throughout life , we are constantly challenged by pathogens , and re - exposure to previously encountered viruses or bacteria happens frequently . homologous re - infections are often effectively controlled by neutralizing antibodies , but pathogens that express altered serological epitopes ( heterosubtypic re - infection ) can bypass antibody - mediated immunity and cause a secondary infection ( corti and lanzavecchia , 2013 ) . right at the beginning of such a re - infection , the t  cell repertoire may already contain large numbers of pathogen - specific t  cells , and some of these can immediately exert effector function ( zhang and bevan , 2011 ) . moreover , preexisting pathogen - specific but insufficiently neutralizing antibodies could strongly alter infection and replication kinetics ( beura et  al . , 2016 ) of a pathogen , and this may significantly alter tissue tropism ( rothman , 2011 ) . it has been shown that cells of the innate arm of the immune system can respond more vigorously ( sun et  al . , 2009 ) , and memory cd8 t  cells can even contribute to controlling pathogen load in the early phase of infection in a non - cognate , innate - like fashion ( chu et  al . , 2013 ) . thus , pathogen spreading , antigen presentation , t  cell activation kinetics , and levels of inflammation and tissue destruction can significantly vary between primary and secondary infections . a particular feature of heterosubtypic re - infections is that many antigens are shared in the primary and secondary infection , but the second pathogen expresses epitopes , which are new to the immune system . such a situation of preexisting partial immunity to a pathogen can also occur in a primary infection when a pathogen happens to share an epitope with a previously encountered unrelated pathogen  a phenomenon known as heterologous immunity ( welsh et  al . , 2010 , che et  al . , 2015 ) . though highly relevant for understanding immunity in humans , interferences between past and present infections ( or vaccinations ) are rarely investigated under well - defined conditions . thus , we still have very limited insight into how existing immunity impacts t  cell activation , expansion , and conversion into effector and memory t  cells . similarly , pre - existing partial immunity can significantly alter the outcome of vaccination ( frahm et  al . , 2012 ) . it is also particularly important to consider that pre - existing immunity does not inevitably result in better immune protection . instead , certain re - infections are known to be accompanied by enhanced pathology , as occurs when a dengue - virus - immune individual is exposed to a different virus serovar ( rothman , 2011 ) . similarly , certain vaccination trials are suspected to have augmented the severity of subsequent infections ( blanco et  al . , 2010 , moore et  al . , 2008 ) . the mechanisms underlying such disease enhancement are not clearly resolved , and it is not known how frequently this may occur . all of these points strongly underline the need to investigate immune responses in immune or partially immune individuals . here , we utilize well - controlled experimental systems to dissect and characterize how different levels of pre - existing immunity influence t  cell responses . we observed that t  cell activation thresholds differ substantially between primary and secondary infections . we report that only the highest - affinity ligands induce t  cell expansion in heterosubtypic re - infections , whereas minor reductions in the levels of tcr stimulation fail to induce t  cell expansion . importantly , we show that neither a shortened inflammatory response nor a globally altered antigen presentation pattern are responsible for raising the threshold . instead , we found that even a single shared epitope recognized by previously formed memory cd8 t  cells is sufficient to increase the t  cell activation threshold of naive t  cells . we noted that this elevated threshold is mediated by a non - transferrable endogenous memory t  cell population . together our data provide relevant insights into t  cell differentiation mechanisms in secondary heterosubtypic infections and for the design of vaccines that are given multiple times . we previously reported that low - affinity t  cells differentiate into effector and memory cells in primary infections ( zehn et  al . , 2009 ) . we established this using recombinant listeria strains , which express full - length ovalbumin [ ova ] that contains either the original high - affinity , h-2k - restricted ot-1 ligand siinfekl ( ova357364 ) or different altered peptide ligands ( apls ) a2  > y3  > q4  > t4 apl ( listed in decreasing ot-1 stimulatory potency ) . we then became interested in addressing the stimulation requirements in secondary heterosubtypic re - infections . to do so , we infected naive or wild - type listeria ( lm - wt)-experienced mice with lm - ova- or apl - expressing listeria ( see figure  1a ) . with this experimental setup , we mimic a frequently occurring situation where an individual is immune to some but not all antigens in a secondary infection . we observed that the high - affinity ot-1 ligand n4 induces similar expansion in mice with or without a previous lm - wt immunization . in contrast , ot-1 expansion in response to listeria expressing the slightly weaker sainfekl ( a2 ) or siynfekl ( y3 ) apl was decreased in lm - wt immune mice ( figure  1b ) and significantly impaired in response to q4 or t4 . similar exclusions of low - affinity t  cells were observed when we transferred ot-1 t  cells before the primary lm - wt infection ( figure  s1a ) and when memory ot-1 t  cells were used instead of naive ot-1 t  cells ( figure  s1b ) . notably , both n4 and t4 induce similar carboxyfluorescein succinimidyl ester ( cfse ) dilution in primary infections , but only n4 induces robust proliferation in immune mice ( figure  1c ) . some ot-1 t  cells proliferate in response to t4 in listeria - immune mice , but this did not increase total ot-1 t  cells ( data not shown ) . we therefore conclude that low - affinity stimulation leads to an abortive t  cell response in heterosubtypic re - infections . to illustrate the relevance of our observation , we transferred low - affinity h-2k / ova - specific ot-3 tcr transgenic t  cells ( enouz et  al . , 2012 ) into naive or lm - wt immune rip - mova mice . the lm - wt immune mice were then infected with the same challenge as in figure  1a , whereas the naive rip - mova mice received a priming dose . using this setup , only naive mice showed diabetic blood glucose levels above 300  mg / dl ( figure  1d ) . next , we wanted to define the difference in the affinity range of t  cells responding in primary versus heterosubtypic infections . we previously showed that the low - affinity v4 ligand activates ot-1 t  cells ( zehn et  al . , 2009 ) , even though it has a 1,000-fold lower ec50 than the n4 ligand ( figure  2a ) . interestingly , the d4 apl has an ec50 value that is 10,000-fold below n4 . to test the ot-1 t  cell response to this very low - affinity ligand we confirmed that the lm - d4 strain is infectious and comparable to lm - n4 ( figure  s2a ) . unexpectedly , even lm - d4 induced rapid initial ot-1 proliferation ( figure  2b ) . d4 activated ot-1 t  cells secrete tnf  and ifn ( figure  2c ) and produce granzyme b ( figure  2d ) , and they differentiate into memory t  cells that can undergo secondary expansion ( figure  2e ) . we illustrated this by transferring ot-1 cells into mice that were either infected with lm - d4 or with lm - wt . four weeks later , we challenged the mice with vesicular stomatits virus expressing ova ( vsv - ova ) . we noticed higher numbers of ot-1 cells in mice primed by d4 in comparison to lm - wt - primed mice that do not stimulate ot-1 t  cells ( figure  2e ) . t  cells indicated that the signal 1 ( tcr stimulation ) activation requirement in primary infections is very low . in contrast , the difference between the normal n4 and the a2 or y3 apl is rather small ( figure  2a ) . nonetheless , this level already strongly impairs t  cell expansion in secondary or cross - reactive infections . we therefore conclude that there is a substantial stimulation threshold difference between primary and secondary infections . to further illustrate the significance of this difference , we marked the known boundaries for ligands which induce positive and negative selection of ot-1 t  cells in figure  2a , whereby t4 is the threshold ligand between positive and negative selection ( figure  2a ) ( daniels et  al . g4 is known to induce positive selection in fetal thymic organ cultures ( yachi et  al . , 2006 ) . given that d4 with a potency much below g4 supports effector t  cell differentiation , we wondered whether even the presumed endogenous positive selecting ligands for ot-1 , isfkfdhl from the f - actin capping protein ( cp1 ) and rtytyekl from -catenin ( hogquist et  al . 2002 ) would drive ot-1 expansion when expressed by listeria , but this was not the case ( figure  2f ) . we confirmed that the lack of an ot-1 response is not due to a lack of bacterial replication and that an lm-cat infection is , in this respect , comparable to an lm - n4 infection ( figure  s2a ) . e1 ova ( eiinfekl ) is the only known apl that supports ot-1-positive selection in  vivo ( stefanski et  al . , 2001 ) . the lack of a peripheral response to e1 ( figure  2f ) suggests that it more closely resembles the quality of ligands ( cp1 and -catenin ) that are thought to naturally support positive selection . given the activation threshold differences in primary versus heterosubtypic infections , we sought to define the responsible mechanism . depletion of cd4 t  cells and nk cells from lm - wt primed mice did not restore low - affinity t  cell activation ( data not shown ) . taking a possible role of listeria - specific memory cd8 t  cells into consideration , we set up a system that allowed us to control the size of the antigen - specific memory population by sharing only one cd8 t  cell epitope between two consecutive infections . we constructed listeria strains that express n4 or t4 plus the lymphocytic choriomeningitis virus ( lcmv)-derived gp33 - 41 epitope . we found that lm - gp33-n4 and lm - gp33-t4 have similar in  vivo growth rates ( figure  s2a ) and induce similarly sized populations of endogenous gp33-specific t  cells ( data not shown and figures s2b and s2c ) . t  cells are primed by these listeria strains in naive or lcmv immune hosts ( figure  3a ) , we observed a 2-fold reduction in ot-1 expansion to n4 and an essentially non - detectable response to t4 in lcmv immune mice ( figure  3b ) . this indicates that  pre - existing antigen - specific memory cd8 t  cells effectively raise the t  cell activation threshold and that only one epitope ( or two if one also considers the h-2k - restricted gp34 epitope ) ( hudrisier et  al . , 1997 ) we considered that rapid listeria clearance in immune hosts  might shorten the duration of antigen - presentation and  lower the magnitude of concomitant inflammation ( zehn et  al . , 2014 , prlic et  al . , 2006 ) . to address this point , we transferred cfse - labeled ot-1 t  cells into naive or lm - wt - primed mice and challenged them with lm - n4 . we observed similar cfse dilution in naive and immune hosts excluding major differences in antigen presentation kinetics in the two hosts ( figures 3c and 3d ) . also anti - cd40-mediated dc maturation or administration of recombinant ifn to increase inflammation left the t4 response unchanged ( data not shown ) . next , we established comparable levels of listeria induced inflammation and tissue destruction in lcmv immune and naive mice by co - injecting lm - wt and lm - gp33-t4 into lcmv immune mice . here , only lm - gp33-t4 are recognized by memory t  cells , whereas the lm - wt infection propagates normally . this resulted in similar absolute listeria titers in lcmv immune and naive mice , but the response to t4 remained unchanged in immune mice ( figure  3e ) . to control for an impact of the different listeria challenge doses in naive or immune mice ( figures 1 , 3a , and 3b ) , we used acta - deficient listeria . these strains can not spread from cell to cell and are rapidly cleared by the host , allowing c57bl/6 mice to be challenged with a high listeria dose . this creates a setup in which antigen levels are primarily dependent on the amount of the initial bacterial challenge dose , but not the in  vivo expansion , of injected bacteria . we observed a lack of low - affinity t  cell expansion in immune mice challenged with acta - deficient lm ( figure  3 g ) . this excludes a principle shortage in antigen presentation and a lower inflammatory response as causes of the observed effect . to determine whether the presence of memory t  cells is sufficient to raise the activation threshold , we transferred gp33-specific memory t  cells into new c57bl/6 hosts along with cd45.1/2 congenic ot-1 t  cells . contrasting our prior observation , the transferred memory p14 t  cells reduced , but did not completely suppress , the ot-1 response to lm - gp33-t4 . in an lm - gp33-n4 infection , the same p14 dose caused only a minor impact on the ot-1 response ( figure  4a ) . similar outcomes were  observed when p14 memory t  cells were transferred 28 or 80  days after the lcmv infection ( figure  s3a ) . we considered the possibility that the number of transferred p14 t  cells was too low to suppress the t4 response effectively and therefore increased the number of transferred p14 to 10  cells . again , the t4 response was incompletely blocked , but surprisingly , this p14 dose already caused a strong decline in the high - affinity ot-1 response ( figure  4b ) . these data suggest that transferring memory t  cells is not sufficient to recapitulate the selective and complete exclusion of low - affinity t  cells , as observed when endogenous memory t  cells respond to a pathogen expressing a shared antigen ( figure  1 ) . to exclude a sole ot-1-dependent phenomenon , we reversed the strategy and used the recently described gp33c6 low - affinity apl for p14 t  cells ( utzschneider et  al . , 2016 ) . we transferred ot-1 memory and naive p14 t  cells into naive b6 mice and infected them with listeria expressing ova and wild - type ( lm - gp33-n4 ) or the low - affinity apl gp33c6 ( lm - gp33c6-n4 ) . again , the memory ot-1 had a minor impact on the p14 response to high - affinity ligands and a much stronger impact on the lower - affinity gp33c6 response ( figure  s3b ) . these data also show that a weak or strong response of de novo primed naive ot-1 or p14 t  cells does not impact the response of memory p14 ( figure  s3a ) or ot-1 t  cells ( figure  s3b ) . we also examined the phenotype of p14 t  cells before ( endogenous situation ) and after the transfer and noticed a larger population of klrg1 t  cells among endogenous compared to  transferred memory t  cells and a higher proportion of cd43cd27 cells ( figures 4c and 4d ) . both populations are thought to have immediate effector function ( olson et  al . , 2013 ) . moreover , listeria monocytogenes enters the spleen via the bloodstream and in a dendritic cell dependent fashion ( neuenhahn et  al . , 2006 ) . at this stage , they will likely be in contact with effector / memory t  cells that are positioned in the spleen in close proximity to the bloodstream . by injecting fluorescently labeled anti - cd8 antibody into mice , we marked the fraction of p14 t  cells that are in close contact with blood ( galkina et  al . , 2005 ) . interestingly , but in line with an earlier publication ( olson et  al . , 2013 ) , we saw that the klrg1 or cd43cd27 t  cells ( figure  4e ) , which show a low engraftment efficacy in our memory transfers , were enriched among the p14 cells with access to the blood stream . therefore , we favor the conclusion that a form of tissue - resident t  cell population is more effective than transferred t  cells in raising the activation threshold in heterosubtypic infections . we show a clear difference in the tcr stimulation threshold between primary and secondary infections . only very high - affinity ligands activate t  cells in immune mice , whereas ligands such as a2 and y3 , whose ec50 for inducing a half - maximum ifn response differed only 2- to 4-fold from the wild - type n4 peptide ( figure  2a ) , trigger a much weaker response in immune compared to naive mice . this is in contrast to the situation in naive mice in which even ligands with 10,000-fold lower ec50  activate ot-1 t  cells ( figure  2a ) . our observations help to understand t  cell responses in heterosubtypic re - infections caused for instance by different influenza strains or dengue virus serotypes . serial infections with the distantly related lymphocytic choriomeningitis virus and pichinde virus were shown to induce a narrowed pathogen - specific t  cell repertoire because of antigens that are shared between these infections ( cornberg et  al . , 2006 , welsh et  al . , 2010 ) . our data indicate that this exclusion is caused by a memory t - cell - mediated elevation of the stimulation threshold and that even minimum cross - reactivity is sufficient to cause this effect . the exclusion of low - affinity t  cells by previously established memory t  cells has consequences for designing vaccine strategies that rely on a prime - boost regimen and intend to elicit a broad t  cell repertoire ( such as hiv vaccine trials ) . several examples underline that optimum protection against pathogens is mediated by highly diverse t  cell populations . this ensures cross - reactivity to pathogens expressing mutated epitopes , and it enables individuals to better handle a heterosubtypic infection with a related but non - identical pathogen . it also prevents the selection of escape variants ( price et  al . , 2004 , meyer - olson et  al . , 2004 , t  cell receptor diversity in the population of antigen - specific t  cells is largely facilitated by expanding t  cells , which suboptimally respond to antigen ( low - affinity t  cells ) ( zehn et  al . , 2009 ) . the focusing of the t  cell repertoire as we observed it may be beneficial for the ongoing secondary infection , but the narrow repertoire may severely diminish the ability to subsequently cross - react to related antigens . similarly , tumor immune responses rely on recruiting t  cells of intermediate or low affinity given that high - affinity t  cells are often eliminated by tolerance mechanisms ( enouz et  al . , 2012 ) . our data imply that vaccination strategies , which involve repetitive injections , will perform suboptimally if the vaccine vector contains previously experienced epitopes . in addition , pre - existing immunity to vaccine vectors were shown to strongly diminish the efficacy of inducing t  cell immunity . specifically , adenovirus - seropositive vaccine recipients had lower hiv - specific responses to an adenovirus 5-vectored hiv vaccine in comparison to adenovirus - seronegative recipients ( frahm et  al . , 2012 ) . in line with this previous study , our data show that a minimal epitope overlap is sufficient to impact not only the quantity but also the breadth of the recruited t  cell repertoire . in contrast , memory t  cells do not seem to impact the clonal - like expansion of antigen - specific nk cells ( johnson et  al . , 2016 ) . our data suggest that populations of memory t  cells , which are difficult to transfer , are more effective than transferrable memory populations in raising the stimulation threshold . these cells may impair the translocation of antigen - loaded dcs from the marginal zone into the t  cell zone , and this barrier function may lead to altered antigen presentation and consequently a higher stimulation threshold . this is in line with a recent report that highlights the presence of a tissue - resident t  cell population in the spleen ( schenkel et  al . , 2014 ) . nonetheless , it remains difficult to precisely distinguish between qualitative and quantitative causes for the observed differences between transferred and endogenous memory t  cells . what supports the notion of a qualitative difference is that the transfer of a large number of memory p14 t  cells , which already diminished a high - affinity response , did not resemble the effect seen with endogenous memory t  cells . c57bl/6 mice were obtained from charles river , and ot-1 , rip - mova , and cd45.1 congenic c57bl/6 mice were obtained from jackson laboratories . low - affinity ot-3 tcr transgenic mice were described previously ( enouz et  al . , 2012 ) . mice were bred in specific pathogen - free ( spf ) facilities and infected in spf or conventional facilities at the university of lausanne . mice that were at least 6  weeks old were used for experiments in compliance with the university of lausanne institutional regulations , and the experiments were approved by the veterinarian authorities of the swiss canton vaud . single - cell suspensions were obtained by mashing spleens through a 100  m nylon cell strainer ( bd falcon ) . red blood cells were lysed with hypotonic ammonium - chloride - potassium ( ack ) lysis buffer . if cells were harvested less than 5  days after the infection , then the spleens were before mashing digested with 150  g / ml dnase i ( roche ) and 200  g / ml liberase tl ( roche ) for 40  min at 37c . the mouse cd8 t  cell enrichment kit ( miltenyi biotech ) was used for cd8 t  cell enrichment . memory p14 cells were isolated by using fluorescein isothiocyanate ( fitc ) , phycoerythrin ( pe ) , or biotin - conjugated cd45.1 antibodies followed by anti - fitc , anti - pe , or anti - biotin microbeads ( miltenyi biotech ) according to the manufacturer s instructions . cells were labeled in serum - free medium with 5  m cfse at 37c for 10  min . mice were infected intraperitoneally ( i.p . ) with 2   10 plaque - forming units ( pfu ) of lymphocytic choriomeningitis virus ( lcmv strain 53b , armstrong ) grown and titrated on vero cells ( utzschneider et  al . , 2013 ) . vesicular stomatitis virus expressing siinfekl ( n4 ) ( kim et  al . , 1998 ) was grown and titrated on bhk cells . recombinant listeria monocytogenes strains expressing ovalbumin that contain k / ova - derived apl were previously described ( zehn et  al . , 2010 ) . 2002 ) , which contains native ovalbumin ( aa134387 ) or ovalbumin with the siitfekl variant ( zehn et  al . , 2009 ) , were digested with sapi . oligonucleotides encoding kavynfatc ( gp33 ) or kavyncatc ( gp33c6 ) plus an alanine on both sides were ligated into the sapi restriction site . the constructs were inserted into wild - type or acta - deficient 10403s listeria by e.  coli conjugation as previously described ( lauer et  al . listeria were grown in brain heart infusion broth ( oxoid , thermo fisher ) to mid - log phase . then , bacterial numbers were determined by measuring the od at 600  nm , and diluted stocks were i.v . naive mice received 1,0003,000 colony - forming units ( cfu ) , mice previously infected with listeria received 5   10 cfu , mice previously infected with lcmv received 2  10 cfu , and , for infection with acta - listeria , 10 cfu were injected . bacterial loads were determined by lysing spleens in pbs supplemented with 0.1% tergitol np-40 ( sigma - aldrich ) . serial dilutions were spread out on brain - heart infusion plates containing streptomycin ( 200  g / ml ) , and colonies were enumerated . a large pool of siinfekl - derived synthetic altered peptide ligands were tested for h-2 kb stabilization in rma - s cells and recognition by ot-1 t  cells as previously described ( zehn et  al . , 2009 ) . the d4 peptide shows similar surface stabilization as the n4 peptide , but it is a weak agonist for ot-1 t  cells ( figure  2a ) . ot-1 cells were stimulated in rpmi ( 10% fcs , 100 iu / ml penicillin , 100 iu / ml streptomycin , 5  m 2-me , 5  mm hepes ; invitrogen ) with anti - cd3/cd28-coated beads ( dynabeads , invitrogen ) and cultured with 50  u / ml human il-2 ( chiron ) in 7% co2 . 6  days after activation , 2   10 ot-1 cells and 1  10 rma cells were mixed in 96-well plates , and titrated concentrations of siinfekl or apl peptides ( emc microcollections ) were added . after 30  min , 7  m brefeldin a ( sigma - aldrich ) was added , and cultures were incubated for another 3.5  hr . up to 4   10 cells were plated in 96-well plates in pbs supplemented with 2% fcs and 0.01% azide . cells were surface stained for 20  min at 4c with fluorescently labeled anti - cd8 ( 536.7 ) , cd127 ( a7r34 ) , klrg1 ( 2f1 ) , cd27 ( lg7.f9 ) , cd43 glyco ( 1b11 ) , cd4 ( gk1.5 ) , cd45.1 ( a20 ) , and cd45.2 ( 104 ) antibodies and fixed in pbs supplemented with 1% formaldehyde , 2% glucose , and 0.03% azide . for intracellular cytokine staining , cells were fixed in pbs 2% formaldehyde and permeabilized in pbs with 0.25% saponin and 0.25% bsa ( perm buffer ) . cells were stained in perm buffer for ifn ( xmg1.2 ) , tnf ( mp6-xt22 ) , or granzyme b ( gb12 ) . fluorescently labeled mhc - i - gp33 multimers were used for detecting gp33-specific t  cells ( tc - metrix ) . for in  vivo staining experiments , we injected mice i.v . with fluorescently labeled anti - cd8 antibodies . mice were sacrificed 3  min later , and harvested organs were processed as indicated above . flow cytometry data were obtained on a bd facs lsrii machine and analyzed using flowjo software ( tree star ) . graphpad prism was used for graphic representation of data , calculation of ec50 values , and statistical calculations . p values  0.05 were considered significant ( p  < 0.05 ; p  < 0.01 ; p  < 0.001 ) , and p  values  > 0.05 were considered non - significant ( ns ) .
summarymany infections are caused by pathogens that are similar , but not identical , to previously encountered viruses , bacteria , or vaccines . 
 in such re - infections , pathogens introduce known antigens , which are recognized by memory t  cells and new antigens that activate naive t  cells . 
 how preexisting memory t  cells impact the repertoire of t  cells responding to new antigens is still largely unknown . 
 we demonstrate that even a minimum epitope overlap between infections strongly increases the activation threshold and narrows the diversity of t  cells recruited in response to new antigens . 
 thus , minimal cross - reactivity between infections can significantly impact the outcome of a subsequent immune response . 
 interestingly , we found that non - transferrable memory t  cells are most effective in raising the activation threshold . 
 our findings have implications for designing vaccines and suggest that vaccines meant to target low - affinity t  cells are less effective when they contain a strong cd8 t  cell epitope that has previously been encountered .