nod / ltj female mice were purchased from charles river ( calco , italy ) . animal care procedures were performed according to protocols approved by the hospital san raffaele institutional animal care and use committee ( iacuc no . blood glucose was measured in the morning three times a week using a glucometer ascensia breeze 2 glucose meter ( bayer , leverkusen , germany ) . a diagnosis of diabetes was made after a glucose measurement of 300 mg / dl to ensure no spontaneous disease reversal . a relapse of disease in treated animals was considered following two consecutive glucose measurements of 200 mg / dl , levels at which spontaneous diabetes reversal never occurred in any of the tested mice ( data not shown ) . after diabetes onset , female nod mice ( aged 22  7 weeks ) were treated with varying doses of the non  fc - binding anti - cd3 f(ab)2 clone 145 - 2c11 ( bio express , west lebanon , nh ) or mab isotype control ( golden syrian hamster igg ; ebioscience , san diego , ca ) , according to the glycemia levels , the same day they were found to have diabetes . rapamycin ( rapamune ; wyeth europe , taplow , u.k . ) was diluted in water and administered by gavage once a day at 1 mg / kg , a dose that we and others previously demonstrated not to be toxic to pancreatic islets ( 14,16 ) . recombinant human il-10 ( bd biosciences , mountain view , ca ) was diluted in pbs and administered twice a day at a dose of 0.05 mg / kg i.p . glucose tolerance was monitored via tail - vein sampling at time 0 ( just before glucose solution injection ) and 5 , 10 , 15 , 20 , 25 , 30 , 45 , 60 , 75 , 90 , and 120 min after glucose solution injection . all statistical analyses were performed using a two - tailed student 's t test . a p value of < 0.05 was deemed significant . nod / ltj female mice were purchased from charles river ( calco , italy ) . animal care procedures were performed according to protocols approved by the hospital san raffaele institutional animal care and use committee ( iacuc no . blood glucose was measured in the morning three times a week using a glucometer ascensia breeze 2 glucose meter ( bayer , leverkusen , germany ) . a diagnosis of diabetes was made after a glucose measurement of 300 mg / dl to ensure no spontaneous disease reversal . a relapse of disease in treated animals was considered following two consecutive glucose measurements of 200 mg / dl , levels at which spontaneous diabetes reversal never occurred in any of the tested mice ( data not shown ) . after diabetes onset , female nod mice ( aged 22  7 weeks ) were treated with varying doses of the non  fc - binding anti - cd3 f(ab)2 clone 145 - 2c11 ( bio express , west lebanon , nh ) or mab isotype control ( golden syrian hamster igg ; ebioscience , san diego , ca ) , according to the glycemia levels , the same day they were found to have diabetes . rapamycin ( rapamune ; wyeth europe , taplow , u.k . ) was diluted in water and administered by gavage once a day at 1 mg / kg , a dose that we and others previously demonstrated not to be toxic to pancreatic islets ( 14,16 ) . recombinant human il-10 ( bd biosciences , mountain view , ca ) was diluted in pbs and administered twice a day at a dose of 0.05 mg / kg i.p . glucose tolerance was monitored via tail - vein sampling at time 0 ( just before glucose solution injection ) and 5 , 10 , 15 , 20 , 25 , 30 , 45 , 60 , 75 , 90 , and 120 min after glucose solution injection . all statistical analyses were performed using a two - tailed student 's t test . a p value of < 0.05 was deemed significant . to define a suboptimal dose of anti - cd3 amenable to combinational therapy studies ( i.e. , having a second agent that improves the action of the first ) and to identify the influence of starting glycemia on the ability to reverse disease , we first grouped nod mice based on degree of hyperglycemia and treated with various dosages of anti - cd3 ( fig . using this strategy , several suboptimal anti - cd3 dosages leading to a wide range of diabetes reversal rates ( i.e. , from 35 to 75% ) were identified . the efficacy of the anti - cd3 treatment was strictly dependent on the dosage , as previously shown ( 17,18 ) , but was also influenced by glucose levels at time of treatment . figure 1b shows the glucose levels of each of the animals treated with the best effective anti - cd3 dosage ( i.e. , 50 g  3 doses in mice with 300349 mg / dl glycemia levels ) , demonstrating a rapid and uniform diabetes reversal in six of eight animals treated ( fig female diabetic nod mice were grouped based on glycemia levels ( 300349 [ gray bar ] and 350400 mg / dl [ white bar ] ) and treated with various anti - cd3 dosages . percentages of diabetes reversal and number of mice in each group are shown ( a ) . glucose levels of the eight nod mice with 300349 mg / dl glycemia and treated with three 50-g doses of anti - cd3 ( 75% diabetes reversal ) are shown ( b ) . female diabetic nod mice with 300400 mg / dl glycemia were treated with rapamycin alone ( 1 mg / kg daily during the entire time of observation [  ] ) or anti - cd3 alone ( 50 g  3 doses at days 0 , 1 , and 2 [  ] ) . although rapamycin has a strong immunomodulatory capacity in pre - diabetic nod mice ( 14,16 ) , rapamycin monotherapy did not lead to disease reversal in any of the diabetic mice ( fig . 1c ) . to test whether rapamycin cooperates with anti - cd3 to reverse diabetes and reinforce the development of long - term tolerance , diabetic nod mice were treated with rapamycin and the anti - cd3 dosage that showed the weaker ability to reverse disease ( i.e. , 35% diabetes reversal ) . the concomitant administration of rapamycin and anti - cd3 did not lead to enhanced diabetes reversal in any of the animals tested ( fig . 2a ) ; rather , the inclusion of rapamycin blocked the ability of anti - cd3 to impart its beneficial effect . the deleterious influence of rapamycin on the anti - cd3 reversal capacity was even observed when rapamycin was coadministered with dosages of anti - cd3 able to cure 65% of diabetic nod mice ( fig . of 10 diabetic nod mice , 9 remained diabetic when treated with rapamycin and anti - cd3 at the highest effective dose ( i.e. , 70% diabetes reversal ) ( fig . 2c ) . female diabetic nod mice were treated with anti - cd3 alone or in conjunction with rapamycin ( 1 mg / kg ) or il-10 ( 0.05 mg / kg ) during the entire time of observation . mice with 350400 mg / dl glycemia were treated with 25 g anti - cd3 ( one dose ) alone (  ) or in conjunction with rapamycin (  ) ( a ) . mice with 300349 mg / dl glycemia were treated with 18 g anti - cd3 ( one dose ) alone (  ) or in conjunction with rapamycin (  ) ( b ) . mice with 300400 mg / dl glycemia were treated with 50 g anti - cd3 ( three doses ) alone (  ) or in conjunction with rapamycin (  ) ( c ) . mice with 300400 mg / dl glycemia were treated with 50 g anti - cd3 ( three doses ) alone (  ) or in conjunction with il-10 (  ) ( d ) . percentages of diabetes reversal and number of mice are shown . to evaluate whether other pro - tolerogenic compounds acted similarly to rapamycin when combined with anti - cd3 , we tested il-10 , an immunomodulatory cytokine with known tolerogenic potential in vivo ( 14,19 ) . when coadministered with anti - cd3 in diabetic nod mice , the addition of il-10 did not interfere with the anti - cd3 therapeutic activity ( fig . these data demonstrate that the tolerogenic capacity of anti - cd3 during the first induction phase is completely halted by rapamycin , but not by il-10 therapy , in diabetic nod mice . to define whether the detrimental effect of rapamycin on anti - cd3 therapy was related to changes in the t - cell compartment , we tested the percentages of circulating cd4 and cd8 t - cells and the frequency of cd4cd25foxp3 t - cells in the pancreatic lymph nodes of nod mice treated with anti - cd3 alone or in combination with rapamycin . circulating cd4 t - cells were depleted upon anti - cd3 treatment irrespective of the presence of rapamycin , whereas cd8 t - cells were only partially affected ( fig . similarly , rapamycin did not alter the frequency of cd4cd25foxp3 t - cells in the pancreatic lymph nodes 2 weeks after anti - cd3 treatment ( fig . contrary to what was previously demonstrated ( 6 ) , anti - cd3treated nod mice did not show a selective increase of cd4cd25 t - cells in the pancreatic lymph nodes compared with control nod mice . this might be due to the reduced anti - cd3 dosage used in our study ( i.e. , 50 g  3 doses ) compared with that used by belghith et al . ( i.e. , 50 g  5 doses ) ( 6 ) , which might lead to different kinetics in the expansion of cd4cd25 t - cells . t - cell frequency and phenotype in nod mice treated with anti - cd3 or anti - cd3 and rapamycin . diabetic nod mice were treated with anti - cd3 alone ( 50 g  3 doses , n = 6 , [  ] ) or in combination with rapamycin ( 1 mg / kg per day , n = 5 , [  ] ) . peripheral blood was collected at different time points after treatment , and circulating cd4 ( left ) and cd8 ( right ) t - cells were analyzed by a fluorescence - activated cell sorter . (  ) , mean  sd of peripheral cd4 and cd8 t - cells in nondiabetic nod mice within cd45 cells ( n = 10 ) ( a ) . pancreatic lymph nodes from normoglycemic untreated ( n = 4 ) , diabetic untreated ( n = 5 ) , anti - cd3treated ( 50 g  3 doses ) ( n = 2 ) , and anti - cd3 ( 50 g  3 doses ) plus rapamycin treated ( n = 5 ) nod mice were analyzed by a fluorescence - activated cell sorter 3 weeks after treatment . one representative plot for each group , after cd4 t - cell gating , is shown . the big gate includes all cd25 t - cells , whereas the small gate includes only cd25foxp3 t - cells . percentages of means  sd of cd25 t - cells ( left panel ) and cd25foxp3 t - cells ( right panel ) within cd4 t - cells in the pancreatic lymph nodes are shown ( c ) . the anti - cd3 maintenance phase in nod mice is a stable condition of tolerance that is no longer dependent on the presence of the antibody . given our previous results , we tested whether rapamycin negatively affects this stable condition of reversed type 1 diabetes . five weeks after anti - cd3mediated diabetes reversal , normoglycemic nod mice were treated with rapamycin . quite remarkably , all previously cured mice returned to a state of hyperglycemia within 7 weeks of rapamycin administration , whereas rapamycin - untreated animals showed no signs of diabetes recurrence ( fig . 4a ) . to further delay the interval between anti - cd3 intervention and rapamycin administration , normoglycemic nod mice were treated with rapamycin 30 weeks after anti - cd3mediated diabetes reversal . consistent with the aforementioned observations , two of three anti - cd3cured nod mice returned to a diabetic state within 10 weeks of rapamycin administration , whereas all rapamycin - untreated animals remained normoglycemic ( fig . this phenomenon was reversible and strictly dependent on the presence of rapamycin , as all mice treated for 230 days with rapamycin promptly returned to a normoglycemic state upon drug removal . delayed rapamycin administration in anti - cd3treated nod mice . according to their glycemia levels , thirty - five days after anti - cd3 mab  mediated diabetes reversal , four mice were treated with rapamycin (  ) and 24 were left untreated (  ) . percentages of diabetes reversal and number of mice are shown ( a ) . according to their glycemia levels , one hundred and eighty days after anti - cd3mediated diabetes reversal , three mice were treated with rapamycin (  ) and 21 were left untreated (  ) . intraperitoneal glucose tolerance test was performed in three normoglycemic untreated nod mice , three diabetic nod mice , six normoglycemic anti - cd3cured mice , and three mice previously cured with anti - cd3 but again diabetic upon rapamycin treatment . intraperitoneal glucose tolerance test was performed in the latter group during rapamycin treatment ( i.e. , 320 days after anti - cd3 mab treatment ) . the average glucose levels per each time point and the best - fitting curve for each group are shown ( c ) . the area under the curve ( auc ) from time 0 to 120 min after glucose injection for all the animals included in panel b is shown ( d ) . to evaluate the metabolic parameters underlying this finding , a glucose tolerance test was performed in nod mice previously treated with anti - cd3 and under rapamycin treatment . although two of three rapamycin - treated mice were hyperglycemic at the time of analysis ( as shown in fig . 4b ) , all three demonstrated a glucose response similar to that observed in diabetic untreated nod mice , while control anti - cd3cured nod mice showed a glucose response superimposable to that of normoglycemic untreated animals ( fig . overall , rapamycin reverts the stable tolerance condition established in anti - cd3cured nod mice while it is administered . with prior data demonstrating that rapamycin is a protolerogenic compound both in vitro and in vivo ( 12,13,15 ) as well as information from the first clinical trials with anti - cd3 in recent - onset type 1 diabetic patients suggesting that this form of therapy can be improved ( 8,9 ) , we tested the specific hypothesis that rapamycin would augment the therapeutic effectiveness of anti - cd3mediated type 1 diabetes reversal in nod mice . against all expectations , we observed that rapamycin not only blocks the ability of anti - cd3 treatment to cure overt hyperglycemia in nod mice but also breaks its curative effect while it is administered . ctla-4 neutralizing antibodies ( 6 ) block the reversal capacity of anti - cd3 in the induction phase while cyclophosphamide breaks anti - cd3mediated tolerance during the maintenance phase ( 20 ) . the mechanisms by which these compounds counteract the anti - cd3 effect have been elucidated . cyclosporine a blocks t - cell activation and t - cell depletion mediated by activation - induced cell death , and anti ctla-4 monoclonal antibodies impede the generation and/or function of inducible tgf-dependent tregs , whereas cyclophosphamide depletes tregs . in addition , rapamycin allows t - cell activation and activation - induced cell death ( 11 ) , permits generation of inducible tregs ( 2123 ) , and does not selectively deplete tregs ( 12,24,25 ) . in fact , t - cell frequency and phenotype in anti - cd3rapamycin  treated mice were identical to those of anti - cd3treated nod mice . it is therefore unlikely that rapamycin alters the anti - cd3 activity through the same inhibitory mechanisms demonstrated for the abovementioned compounds . it has been recently proposed that the reduced il-2 production by t effector cells in nod mice is the root cause of the progressive loss of treg t effector cell balance in the islets , leading to -cell destruction ( 26 ) . rapamycin , by inhibiting signal transduction delivered by il-2 , might directly interfere with this pathway , nowadays considered so crucial for maintaining immunological tolerance in nod mice . . alternative mechanisms that can explain the unique effect of rapamycin in anti - cd3treated nod mice may be related to -islet physiology . it has recently been demonstrated that rapamycin induces fulminant diabetes in the psammomys obesus mouse model of nutrition - dependent type 2 diabetes by increasing insulin resistance and reducing -cell function and mass through increased apoptosis ( 27 ) . the fundamental function of mammalian target of rapamycin  signaling in -cells , which is blocked by rapamycin , has been confirmed by others ( 28,29 ) . rapamycin might therefore have a negative effect directly on the islets rather than blocking the activity of anti - cd3 in nod mice . however , this hypothesis is in contrast to previous observations by our group ( 14 ) and others ( 16 ) in pre - diabetic nod mice wherein rapamycin monotherapy significantly protected animals from disease development . in addition , diabetic nod mice treated with rapamycin did not develop a more aggressive disease , in terms of glycemia , than untreated mice ( a.v . , unpublished data ) . an alternative hypothesis is that rapamycin interferes with -cell proliferation , as demonstrated in specific experimental settings such as pregnancy ( 30 ) and transgenic mice ( 31 ) . however , at this time , there are no data indicating that anti - cd3 leads to -cell proliferation . indeed , currently available data suggest the opposite : recovery of metabolic control following anti - cd3 therapy may be due to mending of -cells that had been already present but not functional in the pancreas at the moment of hyperglycemia rather than -cell proliferation ( 18,32 ) . future experiments will investigate the pancreata of nod mice treated with rapamycin , with or without anti - cd3 , in order to further understand the mechanisms underlying its deleterious action . rapamycin monotherapy in long - lasting type 1 diabetic patients does not aggravate the autoimmune disease but , rather , improves the suppressive function of ntregs ( 15 ) . one should therefore expect that rapamycin behaves similarly in the case of new - onset type 1 diabetes . however , the disease in early - onset type 1 diabetic subjects appears both metabolically and immunologically different from that observed in long - lasting patients , and rapamycin might have a different outcome in diverse patient populations . a clinical trial combining rapamycin and il-2 recently began with new - onset type 1 diabetic patients ( http://www.clinicaltrials.gov/ct2/show/nct00525889?term=rapamune+and+il-2&rank;=1 ) . our data suggest caution in designing rapamycin - based combinational treatments because the addition of rapamycin to anti - cd3 therapy in early - onset diabetic nod mice completely abolished the anti - cd3 therapeutic effect .