toxoplasma gondii ( t. gondii ) is an intracellular parasite and the etiological agent of toxoplasmosis . although the infection is asymptomatic in most immunocompetent individuals , toxoplasmosis may cause severe complications in immunocompromised individuals [ 1 , 2 ] .   if t. gondii infection occurs during pregnancy , transplacental transmission can occur , leading to abortion or congenital malformations [ 25 ] . the immune response against t. gondii has been largely characterized and it has been demonstrated that cell mediated immunity is essential to control infection [ 2 , 6 , 7 ] , involving synergy between cd4 and cd8 t cells [ 8 , 9 ] . t. gondii triggers the production of il-12 , mainly by dendritic cells [ 1012 ] , which stimulate nk cells and t lymphocytes to secrete large amounts of ifn- , a key cytokine for protection against this parasite [ 10 , 13 , 14 ] . regulatory t cells ( tregs ) are a subset of cd4 t cells that control the immune response by suppressing many lymphocyte effector functions [ 1719 ] . natural tregs constitutively express cd25 ( the  chain of the il-2 receptor ) , ctla-4 , and the forkhead family transcription factor foxp3 [ 22 , 23 ] , which is required for their development and function . although initially described for preventing autoimmune responses [ 20 , 24 , 25 ] , it has also been demonstrated that they can regulate the immune response against infectious agents [ 2629 ] . for example , in vivo depletion of cd25 cells leads to an increase in the production of ifn- in animals infected with plasmodium chabaudi adami    and trypanosoma congolense , or in animals infected with schistosoma mansoni to an increased production of ifn- , il-4 , il-5 , and il-13 , indicating that tregs can control both th1 and th2 responses . during infection with plasmodium berghei   or litomosoides sigmodontis , the aim of this paper is to study the role of tregs during the acute infection of t. gondii in the resistant balb / c strain of mice . we carried out depletion experiments by injection of the pc61 mab in mice followed by infection with the type ii strain me49 , and analyzed mortality . since pc61 mab injection could also eliminate other cell subtypes expressing cd25 , mainly activated t cells ( tact ) , we also studied the cd4 t cell subsets affected by injection of the pc61 mab . six  eight - week - old female balb / cann mice , weighing 1820  g , and swiss mice , were bred in our animal house and maintained in pathogen - free conditions . all protocols depicted in this paper were approved by the local bioethics committee for animal research . the me49 strain of t. gondii was maintained in swiss mice as previously described . briefly , brains from infected mice were removed and homogenized in dulbecco 's phosphate buffered saline ( dpbs ) ; the number of cysts was enumerated and mice were infected intraperitoneally with 10 cysts ; this procedure was carried out every 24 months .   for peroral infection , mice anesthetized with sevorane ( abbott , mexico city , mexico ) were infected with 20 cysts by gavage in 0.1  ml dpbs . the pc61 hybridoma secreting rat igg1 against murine cd25 was obtained from the american type culture collection ( atcc , manassas , va ) . the f41d1 hybridoma , secreting an unrelated rat igg1 mab ( isotype control ) , was a kind gift of dr . hybridomas were grown on cd hybridoma medium ( gibco , grand island , ny , usa ) and mabs were obtained after ammonium sulfate ( 45%  w / v ) precipitation . after extensive dialysis against pbs , antibody concentration was determined by spectrophotometry at 280  nm . antibodies were resuspended in pbs at 1 - 2  mg / ml and stored at   20c until used . unless otherwise stated , mice were injected intraperitoneally ( ip ) with 200  g of purified pc61 mab or control isotype at day   2 . two days later ( day 0 ) , mice were infected perorally as described above . depletion was confirmed by analyzing cd4cd25 or cd4cd25foxp3 cells , in peripheral blood samples when mice were infected . weight and cumulative mortality were recorded daily ; survival curves were compared by the logrank test using the prism software ( graphpad , san diego , ca ) . spleen cells ( 1  10 ) were incubated ( 30 minutes , 4c ) with anti - cd4-fitc or -tc ( clone rm4 - 5 , caltag , burlingame , ca ) , anti - cd4-percp ( biolegend , san diego ca ) , anti - cd25-pe or apc ( clone pc61 5.3 , caltag ) , or anti - cd25-pe ( clone 7d4 , miltenyi biotec , auburn , ca ) in 100  l of washing buffer ( dpbs + 1% fcs + 0.1% nan3 ) . after washing 3 times , cells were suspended in dpbs and analyzed immediately .   for foxp3 detection , we used the foxp3 staining buffer set ( ebioscience , san diego , ca ) with anti - foxp3-alexa fluor 488 ( clone fjk-16s , ebioscience ) following the indications provided by the manufacturer . flow cytometry analysis was performed on a facscalibur or a facscan cytometer ( becton dickinson , san jose , ca ) , running the cell quest program ( becton dickinson ) .   lymphocytes were identified by forward scatter ( fsc ) and side scatter ( ssc ) characteristics , gated and further analyzed . samples were analyzed using the flowjo software v. 5.7.2   ( tree star , ashland or ) . we carried out depletion studies by injection of the pc61 mab to analyze the role of tregs in the resistant balb / c strain of mice during the infection with the type ii strain me49 of t. gondii .   in the literature we found many protocols for tregs depletion using the pc61 mab in different models , using a wide range of mab concentrations , from 100  g to 1  mg [ 32 , 33 , 3641 ] , or even several injections of the pc61 mab before and during infection [ 30 , 42 ] . to reduce the possibility of tact elimination due to high antibody concentrations , we tested the lowest doses reported of pc61 mab ( 100  g and 200  g ) by ip injection . two days later we analyzed cd4cd25 cells to determine if depletion was achieved , since mice would be infected at this time point . we used the pc61 mab in the facs analysis because mcneill et al . , using foxp3-gfp transgenic mice , demonstrated that detection of cd25 cells in pc61-depleted mice using either the 7d4 or the pc61 mabs showed similar results . as can be seen in figure 1 , injection of either 100 ( figure 1(b ) ) or 200  g ( figure 1(c ) ) of the pc61 mab leads to similar levels of depletion of cd4cd25 cells in the spleen . even though depletion efficiency was the same by the day we intended to infect the animals , we were concerned about how long would the depletion lasted . during the first week after injection of the pc61 mab ( figure 2 ) , depletion efficiency was very similar using both doses , but at 10 days post - depletion , a higher number of cd4cd25 cells were observed in animals treated with 100  g when compared to cells from animals treated with 200  g .   at 14 days post depletion , about 83% of cells these results show that injection with 200  g leads to a depletion of cd25 cells for at least 10 days . recovery of cd4cd25 is slightly faster in animals treated with 100  g mab ; at 14 days , cd4cd25 cells from both groups reached almost normal levels . we thus chose 200  g of pc61 mab for the subsequent experiments .       in order to study the role of tregs during infection with t. gondii , resistant balb / c mice were depleted of cd25 cells , and 2 days later they were perorally infected with 20 cysts of the me49 strain . since cd25 is a molecule expressed by other cell types , including activated cd4 t cells , and foxp3 is exclusively expressed by tregs and is required for their development and function , we thus analyzed the depletion of foxp3 cells ( tregs ) the day of infection ( 2 days after depletion ) . we found that only 53% of foxp3 cells were eliminated by injection of the pc61 mab ( figure 3 ) , confirming results previously reported . weight changes and mortality were analyzed for 25 days after depletion and infection ( figure 4 ) . all mice lost up to 25% weight due to the parasite ; it has to be noted that depleted mice lost 5% less weight compared to isotype treated mice . after 15 days the latter started to gain weight ( figures 4(a ) and 4(b ) ) , but did not reach their initial weight .             when mortality was analyzed , isotype - treated mice survived during the 25 day experiment , but 50% of pc61-treated mice died   < 13 days post infection ( dpi ) ( figure 4(c ) ) , although this result was not statistically significant ( p = .0549 ) . a similar result was observed when mice were infected with 50 cysts , although we observed a higher mortality rate ( data not shown ) . analysis of blood samples of the same animals from this experiment showed that although cd25 cells were eliminated , including foxp3 and foxp3 cells , at later time points , a marked increase in cd25foxp3 cells ( tact ) was observed in depleted animals , while the percentage of foxp3 cells ( tregs ) was still decreased ( data not shown ) . we depleted and infected mice as described above , they were killed 10 days pi , when animals showed symptoms of toxoplasmosis ( 24 days before death ) and an exhaustive analysis of spleen cells was performed . analysis of tregs and tact cells from infected animals at this time point ( figure 5 ) showed that infection induced an expansion of tact cells ( 3.76 versus 24.79 ) . depleted infected animals , however , showed a dramatic expansion of tact when compared to depleted / noninfected animals ( 0.94 versus 25.79 ) , but no difference was detected between tact from infected nondepleted or depleted mice ( 24.79 versus 25.79 ) , demonstrating that depletion did not prevent activation of t cells .   on the other hand , a nearly 50% reduction in percentage of tregs was observed in infected - nondepleted animals , when compared to control mice ( 8.32 versus 16.35 ) ; depleted noninfected animals still showed a 50% decrease in tregs at this time point ( 8.34 versus 16.35 ) , while depleted infected animals had 4.85% of tregs , which represents a 50% reduction when compared to noninfected - depleted mice ( 4.85 versus 8.34 ) . all these results were similar when mab 7d4 was used to detect cd25 cells ( figure 5 ) , demonstrating no interference in the detection of cd25 with pc61 mab . a summary of the results obtained from all mice studied is depicted in figure 6 . analysis shows that at 10  dpi ( 12 days post depletion ) 50% of tregs are still lacking in depleted noninfected animals . interestingly , infection induces a 50% reduction of tregs , but depleted infected animals have 50% less tregs than nondepleted infected mice , and only 25% of tregs when compared to control noninfected mice . analysis of tact cells confirmed that infection leads to a dramatic expansion of this subset , which is not altered by depletion , since mean percentage of tact cells was unaffected , although a high dispersion in data was observed ( figure 6 ) . when the ratio of tact / tregs cells was calculated ( figure 7 ) , we found that in noninfected animals , whether depleted or nondepleted , ratios remained nearly unchanged ( 0.28 ) .   in infected mice , a higher ratio was observed ( 1.24.0 ) , indicating a expansion of tact cells due to the parasite .   in depleted / infected mice , however , the ratio was 0.512.7 ( figure 7(a ) ) : a group of 4 mice showed a very high ratio ( > 5.0 ) , while another group presented a low ratio ( 0.51.15 ) . the day we carried out this experiment , one animal was moribund , and its spleen cells showed the highest proportion of tact / tregs ( ratio = 12.7 , figure 7(a ) ) . it has to be noted that in this experiment , an additional group of mice was used to evaluate mortality ( figure 7(b ) ) , and we found that 60% of animals died 15 days pi , which correlates to the percentage of mice with the higher proportion of tact / tregs ( figure 7(a ) ) . all these experiments show that although the generation of tact cells due to infection is not altered by depletion , the percentage of tregs remained very low , turning susceptible a resistant strain of mice .          in the present work we analyzed the role of tregs during infection with t. gondii in the resistant balb / c strain of mice . we observed that infected animals showed a decreased number of treg cells , a result which agrees with that reported by ge et al . , who showed that in pregnant and nonpregnant mice , infection with t. gondii induced a reduction in foxp3 mrna expression levels in spleen and a reduction in both percentage and absolute number of tregs . these results differ from those observed during the acute phase of other infections ( p. berghei , s. mansoni , heligmosomoides polygyrus , mycobacterium tuberculosis , plasmodium yoelii , brugia malayi , and l. sigmodontis ) , in which an increase in foxp3 cell number is observed [ 32 , 39 , 4448 ] .   in t. gondii infection , it is possible that tregs die or migrate to other sites in order to control the immune response locally , as it has been shown during p. berghei infection . it has to be noted that t. gondii can infect all cell types and disseminates to most organs [ 49 , 50 ] . the fate of tregs during t. gondii infection , however , remains currently unknown . injection of pc61 to deplete cd25 cells has been widely used to study the role of tregs in different models [ 32 , 36 , 37 , 39 , 42 , 51 , 52 ] .   in our depletion experiments , we observed that 5060% of balb / c mice died during the acute phase of infection , an observation that suggests that tregs play an important role in toxoplasmosis . however , since cd25 is also expressed by other cell types , injection of the mab leads also to the depletion of tact cells and interpretation of results should be taken carefully . we used a low single dose ( 200  g ) of pc61 mab , which was enough to eliminate 50% of tregs . these observations agree with those reported previously , in which a partial depletion of tregs cells is observed after injection of pc61 mab . injection of pc61 mab initially eliminates 50% of tregs , but in infected mice this reduction is exacerbated due to the infection , reaching 75% tregs reduction in these animals at 10 days pi , when compared to nondepleted - noninfected mice .   however , depletion did not hinder the activation of t cells after t. gondii infection . in fact , levels of cd69 expression and percentage of cd69 cells were similar in foxp3 cells from nondepleted infected and depleted infected mice ( data not shown ) , demonstrating that both groups generate the same proportion of tact cells .         while this work was carried out , couper et al .   reported that depletion using 1  mg of the pc61 mab leads to an increased susceptibility of male c57bl/6j mice to the me49 strain of t. gondii . although the results in mortality obtained in our work are similar , we used the highly resistant balb / c strain of mice , in contrast to the highly susceptible c57bl/6j strain . moreover , we depleted animals with a lower concentration of pc61 mab to lessen undesirable effects in other cell populations ; a single dose of mab ( 200  g ) , 2 days before infection was enough to achieve a 50% elimination of tregs . we avoided the use of higher concentrations of pc61 mab because we think that this would induce a broad and long lasting depletion of cd25 cells ( both tact and tregs ) making more difficult the interpretation of the results . the use of a suitable concentration of the pc61 mab allowed us to detect a difference in the proportions of tact and treg cells 10 days pi . thus , the amount of pc61 antibody did not prevent the generation of tact cells due to the infection . the levels of treg , however , remained low during infection , leading to the loss of resistance of the balb / c strain . these results show that the absence of tregs alters the protective immune response against t. gondii and thus mice become susceptible . t. gondii induces the activation of cd4 and cd8 t cells that are crucial to control infection . these cells could in turn be controlled by tregs , as it has been demonstrated in the t. congolense infection . our results show that tregs play an important role in the modulation of the protective immune response against t. gondii . since infection with this parasite drives a powerful th1 immune response , it is tempting to speculate that the absence of tregs induces an uncontrolled inflammatory response by tact cells . the specific molecules and cells directly responsible for the death of animals , however , remain to be established .
regulatory t cells ( tregs ) are cd4+foxp3 + cells that modulate autoimmune responses . 
 tregs have been shown to be also involved during the immune response against infectious agents . 
 the aim of this work is to study the role of tregs during the infection with the intracellular protozoan toxoplasma gondii . 
 resistant balb / c mice were injected with 200  g of anti - cd25  mab ( clone pc61 ) and 2 days later they were infected with 20 cysts of the me49 strain of t. gondii . 
 we observed that depleted mice showed 5060% mortality during the acute infection . when facs analysis was carried out 
 , we observed that although injection of pc61  mab eliminated 50% of tregs , infected - depleted mice showed a similar percentage of cd25+foxp3 ( activated t cells , tact ) to those observed in infected nondepleted animals , demonstrating that in our depletion / infection system , injection of pc61  mab did not hamper t cell activation while percentage of tregs was reduced by 75% 10 days post infection . 
 we concluded that tregs are essential during protection in the acute phase of t. gondii infection .