to  t cells , they lack receptor diversity yet demonstrate remarkable plasticity in response to both infectious and noninfectious stimuli . as an extension of their dichotomous nature ,
 t cells have been held liable for both instigating over - zealous immune responses [ 13 ] as well as reigning in inflammation by controlling activated macrophages and promoting wound healing [ 46 ] . in humans ,
the main subset of peripheral blood  t cells usually comprises between 1 and 5% of circulating t cells and bears the canonical v9v2 t cell receptor .
these v9v2 t cells are unique to primates , and they respond to endogenous and exogenous stress - induced molecules that are nonpeptidic in nature and preclude the requirement for processing or presentation by classical mhc molecules [ 79 ] .
isopentylpyrophosphate ( ipp ) is the prototypical phosphoantigen recognized by v9v2 t cells that was first isolated from lysates of mycobacteria .
it was later determined to be a metabolite in the mevalonate pathway in all eukaryotic and some prokaryotic cells , including staphylococcus aureus [ 10 , 11 ] . during the initial phase of infection with low bacterial inocula of s. aureus ,
v9v2 t cells are activated by endogenous mevalonate metabolites through the accumulation and dephosphorylation of the hydroxymethylglutaryl - coenzyme a reductase , the rate - limiting enzyme of the mevalonate pathway .
therefore , primates have evolved the ability to readily respond to bacterial infection as well as endogenous damage by sensing the dysregulation of the mevalonate pathway , which may be considered a stress - associated  alarmin  for these primordial lymphocytes .
we have shown that stimulating the small pool of human  t cells present in pbmc with ipp markedly augmented the early proinflammatory response to toxic shock syndrome toxin-1 ( tsst-1 ) .
tsst-1 is a staphylococcal superantigen that exerts a potent inflammatory response by bridging v2 specific  t cell receptors with conserved regions of the mhc class ii molecules of antigen presenting cells ( apcs ) outside the peptide - binding groove .
demonstrated the presence of specific v t cell signatures of various superantigenic toxins , including tsst-1 , in the blood of patients with staphylococcal tss .
we previously demonstrated that ipp - activated  t cells augment the levels of proinflammatory cytokines within the first 6 hours post - tsst-1 treatment , thereby changing the kinetics and magnitude of the immune response to the superantigen .  
we have also shown that high mobility group box protein-1 ( hmgb-1 ) is released following tsst-1 stimulation .
hmgb-1 is a highly conserved nonhistone dna - binding protein found in virtually all nucleated cells and is regarded as being the late mediator of sepsis . activated monocytes secrete hmgb-1 after exposure to endotoxin , and it is detectable in the serum of septic patients within 24 hours .
importantly , neutralizing the effects of hmgb-1 at this late time point , either by anti - hmgb-1 antibodies or antagonists , rescued mice from sepsis - induced mortality .
in contrast to endotoxin , which activates only macrophages to secrete hmgb-1 , both t cells and macrophages participate in the nuclear translocation and subsequent release of hmgb-1 into the extracellular environment following tsst-1 stimulation .   the first aim of the present study was to evaluate the influence exerted by ipp - primed v9v2 t cells on the change in costimulatory molecule expression that could explain their ability to enhance the immune response to tsst-1 .
the second objective was to determine the consequence of  t cell mediated immune modulation on the expression and secretion of hmgb-1 .
recombinant tsst-1 was purified from culture supernatants of s. aureus strain rn4220 previously transformed to carry the tst gene , using both preparative isoelectric focusing and chromatofocusing .
toxin purity was assessed by silver staining after sodium dodecyl sulfate - polyacrylamide gel electrophoresis on 14% acrylamide gels , and lps activity was undetectable by the limulus amoebocyte lysate gelation ( sensitivity limit , 10 pg / ml ) .
fresh human peripheral blood mononuclear cells ( pbmc ) from healthy donors were obtained by ficoll - paque plus ( amersham biosciences corp . ,
piscataway , nj ) density centrifugation , and cultured in 96-well u - bottom plates at 1.5  10 cells / ml in complete culture medium consisting of rpmi 1640 ( stemcell technologies inc . ,
vancouver , bc , canada ) , 10% heat - inactivated fetal bovine serum ( hyclone laboratories inc . ,
logan , ut ) , 2 mm l - glutamine ( stemcell ) , 25 mm hepes buffer ( stemcell ) , and 2 ug / ml of polymyxin b sulphate ( sigma - aldrich corp . , st .
depletion of  t cells was accomplished by immunomagnetic positive cell selection with an anti- tcr mab ( clone immu510 , bd biosciences pharmingen inc . , san diego , ca ) conjugated to an antidextran mab tetramer ( stemcell ) , which left the rest of the pbmc untouched , using the stemsep protocol ( stemcell ) .
purity of the  t cell - depleted pbmc was analyzed by flow cytometry using phycoerythrin-(pe- ) conjugated anti - v2 antibody ( bd biosciences pharmingen ) .
figure 1 shows the population of v9v2 t cells in pbmcs before ( which made up 1.5% of live cells ) and after depletion ( 0.1% ) .
pbmcs with or without  t cell depletion were treated with either 45 m ipp ( sigma - aldrich ) or left untreated in complete growth medium for 1618 hours ( overnight ) at 37c , 5% co2 prior to treatment with 1 nm tsst-1 . for
secreted hmgb-1 detection , culture supernatants were microcentrifuged at 800  g for 5 minutes and frozen at   70c until analysis .
surface expressed hmgb-1 was analyzed by flow cytometry ( facscalibur flow cytometry system , bd biosciences pharmingen ) using the anti - hmgb-1 antibody and secondary alexa488-conjugated antibody ( as described below ) .
costimulatory molecule cell surface expression was performed by incubating cells at 4c ( protected from light ) for 30 minutes with the following fluorescently - conjugated antibodies : apc antihuman hla - dr ( ln3 ) , pe - cy5 anti - human cd86 ( b7 - 2 ) ( ebioscience , inc ) , fitc anti - cd40 ( 5c3 ) , pe - cy5   anti - cd80 ( b7 - 1 ) , pe - cy5 anti - cd28 ( cd28.2 ) , and apc anti - cd25 ( m - a251 ) ( bd biosciences , pharmingen ) .
cells were subsequently washed , resuspended in buffer containing 2 mm edta ( to facilitate detachment of adherent cells ) , and transferred to facs to analyze by flow cytometry ( a minimum of 10 000 events were collected ) .
surface expression of hmgb-1 on pbmc by fluorescent microscopy has been previously described in detail . in brief , pbmcs were cultured ( 37c , 5% co2 ) directly on sterile cover slips to allow attachment of adherent cells overnight before stimulation with 1 nm of tsst-1 . for hmgb-1 expression
, cells were incubated with rabbit anti - human hmgb-1 igg as previously described , diluted in blocking buffer ( pbs , 3% fbs ) , followed by a secondary alexa488-conjugated goat antirabbit igg antibody .
fluorescently labelled pbmcs were then stained with hoechst 3342 nuclear dye ( molecular probes ) according to manufacturer 's directions , and mounted on slides using prolong anti - fade reagent ( molecular probes ) .
cells were visualized with an axioplan ii fluorescence microscope equipped with a ccd camera using northern eclipse software ( epix ) for acquisition of images .
images were taken with the 63 x oil immersion objective lens , and adobe photoshop 6.0 software was used for image layout .
pbmc culture supernatants were concentrated ( ~10-fold ) from the original volume of 1.5 ml using amicon ultra centrifugal filters with a molecular weight cutoff of 10 kda ( millipore ) .  
the supernatants then had been further prepared using the sds - page clean - up kit ( amersham ) according to the manufacturer 's directions prior to running on a 12% polyacrylamide gel .
western blotting was performed by semidry transfer of proteins ( trans - blot sd semi - dry electrophoretic transfer cell , biorad ) onto an immobilon - p pvdf membrane ( millipore ) which was blocked for 1 hour at room temperature with 1% bsa , 0.5% tween in tbs prior to overnight incubation ( at 4c ) with rabbit polyclonal anti - hmgb-1 antibody .
the membrane was subsequently incubated with anti - rabbit igg horse - radish - peroxidase-(hrp- ) conjugated secondary antibody for 1 hour at room temperature on a shaker .
hmgb-1 detection was performed using super signal substrate ( pierce ) and developed as well as analyzed using the alpha innotech 3400 gel documentation system ( alpha innotech ) .
student t - tests ( one - tail ) were used to assess the significance of differences in hmgb-1 and co - stimulatory molecule expression between ipp treated and untreated pbmc at different times post - tsst-1 stimulation .
the effects of ipp over time on co - stimulatory molecule expression by apc ( cd40 , cd80 , cd86 , hla - dr ) and  t cells ( cd25 , cd28 ) were further examined by 2-way anova .
differences were considered significant if the probability of the null hypothesis was less than five percent ( p < .05 ) .
following overnight treatment with ipp , cd40 expression was upregulated in comparison to those that were untreated ( ipp + tsst-1 versus tsst-1 alone ; p < .05 , student 's t - test ) ( figure 2(a ) ) .
the elevated cd40 expression was observed up to 6 hours postsuperantigen stimulation , after which time cd40 was downregulated or internalized .
we previously demonstrated that cd40l ( cd154 ) is expressed on t cells within 3 hours following exposure to tsst-1 , therefore , the down - regulation of cd40 beyond 6 hours is likely a consequence of cd40 binding to cd40l on tsst-1-activated t cells .
we verified that up - regulation of cd40 expression in populations exposed to ipp was solely attributed to the presence of activated  t cells , since removal of  t cells completely abrogated the effect of ipp ( figure 2(b ) ) .
the effect of ipp - activated  t cells over time on cd40 expression following tsst-1 stimulation was further verified using 2-way anova ( ipp x time interaction , p < .05 ) .
in contrast to cd40 , the expression of cd86 was down - regulated early in ipp treated pbmc at 3 hours following tsst-1 stimulation ( ipp + tsst-1 versus tsst-1 alone ; p < 0.05 , student 's t - test ) ( figure 2(a ) ) .
again , this effect was abrogated following depletion of  t cells ( figure 2(b ) ) .
two - way anova confirmed an interaction effect ( ipp x time interaction , p < .05 ) .
there was a similar trend for decreased expression of hla - dr ( the mhc class ii molecule to which tsst-1 binds on apc ) and cd80 by ipp treatment , but the differences were not statistically significant .
in contrast to the direct affect of ipp on monocyte function , there was little notable direct influence on the activation state of  t cells as evaluated by the expression of cd25 or cd28 , which remained largely unchanged in response to ipp pretreatment ( figure 3 ) .
the upregulation of cd25 on  t cells at 24 hours was solely dependent on tsst-1 stimulation regardless of ipp , as shown by the comparison of ipp + tsst-1 versus ipp alone ( p = .001 , student 's t - test , figure 3 ) .
two - way anova confirmed this effect ( tsst-1 effect , p < .01 ; tsst-1 x time interaction , p < .005 ) .
established that monocytes that had become adherent export hmgb-1 to the cell surface where it serves a function for process extension , migration , and cell - cell interaction .
extracellular cell - surface expressed hmgb-1 has been called amphoterin to differentiate it from intracellular hmgb-1 found in the nucleus .
given the direct influence of  t cells on the activation , maturation , and regulation of cells of monocytic lineage [ 20 , 21 ] , we wanted to determine how ipp stimulation would influence the surface manifestation of hmgb-1 on cultured monocytes present in pbmc .
figure 4(a ) shows that resting adherent cells have hmgb-1 ( stained in green ) localized at focal points around the perimeter in vesicle - like pockets ( top left corner ; rpmi ) .
t cells , which are nonadherent cells in suspension ( stained in red ) , do not express hmgb-1 on the surface in this manner .  
adherent monocytes bearing surface expressed hmgb-1 subsequently released it following tsst-1 stimulation ( figure 4(a ) ; top right panel ) .
overnight treatment with ipp alone also led to a change in extracellular hmgb-1 expression in pbmc cultures ( figure 4(a ) ; bottom left panel ) .
there was an observable modification on both the morphological appearance of macrophages as well as the distribution of hmgb-1 around the perimeter , which became less concentrated in vesicle - like foci and more dispersed .
overnight incubation of pbmcs with ipp followed by tsst-1 stimulation resulted in significantly more hmgb-1 being released into the extracellular environment compared to tsst-1 alone ( figure 4(a ) , upper and lower right panel ) .
this change in surface expressed hmgb-1 was assessed by flow cytometry , which enabled us to both confirm and quantify the release of hmgb-1 from differentiated monocytes following tsst-1 stimulation .
figure 4(b ) displays the difference between the various treatment conditions on the loss of cell - surface expressed hmgb-1 assessed by flow cytometry .
ipp - pretreated pbmcs that were subsequently stimulated with tsst-1 released more hmgb-1 from the cell surface ( i.e. , less hmgb-1 expression on the cell surface ) than those cells that were left untreated with ipp prior to superantigen stimulation ( ipp + tsst-1 versus tsst-1 alone , p < .05 , paired student 's t - test ) .
the actual secretion of hmgb-1 into the extracellular milieu from tsst-1 stimulated pbmc was further verified by western blot of cell culture supernatants ( figure 4(c ) ) , which again substantiated that ipp pretreatment followed by tsst-1 stimulation led to its greater release than pbmc stimulated with tsst-1 without ipp - activation of  t cells ( ipp + tsst-1 versus tsst-1 alone , p < .05 , paired student 's t - test ) .
overnight treatment with ipp resulted in a notable up - regulation of cd40 on monocytes in the presence of  t cells .
we previously demonstrated that impeding the cd40-cd40l interaction with cd40 monoclonal antibodies ( but not the cd86 or cd80 interaction ) completely blocks tsst-1-induced inflammation .
the early up - regulation of cd40 on monocytes by ipp - primed  t cells in the current study provides a basis for the prior observation that tnf and ifn levels increased 3-to 4-fold during the early phase of the immune response to tsst-1 in pbmc containing activated  t cells .
this study also demonstrates that the exaggerated response to tsst-1 by activated  t cells is through the direct modulation of the responsiveness of monocytes and not  t cells since we found that the upregulation of cd25 on  t cells was affected only by stimulation with tsst-1 and not ipp .
the regulation of cd40 expression by activated  t cells appears to parallel the mechanism of linking innate to adaptive immunity by glycolipid responsive v14 + nk t cells stimulated with -galactosylceramide .
it was found that the rapid release of tnf and ifn to ovalalbumin , acquisition of cd4 + and cd8 + t cell immunity , and maturation of dendritic cells were all dependent on the cd40-cd40l interaction induced by the priming of v14 + nk t cells by -galactosylceramide .
in contrast to cd40 , cd86 expression was found to be reduced in pbmc treated with ipp overnight .
this monocytic phenotype may be reflective of a state described as  immature dendritic cells  in which cycling of the hla - dr molecule is rapid and levels of cd86 are relatively low .
alarmins  influence the ability of apc to effectively stimulate cells of adaptive immunity , and the cd40-cd40l interaction likely plays a pivotal role in this process .
this scenario is in agreement with recent demonstrations that  t cells induce early dendritic cell maturation and share an intimate relationship with apc [ 21 , 24 , 25 ] .
further provided evidence that human  t cells are activated by microbial mevalonate metabolites and interact with monocytes and local macrophages to drive the acute inflammation in bacterial infections .
. it should be noted , however , that these studies primarily used either expanded  t cell lines or a large number of purified  t cells in coculture conditions .
the conditions used in this study may be more reflective of the physiological state in which a small number of  t cells present in peripheral blood would be able to immediately respond to their stress - associated antigens , such as ipp .
this early event would preclude the possibility of any expansion of  t cells , but it would lead to the efficient priming of apc .
this study also provides the first demonstration that ipp - primed human peripheral blood  t cells influence the secretion of hmgb-1 by macrophages .
this finding is similar to the work by semino et al .   who demonstrated that nk cells reciprocally activate autologous dendritic cells , and this interaction was also mediated through the secretion of hmgb-1 .
it is feasible that the enhanced hmgb-1 secretion observed in cells containing activated  t cells could be in response to an increase in apoptosis   due to the fact that ipp - stimulated  t cells are particularly susceptible to activation - induced cell death [ 29 , 30 ] as a self - limiting precaution . during apoptosis
, hmgb-1 is normally tightly bound to nucleosomes   as opposed to necrosis where hmgb-1 is released from cells
one of the main ligands for extracellular hmgb-1 is the receptor for advanced glycation end ( rage ) products , a member of the ig superfamily .
the expression of rage increases following activation on both dendritic cells and antigen stimulated t cells .
the release of hmgb-1 and its subsequent binding to rage is required for the clonal expansion , survival , and functional polarization of naive t cells .  
both dendritic cell maturation and th1 polarization are events that have been attributed to the activity of  t cells and hmgb-1 , respectively [ 21 , 3436 ] .
our findings connect these two previously unrelated concepts and brings forth the suggestion that  t cells mediate this effect and link innate and adaptive immunity by influencing the early upregulation of cd40 expression and release of hmgb-1 from apc , thereby priming the immune response to subsequent stimuli .
 
  t cells play an important role in regulating the immune response to stress stimuli ; however , the mean by which these innate lymphocytes fulfill this function remains 
 poorly defined . 
 the main subset of human peripheral blood  t cells responds to 
 nonpeptidic antigens , such as isopentylpyrophosphate ( ipp ) , a metabolite in the 
 mevalonate pathway for both eukaryote and prokaryote cells . 
 ipp - primed  t cells 
 significantly augment the inflammatory response mediated by monocytes and  t cells 
 to tsst-1 , the staphylococcal superantigen that is the major causative agent of toxic 
 shock syndrome . here 
 we show that the small pool of activated peripheral  t cells 
 induces an early upregulation of cd40 on monocytes and the local release of high 
 mobility group box-1 ( hmgb-1 ) , the molecule designated as the late mediator of 
 systemic inflammation . 
 this finding provides a new basis for how  t cells may serve 
 as influential modulators of both endogenous and exogenous stress stimuli . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion