BACKGROUND
peripheral residence of naïve cd <dig> t cells is marked by many complex microenvironmental interactions.
clonal t cell receptors  expressed on individual t cells are likely to have variable avidity for the self-peptide mhc class ii  complexes they are positively selected on in the thymus, and this avidity leads to tuning of the response of individual t cells  <cit> .
naïve cd <dig> t cells survive because they receive tonic signals through these spmhcii complexes  <cit> .
the levels and availability of these spmhcii complexes are variable  <cit> , plausibly leading to variations in the frequencies of tonic signaling for individual cd <dig> t cells.
notably, tcr-mediated signal sensitivity in mouse t cells is also modulated by mirna-181a  <cit> , which represses expression of dual-specific phosphatase- <dig> , a molecule linked to human longevity  <cit>  which suppresses phosphorylation of erk in human t cells  <cit> .
the levels of cd <dig>  a negative regulator of tcr signaling  <cit> , show modulations correlated with the efficiency of tcr-mediated tonic signaling required for peripheral t cell survival  <cit> .
there is also evidence that persistent tonic signaling itself may lead to hyporesponsiveness and reduction in co-receptor levels in naive cd <dig> t cells  <cit> .
further, naïve t cells can proliferate in the periphery in a cognate ligand-independent fashion if they are in lymphocyte-depleted environments such as during early fetal-neonatal development in the neonatal mouse or in the aged animal with reduced thymic output  <cit> , although the precise functional consequences of such homeostatic proliferation are not yet clear  <cit> .
cytokines such as il- <dig>  − <dig>  − <dig> and their receptors have been implicated in peripheral t cell homeostasis and survival  <cit> , and common receptor gamma-chain-deficient naive cd <dig> cells in the periphery are smaller in size  <cit> , consistent with the possibility that cellular metabolism may be a major variable in determining peripheral survival and functionality of naïve t cells  <cit> .
finally, stochasticity in levels of gene expression and responsiveness is expected to mark any apparently homogenous population of cells including t cells  <cit> , although it is difficult to distinguish between subset-dependent variations and true statistical noise.
the thymus continuously exports naïve mature t cells to the periphery with declining output due to age-associated involution.
many of these naïve t cells in the periphery remain naïve until their eventual loss from the peripheral pool  <cit> .
thus, the post-thymic, peripheral residence times of naive t cells at any given point in time would be variable.
as thymic output decreases with age, naïve t cells in the periphery also appear to be longer-lived  <cit> .
thus, it is likely that the naïve t cells from aged animals will show a longer average peripheral residence time than those from young individuals  <cit> , and this may be connected to the functional cd <dig> t cell defects seen in aged animals  <cit> .
while there are some markers to identify recent thymic emigrants  <cit> , there are currently no phenotypic modalities to measure peripheral residence time in individual naïve t cells in heterogeneous populations.
on this background, we began looking for variable functional behavior in mouse  peripheral naïve cd <dig>  t cells differing in properties that were apparently unimodally distributed, with the initial expectation that these differences would be stochastic in nature.
cd <dig> was chosen since it shows low variance in its distribution and is uniformly present without major alterations through the entire lifespan of cd <dig> t cells.
we find that the top and bottom decile of cd <dig> levels in ncd <dig> t cells define cells that are both phenotypically and functionally different, with ncd4lo cells having smaller sizes, higher cd <dig> levels, poor responses, lower levels of the dusp6-suppressing micro-rna181a, and greater susceptibility to death and to th2-skewing.
however, we find that these differences are not stochastic; they are not found in ncd4lo and ncd4hi t cells in the thymus, but they persist even when the tcr repertoire of the cd <dig> t cell population is homogenized as in ncd <dig> t cells from tcr-transgenic mice, and they can be induced by exposure in vivo to mhcii-mediated tonic signals during a few days of peripheral residence, indicating that unimodal distribution of a variable does not necessarily mean that the variability is stochastic.
dusp and reactive oxygen species  appear to mediate the mhcii-induced hyporesponsiveness of ncd <dig> t cells, since ros scavenging or dusp inhibition ameliorate it.
finally, consistent with the greater average time of peripheral residence of ncd <dig> t cells in aged animals  <cit> , we find that the properties found in ncd4lo t cells from young mice are also found in the ncd <dig> t cells of aged mice, making ros and dusp potential targets for intervention for successful vaccination in the older population.
RESULTS
despite unimodal distribution, cd <dig> levels on naïve cd <dig> t cells are correlated with responsiveness
ncd <dig> cells show unimodal distribution of cd <dig> levels.
to examine whether this apparently homogenous population has any functional consequences, we sorted mouse splenic ncd <dig> cells  into the brightest  and dullest  deciles of cd <dig> levels .
there was no overlap in the cd <dig> levels of these sorted populations, which typically differed by approximately two-fold .
we next characterized these sorted ncd4hi and ncd4lo cells in functional terms.
purified cells were activated with plate-coated anti-cd3 + anti-cd <dig> monoclonal antibodies  for 18 hours and the frequency of cells showing induction of cd <dig> as an early activation marker was estimated.
smaller proportions of ncd4lo cells than of ncd4hi cells expressed cd <dig> at multiple anti-cd <dig> concentrations .
further, ncd4lo cells produced less il- <dig> at 48 hours  and incorporated less -thymidine at 60 hours post-activation .
poor proliferation of activated ncd4lo cells was also confirmed in a carboxyfluorescein succinimidyl ester  dilution assay .
we examined the possibility that anti-cd <dig> antibody bound during sorting signals differentially to the ncd4hi and ncd4lo cells, by resting the sorted cells for 24 hours in il- <dig> before stimulating them with anti-cd3 + anti-cd <dig> for 48 hours.
the difference in their proliferative responses persisted, indicating that it was not related to any anti-cd4-mediated signaling artifact .figure  <dig>
despite unimodal distribution, cd <dig> levels on naïve cd <dig> t cells are correlated with responsiveness.
a. gating strategy used to sort ncd4lo and ncd4hi cells from six- to eight-week-old mice.
right panel shows sort profile for ncd4hi and ncd4lo cells.
b. cd <dig> expression on anti-cd3 + anti-cd <dig>  stimulated ncd4lo and ncd4hi cells 16 hours post-activation.
numbers indicate representative proportions of cd69+ cells in ncd4hi  and ncd4lo  cells.
c. proportions of cd69+ cells in a dose–response curve with 3 μg/ml of anti-cd <dig> and titrating anti-cd <dig> concentrations as shown.
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d. amount of il- <dig> detected 48 hours post-stimulation with anti-cd3 + anti-cd <dig> in culture supernatants.
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e. 3h-thymidine incorporation assay to measure proliferation 60 hours post stimulation with anti-cd3 + anti-cd <dig>  .
f. 3h-thymidine incorporation assay on sorted ncd4hi and ncd4lo cells cultured in vitro with il- <dig> for 24 hours prior to stimulation with anti-cd3 + anti-cd <dig> for 60 hours.
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g. proportion of trypan blue-positive cells 16 hours post-activation .
h. cytokine levels in supernatants of in vitro primed and recalled ncd4hi and ncd4lo cells.
i. ifnγ/il- <dig> ratios and mean ± se calculated based on absorbance values.
se, standard error.
since il- <dig> induction was different between ncd4lo and ncd4hi cells, we tested if exogenous il- <dig> supplementation could abolish the difference.
when exogenous il- <dig>  was added during activation, ncd4lo cells showed some improvement in proliferative responses at lower anti-cd <dig> concentration, but at no point did their responses reach levels achieved by ncd4hi cells , suggesting that, while differential il- <dig> production may contribute to the difference, it was unlikely to represent the sole explanation.
there was more prominent activation-induced cell death in ncd4lo cells at 24 hours post-activation, as scored by trypan blue staining, than in ncd4hi cells .
we also tested the ability of sorted ncd4hi and ncd4lo cells to differentiate in vitro in response to non-polarizing activating conditions .
supernatants from cultures of ncd4hi and ncd4lo cells primed in vitro with anti-cd3 + anti-cd <dig> for three days and then restimulated with titrating doses of anti-cd <dig> were analyzed for ifnγ and il- <dig>  effector cells generated from ncd4lo cells produced more il- <dig> as compared to those from ncd4hi cells .
the relative th1/th <dig> dominance, indicated by the ifnγ/il- <dig> ratio, was significantly lower in differentiated ncd4lo cells .
ncd4lo cells show complex phenotypic modifications, but no differences in proximal signaling events
cd <dig> is a negative regulator of tcr signaling  <cit>  and cd <dig> expression has been inversely correlated with t cell responsiveness  <cit> .
we, therefore, tested the possibility that lower cd <dig> levels might correlate with higher cd <dig> expression.
ncd4hi and ncd4lo cells from young b <dig> mice  were tested for the levels of cd <dig>  when gated ncd4hi and ncd4lo cells were analyzed for cd <dig> expression, it was apparent that ncd4lo cells had higher cd <dig> levels  in multiple mice analyzed .
this was despite the finding that ncd4hi cells were somewhat larger than ncd4lo cells .
we also examined the levels of a number of other molecular markers in these populations.
ncd4lo cells showed lower tcrβ  levels that ncd4hi cells, although both ncd4lo and ncd4hi cells were confirmed to be conventional cd <dig> t cells expressing tcrα/β.
ncd4lo cells also expressed somewhat lower levels of mhc class i and cd <dig>  but higher levels of cd <dig>  in multiple mice examined.
another feature tested based on differences in cell size between ncd4lo and ncd4hi cells was mitochondrial mass and potential using indicator dyes, mitotracker green  and mitotracker red   <cit> .
however, ncd4lo cells did not show any striking differences from ncd4hi cells in mitochondrial content or potential .
these data indicated that the phenotypic differences between ncd4lo and ncd4hi cells were likely to be complexly regulated.figure  <dig>
ncd4lo cells show higher cd <dig> levels but no difference in proximal signaling.
a. ex vivo analysis of stained splenic cells to show gated ncd4lo and ncd4hi cells.
b. comparison of cd <dig> levels as log  and linear  plots between ncd4hi and ncd4lo cells.
c. cd <dig> mfi values for ncd4hi and ncd4lo cells .
d. comparison of forward scatter between ncd4hi and ncd4lo cells.
e. forward scatter values on ncd4hi and ncd4lo cells .
f. comparison of tcrβ levels as log  and linear  plots between ncd4hi and ncd4lo cells.
g. tcrβ mfi values for ncd4hi and ncd4lo cells .
h. representative profile of fluo-3/fura-red ratio in ncd4hi and ncd4lo cells at rest and post-activation.
i. pooled data from independent mouse spleens showing increase in fluo-3/fura-red ratio with anti-cd <dig>  + anti-cd <dig>  treatment j. pooled data for fold increase over background in pzap- <dig> staining mfi in ncd4hi and ncd4lo cells activated with anti-cd3 + anti-cd <dig>  mean ± se.
mfi, mean fluorescence intensity; ncd <dig>  naïve cd <dig> t cells; se, standard error; tcr, t cell receptor.
since far-downstream proliferative outcomes of tcr activation differed between ncd4lo and ncd4hi cells, we next examined proximal signaling events after ligation with anti-cd3 + anti-cd <dig>  activation-induced calcium flux  was indistinguishable between ncd4hi and ncd4lo cells .
similarly, total zap- <dig> levels were comparable between ncd4hi and ncd4lo cells , and the induction of zap- <dig> phosphorylation upon activation for five minutes did not differ between ncd4lo and ncd4hi cells .
similarly, total lck levels were comparable in ncd4lo and ncd4hi cells, and the extent of p-lck induction at five minutes post-activation was similar between them .
together, these data suggest that proximal signaling is not different between ncd4hi and ncd4lo cells, and that the differences between them may lie further downstream.
hypo-responsiveness associated with ncd4lo cells develops exclusively in the periphery and is also seen in monoclonal t cells
in order to see if these properties of ncd4hi and ncd4lo cells is an intrinsic characteristic in developing ncd <dig> populations, we tested thymic mature single-positive  cd8-cd4 + cd24-qa2+ ncd <dig> cells.
when these cells were gated to identify cd4lo and cd4hi cells , it was apparent that they show no differences in either cell size  or cd <dig> levels .
when these cd4lo and cd4hi cd4sp thymocytes were sorted and activated with plate-coated anti-cd3 + anti-cd <dig> they responded comparably  unlike ncd4lo and ncd4hi cells from the periphery .
these data indicate that the functional and phenotypic differences observed between peripheral ncd4lo and ncd4hi cells are not thymic in origin.figure  <dig>
hypo-responsiveness associated with ncd4lo cells develops exclusively in periphery and is also seen in monoclonal t cells.
a. ex vivo analysis of stained thymic cells to show single positive mature cd4lo and cd4hi cells , and corresponding forward scatter  .
b. levels of cd <dig> in log  and linear  scale from the same experiment as in a. c. 3h-thymidine incorporation assay to measure proliferation of sorted thymic single positive mature cd4lo and cd4hi cells 60 hours post-activation with anti-cd <dig>  and anti-cd <dig>  .
d. 3h-thymidine incorporation at 60 hours of sorted ncd4lo and ncd4hi cells from tcr transgenic ot-ii mice to titrating concentrations of ova-ii peptide with irradiated b <dig> dcs as apcs .
e. amount of il- <dig> estimated from supernatants 48 hours post stimulation.
data from one of four experiments.
apcs, antigen-presenting cells; dcs, dendritic cells; ncd <dig>  naïve cd <dig> t cells; se, standard error; tcr, t cell receptor.
in order to examine if the hyporesponsiveness of ncd4lo cells could be plausibly ascribed to differences in the normal polyclonal tcr repertoire  <cit> , we sorted splenic ncd4hi and ncd4lo cells from mice transgenic for an mhcii-restricted tcr  and stimulated these sorted cells with titrating concentrations of the cognate ovalbumin peptide  presented by bone-marrow derived dendritic cells  as antigen-presenting cells .
ncd4lo ot-ii cells responded poorly to cognate peptide and produced less il- <dig> than ncd4hi ot-ii cells did , showing that even in naïve t cells with a single tcr, cd <dig> levels were correlated with responsiveness.
human ncd4lo and ncd4hi cells show properties similar to those from mice
we tested if the distinctions observed between ncd4lo and ncd4hi mouse cells were also found in human cd <dig> cells.
for this, we examined ncd <dig> cells  from human peripheral blood mononuclear cells  from healthy donors.
when gated ncd4hi and ncd4lo populations  were examined, ncd4lo cells were smaller in size in multiple donors , consistent with the mouse data.
sorted human ncd4hi and ncd4lo cells showed about two-fold difference in cd <dig> mean fluorescence intensity  values  similar to mouse cells.
upon activation with plate-coated anti-cd3 + anti-cd <dig> mabs, ncd4hi cells proliferated much more than ncd4lo cells did .
mitochondrial mass and membrane potential were also evaluated in these cells using mg and mr  as for mouse cells.
similar to mouse ncd4lo cells, human ncd4lo cells, too, showed no striking differences from ncd4hi cells in mitochondrial mass and potential .figure  <dig>
ncd4lo and ncd4hi cells from human pbmcs show properties similar to mouse cells.
a. a representative gating strategy for ncd4lo and ncd4hi cells , histogram overlay of sorted ncd4lo and ncd4hi cells  and analysis of the same cells for size .
b. forward scatter for ncd4hi and ncd4lo cells from individual donors .
c. 3h-thymidine incorporation in response to titrating doses of anti-cd <dig> and 3 μg/ml of anti-cd <dig> 60 hours post-activation .
d. histogram overlays for mg staining in log  and linear  scale in ncd4lo and ncd4hi.
e. mfi for mg on ncd4hi and ncd4lo cells from six individual donors .
f. histogram overlays for mr staining in log  and linear  scale in ncd4lo and ncd4hi.
g. mfi for mr on ncd4hi and ncd4lo cells from six individual donors .
mfi, mean fluorescence intensity; mg, mitotracker green; mr, mitotracker red; ncd <dig>  naïve cd <dig> t cells; pbmc, peripheral blood mononuclear cells; se, standard error.
consequences of peripheral residence on ncd <dig> t cells
the data so far suggest that the association between cd <dig> levels and functional phenotype was a peripherally induced characteristic.
in order to probe this further, we began by sorting splenic ncd4lo and ncd4hi cells.
we labelled these cells with green and violet dyes for identification, mixed them in 1: <dig> proportion and transferred them intravenously  to wild type  recipients.
on day  <dig> post-transfer, donor ncd <dig> cells were identified in recipient spleens and analyzed for their cd <dig> levels.
comparison of cd <dig> levels on ncd4hi and ncd4lo cells before  and after transfer , done in parallel, showed that adoptively transferred ncd4lo cells continued to show nearly unchanged cd <dig> levels which were lower than transferred ncd4hi cells .
cd <dig> levels on ncd4hi cells declined from d <dig> levels to day  <dig> after transfer, indicating that naïve cd <dig> t cells lose cd <dig> levels over time in the periphery.
however, despite this decline, ncd4hi cells continued to have higher cd <dig> levels than ncd4lo cells up to day  <dig> after transfer .
notably, there was no appreciable difference in the ability of transferred cells to survive in vivo, at least for a period of one week .
these data suggest that cd <dig> levels do not change randomly in vivo but are likely to be reduced over time, perhaps with longer residence.figure  <dig>
consequences of peripheral residence in ncd <dig> t cells.
a. cd <dig> levels on sorted, and differentially labeled ncd4lo  and ncd4hi  cells prior to  and four days after transfer.
left and middle panel – log plots, right panel – an overlay in linear scale .
b. cd <dig> mfi values for ncd4lo and ncd4hi cells seven days post-transfer.
each circle represents an individual recipient mouse and the horizontal line indicates mean ± se.
c. number of transferred ncd4hi and ncd4lo cells recovered from individual recipient mouse spleens seven days post-transfer shown as mean ± se .
d. 3h-thymidine incorporation in ot-ii cells parked in wt mice for one or five weeks in response to titrating concentrations of ova-ii peptide.
data normalized for  <dig> ot-ii cells .
horizontal line indicates background cpm.
e. levels of il- <dig> in culture supernatants from a representative experiment as in panel d. horizontal line indicates background il- <dig> levels .
f. histogram overlays showing cd <dig> levels as log and linear plots of one-week-parked or eight-week-parked ot-ii cells.
g. cd <dig> mfi levels from long-parked  and short-parked  ot-ii cells .
mfi, mean fluorescence intensity; ncd <dig>  naïve cd <dig> t cells; se, standard error; wt, wild type.
we also used the lymphoreplete environment of wt congenic mice to analyze the consequences of prolonged peripheral residence on ncd <dig> t cell function.
b <dig> sjl mice were given  <dig> ×  <dig> ot-ii cells and the proliferative responses of these donor cells in vitro were analyzed one to eight weeks later.
ot-ii cells parked for five weeks in vivo responded poorly in terms of proliferation and il- <dig> production than those parked for one week .
when cd <dig> levels on these parked ot-ii cells were analyzed, ot-ii cells parked for one week showed higher cd <dig> levels compared to cells parked for eight weeks .
when ot-ii cells parked for less than two weeks were compared with those parked for more than four weeks using data from multiple experiments, significant differences were found in their relative cd <dig> levels .
do <dig>  cells parked in balb.c mice for six months showed lower cd <dig> levels compared to those parked for two weeks .
consequences of prolonged tonic signaling and absence of tonic signaling in naïve t cells
the data so far suggested the hypothesis that cd <dig> levels and the responsiveness of ncd <dig> cells were reduced with increasing duration of peripheral residence.
during peripheral residence, mhcii-mediated tonic signals are received by ncd <dig> cells.
to test if the reduction in cd <dig> levels and the loss of responsiveness were related to mhcii-mediated tonic signaling, we ‘parked’ purified ncd <dig> ot-ii cells by adoptive transfer into either wt or mhcii-null recipient mice.
when recipients were euthanized on day  <dig> post-transfer, the numbers of ot-ii cells recovered per spleen from wt and mhcii-null recipients were comparable  suggesting that there is unlikely to be a major difference in cell survival or homeostatic proliferation in the two groups at this time post-transfer.
however, ncd <dig> ot-ii cells recovered from wt recipients had lower cd <dig> levels  and higher cd <dig> levels  than cells recovered from mhcii-null recipients.
when these cells were stimulated with the cognate ova-ii peptide, ot-ii cells parked in mhcii-null recipients responded better than the corresponding cells parked in wt recipients .figure  <dig>
consequences of prolonged tonic signaling and absence of tonic signaling in naïve t cells.
a. numbers of adoptively transferred ncd <dig> ot-ii cells recovered from spleens three days after transfer into wt and mhcii-null mice .
b. comparison of cd <dig> levels  of donor ncd <dig> ot-ii cells after three days in wt and mhcii-null mice.
c. pooled data, of paired samples showing cd <dig> mfi and mean ± se .
d. comparison of cd <dig> levels  of donor ncd <dig> ot-ii cells after three days in wt and mhcii-null mice.
e. pooled data, of paired samples showing cd <dig> mfi and mean ± se .
f. proliferation assay of donor ncd <dig> ot-ii cells from wt or mhcii-null recipient mice four days post-transfer in response to ova-ii peptide in the presence of irradiated dcs from wt mice.
data were normalized for  <dig>  donor ncd <dig> ot-ii cells .
g. representative dcfda staining  for donor ot-ii cells from wt or mhcii-null spleen cells seven days post-transfer.
h. pooled data, of paired samples showing dcfda mfi values  in wt or mhcii-null recipients seven days after transfer.
dcs, dendritic cells; dcfda, 2-7-dichlorofluorescin diacetate; mfi, mean fluorescence intensity; ncd <dig>  naïve cd <dig> t cells; se, standard error; wt, wild type.
since accumulation of ros is a common hallmark of senescent cells  <cit> , we examined the possibility that longer parking of ot-ii ncd <dig> cells in wt versus mhcii-null recipients may also lead to differences in ros levels in them.
when ncd <dig> ot-ii cells were parked for ten days in wt or mhcii-null recipients, and their levels of staining intensity for the ros indicator dye 2-7-dichlorofluorescin diacetate  examined, cells parked in wt mice showed higher ros levels than cells parked in mhcii-null recipients .
prolonged residence in vivo leads to loss of functionality and accumulation of ros in ncd <dig> cells
we next examined if ros levels differed in parked cells in normal lymphoreplete mice depending on the duration of parking.
ot-ii cells parked for eight weeks in wt congenic lymphoreplete mice showed higher intensities of dcfda staining as compared to cells parked for one week .
in multiple experiments when ot-ii cells parked for less than two weeks were compared with those parked for more than four weeks, significant differences were found in their relative dcfda staining intensities .figure  <dig>
prolonged residence
in vivo
leads to loss of functionality and accumulation of ros in ncd <dig> cells.
a. histogram overlays  for dcfda from ot-ii cells parked for one or eight weeks in wt mice.
b. dcfda mfi values from long-parked  and short-parked  ot-ii cells .
c. 3h-thymidine incorporation in response to titrating concentrations of ova-ii peptide in ot-ii cells parked for one or six weeks in wt mice .
data normalized to  <dig> ot-ii cells .
horizontal line indicates background cpm.
dcfda, 2-7-dichlorofluorescin diacetate; mfi, mean fluorescence intensity; ncd <dig>  naïve cd <dig> t cells; ros, reactive oxygen species; se, standard error; wt, wild type.
we next tested if ros scavenging could ameliorate the relative hyporesponsiveness induced in ot-ii cells by prolonged parking.
ot-ii cells were adoptively transferred into wt recipients that were given alternate-day treatment with either vehicle alone or a superoxide dismutase /catalase mimic, euk- <dig>  <cit> , for five weeks following adoptive transfer.
wt recipients given ot-ii cells one week previously were also used as a control group.
ot-ii cells from euk-134-treated recipients showed a better proliferative response at the end of six weeks of parking compared to cells from vehicle- treated recipients, although their responses still remained poorer than those of ot-ii cells parked for only one week .
since ros level differences had become evident by 10 days or so, we tested if a shorter period of euk- <dig> treatment was sufficient to improve reactivity of parked ot-ii cells.
however, 15-day euk- <dig> treatment did not result in any improvement of reactivity .
these data suggest that the ros-mediated contribution to hyporeactivity requires fairly prolonged ros exposure, and, thus, ros is clearly not the major factor imposing hyporeactivity on ncd <dig> cells upon prolonged peripheral residence.
the erk-dusp6-mir-181a axis contributes to hyporesponsiveness of ncd <dig> t cells during peripheral residence
phosphorylation of mitogen-activated protein  family tyrosine kinase erk has been shown to be poor in many aging cells, including t cells  <cit>  suggesting that t cell age may also be correlated with erk phosphorylation.
we examined baseline p-erk levels ex vivo in ncd4hi and ncd4lo cells.
ncd4lo cells show significantly lower levels compared to ncd4hi cells .
the defective phosphorylation of erk in human t cells is regulated by the activity of dusp <dig>  which in turn is controlled by mir-181a  <cit> .
ncd <dig> cells from older people are reported to express lower levels of mir-181a  than ncd <dig> cells from young people  <cit> .
we therefore estimated the levels of mir-181a and mir- <dig> transcripts in mouse t cells using real-time rt-pcr assays.
levels of mir-181a transcripts  were lower in ncd4lo cells compared to ncd4hi cells , further suggesting that because of longer peripheral residence ncd4lo cells show a pattern similar to that reported in ncd <dig> t cells from older individuals  <cit> .
consistent with the role of mirna-181a in suppressing dusp <dig> expression  <cit> , when ncd4lo and ncd4hi cells were treated with a pharmacological inhibitor of dusp, -2-benzylidene-3-- <dig> -dihydro-1h-inden-1-one  prior to activation, ncd4lo cells responded better following treatment with bci , while ncd4hi cells showed no effect of bci.
further, analysis of cytokines secreted by ncd4lo and ncd4hi cells treated with bci and differentiated in vitro showed that the relative th <dig> tendency of ncd4lo cells was partially reversed by bci treatment .figure  <dig>
the erk-dusp6-mir181a axis contributes to hyporesponsiveness of ncd4lo t cells.
a. histogram overlays of baseline ex vivo p-erk  in ncd4lo and ncd4hi cells.
b. pooled data of paired samples showing baseline p-erk mfi values  in ncd4lo and ncd4hi cells.
c. ΔΔct values of real time rt-pcr for mir-181a normalized to mir- <dig> on ncd4hi and ncd4lo cells .
d. 3h-thymidine incorporation of ncd4lo and ncd4hi cells treated or not with bci prior to activation .
data representative of five mice, one of three experiments.
e. relative ifnγ/il- <dig> and ifnγ/il- <dig> ratios for bci treated or untreated ncd4lo cells during recall response.
data from three independent experiments shown normalized to respective ratios for ncd4hi cells as 100%, shown as a dotted line.
bci, -2-benzylidene-3-- <dig> -dihydro-1h-inden-1-one; dusp <dig>  dual-specific phosphatase 6; mfi, mean fluorescence intensity; ncd <dig>  naïve cd <dig> t cells; se, standard error.
phenotypic and functional features of ncd <dig> cells from young mice with longer peripheral residence are similar to ncd <dig> cells from aged mice
average peripheral residence time of naive t cells in aged mice and older people is reported to be longer than their younger counterparts due to decreasing thymic output with age  <cit> .
since our data above suggest that ncd4lo cells from young mice are likely to be those with longer peripheral residence, we compared ncd <dig> cells from young  and aged  mice for functional and phenotypic features.
we have already shown that ancd <dig> cells respond poorly and die more easily post-activation  <cit> .
we observed that ancd <dig> cells had modestly but consistently lower cd <dig> levels than yncd <dig> cells .
ancd <dig> cells were also consistently smaller than yncd <dig> cells .
we also looked at the ability of sorted ancd <dig> and yncd <dig> cells to differentiate in vitro in response to non-polarizing activating conditions.
supernatants from cultures of ancd <dig> and yncd <dig> cells primed in vitro with anti-cd3 + anti-cd <dig> for three days and then restimulated with titrating doses of anti-cd <dig> were analyzed for ifnγ, il- <dig> and il- <dig>  ancd <dig> cells thus differentiated to effector cells produced more il- <dig> and il- <dig> compared to yncd <dig> cells .
the relative th <dig> dominance, indicated by the ifnγ/il- <dig> ratio, was significantly lower in differentiated ancd <dig> cells .
we also examined total and p-erk levels.
while total erk levels  were not different between ancd <dig> and yncd <dig> cells ex vivo, p-erk levels in the resting state were lower in ancd <dig> cells .
differences in the perk levels were significant .
in multiple independent experiments, relative levels of mir-181a were lower in ancd <dig> cells compared to yncd <dig> cells  extending observations reported on human cells  <cit> .
consistent with the role of mirna-181a in suppressing dusp <dig> expression  <cit> , ancd <dig> cells responded better when treated with a pharmacological inhibitor of dusp, bci, prior to activation .
it is noteworthy that there was no apparent effect of bci on yncd <dig> cells, despite the fact that some of the yncd <dig> cells would be expected to be cd4lo and would, therefore, have shown an effect of bci.
it is likely that the contribution of ncd4lo cells in the bulk yncd <dig> cells may not be statistically apparent.
further, analysis of cytokines secreted by ancd <dig> and yncd <dig> cells treated with bci and differentiated in vitro showed that the relative th <dig> tendency of ancd <dig> cells was partially reversed by bci treatment .figure  <dig>
ncd4lo cells resemble naïve cd <dig> t cells from aged mice in their phenotypic and functional attributes.
a. cd <dig> levels on ancd <dig> and yncd <dig> cells  stained as a mixture in a single well.
b. representative forward scatter histograms for ancd <dig> and yncd <dig> cells.
c. comparison of cd <dig> mfi values of ancd <dig> and yncd <dig> cells .
d. data for forward scatter from individual mice .
e. representative data showing levels of cytokines in supernatants of in vitro primed and recalled yncd <dig> and ancd <dig> cells.
f. ifnγ/il- <dig> ratios and mean ± se calculated based on absorbance values.
g. histogram overlays of baseline ex vivo p-erk  and total erk  in ancd <dig> and yncd <dig> cells .
h. pooled data, of paired samples showing baseline p-erk mfi values  in ancd <dig> and yncd <dig> cells.
i. ΔΔct values of real time rt-pcr for mir-181a normalized to mir- <dig> on ancd <dig> and yncd <dig> cells .
j.
3h-thymidine incorporation of ancd <dig> and yncd <dig> cells treated or not with bci prior to activation .
data representative of six mice, one of three experiments.
k. ifnγ/il- <dig> and ifnγ/il- <dig> ratio for yncd <dig>  bci treated ancd <dig> or bci untreated ancd <dig> cells during recall response.
data representing one of four independent experiments.
ancd <dig>  naïve cd <dig> t cells from aged mice; bci, -2-benzylidene-3-- <dig> -dihydro-1h-inden-1-one; mfi, mean fluorescence intensity; se, standard error; yncd <dig>  naïve cd <dig> t cells from young mice.
discussion
functional variations in apparently homogeneous cell populations have been recognized based on gene induction  <cit>  as well as expression of protein levels  <cit> .
the origins of such variation can be either stochastic and/or can lie in the different experiential histories of individual cells.
our data here show that differences in cd <dig> levels in an ncd <dig> cell population showing unimodal distribution are associated with differences in cell size, cd <dig> expression levels, and ability to respond, differentiate and die post-activation.
our data also indicate that these differences are not purely stochastic but are influenced by microenvironmental mhcii-mediated signals received by peripheral ncd <dig> cells.
finally, we also show that the resultant variations in responsiveness are influenced by ros accumulation and dusp function, providing entry points for envisaging immunotherapeutic interventions in older people.
despite the unimodal distribution, the gated top and bottom cd <dig> level deciles of ncd <dig> cells show distinct differences in both mice and humans.
the ncd4lo cells respond poorly to activation, and their response matures in a more th2-skewed direction.
cd <dig> is a co-receptor and signals mediated by cd4-mhcii interaction can contribute to peptide-mhcii-mediated signaling for activation in naive t cells  <cit> .
however, the anti-cd3 + anti-cd <dig> activation we have used would be expected to bypass any direct contribution of cd <dig>  the cd <dig> molecules on responding ncd <dig> cells are antibody-engaged in our purification protocols, and there is some evidence for tcr-independent signaling capabilities of cd <dig> molecules  <cit> .
however, our data on proximal signaling events such as zap- <dig> or lck phosphorylation and calcium flux induction show no evidence for their modification during activation of ncd4lo and ncd4hi cells, indicating that differential cd4-mediated signals induced by the anti-cd <dig> antibody are not responsible for the differences we observe, and that the differences in cd <dig> levels per se are unlikely to be directly involved in the hyporesponsiveness of ncd4lo cells.
it is more likely that cd <dig> levels are simply correlates of global alterations in these populations.
notably, ncd4lo and ncd4hi cells differ in their il- <dig> production, a characteristic property of ‘anergic’ cd <dig> t cells  <cit> .
however, exogenous il- <dig> supplementation only partially corrects the proliferative deficit in ncd4lo cells, and ncd4lo cells remain quite capable of generating cd <dig> t cell ‘effectors’ on activation, albeit with an altered cytokine balance, indicating that simple anergy is unlikely to be the major modification involved.
our data not only show that the unimodal distribution contains functionally distinct subsets but that they differ phenotypically as well.
the ncd4lo cells have lower cd <dig> and mhci levels, but higher cd <dig> and cd <dig> levels.
they are smaller, too, which may indicate some metabolic downmodulation, although mitochondrial mass and transmembrane potential are not notably altered in them, suggesting that mitochondrially mediated cell size determining factors may not be causal  <cit> .
non-mitochondrial possibilities, such as some potassium channels that are reported to regulate homeostatic volume in t cells, would need to be examined  <cit> .
notably, none of the functional and phenotypic differences between peripheral ncd4lo and ncd4hi t cells are visible in thymic cd4sp cell subsets, suggesting that the differences arise peripherally rather than being programmed during thymic development.
there are interesting implications of the observation that even the mature cd24- thymic cd4sp cells do not show functional distinctions between their cd4lo and cd4hi sub-populations.
this indicates that, in thymic cd4sp cells, variation in cd <dig> levels is indeed likely to be merely stochastic and without functional consequences, as would be expected if the normal distribution of cd <dig> levels in a cell population was due to biological noise and not any functional heterogeneity.
these findings lend interpretative weight to our data with peripheral ncd <dig> cells which do show that their cd <dig> levels correlate with functional heterogeneity and time of peripheral residence.
it may be noted that any more direct functional comparisons between cd4sp thymocytes and peripheral ncd4hi cells are difficult to make, since it is quite likely that ncd <dig> reactivity is low in mature thymocytes for reasons unrelated to cellular age, because recent thymic emigrants in the periphery are poorly responsive and take some time to reach peak reactivity  <cit> .
one obvious distinction between lymphocyte subpopulations can be envisaged based on clonal repertoire diversity.
however, relatively monoclonal tcr-transgenic ncd <dig> cells also show similar differences.
thus, clonal repertoire diversity is not an absolute prerequisite for these differences.
cd <dig> levels on t cells have been linked to adaptation and fine-tuning of t cell responsiveness  <cit> , possibly via tonic signaling through the restricting mhc element which has been shown to be required for naïve t cell survival, maintenance and homeostatic proliferation in lymphopenic environments  <cit> .
the correlation between cd <dig> levels on naive t cells and their responsiveness is somewhat controversial.
recently, higher cd <dig> levels have been correlated with better responsiveness of ncd <dig>  <cit> .
on the other hand, cd <dig> has been shown to be a negative regulator of tcr-mediated activation  <cit> .
there are also other reports, consistent with our findings, that tonic spmhc contact for both cd <dig> and cd <dig> t cells leads to both upregulation of cd <dig> levels and to induction of hyporeactivity  <cit> .
the differences in the correlation of cd <dig> levels on t cells with higher or lower reactivity may be related to the origin of the differences in cd <dig> levels; thus, it is possible that between naive t cells that have received tonic signals for comparable durations, higher cd <dig> levels may identify better-responding t cells as reported  <cit> .
however, in some situations, cd <dig> levels may predominantly reflect the duration of tonic signaling as noted in previous literature  <cit>  and in our data here, and in that context, cd <dig> levels may be inversely correlated with reactivity.
notwithstanding these issues, it is currently plausible to hypothesize that ncd4lo cells, which have higher levels of cd <dig>  may have received much more tonic signal, possibly simply by having spent more time in the periphery.
consistent with this possibility, when ncd4hi cells are purified and transferred in vivo, their cd <dig> levels slowly go down.
this does not happen to ncd4lo cells, possibly indicating that they have already gone as low as possible in their cd <dig> levels.
further, such parked cells steadily become hyporesponsive over time, again implicating time of peripheral residence as a regulatory influence.
thus, within a unimodal population of ncd <dig> cells, it is likely that there are multiple cohorts of cells with varying peripheral residence times and, as a result, varying functionality.
an obvious possibility regulating this global alteration over residence time in vivo is mhc-mediated tonic signaling which, while it is necessary for the survival of naive t cells, has also been reported to be responsible for attenuation of the response potential of these cells, possibly through the negative regulatory function of cd <dig>  <cit> .
indeed, parking ncd <dig> t cells in a mhcii-deficient environment with an acute lack of tonic signaling, for even a short three-day period of parking, reveals mhcii-dependent differences in cd <dig> levels, cd <dig> levels and in responsiveness.
however, cell size does not change over this time window, indicating that it is not necessary for the hyporesponsiveness.
we observe that ros levels go up in ncd <dig> cells as time of peripheral residence increases, whether in the severe situation of parking in mhcii-deficient mice, or in the more physiological situation of parking in wt lymphoreplete mice.
in t cells, the mammalian ste20-like protein kinase mst- <dig> is known to regulate sod- <dig> and catalase levels  <cit> , and mst-1-deficient t cells, in addition to showing lower levels of sod <dig> and catalase, have higher ros levels and are sensitive to apoptosis.
the synthetic mimetic of catalase and sod used here, euk <dig>  has been used as an anti-oxidant in vivo for a variety of outcomes ranging from prevention of heart failure post-hypertension  <cit>  to provision of pain relief  <cit>  in situations where ros accumulation has been implicated in the pathology.
in ncd <dig> cells, ros does contribute to the functional phenotype, since ros scavenging significantly  protects ncd <dig> cells from prolonged six-week parking-induced hyporesponsiveness.
however, this ros-mediated contribution to hyporesponsiveness requires fairly prolonged ros exposure since 15-day anti-oxidant treatment does not improve reactivity.
thus, while ros does make a contribution at late stages, it is clearly not the major factor imposing hyporeactivity on ncd <dig> cells upon prolonged peripheral residence.
if tonic signaling eventually leads to hyporesponsiveness, indications of such a change may be detectable as altered baseline signaling activity  <cit> .
in keeping with this, baseline p-erk levels are lower in ncd4lo t cells.
since erk phosphorylation has been reported to be controlled by dusp <dig> in human t cells, where dusp <dig> is regulated by the mir-181a, it was plausible to examine the mir-181a-dusp axis for a role in the functional differences between ncd4lo and ncd4hi cells.
indeed, mir-181a levels are lower in ncd4lo cells, and a dusp inhibitor rescues both the poor responsiveness and the th2-skewing of these cells.
thus, the mir-181a-dusp axis is at least one mechanism contributing to the alteration of functionality over peripheral residence time in ncd <dig> cells.
it is noteworthy that ncd <dig> t cells from aged animals also show hyporesponsiveness similar to that of ncd4lo cells from young mice that we observed.
as thymic output decreases with age, naïve t cells in the periphery appear to be longer-lived  <cit> , and the average peripheral residence time of naïve t cells is greater in aged than in young individuals  <cit> .
as would be expected from this, the functional and phenotypic characteristics related to peripheral residence time that our data identified, low cd <dig> levels, small cell size, poor responses and th2-skewing, were more prominent in ncd <dig> t cells from aged mice.
while the cd <dig> t cell defect in aged animals is likely to be multi-factorial in origin  <cit> , our data indicate that increased average peripheral residence time of the cells is likely to be one contributory factor.
finally, our data also show that another molecular component regulating the hyporesponsiveness shared by both ancd <dig> and ncd4lo cells is the erk-dusp6-mir-181a axis.
while tcr-mediated activation of t cells from older humans and mice show many early-stage defects in synapse formation and in phosphorylation-mediated activation of critical kinases  <cit> , recent data suggest that increased dusp <dig> activity in naïve cd <dig> t cells from older humans may be important in reducing their ability to respond to tcr-mediated stimulation  <cit> .
our data show that mir-181a levels are lower in both ancd <dig> cells and in ncd4lo cells, and the dusp inhibitor enhances proliferative responses of both ancd <dig> and ncd4lo cells.
it is interesting to note that, although yncd <dig> cells would consist of ncd4hi, ncd4lo and ncd4-intermediate cells, the dusp inhibitor does not show any significant improvement in proliferative responses of bulk yncd <dig> cells.
it is plausible that the contribution of ncd4lo cells in the bulk yncd <dig> cells may not be statistically apparent in the assays we have used.
our findings extend the reported findings from the human system to the mouse system, and establish further concordance between ancd <dig> and ncd4lo t cells and confirm that this axis may indeed be specifically affected during cellular aging.
while linkages between increased ros levels and phosphatase activity of dusp <dig> have been shown in non-t cells  <cit>  whether such a link exists in aging ncd <dig> t cells is not yet clear.
CONCLUSIONS
our data show that, despite unimodal distribution of cd <dig> on ncd <dig> cells, there are subsets of ncd <dig> cells that differ in their peripheral residence time.
during this period, they are in receipt of mhcii-mediated signals and show alteration of phenotype and functionality via ros and dusp activity.
our findings thus indicate the feasibility of potential pharmacological interventions via small molecules serving either as anti-oxidants or as dusp inhibitors, directed specifically at cd <dig> t cells during vaccination of older people.

 peripheral residence of naïve cd t cells is marked by many complex microenvironmental interactions. clonal t cell receptors expressed on individual t cells are likely to have variable avidity for the self-peptide mhc class ii complexes they are positively selected on in the thymus, and this avidity leads to tuning of the response of individual t cells. naïve cd t cells survive because they receive tonic signals through these spmhcii complexes. the levels and availability of these spmhcii complexes are variable, plausibly leading to variations in the frequencies of tonic signaling for individual cd t cells. notably, tcr-mediated signal sensitivity in mouse t cells is also modulated by mirna-181a, which represses expression of dual-specific phosphatase-, a molecule linked to human longevity which suppresses phosphorylation of erk in human t cells. the levels of cd a negative regulator of tcr signaling, show modulations correlated with the efficiency of tcr-mediated tonic signaling required for peripheral t cell survival. there is also evidence that persistent tonic signaling itself may lead to hyporesponsiveness and reduction in co-receptor levels in naive cd t cells. further, naïve t cells can proliferate in the periphery in a cognate ligand-independent fashion if they are in lymphocyte-depleted environments such as during early fetal-neonatal development in the neonatal mouse or in the aged animal with reduced thymic output, although the precise functional consequences of such homeostatic proliferation are not yet clear. cytokines such as il- − − and their receptors have been implicated in peripheral t cell homeostasis and survival, and common receptor gamma-chain-deficient naive cd cells in the periphery are smaller in size, consistent with the possibility that cellular metabolism may be a major variable in determining peripheral survival and functionality of naïve t cells. finally, stochasticity in levels of gene expression and responsiveness is expected to mark any apparently homogenous population of cells including t cells, although it is difficult to distinguish between subset-dependent variations and true statistical noise. the thymus continuously exports naïve mature t cells to the periphery with declining output due to age-associated involution. many of these naïve t cells in the periphery remain naïve until their eventual loss from the peripheral pool. thus, the post-thymic, peripheral residence times of naive t cells at any given point in time would be variable. as thymic output decreases with age, naïve t cells in the periphery also appear to be longer-lived. thus, it is likely that the naïve t cells from aged animals will show a longer average peripheral residence time than those from young individuals, and this may be connected to the functional cd t cell defects seen in aged animals. while there are some markers to identify recent thymic emigrants, there are currently no phenotypic modalities to measure peripheral residence time in individual naïve t cells in heterogeneous populations. on this background, we began looking for variable functional behavior in mouse peripheral naïve cd t cells differing in properties that were apparently unimodally distributed, with the initial expectation that these differences would be stochastic in nature. cd was chosen since it shows low variance in its distribution and is uniformly present without major alterations through the entire lifespan of cd t cells. we find that the top and bottom decile of cd levels in ncd t cells define cells that are both phenotypically and functionally different, with ncd4lo cells having smaller sizes, higher cd levels, poor responses, lower levels of the dusp6-suppressing micro-rna181a, and greater susceptibility to death and to th2-skewing. however, we find that these differences are not stochastic; they are not found in ncd4lo and ncd4hi t cells in the thymus, but they persist even when the tcr repertoire of the cd t cell population is homogenized as in ncd t cells from tcr-transgenic mice, and they can be induced by exposure in vivo to mhcii-mediated tonic signals during a few days of peripheral residence, indicating that unimodal distribution of a variable does not necessarily mean that the variability is stochastic. dusp and reactive oxygen species appear to mediate the mhcii-induced hyporesponsiveness of ncd t cells, since ros scavenging or dusp inhibition ameliorate it. finally, consistent with the greater average time of peripheral residence of ncd t cells in aged animals, we find that the properties found in ncd4lo t cells from young mice are also found in the ncd t cells of aged mice, making ros and dusp potential targets for intervention for successful vaccination in the older population.  despite unimodal distribution, cd levels on naïve cd t cells are correlated with responsiveness ncd cells show unimodal distribution of cd levels. to examine whether this apparently homogenous population has any functional consequences, we sorted mouse splenic ncd cells into the brightest and dullest deciles of cd levels. there was no overlap in the cd levels of these sorted populations, which typically differed by approximately two-fold. we next characterized these sorted ncd4hi and ncd4lo cells in functional terms. purified cells were activated with plate-coated anti-cd3 + anti-cd monoclonal antibodies for 18 hours and the frequency of cells showing induction of cd as an early activation marker was estimated. smaller proportions of ncd4lo cells than of ncd4hi cells expressed cd at multiple anti-cd concentrations. further, ncd4lo cells produced less il- at 48 hours and incorporated less -thymidine at 60 hours post-activation. poor proliferation of activated ncd4lo cells was also confirmed in a carboxyfluorescein succinimidyl ester dilution assay. we examined the possibility that anti-cd antibody bound during sorting signals differentially to the ncd4hi and ncd4lo cells, by resting the sorted cells for 24 hours in il- before stimulating them with anti-cd3 + anti-cd for 48 hours. the difference in their proliferative responses persisted, indicating that it was not related to any anti-cd4-mediated signaling artifact.figure despite unimodal distribution, cd levels on naïve cd t cells are correlated with responsiveness. a. gating strategy used to sort ncd4lo and ncd4hi cells from six- to eight-week-old mice. right panel shows sort profile for ncd4hi and ncd4lo cells. b. cd expression on anti-cd3 + anti-cd stimulated ncd4lo and ncd4hi cells 16 hours post-activation. numbers indicate representative proportions of cd69+ cells in ncd4hi and ncd4lo cells. c. proportions of cd69+ cells in a dose–response curve with 3 μg/ml of anti-cd and titrating anti-cd concentrations as shown.. d. amount of il- detected 48 hours post-stimulation with anti-cd3 + anti-cd in culture supernatants.. e. 3h-thymidine incorporation assay to measure proliferation 60 hours post stimulation with anti-cd3 + anti-cd. f. 3h-thymidine incorporation assay on sorted ncd4hi and ncd4lo cells cultured in vitro with il- for 24 hours prior to stimulation with anti-cd3 + anti-cd for 60 hours.. g. proportion of trypan blue-positive cells 16 hours post-activation. h. cytokine levels in supernatants of in vitro primed and recalled ncd4hi and ncd4lo cells. i. ifnγ/il- ratios and mean ± se calculated based on absorbance values. se, standard error. since il- induction was different between ncd4lo and ncd4hi cells, we tested if exogenous il- supplementation could abolish the difference. when exogenous il- was added during activation, ncd4lo cells showed some improvement in proliferative responses at lower anti-cd concentration, but at no point did their responses reach levels achieved by ncd4hi cells, suggesting that, while differential il- production may contribute to the difference, it was unlikely to represent the sole explanation. there was more prominent activation-induced cell death in ncd4lo cells at 24 hours post-activation, as scored by trypan blue staining, than in ncd4hi cells. we also tested the ability of sorted ncd4hi and ncd4lo cells to differentiate in vitro in response to non-polarizing activating conditions. supernatants from cultures of ncd4hi and ncd4lo cells primed in vitro with anti-cd3 + anti-cd for three days and then restimulated with titrating doses of anti-cd were analyzed for ifnγ and il- effector cells generated from ncd4lo cells produced more il- as compared to those from ncd4hi cells. the relative th1/th dominance, indicated by the ifnγ/il- ratio, was significantly lower in differentiated ncd4lo cells. ncd4lo cells show complex phenotypic modifications, but no differences in proximal signaling events cd is a negative regulator of tcr signaling and cd expression has been inversely correlated with t cell responsiveness. we, therefore, tested the possibility that lower cd levels might correlate with higher cd expression. ncd4hi and ncd4lo cells from young b mice were tested for the levels of cd when gated ncd4hi and ncd4lo cells were analyzed for cd expression, it was apparent that ncd4lo cells had higher cd levels in multiple mice analyzed. this was despite the finding that ncd4hi cells were somewhat larger than ncd4lo cells. we also examined the levels of a number of other molecular markers in these populations. ncd4lo cells showed lower tcrβ levels that ncd4hi cells, although both ncd4lo and ncd4hi cells were confirmed to be conventional cd t cells expressing tcrα/β. ncd4lo cells also expressed somewhat lower levels of mhc class i and cd but higher levels of cd in multiple mice examined. another feature tested based on differences in cell size between ncd4lo and ncd4hi cells was mitochondrial mass and potential using indicator dyes, mitotracker green and mitotracker red. however, ncd4lo cells did not show any striking differences from ncd4hi cells in mitochondrial content or potential. these data indicated that the phenotypic differences between ncd4lo and ncd4hi cells were likely to be complexly regulated.figure ncd4lo cells show higher cd levels but no difference in proximal signaling. a. ex vivo analysis of stained splenic cells to show gated ncd4lo and ncd4hi cells. b. comparison of cd levels as log and linear plots between ncd4hi and ncd4lo cells. c. cd mfi values for ncd4hi and ncd4lo cells. d. comparison of forward scatter between ncd4hi and ncd4lo cells. e. forward scatter values on ncd4hi and ncd4lo cells. f. comparison of tcrβ levels as log and linear plots between ncd4hi and ncd4lo cells. g. tcrβ mfi values for ncd4hi and ncd4lo cells. h. representative profile of fluo-3/fura-red ratio in ncd4hi and ncd4lo cells at rest and post-activation. i. pooled data from independent mouse spleens showing increase in fluo-3/fura-red ratio with anti-cd  + anti-cd treatment j. pooled data for fold increase over background in pzap- staining mfi in ncd4hi and ncd4lo cells activated with anti-cd3 + anti-cd mean ± se. mfi, mean fluorescence intensity; ncd naïve cd t cells; se, standard error; tcr, t cell receptor. since far-downstream proliferative outcomes of tcr activation differed between ncd4lo and ncd4hi cells, we next examined proximal signaling events after ligation with anti-cd3 + anti-cd activation-induced calcium flux was indistinguishable between ncd4hi and ncd4lo cells. similarly, total zap- levels were comparable between ncd4hi and ncd4lo cells, and the induction of zap- phosphorylation upon activation for five minutes did not differ between ncd4lo and ncd4hi cells. similarly, total lck levels were comparable in ncd4lo and ncd4hi cells, and the extent of p-lck induction at five minutes post-activation was similar between them. together, these data suggest that proximal signaling is not different between ncd4hi and ncd4lo cells, and that the differences between them may lie further downstream. hypo-responsiveness associated with ncd4lo cells develops exclusively in the periphery and is also seen in monoclonal t cells in order to see if these properties of ncd4hi and ncd4lo cells is an intrinsic characteristic in developing ncd populations, we tested thymic mature single-positive cd8-cd4 + cd24-qa2+ ncd cells. when these cells were gated to identify cd4lo and cd4hi cells, it was apparent that they show no differences in either cell size or cd levels. when these cd4lo and cd4hi cd4sp thymocytes were sorted and activated with plate-coated anti-cd3 + anti-cd they responded comparably unlike ncd4lo and ncd4hi cells from the periphery. these data indicate that the functional and phenotypic differences observed between peripheral ncd4lo and ncd4hi cells are not thymic in origin.figure hypo-responsiveness associated with ncd4lo cells develops exclusively in periphery and is also seen in monoclonal t cells. a. ex vivo analysis of stained thymic cells to show single positive mature cd4lo and cd4hi cells, and corresponding forward scatter. b. levels of cd in log and linear scale from the same experiment as in a. c. 3h-thymidine incorporation assay to measure proliferation of sorted thymic single positive mature cd4lo and cd4hi cells 60 hours post-activation with anti-cd and anti-cd. d. 3h-thymidine incorporation at 60 hours of sorted ncd4lo and ncd4hi cells from tcr transgenic ot-ii mice to titrating concentrations of ova-ii peptide with irradiated b dcs as apcs. e. amount of il- estimated from supernatants 48 hours post stimulation. data from one of four experiments. apcs, antigen-presenting cells; dcs, dendritic cells; ncd naïve cd t cells; se, standard error; tcr, t cell receptor. in order to examine if the hyporesponsiveness of ncd4lo cells could be plausibly ascribed to differences in the normal polyclonal tcr repertoire, we sorted splenic ncd4hi and ncd4lo cells from mice transgenic for an mhcii-restricted tcr and stimulated these sorted cells with titrating concentrations of the cognate ovalbumin peptide presented by bone-marrow derived dendritic cells as antigen-presenting cells. ncd4lo ot-ii cells responded poorly to cognate peptide and produced less il- than ncd4hi ot-ii cells did, showing that even in naïve t cells with a single tcr, cd levels were correlated with responsiveness. human ncd4lo and ncd4hi cells show properties similar to those from mice we tested if the distinctions observed between ncd4lo and ncd4hi mouse cells were also found in human cd cells. for this, we examined ncd cells from human peripheral blood mononuclear cells from healthy donors. when gated ncd4hi and ncd4lo populations were examined, ncd4lo cells were smaller in size in multiple donors, consistent with the mouse data. sorted human ncd4hi and ncd4lo cells showed about two-fold difference in cd mean fluorescence intensity values similar to mouse cells. upon activation with plate-coated anti-cd3 + anti-cd mabs, ncd4hi cells proliferated much more than ncd4lo cells did. mitochondrial mass and membrane potential were also evaluated in these cells using mg and mr as for mouse cells. similar to mouse ncd4lo cells, human ncd4lo cells, too, showed no striking differences from ncd4hi cells in mitochondrial mass and potential.figure ncd4lo and ncd4hi cells from human pbmcs show properties similar to mouse cells. a. a representative gating strategy for ncd4lo and ncd4hi cells, histogram overlay of sorted ncd4lo and ncd4hi cells and analysis of the same cells for size. b. forward scatter for ncd4hi and ncd4lo cells from individual donors. c. 3h-thymidine incorporation in response to titrating doses of anti-cd and 3 μg/ml of anti-cd 60 hours post-activation. d. histogram overlays for mg staining in log and linear scale in ncd4lo and ncd4hi. e. mfi for mg on ncd4hi and ncd4lo cells from six individual donors. f. histogram overlays for mr staining in log and linear scale in ncd4lo and ncd4hi. g. mfi for mr on ncd4hi and ncd4lo cells from six individual donors. mfi, mean fluorescence intensity; mg, mitotracker green; mr, mitotracker red; ncd naïve cd t cells; pbmc, peripheral blood mononuclear cells; se, standard error. consequences of peripheral residence on ncd t cells the data so far suggest that the association between cd levels and functional phenotype was a peripherally induced characteristic. in order to probe this further, we began by sorting splenic ncd4lo and ncd4hi cells. we labelled these cells with green and violet dyes for identification, mixed them in 1: proportion and transferred them intravenously to wild type recipients. on day post-transfer, donor ncd cells were identified in recipient spleens and analyzed for their cd levels. comparison of cd levels on ncd4hi and ncd4lo cells before and after transfer, done in parallel, showed that adoptively transferred ncd4lo cells continued to show nearly unchanged cd levels which were lower than transferred ncd4hi cells. cd levels on ncd4hi cells declined from d levels to day after transfer, indicating that naïve cd t cells lose cd levels over time in the periphery. however, despite this decline, ncd4hi cells continued to have higher cd levels than ncd4lo cells up to day after transfer. notably, there was no appreciable difference in the ability of transferred cells to survive in vivo, at least for a period of one week. these data suggest that cd levels do not change randomly in vivo but are likely to be reduced over time, perhaps with longer residence.figure consequences of peripheral residence in ncd t cells. a. cd levels on sorted, and differentially labeled ncd4lo and ncd4hi cells prior to and four days after transfer. left and middle panel – log plots, right panel – an overlay in linear scale. b. cd mfi values for ncd4lo and ncd4hi cells seven days post-transfer. each circle represents an individual recipient mouse and the horizontal line indicates mean ± se. c. number of transferred ncd4hi and ncd4lo cells recovered from individual recipient mouse spleens seven days post-transfer shown as mean ± se. d. 3h-thymidine incorporation in ot-ii cells parked in wt mice for one or five weeks in response to titrating concentrations of ova-ii peptide. data normalized for ot-ii cells. horizontal line indicates background cpm. e. levels of il- in culture supernatants from a representative experiment as in panel d. horizontal line indicates background il- levels. f. histogram overlays showing cd levels as log and linear plots of one-week-parked or eight-week-parked ot-ii cells. g. cd mfi levels from long-parked and short-parked ot-ii cells. mfi, mean fluorescence intensity; ncd naïve cd t cells; se, standard error; wt, wild type. we also used the lymphoreplete environment of wt congenic mice to analyze the consequences of prolonged peripheral residence on ncd t cell function. b sjl mice were given × ot-ii cells and the proliferative responses of these donor cells in vitro were analyzed one to eight weeks later. ot-ii cells parked for five weeks in vivo responded poorly in terms of proliferation and il- production than those parked for one week. when cd levels on these parked ot-ii cells were analyzed, ot-ii cells parked for one week showed higher cd levels compared to cells parked for eight weeks. when ot-ii cells parked for less than two weeks were compared with those parked for more than four weeks using data from multiple experiments, significant differences were found in their relative cd levels. do cells parked in balb.c mice for six months showed lower cd levels compared to those parked for two weeks. consequences of prolonged tonic signaling and absence of tonic signaling in naïve t cells the data so far suggested the hypothesis that cd levels and the responsiveness of ncd cells were reduced with increasing duration of peripheral residence. during peripheral residence, mhcii-mediated tonic signals are received by ncd cells. to test if the reduction in cd levels and the loss of responsiveness were related to mhcii-mediated tonic signaling, we ‘parked’ purified ncd ot-ii cells by adoptive transfer into either wt or mhcii-null recipient mice. when recipients were euthanized on day post-transfer, the numbers of ot-ii cells recovered per spleen from wt and mhcii-null recipients were comparable suggesting that there is unlikely to be a major difference in cell survival or homeostatic proliferation in the two groups at this time post-transfer. however, ncd ot-ii cells recovered from wt recipients had lower cd levels and higher cd levels than cells recovered from mhcii-null recipients. when these cells were stimulated with the cognate ova-ii peptide, ot-ii cells parked in mhcii-null recipients responded better than the corresponding cells parked in wt recipients.figure consequences of prolonged tonic signaling and absence of tonic signaling in naïve t cells. a. numbers of adoptively transferred ncd ot-ii cells recovered from spleens three days after transfer into wt and mhcii-null mice. b. comparison of cd levels of donor ncd ot-ii cells after three days in wt and mhcii-null mice. c. pooled data, of paired samples showing cd mfi and mean ± se. d. comparison of cd levels of donor ncd ot-ii cells after three days in wt and mhcii-null mice. e. pooled data, of paired samples showing cd mfi and mean ± se. f. proliferation assay of donor ncd ot-ii cells from wt or mhcii-null recipient mice four days post-transfer in response to ova-ii peptide in the presence of irradiated dcs from wt mice. data were normalized for donor ncd ot-ii cells. g. representative dcfda staining for donor ot-ii cells from wt or mhcii-null spleen cells seven days post-transfer. h. pooled data, of paired samples showing dcfda mfi values in wt or mhcii-null recipients seven days after transfer. dcs, dendritic cells; dcfda, 2-7-dichlorofluorescin diacetate; mfi, mean fluorescence intensity; ncd naïve cd t cells; se, standard error; wt, wild type. since accumulation of ros is a common hallmark of senescent cells, we examined the possibility that longer parking of ot-ii ncd cells in wt versus mhcii-null recipients may also lead to differences in ros levels in them. when ncd ot-ii cells were parked for ten days in wt or mhcii-null recipients, and their levels of staining intensity for the ros indicator dye 2-7-dichlorofluorescin diacetate examined, cells parked in wt mice showed higher ros levels than cells parked in mhcii-null recipients. prolonged residence in vivo leads to loss of functionality and accumulation of ros in ncd cells we next examined if ros levels differed in parked cells in normal lymphoreplete mice depending on the duration of parking. ot-ii cells parked for eight weeks in wt congenic lymphoreplete mice showed higher intensities of dcfda staining as compared to cells parked for one week. in multiple experiments when ot-ii cells parked for less than two weeks were compared with those parked for more than four weeks, significant differences were found in their relative dcfda staining intensities.figure prolonged residence in vivo leads to loss of functionality and accumulation of ros in ncd cells. a. histogram overlays for dcfda from ot-ii cells parked for one or eight weeks in wt mice. b. dcfda mfi values from long-parked and short-parked ot-ii cells. c. 3h-thymidine incorporation in response to titrating concentrations of ova-ii peptide in ot-ii cells parked for one or six weeks in wt mice. data normalized to ot-ii cells. horizontal line indicates background cpm. dcfda, 2-7-dichlorofluorescin diacetate; mfi, mean fluorescence intensity; ncd naïve cd t cells; ros, reactive oxygen species; se, standard error; wt, wild type. we next tested if ros scavenging could ameliorate the relative hyporesponsiveness induced in ot-ii cells by prolonged parking. ot-ii cells were adoptively transferred into wt recipients that were given alternate-day treatment with either vehicle alone or a superoxide dismutase /catalase mimic, euk-, for five weeks following adoptive transfer. wt recipients given ot-ii cells one week previously were also used as a control group. ot-ii cells from euk-134-treated recipients showed a better proliferative response at the end of six weeks of parking compared to cells from vehicle- treated recipients, although their responses still remained poorer than those of ot-ii cells parked for only one week. since ros level differences had become evident by 10 days or so, we tested if a shorter period of euk- treatment was sufficient to improve reactivity of parked ot-ii cells. however, 15-day euk- treatment did not result in any improvement of reactivity. these data suggest that the ros-mediated contribution to hyporeactivity requires fairly prolonged ros exposure, and, thus, ros is clearly not the major factor imposing hyporeactivity on ncd cells upon prolonged peripheral residence. the erk-dusp6-mir-181a axis contributes to hyporesponsiveness of ncd t cells during peripheral residence phosphorylation of mitogen-activated protein family tyrosine kinase erk has been shown to be poor in many aging cells, including t cells suggesting that t cell age may also be correlated with erk phosphorylation. we examined baseline p-erk levels ex vivo in ncd4hi and ncd4lo cells. ncd4lo cells show significantly lower levels compared to ncd4hi cells. the defective phosphorylation of erk in human t cells is regulated by the activity of dusp which in turn is controlled by mir-181a. ncd cells from older people are reported to express lower levels of mir-181a than ncd cells from young people. we therefore estimated the levels of mir-181a and mir- transcripts in mouse t cells using real-time rt-pcr assays. levels of mir-181a transcripts were lower in ncd4lo cells compared to ncd4hi cells, further suggesting that because of longer peripheral residence ncd4lo cells show a pattern similar to that reported in ncd t cells from older individuals. consistent with the role of mirna-181a in suppressing dusp expression, when ncd4lo and ncd4hi cells were treated with a pharmacological inhibitor of dusp, -2-benzylidene-3-- -dihydro-1h-inden-1-one prior to activation, ncd4lo cells responded better following treatment with bci, while ncd4hi cells showed no effect of bci. further, analysis of cytokines secreted by ncd4lo and ncd4hi cells treated with bci and differentiated in vitro showed that the relative th tendency of ncd4lo cells was partially reversed by bci treatment.figure the erk-dusp6-mir181a axis contributes to hyporesponsiveness of ncd4lo t cells. a. histogram overlays of baseline ex vivo p-erk in ncd4lo and ncd4hi cells. b. pooled data of paired samples showing baseline p-erk mfi values in ncd4lo and ncd4hi cells. c. ΔΔct values of real time rt-pcr for mir-181a normalized to mir- on ncd4hi and ncd4lo cells. d. 3h-thymidine incorporation of ncd4lo and ncd4hi cells treated or not with bci prior to activation. data representative of five mice, one of three experiments. e. relative ifnγ/il- and ifnγ/il- ratios for bci treated or untreated ncd4lo cells during recall response. data from three independent experiments shown normalized to respective ratios for ncd4hi cells as 100%, shown as a dotted line. bci, -2-benzylidene-3-- -dihydro-1h-inden-1-one; dusp dual-specific phosphatase 6; mfi, mean fluorescence intensity; ncd naïve cd t cells; se, standard error. phenotypic and functional features of ncd cells from young mice with longer peripheral residence are similar to ncd cells from aged mice average peripheral residence time of naive t cells in aged mice and older people is reported to be longer than their younger counterparts due to decreasing thymic output with age. since our data above suggest that ncd4lo cells from young mice are likely to be those with longer peripheral residence, we compared ncd cells from young and aged mice for functional and phenotypic features. we have already shown that ancd cells respond poorly and die more easily post-activation. we observed that ancd cells had modestly but consistently lower cd levels than yncd cells. ancd cells were also consistently smaller than yncd cells. we also looked at the ability of sorted ancd and yncd cells to differentiate in vitro in response to non-polarizing activating conditions. supernatants from cultures of ancd and yncd cells primed in vitro with anti-cd3 + anti-cd for three days and then restimulated with titrating doses of anti-cd were analyzed for ifnγ, il- and il- ancd cells thus differentiated to effector cells produced more il- and il- compared to yncd cells. the relative th dominance, indicated by the ifnγ/il- ratio, was significantly lower in differentiated ancd cells. we also examined total and p-erk levels. while total erk levels were not different between ancd and yncd cells ex vivo, p-erk levels in the resting state were lower in ancd cells. differences in the perk levels were significant. in multiple independent experiments, relative levels of mir-181a were lower in ancd cells compared to yncd cells extending observations reported on human cells. consistent with the role of mirna-181a in suppressing dusp expression, ancd cells responded better when treated with a pharmacological inhibitor of dusp, bci, prior to activation. it is noteworthy that there was no apparent effect of bci on yncd cells, despite the fact that some of the yncd cells would be expected to be cd4lo and would, therefore, have shown an effect of bci. it is likely that the contribution of ncd4lo cells in the bulk yncd cells may not be statistically apparent. further, analysis of cytokines secreted by ancd and yncd cells treated with bci and differentiated in vitro showed that the relative th tendency of ancd cells was partially reversed by bci treatment.figure ncd4lo cells resemble naïve cd t cells from aged mice in their phenotypic and functional attributes. a. cd levels on ancd and yncd cells stained as a mixture in a single well. b. representative forward scatter histograms for ancd and yncd cells. c. comparison of cd mfi values of ancd and yncd cells. d. data for forward scatter from individual mice. e. representative data showing levels of cytokines in supernatants of in vitro primed and recalled yncd and ancd cells. f. ifnγ/il- ratios and mean ± se calculated based on absorbance values. g. histogram overlays of baseline ex vivo p-erk and total erk in ancd and yncd cells. h. pooled data, of paired samples showing baseline p-erk mfi values in ancd and yncd cells. i. ΔΔct values of real time rt-pcr for mir-181a normalized to mir- on ancd and yncd cells. j. 3h-thymidine incorporation of ancd and yncd cells treated or not with bci prior to activation. data representative of six mice, one of three experiments. k. ifnγ/il- and ifnγ/il- ratio for yncd bci treated ancd or bci untreated ancd cells during recall response. data representing one of four independent experiments. ancd naïve cd t cells from aged mice; bci, -2-benzylidene-3-- -dihydro-1h-inden-1-one; mfi, mean fluorescence intensity; se, standard error; yncd naïve cd t cells from young mice. discussion functional variations in apparently homogeneous cell populations have been recognized based on gene induction as well as expression of protein levels. the origins of such variation can be either stochastic and/or can lie in the different experiential histories of individual cells. our data here show that differences in cd levels in an ncd cell population showing unimodal distribution are associated with differences in cell size, cd expression levels, and ability to respond, differentiate and die post-activation. our data also indicate that these differences are not purely stochastic but are influenced by microenvironmental mhcii-mediated signals received by peripheral ncd cells. finally, we also show that the resultant variations in responsiveness are influenced by ros accumulation and dusp function, providing entry points for envisaging immunotherapeutic interventions in older people. despite the unimodal distribution, the gated top and bottom cd level deciles of ncd cells show distinct differences in both mice and humans. the ncd4lo cells respond poorly to activation, and their response matures in a more th2-skewed direction. cd is a co-receptor and signals mediated by cd4-mhcii interaction can contribute to peptide-mhcii-mediated signaling for activation in naive t cells. however, the anti-cd3 + anti-cd activation we have used would be expected to bypass any direct contribution of cd the cd molecules on responding ncd cells are antibody-engaged in our purification protocols, and there is some evidence for tcr-independent signaling capabilities of cd molecules. however, our data on proximal signaling events such as zap- or lck phosphorylation and calcium flux induction show no evidence for their modification during activation of ncd4lo and ncd4hi cells, indicating that differential cd4-mediated signals induced by the anti-cd antibody are not responsible for the differences we observe, and that the differences in cd levels per se are unlikely to be directly involved in the hyporesponsiveness of ncd4lo cells. it is more likely that cd levels are simply correlates of global alterations in these populations. notably, ncd4lo and ncd4hi cells differ in their il- production, a characteristic property of ‘anergic’ cd t cells. however, exogenous il- supplementation only partially corrects the proliferative deficit in ncd4lo cells, and ncd4lo cells remain quite capable of generating cd t cell ‘effectors’ on activation, albeit with an altered cytokine balance, indicating that simple anergy is unlikely to be the major modification involved. our data not only show that the unimodal distribution contains functionally distinct subsets but that they differ phenotypically as well. the ncd4lo cells have lower cd and mhci levels, but higher cd and cd levels. they are smaller, too, which may indicate some metabolic downmodulation, although mitochondrial mass and transmembrane potential are not notably altered in them, suggesting that mitochondrially mediated cell size determining factors may not be causal. non-mitochondrial possibilities, such as some potassium channels that are reported to regulate homeostatic volume in t cells, would need to be examined. notably, none of the functional and phenotypic differences between peripheral ncd4lo and ncd4hi t cells are visible in thymic cd4sp cell subsets, suggesting that the differences arise peripherally rather than being programmed during thymic development. there are interesting implications of the observation that even the mature cd24- thymic cd4sp cells do not show functional distinctions between their cd4lo and cd4hi sub-populations. this indicates that, in thymic cd4sp cells, variation in cd levels is indeed likely to be merely stochastic and without functional consequences, as would be expected if the normal distribution of cd levels in a cell population was due to biological noise and not any functional heterogeneity. these findings lend interpretative weight to our data with peripheral ncd cells which do show that their cd levels correlate with functional heterogeneity and time of peripheral residence. it may be noted that any more direct functional comparisons between cd4sp thymocytes and peripheral ncd4hi cells are difficult to make, since it is quite likely that ncd reactivity is low in mature thymocytes for reasons unrelated to cellular age, because recent thymic emigrants in the periphery are poorly responsive and take some time to reach peak reactivity. one obvious distinction between lymphocyte subpopulations can be envisaged based on clonal repertoire diversity. however, relatively monoclonal tcr-transgenic ncd cells also show similar differences. thus, clonal repertoire diversity is not an absolute prerequisite for these differences. cd levels on t cells have been linked to adaptation and fine-tuning of t cell responsiveness, possibly via tonic signaling through the restricting mhc element which has been shown to be required for naïve t cell survival, maintenance and homeostatic proliferation in lymphopenic environments. the correlation between cd levels on naive t cells and their responsiveness is somewhat controversial. recently, higher cd levels have been correlated with better responsiveness of ncd. on the other hand, cd has been shown to be a negative regulator of tcr-mediated activation. there are also other reports, consistent with our findings, that tonic spmhc contact for both cd and cd t cells leads to both upregulation of cd levels and to induction of hyporeactivity. the differences in the correlation of cd levels on t cells with higher or lower reactivity may be related to the origin of the differences in cd levels; thus, it is possible that between naive t cells that have received tonic signals for comparable durations, higher cd levels may identify better-responding t cells as reported. however, in some situations, cd levels may predominantly reflect the duration of tonic signaling as noted in previous literature and in our data here, and in that context, cd levels may be inversely correlated with reactivity. notwithstanding these issues, it is currently plausible to hypothesize that ncd4lo cells, which have higher levels of cd may have received much more tonic signal, possibly simply by having spent more time in the periphery. consistent with this possibility, when ncd4hi cells are purified and transferred in vivo, their cd levels slowly go down. this does not happen to ncd4lo cells, possibly indicating that they have already gone as low as possible in their cd levels. further, such parked cells steadily become hyporesponsive over time, again implicating time of peripheral residence as a regulatory influence. thus, within a unimodal population of ncd cells, it is likely that there are multiple cohorts of cells with varying peripheral residence times and, as a result, varying functionality. an obvious possibility regulating this global alteration over residence time in vivo is mhc-mediated tonic signaling which, while it is necessary for the survival of naive t cells, has also been reported to be responsible for attenuation of the response potential of these cells, possibly through the negative regulatory function of cd. indeed, parking ncd t cells in a mhcii-deficient environment with an acute lack of tonic signaling, for even a short three-day period of parking, reveals mhcii-dependent differences in cd levels, cd levels and in responsiveness. however, cell size does not change over this time window, indicating that it is not necessary for the hyporesponsiveness. we observe that ros levels go up in ncd cells as time of peripheral residence increases, whether in the severe situation of parking in mhcii-deficient mice, or in the more physiological situation of parking in wt lymphoreplete mice. in t cells, the mammalian ste20-like protein kinase mst- is known to regulate sod- and catalase levels, and mst-1-deficient t cells, in addition to showing lower levels of sod and catalase, have higher ros levels and are sensitive to apoptosis. the synthetic mimetic of catalase and sod used here, euk has been used as an anti-oxidant in vivo for a variety of outcomes ranging from prevention of heart failure post-hypertension to provision of pain relief in situations where ros accumulation has been implicated in the pathology. in ncd cells, ros does contribute to the functional phenotype, since ros scavenging significantly protects ncd cells from prolonged six-week parking-induced hyporesponsiveness. however, this ros-mediated contribution to hyporesponsiveness requires fairly prolonged ros exposure since 15-day anti-oxidant treatment does not improve reactivity. thus, while ros does make a contribution at late stages, it is clearly not the major factor imposing hyporeactivity on ncd cells upon prolonged peripheral residence. if tonic signaling eventually leads to hyporesponsiveness, indications of such a change may be detectable as altered baseline signaling activity. in keeping with this, baseline p-erk levels are lower in ncd4lo t cells. since erk phosphorylation has been reported to be controlled by dusp in human t cells, where dusp is regulated by the mir-181a, it was plausible to examine the mir-181a-dusp axis for a role in the functional differences between ncd4lo and ncd4hi cells. indeed, mir-181a levels are lower in ncd4lo cells, and a dusp inhibitor rescues both the poor responsiveness and the th2-skewing of these cells. thus, the mir-181a-dusp axis is at least one mechanism contributing to the alteration of functionality over peripheral residence time in ncd cells. it is noteworthy that ncd t cells from aged animals also show hyporesponsiveness similar to that of ncd4lo cells from young mice that we observed. as thymic output decreases with age, naïve t cells in the periphery appear to be longer-lived, and the average peripheral residence time of naïve t cells is greater in aged than in young individuals. as would be expected from this, the functional and phenotypic characteristics related to peripheral residence time that our data identified, low cd levels, small cell size, poor responses and th2-skewing, were more prominent in ncd t cells from aged mice. while the cd t cell defect in aged animals is likely to be multi-factorial in origin, our data indicate that increased average peripheral residence time of the cells is likely to be one contributory factor. finally, our data also show that another molecular component regulating the hyporesponsiveness shared by both ancd and ncd4lo cells is the erk-dusp6-mir-181a axis. while tcr-mediated activation of t cells from older humans and mice show many early-stage defects in synapse formation and in phosphorylation-mediated activation of critical kinases, recent data suggest that increased dusp activity in naïve cd t cells from older humans may be important in reducing their ability to respond to tcr-mediated stimulation. our data show that mir-181a levels are lower in both ancd cells and in ncd4lo cells, and the dusp inhibitor enhances proliferative responses of both ancd and ncd4lo cells. it is interesting to note that, although yncd cells would consist of ncd4hi, ncd4lo and ncd4-intermediate cells, the dusp inhibitor does not show any significant improvement in proliferative responses of bulk yncd cells. it is plausible that the contribution of ncd4lo cells in the bulk yncd cells may not be statistically apparent in the assays we have used. our findings extend the reported findings from the human system to the mouse system, and establish further concordance between ancd and ncd4lo t cells and confirm that this axis may indeed be specifically affected during cellular aging. while linkages between increased ros levels and phosphatase activity of dusp have been shown in non-t cells whether such a link exists in aging ncd t cells is not yet clear.  our data show that, despite unimodal distribution of cd on ncd cells, there are subsets of ncd cells that differ in their peripheral residence time. during this period, they are in receipt of mhcii-mediated signals and show alteration of phenotype and functionality via ros and dusp activity. our findings thus indicate the feasibility of potential pharmacological interventions via small molecules serving either as anti-oxidants or as dusp inhibitors, directed specifically at cd t cells during vaccination of older people. 
our data show that, underlying a unimodally distributed property, the cd level, there are subsets of naïve cd cells that vary in the time spent in the periphery receiving mhcii-mediated signals and show resultant alteration of phenotype and functionality via ros and dusp activity. our findings also suggest the feasibility of potential pharmacological interventions for improved cd t cell responses during vaccination of older people via either anti-oxidant or dusp inhibitor small molecules. human naïve cd4lo and cd4hi cells showed similar differences. naïve cd t cells show unimodal distribution for many phenotypic properties, suggesting that the variation is caused by intrinsic stochasticity, although underlying variation due to subsets created by different histories of microenvironmental interactions remains possible.
 as individual naïve cd t lymphocytes circulate in the body after emerging from the thymus, they are likely to have individually varying microenvironmental interactions even in the absence of stimulation via specific target recognition. it is not clear if these interactions result in alterations in their activation, survival and effector programming. naïve cd t cells show unimodal distribution for many phenotypic properties, suggesting that the variation is caused by intrinsic stochasticity, although underlying variation due to subsets created by different histories of microenvironmental interactions remains possible. to explore this possibility, we began examining the phenotype and functionality of naïve cd t cells differing in a basic unimodally distributed property, the cd levels, as well as the causal origin of these differences.  we examined separated cd4hi and cd4lo subsets of mouse naïve cd cells. cd4lo cells were smaller with higher cd levels and lower levels of the dual-specific phosphatase 6-suppressing micro-rna mir181a, and responded poorly with more th2-skewed outcomes. human naïve cd4lo and cd4hi cells showed similar differences. naïve cd4lo and cd4hi subsets of thymic single-positive cd t cells did not show differences whereas peripheral naïve cd4lo and cd4hi subsets of t cell receptor -transgenic t cells did. adoptive transfer-mediated parking of naïve cd cells in vivo lowered cd levels, increased cd and reactive oxygen species levels and induced hyporesponsiveness in them, dependent, at least in part, on availability of major histocompatibility complex class ii molecules. ros scavenging or dusp inhibition ameliorated hyporesponsiveness. naïve cd cells from aged mice showed lower cd levels and cell sizes, higher cd levels, and hyporesponsiveness and th2-skewing reversed by dusp inhibition.  our data show that, underlying a unimodally distributed property, the cd level, there are subsets of naïve cd cells that vary in the time spent in the periphery receiving mhcii-mediated signals and show resultant alteration of phenotype and functionality via ros and dusp activity. our findings also suggest the feasibility of potential pharmacological interventions for improved cd t cell responses during vaccination of older people via either anti-oxidant or dusp inhibitor small molecules. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords cd t cellnaïveperipheral residencemhc class iith1th2issue-copyright-statement© the author 2014 