human multipotent mesenchymal stromal cells ( msc ) , known for their multilineage differentiation potential , possess pleiotropic immunosuppressive functions that are partly mediated by expression of the tryptophan - catabolizing enzyme indoleamine-2,3-dioxygenase ( ido ) [ 14 ] . upon stimulation with inflammatory cytokines ,
msc exhibit broad - spectrum antimicrobial effector functions directed against various clinically relevant pathogens , and these effects are dependent on ido and/or the antimicrobial peptide ll-37 [ 5 , 6 ] .
these dual immunosuppressive and antimicrobial properties render msc a promising novel cellular immunosuppressant which is currently under intensive clinical investigation for various auto- and alloimmune diseases such as steroid - refractory graft versus host disease ( gvhd ) after allogeneic hematopoietic stem cell transplantation ( hsct ) , crohn 's disease , and multiple sclerosis [ 710 ] .
emerging data indicate that signals from the microenvironment including those induced by hypoxia [ 11 , 12 ] or derived from microbes may critically affect ido and consequently msc effector functions [ 1315 ] . as the cytomegalovirus ( cmv ) represents a prominent pathogen in immunocompromised hosts in particular in patients suffering from gvhd after hsct , we initiated studies investigating the impact of cmv infection on msc - mediated effects . during coevolution with its specific host ,
for example , cmv has been reported to inhibit the upregulation of mhc class ii antigens .
furthermore , it was found that cmv generally inhibits signalling via the ifn- receptor and that this is mediated via a reduced phosphorylation of stat1 and an enhanced degradation of jak1 [ 1921 ] .
mesenchymal stromal cells and embryonic stem cells are able to inhibit t - cell responses and several mechanisms including the production of prostaglandins , of immunosuppressive cytokines , of arginase i , or of adenosine [ 23 , 24 ] appear to be involved in this effect .
in addition , we and others reported that the immunoregulatory effects of mesenchymal stromal cells are , at least in part , brought about by the induction of the tryptophan degrading enzyme indoleamine 2,3-dioxygenase .
we report here that cmv is a major negative regulator of ido activity in human msc , dramatically reducing their immunosuppressive and antimicrobial properties , thereby implicating that active cmv infections may undermine the clinical efficacy of msc treatment .
human bone marrow - derived msc were prepared , propagated , and characterized as previously described .
bone marrow aspirates for the generation of msc were obtained from healthy volunteer donors who had provided written informed consent ; the study was conducted according to the declaration of helsinki principles and approved by the ethics committee of the medical faculty of the heinrich - heine - university , dsseldorf , germany .
okt3 producing hybridoma cells were obtained from the american type culture collection ( rockville , usa ) .
l - tryptophan , l - kynurenine , 1-l - methyl - tryptophan ( 1-mt ) , and ehrlich 's reagent were ordered from sigma - aldrich ( deisenhofen , germany ) .
cmv strains ad169 and tb40e were kindly provided by c. sinzger ( institute for medical virology , ulm , germany ) and a. zimmermann ( institute for virology , dsseldorf , germany ) . before infection of msc ,
the virus - containing solution was thawed and diluted in tryptophan - free rpmi 1640 medium to reach a multiplicity of infection ( moi ) of 0.110 . in some experiments
the enzymatic activity of ido directly correlates with the concentration of kynurenine in supernatants of tissue culture cells and therefore , the measurement of kynurenine can be used to determine ido activity .
msc ( 2  10 per well ) were plated in 96-well flat - bottomed microtiter plates in imdm containing 5% fcs and 0.6  mm l - tryptophan .
the plates were incubated at 37c and after 72  h 160  l were removed from each well and transferred to a 96-well v - bottomed plate .
after the addition of 10  l 30% trichloroacetic acid to each well , the plates were incubated at 50c for 30 minutes to hydrolyze n - formyl - kynurenine to kynurenine .
after centrifugation for 10  min at 600  g , 100  l supernatant was transferred to 96-well flat - bottomed plates and 100  l 1.2% ( w / v ) 4-(dimethylamino ) benzaldehyde ( ehrlich 's reagent ) in glacial acetic acid was added .
after 10 minutes at room temperature , the extinction was determined at 492  nm with a microplate reader ( tecan , crailsheim , germany ) .
in some experiments ido was induced by coculturing msc with okt3-stimulated peripheral blood lymphocytes for three days . as a control 1-l - mt ( 1.5  mm ) or a neutralizing anti - ifn- antibody ( 10  ng / ml ) was added at the time point of msc stimulation .
in addition , ido protein was detected in stimulated msc using western blot analysis as described .
1  10 peripheral blood lymphocytes ( pbl ) , obtained from heparinised blood of healthy donors after ficoll purification , were stimulated with a monoclonal anti - cd3 antibody ( okt3 , american type culture collection , rockville , usa ) in the presence of different amounts of msc as described . in some experiments
msc ( 0.52  10 per well ) were infected with cmv and/or stimulated with ifn- at the start of the culture .
after three days the cultures were pulsed with 0.2  ci [ h ] thymidine for 24 hours .
t - cell proliferation was measured by [ h ] thymidine incorporation using liquid scintillation spectrometry ( 1205 betaplate , perkinelmer , jugesheim , germany ) .
bacterial growth was monitored after further incubation of 16  h by measuring optical density at 620  nm . all data are given as mean  sem of at least 3 independent experiments and each experiment was performed in triplicate .
data of representative experiments , also performed in triplicate , were given as mean  sd . for the comparison of different data student 's t - test for unpaired groups was used and the p value was calculated using graphpad prism software .
the profound t - cell inhibitory capacity of human multipotent mesenchymal stromal cells has raised much interest promting studies investigating msc as a novel cellular immunosuppressant , in particular in steroid - refractory gvhd after hsct . to explore the potential impact of cmv infections on the therapeutic efficacy of msc in this setting
, we set up a three - party cell culture system , in which okt3-induced t - cell proliferation was assessed in the presence or absence of human msc that were infected with cmv at a multiplicity of infection 5 ( moi 5 ) or left untreated .
as shown in figure 1 we found a profound t - cell inhibitory effect of msc , which was partially but significantly reversed in the presence of cmv .
this effect , however , did not rely on cmv - directed t - cell responses as cmv - infected msc did not induce any t - cell proliferation in the absence of okt3 stimulation ( figure 1 ) .
in addition , t cells from our donors did not proliferate in the presence of cmv and cmv did not significantly alter the t - cell response after stimulation with anti - cd3 antibodies as shown in figure 2(a ) .
we have previously shown that the induction of ido is , at least in part , responsible for the reduced t - cell proliferation induced by msc . to prove this finding we used the ido inhibitor 1-l - methyl - tryptophan ( 1mt ) .
as shown in figure 2(b ) human mscs inhibited t - cell responses and this inhibitory effect could be abrogated by the addition of cmv or of 1-mt . to substantiate these experimental findings
, we performed a series of 15 subsequent experiments employing various numbers of cmv - infected msc .
we found that 2  10 msc provided substantial suppression of t - cell proliferation and the addition of cmv consistently antagonizes this t - cell inhibitory effect . in all 15 experiments performed an inhibitory effect of cmv on msc - mediated t - cell suppression was observed ; however , the magnitude of this effect varied ( figure 3(a ) ) .
in contrast , 5  10 msc were unable to restrict t - cell proliferation and , under these conditions , the same amount of cmv that was employed in the experiments shown in figure 3(b ) did not have any impact on t cells , thus ruling out an unspecific effect of cmv .
taken together , this data demonstrates that cmv infection of human msc substantially impedes their t - cell inhibitory effector function . in previous studies
we and others have identified a significant role of the ifn--inducible tryptophan - catabolizing enzyme indoleamine-2,3-dioxygenase ( ido ) in msc - mediated t - cell inhibition [ 4 , 28 ] . in additional studies ,
the role of ido in tolerance induction was demonstrated in in vivo studies using ido deficient animals .
based on these findings we went on to assess the impact of cmv infection on cytokine - induced ido activity of human mcs . as shown in figure 4(a ) , we observed a substantial ido - mediated kynurenine production when msc were cultured in the presence of okt3-activated t cells . as expected , ido activity induced in msc by activated t cells could be blocked by neutralising antibodies directed against ifn- as well as by the ido - specific inhibitor 1-l - methyl - tryptophan .
however , it is of particular interest that cmv - infected msc were unable to express ido activity in the presence of activated t cells , while uv - inactivated cmv preparations had no impact on ido activity .
thus , an infection with replication - competent cmv substantially impairs ifn--induced ido expression in human msc .
in addition to their immunosuppressive capacity , msc have recently been shown to possess cell - autonomous antimicrobial effects directed against various clinically relevant pathogens [ 5 , 6 , 15 ] .
we therefore proceeded to analyse the potential impact of cmv on msc - mediated antibacterial effects induced by cytokines released from okt3-stimulated t cells .
as shown in figure 4(b ) , msc cocultured with activated t cells are able to restrict bacterial growth and cmv infection of msc abrogated their antimicrobial effect against staphylococcus aureus .
the functional relevance of ido - mediated antibacterial effects was confirmed by demonstrating that addition of excess amounts of tryptophan completely abolished the antimicrobial effector function of msc ( figure 4(b ) ) . in additional experiments we analysed cmv - mediated inhibition of
we found that cmv infection impairs cytokine - induced ido activity of human msc in a dose - dependent manner with significant inhibition observed at cmv doses as low as a moi of 0.6 and with a maximum effect at a moi of 5 ( figure 5(a ) ) .
furthermore , western blot analysis , depicted in figure 5(b ) , showed that cmv infection of msc results in a reduced expression of ido protein . in our present work
we describe for the first time that a cmv infection critically impairs the immunosuppressive and antimicrobial effector functions of human msc possibly via an interaction with the ifn--induced ido pathway .
we demonstrate that this interaction between cmv and ido - mediated effector functions of stromal cells critically depends on intact virus as well as the number of host cells and virus employed .
we have recently reported that human fibroblasts lose their immunosuppressive capacity after a cmv infection and that this finding might be relevant for host - versus - graft reaction disease triggered by cmv infection / reactivation after solid organ transplantation . here
we show that a similar cmv - mediated effect on msc might influence the clinical effectivity of these cells as immunosuppressant .
we are aware of the fact that our observations are derived from in vitro cell culture experiments . in the in vivo situation an immunosuppressive therapy , necessary in transplant patients , might result in an inhibition of ifn- production and therefore inhibit ido induction .
immunosuppressive substances such as glucocorticoides at high concentrations are able to enhance ido activity [ 31 , 32 ] .
furthermore the broad organ tropism , different cell types , and differences in cell tropism might have an impact on cmv - mediated inhibition of ido activation .
however , based on the species specificity of cmv infection as well as ido expression [ 5 , 34 ] , murine in vivo experiments including humanized xenograft models will unfortunately not be informative with regard to the in vivo impact of a cmv infection on msc - mediated immunosuppressive and antimicrobial effects .
we put a particular focus on msc in our study as these cells represent a promising novel therapeutic approach for a variety of clinical applications ranging from tissue engineering and regenerative medicine to the treatment of auto- and alloimmune diseases refractory to conventional pharmacologic immunosuppression [ 35 , 36 ] .
one might speculate that the inhibitory interaction of cmv with ido - mediated immunosuppressive effects may explain the consistent clinical observation that cmv infection frequently triggers gvhd following hematopoietic stem cell transplantation .
the findings presented here may have implications for transplantation medicine and the future clinical use of msc .
we found that msc and fibroblasts are permissive for cmv infection and are able to inhibit viral growth due to ido - dependent mechanisms .
we suggest that the balance between ifn- dose - dependent ido induction and cmv dose - dependent ido inhibition might influence the clinical outcome of organ or stem cell transplantation in cmv - infected patients .
we are aware that the magnitude of virus load and of ifn- concentrations in virus plaques within infected human tissues is unknown .
however we recommend that patients scheduled for msc therapy should undergo thorough evaluation for an active cmv infection and receive cmv - directed antiviral therapy prior to administration of msc , if appropriate , since overt cmv infection of msc recipients may undermine the clinical efficacy of the msc treatment .
thus , strategies that aim at restoring ido expression in cmv - infected msc may prove beneficial in certain clinical scenarios .
further studies characterizing the distinct signalling pathways that are targeted by cmv and result in modulation of ido expression in msc are necessary to identify molecular targets for therapeutic intervention .
 human mesenchymal stromal cells ( msc ) possess immunosuppressive and antimicrobial effects that are partly mediated by the tryptophan - catabolizing enzyme indoleamine-2,3-dioxygenase ( ido ) . 
 therefore msc represent a promising novel cellular immunosuppressant which has the potential to control steroid - refractory acute graft versus host disease ( gvhd ) . 
 in addition , msc are capable of reducing the risk of infection in patients after haematopoietic stem cell transplantation ( hst ) . 
 recent data indicate that signals from the microenvironment including those from microbes may modulate msc effector functions . as cytomegalovirus ( cmv ) represents a prominent pathogen in immunocompromised hosts , especially in patients following hst , we investigated the impact of cmv infection on msc - mediated effects on the immune system . we demonstrate that cmv - infected msc lose their cytokine - induced immunosuppressive capacity and are no longer able to restrict microbial growth . 
 ido expression is substantially impaired following cmv infection of msc and this interaction critically depends on intact virus and the number of msc as well as the viral load . since overt cmv infection may undermine the clinical efficacy of msc in the treatment of gvhd in transplant patients , we recommend that patients scheduled for msc therapy should undergo thorough evaluation for an active cmv infection and receive cmv - directed antiviral therapy prior to the administration of msc . 
 
1. Introduction
2. Materials and Methods
3. Results and Discussion