Patent Application: US-51898207-A

Abstract:
an ex vivo method of expanding a population of regulatory t - cells includes culturing a starting population of cells containing cd4 + cd25 − t - cells in a growth medium ; introducing nitric oxide into the growth medium sufficient to potentiate switching of the cd4 + cd25 − t - cells to cd4 + cd25 + regulatory t - cells , whereby a subpopulation of treg cells is produced ; and allowing the treg cells to proliferate in culture , to provide a final population of t - cells containing more treg cells than were present in the original t - cells . the resulting expanded population of treg cells are used to deter or decrease an undesired t - cell mediated immune response , e . g ., allograft rejection , in a mammalian host by transplanting the treg cells at a site of a potential or existing undesired immune response , whereby the undesired immune response is deterred or decreased .

Description:
no at low concentrations can also act as an immunomodulator to regulate t cell - mediated immune response . our studies revealed that no at low concentrations may inhibit t cell proliferation by increasing the generation of treg cells . it was also documented that no potentiates switching of cd4 + cd 25 − cells into cd4 + cd25 + treg cells . natural cd4 + cd25 + regulatory t ( treg ) cells are pivotal in self - tolerance to prevent autoimmune diseases as well as in maintaining tolerance to allografts . it was examined whether nitric oxide ( no ) regulates function of treg cells . our results reveal that low no concentrations ( 10 - 100 μm nitrite ), released from s - nitroso - n - acetyl - dl - penicillamine ( snap ) or sodium nitroprusside ( snp ), increased 3 - fold generation of cd4 + cd25 + cd127 − foxp3 + treg cells , which inhibited proliferation of t cells in response to phytohemagglutinin ( pha ) or alloantigens . in the presence of treg cells , snap - released no elevated switching of cd4 + cd25 − cells into cd4 + cd25 + cd127 − foxp3 + t regulatory ( tr1 ) cells . when tested in vivo , snap - induced treg / tr1 cells protected long - term survivals of skin allografts otherwise rejected by cd4 + cd45 + cells in scid mice . thus , no modulates function of treg and tr1 cells . the rpmi 1640 medium ( hyclone , logan , utah ) was supplemented with 2 mm l - glutamine , 1 % mem vitamin solution , 0 . 01 m hepes buffer , 2 × 10 − 5 m 2 - mercaptoethanol , 1 % penicillin - streptomycin ( all from gibco brl , invitrogen , carlsbad , calif .) and 10 % fetal calf serum ( fcs ; hyclone ) ( rpmi - 10 ). the anti - human cd25 - fluorescein isothiocyanate ( fitc ) antibody ( ab ) ( cat no . 555431 , clone m - a251 , mouse iggi ), anti - human cd25 - allophycocyanin ( apc ) ab ( cat no 555434 , clone m - a251 , mouse iggi ), anti - human cd4 - phycoerythrin ( pe ) ab ( cat no 555347 , clone rpa - t4 , mouse iggi ), anti - human cd4 peridin chlorophyll protein ( percp ) ab , anti - human cd127 - pe ab ( cat no 557938 , clone hil - 7r - m21 , mouse igg1 ) were all purchased from bd pharmingen ( san diego , calif .). s - nitroso - n - acetyl - dl - penicillamine ( snap ), sodium nitroprusside ( snp ) and dimethyl sulfoxide ( dmso ) were purchased from sigma - aldrich , ( st . louis , mo .). as no generator , snap was dissolved in dmso ( 1 m stock solution ) and kept frozen at 20 ° c . until used ; the same dmso volumes were always added to control cultures . in some experiments , another no generator snp dissolved in water was added . peripheral blood lymphocytes ( pbls ) were isolated from a buffy coat from blood of healthy volunteers or from blood bank . t cells ( 95 % purity ) were obtained by t cell negative isolation kit ( invitrogen ; cat no : 113 . 11 ). cd4 + cd25 high , cd4 + cd 25 intermediate , or cd4 + cd25 negative cells were isolated by cell sorting on facsaria ( becton dickinson , san diego , calif .) using anti - human cd4 - fitc and anti - human cd25 - pe abs . the cells were immediately used after isolation . identification of cd4 + cd25 high cd127 low t cell population pbls or purified t cells were either left untreated or treated with 10 μg / ml phytohemagglutanin ( pha , sigma - aldrich ) at 1 × 10 6 cells / 2 ml in 24 - well plates in the absence or presence of various concentrations of snap - dmso , dmso or snp and cultured in rpmi - 10 for 5 days . cells were then harvested and stained with anti - human cd4 - percp , anti - human cd25 - apc and anti - human cd127 - pe abs . flow cytometry analysis measured cd4 + cd25 high cd127 low versus cd4 + cd25 high cd127 high populations using facsaria with diva program or facscalibur with cellquest software ( bd pharmingen , san diego , calif .). foxp3 intracellular staining was carried out using the fitc anti - human foxp3 staining kit ( bdbiosciences , san diego , calif . ; cat no 71 - 5776 - 40 ). in brief , pbls from various groups were incubated with anti - human cd4 - percp , anti - human cd25 - apc and anti - human cd127 - pe abs for 20 minutes at room temperature for surface staining of cd4 , cd25 and cd127 molecules , respectively . cells were then washed , and fixed with fix / perm solution ( 30 minutes at 4 ° c .) and permeabilized with permeabilization buffer followed by blocking with rat serum ( 15 minutes at 4 ° c .). cells were then stained with fitc - conjugated anti - foxp3 or isotype control ab ( 30 minutes at 4 ° c .). cells were analyzed by flow cytometry for intracellular foxp3 levels in cd4 + cd25 high / cd127 low versus cd4 + cd25 high / cd127 high cells on a facsaria . pbls ( 3 × 10 5 / well / 200 μl ) were left untreated or treated with pha in the presence of various concentrations of snap for 24 hours , and 5 mg / ml mtt was added to triplicate samples for 4 hours . cells were lysed overnight with 100 gl lysing buffer ( mukhopadhyay et al ., 1999b ) and the absorbance was determined at 570 nm ( each point represents mean value of 3 wells ). an apo - brdu ™ kit ( phoenix flow systems , san diego , calif .) was used to measure apoptotic cells . pbls ( 1 × 10 6 ) were either left untreated or treated with various snap concentrations for 24 hours . harvested cells ( 1 × 10 6 ) were fixed in 1 % paraformaldehyde ( 15 minutes at 4 ° c .) and resuspended in 50 μl of dna labeling solution containing 5 - bromo - deoxyuridine triphosphate ( br - dutp ) and terminal deoxynucleotidyl transferase ( tdt ) enzyme ( 60 minutes at 37 ° c .). cells were rinsed and resuspended in 100 μl of anti - brdu - fitc monoclonal ab ( 30 minutes at room temperature ). cell suspensions were mixed with 0 . 9 ml pbs containing 2 μg / ml propidium iodide ( sigma - aldrich ) and 50 μg / ml rnase a and analyzed immediately by flow cytometry . the accumulated nitrite ( from no in snap / dmso , dmso or snp cultures ) was measured by griess method ( mukhopadhyay et al ., 2004 ). the 96 - well plates were filled with sample and equal volume of griess reagent ( 1 % sulfanilamide and 0 . 1 % naphthylethylenediamine ( 1 : 1 ) in 2 . 5 % orthophosphoric acid ). the plates were read at 550 nm absorbance and nitrite concentrations were calculated based on a standard curve from a prepared standard solution of sodium nitrite . alloactivation was set up by co - culturing responder pbls ( 1 - 2 × 10 6 cells / ml ) in a total volume of 35 ml of rpmi - 10 with fully hla mismatched gamma - irradiated ( 30 gy ) stimulators ( 1 : 1 ratio ) in the presence of snap / dmso . after 5 days , cells were washed and stained with anti - cd4 - fitc and anti - cd25 - pe abs to isolate cd4 + cd25 high and cd4 + cd25 intermediate populations by facs sorting . these cell populations were then added to fresh syngeneic pbls ( 1 × 10 5 ) at different responder / regulator cell ratios ( 1 / 0 , 1 / 0 . 005 , 1 / 0 . 01 , 1 / 0 . 05 , 1 / 0 . 1 , 1 / 0 . 5 , and 1 / 1 ) with the same hla - mismatched irradiated stimulators ( 1 × 10 5 ). cells cultured in a 200 μl volume of rpmi - 10 in 96 - well round - bottomed plates for 5 days and pulsed with 1 μci of [ 3 h ]- thymidine for last 16 hours before harvesting . cells were collected onto glass filters using an automated multi - sample harvester and the amount of [ 3 h ]- thymidine incorporation was measured with a scintillation counter ( packard , meridan , conn .). proliferative responses are expressed as the mean [ 3 h ]- thymidine incorporation ( counts per minute [ cpm ]) of triplicate wells ± sd . in identical experiments , pbls were activated with pha ( 10 μg / ml ) to examine the ability of no - induced cd4 + cd25 high population to suppress proliferation of alloantigen - specific mlc . naïve scid mice with a balb / c background served as recipients and c57bl / 6 mice as skin donors . spleens from balb / c mice were lysed of red blood cells and splenocytes were enriched for t cells using mouse t cell negative isolation kit ( invitrogen ). these t cells were cultured with irradiated c57bl / 6 spleen cells in the presence of 100 μm snap . after 5 days , cells were harvested and stained with anti - mouse cd4 - fitc and anti - mouse cd25 - pe abs ( bd pharmingen ) to purify cd4 + cd25 high cells by sorting ( facsaria , beckton dickinson ). pure cd4 + cd25 high cells were injected i . v . in scid mice with c57bl / 6 skin allografts ; grafts were ˜ 2 × 2 cm . within 7 days , the same recipients received 1 × 10 6 cd4 + cd45ra high cells purified by cell sorter and grafts were assessed daily for signs of rejection ; 50 % of the graft damage was considered as rejection . the cd4 + cd25 negative and cd4 + cd25 high t cell populations were isolated from t cells by cell sorting ( facsaria ). next , cd4 + cd25 negative cells were labeled with carboxyfluorescein diacetate succinimidyl ester ( cfda - se ; cat no c1157 , invitrogen ) as described elsewhere ( hans et al ., 2005 ). a stock solution of cfse ( 5 mm ) was diluted with pbs and added to the cd4 + cd25 negative cells at final concentration of 1 gm per 1 × 10 6 cells ( 20 minutes at 37 ° c .). after three washings , cfse - labeled cd4 + cd25 negative cells ( 1 × 10 6 ) were co - cultured without or with cd4 + cd25 high cells at 10 : 1 ratio without or with titrated snap or dmso and 6 hours later cells were activated with pha ( 10 μg / ml ). following 5 days cells were harvested and stained with anti - human cd25 - apc and anti - human cd127 - pe abs to measure transformation of cd 4 + cd25 negative cells to cd25 + cd127 negative cells by facs . the same cells were added to a primary syngeneic mlc at responder / regulator cell ratios ( 1 / 0 , 1 / 0 . 25 , 1 / 0 . 5 , and 1 / 1 ). cells were cultured for 5 days and the [ 3 h ]- thymidine incorporation was measured as described above . a paired , 2 - tailed student t test was used to determine the statistical significance of differences between proliferative responses : p & lt ; 0 . 05 were considered significant . no increases cd4 + cd25 high cd127 − treg cells in activated pbls culture previous studies showed that s - nitroso - n - acetylpenicillamine ( snap ) or sodium nitroprusside ( snp ) serve as chemical source of no ( hogan et al ., 1992 , mukhopadhyay et al ., 1999b ). cell viability assay ( with 3 -( 4 , 5 - dimethyl thiazol - 2 - yl )- 2 , 5 - diphenyl tetrazolium bromide ; mtt ) revealed that snap - released no was significantly cytotoxic to peripheral blood lymphocytes ( pbls ) only when increased to 300 μm and above ( fig1 a ). in fact , the 5 - bromo - deoxyuridine triphosphate ( apo - brdu ) apoptosis assay showed that snap - released no concentrations between 10 to 100 μm produced less than 10 % apoptotic cells among pha - activated t cells in 5 - day cultures ( fig1 b ). at the same time snap - released no to the medium in a dose - dependent fashion , as shown by the elevated levels of nitrites ( fig1 c ). the non - activated treg cells are characterized by higher membrane expression of il - 2rα ( cd25 ) and elevated intracellular expression of foxp3 ( sakaguchi s , 2004 ). since conventionally activated cd4 + t cells also express higher levels of cd25 , identification of treg cells must rely on different markers . recently , lack of il - 7r has been confirmed as a potent phenotypic marker for cd4 + cd25 + treg for population with potent inhibitory functions ( seddiki et al ., 2006 ). therefore , the present experiments examined whether snap - released no may affect generation of cd4 + cd25 + cd127 − treg cells ( fig2 a - b ). the non - activated pbls exposed to different snap concentrations ( fig2 a - 1 top , middle and bottom panels ) were compared with pha - activated pbls cultured for 5 days without ( fig2 a - 2 , top , middle and bottom panels ) or with different snap concentrations ( fig2 a , top , middle and bottom panels ). after culture , facs was used to first select cd4 + cd25 high population and then to examine it for the expression of cd127 to identify cd4 + cd25 + cd127 − treg cells ( fig2 a - 1 , 2 a - 2 and 2 a - 3 ). comparison of these populations showed that exposure to no of pha - activated pbls significantly increased levels of cd4 + cd25 + cd127 − treg cells ( fig2 b ). since no did not affect the number of cd4 + cd25 + cd127 − treg cells among non - activated pbls , activation step was required . furthermore , elevated treg numbers were also generated by another no - generator , snp , confirming that this phenomenon is caused by increased no concentrations ( not shown ). the master regulator foxp3 expression inversely correlated with cd127 expression and in vitro suppression by cd4 + cd25 + cd127 − treg cells ( seddiki et al ., 2006 ). pha - activated pbls cultured without ( fig3 a ) or with snap ( fig3 b ) for 5 days confirmed that no levels elevated numbers of cd4 + cd25 + cd127 − treg cells . top panel : 30 μm snap ; middle panel : 50 μm snap ; bottom panel : 100 μm snap . intracellular staining for foxp3 revealed that similar majority of foxp3 + cells were among cd4 + cd25 + cd127 − treg cells in all experimental groups . thus , no induces generation of foxp3 - positive cd4 + cd25 + cd127 − treg cells . as a functional assay , it was examined whether no - induced treg cells may inhibit proliferation of t cells in response to alloantigen or pha ( fig4 a - b ). population of treg cells was always generated by culturing pbls with hla - mismatched irradiated stimulators for 5 days without or with 100 μm snap . following culture , cd4 + cd25 low and cd4 + cd25 high cells were added at different responder / regulator ratios to the same primary mlc ( fig4 a ) or to the same responder stimulated with pha ( fig4 b ). top panels : cd4 + cd25 low ; bottom panels : cd4 + cd25 high . the results demonstrated that cd4 + cd25 high cells inhibited t cell proliferation in response to alloantigen or pha . in contrast , cd4 + cd25 low cells enhanced proliferative response , suggesting that this population may contain primed memory cells . the beneficial effect produced by adding of 100 μm snap to 60 × 10 6 pbls challenged with hla - mismatched alloantigen was also calculated ( fig5 a ). while 5 - day culture generated approximately 1 × 10 6 treg cells , almost 3 - fold that number was generated in the presence of snap - released no . thus , no significantly increases the number of functional cd4 + cd 25 high treg . an in vivo model was established to test whether no - induced treg cells may block allograft rejection or even induce permanent acceptance of allografts . spleen balb / c cells were cultured for 5 days with irradiated c57bl / 6 splenocytes without and with 100 μm snap to obtain donor - specific treg cells . following 5 - day culture , cd4 + cd25 high cells were isolated by cell sorting and 2 - 5 × 10 5 injected to scid recipients transplanted with c57bl / 6 skin allografts . within 7 days all recipients were injected with 1 × 10 5 cd4 + cd45 + cells . the results revealed that recipients injected just with cd4 + cd45 + cells all acutely rejected skin allografts ( fig5 c and 5d ). in contrast , prior injection of cd4 + cd25 high cells cultured without or with snap always induced long - term skin allograft survivals ( fig5 c and 5d ). these results showed that no - induced treg cells are potent in vivo to prevent rejection and may be used for expansion of treg population . no - promotes switching of cd4 + cd25 − cells into cd4 + cd25 + cd127 − cells experiments were performed to understand the mechanism by which no increase the number of treg cells . isolated cd4 + cd25 − cells labeled with carboxyfluorescein diacetate succinimidyl ester ( cfse ) were co - cultured with cd4 + cd25 + cells and stimulated with irradiated hla - mismatched cells . following 5 day - culture , cfse + cd25 high cells were identified by facs ( fig6 ). the results showed that no increased switching of cd4 + cd25 − cells into cd4 + cd25 + regulatory t ( tr1 ) cells . thus , no promotes switching of naive cd4 + cells into next generation of tr1 population . in addition to s - nitroso - n - acetyl - dl - penicillamine ( snap ) or sodium nitroprusside ( snp ) other agents generate nitric oxide ( no ) and these agents may also increase generation of treg cells . for example , 3 - morpholynosydnonimine ( sin - 1 , naproxen ( hct - 3012 [( s )- 6 - methoxy - α - methyl - 2 - naphthaleneacetic acid 4 -( nitrooxy ) butyl ester ]), sodium nitroprusside , 2 - 2 -( hydroxynitrosohydrazino ) bis - ethamine ( noc - 18 ), arginine , and others . to prevent apoptosis of treg cells during culture in the presence of no - releasing agents , phorbol 12 - myristate 13 - acetate ( pma ) will be used to increase the generation of treg cells . this agent prevents no - induced apoptosis . sakaguchi s . naturally arising cd4 regulatory t cells for immunologic self - tolerance and negative control of immune responses . annu rev immunol . 2004 ; 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