect in the function of the cd28 co - receptor . peripheral blood lymphocytes and / or purified t - cells purified from human blood , exposed in vitro to various concentrations of a drug that inhibits gsk - 3a / b . cells are left for various times at 37 ° c . or rt , and re - infused into the patient at various cell concentrations . the invention may be used to reverse the development of non - responsiveness or anergy in the context of infection . peripheral blood lymphocytes and / or purified t - cells purified from human blood may be exposed in vitro to various concentrations of a drug that inhibits gsk - 3a / b , either in the absence or presence of reagents that ligate cd28 such as anti - cd28 , or cd80 / 86 . cells are left for various times at 37 ° c . or rt , and re - infused into the patient at various cell concentrations . the invention provides the development of transgenic nonhuman animals that comprise transgenes that alter cd28 inactivation of gsk - 3β or β and their use in investigating infection . the invention also provides methods for the identification of novel drugs or peptides useful in treating infections that interfere or augment the cd28 inactivation of the gsk - 3α , β pathway . also within the invention is the use of isolated nucleic acid sequences that encode the peptides or proteins that regulate the cd28 - gsk - 3 pathway . for example , the over - expression of a protein will interfere with cd28 function , or gene knock - out technology is used to delete a gene that affects cd28 function . alternatively , gene knock - out technology is used to delete a gene that affects tcr signaling , thereby necessitating the engagement of cd28 to provide signals needed for the response of the t - cell to antigen , or a foreign graft . using gene therapy techniques , dna encoding such proteins and peptides is taken up by cells and expressed in the cytoplasm . the dna may be introduced into target cells in the bloodstream or other tissues of the patient by standard vectors and / or gene delivery systems . suitable gene delivery systems may include liposomes , receptor - mediated delivery systems , naked dna , and viral vectors such as herpes viruses , retroviruses , and adenoviruses , among others . the invention provides screens for therapeutically useful modulators for treating infections by a virus , bacterium , fungi or prions . a screening method for identifying compounds useful in treating infections by identifying compounds capable of modulating cd28 inactivation of gsk - 3 can be carried out . the assay utilizes a cell that expresses cd28 and gsk - 3 . the cell is most preferably a t cell such as hpb - all or jurkat , but may be any type of cell which expresses cd28 on its surface and gsk - 3 in its cytoplasm , e . g ., a cell transfected with cdnas encoding cd28 and / or gsk - 3 . a sample of cells is incubated in the presence or in the absence of a candidate compound . a reference point could be established under standard conditions and the results from any assay compared to the pre - established standard as the control . alternatively , controls could be run in parallel with each screening assay . cell surface cd28 is cross - linked with , e . g ., a cd28 - specific antibody or a cd28 ligand , such as cd80 / 86 . cell lysates would be subject to immunoblotting using a phospho - specific gsk - 3 antibody to ser - 9 , or the equivalent residues on other gsk - 3 family members . also , gsk - 3 is precipitated from a cell lysate and by immunoblotting with an anti - gsk - 3 antibody specific for phospho specific forms of gsk - 3 . the intensity of staining can be quantitated by means of standard densitometric techniques . a method which measures the substitution of cd28 mediated function by inhibiting of gsk - 3 by a given compound can also be used to identify compounds capable of modulating t cell activation and useful in treating infections . using cells which express cd28 and gsk - 3 , cell surface cd28 is crosslinked with anti - cd28 or forms of cd80 or cd86 and followed by immunoblotting with an anti - gsk - 3 antibody specific for phospho specific forms of gsk - 3 . also , gsk - 3 is precipitated from a cell lysate and by immunoblotting with an anti - gsk - 3 antibody specific for phospho specific forms of gsk - 3 . treatment as indicated in the preceding examples may also be carried out by administering to the patient a therapeutically effective amounts of an inhibitor of gsk - 3 . beta ( or alpha ), either alone as a cd28 - substitute signal , or in conjunction with reagents ( i . e . anti - cd28 / cd80 / cd86 ) that ligate cd28 . the invention provides a method for expanding lymphoid cells that could be grown from organs , for example tissue - infiltrating lymphocytes . organs include lymph - nodes , liver , bone marrow , lung , the pancreas and others . the cells are administered an inhibitor of gsk - 3 . beta ( or alpha ), either alone as a cd28 - substitute signal , or in conjunction with reagents ( i . e . anti - cd28 / cd80 / cd86 ) that ligate cd28 . this could be done together with the presentation of specific peptide antigens in vitro and the treated cells may be administered to patients . aruffo et al . molecular cloning of a cd28 by a high - 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( jul . 6 , 2000 ), pp 86 - 90 . holdorf et al . proline residues in cd28 and src homolgy ( sh ) 3 domain of lck are required for t cell costimulation . j . exp . med . 190 : 375 - 384 , 1999 . iwakami et al . replication - deficient adenovirus - mediated transfer of b7 - 1 ( cd80 ) cdna induces anti - tumour immunity in isolated human lung cancer . respirology 6 : 135 - 144 , 2001 . kane et al . akt provides the cd28 costimulatory signal for up - regulation of il - 2 and ifn - gamma but not th2 cytokines . nat . immunol . 2 : 37 - 44 , 2001 . kim et al . growth factor receptor - bound protein 2 sh2 / sh3 domain binding to cd28 and its role in co - signaling . j . biol . chem . 273 : 296 - 301 , 1998 . linsley et al . the role of the cd28 receptor during t cell responses to antigen . annu . rev . immunol . 11 : 191 - 212 ( 1993 ). lu et al . cd28 - induced t cell activation - evidence for a protein - tyrosine kinase signal transduction pathway . j . immunol . 149 : 24 - 29 ( 1992 ). murphy et al . endothelial cells stimulate t cell nfat nuclear translocation i the presence of cyclosporin a : involvement of the wnt / glycogen synthase kinase - 3 beta pathway . j . immunol . 169 : 3717 - 3725 , 2002 . ohteki et al . negative regulation of t cell proliferation and interleukin - 2 production by the serine threonine kinase gsk - 3 . j . exp . med . 192 : 99 - 104 , 2000 . okkenhaug et al . a point mutation in cd28 distinguishes proliferative signals from survival signals . nat immunol 325 : 325 - 332 , 2001 . pages et al . binding of phosphatidylinositol - 3 - oh kinase to cd28 is required for t - cell signaling . nature 369 : 327 - 329 , 1994 . prasad et al . src - homology 3 domain of protein kinase p . 59 . sup . fyn mediates binding to phosphatidylinositol 3 - kinase in t cells . proc . natl . acad . sci . usa 90 : 7366 - 7370 ( 1993 ). prasad et al . t - cell antigen cd28 interacts with the lipid kinase phosphatidylinositol 3 - kinase by a cytoplasmic tyr ( p )- met - xaa - met motif . proc . natl . acad . sci . usa 91 : 2834 - 2838 , 1994 . raab et al . cd28 signaling via vav / slp - 76 adaptors : regulation of cytokine transcription independent of tcr ligation . immunity 15 : 921 - 933 , 2001 . rudd . upstream - downstream : cd28 cosignaling pathways and t cell function . immunity 4 : 1 - 20 , 1996 . schwartz et al . a cell culture model for t lymphocyte clonal anergy . science 248 : 1349 - 1356 , 1990 . truitt et al . stimulation of cd28 triggers an association between cd28 and phosphatidylinositol - 3 - kinase in jurkat cells j . exp . med . 179 : 1071 - 1076 , 1994 . wang et al . regulation of trail expression by the phosphatidylinositol 3 - kinase / akt / gsk - 3 pathway in human colon cancer cells . j . biol . chem . 277 : 36602 - 36610 , 2002 . zhen et al . lithium regulates protein tyrosine phosphatase activity in vitro and in vivo . psychopharmacology 162 : 379 - 384 , 2002 .