Developmental regulation of p53 dependent radiation induced thymocyte apoptosis in mice
Original Article
Developmental regulation of p53-dependent radiation-induced thymocyte apoptosis in mice
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A.Gentil Dit Maurin,*??
C.Lemercier,*??V.Collin-Faure,?§?P.N.Marche,?**??
E.Jouvin-Marche?**??and
S.M.Candéias?§?
*CEA,DSV,iRTSV-BGE,?INSERM U1038,
?Grenoble Alpes Université,§CEA,DSV, iRTSV-CBM,?UMR5249CEA-CNRS-GAU, **INSERM U823,and??Institut Albert Bonniot, UMR_S823,GAU,Grenoble,France Summary
The production of T cell receptorαβ+(TCRαβ+)T lymphocytes in the thymus is a tightly regulated process that can be monitored by the regulated expression of several surface molecules,including CD4,CD8,cKit,CD25and the TCR itself,after TCR genes have been assembled from discrete V,D(for TCR-β)and J gene segments by a site-directed genetic recombination. Thymocyte differentiation is the result of a delicate balance between cell death and survival:developing thymocytes die unless they receive a positive signal to proceed to the next stage.This equilibrium is altered in response to various physiological or physical stresses such as ionizing radiation,which induces a massive p53-dependent apoptosis of CD4+CD8+double-positive (DP)thymocytes.Interestingly,these cells are actively rearranging their TCR-αchain genes.To unravel an eventual link between V(D)J recombina-tion activity and thymocyte radio-sensitivity,we analysed the dynamics of thymocyte apoptosis and regeneration following exposure of wild-type and p53-de?cient mice to different doses ofγ-radiation.p53-dependent radio-sensitivity was already found to be high in immature CD4?CD8?(double-negative,DN)cKit+CD25+thymocytes,where TCR-βgene rearrangement is initiated.However,TCR-αβ?CD8+immature single-positive thymocytes,an actively cycling intermediate population between the DN and DP stages,are the most radio-sensitive cells in the thymus,even though their apoptosis is only partially p53-dependent.Within the DP population,TCR-αβ+thymo-cytes that completed TCR-αgene recombination are more radio-resistant than their TCR-αβ?progenitors.Finally,we found no correlation between p53activation and thymocyte sensitivity to radiation-induced apoptosis. Keywords:apoptosis,p53,radiation,thymocyte differentiation
Accepted for publication13March2014
Correspondence:S.M.Candéias,
LCBM/ProMD,iRTSV,17rue des martyrs,3854
Grenoble,France.
E-mail:serge.candeias@cea.fr
IMMUNE SYSTEM
Introduction
T cell receptor-αβ+(TCR-αβ+)T lymphocyte development is the result of a complex programme initiated in the thymus by blood-borne haematopoietic stem cell-derived progenitors.The regulated expression of several surface molecules,including the TCR chains,marks the progression of developing thymocytes through the different maturation stages.The most immature expresses neither CD4nor CD8 (CD4?CD8?,DN).Within this population,thymocytes pro-gress from the CD44+cKit+CD25?(DN1),to the CD44+cKit+ CD25+(DN2),CD44?cKit?CD25+(DN3)and cD44?cKit?CD25?(DN4)subpopulations as they mature[1,2].TCR-βexpression in a pre-TCR complex on DN3thymocytes is required for differentiation to the DN4stage[3],and then through the rapidly cycling CD8+TCR-αβ?immature single-positive(ISP)stage[4,5],to the CD4+CD8+double-positive(DP)population.DP thymocytes?rst express only low levels of TCR-αβ,until their antigenic speci?city is tested through positive and negative selection[6–8].At that stage,they up-regulate TCR-αβexpression and transiently express CD69[9,10]before being sorted into mature TCR-αβ+CD4+and TCR-αβ+CD8+single-positive(SP)thymo-cytes and exported to the periphery.At each step of differentiation,thymocytes must receive a positive cue,?rst from cell–cell interactions and cytokine signalling and then from pre-TCR-and TCR-derived signals,to progress to the next stage,otherwise they die[11–14].Thymocytes are extremely sensitive to various kinds of physiological and physical stresses that induce a massive thymic involution [15].
Before being expressed,the TCR chain genes must be rearranged from discrete V,D(for TCR-β)and J gene seg-ments by a site-directed genetic recombination mechanism. During V(D)J recombination,the lymphoid-speci?c recombination-activating gene(RAG1)/RAG2introduces DNA double-stranded breaks(DSBs)at the border of each gene[16–18],which are then repaired by non-homologous end joining[19].The creation of these DSBs,even in this physiological context,activates some of the actors of the checkpoints elicited in response to genotoxic stress[20–22]. This‘pre-activation’state could explain the well-documented high p53-dependent radio-sensitivity of DP thymocytes[23–25].To unravel an eventual link between V(D)J recombination and p53-dependent thymocyte radio-sensitivity,we analysed the dynamics of thymocyte apoptosis following exposure of wild-type(WT)and p53-de?cient mice to different doses ofγ-radiation.We found that before reaching the DP compartment,DN2,in which TCR-βgene rearrangement is initiated[26–28],and ISP thymocytes are already extremely radio-sensitive.ISP apoptosis can be observed as early as3h post-exposure and is only partially p53-dependent.In the DP population,TCR selection marks the end of the high sensitivity to IR expo-sure.Unexpectedly,the highest susceptibility to p53-dependent radiation-induced apoptosis does not depend upon the higher level of p53.
Materials and methods
Mice
C57BL/6WT and p53-de?cient[23–25]mice bred under speci?c pathogen-free conditions in the animal facility of CEA-Grenoble were exposed to a60Co source with a dose rate of2Gy/min in the Anemome irradiator of the ARC-Nucleart Facility(Grenoble,France).Mice were aged between7and13weeks at the time of irradiation.These experiments were performed in accordance with French regulations.
Fluorescence activated cell sorter(FACS)analysis
Single-cell thymocyte suspensions were prepared by disso-ciation of thymi for30min at37°C in collagenase B(2mg/ ml)and DNase I(0·4mg/ml)in phosphate-buffered saline (PBS)–10%fetal calf serum(FCS)[29].Labelling was performed as described[30].Data were acquired on a FACSCalibur?ow cytometer(Becton Dickinson,Franklin Lakes,NJ,USA)and analysed with CellQuest software. Antibodies used in this study were anti-CD4(GK1·5or H129·19),anti-CD8(53–6·7),anti-TCR-β(H57-597),anti-CD69(H1·2F3),anti-CD3ε(145-2C.11),anti-cKit(2B8), anti-CD44(IM7)and anti-CD25(7D4)conjugated to?uo-rescein isothiocyanate(FITC),Cy-chrome,phycoerythin (PE),allophycocyanin(APC)or biotin.Biotinylated anti-bodies were revealed with APC-coupled streptavidin.All reagents were from BD Pharmingen(San Diego,CA,USA). Immuno?uorescence staining and confocal microscopy
Thymocytes were labelled with APC-conjugated anti-CD4 and biotinylated anti-CD8as above and biotin was detected with Alexa546-coupled streptavidin(Molecular Probes, Eugene,OR,USA).Thymocytes were then attached to poly-L-lysine-coated slides,?xed and permeabilized as described [31].p53was detected with a rabbit polyclonal anti-p53 antibody(CM5;Novocastra,Newcastle upon Tyne,UK) revealed by an Alexa488-conjugated anti-rabbit polyclonal antibody(Molecular Probes).Nuclei were counterstained with4′,6-diamidino-2-phenylindole(DAPI)just before mounting the slides in Vectashield(Vector Laboratories, Burlingame,CA,USA).Images were collected under the ×100objective(numerical aperture=1·4)of a Leica TSC-P2confocal microscope in sequential mode for four-colour acquisitions(laser excitation at488,543and633nm and a UV source for DAPI).All conditions were compared under identical settings.Images were imported in Adobe Photoshop for?gure preparation.
FOCUS ON DYING AUTOLOGOUS CELLS AS INSTRUCTORS OF THE IMMUNE SYSTEM p53in radiation-induced thymocyte apoptosis
Western blotting
Total cell extracts were prepared from thymocytes by direct lysis of the cells in Laemmli’s sample buffer and processed for Western blotting,as described previously [31].Primary antibodies directed against p53(CM5;Novocastra),p53phosphorylated on Ser15(Cell Signaling,Beverly,MA,USA),γH2AX (Upstate,Billerica,MA,USA)and βtubulin (sc-5274;Santa Cruz Biotechnology,Santa Cruz,CA,USA)were detected with the appropriate horseradish peroxidase-conjugated secondary polyclonal anti-rabbit or anti-mouse,respectively.Membranes were then revealed by chemolu-minescence with the enhanced chemiluminescence (ECL)reagent (GE Healthcare,Little Chalfont,UK).
Results
Dynamics of radiation-induced unselected and selected DP thymocyte apoptosis
T o analyse the dynamics of the DP thymocyte population fol-lowing radiation exposure,groups of mice received 1·5,4or 6Gy of γ-radiation (total body irradiation)and were sacri?ced 1,2and 4days later to analyse thymic involution.The dose-
dependence of the DP thymocyte apoptotic response was evident from the number and phenotype of the surviving thymocytes (Fig.1).Exposure to 1·5Gy induces a 75%reduc-tion of thymocyte numbers at day 1post-exposure.This decrease is slightly accentuated at day 2,but by day 4the recov-ery is already apparent.The frequency of DP thymocytes in the thymus of irradiated animals follows this kinetics:the decrease observed at day 1post-exposure is accentuated from days 1to 2,before the proportion of DP thymocytes returns to normal at day 4,even if the cellularity is only one-third that of control animals (Fig.1and T able 1).Exposure to 4and 6Gy produces more severe effects:they induce a decrease of more than 90%of the thymocyte numbers at day 1,which reaches 97%by day 2,with no apparent recovery at day 4.FACS analysis shows a more contrasted situation,and reveals different kinetics for the DP thymocytes.The proportion of DP thymocytes,strongly reduced at day 1,decreases further from days 1to 2post-4and -6Gy,when it represents fewer than 5%of the living cells,but by day 4a reprise of DP thymocyte differentia-tion is visible in irradiated animals,whereas the cell number does not increase.The restoration of the DP pool is very partial (approximately 30and 7%of thymocytes at 4and 6Gy,respectively)when compared to mice exposed to 1·5Gy (80%)(T able 1).
Control
d1
d2
d4
138·56 ± 28·22
36·10 ±7·01
26·64 ±12·24
50·79 ±13·02
101010101001234
Fig.1.Thymus involution following radiation exposure.Fluorescence activated cell sorter (FACS)analysis of CD4and CD8expression on thymocytes from C57BL/6mice exposed to 1·5Gy,4Gy or 6Gy and killed at the indicated times.Live thymocytes were gated on the basis of forward-and side-scatter.The number above each plot is the average (±standard error)number of cells per thymus.The number of mice analysed in each group is as follows:n =7for control;n =3for day 1,n =4for day 2and n =2for day 4.
Table 1.Frequency (±standard error)of the double-negative (DN),double-positive (DP)and single-positive (SP)populations in the thymus of control and irradiated mice shown in Fig.1.
0Gy
1·5Gy
4Gy 6Gy Day 1
Day 2
Day 4
Day 1
Day 2
Day 4
Day 1
Day 2
Day 4
DN 3·38±0·525·76±0·5010·02±1·864·71±0·3512·7±1·229·35±1·0916·65±2·6415·33±2·087·67±0·9024·32±1·23DP 83·91±1·1073·43±1·0449·11±1·6980·24±2·7644·82±4·224·68±0·9031·17±12·4023·62±1·411·56±0·347·06±3·55SP CD47·31±0·3412·48±0·9423·72±0·545·72±0·7425·06±2·8454·16±1·7822·03±4·5838·1±2·5661·49±2·7331·68±0·41SP CD82·73±0·644·44±0·8011·87±3·135·29±0·839·17±1·1624·58±6·1719·05±2·9611·66±4·2022·22±5·8521·27±0·44
FOCUS ON DYING AUTOLOGOUS CELLS AS INSTRUCTORS OF THE IMMUNE SYSTEM
A.Gentil Dit Maurin et al .
The DP population is not homogeneous.The levels of expression of TCR and CD69allows discrimination of TCR low CD69?‘immature’DP from selected TCR high CD69+and TCR high CD69?DP ,the immediate precursors of SP thymocytes (Fig.2a).As shown in Fig.2b,we observed a relative increase (2·2–3·2-fold over control)in the proportion of selected DP thymocytes at day 1for all doses.This proportion is main-tained at day 2for 1·5Gy-irradiated mice,but is increased dramatically in mice exposed to 4Gy (7·7-fold)and 6Gy (11·8-fold),where selected DP represent 60and 90%of the DP population,respectively.Four days after 1·5Gy and 4Gy,the frequency of selected DP drops to values lower than those of control mice,whereas it is still somewhat elevated (1·8-fold)after exposure to 6Gy.The relative increase in selected DP at days 1and 2shows clearly that these cells are more radio-resistant than their TCR low DP precursors.The diminu-tion of their frequency between days 2and 4suggests either that they take longer to die,or that they resume differentiation and progress to the SP compartment.
ISP thymocytes are very radio-sensitive
The observation that it takes 2days for the DP population to reach its minimum following radiation exposure (Fig.1)
suggests that,in addition to DP cell death and eventual pro-gression of selected DP to the SP compartment,the in?ux of ISP cells may be stopped.Indeed,we observed a very strong reduction of the frequency of TCR ?CD8+ISP cells in irradiated mice at day 1for all doses (Fig.2c).Whereas ISP frequency already increases at day 2in mice exposed to 1·5Gy,it remains at the same low level in mice exposed to higher doses.We then observed a very strong rebound in ISP frequency at day 4for all doses to levels higher than in control animals,indicating a strong compensatory homeo-static mechanism.The frequency of ISP thymocytes was found to be reduced already by 75%3h after a 6Gy irradia-tion (Fig.2c),when no change in the frequency of the DN,DP and SP populations is detectable (data not shown).Thus,ISP thymocytes are more radio-sensitive than TCR low DP ,die earlier,but are also regenerated faster.
p53-dependency of DN2,ISP and selected DP radiation-induced apoptosis
As it is well established that DP thymocyte apoptosis requires the activity of the p53tumour suppressor [23–25],we compared the fate of ISP and TCR high DP in WT and
ISP 27·81%
Selected DP 6·01%
Selected DP 54·29%
ISP 0·67%
Ctrl
4 Gy, d20
204060801000
102030400
10203040Ctrl
6 Gy
% T C R h i g h D P
% I S P i n C D 8+S P
% I S P i n C D 8+S P
100
100101
102
103
104
101102103104100
100101
102
103
104
101102103104100
100
101
102
103
104
101102
10310
4100
100
101
102
103
104
101
102103104(a)
(b)
(c)Fig.2.Immature single-positive (ISP)and selected double-positive (DP)populations in irradiated mice.(a)Gating used to identify ISP (right)and selected DP (left)thymocyte populations from gated DP and
SPCD8+thymocytes (regions R3and R2,
respectively,in Fig.1)in a control (top)and an irradiated (4Gy,day 2,bottom)animal
following four-colour CD4/CD8/CD69/T cell receptor (TCR)-βlabelling.(b)Graph bar showing the average (±standard error)
frequency of selected DP thymocytes in mice shown in Fig.1.(c)Graph bar showing the average (±standard error)frequency of ISP in mice shown in Fig.1(left)and in C57BL/6mice unirradiated and 3h after exposure to 6Gy (right).For the right panel,n =4for control mice,and n =5for irradiated mice.
FOCUS ON DYING AUTOLOGOUS CELLS AS INSTRUCTORS OF THE IMMUNE SYSTEM
p53in radiation-induced thymocyte apoptosis
p53-de?cient mice 1day after exposure to 6Gy to deter-mine if they share the same p53dependency.We also included earlier immature DN thymocyte populations in our analysis.
As expected,the important diminution of DP frequency and the corresponding compensatory increase in the fre-quencies of DN and SP populations observed in irradiated WT mice do not take place in irradiated p53-de?cient animals (Fig.3a).However,even if the DN population,as a whole,is relatively less radio-sensitive than the DP ,the DN subpopulations are differentially affected.Radiation expo-sure induces an 85%reduction of the frequency of CD44+cKit +CD25+DN2thymocytes in WT mice (Fig.3b,inset),indicating that they are more radio-sensitive than their DN1precursors or their DN3/DN4descendants.This preferential death is not observed in p53-de?cient mice.As previously,we found a strong reduction of the ISP fre-quency in irradiated WT mice but,in contrast to DN2thymocytes,radiation-induced ISP cell death is only par-tially p53-dependent,as the frequency of the ISP popula-tion in irradiated p53?/?mice is reduced to 60%of that found in control mice (Fig.3c).Finally,the proportion of selected DP in the thymus of p53?/?mice does not change following irradiation,indicating that the higher proportion observed in irradiated WT mice results from TCR low DP radio-sensitivity (Fig.3c).
p53is differentially expressed and up-regulated in radio-sensitive and radio-resistant thymocytes Radiation-induced p53activation results from phosphory-lation and stabilization after dissociation from the com-plexes it forms with MDM2in steady state conditions [32–34].Hence,increased levels of phosphorylated,acti-vated p53protein are found in irradiated thymocytes (Fig.4a).As DN,DP and SP thymocytes exhibit a clear dif-ference in radio-sensitivity,we wanted to determine whether these differences would be correlated with the level of p53up-regulation/accumulation in these cells following exposure.We analysed p53expression by confocal micros-copy in thymocytes prepared from mice 3h after a 6Gy irradiation (Fig.4b).Interestingly,in thymocytes from unirradiated mice,p53seems to be less expressed in DP than in the other populations.In thymocytes from irradi-ated animals we clearly observed an increase of the level of p53expression in all populations,but it stays higher in DN and SP cells.Some apoptotic cells are also already visible,but they do not express high levels of p53.
Discussion
Altogether,our results show that high radiation doses induce a more severe thymic involution and a longer block of differentiation than an intermediate dose (1·5Gy),which only transiently affects thymocyte development.This differ-ent behaviour most probably re?ects different p53radiation-induced levels/activation states,which result in radiation-dose-dependent transcriptional activation of pro-apoptotic p53target genes [35].A clearer sign of this involution is the disappearance of the prominent DP popu-lation.However,rapidly cycling ISP thymocytes,which con-stitute a much smaller population (approximately 20%of SPCD8thymocytes,i.e.>1%of total thymocytes),are more radio-sensitive:their death is evident as early as 3h post-irradiation,a time at which the DP population is not yet affected.The in?ux of ISP into the DP compartment is
(a)(b)
(c)% o f t h y m o c y t e s
% o f D N
% o f C D 8+S P
100806040200
806040200
30
20100
Fig.3.Fluorescence activated cell sorter (FACS)analysis of unirradiated and irradiated (6Gy,day 1)wild-type and p53
thymocytes.(a)Bar graph showing the average frequency (±standard error)of double-negative (DN),double-positive (DP)and
single-positive (SP)populations in the indicated mice.(b)Bar graph showing the average frequency (±standard error)of DN1,DN2,DN3and DN4populations in the DN thymocytes of mice shown in (a).(c)Bar graph showing the average frequency (±standard error)of immature single-positive (ISP)(left)and selected DP (right)
thymocytes in the SPCD8and DP populations (regions R2and R3,respectively,in Fig.1)of mice shown in (a).Solid dark grey bars:unirradiated p53wild-type,solid light grey bars:irradiated p53wild-type,hatched dark grey bars;unirradiated p53?/?,hatched light grey bars:irradiated p53?/?.The number of mice in each group is as follows:n =6for unirradiated wild-type,n =9for irradiated
wild-type,n =4for unirradiated p53?/?and n =6for irradiated p53?/?.
FOCUS ON DYING AUTOLOGOUS CELLS AS INSTRUCTORS OF THE IMMUNE SYSTEM
A.Gentil Dit Maurin et al .
therefore arrested even before the DP thymocytes begin to die.ISP thymocytes are generated after developing thymocytes traverse the pre-TCR checkpoint.The prolifera-tion signals received ensure a large expansion of the pool of thymocytes that succeed in rearranging and expressing a TCR-βchain and,consequently,will be available to rear-range a TCR-αchain and express a TCR-αβin the DP com-partment.The disruption of this developmental pathway by the early death of ISP therefore participates in the reduction of the number of thymocytes trying to pass positive and negative selection in a highly disturbed environment,where these selection processes may be affected by the presence of cellular debris and/or byproducts,including damage-associated molecular pattern (DAMPs)molecules,resulting from radiation-induced cell death.Interestingly,1·5Gy induces only a mild and very transitory thymic involution.In mice exposed to low-dose radiation,a signi?cant fraction of DP thymocytes will therefore survive and develop in a perturbed environment,rich in DAMPs.It will be of inter-est to determine if the presence of these usually intracellular molecules affects DP selection and modi?es the expressed TCR repertoire.
The dynamics of ISP and DP populations are not syn-chronous.Regeneration of the ISP compartment is already in progress at day 2after exposure to 1·5Gy and is massive at day 4,irrespective of the dose and the resulting level of DP involution at day 2.This kinetics probably denotes an attempt to compensate for radiation-induced DP death and replenish the DP compartment as quickly as possible,once most of the radiation effects have been eliminated.Impor-tantly,in mice exposed to 4and 6Gy,this regeneration takes place in the absence of an increase in the total thymocyte number.This dichotomy suggests that there is no co-ordination of proliferation and differentiation of the different thymocyte populations (at least ISP and DP)fol-lowing radiation exposure,and that the different steps of the thymocyte differentiation programme are uncoupled.In addition,the rapid restoration of the proliferation/differentiation of ISP thymocytes suggests that they are gen-erated from intrathymic radio-resistant precursors,as described previously [36,37].
Our results also show that DP thymocytes respond differ-ently to radiation depending on their differentiation stage.Selected TCR high DP thymocytes are more radio-resistant than their unselected TCR low DP precursors.The transition from TCR low to TCR high follows positive and negative selec-tion of developing thymocytes.Thus,TCR signalling in DP thymocytes modulates radiation sensitivity.This may be due to epigenetic and/or transcriptional changes during dif-ferentiation to the SPCD4or SPCD8compartment [38].However,DP thymocyte selection also results in down-regulation of RAG-1and RAG-2gene expression and the cessation of V(D)J recombination [39–41].Hence,when their TCR speci?city is ?xed and they are no longer per-forming ‘dangerous’genomic rearrangements requiring the generation of ‘physiological’DSBs in a controlled setting [13,16–18],DP thymocytes become less prone to die when more DSBs are generated by radiation.One interpretation could be that rearranging TCR low DP are ‘sensitized’to DSBs,i.e.that their DSB checkpoints are pre-activated [20–22,42]and,therefore,they might be more susceptible to ini-tiate apoptosis when additional radiation-induced DSBs are generated.The ?nding that DN2thymocytes,the popula-tion in which TCR-βgene rearrangement is initiated [26–28],is more radio-sensitive than the other DN populations supports this hypothesis.We would therefore like to propose that the high level of radio-sensitivity observed in DN2and TCR low DP thymocytes is correlated to their V(D)J recombination activity.
Finally,it is well known that radiation-induced p53acti-vation and its outcomes,i.e.induction of apoptosis,cell cycle arrest or senescence,varies largely according to the tissular and/or cellular context [43,44],probably re?ecting both the cell-speci?city of radiation-induced p53activation
(a)(b)p53p53+/+p53–/–
p53S15γH2Ax Tubulin
C o n t r o l
6 G y
C o n t r o l
6 G y
I r r a d i a t i o n
C o n t r o l
CD4/CD8/p53
DNA
CD8
CD4
Fig.4.Up-regulation of p53expression in
irradiated thymocytes.(a)Western blot analysis of p53expression,p53phosphorylation and γH2AX in thymocytes following radiation exposure of wild-type and p53-de?cient
thymocytes.(b)Immuno?uorescent labelling and confocal microscopy of p53expression (green)in CD4(red)and CD8(yellow)labelled thymocytes prepared from control (top)or 6Gy irradiated (bottom)mice (left panels).DNA staining by 4′,6-diamidino-2-phenylindole (DAPI)(blue)is shown in the right panels.Single optical sections are shown with
individual or superposed staining.The CD8labelling is punctuated,because
streptavidin-coupled Alexa 546was used to reveal the anti-CD8antibody binding.*Apoptotic cells;scale bar =10μm.
FOCUS ON DYING AUTOLOGOUS CELLS AS INSTRUCTORS OF THE IMMUNE SYSTEM
p53in radiation-induced thymocyte apoptosis
and extracellular survival/differentiation signals received by the irradiated cells.Bax and p21proteins were,for example, found to be induced differentially in the thymus of irradi-ated mice(5Gy).Bax was more induced in the medulla,the anatomical location of mature SP thymocytes,than in the cortex,where DN and DP thymocytes reside and,even though both Bax and p21induction were heterogeneous, the patterns of their induction were different.This work illustrates the speci?city of p53transcriptional activity in the same organ[45].In this study,we now show that whereas DN2and TCR low DP,both actively rearranging TCR genes,undergo p53-dependent apoptosis following radia-tion exposure,p53-independent radiation-induced cell death occurs in ISP(Fig.3).The high proliferation rate of ISPs cannot explain this different behaviour,as DN2are also cycling[46,47].This difference in p53dependence may re?ect changes in pro-apopotic/survival factors resulting from pre-TCR signalling[48–50].p53has been proposed to enforce thymocyte differentiation checkpoints[20,51,52], and its activity has been shown to be regulated by different pathways in developing thymocytes[50,53].We now show that the basal level of p53expression is regulated develop-mentally during thymocyte maturation in unirradiated mice.Interestingly,it is lower in DP than in the other popu-lations.Radiation induces up-regulation of p53expression as early as3h post-exposure in all the populations,but remains lower in DP thymocytes than in DN and SP cells. Thus,there is clearly no correlation between radio-sensitivity and the level of p53expression,as those cells that will preferentially survive express the highest level of p53,even if all thymocytes were exposed to the same dose of genotoxic radiation.This higher level of p53expression was already present in the steady state.Our results showing that DP radio-sensitivity is regulated by TCR signalling underscore the crucial importance of the cellular context in the determination of cell fate following radiation exposure.
A similar phenomenon can also be observed in periph-eral T cells:apoptosis induction is different in quiescent human naive,effector,central memory and effector memory CD4and CD8human T lymphocytes irradiated ex vivo[54].It is therefore likely that,in vivo,these different T lymphocyte subsets will also respond differently to radia-tion exposure,for example during radiotherapy.In this situ-ation,an eventual difference in radio-sensitivity between effector and regulatory T cells(T regs)could result in critical changes in the tumour-speci?c immune response.In tumour-bearing mice,T regs were found to be relatively more radio-resistant,and therefore enriched both in situ and in the spleen,following irradiation(10Gy)of the tumour [55].In apparent contradiction with these?ndings,pre-operative radiotherapy was found to promote a local increase in proliferating CD8+T cells[56]and radio-chemotherapy to lead to a higher cytotoxic/forkhead box protein3(FoxP3+)T cell ratio due to a decrease in regula-tory T cells[57]in patients with head and neck cancer.T regs were also found to be the more affected intratumoral lymphocyte subset in oesophageal cancer following radio-chemotherapy[58,59].Therefore,in these studies,cumula-tive doses around50Gy,with the eventual administration of chemotherapeutic drugs,result in a relative decrease in T regs.Several factors affecting one or more lymphocyte subsets including,for example,preferential apoptosis, activation/proliferation or intratumoral recruitment may contribute to this effect,and more studies will be needed to determine the role,if any,of differential radio-sensitivity on the dynamics of intratumoral lymphocytes following local irradiation.
Disclosure
None.
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