BACKGROUND
granzyme b  is a serine proteinase important for its role in mediating cellular apoptosis as well as acting as an extracellular protease.
grzb is expressed primarily by activated memory cd <dig> and memory cd <dig> t cells, and nk and nkt cells during infections and inflammation.
other leukocytes such as dendritic cells, macrophages, b cells, and mast cells can express grzb but such expression is more limited  <cit> .
grzb is upregulated in cd <dig> t cells after cd3/tcr activation, as well as by common γ-chain cytokines including il <dig> and il <dig>  in memory and effector cd <dig> t cells, treg, th <dig>  and th <dig> cells, grzb is also induced after tcr activation and similar cytokines, as well as by tlr ligands  <cit> .
similarly to memory cd <dig> t cells, memory cd <dig> t cells also kill virally-infected or tumor cells via grzb  <cit> .
grzb expression and bioactivity appears to be comparable amongst cd <dig> and cd <dig> t cells, but no studies have directly compared grzb production between human cd <dig> and cd <dig> t cells.
differences in grzb expression, storage, and secretion suggest that grzb functions may differ between cd <dig> and cd <dig> t cells in immunity and disease.
studies examining expression and functional activity of grzb or grzb-associated molecules such as perforin or cd107a  in cd <dig> and cd <dig> t cells utilize mainly western blot, flow cytometry, and ctl killing assays.
for example, previous comparison of grzb expression in human cd <dig> and cd <dig> t cells by flow cytometry showed that cd <dig> t cells express more intracellular grzb protein, however, comparison of extracellular grzb between cd <dig> and cd <dig> t cells was not examined  <cit> .
our previous work directly compared human memory cd <dig> and memory cd <dig> t cells by flow cytometry and we found that resting and activated memory cd <dig> t cells store little to no grzb protein intracellularly, whereas resting and activated memory cd <dig> t cells store substantially more grzb  <cit> .
however, elisa showed that activated memory cd <dig> and memory cd <dig> t cells secreted similar amounts of grzb.
in another study, using immortalized human hsv- and ebv-specific cd <dig> ctl clones, cd <dig> ctl’s were shown to express significantly more perforin mrna compared to cd <dig> ctl’s, and target cell killing was similar between cd <dig> and cd <dig> ctl’s   <cit> .
in a mouse model of lcmv infection, direct comparison of antigen-specific cd <dig> and cd <dig> ctl’s by flow cytometry showed that cd <dig> t cells express more grzb and cd107a.
however, in vivo ctl killing measurements showed that cd <dig> t cells eliminate target cells with comparable efficiency and magnitude as cd <dig> t cells  <cit> .
thus, cd <dig> and cd <dig> t cells may differ in grzb synthesis, storage and secretion, but clarification of discrepancies between methods to measure intracellular and extracellular grzb is necessary to better understand the roles of grzb in effector functions and tissue pathology mediated by cd <dig> t cells.
the goal of the present study was to directly compare grzb production by human memory cd <dig> and memory cd <dig> t cells  with the common assays of flow cytometry, elispot, and elisa.
each of these gives different information about the effector function of a t cell.
flow cytometry indicates the intracellular content of single cells, and elispot measures how much is secreted from individual cells and is thus conceptually similar to flow cytometry measurement.
lastly, elisa indicates how much grzb is secreted globally, but does not indicate how many cells the material came from.
our studies seek to clarify differences of intracellular grzb expression vs. actual secretion by memory t cells.
differences in grzb protein storage and secretion were observed between resting and activated memory cd <dig> and memory cd <dig> t cells.
activated memory cd <dig> t cells usually secreted similar amounts of grzb compared to memory cd <dig> t cells , despite memory cd <dig> t cells storing significantly more grzb as indicated by flow cytometry.
thus, activated memory cd <dig> t cells are major sources of extracellular grzb, and the use of flow cytometry or western blot alone to measure intracellular grzb does not accurately predict secretion levels.
methods
cells and culture
human memory cd4+cd45ro+ and memory cd8+cd45ro+ t cells were purified from pbmc of healthy buffy coat donors.
pbmc were first isolated from buffy coats  by using ficoll-paque plus .
memory cd <dig> and memory cd <dig> t cells were then negatively selected from pbmc with magnetic bead-based easysep kits .
purities of memory cd4+cd45ro+ t cells were usually >90% and memory cd8+cd45ro+ t cells >80% as determined by flow cytometry.
natural killer cells were purified from pbmc with negative selection kits , and cd3-cd56+ purity was >90%.
cells were cultured at 37°c + 5%co <dig> in complete rpmi- <dig> medium, supplemented with 10% fbs,  <dig>  mm mem nonessential amino acids, 2mm sodium pyruvate, 2mm hepes, 1× antibiotic-antimycotic and 2mm l-glutamine.
most experiments involved no stimulation  or stimulation of 1-5× <dig> memory cd <dig> and memory cd <dig> t cells, or nk cells  for  <dig> hrs with  <dig> ml/ml coated cd <dig>  or soluble cd <dig>  +  <dig> μg/ml cd <dig>  mabs.
for some experiments, cells were stimulated with  <dig> ng/ml recombinant il <dig>  or soluble cd <dig> mab alone for  <dig> hrs with or without brefeldin .
flow cytometry
at appropriate time points, memory cd <dig> and memory cd <dig> t cells were examined for activation  and intracellular grzb expression by flow cytometry.
cells were washed with pbs/2%fbs and incubated with cd69-apc or -pe mabs  for  <dig> mins at 4°c.
cells were then washed and fixed and permeabilized with cytofix/cytoperm solution  for  <dig> mins at 4°c.
after fixation, cells were washed with perm/wash solution , then incubated with grzb-alexafluor- <dig>  -pe, or –apc mabs  for  <dig> mins at 4°c.
cells were washed and analyzed with flow cytometer.
cells were also stained with appropriate isotype controls to assess nonspecific binding.
data was acquired with gallios flow cytometer and analyzed with kaluza <dig>  software .
for measurement of cd107a coexpression with grzb, cd107a-fitc  was added to cells at the start of the experiment.
cells were then cultured with brefeldin for 3- <dig> hrs.
cells were then harvested and stained for grzb as described above.
for measurement of intracellular perforin expression with grzb, cells  were fixed/permeabilized with cytofix/cytoperm solutions, and stained with perforin-pe  and grzb-apc mabs.
for measurement of intracellular cytokine expression with grzb, memory t cells were fixed/permeabilized with cytofix/cytoperm solutions, and stained with grzb-pe and either ifnγ-fitc, il4-fitc, or il17a-pacific blue mabs .
for measurement of grzb expression by tregs, memory cd <dig> t cells were stained using foxp <dig> fixation/permeabilization solutions  and cd25-v <dig> , foxp3-pe , and grzb-apc.
elispot
grzb elispot’s were performed in accordance with manufacturer instructions .
1× <dig> memory cd <dig> and memory cd <dig> t cells were cultured  for  <dig> hrs in 96-well flat-bottomed nitrocellulose plates .
wells were first prepared by treating with 70% ethanol for  <dig> mins and rinsing with sterile h <dig>  wells were then coated overnight at 4°c with  <dig> μg/ml grzb mab .
after gb <dig> coating, wells were washed with pbs, and pre-incubated with cell culture rpmi- <dig> medium  for  <dig> mins at room temp.
cells were then added to appropriate wells and either unstimulated  or costimulated with  <dig> μg/ml soluble cd <dig>  + soluble cd <dig> mabs  for  <dig> hrs wrapped in aluminum foil at 37°c + 5%co <dig>  after the treatment period, cells were gently harvested for flow cytometry staining, and wells were washed with pbs.
<dig> μg/ml biotinylated grzb detection mabs  were added for  <dig> hrs at room temp.
wells were washed and streptavidin-hrp added for  <dig> hr.
wells were washed and tmb substrate added until spot development .
color development was stopped with h <dig>  and plates were air-dried, wrapped, and stored at room temp.
until spot analysis.
spots were analyzed using ctl-immunospot s <dig> analyzer .
elisa
at appropriate time points, culture supernatants were harvested, spun free of cells, and frozen at -20°c for extracellular grzb or perforin measurements by sandwich elisa kit .
grzb elisa was performed according to manufacturer instructions.
maxisorp flat-bottom 96-well plates  were first coated with  <dig> μg/ml grzb mab  overnight at 4°c.
wells were then washed 2x with pbs, then blocked for  <dig> hr at room temperature with pbs/ <dig> %tween-20/ <dig> %bsa.
samples were added for  <dig> hrs at room temp., wells washed, then  <dig> μg/ml grzb-biotin  added for  <dig> hr.
wells were then washed, and  <dig> μg/ml streptavidin-hrp added for  <dig> hr.
after streptavidin-hrp incubation, wells were washed and tmb substrate added for up to  <dig> mins .
color development was stopped with tmb stop solution and od  measured with plate reader .
sample concentrations were determined with standard curve  after blank subtraction.
for measurement of extracellular perforin, an elisa kit was used in accordance with manufacturer instructions .
samples were added to perforin mab-precoated plates for  <dig> hrs.
wells were then washed and detection mab added for  <dig> mins.
wells were washed and streptavidin-hrp added for  <dig> mins.
wells were washed and tmb substrate added for  <dig> mins, followed by addition of stop solution.
od was measured at  <dig> nm absorbance and concentrations determined with standard curve .
statistics
the values were presented by mean and standard error.
wilcoxon signed-rank tests were used to compare the difference between untreated and costimulated conditions, and between cd <dig> and cd <dig> cells for each assay, respectively.
the spearman correlation coefficients were used to assess the correlation among continuous variables.
a p-value < <dig>  was considered statistically significant.
sas software version  <dig>   was used for analyses.
RESULTS
differential storage and secretion of granzyme b between memory cd <dig> and memory cd <dig> t cells
both memory cd <dig> and cd <dig> t cells express grzb and utilize grzb for ctl functions.
the use of flow cytometry for measurement of grzb, perforin, or cd107a suggests that cd <dig> t cells produce more grzb and may be more potent ctl effectors than cd <dig> t cells  <cit> .
however, murine ctl lysis assays show that cd <dig> and cd <dig> t cells kill target cells with similar efficacy, indicating lack of correlation between intracellular grzb expression and extracellular grzb secretion by cd <dig> and cd <dig> t cells  <cit> .
to further clarify these differences, we directly compared resting or activated  1× <dig>  purified human memory cd4+cd45ro+ and cd8+cd45ro+ t cells from the same donor using in parallel multiparameter flow cytometry, elispot, and elisa.
multiparameter flow cytometry measures intracellular grzb protein which can be examined in conjunction with other cellular markers, elispot measures individual grzb-secreting cells, and elisa measures total amounts of grzb released by cells in a specified volume.
measurement of intracellular grzb by flow cytometry showed that resting and activated memory cd <dig> t cells stored significantly more grzb compared to memory cd <dig> t cells .
resting memory cd <dig> t cells expressed  <dig>  ±  <dig> % intracellular grzb protein, which did not significantly increase after  <dig> hrs costimulation to  <dig>  ±  <dig> % .
resting memory cd <dig> t cells expressed little to no intracellular grzb , but grzb expression was significantly increased by costimulation to  <dig>  ±  <dig> % .
additionally, resting and activated memory cd <dig> t cells expressed significantly more intracellular grzb compared to resting and activated memory cd <dig> t cells .
these data corroborate previous reports of intracellular grzb protein measurements by flow cytometry or western blot showing higher expression by cd <dig> compared to cd <dig> t cells  <cit> .
however, unlike memory cd <dig> t cells, grzb synthesis is actually upregulated by memory cd <dig> t cells during t cell activation.
these data suggest that activated memory cd <dig> t cells secrete pre-synthesized grzb  directly from granules, whereas activated memory cd <dig> t cells secrete pre-stored and newly synthesized grzb.
this grzb synthesis step by memory cd <dig> t cells may also be consistent with a previously reported observation of a less rapid, but ultimately comparable, ctl activity  by antigen-specific cd <dig> t cells compared to cd <dig> t cells in a murine model of lcmv infection  <cit> .figure  <dig>
comparison of intracellular and extracellular grzb protein levels by memory cd <dig> and memory cd <dig> t cells.
representative flow cytometry dotplots and mean ± sem intracellular grzb protein expression by 1× <dig> untreated  or activated  memory t cells after  <dig> hrs culture .
representative elispot wells and mean ± sem grzb spots  of untreated or activated  memory t cells after  <dig> hrs culture .
extracellular grzb production  by 1× <dig> untreated or activated  memory t cells after  <dig> hrs culture .
although the use of elispot to examine grzb release by cd <dig> t cells is well-described  <cit> , less clear is how well this method measures grzb release by memory cd <dig> t cells.
by contrast to flow cytometry data, measurement of secreted grzb with elispot showed that the number of grzb-secreting cells was comparable between activated memory cd <dig> and memory cd <dig> t cells .
elispot assays showed that resting memory cd <dig> t cells did not secrete grzb after  <dig> hrs culture.
however, some resting memory cd <dig> t cells constitutively secreted grzb , consistent with more storage of grzb and low levels of stimulation in vitro.
after  <dig> hrs costimulation, similar numbers of grzb spots were detected by activated memory cd <dig>  and memory cd <dig> t cells .
although elispot sensitivity may more be limited beyond a particular range of spot formation, these data suggest that similar numbers of activated memory cd <dig> and memory cd <dig> t cells secreted grzb.
consistent with the elispot assays, measurement of bulk supernatant grzb by elisa showed that activated memory cd <dig> and memory cd <dig> t cells secrete similar amounts of grzb .
extracellular grzb was not detected by resting memory cd <dig> t cells after  <dig> hrs culture, but ~ <dig> pg/ml grzb was detected by resting memory cd <dig> t cells.
after  <dig> hrs costimulation, extracellular grzb was  <dig>  ±  <dig> pg/ml and 627 ±  <dig> pg/ml for activated memory cd <dig> and memory cd <dig> t cells, respectively.
in conjunction with the elispot data, these elisa data indicate that at a single cell level, similar amounts of grzb are secreted per activated memory cd <dig>  and memory cd <dig>  t cell.
these extracellular grzb data may also suggest that much of the pre-stored intracellular grzb in memory cd <dig> t cells indicated by flow cytometry may not actually be secreted.
these similarities of extracellular grzb production between memory cd <dig> and cd <dig> t cells are consistent with previous reports of grzb effector function demonstrating comparable ctl activity by cd <dig> and cd <dig> t cells using in vitro-generated virus-specific human ctl clones  <cit> , and in vivo lcmv-specific ctl assays with mice  <cit> .
thus, the simultaneous use of these intracellular and extracellular grzb measurements confirm that resting memory cd <dig> t cells store more grzb compared to memory cd <dig> t cells.
however, similar numbers of activated memory cd <dig> t cells secrete similar amounts of grzb as memory cd <dig> t cells, which are likely achieved by higher levels of de novo grzb synthesis.
memory t cell granzyme b assay correlations, and lack of correlation of grzb production between memory cd <dig> and memory cd <dig> t cells within single donors
we examined correlations between each grzb assay for memory cd <dig> or memory cd <dig> t cells .
inter-assay correlations of grzb measurement between flow cytometry, elispot, and elisa suggested that intracellular grzb measured by flow cytometry correlates with extracellular grzb as measured by elispot and elisa for memory cd <dig> t cells, but not for memory cd <dig> t cells.
for memory cd <dig> t cells, the spearman correlation coefficient between grzb flow cytometry and elisa was r =  <dig>  , and r =  <dig>   between grzb flow cytometry and elispot .
however, because memory cd <dig> t cells store more intracellular grzb compared to memory cd <dig> t cells without proportionally secreting higher amounts of grzb, no correlation was observed between grzb flow cytometry and elisa , or between grzb flow cytometry and elispot  .
additionally, these weak correlations between intracellular and extracellular grzb of memory cd <dig> t cells are unlike those for cytokines such as ifnγ or il <dig> which strongly correlate between intracellular and extracellular levels for cd <dig> and cd <dig> t cells.
for example, human resting t cells do not express or pre-store intracellular ifnγ or il <dig> , but upregulate these cytokines intracellularly and extracellularly  upon t cell activation, and cytokine detection between flow cytometry and either elispot or elisa significantly correlate   <cit> .
since memory cd <dig> t cells do not pre-store grzb at levels similar to memory cd <dig> t cells, these inter-assay correlations between intracellular and extracellular grzb of memory cd <dig> t cells resemble the correlations of other secreted factors that are not pre-stored, such as cytokines.
lastly, the correlation between grzb elispot and elisa was comparable for memory cd <dig>  and memory cd <dig>  t cells.
thus, the correlation between intracellular and extracellular grzb is stronger for memory cd <dig> t cells compared to memory cd <dig> t cells.figure  <dig>
inter-assay correlations between intracellular and extracellular grzb assays of memory t cells, and intra-donor correlations of grzb production between memory t cells.
spearman correlations between intracellular  and extracellular  grzb measurements of 1× <dig> untreated  or activated  memory cd <dig>  or memory cd <dig>  t cells .
spearman correlations comparing grzb production  of memory cd <dig> vs. memory cd <dig> t cells within single donors as measured by flow cytometry, elispot, or elisa .
the bulk data in figure  <dig> suggests that intracellular grzb is increased by activated memory cd <dig> but not by memory cd <dig> t cells , whereas extracellular grzb is increased by both activated memory cd <dig> and memory cd <dig> t cells .
however, when we examined grzb production by memory cd <dig> and memory cd <dig> t cells within individual donors to determine if increased grzb production by memory cd <dig> t cells is also associated with increased grzb production by memory cd <dig> t cells, and vice versa , correlations of grzb production between memory cd <dig> and memory cd <dig> t cells from the same donor were weak .
spearman correlations of intracellular and extracellular grzb production  also indicated that no significant correlations exist between memory cd <dig> and memory cd <dig> t cells for any assay .
the use of grzb absolute or percent changes  also yielded no significant correlations between memory cd <dig> and memory cd <dig> t cells for each assay .
although figure  <dig> shows that grzb production by memory t cells is generally increased by t cell activation, these data in figure  <dig> suggest that within an individual, the induction and secretion levels of grzb are different and unique between memory cd <dig> and memory cd <dig> t cells.
association of cd107a degranulation marker with extracellular, but not intracellular, granzyme b of memory t cells
we examined the association of memory t cell grzb with activation and degranulation markers, which more accurately indicates grzb-expressing cells that are also actively secreting grzb.
cd <dig> is an early t cell activation marker that is transiently upregulated by activated t cells, and cd107a  is a lysosomal protein detectable by flow cytometry surface staining after transient lysosomal fusion to the cellular membrane and release of granule proteins during t cell activation.
surface expression of cd107a is also associated with perforin release by human cd <dig> t cells, and correlates with ctl effector function  <cit> .
after  <dig> hrs costimulation, total cd <dig> expression by memory cd <dig>  and memory cd <dig>  t cells were similar .
if gated on total memory cd <dig> or memory cd <dig> t cells, cd <dig> coexpression with grzb  was higher by activated memory cd <dig>  compared to memory cd <dig>  t cells .
however, out of the grzb+ memory cd <dig> or memory cd <dig> t cells, cd <dig> expression was higher by memory cd <dig>  compared to memory cd <dig> t cells , indicating that more grzb-expressing memory cd <dig> t cells are also activated compared to memory cd <dig> t cells.figure  <dig>
association of memory t cell grzb secretion with activation and degranulation markers.
memory t cell flow cytometry dotplots and mean ± sem  total surface cd <dig> expression or cd69+/grzb+ coexpression , or cd <dig> expression gated on grzb+ memory cd <dig> or memory cd <dig> t cells.
flow cytometry dotplots and mean ± sem  total surface cd107a expression or cd107a+/grzb+ coexpression , or cd107a expression gated on grzb+ memory cd <dig> or memory cd <dig> t cells.
grzb elisa of memory t cells conducted in parallel with cd107a experiments.
spearman correlations between cd107a and intracellular  or extracellular  grzb of memory t cells .
after  <dig> hrs costimulation, total cd107a expression was  <dig>  ±  <dig> % in activated memory cd <dig>  and  <dig>  ±  <dig> % in memory cd <dig> t cells .
if gated on total memory cd <dig> or memory cd <dig> t cells, cd107a+/grzb+ coexpression was  <dig>  ±  <dig> % by memory cd <dig>  but higher at  <dig>  ±  <dig> % by memory cd <dig> t cells , which may more accurately corroborate the frequency of less than 1% activated memory t cells that secrete grzb as indicated by elispot in figure 1b.
but unlike the difference of cd <dig> expression  between activated memory t cells shown in figure 3d, cd107a expression by activated grzb+ memory t cells was similar .
additionally, the parallel extracellular grzb levels  for these experiments were similar between activated memory t cells , suggesting that cd107a is more accurate than cd <dig> as an indicator of grzb secretion by activated memory t cells.
for memory cd <dig> t cells, the spearman correlation of cd107a with intracellular grzb was r =  <dig>  , and r =  <dig>   with extracellular grzb .
similarly for memory cd <dig> t cells, the correlation of cd107a with intracellular grzb was r = - <dig>  , and r =  <dig>   with extracellular grzb .
despite increased surface expression of cd107a and release of grzb by activated memory cd <dig> t cells , intracellular grzb of memory cd <dig> t cells does not significantly change after activation as shown by flow cytometry in figure 1a, which may be consistent with an earlier report that demonstrates by flow cytometry that increased surface expression of cd107a and cd107b by human cd <dig> t cells is associated with loss of intracellular perforin  <cit> .
for memory cd <dig> t cells, both cd107a and intracellular grzb are associated with grzb secretion to similar degrees, and increased surface expression of cd <dig> and cd107a by activated memory cd <dig> t cells is further associated with the increased intracellular grzb expression shown by flow cytometry in figure 1a.
these data further corroborate figure  <dig>  in which activated memory cd <dig> t cells secrete grzb mainly from pre-stored granules, whereas grzb secretion by memory cd <dig> t cells is likely derived from pre-stored and newly synthesized grzb to achieve the similar extracellular grzb levels.
despite higher intracellular storage of grzb by activated memory cd <dig> compared to memory cd <dig> t cells , cd107a expression by activated grzb+ memory t cells, as well as the number of grzb-secreting cells , were similar.
this may suggest that a higher proportion of activated grzb+ memory cd <dig> t cells secrete and deplete their grzb stores.
this contrasts with higher retention of intracellular grzb by activated memory cd <dig> t cells, since elispot numbers would be substantially higher if all activated cd <dig> t cells released their intracellular grzb stores.
these differences may allow memory cd <dig> t cells to be more sustained serial killers of target cells, whereas memory cd <dig> t cells may express grzb for shorter-term functions  <cit> .
less association of granzyme b with perforin by memory cd <dig> t cells compared to memory cd <dig> and natural killer cells
we next examined grzb production by memory t cells in conjunction with perforin, and also in comparison with natural killer  cells, abundant producers of grzb and perforin.
in addition to grzb, cd <dig> t cells express perforin to eliminate target cells  <cit> .
memory cd4+cd45ro+ and memory cd8+cd45ro+ t cells, and cd56+ nk cells, were purified from peripheral blood of the same donor and cultured in parallel, with or without the intracellular protein transport inhibitor brefeldin.
memory t cells and nk cells  were stimulated for  <dig> hrs with il <dig> .
additionally, memory t cells were alternatively stimulated with soluble anti-cd <dig> mabs only , as cd <dig> expression by memory cd <dig> t cells  was modestly lower compared to memory cd <dig> t cells , which may result in memory cd <dig> t cells receiving stronger stimulation by cd3/cd <dig> costimulation compared to memory cd <dig> t cells.
agonism of other costimulatory receptors such as icos or tnf superfamily members may further yield different results.
intracellular and extracellular perforin and grzb were measured by flow cytometry and elisa.
figure 4a-b shows representative perforin/grzb flow cytometry dotplots  and the mean distribution of intracellular perforin/grzb subpopulations  for memory t cells and nk cells .
for memory cd <dig> t cells, only perforin-/grzb+ cells were mainly observed in all conditions , and a small percentage of perforin+/grzb+ cells were observed by il <dig> stimulation .
however, memory cd <dig> t cells expressed more intracellular perforin and grzb.
in the absence of brefeldin, ut memory cd <dig> t cells were  <dig>  ±  <dig> % perforin+/grzb-,  <dig>  ±  <dig> % perforin-/grzb+, and  <dig>  ±  <dig> % perforin+/grzb+.
il <dig> stimulation changed these perforin/grzb distributions by increasing perforin expression , whereas cd <dig> stimulation did not affect the perforin/grzb expression patterns.
in the presence of brefeldin, perforin expression was mitigated and memory cd <dig> t cells were mostly perforin-/grzb+ .
as expected, intracellular perforin and grzb expression was substantially higher for nk cells compared to memory t cells.
in the absence of brefeldin, nk cells were mostly perforin+/grzb+ , by contrast to memory t cells which were mostly perforin-/grzb+.
but in the presence of brefeldin, this distribution changed to mostly perforin-/grzb+ , which was similar to the perforin mitigation by brefeldin of memory cd <dig> t cells.
thus, intracellular perforin is marginally expressed with grzb by stimulated memory cd <dig> t cells, whereas perforin is more associated with grzb in memory cd <dig> t cells and nk cells.figure  <dig>
comparison of grzb and perforin expression between memory cd <dig>  memory cd <dig>  and natural killer cells.
representative grzb/perforin dotplots of memory cd <dig>  memory cd <dig>  and nk cells.
cells were purified from peripheral blood, and 5× <dig> cells  were untreated  or stimulated with il <dig>  or anti-cd <dig> only mabs  for  <dig> hrs +/- brefeldin.
mean  distribution of perforin/grzb subsets of memory cd <dig>  memory cd <dig>  and nk cells.
extracellular grzb and perforin production by memory cd <dig>  memory cd <dig>  and nk cells after  <dig> hrs culture +/- brefeldin .
despite little to no intracellular perforin expression by memory cd <dig> t cells, perforin was secreted with grzb by stimulated memory cd <dig> t cells as determined by elisa, but in a pattern distinct from memory cd <dig> t cells .
in the absence of brefeldin, il2- and cd3-stimulated memory cd <dig> t cells released  <dig>  ±  <dig> pg/ml and 336 ±  <dig> pg/ml grzb, respectively, more than memory cd <dig> t cells which released 249 ±  <dig> pg/ml and 86 ±  <dig> pg/ml grzb .
however, ut and il2-stimulated memory cd <dig> t cells released 372 ±  <dig> pg/ml and  <dig>  ±  <dig> pg/ml perforin, which was more than memory cd <dig> t cells which released 159 ±  <dig> pg/ml and 521 ±  <dig> pg/ml perforin .
perforin release by cd3-stimulated memory t cells was similar .
as expected, ut and il2-stimulated nk cells secreted substantially more perforin  and grzb  compared to memory t cells in the absence of brefeldin .
in the presence of brefeldin, perforin and grzb release by stimulated memory t cells was reduced nearly to baseline  levels, and reduced ~2- <dig> fold by nk cells.
an unclear effect of brefeldin upon resting memory t cells and nk cells was observed, in which a modest decrease of constitutive perforin release, but an increase of grzb release, by ut memory t cells and nk cells occurred.
it is possible that intracellular storage of grzb may be more disassociated with perforin by resting t cells and nk cells compared to activated states.
additionally, il2-stimulated memory cd <dig> t cells and nk cells appear to secrete more grzb than perforin, whereas memory cd <dig> t cells secrete more perforin than grzb.
lastly, despite the release of perforin and grzb by stimulated memory t cells and nk cells , intracellular accumulation  of perforin and grzb in stimulated cells did not occur in the presence of brefeldin .
this is likely due to glycosylation requirements of perforin and grzb for intracellular transport and secretion  <cit> , which would be inhibited by brefeldin.
memory cd <dig> t cells with granzyme b production capacity include tregs and th <dig> cells, with potentially more distinct grzb vs. cytokine secretory mechanisms compared to cd <dig> t cells
we lastly examined memory cd <dig> t cell subsets that produce grzb such as treg, th <dig>  th <dig>  and th <dig> cells.
memory cd4+cd45ro+ t cells were purified from peripheral blood and activated  or ut for  <dig> hrs.
cells were then analyzed by flow cytometry for phenotype  and function  in conjunction with grzb expression.
<dig> hrs costimulation or pma/io treatment of memory cd <dig> t cells resulted in grzb expression by phenotypes consistent with treg and th <dig> cells .
tregs were examined by measuring intracellular grzb expression by cd25+/foxp3+ costimulated memory cd <dig> t cells.
activated memory cd <dig> t cells expressed ~18% cd <dig> .
grzb was coexpressed with foxp <dig> by cd25-dim cells , and more so by cd25-bright cells .
this is consistent with reports that demonstrate treg expression of grzb for suppressive functions  <cit> .
ifnγ was modestly induced and coexpressed with grzb by costimulated memory cd <dig> t cells , but more robustly by pma/io treatment , indicating that memory cd <dig> th <dig> cells express grzb, consistent with other reports  <cit> .
by contrast, il <dig> was not induced by costimulated or pma/io-treated memory cd <dig> t cells , suggesting that grzb is not expressed by memory cd <dig> th <dig> cells.
il17a was induced by costimulation and pma/io, but not coexpresssed with grzb , suggesting that in these short-term conditions memory cd <dig> th <dig> cells may not express grzb.
th <dig> cells are reported to express grzb in vivo  <cit> , which likely involves more complex stimuli, timing or differentiation, and more specific phenotypic characterization .
thus, tregs and th <dig> cells are subsets of peripheral blood memory cd <dig> t cells that produce grzb in the present study, although other grzb-producing subsets, particularly cd <dig> ctl’s, are likely involved as well  <cit> .figure  <dig>
grzb production by memory cd <dig> t cell subsets and coexpression with cytokines.
grzb expression by tregs.
memory cd <dig> t cells were purified from peripheral blood, and 5× <dig> cells  were untreated  or activated  for  <dig> hrs.
cells were then stained for cd <dig>  foxp <dig>  and grzb.
cd <dig> expression levels  by memory cd <dig> t cells after  <dig> hrs culture .
foxp3/grzb expression by cd25-negative, cd25-dim, or cd25-bright memory cd <dig> t cells after  <dig> hrs culture.
graph shows the mean  distribution of foxp3/grzb subsets gated on cd25-negative, -dim, or -bright cells.
grzb coexpression with ifnγ, il <dig>  or il17a by memory cd <dig> t cells.
memory cd <dig> t cells  were untreated for activated  for  <dig> hrs, then stained for grzb and either ifnγ, il <dig>  or il17a.
graphs show the mean  distribution of cytokine/grzb subsets.
grzb/ifnγ coexpression by memory cd <dig> t cells.
purified memory cd <dig> t cells  were untreated or activated  for  <dig> hrs.
graph shows the mean  distribution of ifnγ/grzb subsets.
for comparison of cytokine/grzb coexpression by memory cd <dig> th <dig> cells, purified memory cd8+cd45ro+ t cells were examined for ifnγ and grzb coexpression as well .
compared to cd <dig> t cells or nk cells, cytokine secretion pathways of cd <dig> t cells may be more diversified and distinct from those of cytotoxic mediators such as granzymes  <cit> .
for example, in activated murine cd <dig> t cells, il <dig> and ifnγ colocalize and are secreted by a more polarized mechanism compared to other secreted mediators such as tnfα and chemokines  <cit> .
additionally in nk cells, ifnγ and tnfα are stored and packaged in vesicles distinct from perforin-containing granules, and secreted in a more nonpolarized fashion compared to perforin  <cit> .
in the present study,  <dig> hrs cd3/cd <dig> costimulation of memory cd <dig> t cells induced  <dig>  ±  <dig> % ifnγ+/grzb- and  <dig>  ±  <dig> % ifnγ+/grzb+ expression, and pma/io treatment induced  <dig>  ±  <dig> % ifnγ+/grzb- and  <dig>  ±  <dig> % ifnγ+/grzb+ expression.
thus, compared to pma/io-treated memory cd <dig> t cells , ifnγ+/grzb+ expression by pma/io-treated memory cd <dig> t cells was significantly higher , suggesting that cytokine, as well as perforin, secretion pathways of memory cd <dig> t cells may be more disassociated with grzb secretion compared to memory cd <dig> t cells.
these differences may be relevant to the weak correlations of grzb expression and secretion between memory cd <dig> and memory cd <dig> t cells within single donors shown in figure 2g-i.
CONCLUSIONS
in the present study, grzb expression and secretion was directly compared between human memory cd <dig> and memory cd <dig> t cells using flow cytometry, elispot, and elisa.
the results show that memory cd <dig> t cells store more grzb, but do not secrete more grzb, compared to memory cd <dig> t cells during short-term activation, suggesting that activated cd <dig> t cells regulate their grzb release more than cd <dig> t cells.
consequently, the correlation between intracellular and extracellular grzb for memory cd <dig> t cells is weaker compared to memory cd <dig> t cells.
additionally, cd107a is a better indicator of grzb secretion by memory t cells than intracellular grzb itself.
these results corroborate and further clarify previous studies examining grzb production by human or mouse cd <dig> and cd <dig> t cells, which utilized either flow cytometry, elispot, or elisa, but not all three methods simultaneously.
distinct patterns of grzb production between memory t cells may further be due to different associations with other secreted factors such as perforin or cytokines.
abbreviations
ctlcytotoxic t lymphocyte
lamp-1lysosomal-associated membrane protein1
tcrt cell receptor
lin lin and jacob couturier contributed equally to this work.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
ll, jc, and mam performed experiments, analyzed data, and wrote the manuscript.
xy and cak analyzed data.
del supervised the project and reviewed the manuscript.
all authors read and approved the final manuscript.
this work was supported by nih/niaid baylor college of medicine/ut-health center for aids research  grant ai ro <dig>  <dig> , and shandong medicine and health technology development program grant 2011hw <dig> .

 granzyme b is a serine proteinase important for its role in mediating cellular apoptosis as well as acting as an extracellular protease. grzb is expressed primarily by activated memory cd and memory cd t cells, and nk and nkt cells during infections and inflammation. other leukocytes such as dendritic cells, macrophages, b cells, and mast cells can express grzb but such expression is more limited. grzb is upregulated in cd t cells after cd3/tcr activation, as well as by common γ-chain cytokines including il and il in memory and effector cd t cells, treg, th and th cells, grzb is also induced after tcr activation and similar cytokines, as well as by tlr ligands. similarly to memory cd t cells, memory cd t cells also kill virally-infected or tumor cells via grzb. grzb expression and bioactivity appears to be comparable amongst cd and cd t cells, but no studies have directly compared grzb production between human cd and cd t cells. differences in grzb expression, storage, and secretion suggest that grzb functions may differ between cd and cd t cells in immunity and disease. studies examining expression and functional activity of grzb or grzb-associated molecules such as perforin or cd107a in cd and cd t cells utilize mainly western blot, flow cytometry, and ctl killing assays. for example, previous comparison of grzb expression in human cd and cd t cells by flow cytometry showed that cd t cells express more intracellular grzb protein, however, comparison of extracellular grzb between cd and cd t cells was not examined. our previous work directly compared human memory cd and memory cd t cells by flow cytometry and we found that resting and activated memory cd t cells store little to no grzb protein intracellularly, whereas resting and activated memory cd t cells store substantially more grzb. however, elisa showed that activated memory cd and memory cd t cells secreted similar amounts of grzb. in another study, using immortalized human hsv- and ebv-specific cd ctl clones, cd ctl’s were shown to express significantly more perforin mrna compared to cd ctl’s, and target cell killing was similar between cd and cd ctl’s. in a mouse model of lcmv infection, direct comparison of antigen-specific cd and cd ctl’s by flow cytometry showed that cd t cells express more grzb and cd107a. however, in vivo ctl killing measurements showed that cd t cells eliminate target cells with comparable efficiency and magnitude as cd t cells. thus, cd and cd t cells may differ in grzb synthesis, storage and secretion, but clarification of discrepancies between methods to measure intracellular and extracellular grzb is necessary to better understand the roles of grzb in effector functions and tissue pathology mediated by cd t cells. the goal of the present study was to directly compare grzb production by human memory cd and memory cd t cells with the common assays of flow cytometry, elispot, and elisa. each of these gives different information about the effector function of a t cell. flow cytometry indicates the intracellular content of single cells, and elispot measures how much is secreted from individual cells and is thus conceptually similar to flow cytometry measurement. lastly, elisa indicates how much grzb is secreted globally, but does not indicate how many cells the material came from. our studies seek to clarify differences of intracellular grzb expression vs. actual secretion by memory t cells. differences in grzb protein storage and secretion were observed between resting and activated memory cd and memory cd t cells. activated memory cd t cells usually secreted similar amounts of grzb compared to memory cd t cells, despite memory cd t cells storing significantly more grzb as indicated by flow cytometry. thus, activated memory cd t cells are major sources of extracellular grzb, and the use of flow cytometry or western blot alone to measure intracellular grzb does not accurately predict secretion levels. methods cells and culture human memory cd4+cd45ro+ and memory cd8+cd45ro+ t cells were purified from pbmc of healthy buffy coat donors. pbmc were first isolated from buffy coats by using ficoll-paque plus. memory cd and memory cd t cells were then negatively selected from pbmc with magnetic bead-based easysep kits. purities of memory cd4+cd45ro+ t cells were usually >90% and memory cd8+cd45ro+ t cells >80% as determined by flow cytometry. natural killer cells were purified from pbmc with negative selection kits, and cd3-cd56+ purity was >90%. cells were cultured at 37°c + 5%co in complete rpmi- medium, supplemented with 10% fbs, mm mem nonessential amino acids, 2mm sodium pyruvate, 2mm hepes, 1× antibiotic-antimycotic and 2mm l-glutamine. most experiments involved no stimulation or stimulation of 1-5× memory cd and memory cd t cells, or nk cells for hrs with ml/ml coated cd or soluble cd  +  μg/ml cd mabs. for some experiments, cells were stimulated with ng/ml recombinant il or soluble cd mab alone for hrs with or without brefeldin. flow cytometry at appropriate time points, memory cd and memory cd t cells were examined for activation and intracellular grzb expression by flow cytometry. cells were washed with pbs/2%fbs and incubated with cd69-apc or -pe mabs for mins at 4°c. cells were then washed and fixed and permeabilized with cytofix/cytoperm solution for mins at 4°c. after fixation, cells were washed with perm/wash solution, then incubated with grzb-alexafluor- -pe, or –apc mabs for mins at 4°c. cells were washed and analyzed with flow cytometer. cells were also stained with appropriate isotype controls to assess nonspecific binding. data was acquired with gallios flow cytometer and analyzed with kaluza software. for measurement of cd107a coexpression with grzb, cd107a-fitc was added to cells at the start of the experiment. cells were then cultured with brefeldin for 3- hrs. cells were then harvested and stained for grzb as described above. for measurement of intracellular perforin expression with grzb, cells were fixed/permeabilized with cytofix/cytoperm solutions, and stained with perforin-pe and grzb-apc mabs. for measurement of intracellular cytokine expression with grzb, memory t cells were fixed/permeabilized with cytofix/cytoperm solutions, and stained with grzb-pe and either ifnγ-fitc, il4-fitc, or il17a-pacific blue mabs. for measurement of grzb expression by tregs, memory cd t cells were stained using foxp fixation/permeabilization solutions and cd25-v, foxp3-pe, and grzb-apc. elispot grzb elispot’s were performed in accordance with manufacturer instructions. 1× memory cd and memory cd t cells were cultured for hrs in 96-well flat-bottomed nitrocellulose plates. wells were first prepared by treating with 70% ethanol for mins and rinsing with sterile h wells were then coated overnight at 4°c with μg/ml grzb mab. after gb coating, wells were washed with pbs, and pre-incubated with cell culture rpmi- medium for mins at room temp. cells were then added to appropriate wells and either unstimulated or costimulated with μg/ml soluble cd  + soluble cd mabs for hrs wrapped in aluminum foil at 37°c + 5%co after the treatment period, cells were gently harvested for flow cytometry staining, and wells were washed with pbs. μg/ml biotinylated grzb detection mabs were added for hrs at room temp. wells were washed and streptavidin-hrp added for hr. wells were washed and tmb substrate added until spot development. color development was stopped with h and plates were air-dried, wrapped, and stored at room temp. until spot analysis. spots were analyzed using ctl-immunospot s analyzer. elisa at appropriate time points, culture supernatants were harvested, spun free of cells, and frozen at -20°c for extracellular grzb or perforin measurements by sandwich elisa kit. grzb elisa was performed according to manufacturer instructions. maxisorp flat-bottom 96-well plates were first coated with μg/ml grzb mab overnight at 4°c. wells were then washed 2x with pbs, then blocked for hr at room temperature with pbs/ %tween-20/ %bsa. samples were added for hrs at room temp., wells washed, then μg/ml grzb-biotin added for hr. wells were then washed, and μg/ml streptavidin-hrp added for hr. after streptavidin-hrp incubation, wells were washed and tmb substrate added for up to mins. color development was stopped with tmb stop solution and od measured with plate reader. sample concentrations were determined with standard curve after blank subtraction. for measurement of extracellular perforin, an elisa kit was used in accordance with manufacturer instructions. samples were added to perforin mab-precoated plates for hrs. wells were then washed and detection mab added for mins. wells were washed and streptavidin-hrp added for mins. wells were washed and tmb substrate added for mins, followed by addition of stop solution. od was measured at nm absorbance and concentrations determined with standard curve. statistics the values were presented by mean and standard error. wilcoxon signed-rank tests were used to compare the difference between untreated and costimulated conditions, and between cd and cd cells for each assay, respectively. the spearman correlation coefficients were used to assess the correlation among continuous variables. a p-value < was considered statistically significant. sas software version was used for analyses.  differential storage and secretion of granzyme b between memory cd and memory cd t cells both memory cd and cd t cells express grzb and utilize grzb for ctl functions. the use of flow cytometry for measurement of grzb, perforin, or cd107a suggests that cd t cells produce more grzb and may be more potent ctl effectors than cd t cells. however, murine ctl lysis assays show that cd and cd t cells kill target cells with similar efficacy, indicating lack of correlation between intracellular grzb expression and extracellular grzb secretion by cd and cd t cells. to further clarify these differences, we directly compared resting or activated 1× purified human memory cd4+cd45ro+ and cd8+cd45ro+ t cells from the same donor using in parallel multiparameter flow cytometry, elispot, and elisa. multiparameter flow cytometry measures intracellular grzb protein which can be examined in conjunction with other cellular markers, elispot measures individual grzb-secreting cells, and elisa measures total amounts of grzb released by cells in a specified volume. measurement of intracellular grzb by flow cytometry showed that resting and activated memory cd t cells stored significantly more grzb compared to memory cd t cells. resting memory cd t cells expressed  ±  % intracellular grzb protein, which did not significantly increase after hrs costimulation to  ±  %. resting memory cd t cells expressed little to no intracellular grzb, but grzb expression was significantly increased by costimulation to  ±  %. additionally, resting and activated memory cd t cells expressed significantly more intracellular grzb compared to resting and activated memory cd t cells. these data corroborate previous reports of intracellular grzb protein measurements by flow cytometry or western blot showing higher expression by cd compared to cd t cells. however, unlike memory cd t cells, grzb synthesis is actually upregulated by memory cd t cells during t cell activation. these data suggest that activated memory cd t cells secrete pre-synthesized grzb directly from granules, whereas activated memory cd t cells secrete pre-stored and newly synthesized grzb. this grzb synthesis step by memory cd t cells may also be consistent with a previously reported observation of a less rapid, but ultimately comparable, ctl activity by antigen-specific cd t cells compared to cd t cells in a murine model of lcmv infection.figure comparison of intracellular and extracellular grzb protein levels by memory cd and memory cd t cells. representative flow cytometry dotplots and mean ± sem intracellular grzb protein expression by 1× untreated or activated memory t cells after hrs culture. representative elispot wells and mean ± sem grzb spots of untreated or activated memory t cells after hrs culture. extracellular grzb production by 1× untreated or activated memory t cells after hrs culture. although the use of elispot to examine grzb release by cd t cells is well-described, less clear is how well this method measures grzb release by memory cd t cells. by contrast to flow cytometry data, measurement of secreted grzb with elispot showed that the number of grzb-secreting cells was comparable between activated memory cd and memory cd t cells. elispot assays showed that resting memory cd t cells did not secrete grzb after hrs culture. however, some resting memory cd t cells constitutively secreted grzb, consistent with more storage of grzb and low levels of stimulation in vitro. after hrs costimulation, similar numbers of grzb spots were detected by activated memory cd and memory cd t cells. although elispot sensitivity may more be limited beyond a particular range of spot formation, these data suggest that similar numbers of activated memory cd and memory cd t cells secreted grzb. consistent with the elispot assays, measurement of bulk supernatant grzb by elisa showed that activated memory cd and memory cd t cells secrete similar amounts of grzb. extracellular grzb was not detected by resting memory cd t cells after hrs culture, but ~ pg/ml grzb was detected by resting memory cd t cells. after hrs costimulation, extracellular grzb was  ±  pg/ml and 627 ±  pg/ml for activated memory cd and memory cd t cells, respectively. in conjunction with the elispot data, these elisa data indicate that at a single cell level, similar amounts of grzb are secreted per activated memory cd and memory cd t cell. these extracellular grzb data may also suggest that much of the pre-stored intracellular grzb in memory cd t cells indicated by flow cytometry may not actually be secreted. these similarities of extracellular grzb production between memory cd and cd t cells are consistent with previous reports of grzb effector function demonstrating comparable ctl activity by cd and cd t cells using in vitro-generated virus-specific human ctl clones, and in vivo lcmv-specific ctl assays with mice. thus, the simultaneous use of these intracellular and extracellular grzb measurements confirm that resting memory cd t cells store more grzb compared to memory cd t cells. however, similar numbers of activated memory cd t cells secrete similar amounts of grzb as memory cd t cells, which are likely achieved by higher levels of de novo grzb synthesis. memory t cell granzyme b assay correlations, and lack of correlation of grzb production between memory cd and memory cd t cells within single donors we examined correlations between each grzb assay for memory cd or memory cd t cells. inter-assay correlations of grzb measurement between flow cytometry, elispot, and elisa suggested that intracellular grzb measured by flow cytometry correlates with extracellular grzb as measured by elispot and elisa for memory cd t cells, but not for memory cd t cells. for memory cd t cells, the spearman correlation coefficient between grzb flow cytometry and elisa was r = , and r =  between grzb flow cytometry and elispot. however, because memory cd t cells store more intracellular grzb compared to memory cd t cells without proportionally secreting higher amounts of grzb, no correlation was observed between grzb flow cytometry and elisa, or between grzb flow cytometry and elispot. additionally, these weak correlations between intracellular and extracellular grzb of memory cd t cells are unlike those for cytokines such as ifnγ or il which strongly correlate between intracellular and extracellular levels for cd and cd t cells. for example, human resting t cells do not express or pre-store intracellular ifnγ or il, but upregulate these cytokines intracellularly and extracellularly upon t cell activation, and cytokine detection between flow cytometry and either elispot or elisa significantly correlate. since memory cd t cells do not pre-store grzb at levels similar to memory cd t cells, these inter-assay correlations between intracellular and extracellular grzb of memory cd t cells resemble the correlations of other secreted factors that are not pre-stored, such as cytokines. lastly, the correlation between grzb elispot and elisa was comparable for memory cd and memory cd t cells. thus, the correlation between intracellular and extracellular grzb is stronger for memory cd t cells compared to memory cd t cells.figure inter-assay correlations between intracellular and extracellular grzb assays of memory t cells, and intra-donor correlations of grzb production between memory t cells. spearman correlations between intracellular and extracellular grzb measurements of 1× untreated or activated memory cd or memory cd t cells. spearman correlations comparing grzb production of memory cd vs. memory cd t cells within single donors as measured by flow cytometry, elispot, or elisa. the bulk data in figure  suggests that intracellular grzb is increased by activated memory cd but not by memory cd t cells, whereas extracellular grzb is increased by both activated memory cd and memory cd t cells. however, when we examined grzb production by memory cd and memory cd t cells within individual donors to determine if increased grzb production by memory cd t cells is also associated with increased grzb production by memory cd t cells, and vice versa, correlations of grzb production between memory cd and memory cd t cells from the same donor were weak. spearman correlations of intracellular and extracellular grzb production also indicated that no significant correlations exist between memory cd and memory cd t cells for any assay. the use of grzb absolute or percent changes also yielded no significant correlations between memory cd and memory cd t cells for each assay. although figure  shows that grzb production by memory t cells is generally increased by t cell activation, these data in figure  suggest that within an individual, the induction and secretion levels of grzb are different and unique between memory cd and memory cd t cells. association of cd107a degranulation marker with extracellular, but not intracellular, granzyme b of memory t cells we examined the association of memory t cell grzb with activation and degranulation markers, which more accurately indicates grzb-expressing cells that are also actively secreting grzb. cd is an early t cell activation marker that is transiently upregulated by activated t cells, and cd107a is a lysosomal protein detectable by flow cytometry surface staining after transient lysosomal fusion to the cellular membrane and release of granule proteins during t cell activation. surface expression of cd107a is also associated with perforin release by human cd t cells, and correlates with ctl effector function. after hrs costimulation, total cd expression by memory cd and memory cd t cells were similar. if gated on total memory cd or memory cd t cells, cd coexpression with grzb was higher by activated memory cd compared to memory cd t cells. however, out of the grzb+ memory cd or memory cd t cells, cd expression was higher by memory cd compared to memory cd t cells, indicating that more grzb-expressing memory cd t cells are also activated compared to memory cd t cells.figure association of memory t cell grzb secretion with activation and degranulation markers. memory t cell flow cytometry dotplots and mean ± sem total surface cd expression or cd69+/grzb+ coexpression, or cd expression gated on grzb+ memory cd or memory cd t cells. flow cytometry dotplots and mean ± sem total surface cd107a expression or cd107a+/grzb+ coexpression, or cd107a expression gated on grzb+ memory cd or memory cd t cells. grzb elisa of memory t cells conducted in parallel with cd107a experiments. spearman correlations between cd107a and intracellular or extracellular grzb of memory t cells. after hrs costimulation, total cd107a expression was  ±  % in activated memory cd and  ±  % in memory cd t cells. if gated on total memory cd or memory cd t cells, cd107a+/grzb+ coexpression was  ±  % by memory cd but higher at  ±  % by memory cd t cells, which may more accurately corroborate the frequency of less than 1% activated memory t cells that secrete grzb as indicated by elispot in figure 1b. but unlike the difference of cd expression between activated memory t cells shown in figure 3d, cd107a expression by activated grzb+ memory t cells was similar. additionally, the parallel extracellular grzb levels for these experiments were similar between activated memory t cells, suggesting that cd107a is more accurate than cd as an indicator of grzb secretion by activated memory t cells. for memory cd t cells, the spearman correlation of cd107a with intracellular grzb was r = , and r =  with extracellular grzb. similarly for memory cd t cells, the correlation of cd107a with intracellular grzb was r = -, and r =  with extracellular grzb. despite increased surface expression of cd107a and release of grzb by activated memory cd t cells, intracellular grzb of memory cd t cells does not significantly change after activation as shown by flow cytometry in figure 1a, which may be consistent with an earlier report that demonstrates by flow cytometry that increased surface expression of cd107a and cd107b by human cd t cells is associated with loss of intracellular perforin. for memory cd t cells, both cd107a and intracellular grzb are associated with grzb secretion to similar degrees, and increased surface expression of cd and cd107a by activated memory cd t cells is further associated with the increased intracellular grzb expression shown by flow cytometry in figure 1a. these data further corroborate figure  in which activated memory cd t cells secrete grzb mainly from pre-stored granules, whereas grzb secretion by memory cd t cells is likely derived from pre-stored and newly synthesized grzb to achieve the similar extracellular grzb levels. despite higher intracellular storage of grzb by activated memory cd compared to memory cd t cells, cd107a expression by activated grzb+ memory t cells, as well as the number of grzb-secreting cells, were similar. this may suggest that a higher proportion of activated grzb+ memory cd t cells secrete and deplete their grzb stores. this contrasts with higher retention of intracellular grzb by activated memory cd t cells, since elispot numbers would be substantially higher if all activated cd t cells released their intracellular grzb stores. these differences may allow memory cd t cells to be more sustained serial killers of target cells, whereas memory cd t cells may express grzb for shorter-term functions. less association of granzyme b with perforin by memory cd t cells compared to memory cd and natural killer cells we next examined grzb production by memory t cells in conjunction with perforin, and also in comparison with natural killer cells, abundant producers of grzb and perforin. in addition to grzb, cd t cells express perforin to eliminate target cells. memory cd4+cd45ro+ and memory cd8+cd45ro+ t cells, and cd56+ nk cells, were purified from peripheral blood of the same donor and cultured in parallel, with or without the intracellular protein transport inhibitor brefeldin. memory t cells and nk cells were stimulated for hrs with il. additionally, memory t cells were alternatively stimulated with soluble anti-cd mabs only, as cd expression by memory cd t cells was modestly lower compared to memory cd t cells, which may result in memory cd t cells receiving stronger stimulation by cd3/cd costimulation compared to memory cd t cells. agonism of other costimulatory receptors such as icos or tnf superfamily members may further yield different results. intracellular and extracellular perforin and grzb were measured by flow cytometry and elisa. figure 4a-b shows representative perforin/grzb flow cytometry dotplots and the mean distribution of intracellular perforin/grzb subpopulations for memory t cells and nk cells. for memory cd t cells, only perforin-/grzb+ cells were mainly observed in all conditions, and a small percentage of perforin+/grzb+ cells were observed by il stimulation. however, memory cd t cells expressed more intracellular perforin and grzb. in the absence of brefeldin, ut memory cd t cells were ± % perforin+/grzb-, ± % perforin-/grzb+, and ± % perforin+/grzb+. il stimulation changed these perforin/grzb distributions by increasing perforin expression, whereas cd stimulation did not affect the perforin/grzb expression patterns. in the presence of brefeldin, perforin expression was mitigated and memory cd t cells were mostly perforin-/grzb+. as expected, intracellular perforin and grzb expression was substantially higher for nk cells compared to memory t cells. in the absence of brefeldin, nk cells were mostly perforin+/grzb+, by contrast to memory t cells which were mostly perforin-/grzb+. but in the presence of brefeldin, this distribution changed to mostly perforin-/grzb+, which was similar to the perforin mitigation by brefeldin of memory cd t cells. thus, intracellular perforin is marginally expressed with grzb by stimulated memory cd t cells, whereas perforin is more associated with grzb in memory cd t cells and nk cells.figure comparison of grzb and perforin expression between memory cd memory cd and natural killer cells. representative grzb/perforin dotplots of memory cd memory cd and nk cells. cells were purified from peripheral blood, and 5× cells were untreated or stimulated with il or anti-cd only mabs for hrs +/- brefeldin. mean distribution of perforin/grzb subsets of memory cd memory cd and nk cells. extracellular grzb and perforin production by memory cd memory cd and nk cells after hrs culture +/- brefeldin. despite little to no intracellular perforin expression by memory cd t cells, perforin was secreted with grzb by stimulated memory cd t cells as determined by elisa, but in a pattern distinct from memory cd t cells. in the absence of brefeldin, il2- and cd3-stimulated memory cd t cells released  ±  pg/ml and 336 ±  pg/ml grzb, respectively, more than memory cd t cells which released 249 ±  pg/ml and 86 ±  pg/ml grzb. however, ut and il2-stimulated memory cd t cells released 372 ±  pg/ml and  ±  pg/ml perforin, which was more than memory cd t cells which released 159 ±  pg/ml and 521 ±  pg/ml perforin. perforin release by cd3-stimulated memory t cells was similar. as expected, ut and il2-stimulated nk cells secreted substantially more perforin and grzb compared to memory t cells in the absence of brefeldin. in the presence of brefeldin, perforin and grzb release by stimulated memory t cells was reduced nearly to baseline levels, and reduced ~2- fold by nk cells. an unclear effect of brefeldin upon resting memory t cells and nk cells was observed, in which a modest decrease of constitutive perforin release, but an increase of grzb release, by ut memory t cells and nk cells occurred. it is possible that intracellular storage of grzb may be more disassociated with perforin by resting t cells and nk cells compared to activated states. additionally, il2-stimulated memory cd t cells and nk cells appear to secrete more grzb than perforin, whereas memory cd t cells secrete more perforin than grzb. lastly, despite the release of perforin and grzb by stimulated memory t cells and nk cells, intracellular accumulation of perforin and grzb in stimulated cells did not occur in the presence of brefeldin. this is likely due to glycosylation requirements of perforin and grzb for intracellular transport and secretion, which would be inhibited by brefeldin. memory cd t cells with granzyme b production capacity include tregs and th cells, with potentially more distinct grzb vs. cytokine secretory mechanisms compared to cd t cells we lastly examined memory cd t cell subsets that produce grzb such as treg, th th and th cells. memory cd4+cd45ro+ t cells were purified from peripheral blood and activated or ut for hrs. cells were then analyzed by flow cytometry for phenotype and function in conjunction with grzb expression. hrs costimulation or pma/io treatment of memory cd t cells resulted in grzb expression by phenotypes consistent with treg and th cells. tregs were examined by measuring intracellular grzb expression by cd25+/foxp3+ costimulated memory cd t cells. activated memory cd t cells expressed ~18% cd. grzb was coexpressed with foxp by cd25-dim cells, and more so by cd25-bright cells. this is consistent with reports that demonstrate treg expression of grzb for suppressive functions. ifnγ was modestly induced and coexpressed with grzb by costimulated memory cd t cells, but more robustly by pma/io treatment, indicating that memory cd th cells express grzb, consistent with other reports. by contrast, il was not induced by costimulated or pma/io-treated memory cd t cells, suggesting that grzb is not expressed by memory cd th cells. il17a was induced by costimulation and pma/io, but not coexpresssed with grzb, suggesting that in these short-term conditions memory cd th cells may not express grzb. th cells are reported to express grzb in vivo, which likely involves more complex stimuli, timing or differentiation, and more specific phenotypic characterization. thus, tregs and th cells are subsets of peripheral blood memory cd t cells that produce grzb in the present study, although other grzb-producing subsets, particularly cd ctl’s, are likely involved as well.figure grzb production by memory cd t cell subsets and coexpression with cytokines. grzb expression by tregs. memory cd t cells were purified from peripheral blood, and 5× cells were untreated or activated for hrs. cells were then stained for cd foxp and grzb. cd expression levels by memory cd t cells after hrs culture. foxp3/grzb expression by cd25-negative, cd25-dim, or cd25-bright memory cd t cells after hrs culture. graph shows the mean distribution of foxp3/grzb subsets gated on cd25-negative, -dim, or -bright cells. grzb coexpression with ifnγ, il or il17a by memory cd t cells. memory cd t cells were untreated for activated for hrs, then stained for grzb and either ifnγ, il or il17a. graphs show the mean distribution of cytokine/grzb subsets. grzb/ifnγ coexpression by memory cd t cells. purified memory cd t cells were untreated or activated for hrs. graph shows the mean distribution of ifnγ/grzb subsets. for comparison of cytokine/grzb coexpression by memory cd th cells, purified memory cd8+cd45ro+ t cells were examined for ifnγ and grzb coexpression as well. compared to cd t cells or nk cells, cytokine secretion pathways of cd t cells may be more diversified and distinct from those of cytotoxic mediators such as granzymes. for example, in activated murine cd t cells, il and ifnγ colocalize and are secreted by a more polarized mechanism compared to other secreted mediators such as tnfα and chemokines. additionally in nk cells, ifnγ and tnfα are stored and packaged in vesicles distinct from perforin-containing granules, and secreted in a more nonpolarized fashion compared to perforin. in the present study, hrs cd3/cd costimulation of memory cd t cells induced  ±  % ifnγ+/grzb- and  ±  % ifnγ+/grzb+ expression, and pma/io treatment induced  ±  % ifnγ+/grzb- and  ±  % ifnγ+/grzb+ expression. thus, compared to pma/io-treated memory cd t cells, ifnγ+/grzb+ expression by pma/io-treated memory cd t cells was significantly higher, suggesting that cytokine, as well as perforin, secretion pathways of memory cd t cells may be more disassociated with grzb secretion compared to memory cd t cells. these differences may be relevant to the weak correlations of grzb expression and secretion between memory cd and memory cd t cells within single donors shown in figure 2g-i.  in the present study, grzb expression and secretion was directly compared between human memory cd and memory cd t cells using flow cytometry, elispot, and elisa. the results show that memory cd t cells store more grzb, but do not secrete more grzb, compared to memory cd t cells during short-term activation, suggesting that activated cd t cells regulate their grzb release more than cd t cells. consequently, the correlation between intracellular and extracellular grzb for memory cd t cells is weaker compared to memory cd t cells. additionally, cd107a is a better indicator of grzb secretion by memory t cells than intracellular grzb itself. these results corroborate and further clarify previous studies examining grzb production by human or mouse cd and cd t cells, which utilized either flow cytometry, elispot, or elisa, but not all three methods simultaneously. distinct patterns of grzb production between memory t cells may further be due to different associations with other secreted factors such as perforin or cytokines. abbreviations ctlcytotoxic t lymphocyte lamp-1lysosomal-associated membrane protein1 tcrt cell receptor lin lin and jacob couturier contributed equally to this work. competing interests the authors declare that they have no competing interests. authors’ contributions ll, jc, and mam performed experiments, analyzed data, and wrote the manuscript. xy and cak analyzed data. del supervised the project and reviewed the manuscript. all authors read and approved the final manuscript. this work was supported by nih/niaid baylor college of medicine/ut-health center for aids research grant ai ro, and shandong medicine and health technology development program grant 2011hw.
although measurement of intracellular grzb by flow cytometry suggests that a larger proportion of cd t cells have higher capacity for grzb production compared to cd t cells, elispot and elisa show that similar numbers of activated cd and cd t cells secrete similar amounts of grzb. granzyme b is a serine proteinase expressed by memory t cells and nk cells. elisaelispotflow cytometrygranzyme bmemory t cellsperforinissue-copyright-statement© the author 2014 expression of cd107a further indicated that grzb is secreted similarly by activated cd and cd t cells, consistent with the elisa and elispot results. memory cd and memory cd t cells were purified from human peripheral blood of healthy donors, and production of grzb was directly compared between memory cd and memory cd t cells from the same donors using parallel measurements of flow cytometry, elispot, and elisa. memory cd t cells constitutively stored significantly more grzb protein compared to memory cd t cells as determined by flow cytometry, and this difference remained stable after hrs of activation. because grzb is an important mediator of cell death, tissue pathology and disease, clarification of differences of grzb expression and secretion between cd and cd t cells is important for understanding effector functions of these cells. however, measurement of extracellular grzb by elisa revealed that activated memory cd t cells secrete similar amounts of grzb compared to memory cd t cells. measurement of individual grzb-secreting cells by elispot also indicated that similar numbers of activated memory cd and memory cd t cells secreted grzb. methods to measure grzb protein usually involve intracellular and extracellular assays.
 granzyme b is a serine proteinase expressed by memory t cells and nk cells. methods to measure grzb protein usually involve intracellular and extracellular assays. cd t cells are the main source of grzb during immunological reactions, but activated cd t cells deploy grzb as well. because grzb is an important mediator of cell death, tissue pathology and disease, clarification of differences of grzb expression and secretion between cd and cd t cells is important for understanding effector functions of these cells.  memory cd and memory cd t cells were purified from human peripheral blood of healthy donors, and production of grzb was directly compared between memory cd and memory cd t cells from the same donors using parallel measurements of flow cytometry, elispot, and elisa. memory cd t cells constitutively stored significantly more grzb protein compared to memory cd t cells as determined by flow cytometry, and this difference remained stable after hrs of activation. however, measurement of extracellular grzb by elisa revealed that activated memory cd t cells secrete similar amounts of grzb compared to memory cd t cells. measurement of individual grzb-secreting cells by elispot also indicated that similar numbers of activated memory cd and memory cd t cells secreted grzb. expression of cd107a further indicated that grzb is secreted similarly by activated cd and cd t cells, consistent with the elisa and elispot results. however, memory cd t cells expressed and secreted more perforin compared to memory cd t cells, suggesting that perforin may be less associated with grzb function for memory cd t cells.  although measurement of intracellular grzb by flow cytometry suggests that a larger proportion of cd t cells have higher capacity for grzb production compared to cd t cells, elispot and elisa show that similar numbers of activated cd and cd t cells secrete similar amounts of grzb. secretion of grzb by activated cd t cells may be more tightly controlled compared to cd t cells. keywords elisaelispotflow cytometrygranzyme bmemory t cellsperforinissue-copyright-statement© the author 2014 