cd8 cytotoxic t lymphocytes ( ctls ) play an essential role in combating viral infections ( zhang and bevan , 2011 ) . during activation by antigen - presenting cells ,
ctls integrate t  cell receptor ( tcr ) , co - stimulatory , and cytokine receptor signaling to fine - tune proliferation and differentiation and establish various effector cell subtypes characterized by the expression of different surface markers and cytokine production abilities ( marchingo et  al . , 2014 ) .
together , these mechanisms allow the generation of ctl responses that can defend the host organism during primary infection and provide protective immunity against reinfection .
primed ctls are able to detect viral peptides restricted to major histocompatibility complex class i ( mhc - i ) and establish a tcr - triggered immunological synapse with their targets to  secrete the content of their cytotoxic granules toward the infected cell ( dustin , 2008 ) .
the targeted secretion of several effector proteins , such as granzymes and perforin , triggers the cell - death machinery in the infected cell while leaving antigen - negative bystander cells intact ( lopez et  al . , 2012 ) .
furthermore , ctls secrete various cytokines that contribute to antiviral immunity . however , it remains unclear how important the contact - dependent killing of target cells is in relation to these indirect mechanisms of control .
the efficiency of ctl - mediated contact - dependent killing of  different cell types has been studied extensively in  vitro .
such studies have suggested that ctls can rapidly , serially , and even simultaneously kill multiple target cells within minutes ( wiedemann et  al . , 2006 ) .
however , assays of in  vitro killing have a limited capacity to reflect the situation of how ctls sense , reach , and interact with infected cells in a three - dimensional tissue in  vivo .
whereas many assays of in  vitro killing involve ctls and targets co - cultured in suspension or as cell pellets , access to infected cells is likely to be limited in the intact tissue in which only selected cells are infected with viruses . also , the extracellular matrix and bystander cells might exert multiple , often suppressive effects on ctl function ( zhang and bevan , 2011 ) .
in addition , in co - culture killing assays , ctls are brought passively together with target cells , whereas in  vivo killing requires active ctl sensing and migration ( germain et  al . , 2012 ) .
thus , it remains unclear how fast and how robustly virus - infected cells are killed by single ctls in different virus - infected tissues ( elemans et  al .
, 2014 , elemans et  al . , 2012 , hickman et  al . , 2015 , hogan et  al . , 2014 ) .
in the current study , we quantified ctl killing kinetics by two - photon microscopy in mice infected with murine cytomegalovirus ( mcmv ) or modified vaccinia virus ankara ( mva ) . to this end , we used ex  vivo two - photon imaging of explanted lymph nodes and in  vivo imaging of intact skin together with transgenic and natural ctls and virus - expressed functional reporter systems .
importantly , we found that not every contact between ctls and target cells led to a perforin - dependent ca flux and target - cell death . using datasets on single - cell tracking , we estimated the average per capita killing rates ( pckrs : the number of targets killed per ctl per day ) of transgenic and endogenous ctls that kill different types of cells infected with several strains and species of viruses .
in contrast to the conventional theory of  highly efficient  killing , our results consistently showed that pckrs in  vivo were overall limited to a value  of about 216 infected cells killed per ctl per day .
furthermore , we observed that viral mhc - i immune evasion strongly reduced ctl - mediated antigen - specific contact - dependent killing in  vivo .
finally , we showed that by increasing the probability of target - cell death after multiple encounters , ctls could cooperate during killing of virus - infected cells .
to determine killing kinetics of ctls in  vivo , we infected mice with mcmv reporter strains ( marquardt et  al . , 2011 ) .
mcmv-2d expresses the red fluorescent protein ( fp ) mcherry and a  secretable gaussia luciferase , whereas mcmv-3d additionally  expresses the ovalbumin ( ova)-derived siinfekl peptide epitope .
mcmv downmodulates surface mhc - i expression in infected cells , possibly leaving ctls incapable of recognizing their targets ( gold et  al . , 2004 , hansen et  al . , 2010 , krmpotic et  al . , 1999 ) .
we therefore also used mcmv-3d-vrap , which lacks m06 and m152 , two viral regulators of antigen presentation ( vrap ) genes interfering with mhc - i recognition ( ziegler et  al . , 1997 ) , to study the effect of mhc - i downregulation on ctl effector function . to determine mcmv tropism after subcutaneous ( s.c . ) footpad infection of c57bl/6 ( b6 ) mice with mcmv-3d , we used fluorescence microscopy to detect bright - mcherry - expressing cells in the draining popliteal lymph node at days 1 and 2 after infection .
all mcmv - infected cells lacked expression of cd45 and cd169 and were located below the subcapsular sinus floor on the afferent side of the lymph node , approximately 2030  m beneath the capsule ( figures 1a1e ) .
some mcmv - infected cells expressed the fibroblast markers podoplanin ( gp38 ) and er - tr7-antigen , but not the cell - adhesion molecule madcam ( data not shown ) . thus , the mcmv strains used in this study initially infect fibroblast- or pericyte - like stromal cells , but not macrophages or dendritic cells .
the number of mcherry virus - infected cells as determined by microscopy strongly correlated with the mcmv - encoded gaussia luciferase activity ( figure  1f and data not shown ) .
therefore , we used the number of intact virus - infected cells to determine infected cell densities throughout this study . taken together , different microscopy techniques allowed for a clear and unbiased quantification of  the density of intact virus - infected cells in defined micro - anatomical regions .
we tested the ability of ctls to directly detect and kill cells infected by different variants of mcmv after adoptive transfer of tcr - transgenic , fp - expressing , siinfekl - k - specific ot - i cells into b6 recipients .
these mice were immunized with siinfekl peptide and poly(i : c ) or with ova protein together with mva .
both procedures induced expansion and maturation of ot - i ctls within 46  days ( figures 2a and 2b ) .
the percentage of expanded cd44 ot - i ctls present in blood reliably predicted the number of ot - i ctls in lymph nodes 2  days later ( figure  2c ) , allowing the analysis of mcmv infection in mice harboring defined numbers of virus - specific ctls .
one day after mcmv-2d infection , ctls expectedly did not express the activation marker cd69 , and ex  vivo two - photon microscopy showed that ctls failed to attach to or kill infected cells ( figures 2d and 2e ; movie s1 ) . upon infection with mcmv-3d , ot - i ctls
strongly upregulated the activation marker cd69 but remained highly motile and established only a few contacts with infected cells .
consequently , most mcmv-3d - infected cells remained intact , even in the presence of high numbers of activated ctls ( figures 2d and 2f ; movie s1 ) .
in contrast , in lymph nodes with high ctl densities , most mcmv-3d-vrap - infected cells were killed within 24  hr after infection ( figure  2 g ; movie s1 ) .
together , these findings demonstrate that viral mhc - i immune evasion protects mcmv-3d - infected cells from ctl - mediated killing and that in the absence of viral immune evasion , high ctl densities correlate with local eradication of infected cells .
next , we characterized migration parameters of ctls within the first 1420  hr after infection .
compared to ctls in mock , mcmv-2d , or mcmv-3d infection , ctls that attacked mcmv-3d-vrap - infected cells showed significantly reduced track speeds ( figure  3a ) .
furthermore , ctls showed very low motility coefficients , a measure for ctl displacement over time , according to the random - walk - hypothesis framework ( beltman et  al . , 2009 ) .
also , ctl turning angles were increased during local confinement to regions with infected cells ( figures 3b3d ) .
these findings demonstrate that ctls migrate at low velocities and stay confined in small tissue volumes while recognizing virus - infected cells and that viral immune evasion significantly alters ctl migration behavior and killing of virus - infected cells .
mcmv-3d-vrap - infected targets usually remained intact after contact by a single ot - i ctl ( figures s1a and s1b ; movie s1 ) .
in contrast , when numerous ctls interacted simultaneously or  serially with mcmv-3d-vrap - infected cells , we frequently observed target cell disruption ( figure  4a ; movie s2 ) .
the killing process of the virus - infected cells took some time , and morphological changes were observed over 1040  min , long before the  target cells finally disappeared . typically , we observed initial  morphological disturbances followed by the formation of blebs and later by the shedding of larger portions , resembling apoptotic bodies , of the mcherry - labeled cell body ( figure  4a [ lower panel ] , figure  4b ) .
the cognate interaction between ctls and their targets rarely led to stable synapses with complete arrest of ctls .
instead , while contacting their targets , ctls remained motile with an instantaneous velocity of 24  m / min for periods of approximately 1015  min ( figure  4c ) before regaining velocity .
kinapses  observed between t  cells and dendritic cells presenting antigen with intermediate or low affinity during t  cell priming ( dustin , 2008 ) . throughout the manuscript , we will use the term  kinapse  to describe this type of migratory interaction between ctls and virus - infected cells during target - cell killing .
direct observation of ctl - target - cell interaction behavior and quantification of target - cell fate revealed that killed virus - infected cells experienced a median of 3.5 distinct ctl contacts , whereas surviving cells were rarely targeted ( 0 median contacts ; figure  4d ) .
killed targets encountered a cumulative median contact time of 50  min ( figure  4e ) .
individual contacts between ctls and surviving targets lasted 8.5  min ( figure  4f ) , and individual contacts between ctls and killed targets lasted for approximately 9.0  min ( median ; figure  4f ) .
these observations indicate that successful killing of infected cells is not simply determined by the duration of individual ctl contacts . when comparing the different mcmv strains , we observed that 1% and 8% of ctls established contacts with mcmv-2d- and mcmv-3d - infected cells , respectively , whereas 38% of the ctls contacted mcmv-3d-vrap infected cells ( figures 4 g and 4h ) .
furthermore , we found that virus - infected cells were usually disrupted within 2060  min ( but rarely within 10  min ) after  the first observed ctl contact ( figure  4i ) .
together , these findings indicate that induction and execution of pro - apoptotic signaling cascades in virus - infected cells does not ensue instantaneously after a single ctl contact but that the accumulation of multiple ctl contacts determines the fate of virus - infected cells . to study
ctl - mediated killing in another infection model , we generated two reporter strains of the poxvirus mva ( kremer et  al . , 2012 ) .
injection of mva - mcherry into the footpad led to  infection of cd169 subcapsular sinus macrophages in the draining lymph node ( data not shown ) . ot - i
ctls killed mva - ova - mcherry- but not mva - mcherry - infected cells ( figure  5a ; movie s3 ) .
as observed during mcmv-3d-vrap infection , ctls within the mva - ova - mcherry - infected lymph nodes showed reduced track speed ( figure  5b ) , antigen - triggered dynamic contacts ( figure  5c ) , and disruption of targets that depended on ctl density at the site of infection ( figure  5d ) .
to quantify ctl - mediated killing of the different virus - infected cells , we analyzed the expression kinetics of the mcherry reporter to test whether detection of all infected cells can be expected during the two - photon imaging time windows . in  vitro infection revealed stronger and faster mcherry expression by mva at 48  hr after infection ( figures s2a  s2d ) , whereas after 12  hr , both mva and mcmv were robustly detectable both in  vitro and in the infected lymph node ( figures s3a  s3d ; movie s4 ) .
furthermore , virus - specific ctls could already recognize their specific virus - encoded peptide at 8  hr after infection ( figures s3d and s3e ) .
thus , direct observation of single ctls and single virus - infected cells was feasible from 1224  hr after infection .
next , we used two - photon - microscopy - derived datasets from the different virus - infection models to calculate pckrs of ot - i ctls , i.e. , the theoretical average number of infected cells killed per ctl per day ( elemans et  al . , 2012 ) .
pckr values reported in the literature are highly variable and were calculated on the basis of indirect assays or on assays relying on adoptively transferred artificial targets ( elemans et  al . , 2012 , regoes et  al . , 2007 ) . on the  basis of our direct observation of endogenous virus - infected cells
killed , here we calculated pckrs of 4.8 ( median , mcmv-3d-vrap ) and 4.2 ( median , mva - ova - mcherry ; figure  5e ) .
notably , viral mhc - i immune evasion of mcmv-3d reduced the pckr to 0.0 ( median ) , thus resembling infection with mcmv-2d ( figure  5e ) . together
, direct visualization and quantification revealed that the ot - i ctl population showed a limited killing efficiency in different infection models and that viral mhc - i immune evasion dramatically reduced ctl killing efficiency in  vivo . to determine whether ctls generated from the endogenous pool of cd8 t  cells show killing efficiencies similar to those of tcr - transgenic cd8 t  cells , we generated ctls in wild - type or perforin - deficient b6 mice ( prf ) by intraperitoneal infection with mcmv-3d .
after 68  days , mcmv - specific ctls were isolated by tetramer staining ( altman et  al . , 1996 ) .
employing our previously developed technique of intralymphatic injection ( braun et  al . , 2011 ) , we delivered tetramer - enriched ( 60% purity ) or tetrameter - sorted ( 90% purity ) ctls or negatively enriched ctls ( depletion of cd62l and non - cd8 t  cells ) into the afferent lymphatic vessel draining toward the popliteal lymph node of mcmv-3d-vrap - infected mice .
one day after intralymphatic transfer , we found that the reduction of virus - infected cell numbers , i.e. , killing of targets , was dependent on the local number of b6 ctls ( figures 6a and 6b ) .
similar to b6 ctls , tetramer - sorted prf ctls showed the typical migration behavior during target cell attack but were unable to disrupt virus - infected cells ( figure  6c ; movie s4 ) .
to quantify ctl killing efficiencies after intralymphatic injection , we used a mathematical model to describe the killing of virus - infected cells at different ctl densities 024  hr after infection ( supplemental experimental procedures ; figure  6d ) .
this model makes no assumptions on ctl killing mechanisms and has been developed in accordance with published modeling approaches ( reviewed in elemans et  al . , 2012 , and regoes et  al . , 2007 ) .
furthermore , this model calculates average pckr values for the entire ctl population , and the results obtained can be directly compared to the counting approach described in figure  5 . applying this simple mathematical model , we calculated pckrs from 2.0 to 10.4 for tetramer - enriched , tetramer - sorted , or negatively selected polyclonal ctls ( figure  6e ) . in contrast
thus , pckrs obtained by intralymphatic transfer of in - vivo - primed ctls and mathematical modeling were in agreement with the pckrs obtained by real - time two - photon imaging , together arguing for a limited average pckr for ctls attacking virus - infected cells . because ctl killing efficiency might be different in a non - lymphoid organ , we next studied ctl killing in the dermis of t - cell - deficient mice ( cd3e ; rag2 ) .
these animals were reconstituted with fp - ot - i or fp - cd8 t  cells .
intraperitoneal virus infection was then used to prime and expand t  cells ( figure  s4a ) . in  vivo two - photon microscopy showed that 1  day after secondary s.c .
ear infection with mcmv-3d-vrap , but not after infection with mcmv-3d , ot - i ctls migrating around infected dermal fibroblasts were found to exhibit the typical dynamic scanning behavior characterized by low track speed and low motility coefficients ( figures s4b  s4d ) .
likewise , mice reconstituted with polyclonal cd8 t  cells and primed intraperitoneally with mcmv-3d displayed ctls characteristically scanning infected cells in the dermis , as well as local eradication of mcmv-3d-vrap - infected targets ( figures s4e  s4 g ; movie s5 ) . in the skin , we observed median pckrs of 16.0 and 12.5 for ot - i and polyclonal ctls , respectively ( figure  s4h ) .
thus , two - photon in  vivo imaging of the infected skin supports the conclusion that ctls show limited killing efficiency when attacking virus - infected stromal cells .
it is currently unclear whether all ctls specific to the same epitope show the same killing rate or whether some ctls kill more efficiently than others .
thus , we first tested whether all ctls that are in contact with infected cells show signs of activation . using ot - i ctls expressing retrovirally transduced nuclear factor of activated t  cells ( nfat)-gfp ( aramburu et  al .
, 1998 ) and h2b - morange reporter constructs , and assuming that cognate recognition results in nuclear nfat translocation within 13  min ( marangoni et  al . , 2013 )
, we addressed whether ctls in contact with infected cells can readily recognize the target .
after mcmv-2d or -3d infection , the nfat - gfp fluorescent signals remained in the ctl cytoplasm , which is in agreement with the absence of killing of these virus variants .
in contrast , after mcmv-3d-vrap infection , 80% of ctls in contact with infected cells  but also some ctls not in contact with targets  showed nuclear nfat - gfp ( figures s5a  s5d ; movie s6 ; data not shown ) .
thus , ctls were usually activated during target - cell contact . however , because ctls frequently retained the nfat signal in the nucleus after target disengagement ( marangoni et  al . , 2013 ) ,
perforin pores in the plasma membrane have been suggested to trigger a transient calcium ( ca ) flux in the target cell ( keefe et  al . , 2005 ) .
to gain further insights into the functional heterogeneity of ctls , we therefore qualitatively and quantitatively analyzed ctl - induced ca influx in infected targets . to study ca fluxes in virus - infected cells , we replaced the luciferase - encoding sequence of mcmv-3d and mcmv-3d-vrap with a sequence encoding the ultra - sensitive ca sensor gcamp6s ( chen et  al . , 2013 ) , resulting in the reporter viruses mcmv-3d - ca and mcmv-3d-vrap - ca , respectively ( figures s6a  s6c ) .
after footpad infection of non - immunized b6 mice with mcmv-3d - ca or mcmv-3d-vrap - ca , most infected cells showed spontaneous short ca fluxes that lasted on average 6  s ( median ; figures 7a7d ; movie s7 ) . after infection with mcmv-3d-vrap - ca , ot - i
ctls dynamically interacted with and triggered long - lasting ca fluxes in infected targets ( figures 7e7 g ; movie s7 ) .
ctl - induced ca fluxes lasted for 80  s ( median ; interquartile range [ iqr ]  = 40240 s ; figures 7e7 g ) and started 480  s ( median ) after ctl encounter ( iqr  = 60820 s ; figure  7h ) .
although ctls intensively contacted infected cells , 40% failed to induce a long - lasting ca flux ( figure  7i ) , and only 10% of ctls induced three or four death - associated ca fluxes ( figure  7j ) .
perforin - deficient ctls failed to elicit long - lasting ca fluxes and cell death ( figure  7k ; figure  s6d ) .
together , these findings reveal that individual ctls and ctl contacts targeting virus - infected cells show strong functional heterogeneity .
because single ctl contacts frequently failed to trigger long - lasting ca influx or death of target cells , we next addressed whether ctls cooperate while killing virus - infected cells . in all of the above - described imaging models ,
virus - infected cells were disrupted more frequently when they were contacted by several rather than single ctls . to test for ctl cooperativity , we choose a null hypothesis that assumed that every ctl contact is an independent event and that previous contacts do not influence the target cell s death probability . this bernoulli - series null hypothesis states that the probability p(n ) that a target cell  will die after n interactions is given by the term p(n )  = 1  ( 1  
data from 660 individual mcmv-3d-vrap - infected target cells were grouped according to the total number of ctl contacts observed per target cell .
targets without any observed ctl interactions died with a probability of p(0 )  = 0.01 , whereas targets with a single or two ctl contacts died with a probability of p(1 )  = 0.15 or p(2 )  = 0.28 , respectively . in the case of three or five ctl contacts , the probability that an infected cell would die was significantly higher than predicted from the null hypothesis of independent interaction outcomes ( figures 7l and 7 m ) .
thus , ctls were able to cooperate to kill virus - infected cells by increasing the probability of target cell death after multiple ctl encounters .
in the present study , we visualized how ctls killed virus - infected cells in  vivo .
injection , infected draining lymph node stromal cells and subcapsular sinus macrophages , respectively . because all reporter viruses encoded mcherry , we could clearly identify single infected cells and use time - lapse ex  vivo and in  vivo two - photon microscopy to determine their fates after they were contacted by effector cd8 t  cells .
two - photon imaging revealed that in most situations , migrating ctls do not come to a complete arrest to establish a static synapse with their target .
stable and static synapses have been described in lymph nodes ( 1 ) in  vivo between antigen - presenting cells and t  cells during defined stages of t  cell priming and ( 2 ) in  vitro on coated surfaces between ctls and targets ( mempel et  al . , 2004 , ritter et  al . , 2015 ) .
as described for static synapses , the formation of dynamic kinapses observed in the present study also relied on the cognate interaction between tcr and mhc - i - presented antigen , because kinapses do not form between ot - i ctls and cells infected with mcmv-2d ( no cognate antigen ) or mcmv-3d ( low surface mhc - i expression ) . among other factors ,
the formation of static immunological synapses has been shown to depend on integrin activation , which allows firm adhesion and complete arrest of  migrating cells ( liu et  al .
interestingly , treatment of mice with neutralizing antibodies against 1- and 2-integrin or injection of ctls lacking the tcr adaptor protein adap , involved in tcr signaling leading to integrin activation , did not  show differences in the slow but dynamic ctl migration behavior during the attack on infected cells or during the faster migration when ctls were not contacting infected target cells ( figure  s7 ) .
therefore , integrin - independent formation of dynamic kinapses might represent a mechanism that not only allows ctls to deliver the content of their cytotoxic granules but also keeps them in a motile state to successfully search for further targets . by recruiting effector cells to the site of infection , chemokine receptors such as cxcr3 and ccr5 have been shown to contribute to ctl function ( hickman et  al . , 2015 , kastenmller et  al . , 2013 ) .
in the present study , we observed no significant difference between wild - type and cxcr3- or ccr5-deficient ctls regarding killing efficiencies in the intralymphatic transfer model ( data not shown ) .
these findings indicate that the mode of ctl delivery , the strain of virus investigated , and the cell type infected might affect the differential requirement for chemokine receptors for ctl recruitment . in our study ,
ctl attack on virus - infected cells resembled the behavior of nk cells attacking tumor cells ( deguine and bousso , 2013 ) .
in addition to assessing cell disruption on the basis of changes in cell morphology , we used the genetic ca sensor gcamp6s expressed in the target cell to better understand ctl - mediated killing .
earlier in  vitro studies have suggested that perforin pores in the plasma membrane of target cells cause a transient ca influx that is sensed by the cells as a sign to repair plasma - membrane damage ( keefe et  al .
in virus - infected cells , expression of gcamp6s proved a sensitive and reliable indicator of ca fluctuations . in the absence of natural killer cells and ctls , virus - infected cells showed spontaneous short ca fluxes ( data not shown ) , whereas ctl - induced fluxes could last for several minutes and reliably indicated subsequent cell death .
our study revealed that , on average , 8  min elapsed between ctl contact and ca influx , whereas subsequent cell disruption occurred in most situations between 20 and 120  min after the first ctl contact . in one study , in  vivo imaging revealed killing of lymphoid cells within 1020  min of ctl contact ( mempel et  al . , 2006 ) , whereas other studies failed to observe rapid killing of virus - infected keratinocytes , malaria - infected hepatocytes , or tumor cells ( breart et  al .
, 2008 , cockburn et  al . , 2013 , hickman et  al . , 2013 ) .
notably , when ctls attack peptide - pulsed b cells , additional b cell surface molecules ( such as slamf7 ) could strengthen the ctl attachment and affect ctl behavior during killing .
together , the specific tropism of the pathogen and the targeted cell type essentially contribute to the outcome of ctl - mediated immune reactions . the control of virus infection is critically determined by the quality of the ctls generated ( plotkin , 2013 , varadarajan et  al . , 2011 ) .
ctls can be heterogeneous with regard to the expression of effector molecules as well as the expression of co - stimulatory or inhibitory surface molecules ( jenkins et  al .
the data shown here also reveal that ctls are heterogeneous with regard to nuclear translocation of the transcription factor nfat after cognate antigen recognition .
furthermore , ctls are highly diverse with regard to their ability to induce death - associated ca influx in target cells ; for example , in movies with an average duration of 66  min , 40% of the ctls were unable to trigger ca influx , whereas 10% induced three or four ca fluxes .
the local presence of antigen - specific regulatory t  cells can affect ctl killing efficiency ( mempel et  al .
the presence of immune - suppressive cells might also explain why intralymphatically delivered tetramer - enriched cells kill slightly less efficiently than very pure tetramer - sorted ctls .
two - photon microscopy allowed us to analyze the fate of single infected cells with regard to their ctl - contact history .
this approach revealed that ctls exhibited cooperativity in killing targets when more than two ctls contacted the infected cell .
the underlying mechanisms for this observation are unclear but could be explained by the differential expression levels of granzymes and perforin ( and potentially other molecules ) in individual ctls ( jenkins et  al . , 2008 ) .
two ctl contacts did not result in detectable signs of cooperativity , maybe because the effect of two ctl contacts is too small to be revealed in the assay setup used .
in general , if we assume the existence of ctls with a high or low killing capacity , it is plausible that multiple contacts increase the probability that a fully armed ctl actually disintegrates the infected cell . alternatively or additionally , it seems possible that the accumulation of sub - lethal pro - death signals delivered by different single ctls might lead to faster apoptosis of the target .
the average killing efficacy of individual ctls crucially affects the outcome of infectious diseases relying on ctl - mediated killing as a main control mechanism .
there is currently no consensus on even the order of magnitude of in  vivo ctl killing rates in either experimental models or human diseases ( elemans et  al . , 2014 , elemans et  al . , 2012 , hickman et  al . , 2013 ) .
estimated killing rates of mouse and human ctls vary considerably , and it is unknown how much the different viruses studied , the vaccination protocols applied to ctl generation , or the killing assays used affect the killing estimates ( garcia et  al . , 2015 ) . further complicating matters
, the definition of killing rates often relies on different rate constants , not taking into account the number of ctls that might be responsible for the killing ( ganusov and de boer , 2008 ) .
we therefore chose to calculate the average number of virus - infected cells killed per ctl per 24  hr as a simple measure of the  ctl - mediated killing to allow direct comparison between different models .
we determined killing rates either by directly observing disintegration of infected cells over time by a defined ctl population size or by determining the ratio of ctls and infected cells at given time points after infection .
the pckrs calculated by these approaches give an average value that describes the killing efficiency of the whole ctl population ( and not of individual ctls ) without assumptions about ctl cooperation or the percentage of non - active ctls .
strikingly , our data show a consistent range of 216 infected cells killed per ctl per day , irrespective of the investigated epitopes recognized by the ctl , the method of calculation ( counting or mathematical modeling ) , the type of cell infected ( macrophages or stromal cells ) , or the virus used ( mva or mcmv-3d-vrap ) . because two - photon imaging can not detect killing events that might occur before imaging is started
, the pckr values from direct imaging - based counting might slightly underestimate killing efficiencies .
however , killing of  non - visible  cells does not affect our mathematical model , because the time point of killing is not important in this experimental setup .
both direct counting and mathematical modeling might have limitations , but all pckr estimates obtained here ( from different viruses infecting different cells , different organs , and by different methods ) are consistent in that they range from 2 to 16 target cells killed per ctl per day .
it remains unknown for how long a single ctl can maintain a certain killing rate , and it will be interesting to measure whether single ctl killing rates increase or decrease after sequential target - cell encounter . all together , our data indicate that the average in  vivo ctl killing capacity is rather limited and not in agreement with the assumption that all ctls act as 
rapid  and  serial  killers that destroy hundreds or thousands of targets per day ( elemans et  al . ,
this can possibly explain the failure of some ctl - based vaccines ( plotkin , 2013 , yewdell , 2010 ) or the development of chronic viral infections ( west et  al . , 2011 ) ,
in which functional ctls are initially present but fail to eradicate all virus - infected cells .
all mcmv strains were derived from the psm3fr smith strain ( cloned as a bacterial artificial chromosome ) and were produced and titrated in  vitro according to standard techniques ( marquardt et  al . , 2011 ) .
gcamp6s - expressing mcmv strains were designed with plasmid pgp - cmv - gcamp6s ( chen et  al . , 2013 ) obtained from addgene ( plasmid 40753 ) .
mva - mcherry and mva - ova - mcherry were generated by homologous recombination using the plasmid vectors piiidhr - p7.5 and plw-73 according to standard procedures .
c57bl/6 ( b6 ) mice were bred at the central animal facility at hannover medical school under specific - pathogen - free conditions or purchased from charles river or the jackson laboratory ( jax ) .
the following mutant b6 mouse strains were used : c57bl/6-tg(cag - egfp ) ( jax 003291 ) mice expressing gfp under an artificial cmv - actin - globin promoter ( here named gfp ) , c57bl/6-tg(cag - ecfp ) mice expressing cfp under the beta - actin promoter ( jax 004218 ) , tcr - transgenic ot - i cd45.1 mice ( derived from c57bl/6-tg(tcratcrb)1100mjb / j ; jax 003831 ) , f1-cross between gfp and ot - i cd45.1 ( named fp - ot - i ) , adap mice ( c57bl/6j fyb  b6.pl-thy1a/cyj  b6-tg(tcratcrb)1100mjb ) , cd3e mice ( c57bl/6-cd3etm1mal / orl ) , prf1 mice ( jax 002407 ) , and rag2 mice ( jax 008449 ) .
all experiments were conducted in accordance with the local animal welfare regulations reviewed by the institutional review board and the lower saxony state office for consumer protection and food safety ( laves ) .
cd8 t  cells from ot - i mice , containing 10 cd8 tcr v5 ot - i cells , were transferred into recipient mice .
two to six hours after mcmv-3d-vrap infection , 2.5   10 to 1   10 ctls diluted in 5  l pbs were injected in 90  s into the afferent lymph vessel draining toward the popliteal lymph node , as described before ( braun et  al . , 2011 ) .
ex  vivo two - photon imaging was performed with custom - build chambers for  imaging explanted lymph nodes , as described in halle et  al .
the skin at the site of infection in the ear of anesthetized mice was imaged directly in  vivo .
we calculated the number of infected cells killed per t  cell per day on the basis of the observed time course of the number of killed virus - infected cells and the average number of ctls in two - photon microscopy movies .
data from independent experiments were pooled , and average killing rates were reported . alternatively , we used the single - time - point imaging data from the intralymphatic injection together with the mathematical model to calculate average ctl killing rates and 95% confidence intervals .
statistical analysis was performed with graphpad prism 4 . when comparing two groups , we calculated p values with the nonparametric mann - whitney test . to compare multiple groups , we used the kruskal - wallis test with dunn s test .
a. braun . , k. werth , and r.a . performed the experiments ; k.a.k .
, h.k . , and m.m .- h . designed and applied the mathematical models ; and s.h . and r.f .
 summaryaccording to in  vitro assays , t  cells are thought to kill rapidly and efficiently , but the efficacy and dynamics of cytotoxic t lymphocyte ( ctl)-mediated killing of  virus - infected cells in  vivo remains elusive . 
 we used two - photon microscopy to quantify ctl - mediated killing in mice infected with herpesviruses or poxviruses . on average , 
 one ctl killed 216 virus - infected cells per day as determined by real - time imaging and by mathematical modeling . 
 in contrast , upon virus - induced mhc class i downmodulation , ctls failed to destroy their targets . during killing , ctls remained migratory and formed motile kinapses rather than static synapses with targets . 
 viruses encoding the calcium sensor gcamp6s revealed strong heterogeneity in individual ctl functional capacity . 
 furthermore , the probability of death of infected cells increased for those contacted by more than two ctls , indicative of ctl cooperation . 
 thus , direct visualization of ctls during killing of virus - infected cells reveals crucial parameters of cd8 + t  cell immunity . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions